Teschovirus Research Articles

Genetic Characterization and Pathology of Porcine Teschovirus (PTV) in India

Background: The porcine teschovirus (PTV) is common swine pathogen which causes a wide range of illnesses ranging from asymptomatic infection to acute fatal encephalomyelitis, diarrhoea and pneumonia. Despite of its economical importance very limited studies are available on the pathology of PTV. The present study was conducted with the objective to investigate the PTV infection and associated pathology in piglets. Methods: A total of 78 piglets below 3 months of age were necropsied and representative tissue samples along with intestinal content were collected for histopathological examination and molecular investigation during April 2019 to November 2020. The 5' UTR region of PTV was amplified via RT-PCR and further confirmed by sequencing. Thereafter, genetic characterization of the Indian PTV isolate was done on the basis of 5' UTR gene. Result: A total of seven out of 78 cases were found positive for the PTV. On necropsy the intestinal wall was thinned and distended with yellowish coloured diarrhoeic content. Congestion of serosal and mucosal vessels along with severe meningeal congestion was observed. Microscopic lesions included congestion of mucosal vessels and destruction of villous structure of jejunum along with perivascular cuffing, focal gliosis, neuronophagia, congestion of meningeal and cerebral vessels and interstitial pneumonia. The phylogenetic analysis revealed that the isolate PTV/India/IVRI-381/2020 (MW022462) and PTV/India/IVRI-1093/2020 (MW022463) were clustered with PTV-13 strain wild boar/WB2C-TV/2011/HUN (JQ429405) and PTV-2 strain Vir 6711-12/83 (AF296107) of Hungary and Germany respectively. This study reported the genotypic and pathological investigation of PTV from Indian piglets. Further explorative surveillance along with studies in suitable cell lines and animal model will pave the way for better understanding of PTV among Indian pigs.

Antiviral properties of titanium nanoparticles and features of their influence on the morphology of virions

The search for new antiviral substances and the development of modern antiviral preparations and disinfectants is an important area of modern science. Nanoparticles (NPs) and nanomaterials (NMs) have received increasing attention from scientists as promising antiviral substances recently. Our study aimed to investigate the antiviral activity of citrate-stabilized titanium nanoparticles (Ti NPs) against Teschovirus A (TV-A) and Potato virus Y (PVY). The biological activity of PVY has been tested using cultivated tobacco plants (Nicotiana tabacum L.) and bell pepper plants (Capsicum annum L.). It has been found that injection of plants with the virus has led to the appearance of PVY infection symptoms with further death of the plants. The threshold limit value (TLV) for Ti NPs in embryonic pig kidney cell line culture is 12.5 µg/cm3. According to the prophylactic and treatment plots, Ti NPs have slight antiviral activity against TV-A strain Dniprovskyi 34, decreasing its titer according to both plots by 0.5 lg TCD50/cm3. However, according to the virucidal plot at TLV concentration, Ti NPs show high antiviral activity against TV-A strain Dniprovskyi 34, significantly decreasing its titer in embryonic pig kidney cell line culture by 4.46 lg TCD50/cm3. Ti NPs do not have antiviral activity against PVY, according to the prophylactic and treatment plots. Nevertheless, according to the virucidal plot Ti NPs showed high antiviral activity against PVY, inactivating the virus in the reaction mixture in vitro. According to the results of TEM, Ti NPs bind to virus particles. Virus particles with NPs attached to them, deformed and partially destroyed virus particles have been found in samples of virus-containing suspension, which were incubated with Ti NPs. We suggest that Ti NPs are useful as disinfectants to control viral infections, particularly TV-A and PVY.

The VP1 Protein of Porcine Teschovirus Inhibits the Innate Immune Response to Viral Infection by Blocking MDA5 Activation

Porcine teschovirus (PTV) can cause reproductive dysfunction and respiratory diseases, with high mortality rates for sick pigs. Among Picornaviridae family virus-encoded proteins, the VP1 structural protein is critical for viral immune evasion. However, whether PTV VP1 inhibits the type I interferon (IFN) response remains unknown. Here, it shows that the PTV VP1 protein significantly hinders the activation of NF-κB, impairing Sendai virus-induced expression of beta IFN (IFN-β). Further studies revealed that VP1 targets and interacts with the MDA5 factor in the RIG-I like receptors pathway. More importantly, the VP1 protein interacts with the caspase activation and recruitment domain and Hel domains of MDA5, blocking IFN-β expression. Our findings provide evidence about the VP1 protein of PTV hinders MDA5 activation and may represent a viral mechanism to escape the innate immune response.

Detection and Molecular Characterization of Porcine Teschoviruses in India: Identification of New Genotypes.

Porcine Teschoviruses (PTVs) are ubiquitous enteric viral pathogens that infect pigs and wild boars worldwide. PTVs have been responsible for causing the severe clinical disease (Teschen disease) to asymptomatic infections. However, to date, limited information is available on large-scale epidemiological data and molecular characterization of PTVs in several countries. In this study, we report epidemiological data on PTVs based on screening of 534 porcine fecal samples from different states of India and a RT-PCR based detection of PTVs shows a percent positivity of 8.24% (44/534). The PTV prevalence varied among different regions of the country with the highest detection rates observed in the state of Karnataka (38.1%). Phylogenetic analysis based on VP1 gene reveals the presence of PTV genotype 6 and 13 along with some unassigned novel genotypes which did not cluster with any of the established PTV genotypes (PTV 1-PTV 13). Indian PTV 6 strains are genetically closest to the Spanish strains (85.7-94.4%) whereas PTV 13 and novel genotype strains were found to be more similar to the Chinese strains (88.1-99.1%). Using recombination detection software, no Indian PTVs found to be recombinant on VP1 gene and selection pressure analysis revealed the purifying selection in the several sites of the VP1 gene of PTVs. The Bayesian analysis of Indian PTVs shows 1.16 × 10-4 substitution/site/year as the mean evolutionary rate. Further, isolation of the novel PTV strains from India and more detailed investigation much needed to know the evolutionary history of PTV strains circulating in porcine populations in India.

Identification of a Novel Porcine Teschovirus Subtype 19 within the Species Teschovirus A

Porcine teschovirus (PTV) is a member of the genus Teschovirus in the family Picornaviridae. Several novel PTVs in Teschovirus were identified in our previous study. To elucidate the taxonomic status of the species and subtypes, PTV-positive samples were inoculated into porcine kidney (PK-15) and swine testicular (ST) cells. A total of 58 PTV strains were successfully isolated and 21 different VP1 gene sequences were obtained. A phylogenetic analysis of the VP1 gene revealed that these PTV isolates comprised 11 genotypes, including 10 known genotypes (PTV 1–6, PTV 8–9, PTV 11, and Teschovirus B3), and a newly identified genotype. A nearly complete genome sequence of the novel PTV genotype isolate, PTV-SG2, was obtained. The homologies of the nucleotides and amino acids between PTV-SG2 and other PTVs were 69.7%–85.0% and 76.1%–90.4%, respectively. A phylogenetic analysis of the P1, polyprotein, and 3 CD genes revealed that PTV-SG2 belonged to a novel genotype within Teschovirus A, and a genetic divergence analysis of the novel PTV isolate further verified its taxonomic status. Based on the PTV naming convention, the novel PTV genotype identified in the present study was provisionally named PTV 19. Serum neutralization tests indicated that PTV 19 is a serotype-specific and completely different from previously reported PTV serotypes. Therefore, PTV 19 is a novel PTV serogroup. In conclusion, this study identified a PTV genotype and serotype within Teschovirus A.

Metatranscriptomic assessment of diarrhoeic faeces reveals diverse RNA viruses in rotavirus group A infected piglets and calves from India.

RNA viruses are a major group contributing to emerging infectious diseases and neonatal diarrhoea, causing morbidity and mortality in humans and animals. Hence, the present study investigated the metatranscriptomic-derived faecal RNA virome in rotavirus group A (RVA)-infected diarrheic piglets and calves from India. The viral genomes retrieved belonged to Astroviridae in both species, while Reoviridae and Picornaviridae were found only in piglets. The nearly complete genomes of porcine RVA (2), astrovirus (AstV) (6), enterovirus G (EVG) (2), porcine sapelovirus (PSV) (2), Aichivirus C (1), and porcine teschovirus (PTV) (1) were identified and characterised. In the piglet, AstVs of PAstV2 (MAstV-26) and PAstV4 (MAstV-31) lineages were predominant, followed by porcine RVA, EVG, PSV, Aichivirus C, teschovirus (PTV-17) in decreasing order of sequence reads. In contrast, AstV accounted for the majority of reads in bovines and belonged to MAstV-28 and a proposed MAstV-35. Both RVA G4P[6] strains exhibited prototype Gottfried strains like a genotypic constellation of G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1. Ten out of eleven genes were of porcine origin, while the VP7 gene clustered with G4-lineage-1, consisting of human strains, suggesting a natural porcine-human reassortant. In the recombination analysis, multiple recombination events were detected in the PAstV4 and PAstV2 genomes, pointing out that these viruses were potential recombinants. Finally, the study finds diverse RNA virome in Indian piglets and calves for the first time, which may have contributed to diarrhoea. In the future, the investigation of RNA virome in animals will help in revealing pathogen diversity in multifactorial diseases, disease outbreaks, monitoring circulating viruses, viral discovery, and evaluation of their zoonotic potential.

Analysis on the genome of a teschovirus type 1 isolates with swine diarrhea

Porcine Teschoviruses (PTVs) are associated with polioencephalomyelitis and various diseases, including reproductive and gastrointestinal disorders of pigs and wild boars, but rarely detected in the feces of pigs. In this study, a sample of swine diarrhea that tested positive for PTVs is subjected to high-throughput sequencing. The viral genome was 7221 nucleotides (nt) in length, which was consisted of twelve genes. Phylogenetic analysis showed and it was closely related to the PTV-HNMY(MG755212.1). The nucleotide homology of VP1 gene of PTVs JS2021 with PTV-1AF 296102.1 reached 82.97%, belonging to a branch of PTV-1 serotype. The nucleotide homology of VP1 protein with other serotypes of PTV is quite different from that of other serotypes of PTV. Bioinformatics analysis showed that PTVs have four capsid proteins, namely VP1, VP2, VP3 and VP4. The VP1 encodes a 29 kDa protein, which is the main protective antigen, a theoretical isoelectric point of 6.73, no transmembrane domain, no signal peptide and potential phosphorylation site. The VP1 protein is an unstable hydrophilic intracellular protein, which contains four secondary structures: irregular curl (c), extended chain (e), α-helix (h) and β-folded (t). The tertiary structure is heart-shaped and has multiple B cell epitopes. By analyzing the tertiary structure, we found that the amino acid at position 129 of VP1 mutated and reduction a larger alpha helix. This may lead to the main cause of piglet diarrhea. These findings enriched our knowledge of the viruses in the role of swine diarrhea, and help to develop an effective strategy for disease prevention and control.

Identification of a novel porcine Teschovirus 2 strain as causative agent of encephalomyelitis in suckling piglets with high mortality in China

BackgroundPorcine Teschovirus (PTV), also named Teschovirus A, is prevalent in pig populations, mainly causing neurological symptoms, diarrhea, pneumonia, and reproductive failure, however the morbidity and mortality are usually low in pig farms.Case presentationIn this study, we reported a PTV outbreak investigation in one large-scale pig farm in China with severe symptoms including diarrhea, lethargy, locomotor ataxia, nystagmus, paralysis of the hind limbs, and coma in piglets. More importantly, the mortality reached 38% in suckling pigs, which is remarkably high in PTV history. A novel PTV strain, named HeNZ1, was isolated from cerebral samples of one suckling pig and the genome sequence was obtained by NGS sequencing. Phylogenetic and evolutionary divergence analyses revealed that HeNZ1 belongs to PTV genotype 2. Surprisingly, the VP1 coding region of HeNZ1 shares the highest sequence similarity with European PTV-2 strains, instead of China domestic PTV-2 strains, implying it may not derive from China local PTV-2 strains. Multiple sequence alignment and B cell epitope prediction of PTV VP1 and VP2 protein revealed 10 B cell epitopes, 5 mutant clusters and 36 unique mutation sites, of which 19 unique mutation sites are located in B cell epitopes and exposed on the surface of VP1 or VP2, implying significant antigenic drift potential of HeNZ1.ConclusionThese results indicate that HeNZ1 is a highly virulent PTV-2 strain, which capable of causing severe neurological symptoms and high mortality in piglets. Bioinformatic analysis suggest that HeNZ1 is genetically and antigenically different from other Chinese PTV-2 strains. Overall, current case expanded our understanding of PTV-2 clinical spectrum and revealed the emergence of a highly virulent PTV-2 strain with substantial genetic diversity and antigenic drift potential in VP1 and VP2.

Protease-Independent Production of Poliovirus Virus-like Particles in Pichia pastoris: Implications for Efficient Vaccine Development and Insights into Capsid Assembly.

The production of enterovirus virus-like particles (VLPs) that lack the viral genome have great potential as vaccines for a number of diseases, such as poliomyelitis and hand, foot, and mouth disease. These VLPs can mimic empty capsids, which are antigenically indistinguishable from mature virions, produced naturally during viral infection. Both in infection and in vitro, capsids and VLPs are generated by the cleavage of the P1 precursor protein by a viral protease. Here, using a stabilized poliovirus 1 (PV-1) P1 sequence as an exemplar, we show the production of PV-1 VLPs in Pichia pastoris in the absence of the potentially cytotoxic protease, 3CD, instead using the porcine teschovirus 2A (P2A) peptide sequence to terminate translation between individual capsid proteins. We compare this to protease-dependent production of PV-1 VLPs. Analysis of all permutations of the order of the capsid protein sequences revealed that only VP3 could be tagged with P2A and maintain native antigenicity. Transmission electron microscopy of these VLPs reveals the classic picornaviral icosahedral structure. Furthermore, these particles were thermostable above 37°C, demonstrating their potential as next generation vaccine candidates for PV. Finally, we believe the demonstration that native antigenic VLPs can be produced using protease-independent methods opens the possibility for future enteroviral vaccines to take advantage of recent vaccine technological advances, such as adenovirus-vectored vaccines and mRNA vaccines, circumventing the potential problems of cytotoxicity associated with 3CD, allowing for the production of immunogenic enterovirus VLPs in vivo. IMPORTANCE The widespread use of vaccines has dramatically reduced global incidence of poliovirus infections over a period of several decades and now the wild-type virus is only endemic in Pakistan and Afghanistan. However, current vaccines require the culture of large quantities of replication-competent virus for their manufacture, thus presenting a potential risk of reintroduction into the environment. It is now widely accepted that vaccination will need to be extended posteradication into the foreseeable future to prevent the potentially catastrophic reintroduction of poliovirus into an immunologically naive population. It is, therefore, imperative that novel vaccines are developed which are not dependent on the growth of live virus for their manufacture. We have expressed stabilized virus-like particles in yeast, from constructs that do not require coexpression of the protease. This is an important step in the development of environmentally safe and commercially viable vaccines against polio, which also provides some intriguing insights into the viral assembly process.

Construction and characterization of the full-length cDNA of an infectious clone of emerging porcine teschovirus-2.

Porcine teschovirus (PTV) is a causative agent of polioencephalomyelitis, encephalomyelitis, reproductive disorders and gastrointestinal and respiratory diseases in swine. In the present study, the PTV2 GX/2020 strain was isolated from pig intestinal tissue through the use of ST cells. Phylogenetic analysis of VP1 nucleotide sequences indicated that the GX/2020 isolate is closely related to PTV2. Furthermore, the full-length cDNA of an infectious GX/2020 clone was constructed using seamless ligation technology. The genome sequence of the rescued virus is largely consistent with the sequence of the parental virus, and it exhibits viral growth properties. The PTV2 virus was successfully isolated in the present study, and the reverse-genetic platform provides a foundation for studies of the pathogenic mechanisms of porcine teschovirus.

Antiviral Properties of Cerium Nanoparticles

According to the results of the study, the threshold limit values (TLVs) of CeO2, and Ce nanoparticles (NPs) for the culture of swine kidney embryonic cell line (SKECL) have been estimated. The TLVs are 0.1 μg/cm3 for Ce NPs, and 1 μg/cm3 for CeO2 NPs. All NPs were non-toxic for white mice at concentration of 2000 mg/kg, which opens up a perspective for the further use of these NPs in the development of antiviral substances. CeO2, and Ce NPs have decreased the titer of Teschovirus by 1.46-2 lg10 TCD50/ cm3 at TLVs at all stages of virus reproduction. Sensitivity of the strain Dniprovskyi-34 of species Teschovirus A, serotype Porcine teschovirus 1 (PTV-1), to solvents, proteolytic enzyme trypsin under the presence of CeO2, and Ce NPs has been studied. Under the presence of solvents, and trypsin, NPs decreased the infectious titer of the virus slightly by only 0.23 lg10 TCD50/cm3. CeO2, and Ce NPs have not shown antiviral activity under heating to 50 °C, either with the addition of 1 M solution of MgCl2 or without it, and did not cause significant decrease of infectious activity of the virus under pH values 2.0, 7.2, and 11.0 within 10 minutes exposition. Ability of CeO2, and Ce NPs to interact with viral particles, change their morphology, which can affect infective activity of the virus significantly, has been shown. Addition of CeO2, and Ce NPs to the antigen has caused the decrease of the titer of virus-neutralizing antibodies, though it remained high. The results of the studies can be used in development of disinfectants, and antiviral drugs.

Development and Large-Scale Testing of a Novel One-Step Triplex RT-qPCR Assay for Simultaneous Detection of "Neurotropic" Porcine Sapeloviruses, Teschoviruses (Picornaviridae) and Type 3 Porcine Astroviruses (Astroviridae) in Various Samples including Nasal Swabs.

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.

Hind limb paralysis in fattening pigs due to a new strain of porcine Teschovirus A11

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.

Detection of porcine enteric picornaviruses from faecal samples of Indian pigs.

The online version contains supplementary material available at 10.1007/s13337-022-00756-0.

COLLECTION OF THE STRAINS TESCHOVIRUS A, SAPELOVIRUS A, ENTEROVIRUS G OF THE INSTITUTE OF AGRICULTURAL MICROBIOLOGY AND AGROINDUSTRIAL MANUFACTURE OF THE NAAS

Objective. Arrangement of the collection of porcine enteroviruses (PEV) strains isolated on the territory of Ukraine in accordance with the requirements of the International Committee on Virus Taxonomy and supplementing it with new strains Teschovirus A (TV-A), Sapelovirus A (SV-A), Enterovirus G (EV-G). Methods. Virological, serological, molecular genetic, instrumental and statistical. Isolation, cultivation of viruses and determination of their biological activity were performed in passaged culture of porcine embryonic kidney cells (СНЕВ). The viral titre was calculated by the method of Reed and Muench. The typical affiliation of viruses was determined in the virus neutralization reaction in СНЕВ cell culture. Species affiliation was determined by reverse transcription polymerase chain reaction (RT PCR) using species-specific primers for TV-A, SV-A and EV-G, developed by us. Electron microscopy of viruses was performed on a transmission electron microscope by negative contrast enhancement method. Statistical processing was performed in Microsoft Office Excel and StatSoft STATISTICA 12. Results. As a result of the epizootic survey during 2002– 2019, 1,216 samples for virological testing were selected. Successive passages of СНЕВ cell culture resulted in obtaining 274 viral isolates. According to the results of studying physicochemical, morphological, biological properties of these isolated, they are classified as PEV. In connection with the change of taxonomy and nomenclature of PEV, serological and genetic reclassification of 30 strains of viruses isolated in Ukraine, including 14 reference strains according to the classification of V. P. Romanenko, 7 production strains, 9 strains with polyantigenic properties and 4 strains that did not have antigenic affinity with viruses of known PEV serotypes according to the classification of V. P. Romanenko was performed. It has been established that the reference strains of PEV according to the trivial classification of V. P. Romanenko belong to the species TV-A of the genus Teschovirus. As a result of conducted serological testing, PEV-10 M 2323, PEV-12 K 22, PEV-13 L 90, PEV-14 M 116, PEV-16 G 95, PEV-17 V 111, PEV-18 Ch 184, PEV-19 D 227, PEV-20 I 249, PEV-23 I 393 were classified as TV-A1; PEV-11 K 9, PEV-15 Ch 73 — as TV-A3, PEV-22 V 151 — as TV-A6. PEV-21 P 142 did not have antigen affinity with reference strains TV-A, SV-A and EV-G and belongs to a new serotype. Production strains of PEV-1 Perechynskyi 642, Bereznianskyi 652, Chernihivskyi 2372 were reclassified as TV-A1. PEV strains with polyantigenic properties such as G 31 and L 2661 have intertypic antigens with TV-A 1, 10, 11 and TV-A 3, 6, 10 serotypes, respectively. PEV strain of a new serotype Ch 881 was reclassified as SV-A. PEV strains Т 3, Ch 863, Ch 878 are the new serotypes of TV-A. Conclusion. As a result of studies, 274 viral isolates were isolated from 1,216 samples of material. The collection was supplemented with 20 reference strains of Teschovirus A, Sapelovirus A and Enterovirus G. Genetic and serological reclassification of 30 PEV strains isolated in Ukraine was performed. The collection of viral strains has been arranged in accordance with modern taxonomy and nomenclature. Seven viral strains were deposited. The collection of viruses has been supplemented with 4 strains of new serotypes of Teschovirus A.

THE FEASIBILITY OF CONSTRUCTING A PORCINE TESCHOVIRUS INFECTIOUS cDNA CLONE TAGGED WITH iLOV FLUORESCENT PROTEIN RESCUED AS A VISIBLE MARKER VIRUS

Viral vectors serve as promising tools for the development of novel multivalent or multipathogen vaccines. Poliovirus (of the family Picornaviridae) vectors offer the advantages of small genome size, ease of manipulation, inherent stability in the intestinal tract, and induction of potent mucosal immunity through oral administration. Porcine teschovirus (PTV), also belonging to Picornaviridae, generally causes asymptomatic infections in pigs. PTV’s wide tissue tropism suggests that it can act as a potential vector for vaccine development; however, no infectious PTV cDNA clone has been reported yet. In this study, infectious PTV cDNA was cloned and recombinant porcine teschovirus (rPTV) was constructed with a unique XhoI site introduced into 2A, as well as substitution of the G–H loop sequence “RNNQIPQDF” of VP1 by an 8-histidine marker which helps to differentiate it from the parental PTV. Subsequently, the coding sequence of a small fluorescent protein, iLOV, was incorporated into the XhoI site to rescue the recombinant rPTV-iLOV virus, allowing for direct visualization of the viral infection. These rescued viruses were replication-competent and antigenically identical to the parental virus, but showed attenuation due to an impaired self-cleaving function caused by the insertion of iLOV into the 2A protease site, as assessed by a double reporter expressing system. This rescued recombinant virus shows potential for the development of attenuated vaccines with few safety concerns and may serve as an important tool to visually study virus–cell interactions.

Exploring the Cause of Diarrhoea and Poor Growth in 8-11-Week-Old Pigs from an Australian Pig Herd Using Metagenomic Sequencing.

Diarrhoea and poor growth among growing pigs is responsible for significant economic losses in pig herds globally and can have a wide range of possible aetiologies. Next generation sequencing (NGS) technologies are useful for the detection and characterisation of diverse groups of viruses and bacteria and can thereby provide a better understanding of complex interactions among microorganisms potentially causing clinical disease. Here, we used a metagenomics approach to identify and characterise the possible pathogens in colon and lung samples from pigs with diarrhoea and poor growth in an Australian pig herd. We identified and characterized a wide diversity of porcine viruses including RNA viruses, in particular several picornaviruses—porcine sapelovirus (PSV), enterovirus G (EV-G), and porcine teschovirus (PTV), and a porcine astrovirus (PAstV). Single stranded DNA viruses were also detected and included parvoviruses like porcine bocavirus (PBoV) and porcine parvovirus 2 (PPV2), porcine parvovirus 7 (PPV7), porcine bufa virus (PBuV), and porcine adeno-associated virus (AAV). We also detected single stranded circular DNA viruses such as porcine circovirus type 2 (PCV2) at very low abundance and torque teno sus viruses (TTSuVk2a and TTSuVk2b). Some of the viruses detected here may have had an evolutionary past including recombination events, which may be of importance and potential involvement in clinical disease in the pigs. In addition, our metagenomics data found evidence of the presence of the bacteria Lawsonia intracellularis, Brachyspira spp., and Campylobacter spp. that may, together with these viruses, have contributed to the development of clinical disease and poor growth.

Porcine teschovirus, sapelovirus, and enterovirus in Swiss pigs: multiplex RT-PCR investigation of viral frequencies and disease association.

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.

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.

A Highly Conserved Epitope (RNNQIPQDF) of Porcine teschovirus Induced a Group-Specific Antiserum: A Bioinformatics-Predicted Model with Pan-PTV Potential.

Porcine teschovirus (PTV) is an OIE-listed pathogen with 13 known PTV serotypes. Heterologous PTV serotypes frequently co-circulate and co-infect with another swine pathogen, causing various symptoms in all age groups, thus highlighting the need for a pan-PTV diagnostic tool. Here, a recombinant protein composed of a highly conserved “RNNQIPQDF” epitope on the GH loop of VP1, predicted in silico, and a tandem repeat of this epitope carrying the pan DR (PADRE) and Toxin B epitopes was constructed to serve as a PTV detection tool. This recombinant GST-PADRE-(RNNQIPQDF)n-Toxin B protein was used as an immunogen, which effectively raised non-neutralizing or undetectable neutralizing antibodies against PTV in mice. The raised antiserum was reactive against all the PTV serotypes (PTV–1–7) tested, but not against members of the closely related genera Sapelovirus and Cardiovirus, and the unrelated virus controls. This potential pan-PTV diagnostic reagent may be used to differentiate naturally infected animals from vaccinated animals that have antibodies against a subunit vaccine that does not contain this epitope or to screen for PTV before further subtyping. To our knowledge, this is the first report that utilized in silico PTV epitope prediction to find a reagent broadly reactive to various PTV serotypes.