| In 2010,an infectious disease caused by Duck Tembusu virus has outbroken in the main laying duck farming areas in China,the clinical symptoms of which are decline in egg production and the main pathological features are ovarian inflammation and viral encephalitis.The disease brought a huge loss for the duck industry.At present,the main ways of cultivating DTMUV in China are chicken or duck embryos and adherent cell cultures,which have some limitations.In order to study the large-scale suspension culture technology of DTMUV,a strain of Duck Tembusu virus was isolated and identified in this study.Conducting the research of serum-free total suspension culture technology through duck embryonic stem cell derived cell EB66,it could provide a new idea for further exploring the culture technology of Duck Tembusu virus and the development of Duck Tembusu vaccine.In December 2017,a certain amount of suspicious materials were collected from a large-scale laying duck farm in Guangdong province,and it was sterile disposed and inoculated into a 9 day-old duck embryo.As a result,the duck embryo died after 3 days.The embryo body became small and its liver and brain were swelling bleeding.The allantoic fluid had no hemagglutination.The RNA extracted from collected allantoic fluid was identified as positive by RT-PCR,and the common exogenous viruses are negative.This virus can proliferate on DEF cells.Pathological changes would appear 48 hours later after inoculation,which ELD50and TCID50 were 10-3.67/0.2 m L and 10-4.73/0.2 m L,respectively.In order to obtain the full-length genome and genetic evolution of the isolates,11 pairs of primers were designed for the virus in this study.Finally,the whole genome sequence of the isolate was obtained and size of which was10990 bp.The viru was named DTMUV QY17.The highest homology was found between the virus and STIAVAN virus of flavivirus genus,with 86.9%homology.In the homology analysis among this virus and 19 Tembusu virus strains isolated from goose,mosquito,chicken and sparrow sources from 2010 to 2016,the homology was over 96.2%.Therefor,it can be seen that the genomic variation of Duck Tembusu virus is not significant and the virus is relatively stable.To study the pathogenicity of the DTMUV QY17,15 1-day-old Cherry Valley ducks were selected,with each duck's leg injected 0.2 m L virus(containing 1000 ELD50).Three days later,death occurred in the experimental group,with a mortality rate of 20%.After dissection,swollen bleeding was found in the brain,liver,spleen and other organs of the dead ducks.Pathological sections showed viral encephalitis,pancreatic granular necrosis,necrosis of liver and kidney cells,lymphocyte infiltration and proliferation,etc.Through q PCR detection,the results showed that ducks had the highest viral contents in liver,spleen,pancreas and brain at 3 days after challenging,and the viral content in tissues and organs began to decrease.To study whether the isolated strain can be stably passaged in EB66 cells,in this research,the DTMUV QY17 strain was inoculated into EB66 cells and tamed after blind transmission to the twentieth generation.The results showed the ELD50can be stabilized at about 10-5.0/0.2m L.In the nucleotide and amino acid analysis of NS1 and E protein genes of cultured virus,the homology was over 99.1%.Only one had amino acid mutation.It was proved that DTMUV QY17 strain could be stably passaged in serum-free suspension culture of EB66cells,and the viral titer increased with the passage times.To study the technology of enlarging DTMUV QY17 strain by serum-free suspension culture of EB66 cells,firstly,the optimum inoculation concentration of EB66 was 1.0×106cells/m L and TOI was 60 h.Secondly,DTMUV QY17 strains were cultured in serum-free suspension with different MOI and harvesting time respectively.Throug the detection of ELD50,it showed that the optimum MOI was 10-5 and the optimum harvest time was 72 hours.The optimized conditions in shaking flask were replicated in a 5 L bioreactor.The highest ELD50could reach 10-5.67/0.2 m L,which was higher than the highest ELD50(10-5.33/0.2 m L)in shaking flask under the same conditions.The experiment of enlarging culture technology has been preliminarily realized.The virus in serum-free suspension culture was inactivated by 2/1000 formaldehyde for24 hours.The 14-day-old Cherry Valley ducks were immunized by the inactivated emulsification vaccine.The second immunization was performed 14 days after the first immunization.The results of commercial ELISA kit showed that the level of antibody was low,vaccine preparation and serological detection in this study need further study. |
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