Pathological and immunohistochemical assessment of the impact of three different strains of swine enteric coronaviruses in the intestinal barrier.

Swine enteric coronaviruses, such as porcine epidemic diarrhea virus (PEDV) or transmissible gastroenteritis virus (TGEV), have risen concern for the porcine industry and research community due to the increase in their virulence, their potential recombination capacity and the emergence of new variants. This in vivo study aims to compare the impact of three different strains of swine enteric coronaviruses [(two G1b (S-INDEL) PEDV strains and a recombinant TGEV-PEDV or Swine enteric coronavirus (SeCoV)] in the intestine of 3-weeks-old infected piglets, focusing on the pathology and main components of the intestinal barrier, including the number of goblet cells, and the expression of IgA as well as FoxP3, a regulatory T cell marker. Severity of lesions was evidenced in the three infected groups and was highly correlated with the viral load in feces and the frequency of viral antigen-positive cells. Furthermore, higher cellular death together with an increase in the expression of the FoxP3 marker was detected in the duodenum and jejunum of infected animals at 3 days post-infection. Our results highlight a recruitment of FoxP3+ cells in the small intestine of infected animals which may represent a response to the tissue damage caused by viral replication and cell death. Further studies should be addressed to determine the potential role of these cells during swine enteric coronavirus infections.

Risk Assessment of the Possible Intermediate Host Role of Pigs for Coronaviruses with a Deep Learning Predictor.

Swine coronaviruses (CoVs) have been found to cause infection in humans, suggesting that Suiformes might be potential intermediate hosts in CoV transmission from their natural hosts to humans. The present study aims to establish convolutional neural network (CNN) models to predict host adaptation of swine CoVs. Decomposing of each ORF1ab and Spike sequence was performed with dinucleotide composition representation (DCR) and other traits. The relationship between CoVs from different adaptive hosts was analyzed by unsupervised learning, and CNN models based on DCR of ORF1ab and Spike were built to predict the host adaptation of swine CoVs. The rationality of the models was verified with phylogenetic analysis. Unsupervised learning showed that there is a multiple host adaptation of different swine CoVs. According to the adaptation prediction of CNN models, swine acute diarrhea syndrome CoV (SADS-CoV) and porcine epidemic diarrhea virus (PEDV) are adapted to Chiroptera, swine transmissible gastroenteritis virus (TGEV) is adapted to Carnivora, porcine hemagglutinating encephalomyelitis (PHEV) might be adapted to Primate, Rodent, and Lagomorpha, and porcine deltacoronavirus (PDCoV) might be adapted to Chiroptera, Artiodactyla, and Carnivora. In summary, the DCR trait has been confirmed to be representative for the CoV genome, and the DCR-based deep learning model works well to assess the adaptation of swine CoVs to other mammals. Suiformes might be intermediate hosts for human CoVs and other mammalian CoVs. The present study provides a novel approach to assess the risk of adaptation and transmission to humans and other mammals of swine CoVs.

Assessment and sero-diagnosis for coronaviruses with risk of human spillover.

Zoonotic coronaviruses (CoVs) caused major human outbreaks in the last two decades. One of the biggest challenges during future CoV disease is ensuring rapid detection and diagnosis at the early phase of a zoonotic event, and active surveillance to the zoonotic high-risk CoVs appears the best way at the present time to provide early warnings. However, there is neither an evaluation of spillover potential nor diagnosis tools for the majority of CoVs. Here, we analyzed the viral traits, including population, genetic diversity, receptor and host species for all 40 alpha- and beta-CoV species, where the human-infecting CoVs are from. Our analysis proposed 20 high-risk CoV species, including 6 of which jumped to human, 3 with evidence of spillover but not to human and 11 without evidence of spillover yet, which prediction were further supported by an analysis of the history of CoV zoonosis. We also found three major zoonotic sources: multiple bat-origin CoV species, the rodent-origin sub-genus Embecovirus and the CoV species AlphaCoV1. Moreover, the Rhinolophidae and Hipposideridae bats harbour a significantly higher proportion of human-threatening CoV species, whereas camel, civet, swine and pangolin could be important intermediate hosts during CoV zoonotic transmission. Finally, we established quick and sensitive serologic tools for a list of proposed high-risk CoVs and validated the methods in serum cross-reaction assays using hyper-immune rabbit sera or clinical samples. By comprehensive risk assessment of the potential human-infecting CoVs, our work provides a theoretical or practical basis for future CoV disease preparedness.

Urgent Need for Field Surveys of Coronaviruses in Southeast Asia to Understand the SARS-CoV-2 Phylogeny and Risk Assessment for Future Outbreaks.

Phylogenetic analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is focused on a single isolate of bat coronaviruses (bat CoVs) which does not adequately represent genetically related coronaviruses (CoVs) [...].

Theoretical Analysis Reveals the Cost and Benefit of Proofreading in Coronavirus Genome Replication.

Severe acute respiratory syndrome coronaviruses have unusually large RNA genomes replicated by a multiprotein complex containing an RNA-dependent RNA polymerase (RdRp). Exonuclease activity enables the RdRp complex to remove wrongly incorporated bases via proofreading, a process not utilized by other RNA viruses. However, it is unclear why the RdRp complex needs proofreading and what the associated trade-offs are. Here we investigate the interplay among the accuracy, speed, and energetic cost of proofreading in the RdRp complex using a kinetic model and bioinformatics analysis. We find that proofreading nearly optimizes the rate of functional virus production. However, we find that further optimization would lead to a significant increase in the proofreading cost. Unexpected importance of the cost minimization is further supported by other global analyses. We speculate that cost optimization could help avoid cell defense responses. Thus, proofreading is essential for the production of functional viruses, but its rate is limited by energy costs.

Similarities and Dissimilarities of COVID-19 and Other Coronavirus Diseases.

In less than two decades, three deadly zoonotic coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2, have emerged in humans, causing SARS, MERS, and coronavirus disease 2019 (COVID-19), respectively. The current COVID-19 pandemic poses an unprecedented crisis in health care and social and economic development. It reinforces the cruel fact that CoVs are constantly evolving, possessing the genetic malleability to become highly pathogenic in humans. In this review, we start with an overview of CoV diseases and the molecular virology of CoVs, focusing on similarities and differences between SARS-CoV-2 and its highly pathogenic as well as low-pathogenic counterparts. We then discuss mechanisms underlying pathogenesis and virus-host interactions of SARS-CoV-2 and other CoVs, emphasizing the host immune response. Finally, we summarize strategies adopted for the prevention and treatment of CoV diseases and discuss approaches to develop effective antivirals and vaccines.

Current perspective on diagnosis, epidemiological assessment, prevention strategies, and potential therapeutic interventions for severe acute respiratory infections caused by 2019 novel coronavirus (SARS-CoV-2).

Coronaviruses are single-stranded RNA viruses that cause severe respiratory, enteric, and systemic infections in a vast range of hosts, including man, fish, mammals, and avian. Scientific interest has heightened on coronaviruses after the emergence of the 2019 novel Coronavirus (SARS-CoV-2). This review provides current perspectives on morphology, genetic diversity, transmission characteristics, replication cycle, diagnostic approaches, epidemiological assessment, and prevention strategies against the SARS-CoV-2. Moreover, different potential biotherapeutics such as small drug molecules, different vaccines, and immunotherapies to control severe acute respiratory infections caused by 2019 novel coronavirus (SARS-CoV-2) are repurposed and discussed with different mechanistic approaches. The current growth trends of the SARS-CoV-2/COVID-19 outbreak globally and preventive measures are briefly discussed. Furthermore, the lessons learned from the COVID-19 outbreak, so far, concluding remarks and future directions for controlling for COVID-19, are also recommended for a safer tomorrow.

Rapid assessment of regional SARS-CoV-2 community transmission through a convenience sample of healthcare workers, the Netherlands, March 2020.

To rapidly assess possible community transmission in Noord-Brabant, the Netherlands, healthcare workers (HCW) with mild respiratory complaints and without epidemiological link (contact with confirmed case or visited areas with active circulation) were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Within 2 days, 1,097 HCW in nine hospitals were tested; 45 (4.1%) were positive. Of six hospitals with positive HCW, two accounted for 38 positive HCW. The results informed local and national risk management.

Burden and Risk Factors for Coronavirus Infections in Infants in Rural Nepal.

Background: Knowledge of risk factors for symptomatic human coronavirus (HCoV) infections in children in community settings is limited. We estimated the disease burden and impact of birth-related, maternal, household, and seasonal factors on HCoV infections among children from birth to 6 months old in rural Nepal. Methods: Prospective, active, weekly surveillance for acute respiratory infections (ARIs) was conducted in infants over a period of 3 years during 2 consecutive, population-based randomized trials of maternal influenza immunization. Midnasal swabs were collected for acute respiratory symptoms and tested for HCoV and other viruses by reverse-transcription polymerase chain reaction. Association between HCoV incidence and potential risk factors was modeled using Poisson regression. Results: Overall, 282 of 3505 (8%) infants experienced an HCoV ARI within the first 6 months of life. HCoV incidence overall was 255.6 (95% confidence interval [CI], 227.3-286.5) per 1000 person-years, and was more than twice as high among nonneonates than among neonates (incidence rate ratio [IRR], 2.53; 95% CI, 1.52-4.21). HCoV ARI incidence was also positively associated with the number of children <5 years of age per room in a household (IRR, 1.13; 95% CI, 1.01-1.28). Of the 296 HCoV infections detected, 46% were coinfections with other respiratory viruses. While HCoVs were detected throughout the study period, seasonal variation was also observed, with incidence peaking in 2 winters (December-February) and 1 autumn (September-November). Conclusions: HCoV is associated with a substantial proportion of illnesses among young infants in rural Nepal. There is an increased risk of HCoV infection beyond the first month of life.

Recommendations for a standardized avian coronavirus (AvCoV) nomenclature: outcome from discussions within the framework of the European Union COST Action FA1207: "towards control of avian coronaviruses: strategies for vaccination, diagnosis and surveillance".

Viruses within the Coronaviridae family show variations within their genome sequences, especially within the major structural protein the Spike (S) glycoprotein gene. Therefore, many different antigenic forms, serotypes or variant strains of avian coronaviruses (AvCoV) exist worldwide. Only a few of them, the so called protectotypes, cross protect against different serotypes. New serotypes arise by recombination or spontaneous mutations. From time to time, antigenic virus variants appear, which differ significantly from known serotypes. The result of this variability is an inconsistent nomenclature and classification of virus strains. Furthermore, there are currently no standard classification methods defined. Within the framework of the COST Action FA1207 "Towards control of avian coronaviruses: strategies for diagnosis, surveillance and vaccination" (working groups "Molecular virology" and "Epidemiology"), we aimed at defining and developing a unified and internationally standardized nomenclature and classification of AvCoVs. We recommend the use of "CoV Genus/AvCov/host/country/specimen id/year" to refer to AvCoV strains.

Preliminary epidemiological assessment of MERS-CoV outbreak in South Korea, May to June 2015.

South Korea is experiencing the largest outbreak of Middle East respiratory syndrome coronavirus infections outside the Arabian Peninsula, with 166 laboratory-confirmed cases, including 24 deaths up to 19 June 2015. We estimated that the mean incubation period was 6.7 days and the mean serial interval 12.6 days. We found it unlikely that infectiousness precedes symptom onset. Based on currently available data, we predict an overall case fatality risk of 21% (95% credible interval: 14­31).

Coronaviruses resistant to a 3C-like protease inhibitor are attenuated for replication and pathogenesis, revealing a low genetic barrier but high fitness cost of resistance.

Viral protease inhibitors are remarkably effective at blocking the replication of viruses such as human immunodeficiency virus and hepatitis C virus, but they inevitably lead to the selection of inhibitor-resistant mutants, which may contribute to ongoing disease. Protease inhibitors blocking the replication of coronavirus (CoV), including the causative agents of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), provide a promising foundation for the development of anticoronaviral therapeutics. However, the selection and consequences of inhibitor-resistant CoVs are unknown. In this study, we exploited the model coronavirus, mouse hepatitis virus (MHV), to investigate the genotype and phenotype of MHV quasispecies selected for resistance to a broad-spectrum CoV 3C-like protease (3CLpro) inhibitor. Clonal sequencing identified single or double mutations within the 3CLpro coding sequence of inhibitor-resistant virus. Using reverse genetics to generate isogenic viruses with mutant 3CLpros, we found that viruses encoding double-mutant 3CLpros are fully resistant to the inhibitor and exhibit a significant delay in proteolytic processing of the viral replicase polyprotein. The inhibitor-resistant viruses also exhibited postponed and reduced production of infectious virus particles. Biochemical analysis verified double-mutant 3CLpro enzyme as impaired for protease activity and exhibiting reduced sensitivity to the inhibitor and revealed a delayed kinetics of inhibitor hydrolysis and activity restoration. Furthermore, the inhibitor-resistant virus was shown to be highly attenuated in mice. Our study provides the first insight into the pathogenicity and mechanism of 3CLpro inhibitor-resistant CoV mutants, revealing a low genetic barrier but high fitness cost of resistance. Importance: RNA viruses are infamous for their ability to evolve in response to selective pressure, such as the presence of antiviral drugs. For coronaviruses such as the causative agent of Middle East respiratory syndrome (MERS), protease inhibitors have been developed and shown to block virus replication, but the consequences of selection of inhibitor-resistant mutants have not been studied. Here, we report the low genetic barrier and relatively high deleterious consequences of CoV resistance to a 3CLpro protease inhibitor in a coronavirus model system, mouse hepatitis virus (MHV). We found that although mutations that confer resistance arise quickly, the resistant viruses replicate slowly and do not cause lethal disease in mice. Overall, our study provides the first analysis of the low barrier but high cost of resistance to a CoV 3CLpro inhibitor, which will facilitate the further development of protease inhibitors as anti-coronavirus therapeutics.

The autocatalytic release of a putative RNA virus transcription factor from its polyprotein precursor involves two paralogous papain-like proteases that cleave the same peptide bond.

The largest replicative protein of coronaviruses is known as p195 in the avian infectious bronchitis virus (IBV) and p210 (p240) in the mouse hepatitis virus. It is autocatalytically released from the precursors pp1a and pp1ab by one zinc finger-containing papain-like protease (PLpro) in IBV and by two paralogous PLpros, PL1pro and PL2pro, in mouse hepatitis virus. The PLpro-containing proteins have been recently implicated in the control of coronavirus subgenomic mRNA synthesis (transcription). By using comparative sequence analysis, we now show that the respective proteins of all sequenced coronaviruses are flanked by two conserved PLpro cleavage sites and share a complex (multi)domain organization with PL1pro being inactivated in IBV. Based upon these predictions, the processing of the human coronavirus 229E p195/p210 N terminus was studied in detail. First, an 87-kDa protein (p87), which is derived from a pp1a/pp1ab region immediately upstream of p195/p210, was identified in human coronavirus 229E-infected cells. Second, in vitro synthesized proteins representing different parts of pp1a were autocatalytically processed at the predicted site. Surprisingly, both PL1pro and PL2pro cleaved between p87 and p195/p210. The PL1pro-mediated cleavage was slow and significantly suppressed by a non-proteolytic activity of PL2pro. In contrast, PL2pro, whose proteolytic activity and specificity were established in this study, cleaved the same site efficiently in the presence of the upstream domains. Third, a correlation was observed between the overlapping substrate specificities and the parallel evolution of PL1pro and PL2pro. Collectively, our results imply that the p195/p210 autoprocessing mechanisms may be conserved among coronaviruses to an extent not appreciated previously, with PL2pro playing a major role. A large subset of coronaviruses may employ two proteases to cleave the same site(s) and thus regulate the expression of the viral genome in a unique way.