Isolation And Identification Of Two MDPV Strains And Establishment Of RAA Diagnostic Method

Muscovy duck parvovirus（MDPV）is one of the main pathogens that endanger the waterfowl industry in China.In Clinically,it mainly infects young muscovy ducklings within 20 days of age.Muscovy duck parvovirus disease,as an acute viral infectious disease,has high morbidity and mortality,and ultimately resistant ducks will become stiff ducks,causing significant economic loss.In this study,the isolation and identification of MDPV in Guangxi and its whole genome sequencing analysis were carried out,and the RAA diagnosis method for MDPV was established,which provided a theoretical basis for the accurate prevention and control of MDPV in duck industry in Guangxi and the rapid diagnosis of MDPV in the field.The specific research contents are as follows:1.Isolation,identification and animal regression test of two MDPV strainsIn this study,clinical samples suspected to be infected with MDPV were collected in Hepu area of Guangxi.The positive samples detected by PCR were treated with conventional methods and inoculated into 11-day-old muscovy duck embryos for virus isolation and identification.MDPV isolates were inoculated into DEF cells to observe cytopathic effect.At the same time,the half lethal dose（ELD50）of MDPV isolates was determined and animal regression test was carried out.The results showed that two MDPV strains were successfully isolated in this study,named as HP200620 and HP200107.After inoculating DEF cells with the third passage virus solution of the isolated strain,the cytopathic effect with obvious shrinkage,rounding and shedding were observed.The ELD50 of HP200107 and HP200620 were 10³/0.2 m L and 10^3.5/0.2 m L,respectively.The results of animal regression test showed that the ducks infected with two isolates had typical clinical symptoms and characteristic pathological changes,mainly manifested as mouth opening breathing,pancreatic gray necrosis and intestinal mucosal bleeding.The mortality rates of HP200620 strain and HP200107 strain were 13%（2/15）and 20%（3/15）,respectively.The results of virus shedding test showed that the virus shedding rate of the isolates increased first and then decreased,and the virus shedding of pharyngeal and cloacal samples from diseased Muscovy ducks could not be detected on the 21 st day post-infection（DPI）.The viral load analysis showed that the viral load in the spleen of diseased ducks remained at a high level during the test period.The peak period of viral load was from 3DPI to 7DPI,and the viral load gradually decreased from the 14 DPI.In general,the animal regression test of the isolates showed the characteristics of early onset and acute course of disease.2.Whole genome sequencing and analysis of MDPV HP200620 and HP200107 strainsBy analyzing the whole genome sequence of MDPV reference strains,specific primers were designed to amplify the whole sequence of MDPV.The gene cloning,sequencing,sequence splicing and analysis of two MDPV isolates in this study were conducted.The results of sequencing showed that the genome length of HP200620 and HP200107 were 5131 bp and 5141 bp,respectively.The difference in length was mainly due to the nucleotide insertion of ITR gene.The nucleotide similarity of the whole genome between the above two isolates and the22 reference strains was 80.6-99.3%,which was the highest with the typical MDPV YY strain.The construction of genome-wide genetic evolution tree showed that the isolates were in a typical MDPV branch and had close genetic relationship with the reference strains YY of Fujian Province,China,with similarity of 99.3%.The sequences of NS1,VP1,VP3 and ITR genes of the isolates were analyzed and the genetic evolution tree was constructed.The results showed that the two isolates had high nucleotide and amino acid homology,and the homology was the highest with the reference strains YY and P1.The genetic evolution tree of each gene showed that the two isolates were in a separate small branch except for ITR gene,indicating that the two isolates were different from other typical MDPV reference strains to some extent,and they were representative for MDPV epidemic strains in Guangxi.3.Establishment a diagnostic method recombinase aided amplification（RAA）of MDPVSpecific primers were designed for the conserved region of VP1 gene of MDPV.The RAA detection method of MDPV was established by primer screening and RAA reaction conditions optimization.The results showed that the shortest reaction time of RAA for MDPV was 20 min at 37℃.The established RAA detection method and conventional PCR method were used to detect 55clinical samples simultaneously.The results showed that the detection rates of RAA diagnosis method and PCR method were 47.3%（26/55）,indicating that this method was rapid,simple and reliable in clinical practice.In summary,two MDPV strains,HP200620 and HP200107,were isolated and identified in Guangxi,and the whole genome sequencing and analysis were completed.These two isolates may come from Fujian Province and have genetic variation on this basis.At the same time,the RAA detection method of MDPV is successfully established,which is convenient,rapid and accurate,and is suitable for detection of MDPV in the field and laboratories of grassroots units.