Biology subjects

Martinez-Sobrido, L.

Publications and source records attributed to Martinez-Sobrido, L..

At least 19 recordsLinked to original sources

bioRxiv2026

Mice immunized with N1 neuraminidase in a single cycle infectious virus or in a recombinant nanoparticle vaccine are protected against lethal challenge with highly pathogenic avian influenza H5N1 influenza

The recent spillover of highly pathogenic (HP) H5N1 clade 2.3.4.4b into dairy cattle and other mammalian species has been concerning. Vaccine candidates that can be rapidly synthesized and implemented are key countermeasures for protecting the human population against emerging pathogens. Here we examine the use of a pH1N1 virus lacking the genetic material for hemagglutinin (HA), limiting the infection to a single cycle (sciIAV) or Cobalt lipid-porphyrin nanoparticles decorated with neuraminidase (NA) of HPAI H5N1 (N1-CoPoP) as vaccine candidates. C57BL/6 mice that were inoculated intranasally with a single dose of pH1N1 sciIAV did not differ in serum NA antibody compared to uninoculated mice. However, mice that were inoculated intramuscularly with the N1-CoPoP alone or in combination with pH1N1 sciIAV had increased N1 IgG antibodies. Upon lethal challenge with HPAIV H5N1, all mice inoculated with either pH1N1 sciIAV or N1-CoPoP were protected from weight loss, unlike unvaccinated mice with substantial body weight losses that succumb to viral infection between 7 and 10 days post infection (dpi). Both pH1N1 sciIAV and N1-CoPoP immunization minimized viral infection such that only one mouse receiving pH1N1 sciIAV had detectable viral titer in the lungs at 4 dpi. By 6 dpi, no mouse inoculated with pH1N1 sciIAV and/or N1-CoPoP had detectable virus in nasal turbinates, lungs or the brain, unlike the high viral titers found in uninoculated mice. Both pH1N1 sciIAV and N1-CoPoP were protective and should be investigated further as alternative vaccine candidates.

microbiology↗

bioRxiv2026

Scarless SARS-CoV-2 Genome Engineering and Variant Analysis

In addition to causing cold and flu-like symptoms, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can also cause chronic longer-term diseases. Antiviral drugs, especially used combinatorially, have the potential to reduce the severity of individual infections and prevent the development of chronic disease. One of the safest and most versatile reverse genetics systems for SARS-CoV-2 studies is a bacterial artificial chromosome (BAC)-based system harboring the WA1 strain full-length genome and attenuating deletions in the accessory open reading frame 3a and 7b proteins (ORF3a and ORF7b, respectively). Here, a scarless genome engineering technique called En Passant mutagenesis was used to change one amino acid in the viral main protease (Mpro P132) into the residue present in contemporary Omicron strains (H132), in order to more accurately study protease inhibitors and resistance mechanisms. This recombinant, attenuated viral system yields antiviral EC50 values for the active component of approved drugs including nirmatrelvir (Paxlovid) and ensitrelvir (Xocova) and, importantly, also enables a parallel assessment of drug efflux. For instance, the antiviral potency of nirmatrelvir improves 50-fold by inhibiting the P-Glycoprotein (P-Gp) transporter with ritonavir or tariquidar, whereas the potency of ensitrelvir is unaffected. This system also enables the safe isolation and characterization of viral variants with reduced sensitivity to drugs, as evidenced by Mpro M49L compromising the efficacy of ensitrelvir. Together, these systems combine to provide safe, reliable, and quantitative approaches for Mpro variant analysis and drug testing without the biosafety concerns of conducting these experiments using wildtype isolates.

microbiology↗

bioRxiv2026

Genome-Scale Codon Deoptimization Enables Attenuation of Rift Valley Fever Virus

Rift Valley Fever Virus (RVFV) is a mosquito-borne zoonotic pathogen responsible for severe disease in domestic and wild ungulates as well as humans, representing a major threat to livestock production and human public health. RVFV is endemic in many African countries and has the potential to spread to new geographical regions. Current vaccines have limitations in safety and efficacy, highlighting the need for strategies to develop new vaccines candidates. In this study, we explored the use of codon deoptimization (CD) as a novel attenuation approach for the development of live-attenuated vaccine (LAV) against RVFV. CD exploits the redundancy of the genetic code by replacing frequently used codons with synonymous, less-preferred codons, thereby reducing translational efficiency without altering the amino acid sequence. We recoded parts of the M and S genome segments of RVFV using the least frequently used codons in mammalian cells, ensuring complete preservation of protein functionality and immunogenicity. Using reverse genetics, we rescued a panel of recombinant (r)RVFV encoding codon-deoptimized S-segment NSs gene (rNScd), M-segment Gn/Gc genes (rMcd), or both (rMcd/NScd). These recombinant CD viruses were characterized in vitro in mammalian and insect cell lines and in vivo using wild-type and immunocompromised mice. Results demonstrated varying degrees of attenuation among the three CD rRVFV, with the one deoptimized in both viral segments, rMcd/NScd, as a promising LAV based on the safety profiles. This study provides proof of concept for the use of CD as a rational strategy to generate attenuated RVFV, for the development of next-generation vaccines against this zoonotic threat.

microbiology↗

bioRxiv2026

Enhanced Pathogenicity and Contact Transmissibility of Human-origin Avian Influenza H5N1 Clade 2.3.4.4b Genotype B3.13 Compared to D1.1 in Ferrets

Since its emergence in 2020, multiple genotypes of the H5N1 clade 2.3.4.4b have been identified, with B3.13 and D1.1 emerging in the USA as two major and concerning genotypes. However, their relative pathogenicity and transmissibility in mammals have not been fully elucidated. We compared the pathogenicity and transmissibility of the first two human H5N1 clade 2.3.4.4b cases caused by B3.13 in Texas (A/Texas/37/2024; HPhTX B3.13) and D1.1 in Louisiana (A/Louisiana/12/2024; HPhLA D1.1) in a ferret model of infection and transmission. HPhTX B3.13 infection resulted in more severe clinical disease and enhanced viral shedding, with evidence of increased transmission relative to HPhLA D1.1. Histopathological analysis revealed more extensive lung pathology in animals infected with HPhTX B3.13, consistent with increased viral loads and inflammatory responses. Importantly, both genotypes showed no significant differences in reactivity to ferret sera raised against candidate vaccine virus (CVV) strains, receptor binding properties, or neuraminidase (NA) activity and thermostability. Whole-genome sequencing revealed no adaptive mutations in HPhTX B3.13 following infection or transmission. In contrast, HPhLA D1.1 showed rapid acquisition of the mammalian-adaptive mutation E627K in infected ferrets and both E627K and Q194K in the only fatal contact animal. Both mutations were associated with enhanced polymerase activity and computational analyses suggested that they enhance interactions with the mammalian host factors ANP32A and B. Our findings indicate that B3.13 is already well adapted for mammalian infection and transmission whereas D1.1 retains evolutionary potential through the rapid acquisition of adaptive mutations, highlighting important genotype-specific differences relevant to zoonotic risk assessment and pandemic preparedness. SignificanceInfluenza H5N1 viruses continue to diversify genetically while expanding into mammalian hosts, increasing opportunities for viral adaptation and zoonotic transmission, including humans. However, whether the predominant clade 2.3.4.4b genotype differs in its capacity to infect, transmit, and evolve in mammals remains poorly understood. Using the ferret model of influenza infection and transmission, we demonstrated that the currently circulating B3.13 and D1.1 genotypes exhibit distinct pathogenic and transmission characteristics despite retaining similar receptor-binding characteristics, NA functions, and antigenic profiles. While B3.13 readily infects and transmits in ferrets and does not develop further adaptive mutations associated with increased replication and transmission, D1.1 rapidly acquires mammalian-adaptive mutations after a single infection and/or transmission event, highlighting its evolutionary potential. These findings show that genotype-specific biological properties can influence zoonotic risk independently of antigenic similarity and emphasize the importance of integrating phenotypic characterization with genomic surveillance to improve pandemic preparedness and guide public health risk assessment.

microbiology↗

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

A One-Shot Multivalent Live-Attenuated Candidate Influenza Vaccine against Divergent Zoonotic H5N1 Clades

The continued emergence of genetically diverse high pathogenicity avian influenza (HPAI) H5N1 viruses with zoonotic potential highlights the urgent need for developing vaccines capable of providing broad protection against multiple circulating clades. Here, we developed a one-shot, multivalent, live-attenuated influenza vaccine (LAIV) based on the temperature-sensitive (ts), cold-adapted (ca), and attenuated (att) influenza A/Ann Arbor/6/1960 master donor virus (MDV) that incorporates the hemagglutinin (HA) and neuraminidase (NA) glycoproteins from representative clades 2.3.4.4b (A/Louisiana/12/2024), 2.3.2.1a (A/Victoria/149/2024), and 2.3.2.1e (A/Cambodia/2302009/2023) H5N1 viruses. A single intranasal (IN) immunization of C57BL/6 mice with the multivalent LAIV elicited robust humoral immune responses, with immune sera exhibiting broad cross-reactivity against antigens from all three H5N1 clades included in the vaccine. Following homologous viral challenge, vaccinated C57BL/6 mice were completely protected from disease, demonstrating the immunogenicity and protective efficacy of the multivalent LAIV. By simultaneously targeting antigenically distinct H5N1 lineages with pandemic potential, this strategy expands antigenic coverage within a single LAIV to confirm pan-H5N1 protection. Together, these findings support the development and implementation of this multivalent LAIV as a broadly protective pan-H5N1 LAIV for pandemic preparedness. SignificanceThe increasing genetic diversity of zoonotic H5N1 viruses complicates vaccine design. We developed a multivalent live-attenuated influenza vaccine (LAIV) based on the temperature-sensitive, cold-adapted, and attenuated (ts, ca, att) master donor virus (MDV) influenza A/Ann Arbor/6/1960 backbone that expresses the hemagglutinin (HA) and neuraminidase (NA) glycoproteins of H5N1 clades 2.3.4.4b, 2.3.2.1a, and 2.3.2.1e. A single intranasal (IN) immunization with the multivalent LAIV induced broadly cross-reactive neutralizing antibody (NAb) responses and protected experimental vaccinated animals against homologous lethal viral challenge, demonstrating the feasibility of the multivalent LAIV to protect against H5N1 clades of highest concern to humans. These findings demonstrate the feasibility of developing and implementing this multivalent LAIV as a broad protective pan-H5N1 LAIV against divergent H5N1 viruses for human use.

microbiology↗

bioRxiv2026

Neuraminidase-Based Cross-Protective Immunity against H5N1 Influenza Viruses in Humans

Highly pathogenic avian influenza A(H5N1) virus continues to expand globally across wild birds, poultry, and multiple mammalian hosts, and the detection of genetic features associated with mammalian adaptation underscores the need to better define preexisting human immunity relevant to pandemic preparedness. Although serologic and vaccine studies have historically emphasized hemagglutinin (HA), neuraminidase (NA) is a key antigen capable of providing protective immunity. Here, we performed a retrospective analysis of NA-specific antibodies in human sera collected across ten influenza seasons spanning the pre- and post-2009 pandemic periods. A total of 749 paired human serum samples (1,498 total) were obtained prior to seasonal inactivated influenza vaccination (T1) and 28 days after vaccination (T2). NA-inhibiting antibodies were measured by enzyme-linked lectin assay (ELLA) using N1 antigens representing three genotypes (B.3.13, D1.1, and EA-2021-AB) from clade 2.3.4.4b A(H5N1) viruses. In parallel, HA-directed responses were assessed by hemagglutination inhibition (HAI). In addition, luciferase-based microneutralization assays were performed to assess the presence of neutralizing antibodies. NA-inhibiting antibodies were detectable against all three genotypes across seasons. Seasonal vaccination was associated with a modest but reproducible increase in NA-inhibiting titers, most apparent from the 2009-2010 season onward, coincident with the introduction of A(H1N1)pdm09 into seasonal vaccine formulations. In contrast, HAI activity against H5 was generally low or undetectable, despite detectable HA-reactive antibodies by ELISA. These data indicate that cross-reactive NA-specific antibodies are present in human sera and can be boosted by seasonal vaccination, supporting increased consideration of NA in influenza serosurveillance and vaccine evaluation. IMPORTANCEThe continued spread of A(H5N1) viruses and reports of mammalian adaptation markers heighten concerns about potential zoonotic transmission into humans. Most studies of human antibody responses focus on influenza HA, but NA is also an important immune target. By analyzing paired sera from humans collected over ten influenza seasons, we show that NA-inhibiting antibodies cross-reacting with multiple contemporary genotypes from clade 2.3.4.4b A(H5N1) viruses are detectable and can be modestly boosted seasonal inactivated influenza vaccination, particularly after the 2009 pandemic. These findings highlight the importance of inducing NA-specific antibodies during influenza vaccination and in including NA in surveillance and in the assessment of vaccines intended to improve preparedness for emerging influenza viruses.

immunology↗

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

Recovery of proofreading-impaired SARS-CoV-2 reveals a mutator phenotype and an ExoN activity threshold for viability

Coronaviruses (CoVs) replicate unusually large RNA genomes that necessitate proofreading by the 3'-to-5' exoribonuclease (ExoN) formed by nonstructural proteins 14 (nsp14) and 10 (nsp10). Previous studies suggested that inactivation of the ExoN catalytic site in severe acute respiratory syndrome CoV 2 (SARS-CoV-2) is lethal, leaving unresolved whether the virus can tolerate impaired proofreading activity. Here, we investigated the functional requirement for ExoN in SARS-CoV-2 replication by combining a continuous fluorescence-based biochemical assay with an optimized single-bacmid reverse genetics system. Mutational analysis of residues involved in RNA binding or catalysis revealed graded effects on ExoN activity in vitro. Alanine substitution of Lys9, a residue positioned near the RNA-binding interface, did not reduce ExoN activity, whereas charge reversal at this position (K9E) impaired activity more strongly than alanine substitutions of the catalytic motif I residues D90 and E92 (D90A/E92A). Correspondingly, recombinant SARS-CoV-2 carrying K9A was readily recovered, whereas the D90A/E92A mutant was recovered only after an extended delay and K9E could not be rescued despite repeated attempts. The D90A/E92A mutant exhibited reduced replication while maintaining the engineered ExoN substitutions during serial passage. Deep sequencing of viral populations revealed a marked increase in genome-wide sequence variation in the D90A/E92A mutant, demonstrating a stable mutator phenotype. Together, these findings indicate that SARS-CoV-2 can tolerate substantial impairment of ExoN activity but depends on a minimal activity threshold for viability. This system provides a platform for defining how SARS-CoV-2 proofreading controls genome stability, viral fitness, and sensitivity to antiviral strategies that exploit reduced replication fidelity. ImportanceCoronaviruses have unusually large RNA genomes because they encode a proofreading enzyme that removes copying errors during replication. It has been unclear whether SARS-CoV-2 can survive when this proofreading function is strongly weakened, because earlier studies suggested that loss of the enzymes catalytic activity is lethal. We show that SARS-CoV-2 can tolerate substantial impairment of proofreading, but only when residual exonuclease activity remains above a minimal threshold. A virus with impaired proofreading replicates less efficiently and accumulates mutations across its genome, whereas a more severe defect prevents virus recovery. These findings clarify how coronavirus proofreading balances genome stability with viral fitness and provide a useful system for studying how reduced replication fidelity affects viral evolution, antiviral sensitivity, and attenuation. Defining this activity threshold may also help guide antiviral strategies that target coronavirus proofreading.

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

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

A bireporter recombinant SARS-CoV-2 Omicron BA.5 for in vitro and in vivo studies

The continuous emergence of variants of concern (VoC) represent a significant challenge to effectively control SARS-CoV-2. Although FDA-approved vaccines and antivirals have been successfully developed and implemented for the prophylactic and therapeutic intervention of SARS-CoV-2 infection, recent VoC could escape protection garnered by previous vaccine and antiviral approaches. Determining the efficacy of prophylactics and/or therapeutics against recent VoC will assist in efficiently controlling currently circulating SARS-CoV-2 strains. We used our previously described bacterial artificial chromosome (BAC)-based reverse genetics approach for Omicron BA.5 to generate a recombinant SARS-CoV-2 BA.5 encoding a fusion of ZsGreen to Nanoluciferase (rBA.5 ZsG-Nluc) from the locus of the viral nucleocapsid (N) protein separated by the porcine teschovirus-1 (PTV-1) 2A proteolytic cleavage site. The rBA5 ZsG-Nluc replicates to levels comparable to recombinant BA.5 wild-type (rBA.5 WT) and expresses high levels of ZsG and Nluc in cultured cells. This facilitates tracking viral infection and the identification of antivirals and neutralizing antibodies (NAbs) with EC50 and NT50 values, respectively, similar to those obtained with rBA.5 WT. Importantly, in K18 hACE2 mice, rBA.5 ZsG-Nluc retains the same pathogenicity and ability to replicate in the lungs of infected mice as rBA.5 WT. Using rBA.5 ZsG-Nluc, we detected Nluc activity systemically and Nluc and ZsG expression in the lungs of infected mice using an in vivo imaging system (IVIS). Our results demonstrate the feasibility of using rBA.5 ZsG-Nluc to track viral infections and identify prophylactics and therapeutics against recent SARS-CoV-2 VoC in vitro, ex vivo, and in vivo. IMPORTANCESARS-CoV-2, the causative virus of the COVID-19 pandemic, is continually evolving to escape immunity acquired by previous natural infections or vaccinations. Moreover, recent SARS-CoV-2 variants of concern (VoC) have acquired antiviral resistant mutations to FDA-approved drugs. The emergence of these VoC highlights the importance of identifying new prophylactics and therapeutics against currently circulating SARS-CoV-2 strains. We generated a recombinant bireporter Omicron BA.5 SARS-CoV-2 (rBA.5 ZsG-Nluc) that expresses reporter proteins, which are useful for cellular and whole animal studies and has similar viral replication and pathogenicity to a wild-type (WT) recombinant Omicron BA.5 SARS-CoV-2 (rBA.5 WT). In K18 hACE2 mice, rBA.5 ZsG-Nluc infection can be tracked systemically or in the lungs of infected mice using an in vivo imaging system (IVIS). We establish a proof-of-concept platform of rBA.5 ZsG-Nluc in combination with an ancestral SARS-CoV-2 strain expressing mCherry to simultaneously identify antivirals and neutralizing antibodies against original and recent SARS-CoV-2 strains.

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

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

Identification of protective human monoclonal antibodies using a K18 hACE2 transgenic mouse SARS-CoV-2 challenge model

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), uses human angiotensin converting enzyme 2 (hACE2) as its obligate receptor for cell entry. The K18 hACE2 transgenic mouse line, which expresses hACE2 under control of the human keratin 18 (K18) promoter, is used as an animal model for the study of COVID-19 pathogenesis. Here, we evaluate this model in the screening of human monoclonal antibody (hmAb) therapies against SARS-CoV-2. We included 206 hmAbs from the Coronavirus Immunotherapeutic Consortium Database (CoVIC-DB) and identified many that protected against a lethal challenge with the virus. Our data showed that mouse weight change from day 5 onward highly correlated with survival. Many of the protective hmAb candidates we identified also showed strong viral neutralization and spike protein (SP) binding when measured in vitro; however, in many cases, in vitro assays failed to identify protective hmAbs, suggesting that the mouse model may capture characteristics of the hmAbs that other methods cannot. Our findings demonstrate the relevance of including in vivo models for the characterization of therapeutics against SARS-CoV-2, as these improve both accuracy and expediency in the screening process.

immunology↗