Diversity And Evolution Of Picornaviruses In Human And Rodents Sampled From Zhejiang Province, China

Hand, foot, and mouth disease (HFMD) is caused by human enteroviruses in children, particularly in those younger than 5 years. In China, HFMD was a severe public health problem during recent years with several million cases reported each year. Previous studies showed that enterovirus A-71 (EV-A71) and Coxsackievirus A16 (CV-A16) were responsible for most cases of HFMD. But, the cases caused by other types of human enteroviruses increased in some regions of China. Although Wenzhou city has been severely affected by HFMD, the prevalence and clinical symptoms associated with the different agents of HFMD in this city remain poorly understood.Rodentia can harbor a great number of viruses. Studies indicate they have played an important role in the origin and evolution of viruses, expecially for thoses which are pathogenic to human. Although many picornavirues can cause human diseases, less is known about their prevelence and diversity in rodents in China. Hence, identification and characterization of unrecognizned picornaviruses in rodents will be helpful to understand their evolution and prevent their emergence in humans.In this study, stool and cerebrospinal fluid specimens were colected from patients with clinically diagnosed HFMD admitted to the Yuying Children’s Hospital in Wenzhou during 2013. Total RNA was extrancted, and RT-PCR was used to identify the specific viral RNA. The whole viral genomes were obtained for each type of enterovirus. Based on the viral sequences recovered in this study, the genetic analysis was performed. Of 955 specimens from clinically diagnosed HFMD cases,755 were confirmed as HFMD used RT-PCR, and 14 types of enteroviruses belonging to human enterovirus A and B were identified. Notably, CV-A6 was the most common agent (77.8%), followed by EV-A71 (8.2%) and CV-A10 (8.1%). Phylogenetic analysis suggested these viruses emerged in Wenzhou through multiple independent introductions. In addition, recombination events occurred in the genomes of all enteroviruses in Wenzhou, most strains with two recombination breakpoints and several with three. Compared with the disease caused by EV-A71, the illness associated with CV-A6 was milder. Additionlly, CV-A6 infection presented atypic HFMD, including more widespread rash, larger blisters, and subsequent skin peeling and/or nail shedding. Although severe HFMD cases were mainly cuased by EV-A71, the agent of some severe cases was CV-A6. This work highlights the need to perform larger-scale studies on etiology and molecular epidemiology to control its pandemic.Herein, rodents were traped in Zhejiang province (Longquan city, Wencheng county, Longwan district and Ruian city). The captured animals were initially identified by morphology. They were dissected under aseptic conditions, and tissue samples of lung and rectum were colected. The total DNA was extracted from lung, and mitochondrial cytochrome b (mt-cytb) gene was amplified for further sepcies confirmation. The viral RNA was detected by RT-PCR with primers targing 5’NCR. As a result, picornaviruses were identified in these rodents. The nearly complete genome sequences were obtained from positive samples by genome walking strategy. 3’and 5’RACE, and RNA circularization were used to amplify the terminal ends. Phylogenetic analysis revealed that the rodent viruses identified here belonged to cardioviruses. In addition, different virus variants could co-infect an individual. The animo acid sequences of polyprotein, P1,2C+3CD of Ruian-Rn93-1, Longquan-Aa3-1, Ruian-Rn93-3 shared 71.9%-85.4%,65.2%-89.8%,78.3%-95.4% identities with other strains of Cardiovirus B respectively, while Wencheng-Rn416 shared 92.5%,85.3% and 96.9% identities with Boone cardiovirus in polyprotein, P1 and 2C+3CD, indicating the co-circulation of Cardioviruses B and C. Thus, more epidemiological investigations are needed to known whether rodent cardioviruses infect human.