Mutation of the sixth amino acid of the Rep protein has no effect on porcine circovirus 2b but enhances porcine circovirus 2d replication in vitro.

Porcine circovirus type 2 (PCV2) is the etiological agent of post-weaning multisystemic wasting syndrome (PMWS). The original prevalent genotype, PCV2a, has been replaced by genotypes 2b and 2d in the swine population worldwide. The Rep protein is critical for viral replication. Comparison of a large number of Rep protein amino acid (aa) sequences showed that three sites distinguish genotype 2b from genotype 2d. In order to analyze the effect of exchanging the amino acids (asparagine and serine) at position 6 in the Rep proteins of PCV2b and PCV2d, two wild-type and two mutant viruses were rescued. Real-time quantitative PCR and a one-step growth curve were used to determine the viral load to assess the replication ability of the rescued viruses. The results showed that there was no significant difference in in vitro performance between the wild-type PCV2b and the mutated virus, while the mutation of PCV2d enhanced viral replication.

Efficient influence of ssDNA virus PCV2 replication by CRISPR/Cas9 targeting of the viral genome.

Porcine circovirus type 2 (PCV2), a ubiquitous pathogen that primary cause of postweaning multisystemic wasting syndrome (PMWS), had caused significant morbidity and mortality in swine populations with huge economic losses in the worldwide swine industry. Currently, looking for effective antiviral drugs for PCV2 infection remains an important works. In our study, CRISPR/Cas9 system was used to further detected the key sites of PCV2 replication. We designed 8 single guide RNAs (sgRNA) by targeting essential genes across the genome of PCV2. Western-blot(WB), Cell counting kit-8 for high-throughput sgRNA screening were applied to detect PCV2 replication levels. The results showed that Oc8, O13, O134, NQT and NPS sgRNAs can edit the PCV2 genome efficiently and inhibit PCV2 replication in PK-15 cell; H3 sgRNA cannot edit the PCV2 genome successfully; NAT sgRNA can edit the PCV2 genome efficiently to improve the PCV2 replication in PK-15 cell; O26 sgRNA can edit the PCV2 genome successfully but it is not known yet of its effect on PCV2 replication, besides the Cas9 expression had no effect on cell viability. These data suggest that CRISPR/Cas9 system targeting PCV2 essential genes may serve as a novel therapeutic agent against PCV2 infection in the future.

Genetic characterization of Porcine circovirus type 2 from 2013 to 2018 in Shandong Province, China.

PCV2 belongs to the genus Circovirus, family Circoviridae, who is recognized as the causative agents of postweaning multisystemic wasting syndrome. Since being found to China in 2000, it has caused serious damage to the pig industry. In this study, we downloaded 40 PCV2 genome-wide sequences uploaded to GenBank from 2013 to 2018 in Shandong Province, including 23 uploaded by our laboratory. Construction of a genome-wide evolution tree using MEGA V5.0 software. Phylogenetic tree analysis indicated that the genotype of PCV2 in Shandong Province was: three genotypes coexisted (2a, 2b, 2d); among them, PCV2d has become the main genotype in the province due to its number and spread range. Amino acid sequence analysis of different genotypes of ORF2 showed that specific amino acid sites exist in different genotypes, with the most significant range of 81-160; different genotypes of PCV2 can be distinguished at the molecular level. This study found that due to the increase in infections of the PCV2d genotype in recent years, it may replace PCV2b as the dominant base in Shandong.

The correlation between the mutual deletions of amino acids within porcine circovirus rep protein and the discrepancy of replication.

Porcine circovirus (PCV) has two potential open reading frames, ORF1 and ORF2. ORF1 is predicted to encode a replication-associated protein (Rep) essential for replication of viral DNA. In some studies, PCV1 replicated more efficiently in PK-15 cells than PCV2 was elucidated. PCV1 compared with PCV2; there is some amino acids' deficiency on Rep protein. To identify whether the above amino acids deletion affects the replication of PCV1 and PCV2, we constructed three double copy clones by overlap extension PCR. The 2PCV2(vV) clone deleted the valine of Rep protein in the backbone of PCV2 genome. The 2PCV2(dSGR) clone inserted serine, glycine and arginine of Rep protein successively in the backbone of PCV2 genome. The 2PCV2(dSGR&vV) clone inserted serine, glycine and arginine as well as deleted the valine of Rep protein in the backbone of PCV2 genome. These clones we constructed with amino acid mutations and parental DNA clones were all transfected in PK-15 cells that free of PCV contamination, and their growth characteristics in vitro were determined and compared, to evaluating the replication of the mutant infectious DNA clones. Our results showed that the double copy infectious clones with amino acid mutations could be rescued in vitro. The 2PCV2(vV) replicated more efficiently than parental viruses 2PCV2 and 2PCV1 but the replicated ability of 2PCV2(dSGR) and 2PCV2(dSGR&vV) is attenuated than parental viruses 2PCV2 and 2PCV1. We can determine the valine is the important amino acid that cause PCV1 replicated more efficiently in PK-15 cells than PCV2 primarily. These findings are benefit for exploring the mechanisms of viral replication in pigs and important implications for PCV2 vaccine development.