Discovery and Genetic Characterization of Novel Paramyxoviruses Related to the Genus Henipavirus in Crocidura Species in the Republic of Korea.

Paramyxoviruses, negative-sense single-stranded RNA viruses, pose a critical threat to human public health. Currently, 78 species, 17 genera, and 4 subfamilies of paramyxoviruses are harbored by multiple natural reservoirs, including rodents, bats, birds, reptiles, and fish. Henipaviruses are critical zoonotic pathogens that cause severe acute respiratory distress and neurological diseases in humans. Using reverse transcription-polymerase chain reaction, 115 Crocidura species individuals were examined for the prevalence of paramyxovirus infections. Paramyxovirus RNA was observed in 26 (22.6%) shrews collected at five trapping sites, Republic of Korea. Herein, we report two genetically distinct novel paramyxoviruses (genus: Henipavirus): Gamak virus (GAKV) and Daeryong virus (DARV) isolated from C. lasiura and C. shantungensis, respectively. Two GAKVs and one DARV were nearly completely sequenced using next-generation sequencing. GAKV and DARV contain six genes (3'-N-P-M-F-G-L-5') with genome sizes of 18,460 nucleotides and 19,471 nucleotides, respectively. The phylogenetic inference demonstrated that GAKV and DARV form independent genetic lineages of Henipavirus in Crocidura species. GAKV-infected human lung epithelial cells elicited the induction of type I/III interferons, interferon-stimulated genes, and proinflammatory cytokines. In conclusion, this study contributes further understandings of the molecular prevalence, genetic characteristics and diversity, and zoonotic potential of novel paramyxoviruses in shrews.

Best Molecular Tools to Investigate Coronavirus Diversity in Mammals: A Comparison.

Coronaviruses (CoVs) are widespread and highly diversified in wildlife and domestic mammals and can emerge as zoonotic or epizootic pathogens and consequently host shift from these reservoirs, highlighting the importance of veterinary surveillance. All genera can be found in mammals, with α and ß showing the highest frequency and diversification. The aims of this study were to review the literature for features of CoV surveillance in animals, to test widely used molecular protocols, and to identify the most effective one in terms of spectrum and sensitivity. We combined a literature review with analyses in silico and in vitro using viral strains and archive field samples. We found that most protocols defined as pan-coronavirus are strongly biased towards α- and ß-CoVs and show medium-low sensitivity. The best results were observed using our new protocol, showing LoD 100 PFU/mL for SARS-CoV-2, 50 TCID50/mL for CaCoV, 0.39 TCID50/mL for BoCoV, and 9 ± 1 log2 ×10-5 HA for IBV. The protocol successfully confirmed the positivity for a broad range of CoVs in 30/30 field samples. Our study points out that pan-CoV surveillance in mammals could be strongly improved in sensitivity and spectrum and propose the application of a new RT-PCR assay, which is able to detect CoVs from all four genera, with an optimal sensitivity for α-, ß-, and γ-.

Common Themes in Zoonotic Spillover and Disease Emergence: Lessons Learned from Bat- and Rodent-Borne RNA Viruses.

Rodents (order Rodentia), followed by bats (order Chiroptera), comprise the largest percentage of living mammals on earth. Thus, it is not surprising that these two orders account for many of the reservoirs of the zoonotic RNA viruses discovered to date. The spillover of these viruses from wildlife to human do not typically result in pandemics but rather geographically confined outbreaks of human infection and disease. While limited geographically, these viruses cause thousands of cases of human disease each year. In this review, we focus on three questions regarding zoonotic viruses that originate in bats and rodents. First, what biological strategies have evolved that allow RNA viruses to reside in bats and rodents? Second, what are the environmental and ecological causes that drive viral spillover? Third, how does virus spillover occur from bats and rodents to humans?

A Novel Potentially Recombinant Rodent Coronavirus with a Polybasic Cleavage Site in the Spike Protein.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reignited global interest in animal coronaviruses and their potential for human transmission. While bats are thought to be the wildlife reservoir of SARS-CoV and SARS-CoV-2, the widespread human coronavirus OC43 is thought to have originated in rodents. Here, we sampled 297 rodents and shrews, representing eight species, from three municipalities of southern China. We report coronavirus prevalences of 23.3% and 0.7% in Guangzhou and Guilin, respectively, with samples from urban areas having significantly higher coronavirus prevalences than those from rural areas. We obtained three coronavirus genome sequences from Rattus norvegicus, including a Betacoronavirus (rat coronavirus [RCoV] GCCDC3), an Alphacoronavirus (RCoV-GCCDC5), and a novel Betacoronavirus (RCoV-GCCDC4). Recombination analysis suggests that there was a potential recombination event involving RCoV-GCCDC4, murine hepatitis virus (MHV), and Longquan Rl rat coronavirus (LRLV). Furthermore, we uncovered a polybasic cleavage site, RARR, in the spike (S) protein of RCoV-GCCDC4, which is dominant in RCoV. These findings provide further information on the potential for interspecies transmission of coronaviruses and demonstrate the value of a One Health approach to virus discovery. IMPORTANCE Surveillance of viruses among rodents in rural and urban areas of South China identified three rodent coronaviruses, RCoV-GCCDC3, RCoV-GCCDC4, and RCoV-GCCDC5, one of which was identified as a novel potentially recombinant coronavirus with a polybasic cleavage site in the spike (S) protein. Through reverse transcription-PCR (RT-PCR) screening of coronaviruses, we found that coronavirus prevalence in urban areas is much higher than that in rural areas. Subsequently, we obtained three coronavirus genome sequences by deep sequencing. After different method-based analyses, we found that RCoV-GCCDC4 was a novel potentially recombinant coronavirus with a polybasic cleavage site in the S protein, dominant in RCoV. This newly identified coronavirus RCoV-GCCDC4 with its potentially recombinant genome and polybasic cleavage site provides a new insight into the evolution of coronaviruses. Furthermore, our results provide further information on the potential for interspecies transmission of coronaviruses and demonstrate the necessity of a One Health approach for zoonotic disease surveillance.

Exploring the potential roles of some rodents in the transmission of the Middle East respiratory syndrome coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) is one of the recently identified zoonotic coronaviruses. The one-hump camels are believed to play important roles in the evolution and transmission of the virus. The animal-to-animal, as well as the animal-to-human transmission in the context of MERS-CoV infection, were reported. The camels shed the virus in some of their secretions, especially the nasal tract. However, there are many aspects of the transmission cycle of the virus from animals to humans that are still not fully understood. Rodents played important roles in the transmission of many pathogens, including viruses and bacteria. They have been implicated in the evolution of many human coronaviruses, especially HCoV-OC43 and HCoV-HKU1. However, the role of rodents in the transmission of MERS-CoV still requires more exploration. To achieve this goal, we identified MERS-CoV that naturally infected dromedary camel by molecular surveillance. We captured 15 of the common rodents (rats, mice, and jerboa) sharing the habitat with these animals. We collected both oral and rectal swabs from these animals and then tested them by the commercial MERS-CoV real-time-PCR kits using two targets. Despite the detection of the viral shedding in the nasal swabs of some of the dromedary camels, none of the rodents tested positive for the virus during the tenure of this study. We concluded that these species of rodents did not harbor the virus and are most unlikely to contribute to the transmission of the MERS-CoV. However, further large-scale studies are required to confirm the potential roles of rodents in the context of the MERS-CoV transmission cycle, if any.

Coronavirus surveillance in wildlife from two Congo basin countries detects RNA of multiple species circulating in bats and rodents.

Coronaviruses play an important role as pathogens of humans and animals, and the emergence of epidemics like SARS, MERS and COVID-19 is closely linked to zoonotic transmission events primarily from wild animals. Bats have been found to be an important source of coronaviruses with some of them having the potential to infect humans, with other animals serving as intermediate or alternate hosts or reservoirs. Host diversity may be an important contributor to viral diversity and thus the potential for zoonotic events. To date, limited research has been done in Africa on this topic, in particular in the Congo Basin despite frequent contact between humans and wildlife in this region. We sampled and, using consensus coronavirus PCR-primers, tested 3,561 wild animals for coronavirus RNA. The focus was on bats (38%), rodents (38%), and primates (23%) that posed an elevated risk for contact with people, and we found coronavirus RNA in 121 animals, of which all but two were bats. Depending on the taxonomic family, bats were significantly more likely to be coronavirus RNA-positive when sampled either in the wet (Pteropodidae and Rhinolophidae) or dry season (Hipposideridae, Miniopteridae, Molossidae, and Vespertilionidae). The detected RNA sequences correspond to 15 alpha- and 6 betacoronaviruses, with some of them being very similar (>95% nucleotide identities) to known coronaviruses and others being more unique and potentially representing novel viruses. In seven of the bats, we detected RNA most closely related to sequences of the human common cold coronaviruses 229E or NL63 (>80% nucleotide identities). The findings highlight the potential for coronavirus spillover, especially in regions with a high diversity of bats and close human contact, and reinforces the need for ongoing surveillance.

Natural killer cell responses to emerging viruses of zoonotic origin.

Emerging viral diseases pose a major threat to public health worldwide. Nearly all emerging viruses, including Ebola, Dengue, Nipah, West Nile, Zika, and coronaviruses (including SARS-Cov2, the causative agent of the current COVID-19 pandemic), have zoonotic origins, indicating that animal-to-human transmission constitutes a primary mode of acquisition of novel infectious diseases. Why these viruses can cause profound pathologies in humans, while natural reservoir hosts often show little evidence of disease is not completely understood. Differences in the host immune response, especially within the innate compartment, have been suggested to be involved in this divergence. Natural killer (NK) cells are innate lymphocytes that play a critical role in the early antiviral response, secreting effector cytokines and clearing infected cells. In this review, we will discuss the mechanisms through which NK cells interact with viruses, their contribution towards maintaining equilibrium between the virus and its natural host, and their role in disease progression in humans and other non-natural hosts.

Infectivity, virulence, pathogenicity, host-pathogen interactions of SARS and SARS-CoV-2 in experimental animals: a systematic review.

The outbreak of the SARS-CoV-2 in mainland China with subsequent human to human transmission worldwide had taken up the shape of a devastating pandemic. The ability of the virus to infect multiple species other than humans has currently been reported in experimental conditions. Non-human primates, felines, ferrets, rodents and host of other animals could previously be infected in experimental conditions with SARS-CoV and recently with SARS-CoV-2, both virus using Angiotensin-converting-enzyme 2 receptor for cellular entry. The variations in sequence homology of ACE2 receptor across species is identified as one of the factors determining virulence and pathogenicity in animals. The infection in experimental animals with SARS-CoV or SARS-CoV-2 on most occasions are asymptomatic, however, the virus could multiply within the respiratory tract and extra-pulmonary organs in most of the species. Here, we discuss about the pathogenicity, transmission, variations in angiotensin-converting-enzyme 2 receptor-binding across species and host pathogen interactions of SARS and SARS-CoV-2 in laboratory animals used in research.

MERS-CoV and SARS-CoV infections in animals: a systematic review and meta-analysis of prevalence studies.

INTRODUCTION: Coronaviruses are zoonotic viruses that include human epidemic pathogens such as the Middle East Respiratory Syndrome virus (MERS-CoV), and the Severe Acute Respiratory Syndrome virus (SARS-CoV), among others (e.g., COVID-19, the recently emerging coronavirus disease). The role of animals as potential reservoirs for such pathogens remains an unanswered question. No systematic reviews have been published on this topic to date. METHODS: We performed a systematic literature review with meta-analysis, using three databases to assess MERS-CoV and SARS-CoV infection in animals and its diagnosis by serological and molecular tests. We performed a random-effects model meta-analysis to calculate the pooled prevalence and 95% confidence interval (95%CI). RESULTS: 6,493articles were retrieved (1960-2019). After screening by abstract/title, 50 articles were selected for full-text assessment. Of them, 42 were finally included for qualitative and quantitative analyses. From a total of 34 studies (n=20,896 animals), the pool prevalence by RT-PCR for MERS-CoV was 7.2% (95%CI 5.6-8.7%), with 97.3% occurring in camels, in which pool prevalence was 10.3% (95%CI 8.3-12.3). Qatar was the country with the highest MERS-CoV RT-PCR pool prevalence: 32.6% (95%CI 4.8-60.4%). From 5 studies and 2,618 animals, for SARS-CoV, the RT-PCR pool prevalence was 2.3% (95%CI 1.3-3.3). Of those, 38.35% were reported on bats, in which the pool prevalence was 14.1% (95%CI0.0-44.6%). DISCUSSION: A considerable proportion of infected animals tested positive, particularly by nucleic acid amplification tests (NAAT). This essential condition highlights the relevance of individual animals as reservoirs of MERS-CoV and SARS-CoV. In this meta-analysis, camels and bats were found to be positive by RT-PCR in over 10% of the cases for both; thus, suggesting their relevance in the maintenance of wild zoonotic transmission.

Coronavirus surveillance of wildlife in the Lao People's Democratic Republic detects viral RNA in rodents.

Coronaviruses can become zoonotic, as in the case of COVID-19, and hunting, sale, and consumption of wild animals in Southeast Asia increases the risk for such incidents. We sampled and tested rodents (851) and other mammals and found betacoronavirus RNA in 12 rodents. The sequences belong to two separate genetic clusters and are closely related to those of known rodent coronaviruses detected in the region and distantly related to those of human coronaviruses OC43 and HKU1. Considering the close human-wildlife contact with many species in and beyond the region, a better understanding of virus diversity is urgently needed for the mitigation of future risks.

Zoonotic origins of human coronaviruses.

Mutation and adaptation have driven the co-evolution of coronaviruses (CoVs) and their hosts, including human beings, for thousands of years. Before 2003, two human CoVs (HCoVs) were known to cause mild illness, such as common cold. The outbreaks of severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome (MERS) have flipped the coin to reveal how devastating and life-threatening an HCoV infection could be. The emergence of SARS-CoV-2 in central China at the end of 2019 has thrusted CoVs into the spotlight again and surprised us with its high transmissibility but reduced pathogenicity compared to its sister SARS-CoV. HCoV infection is a zoonosis and understanding the zoonotic origins of HCoVs would serve us well. Most HCoVs originated from bats where they are non-pathogenic. The intermediate reservoir hosts of some HCoVs are also known. Identifying the animal hosts has direct implications in the prevention of human diseases. Investigating CoV-host interactions in animals might also derive important insight on CoV pathogenesis in humans. In this review, we present an overview of the existing knowledge about the seven HCoVs, with a focus on the history of their discovery as well as their zoonotic origins and interspecies transmission. Importantly, we compare and contrast the different HCoVs from a perspective of virus evolution and genome recombination. The current CoV disease 2019 (COVID-19) epidemic is discussed in this context. In addition, the requirements for successful host switches and the implications of virus evolution on disease severity are also highlighted.