Detection of porcine enteric viruses (Kobuvirus, Mamastrovirus and Sapelovirus) in domestic pigs in Corsica, France

Many enteric viruses are found in pig farms around the world and can cause death of animals or important production losses for breeders. Among the wide spectrum of enteric viral species, porcine Sapelovirus (PSV), porcine Kobuvirus (PKoV) and porcine Astrovirus (PAstV) are frequently found in pig feces. In this study we investigated sixteen pig farms in Corsica, France, to evaluate the circulation of three enteric viruses (PKoV, PAstV-1 and PSV). In addition to the three viruses studied by RT–qPCR (908 pig feces samples), 26 stool samples were tested using the Next Generation Sequencing method (NGS). Our results showed viral RNA detection rates (i) of 62.0% [58.7–65.1] (n = 563/908) for PSV, (ii) of 44.8% [41.5–48.1] (n = 407/908) for PKoV and (iii) of 8.6% [6.8–10.6] (n = 78/908) for PAstV-1. Significant differences were observed for all three viruses according to age (P-value = 2.4e–13 for PAstV-1; 2.4e–12 for PKoV and 0.005 for PSV). The type of breeding was significantly associated with RNA detection only for PAstV-1 (P-value = 9.6e–6). Among the 26 samples tested with NGS method, consensus sequences corresponding to 10 different species of virus were detected. This study provides first insight on the presence of three common porcine enteric viruses in France. We also showed that they are frequently encountered in pigs born and bred in Corsica, which demonstrates endemic local circulation.

Epidemiology of Astrovirus, Norovirus and Sapovirus in Greek pig farms indicates high prevalence of Mamastrovirus suggesting the potential need for systematic surveillance

Backround Astrovirus, Norovirus and Sapovirus exhibit a wide distribution in swine pig herds worldwide. However, the association of porcine Astrovirus (PAstV), porcine Norovirus (PoNoV) and porcine Sapovirus (PoSaV) with disease in pigs remains uncertain. In this study, we investigated the prevalence of PAstV, PoNoV and PoSaV in Greek pig farms using both conventional RT-PCR and SYBR-Green Real-time RT-PCR in an effort to compare the sensitivity of the two methods. We examined 1400 stool samples of asymptomatic pigs originating from 28 swine farms throughout Greece in pools of five. Results PAstV was detected in all 28 swine farms examined, with an overall prevalence of 267/280 positive pools (95.4%). Porcine Caliciviruses prevalence was found at 36 and 57 out of the 280 examined samples, by the conventional and SYBR-Green Real time RT-PCR, respectively. Sequencing and phylogenetic analysis of the positive samples revealed that the detected PAstV sequences are clustered within PAstV1, 3 and 4 lineages, with PAstV3 being the predominant haplotype (91.2%). Interestingly, sequencing of the Calicivirus positive samples demonstrated the presence of non-target viruses, i.e. Sapovirus, Kobuvirus and Sapelovirus sequences and one sequence highly similar to bat Astrovirus, while no Norovirus sequence was detected. Conclusions The high prevalence of PAstV in Greek pig farms poses a necessity for further investigation of the pathogenicity of this virus and its inclusion in surveillance programs in case that it proves to be important. To our knowledge, this is the first epidemiological study of these viruses in pig farms in Greece.

Molecular Detection and Characterization of Porcine Astrovirus on the basis of Partial ORF1b/ORF2 Region

The porcine astrovirus (PAstV) is distributed globally and exists as five distinct lineages (PAstV1-PAstV5). PAstV is considered one of the important pathogen associated with diarrhea among pigs. In the present study, the PAstV was detected in 13.4% (19/141) of fecal samples including 14.4% (16/111) diarrheic and 10% (3/30) non-diarrheic samples by RT-PCR based on partial ORF1b/ORF2 gene from Haryana, India. The results indicated that the weaning piglets were more susceptible to PAstV infection followed by suckling piglets. The phylogenetic analysis of the viral strains revealed the circulation ofPAstV4 (55.5%) and PAstV2 (44.4%) lineages with PAstV4 being the predominant lineage. To conclude, RT-PCR screening followed by sequencing of PAstV revealed high genetic diversity among the PAstV strains suggesting the wide range of heterogeneity and possible recombination events of viral strains in the state.

Dynamics of the Enteric Virome in a Swine Herd Affected by Non-PCV2/PRRSV Postweaning Wasting Syndrome

A commercial pig farm with no history of porcine circovirus 2 (PCV2) or porcine reproductive and respiratory syndrome virus (PRRSV) repeatedly reported a significant reduction in body weight gain and wasting symptoms in approximately 20–30% of the pigs in the period between three and six weeks after weaning. As standard clinical interventions failed to tackle symptomatology, viral metagenomics were used to describe and monitor the enteric virome at birth, 3 weeks, 4 weeks, 6 weeks, and 9 weeks of age. The latter four sampling points were 7 days, 3 weeks, and 6 weeks post weaning, respectively. Fourteen distinct enteric viruses were identified within the herd, which all have previously been linked to enteric diseases. Here we show that wasting is associated with alterations in the enteric virome of the pigs, characterized by: (1) the presence of enterovirus G at 3 weeks of age, followed by a higher prevalence of the virus in wasting pigs at 6 weeks after weaning; (2) rotaviruses at 3 weeks of age; and (3) porcine sapovirus one week after weaning. However, the data do not provide a causal link between specific viral infections and the postweaning clinical problems on the farm. Together, our results offer evidence that disturbances in the enteric virome at the preweaning stage and early after weaning have a determining role in the development of intestinal barrier dysfunctions and nutrient uptake in the postweaning growth phase. Moreover, we show that the enteric viral load sharply increases in the week after weaning in both healthy and wasting pigs. This study is also the first to report the dynamics and co-infection of porcine rotavirus species and porcine astrovirus genetic lineages during the first 9 weeks of the life of domestic pigs.

Molecular Identification and Characterization of a Genotype 3 Hepatitis E Virus (HEV) Strain Detected in a Wolf Faecal Sample, Italy

Hepatitis E virus (HEV) infection is a major health problem worldwide. In developed countries, zoonotic transmission of HEV genotypes (Gt) 3 and 4 is caused by the ingestion of raw or undercooked meat of infected pigs and wild boars, the main reservoirs of HEV. However, additional animals may harbour HEV or HEV-related strains, including carnivores. In this study, we investigated the molecular epidemiology of orthohepeviruses in wild canids by screening a total of 136 archival faecal samples, collected from wolves (42) and red foxes (94) in Northwestern Italy. Orthohepevirus RNA was identified in a faecal specimen, collected from a wolf carcass in the province of La Spezia (Liguria Region, Italy). The nearly full-length (7212 nucleotides) genome of the strain HEV/81236/Wolf/2019/ITA (GenBank accession no. MZ463196) was determined by combining a sequence-independent single-primer amplification (SISPA) approach with the Oxford Nanopore Technologies sequencing platform. Upon phylogenetic analysis, the HEV detected in wolf was segregated into clade HEV-3.1, displaying the highest nucleotide (nt) identity (89.0–93.3%) to Gt3 strains belonging to subtype c. Interestingly, the wolf faecal sample also contained porcine astrovirus sequences, endorsing the hypothesis of a dietary origin of the HEV strain due to preying habits.

Case Report: First Report and Phylogenetic Analysis of Porcine Astroviruses in Chile

Porcine Astrovirus (PoAstV) causes mild diarrhea in young pigs and is considered an emerging virus in the swine industry worldwide. PoAstV has high genetic diversity and has been classified into five genetic lineages, PoAstV1–5. In Chile, only human astroviruses have been reported. This study aimed to determine the presence and genetic diversity of PoAstV circulating in intensive pig farms in Chile. Seventeen Chilean intensive swine farms from Valparaíso, Metropolitana, O'Higgins, Ñuble and Araucanía regions were sampled. A selection of oral fluid and fecal material samples from 1–80 days-old pigs were collected and analyzed using next-generation sequencing. The circulation of PoAstV was confirmed in all studied farms. We obtained complete or partial sequences of PoAstV-2 (n = 3), PoAstV-4 (n = 2), and PoAstV-5 (n = 7). In 15 out of 17 farms, we detected more than one lineage co-circulating. Phylogenetic analyses grouped the seven PoAstV-5 strains in a monophyletic cluster, closely related to the United States PoAstV-5 strains. The three PoAstV-2 were located into two separate sub-clusters. PoAstV-4 sequences are also grouped in two different clusters, all related to Japanese strains. Thus, our results indicate that PoAstV circulates in Chile with high frequency and diversity. However, the lack of reference sequences impairs local evolution patterns establishment and regional comparisons. This is the first contribution of PoAstV genomes in Latin America; more studies are needed to understand the diversity and impact of PoAstV on swine health.

Insertion of Exogenous Genes within the ORF1a Coding Region of Porcine Astrovirus

A tagged or reporter astrovirus can be a valuable tool for the analysis of various aspects of the virus life cycle, and to aid in the development of genetically engineered astroviruses as vectors. Here, transposon-mediated insertion mutagenesis was used to insert a 15-nucleotide (nt) sequence into random sites of open reading frame 1a (ORF1a) based on an infectious full-length cDNA clone of porcine astrovirus (PAstV). Five sites in the predicted coiled-coil structures (CC), genome-linked protein (VPg), and hypervariable region (HVR) in ORF1a of the PAstV genome were identified that could tolerate random 15 nt insertions. Incorporation of the commonly used epitope tags, His, Flag, and HA, into four of the five insertion sites permitted the production of infectious viruses and allowed recognition by specifically tagged monoclonal antibodies. The results of immuno-fluorescent assays showed that Flag-tagged ORF1a protein overlapped partially with capsid and ORF2b proteins in the cytoplasm. Improved light-oxygen-voltage (iLOV) gene was also introduced at the insertion sites of CC, VPg, and HVR. Only one viable recombinant reporter PAstV expressing iLOV inserted in HVR was recovered. Biological analysis of the reporter virus showed that it displayed similar growth characteristics, and yet produced less infectious virus particles, when compared with the parental virus. The recombinant virus carrying the iLOV fused with the HVR of ORF1a protein maintained its stability and showed green fluorescence after 15 passages in cell cultures. The resultant fluorescently tagged virus could provide a promising tool for the rapid screening of antiviral drugs as well as allowing the visualization of PAstV infection and replication in living cells.

Experimental porcine astrovirus type 3-associated polioencephalomyelitis in swine

Porcine astrovirus type 3 (PoAstV3) is an emerging virus in the family Astroviridae that has been recently associated with polioencephalomyelitis/encephalitis. Herein, we describe the experimental oral and intravenous inoculation of an infectious central nervous system (CNS) tissue homogenate containing PoAstV3 to cesarean-derived, colostrum-deprived pigs, and the subsequent development of clinical signs, histologic lesions, specific humoral immune response, and detection of viral particles by electron microscopy (EM) and viral RNA by RT-qPCR (reverse transcriptase quantitative polymerase chain reaction) and in situ hybridization (ISH). IgG against a portion of the PoAstV3 ORF2 capsid was first detected at 7 days post-inoculation (DPI) in 2 of 4 inoculated animals and in all inoculated animals by 14 DPI. At 21 and 28 DPI, 2 of 4 inoculated animals developed ataxia, tetraparesis, and/or lateral recumbency. All inoculated animals had histologic lesions in the CNS including perivascular lymphoplasmacytic cuffs, multifocal areas of gliosis with neuronal necrosis, satellitosis, and radiculoneuritis, and PoAstV3 RNA as detected by RT-qPCR within multiple anatomic regions of the CNS. Consistent viral structures were within the soma of a spinal cord neuron in the single pig examined by EM. Of note, PoAstV3 was not only detected by ISH in neurons of the cerebrum and spinal cord but also neurons of the dorsal root ganglion and nerve roots consistent with viral dissemination via axonal transport. This is the first study reproducing CNS disease with a porcine astrovirus strain consistent with natural infection, suggesting that pigs may serve as an animal model to study the pathogenesis of neurotropic astroviruses.