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Porcine sapeloviruses, teschoviruses of family Picornaviridae and type 3 porcine astroviruses of family Astroviridae are (re-)emerging enteric pathogens that could be associated with severe, disseminated infections in swine, affecting multiple organs including the central nervous system (CNS). Furthermore, small-scale pioneer studies indicate the presence of these viruses in porcine nasal samples to various extents. The laboratory diagnostics are predominantly based on the detection of the viral RNA from faecal and tissue samples using different nucleic-acid-based techniques such as RT-qPCR. In this study, a novel highly sensitive one-step triplex RT-qPCR assay was introduced which can detect all known types of neurotropic sapelo-, tescho- and type 3 astroviruses in multiple types of samples of swine. The assay was evaluated using in vitro synthesized RNA standards and a total of 142 archived RNA samples including known sapelo-, tescho- and type 3 astrovirus positive and negative CNS, enteric and nasal specimens. The results of a large-scale epidemiological investigation of these viruses on n = 473 nasal swab samples from n = 28 industrial-type swine farms in Hungary indicate that all three neurotropic viruses, especially type 3 astroviruses, are widespread and endemically present on most of the investigated farms.

In a fattening farm in southern Germany, paralysis of the hind limbs was observed in 2 age groups (50 kg as well as 60 kg) during a 4 week period. Despite a low morbidity of 3.3 % the majority of the affected animals needed to be euthanized in consequence to the progression of their hind limb paralysis. During pathomorphological examinations of 2 affected fattening pigs severe lymphohistiocytic meningoencephalomyelitis and vasculitis were detected. Immunhistochemistry revealed the presence of Porcine Teschovirus antigen in all parts of the central nervous system as well as in several cell types (neurons, glia cells, endothelial cells, mononuclear cells). Porcine Teschovirus was detected by PCR in spinal cord samples. The subsequently performed phylogenetic analysis PCR revealed a close relation (88 % full genome sequence) to porcine Teschovirus A11 strain "Dresden". Other swine relevant pathogens were excluded by PCR, bacteriologic examination and sequencing. Following a period of 4 weeks no additional cases of hind limb paralysis were observed in the fattening farm., Competing Interests: Die Autoren bestätigen, dass kein Interessenskonflikt in wirtschaftlicher oder persönlicher Hinsicht bezüglich der vorliegenden Arbeit besteht., (Thieme. All rights reserved.)

Catalase is one of the most abundant enzymes on Earth. It decomposes hydrogen peroxide, thus protecting cells from dangerous reactive oxygen species. The catalase-encoding gene is conspicuously absent from the genome of most representatives of the family Trypanosomatidae. Here, we expressed this protein from the Leishmania mexicana Β-TUBULIN locus using a novel bicistronic expression system, which relies on the 2A peptide of Teschovirus A . We demonstrated that catalase-expressing parasites are severely compromised in their ability to develop in insects, to be transmitted and to infect mice, and to cause clinical manifestation in their mammalian host. Taken together, our data support the hypothesis that the presence of catalase is not compatible with the dixenous life cycle of Leishmania , resulting in loss of this gene from the genome during the evolution of these parasites.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, is one of the biggest threats to public health. However, the dynamic of SARS-CoV-2 infection remains poorly understood. Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing recombinant (r)SARS-CoV-2 in the locus of the open reading frame (ORF)7a protein have jeopardized their use to monitor the dynamic of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the highly expressed viral nucleocapsid (N) gene followed by a porcine tescherovirus (PTV-1) 2A proteolytic cleavage site. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and excised lungs or whole organism of infected K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice. Importantly, real-time viral infection was readily tracked using a noninvasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2, in which a viral gene was not deleted, not only retained wild-type (WT) virus-like pathogenicity in vivo but also exhibited high stability in vitro and in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis, and therapeutic interventions for the treatment of SARS-CoV-2 in vivo., Competing Interests: Competing interest statement: J.-G.P., J.J.K., M.R.W., and L.M.-S. are coinventors on a patent that includes claims related to some of the NAbs described., (Copyright © 2021 the Author(s). Published by PNAS.)

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., (© 2021. Sociedade Brasileira de Microbiologia.)

Porcine teschovirus (PTV), sapelovirus (PSV-A), and enterovirus (EV-G) are enteric viruses that can infect pigs and wild boars worldwide. The viruses have been associated with several diseases, primarily gastrointestinal, neurologic, reproductive, and respiratory disorders, but also with subclinical infections. However, for most serotypes, proof of a causal relationship between viral infection and clinical signs is still lacking. In Switzerland, there has been limited investigation of the occurrence of the 3 viruses. We used a modified multiplex reverse-transcription PCR protocol to study the distribution of the viruses in Swiss pigs by testing 363 fecal, brain, and placental or abortion samples from 282 healthy and diseased animals. We did not detect the 3 viruses in 94 placental or abortion samples or in 31 brain samples from healthy pigs. In brain tissue of 81 diseased pigs, we detected 5 PSV-A and 4 EV-G positive samples. In contrast, all 3 viruses were detected at high frequencies in fecal samples of both healthy and diseased pigs. In healthy animals, PTV was detected in 47%, PSV-A in 51%, and EV-G in 70% of the 76 samples; in diseased animals, frequencies in the 81 samples were 54%, 64%, and 68%, respectively. The viruses were detected more frequently in fecal samples from weaned and fattening pigs compared to suckling piglets and sows. Co-detections of all 3 viruses were the most common finding. Based on clinical and pathology data, statistical analysis yielded no evidence for an association of virus detection and disease. Further research is required to determine if pathogenicity is linked to specific serotypes of these viruses.

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.

The segmented 18.5-kbp dsRNA genome of rotavirus expresses 6 structural and 6 nonstructural proteins. We investigated the possibility of using the recently developed plasmid-based rotavirus reverse genetics (RG) system to generate recombinant viruses that express a separate heterologous protein in addition to the 12 viral proteins. To address this, we replaced the NSP3 open reading frame (ORF) of the segment 7 (pT7/NSP3) transcription vector used in the RG system with an ORF encoding NSP3 fused to a fluorescent reporter protein (i.e., UnaG, mRuby, mKate, or TagBFP). Inserted at the fusion junction was a teschovirus translational 2A stop-restart element designed to direct the separate expression of NSP3 and the fluorescent protein. Recombinant rotaviruses made with the modified pT7/NSP3 vectors were well growing and generally genetically stable, and they expressed NSP3 and a separate fluorescent protein detectable by live cell imaging. NSP3 made by the recombinant viruses was functional, inducing nuclear accumulation of cellular poly(A)-binding protein. Further modification of the NSP3 ORF showed that it was possible to generate recombinant viruses encoding 2 heterologous proteins (mRuby and UnaG) in addition to NSP3. Our results demonstrate that, through modification of segment 7, the rotavirus genome can be increased in size to at least 19.8 kbp and can be used to produce recombinant rotaviruses expressing a full complement of viral proteins and multiple heterologous proteins. The generation of recombinant rotaviruses expressing fluorescent proteins will be valuable for the study of rotavirus replication and pathogenesis by live cell imagining and suggest that rotaviruses will prove useful as expression vectors. IMPORTANCE Rotaviruses are a major cause of severe gastroenteritis in infants and young children. Recently, a highly efficient reverse genetics system was developed that allows genetic manipulation of the rotavirus segmented double-stranded RNA genome. Using the reverse genetics system, we show that it is possible to modify one of the rotavirus genome segments (segment 7) such that virus gains the capacity to express a separate heterologous protein in addition to the full complement of viral proteins. Through this approach, we have generated wild-type-like rotaviruses that express various fluorescent reporter proteins, including UnaG (green), mRuby (far red), mKate (red), and TagBFP (blue). Such strains will be of value in probing rotavirus biology and pathogenesis by live cell imagining techniques. Notably, our work indicates that the rotavirus genome is remarkably flexible and able to accommodate significant amounts of heterologous RNA sequence, raising the possibility of using the virus as a vaccine expression vector., (Copyright © 2020 American Society for Microbiology.)

Antibody Fab fragments consist of heavy chain (Hc) and light chain (Lc) polypeptides assembled with a disulphide bond. The production of a recombinant Fab fragment requires the simultaneous expression of two genes encoding both an Hc and an Lc in the same host cell. In the present study, we investigated the production of Fab fragments in lepidopteran insect cells using a bicistronic plasmid vector carrying the Hc and Lc genes linked with a 2A self-cleaving peptide sequence from the porcine teschovirus-1. We also examined the arrangement of a GSG spacer sequence and a furin cleavage site sequence with the 2A sequence. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) of culture supernatants showed that Trichoplusia ni BTI-TN-5B1-4 (High Five) cells transfected with a plasmid in which the Hc and Lc genes were joined by the 2A sequence successfully secreted Fab fragments with antigen-binding activity after self-cleavage of the 2A peptide. The GSG linker enhanced 2A cleavage efficiency, and the furin recognition site was useful for removal of 2A residues from the Hc. Transfection with a single plasmid that contained sequences for GSG, the furin cleavage site, GSG, and the 2A peptide between the Hc and Lc genes exhibited a higher productivity than co-transfection with a set of plasmids separately carrying the Hc or Lc gene. These results demonstrate that bicistronic expression with the appropriate combination of a furin recognition site, GSG linkers, and a 2A peptide may be an effective way to efficiently produce recombinant antibody molecules in insect cells., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

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., (© FEMS 2020.)

Several emerging viral agents related to gastroenteritis are distributed in human and animal populations and may contaminate the environment due to anthropic activities. The objective of this study was to analyze the seasonal contamination by enteric virus and coliforms in water from streams in the Vale do Taquari, draining a large number of pig farms. Microbiological contamination was evidenced by the detection of total and thermotolerant coliforms, reaching their peak in December. Hepatitis E virus (HEV), Enterovirus-G (EV-G) genome, and Sapelovirus-A (SV-A) genome were not detected. On the other hand, Rotavirus (RV) was detected in 3% (1/32) of the samples, whereas Teschovirus-A (PTV) was detected in 6% (2/32). This is the first detection of PTV in environmental samples in Brazil, pointing that the virus is being shedded from swine herds to watersheds. Human mastadenovirus (HAdV) was the most frequent detected viral agent in 9.3% (3/32) with values of 2.54 × 10 5 , 7.13 × 10 4 , and 3.09 × 10 5 genome copies/liter (gc/L). The circulation of coliforms and viral pathogens is noticeable due to anthropic activities and to the management of animal waste from the pig farming. In this way, enteric viruses can assist in monitoring the quality of watersheds and in tracking sources of contamination.

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.

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.

Conventionally, Porcine teschovirus (PTV) consists of 13 genotypes (PTV 1-13, which belong to Teschovirus A); however, several novel members including PTV 14-22 have been discovered recently. PTV 21 was identified as a novel Teschovirus species named Teschovirus B. In this study, almost all 22 reported PTV genotypes except PTV 6, 7, 12 and 20 were identified in the pig populations of western Jiangxi, China, which reflects the high genotype diversity. Moreover, to the best of our knowledge, the nearly complete genome of PTV 22-JiangX1 was first sequenced in the present study. The homology comparison of the polyprotein genes showed that PTV 22-JiangX1 shared a relatively high nucleotide and deduced amino acid sequence identities ranging from 78.3% to 82.0% and 84.6% to 89.3%, respectively, with PTV 19 and 21. Additionally, the PTV strain of JiangX1 represents genotype 22 with the PTV 19, and 21 strains proved to be members of Teschovirus B based on the phylogenetic and evolutionary divergence analyses. Finally, we determined that the novel Teschovirus B species comprises at least three PTV genotypes., (© 2019 Blackwell Verlag GmbH.)

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.

Multicistronic elements, such as the internal ribosome entry site (IRES) and 2A-like cleavage sequence, serve crucial roles in the eukaryotic ectopic expression of exogenous genes. For utilization of multicistronic elements, the cleavage efficiency and order of elements in multicistronic vectors have been investigated; however, the dynamics of multicistronic element-mediated expression remains unclear. Here, we investigated the dynamics of encephalomyocarditis virus (EMCV) IRES- and porcine teschovirus-1 2A (p2A)-mediated expression. By utilizing real-time fluorescent imaging at a minute-level resolution, we monitored the expression of fluorescent reporters bridged by either EMCV IRES or p2A in two independent cultured cell lines, HEK293 and Neuro2a. We observed significant correlations for the two fluorescent reporters in both multicistronic elements, with a higher correlation coefficient for p2A in HEK293 but similar coefficients for IRES-mediated expression and p2A-mediated expression in Neuro2a. We further analyzed the causal relationship of multicistronic elements by convergent cross mapping (CCM). CCM revealed that in all four conditions examined, the expression of the preceding gene causally affected the dynamics of the subsequent gene. As with the cross correlation, the predictive skill of p2A was higher than that of IRES in HEK293, while the predictive skills of the two multicistronic elements were indistinguishable in Neuro2a. To summarize, we report a significant temporal correlation in both EMCV IRES- and p2A-mediated expression based on the simple bicistronic vector and real-time fluorescent monitoring. The current system also provides a valuable platform to examine the dynamic aspects of expression mediated by diverse multicistronic elements under various physiological conditions.

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.

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., (Copyright © 2018. Published by Elsevier B.V.)

Porcine teschovirus (PTV) comprises at least 13 genotypes (PTV 1-13). Here, the genotypes of field strains prevalent among pig populations in Hunan Province, China, were identified. Multiple PTV genotypes, including all genotypes except PTV 7 and 8, were found co-circulating in the pig populations, reflecting a high genetic diversity. Moreover, we identified nine novel PTV genotypes, provisionally designated as PTV 14-22. PTV 21-HuN41 and PTV 21-HuN42 were successfully isolated, and their nearly complete genomes were sequenced. Homology comparison of the polyprotein genes of PTV 21-HuN41-42 to those of other known PTVs revealed low identities, ranging from 70.1 to 71.9 % (nucleotide identity) and 75.4 to 77.6 % (amino acid identity). Moreover, PTV 21-HuN41-42 were identified as a novel teschovirus species (tentatively Teschovirus B), based on the analyses of phylogenetics and evolutionary divergence. The findings of this study are expected to greatly enrich our knowledge of PTV genotypes.

Background: Porcine teschoviruses (PTVs) are small non-enveloped viruses with single-stranded, positive sense genomic RNA, belonging to the family Picornaviridae. Natural infections of teschoviruses are limited to pigs., Results: In this study, a PTV HuN-1 was found that it could be proliferated in PK-15 cell, and it came from the pig fecal samples from Hunan province, in central China. The complete genome of the HuN-1 was amplified by RT-PCR and sequenced. The complete genome of HuN-1 isolate is 7098 nt, which shares the highest sequence identity (85.9%) with the PTV 8 strain of Jilin/2003/2 and Fuyu/2009/2. The HuN-1 isolate contains only one ORF (from 320 to 7039 nt) coding a 2240 amino acid polyprotein. Aligned sequences show that more mutations occurred in the structural region than in the nonstructural region. Phylogenetic analysis showed that HuN-1 isolate did not clustered with the hitherto reported strains, according to P1 sequences, forming a subgroup in the PTV cluster., Conclusion: In this study, complete genome of PTV HuN-1 was cloned and sequenced. Detection and characterization of further PTV strains from different geographic areas are important to understand the worldwide distribution and heterogeneity (serotype) of PTVs and their association with symptomatic infections in pigs.

Porcine teschoviruses (PTVs) have been shown to be widely distributed in pig populations. In this study, 261 faecal and 91 intestinal content samples collected from pigs at 29 farms in Hunan, China, were tested for the presence of PTV by reverse transcription-polymerase chain reaction (RT-PCR). An overall PTV-positivity rate of 19.03% was detected by RT-PCR, and a high PTV infection rate was circulating in asymptomatic fattening and nursery pigs. In total, 40 PTV isolates (PTV-HuNs) were obtained. Alignment of their coding sequences with those of other known PTVs revealed that the genomic sequence of the polyprotein contains 6,606-6,621 nucleotides, encoding a 2,202-2,207-amino acid sequence. Phylogenetic analyses based on the VP1 gene and capsid protein gene exhibited 13 main lineages corresponding to PTV serotypes 1-13, and seven PTV serotypes (PTV 2-6, 9, and 11) were identified in the isolates obtained in our study; this is the first report of PTV 5, 9 and 11 in China. Recombination analysis among the PTV-HuNs indicated that nine recombination events have occurred, including both inter- and intraserotype events. In addition, results demonstrated that only limited positive selection is acting on the global population of PTV isolates, and purifying selection is predominant. In conclusion, this study revealed a high infection rate of PTVs circulating in asymptomatic fattening and nursery pigs. The 40 PTV-HuNs showed high genetic diversity, and genetic analysis of all available PTV sequences revealed that strong purifying selection and recombination play important roles in the genetic diversity and evolution of the virus., (© 2017 Blackwell Verlag GmbH.)

Autophagy is a homeostatic process that has been shown to be vital in the innate immune defense against pathogens. However, little is known about the regulatory role of autophagy in porcine teschovirus 2 (PTV-2) replication. In this study, we found that PTV-2 infection induces a strong increase in GFP-LC3 punctae and endogenous LC3 lipidation. However, PTV-2 infection did not enhance autophagic protein degradation. When cellular autophagy was pharmacologically inhibited by wortmannin or 3-methyladenine, PTV-2 replication increased. The increase in virus yield via autophagy inhibition was further confirmed by silencing atg5, which is required for autophagy. Furthermore, PTV-2 replication was suppressed when autophagy was activated by rapamycin. Together, the results suggest that PTV-2 infection activates incomplete autophagy and that autophagy then inhibits further PTV-2 replication.

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.

There are concerns about the zoonotic transmission of viruses through undercooked pork products. There is a lack of information on suitable indicator viruses for fecal contamination with pathogenic enteric viruses in the meat processing chain. The study compared the incidence and levels of contamination of hog carcasses with F-coliphages, porcine teschovirus (PTV), and porcine adenovirus (PAdV) at different stages of the dressing process to assess their potential as indicator viruses of fecal contamination. One hundred swab samples (200cm 2 ) were collected from random sites on hog carcasses at 4 different stages of the dressing process and from retail pork over the span of a year from 2 pork processing plants (500/plant). Viable F-coliphages, PAdV DNA and PTV RNA were each detected on ≥99% of the incoming carcasses at both plants and were traceable through the pork processing chain. Significant correlations were observed between viable F-coliphages and PAdV DNA and between F-coliphages and PTV RNA but not between PAdV DNA and PTV RNA at the various stages of pork processing. Detection of viable F-coliphages was more sensitive than genomic copies of PAdV and PTV at low levels of contamination, making F-coliphages a preferred indicator in the pork slaughter process as it also provides an indication of infectivity. For plant A, F-RNA coliphages were detected in 25%, 63%, and 21% of carcass swabs after pasteurization, evisceration, and retail pork products, respectively. For plant B, F-coliphages were detected in 33%, 25%, and 13% of carcass swabs after skinning, evisceration, and retail pork samples, respectively. Viable F-RNA coliphages were genotyped. Viable F-RNA GII and GIII were generally not detected at the earlier stages of the slaughter process but they were detected on 13% of carcasses after evisceration and 2% of retail pork samples at plant A, which raises concerns of potential food handler contamination during pork processing. Consumers could be at risk when consuming undercooked meat contaminated with pathogenic enteric viruses., (Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.)

The "self-cleaving" 2A sequence of picornavirus, which mediates ribosome-skipping events, enables the generation of two or more separate peptide products from one mRNA containing one or more "self-cleaving" 2A sequences. In this study, we introduced a single 2A sequence of porcine teschovirus-1 (P2A) linked to two fluorescent protein genes, the enhanced yellow fluorescent protein (EYFP) gene and the red fluorescent protein (RFP) gene, in a single cassette into transgenic Eimeria tenella (EtER). As expected, we obtained two separated protein molecules rather than a fused protein, although the two molecules were translated from the same mRNA carrying a single "self-cleaving" 2A sequence. Importantly, RFP led by a secretion signal was secreted into parasitophorous vacuoles, while EYFP localized mainly to the nucleus of EtER. Our results demonstrate that the "self-cleaving" 2A sequence actively mediated cleavage of polyproteins in the apicomplexan parasite E. tenella.

To study the outcome of natural selection using phylogenetic trees, we analyzed full-length genome sequences of porcine teschovirus (PTV). PTV belongs to the family Picornaviridae and has a positive-stranded RNA genome, the replication of which is carried out by the error-prone viral RNA-dependent RNA polymerase. The viral RNA encodes a single polyprotein that is cleaved into structural (i.e., L, VP4, VP2, VP3 and VP1) and nonstructural proteins (i.e., 2A, 2B, 2C, 3A, 3B, and 3C). A high degree of genetic diversity was found based on the pairwise nucleotide distances and on the mean ratio of the number of nonsynonymous (dN) and synonymous (dS) substitutions (dN/dS) in the structural genes. Conversely, the diversity of the nonstructural genes was lower. The differences in genetic diversity between the structural and nonstructural genomic regions were likely due to strong purifying selection; consequently, the estimates of phylogenies were also discordant among these genes. In particular, maximum-likelihood and Bayesian methods generated short-branched trees when loci that are under strong purifying selection were used. These findings indicate that even in an RNA virus with an intrinsically high mutation rate, a strong purifying selection will curb genetic diversity and should be considered an important source of bias in future studies based on phylogenetic methods.

Porcine teschoviruses (PTVs) belong to the genus Teschovirus within the family Picornaviridae. PTVs are universal contaminants in pig herds in endemic and multi-infection statuses. Previous research has demonstrated PTV antigens and nucleic acid in renal glomeruli and tubular epithelia, suggesting the possibility that PTVs might be shed and transmitted via urine. The study aimed to demonstrate, in the context of pathogenesis, the presence of PTVs in the urine of naturally infected pigs. Viral loads of fluid and tissue samples quantified by an established qRT-PCR showed detection rates of 100% by head and in urine, feces, plasma and nasal swabs, and 38% in kidney. As predicted, PTVs were present in urine at 10(4.02 ± 1.45) copies/100 μl volume, equivalent to 17% of that in plasma. No significant differences were observed between healthy and culled pigs or among the 7 sampled herds. The presence of PTVs in urine was further substantiated by molecular serotyping. In particular, PTV-10 was identified in the urine of 3 piglets from 3 separate herds, consistent with the most prevalent serotype found in this study, and in plasma. The urine mixes with feces to form slurry making it easier for PTV to spread and contaminate the environment., (Copyright © 2015 Elsevier B.V. All rights reserved.)

No commercial vaccines are currently available for enterovirus 71 (EV71) infection. Oral virus-like particle (VLP) vaccines are regarded as a better choice for prevention from food-borne diseases compared with injected whole virus vaccines. Unfortunately, the application of oral VLP vaccines produced from transgenic plants was limited due to the concerns of gene contamination. Alternatively, using transgenic mushrooms retains the advantages of transgenic plants and tremendously reduce risks of gene contamination. Polycistronic expression vectors harboring the glyceraldehyde-3-phospho-dehydrogenase promoter to codrive EV71 structural protein P1 and protease 3C using the 2A peptide of porcine teschovirus-1 were constructed and introduced into Flammulina velutipes via Agrobacterium tumefaciens-mediated transformation. The analyses of the genomic PCR, Southern blotting, and RT-PCR showed that the genes of P1 and 3C were integrated into the chromosomal DNA through a single insertion, and their resulting mRNAs were transcribed. The Western blotting analysis combined with LC-MS/MS demonstrated that EV71 VLPs were composed of the four subunit proteins digested from P1 polyprotein by 3C protease. Through the use of a single particle electron microscope, images of 1705 particles with diameter similar to the EV71 viron were used for 3D reconstruction. Protrusions were observed on the surface in the 2D class averages, and a 3D reconstruction of the VLPs was obtained. In conclusion, EV71 VLPs were successfully produced in transgenic F. velutipes using a polycistronic expression strategy, which indicates that this approach is promising for the development of oral vaccines produced in mushrooms.

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.

Marek's disease virus (MDV) is a growing threat for the poultry industry. Unfortunately, despite successful vaccination against the disease, MDV remains in circulation within vaccinated flocks, leading to the selection of increasingly virulent pathotypes. Detailed knowledge of the virus biology and the host-virus interaction is required to improve the vaccine efficiency. In the present study, I engineered an original, dual-reporter MDV to track and quantify virus replication in vitro and in vivo., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Increasing attention is being paid to the impact of agricultural activities on water quality to understand the impact on public health. F-RNA coliphages have been proposed as viral indicators of fecal contamination while porcine teschovirus (PTV) and porcine adenovirus (PAdV) are proposed indicators of fecal contamination of swine origin. Viruses and coliphages are present in water in very low concentrations and must be concentrated to permit their detection. There is little information comparing the effectiveness of the methods for concentrating F-RNA coliphages with concentration methods for other viruses and vice versa. The objective of this study was to compare 5 current published methods for recovering F-RNA coliphages, PTV and PAdV from river water samples concentrated by electronegative nitrocellulose membrane filters (methods A and B) or electropositive Zeta Plus 60S filters (methods C-E). Method A is used routinely for the detection of coliphages (Méndez et al., 2004) and method C (Brassard et al., 2005) is the official method in Health Canada's compendium for the detection of viruses in bottled mineral or spring water. When river water was inoculated with stocks of F-RNA MS2, PAdV, and PTV to final concentrations of 1×10(6) PFU/100 mL, 1×10(5) gc/100 mL and 3×10(5) gc/100 mL, respectively, a significantly higher recovery for each virus was consistently obtained for method A with recoveries of 52% for MS2, 95% for PAdV, and 1.5% for PTV. When method A was compared with method C for the detection of F-coliphages, PAdV and PTV in river water samples, viruses were detected with higher frequencies and at higher mean numbers with method A than with method C. With method A, F-coliphages were detected in 11/12 samples (5-154 PFU/100 mL), PTV in 12/12 samples (397-10,951 gc/100 mL), PAdV in 1/12 samples (15 gc/100 mL), and F-RNA GIII in 1/12 samples (750 gc/100 mL) while F-RNA genotypes I, II, and IV were not detected by qRT-PCR., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

Expression of multiple reporter or effector transgenes in the same cell from a single construct is increasingly necessary in various experimental paradigms. The discovery of short, virus-derived peptide sequences that mediate a ribosome-skipping event enables generation of multiple separate peptide products from one mRNA. Here we describe methods and vectors to facilitate easy production of polycistronic-like sequences utilizing these 2A peptides tailored for expression in Drosophila both in vitro and in vivo. We tested the separation efficiency of different viral 2A peptides in cultured Drosophila cells and in vivo and found that the 2A peptides from porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) worked best. To demonstrate the utility of this approach, we used the P2A peptide to co-express the red fluorescent protein tdTomato and the genetically-encoded calcium indicator GCaMP5G in larval motorneurons. This technique enabled ratiometric calcium imaging with motion correction allowing us to record synaptic activity at the neuromuscular junction in an intact larval preparation through the cuticle. The tools presented here should greatly facilitate the generation of 2A peptide-mediated expression of multiple transgenes in Drosophila.

Teschovirus and Sapelovirus are two genera of the Picornaviridae family, comprising highly variable and heterogeneous enteric viruses, commonly found in faecal samples from domestic pigs. Although both of them are also known to infect wild boar, studies on their presence in these wild suids are scarce. The present study aimed at determining the presence of porcine teschovirus (PTV) and sapelovirus (PSV) in free-living wild boar populations, as well as to study their relationships with similar viruses present in pigs. Fresh faecal samples (n=63) from wild boar were collected in Doñana Biological Reserve (SW Spain) during 2007 and 2011, and analysed using multiplex RT-PCR for the simultaneous detection and differentiation of PTV and PSV. A total of 32 samples (50.8%) presented positive PTV bands, while PSV amplicons were detected in 4 samples (6.4%). All PSV-positive samples were also positive for PTV, which indicated co-infection with both viruses. Virus isolation was successful from 6 samples, 4 of which were identified as PTV by RT-PCR, and three of these were further characterized by sequencing of the VP1 capsid protein. The remaining two isolates were negative for PTV or PSV. Genetic characterization of PSV-positive faecal samples, using the VP4 protein coding gene, was successful in 4 stool samples. Close phylogenetic relationship was found among wild boar and domestic pig strains in both PTV and PSV. More studies are needed to ascertain the epizootiological significance of these findings., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Simultaneous expression of multiple target genes is often required in biotechnology. Multicistronic vectors coding for several proteins are being actively developed for this purpose. In commercially available vectors different variants ofencephalomyocarditis virus internal ribosome entry site (IRES EMCV) are used most often. However, many researchers consider that utilization ofself-cleaving 2A peptides sequences within multi- and bicistronic vectors is more promising. In this work, we compared the efficiency of gene expression in cells transfected with bicistronic vectors based on IRES EMCV and 2A peptide sequence derived form porcine teschovirus-1 (P2A). Efficiency ofgene expression was determined in three mammalian cell lines by measurement of co-expression levels of genes coding for RFP and EGFP proteins linked by IRES or P2A sequence. Higher level oftransgene expression was exhibited by cells transfected with the vector containing the 2A peptide sequence.

A multiplex PCR assay was developed and evaluated for its ability to simultaneously detect three viral infections of swine. Specific primers were carefully selected from articles published for each of the following three viruses: porcine circovirus type II (PCV2), porcine teschovirus (PTV) and porcine transmissible gastroenteritis virus (TGEV). Each target produced a specific amplicon with a size of 353 bp (PCV2), 168 bp (PTV) and 499 bp (TGEV). The sensitivity of the multiplex PCR using purified plasmid constructs containing the specific viral target fragments was 6.60 × 10(2), 8.43 × 10(2) and 7.30 × 10(2) copies for PCV2, PTV and TGEV, respectively. Among 127 samples which were collected from Heilongjiang, Jilin, Henan and Guangxi provinces, the single infection of PCV2, PTV and TGEV was 99.21, 46.88 and 65.35 %, respectively, and co-infection of the three viruses was 26.77 %. In conclusion, the multiplex PCR has the potential to be useful for routine molecular diagnosis and epidemiology.

A real-time reverse transcription polymerase chain reaction (RT-PCR) based on TaqMan was established and evaluated for quantitative detection of porcine teschoviruses (PTVs). A pair of primers and a TaqMan probe targeting on the highly conserved sequence of the 5'-untranslated region (5'-UTR) of one to 11 serotypes of PTV were designed. Standard plasmid DNA containing PCR amplification of the 5'-UTR were constructed and used to develop the real-time RT-PCR. The results indicated that the real-time RT-PCR was specific for detection of PTV with a detection limit of 10 copies/μL, but not for porcine parvovirus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, pseudorabies virus, classical swine fever virus. The coefficient of variation of inter-assay and intra-assay were less than 3 %. A total of 91 clinical samples were tested by the real-time RT-PCR and virus isolation (OIE 2008) and positive rates were 79.12 % (72/91) and 57.14 % (48/91), respectively. In conclusion, the developed real-time RT-PCR assay was an effective method for detection and quantification of PTV in fields or organs of infected pigs.

Porcine teschoviruses (PTVs) belong to the genus Teschovirus within the family Picornaviridae. Hitherto, PTVs have had 13 serotypes associated with a variety of clinical diseases. The virulent PTV-1 strains were associated with highly fatal, nonsuppurative encephalomyelitis of pigs (Teschen disease) in the 1930-1950s. Today, less virulent Talfan strains of PTV-1 are more widespread, and PTVs have contaminated swine herds worldwide (endemic or enzootic) together with a variety of common swine pathogens (multi-infection status). The aim of this study was to investigate the extent to which PTVs play a role in causing diseases in the field, under the endemic and multi-infection situation, when most pigs in the herds are infected and immune. Based on the fecal-oral model of pathogenesis, a set of 15 organs were collected from 30 culled post-weanling piglets of 4-8 weeks old. For nested RT-PCR targeted on the 5'-NTR, the PTV detection rate was 96.7% (by heads), confirming the endemic status, and infection was most commonly detected in the intestines (averaged 61%) and lymphoid organs (averaged 59%), followed by visceral organs (averaged 37%) and the CNS (different parts varied from 17 to 47%). The correlation of PTVs detected by nested RT-PCR and a histological lesion were analyzed by Chi-square test showing that in the field situation only non-suppurative encephalitis in the caudal part of the brain (P=0.054) may be marginal significantly attributed to infection by PTVs. By genotyping based on partial VP1 sequences, 5 serotypes, namely PTV-1, -4, -6, -7, and -11, were identified, with some animals having two serotypes co-existed in different organs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

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.

In February and March 2009, approximately 1,500 backyard pigs of variable age became sick, and approximately 700 of them died or were euthanized in the Lower Artibonite Valley and the Lower Plateau of the Republic of Haiti. The main clinical sign was posterior ataxia followed by paresis and/or paralysis on the second or third day of illness. No gross lesions were observed at postmortem examinations. The morbidity and mortality were approximately 60% and 40%, respectively. Diagnostic samples (whole blood, brain, tonsil, lymph nodes, spleen, and lung) were negative for Classical swine fever virus and African swine fever virus. Porcine teschovirus type 1 was detected by reverse transcription polymerase chain reactions in brain samples. Results of virus isolation, electron microscopy of virus particles, histopathological analysis on brain tissues, nucleic acid sequencing, and phylogenetic analysis of the viral isolate supported the diagnosis of teschovirus encephalomyelitis. The outbreak of the disease in Haiti is the first appearance of the severe form of teschovirus encephalomyelitis in the Americas. This disease poses a potential threat to the swine industries in other Caribbean countries, as well as to Central and North American countries.

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Loop-mediated isothermal amplification (LAMP) is a sensitive method for DNA amplification. In the present report, the development of a single-tube, one-step, real-time accelerated reverse transcription (RT)-LAMP for the detection of Porcine teschovirus (PTV) is described. Six designed primers amplified target gene sequences successfully at constant temperature (65 °C) within 1 hr, and the amplification results could be visualized directly by the naked eye. The sensitivity of the LAMP was 10 times higher than that of conventional polymerase chain reaction, and no cross-reactivity was found when the genomes of other common swine pathogens were subjected to the RT-LAMP system. When 43 clinical samples were tested by the RT-LAMP method, results indicated that the test is simple, rapid, accurate, and sensitive for the detection of PTV., (© 2011 The Author(s))

A multiplex RT-PCR (mRT-PCR) assay was developed and evaluated for its ability to detect multiple viral infections of swine simultaneously. One pair of primers was selected carefully for each of the following three RNA viruses: porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and porcine teschovirus (PTV). Each target produced a specific amplicon with a size of 451bp (PRRSV), 343bp (CSFV), or 163bp (PTV). The sensitivity of the mRT-PCR using purified plasmid constructs containing the specific viral target fragments was 2.02 x 10², 2.90 x 10³, and 6.16 x 10³ copies for PRRSV, CSFV, and PTV, respectively. Among 69 clinical samples from Heilongjiang, Jilin, and Henan provinces, co-infection by PRRSV and CSFV was 4.4%, co-infection by PRRSV and PTV was 11.6%, co-infection by PTV and CSFV was 13.0%, and co-infection by the three viruses was 8.7%. In conclusion, the mRT-PCR should be useful for routine molecular diagnosis and epidemiology., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Beginning in 2002, a small number of pig farms in western Canada began reporting 4-7-week-old pigs with bilateral hind-end paresis or paralysis. Low numbers of pigs were affected, some died, most had to be euthanized, and those that survived had reduced weight gains and neurological deficits. Necropsies revealed no gross lesions, but microscopic lesions consisted of a nonsuppurative polioencephalomyelitis, most severe in the brain stem and spinal cord. The lesions were most consistent with a viral infection. Tests for circovirus, Porcine reproductive and respiratory syndrome virus, coronavirus, Rabies virus, and Pseudorabies virus were negative. Using immunohistochemistry, virus neutralization, fluorescent antibody test, and nested reverse transcription polymerase chain reaction, Porcine teschovirus was identified in tissues from affected individuals. To the authors' knowledge, this is the first report of teschovirus encephalitis in western Canada and the first reported case of polioencephalomyelitis in pigs in Canada, where teschovirus was confirmed as the cause.

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Investigations were carried out to identify the causal agent of acute diarrhea, respiratory distress, and death of pigs on a swine farm in Jilin Province, northern China. Only porcine Teschovirus (PTV, designated as PTV-8 Jilin/2003) was isolated from samples of organs. The presence of PTV was confirmed by the production of a specific cytopathic effect on susceptible cells and by the results of the immunoperoxidase monolayer assay (IPMA), polymerase chain reaction, and electron microscopy. Other pathogenic agents causing diarrhea, respiratory distress, and death (including porcine rotavirus, transmissible gastroenteritis virus of swine, porcine epidemic diarrhea virus, classical swine fever virus, pseudorabies virus, porcine circovirus, porcine reproductive and respiratory syndrome virus, Japanese encephalitis virus, Mycoplasma, Leptospira, Streptococcus, Listeria, and Brucella species) were excluded as possible causal agents because they were not associated consistently with the disease of the pigs. PTV-8 Jilin/2003 was adapted to grow in swine primary kidney (PK-15) cells and in a swine testicular cell line (ST cells). When inoculated into healthy pigs, PTV-8 Jilin/2003 caused the same symptoms as those observed in the affected herd. It is concluded that PTV-8 Jilin/2003 was the causal agent of this disease., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Porcine teschovirus (PTV) was isolated in cell culture and/or demonstrated by polymerase chain reaction in samples of brain and/or spinal cord in pigs in Indiana during the 2002-2007 period. Testing was initiated on pigs originating from populations exhibiting nervous clinical disease and/or pigs with microscopic lesions in central nervous tissues, indicating viral encephalitis and/or myelitis. Virus was demonstrated in pigs with and without lesions as well as with and without nervous clinical disease. Nucleotide sequence analysis of the 5'-nontranslated region of the viral genome revealed that these isolates had low-level genetic heterogeneity but were homologous to porcine PTV serotype 1 (PTV-1). These findings indicate that low-to-moderate virulence strains of PTV with some homology to PTV-1 are endemic in many swineherds of Indiana and are associated with subclinical and clinical nervous disease in weaned pigs.

Translation of hepatitis C virus (HCV) genomic RNA is directed by an internal ribosome entry site (IRES) in the 5'-untranslated region (5'-UTR), and the HCV 3'-UTR enhances IRES activity. Since the HCV 3'-UTR has a unique structure among 3'-UTRs, we checked possible communication between the 5'- and the 3'-UTR of HCV during translation using chimeric reporter RNAs. We show that translation directed by the HCV IRES and by the HCV-like IRES of porcine teschovirus (PTV) which belongs to a quite distinct family of viruses (picornaviruses) or by the EMCV IRES is also enhanced by the HCV 3'-UTR or by a poly(A)-tail in different cell types., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)