| Duck Viral Hepatitis(DVH) is an acute, highly contagious and lethal disease within 3weeks of age ducklings.The main symptoms of diseased ducklings are hepatitis, and the pathogen is duck hepatitis virus. The characteristics of the DVH are short course, high mortality, high mortality rate, and usually onset small ages. Adult ducks can be infected but do not fall ill generally, so it is also known as duckling viral hepatitis. Duck Viral Hepatitis can be caused by small RNA virus, avian virus duck hepatitis virus and duck astrovirus.DHV was divided into type1, type 2 and type 3. At present, the mainly popular duck hepatitis virus is DHAV-1 in china, DHAV-3 was found and burst out in many areas of our country since 2012. In recent years, type 1 and type 3 are Popular at the same time. in our country,vaccination for duck hepatitis is the most fundamental and effective measures to prevent, but conventional vaccines often fail to achieve satisfactory results in production. The situation to prevent and control the DVH is not optimistic as DHAV-3 sparked more widespread harm in duck industry. Therefore, accelerate the in-depth study of multivalent vaccine about DVH is particularly important.As the major capsid protein in the genome of DHAV-3, VP1 can code the major protective antigenic sites of virus and identify the corresponding receptors. In different strains, VP1 varied greatly, and with maximum genetic diversity. The biological significance of this variability is worth a further study. In order to domesticated DHAV-3 wild strain and attenuated vaccine strain, this study selected five strains of DHAV-3 virus and passaged in SPF chicken embryos, cloning and sequencing of VP1 gene and sequence alignment analysis,summing up the relevant rules to provide the theoretical basis for the genetic variation of DHAV-3.This study was divided into two parts:The first part, 5 strains of DHAV-3 virus were cultured on SPF chicken embryos.Choose 5 virulent of DHAV-3( 4 wild virus 1C、2C、3C、4C and molecular cloning of the virus 5C) treated as primary poison, and were inoculated into the allantoic cavitythe allantoic of SPF chicken embryos, inoculation amount was 0.2 ml/embryo. Retained 24 hours after the death of chicken embryos, and store at 4 refrigerator freezing for 4-12 h, then collected allantoic fluid of chick embryo in the sterile situation, and corresponding access the next batch of chicken embryos till 80 generation. The results shows : before the 5generations ago, between 24-96 h the chicken embryos had no death phenomenon, embryo body without obvious bleeding points also; Continuous spread 6th generation, chicken embryos began to dead, when reached 19 th generation, five chicken embryos inoculated with the virus appears all died, but the time of death is not the law; After 36 generations, the time of chick death controlled in the basic 120h; After 55 generations, the death time of the embryo controlled between 48 h and 72h; After 73 generations, the time of chick death controlled in the between 36 h and 48h; The embryo had significant pathological changes and the time of death tends to stable at 48 h until reached 80 generations. In addition, in the process of passage, by observing each strain and each generation of the death chick, found allantoic fluid was translucent. With the passage number increased, embryo death time is shortened, the pathological changes of the embryo become more and more obvious. The development of embryo become slow gradually,and the embryo congestion serious. The head,body and limbs of embryo appear a large number of surface spotting. liver present green and yellow or yellow, and had punctate or punctate bleeding. Besides the size of the embryo is smaller than normal embryos.Choose the allantoic fluid taken from the 2nd、3rd、4th、5th、10th、15th、20th、30th、40th、50th、60th、70th、80th virus came from the passage of two strains of the virus 4C and 5C. Experimental results show that, the first 10 generations of 4C and 5C strains of virus can cause the morbidity and mortality of ducklings, and organ tissues have significant pathological changes. Chicken embryo virus of the 15 th generation to the 80 th generation did not cause morbidity and mortality in Ducklings, but the viruses of the 15 th generation to the 20 th generation can cause slight liver lesions in ducklings. The liver of duck did not occur tissue lesions from the beginning of the 30 th generation no matter shows that after the passage of SPF chick embryo, the virulence of DHAV-3 virus gradually attenuated,and no longer pathogenic after the 30 generation until the 80 th generation of virulence of the virus.The second part: the sequencing and sequence analysis of the VP1 gene of DHAV-3strain of chicken embryo.Cloning and sequencing the strains of the virus every 5-10 generations of VP1 gene,comparing the gene sequence obtained gene sequence in the horizontal and vertical direction.After the comparison, we can found that: 5 strains of DHAV-3 isolates in SPF chicken embryo variation rules are not all the same. In the process of the passaging in SPF chicken embryos, viru’s virulence of the 5 strains of DHAV-3 were weakened. Different strains of different generations of the VP1 protein amino acid mutation sites have undergone repeated variation and synchronous variation phenomenon, VP1 protein of different strains have different numbers of amino acids in the high variable region. It can’t be summed up the same rules showed that there was no direct relationship between the change of DHAV-3 and the mutation of VP1 protein. |
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