The coronavirus recombination pathway.

Recombination is thought to be a mechanism that facilitates cross-species transmission in coronaviruses, thus acting as a driver of coronavirus spillover and emergence. Despite its significance, the mechanism of recombination is poorly understood, limiting our potential to estimate the risk of novel recombinant coronaviruses emerging in the future. As a tool for understanding recombination, here, we outline a framework of the recombination pathway for coronaviruses. We review existing literature on coronavirus recombination, including comparisons of naturally observed recombinant genomes as well as in vitro experiments, and place the findings into the recombination pathway framework. We highlight gaps in our understanding of coronavirus recombination illustrated by the framework and outline how further experimental research is critical for disentangling the molecular mechanism of recombination from external environmental pressures. Finally, we describe how an increased understanding of the mechanism of recombination can inform pandemic predictive intelligence, with a retrospective emphasis on SARS-CoV-2.

Porcine Deltacoronaviruses: Origin, Evolution, Cross-Species Transmission and Zoonotic Potential.

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus of swine that causes acute diarrhoea, vomiting, dehydration and mortality in seronegative neonatal piglets. PDCoV was first reported in Hong Kong in 2012 and its etiological features were first characterized in the United States in 2014. Currently, PDCoV is a concern due to its broad host range, including humans. Chickens, turkey poults, and gnotobiotic calves can be experimentally infected by PDCoV. Therefore, as discussed in this review, a comprehensive understanding of the origin, evolution, cross-species transmission and zoonotic potential of epidemic PDCoV strains is urgently needed.

Corona Viruses: Emergence, Evolution, and Recurrence

Corona viruses (CoVs) are enveloped RNA viruses that infect a broad array of avian and mammalian species, including humans. The existence of these viruses is believed to have occurred thousands of years ago as animal CoVs;bats, birds, rodents were reported to be natural reservoirs. They garnered scientific attention after their emergence as human pathogens, till date, seven corona viruses were reported to infect humans, with mild to moderate and/or severe respiratory illness. The ongoing pandemic COVID-19 is caused by one of such Corona viruses named Severe Acute Respiratory Syndrome Corona Virus -2 (SARS-CoV-2), which surprised all with its unprecedented transmission dynamics and etiology. This virus surged twice within a gap of a year all over the world and became a major health concern to many nations. Most of these Corona viruses transferred to humans through intermediate hosts. Here, in this chapter, we summarized the structural and genomic features of the Coronaviruses in general and emphasizing the SARS CoV-2 and added an account of the different vaccines and their production platforms in combating the pandemic. We briefly discussed the evolution of new variants of SARS-CoV-2 and their role in the surge of COVID-19 infections. We tried to give a brief account of the historical aspects, cross-species transmission, mutations/recombinations scenarios of CoVs with a note on their emergence as human pathogens and future prospects of recurrence. © The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2021.

Development of a colloidal gold immunochromatographic assay strip using monoclonal antibody for rapid detection of porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) cause diarrhea and dehydration in newborn piglets and has the potential for cross-species transmission. Rapid and early diagnosis is important for preventing and controlling infectious disease. In this study, two monoclonal antibodies (mAbs) were generated, which could specifically recognize recombinant PDCoV nucleocapsid (rPDCoV-N) protein. A colloidal gold immunochromatographic assay (GICA) strip using these mAbs was developed to detect PDCoV antigens within 15 min. Results showed that the detection limit of the GICA strip developed in this study was 103 TCID50/ml for the suspension of virus-infected cell culture and 0.125 µg/ml for rPDCoV-N protein, respectively. Besides, the GICA strip showed high specificity with no cross-reactivity with other porcine pathogenic viruses. Three hundred and twenty-five fecal samples were detected for PDCoV using the GICA strip and reverse transcription-quantitative real-time PCR (RT-qPCR). The coincidence rate of the GICA strip and RT-qPCR was 96.9%. The GICA strip had a diagnostic sensitivity of 88.9% and diagnostic specificity of 98.5%. The specific and efficient detection by the strip provides a convenient, rapid, easy to use and valuable diagnostic tool for PDCoV under laboratory and field conditions.

Porcine Epidemic Diarrhea Virus Replication in Human Intestinal Cells Reveals Potential Susceptibility to Cross-Species Infection.

Various coronaviruses have emerged as a result of cross-species transmission among humans and domestic animals. Porcine epidemic diarrhea virus (PEDV; family Coronaviridae, genus Alphacoronavirus) causes acute diarrhea, vomiting, dehydration, and high mortality in neonatal piglets. Porcine small intestinal epithelial cells (IPEC-J2 cells) can be used as target cells for PEDV infection. However, the origin of PEDV in pigs, the host range, and cross-species infection of PEDV remain unclear. To determine whether PEDV has the ability to infect human cells in vitro, human small intestinal epithelial cells (FHs 74 Int cells) were inoculated with PEDV LJX and PEDV CV777 strains. The results indicated that PEDV LJX, but not PEDV CV777, could infect FHs 74 Int cells. Furthermore, we observed M gene mRNA transcripts and N protein expression in infected FHs 74 Int cells. A one-step growth curve showed that the highest viral titer of PEDV occurred at 12 h post infection. Viral particles in vacuoles were observed in FHs 74 Int cells at 24 h post infection. The results proved that human small intestinal epithelial cells are susceptible to PEDV infection, suggesting the possibility of cross-species transmission of PEDV.

SARS-CoV-2 does not infect pigs, but this has to be verified regularly.

For successful xenotransplantation, freedom of the xenocraft donor from certain viral infections that may harm the organ recipient is important. A novel human coronavirus (CoV) with a respiratory tropism, designated as SARS-CoV-2, was first identified in January 2020 in China, but likely has been circulating unnoticed for some time before. Since then, this virus has reached most inhabited areas, resulting in a major global pandemic which is still ongoing. Due to a high number of subclinical infections, re-infections, geographic differences in diagnostic tests used, and differences in result reporting programs, the percentage of the population infected with SARS-CoV-2 at least once has been challenging to estimate. With continuous ongoing infections in people and an overall high viral load, it makes sense to look into possible viral spillover events in pets and farm animals, who are often in close contact with humans. The pig is currently the main species considered for xenotransplantation and hence there is interest to know if pigs can become infected with SARS-CoV-2 and if so what the infection dynamics may look like. This review article summarizes the latest research findings on this topic. It would appear that pigs can currently be considered a low risk species, and hence do not pose an immediate risk to the human population or xenotransplantation recipients per se. Monitoring the ever-changing SARS-CoV-2 variants appears important to recognize immediately should this change in the future.

Cross-species transmission, evolution and zoonotic potential of coronaviruses.

Coronaviruses (CoVs) continuously evolve, crossing species barriers and spreading across host ranges. Over the last two decades, several CoVs (HCoV-229E, HCoV-NL63, HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, and SARS-CoV-2) have emerged in animals and mammals, causing significant economic and human life losses. Due to CoV cross-species transmission and the evolution of novel viruses, it is critical to identify their natural reservoiurs and the circumstances under which their transmission occurs. In this review, we use genetic and ecological data to disentangle the evolution of various CoVs in wildlife, humans, and domestic mammals. We thoroughly investigate several host species and outline the epidemiology of CoVs toward specific hosts. We also discuss the cross-species transmission of CoVs at the interface of wildlife, animals, and humans. Clarifying the epidemiology and diversity of species reservoirs will significantly impact our ability to respond to the future emergence of CoVs in humans and domestic animals.

Cross-species transmission of coronaviruses with a focus on severe acute respiratory syndrome coronavirus 2 infection in animals: a review for the veterinary practitioner.

In 2019 a novel coronavirus termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged from an unidentified source and spread rapidly among humans worldwide. While many human infections are mild, some result in severe clinical disease that in a small proportion of infected people is fatal. The pandemic spread of SARS-CoV-2 has been facilitated by efficient human-to-human transmission of the virus, with no data to indicate that animals contributed to this global health crisis. However, a range of domesticated and wild animals are also susceptible to SARS-CoV-2 infection under both experimental and natural conditions. Humans are presumed to be the source of most animal infections thus far, although natural transmission between mink and between free-ranging deer has occurred, and occasional natural transmission between cats cannot be fully excluded. Considering the ongoing circulation of the virus among people, together with its capacity to evolve through mutation and recombination, the risk of the emergence of animal-adapted variants is not negligible. If such variants remain infectious to humans, this could lead to the establishment of an animal reservoir for the virus, which would complicate control efforts. As such, minimising human-to-animal transmission of SARS-CoV-2 should be considered as part of infection control efforts. The aim of this review is to summarise what is currently known about the species specificity of animal coronaviruses, with an emphasis on SARS-CoV-2, in the broader context of factors that facilitate cross-species transmission of viruses.

Animal Models, Zoonotic Reservoirs, and Cross-Species Transmission of Emerging Human-Infecting Coronaviruses.

Over the past three decades, coronavirus (CoV) diseases have impacted humans more than any other emerging infectious disease. The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 (coronavirus disease 2019), has resulted in huge economic disruptions and loss of human lives. The SARS-CoV-2 genome was found to mutate more rapidly due to sustained transmission in humans and potentially animals, resulting in variants of concern (VOCs) that threaten global human health. However, the primary difficulties are filling in the current knowledge gaps in terms of the origin and modalities of emergence for these viruses. Because many CoVs threatening human health are suspected to have a zoonotic origin, identifying the animal hosts implicated in the spillover or spillback events would be beneficial for current pandemic management and to prevent future outbreaks. In this review, wesummarize the animal models, zoonotic reservoirs, and cross-species transmission of the emerging human CoVs. Finally, we comment on potential sources of SARS-CoV-2 Omicron VOCs and the new SARS-CoV-2 recombinants currently under investigation.

Enveloped viruses show increased propensity to cross-species transmission and zoonosis.

The transmission of viruses between different host species is a major source of emerging diseases and is of particular concern in the case of zoonotic transmission from mammals to humans. Several zoonosis risk factors have been identified, but it is currently unclear which viral traits primarily determine this process as previous work has focused on a few hundred viruses that are not representative of actual viral diversity. Here, we investigate fundamental virological traits that influence cross-species transmissibility and zoonotic propensity by interrogating a database of over 12,000 mammalian virus-host associations. Our analysis reveals that enveloped viruses tend to infect more host species and are more likely to be zoonotic than nonenveloped viruses, while other viral traits such as genome composition, structure, size, or the viral replication compartment play a less obvious role. This contrasts with the previous notion that viral envelopes did not significantly impact or even reduce zoonotic risk and should help better prioritize outbreak prevention efforts. We suggest several mechanisms by which viral envelopes could promote cross-species transmissibility, including structural flexibility of receptor-binding proteins and evasion of viral entry barriers.

The Role of Molossidae and Vespertilionidae in Shaping the Diversity of Alphacoronaviruses in the Americas.

Bats are reservoirs of diverse coronaviruses (CoVs), including progenitors of severe acute respiratory syndrome CoV (SARS-CoV) and SARS-CoV-2. In the Americas, there is a contrast between alphacoronaviruses (alphaCoVs) and betaCoVs: while cospeciation prevails in the latter, alphaCoV evolution is dominated by deep and recent host switches. AlphaCoV lineages are maintained by two different bat family groups, Phyllostomidae and Vespertilionidae plus Molossidae. In this study, we used a Bayesian framework to analyze the process of diversification of the lineages maintained by Molossidae and Vespertilionidae, adding novel CoV sequences from Argentina. We provide evidence that the observed CoV diversity in these two bat families is shaped by their geographic distribution and that CoVs exhibit clustering at the level of bat genera. We discuss the causes of the cocirculation of two independent clades in Molossus and Tadarida as well as the role of Myotis as the ancestral host and a major evolutionary reservoir of alphaCoVs across the continent. Although more CoV sampling efforts are needed, these findings contribute to a better knowledge of the diversity of alphaCoVs and the links between bat host species. IMPORTANCE Bats harbor the largest diversity of coronaviruses among mammals. In the Americas, seven alphacoronavirus lineages circulate among bats. Three of these lineages are shared by members of two bat families: Vespertilionidae and Molossidae. Uncovering the relationships between these coronaviruses can help us to understand patterns of cross-species transmission and, ultimately, which hosts are more likely to be involved in spillover events. We found that two different lineages cocirculate among the bat genera Molossus and Tadarida, which share roosts and have common viral variants. The bat genus Myotis functions as a reservoir of coronavirus diversity and, as such, is a key host. Although there were some spillovers recorded, there is a strong host association, showing that once a successful host jump takes place, it is transmitted onward to members of the same bat genus.

Human coronaviruses: The emergence of SARS-CoV-2 and management of COVID-19.

To date, a total of seven human coronaviruses (HCoVs) have been identified, all of which are important respiratory pathogens. Recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has led to a global pandemic causing millions of infections and deaths. Here, we summarize the discovery and fundamental virology of HCoVs, discuss their zoonotic transmission and highlight the weak species barrier of SARS-CoV-2. We also discuss the possible origins of SARS-CoV-2 variants of concern identified to date and discuss the experimental challenges in characterizing mutations of interest and propose methods to circumvent them. As the COVID-19 treatment and prevention landscape rapidly evolves, we summarize current therapeutics and vaccines, and their implications on SARS-CoV-2 variants. Finally, we explore how interspecies transmission of SARS-CoV-2 may drive the emergence of novel strains, how disease severity may evolve and how COVID-19 will likely continue to burden healthcare systems globally.

Coronavirus Infections in Animals: Risks of Direct and Reverse Zoonoses.

The publications on animal coronavirus infections that have the greatest emerging potential, as well as official data from the World Organization for Animal Health (OIE) on cases of animal infection with COVID-19, are analyzed. Like most infectious diseases common to humans, coronavirus infections were first discovered in animals. Due to the increased rate of replication and recombination activity compared to other viruses, mutations occur more often in the genome of coronaviruses, which contribute to the acquisition of new qualities in order to consolidate in the host organism. Examples of cross-species transmission are not only SARS-CoV, MERS-CoV, and SARS-CoV-2, which are dangerous to humans, but also coronaviruses of agricultural and domestic animals, between which there is a genetic relationship. There are several known cases of zoo, wild, domestic, and farm animals displaying symptoms characteristic of COVID-19 and identification of the genome of the SARS-CoV-2 virus in them. The issue of cross-species transmission of coronavirus infections, in particular the reverse zoonosis of SARS-CoV-2 from animals to humans, is widely discussed. According to the conclusions of many researchers, including OIE experts, there is no direct evidence base for infection of humans with COVID-19 from animals. However, people with suspected COVID-19 and with a confirmed diagnosis are still advised to isolate not only from people but also from animals. A number of methods for specific prevention, diagnosis, and immunization against a wide range of coronavirus infections are being developed at the All-Russia Research Institute for Animal Protection.

Effect of human aminopeptidase N (hAPN) on porcine deltacoronavirus infecting HEK293 cells

HEK293 cells were used as the cell model to investigate the role of human aminopeptidase N (hAPN) in the invasion of porcine deltacoronavirus (PDCoV) into human cells. The proliferation of PDCoV on HEK293 cells was firstly identified by RT-qPCR/RT-PCR. And then, hAPN knockout cell line was constructed by CRISPR/Cas9 technology and cell viability of HEK293 hAPN knockout and wild-type cells was verified by CCK-8 assay. Effect of hAPN knockout and overexpression on PDCoV replication was detected by RT-qPCR and Western blot. Meanwhile, interaction of PDCoV S protein and hAPN protein was analyzed by homology modeling and molecular docking. Results showed that PDCoV virus copies rapidly increased at 12-36 h and reached peak level at 36 h, it could propagate at least for passage 2 on HEK293 cells. There was no significant difference in cell viability between hAPN knockout cells and wild-type cells. Knockout of hAPN inhibit PDCoV replication and overexpression of hAPN enhance PDCoV replication. Homology modeling and molecular docking analysis showed S1 protein could bind hAPN domain II. Residues TYR92, THR51, THR48, PHE16 and MET14of S1 protein receptor binding motif 1 (RBM1) can form hydrogen bonds with residues PHE490, GLN531, ARG528 and SER529 of hAPN. This study indicates that hAPN plays a critical role in HEK293 cells during PDCoV infection, which provides new theoretical evidence for further studies on the mechanism of PDCoV entry into host cells and cross-species transmission.

Susceptibility to mice and potential evolutionary characteristics of porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is a novel coronavirus that causes diarrhea in suckling piglets and has the potential for cross-species transmission, posing a threat to animal and human health. However, the susceptibility profile of different species of mice to PDCoV infection and its evolutionary characteristics are still unclear. In the current study, we found that BALB/c and Kunming mice are susceptible to PDCoV. Our results showed that there were obvious lesions in intestinal and lung tissues from the infected mice. PDCoV RNAs were detected in the lung, kidney, and intestinal tissues from the infected mice of both strains, and there existed wider tissue tropism in the PDCoV-infected BALB/c mice. The RNA and protein levels of aminopeptidase N from mice were relatively high in the kidney and intestinal tissues and obviously increased after PDCoV infection. The viral-specific IgG and neutralizing antibodies against PDCoV were detected in the serum of infected mice. An interesting finding was that two key amino acid mutations, D138H and Q641K, in the S protein were identified in the PDCoV-infected mice. The essential roles of these two mutations for PDCoV-adaptive evolution were confirmed by cryo-electron microscope structure model analysis. The evolutionary characteristics of PDCoV among Deltacoronaviruses (δ-CoVs) were further analyzed. δ-CoVs from multiple mammals are closely related based on the phylogenetic analysis. The codon usage analysis demonstrated that similar codon usage patterns were used by most of the mammalian δ-CoVs at the global codon, synonymous codon, and amino acid usage levels. These results may provide more insights into the evolution, host ranges, and cross-species potential of PDCoV.

Advances in Bovine Coronavirus Epidemiology.

Bovine coronavirus (BCoV) is a causative agent of enteric and respiratory disease in cattle. BCoV has also been reported to cause a variety of animal diseases and is closely related to human coronaviruses, which has attracted extensive attention from both cattle farmers and researchers. However, there are few comprehensive epidemiological reviews, and key information regarding the effect of S-gene differences on tissue tendency and potential cross-species transmission remain unclear. In this review, we summarize BCoV epidemiology, including the transmission, infection-associated factors, co-infection, pathogenicity, genetic evolution, and potential cross-species transmission. Furthermore, the potential two-receptor binding motif system for BCoV entry and the association between BCoV and SARS-CoV-2 are also discussed in this review. Our aim is to provide valuable information for the prevention and treatment of BCoV infection throughout the world.

Cross-Species Transmission of Bat Coronaviruses in the Americas: Contrasting Patterns between Alphacoronavirus and Betacoronavirus.

Bats harbor the largest number of coronavirus (CoV) species among mammals, serving as major reservoirs of alphaCoVs and betaCoVs, which can jump between bat species or to different mammalian hosts, including humans. Bat-CoV diversity is correlated with host taxonomic diversity, with the highest number of CoV species found in areas with the highest levels of bat species richness. Although the Americas harbor a unique and distinctive CoV diversity, no cross-species transmission (CST) or phylogeographic analysis has yet been performed. This study analyzes a large sequence data set from across the Americas through a Bayesian framework to understand how codivergence and cross-species transmission have shaped long-term bat-CoV evolution and ultimately identify bat hosts and regions where the risk of CST is the highest. Substantial levels of CST were found only among alphaCoVs. In contrast, cospeciation prevailed along the evolution of betaCoVs. Brazil is the center of diversification for both alpha and betaCoVs, with the highest levels of bat species richness. The bat family Phyllostomidae has played a key role in the evolution of American bat-CoVs, supported by the highest values of host transition rates. Although the conclusions drawn from this study are supported by biological/ecological evidence, it is likely that novel lineages will be discovered, which could also reveal undetected CSTs given that sequences are available from 11 of the 35 countries encompassing the Americas. The findings of this study can be useful for conducting targeted discovery of bat-CoVs in the region, especially in countries of the Americas with no reported sequences. IMPORTANCE Coronaviruses (CoVs) have a strong zoonotic potential due to their high rates of evolvability and their capacity for overcoming host-specific barriers. Bats harbor the largest number of CoV species among mammals, with the highest CoV diversity found in areas with the highest levels of bat species richness. Understanding their origin and patterns of cross-species transmission is crucial for pandemic preparedness. This study aims to understand how bat-CoVs diversify in the Americas, circulate among and transmit between bat families and genera, and ultimately identify bat hosts and regions where the risk of CoV spillover is the highest.

Long-range enhancement of N501Y-endowed mouse infectivity of SARS-CoV-2 by the non-RBD mutations of Ins215KLRS and H655Y.

BACKGROUND: Rodents, such as mice, are vulnerable targets, and potential intermediate hosts, of SARS-CoV-2 variants of concern, including Alpha, Beta, Gamma, and Omicron. N501Y in the receptor-binding domain (RBD) of Spike protein is the key mutation dictating the mouse infectivity, on which the neighboring mutations within RBD have profound impacts. However, the impacts of mutations outside RBD on N501Y-mediated mouse infectivity remain to be explored. RESULTS: Herein, we report that two non-RBD mutations derived from mouse-adapted strain, Ins215KLRS in the N-terminal domain (NTD) and H655Y in the subdomain linking S1 to S2, enhance mouse infectivity in the presence of N501Y mutation, either alone or together. This is associated with increased interaction of Spike with mouse ACE2 and mutations-induced local conformation changes in Spike protein. Mechanistically, the H655Y mutation disrupts interaction with N657, resulting in a less tight loop that wraps the furin-cleavage finger; and the insertion of 215KLRS in NTD increases its intramolecular interaction with a peptide chain that interfaced with the RBD-proximal region of the neighboring protomer, leading to a more flexible RBD that facilitates receptor binding. Moreover, the Omicron Spike that contains Ins214EPE and H655Y mutations confer mouse infectivity > 50 times over the N501Y mutant, which could be effectively suppressed by mutating them back to wild type. CONCLUSIONS: Collectively, our study sheds light on the cooperation between distant Spike mutations in promoting virus infectivity, which may undermine the high infectiousness of Omicron variants towards mice.

Presence of Recombinant Bat Coronavirus GCCDC1 in Cambodian Bats.

Bats have been recognized as an exceptional viral reservoir, especially for coronaviruses. At least three bat zoonotic coronaviruses (SARS-CoV, MERS-CoV and SARS-CoV-2) have been shown to cause severe diseases in humans and it is expected more will emerge. One of the major features of CoVs is that they are all highly prone to recombination. An extreme example is the insertion of the P10 gene from reoviruses in the bat CoV GCCDC1, first discovered in Rousettus leschenaultii bats in China. Here, we report the detection of GCCDC1 in four different bat species (Eonycteris spelaea, Cynopterus sphinx, Rhinolophus shameli and Rousettus sp.) in Cambodia. This finding demonstrates a much broader geographic and bat species range for this virus and indicates common cross-species transmission. Interestingly, one of the bat samples showed a co-infection with an Alpha CoV most closely related to RsYN14, a virus recently discovered in the same genus (Rhinolophus) of bat in Yunnan, China, 2020. Taken together, our latest findings highlight the need to conduct active surveillance in bats to assess the risk of emerging CoVs, especially in Southeast Asia.