RU2788130C2 - Living attenuated strain of yellow fever virus, adapted to growth in vero cells, and vaccine composition containing it - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to a living attenuated strain of a yellow fever virus, adapted to growth in Vero cells, obtained from a source 17D sub-strain of the yellow fever virus, which is not adapted to growth in Vero cells, where the specified living attenuated strain of the yellow fever virus is less neurovirulent than the specified source 17D sub-strain of the yellow fever virus.

EFFECT: invention expands the arsenal of remedies for protection of patients from an infection of a yellow fever virus.

17 cl, 15 dwg, 18 tbl, 7 ex

Description

FIELD OF THE INVENTION

The present invention relates to a live attenuated strain of yellow fever virus (YFV) and methods of using it to obtain a vaccine composition against infection caused by YFV.

In particular, a live attenuated YFV strain adapted to grow in Vero cells was derived from a parent live attenuated YFV strain that is not adapted to grow in Vero cells, but rather adapted to grow in embryonated eggs. The live attenuated YFV strain is further characterized by reduced neurovirulence compared to the original live attenuated YFV strain.

BACKGROUND OF THE INVENTION

Yellow fever is a virus-mediated and fatal disease that is prevalent in more than 50 countries located in tropical regions of Africa, Central and South America.

Yellow fever is an acute viral hemorrhagic disease, with some patients experiencing jaundice, which explains the use of the term "yellow". Signs of yellow fever may include fever, headache, jaundice, muscle pain, nausea, vomiting, and fatigue. In addition, a small proportion of patients who are infected with the virus develop severe symptoms and about half of them die within 7 to 10 days.

Yellow fever virus (YFV) belongs to the Flaviviruses family, among which other members are dengue fever virus (DV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), West Nile virus (WNV) and Zika virus (ZV) . YFV consists of a lipoprotein envelope surrounding a nucleocapsid consisting of a capsid protein and a single-stranded, positively polar RNA that is 10,862 nucleotides long. Between the 5'-untranslated (5'UTR) and 3'-untranslated regions (3'UTR), the RNA encodes, from the 5'- to the 3'-end, three structural proteins, namely, the capsid protein (protein C), premembrane / membrane protein (prM/M protein), envelope protein (E protein), and eight non-structural (NS) proteins, namely NS1, NS2A, NS2B, NS3, NS4A, P2k peptide, NS4B and NS5 proteins.

Wild-type YFV is mainly carried by mosquitoes Aedes spp. in Africa and Haemogogus and Sabethes spp. in South America, and there are non-human primate hosts that vary by geographic region. Transmission of YFV generally follows two epidemiological patterns: (1) the urban pattern and (2) the forest pattern (also known as the jungle cycle or woodland). Despite two patterns of transmission, only one clinically significant disease has been identified that causes infection with the same virus. In the Americas, YFV is currently circulating in an endemic forest pattern, resulting in several hundred reports per year of infection in non-immune forest workers. In parallel, the virus circulates in Africa through both urban and forested patterns, and periodically moves beyond its endemic pattern, infecting large numbers of non-immune people in major epidemics.

There are currently no antiviral drugs available to treat yellow fever, and vaccination is critical in preventing the disease. In this regard, two types of live attenuated vaccines against YFV were developed back in the 1930s.

Mayali в Сенегале в 1928 году), и он был пассирован в головном мозге мыши. Однако быстро было доказано, что FNV является слишком нейровирулентным, что привело к обострению заболеваемости поствакцинальным энцефалитом у детей, и от него отказались в начале 1980-х годов (Barrett, 2017).The first corresponds to the French Neurotropic Vaccine (FNV), which was derived from wild-type French viscerotropic virus (FVV isolated from

Mayali in Senegal in 1928), and it was passaged in the brain of a mouse. However, FNV was quickly proven to be too neurovirulent, leading to an exacerbation of the incidence of post-vaccination encephalitis in children, and was abandoned in the early 1980s (Barrett, 2017).

The second approach corresponds to the "17D" strain, which was obtained from a wild-type strain of Asibi (isolated from a mildly diseased human, "Mr. Asibi", in Ghana in 1927) and passaged in mouse and chicken tissues. The 17D vaccine strain lost both viscerotropism and neurovirulence (Monath, 2005).

Currently, six countries are producing vaccine formulations of live attenuated YFV from substrains derived from strain 17D, namely Brazil (substrain 17DD), China (substrain 17D-204), France (substrain 17D-204 Stamaril®), Russia (substrain 17D -213), Senegal (substrain 17D-204) and USA (substrain 17D-204 YF-VAX®) (Barrett, 2017).

To date, all commercially available vaccines have been produced in embryonated hen eggs, a manufacturing process that has been plagued by reliability issues in the past (Barrett, 2017). In particular, there is a shortage of YFV vaccines due to manufacturing problems. Indeed, during the 2016 epidemics in Angola and the Democratic Republic of the Congo, the lack of available vaccine lots necessitated for the first time the need to split doses to adapt to emergency conditions (Barrett; 2017). In addition, the YFV vaccine produced in developing embryonated hen eggs is contraindicated in people who are allergic to eggs.

An alternative to the production of vaccines based on embryonated eggs is the use of suitable cell lines for virus passage, such as mammalian cell lines. Among mammalian cell lines, the Vero cell line is one of the best studied, while providing stability and well-established indicators of the quality and quantity of viral output. Vero cells have received FDA approval and are used worldwide. For example, Vero cells have been used to produce a vaccine against Japanese encephalitis (marketed under the brand name IXIARO®), influenza virus, poliovirus, and rabies virus.

Strategies have emerged in the past and currently to use Vero cells to produce YFV vaccines, and it should be noted that these strategies only focus on the possibility of producing YFV vaccines based on inactivated virus (Hayes, 2010; Beasley et al., 2013; Pereira et al. al., 2015). However, while an inactivated yellow fever vaccine may theoretically appear to be safer, it is unlikely to fully match the long-term protection provided by a single dose of current live attenuated yellow fever vaccines (Hayes, 2010). In addition, in the context of recent yellow fever epidemics, live attenuated vaccines seem to be more appropriate to provide high population coverage with long-term protective immunity against yellow fever in endemic areas.

Specific limitations of live attenuated virus for use in a vaccine are to maintain its attenuation, i.e. that the yellow fever virus be at least as attenuated in terms of neurovirulence and viscerotropism as the live attenuated yellow fever vaccines available on the market; while also being immunogenic enough to protect patients from the underlying disease. In this regard, the achievement of both characteristics, i.e. attenuation and immunogenicity for a given yellow fever strain was not easily feasible, as can be seen, for example, in Monath, 2005.

Accordingly, due to the various obstacles associated with the production of live attenuated YFV vaccines from embryonated hen eggs, there remains a need for alternative methods for the production of live attenuated YFV vaccines.

BRIEF DESCRIPTION OF THE INVENTION

In one aspect, the present invention relates to a live attenuated strain of yellow fever virus adapted to grow in Vero cells, derived from the original substrain of yellow fever virus 17D, which is not adapted to grow in Vero cells, where said live attenuated strain of yellow fever virus is less neurovirulent than the specified parent substrain of yellow fever virus 17D.

In another aspect, the present invention further relates to a live attenuated strain of yellow fever virus containing a nucleic acid that contains:

i) a mutation in the codon of the amino acid at position 480 of the coat protein (E), which results in the substitution of the amino acid valine for leucine, or

ii) a mutation in the codon of the amino acid at position 65 of the non-structural protein 2A (NS2a), which results in the substitution of the amino acid methionine for valine.

In another aspect, the present invention relates to a live attenuated strain of yellow fever virus that contains an envelope protein containing a mutation at position 480 that results in a change from the amino acid valine to leucine.

In another aspect, the present invention relates to a live attenuated strain of yellow fever virus that contains an envelope protein containing a leucine residue at a position in the protein that corresponds to position 480 in SEQ ID NO. 15.

In another aspect, the present invention further relates to a live attenuated yellow fever virus strain comprising a nucleic acid molecule encoding:

(i) an envelope protein containing a mutation at position 480 that results in a change from the amino acid valine to leucine, and

(ii) an NS2a protein containing a mutation at position 65 that results in the substitution of the amino acid methionine for valine.

In another aspect, the present invention relates to a live attenuated strain of yellow fever virus containing a nucleic acid molecule encoding:

(i) an envelope protein that contains a leucine residue at a position in the protein that corresponds to position 480 in SEQ ID NO. 15, and

(ii) an NS2a protein that contains a valine residue at a position in the protein that corresponds to position 65 in SEQ ID NO. sixteen.

In another aspect, the present invention also relates to an immunogenic composition comprising a live attenuated strain of yellow fever virus in accordance with the present invention and a pharmaceutically acceptable carrier.

In yet another aspect, the present invention further relates to a method for producing a live attenuated strain of yellow fever virus adapted to grow in Vero cells, comprising the steps of:

- a) purification of viral genomic RNA of the original live attenuated strain of yellow fever virus, which is not adapted to growth in Vero cells and which is not necessarily adapted to growth in eggs;

- b) transfection of Vero cells with viral genomic RNA purified in step a), resulting in transfected Vero cells;

c) growing the transfected Vero cells obtained in step b) in a culture medium, whereby a first population of yellow fever virus is obtained and then isolated;

d) amplifying the isolated first population of yellow fever virus obtained at the end of step c) by 2-fold or more in fresh Vero cells, resulting in a second population of yellow fever virus;

e) cloning the second yellow fever virus population obtained in step d) by two or more successive plaque clonings using Vero cells, resulting in a plurality of yellow fever virus clones;

- f) amplifying separately each of the isolated clones of yellow fever virus obtained at the end of step e), 2-fold or more in fresh Vero cells, resulting in a plurality of strains of yellow fever virus; and

- g) selecting from said plurality of yellow fever virus strains isolated in step f), one or more live attenuated yellow fever virus strains that are less neurovirulent than the original live attenuated yellow fever virus strain, according to a lethal dose test. 50% mice (MLD ₅₀ ).

In another aspect, the present invention also relates to a live attenuated yellow fever virus strain obtainable by the method of the present invention.

In another aspect, the present invention also relates to a live attenuated strain of yellow fever virus according to the present invention for use in preparing a vaccine.

In another aspect, the present invention relates to a vaccine comprising a live attenuated strain of yellow fever virus according to the present invention for use in the prevention of yellow fever virus infection.

DESCRIPTION OF GRAPHICS

Figure 1: Diagram illustrating the production of a live attenuated strain of yellow fever virus adapted to grow in Vero (vYF) cells at the stage of the home seeding series (pMSL).

Figure 2: Diagram illustrating the analysis of viscerotropism in the A129 mouse model.

Figure 3: graphs illustrating viremia measured by qRT-PCR YF-NS5 in sera collected at D4 and D6 (day 4 and day 6) from A129 mice immunized in D0 PBS (white bar); Stamaril® reference vaccine (black bars) or candidate vYF pMSL vaccines derived from the Stamaril® lineage (TV2212, TV2232 and TV2241; dark gray bars) or from the YF-VAX® lineage (TV3111, TV3112 and TV4221; light gray columns).

Figure 4: Graphs illustrating viral load measured by YF-NS5 qRT-PCR in liver samples collected at D6 and D11 from A129 mice immunized with D0 PBS (white bar); Stamaril® reference vaccine (black bars) or candidate vYF pMSL vaccines derived from the Stamaril® lineage (TV2212, TV2232 and TV2241; dark gray bars) or from the YF-VAX® lineage (TV3111, TV3112 and TV4221; light gray columns).

Figure 5: Graphs illustrating viral load measured by qRT-PCR YF-NS5 in brain samples collected at D6 and D11 from A129 mice immunized with D0 PBS (white bar); Stamaril® reference vaccine (black bars) or candidate vYF pMSL vaccines derived from the Stamaril® lineage (TV2212, TV2232 and TV2241; dark gray bars) or from the YF-VAX® lineage (TV3111, TV3112 and TV4221; light gray columns).

Figure 6: Graphs illustrating viral load measured by YF-NS5 qRT-PCR in spleen samples collected at D6 and D11 from A129 mice immunized with D0 PBS (white bar); Stamaril® reference vaccine (black bars) or candidate vYF pMSL vaccines derived from the Stamaril® lineage (TV2212, TV2232 and TV2241; dark gray bars) or from the YF-VAX® lineage (TV3111, TV3112 and TV4221; light gray columns).

Figure 7: Graphs illustrating Kaplan-Meier survival curves after a single immunization of A129 Stamaril® mice with vYF strains TV221, TV2241, TV3111, TV3112, TV4221 (dashed lines) or TV2232 (solid line).

Figure 8: Diagram illustrating the immunogenicity assay in a hamster model.

Figure 9: Graphs illustrating neutralizing antibody titers specific for a live attenuated yellow fever virus strain as measured by a seroneutralization assay on Vero cells in serum collected at D26 from hamsters immunized at D0 2.5 or 5.5 log ₁₀ CCID ₅₀ /dose of vYF strains (TV2212, TV2232, TV2241, TV3111, TV3112 and TV4221) or Stamaril® reference vaccine. The horizontal line represents the threshold of those who responded to the treatment.

Figure 10: Graphs illustrating neutralizing antibody titers specific for a live attenuated strain of yellow fever virus as measured by a seroneutralization assay on Vero cells in serum collected at D41 from hamsters immunized at D0 and D26 2.5 or 5.5 log ₁₀ CCID ₅₀ /dose of vYF strains (TV2212, TV2232, TV2241, TV3111, TV3112 and TV4221) or Stamaril® reference vaccine. The horizontal line represents the threshold of those who responded to the treatment.

Figure 11: Graphs illustrating the response of neutralizing antibodies in monkeys vaccinated with vYF strain TV3112 compared to existing Stamaril® and YF-VAX® vaccines. The horizontal line represents the detection limit.

Figure 12: Graphs illustrating YF-specific IgM responses from memory B cells in peripheral blood of monkeys vaccinated with a live attenuated strain of vYF TV3112 compared to existing reference vaccines Stamaril® and YF-VAX®. Results are expressed as the percentage of cells secreting IgM antibodies in the total IgM population.

Figure 13: Graphs illustrating YF-specific IgG responses from memory B cells in peripheral blood of monkeys vaccinated with live attenuated strain vYF TV3112 compared to existing reference vaccines Stamaril® and YF-VAX®. Results are expressed as the percentage of cells secreting IgG antibodies in the total IgG population.

Figure 14: Graphs illustrating IFN-γ (upper panels) and IL-2 (lower panels) specific T cell responses in peripheral blood of monkeys vaccinated with vYF strain TV3112 when stimulated with envelope protein (ENV; left panels) or stimulated with non-structural protein 3 (NS3; right panels) and comparison with existing Stamaril® and YF-VAX® vaccines.

Figure 15: Graphs illustrating the viral load in the organs of monkeys vaccinated with the live attenuated strain vYF TV3112 compared to existing reference vaccines Stamaril® and YF-VAX®. Light gray bars and circles represent results obtained with Stamaril® vaccinated monkeys; medium gray bars and squares represent the results of monkeys vaccinated with YF-VAX®; dark gray bars and triangles represent results from monkeys vaccinated with live attenuated strain vYF TV3112. The horizontal line represents the detection limit.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides live attenuated YFV strains adapted to grow in Vero cells that were derived from a parent live attenuated YFV strain that is adapted to grow in embryonated eggs. Live attenuated YFV strains were selected for their reduced neurovirulence in the mouse LD ₅₀ test (MLD ₅₀ ) compared to the original live attenuated YFV strain.

As will become clear from the present invention, the production of YFV by passaging on Vero cells allows the production of stable, highly reproducible, high standard quality and quantity live attenuated YFV strains, which are subsequently suitable for the production of a vaccine against YF infection.

• Various definitions

Within the scope of the present invention, "YFV" refers to yellow fever virus, while the term "vYF" refers to Vero cell-adapted yellow fever virus, i. yellow fever virus adapted to grow in Vero cells.

Therefore, within the scope of the present invention, "yellow fever virus adapted to grow in Vero cells" (vYV) and "yellow fever virus adapted to grow in Vero cells" are intended to be interchangeable terms.

Within the scope of the present invention, a virus adapted to grow in Vero cells is one that has undergone at least 3 consecutive passages on Vero cells. In some embodiments, the virus has undergone approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive passages on Vero cells.

By "passage" can be understood any step in which the virus undergoes at least one replication cycle in Vero cells, in particular any step of transfection, amplification or cloning of the virus in Vero cells.

The expression "live attenuated yellow fever virus" as used herein has the conventional meaning known to one of skill in the art. In some embodiments, this expression refers to a live yellow fever virus having attenuated neurovirulence and/or attenuated viscerotropism.

Within the scope of the present invention, the term "neurovirulence" is intended to refer to the ability of a virus to cross the blood-brain barrier (neuroinvasiveness), replicate in brain tissue (neurotropism), and cause inflammation, neuronal damage and encephalitis (neurovirulence stricto sensu).

Within the scope of the present invention, the term "viscerotropism" refers to the ability of a virus to replicate in extraneural tissues, cause viremia, and damage vital organs, including the liver (Monath, 2005).

In some embodiments, said live attenuated yellow fever virus is at least as attenuated as one of the commercially available live attenuated yellow fever vaccine strains, such as Stamaril® or YF-VAX®.

In some embodiments, said live attenuated yellow fever virus has neurovirulence at least as attenuated as one of the commercially available live attenuated yellow fever vaccine strains, such as Stamaril® or YF-VAX®.

In some embodiments, said live attenuated yellow fever virus has viscerotropism at least as attenuated as one of the commercially available live attenuated yellow fever vaccine strains, such as Stamaril® or YF-VAX®.

In some embodiments, said live attenuated yellow fever virus has neurovirulence and viscerotropism at least as attenuated as one of the commercially available live attenuated yellow fever vaccine strains, such as Stamaril® or YF-VAX®.

The terms "comprising"/"comprises"/"comprise"/"contained" include, respectively, "comprising"/"includes"/"include"/"included", and also "consisting"/"consisting"/"consisting"/"respectively" consisting of", for example, a composition "comprising" an X component may consist solely of an X component, or may include one or more additional components, such as an X component and a Y component.

In this context, "CCID ₅₀ " refers to the 50% cell culture infection dose, i.e., the amount of virus sufficient to induce a cytopathic effect in 50% of inoculated replicated cell cultures, as determined in the dilution endpoint assay in single layer cell cultures. cultures.

In accordance with the standard definitions of the World Health Organization (WHO), the present invention relates to the following definitions (WHO Technical report series, No. 872, 1998).

The terms "Master Seed Lot" ("MSL") or "Primary Seed Lot" as used herein refers to the amount of virus suspension that has been processed in a single production run and has a uniform composition.

The terms "working seed lot" ("WSL") or "secondary seed lot" as used herein refers to the amount of viral suspension that has been processed in one production run and that is uniform in composition, fully characterized, and represents only one passage from MSL. Within the scope of the present invention, material recovered from WSL is used to inoculate embryonated eggs or suitable cell lines in the preparation of a vaccine.

The term "plaque forming unit" (PFU) as used herein refers to the smallest amount of viral suspension that will form plaques in single layer cell cultures.

The term "median mouse lethal dose" (LD ₅₀ or MLD ₅₀ mouse) as used herein refers to the amount of virus suspension that will kill 50% of mice injected with it.

Live attenuated YFV adapted to grow in Vero cells (also referred to as "vYF virus" in the case of YF virus adapted to Vero cells)

In one aspect, the present invention relates to a live attenuated strain of yellow fever virus adapted to grow in Vero cells, derived from the original substrain of yellow fever virus 17D, which is not adapted to grow in Vero cells. In various embodiments, said live attenuated yellow fever virus strain is less neurovirulent than said parent 17D yellow fever virus substrain.

In some embodiments, the parent strain of yellow fever virus is a live attenuated strain of yellow fever virus adapted to grow in eggs.

In some embodiments, the eggs are embryonated chicken eggs.

"Substrain 17D" is a yellow fever strain having strain 17D among its progenitors.

The term "strain 17D" has a common meaning known to a person skilled in the art. In some embodiments, "strain 17D" refers to a yellow fever strain that was isolated from a mildly ill human, "Mr. Asibi", in Ghana in 1927, and passaged 18 times in minced mouse embryonic tissue, and then passaged 58 times in minced chick embryo tissue as described in Monath (2005).

In some embodiments, the 17D substrain encompasses the 17D-204 substrain, the 17DD substrain, and/or the 17D-213 substrain, as described in Monath (2005). In an exemplary embodiment, the RNA sequence of YFV strain 17D-204 (Genbank accession number X03700) previously disclosed by Rice et al. in 1985, may be represented by the RNA sequence under SEQ ID NO. 1.

In some embodiments, the parent strain of yellow fever virus is a substrain of yellow fever virus 17D-204.

In some embodiments, the YFV parent strain is YF-VAX® derived from YFV 17D-204, which is the YFV reference strain used in the commercially available YF-VAX® vaccine.

In an exemplary embodiment, the RNA sequence of the YF-VAX® strain derived from YFV 17D-204 can be represented by the RNA sequence of SEQ ID NO. 2.

In some embodiments, the YFV parent strain is a Stamaril® strain derived from YFV 17D-204, which is the YFV reference strain used in the commercially available Stamaril® vaccine.

In an exemplary embodiment, the RNA sequence of the Stamaril® strain derived from YFV 17D-204 may be the RNA sequence of SEQ ID NO. 3.

In an exemplary embodiment, the original 17D yellow fever virus substrain contains the RNA sequence of SEQ ID NO. 2.

In an exemplary embodiment, the original 17D yellow fever virus substrain contains the RNA sequence of SEQ ID NO. 3.

In an exemplary embodiment, the RNA sequence of YFV strain 17D-213 (Genbank accession number U17067) previously disclosed by Dos Santos et al. in 1995, may be represented by the RNA sequence under SEQ ID NO. 4 and the RNA sequence of YFV strain 17DD (Genbank accession number U17066), also previously disclosed by Dos Santos et al. in 1995, may be represented by the RNA sequence under SEQ ID NO. five.

In an exemplary embodiment, the Asibi strain RNA sequence (Genbank accession number KF769016) can be represented by the RNA sequence of SEQ ID NO. 6.

In some embodiments, the live attenuated yellow fever virus strain is less neurovirulent than the parent 17D yellow fever virus substrain in a 50% mouse lethal dose (MLD ₅₀ ) test.

In some embodiments, an appropriate mouse lethal dose 50 (MLD ₅₀ ) test is performed according to the protocol disclosed on page 68 of WHO Technical report series No. 872, 1998 (incorporated by reference).

Within the scope of the present invention, MLD ₅₀ is the amount of viral suspension estimated to cause fatal specific encephalitis in 50% of intracerebral vaccinated mice.

In some embodiments, appropriate serial dilutions of the reconstituted vaccine are made in phosphate buffer, 0.75% serum albumin.

In an exemplary embodiment, mice aged 4-6 weeks are administered intracerebral under anesthesia with an extemporaneous dilution of the vaccine. Groups of at least 6 mice each are used for each dilution, and the series of dilutions should result in post-inoculation mortality ranging from 0-100%. Deaths are recorded within 21 days. Mice dying from unrelated causes are excluded from both the numerator and denominator of mortality calculations. Mice that are paralyzed on the twenty-first day are considered alive.

In certain embodiments, neurovirulence according to the 50% mouse lethal dose test (MLD ₅₀ ) can be measured using the log ₁₀ MLD ₅₀ /ml parameter.

In some embodiments, a live attenuated YFV strain according to the present invention achieves a log ₁₀ MLD ₅₀ /mL that is 4 or less, 3.5 or less, 3 or less, or 2.5 or less in a dose determination test. , lethal for 50% of mice (MLD ₅₀ ).

In one embodiment, the live attenuated yellow fever virus strain of the present invention is adapted to grow in VERO cells, is less neurovirulent than its parent 17D yellow fever virus substrain, and is at least as attenuated for viscerotropism as its parent substrain. yellow fever virus 17D.

In one embodiment, the live attenuated yellow fever virus strain of the present invention is adapted to grow in VERO cells, is less neurovirulent than its parent 17D yellow fever virus substrain, is at least as attenuated for viscerotropism as its parent virus substrain. yellow fever virus 17D, and is at least as immunogenic as its parent substrain of yellow fever virus 17D.

In various embodiments, the present invention provides a live attenuated YFV strain comprising the RNA sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5, where one or more nucleotides are mutated.

Within the scope of the present invention, the expression "one or more nucleotides" encompasses 2, 3, 4, 5 or more nucleotides.

In other words, the expression "one or more nucleotides" is intended to cover 1 nucleotide, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 or more nucleotides.

In some embodiments, the mutation is a nucleotide change.

In some other embodiments, the implementation of the mutation does not include the insertion of a nucleotide and the deletion of a nucleotide.

In some embodiments, the nucleotide substitution is silent. Alternatively, a nucleotide substitution may promote amino acid substitution.

In one embodiment, two nucleotides are mutated in the RNA sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. five.

In another embodiment, three nucleotides are mutated in the RNA sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. five.

In an additional embodiment, four nucleotides are mutated in the RNA sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. five.

In an additional embodiment, five nucleotides are mutated in the RNA sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. five.

In another aspect, the present invention also relates to a live attenuated strain of yellow fever virus that contains an RNA sequence selected from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, where at least the nucleotide at position 2411, position 3701, or position 6496 is mutated.

In some embodiments, the live attenuated YFV strain contains an RNA sequence selected from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, where at least the nucleotide at position 2411 and the nucleotide at position 3701 are mutated.

In some embodiments, the live attenuated YFV strain contains an RNA sequence selected from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, where at least the nucleotide at position 2411 and the nucleotide at position 6496 are mutated.

In some embodiments, the live attenuated YFV strain contains an RNA sequence selected from SEQ ID NO. 1, SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, where at least the nucleotide at position 3701 and the nucleotide at position 6496 are mutated.

In some embodiments, the live attenuated YFV strain contains an RNA sequence selected from SEQ ID NO. 1, SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, where at least the nucleotide at position 2411, the nucleotide at position 3701 and the nucleotide at position 6496 are mutated.

In certain embodiments, nucleotide G (guanosine) at position 2411 of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 is substituted by nucleotide U (uridine).

In certain embodiments, nucleotide A (adenosine) at position 3701 of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 is substituted with nucleotide G (guanosine).

In certain embodiments, nucleotide A (adenosine) at position 6496 of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 is substituted with nucleotide G (guanosine).

In some embodiments, the live attenuated YFV strain is characterized as follows:

- (i) nucleotide G (guanosine) at position 2411 of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 is substituted with a U nucleotide (uridine), and

- (ii) nucleotide A (adenosine) at position 3701 of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 is substituted with nucleotide G (guanosine).

In some other embodiments, the live attenuated YFV strain is characterized as follows:

- (i) nucleotide G (guanosine) at position 2411 of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 is substituted with a U nucleotide (uridine), and

- (ii) nucleotide A (adenosine) at position 6496 of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 is substituted with nucleotide G (guanosine).

In some other embodiments, the live attenuated YFV strain is characterized as follows:

- (i) nucleotide A (adenosine) at position 3701 of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 is substituted with nucleotide G (guanosine), and

- (ii) nucleotide A (adenosine) at position 6496 of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 is substituted with nucleotide G (guanosine).

In certain embodiments, the live attenuated YFV strain is characterized as follows:

- (i) nucleotide G (guanosine) at position 2411 of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 is substituted by nucleotide U (uridine),

- (ii) nucleotide A (adenosine) at position 3701 of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 is substituted with nucleotide G (guanosine); and

- (iii) nucleotide A (adenosine) at position 6496 of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 is substituted with nucleotide G (guanosine).

In some embodiments, the live attenuated YFV strain further comprises a mutation located at position 1408 of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. five.

In some embodiments, the live attenuated YFV strain contains an RNA sequence selected from the group containing SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5, where at least the nucleotide at position 1408 and the nucleotide at position 2411 are mutated.

In some embodiments, the live attenuated YFV strain contains an RNA sequence selected from the group containing SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, wherein at least the nucleotide at position 1408 and the nucleotide at position 3701 are mutated.

In some embodiments, the live attenuated YFV strain contains an RNA sequence selected from the group containing SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, where at least the nucleotide at position 1408 and the nucleotide at position 6496 are mutated.

In some embodiments, the live attenuated YFV strain contains an RNA sequence selected from the group containing SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, where at least the nucleotide at position 1408, the nucleotide at position 2411 and the nucleotide at position 3701 are mutated.

In some embodiments, the live attenuated YFV strain contains an RNA sequence selected from the group containing SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, where at least the nucleotide at position 1408, the nucleotide at position 2411 and the nucleotide at position 6496 are mutated.

In some embodiments, the live attenuated YFV strain contains an RNA sequence selected from the group containing SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, where at least the nucleotide at position 1408, the nucleotide at position 3701 and the nucleotide at position 6496 are mutated.

In some embodiments, the live attenuated YFV strain contains an RNA sequence selected from the group containing SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, where at least the nucleotide at position 1408, the nucleotide at position 2411, the nucleotide at position 3701 and the nucleotide at position 6496 are mutated.

In some embodiments, nucleotide A (adenine) at position 1408 of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 is substituted by nucleotide U (uridine).

In certain embodiments, the live attenuated YFV strain is characterized as follows:

- (i) nucleotide G (guanosine) at position 2411 of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 is substituted by nucleotide U (uridine),

- (ii) nucleotide A (adenosine) at position 3701 of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 is substituted with nucleotide G (guanosine);

- (iii) nucleotide A (adenosine) at position 6496 of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 is substituted with nucleotide G (guanosine); and

- (iv) nucleotide A (adenosine) at position 1408 of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 is substituted by nucleotide U (uridine).

In some embodiments, a live attenuated YFV strain of the present invention contains an RNA sequence selected from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, where at least one or more nucleotides at position 2411, 3701, 6496 and optionally 1408 is/are mutated, provided that none of the nucleotides is mutated in such a way as to result in a reversal of the genotype Asibi (which may be represented by the RNA sequence of SEQ ID NO. 6). In other words, if the nucleotide in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 is different from the nucleotide at the same position in the Asibi genome (in SEQ ID NO. 6), then that nucleotide in the RNA sequence of the live attenuated YFV strain according to the present invention is not mutated to become the nucleotide at the same position in the genome Asibi (in SEQ ID NO. 6). Nucleotides from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 that differ from nucleotides at the same positions in the Asibi genome can be easily identified by sequence alignment (Needleman and Wunsch, (1970)) between SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 and the Asibi sequence (SEQ ID NO. 6).

In some embodiments, a live attenuated YFV strain of the present invention comprises an RNA sequence comprising SEQ ID NO. 2, where at least one or more nucleotides at position 2411, 3701, 6496 and optionally 1408 is/are mutated(s), provided that the nucleotides at the following positions in SEQ ID NO. 2 are not mutated so as to result in reversion to the Asibi genotype (SEQ ID NO. 6): 304, 370, 854, 883, 1127, 1140, 1431, 1482, 1491, 1572, 1750, 1819, 1870, 1887, 1946, 1965, 2112, 2193, 2219, 2356, 2687, 3371, 3613, 3817, 3860, 3925, 4007, 4013, 4022, 4054, 4056, 4289, 4387, 4505, 4507, 4612, 4864, 4873. 5153, 5194, 5362, 5431, 5473, 5926, 6023, 6448, 6876, 7171, 7496, 7571, 7580, 7642, 7701, 7945, 8008, 8629, 10142, 10285, 10312, 10338, 10367, 10418, 10550 и 10800.

Table 1. Nucleotide differences between SEQ ID NO. 2 and the RNA sequence of the Asibi genome (SEQ ID NO. 6)

The position of the nucleotide in SEQ ID NO. 2 Nucleotide position in Asibi KF769016 Nucleotide in SEQ ID NO. 2 The position of an amino acid in a protein Amino acid in Asibi KF769016 The amino acid in SEQ ID NO. 2 304 G A C-62 T T 370 U C C-84 V V 854 C U M-36 L F 883 A G M-45 T T 1127 G A E-52 G R 1140 C U E-56 A V 1431 A C E-153 N T 1482 C U E-170 A V 1491 C U E-173 T I 1572 A C E-200 L T 1750 C U E-258 T T 1819 C U E-281 S S 1870 G A E-299 M I 1887 C U E-305 S F 1946 C U E-325 P S 1965 A G E-331 K R 2112 C G E-380 T R 2193 C U E-407 A V 2219 G A E-416 A T 2356 C U E-460 L L 2687 C U NS1-79 L F 3371 A G NS1-307 I V 3613 G A NS2a-35 V V 3817 A G NS2a-103 V V 3860 A G NS2a-118 R V 3925 A U NS2a-139 V V 4007 A G NS2a-167 T A 4013 C U NS2a-169 P F 4022 A G NS2a-172 T A 4054 C U NS2a-182 N N 4056 C U NS2a-183 S F 4289 A C NS2b-37 I L 4387 A G NS2b-69 G G 4505 A C NS2b-108 I L 4507 U C 4612 U C NS3-14 I I 4864 A G NS3-98 Q Q 4873 U G NS3-101 A A 5153 A G NS3-195 I V 5194 U C NS3-208 F F 5362 C U NS3-264 A A 5431 C U NS3-287 I I 5473 C U NS3-301 A A 5926 C U NS3-452 R R 6023 G A NS3-485 D N 6448 G U NS4a-3 A A 6876 U C P2k-20 V A 7171 A G NS4b-95 I M 7496 U C NS4b-204 L L 7571 C A NS4b-229 R R 7580 U C NS4b-232 Y H 7642 U C NS5-2 S S 7701 A G NS5-22 Q R 7945 C U NS5-103 F F 8008 U C NS5-124 I I 8629 C U NS5-331 Y Y 10142 G A NS5-836 E K 10285 U C NS5-883 Y Y 10312 A G NS5-892 R R 10338 C U NS5-901 P L 10367 U C 3'UTR - - 10418 U C 3'UTR - - 10550 U C 3'UTR - - 10800 G A 3'UTR - -

In some embodiments, the live attenuated YFV strain of the present invention contains the RNA sequence of SEQ ID NO. 7. Preferably, the live attenuated YFV strain of the present invention contains an RNA sequence that differs by a limited number of mutations, such as no more than 5, no more than 4, no more than 3, or no more than 2 of SEQ ID NO. 7. Preferably, the live attenuated YFV strain of the present invention contains an RNA sequence that differs by a limited number of mutations, such as no more than 5, no more than 4, no more than 3, or no more than 2 of SEQ ID NO. 7 provided that none of the nucleotides is mutated in such a way as to result in reversion to the Asibi genotype. In an exemplary embodiment, the genomic RNA sequence of a live attenuated YFV strain according to the present invention may consist of the nucleotide sequence of SEQ ID NO. 7.

In some embodiments, the live attenuated YFV strain of the present invention contains the RNA sequence of SEQ ID NO. 8. Preferably, the live attenuated YFV strain of the present invention contains an RNA sequence that differs by a limited number of mutations, such as no more than 5, no more than 4, no more than 3, or no more than 2 of SEQ ID NO. 8. Preferably, the live attenuated YFV strain of the present invention contains an RNA sequence that differs by a limited number of mutations, such as no more than 5, no more than 4, no more than 3, or no more than 2 of SEQ ID NO. 8, provided that none of the nucleotides is mutated in such a way as to result in a reversion to the Asibi genotype. In an exemplary embodiment, the genomic RNA sequence of a live attenuated YFV strain according to the present invention may consist of the nucleotide sequence of SEQ ID NO. 8.

As mentioned above, the YFV nucleic acid encodes 11 proteins, as shown below:

- capsid protein (protein C), the length of the precursor of which is 121 a.a., and the length of the mature protein is 101 a.k.,

- premembrane protein (protein prM) 164 a.a. long, which is the precursor of the membrane protein (protein M), which is 75 a.a. long,

- shell protein (protein E), the length of which is 493 a.k.,

- non-structural protein 1 (NS1), the length of which is 352 a.k.,

- non-structural protein 2a (NS2a), the length of which is 224 a.k.,

- non-structural protein 2b (NS2b), whose length is 130 a.a.,

- non-structural protein 3 (NS3), the length of which is 623 a.k.,

- non-structural protein 4a (NS4a), the length of which is 126 a.k.,

- non-structural peptide P2k, the length of which is 23 a.k.,

- non-structural protein 4b (NS4b), the length of which is 250 a.a.,

- non-structural protein 5 (NS5), the length of which is 905 a.k.

In some embodiments, a live attenuated strain of yellow fever virus comprises a nucleic acid containing a mutation at the amino acid codon at position 480 of the envelope protein (E), a mutation at the amino acid codon at position 65 of the nonstructural protein 2A (NS2a), a mutation at the amino acid codon at position 19 of the nonstructural protein 4A (NS4a) and/or a mutation in the amino acid codon at position 145 of the envelope protein (E).

In some embodiments, the live attenuated strain of yellow fever virus comprises a nucleic acid containing a mutation at the amino acid codon at position 480 of the envelope protein (E) and a mutation at the amino acid codon at position 65 of the nonstructural protein 2A (NS2a).

In some embodiments, a live attenuated strain of yellow fever virus comprises a nucleic acid containing a mutation at the amino acid codon at position 480 of the envelope protein (E), a mutation at the amino acid codon at position 65 of the nonstructural protein 2A (NS2a), and a mutation at the amino acid codon at position 19 of the nonstructural protein 4A (NS4a).

In some embodiments, a live attenuated strain of yellow fever virus comprises a nucleic acid containing a mutation at the amino acid codon at position 480 of the envelope protein (E), a mutation at the amino acid codon at position 65 of the nonstructural protein 2A (NS2a), a mutation at the amino acid codon at position 19 of the nonstructural protein 4A (NS4a) and a mutation in the amino acid codon at position 145 of the envelope protein (E).

In some embodiments, the live attenuated strain of yellow fever virus contains a nucleic acid comprising:

i) a mutation in the codon of the amino acid at position 480 of the coat protein (E), which results in the substitution of the amino acid valine for leucine, or

ii) a mutation in the codon of the amino acid at position 65 of the non-structural protein 2A (NS2a), which results in the substitution of the amino acid methionine for valine.

In some embodiments, a live attenuated strain of yellow fever virus according to the present invention comprises a nucleic acid comprising:

i) a mutation in the codon of the amino acid at position 480 of the coat protein (E), which results in the substitution of the amino acid valine for leucine, and

ii) a mutation in the codon of the amino acid at position 65 of the non-structural protein 2A (NS2a), which results in the substitution of the amino acid methionine for valine.

In some embodiments, the nucleic acid further comprises a mutation at the amino acid codon at position 19 of non-structural protein 4A (NS4a), which results in a codon change from AAA to AAG.

In some embodiments, a mutation in the amino acid codon at position 480 of the envelope protein (E) results in a codon change from GUA to UUA, UUG, CUU, CUC, CUA, or CUG. In an embodiment, there is a codon change from GUA to UUA.

In some embodiments, a mutation in the amino acid codon at position 65 of non-structural protein 2A (NS2a) results in a codon change from AUG to GUG, GUU, GUC, or GUA. In an embodiment, there is a codon change from AUG to GUG.

In some embodiments, the nucleic acid further comprises a mutation in the amino acid codon at position 145 of the envelope protein (E), which results in the GUA codon being changed to GUU.

In some embodiments, a live attenuated strain of yellow fever virus according to the present invention comprises a nucleic acid comprising:

i) a mutation in the codon of the amino acid at position 480 of the envelope protein (E), which results in the substitution of the amino acid valine for leucine;

ii) a mutation in the codon of the amino acid at position 65 of the non-structural protein 2A (NS2a), which results in the substitution of the amino acid methionine for valine; and

iii) a mutation in the amino acid codon at position 19 of the non-structural protein 4A (NS4a), which results in a codon change from AAA to to AAG.

In some embodiments, a live attenuated strain of yellow fever virus according to the present invention comprises a nucleic acid comprising:

i) a mutation in the codon of the amino acid at position 480 of the envelope protein (E), which results in the substitution of the amino acid valine for leucine;

ii) a mutation in the codon of the amino acid at position 65 of the non-structural protein 2A (NS2a), which results in the substitution of the amino acid methionine for valine;

iii) a mutation in the amino acid codon at position 19 of the non-structural protein 4A (NS4a), which results in a codon change from AAA to AAG; and

iv) a mutation in the amino acid codon at position 145 of the envelope protein (E), which results in a change from the GUA codon to GUU.

In some embodiments, a live attenuated strain of yellow fever virus contains an envelope protein containing a mutation at position 480. In particular, a live attenuated strain of yellow fever virus in accordance with the present invention contains an envelope protein containing a mutation at position 480, which results in valine amino acids to leucine.

In some embodiments, the live attenuated strain of yellow fever virus comprises an envelope protein containing a leucine residue at a position in the protein that corresponds to position 480 in SEQ ID NO. 15. In particular, said envelope protein contains a sequence that is at least 90%, 95%, 98%, or 100% identical to the sequence under SEQ ID NO. 15.

In particular, in the nucleic acid of the live attenuated yellow fever virus strain of the present invention, no nucleotide is mutated in such a way as to result in reversal of the Asibi genotype (SEQ ID NO. 6). For example, the nucleic acid of a live attenuated strain of yellow fever virus does not contain nucleotide mutations at the following positions in SEQ ID NO. 2 so as to result in reversion to the Asibi genotype (SEQ ID NO. 6): 304, 370, 854, 883, 1127, 1140, 1431, 1482, 1491, 1572, 1750, 1819, 1870, 1887, 1946 1965, 2112, 2193, 2219, 2356, 2687, 3371, 3613, 3817, 3860, 3925, 4007, 4013, 4022, 4054, 4056, 4289, 4387, 4505, 4507, 4612, 4864, 4873, 5153, 5194, 5194, 5194, 5194 , 5362, 5431, 5473, 5926, 6023, 6448, 6876, 7171, 7496, 7571, 7580, 7642, 7701, 7945, 8008, 8629, 10142, 10285, 10312, 10338, 10367, 10418, 10550 and 10800.

In some embodiments, the live attenuated strain of yellow fever virus contains a nucleic acid molecule encoding:

(i) an envelope protein containing a mutation at position 480, and

(ii) an NS2a protein containing a mutation at position 65.

In particular, the nucleic acid molecule of a live attenuated yellow fever virus strain additionally contains a mutation in the amino acid codon at position 19 of the non-structural protein 4A (NS4a) and/or a mutation in the amino acid codon at position 145 of the envelope protein (E).

In some embodiments, a live attenuated strain of yellow fever virus according to the present invention comprises a nucleic acid molecule encoding:

(i) an envelope protein containing a mutation at position 480 that results in a change from the amino acid valine to leucine, and

(ii) an NS2a protein containing a mutation at position 65 that results in the substitution of the amino acid methionine for valine.

In particular, the nucleic acid further comprises a mutation at the amino acid codon at position 19 of the non-structural protein 4A (NS4a), which results in a codon change from AAA to AAG, and/or a mutation at the amino acid codon at position 145 of the envelope protein (E), which results in resulting in a GUA to GUU codon change.

In particular, in the nucleic acid of the live attenuated yellow fever virus strain of the present invention, no nucleotide is mutated in such a way as to result in reversal of the Asibi genotype (SEQ ID NO. 6). For example, the nucleic acid of a live attenuated strain of yellow fever virus does not contain nucleotide mutations at the following positions in SEQ ID NO. 2 so as to result in reversion to the Asibi genotype (SEQ ID NO. 6): 304, 370, 854, 883, 1127, 1140, 1431, 1482, 1491, 1572, 1750, 1819, 1870, 1887, 1946 1965, 2112, 2193, 2219, 2356, 2687, 3371, 3613, 3817, 3860, 3925, 4007, 4013, 4022, 4054, 4056, 4289, 4387, 4505, 4507, 4612, 4864, 4873, 5153, 5194, 5194, 5194, 5194 , 5362, 5431, 5473, 5926, 6023, 6448, 6876, 7171, 7496, 7571, 7580, 7642, 7701, 7945, 8008, 8629, 10142, 10285, 10312, 10338, 10367, 10418, 10550 and 10800.

In some embodiments, a live attenuated strain of yellow fever virus according to the present invention comprises a nucleic acid molecule encoding:

(i) an envelope protein that contains a leucine residue at a position in the protein that corresponds to position 480 in SEQ ID NO. 15, and

(ii) an NS2a protein that contains a valine residue at a position in the protein that corresponds to position 65 in SEQ ID NO. sixteen.

In particular, said envelope protein contains a sequence that is at least 90%, 95%, 98%, or 100% identical to the sequence under SEQ ID NO. 15, and said NS2a protein contains a sequence that is at least 90%, 95%, 98%, or 100% identical to the sequence under SEQ ID NO. sixteen.

The nucleic acid of the live attenuated yellow fever virus strain of the present invention may further comprise a G nucleotide at a position in the nucleic acid encoding non-structural protein 4A (NS4a) that corresponds to position 57 in SEQ ID NO. 17 and/or a U nucleotide at the position in the nucleic acid encoding the envelope protein (E) which corresponds to position 435 in SEQ ID NO. 18. In particular, the live attenuated strain of yellow fever virus of the present invention may comprise a nucleic acid molecule containing a nucleic acid encoding a non-structural protein 4A (NS4a) that contains a sequence of at least 90%, 95%, 98%, or 100 % identical to the sequence under SEQ ID NO. 17 and/or a nucleic acid encoding an envelope protein (E) which contains a sequence at least 90%, 95%, 98% or 100% identical to the sequence under SEQ ID NO. eighteen.

In particular, in the nucleic acid of the live attenuated yellow fever virus strain of the present invention, no nucleotide is mutated in such a way as to result in reversal of the Asibi genotype (SEQ ID NO. 6). For example, the nucleic acid of a live attenuated strain of yellow fever virus does not contain nucleotide mutations at the following positions in SEQ ID NO. 2 so as to result in reversion to the Asibi genotype (SEQ ID NO. 6): 304, 370, 854, 883, 1127, 1140, 1431, 1482, 1491, 1572, 1750, 1819, 1870, 1887, 1946 1965, 2112, 2193, 2219, 2356, 2687, 3371, 3613, 3817, 3860, 3925, 4007, 4013, 4022, 4054, 4056, 4289, 4387, 4505, 4507, 4612, 4864, 4873, 5153, 5194, 5194, 5194, 5194 , 5362, 5431, 5473, 5926, 6023, 6448, 6876, 7171, 7496, 7571, 7580, 7642, 7701, 7945, 8008, 8629, 10142, 10285, 10312, 10338, 10367, 10418, 10550 and 10800.

In some embodiments, the implementation of the nucleic acid contains the RNA sequence of the 17D substrain containing mutations in accordance with the present invention, as described above.

In some embodiments, the implementation of the nucleic acid contains the RNA sequence of the 17D-204 substrain containing mutations in accordance with the present invention, as described above.

In some embodiments, the implementation of the nucleic acid contains an RNA sequence under SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO.5 containing mutations in accordance with the present invention, as described above.

In an exemplary embodiment, the nucleic acid comprises an RNA sequence under SEQ ID NO. 2 containing mutations in accordance with the present invention, as described above.

As will be seen in the examples section of this document below, the above-defined mutations produce YFV strains that are adapted to grow in VERO cells and have attenuated virulence, such as attenuated neurovirulence, compared to the original YFV strain, and whose virulence is compatible with the use of these strains as vaccines or in vaccine compositions. In an embodiment, the above-defined mutations produce YFV strains adapted to grow in VERO cells that are less neurovirulent than the parental YFV strain and at least as attenuated in viscerotropism as the parental YFV strain. In an embodiment, the above-defined mutations produce YFV strains adapted to grow in VERO cells that are less neurovirulent than the original YFV strain, at least as viscerotropism attenuated as the original YFV strain, and at least as immunogenic. , like the original YFV strain.

• Immunogenic, vaccine and pharmaceutical compositions

In another aspect, the present invention also relates to an immunogenic composition containing a live attenuated YFV strain in accordance with the present invention.

Within the scope of the present invention, the term "immunogenic" refers to the ability of a composition to stimulate antibody-mediated and/or cell-mediated immunity and/or immunological memory.

In some embodiments, the implementation of the immunogenic composition can be used to generate neutralizing antibodies against yellow fever virus.

In another aspect, the present invention further relates to an immunogenic composition comprising a live attenuated strain of yellow fever virus according to the present invention and a pharmaceutically acceptable carrier.

In some embodiments, the implementation of the present invention also relates to a vaccine composition containing a live attenuated strain of the virus in accordance with the present invention and/or a vaccine composition containing an immunogenic composition in accordance with the present invention.

In some embodiments, the implementation of the vaccine composition may not contain any adjuvant.

Within the scope of the present invention, "adjuvant" refers to any substance intended to enhance the respective immune response and subsequent clinical efficacy of the vaccine.

Alternatively, the vaccine composition may further comprise one or more adjuvants.

In some embodiments, the adjuvant may comprise a mineral salt, an emulsion, a natural or synthetic derivative derived from microorganisms, a combination adjuvant, an adjuvant derived from cytokines, or an adjuvant derived from additional molecules, a particulate formulation, and the like. . The preparation and use of aids is well known in the art.

In some embodiments, the present invention provides an immunogenic composition comprising a live attenuated YFV strain as described herein and a pharmaceutically acceptable carrier.

In some embodiments, the present invention provides a pharmaceutical composition comprising a live attenuated YFV strain as described herein and a pharmaceutically acceptable carrier.

In the context of the present invention, the expression "pharmaceutically acceptable carrier" refers to a carrier that is physiologically acceptable for administration to a human, while maintaining the physiological activity of the immunogenic composition in accordance with the present invention, i. its ability to induce an immune response. One illustrative example of a pharmaceutically acceptable carrier is physiological saline buffer. Other physiologically acceptable carriers are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences ( ^18th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, PA, USA. The immunogenic composition described herein may optionally contain pharmaceutically acceptable adjuvants necessary to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity agents, wetting agents, and the like. In addition, the vaccine composition may optionally contain pharmaceutically acceptable additives, including, for example, diluents, binders, stabilizers, and preservatives.

In various embodiments, the implementation of the pH of the immunogenic composition is from 5.5 to 8, such as from 6.5 to 7.5 (eg, about 7). A stable pH can be maintained with a buffer. Thus, in some embodiments, the implementation of the immunogenic composition contains a buffer. The immunogenic compositions may be isotonic with respect to the human body. The immunogenic composition may also contain one or more additional salts such as NaCl. The preparation and use of pharmaceutically acceptable carriers is well known in the art.

In practice, an immunogenic composition and/or a vaccine composition and/or a pharmaceutical composition containing a live attenuated YFV strain of the present invention can be prepared using conventional and best practices in the art.

In some embodiments, the implementation of the immunogenic composition, vaccine composition and/or pharmaceutical composition in accordance with the present invention may contain one or more suitable diluents and/or excipients.

In various embodiments, pharmaceutical compositions, immunogenic compositions, and vaccine compositions may be sterilized using conventional sterilization techniques, or may be sterilized by filtration. The resulting aqueous solutions can be packaged and stored in liquid form or lyophilized, wherein the lyophilized preparation is reconstituted with a sterile aqueous vehicle prior to administration. In an exemplary embodiment, pharmaceutical compositions, immunogenic compositions, and vaccine compositions are packaged and stored as micropellets via the prilling process described in WO 2009/109550. In an embodiment, the pharmaceutical compositions, immunogenic compositions, and/or vaccine compositions are lyophilized or spray-freeze dried.

Method for obtaining a live attenuated YFV strain

In a further aspect, the present invention relates to a method for producing a live attenuated strain of yellow fever virus adapted to grow in Vero cells, comprising the steps of:

- a) purification of viral genomic RNA of the original live attenuated strain of yellow fever virus, which is not adapted to growth in Vero cells and which is not necessarily adapted to growth in eggs;

- b) transfection of Vero cells with viral genomic RNA purified in step a), resulting in transfected Vero cells;

c) growing the transfected Vero cells obtained in step b) in a culture medium, whereby a first population of yellow fever virus is obtained and then isolated;

d) amplifying the isolated first population of yellow fever virus obtained at the end of step c) by 2-fold or more in fresh Vero cells, resulting in a second population of yellow fever virus;

e) cloning the second yellow fever virus population obtained in step d) by two or more successive plaque clonings using Vero cells, resulting in a plurality of yellow fever virus clones;

- f) amplifying separately each of the isolated clones of yellow fever virus obtained at the end of step e), 2-fold or more in fresh Vero cells, resulting in a plurality of strains of yellow fever virus; and

- g) selecting from said plurality of yellow fever virus strains isolated in step f), one or more live attenuated yellow fever virus strains that are less neurovirulent than the original live attenuated yellow fever virus strain, according to a lethal dose test. 50% mice (MLD ₅₀ ).

In some embodiments, step d) of the above method of the present invention is performed 2, 3, 4, 5, 6 or more times. In some embodiments, the cloning in step e) of the above method of the present invention is performed with 2, 3, 4, 5, 6 or more consecutive plaque clonings using Vero cells. In some embodiments, step f) of the above method of the present invention is performed 2, 3, 4, 5, 6 or more times.

In a further aspect, the present invention relates to a method for producing a live attenuated strain of yellow fever virus adapted to grow in Vero cells, comprising the steps of:

- a) purification of viral genomic RNA of the original live attenuated strain of yellow fever virus, which is not adapted to growth in Vero cells and which is not necessarily adapted to growth in eggs;

- b) transfection of Vero cells with viral genomic RNA purified in step a), resulting in transfected Vero cells;

c) growing the transfected Vero cells obtained in step b) in a culture medium, whereby a first population of yellow fever virus is obtained and then isolated;

d) amplifying the isolated first population of yellow fever virus obtained at the end of step c) by 2-fold or more in fresh Vero cells, resulting in a second population of yellow fever virus;

e) cloning the second yellow fever virus population obtained in step d) by two or more successive plaque clonings using Vero cells, resulting in a plurality of yellow fever virus clones;

- f) amplifying separately each of the isolated clones of yellow fever virus obtained at the end of step e), 2-fold or more in fresh Vero cells, resulting in a plurality of strains of yellow fever virus; and

- g) selecting from said plurality of yellow fever virus strains isolated in step f), one or more yellow fever virus strains containing a nucleic acid that contains:

i) a mutation in the codon of the amino acid at position 480 of the coat protein (E), which results in the substitution of the amino acid valine for leucine, or

ii) a mutation in the codon of the amino acid at position 65 of the non-structural protein 2A (NS2a), which results in the substitution of the amino acid methionine for valine. Such selection is easily accomplished using sequencing methods well known in the art.

In some embodiments, step g) may involve selecting one or more live attenuated yellow fever virus strains containing a nucleic acid that contains:

i) a mutation in the codon of the amino acid at position 480 of the coat protein (E), which results in the substitution of the amino acid valine for leucine, and

ii) a mutation in the codon of the amino acid at position 65 of the non-structural protein 2A (NS2a), which results in the substitution of the amino acid methionine for valine.

In some embodiments, step g) may involve selecting one or more of the live attenuated yellow fever virus strains described above comprising a nucleic acid further containing a mutation at the amino acid codon at position 19 of non-structural protein 4A (NS4a) that results in a codon change from AAA to AAG.

In some embodiments, step g) may involve selecting one or more of the live attenuated yellow fever virus strains described above, comprising a nucleic acid further containing a mutation at the amino acid codon at position 145 of the envelope protein (E), which results in a codon change from GUA to GUU .

In some embodiments, step g) may involve selecting one or more live attenuated yellow fever virus strains containing a nucleic acid that contains:

i) a mutation in the codon of the amino acid at position 480 of the envelope protein (E) which results in the substitution of the amino acid valine for leucine,

ii) a mutation in the codon of the amino acid at position 65 of the non-structural protein 2A (NS2a), which results in the substitution of the amino acid methionine for valine,

iii) a mutation in the amino acid codon at position 19 of the non-structural protein 4A (NS4a), which results in a codon change from AAA to to AAG, and/or

iv) a mutation in the amino acid codon at position 145 of the envelope protein (E), which results in a change from the GUA codon to GUU.

In one embodiment, the original live attenuated yellow fever virus strain from step a) is a 17D yellow fever virus substrain, such as the 17D-204 yellow fever virus substrain.

In practice, Vero cells are available from cell collections such as ATCC. Methods suitable for growing Vero cells in cell culture in vitro, including methods using serum-free medium, are well known to the person skilled in the art (Kolell K. et al., 2007). In one embodiment, the Vero cells are adapted to grow on a serum-free medium prior to any virus culture.

In some embodiments, the culture medium used to grow Vero cells is serum-free and optionally free of any substance of human or animal origin.

Within the scope of the present invention, the expression "substance of human or animal origin" refers to a substance, such as a protein, lipid, glycoprotein, lipoprotein, glycolipid, monosaccharide or polysaccharide, derived from the body of a human or non-human animal, for example, a growth factor, a hormone that obtained, for example extracted, from a human body or an animal body other than a human. Recombinant molecules are not considered to be of human or animal origin. Such serum-free media and/or media free of any substance of human or animal origin are readily available from vendor catalogs (eg THERMOFISHER SCIENTIFIC® catalogue).

In some embodiments, the culture medium used to grow Vero cells also does not contain antibiotics.

In some embodiments, the culture medium used to grow Vero cells may contain one or more extracts derived from bacteria, yeast, and/or plants.

In some embodiments, the genome of the original live attenuated strain of yellow fever virus not adapted to grow in Vero cells may be in the form of a cDNA encoding a genomic RNA.

In certain embodiments, the implementation of the cDNA is carried by a suitable vector, such as, for example, a plasmid.

In some other aspects, the present invention relates to a vector containing a nucleic acid that contains the RNA sequence under SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 in which the mutation(s) described herein is/are present.

In some other aspects, the present invention relates to a vector containing a nucleic acid that contains a cDNA sequence corresponding to the sequence under SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5, in which there is/are mutations corresponding to the mutation(s) described herein.

In a further aspect, a live attenuated strain of yellow fever virus according to the present invention is obtained by mutating the genomic sequence of yellow fever virus so as to introduce into said genomic sequence the mutations described in the present invention. In some embodiments, the genomic sequence of the 17D yellow fever virus substrain can be mutated to introduce into said genomic sequence the mutations described herein. In some embodiments, the implementation of the nucleic acid containing the RNA sequence under SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 or the corresponding cDNA sequence may be mutated to introduce the mutations described in the present invention. Mutations can be introduced into the genomic sequence by site-directed mutagenesis using methods well known to those skilled in the art, including the use of any suitable genome editing technology. The genomic sequence into which the mutations described in the present invention are introduced may be a cDNA encoding a yellow fever virus genomic RNA, for example, a cDNA encoding a yellow fever virus substrain 17D genomic RNA, for example, a cDNA encoding SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5. In some embodiments, the cDNA is transferred with a suitable plasmid. The mutations described in the present invention that can be introduced into the yellow fever virus genomic sequence are selected from a nucleotide mutation at position 2411, position 3701, or position 6496 of the genomic sequence; or any combination of them. In some embodiments, these mutations may comprise replacing the G (guanosine) nucleotide at position 2411 of the genomic sequence with a U (uridine) nucleotide, wherein the A (adenosine) nucleotide at position 3701 of the genomic sequence is replaced with a G (guanosine) nucleotide; or nucleotide A (adenosine) at position 6496 of the genomic sequence is replaced by nucleotide G (guanosine) or any combination thereof. In some embodiments, an additional mutation is introduced located at position 1408 of the genomic sequence. In some embodiments, this additional mutation is a change from the A (adenine) nucleotide at position 1408 of the genomic sequence to a U (uridine) nucleotide. In some embodiments, other mutations may be introduced into the yellow fever virus genomic sequence, provided that no nucleotide is mutated in a manner that results in reversion to the Asibi genotype. In another aspect, the mutations described herein that can be introduced into the genomic sequence of yellow fever virus are selected from a mutation at the codon of the amino acid at position 480 of the envelope protein (E), which results in a change in the amino acid valine to leucine, a mutation at the codon amino acid at position 65 of non-structural protein 2A (NS2a), which results in a change from the amino acid methionine to valine, or a mutation in the amino acid codon at position 19 of non-structural protein 4A (NS4a), which results in a codon change from AAA to AAG, or any their combinations. In some embodiments, an additional mutation is introduced at the amino acid codon at position 145 of the envelope protein (E), which results in the GUA codon being changed to GUU. In some embodiments, other mutations may be introduced into the yellow fever virus genomic sequence, provided that no nucleotide is mutated in a manner that results in reversion to the Asibi genotype. In particular, the mutations described in the present invention, which can be introduced into the genomic sequence of yellow fever virus, are a mutation in the amino acid codon at position 480 of the envelope protein (E), which results in a change in the amino acid valine to leucine, and a mutation in amino acid codon at position 65 of non-structural protein 2A (NS2a), which results in the substitution of the amino acid methionine for valine.

In a further aspect, the present invention also relates to a live attenuated yellow fever virus strain obtainable by the method of the present invention.

This document also discloses a live attenuated strain of yellow fever virus obtained in accordance with the present invention.

Other methods and uses

The present invention also relates to a method of immunizing an individual in need thereof against a YFV infection, comprising administering to said individual a vaccine composition in accordance with the present invention.

Within the scope of the present invention, the term "individual in need thereof" is intended to refer to an individual at risk of being infected with YFV.

In a further aspect, the present invention also relates to the use of a live attenuated YFV strain according to the present invention for the preparation of a vaccine. In some embodiments, the present invention also relates to the use of a live attenuated YFV strain of the present invention as pMSL, as MSL, or as WSL. In particular, the present invention also relates to the use of a live attenuated YFV strain according to the present invention as pMSL, as MSL or as WSL during the preparation of a vaccine.

In another aspect, the present invention relates to a live attenuated YFV strain according to the present invention for use in preparing a vaccine.

In another additional aspect, the present invention also relates to a vaccine composition in accordance with the present invention for use in the prevention of YFV infection.

In some embodiments, the present invention relates to a method of preventing YFV infection in an individual, comprising administering to said individual an effective amount of a live attenuated YFV, an immunogenic composition, a pharmaceutical composition, or a vaccine composition in accordance with the present invention.

In some embodiments, the present invention relates to a method for generating neutralizing antibodies against yellow fever virus in an individual, comprising administering to said individual an effective amount of a live attenuated YFV, an immunogenic composition, a pharmaceutical composition, or a vaccine composition in accordance with the present invention.

In some embodiments, the present invention relates to the use of a live attenuated virus according to the present invention for the preparation of a medicament for the prevention of YFV infection.

In some embodiments, the present invention provides a live attenuated virus of the present invention for use in preventing YFV infection.

In some embodiments, the present invention relates to the use of an immunogenic composition according to the present invention for the preparation of a medicament for the prevention of YFV infection.

In some embodiments, the present invention relates to the use of an immunogenic composition according to the present invention for the preparation of a vaccine composition for preventing YFV infection.

In some embodiments, the present invention relates to an immunogenic composition according to the present invention for use in preventing YFV infection.

The vaccine composition and immunogenic composition according to the present invention may be administered to an individual in need thereof by any suitable route of administration.

The immunogenic composition or vaccine of the present invention may be administered by any suitable route, such as mucosal administration (eg, intranasal or sublingual), parenteral administration (eg, intramuscular, subcutaneous, transdermal, or intradermal route), or oral administration. As will be appreciated by one of skill in the art, the vaccine of the present invention is suitably formulated to be compatible with the intended route of administration. In exemplary embodiments, the composition of the present invention is administered intramuscularly or subcutaneously.

The vaccine of the present invention can be administered in multiple doses. For example, the vaccine of the present invention can be administered in one, two or three doses. In an embodiment, the vaccine according to the present invention is administered in a single dose.

The vaccine according to the present invention can be administered in amounts that can be easily determined by those of ordinary skill in the art. In some embodiments, the vaccine dose is 4 to 6 log ₁₀ CCID ₅₀ .

EXAMPLES

EXAMPLE 1 Production of Live Attenuated YFV Strains by Adaptation to Vero Cells (Home Seed Series (pMSL))

1.1. Choice of method - principles

The general strategy for pre-primary seed lots (pMSL) is shown in figure 1.

The YF-VAX® and Stamaril® vaccines were developed from preparations of the non-cloned YF17D-204 strain and contained heterologous virus populations as visualized by plaque size phenotype. In addition, both vaccines were obtained in eggs.

To generate homogeneous, well-defined virus strains adapted to grow in Vero cells, and to ensure sterility and the absence of introduced agents in the final pMSL:

- (1) the viral genomic RNA of the YF-VAX® and Stamaril® viruses was purified;

- (2) and then transfected into Vero cells to isolate yellow fever viruses, which were then amplified twice on Vero cells to adapt the viruses to grow on this cell substrate;

- (3) the viruses were then cloned using two rounds of plaque purification. To do this, the viral preparation was diluted to infect Vero cells and grown under a semi-solid layer to obtain well-separated viral plaques. For each transfection, 2 individual plaques, each corresponding to a different viral population, were sampled through the top layer, diluted, and used for a second round of plaque purification resulting in viral clones;

- (4) then these clones were amplified to obtain a sufficient supply of virus to obtain pMSL.

All media and solutions used to obtain pMSL did not contain components of animal and human origin.

1.2. Ways

1.2.1. In vitro transcription from YF-VAX® genomic cDNA

In vitro transcription of the YFV genomic cDNA from YF-VAX® (plasmid pJSY2374.5 disclosed in WO 2014/016360) is carried out using the mMessage mMachine™ SP6 kit (AMBION®, reference AM1340) according to the supplier's recommendations. Simultaneously, in vitro transcriptions were carried out from plasmid pJSY2374.5 2 .

Briefly, after thawing at room temperature, 10 μg of the plasmid was linearized by digestion for 2 hours at 37 ± 2°C with restriction enzyme NruI (30 U/10 μg). The enzyme was then inactivated by incubation at 65° C. for 20 minutes. Plasmid linearization was confirmed by 0.5% agarose gel electrophoresis. Received 40 μl of the reaction mixture containing the reaction buffer kit, ribonucleotides (ATP, CTP, UTP and a mixture of GTP and 7-methyl-GTP), enzyme and 1 μg of the plasmid. The resulting mixture was incubated for 2 hours at 37 ± 2°C.

1.2.2. RNA purification

a) From a working seed lot of Stamaril® vaccine

Simultaneously carried out two purification of viral RNA.

Four vials of Stamaril® vaccine working seed lot (batch # FA238667, infectious titer 6.38 log ₁₀ pfu/vial) were suspended in 200 μl of RNeasy® kit lysis buffer (QIAGEN®) and then pooled. The RNA was then purified by two series of phenol/chloroform/isoamyl alcohol (125:24:1; pH 4.5) extractions.

2 ml Phase Lock Gel Heavy tubes (5PRIME®) were centrifuged for 30 seconds at 11,000 x g. Each tube was injected with 750 μl of RNA/lysis buffer mixture. An equal volume (750 µl) of the phenol/chloroform/IAA solution was then added to each tube. After vigorously mixing the organic and aqueous phases to form a homogeneous temporary suspension, the tubes were centrifuged at 11,000×g for 5 minutes to separate the phases. The upper phase (aqueous phase) was then recovered. The operation was restarted in new 2 ml Phase Lock Gel tubes. The operation was then carried out again with a mixture of chloroform and isoamyl alcohol (24:1) to remove all phenol impurities. The RNA was then concentrated and purified of any organic solvent impurities by silica column purification using the RNeasy® (QIAGEN®) kit according to the supplier's recommendations. The purified RNA was then eluted with nuclease-free water.

b) From YF-VAX® genomic cDNA derived from in vitro transcription

Plasmid DNA contaminating RNA obtained by in vitro transcription (see above) was removed with 4 U. DNase for 15 minutes at 37 ± 2°C. Then the SP6 polymerase was inactivated by incubation for 10 minutes at 70°C.

RNA obtained by in vitro transcription was mixed with 60 μl of RNase-free water and 350 μl of RNeasy® lysis buffer (QIAGEN®). The RNA was then purified by two series of phenol/chloroform/isoamyl alcohol (125:24:1; pH 4.5) extractions. To do this, 1.5 ml Phase Lock Gel Heavy tubes were centrifuged for 30 seconds at 11,000 × g. 750 μl of RNA/lysis buffer mixture was injected into each tube. Then an equal volume (750 µl) of a phenol/chloroform/isoamyl alcohol solution was added to each tube. After vigorously mixing the organic and aqueous phases to form a homogeneous temporary suspension, the tubes were centrifuged at 11,000×g for 5 minutes to separate the phases. The upper phase (aqueous phase) was then recovered. The operation was restarted in new 1.5 ml Phase Lock Gel tubes. The operation was then carried out again with a mixture of chloroform and isoamyl alcohol (24:1) to remove all phenol impurities. The RNA was then purified on a silica column using the RNeasy® (QIAGEN®) kit as recommended by the supplier. The purified RNA was then eluted with nuclease-free water.

1.2.3. Transfection

For each RNA purification, two transfections were performed simultaneously.

a) Preparation of RNA/Lipofectamine™ mixture

10 or 15 µl of Lipofectamine™ 2000 CD (LIFE TECHNOLOGIES®) was mixed with 1 ml of OptiPro SFM medium (LIFE TECHNOLOGIES®) and incubated for 5 minutes at room temperature. Approximately 10 log ₁₀ Geq was then added. (genome equivalent titer determined by qRT-PCR YF-NS5 as described in Mantel et al. (2008)) of purified RNA. These mixtures were incubated for 10 minutes at room temperature.

b) Preparation of Vero cells

Prior to transfection, serum-free Vero cells from the GMP Sanofi Pasteur bank, pre-seeded in 6-well plates (9.105 cells in 3 ml VP-SFM ( ^THERMOFISHER SCIENTIFIC) per well), were washed with OptiPro SFM medium at 2 ml per well.

c) Transfection reaction

In a 6-well plate, after removal of the washing medium from the cells, the transfection mixtures containing RNA were deposited in two wells (1 ml/well) for each preparation. The well was brought into contact with RNA-free OptiPro SFM/Lipofectamine™ medium and the last well was left as a cell control in OptiPro SFM medium only. Two plates were received simultaneously: one with mixtures containing 10 μl of Lipofectamine™ and one with mixtures containing 15 μl of Lipofectamine™. The mixture containing Lipofectamine™ and RNA was left in contact with Vero cells for 4 hours at 37 ± 2°C, at 5 ± 2% CO ₂ and then 2 ml of prewarmed VP-SFM medium was added to each well. 6-well plates were incubated at 37 ± 2°C; 5 ± 2% CO ₂ for 16 hours. Then the medium was renewed and the plates were re-incubated at 37 ± 2°C, 5 ± 2% CO ₂ . Transfection supernatants were harvested when a cytopathic effect (cell lysis) was seen and when the genomic titer, determined by qRT-PCR YF-NS5 from culture supernatant (as described in Mantel et al. (2008)), was greater than 8.0 log ₁₀ Geq/ml. Replacement of the culture medium with fresh medium was performed at D5 and D8 if the culture time required to collect the material was longer than this time. The collected supernatants were divided into aliquots.

1.2.4. Virus amplification

a) Amplification No. 1 (virus passage No. 2)

Two days prior to virus #1 amplification, 2.10 ⁵ Vero cells were seeded in 25 cm ² flasks containing 5 ml of VP-SFM medium. The transfected virus suspensions were then diluted in VP-SFM to give a genome multiplicity (mog) of 2 (i.e. 2 Geq per cell calculated from qRT-PCR RNA concentration) . The culture medium from the previously seeded Vero cells was removed and the cells were brought into contact with 1 ml of the diluted virus suspension or 1 ml of VP-SFM medium alone (cell control). The flasks were incubated for 2 h at 37 ± 2°C; 5 ± 2% CO ₂ . Then the viral inoculum was removed and replaced with 10 ml of VP-SFM medium, and the cells were incubated at 37 ± 2°C; 5 ± 2% CO ₂ for 2 days. The culture medium was then renewed with new VP-SFM medium pre-warmed to 37 ± 2°C, and the flasks were re-incubated at 37 ± 2°C; 5 ± 2% CO ₂ for 2-3 days. After 4-5 days of incubation, the culture supernatant containing the virus was isolated. The virus suspension was clarified by centrifugation for 10 minutes at 1200 rpm. at 4°C, and then distributed into aliquots. 140 µl of this virus suspension was used for total RNA extraction with a QiaAmp Virus Mini-Kit (QIAGEN®; according to supplier's protocol) and for viral RNA quantification with YF-NS5 qRT-PCR (as described in Mantel et al. (2008)).

One or more aliquots, depending on the titer of viral RNA, were stored for the second stage of amplification, if it was carried out on the same day, the rest were frozen at ≤ -70°C in the presence of 10% final sorbitol solution.

b) Amplification No. 2 (virus passage No. 3)

Two days before the amplification of virus No. 2, 5×10 ⁵ Vero cells were seeded in 75 cm ² flasks containing 20 ml of VP-SFM medium. The virus suspension from the first amplification was then diluted in VP-SFM to infect Vero cells at a mog of 2 (i.e. Geq per cell calculated from qRT-PCR RNA concentration).

The other steps of amplification #2 were performed as detailed for amplification #1 (see section a) above).

c) Virus cloning - plate purification (virus passages 4 and 5)

Two 6-well plates were required for each virus suspension obtained after transfection and amplification.

An aliquot of the viral suspension from amplification #2 was diluted to give a suspension of approximately 2.0 log ₁₀ PFU/mL and a suspension of 1.7 log ₁₀ PFU/mL. Vero cells pre-seeded in 6-well plates (9.10 ⁵ cells in 3 ml VP-SFM per well) were observed to check for cell integrity and absence of contamination, then the culture medium was removed. For each dilution, 5 wells of the plate were infected with 500 μl of diluted virus per well (2.0 log ₁₀ pfu/ml or 1.7 log ₁₀ pfu/ml dilutions) and the cell control well contained only 500 μl VP-SFM. The plates were incubated for 2 hours at 37 ± 2°C; 5 ± 2% CO ₂ . The inoculum was then removed and replaced with 4 ml top layer mix, i.e. VP-SFM 2X solution preheated to 42° C. and mixed extemporaneously v/v with 2% agarose solution. After the mixture for the top layer had solidified, the plates were incubated in the reverse position (lid down) for 3-6 days at 37 ± 2°C; 5 ± 2% CO ₂ . The tablets were monitored every day. Once the cytopathic effect was evident, a second topcoat mix, which was identical to the first but additionally containing 0.008% neutral red, was added to each well (2 ml) and the plates were incubated in reverse for 1-2 days at 37 ± 2°C. FROM; 5 ± 2% CO ₂ .

Infection of the cell with a viral particle (clone) remained under these conditions limited to the immediate surrounding cells and caused local lysis, creating a virus-rich white spot (lysis plaque) over the otherwise red-stained cell monolayer. For each amplified viral dilution, two clones were isolated through cover medium using a micropipette and a 1000 μl cone. The virus clone thus obtained was suspended in 1 ml of VP-SFM medium and then vigorously stirred.

Each of these suspensions was diluted in cascade steps from 1:2 to 1:200,000 to perform a second series of plate purifications. At the end of this second cloning cycle, two clones per plate were again harvested. Received a maximum of sixteen clones per lineage, ie. 16 clones from the original Stamaril® strain and 16 clones from the original YF-VAX® strain.

d) Amplification #3 (virus passage #6)

For each clone (up to and including 32), the virus suspension obtained by resuspension of the viral material contained in agarose was diluted 1/4 or 1/2 (depending on the size of the collected plaque) in VP-SFM.

Amplification #3 was performed according to the same protocol as amplification #1 (see section a) above). Amplified virus was harvested when a cytopathic effect was evident and the qRT-PCR genomic titer was greater than 8.0 log ₁₀ Geq/ml.

After 4-5 days of incubation, the culture supernatant containing the virus was isolated and aliquoted. 140 µl of this virus suspension was used for total RNA extraction with the QiaAmp Virus Mini-Kit (QIAGEN®) according to the vendor's protocol and for RNA quantification with YF-NS5 qRT-PCR (as described in Mantel et al. (2008 )). One or more aliquots, depending on the titer of viral RNA, were stored for the next stage of amplification, the rest were frozen at ≤ -70°C in the presence of 10% of the final solution of sorbitol.

e) Amplification No. 4 (virus passage No. 7)

The virus suspension from amplification #3 (see section d) above) was diluted in VP-SFM to infect Vero cells previously seeded at an m.o.g. of 2 and further processed according to the same protocol, as in amplification #2 (see section b) above).

Amplified virus was harvested when a cytopathic effect was evident and the qRT-PCR genomic titer was greater than 8.0 log ₁₀ Geq/ml. After a total of 4-5 days of incubation, the supernatant was collected, the virus suspension was clarified by centrifugation for 10 minutes at 1200 rpm. at 4°C, and then divided into frozen aliquots at ≤ -70°C in the presence of 10% final solution of sorbitol.

The virus suspension thus obtained was used to perform infection titration and viral genome sequencing.

Based on these data, three strains from each lineage (i.e., three strains TV2212, TV2232 and TV2241 from the original strain Stamaril® and three strains TV3111, TV3112 and TV4221 from the original strain YF-VAX®) were selected according to the following criteria: infectious titer ≥ 6 log ₁₀ CCID ₅₀ /ml and genomic sequence showing no reversion to the sequence of the original Asibi strain.

Infection titration of the virus suspension was performed by the CCID ₅₀ method on Vero cells. Briefly, virus suspensions were serially diluted 4-fold in 96-well plate in IMDM (THERMOFISHER SCIENTIFIC) + 4% FCS starting at -4.6 log10 to -8 log10. A control virus (Stamaril® virus amplified once on Vero cells, lot MLE-JPO-000089) was included in each assay as a positive reference. One hundred microliters of each viral dilution was added to 10 wells containing Vero cells seeded in 96-well flat bottom plates three days prior to analysis (8000 cells/well). 4 days after incubation at +37°C, 5% CO ₂ , the supernatants were removed and the cells were fixed for 15 minutes at -20 ± 3°C with 150 µl of 85% acetone, then saturated with 2.5% lactic buffer solution PBS-Tween before immunostaining with mouse monoclonal antibody 4G2, specific for all flaviviruses E (RD BIOTECH®, lot # 130726-4G2), at a concentration of 2 μg/ml (dilution 1/2000). Infected lesions stained with 4G2 antibody were then detected after incubation with alkaline phosphatase-conjugated goat anti-mouse IgG (CLINISCIENCES® SA, ref #1030-04, batch #A7013-Z145) diluted to 1/1000 and then with substrate alkaline phosphatase (BCIP/NBT, SIGMA-ALDRICH® Ref. No. B5655, Lot No. SLBN0689V, and Levamisole, SIGMA-ALDRICH®, Ref. No. L9756, Lot No. 091M1227V).

Positive wells were counted, ie. wells containing at least one plaque stained black, and the final titer was calculated using the least squares regression method.

f) Amplification No. 5 (Virus Passage No. 8) - Candidate for Home Seed Lot (pMSL)

The virus suspension from each of the 6 strains selected after amplification #4 (see section e) above) was diluted in VP-SFM to infect Vero cells at an m.o.i. of 0.01.

Two days prior to virus #5 amplification, 12.10 ⁶ Vero cells were seeded in 175 cm ² flasks containing 30 ml of VP-SFM medium. As was done previously, the culture medium was removed and replaced with 12 ml of diluted virus suspension or VP-SFM alone (control cells). The flasks were incubated for 2 h at 37 ± 2°C; 5 ± 2% CO ₂ . The viral inoculum was then removed and replaced with 50 ml of VP-SFM medium. The flasks were incubated for 2 days at 37 ± 2°C; 5 ± 2% CO ₂ . The culture medium was then renewed with fresh VP-SFM medium preheated to 37 ± 2°C, and the flasks were re-incubated at 37 ± 2°C; 5 ± 2% CO ₂ for 1-3 days. Amplified virus was harvested when a cytopathic effect was evident and the qRT-PCR genomic titer was greater than 8.0 log ₁₀ Geq/ml.

After a total of 3-5 days of incubation, the supernatant was collected, the virus suspension was clarified by centrifugation for 10 minutes at 1200 rpm. at 4°C, then divided into aliquots, which were frozen at ≤ -70°C in the presence of 10% final solution of sorbitol. Amplified viruses derived from 6 selected strains were 6 candidates for pMSL.

EXAMPLE 2 Neurovirulence of Candidates in a Mouse Model

2.1. Neurovirulence of candidates for pMSL in a mouse model

The neurovirulence of vYF home seed lot (pMSL) candidate vYF (Vero-adapted yellow fever virus) candidates was assessed by determining the 50% mouse lethal dose (MLD ₅₀ ) as described in WHO TRS 872 Appendix 2 (1998).

To study the neurovirulence of pMSL candidates, groups of 8 female OF1 mice (4 weeks old at the time of inoculation) were intracerebral injected with 30 μl of 5 to 7 viral dilutions in 0.4% NaCl 2.5% human serum albumin (HSA) buffer. Four candidates for vYF pMSL TV2212, TV3111, TV3112 and TV4221 were evaluated for their neurovirulence and compared with the reference vaccines Stamaril® and YF-VAX®. The mice were observed for 21 days and on day 21 the number of surviving mice was recorded. Three independent experiments were carried out with a random distribution of samples. For each experiment, the amounts administered were checked by back titration using the CCID ₅₀ method on the day of inoculation.

Clinical controls were performed daily for daily recording of survival. MLD ₅₀ was calculated as the dose resulting in 50% survival of mice using the least squares regression method and expressed as log ₁₀ MLD ₅₀ /ml. The MLD ₅₀ of each strain was determined as the weighted average of 3 determinations and 95% confidence intervals, with the exception of TV3111 and TV3112, for which MLD ₅₀ could not be calculated because 100% mouse survival was recorded for the groups injected with strains TV3111 and TV3112 even with the highest dose (30 µl 0.7 log ₁₀ dilution).

The results are presented in table 2 below.

Table 2. Characterization of vYF strains at the pMSL stage in the mouse neurovirulence test (MLD ₅₀ test)

YFV strain log ₁₀ CCID ₅₀ /ml log ₁₀ MLD ₅₀ /ml Stamaril® 6.3 6.0 TV2212 6.8 6.7 YF-VAX® 7.5 4.7 TV3111 7.2 < 2.2 TV3112 7.2 < 2.2 TV4221 7.9 4.8

As for the vYF TV2212 strain derived from the Stamaril® lineage, it exhibited similar neurovirulence compared to the Stamaril® reference vaccine.

The vYF TV4221 strain derived from the YF-VAX® lineage showed similar neurovirulence compared to the YF-VAX® reference vaccine. Finally, both vYF strains TV3111 and TV3112, derived from the YF-VAX® lineage, showed no neurovirulent effect compared to the YF-VAX® reference vaccine. For these 2 vYF strains, it is not possible to calculate the MLD ₅₀ titer (at least <2.2 log ₁₀ MLD ₅₀ /ml).

As a result, the 2 vYF strains TV2212 and TV4221 showed a similar neurovirulence profile and MLD ₅₀ titers as their respective parent reference strains Stamaril® and YF-VAX®. Two other vYF strains, both TV3111 and TV3112, derived from the YF-VAX® lineage, showed marked neurovirulence attenuation compared to their parental YF-VAX® strain, and their MLD ₅₀ titers could not be assessed.

2.2. Neurovirulence of MSL and WSL strain TV3112 in a mouse model

2.2.1. MSL and WSL strain TV3112

All media and solutions used to obtain MSL and WSL did not contain components of animal and human origin.

After amplification of Vero cells under static conditions, the cells were seeded into a bioreactor. After 3 days of cell growth in the bioreactor, the medium was changed from cell growth medium to virus production medium. The virus was inoculated by adding a seed lot (pMSL TV3112 to produce MSL TV3112 or MSL TV3112 to produce WSL TV3112) to the bioreactor. After 2 days of virus propagation, the virus production medium was removed and replaced with the same volume of fresh virus production medium. 4 days after virus inoculation, the contents of the bioreactor were collected, clarified, stabilized, filled and stored frozen.

2.2.2. Neurovirulence of MSL and WSL strain TV3112

The same protocol described in example 2, subsection 2.1, above was used.

Table 3. Characterization of the TV3112 strain at the MSL and WSL stages in the mouse virulence test (MLD ₅₀ test)

YFV strain log ₁₀ CCID ₅₀ /ml log ₁₀ MLD ₅₀ /ml MSL TV3112 7.0 < 2.2 WSL TV3112 8.1 < 2.2

As for pMSL TV3112, MSL TV3112 and WSL TV3112 did not show any neurovirulence effect. For MSL TV3112 and WSL TV3112 it is not possible to calculate the MLD ₅₀ titer (at least <2.2 log ₁₀ MLD ₅₀ /ml).

MSL TV3112 and WSL TV3112 showed a marked attenuation of neurovirulence compared to their parent YF-VAX® strain and their MLD ₅₀ titers could not be assessed.

EXAMPLE 3 Viscerotropism and Neurotropism of vYF Strain Candidates in a Mouse Model

Viscerotropism and neurotropism of 6 home seed lot (pMSL) candidate vYF (yellow fever virus adapted to Vero cells) were evaluated in assays based on inoculation of type I IFN receptor deficient mice that were designed to allow differentiation of pathogenic and attenuated vaccine strains (Meier et al ., 2009; Erickson and Pfeiffer, 2015). A129 immunodeficient mice with KO type I IFN receptors have been described to mimic wild-type YF virus infection in primates and humans (Meier et al., 2009). Thus, such a mouse model appears to be suitable for studying viscerotropic disease caused by non-attenuated yellow fever viruses.

3.1. Ways

3.1.1. Group Definition

Fifteen groups (Groups A to O) of six female A129 mice aged 4-8 weeks were administered 4 log ₁₀ CCID _CCID50 /dose each of 6 candidates for pMSL or Stamaril® reference vaccine as described in Table 4 below (without adjuvant ; subcutaneous route of administration; 200 µl in D0).

Table 4. Definition of groups

Group Number of mice per group Tested product Active substance Name Dose A 6 PBS 1X - B 6 Stamaril® 4 log PFU C 6 Stamaril® 4 log PFU D 6 TV2212 4 log ₁₀ CCID ₅₀ E 6 TV2212 4 log ₁₀ CCID ₅₀ F 6 TV2232 4 log ₁₀ CCID ₅₀ G 6 TV2232 4 log ₁₀ CCID ₅₀ H 6 TV2241 4 log ₁₀ CCID ₅₀ I 6 TV2241 4 log ₁₀ CCID ₅₀ J 6 TV3111 4 log ₁₀ CCID ₅₀ K 6 TV3111 4 log ₁₀ CCID ₅₀ L 6 TV3112 4 log ₁₀ CCID ₅₀ M 6 TV3112 4 log ₁₀ CCID ₅₀ N 6 TV4221 4 log ₁₀ CCID ₅₀ O 6 TV4221 4 log ₁₀ CCID ₅₀

3.1.2. Study scheme

The study scheme is described in figure 2.

6 mice from groups C, E, G, I, K, M, O were sacrificed and their organs were sampled in D6, and 6 mice from groups B, D, F, H, J, L, N were sacrificed and their organs were sampled in D11. Intermediate blood sampling was performed in groups A, B, D, F, H, J, L and N in D4. For PBS control, only 6 mice were included and samples were taken in D11 (Group A).

3.1.3. Clinical Observations and Evaluation of Mice

Animals were observed daily for 11 days post-inoculation according to the scoring grid described in Table 5 below. Body temperature was monitored and recorded every day from D3 until the end of the experiment at D11.

Table 5. Evaluation table

Options Description score General aspect
(GA) Normal 0 Wool/ruffled 1 arched back 2 Shiver 3 Neurological signs
(NS) Normal 0 On tiptoe 1 Complication of movements 2 Spasm 3 Reaction to stimuli
(RS) Normal 0 Unchanging 1 Reduced 2 Excess/prostration 3 Lack of response four Breath
(B) Normal 0 Fast or erratic 1 Respiratory distress syndrome 2

During the experiment, animals were sacrificed upon the occurrence of any of the following events:

- signs of suffering (cachexia, weakness, difficulty moving or eating);

- mixed toxicity (stoop, convulsions);

- evaluation of general aspects=3+response to stimuli=3;

- body weight loss >20%.

Any animal found dead was dissected.

3.1.4. Biological sampling

a) at D4, under anesthesia, intermediate blood samples were taken from the submandibular vein. Approximately 200 μl of blood was collected into vials containing a clotting activator and a serum separator (BD Microtainer SST).

b) on day 6 and D11, blood samples were taken from all animals under anesthesia after bloodletting through an incision in the carotid artery. About 1 ml of blood was collected in vials containing a clotting activator and a serum separator (BD Vacutainer SST).

c) Organ collections were performed under sterile conditions. Instruments used for animal dissection were pre-washed with RNaseZap™ decontamination solution. All organs listed below were harvested from all mice as soon as possible after bloodletting, followed by euthanasia of the animal by dislocation of the cervical vertebrae under anesthesia: brain, liver and spleen.

For the liver, two 7 mm biopsy needles designed to detect viral load were placed in vials containing 1 ml of RNAlater™ solution.

For the brain and spleen, 2 half sections for viral load detection were placed in vials containing 1 ml of RNAlater™ solution.

3.1.7. Analytical tests

a) Viremia

Total genomic RNA was extracted from 140 µl of each individual serum sample using the Macherey Nagel NucleoSpin®96 Virus Kit on a Tecan Evoware automated RNA extraction workstation according to the manufacturer's instructions and eluted in two steps to a final volume of 140 µl of nuclease-free water. .

Immediately after extraction, RNA quantitation was performed using qRT-PCR YF-NS5 (as described in Mantel et al. (2008)). QRT-PCR targets the conserved region of the YF NS5 gene to detect the presence of the YF viral genome.

b) Viral load in organs

Biopsy needles were frozen at -80° C. in RNA later™ solution. During thawing, each organ sample was weighed.

Total RNA was extracted from organ biopsy needles using the combined Trizol™ (Invitrogen®)/RNeasy™ (Qiagen®) method according to the supplier's recommendations.

The presence of viral RNA in the purified RNA samples was then quantified using the YF-NS5 qRT-PCR assay described in Mantel et al. (2008). qRT-PCR targets the conserved region of the YF NS5 gene to detect the presence of the YF viral genome.

Each qRT-PCR cycle included two non-matrix controls (negative qRT-PCR controls) and two positive controls based on CYD-3 virus suspension.

For cycle confirmation, all negative controls must be below the limit of detection (LOD) and positive controls must be included in the control charts.

Because of the dilution factors and for a 100 mg organ sample, the limit of detection was calculated at 1 Geq/mg organ.

3.2. results

3.2.1. Clinical signs

All animals were observed daily after inoculation according to the scoring grid described in Table 5 above: all mice from groups A to O were assessed daily from day 3 to day 6, and all mice from groups A, B, D, F, H , J, L and N were additionally assessed daily up to and including 11 days.

Mean scores were calculated for each criterion, i.e., general aspect (GA), response to stimuli (RS), neurological signs (NS), and respiration (B), from day 3 to day 11 for each group at each time point. As expected, for all A129 mice injected with control saline (PBS, group A), no specific clinical signs were recorded for any animal during the entire control period.

For all A129 arms administered with either the Stamaril® reference vaccine or one of the pMSL vYF candidate vaccines, clinical signs were mild with a mean score for each criterion below 1.5 regardless of time and criterion (GA <1.5; RS, NS and B<1).

No specific clinical evaluation was recorded during days 3, 4 and 5; then some scores of 1/0/0/0 or 2/0/0/0 (GA/RS/NS/B) were recorded for several mice on days 6 and 7. On days 10 and 11, all A129 mice treated with the reference vaccine Stamaril®, or one of the candidates for pMSL vYF, scored low (some mice with GA=1 or 2 and RS, NS, and B=1) except for one mouse treated with TV2232 (Group F), which on day 10 exhibited a shivering phenotype, difficulty moving, prostration, and respiratory distress (score 3/2/3/2), and was euthanized for ethical reasons.

3.2.2. Weight control

All mice (groups A to O) were weighed on days 0, 3, 4, 5 and 6; on days 7, 10 and 11 all mice from the remaining groups (A, B, D, F, H, J, L and N) were weighed. Percentage weight loss compared to day 0 was calculated for each individual mouse at each time point.

After immunization with the Stamaril® reference vaccine, little weight loss was observed over a control period of 11 days (mean less than 5% weight loss in D11).

Following immunization with candidates for pMSL vYF derived from the Stamaril® lineage, as with the Stamaril control, no dramatic weight loss was observed, except for one mouse immunized with clone TV2232 on day 10, which lost over 20% of its weight. This mouse had to be sacrificed for ethical reasons (see 3.2.1 above).

After immunization with candidates for pMSL vYF derived from the YFVAX® lineage, TV3111, TV3112 and TV4221, stable weight was observed and recorded up to days 5-6, and a slight increase in weight was observed until the end of the control period (mean value less than 5% weight gain in D11).

3.2.3. Viral load in serum and organs

a) In serum - figure 3

Individual viremia as well as geometric mean titers (GMT) and standard deviations calculated for each group and time points are shown in Figure 3.

As expected, no viremia was detected on day 4 in A129 mice treated with PBS on day 0 (<LOD 3 log ₁₀ Geq/ml), while geometric mean viremia titers were between 4 and 5 log ₁₀ Geq/ml on days 4 and 6 were detected in A129 mice treated with the reference Stamaril® vaccine.

After immunization with pMSL vYF candidates, no significant excess of viremia was observed compared to viremia induced after immunization with the control Stamaril® strain (all p-values > 0.2 for TV2212, TV2232, TV2241, TV3111 and TV3112 at any time point), except for TV4221, derived from the YF-VAX® lineage, which induced significantly higher viremia than the Stamaril® control on day 4 post-injection (p-value=0.001).

b) In the liver - figure 4

Results are expressed as log ₁₀ Geq/mg organ. Individual viral load, as well as geometric means and standard deviations calculated for each group and time points, are shown in Figure 4.

As expected, liver viral load was not detected on day 11 in A129 mice treated with PBS on day 0 (<LOD 1 log ₁₀ Geq/mg). Similarly, no or low viral loads were detected in A129 mice treated with the reference Stamaril® vaccine (GMT=0.8 on day 6, <LOD on day 11).

After immunization with pMSL vYF candidates, no significant excess of liver viral load was observed compared to liver viral load induced after immunization with the Stamaril® control strain (all p-values >0.1 for TV2212, TV2232, TV2241, TV3111, TV3112 and TV4221 per day 6, statistical analysis was not performed on day 11 due to the large number of treatment non-responders <LOD).

c) In the brain - figure 5

Results are expressed as log ₁₀ Geq/mg organ. The individual viral load as well as the geometric mean titers and standard deviations calculated for each group and time points are shown in Figure 5.

As expected, brain viral loads were not detected on day 11 in A129 mice treated with PBS (<LOD 1 log ₁₀ Geq/mg) on day 0, while brain viral loads were detected in A129 mice, who received the reference vaccine Stamaril® (GMT=0.6 on day 6, 3.7 on day 11).

After immunization with candidates for pMSL vYF, no significant excess of viral load in the brain was observed compared to the viral load in the brain induced after immunization with the control Stamaril® strain (p-values > 0.06 for TV2212 and TV2232). In addition, TV2241, TV3111, TV3112 and TV4221 induced significantly lower viral load in the brain at day 11 than the Stamaril® control (p values ≤0.003).

d) In the spleen - figure 6

Results are expressed as log ₁₀ Geq/mg organ. The individual viral load as well as the geometric mean titers and standard deviations calculated for each group and time points are shown in Figure 6.

As expected, spleen viral loads were not detected on day 11 in A129 mice treated with PBS (<LOD 1 log ₁₀ Geq/mg) on day 0, while spleen viral loads were detected in A129 mice treated with Stamaril® reference vaccine (GMT=4.1 on day 6, 2 on day 11).

After immunization with pMSL vYF candidates, no significant excess of viral load in the spleen was observed compared to the viral load in the spleen induced after immunization with the control Stamaril® strain (all p-values > 0.09 for TV2212, TV2232, TV2241, TV3111, TV3112 and TV4221 regardless of the time point).

3.2.4. Survival

To calculate survival for each group (for groups A, B, D, F, H, J, L and N), the number of surviving mice was recorded daily for 11 days after subcutaneous immunization with 4 log ₁₀ CCID ₅₀ /dose of Stamaril® or one of 6 candidates for pMSL vYF.

As shown in the Kaplan-Meier curves (Figure 7), 100% (6 mice out of 6) mice survived throughout the course of the study with the introduction of either PBS buffer, Stamaril®, or one of the five strains of vYF TV2212, TV2241, TV3111, TV3112 and TV4221.

In contrast, only 80% of the mice survived in group F treated with the TV2232 strain derived from the Stamaril® lineage, as one mouse was euthanized on day 10 for ethical reasons (see 3.2.1 above).

EXAMPLE 4 Immunogenicity of Candidates for the vYF Strain in a Hamster Model

The immunogenicity of 6 candidates for pMSL vYF in a hamster model was evaluated and compared to the reference Stamaril® vaccine.

4.1. Ways

4.1.1. Group Definition

Fifteen female Golden Syrian hamsters aged 5-6 weeks were included in each group and 2 doses were administered for each of the 6 pMSL candidates, i.e. a low suboptimal dose of 2.5 log ₁₀ CCID ₅₀ /dose and a high dose of 5.5 log ₁₀ CCID ₅₀ /dose.

For the Stamaril® reference strain, only 10 hamsters per group were included for the 2 test doses described above.

A total of 200 female Golden Syrian hamsters were randomly assigned to one of the following 14 groups (groups A to N) described in Table 6 below (no adjuvant; subcutaneous route; 200 μl in D0 and D26).

Table 6. Definition of groups

Group Number of hamsters per group Tested product Active substance Name Dose A 10 Stamaril® 2.5 log PFU B 10 5.5 log PFU C 15 TV2212 2.5 log ₁₀ CCID ₅₀ D 15 5.5 log ₁₀ CCID ₅₀ E 15 TV2232 2.5 log ₁₀ CCID ₅₀ F 15 5.5 log ₁₀ CCID ₅₀ G 15 TV2241 2.5 log ₁₀ CCID ₅₀ H 15 5.5 log ₁₀ CCID ₅₀ I 15 TV3111 2.5 log ₁₀ CCID ₅₀ J 15 5.5 log ₁₀ CCID ₅₀ K 15 TV3112 2.5 log ₁₀ CCID ₅₀ L 15 5.5 log ₁₀ CCID ₅₀ M 15 TV4221 2.5 log ₁₀ CCID ₅₀ N 15 5.5 log ₁₀ CCID ₅₀

4.1.2. Study scheme

The study design is summarized in Figure 8.

Procedure planning and procedure details are described in Table 7 below.

Table 7 Study Design

Day Group
Quantity Quantity
Animals Procedures Specific Features D0 A to N 200 Collection of blood samples
Immunization (first
injection) Chemical anesthesia (IP route)
Interim blood sampling
SC immunization pathway D26 A to N 200 Collection of blood samples
Immunization (second
injection) Chemical anesthesia (IP route)
Interim blood sampling
SC immunization pathway D41 A to N 200 Collection of blood samples Chemical anesthesia (IP route)
Interim blood sampling D55 A to N 200 Collection of blood samples Chemical anesthesia (IP route)
Final blood sample collection
Euthanasia

4.1.3. Biological sampling and seroneutralization assays

a) Collection of biological samples

Intermediate blood samples were taken under anesthesia from the retroorbital sinus (ROS) at D0, D26 and D41 from all animals. The last blood draw was taken under anesthesia by intracardiac puncture. Anesthesia was performed using Imalgène (150 mg/kg) and Rompun (10 mg/kg) administered in a volume of 200 μl by the intraperitoneal route.

Blood was collected in vials containing a clotting activator and a serum separator (BD Microtainer SST). After overnight at +4°C or 2 hours at 37°C, blood was centrifuged at 2000×g for 10 minutes and serum was collected and stored at -20°C until analysis.

b) Neutralization assays

Functional neutralizing antibodies present in the sera of immunized animals were titrated at D0, D26 and D41 after the first injection.

Briefly, heat-inactivated sera were serially diluted 2-fold in IMDM+4% fetal calf serum (FCS) starting at 1:5. Stamaril® YF-17D virus grown on Vero cells was diluted to 4000 µFFU/ml in IMDM and incubated for 90 minutes with 2-fold diluted serum samples (v/v). The virus/serum mixture was then added to Vero cells in 96-well plates and incubated for 45+/-2 hours. After incubation, cells were fixed with 85% acetone before immunostaining. The plates were blocked with PBS+0.05% Tween 20+2.5% skim milk and incubated first with anti-flavivirus monoclonal antibody 4G2 and then with goat anti-mouse IgG HRP conjugate. Finally, the plates were stained with Trueblue™ Chromogen. Plaques were counted using a Viruscope reader from Microvision™.

The final titer of seroneutralizing antibodies was calculated using the least squares method and was consistent with a back dilution demonstrating 50% neutralization of viral plaques. The LOD of the assay was 10, corresponding to the first back dilution in the final volume.

An arbitrary titer of 5 (half LOD) was assigned per group for all titers below 10 to calculate mean values.

4.2. Results - seroneutralization

Neutralizing activity against the yellow fever virus vaccine strain 17D on Vero cells was monitored by seroneutralization assays in individual serum samples collected from all animals at baseline (D0), four weeks after one immunization (D26) and two weeks after two immunizations (D41). Geometric mean titers (GMT) as well as individual neutralizing titers and 95% confidence interval (CI) are shown in Figure 9 and Figure 10.

As expected, untreated hamsters at baseline (D0) in the GMT ≤12 group, regardless of the use of candidates for pMSL vYF or the Stamaril® reference strain, showed no or low titers of neutralizing antibodies (≤40). The response threshold for treatment was determined at 20.87 (1.32 log ₁₀ ) by statistical analysis of all individual data obtained in D0 (increased tolerance interval with a proportion of 0.99 and an alpha risk of 5%).

With respect to response kinetics, one month after 1 immunization (D26; FIG. 9), a marked increased neutralizing response was observed for all immunized groups with at least a 10-850-fold increase in neutralizing GMT from baseline in D0. Two weeks after the second immunization (D41; figure 10) neutralizing GMT increased further for all groups with a 0.8-7.5-fold increase in neutralizing GMT compared to D26.

The neutralizing antibody response induced by the Stamaril® reference strain was significantly lower (p-values=0.007 and 0.023 in D26 and D41, respectively) for a dose of 2.5 log ₁₀ CCID ₅₀ (GMT 281 and 544 in D26 and D41, respectively), than for 5.5 log ₁₀ CCID ₅₀ (GMT 5061 and 11714 in D26 and D41, respectively). Of note, 100% of the hamsters in the 5.5 log ₁₀ CCID ₅₀ dose group were defined as treatment responders (>20.87 threshold) immediately after the first immunization (at D26), while only 60% and 70% of the hamsters in the 2.5 log ₁₀ CCID ₅₀ dose group were found to respond to treatment at D26 and D41, respectively.

For pMSL vYF candidates from the Stamaril® lineage TV2212, TV2232 and TV2241, no significant difference was observed between the two doses tested (all p-values > 0.07 regardless of pMSL vYF candidates and time point), with GMT ranging from 144 to 505 in D26 and 115 to 1159 in D41 for a dose of 2.5 log ₁₀ CCID ₅₀ compared with GMT ranging from 115 to 373 in D26 and 465 to 955 in D41 for 5.5 log ₁₀ CCID ₅₀ . None of the candidates for pMSL vYF from the Stamaril® lineage was able to induce a sustained neutralizing antibody response in all immunized animals, regardless of the dose tested and regardless of the immunization schedule (after 1 or 2 immunizations). The percentage of hamsters that responded to treatment was found to range from 53% to 93% after one immunization and from 43% to 93% after two TV2212, TV2232 and TV2241 immunizations, regardless of dose.

For vYF pMSL candidates from the YF-VAX® lineage TV3111, TV3112, and TV4221, no significant difference was observed between the two doses tested (all p-values > 0.06 regardless of vYF strains and time point), except for TV3111, for which the dose 2.5 log ₁₀ CCID ₅₀ induced significantly higher neutralizing antibody titers than 5.5 log ₁₀ CCID ₅₀ (p-value=0.04 and 0.003 in D26 and D41, respectively). For the 2.5 log ₁₀ CCID ₅₀ dose, the induced GMTs were high and ranged from 3939 to 8898 in D26 and from 3771 to 13674 in D41, while for the 5.5 log ₁₀ CCID ₅₀ dose, the GMTs ranged from 2071 to 5145 in D26 and from 1821 to 6421 in D41. The pMSL vYF candidates from the YF-VAX® lineage were able to induce a sustained neutralizing antibody response in the majority of immunized animals after 1 immunization (93% responders to treatment for the 2.5 log ₁₀ CCID ₅₀ TV3111 dose and 100% responders to treatment for all other candidates for pMSL vYF from the YF-VAX® lineage, regardless of the dose tested). After two immunizations, all pMSL vYF candidates from the YF-VAX® lineage were able to induce a sustained neutralizing antibody response in 100% of immunized hamsters, regardless of dose.

Regarding the comparison of each of the candidates for pMSL vYF with the reference Stamaril® strain, the neutralizing responses induced by a dose of 2.5 log ₁₀ CCID ₅₀ candidates for pMSL vYF derived from the lineage YF-VAX®, TV3111, TV3112 and TV4221, are not significantly inferior to those obtained with the reference vaccine Stamaril® (p-values p ≤ 0.010 and ≤ 0.047 in D26 and D41, respectively). No significant lack of inferior efficacy was demonstrated for either vYF strains derived from the Stamaril® lineage (all p-values ≥ 0.25, regardless of dose and time point) nor for vYF strains derived from the YF-VAX® lineage. administered at 5.5 log ₁₀ CCID ₅₀ (p-values ≥0.49, at any time point).

EXAMPLE 5 Toxicity and Immunogenicity of TV3112 vYF in a Monkey Model

A preliminary toxicity study and an immunogenicity study were performed in non-human primates (NHP). Non-human primates, and in particular rhesus or cynomolgus monkeys, are commonly used to assess the safety and infectivity as measured by viremia, as well as the immunogenicity of vaccine candidates against flaviviruses (dengue, yellow fever...). In the context of yellow fever, monkeys are natural hosts; the virus was first isolated from monkeys, and it was in this model that the attenuation of vaccine strains was evaluated. Since the 2000s, "small animal" models have been described that can be used to evaluate certain properties of candidate vaccines, as has been done to select candidates for pMSL vYF. However, these models (hamster, mouse A129) have limitations, and the macaque remains the most predictive gold standard model compared to humans to date and has been extensively described in the literature, e.g., in Julander (2016), Mason et al. (1973), Monath et al. (2010) and Moulin et al. (2013). In addition, this model is recommended by regulatory documents.

5.1. Ways

5.1.1. Definition of groups and purpose

Three groups of nine 2-year-old male cynomolgus monkeys (Macaca fascicularis) imported from Mauritius were immunized subcutaneously with 500 µl Stamaril® (one human dose corresponded to 4.2 log ₁₀ CCID ₅₀ /dose), one dose of YF-VAX® (6 .2 log ₁₀ CCID ₅₀ ) or 4.2 log ₁₀ CCID ₅₀ candidate for WSL vYF TV3112.

As primary indicators, the candidate vaccine was compared with each of the reference vaccines to assess i) the safety of the vaccine, ii) its ability to induce specific YF viremia and viral load in the organs: liver, spleen, kidney, lymph node and brain (assessed by quantitative viral RNA analysis by qRT-PCR YF-NS5 as described in Mantel et al (2008)), and iii) induction of yellow fever virus-specific seroneutralizing antibodies (assessed by microPRNT ₅₀ assay) as a correlate of protection.

Other parameters were monitored as secondary measures to determine other potential vaccine efficacy biomarkers. These analyzes dealt with: i) robustness of the antibody response and ii) B- and T-cell immune response, including memory responses.

5.1.2. Anesthesia

For immunization and in certain cases (eg, blood sampling in combination with other procedures during the acclimatization period, or if monkeys prevented blood sampling without anesthesia), light anesthesia was performed. Ketamine (Imalgene 1000, MERIAL®) at a dose of 10 mg/kg was injected intramuscularly into the thigh area.

5.1.3. Control

All animals were weighed at D-29 and D0, observed daily for clinical signs up to D7 and their individual body temperature (transponder system) was recorded at D-17 and during the expected period of viremia at D0, D3, D4, D5, D6 and D7. Complete blood count, biochemical analysis of blood and blood viremia were evaluated in all monkeys in D1, D3 and D7. Three monkeys from each group were then euthanized and their organs were harvested at D7 for histopathology and organ viral load assessments. The 6 remaining monkeys in each group at D27, D60, D90, D122, D153 and D181 were additionally weighed and their individual body temperature was additionally recorded during the expected period of viremia at D10 and D14. The 6 remaining monkeys in each group were also observed daily for clinical signs up to and including D221. Blood samples for seroneutralization, T cell and memory B cell analyzes were collected from the 6 remaining monkeys in each group at D27, D60, D90, D122, D153, D181 and D221.

5.1.4. Research methods

YF-specific seroneutralizing antibodies were titrated using the microPRNT50 method on VERO cells. Briefly, heat-inactivated sera were serially diluted 2-fold in IMDM (THERMOFISHER SCIENTIFIC) + 4% FCS starting at 1:5. Stamaril® YF-17D virus grown on Vero cells was diluted to 4000 µFFU/ml in IMDM and incubated for 90 minutes with 2-fold diluted serum samples (v/v). The virus/serum mixture was then added to Vero cells in 96-well plates and incubated for 45+/-2 hours. After incubation, cells were fixed with 85% acetone before immunostaining. The plates were blocked with PBS+0.05% Tween 20+2.5% skim milk and incubated first with anti-flavivirus monoclonal antibody 4G2 and then with goat anti-mouse IgG HRP conjugate. Finally, the plates were stained with Trueblue™ Chromogen. Plaques were counted using a Viruscope reader from Microvision™. The end titer was calculated using the least squares method and was consistent with a back dilution demonstrating 50% plaque neutralization.

The LOD of the microPRNT50 assay was approximately 20 microPRNT50 at the first 1:10 dilution of the test serum. To calculate the mean per group, an arbitrary half-LOD value was assigned to all samples below the LOD, i.e. 10 microPRNT50.

Memory cell responses were measured using ELISPOT. Fluorescent enzyme-linked immunosorbent assay (FLUOROSPOT) was used to detect and enumerate individual antibody-secreting memory B cells, regardless of antigen specificity (total IgM or total IgG).

In D0, frozen PBMCs were thawed in RPMI medium (THERMOFISHER SCIENTIFIC®) supplemented with 10% FBS and 100 μg/ml DNase and incubated for 1 hour at 37°C; ₅ % CO2. After 1 hour, the cells were diluted to 1 million cells/ml and stimulated by incubation for 4 days at 37°C; 5% CO ₂ in RPMI 10% FBS supplemented with rIL2 (10 μg/ml).

In D3, the membrane of 96-well FluoroSpot microplates equipped with a low fluorescent PVDF membrane (MERCK Millipore®) was pre-wetted for 1 minute with 35 µl of 35% ethanol. Each well was washed twice with 200 μl PBS 1X. Microplates were then coated with YF-17D-infected Vero cell lysate (SANOFI PASTEUR®) at a 1:80 dilution or with a mixture of monoclonal antibodies specific for monkey IgG and IgM at a dilution of 15 μg/ml and incubated overnight at 4°C .

In D4, the plates were washed with PBS and then blocked for at least 2 hours at 37° C. with RPMI 10% FBS. After washing the plates, 2 x 10 ⁵ or 4 x 10 ⁵ stimulated PBMCs were added to wells coated with YF-17D-infected Vero cell lysate. To wells coated with anti-IgG and anti-IgM antibodies, a dilution range of stimulated cells (5 x 10 ³ to 6.2 x 10 ² ) was added.

After 5 hours, the plates were washed 3 times with PBS 1X and stored at 4°C overnight.

In D7, the plates were washed 6 times with PBS 1X-BSA 0.5% (150 μl/well). After the washing step, 100 μl/well of anti-monkey IgM-FITC and IgG-CY3 antibodies, respectively, were added at a 1/500 dilution in PBS1X-BSA 0.5% for 2 hours at room temperature in the dark. The plates were washed 6 times again with PBS 1X-BSA 0.5% (150 μl/well). The plates were stored at 5°C ± 3°C in the dark until readings were taken.

Each spot corresponding to an antibody secreting cell was counted using a FLUOROSPOT automatic plate reader (Microvision™). The results were expressed as the number of secreting ACS cells per 10 ⁶ cells.

T cell responses were determined using the IFN-γ IL-2 Dual FluoroSpot Response Kit (FS-2122-10 Monkey IFN-γ/IL-2 FluoroSpot kit from Mabtech®) in isolated PBMCs.

Briefly, microplates equipped with a PVDF FluoroSpot membrane were pretreated with 35% ethanol, washed and coated overnight by incubation with monoclonal antibodies against monkey IFN-γ (clone GZ-4, Mabtech®) and against monkey IL-2 (clones IL2M-I/249, Mabtech®) at a concentration of 15 μg/ml in sterile phosphate-buffered saline (PBS) at 4°C. The plates were washed 3 times with PBS and then blocked by incubation for 2 hours at 37° C. with RPMI 1640 medium (Gibco®) supplemented with 10% FCS. PBMC (4×10 ⁵ ) was added to each well with 0.1 μg/ml CD28-A monoclonal antibody (Mabtech®). Pools of peptides YF-Env and YF-NS3 (15-mer overlapping peptides spanning the amino acid sequences of YF-Env and YF-NS3) were added to a final concentration of each peptide in culture medium of 1 μg/ml. An anti-CD3 mAb (Mabtech®) was used as a positive control at a concentration of 2.5 μg/ml. Tablets were incubated for 24 hours at 37°C in an atmosphere containing 5% CO ₂ . After incubation, the plates were washed 6 times with PBS. FITC anti-IFN-γ antibody (clone 7-B6-1-FS-FITC, Mabtech®) and biotinylated anti-IL-2 antibody (IL2-biotin MT8G10, Mabtech®) were added at concentrations of 1:200 and 1 µg/ml, respectively, in 0.5% BSA in PBS; the plates were incubated for 2 hours at 37°C. After 3 washes with PBS, incubation was performed with anti-FITC-490 antibody (1:200, Mabtech®) and anti-streptavidin SA-550 antibody (1:200, Mabtech®) diluted in 0.5% BSA in PBS for 1 hour at room temperature and washed 6 times with PBS. The plates were stored at 5°C ± 3°C in the dark until readings were taken. Fluorescent spots corresponding to cells secreting IFN-γ or IL-2 (IFN-γ SC or IL5 SC), as well as polyfunctional T cells secreting IFN-γ and IL-2 cytokines, were counted using a FLUOROSPOT automatic plate reader ( Microvision™). The results were expressed as the number of cells secreting IFN-γ or IL-2 per 10 ⁶ PBMC.

YF vaccine viremia and organ viral load were monitored by YF-NS5 qRT-PCR (as described in Mantel et al. (2008)).

5.2. results

The correlate of protection for live attenuated yellow fever vaccines is defined in WHO TRS 978 Annex 5 as the induction of measurable neutralizing antibodies in a previously seronegative individual, eg as PRNT titer > detection limit. Neutralizing antibodies well above the pre-set protective threshold (LOD=20) were detected in all monkeys as early as D14 and for at least 9 months (see Figure 11). The titers of neutralizing antibodies did not differ significantly from the titers detected after immunization with existing vaccines.

This sustained neutralizing antibody response was also maintained by a sustained frequency of memory B cells controlled in peripheral blood from day 14 to day 221 after vaccination against vYF TV3112 (see Figure 12 and Figure 13). These data show that both IgM (FIG. 12) and IgG (FIG. 13) memory B cells developed as early as day 14 post-vaccination and persisted during the study period for at least 221 days. With respect to vYF TV3112, the kinetics and percentage of memory B cells induced were similar to the profile of memory B cells induced by both reference Stamaril® and YF-VAX® vaccines.

Moreover, a specific Th1 cellular response (IFN-γ and IL-2 secreting cells) to YF-ENV and YF-NS3 was induced after vaccination with vYF TV3112 and was similar to the cellular response observed after vaccination with Stamaril® or YF-VAX ® (see figure 14).

This study also demonstrated a preserved safety profile of vYF TV3112 compared to control vaccines: no clinical signs, no body weight loss, no temperature changes, no haematological (leukocytes and erythrocytes; neutrophils; lymphocytes; monocytes; eosinophils; basophils; reticulocytes; platelets; hemoglobin ; hematocrit; mean erythrocyte volume; mean erythrocyte hemoglobin) or biochemical (alkaline phosphatase; alanine transferase; aspartate transferase; gamma-glutamyl transferase; C-reactive protein; bile acids; total bilirubin; albumin; blood urea nitrogen; creatinine) disorders (no statistical differences compared to Stamaril® and YF-VAX® by PLS-DA statistical analysis), no or very low viremia (<4 log ₁₀ Geq/mL in 1 of 9 monkeys), no or very low levels of viral RNA detected in target organs in yellow fever (100-10,000 times lower than viral load) a observed after infection with the wild-type Asibi strain) (see 15), lack of vaccine-related histopathological findings in yellow fever target organs.

EXAMPLE 6 Protection Induced by vYV TV3112 Against Lethal Infection in a Macaque Model

The aim was to evaluate protection against yellow fever virus infection in macaques immunized with vaccine candidate vYF TV3112.

6.1. Ways

6.1.1. Animals

Nine months after immunization with vaccine candidates Stamaril®, YF-VAX® or vYF TV3112, the 6 monkeys remaining in D221 from each of the three animal groups tested in Example 5 above were infected with a virulent strain of Asibi yellow fever virus to evaluate vaccine efficacy. Another group of 6 untreated control monkeys were also challenged.

6.1.2. YFV and buffers

Infection was carried out using the Asibi Yellow Fever Virus (YFV) strain from the University of Texas Medical Branch (UTMB). YFV (batch 19455, infectious titer 7.7 Log ₁₀ CCID ₅₀ /ml on VERO cells) was diluted in NaCl+HSA buffer (NaCl 0.4% + human serum albumin (HSA) 2.5%). Each animal was injected subcutaneously in the upper right posterior region with 10 ³ CCID ₅₀ YFV in 1 ml of NaCl+HSA buffer.

6.1.3. Control

The animals were observed for 28 days after infection with the Asibi strain. The animals were observed daily for food intake and behavior. Rectal temperature and body weight were recorded at each sampling time point. Blood sampling was performed as described in Table 8 below.

Table 8. Timeline

days after infection -8 0 2 3 four five 7 10 fourteen 28 Control infection X Euthanasia X Observations ¹ X X X X X X X X X X Local assessment X X X X X X X X X X Blood for complete blood count ² X X X X X X X X X Blood for plasma analysis X X X X X X X X X Serum for detection of neutralizing antibodies ³ and antibody binding X X X X Serum for biochemical analysis ⁴ X X X X X X X X X

¹ Clinical observation, body weight, rectal temperature. ² Leukocytes and erythrocytes; neutrophils; lymphocytes; monocytes; eosinophils; basophils; reticulocytes; platelets; hemoglobin; hematocrit; mean erythrocyte volume; mean hemoglobin of erythrocytes. ³ Neutralization analysis. ^4. Alkaline phosphatase; alanine transferase; aspartate transferase; gamma-glutamyltransferase; C-reactive protein; bile acids; total bilirubin; albumen; the content of urea nitrogen in the blood; creatinine.

6.1.4. Intravital observations

Animals were observed 7 days a week. During each blood draw, a clinical examination was performed as described in Table 9 below.

Table 9. Animal Observation Map

Mortality Daily Overt signs of illness such as diarrhea, loss of appetite, and lethargy Daily Body weight On the day of sampling ¹ Body temperature (rectal) On the day of sampling ¹ Food and water intake Daily score

¹ See table 8.

6.2. results

All vaccinated monkeys were protected from the effects of infection: viremia (low viremia only, i.e. <3.6 log ₁₀ Geq/ml in 2/6 monkeys for only one day, measured by qRT-PCR YF-NS5, as described in Mantel et al (2008)), hematological disorders, biochemical disorders and death.

In this study, 3 of 6 NHPs in the unvaccinated control group survived infection, however, all 6 NHP controls showed viremia (>8 log ₁₀ Geq/mL), lymphopenia, thrombocytopenia, and abnormal blood chemistry with high elevations in transaminases, CRP, bilirubin and bile acids.

Accordingly, vaccine candidate vYF TV3112 was able to protect cynomolgus monkeys, one of the most effective animal models for predicting yellow fever vaccines, from infection with the wild-type Asibi strain, as were the currently available Stamaril® and YF-VAX® vaccines.

EXAMPLE 7 Sequence Analysis

RNA viruses in nature have high levels of genetic variability, which are responsible for the quasi-species internal nature of these viruses. Even if the yellow fever virus polymerase error rate is described as low for an RNA virus, this polymerase error rate is about 10 ^-6 substitutions per genome per infectious cycle.

A well-defined production process is set up to limit this phenomenon to a strict minimum by constantly maintaining the same virus growth conditions. However, statistically, virus quasi-species are continuously formed each time a virus replicates in a cell, and whenever a variant gives the virus a growth advantage, it will persist and amplify in the long term, gradually replacing the original population.

Also, as the new viral growth system moves from egg to Vero cell culture, some adaptive mutation is likely to occur. In particular, several mutations in NS4B have been described in various flavivirus models as a positive adaptation of the virus to growth in Vero cells (Blaney et al., 2003; Tang et al., 2005; Beasley et al., 2013).

In addition, the real seed has never been cloned, so a mixture of quasi-species coexists in existing vaccine strains. Reference sequences will first be established by high-throughput sequencing of the genomes of the YF-VAX® and Stamaril® vaccines, and then the new pMSL candidate genomes will be compared against them.

Since new pMSL candidates are obtained after 2 cloning steps, they represent homogeneous viral populations.

7.3. Ways

7.3.1. Principles

Yellow fever virus sequencing is carried out after the isolation and purification of the viral RNA.

The RNA is then reverse transcribed into complementary DNA and then the genome is fully amplified by PCR using specific primers. The PCR products are then used to form a library using the Nextera® XT DNA Sample Preparation Kit (Illumina, Inc.). The formation of a library occurs in several stages. First, amplicons are collected by an equimolar method. They are then fragmented using transposomes (tagmentase). The transposomes cut the DNA and add adapters. The amplification step was then performed by PCR using primers complementary to the adapters. This step made it possible to add indexes (used to label the sample) on both sides of the fragments and attach the fragments to the sequencing support. Finally, the library was purified using Agencourt® beads (AMPure® XP, Beckman Coultern Genomics, Inc.) and sequenced using a MiSeq sequencer (Illumina®, Inc.).

Once the sequences were obtained, analysis was performed using the "Quality Based Variant Detection (Legacy)" analysis module of the CLC Genomics Workbench (QIAGEN®) software.

7.3.2. RNA extraction

Viral RNA was extracted from 140 μl of viral suspension at a minimum concentration of 10 ⁸ Geq/ml (quantification by qRT-PCR YF-NS5) using the Qiamp Viral kit (QIAGEN®) according to the supplier's recommendations. Purified viral RNA was eluted in 140 µl of nuclease-free water.

7.3.3. RT-PCR

First, a specific reverse transcription (RT) step of RNA to cDNA was performed using three antisense primers designed to span the yellow fever virus genome. PCR amplification was then carried out using the three primer pairs described in Table 10.

Table 10. Primer sequences (MWG®)

SEQID NO. Sequence (5' -> 3') But. from nucl. Amplicon size Position in the genome nine GCTAGGCAATAAACACATTTGGA 23 4146 49-4195 10 TTCACTGGGATACTCCTTTCGC 21 eleven ATCAAATACCATCTTGCCCCTC 22 4009 3940-7949 12 AGTAAATCCTTTGACCCCACT 21 13 GGCTTACCGCAATGCACT eighteen 4235 6553-10788 fourteen CAGAGAACCACTCCGGTC eighteen

Three mixtures were prepared containing one of the three antisense primers (SEQ ID NO. 10; SEQ ID NO. 12 or SEQ ID NO. 14) (see Table 11 below).

Table 11 Mixture for one sample

Reverse primer mix (10 µM) 1 µl dNTP (10 mM) 1 µl Nuclease free water 5 µl Extracted RNA 5 µl

The samples were heated in a thermal cycler for 5 minutes at +65°C and heat shock was immediately performed by incubating the tube for 3 minutes on ice.

The mixture was then prepared as described in Table 12 below.

Table 12. Mixture for one viral RNA sample (4 reactions with RT)

Buffer with RT 16 µl DTT(0.1M) 4 µl RNase OUT (LIFE TECHNOLOGIES®) 2 µl Superscript III (LIFE TECHNOLOGIES®) 2 µl Nuclease free water 8 µl

12 μl of this mixture was added to each of the three RNA/primer tubes and the reverse transcription cycle was performed as described in Table 13 below.

Table 13. RT Program

Stage Time (min.) Temperature (°C) Reverse transcription step 60 min +50°C Enzyme inactivation 5 minutes 85°C Storage - 10°C

1 μl of RNase H was added to each of the tubes and the tubes were incubated for 20 minutes at 37°C in a thermal cycler.

From the resulting cDNA, amplification was performed on three amplicons by PCR.

Three PCR mixtures were prepared as described in Table 14 below, containing one of three primer pairs (SEQ ID NO. 9 and SEQ ID NO. 10; SEQ ID NO. 11 and SEQ ID NO. 12; SEQ ID NO. 13 and SEQ ID No. 14).

Table 14. PCR mix for one sample

Buffer 5X Phusion HF (BioLabs®) 5 µl dNTP 10 mM 1 µl Forward primer 10 µM 1 µl Reverse primer 10 µM 1 µl Nuclease free water 12.5 µl Phusion HF DNA polymerase (BioLabs®) 0.5 µl

20 µl of each mixture was added to 5 µl of the corresponding cDNA.

The PCR program is described in Table 15 below.

Table 15. PCR program

Stage Time (s or min) Temperature (°C) Initial denaturation 30 s +98°C 35 cycles
- Denaturation
- Primer hybridization
- Elongation 10 s +98°C 20 s +58°C 4 min +72°C Terminal elongation 5 minutes +72°C Storage - +10°C

7.3.5. Analysis and purification of amplicons

All amplicons were analyzed on a 1.2% agarose gel to check the quality of the amplification. Amplicons were manually purified using the QIAQuick® PCR Purification Kit (QIAGEN®) as recommended by the supplier.

7.3.6. Building a Library with the Nextera® XT Kit (Illumina, Inc.)

Purified amplicons were quantified using a Qubit® 2.0 fluorimeter (LIFE TECHNOLOGIES®) using a dsDNA HS Qubit® assay kit according to the supplier's recommendations.

After analysis, the amplicons were serially diluted in nuclease-free water to a final concentration of 0.2 ng/µl. Three amplicons were then mixed for each sample to obtain one concentrated PCR pool at 0.6 ng/µl.

The PCR program is described in Table 16 below.

Table 16. PCR program

Stage Time (s or min) Temperature (°C) DNA polymerase activation 3 min +72°C Initial denaturation 30 s +95°C 12 cycles
- Denaturation
- Primer hybridization
- Elongation 10 s +95°C 30 s +55°C 30 s +72°C Terminal elongation 5 minutes +72°C Storage - +10°C

Amplicons were purified and calibrated with the Agencourt® AMPure® XP kit (BECKMAN COULTER®) according to the supplier's recommendations. The library was stable at -20°C for a week.

7.3.7. Library Analysis

The library was quantitated using a Qubit® 2.0 fluorimeter (LIFE TECHNOLOGIES®) using a dsDNA HS Qubit® assay kit as recommended by the supplier.

7.3.8. Library sequencing

Library sequencing was performed using a MiSeq system (ILLUMINA®) according to the supplier's recommendations. Sequences were analyzed using the ILLUMINA® Sequencing Analysis Viewer (Illumina, Inc.) as recommended by the supplier.

Analysis of generated sequences was performed using CLC Genomics Workbenck 7.5.2 software (QIAGEN®) according to the supplier's recommendations.

7.4. results

7.4.1. Reference sequences for YF-VAX® and Stamaril® vaccines

The reference sequence of the YF-VAX® vaccine was presented as SEQ ID NO. 2. The reference sequence of the Stamaril® vaccine was found as SEQ ID NO. 3.

7.4.1.1. pMSL derived from Stamaril®

The pMSL candidate genomes (passage #8) were sequenced and compared with the genome of their parent strain. Table 17 below shows the high throughput sequencing results for three strains from the Stamaril® lineage.

Table 17. Sequencing results from the Stamaril® lineage

Strain no. ¹ Reference. nucl. (Stamaril®) ² Nucle. afterbirth. ³ Reference. codon (Stamaril®) Last codon Frequency (%) Abstract ⁴ Replacement of a.k. TV2212 2524 C U GAC GAU one hundred% NS1-24 - TV2232 5590 U G GUU GUG one hundred% NS3-340 - 5695 C U GUC GUU one hundred% NS3-375 - 7766 U C UUG CUG one hundred% NS5-47 - 8404 C U GAC GAU one hundred% NS5-256 - 6379 A G GAA GAG 99% NS3-603 - TV2241 2524 C U GAC GAU one hundred% NS1-24 -

¹ Nucleotide position from the 5' end of the first nucleotide. ² Nucleotide of the genome of the Stamaril® reference strain. ³ Mutated nucleotide compared to the corresponding nucleotide from the genome of the Stamaril® reference strain. ⁴ Corresponding YFV protein and corresponding amino acid position in the protein.

TV2241 and TV2212 represent one mutation compared to the original Stamaril® strain used as a reference (nucleotide 2524 located in the NS1 coding region, silent at the amino acid level). TV2232 shows a different profile, having five mutations in NS3 and NS5, all silent.

7.4.1.2. pMSL derived from YF-VAX®

Table 18 below shows high throughput sequencing results for three pMSL candidates (passage #8) from the YF-VAX® lineage.

Table 18. Results of sequencing of strains derived from YF-VAX®

Strain no. ¹ Reference. nucl. (YF-VAX®) ² Nucle. afterbirth. ³ Reference. codon (YF-VAX®) Last codon Frequency (%) Abstract ⁴ Replacement of a.k. ^five TV3111 2411 G U GUA UUA one hundred% E-480 Val480Leu 3701 A G AUG GUG one hundred% NS2a-65 Met65Val 6496 A G AAA AAG one hundred% NS4a-19 - TV3112 3701 A G ATG GUG one hundred% NS2a-65 Met65Val 2411 G U GUA UUA one hundred% E-480 Val480Leu 6496 A G AAA AAG one hundred% NS4a-19 - 1408 A U GUA GUU one hundred% E-145 - TV4221 No options found.

¹ Nucleotide position from the 5' end of the first nucleotide. ² Nucleotide of the genome of the YF-VAX® reference strain. ³ Mutated nucleotide compared to the corresponding nucleotide from the genome of the YF-VAX® reference strain. ⁴ Corresponding YFV protein and corresponding amino acid position in the protein. ⁵ Mutated amino acid and protein position compared to the corresponding amino acid from the YF-VAX® reference strain.

TV4221 is identical to the reference sequence of the YF-VAX® vaccine strain.

TV3111 has 3 mutations at position 2411 (E-480, Val to Leu), 3701 (NS2a-65, Met to Val), and 6496 (NS4a-19, silent).

TV3112 has the same mutations as TV3111, along with one additional mutation at position 1408 (E-145, silent).

Strains TV3112 and TV3111 contain an envelope protein represented by SEQ ID NO 15 (with a leucine residue at position 480). SEQ ID NO 16 (with a leucine residue at position 65) is the NS2a protein sequence from strains TV3112 and TV3111. SEQ ID NO 17 (with nucleotide G at position 57) is the RNA sequence encoding the NS4a protein from strain TV3112. SEQ ID NO 18 (with nucleotide U at position 435) is an RNA sequence encoding an envelope protein from strain TV3112.

The person skilled in the art is well aware that the role of the genome is to maintain information, and that proteins, by their function, play a role in the phenotype of the virus. Silent mutations do not affect the function of proteins. Accordingly, strains TV3112 and TV3111 can be described as live attenuated strains of yellow fever virus containing a nucleic acid molecule encoding:

(i) an envelope protein containing a mutation at position 480 that results in a change from the amino acid valine to leucine, and

(ii) an NS2a protein containing a mutation at position 65 that results in the substitution of the amino acid methionine for valine. Or strains TV3112 and TV3111 can be described as live attenuated strains of yellow fever virus containing a nucleic acid molecule encoding:

(i) an envelope protein that contains a leucine residue at a position in the protein that corresponds to position 480 in SEQ ID NO. 15; and

(ii) an NS2a protein that contains a valine residue at a position in the protein that corresponds to position 65 in SEQ ID NO. sixteen.

7.4.2. YF-VAX®-derived TV3112 strain at MSL and WSL stages

The TV3112 MSL consensus sequence remained identical to that of its parent pMSL (pMSL TV3112). The TV3112 WSL consensus sequence remained identical to its original MSL (MSL TV3112). The TV3112 strain is genetically stable and retains mutations in its consensus sequence at nucleotide positions 1408, 2411, 3701, and 6496 from the pMSL stage to the WSL stage.

REFERENCES TO LITERATURE

Non-Patent Links

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- Dupuy A, Despres P, Cahour A, Girard M, Bouloy M. Nucleotide sequence comparison of the genome of two 17D-204 yellow fever vaccines. Nucleic Acids Res. 1989 May 25; 17(10):3989.

- Erickson AK, Pfeiffer JK. Spectrum of disease outcomes in mice infected with YFV-17D. J Gen Virol. Jun 2015; 96:1328-1339.

- Hayes EB. Is it time for a new yellow fever vaccine? Vaccine. 2010 Nov 29; 28(51):8073-6.

- Julander JG. Animal models of yellow fever and their application in clinical research. Curr Opin Virol. Jun 2016; 18:64-9.

- Kolell K. et al. Virus Production in Vero Cells Using a Serum-free Medium. In: Smith R. (eds) Cell Technology for Cell Products (2007). Springer.

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- Moulin JC, Silvano J, Barban V, Riou P, Allain C. Yellow fever vaccine: comparison of the neurovirulence of new 17D-204 Stamaril™ seed lots and RK 168-73 strain. Biologicals. 2013 Jun; 41(4):238-46.

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Links to patent documents

- WO2009/109550

- WO 2014/016360.

--->

SEQUENCE LIST

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<120> LIVE ATTENUATED YELLOW FEVER VIRUS STRAIN, ADAPTED

FOR GROWTH ON VERO CELLS AND A VACCINE COMPOSITION CONTAINING IT

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<223> RNA sequence of YFV strain 17D204

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uucccaugau guucugacug ugcaauuccu aauuuuggga augcuguuga ugacgggugg 480

agugaccuug gugcggaaaa acagaugguu gcuccuaaau gugacaucug aggaccucgg 540

gaaaacauuc ucugugggca caggcaacug cacaacaaac auuuuggaag ccaaguacug 600

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agcaggcagg ucuaggaggu caagaagggc cauugacuug ccuacgcaug aaaaccaugg 780

uuugaagacc cggcaagaaa aauggaugac uggaagaaug ggugaaaggc aacuccaaaa 840

gauugagaga ugguucguga ggaaccccuu uuuugcagug acggcucuga ccauugccua 900

ccuuguggga agcaacauga cgcaacgagu cgugauugcc cuacugggucu uggcuguugg 960

uccggccuac ucagcucacu gcauuggaau uacugacagg gauuucauug agggggugca 1020

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ugacaagccu ucauuggaca ucucacuaga gacaguagcc auugauagac cugcugaggu 1140

1200 gaggaaagug uguuacaaug caguucucac

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guaugucauc agagcacaau ugcauguagg ggccaagcag gaaaauugga auaccgacau 1440

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auggcagagu ggaaguggcg ggguguggag agagaugcau caucuugucg aauuugaacc 1680

uccgcaugcc gccacuauca gaguacuggc ccugggaaac caggaaggcu ccuugaaaac 1740

agcucuuacu ggcgcaauga ggguuacaaa ggacacaaau gacaacaacc uuuacaaacu 1800

acauggugga cauguuucuu gcagagugaa auugucagcu uugacacuca aggggacauc 1860

cuacaaaaua ugcacugaca aaauguuuuu ugucaagaac ccaacugaca cuggccaugg 1920

cacuguugug augcagguga aagugucaaa aggagccccc ugcaggauuc cagugauagu 1980

agcugaugau cuuacagcgg caaucaauaa aggcauuuug guuacaguua accccaucgc 2040

cucaaccaau gaugaugaag ugcugauuga ggugaaccca ccuuuuggag acagcuacau 2100

uaucguuggg agaggagauu cacgucucac uuaccagugg cacaaagagg gaagcucaau 2160

aggaaaguug uucacucaga ccaugaaagg cguggaacgc cuggccguca ugggagacac 2220

2280

gguguuuggc ucugccuuuc aggggcuauu uggcggcuug aacuggauaa caaaggucau 2340

2400

caugaucuug guaggaguga ucauguguu uuugucucua ggaguugggg cggaucaagg 2460

2520

agacucugau gacuggcuga acaaguacuc auacuaucca gaagauccug ugaagcuugc 2580

2640

ugagcaugag auguggagaa gcagggcaga ugagaucaau gccauuuuug aggaaaacga 2700

gguggacauu ucuguugucg ugcaggaucc aaagaauguu uaccagagag gaacucaucc 2760

auuuuccaga auucgggaug gucugcagua ugguuggaag acuuggggua agaaccuugu 2820

guucucccca gggaggaaga auggaagcuu caucauagau ggaaagucca ggaaagaaug 2880

cccguuuuca aaccgggucu ggaauucuuu cggauagag gaguuuggga cgggaguguu 2940

caccacacgc guguacaugg acgcagucuu ugaauacacc auagacugcg auggaucuau 3000

cuugggugca gcggugaacg gaaaaaagag ugcccauggc ucuccaacau uuuggauggg 3060

3120

gugugagugg cccugacac auacgauugg aacaucaguu gaagagagug aaauguucau 3180

3240

gacgaacgga ccuuggaugc agguaccacu agaagugaag agagaagcuu gcccagggac 3300

uagcgugauc auugauggca acugugaugg acggggaaaa

uagcgggaaa guuauuccug aaugguguug ccgcuccugc acaaugccgc cugugagcuu 3420

ccaugguagu gauggguguu gguaucccau ggaaauuagg ccaaggaaaa cgcaugaaag 3480

ccaucuggug cgcuccuggg uuacagcugg agaaauacau gcugucccuu uugguuuggu 3540

gagcaugaug auagcaaugg aagugguccu aaggaaaaga cagggaccaa agcaaauguu 3600

3660

3720

cauguauaug gcguugauug cugccuuuuc aaucagacca gggcugcuca ucggcuuugg 3780

gcucaggacc cuauggagcc cucgggaacg ccuugugcug acccuagggag cagccauggu 3840

ggagauugcc uuggguggcg ugagggcgg ccuuggaag uaucuaaaug caguuucucu 3900

3960

cauggcucug uugacaccug ucacuauggc ugaggugaga

ugccgugguu aucauagggg uccuucacca gaauuucaag gacaccucca ugcagaagac 4080

uauaccucug guggcccuca cacucacauc uuaccugggc uugacacaac cuuuuuuggg 4140

ccugugugca uuucuggcaa ccgcauauu ugggcgaagg aguaucccag ugaaugaggc 4200

acucgcagca gcuggucuag ugggagugcu ggcaggacug gcuuuucagg agauggagaa 4260

cuuccuuggu ccgauugcag uuggaggacu ccugaugaug cugguuagcg uggcugggag 4320

gguggauggg cuagagcuca agaagcuugg ugaaguuuca ugggaagagg aggcggagau 4380

cagcgggagu uccgcccgcu augauguggc acucagugaa caaggggagu ucaagcugcu 4440

uucugaagag aaagugccau gggaccaggu ugugaugacc ucgcuggccu ugguuggggc 4500

ugcccuccau ccauuugcuc uucugcuggu ccuugcuggg uggcuguuuc augucagggg 4560

4620

ugaacaugg gaggauggga uuuauggcau auuccaguca accuucuugg gggccuccca 4680

gcgaggagug ggaguggcac agggaggggu guuccacaca auguggcaug ucacaagagg 4740

agcuuuccuu gucaggaaug gcaagaaguu gauuccaucu ugggcuucag uaaaggaaga 4800

ccuugucgcc uaugguggcu cauggaaguu ggaaggcaga ugggauggag aggaagggu 4860

ccaguugauc gcggcuguuc caggaaagaa cguggucaac guccagacaa aaccgagcuu 4920

guucaaagug aggaaugggg gagaaaucgg ggcugucgcu cuugacuauc cgaguggcac 4980

uucaggaucu ccuauuguua acaggaacgg agaggugauu gggcuguacg gcaauggcau 5040

ccuugucggu gacaacuccu ucguguccgc cauaucccag acugagguga aggaagaagg 5100

aaaggaggag cuccaagaga ucccgacaau gcuaaagaaa ggaaugacaa cuguccuuga 5160

uuuucauccu ggagcuggga agacaagacg uuuccuccca cagaucuugg ccgagugcgc 5220

acggagacgc uugcgcacuc uuguguuggc ccccaccagg guuguucuuu cugaaaugaa 5280

ggaggcuuuu cacggccugg acgugaaauu ccacacacag gcuuuuuccg cucacggcag 5340

cgggagagaa gucauugaug ccaugugcca ugccacccua acuuacagga uguuggaacc 5400

aacuaggguu guuaacuggg aagugaucau uuggaugaa gcccauuuuu uggauccagc 5460

uagcauagcc gcuagagguu gggcagcgca cagagcuagg gcaaaugaaa gugcaacaau 5520

cuugaugaca gccacaccgc cugggacuag ugaugaauuu ccacauucaa aggugaaau 5580

agaagauguu caaacggaca uacccaguga gcccuggaac acagggcaug acuggauccu 5640

agcugacaaa aggcccacgg caugguuccu uccauccauc agagcugcaa augucauggc 5700

ugccucuuug cguaaggcug gaaagagugu ggugguccug aacaggaaaa ccuuugagag 5760

agaauacccc acgauaaagc agaagaaacc ugacuuuaua uuggcacug acauagcuga 5820

aaugggagcc aaccuuugcg uggagcgagu gcuggauugc aggacggcuu uuaagccugu 5880

gcuuguggau gaagggagga agguggcaau aaaagggcca cuucguaucu cgcauccuc 5940

ugcugcucaa aggagggggc gcauugggag aaaucccaac agagauggag acucauacua 6000

6060

gcucuuggac aacauggagg ugaggggugg aauggucgcc ccacucuaug gcguugaagg 6120

aacuaaaaca ccaguuuccc cuggugaaau gagacugagg gaugaccaga ggaaagucuu 6180

cagagaacua gugaggaauu gugaccugcc cguuuggcuu ucguggcaag uggccaaggc 6240

ugguuugaag acgaaugauc guaaguggug uuuugaaggc ccugggaac augagaucuu 6300

6360

6420

6480

uuuccuggcu aaaaaaggug gagaggcaau ggauaccauc aguguguucc uccacucuga 6540

ggaaggcucu agggcuuacc gcaaugcacu aucaaugaug ccugaggcaa ugacaauagu 6600

caugcuguuu auacuggcug gacuacugac aucgggaaug

caaaggcauc aguagaaugu cuauggcgau gggcacaaug gccggcugug gauaucucau 6720

6780

gauggugguu gugauccccg agccagggca acaaaggucc auccaagaca accaaguggc 6840

auaccucauu auuggcaucc ugacgcuggu uucagcggug gcagccaacg agcuaggcau 6900

gcuggagaaa accaaagagg accucuuugg gaagaagaac uuaauuccau cuagugcuuc 6960

acccuggagu uggccggauc uugaccugaa gccaggagcu gccuggacag uguacguugg 7020

cauuguuaca augcucucuc caauguugca ccacuggauc aaagucgaau auggcaaccu 7080

gucucugucu ggaauagccc agucagccuc aguccuuucu uucauggaca aggggauacc 7140

auucaugaag augaauaucu cggucauaau gcugcugguc aguggcugga auucaauaac 7200

agugaugccu cugcucugug gcauagggug cgccaugcuc cacuggucuc ucauuuuacc 7260

uggaaucaaa gcgcagcagu caaagcuugc acagagaagg guguuccaug gcguugccga 7320

7380

ccuuuaugag aagaaacugg cucuauaucu ccuucuugcu cucagccuag cuucuguugc 7440

caugugcaga acgcccuuuu cauuggcuga aggcauuguc cuagcaucag cugccuuagg 7500

7560

aggagucaug agggggaauc acuaugcuuu ugugggaguc auguacaauc uauggaagau 7620

gaaaacugga cgccggggga gcgcgaaugg aaaaacuuug ggugaagucu ggaagaggga 7680

acugaaucug uuggacaagc gacaguuuga guuguauaaa aggaccgaca uuguggaggu 7740

ggaucgugau acggcacgca ggcauuuggc cgaagggaag guggacaccg ggguggcggu 7800

cuccaggggg accgcaaagu uaaggugguu ccaugagcgu ggcuauguca agcuggaagg 7860

uagggugauu gaccuggggu guggccgcgg aggcuggugu uacuacgcug cugcgcaaaa 7920

ggaagugagu ggggucaaag gauuuacucu uggaagagac ggccaugaga aacccaugaa 7980

ugugcaaagu cugggaugga acaucaucac cuucaaggac aaaacugaua uccaccgccu 8040

agaaccagug aaaugugaca cccuuuugug ugacauugga gagucaucau cgucaucggu 8100

cacagagggg gaaaggaccg ugagaguucu ugauacugua gaaaaauggc uggcuugugg 8160

8220

8280

cacucaugaa auguacuacg ugucuggagc ccgcagcaau gucacauuua cugugaacca 8340

aacaucccgc cuccugauga ggagaaugag gcguccaacu ggaaaaguga cccuggaggc 8400

ugacgucauc cucccaauug ggacacgcag uguugagaca gacaagggac cccuggacaa 8460

agaggccaua gaagaaaggg uugagaggau aaaaucugag uacaugaccu cuugguuuua 8520

ugacaaugac aaccccuaca ggaccuggca cuacuguggc uccuauguca caaaaaccuc 8580

aggaagugcg gcgagcaugg uaaauggugu uauuaaaauu cugacauauc caugggacag 8640

gauagaggag gucacaagaa uggcaaugac ugacacaacc ccuuuuggac agcaaagagu 8700

guuuaaagaa aaaguugaca ccagagcaaa ggauccacca gcgggaacua ggaagaucau 8760

8820

8880

agaacaagaa caguggaaga cugccaauga ggcuguccaa gacccaaagu ucugggaacu 8940

gguggaugaa gaaaggaagc ugcaccaaca aggcaggugu cggacuugug uguacaacau 9000

gauggggaaa agagagaaga agcugucaga guuugggaaa gcaaagggaa gccgugccau 9060

augguauaug uggcugggag cgcgguaucu ugaguuugag gcccugggau uccugaauga 9120

ggaccauugg gcuuccaggg aaaacucagg aggaggagug gaaggcauug gcuuacaaua 9180

ccuagauau gugaucagag accuggcugc aauggauggu gguggauucu acgcggauga 9240

caccgcugga ugggacacgc gcaucacaga ggcagaccuu gaugaugaac aggagaucuu 9300

gaacuacaug agcccacauc acaaaaaacu ggcacaagca gugauggaaa ugacauacaa 9360

gaacaaagug gugaaagugu ugagaccagc cccaggaggg aaagccuaca uggaugucau 9420

aagucgacga gaccagagag gauccgggca gguagugacu uaugcucuga acaccaucac 9480

caacuugaaa guccaauuga ucagaauggc agaagcagag auggugauac aucaccaaca 9540

uguucaagau ugugaugaau caguucugac caggcuggag gcauggcuca cugagcacgg 9600

augugacaga cugaagagga uggcggugag uggagacgac uguguggucc ggcccaucga 9660

ugacagguuc ggccuggccc ugucccaucu caacgccaug uccaagguua gaaaggacau 9720

aucugaaugg cagccauccaa aaggguggaa ugauugggag aaugugcccu ucuguuccca 9780

ccacuuccau gaacuacagc ugaaggaugg caggaggauu guggugccuu gccgagaaca 9840

ggacgagcuc auugggagag gaaggguguc uccaggaaac ggcuggauga ucaaggaaac 9900

agcuugccuc agcaaagccu augccaacau guggucacug auguauuuuc acaaaaggga 9960

caugaggcua cugucauugg cuguuuccuc agcuguuccc accucauggg uuccacaagg 10020

10080

gguguggaac agaguaugga uaaccaacaa cccacacaug caggacaaga caauggugaa 10140

10200

uggaaugacc aauagggcca ccugggccuc ccacauccau uuagucaucc aucguauccg 10260

10320

ggaugcugac cugcaacugg gugagcuuau cugaaacacc aucuaacagg aauaaccggg 10380

auacaaacca cgggguggaga accggacucc ccacaaccug aaaccgggau auaaaccacg 10440

gcuggagaac cgggcuccgc acuuaaaaug aaacagaaac cgggauaaaa acuacggaug 10500

10560

uuugccacug cuaagcugug aggcagugca ggcugggaca gccgaccucc agguugcgaa 10620

aaaccugguu ucugggaccu cccaccccag aguaaaaaga acggagccuc cgcuaccacc 10680

cucccacgug gugguagaaa gacggggucu aggguuaga ggagacccuc cagggaacaa 10740

auagugggac cauauugacg ccagggaaag accggagugg uucucugcuu uuccuccaga 10800

ggucugugag cacaguuugc ucaagaauaa gcagaccuuu ggaugacaaa cacaaaacca 10860

cu 10862

<210> 2

<211> 10862

<212> RNA

<213> Yellow fever virus

<220>

<223> RNA sequence of YFV strain YF-Vax®

<400> 2

aguaaauccu gugugcuaau ugaggugcau uggucugcaa aucgaguugc uaggcaauaa 60

acacauuugg auuaauuuua aucguucguu gagcgauuag cagagaacug accagaacau 120

gucggucgu aaagcucagg gaaaaacccu gggcgucaau augguacgac gaggaguucg 180

cuccuuguca aacaaaauaa aacaaaaaac aaaacaaauu ggaaacagac cuggaccuuc 240

aagagguguu caaggauuua ucuuuuucuu uuuguucaac auuuugacug gaaaaaagau 300

cacagcccac cuaaagaggu uguggaaaau gcuggaccca agacaaggcu uggcuguucu 360

aaggaaaguc aagagagugg uggccaguuu gaugagagga uuguccucaa ggaaacgccg 420

uucccaugau guucugacug ugcaauuccu aauuuuggga augcuguuga ugacgggugg 480

agugaccuug gugcggaaaa acagaugguu gcuccuaaau gugacaucug aggaccucgg 540

gaaaacauuc ucugugggca caggcaacug cacaacaaac auuuuggaag ccaaguacug 600

gugcccagac ucaauggaau acaacugucc caaucucagu ccaagagagg agccagauga 660

cauugauugc uggugcuaug ggguggaaaa cguuagaguc gcauauggua agugugacuc 720

agcaggcagg ucuaggaggu caagaagggc cauugacuug ccuacgcaug aaaaccaugg 780

uuugaagacc cggcaagaaa aauggaugac uggaagaaug ggugaaaggc aacuccaaaa 840

gauugagaga ugguucguga ggaaccccuu uuuugcagug acggcucuga ccauugccua 900

ccuuguggga agcaacauga cgcaacgagu cgugauugcc cuacugggucu uggcuguugg 960

uccggccuac ucagcucacu gcauuggaau uacugacagg gauuucauug agggggugca 1020

uggaggaacu uggguuucag cuacccugga gcaagacaag ugugucacug uuauggcccc 1080

ugacaagccu ucauuggaca ucucacuaga gacaguagcc auugauagac cugcugaggu 1140

1200 gaggaaagug uguuacaaug caguucucac

cacuggagag gcccaccuag cugaagagaa cgaaggggac aaugcgugca agcgcacuua 1260

uucugauaga ggcuggggca auggcugugg ccuauuuggg aaagggagca uuguggcaug 1320

cgccaaauuc acuugugcca aauccaugag uuuguuugag guugaucaga ccaaaauuca 1380

guaugucauc agagcacaau ugcauguagg ggccaagcag gaaaauugga cuaccgacau 1440

uaagacucuc aaguuugaug cccugucagg cucccaggaa gucgaguuca uuggguaugg 1500

aaaagcuaca cuggaaugcc aggugcaaac ugcgguggac uuugguaaca guuacaucgc 1560

ugagauggaa acagaggcu ggauaggga cagacagugg gcccaggacu ugacccugcc 1620

auggcagagu ggaaguggcg ggguguggag agagaugcau caucuugucg aauuugaacc 1680

uccgcaugcc gccacuauca gaguacuggc ccugggaaac caggaaggcu ccuugaaaac 1740

agcucuuacu ggcgcaauga ggguuacaaa ggacacaaau gacaacaacc uuuacaaacu 1800

acauggugga cauguuucuu gcagagugaa auugucagcu uugacacuca aggggacauc 1860

cuacaaaaua ugcacugaca aaauguuuuu ugucaagaac ccaacugaca cuggccaugg 1920

cacuguugug augcagguga aagugucaaa aggagccccc ugcaggauuc cagugauagu 1980

agcugaugau cuuacagcgg caaucaauaa aggcauuuug guuacaguua accccaucgc 2040

cucaaccaau gaugaugaag ugcugauuga ggugaaccca ccuuuuggag acagcuacau 2100

uaucguuggg agaggagauu cacgucucac uuaccagugg cacaaagagg gaagcucaau 2160

aggaaaguug uucacucaga ccaugaaagg cguggaacgc cuggccguca ugggagacac 2220

2280

gguguuuggc ucugccuuuc aggggcuauu uggcggcuug aacuggauaa caaaggucau 2340

2400

caugaucuug guaggaguga ucauguguu uuugucucua ggaguugggg cggaucaagg 2460

2520

agacucugau gacuggcuga acaaguacuc auacuaucca gaagauccug ugaagcuugc 2580

2640

ugagcaugag auguggagaa gcagggcaga ugagaucaau gccauuuuug aggaaaacga 2700

gguggacauu ucuguugucg ugcaggaucc aaagaauguu uaccagagag gaacucaucc 2760

auuuuccaga auucgggaug gucugcagua ugguuggaag acuuggggua agaaccuugu 2820

guucucccca gggaggaaga auggaagcuu caucauagau ggaaagucca ggaaagaaug 2880

cccguuuuca aaccgggucu ggaauucuuu cggauagag gaguuuggga cgggaguguu 2940

caccacacgc guguacaugg acgcagucuu ugaauacacc auagacugcg auggaucuau 3000

cuugggugca gcggugaacg gaaaaaagag ugcccauggc ucuccaacau uuuggauggg 3060

3120

gugugagugg cccugacac auacgauugg aacaucaguu gaagagagug aaauguucau 3180

3240

gacgaacgga ccuuggaugc agguaccacu agaagugaag agagaagcuu gcccagggac 3300

uagcgugauc auugauggca acugugaugg acggggaaaa

uagcgggaaa guuauuccug aaugguguug ccgcuccugc acaaugccgc cugugagcuu 3420

ccaugguagu gauggguguu gguaucccau ggaaauuagg ccaaggaaaa cgcaugaaag 3480

ccaucuggug cgcuccuggg uuacagcugg agaaauacau gcugucccuu uugguuuggu 3540

gagcaugaug auagcaaugg aagugguccu aaggaaaaga cagggaccaa agcaaauguu 3600

3660

3720

cauguauaug gcguugauug cugccuuuuc aaucagacca gggcugcuca ucggcuuugg 3780

gcucaggacc cuauggagcc cucgggaacg ccuugugcug acccuagggag cagccauggu 3840

ggagauugcc uuggguggcg ugagggcgg ccuuggaag uaucuaaaug caguuucucu 3900

3960

cauggcucug uugacaccug ucacuauggc ugaggugaga

ugccgugguu aucauagggg uccuucacca gaauuucaag gacaccucca ugcagaagac 4080

uauaccucug guggcccuca cacucacauc uuaccugggc uugacacaac cuuuuuuggg 4140

ccugugugca uuucuggcaa ccgcauauu ugggcgaagg aguaucccag ugaaugaggc 4200

acucgcagca gcuggucuag ugggagugcu ggcaggacug gcuuuucagg agauggagaa 4260

cuuccuuggu ccgauugcag uuggaggacu ccugaugaug cugguuagcg uggcugggag 4320

gguggauggg cuagagcuca agaagcuugg ugaaguuuca ugggaagagg aggcggagau 4380

cagcgggagu uccgcccgcu augauguggc acucagugaa caaggggagu ucaagcugcu 4440

uucugaagag aaagugccau gggaccaggu ugugaugacc ucgcuggccu ugguuggggc 4500

ugcccuccau ccauuugcuc uucugcuggu ccuugcuggg uggcuguuuc augucagggg 4560

4620

ugaacaugg gaggauggga uuuauggcau auuccaguca accuucuugg gggccuccca 4680

gcgaggagug ggaguggcac agggaggggu guuccacaca auguggcaug ucacaagagg 4740

agcuuuccuu gucaggaaug gcaagaaguu gauuccaucu ugggcuucag uaaaggaaga 4800

ccuugucgcc uaugguggcu cauggaaguu ggaaggcaga ugggauggag aggaagggu 4860

ccaguugauc gcggcuguuc caggaaagaa cguggucaac guccagacaa aaccgagcuu 4920

guucaaagug aggaaugggg gagaaaucgg ggcugucgcu cuugacuauc cgaguggcac 4980

uucaggaucu ccuauuguua acaggaacgg agaggugauu gggcuguacg gcaauggcau 5040

ccuugucggu gacaacuccu ucguguccgc cauaucccag acugagguga aggaagaagg 5100

aaaggaggag cuccaagaga ucccgacaau gcuaaagaaa ggaaugacaa cuguccuuga 5160

uuuucauccu ggagcuggga agacaagacg uuuccuccca cagaucuugg ccgagugcgc 5220

acggagacgc uugcgcacuc uuguguuggc ccccaccagg guuguucuuu cugaaaugaa 5280

ggaggcuuuu cacggccugg acgugaaauu ccacacacag gcuuuuuccg cucacggcag 5340

cgggagagaa gucauugaug cuaugugcca ugccacccua acuuacagga uguuggaacc 5400

aacuaggguu guuaacuggg aagugaucau uuggaugaa gcccauuuuu uggauccagc 5460

uagcauagcc gcuagagguu gggcagcgca cagagcuagg gcaaaugaaa gugcaacaau 5520

cuugaugaca gccacaccgc cugggacuag ugaugaauuu ccacauucaa aggugaaau 5580

agaagauguu caaacggaca uacccaguga gcccuggaac acagggcaug acuggauccu 5640

ggcugacaaa aggcccacgg caugguuccu uccauccauc agagcugcaa augucauggc 5700

ugccucuuug cguaaggcug gaaagagugu ggugguccug aacaggaaaa ccuuugagag 5760

agaauacccc acgauaaagc agaagaaacc ugacuuuaua uuggcacug acauagcuga 5820

aaugggagcc aaccuuugcg uggagcgagu gcuggauugc aggacggcuu uuaagccugu 5880

gcuuguggau gaagggagga agguggcaau aaaagggcca cuucguaucu cgcauccuc 5940

ugcugcucaa aggagggggc gcauugggag aaaucccaac agagauggag acucauacua 6000

6060

gcucuuggac aacauggagg ugaggggugg aauggucgcc ccacucuaug gcguugaagg 6120

aacuaaaaca ccaguuuccc cuggugaaau gagacugagg gaugaccaga ggaaagucuu 6180

cagagaacua gugaggaauu gugaccugcc cguuuggcuu ucguggcaag uggccaaggc 6240

ugguuugaag acgaaugauc guaaguggug uuuugaaggc ccugggaac augagaucuu 6300

6360

6420

6480

uuuccuggcu aaaaaaggug gagaggcaau ggauaccauc aguguguuuc uccacucuga 6540

ggaaggcucu agggcuuacc gcaaugcacu aucaaugaug ccugaggcaa ugacaauagu 6600

caugcuguuu auacuggcug gacuacugac aucgggaaug

caaaggcauc aguagaaugu cuauggcgau gggcacaaug gccggcugug gauaucucau 6720

6780

gauggugguu gugauccccg agccagggca acaaaggucc auccaagaca accaaguggc 6840

auaccucauu auuggcaucc ugacgcuggu uucagcggug gcagccaacg agcuaggcau 6900

gcuggagaaa accaaagagg accucuuugg gaagaagaac uuaauuccau cuagugcuuc 6960

acccuggagu uggccggauc uugaccugaa gccaggagcu gccuggacag uguacguugg 7020

cauuguuaca augcucucuc caauguugca ccacuggauc aaagucgaau auggcaaccu 7080

gucucugucu ggaauagccc agucagccuc aguccuuucu uucauggaca aggggauacc 7140

auucaugaag augaauaucu cggucauaau gcugcugguc aguggcugga auucaauaac 7200

agugaugccu cugcucugug gcauagggug cgccaugcuc cacuggucuc ucauuuuacc 7260

uggaaucaaa gcgcagcagu caaagcuugc acagagaagg guguuccaug gcguugccaa 7320

7380

ccuuuaugag aagaaacugg cucuauaucu ccuucuugcu cucagccuag cuucuguugc 7440

caugugcaga acgcccuuuu cauuggcuga aggcauuguc cuagcaucag cugcccuagg 7500

7560

aggagucaug agggggaauc acuaugcuuu ugugggaguc auguacaauc uauggaagau 7620

gaaaacugga cgccggggga gcgcgaaugg aaaaacuuug ggugaagucu ggaagaggga 7680

acugaaucug uuggacaagc gacaguuuga guuguauaaa aggaccgaca uuguggaggu 7740

ggaucgugau acggcacgca ggcauuuggc cgaagggaag guggacaccg ggguggcggu 7800

cuccaggggg accgcaaagu uaaggugguu ccaugagcgu ggcuauguca agcuggaagg 7860

uagggugauu gaccuggggu guggccgcgg aggcuggugu uacuacgcug cugcgcaaaa 7920

ggaagugagu ggggucaaag gauuuacucu uggaagagac ggccaugaga aacccaugaa 7980

ugugcaaagu cugggaugga acaucaucac cuucaaggac aaaacugaua uccaccgccu 8040

agaaccagug aaaugugaca cccuuuugug ugacauugga gagucaucau cgucaucggu 8100

cacagagggg gaaaggaccg ugagaguucu ugauacugua gaaaaauggc uggcuugugg 8160

8220

8280

cacucaugaa auguacuacg ugucuggagc ccgcagcaau gucacauuua cugugaacca 8340

aacaucccgc cuccugauga ggagaaugag gcguccaacu ggaaaaguga cccuggaggc 8400

ugacgucauc cucccaauug ggacacgcag uguugagaca gacaagggac cccuggacaa 8460

agaggccaua gaagaaaggg uugagaggau aaaaucugag uacaugaccu cuugguuuua 8520

ugacaaugac aaccccuaca ggaccuggca cuacuguggc uccuauguca caaaaaccuc 8580

aggaagugcg gcgagcaugg uaaauggugu uauuaaaauu cugacauauc caugggacag 8640

gauagaggag gucacaagaa uggcaaugac ugacacaacc ccuuuuggac agcaaagagu 8700

guuuaaagaa aaaguugaca ccagagcaaa ggauccacca gcgggaacua ggaagaucau 8760

8820

8880

agaacaagaa caguggaaga cugccaauga ggcuguccaa gacccaaagu ucugggaacu 8940

gguggaugaa gaaaggaagc ugcaccaaca aggcaggugu cggacuugug uguacaacau 9000

gauggggaaa agagagaaga agcugucaga guuugggaaa gcaaagggaa gccgugccau 9060

augguauaug uggcugggag cgcgguaucu ugaguuugag gcccugggau uccugaauga 9120

ggaccauugg gcuuccaggg aaaacucagg aggaggagug gaaggcauug gcuuacaaua 9180

ccuagauau gugaucagag accuggcugc aauggauggu gguggauucu acgcggauga 9240

caccgcugga ugggacacgc gcaucacaga ggcagaccuu gaugaugaac aggagaucuu 9300

gaacuacaug agcccacauc acaaaaaacu ggcacaagca gugauggaaa ugacauacaa 9360

gaacaaagug gugaaagugu ugagaccagc cccaggaggg aaagccuaca uggaugucau 9420

aagucgacga gaccagagag gauccgggca gguagugacu uaugcucuga acaccaucac 9480

caacuugaaa guccaauuga ucagaauggc agaagcagag auggugauac aucaccaaca 9540

uguucaagau ugugaugaau caguucugac caggcuggag gcauggcuca cugagcacgg 9600

auguaacaga cugaagagga uggcggugag uggagacgac uguguggucc ggcccaucga 9660

ugacagguuc ggccuggccc ugucccaucu caacgccaug uccaagguua gaaaggacau 9720

aucugaaugg cagccauccaa aaggguggaa ugauugggag aaugugcccu ucuguuccca 9780

ccacuuccau gaacuacagc ugaaggaugg caggaggauu guggugccuu gccgagaaca 9840

ggacgagcuc auugggagag gaaggguguc uccaggaaac ggcuggauga ucaaggaaac 9900

agcuugccuc agcaaagccu augccaacau guggucacug auguauuuuc acaaaaggga 9960

caugaggcua cugucauugg cuguuuccuc agcuguuccc accucauggg uuccacaagg 10020

10080

gguguggaac agaguaugga uaaccaacaa cccacacaug caggacaaga caauggugaa 10140

10200

uggaugacc aauagggcca ccugggccuc ccacauccau uuggucaucc aucguauccg 10260

10320

ggaugcugac cugcaacugg gugagcuuau cugaaacacc aucuaacagg aauaaccggg 10380

auacaaacca cgggguggaga accggacucc ccacaaccug aaaccgggau auaaaccacg 10440

gcuggagaac cggacuccgc acuuaaaaug aaacagaaac cgggauaaaa acuacggaug 10500

10560

uuugccacug cuaagcugug aggcagugca ggcugggaca gccgaccucc agguugcgaa 10620

aaaccugguu ucugggaccu cccaccccag aguaaaaaga acggagccuc cgcuaccacc 10680

cucccacgug gugguagaaa gacggggucu aggguuaga ggagacccuc cagggaacaa 10740

auagugggac cauauugacg ccagggaaag accggagugg uucucugcuu uuccuccaga 10800

ggucugugag cacaguuugc ucaagaauaa gcagaccuuu ggaugacaaa cacaaaacca 10860

cu 10862

<210> 3

<211> 10862

<212> RNA

<213> Yellow fever virus

<220>

<223> RNA sequence of YFV Stamaril® strain

<400> 3

aguaaauccu gugugcuaau ugaggugcau uggucugcaa aucgaguugc uaggcaauaa 60

acacauuugg auuaauuuua aucguucguu gagcgauuag cagagaacug accagaacau 120

gucggucgu aaagcucagg gaaaaacccu gggcgucaau augguacgac gaggaguucg 180

cuccuuguca aacaaaauaa aacaaaaaac aaaacaaauu ggaaacagac cuggaccuuc 240

aagagguguu caaggauuua ucuuuuucuu uuuguucaac auuuugacug gaaaaaagau 300

cacagcccac cuaaagaggu uguggaaaau gcuggaccca agacaaggcu uggcuguucu 360

aaggaaaguc aagagagugg uggccaguuu gaugagagga uuguccucaa ggaaacgccg 420

uucccaugau guucugacug ugcaauuccu aauuuuggga augcuguuga ugacgggugg 480

agugaccuug gugcggaaaa acagaugguu gcuccuaaau gugacaucug aggaccucgg 540

gaaaacauuc ucugugggca caggcaacug cacaacaaac auuuuggaag ccaaguacug 600

gugcccagac ucaauggaau acaacugucc caaucucagu ccaagagagg agccagauga 660

cauugauugc uggugcuaug ggguggaaaa cguuagaguc gcauauggua agugugacuc 720

agcaggcagg ucuaggaggu caagaagggc cauugacuug ccuacgcaug aaaaccaugg 780

uuugaagacc cggcaagaaa aauggaugac uggaagaaug ggugaaaggc aacuccaaaa 840

gauugagaga ugguucguga ggaaccccuu uuuugcagug acggcucuga ccauugccua 900

ccuuguggga agcaacauga cgcaacgagu cgugauugcc cuacugggucu uggcuguugg 960

uccggccuac ucagcucacu gcauuggaau uacugacagg gauuucauug agggggugca 1020

uggaggaacu uggguuucag cuacccugga gcaagacaag ugugucacug uuauggcccc 1080

ugacaagccu ucauuggaca ucucacuaga gacaguagcc auugauagac cugcugaggu 1140

1200 gaggaaagug uguuacaaug caguucucac

cacuggagag gcccaccuag cugaagagaa cgaaggggac aaugcgugca agcgcacuua 1260

uucugauaga ggcuggggca auggcugugg ccuauuuggg aaagggagca uuguggcaug 1320

cgccaaauuc acuugugcca aauccaugag uuuguuugag guugaucaga ccaaaauuca 1380

guaugucauc agagcacaau ugcauguagg ggccaagcag gaaaauugga auaccgacau 1440

uaagacucuc aaguuugaug cccugucagg cucccaggaa gucgaguuca uuggguaugg 1500

aaaagcuaca cuggaaugcc aggugcaaac ugcgguggac uuugguaaca guuacaucgc 1560

ugagauggaa acagaggcu ggauaggga cagacagugg gcccaggacu ugacccugcc 1620

auggcagagu ggaaguggcg ggguguggag agagaugcau caucuugucg aauuugaacc 1680

uccgcaugcc gccacuauca gaguacuggc ccugggaaac caggaaggcu ccuugaaaac 1740

agcucuuacu ggcgcaauga ggguuacaaa ggacacaaau gacaacaacc uuuacaaacu 1800

acauggugga cauguuucuu gcagagugaa auugucagcu uugacacuca aggggacauc 1860

cuacaaaaua ugcacugaca aaauguuuuu ugucaagaac ccaacugaca cuggccaugg 1920

cacuguugug augcagguga aagugucaaa aggagccccc ugcaggauuc cagugauagu 1980

agcugaugau cuuacagcgg caaucaauaa aggcauuuug guuacaguua accccaucgc 2040

cucaaccaau gaugaugaag ugcugauuga ggugaaccca ccuuuuggag acagcuacau 2100

uaucguuggg agaggagauu cacgucucac uuaccagugg cacaaagagg gaagcucaau 2160

aggaaaguug uucacucaga ccaugaaagg cguggaacgc cuggccguca ugggagacac 2220

2280

gguguuuggc ucugccuuuc aggggcuauu uggcggcuug aacuggauaa caaaggucau 2340

2400

caugaucuug guaggaguga ucauguguu uuugucucua ggaguugggg cggaucaagg 2460

2520

agacucugau gacuggcuga acaaguacuc auacuaucca gaagauccug ugaagcuugc 2580

2640

ugagcaugag auguggagaa gcagggcaga ugagaucaau gccauuuuug aggaaaacga 2700

gguggacauu ucuguugucg ugcaggaucc aaagaauguu uaccagagag gaacucaucc 2760

auuuuccaga auucgggaug gucugcagua ugguuggaag acuuggggua agaaccuugu 2820

guucucccca gggaggaaga auggaagcuu caucauagau ggaaagucca ggaaagaaug 2880

cccguuuuca aaccgggucu ggaauucuuu cggauagag gaguuuggga cgggaguguu 2940

caccacacgc guguacaugg acgcagucuu ugaauacacc auagacugcg auggaucuau 3000

cuugggugca gcggugaacg gaaaaaagag ugcccauggc ucuccaacau uuuggauggg 3060

3120

gugugagugg cccugacac auacgauugg aacaucaguu gaagagagug aaauguucau 3180

3240

gacgaacgga ccuuggaugc agguaccacu agaagugaag agagaagcuu gcccagggac 3300

uagcgugauc auugauggca acugugaugg acggggaaaa

uagcgggaaa guuauuccug aaugguguug ccgcuccugc acaaugccgc cugugagcuu 3420

ccaugguagu gauggguguu gguaucccau ggaaauuagg ccaaggaaaa cgcaugaaag 3480

ccaucuggug cgcuccuggg uuacagcugg agaaauacau gcugucccuu uugguuuggu 3540

gagcaugaug auagcaaugg aagugguccu aaggaaaaga cagggaccaa agcaaauguu 3600

3660

3720

cauguauaug gcguugauug cugccuuuuc aaucagacca gggcugcuca ucggcuuugg 3780

gcucaggacc cuauggagcc cucgggaacg ccuugugcug acccuagggag cagccauggu 3840

ggagauugcc uuggguggcg ugagggcgg ccuuggaag uaucuaaaug caguuucucu 3900

3960

cauggcucug uugacaccug ucacuauggc ugaggugaga

ugccgugguu aucauagggg uccuucacca gaacuucaag gacaccucca ugcagaagac 4080

uauaccucug guggcccuca cacucacauc uuaccugggc uugacacaac cuuuuuuggg 4140

ccugugugca uuucuggcaa ccgcauauu ugggcgaagg aguaucccag ugaaugaggc 4200

acucgcagca gcuggucuag ugggagugcu ggcaggacug gcuuuucagg agauggagaa 4260

cuuccuuggu ccgauugcag uuggaggacu ccugaugaug cugguuagcg uggcugggag 4320

gguggauggg cuagagcuca agaagcuugg ugaaguuuca ugggaagagg aggcggagau 4380

cagcgggagu uccgcccgcu augauguggc acucagugaa caaggggagu ucaagcugcu 4440

uucugaagag aaagugccau gggaccaggu ugugaugacc ucgcuggccu ugguuggggc 4500

ugcccuccau ccauuugcuc uucugcuggu ccuugcuggg uggcuguuuc augucagggg 4560

4620

ugaacaugg gaggauggga uuuauggcau auuccaguca accuucuugg gggccuccca 4680

gcgaggagug ggaguggcac agggaggggu guuccacaca auguggcaug ucacaagagg 4740

agcuuuccuu gucaggaaug gcaagaaguu gauuccaucu ugggcuucag uaaaggaaga 4800

ccuugucgcc uaugguggcu cauggaaguu ggaaggcaga ugggauggag aggaagggu 4860

ccaguugauc gcggcuguuc caggaaagaa cguggucaac guccagacaa aaccgagcuu 4920

guucaaagug aggaaugggg gagaaaucgg ggcugucgcu cuugacuauc cgaguggcac 4980

uucaggaucu ccuauuguua acaggaacgg agaggugauu gggcuguacg gcaauggcau 5040

ccuugucggu gacaacuccu ucguguccgc cauaucccag acugagguga aggaagaagg 5100

aaaggaggag cuccaagaga ucccgacaau gcuaaagaaa ggaaugacaa cuguccuuga 5160

uuuucauccu ggagcuggga agacaagacg uuuccuccca cagaucuugg ccgagugcgc 5220

acggagacgc uugcgcacuc uuguguuggc ccccaccagg guuguucuuu cugaaaugaa 5280

ggaggcuuuu cacggccugg acgugaaauu ccacacacag gcuuuuuccg cucacggcag 5340

cgggagagaa gucauugaug ccaugugcca ugccacccua acuuacagga uguuggaacc 5400

aacuaggguu guuaacuggg aagugaucau uuggaugaa gcccauuuuu uggauccagc 5460

uagcauagcc gcuagagguu gggcagcgca cagagcuagg gcaaaugaaa gugcaacaau 5520

cuugaugaca gccacaccgc cugggacuag ugaugaauuu ccacauucaa aggugaaau 5580

agaagauguu caaacggaca uacccaguga gcccuggaac acagggcaug acuggauccu 5640

ggcugacaaa aggcccacgg caugguuccu uccauccauc agagcugcaa augucauggc 5700

ugccucuuug cguaaggcug gaaagagugu ggugguccug aacaggaaaa ccuuugagag 5760

agaauacccc acgauaaagc agaagaaacc ugacuuuaua uuggcacug acauagcuga 5820

aaugggagcc aaccuuugcg uggagcgagu gcuggauugc aggacggcuu uuaagccugu 5880

gcuuguggau gaagggagga agguggcaau aaaagggcca cuucguaucu cgcauccuc 5940

ugcugcucaa aggagggggc gcauugggag aaaucccaac agagauggag acucauacua 6000

6060

gcucuuggac aacauggagg ugaggggugg aauggucgcc ccacucuaug gcguugaagg 6120

aacuaaaaca ccaguuuccc cuggugaaau gagacugagg gaugaccaga ggaaagucuu 6180

cagagaacua gugaggaauu gugaccugcc cguuuggcuu ucguggcaag uggccaaggc 6240

ugguuugaag acgaaugauc guaaguggug uuuugaaggc ccugggaac augagaucuu 6300

6360

6420

6480

uuuccuggcu aaaaaaggug gagaggcaau ggauaccauc aguguguuuc uccacucuga 6540

ggaaggcucu agggcuuacc gcaaugcacu aucaaugaug ccugaggcaa ugacaauagu 6600

caugcuguuu auacuggcug gacuacugac aucgggaaug

caaaggcauc aguagaaugu cuauggcgau gggcacaaug gccggcugug gauaucucau 6720

6780

gauggugguu gugauccccg agccagggca acaaaggucc auccaagaca accaaguggc 6840

auaccucauu auuggcaucc ugacgcuggu uucagcggug gcagccaacg agcuaggcau 6900

gcuggagaaa accaaagagg accucuuugg gaagaagaac uuaauuccau cuagugcuuc 6960

acccuggagu uggccggauc uugaccugaa gccaggagcu gccuggacag uguacguugg 7020

cauuguuaca augcucucuc caauguugca ccacuggauc aaagucgaau auggcaaccu 7080

gucucugucu ggaauagccc agucagccuc aguccuuucu uucauggaca aggggauacc 7140

auucaugaag augaauaucu cggucauaau gcugcugguc aguggcugga auucaauaac 7200

agugaugccu cugcucugug gcauagggug cgccaugcuc cacuggucuc ucauuuuacc 7260

uggaaucaaa gcgcagcagu caaagcuugc acagagaagg guguuccaug gcguugccaa 7320

7380

ccuuuaugag aagaaacugg cucuauaucu ccuucuugcu cucagccuag cuucuguugc 7440

caugugcaga acgcccuuuu cauuggcuga aggcauuguc cuagcaucag cugccuuagg 7500

7560

aggagucaug agggggaauc acuaugcuuu ugugggaguc auguacaauc uauggaagau 7620

gaaaacugga cgccggggga gcgcgaaugg aaaaacuuug ggugaagucu ggaagaggga 7680

acugaaucug uuggacaagc gacaguuuga guuguauaaa aggaccgaca uuguggaggu 7740

ggaucgugau acggcacgca ggcauuuggc cgaagggaag guggacaccg ggguggcggu 7800

cuccaggggg accgcaaagu uaaggugguu ccaugagcgu ggcuauguca agcuggaagg 7860

uagggugauu gaccuggggu guggccgcgg aggcuggugu uacuacgcug cugcgcaaaa 7920

ggaagugagu ggggucaaag gauuuacucu uggaagagac ggccaugaga aacccaugaa 7980

ugugcaaagu cugggaugga acaucaucac cuucaaggac aaaacugaua uccaccgccu 8040

agaaccagug aaaugugaca cccuuuugug ugacauugga gagucaucau cgucaucggu 8100

cacagagggg gaaaggaccg ugagaguucu ugauacugua gaaaaauggc uggcuugugg 8160

8220

8280

cacucaugaa auguacuacg ugucuggagc ccgcagcaau gucacauuua cugugaacca 8340

aacaucccgc cuccugauga ggagaaugag gcguccaacu ggaaaaguga cccuggaggc 8400

ugacgucauc cucccaauug ggacacgcag uguugagaca gacaagggac cccuggacaa 8460

agaggccaua gaagaaaggg uugagaggau aaaaucugag uacaugaccu cuugguuuua 8520

ugacaaugac aaccccuaca ggaccuggca cuacuguggc uccuauguca caaaaaccuc 8580

aggaagugcg gcgagcaugg uaaauggugu uauuaaaauu cugacauauc caugggacag 8640

gauagaggag gucacaagaa uggcaaugac ugacacaacc ccuuuuggac agcaaagagu 8700

guuuaaagaa aaaguugaca ccagagcaaa ggauccacca gcgggaacua ggaagaucau 8760

8820

8880

agaacaagaa caguggaaga cugccaauga ggcuguccaa gacccaaagu ucugggaacu 8940

gguggaugaa gaaaggaagc ugcaccaaca aggcaggugu cggacuugug uguacaacau 9000

gauggggaaa agagagaaga agcugucaga guuugggaaa gcaaagggaa gccgugccau 9060

augguauaug uggcugggag cgcgguaucu ugaguuugag gcccugggau uccugaauga 9120

ggaccauugg gcuuccaggg aaaacucagg aggaggagug gaaggcauug gcuuacaaua 9180

ccuagauau gugaucagag accuggcugc aauggauggu gguggauucu acgcggauga 9240

caccgcugga ugggacacgc gcaucacaga ggcagaccuu gaugaugaac aggagaucuu 9300

gaacuacaug agcccacauc acaaaaaacu ggcacaagca gugauggaaa ugacauacaa 9360

gaacaaagug gugaaagugu ugagaccagc cccaggaggg aaagccuaca uggaugucau 9420

aagucgacga gaccagagag gauccgggca gguagugacu uaugcucuga acaccaucac 9480

caacuugaaa guccaauuga ucagaauggc agaagcagag auggugauac aucaccaaca 9540

uguucaagau ugugaugaau caguucugac caggcuggag gcauggcuca cugagcacgg 9600

auguaacaga cugaagagga uggcggugag uggagacgac uguguggucc ggcccaucga 9660

ugacagguuc ggccuggccc ugucccaucu caacgccaug uccaagguua gaaaggacau 9720

aucugaaugg cagccauccaa aaggguggaa ugauugggag aaugugcccu ucuguuccca 9780

ccacuuccau gaacuacagc ugaaggaugg caggaggauu guggugccuu gccgagaaca 9840

ggacgagcuc auugggagag gaaggguguc uccaggaaac ggcuggauga ucaaggaaac 9900

agcuugccuc agcaaagccu augccaacau guggucacug auguauuuuc acaaaaggga 9960

caugaggcua cugucauugg cuguuuccuc agcuguuccc accucauggg uuccacaagg 10020

10080

gguguggaac agaguaugga uaaccaacaa cccacacaug caggacaaga caauggugaa 10140

10200

uggaugacc aauagggcca ccugggccuc ccacauccau uuggucaucc aucguauccg 10260

10320

ggaugcugac cugcaacugg gugagcuuau cugaaacacc aucuaacagg aauaaccggg 10380

auacaaacca cgggguggaga accggacucc ccacaaccug aaaccgggau auaaaccacg 10440

gcuggagaac cggacuccgc acuuaaaaug aaacagaaac cgggauaaaa acuacggaug 10500

10560

uuugccacug cuaagcugug aggcagugca ggcugggaca gccgaccucc agguugcgaa 10620

aaaccugguu ucugggaccu cccaccccag aguaaaaaga acggagccuc cgcuaccacc 10680

cucccacgug gugguagaaa gacggggucu aggguuaga ggagacccuc cagggaacaa 10740

auagugggac cauauugacg ccagggaaag accggagugg uucucugcuu uuccuccaga 10800

ggucugugag cacaguuugc ucaagaauaa gcagaccuuu ggaugacaaa cacaaaacca 10860

cu 10862

<210> 4

<211> 10862

<212> RNA

<213> Yellow fever virus

<220>

<223> RNA sequence of YFV strain 17D-213

<400> 4

aguaaauccu gugugcuaau ugaggugcau uggucugcaa aucgaguugc uaggcaauaa 60

acacauuugg auuaauuuua aucguucguu gagcgauuag cagagaacug accagaacau 120

gucggucgu aaagcucagg gaaaaacccu gggcgucaau augguacgac gaggaguucg 180

cuccuuguca aacaaaauaa aacaaaaaac aaaacaaauu ggaaacagac cuggaccuuc 240

aagagguguu caaggauuua ucuuuuucuu uuuguucaac auuuugacug gaaaaaagau 300

cacagcccac cuaaagaggu uguggaaaau gcuggaccca agacaaggcu uggcuguucu 360

aaggaaaguc aagagagugg uggccaguuu gaugagagga uuguccucaa ggaaacgccg 420

uucccaugau guucugacug ugcaauuccu aauuuuggga augcuguuga ugacgggugg 480

agugaccuug gugcggaaaa acagaugguu gcuccuaaau gugacaucug aggaccucgg 540

gaaaacauuc ucugugggca caggcaacug cacaacaaac auuuuggaag ccaaguacug 600

gugcccagac ucaauggaau acaacugucc caaucucagu ccaagagagg agccagauga 660

cauugauugc uggugcuaug ggguggaaaa cguuagaguc gcauauggua agugugacuc 720

agcaggcagg ucuaggaggu caagaagggc cauugacuug ccuacgcaug aaaaccaugg 780

uuugaagacc cggcaagaaa aauggaugac uggaagaaug ggugaaaggc aacuccaaaa 840

gauugagaga ugguucguga ggaaccccuu uuuugcagug acggcucuga ccauugccua 900

ccuuguggga agcaacauga cgcaacgagu cgugauugcc cuacugggucu uggcuguugg 960

uccggccuac ucagcucacu gcauuggaau uacugacagg gauuucauug agggggugca 1020

uggaggaacu uggguuucag cuacccugga gcaagacaag ugugucacug uuauggcccc 1080

ugacaagccu ucauuggaca ucucacuaga gacaguagcc auugauagac cugcugaggu 1140

1200 gaggaaagug uguuacaaug caguucucac

cacuggagag gcccaccuag cugaagagaa cgaaggggac aaugcgugca agcgcacuua 1260

uucugauaga ggcuggggca auggcugugg ccuauuuggg aaagggagca uuguggcaug 1320

cgccaaauuc acuugugcca aauccaugag uuuguuugag guugaucaga ccaaaauuca 1380

guaugucauc agagcacaau ugcauguagg ggccaagcag gaaaauugga cuaccgacau 1440

uaagacucuc aaguuugaug cccugucagg cucccaggaa gucgaguuca uuggguaugg 1500

aaaagcuaca cuggaaugcc aggugcaaac ugcgguggac uuugguaaca guuacaucgc 1560

ugagauggaa acagaggcu ggauaggga cagacagugg gcccaggacu ugacccugcc 1620

auggcagagu ggaaguggcg ggguguggag agagaugcau caucuugucg aauuugaacc 1680

uccgcaugcc gccacuauca gaguacuggc ccugggaaac caggaaggcu ccuugaaaac 1740

agcucuuacu ggcgcaauga ggguuacaaa ggacacaaau gacaacaacc uuuacaaacu 1800

acauggugga cauguuucuu gcagagugaa auugucagcu uugacacuca aggggacauc 1860

cuacaaaaua ugcacugaca aaauguuuuu ugucaagaac ccaacugaca cuggccaugg 1920

cacuguugug augcagguga aagugucaaa aggagccccc ugcaggauuc cagugauagu 1980

agcugaugau cuuacagcgg caaucaauaa aggcauuuug guuacaguua accccaucgc 2040

cucaaccaau gaugaugaag ugcugauuga ggugaaccca ccuuuuggag acagcuacau 2100

uaucguuggg agaggagauu cacgucucac uuaccagugg cacaaagagg gaagcucaau 2160

aggaaaguug uucacucaga ccaugaaagg cguggaacgc cuggccguca ugggagacac 2220

2280

gguguuuggc ucugccuuuc aggggcuauu uggcggcuug aacuggauaa caaaggucau 2340

2400

caugaucuug guaggaguga ucauguguu uuugucucua ggaguugggg cggaucaagg 2460

2520

agacucugau gacuggcuga acaaguacuc auacuaucca gaagauccug ugaagcuugc 2580

2640

ugagcaugag auguggagaa gcagggcaga ugagaucaau gccauuuuug aggaaaacga 2700

gguggacauu ucuguugucg ugcaggaucc aaagaauguu uaccagagag gaacucaucc 2760

auuuuccaga auucgggaug gucugcagua ugguuggaag acuuggggua agaaccuugu 2820

guucucccca gggaggaaga auggaagcuu caucauagau ggaaagucca ggaaagaaug 2880

cccguuuuca aaccgggucu ggaauucuuu cggauagag gaguuuggga cgggaguguu 2940

caccacacgc guguacaugg acgcagucuu ugaauacacc auagacugcg auggaucuau 3000

cuugggugca gcggugaacg gaaaaaagag ugcccauggc ucuccaacau uuuggauggg 3060

3120

gugugagugg cccugacac auacgauugg aacaucaguu gaagagagug aaauguucau 3180

3240

gacgaacgga ccuuggaugc agguaccacu agaagugaag agagaagcuu gcccagggac 3300

uagcgugauc auugauggca acugugaugg acggggaaaa

uagcgggaaa guuauuccug aaugguguug ccgcuccugc acaaugccgc cugugagcuu 3420

ccaugguagu gauggguguu gguaucccau ggaaauuagg ccaaggaaaa cgcaugaaag 3480

ccaucuggug cgcuccuggg uuacagcugg agaaauacau gcugucccuu uugguuuggu 3540

gagcaugaug auagcaaugg aagugguccu aaggaaaaga cagggaccaa agcaaauguu 3600

3660

3720

cauguauaug gcguugauug cugccuuuuc aaucagacca gggcugcuca ucggcuuugg 3780

gcucaggacc cuauggagcc cucgggaacg ccuugugcug acccuagggag cagccauggu 3840

ggagauugcc uuggguggcg ugagggcgg ccuuggaag uaucuaaaug caguuucucu 3900

3960

cauggcucug uugacaccug ucacuauggc ugaggugaga

ugccgugguu aucauagggg uccuucacca gaauuucaag gacaccucca ugcagaagac 4080

uauaccucug guggcccuca cacucacauc uuaccugggc uugacacaac cuuuuuuggg 4140

ccugugugca uuucuggcaa ccgcauauu ugggcgaagg aguaucccag ugaaugaggc 4200

acucgcagca gcuggucuag ugggagugcu ggcaggacug gcuuuucagg agauggagaa 4260

cuuccuuggu ccgauugcag uuggaggacu ccugaugaug cugguuagcg uggcugggag 4320

gguggauggg cuagagcuca agaagcuugg ugaaguuuca ugggaagagg aggcggagau 4380

cagcgggagu uccgcccgcu augauguggc acucagugaa caaggggagu ucaagcugcu 4440

uucugaagag aaagugccau gggaccaggu ugugaugacc ucgcuggccu ugguuggggc 4500

ugcccuccau ccauuugcuc uucugcuggu ccuugcuggg uggcuguuuc augucagggg 4560

4620

ugaacaugg gaggauggga uuuauggcau auuccaguca accuucuugg gggccuccca 4680

gcgaggagug ggaguggcac agggaggggu guuccacaca auguggcaug ucacaagagg 4740

agcuuuccuu gucaggaaug gcaagaaguu gauuccaucu ugggcuucag uaaaggaaga 4800

ccuugucgcc uaugguggcu cauggaaguu ggaaggcaga ugggauggag aggaagggu 4860

ccaguugauc gcggcuguuc caggaaagaa cguggucaac guccagacaa aaccgagcuu 4920

guucaaagug aggaaugggg gagaaaucgg ggcugucgcu cuugacuauc cgaguggcac 4980

uucaggaucu ccuauuguua acaggaacgg agaggugauu gggcuguacg gcaauggcau 5040

ccuugucggu gacaacuccu ucguguccgc cauaucccag acugagguga aggaagaagg 5100

aaaggaggag cuccaagaga ucccgacaau gcuaaagaaa ggaaugacaa cuguccuuga 5160

uuuucauccu ggagcuggga agacaagacg uuuccuccca cagaucuugg ccgagugcgc 5220

acggagacgc uugcgcacuc uuguguuggc ccccaccagg guuguucuuu cugaaaugaa 5280

ggaggcuuuu cacggccugg acgugaaauu ccacacacag gcuuuuuccg cucacggcag 5340

cgggagagaa gucauugaug cuaugugcca ugccacccua acuuacagga uguuggaacc 5400

aacuaggguu guuaacuggg aagugaucau uuggaugaa gcccauuuuu uggauccagc 5460

uagcauagcc gcuagagguu gggcagcgca cagagcuagg gcaaaugaaa gugcaacaau 5520

cuugaugaca gccacaccgc cugggacuag ugaugaauuu ccacauucaa aggugaaau 5580

agaagauguu caaacggaca uacccaguga gcccuggaac acagggcaug acuggauccu 5640

ggcugacaaa aggcccacgg caugguuccu uccauccauc agagcugcaa augucauggc 5700

ugccucuuug cguaaggcug gaaagagugu ggugguccug aacaggaaaa ccuuugagag 5760

agaauacccc acgauaaagc agaagaaacc ugacuuuaua uuggcacug acauagcuga 5820

aaugggagcc aaccuuugcg uggagcgagu gcuggauugc aggacggcuu uuaagccugu 5880

gcuuguggau gaagggagga agguggcaau aaaagggcca cuucguaucu cgcauccuc 5940

ugcugcucaa aggagggggc gcauugggag aaaucccaac agagauggag acucauacua 6000

6060

gcucuuggac aacauggagg ugaggggugg aauggucgcc ccacucuaug gcguugaagg 6120

aacuaaaaca ccaguuuccc cuggugaaau gagacugagg gaugaccaga ggaaagucuu 6180

cagagaacua gugaggaauu gugaccugcc cguuuggcuu ucguggcaag uggccaaggc 6240

ugguuugaag acgaaugauc guaaguggug uuuugaaggc ccugggaac augagaucuu 6300

6360

6420

6480

uuuccuggcu aaaaaaggug gagaggcaau ggauaccauc aguguguuuc uccacucuga 6540

ggaaggcucu agggcuuacc gcaaugcacu aucaaugaug ccugaggcaa ugacaauagu 6600

caugcuguuu auacuggcug gacuccugac aucgggaaug

caaaggcauc aguagaaugu cuauggcgau gggcacaaug gccggcugug gauaucucau 6720

6780

gauggugguu gugauccccg agccagggca acaaaggucc auccaagaca accaaguggc 6840

auaccucauu auuggcaucc ugacgcuggu uucagcggug gcagccaacg agcuaggcau 6900

gcuggagaaa accaaagagg accucuuugg gaagaagaac uuaauuccau cuagugcuuc 6960

acccuggagu uggccggauc uugaccugaa gccaggagcu gccuggacag uguacguugg 7020

cauuguuaca augcucucuc caauguugca ccacuggauc aaagucgaau auggcaaccu 7080

gucucugucu ggaauagccc agucagccuc aguccuuucu uucauggaca aggggauacc 7140

auucaugaag augaauaucu cggucauaau gcugcugguc aguggcugga auucaauaac 7200

agugaugccu cugcucugug gcauagggug cgccaugcuc cacuggucuc ucauuuuacc 7260

uggaaucaaa gcgcagcagu caaagcuugc acagagaagg guguuccaug gcguugccaa 7320

7380

ccuuuaugag aagaaacugg cucuauaucu ccuucuugcu cucagccuag cuucuguugc 7440

caugugcaga acgcccuuuu cauuggcuga aggcauuguc cuagcaucag cugccucagg 7500

7560

aggagucaug agggggaauc acuaugcuuu ugugggaguc auguacaauc uauggaagau 7620

gaaaacugga cgccggggga gcgcgaaugg aaaaacuuug ggugaagucu ggaagaggga 7680

acugaaucug uuggacaagc gacaguuuga guuguauaaa aggaccgaca uuguggaggu 7740

ggaucgugau acggcacgca ggcauuuggc cgaagggaag guggacaccg ggguggcggu 7800

cuccaggggg accgcaaagu uaaggugguu ccaugagcgu ggcuauguca agcuggaagg 7860

uagggugauu gaccuggggu guggccgcgg aggcuggugu uacuacgcug cugcgcaaaa 7920

ggaagugagu ggggucaaag gauuuacucu uggaagagac ggccaugaga aacccaugaa 7980

ugugcaaagu cugggaugga acaucaucac cuucaaggac aaaacugaua uccaccgccu 8040

agaaccagug aaaugugaca cccuuuugug ugacauugga gagucaucau cgucaucggu 8100

cacagagggg gaaaggaccg ugagaguucu ugauacugua gaaaaauggc uggcuugugg 8160

8220

8280

cacucaugaa auguacuacg ugucuggagc ccgcagcaau gucacauuua cugugaacca 8340

aacaucccgc cuccugauga ggagaaugag gcguccaacu ggaaaaguga cccuggaggc 8400

ugacgucauc cucccaauug ggacacgcag uguugagaca gacaagggac cccuggacaa 8460

agaggccaua gaagaaaggg uugagaggau aaaaucugag uacaugaccu cuugguuuua 8520

ugacaaugac aaccccuaca ggaccuggca cuacuguggc uccuauguca caaaaaccuc 8580

aggaagugcg gcgagcaugg uaaauggugu uauuaaaauu cugacauauc caugggacag 8640

gauagaggag gucacaagaa uggcaaugac ugacacaacc ccuuuuggac agcaaagagu 8700

guuuaaagaa aaaguugaca ccagagcaaa ggauccacca gcgggaacua ggaagaucau 8760

8820

8880

agaacaagaa caguggaaga cugccaauga ggcuguccaa gacccaaagu ucugggaacu 8940

gguggaugaa gaaaggaagc ugcaccaaca aggcaggugu cggacuugug uguacaacau 9000

gauggggaaa agagagaaga agcugucaga guuugggaaa gcaaagggaa gccgugccau 9060

augguauaug uggcugggag cgcgguaucu ugaguuugag gcccugggau uccugaauga 9120

ggaccauugg gcuuccaggg aaaacucagg aggaggagug gaaggcauug gcuuacaaua 9180

ccuagauau gugaucagag accuggcugc aauggauggu gguggauucu acgcggauga 9240

caccgcugga ugggacacgc gcaucacaga ggcagaccuu gaugaugaac aggagaucuu 9300

gaacuacaug agcccacauc acaaaaaacu ggcacaagca gugauggaaa ugacauacaa 9360

gaacaaagug gugaaagugu ugagaccagc cccaggaggg aaagccuaca uggaugucau 9420

aagucgacga gaccagagag gauccgggca gguagugacu uaugcucuga acaccaucac 9480

caacuugaaa guccaauuga ucagaauggc agaagcagag auggugauac aucaccaaca 9540

uguucaagau ugugaugaau caguucugac caggcuggag gcauggcuca cugagcacgg 9600

auguaacaga cugaagagga uggcggugag uggagacgac uguguggucc ggcccaucga 9660

ugacagguuc ggccuggccc ugucccaucu caacgccaug uccaagguua gaaaggacau 9720

aucugaaugg cagccauccaa aaggguggaa ugauugggag aaugugcccu ucuguuccca 9780

ccacuuccau gaacuacagc ugaaggaugg caggaggauu guggugccuu gccgagaaca 9840

ggacgagcuc auugggagag gaaggguguc uccaggaaac ggcuggauga ucaaggaaac 9900

agcuugccuc agcaaagccu augccaacau guggucacug auguauuuuc acaaaaggga 9960

caugaggcua cugucauugg cuguuuccuc agcuguuccc accucauggg uuccacaagg 10020

10080

gguguggaac agaguaugga uaaccaacaa cccacacaug caggacaaga caauggugaa 10140

10200

uggaaugacc aauagggcca ccugggccuc ccacauccau uuagucaucc aucguauccg 10260

10320

ggaugcugac cugcaacugg gugagcuuau cugaaacacc aucuaacagg aauaaccggg 10380

auacaaacca cgggguggaga accggacucc ccacaaccug aaaccgggau auaaaccacg 10440

gcuggagaac cggacuccgc acuuaaaaug aaacagaaac cgggauaaaa acuacggaug 10500

10560

uuugccacug cuaagcugug aggcagugca ggcugggaca gccgaccucc agguugcgaa 10620

aaaccugguu ucugggaccu cccaccccag aguaaaaaga acggagccuc cgcuaccacc 10680

cucccacgug gugguagaaa gacggggucu aggguuaga ggagacccuc cagggaacaa 10740

auagugggac cauauugacg ccagggaaag accggagugg uucucugcuu uuccuccaga 10800

ggucugugag cacaguuugc ucaagaauaa gcagaccuuu ggaugacaaa cacaaaacca 10860

cu 10862

<210> 5

<211> 10862

<212> RNA

<213> Yellow fever virus

<220>

<223> RNA sequence of YFV strain 17DD

<400> 5

aguaaauccu gugugcuaau ugaggugcau uggucugcaa aucgaguugc uaggcaauaa 60

acacauuugg auuaauuuua aucguucguu gagcgauuag cagagaacug accagaacau 120

gucggucgu aaagcucagg gaaaaacccu gggcgucaau augguacgac gaggaguucg 180

cuccuuguca aacaaaauaa aacaaaaaac aaaacaaauu ggaaacagac cuggaccuuc 240

aagagguguu caaggauuua ucuuuuucuu uuuguucaac auuuugacug gaaaaaagau 300

cacagcccac cuaaagaggu uguggaaaau gcuggaccca agacaaggcu uggcuguucu 360

aaggaaaguu aagagagugg uggccaguuu gaugagagga uuguccucaa ggaaacgccg 420

uucccaugau guucugacug ugcaauuccu aauuuuggga augcuguuga ugacgggugg 480

agugaccuug gugcggaaaa acagaugguu gcuccuaaau gugacaucug aggaccucgg 540

gaaaacauuc ucugugggca caggcaacug cacaacaaac auuuuggaag ccaaguacug 600

gugcccagac ucaauggaau acaacugucc caaucucagu ccgagagagg agccagauga 660

cauugauugc uggugcuaug ggguggaaaa cguuagaguc gcauauggua agugugacuc 720

agcaggcagg ucuaggaggu caagaagggc cauugacuug ccuacgcaug aaaaccaugg 780

uuugaagacc cggcaagaaa aauggaugac uggaagaaug ggugaaaggc aacuccaaaa 840

gauugagaga ugguucguga ggaaccccuu uuuugcagug acagcucuga ccauugccua 900

ccuuguggga agcaacauga cgcaacgagu cgugauugcc cuacugggucu uggcuguugg 960

uccggccuac ucagcucacu gcauuggaau uacugacagg gauuucauug agggggugca 1020

uggaggaacu uggguuucag cuacccugga gcaagacaag ugugucacug uuauggcccc 1080

ugacaagccu ucauuggaca ucucacuaga gacaguagcc auugauagac cugcugaggc 1140

1200 gaggaaagug uguuacaaug caguucucac

cacuggagag gcccaccuag cugaagagaa cgaaggggac aaugcgugca agcgcacuua 1260

uucugauaga ggcuggggca auggcugugg ccuauuuggg aaagggagca uuguggcaug 1320

cgccaaauuc acuugugcca aauccaugag uuuguuugag guugaucaga ccaaaauuca 1380

guaugucauc agagcacaau ugcauguagg ggccaagcag gaaaauugga auaccagcau 1440

uaagacucuc aaguuugaug cccugucagg cucccaggaa gucgaguuca uuggguaugg 1500

aaaagcuaca cuggaaugcc aggugcaaac ugcgguggac uuugguaaca guuacauagc 1560

ugagauggaa acagaggcu ggauaggga cagacagugg gcccaggacu ugacccugcc 1620

auggcagagu ggaaguggcg ggguguggag agagaugcau caucuugucg aauuugaacc 1680

uccgcaugcc gccacuauca gaguacuggc ccugggaaac caggaaggcu ccuugaaaac 1740

agcucuuacu ggcgcaauga ggguuacaaa ggacacaaau gacaacaacc uuuacaaacu 1800

acauggugga cauguuucuu gcagagugaa auugucagcu uugacacuca aggggacauc 1860

cuacaaaaua ugcacugaca aaauguuuuu ugucaagaac ccaacugaca cuggccaugg 1920

cacuguugug augcagguga aagugccaaa aggagccccc ugcaggauuc cagugauagu 1980

agcugaugau cuuacagcgg caaucaauaa aggcauuuug guuacaguua accccaucgc 2040

cucaaccaau gaugaugaag ugcugauuga ggugaaccca ccuuuuggag acagcuacau 2100

uaucguugga agaggagauu cacgucucac uuaccagugg cacaaagagg gaagcucaau 2160

aggaaaguug uucacucaga ccaugaaagg cguggaacgc cuggccguca ugggagacgu 2220

2280

gguguuuggc ucugccuuuc aggggcuauu uggcggcuug aacuggauaa caaaggucau 2340

2400

caugaucuug guaggaguga ucauguguu uuugucucua ggaguugggg cggaucaagg 2460

2520

agacucugau gacuggcuga acaaguacuc auacuaucca gaagauccug ugaagcuugc 2580

2640

ugagcaugag auguggagaa gcagggcaga ugagaucaau gccauuuuug aggaaaacga 2700

gguggacauu ucuguugucg ugcaggaucc aaagaauguu uaccagagag gaacucaucc 2760

auuuuccaga auucgggaug gucugcagua ugguuggaag acuuggggua agaaccuugu 2820

guucucccca gggaggaaga auggaagcuu caucauagau ggaaagucca ggaaagaaug 2880

cccguuuuca aaccgggucu ggaauucuuu cggauagag gaguuuggga cgggaguguu 2940

caccacacgc guguacaugg acgcagucuu ugaauacacc auagacugcg auggaucuau 3000

cuugggugca gcggugaacg gaaaaaagag ugcccauggc ucuccaacau uuuggauggg 3060

3120

gugugagugg cccugacac auacgauugg aacaucaguu gaagagagug aaauguucau 3180

3240

gacgaacgga ccuuggaugc agguaccacu agaagugaag agagaagcuu gcccagggac 3300

uagcgugauc auugauggca acugugaugg acggggaaaa

uagcgggaaa guuauuccug aaugguguug ccgcuccugc acaaugccgc cugugagcuu 3420

ccaugguagu gauggguguu gguaucccau ggaaauuagg ccaaggaaaa cgcaugaaag 3480

ccaucuggug cgcuccuggg uuacagcugg agaaauacau gcugucccuu uugguuuggu 3540

gagcaugaug auagcaaugg aagugguccu aaggaaaaga cagggaccaa agcaaaugcu 3600

3660

3720

cauguauaug gcguugauug cugccuuuuc aaucagacca gggcugcuca ucggcuuugg 3780

gcucaggacc cuauggagcc cucgggaacg ccuugugcug acccuagggag cagccauggu 3840

ggagauugcc uuggguggcg ugagggcgg ccuuggaag uaucuaaaug caguuucucu 3900

3960

uugacaccug ucacuauggc ugaggugaga cuugccgcaa ugcucuuuug 4020

ugccgugguu aucauagggg uccuucacca gaacuucaag gacaccucca ugcagaagac 4080

uauaccucug guggcccuca cacucacauc uuaccugggc uugacacaac cuuuuuuggg 4140

ccugugugca uuucuggcaa ccgcauauu ugggcgaagg aguaucccag ugaaugaggc 4200

acuugcagca gcuggucuag ugggagugcu ggcaggacug gcuuuucagg agauggagaa 4260

cuuccuuggu ccgauugcag uuggaggacu ccugaugaug cugguuagcg uggcugggag 4320

gguggauggg cuagagcuca agaagcuugg ugaaguuuca ugggaagagg aggcggagau 4380

cagcgggagu uccgcccgcu augauguggc acucagugaa caaggggagu ucaagcugcu 4440

uucugaagag aaagugccau gggaccaggu ugugaugacc ucgcuggccu ugguuggggc 4500

ugcccuccau ccauuugcuc uucugcuggu ccuugcuggg uggcuguuuc augucagggg 4560

4620

ugaacaugg gaggauggga uuuauggcau auuccaguca accuucuugg gggccuccca 4680

gcgaggagug ggaguggcac agggaggggu guuccacaca auguggcaug ucacaagagg 4740

agcuuuccuu gucaggaaug gcaagaaguu gauuccaucu ugggcuucag uaaaggaaga 4800

ccuugucgcc uaugguggcu cauggaaguu ggaaggcaga ugggauggag aggaagggu 4860

ccaguugauc gcugcuguuc caggaaagaa cguggucaac guccagacaa aaccgagcuu 4920

guucaaagug aggaaugggg gggaaaucgg ggcuguugcu cuugacuauc caaguggcac 4980

uucaggaucu ccuauuguua acaggaacgg agaggugauu gggcuguacg gcaauggcau 5040

ccuugucggu gacaacuccu ucguguccgc cauaucccag acugagguga aggaagaagg 5100

aaaggaggag cuccgagaga ucccgacaau gcuaaagaaa ggaaugacaa cuauccuuga 5160

uuuucauccu ggagcuggga agacaagacg uuuccuccca cagaucuugg ccgagugcgc 5220

acggcgacgc uugcgcacuc uuguguuggc ccccaccagg guuguucuuu cugaaaugaa 5280

ggaggcuuuu cacggccugg acgugaaauu ccacacacag gcuuuuuccg cucacggcag 5340

cgggagagaa gucauugaug caaugugcca ugccacccua acuuacagga uguuggaacc 5400

aacuaggguu guuaacuggg aagugaucau uuggaugaa gcccauuuuu uggauccagc 5460

uagcauagcc gcuagagguu gggcagcgca cagagcuagg gcaaaugaaa gugcaacaau 5520

cuugaugaca gccacaccgc cugggacuag ugaugaauuu ccacauucaa aggugaaau 5580

agaagauguu caaacggaca uacccaguga gcccuggaac acagggcaug acuggauccu 5640

ggcugacaaa aggcccacgg caugguuccu uccauccauc agagcugcaa augucauggc 5700

ugccucuuug cguaaggcug gaaagagugu ggugguccug aacaggaaaa ccuuugagag 5760

agaauacccc acgauaaagc agaagaaacc ugacuuuaua uuggcacug acauagcuga 5820

aaugggagcc aaccuuugcg uggagcgagu gcuggauugc aggacggcuu uuaagccugu 5880

gcuuguggau gaagggagga agguggcaau aaaagggcca cuucguaucu cgcauccuc 5940

ugcugcucaa aggagggggc gcauugggag aaaucccaac agagauggag acucauacua 6000

6060

gcucuuggau aacauggagg ugaggggugg aauggucgcc ccacucuaug gcguugaagg 6120

aacuaaaaca ccaguuuccc cuggugaaau gagacugagg gaugaccaga ggaaagucuu 6180

cagagaacua gugaggaauu gugaccugcc cguuuggcuu ucguggcaag uggccaaggc 6240

ugguuugaag acgaaugauc guaaguggug uuuugaaggc ccugggaac augagaucuu 6300

6360

6420

6480

uuuccuggcu aaaaaaggug gagaggcaau ggacaccauc aguguguuuc uccacucuga 6540

ggaaggcucu agggcuuacc gcaaugcacu aucaaugaug ccugaggcaa ugacaauagu 6600

caugcuguuu auacuggcug gacuccugac aucgggaaug

caaaggcauc aguagaaugu cuauggcgau gggcacaaug gccggcugug gauaucucau 6720

6780

gauggugguu gugauccccg agccagggca acaaaggucc auccaagaca accaaguggc 6840

auaccucauu auuggcaucc ugacgcuggu uucagcggug gcagccaacg agcuaggcau 6900

gcuggagaaa accaaagagg accucuuugg gaagaagaac uuaauuccau cuagugcuuc 6960

acccuggagu uggccggauc uugaccugaa gccaggagcu gccuggacag uguacguugg 7020

cauuguuaca augcucucuc caauguugca ccacuggauc aaagucgaau auggcaaccu 7080

gucucugucu ggaauagccc agucagccuc aguccuuucu uucauggaca aggggauacc 7140

auucaugaag augaauaucu cggucauaau gcugcugguc aguggcugga auucaauaac 7200

agugaugccu cugcucugug gcauagggug cgccaugcuc cacuggucuc ucauuuuacc 7260

uggaaucaaa gcgcagcagu caaagcuugc acagagaagg guguuccaug gcguugccaa 7320

7380

ccuuuaugag aagaaacugg cucuauaucu ccuucuugcu cucagccuag cuucuguugc 7440

caugugcaga acgcccuuuu cauuggcuga aggcauuguc cuagcaucag cugccuuagg 7500

7560

aggagucaug cgggggaauc acuaugcuuu ugugggaguc auguacaauc uauggaagau 7620

gaaaacugga cgccggggga gcgcgaaugg aaaaacuuug ggugaagucu ggaagaggga 7680

acugaaucug uuggacaagc aacaguuuga guuguauaaa aggaccgaca uuguggaggu 7740

ggaucgugau acggcacgca ggcauuuggc cgaagggaag guggacaccg ggguggcggu 7800

cuccaggggg accgcaaagu uaaggugguu ccaugagcgu ggcuauguca agcuggaagg 7860

uagggugauu gaccuggggu guggccgcgg aggcuggugu uacuacgcug cugcgcaaaa 7920

ggaagugagu ggggucaaag gauuuacucu uggaagagac ggccaugaga aaccuaugaa 7980

ugugcaaagu cugggaugga acaucaucac cuucaaggac aaaacugaca uccaccgccu 8040

agaaccagug aaaugugaca cccuuuugug ugacauugga gagucaucau cgucaucggu 8100

cacagagggg gaaaggaccg ugagaguucu ugauacugua gaaaaauggc uggcuugugg 8160

8220

8280

cacucaugaa auguacuacg ugucuggagc ccgcagcaau gucacauuua cugugaacca 8340

aacaucccgc cuccugauga ggagaaugag gcguccaacu ggaaaaguga cccuggaggc 8400

ugacgucauc cucccaauug ggacacgcag uguugagaca gacaagggac cccuggacaa 8460

agaggccaua gaagaaaggg uugagaggau aaaaucugag uacaugaccu cuugguuuua 8520

ugacaaugac aaccccuaca ggaccuggca cuacuguggc uccuauguca caaaaaccuc 8580

aggaagugcg gcgagcaugg uaaauggugu uauuaaaauu cugacauauc caugggacag 8640

gauagaggag gucacaagaa uggcaaugac ugacacaacc ccuuuuggac agcaaagagu 8700

guuuaaagaa aaaguugaca ccagagcaaa ggauccacca gcgggaacua ggaagaucau 8760

8820

8880

agaacaagaa caguggaaga cugccaauga ggcuguccaa gacccaaagu ucugggaacu 8940

gguggaugaa gaaaggaagc ugcaccaaca aggcaggugu cggacuugug uguacaacau 9000

gauggggaaa agagagaaga agcugucaga guuugggaaa gcaaagggaa gccgugccau 9060

augguauaug uggcugggag cgcgguaucu ugaguuugag gcccugggau uccugaauga 9120

ggaccauugg gcuuccaggg aaaacucagg aggaggagug gaaggcauug gcuuacaaua 9180

ccuagauau gugaucagag accuggcugc aauggauggu gguggauucu acgcggauga 9240

caccgcugga ugggacacgc gcaucacaga ggcagaccuu gaugaugaac aggagaucuu 9300

gaacuacaug agcccacauc acaaaaaacu ggcacaagca gugauggaaa ugacauacaa 9360

gaacaaagug gugaaagugu ugagaccagc cccagggggg aaagccuaca uggaugucau 9420

aagucgacga gaccagagag gauccgggca gguagugacu uaugcucuga acaccaucac 9480

caacuugaaa guccaauuga ucagaauggc agaagcagag auggugauac aucaccaaca 9540

uguucaagau ugugaugaau caguucugac caggcuggag gcauggcuca cugagcacgg 9600

auguaacaga cugaagagga uggcggugag uggagacgac uguguggucc ggcccaucga 9660

ugacagguuc ggccuggccc ugucccaucu caacgccaug uccaagguua gaaaggacau 9720

aucugaaugg cagccauccaa aaggguggaa ugauugggag aaugugcccu ucuguuccca 9780

ccacuuccau gaacuacagc ugaaggaugg caggaggauu guggugccuu gccgagaaca 9840

ggacgagcuc auugggagag gaaggguguc uccaggaaac ggcuggauga ucaaggaaac 9900

agcuugccuc agcaaagccu augccaacau guggucacug auguauuuuc acaaaaggga 9960

caugaggcua cugucauugg cuguuuccuc agcuguuccc accucauggg uuccacaagg 10020

10080

gguguggaac agaguaugga uaaccaacaa cccacacaug caggacaaga caauggugaa 10140

10200

uggaaugacc aauagggcca ccugggccuc ccacauccau uuagucaucc aucguauccg 10260

10320

ggaugcugac cugcaacugg gugagcuuau cugaaacacc aucuaacagg aauaaccggg 10380

auacaaacca cgggguggaga accggacucc ccacaaccug aaaccgggau auaaaccacg 10440

gcuggagaac cggacuccgc acuuaaaaug aaacagaaac cgggauaaaa acuacggaug 10500

10560

uuugccacug cuaagcugug aggcagugca ggcugggaca gccgaccucc agguugcgaa 10620

aaaccugguu ucugggaccu cccaccccag aguaaaaaga acggagccuc cgcuaccacc 10680

cucccacgug gugguagaaa gacggggucu aggguuaga ggagacccuc cagggaacaa 10740

auagugggac cauauugacg ccagggaaag accggagugg uucucugcuu uuccuccaga 10800

ggucugugag cacaguuugc ucaagaauaa gcagaccuuu ggaugacaaa cacaaaacca 10860

cu 10862

<210> 6

<211> 10833

<212> RNA

<213> Yellow fever virus

<220>

<223> RNA sequence of YFV strain Asibi

<400> 6

gugugcuaau ugaggugcau uggucugcaa aucgaguugc uaggcaauaa acacauuugg 60

auuaauuuua aucguucguu gagcgauuag cagagaacug accagaacau gucuggucgu 120

aaagcucagg gaaaaacccu gggcgucaau augguacgac gaggaguucg cuccuuguca 180

aacaaaauaa aacaaaaaac aaaacaaauu ggaaacagac cuggaccuuc aagagguguu 240

caaggauuua ucuuuuucuu uuuguucaac auuuugacug gaaaaaagau cacggcccac 300

cuaaagaggu uguggaaaau gcuggaccca agacaaggcu uggcuguucu aaggaaaguu 360

aagagugggg uggccaguuu gaugagagga uuguccucaa ggaaacgccg uucccaugau 420

guucugacug ugcaauuccu aauuuuggga augcuguuga ugacggggugg agugaccuug 480

gugcggaaaa acagaugguu gcuccuaaau gugacaucug aggaccucgg gaaaacauuc 540

ucugugggca caggcaacug cacaacaaac auuuuggaag ccaaguacug gugcccagac 600

ucauggaau acaacugucc caaucucagu ccaagagagg agccagauga cauugauugc 660

uggugcuaug ggguggaaaa cguuagaguc gcauauggua agugugacuc agcaggcagg 720

ucuaggaggu caagaagggc cauugacuug ccuacgcaug aaaaccaugg uuugaagacc 780

cggcaagaaa aauggaugac uggaagaaug ggugaaaggc aacuccaaaa gauugagaga 840

uggcucguga ggaaccccuu uuuugcagug acagcucuga ccauugccua ccuuguggga 900

agcaacauga cgcaacgagu cgugauugcc cuacuggucu uggcuguugg uccggccuac 960

ucagcucacu gcauuggaau uacugacagg gauuucauug agggggugca uggaggaacu 1020

uggguuucag cuacccugga gcaagacaag ugugucacug uuauggcccc ugacaagccu 1080

ucauuggaca ucucacuaga gacaguagcc auugauggac cugcugaggc gaggaaagug 1140

uguuacaaug caguucucac ucaugugaag auuaaugaca agugccccag cacuggagag 1200

gcccaccuag cugaagagaa cgaaggggac aaugcgugca agcgcacuua uucugauaga 1260

ggcuggggca auggcugugg ccuauuuggg aaagggagca uuguggcaug cgccaaauuc 1320

acuugugcca aauccaugag uuuguuugag guugaucaga ccaaaauuca guaugucauc 1380

agagcacaau ugcauguagg ggccaagcag gaaaauugga auaccgacau uaagacucuc 1440

aaguuugaug cccugucagg cucccaggaa gccgaguuca cuggguaugg aaaagcuaca 1500

cuggaugcc aggugcaaac ugcgguggac uuugguaaca guuacaucgc ugagauggaa 1560

1620

ggaaguggcg ggguguggag agagaugcau caucuugucg aauuugaacc uccgcaugcc 1680

gccacuauca gaguacuggc ccugggaaac caggaaggcu ccuugaaaac agcucuuacc 1740

ggcgcaauga ggguuacaaa ggacacaaau gacaacaacc uuuacaaacu acauggugga 1800

cauguuuccu gcagagugaa auugucagcu uugacacuca aggggacauc cuacaaaaug 1860

ugcacugaca aaaugucuuu ugucaagaac ccaacugaca cuggccaugg cacuguugug 1920

augcagguga aagugccaaa aggagccccc ugcaagauuc cagugauagu agcugaugau 1980

cuuacagcgg caaucaauaa aggcauuuug guuacaguua accccaucgc cucaaccaau 2040

gaugaugaag ugcugauuga ggugaaccca ccuuuuggag acagcuacau uaucguuggg 2100

acaggagauu cacgucucac uuaccagugg cacaaagagg gaagcucaau aggaaaguug 2160

uucacucaga ccaugaaagg cgcggaacgc cuggccguca ugggagacgc cgccugggau 2220

uucagcuccg cuggaggguu cuucacuucg guugggaaag gaauucauac gguguuuggc 2280

ucugccuuuc aggggcuauu uggcggcuug aacuggauaa caaaggucau caugggggcg 2340

2400

guaggaguga ucauguguu uuugucucua ggaguugggg cggaucaagg augcgccauc 2460

2520

2580 gacuggcuga acaaguacuc auacuaucca gaagauccug

2640

2700

ucuguugucg ugcaggaucc aaagaauguu uaccagagag gaacucaucc auuuuccaga 2760

2820

gggaggaaga auggaagcuu caucauagau ggaaagucca ggaaagaaug cccguuuuca 2880

aaccgggucu ggaauucuuu ccagauagag gaguuuggga cgggaguguu caccacacgc 2940

guguacaugg acgcagucuu ugaauacacc auagacugcg auggaucuau cuugggugca 3000

3060

guaaauggga cauggaugau ccacaccuug gaggcauuag auuacaagga gugugagugg 3120

ccacugacac auacgauugg aacaucaguu gaagagagug aaauguucau gccgagauca 3180

3240

ccuuggaugc agguaccacu agaagugaag agagaagcuu gccagggac uagcgugauc 3300

3360

auuauuccug aaugguguug ccgcuccugc acaaugccgc

3480

cgcuccuggg uuacagcugg agaaauacau gcugucccuu uugguuuggu gagcaugaug 3540

auagcaaugg aagugguccu aaggaaaaga cagggaccaa agcaaauguu gguuggagga 3600

guggugcucu ugggagcaau gcuggucggg caaguaacuc uccuugauuu gcugaaacuc 3660

3720

gcguugauug cugccuuuuc aaucagacca gggcugcuca ucggcuuugg gcucaggacc 3780

cuauggagcc cucgggaacg ccuuguacug acccuaggag cagccauggu ggagauugcc 3840

uuggguggca ugagggcgg ccuguggaag uaucuaaaug caguuucucu cugcauccug 3900

acaauaaaug cuguagcuuc uaggaaagca ucaaauacca ucuugccccu cauggcucug 3960

uugacaccug ucacuauggc ugaggugaga cuugccacaa ugcucuuuug uaccgugguu 4020

4080

guggcccuca cacucacauc uuaccugggc uugacacaac cuuuuuuggg ccugugugca 4140

uuucuggcaa ccgcauauu ugggcgaagg aguaucccag ugaaugaggc acucgcagca 4200

4260

ccgauugcag uuggaggaau ccugaugaug cugguuagcg uggcugggag gguggauggg 4320

cuagagcuca agaagcuugg ugaaguuuca ugggaagagg aggcggagau cagcggaagu 4380

uccgcccgcu augauguggc acucagugaa caaggggagu ucaagcugcu uucugaagag 4440

aaagugccau gggaccaggu ugugaugacc ucgcuggccu ugguuggggc ugccauucau 4500

ccauuugcuc uucugcuggu ccuugcuggg uggcuguuuc augucagggg agcuaggaga 4560

4620

gaggauggga uuuauggcau auuccaguca accuucuugg gggccuccca gcgaggagug 4680

ggaguggcac agggaggggu guuccacaca auguggcaug ucacaagagg agcuuuccuu 4740

gucaggaaug gcaagaaguu gauuccaucu ugggcuucag uaaaggaaga ccuugucgcc 4800

uaugguggcu cauggaaguu ggaaggcaga ugggauggag aggaagaggu ccaauugauc 4860

4920

aggaaugggg gagaaaucgg ggcugucgcu cuugacuauc cgaguggcac uucaggaucu 4980

5040

gacaacuccu ucguguccgc cauaucccag acugagguga aggaagaagg aaaggaggag 5100

cuccaagaga ucccgacaau gcuaaagaaa ggaaugacaa cuauccuuga uuuucauccu 5160

ggagcuggga agacaagacg uuuucuccca cagaucuugg ccgagugcgc acggagacgc 5220

uugcgcacuc uuguguuggc ccccaccagg guuguucuuu cugaaaugaa ggaggcuuuu 5280

cacggccugg acgugaaauu ccacacacag gcuuuuuccg cucacggcag cgggagaaa 5340

gucauugaug ccaugugcca ugccacccua acuuacagga uguuggaacc aacuaggguu 5400

guuaacuggg aagugaucau cauggaugaa gcccauuuuu uggauccagc uagcauagcc 5460

gccagagguu gggcagcgca cagagcuagg gcaaaugaaa gugcaacaau cuugaugaca 5520

gccacaccgc cugggacuag ugaugaauuu ccacauucaa auggugaaau agaagauguu 5580

caaacggaca uacccaguga gcccuggaac acagggcaug acuggauccu ggcugacaaa 5640

aggcccacgg caugguuccu uccauccauc agagcugcaa augucauggc ugccucuuug 5700

cguaaggcug gaaagagugu ggugguccug aacaggaaaa ccuuugagag agaauacccc 5760

acgauaaagc agaagaaacc ugacuuuaua uuggcacug acauagcuga aaugggagcc 5820

aaccuuugcg uggagcgagu gcuggauugc aggacggcuu uuaagccugu gcuuguggau 5880

gaagggagga agguggcaau aaaagggcca cuucgcaucu ccgcauccuc ugcugcucaa 5940

aggagggggc gcauugggag aaaucccaac agagauggag acucauacua cuauucugag 6000

ccuacaagug aagauaaugc ccaccguc ugcugguugg aggccucaau gcucuuggac 6060

6120

ccaguuuccc cuggugaaau gagacugagg gaugaccaga ggaaagucuu cagagaacua 6180

gugaggaauu gugaccugcc cguuuggcuu ucguggcaag uggccaaggc ugguuugaag 6240

acgaaugauc guaaguggug uuuugaaggc

ggugaaacag ugaagugcag ggcuccugga ggagcaaaga agccucugcg cccaaggugg 6360

ugugaugaaa gggugucauc ugaccagagu gcgcugucug aauuuauuaa guuugcugaa 6420

6480

6540

agggcuuacc gcaaugcacu aucaaugaug ccugaggcaa ugacaauagu caugcuguuu 6600

auacuggcug gacuacugac aucgggaaug gucaucuuuu ucaugucucc caaaggcauc 6660

aguagaaugu cuauggcgau gggcacaaug gccggcugug gauaucucau guuccuugga 6720

ggcgucaaac ccacucacau cuccuauauc augcucauau ucuuuguccu gauggugguu 6780

gugauccccg agccagggca acaaaggucc auccaagaca accaaguggc auaccucauu 6840

auuggcaucc ugacgcuggu uucaguggug gcagccaacg agcuaggcau gcuggagaaa 6900

accuuuugg accuuugg gaagaagaac uuaauuccau cuagugcuuc acccuggagu 6960

uggccggauc uugaccugaa gccaggagcu gccuggacag uguacguugg cauuguuaca 7020

7080

7140

augaauaucu cggucauaau acugcugguc aguggcugga auucaauaac agugaugccu 7200

7260

7320

7380

7440

acgcccuuuu cauuggcuga aggcauuguc cuagcaucag cugccuuagg gccgcucaua 7500

7560

cgggggaauu acuaugcuuu ugugggaguc auguacaauc uauggaagau gaaaacugga 7620

cgccggggga gugcgaaugg aaaaacuuug ggugaagucu ggaagaggga acugaaucug 7680

uuggacaagc aacaguuuga guuguauaaa aggaccgaca uuguggaggu ggaucgugau 7740

acggcacgca ggcauuuggc cgaagggaag guggacaccg ggguggcggu cuccaggggg 7800

accgcaaagu uaaggugguu ccaugagcgu ggcuauguca agcuggaagg uagggugauu 7860

gaccuggggu guggccgcgg aggcuggugu uacuacgcug cugcgcaaaa ggaagugagu 7920

ggggucaaag gauucacucu uggaagagac ggccaugaga aacccaugaa ugugcaaagu 7980

cugggaugga acaucauuac cuucaaggac aaaacugaua uccaccgccu agaaccagug 8040

aaaugugaca cccuuuugug ugacauugga gagucaucau cgucaucggu cacagagggg 8100

gaaaggaccg ugagaguucu ugauacugua gaaaaauggc uggcuugugg gguugacaac 8160

uucuguguga agguguuagc uccauacaug ccagauguuc ucgagaaacu ggaauugcuc 8220

8280

auguacuacg ugucuggagc ccgcagcaau gucacauuua cugugaacca aacaucccgc 8340

cuccugauga ggagaaugag gcguccaacu ggaaaaguga cccuggaggc ugacgucauc 8400

cucccaauug ggacacgcag uguugagaca gacaagggac cccuggacaa agaggccaua 8460

8520

aaccccuaca ggaccuggca cuacuguggc uccuauguca caaaaaccuc aggaagugcg 8580

gcgagcaugg uaaauggugu uauuaaaauu cugacauacc caugggacag gauagaggag 8640

gucacaagaa uggcaaugac ugacacaacc ccuuuuggac agcaaagagu guuuaaagaa 8700

8760

8820

8880

caguggaaga cugccaauga ggcuguccaa gacccaaagu ucugggaacu gguggaugaa 8940

gaaaggaagc ugcaccaaca aggcaggugu cggacuugug uguacaacau gauggggaaa 9000

agagagaaga agcugucaga guuugggaaa gcaaagggaa gccgugccau augguauaug 9060

uggcugggag cgcgguaucu ugaguuugag gcccugggau uccugaauga ggaccauugg 9120

gcuuccaggg aaaacucagg aggaggagug gaaggcauug

gugaucagag accuggcugc aauggauggu gguggauucu acgcggauga caccgcugga 9240

uggacacgc gcaucacaga ggcagaccuu gaugaugaac aggagaucuu gaacuacaug 9300

agcccacauc acaaaaaacu ggcacaagca gugauggaaa ugacauacaa gaacaaagug 9360

gugaaagugu ugagaccagc cccaggaggg aaagccuaca uggaugucau aagucgacga 9420

gaccagagag gauccgggca gguagugacu uaugcucuga acaccaucac caacuugaaa 9480

guccaauuga ucagaauggc agaagcagag auggugauac aucaccaaca uguucaagau 9540

ugugaugaau caguucugac caggcuggag gcauggcuca cugagcacgg auguaacaga 9600

cugaagagga uggcggugag uggagacgac uguguggucc ggcccaucga ugacagguuc 9660

ggccuggccc ugucccaaucu caacgccaug uccaagguua gaaaggacau aucugaaugg 9720

cagccauccaa aaggguggaa ugauugggag aaugugcccu ucuguuccca ccacuuccau 9780

gaacuacagc ugaaggaugg caggaggauu guggugccuu gccgagaaca ggacgagcuc 9840

auugggagag gaaggguguc uccaggaaac ggcuggauga ucaaggaaac agcuugccuc 9900

agcaaagccu augccaacau guggucacug auguauuuuc acaaaaggga caugaggcua 9960

10020

uggucgauuc augggaaagg ggaguggaug accacggaag acaugcuuga gguguggaac 10080

agaguaugga uaaccaacaa cccacacaug caggacaaga caauggugaa agaauggaga 10140

10200

aauagggcca ccugggccuc ccacauccau uuggucaucc aucguauccg aacgcugauu 10260

10320

cugcaaccgg gugagcuuau cugaaacacc aucuaauagg aauaaccgggg auacaaacca 10380

cgggggaga accggacucc ccacaacuug aaaccgggau auaaaccacg gcuggagaac 10440

10500

cuccacacau ugagacagaa gaaguuguca gcccagaacu ccacacgagu uuugccacug 10560

cuaagcugug aggcagugca ggcugggaca gccgaccucc agguugcgaa aaaccugguu 10620

10680

10740

cauauugacg ccagggaaag accggagugg uucucugcuu uuccuccagg ggucugugag 10800

cacaguuugc ucaagaauaa gcagaccuuu gga 10833

<210> 7

<211> 10862

<212> RNA

<213> Yellow fever virus

<220>

<223> RNA sequence of YFV strain TV3112

<400> 7

aguaaauccu gugugcuaau ugaggugcau uggucugcaa aucgaguugc uaggcaauaa 60

acacauuugg auuaauuuua aucguucguu gagcgauuag cagagaacug accagaacau 120

gucggucgu aaagcucagg gaaaaacccu gggcgucaau augguacgac gaggaguucg 180

cuccuuguca aacaaaauaa aacaaaaaac aaaacaaauu ggaaacagac cuggaccuuc 240

aagagguguu caaggauuua ucuuuuucuu uuuguucaac auuuugacug gaaaaaagau 300

cacagcccac cuaaagaggu uguggaaaau gcuggaccca agacaaggcu uggcuguucu 360

aaggaaaguc aagagagugg uggccaguuu gaugagagga uuguccucaa ggaaacgccg 420

uucccaugau guucugacug ugcaauuccu aauuuuggga augcuguuga ugacgggugg 480

agugaccuug gugcggaaaa acagaugguu gcuccuaaau gugacaucug aggaccucgg 540

gaaaacauuc ucugugggca caggcaacug cacaacaaac auuuuggaag ccaaguacug 600

gugcccagac ucaauggaau acaacugucc caaucucagu ccaagagagg agccagauga 660

cauugauugc uggugcuaug ggguggaaaa cguuagaguc gcauauggua agugugacuc 720

agcaggcagg ucuaggaggu caagaagggc cauugacuug ccuacgcaug aaaaccaugg 780

uuugaagacc cggcaagaaa aauggaugac uggaagaaug ggugaaaggc aacuccaaaa 840

gauugagaga ugguucguga ggaaccccuu uuuugcagug acggcucuga ccauugccua 900

ccuuguggga agcaacauga cgcaacgagu cgugauugcc cuacugggucu uggcuguugg 960

uccggccuac ucagcucacu gcauuggaau uacugacagg gauuucauug agggggugca 1020

uggaggaacu uggguuucag cuacccugga gcaagacaag ugugucacug uuauggcccc 1080

ugacaagccu ucauuggaca ucucacuaga gacaguagcc auugauagac cugcugaggu 1140

1200 gaggaaagug uguuacaaug caguucucac

cacuggagag gcccaccuag cugaagagaa cgaaggggac aaugcgugca agcgcacuua 1260

uucugauaga ggcuggggca auggcugugg ccuauuuggg aaagggagca uuguggcaug 1320

cgccaaauuc acuugugcca aauccaugag uuuguuugag guugaucaga ccaaaauuca 1380

guaugucauc agagcacaau ugcauguugg ggccaagcag gaaaauugga cuaccgacau 1440

uaagacucuc aaguuugaug cccugucagg cucccaggaa gucgaguuca uuggguaugg 1500

aaaagcuaca cuggaaugcc aggugcaaac ugcgguggac uuugguaaca guuacaucgc 1560

ugagauggaa acagaggcu ggauaggga cagacagugg gcccaggacu ugacccugcc 1620

auggcagagu ggaaguggcg ggguguggag agagaugcau caucuugucg aauuugaacc 1680

uccgcaugcc gccacuauca gaguacuggc ccugggaaac caggaaggcu ccuugaaaac 1740

agcucuuacu ggcgcaauga ggguuacaaa ggacacaaau gacaacaacc uuuacaaacu 1800

acauggugga cauguuucuu gcagagugaa auugucagcu uugacacuca aggggacauc 1860

cuacaaaaua ugcacugaca aaauguuuuu ugucaagaac ccaacugaca cuggccaugg 1920

cacuguugug augcagguga aagugucaaa aggagccccc ugcaggauuc cagugauagu 1980

agcugaugau cuuacagcgg caaucaauaa aggcauuuug guuacaguua accccaucgc 2040

cucaaccaau gaugaugaag ugcugauuga ggugaaccca ccuuuuggag acagcuacau 2100

uaucguuggg agaggagauu cacgucucac uuaccagugg cacaaagagg gaagcucaau 2160

aggaaaguug uucacucaga ccaugaaagg cguggaacgc cuggccguca ugggagacac 2220

2280

gguguuuggc ucugccuuuc aggggcuauu uggcggcuug aacuggauaa caaaggucau 2340

2400

2460

2520

agacucugau gacuggcuga acaaguacuc auacuaucca gaagauccug ugaagcuugc 2580

2640

ugagcaugag auguggagaa gcagggcaga ugagaucaau gccauuuuug aggaaaacga 2700

gguggacauu ucuguugucg ugcaggaucc aaagaauguu uaccagagag gaacucaucc 2760

auuuuccaga auucgggaug gucugcagua ugguuggaag acuuggggua agaaccuugu 2820

guucucccca gggaggaaga auggaagcuu caucauagau ggaaagucca ggaaagaaug 2880

cccguuuuca aaccgggucu ggaauucuuu cggauagag gaguuuggga cgggaguguu 2940

caccacacgc guguacaugg acgcagucuu ugaauacacc auagacugcg auggaucuau 3000

cuugggugca gcggugaacg gaaaaaagag ugcccauggc ucuccaacau uuuggauggg 3060

3120

gugugagugg cccugacac auacgauugg aacaucaguu gaagagagug aaauguucau 3180

3240

gacgaacgga ccuuggaugc agguaccacu agaagugaag agagaagcuu gcccagggac 3300

uagcgugauc auugauggca acugugaugg acggggaaaa

uagcgggaaa guuauuccug aaugguguug ccgcuccugc acaaugccgc cugugagcuu 3420

ccaugguagu gauggguguu gguaucccau ggaaauuagg ccaaggaaaa cgcaugaaag 3480

ccaucuggug cgcuccuggg uuacagcugg agaaauacau gcugucccuu uugguuuggu 3540

gagcaugaug auagcaaugg aagugguccu aaggaaaaga cagggaccaa agcaaauguu 3600

3660

3720 gcugaaacuc acaguggcug ugggauugca

cauguauaug gcguugauug cugccuuuuc aaucagacca gggcugcuca ucggcuuugg 3780

gcucaggacc cuauggagcc cucgggaacg ccuugugcug acccuagggag cagccauggu 3840

ggagauugcc uuggguggcg ugagggcgg ccuuggaag uaucuaaaug caguuucucu 3900

3960

cauggcucug uugacaccug ucacuauggc ugaggugaga

ugccgugguu aucauagggg uccuucacca gaauuucaag gacaccucca ugcagaagac 4080

uauaccucug guggcccuca cacucacauc uuaccugggc uugacacaac cuuuuuuggg 4140

ccugugugca uuucuggcaa ccgcauauu ugggcgaagg aguaucccag ugaaugaggc 4200

acucgcagca gcuggucuag ugggagugcu ggcaggacug gcuuuucagg agauggagaa 4260

cuuccuuggu ccgauugcag uuggaggacu ccugaugaug cugguuagcg uggcugggag 4320

gguggauggg cuagagcuca agaagcuugg ugaaguuuca ugggaagagg aggcggagau 4380

cagcgggagu uccgcccgcu augauguggc acucagugaa caaggggagu ucaagcugcu 4440

uucugaagag aaagugccau gggaccaggu ugugaugacc ucgcuggccu ugguuggggc 4500

ugcccuccau ccauuugcuc uucugcuggu ccuugcuggg uggcuguuuc augucagggg 4560

4620

ugaacaugg gaggauggga uuuauggcau auuccaguca accuucuugg gggccuccca 4680

gcgaggagug ggaguggcac agggaggggu guuccacaca auguggcaug ucacaagagg 4740

agcuuuccuu gucaggaaug gcaagaaguu gauuccaucu ugggcuucag uaaaggaaga 4800

ccuugucgcc uaugguggcu cauggaaguu ggaaggcaga ugggauggag aggaagggu 4860

ccaguugauc gcggcuguuc caggaaagaa cguggucaac guccagacaa aaccgagcuu 4920

guucaaagug aggaaugggg gagaaaucgg ggcugucgcu cuugacuauc cgaguggcac 4980

uucaggaucu ccuauuguua acaggaacgg agaggugauu gggcuguacg gcaauggcau 5040

ccuugucggu gacaacuccu ucguguccgc cauaucccag acugagguga aggaagaagg 5100

aaaggaggag cuccaagaga ucccgacaau gcuaaagaaa ggaaugacaa cuguccuuga 5160

uuuucauccu ggagcuggga agacaagacg uuuccuccca cagaucuugg ccgagugcgc 5220

acggagacgc uugcgcacuc uuguguuggc ccccaccagg guuguucuuu cugaaaugaa 5280

ggaggcuuuu cacggccugg acgugaaauu ccacacacag gcuuuuuccg cucacggcag 5340

cgggagagaa gucauugaug cuaugugcca ugccacccua acuuacagga uguuggaacc 5400

aacuaggguu guuaacuggg aagugaucau uuggaugaa gcccauuuuu uggauccagc 5460

uagcauagcc gcuagagguu gggcagcgca cagagcuagg gcaaaugaaa gugcaacaau 5520

cuugaugaca gccacaccgc cugggacuag ugaugaauuu cccaauucaa aggugaaau 5580

agaagauguu caaacggaca uacccaguga gcccuggaac acagggcaug acuggauccu 5640

ggcugacaaa aggcccacgg caugguuccu uccauccauc agagcugcaa augucauggc 5700

ugccucuuug cguaaggcug gaaagagugu ggugguccug aacaggaaaa ccuuugagag 5760

agaauacccc acgauaaagc agaagaaacc ugacuuuaua uuggcacug acauagcuga 5820

aaugggagcc aaccuuugcg uggagcgagu gcuggauugc aggacggcuu uuaagccugu 5880

gcuuguggau gaagggagga agguggcaau aaaagggcca cuucguaucu cgcauccuc 5940

ugcugcucaa aggagggggc gcauugggag aaaucccaac agagauggag acucauacua 6000

6060

gcucuuggac aacauggagg ugaggggugg aauggucgcc ccacucuaug gcguugaagg 6120

aacuaaaaca ccaguuuccc cuggugaaau gagacugagg gaugaccaga ggaaagucuu 6180

cagagaacua gugaggaauu gugaccugcc cguuuggcuu ucguggcaag uggccaaggc 6240

ugguuugaag acgaaugauc guaaguggug uuuugaaggc ccugggaac augagaucuu 6300

6360

6420

6480

uuuccuggcu aaaaagggug gagaggcaau ggauaccauc aguguguuuc uccacucuga 6540

ggaaggcucu agggcuuacc gcaaugcacu aucaaugaug ccugaggcaa ugacaauagu 6600

caugcuguuu auacuggcug gacuacugac aucgggaaug

caaaggcauc aguagaaugu cuauggcgau gggcacaaug gccggcugug gauaucucau 6720

6780

gauggugguu gugauccccg agccagggca acaaaggucc auccaagaca accaaguggc 6840

auaccucauu auuggcaucc ugacgcuggu uucagcggug gcagccaacg agcuaggcau 6900

gcuggagaaa accaaagagg accucuuugg gaagaagaac uuaauuccau cuagugcuuc 6960

acccuggagu uggccggauc uugaccugaa gccaggagcu gccuggacag uguacguugg 7020

cauuguuaca augcucucuc caauguugca ccacuggauc aaagucgaau auggcaaccu 7080

gucucugucu ggaauagccc agucagccuc aguccuuucu uucauggaca aggggauacc 7140

auucaugaag augaauaucu cggucauaau gcugcugguc aguggcugga auucaauaac 7200

agugaugccu cugcucugug gcauagggug cgccaugcuc cacuggucuc ucauuuuacc 7260

uggaaucaaa gcgcagcagu caaagcuugc acagagaagg guguuccaug gcguugccaa 7320

7380

ccuuuaugag aagaaacugg cucuauaucu ccuucuugcu cucagccuag cuucuguugc 7440

caugugcaga acgcccuuuu cauuggcuga aggcauuguc cuagcaucag cugcccuagg 7500

7560

aggagucaug agggggaauc acuaugcuuu ugugggaguc auguacaauc uauggaagau 7620

gaaaacugga cgccggggga gcgcgaaugg aaaaacuuug ggugaagucu ggaagaggga 7680

acugaaucug uuggacaagc gacaguuuga guuguauaaa aggaccgaca uuguggaggu 7740

ggaucgugau acggcacgca ggcauuuggc cgaagggaag guggacaccg ggguggcggu 7800

cuccaggggg accgcaaagu uaaggugguu ccaugagcgu ggcuauguca agcuggaagg 7860

uagggugauu gaccuggggu guggccgcgg aggcuggugu uacuacgcug cugcgcaaaa 7920

ggaagugagu ggggucaaag gauuuacucu uggaagagac ggccaugaga aacccaugaa 7980

ugugcaaagu cugggaugga acaucaucac cuucaaggac aaaacugaua uccaccgccu 8040

agaaccagug aaaugugaca cccuuuugug ugacauugga gagucaucau cgucaucggu 8100

cacagagggg gaaaggaccg ugagaguucu ugauacugua gaaaaauggc uggcuugugg 8160

8220

8280

cacucaugaa auguacuacg ugucuggagc ccgcagcaau gucacauuua cugugaacca 8340

aacaucccgc cuccugauga ggagaaugag gcguccaacu ggaaaaguga cccuggaggc 8400

ugacgucauc cucccaauug ggacacgcag uguugagaca gacaagggac cccuggacaa 8460

agaggccaua gaagaaaggg uugagaggau aaaaucugag uacaugaccu cuugguuuua 8520

ugacaaugac aaccccuaca ggaccuggca cuacuguggc uccuauguca caaaaaccuc 8580

aggaagugcg gcgagcaugg uaaauggugu uauuaaaauu cugacauauc caugggacag 8640

gauagaggag gucacaagaa uggcaaugac ugacacaacc ccuuuuggac agcaaagagu 8700

guuuaaagaa aaaguugaca ccagagcaaa ggauccacca gcgggaacua ggaagaucau 8760

8820

8880

agaacaagaa caguggaaga cugccaauga ggcuguccaa gacccaaagu ucugggaacu 8940

gguggaugaa gaaaggaagc ugcaccaaca aggcaggugu cggacuugug uguacaacau 9000

gauggggaaa agagagaaga agcugucaga guuugggaaa gcaaagggaa gccgugccau 9060

augguauaug uggcugggag cgcgguaucu ugaguuugag gcccugggau uccugaauga 9120

ggaccauugg gcuuccaggg aaaacucagg aggaggagug gaaggcauug gcuuacaaua 9180

ccuagauau gugaucagag accuggcugc aauggauggu gguggauucu acgcggauga 9240

caccgcugga ugggacacgc gcaucacaga ggcagaccuu gaugaugaac aggagaucuu 9300

gaacuacaug agcccacauc acaaaaaacu ggcacaagca gugauggaaa ugacauacaa 9360

gaacaaagug gugaaagugu ugagaccagc cccaggaggg aaagccuaca uggaugucau 9420

aagucgacga gaccagagag gauccgggca gguagugacu uaugcucuga acaccaucac 9480

caacuugaaa guccaauuga ucagaauggc agaagcagag auggugauac aucaccaaca 9540

uguucaagau ugugaugaau caguucugac caggcuggag gcauggcuca cugagcacgg 9600

auguaacaga cugaagagga uggcggugag uggagacgac uguguggucc ggcccaucga 9660

ugacagguuc ggccuggccc ugucccaucu caacgccaug uccaagguua gaaaggacau 9720

aucugaaugg cagccauccaa aaggguggaa ugauugggag aaugugcccu ucuguuccca 9780

ccacuuccau gaacuacagc ugaaggaugg caggaggauu guggugccuu gccgagaaca 9840

ggacgagcuc auugggagag gaaggguguc uccaggaaac ggcuggauga ucaaggaaac 9900

agcuugccuc agcaaagccu augccaacau guggucacug auguauuuuc acaaaaggga 9960

caugaggcua cugucauugg cuguuuccuc agcuguuccc accucauggg uuccacaagg 10020

10080

gguguggaac agaguaugga uaaccaacaa cccacacaug caggacaaga caauggugaa 10140

10200

uggaugacc aauagggcca ccugggccuc ccacauccau uuggucaucc aucguauccg 10260

10320

ggaugcugac cugcaacugg gugagcuuau cugaaacacc aucuaacagg aauaaccggg 10380

auacaaacca cgggguggaga accggacucc ccacaaccug aaaccgggau auaaaccacg 10440

gcuggagaac cggacuccgc acuuaaaaug aaacagaaac cgggauaaaa acuacggaug 10500

10560

uuugccacug cuaagcugug aggcagugca ggcugggaca gccgaccucc agguugcgaa 10620

aaaccugguu ucugggaccu cccaccccag aguaaaaaga acggagccuc cgcuaccacc 10680

cucccacgug gugguagaaa gacggggucu aggguuaga ggagacccuc cagggaacaa 10740

auagugggac cauauugacg ccagggaaag accggagugg uucucugcuu uuccuccaga 10800

ggucugugag cacaguuugc ucaagaauaa gcagaccuuu ggaugacaaa cacaaaacca 10860

cu 10862

<210> 8

<211> 10862

<212> RNA

<213> Yellow fever virus

<220>

<223> RNA sequence of strain YFV TV3111

<400> 8

aguaaauccu gugugcuaau ugaggugcau uggucugcaa aucgaguugc uaggcaauaa 60

acacauuugg auuaauuuua aucguucguu gagcgauuag cagagaacug accagaacau 120

gucggucgu aaagcucagg gaaaaacccu gggcgucaau augguacgac gaggaguucg 180

cuccuuguca aacaaaauaa aacaaaaaac aaaacaaauu ggaaacagac cuggaccuuc 240

aagagguguu caaggauuua ucuuuuucuu uuuguucaac auuuugacug gaaaaaagau 300

cacagcccac cuaaagaggu uguggaaaau gcuggaccca agacaaggcu uggcuguucu 360

aaggaaaguc aagagagugg uggccaguuu gaugagagga uuguccucaa ggaaacgccg 420

uucccaugau guucugacug ugcaauuccu aauuuuggga augcuguuga ugacgggugg 480

agugaccuug gugcggaaaa acagaugguu gcuccuaaau gugacaucug aggaccucgg 540

gaaaacauuc ucugugggca caggcaacug cacaacaaac auuuuggaag ccaaguacug 600

gugcccagac ucaauggaau acaacugucc caaucucagu ccaagagagg agccagauga 660

cauugauugc uggugcuaug ggguggaaaa cguuagaguc gcauauggua agugugacuc 720

agcaggcagg ucuaggaggu caagaagggc cauugacuug ccuacgcaug aaaaccaugg 780

uuugaagacc cggcaagaaa aauggaugac uggaagaaug ggugaaaggc aacuccaaaa 840

gauugagaga ugguucguga ggaaccccuu uuuugcagug acggcucuga ccauugccua 900

ccuuguggga agcaacauga cgcaacgagu cgugauugcc cuacugggucu uggcuguugg 960

uccggccuac ucagcucacu gcauuggaau uacugacagg gauuucauug agggggugca 1020

uggaggaacu uggguuucag cuacccugga gcaagacaag ugugucacug uuauggcccc 1080

ugacaagccu ucauuggaca ucucacuaga gacaguagcc auugauagac cugcugaggu 1140

1200 gaggaaagug uguuacaaug caguucucac

cacuggagag gcccaccuag cugaagagaa cgaaggggac aaugcgugca agcgcacuua 1260

uucugauaga ggcuggggca auggcugugg ccuauuuggg aaagggagca uuguggcaug 1320

cgccaaauuc acuugugcca aauccaugag uuuguuugag guugaucaga ccaaaauuca 1380

guaugucauc agagcacaau ugcauguagg ggccaagcag gaaaauugga cuaccgacau 1440

uaagacucuc aaguuugaug cccugucagg cucccaggaa gucgaguuca uuggguaugg 1500

aaaagcuaca cuggaaugcc aggugcaaac ugcgguggac uuugguaaca guuacaucgc 1560

ugagauggaa acagaggcu ggauaggga cagacagugg gcccaggacu ugacccugcc 1620

auggcagagu ggaaguggcg ggguguggag agagaugcau caucuugucg aauuugaacc 1680

uccgcaugcc gccacuauca gaguacuggc ccugggaaac caggaaggcu ccuugaaaac 1740

agcucuuacu ggcgcaauga ggguuacaaa ggacacaaau gacaacaacc uuuacaaacu 1800

acauggugga cauguuucuu gcagagugaa auugucagcu uugacacuca aggggacauc 1860

cuacaaaaua ugcacugaca aaauguuuuu ugucaagaac ccaacugaca cuggccaugg 1920

cacuguugug augcagguga aagugucaaa aggagccccc ugcaggauuc cagugauagu 1980

agcugaugau cuuacagcgg caaucaauaa aggcauuuug guuacaguua accccaucgc 2040

cucaaccaau gaugaugaag ugcugauuga ggugaaccca ccuuuuggag acagcuacau 2100

uaucguuggg agaggagauu cacgucucac uuaccagugg cacaaagagg gaagcucaau 2160

aggaaaguug uucacucaga ccaugaaagg cguggaacgc cuggccguca ugggagacac 2220

2280

gguguuuggc ucugccuuuc aggggcuauu uggcggcuug aacuggauaa caaaggucau 2340

2400

2460

2520

agacucugau gacuggcuga acaaguacuc auacuaucca gaagauccug ugaagcuugc 2580

2640

ugagcaugag auguggagaa gcagggcaga ugagaucaau gccauuuuug aggaaaacga 2700

gguggacauu ucuguugucg ugcaggaucc aaagaauguu uaccagagag gaacucaucc 2760

auuuuccaga auucgggaug gucugcagua ugguuggaag acuuggggua agaaccuugu 2820

guucucccca gggaggaaga auggaagcuu caucauagau ggaaagucca ggaaagaaug 2880

cccguuuuca aaccgggucu ggaauucuuu cggauagag gaguuuggga cgggaguguu 2940

caccacacgc guguacaugg acgcagucuu ugaauacacc auagacugcg auggaucuau 3000

cuugggugca gcggugaacg gaaaaaagag ugcccauggc ucuccaacau uuuggauggg 3060

3120

gugugagugg cccugacac auacgauugg aacaucaguu gaagagagug aaauguucau 3180

3240

gacgaacgga ccuuggaugc agguaccacu agaagugaag agagaagcuu gcccagggac 3300

uagcgugauc auugauggca acugugaugg acggggaaaa

uagcgggaaa guuauuccug aaugguguug ccgcuccugc acaaugccgc cugugagcuu 3420

ccaugguagu gauggguguu gguaucccau ggaaauuagg ccaaggaaaa cgcaugaaag 3480

ccaucuggug cgcuccuggg uuacagcugg agaaauacau gcugucccuu uugguuuggu 3540

gagcaugaug auagcaaugg aagugguccu aaggaaaaga cagggaccaa agcaaauguu 3600

3660

3720 gcugaaacuc acaguggcug ugggauugca

cauguauaug gcguugauug cugccuuuuc aaucagacca gggcugcuca ucggcuuugg 3780

gcucaggacc cuauggagcc cucgggaacg ccuugugcug acccuagggag cagccauggu 3840

ggagauugcc uuggguggcg ugagggcgg ccuuggaag uaucuaaaug caguuucucu 3900

3960

cauggcucug uugacaccug ucacuauggc ugaggugaga

ugccgugguu aucauagggg uccuucacca gaauuucaag gacaccucca ugcagaagac 4080

uauaccucug guggcccuca cacucacauc uuaccugggc uugacacaac cuuuuuuggg 4140

ccugugugca uuucuggcaa ccgcauauu ugggcgaagg aguaucccag ugaaugaggc 4200

acucgcagca gcuggucuag ugggagugcu ggcaggacug gcuuuucagg agauggagaa 4260

cuuccuuggu ccgauugcag uuggaggacu ccugaugaug cugguuagcg uggcugggag 4320

gguggauggg cuagagcuca agaagcuugg ugaaguuuca ugggaagagg aggcggagau 4380

cagcgggagu uccgcccgcu augauguggc acucagugaa caaggggagu ucaagcugcu 4440

uucugaagag aaagugccau gggaccaggu ugugaugacc ucgcuggccu ugguuggggc 4500

ugcccuccau ccauuugcuc uucugcuggu ccuugcuggg uggcuguuuc augucagggg 4560

4620

ugaacaugg gaggauggga uuuauggcau auuccaguca accuucuugg gggccuccca 4680

gcgaggagug ggaguggcac agggaggggu guuccacaca auguggcaug ucacaagagg 4740

agcuuuccuu gucaggaaug gcaagaaguu gauuccaucu ugggcuucag uaaaggaaga 4800

ccuugucgcc uaugguggcu cauggaaguu ggaaggcaga ugggauggag aggaagggu 4860

ccaguugauc gcggcuguuc caggaaagaa cguggucaac guccagacaa aaccgagcuu 4920

guucaaagug aggaaugggg gagaaaucgg ggcugucgcu cuugacuauc cgaguggcac 4980

uucaggaucu ccuauuguua acaggaacgg agaggugauu gggcuguacg gcaauggcau 5040

ccuugucggu gacaacuccu ucguguccgc cauaucccag acugagguga aggaagaagg 5100

aaaggaggag cuccaagaga ucccgacaau gcuaaagaaa ggaaugacaa cuguccuuga 5160

uuuucauccu ggagcuggga agacaagacg uuuccuccca cagaucuugg ccgagugcgc 5220

acggagacgc uugcgcacuc uuguguuggc ccccaccagg guuguucuuu cugaaaugaa 5280

ggaggcuuuu cacggccugg acgugaaauu ccacacacag gcuuuuuccg cucacggcag 5340

cgggagagaa gucauugaug cuaugugcca ugccacccua acuuacagga uguuggaacc 5400

aacuaggguu guuaacuggg aagugaucau uuggaugaa gcccauuuuu uggauccagc 5460

uagcauagcc gcuagagguu gggcagcgca cagagcuagg gcaaaugaaa gugcaacaau 5520

cuugaugaca gccacaccgc cugggacuag ugaugaauuu ccacauucaa aggugaaau 5580

agaagauguu caaacggaca uacccaguga gcccuggaac acagggcaug acuggauccu 5640

ggcugacaaa aggcccacgg caugguuccu uccauccauc agagcugcaa augucauggc 5700

ugccucuuug cguaaggcug gaaagagugu ggugguccug aacaggaaaa ccuuugagag 5760

agaauacccc acgauaaagc agaagaaacc ugacuuuaua uuggcacug acauagcuga 5820

aaugggagcc aaccuuugcg uggagcgagu gcuggauugc aggacggcuu uuaagccugu 5880

gcuuguggau gaagggagga agguggcaau aaaagggcca cuucguaucu cgcauccuc 5940

ugcugcucaa aggagggggc gcauugggag aaaucccaac agagauggag acucauacua 6000

6060

gcucuuggac aacauggagg ugaggggugg aauggucgcc ccacucuaug gcguugaagg 6120

aacuaaaaca ccaguuuccc cuggugaaau gagacugagg gaugaccaga ggaaagucuu 6180

cagagaacua gugaggaauu gugaccugcc cguuuggcuu ucguggcaag uggccaaggc 6240

ugguuugaag acgaaugauc guaaguggug uuuugaaggc ccugggaac augagaucuu 6300

6360

6420

6480

uuuccuggcu aaaaagggug gagaggcaau ggauaccauc aguguguuuc uccacucuga 6540

ggaaggcucu agggcuuacc gcaaugcacu aucaaugaug ccugaggcaa ugacaauagu 6600

caugcuguuu auacuggcug gacuacugac aucgggaaug

caaaggcauc aguagaaugu cuauggcgau gggcacaaug gccggcugug gauaucucau 6720

6780

gauggugguu gugauccccg agccagggca acaaaggucc auccaagaca accaaguggc 6840

auaccucauu auuggcaucc ugacgcuggu uucagcggug gcagccaacg agcuaggcau 6900

gcuggagaaa accaaagagg accucuuugg gaagaagaac uuaauuccau cuagugcuuc 6960

acccuggagu uggccggauc uugaccugaa gccaggagcu gccuggacag uguacguugg 7020

cauuguuaca augcucucuc caauguugca ccacuggauc aaagucgaau auggcaaccu 7080

gucucugucu ggaauagccc agucagccuc aguccuuucu uucauggaca aggggauacc 7140

auucaugaag augaauaucu cggucauaau gcugcugguc aguggcugga auucaauaac 7200

agugaugccu cugcucugug gcauagggug cgccaugcuc cacuggucuc ucauuuuacc 7260

uggaaucaaa gcgcagcagu caaagcuugc acagagaagg guguuccaug gcguugccaa 7320

7380

ccuuuaugag aagaaacugg cucuauaucu ccuucuugcu cucagccuag cuucuguugc 7440

caugugcaga acgcccuuuu cauuggcuga aggcauuguc cuagcaucag cugcccuagg 7500

7560

aggagucaug agggggaauc acuaugcuuu ugugggaguc auguacaauc uauggaagau 7620

gaaaacugga cgccggggga gcgcgaaugg aaaaacuuug ggugaagucu ggaagaggga 7680

acugaaucug uuggacaagc gacaguuuga guuguauaaa aggaccgaca uuguggaggu 7740

ggaucgugau acggcacgca ggcauuuggc cgaagggaag guggacaccg ggguggcggu 7800

cuccaggggg accgcaaagu uaaggugguu ccaugagcgu ggcuauguca agcuggaagg 7860

uagggugauu gaccuggggu guggccgcgg aggcuggugu uacuacgcug cugcgcaaaa 7920

ggaagugagu ggggucaaag gauuuacucu uggaagagac ggccaugaga aacccaugaa 7980

ugugcaaagu cugggaugga acaucaucac cuucaaggac aaaacugaua uccaccgccu 8040

agaaccagug aaaugugaca cccuuuugug ugacauugga gagucaucau cgucaucggu 8100

cacagagggg gaaaggaccg ugagaguucu ugauacugua gaaaaauggc uggcuugugg 8160

8220

8280

cacucaugaa auguacuacg ugucuggagc ccgcagcaau gucacauuua cugugaacca 8340

aacaucccgc cuccugauga ggagaaugag gcguccaacu ggaaaaguga cccuggaggc 8400

ugacgucauc cucccaauug ggacacgcag uguugagaca gacaagggac cccuggacaa 8460

agaggccaua gaagaaaggg uugagaggau aaaaucugag uacaugaccu cuugguuuua 8520

ugacaaugac aaccccuaca ggaccuggca cuacuguggc uccuauguca caaaaaccuc 8580

aggaagugcg gcgagcaugg uaaauggugu uauuaaaauu cugacauauc caugggacag 8640

gauagaggag gucacaagaa uggcaaugac ugacacaacc ccuuuuggac agcaaagagu 8700

guuuaaagaa aaaguugaca ccagagcaaa ggauccacca gcgggaacua ggaagaucau 8760

8820

8880

agaacaagaa caguggaaga cugccaauga ggcuguccaa gacccaaagu ucugggaacu 8940

gguggaugaa gaaaggaagc ugcaccaaca aggcaggugu cggacuugug uguacaacau 9000

gauggggaaa agagagaaga agcugucaga guuugggaaa gcaaagggaa gccgugccau 9060

augguauaug uggcugggag cgcgguaucu ugaguuugag gcccugggau uccugaauga 9120

ggaccauugg gcuuccaggg aaaacucagg aggaggagug gaaggcauug gcuuacaaua 9180

ccuagauau gugaucagag accuggcugc aauggauggu gguggauucu acgcggauga 9240

caccgcugga ugggacacgc gcaucacaga ggcagaccuu gaugaugaac aggagaucuu 9300

gaacuacaug agcccacauc acaaaaaacu ggcacaagca gugauggaaa ugacauacaa 9360

gaacaaagug gugaaagugu ugagaccagc cccaggaggg aaagccuaca uggaugucau 9420

aagucgacga gaccagagag gauccgggca gguagugacu uaugcucuga acaccaucac 9480

caacuugaaa guccaauuga ucagaauggc agaagcagag auggugauac aucaccaaca 9540

uguucaagau ugugaugaau caguucugac caggcuggag gcauggcuca cugagcacgg 9600

auguaacaga cugaagagga uggcggugag uggagacgac uguguggucc ggcccaucga 9660

ugacagguuc ggccuggccc ugucccaucu caacgccaug uccaagguua gaaaggacau 9720

aucugaaugg cagccauccaa aaggguggaa ugauugggag aaugugcccu ucuguuccca 9780

ccacuuccau gaacuacagc ugaaggaugg caggaggauu guggugccuu gccgagaaca 9840

ggacgagcuc auugggagag gaaggguguc uccaggaaac ggcuggauga ucaaggaaac 9900

agcuugccuc agcaaagccu augccaacau guggucacug auguauuuuc acaaaaggga 9960

caugaggcua cugucauugg cuguuuccuc agcuguuccc accucauggg uuccacaagg 10020

10080

gguguggaac agaguaugga uaaccaacaa cccacacaug caggacaaga caauggugaa 10140

10200

uggaugacc aauagggcca ccugggccuc ccacauccau uuggucaucc aucguauccg 10260

10320

ggaugcugac cugcaacugg gugagcuuau cugaaacacc aucuaacagg aauaaccggg 10380

auacaaacca cgggguggaga accggacucc ccacaaccug aaaccgggau auaaaccacg 10440

gcuggagaac cggacuccgc acuuaaaaug aaacagaaac cgggauaaaa acuacggaug 10500

10560

uuugccacug cuaagcugug aggcagugca ggcugggaca gccgaccucc agguugcgaa 10620

aaaccugguu ucugggaccu cccaccccag aguaaaaaga acggagccuc cgcuaccacc 10680

cucccacgug gugguagaaa gacggggucu aggguuaga ggagacccuc cagggaacaa 10740

auagugggac cauauugacg ccagggaaag accggagugg uucucugcuu uuccuccaga 10800

ggucugugag cacaguuugc ucaagaauaa gcagaccuuu ggaugacaaa cacaaaacca 10860

cu 10862

<210> 9

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> DNA primer

<400> 9

gctagggcaat aaacacattt gga 23

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> DNA primer

<400> 10

ttcactggga tactccttcg c 21

<210> 11

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> DNA primer

<400> 11

atcaaatacc atcttgcccc tc 22

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> DNA primer

<400> 12

agtaaatcct ttgaccccac t 21

<210> 13

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> DNA primer

<400> 13

ggcttaccgc aatgcact 18

<210> 14

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> DNA primer

<400> 14

cagagaacca ctccggtc 18

<210> 15

<211> 493

<212> PROTEIN

<213> Yellow fever virus

<220>

<223> Envelope protein sequence of YFV strains TV3111 or TV3112

<400> 15

Ala His Cys Ile Gly Ile Thr Asp Arg Asp Phe Ile Glu Gly Val His

1.5 10 15

Gly Gly Thr Trp Val Ser Ala Thr Leu Glu Gln Asp Lys Cys Val Thr

20 25 30

Val Met Ala Pro Asp Lys Pro Ser Leu Asp Ile Ser Leu Glu Thr Val

35 40 45

Ala Ile Asp Arg Pro Ala Glu Val Arg Lys Val Cys Tyr Asn Ala Val

50 55 60

Leu Thr His Val Lys Ile Asn Asp Lys Cys Pro Ser Thr Gly Glu Ala

65 70 75 80

His Leu Ala Glu Glu Asn Glu Gly Asp Asn Ala Cys Lys Arg Thr Tyr

85 90 95

Ser Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser

100 105 110

Ile Val Ala Cys Ala Lys Phe Thr Cys Ala Lys Ser Met Ser Leu Phe

115 120 125

Glu Val Asp Gln Thr Lys Ile Gln Tyr Val Ile Arg Ala Gln Leu His

130 135 140

Val Gly Ala Lys Gln Glu Asn Trp Thr Thr Asp Ile Lys Thr Leu Lys

145 150 155 160

Phe Asp Ala Leu Ser Gly Ser Gln Glu Val Glu Phe Ile Gly Tyr Gly

165 170 175

Lys Ala Thr Leu Glu Cys Gln Val Gln Thr Ala Val Asp Phe Gly Asn

180 185 190

Ser Tyr Ile Ala Glu Met Glu Thr Glu Ser Trp Ile Val Asp Arg Gln

195 200 205

Trp Ala Gln Asp Leu Thr Leu Pro Trp Gln Ser Gly Ser Gly Gly Val

210 215 220

Trp Arg Glu Met His His Leu Val Glu Phe Glu Pro Pro His Ala Ala

225 230 235 240

Thr Ile Arg Val Leu Ala Leu Gly Asn Gln Glu Gly Ser Leu Lys Thr

245 250 255

Ala Leu Thr Gly Ala Met Arg Val Thr Lys Asp Thr Asn Asp Asn Asn

260 265 270

Leu Tyr Lys Leu His Gly Gly His Val Ser Cys Arg Val Lys Leu Ser

275 280 285

Ala Leu Thr Leu Lys Gly Thr Ser Tyr Lys Ile Cys Thr Asp Lys Met

290 295 300

Phe Phe Val Lys Asn Pro Thr Asp Thr Gly His Gly Thr Val Val Met

305 310 315 320

Gln Val Lys Val Ser Lys Gly Ala Pro Cys Arg Ile Pro Val Ile Val

325 330 335

Ala Asp Asp Leu Thr Ala Ala Ile Asn Lys Gly Ile Leu Val Thr Val

340 345 350

Asn Pro Ile Ala Ser Thr Asn Asp Asp Glu Val Leu Ile Glu Val Asn

355 360 365

Pro Pro Phe Gly Asp Ser Tyr Ile Ile Val Gly Arg Gly Asp Ser Arg

370 375 380

Leu Thr Tyr Gln Trp His Lys Glu Gly Ser Ser Ile Gly Lys Leu Phe

385 390 395 400

Thr Gln Thr Met Lys Gly Val Glu Arg Leu Ala Val Met Gly Asp Thr

405 410 415

Ala Trp Asp Phe Ser Ser Ala Gly Gly Phe Phe Thr Ser Val Gly Lys

420 425 430

Gly Ile His Thr Val Phe Gly Ser Ala Phe Gln Gly Leu Phe Gly Gly

435 440 445

Leu Asn Trp Ile Thr Lys Val Ile Met Gly Ala Val Leu Ile Trp Val

450 455 460

Gly Ile Asn Thr Arg Asn Met Thr Met Ser Met Ser Met Ile Leu Leu

465 470 475 480

Gly Val Ile Met Met Phe Leu Ser Leu Gly Val Gly Ala

485 490

<210> 16

<211> 224

<212> PROTEIN

<213> Yellow fever virus

<220>

<223> NS2a protein sequence of YFV strains TV3111 or TV3112

<400> 16

Gly Glu Ile His Ala Val Pro Phe Gly Leu Val Ser Met Met Ile Ala

1.5 10 15

Met Glu Val Val Leu Arg Lys Arg Gln Gly Pro Lys Gln Met Leu Val

20 25 30

Gly Gly Val Val Leu Leu Gly Ala Met Leu Val Gly Gln Val Thr Leu

35 40 45

Leu Asp Leu Leu Lys Leu Thr Val Ala Val Gly Leu His Phe His Glu

50 55 60

Val Asn Asn Gly Gly Asp Ala Met Tyr Met Ala Leu Ile Ala Ala Phe

65 70 75 80

Ser Ile Arg Pro Gly Leu Leu Ile Gly Phe Gly Leu Arg Thr Leu Trp

85 90 95

Ser Pro Arg Glu Arg Leu Val Leu Thr Leu Gly Ala Ala Met Val Glu

100 105 110

Ile Ala Leu Gly Gly Val Met Gly Gly Leu Trp Lys Tyr Leu Asn Ala

115 120 125

Val Ser Leu Cys Ile Leu Thr Ile Asn Ala Val Ala Ser Arg Lys Ala

130 135 140

Ser Asn Thr Ile Leu Pro Leu Met Ala Leu Leu Thr Pro Val Thr Met

145 150 155 160

Ala Glu Val Arg Leu Ala Ala Met Phe Phe Cys Ala Val Val Ile Ile

165 170 175

Gly Val Leu His Gln Asn Phe Lys Asp Thr Ser Met Gln Lys Thr Ile

180 185 190

Pro Leu Val Ala Leu Thr Leu Thr Ser Tyr Leu Gly Leu Thr Gln Pro

195 200 205

Phe Leu Gly Leu Cys Ala Phe Leu Ala Thr Arg Ile Phe Gly Arg Arg

210 215 220

<210> 17

<211> 378

<212> RNA

<213> Yellow fever virus

<220>

<223> RNA sequence encoding NS4a protein of strain YFV TV3112

<400> 17

ggagcugcug aagugcuagu ugugcugagu gaacucccug auuuccuggc uaaaaagggu 60

ggagaggcaa uggauaccau caguguguuu cuccacucug aggaaggcuc uagggcuuac 120

cgcaaugcac uaucaaugau gccugaggca augacaauag ucaugcuguu uauacuggcu 180

ggacuacuga caucgggaau ggucaucuuu uucaugucuc ccaaaggcau caguagaaug 240

ucuauggcga ugggcacaau ggccggcugu ggauauucuca uguuccuugg aggcgucaaa 300

360

gagccagggc aacaaagg 378

<210> 18

<211> 1479

<212> RNA

<213> Yellow fever virus

<220>

<223> RNA sequence encoding the envelope protein of strain YFV TV3112

<400> 18

gcucacugca uuggaauuac ugacagggau uucauugagg gggugcaugg aggaacuugg 60

guuucagcua cccuggagca agacaagugu gucacuguua uggccccuga caagccuuca 120

uuggacaucu cacuagagac aguagccauu gauagaccug cugaggugag gaaagugugugu 180

uacaaugcag uucucacuca ugugaagauu aaugacaagu gccccagcac uggagaggcc 240

caccuagcug aagagaacga aggggacau gcgugcaagc gcacuuauuc ugauagaggc 300

uggggcaaug gcuguggccu auuugggaaa gggagcauug uggcaugcgc caaauucacu 360

ugugccaaau ccaugaguuu guuugagguu gaucagacca aaauucagua ugucaucaga 420

gcacaauugc auguuggggc caagcaggaa aauuggacua ccgacauuaa gacucucaag 480

uuugaugccc ugucaggcuc cggaaguc gaguucauug gguauggaaa agcuacacug 540

gaaugccagg ugcaaacugc gguggacuuu gguaacaguu acaucgcuga gauggaaaca 600

gagagcugga uaguggacag acagugggcc caggacuuga cccugccaug gcagagugga 660

aguggcgggg uguggagaga gaugcaucau cuugucgaau uugaaccucc gcaugccgcc 720

acuaucagag uacuggcccu gggaaaccag gaaggcuccu ugaaaacagc ucuuacuggc 780

gcaaugaggg uuacaaagga cacaaaugac aacaaccuuu acaaacuaca ugguggacau 840

guuucuugca gagugaaauu gucagcuuug acacucaagg ggacauccua caaaauauugc 900

acugacaaaa uguuuuuugu caagaaccca acugacacug gccauggcac uguugugaug 960

1020

acagcggcaa ucaauaaagg cauuuugguu acaguuaacc ccaucgccuc aaccaaugau 1080

1140

ggagauucac gucucacuua ccaguggcac aaagagggaa gcucaauagg aaaguuguuc 1200

acucagacca ugaaaggcgu ggaacgccug gccgucaugg gagacaccgc cugggauuuc 1260

1320

gccuuucagg ggcuauuugg cggcuugaac uggauaacaa aggucaucau gggggcggua 1380

cuuauauggg uuggcaucaa cacaagaaac augacaaugu ccaugagcau gaucuuguua 1440

ggagugauca ugauguuuuu gucucuagga guuggggcg 1479

<---

Claims (25)

1. Live attenuated strain of yellow fever virus to protect patients from infection caused by yellow fever virus, containing a nucleic acid containing: i) a mutation in the codon of the amino acid at position 480 of the coat protein (E), which results in the substitution of the amino acid valine for leucine, or ii) a mutation in the codon of the amino acid at position 65 of the non-structural protein 2A (NS2a), which results in the substitution of the amino acid methionine for valine. 2. Live attenuated strain of yellow fever virus according to claim 1, containing a nucleic acid containing: i) a mutation in the codon of the amino acid at position 480 of the coat protein (E), which results in the substitution of the amino acid valine for leucine, and ii) a mutation in the codon of the amino acid at position 65 of the non-structural protein 2A (NS2a), which results in the substitution of the amino acid methionine for valine. 3. A live attenuated strain of yellow fever virus according to claim 1 or 2, wherein the nucleic acid further comprises a mutation at the amino acid codon at position 19 of the non-structural protein 4A (NS4a), which results in a codon change from AAA to AAG. 4. Live attenuated strain of yellow fever virus according to any one of paragraphs. 1-3, wherein the nucleic acid further contains a mutation in the amino acid codon at position 145 of the envelope protein (E), which results in a GUA to GUU codon change. 5. A live attenuated strain of yellow fever virus to protect patients from yellow fever virus infection that contains an envelope protein containing a mutation at position 480 that results in the substitution of the amino acid valine for leucine. 6. A live attenuated yellow fever virus strain for protecting patients from yellow fever virus infection, which contains an envelope protein containing a leucine residue at a position in the protein that corresponds to position 480 of the envelope protein having SEQ ID NO. 15. 7. A live attenuated strain of yellow fever virus according to claim 6, wherein said envelope protein contains a sequence that is at least 90%, 95%, 98%, or 100% identical to the sequence under SEQ ID NO. 15. 8. Live attenuated strain of yellow fever virus to protect patients from infection caused by yellow fever virus, containing a nucleic acid molecule encoding: (i) an envelope protein containing a mutation at position 480 that results in a change from the amino acid valine to leucine, and (ii) an NS2a protein containing a mutation at position 65 that results in the substitution of the amino acid methionine for valine. 9. The live attenuated strain of yellow fever virus according to claim 8, wherein the nucleic acid further comprises a mutation at the amino acid codon at position 19 of the non-structural protein 4A (NS4a), which results in a codon change from AAA to AAG. 10. The live attenuated strain of yellow fever virus according to claim 8 or 9, wherein the nucleic acid further comprises a mutation at the amino acid codon at position 145 of the envelope protein (E), which results in the GUA codon being changed to GUU. 11. Live attenuated strain of yellow fever virus to protect patients from infection caused by yellow fever virus, containing a nucleic acid molecule encoding: (i) an envelope protein that contains a leucine residue at a position in the protein that corresponds to position 480 of the envelope protein having SEQ ID NO. 15; and (ii) an NS2a protein that contains a valine residue at a position in the protein that corresponds to position 65 of the NS2a protein having SEQ ID NO. sixteen. 12. A live attenuated strain of yellow fever virus according to claim 11, wherein said envelope protein contains a sequence that is at least 90%, 95%, 98%, or 100% identical to the sequence under SEQ ID NO. 15, and said NS2a protein contains a sequence that is at least 90%, 95%, 98%, or 100% identical to the sequence under SEQ ID NO. sixteen. 13. A live attenuated strain of yellow fever virus according to claim 11 or 12, wherein the nucleic acid further comprises a G nucleotide at a position in the nucleic acid encoding non-structural protein 4A (NS4a) that corresponds to position 57 in SEQ ID NO. 17. 14. Live attenuated strain of yellow fever virus according to any one of paragraphs. 11-13, wherein the nucleic acid further comprises a nucleotide U at a position in the nucleic acid encoding the envelope protein (E) which corresponds to position 435 in SEQ ID NO. eighteen. 15. An immunogenic composition for inducing an immune response against yellow fever virus, containing a live attenuated strain of yellow fever virus according to any one of paragraphs. 1-14 and a pharmaceutically acceptable carrier. 16. The use of a live attenuated strain of yellow fever virus according to any one of paragraphs. 1-14 to obtain a vaccine against yellow fever virus. 17. The use of a live attenuated strain of yellow fever virus according to any one of paragraphs. 1-14 to obtain a vaccine for use in the prevention of infection caused by yellow fever virus.

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