| Porcine Circovirus(PCV)is one of the common pathogens in pigs.The spread of the virus is mainly caused by the movement of individual animals between different herds of pigs,Cross-species transmission of PCVs may also occur(e.g.from pigs to rodents,from pigs to cattle,from pigs to fur-bearing animals,etc.).At the same time,the positive rate of PCV in pig population is very high,which is a long-term threat to China's pig industry and hinders the economic development of pig industry.It is a common strategy in virology to use infectious cloning to rescue the virus and to study its replication and pathogenic mechanism.In this study,a novel circovirus,named PCV4,was found and identified in a pig farm in Hunan Province,China,which was significantly different from other circoviruses.The genome was sequenced and found to be 1,770 nucleotides in size.In order to investigate the prevalence of this new virus,Taq Man? probe method for quantitative PCR was established.Clinical samples(including serum samples,tissue samples,nasal swabs and anal swabs)from 187 pigs collected from different pig farms in Hunan province between October 2017 and May 2019 were tested.The results showed that the virus was distributed differently in different samples:Twenty-four out of 187 samples were positive.The positive rate of PCV4 was 12.8%,followed by nasal swab(28.5%,6/21),serum(13.4%,11/82),tissue(8.7%,4/46),and anal swab(7.9%,3/38).PCV4 was also found in pigs coinfected with PRRSV or PCV2.Most of the pigs studied had severe clinical symptoms,including intestinal and respiratory diseases,and porcine dermatitis and nephrotic syndrome.However,PCV4 DNA has also been detected in healthy pigs.The highest homology with PCV4 genome was mink circovirus(66.9%),and the homology between PCV4 and other PCV genomes was43.2%-51.5%.Two major genes were predicted,namely,Replicase(REP)gene 891 nt and Capsid(CAP)gene 687 nt.Further phylogenetic and amino acid sequence analysis of the two genes showed that PCV4 was a new and independent species in the genus Circovirus.In this study,we simultaneously expressed the replicase protein(REP)and capsid protein(CAP)of PCV4 using the PCUE prokaryotic expression system,and obtained polyclonal antibodies against them by immunizing BALB /C mice.Isolating and obtaining viral strains is key to studying the mechanisms of viral replication and infection,However,due to unsuccessful attempts to isolate PCV4,in order to obtain PCV4 virus,an infectious clone of PCV4 was constructed based on the PCV4 genome measured in the laboratory(a single copy genome and a tandem double-copy genome were integrated into p MD19-T vector).BHK-21 cells were transfected,and the Immuno Fluorescence Assay(IFA)was used to evaluate the transfection and virus-saving effects of the prepared mouse polyantibodies.The results showed that the transfection effect of single copy genome was better than that of double copy genome.However,after five generations of blind cell transmission,the fluorescence of the gene Rep protein polyclonal antibody was very weak and almost unobservable,possibly because the amount of virus saved was too low or because the virus could not be passed stably in the cell.This study provides an important basis for further study of the molecular biological characteristics of PCV4 and its replication mechanism in cells. |
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