Isolation And Identification,sequencing And Analysis Of The Genome Of Two Novel NGPV Strains And Establishment Of Fluorescence Quantitative Real-time PCR Detection Methods For The Virus

Waterfowl parvoviruses are classified into Muscovy duck parvovirus(MDPV),Goose parvovirus(GPV)and Novel goose parvovirus(NGPV),and the emerging pathogen NGPV is a new virus strain naturally recombined by MDPV and GPV,which has been widely prevalent in cherry valley duck,semi-Muscovy duck and Muscovy duck in China since the spring of 2015,causing significant economic losses to the duck industry.To study the prevalence of NGPV in Guangxi,the isolation and identification of NGPV,whole genome sequence analysis,and establishment of a quantitative real-time PCR detection method for NGPV were carried out in this study,providing materials,detection methods and theoretical basis for the research,prevention and control of the disease.The research content and results of the paper are as follows:1.Isolation and identification of Muscovy duck-origin NGPV GXYL201903 and GXYL201907 strainsTo understand the prevalence of NGPV in Guangxi,this study conducted routine PCR detection and bacterial isolation and culture from clinical samples from two muscovy duck farms suspected of having NGPV infection in Yulin city of Guangxi in 2019.The results indicated all detected samples were positive for NGPV,and Escherichia coli was isolated from a few diseased duck tissues.The liver,spleen and other tissues of NGPV-positive clinical samples were treated by conventional methods and then inoculated with 9-11-day-old Muscovy duck embryos through the allantoic cavity to isolate the virus.The death of Muscovy duck embryos was observed and recorded,and then the allantoic fluid of virus-killed embryos was collected to carry out PCR detection.Cloning and sequencing of the target gene fragment was also performed.1-day-old healthy Muscovy ducks were inoculated with the virus isolate by intramuscular injection,the clinical symptoms of the infected Muscovy ducks were observed,the death of the Muscovy ducks was counted,and the tissue samples of the infected ducks were detected by PCR.The results indicated that Muscovy duck embryos inoculated with NGPV-positive sample virus fluid showed embryonic body hyperemia and developmental delay.A 409-base-pair-long DNA band was detected from the allantoic fluid of NGPV-infected Muscovy duck embryo by PCR.Sequence analysis of the target fragment proved to be NGPV,and the two NGPV isolates were named GXYL201903 and GXYL201907,respectively.The results of the artificial infection test of the isolates GXYL201903 and GXYL201907 showed that both the two isolates could cause characteristic symptoms and lessons in the test ducklings with growth retardation,opisthotonus and cyanotic beak,bleeding spots in the liver and kidney and fibrosing enteritis.Part of the tissues were collected for PCR test,and the results showed the samples were positive for NGPV,indicating that the infection in the animal test was successful.The GXYL201903 and GXYL201907 isolates had strong pathogenicity to the 1-day-old muscovy ducklings.2.Whole gene sequence analysis of NGPV GXYL201903 and GXYL201907 strainsWhole-genome sequencing of NGPV isolates GXYL201903 and GXYL201907 was performed using Illumina Hi Seq,and the whole genome sequences of the two NGPV isolates were obtained.The homologous analysis of whole genome sequences of GXYL201903and GXYL201907 were performed between the two isolates and the reference strains of waterfowl parvovirus,and a genetic evolutionary tree was constructed.The virus recombination analysis of the isolates was carried out by molecular biology software RDP and Sim Plot.Finally,a Bayesian phylogenetic tree analysis was performed using capsid genes from the isolates and other reference strains of waterfowl parvovirus.The results showed that the genome of GXYL2012903 was 5083bp in length,and the capsid gene was 2406bp～4604bp;the genome of GXYL2012907was 5063bp,and the capsid gene was 2416bp～4616bp.Whole-genome sequence analysis showed that the GXYL201903 and GXYL201907isolates were 88.7%～99.8%similar to the reference strains.GXYL201903 had the highest similar of 98.9%with Fujian GPV D strain,and GXYL201907 had the highest similarity of 99.8%with Guangdong MDPV GD201911 strain and Fujian NGPV FJFZ20812 strain.Virus recombination analysis proved that Muscovy duck-derived NGPV strains GXYL201903 and GXYL201907 were two recombinant strains.The Bayesian phylogenetic tree of waterfowl parvovirus capsid gene showed that the NGPV branch where the two strains are located is in the MDPV branch;The molecular clock time signal assessment found that the evolution rate of the MDPV branch where NGPV is located is constant,and the gene mutation continues to increase;Species diversity showed a certain downward trend after 2010.3.Preliminary establishment of NGPV SYBR Green I fluorescence quantitative PCR methodA specific primer with a length of 244bp was designed according to the nucleotide sequence of the VP3 gene of the GXYL201907 strain.Recombinant plasmids were prepared by PCR amplification,gene cloning and sequencing identification,and fluorescent quantitative PCR amplification was performed with SYBR Green I dye.The optimization of various reaction conditions and tests for specificity,sensitivity,and reproducibility were performed.The results showed that a qRT-PCR detection method for NGPV was established in this study.The established standard curve equation of fluorescence quantitative PCR is y=-3.1671x+31.03 and the linear correlation coefficient R~2is 0.9983,indicating that the linearity is good.The specificity test results show that the established method can only detect NGPV but cannot detect reference strains such as Newcastle disease virus(NDV),duck reovirus(DRV),duck hepatitis virus(DHV),duck Tembusu virus(DTMUV).The sensitivity of the detection result is 9.66×10~0copies/μL,and the sensitivity of fluorescence quantitative PCR is 100 times higher than that of conventional PCR,which can be used for a clinical diagnosis and epidemiological investigation of NGPV.In conclusion,this study successfully isolated and identified two NGPV strains from Muscovy duck in Yulin,Guangxi.Through the whole genome sequencing and similarity and homology analysis of two Muscovy duck-derived NGPV strains,the biological information of NGPV strains was enriched;the phylogeny,virus origin and effective population diversity of isolates were analyzed by the Bayesian method.The changes in the genetic diversity of NGPV strains in China were clarified.A real-time fluorescent quantitative PCR detection method for NGPV was successfully established.This study provides theoretical basis and technical support for the epidemiological investigation of NGPV and the understanding of gene recombination and genetic variation.It provides a certain reference value for the prevention and control of NGPV.