Biology subjects

Plemper, R. K.

Publications and source records attributed to Plemper, R. K..

At least 19 recordsLinked to original sources

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↗

bioRxiv2025

Developmental candidate GHP-88310/EIDD-3608 with high tolerability and oral efficacy in measles and respiratory paramyxovirus models

Orthoparamyxoviruses such as human parainfluenza virus type-3 (HPIV3) and measles virus (MeV), are a major health threat. We discovered an orally efficacious broad-spectrum inhibitor of orthoparamyxovirus polymerases. However, here we found that tolerability in higher mammals was limited. We report development of clinical candidate analog GHP-88310 (EIDD-3608), which combines improved oral efficacy with favorable tolerability in non-rodents (ferrets and dogs). GHP-88310 was active against HPIV3, Sendai virus (SeV), MeV, and related canine distemper virus (CDV). In 7-day tolerability studies, daily doses of 2,000 mg/kg were well tolerated. Pharmacokinetic analysis revealed altered plasma exposure of GHP-88310 compared to the original hit. In HPIV3-infected cotton rats, GHP-88310 lowered respiratory tract viral load. Dosing of ferrets infected with CDV, causing lethal measles-like disease, resulted in complete survival, reduction of viremia and shed viral load, and alleviated lymphocytopenia. Once-daily GHP-88310 was efficacious in the CDV-ferret and HPIV3-cotton rat models. The compound was sterilizing against HPIV3 at physiological concentrations in human airway epithelium organoids.

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 and Characterization of Novel Chikungunya Virus Polymerase Inhibitors

Chikungunya virus (CHIKV) and other alphaviruses in the Togaviridae family are positive-sense RNA viruses and major human pathogens, causing millions of infections worldwide. In humans, alphaviruses such as CHIKV, Mayaro and Ross River viruses typically cause arthritogenic disease characterized by debilitating arthralgia, joint inflammation, fever, and rash. Although a vaccine was recently approved for use against CHIKV, no vaccines are licensed against other alphaviruses. No antiviral treatments are available to prevent or treat infections by any alphavirus. To address this unmet need, we used a CHIKV nanoluciferase reporter virus to develop a high-throughput screening assay for novel small-molecule inhibitors. From this campaign, we identified several unique inhibitors of CHIKV replication. Mechanistic characterization of two inhibitors revealed that both target the nsP4 RNA-dependent RNA polymerase, while susceptibility profiling pinpointed unique nsP4 mutations that specifically confer resistance. In silico docking analyses indicated potential binding poses of the inhibitors near the polymerase active site. Collectively, these results define multiple chemotypes for further development and highlight novel molecular targets within nsP4 for CHIKV inhibition. IMPORTANCEChikungunya virus is a mosquito-borne pathogen that has caused millions of human infections worldwide, producing severe fever, rash, and long-lasting joint pain that can persist for months. Related viruses such as Mayaro and Ross River viruses also cause debilitating disease, yet no antiviral drugs are available to treat any infection caused by this family of viruses. In this study, we developed a high-throughput assay that allowed us to rapidly identify compounds capable of blocking chikungunya virus replication. We discovered new hit compounds that inhibit virus growth. In addition, we determined that two of the most promising hit candidates target the viral nsP4 polymerase. By identifying these novel inhibitors and characterizing both their mechanisms of action and resistance profiles, we have established the groundwork for future efforts to develop much needed therapies against chikungunya virus and related pathogens.

molecular biology↗

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

SARS-CoV-2 causes chronic lung inflammation and impaired respiratory capacity in aged Roborovski dwarf hamsters

Roborovski dwarf hamsters are permissive for SARS-CoV-2 infection and progress to acute viral pneumonia with profound lung tissue injury, recapitulating hallmarks of severe COVID-19 in vulnerable patient groups such as older adults. In this study, we established dwarf hamster whole body plethysmography and assessed disease severity and propensity for long-term compromise of lung recovery from severe COVID-19-like disease in young, adult, and aged animals. Aged dwarf hamsters infected intranasally with variant of concern (VOC) omicron BA.4 experienced more severe clinical signs, carried a higher lung virus load, and had a greater risk of succumbing to infection. Resting airway hypersensitivity was transiently increased in aged, but not young, dwarf hamsters 3-4 days post infection (dpi). Pharmacologically induced respiratory distress revealed compromised lung capacity in animals of both age groups at peak disease. Aged animals showed impaired respiratory function for 45 days, mounted a weaker antiviral response, and developed chronic pneumonia with lasting tissue damage. Treatment of acute disease with approved antivirals, paxlovid-like nirmatrelvir+ritonavir or molnupiravir, prevented long-term respiratory sequelae in aged animals. Nirmatrelvir+ritonavir fully suppressed transient respiratory distress and mediated complete survival of aged animals. This study shows a high positive correlation between host age and SARS-CoV-2 disease severity in dwarf hamsters, establishes a model for chronic pneumonia with impaired respiratory capacity in at-risk hosts, and demonstrates benefit of antiviral therapy of acute disease for long-term respiratory health. Author SummaryIn the COVID-19 pandemic, the frequency of chronic respiratory insufficiency after acute SARS-CoV-2 infection was positively linked to patient age. Roborovski dwarf hamsters recapitulate hallmarks of life-threatening COVID-19 in at-risk patients that present with acute respiratory failure and prolonged respiratory incapacitation. In this study, we monitored disease progression and lung function in young and aged dwarf hamsters infected with a VOC omicron isolate and assessed the effect of antiviral treatment on long-term lung function. We established a strong correlation between host age and SARS-CoV-2 disease severity in dwarf hamsters, identified a high propensity of aged animals to develop chronic lung inflammation, and demonstrated a long-term loss of respiratory capacity in the subset of aged animals that survived the acute infection. Antiviral treatment suppressed the development of late sequelae and preserved lung function. These results have important implications for effective SARS-CoV-2 management in aged hosts at high risk of developing severe viral pneumonia with long-term impaired lung function.

microbiology↗

bioRxiv2025

Reprogramming of alveolar macrophages by intestinal segmented filamentous bacteria protects mice from lethal bacterial pneumoniae following influenza infection

The most severe outcomes of respiratory viral infection (RVI) result from secondary bacterial infection, which RVI promotes via depletion of alveolar macrophages (AM). Colonization of the intestine by the common but non-ubiquitous commensal, segmented filamentous bacteria (SFB), reprograms AM to resist RVI-induced depletion. Hence, we examined if SFB against secondary infection by S. pneumoniae, H. influenzae, or S. aureus, following primary infection by influenza virus (IAV). Indeed, SFB colonization conferred strong post-IAV protection against these lethal bacterial pathogens. AM depletion and transplant studies indicated that SFB reprogramming these cells was necessary and sufficient for such protection. Assay of AM, ex vivo, from SFB-colonized mice argued their protection against secondary bacterial infection was not only due to their withstanding IAV-induced depletion. Rather, AM from SFB-colonized mice displayed complement-dependent increases in phagocytosis and killing of these bacteria. Furthermore, AM from SFB-colonized mice stably held their enhanced anti-bacterial phenotype even when transplanted into an inflamed interferon-rich post IAV-environment. Thus, SFB, and perhaps gut microbiota composition in general influences proneness to bacterial pneumonia, especially post-RVI. One Sentence summarySFB colonization stably changed the phenotype of alveolar macrophages resulting in sustained clearance of bacterial pathogens even amidst an inflamed interferon-rich immune suppressed lung.

immunology↗

bioRxiv2024

Efficacy of late-onset antiviral treatment in immune-compromised hosts with persistent SARS-CoV-2 infection

The immunocompromised are at high risk of prolonged SARS-CoV-2 infection and progression to severe COVID-19. However, efficacy of late-onset direct-acting antiviral (DAA) therapy with therapeutics in clinical use and experimental drugs to mitigate persistent viral replication is unclear. In this study, we employed an immunocompromised mouse model, which supports prolonged replication of SARS-CoV-2 to explore late-onset treatment options. Tandem immuno-depletion of CD4+ and CD8+ T cells in C57BL/6 mice followed by infection with SARS-CoV-2 variant of concern (VOC) beta B.1.351 resulted in prolonged infection with virus replication for five weeks after inoculation. Early-onset treatment with nirmatrelvir/ritonavir (paxlovid) or molnupiravir was only moderately efficacious, whereas the experimental therapeutic 4-fluorourdine (4-FlU, EIDD-2749) significantly reduced virus load in upper and lower respiratory compartments four days post infection (dpi). All antivirals significantly lowered virus burden in a 7-day treatment regimen initiated 14 dpi, but paxlovid-treated animals experienced rebound virus replication in the upper respiratory tract seven days after treatment end. Viral RNA was detectable 28 dpi in paxlovid-treated animals, albeit not in the molnupiravir or 4-FlU groups, when treatment was initiated 14 dpi and continued for 14 days. Low-level virus replication continued 35 dpi in animals receiving vehicle but had ceased in all treatment groups. These data indicate that late-onset DAA therapy significantly shortens the duration of persistent virus replication in an immunocompromised host, which may have implications for clinical use of antiviral therapeutics to alleviate the risk of progression to severe disease in highly vulnerable patients. ImportanceFour years after the onset of the global COVID-19 pandemic, the immunocompromised are at greatest risk of developing life-threatening severe disease. However, specific treatment plans for this most vulnerable patient group have not yet been developed. Employing a CD4+ and CD8+ T cell-depleted immunocompromised mouse model of SARS-CoV-2 infection, we explored therapeutic options of persistent infections with standard-of-care paxlovid, molnupiravir, and the experimental therapeutic 4-FlU. Late-onset treatment initiated 14 days after infection was efficacious, but only 4-FlU was rapidly sterilizing. No treatment-experienced viral variants with reduced susceptibility to the drugs emerged, albeit virus replication rebounded in animals of the paxlovid group after treatment end. This study supports the use of direct-acting antivirals for late-onset management of persistent SARS-CoV-2 infection in immunocompromised hosts. However, treatment courses likely require to be extended for maximal therapeutic benefit, calling for appropriately powered clinical trials to meet the specific needs of this patient group.

microbiology↗

bioRxiv2024

Tetracistronic Minigenomes Elucidate a Functional Promoter for Ghana Virus and Unveils Cedar Virus Replicase Promiscuity for all Henipaviruses

Batborne henipaviruses, such as Nipah virus and Hendra virus, represent a major threat to global health due to their propensity for spillover, severe pathogenicity, and high mortality rate in human hosts. Coupled with the absence of approved vaccines or therapeutics, work with the prototypical species and uncharacterized, emergent species is restricted to high biocontainment facilities. There is a scarcity of such specialized spaces for research, and often the scope and capacity of research which can be conducted at BSL-4 is limited. Therefore, there is a pressing need for innovative life-cycle modeling systems to enable comprehensive research within lower biocontainment settings. This work showcases tetracistronic, transcription and replication competent minigenomes for Nipah virus, Hendra virus, Cedar virus, and Ghana virus, which encode viral proteins facilitating budding, fusion, and receptor binding. We validate the functionality of all encoded viral proteins and demonstrate a variety of applications to interrogate the viral life cycle. Notably, we found that the Cedar virus replicase exhibits remarkable promiscuity, efficiently rescuing minigenomes from all tested henipaviruses. We also apply this technology to GhV, an emergent species which has so far not been isolated in culture. We demonstrate that the reported sequence of GhV is incomplete, but that this missing sequence can be substituted with analogous sequences from other henipaviruses. Use of our GhV system establishes the functionality of the GhV replicase and identifies two antivirals which are highly efficacious against the GhV polymerase. Author SummaryHenipaviruses, such as the prototypical Nipah virus and Hendra virus, are recognized as significant global health threats due to their high mortality rates and lack of effective vaccines or therapeutics. Due to the requirement for high biocontainment facilities, the scope of research which may be conducted on henipaviruses is limited. To address this challenge, we developed innovative tetracistronic, transcription and replication competent minigenomes for Nipah virus, Hendra virus, Cedar virus, as well as for the emergent species, Ghana virus. We demonstrate that these systems replicate key aspects of the viral life cycle, such as budding, fusion, and receptor binding, and are safe for use in lower biocontainment settings. Importantly, application of this system to Ghana virus revealed that its known sequence is incomplete; however, substituting the missing sequences with those from other henipaviruses allowed us to overcome this challenge. We demonstrate that the Ghana virus replicative machinery is functional and identify two orally-efficacious antivirals effective against it. Further, we compare the compatibility of divergent henipavirus replicases with heterotypic viral genetic elements, providing valuable insights for how these species have evolved. Our research offers a versatile system for life-cycle modeling of highly pathogenic henipaviruses at low biocontainment.

microbiology↗

bioRxiv2023

Influenza A virus resistance to 4-fluorouridine coincides with viral attenuation in vitro and in vivo

Pre-existing or rapidly emerging resistance of influenza viruses to approved antivirals makes the development of novel therapeutics to mitigate seasonal influenza and improve preparedness against future influenza pandemics an urgent priority. We have recently identified the chain-terminating broad-spectrum nucleoside analog clinical candidate 4-fluorouridine (4-FlU) and demonstrated oral efficacy against seasonal, pandemic, and highly pathogenic avian influenza viruses in the mouse and ferret model. Here, we have resistance-profiled 4-FlU against a pandemic A/CA/07/2009 (H1N1) (CA09). In vitro viral adaptation yielded six independently generated escape lineages with distinct mutations that mediated moderate resistance to 4-FlU in the genetically controlled background of recombinant CA09 (recCA09). Mutations adhered to three distinct structural clusters that are all predicted to affect the geometry of the active site of the viral RNA-dependent RNA polymerase (RdRP) complex for phosphodiester bond formation. Escape could be achieved through an individual causal mutation, a combination of mutations acting additively, or mutations functioning synergistically. Fitness of all resistant variants was impaired in cell culture, and all were attenuated in the mouse model. Oral 4-FlU administered at lowest-efficacious (2 mg/kg) or elevated (10 mg/kg) dose overcame moderate resistance when mice were inoculated with 10 LD50 units of parental or resistant recCA09, demonstrated by significantly reduced virus load and complete survival. In the ferret model, invasion of the lower respiratory tract by variants representing four adaptation lineages was impaired. Resistant variants were either transmission-incompetent, or spread to untreated sentinels was fully blocked by therapeutic treatment of source animals with 4-FlU. Author SummaryReduced sensitivity to FDA-approved influenza drugs is a major obstacle to effective antiviral therapy. We have previously demonstrated oral efficacy of a novel clinical candidate drug, 4-FlU, against seasonal, pandemic, and highly pathogenic avian influenza viruses. In this study, we have determined possible routes of influenza virus escape from 4-FlU and addressed whether resistance imposes a viral fitness penalty, affecting pathogenicity or ability to transmit. We identified three distinct clusters of mutations that lead to moderately reduced viral sensitivity to the drug. Testing of resistant variants against two chemically unrelated nucleoside analog inhibitors of influenza virus, conditionally approved favipiravir and the broad-spectrum SARS-CoV-2 drug molnupiravir, revealed cross-resistance of one cluster with favipiravir, whereas no viral escape from molnupiravir was noted. We found that the resistant variants are severely attenuated in mice, impaired in their ability to invade the lower respiratory tract and cause viral pneumonia in ferrets, and transmission-defective or compromised. We could fully mitigate lethal infection of mice with the resistant variants with standard or 5-fold elevated oral dose of 4-FlU. These results demonstrate that partial viral escape from 4-FlU is feasible in principle, but escape mutation clusters are unlikely to reach clinical significance or persist in circulating influenza virus strains.

microbiology↗

bioRxiv2023

Intestinal microbiota programming of alveolar macrophages influences severity of respiratory viral infection

Susceptibility to respiratory virus infections (RVIs) varies widely across individuals. Because the gut microbiome impacts immune function, we investigated the influence of intestinal microbiota composition on RVI and determined that segmented filamentous bacteria (SFB), naturally acquired or exogenously administered, protected mice against influenza virus (IAV) infection. Such protection, which also applied to respiratory syncytial virus and SARS-CoV-2, was independent of interferon and adaptive immunity but required basally resident alveolar macrophages (AM). In SFB-negative mice, AM were quickly depleted as RVI progressed. In contrast, AM from SFB-colonized mice were intrinsically altered to resist IAV-induced depletion and inflammatory signaling. Yet, AM from SFB-colonized mice were not quiescent. Rather, they directly disabled IAV via enhanced complement production and phagocytosis. Accordingly, transfer of SFB-transformed AM into SFB-free hosts recapitulated SFB-mediated protection against IAV. These findings uncover complex interactions that mechanistically link the intestinal microbiota with AM functionality and RVI severity. One sentence summaryIntestinal segmented filamentous bacteria reprogram alveolar macrophages promoting nonphlogistic defense against respiratory viruses.

immunology↗

bioRxiv2023

Therapeutic mitigation of measles-like immune amnesia and exacerbated disease after prior respiratory virus infections in ferrets

After years of the COVID-19 pandemic, over 40 million children worldwide are at risk of measles due to delayed vaccination1 and temporary SARS-CoV-2 viral dominance2. Acute measles has a case-fatality rate of [~]1%, but most morbidity and mortality arise post-measles due to destruction of pre-existing immune memory by lymphotropic measles virus (MeV)3,4, a paramyxovirus of the Morbillivirus genus. MeV-induced immune amnesia is not mitigated by post-exposure vaccination and the impact of unrelated respiratory virus disease history on measles severity has not been defined. We used a lethal canine distemper virus (CDV)-ferret model as surrogate for human morbillivirus disease5 and employed the orally efficacious broad-spectrum paramyxovirus polymerase inhibitor GHP-883096 to establish measles treatment paradigms. Applying a receptor tropism-intact recombinant CDV with low lethality, we provide in vivo confirmation of the morbillivirus immune amnesia hypothesis and reveal an 8-day advantage of antiviral treatment versus therapeutic vaccination in preserving immune memory. Infection of ferrets with non-lethal influenza A virus (IAV) A/CA/07/2009 (H1N1) or respiratory syncytial virus (RSV) four weeks prior to CDV caused exacerbated CDV disease that rapidly advanced to fatal hemorrhagic pneumonia associated with lung onslaught by commensal bacteria. RNAseq of BAL samples and lung tissue identified CDV-induced expression of trefoil factor (TFF) peptides, which was absent in animals pre-infected with IAV, thus highlighting that immune priming by unrelated respiratory viruses influences morbillivirus infection outcome. Non-invasive pulmonary ferret MRI revealed that severe outcomes of consecutive IAV/CDV infections were prevented by oral GHP-88309 treatment even when initiated after peak clinical signs of CDV. These findings validate the morbillivirus immune amnesia hypothesis, define treatment paradigms for measles, identify prior disease history as risk factor for exacerbated morbillivirus disease, and demonstrate that treating morbillivirus infection with direct-acting oral antivirals provides therapeutic benefit regardless of whether the time window to mitigate primary clinical signs of infection has closed.

microbiology↗

bioRxiv2022

Paxlovid-like nirmatrelvir/ritonavir fails to block SARS-CoV-2 transmission in ferrets

Despite the continued spread of SARS-CoV-2 and emergence of variants of concern (VOC) that are capable of escaping preexisting immunity, therapeutic options are underutilized. In addition to preventing severe disease in high-risk patients, antivirals may contribute to interrupting transmission chains. The FDA has granted emergency use authorizations for two oral drugs, molnupiravir and paxlovid. Initial clinical trials suggested an efficacy advantage of paxlovid, giving it a standard-of-care-like status in the United States. However, recent retrospective clinical studies suggested a more comparable efficacy of both drugs in preventing complicated disease and case-fatalities in older adults. For a direct efficacy comparison under controlled conditions, we assessed potency of both drugs against SARS-CoV-2 in two relevant animal models; the Roborovski dwarf hamster model for severe COVID-19 in high-risk patients and the ferret model of upper respiratory tract disease and transmission. After infection of dwarf hamsters with VOC omicron, paxlovid and molnupiravir were efficacious in mitigating severe disease and preventing death. However, a pharmacokinetics-confirmed human equivalent dose of paxlovid did not significantly reduce shed SARS-CoV-2 titers in ferrets and failed to block virus transmission to untreated direct-contact ferrets, whereas transmission was fully suppressed in a group of animals treated with a human-equivalent dose of molnupiravir. Prophylactic administration of molnupiravir to uninfected ferrets in direct contact with infected animals blocked productive SARS-CoV-2 transmission, whereas all contacts treated with prophylactic paxlovid became infected. These data confirm retrospective reports of similar therapeutic benefit of both drugs for older adults, and reveal that treatment with molnupiravir, but not paxlovid, may be suitable to reduce the risk of SARS-CoV-2 transmission.

microbiology↗

bioRxiv2022

4-Fluorouridine mitigates lethal infection with pandemic human and highly pathogenic avian influenza viruses

Influenza outbreaks are associated with substantial morbidity, mortality and economic burden. Next generation antivirals are needed to treat seasonal infections and prepare against zoonotic spillover of avian influenza viruses with pandemic potential. Having previously identified oral efficacy of the nucleoside analog 4-Fluorouridine (4-FlU, EIDD-2749) against SARS-CoV-2 and respiratory syncytial virus, we explored activity of the compound against seasonal and highly pathogenic influenza (HPAI) viruses in cell culture, human airway epithelium organoids, and/or two animal models, ferrets and mice, that assess IAV transmission and lethal viral pneumonia, respectively. 4-FlU inhibited a panel of relevant influenza A and B viruses with nanomolar potency in organoids. In vitro polymerase assays revealed immediate chain termination of IAV polymerase after 4-FlU incorporation, in contrast to delayed chain termination of SARS-CoV-2 and RSV polymerase. Once-daily oral treatment of ferrets with 2 mg/kg 4-FlU initiated 12 hours after infection rapidly stopped virus shedding and prevented direct-contact transmission to untreated sentinels. Treatment of mice infected with a lethal inoculum of pandemic A/CA/07/2009 (H1N1)pdm09 (Ca09) with 2 mg/kg 4-FlU alleviated pneumonia. Three doses mediated complete survival when treatment was initiated up to 60 hours after infection, indicating an unusually broad window for effective intervention. Therapeutic oral 4-FlU ensured survival of animals infected with HPAI A/VN/12/2003 (H5N1) and of immunocompromised mice infected with pandemic Ca09. Recoverees were fully protected against homologous reinfection. This study defines the mechanistic foundation for high sensitivity of influenza viruses to 4-FlU and supports 4-FlU as developmental candidate for the treatment of seasonal and pandemic influenza. Author SummaryNext-generation antiviral therapeutics are needed to better mitigate seasonal influenza and prepare against zoonotic virus spillover from animal reservoirs. At greatest risk are the immunocompromised and patients infected with highly pathogenic influenza viruses. In this study, we have demonstrated efficacy of a broad-spectrum nucleoside analog, 4-fluorouridine, against a representative panel of influenza viruses in cell culture, human organoids, and two animal models, ferrets and mice. Acting as an immediate chain terminator of the influenza virus polymerase, once-daily oral treatment protected against lethal infection with seasonal and highly pathogenic avian influenza viruses, prevented direct-contact transmission to untreated sentinels, and mitigated lethal infection of immunocompromised hosts. These results support the developmental potential of 4-fluorouridine for treatment of vulnerable patient groups and mitigation of pandemic influenza, providing a much-needed additional therapeutic option for improved disease management.

microbiology↗

bioRxiv2022

Monitoring SARS-CoV-2 infection using a double reporter-expressing virus

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the highly contagious agent responsible for the coronavirus disease 2019 (COVID-19) pandemic. An essential requirement for understanding SARS-CoV-2 fundamental biology and the impact of anti-viral therapeutics are robust methods to detect for the presence of the virus in infected cells or animal models. Despite the development and successful generation of recombinant (r)SARS-CoV-2 expressing fluorescent or luciferase reporter genes, knowledge acquired from their use in in vitro assays and/or in live animals are limited to the properties of the fluorescent or luciferase reporter genes. Herein, for the first time, we engineered a replication-competent rSARS-CoV-2 that expresses both fluorescent (mCherry) and luciferase (Nluc) reporter genes (rSARS-CoV-2/mCherry-Nluc) to overcome limitations associated with the use of a single reporter gene. In cultured cells, rSARS-CoV-2/mCherry-Nluc displayed similar viral fitness as rSARS-CoV-2 expressing single reporter fluorescent and luciferase genes (rSARS-CoV-2/mCherry and rSARS-CoV-2/Nluc, respectively), or wild-type (WT) rSARS-CoV-2, while maintaining comparable expression levels of both reporter genes. In vivo, rSARS-CoV-2/mCherry-Nluc has similar pathogenicity in K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice than rSARS-CoV-2 expressing individual reporter genes, or WT rSARS-CoV-2. Importantly, rSARS-CoV-2/mCherry-Nluc facilitates the assessment of viral infection and transmission in golden Syrian hamsters using in vivo imaging systems (IVIS). Altogether, this study demonstrates the feasibility of using this novel bireporter-expressing rSARS-CoV-2 for the study SARS-CoV-2 in vitro and in vivo. IMPORTANCEDespite the availability of vaccines and antivirals, the coronavirus disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to ravage health care institutions worldwide. Previously, we have generated replication-competent recombinant (r)SARS-CoV-2 expressing fluorescent or luciferase reporter proteins to track viral infection in vitro and/or in vivo. However, these rSARS-CoV-2 are restricted to express only a single fluorescent or a luciferase reporter gene, limiting or preventing their use to specific in vitro assays and/or in vivo studies. To overcome this limitation, we have engineered a rSARS-CoV-2 expressing both fluorescent (mCherry) and luciferase (Nluc) genes and demonstrated its feasibility to study the biology of SARS-CoV-2 in vitro and/or in vivo, including the identification and characterization of neutralizing antibodies and/or antivirals. Using rodent models, we visualize SARS-CoV-2 infection and transmission through in vivo imaging systems (IVIS).

microbiology↗

bioRxiv2022

Immunization with recombinant accessory protein-deficient SARS-CoV-2 protects against lethal challenge and viral transmission

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to a worldwide Coronavirus Disease 2019 (COVID-19) pandemic. Despite high efficacy of the authorized vaccines, protection against the surging variants of concern (VoC) was less robust. Live-attenuated vaccines (LAV) have been shown to elicit robust and long-term protection by induction of host innate and adaptive immune responses. We sought to develop a COVID-19 LAV by generating 3 double open reading frame (ORF)-deficient recombinant (r)SARS-CoV-2 simultaneously lacking two accessory open reading frame (ORF) proteins (ORF3a/ORF6, ORF3a/ORF7a, and ORF3a/ORF7b). Here, we report that these double ORF-deficient rSARS-CoV-2 have slower replication kinetics and reduced fitness in cultured cells as compared to their parental wild-type (WT) counterpart. Importantly, these double ORF-deficient rSARS-CoV-2 showed attenuation in both K18 hACE2 transgenic mice and golden Syrian hamsters. A single intranasal dose vaccination induced high levels of neutralizing antibodies against different SARS-CoV-2 VoC, and also activated viral component-specific T-cell responses. Notably, the double ORF-deficient rSARS-CoV-2 were able to protect, as determined by inhibition of viral replication, shedding, and transmission, against challenge with SARS-CoV-2. Collectively, our results demonstrate the feasibility to implement these double ORF-deficient rSARS-CoV-2 as safe, stable, immunogenic and protective LAV for the prevention of SARS-CoV-2 infection and associated COVID-19 disease.

microbiology↗

bioRxiv2022

SARS-CoV-2 variant of concern type and biological sex affect efficacy of molnupiravir in dwarf hamster model of severe COVID-19

Summary ParagraphSARS-CoV-2 variants of concern (VOC) have triggered distinct infection waves in the coronavirus disease 2019 (COVID-19) pandemic, culminating in currently all-time high incidence rates of VOC omicron. Orally available direct-acting antivirals such as molnupiravir promise to improve disease management and limit SARS-CoV-2 spread. However, molnupiravir efficacy against VOC delta was questioned based on clinical trial results and its potency against omicron is unknown. This study evaluates molnupiravir against a panel of relevant VOC in three efficacy models: primary human airway epithelium organoids, the ferret model of upper respiratory disease, and a lethal Roborovski dwarf hamster efficacy model of severe COVID-19-like acute lung injury. All VOC were equally efficiently inhibited by molnupiravir in cultured cells and organoids. Treatment consistently reduced upper respiratory VOC shedding in ferrets and prevented viral transmission. Pathogenicity in the dwarf hamsters was VOC-dependent and highest for gamma, omicron, and delta with fulminant lung histopathology. Oral molnupiravir started 12 hours after infection resulted in complete survival of treated dwarf hamsters independent of challenge VOC. However, reduction in lung virus differed VOC-dependently, ranging from one (delta) to four (gamma) orders of magnitude compared to vehicle-treated animals. Dwarf hamsters infected with VOC omicron showed significant individual variation in response to treatment. Virus load reduction was significant in treated males, but not females. The dwarf hamster model recapitulates mixed efficacy of molnupiravir seen in human trials and alerts that therapeutic benefit of approved antivirals must be continuously reassessed in vivo as new VOC emerge.

microbiology↗