Isolation And Identification,whole Genome Sequencing And Animal Trial Of Avipoxvirus In Domestic Mallard Ducks

Avian pox is a viral disease of captive and wild birds caused by avipoxvirus and affected about 278 species of birds.Avipoxvirus mainly infects chicken,pigeon and turkey,causing the survival crisis of some endangered rare birds.Cutaneous or diphtheric or both forms of disease are associated with different routes of infection.The mortality rates of the diphtheric and both forms are higher than the cutaneous form.There are rarely reports about waterfowl pox in the world,and the knowledge about waterfowl poxvirus is little.In recent years,some cases of avian poxvirus in mallard duck were occasionally reported in Guangxi.In this study,the diagnosis,identification,genome sequencing and a series of animal trials about avianpoxvirus in mallard duck were carried out.In 2016,an occasional pox in domestic mallard duck and Yangjiang goose were occurred in Guangxi.Clinical signs included cutaneous nodules on the birds' eyelids,beaks,and legs.The incidence was about 20%.No deaths were observed.The disease was diagnosed as avipoxvirus by q PCR.Virus particles is oval-shaped 330 nm × 280 nm × 200 nm with irregular pipe-shaped surface structure by electron microscopy.Ultrastructurall examination revealed that the particles had a dumbbell-shaped central core and lateral bodies.Histopathologic examination of skin nodules using hematoxylin and eosin stain showed proliferative and necrotic dermatitis,with ballooning degeneration of keratinocytes,and large,eosinophilic ring-shaped Bollinger bodies.All of these were consistent with those of members of genus Avipoxvirus.Virus isolation from skin nodules was conducted on duck chorioallantoic membranes of specific pathogen-free embryonated from 9 to 12 days duck eggs.At 64 hours after infection,chorioallantoic membrane thickening and white spot in embryo eyes were observed.DEF cells were inoculated with thicken chorioallantoic membrane,and 50% CPE was observed after 3 days.The specific green fluorescence in cells was detected by IFA.After three generations,it was identified as avipoxvirus by PCR.Worldwide phylogenetic relationship of avain poxvirus was constructed by conserved P4 b gene and DNA polymerase gene.According to this method,four samples were clonedand sequenced.YNY and YNE were from mallard duck and Yangjiang goose in Guangxi Yongning,and MDUPV1-GX01/2015 was from mallard duck in Guangxi Baise,the other was Chinese fowl poxvirus vaccine stain 282E4.The evolutionary tree was constructed by homologous comparison with 62 referenced sequence of avian poxvirus.YNY,YNE and MDUPV01/GX-2015 were divided into A5 subclade of waterfowl source.The A5 subclade was divided into two branches.MDUPV01/GX-2015 and three mallard poxvirus from Guangxi Baise and Chongzuo from 2013 to 2014 belong to A5.1,and the nucleotide identity was from 99.9% to100%.YNY and YNE strains belong to 5.2 branches,and their nucleotide identity was 99.9%.At least two species of waterfowl povirus have firstly been identified in China.The nucleotide identity of P4 b and Popr gene about Guangxi starins between A5.2 branch and A5.1 branch were 100% and from 87.4% to 87.6% respectively.The nucleotide identity of Popr gene about Guangxi starins between A5.2 branch and A1 were from 99.5% to 99.8%.It speculated that A5.2 branch were genetic recombination between A5.1 branch and A1 group.In 2015,the sample MDUPV01/GX-2015 collected from mallard duck pox in Guangxi Baise was purified by sucrose density gradient centrifugation method.The genome of the poxviruse was sequenced by illumina high-throughput sequencing technology.The genome of duckpox virus(DPV),was first assembled into a contiguous sequence of 230805 bp.The nucleotide composition is 31.02% G+C.Comparison of the genome with other six avipoxviruses which have been published in Vi PR,the 201 open reading frames were annotation and predicted their protein function.Comparison of the DPV genome with other Ch PVs,it revealed 90 conserved gene homologues,locating in the central coding region of the genome,accounting for 38.3% of the genome and encoding proteins involved in transcription and m RNA biogenesis,nucleotide metabolism,DNA replication and repair,protein processing,and virion structure.There were 56 members of the gene families,accounting for 31.7% of the genome,and located mostly at the end of the central coding region.These genes may function in the host cell functions,involving the host's immune escape,immunity,virus in cell or tissue tropism and other cellular functions.DPV could infect Cherry Valley Duck,mallard duck and Yangjiang goose in field through cohabitation with infeced mallard ducks,but not in Muscovy Duck and chicken.The course of mallard duck pox was clear.The incubation period was 5 to7 days,the obvious period began at 10 days and the transition period began at 19 days after infection.It lasted about one month.Under experimental conditions,the cutaneous form mainlyappeared in mallard duck.The nodules appeared in the nose,beak,eyelid,wings and flipper.Some ducks showed cutaneous or diphtheric forms,adding pox in oral mucosa,tip of tongue and throat.The morbidity and the mortality was 84.0% and 8.0%,respectively.A total of 597 tissue samples of the infected mallard duck were detected by q PCR.Virus DNA was detected in multiple tissues,and the highest levels was in nodules,followed by the esophagus,larynx and trachea.It was confirmed that chicken pox vaccine had a certain protective effect on DPV,and the protection rate was 64.7%.The results supplemented our knowledge of the morphology and gonome of DPV,pathogenesis and epidemiology of duck pox.It establishes the foundation for the study of the taxonomy,genetic evolution,pathogenic mechanism and prevention measures of waterfowl poxvirus.