Identification And Prevalence Investigation Of A Novel Tilapia Parvovirus In Hainan Island

Research Objective:In this study,a novel tilapia parvovirus HMU-HKU was indentified in Hainan Island.Whole genome amplification,genetic evolution analysis,prevalence investigation and pathogenicity study were performed and detection methods were established to provide a research basis for the basic research and prevalence investigation of novel parvovirus derived from tilapia in Hainan Island.Research methods:By analyzing the results of high-throughput genome sequencing of crocodile feces,a novel parvovirus with identity to existing parvoviruses with a nucleotide level of less than 10%was found.Specific primers were designed based on the results of high-throughput sequencing.The sequence of the virus was verified,and the whole genome of the novel parvovirus was spliced using PCR,3`RACE and 5`RACE methods.Collect crocodile and tilapia fecal samples,design specific nested PCR primers,establish a novel parvovirus detection method,and trace the natural host of the novel parvovirus,establish a novel parvovirus real-time PCR detection method,the viral load of novel parvovirus carried by various organs of tilapia was quantified.Research results:Through high-throughput genome sequencing of crocodile feces,a novel parvovirus was found,and the whole genome of the novel parvovirus was successfully spliced.The novel parvovirus genome is 3566bp in length,contains two open reading frames,non-structural proteins NS and capsid proteins VP1 and VP2 were encoded respectively.The G+C content is 43.89%.The whole genome sequence of the novel tilapia parvovirus has been submitted to Gen Bank with accession number MK940538.The results of genetic and evolutionary analysis showed that the nucleotide identity of the novel parvovirus and other viruses in the parvoviridae family was very low and less than 8%,and the identity of NS1 and VP1 with other parvoviruses at the amino acid level is less than 45.50%and 40.38%,respectively.According to the latest classification criteria of ICTV parvoviruses,the novel parvovirus belongs to a new species of the genus Chaphamaparvovirus in the parvoviridae family.This study further traced the natural host of this novel parvovirus and conducted the prevalence investigation of crocodile stool samples from two crocodile farms in Wenchang City,Hainan Island.The results showed that the positive rate of crocodile stool fed tilapia was as high as 86%（43/50）,and the positive rate of fecal crocodile feces was 4%（2/50）.It is speculated that the host of the novel parvovirus may be tilapia.The prevalence investigation was conducted on tilapia feces samples in six areas of Hainan Island,the results showed that the positive rates were:Haikou 40%（8/20）,Ding’an 80%（16/20）,Qionghai 90%（18/20）,Ledong 0%（0/20）,Dongfang 80%（8/10）,0%in Qiongzhong（0/10）,indicating that the host of the novel parvovirus is tilapia,and further studies have shown that the collected 4 kinds of marine fish,5kinds of freshwater fish and 1 kind of freshwater shrimp were not detected to this virus,we named this novel parvovirus as a novel tilapia parvovirus.In this study,a real-time PCR detection method for novel tilapia parvovirus was established.The linear range is 1×10²-1×10⁸copies/μl,the amplification coefficient is 0.999,and only a specific peak appears in the melt curve,the detection sensitivity reached 1×10²copies/μl.Quantitative results of tilapia organs show that the spleen virus content is the highest,which can reach 1×10¹¹copies/g,and the fish seed is also infected,indicating that the novel tilapia parvovirus may be vertically transmitted to the progeny through the fish eggs.Conclusion:This study found a novel tilapia parvovirus HMU-HKU,which belongs to a new species of the genus Chaphamaparvovirus in the Parvoviridae family.The virus is widespread in tilapia farmed in Hainan Island,but not detected in marine fish,freshwater fish and freshwater shrimp detected,indicating that the virus mainly infects tilapia.The novel tilapia parvovirus can infect most tissues and organs in tilapia,with the highest content of spleen virus,which can be vertically transmitted through fish eggs.