Observation On Biological Characteristics Of Recombinant Duck Plague Virus Infected Host

Bacterial artificial chromosome?BAC?is the second generation cloning vector system of large fragments of DNA.At present,our laboratory has successfully inserted BAC vector into Thymidine kinase?TK?gene sequence of duck plague virus Chinese virulent strain?DPV CHv?genome,thereby obtaining recombinant duck plague virus DPV CHv-BAC-G.However,the biological characteristics of the recombinant virus itself have not been fully clarified.However,previous studies only preliminarily studied some characteristics of DPV CHv-BAC-G in vitro cell culture.Based on this situation,the biological characteristics of DPV CHv-BAC-G were studied comprehensively by poultry embryos and ducklings,and the effects of transient expression of DPV CHv-TK on the proliferation of DPV CHv-BAC-G were analyzed in vitro.The main contents and results were as follows:1.Biological characteristics of DPV CHv-BAC-G virus in chicken embryosThe DPV CHv-BAC-G strain?virus copy number:10⁷.0.0 copies/0.2mL;dose:0.2mL/embryo?was inoculated into the allantoic cavity of 10-day-old SPF chicken embryos.All chicken embryos survived normally after 10 days,indicating that DPV CHv-BAC-G was not lethal to chicken embryos.The viral load in brain,liver and intestine tissues was detected by real-time quantitative PCR.The results showed that the viral copy number in each tissue sample was extremely low,and the viral genome was not detected in individual samples,indicating that the proliferation ability of DPV CHv-BAC-G in chicken embryos was extremely weak.2.Biological characteristics of DPV CHv-BAC-G virus in duck embryosThe DPV CHv-BAC-G strain?virus copy number:10⁷.0.0 copies/0.2mL;dose:0.2mL/embryo?was inoculated into the allantoic cavity of 10-day-old duck embryos.The mortality rate of duck embryos was 100%after 144h,which indicated that DPV CHv-BAC-G had high lethality to duck embryos.The pathological changes of brain and liver caused by DPV CHv-BAC-G were observed to be significant from three aspects:anatomical changes,histopathological changes and ultrastructural pathology,and the degree of lesions increased with the inoculation time,indicating DPV CHv-BAC-G is highly pathogenic to duck embryos.The morphological structure of DPV CHv-BAC-G virus particles was similar to that of DPV CHv parental strains by transmission electron microscopy,indicating that the functional defects of TK gene and the insertion of BAC vector did not affect the structure and assembly of DPV CHv virus particles.The dynamic distribution of DPV CHv-BAC-G in duck embryos was detected by fluorescence quantitative PCR.The results showed that the proliferation of DPV CHv-BAC-G in the mixture of allantoic fluid and amniotic fluid was not significantly different from that of DPV CHv parent strains.In the brain and body?removal of the head,limbs and internal organs,etc.?,the proliferation of DPV CHv-BAC-G is about 24 h and 12 h earlier than the DPV CHv parent strain,respectively.This indicated that DPV CHv-BAC-G It has a faster proliferation characteristic in the embryo than the parental virus DPV CHv.The transcription levels of some innate immune-related genes in brain,liver and trunk tissues of duck embryos inoculated with fluorescent quantitative PCR were detected.The results showed that the transcription levels of some pattern recognition receptors?PRR?and antiviral factors were up-regulated in varying degrees,indicating that replication of the viral genome can effectively induce the occurrence of innate immune antiviral responses in the embryo.3.Biological characteristics of DPV CHv-BAC-G virus on ducklingsThe DPV CHv-BAC-G strain(virus copy number:10^7.0copies/0.2mL;dose:1.0mL/ducklings)was injected intramuscularly into 1-day-old ducklings.After 2 weeks of inoculation,all ducklings survived normally,indicating that DPV CHv-BAC-G was not lethal to ducklings.Virus detection of ducks at 1dpi,3dpi and 5dpi by conventional PCR showed that DPV CHv-BAC-G was not detected in the blood,small intestine and liver of each duckling,indicating that DPV CHv-BAC-G could not grow and proliferate in ducklings.The results of fluorescence quantitative PCR showed that the transcription levels of some natural immune antiviral factors in the brain tissues of ducklings with 1dpi and 5dpi did not change significantly,suggesting that the replication deficiency of DPV CHv-BAC-G in ducklings was not related to the innate immune antiviral mechanism.4.Effect of transient expression of DPV CHv-TK on proliferation of recombinant duck plague virus DPV CHv-BAC-G in vitroThe pCAGGS-DPV CHv-TK eukaryotic expression vector was constructed and transfected on duck embryo fibroblast?DEF?to express DPV CHv-TK,and then the DEF culture was used to culture the recombinant virus DPV CHv-BAC-G.The results of fluorescence quantitative PCR showed that there was no significant change in the proliferation of DPV cultures in DEF cultures expressing viral TK compared to the proliferation performance of DPV CHv-BAC-G in DEF cultures transfected with pCAGGS empty plasmids.It indicated that the TK expression of exogenous virus had no significant effect on the proliferation of DPV CHv-BAC-G in vitro,further confirming that the TK gene is a non-essential gene for DPV CHv proliferation in vitro.In summary,DPV CHv-BAC-G was not lethal to chicken embryos,and its proliferation ability in chicken embryos was very weak.DPV CHv-BAC-G had high lethality and pathogenicity to duck embryos and had a faster proliferation characteristic in embryos than parent virus DPV CHv,and the replication of virus genome could effectively induce significant changes in transcription levels of innate immune-related genes in embryos.CHv-BAC-G could not grow and proliferate in ducklings.In vitro cells,transient expression of complemented DPV CHv-TK had no significant effect on the proliferation of DPV CHv-BAC-G.