Construction Of Recombinant Fowl Adenovirus 4 And Recombinant Duck Adenovirus 3

Fowl adenovirus serotype 4(FAdV-4)is the main pathogen of Hepatitis-hydropericardium syndrome(HHS).HHS is a viral disease characterized by pericardial effusion and liver necrosis with high incidence,no seasonality,and high mortality.Recently,it has been reported in many provinces of China with increasing infection rate.Therefore,study of the gene function,pathogenic mechanism and attenuation metod of FAdV-4 is of practical significance for constructing of safe and effective fowl adenovirus-based vaccine.In this study,the gene of interleukin-1 receptor antagonist was inserted at the 1966 bp deletion mutatant of the highly pathogenic strain of FAdV-4 CH/HNJZ/2015.The recombinant plasmid was transfected into LMH.The recombinant virus rHNJZ-IL-1Ra-1966 was successfully rescued.Comparing the replication dynamics of recombinant virus rHNJZ-IL-1Ra-1966 and its wild type,the insertion of IL-1Ra gene did not affect the replication in vitro.The results of Western Blotting and serial passage showed that recombinant fowl adenovirus rHNJZ-IL-1Ra-1966 being expressing IL-1Ra with genetic stability.In 2014,a new disease with symptom of characterized by liver hemorrhage,enlargement and necrosis outbroke in a Muscovy duck factory in Guangdong.The virus CH-GD-12-2014 was isolated from the diseased Muscovy ducks.The genome sequence and evolutionary analysis indicated that the virus belonged to avian adenovirus genus.Sequence alimentment of the fiber and hexon showed that the virus was a novel duck adenovirus,which was named duck adenovirus type 3(DAdV-3).In recent years,DAdV-3 has been spreading in many areas of China.It has high infection rate but low mortality.It is prone to secondary infection,causing significant economic losses.Therefore,it is urgent to develop safe and effective prevention and control measuresDuck virus hepatitis A(DVHA)is caused by duck hepatitis A virus(DHAV).DHAV infection is complicated and often mixed with other virues.Spreding of DVHA is accompanied with constantly mutating,which greatly challenge the control of DVHA.At moment the control of the DVHA being rely on the yolk antibody after infection.Vaccines are rarely used.Therefore,developing a safe and effective viral vector-based vaccine is demanding.In this study,the DHAV-VP1 gene was inserted into the genome of the DAdV-3 strain CH-GD-12-2014 by infectious clone engineering.The recombi-nant plasmid was transfected into DEF and the recombinant virus rDAdV3-DHAV-VP1 was successfully rescued.To compare the growth curves of rDAdV3-DHAV-VP1 and its wild type,the insertion of DHAV-VP1 reduced the replication rate at the early stage.This need to be further investigated in vivo.The expression of DHAV-VP1 protein was verified by Western Blot.The serial passage results proved that the recombinant virus rDAdV3-DHAV-VP 1 is of genetic stability.Muscovy duck parvovirus(MDPV)causes disease of young duck in many provinces of South China.Infected ducklings are stunted,causing huge economic losses.At present the control of MPVD is rely on strict biosafety measures,inactivated vaccine or attenuated vaccine.A vector based vaccine may overcome the disadvantages of the tradetional vaccines.In this study,the MDPV-VP3 gene was inserted into the genome of the DAdV-3 strain CH-GD-12-2014 by infectious clone engineeing.The recom-binant plasmid was transfected into DEF and the recombinant virus rDAdV3-MDPV-VP3 was successfully rescued.The titer of the recombinant virus rDAdV3-MDPV-VP3 is comparable to its wild.Western blot verified the expression of MDPV-VP3 protein.The results showed that the recombinant duck adenovirus expressing MDPV-VP3 protein was successfully constructed with high genetic stability.