Etiology Of Hand, Foot And Mouth Disease In Luoyang And Kaifeng City, Henan Province, China,2009

Hand, foot, and mouth disease (HFMD), a highly contagious enteroviral infection, usually affects infants and children.Generally, HFMD is mild and self-limiting in children, the clinical symptoms showed vesicular exanthema on their hands, feet, mouths and buttocks. But severe cases usually presented with neurologic complications, brainstem encephalitis or aseptic meningitis; continuous high fever (temperature of at least38℃); weakness, vomiting, irritability, myoclonus and acute flaccid paralysis; pulmonary edema or hemorrhage, heart and lung failure. And Human enterovirus71(HEV71) and Coxsackievirus A16(CVA16) are the major causative agents of this disease.In recent years, numerous large outbreaks of HFMD have been reported in Eastern and Southeastern Asian countries, including Singapore, Korea, Malaysia, Japan, Vietnam, and China. An outbreak of HFMD had occurred in Fuyang City of Anhui Province, China,2008. A total of6,049HFMD cases were reported between1March and9May in this outbreak, which included22fatal cases. Since the epidemic developed over a relatively short time span and was associated with high morbidity and mortality, HFMD had received considerable attention from clinicians and public health officials, and HFMD was classified as a category C notifiable infectious disease by the Ministry of Health of P. R. China on May2,2008. After20083, the outbreak of HFMD continues in China, and the severe or fatal cases were reported year by year.In order to analyze the pathogens of HFMD, set up the database of their molecular character in Luoyang and Kaifeng city,327stool specimens were collected from327hospitalized children with characteristic symptoms of HFMD within7days of onset from March to June in Luoyang and Kaifeng city, the peak time of this outbreak.The cell lines used to isolate viruses from the specimens were Human rhabdomyosarcoma (RD) cells and human laryngeal carcinoma (HEp-2) cells. If the second-generation cultures showed negative results, the nucleic acid hybridization was performed. The positive results of the hybridization led to the third-generation culture. The results showed that introduction of the nucleic acid hybridization had increased the rate of virus isolation. Finally, a total of230isolates from RD cell lines and8isolates from HEp-2cell lines were obtained.The complete VP1or VP4region of the isolates was amplified by RT-PCR with specific primers, and serotype was identified by sequence analysis.Among the isolates from Luoyang,118isolates were identified as HEV71, and26isolates were identified as CVA16. In addition, three isolates as CVA6, one isolate as Echovirus30(ECHO30), one isolate as CVA2, one isolate as CVA4, one isolate as CVA24and one isolate as CVB2were identified. And in Kaifeng’s isolates,66isolates were identified as HEV71,10isolates as CVA16,2isolates as CVA24,2isolates as CVB5, one isolate as ECHO7and isolate as CVA4and three human adenoviruses (HAdV) were found in the isolates.Exclude the interference of co-infection and non-HEV HFMD pathogens identified, the etiology of Luoyang City, Henan Province in2009included the HEV71(78.76%), CVA16(17.33%), CVA6(2.00%), CVA4(0.67%), CVA2(0.67%), CVB2(0.67%), in Kaifeng, HEV71(81.48%), CVA16(12.35%), CVA4(1.23%), CVB5(1.23%), ECHO7(1.23%), CVA24(1.23%).Alignment of the nucleotide sequences of the isolates was performed, and a phylogenetic dendrogram was constructed. The complete VP1region sequences of the HEV71strains isolated from the patients were used for phylogenetic analysis. There were4transmission chains in Luoyang and2in Kaifeng at least. It was shown that all the HEV71strains isolated belonged to branch C4a of the subgenotype C4, similar to the HEV71sequences isolated from China during2003-2008. The phylogenetic analysis of VP1region of CVA16showed that,2genotypes can be assigned, A and B. The genotype B could be divided into B1and B2, and subgenotype B1could be further divided into clusters B1a and B1b. All CVA16strains isolated in this study belonged to clusters B1a and B1b within the subgenotype B1. There were4transmission chains co-circulated in Luoyang and7in Kaifeng at least. It has high amino acid identity with the strains isolated from Luoyang and Kaifeng city.The entire VP1sequences of CVA4were used for phylogenetic analysis. The results indicated that5genotypes could be divided, and the strains isolated from Luoyang and Kaifeng were in the same genotype:C. It indicated they maintained high homology of nucleotide and amino acid.Based on complete VP1sequences of CVA6, it should be divided into4genotypes:A, B, C and D. The Luoyang strains were in the C genotype, and kept the high homology of nucleotide and amino acid.Because of the absence of entire VP1sequences of CVA2in the Genbank database, the partial VP1sequences were used for constructing phylogenetic dendrogram. The results showed that they were divided into4clusters. The CVA2strain isolated from Luoyang was belonged to cluster2.The phylogenetic analysis of VP1region of CVB2showed that,3genotypes could be assigned, Luoyang CVB2belonged to genotype C, and had high nucleotide similarity and amino acid similarity with strains isolated from Shandong province in2004. Although it had low nucleotide identity with genotype A viruses, it had high amino acid identity with them, which indicated that their antigenicity may not alter so much due to big differences in nucleotide.6genotypes within CVB5could be assigned based on their complete VP1sequences, and Kaifeng CVB5belonged to genotype D, which clustered with other CVB5isolated in China. It had low nucleotide similarity and amino acid similarity with strains isolated in other countries, and showed big variation.The phylogenetic analysis of complete VP1region of ECHO7showed that,4genotypes could be assigned. Kaifeng ECHO7belonged to genotype C, and had high nucleotide similarity and amino acid similarity with strains isolated from Shandong province in2008.As a member of HEV-C, CVA24was reported to be a pathogen of acute hemorrhagic conjunctivitis (AHC) usually. But2isolates from the HFMD cases indicated it was probable agent of HFMD. In fact, CVA24was isolated in other outbreaks of HFMD, so it needs to be further confirmed.