Development of a SYBR green I-based duplex real-time PCR assay for detection of pseudorabies virus and porcine circovirus 3.

In the present study, a specific and reliable duplex SYBR green I-based quantitative real-time polymerase chain reaction assay was established to detect pseudorabies virus (PRV) and porcine circovirus 3 (PCV3) simultaneously. Viral genomes of PRV and PCV3 in one specimen were identified by their different melting temperatures with melting peaks at 87 °C and 81 °C for PRV and PCV3 respectively, whilst other non-targeted swine pathogens exhibited no fluorescent signals. The assay displayed a high degree of linearity (R2 > 0.997), and the limits of detection were 37.8 copies/µL, 30.6 copies/µL and 60 copies/µL for PRV, PCV3 and the mixture of two recombinant plasmids, respectively. It had good repeatability and reproducibility, and the coefficients of variation in intra-batch and inter-batch assays were all less than 2.0%. In this research, the duplex assay was further evaluated using 117 clinical tissue specimens from diseased pigs in the field. The results revealed the infection rates of PRV and PCV3 were 23.08% (27/117) and 55.56% (65/117) respectively, and PRV and PCV3 co-infection rate was 14.53% (17/117). The assay could be utilized as a diagnostic tool with specificity, sensitivity, and reliability for molecular epidemiological surveillance of PRV and PCV3.

Simultaneous detection of porcine reproductive and respiratory syndrome virus and porcine circovirus 3 by SYBR Green І-based duplex real-time PCR.

The SYBR Green І-based duplex real-time PCR assay was developed for simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus 3 (PCV-3) genomes. PRRSV and PCV-3 were distinguished in the same sample by their distinctive melting temperature (Tm) which was 84 °C for PRRSV and 81.5 °C for PCV-3, and other non-targeted swine viruses showed no specific melting peaks. The detection limits of this assay were 46.1copies/µL for PRRSV and 49.3copies/µL for PCV-3, respectively. Thirty-three lung samples of porcine with respiratory and reproductive failure symptoms were collected and confirmed by the SYBR Green І-based real-time PCR assay and conventional PCR assay. The real-time PCR detection results showed that the PRRSV positive rate was 45.45%, the PCV-3 positive rate was 63.63%, the PRRSV and PCV-3 co-infection positive rate was 36.36%, which were more sensitive than conventional PCR detection. This duplex real-time PCR assay could be a rapid, sensitive and reliable method for the detection of PRRSV and PCV-3 co-infection.

Development of a SYBR green I-based duplex real-time fluorescence quantitative PCR assay for the simultaneous detection of porcine epidemic diarrhea virus and porcine circovirus 3.

The development of a rapid, specific, and sensitive SYBR Green I-based duplex real-time quantitative PCR assay is described for the simultaneous detection of porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 3 (PCV3). The assay specifically detected PEDV and PCV3, with no fluorescence detected for other non-targeted pig pathogens. The assay showed a good linear relationship, and the limits of detection for this assay were 34.6 copies/µL and 61.2 copies/µL for PEDV and PCV3, respectively. The assay exhibited high repeatability and reproducibility, with intra-assay and inter-assay variation coefficients less than 2.0%. A clinical evaluation using intestinal tissue and fecal samples from piglets suffering from diarrhea at different pig farms in China revealed that the singular infection rates of PEDV and PCV3 were 43.94% (29/66) and 16.67% (11/66), respectively, while the co-infection rate of PCV3 with PEDV was 27.27% (18/66). The results indicate this assay is a rapid and reliable diagnostic tool for PEDV and PCV3 monitoring and surveillance in the field, and provides technical support for the quantitative detection of clinical samples infected or co-infected with PEDV and PCV3.

Development of a TB green II-based duplex real-time fluorescence quantitative PCR assay for the simultaneous detection of porcine circovirus 2 and 3.

Porcine circovirus 3 (PCV3), as a newly emerged circovirus, is widely distributed in pig populations worldwide. Co-infection of PCV2 and PCV3 has been reported frequently in clinical samples. In the present study, a TB Green II-based duplex real-time polymerase chain reaction (qPCR) was developed to rapidly and differentially detect PCV2 and PCV3. The assay specifically detected PCV2 and PCV3, with no fluorescence signals being detected for other non-targeted pig pathogens. The duplex qPCR showed a high degree of linearity (R2 > 0.998), and its limits of detection were 10 and 78 copies/µL for PCV2 and PCV3, respectively. The duplex qPCR could detect and differentiate PCV2 (melting peaks at 85.5 °C) and PCV3 (melting peaks at 82.5 °C), and showed high repeatability and reproducibility, with intra- and inter-assay coefficients of variation of less than 2.0%. Fifty-six tissue samples from 18 pig farms were used to evaluate the duplex qPCR method. The results revealed infection rates of 66.07% (37/56) and 39.28% (22/56) for PCV2 and PCV3, respectively. The PCV2 + PCV3 co-infection rate was 39.28% (22/56). The developed method could be used as an efficient molecular biology tool for epidemiological investigations of PCV2 and PCV3.

Analysis of genetic variation of porcine circovirus type 2 within pig populations in central China.

In order to investigate the genetic diversity of porcine circovirus type 2 (PCV2), 284 clinical tissue samples were collected from different pig farms in central China from 2015 to 2017. A total of 162 tissue samples (162/284, 57.04%) were positive for PCV2 by PCR, and subsequently, the complete genome of 36 of these PCV2 samples was cloned and sequenced. The sequencing results showed that 37 complete PCV2 sequences were obtained from 36 PCV2-positive clinical samples. These PCV2 strains were relatively conserved and extremely homologous to the representative classical PCV2 strains. Of these, 20 PCV2 strains belonged to genotype PCV2d, 14 belonged to PCV2b, and three others belonged to PCV2a. Coinfection with PCV2b and PCV2d was identified in one sample (DF-2). These results show that PCV2d may be gradually replacing PCV2b as the predominant PCV2 genotype in central China, and that other genotypes also exist in individual regions. The results of this study will aid in our understanding of the molecular epidemiology of PCV2.

Characterization of a gE/gI/TK gene-deleted pseudorabies virus variant expressing the Cap protein of porcine circovirus type 2d.

Porcine circovirus type 2 (PCV2) plays a key role in the etiology of PCV2-associated disease (PCVAD), and its predominant strain is PCV2d which is not completely controlled by most commercially available vaccines against PCV2a strains. Pseudorabies (PR) caused by pseudorabies virus (PRV) variants re-emerged in Bartha-K61 vaccine-immunized swine herds in late 2011, which brought considerable losses to the global pig husbandry. Therefore, it is significantly important to develop a safe and effective vaccine against both PCV2d and PRV infection. In the present study, the PCV2d ORF2 gene was amplified by PCR, and cloned into the BamHI site of PRV transfer plasmid pG vector to obtain the recombinant transfer plasmid pG-PCV2dCap-EGFP. Subsequently, it was transfected into ST cells infected with the three gene deleted PRV variant strain NY-gE-/gI-/TK- to generate a recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+/EGFP+, and then the EGFP gene was knocked out to harvest the rPRV NY-gE-/gI-/TK-/PCV2dCap+ using gene-editing technology termed CRISPR/Cas9 system. The recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+ had similar genetic stability and proliferation characteristics to the parental PRV as indicated by PCR and one-step growth curve test, and the expression of Cap was validated by Western blot. In animal experiment, higher PCV2-specific ELISA antibodies and detectable PCV2-specific neutralizing antibodies could be elicited in mice immunized with rPRV NY-gE-/gI-/TK-/PCV2dCap+ compared to commercial PCV2 inactivated vaccine. Moreover, the recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+ significantly reduced the viral loads in the hearts, livers, spleens, lungs, and kidneys in mice following a virulent PCV2d challenge. Mice immunized with rPRV NY-gE-/gI-/TK-/PCV2dCap+ developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal PRV challenge. Together, the rPRV NY-gE-/gI-/TK-/PCV2dCap+ recombinant strain has strong immunogenicity.

Prevalence and genetic analysis of porcine circovirus type 2 (PCV2) and type 3 (PCV3) between 2018 and 2020 in central China.

Porcine circovirus type 2 (PCV2) is the causative agent of porcine circovirus-associated diseases (PCVAD), causing substantial economic losses to the swine industry worldwide. PCV3, as a recently discovered virus, is associated with porcine dermatitis, nephropathy syndrome, reproductive failure, congenital tremors, and other clinical symptoms. To further investigate the epidemic profile and genetic diversity of the two viruses, a total of 198 samples from swine at various growth stages suspected for PCVAD on 55 different pig farms between 2018 and 2020 were analyzed for presence of PCV2 and PCV3 by using a multiplex real-time PCR assay. Among the 198 samples, 113 (57.07%) and 72 (36.36%) were positive for PCV2 and PCV3 respectively, and 39 (19.7%) were positive for PCV2 and PCV3 co-infection. Subsequently, whole genome sequences of 34 PCV2 and 19 PCV3 strains were obtained from 30 and 19 clinical samples, respectively. Of these, 8 PCV2 strains belonged to PCV2a, 10 belonged to PCV2b and 16 belonged to PCV2d, indicating PCV2d was the predominant PCV2 genotype circulating in central China. Furthermore, co-infection of different PCV2 genotype strains was identified in three samples (JZ-4, KF-2 and JY-1), and a cross-recombination was found in the ORF2 region of the sequenced 13 PCV2d strains whose putative parental strains were LN6/1999 (MF278777) and MEX/41238/2014 (KT795287) strains. The phylogenetic analysis of PCV3 showed high nucleotide identity (>98%) among sequences obtained in this study and reference sequences. These data will aid our understanding of the molecular epidemiology and evolution of PCV2 and PCV3.

Molecular detection and phylogenetic analysis of Porcine circovirus 4 in Henan and Shanxi Provinces of China.

Porcine circovirus 4 (PCV4), a new circovirus with a distinct relationship to other circoviruses, was identified in 2019 in several pigs with severe clinical disease in Hunan Province, China. To investigate the epidemic profile and genetic diversity of the virus, 63 clinical samples were collected from 24 different pig farms in 14 cities in Henan and Shanxi Provinces, China, between February 2018 and December 2019, and the partial Cap gene of PCV4 was amplified by PCR. Among the 63 samples, 16 (25.40%) were positive for PCV4, and 50% (12/24) of the pig farms were positive for PCV4. PCV4 was detected in samples from pigs with different clinical presentations. One PCV4 strain (Henan-LY1-2019) was sequenced in this study, and shared 98.4% genomic nucleotide identity with PCV4 strain HNU-AHG1-2019 (accession no. MK986820) detected on a pig farm in Hunan Province in 2019. A phylogenetic analysis based on the genomes of Henan-LY1-2019 and 31 reference strains showed that the Henan-LY1-2019 strain together with PCV4 strain HNU-AHG1-2019 was grouped in a relatively independent sub-branch, and separated from other viruses in the genus Circovirus. The results of this study extend our understanding of the molecular epidemiology of PCV4.