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.

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.

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.

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.

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.

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.

[New classification of enteroviruses of pigs: serological and molecular genetic aspects].

Due to changes in modern classification and nomenclature family Picornaviridae made by the International Committee of Taxonomy of Viruses, conducted genetic and serological reclassification of 36 industrial, typical and laboratory strains porcine enteroviruses isolated in Ukraine. The results of molecular genetic studies of 34 strains assigned to the family Picornaviridae, genus Teschovirus, species Porcine teschovirus, 2 strains of virus did not engage in polymerase chain reaction with species specific primer. In the neutralization reaction of the virus revealed that 23 strains belonging to 1 serotype Porcine teschovirus, 4 strain--PTV-3, 1--to PTV-6, 1 strain--to Porcine sapelovirus, three strains have between typical antigenic properties, and 4 strains--antigenically different from reference strains Porcine teschovirus, Enterovirus G and Porcine sapelovirus.

First identification and characterization of porcine enterovirus G in the United States.

Porcine enterovirus G (EV-G) is a member of the family Picornavirdae, genus Enterovirus. To date, eleven EV-G types (EV-G1 through EV-G11) have been identified in pigs from Asia and Europe however they have never been reported in North America. In this study, we isolated and characterized the complete genome of NP/2013/USA, an EV-G from a porcine diarrhea sample from the United States. The complete genome consists of 7,390 nucleotides excluding the 3' poly(A) tail, and has an open reading frame that encodes a 2,169 amino acid polyprotein. NP/2013/USA was most similar at the nucleotide (84%) and amino acid (95%) level to the HM131607, an EV-G1 type isolated from China in 2012.

Prevalence, genetic diversity and recombination of species G enteroviruses infecting pigs in Vietnam.

Picornaviruses infecting pigs, described for many years as 'porcine enteroviruses', have recently been recognized as distinct viruses within three distinct genera (Teschovirus, Sapelovirus and Enterovirus). To better characterize the epidemiology and genetic diversity of members of the Enterovirus genus, faecal samples from pigs from four provinces in Vietnam were screened by PCR using conserved enterovirus (EV)-specific primers from the 5' untranslated region (5' UTR). High rates of infection were recorded in pigs on all farms, with detection frequencies of approximately 90% in recently weaned pigs but declining to 40% in those aged over 1 year. No differences in EV detection rates were observed between pigs with and without diarrhoea [74% (n = 70) compared with 72% (n = 128)]. Genetic analysis of consensus VP4/VP2 and VP1 sequences amplified from a subset of EV-infected pigs identified species G EVs in all samples. Among these, VP1 sequence comparisons identified six type 1 and seven type 6 variants, while four further VP1 sequences failed to group with any previously identified EV-G types. These have now been formally assigned as EV-G types 8-11 by the Picornavirus Study Group. Comparison of VP1, VP4/VP2, 3D(pol) and 5' UTRs of study samples and those available on public databases showed frequent, bootstrap-supported differences in their phylogenies indicative of extensive within-species recombination between genome regions. In summary, we identified extremely high frequencies of infection with EV-G in pigs in Vietnam, substantial genetic diversity and recombination within the species, and evidence for a much larger number of circulating EV-G types than currently described.

Prevalence of porcine enterovirus 9 in pigs in middle and eastern China.

Little information on the epidemiology and pathogenicity of porcine enterovirus 9 (PEV-9) is available. The present study investigated the prevalence of PEV-9 in pig populations in middle and eastern China using reverse transcriptase (RT)-PCR. All 14 sampled farms were positive for PEV-9 and the overall prevalence of infection in the studied pigs was 8.3% (37/447). There was a higher frequency of infection in pigs aged 10-15 weeks (12/119, 10.1%) than in pigs aged >20 weeks (5/103, 4.9%). A 313 nucleotide sequence from the 5'-UTR region of 37 Chinese PEV-9 positive samples had 96.1-100% sequence homology. On phylogenetic analysis, sequences clustered into two major groups, from which two representative strains were selected to determine the complete RNA-dependent RNA polymerase (RdRp) gene sequence. Phylogenetic analysis based on the RdRp gene suggested that PEV-9 strains from China formed a new subgroup. Piglets were inoculated orally with the PEV-9 strain identified in this study. Although most experimental pigs showed no clinical signs, almost all carried PEV-9 in one or more tissues after 6 days post-inoculation. The results of tissue histologic examination suggested that PEV9 can cause pathological changes in cerebrum and lung.

Complete genome analysis of porcine enterovirus B isolated in Korea.

The complete genome sequence of porcine enterovirus B (PEV-B) from a Korean isolate was analyzed. The genome size was 7,393 bp. Previously, full genome sequences of PEV-B had been reported from the United Kingdom, Hungary, and China. The Korean PEV-B isolate presented polyprotein gene nucleotide sequence similarities of 77.9, 73.7, 78.9, and 80.3%, respectively, to PEV-B UKG/410/73, LP54, PEV15, and Chinese strains (Ch-ah-f1).

Natural interspecies recombinant bovine/porcine enterovirus in sheep.

Members of the genus Enterovirus (family Picornaviridae) are believed to be common and widespread among humans and different animal species, although only a few enteroviruses have been identified from animal sources. Intraspecies recombination among human enteroviruses is a well-known phenomenon, but only a few interspecies examples have been reported and, to our current knowledge, none of these have involved non-primate enteroviruses. In this study, we report the detection and complete genome characterization (using RT-PCR and long-range PCR) of a natural interspecies recombinant bovine/porcine enterovirus (ovine enterovirus type 1; OEV-1) in seven (44 %) of 16 faecal samples from 3-week-old domestic sheep (Ovis aries) collected in two consecutive years. Phylogenetic analysis of the complete coding region revealed that OEV-1 (ovine/TB4-OEV/2009/HUN; GenBank accession no. JQ277724) was a novel member of the species Porcine enterovirus B (PEV-B), implying the endemic presence of PEV-B viruses among sheep. However, the 5' UTR of OEV-1 showed a high degree of sequence and structural identity to bovine enteroviruses. The presumed recombination breakpoint was mapped to the end of the 5' UTR at nucleotide position 814 using sequence and SimPlot analyses. The interspecies-recombinant nature of OEV-1 suggests a closer relationship among bovine and porcine enteroviruses, enabling the exchange of at least some modular genetic elements that may evolve independently.

The survey of porcine teschoviruses, sapeloviruses and enteroviruses B infecting domestic pigs and wild boars in the Czech Republic between 2005 and 2011.

This study presents results of epidemiological survey and genetic characterisation of porcine enteric picornaviruses belonging to the genera Teschovirus, Sapelovirus, and Porcine enterovirus B. Faecal or gut content samples from domestic pigs (Sus scrofa f. domestica) and the cecal content of wild boars (Sus scrofa) of different ages (collected between 2005 and 2011) were analysed by molecular methods. Porcine enterovirus B was the most prevalent virus detected in both domestic pigs and wild boars (50.2% and 69.4%, respectively), followed by Porcine teschovirus and Porcine sapelovirus. The majority of positive domestic pigs (69.4%) and wild boars (64.3%) were infected with two or three tested viruses. There was no significant difference in prevalences of teschoviruses, sapeloviruses, and enteroviruses among healthy and diarrhoeic pigs. Results of epidemiological survey demonstrated that all target viral genera are common in Czech farms producing pigs and wild boars. Amplified nucleotide fragments of VP2 region obtained from randomly selected both historical and recent Teschovirus isolates were sequenced. Based on sequence data, historical Porcine teschovirus isolate CAPM V-180, previously determined as serotype 1 was reclassified into serotype 11. Moreover, another recent Porcine teschovirus isolate OH264/2010 was described and classified into serotype 11. Four nontypeable PTV strains (historical isolate CAPM V-182/1976 and recent isolates JA247/2010, NI429/2010, and BR1576/2007) identified in this study might represent novel serotypes. To the best of our knowledge, our study represents the first description of this serotype in the Czech Republic.

Complete genome sequence of a novel porcine enterovirus strain in China.

The porcine enteroviruses (PEVs) belong to the family Picornaviridae. We report a complete genome sequence of a novel PEV strain that is widely prevalent in pigs at least in central and eastern China. The complete genome consists of 7,390 nucleotides, excluding the 3' poly(A) tail, and has an open reading frame that maps between nucleotide positions 812 and 7318 and encodes a 2,168-amino-acid polyprotein. Phylogenetic analysis based on the 3CD and VP1 regions reveals that this PEV strain belongs to a species of PEV9 but may represent a novel sero-/genotype in CPE group III. We also report the major findings from bootscan analysis based on the whole genomes of PEVs in the present study and those available in GenBank.

Characterization of a novel porcine enterovirus in wild boars in Hungary.

Porcine enteroviruses (PEVs) are members of the family Picornaviridae, genus Enterovirus. Until now, only three different PEV genotypes (PEV-9 and -10, and PEV-3H/PEV-14) have been detected in domestic pigs, and there is no information about the presence of PEVs in wild animals. Here, we identify and characterize the complete genomes of PEV originated from 5 of 10 (50%) of wild boar (Sus scrofa) piglets by RT-PCR and pyrosequencing. Wild boar/WBD/2011/HUN (JN807387) PEV showed only 67% amino acid identity in VP1 compared to the most closely related prototype PEV-3H/PEV-14. Wild boar enterovirus represents a novel PEV genotype, provisionally called PEV-15.

Characterization of a novel porcine enterovirus in domestic pig in Hungary.

Porcine enteroviruses (PEVs) of genus Enterovirus are small, non-enveloped viruses with single-stranded, positive sense genomic RNA, belonging to the family Picornaviridae. The discovery of two distinct serotypes (PEV9 and 10) was first reported in 1979. Despite the sporadic detection and partial genome sequences of these viruses our knowledge about the prevalence and molecular epidemiology of PEV types in domestic pigs is very deficient. In this study, we identified a novel PEV from fecal samples of clinically healthy pigs (Sus scrofa domestica) in Hungary by RT-PCR using human enterovirus generic primer pairs for 5'UTR region, with subsequent partial VP1 and complete genome sequencing and phylogenetic analysis. Among 45 fecal and blood sample pairs collected at the same farm from domestic pigs divided into three age groups (10 days, 4 weeks, and 3 months of age, N = 15 each group) six (40%) of the 15 fecal samples of 10-day-old pigs were enterovirus-positive. PEV was not detected in serum samples. Sequence- and phylogenetic analysis of the complete genome of swine/K23/2008/HUN (HQ702854) show relationship to PEV strains but it is separated from the PEV9 and 10, especially in structural regions. Swine/K23/2008/HUN has average of 77 and 75% amino acid identity in the P1 region, and only 61% in VP1 region to PEV9 and 10, respectively. The partial VP1 sequences of the Hungarian PEV strains show 99% nucleotide identity compared to each other. PEVs could be capable of at least local endemic spread among newborn piglets and cause no clinical symptoms or viraemia. Sequence data indicates that the Hungarian PEV strain belongs to a novel PEV. To clarify the taxonomic confusion related to PEV--as a consequence of recent extensive taxonomic changes among porcine enteric picornaviruses--we propose that PEV9 and PEV10 should be reclassified as PEV1 and PEV2. In this classification swine/K23/2008/HUN represents PEV3.

Sequence analysis of a duck picornavirus isolate indicates that it together with porcine enterovirus type 8 and simian picornavirus type 2 should be assigned to a new picornavirus genus.

In a 1990 outbreak, a virus isolated in Taiwan from the intestines of ducks showing signs of hepatitis was tentatively classified as a picornavirus on the basis of physical, chemical, and morphological characteristics. The virus was cloned and then found not to be type 1 duck hepatitis virus (DHV-1) or a new serotype of duck hepatitis virus (N-DHV) by serum neutralization. Complete genome sequencing indicated that the virus genome had 8351 nucleotides and the typical picornavirus genome organization (i.e., 5' untranslated region (UTR)-L-P1 (VP 4-2-3-1)-P2 (2A-B-C)-P3 (3A-B-C-D)-3' UTR-poly A). One open reading frame encoded 2521 amino acids, which makes this virus one of the largest picornaviruses, second only to equine rhinitis B virus of the genus Erbovirus. Its L protein was the largest within the family Picornaviridae (451 amino acids) and suspected to be a trypsin-like protease. The 235-nucleotide 3' UTR region was of intermediate size, quite long compared to other picornaviruses but shorter than other picornaviruses of duck-origin (DHV-1 and N-DHV) and had four regions of secondary structure. The 2A protein was composed of only 12 amino acids, which is the shortest of any member of the family Picornaviridae. Phylogenetic analysis of the polyprotein and 3D sequences indicated that this virus (named duck picornavirus [DPV]) together with porcine enterovirus type 8 virus and several simian picornaviruses form a distinct branch of the family Picornaviridae and should be assigned to a new picornavirus genus.

John Burns Brooksby CBE: 25 December 1914 - 17 December 1998.

John Brooksby was an outstanding veterinary virologist, who worked at the Animal Virus Disease Research Institute, Pirbright, for 40 years, for 16 of which he was Director of the Institute. He will be remembered for his contributions to the diagnosis of foot-and-mouth disease, for his discovery of four new types, for the classification of subtypes and for fundamental studies of the virus. As Deputy Director and Director he was responsible for programmes on fundamental investigations of foot-and-mouth disease virus and other viruses exotic to the UK and for the application of the results both in the UK and worldwide. His advice on the distribution and the control of foot-and-mouth disease was sought by international organizations and by individual countries and was responsible for reducing the risk of spread of disease.