Studies On Duck Hepatitis A Virus Serotype 3(Strain HuB60) Safety And Genetic Stability

Duck hepatitis A virus serotype 3(DHAV-3)is one of the main pathogens causing hepatitis of ducklings in recent years.Therefore,it is imperative to develop a safe and effective vaccine for serotype 3 duck hepatitis A virus.Currently,a virulent strain HuB of DHAV-3 has been isolated,and was attenuated by serial passages in SPF chicken embryos60 times,the attenuated strain named HuB60.To figure out safety and genetic stability of the attenuated strain,this study of the strain of HuB60 continuous regression on SPF duckling virulence reversion,from clinical and anatomical observation,test of pathological histology and liver function,and the virus gene sequencing analysis,provide theory basis for breeding a promising vaccine candidate strain.The main research contents include:1.The clinical and anatomical observation of ducklings about HuB60 virulence reversionThere results of HuB60 virulence reversion ducklings for the clinical and anatomical observation show that the drinking water,food intake,growth conditions and the spirit of the ducklings are normal.There were no visible pathological changes on the liver,spleen,kidney,pancreas,lungs,brain,bowel by necropsy observation;The results of the ducklingsc1 inical symptoms showed that inoculated virulent strain(DHAV-3 SD)after24-48 h began to appear neurological symptoms such as sensitive to external stimuli,ataxia,heads drawn back and leg stretched,and then died with obvious opisthotonus posture.Some duckling suddenly fell spasm of death on acute process with no any abnormal behavior;there were some serous hepatic pathological lesion on hepatomegaly liver with very brittle,dotted or strip bleeding by necropsy observation;The results show that HuB60 has the characters of safety and stable virulence.2.The detection of the liver virus each generation of virulence reversion bout HuB60The results show DHAV-3 can be detected in the duckling liver tissues of each regressive passage by RT-PCR and the DHAV-3 can be isolated from the duckling liver tissues of each generation of virulence reversion by the method of inoculated chicken embryos;the viral titer of the former second regressive passage is low at the range of 10-4.70 and 10-4.92 ELD50/ml,the viral titer is relatively stable at the range of 10-7.02 and 10-8.08ELD50/ml after the third regressive passage.3.The study of the effects of HuB60 of duckling liver functionBlood samples were taken,and then serums of blood samples were separated at the48 th hour after inoculation HuB60 for ducklings of each generation of virulence reversion,Alanine aminotransferase(ALT),Aspartate aminotransferase(AST),Glut aminetransferase(GGT),Alkaline phosphatase(ALP)and other serum enzymes and bilirubin were detected to reflect the liver function,The results show that four serum enzymes and total bilirubin(TBIL)of the test ducklings compared with healthy group,there have no any obvious changes.But the content of TBIL,ALT,ALP and AST increased significantly in the ducklings of virulent group at 48 h compared with healthy control and test group.The experimental results showed that HuB60 strains have no damage to ducklings liver cells.4.The study of damage to duckling tissues and organs of HuB60Pathological histology examination show that there have no any abnormal changes on organization structure for livers and the main internal organization of 5th and 8th generation of virulence reversion.As for the test group ducklings,the dividing lines of liver hepatic cords and liver lobules are clear;there is no foreign matter in the alveoli,glomerular structure clear,no swelling and no hemorrhage.At same time,marked historical change occur at various tissues of ducklings inoculated virulent strain(DHAV-3 SD).For example,liver congestion,liver cell swelling and necrosis,accompanied by infiltration of lymphocytes,spleen splenic lymphocytes sparse,disappearance or obvious germinal center,a large number of apoptotic cells in the spleen,kidney glomeruli swelling,renal cysts disappeared,heart muscle fiber rupture,heart hemorrhage.The organization pathology test proved that the virulence of HuB60 is not easy to reverse.5.The research on the genetic stability of HuB60The full length of HuB60 has 7773 nt,including the 651 nt of 5'UTR,366 nt of3'UTR and 6756 nt of ORF,not including the Poly(A)tail,where in the ORF may encode2252 amino acids poly protein.The results show that there is no nucleic acid bases insertion or deletion in whole genome sequence by HuB60 and HuB60-F5 of the whole sequence alignment and analysis.There are only 6 nucleic acid bases displacement but no amino acid change,that is to say,these nucleic acid bases changes make no difference for pathogenicity of HuB60.Compared with HuB60-F8,2 bases mutation in the ORF of HuB60 caused the amino acid change,there are two amino acid sites located in 2C polyprotein.The proline(P)at 1242 site and glutamine(Q)at 1418 site of HuB60,turn into leucine(L)and arginine(R)respectively of HuB60-F5.The result suggest that amino acid change in this site may be associated with the change the virulence of HuB60.But the twoloci amino acid change will cause HuB60 virulence return whether or not needs further study.In the test of eight generations of virulence reversion experiment,the results indicate that the HuB60 has the character of genetic stability and virulence return less risk from clinical and anatomical observation,liver function index determination of serum enzymes,histologic examination and molecular biology.