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Host genetic variation is known to contribute to differential pathogenesis following infection. Mouse models allow direct assessment of host genetic factors responsible for susceptibility to Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). Based on an assessment of early stage lines from the Collaborative Cross mouse multi-parent population, we identified two lines showing highly divergent susceptibilities to SARS-CoV: the resistant CC003/Unc and the susceptible CC053/Unc. We generated 264 F2 mice between these strains, and infected them with SARS-CoV. Weight loss, pulmonary hemorrhage, and viral load were all highly correlated disease phenotypes. We identified a quantitative trait locus of major effect on chromosome 18 (27.1–58.6 Mb) which affected weight loss, viral titer and hemorrhage. Additionally, each of these three phenotypes had distinct quantitative trait loci [Chr 9 (weight loss), Chrs 7 and 12 (virus titer), and Chr 15 (hemorrhage)]. We identified Ticam2, an adaptor protein in the TLR signaling pathways, as a candidate driving differential disease at the Chr 18 locus. Ticam2−/− mice were highly susceptible to SARS-CoV infection, exhibiting increased weight loss and more pulmonary hemorrhage than control mice. These results indicate a critical role for Ticam2 in SARS-CoV disease, and highlight the importance of host genetic variation in disease responses.

Using incipient lines of the Collaborative Cross (CC), a murine genetic reference population, we previously identified a quantitative trait loci (QTL) associated with low SARS-CoV titer. In this study, we integrated sequence information and RNA expression of genes within the QTL to identify mucin 4 (Muc4) as a high priority candidate for controlling SARS-CoV titer in the lung. To test this hypothesis, we infected Muc4-/- mice and found that female, but not male, Muc4-/- mice developed more weight loss and disease following infection with SARS-CoV. Female Muc4-/- mice also had more difficulty breathing despite reduced lung pathology; however, no change in viral titers was observed. Comparing across viral families, studies with chikungunya virus, a mosquito-borne arthralgic virus, suggests that Muc4’s impact on viral pathogenesis may be widespread. Although not confirming the original titer QTL, our data identifies a role for Muc4 in the SARS-CoV disease and viral pathogenesis.ImportanceGiven the recent emergence of SARS-CoV-2, this work suggest that Muc4 expression plays a protective role in female mice not conserved in male mice following SARS-CoV infection. With the SARS-CoV-2 outbreak continuing, treatments that modulate or enhance Muc4 activity may provide an avenue for treatment and improved outcomes. In addition, the work highlights the importance of studying host factors including host genetics and biological sex as key parameters influencing infection and disease outcomes.

Emerging zoonotic viral diseases remain a challenge to global public health. Recent surveillance studies have implicated bats as potential reservoirs for a number of viral pathogens, including coronaviruses and Ebola viruses. Caliciviridae represent a major viral family contributing to emerging diseases in both human and animal populations and have been recently identified in bats. In this study, we blended metagenomics, phylogenetics, homology modeling, and in vitro assays to characterize two novel bat calicivirus (BtCalV) capsid sequences, corresponding to strain BtCalV/A10/USA/2009, identified in Perimyotis subflavus near Little Orleans, MD, and bat norovirus. We observed that bat norovirus formed virus-like particles and had epitopes and receptor-binding patterns similar to those of human noroviruses. To determine whether these observations stretch across multiple bat caliciviruses, we characterized a novel bat calicivirus, BtCalV/A10/USA/2009. Phylogenetic analysis revealed that BtCalV/A10/USA/2009 likely represents a novel Caliciviridae genus and is most closely related to "recoviruses." Homology modeling revealed that the capsid sequences of BtCalV/A10/USA/2009 and bat norovirus resembled human norovirus capsid sequences and retained host ligand binding within the receptor-binding domains similar to that seen with human noroviruses. Both caliciviruses bound histo-blood group antigens in patterns that overlapped those seen with human and animal noroviruses. Taken together, our results indicate the potential for bat caliciviruses to bind histo-blood group antigens and overcome a significant barrier to cross-species transmission. Additionally, we have shown that bat norovirus maintains antigenic epitopes similar to those seen with human noroviruses, providing further evidence of evolutionary descent. Our results reiterate the importance of surveillance of wild-animal populations, especially of bats, for novel viral pathogens., IMPORTANCE Caliciviruses are rapidly evolving viruses that cause pandemic outbreaks associated with significant morbidity and mortality globally. The animal reservoirs for human caliciviruses are unknown; bats represent critical reservoir species for several emerging and zoonotic diseases. Recent reports have identified several bat caliciviruses but have not characterized biological functions associated with disease risk, including their potential emergence in other mammalian populations. In this report, we identified a novel bat calicivirus that is most closely related to nonhuman primate caliciviruses. Using this new bat calicivirus and a second norovirus-like bat calicivirus capsid gene sequence, we generated virus-like particles that have host carbohydrate ligand binding patterns similar to those of human and animal noroviruses and that share antigens with human noroviruses. The similarities to human noroviruses with respect to binding patterns and antigenic epitopes illustrate the potential for bat caliciviruses to emerge in other species and the importance of pathogen surveillance in wild-animal populations.

While dispensable for viral replication, coronavirus (CoV) accessory open reading frame (ORF) proteins often play critical roles during infection and pathogenesis. Utilizing a previously generated mutant, we demonstrate that the absence of all four Middle East respiratory syndrome CoV (MERS-CoV) accessory ORFs (deletion of ORF3, -4a, -4b, and -5 [dORF3-5]) has major implications for viral replication and pathogenesis. Importantly, attenuation of the dORF3-5 mutant is primarily driven by dysregulated host responses, including disrupted cell processes, augmented interferon (IFN) pathway activation, and robust inflammation. In vitro replication attenuation also extends to in vivo models, allowing use of dORF3-5 as a live attenuated vaccine platform. Finally, examination of ORF5 implicates a partial role in modulation of NF-κB-mediated inflammation. Together, the results demonstrate the importance of MERS-CoV accessory ORFs for pathogenesis and highlight them as potential targets for surveillance and therapeutic treatments moving forward., IMPORTANCE The initial emergence and periodic outbreaks of MERS-CoV highlight a continuing threat posed by zoonotic pathogens to global public health. In these studies, mutant virus generation demonstrates the necessity of accessory ORFs in regard to MERS-CoV infection and pathogenesis. With this in mind, accessory ORF functions can be targeted for both therapeutic and vaccine treatments in response to MERS-CoV and related group 2C coronaviruses. In addition, disruption of accessory ORFs in parallel may offer a rapid response platform to attenuation of future emergent strains based on both SARS- and MERS-CoV accessory ORF mutants.

Emergent and preemergent coronaviruses (CoVs) pose a global threat that requires immediate intervention. Rapid intervention necessitates the capacity to generate, grow, and genetically manipulate infectious CoVs in order to rapidly evaluate pathogenic mechanisms, host and tissue permissibility, and candidate antiviral therapeutic efficacy. CoVs encode the largest viral RNA genomes at about 28-32,000 nucleotides in length, and thereby complicate efficient engineering of the genome. Deconstructing the genome into manageable fragments affords the plasticity necessary to rapidly introduce targeted genetic changes in parallel and assort mutated fragments while maximizing genome stability over time. In this protocol we describe a well-developed reverse genetic platform strategy for CoVs that is comprised of partitioning the viral genome into 5-7 independent DNA fragments (depending on the CoV genome), each subcloned into a plasmid for increased stability and ease of genetic manipulation and amplification. Coronavirus genomes are conveniently partitioned by introducing type IIS or IIG restriction enzyme recognition sites that confer directional cloning. Since each restriction site leaves a unique overhang between adjoining fragments, reconstruction of the full-length genome can be achieved through a standard DNA ligation comprised of equal molar ratios of each fragment. Using this method, recombinant CoVs can be rapidly generated and used to investigate host range, gene function, pathogenesis, and candidate therapeutics for emerging and preemergent CoVs both in vitro and in vivo.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel virus that emerged in 2012, causing acute respiratory distress syndrome (ARDS), severe pneumonia-like symptoms and multi-organ failure, with a case fatality rate of ∼36%. Limited clinical studies indicate that humans infected with MERS-CoV exhibit pathology consistent with the late stages of ARDS, which is reminiscent of the disease observed in patients infected with severe acute respiratory syndrome coronavirus. Models of MERS-CoV-induced severe respiratory disease have been difficult to achieve, and small-animal models traditionally used to investigate viral pathogenesis (mouse, hamster, guinea-pig and ferret) are naturally resistant to MERS-CoV. Therefore, we used CRISPR-Cas9 gene editing to modify the mouse genome to encode two amino acids (positions 288 and 330) that match the human sequence in the dipeptidyl peptidase 4 receptor, making mice susceptible to MERS-CoV infection and replication. Serial MERS-CoV passage in these engineered mice was then used to generate a mouse-adapted virus that replicated efficiently within the lungs and evoked symptoms indicative of severe ARDS, including decreased survival, extreme weight loss, decreased pulmonary function, pulmonary haemorrhage and pathological signs indicative of end-stage lung disease. Importantly, therapeutic countermeasures comprising MERS-CoV neutralizing antibody treatment or a MERS-CoV spike protein vaccine protected the engineered mice against MERS-CoV-induced ARDS.

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic alphacoronavirus. In the United States, highly virulent PEDV strains cause between 80 and 100% mortality in suckling piglets and are rapidly transmitted between animals and farms. To study the genetic factors that regulate pathogenesis and transmission, we developed a molecular clone of PEDV strain PC22A. The infectious-clone-derived PEDV (icPEDV) replicated as efficiently as the parental virus in cell culture and in pigs, resulting in lethal disease in vivo. Importantly, recombinant PEDV was rapidly transmitted to uninoculated pigs via indirect contact, demonstrating virulence and efficient transmission while replicating phenotypes seen in the wild-type virus. Using reverse genetics, we removed open reading frame 3 (ORF3) and replaced this region with a red fluorescent protein (RFP) gene to generate icPEDV-ΔORF3-RFP. icPEDV-ΔORF3-RFP replicated efficiently in vitro and in vivo, was efficiently transmitted among pigs, and produced lethal disease outcomes. However, the diarrheic scores in icPEDV-ΔORF3-RFP-infected pigs were lower than those in wild-type-virus- or icPEDV-infected pigs, and the virus formed smaller plaques than those of PC22A. Together, these data describe the development of a robust reverse-genetics platform for identifying genetic factors that regulate pathogenic outcomes and transmission efficiency in vivo, providing key infrastructural developments for developing and evaluating the efficacy of live attenuated vaccines and therapeutics in a clinical setting., IMPORTANCE Porcine epidemic diarrhea virus (PEDV) emerged in the United States in 2013 and has since killed 10% of U.S. farm pigs. Though the disease has been circulating internationally for decades, the lack of a rapid reverse-genetics platform for manipulating PEDV and identifying genetic factors that impact transmission and virulence has hindered the study of this important agricultural disease. Here, we present a DNA-based infectious-clone system that replicates the pathogenesis of circulating U.S. strain PC22A both in vitro and in piglets. This infectious clone can be used both to study the genetics, virulence, and transmission of PEDV coronavirus and to inform the creation of a live attenuated PEDV vaccine.