Differential role of NSs genes in the neurovirulence of two genogroups of Akabane virus causing postnatal encephalomyelitis.

Akabane virus (AKAV) is a member of the genus Orthobunyavirus, family Peribunyaviridae. In addition to AKAV strains that cause fetal Akabane disease, which is characterized by abortion in ruminants, some AKAV strains cause postnatal infection characterized by nonsuppurative encephalomyelitis in ruminants. Here, we focused on the NSs protein, a virulence factor for most viruses belonging to the genus Orthobunyavirus, and we hypothesized that this protein would act as a neurovirulence factor in AKAV strains causing postnatal encephalomyelitis. We generated AKAV strains that were unable to produce the NSs protein, derived from two different genogroups, genogroups I and II, and then examined the role of their NSs proteins by inoculating mice intracerebrally with these modified viruses. Our results revealed that the neurovirulence of genogroup II strains is dependent on the NSs protein, whereas that of genogroup I strains is independent of this protein. Notably, infection of primary cultured bovine cells with these viruses suggested that the NSs proteins of both genogroups suppress innate immune-related gene expression with equal efficiency. These results indicate differences in the determinants of virulence of orthobunyaviruses.

Characterization of an avian rotavirus A strain isolated from a velvet scoter (Melanitta fusca): implication for the role of migratory birds in global spread of avian rotaviruses.

Avian G18P[17] rotaviruses with similar complete genome constellation, including strains that showed pathogenicity in mammals, have been detected worldwide. However, it remains unclear how these strains spread geographically. In this study, to investigate the role of migratory birds in the dispersion of avian rotaviruses, we analysed whole genetic characters of the rotavirus strain RK1 that was isolated from a migratory species of birds [velvet scoter (Melanitta fusca)] in Japan in 1989. Genetic analyses revealed that the genotype constellation of the RK1 strain, G18-P[17]-I4-R4-C4-M4-A21-N4-T4-E4-H4, was highly consistent with those of other G18P[17] strains detected in various parts of the world, supporting the possibility that the G18P[17] strains spread via migratory birds that move over a wide area. Furthermore, the RK1 strain induced diarrhoea in suckling mice after oral gastric inoculation, indicating that at least some of the rotaviruses that originated from migratory birds are infectious to and pathogenic in mammals. In conclusion, it was demonstrated that migratory birds may contribute to the global spread of avian rotaviruses that are pathogenic in mammalian species.

Molecular characterisation of a novel avian rotavirus A strain detected from a gull species (Larus sp.).

A recent study demonstrated the possibility that migratory birds are responsible for the global spread of avian rotavirus A (RVA). However, little is known about what types of RVAs are retained in migratory birds. In this study, to obtain information on RVA strains in migratory birds, we characterised an RVA strain, Ho374, that was detected in a faecal sample from a gull species (Larus sp.). Genetic analysis revealed that all 11 genes of this strain were classified as new genotypes (G28-P[39]-I21-R14-C14-M13-A24-N14-T16-E21-H16). This clearly indicates that the genetic diversity of avian RVAs is greater than previously recognised. Our findings highlight the need for investigations of RVA strains retained in migratory birds, including gulls.

Establishment of a reverse genetics system for avian rotavirus A strain PO-13.

Avian rotavirus A (RVA) is one of major enteric pathogens that cause diarrhoea in young avian individuals. Importantly, some of the avian RVA strains of G18P[17] genotype are naturally transmitted to and cause clinical diseases in mammalian species, indicating their potential risks to animal health. Although molecular information on the pathogenesis by avian RVA strains will be useful for estimating their risks, the absence of a reverse genetics (RG) system for these strains has hindered the elucidation of their pathogenic mechanisms. In this study, we aimed to establish an RG system for the avian G18P[17] prototype strain PO-13, which was isolated from a pigeon in Japan in 1983 and was experimentally shown to be pathogenic in suckling mice. Transfection with plasmids expressing 11 genomic RNA segments of the strain resulted in rescue of the infectious virus with an artificially introduced genetic marker on its genome, indicating that an RG system for the PO-13 strain was successfully established. The rescued recombinant strain rPO-13 had biological properties almost identical to those of its wild-type strain (wtPO-13). Notably, both rPO-13 and wtPO-13 induced diarrhoea in suckling mice with similar efficiencies. It was thus demonstrated that the RG system will be useful for elucidating the pathogenic mechanisms of the PO-13 strain at the molecular level. This is the first report of the establishment of an RG system for an avian RVA strain.

Y-shaped RNA secondary structure of a noncoding region in the genomic RNA of tick-borne encephalitis virus affects pathogenicity.

Tick-borne encephalitis virus (TBEV) is a zoonotic virus that causes encephalitis in humans. Various deletions have been reported in a variable region of the 3' untranslated region of the TBEV genome. This study analyzed the role of a Y-shaped secondary structure in the pathogenicity of TBEV by using reverse genetics. Deletion of the structure increased the mortality rate of virus-infected mice but did not affect the virus multiplication in cultured cells and organs. The results indicate that the secondary structure is involved in the regulation of TBEV pathogenesis.

Characterization of tick-borne encephalitis virus isolated from a tick in central Hokkaido in 2017.

Tick-borne encephalitis virus (TBEV) is a zoonotic virus in the genus Flavivirus, family Flaviviridae. TBEV is widely distributed in northern regions of the Eurasian continent, including Japan, and causes severe encephalitis in humans. Tick-borne encephalitis (TBE) was recently reported in central Hokkaido, and wild animals with anti-TBEV antibodies were detected over a wide area of Hokkaido, although TBEV was only isolated in southern Hokkaido. In this study, we conducted a survey of ticks to isolate TBEV in central Hokkaido. One strain, designated Sapporo-17-Io1, was isolated from ticks (Ixodes ovatus) collected in Sapporo city. Sequence analysis revealed that the isolated strain belonged to the Far Eastern subtype of TBEV and was classified in a different subcluster from Oshima 5-10, which had previously been isolated in southern Hokkaido. Sapporo-17-Io1 showed similar growth properties to those of Oshima 5-10 in cultured cells and mouse brains. The mortality rate of mice infected intracerebrally with each virus was similar, but the survival time of mice inoculated with Sapporo-17-Io1 was significantly longer than that of mice inoculated with Oshima 5-10. These results indicate that the neurovirulence of Sapporo-17-Io1 was lower than that of Oshima 5-10. Using an infectious cDNA clone, the replacement of genes encoding non-structural genes from Oshima 5-10 with those from Sapporo-17-Io1 attenuated the neuropathogenicity of the cloned viruses. This result indicated that the non-structural proteins determine the neurovirulence of these two strains. Our results provide important insights for evaluating epidemiological risk in TBE-endemic areas of Hokkaido.

The Lethal(2)-Essential-for-Life [L(2)EFL] Gene Family Modulates Dengue Virus Infection in Aedes aegypti.

Efforts to determine the mosquito genes that affect dengue virus replication have identified a number of candidates that positively or negatively modify amplification in the invertebrate host. We used deep sequencing to compare the differential transcript abundances in Aedes aegypti 14 days post dengue infection to those of uninfected A. aegypti. The gene lethal(2)-essential-for-life [l(2)efl], which encodes a member of the heat shock 20 protein (HSP20) family, was upregulated following dengue virus type 2 (DENV-2) infection in vivo. The transcripts of this gene did not exhibit differential accumulation in mosquitoes exposed to insecticides or pollutants. The induction and overexpression of l(2)efl gene products using poly(I:C) resulted in decreased DENV-2 replication in the cell line. In contrast, the RNAi-mediated suppression of l(2)efl gene products resulted in enhanced DENV-2 replication, but this enhancement occurred only if multiple l(2)efl genes were suppressed. l(2)efl homologs induce the phosphorylation of eukaryotic initiation factor 2α (eIF2α) in the fruit fly Drosophila melanogaster, and we confirmed this finding in the cell line. However, the mechanism by which l(2)efl phosphorylates eIF2α remains unclear. We conclude that l(2)efl encodes a potential anti-dengue protein in the vector mosquito.

The L, M, and S Segments of Rift Valley Fever Virus MP-12 Vaccine Independently Contribute to a Temperature-Sensitive Phenotype.

UNLABELLED: Rift Valley fever (RVF) is endemic to Africa, and the mosquito-borne disease is characterized by "abortion storms" in ruminants and by hemorrhagic fever, encephalitis, and blindness in humans. Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) has a tripartite negative-stranded RNA genome (L, M, and S segments). A live-attenuated vaccine for RVF, the MP-12 vaccine, is conditionally licensed for veterinary use in the United States. MP-12 is fully attenuated by the combination of the partially attenuated L, M, and S segments. Temperature sensitivity (ts) limits viral replication at a restrictive temperature and may be involved with viral attenuation. In this study, we aimed to characterize the ts mutations for MP-12. The MP-12 vaccine showed restricted replication at 38°C and replication shutoff (100-fold or greater reduction in virus titer compared to that at 37°C) at 39°C in Vero and MRC-5 cells. Using rZH501 reassortants with either the MP-12 L, M, or S segment, we found that all three segments encode a temperature-sensitive phenotype. However, the ts phenotype of the S segment was weaker than that of the M or L segment. We identified Gn-Y259H, Gc-R1182G, L-V172A, and L-M1244I as major ts mutations for MP-12. The ts mutations in the L segment decreased viral RNA synthesis, while those in the M segment delayed progeny production from infected cells. We also found that a lack of NSs and/or 78kD/NSm protein expression minimally affected the ts phenotype. Our study revealed that MP-12 is a unique vaccine carrying ts mutations in the L, M, and S segments. IMPORTANCE: Rift Valley fever (RVF) is a mosquito-borne viral disease endemic to Africa, characterized by high rates of abortion in ruminants and severe diseases in humans. Vaccination is important to prevent the spread of disease, and a live-attenuated MP-12 vaccine is currently the only vaccine with a conditional license in the United States. This study determined the temperature sensitivity (ts) of MP-12 vaccine to understand virologic characteristics. Our study revealed that MP-12 vaccine contains ts mutations independently in the L, M, and S segments and that MP-12 displays a restrictive replication at 38°C.

Identification of novel anelloviruses with broad diversity in UK rodents.

Anelloviruses are a family of small circular ssDNA viruses with a vast genetic diversity. Human infections with the prototype anellovirus, torque teno virus (TTV), are ubiquitous and related viruses have been described in a number of other mammalian hosts. Despite over 15 years of investigation, there is still little known about the pathogenesis and possible disease associations of anellovirus infections, arising in part due to the lack of a robust cell culture system for viral replication or tractable small-animal model. We report the identification of diverse anelloviruses in several species of wild rodents. The viruses are highly prevalent in wood mice (Apodemus sylvaticus) and field voles (Microtus agrestis), detectable at a low frequency in bank voles (Myodes glareolus), but absent from house mice (Mus musculus). The viruses identified have a genomic organization consistent with other anelloviruses, but form two clear phylogenetic groups that are as distinct from each other as from defined genera.

Molecular characterization of an avian rotavirus a strain detected from a large-billed crow (Corvus macrorhynchos) in Japan.

Avian rotaviruses A (RVAs) are occasionally transmitted to animals other than the original hosts across species barriers. Information on RVAs carried by various bird species is important for identifying the origin of such interspecies transmission. In this study, to facilitate an understanding of the ecology of RVAs from wild birds, we characterized all of the genes of an RVA strain, JC-105, that was detected in a fecal sample of a large-billed crow (Corvus macrorhynchos) in Japan. All of the genes of this strain except for the VP4 and VP7 genes, which were classified as novel genotypes (P[56] and G40, respectively), were closely related to those of the avian-like RVA strain detected from a raccoon, indicating the possibility that crows had been involved in the transmission of avian RVAs to raccoons. Our findings highlight the need for further viral investigations in wild birds and mammals to understand the mechanisms of avian-to-mammal RVA transmission.

Generation and characterization of a genetically modified live rabies vaccine strain with attenuating mutations in multiple viral proteins and evaluation of its potency in dogs.

Live rabies vaccines have advantageous features that can facilitate mass vaccination for dogs, the most important reservoirs/transmitters of rabies. However, some live vaccine strains have problems in their safety, namely, risks from the residual pathogenicity and the pathogenic reversion of live vaccine strains. The reverse genetics system of rabies virus provides a feasible option to improve the safety of a live vaccine strain by, for example, artificially introducing attenuating mutations into multiple viral proteins. It was previously demonstrated in separate studies that introduction of amino acid residues Leu at position 333 in the viral glycoprotein (G333), Ser at G194, and Leu/His at positions 273/394 in the nucleoprotein (N273/394) enhance the safety of a live vaccine strain. In this study, to test our hypothesis that combinational introduction of these residues would significantly increase the safety level of a vaccine strain, we generated a novel live vaccine candidate, ERA-NG2, that is attenuated by mutations at N273/394 and G194/333, and we examined its safety and immunogenicity in mice and dogs. ERA-NG2 did not cause any clinical signs in mice after intracerebral inoculation. After 10 passages in suckling mouse brains, ERA-NG2 retained all of the introduced mutations except the mutation at N394 and the highly attenuated phenotype. These findings indicate that the ERA-NG2 is highly and stably attenuated. After confirming that ERA-NG2 induced a virus-neutralizing antibody (VNA) response and protective immunity in mice, we immunized dogs intramuscularly with a single dose (105-7 focus-forming units) of ERA-NG2 and found that, at all of the tested doses, the strain induced a VNA response in dogs without inducing any clinical signs. These findings demonstrate that ERA-NG2 has a high level of safety and a substantial level of immunogenicity in dogs and thus is a promising live vaccine candidate that can facilitate vaccination in dogs.

Establishment of a reverse genetics system for rabies virus strain Komatsugawa.

The rabies virus strain Komatsugawa isolated from a dog in Tokyo in the 1940s retains biological properties as a field strain, providing an effective model for studying rabies pathogenesis. To facilitate molecular studies on the pathogenesis, this study aimed to establish a reverse genetics system for the Komatsugawa strain. By transfecting the full-length genome plasmid of this strain, infectious virus with artificially introduced genetic markers in its genome was rescued. The recombinant strain had biological properties similar to those of the original strain. These findings indicate that a reverse genetics system for the Komatsugawa strain has successfully been established.

Congenital cutaneous fibropapillomatosis with no evidence of papillomavirus infection in a piglet.

Multiple yellowish-white, cauliflower-like mass lesions on the skin of the head and back in a 4-month-old piglet were pathologically examined. These lesions had developed before the weaning period. Histologically, the cutaneous neoplasms were characterized by papillary outgrowth of connective tissue covered by thick epidermis. Hyperplasia of the epidermis was corresponded with proliferation of capillaries, lympho-plasmacytic infiltration, and proliferation of fibroblasts in the dermal stroma. There were no inclusion bodies and significant degeneration in the keratinocytes. Papillomavirus antigen and DNA were not detected in these lesions by immunohistochemistry and polymerase chain reaction, respectively. Accordingly, the fibropapillomatosis of the present case might be hamartomatous rather than infectious.

Safety enhancement of a genetically modified live rabies vaccine strain by introducing an attenuating Leu residue at position 333 in the glycoprotein.

To improve the safety of genetically modified live rabies vaccine strains, most studies have utilized an attenuating Arg-to-Glu mutation at position 333 in the glycoprotein (G333), which is responsible for attenuation of the live vaccine strain SAG2. The Glu residue requires two nucleotide substitutions to revert to pathogenic Arg, thus significantly lowering the probability of pathogenic reversion caused by the Glu-to-Arg mutation at G333. However, only one nucleotide substitution is sufficient to convert the Glu residue to another pathogenic residue, Lys, and thereby to cause pathogenic reversion. This indicates a potential safety problem of SAG2 and the live vaccine candidates attenuated by Glu at G333. In this study, aiming to solve this problem, we examined the utility of a Leu residue, which requires two nucleotide substitutions to be both Arg and Lys, as an attenuating mutation at G333. Using a reverse genetics system of the live vaccine strain ERA, we generated ERA-G333Leu by introducing an Arg-to-Leu mutation at G333. Similar to ERA-G333Glu, which is attenuated by an Arg-to-Glu mutation at G333, ERA-G333Leu did not cause obvious clinical signs in 6-week-old mice after intracerebral inoculation. Importantly, after 10 passages in suckling mouse brains, ERA-G333Glu acquired a pathogenic Lys or Arg at G333 and a high level of lethality in mice, whereas ERA-G333Leu retained the attenuating Leu at G333 and only showed a modest level of virulence probably caused by a mutation at G194. In addition, ERA-G333Leu and ERA-G333Glu induced neutralizing antibody response and protective immunity in mice with similar efficiencies. The results demonstrate that, compared to ERA-G333Glu, ERA-G333Leu is more stably attenuated, also indicating the high utility of a Leu residue as an attenuating mutation at G333 in the development of live rabies vaccine strains with a high level of safety.

Long-term shedding of hepatitis E virus in the feces of pigs infected naturally, born to sows with and without maternal antibodies.

Pigs are presumed reservoirs for hepatitis E virus (HEV) transmission to humans. To examine infection kinetics, two litters of domestic pigs (A and B, each containing 10 piglets) infected naturally with HEV were studied until pigs were 6 months old. Maternal IgG and IgA antibodies were detected in litter A piglets, but not in litter B ones. All pigs shed HEV in feces when they were 30-110 days old, and 17 developed viremia at 40-100 days of age. Phylogenetic analysis revealed a highly close sequence of HEV genotype 3 in all pigs. The serum levels of specific IgG and IgA were similar in all pigs, although IgA was not detected in the feces. Interestingly, the onset of both viremia and seroconversion was delayed significantly in litter A pigs. The kinetics of fecal virus shedding was similar in both litters; shedding was not detected after the pigs were 120 days old. The differences in the infection kinetics between litters A and B suggested that maternal antibodies delayed the onset of viremia and seroconversion. Quantitative real-time reverse transcriptase-polymerase chain reaction revealed that HEV RNA in feces peaked 10 days after initial shedding of approximately 10(6.0) copies/g. The viral load was much lower in the serum than in the feces. At 200 days of age, HEV RNA was found in the internal organs of 3 out of 13 pigs. These study findings improve the understanding of the dynamics of natural HEV transmission in pigs, which could help in controlling virus transmission from pigs to humans.

Genetic and Phenotypic Characterization of a Rabies Virus Strain Isolated from a Dog in Tokyo, Japan in the 1940s.

The rabies virus strain Komatsugawa (Koma), which was isolated from a dog in Tokyo in the 1940s before eradication of rabies in Japan in 1957, is known as the only existent Japanese field strain (street strain). Although this strain potentially provides a useful model to study rabies pathogenesis, little is known about its genetic and phenotypic properties. Notably, this strain underwent serial passages in rodents after isolation, indicating the possibility that it may have lost biological characteristics as a street strain. In this study, to evaluate the utility of the Koma strain for studying rabies pathogenesis, we examined the genetic properties and in vitro and in vivo phenotypes. Genome-wide genetic analyses showed that, consistent with previous findings from partial sequence analyses, the Koma strain is closely related to a Russian street strain within the Arctic-related phylogenetic clade. Phenotypic examinations in vitro revealed that the Koma strain and the representative street strains are less neurotropic than the laboratory strains. Examination by using a mouse model demonstrated that the Koma strain and the street strains are more neuroinvasive than the laboratory strains. These findings indicate that the Koma strain retains phenotypes similar to those of street strains, and is therefore useful for studying rabies pathogenesis.

Cutaneous clear cell adnexal carcinoma in a dog: special reference to cytokeratin expression.

A 5-year-old, male Bichon-Frise dog presented with a cutaneous mass in the basal region of the auricle. Histologically, the cutaneous neoplasm was comprised of lobules with solid cellular proliferation separated by thin fibrous septa. Neoplastic cells varied in size, with moderate to abundant amounts of PAS-positive cytoplasm, large nuclei and prominent nucleoli. Immunohistochemical examinations showed that tumor cells were positive for pan-cytokeratin (CK) (AE1/AE3 and CAM5.2), CK8 and CK18, but negative for pan-CK (KL1), CK7, CK14, CK16 and CK20. Double-labeled immunofluorescence testing indicated that neoplastic cells frequently co-expressed CK and vimentin, suggesting divergent differentiation of tumor cells. Based on these findings, the tumor was diagnosed as canine clear cell adnexal carcinoma.

Risk analysis of inter-species reassortment through a Rift Valley fever phlebovirus MP-12 vaccine strain.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and the Arabian Peninsula. The causative agent, Rift Valley fever phlebovirus (RVFV), belongs to the genus Phlebovirus in the family Phenuiviridae and causes high rates of abortions in ruminants, and hemorrhagic fever, encephalitis, or blindness in humans. Viral maintenance by mosquito vectors has led to sporadic RVF outbreaks in ruminants and humans in endemic countries, and effective vaccination of animals and humans may minimize the impact of this disease. A live-attenuated MP-12 vaccine strain is one of the best characterized RVFV strains, and was conditionally approved as a veterinary vaccine in the U.S. Live-attenuated RVF vaccines including MP-12 strain may form reassortant strains with other bunyavirus species. This study thus aimed to characterize the occurrence of genetic reassortment between the MP-12 strain and bunyavirus species closely related to RVFV. The Arumowot virus (AMTV) and Gouleako goukovirus (GOLV), are transmitted by mosquitoes in Africa. The results of this study showed that GOLV does not form detectable reassortant strains with the MP-12 strain in co-infected C6/36 cells. The AMTV also did not form any reassortant strains with MP-12 strain in co-infected C6/36 cells, due to the incompatibility among N, L, and Gn/Gc proteins. A lack of reassortant formation could be due to a functional incompatibility of N and L proteins derived from heterologous species, and due to a lack of packaging via heterologous Gn/Gc proteins. The MP-12 strain did, however, randomly exchange L-, M-, and S-segments with a genetic variant strain, rMP12-GM50, in culture cells. The MP-12 strain is thus unlikely to form any reassortant strains with AMTV or GOLV in nature.

Attenuation and protective efficacy of Rift Valley fever phlebovirus rMP12-GM50 strain.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and the Arabian Peninsula that affects sheep, cattle, goats, camels, and humans. Effective vaccination of susceptible ruminants is important for the prevention of RVF outbreaks. Live-attenuated RVF vaccines are in general highly immunogenic in ruminants, whereas residual virulence might be a concern for vulnerable populations. It is also important for live-attenuated strains to encode unique genetic markers for the differentiation from wild-type RVFV strains. In this study, we aimed to strengthen the attenuation profile of the MP-12 vaccine strain via the introduction of 584 silent mutations. To minimize the impact on protective efficacy, codon usage and codon pair bias were not de-optimized. The resulting rMP12-GM50 strain showed 100% protective efficacy with a single intramuscular dose, raising a 1:853 mean titer of plaque reduction neutralization test. Moreover, outbred mice infected with one of three pathogenic reassortant ZH501 strains, which encoded rMP12-GM50 L-, M-, or S-segments, showed 90%, 50%, or 30% survival, respectively. These results indicate that attenuation of the rMP12-GM50 strain is significantly attenuated via the L-, M-, and S-segments. Recombinant RVFV vaccine strains encoding similar silent mutations will be also useful for the surveillance of reassortant strains derived from vaccine strains in endemic countries.

Distinct virulence of Rift Valley fever phlebovirus strains from different genetic lineages in a mouse model.

Rift Valley fever phlebovirus (RVFV) causes high rates of abortions and fetal malformations in ruminants, and hemorrhagic fever, encephalitis, or blindness in humans. Viral transmission occurs via mosquito vectors in endemic areas, which necessitates regular vaccination of susceptible livestock animals to prevent the RVF outbreaks. Although ZH501 strain has been used as a challenge strain for past vaccine efficacy studies, further characterization of other RVFV strains is important to optimize ruminant and nonhuman primate RVFV challenge models. This study aimed to characterize the virulence of wild-type RVFV strains belonging to different genetic lineages in outbred CD1 mice. Mice were intraperitoneally infected with 1x103 PFU of wild-type ZH501, Kenya 9800523, Kenya 90058, Saudi Arabia 200010911, OS1, OS7, SA75, Entebbe, or SA51 strains. Among them, mice infected with SA51, Entebbe, or OS7 strain showed rapid dissemination of virus in livers and peracute necrotic hepatitis at 2-3 dpi. Recombinant SA51 (rSA51) and Zinga (rZinga) strains were recovered by reverse genetics, and their virulence was also tested in CD1 mice. The rSA51 strain reproduced peracute RVF disease in mice, whereas the rZinga strain showed a similar virulence with that of rZH501 strain. This study showed that RVFV strains in different genetic lineages display distinct virulence in outbred mice. Importantly, since wild-type RVFV strains contain defective-interfering RNA or various genetic subpopulations during passage from original viral isolations, recombinant RVFV strains generated by reverse genetics will be better suitable for reproducible challenge studies for vaccine development as well as pathological studies.