| In 2016,a novel porcine circovirus,designated porcine circovirus 3(PCV3),was first identified in diseased sows with porcine dermatitis and nephropathy syndrome(PDNS)-like clinical signs as well as in the aborted fetuses by researchers from the Kansas State University using metagenomic sequencing technology.Epidemiological survey further demonstrated that PCV3 infection was mainly distributed in the pig herds exhibiting PDNS and reproductive failure symptoms and was widely prevalent in the swine population in several states of the USA.The Blood Systems Research Institute of San Francisco also discovered PCV3 in three sick piglets with cardiac and multi-systemic inflammation of unknown etiology.PCV3 was subsequently detected in the Chinese pig herds in Hubei and Guangdong Provinces,etc.These results suggest that PCV3 may be highly prevalent worldwide,and how can we detect pigs antibody levels fast and accurately has became an important step for prevention and control of this disease.This study aimed to identify PCV3 in clinical samples from pigs collected during 2008 to 2017 in Jiangsu province,China.A total of 272 pig tissue samples from 141 pig farms in Jiangsu province were examined and analyzed.Most of the diseased animals were weaned pigs that exhibited some clinical signs of PCV disease(e.g.,wasting,skin pallor,respiratory distress,diarrhea,and/or icterus).Besides PCV3,the common porcine viruses,such as PCV2,porcine parvovirus(PPV),pseudorabies virus(PRV),classical swine fever virus(CSFV)and porcine reproductive and respiratory syndrome virus(PRRSV)were also examined by PCR or RT-PCR.Forty of the 272(14.7%)samples tested were positive for PCV3.The four complete genome sequences,which were identified as PCV3 by PCR amplification and DNA sequencing.Our results indicate that PCV3 co-infections with other pathogens have occurred widely in the pig herds in Jiangsu province from 2013 to 2017.The PCR products of PCV3 positive from this study were sequenced directly in opposite directions at a commercial sequencing facility,and phylogenetic trees representing the complete genomes,rep genes and capsid genes were constructed by MEGA 7.0.It is imperative,therefore,to gain better understanding of the pathogenicity of PC V3 and control its further dissemination in this region.In this study,the gene encoding the capsid protein free of nuclear localization signal was cloned into the prokaryotic expression vector pET-28a,resulting in a fusion protein with immunogenicity induced by IPTG By optimizing the expression conditions,the protein was also present in the form of inclusion body.Through the steps of denatured by Urea,dialysis renaturation and purificated with cation exchange column,the protein was used to immune 6-8 week-old BALB/c mice as an immunogen,after fusion and detection,hybridoma were selected by indirect enzyme-linked immuneosorbent assay(ELISA),and the positive clones were subcloned.For hybridoma designated as 1D3-6-25,1D3-11-39,1D3-5-10,1D3-32-61,1D3-29-72,1D3-14-57,1D3-38-83 and 1D3-40-92 which stably secret monoclonal antibodies against the capsid protein of PCV3 were obtained.Western Blot and IFA results showed that 8 hybridoma cells secreted monoclonal antibodies were obtained.Of 8 monoclonal antibodies,3 were selected to prepare ascites(1D3-14-57,1D3ยท29-72,1D3-11-39),and the highest dilution multiple of the positive reaction was determined by indirect ELISA,and the titer of the ascites of 3 monoclonal antibodies were 1:409600,1204800 and 1:409600 respectively.Immunohistochemical study of the pig inguinal lymph nodes infected with PCV3,and the serum of unimmunized mice was used as negative control.At the same time,the monoclonal antibody ascites diluted 1000 times as first antibody,1:8000 diluted HRP labeled Goat anti mouse IgG was the second antibody,and the specificity of the monoclonal antibody was identified.The results showed that the ascites of 3 McAbs had good reactivity. |
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