High frequency of enterovirus serotype circulation in a densely populated area of India.

INTRODUCTION: In the state of Uttar Pradesh in India, enteroviruses are a significant cause of infection presenting in endemic or epidemic forms. The present study aimed to use molecular methods to identify enterovirus serotypes in clinical specimens to determine their circulation in the community. METHODOLOGY: A total of 320 clinical specimens were collected between January 2009 and December 2010 from children younger than 15 year of age in northern India. Reverse- transcription (RT) real time PCR and semi-nested RT PCR targeting the 5'untranslated region and VP1 region was used for the detection and identification of enterovirus serotypes. RESULTS: The enterovirus genome was detected in 79 (24.7%) of 320 clinical specimens by real time PCR. Central nervous system syndrome (CNS) was the most common clinical manifestation (n=32, 62.74%), followed by respiratory tract infection (n=8, 15.69%), acute febrile illness (n=7, 13.73%), and gastrointestinal disease (n=4, 7.84%). A total of 32 different serotypes were identified with the predominance of coxsackievirus B5 and echovirus 6. Phylogenetic analysis of partial VP1 gene sequences from this study showed that many enterovirus serotypes showed good similarity with strains from America and Europe in comparison to neighbouring Asian countries. CONCLUSIONS: To our knowledge this is the first study of enterovirus prevalence from northern India based on unbiased molecular methods which leads to the identification of fifteen different enterovirus serotypes. The high frequency of enterovirus B species serotypes circulation may be an important cause of CNS infection in the children of this region.

Environmental surveillance of enterovirus in Northern India using an integrated shell vial culture with a semi-nested RT PCR and partial sequencing of the VP1 gene.

Enteroviruses have been reported in epidemic form during last 10 years in northern India. Environmental surveillance of sewage is the method of choice in limited resources countries for detection of enterovirus serotypes circulating in the community. Twenty-four sewage samples collected between January, 2009 and December, 2010 were tested for enterovirus by using a new modified integrated shell vial culture (ISVC) with a semi-nested RT-PCR of a partial VP1 gene and virus isolation integrated with semi-nested RT-PCR of a partial VP1 gene. Twenty-one (87.5%) out of 24 samples were positive for enterovirus by the conventional method and all samples (100%) by the ISVC-RT-PCR. The additional positive samples detected by ISVC-RT-PCR was typed as six different enterovirus serotypes (Sabin poliovirus 3, Coxsackievirus B3, Coxsackievirus A13, Coxsackievirus A17, Echovirus 33, and Enterovirus 75). Phylogenetic analysis of a partial VP1 gene of Echovirus 19 showed that one genetic lineage clustered with isolates from Georgia suggesting their importation into northern India. Detection of wild poliovirus in the absence of clinical cases with 16 different co-circulating enterovirus serotypes supports the need of increased molecular surveillance of sewage. Rapid identification and characterization of enterovirus serotypes is necessary to study their transmission and evolution in different geographical regions to prevent future outbreak.

Molecular identification of enteroviruses associated with aseptic meningitis in children from India.

We identified and characterized enteroviruses associated with aseptic meningitis in children between April 2009 and March 2010. Enterovirus RNA was detected in 51 (45.5 %) of 112 CSF samples. Molecular typing by RT-PCR and sequencing of a partial VP1 region revealed the predominance of echovirus (ECV) 32 (n = 20), followed by ECV 11 (n = 10), ECV 13 and ECV 14 (n = 5 each), coxsackievirus (CV) B3 and CV B6 (n = 3 each), CV A2, CV A10 and ECV 30 (n = 1 each). Phylogenetic analysis of ECV 32 showed 0 to 4 % sequence divergence among strains of the present study and 20-23 % from the prototype Puerto Rico strain at the nucleotide level. This is the first report of ECV 32 associated with an aseptic meningitis epidemic and identification of seven different enterovirus serotypes (CV A2, CV A10, CV B3, CV B6, ECV 13, ECV 14 and ECV 32) in meningitis cases from India.

Molecular epidemiological study of enteroviruses associated with encephalitis in children from India.

Enteroviruses have been reported in encephalitis cases. However, clinical and epidemiological characteristics of enteroviruses in encephalitis are not fully established. We prospectively investigated 204 children with encephalitis over a period of 2 years (2009 to 2010) for enterovirus. Enterovirus was detected in 45 specimens (22.1%); of these, 40 were typed by seminested reverse transcription-PCR (RT-PCR) and sequencing of the VP1 gene. Molecular typing of enterovirus revealed the predominance of echovirus 21 associated with an epidemic during the rainy seasons of 2010 and the circulation of echovirus 1, coxsackievirus B1, enterovirus 75, enterovirus 76, coxsackievirus B5, and echovirus 19. The nucleotide divergence among echovirus 21 strains was 0 to 2% at the nucleotide level. This study suggests that enterovirus is an important cause of encephalitis in children from India. To our knowledge, this is the first report of echovirus 21 in encephalitis cases worldwide.

An epidemic of encephalitis associated with human enterovirus B in Uttar Pradesh, India, 2008.

BACKGROUND: Human enteroviruses (HEVs) are a rare cause of encephalitis, presenting in endemic or epidemic form. OBJECTIVES: The aim of the study is to identify and characterise the causative agent of the encephalitis epidemic, which occurred in Uttar Pradesh, India during the summer of 2008. STUDY DESIGN: A total of 90 cerebrospinal fluid (CSF) specimens were collected between June and October 2008 from children with symptoms of encephalitis admitted to Chhatrapati Shahuji Maharaj Medical University and Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India. Conventional and molecular methods were used to identify and characterise the viral agent associated with the epidemic. RESULTS: Enterovirus RNA was detected in 37 (41.11%) of 90 CSF samples by real-time polymerase chain reaction (PCR). Seroneutralisation, amplification and sequencing of the 3'-end of the VP1 region of EV isolates revealed coxsackievirus B5 (CBV) and echovirus 19 (ECV) as the main serotypes causing this epidemic. Phylogenetic analysis showed that sequence divergence among the same serotypes was 0-4% at the nucleotide level. CONCLUSIONS: This is the first report suggesting that CBV 5 and ECV 19 may be responsible for an epidemic of encephalitis in India. These serotypes were variant and evolved within the studied area.