| Hydropericardium syndrome(HPS)is an infectious viral disease caused by FAd V-4(serotype 4 fowl adenovirus),and It was also known as “Angara Diesase” because its first outbreak was observed in the Angara Goth,Pakistan in 1987.FAd V-4 is non-enveloped double stranded DNA-viruses,measuring 70-90 nm in size and belongs to the species Fowl Adenovirus C,genus Aviadenovirus,family Adenoviridaethe.The disease occurs predominantly in broilers and layers at 3-5 weeks,characterized by yellow or canary yellow hydropericardium and always with hepatomegaly.Since July 2015,there have been a large number of FAd V-4 outbreaks in small and medium chickens farms,in several parts of China,not only in broilers also in layers,causing considerable economic losses.However,to date,there have not been effective prevention and control measures due to lack of the molecular and genetic evolution characteristics and pathogenic characteristics of FAd V-4strains that remain circulating in China.Hence,in this study,we collected samples from flocks where HPS outbroke in 12 different regions of Henan,central China,and conducted the isolation and identification and molecular epidemiology,also investigated the pathogenicity of the strains,aimed to provide new clues to prevent and control the FAd V-4 strains.We collected samples from 12 different regions in central China,Henan,such as Anyang,Hebi,Puyang,Xinxiang,Jiaozuo,Zhengzhou,Kaifeng,Shangqiu,Xuchang,Zhoukou,Pingdingshan,Luohe,and isolated the strains via the chorioallantoic membrane(CAM)route,then identified the strains by PCR and sequencing.Luckily,12 strains from different regions were isolated and proliferated stably in vitro via the CEL cells,which laid the foundation for further study of the FAd V-4 strains.We conducted the molecular phylogenetic analyses which based on the hexon genes and constructed the phylogenetic trees including 12 FAd V-4 isolates from this study and 24 reference strains.It revealed that these outbreak-associated FAd V-4 strains all belonged to serotype FAd V-4,species FAd V-C.And the genetic evolutionary relationship between the isolates from this study and these before 2015 is very relatively far,on the contrary,the genetic evolutionary relationship between the isolates from this study and the two India strains PK-01 and PJ-06 is very close.At the same time,the alignment of nucleic acids and amino acids revealed that: in comparison with ON1,KR5,MX-SHP95,PK-01,PJ-06 strains within the same cluster,the nucleic acids divergence of the new isolates from this study were 1.31,1.10,1.42,2.77 and 2.84%,respectively.Finally,we postulate that ancestors of the new strains is not the local strains but earlier strains in India.Basing on the sequence of hexon gene,the primers were designed,and the standard plasmid was constructed,and then,we developed a real-time polymerase chain reaction(PCR)assay to detect and quantitate FAd V strains.There was a good linear relationship between the Ct values and the copies of standard plasmids(102-109).The experiments proved that the specificity and reproducibility of this assaywas high.At the same moment,the HNZZ strains and a HPS-positive liver supernatant were selected and injected SPF chickens at 21 days by intramuscular injection,and the control group were injected isodose sterilized PBS by the same method.The mental state,incidence,diet,drinking and survival time were observed and recorded daily.After injection,the injected groups appeared to be depression,inappetence,drowsiness,lazy and all died within 2 to 3 days.What's more,the typical symptom: yellow hydropericardium and hepatomegaly were observed after dissection.In contrast,the control group appeared orthophrenia,normal drinking and diet,without death and after dissection,obvious clinical symptoms and pathological damage were not observed.In conclusion,the isolates were highly pathogenic and consistent with the clinical symptoms. |
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