Isolation And Identification Of Avian Polyomavirus And Establishment Of Fluorescent Quantitative PCR Detection Method For Avian Polyomavirus

Avian polyomavirus disease,also named budgerigar fledgling disease(BFD),is an acute viral infection caused by avian polyomavirus(APV),which can infect multiple species of birds,but mainly attack parrot chicks and induce difficulty in breathing,impaired feathers,growth retardation,weight loss and eventually death.The polyomavirus disease was introduced to China at the end of the 20 th century,and causing huge damage to local parrot farming enterprises once broke out in Hubei,Shandong,Fujian and other provinces in China.There are no effective vaccines and treatments for Avian polyomavirus disease until now,therefore,it is of great significance to carry out research on APV.This experiment isolated a virus from the liver of the diseased budgerigar that suspected avian polyomavirus disease.Genetic analysis and animal regression assay demonstrated that the isolated virus was avian polyomavirus.A SYBR Green fluorescent quantitative PCR(qPCR)method was established in the experiment to enable the diagnosis of avian polyomavirus disease.In addition,our experiment also carried out the qPCR detection method to study the proliferation of APV on budgerigar embryo fibroblasts,and mapped the virus proliferation curve.The specific test results are as follows:1.The disease material suspected avian polyomavirus infection was collected in Lintong area of Shaanxi Province.The budgerigar embryonic fibroblast(BEF)primary cells were made from budgerigar embryos of 15 days old.A virus was successfully isolated by inoculating the diseased material into BEF cells.The isolated virus was found to be more than 99% similar to the nucleotide and amino acid sequence of 11 avian polyomaviruses published from NCBI,and the homology with the isolates of Shandong and Japan was as high as 100% through the sequence analysis of the VP1 gene.The isolated virus was inoculated into healthy budgerigar fledgling,and the clinical symptoms and pathological changes were similar to those of avian polyomavirus,which demonstrated that the isolated virus was avian polyomavirus.2.A 209 bp positive standard plasmid of APV was constructed,and then the standard plasmid diluted by 10-fold gradient dilution was determined by fluorescent quantitative PCR to obtain the standard curve Y=-3.255X+40.867.Finally,a SYBR Green Fluorescent Quantitative PCR was established.To evaluate various aspects of the method,the results revealed that there are no effective fluorescence signal when the type 4 Fowel Adenovirus,H9N2 avian influenza virus and newcastle disease virus were used as templates.Only the avian polyomavirus showed a specific amplification curve.The method of qPCR that we established is 100 times more sensitive than ordinary PCR in the sensitivity test.In repeatability test,the coefficient of variation of intra reduplicative test and inter reduplicative test was less than 0.5% and 1.5%,respectively.Using this method to detect 30 clinical samples,24 samples were detected positive,and the detection rate was higher than that of conventional PCR.In summary,the fluorescent quantitative PCR assay for avian polyomavirus established in this experiment has the characteristics of high specificity,high sensitivity and good reproducibility.3.The virus copy number of avian polyomavirus at different time points after inoculation on budgerigar embryo fibroblasts was determined by qPCR.The virus proliferation curve was drawn based on the measured data.It was found that the virus replicated efficiently on budgerigar embryo fibroblasts starting 12 h after inoculation.The highest peak of virus concentration was reached at 60 h after inoculation.This result provides an important reference for large-scale proliferation of avian polyomavirus.