Porcine Astrovirus Type Research Articles

Isolation and Pathogenicity of a Chinese Porcine Astrovirus Type 5 Strain HNPDS-01 and Its Influence on Cecum Microbiota in Piglets

Astroviruses have frequently been found in mammals and poultry, but only a few have been successfully isolated for extensive research. Here, we isolated a strain of porcine astrovirus type 5 (PAstV 5) on LLC-porcine kidney (LLC-PK) cells, from the intestinal contents of diarrhea piglets, namely PAstV 5-HNPDS-01. The complete genome sequence length of this strain was 6,419 nt, which has 77.2%–91.1% nucleotide homology with other PAstV 5 strains and 45.0%–50.0% nucleotide homology with other mammalian astroviruses. The recombination analysis indicated that the recombination events were occurred in ORF 2 region (4,444–5,323 nt) in PAstV 5-HNPDS-01 strain. Subsequently, the pathogenicity of PAstV 5-HNPDS-01 was evaluated in 5-day-old piglets. It showed that the PAstV 5-HNPDS-01 could cause mild diarrhea, growth retardation, minor damage to intestinal villi clinically. Meanwhile, PAstV 5-HNPDS-01 infection could affect the microbiota diversity and composition of cecum in piglet from phylum to genus level. After infected with PAstV 5, there was a significant downregulation of beneficial bacteria, including Faecalibacterium, Bacteroides, Lactobacillus, and Prevotella, while harmful bacteria such as Subdoligranulun showed a significant upregulation. These results provided a research basis for pathogenic mechanisms, vaccine development, and beneficial symbiotic bacteria development for PAstV 5 infection.

Replication of Porcine Astrovirus Type 1-Infected PK-15 Cells In Vitro Affected by RIG-I and MDA5 Signaling Pathways.

The interferon (IFN) system is an extremely powerful antiviral response in animal cells. The subsequent effects caused by porcine astrovirus type 1 (PAstV1) IFN activation are important for the host's response to viral infections. Here, we show that this virus, which causes mild diarrhea, growth retardation, and damage of the villi of the small intestinal mucosa in piglets, induces an IFN response upon infection of PK-15 cells. Although IFN-β mRNA was detected within infected cells, this response usually occurs during the middle stages of infection, after genome replication has taken place. Treatment of PAstV1-infected cells with the interferon regulatory factor 3 (IRF3) inhibitor BX795 decreased IFN-β expression, whereas the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) inhibitor BAY11-7082 did not. These findings indicate that PAstV induced the production of IFN-β via IRF3-mediated rather than NF-κB-mediated signaling pathways in PK-15 cells. Moreover, PAstV1 increased the protein expression levels of retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) in PK-15 cells. The knockdown of RIG-I and MDA5 decreased the expression levels of IFN-β and the viral loads and increased the infectivity of PAstV1. In conclusion, PAstV1 induced the production of IFN-β via the RIG-I and MDA5 signaling pathways, and the IFN-β produced during PAstV1 infection inhibited viral replication. These results will help provide new evidence that PAstV1-induced IFNs may protect against PAstV replication and pathogenesis. IMPORTANCE Astroviruses (AstVs) are widespread and can infect multiple species. Porcine astroviruses produce mainly gastroenteritis and neurological diseases in pigs. However, astrovirus-host interactions are less well studied, particularly with respect to their antagonism of IFN. Here, we report that PAstV1 acts via IRF3 transcription pathway activation of IFN-β. In addition, the knockdown of RIG-I and MDA5 attenuated the production of IFN-β induced by PAstV1 in PK-15 cells and increased efficient viral replication in vitro. We believe that these findings will help us to better understand the mechanism of how AstVs affect the host IFN response.

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.

Comparative Analysis of Novel Strains of Porcine Astrovirus Type 3 in the USA.

Porcine astrovirus type 3 (PoAstV3) has been previously identified as a cause of polioencephalomyelitis in swine and continues to cause disease in the US swine industry. Herein, we describe the characterization of both untranslated regions, frameshifting signal, putative genome-linked virus protein (VPg) and conserved antigenic epitopes of several novel PoAstV3 genomes. Twenty complete coding sequences (CDS) were obtained from 32 diagnostic cases originating from 11 individual farms/systems sharing a nucleotide (amino acid) percent identity of 89.74–100% (94.79–100%), 91.9–100% (96.3–100%) and 90.71–100% (93.51–100%) for ORF1a, ORF1ab and ORF2, respectively. Our results indicate that the 5′UTR of PoAstV3 is highly conserved highlighting the importance of this region in translation initiation while their 3′UTR is moderately conserved among strains, presenting alternative configurations including multiple putative protein binding sites and pseudoknots. Moreover, two predicted conserved antigenic epitopes were identified matching the 3′ termini of VP27 of PoAstV3 USA strains. These epitopes may aid in the design and development of vaccine components and diagnostic assays useful to control outbreaks of PoAstV3-associated CNS disease. In conclusion, this is the first analysis predicting the structure of important regulatory motifs of neurotropic mamastroviruses, which differ from those previously described in human astroviruses.

Porcine Astrovirus Type 5-Associated Enteritis in Pigs

Astroviruses (AstVs) are emerging, potentially zoonotic pathogens, associated with enteric and neurological signs in various animal species. In pigs, five lineages have been identified, porcine AstVs (PoAstVs) 1-5. We now report PoAstV5-associated atrophic enteritis in a colostrum-deprived (CD) pig and a similar disease in field cases of enteritis. Four newborn CD piglets were housed under biosafety level 2 conditions and monitored for the presence of PoAstV RNA in rectal swabs and tissues. PoAstV4 RNA was detected in low numbers (0.9-5.3 log10 genomic copies/rectal swab) in rectal swabs from all of these pigs while all animals were negative for PoAstV1, PoAstV2, PoAstV3 and PoAstV5. At 19 and 20 days of age, high levels of PoAstV5 RNA (7.4-7.5 log10 genomic copies/rectal swab) were detected in one pig, which had developed enteritis. At necropsy 1 day after the first evidence of PoAstV5 shedding, this pig was strongly positive for PoAstV5 RNA in most tissues with strong immunolabelling of PoAstV5 in enterocytes. Gene sequencing confirmed PoAstV5 infection. A retrospective investigation of PoAstVs 3, 4 and 5 in archival tissues from field cases of post-weaning enteritis identified high PoAstV4 or PoAstV5 antigen levels in intestinal tissues. These pigs were often concurrently infected with porcine rotavirus. Our findings suggest that PoAstV5 may contribute to the pathogenesis of enteritis in young pigs.

Ecology of Porcine Astrovirus Type 3 in a Herd with Associated Neurologic Disease.

Astroviruses (AstVs) cause disease in a wide variety of species. Porcine AstVs are highly genetically diverse and conventionally assigned to five genetic lineages (PoAstV1-5). Due to the increasing evidence that porcine astrovirus type 3 (PoAstV3) is a cause of encephalomyelitis in swine and to elucidate important ecologic characteristics, the infection dynamics and environmental distribution of PoAstV3 were investigated in a herd with PoAstV3-associated neurologic disease. Over a 22 week period, the frequency of PoAstV3 fecal shedding varied by pig and age. The peak detection by RT-qPCR of PoAstV3 on fecal swabs (95%; 61 of 64) occurred at 3 weeks of age. The lowest frequency of detection was at 21 weeks of age (4%; 2 of 47); however, the frequency increased to 41% (19 of 46) at the final sampling time point (25 weeks of age). Viremia was rare (0.9%: 4 of 433). Detection in oral fluid was consistent with 75% to 100% of samples positive at each time point. Pens and feeders also had a high rate of detection with a majority of samples positive at a majority of sampling time points. Based on the data presented, PoAstV3 can be consistently detected in the environment with a majority of pigs being infected and a subset intermittently shedding the virus in feces out to 25 weeks of age. These findings suggest the importance of as-yet unidentified risk factors associated with the development of PoAstV3-associated polioencephalomyelitis.

Genetic characterization and phylogenetic analysis of feline astrovirus from Anhui province in eastern China

This study aimed to explore the phylogenetic and molecular characteristics of feline astrovirus. A total of 33 fecal samples of domestic cats with or without diarrhea were collected from the Anhui province, and two positive samples were detected. The complete genome and ORF2 of the two strains were sequenced and phylogenetically analyzed. AH-1-2020 and AH-2-2020 displayed 83.4% homology, and their homologies with other reference strains were 75.3%-83.4% and 83.4%-95.0%, respectively. Phylogenetic tree analysis revealed that all strains could be classified into three different clusters; therefore, the mean amino acid genetic distances (p-dist) among the three clusters were estimated. The results suggested that the two strains and other FeAstV strains were grouped into three genotypes, with AH-1-2020 belonging to a novel genotype. High similarity was observed (65.9%-66.5% nucleotide identity and 63.8%-64.8% amino acid identity) in ORF2 between porcine astrovirus type 1 and AH-1-2020. Furthermore, inter-specific recombination between porcine astrovirus type 1 and FeAstV was observed. We, therefore, inferred that inter-specific transmission may exist between pigs and cats; however, further studies are required to verify this. This is the first report on the genetic characterization and phylogenetic analysis of FeAstVs in the Anhui province and would further the current understanding of the genetic diversity and epidemiology of FeAstVs.

Detection and Cellular Tropism of Porcine Astrovirus Type 3 on Breeding Farms.

Astroviruses cause disease in a variety of species. Yet, little is known about the epidemiology of a majority of astroviruses including porcine astrovirus type 3 (PoAstV3), which is a putative cause of polioencephalomyelitis in swine. Accordingly, a cross-sectional study was conducted on sow farms with or without reported PoAstV3-associated neurologic disease in growing pigs weaned from those farms. Additionally, a conveniently selected subset of piglets from one farm was selected for gross and histologic evaluation. The distribution of PoAstV3 in the enteric system was evaluated through in situ hybridization. PoAstV3, as detected by RT-qPCR on fecal samples, was frequently detected across sows and piglets (66–90%) on all farms (65–85%). PoAstV3 was detected subsequently at a similar detection frequency (77% vs 85%) on one farm after three months. Viral shedding, as determined by the cycle quantification value, suggests that piglets shed higher quantities of virus than adult swine. No link between gastrointestinal disease and PoAstV3 was found. However, PoAstV3 was detected by in situ in myenteric plexus neurons of piglets elucidating a possible route of spread of the virus from the gastrointestinal tract to the central nervous system. These data suggest PoAstV3 has endemic potential, is shed in the feces at greater quantities by suckling piglets when compared to sows, and infection is widespread on farms in which it is detected.

Outbreaks of Neuroinvasive Astrovirus Associated with Encephalomyelitis, Weakness, and Paralysis among Weaned Pigs, Hungary.

A large, highly prolific swine farm in Hungary had a 2-year history of neurologic disease among newly weaned (25- to 35-day-old) pigs, with clinical signs of posterior paraplegia and a high mortality rate. Affected pigs that were necropsied had encephalomyelitis and neural necrosis. Porcine astrovirus type 3 was identified by reverse transcription PCR and in situ hybridization in brain and spinal cord samples in 6 animals from this farm. Among tissues tested by quantitative RT-PCR, the highest viral loads were detected in brain stem and spinal cord. Similar porcine astrovirus type 3 was also detected in archived brain and spinal cord samples from another 2 geographically distant farms. Viral RNA was predominantly restricted to neurons, particularly in the brain stem, cerebellum (Purkinje cells), and cervical spinal cord. Astrovirus was generally undetectable in feces but present in respiratory samples, indicating a possible respiratory infection. Astrovirus could cause common, neuroinvasive epidemic disease.

Porcine Astrovirus Type 3 in Central Nervous System of Swine with Polioencephalomyelitis.

Using next-generation sequencing, we identified and genetically characterized a porcine astrovirus type 3 strain found in tissues from the central nervous system of 1 piglet and 3 sows with neurologic signs and nonsuppurative polioencephalomyelitis. Further studies are needed to understand the potential for cross-species transmission and clinical impact.

No evidence of enteric viral involvement in the new neonatal porcine diarrhoea syndrome in Danish pigs

BackgroundThe aim of this study was to investigate whether the syndrome New Neonatal Porcine Diarrhoea Syndrome (NNPDS) is associated with a viral aetiology. Four well-managed herds experiencing neonatal diarrhoea and suspected to be affected by NNPDS were included in a case-control set up. A total of 989 piglets were clinically examined on a daily basis. Samples from diarrhoeic and non-diarrhoeic piglets at the age of three to seven days were selected for extensive virological examination using specific real time polymerase chain reactions (qPCRs) and general virus detection methods.ResultsA total of 91.7% of the animals tested positive by reverse transcription qPCR (RT-qPCR) for porcine kobuvirus 1 (PKV-1) while 9% and 3% were found to be positive for rotavirus A and porcine teschovirus (PTV), respectively. The overall prevalence of porcine astrovirus (PAstV) was 75% with 69.8% of the PAstV positive pigs infected with PAstV type 3. No animals tested positive for rotavirus C, coronavirus (TGEV, PEDV and PRCV), sapovirus, enterovirus, parechovirus, saffoldvirus, cosavirus, klassevirus or porcine circovirus type 2 (PCV2). Microarray analyses performed on a total of 18 animals were all negative, as were eight animals examined by Transmission Electron Microscopy (TEM). Using Next Generation de novo sequencing (de novo NGS) on pools of samples from case animals within all herds, PKV-1 was detected in four herds and rotavirus A, rotavirus C and PTV were detected in one herd each.ConclusionsOur detailed analyses of piglets from NNPDS-affected herds demonstrated that viruses did not pose a significant contribution to NNPDS. However, further investigations are needed to investigate if a systemic virus infection plays a role in the pathogenesis of NNPDS.

Molecular investigations on the prevalence and viral load of enteric viruses in pigs from five European countries

Enteric viral infections in pigs may cause diarrhea resulting in ill-thrift and substantial economic losses. This study reports the enteric infections with porcine astrovirus type 4 (PAstV4), porcine group A rotavirus (GARV), porcine group C rotavirus (GCRV), porcine circovirus type 2 (PCV2) and porcine kobuvirus (PKoV) in 419 pigs, comprising both healthy and diarrheic animals, from 49 farms in five European countries (Austria, Germany, Hungary, Spain and Sweden). Real-time RT-PCR assays were developed to test fecal samples and to compare the prevalence and viral load in relation to health status, farms of origin and age groups. The results showed that PAstV4 (70.4%) was the dominant virus species, followed by PKoV (56.7%), PCV2 (42.2%), GCRV (3%) and GARV (0.9%). Diarrheic pigs had a higher viral load of PAstV4 in the nursery and growing-finishing groups. Rotaviruses were mainly detected in diarrheic pigs, whereas PCV2 was more often detected in clinically healthy than in diarrheic pigs, suggesting that most PCV2 infections were subclinical. PAstV4, PCV2 and PKoV were considered ubiquitous in the European pig livestock and co-infections among them were frequent, independently of the disease status, in contrast to a low prevalence of classical rotavirus infections.

Full genome analysis of bovine astrovirus from fecal samples of cattle in Japan: identification of possible interspecies transmission of bovine astrovirus.

A viral metagenomics approach was used to investigate fecal samples of Japanese calves with and without diarrhea. Of the different viral pathogens detected, read counts gave nearly complete astrovirus-related RNA sequences in 15 of the 146 fecal samples collected in three distinct areas (Hokkaido, Ishikawa, and Kagoshima Prefectures) between 2009 and 2015. Due to the lack of genetic information about bovine astroviruses (BoAstVs) in Japan, these sequences were analyzed in this study. Nine of the 15 Japanese BoAstVs were closely related to Chinese BoAstVs and clustered into a lineage (tentatively named lineage 1) in all phylogenetic trees. Three of 15 strains were phylogenetically separate from lineage 1, showing low sequence identities, and clustered instead with an American strain isolated from cattle with respiratory disease (tentatively named lineage 2). Interestingly, two of 15 strains clustered with lineage 1 in the open reading frame (ORF)1a and ORF1b regions, while they clustered with lineage 2 in the ORF2 region. Remarkably, one of 15 strains exhibited low amino acid sequence similarity to other BoAstVs and was clustered separately with porcine astrovirus type 5 in all trees, and ovine astrovirus in the ORF2 region, suggesting past interspecies transmission.

Detection of diverse novel astroviruses from small mammals in China.

Astroviruses infect humans and many animal species and cause gastroenteritis. To extensively understand the distribution and genetic diversity of astrovirus in small mammals, we tested 968 anal swabs from 39 animal species, most of which were bats and rodents. We detected diverse astroviruses in 10 bat species, including known bat astroviruses and a large number of novel viruses. Meanwhile, novel groups of astroviruses were identified in three wild rodent species and a remarkably high genetic diversity of astrovirus was revealed in Eothenomys cachinus. We detected astroviruses in captive-bred porcupines and a nearly full-length genome sequence was determined for one strain. Phylogenetic analysis of the complete ORF2 sequence suggested that this strain may share a common ancestor with porcine astrovirus type 2. Moreover, to our knowledge, this study reports the first discovery of astroviruses in shrews and pikas. Our results provide new insights for understanding these small mammals as natural reservoirs of astroviruses.

Detection and molecular characterization of Porcine astrovirus strains associated with swine diarrhea

Astrovirus has been reported to be associated with diarrhea in pigs. The current study was conducted for the detection and molecular characterization of astroviruses in diarrheic pigs submitted to the Veterinary Diagnostic Laboratory, University of Minnesota. Intestinal contents from 269 pigs were examined by reverse transcription polymerase chain reaction (RT-PCR), and 62% were found positive for astroviruses. Of the positive samples, 20% were positive for astrovirus alone while astrovirus with rotavirus was detected in 58% of the samples. The remaining 22% revealed the presence of astrovirus along with Porcine hemagglutinating encephalomyelitis virus, Transmissible gastroenteritis virus, or Porcine circovirus-2. Sequencing the capsid gene of 56 randomly selected samples confirmed them to be Porcine astrovirus type 4 (PAstV-4) with 58-100% nucleotide identity within these viruses. Phylogenetic analysis revealed 2 possible subgroups. The results indicate that PAstV is present on swine farms in the United States and that it may be associated with diarrhea either alone or in combination with other enteric viruses. Further studies are needed to determine strain diversity among porcine astroviruses so that appropriate control strategies can be devised and implemented.

Identification of a novel astrovirus in a domestic pig in Hungary

The family Astroviridae consists of two genera, Avastrovirus and Mamastrovirus, whose members are associated with gastroenteritis in avian and mammalian hosts, respectively. We serendipitously identified a novel porcine astrovirus in a fecal specimen from a domestic pig (Sus scrofa domestica) in Hungary. Sequencing of a fragment indicated that it was an ORF1b/ORF2/3′UTR sequence, and it has been submitted to the database as porcine astrovirus type 2 (PAstV-2/Hungary/2007) with accession number GU562296. Its unique sequence characteristics and its phylogenetic position suggest that PAstV-2 could be an important link between previously reported astroviruses and that a genetically divergent lineage of astroviruses exist in piglets.