Biological Characteristics And Pathogenicity Of Avipoxviruses From Water Fowl

Avian pox is an acute contagious infectious disease caused by members of the Avipoxvirus(APV)in poultry,wild birds and all kinds of birds,which is prevalent all over the world.The clinical symptoms of avian pox are mainly manifested as pox rash type lesions on the skin without or with a little feather in the skin,or as mucosa type lesions on the oral,throat or tracheal mucosa with scabby membrane.Avipoxviruses are mainly transmitted by insects,aerosols,broken skin and mucous membranes.Avian pox has huge impact on the poultry industry and poses a great threat to wild birds,especially endangered rare birds.Avian pox is rare in waterfowl such as ducks and geese,and the related researches are rare and poor.Our research group has previously detected avian poxvirus in domestic ducks and geese.In this study we have an attempt to explore the biological and pathogenic characteristics of an APV strain YNE isolated from goose and the other one strain MDUPV01/GX-2015 isolated from mallard duck,respectively.Skin nodule suspension samples of YNE and MDUPV01/GX-2015 were collected from natural infected geese and ducks were separately inoculated in the chorioallantoic membrane(CAM)of 9-12 days old SPF chicken embryo and continuously passaged.PCR amplification,pathological examination and electron microscopy were employed to investigate the culture characteristics of two APVs.PCR detection of APV P4b gene in CAM was positive.The eosinophilic inclusion bodies were observed in histopathology examination and the typical particle structure of poxvirus was observed in electron microscopy.These results confirmed that YNE and MDUPV01/GX-2015 could proliferated in the CAM of chicken embryo.The physicochemical characteristics of the two viruses were determined.YNE and MDUPV01/GX-2015 lost their infectivity after treatment at 55?for 30 min and 60 min,respectively.The EID₅₀ of two viruses were decreased by more than 100 fold after treated with trypsin(0.5%),p H=5 or p H=10.Two viruses also lost their infectivity after treated with chloroform(5%).The results showed two viruses were sensitive to heat,acid(p H=5),alkali(p H=10),trypsin and chloroform.Hemagglutination test showed that two viruses had moderate agglutination activity to erythrocytes of geese with the agglutination titers of1:16 and 1:64,respectively,and the hemagglutination activity could be inhibited by positive serum.The egg infectious dose(EID₅₀) of the 5^th generation virus passaged on the chicken embryo of YNE and MDUPV01/GX-2015 were 10^4.14 EID₅₀/0.1 m L and 10⁴ EID₅₀/0.1 m L,respectively,and virus content were 10^4.89 Copies/?L and10^4.62 Copies/?L,respectively.Furthermore,SPF chicken embryos were inoculated with YNE and MDUPV01/GX-2015 to determinate virus growth trend.The virus content in the infected CAM peak appeared at 4 and 5 days post infection(DPI),respectively,then came into the platform stage.The proteome of MDUPV01/GX-2015 strain was examined by liquid chromatography and mass spectrometry.Based on bioinformatics analysis and using avian poxvirus protein database as reference,108 APV protein homologues were identified,including 14 structural proteins,9 enzymes related to virus replication or virulence,4 virus transcription factor and 12 protein family members.The pathogenicity of YNE and MDUPV01/GX-2015 strains on chicken embryos was reflected in their influence on the incubation rate of chicken embryos and the lesions of CAM.The pathological changes on the infected CAM were the edema and thickening and the appearance of pox spots.The CAM of chicken embryo was thickened on the 2 DPI,the white nodule appeared on the 3 DPI.After that,the infected CAM showed continuous edema and thickening and increasing nodules.The virus in the chorioallantoic membrane,allantoic fluid,beak,larynx,trachea,heart,lung and spleen of the chicken embryo on the 9DPI were detected.The virus was only detected in the chorioallantoic membrane,but not in other tissues.Finally,the pathogenicity of MDUPV01/GX-2015 on chicken,pigeon and duck were performed.Firstly,the best way of inoculation was explored,and found that only the pricking method could produce typical clinical symptoms after inoculation;whilethe other inoculation methods,including nose drip,eye drip,nose drip plus eye drip and feeding,were all failed.2×10₄ ELD⁵⁰/0.1 m L virus was inoculated into mallar duck,chicken and pigeon by pricking method.Quantitative Real-time PCRwas used to detect the distribution of virus in the tissues and organs of mallar dduck on day 3,7 and 15 DPI.Virus was detected in palate,periocular and beak areas on the 3 DPI and 7 DPI.Virus titer was highest in all skin pox on the 15 DPI.Moreover,virus was also detected in larynx,trachea,heart and lung,but not in serum,brain,liver,spleen and kidney.Chickens and pigeons inoculated with MDUPV01/GX-2015 strain were observed for 15 days,and the tissues were dissected and collected for detection on 3,7 and 15 DPI.No clinical symptoms could be found in the chicken and pigeon inoculated with MDUPV01/GX-2015,and no virus was detected in the tissues and organs of the chicken and pigeon.In summary,these results fill in the knowledge gap of APV from waterfowl,and provide basic data for improving the taxonomy,genetic evolution and pathogenesis of APV.