Mammarenaviruses include several known human pathogens, such as the prototypic lymphocytic choriomeningitis virus (LCMV) that can cause neurological diseases and Lassa virus (LASV) that causes endemic hemorrhagic fever infection. LASV-infected patients show diverse clinical manifestations ranging from asymptomatic infection to hemorrhage, multi-organ failures and death, the mechanisms of which have not been well characterized. We have previously shown that the matrix protein Z of pathogenic arenaviruses, including LASV and LCMV, can strongly inhibit the ability of the innate immune protein RIG-I to suppress type I interferon (IFN-I) expression, which serves as a mechanism of viral immune evasion and virulence. Here, we show that Z proteins of diverse LASV isolates derived from rodents and humans have a high degree of sequence variations at their N- and C-terminal regions and produce variable degrees of inhibition of human RIG-I (hRIG-I) function in an established IFN-β promoter-driven luciferase (LUC) reporter assay. Additionally, we show that Z proteins of four known LCMV strains can also inhibit hRIG-I at variable degrees of efficiency. Collectively, our results confirm that Z proteins of pathogenic LASV and LCMV can inhibit hRIG-I and suggest that strain variations of the Z proteins can influence their efficiency to suppress host innate immunity that might contribute to viral virulence and disease heterogeneity.

Vaccination may be an effective way to reduce turkey arthritis reovirus (TARV)-induced lameness in turkey flocks. However, there are currently no commercial vaccines available against TARV infection. Here, we describe the use of reverse genetics technology to generate a recombinant Pichinde virus (PICV) that expresses the Sigma C and/or Sigma B proteins of TARV as antigens. Nine recombinant PICV-based TARV vaccines were developed carrying the wild-type S1 (Sigma C) and/or S3 (Sigma B) genes from three different TARV strains. In addition, three recombinant PICV-based TARV vaccines were produced carrying codon-optimized S1 and/or S3 genes of a TARV strain. The S1 and S3 genes and antigens were found to be expressed in virus-infected cells via reverse transcriptase polymerase chain reaction (RT-PCR) and the direct fluorescent antibody (DFA) technique, respectively. Turkey poults inoculated with the recombinant PICV-based TARV vaccine expressing the bivalent TARV S1 and S3 antigens developed high anti-TARV antibody titers, indicating the immunogenicity (and safety) of this vaccine. Future in vivo challenge studies using a turkey reovirus infection model will determine the optimum dose and protective efficacy of this recombinant virus-vectored candidate vaccine.

Influenza A virus of swine (IAV-S) is an economically important swine pathogen. The IAV-S hemagglutinin (HA) surface protein is the main target for vaccine development. In this study, we evaluated the feasibility of using the recombinant tri-segmented Pichinde virus (rPICV) as a viral vector to deliver HA antigen to protect pigs against IAV-S challenge. Four groups of weaned pigs (T01–T04) were included in the study. T01 was injected with PBS to serve as a non-vaccinated control. T02 was inoculated with rPICV expressing green fluorescence protein (rPICV-GFP). T03 was vaccinated with rPICV expressing the HA antigen of the IAV-S H3N2 strain (rPICV-H3). T04 was vaccinated with the recombinant HA protein antigen of the same H3N2 strain. Pigs were vaccinated twice at day 0 and day 21 and challenged at day 43 by intra-tracheal inoculation with the homologous H3N2 IAV-S strain. After vaccination, all pigs in T03 and T04 groups were seroconverted and exhibited high titers of plasma neutralizing antibodies. After challenge, high levels of IAV-S RNA were detected in the nasal swabs and bronchioalveolar lavage fluid of pigs in T01 and T02 but not in the T03 and T04 groups. Similarly, lung lesions were observed in T01 and T02, but not in the T03 and T04 groups. No significant difference in terms of protection was observed between the T03 and T04 group. Collectively, our results demonstrate that the rPICV-H3 vectored vaccine elicited protective immunity against IAV-S challenge. This study shows that rPICV is a promising viral vector for the development of vaccines against IAV-S.

Current antiviral treatments often target specific viral components, which can lead to the rapid emergence of drug-resistant mutants. Targeting host signaling pathways, including their associated cellular factors, that are important for virus replication is a novel approach toward the development of next-generation antivirals to overcome drug resistance. Various cellular receptor tyrosine kinases (RTKs) have previously been shown to play important roles in mediating viral replication including coronaviruses. In this study, we examined the roles of RTKs in SARS-CoV-2 replication in two cell lines, A549-ACE2 (human lung epithelial cells) and Vero-E6 (African Green Monkey kidney cell), via chemical inhibitors. We showed that the HER2 inhibitor Lapatinib significantly reduced viral replication in both cell lines, the TrkA inhibitor GW441756 was effective only in A549-ACE2 cells, while the EGFR inhibitor Gefitinib had little effect in either cell line. Lapatinib and GW441756 exhibited a high therapeutic index (CC₅₀/EC₅₀ > 10) in A549-ACE2 cells. Time-of-addition experiments indicated that Lapatinib may inhibit the early entry step, whereas GW441756 can affect post-entry steps of the viral life cycle. These findings suggest the important roles of HER2 and TrkA signaling in SARS-CoV-2 infection in human lung epithelial cells and support further investigation of RTK inhibitors as potential COVID-19 treatments.

Several arenaviruses, such as the Old World (OW) Lassa virus (LASV) and the New World (NW) Junin virus (JUNV), can cause severe and lethal viral hemorrhagic fevers in humans. Currently, no vaccines or specific antiviral therapies are FDA-approved for treating arenavirus infections. One major challenge for the development of new therapeutic candidates against these highly pathogenic viruses is that they are BSL-3/4 pathogens that need to be handled in high biocontainment laboratories. In this work, a recombinant non-pathogenic New World arenavirus, Pichinde virus (rPICV), was used for the development of a high-throughput screening (HTS) assay in the BSL-2 laboratory for the screening and identification of small molecule inhibitors against arenaviruses. The rPICV is a replication-competent virus expressing the firefly luciferase reporter gene in the infected cells proportionally to the infection rate. rPICV infection was optimized for an automated HTS in 384-well format with robust Z' scores, high signal-to-background ratios, and low intrinsic variance. Screening an established library allowed for the identification of five top hit compounds, which included ribavirin, a known inhibitor of arenaviral RNA synthesis, showing good potency and selectivity in inhibiting rPICV replication. The antiviral activity of the top hit compounds was further validated against another recombinant arenavirus, the OW lymphocytic choriomeningitis virus (rLCMV) and against laboratory strains of LASV (Josiah) and JUNV (Romero). The use of rPICV in the HTS-based antiviral assay under BSL-2 condition has proven to be safe and suitable for the identification of broad-spectrum small molecule inhibitors against highly pathogenic arenaviruses.

Pichinde virus (PICV) can infect several animal species and has been developed as a safe and effective vaccine vector. Our previous study showed that pigs vaccinated with a recombinant PICV-vectored vaccine expressing the hemagglutinin (HA) gene of an H3N2 influenza A virus of swine (IAV-S) developed virus-neutralizing antibodies and were protected against infection by the homologous H3N2 strain. The objective of the current study was to evaluate the immunogenicity and protective efficacy of a trivalent PICV-vectored vaccine expressing HA antigens from the three co-circulating IAV-S subtypes: H1N1, H1N2, and H3N2. Pigs immunized with the trivalent PICV vaccine developed virus-neutralizing (VN) and hemagglutination inhibition (HI) antibodies against all three matching IAV-S. Following challenge infection with the H1N1 strain, five of the six pigs vaccinated with the trivalent vaccine had no evidence of IAV-S RNA genomes in nasal swabs and bronchoalveolar lavage fluid, while all non-vaccinated control pigs showed high number of copies of IAV-S genomic RNA in these two types of samples. Overall, our results demonstrate that the trivalent PICV-vectored vaccine elicits antibody responses against the three targeted IAV-S strains and provides protection against homologous virus challenges in pigs. Therefore, PICV exhibits the potential to be explored as a viral vector for delivering multiple vaccine antigens in swine.