Genetic and Antigenic Characterization of Enterovirus 71 in Ho Chi Minh City, Vietnam, 2011

Enterovirus 71 (EV71) frequently causes fatal infections in young children in Asia. In 2011, EV71 epidemics occurred in southern Vietnam. We conducted genetic and antigenic analysis of the EV71 isolates and found that 94% of them were genotype C4a related to two lineages circulating in China and 6% were genotype C5 which have circulated in Vietnam since 2003. Antigenic variants were not detected. EV71 vaccines are being developed. Longitudinal enterovirus surveillance data are critical to formulate vaccination policy in Vietnam.


Introduction
Enterovirus 71 (EV71) is a member of genus Enterovirus within the family Picornaviridae. Although EV71 was first described in 1969, a retrospective analysis shows that this virus circulated in the Netherlands as early as 1963 [1]. Recent molecular evolution studies predicted that EV71 could emerge in human population around 1941 and seems to evolve more quickly through gene recombination in the past 15 years [2,3]. Based on VP1 genotyping, EV71 could be classified into 3 genogroups (A, B and C) including 11 genotypes (A, B1 ~ B5, C1 ~ C5). The C4 genotype could be further classified into C4a and C4b [3]. Recently, three new genogroups (genogroup D, E, and F) were identified in India [4]. Since 1997, EV71 has repeatedly caused life-threatening outbreaks of hand-footmouth disease (HFMD) with neurological complications in Asian children. The neurological manifestations progress very quickly and range from aseptic meningitis to acute flaccid paralysis and brainstem encephalitis, which frequently cause societal panics [3].
In Vietnam, EV71 was first identified in 2003 and a large EV71 epidemic was documented in southern Vietnam in 2005, which reported 173 EV71 cases including 51 with neurological complications and 3 fatal cases [5]. Based on phylogenetic analysis of complete VP4 and partial VP1 genes, 94% (162/173) of the 2005 isolates were attributable to a novel genotype C5, 5% (9/173) were genotype C4, and 1% (2/173) were genotype C1. Twenty-three strains (16 C5, 5 C4 and 2 C1) were further confirmed with analysis of complete VP1 gene. However, full genome of C5 viruses isolated in Vietnam is not available in public domain, which is critical to elucidate its molecular evolution. From 2006 to 2010, only sporadic EV71 cases were detected in southern Vietnam [6]. In 2011, EV71 epidemics occurred again in southern Vietnam. Clinical characterization of the 2011 epidemic has recently been described [7]. In this study, we conducted genetic and antigenic analysis of the EV71 strains isolated in a children hospital in Ho Chi Minh City, Vietnam, 2011.

Study populations
Children's Hospital No. 1 (CH1), Ho Chi Minh (HCM) City, is a major pediatric hospital serving children in HCM City and southern Vietnam. In CH1-HCM, clinical specimens from hospitalized children with HFMD were collected for virus isolation. Institutional review board approvals were obtained from CH1-HCM following the Helsinki Declaration; and written informed consent was obtained from guardians of participating children.

Clinical and laboratory definitions
Clinical stages of enterovirus cases in Vietnam are defined as follow [7]: Stage I is uncomplicated disease such as herpangina or HFMD; IIA is myoclonus reported by the caregiver; IIB is myoclonus observed by a physician; III is autonomic dysfunction with fever that is not responsive to antipyretics and with hypertension and persistent tachycardia; IV is cardiopulmonary compromise with pulmonary edema or hemorrhage. Laboratory evidence of EV71 infection was defined as the isolation of EV71 from a throat swab, a rectal swab, or a stool sample.

Virologic analysis
In CH1-HCM, clinical samples (throat swabs, or rectal swabs) were routinely collected for virus isolation from hospitalized pediatric patients with suspected enterovirus infections (HFMD or encephalitis). Samples were inoculated into rhabdomyosarcoma cells. When enteroviral cytopathic effect involved more than 50% of the cell monolayer, cells were scraped and subjected to indirect fluorescent antibody staining with pan-enterovirus and EV71-specific monoclonal antibodies (3360, 3324, Millipore, USA). VP1 genes of isolated EV71 viruses were sequenced and genotyped by phylogenetic analysis using the Neighbor-joining method in MEGA 4 software as described previously [8]. Full genomes of selected EV71 viruses were sequenced for phylogenetic analysis and detection of gene recombination [9][10][11]. Backgrounds of reference virus sequences used in the phylogenetic analysis were listed in the Table S1. Primers for amplifying and sequencing VP1 genes and complete genome were listed in Table S2.

Serologic assays
Post-infection sera were collected from Taiwanese children infected with EV71 for measuring neutralizing antibody titers [12,13] (Table 1). Laboratory methods for measuring EV71 serum neutralizing antibody titers followed standard protocols [14]. Twofold serially diluted sera (1:8 -1:512) and virus working solution containing 100 TCID 50 of EV71 strain, were mixed on 96-well microplates and incubated with rhabdomyosarcoma cells. A cytopathic effect was observed in a monitor linked with an inverted microscope after an incubation period of 4 to 5 days. The neutralization titers were read as the highest dilution that could result in a 50% reduction in the cytopathic effect. Each test sample was run simultaneously with cell control, serum control, and virus back titration. The starting dilution was

Results
In 2011, 148 HFMD inpatients were tested and 65 of them (43%) were positive for enterovirus by immunofluorescience assay, including 36 EV71 patients. Complete VP1 genes (891 nucleotides) of 18 EV71 isolates were sequenced for phylogenetic analysis. Based on phylogenetic analysis of complete VP1 genes, 94% (17/18) and 6% (1/18) of EV71 isolates are genotype C4a and C5, respectively (Table 2). Overall, the C4a viruses are phylogenetically related to the C4a viruses circulating in China and the only C5 virus is related to the C5 viruses isolated in Vietnam in 2005 and Taiwan in 2007 ( Figure 1). Interestingly, 17 C4a virus strains isolated in Ho Chi Minh City could be classified into 2 groups including 15 C4a viruses in group one and 2 C4a viruses (virus ID HCM52 and HCM120) in group two. Genetic differences between and within VP1 genes of these two C4a groups were 2.6~2.9% and 0.2~0.3%, respectively. Therefore, full genome analysis (~7000 nucleotides) of three EV71 isolates including one virus from C4a group one (virus ID HCM82), one virus from C4a group two (virus ID HCM120) and the only C5 virus (virus ID HCM84), were further conducted to understand molecular evolution of the 2011 EV71 isolates in southern Vietnam. Based on BLAST analysis of full genome, the HCM82 virus is most similar to a C4a virus isolated in Henan China in 2009 (accession no. JN252063, nucleotide identity 99%), the HCM120 virus is most similar to a C4a virus isolated in Beijing, China in 2008 (accession no. FJ606448, nucleotide identity 98%), and the Phylogenetic analysis of full genome revealed similar findings to that of the BLAST analysis ( Figure 1).
EV71 genotype C4 viruses could be classified into C4a and C4b. The EV71 genotype C4b viruses were first detected in China and Taiwan in 1998 with unknown origins and were later identified as recombinants among multiple enteroviruses in  Table  1 and Table S1 [15], respectively. The prototype coxsackievirus A16 (CA16G10) was used as the outgroup virus. The phylogenetic tree was constructed using the neighbor-joining method. Bootstrap values (>70%) are shown as percentage derived from 1,000 sampling at the nodes of the tree. Scale bar denotes number of nucleotide substitutions per site along the branches. Red dot indicates the viruses isolated and sequenced in this study.   [15]. Based on phylogenetic analysis of VP1 genes and full genomes, the C4a viruses detected in HCM in 2011 belong to two lineages which could have been separately introduced from China. So far, only two C5 viruses isolated in Taiwan in 2006 and 2007 have full genome sequence available in public domain, which were highly identical to the C5 virus isolated in Vietnam in 2011. The evolution pathway of C5 viruses has not been well characterized ant it is very likely through gene recombination. To further detect occurrence of genetic recombination, full genome sequence of the Vietnam C5 isolate was divided into P1 (nucleotide 744~3329), P2 (nucleotide 3330~5063), and P3 segments (nucleotide 5064~7322) for BLAST and phylogenetic analysis. These analyses indicated that P1 and P2 genes of the C5 viruses are closed to that of EV71 genotypes C1, C2 and C3 viruses but source of P3 gene could not be identified (Table 3). SimPlot analysis of full genome further confirmed the possibility of gene recombination at P3 gene ( Figure 2). P3 gene of the C5 viruses could be derived from other human enterovirus specie A viruses such as Coxsakievirus A8 but it needs more full genome sequence data of early isolates to confirm.
To characterize antigenic variations among genotypes C4a, and C5 viruses isolated in Vietnam and Taiwan, sera collected Table 4. Cross-reactive neutralizing antibody titers in postinfection children sera against EV71 viruses isolated in Taiwan and Vietnam. from Taiwanese children infected with C4a, B4 and B5 were employed to measure cross-reactive neutralizing antibody titers against these EV71 viruses. As shown in Table 4, the C4a and C5 viruses isolated in Vietnam and Taiwan have similar antigenic profiles and could be neutralized by the post-infection children sera.

Discussion
Since 1997, several EV71 genotypes have caused largescale epidemics with fatal cases in Asian countries [3]. The  [5,16,17]. The reason for this phenomenon is not clear and needs international collaborations to clarify. In addition, origin and evolution of EV71 genotype C5 viruses have not been well understood and comprehensive genome studies are desirable.
In a clinical investigation of HFMD epidemic in Southern Vietnam in 2011, partial VP1 sequences of 15 EV71 isolates were analyzed and 13 of them (87%) were C4a genotype and the remain 2 strains were C5 genotype, which are similar to the finding in our study [7]. However, complete genome sequencing and antigenic analysis were not conducted in the study. In addition, this clinical investigation did not conduct antigenic analysis which has been performed in our study. Although, large-scale EV71 epidemics in southern Vietnam were only reported in 2005 and 2011, EV71 were active in young children in HCM City during 2006~2009 based on serological and hospital-based enterovirus surveillance studies [18,19]. Moreover, it is well documented that molecular methods are much more sensitive than virus isolation for detecting enterovirus infections [20,21]. Therefore, enterovirus surveillance in endemic countries should combine virus culture, molecular methods and serology to well characterize disease burden.
International spreading of EV71 is common and may play an important role for reemergence of large-scale epidemics in Asia [3]. For example, EV71 genotype B5 viruses spread from Southeastern Asia to Taiwan in 2007 and caused a nation-wide epidemic in 2008 [9,13]. In the current study, EV71 genotype C4a viruses spread from China to Vietnam and caused a largescale epidemic in HCM City and southern Vietnam in 2011. Recently, Western Pacific Regional Office of the World Health Organization has established a HFMD reporting system to collect monthly HFMD cases no. from China, Hong Kong, Macao, Japan, Republic of Korea, Singapore and Vietnam (www.wpro.who.int). However, the reporting systems vary country by country and do not include virus identification data, which make international comparisons impossible. As learnt from global influenza surveillance system, an international enterovirus surveillance system with harmonized clinical definitions and virus identification data is urgently desirable to understand epidemiology and disease burden of EV71 in Asia.
Based on serological analysis using hyperimmune animal antisera, EV71 has only one serotype. Recently, antigenic variations between different genotypes could be detected using post-infection human sera but did not have a clear pattern [9,13]. In this study, genotype C4a and B5 viruses isolated in Vietnam and Taiwan have similar antigenic profiles. Currently, different vaccine strains are being evaluated in China (genotype C4a), Singapore (genotype B2) and Taiwan (genotype B4) [3]. It would be important to monitor crossreactive neutralizing antibody profiles using sera collected from vaccinated children in the future. Moreover, longitudinal enterovirus surveillance data are critical to formulate vaccination policy [9,14] and are warranted in Vietnam.