| Influenza D virus is a newly discovered influenza virus in recent years.It has a wide host range and geographical distribution.It can cause serious respiratory diseases with other respiratory viruses in bovine,posing a threat to the breeding industry,especially the cattle,and there is a risk of spreading to humans across the interspecies barrier.In abroad,they have been involved in both inactivated vaccines and DNA vaccines.Until now,there are no reports about influenza D vaccine in China.Influenza D virus’ s genome consists of 7 RNA segments,which code 9 proteins in total.Hemagglutinin esterase fusion(HEF)protein is the only glycoprotein of influenza D virus.It comprises the functions of influenza A virus hemagglutinin(HA)and neuraminidase(NA)proteins.HEF protein as immunogen is a key for the development of preventive vaccines.In this study,two recombinant newcastle disease viruses expressing HEF protein were rescued by reverse genetics technology and named NDV-F-HEF(NDV97’s F cleavage site replaced with Lasota cleavage site,insert HEF gene),MG7-HEF(NDV97’s F cleavage site replaced with Lasota cleavage site,inserted HEF gene,and knocked 18 amino acids of the NP protein).We have evaluated the biological characteristics and immune effects of the two rescued viruses.Using confocal microscopy and western blot experiments,we confirmed that the recombinant newcastle disease virus can express the HEF protein of influenza D virus.The results of next-generation sequencing verified the genetic stability of the recombinant virus,and the results showed that the HEF gene can be stably inherited in the recombinant newcastle disease viruses MG7-HEF and NDV-F-HEF until generation 15 in embryonated egg.We also analyzed the mean death time(MDT),the intracerebral pathogenicity index(ICPI),intravenous pathogenicity index(IVPI),and the growth ability of two recombinant newcastle disease viruses and its parental strains.The results showed that the insertion of the foreign gene HEF weakened the virulence of the strain NDV-F-HEF,and did not show significant weakness on the virulence of the strain MG7-HEF.The growth ability and growth trendency of the two recombinant viruses and its parental strains showed no significant difference in chicken embryo fibroblast cells.The immune effect of the two recombinant viruses was further evaluated.Two strains of recombinant virus MG7-HEF and NDV-F-HEF were immunized to BALB/c mice by Intramuscular injection,and the mouse serum was collected at post immunization.We can also detect HEF-specific IgG antibodies and their subtypes IgG1 and IgG2 a antibodies in mouse serum.After the fourth immunization,the titer of HEF-specific IgG antibodies in the serum reached 2800 ng/m L.By calculation and analysis,it was found that the Th2/Th1 ratio in the immune response of the two recombinant viruses was about 2.Using enzyme-linked immunospot assay,we found the cell proliferation capacity and the number of IL-4 and IFN-γ secreting cells in the recombinant virus MG7-HEF and NDV-F-HEF immunized group were higher than those in the parental control group and the PBS control group.Among the two recombinant viruses,the test results of the NDV-F-HEF immunization group were slightly higher than those of the MG7-HEF immunization group,but the difference was not significant.That both recombinant virues have the potential as vaccine strains.In summary,we rescued the recombinant newcastle disease viruses MG7-HEF and NDV-F-HEF expressing the HEF protein of influenza D virus,and determined that the two recombinant viruses are attenuated strains.The safety of the two recombinant viruses were verified to meet the requirements for vaccine strains.Evaluation of its immunogenicity through animal experiments proved that it can stimulate mice to produce humoral and cellular immunity.This research has enriched the domestic related content on the research and development of influenza D vaccines,and conducted preliminary explorations on the research of influenza D vaccines. |
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