Development of Duck Viral Hepatitis Living Vaccine(A66Strain)
|Nanjing Agricultural College
|duck viral hepatitis living vaccine A66strain research anddevelopment
Duck viral hepatitis (DVH) is a highly lethal acute infectious disease of ducklings; its main characteristics is hepatitis and the pathogen is Duck hepatitis virus (DHV). DHV1is the mainly epidemic in China. Vaccination is the most effective measure to prevent the disease, and there is no commercial vaccine in China. In the study, In the study, we selected the60passages virus (E60) of DHV1A66attenuated strain by chicken embryo passage as the origin virus, by cloning selection on SPF chicken embryo acquired DHV A66strain. On the basis of the detection of the safety, immunogenicity, purity and the genetic stability of A66strain, we do further research of the safety, production technology and trial production of the vaccine, and completed the clinical test research of the vaccine after the permission of the Ministry of Agriculture, finally, we developed DHV living vaccine (A66strain).1. Cloning selection and purification of DHV1A66attenuated strainTreating the E60of DHV1A66attenuated strain by chicken embryo passage as the origin virus, cloning selection of inoculation on SPF chicken embryos by the limited dilution for3times, purified cultivation for2times and1times expanding cultivation till66passages(E66), detecting the virus titre,virulence,immunogenicity, purity and exogenous virus, the results showed that the E66has met the requirements of producing vaccine. Therefore, we determined the chicken embryo virus E66of DHV as the origin virus for the development of DHV living vaccine, which we named DHV A66strain.2. The safety, genetic stability, immunogenicity and purity of DHV A66strain1-day-old ducklings were inoculated by subcutaneous injection and oral route with the E66passage virus of DHV A66; And the clinical observation of the ducklings was normal; there were no visible pathological changes on liver and other visceral organs by necropsy observation; histological examination of liver and other tissues was normal. Then,201-day-old ducklings inoculated by subcutaneous injection with a large dose of this virus were collated blood to do cell count, erythrocyte sedimentation rate, to determine the hemoglobin and other blood tests at different time after inoculation, at the same time, to detect alanine aminotransferase(ALT), aspartate aminotransferase(AST), glutamine transferase (GGT), alkaline phosphatase (ALP) and other serum enzymes and bilirubin to reflect the liver function by separated serum. The results showed:the measured values of blood routine, four serum enzymes and total bilirubin (TBIL). of the test ducklings have no obvious difference with healthy control ducklings, but there were significant (P<0.05) or extremely significant difference (P<0.01) between the measured values of the virulent control ducklings after24-36h inoculation and healthy10ducklings.In terms of genetic stability of strains, using101-3days old ducklings infected by the E66passage virus of DHV1A66each time, we did the test of virulence rejuvenation by the continuous passage for10times, the duckling liver tissues of each regressive passage can be detected DHV, the virus titre of which is relatively stable at the range of103.5-4.8ELD50/ml; the ducklings of each regressive generation are healthy, pathological observation and pathological examination are all normal; the measured values of blood routine, four serum enzymes and total bilirubin (TBIL) of ducklings for each regressive generation are all normal. Compared and analyzed the the whole genome sequence between A66-F10and F0of DHV1, the results show that there is not insertion or deletion in whole genome sequence, with the difference of only6nucleotides, more than99.9%homology, and the sequences of ORF amino acid don’t change. The above prove DHV1A66attenuated strain has a good safety and genetic stability.Continuous passage on9-10days old SPF chicken embryos inoculated with the virus of DHV1A66till E80generation, we determined the content of each generation virus and the immune ability of1-day-old ducklings, which showed that:the virus titre of different generations is in10ELD50/lml to10ELD50/lml, the IMD50of subcutaneous immunization is in106.10IMD50/lml to10640IMD50/1ml, and indicated that the immunity of strain A66is stable and good. Accordingly E66is the original seed passage, E67-72are basic seed passages, the sub-cultivation of the seeds is not more that3generations, that is E75is the highest passage generation of the antigen used in production. By the determination of the minimum immune amount of the mix virus of E66, E70and E75chick embryo passages of A66strain, and the immune test with the minimum immune dose of different times taken by the high chicken embryo passages E75of A66strain, we determined that the minimum immune of A66strain is103.3ELD5o; we determined that the immune dose of A66strain should be higher than104.3ELD50, according to the results of immune tests and integrated consideration of other factors. Under the current provisions of "China Veterinary Pharmacopoeia", to determine the bacteria and the exogenous virus, the result was negative for bacteria, mycolasma,12types of exogenous virus need to determine by the current provisions of "China Veterinary Pharmacopoeia" and chicken infectious anemia virus (CIAV), which indicated that the virus seed of DHV A66strain is purified.3. System identification of duck hepatitis virulent strain (W strain) used for detectionAfter the rejuvenation of DHV1W virulent strain inoculated ducklings, we did the virus passage, detected the virulence, stability and serological specificity, and compared the virulence with the virulent DHV1R85952, etc, in order to determine DHV1W suitable for detection. The text of the dose of attacking virus and retention period of W strain show that1-7day-old ducklings immunized10LD50/duck and7-10day-old ducklings immunized104LD50/duck; the retention period of the liver tissue freeze-dried of W strain, stored at-20℃and-70℃,are respectively for5and8years.4. The safety, effectiveness and retention period of the duck virual hepatitis living vaccine (A66strain)5batches A66vaccine made in laboratory, respectively, by subcutaneous injection and oral route on1day-old ducklings, are done the safety texts of a single dose of vaccination, a single dose of repeat vaccination and a large dose of vaccination, the spirit, feeding, drinking, growth and weight gain of the ducklings were all normal by clinical observation; the results of liver function test, anatomical observation of liver and other organs, and the liver histological examination were normal. The results showed that:A66live vaccine is safe and has no side effects for young duckling.Using the virus seeds of the highest passages of attenuated strain A66(E75) for the production of vaccine to prepare three batches, each batch of vaccine was treated with subcutaneous injection, intramuscular injection, drinking water and oral route on10susceptible ducklings. The results showed that:the ducklings immunized by subcutaneous and intramuscular injection can generate protective immunity in2to3days; the ducklings immunized by oral, intranasal and drinking water means, produce good protective effect in5days. Using1/10dose of the three batches vaccines to immune susceptible1-day ducklings, which are attacked with a virulent virus after5days (120h), immuned ducklings are all protected, and this indicates that the vaccines produced by the highest passages of A66have good immune efficacy. The immune period of the1-day-old ducklings inoculated the immune dose of vaccine is more than60days, that means once immunization can effectively protect the duckling to live through susceptible period (less than6-weeks-old ducklings.)Each201-day-old ducklings immunized with with10-time dose of A66vaccine, respectively by the oral route and subcutaneous injection, after5days,10of which selected randomly cohabit and feed for5days with201-day-old susceptible ducklings for the1st generation;10of1st generation cohabitation infected ducklings cohabit with201-day-old susceptible ducklings for the2st generation; in this way,5consecutive passages of cohabitation infection experiments are done. The anal swab samples of inoculated ducks and cohabited ducks collected regularly are done PCR, to determine the infection and detoxification of DHV1A66inoculated ducks or5-day cohabited ducks are done the attacking-virus protection text to determine the conservation status of cohabitation infection. The results showed that:the ducklings immunized A66vaccine by subcutaneous injection and oral route can both excrete the virus with the feces; the cohabited susceptible ducklings can be infected by the fecal-oral route, infected ducklings can acquire the immune protection; with the increase of cohabited generations, the excretion time of virus is delayed gradually, the infectious rate is declined gradually, the phenomenon of cohabited infection ends basically till5st generational cohabitation. immune protection; with the increase of cohabited generations, the excretion time of virus is delayed gradually, the infectious rate is declined gradually, the phenomenon of cohabited infection ends basically till5st generational cohabitation.The ducklings from hatching eggs produced by ducklings immunized by DHV vaccine were detected the level of maternal antibody of DHV using chick embryo neutralization test; the ducklings with different levels of maternal antibodies were immunized with1plume dose at1day old, and then were attacked the virulent DHV1W on second day and5st day after immunization; the results showed that the level of maternal antibodies has no obvious effect on immune effect of the vaccine.3batches A66strain live vaccine prepared, saved at different temperatures and period, were detected the changes of the half chicken embryo lethal dose(ELD50) to judge the stability of vaccine; the retention period of vaccines was determined by attacking-virus immune protection test, observation of physical properties, the measurement of water and vacuum.The results showed that:A66live vaccine can be stored at-15℃in2years,2～8℃in1year; the titre of vaccine diluted with saline and stored for8h at25℃isn’t declined obviously. 5. Production technology and intermediate test production of the duck virual hepatitis living vaccine (A66strain)The test of level orthogonal design is to select the freeze-dried protectant and to optimize the freeze-dried curve, by selecting sucrose, gelatin, protein (amino acids) and other components, and by controlling the degree of cacuum, heating rate and other factors. According to the results of the tests, the productive technology of A66strain live vaccine is that, Dilution of strain virus by100-1000times, inoculation of9-11days SPF chicken embryos by urine cysts,0.2ml/embryo, harvest of dead chicken organization (body+membrane+liquid) after36-72h inoculation, homogenate of the tissue for3-5min and save; The formula of optimal protectant and freeze-dried condition is:5%sucrose,1.5%gelatin,6%glycine protection agents, vacuum0.02mbar, heating rate of1.5℃/min.1:1ratio of antigen and protective agents, in the freeze-drying curve:-40℃3h;-25℃13h;-10℃4h;25℃4h, which is the best freeze-dried vaccine. To verify the three batches by the identified protectant and the freeze-dried condition, its physical properties, vacuum, residual water content are in line with the "China Veterinary Pharmacopoeia" and the loss of virus titre is low to100.6.According to this study, we developed a draft "Duck hepatitis vaccine trial procedures and quality standards ", and produced5batches of vaccine A66, a total of6.05million doses in Nanjing Tianbang Biotechnology Co., Ltd. To detect5batched of vaccine by trial procedures and quality standards, the results of physical traits, sterility test, Mycoplasma test, virus content, security testing, effectiveness testing, residual moisture content, determination of the vacuum are all in line with the standards. Which indicated that the production technology is reasonable; the production is stable and is suitable for large-scale production.6. The duck virual hepatitis living vaccine (A66strain) in clinical trialsAfter the acquirement of the approval document of clinical trials of veterinary biological products of the Ministry Agriculture (number:200844) on27December2008, we had applied5batch products for clinical trials to test a total of59,000young ducklings in5duck farms of Anhui Province since February to June2009.