Development of a live attenuated vaccine candidate against duck Tembusu viral disease
Guoxin LiXuyuan GaoYali XiaoShaoqiong LiuShan PengXuesong LiYing ShiYuee ZhangLei YuXiaogang WuPixi YanLiping YanQiaoyang TengGuangzhi TongZejun Li
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Embryonated
Attenuated vaccine
Flavivirus
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Strain (injury)
This study looks into optimal conditions for cultivating the recombinant strains of subtype H5 influenza virus. The study results in establishing optimal conditions (inoculation dose, incubation temperature, incubation time, and chicken embryos’ age) for growing the influenza virus. This study establishes optimum conditions for cultivating the A/Sichuan/26221/2014(H5N6)-PR8-IDCDC-RG42A and A/gyrfalcon/Washington/41088-6/2014(H5N8)-PR8-IDCDC-RG43A subtype H5 influenza virus recombinant strains in embryonated chicken eggs. Data on culturing influenza virus recombinant strains presented herein indicates that they can be used in developing subtype H5 highly-pathogenic avian influenza vaccines. The results of this research will serve as a basis for developing a new inactivated emulgated vaccine following the process previously used by RIBSP to design its commercial vaccine. These optimum conditions are an infective dose of 10000 EID50/0.2 cm3, an incubation temperature of 36±0.50С, an embryo age of 10 days for cultivating the recombinant strain A/Sichuan/26221/2014(H5N6)-PR8-IDCDC-RG42A. Using these culturing conditions allows a stable production of virus-containing materials with an infectivity level of not less than 8.45±0.24 log EID50/cm3, which is fully consistent with requirements for producing inactivated vaccines for avian influenza. And optimal conditions for growing recombinant strain A/gyrfalcon/Washington/41088-6/2014(H5N8)-PR8-IDCDC-RG43A in 10 days embryonated chicken eggs with the infective dose 100000 EID50 in the incubation temperature 350С. These optimum conditions are helping culturing a stable production of virus-containing materials with an infectivity level of not less than 8.74±0.06 log EID50/cm3, which is fully consistent with requirements for producing inactivated vaccines for recombinant strain A/gyrfalcon/Washington/41088-6/2014(H5N8)-PR8-IDCDC-RG43A. Аn optimum incubation time of 48 hours, and relative air humidity of 55±5% for cultivating both recombinant strains.
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Bluetongue is a major infectious disease of ruminants that is caused by bluetongue virus (BTV). In this study, we analyzed virulence and genetic differences of (i) three BTV field strains from Italy maintained at either a low (L strains) or high (H strains) passage number in cell culture and (ii) three South African "reference" wild-type strains and their corresponding live attenuated vaccine strains. The Italian BTV L strains, in general, were lethal for both newborn NIH-Swiss mice inoculated intracerebrally and adult type I interferon receptor-deficient (IFNAR(-/-)) mice, while the virulence of the H strains was attenuated significantly in both experimental models. Similarly, the South African vaccine strains were not pathogenic for IFNAR(-/-) mice, while the corresponding wild-type strains were virulent. Thus, attenuation of the virulence of the BTV strains used in this study is not mediated by the presence of an intact interferon system. No clear distinction in virulence was observed for the South African BTV strains in newborn NIH-Swiss mice. Full genomic sequencing revealed relatively few amino acid substitutions, scattered in several different viral proteins, for the strains found to be attenuated in mice compared to the pathogenic related strains. However, only the genome segments encoding VP1, VP2, and NS2 consistently showed nonsynonymous changes between all virulent and attenuated strain pairs. This study established an experimental platform for investigating the determinants of BTV virulence. Future studies using reverse genetics will allow researchers to precisely map and "weight" the relative influences of the various genome segments and viral proteins on BTV virulence.
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For find out the reason of egg drops on breeder and egg-laying ducks,the partial sequence of a new flavivirus isolated from the oviduct of egg droping duck was determined and analyzed.Allantoic fluid on SPF embryonated egg was observed under electron microscope and histopathology was study.Sequence analysis revealed the strain was most closely related to Ntaya virus group in the gene Flavivirus of the family Flaviviridae.Electron microscopic observation revealed that the virion with capsul in the isolate was 40 nm-60 nm in diameter.The histopathological tests showed the heavy hyperemia and hemorrhage in oviduct,and the hyperemia and inflammatory reaction in brain.Inflammatory infiltration was also observed in the heart.
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1) In comparative experiments with Enders strain adapted to the chorio-allantoic cavity of the developing chick embryo and the newly isolated Towata strain of mumps virus, it was noticed that Enders strain was considerably attenuated. Towata strain showed a wide host range of tissue culture cells, mild virus growth in suckling mouse brains and high pathogenicity for the parotid glands of monkeys.2) Attenuated Enders Strain was used for an inhalation test with monkeys as the live mumps vaccine. All monkeys showed good antibody responses without clinical manifestations.3) Passage of the virulent Towata strain in the chorio-allantoic cavity of the embryonated egg was successful. During many passages, Towata strain lost the infectivity to various cells, acquiring biological characteristics similar to Enders strain.
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Avian influenza A H9N2 viruses are circulating in domestic poultry worldwide. In this research a low-pathogenic AIV H9N2 was multiplied on MDCK cell line and SPF eggs and irradiated by a Nordian gamma cell instrument. Irradiated and non-irradiated AIV samples were titrated by TCID50 and EID50 methods, respectively. Haemagglutinin antigen was analysed by Haemagglutinin test. Infectivity of irradiated virus samples was determined by cell culture and egg inoculation methods. The virus titration decreased as the dose of gamma radiation increased. AIV proliferation on cell culture can be inactivated by gamma irradiation at a lower dose of gamma-ray (20 kGy) than the virus inactivation on embryonated eggs (30 kGy). The safety test showed complete inactivation of AIV on allantoic fluid with gamma-ray doses: 30 kGy and 20 kGy for virus on MDCK cells after four blind cultures.
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In the present study we describe the rapid development of an attenuated live vaccine for GA08, a new serotype of infectious bronchitis virus, using a heat-treatment method. Incubation of the GA08 strain of IBV at 56 degrees C and passage in embryonated eggs was used as a method to fast track the attenuation process. The virus was incubated in a 56 degrees C water bath and aliquots were removed every 5 min for up to 1 h, and then each aliquot was inoculated into 10-day-old embryonated eggs. Virus with the longest incubation time that produced lesions in the embryos was harvested, again incubated at 56 degrees C as described and passaged in embryonated eggs. Attenuation of the virus, designated GA08/GA08HSp16/08, was verified in 1-day-old specific pathogen free chicks. A 10x dose of the vaccine was found to be safe for 2-week-old broiler chicks of commercial origin. The efficacy of the heat-treated attenuated virus was determined by vaccinating broiler chicks of commercial origin at 1 and 14 days of age intraocularly/intranasally. Vaccinated birds that were challenged with 10(4.5) median embryo infectious doses of pathogenic GA08 virus/bird at 28 days of age were protected from the disease, and challenge virus was only detected in the trachea of one of 21 birds by real-time reverse transcriptase-polymerase chain reaction at 5 days post challenge. The attenuation process took 10 weeks to complete, which is a substantially shorter time than required to attenuate infectious bronchitis virus by serial passage in embryonated eggs without heat treatment (38 weeks or more).
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ABSTRACT The propensity of RNA viruses to revert attenuating mutations contributes to disease and complicates vaccine development. Despite the presence of virulent revertant viruses in some live-attenuated vaccines, disease from vaccination is rare. This suggests that in mixed viral populations, attenuated viruses may limit the pathogenesis of virulent viruses, thus establishing a virulence threshold. Here we examined virulence thresholds using mixtures of virulent and attenuated viruses in a transgenic mouse model of poliovirus infection. We determined that a 1,000-fold excess of the attenuated Sabin strain of poliovirus was protective against disease induced by the virulent Mahoney strain. Protection was induced locally, and inactivated virus conferred protection. Treatment with a poliovirus receptor-blocking antibody phenocopied the protective effect of inactivated viruses in vitro and in vivo , suggesting that one mechanism controlling virulence thresholds may be competition for a viral receptor. Additionally, the type I interferon response reduces poliovirus pathogenesis; therefore, we examined virulence thresholds in mice lacking the alpha/beta interferon receptor. We found that the attenuated virus was virulent in immunodeficient mice due to the enhanced replication and reversion of attenuating mutations. Therefore, while the type I interferon response limits the virulence of the attenuated strain by reducing replication, protection from disease conferred by the attenuated strain in immunocompetent mice can occur independently of replication. Our results identified mechanisms controlling the virulence of mixed viral populations and indicate that live-attenuated vaccines containing virulent virus may be safe, as long as virulent viruses are present at levels below a critical threshold.
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