Large outbreak of herpangina in children caused by enterovirus in summer of 2015 in Hangzhou, China

Herpangina, usually caused by coxsackie virus A, is prevalent in children spreading through the fecal-oral transmission and the respiratory droplets dissemination. Also, it is mostly asymptomatic and self-limiting. In our study, we found that large outbreak of herpangina in children occurred in the summer of 2015 in Hangzhou, China. From May 1th to August 31th, a total of 10 210 children were diagnosed with herpangina in Children’s Hospital of Zhejiang University School of Medicine. 2 310 throat swabs were collected and tested for enterovirus detection by real-time RT-PCR, while 1 651 cases were positive with the rate of 71.5%. Based on VP1 gene or 5′UTR region sequences, Coxsackievirus A2, A4, A6, A10, B2, B4 and echovirus 30 were detected in these cases. More importantly, Coxsackievirus A2 may be the major subtype of enterovirus resulting in children with herpangina in hangzhou, China.

Results of enterovirus assay. To confirm the major pathogen causing herpangina, 2 310 throat swabs were randomly collected from patients and tested by enterovirus one-step real-time RT-PCR. As a result, 1 651 samples were tested positive for enterovirus with the rate of 71.5%. Positive rate for enterovirus was the highest among children aged 1-3 years (76.4%), and steadily declined with age increase or decrease (Fig. 1). However, children in other age groups, the detection rates of enterovirus infection were still as high as 62.4%, 72.5%, 66.4%,57.8% and 58.7% in < 1 year, 3-5 years, 5-7 years, 7-9 years, and > 9 years, respectively.

VP1 gene based phylogenetic analysis.
To confirm the subtypes of enterovirus, 35 clinical strains were randomly collected and the VP1 and 5′ UTR gene of the EV were amplified by the conventional RT-PCR ( Figure S1). The amplification products were purified, sequenced and then used for phylogenetic analysis. After comparing the VP1 and 5′ UTR genes withdifferent referenced EV strains, the sequence homology was displayed as 90.9-99.5% (nucleotide), which met the serotype identification criteria for homologous serotypes. The molecular typing results showed that the major EV isolates belonged to the human EVA and EVB species, including 22 samples with Coxsackievirus A2, 3 samples with Coxsackievirus A10, 1 sample with Coxsackievirus A6, 2 samples with Coxsackievirus B2, 1 sample with Coxsackievirus B4 and 3 samples with Echovirus 30. The last 3 samples were negative for VP1 PCR and were identified as Coxsackievirus A4 by 5′ UTR PCR and sequencing. Based on the partial VP1 gene and 5′ UTR region, the homologous comparison results for the isolates from the same serotypes were 92.5-99.4% (nucleotide) ( Table S1).
Based on the VP1 gene and 5′ UTR region sequences, phylogenetic analysis for the EVs of this study was done by comparison with all available VP1 gene sequences from the Genbank. From the constructed phylogenetic tree (Fig. 2), we can find all clinical isolates from Zhejiang belonged to the human EVA and EVB species. The CVA2, CVA10 and CVB4 isolates were most closely related to the stains from Shenzhen (Southern China). CVB2 isolate was most closely related to the stains from Shanghai (Southern China) and E30 isolates were most closely related to the stains from Shandong (Northern China).

Discussion
Herpangina, associated with various enterovirus serotypes, is a commonly prevalent illness in young children 13,14 . In the summer of 2015, large outbreak of herpangina happened in Hangzhou, China. From May to August, 10 210 children were diagnosed with herpangina with the highest prevalence rate in June. Among the infected children, 91.6% were less than 5 years with high rate in 1-3 years children. We also found that the level of CRP slightly increased in children with herpangina during the study period.
Enteroviruses infection occurred in summer throughout the world 15 . To confirm whether enteroviruses are the major pathogens causing herpangina in Hangzhou, China, real-time RT-PCR targeting to highly conserved region in the human enteroviruses (HEV) genome was performed to detect the enterovirus. Among 2 310 throat swabs, 1 651(71.5%) were tested positive for enterovirus. The children aged 1 to 5 years old were the most susceptible population with a peak incidence in 1-3 years, which was similar to all herpangina aging incidence. Most importantly, children older than 3 years also exhibited high infection rate in Hangzhou. Amongpatients belonged to kindergarten and primary school age, cross infections occurred. To control infection, a short-term suspension was conducted in someschools.
As reported in a previous study, herpangina was associated with different strains of enteroviruses, such as CVA2 in Taipei in 2008 16 19 . In our surveillance, we didn't find death cases among children for diagnosis of herpangina. Besides surveillance of herpangina, our hospital has also conducted the surveillance of enterovirus-associated encephalitis. Different from herpangina, we found E30 was the major pathogen causing enterovirus-associated encephalitis and none of CVA2 samples were found (data not shown). These results indicated that E30 may more easily invade to brain system of children. This is the first report about the large outbreak of enterovirus herpangina among children in hangzhou, China. And the associated major subtype of enterovirus may be HEV A with high incidence of CVA2. In further studies, we will sequence more clinical enterovirus samples from EV-positive children with herpangina which would be helpful to further surveillance of enterovirus.

Materials and Methods
Patients and definitions. The Children's Hospital of Zhejiang University School of Medicine is the largest comprehensive center for pediatric health care in Zhejiang province. This was a retrospective study conducted from May 2015 to August 2015 in the hospital. Patients who met the following criteria were enrolled: (1) age less than 14 years old, (2) all children who visit our hospital in the study period, (3) all children who were diagnosed with herpangina (well-characterized multiple vesicular exanthema and ulcers of the soft palate with presentation of fever, sore throat and anorexia) in our hospital in the study period.
Ethical approval and informed consent. This retrospective study and method were approved by the medical ethics committee of the Children's Hospital of Zhejiang University School of Medicine (NO. 2015-PRIB008) and all experiments were performed in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects.

Routine blood test and C-reactive protein.
White blood cell (WBC) counting, the proportion of lymphocyte and neutrophils were measured by Sysmex blood cell instrument (Sysmex 800i, Janpan). Concentrations of C-reactive protein (CRP) were measured by the QuikRead go instrument with QuikRead go CRP kits (Orion Diagnostica, Finland). . For VP1 PCR negative samples, we used 5′ UTR primers (Zhi-jiang company, Shanghai, China) to amplificate and sequence 5′ UTR region. The protocol is the same to VP1 PCR. VP1 gene and 5′ UTR DNA sequences of the EV isolated were contrasted with the National Center for Biotechnology Information (NCBI) database through BLAST. Based on the sequences of the VP1gene, phylogenetic analysis was done by using the Mega 5.1software. The tree was constructed by using the neighbor-joining method. Significance of phylogenies was investigated by bootstrap analysis with 1,000 pseudoreplicate data sets. Bootstrap values of are indicated on the tree.