Patrick C. Reading

Highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b viruses have recently infected dairy cows in the United States, raising concerns about human spillover. The antiviral roles of bovine host defense factors, including myxovirus resistance proteins (bMx1 and bMx2), remain relatively understudied. We examined bMx1/bMx2 induction in differentiated bovine cells following HPAI H5N1 infection and assessed their antiviral activity using overexpression, knockdown, and mutagenesis approaches. Both bMx1 and bMx2 were more strongly induced in mammary cells than in tracheal cells. Overexpression of bMx1, but not bMx2, significantly inhibited seasonal influenza A viruses (IAVs), low-pathogenic, and highly pathogenic avian influenza viruses, including H5N1 2.3.4.4b, but not influenza B viruses. Conversely, knockdown of bMx1 resulted in enhanced IAV growth. Further, bMx1 inhibited early stages of viral replication, acting to reduce viral transcription and subsequent steps in the viral replication cycle. Known mutations that reduce human MxA (hMxA) activity also impaired bMx1 function, and mutations in the viral nucleoprotein (NP) associated with resistance to hMxA also reduced virus sensitivity to bMx1. Furthermore, phylogenetic analysis showed that a small minority (>0.03%) of avian H5N1 2.3.4.4b viruses already carry some of these NP mutations. These findings suggest that while current H5N1 viruses largely remain susceptible to bMx1, viral evolution or opportunistic reassortment could drive Mx resistance and increase pandemic risk. IMPORTANCE: Highly pathogenic avian influenza viruses have spilled over into dairy cows and have been circulating on dairy farms in the United States since 2024. The innate antiviral immune responses of cows to influenza viruses have not been extensively studied. In this study, we focused on Mx proteins, a key part of the innate immune defense system toward viruses. We specifically looked at bovine Mx proteins and demonstrated antiviral activity toward different influenza viruses, including the highly pathogenic strains. We also showed that the mechanism of antiviral activity of bovine Mx1 was very similar to that of the human equivalent MxA protein. Further, we found that a small minority of these avian viruses have mutations associated with resistance to both bovine Mx1 and human MxA proteins. Overall, this work highlights the elevated pandemic risk for highly pathogenic influenza viruses through continual circulation in cows, a mammalian species in close proximity to humans.

Background: SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase protein 1 (SAMHD1) is an intracellular protein that regulates a stable deoxynucleoside triphosphates (dNTPs) for normal cellular function. Many studies have documented SAMHD1-mediated restriction of RNA retroviruses and different DNA viruses in non-dividing myeloid cells, where it acts to deplete cellular dNTP pools to inhibit replication of the viral genome. Less is currently known regarding its ability to restrict the replication of other RNA viruses in different cell types. In this study, we investigate the role of SAMHD1 in modulating the replication of RNA viruses associated with respiratory virus infection, with a particular focus on seasonal influenza A virus (IAV). Moreover, our studies have assessed the ability of SAMHD1 to modulate IAV replication in two key cell types associated with the pathogenesis of IAV, namely epithelial and macrophage-like cells. Methods: qPCR was used to examine expression of endogenous SAMHD1 in response to IAV infection and/or treatment with recombinant type I interferon (IFN) in epithelial (A549) and macrophage-like (THP-1) cells. CRISPR/Cas9 knockout (KO) of endogenous SAMHD1 was performed to assess the role of SAMHD1 in modulating IAV replication in A549 cells or in differentiated (d)THP-1. IAV infection and replication were also assessed in A549 and dTHP-1 SAMHD1 KO cells with stable doxycycline (DOX)-induced expression of SAMHD1 or of phosphorylation mutants of SAMHD1. Results: CRISPR/Cas9 KO of endogenous SAMHD1 in dTHP-1 or A549 cells did not enhance virus release following infection with seasonal IAV. Moreover, reconstitution of KO cells with DOX-inducible SAMHD1 did not restrict the growth of seasonal IAV in dTHP-1 or A549 cells, despite SAMHD1-mediated restriction of a DNA virus (HSV-1) in both cell types. In contrast to seasonal IAV, we also observed restriction of highly pathogenic avian influenza (HPAI) A(H5N1) IAV replication in dTHP-1 cells expressing DOX-inducible SAMHD1. Conclusions: Together, these studies indicate that neither endogenous nor DOX-inducible SAMHD1 restricts the growth of seasonal IAV in macrophage-like or epithelial cells.

Background: A coronavirus disease 2019 (COVID-19) subunit vaccine developed with the molecular clamp platform was among the first to enter clinical trials in 2020. While the vaccine was well suited to large-scale manufacture and demonstrated a favorable safety and immunogenicity profile, it did not progress into further clinical testing due to the human immunodeficiency virus 1 (HIV-1)–derived sequence in the molecular clamp, interfering with certain point-of-care HIV-1 diagnostic tests. A second-generation molecular clamp (MC2) has since been developed. Here, we describe a phase I clinical trial of an MC2-stabilized severe acute respiratory syndrome coronavirus (SARS-CoV-2) spike subunit vaccine, UQSC2. Methods: A phase I, double-blind, active comparator-controlled trial was conducted in Australia (ClinicalTrials.gov NCT05775887). Healthy adults (aged 18–50 years), who had previously received ≥3 doses of an approved messenger RNA SARS-CoV-2 vaccine, received a single booster dose of either UQSC2 or the approved comparator NVX-CoV2373 (Nuvaxovid; Novavax) (n = 70; 35 per group). Safety, humoral immunogenicity (including against virus variants), and cellular immunogenicity were assessed through day 183. Cases of confirmed coronavirus disease 2019 (COVID-19) infection during the study were also examined. Results: Both vaccines were equally well tolerated and elicited comparable increases in neutralizing humoral responses against the vaccine-matched prototypic, Wuhan strain of SARS-CoV-2. Conclusions: UQSC2 was as well tolerated as the authorized comparator vaccine and produced an equally robust boost in neutralizing immune response. These findings support the effectiveness of the MC2 platform in producing vaccines against newly emerging variants of SARS-CoV-2 and other respiratory viruses, as well as the platform's potential use in emergency response to novel viruses.

Severe respiratory syndrome coronavirus 2 (SARS-CoV-2) infections are associated with significant morbidity and mortality worldwide. Identification and characterization of intracellular proteins with antiviral activity (known as restriction factors) is a key first step towards the future development of novel host-directed antiviral therapies. In this study, we investigated the antiviral activity of 14 different interferon-stimulated gene (ISG) proteins against SARS-CoV-2. Overexpression of human guanylate binding protein (GBP) 1 resulted in potent inhibition of the ancestral SARS-CoV-2 strain, as well as against Alpha, Beta, Delta, Omicron BA.1, and Omicron BA.2 variants of concern (VOCs). Moreover, knockdown or knockout of endogenous human GBP1 resulted in enhanced titers of SARS-CoV-2. While hGBP1 can restrict some viruses via actin remodeling, our data indicate that this is not the mechanism here. Moreover, we show that unlike GBP2 and GBP5, which impair spike protein processing, GBP1 did not reduce infectivity of spike-pseudotyped viruses following titration on Caco-2 cells. Multiple human GBPs have been reported to inhibit SARS-CoV-2 in vitro ; however, we report no significant differences in virus titers recovered from the upper or lower airways of SARS-CoV-2-infected wild-type mice compared to from mice lacking the chromosome 3 cluster of mouse GBPs (GBP1/2/3/5/7). Together, our studies describe the ability of human GBP1 to inhibit SARS-CoV-2 replication, highlighting this host protein as a potential target for the development of host-directed antiviral therapies against this virus. IMPORTANCE: Viruses like SARS-CoV-2 can cause widespread illness and death. While currently licensed antiviral drugs are critical tools, drug resistance can develop. Our immune system produces intracellular proteins called “restriction factors” that can limit virus replication within cells. These proteins are promising targets for developing new antiviral therapies. In this study, we identified one such protein, human GBP1, which inhibited a range of SARS-CoV-2 variants in vitro , including Delta and Omicron. Interestingly, GBP1 inhibited SARS-CoV-2 through a different mechanism to that of other human GBPs as it did not interfere with prosessing of the viral spike protein. Of interest, a cluster of mouse GBPs, including GBP1, did not demontrate significant antiviral activity in a mouse model of infection. Overall, our findings suggest that human GBP1 could be a valuable target for host-directed antiviral strategies and highlight the limitations of using mouse models to study certain aspects of human innate immunity.

The European domestic ferret ( Mustela putorius furo ) is considered the gold standard small animal model for studying human and avian influenza virus infections. However, experimental characterisation of the transcriptomic response to interferon (IFN) stimulation and/or influenza virus infection has been limited, particularly in defining the induction of interferon‐stimulated genes (ISGs), with most being computationally predicted. In this study, we present a comprehensive transcriptome‐wide assessment of the ferret transcriptome following IFN‐α treatment of a ferret lung (FRL) cell line, as well as in nasal turbinates from influenza A virus (IAV)‐infected ferrets using long‐read RNA sequencing. We have identified a panel of ferret genes orthologous to human ISGs that are upregulated both in response to IFN‐α stimulation in vitro and IAV infection in vivo. We have also identified novel IFN‐stimulated genes and transcripts. Furthermore, we observed elongation of the poly(A) tails of genes in the ribosome and Coronavirus Disease‐19 pathways in response to IFN‐α treatment in vitro , suggesting a relationship between poly(A) elongation and the antiviral responses of the host. These results illuminate the dynamics of the transcriptional innate immune response of the domestic ferret and provide an important resource for better utilising ferrets as a small animal model to study influenza virus infections.

Nipah virus (NiV) and Hendra virus (HeV) are highly pathogenic henipaviruses without approved human vaccines or therapies. Here, we report on a highly potent bispecific therapeutic that combines an anti-fusion glycoprotein nanobody with an anti-receptor-binding glycoprotein (RBP) antibody to deliver a dual-targeting biologic that is resistant to viral escape. We show that the nanobody, DS90, engages a unique, conserved site within the fusion glycoprotein of NiV and HeV and provides neutralization and complete protection from NiV disease. Bispecific engineering of DS90 with the anti-RBP monoclonal antibody m102.4 results in neutralization, elimination of viral escape and superior protection from NiV disease compared to leading monovalent approaches. These findings carry implications for the development of cross-neutralizing immunotherapies that limit the emergence of henipaviral escape mutants.

Dynamin-like GTPase proteins, including myxoma (Mx) and guanylate-binding proteins (GBPs), are among the many interferon stimulated genes induced following viral infections. While studies report that human (h)GBPs inhibit different viruses in vitro, few have convincingly demonstrated that mouse (m)GBPs mediate antiviral activity, although mGBP-deficient mice have been used extensively to define their importance in immunity to diverse intracellular bacteria and protozoa. Herein, we demonstrate that individual (overexpression) or collective (knockout (KO) mice) mGBPs of the chromosome 3 cluster (mGBPchr3) do not inhibit replication of five viruses from different virus families in vitro, nor do we observe differences in virus titres recovered from wild type versus mGBPchr3 KO mice after infection with three of these viruses (influenza A virus, herpes simplex virus type 1 or lymphocytic choriomeningitis virus). These data indicate that mGBPchr3 do not appear to be a major component of cell-intrinsic antiviral immunity against the diverse viruses tested in our studies.

Myxovirus resistance (Mx) proteins are products of interferon stimulated genes (ISGs) and Mx proteins of different species have been reported to mediate antiviral activity against a number of viruses, including influenza A viruses (IAV). Ferrets are widely considered to represent the ‘gold standard’ small animal model for studying pathogenesis and immunity to human IAV infections, however little is known regarding the antiviral activity of ferret Mx proteins. Herein, we report induction of ferret (f)Mx1/2 in a ferret lung cell line and in airway tissues from IAV-infected ferrets, noting that fMx1 was induced to higher levels that fMx2 both in vitro and in vivo. Overexpression confirmed cytoplasmic expression of fMx1 as well as its ability to inhibit infection and replication of IAV, noting that this antiviral effect of fMx1was modest when compared to cells overexpressing either human MxA or mouse Mx1. Together, these studies provide the first insights regarding the role of fMx1 in cell innate antiviral immunity to influenza viruses. Understanding similarities and differences in the antiviral activities of human and ferret ISGs provides critical context for evaluating results when studying human IAV infections in the ferret model.