Publications

Minigenomes (MGs) have greatly advanced research on the viral life cycle, including viral replication and transcription, virus‒host interactions, and the discovery of antivirals against RNA viruses. However, an MG for infectious bursal disease virus (IBDV) has not been well established. Here, we describe the development of IBDV MG, in which the entire coding sequences of viral genomic segments A and B are replaced with Renilla luciferase (Rluc) or enhanced green fluorescent protein (EGFP) reporter genes. Under the control of the RNA polymerase I promoter, the translation of IBDV MG is controlled by the viral proteins VP1 and VP3. Interestingly, IBDV B MG shows greater activity than does IBDV A MG. Moreover, the sense IBDV B MG was expressed at a higher level than the antisense IBDV B MG. In agreement with our previous findings, the translation of IBDV B MG controlled by VP1 and VP3 is independent of the cellular translation machinery components eukaryotic initiation factor (eIF)4E and eIF4G, but intact VP1 polymerase activity, VP3 dsRNA-binding activity, and the interaction between VP1 and VP3 are indispensable for both sense and antisense IBDV B MG activity. In addition, ribavirin, which inhibits IBDV replication, inhibits IBDV B MG activity in a dose-dependent manner. Collectively, the IBDV MG established in this study provides a powerful tool to investigate IBDV intracellular replication and transcription and virus‒host interactions and facilitates high-throughput screening for the identification of IBDV antivirals.

Since the emergence of highly pathogenic avian influenza virus (HPAIV) H5N1 of clade 2.3.4.4b as a novel reassortant virus from subtype H5N8, the virus has led to a massive number of outbreaks worldwide in wild and domestic birds. Compared to the parental HPAIV H5N8 clade 2.3.4.4b, the novel reassortant HPAIV H5N1 displayed an increased ability to escape species barriers and infect multiple mammalian species, including humans. The virus host range has been recently expanded to include ruminants, particularly dairy cattle in the United States, where cattle-to-cattle transmission was reported. As with the avian 2.3.4.4.b H5N1 viruses, the cattle-infecting virus was found to transmit from cattle to other contact animals including cats, raccoons, rodents, opossums, and poultry. Although replication of the virus in cows appears to be mainly confined to the mammary tissue, with high levels of viral loads detected in milk, infected cats and poultry showed severe respiratory disease, neurologic signs, and eventually died. Furthermore, several human infections with HPAIV H5N1 have also been reported in dairy farm workers and were attributed to exposures to infected dairy cattle. This is believed to represent the first mammalian-to-human transmission report of the HPAIV H5N1. Fortunately, infection in humans and cows, as opposed to other animals, appears to be mild in most cases. Nevertheless, the H5N1 bovine outbreak represents the largest outbreak of the H5N1 in a domestic mammal close to humans, increasing the risk that this already mammalian adapted H5N1 further adapts to human-to-human transmission and starts a pandemic. Herein, we discuss the epidemiology, evolution, pathogenesis, and potential impact of the recently identified HPAIV H5N1 clade 2.3.4.4b in dairy cattle in the United States. Eventually, interdisciplinary cooperation under a One Health framework is required to be able to control this ongoing HPAIV H5N1 outbreak to stop it before further expansion of its host range and geographical distribution.

SARS-CoV-2 continues to be a public health burden, driven in-part by its continued antigenic diversification and resulting emergence of new variants. By increasing herd immunity, current vaccines have improved infection outcomes for many. However, prophylactic and treatment interventions that are not compromised by viral evolution of the Spike protein are still needed. Using a differential staining strategy with a rationally designed SARS-CoV-2 Receptor Binding Domain (RBD) - ACE2 fusion protein and a native Omicron RBD protein, we developed a recombinant human monoclonal antibody (hmAb) from a convalescent individual following SARS-CoV-2 Omicron infection. The resulting hmAb, 1301B7 potently neutralized a wide range of SARS-CoV-2 variants including the original Wuhan-1, the more recent Omicron JN.1 strain, and SARS-CoV. 1301B7 contacts the ACE2 binding site of RBD exclusively through its VH1-69 heavy chain. Broad specificity is achieved through 1301B7 binding to many conserved residues of Omicron variants including Y501 and H505. Consistent with its extensive binding epitope, 1301B7 is able to potently diminish viral burden in the upper and lower respiratory tract and protect mice from challenge with Omicron XBB1.5 and Omicron JN.1 viruses. These results suggest 1301B7 has broad potential to prevent or treat clinical SARS-CoV-2 infections and to guide development of RBD-based universal SARS-CoV-2 prophylactic vaccines and therapeutic approaches.

Lassa virus (LASV) is a rodent-borne mammarenavirus that causes tens to hundreds of thousands of human infections annually in Western Africa. Approximately 20% of these infections progress to Lassa fever (LF), an acute disease with case-fatality rates from ≈20-70%. Currently, there are no approved vaccines or specific therapeutics to prevent or treat LF. The LASV genome consists of a small (S) segment that has two genes, GP and NP, and a large (L) segment that has two genes, L and Z. In both segments, the two genes are separated by non-coding intergenic regions (IGRs). Recombinant LASVs (rLASVs), in which the L segment IGR was replaced with the S segment IGR or in which the GP gene was codon-deoptimized, lost fitness in vitro, were highly attenuated in vivo, and, when used as vaccines, protected domesticated guinea pigs from otherwise lethal LASV exposure. Here, we report the generation of rLASV/IGR-CD, which includes both determinants of attenuation and further enhances the safety of the vaccine compared with its predecessors. rLASV/IGR-CD grew to high titers in Vero cells, which are approved for human vaccine production, but did not cause signs of disease or pathology in guinea pigs. Importantly, guinea pigs vaccinated with rLASV/IGR-CD were completely protected from disease and death after a typically lethal exposure to wild-type LASV. Our data support the development of rLASV/IGR-CD as a live-attenuated LF vaccine with stringent safety features.

The SARS-CoV-2 pandemic has caused unprecedented worldwide infections from persistent mutant variants with various degrees of infectivity and virulence. The elusiveness of a highly penetrant, worldwide vaccination strategy suggests that the complete eradication of SARS-CoV-2 is unlikely. Even with the advent of new antiviral agents, the disease burden worldwide continues to exceed current preventative and therapeutic strategies. Greater interest has been placed towards the development of affordable,broadly effective antiviral therapeutics. Here, we report that the small branched-chain fatty acid Valproic acid (VPA), approved for maintenance of seizure and bipolar disorder, has a novel anti- coronavirus activity that can be augmented with the addition of a long-chain, polyunsaturated omega-3 fatty acid, Docosahexaenoic acid (DHA). An EMR-based epidemiological study of patients tested for COVID-19 demonstrated a correlation exists between a reduced infection rate in patients treated withVPA of up to 25%, as well as a decreased risk of emergency room visits, hospitalization, ICU admission,and use of mechanical ventilation. In vitro studies have demonstrated that VPA modifies gene expression in MRC5 cells. Interestingly, VPA correlates with the inhibition of several SARS-CoV2 interacting genes and the greater inhibition of alpha-coronavirus HCoV-229E (a "common cold" virus) and SARS-CoV2. The VPA-DHA combination activates pre-existing intracellular antiviral mechanisms normally repressed by coronaviruses. Gene expression profiles demonstrate subtle differences in overall gene expression between VPA-treated and VPA-DHA-treated cells. HCoV-229E infection caused an intensely different response with a marked induction of multiple intracellular inflammatory genes. Changes in gene expression took at least 24 hours to manifest and most likely why prior drug screens failed to identify any antiviral VPA activity despite in silico predictions. This report demonstrates an interaction between HDAC inhibition and the potent activation of cellular antiviral responses. A foundation now exists for a low-cost, highly effective antiviral strategy when supplemented with DHA.

The mammarenavirus matrix Z protein plays critical roles in virus assembly and cell egress. Meanwhile, heterotrimer complexes of a stable signal peptide (SSP) together with glycoprotein subunits GP1 and GP2, generated via co-and post-translational processing of the surface glycoprotein precursor GPC, form the spikes that decorate the virion surface and mediate virus cell entry via receptor-mediated endocytosis. The Z protein and the SSP undergo N-terminal myristoylation by host cell N-myristoyltransferases (NMT1 and NMT2), and G2A mutations that prevent myristoylation of Z or SSP have been shown to affect the Z-mediated virus budding and GP2-mediated fusion activity that is required to complete the virus cell entry process. In the present work, we present evidence that the validated on-target specific pan-NMT inhibitor DDD85646 exerts a potent antiviral activity against the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) that correlates with reduced Z budding activity and GP2-mediated fusion activity as well as with proteasome-mediated degradation of the Z protein. The potent anti-mammarenaviral activity of DDD85646 was also observed with the hemorrhagic-fever-causing Junin (JUNV) and Lassa (LASV) mammarenaviruses. Our results support the exploration of NMT inhibition as a broad-spectrum antiviral against human pathogenic mammarenaviruses.

As the new SARS-CoV-2 Omicron variants and subvariants emerge, there is an urgency to develop intranasal, broadly protective vaccines. Here, we developed highly efficacious, intranasal trivalent SARS-CoV-2 vaccine candidates (TVC) based on three components of the MMR vaccine: measles virus (MeV), mumps virus (MuV) Jeryl Lynn (JL1) strain, and MuV JL2 strain. Specifically, MeV, MuV-JL1, and MuV-JL2 vaccine strains, each expressing prefusion spike (preS-6P) from a different variant of concern (VoC), were combined to generate TVCs. Intranasal immunization of IFNAR1-/- mice and female hamsters with TVCs generated high levels of S-specific serum IgG antibodies, broad neutralizing antibodies, and mucosal IgA antibodies as well as tissue-resident memory T cells in the lungs. The immunized female hamsters were protected from challenge with SARS-CoV-2 original WA1, B.1.617.2, and B.1.1.529 strains. The preexisting MeV and MuV immunity does not significantly interfere with the efficacy of TVC. Thus, the trivalent platform is a promising next-generation SARS-CoV-2 vaccine candidate.

The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm. Based on Nsp1 crystal structure, we generated mutants on Nsp1 surfaces and identified an acidic N-terminal patch that is critical for interaction with NXF1-NXT1. Photoactivatable Nsp1 probe reveals the RNA Recognition Motif (RRM) domain of NXF1 as an Nsp1 N-terminal binding site. By mutating the Nsp1 N-terminal acidic patch, we identified a separation-of-function mutant of Nsp1 that retains its translation inhibitory function but substantially loses its interaction with NXF1 and reverts Nsp1-mediated mRNA export inhibition. We then generated a recombinant (r)SARS-CoV-2 mutant on the Nsp1 N-terminal acidic patch and found that this surface is key to promote NXF1 binding and inhibition of host mRNA nuclear export, viral replication, and pathogenicity in vivo. Thus, these findings provide a mechanistic understanding of Nsp1-mediated mRNA export inhibition and establish the importance of this pathway in the virulence of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has caused millions of deaths since its emergence in 2019. Innate immune antagonism by lethal CoVs such as SARS-CoV-2 is crucial for optimal replication and pathogenesis. The conserved nonstructural protein 15 (nsp15) endoribonuclease (EndoU) limits activation of double-stranded (ds)RNA-induced pathways, including interferon (IFN) signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L) during diverse CoV infections including murine coronavirus and Middle East respiratory syndrome (MERS)-CoV. To determine how nsp15 functions during SARS-CoV-2 infection, we constructed a recombinant SARS-CoV-2 (nsp15mut) expressing catalytically inactivated nsp15, which we show promoted increased dsRNA accumulation. Infection with SARS-CoV-2 nsp15mut led to increased activation of the IFN signaling and PKR pathways in lung-derived epithelial cell lines and primary nasal epithelial air-liquid interface (ALI) cultures as well as significant attenuation of replication in ALI cultures compared to wild-type virus. This replication defect was rescued when IFN signaling was inhibited with the Janus activated kinase (JAK) inhibitor ruxolitinib. Finally, to assess nsp15 function in the context of minimal (MERS-CoV) or moderate (SARS-CoV-2) innate immune induction, we compared infections with SARS-CoV-2 nsp15mut and previously described MERS-CoV nsp15 mutants. Inactivation of nsp15 had a more dramatic impact on MERS-CoV replication than SARS-CoV-2 in both Calu3 cells and nasal ALI cultures suggesting that SARS-CoV-2 can better tolerate innate immune responses. Taken together, SARS-CoV-2 nsp15 is a potent inhibitor of dsRNA-induced innate immune response and its antagonism of IFN signaling is necessary for optimal viral replication in primary nasal ALI cultures.

INTRODUCTION: Influenza A virus (IAV) infection can cause the often-lethal acute respiratory distress syndrome (ARDS) of the lung. Concomitantly, acute kidney injury (AKI) is frequently noticed during IAV infection, correlating with an increased mortality. The aim of this study was to elucidate the interaction of IAV with human kidney cells and, thereby, to assess the mechanisms underlying IAV-mediated AKI.

METHODS: To investigate IAV effects on nephron cells we performed infectivity assays with human IAV, as well as with human isolates of either low or highly pathogenic avian IAV. Also, transcriptome and proteome analysis of IAV-infected primary human distal tubular kidney cells (DTC) was performed. Furthermore, the DTC transcriptome was compared to existing transcriptomic data from IAV-infected lung and trachea cells.

RESULTS: We demonstrate productive replication of all tested IAV strains on primary and immortalized nephron cells. Comparison of our transcriptome and proteome analysis of H1N1-type IAV-infected human primary distal tubular cells (DTC) with existing data from H1N1-type IAV-infected lung and primary trachea cells revealed enrichment of specific factors responsible for regulated cell death in primary DTC, which could be targeted by specific inhibitors.

DISCUSSION: IAV not only infects, but also productively replicates on different human nephron cells. Importantly, multi-omics analysis revealed regulated cell death as potential contributing factor for the clinically observed kidney pathology in influenza.