Obesity fosters severe disease outcomes in a mouse model of coronavirus infection associated with transcriptomic abnormalities.

Obesity has been identified as an independent risk factor for severe outcomes in humans with coronavirus disease 2019 (COVID-19) and other infectious diseases. Here, we established a mouse model of COVID-19 using the murine betacoronavirus, mouse hepatitis virus 1 (MHV-1). C57BL/6 and C3H/HeJ mice exposed to MHV-1 developed mild and severe disease, respectively. Obese C57BL/6 mice developed clinical manifestations similar to those of lean controls. In contrast, all obese C3H/HeJ mice succumbed by 8 days postinfection, compared to a 50% mortality rate in lean controls. Notably, both lean and obese C3H/HeJ mice exposed to MHV-1 developed lung lesions consistent with severe human COVID-19, with marked evidence of diffuse alveolar damage (DAD). To identify early predictive biomarkers of worsened disease outcomes in obese C3H/HeJ mice, we sequenced RNA from whole blood 2 days postinfection and assessed changes in gene and pathway expression. Many pathways uniquely altered in obese C3H/HeJ mice postinfection aligned with those found in humans with severe COVID-19. Furthermore, we observed altered gene expression related to the unfolded protein response and lipid metabolism in infected obese mice compared to their lean counterparts, suggesting a role in the severity of disease outcomes. This study presents a novel model for studying COVID-19 and elucidating the mechanisms underlying severe disease outcomes in obese and other hosts.

Persistence and free chlorine disinfection of human coronaviruses and their surrogates in water.

The coronavirus disease 2019 pandemic illustrates the importance of understanding the behavior and control of human pathogenic viruses in the environment. Exposure via water (drinking, bathing, and recreation) is a known route of transmission of viruses to humans, but the literature is relatively void of studies on the persistence of many viruses, especially coronaviruses, in water and their susceptibility to chlorine disinfection. To fill that knowledge gap, we evaluated the persistence and free chlorine disinfection of human coronavirus OC43 (HCoV-OC43) and its surrogates, murine hepatitis virus (MHV) and porcine transmissible gastroenteritis virus (TGEV), in drinking water and laboratory buffer using cell culture methods. The decay rate constants of human coronavirus and its surrogates in water varied, depending on virus and water matrix. In drinking water without disinfectant addition, MHV showed the largest decay rate constant (estimate ± standard error, 2.25 ± 0.09 day-1) followed by HCoV-OC43 (0.99 ± 0.12 day-1) and TGEV (0.65 ± 0.06 day-1), while in phosphate buffer without disinfectant addition, HCoV-OC43 (0.51 ± 0.10 day-1) had a larger decay rate constant than MHV (0.28 ± 0.03 day-1) and TGEV (0.24 ± 0.02 day-1). Upon free chlorine disinfection, the inactivation rates of coronaviruses were independent of free chlorine concentration and were not affected by water matrix, though they still varied between viruses. TGEV showed the highest susceptibility to free chlorine disinfection with the inactivation rate constant of 113.50 ± 7.50 mg-1 min-1 L, followed by MHV (81.33 ± 4.90 mg-1 min-1 L) and HCoV-OC43 (59.42 ± 4.41 mg-1 min-1 L). IMPORTANCE: This study addresses an important knowledge gap on enveloped virus persistence and disinfection in water. Results have immediate practical applications for shaping evidence-based water policies, particularly in the development of disinfection strategies for pathogenic virus control.

Therapeutic Effects of a Novel Aptamer on Coronaviral Infection-Induced Lung Injury and Systemic Inflammatory Responses.

BACKGROUND: Coronaviral infection-induced acute lung injury has become a major threat to public health, especially through the ongoing pandemic of COVID-19. Apta-1 is a newly discovered Aptamer that has anti-inflammatory effects on systemic septic responses. The therapeutic effects of Apta-1 on coronaviral infection-induced acute lung injury and systemic responses were evaluated in the present study. METHODS: Female A/J mice (at 12-14 weeks of age) were challenged with murine hepatitis virus 1 (MHV-1), a coronavirus, at 5000 PFU intranasally, followed by Apta-1 intravenously administered (100 mg/kg, twice) 1.5 h or 2 days after viral delivery. Animals were sacrificed at Day 2 or Day 4. Lung tissues were examined with H&E, immunohistochemistry staining, and western blotting. RT-qPCR was used for cytokine gene expression. Serum and plasma were collected for laboratory assessments. RESULTS: Apta-1 treatment reduced viral titers, prevented MHV-1-induced reduction of circulating blood volume and hemolysis, reduced alveolar space hemorrhage, and protease-activated receptor 1 (PAR-1) cleavage. Apta-1 treatment also significantly reduced chemokine (MKC, MCP-1, and RANTES) levels, as well as AST, ALT, total bilirubin, and reduced unconjugated bilirubin levels in the serum. CONCLUSION: Apta-1 showed therapeutic benefits in coronaviral infection-induced hemorrhage and PAR-1 cleavage in the lung. It also has anti-inflammatory effects systemically.

Neurons cytoskeletal architecture remodeling during the replication cycle of mouse coronavirus MHV-JHM: a morphological in vitro study.

Nowadays, the population is still struggling with a post-COVID19 syndrome known as long COVID, including a broad spectrum of neurological problems. There is an urgent need for a better understanding and exploration of the mechanisms of coronavirus neurotropism. For this purpose, the neurotropic strain of mouse hepatitis virus (MHV-JHM) originating from the beta-coronavirus genus, the same as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been used. The role of the cytoskeleton during virus replication in neurons in vitro was determined to understand the mechanisms of MHV-JHM neuroinfection. We have described for the first time the changes of actin filaments during MHV-JHM infection. We also observed productive replication of MHV-JHM in neurons during 168 h p.i. and syncytial cytopathic effect. We discovered that the MHV-JHM strain modulated neuronal cytoskeleton during infection, which were manifested by: (i) condensation of actin filaments in the cortical layer of the cytoplasm, (ii) formation of microtubule cisternae structures containing viral antigen targeting viral replication site (iii) formation of tunneling nanotubes used by MHV-JHM for intercellular transport. Additionally, we demonstrated that the use of cytoskeletal inhibitors have reduced virus replication in neurons, especially noscapine and nocodazole, the microtubule shortening factors.

Coronavirus M Protein Trafficking in Epithelial Cells Utilizes a Myosin Vb Splice Variant and Rab10.

The membrane (M) glycoprotein of coronaviruses (CoVs) serves as the nidus for virion assembly. Using a yeast two-hybrid screen, we identified the interaction of the cytosolic tail of Murine Hepatitis Virus (MHV-CoV) M protein with Myosin Vb (MYO5B), specifically with the alternative splice variant of cellular MYO5B including exon D (MYO5B+D), which mediates interaction with Rab10. When co-expressed in human lung epithelial A549 and canine kidney epithelial MDCK cells, MYO5B+D co-localized with the MHV-CoV M protein, as well as with the M proteins from Porcine Epidemic Diarrhea Virus (PEDV-CoV), Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome 2 (SARS-CoV-2). Co-expressed M proteins and MYO5B+D co-localized with endogenous Rab10 and Rab11a. We identified point mutations in MHV-CoV M that blocked the interaction with MYO5B+D in yeast 2-hybrid assays. One of these point mutations (E121K) was previously shown to block MHV-CoV virion assembly and its interaction with MYO5B+D. The E to K mutation at homologous positions in PEDV-CoV, MERS-CoV and SARS-CoV-2 M proteins also blocked colocalization with MYO5B+D. The knockdown of Rab10 blocked the co-localization of M proteins with MYO5B+D and was rescued by re-expression of CFP-Rab10. Our results suggest that CoV M proteins traffic through Rab10-containing systems, in association with MYO5B+D.

Retinoic Acid-Mediated Inhibition of Mouse Coronavirus Replication Is Dependent on IRF3 and CaMKK.

The ongoing COVID-19 pandemic has revealed the shortfalls in our understanding of how to treat coronavirus infections. With almost 7 million case fatalities of COVID-19 globally, the catalog of FDA-approved antiviral therapeutics is limited compared to other medications, such as antibiotics. All-trans retinoic acid (RA), or activated vitamin A, has been studied as a potential therapeutic against coronavirus infection because of its antiviral properties. Due to its impact on different signaling pathways, RA's mechanism of action during coronavirus infection has not been thoroughly described. To determine RA's mechanism of action, we examined its effect against a mouse coronavirus, mouse hepatitis virus strain A59 (MHV). We demonstrated that RA significantly decreased viral titers in infected mouse L929 fibroblasts and RAW 264.7 macrophages. The reduced viral titers were associated with a corresponding decrease in MHV nucleocapsid protein expression. Using interferon regulatory factor 3 (IRF3) knockout RAW 264.7 cells, we demonstrated that RA-induced suppression of MHV required IRF3 activity. RNA-seq analysis of wildtype and IRF3 knockout RAW cells showed that RA upregulated calcium/calmodulin (CaM) signaling proteins, such as CaM kinase kinase 1 (CaMKK1). When treated with a CaMKK inhibitor, RA was unable to upregulate IRF activation during MHV infection. In conclusion, our results demonstrate that RA-induced protection against coronavirus infection depends on IRF3 and CaMKK.

Analysis of the molecular determinants for furin cleavage of the spike protein S1/S2 site in defined strains of the prototype coronavirus murine hepatitis virus (MHV).

We analyzed the spike protein S1/S2 cleavage of selected strains of a prototype coronavirus, mouse hepatitis virus (MHV) by the cellular protease furin, in order to understand the structural requirements underlying the sequence selectivity of the scissile segment. The probability of cleavage of selected MHV strains was first evaluated from furin cleavage scores predicted by the ProP computer software, and then cleavage was measured experimentally with a fluorogenic peptide cleavage assay consisting of S1/S2 peptide mimics and purified furin. We found that in vitro cleavability varied across MHV strains in line with predicted results-but with the notable exception of MHV-A59, which was not cleaved despite a high score predicted for its sequence. Using the known X-Ray structure of furin in complex with a substrate-like inhibitor as an initial structural reference, we carried out molecular dynamics (MD) simulations to learn the modes of binding of the peptides in the furin active site, and the suitability of the complex for initiation of the enzymatic cleavage. We identified the 3D structural requirements of the furin active site configuration that enable bound peptides to undergo cleavage, and the way in which the various strains tested experimentally are fulfilling these requirements. We find that despite some flexibility in the organization of the peptide bound to the active site of the enzyme, the presence of a histidine at P2 of MHV-A59 fails to properly orient the sidechain of His194 of the furin catalytic triad and therefore produces a distortion that renders the peptide/complex structural configuration in the active site incompatible with requirements for cleavage initiation. The Ser/Thr in P1 of MHV-2 and MHV-S has a similar effect of distorting the conformation of the furin active site residues produced by the elimination of the canonical salt-bridge formed by arginine in P1 position. This work informs a study of coronavirus infection and pathogenesis with respect to the function of the viral spike protein, and suggests an important process of viral adaptation and evolution within the spike S1/S2 structural loop.

Defining Parameters That Modulate Susceptibility and Protection to Respiratory Murine Coronavirus MHV1 Infection.

Patients infected with SARS-CoV-2 experience variable disease susceptibility, and patients with comorbidities such as sepsis are often hospitalized for COVID-19 complications. However, the extent to which initial infectious inoculum dose determines disease outcomes and whether this can be used for immunological priming in a genetically susceptible host has not been completely defined. We used an established SARS-like murine model in which responses to primary and/or secondary challenges with murine hepatitis virus type 1 (MHV-1) were analyzed. We compared the response to infection in genetically susceptible C3H/HeJ mice, genetically resistant C57BL/6J mice, and genetically diverse, variably susceptible outbred Swiss Webster mice. Although defined as genetically susceptible to MHV-1, C3H/HeJ mice displayed decreasing dose-dependent pathological changes in disease severity and lung infiltrate/edema, as well as lymphopenia. Importantly, an asymptomatic dose (500 PFU) was identified that yielded no measurable morbidity/mortality postinfection in C3H/HeJ mice. Polymicrobial sepsis induced via cecal ligation and puncture converted asymptomatic infections in C3H/HeJ and C57BL/6J mice to more pronounced disease, modeling the impact of sepsis as a comorbidity to ß-coronavirus infection. We then used low-dose infection as an immunological priming event in C3H/HeJ mice, which provided neutralizing Ab-dependent, but not circulating CD4/CD8 T cell-dependent, protection against a high-dose MHV-1 early rechallenge. Together, these data define how infection dose, immunological status, and comorbidities modulate outcomes of primary and secondary ß-coronavirus infections in hosts with variable susceptibility.

DYRK1A is a multifunctional host factor that regulates coronavirus replication in a kinase-independent manner.

Coronaviruses (CoVs) pose a major threat to human and animal health worldwide, which complete viral replication by hijacking host factors. Identifying host factors essential for the viral life cycle can deepen our understanding of the mechanisms of virus-host interactions. Based on our previous genome-wide CRISPR screen of α-CoV transmissible gastroenteritis virus (TGEV), we identified the host factor dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), but not DYRK1B, as a critical factor in TGEV replication. Rescue assays and kinase inhibitor experiments revealed that the effect of DYRK1A on viral replication is independent of its kinase activity. Nuclear localization signal modification experiments showed that nuclear DYRK1A facilitated virus replication. Furthermore, DYRK1A knockout significantly downregulated the expression of the TGEV receptor aminopeptidase N (ANPEP) and inhibited viral entry. Notably, we also demonstrated that DYRK1A is essential for the early stage of TGEV replication. Transmission electron microscopy results indicated that DYRK1A contributes to the formation of double-membrane vesicles in a kinase-independent manner. Finally, we validated that DYRK1A is also a proviral factor for mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. In conclusion, our work demonstrated that DYRK1A is an essential host factor for the replication of multiple viruses, providing new insights into the mechanism of virus-host interactions and facilitating the development of new broad-spectrum antiviral drugs.IMPORTANCECoronaviruses, like other positive-sense RNA viruses, can remodel the host membrane to form double-membrane vesicles (DMVs) as their replication organelles. Currently, host factors involved in DMV formation are not well defined. In this study, we used transmissible gastroenteritis virus (TGEV) as a virus model to investigate the regulatory mechanism of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) on coronavirus. Results showed that DYRK1A significantly inhibited TGEV replication in a kinase-independent manner. DYRK1A knockout (KO) can regulate the expression of receptor aminopeptidase N (ANPEP) and endocytic-related genes to inhibit virus entry. More importantly, our results revealed that DYRK1A KO notably inhibited the formation of DMV to regulate the virus replication. Further data proved that DYRK1A is also essential in the replication of mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. Taken together, our findings demonstrated that DYRK1A is a conserved factor for positive-sense RNA viruses and provided new insights into its transcriptional regulation activity, revealing its potential as a candidate target for therapeutic design.

Dietary Vitamin D Mitigates Coronavirus-Induced Lung Inflammation and Damage in Mice.

The COVID-19 pandemic caused by the SARS-CoV-2 (ß-CoV) betacoronavirus has posed a significant threat to global health. Despite the availability of vaccines, the virus continues to spread, and there is a need for alternative strategies to alleviate its impact. Vitamin D, a secosteroid hormone best known for its role in bone health, exhibits immunomodulatory effects in certain viral infections. Here, we have shown that bioactive vitamin D (calcitriol) limits in vitro replication of SARS-CoV-2 and murine coronaviruses MHV-3 and MHV-A59. Comparative studies involving wild-type mice intranasally infected with MHV-3, a model for studying ß-CoV respiratory infections, confirmed the protective effect of vitamin D in vivo. Accordingly, mice fed a standard diet rapidly succumbed to MHV-3 infection, whereas those on a vitamin D-rich diet (10,000 IU of Vitamin D3/kg) displayed increased resistance to acute respiratory damage and systemic complications. Consistent with these findings, the vitamin D-supplemented group exhibited lower viral titers in their lungs and reduced levels of TNF, IL-6, IL-1ß, and IFN-γ, alongside an enhanced type I interferon response. Altogether, our findings suggest vitamin D supplementation ameliorates ß-CoV-triggered respiratory illness and systemic complications in mice, likely via modulation of the host's immune response to the virus.

The survival of murine hepatitis virus (a surrogate of SARS-CoV-2) on conventional packaging materials under cold chain conditions.

Introduction: The cold chain conditions have been suggested to facilitate long-distance transmission of SARS-CoV-2, but it is unclear how viable the virus is on cold chain packaging materials. Methods: This study used the MHV-JHM strain of murine hepatitis virus as a model organism to investigate the viability of SARS-CoV-2 on foam, plastic, cardboard, and wood sheets at different temperatures (-40°C, -20°C, and 4°C). In addition, the ability of peracetic acid and sodium hypochlorite to eliminate the MHV-JHM on plastic and cardboard sheets were also evaluated. Results: The results indicate that MHV-JHM can survive on foam, plastic, or cardboard sheets for up to 28 days at -40°C and -20°C, and up to 14 days on foam and plastic surfaces at 4°C. Although viral nucleic acids were still detectable after storing at 4°C for 28 days, the corresponding virus titer was below the limit of quantification (LOQ). Discussion: The study highlights that a positive nucleic acid test result may not indicate that the virus is still viable, and confirms that peracetic acid and sodium hypochlorite can effectively eliminate MHV-JHM on packaging materials under cold chain conditions.

Targeting the conserved coronavirus octamer motif GGAAGAGC is a strategy for the development of coronavirus vaccine.

BACKGROUND: Coronaviruses are pathogens of humans and animals that cause widespread and costly diseases. The development of effective strategies to combat the threat of coronaviruses is therefore a top priority. The conserved coronavirus octamer motif 5'GGAAGAGC3' exists in the 3' untranslated region of all identified coronaviruses. In the current study, we aimed to examine whether targeting the coronavirus octamer motif GGAAGAGC is a promising approach to develop coronavirus vaccine. METHODS: Plaque assays were used to determine the titers of mouse hepatitis virus (MHV)-A59 octamer mutant (MHVoctm) and wild-type (wt) MHV-A59 (MHVwt). Western blotting was used for the determination of translation efficiency of MHVoctm and MHVwt. Plaque assays and RT-qPCR were employed to examine whether MHVoctm was more sensitive to interferon treatment than MHVwt. Weight loss, clinical signs, survival rate, viral RNA detection and histopathological examination were used to evaluate whether MHVoctm was a vaccine candidate against MHVwt infection in BALB/c mice. RESULTS: In this study, we showed that (i) the MHVoctm with mutation of coronavirus octamer was able to grow to high titers but attenuated in mice, (ii) with the reduced multiplicity of infection (MOI), the difference in gene expression between MHVoctm and MHVwt became more evident in cultured cells, (iii) MHVoctm was more sensitive to interferon treatment than MHVwt and (iv) mice inoculated with MHVoctm were protected from MHVwt infection. CONCLUSIONS: Based on the results obtained from cultured cells, it was suggested that the synergistic effects of octamer mutation, multiplicity of infection and immune response may be a mechanism explaining the distinct phenotypes of octamer-mutated coronavirus in cell culture and mice. In addition, targeting the conserved coronavirus octamer motif is a strategy for development of coronavirus vaccine. Since the conserved octamer exists in all coronaviruses, this strategy of targeting the conserved octamer motif can also be applied to other human and animal coronaviruses for the development of coronavirus vaccines, especially the emergence of novel coronaviruses such as SARS-CoV-2, saving time and cost for vaccine development and disease control.

The role of lysosomes as intermediates in betacoronavirus PHEV egress from nerve cells.

IMPORTANCE: Betacoronaviruses, including severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and mouse hepatitis virus (MHV), exploit the lysosomal exocytosis pathway for egress. However, whether all betacoronaviruses members use the same pathway to exit cells remains unknown. Here, we demonstrated that porcine hemagglutinating encephalomyelitis virus (PHEV) egress occurs by Arl8b-dependent lysosomal exocytosis, a cellular egress mechanism shared by SARS-CoV-2 and MHV. Notably, PHEV acidifies lysosomes and activates lysosomal degradative enzymes, while SARS-CoV-2 and MHV deacidify lysosomes and limit the activation of lysosomal degradative enzymes. In addition, PHEV release depends on V-ATPase-mediated lysosomal pH. Furthermore, this is the first study to evaluate ßCoV using lysosome for spreading through the body, and we have found that lysosome played a critical role in PHEV neural transmission and brain damage caused by virus infection in the central nervous system. Taken together, different betacoronaviruses could disrupt lysosomal function differently to exit cells.

C-C chemokine receptor 5 is essential for conventional NK cell trafficking and liver injury in a murine hepatitis virus-induced fulminant hepatic failure model.

BACKGROUND: Previous studies have demonstrated that natural killer (NK) cells migrated into the liver from peripheral organs and exerted cytotoxic effects on hepatocytes in virus-induced liver failure. AIM: This study aimed to investigate the potential therapeutic role of chemokine receptors in the migration of NK cells in a murine hepatitis  virus strain 3 (MHV-3)-induced fulminant hepatic failure (MHV-3-FHF) model and its mechanism. RESULTS: By gene array analysis, chemokine (C-C motif) receptor 5 (CCR5) was found to have remarkably elevated expression levels in hepatic NK cells after MHV-3 infection. The number of hepatic CCR5+ conventional NK (cNK) cells increased and peaked at 48 h after MHV-3 infection, while the number of hepatic resident NK (rNK) cells steadily declined. Moreover, the expression of CCR5-related chemokines, including macrophage inflammatory protein (MIP)-1α, MIP-1ß and regulated on activation, normal T-cell expressed and secreted (RANTES) was significantly upregulated in MHV-3-infected hepatocytes. In an in vitro Transwell migration assay, CCR5-blocked splenic cNK cells showed decreased migration towards MHV-3-infected hepatocytes, and inhibition of MIP-1ß or RANTES but not MIP-1α decreased cNK cell migration. Moreover, CCR5 knockout (KO) mice displayed reduced infiltration of hepatic cNK cells after MHV-3 infection, accompanied by attenuated liver injury and improved mouse survival time. Adoptive transfer of cNK cells from wild-type mice into CCR5 KO mice resulted in the abundant accumulation of hepatic cNK cells and aggravated liver injury. Moreover, pharmacological inhibition of CCR5 by maraviroc reduced cNK cell infiltration in the liver and liver injury in the MHV-3-FHF model. CONCLUSION: The CCR5-MIP-1ß/RANTES axis played a critical role in the recruitment of cNK cells to the liver during MHV-3-induced liver injury. Targeted inhibition of CCR5 provides a therapeutic approach to ameliorate liver damage during virus-induced acute liver injury.

A suitable model to investigate acute neurological consequences of coronavirus infection.

OBJECTIVE AND DESIGN: The present study aimed to investigate the neurochemical and behavioral effects of the acute consequences after coronavirus infection through a murine model. MATERIAL: Wild-type C57BL/6 mice were infected intranasally (i.n) with the murine coronavirus 3 (MHV-3). METHODS: Mice underwent behavioral tests. Euthanasia was performed on the fifth day after infection (5 dpi), and the brain tissue was subjected to plaque assays for viral titration, ELISA, histopathological, immunohistochemical and synaptosome analysis. RESULTS: Increased viral titers and mild histological changes, including signs of neuronal degeneration, were observed in the cerebral cortex of infected mice. Importantly, MHV-3 infection induced an increase in cortical levels of glutamate and calcium, which is indicative of excitotoxicity, as well as increased levels of pro-inflammatory cytokines (IL-6, IFN-γ) and reduced levels of neuroprotective mediators (BDNF and CX3CL1) in the mice brain. Finally, behavioral analysis showed impaired motor, anhedonia-like and anxiety-like behaviors in animals infected with MHV-3. CONCLUSIONS: In conclusion, the data presented emulate many aspects of the acute neurological outcomes seen in patients with COVID-19. Therefore, this model may provide a preclinical platform to study acute neurological sequelae induced by coronavirus infection and test possible therapies.

A Mouse Model of MHV-1 Virus Infection for Study of Acute and Long COVID Infection.

COVID-19, caused by SARS-CoV-2, has had a significant global impact. While vaccines and treatments have reduced severe cases and deaths, the long-term effects are not yet well understood. Current models used for research, such as non-human primates and transgenic mice, are expensive and require scarce Biosafety Level-3 (BSL-3) laboratories, thereby limiting their practicality. However, the mouse hepatitis virus 1 (MHV-1) mouse model offers a promising alternative. This surrogate model can be investigated in more widely available Biosafety Level-2 (BSL-2) laboratories. Furthermore, mice are affordable and easy to handle, and utilizing MHV-1 as a surrogate for SARS-CoV-2 eliminates the need for costly transgenic mice. Importantly, the MHV-1 model successfully recapitulates COVID-19-related clinical symptoms, weight loss, multiorgan pathological changes and failure in acute stages, irreversible neurological complications, and other long-term organ dysfunction post-infection, which are similar to available human data post-COVID-19. To assist researchers in establishing and using the MHV-1 mouse model, this protocol offers comprehensive guidance encompassing procedures for animal preparation, induction of viral infection, clinical observation, pathological changes, and tissue analysis for mechanistic studies, thereby yielding valuable insights into disease mechanisms and progression. By adopting the MHV-1 model and the provided protocols, researchers can effectively circumvent financial constraints and the limited availability of BSL-3 laboratories, thus facilitating a more accessible and cost-effective approach to investigating the underlying mechanisms of SARS-CoV-2 pathophysiology and exploring potential therapeutic interventions. © 2023 Wiley Periodicals LLC. Basic Protocol: Induction of mouse hepatitis virus 1 (MHV-1) infection in A/J mice Support Protocol 1: Histological evaluation Support Protocol 2: Liver enzyme measurement Support Protocol 3: Western blot analysis of aquaporin expression Support Protocol 4: mRNA measurement Support Protocol 5: Immunohistochemistry/immunofluorescence Support Protocol 6: Tissue water measurement.

Unveiling the biology of defective viral genomes in vitro and in vivo: implications for gene expression and pathogenesis of coronavirus.

BACKGROUND: Defective viral genome (DVG) is a truncated version of the full-length virus genome identified in most RNA viruses during infection. The synthesis of DVGs in coronavirus has been suggested; however, the fundamental characteristics of coronavirus DVGs in gene expression and pathogenesis have not been systematically analyzed. METHODS: Nanopore direct RNA sequencing was used to investigate the characteristics of coronavirus DVGs in gene expression including reproducibility, abundance, species and genome structures for bovine coronavirus in cells, and for mouse hepatitis virus (MHV)-A59 (a mouse coronavirus) in cells and in mice. The MHV-A59 full-length genomic cDNAs (~ 31 kilobases) were in vitro constructed to experimentally validate the origin of coronavirus DVG. The synthesis of DVGs was also experimentally identified by RT-PCR followed by sequencing. In addition, the alterations of DVGs in amounts and species under different infection environments and selection pressures including the treatment of antiviral remdesivir and interferon were evaluated based on the banding patterns by RT-PCR. RESULTS: The results are as follows: (i) the structures of DVGs are with diversity, (ii) DVGs are overall synthesized with moderate (MHV-A59 in cells) to high (BCoV in cells and MHV-A59 in mice) reproducibility under regular infection with the same virus inoculum, (iii) DVGs can be synthesized from the full-length coronavirus genome, (iv) the sequences flanking the recombination point of DVGs are AU-rich and thus may contribute to the recombination events during gene expression, (v) the species and amounts of DVG are altered under different infection environments, and (vi) the biological nature of DVGs between in vitro and in vivo is similar. CONCLUSIONS: The identified biological characteristics of coronavirus DVGs in terms of abundance, reproducibility, and variety extend the current model for coronavirus gene expression. In addition, the biological features of alterations in amounts and species of coronavirus DVGs under different infection environments may assist the coronavirus to adapt to the altered environments for virus fitness and may contribute to the coronavirus pathogenesis. Consequently, the unveiled biological features may assist the community to study the gene expression mechanisms of DVGs and their roles in pathogenesis, contributing to the development of antiviral strategy and public health.

Biological characterization of coronavirus noncanonical transcripts in vitro and in vivo.

BACKGROUND: In addition to the well-known coronavirus genomes and subgenomic mRNAs, the existence of other coronavirus RNA species, which are collectively referred to as noncanonical transcripts, has been suggested; however, their biological characteristics have not yet been experimentally validated in vitro and in vivo. METHODS: To comprehensively determine the amounts, species and structures of noncanonical transcripts for bovine coronavirus in HRT-18 cells and mouse hepatitis virus A59, a mouse coronavirus, in mouse L cells and mice, nanopore direct RNA sequencing was employed. To experimentally validate the synthesis of noncanonical transcripts under regular infection, Northern blotting was performed. Both Northern blotting and nanopore direct RNA sequencing were also applied to examine the reproducibility of noncanonical transcripts. In addition, Northern blotting was also employed to determine the regulatory features of noncanonical transcripts under different infection conditions, including different cells, multiplicities of infection (MOIs) and coronavirus strains. RESULTS: In the current study, we (i) experimentally determined that coronavirus noncanonical transcripts were abundantly synthesized, (ii) classified the noncanonical transcripts into seven populations based on their structures and potential synthesis mechanisms, (iii) showed that the species and amounts of the noncanonical transcripts were reproducible during regular infection but regulated in altered infection environments, (iv) revealed that coronaviruses may employ various mechanisms to synthesize noncanonical transcripts, and (v) found that the biological characteristics of coronavirus noncanonical transcripts were similar between in vitro and in vivo conditions. CONCLUSIONS: The biological characteristics of noncanonical coronavirus transcripts were experimentally validated for the first time. The identified features of noncanonical transcripts in terms of abundance, reproducibility and variety extend the current model for coronavirus gene expression. The capability of coronaviruses to regulate the species and amounts of noncanonical transcripts may contribute to the pathogenesis of coronaviruses during infection, posing potential challenges in disease control. Thus, the biology of noncanonical transcripts both in vitro and in vivo revealed here can provide a database for biological research, contributing to the development of antiviral strategies.

PARP12 is required to repress the replication of a Mac1 mutant coronavirus in a cell- and tissue-specific manner.

ADP-ribosyltransferases (ARTs) mediate the transfer of ADP-ribose from NAD+ to protein or nucleic acid substrates. This modification can be removed by several different types of proteins, including macrodomains. Several ARTs, also known as PARPs, are stimulated by interferon indicating ADP-ribosylation is an important aspect of the innate immune response. All coronaviruses (CoVs) encode for a highly conserved macrodomain (Mac1) that is critical for CoVs to replicate and cause disease, indicating that ADP-ribosylation can effectively control coronavirus infection. Our siRNA screen indicated that PARP12 might inhibit the replication of a murine hepatitis virus (MHV) Mac1 mutant virus in bone-marrow-derived macrophages (BMDMs). To conclusively demonstrate that PARP12 is a key mediator of the antiviral response to CoVs both in cell culture and in vivo, we produced PARP12-/-mice and tested the ability of MHV A59 (hepatotropic/neurotropic) and JHM (neurotropic) Mac1 mutant viruses to replicate and cause disease in these mice. Notably, in the absence of PARP12, Mac1 mutant replication was increased in BMDMs and mice. In addition, liver pathology was also increased in A59-infected mice. However, the PARP12 knockout did not restore Mac1 mutant virus replication to WT virus levels in all cell or tissue types and did not significantly increase the lethality of Mac1 mutant viruses. These results demonstrate that while PARP12 inhibits MHV Mac1 mutant virus infection, additional PARPs or innate immune factors must contribute to the extreme attenuation of this virus in mice. IMPORTANCE Over the last decade, the importance of ADP-ribosyltransferases (ARTs), also known as PARPs, in the antiviral response has gained increased significance as several were shown to either restrict virus replication or impact innate immune responses. However, there are few studies showing ART-mediated inhibition of virus replication or pathogenesis in animal models. We found that the CoV macrodomain (Mac1) was required to prevent ART-mediated inhibition of virus replication in cell culture. Using knockout mice, we found that PARP12, an interferon-stimulated ART, was required to repress the replication of a Mac1 mutant CoV both in cell culture and in mice, demonstrating that PARP12 represses coronavirus replication. However, the deletion of PARP12 did not fully rescue Mac1 mutant virus replication or pathogenesis, indicating that multiple PARPs function to counter coronavirus infection.

SARS-CoV-2 Mac1 is required for IFN antagonism and efficient virus replication in cell culture and in mice.

Several coronavirus (CoV) encoded proteins are being evaluated as targets for antiviral therapies for COVID-19. Included in these drug targets is the conserved macrodomain, or Mac1, an ADP-ribosylhydrolase and ADP-ribose binding protein encoded as a small domain at the N terminus of nonstructural protein 3. Utilizing point mutant recombinant viruses, Mac1 was shown to be critical for both murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV virulence. However, as a potential drug target, it is imperative to understand how a complete Mac1 deletion impacts the replication and pathogenesis of different CoVs. To this end, we created recombinant bacterial artificial chromosomes (BACs) containing complete Mac1 deletions (ΔMac1) in MHV, MERS-CoV, and SARS-CoV-2. While we were unable to recover infectious virus from MHV or MERS-CoV ΔMac1 BACs, SARS-CoV-2 ΔMac1 was readily recovered from BAC transfection, indicating a stark difference in the requirement for Mac1 between different CoVs. Furthermore, SARS-CoV-2 ΔMac1 replicated at or near wild-type levels in multiple cell lines susceptible to infection. However, in a mouse model of severe infection, ΔMac1 was quickly cleared causing minimal pathology without any morbidity. ΔMac1 SARS-CoV-2 induced increased levels of interferon (IFN) and IFN-stimulated gene expression in cell culture and mice, indicating that Mac1 blocks IFN responses which may contribute to its attenuation. ΔMac1 infection also led to a stark reduction in inflammatory monocytes and neutrophils. These results demonstrate that Mac1 only minimally impacts SARS-CoV-2 replication, unlike MHV and MERS-CoV, but is required for SARS-CoV-2 pathogenesis and is a unique antiviral drug target.