dEbola virus (EBOV) infections are characterized by deficient T lymphocyte responses, T lymphocyte apoptosis, and lymphopenia in the absence of direct infection of T lymphocytes. In contrast, dendritic cells (DC) are infected but fail to mature appropriately, thereby impairing the T cell response. We investigated the contributions of EBOV proteins in modulating DC maturation by generating recombinant viruses expressing enhanced green fluorescent protein and carrying mutations affecting several potentially immunomodulating domains. They included envelope glycoprotein (GP) domains, as well as innate response antagonist domains (IRADs) previously identified in the VP24 and VP35 proteins. GP expressed by an unrelated vector, but not the wild-type EBOV, was found to strongly induce DC maturation, and infections with recombinant EBOV carrying mutations disabling GP functional domains did not restore DC maturation. In contrast, each of the viruses carrying mutations disabling any IRAD in VP35 induced a dramatic upregulation of DC maturation markers. This was dependent on infection, but not interaction with GP. Disabling of IRADs also resulted in up to a several hundredfold increase in secretion of cytokines and chemokines. Furthermore, these mutations induced formation of homotypic DC clusters, which represent close correlates of their maturation and presumably facilitate transfer of antigen from migratory DC to lymph node DC. Thus, an individual IRAD is insufficient to suppress DC maturation; rather, the suppression of DC maturation and the "immune paralysis" observed during EBOV infections results from a cooperative effect of two or more individual IRADs.

Background:The Ebola VP30 is required for viral transcription and can exist in phosphorylated (inactive) and dephosphorylated (active) forms. Results: VP30 is dephosphorylated by PP1; the small PP1-targeting molecule 1E7-03 inhibits VP30 dephosphorylation and viral transcription, thereby blocking viral replication. Conclusion: PP1 plays an important role in Ebola virus transcription. Significance: Targeting PP1 is a feasible approach for inhibition of Ebola virus.

Ebola virus (EBOV) protein VP35 inhibits production of interferon alpha/beta (IFN) by blocking RIG-I-like receptor signaling pathways, thereby promoting virus replication and pathogenesis. A high-throughput screening assay, developed to identify compounds that either inhibit or bypass VP35 IFN-antagonist function, identified five DNA intercalators as reproducible hits from a library of bioactive compounds. Four, including doxorubicin and daunorubicin, are anthracycline antibiotics that inhibit topoisomerase II and are used clinically as chemotherapeutic drugs. These compounds were demonstrated to induce IFN responses in an ATM kinase-dependent manner and to also trigger the DNA-sensing cGAS-STING pathway of IFN induction. These compounds also suppress EBOV replication in vitro and induce IFN in the presence of IFN-antagonist proteins from multiple negative-sense RNA viruses. These findings provide new insights into signaling pathways activated by important chemotherapy drugs and identify a novel therapeutic approach for IFN induction that may be exploited to inhibit RNA virus replication.

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.

BackgroundJunín 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 FindingsTo 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/SignificanceThe 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.