Publications

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: 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.

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: 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.

Highly pathogenic avian influenza viruses (HPAIV) pose a serious public health concern. In March 2024, a first-time outbreak of HPAIV H5N1 in dairy cattle herds was reported in the United States (US). Since then, the virus has continued to spread in cattle herds and spilt over into humans. We recently showed that the first human isolate reported in the US in Texas (HPhTX) from a dairy worker in an affected cattle farm has enhanced replication kinetics and pathogenicity in mice compared to a closely related bovine isolate (HPbTX). However, the molecular determinants of differential pathogenicity have not yet been identified. Herein, we show that HPhTX has enhanced polymerase activity, compared with HPbTX, in human cells and that the polymerase basic 2 (PB2) protein is the main factor responsible for this difference. Through single and combined site-directed mutagenesis and swapping the three amino acids different between HPhTX and HPbTX, we found that PB2 mutation E627 K is the major contributor to the enhanced polymerase activity of HPhTX. E362G substitution in HPhTX PB2 affected the polymerase, although to a lesser extent than E627 K. Moreover, M631L mutation in HPhTX PB2 enhanced polymerase activity. Rescue of a loss-of-function recombinant HPhTX (rHPhTX) containing mutations at residues 627 and 362, alone or in combination, revealed a contribution of PB2 E362G and K627E in morbidity, mortality, and viral replication as compared to rHPhTX wild-type (WT), and significantly reduced viral pathogenicity to levels comparable to rHPbTX WT. These findings indicate that HPAIV H5N1 of cattle origin isolated from the first human case has post-transmission amino acid changes that increase viral replication in human cells and pathogenicity in mice.

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed unprecedented challenges to public health and economic stability. Central to SARS-CoV-2 pathogenesis is its ability to evade the host immune response by hijacking host pathways via the interaction between viral and host proteins. We identified Ras-GTPase-activating protein SH3 domain-binding protein 1/2 (G3BP1/G3BP2) as a critical host factor that interacts with the viral nucleocapsid (N) protein, emerging from a comparative analysis of proteomic data from multiple studies. We revisited the underlying molecular mechanisms by confirming the residues required for the interaction between G3BP1/G3BP2 and SARS-CoV-2 N protein and showed that this interaction disrupts stress granule formation. Intriguingly, we observed that the ablation of both G3BP1 and G3BP2 enhanced SARS-CoV-2 replication. Our data collectively supports the notion that G3BP1 and G3BP2 play a critical role in modulating the host-virus interface during SARS-CoV-2 infection, and that their multifaceted function in cellular defense extends beyond the stress granule pathway.

Background/Objectives: Co-transfection of multiple DNAs is important to many research and therapeutic applications. While the optimization of single plasmid transfection is common, multiple plasmid co-transfection analyses are limited. Here we provide empirical data regarding multiple plasmid co-transfection while altering the number of species of plasmids transfected (up to four different plasmids) and the amount of plasmids/cell using the two most common non-viral techniques, electroporation and lipofection. Methods: A549 human lung epithelial cells were transfected using lipofectamine 2000 or electroporation with combinations of plasmids, each expressing one of four different fluorescent proteins from the CAGG promoter. Twenty-four hours later, cells were analyzed by spectral flow cytometry to determine the number of cells expressing each fluorescent protein and the amount of fluorescent signal of each protein in a cell. Results and Conclusions: For electroporation, while the fraction of cells expressing plasmids increased with increasing amounts of DNA, increasing the number of plasmid species did not alter the fraction of expressing cells and had no effect on levels of expression in individual cells. By contrast, for lipofection, the fraction of cells expressing plasmids was not affected by the amount of DNA added but both the fraction of cells expressing and the level of protein produced in these cells decreased for each plasmid species as the number of delivered species increased. For both lipofection and electroporation after single plasmid transfection, the expressing cells had greater numbers of plasmid copies/cell than non-expressing cells. Multiple plasmid lipofection resulted in more plasmid copies/cell in co-expressing than non-expressing cells. Multiple plasmid electroporation was the inverse of this with fewer plasmid copies/cell in co-expressing than non-expressing cells.

Avian Influenza viruses (AIVs) present a public health risk, especially with seasonal vaccines offering limited protection. AIV H5N1 clade 2.3.4.4b has caused a multi-state outbreaks in the United States (US) poultry and cattle since March 2024, raising pandemic concerns. We developed a nonstructural protein 1 (NS1)-deficient mutant of a low pathogenic version of the cattle-origin human influenza A/Texas/37/2024 H5N1, namely LPhTXdNS1, and assessed its safety, immunogenicity, and protection efficacy. LPhTXdNS1 is attenuated in vitro, showing reduced replication efficiency in Vero cells and inability to control IFNβ promoter activation. The LPhTXdNS1-immunized C57BL/6 J mice exhibit significantly reduced viral replication and pathogenicity compared to those infected with the low pathogenic version expressing NS1, namely LPhTX. Notably, a single intranasal dose of LPhTXdNS1 elicited protective immune responses, providing robust protection against lethal wild-type H5N1 challenge. These results demonstrate that LPhTXdNS1 is safe and able to induce protective immune responses against H5N1.

Since Spring 2024, new genotypes of highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b have been identified in the United States (US). These HPAI H5N1 genotypes have caused unprecedented multi-state outbreaks in poultry and dairy farms, and human infections. Here, we discuss the current situation of this outbreak and emphasizes the need for pre-pandemic preparedness to control HPAI H5N1 in both poultry and dairy farms in the US.