| Bovine enterovirus(BEV)are members of the genus Enterovirus of the family Picornaviridae,are the pathogens that cause clinically characterized by digestive and respiratory signs in bovine.Although BEV usually shows low pathogenic and cause mild clinical signs after infection,while it has mixed infections and the secondary infections with other pathogens,resulting in higher mortality of cattle and serious damage to the healthy development of the cattle.Since BEV is a new disease in China,many problems related to the pathogenesis and prevention of the disease are still unclear.In 2012,the laboratory isolated and identified of the first EV-E HY12 from a cattle herd with an outbreak epidemic of cough,dyspnea and severe diarrhea as the main features with the morbidity and mortality rate of up to 50% in China.Studies on the HY12 virus have found that the virus has unique biological characteristics,and its 5'-UTR secondary structure is significantly different from other enteroviruses,thereby providing potential possibilities for the virus to be delivered as a live vector and to express foreign antigens.In order to explore the potential insertion sites of foreign antigens in the HY12 viral genome,which is provide an effective technical method and basis for the study of HY12 chimeric virus.In this study,molecular biology,reverse genetics,virology and mutant PCR techniques were used to successfully screen and identify HY12 suitable for expression of foreign sequences by constructing c DNA infectious clones,which were rescued according to manufacturer's instructions.Furthermore,we are used reverse genetics and mutant PCR to identify the 5'-UTR key nucleotide sequence that affects HY12 replication.These results not only lay a theoretical foundation for studying replication and pathogenic mechanism of BEV,but also lay a foundation development of HY12 used as a new live vector vaccine based on artificial mutation to attenuate HY12 virulence.Specific research and results are as follows:First,Preparation and epitope analysis of monoclonal antibodies against HY12-VP2 proteinIn this study,the GST-VP2 fusion protein was expressed in the prokaryotic expression system as an immunogen to immunize BALB/c mice,and three monoclonal antibodies against HY12-VP2 protein were obtained by lymphocyte hybridoma technique,which were named 3H4,1C2 and 1E10.The results showed that the prepared monoclonal antibodies 3H4 and 1E10 were positively reacted with MDBK cells infected with HY12 strain.In addition,all three monoclonal antibodies produced a good immune response with natural HY12-VP2 protein.Moreover,the sub-cellular localization of HY12-VP2 protein was determined using monoclonal antibodies 3H4 and 1E10 as detection antibodies,indicating that VP2 protein expression was detected in the cytoplasm 2 h after viral infection,and it is still visible when 16 h after infection.The epitope identification of the three monoclonal antibodies showed that they were different the epitopes,namely 152FQEAFWLEDG161(3H4),168LIYPHQ173(1C2)and 46DATSVD51(1E10).By aligning the amino acid sequences of the epitopes that targeted by the three monoclonal antibodies and expressing the differential amino,indicated that epitope 168LIYPHQ173 is completely conserved among all BEV strains,and epitope 46DATSVD51 is highly conservative in all BEV-E strains and a part of BEV-F strains.In this study,three monoclonal antibodies against HY12-VP2 protein were successfully prepared,which laid a foundation for the detection of subsequent recombinant VP2 protein antigen,sub-cellular localization of HY12-VP2 protein and differential diagnosis of BEV.Second,Recombinant virus rescue of HA/Flag tag introducing HY12 viral structural protein and its biological characterizationThis study successfully introduce the HA/Flag tag into 8 sites of HY12 structural protein by the mutant PCR method and reverse genetics technology.Sixteen full-length c DNA clones were constructed and three recombinant marker viruses were successfully rescued,named r HY12-VP4-N-HA,r HY12-VP4-N-Flag and r HY12-VP1-127 HA.It was identified by immunoperoxidase monolayer assay(IPMA),and the results showed that r HY12-VP1-127 HA virus infected cells could strongly positively react with anti-HY12-VP2 m Ab and anti-HA m Ab,while r HY12-VP4-N-HA and r HY12-VP4-N-Flag virus infected cells with weakly positive reaction with anti-HA m Ab or anti-Flag MAb.Further biological characterization of the r HY12-VP1-127 HA virus by Western Blot,immunoelectron microscopy,plaque assay and serum neutralization assay showed that the marker virus successfully expressed HA protein,and the virion diameter,plaque size and neutralization activity is basically consistent with the r HY12 parental virus.In addition,confocal experiments showed that the introduction of the HA between VP1 amino acids 127/128 did not significantly affect the replication cycle of the marker virus.Nucleic acid sequence sequencing analysis showed that the HA was stably retained after 15 th in the marker viruse infected MDBK cells.It was confirmed by ICR mouse infection experiment that both the recombinant marker virus and the r HY12 parental virus can cause different degrees of pathological changes in the brain,lung,liver and intestine of mice;both the recombinant marker virus and the parental virus can induce anti-HY12 antibody in mice.And the marker virus can induce antibodies that produce anti-HA.The above results lay the foundation for further study of the interaction between viruses and viruses or viruses and hosts and the development of new viral live vector vaccines.Third,Recombinant virus rescue of HA tag introducing HY12 viral non-structural protein and its biological characterizationTo further explore the possibility of introducing exogenous genes into non-structural proteins.In this study,HA was successfully introduced into 6 sites of HY12 non-structural proteins 2A,2B and 3A using the above biological methods.6 full-length c DNA clones were constructed,and three recombinant marker viruses were successfully rescued,which were named r HY12-3A-2-HA?r HY12-3A-3-HA and r HY12-3A-9-HA respectively.The results of IPMA and Western Blot showed that the HA can be expressed correctly three recombinant marker virus and the HA virus can be distinguished from parental virus.In addition,the immune electron microscopy,virus one-step growth curve,plaque experiment and nucleic acid sequencing showed that the three recombinant marker viruses have similar particle diameter,plaque size and growth characteristics compared with parental virus.HA in three virus strains can be stable in infected MDBK cells in 15 passages.Then,we used the three recombinant marker virus and parental virus to infect 3-weeks old ICR mouse.The results showed that three recombinant marker virus and parental virus could induce anti-HY12 antibody in mice,and three marker viruses could induce anti-HA tag antibody.These results showed that the non-structural protein 3A of BEV can also be used to express foreign genes,providing a new technical platform for further research on virus replication mechanism,protein interaction and to develop vector vaccine.Fourth,The effect of 5'-UTR for HY12 virus replicationIn this study,38 full-length c DNA clones of 5'-UTR mutant viruses were successfully constructed by mutating the key nucleotides of 5'-UTR using mutant PCR and reverse genetics technique.By transfecting the infecting the infectious clone into BHK-21,it was found that simultaneous mutation of 5'-UTR 528/529 GGcould not rescue the live virus,indicating that these two nucleotide sites play an important role in viral replication.TCID50 of the recued 38 strains of 5'-UTR mutant virus and r HY12 parental virus was determined in BHK-21 cells.The results showed that the 562 C mutation of 5'-UTR affected the replication of the virus in BHK-21 cells.Western blot analysis and morphological observation of plaque further confirmed that the 562 C mutation in the 5'-UTR affected the replication of the virus significantly.These results lay the theoretical foundation for further research on the replication and pathogenesis of BEV. |
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