Sequence Analysis Of H Gene And Construction Of Sensitive Cell Line For Canine Distemper Virus

Canine distemper(CD)is an infectious disease caused by canine distemper virus(CDV)infection that causes high morbidity and mortality in predatory animals.CDV is a large ssRNA virus belonging to the genus Morbillivirus of the family Paramyxoviridae and is closely related to measles virus and rinderpest virus.Clinically,it usually manifests as fever,anorexia,runny nose,conjunctivitis and diarrhea,and there are also signs of hyperkeratosis and central nervous system symptoms,leading to decreased lymphocytes and inhibition of lymphocyte proliferation in the acute phase.The disease caused by CDV has a history of several hundred years.Its natural host has been expanded from the traditional canine family,raccoonidae and cockroaches to all terrestrial carnivores such as cats and privets.In addition,it has cloven-hoofed animals,seals and inon-human primates.CDV has caused great losses and damages to the animal husbandry industry and rare wild animals in the world.Although live vaccines have effectively reduced the incidence of diseases,the highly contagious CDV remains an important issue for humans and veterinary medicine.The use of MDCK or African green monkey kidney cell lines(CDV viral receptors are not present on cell membranes)is generally difficult to isolate and culture viruses,leading to a relatively slow progress in the study of the etiology and immunity of CDV.Therefore,it is necessary to construct high-sensitivity cell lines,analyze the characteristics and differences of H proteins of wild-type strains and vaccine strains,and construct eukaryotic expression vectors of canine N1 ICD gene.It is of great significance for the further study of the pathogenic mechanism and treatment plan of canine distemper virus in the future,and has important practical significance for preventing and controlling the spread of canine distemper fever in China.In this study,the wild-type strain of CDV H gene was amplified from the clinicalsamples and analyzed for genetic characteristics.A signalling lymphocyte activation molecule(SLAM)eukaryotic expression plasmid PWPI-SLAM was constructed.This plasmid contains the EGFP-tagged gene that expresses the green fluorescent protein.The MDCK-SLAM,a positive MDCK expressing green fluorescent protein,was screened by limiting dilution.The MDCK-SLAM,a green fluorescent protein-tagged MDCK-positive cell,was screened by limiting dilution.RT-PCR,indirect immunofluorescence and Western Blotting were used to identify whether the MDCK-SLAM cell line could transcribe or translate SLAM gene.RT-PCR assay,indirect immunofluorescence assay,and Western Blotting assay were used to determine whether the MDCK-SLAM cell line could transcribe and translate the SLAM gene.The canine N1 ICD gene fragment was amplified from canine peripheral lymphocyte cDNA and cloned into p3×FLAG-CMV10 and EGFP-N1 eukaryotic vectors.The results showed that the MDCK-SLAM cells screened by the limiting dilution method high green fluorescence expression rate and strong fluorescence.After many passages,the shape and state of the cells did not change,and the SLAM receptor could be stably expressed and anchored on the cell membrane surface.The sensitive cell could present typical CPE-syncytium after being vaccinated with CDV-11 attenuated vaccine for36h~48h,shortening the virus isolation time and increasing the separation rate,which will strongly promote the study of molecular characteristics of CDV.Compared with the traditional vaccine strains,the results showed that the homology of the H strain in the epidemic strains in Guangdong Province was only 89.3% to 91.8%.The vaccine strain H protein has 4~7 N-glycosylation sites,whereas the field strain has evolved 9N-glycosylation sites.The diversity of the H gene sequence may affect the cell tropism of CDV,thereby determining the distribution,pathogenesis and clinical symptoms of the virus in the body.This study cloned and obtained the canine N1 ICD gene for the first time.The bioinformatic analysis showed that the 2 469 nucleotides of N1 ICD DNA sequence encode822 amino acids whose predicted protein size is 91.2 kDa.The p3×FLAG-CMV10-N1 ICD and EGFP-N1 ICD eukaryotic expression vectors were successfully constructed.This study lay the foundation for further research on the roles of canine N1 ICD and canine infectious diseases.