SARS-CoV-2 entry into and evolution within a skilled nursing facility.

SARS-CoV-2 belongs to the family Coronaviridae which includes multiple human pathogens that have an outsized impact on aging populations. As a novel human pathogen, SARS-CoV-2 is undergoing continuous adaptation to this new host species and there is evidence of this throughout the scientific and public literature. However, most investigations of SARS-CoV-2 evolution have focused on large-scale collections of data across diverse populations and/or living environments. Here we investigate SARS-CoV-2 evolution in epidemiologically linked individuals within a single outbreak at a skilled nursing facility beginning with initial introduction of the pathogen. The data demonstrate that SARS-CoV-2 was introduced to the facility multiple times without establishing an interfacility transmission chain, followed by a single introduction that infected many individuals within a week. This large-scale introduction by a single genotype then persisted in the facility. SARS-CoV-2 sequences were investigated at both the consensus and intra-host variation levels. Understanding the variability in SARS-CoV-2 during transmission chains will assist in understanding the spread of this disease and can ultimately inform best practices for mitigation strategies.

The Incompetence of Mosquitoes-Can Zika Virus Be Adapted To Infect Culex tarsalis Cells?

The molecular evolutionary mechanisms underpinning virus-host interactions are increasingly recognized as key drivers of virus emergence, host specificity, and the likelihood that viruses can undergo a host shift that alters epidemiology and transmission biology. Zika virus (ZIKV) is mainly transmitted between humans by Aedes aegypti mosquitoes. However, the 2015 to 2017 outbreak stimulated discussion regarding the role of Culex spp. mosquitoes in transmission. Reports of ZIKV-infected Culex mosquitoes, in nature and under laboratory conditions, resulted in public and scientific confusion. We previously found that Puerto Rican ZIKV does not infect colonized Culex quinquefasciatus, Culex pipiens, or Culex tarsalis, but some studies suggest they may be competent ZIKV vectors. Therefore, we attempted to adapt ZIKV to Cx. tarsalis by serially passaging virus on cocultured Ae. aegypti (Aag2) and Cx. tarsalis (CT) cells to identify viral determinants of species specificity. Increasing fractions of CT cells resulted in decreased overall virus titer and no enhancement of Culex cell or mosquito infection. Next-generation sequencing of cocultured virus passages revealed synonymous and nonsynonymous variants throughout the genome that arose as CT cell fractions increased. We generated nine recombinant ZIKVs containing combinations of the variants of interest. None of these viruses showed increased infection of Culex cells or mosquitoes, demonstrating that variants associated with passaging were not specific to increased Culex infection. These results reveal the challenge of a virus adapting to a new host, even when pushed to adapt artificially. Importantly, they also demonstrate that while ZIKV may occasionally infect Culex mosquitoes, Aedes mosquitoes likely drive transmission and human risk. IMPORTANCE ZIKV is mainly transmitted between humans by Aedes mosquitoes. In nature, ZIKV-infected Culex mosquitoes have been found, and ZIKV infrequently infects Culex mosquitoes under laboratory conditions. Yet, most studies show that Culex mosquitoes are not competent vectors for ZIKV. We attempted to adapt ZIKV to Culex cells to identify viral determinants of species specificity. We sequenced ZIKV after it was passaged on a mixture of Aedes and Culex cells and found that it acquired many variants. We generated recombinant viruses containing combinations of the variants of interest to determine if any of these changes enhance infection in Culex cells or mosquitoes. Recombinant viruses did not show increased infection in Culex cells or mosquitoes, but some variants increased infection in Aedes cells, suggesting adaptation to those cells instead. These results reveal that arbovirus species specificity is complex, and that virus adaptation to a new genus of mosquito vectors likely requires multiple genetic changes.

Detection of SARS-CoV-2 in exhaled air using non-invasive embedded strips in masks.

BACKGROUND: SARS-CoV-2 emerged in 2019 and resulted in a pandemic causing millions of infections worldwide. Gold-standard for SARS-CoV-2 detection uses quantitative RT-qPCR on respiratory secretions to detect viral RNA (vRNA). Acquiring these samples is invasive, can be painful for those with xerostomia and other health conditions, and sample quality can vary greatly. Frequently only symptomatic individuals are tested even though asymptomatic individuals can have comparable viral loads and efficiently transmit virus. METHODS: We utilized a non-invasive approach to detect SARS-CoV-2 in individuals, using polyvinyl alcohol (PVA) strips embedded in KN95 masks. PVA strips were tested for SARS-CoV-2 vRNA via qRT-PCR and infectious virus. RESULTS: We show efficient recovery of vRNA and infectious virus from virus-spiked PVA with detection limits comparable to nasal swab samples. In infected individuals, we detect both human and SARS-CoV-2 RNA on PVA strips, however, these levels are not correlated with length of time mask was worn, number of times coughed or sneezed, or level of virus in nasal swab samples. We successfully cultured and deep-sequenced PVA-associated virus. CONCLUSIONS: These results demonstrate the feasibility of using PVA-embedded masks as a non-invasive platform for detecting SARS-CoV-2 in exhaled air in COVID-positive individuals regardless of symptom status.

Early Adoption of Longitudinal Surveillance for SARS-CoV-2 among Staff in Long-Term Care Facilities: Prevalence, Virologic and Sequence Analysis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 and has become a major global pathogen in an astonishingly short period of time. The emergence of SARS-CoV-2 has been notable due to its impacts on residents in long-term care facilities (LTCFs). LTCF residents tend to possess several risk factors for severe outcomes of SARS-CoV-2 infection, including advanced age and the presence of comorbidities. Indeed, residents of LTCFs represent approximately 40% of SARS-CoV-2 deaths in the United States. Few studies have focused on the prevalence and transmission dynamics of SARS-CoV-2 among LTCF staff during the early months of the pandemic, prior to mandated surveillance testing. To assess the prevalence and incidence of SARS-CoV-2 among LTCF staff, characterize the extent of asymptomatic infections, and investigate the genomic epidemiology of the virus within these settings, we sampled staff for 8 to 11 weeks at six LTCFs with nasopharyngeal swabs from March through June of 2020. We determined the presence and levels of viral RNA and infectious virus and sequenced 54 nearly complete genomes. Our data revealed that over 50% of infections were asymptomatic/mildly symptomatic and that there was a strongly significant relationship between viral RNA (vRNA) and infectious virus, prolonged infections, and persistent vRNA (4+ weeks) in a subset of individuals, and declining incidence over time. Our data suggest that asymptomatic SARS-CoV-2-infected LTCF staff contributed to virus persistence and transmission within the workplace during the early pandemic period. Genetic epidemiology data generated from samples collected during this period support that SARS-CoV-2 was commonly spread between staff within an LTCF and that multiple-introduction events were less common. IMPORTANCE Our work comprises unique data on the characteristics of SARS-CoV-2 dynamics among staff working at LTCFs in the early months of the SARS-CoV-2 pandemic prior to mandated staff surveillance testing. During this time period, LTCF residents were largely sheltering-in-place. Given that staff were able to leave and return daily and could therefore be a continued source of imported or exported infection, we performed weekly SARS-CoV-2 PCR on nasal swab samples collected from this population. There are limited data from the early months of the pandemic comprising longitudinal surveillance of staff at LTCFs. Our data reveal the surprisingly high level of asymptomatic/presymptomatic infections within this cohort during the early months of the pandemic and show genetic epidemiological analyses that add novel insights into both the origin and transmission of SARS-CoV-2 within LTCFs.

Durable Antibody Responses in Staff at Two Long-Term Care Facilities, during and Post SARS-CoV-2 Outbreaks.

SARS-CoV-2 has had a disproportionate impact on nonhospital health care settings, such as long-term-care facilities (LTCFs). The communal nature of these facilities, paired with the high-risk profile of residents, has resulted in thousands of infections and deaths and a high case fatality rate. To detect presymptomatic infections and identify infected workers, we performed weekly surveillance testing of staff at two LTCFs, which revealed a large outbreak at one of the sites. We collected serum from staff members throughout the study and evaluated it for binding and neutralization to measure seroprevalence, seroconversion, and type and functionality of antibodies. At the site with very few incident infections, we detected that over 40% of the staff had preexisting SARS-CoV-2 neutralizing antibodies, suggesting prior exposure. At the outbreak site, we saw rapid seroconversion following infection. Neutralizing antibody levels were stable for many weeks following infection, suggesting a durable, long-lived response. Receptor-binding domain antibodies and neutralizing antibodies were strongly correlated. The site with high seroprevalence among staff had two unique introductions of SARS-CoV-2 into the facility through seronegative infected staff during the period of study, but these did not result in workplace spread or outbreaks. Together, our results suggest that a high seroprevalence rate among staff can contribute to immunity within a workplace and protect against subsequent infection and spread within a facility. IMPORTANCE Long-term care facilities (LTCFs) have been disproportionately impacted by COVID-19 due to their communal nature and high-risk profile of residents. LTCF staff have the ability to introduce SARS-CoV-2 into the facility, where it can spread, causing outbreaks. We tested staff weekly at two LTCFs and collected blood throughout the study to measure SARS-CoV-2 antibodies. One site had a large outbreak and infected individuals rapidly generated antibodies after infection. At the other site, almost half the staff already had antibodies, suggesting prior infection. The majority of these antibodies bind to the receptor-binding domain of the SARS-CoV-2 spike protein and are potently neutralizing and stable for many months. The non-outbreak site had two unique introductions of SARS-CoV-2 into the facility, but these did not result in workplace spread or outbreaks. Our results reveal that high seroprevalence among staff can contribute to immunity and protect against subsequent infection and spread within a facility.

SARS-CoV-2 infection, neuropathogenesis and transmission among deer mice: Implications for spillback to New World rodents.

Coronavirus disease-19 (COVID-19) emerged in late 2019 in China and rapidly became pandemic. As with other coronaviruses, a preponderance of evidence suggests the virus originated in horseshoe bats (Rhinolophus spp.) and may have infected an intermediate host prior to spillover into humans. A significant concern is that SARS-CoV-2 could become established in secondary reservoir hosts outside of Asia. To assess this potential, we challenged deer mice (Peromyscus maniculatus) with SARS-CoV-2 and found robust virus replication in the upper respiratory tract, lungs and intestines, with detectable viral RNA for up to 21 days in oral swabs and 6 days in lungs. Virus entry into the brain also occurred, likely via gustatory-olfactory-trigeminal pathway with eventual compromise to the blood-brain barrier. Despite this, no conspicuous signs of disease were observed, and no deer mice succumbed to infection. Expression of several innate immune response genes were elevated in the lungs, including IFNα, IFNß, Cxcl10, Oas2, Tbk1 and Pycard. Elevated CD4 and CD8ß expression in the lungs was concomitant with Tbx21, IFNγ and IL-21 expression, suggesting a type I inflammatory immune response. Contact transmission occurred from infected to naive deer mice through two passages, showing sustained natural transmission and localization into the olfactory bulb, recapitulating human neuropathology. In the second deer mouse passage, an insertion of 4 amino acids occurred to fixation in the N-terminal domain of the spike protein that is predicted to form a solvent-accessible loop. Subsequent examination of the source virus from BEI Resources determined the mutation was present at very low levels, demonstrating potent purifying selection for the insert during in vivo passage. Collectively, this work has determined that deer mice are a suitable animal model for the study of SARS-CoV-2 respiratory disease and neuropathogenesis, and that they have the potential to serve as secondary reservoir hosts in North America.

Genome Number and Size Polymorphism in Zika Virus Infectious Units.

Zika virus (ZIKV; Flaviviridae, Flavivirus) is an arthropod-borne infection that can result in severe outcomes, particularly in fetuses infected in utero It has been assumed that infection by ZIKV, as well as other viruses, is largely initiated by individual virus particles binding to and entering a cell. However, recent studies have demonstrated that multiple virus particles are frequently delivered to a cell simultaneously and that this collective particle delivery enhances infection. ZIKV is maintained in nature between Aedes aegypti mosquitos and vertebrate hosts, including humans. Human infection is initiated through the injection of a relatively small initial inoculum comprised of a genetically complex virus population. Since most mutations decrease virus fitness, collective particle transmission could benefit ZIKV and other arthropod-borne diseases by facilitating the maintenance of genetic complexity and adaptability during infection or through other mechanisms. Therefore, we utilized a barcoded ZIKV to quantify the number of virus genomes that initiate a plaque. We found that individual plaques contain a mean of 10 infecting viral genomes (range, 1 to 212). Few plaques contained more than two dominant genomes. To determine whether multigenome infectious units consist of collectively transmitting virions, infectious units of ZIKV were then separated mechanically by centrifugation, and heavier fractions were found to contain more genomes per plaque-forming unit, with larger diameters. Finally, larger/heavier infectious units reformed after removal. These data suggest that ZIKV populations consist of a variety of infectious unit sizes, likely mostly made up of aggregates, and only rarely begin with a single virus genome.IMPORTANCE The arthropod-borne Zika virus (ZIKV) infects humans and can cause severe neurological sequelae, particularly in fetuses infected in utero How this virus has been able to spread across vast geological ranges and evolve in new host populations is not yet understood. This research demonstrates a novel mechanism of ZIKV transmission through multigenome aggregates, providing insight into ZIKV evolution, immunologic evasion, and better future therapeutic design. This study shows that ZIKV plaques result from collections of genomes rather than individual genomes, increasing the potential for interactions between ZIKV genotypes.

SARS-CoV-2 infection, neuropathogenesis and transmission among deer mice: Implications for reverse zoonosis to New World rodents.

Coronavirus disease-19 (COVID-19) emerged in November, 2019 in China and rapidly became pandemic. As with other coronaviruses, a preponderance of evidence suggests the virus originated in horseshoe bats (Rhinolophus spp.) and likely underwent a recombination event in an intermediate host prior to entry into human populations. A significant concern is that SARS-CoV-2 could become established in secondary reservoir hosts outside of Asia. To assess this potential, we challenged deer mice (Peromyscus maniculatus) with SARS-CoV-2 and found robust virus replication in the upper respiratory tract, lungs and intestines, with detectable viral RNA for up to 21 days in oral swabs and 14 days in lungs. Virus entry into the brain also occurred, likely via gustatory-olfactory-trigeminal pathway with eventual compromise to the blood brain barrier. Despite this, no conspicuous signs of disease were observed and no deer mice succumbed to infection. Expression of several innate immune response genes were elevated in the lungs, notably IFNα, Cxcl10, Oas2, Tbk1 and Pycard. Elevated CD4 and CD8ß expression in the lungs was concomitant with Tbx21, IFNγ and IL-21 expression, suggesting a type I inflammatory immune response. Contact transmission occurred from infected to naive deer mice through two passages, showing sustained natural transmission. In the second deer mouse passage, an insertion of 4 amino acids occurred to fixation in the N-terminal domain of the spike protein that is predicted to form a solvent-accessible loop. Subsequent examination of the source virus from BEI Resources indicated the mutation was present at very low levels, demonstrating potent purifying selection for the insert during in vivo passage. Collectively, this work has determined that deer mice are a suitable animal model for the study of SARS-CoV-2 pathogenesis, and that they have the potential to serve as secondary reservoir hosts that could lead to periodic outbreaks of COVID-19 in North America.

A Hyperactive Kunjin Virus NS3 Helicase Mutant Demonstrates Increased Dissemination and Mortality in Mosquitoes.

The unwinding of double-stranded RNA intermediates is critical for the replication and packaging of flavivirus RNA genomes. This unwinding activity is achieved by the ATP-dependent nonstructural protein 3 (NS3) helicase. In previous studies, we investigated the mechanism of energy transduction between the ATP and RNA binding pockets using molecular dynamics simulations and enzymatic characterization. Our data corroborated the hypothesis that motif V is a communication hub for this energy transduction. More specifically, mutations T407A and S411A in motif V exhibit a hyperactive helicase phenotype, leading to the regulation of translocation and unwinding during replication. However, the effect of these mutations on viral infection in cell culture and in vivo is not well understood. Here, we investigated the role of motif V in viral replication using West Nile virus (Kunjin subtype) T407A and S411A mutants (T407A and S411A Kunjin, respectively) in cell culture and in vivo We were able to recover S411A Kunjin but unable to recover T407A Kunjin. Our results indicated that S411A Kunjin decreased viral infection and increased cytopathogenicity in cell culture compared to wild-type (WT) Kunjin. Similarly, decreased infection rates in surviving S411A Kunjin-infected Culex quinquefasciatus mosquitoes were observed, but S411A Kunjin infection resulted in increased mortality compared to WT Kunjin infection. Additionally, S411A Kunjin infection increased viral dissemination and saliva positivity rates in surviving mosquitoes compared to WT Kunjin infection. These data suggest that S411A Kunjin increases viral pathogenesis in mosquitoes. Overall, these data indicate that NS3 motif V may play a role in the pathogenesis, dissemination, and transmission efficiency of Kunjin virus.IMPORTANCE Kunjin and West Nile viruses belong to the arthropod-borne flaviviruses, which can result in severe symptoms, including encephalitis, meningitis, and death. Flaviviruses have expanded into new populations and emerged as novel pathogens repeatedly in recent years, demonstrating that they remain a global threat. Currently, there are no approved antiviral therapeutics against either Kunjin or West Nile viruses. Thus, there is a pressing need for understanding the pathogenesis of these viruses in humans. In this study, we investigated the role of the Kunjin virus helicase on infection in cell culture and in vivo This work provides new insight into how flaviviruses control pathogenesis and mosquito transmission through the nonstructural protein 3 helicase.

Longitudinal Surveillance for SARS-CoV-2 Among Staff in Six Colorado Long-Term Care Facilities: Epidemiologic, Virologic and Sequence Analysis.

BACKGROUND: SARS-CoV-2 emerged in 2019 and has become a major global pathogen. Its emergence is notable due to its impacts on individuals residing within long term care facilities (LTCFs) such as rehabilitation centers and nursing homes. LTCF residents tend to possess several risk factors for more severe SARS-CoV-2 outcomes, including advanced age and multiple comorbidities. Indeed, residents of LTCFs represent approximately 40% of SARS-CoV-2 deaths in the United States. METHODS: To assess the prevalence and incidence of SARS-CoV-2 among LTCF workers, determine the extent of asymptomatic SARS-CoV-2 infection, and provide information on the genomic epidemiology of the virus within these unique care settings, we collected nasopharyngeal swabs from workers for 8-11 weeks at six Colorado LTCFs, determined the presence and level of viral RNA and infectious virus within these samples, and sequenced 54 nearly complete genomes. FINDINGS: Our data reveal a strikingly high degree of asymptomatic/mildly symptomatic infection, a strong correlation between viral RNA and infectious virus, prolonged infections and persistent RNA in a subset of individuals, and declining incidence over time. INTERPRETATION: Our data suggest that asymptomatic SARS-CoV-2 infected individuals contribute to virus persistence and transmission within the workplace, due to high levels of virus. Genetic epidemiology revealed that SARS-CoV-2 likely spreads between staff within an LTCF. FUNDING: Colorado State University Colleges of Health and Human Sciences, Veterinary Medicine and Biomedical Sciences, Natural Sciences, and Walter Scott, Jr. College of Engineering, the Columbine Health Systems Center for Healthy Aging, and the National Institute of Allergy and Infectious Diseases.

Effects of Arbovirus Multi-Host Life Cycles on Dinucleotide and Codon Usage Patterns.

Arthropod-borne viruses (arboviruses) of vertebrates including dengue, zika, chikungunya, Rift Valley fever, and blue tongue viruses cause extensive morbidity and mortality in humans, agricultural animals, and wildlife across the globe. As obligate intercellular pathogens, arboviruses must be well adapted to the cellular and molecular environment of both their arthropod (invertebrate) and vertebrate hosts, which are vastly different due to hundreds of millions of years of separate evolution. Here we discuss the comparative pressures on arbovirus RNA genomes as a result of a dual host life cycle, focusing on pressures that do not alter amino acids. We summarize what is currently known about arboviral genetic composition, such as dinucleotide and codon usage, and how cyclical infection of vertebrate and invertebrate hosts results in different genetic profiles compared with single-host viruses. To serve as a comparison, we compile what is known about arthropod tRNA, dinucleotide, and codon usages and compare this with vertebrates. Additionally, we discuss the potential roles of genetic robustness in arboviral evolution and how it may vary from other viruses. Overall, both arthropod and vertebrate hosts influence the resulting genetic composition of arboviruses, but a great deal remains to be investigated.

Fitness Barriers Limit Reversion of a Proofreading-Deficient Coronavirus.

The 3'-to-5' exoribonuclease in coronavirus (CoV) nonstructural protein 14 (nsp14-ExoN) mediates RNA proofreading during genome replication. ExoN catalytic residues are arranged in three motifs: I (DE), II (E), and III (D). Alanine replacement of the motif I residues (AA-E-D; four nucleotide substitutions) in murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV yields viable mutants with impaired replication and fitness, increased mutation rates, and attenuated virulence in vivo Despite these impairments, MHV- and SARS-CoV ExoN motif I AA mutants (ExoN-AA) have not reverted at motif I in diverse in vitro and in vivo environments, suggesting that profound fitness barriers prevent motif I reversion. To test this hypothesis, we engineered MHV-ExoN-AA with 1, 2, or 3 nucleotide mutations along genetic pathways to AA-to-DE reversion. We show that engineered intermediate revertants were viable but had no increased replication or competitive fitness compared to that of MHV-ExoN-AA. In contrast, a low-passage-number (passage 10 [P10]) MHV-ExoN-AA showed increased replication and competitive fitness without reversion of ExoN-AA. Finally, engineered reversion of ExoN-AA to ExoN-DE in the presence of ExoN-AA passage-adaptive mutations resulted in significant fitness loss. These results demonstrate that while reversion is possible, at least one alternative adaptive pathway is more rapidly advantageous than intermediate revertants and may alter the genetic background to render reversion detrimental to fitness. Our results provide an evolutionary rationale for lack of ExoN-AA reversion, illuminate potential multiprotein replicase interactions and coevolution, and support future studies aimed at stabilizing attenuated CoV ExoN-AA mutants.IMPORTANCE Coronaviruses encode an exoribonuclease (ExoN) that is important for viral replication, fitness, and virulence, yet coronaviruses with a defective ExoN (ExoN-AA) have not reverted under diverse experimental conditions. In this study, we identify multiple impediments to MHV-ExoN-AA reversion. We show that ExoN-AA reversion is possible but evolutionarily unfavorable. Instead, compensatory mutations outside ExoN-AA motif I are more accessible and beneficial than partial reversion. We also show that coevolution between replicase proteins over long-term passage partially compensates for ExoN-AA motif I but renders the virus inhospitable to a reverted ExoN. Our results reveal the evolutionary basis for the genetic stability of ExoN-inactivating mutations, illuminate complex functional and evolutionary relationships between coronavirus replicase proteins, and identify potential mechanisms for stabilization of ExoN-AA coronavirus mutants.

Murine Hepatitis Virus nsp14 Exoribonuclease Activity Is Required for Resistance to Innate Immunity.

Coronaviruses (CoVs) are positive-sense RNA viruses that infect numerous mammalian and avian species and are capable of causing severe and lethal disease in humans. CoVs encode several innate immune antagonists that counteract the host innate immune response to facilitate efficient viral replication. CoV nonstructural protein 14 (nsp14) encodes 3'-to-5' exoribonuclease activity (ExoN), which performs a proofreading function and is required for high-fidelity replication. Outside of the order Nidovirales, arenaviruses are the only RNA viruses that encode an ExoN, which functions to degrade double-stranded RNA (dsRNA) replication intermediates. In this study, we tested the hypothesis that CoV ExoN also functions to antagonize the innate immune response. We demonstrate that viruses lacking ExoN activity [ExoN(-)] are sensitive to cellular pretreatment with interferon beta (IFN-ß) in a dose-dependent manner. In addition, ExoN(-) virus replication was attenuated in wild-type bone marrow-derived macrophages (BMMs) and partially restored in interferon alpha/beta receptor-deficient (IFNAR-/-) BMMs. ExoN(-) virus replication did not result in IFN-ß gene expression, and in the presence of an IFN-ß-mediated antiviral state, ExoN(-) viral RNA levels were not substantially reduced relative to those of untreated samples. However, ExoN(-) virus generated from IFN-ß-pretreated cells had reduced specific infectivity and decreased relative fitness, suggesting that ExoN(-) virus generated during an antiviral state is less viable to establish a subsequent infection. Overall, our data suggest murine hepatitis virus (MHV) ExoN activity is required for resistance to the innate immune response, and antiviral mechanisms affecting the viral RNA sequence and/or an RNA modification act on viruses lacking ExoN activity.IMPORTANCE CoVs encode multiple antagonists that prevent or disrupt an efficient innate immune response. Additionally, no specific antiviral therapies or vaccines currently exist for human CoV infections. Therefore, the study of CoV innate immune antagonists is essential for understanding how CoVs overcome host defenses and to maximize potential therapeutic interventions. Here, we sought to determine the contributions of nsp14 ExoN activity in the induction of and resistance to the innate immune response. We show that viruses lacking nsp14 ExoN activity are more sensitive than wild-type MHV to restriction by exogenous IFN-ß and that viruses produced in the presence of an antiviral state are less capable of establishing a subsequent viral infection. Our results support the hypothesis that murine hepatitis virus ExoN activity is required for resistance to the innate immune response.

Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations.

The coronavirus (CoV) RNA genome is the largest among the single-stranded positive-sense RNA viruses. CoVs encode a proofreading 3'-to-5' exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is responsible for CoV high-fidelity replication. Alanine substitution of ExoN catalytic residues [ExoN(-)] in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and murine hepatitis virus (MHV) disrupts ExoN activity, yielding viable mutant viruses with defective replication, up to 20-fold-decreased fidelity, and increased susceptibility to nucleoside analogues. To test the stability of the ExoN(-) genotype and phenotype, we passaged MHV-ExoN(-) 250 times in cultured cells (P250), in parallel with wild-type MHV (WT-MHV). Compared to MHV-ExoN(-) P3, MHV-ExoN(-) P250 demonstrated enhanced replication and increased competitive fitness without reversion at the ExoN(-) active site. Furthermore, MHV-ExoN(-) P250 was less susceptible than MHV-ExoN(-) P3 to multiple nucleoside analogues, suggesting that MHV-ExoN(-) was under selection for increased replication fidelity. We subsequently identified novel amino acid changes within the RNA-dependent RNA polymerase and nsp14 of MHV-ExoN(-) P250 that partially accounted for the reduced susceptibility to nucleoside analogues. Our results suggest that increased replication fidelity is selected in ExoN(-) CoVs and that there may be a significant barrier to ExoN(-) reversion. These results also support the hypothesis that high-fidelity replication is linked to CoV fitness and indicate that multiple replicase proteins could compensate for ExoN functions during replication.IMPORTANCE Uniquely among RNA viruses, CoVs encode a proofreading exoribonuclease (ExoN) in nsp14 that mediates high-fidelity RNA genome replication. Proofreading-deficient CoVs with disrupted ExoN activity [ExoN(-)] either are nonviable or have significant defects in replication, RNA synthesis, fidelity, fitness, and virulence. In this study, we showed that ExoN(-) murine hepatitis virus can adapt during long-term passage for increased replication and fitness without reverting the ExoN-inactivating mutations. Passage-adapted ExoN(-) mutants also demonstrate increasing resistance to nucleoside analogues that is explained only partially by secondary mutations in nsp12 and nsp14. These data suggest that enhanced resistance to nucleoside analogues is mediated by the interplay of multiple replicase proteins and support the proposed link between CoV fidelity and fitness.

Homology-Based Identification of a Mutation in the Coronavirus RNA-Dependent RNA Polymerase That Confers Resistance to Multiple Mutagens.

UNLABELLED: Positive-sense RNA viruses encode RNA-dependent RNA polymerases (RdRps) essential for genomic replication. With the exception of the large nidoviruses, such as coronaviruses (CoVs), RNA viruses lack proofreading and thus are dependent on RdRps to control nucleotide selectivity and fidelity. CoVs encode a proofreading exonuclease in nonstructural protein 14 (nsp14-ExoN), which confers a greater-than-10-fold increase in fidelity compared to other RNA viruses. It is unknown to what extent the CoV polymerase (nsp12-RdRp) participates in replication fidelity. We sought to determine whether homology modeling could identify putative determinants of nucleotide selectivity and fidelity in CoV RdRps. We modeled the CoV murine hepatitis virus (MHV) nsp12-RdRp structure and superimposed it on solved picornaviral RdRp structures. Fidelity-altering mutations previously identified in coxsackie virus B3 (CVB3) were mapped onto the nsp12-RdRp model structure and then engineered into the MHV genome with [nsp14-ExoN(+)] or without [nsp14-ExoN(-)] ExoN activity. Using this method, we identified two mutations conferring resistance to the mutagen 5-fluorouracil (5-FU): nsp12-M611F and nsp12-V553I. For nsp12-V553I, we also demonstrate resistance to the mutagen 5-azacytidine (5-AZC) and decreased accumulation of mutations. Resistance to 5-FU, and a decreased number of genomic mutations, was effectively masked by nsp14-ExoN proofreading activity. These results indicate that nsp12-RdRp likely functions in fidelity regulation and that, despite low sequence conservation, some determinants of RdRp nucleotide selectivity are conserved across RNA viruses. The results also indicate that, with regard to nucleotide selectivity, nsp14-ExoN is epistatic to nsp12-RdRp, consistent with its proposed role in a multiprotein replicase-proofreading complex. IMPORTANCE: RNA viruses have evolutionarily fine-tuned replication fidelity to balance requirements for genetic stability and diversity. Responsibility for replication fidelity in RNA viruses has been attributed to the RNA-dependent RNA polymerases, with mutations in RdRps for multiple RNA viruses shown to alter fidelity and attenuate virus replication and virulence. Coronaviruses (CoVs) are the only known RNA viruses to encode a proofreading exonuclease (nsp14-ExoN), as well as other replicase proteins involved in regulation of fidelity. This report shows that the CoV RdRp (nsp12) likely functions in replication fidelity; that residue determinants of CoV RdRp nucleotide selectivity map to similar structural regions of other, unrelated RNA viral polymerases; and that for CoVs, the proofreading activity of the nsp14-ExoN is epistatic to the function of the RdRp in fidelity.

Thinking Outside the Triangle: Replication Fidelity of the Largest RNA Viruses.

When judged by ubiquity, adaptation, and emergence of new diseases, RNA viruses are arguably the most successful biological organisms. This success has been attributed to a defect of sorts: high mutation rates (low fidelity) resulting in mutant swarms that allow rapid selection for fitness in new environments. Studies of viruses with small RNA genomes have identified fidelity determinants in viral RNA-dependent RNA polymerases and have shown that RNA viruses likely replicate within a limited fidelity range to maintain fitness. In this review we compare the fidelity of small RNA viruses with that of the largest RNA viruses, the coronaviruses. Coronaviruses encode the first known viral RNA proofreading exoribonuclease, a function that likely allowed expansion of the coronavirus genome and that dramatically increases replication fidelity and the range of tolerated variation. We propose models for regulation of coronavirus fidelity and discuss the implications of altered fidelity for RNA virus replication, pathogenesis, and evolution.

Chimeric exchange of coronavirus nsp5 proteases (3CLpro) identifies common and divergent regulatory determinants of protease activity.

Human coronaviruses (CoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) cause epidemics of severe human respiratory disease. A conserved step of CoV replication is the translation and processing of replicase polyproteins containing 16 nonstructural protein domains (nsp's 1 to 16). The CoV nsp5 protease (3CLpro; Mpro) processes nsp's at 11 cleavage sites and is essential for virus replication. CoV nsp5 has a conserved 3-domain structure and catalytic residues. However, the intra- and intermolecular determinants of nsp5 activity and their conservation across divergent CoVs are unknown, in part due to challenges in cultivating many human and zoonotic CoVs. To test for conservation of nsp5 structure-function determinants, we engineered chimeric betacoronavirus murine hepatitis virus (MHV) genomes encoding nsp5 proteases of human and bat alphacoronaviruses and betacoronaviruses. Exchange of nsp5 proteases from HCoV-HKU1 and HCoV-OC43, which share the same genogroup, genogroup 2a, with MHV, allowed for immediate viral recovery with efficient replication albeit with impaired fitness in direct competition with wild-type MHV. Introduction of MHV nsp5 temperature-sensitive mutations into chimeric HKU1 and OC43 nsp5 proteases resulted in clear differences in viability and temperature-sensitive phenotypes compared with MHV nsp5. These data indicate tight genetic linkage and coevolution between nsp5 protease and the genomic background and identify differences in intramolecular networks regulating nsp5 function. Our results also provide evidence that chimeric viruses within coronavirus genogroups can be used to test nsp5 determinants of function and inhibition in common isogenic backgrounds and cell types.

A neutralizing monoclonal antibody targeting the acid-sensitive region in chikungunya virus E2 protects from disease.

The mosquito-borne alphavirus, chikungunya virus (CHIKV), has recently reemerged, producing the largest epidemic ever recorded for this virus, with up to 6.5 million cases of acute and chronic rheumatic disease. There are currently no licensed vaccines for CHIKV and current anti-inflammatory drug treatment is often inadequate. Here we describe the isolation and characterization of two human monoclonal antibodies, C9 and E8, from CHIKV infected and recovered individuals. C9 was determined to be a potent virus neutralizing antibody and a biosensor antibody binding study demonstrated it recognized residues on intact CHIKV VLPs. Shotgun mutagenesis alanine scanning of 98 percent of the residues in the E1 and E2 glycoproteins of CHIKV envelope showed that the epitope bound by C9 included amino-acid 162 in the acid-sensitive region (ASR) of the CHIKV E2 glycoprotein. The ASR is critical for the rearrangement of CHIKV E2 during fusion and viral entry into host cells, and we predict that C9 prevents these events from occurring. When used prophylactically in a CHIKV mouse model, C9 completely protected against CHIKV viremia and arthritis. We also observed that when administered therapeutically at 8 or 18 hours post-CHIKV challenge, C9 gave 100% protection in a pathogenic mouse model. Given that targeting this novel neutralizing epitope in E2 can potently protect both in vitro and in vivo, it is likely to be an important region both for future antibody and vaccine-based interventions against CHIKV.

Filoviruses utilize glycosaminoglycans for their attachment to target cells.

Filoviruses are the cause of severe hemorrhagic fever in human and nonhuman primates. The envelope glycoprotein (GP), responsible for both receptor binding and fusion of the virus envelope with the host cell membrane, has been demonstrated to interact with multiple molecules in order to enhance entry into host cells. Here we have demonstrated that filoviruses utilize glycosaminoglycans, and more specifically heparan sulfate proteoglycans, for their attachment to host cells. This interaction is mediated by GP and does not require the presence of the mucin domain. Both the degree of sulfation and the structure of the carbohydrate backbone play a role in the interaction with filovirus GPs. This new step of filovirus interaction with host cells can potentially be a new target for antiviral strategies. As such, we were able to inhibit filovirus GP-mediated infection using carrageenan, a broad-spectrum microbicide that mimics heparin, and also using the antiviral dendrimeric peptide SB105-A10, which interacts with heparan sulfate, antagonizing the binding of the virus to cells.