In order to understand and grasp the latest epidemic characteristics of a new type of duck reovirus(NDRV),liver samples of ducks with clinical symptoms such as liver swelling and bleeding were collected from a duck farm in Anhui Province,and NDRV was detected by RT-PCR.Positive samples were inoculated into SPF chicken embryo to isolate the virus,and the third generation allantoic fluid was harvested for PCR detection and virus purity identification.Meanwhile,the full-length σC gene of NDRV isolates was cloned and sequenced.The σC gene sequence of NDRV AH strain was compared with the gene sequence and derived amino acid sequence of 21 avian reovirus reference strains downloaded from GenBank.Meanwhile,the phylogenetic tree of isolates was constructed using the σC gene sequence as the object by using Mega 7.0 software,referencing sequences from GenBank.Finally,the isolates were inoculated with Vero cells to observe their culture characteristics in vitro,and the chicks were infected for pathogenicity test.The result showed,inoculated of chicken embryoswith positive material resulted in embryos death within 3 days,accompanied by edema and severe congestion.Virus purity test results confirmed that the samples were positive for NDRV,but negative for other pathogens such as duck hepatitis A virus(DHV),duck Tambusu virus(DTMUV),avian reovirus(ARV),Muscovy duck reovirus(MDRV),H9 subtype avian influenza virus(H9N2 AIV)and avian adenovirus(FAdV).Sequencing and analysis showed that the three isolates had identical target genes and belonged to one strain,named NDRV AH strain.When Vero cells was inoculated with the strain,the pathological changes such as morphological rounding and nucleus aggregation were observed.Genetic evolution analysis of σC gene of NDRV AH strain showed a similarity of 89.5％-99.0％σC in gene sequence and 85.3％-98.9％in amino acid sequence compared to other NDRV reference strains.The evolutionary tree of the σC gene showed that the NDRV AH strain isolated in this study belonged to genotype 2.Compared with the inactivated vaccine TH11 strain(KC493571.1),NDRV AH strain had mutations at amino acids aa67,aa93,aa100,aa132,aa151,aa158,aa212,aa253 and aa298.The results of pathogenicity test showed that the mortality rate of infected ducks was 40％.Necropsy showed bleeding of kidneys and liver,enlargement of spleen,slight necrotic focus or large necrotic focus,some of which were hard in texture.This study revealed the epidemic trend of NDRV in Anhui Province of China and laid the foundation for the development of mutant vaccine,which has important practical significance for the prevention and control of NDRV infection and reducing the loss of poultry industry.
novel duck reovirusisolation and identificationσC genephylogenetic analysispathogenic test
维普
万方数据
