Biology subjects

Jackson, N.

Publications and source records attributed to Jackson, N..

At least 19 recordsLinked to original sources

bioRxiv2026

Mutations in SARS-CoV-2 Nsp1 are critical determinants of viral pathogenicity in mice

Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) nonstructural protein 1 (Nsp1) dampens the host immune response by shutting off host gene expression, a strategy that has remained evolutionary conserved among a diverse range of coronaviruses (CoVs). Residues important for SARS-CoV-2 Nsp1 mediated host shutoff have been incompletely defined. We have generated and characterized the ability of four Nsp1 mutants to inhibit host gene expression in both plasmid-based overexpression assays and utilized reverse genetic approaches to generate recombinant (r)SARS-CoV-2 expressing each Nsp1 mutant to investigate their impact on viral infection. Infection of K18-hACE2 transgenic mice with the rSARS-CoV-2 Nsp1 mutants resulted in reduced pathogenicity as determined by body weight maintenance and survival, attenuated viral replication in lung and nasal turbinate, distinct immune signatures in lung, and less severe lung pathology in comparison to wild-type (WT) virus-infected mice. Our data suggests amino acid residues in the C-terminal domain, in addition to the linker domain, of SARS-CoV-2 Nsp1 are critical determinants of viral pathogenicity as a result of their role in disrupting host gene expression. The reduced pathogenicity of rSARS-CoV-2 Nsp1 mutants highlights the potential for targeting Nsp1 for rational design of viral inhibitors and development of live-attenuated vaccine strategies as effective prophylactic and therapeutic treatments, respectively, to combat SARS-CoV-2 and possibly other CoV infections. IMPORTANCETo mitigate the ongoing public health threat posed by Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) and prepare for future coronavirus (CoV) outbreaks, there is an urgent need for effective prophylactic and therapeutic strategies, including vaccines and antivirals. The nonstructural protein 1 (Nsp1) is a conserved CoV virulence factor that suppresses host gene expression and disrupts immune responses. However, the contribution of specific Nsp1 residues to CoV pathogenesis remains unclear. Here, we identify residues within the C-terminal and linker regions of Nsp1 as critical determinants of SARS-CoV-2 pathogenicity in vivo. These findings advance our understanding of CoV host shutoff mechanisms and support Nsp1 as a promising target for the development of live-attenuated vaccines and antiviral therapeutics.

microbiology↗

bioRxiv2026

Substrate and target selectivity of 4'-fluoroadenosine against viral and host polymerases

Developing safe and effective treatments against emerging RNA viruses is an important goal in pandemic preparedness efforts. 4'-fluorouridine (4'-FlU) is a broad-spectrum antiviral that was shown to inhibit viral RNA-dependent RNA polymerases (RdRps). Given its notable range of antiviral activity, this class of nucleoside analogs warrants further investigation. Here, we studied the antiviral activity and underlying mechanism of inhibition of 4'-fluoroadenosine (4'-FlA). Like 4'-FlU, 4'-FlA demonstrates a broad-spectrum of antiviral activity against eight prototypic viruses representing diverse families. Enzyme kinetics show that the triphosphate (4'-FlA-TP) is efficiently incorporated by viral RdRps. A cryo-EM structure of RdRp of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in complex with double-stranded RNA and the incorporated monophosphate (4'-FlA-MP) characterizes interactions at the active site. The incorporated analog elicits heterogeneous inhibition patterns in primer extension reactions. In contrast, templates with embedded 4'-FlA-MP inhibit incorporation of complementary UTP across the viral RdRps. However, incorporation of 4'-FIA-TP is not limited to viral polymerases and likewise includes human mitochondrial RNA polymerase. These results demonstrate the general potential for 4'-fluorinated nucleotides as antiviral drugs and guide the development of more selective derivatives for medical use in appropriate settings.

microbiology↗

bioRxiv2026

A single PA-X mutation in bovine-origin H5N1 influenza virus reduces pathogenicity in mice

Dairy cows have emerged as a reservoir for human infection with highly pathogenic avian influenza (HPAI) H5N1. At the bovine-human interface, H5N1 strains may acquire adaptive mutations that influence their zoonotic potential. Sequence analysis identified a K142E substitution (bovine to human) in the PA and PA-X proteins, with the potential to affect both polymerase activity and host shutoff. Here, we used a loss-of-function approach to investigate how the bovine substitution (E142K) in PA/PA-X impacts viral replication, host shutoff activity, and pathogenicity in the human H5N1 background. Viral growth kinetics demonstrated that the virus containing the E142K substitution is attenuated, with reduced replication compared to wild-type (WT) virus. Consistently, PA-X-mediated host shutoff activity was reduced, resulting in increased induction of interferon (IFN) responses relative to WT. In vivo, mice infected with the E142K mutant virus survived, whereas infection with the WT virus was uniformly lethal. Despite comparable viral titers and inflammation score in mouse lungs, cytokine and chemokine profiling revealed distinct immune responses, with reduced CCL2 and increased CCL5 and IFN-{gamma} in mice infected with the E142K mutant virus compared to mice infected with the WT virus. These findings indicate that increased virulence of the human-adapted strain is driven by a PA-X mutation that modulates inflammatory responses, producing distinct immune signatures linked to host survival or viral lethality rather than changes in polymerase activity by PA. Collectively, these results highlight PA-X as a key determinant of pathogenicity of H5N1 and a potential target for the rational design of antiviral strategies. IMPORTANCEHighly pathogenic avian influenza (HPAI) H5N1 viruses have recently expanded beyond their traditional avian hosts to infect mammals, where they are acquiring mutations associated with mammalian adaptation. These changes raise the concern that influenza H5N1 viruses could evolve the capacity for sustained human infection and human-to-human transmission and pose a pandemic threat. Therefore, it is critical to identify and functionally characterize emerging mutations that influence viral pathogenicity and host interactions. Such studies will enhance our understanding of the requirements for efficient infection and disease in mammalian hosts and inform the rational design of antiviral strategies. In this study, we present data characterizing a bovine-to-human substitution (K142E) in the viral PA-X impacting viral replication, host shutoff activity, and pathogenicity. Our results demonstrate the key role of PA-X in H5N1 viral pathogenicity and the feasibility of targeting PA-X for the rational design of antivirals to control influenza infections.

microbiology↗

bioRxiv2026

Patient CACNA1A variant uncouples calcium channel conductance from synaptic output

Channelopathies are a class of neurodevelopmental disorders with often devastating consequences, and effective therapies depend on understanding how patient variants alter channel function. These effects are typically assessed by biophysical characterization in heterologous expression systems. Mutations in CACNA1A, which encodes the P/Q-type calcium channel CaV2.1, underlie a spectrum of neurological disorders in which symptoms are generally classified as loss-of-function (LoF) or gain-of-function (GoF). However, some patients present with overlapping phenotypes that defy this binary framework. Here we describe a CACNA1A variant for which heterologous assays fail to capture a key in vivo functional effect. We characterize a de novo variant of a highly conserved residue, D1634N, identified in a patient with a mixed clinical presentation that includes both LoF- and GoF-associated symptoms. Biophysical characterization in HEK293T cells supports a classic and severe LoF effect, including reduced current density and a right-shifted current-voltage relationship. In contrast, in vivo analysis of the corresponding endogenous variant in the C. elegans homolog reveals a paradoxical increase in spontaneous synaptic vesicle release, despite reduced channel expression. Molecular dynamics simulations predict that the mutation prolongs dwell time in a partially open state, potentially increasing calcium influx at rest. This model is supported by biophysical recordings of the human channel showing increased current at hyperpolarized potentials and by rescue of the C. elegans phenotype through genetic elevation of resting membrane potential. Together, these findings reconcile the patients clinical presentation by describing a complex, mixed-function variant, highlight the importance of cellular context in variant interpretation and therapeutic development, and establish C. elegans as a powerful in vivo platform for evaluating the functional consequences of pathogenic ion channel variants.

neuroscience↗

bioRxiv2026

Social Distancing Responses to Fungal Disease in an Australian Wild Lizard Population

Social living often confers substantial fitness benefits; however, close spatial association among individuals can also elevate opportunities for pathogen transmission, especially where the populations are dense. Despite this, the extent to which avoidance behaviours are expressed by wild reptiles facing fungal disease remains unclear. We examined Eastern Water Dragons (Intellagama lesueurii) in Roma Street Parklands, Brisbane, Australia, where a population is affected by the emerging fungal pathogen Nannizziopsis barbatae (Nb). Using a five-year dataset (2018-2023) spanning 146 individuals, we quantified social distance, as the minimum distance to the nearest neighbour, in relation to the number of diseased conspecifics that overlapped each individual seasonal core home area. Social distance was negatively associated with the number of diseased conspecifics, indicating a strong crowding effect; however, this association was weaker for diseased individuals, which were observed at larger distances than healthy individuals even when the number of diseased conspecifics was high. Together, these patterns indicate a context-dependent spacing response associated with disease status and local disease context, suggesting that infection risk constrains density-driven proximity. Our findings provide new insights into how disease pressure shapes social spacing in reptiles and contribute to a broader understanding of behavioural responses to emerging infectious fungal diseases.

animal behavior and cognition↗

bioRxiv2026

Coordinated IL-2 and TGF-β Signaling via a Novel Fusion Protein Selectively Expands and Activates Regulatory T Cells

Establishing immune tolerance requires the integration of cytokine signals that favor regulatory T cell (Treg) function over effector differentiation, yet how such coordination can be achieved selectively in vivo remains incompletely defined. Regulatory T cells are essential for maintaining immune homeostasis and peripheral tolerance, and both interleukin-2 (IL-2) and transforming growth factor-{beta} (TGF-{beta}) contribute to Treg activation, differentiation, and stability in a highly context-dependent manner. Here, we examine the consequences of coordinated IL-2 and TGF-{beta}3 signaling using an engineered cytokine construct combining attenuated IL-2 with receptor-masked TGF-{beta}3. In vitro, attenuated IL-2/TGF-{beta}3 signaling promoted conversion of naive CD4 T cells into Foxp3 induced Tregs, expanded endogenous Tregs, and suppressed inflammatory cytokine production by memory and effector CD4 T cells. In vivo, a single administration selectively increased the frequency of CD4Foxp3 regulatory T cells exhibiting an activated and stable phenotype, while minimally activating Foxp3- conventional CD4 T cells relative to IL-2 alone. In a Treg-deficiency-driven adoptive transfer model of autoimmunity, early exposure to coordinated IL-2/TGF-{beta}3 signaling enhanced Treg activation, reduced tissue infiltration by autoreactive T cells, and conferred sustained protection from autoimmune gastritis in the absence of continued treatment. Together, these findings identify a mode of coordinated IL-2 and TGF-{beta}3 signaling sufficient to stabilize regulatory T cell responses and promote immune tolerance while limiting activation of conventional CD4 T cells, highlighting signal integration as a determinant of tolerogenic immune regulation in vivo. One Sentence SummaryCUE-401 coordinates IL-2 and TGF-{beta} signaling to expand and activate regulatory T cells and restrain autoimmune responses.

immunology↗

bioRxiv2026

Fluorescent-expressing Modified Vaccinia Ankara Encoding T7 RNA Polymerase

Efficient recovery of negative-stranded RNA viruses from plasmid cDNA requires robust bacteriophage T7 RNA polymerase expression, commonly supplied by Modified Vaccinia Ankara expressing T7 polymerase (MVA T7). Here, we describe the generation and characterization of two fluorescent MVA T7 viruses expressing either enhanced green fluorescent protein (EGFP) or monomeric red fluorescent protein 1 (mRFP1). Both fluorescent-expressing MVA T7 viruses retain plaque morphology, growth kinetics, and transgene expression comparable to parental MVA T7. Importantly, both fluorescent-expressing MVA T7 viruses support rescue of replication-competent recombinant Vesicular Stomatitis Virus (rVSV) while enabling direct visualization of MVA T7 infection. Fluorescent protein expression from MVA T7 viruses facilitates stock generation and titration and allows detection of remaining helper virus during rVSV recovery and amplification. Altogehter, EGFP- and mRFP1-expressing MVA T7 viruses provide a practical tool for monitoring T7-driven rescue of recombinant RNA viruses and identifying helper virus carryover. IMPORTANCEReverse genetics systems for negative-stranded RNA viruses rely on robust bacteriophage T7 RNA polymerase expression, commonly provided by a Modified Vaccinia Ankara virus expressing T7 polymerase (MVA T7). However, generation of virus stocks, monitoring MVA T7 helper virus infection and detecting residual virus during rescue of negative-stranded RNA viral can be challenging. We generated fluorescent-expressing MVA T7 variants that enable direct visualization of helper virus infection without compromising viral growth kinetics, transgene expression, or efficient rescue of recombinant Vesicular Stomatitis Virus (rVSV) while enabling direct visualization of MVA T7 infection. MVA T7 viruses expressing fluorescent proteins simplify stock generation and titration, facilitate optimization of negative-stranded RNA rescue conditions, and allow rapid identification of helper virus carryover during generation and amplification of recombinant virus, improving the efficiency, reproducibility, and quality control in reverse genetics systems relying on T7 expression widely used across virology.

microbiology↗

bioRxiv2026

Development and characterization of mouse-adapted recombinant SARS-CoV-2 expressing reporter genes

Transgenic K18-hACE2 mice are a standard model for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), albeit with limitations. A mouse-adapted 30 (MA30) SARS-CoV-2 has been developed to allow infection of wild-type (WT) mice strains. However, SARS-CoV-2 MA30 cannot be easily tracked in vitro, ex vivo, or in vivo. To address the problem, we developed a recombinant (r)SARS-CoV-2 based on the MA30 strain expressing fluorescent (mCherry) and luciferase (nanoluciferase, Nluc) reporter genes, alone or in combination, that enable tracking of viral infection in WT C57BL/6 and BALB/c mice. Insertion of the reporter genes resulted in minor viral attenuation in vitro, with [~]0.5-1.0-log lower titers than rSARS-CoV-2 MA30 WT in A549 hACE2 cells, while maintain similar plaque morphology and replication kinetics in Vero AT cells. In vivo, reporter-expressing rSARS-CoV-2 MA30 caused transient weight loss, contrasting with lethal rSARS-CoV-2 MA30 WT infection. Bioluminescence imaging of rSARS-CoV-2 MA30 Nluc in C57BL/6 and BALB/c mice revealed peak pulmonary replication at 2 days post-infection, with resolution by day 4, and correlated with tissue viral loads. Our results demonstrate the feasibility of using rSARS-CoV-2 MA30 expressing reporter genes to track viral infection in vitro, ex vivo, and in vivo without a need for secondary approaches to monitor viral infection as are required for rSARS-CoV-2 MA30 WT. Our system is highly suitable to evaluate prophylactic vaccines and therapeutic antibodies or antiviral approaches in WT or transgenic C57BL/6 and BALB/c mice without the shortcomings of K18-hACE2 mice and with the added advantage of non-invasive monitoring of treatment efficacy. ImportanceThe K18-hACE2 transgenic mouse model limits the capability to study SARS-CoV-2. While a mouse adapted 30 (MA30) has been developed to study SARS-CoV-2 in wild-type (WT) mice, it does not allow non-invasive tracking of viral infections. Recombinant viruses expressing reporter genes enable real-time monitoring of infection dynamics, opening an avenue to study viral tropism and easily evaluate prophylactic and therapeutic approaches. They furthermore support longitudinal studies, which reduces the number of research animals required. Here, we show that a recombinant (r)SARS-CoV-2 expressing fluorescent (mCherry) and nanoluciferase (Nluc) reporter genes, alone or in combination, can be used to track viral infections in vitro, ex vivo, and in vivo without the need for secondary approaches that are required to detect SARS-CoV-2 MA30 in WT mice. These reporter-expressing rSARS-CoV-2 MA30 may accelerate vaccine development and antiviral drug discovery in WT or transgenic mice bypassing the need for hACE2 overexpression in K18-hACE2 transgenic mice.

microbiology↗

bioRxiv2026

Prolyl tRNA Synthetase Is Required for Mammarenavirus Multiplication

Several mammarenaviruses (MaAv), chiefly Lassa virus (LASV) in Western Africa and Junin virus (JUNV) in the Argentinean Pampas, cause severe disease in humans and pose important public health problems in their endemic regions. In addition, the globally distributed MaAv lymphocytic choriomeningitis virus (LCMV) is an underrecognized human pathogen of clinical significance especially in congenital infections and LCMV poses a serious risk for immunocompromised individuals. There are no FDA-approved MaAv vaccines or antivirals and current anti-MaAv therapy is limited to an off-label use of ribavirin whose efficacy remains controversial. This highlights an urgent unmet need for developing antivirals against human pathogenic MaAv. Halofuginone (HF), a derivative of the natural alkaloid febrifugine, has been shown to exhibit antiviral activity against several RNA viruses. Here, we present evidence that HF exhibits a potent dose-dependent antiviral activity against LCMV, and the hemorrhagic fever causing MaAv LASV and JUNV. HF binds to the bifunctional enzyme glutamyl-prolyl-tRNA synthetase 1 (EPRS1) and specifically inhibits its prolyl-tRNA synthetase (PRS) activity, resulting in translation inhibition via the amino acid starvation (AAS) response with preferential impact on proline-rich proteins. HF anti-LCMV activity was prevented by the addition of exogenous proline supporting that inhibition of PRS activity plays a critical role on the anti-MaAv activity of HF. We found that HF did not affect LCMV cell entry, modestly (twofold) reduced the activity of the virus ribonucleoprotein (vRNP) but strongly inhibited (>90%) Z budding activity, a process involving the Z proline-rich late domain motifs.

microbiology↗

bioRxiv2025

SARS-CoV-2 and MERS-CoV disrupt host protein synthesis via nsp1 with differential effects on the integrated stress response

Coronaviruses pose a serious threat to public health, driving the need for antiviral therapeutics and vaccines. Therefore, it is paramount to understand how this family of viruses evades cellular antiviral responses and establishes productive infection. The conserved coronavirus non-structural protein (nsp)1 has been shown to inhibit host protein synthesis and promote host mRNA degradation while viral mRNAs are protected. We showed previously that SARS-CoV-2 induces activation of host integrated stress response (ISR) kinases PKR and PERK, which promote phosphorylation of eIF2 and consequent inhibition of host protein synthesis. In contrast, eIF2 remains unphosphorylated during MERS-CoV infection. To investigate the interactions of nsp1 and the ISR kinases, we utilized recombinant SARS-CoV-2 and MERS-CoV expressing nsp1 with mutations in each of two conserved domains. Upon infection with SARS-CoV-2 nsp1 mutants, translation was shut down in wildtype (WT) and PKR knockout (KO) cells but rescued in PERK KO cells, likely due to reduced p-eIF2. In contrast, translation was rescued during infection with the analogous MERS-CoV nsp1 mutants even in WT cells. Moreover, SARS-CoV-2 WT suppressed expression of GADD34, a negative regulator of eIF2 phosphorylation, while SARS-CoV-2 nsp1 mutants induced GADD34. In contrast MERS-CoV WT induced GADD34. Utilizing single-molecule fluorescence in situ hybridization, we found that SARS-CoV-2 and MERS-CoV nsp1 promote host mRNA degradation during WT, but not nsp1 mutant, infection. Finally, while SARS-CoV-2 WT suppressed stress granule formation, nsp1 mutants induced stress granules containing host RNA. Thus, SARS-CoV-2 and MERS-CoV differ in interactions with the ISR and nsp1 control of host protein synthesis. SignificanceCoronaviruses cause disease across a wide range of animal species, and the human coronaviruses SARS-CoV-2 and MERS-CoV have caused epidemics of severe respiratory illness. Thus, it is imperative to understand how these viruses antagonize host responses and cause lethal disease. We show here that the betacoronavirus non-structural protein (nsp)1 promotes shutdown of host protein synthesis while preserving viral protein synthesis and, in addition, promotes degradation of host mRNAs. However, SARS-CoV-2 and MERS-CoV differ in their ability to manipulate the host integrated stress response, indicating that it is important to understand detailed coronavirus-host interactions and how they differ even between lethal coronaviruses. Such insights will inform the development of antiviral therapeutics to treat and prevent current and future coronavirus outbreaks.

microbiology↗

bioRxiv2025

The future is faeces: Using faecal genomic sequencing to understand dietary choices of an endangered arboreal marsupial

1. Understanding critical components of habitat use, such as dietary preferences, is crucial for creating and implementing effective conservation plans. This becomes challenging when species exhibit cryptic behaviours, such as arboreality and nocturnality, as seen in the endangered greater glider (Petauroides spp). Existing methods for determining greater glider diet are inadequate, time consuming and prone to human error. Furthermore, current literature lacks specificity, with most research stating simply that greater gliders are dietary eucalypt specialists, without providing additional insight into which species are eaten. 2. Here, we tested a non-invasive technique using targeted sequencing of species-specific single nucleotide polymorphisms from greater glider faecal samples to identify dietary components across multiple locations in Southeast Queensland. 3. Sequencing identified 17 plant species present in the faecal samples, including Angophora, Acacia and Casuarina spp. that were previously unknown to be feed tree taxa, profoundly increasing our understanding of the habitat requirements of this endangered marsupial. Four of these identified species were out of range of their natural occurrence, suggesting limitations to the accuracy of this technique. 4. This study demonstrates the value of using genomic sequencing for analysing diets of arboreal mammals and makes recommendations for improving the accuracy of this methodology for future studies. Our findings highlight key tree species to be considered important for future greater glider conservation plans and raises the question whether there are local drivers behind the differences in dietary choices across the landscape. Such information will be critical for greater glider conservation, particularly when management planning across multiple habitats.

molecular biology↗

bioRxiv2025

A cocktail of SARS-CoV-2 spike stem helix domain and receptor binding domain human monoclonal antibodies prevent the emerge of viral escape mutants

Neutralizing antibodies (NAbs) targeting the spike (S) glycoprotein remain a crucial therapeutic strategy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, emerging viral variants have escaped all Food and Drug Administration (FDA)-approved NAb treatments, underscoring the urgent need for effective therapeutic alternatives. Using a nanoluciferase (Nluc)-expressing attenuated recombinant SARS-CoV-2 lacking the open reading frames (ORF) 3a and 7b ({Delta}3a7b-Nluc), we characterized resistance profiles of two broadly protective NAbs targeting the S receptor binding domain (RBD) in S1 (1301B7) and the stem helix domain (SH) in S2 (1249A8). Serial passaging of {Delta}3a7b-Nluc under selective pressure identified a 1301B7 antibody-resistant mutants (ARM-B7) harboring an RBD mutation (S371F) that conferred resistance to 1301B7 and other RBD-directed NAbs (Casirivimab, SC27 and Sotrovimab). In contrast, no ARM emerged under treatment with 1249A8, or an antibody cocktail of 1301B7 (RBD) + 1249A8 (SH). These findings demonstrate that S2 SH-targeting NAbs shows higher genetic barrier to resistance than S1 RBD-targeting NAbs, and that a NAbs cocktail therapy targeting the SARS-CoV-2 S1 RBD and S2 SH offers the most effective strategy to prevent the emergence of escape mutations. Together, our findings provide critical insights into developing next-generation resistance-evading NAb therapies against SARS-CoV-2, and potentially other coronaviruses, and demonstrate the value of using our attenuated viral platforms for the safe identification of ARM without the potential biosafety concerns of doing these experiments using wild-type (WT) forms of SARS-CoV-2. SIGNIFICANCEThe clinical efficacy of early SARS-CoV-2 NAbs has been challenged by the emergence of escape viral variants, highlighting an urgent need to anticipate resistance. Using a luminescent attenuated SARS-CoV-2 platform, we profiled resistant mutations against two broadly protective SARS-CoV-2 NAbs. Passage of {Delta}3a7b-Nluc in the presence of a NAb targeting the RBD S1 domain (1301B7) readily selected for an ARM, whereas passage in the presence of an SH S2 domain NAb (1249A8) did not. Notably, a cocktail of 1301B7 and 1249A8 created a high barrier of selecting SARS-CoV-2 ARM, preventing the emergence of resistant variants. We identified an S371F mutation in the S1 RBD of ARM-B7 that confers resistance to 1301B7 and other S1 RBD-targeting NAbs. These results highlight the importance of combination therapies targeting both variable RBD S1 and conserved SH S2 domain for the efficient treatment of SARS-CoV-2 and to prevent the emerge of NAb-induced escape mutations.

microbiology↗

bioRxiv2025

Identification and characterization of a SARS-CoV-2 Mpro G23 deletion ensitrelvir-resistant mutant

Ensitrelvir is an antiviral drug that specifically targets the main protease (Mpro) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been approved for the treatment of coronavirus disease 2019 (COVID-19) due to the conservation of its target protein which is essential in the viral lifecycle. However, SARS-CoV-2 could introduce mutations in the viral proteins to confer resistance to antivirals. Thus, screening for drug-resistant SARS-CoV-2 mutants and elucidating their resistant mechanisms are critical for guiding the selection of effective antiviral therapies. Here, we utilized a luminescent attenuated SARS-CoV-2 ({Delta}3a7b-Nluc WT) to safely identify ensitrelvir drug-resistant mutants (DRM-E) without the need of using virulent forms of SARS-CoV-2. We isolated a DRM-E containing a G23 deletion (G23del) in Mpro with high resistance (>1,000 fold) to ensitrelvir, but not to the other Mpro inhibitor (nirmatrelvir) or to the RNA-dependent RNA polymerase (RdRp) inhibitor remdesivir. The contribution of G23del was confirmed by generating a recombinant luminescent attenuated SARS-CoV-2 containing G23del in the non-structural protein 5 (NSP5) gene ({Delta}3a7b-Nluc G23del). {Delta}3a7b-Nluc G23del exhibited significant resistance to ensitrelvir in both cultured cells an in K18 hACE2 transgenic mice. Binding affinity revealed that G23del mutation substantially altered Mpro binding affinity for ensitrelvir but not nirmatrelvir. In conclusion, our results demonstrate that G23del in Mpro can confer high resistance to ensitrelvir. Positively, G23del in Mpro does not render SARS-CoV-2 resistant to nirmatrelvir or remdesivir, suggesting the feasibility of treating infections with SARS-CoV-2 containing G23del with these other approved antivirals. SIGNIFICANCEThe clinical use of SARS-CoV-2 antiviral drugs is increasingly challenged by the emergence of drug-resistant mutants. Thus, there is a pressing need to identify and characterize antiviral escape SARS-CoV-2 variants, particularly for FDA-approved antivirals. Our study addresses this by employing a luminescent attenuated virus platform ({Delta}3a7b-Nluc WT) to safely identify and characterize resistance mutations without the concern of using virulent forms of SARS-CoV-2. Using this safe approach, we have identified a G23 deletion (G23del) in SARS-CoV-2 Mpro, which mediates resistance to ensitrelvir in vitro and in vivo. Importantly, while G23del was able to confer more than 1,000-fold increased resistance to ensitrelvir, SARS-CoV-2 containing G23del remained sensitive to other Mpro (nirmatrelvir) and RdRp (remdesivir) inhibitors. Altogether, this study demonstrates the feasibility of using {Delta}3a7b-Nluc to safely identify and characterize drug resistant viruses without the biosafety concern of using virulent SARS-CoV-2 and advance the design of next-generation antiviral drugs.

microbiology↗

bioRxiv2025

A luminescent attenuated SARS-CoV-2 for the identification and validation of drug-resistant mutants

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 {Delta}3a7b-Nluc, to facilitate tracking viral infection. Using this virus, we selected drug-resistant mutants to the main viral protease (Mpro) inhibitor nirmatrelvir. After passaging {Delta}3a7b-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 {Delta}3a7b-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 {Delta}3a7b-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. IMPORTANCESmall-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 ({Delta}3a7b-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 {Delta}3a7b-Nluc, we have selected variants with reduced sensitivity to nirmatrelvir that were validated by the generation of rSARS-CoV-2 {Delta}3a7b-Nluc containing the candidate L50F and R188G mutations in Mpro. These results demonstrate the feasibility of using rSARS-CoV-2 {Delta}3a7b-Nluc to safely identify and validate drug-resistant mutants overcoming concerns originating from adaptation studies using WT SARS-CoV-2.

microbiology↗

bioRxiv2025

Deletion of a KSF Motif Attenuates NSP1 Host Cell Translation Shutoff and Impairs SARS-CoV-2 Virulence

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), triggered a global pandemic with profound social and economic consequences. The viral spike (S) protein has been identified as a key determinant of SARS-CoV-2 pathogenicity. In this study, we demonstrate that the Omicron BA.4 and BA.5 variants, which have closely related S proteins, exhibit different virulence in K18-hACE2 transgenic mice. A comparison of genomic sequences revealed key differences between variants BA.4 and BA.5, including a three amino acid deletion ({Delta}KSF) in the linker region of the non-structural protein 1 (NSP1) in BA.4. Using reverse genetic systems, we engineered a recombinant (r)SARS-CoV-2 BA.5 expressing BA.4 NSP1, which was significantly attenuated in vivo, similar to the natural BA.4 isolate, compared to rBA.5 wild-type (WT). This finding indicates that NSP1 is responsible, at least in part, for the differences in virulence between BA.4 and BA.5. Mechanistically, BA.4 NSP1 showed a reduced ability to inhibit host gene translation compared to BA.5 NSP1. Notably, a rSARS-CoV-2 WA1 original strain containing the same {Delta}KSF in NSP1 was also attenuated in vivo compared to rWA1 WT. Together, these findings highlight the contributions of the NSP1 linker region to inhibiting host gene expression and SARS-CoV-2 pathogenicity, as well as the feasibility of targeting NSP1 for the rational design of live-attenuated vaccines and/or antivirals. IMPORTANCEUnderstanding why some SARS-CoV-2 variants cause more severe disease than others is crucial for proper management or for the rational design of prophylactic and therapeutic treatments. While most studies focus on the spike (S) protein, we found that another viral protein, NSP1, also plays a key role in disease severity among SARS-CoV-2 variants. A small deletion in NSP1, present in the Omicron BA.4 variant, weakens the virus ability to shut down the hosts immune response, making BA.4 less severe than BA.5. When we introduced the same deletion into the original SARS- CoV-2 WA1 strain, the virus also became less harmful. This discovery suggests that NSP1 is an important virulence factor and supports the feasibility of targeting NSP1 for the development of new prophylactic and therapeutic treatments against SARS-CoV-2. By uncovering NSP1s role in pathogenesis, our study provides insights that could help in designing better strategies to combat future variants of SARS-CoV-2.

microbiology↗

bioRxiv2025

A Live Attenuated Vaccine Candidate against Emerging Highly Pathogenic Cattle-Origin 2.3.4.4b H5N1 Viruses

Influenza viruses present a significant public health risk, causing substantial illness and death in humans each year. Seasonal flu vaccines must be updated regularly, and their effectiveness often decreases due to mismatches with circulating strains. Furthermore, inactivated vaccines do not provide protection against shifted influenza viruses that have the potential to cause a pandemic. The highly pathogenic avian influenza H5N1 clade 2.3.4.4b is prevalent among wild birds worldwide and is causing a multi-state outbreak affecting poultry and dairy cows in the United States (US) since March 2024. In this study, we have generated a NS1 deficient mutant of a low pathogenic version of the cattle-origin human influenza A/Texas/37/2024 H5N1, namely LPhTXdNS1, and validated its safety, immunogenicity, and protection efficacy in a prime vaccination regimen against wild-type (WT) A/Texas/37/2024 H5N1. The attenuation of LPhTXdNS1 in vitro was confirmed by its reduced replication in cultured cells and inability to control IFN{beta} promoter activation. In C57BL/6J mice, LPhTXdNS1 has reduced viral replication and pathogenicity compared to WT A/Texas/37/2024 H5N1. Notably, LPhTXdNS1 vaccinated mice exhibited high immunogenicity that reach its peak at weeks 3 and 4 post-immunization, leading to robust protection against subsequent lethal challenge with WT A/Texas/37/2024 H5N1. Altogether, we demonstrate that a single dose vaccination with LPhTXdNS1 is safe and able to induce protective immune responses against H5N1. Both safety profile and protection immunity suggest that LPhTXdNS1 holds promise as a potential solution to address the urgent need for an effective vaccine in the event of a pandemic for the treatment of infected animals and humans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=143 HEIGHT=200 SRC="FIGDIR/small/646033v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1ca0d0borg.highwire.dtl.DTLVardef@1e5232org.highwire.dtl.DTLVardef@a6694corg.highwire.dtl.DTLVardef@14b0832_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

A Human H5N1 Influenza Virus Expressing Bioluminescence for Evaluating Viral Infection and Identifying Therapeutic Interventions

A multistate outbreak of highly pathogenic avian influenza virus (HPAIV) H5N1 in dairy cows was first reported on March 25, 2024, in the United States (US), marking the first discovery of HPAIV H5N1 in cattle. Soon after, a dairy worker on an affected dairy farm became the first human case linked directly to this outbreak. Studies with influenza A virus (IAV) require secondary methods to detect the virus in infected cells or animal models of infection. We modified the non-structural (NS) genome segment of the human A/Texas/37/2024 (HPhTX) H5N1 virus to create a recombinant virus expressing nanoluciferase (HPhTX NSs-Nluc), enabling the tracking of virus in cultured cells and mice via in vitro, ex vivo, and in vivo imaging systems (IVIS). In vitro, HPhTX NSs-Nluc showed growth and plaque characteristics similar to its wild-type (WT) counterpart. In vivo, HPhTX NSs-Nluc allowed tracking viral infection in the entire animals and in the organs of infected animals using in vivo and ex vivo IVIS, respectively. Importantly, the morbidity, mortality, and replication titers of HPhTX NSs-Nluc were comparable to those of the WT HPhTX. In vitro, HPhTX NSs-Nluc was inhibited by Baloxavir acid (BXA) to levels observed with WT HPhTX. We also demonstrate the feasibility of using HPhTX NSs-Nluc to evaluate the antiviral activity of BXA in vivo. Our findings support that HPhTX NSs-Nluc represents an excellent tool for tracking viral infections, including the identification of prophylactics or therapeutics for the treatment of the HPAIV H5N1 responsible of the outbreak in dairy cows.

microbiology↗

bioRxiv2025

Identification of Amino Acid Residues Responsible for Differential Replication and Pathogenicity of Avian Influenza Virus H5N1 Isolated from Human and Cattle in Texas, US

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 E627K 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 E627K. 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.

microbiology↗