ATTENUATED VARIANT OF THE RIFT VALLEY FEVER VIRUS, COMPOSITION COMPRISING SAME, AND USES THEREOF

Abstract

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.

Claims

1. An attenuated variant of the Rift Valley Fever virus (RVFV), wherein the substitutions in the RdRp protein encoded by the L segment of the RNA of said variant consists of: the amino acid at position 924 is serine (L[Gly924Ser]); the amino acid at position 1303 is threonine (L[Alal303Thr]); 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 substitution in the NSs protein encoded by the S segment of the RNA of said variant consists of: 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. 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.

3. The RVFV variant according to claim 1, wherein 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.

4. The RVFV variant according to claim 1, containing an RNA encoding said variant, wherein 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.

5. A pharmaceutical or veterinary composition comprising the RVFV variant according to claim 1, together with at least one pharmaceutically acceptable excipient or an excipient for veterinary use.

6. (canceled)

7. A method of preventing Rift Valley fever in animals, said method comprising administering to the animals an effective amount of the RVFV variant according to claim 1.

8. The method of claim 7, wherein the route of administration of the RVFV variant is subcutaneous, intravenous or intramuscular.

9. The method of claim 7, wherein said animals are ruminants.

10. The method of claim 9, wherein said ruminants are selected from: cows, sheep, goats, camels and buffalo.

11. The method of claim 7, wherein said animals are in humans.

12. A Rift Valley Fever vaccine comprising the RVFV variant of claim 1.

13. The vaccine according to claim 12, further comprising at least one pharmaceutically acceptable excipient or an excipient for veterinary use.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0114] 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.

[0115] 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 10.sup.2; 3, sample FMH-P8 10.sup.2; 4, sample FMH-P8 10.sup.3; 5, sample FMH-P8 10.sup.4. dpi represents days post infection.

[0116] 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 10.sup.3 pfu (triangles) and 10.sup.4 pfu (squares) and MP-12 RVF virus (doses 10.sup.4 pfu, circles). dpi represents days post infection.

[0117] 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 129 Sv/Ev mice (n=9) were inoculated intraperitoneally with 10.sup.4 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 ⅒ 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: ⅒ (cut); “neg” samples (CPE in all wells in the first dilution analyzed) arbitrarily represented as ⅕. 1, “neg” samples; 2, samples with control mice inoculation; 3, FMH-P8 samples. B. Survival of 129 Sv/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 Iog10 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.

[0118] 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 10.sup.7 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

[0119] 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% CO.sub.2 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 .Math.g/ml) and vitamin MEM solution (Sigma) in a humid atmosphere of 5% CO.sub.2 at 28° C.

[0120] 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

[0121] 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-US-00001 Name SEQ ID NO Position/L 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-US-00002 Name SEQ ID NO Position/ M sement 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-US-00003 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-US-00004 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

[0122] Groups of transgenic 129Sv/Ev IFNAR.sup.-/- 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

[0123] Two ewes were inoculated with a dose of 10.sup.7 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

[0124] 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 ⅒ dilution in DMEM medium containing 2% fetal bovine serum, mixed with an equal volume of infectious virus containing 100 TCID.sub.50 (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.

[0125] 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 H.sub.2SO.sub.4). The optical densities were measured at 450 nm (OD.sub.450).

Statistical Analysis

[0126] Data analysis was performed with GraphPad prism version 6 software.

Example 1. Obtaining FMH-P8 Attenuated RVF Virus

[0127] The parental virus isolate RVFV 56/74 was subjected to serial passages in Vero cells in the presence of 40 .Math.M 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 .Math.M 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

[0128] 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-US-00005 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

[0129] 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-US-00006 RNA segment Protein/region Change in nucleotide(s) position Change in nucleotide(s) (codon) Amino acid position Amino acid substitution L N-TERM 5′ end 198 C.fwdarw.T 60 (Gly) - 317 ATG.fwdarw.ACG 100 Met.fwdarw.Thr 396 C.fwdarw.T 126 (Phe) - 1120 C.fwdarw.T 368 (Leu) - 1141 CAC.fwdarw.TAC 375 His.fwdarw.Tyr RdRp nucleus 2757 C.fwdarw.T 913 (His) - 2788 GGT.fwdarw.AGT 924 Gly.fwdarw.Ser 3166 ATT.fwdarw.GTT 1050 lle.fwdarw.Val C-TERM 3′ end 3925 GCC.fwdarw.ACC 1303 Ala.fwdarw.Thr 4110 G.fwdarw.A 1364 (Leu) - 4903 CTC.fwdarw.TTC 1629 Leu.fwdarw.Phe 4992 G.fwdarw.A 1658 (Lys) - 5025 G.fwdarw.A 1669 (Val) - 5178 G.fwdarw.A 1720 (Lys) - 5193 A.fwdarw.G 1725 (Lys) - 5229 C.fwdarw.T 1737 (Phe) - 6229 GAG.fwdarw.AAG 2071 Glu.fwdarw.Lys Total number of changes 17 7 M NSm 97 AGA.fwdarw.AAA 26 Arg.fwdarw.Lys 342 CAC.fwdarw.TAC 108 His.fwdarw.Tyr 372 GAG.fwdarw.AAA 118 Glu.fwdarw.Lys 374 Gn 649 (mixture) AGA.fwdarw.AAA 210 Arg.fwdarw.Lys 716 CAG.fwdarw.CAA 232 (Gln) - 1017 GAT.fwdarw.AAT 333 Asp.fwdarw.Asn 1299 GCT.fwdarw.ACT 427 Ala.fwdarw.Thr 1315 GCC.fwdarw.GTC 432 Ala.fwdarw.Val 1337 GGT.fwdarw.GGA 439 (Gly) - 1480 GAG.fwdarw.GGG 487 Glu.fwdarw.Gly 1638 CAC.fwdarw.TAC 540 His.fwdarw.Tyr 1742 CTG.fwdarw.CTA 574 (Leu) - 1764 GCT.fwdarw.ACT 582 Ala.fwdarw.Thr 1779 GTT.fwdarw.ATT 587 Val.fwdarw.Ile Gc 2324 AGC.fwdarw.AGT 768 (Ser) - 2869 GCA.fwdarw.GTA 950 Ala.fwdarw.Val 3288 GTA.fwdarw.ATA 1090 Val.fwdarw.Ile 3359 ACC.fwdarw.ACT 1113 (Thr) - 3367 GCT.fwdarw.GTT 1116 Ala.fwdarw.Val 3565 AGA.fwdarw.AAA 1182 Arg.fwdarw.Lys 3′NCR 3821 A.fwdarw.G --- --- 3823 T.fwdarw.A --- --- Total number of changes 23 15 S NSs 124 AGG.fwdarw.AGA 30 (Arg) - 188 GTT.fwdarw.ATT 52 Val.fwdarw.Ile 279 CCA.fwdarw.CTA 82 Pro.fwdarw.Leu 598 GAG.fwdarw.GAA 188 (Glu) - Intergenic region 887 C.fwdarw.T --- --- N 952 GTC.fwdarw.GTT 234 (Val) - 1645 AAC.fwdarw.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.

[0130] Nucleotide changes found in segment S led to only 2 amino acid substitutions, both in the NSs protein: Val52lle 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.

[0131] 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, Val587lle) and four in the Gc protein (Ala950Val, Val1090lle, Ala1116Val and Arg1182Lys). The Arg1182Gly change in Gc has been identified as an attenuation marker for MP-12 virus (Ikegami et al., 2015).

[0132] 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.

[0133] 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-US-00007 Virus Amino Acid Positions in RVFV Isolates 100 375 924 1050 1303 1629 2071 Nucleotide 2788 3166 3925 RVFV 56/74 Met His Gly Ile Ala Leu Glu FMH-P8 RVFV Thr Tyr Ser Val Thr Phe Lys RVFV (60) - - - - - - - Phlebovirus Bujaru (2) Ile/Val Tyr - Val - Ile/Leu Thr/Ser Phlebovirus Candiru (2) Val Asp/Glu - -/Val - Ile/Val Asp/Arg Phlebovirus Frijoles (1) Ser Asn - - - - Arg Phlebovirus Punta Toro (3) Ile/Val Tyr - Val - Ile/Ser Asn/Asp/Ser Phlebovirus Salehabad (2) Val Asp - -/Val - Glu Ala/Thr Phlebovirus Naples Fly Fever (3) Val* -/Asn - -/Val - Glu Ser Phlebovirus SFTS (2) Ile Val - Ser - - Asp Phlebovirus Uukuniemi (2) Leu/Ile Asp/Val - Thr -/Thr Glu/Ser Ser/Thr 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

[0134] 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).

[0135] 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).

[0136] 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 10.sup.4 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 10.sup.7 pfu/mL, were below those reached by the parental virus RVFV 56/74 (> 10.sup.8 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.

[0137] 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).

[0138] Example 4. FMH-P8 Attenuated RVFV Infectivity in IFNAR Immunodeficient Mice.sup.-/- To check in vivo the attenuation of the FMH-P8 RVF virus, an infection experiment was performed using the A129 mouse strain (IFNAR.sup.-/-). 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.

[0139] 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 10.sup.2 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 10.sup.3 pfu; 90% in those inoculated with 10.sup.4 pfu, with no survivors at day 10.

[0140] In contrast, animals inoculated with the FMH-P8 RVF virus showed survival rates above 70% even with a high challenge dose (10.sup.4 pfu), with a significant number of survivors at the end of the experiment: ⅚ (83%) in those who received 10.sup.3 pfu and ⅘ (80%) in those inoculated with 10.sup.4 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).

[0141] 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, 10.sup.2 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; ¼ in mice inoculated with FMH-P8 virus). All animals inoculated with 10.sup.3 and 10.sup.4 pfu of FMH-P8 RVF virus, as well as three of the group inoculated with 10.sup.2 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.

[0142] 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 (10.sup.4 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

[0143] FMH-P8 attenuated RVF virus was assayed in immunocompetent mice. To do this, wild 129Sv/Ev mice were inoculated intraperitoneally with 10.sup.4 pfu of the FMH-P8 RVF virus, and 4 weeks later were challenged with a lethal dose (10.sup.4 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).

[0144] 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

[0145] 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 10.sup.7 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

[0146] 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).

[0147] 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. [0148] Mice of group C1 were inoculated with strain ZH548 of the RVF virus, a wild virulent control. [0149] 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. [0150] 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. [0151] Mice of group B3 were 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.

[0152] 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-US-00008 After inoculation of the ZH548 strain or variant of the RVF virus After challenge Group % survival (15 dpi) Viraemia (day 3) Neutralizing antibodies (12 dpi) % survival (15 dpi) Viraemia (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 (log10). 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; log10 = 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 (⅙), with a Cq = 33.69.

[0153] 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.

[0154] 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-US-00009 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

[0155] A group of two sheep were inoculated with a lethal dose of the RVF virus strain ZH548.

[0156] 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.

[0157] 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.

[0158] 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

[0159] The sequence listing free text is reproduced in Table 7.

TABLE-US-00010 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 NS2q primer 32 R-S primer 33 F-S primer 34 NScag primer 35 SS1 primer 36 RTss1 primer 37 NP0ag primer 38 LseqcARN 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

[0160] Borrego et al. (2019). Lethal Mutagenesis of Rift Valley Fever Virus Induced by Favipiravir. Antimicrob Agents Chemother, 63(8), PII: e00669-19. https://doi.org/10.1128/AAC.00669-19

[0161] 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. https://doi.org/10.1089/vbz.2009.0205

[0162] 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. https://doi.org/10.1128/JVI.00135-15