Genetic diversity and recombination of bovine enterovirus strains in China.

Bovine enterovirus (BEV) consisting of enterovirus species E (EV-E) and F (EV-F) is the causative agent associated with respiratory and gastrointestinal diseases in cattle. Here, we reported the characterization, genetic diversity, and recombination of novel BEV strains isolated from the major cattle-raising regions in China during 2012-2018. Twenty-seven BEV strains were successfully isolated and characterized. Molecular characterization demonstrated that the majority of these novel BEV strains (24/27) were EV-E, while only few strains (3/27) were EV-F. Sequence analysis revealed the diversity of the circulating BEV strains such as species and subtypes where different species or subtype coinfections were detected in the same regions and even in the same cattle herds. For the EV-E, two novel subtypes, designated as EV-E6 and EV-E7, were revealed in addition to the currently reported EV-E1-EV-E5. Comparative genomic analysis revealed the intraspecies and interspecies genetic exchanges among BEV isolates. The representative strain HeN-B62 was probably from AN12 (EV-F7) and PS-87-Belfast (EV-F3) strains. The interspecies recombination between EV-E and EV-F was also discovered, where the EV-F7-AN12 might be from EV-E5 and EV-F1, and EV-E5-MexKSU/5 may be recombined from EV-F7 and EV-E1. The aforementioned results revealed the genetic diversity and recombination of novel BEV strains and unveiled the different BEV species or subtype infections in the same cattle herd, which will broaden the understanding of enterovirus genetic diversity, recombination, pathogenesis, and prevention of disease outbreaks. IMPORTANCE: Bovine enterovirus (BEV) infection is an emerging disease in China that is characterized by digestive, respiratory, and reproductive disorders. In this study, we first reported two novel EV-E subtypes detected in cattle herds in China, unveiled the coinfection of two enterovirus species (EV-E/EV-F) and different subtypes (EV-E2/EV-E7, EV-E1/EV-E7, and EV-E3/EV-E6) in the same cattle herds, and revealed the enterovirus genetic exchange in intraspecies and interspecies recombination. These results provide an important update of enterovirus prevalence and epidemiological aspects and contribute to a better understanding of enterovirus genetic diversity, evolution, and pathogenesis.

Emerging porcine Enterovirus G infections, epidemiological, complete genome sequencing, evolutionary and risk factor analysis in India.

The current study reports the in-depth analysis of the epidemiology, risk factors, and molecular characterization of a complete genome of Enterovirus G (EV-G) isolated from Indian pigs. We analysed several genes of EV-G isolates collected from various provinces in India, using phylogenetic analysis, recombination detection, SimPlot, and selection pressure analyses. Our analysis of 534 porcine faecal samples revealed that 11.61% (62/534) of the samples were positive for EV-G. While the G6 genotype was the most predominant, our findings showed that Indian EV-G strains also clustered with EV-G types G1, G6, G8, and G9. Furthermore, Indian EV-G strains exhibited the highest nucleotide similarity with Vietnamese (81.3%) and Chinese EV-G isolates (80.3%). Moreover, we identified a recombinant Indian EV-G strain with a putative origin from a Japanese isolate and South Korean EV-G isolate. In summary, our findings provide significant insights into the epidemiology, genetic diversity, and evolution of EV-G in India.

A novel VP1-based enzyme-linked immunosorbent assay revealed widespread Enterovirus G infections in Guangxi, China.

Enterovirus G (EV-G) has recently been shown to affect weight gain and cause neurological symptoms in piglets. However, the serological investigation of EV-G is limited. In this study, we developed a novel serological detection method based on the structural protein, VP1 of EV-G. The intra-assay and inter-assay coefficient variations were 3.2-8.9% and 2.6-8.0%, respectively. There was no cross-reaction of the VP1-based enzyme-linked immunosorbent assay (ELISA) with antisera against the other known porcine viruses. In addition, a comparison was made with other methods including the developed indirect ELISAs based on VP2 and VP3 proteins and western blot (WB) analysis, which demonstrated the reliability of the novel method. Using the VP1-based ELISA, we carried out the first seroepidemiological survey of EV-G in China by testing 1041 serum samples collected from different pig farms in Guangxi from 2019 to 2021. Our results showed that 68.78% of the serum samples and 100% of the pig farms were positive for EV-G, with a relatively high incidence of seropositivity in pigs of different ages. This was specifically evident in fattening pigs and sows, which may suggest that the piglets have experienced an infection with EV-G during their growth process. Our data provide the first serological evidence of EV-G infections in pigs from China and reveal the widespread presence of EV-G infections in Guangxi, China.

Estimating prevalence of Enterovirus D111 in human and non-human primate populations using cross-sectional serology.

Enteroviruses primarily affect young children with a varying severity of disease. Recent outbreaks of severe respiratory and neurological disease due to EV-D68 and EV-A71, as well as atypical hand-foot-and-mouth-disease due to CVA6, have brought to light the potency of enteroviruses to emerge as severe human pathogens. Enterovirus D111 (EV-D111) is an enteric pathogen initially detected in Central Africa in human and wildlife samples and was recently detected in environmental samples. The natural history and epidemiology of EV-D111 are poorly studied. Here, the presence of serum neutralizing antibodies to EV-D111 was estimated in human and wildlife samples from five countries. We report high prevalence of neutralizing antibodies measured against EV-D111 in human populations (range, 55-83 %), a proxy for previous infection, which indicates active virus circulation in absence of detection in clinical cases and a high number of undiagnosed infections. Notably, seroprevalence in samples from the UK varied by age and was higher in children and older adults (1-5 and >60 years old), but lower in ages 11-60. EV-D111 seroprevalence in apes and Old World monkeys was 50 % (33-66 %), which also suggests prior exposure and supports existing knowledge of enterovirus circulation in wild and captive apes and Old World monkeys. Generally, reported cases of infection likely underestimate the prevalence of infection particularly when the knowledge of community transmission is limited. Continued serologic surveillance and detection of EV-D111 in clinical and environmental samples will allow for a more robust assessment of EV-D111 epidemiology.

Molecular characterization and pathogenicity evaluation of enterovirus G isolated from diarrheic piglets.

IMPORTANCE: Enterovirus G is a species of positive-sense single-stranded RNA viruses associated with several mammalian diseases. The porcine enterovirus strains isolated here were chimeric viruses with the PLCP gene of porcine torovirus, which grouped together with global EV-G1 strains. The isolated EV-G strain could infect various cell types from different species, suggesting its potential cross-species infection risk. Animal experiment showed the pathogenic ability of the isolated EV-G to piglets. Additionally, the EV-Gs were widely distributed in the swine herds. Our findings suggest that EV-G may have evolved a novel mechanism for broad tropism, which has important implications for disease control and prevention.

Animal enteroviruses: a glimpse of a wide evolutionary landscape.

The genus Enterovirus (family Picornaviridae) contains numerous viruses, most of which have been identified in humans. Among them, the three serotypes of poliovirus, coxsackieviruses A and B, echoviruses, rhinoviruses and other enteroviruses (EVs) responsible in humans for a wide spectrum of clinical manifestations. There are also 60 identified EVs in different mammals. Some have been found in both humans and animals, demonstrating the possibility of zoonotic transmission of certain EVs. Compared to human EVs, genetic and epidemiological data about animal EVs are scarce. However, the detection of EVs in various species of mammals and their presence on all continents suggest that the number of EVs still to be discovered is very important. Some EVs found in animals have characteristics never seen in human EVs. Furthermore, the unique phylogenetic relationships observed between some animal EVs raise interesting questions about the rules that govern the evolution of these viruses. The aim of this review is to present the salient data on animal EVs and to highlight the questions they raise.

[Animal enteroviruses: a glimpse of a wide evolutionary landscape]. / Entérovirus des animaux : lumière sur l'immensité de l'espace évolutif des entérovirus.

The genus Enterovirus (family Picornaviridae) contains numerous viruses, most of which have been identified in humans. Among them, the three serotypes of poliovirus, coxsackieviruses A and B, echoviruses, rhinoviruses and other enteroviruses (EVs) responsible in humans for a wide spectrum of clinical manifestations. There are also 60 identified EVs in different mammals. Some have been found in both humans and animals, demonstrating the possibility of zoonotic transmission of certain EVs. Compared to human EVs, genetic and epidemiological data for animal EVs are scarce. However, the detection of EV in various species of mammals and their presence on all continents suggest that the number of EV still to be discovered is very important. Some EVs found in animals have characteristics never seen in human EVs. Furthermore, the unique phylogenetic relationships observed between animal EVs raise interesting questions about the rules that govern the evolution of these viruses. The aim of this review is to present the salient data on animal EVs and to highlight the questions they raise.

A Neonatal Murine Model for Caprine Enterovirus Infection and the Viral Tissue Tropism.

As the first caprine enterovirus identified from goat herds characterized by severe diarrhea with a high morbidity and mortality rate, the underlying pathogenesis and tissue tropism for CEV-JL14 remains largely unknown. Here, we reported the establishment of a neonatal murine model for caprine enterovirus and the unveiling of the tissue tropism and underlying pathogenesis for CEV-JL14 enterovirus. Susceptible murine strains, the infective dose, the infective routes, viral loads, and tissue tropism for CEV-JL14 infection were determined. The findings showed that ICR mice were susceptible to CEV-JL14 infection via all infection routes. Tissue viral load analysis showed that CEV-JL14 was detected in almost all tissues including the heart, liver, spleen, lung, kidney, intestine, brain, and muscle, with significantly higher viral loads in the heart, liver, lung, kidney, and intestine. These results revealed the pattern of viral load and tropism for CEV-JL14 and provided a model system for elucidating the pathogenesis of CEV-JL14 viruses.

Novel recombinant porcine enterovirus G viruses lacking structural proteins are maintained in pig farms in Japan.

Type 1 recombinant enterovirus G (EV-G), which carries the papain-like cysteine protease (PLCP) gene of torovirus between its 2C/3A regions, and type 2 recombinant EV-G, which carries the torovirus PLCP gene with its flanking regions having non-EV-G sequences in place of the viral structural genes, have been detected in pig farms in several countries. In a previous study, we collected 222 fecal samples from 77 pig farms from 2104 to 2016 and detected one type 2 recombinant EV-G genome by metagenomics sequencing. In this study, we reanalyzed the metagenomic data and detected 11 type 2 recombinant EV-G genomes. In addition, we discovered new type 2 recombinant EV-G genomes of the two strains from two pig farms samples in 2018 and 2019. Thus, we identified the genomes of 13 novel type 2 recombinant EV-Gs isolated from several pig farms in Japan. Type 2 recombinant EV-G has previously been detected only in neonatal piglets. The present findings suggest that type 2 recombinant EV-G replicates in weaning piglets and sows. The detection of type 1 recombinant EV-Gs and type 2 recombinant EV-Gs at 3-year and 2-year intervals, respectively, from the same pig farm suggests that the viruses were persistently infecting or circulating in these farms.

Isolation and Identification of Two Clinical Strains of the Novel Genotype Enterovirus E5 in China.

Most enterovirus (EV) infections are subclinical but, occasionally, can cause severe and potentially fatal diseases in humans and animals. Currently, EVs are divided into 12 types (A to L) based on phylogenetic analysis and on their natural hosts. Bovine enterovirus (BEV) is an essential member of the enterovirus belonging to the types E and F that attacks cattle as its natural host and causes clinical disorders in the digestive, respiratory, and reproductive tracts. In 2020, several dairy farms in China experienced cow mortality with acute clinical signs, including fever, and diarrhea. In these cases, GX20-1 and JS20-1 virus strains were isolated and sequenced. Cellular adaptation of these two strains showed efficient replications on Madin-Darby bovine kidney (MDBK) cells and produced a significant cytopathogenic effect (CPE). However, on baby hamster kidney (BHK-21) and Vero cells, viral replication was inefficient and did not produce CPE. As noted in comparative genomics analysis, these two strains showed distant evolutionary relationships with the well-known E1 to E4 and F1 to F4 subtypes of BEV and high sequence identities with the candidate type Enterovirus E5, a novel genotype recently identified based on the genomic data of three strains, including the GX20-1 and JS20-1 strains. This study provides the first evidence of a novel genotype bovine enterovirus infection in Chinese cattle herds, a potential threat to the cattle industry in China. IMPORTANCE Bovine enterovirus (BEV) is a cattle-infecting pathogen. This study is the first report of natural infection of a novel genotype of enterovirus in herds of cattle in China. The homology of the novel enterovirus is far different from the structural protein of other enteroviruses and has different cellular adaptations. This study provides a reference for the biological characteristics and prevalence of the novel enterovirus in Chinese cattle populations.

Molecular Analysis of Caprine Enterovirus Circulating in China during 2016-2021: Evolutionary Significance.

Here, we report the characterization of 13 novel caprine/ovine enterovirus strains isolated from different regions in China during 2016-2021. Immunoperoxidase monolayer assay showed that these viral strains shared strong cross-reaction with the previously reported caprine enterovirus CEV-JL14. Alignment analysis of the complete nucleotide sequences revealed 79.2%-87.8% and 75.0%-76.7% sequence identity of these novel caprine enterovirus strains to CEV-JL14 and TB4-OEV, respectively. Phylogenetic analyses clustered these novel strains to EV-G based on the amino acid sequences of P1 and 2C+3CD. Moreover, phylogenetic analysis of these caprine enterovirus strains identified three new EV-G types using VP1 sequences. These results demonstrate the genetic variations and the evolution of caprine enterovirus.

Cryo-EM Structure of a Possum Enterovirus.

Enteroviruses (EVs) represent a substantial concern to global health. Here, we present the cryo-EM structure of a non-human enterovirus, EV-F4, isolated from the Australian brushtail possum to assess the structural diversity of these picornaviruses. The capsid structure, determined to ~3 Å resolution by single particle analysis, exhibits a largely smooth surface, similar to EV-F3 (formerly BEV-2). Although the cellular receptor is not known, the absence of charged residues on the outer surface of the canyon suggest a different receptor type than for EV-F3. Density for the pocket factor is clear, with the entrance to the pocket being smaller than for other enteroviruses.

A novel enterovirus in lambs with poliomyelitis and brain stem encephalitis.

An Austrian organic dairy sheep farm experienced cases of recumbency and sudden deaths in 3- to 4-week-old lambs. Two animals were subjected to thorough clinical and pathological investigations. Pathohistological analysis identified severe nonsuppurative myelitis and mild nonsuppurative encephalitis. A reverse-transcription quantitative PCR (RT-qPCR) assay for the recently discovered ovine picornavirus causing comparable lesions scored negative. By next-generation sequencing-based metagenomics, a nearly complete genome of a novel enterovirus could be detected and assembled. In situ hybridization using a specifically designed probe revealed robust signals in affected motoneurons of the spinal cord suggesting a causative role of the novel virus.

Molecular characterization of a porcine sapelovirus strain isolated in China.

Porcine sapelovirus (PSV) infections have been associated with a wide spectrum of symptoms, ranging from asymptomatic infection to clinical signs including diarrhoea, pneumonia, reproductive disorders, and polioencephalomyelitis. Although it has a global distribution, there have been relatively few studies on PSV in domestic animals. We isolated a PSV strain, SHCM2019, from faecal specimens from swine, using PK-15 cells. To investigate its molecular characteristics and pathogenicity, the genomic sequence of strain SHCM2019 was analysed, and clinical manifestations and pathological changes occurring after inoculation of neonatal piglets were observed. The virus isolated using PK-15 cells was identified as PSV using RT-PCR, transmission electron microscopy (TEM), and immunofluorescence assay (IFA). Sequencing results showed that the full-length genome of the SHCM2019 strain was 7,567 nucleotides (nt) in length, including a 27-nucleotide poly(A) tail. Phylogenetic analysis demonstrated that this virus was a PSV isolate belonging to the Chinese strain cluster. Recombination analysis indicated that there might be a recombination breakpoint upstream of the 3D region of the genome. Pathogenicity experiments demonstrated that the virus isolate could cause diarrhoea and pneumonia in piglets. In breif, a recombinant PSV strain, SHCM2019, was isolated and shown to be pathogenic. Our results may provide a reference for future research on the pathogenic mechanism and evolutionary characteristics of PSV.

Multiple genotypes of enterovirus G carrying a papain-like cysteine protease (PL-CP) sequence circulating on two pig farms in Japan: first identification of enterovirus G10 carrying a PL-CP sequence.

Two and three genotypes of enterovirus G (EV-G) carrying a papain-like cysteine protease (PL-CP) sequence were detected on two pig farms and classified into genotypes G1 and G10, and G1, G8, and G17, respectively, based on VP1 sequences. A G10 EV-G virus bearing a PL-CP sequence was detected for the first time. Phylogenetic analysis of the P2 and P3 regions grouped the viruses by farm with high sequence similarity. Furthermore, clear recombination break points were detected in the 2A region, suggesting that PL-CP EV-G-containing strains gained sequence diversity through recombination events among the multiple circulating EV-G genotypes on the farms.

Detection and molecular characterization of porcine enterovirus G15 and teschovirus from India.

Porcine enterovirus G (EV-G) and teschovirus (PTV) generally cause asymptomatic infections. Although both viruses have been reported from various countries, they are rarely detected from India. To detect these viruses in Western India, fecal samples (n = 26) of diarrheic piglets aged below three months from private pig farms near Pune (Maharashtra) were collected. The samples were screened by reverse transcription-polymerase chain reaction using conserved enterovirus specific primers from 5' untranslated region. For genetic characterization of detected EV-G strain, nearly complete genome, and for PTV, partial VP1 gene were sequenced. EV-G strain showed the highest identity in a VP1 gene at nucleotide (78.61%) and amino acid (88.65%) level with EV-G15, prototype strain. However, its complete genome was homologous with the nucleotide (78.38% identity) and amino acid (91.24% identity) level to Ishi-Ka2 strain (LC316832), unassigned EV-G genotype detected from Japan. The nearly complete genome of EV-G15 consisted of 7398 nucleotides excluding the poly(A) tail and has an open reading frame that encodes a 2170 amino acid polyprotein. Genetic analysis of the partial VP1 gene of teschovirus identified porcine teschovirus 4 (PTV-4) and putative PTV-17 genotype. To the best of our knowledge, this is the first report on nearly full genome characterization of EV-G15, and detection of PTV-4 and putative PTV-17 genotypes from India. Further, detection and characterization of porcine enteroviruses are needed for a comprehensive understanding of their genetic diversity and their association with symptomatic infections from other geographical regions of India.

Experimental immunization of mice with a recombinant bovine enterovirus vaccine expressing BVDV E0 protein elicits a long-lasting serologic response.

BACKGROUND: Bovine viral diarrhea virus (BVDV) is a cause of substantial economic loss to the cattle industry worldwide, and there are currently no effective treatment or preventive measures. Bovine enterovirus (BEV) has a broad host range with low virulence and is a good candidate as a viral vaccine vector. In this study, we explored new insertion sites for the expression of exogenous genes in BEV, and developed a recombinant infectious cDNA clone for BEV BJ101 strain expressing BVDV E0 protein. METHODS: A recognition site for the viral proteinase 3Cpro was inserted in the GpBSK-BEV plasmid at the 2C/3A junction by overlapping PCR. Subsequently, the optimized full-length BVDV E0 gene was inserted to obtain the recombinant infectious plasmid GpBSK-BEV-E0. The rescued recombinant virus was obtained by transfection with linearized plasmid. Expression of BVDV E0 in the recombinant virus was confirmed by PCR, western blotting, and immunofluorescence analysis, and the genetic stability was tested in MDBK cells over 10 passages. We further tested the ability of the recombinant virus to induce an antibody response in mice infected with BVDV and immunized them with the recombinant virus and parental strain. RESULTS: The rescued recombinant virus rBEV-E0 was identified and confirmed by western blot and indirect immunofluorescence. The sequencing results showed that the recombinant virus remained stable for 10 passages without genetic changes. There was also no significant difference in growth dynamics and plaque morphology between the recombinant virus and parental virus. Mice infected with both recombinant and parental viruses produced antibodies against BEV VP1, while the recombinant virus also induced antibodies against BVDV E0. CONCLUSION: A new insertion site in the BEV vector can be used for the prevention and control of both BEV and BVDV, providing a useful tool for future research on the development of viral vector vaccines.

Novel enteric viruses in fatal enteritis of grey squirrels.

Astro- and kobuviruses infect both humans and animals. Here, we report on the disease history, detection and genomic characterization of novel astro- and kobuviruses from fatal diarrhoea of two juvenile grey squirrels. The virus particles had enterovirus-like morphology and a diameter of 28-32 nm. Next-generation sequencing confirmed astro- and kobuviruses and sequence analysis revealed typical astrovirus and picornavirus genome organizations. The astrovirus ORF2 sequence clustered with a clade of unassigned astroviruses, with marmot and rodent mamastroviruses as closest relatives. For the kobuvirus, divergences greater than 49.4 % for P1 and 43.5 % in the non-structural proteins indicated a novel species. However, phylogenetic analysis of the 3D polymerase showed that it clustered with that of the newly classified ludopivirus A1, suggesting a previous recombination event in the evolution of the kobuvirus. Our data provide further insights into the diversity of astro- and kobuviruses and broaden the spectrum of viruses infecting grey squirrels.

Virome diversity analysis reveals novel enteroviruses and a human picobirnavirus in stool samples from African green monkeys with diarrhea.

It is important to identify viruses in animals because most infectious diseases in humans are caused by viruses of zoonotic origin. African green monkey is a widely used non-human primate model in biomedical investigations. In this study, total RNAs were extracted from stool samples of 10 African green monkeys with diarrhea. High-throughput sequencing was used to characterize viromes. PCR and Sanger sequencing were used to determine the full genome sequences. Great viral diversity was observed. The dominant viruses were enteroviruses and picobirnaviruses. Six enterovirus genomes and a picobirnavirus RNA-dependent RNA polymerase sequence were characterized. Five enteroviruses belonged to two putative new genotypes of species Enterovirus J. One enterovirus belonged to EV-A92. The picobirnavirus RNA-dependent RNA polymerase sequence had the highest nucleotide similarity (93.48%) with human picobirnavirus isolate GPBV6C2. The present study helped to identify the potential zoonotic viruses in African green monkeys. Further investigations are required to elucidate their pathogenic roles in animals and humans.

Structural basis for hijacking of the host ACBD3 protein by bovine and porcine enteroviruses and kobuviruses.

Picornaviruses infect a wide range of mammals including livestock such as cattle and swine. As with other picornavirus genera such as Aphthovirus, there is emerging evidence of a significant economic impact of livestock infections caused by members of the genera Enterovirus and Kobuvirus. While the human-infecting enteroviruses and kobuviruses have been intensively studied during the past decades in great detail, research on livestock-infecting viruses has been mostly limited to the genomic characterization of the viral strains identified worldwide. Here, we extend our previous studies of the structure and function of the complexes composed of the non-structural 3A proteins of human-infecting enteroviruses and kobuviruses and the host ACBD3 protein and present a structural and functional characterization of the complexes of the following livestock-infecting picornaviruses: bovine enteroviruses EV-E and EV-F, porcine enterovirus EV-G, and porcine kobuvirus AiV-C. We present a series of crystal structures of these complexes and demonstrate the role of these complexes in facilitation of viral replication.