Isolation,Identification And Sequence Analysis Of Common Viruses Isolated From Dead Duck Embryos

In recent years,duck eggs in the incubation process,the rate of death continues to rise.During the incubation processoccurs embryonic lethal,duck embryo burst,stench and other phenomena,just hatched ducklings showed weak young,malnutrition and so on,seriously restricting the development of duck industry in China.In this study,a duck embryo was isolated from a hatcheries in Linyi,Shandong,and the pathogens causing duck embryo death were identified.,and the target gene was amplified and analyzed by genetic evolution.Now the results reported as follows:1?Virus isolation and identification:Allantoic fluid collected from the dead duck embryo used for PCR detection of Tembusu virus,Novel Goose Parvovirus,H9 Subtype Avian Influenza Virus,Serotype 4 Group I Avian Adenovirus and Novel Duck Reovirus.The mixed infection samples were neutralized to make the isolates into a single strain,after filtration sterilization,inoculated at 9 days of healthy duck embryo,later use allantoic fluid collected according to conventional virus isolation method blind pass 3 generations.Since the blind pass to 3 generations,virus solution can cause duck embryo die within 3 to 4 days,ccompanied by duck embryo body edema,bleeding.Soon after,Allantoic fluid collected was detected by PCR or RT-PCR,and the amplified fragments were sequenced for genetic evolution analysis.2?Amplification and genetic evolution analysis of target genes in different isolates:Specific primers were designed for Tembusuvirus,novel goose parvovirus,H9 subtype avian influenza virus,serotype 4 group I avian adenovirus and novel duck reovirus.And then the target fragment was amplified and sequenced.The genetic evolution analysis of the target fragment was performed that the nucleotide sequence homology of TMUV-LY strain was higher than 98.1%,Indicating that the variation of Tembusu virus is small.The nucleotide sequence homology of NGPV-LY strain and GPV isolate was 93.3%-96.3%,while that of MDPV was 81.9%-90.8%,the results showed that the isolate had close homology with goose parvovirus and had far nucleotide homology with Muscovy duck parvovirus,illustrated that the Isolates and goose parvovirus in Europe on the same branch,and Muscovy duck parvovirus in different branches.It is likely that the virus was replayed from goose to duck and caused variation.The H9N2-LY strain was highly homologous to the A/Duck/Hong Kong/Y439/97 strain,(AF156376),A/Quail/Hong Kong/G1/97 strain(AF156378)had a nucleotide homology of only 84%with A/Duck/Hong Kong/Y280/,Indicating that the current strain of H9 is likely to be Y439 strain.FAV4-LY strain has close phylogenetic relationship with Indian isolates,the nucleotide homology is 99.6%-99.8%,and the nucleotide homology is 98.7%-99.1%between European strain and American strain,the nucleotide homology between the isolates and the Korean isolates was 98.6%.The results of the phylogenetic tree showed that the isolate was on the same branch as the Indian strain,and on the same large branch with the Russian strain,the Korean strain and the European and American strains were located on the other branch.The NDRV-LY strain has 95.5%-97.6%homology with the published NDRV in Genbank,and the smaller variation of the nucleotide variation is probably caused by the variation in the use of the vaccine.This study was to isolate and identify the virus that caused the death of duck embryo in clinical practice,and determined the main viral infectious diseases.It provided the epidemiological basis for the purification of duck virus in China.