Epidemics and Frequent Recombination within Species in Outbreaks of Human Enterovirus B-Associated Hand,Foot and Mouth Disease in Shandong China in 2010 and 2011

The epidemiology and molecular characteristics of human enterovirus B (HEV-B) associated with hand, foot and mouth disease (HFMD) outbreaks in China are not well known. In the present study, we tested 201 HEV isolates from 233 clinical specimens from patients with severe HFMD during 2010–2011 in Linyi, Shandong, China. Of the 201 isolates, 189 were fully typed and 18 corresponded to HEV-B species (six serotypes CVA9, CVB1, CVB4, Echo 6, Echo 25 and Echo 30) using sensitive semi-nested polymerase chain reaction analysis of VP1 gene sequences. Phylogenetic analysis based on the VP1 region showed that eight E30SD belonged to a novel sub-genogroup D2; E25SD belonged to a novel sub-genogroup D6; E6SD belonged to sub-lineage C6 and five CVB1SD belonged to subgroup 4C; and B4SD belonged sub-lineage D2. The full viral genomes of the CVB1SD, E6SD, E25SD and E30SD isolates were sequenced. Analysis of phylogenetic and similarity plots indicated that E25SD recombined with E25-HN-2, E30FDJS03 and E4AUS250 at noncontiguous P2A–P3D regions, while E30SD, E30FDJ03, E25-HN-2 and E9 DM had shared sequences in discrete regions of P2 and P3. Both E6SD and B1SD shared sequences with E1-HN, B4/GX/10, B5-HN, and A9-Alberta in contiguous regions of most of P2 and P3. Genetic algorithm recombination detection analysis further confirmed the existence of multiple potential recombination points. In conclusion, analysis of the complete genomes of E25SD, E30SD, CVB1SD and E6SD isolated from HFMD patients revealed that they formed novel subgenogroup. Given the prevalence and recombination of these viruses in outbreaks of HFMD, persistent surveillance of HFMD-associated HEV-B pathogens is required to predict potential emerging viruses and related disease outbreaks.