Isolation and analysis of genetic characteristics of a new strain of enterovirus EV-C99 from a healthy child in Yunnan Province

Abstract

Objective To isolate and analyze the genetic characteristics of a new strain of enterovirus EV-C99 from a healthy child in Yunnan Province in 2016. Methods Virus isolation was performed according to the World Health Organization (WHO) recommended procedures.Viral RNA was extracted from the supernatant of cell culture.RT-PCR and sequencing analysis of VP1 gene were used for virus identification.VP1 sequence was edited and stitched by Sequencher 5.0 software.The edited sequence was searched in GenBank and the preliminary result indicated that it was an EV-C99 virus.Nucleotide (nt) and amino acid (aa) identities were calculated by MEGA5.2 software.Serotype of the virus was identified according to the standard for enterovirus sequence typing. Results The virus could be amplified by 494/496 and 495/497 primer pairs and the edited sequence was about 1 200 bp.Result of the BLAST search showed that it was a new strain of enterovirus EV-C99.Comparative analysis with the prototype strain BAN00-10461 (GenBank ID: EF015008) by MEGA5.2 software showed that the VP1 gene of that virus was 909 bp and the identities between them were 77.05% in nt sequence and 90.04% in aa sequence.According to the standard for enterovirus sequence typing, the virus was a new strain of enterovirus EV-C99 belonging to human enterovirus species C (EV-C). Conclusion A new strain of enterovirus EV-C99 was isolated during an investigation of enteroviruses among healthy children in Yunnan Province in 2016.To our knowledge, this is the third report of EV-C99 in China.Phylogenetic analysis reveals that the EV-C99 Yunnan strain, Xinjiang strains and Shandong strains all belong to genotype B, but group into different clusters, indicating that Chinese strains have diverse genetic characteristics. Key words: Healthy children; EV-C99; Sequence typing; Genetic characteristics