Replication models have been developed to describe the replication dynamics of a variety of viruses to better understand the kinetics and key contributing factors affecting infectivity and spread. However, accurate representations of the dynamics of virus replication observed in vitro and in vivo are often limited due to the failure of these models to account for both environmental influences, such as temperature, and the variety of possible mechanisms employed by viruses to spread. Several major families of viruses including paramyxoviruses, pneumoviruses and coronaviruses, induce and use the formation of syncytia, large multinucleated cell masses formed through fusion of cells, to aid in spread to neighbouring susceptible cells. In this study, we evaluate and compare both the dynamics and roles of temperature and syncytia formation on the replication of two different fusogenic viruses in vitro : human respiratory syncytial virus (hRSV) and a murine coronavirus, mouse hepatitis virus (MHV). Thermal stability, replication kinetics and both the rates and dynamics of syncytia formation were evaluated for hRSV and MHV. These data were then incorporated into a novel and improved replication model for each of the two viruses, which provides new insights into the contributions of both temperature and syncytia formation in the replication of fusogenic viruses.

The recent emergence of SARS-CoV-2 in 2019 has highlighted the necessity of antiviral therapeutics for current and future emerging coronaviruses. Recently, the traditional herbal medicines baicalein, baicalin, and andrographolide have shown inhibition against the main protease of SARS-CoV-2. This provides a promising new direction for COVID-19 therapeutics, but it remains unknown whether these three substances inhibit other human coronaviruses. In this study, we describe the development of novel chimeric mouse hepatitis virus (MHV) reporters that express firefly luciferase (FFL) and the 3CLpro proteases of human coronaviruses HKU1 and OC43. These chimeric viruses were used to determine if the phytochemicals baicalein, baicalin, and andrographolide are inhibitory against human coronavirus strains HKU1 and OC43. Our data show that both baicalein and baicalin exhibit inhibition towards the chimeric MHV strains. However, andrographolide induces cytotoxicity and failed to demonstrate selective toxicity towards the viruses. This study reports the development and use of a safe replicating reporter platform to investigate potential coronavirus 3CLpro inhibitors against common-cold human coronavirus strains HKU1 and OC43.

Sin Nombre virus (SNV) is an emerging virus that was first discovered in the Four Corners region of the United States in 1993. The virus causes a disease known as Hantavirus Pulmonary Syndrome (HPS), sometimes called Hantavirus Cardiopulmonary Syndrome (HCPS), a life-threatening illness named for the predominance of infection of pulmonary endothelial cells. SNV is one of several rodent-borne hantaviruses found in the western hemisphere with the capability of causing this disease. The primary reservoir of SNV is the deer mouse (Peromyscus maniculatus), and the virus is transmitted primarily through aerosolized rodent excreta and secreta. Here, we review the history of SNV emergence and its virus biology and relationship to other New World hantaviruses, disease, treatment, and prevention options.

Approximately 15–30% of all cases of the common cold are due to human coronavirus infections. More recently, the emergence of the more severe respiratory coronaviruses, SARS-CoV and MERS-CoV, have highlighted the increased pathogenic potential of emergent coronaviruses. Lastly, the current emergence of SARS-CoV-2 has demonstrated not only the potential for significant disease caused by emerging coronaviruses, but also the capacity of novel coronaviruses to promote pandemic spread. Largely driven by the global response to the COVID-19 pandemic, significant research in coronavirus biology has led to advances in our understanding of these viruses. In this review, we evaluate the virology, emergence, and evolution of the four endemic coronaviruses associated with the common cold, their relationship to pandemic SARS-CoV-2, and discuss the potential for future emergent human coronaviruses.

Significance The viral RNA sensor RIG-I initiates the antiviral innate immune response by activating a signaling cascade that induces interferon. Activation of the RIG-I signaling pathway is highly regulated to quickly mount a protective immune response while preventing dysregulation that can lead to excessive inflammation or autoimmune disorders. Here, we characterize one such mechanism of regulation. We describe that UFL1, an E3 ligase for the ubiquitin-like modifier conjugation system called ufmylation, is important to promote RIG-I signaling. Using molecular approaches, we show that ufmylation promotes RIG-I interaction with the membrane-targeting protein 14-3-3ε. As such, ufmylation positively regulates RIG-I recruitment to its signaling adaptor protein MAVS for induction of interferon in response to RNA virus infection.

Coronavirus protease nsp5 (Mpro, 3CLpro) remains a primary target for coronavirus therapeutics due to its indispensable and conserved role in the proteolytic processing of the viral replicase polyproteins. In this review, we discuss the diversity of known coronaviruses, the role of nsp5 in coronavirus biology, and the structure and function of this protease across the diversity of known coronaviruses, and evaluate past and present efforts to develop inhibitors to the nsp5 protease with a particular emphasis on new and mostly unexplored potential targets of inhibition. With the recent emergence of pandemic SARS-CoV-2, this review provides novel and potentially innovative strategies and directions to develop effective therapeutics against the coronavirus protease nsp5.

Nucleic acid-based antiviral innate immunity activates a signaling cascade that induces type I and type III interferons (IFNs), and other cytokines. This signaling, which is highly regulated, is initiated by pattern recognition receptors, such as RIG-I, that sense viral RNA and then signal to the adaptor protein, MAVS. This adaptor protein then recruits additional signaling proteins, including TRAF3 and TBK1, to form a signaling complex that results in IRF3 activation for transcriptional induction of IFN. Here, we show that the GTPase trafficking protein RAB1B positively regulates RIG-I signaling to promote IFN-β induction and the antiviral response. Over-expression of RAB1B increases RIG-I-mediated signaling to IFN-β, while deletion results in reduced signaling of this pathway. Additionally, this loss of RAB1B results in a dampened antiviral response, as Zika virus infection is enhanced in the absence of RAB1B. Importantly, we identified the mechanism of RAB1B action by determining that it interacts with TRAF3 to facilitate the interaction of TRAF3 with MAVS. Thus, we identified RAB1B as a regulator of TRAF3 to promote the formation of innate immune signaling complexes in response to nucleic acid sensing.

Innate immune detection of viral nucleic acids during viral infection activates a signaling cascade that induces type I and type III IFNs as well as other cytokines, to generate an antiviral response. This signaling is initiated by pattern recognition receptors, such as the RNA helicase retinoic acid-inducible gene I (RIG-I), that sense viral RNA. These sensors then interact with the adaptor protein mitochondrial antiviral signaling protein (MAVS), which recruits additional signaling proteins, including TNF receptor-associated factor 3 (TRAF3) and TANK-binding kinase 1 (TBK1), to form a signaling complex that activates IFN regulatory factor 3 (IRF3) for transcriptional induction of type I IFNs. Here, using several immunological and biochemical approaches in multiple human cell types, we show that the GTPase-trafficking protein RAB1B up-regulates RIG-I pathway signaling and thereby promotes IFN-β induction and the antiviral response. We observed that RAB1B overexpression increases RIG-I-mediated signaling to IFN-β and that RAB1B deletion reduces signaling of this pathway. Additionally, loss of RAB1B dampened the antiviral response, indicated by enhanced Zika virus infection of cells depleted of RAB1B. Importantly, we identified the mechanism of RAB1B action in the antiviral response, finding that it forms a protein complex with TRAF3 to facilitate the interaction of TRAF3 with mitochondrial antiviral signaling protein. We conclude that RAB1B regulates TRAF3 and promotes the formation of innate immune signaling complexes in response to nucleic acid sensing during RNA virus infection.

The mitochondrial antiviral signaling (MAVS) protein is a central adaptor protein required for antiviral innate immune signaling. To facilitate its roles in innate immunity, MAVS localizes to multiple intracellular membranous compartments, including the mitochondria, the mitochondrial-associated ER membrane (MAM), and peroxisomes. Studies of MAVS function therefore often require an analysis of MAVS localization. To detect MAVS protein on intracellular membranes, biochemical fractionation to isolate MAMs, mitochondria, or peroxisomes can be used. Further, immunofluorescence with antibodies against specific membrane markers can be used to visualize MAVS distribution throughout the cell. Here, we describe the biochemical fractionation and immunofluorescence protocols used to detect MAVS subcellular localization.

Upon infection, both DNA and RNA viruses can be sensed by pattern recognition receptors (PRRs) in the cytoplasm or the nucleus to activate antiviral innate immunity. Sensing of viral products leads to the activation of a signaling cascade that ultimately results in transcriptional activation of type I and III interferons, as well as other antiviral genes that together mediate viral clearance and inhibit viral spread. Therefore, in order for viruses to replicate and spread efficiently, they must inhibit the host signaling pathways that induce the innate antiviral immune response. In this review, we will highlight recent advances in the understanding of the mechanisms by which viruses evade PRR detection, intermediate signaling molecule activation, transcription factor activation, and the actions of antiviral proteins.

ABSTRACT A common feature of infection by positive-sense RNA virus is the modification of host cell cytoplasmic membranes that serve as sites of viral RNA synthesis. Coronaviruses induce double-membrane vesicles (DMVs), but the role of DMVs in replication and virus fitness remains unclear. Coronaviruses encode 16 nonstructural proteins (nsps), three of which, nsp3, nsp4, and nsp6, are necessary and sufficient for DMV formation. It has been shown previously that mutations in murine hepatitis virus (MHV) nsp4 loop 1 that alter nsp4 glycosylation are associated with disrupted DMV formation and result in changes in virus replication and RNA synthesis. However, it is not known whether DMV morphology or another function of nsp4 glycosylation is responsible for effects on virus replication. In this study, we tested whether mutations across nsp4, both alone and in combination with mutations that abolish nsp4 glycosylation, affected DMV formation, replication, and fitness. Residues in nsp4 distinct from glycosylation sites, particularly in the endoplasmic reticulum (ER) luminal loop 1, independently disrupted both the number and morphology of DMVs and exacerbated DMV changes associated with loss of glycosylation. Mutations that altered DMV morphology but not glycosylation did not affect virus fitness while viruses lacking nsp4 glycosylation exhibited a loss in fitness. The results support the hypothesis that DMV morphology and numbers are not key determinants of virus fitness. The results also suggest that nsp4 glycosylation serves roles in replication in addition to the organization and stability of MHV-induced double-membrane vesicles. IMPORTANCE All positive-sense RNA viruses modify host cytoplasmic membranes for viral replication complex formation. Thus, defining the mechanisms of virus-induced membrane modifications is essential for both understanding virus replication and development of novel approaches to virus inhibition. Coronavirus-induced membrane changes include double-membrane vesicles (DMVs) and convoluted membranes. Three viral nonstructural proteins (nsps), nsp3, nsp4, and nsp6, are known to be required for DMV formation. It is unknown how these proteins induce membrane modification or which regions of the proteins are involved in DMV formation and stability. In this study, we show that mutations across nsp4 delay virus replication and disrupt DMV formation and that loss of nsp4 glycosylation is associated with a substantial fitness cost. These results support a critical role for nsp4 in DMV formation and virus fitness.

Coronavirus replicase nsp4 is critical for virus-induced membrane modifications. An nsp4 mutant (N258T) of murine hepatitis virus (MHV) has been reported to be temperature-sensitive (ts) and to alter membrane targeting. We engineered and recovered all four possible codon variants of N258T in the cloned MHV-A59 background. All mutant viruses demonstrated impaired replication compared to wildtype MHV, but no nsp4 N258T mutant virus was ts, and all variants colocalized with viral protein markers for replication complexes, but not with markers for mitochondria. This study emphasizes that complete genome sequencing may be necessary, even with directed and confirmed reverse genetic mutants.

Sarcoidosis is a non-caseating granulomatous disease for which a role for infectious antigens continues to strengthen. Recent studies have reported molecular evidence of mycobacteria or propionibacteria. We assessed for immune responses against mycobacterial and propionibacterial antigens in sarcoidosis bronchoalveolar lavage (BAL) using flow cytometry, and localized signals consistent with microbial antigens with sarcoidosis specimens, using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS).

Introduction Sarcoidosis is a multisystem granulomatous disease for which the association with mycobacteria continues to strengthen. It is hypothesized that a single, poorly degradable antigen is responsible for sarcoidosis pathogenesis. Several reports from independent groups support mycobacterial antigens having a role in sarcoidosis pathogenesis. To identify other microbial targets of the adaptive immune response, we tested the ability of CD4+ and CD8+ T cells to recognize multiple mycobacterial antigens. Methods Fifty-four subjects were enrolled in this study: 31 sarcoidosis patients, nine non-tuberculosis mycobacterial (NTM) infection controls, and 14 PPD- controls. Using flow cytometry, we assessed for Th1 immune responses to ESAT-6, katG, Ag85A, sodA, and HSP. Results Alveolar T-cells from twenty-two of the 31 sarcoidosis patients produced a CD4+ response to at least one of ESAT-6, katG, Ag85A, sodA, or HSP, compared to two of 14 PPD- controls (p = 0.0008) and five of nine NTM controls (p = 0.44), while eighteen of the 31 sarcoidosis subjects tested produced a CD8+ response to at least one of the mycobacterial antigens compared to two of 14 PPD- controls (p = 0.009) and three of nine NTM controls (0.26). Not only did the BAL-derived T cells respond to multiple virulence factors, but also to multiple, distinct epitopes within a given protein. The detection of proliferation upon stimulation with the mycobacterial virulence factors demonstrates that these responses are initiated by antigen specific recognition. Conclusions Together these results reveal that antigen-specific CD4+ and CD8+ T cells responses to multiple mycobacterial epitopes are present within sites of active sarcoidosis involvement, and that these antigen-specific responses are present at the time of diagnosis.

Positive-strand RNA viruses induce modifications of cytoplasmic membranes to form replication complexes. For coronaviruses, replicase nonstructural protein 4 (nsp4) has been proposed to function in the formation and organization of replication complexes. Murine hepatitis virus (MHV) nsp4 is glycosylated at residues Asn176 (N176) and N237 during plasmid expression of nsp4 in cells. To test if MHV nsp4 residues N176 and N237 are glycosylated during virus replication and to determine the effects of N176 and N237 on nsp4 function and MHV replication, alanine substitutions of nsp4 N176, N237, or both were engineered into the MHV-A59 genome. The N176A, N237A, and N176A/N237A mutant viruses were viable, and N176 and N237 were glycosylated during infection of wild-type (wt) and mutant viruses. The nsp4 glycosylation mutants exhibited impaired virus growth and RNA synthesis, with the N237A and N176A/N237A mutant viruses demonstrating more profound defects in virus growth and RNA synthesis. Electron microscopic analysis of ultrastructure from infected cells demonstrated that the nsp4 mutants had aberrant morphology of virus-induced double-membrane vesicles (DMVs) compared to those infected with wt virus. The degree of altered DMV morphology directly correlated with the extent of impairment in viral RNA synthesis and virus growth of the nsp4 mutant viruses. The results indicate that nsp4 plays a critical role in the organization and stability of DMVs. The results also support the conclusion that the structure of DMVs is essential for efficient RNA synthesis and optimal replication of coronaviruses.

The structure of mycolactone E, isolated from the frog pathogen Mycobacterium liflandii, was established via organic synthesis. Within the mycolactone family of metabolites, a structural variation has been seen only at the unsaturated fatty acid moiety thus far, and mycolactone E follows this observation. Interestingly, the absolute configuration of its unsaturated fatty acid matches that of the mycolactones from human mycobacteria, rather than the structurally more closely related mycolactone F from fish mycobacteria.

Mycobacterium ulcerans, the causative agent of Buruli ulcer, is an emerging environmental bacterium in Australia and West Africa. The primary risk factor associated with Buruli ulcer is proximity to slow moving water. Environmental constraints for disease are shown by the absence of infection in arid regions of infected countries. A particularly mysterious aspect of Buruli ulcer is the fact that endemic and non-endemic villages may be only a few kilometers apart within the same watershed. Recent studies suggest that aquatic invertebrate species may serve as reservoirs for M. ulcerans, although transmission pathways remain unknown. Systematic studies of the distribution of M. ulcerans in the environment using standard ecological methods have not been reported. Here we present results from the first study based on random sampling of endemic and non-endemic sites. In this study PCR-based methods, along with biofilm collections, have been used to map the presence of M. ulcerans within 26 aquatic sites in Ghana. Results suggest that M. ulcerans is present in both endemic and non-endemic sites and that variable number tandem repeat (VNTR) profiling can be used to follow chains of transmission from the environment to humans. Our results suggesting that the distribution of M. ulcerans is far broader than the distribution of human disease is characteristic of environmental pathogens. These findings imply that focal demography, along with patterns of human water contact, may play a major role in transmission of Buruli ulcer.