Detection and Identification of Human Enteroviruses among Healthy Children in Antajaya, Bogor

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1 Detection and Identification of Human Enteroviruses among Healthy Children in Antajaya, Bogor Rifqiyah Nur Umami 1 *, Rama Dhenni 1,2, Ahmad Jajuli 1,3, Yorihiro Nishimura 4, Hiroyuki Shimizu 4, Andi Utama 1 1 Research Center for Biotechnology, Indonesian Institute of Sciences, Jalan Raya Bogor Km. 46 Cibinong, Bogor Department of Biology, Faculty of Mathematic and Natural Sciences, University of Indonesia 3 Bogor Industrial Technology and Pharmaceutical Institute 4 Department of Virology II, National Institute of Infectious Diseases, Japan Abstract Human Enteroviruses (HEVs), members of the genus Enterovirus, family Picornaviridae, are the major cause of variety illnesses, such as poliomyelitis, acute flaccid paralysis, aseptic meningitis and handfoot-and-mouth disease, especially in young children. Virus isolation and neutralization tests are usually performed to identify the serotype of HEVs, but these tests are labor intensive and time consuming. At present, molecular methods allowing the rapid and specific detection of HEVs are being used. Since the sequence identity of HEVs VP1 region has been shown to be well correlated with the serotype, this VP1 sequence is often used for serotyping of HEVs. This study was aimed to identify the circulating HEVs among healthy children in Antajaya, one of the regions with poor sanitation in Bogor, directly from stool samples. The Reverse- Transcription seminested Polymerase Chain Reaction (RT-snPCR) and CODEHOP specific primers which able to amplify almost all of HEVs VP1 region, was used. The phylogenetic tree was constructed by the neighbor-joining method on the basis of VP1 sequences. A hundred and two samples were collected and 53 (52.0%) samples were positive for PCR. Thirty of 53 (56.6%) EVs-positive samples were analyzed and identified as Echovirus 21 (30.0%), Coxsackievirus A24 (23.3%), Coxsackievirus B4 (10.0%), Coxsackievirus B3 (10.0%), Echovirus 25 (16.6%), Echovirus 9 (3.3%), Echovirus 14 (3.3%) and Coxsackievirus A10 (3.3%). Thus, Coxsackievirus A24 that might be able to recombine with polioviruses causing vaccine associated paralytic poliomyelitis was dominantly circulating in the population. Keywords: Human Enteroviruses, CODEHOP, VP1, vaccine associated paralytic poliomyelitis. *Correspondence to: Dr. Andi Utama andiutama2002@yahoo.com INTRODUCTION Enteroviruses (EVs) are members of the genus Enterovirus, family Picornaviridae. Although most of EVs infections are asymptomatic, EVs infection can cause serious illnesses, including poliomyelitis, acute flaccid paralysis, aseptic meningitis and handfoot-and-mouth disease. The genus Enterovirus contains 5 species comprising of 85 serotypes with strains isolated from humans: Human Enterovirus A (HEV-A), HEV-B, HEV-C and HEV-D (Pallansch & Roos, 2006). The EVs genome is a singlestranded RNA molecule of positive polarity that is ~7,500 nucleotides long. Genetic recombination can play a role in the evolution of EVs and has been reported to occur between vaccine strains and/or wild polioviruses (Dahourou et al., 2002; Guillot et al., 2002) and between non-poliovirus EVs (NPEV) belonging to the same or different serotypes (Oprisan et al., 2002). The species HEV-C includes Polioviruses (PVs), a major cause of poliomyelitis that has been eradicated worldwide, with only a few remaining countries of endemic with reservoirs of wild PV (CDC, 2004). However, poliomyelitis associated with mutated and pathogenic PV strains derived from the oral polio vaccine (OPV) resulting vaccine-derived poliovirus (VDPV) causes increasing concern. Outbreaks of poliomyelitis associated with VDPV recently occurred in Egypt (Yang et al., 2003), Hispaniola (Kew et al., 2002), Philippines (Shimizu et al., 2004) and Madagascar (Rousset et al., 2003). The strains implicated in these 4 outbreaks were recombinant between vaccine polioviruses and unknown EVs that could be either wild polioviruses or HEV- C. More recently, artificial recombination between PV and some EVs belong to the HEV-C (Coxsackievirus A11, A17 and A18) resulted in recombinant virus exhibited similar characteristics with the wildtype PV (Utama & Shimizu, 2005; 2006; 2008). Detection and Identification of Human Enteroviruses among Healthy Children in Antajaya, Bogor 1

2 Molecular methods that allow the rapid and specific detection of EVs are currently available. Amplification of the genome by Reverse-Transcription Polymerase Chain Reaction (RT-PCR), determination of the nucleotide sequences and phylogenetic analysis are standardized techniques for both classification and identification of EVs. The protein capsid VP1 is the major surface-accessible protein in the mature EVs virion and carrying important serotype-specific neutralization epitopes. Therefore, its sequence has been shown to correlate very well with the classical serotype classification (Oberste et al., 1999b). The partial or complete VP1 nucleotide sequences of homologous strains from the same serotype were at least 75% identical to one another (>88% amino acid identity), whereas strain with <70% VP1 sequence identity to that of each of the EVs prototype may represent new EVs serotypes. We have adapted a method called COnsensus- DEgenerate Hybrid Oligonucleotide Primer (CO- DEHOP) VP1 RT-seminested PCR that broaden target specificity and amplified all recognized and proposed EVs serotypes and other antigenic variant strains tested (Nix et al., 2006), thus, this method has extended the prospective of EVs surveillance and epidemiological studies by facilitating the identification of serotypes directly from clinical specimens. Circulating-VDPV has been found in Indonesia in 2005, but has not been characterized (Pallansch & Roos, 2006). However, little is known about the circulation and epidemiology of either HEV-C or other EVs in the regions where VDPV outbreaks occurred. A study for EVs surveillance conducted in Ghana indicated that EVs isolations are more prevalent among children in areas with poorer sanitation and in urban area during both rainy and dry season (Otatume & Addy, 1975), therefore, we targeted several regions in West Java province that lack of sanitation. Local health data sheet of Kabupaten Bogor showed that 85% of the Desa Antajaya households have no access to safe water and good sanitation (PKK Kecamatan Tanjungsari, Kabupaten Bogor, 2007). The aim of this study was to detect and identify circulating EVs from healthy children aged less than 5 years, living in Desa Antajaya, Kabupaten Bogor. MATERIALS AND METHODS Subjects and samples: One hundred and two healthy children were purposively enrolled from their respective residence located in Kampung Campedak, Cikembar Hilir, Cikembar Kaler, Kebon Jambe and Pasir Santri, Desa Antajaya, Bogor. All subjects were less than 5 years old and had received OPV vaccination according to the schedules. The sample number was calculated to achieve the children s representative population, which is 20% of 456 children. A single stool sample was collected from each child and these samples were directly used for viral RNA extraction. The preparation of stool samples and viral RNA extraction: Stool suspensions were prepared according to the standard methods recommended by the WHO (WHO, 2004). One gram of stool sample was mixed with 5 ml of phosphate-buffered saline and 0.5 ml of chloroform. The mixtures were vortexed vigorously for 10 min, centrifuged at 1500 g for 15 min at 4 C, and the supernatant was transferred to a fresh collection tube. Viral RNA was extracted from the supernatant with the High Pure Viral RNA Kit (Roche Applied Science) according to the manufacturer s instructions. The eluted RNAs were directly used for VP1 COnsensus-DEgenerate Hybrid Oligonucleotide Primer (CODEHOP) Reverse Transcription Seminested-Polymerase Chain Reaction (RT-snPCR) or stored at -20 C until used. CODEHOP VP1 RT-snPCR and sequencing: The VP1 CODEHOP RT-snPCR method was performed as described previously (Nix et al., 2006), which comprise of cdna synthesis, first PCR (PCR1) and second PCR (snpcr2). Synthesis of cdna was carried out in a 10 µl reaction mixture containing 5 µl of viral RNA template and 5 µl master mix (20 mm deoxynucleoside triphosphate (dntp; GE Healthcare), 5 RT buffer (Invitrogen), 0.01 M dithiothreitol (DTT; GE Healthcare), 10 µm each cdna primer (primers AN32, AN33, AN34 and AN35; Table 1), 20 U of RNasin (Promega) and 100 U of Super- Script II reverse transcriptase (Invitrogen). The mixture was put in the following incubation: 22 C for 10 min, 42 C for 60 min and 95 C for 5 min. The entire 10 µl cdna synthesis reaction mixture was used in the PCR1 (final volume, 50 µl), consisting of 10 PCR + Mg2+ buffer (Roche Applied Science), 20 mm dntp, 10 µm each of primers 224 and 222 (Table 1) and 2.5 U of Taq DNA polymerase (Roche Applied Science), with 40 cycles of amplification (at 95 C for 30 sec, at 42 C for 30 sec, at 60 C for 45 sec). One microliter of the first PCR product was added to a snpcr2 reaction mixture contained 10 µm each of primers AN89 and AN88 (Table 1), 20 mm dntp, 10 FastStart Taq buffer (Roche Applied Science) and 2.5 U of FastStart Taq DNA polymerase (Roche Applied Science) in a final volume of 50 µl. The FastStart Taq polymerase was activated by incubation at 95 C for 6 min prior to 40 amplification cycles at 95 C for 30 sec, at 60 C for 20 sec and at 72 C for 15 sec. The schematic steps of the CODEHOP VP1 RT-snPCR Umami, et.al. 2

3 method were shown in Fig. 1. The reaction products were separated and visualized on 1% agarose gels containing 0.5 µg ethidium bromides per ml and the EVs-positive result (noted as a single band at position ~350 to 400 bp) were purified by using a Wizard SV Gel & PCR Clean-Up System (Promega) according to the manufacturer s instructions. Slight variations in the sizes of the PCR products were observed due to VP1 gene length differences in the different serotypes, as described previously (Oberste et al., 1999a; 1999b; 2000). The DNA templates results were sequenced with a BigDye Terminator v1.1 ready reaction cycle sequencing kit on an ABI Prism 3130xl Genetic Analyzer (both from Applied Biosystems) by using primers AN89 and AN88 (Table 1). Table 1. Primers used for cdna synthesis, PCR1, snpcr2 and sequencing (Nix et al., 2006). Primers 5 3 Sequences a Amino acid motif Location c AN32 GTYTGCCA WQT AN33 GAYTGCCA WQS AN34 CCRTCRTA YDG AN35 RCTYTGCCA WQS SO224 GCIATGYTIGGIACICAYRT AMLGTH(I/L/M) SO222 CICCIGGIGGIAYRWACAT M(F/Y)(I/V)PPG(A/G) AN89 CCAGCACTGACAGCAGYNGARAYNGG b PALTA(A/V)E(I/T)G AN88 TACTGGACCACCTGGNGGNAYRWACAT b M(F/Y)(I/V)PPGGPV a Ambiguity codes based on Nomenclature Committee of the International Union of Biochemistry (NC-IUB) are as follows: G, guanine; A, adenine; C, cytosine; T, thymine; R, A/G; Y, C/T; W, A/T; N, A/C/G/T; I, inosine b The nondegenerate clamp regions within AN88 and AN89 sequences are underlined c The locations of all primers are those relative to the genome of PV1 Mahoney strain (GenBank accession number J02281) Fig. 1. Schematic of the locations of the primers used and the representation of the steps in the CODEHOP VP1 RT-snPCR method. Nucleotide sequence and phylogenetic analyses: Raw sequence data were first analysed with Chromas Lite version 2.01 (Technelysium Pty Ltd), the forward and reverse sequence data for each sample were then assembled by using BioEdit version to obtain the final contig sequence (Hall, 1999). The partial VP1 coding sequences obtained were compared with all of the EVs reference strain sequences available in GenBank using BLAST ( BLAST) (Altschul et al., 1990) and each sample was assigned the serotype of the strain that gave the highest identity score. The result was confirmed by phylogenetic analysis by including prototype strain of all EVs serotypes and VP1 coding sequences determined in this study for multiple sequence alignment with Detection and Identification of Human Enteroviruses among Healthy Children in Antajaya, Bogor 3

4 ClustalX version 1.83 (Thompson et al., 1997). Kimura two-parameter was used as a model of nucleotide substitution and the neighbor-joining (NJ) method was used to reconstruct the phylogenetic trees (Kimura, 1980; Saitou & Nei, 1987). The reliability of the tree established with the NJ method was estimated by bootstrap analysis with 1,000 pseudoreplicate data sets (Felsenstein, 1985) and the tree was viewed using NJplot or TreeView software. RESULTS AND DISCUSSION From 102 stool samples, 53 (52%) EVs could be amplified by the CODEHOP VP1 RT-snPCR and partial VP1 coding sequences were successfully identify from 30 samples (56.6%). Analysis of each sequences determined by BLAST were assigned the viruses into 8 different serotypes within 3 different species: HEV-A (Coxsackievirus A10 (CVA10)); HEV-B (Coxsackievirus B3 (CVB3), CVB4, Echovirus 9 (E9), E14, E21 and E25) and HEV-C (CVA24) species. Echovirus 21 was the most frequently identified serotype, accounting for 30% of the positive samples, followed by CVA24 (23.3%), E25 (16.6%), CVB4 (10.0%), CVB3 (10.0%), CVA10, E14 and E9 (3.3% each) (Table 2). These results showed that HEV-C (CVA24 serotype) were the second-most dominant species circulating in the area, accounting for 23.3% of the positive samples. No Poliovirus or HEV-D species were detected in this study. Table 3 shows the Enteroviruses detected among healthy children in Desa Antajaya, Bogor. Table 2. Enterovirus serotypes and species detected in stool samples taken from healthy children in Desa Antajaya. Species Serotypes % (n= 30) HEV-A CVA HEV-B CVB CVB E9 3.3 E E E HEV-C CVA Phylogenetic analysis was performed with partial VP1 coding sequences (obtained from 30 positive samples) with homologous sequences of all EVs prototype strains to confirm the serotyping results. The phylogenetic relationships between the samples and the EVs prototype strains are shown in Fig. 2. Samples that were assigned the same serotype clustered together with a >99% bootstrap value with their respective prototype strain with most of bootstrap value higher than 61%. Therefore, the phylogenetic analysis definitely confirmed serotyping with the VP1 sequences. Samples assigned as serotype E14 (H91) and E21 (H82, H116, H118, H121, H123, H125 and H127) showed low bootstrap values (<60%) with their respective prototype strains. The low bootstrap values indicated that the position of these strains were not stable in the tree. Since the phylogenetic signal was influenced by two factors, degree of conservation and length of sequence, these samples need to be confirmed by using longer nucleotide sequences. Table 3. Enteroviruses detected among healthy children in Desa Antajaya, Bogor. Age (years) Campedak Cikembar Hilir Cikembar Kaler Kebon Jambe Pasir Santri Campedak N.S. EVs N.S. EVs N.S. EVs N.S. EVs N.S. EVs N.S. EVs < > N.D Total N.S.: number of subjects; N.D.: not determined; EVs: Enteroviruses. Umami, et.al. 4

5 Fig. 2. A rooted phylogenetic tree of field isolates partial VP1 coding sequences and prototypes from GenBank for each of the serotypes. The evolutionary distances were calculated using the Kimura twoparameter model as a model of nucleotide substitution and the neighbor-joining method to reconstruct the phylogenetic tree. The bootstrap values (1,000 pseudoreplicate data sets) supporting each cluster are shown at the nodes; for clarity, only values of >60% are shown. The outgroup taxa are the Porcine Enterovirus 8 (PEV8) prototype strains. Sequence names for samples studied (in boldface) are constructed as assigned serotype and sample number. The prototype strain sequences have GenBank accession numbers. The scale bar represents the genetic distance (the number of nucleotide substitutions per site). The use of molecular serotyping strategy has improved the ability for rapid detection of EVs directly from human stool samples. Molecular methods based on the correlation between the nucleotide VP1 coding sequence and those of prototype EVs strains allowed us to search almost all EVs. To date, this is Detection and Identification of Human Enteroviruses among Healthy Children in Antajaya, Bogor 5

6 the first study of EVs detection and identification among healthy children in Indonesia based on molecular serotyping strategy. CODEHOP VP1 RT-snPCR method which based on the degenerate primer design was used in the first PCR primers to broaden the target specificity of amplification, allowing amplification of all EV serotypes and increasing the absolute concentration of virus-specific product to be used as the template for the second PCR (snpcr2). This method combined with minimally degenerate primers used for snpcr2 which maintains the broad viral target specificity of the PCR1 that use inosine-containing primers (Nix et al., 2006). As a result, we were able to detect and identify 8 different serotypes from 30 EVs-positive samples. The variability of EVs detected in this finding was consistent with earlier studies which reported various EVs infections in symptomatic case or from healthy children. In a study of clinical samples (primarily stool samples) obtained in from patient in Tunisia, 29 different serotypes of NPEV were detected in the country during the study period with E6, E11 and E30 serotypes were the most frequently isolated (Bahri et al., 2005). Similarly, in surveillance of EVs in France from clinical samples and environment identified 27 different serotypes; with the 10 main of NPEVs were E30, E13, E6, CVB5, E11, CVB4, E9, E7, CVB1 and CVB2 (Antona et al., 2007). These results support the understanding that variation by location is a major characteristic of EVs. Therefore, outbreaks or infections of EVs can be restricted to small groups or to select communities or they may become widespread at the regional, national or even international level (Pallansch & Roos, 2006). The discovery of circulating VDPV bearing NPEV sequences thought to be derived from HEV- C has increased interest in the epidemiology and circulation of these EVs species. In Madagascar where outbreaks of poliomyelitis associated with VDPV bearing NPEV sequences from HEV-C occurred, there were high frequency of HEV-C circulation identified as CVA13, CVA17, CVA18, CVA20, CVA21 and CVA24 (Andrianarivelo et al., 2005). Our data showed that CVA24, the only HEV- C serotype detected, were the second most frequently EVs circulating in Desa Antajaya. The possibilities of circulating CVA24 to recombine with PV from oral polio vaccine (OPV) should be well recognized. Therefore, this study might provide information for the emergence possibilities of recombination between OPV and HEV-C that resulting VDPV. Coxsackievirus A24 strains have been involved in several outbreaks of acute hemorrhagic conjunctivitis worldwide (Ishiko et al., 1992; Oh et al., 2003; Yeo et al., 2005). However, little attention has been paid to NPEV and to other related diseases in tropical countries and even in Indonesia. The most dominant serotype detected in this study were E21, accounting for 30% of the 30 EVspositive samples analyzed. This finding contrast with recent studies of EVs surveillance from France over that reported the presence of E21 only 0.8% of 2,757 clinical specimens (Antona et al., 2007). A surveillance of EVs in Tunisia reported the presence of E21 only one patient from 525 paralytic cases (Bahri et al., 2004). This difference might be caused by methodological differences, whereas the two above studies were only detected EVs in clinical specimens from symptomatic patient, our study included only healthy children without any medical records. Nevertheless, to our knowledge, there are little studies reporting the prevalence and associated disease caused by E21. In conclusions, we have detected and identified EVs among healthy children from one specific region in Indonesia and this study would be the first step toward identifying EVs that could recombine with PV in Indonesia. 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