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.

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.

Detection and molecular characterization of porcine enterovirus G15 and teschovirus from India

ABSTRACT 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.

Polycistronic gene expression in the model micro-organism Ustilago maydis

AbtractEukaryotic microorganisms transcribe monocistronic mRNAs to encode proteins. For synthetic biological approaches like metabolic engineering, precise co-expression of several proteins in space and time is advantageous. A straightforward approach is the application of viral 2A peptides to design synthetic polycistronic mRNAs in eukaryotes. Here, we establish such a system in the well-studied model microorganism Ustilago maydis. Using two fluorescence reporter proteins, we compared the activity of five viral 2A peptides. Their activity was evaluated in vivo using fluorescence microscopy and validated using fluorescence resonance energy transfer (FRET). Activity ranged from 20 to 100% and the best performing 2A peptide was P2A from porcine teschovirus-1. As proof of principle, we followed regulated gene expression efficiently over time and synthesised a tri-cistronic mRNA encoding biosynthetic enzymes to produce mannosylerythritol lipids (MELs). In essence, we evaluated 2A peptides in vivo and demonstrated the applicability of 2A peptide technology for U. maydis in basic and applied science.

Detection of atypical porcine pestivirus genome in newborn piglets affected by congenital tremor and high preweaning mortality1

Recently, piglets from a high-health status farm began exhibiting congenital tremors, high preweaning mortality and incidence of splayed legs. Postmortem histological examination identified a small number of scattered white matter vacuoles in the cerebellum and underlying brainstem of affected piglets. Presence of potential viral sources associated with this neurologic condition was initially infirmed using quantitative PCR for atypical porcine pestivirus (APPV), porcine teschovirus, and porcine sapelovirus. Using metagenomic analysis, APPV was identified as the main microbial species in serum obtained from piglets affected by congenital tremor. These piglets had higher preweaning mortality rates (46.4% vs. 15.3%) and incidence of splayed legs (33.0% vs. 0.8 %) compared to unaffected piglets. Piglets affected by congenital tremor had higher viral titer (P < 0.15) and larger birth weights (P < 0.05) compared to normal litter mates. Whole-genome sequencing and genome assembly of the novel APPV strain (MK728876) was carried out using Oxford Nanopore and related bioinformatics pipelines. Phylogenic analysis demonstrated that this strain along with other completely sequenced APPV strains were grouped into 2 clades, both including strains-inducing congenital tremor. Strains appear to cluster based on region but there were still significant differences within regions. Future research needs to address potential underdiagnosis due to genetic diversity but also to understand mode of transmission, variation in virulence, and the role of host genetics in APPV susceptibility.

Congenital Microphthalmic Syndrome in a Swine

A 17-week-old crossbred finishing pig was presented for lameness of approximately one week. Clinical evaluation, including ophthalmologic examination, revealed ataxia, partial flaccid paresis of the pelvic limbs, skin lesions at feet and claws, and severely reduced vision/blindness. Both eyes had multiple persistent pupillary membranes (iris-to-iris and iris-to-lens) and hypermature cataracts. Histopathological examination of the eyes revealed microphthalmia, microphakia with cataract formation, myovascularised membrane in the vitreous, retinal detachment, and retinal dysplasia. Microscopic examination of tissues collected postmortem demonstrated nonsuppurative polioencephalomyelitis with the most prominent inflammatory lesions in the lumbar spinal cord. Subsequently, presumed Teschen/Talfan disease was confirmed by porcine teschovirus identification in the spinal cord using the reverse transcription-polymerase chain reaction (RT-PCR). To the authors’ knowledge, this is the first case report describing in detail histopathological changes in the porcine congenital microphthalmic syndrome.