Epidemiological Investigation Of Feline Morbillivirus And Determination Of N Protein Nuclear Export Signal

Feline morbillivirus(Fe MV)is the seventh species of the genus Paramyxoviridae named after the 2016 International Committee for Classification of Viruses.The majority of members of the measles virus are important pathogens of humans and animals.The measles virus is highly pathogenic and causes death in animals and humans.Since Fe MV was first reported in Hong Kong in 2012,it has been detected in many countries and regions around the world.So far,there has been no report on the epidemiological examination of Fe MV in China's mainland.In order to detect the prevalence of measles virus in cats,64 cat urine samples were collected from Guangzhou,Guangdong Province from 2017 to 2018.The nested PCR was used to specifically amplify the more conserved L gene,6 of cat urine samples were tested positive for Fe MV,with a positive sample ratio of 9.4%.The six cat measles viruses were named GD1,GD2,GD3,GD4,GD5 and GD6,respectively.According to the sequencing results and based on the genetic analysis of the cat measles virus L gene,these six viruses are in the same large branch as the 776 U and 761 U reported in Hong Kong in 2012.The viral RNA was extracted from the positive cat urine sample,and the N gene was amplified by RT-PCR.The prokaryotic expression plasmid p MAL-c5X-N was cloned into p MAL-c5 X,and the correctly sequenced plasmid was transformed into the expression strain Rosetta(DE3),induced and purified by IPTG to obtain recombinant N protein,Western-blot method for analysis of antigenic properties of N protein;purified recombinant N protein as antigen,multiple immunization of New Zealand after complete emulsification by mixing Gel 01 ST After the rabbit was obtained,the polyclonal antibody against the cat measles virus N protein was obtained,and the polyclonal antibody against the cat measles virus N protein obtained by indirect ELISA was more than 1:1,000,000.The polyclonalantibody against cat measles virus N protein has good sensitivity and specificity,and provides materials for subsequent sub-localization of cat measles virus N protein.During paramyxovirus replication,N protein may shuttle in the cytoplasm and nucleus,and nuclear transport of N protein may be involved in measles virus-specific pathogenicity.According to the existing research on measles virus,it is predicted that the nuclear export signal of cat measles virus N protein is located in the first 20 amino acids of the C-terminus of N protein.In order to determine the specific amino acid sequence of the cat's measles virus nuclear export signal,the first 20 amino acids of the C-terminal end of the N protein were truncated 10 amino acids one by one,and the corresponding nucleotide sequence was constructed with the p EGFP-C3 eukaryotic expression system.An eukaryotic expression plasmid was observed by transfecting 293 T human renal epithelial cells to observe the sublocalization of the expressed green fluorescent protein in the cells.The amino acid of the N-core nuclear export signal of cat measles virus was located at 3 to 11 positions,and the eukaryotic expression plasmid was constructed by cloning the nucleotide sequence corresponding to the amino acid of 3 to 11 and p EGFP-C3 to verify its export capacity..This study identified the amino acid site of the nuclear export signal of cat measles virus N protein,which laid a foundation for the study of the transport mechanism of cat measles virus N protein and the pathogenicity of the virus.