Establishment Of A Nested PCR Assay And Analysis Of The Full Genome Of HUMAN COSAVIRUS

Picornaviruses are non-enveloped, positive-stranded RNA viruses with an icosahedral capsid. Human cosavirus is a newly described candidate picornavirus genus consisting of five species making up a new genus in the Picornaviridae family that make the family Picornaviridae consists of 14 genera: Aphthovirus, Avihepatovirus, Cardiovirus, Enterovirus, Erbovirus, Hepatovirus, Kobuvirus, Parechovirus, Sapelovirus, Senecavirus, Teschovirus, Tremovirus, Cosavirus and Seal Picornavirus 1. Human Cosaviruses are most closely related to members of the Cardiovirus and Senecavirus genera, but they lack a leader polypeptide. Although the relationship between the human cosavirus and clinical symptom is unclear, the high detection rate of cosavirus in humans across the world suggested that human cosavirus may bring potential harm to the healthy of humans or may be a factor that inducing some diseases including diarrhea. So, it is important to develop a method for human cosavirus detection. About the nested PCR detection method of human cosavirus, a pair primers was designed to the 5'UTR and the best conditions of the method is as follows: the optimal concentration of Mg2+ was 1.25mmol?L-1, the optimal concentration of Taq enzyme is 0.1U/ul and the optimal annealing temperature is 55℃. Stool specimens were collected from 188 hospitalized children with diarrhea and 60 healthy controls in Shanghai were detected by using this method, 6 (3.2%) specimens from the 188 hospitalized children and 1 (1.6%) from the 60 healthy children were found to be positive for HCoSV and the total masculine ratio was 2.8%. When compared with available cosavirus strains in GenBank on the basis of the sequences of 5'UTR, the majority of positive samples (5 of 7) could be classified within cosavirus species A, indicating that HCoSV-A may be the dominant species in circulation in China. The result showed that the nested PCR assay had the advantage such as high sensibility, strong specificity and this method can be used for the epidemiological investigation and diagnosis of human cosavirus.Using a combination of mass sequencing, RT-PCR and Genome Walking and 10 pairs of primers are devised based on those sequences available in GenBank, the nearly-full genome of human cosavirus was then determined which is 7262nt, encoding a putative polyprotein of 2125 aa. Then VP3 gene was amplified by nested PCR, and inserted into the prokaryotic expression vector pET-30a. After identified by restrict enzyme and sequencing, the constructed recombinant expression plasmid was transformed into the receptive cells of E. coli BL21 and induced by IPTG with a finial concentration 1mmol?L-1, and the recombinant protein was expressed in the form of inclusion bodies. After the bacterium cell walls were disrupted by ultrasonication, the inclusion bodies of recombinant proteins were dissolved, and then were purified by Ni2+ affinity columns following with annealing. The purified protein may make senses in the coming study of human cosavirus.