Circulation of multiple enterovirus serotypes causing hand, foot and mouth disease in India

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1 Journal of Medical Microbiology (2012), 61, DOI /jmm Circulation of multiple enterovirus serotypes causing hand, foot and mouth disease in India V. Gopalkrishna, Pooja R. Patil, Gajanan P. Patil and Shobha D. Chitambar Correspondence V. Gopalkrishna Received 23 July 2011 Accepted 27 October 2011 Enteric Viruses Group, National Institute of Virology, Pune, Maharashtra, India Hand, foot and mouth disease (HFMD), a common contagious disease that usually affects children, can be caused by enteroviruses. Coxsackievirus A16 (CV-A16) and enterovirus 71(EV- 71) are the major aetiological agents of HFMD. Other EV serotypes, CV-A4-7, CV-A9-10, CV- B1-3, CV-B5, E-4 and E-19, have also been found associated with both sporadic infections and outbreaks of HFMD. In India, outbreaks of HFMD have been documented; however, molecular characterization of the aetiological agents has rarely been reported. Cases of HFMD were identified during on the basis of clinical features in southern and eastern parts of India. The aim of the present study was to detect and characterize the aetiological agents associated with the disease. A total of 89 specimens consisting of 41 sera, 24 vesicular fluids, 18 stools and 6 throat swabs were collected from 61 clinically diagnosed HFMD cases from southern and eastern parts of India. RT-PCR followed by sequencing of PCR amplicons and phylogenetic analysis were performed on all specimens for detection of EV RNA and identification of EV types. EV RNA was detected in 47.1 % (42/89) of the specimens collected from 57.4 % (35/61) of the HFMD cases. Thirty-six of 42 EV strains showed amplification of the VP1/2A junction or VP1 regions. Sequence analysis of the amplicons identified the presence of CV-A16 (54.8 %), CV-A6 (38.1 %), EV-71 (2.4 %), CV-A10 (2.4 %) and E-9 (2.4 %) serotypes in the HFMD cases. The study documents CV-A16 and CV-A6 as major and CV-A10, EV-71 and E-9 as rare viral pathogens of HFMD in India. INTRODUCTION Hand, foot and mouth disease (HFMD) is caused by an acute enterovirus infection in humans. The disease is manifested by fever followed by pharyngitis, mouth ulcers and rashes on the hands and feet and is usually selflimiting, with occasional complications leading to pneumonia, meningitis and encephalitis (Österback et al., 2009). Viral transmission occurs through direct contact with saliva, faeces, respiratory droplets or vesicular fluid of an infected person, or indirectly via contaminated objects (Ang et al., 2009). Human enteroviruses (HEV) of the family Picornaviridae belonging to the HEV-A species have been reported frequently in association with HFMD (Zhang et al., 2009). Coxsackievirus A16 (CV-A16) and enterovirus 71 (EV-71) usually cause outbreaks of HFMD (McMinn, 2002), while other EV types such as CV-A4 CV- A7, CV-A9-10, CV-B1-3, CV-B5, E-4 and E-19 have also been detected in both sporadic infections and outbreaks of HFMD (Russo et al., 2006; Zhu et al., 2007). Abbreviations: CV-A6, coxsackievirus A6; CV-A16, coxsackievirus A16; EV, enterovirus; EV-71, enterovirus 71; HFMD, hand, foot and mouth disease; 59NCR, 59 non-coding region. The GenBank/DDBJ/EMBL accession numbers for the VP1 sequences of CV-A6 and CV-A16 are HM HM and HM HM190298, respectively. Investigations of two outbreaks of HFMD that occurred in the Kerala and West Bengal states of India in 2003 and 2007, respectively, have been reported (Sasidharan et al., 2005; Sarma et al., 2009). These investigations identified 119 cases of HFMD on the basis of clinical examination and serological tests. No fatal cases were reported during these outbreaks. Using a molecular approach, the present study reports the investigation of EV aetiology in cases of HFMD that occurred during June to October 2009 and January to September 2010, respectively, in southern (Kerala)/eastern (West Bengal and Orissa) and southern (Tamil Nadu and Kerala) states of India. METHODS Clinical specimens. A total of 89 specimens consisting of 41 sera, 24 vesicular fluids, 18 stools and 6 throat swabs were collected from 61 HFMD cases identified in the Allapuzha and Pathaam thitta districts, central Kerala (n537, 30 from 2009 and 7 from 2010), Kolkata, West Bengal (n58, 2009), Bhubaneswar, Orissa (n57, 2009) and Ooty, Tamil Nadu (n59, 2010). The patients belonged to the paediatric age group, ranging from 4 months to 10 years, with a median age of 5.1 years. Multiple specimens were collected from 23 cases and at least one sample was collected from the remaining cases. Clinical features of the patients included rashes on the face, hands, feet and buttocks, with mouth ulcerations. All specimens were transported to the G 2012 SGM Printed in Great Britain

2 Multiple enterovirus serotypes causing HFMD in India National Institute of Virology, Pune, on ice (+4 uc) and stored at 220 uc until tested. Detection of EV RNA. Viral nucleic acids were extracted from neat serum and vesicular fluid samples and from 30 % (w/v) stool samples and throat swabs suspended in 0.01 M PBS ph 7.5, using the QIAamp viral RNA mini kit (Qiagen) according to the manufacturer s instructions. The presence of EV in clinical specimens was determined by RT-PCR using primers S1 (59-CGGTACCTTTGTAC- GCCTGT-39, 64 84) and AS1 (59-ATTGTCACCATAAGCAGCCA-39, ), targeting 537 bp of the 59 non-coding region (59NCR) followed by nested PCR amplifying a 400 bp region by using primers S2 (59-CAAGCACTTCTGTTTCCCCGG-39, ) and AS2 (59- GAAACACGGACACCCAAAGTA-39, ) as described previously (Sapkal et al., 2009). The amplicons were sequenced and sequence identity was determined by BLAST ( blast). Molecular typing of EV. To identify the EV type in the specimens, an RT-PCR-based amplification of the VP1/2A junction region was performed by using the forward primers 012 (59-ATGTAYGTICCI- CCIGGIGG-39) and 040 (59ATGTAYRTICCIMCIGGIGC-39) and reverse primer 011 (59-GCICCIGAYTGITGICCRAA-39) or of the VP1 gene using primers specific for CV-A16 [CA16F (59-TTGCAG- ACATGATTGACCAG-39, ) and CA16R (59-GAGTGAT- GGTTCAACACACA-39, )] and EV-71 [71F (59-GTGGC- AGATGTGATTGAGAG-39, ) and 71R (59-GTTATGTC- TATGTCCCAGTT-39, )] as described previously (Oberste et al., 1999; Yan et al., 2001). PCR conditions involved an initial reverse transcription step of 1 h at 42 uc, followed by PCR activation at 95 uc for 15 min, 35 cycles of amplification (94 uc for 1 min, 50 uc for 1 min, 72 uc for 1 min) and a final extension of 5 min at 72 uc. The PCR products of ~434 bp (for CV-A6, CV-A10 and E-9)/ 211 bp (for CV-A16)/332 bp (for EV-71) in size were visualized in 2 % agarose gel containing 0.5 mg ethidium bromide ml 21 under a UV transilluminator. Nucleotide sequencing. PCR products were purified by using the QIAquick Gel Extraction kit (Qiagen) and both the strands were sequenced using Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems) in an ABI PRISM 3130XL Genetic Analyzer (Applied Biosystems). Phylogenetic analysis. Sequence identity was determined by BLAST ( Sequences were curated from both ends and aligned by CLUSTAL W. Phylogenetic trees were constructed using the Kimura two-parameter algorithm and neighbour-joining method using MEGA4 software, and reliability of the analysis was tested by applying bootstrap analysis with 1000 bootstrap replications (Tamura et al., 2007). RESULTS Circulating virus strains Of the 89 specimens, 42 (47.1 %), which included 18 vesicular swabs, 8 sera, 2 throat swabs and 14 stool samples from 35 of 61 (57.3 %) HFMD patients, showed presence of EV RNA by RT-PCR targeted against the 59NCR (400 bp) (Table 1). EV RNA positivity detected in patients from Kerala, West Bengal and Orissa states during 2009 was 53.3 % (16/30), 62.5 % (5/8) and 71.4 % (5/7), respectively, while it was 42.8 % (3/7) and 66.6 % (6/9), respectively, during 2010 from Kerala and Tamil Nadu states. All of the amplicons obtained in RT-PCR of a single or multiple specimens from individual patients were sequenced. BLAST analysis of the sequences indicated homology of the Kerala strains to CV-A6 (n516), CV- A16 (n52), CV-A10 (n51), EV-71 (n51), E-9 (n51) and of the West Bengal (n59), Orissa (n55) and Tamil Nadu (n57) strains to CV-A16 (n521). Thirty-six of the 42 (85.7 %) strains amplified in the 59NCR also showed amplification of the VP1/2A junction region or VP1 region. Sequence analysis of the PCR products revealed the presence of CV-A16 in West Bengal (n59, 2009), Orissa (n55, 2009), Kerala (n52, 2010) and Tamil Nadu (n57, 2010) and that of CV-A6 (n510, 2009), EV-71 (n51, 2009), CV-A10 (n51, 2009) and E-9 (n51, 2009) in Kerala. Genogroupwise distribution showed predominance of HEV-A (97.2 %, CV-A16, CV-A6, CV- A10 and EV-71) followed by HEV-B (2.8 %, E-9). Phylogenetic analysis A phylogenetic tree constructed based on partial VP1gene sequences of CV-A16 strains (n523) of the present study grouped separately from those available in GenBank (Fig. 1). However, three distinct subclusters indicating % nucleotide divergence were identified for the strains from West Bengal/Orissa, Kerala and Tamil Nadu. The nucleotide identities exhibited by these strains were highest ( %) with a Malaysian CV-A16 strain (AM292476) and lowest ( %) with an EV-71 (BrCr, U22521) strain. Table 1. Distribution of positivity to EV RNA in HFMD patients by state and specimen type NA, Specimens not available. State (no. of cases) No. positive/no. tested (%) in Total Vesicular fluid Serum Throat swabs Stool Kerala (n537) 7/10 (70 %) 1/22 (4.5 %) NA 13/16 (81.25 %) 21/48 (43.75 %) Orissa (n57) NA 5/7 (71.4 %) NA NA 5/7 (71.4 %) West Bengal (n58) 5/7 (71.4 %) 1/3 (33.3 %) 2/6 (33.3 %) 1/2 (50 %) 9/18 (50 %) Tamil Nadu (n59) 6/7 (85.7 %) 1/9 (11.1 %) NA NA 7/16 (43.7 %) Total (n561) 18/24 (75 %) 8/41 (19.5 %) 2/6 (33.33 %) 14/18 (77.7 %) 42/89 (47.1 %) 421

3 V. Gopalkrishna and others Fig. 1. Phylogenetic tree based on partial VP1 gene sequences ( ) of CV-A16 strains. EV-71 strains are included as outgroups. Indian strains are in heavier type and are identified by abbreviations: name of state, sample number, country and year of illness. The scale bar denotes number of nucleotide substitutions per site. Phylogenetic analysis of the VP1/2A junction region (420 bp) of 10 CV-A6 strains showed clustering with two Japanese CV-A6 strains (AB and AB114111) with % nucleotide identity. With CV-A16 (G-10, U05876) and EV-71 (BrCr, U22521) strains, the nucleotide identities were found to be % and % respectively (Fig. 2). 422 Journal of Medical Microbiology 61

4 Multiple enterovirus serotypes causing HFMD in India Fig. 2. Phylogenetic tree based on VP1/2A gene sequences ( ) of CV-A6, CV- A10 and E-9 strains. Indian strains are in heavier type and are identified by abbreviations: name of state, sample number, country and year of illness. The scale bar denotes number of nucleotide substitutions per site. Comparative analysis of the VP1 gene sequence of the EV-71 strain detected in only one of the samples showed higher ( %) nucleotide identity with Australian strains (AF135946, AY722909) than with the BrCr strain isolated from an encephalitis case in California and strain R13223-IND-01 recovered from a case of acute flaccid paralysis in India ( %). With G-10 of CV-A16 type and Gdula of CV-A6 type strains, the nucleotide identities were 57.2 % and 51 % only, respectively. An E-9 strain identified in the study showed 94.4 % and 75.6 % nucleotide identity respectively with the E-9 strains (AM and AM711072) isolated from meningitis cases in France and E-9 strain-hill (X84981) from the USA in the VP1/2A junction region. A CV-A10 strain identified in the study showed 95.1 % nucleotide identity with strain AY from Germany

5 V. Gopalkrishna and others DISCUSSION Aetiology of multiple enterovirus serotypes has been reported for HFMD worldwide (Cherry, 1992; Chang et al., 2010). Serotypic identification and classification of EV rely on virus isolation in cell culture. In India, the role of enteroviruses in causing HFMD is not very clear on account of lack of accurate diagnosis, as most of the cases reported earlier were diagnosed on the basis of clinical signs compatible with HFMD (Sarma et al., 2009). A single study conducted to investigate an outbreak of HFMD in Calicut, Kerala, identified association of EV-71 by an immunological approach (Sasidharan et al., 2005). In the present study, use of sensitive molecular methods, RT-PCR and sequencing targeted against conserved and variable regions of the EV genome revealed the presence of significant EV RNA positivity as well as multiple types in the clinical specimens. To date, EV-71 and/or CV-A16 have been described as predominant causative serotypes of HFMD all over the world (Ang et al., 2009; Hosoya et al., 2007; Solomon et al., 2010) while infections with CV-A6 and CV-A10 have been mainly detected in Finland (Blomqvist et al., 2010). China and Singapore have reported circulation of multiple EV serotypes along with coinfection in HFMD cases (Shah et al., 2003; De et al., 2011). Interestingly, the present study demonstrated association of CV-A16 and CV-A6 as major and CV-A10, EV-71 and E-9 as rare pathogens of HFMD in India. The virus strains detected in different specimens from the same patient represented the same type, with no co-infections. Of the 16 strains from Kerala identified as CV-A6 on the basis of the 59NCR, only 10 could be typed using primers specific to the VP1 region. The failure of amplification of typing regions in six specimens may have been due to a low viral load. It may be noted that the analysis of data obtained in this study was limited due to the small size of samples available from both years, 2009 and Despite these constraints, the yearwise distribution of EV serotypes in Kerala may suggest cyclic predominance of CV-A6 and CV-A16. However, in order to ascertain this pattern continued surveillance is necessary. The majority of the HFMD patients of the present study were school children, aged 10 years. This could be due to the lack of immune response of this population resulting from the absence of previous exposure to the circulating EV types or recent introduction of causative agents in the environment from a distant source. EV infections have been described to be associated with climatic conditions, especially temperature (Urashima et al., 2003). The high humidity (.60 %) and high average temperature (.28 uc) in the southern and eastern states during the study period might have contributed to the occurrence of HFMD infections. Recently, nail shedding has been reported in HFMD patients within 1 2 months after the onset of disease (Österback et al., 2009; Davia et al., 2011). However, in the present study this observation could not be made as the patients were from remote rural areas, and no complaints were received from the parents. In summary, the present investigation highlights the cocirculation of CV-A16, CV-A6, CV-A10, EV-71 and E-9 serotypes causing HFMD in India. These results emphasize the need for continuous monitoring of HFMD in India and facilitation of the diagnosis of the associated EV infections using molecular and/or serological approaches. ACKNOWLEDGEMENTS The authors thank Dr A. C. Mishra, Director, National Institute of Virology, Pune (India) for constant support and valuable suggestions. They also thank Dr Prakash Loganathan, Sree Narayani Institute of Medical Science, Chalaka, Kerala, Dr Soorya Gayatri, T. D. Medical College, Alapuzzha, Kerala, Dr Rigvardhan, Military Hospital Wellington, Ooty, Tamil Nadu, Dr Nilendu Sarma, NRS Medical College, Kolkata, West Bengal, and Dr B. Dwibedi, Regional Medical Research Centre, Bhubhaneshwar, Orissa, for extending their cooperation in collection of clinical samples from southern/eastern regions of India and providing them for analysis. REFERENCES Ang, L. W., Koh, B. K. W., Chan, K. P., Chua, L. T., James, L. & Goh, K. T. (2009). Epidemiology and control of hand, foot and mouth disease in Singapore, Ann Acad Med Singapore 38, Blomqvist, S., Klemola, P., Kaijalainen, S., Paananen, A., Simonen, M. L., Vuorinen, T. & Roivainen, M. (2010). Co-circulation of coxsackieviruses A6 and A10 in hand, foot and mouth disease outbreak in Finland. J Clin Virol 48, Chang, G. H., Lin, L., Luo, Y. J., Cai, L. J., Wu, X. Y., Xu, H. M. & Zhu, Q. Y. (2010). Sequence analysis of six enterovirus 71 strains with different virulences in humans. Virus Res 151, Cherry, J. D. (1992). Enteroviruses: polioviruses (poliomyelitis), coxsackieviruses, echoviruses and enteroviruses. In Textbook of Pediatric Infectious Diseases, 3rd edn, pp Edited by R. D. Feigin & J. D. Cherry. Philadelphia, PA: W. B. Saunders. Davia,J.L., Bel,P. H.,Ninet,V.Z.,Bracho,M.A.,González-Candelas, F., Salazar, A., Gobernado, M. & Bosch, I. F. (2011).Onychomadesis outbreak in Valencia, Spain associated with hand, foot, and mouth disease caused by enteroviruses. Pediatr Dermatol 28, 1 5. De, W., Changwen, K., Wei, L., Monagin, C., Jin, Y., Cong, M., Hanri, Z. & Jun, S. (2011). A large outbreak of hand, foot, and mouth disease caused by EV71 and CAV16 in Guangdong, China, Arch Virol 156, Hosoya, M., Kawasaki, Y., Sato, M., Honzumi, K., Hayashi, A., Hiroshima, T., Ishiko, H., Kato, K. & Suzuki, H. (2007). Genetic diversity of coxsackievirus A16 associated with hand, foot, and mouth disease epidemics in Japan from 1983 to J Clin Microbiol 45, McMinn, P. C. (2002). An overview of the evolution of enterovirus 71 and its clinical and public health significance. FEMS Microbiol Rev 26, Oberste, M. S., Maher, K., Kilpatrick, D. R., Flemister, M. R., Brown, B. A. & Pallansch, M. A. (1999). Typing of human enteroviruses by partial sequencing of VP1. J Clin Microbiol 37, Österback, R., Vuorinen, T., Linna, M., Susi, P., Hyypiä, T. & Waris, M. (2009). Coxsackievirus A6 and hand, foot, and mouth disease, Finland. Emerg Infect Dis 15, Journal of Medical Microbiology 61

6 Multiple enterovirus serotypes causing HFMD in India Russo, D. H., Luchs, A., Machado, B. C., Carmona, R. C. & Timenetsky, M. C. (2006). Echovirus 4 associated to hand, foot and mouth disease. Rev Inst Med Trop Sao Paulo 48, Sapkal, G. N., Bondre, V. P., Fulmali, P. V., Patil, P., Gopalkrishna, V., Dadhania, V., Ayachit, V. M., Gangale, D., Kushwaha, K. P. & other authors (2009). Enteroviruses in patients with acute encephalitis, Uttar Pradesh, India. Emerg Infect Dis 15, Sarma, N., Sarkar, A., Mukherjee, A., Ghosh, A., Dhar, S. & Malakar, R. (2009). Epidemic of hand, foot and mouth disease in West Bengal, India in August, 2007: a multicentric study. Indian J Dermatol 54, Sasidharan, C. K., Sugathan, P., Agarwal, R., Khare, S., Lal, S. & Jayaram Paniker, C. K. (2005). Hand-foot-and-mouth disease in Calicut. Indian J Pediatr 72, Shah, V. A., Chong, C. Y., Chan, K. P., Ng, W. & Ling, A. E. (2003). Clinical characteristics of an outbreak of hand, foot and mouth disease in Singapore. Ann Acad Med Singapore 32, Solomon, T., Lewthwaite, P., Perera, D., Cardosa, M. J., McMinn, P. & Ooi, M. H. (2010). Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis 10, Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: Molecular Evolutionary Genetic Analysis (MEGA) software version 4.0. Mol Biol Evol 24, Urashima, M., Shindo, N. & Okabe, N. (2003). Seasonal models of herpangina and hand-foot-mouth disease to simulate annual fluctuations in urban warming in Tokyo. Jpn J Infect Dis 56, Yan, J. J., Su, I. J., Chen, P. F., Liu, C. C., Yu, C. K. & Wang, J. R. (2001). Complete genome analysis of enterovirus 71 isolated from an outbreak in Taiwan and rapid identification of enterovirus 71 and coxsackievirus A16 by RT-PCR. J Med Virol 65, Zhang, Y., Tan, X. J., Wang, H. Y., Yan, D. M., Zhu, S. L., Wang, D. Y., Ji, F., Wang, X. J., Gao, Y. J. & other authors (2009). An outbreak of hand, foot, and mouth disease associated with subgenotype C4 of human enterovirus 71 in Shandong, China. J Clin Virol 44, Zhu, Z., Xu, W. B., Xu, A. Q., Wang, H. Y., Zhang, Y., Song, L. Z., Yang, H. L., Li, Y. & Ji, F. (2007). Molecular epidemiological analysis of echovirus 19 isolated from an outbreak associated with hand, foot, and mouth disease (HFMD) in Shandong Province of China. Biomed Environ Sci 20,