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.

Evaluation of packaging capacity at the genomic 2C/3A junction region in Porcine enterovirus G.

Porcine enterovirus G (EV-G) is endogenous to most pig farming countries worldwide. Reports that a papain-like protease (PLP) gene has been naturally inserted into the 2C/3A junction region of the EV-G genome, has increased the potential public health threats from this virus. We constructed a full-length infectious cDNA clone of EV-G, CH/17GXQZ/2017, in order to determine the packaging capacity at the 2C/3A insertion site. Subsequently, recombinants viruses containing the coding tags, GFP, iLOV and His at the 2C/3A junction region, were synthesized. The infectious virus was successfully rescued only with the insertion of the His-tag, which displayed similar virological and molecular properties to its parental strain. This study determined the packaging capacity of the 2C/3A insertion site, and it provides a practical tool for studying the functions and pathogenic mechanisms of EV-G in pigs.

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.

Characterization, phylogenetic analysis, and pathogenicity of a novel genotype 2 porcine Enterovirus G.

Enterovirus G belongs to the family Picornaviridae and are associated with a variety of animal diseases. We isolated and characterized a novel EV-G2 strain, CHN-SCMY2021, the first genotype 2 strain isolated in China. CHN-SCMY2021 is about 25 nm diameter with morphology typical of picornaviruses and its genome is 7341 nucleotides. Sequence alignment and phylogenetic analysis based on VP1 indicated that this isolate is a genotype 2 strain. The whole genome similarity between CHN-SCMY2021 and other EV-G genotype 2 strains is 78.3-86.4%, the greatest similarity is to EVG/Porcine/JPN/Iba26-506/2014/G2 (LC316792.1). Recombination analysis indicated that CHN-SCMY2021 resulted from recombination between 714,171/CaoLanh_VN (KT265894.2) and LP 54 (AF363455.1). Except for ST cells, CHN-SCMY2021 has a broad spectrum of cellular adaptations, which are susceptible to BHK-21, PK-15, IPEC-J2, LLC-PK and Vero cells. In piglets, CHN-SCMY2021 causes mild diarrhea and thinning of the intestinal wall. The virus was mainly distributed to intestinal tissue but was also found in heart, liver, spleen, lung, kidney, brain, and spinal cord. CHN-SCMY2021 is the first systematically characterized EV-G genotype 2 strain from China, our results enrich the information on the epidemiology, molecular evolution and pathogenicity associated with EV-G.

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.

Distribution and genetic diversity of Enterovirus G (EV-G) on pig farms in Thailand.

BACKGROUND: Enterovirus G (EV-G) causes subclinical infections and is occasionally associated with diarrhea in pigs. In this study, we conducted a cross-sectional survey of EV-G in pigs from 73 pig farms in 20 provinces of Thailand from December 2014 to January 2018. RESULTS: Our results showed a high occurrence of EV-Gs which 71.6 % of fecal and intestinal samples (556/777) and 71.2 % of pig farms (52/73) were positive for EV-G by RT-PCR specific to the 5'UTR. EV-Gs could be detected in all age pig groups, and the percentage positivity was highest in the fattening group (89.7 %), followed by the nursery group (89.4 %). To characterize the viruses, 34 EV-G representatives were characterized by VP1 gene sequencing. Pairwise sequence comparison and phylogenetic analysis showed that Thai-EV-Gs belonged to the EV-G1, EV-G3, EV-G4, EV-G8, EV-G9 and EV-G10 genotypes, among which the EV-G3 was the predominant genotype in Thailand. Co-infection with different EV-G genotypes or with EV-Gs and porcine epidemic diarrhea virus (PEDV) or porcine deltacoronavirus (PDCoV) on the same pig farms was observed. CONCLUSIONS: Our results confirmed that EV-G infection is endemic in Thailand, with a high genetic diversity of different genotypes. This study constitutes the first report of the genetic characterization of EV-GS in pigs in Thailand.

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.

Swine polioencephalomyelitis in Brazil: identification of Teschovirus A, Sapelovirus A, and Enterovirus G in a farm from Southern Brazil.

Porcine encephalomyelitis can be associated with many etiologies, including viral agents, such as Porcine teschovirus (PTV), Porcine sapelovirus (PSV), and Porcine astrovirus (PoAstV). In this study, we investigated the presence of these viruses in a neurological disease outbreak in a swine farm in Southern Brazil. The piglet production farm unity had 1200 weaning piglets, and 40 piglets with neurological signs such as motor incoordination, paresis, and paralysis of hind limbs, with an evolution time of approximately 4 days. Among these, 10 piglets were submitted to postmortem examination. Gross lesions were restricted to a mild enlargement of the nerve roots and ganglia of spinal cord segments. The microscopic lesions were characterized by nonsuppurative encephalomyelitis and ganglioneuritis with evident neuronal degeneration and necrosis. Samples of the central nervous system (CNS), cerebrospinal fluid, and feces were collected and submitted to molecular analysis. PTV was identified in all samples of the CNS, while eight of the piglets were also positive for PSV, and seven were positive for Porcine enterovirus (EV-G). PoAstV was identified in a pool of feces of healthy animals used as controls. This study demonstrates the occurrence of encephalomyelitis associated with PTV on a swine farm in Southern Brazil, as well as the presence of other viruses such as PSV, EV-G, and PoAstV in the swineherd. Sequences of the fragments that were previously amplified by PCR showed a high similarity to PTV 6. Herein, we describe the first case report of severe swine polioencephalomyelitis associated with PTV in South America.

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.

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.

A novel defective recombinant porcine enterovirus G virus carrying a porcine torovirus papain-like cysteine protease gene and a putative anti-apoptosis gene in place of viral structural protein genes.

Enterovirus G (EV-G) belongs to the family of Picornaviridae. Two types of recombinant porcine EV-Gs carrying papain-like cysteine protease (PLCP) gene of porcine torovirus, a virus in Coronaviridae, are reported. Type 1 recombinant EV-Gs are detected in pig feces in Japan, USA, and Belgium and carry the PLPC gene at the junction site of 2C/3A genes, while PLPC gene replaces the viral structural genes in type 2 recombinant EV-G detected in pig feces in a Chinese farm. We identified a novel type 2 recombinant EV-G carrying the PLCP gene with flanking sequences in place of the viral structural genes in pig feces in Japan. The ~0.3 kb-long upstream flanking sequence had no sequence homology with any proteins deposited in GenBank, while the downstream ~0.9 kb-long flanking sequence included a domain having high amino acid sequence homology with a baculoviral inhibitor of apoptosis repeat superfamily. The pig feces, where the novel type 2 recombinant EV-G was detected, also carried type 1 recombinant EV-G. The amount of type 1 and type 2 recombinant EV-G genomes was almost same in the pig feces. Although the phylogenetic analysis suggested that these two recombinant EV-Gs have independently evolved, type 1 recombinant EV-G might have served as a helper virus by providing viral structural proteins for dissemination of the type 2 recombinant EV-G.

Longitudinal survey of Teschovirus A, Sapelovirus A, and Enterovirus G fecal excretion in suckling and weaned pigs.

Fecal samples from 27 pigs were longitudinally analyzed for Teschovirus A (TV-A), Sapelovirus A (SV-A), and Enterovirus G (EV-G) RNA presence. Suckling piglet fecal samples were negative for the three enteric picornaviruses. However, these picornaviruses were detected in 22/27 weaned pig fecal samples. This study provides new data on TV-A, SV-A, and EV-G infection dynamics.

First detection of novel enterovirus G recombining a torovirus papain-like protease gene associated with diarrhoea in swine in South Korea.

Enterovirus species G (EV-G) comprises a highly diversity of 20 genotypes that is prevalent in pig populations, with or without diarrhoea. In the present study, a novel EV-G strain (KOR/KNU-1811/2018) that resulted from cross-order recombination was discovered in diagnostic faecal samples from neonatal pigs with diarrhoea that were negative for swine enteric coronaviruses and rotavirus. The recombinant EV-G genome possessed an exogenous 594-nucleotide (198-amino acid) sequence, flanked by two viral 3Cpro cleavage sites at the 5' and 3' ends in its 2C/3A junction region. This insertion encoded a predicted protease similar to the porcine torovirus papain-like cysteine protease (PLCP), which was recently found in the EV-G1, -G2, and -G17 genomes. The complete KNU-1811 genome shared 73.7% nucleotide identity with a prototype EV-G1 strain, but had 83.9%-86.7% sequence homology with the global EV-G1-PLCP strains. Genetic and phylogenetic analyses demonstrated that the Korean recombinant EV-G's own VP1 and inserted foreign PLCP genes are most closely related independently to contemporary chimeric G1-PLCP and G17-PLCP strains respectively. These results implied that the torovirus-derived PLCP gene might have undergone continuous nucleotide mutations in the respective EV-G genome following its independent acquisition through naturally occurring recombination. Our results advance the understanding of the genetic evolution of EV-G driven by infrequent viral recombination events, by which EV-G populations laterally gain an exotic gene encoding a virulence factor from heterogeneous virus families, thereby causing clinical disease in swine.

Molecular Identification of Enteroviruses from Cattle and Goat Feces and Environment in Thailand.

The identification and characterization of viruses of the genus Enterovirus in healthy and infected livestock, including cattle and goats, have been increasing. Enterovirus E (EV-E) and Enterovirus F (EV-F) are commonly found in cattle, whereas Enterovirus G (EV-G) is found in goats. In this study, molecular and phylogenetic analyses were performed to determine the prevalence of EVs in cattle and goat feces from Kanchanaburi Province, Thailand. The presence of EVs in water samples and the feces of other animals collected from the areas surrounding cattle and goat farms was also investigated. By use of 5'-untranslated region (5' UTR) real-time reverse transcription-PCR (RT-PCR), EVs were detected in 39.5% of cattle samples, 47% of goat samples, 35.3% of water samples, and one pool of chicken feces. Phylogenetic analysis revealed the presence of EV-E and EV-F in cattle, EV-E and EV-G in goats, and EV-F in water samples and chicken feces. Analysis of enteroviral VP1 sequences from cattle revealed that the EV-E genotypes circulating in the study region were EV-E1, with a possible new genotype that is closely related to EV-E2. Analysis of enteroviral VP1 sequences from goats suggested the circulation of EV-G5 and a possible new genotype that is closely related to EV-G20. Sequence analyses also suggested that although the VP1 sequences from goats were closely related to those of EV-G, which were considered porcine enterovirus sequences, their 5' UTRs form a separated cluster with sequences of sheep and goat origin, suggesting a new classification of the ovine/caprine-specific enterovirus group.IMPORTANCE Possible new EV-E and EV-G genotypes were identified for EVs detected in this study. The EV-E viruses were also successfully isolated from MDBK cells. The goat EV sequence analysis suggested the presence of an ovine/caprine-specific EV group that is different from EV-G of porcine origin. The significance of our research is that it identifies and characterizes possible novel EVs, thereby indicating that enteroviruses in animals are continually evolving. The facts that enteroviruses can persist in the environment, contaminate it for long periods, and be transmitted between animals raise serious concerns regarding this group of viruses as emerging livestock pathogens.

Full-length and defective enterovirus G genomes with distinct torovirus protease insertions are highly prevalent on a Chinese pig farm.

Recombination occurs frequently between enteroviruses (EVs) which are classified within the same species of the Picornaviridae family. Here, using viral metagenomics, the genomes of two recombinant EV-Gs (strains EVG 01/NC_CHI/2014 and EVG 02/NC_CHI/2014) found in the feces of pigs from a swine farm in China are described. The two strains are characterized by distinct insertion of a papain-like protease gene from toroviruses classified within the Coronaviridae family. According to recent reports the site of the torovirus protease insertion was located at the 2C/3A junction region in EVG 02/NC_CHI/2014. For the other variant EVG 01/NC_CHI/2014, the inserted protease sequence replaced the entire viral capsid protein region up to the VP1/2A junction. These two EV-G strains were highly prevalent in the same pig farm with all animals shedding the full-length genome (EVG 02/NC_CHI/2014) while 65% also shed the capsid deletion mutant (EVG 01/NC_CHI/2014). A helper-defective virus relationship between the two co-circulating EV-G recombinants is hypothesized.

Genetic diversity and recombination of enterovirus G strains in Japanese pigs: High prevalence of strains carrying a papain-like cysteine protease sequence in the enterovirus G population.

To study the genetic diversity of enterovirus G (EV-G) among Japanese pigs, metagenomics sequencing was performed on fecal samples from pigs with or without diarrhea, collected between 2014 and 2016. Fifty-nine EV-G sequences, which were >5,000 nucleotides long, were obtained. By complete VP1 sequence analysis, Japanese EV-G isolates were classified into G1 (17 strains), G2 (four strains), G3 (22 strains), G4 (two strains), G6 (two strains), G9 (six strains), G10 (five strains), and a new genotype (one strain). Remarkably, 16 G1 and one G2 strain identified in diarrheic (23.5%; four strains) or normal (76.5%; 13 strains) fecal samples possessed a papain-like cysteine protease (PL-CP) sequence, which was recently found in the USA and Belgium in the EV-G genome, at the 2C-3A junction site. This paper presents the first report of the high prevalence of viruses carrying PL-CP in the EV-G population. Furthermore, possible inter- and intragenotype recombination events were found among EV-G strains, including G1-PL-CP strains. Our findings may advance the understanding of the molecular epidemiology and genetic evolution of EV-Gs.

High genetic diversity of porcine enterovirus G in Schleswig-Holstein, Germany.

Between 2012 and 2015, 495 pooled snout swabs from fattening pigs raised in Schleswig-Holstein, Germany, were screened for the presence of enterovirus G (EV-G) RNA. Nucleic acids were tested in diverse reverse transcription polymerase chain reaction assays applying published oligonucleotide primers specific for the viral protein (VP) 1 and 2/4 encoding regions as well as for 3D polymerase. Phylogenetic analyses of VP1 revealed the presence of 12 EV-G types, three of which had highly divergent sequences suggesting putative new types. Co-circulation of EV-G types was observed in several pigsties. Thus, genetic diversity of EV-G was demonstrated in this small geographic area.

A porcine enterovirus G associated with enteric disease contains a novel papain-like cysteine protease.

Identification of unknown pathogens in pigs displaying enteric illness is difficult due to the large diversity of bacterial and viral species found within faecal samples. Current methods often require bacterial or viral isolation, or testing only a limited number of known species using quantitative PCR analysis. Herein, faeces from two 25-day-old piglets with diarrhoea from Texas, USA, were analysed by metagenomic next-generation sequencing to rapidly identify possible pathogens. Our analysis included a bioinformatics pipeline of rapid short-read classification and de novo genome assembly which resulted in the identification of a porcine enterovirus G (EV-G), a complete genome with substantial nucleotide differences (>30 %) among current sequences, and a novel non-structural protein similar in sequence to the Torovirus papain-like cysteine protease (PLpro). This discovery led to the identification and circulation of an EV-G with a novel PLpro in the USA that has not been previously reported.

Dynamics of Virus Distribution in a Defined Swine Production Network Using Enteric Viruses as Molecular Markers.

Modern swine production systems represent complex and dynamic networks involving numerous stakeholders. For instance, livestock transporters carry live animals between fattening sites, abattoirs, and other premises on a daily basis. This interconnected system may increase the risk of microbial spread within and between networks, although little information is available in that regard. In the present study, a swine network composed of 10 finishing farms, one abattoir, and three types of stakeholders (veterinarians, livestock transporters, and nutritional technicians) in Quebec, Canada, was selected to investigate specific vectors and reservoirs of enteric viruses. Environmental samples were collected from the premises over a 12-month period. Samples were screened using targeted reverse transcription-PCR and sequencing of two selected viral markers, group A rotaviruses (RVA) and porcine astroviruses (PoAstV), both prevalent and genetically heterogeneous swine enteric viruses. The results revealed frequent contamination of farm sites (21.4 to 100%), livestock transporter vehicles (30.6 to 68.8%) and, most importantly, the abattoir yard (46.7 to 94.1%), depending on the sample types. Although high levels of strain diversity for both viruses were found, identical PoAstV and RVA strains were detected in specific samples from farms, the abattoir yard, and the livestock transporter vehicle, suggesting interconnections between these premises and transporters. Overall, the results from this study underscore the potential role of abattoirs and livestock transport as a reservoir and transmission route for enteric viruses within and between animal production networks, respectively. IMPORTANCE: Using rotaviruses and astroviruses as markers of enteric contamination in a swine network has revealed the potential role of abattoirs and livestock transporters as a reservoir and vectors of enteric pathogens. The results from this study highlight the importance of tightening biosecurity measures. For instance, implementing sanitary vacancy between animal batches and emphasizing washing, disinfection, and drying procedures on farms and for transportation vehicles, as well as giving limited access and circulation of vehicles throughout the production premises, are some examples of measures that should be applied properly. The results also emphasize the need to closely monitor the dynamics of enteric contamination in the swine industry in order to better understand and potentially prevent the spread of infectious diseases. This is especially relevant when a virulent and economically damaging agent is involved, as seen with the recent introduction of the porcine epidemic diarrhea virus in the country.