Coinfection of Chickens with H9N2 and H7N9 Avian Influenza Viruses Leads to Emergence of Reassortant H9N9 Virus with Increased Fitness for Poultry and a Zoonotic Potential.

An H7N9 low-pathogenicity avian influenza virus (LPAIV) emerged in 2013 through genetic reassortment between H9N2 and other LPAIVs circulating in birds in China. This virus causes inapparent clinical disease in chickens, but zoonotic transmission results in severe and fatal disease in humans. To examine a natural reassortment scenario between H7N9 and G1 lineage H9N2 viruses predominant in the Indian subcontinent, we performed an experimental coinfection of chickens with A/Anhui/1/2013/H7N9 (Anhui/13) virus and A/Chicken/Pakistan/UDL-01/2008/H9N2 (UDL/08) virus. Plaque purification and genotyping of the reassortant viruses shed via the oropharynx of contact chickens showed H9N2 and H9N9 as predominant subtypes. The reassortant viruses shed by contact chickens also showed selective enrichment of polymerase genes from H9N2 virus. The viable "6+2" reassortant H9N9 (having nucleoprotein [NP] and neuraminidase [NA] from H7N9 and the remaining genes from H9N2) was successfully shed from the oropharynx of contact chickens, plus it showed an increased replication rate in human A549 cells and a significantly higher receptor binding to α2,6 and α2,3 sialoglycans compared to H9N2. The reassortant H9N9 virus also had a lower fusion pH, replicated in directly infected ferrets at similar levels compared to H7N9 and transmitted via direct contact. Ferrets exposed to H9N9 via aerosol contact were also found to be seropositive, compared to H7N9 aerosol contact ferrets. To the best of our knowledge, this is the first study demonstrating that cocirculation of H7N9 and G1 lineage H9N2 viruses could represent a threat for the generation of novel reassortant H9N9 viruses with greater virulence in poultry and a zoonotic potential. IMPORTANCE We evaluated the consequences of reassortment between the H7N9 and the contemporary H9N2 viruses of the G1 lineage that are enzootic in poultry across the Indian subcontinent and the Middle East. Coinfection of chickens with these viruses resulted in the emergence of novel reassortant H9N9 viruses with genes derived from both H9N2 and H7N9 viruses. The "6+2" reassortant H9N9 (having NP and NA from H7N9) virus was shed from contact chickens in a significantly higher proportion compared to most of the reassortant viruses, showed significantly increased replication fitness in human A549 cells, receptor binding toward human (α2,6) and avian (α2,3) sialic acid receptor analogues, and the potential to transmit via contact among ferrets. This study demonstrated the ability of viruses that already exist in nature to exchange genetic material, highlighting the potential emergence of viruses from these subtypes with zoonotic potential.

Genetic Characterization and Pathogenesis of Three Novel Reassortant H5N2 Viruses in South Korea, 2018.

The outbreaks of H5N2 avian influenza viruses have occasionally caused the death of thousands of birds in poultry farms. Surveillance during the 2018 winter season in South Korea revealed three H5N2 isolates in feces samples collected from wild birds (KNU18-28: A/Wild duck/South Korea/KNU18-28/2018, KNU18-86: A/Bean Goose/South Korea/KNU18-86/2018, and KNU18-93: A/Wild duck/South Korea/KNU18-93/2018). Phylogenetic tree analysis revealed that these viruses arose from reassortment events among various virus subtypes circulating in South Korea and other countries in the East Asia-Australasian Flyway. The NS gene of the KNU18-28 and KNU18-86 isolates was closely related to that of China's H10N3 strain, whereas the KNU18-93 strain originated from the H12N2 strain in Japan, showing two different reassortment events and different from a low pathogenic H5N3 (KNU18-91) virus which was isolated at the same day and same place with KNU18-86 and KNU18-93. These H5N2 isolates were characterized as low pathogenic avian influenza viruses. However, many amino acid changes in eight gene segments were identified to enhance polymerase activity and increase adaptation and virulence in mice and mammals. Experiments reveal that viral replication in MDCK cells was quite high after 12 hpi, showing the ability to replicate in mouse lungs. The hematoxylin and eosin-stained (H&E) lung sections indicated different degrees of pathogenicity of the three H5N2 isolates in mice compared with that of the control H1N1 strain. The continuing circulation of these H5N2 viruses may represent a potential threat to mammals and humans. Our findings highlight the need for intensive surveillance of avian influenza virus circulation in South Korea to prevent the risks posed by these reassortment viruses to animal and public health.

Molecular Analysis of the Avian H7 Influenza Viruses Circulating in South Korea during 2018-2019: Evolutionary Significance and Associated Zoonotic Threats.

Avian influenza virus (AIV) subtypes H5 and H7, possessing the ability to mutate spontaneously from low pathogenic (LP) to highly pathogenic (HP) variants, are major concerns for enormous socio-economic losses in the poultry industry, as well as for fatal human infections. Through antigenic drift and shift, genetic reassortments of the genotypes pose serious threats of increased virulence and pathogenicity leading to potential pandemics. In this study, we isolated the H7-subtype AIVs circulating in the Republic of Korea during 2018-2019, and perform detailed molecular analysis to study their circulation, evolution, and possible emergence as a zoonotic threat. Phylogenetic and nucleotide sequence analyses of these isolates revealed their distribution into two distinct clusters, with the HA gene sharing the highest nucleotide identity with either the A/common teal/Shanghai/CM1216/2017, isolated from wild birds in Shanghai, China, or the A/duck/Shimane/2014, isolated from Japan. Mutations were found in HA (S138A (H3 numbering)), M1 (N30D and T215A), NS1 (P42S), PB2 (L89V), and PA (H266R and F277S) proteins-the mutations had previously been reported to be related to mammalian adaptation and changes in the virulence of AIVs. Taken together, the results firmly put forth the demand for routine surveillance of AIVs in wild birds to prevent possible pandemics arising from reassortant AIVs.

The C-terminus of non-structural protein 1 (NS1) in H5N8 clade 2.3.4.4 avian influenza virus affects virus fitness in human cells and virulence in mice.

Avian influenza viruses (AIV) H5N8 clade 2.3.4.4 pose a public health threat but the viral factors relevant for its potential adaptation to mammals are largely unknown. The non-structural protein 1 (NS1) of influenza viruses is an essential interferon antagonist. It commonly consists of 230 amino acids, but variations in the disordered C-terminus resulted in truncation or extension of NS1 with a possible impact on virus fitness in mammals. Here, we analysed NS1 sequences from 1902 to 2020 representing human influenza viruses (hIAV) as well as AIV in birds, humans and other mammals and with an emphasis on the panzootic AIV subtype H5N8 clade 2.3.4.4A (H5N8-A) from 2013 to 2015 and clade 2.3.4.4B (H5N8-B) since 2016. We found a high degree of prevalence for short NS1 sequences among hIAV, zoonotic AIV and H5N8-B, while AIV and H5N8-A had longer NS1 sequences. We assessed the fitness of recombinant H5N8-A and H5N8-B viruses carrying NS1 proteins with different lengths in human cells and in mice. H5N8-B with a short NS1, similar to hIAV or AIV from a human or other mammal-origins, was more efficient at blocking apoptosis and interferon-induction without a significant impact on virus replication in human cells. In mice, shortening of the NS1 of H5N8-A increased virus virulence, while the extension of NS1 of H5N8-B reduced virus virulence and replication. Taken together, we have described the biological impact of variation in the NS1 C-terminus in hIAV and AIV and shown that this affects virus fitness in vitro and in vivo.

Key Amino Acids of M1-41 and M2-27 Determine Growth and Pathogenicity of Chimeric H17 Bat Influenza Virus in Cells and in Mice.

Based on our previous studies, we show that the M gene is critical for the replication and pathogenicity of the chimeric H17 bat influenza virus (Bat09:mH1mN1) by replacing the bat M gene with those from human and swine influenza A viruses. However, the key amino acids of the M1 and/or M2 proteins that are responsible for virus replication and pathogenicity remain unknown. In this study, replacement of the PR8 M gene with the Eurasian avian-like M gene from the A/California/04/2009 pandemic H1N1 virus significantly decreased viral replication in both mammalian and avian cells in the background of the chimeric H17 bat influenza virus. Further studies revealed that M1 was more crucial for viral growth and pathogenicity than M2 and that the amino acid residues M1-41V and M2-27A were responsible for these characteristics in cells and in mice. These key residues of the M1 and M2 proteins identified in this study might be important for influenza virus surveillance and could be used to produce live attenuated vaccines in the future. IMPORTANCE The M1 and M2 proteins influence the morphology, replication, virulence, and transmissibility of influenza viruses. Although a few key residues in the M1 and M2 proteins have been identified, whether other residues of the M1 and M2 proteins are involved in viral replication and pathogenicity remains to be discovered. In the background of the chimeric H17 bat influenza virus, the Eurasian avian-like M gene from the A/California/04/2009 virus significantly decreased viral growth in mammalian and avian cells. Further study showed that M1 was implicated more than M2 in viral growth and pathogenicity in vitro and in vivo and that the key amino acid residues M1-41V and M2-27A were responsible for these characteristics in cells and in mice. These key residues of the M1 and M2 proteins could be used for influenza virus surveillance and live attenuated vaccine applications in the future. These findings provide important contributions to knowledge of the genetic basis of the virulence of influenza viruses.

Novel reassortant 2.3.4.4B H5N6 highly pathogenic avian influenza viruses circulating among wild, domestic birds in Xinjiang, Northwest China.

BACKGROUND: The H5 avian influenza viruses (AIVs) of clade 2.3.4.4 circulate in wild and domestic birds worldwide. In 2017, nine strains of H5N6 AIVs were isolated from aquatic poultry in Xinjiang, Northwest China. OBJECTIVES: This study aimed to analyze the origin, reassortment, and mutations of the AIV isolates. METHODS: AIVs were isolated from oropharyngeal and cloacal swabs of poultry. Identification was accomplished by inoculating isolates into embryonated chicken eggs and performing hemagglutination tests and reverse transcription polymerase chain reaction (RT-PCR). The viral genomes were amplified with RT-PCR and then sequenced. The sequence alignment, phylogenetic, and molecular characteristic analyses were performed by using bioinformatic software. RESULTS: Nine isolates originated from the same ancestor. The viral HA gene belonged to clade 2.3.4.4B, while the NA gene had a close phylogenetic relationship with the 2.3.4.4C H5N6 highly pathogenic avian influenza viruses (HPAIVs) isolated from shoveler ducks in Ningxia in 2015. The NP gene was grouped into an independent subcluster within the 2.3.4.4B H5N8 AIVs, and the remaining six genes all had close phylogenetic relationships with the 2.3.4.4B H5N8 HPAIVs isolated from the wild birds in China, Egypt, Uganda, Cameroon, and India in 2016-2017, Multiple basic amino acid residues associated with HPAIVs were located adjacent to the cleavage site of the HA protein. The nine isolates comprised reassortant 2.3.4.4B HPAIVs originating from 2.3.4.4B H5N8 and 2.3.4.4C H5N6 viruses in wild birds. CONCLUSIONS: These results suggest that the Northern Tianshan Mountain wetlands in Xinjiang may have a key role in AIVs disseminating from Central China to the Eurasian continent and East African.

Pathogenicity of novel reassortant Eurasian avian-like H1N1 influenza virus in pigs.

Reassortant Eurasian avian-like (EA) H1N1 virus, possessing 2009 pandemic (pdm/09) and triple-reassortant (TR)-derived internal genes, namely G4 genotype, has replaced the G1 genotype EA H1N1 virus (all the genes were of EA origin) and become predominant in swine populations in China. Understanding the pathogenicity of G4 viruses in pigs is of great importance for disease control. Here, we conducted comprehensive analyses of replication and pathogenicity of G4 and G1 EA H1N1 viruses in pigs. G4 virus exhibited enhanced replication, increased duration of virus shedding, and caused more severe respiratory lesions in pigs compared with G1 virus. G4 virus, with viral ribonucleoprotein (vRNP) complex genes of pdm/09 origin, exhibited higher levels of nuclear accumulation and higher polymerase activity, which is essential for improved replication of G4 virus. These findings indicate that G4 virus poses a great threat to both swine industry and public health, and control measures should be urgently implemented.

Intertypic reassortment of mammalian orthoreovirus identified in wastewater in Japan.

Mammalian orthoreovirus (MRV), a non-enveloped virus with a ten-segmented double-stranded RNA genome, infects virtually all mammals, including humans. Human infection with MRV seems to be common in early childhood, but is rarely symptomatic. Despite the ubiquitous presence of MRV in mammals as well as in environmental waters, the molecular characterisation of the MRV genome remains to be fully elucidated. In this study, two novel strains, MRV-2 THK0325 and MRV-1 THK0617, were unintentionally isolated from wastewater in Japan via an environmental surveillance of enteric viruses. Homology and phylogenetic analysis demonstrated that all the segments of THK0325 were closely related to the MRV-2 Osaka strains, which were recently proposed to have existed for at least two decades in Japan. Most of the segments in THK0617 also showed a close relationship with the MRV-2 Osaka strains, but the M2, S1, and S3 segments belong to another MRV cluster. According to the S1 sequence, the determinant of serotype THK0617 was classified as MRV-1, and both the M2 and S3 segments were closely related to MRV-1 and -3 from the tree shrew in China. These results suggest that the MRV-2 Osaka-like strain spread widely throughout Japan, accompanied by intertypic reassortment occurring in East Asia.

Mink is a highly susceptible host species to circulating human and avian influenza viruses.

Pandemic influenza, typically caused by the reassortment of human and avian influenza viruses, can result in severe or fatal infections in humans. Timely identification of potential pandemic viruses must be a priority in influenza virus surveillance. However, the range of host species responsible for the generation of novel pandemic influenza viruses remains unclear. In this study, we conducted serological surveys for avian and human influenza virus infections in farmed mink and determined the susceptibility of mink to prevailing avian and human virus subtypes. The results showed that farmed mink were commonly infected with human (H3N2 and H1N1/pdm) and avian (H7N9, H5N6, and H9N2) influenza A viruses. Correlational analysis indicated that transmission of human influenza viruses occurred from humans to mink, and that feed source was a probable route of avian influenza virus transmission to farmed mink. Animal experiments showed that mink were susceptible and permissive to circulating avian and human influenza viruses, and that human influenza viruses (H3N2 and H1N1/pdm), but not avian viruses, were capable of aerosol transmission among mink. These results indicate that farmed mink could be highly permissive "mixing vessels" for the reassortment of circulating human and avian influenza viruses. Therefore, to reduce the risk of emergence of novel pandemic viruses, feeding mink with raw poultry by-products should not be permitted, and epidemiological surveillance of influenza viruses in mink farms should be urgently implemented.

Isolation and characterization of two novel reassortant H5N6 avian influenza viruses from waterfowl in eastern China.

Waterfowl are considered to be the natural hosts of avian influenza virus. In 2017, two reassortant highly pathogenic H5N6 avian influenza viruses of clade 2.3.4.4, subclade II, were identified in wild birds in eastern China. Genome sequencing and phylogenetic and antigenicity analysis showed that the viruses originated from multiple reassortments. To evaluate their pathogenicity in mammals, 15 BALB/c mice were infected with these viruses, and survival and weight loss were monitored for 14 days. Infection was associated with moderate pathogenicity in the mice, and the viruses could replicate in the lungs without prior adaptation. Thus, the existence of these viruses poses a continuous threat to both birds and humans.

The inactivated vaccine of reassortant H3N2 canine influenza virus based on internal gene cassette from PR8 is safe and effective.

Canine influenza (CI) is a contagious respiratory disease in dogs, which poses a threat to canine health. A safe, high-yield vaccine seed virus is critical for CI vaccine development. We developed a PR8-based reassortant H3N2 canine influenza virus (RT CIV) using the reverse genetic method and evaluated its yield in canine kidney epithelial (MDCK) cells, Vero cells, and specific pathogen-free (SPF) chicken embryos. Mice and dogs were infected with RT CIV, and the pathogenicity was evaluated. The viral titers of RT CIV increased in MDCK cells, Vero cells, and SPF chicken embryos; the HA yield in SPF chicken embryos increased 4-fold. However, RT CIV was not lethal to mice, and it showed similar virulence as wild-type CIV. RT CIV also showed minimal pathogenicity in dogs, which manifested as mild fever and rhinorrhea for the first two days post-infection. Thus, RT CIV carrying the internal gene cassette from PR8 showed almost no pathogenicity in dogs. And the reassortant virus inactivated vaccine could provide complete protection against H3N2 CIV. To our knowledge, this is the first report on the pathogenicity of PR8-based reassortant H3N2 CIV in dogs. These studies are relevant for developing a high-yield and safe CI vaccine.

Genetic characteristics and pathogenicity of novel reassortant H6 viruses isolated from wild birds in China.

During our routine surveillance, we isolated seven H6 avian influenza virus (AIV) strains, including three H6N1 strains, three H6N2 strains, and one H6N8 strain, from 3667 fresh fecal samples that were collected from wild bird habitats in China from March 2017 and May 2019. Phylogenetic analysis revealed that these viruses formed five different genotypes and have undergone complicate reassortment during their evolution by acquiring genes from AIVs of both Eurasian and North American lineages that have been previously detected in migrating waterfowl and poultry. Viral pathogenesis in mice showed that these H6 viruses replicated efficiently in both the nasal turbinates and lungs of mice without pre-adaptation, but none of them were lethal for mice. We studied the genetic characteristic and biological property of novel reassortant H6 viruses isolated from wild birds in China. It also highlights the need for continued surveillance of H6 AIVs circulating in nature.

The genetics of highly pathogenic avian influenza viruses of subtype H5 in Germany, 2006-2020.

The H5 A/Goose/Guangdong/1/1996 (gs/GD) lineage emerged in China in 1996. Rooted in the respective gs/GD lineage, the hemagglutinin (HA) gene of highly pathogenic avian influenza viruses (HPAIV) has genetically diversified into a plethora of clades and subclades and evolved into an assortment of sub- and genotypes. Some caused substantial losses in the poultry industry and had a major impact on wild bird populations alongside public health implications due to a zoonotic potential of certain clades. After the primary introduction of the HPAI H5N1 gs/GD lineage into Europe in autumn 2005 and winter 2005/2006, Germany has seen recurring incursions of four varying H5Nx subtypes (H5N1, H5N8, H5N5, H5N6) carrying multiple distinct reassortants, all descendants of the gs/GD virus. The first HPAIV H5 epidemic in Germany during 2006/2007 was caused by a clade 2.2 subtype H5N1 virus. Phylogenetic analysis confirmed three distinct clusters belonging to clades 2.2.1, 2.2.2 and 2.2, concurring with geographic and temporal structures. From 2014 onwards, HPAIV clade 2.3.4.4 has dominated the epidemiological situation in Germany. The initial clade 2.3.4.4a HPAIV H5N8, reaching Germany in November 2014, caused a limited epidemic affecting five poultry holdings, one zoo in Northern Germany and few wild birds. After November 2016, HPAIV of clade 2.3.4.4b have dominated the situation to date. The most extensive HPAIV H5 epidemic on record reached Germany in winter 2016/2017, encompassing multiple incursion events with two subtypes (H5N8, H5N5) and entailing five reassortants. A novel H5N6 clade 2.3.4.4b strain affected Germany from December 2017 onwards, instigating low-level infection in smallholdings and wild birds. Recently, in spring 2020, a novel incursion of a genetically distinct HPAI clade 2.3.4.4b H5N8 virus caused another epidemic in Europe, which affected a small number of poultry holdings, one zoo and two wild birds throughout Germany.

Phylogenetic tracing and biological characterization of a novel clade 2.3.2.1 reassortant of H5N6 subtype avian influenza virus in China.

In recent years in China, clade 2.3.4.4 H5N6 plus clade 2.3.2.1 H5N1 subtype highly pathogenic avian influenza (HPAI) viruses have gradually become endemic in poultry, and their co-circulation could inevitably facilitate the gene reassortment between each other. During our routine surveillance in live poultry markets (LPMs) in eastern China in 2017-2018, a novel reassortant H5N6 strain with the HA gene derived from clade 2.3.2.1 was isolated from the cloacal swabs of apparently healthy ducks. Phylogenetic tracing analysis indicated that another two clade 2.3.2.1 H5N1 strains with divergent lineages of PB1 gene and one clade 2.3.4.4 H5N6 isolate of the dominant genotype sharing spatio-temporal proximity were intimately involved in the generation of this rarely reported clade 2.3.2.1 H5N6 reassortant. Distinct with the other three HPAI H5 viruses showing moderate virulence in mice, the H5N1 strain of the homologous internal gene constellation against the clade 2.3.2.1 H5N6 reassortant was highly pathogenic, which might probably attribute to the H3 subtype-derived PB1 gene. However, as compared to the clade 2.3.4.4 H5N6 ancestor, the clade 2.3.2.1 H5N6 reassortant displayed a broader tissue distribution and higher viral titres in mice, which could likely facilitate the viral maintenance and spread in nature. Therefore, our results highlight that continuous epidemiological survey of H5 subtype HPAI viruses in LPMs needs to be strengthened to prevent the potential poultry or even public health threat of the novel reassortants from endemic viruses.

Refined experimental design may increase the value of murine models for estimation of bluetongue virus virulence.

Bluetongue is a serious non-contagious vector-borne viral disease in ruminants, causing poor animal welfare and economic consequences globally. Concern has been raised about the development of novel bluetongue virus (BTV) strains and their possibly altered virulence through the process of viral reassortment. Virulence is traditionally estimated in lethal dose 50 (LD50) studies in murine models, but agreement with both in vitro and virulence in ruminants is questionable, and a refined experimental design is needed. Specific reassortants between wild-type and vaccine strains of BTV-1, -6 and -8 have previously been developed by reverse genetics. The aim of the present study was to rank the in vivo virulence of these parental and reassortant BTV strains by calculating LD50 in a murine model by using an experimental design that is new to virology: a between-patient optimised three-level response surface pathway design. The inoculation procedure was intracranial. Fifteen suckling mice were used to establish LD50 for each strain. Three parental and five reassortant virus strains were included. The LD50s varied from of 0.1 (95% confidence interval (CI) 0-0.20) to 3.3 (95% CI 2.96-3.72) tissue culture infectious dose 50/ml. The results support the hypothesis that reassortment in BTV may lead to increased virulence in mice with potential negative consequences for the natural ruminant host. The ranking showed low agreement with in vitro properties and virulence in ruminants according to existing literature. Refined design such as response surface pathway design was found suitable for use in virology, and it introduces significant ethical and scientific improvements.

Characterization of contemporary 2010.1 H3N2 swine influenza A viruses circulating in United States pigs.

In 2012, swine influenza surveillance detected a novel reassorted influenza A virus (IAV) strain containing human-seasonal hemagglutinin (HA) and neuraminidase (NA). Subsequently, these viruses reassorted, maintaining only the human-origin H3, which resulted in a new lineage of viruses that became the most frequently detected H3 clade in US swine (2010.1 HA clade). Here, we assessed the antigenic phenotype, virulence, and transmission characteristics of this virus lineage following its introduction to swine. Relative to 2010.1 viruses from 2012 and 2014, recent 2010.1 contemporary strains from 2015 to 2017 resulted in equivalent macroscopic lung lesions and transmission in pigs. A single mutation at amino acid residue 145 within the previously defined HA antigenic motif was associated with a change of antigenic phenotype, potentially impairing vaccine efficacy. Contemporary 2010.1 viruses circulating in swine since 2012 were significantly different from both pre-2012H3N2 in swine and human-seasonal H3N2 viruses and demonstrated continued evolution within the lineage.

The genetic structure of SARS-CoV-2 does not rule out a laboratory origin: SARS-COV-2 chimeric structure and furin cleavage site might be the result of genetic manipulation.

Severe acute respiratory syndrome-coronavirus (SARS-CoV)-2's origin is still controversial. Genomic analyses show SARS-CoV-2 likely to be chimeric, most of its sequence closest to bat CoV RaTG13, whereas its receptor binding domain (RBD) is almost identical to that of a pangolin CoV. Chimeric viruses can arise via natural recombination or human intervention. The furin cleavage site in the spike protein of SARS-CoV-2 confers to the virus the ability to cross species and tissue barriers, but was previously unseen in other SARS-like CoVs. Might genetic manipulations have been performed in order to evaluate pangolins as possible intermediate hosts for bat-derived CoVs that were originally unable to bind to human receptors? Both cleavage site and specific RBD could result from site-directed mutagenesis, a procedure that does not leave a trace. Considering the devastating impact of SARS-CoV-2 and importance of preventing future pandemics, researchers have a responsibility to carry out a thorough analysis of all possible SARS-CoV-2 origins.

Molecular characterization identifies intra-host recombination and zoonotic potential of canine rotavirus among dogs from Thailand.

From September 2016 to January 2019, we collected 710 rectal swabs from both healthy and sick dogs from small animal hospitals in 5 provinces of Thailand. The samples were tested for canine rotavirus group A (CRV) by using one-step RT-PCR specific to the VP6 gene. Our results showed that 0.70% (5/710) were positive for CRV. The five CRVs were then characterized by whole-genome sequencing. Our results showed that the genotype of Thai CRVs is G3P[3], which is the predominant genotype reported in dogs. The Thai CRVs posed a novel genetic constellation 'G3-P[3]-I3-R3-C3-M3-A9-N2-T3-E3-H6', which has never been reported in CRVs from dogs but has been reported in rotaviruses from humans. Based on phylogenetic analysis, the Thai CRVs are the result of multiple reassortments in which gene segments might have originated from human and bat rotaviruses and suggests the zoonotic potential of the virus.

Where Did SARS-CoV-2 Come From?

Identifying the origin of SARS-CoV-2, the etiological agent of the current COVID-19 pandemic, may help us to avoid future epidemics of coronavirus and other zoonoses. Several theories about the zoonotic origin of SARS-CoV-2 have recently been proposed. Although Betacoronavirus found in Rhinolophus bats from China have been broadly implicated, their genetic dissimilarity to SARS-CoV-2 is so high that they are highly unlikely to be its direct ancestors. Thus, an intermediary host is suspected to link bat to human coronaviruses. Based on genomic CpG dinucleotide patterns in different coronaviruses from different hosts, it was suggested that SARS-CoV-2 might have evolved in a canid gastrointestinal tract prior to transmission to humans. However, similar CpG patterns are now reported in coronaviruses from other hosts, including bats themselves and pangolins. Therefore, reduced genomic CpG alone is not a highly predictive biomarker, suggesting a need for additional biomarkers to reveal intermediate hosts or tissues. The hunt for the zoonotic origin of SARS-CoV-2 continues.