Publications

The host range of HPAIV H5N1 was recently expanded to include ruminants, particularly dairy cattle in the United States (US). Shortly after, human H5N1 infection was reported in a dairy worker in Texas following exposure to infected cattle. Herein, we rescued the cattle-origin influenza A/bovine/Texas/24-029328-02/2024(H5N1, rHPbTX) and A/Texas/37/2024(H5N1, rHPhTX) viruses, identified in dairy cattle and human, respectively, and their low pathogenic forms, rLPbTX and rLPhTX, with monobasic HA cleavage sites. Intriguingly, rHPhTX replicated more efficiently than rHPbTX in mammalian and avian cells. Still, variations in the PA and NA proteins didn't affect their antiviral susceptibility to PA and NA inhibitors. Unlike rHPbTX and rLPbTX, both rHPhTX and rLPhTX exhibited higher pathogenicity and efficient replication in infected C57BL/6J mice. The lungs of rHPhTX-infected mice produced higher inflammatory cytokines/chemokines than rHPbTX-infected mice. Our results highlight the potential risk of HPAIV H5N1 virus adaptation in human and/or dairy cattle during the current multistate/multispecies outbreak in the US.

The current situation with H5N1 highly pathogenic avian influenza virus (HPAI) is causing a worldwide concern due to multiple outbreaks in wild birds, poultry, and mammals. Moreover, multiple zoonotic infections in humans have been reported. Importantly, HPAI H5N1 viruses with genetic markers of adaptation to mammals have been detected. Together with HPAI H5N1, avian influenza viruses H7N9 (high and low pathogenic) stand out due to their high mortality rates in humans. This raises the question of how prepared we are serologically and whether seasonal vaccines are capable of inducing protective immunity against these influenza subtypes. An observational study was conducted in which sera from people born between years 1925-1967, 1968-1977, and 1978-1997 were collected before or after 28 days or 6 months post-vaccination with an inactivated seasonal influenza vaccine. Then, hemagglutination inhibition, viral neutralization, and immunoassays were performed to assess the basal protective immunity of the population as well as the ability of seasonal influenza vaccines to induce protective responses. Our results indicate that subtype-specific serological protection against H5N1 and H7N9 in the representative Spanish population evaluated was limited or nonexistent. However, seasonal vaccination was able to increase the antibody titers to protective levels in a moderate percentage of people, probably due to cross-reactive responses. These findings demonstrate the importance of vaccination and suggest that seasonal influenza vaccines could be used as a first line of defense against an eventual pandemic caused by avian influenza viruses, to be followed immediately by the use of more specific pandemic vaccines.IMPORTANCEInfluenza A viruses (IAV) can infect and replicate in multiple mammalian and avian species. Avian influenza virus (AIV) is a highly contagious viral disease that occurs primarily in poultry and wild water birds. Due to the lack of population immunity in humans and ongoing evolution of AIV, there is a continuing risk that new IAV could emerge and rapidly spread worldwide, causing a pandemic, if the ability to transmit efficiently among humans was gained. The aim of this study is to analyze the basal protection and presence of antibodies against IAV H5N1 and H7N9 subtypes in the population from different ages. Moreover, we have evaluated the humoral response after immunization with a seasonal influenza vaccine. This study is strategically important to evaluate the level of population immunity that is a major factor when assessing the impact that an emerging IAV strain would have, and the role of seasonal vaccines to mitigate the effects of a pandemic.

2'-O-ribose methylation of the first transcribed base (adenine or A1 in SARS-CoV-2) of viral RNA mimics host RNAs and subverts the innate immune response. How nsp16, with partner nsp10, assembles on the 5'-end of SARS-CoV-2 mRNA to methylate A1 is not fully understood. We present a ∼2.4 Å crystal structure of the heterotetrameric complex formed by the cooperative assembly of two nsp16/nsp10 heterodimers with one 10-mer Cap-1 RNA (product) bound to each. An aromatic zipper-like motif in nsp16 and the N-terminal regions of nsp10 and nsp16 orchestrate oligomeric assembly for efficient methylation. The front catalytic pocket of nsp16 stabilizes the upstream portion of the RNA while downstream RNA remains unresolved, likely due to flexibility. An inverted nsp16 dimer extends the positively charged surface for longer RNA to influence catalysis. Additionally, a non-specific nucleotide-binding pocket on the backside of nsp16 plays a critical role in catalysis, contributing to enzymatic activity.

Monoclonal antibodies (mAbs) that recognize and inhibit a diverse range of influenza viruses, although relatively rare, have been isolated following infection or vaccination. Study of their ontology and mechanisms of action informs universal vaccine and therapeutic development. We have previously described a potent and broad neuraminidase (NA)-neutralizing human mAb, 1122A11, that neutralizes a wide range of H3N2 viruses. Here, further characterization of 1122A11 reveals reactivity to cross-group influenza A virus NAs, including group-1 N1 and N8, and group-2 N2 and N3 NAs. Recent H3N2 viruses have acquired Asn245 glycosylation on the active site rim. Crystal structures of an N2 NA from A/Singapore/INFIMH-16-0019/2016 (H3N2) at 2.3 Å (apo) and 2.2 Å (Fab bound) resolution showed that 1122A11 binding causes local changes to the periphery of NA active site to accommodate the glycan. The CDRH3 of 1122A11 inserts into the active site and mimics the substrate sialic acid. We then determined that the ability of 1122A11 to protect from lethal challenge in mice is not dependent on Fc-effector function. These results highlight the therapeutic potential of 1122A11 as a broad protective anti-viral and reinforce pursuit of immunogen development of NA antibodies toward achieving more universal influenza protection.

The ongoing outbreak of highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b has affected at least 989 dairy herds across 17 states in the United States (U.S.) and resulted in 70 confirmed human infections, underscoring the urgent need to understand the pathogenesis and therapeutic interventions of emerging H5N1 viruses. In this study, we modelled infection with a highly pathogenic recombinant human A/Texas/37/2024 H5N1 (rHPh-TX H5N1) strain using human airway organoids (HAO) to investigate viral replication, innate immune response, infection-induced fibrogenesis, and potential therapeutic interventions. rHPh-TX H5N1 replicated efficiently in HAO, eliciting a robust interferon (IFN) response and pro-inflammatory cytokine production. Prolonged infection led to the accumulation of fibroblast-like cells surrounding infected regions, marked by increased alpha-smooth muscle actin (α-SMA) expression and upregulation of transforming growth factor-beta (TGF-β), indicative of fibroblast activation and extracellular matrix (ECM) remodelling. Compared to organoids infected with the pandemic A/California/04/09 H1N1 (pH1N1) strain, rHPh-TX H5N1 induced significantly higher expression of fibrosis-associated markers, including fibronectin (FN), collagen 1A (COL1A), collagen 3A (COL3A), metalloproteinases 2 and 9 (MMP2, and MMP9). Notably, the inhibition of Rho-associated coiled-coil-forming protein kinases (ROCK) signalling reduced fibrogenesis, with ROCK1 inhibition being more effective than ROCK2 inhibition. These findings highlight the potential of targeting ROCK signalling to mitigate H5N1-induced lung fibrosis, informing therapeutic strategies for severe influenza infections.

UNLABELLED: The emergence of SARS-CoV-2 variants has necessitated continuous updating of vaccines. In contrast, antivirals remained effective as they target conserved viral proteins that are essential for the viral life cycle. However, several mutations in SARS-CoV-2 that may affect the efficacy of United States (US) Food and Drug Administration (FDA)-approved antivirals have been recently identified. Detecting drug-resistant SARS-CoV-2 mutants and investigating their escape mechanism(s) are critical to guide the selection of effective antiviral therapies. In this study, we constructed an attenuated recombinant (r)SARS-CoV-2 lacking the open reading frame (ORF) proteins 3a and 7b but expressing nanoluciferase (Nluc), rSARS-CoV-2 Δ3a7b-Nluc, to facilitate tracking viral infection. Using this virus, we selected drug-resistant mutants to the main viral protease (Mpro) inhibitor nirmatrelvir. After passaging Δ3a7b-Nluc 10 times in the presence of increasing concentrations of nirmatrelvir, a virus population with enhanced resistance was selected. We identified two non-synonymous mutations (L50F and R188G) in Mpro, encoded by the non-structural protein 5 (NSP5) gene. Using reverse genetics, we generated rSARS-CoV-2 Δ3a7b-Nluc containing the identified L50F and R188G mutations, individually or in combination, and assessed their contribution to nirmatrelvir resistance. Our results indicate that both mutations are involved in escaping from nirmatrelvir. Altogether, our results demonstrate the feasibility of using rSARS-CoV-2 Δ3a7b-Nluc variant to identify and validate mutations that confer resistance to FDA-approved antiviral drugs without the concern of conducting gain of function (GoF) experiments with wild-type (WT) forms of SARS-CoV-2.

IMPORTANCE: Small-molecule antiviral drugs have been used for the treatment of SARS-CoV-2 infections. However, drug-resistant SARS-CoV-2 mutants to currently US FDA-approved Mpro targeting antivirals have been identified. Information on SARS-CoV-2 escape mutants and mutations affecting the antiviral activity of licensed antivirals remain limited. In this study, we developed a nanoluciferase (Nluc)-expressing attenuated recombinant (r)SARS-CoV-2 lacking the ORF 3a and 7b proteins (Δ3a7b-Nluc) to identify nirmatrelvir resistant mutants without the biosafety concerns associated with gain-of-function (GoF) research using wild-type (WT) SARS-CoV-2. Using Δ3a7b-Nluc, we have selected variants with reduced sensitivity to nirmatrelvir that were validated by the generation of rSARS-CoV-2 Δ3a7b-Nluc containing the candidate L50F and R188G mutations in Mpro. These results demonstrate the feasibility of using rSARS-CoV-2 Δ3a7b-Nluc to safely identify and validate drug-resistant mutants overcoming concerns originating from adaptation studies using WT SARS-CoV-2.

Coronaviruses, including SARS-CoV-2, can cause severe disease in humans, whereas reservoir hosts like Rhinolophus bats remain asymptomatic. To investigate how host-specific protein-protein interactions (PPIs) influence infection, we generated comparative PPI maps for SARS-CoV-2 and its bat-origin relative RaTG13 using affinity purification-mass spectrometry (AP-MS) in human and Rhinolophus ferrumequinum (RFe) bat cells. This approach identified both conserved and virus- and host-specific interactions that regulate infection dynamics. Notably, SARS-CoV-2 required a non-synonymous mutation in nucleocapsid to replicate in bat cells expressing human ACE2 and TMPRSS2. Analysis of the viral protein Orf9b revealed differential interactions with mitochondrial proteins Tom70 and MTARC2. A single residue difference in Orf9b between SARS-CoV-2 and RaTG13 functions as a molecular switch, weakening Tom70 binding and immune evasion in human cells while enhancing interaction with the bat-specific restriction factor MTARC2. These findings demonstrate how a single-residue substitution can reshape virus-host interactions and contribute to immune evasion and host adaptation.

UNLABELLED: Influenza A viruses (IAV) infect a wide range of mammal and bird species and are responsible for seasonal outbreaks and occasional pandemics. Studying IAV requires methods to detect the presence of the virus in infected cells or animal models. Recombinant IAV expressing fluorescent proteins have allowed monitoring viral infection in cultured cells and ex vivo in the organs of infected animals. However, fluorescent-expressing IAV are often attenuated and are not suited for the imaging of infected animals using in vivo imaging systems (IVIS). To overcome this limitation, we generated a recombinant A/California/04/2009 H1N1 (pH1N1) expressing nanoluciferase (Nluc) from the non-structural (NS) viral segment (pH1N1-Nluc) that replicates efficiently in vitro , with growth kinetics and plaque morphology comparable to wild-type pH1N1 (pH1N1-WT). We used this pH1N1-Nluc to demonstrate its ability to identify neutralizing antibodies and antivirals, with neutralization and inhibition results comparable to pH1N1-WT. In mice, pH1N1-Nluc was able to induce similar body weight loss and mortality, and viral titers comparable to pH1N1-WT, results that were recapitulated in a ferret model of IAV infection. Using IVIS, pH1N1-Nluc enabled non-invasive, real-time tracking of viral infection in vivo and ex vivo following infection of mice with viral titers comparable to pH1N1-WT. The flexibility of this approach was further demonstrated by the generation of a Nluc-expressing recombinant A/Puerto Rico/8/1934 H1N1 (PR8-Nluc). Altogether, our results demonstrate that Nluc-expressing recombinant IAV represent a valuable tool for in vitro and in vivo studies, including the identification of antivirals and/or neutralizing antibodies, and to assess protective efficacy of vaccines.

IMPORTANCE: Despite the availability of recombinant influenza A viruses (IAV) expressing fluorescent reporter genes to track viral infections in vitro and ex vivo , these viruses are often attenuated and do not represent the best option for imaging entire animals using in vivo imaging systems (IVIS). To solve this limitation, we generated recombinant influenza pandemic A/California/04/2009 H1N1 expressing nanoluciferase (pH1N1-Nluc) from the viral non-structural (NS) segment and demonstrate how expression of Nluc does not affect viral replication in vitro or viral pathogenesis in vivo. Importantly, we demonstrate the feasibility of detecting pH1N1-Nluc infection in vivo using IVIS. We also validate the flexibility of this approach by generating an influenza A/Puerto Rico/8/1934 H1N1 (PR8-Nluc). Our results support the feasibility of using these recombinant IAVs expressing Nluc from the NS segment for in vitro and in vivo studies, including the identification of neutralizing antibodies and/or antivirals, and to assess protective efficacy of vaccines.

UNLABELLED: The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has necessitated a continuous updating of vaccines. In contrast, antivirals remained effective as they target conserved viral proteins that are essential for the viral life cycle. However, several mutations in SARS-CoV-2 that may affect the efficacy of United States (US) Food and Drug Administration (FDA)-approved antivirals have been recently identified. Detecting drug-resistant SARS-CoV-2 mutants and investigating their escape mechanism(s) are critical to guide the selection of effective antiviral therapies. In this study, we constructed an attenuated recombinant (r)SARS-CoV-2 lacking the open reading frame (ORF) proteins 3a and 7b but expressing nanoluciferase (Nluc), rSARS-CoV-2 Δ3a7b-Nluc, to facilitate tracking viral infection. Using this virus, we selected drug-resistant mutants to the main viral protease (Mpro) inhibitor nirmatrelvir. After passaging Δ3a7b-Nluc 10 times in the presence of increasing concentrations of nirmatrelvir, a virus population with enhanced resistance was selected. We identified two non-synonymous mutations (L50F and R188G) in Mpro encoded by the non-structural protein 5 (NSP5) gene. Using reverse genetics, we generated rSARS-CoV-2 Δ3a7b-Nluc containing the identified L50F and R188G mutations, individually or in combination, and assessed their contribution to nirmatrelvir resistance. Our results indicate that both mutations are involved in escaping from nirmatrelvir. Altogether, our results demonstrate the feasibility of using the rSARS-CoV-2 Δ3a7b-Nluc variant to identify and validate mutations that confer resistance to FDA-approved antiviral drugs without the concern of conducting gain of function (GoF) experiments with wild-type (WT) forms of SARS-CoV-2.

IMPORTANCE: Small-molecule antiviral drugs have been used for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. However, drug-resistant SARS-CoV-2 mutants to currently United States Food and Drug Administration-approved Mpro targeting antivirals have been identified. Information on SARS-CoV-2 escape mutants and mutations affecting the antiviral activity of licensed antivirals remains limited. In this study, we developed a nanoluciferase (Nluc)-expressing attenuated recombinant (r)SARS-CoV-2 lacking the ORF 3a and 7b proteins (Δ3a7b-Nluc) to identify nirmatrelvir-resistant mutants without the biosafety concerns associated with gain-of-function (GoF) research using wild-type (WT) SARS-CoV-2. Using Δ3a7b-Nluc, we have selected variants with reduced sensitivity to nirmatrelvir that were validated by the generation of rSARS-CoV-2 Δ3a7b-Nluc containing the candidate L50F and R188G mutations in Mpro. These results demonstrate the feasibility of using rSARS-CoV-2 Δ3a7b-Nluc to safely identify and validate drug-resistant mutants overcoming concerns originating from adaptation studies using WT SARS-CoV-2.

The emergence of immune-evasive SARS-CoV-2 Omicron subvariants highlights the need to develop a mucosal SARS-CoV-2 vaccine that can provide broad protection against virus infection and transmission. Here, we developed an intranasal monovalent SARS-CoV-2 vaccine expressing the six-proline-stabilized prefusion spike proteins (preS-6P) of Omicron XBB.1.5 based on the attenuated mumps virus (MuV) Jeryl Lynn (JL1) vaccine strain. We also developed an intranasal trivalent vaccine expressing the preS-6P of ancestral SARS-CoV-2 WA1 and two Omicron subvariants, BA.1 and XBB.1.5, using the attenuated measles virus (MeV) and MuV-JL1 and JL2 vaccine strains, respectively. Intranasal immunization of hamsters with the monovalent rMuV-JL1-XBB.1.5 or the trivalent vaccine induced high levels of neutralizing antibodies (NAbs) that efficiently neutralized Omicron subvariants XBB.1.5, EG.5, and JN.1, providing complete protection against these Omicron subvariants. Similar levels of Omicron XBB.1.5 NAbs were detected in monovalent rMuV-JL1-XBB.1.5 and trivalent vaccine groups even when hamsters had been preimmunized with the rMuV-JL2-WA1 vaccine, suggesting that both intranasal vaccines are effective in the presence of immune imprinting induced by the spike of SARS-CoV-2 WA1. Intranasal, but not subcutaneous, immunization generated high levels of S-specific mucosal IgA antibodies as well as lung-resident memory T cells in IFNAR1-/- mice. Finally, intranasal immunization with the trivalent vaccine efficiently blocked transmission of SARS-CoV-2 WA1 and Omicron XBB.1.5 among hamsters in a direct contact transmission setting. In summary, we have developed intranasal MeV and MuV-based trivalent vaccines that induce broad NAbs, robust mucosal immunity, and strong protection against both virus challenge and virus transmission.