Isolation and genetic characteristics of a neurotropic teschovirus variant belonging to genotype 1 in northeast China.

Porcine teschovirus (PTV) is a causative agent of reproductive disorders, encephalomyelitis, respiratory diseases, and diarrhea in swine, with a worldwide distribution. In this work, we identified PTV-associated nonsuppurative encephalitis as a potential cause of posterior paralysis in neonatal pigs in northeast China. Using indirect immunofluorescence assay, western blot, electron microscopy, and genome sequencing, we identified a neurotropic PTV strain, named CHN-NP1-2016, in the supernatants of pooled cerebrum and cerebellum samples from an affected piglet. Nucleotide sequence alignment revealed that the whole genome of CHN-NP1-2016 shared the highest sequence similarity (86.76% identity) with PTV 1 strain Talfan. A combination of phylogenetic and genetic divergence analysis was applied based on the deduced amino acid sequence of the P1 gene with a cutoff value of the genetic distance (0.102 ± 0.008) for defining PTV genotypes, and this showed that CHN-NP1-2016 is a variant of genotype 1. In total, 16 unique mutations and five mutant clusters were detected in the capsid proteins VP1 and VP2 of CHN-NP1-2016 when compared to other PTV1 isolates. Importantly, we detected three mutant clusters located in the exposed surface loops of the capsid protein, potentially indicating significant differences in major neutralization epitopes. Moreover, a potential recombination event in the P1 region of PTV CHN-NP1-2016 was detected. These findings provide valuable insights into the role of recombination in the evolution of teschoviruses. To our knowledge, this is the first case report of PTV-1-associated encephalitis in northeast China. Future investigations will narrow on the serology and pathogenicity of this novel isolate.

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.

Identification of novel genotypes belonging to the species Teschovirus A from indigenous pigs in western Jiangxi, China.

Teschovirus A is currently the sole species in the genus Teschovirus, whose members are divided into 13 subtypes: porcine teschovirus (PTV) 1-13. However, recent discoveries of novel PTV genotypes have suggested that a new species, "Teschovirus B", should be established. Here, we have identified six of the 19 known genotypes and two novel genotypes (PTV 17-18), revealing the high genetic diversity of the PTV subpopulation in indigenous pigs of western Jiangxi, China. Moreover, we determined the nearly complete genome sequences of PTV 17-SG9 and PTV 18-SG10. Together with PTV 1-13, these novel genotypes were confirmed to be members of the species Teschovirus A based on phylogenetic and genetic divergence analysis. Consequently, the species Teschovirus A now includes at least 15 PTV genotypes.

Two novel porcine teschovirus strains as the causative agents of encephalomyelitis in the Netherlands.

BACKGROUND: Porcine teschovirus (PTV) circulates among wild and domesticated pig populations without causing clinical disease, however neuroinvasive strains have caused high morbidity and mortality in the past. In recent years, several reports appeared with viral agents as a cause for neurologic signs in weanling and growing pigs among which PTV and new strains of PTV were described. CASE PRESENTATION: On two unrelated pig farms in the Netherlands the weanling pig population showed a staggering gate, which developed progressively to paresis or paralysis of the hind legs with a morbidity up to 5%. After necropsy we diagnosed a non-suppurative encephalomyelitis on both farms, which was most consistent with a viral infection. PTV was detected within the central nervous system by qPCR. From both farms PTV full-length genomes were sequenced, which clustered closely with PTV-3 (98%) or PTV-11 (85%). Other common swine viruses were excluded by qPCR and sequencing of the virus. CONCLUSION: Our results show that new neuroinvasive PTV strains still emerge in pigs in the Netherlands. Further research is needed to investigate the impact of PTV and other viral agents causing encephalomyelitis within wild and domestic pig populations supported by the awareness of veterinarians.

Identification and genotypic characterization of porcine teschovirus from selected pig populations in India.

Porcine teschovirus (PTV) previously classified as porcine enteroviruses in the family Picornaviridae are associated with a wide range of illnesses in swine ranging from asymptomatic infection to acute fatal encephalomyelitis, diarrhea, and pneumonia. This study was planned to investigate whether porcine teschovirus is prevalent among pigs in India and to characterize the PTV identified in the study population. The study conducted in certain farms of North India revealed that 13 of 190 (6.84%) fecal samples were PTV positive by RT-PCR. Three viruses were successfully isolated from fecal samples using IB-RS-2 cell lines which were confirmed by RT-PCR and sequencing. Molecular characterization based on the VP1 region of the viral genome identified the isolated viruses as serotype 5 and serotype 8 of PTV. A new variant of teschovirus was also identified which showed significant nucleotide diversity from the known serotypes of the teschoviruses. This is the first report of isolation, identification, and characterization of porcine teschoviruses in 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.

Metagenomic identification and sequence analysis of a Teschovirus A-related virus in porcine feces in Japan, 2014-2016.

Porcine Teschoviruses (PTVs) are associated with polioencephalomyelitis and various diseases, including reproductive and gastrointestinal disorders, of pigs and wild boars, and are also detected in the feces of healthy pigs. The genus Teschovirus contains a single species Teschovirus A that currently includes 13 serotypes. In the present study, we identified novel PTVs that are distantly related to Teschovirus A and were found in fecal samples of pigs with or without diarrhea in Japan. Phylogenetic analysis of amino acid (aa) sequences of the complete coding region revealed that these newly identified viruses did not cluster with any strains of PTVs or other strains within the picornavirus supergroup 1, suggesting that the viruses may not belong to Teschovirus A or any genus of the family Picornaviridae. These novel PTVs share a type IV internal ribosomal entry site and conserved characteristic motifs in the coding region, yet exhibit 62.2-79.0%, 86.6-92.8%, 77.1-81.0%, and 84.3-86.7% aa identities to PTV strains in P1, 2C, 3C, and 3D regions, respectively. In contrast, PTV 1-13 strains of the Teschovirus A share 76.5-92.1%, 88.1-99.7%, 93.2-100%, and 95.8-100% aa identities in the P1, 2C, 3C, and 3D, respectively, within the species. These data imply that the newly identified viruses belong to teschoviruses, and may represent a novel species in the genus Teschovirus.

Characterization of PTV-12, a newly described porcine teschovirus serotype: in vivo infection and cross-protection studies.

Porcine teschoviruses (PTVs) constitute 1 of the 31 genera within the Picornaviridae family, comprising at least 13 genetic types (PTV-1 to PTV-13), of which only 11 (PTV-1 to PTV-11) have been recognized as serotypes to date. Specific for swine and wild boars, most PTVs are usually non-pathogenic, but some viral variants cause severe disorders in the central nervous system (Teschen disease) or milder signs (Talfan disease), as well as reproductive, digestive and respiratory disorders and skin lesions. Previous studies revealed a high diversity of teschoviruses circulating in Spanish pig populations. Phylogenetic analysis performed with these sequences and others available in GenBank disclosed 13 clusters, 11 of which corresponded to the known PTV serotypes, and 1 of 2 additional groups is represented by isolate CC25, whose full-length genomic sequence has been obtained. This group is new to science, and was putatively named PTV-12. Here, a complete characterization of this isolate is presented, including the experimental infection of minipigs to assess tissue tropism and possible pathogenicity in vivo in the host species. In addition, using this experimental animal model, we investigated whether a pre-existing infection with this PTV-12 isolate could confer cross-protection against infection with a heterotypic PTV-1 virulent strain. Based on phylogenetic analysis and serological data, we propose CC25 as the prototype strain of a new teschovirus serotype, PTV-12.

New serotypes of porcine teschovirus identified in Shanghai, China.

Teschoviruses are widely endemic and commonly found in pig fecal samples. In this study, we collected fecal specimens from various pig herds and genotyped them based on the VP1 gene. Of 322 samples, 276 were positive, giving a PTV infectivity rate of 85.7 %. PTV4 was the most common serotype found in Shanghai, followed by PTV8 and PTV10. Interestingly, Some Shanghai strains belonging to a new PTV serotype were also isolated. In phylogenetic analysis, PTV SH8 did not correspond to any known serotype. PTV4 and PTV6 showed similar levels of sequence identity to PTV SH8. These data suggest that PTV SH8 is a new serotype, distinct from the new serotype PTV wild boar/WB2C-TV/2011/HUN, which clusters with PTV SH2, SH10, and SH25.

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.

Porcine teschovirus in wild boars in Hungary.

The genus Teschovirus, family Picornaviridae, currently includes 12 serotypes (PTV 1 to 12) isolated from swine. PTVs have been well studied in domestic pigs, but knowledge about PTVs in wild boars is deficient. Here, we report the first complete PTV genome sequence from 7 (70 %) of 10 fecal samples of wild boar piglets (Sus scrofa) by RT-PCR and pyrosequencing. Analysis of the wild boar PTV strain WB2C-TV/2011/HUN (JQ429405) showed considerable difference, especially in VP1 (66-74 % amino acid identity) compared to the available PTVs. PTV is present in wild boars, and WB2C-TV/2011/HUN represents a novel PTV genotype, provisionally named PTV-13.

Phylogeny and evolution of porcine teschovirus 8 isolated from pigs in China with reproductive failure.

A porcine teschovirus (PTV) was isolated from a dead piglet from a herd of 200 sows showing reproductive failure in Fuyu, Heilongjiang Province, China. Sequencing of most of the genome of this isolate, designated Fuyu/2009, revealed that is was a PTV-8 isolate, closely related to a previously identified Chinese PTV-8 strain (Jilin/2003). Both Chinese strains vary from the European PTV-8 strains by different clustering of the 3CD sequences. Infection with these viruses may be associated with pronounced diseases.

Analyzing the genetic diversity of teschoviruses in Spanish pig populations using complete VP1 sequences.

Porcine teschoviruses (PTVs) have been previously shown to be the most abundant cytopathic viruses found in swine feces. In the present study, the diversity of PTVs was studied, using PTV isolates collected between 2004 and 2009 in a wide territory in Spain. In order to characterize genetically the isolates, phylogeny reconstructions were made using maximum likelihood and Bayesian inference methods, based on the 1D (VP1) gene, and including sequences available in public databases. The phylogenetic trees obtained indicated that PTVs present 12 main lineages, 11 corresponding to the PTV serotypes described to date, and one lineage distinct from the rest. The geographic distribution of the different lineages does not seem to be strongly associated to particular territories, and co-circulation of multiple lineages was found in the same geographic areas. Nevertheless, some spatial structuring of the viral populations studied is indicated by the differences found between Spanish samples with respect to other European countries. A coalescent-based approach indicated that mutation may have been the main factor in originating the genetic diversity observed in the VP1 gene region. This study revealed a high diversity of teschoviruses circulating in the pig populations studied, and showed that molecular analysis of the complete VP1 protein is a suitable method for the identification of members of the porcine teschovirus group. However, further analyses are needed to clarify the geographical structuring of the different PTV populations.

Evaluation of a fluorogenic real-time reverse transcription-polymerase chain reaction method for the specific detection of all known serotypes of porcine teschoviruses.

Performance of a real-time reverse-transcription polymerase chain reaction method for the rapid, simple and reliable detection of porcine teschovirus (PTV) was assessed. The method was based on the use of a set of oligonucleotides consisting of two specific primers and a fluorogenic TaqMan-MGB probe. Reverse transcription and PCR reactions were performed sequentially in one step. As a result the whole procedure was simple and rapid, taking less than 3h for completion. The method reacted in a dose-dependent manner with prototype strains for the eleven known PTV serotypes (PTV1-11), with higher analytical sensitivity than other gel-based RT-PCR methods described, which were performed in parallel to allow for a comparison. The assay did not cross-react with other related viruses or porcine viruses tested. The diagnostic performance of the method was analyzed using a panel of field samples consisting of pig fecal and pig slurry samples. As a conclusion, this technique is adequate and convenient for porcine teschovirus detection, both for diagnosis as well as in environmental investigations.

A survey of porcine picornaviruses and adenoviruses in fecal samples in Spain.

In the course of an epidemiologic surveillance program for swine diseases carried out in Spain, 206 cytopathic viruses were isolated from 600 porcine fecal samples between 2004 and 2005. The virus isolates were examined using reverse transcription polymerase chain reaction (RT-PCR) methods specific for different types of porcine picornaviruses, including members of the Teschovirus, Enterovirus, and Sapelovirus genera, and PCR for porcine adenoviruses. Of the 206 isolates, 97 (47%) were identified as teschoviruses, 18 (9%) as sapeloviruses, and 7 (3%) as porcine adenoviruses. Neither Porcine enterovirus B nor Swine vesicular disease virus was found among the isolates. The present study confirms that teschoviruses are highly prevalent in porcine fecal samples, at least in Spain. It also reveals that these viruses commonly circulate among apparently healthy pigs.

Isolation of serotype 2 porcine teschovirus in China: evidence of natural recombination.

Porcine teschovirus (PTV), the pathogen of porcine polioencephalomyelitis, is a member of the family Picornaviridae. In this study, a new PTV strain (designated as JF613) was isolated from pigs in China. It was confirmed by the specific CPE on susceptible cells, RT-PCR and nucleotide sequencing. Analysis of its amino acids sequence of complete polyprotein indicated that the isolate belongs to serotype 2. Genetic recombination is a well-known phenomenon for picornavirus which has been demonstrated in many other members of the family, but it remains so far unclear whether recombination occurs in PTV. To detect possible recombination events, 30 sequences of complete coding regions of PTV strains accessible in GenBank were examined. Putative recombinant sequence was identified with the use of SimPlot program. The result showed that the genomic sequence of our isolate exhibited highest similarities with strains of serotypes 2 and 5, respectively, in two crossover regions, suggesting the recombination event in PTV. Then the mosaic structure of viral genome was confirmed by bootscanning and genetic algorithm for recombination detection (GARD). This represents the first PTV-2 isolate in China. Furthermore, our study provided the first evidence of natural recombination in PTV and indicated that homologous recombination may be a driving force in PTV evolution.

Isolation and molecular characterization of a porcine teschovirus 1 isolate from China.

Porcine teschovirus 1 (PTV-1) (Swine/CH/IMH/03) was isolated from piglets in a farm in Inner Mongolia Province, P.R. China. It was confirmed by electron microscopy, RT-PCR, and sequencing. Comparison of the sequences of the amino acid and nucleotides and phylogenetic analysis of the polyprotein showed that PTV Swine/CH/IMH/03 strain is PTV-1. The isolated virus has closest relationship with Talfan strain, they shared 98.9% and 99.5% homology of amino acids and nucleotides, respectively, in the ORF of polyprotein. To our knowledge, this is the first report about isolation and identification of a PTV in China.

Antigenic properties of porcine teschovirus 1 (PTV-1) Talfan strain and molecular strategy for serotyping of PTVs.

For reliable diagnosis of porcine teschovirus (PTV) infection we created an RT-PCR-based molecular strategy for serotyping that encompassed the dominant neutralizing antigenic site of PTV, followed by phylogenetic analyses of amplicons. We identified neutralizing antigenic sites of PTV-1 Talfan strain through epitope mapping of neutralizing monoclonal antibodies (MAbs), using synthetic peptides spanning the capsid proteins. All 11 MAbs obtained recognized peptides in the EF loop ("puff") of VP2 protein. Two MAbs concurrently reacted to peptides, one in the GH loop of VP1 and one in the VP1 C terminus. Three-dimensional modeling of Talfan capsid protein predicted exposure of all these sites on the virion surface in a close line centered around puff. We then designed a single pair of degenerate primers to VP2 and amplified the region of approximately 320 bp encompassing puff in 8 PTV prototype strains and 6 field isolates. Phylogenetic analyses of the puff sequences of 11 prototype strains and 34 field isolates obtained from databanks showed that all homotypic strains (both field and prototype) were always monophyletic, except for one 'untypable' Japanese strain. This RT-PCR-based strategy appears to be a reliable surrogate for serotyping and could facilitate the diagnosis and epidemiological study of PTV infection.