Attenuated variant of the rift valley fever virus, composition comprising same, and uses thereof

The invention relates to an attenuated variant of the Rift Valley Fever Virus (RVFV) with mutations in the amino acid sequence coded by segments L, M and S of RVFV RNA; a pharmaceutical or veterinary composition comprising same; an attenuated RVFV variant for use in the prevention of Rift Valley Fever, and a vaccine against Rift Valley Fever comprising the attenuated RVFV variant. Attenuated RVFV variants with the mutations Gly924Ser and Ala303Thr in protein L, and the Pro82Leu substitution in protein NSs, are also included.

1. 11. An attenuated variant of the Rift Valley Fever virus (RVFV), wherein the RNA-dependent RNA polymerase (RdRp) protein encoded by the L segment of the RNA of said variant comprises the substitutions: the amino acid at position 924 is serine (L[Gly924Ser]); the amino acid at position 1303 is threonine (L[Ala1303Thr]); wherein the sequence SEQ ID NO: 47 of wild strain 56/74 of the RVF virus or the sequence SEQ ID NO: 54 of the wild type ZH548 strain of the RVF virus, are the reference sequences for the numbering of the amino acids of said protein; and the non-structural (NSs) protein encoded by the S segment of the RNA of said variant comprises the substitution: the amino acid at position 82 is leucine (NSs[Pro82Leu]); wherein the sequence SEQ ID NO: 49 of wild strain 56/74 of the RVF virus or the sequence SEQ ID NO: 56 of the wild type ZH548 strain of the RVF virus, are the reference sequences for the numbering of the amino acids of said protein.
2. 22. The RVFV variant according to claim 1 wherein, further, in the RdRp protein encoded by the L segment of the RNA of said variant: the amino acid at position 100 is threonine (L[Met100Thr]); the amino acid at position 375 is tyrosine (L[His375Tyr]); the amino acid at position 1050 is valine (L[Ile1050Val]); the amino acid at position 1629 is phenylalanine (L[Leu1629Phe]); and the amino acid at position 2071 is lysine (L[Glu2071Lys]); wherein the sequence SEQ ID NO: 47 of wild strain 56/74 of the RVF virus or the sequence SEQ ID NO: 54 of the wild type ZH548 strain of the RVF virus, are the reference sequences for the numbering of the amino acids of said protein; in the amino acid sequence encoded by the M segment of the RNA of said variant: the amino acid at position 26 is lysine (M[Arg26Lys]); the amino acid at position 108 is tyrosine (M[His108Tyr]); the amino acid at position 118 is lysine (M[Glu118Lys]); the amino acid at position 210 is lysine (M[Arg210Lys]); the amino acid at position 333 is asparagine (M[Asp333Asn]); the amino acid at position 427 is threonine (M[Ala427Thr]); the amino acid at position 432 is valine (M[Ala432Val]); the amino acid at position 487 is glycine (M[Glu487Gly]); the amino acid at position 540 is tyrosine (M[His540Tyr]); the amino acid at position 582 is threonine (M[Ala582Thr]); the amino acid at position 587 is isoleucine (M[Val587Ile]); the amino acid at position 950 is valine (M[Ala950Val]); the amino acid at position 1090 is isoleucine (M[Val1090Ile]); the amino acid at position 1116 is valine (M[Ala1116Val]); and the amino acid at position 1182 is lysine (M[Arg1182Lys]); wherein the sequence SEQ ID NO: 48 of wild strain 56/74 of the RVF virus or the sequence SEQ ID NO: 55 of the wild strain ZH548 of the RVF virus, are the reference sequences for amino acid numbering of said amino acid sequence encoded by the M segment of the RNA of said variant; in the NSs protein encoded by the S segment of the RNA of said variant: the amino acid at position 52 is isoleucine (NSs[Val52Ile]); wherein the sequence SEQ ID NO: 49 of wild strain 56/74 of the RVF virus or the sequence SEQ ID NO: 56 of the wild type ZH548 strain of the RVF virus, are the reference sequences for the numbering of the amino acids of said protein.

CROSS-REFERENCE TO RELATED APPLICATIONS
This application is filed under the provisions of 35 U.S.C. § 371 and claims the priority of International Patent Application No. PCT/ES2021/070403, filed on 3 Jun. 2021 entitled “ATTENUATED VARIANT OF THE RIFT VALLEY FEVER VIRUS, COMPOSITION COMPRISING SAME, AND USES THEREOF” in the name of Alejandro BRUN TORRES, et al., which claims priority to Spanish Patent Application No. P202030529 filed on 4 Jun. 2020, both of which are hereby incorporated by reference herein in their entirety.
TECHNICAL FIELD OF THE INVENTION
The present invention relates to live vaccines for attenuated Rift Valley Fever (RVF), a disease caused by the Rift Valley Fever virus (RVFV). In particular, the invention relates to an attenuated variant of RVFV obtained by mutagenic treatment with favipiravir.
BACKGROUND OF THE INVENTION
The Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that belongs to the Phenuiviridae family of the genus Phlebovirus. RVFV causes Rift Valley Fever (RVF) in ruminants, which after epizootic outbreaks, is transmitted to humans mainly through mosquito bites.
Rift Valley Fever is currently present in the African continent, in the southern Arabian Peninsula and in the Indian Ocean islands. In addition, RVF has the potential to extend to other geographical areas, in particular in relation to climate change and globalization. Currently, there is no treatment or vaccine against Rift Valley Fever on the market.
RVFV is an RNA virus. The structure of the RVFV virion consists of a lipid envelope with two membrane glycoproteins (Gn and Gc) arranged in an icosahedral lattice that protects an internal nucleocapsid composed of the viral nucleoprotein (N) and an RNA-dependent polymerase (RdRp) linked to the viral RNA.
The RVFV genome is made up of three segments of single-stranded RNA of different size (large (L), medium (M), small(S)) with negative polarity (L and M) or bipolarity(S). The L segment encodes the RNA-dependent RNA polymerase (RdRp). The M segment encodes for two 78 kDa and 13-14 kDa non-structural proteins (NSm) and for Gn and Gc membrane glycoproteins. The S segment encodes a 27 kDa non-structural protein (NSs), considered the primary virulence factor of the virus, as well as the N protein.
Live attenuated vaccines induce long-lasting and highly protective immunity after single-dose administration in both animals and humans. They are called live vaccines because they contain the microorganism causing the infection in a live or viable form, but with a very reduced (attenuated) capacity for infection, reproduction or, in general, virulence. This makes live attenuated vaccines an excellent basis for the development of successful immunization programs in the affected countries or to implement preventive control measures in countries with a higher risk of introduction or expansion of the disease.
Mutagenic treatments have frequently been used as viral attenuation methods. One example of this is the MP-12 live attenuated FVR vaccine (Ikegami et al., 2015), obtained by mutagenic treatment with 5-fluorouracil. Currently, the MP-12 vaccine is in phase II trials as a vaccine candidate for humans.
Nucleoside analogues with antiviral activity against RVFV have been described, such as ribavirin (1-β-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxyamide), favipiravir (6-fluoro-3-hydroxy-2-pyrazinecarboxyamide) and BCX4430 [(2S,3S,4R,5R)-2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(hydroxymethyl) pyrrolidine-3,4-diol] (galidesivir). Favipiravir is a pyrazine derivative (6-fluoro-3-hydroxy-2-pyrazinecarboxyamide) that acts as a pyrimidine analogue and has potent antiviral activity against different RNA viruses.
Favipiravir has mutagenic activity on several RNA viruses, including hepatitis C virus, foot-and-mouth disease virus, West Nile fever virus, norovirus, influenza virus and RVFV. It has been described in the prior art that the mechanism of action of favipiravir against RVFV is due to the accumulation of mutations in the viral genome leading to a progressive decrease of viable viral progeny (Borrego et al., 2019).
The development of live attenuated RVF vaccines is an active field of research and there is a need to develop new live attenuated RVF vaccines, which can serve to develop safe and effective RVF control strategies, for animal and/or human use.
DESCRIPTION OF THE INVENTION
For the purposes of the present invention, the amino acids are mentioned by their full name or are represented using the three-letter symbols of the IUPAC nomenclature, also used in the ST.25 standard for the presentation of lists of nucleotide and amino acid sequences in patent applications of the World Intellectual Property Organization (WIPO). Thus, for example, the amino acid alanine is represented by the symbol “Ala”, the amino acid aspartic acid is represented by “Asp”, etc.
For the purposes of the present invention, nucleotide sequences of RNA molecules are described by the corresponding nucleotide sequence of DNA molecules. It is known in the art how to determine RNA sequences from the corresponding DNA sequences, which is performed by replacing the thymine nucleotides with uracil nucleotides.
For the purposes of the present invention, “multiplicity of infection (MOI)” refers to the ratio of agents (e.g., viruses) to infection targets (e.g., cells). By way of example, when referring to a group of cells inoculated with virus particles, the multiplicity of infection or MOI is the ratio between the number of virus particles and the number of target cells present.
For the purposes of the present invention, the term “ELISA” refers to enzyme-linked immunoabsorbent assay, which is an immunoassay technique in which an immobilized antigen is detected by an antibody bound to an enzyme (peroxidase, alkaline phosphatase, etc.,) capable of generating a detectable product from a substrate by a colour change or some other type of change caused by the enzymatic action on said substrate. In said technique, there may be a primary antibody that recognizes the antigen and that in turn is recognized by a secondary antibody bound to said enzyme. The antigen can be detected indirectly in the sample by colour changes measured by spectrophotometry.
For the purposes of the present invention, “challenge”, in the context of immunology, refers to the deliberate exposure of an animal to an infectious agent, e.g., a virus, to study the response of the animal to exposure to said infectious agent. The dose used in the challenge is called the “challenge dose”.
For the purposes of the present invention, RACE (Rapid amplification of cDNA ends) refers to a technique of rapid amplification of complementary DNA (cDNA) ends. RACE is a technique used in molecular biology to obtain the full-length sequence of an RNA molecule, in which a cDNA copy of the RNA sequence of interest is produced by reverse transcription, followed by polymerase chain reaction (PCR) amplification of the cDNA copies.
For the purposes of the present invention, “ANOVA” or “analysis of variance” is a set of statistical models used to analyze differences between means within a statistical sample. ANOVA is based on the law of total variance, where the variance observed in a particular variable is divided into components attributable to different sources of variation. ANOVA provides a statistical test to determine if two or more population means are the same.
The present invention provides an attenuated variant of Rift Valley Fever Virus (RVFV), wherein:

    
    
        in the RdRp protein encoded by the L segment of the RNA of said variant:
        
            the amino acid of position 924 is serine (L[Gly924Ser]); and
            the amino acid at position 1303 is threonine (L[Ala1303Thr]);
            wherein the sequence SEQ ID NO: 47 of wild strain 56/74 of the RVF virus or the sequence SEQ ID NO: 54 of wild-type strain ZH548 of the RVF virus, are the reference sequences for amino acid numbering of said protein; and
        
        
        in the NSs protein encoded by the S segment of the RNA of said variant:
        
            the amino acid at position 82 is leucine (NSs[Pro82Leu]);
            wherein the sequence SEQ ID NO: 49 of wild strain 56/74 of the RVF virus or the sequence SEQ ID NO: 56 of the wild type ZH548 strain of the RVF virus, are the reference sequences for the numbering of the amino acids of said protein.
        
        
    
    


In one embodiment of the variant of the RVFV of the invention, further:

    
    
        in the RdRp protein encoded by the L segment of the RNA of said variant:
        
            the amino acid at position 100 is threonine (L[Met100Thr]);
            the amino acid at position 375 is tyrosine (L[His375Tyr]);
            the amino acid at position 1050 is valine (L[Ile1050Val]);
            the amino acid at position 1629 is phenylalanine (L[Leu1629Phe]); and
            the amino acid at position 2071 is lysine (L[Glu2071Lys]);
            wherein the sequence SEQ ID NO: 47 of wild strain 56/74 of the RVF virus or the sequence SEQ ID NO: 54 of the wild type ZH548 strain of the RVF virus, are the reference sequences for the numbering of the amino acids of said protein;
        
        
        in the amino acid sequence encoded by the M segment of the RNA of said variant:
        
            the amino acid at position 26 is lysine (M[Arg26Lys]);
            the amino acid at position 108 is tyrosine (M[His108Tyr]);
            the amino acid at position 118 is lysine (M[Glu118Lys]);
            the amino acid at position 210 is lysine (M[Arg210Lys]);
            the amino acid at position 333 is asparagine (M[Asp333Asn]);
            the amino acid at position 427 is threonine (M[Ala427Thr]);
            the amino acid at position 432 is valine (M[Ala432Val]);
            the amino acid at position 487 is glycine (M[Glu487Gly]);
            the amino acid at position 540 is tyrosine (M[His540Tyr]);
            the amino acid at position 582 is threonine (M[Ala582Thr]);
            the amino acid at position 587 is isoleucine (M[Val587Ile]);
            the amino acid at position 950 is valine (M[Ala950Val]);
            the amino acid at position 1090 is isoleucine (M[Val1090Ile]);
            the amino acid at position 1116 is valine (M[Ala1116Val]); and
            the amino acid at position 1182 is lysine (M[Arg1182Lys]);
            wherein the sequence SEQ ID NO: 48 of wild strain 56/74 of the RVF virus or the sequence SEQ ID NO: 55 of the wild strain ZH548 of the RVF virus, are the reference sequences for amino acid numbering of said amino acid sequence encoded by the M segment of the RNA of said variant;
        
        
        in the NSs protein encoded by the S segment of the RNA of said variant:
        
            the amino acid at position 52 is isoleucine (NSs[Val52Ile]);
            wherein the sequence SEQ ID NO: 49 of wild strain 56/74 of the RVF virus or the sequence SEQ ID NO: 56 of the wild type ZH548 strain of the RVF virus, are the reference sequences for the numbering of the amino acids of said protein.
        
        
    
    


In one embodiment of the attenuated variant Rift Valley fever virus (RVFV):

    
    
        in the amino acid sequence encoded by the L segment of the RNA of said variant:
        
            the amino acid at position 100 is threonine;
            the amino acid at position 375 is tyrosine;
            the amino acid at position 924 is serine;
            the amino acid at position 1050 is valine;
            the amino acid at position 1303 is threonine;
            the amino acid at position 1629 is phenylalanine; and
            the amino acid at position 2071 is lysine;
        
        
        in the amino acid sequence encoded by the M segment of the RNA of said variant:
        
            the amino acid at position 26 is lysine;
            the amino acid at position 108 is tyrosine;
            the amino acid at position 118 is lysine;
            the amino acid at position 210 is lysine;
            the amino acid at position 333 is asparagine;
            the amino acid at position 427 is threonine;
            the amino acid at position 432 is valine;
            the amino acid at position 487 is glycine
            the amino acid at position 540 is tyrosine;
            the amino acid at position 582 is threonine;
            the amino acid at position 587 is isoleucine;
            the amino acid at position 950 is valine;
            the amino acid at position 1090 is isoleucine;
            the amino acid at position 1116 is valine; and
            the amino acid at position 1182 is lysine;
        
        
        in the amino acid sequence encoded by the S segment of the RNA of said variant:
        
            the amino acid at position 52 is isoleucine; and
            the amino acid at position 82 is leucine;
        
        
        wherein said amino acids correspond to amino acid substitutions of the sequence of wild strain 56/74 of the RVF virus.
    
    


In one embodiment of the RVFV variant of the invention, the amino acid sequence encoded by the L segment of the RNA of said variant is SEQ ID NO: 4; the amino acid sequence encoded by the M segment of the RNA of said variant is SEQ ID NO: 5; the NSs protein consists of the sequence SEQ ID NO: 6; and the N protein consists of the sequence SEQ ID NO: 7.
In the present disclosure, the amino acid sequence encoded by the L segment of the RNA of wild strain 56/74 of the RVF virus is SEQ ID NO: 47; the amino acid sequence encoded by the M segment of the RNA of wild strain 56/74 of the RVF virus is SEQ ID NO: 48; the NSs protein of the wild type 56/74 strain of the RVF virus consists of the sequence SEQ ID NO: 49; and the N protein of wild-type strain 56/74 of the RVF virus consists of the sequence SEQ ID NO: 50.
In the present disclosure, the amino acid sequence encoded by the L segment of the RNA of wild strain ZH548 of the RVF virus is SEQ ID NO: 54; the amino acid sequence encoded by the M segment of the RNA of the ZH548 wild strain of the RVF virus is SEQ ID NO: 55; the NSs protein of the wild type ZH548 strain of the RVF virus consists of the sequence SEQ ID NO: 56; and the N protein of the wild type ZH548 strain of the RVF virus consists of the sequence SEQ ID NO: 57.
In one embodiment of the RVFV variant of the invention, comprising an RNA encoding said variant, the L segment of said RNA consists of the sequence SEQ ID NO: 1; and the M segment of said RNA consists of the sequence SEQ ID NO: 2; and the S segment of said RNA consists of the sequence SEQ ID NO: 3.
In the present disclosure, the wild strain 56/74 of the RVF virus comprises an RNA encoding said wild strain, wherein the L segment of said RNA consists of the sequence SEQ ID NO: 44; and the M segment of said RNA consists of the sequence SEQ ID NO: 45; and the S segment of said RNA consists of the sequence SEQ ID NO: 46.
In the present disclosure, wild type strain ZH548 of the RVF virus comprises an RNA encoding said wild type strain, wherein the L segment of said RNA consists of the sequence SEQ ID NO: 51; and the M segment of said RNA consists of the sequence SEQ ID NO: 52; and the S segment of said RNA consists of the sequence SEQ ID NO: 53.
The present invention also provides a pharmaceutical or veterinary composition comprising the RVFV variant of the invention, together with at least one pharmaceutically acceptable excipient or for veterinary use.
The present invention also provides the RVFV variant of the invention for use as a medicament.
In one embodiment, the present invention provides the use of the RVFV variant of the invention for the manufacture of a medicament.
In another embodiment, the present invention provides a therapeutic method for preventing Rift Valley fever comprising administering to an animal an effective amount of the attenuated variant of the RVFV of the invention. Preferably, said animal is a human or a ruminant.
For the purposes of the present invention, “effective amount” refers to the amount of the RVFV variant of the invention that provides an objectively identifiable improvement in the state of the animal, recognized by a qualified observer, and wherein said animal is treated with a pharmaceutical composition comprising said amount of the RVFV variant.
Additionally, the present invention provides the RVFV variant of the invention for use in the prevention of Rift Valley fever.
In one embodiment, the present invention provides the use of the RVFV variant of the invention for the manufacture of a medicament for the prevention of Rift Valley fever. Preferably, said medicament is a vaccine.
In one embodiment, the RVFV variant of the invention is for use in animals.
In a preferred embodiment of the variant of the RVFV for use of the invention, said animals are ruminants. Preferably, said ruminants are selected from: cows, sheep, goats, camels and buffaloes.
The RVFV variant of the invention is for use in domestic ruminants and wild ruminants. In the use in wild ruminants, the RVFV variant of the invention is useful for preventing RVF in wild animals in reserves, zoos, etc. In fact, it is suspected that the African buffalo may be a carrier of the virus.
Thus, in one embodiment of the variant of the RVFV for use of the invention, such ruminants are domestic or wild ruminants.
In a preferred embodiment, said domestic ruminants are selected from: cows, sheep, goats and camels.
In another preferred embodiment, said wild ruminants are buffalo.
In another preferred embodiment, the RVFV variant of the invention is for use in humans.
The present invention also provides a Rift Valley fever vaccine comprising the RVFV variant of the invention.
In a preferred embodiment, the vaccine of the invention comprises at least one pharmaceutically acceptable excipient or for veterinary use.
For the purposes of the present invention, inert ingredients such as, but not limited to, buffers, co-solvents, surfactants, oils, humectants, emollients, preservatives, stabilizers, antioxidants, dyes, air protectors and/or moisture and binders are pharmaceutically acceptable excipients or for veterinary use. An example of a buffer is phosphate buffered saline (PBS).
In one embodiment, said pharmaceutical or veterinary composition comprises Dulbecco's Modified Eagle Medium (DMEM).
The pharmaceutical, veterinary composition or vaccine of the invention can be formulated with pharmaceutically acceptable excipients or for veterinary use, as well as with any other type of pharmaceutically acceptable carriers or diluents or for veterinary use, according to conventional techniques in pharmaceutical or veterinary practice.
The pharmaceutical, veterinary composition or the vaccine of the invention can be administrated in single or multiple doses.
The pharmaceutical, veterinary composition or the vaccine of the invention can be administered by any route of administration for which said composition will be formulated in the pharmaceutical form suitable for the chosen route of administration. Examples of routes of administration include, but are not limited to, subcutaneous, intravenous and intramuscular.
The present specification also provides primers for amplification of genomic regions of the RVF virus RNA comprising nucleic acids at least 80% identical to any of the sequences selected from: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43.
In a preferred disclosure, said sequence identity is selected from: 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%.
In a more preferred disclosure, said primers of the invention consist of nucleic acids consisting of the sequences selected from: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43.
In one disclosure of such primers of the invention, said RNA is from the RVFV variant of the invention or from the wild strain 56/74 of the RVFV.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Growth kinetics in mammalian and insect cells. Vero (A) cells and C6/36 mosquito cells (B) were infected with a multiplicity of infection (MOI) of 0.01 or 0.005 respectively, with the FMH-P8 RVF virus (squares), the parental RVF virus 56/74 after 8 passes (open circles) and before such passes (closed circles). hpi represents hours post infection and dpi represents days post infection. After one hour of adsorption the inoculum was removed, the cells washed and new medium was added. The supernatants were collected at different times post infection (pi) and assayed in Vero cell monolayers by a standard plate assay using semi-solid medium. The monolayers were fixed and stained 4 days post infection. Assessments were performed at least twice. The mean values plus the standard deviation represented by an asterisk have p<0.05 (multiple t-test). The data shown correspond to a representative experiment. C. Plate phenotype of the indicated viruses in Vero cell monolayers with the parental virus RVFV 56/74 after 8 passes (center), before said passes (left) and with the FMH-P8 RVF virus (right). The infection was performed in semi-solid medium. The monolayers were fixed and stained 4 days post infection.
FIG. 2. In vivo infectivity analysis of the FMH-P8 RVF virus in A129 mice (IFNAR−/−). Male mice 5-6 months of age were inoculated with the indicated doses of the FMH-P8 RVF virus, the parental RVF virus 56/74 after 8 passes (56/74 p8) and before said passes (56/74). Animals were monitored daily for 14 days. Survival rates (A) and body weight change (B) in challenged mice at different doses. C. Detection of nucleoprotein-specific antibodies by indirect ELISA assay. 1, control sample; 2, sample 56/74 102; 3, sample FMH-P8 102; 4, sample FMH-P8 103; 5, sample FMH-P8 104. dpi represents days post infection.
FIG. 3. Comparison of survival rates of A129 mice (IFNAR−/−) inoculated with FMH-P8 RVF virus and MP-12 RVF virus (Ikegami et al., 2015). Mice were inoculated with FMH-P8 RVF virus doses 103 pfu (triangles) and 104 pfu (squares) and MP-12 RVF virus (doses 104 pfu, circles). dpi represents days post infection.
FIG. 4. Analysis of the immunogenicity and efficacy of FMH-P8 attenuated virus to a lethal challenge with RVF virus 56/74. Wild 11-month-old 129Sv/Ev mice (n=9) were inoculated intraperitoneally with 104 pfu FMH-P8 RVFV. A group of control mice was inoculated (n=4). A. Microneutralization test. Serum samples were taken on day 24 (prechallenge samples) and assayed for neutralizing antibodies. Serums were analyzed in dilutions in base 2 from 1/10 to 1/1280. The titer is expressed as the last dilution of serum that results in a reduction of cytopathic effect (CPE) in 50% of wells. First dilution tested: 1/10 (cut); “neg” samples (CPE in all wells in the first dilution analyzed) arbitrarily represented as 1/5. 1, “neg” samples; 2, samples with control mice inoculation; 3, FMH-P8 samples. B. Survival of 129Sv/Ev mice in challenge with FMH-P8 RVFV (squares) and with control mice inoculation (circles). C. Morbidity after exposure in vaccinated mice. D, S and H represent the clinical status of each mouse (D: dead, S: sick, H: healthy). Lower panel C, mice in challenge with FMH-P8 RVFV; upper panel C, control mice inoculation. D. Detection of anti-nucleoprotein antibodies by ELISA. Each symbol corresponds to an individual animal, except for the “neg” samples (groups of pre-immune sera). The titer is represented as log 10 of the last dilution of serum providing an optical density (OD) greater than 0.250 (control “neg” samples giving readings of 0.07 as an average; blank wells mean 0.04). 1, “neg” samples; 2, control group samples; 3, FMH-P8 samples; 4, post-challenge FMH-P8 samples. Serums were analyzed in dilutions in base 3 from 1/50 to 1/109350. First dilution analyzed: 1/50 (titre 1.7 (cut-off value); samples “neg” 1/25 (log 1.4)). dpi represents days post infection.
FIG. 5. Clinical signs in sheep inoculated with FMH-P8 RVF virus and parental virus 56/74. Rectal temperature (A), liver enzymes (B) and neutralizing antibody production (C) are compared in inoculated sheep with 107 pfu of FMH-P8 RVF virus (squares, n=2) and parental RVF virus 56/74 (circles, n=3).



DESCRIPTION OF EMBODIMENTS
Materials and Methods
Cells, Viruses and Infections
Vero cells (ATCC No. Catalogue CCL-81) were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% to 10% fetal calf serum (FCS) and L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 g/ml) in a humid atmosphere of 5% CO2 at 37° C. Insect cells of Aedes albopictus C6/36 (ATCC No. Catalogue CRL1660) were grown in Eagle minimal essential medium (EMEM) supplemented with 10% fetal calf serum (FCS), L-glutamine (2 mM), gentamicin (50 μg/ml) and vitamin MEM solution (Sigma) in a humid atmosphere of 5% CO2 at 28° C.
The starting parental virus originated in a sheep experimentally infected with wild strain 56/74 isolate of the VRVR 56/74 virus (parental virus) (Borrego et al., 2019); (Busquets et al., 2010). The virus was re-isolated from infected sheep plasma and cultured in a C6/36 mosquito cell line (ATCC CRL-1660). Assays to quantify plate-forming units (pfu) were performed in semi-solid medium including 1% carboxymethylcellulose (CMC; Sigma). pfu units are used in virology to describe the number of virus particles capable of forming plates per unit volume. Viral particles that are defective or that fail to infect their target cell will not form a plaque, and are not counted.
RNA Extraction, RT-PCR and Nucleotide Sequencing
RNA was extracted from the supernatants of the infected cells using the Speedtools RNA virus extraction kit (Biotools B&M Labs, S.A., Madrid, Spain) according to the manufacturer's instructions. Reverse transcription polymerase chain reaction (RT-PCR) was performed using SuperScript IV reverse transcriptase (Invitrogen) and Phusion high-fidelity DNA polymerase (Finnzymes), as directed by the manufacturers, using primers designed to amplify the L segments (Table 1A), M segments (Table 1B), and S segments (Table 1C) of the viral genome. Table 1D shows the primers used for the amplification of the genomic ends by the RACE technique. The overlapping PCR amplicons were purified and subjected to automatic Sanger sequencing. The Laser Gene software was used for the analysis of the results.










TABLE 1A







Position/L



Name
SEQ ID NO
segment
Orientation







5′ end L segment
 8
 1-30
Antigenomics


716F
 9
716-732
Antigenomics


L-F segment 1028ag
10
1028-1044
Antigenomics


L-R 2300g
11
2281-2300
Genomics


RdRp central-F
12
2701-2723
Antigenomics


L-F segment
13
2872-2894
Antigenomics


L-R segment
14
3006-2984
Antigenomics


Central RdRp-R
15
3938-3960
Antigenomics


3817 F
16
3817-3833
Antigenomics


4553 F
17
4553-4569
Antigenomics


5455 F
18
5455-5477
Antigenomics


5583 R
19
5583-5567
Antigenomics


Q3′25nts
20
6361-6385
Genomics


End q3′R L segment
21
6369-6385
Genomics



















TABLE 1B







Position/M



Name
SEQ ID NO
segment
Orientation







(−2)RTsm1
22
27-53
Antigenomics


MRV1ag
23
772-790
Antigenomics


RTsm2
24
1953-1976
Antigenomics


Sm2
25
2072-2095
Genomics


Sm3
26
3200-3223
Genomics


Sm4
27
3817-3838
Genomics


EM-RVFV-R
28
3867-3884
Genomics


EM-RVFV-F
29
3405-3424
Antigenomics





















TABLE 1C







Name
SEQ ID NO
Position/S segment
Orientation









NS0g
30
 1-19
Antigenomics



NS2g
31
61-80
Antigenomics



R-S
32
241-262
Antigenomics



F-S
33
338-361
Genomics



NScag
34
824-841
Genomics



SS1
35
909-931
Antigenomics



RTss1
36
1634-1663
Genomics



NP0ag
37
1670-1690
Genomics





















TABLE 1D





Name
SEQ ID NO
Position
Segment
Orientation







LsegcRNA
38
349-366
L
Genomics


LsegvRNA
39
6011-6026
L
Antigenomics


MsegcRNA
40
393-409
M
Genomics


MsegvRNA
41
3544-3561
M
Antigenomics


SsegcRNA
42
296-313
S
Genomics


SegvRNA
43
1421-1437
S
Antigenomics









Experiments with Mice

Groups of transgenic 129Sv/Ev IFNAR−/− mice of 5-6 months of age or wild 129Sv/Ev mice of 11 months of age (B&K Universal) were inoculated intraperitoneally with different doses of the viruses, as indicated in the corresponding experiments. After viral inoculation, animals were monitored daily for weight and development of clinical signs, including signs on the coat, hunched posture, reduced activity, and conjunctivitis. At the indicated times, the animals were bled through the maxillary vein. Serums were inactivated by heat at 56° C. for 30 minutes and maintained at −20° C. until use. All mice were housed in a BSL-3 containment area with food and water supplied ad libitum. All experimental procedures were managed in accordance with the guidelines of the EU Directive 2010/63/EU for animal experiments and protocols approved by the Biosafety and Ethics Committees for Animal Experiments of INIA (EAEC permit codes 2012/014 and CBS 2012/017).
Sheep Experiments
Two ewes were inoculated with a dose of 107 pfu of FMH-P8 and compared to three additional ewes inoculated with virus 56/74 (control group). One sheep from each group was slaughtered on day 4 post infection to analyze the degree of liver injury caused by the infection. Rectal temperature was taken daily after the challenge and blood and serum samples were taken daily for at least 8 consecutive days. The blood and serum samples obtained were used to perform a quantification of liver transaminase levels, as well as in vitro tests for neutralizing antibodies.
Antibody Assays
Neutralization tests were performed on 96-well culture plates following the test prescribed by the OIE (Chapter 2.1.14 OIE Terrestrial Manual 2012). Briefly, sera were diluted in base 2 from an initial 1/10 dilution in DMEM medium containing 2% fetal bovine serum, mixed with an equal volume of infectious virus containing 100 TCID50 (50% infectious tissue culture dose) and incubated 30 minutes at 37° C. A suspension of Vero cells was then added and the plates were incubated for 4 days. The monolayers were controlled for the development of cytopathic effect, fixed and stained. Each sample was tested in 4 wells. The titer is expressed as the last dilution of serum that gives a reduction of the cytopathic effect in 50% of the wells.
For the detection of antibodies against the nucleoprotein (N protein), an ELISA assay was performed. The ELISA plates were adsorbed with 100 ng/well of recombinant N-protein produced in E. coli and purified, diluted in carbonate buffer (pH 9.6). After blocking with 5% skimmed milk-PBS-0.05% Tween 20, the sera were analyzed in duplicate in serial dilutions in base 3 starting at 1/50. The bound antibodies were detected with goat antibodies conjugated to horseradish peroxidase (HRP), mouse-HRP anti-IgG (H+L) (BioRad) and the bound conjugate was detected using 3,3′,5,5′-tetramethylbenzidine (TMB, Invitrogen/Life Technologies) for 10 minutes, followed by a volume of stop solution (3N H2SO4). The optical densities were measured at 450 nm (OD450).
Statistical Analysis
Data analysis was performed with GraphPad prism version 6 software.
Example 1. Obtaining FMH-P8 Attenuated RVF Virus
The parental virus isolate RVFV 56/74 was subjected to serial passages in Vero cells in the presence of 40 μM favipiravir. Viral titration of the culture supernatants indicated that the production of viral progeny progressively decreased, being undetectable in steps 5, 6 and 7. However, in steps 8 and 9 infectivity was recovered with normal viral titers, indicating the generation of a virus resistant to favipiravir, which was called FMH-P8 (from “Favipiravir-Mutagenized Hyperattenuated Passage 8”). The viral production of FMH-P8 virus was analyzed in the presence of different concentrations of favipiravir, obtaining a 50% reduction in viral production at a concentration of 80 UM of favipiravir. These results indicated that FMH-P8 virus was more resistant to favipiravir compared to parental virus.
Example 2. Genetic Changes in FMH-P8 Attenuated RVF Virus
Overlapping RT-PCR reactions were performed from RNA extracted from infection supernatants of attenuated RVF virus of the invention, FMH-P8, obtained in the previous example. Amplicons were produced in these reactions, covering all 3 segments of the viral genome. We proceeded to sequence these amplicons by automatic sequencing (Sanger sequencing). The deduced amino acid sequences were aligned and compared to those of the parental virus RVFV 56/74. The description of the sequences of the attenuated RVF virus of the invention FMH-P8 and the parental virus RVFV 56/74 are shown in Table 2.










TABLE 2







Description
SEQ ID NO









FMH-P8 RVF virus L segment
 1



FMH-P8 RVF virus M segment
 2



FMH-P8 RVF virus S segment
 3



FMH-P8 RVFV L protein
 4



FMH-P8 RVFV M (poly)protein
 5



FMH-P8 RVFV NSs protein
 6



FMH-P8 RVFV N protein
 7



RVFV 56/74 L segment
44



RVFV 56/74 M segment
45



RVFV 56/74 S segment
46



RVFV 56/74 L protein
47



RVFV 56/74 M poly(protein)
48



RVFV 56/74 NSs protein
49



RVFV 56/74 N protein
50










Comparing the sequences of the attenuated RVF virus of the invention FMH-P8 with that of the parental virus RVFV 56/74, a total of 47 nucleotide changes have been found that result in 24 amino acid changes. In particular, in the L segment, which encodes the viral polymerase, the target of favipiravir, 17 nucleotide changes were identified, with 7 amino acid changes. The distribution of all changes found in the 3 genomic segments is shown in Table 3.












TABLE 3







Change in
Change in
Amino



RNA
Protein/
nucleotide(s)
nucleotide(s)
acid
Amino acid


segment
region
position
(codon)
position
substitution




















L
N-TERM
198
C→T
60
(Gly) -



5′ end
317
ATG→ACG
100
Met→Thr




396
C→T
126
(Phe) -




1120
C→T
368
(Leu) -




1141
CAC→TAC
375
His→Tyr



RdRp 
2757
C→T
913
(His) -



nucleus
2788
GGT→AGT
924
Gly→Ser




3166
ATT→GTT
1050
Ile→Val



C-TERM
3925
GCC→ACC
1303
Ala→Thr



3′ end
4110
G→A
1364
(Leu) -




4903
CTC→TTC
1629
Leu→Phe




4992
G→A
1658
(Lys) -




5025
G→A
1669
(Val) -




5178
G→A
1720
(Lys) -




5193
A→G
1725
(Lys) -




5229
C→T
1737
(Phe) -




6229
GAG→AAG
2071
Glu→Lys









Total number 
17
 7


of changes














M
NSm
97
AGA→AAA
26
Arg→Lys




342
CAC→TAC
108
His→Tyr




372
GAG→AAA
118
Glu→Lys




374






Gn
649
AGA→AAA
210
Arg→Lys




(mixture)







716
CAG→CAA
232
(Gln) -




1017
GAT→AAT
333
Asp→Asn




1299
GCT→ACT
427
Ala→Thr




1315
GCC→GTC
432
Ala→Val




1337
GGT→GGA
439
(Gly) -




1480
GAG→GGG
487
Glu→Gly




1638
CAC→TAC
540
His→Tyr




1742
CTG→CTA
574
(Leu) -




1764
GCT→ACT
582
Ala→Thr




1779
GTT→ATT
587
Val→Ile



Gc
2324
AGC→AGT
768
(Ser) -




2869
GCA→GTA
950
Ala→Val




3288
GTA→ATA
1090
Val→Ile




3359
ACC→ACT
1113
(Thr) -




3367
GCT→GTT
1116
Ala→Val




3565
AGA→AAA
1182
Arg→Lys



3′NCR
3821
A→G
—
—




3823
T→A
—
—









Total number 
23
15


of changes














S
NSs
124
AGG→AGA
30
(Arg) -




188
GTT→ATT
52
Val→Ile




279
CCA→CTA
82
Pro→Leu




598
GAG→GAA
188
(Glu) -



Intergenic
887
C→T
—
—



region







N
952
GTC→GTT
234
(Val) -




1645
AAC→AAT
3
(Asn) -











Total number 













of changes
 7
 2


TOTAL
47
24





Amino acids between parentheses followed by a dash indicate that there is no amino acid substitution in the FMH-P8 RVF virus relative to the parental RVFV 56/74 virus.






Nucleotide changes found in segment S led to only 2 amino acid substitutions, both in the NSs protein: Val52Ile and Pro82Leu. Pro82 belongs to the second Pro-X-X-Pro motif involved in the nuclear localization of the NSs protein and the activation of Interferon-β (IFN-β). The N nucleoprotein was the only FMH-P8 virus protein that showed an amino acid sequence identical to that of the parental virus, with only two (silent) nucleotide substitutions.
In the sequence corresponding to the M segment of the FMH-P8 virus, a total of 15 amino acid substitutions were identified, three in the NSm protein (Arg26Lys, His108Tyr, Glu118Lys), eight in the Gn protein (Arg210Lys-mix-, Asp333Asn, Ala427Thr, Ala432Val, Glu487Gly, His540Lys, Ala582Thr, Val587Ile) and four in the Gc protein (Ala950Val, Val1090Ile, Ala1116Val and Arg1182Lys). The Arg1182Gly change in Gc has been identified as an attenuation marker for MP-12 virus (Ikegami et al., 2015).
Seven amino acid substitutions were identified in the FMH-P8 virus L protein, distributed throughout the sequence. Two changes were located in the N-terminal region of the L protein (Met100Thr and His375Tyr); two in the C-terminal region (Leu1629Phe and Glu2071Lys), the three remaining substitutions (Gly924Ser, Ile1050Val and Ala1303Thr) in the central region of the protein. Positions 924 and 1050 are located within the RdRp core, where the conserved catalytic motifs A to H of the polymerase reside.
Since viral RNA polymerase is known to be a target of favipiravir, the conservation level of mutated residues of FMH-P8 RVF virus located within the catalytic nucleus of RdRp has been evaluated. The L-protein sequences corresponding to 60 different strains of RVFV have been compared and several virus species belonging to the genus phlebovirus have also been included (Table 4). Residues Gly924 and Ala1303 were found to be extremely conserved in all viruses included in the alignment. Position 1050 showed only conservative changes, mainly showing isoleucine (such as parental virus 56/74) or valine (such as attenuated FMH-P8 RVFV), while the other substituted positions were conserved among the RVFV strains but varied in other viruses of the phlebovirus genus.








TABLE 4








Amino Acid Positions in RVFV Isolates














Virus
100
375
924
1050
1303
1629
2071





Nucleotide


2788
3166
3925




RVFV 
Met
His
Gly
Ile
Ala
Leu
Glu


56/74









FMH-P8
Thr
Tyr
Ser
Val
Thr
Phe
Lys


RVFV









RVFV (60)
—
—
—
—
—
—
—


Phlebovirus
Ile/
Tyr
—
Val
—
Ile/
Thr/


Bujaru (2)
Val




Leu
Ser


Phlebovirus
Val
Asp/
—
—/
—
Ile/
Asp/


Candiru (2)

Glu

Val

Val
Arg


Phlebovirus
Ser
Asn
—
—
—
—
Arg


Frijoles (1)









Phlebovirus
Ile/
Tyr
—
Val
—
Ile/
Asn/


Punta Toro
Val




Ser
Asp/


(3)






Ser


Phlebovirus
Val
Asp
—
—/
—
Glu
Ala/


Salehabad



Val


Thr


(2)









Phlebovirus
 Val*
—/
—
—/
—
Glu
Ser


Naples Fly

Asn

Val





Fever (3)









Phlebovirus
Ile
Val
—
Ser
—
—
Asp


SFTS (2)









Phlebovirus
Leu/
Asp/
—
Thr
—/
Glu/
Ser/


Uukuniemi
Ile
Val


Thr
Ser
Thr


(2)





The amino acid residues at the L protein position of the indicated viruses are shown.


The dash means that the residue matches that of the parental virus RVFV 56/74.






Example 3. Infectivity of FMH-P8 Attenuated RVF Virus in Vero and C6/36 Cells
The kinetics and total yield of FMH-P8 attenuated RVF virus were analyzed. For comparison purposes, parental virus RVFV 56/74 was also tested before and after spread over 8 passes, in the absence of favipiravir. As mosquitoes play an important role in the natural transmission cycle of RVFV, infections were also carried out in the C6/36 cell line derived from Aedes albopictus (ATCC CRL1660).
Infections performed on Vero cells showed similar growth curves for all three viruses (FIG. 1A). Titration of supernatants collected at different times post infection in several independent experiments showed only small differences between the three viruses. The growth pattern of parental virus RVFV 56/74 after 8 passes showed no differences with parental virus RVFV 56/74 prior to such passes. FMH-P8 RVFV grew a little faster and with higher viral production yields 3-4 days post infection, although the differences were not statistically significant (multiple t-test).
Both viral growth and final yield in C6/36 mosquito cells were clearly affected by FMH-P8 RVF virus (FIG. 1B). Infected C6/36 mosquito cells remain viable for longer in cell culture than Vero cells and the analysis was extended up to 9 days. In the insect cells, the growth of the FMH-P8 RVF virus was significantly delayed, with viral titers of 104 pfu/ml to 2-4 days post infection, at least 3 log units lower than those produced by the control viruses. The total virus yields at the last points analyzed (7-9 days post infection), although they reached a titer of 107 pfu/mL, were below those reached by the parental virus RVFV 56/74 (>108 pfu/mL). No significant changes were found for the parental virus RVFV 56/74 after 8 passes with respect to the parental virus RVFV 56/74 before said passes.
The phenotype of the Vero cell plate in the presence of the FMH-P8 RVF virus differed substantially from the parental virus, producing plates smaller than those produced by the RVFV 56/74 parental virus before or after 8 passes (FIG. 1C).
Example 4. FMH-P8 Attenuated RVFV Infectivity in IFNAR Immunodeficient Mice−/−
To check in vivo the attenuation of the FMH-P8 RVF virus, an infection experiment was performed using the A129 mouse strain (IFNAR−/−). A129 mice cannot cope with acute viral infection and are highly susceptible to RVFV infection and offer a highly sensitive assessment of FMH-P8 RVFV attenuation.
Different doses of virus were inoculated intraperitoneally to groups of 5-6 mice and were monitored daily for 2 weeks to check the development of signs of disease and survival (FIG. 2A). After 8 passes the parental virus RVFV 56/74 caused 100% mortality 4 days post inoculation with 102 pfu, while mice inoculated with the same dose of the parental virus RVFV 56/74 before such passes showed a survival rate of 40% (⅖). Although these data suggest increased virulence of the parental virus RVFV 56/74 after 8 passes, the results were not statistically significant. Higher doses of the parental virus RVFV 56/74 caused death of inoculated animals in the first 4 days post infection: 100% in those inoculated with 103 pfu; 90% in those inoculated with 104 pfu, with no survivors at day 10.
In contrast, animals inoculated with the FMH-P8 RVF virus showed survival rates above 70% even with a high challenge dose (104 pfu), with a significant number of survivors at the end of the experiment: ⅚ (83%) in those who received 103 pfu and ⅘ (80%) in those inoculated with 104 pfu. No signs of disease were seen in any of these animals, except for slight weight loss on days 3-5 post infection (FIG. 2B).
Serum samples collected on day 14 (end of experiment) were analyzed by ELISA for the presence of N-nucleoprotein (anti-N) antibodies in survivors, indicative of viral replication (FIG. 2C). In some animals within the groups that received the lowest viral dose, 102 pfu, anti-N antibodies were undetectable, probably reflecting low or zero levels of viral replication (2/2 in mice inoculated with RVFV 56/74 virus; 1/4 in mice inoculated with FMH-P8 virus). All animals inoculated with 103 and 104 pfu of FMH-P8 RVF virus, as well as three of the group inoculated with 102 pfu of FMH-P8 RVF virus developed specific anti-N antibodies. The titers of anti-N antibodies showed no significant differences (ordinary one-way ANOVA) within the groups inoculated with FMH-P8 RVF virus, regardless of the dose received.
An in vivo infectivity assay was performed in A129 mice with FMH-P8 RVF virus and with MP-12 live attenuated vaccine (Ikegami et al., 2015). The MP-12 vaccine administered in IFNAR−/− mice at the same dose (104 cfu) causes the death of 100% of the mice of the strain within 5 days (FIG. 3). These results demonstrate the high vaccine potential of the FMH-P8 RVF virus.
Example 5. Immunogenicity and Efficacy of FMH-P8 Attenuated RVF Virus in Immunocompetent Mice
FMH-P8 attenuated RVF virus was assayed in immunocompetent mice. To do this, wild 129Sv/Ev mice were inoculated intraperitoneally with 104 pfu of the FMH-P8 RVF virus, and 4 weeks later were challenged with a lethal dose (104 pfu) of the parental RVF virus 56/74. After inoculation with FMH-P8 RVF virus, mice showed no signs of disease, not even significant weight variations. In serum samples collected 24 days after inoculation (samples prior to lethal challenge with parental virus RVFV 56/74), seven out of nine mice showed a strong neutralizing antibody response (FIG. 4A). N-nucleoprotein antibodies were detected in all these samples by indirect ELISA, including two samples that were negative in the neutralization assay, although their anti-N antibody titers were slightly lower (FIG. 4D). This indicates that the FMH-P8 RVF virus replicated in all inoculated mice at least to an extent sufficient to elicit an immune response. When mice were subjected to a lethal challenge with the virulent strain RVFV 56/74, 100% of the mice survived to the end of the experiment (FIG. 4B, FIG. 4C) without apparent clinical manifestation, including those in which no neutralizing antibody titers had been detected. In contrast, all mice in the control group became ill and died on day 4 (FIG. 4B, FIG. 4C).
Anti-N antibody titers increased following lethal challenge with parental virus RVFV 56/74 (FIG. 4D), indicating a booster effect of attenuated virus FMH-P8 RVFV in mice. Taken together, these results show that, despite its highly attenuated phenotype, the FMH-P8 RVF virus could be replicated in immunocompetent 129Sv/Ev mice at levels that allow for the induction of protective immune responses, even when no neutralizing antibodies are detected.
Example 6. Immunogenicity of FMH-P8 Attenuated RVF Virus in Sheep
The attenuated FMH-P8 RVF virus was inoculated into ewes to assess its attenuation and immunogenicity in a natural host of the RVF virus. Animals received an elevated dose of 107 pfu of the attenuated FMH-P8 RVF virus and clinical signs were monitored daily and daily sampling was performed. Fever was not recorded in any of the animals on the days immediately following inoculation of the attenuated FMH-P8 RVF virus, and liver enzyme titration did not indicate alterations in sheep inoculated with FMH-P8 unlike control sheep that had been inoculated with parental virus 56/74 which showed a spike in fever from day 2 post infection. Even in the absence of clinical signs, seroconversion was observed, reaching a significant titer of neutralizing antibodies at day 8 post-inoculation. Although the neutralizing antibody titer obtained with RVFV is lower than that obtained with parental virus 56/74, it is concluded that said neutralizing antibody titer is significant and sufficient to provide protection to sheep. The results of this example also demonstrate the safety provided by the attenuated FMH-P8 RVF virus and support the vaccine viability of the FMH-P8 RVF virus (FIG. 5).
Example 7. Assay in Mice Inoculated with Variants of the RVF Virus and after Challenge with a Lethal Dose of the ZH548 Strain of the RVF Virus
Wild mice 129 were inoculated with variants of the RVF virus and subsequently challenged with a lethal dose of the wild strain ZH548 of the RVF virus (a virulent strain).
Four groups of mice were inoculated with either the ZH548 strain of the RVF virus or different variants of the ZH548 strain, as indicated below.

    
    
        Mice of group C1 were inoculated with strain ZH548 of the RVF virus, a wild virulent control.
        Mice of group G1 were inoculated with the ZH548_ΔNSs:gfp variant of the RVF virus, an attenuated control with the region of the RNA encoding the deleted NSs protein.
        Mice of group A2 were inoculated with the variant ZH548_L[Gly924Ser]_L[Ala1303Thr]_NSs[Pro82Leu] of the RVF virus, a variant with Gly924Ser and Ala1303Thr substitutions in the protein encoded by the RdRp gene of the L segment of the viral RNA and with Pro82Leu substitution in the protein encoded by the NSs gene of the S segment of the viral RNA.
        Mice of group were B3 inoculated with variant ZH548_L [Gly924Ser]_L[Ala1303Thr], a variant with Gly924Ser and Ala1303Thr substitutions in the protein encoded by the RdRp gene of the L segment of the viral RNA.
    
    


Table 5 shows the results of viraemia, survival and seroconversion after inoculation of the different variants and after challenge with a lethal dose of the ZH548 strain of the RVF virus.










TABLE 5








After inoculation of the ZH548





strain or variant of the RVF virus
















Neutralizing
After challenge













% survival
Viraemia
antibodies
% survival
Viraemia


Group
(15 dpi)
(day 3)
(12 dpi)
(15 dpi)
(day 3)





C1
  0
25.10
nd
—
—


G1
100
NEG
1.98
100
NEG


A2
100
NEG
 2.60*
100
NEG


B3
100
NEG
3.27
100
NEG**









The viraemia values and neutralizing antibody titers correspond to the group means (n=6, except G1 n=5).
dpi: days post-infection.
viraemia: Cq value (quantification cycle) by RT-qPCR technique (reverse transcriptase quantitative polymerase chain reaction).
NEG: Values below the sensitivity level of the test (Cq=37).
Neutralizing antibody titer=PRNT80 (log 10). In group C1, no survivors were recorded at that time post-infection. In groups G1 and B3 all animals were positive, while in group A2 there were 2 animals (2/6) with values below the limit of detection of the test (dilution 1/50; log 10=1.70). * The indicated mean excludes these 2 negative values.
** After the challenge, in group B3, viraemia was detected at day 3 in a single animal (1/6), with a Cq=33.69.
The results of this example demonstrate a very clear virus attenuation effect of three amino acid substitutions in the ZH548 strain of the RVF virus: the Pro82Leu substitution in the protein encoded by the NSs gene of the S segment of the viral RNA and the Gly924Ser and Ala1303Thr substitutions in the RdRp protein of the L segment of the viral RNA.
The sequences of strain ZH548 are accessible through GenBank with the access codes DQ375403 (segment L), DQ380206 (segment M) and DQ380151 (segment S). The description of the sequences of strain ZH548 is shown in Table 6.










TABLE 6







Description
SEQ ID NO









ZH548 RVF virus L segment
51



ZH548 RVF virus M segment
52



ZH548 RVF virus S segment
53



ZH548 RVFV L protein
54



ZH548 RVFV M (poly)protein
55



ZH548 RVFV NSs protein
56



ZH548 RVFV N protein
57










Example 8. Assay in Sheep Inoculated with Variants of the RVF Virus and after Challenge with a Lethal Dose of the ZH548 Strain of the RVF Virus
A group of two sheep were inoculated with a lethal dose of the RVF virus strain ZH548.
A group of four sheep were inoculated with variant ZH548_L[Gly924Ser]_L[Ala1303Thr]_NSs[Pro82Leu] of the RVF virus (variant with Gly924Ser and Ala1303Thr substitutions in the protein encoded by the RdRp gene of viral RNA segment L and with Pro82Leu substitution in the protein encoded by the NSs gene of viral RNA segment S. Two sheep in this group were challenged with the ZH548 strain of the RVF virus three weeks later. The other two sheep in the group were sacrificed in a short time to compare the possible lesions with the control sheep given the lethal dose of the ZH548 strain of the RVF virus.
Ewes inoculated with the RVF virus strain ZH548_L [G924S/A1303T]_NSs[P82L] produced neutralizing antibodies and showed no lesions compared to those inoculated with the control virus. It was also not possible to detect infectious virus in the blood of immunized sheep compared to control.
Results of this example demonstrate the attenuation conferred by the three substitutions L[Gly924Ser], L[Ala1303Thr] and NSs[P82L]. They also confirm that said variant has the ability to induce an immune response capable of protecting sheep from a challenge with the wild strain ZH548.
SEQUENCE LISTING FREE TEXT
The sequence listing free text is reproduced in Table 7.









TABLE 7





SEQ 




ID NO
Position
Free text

















 1

FMH-P8 RVF virus L segment



  19-6297
FMH-P8 RVF virus L-segment open reading frame


 2

FMH-P8 RVF virus M segment



  21-3614
FMH-P8 RFV virus gene encoding M (poly)protein


 3

FMH-P8 RVF virus S segment



 35-832
FMH-P8 RVF virus gene encoding NSs protein



 916-1653
FMH-P8 RVFV gene encoding N protein (complementary)


 4

FMH-P8 RVFV L protein



100
Thr



375
Tyr



924
Ser



1050
Val



1303
Thr



1629
Phe



2071
Lys


 5

FMH-P8 RVFV M (poly)protein



26
Lys



108
Tyr



118
Lys



210
Lys



333
Asn



427
Thr



432
Val



487
Gly



540
Tyr



582
Thr



587
Ile



950
Val



1090
Ile



1116
Val



1182
Lys


 6

FMH-P8 RVFV NSs protein



52
Ile



82
Leu


 7

FMH-P8 RVFV N protein


 8

5′ end L segment primer


 9

716F primer


10

L-F segment 1028ag primer


11

L-R 2300g primer


12

RdRp central-F primer


13

L-F segment primer


14

L-R segment primer


15

Central-R RdRp primer


16

3817 F primer


17

4553 F primer


18

5455 F primer


19

R 5583 primer


20

Q3′25nts primer


21

L-segment end q3′R primer


22

(−2)Rtsm1 primer


23

MRV1ag primer


24

RTsm2 primer


25

Sm2 primer


26

Sm3 primer


27

Sm4 primer


28

EM-RVFV-R primer


29

EM-RVFV-F primer


30

NS0g primer


31

NS2g primer


32

R-S primer


33

F-S primer


34

NScag primer


35

SS1 primer


36

RTss1 primer


37

NP0ag primer


38

LsegcARN primer


39

LsegvARN primer


40

MsegcARN primer


41

MsegvARN primer


42

SsegcARN primer


43

SsegvARN primer


44

RVFV 56/74 L segment



  19-6297
RVFV 56/74 L segment open reading frame


45

RVFV 56/74 M segment



  21-3614
RVFV 56/74 gene encoding M (poly)protein


46

RVFV 56/74 S segment



 35-832
RVFV 56/74 gene encoding NSs protein



916 . . . 1653
RVF virus gene encoding N protein 56/74


47

RVFV 56/74 L protein


48

RVFV 56/74 M poly(protein)


49

RVFV 56/74 NSs protein


50

RVFV 56/74 N protein


51

ZH548 RVF virus L segment



  19-6297
ZH548 RVF virus L-segment open reading frame


52

ZH548 RVF virus M segment



  21-3614
ZH548 RFV virus gene encoding M (poly)protein


53

ZH548 RVF virus S segment



 35-832
ZH548 RVF virus gene encoding NSs protein



915 . . . 1652
ZH548 RVF virus gene encoding N protein


54

ZH548 RVFV L protein


55

ZH548 RVFV M (poly)protein


56

ZH548 RVFV NSs protein


57

ZH548 RVFV N protein









REFERENCES


    
    
        Borrego et al. (2019). Lethal Mutagenesis of Rift Valley Fever Virus Induced by Favipiravir. Antimicrob Agents Chemother, 63 (8), PII: e00669-19.
        Busquets et al. (2010). Experimental infection of young adult European breed sheep with Rift Valley fever virus field isolates. Vector Borne Zoonotic Dis, 10 (7), 689-696.
        Ikegami et al. (2015). Rift Valley Fever Virus MP-12 Vaccine Is Fully Attenuated by a Combination of Partial Attenuations in the S, M, and L Segments. J Virol, 89 (14), 7262-7276.