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Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus with a wide host range including ruminants and humans. RVFV outbreaks have had devastating effects on public health and the livestock industry in African countries. However, there is no approved RVFV vaccine for human use in non-endemic countries and no FDA-approved antiviral drug for RVFV treatment. The RVFV 78kDa protein (P78), which is a membrane glycoprotein, plays a role in virus dissemination in the mosquito host, but its biological role in mammalian hosts remains unknown. We generated an attenuated RVFV MP-12 strain-derived P78-High virus and a virulent ZH501 strain-derived ZH501-P78-High virus, both of which expressed a higher level of P78 and carried higher levels of P78 in the virion compared to their parental viruses. We also generated another MP-12-derived mutant virus (P78-KO virus) that does not express P78. MP-12 and P78-KO virus replicated to similar levels in fibroblast cell lines and Huh7 cells, while P78-High virus replicated better than MP-12 in Vero E6 cells, fibroblast cell lines, and Huh7 cells. Notably, P78-High virus and P78-KO virus replicated less efficiently and more efficiently, respectively, than MP-12 in macrophage cell lines. ZH501-P78-High virus also replicated poorly in macrophage cell lines. Our data further suggest that inefficient binding of P78-High virus to the cells led to inefficient virus internalization, low virus infectivity and reduced virus replication in a macrophage cell line. P78-High virus and P78-KO virus showed lower and higher virulence than MP-12, respectively, in young mice. ZH501-P78-High virus also exhibited lower virulence than ZH501 in mice. These data suggest that high levels of P78 expression attenuate RVFV virulence by preventing efficient virus replication in macrophages. Genetic alteration leading to increased P78 expression may serve as a novel strategy for the attenuation of RVFV virulence and generation of safe RVFV vaccines., Author summary Rift Valley fever virus (RVFV) is the causative agent of Rift Valley fever, which is primarily endemic in African countries. The virus infects a wide variety of mammalian species, including ruminants and humans, and causes devastating effects on public health and the livestock industry. Because RVFV is transmitted by several mosquito species that are ubiquitous in non-endemic areas, there is a potential risk that the virus will spread outside of the current endemic areas. However, there are no effective therapeutics nor commercially available vaccines for human use. RVFV encodes a 78kDa protein (P78) of unidentified biological function in mammalian cells. We found that a mutant RVFV expressing a higher level of P78 (P78-High virus) showed lower infectivity and less efficient replication than parental RVFV in macrophage cell lines. P78-High virus and a mutant RVFV lacking P78 expression showed lower and higher virulence than the parental virus, respectively, in mice, suggesting that high levels of P78 expression prevent efficient virus replication in macrophages, leading to attenuation of virus virulence. Genetic alteration leading to increased P78 expression may serve as a novel strategy for the generation of attenuated RVFV as vaccine candidates.

Global Zika virus (ZIKV) outbreaks and their strong link to microcephaly have raised major public health concerns. ZIKV has been reported to affect the innate immune responses in neural stem/progenitor cells (NS/PCs). However, it is unclear how these immune factors affect neurogenesis. In this study, we used Asian-American lineage ZIKV strain PRVABC59 to infect primary human NS/PCs originally derived from fetal brains. We found that ZIKV overactivated key molecules in the innate immune pathways to impair neurogenesis in a cell stage-dependent manner. Inhibiting the overactivated innate immune responses ameliorated ZIKV-induced neurogenesis reduction. This study thus suggests that orchestrating the host innate immune responses in NS/PCs after ZIKV infection could be promising therapeutic approach to attenuate ZIKV-associated neuropathology., Author summary ZIKV has been reported to affect the innate immune responses in neural stem cells. We found that inhibiting the overactivated innate immune responses ameliorated Zika virus-induced neurogenesis reduction in human neural stem cells, which are the origin of the brain. This study suggests that coordinating the host innate immune responses in neural stem cells after ZIKV infection could be promising therapeutic approach to attenuate ZIKV-associated neuropathology.

Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever, neurological disorders or blindness in humans, and a high rate of abortion in ruminants. MP-12 strain, a live-attenuated candidate vaccine, is attenuated in the M- and L-segments, but the S-segment retains the virulent phenotype. MP-12 was manufactured as an Investigational New Drug vaccine by using MRC-5 cells and encodes a functional NSs gene, the major virulence factor of RVFV which 1) induces a shutoff of the host transcription, 2) inhibits interferon (IFN)-β promoter activation, and 3) promotes the degradation of dsRNA-dependent protein kinase (PKR). MP-12 lacks a marker for differentiation of infected from vaccinated animals (DIVA). Although MP-12 lacking NSs works for DIVA, it does not replicate efficiently in type-I IFN-competent MRC-5 cells, while the use of type-I IFN-incompetent cells may negatively affect its genetic stability. To generate modified MP-12 vaccine candidates encoding a DIVA marker, while still replicating efficiently in MRC-5 cells, we generated recombinant MP-12 encoding Punta Toro virus Adames strain NSs (rMP12-PTNSs) or Sandfly fever Sicilian virus NSs (rMP12-SFSNSs) in place of MP-12 NSs. We have demonstrated that those recombinant MP-12 viruses inhibit IFN-β mRNA synthesis, yet do not promote the degradation of PKR. The rMP12-PTNSs, but not rMP12-SFSNSs, replicated more efficiently than recombinant MP-12 lacking NSs in MRC-5 cells. Mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs induced neutralizing antibodies at a level equivalent to those vaccinated with MP-12, and were efficiently protected from wild-type RVFV challenge. The rMP12-PTNSs and rMP12-SFSNSs did not induce antibodies cross-reactive to anti-RVFV NSs antibody and are therefore applicable to DIVA. Thus, rMP12-PTNSs is highly efficacious, replicates efficiently in MRC-5 cells, and encodes a DIVA marker, all of which are important for vaccine development for Rift Valley fever., Author Summary Upon outbreak of zoonotic viral diseases in herds of animals, early detection of naturally infected animals and prevention of further viral spread are important for minimizing the impact of outbreak in the society. Vaccination may compromise the identification of infected animals since both natural infection and vaccination induce antibodies specific to the pathogen. Therefore, new generation vaccines should have a marker to differentiate infected from vaccinated animals (DIVA). Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever, neurological disorders or blindness in humans and a high-rate abortion in ruminants. MP-12 strain, a live-attenuated candidate vaccine, is safe and immunogenic, but lacks a DIVA marker. In this study, we developed and characterized improved MP-12 viruses which encode a DIVA marker by replacing the virulence gene with that of serologically distinct viruses belonging to the same genera. The novel MP-12 variant with such DIVA marker was highly efficacious and replicated efficiently in human diploid cells for vaccine production, and will become alternative candidate vaccines of MP-12 for veterinary applications.

Rift Valley fever virus (RVFV) is a major human and animal pathogen associated with severe disease including hemorrhagic fever or encephalitis. RVFV is endemic to parts of Africa and the Arabian Peninsula, but there is significant concern regarding its introduction into non-endemic regions and the potentially devastating effect to livestock populations with concurrent infections of humans. To date, there is little detailed data directly comparing the host response to infection with wild-type or vaccine strains of RVFV and correlation with viral pathogenesis. Here we characterized clinical and systemic immune responses to infection with wild-type strain ZH501 or IND vaccine strain MP-12 in the C57BL/6 mouse. Animals infected with live-attenuated MP-12 survived productive viral infection with little evidence of clinical disease and minimal cytokine response in evaluated tissues. In contrast, ZH501 infection was lethal, caused depletion of lymphocytes and platelets and elicited a strong, systemic cytokine response which correlated with high virus titers and significant tissue pathology. Lymphopenia and platelet depletion were indicators of disease onset with indications of lymphocyte recovery correlating with increases in G-CSF production. RVFV is hepatotropic and in these studies significant clinical and histological data supported these findings; however, significant evidence of a pro-inflammatory response in the liver was not apparent. Rather, viral infection resulted in a chemokine response indicating infiltration of immunoreactive cells, such as neutrophils, which was supported by histological data. In brains of ZH501 infected mice, a significant chemokine and pro-inflammatory cytokine response was evident, but with little pathology indicating meningoencephalitis. These data suggest that RVFV pathogenesis in mice is associated with a loss of liver function due to liver necrosis and hepatitis yet the long-term course of disease for those that might survive the initial hepatitis is neurologic in nature which is supported by observations of human disease and the BALB/c mouse model., Author Summary Rift Valley fever virus (RVFV) is a deadly virus that is found primarily in sub-Saharan Africa. However, mosquitoes that are capable of transmitting RVFV have a worldwide distribution. RVF affects a broad range of animal species and is known to cause encephalitis or hemorrhagic fever in humans with survivors having long-term health effects such as a loss of vision. There is no vaccine currently approved for use in humans. In this study we evaluated the host response to infection with either a RVFV vaccine strain or a wild-type strain to identify similarities or differences that could be exploited for development of therapeutics or improved vaccines. While mice infected with the vaccine strain did not develop disease and survived, infection with the wild-type strain caused severe disease with a fatal outcome. Analyzing multiple clinical factors and the host immune response over the course of infection allowed us to identify potential host factors associated with disease progression during wild-type virus infection. This work also provides support for a current vaccine candidate, MP-12, in demonstrating a limited and protective host response to infection.

Background and purpose The ability to track changes in gene expression following viral infection is paramount to understanding viral pathogenesis. This study was undertaken to evaluate the nCounter, a high throughput digital gene expression system, as a means to better understand West Nile virus (WNV) dissemination and the inflammatory response against WNV in the outbred Swiss Webster (SW) mouse model over the course of infection. Methodology The nCounter Mouse Inflammation gene expression kit containing 179 inflammation related genes was used to analyze gene expression changes in multiple tissues over a nine day course of infection in SW mice following intraperitoneal injection with WNV. Protein expression levels for a subset of these cytokine/chemokine genes were determined using a multiplex protein detection system (BioPlex) and comparisons of protein/RNA expression levels made. Results Expression analysis of spleen, lung, liver, kidney and brain of SW mice infected with WNV revealed that Cxcl10 and Il12b are differentially expressed in all tissues tested except kidney. Data stratification of positively confirmed infected (WNV (+)) versus non-infected (WNV (−) tissues allowed differentiation of the systemic inflammatory gene response from tissue-specific responses arising from WNV infection. Significant (p, Author Summary The nCounter, a high throughput digital molecular detection and quantitation system, was used to define the inflammatory response of SW mice to WNV infection following intraperitoneal injection in multiple tissues over a 9 day course of viral infection. Data analysis identified expression profiles common and unique to major tissues. Complementary protein suspension array analysis of a subset of genes demonstrated the expression kinetics of RNA and protein expression compared to infectious virus production. Data stratification of WNV (+) versus WNV (−) tissues allowed differentiation of the systemic inflammatory gene response versus tissue-specific responses due to infection with WNV over time. The results generated from this study will allow for further hypothesis generation and testing.

Background Junín virus (JUNV), the etiologic agent of Argentine hemorrhagic fever (AHF), is classified by the NIAID and CDC as a Category A priority pathogen. Presently, antiviral therapy for AHF is limited to immune plasma, which is readily available only in the endemic regions of Argentina. T-705 (favipiravir) is a broadly active small molecule RNA-dependent RNA polymerase inhibitor presently in clinical evaluation for the treatment of influenza. We have previously reported on the in vitro activity of favipiravir against several strains of JUNV and other pathogenic New World arenaviruses. Methodology/Principal Findings To evaluate the efficacy of favipiravir in vivo, guinea pigs were challenged with the pathogenic Romero strain of JUNV, and then treated twice daily for two weeks with oral or intraperitoneal (i.p.) favipiravir (300 mg/kg/day) starting 1–2 days post-infection. Although only 20% of animals treated orally with favipiravir survived the lethal challenge dose, those that succumbed survived considerably longer than guinea pigs treated with placebo. Consistent with pharmacokinetic analysis that showed greater plasma levels of favipiravir in animals dosed by i.p. injection, i.p. treatment resulted in a substantially higher level of protection (78% survival). Survival in guinea pigs treated with ribavirin was in the range of 33–40%. Favipiravir treatment resulted in undetectable levels of serum and tissue viral titers and prevented the prominent thrombocytopenia and leucopenia observed in placebo-treated animals during the acute phase of infection. Conclusions/Significance The remarkable protection afforded by i.p. favipiravir intervention beginning 2 days after challenge is the highest ever reported for a small molecule antiviral in the difficult to treat guinea pig JUNV challenge model. These findings support the continued development of favipiravir as a promising antiviral against JUNV and other related arenaviruses., Author Summary Argentine hemorrhagic fever (AHF) is a severe and often-fatal disease caused by infection with Junín virus (JUNV). Presently, there is an unmet need to develop new therapeutics to address current medical, public health and national security concerns, as JUNV is considered a potential bioterror agent amenable to aerosolization and intentional release. In the present study, favirpiravir, a promising anti-JUNV drug in clinical development for the treatment of influenza, was evaluated in an experimental small animal model of AHF. Guinea pigs challenged with JUNV were treated with favipiravir twice daily for two weeks starting 1–2 days after infection. Consistent with pharmacokinetic analysis that showed greater plasma levels of favipiravir in animals dosed by intraperitoneal injection, administration by this route resulted in a dramatic protective effect as 78% animals survived the infection compared to 11% in the placebo-treated group. Favipiravir treatment inhibited JUNV replication and prevented the development of disease observed in animals receiving placebo during the acute stage of infection. The high level efficacy observed following post-exposure prophylaxis with favipiravir is the highest ever reported for a small molecule antiviral in the guinea pig JUNV challenge model and thus supports its continued development as a promising antiviral therapy for the treatment of AHF.