Porcine Circovirus-like Virus P1 Research Articles

Vertical transmission of porcine circovirus-like virus P1 in BALB/c mice

BackgroundPorcine circovirus-like virus P1 is the animal virus with the smallest genome discovered so far, and it has become widely distributed in the Chinese mainland in recent years.ResultsIn this study, a BALB/c mouse model was used to reveal P1 infection in female reproductive systems and the vertical transmission of the virus. The female reproductive system, including the ovary and uterus, was harvested on day 14 postinfection and examined for pathological lesions. One-day-old mice without colostrum born from infected or uninfected mothers were collected, and P1 virus distribution in the different organs was investigated. During the trials, all the mice showed no clinical symptoms or gross lesions. However, stillbirth did occur in groups infected with the P1 virus. P1 nucleic acid was detected in the heart, liver, spleen, lung, kidney, and brain tissues of 1-day-old mice born from infected mice. Microscopic lesions in P1-infected female mice were characterized by necrosis of the ovarian follicular granulosa cells and abscission, follicular atresia, necrosis of the endometrial epithelial and uterine glandular epithelial cells, and hyperplasia of the squamous endometrial epithelium. The spermatocytes in the seminiferous tubules of the infected male mice were disorderly arranged, and the germ and Sertoli cells were shed, necrotic, and decreased in number. Immunohistochemical results identified P1-positive particles in the nucleus and cytoplasm of cells from the ovary and uterus of female mice.ConclusionsThis study shows that the P1 virus could cause pathological damage to the reproductive system of female mice and could be transmitted vertically.

Porcine circovirus-like virus P1 activates pancreatic secretion pathway by interacting with CHRM3 protein

The porcine circovirus-like virus P1, a member of the circovirus family, causes post-weaning multisystemic wasting syndrome (PMWS) in weaned piglets with progressive wasting as the main clinical symptom. The pancreatic secretion pathway induces pancreatic acinar cells to secrete various digestive enzymes and as such is an important signaling pathway for the digestive system and somatic growth. This study examined the effects and mechanism of P1 virus infection on the pancreatic secretion pathway. The experiment was conducted by transfecting double-copy plasmid P1 into PK-15 and 3D4 cells and by infecting cells with the P1 virus. Samples were collected at various times after transfection or infection. The pathway's transcription and translation levels of CHRM3, Gq, PLC-β2, PRKCA, Rab3D, RhoA, Rac1, and amyA proteins were detected by real-time PCR and Western blots; these analyses confirmed that the P1 virus infection could upregulate the expression level of key pancreatic secretion signaling molecules. Then, we confirmed that the VP1 protein of the P1 virus could interact with the pathway initiation protein CHRM3 using Co-IP, pull-downs, and confocal fluorescence microscopy. Finally, we demonstrated that the VP1 protein activates the pancreatic secretory pathway through the CHRM3 protein. In conclusion, this study demonstrated that the P1 virus can interact with the CHRM3 receptor protein to activate the pancreatic secretion pathway and promote the secretion of various digestive enzymes downstream of the pathway, thereby providing a basis for P1 virus pathogenesis.

Nearly 20 Years of Genetic Diversity and Evolution of Porcine Circovirus-like Virus P1 from China.

Porcine circovirus-like virus P1 can infect many kinds of animals and mainly causes postweaning multisystemic wasting syndrome. In China, the genetic diversity, variation, and evolutionary processes of this virus have not been described yet. To improve our knowledge of its genetic diversity, evolution, and gene flow, we performed a bioinformatics analysis using the available nucleotide sequences of the P1 virus; among them, 12 nucleotide sequences were from ten pig farms in Jiangsu Province in this epidemiological survey, and 84 sequences were downloaded from GenBank. The P1 sequences showed a rich composition of AT nucleotides. Analyses of the complete genomic sequences were polymorphic and revealed high haplotype (gene) diversity and nucleotide diversity. A phylogenetic analysis based on the NJ method showed that all P1 virus sequences formed two distinct groups: A and B. High genetic differentiation was observed between strains from groups A and B. The codon usage pattern of P1 was affected by dinucleotide compositions. Dinucleotide UU/CC was overrepresented, and dinucleotide CG was underrepresented. The mean evolutionary rate of the P1 virus was estimated to be 3.64 × 10−4 nucleotide substitutions per site per year (subs/site/year). The neutrality tests showed negative values. The purifying selection and recombination events may play a major driving role in generating the genetic diversity of the P1 population. The information from this research may be helpful to obtain new insights into the evolution of P1.

Genomic Rearrangement and Recombination of Porcine Circovirus Type 2 and Porcine Circovirus-Like Virus P1 in China.

Porcine circovirus type 2 (PCV2) belongs to the genus Circovirus of the family Circoviridae, and it has been associated with porcine circovirus (associated) disease (PCVD or PCVAD) in pigs. PCVAD is the generic term for a series of disease syndromes that have caused economic losses to the pig industry worldwide. Since the discovery of PCV2 in the late 1990s, the virus has continued to evolve, and novel genotypes have continued to appear. Moreover, there has been recombination between different genotypes of PCV2. This review attempts to illustrate some progress concerning PCV2 in genome rearrangement and genomic recombination with non-PCV2-related nucleic acids, particularly focusing on the porcine circovirus-like virus P1 formed by the recombination of PCV2. The presence of rearranged PCV2 genomes can be demonstrated both in vivo and in vitro, and these subviral molecules ranged from 358 to 1,136 bp. Depending on whether it has the ability to encode a protein, the agents formed by PCV2 recombination can be divided into two categories: porcine circovirus-like viruses and porcine circovirus-like mini agents. We mainly discuss the porcine circovirus-like virus P1 regarding genomic characterization, etiology, epidemiology, and pathogenesis. Further research needs to be conducted on the pathogenicity of other porcine circovirus-like viruses and porcine circovirus-like mini agents and the effects of their interactions with PCV2, especially for the porcine circovirus-like mini agents that do not have protein-coding functions in the genome.

Interaction of porcine circovirus-like virus P1 capsid protein with host proteins

BackgroundPorcine circovirus-like virus P1 is a relatively new kind of virus that is closely related to the post-weaning multisystemic wasting syndrome, congenital tremors, and abortions in swine. The molecular mechanisms of P1 virus infection and pathogenesis are fully unknown. To analyze P1 and its host interactions, we used a yeast two-hybrid (Y2H) assay to identify cellular proteins interacting with the Cap of the P1 virus. In this study, the Cap of the P1 virus exhibited no self-activation and toxicity to yeast cells and was used as bait to screen the Y2H library prepared from the pancreas tissue.ResultsFive cellular proteins (EEP, Ral GDS, Bcl-2-L-12, CPS1, and one not identified) were found to interact with P1 Cap. The interaction between Cap and Ral GDS was confirmed by co-immunoprecipitation.ConclusionsOur data are likely to support the future investigation of the underlying mechanism of P1 infection and pathogenesis.

Characterization of porcine circovirus-like virus P1 replication and lesions in BALB/c mice

Five-week-old male BALB/c mice were inoculated intraperitoneally with a single (sP1) or multiple doses (mP1) of porcine circovirus-like virus P1 or cell culture medium. None of the mice exhibited clinical signs or gross lesions throughout the study. However, the body weights of the mP1 mice were significantly decreased, and the mice inoculated with P1 exhibited viral replication, seroconversion, and microscopic lesions. P1 nucleic acid was detected in the heart, liver, spleen, lung, bladder, testis, brain, thymus, and pancreatic tissues. Special P1 antibody was found in the P1-inoculated mice. Microscopic lesions in the sP1 and mP1 mice were characterized by interstitial pneumonia, including edema in the connective tissue around the pulmonary vessels, mild inflammatory cell infiltrate, thickened alveolar walls, myocardial necrosis, and dissolution of Purkinje cell nuclei. The results showed that the P1 virus could infect BALB/c mice. Thus, BALB/c mice may serve as models for P1 research.

Complete genome sequences of porcine circovirus-like virus P1 mutants with 163 amino acids in the capsid protein.

Porcine circovirus-like virus P1 is a novel circovirus that was originally detected in China in 2005. Here, we report the genome sequences of P1 isolates JS02, JS03, and HuN06, each with 163 amino acids in its capsid protein. The complete genome of each of these isolates contains 649 nucleotides and has a T insertion at position 207. Phylogenetic analysis based on complete genome sequences of 18 P1 reference strains grouped 16 P1 sequences from this study into one cluster, with the JS02, JS03, and HuN06 isolates forming an independent clade. However, phylogenetic analysis based on amino acid sequences of the capsid protein showed that the JS02, JS03, and HuN06 strains were on the same large branch with PCV2, distinct from other P1 isolates. These results help us to understand the origin and evolution of P1.

Detection of porcine circovirus-like virus P1 in Hebei, China.

Porcine circovirus-like virus P1 is a novel unclassified circovirus that was first detected in China and may be associated with post-weaning multisystemic wasting syndrome (PMWS) and congenital tremor. In this study, we detected P1 infection in pigs in Hebei Province, China, in 2017. One hundred and forty of 500 (28.0%) serum samples from 25 pig farms with different PMWS status in seven cities were P1 positive on PCR. Twelve P1 strains were sequenced, and the complete genomes of 11 P1 strains were 648 nucleotides (nt) in length, whereas that of strain ZJK02 was 647 nt, with a G deletion at position of 183 in its genome. The complete genomic and capsid protein sequences of the 12 P1 strains analysed in this study shared 98.8%-100.0% and 86.5%-100.0% identity, respectively. A phylogenetic analysis based on the complete genomic and capsid sequences of 26 P1 strains showed that the 12 P1 sequences from Hebei Province clustered on two small branches. Further studies of the evolution and pathogenesis of P1 are required.

Porcine Circovirus-Like Virus P1 Inhibits Wnt Signaling Pathway in Vivo and in Vitro.

Porcine circovirus-like virus P1 is an important pathogen of the current pig industry, the infection mechanism is not entirely clear. Wnt signaling pathway plays an important role in the growth of young animals and infection of some viruses. This study was designed to demonstrate the effects of P1 infection on the Wnt signaling pathway. In vivo experiments, we demonstrated the down-regulatory effects of P1 infection in piglets and mice on the downstream components expression levels of Wnt signaling pathway, and the effects of Wnt signaling pathway activation on the pathogenesis of P1. In vitro studies, we found P1 infection down-regulated protein level of β-catenin and mRNA level of mmp2, prevented the β-catenin from entering into nucleus, abolished the TCF/LEF promoter activity, proved that P1 could inhibit the activation of Wnt signaling pathway in vitro. Finally, we found that VP1 of P1 virus also had the inhibitory effects on Wnt signaling pathway in vitro, elucidated the mechanism of P1’s inhibitory effects on the Wnt signaling pathway and offered the possibility that the suppression of Wnt signaling pathway was involved in the post-weaning multisystemic wasting syndrome (PMWS), laying a foundation for elucidating the pathogenesis of P1.

Protective efficacy of a DNA vaccine encoding capsid protein of porcine circovirus-like virus P1 against porcine circovirus 2 in mice.

The capsid protein is the major immunogenic protein of porcine circovirus 2 (PCV2). The nucleotide sequence of porcine circovirus-like virus P1 shares high homology with open reading frame (ORF) 2 of PCV2, and ORF1 of P1 encodes its structural protein. Mice were vaccinated twice intramuscularly with a plasmid expressing the P1 ORF1 protein (pcDNA3.1(+)-ORF1) at 2-week intervals. All animals vaccinated with pcDNA3.1(+)-ORF1 developed higher specific anti-P1 antibody levels, and had less PCV2 viremia and milder histopathological changes than PCV2-challenged mice in the control group. Our results show that the P1 DNA vaccine elicited immune responses against PCV2 infection in a mouse model.

Evidence of porcine circovirus-like virus P1 in piglets with an unusual congenital tremor.

Outbreaks of trembling and shaking were reported among pigs at two pig farms in Jiangsu Province, China. Serum and tissue samples tested positive for porcine circovirus-like virus P1 and negative for classical swine fever virus, porcine circovirus type 2, astrovirus and porcine pestivirus using PCR/RT-PCR and immunohistochemical techniques. High P1 viral genome loads were identified in sera, brain and lymph node tissue samples by qPCR. In addition, one of the most notable pathological changes was dissolution of the nucleus in Purkinje cells. The results of this study provide molecular evidence of an association between congenital tremor in pigs and P1 virus.

Porcine circovirus-like virus P1 in cattle, goats and rabbits in China.

Porcine circovirus-like virus P1, a novel single-stranded circular DNA virus, is a potential causative agent of post-weaning multisystemic wasting syndrome in swine. In this study, we detected P1 strains in serum samples from cattle, goats and rabbits suffering different clinical symptoms, in Jiangsu Province, China. We sequenced the whole genomes of six P1 strains, which showed 99.4%-100% nucleotide sequence similarity and were closely related to other porcine-origin P1 isolates. The sequences of six non-porcine-origin P1 viruses were most closely phylogenetically related to other porcine-origin P1 sequences. Therefore, the P1 virus has a wide host range in China.

Molecular characterization of porcine circovirus-like virus P1 in eastern China

A large-scale epidemiological study of porcine circovirus-like virus P1 was carried out from 2007 to 2015 in China, which revealed a high level of P1 infection. Thirty-nine sequences obtained from pigs in eastern China showed remarkable genetic diversity, with the P1 predominant sequences and the mutant P1 with ORF1 or ORF2 containing an additional amino-acid extension at the C-terminus. This information may be useful for understanding the evolution of P1 circovirus-like virus.

Genome sequence of a porcine circovirus-like virus P1 mutant in China.

A new porcine circovirus-like virus P1 mutant was isolated and sequenced in China in 2015. This P1 mutant has a mutation in its ORF1-encoded capsid protein that results in its elongation by eight amino acids.

Development of real-time PCR assay for detection of porcine circovirus-like virus P1 in domestic pigs in China.

BackgroundThe porcine circovirus-like agent P1 is a newly discovered DNA virus with a single-stranded circular genome that is highly homologous to that of porcine circovirus type 2. P1 infection can cause symptoms resembling postweaning multisystemic wasting syndrome. This study aims to develop a rapid, sensitive and specific method to detect P1.ResultsA pair of primers was designed and used to amplify a 119 bp DNA fragment to generate a recombinant plasmid which was served as the standard. A SYBR I qPCR protocol was established using the P1 recombinant plasmid standard and the sensitivity, specificity and stability of this method was analyzed. The results demonstrate a strong correlation with P1 recombinant plasmid titers when virus DNA copy numbers fall in between 100 ~ 109 copies/μL. This method doesn’t detect pseudo rabies, porcine parvovirus or porcine reproductive and respiratory syndrome virus; moreover it can distinguish porcine circovirus type 2 from P1 by melting temperature analysis. Coefficient of variation for each batch of reaction is less than 5 %. The serum virus titers of P1 positive in this study were measured by this protocol to be 103 to 107 copies/mL.ConclusionsThe established qPCR is sensitive, specific, and reliable, which could be a useful tool when applied to quantification of P1 in a variety of samples from infected pigs.

Function analysis of proteins encoded by ORFs 1 to 8 of porcine circovirus-like virus P1 by microarray assay.

Porcine circovirus-like agent P1 is a newly discovered virus containing a single-strand circular genome. The genome of P1 is a DNA molecule of 648 nucleotides which contains eight open reading frames (ORFs) that probably encode potential proteins or polypeptides. Thus it is very important to clarify these proteins' function. Here we provide the methods and analysis of microarray data in detail to characterize the transcriptome profile of P1 with and without the ORF. The relevant microarray data sets have been deposited in Gene Expression Omnibus (GEO) database under accession number GSE71945.

Transcriptional analysis of porcine circovirus-like virus P1.

BackgroundRecently identified porcine circovirus-like virus P1 has the smallest DNA viral genome. In this study, we identified the viral genes and their corresponding mRNA transcripts.ResultsThe RNAs of P1, synthesized in porcine kidney cells, were examined with northern blotting and PCR analyses.Eight virus-specific RNAs were detected. Four mRNAs (open reading frames (ORFs) 1, 2, 4, and 5) are encoded by the viral (−) strand and four (ORFs 3, 6, 7, and 8) are encoded by the viral (+) strand. All proteins encoded by the ORFs of the P1 virus are less than 50 amino acids in length, except that encoded by ORF1 (113 amino acids).ConclusionsWe show a very complex viral transcription pattern in P1-infected cells.