Epidemiology of rotavirus diarrhea among children less than 5 years hospitalized with acute gastroenteritis prior to rotavirus vaccine introduction in India.

BACKGROUND: Rotavirus is an important cause of severe diarrhea requiring hospitalization, accounting for approximately 78,000 deaths annually in Indian children below 5 years of age. We present epidemiological data on severe rotavirus disease collected during hospital-based surveillance in India before the introduction of the oral rotavirus vaccine into the national immunization schedule. METHODS: The National Rotavirus Surveillance Network was created involving 28 hospital sites and 11 laboratories across the four geographical regions of India. From September 2012 to August 2016 children less than 5 years of age hospitalized for diarrhea for at least 6 h, were enrolled. After recording clinical details, a stool sample was collected from each enrolled child, which was tested for rotavirus antigen using enzyme immunoassay (EIA). Nearly 2/3rd of EIA positive samples were genotyped using reverse transcription polymerase chain reaction to identify the G and P types. RESULTS: Of the 21,421 children enrolled during the 4 years surveillance, 36.3% were positive for rotavirus. The eastern region had the highest proportion of rotavirus associated diarrhea (39.8%), while the southern region had the lowest (33.8%). Rotavirus detection rates were the highest in children aged 6-23 months (41.8%), and 24.7% in children aged < 6 months. Although rotavirus associated diarrhea was seen throughout the year, the highest positivity was documented between December and February across all the regions. The most common rotavirus genotype was G1P[8] (52.9%), followed by G9P4 (8.7%) and G2P4 (8.4%). CONCLUSIONS: There is high burden of rotavirus gastroenteritis among Indian children below 5 years of age hospitalized for acute diarrhea thereby highlighting the need for introduction of rotavirus vaccine into the national immunization program and also for monitoring circulating genotypes.

Diversity of rotavirus genotypes circulating in children < 5 years of age hospitalized for acute gastroenteritis in India from 2005 to 2016: analysis of temporal and regional genotype variation.

BACKGROUND: From 2016, the Government of India introduced the oral rotavirus vaccine into the national immunization schedule. Currently, two indigenously developed vaccines (ROTAVAC, Bharat Biotech; ROTASIIL, Serum Institute of India) are included in the Indian immunization program. We report the rotavirus disease burden and the diversity of rotavirus genotypes from 2005 to 2016 in a multi-centric surveillance study before the introduction of vaccines. METHODS: A total of 29,561 stool samples collected from 2005 to 2016 (7 sites during 2005-2009, 3 sites from 2009 to 2012, and 28 sites during 2012-2016) were included in the analysis. Stools were tested for rotavirus antigen using enzyme immunoassay (EIA). Genotyping was performed on 65.8% of the EIA positive samples using reverse transcription- polymerase chain reaction (RT-PCR) to identify the G (VP7) and P (VP4) types. Multinomial logistic regression was used to quantify the odds of detecting genotypes across the surveillance period and in particular age groups. RESULTS: Of the 29,561 samples tested, 10,959 (37.1%) were positive for rotavirus. There was a peak in rotavirus positivity during December to February across all sites. Of the 7215 genotyped samples, G1P[8] (38.7%) was the most common, followed by G2P[4] (12.3%), G9P[4] (5.8%), G12P[6] (4.2%), G9P[8] (4%), and G12P[8] (2.4%). Globally, G9P[4] and G12P[6] are less common genotypes, although these genotypes have been reported from India and few other countries. There was a variation in the geographic and temporal distribution of genotypes, and the emergence or re-emergence of new genotypes such as G3P[8] was seen. Over the surveillance period, there was a decline in the proportion of G2P[4], and an increase in the proportion of G9P[4]. A higher proportion of mixed and partially typed/untyped samples was also seen more in the age group 0-11 months. CONCLUSIONS: This 11 years surveillance highlights the high burden of severe rotavirus gastroenteritis in Indian children < 5 years of age before inclusion of rotavirus vaccines in the national programme. Regional variations in rotavirus epidemiology were seen, including the emergence of G3P[8] in the latter part of the surveillance. Having pre-introduction data is important to track changing epidemiology of rotaviruses, particularly following vaccine introduction.

Evaluation of different genomic regions of Rotavirus A for development of real time PCR.

The nucleotide alignment of all 11 genes of human Rotavirus A (RVA) strains revealed suitability of NSP2, NSP3 and VP6 genes for the development of real time PCR (qRT-PCR). Evaluation of qRT-PCR assays using known rotavirus ELISA positive and negative fecal specimens showed non-overlapping ranges of Mean ±3SD cycle threshold (Ct) values for NSP3 and VP6 based assays. Using serial dilutions of purified RVA, high sensitivity of VP6 qRT-PCR assay (1.95 × 10-5 pg/µL of RNA) was recorded as compared to NSP2 and NSP3 qRT-PCR assays (1.95 × 10-4 pg/µL of RNA). Further, evaluation of the VP6 qRT-PCR assay involving 266 fecal specimens and frequency polygon analysis of the data indicated cut-off value of 35 for Ct with high sensitivity (126/131, 96%) and specificity (12/12, 100%). This VP6 qRT-PCR assay will be a useful diagnostic tool to evaluate clinical presentations in rotaviral gastroenteritis under different conditions such as breast feeding and administration of rotavirus vaccines.

Genetic analysis of rotavirus G2P[4] strains in Pune, Western India: circulation of a novel reassortant bearing E6 NSP4 genotype.

In India, G2P[4] strains are known to be the second most predominant group A rotaviruses causing acute gastroenteritis among children. This study was performed to determine the diversity within VP7(G), VP4(P), VP6(I) and NSP4(E) genes of 16 G2P[4] rotavirus strains detected in children hospitalized for acute gastroenteritis in Pune, Western India during 2009-2013. Fourteen strains showed G2-P[4]-I2-E2 and two strains showed G2-P[4]-I2-E6 genotype constellation. Phylogenetic analysis showed their clustering into G2-IV-a3, P[4]-5bi/ii, I2-3ii and E2-4i/ii or E6 genotypes/lineages. These data reveal inter- and/or intra-genotypic variations in a genogroup-2 constellation of G2P[4] rotavirus strains circulating in Pune, Western India, providing evidence of a novel G2P[4] reassortant bearing a rare NSP4 genotype, E6 during 2009-2013.

Full genomic analysis of G1P[8] rotavirus strains recovered from rotavirus vaccinated and non-vaccinated children hospitalized for acute gastroenteritis in Pune, western India.

G1P[8] rotaviruses are predominant in causing diarrheal infections in humans all over the world. This study reports the analysis of complete genomes of G1P[8] strains, two each recovered from Rotarix™ vaccine recipients and non-recipients hospitalized for acute gastroenteritis in Pune, western India. All four strains showed a genogroup-1 backbone with intra-genotypic diversity in the VP7 and VP4 gene segments and a homogeneous constellation of the internal gene segments. A divergence in the range of 1.4-17.3% from Rotarix™ vaccine strain was revealed by structural and non-structural genes of the strains at nucleotide and amino acid level. These data reflect ability of such G1P[8] strains to cause rotavirus infections in humans.

Prevalence and characterization of rotaviruses in children hospitalized for diarrheal disease in a tertiary care hospital, Pune.

BACKGROUND: Diarrhoea remains the second most common cause of death among children below 5 years globally. Among various enteric pathogens, rotavirus appears to be the most important aetiological agent of acute gastroenteritis in infants and young children. Increased understanding of epidemiology of rotavirus infections is needed to improve the vaccine efficacy. AIM: This study aims to determine prevalence rotavirus infection and prevalent circulating strains of rotavirus in and around Pune. SETTING AND DESIGN: Prospective hospital-based study. The study was approved by Institutional Ethical Committee. MATERIALS AND METHODS: Stool samples (n = 100) were collected from children aged <5 years, hospitalised for acute diarrhoea in paediatric ward at a tertiary care hospital. Samples were subjected for rotavirus antigen capture ELISA. The viral RNA was subjected to multiplex reverse transcription polymerase chain reaction to amplify VP7 genotypes G1-G4, G8-G10 and G12 and VP4 genotypes P[4], P[6], P[8], P[9], P[10] and P[11]. Nontypable rotavirus strains were sequenced. RESULTS: About 35% stool samples were positive for rotavirus antigen by ELISA. G9P[4] (28.6%) was found to be the most prevalent rotavirus strain. The detection of emerging strain G12P[6] (14.3%) and rare reassortant strain G9P[4] was the significant finding. CONCLUSION: Genotypes found in circulation are not present in the currently used vaccine. Thus, an emergence of newer genotypes over a period calls for the continued surveillance and genomic characterisation of rotaviruses to improve the vaccine efficacy.

Molecular characterization of emerging G9P[4] rotavirus strains possessing a rare E6 NSP4 or T1 NSP3 genotype on a genogroup-2 backbone using a refined classification framework.

Rotavirus infections associated with unusual strains are an emerging concern in rotavirus vaccination programmes. Recently, an increase in circulation of unusual G9P[4] strains was reported from different regions of India, placing this genotype in third position, after G1P[8] and G2P[4], of the most common rotavirus strains. The aim of the present study was to analyse the complete genomic constellation of three G9P[4] strains (RV09, RV10 and RV11), determine their genetic relatedness to other genogroup-2 strains and understand the evolution of a rare E6 and other NSP4 genotypes. All strains revealed the presence of a genogroup-2 backbone, with RV09 constituting the NSP3 T1 genotype and RV10 and RV11 bearing the NSP4 E6 genotype. A refined criterion adopted to classify the nine internal gene segments of G2P[4] and non-G2P[4] strains with the genogroup-2 backbone into lineages and sub-lineages indicated divergence of >8 % (except NSP1: >5.5 %) for lineages and >3 % for sub-lineages. The VP1 and/or VP3 genes of study strains showed close relationships with animal-like human rotaviruses. The estimated evolutionary rate for the NSP4 E6 genotype was marginally higher (3.78×10-3 substitutions per site per year) than that of genotypes E1 (2.6×10-3 substitutions per site per year) and E2 (3.06×10-3 substitutions per site per year), suggesting a step towards adaptation of E6 on a genogroup-2 backbone. The time and origin of the most recent common ancestor of E6 genotype were estimated to be 1981 and South Asia, respectively. Full-genome and evolutionary analyses performed in this study for G9P[4] strains will help better understand the extent of gene reassortment and origin in unusual rotavirus strains that may remain viable and cause infections in humans.

Rotavirus Infections in Children Vaccinated Against Rotavirus in Pune, Western India.

OBJECTIVE: To characterize rotavirus infections detected in rotavirus vaccinated children hospitalized for acute gastroenteritis. DESIGN: Observational, hospital-based study. SETTING: Three hospitals in Pune, Western India. PARTICIPANTS: Children aged <5 years hospitalized for acute gastroenteritis during 2013-14. METHODS: Rotavirus capture ELISA was performed on all stool samples that were collected from patients following informed consent from parents. VP7 and VP4 genes of rotavirus strains were genotyped by multiplex RT-PCR. Stool samples from vaccinated children were tested for other enteric viruses. RESULTS: Among the 529 children, 53 were vaccinated with at least one dose of the rotavirus vaccine. There was no difference in the mean (SD) (months) age of vaccinated [14.8 (10.6)] and unvaccinated [14.4 (10.5)] children. Rotavirus positivity was significantly higher (47%) in unvaccinated than in vaccinated (28.3%) children (P=0.01). Mean Vesikari score and severe cases were significantly more in rotavirus positive than in negative children within unvaccinated group (P<0.001), while these did not differ within the vaccinated group. Rotavirus strain G1P[8] was identified as the most prevalent strain in both, vaccinated (60%) and unvaccinated (72.8%) groups. No association was found between mean Vesikari score and viral coinfections. CONCLUSIONS: This study suggests decline in rotavirus positivity in rotavirus-vaccinated children hospitalized for acute gastroenteritis and high prevalence of G1P[8] and non-rotaviral co-infections in Pune, Western India.

Rotavirus Infection in Children with Acute Gastroenteritis in Aurangabad, Central Maharashtra.

OBJECTIVE: To determine the prevalence of rotavirus diarrhea and its genotypes in children from Aurangabad, India. METHODS: Stool samples collected during 2012-2013 from 168 children, aged ?3 years, were tested by ELISA to detect rotavirus. Rotavirus strains were genotyped by multiplex reverse-transcription polymerase chain reaction. RESULTS: Stool samples from 20 (11.9%) children tested positive for rotavirus. Rotavirus positivity was higher among the children aged 0-12 months than those in 13-24 and 25-36 months. Severity of disease was moderate in both rotavirus-infected and uninfected children. Genotype G1P[8] combination was detected predominantly in circulation. CONCLUSIONS: Rotavirus diarrhea was caused mainly by G1P[8] strains during 2012-2013 in Aurangabad, Central Maharashtra, India.

Seroprevalence of antibodies against GII.4 norovirus among children in Pune, India.

This study reports the seroprevalence of antibodies against GII.4 norovirus among children (≤5 years) in Pune, India. Of 191 serum specimens, 98 (51.3%) tested positive with 61, 34 and 3 having IgG, IgG-IgA and IgG-IgA-IgM, respectively. Histoblood group antigen (HBGA)-blocking antibodies were detected in 33 of the 54 tested positive specimens. IgG and blocking antibody prevalence and titer varied with age and was lowest among children aged 6-23 months. Antibody-positive children, suggesting past norovirus exposure, showed significantly lower faecal norovirus RNA detection rate than antibody-negative children. Further investigation of the seroepidemiology of norovirus infections in India is warranted. J. Med. Virol. 88:1636-1640, 2016. © 2016 Wiley Periodicals, Inc.

Genomic characterization of coxsackievirus type B3 strains associated with acute flaccid paralysis in south-western India.

Acute flaccid paralysis (AFP) associated with coxsackievirus type B3 (CV-B3) of the species Enterovirus B is an emerging concern worldwide. Although CV-B3-associated AFP in India has been demonstrated previously, the genomic characterization of these strains is unreported. Here, CV-B3 strains detected on the basis of the partial VP1 gene in 10 AFP cases and five asymptomatic contacts identified from different regions of south-western India during 2009-2010 through the Polio Surveillance Project were considered for complete genome sequencing and characterization. Phylogenetic analysis of complete VP1 gene sequences of global CV-B3 strains classified Indian CV-B3 strains into genogroup GVI, along with strains from Uzbekistan and Bangladesh, and into a new genogroup, GVII. Genomic divergence between genogroups of the study strains was 14.4 % with significantly lower divergence (1.8 %) within GVI (n = 12) than that within GVII (8.5 %) (n = 3). The strains from both AFP cases and asymptomatic contacts, identified mainly in coastal Karnataka and Kerala, belonged to the dominant genogroup GVI, while the GVII strains were recovered from AFP cases in north interior Karnataka. All study strains carried inter-genotypic recombination with the structural region similar to reference CV-B3 strains, and 5' non-coding regions and non-structural regions closer to other enterovirus B types. Domain II structures of 5' non-coding regions, described to modulate virus replication, were predicted to have varied structural folds in the two genogroups and were attributed to differing recombination patterns. The results indicate two distinct genomic compositions of CV-B3 strains circulating in India and suggest the need for concurrent analysis of viral and host factors to further understand the varied manifestations of their infections.

Characterization of GII.4 noroviruses circulating among children with acute gastroenteritis in Pune, India: 2005-2013.

Genogroup II genotype 4 noroviruses (GII.4 NoVs), an important cause of sporadic childhood gastroenteritis worldwide, undergo continuous evolution leading to the periodic emergence of novel variants. The present study was undertaken for surveillance of GII.4 NoVs and identification and characterization of GII.4 variants circulating among children with sporadic gastroenteritis in Pune, India during 2005-2013. Among the 12 GII genotypes detected in the study, GII.4 was predominant. Sequencing and phylogenetic analysis of ORF2 (major capsid protein VP1 gene) of the GII.4 NoVs revealed circulation of seven GII.4 variants, Hunter_2004 (2005-2007), Yerseke_2006a (2006), DenHaag_2006b (2007), Osaka_2007 (2007-2009), Apeldoorn_2007 (2008), New Orleans_2009 (2008-2012) and Sydney_2012 (2013), with the Pune strains grouping with the contemporary global reference strains. The Hunter_2004, Osaka_2007 and New Orleans_2009 variants showed prolonged circulation, with the Hunter_2004 and New Orleans_2009 variants differentiating into temporally separated sub-clusters. Analysis of VP1 sequences and predicted structures of the GII.4 variants identified variant specific amino acid positions, particularly in and near (within 8A(°)) the epitopes A-E, displaying differences in the sequence and physicochemical characteristics of the different variants. Comparison with the reference strains of each of the GII.4 variants revealed up to 11 amino acid substitutions at the variant specific positions in the GII.4 strains from Pune. Amino acid variations were also noted among the strains of the same GII.4 variant in Pune. The strains of different sub-clusters identified in the Hunter_2004 and New Orleans_2009 variants showed differences in sequence and physicochemical properties of either or all of the epitopes A, C and E. The study thus describes the temporal variations and diversity of the GII.4 strains in Pune and emphasizes continuous monitoring and analysis of the GII.4 variants.

Emerging OP354-Like P[8] Rotaviruses Have Rapidly Dispersed from Asia to Other Continents.

The majority of human group A rotaviruses possess the P[8] VP4 genotype. Recently, a genetically distinct subtype of the P[8] genotype, also known as OP354-like P[8] or lineage P[8]-4, emerged in several countries. However, it is unclear for how long the OP354-like P[8] gene has been circulating in humans and how it has spread. In a global collaborative effort 98 (near-)complete OP354-like P[8] VP4 sequences were obtained and used for phylogeographic analysis to determine the viral migration patterns. During the sampling period, 1988-2012, we found that South and East Asia acted as a source from which strains with the OP354-like P[8] gene were seeded to Africa, Europe, and North America. The time to the most recent common ancestor (TMRCA) of all OP354-like P[8] genes was estimated at 1987. However, most OP354-like P[8] strains were found in three main clusters with TMRCAs estimated between 1996 and 2001. The VP7 gene segment of OP354-like P[8] strains showed evidence of frequent reassortment, even in localized epidemics, suggesting that OP354-like P[8] genes behave in a similar manner on the evolutionary level as other P[8] subtypes. The results of this study suggest that OP354-like P[8] strains have been able to disperse globally in a relatively short time period. This, in combination with a relatively large genetic distance to other P[8] subtypes, might result in a lower vaccine effectiveness, underscoring the need for a continued surveillance of OP354-like P[8] strains, especially in countries where rotavirus vaccination programs are in place.

Molecular surveillance of non-polio enterovirus infections in patients with acute gastroenteritis in Western India: 2004-2009.

Acute gastroenteritis is a major cause of childhood morbidity and mortality worldwide. Rotavirus (RV) and Norovirus (NoV) are the leading cause of the disease. Despite the use of improved diagnostic methods a significant proportion of gastroenteritis cases remained undiagnosed. Though nonpolio enteroviruses (NPEVs) have been reported frequently in children with acute gastroenteritis, their etiologic role has not been established. To investigate the epidemiology of NPEVs in gastroenteritis cases which remained negative for leading causative agents, 955 RV and NoV negative stool specimens from children hospitalized for acute gastroenteritis were included in the study. A case control study was conducted which includes stool specimens from 450 children with gastroenteritis and 162 asymptomatic control subjects to determine the association of NPEVs with the disease. NPEV detection and typing was carried out by RT-PCR and sequencing. Presence of RV, NoV, Adenovirus, and Astrovirus was confirmed by ELISA or PCR/RT-PCR. Overall 14% NPEV prevalence was noted. The percentage of children with NPEV infection differed significantly between gastroenteritis and non-gastroenteritis patients (13.7% vs. 4.9%). NPEV was more prevalent among patients with gastroenteritis of undetectable etiology as compared to those detected positive for other viruses (17.9% vs. 7%) (P < 0.01). Genotyping of NPEV identified predominance of EV-B species (56.5%) followed by EV-C (16.7%), EV-A (13.8%) species and mixed NPEV infections (13%). These data support the association of NPEVs with acute gastroenteritis and highlights the clinical and epidemiological features of NPEV infections in patients with acute gastroenteritis from western India.

Molecular characterization of group B rotavirus circulating in pigs from India: identification of a strain bearing a novel VP7 genotype, G21.

The occurrence of group B rotavirus (RVB) infections in pigs has been reported from different parts of world. However, such infection in the pig population maintained in Indian farms has not been investigated as yet. A total of 187 faecal specimens were collected from pigs reared in different pig farms/pigsties located in western and northern regions of India and tested for the presence of porcine RVB by amplification of the NSP2 gene using conventional RT-PCR. Nine specimens (4.8%) were shown to contain RVB RNA. N2 and N4 genotypes of NSP2 gene were detected in three and six RVB strains respectively. VP7 (G-type) and NSP5 (H-type) genes of selected six RVB strains were characterized to identify the genotypes. Multiple G (G7, G19 and G20) and H (H4 and H5) genotypes detected in the RVB strains indicated circulation of heterogeneous population of RVB strains in pigs of India. Additionally, one strain was proposed to belong to a novel RVB genotype designated as G21 on account of <80% identity of VP7 gene sequence with its counterpart in RVB strains from 20 established genotypes. Deduced amino acid sequence of VP7 gene also displayed the presence of seven unique substitutions in the strain. The study reports for the first time the occurrence of RVB infections in Indian pig herds and provides important epidemiological data useful for better understanding of ecology and evolution of porcine RVBs.

Genomic characterization of coxsackievirus type A24 strains associated with acute flaccid paralysis and rarely identified Hopkins syndrome.

The full-length genome sequence analysis of four coxsackievirus A24 (CV-A24) strains, detected in three paralytic and one post-asthmatic paralytic (Hopkins syndrome) cases, is reported here for the first time. A phylogenetic tree constructed on the basis of entire genomes displayed topology similar to that of the full-VP1 tree, classifying the study strains in genogroup CV-A24vGIV along with their temporal counterparts in strains from non-paralytic cases. The strains of the study formed a single genetic cluster C4 within CV-A24vGIV and showed 3.5-19.4 % nucleotide sequence divergence, with 2-4 novel nucleotide mutations in the 5'NCR and 3-8 unique amino acid substitutions in the polyprotein, with respect to the CV-A24 strains associated with non-paralytic cases. Among the nucleotide mutations, A299U was identified in the 5'NCRs of all of the study strains. CV-A24v strains of the same genogroup with few genomic variations but different disease manifestations need to be explored to investigate the molecular basis of evolution of neurovirulence.

Changing trends in circulating rotavirus strains in Pune, western India in 2009-2012: emergence of a rare G9P[4] rotavirus strain.

BACKGROUND: A vast diversity in rotaviruses at inter- and intra-genotypic level underscores the need for monitoring of circulating rotavirus strains. The aim of this study was to update the data on rotavirus disease and strains for the period from January 2009 to December 2012 in Pune, western India which has been one of the sites of the Indian Rotavirus Strain Surveillance Network since November 2005. METHODS: Children aged <5 years admitted for acute gastroenteritis in three different hospitals from Pune city were included in the study. The stool specimens were collected and tested for rotavirus antigen by a commercial enzyme immunoassay. The rotavirus strains were genotyped by multiplex reverse transcription polymerase chain reaction. RESULTS: During the study period, we found 35.1% of 685 stool specimens contained rotavirus antigen. Frequency of rotavirus detection was greatest (58.5%) among children aged 7-12 months. The G1P[8] (31.4%), G2P[4] (20.2%) and G9P[8] (11.8%) strains were the most common types. We noted predominance of G1P[8] strains (39.6%-46.1%) in all the years of study except 2009 wherein G9P[8] strains scored highest level (15.3%). Subsequent to this, we identified G9P[8] strains at the second highest position in 2010, their sudden decline and rise in G9P[4] strains in 2011-2012. We detected G12 strains in combination with P[6] and P[8] at variable rates (0-10.2%) and highest level (27.1%) of mixed rotavirus infections in 2009 as compared to 2010-2012 (0-3.8%). CONCLUSION: The study highlights the huge burden of rotavirus disease and changing profile of circulating rotavirus strains displaying emergence of G9P[4] reassortant strains in Pune, western India and emphasizes the need to analyze the entire genomic constellation of rotavirus strains for better evaluation of the impact of rotavirus.

Characterisation of rotavirus strains identified in adolescents and adults with acute gastroenteritis highlights circulation of non-typeable strains: 2008-2012.

BACKGROUND: Group-A Rotavirus (RV) is the main causative agent of acute gastroenteritis in children < 5 years of age. Its role as a pathogen in adults needs to be monitored. The aim of this study was to characterise the group-A RV strains that cause infections of acute gastroenteritis in adolescents and adults and determine the temporal variations in the circulating strains during 2008-2012 in continuation of an earlier study conducted in 2004-2007, in Pune, India. METHODS: A total of 371 stool samples were tested by RV antigen capture ELISA. VP4, VP6, VP7 and NSP4 genes of all of the RV strains detected in the study were analysed using reverse transcription PCR, multiplex PCR and sequencing. RESULTS: Group-A RV was detected in 9.4% (35/371) of the stool samples examined in the study period. The frequency of detection of RV was found to decline from 18.0% (16/90) in 2008 to 3.8% (2/52) in 2012. Of the 6 strains typed for both VP7 and VP4 genes, G2P[4], G1P[8] and G9P[4] were detected in 3, 1 and 2 samples, respectively. Sequencing and phylogenetic analysis of the VP4, VP6, VP7 and NSP4 genes revealed an infrequently reported NSP4-E6 genotype and circulation of heterogenous [G2 (lineage IIC and IID), G9 (lineage 3), P[4] (lineage P[4]-5), P[8] (lineage P[8]-3), VP6 I1 / I2 and NSP4 E2] genotypes/lineages in the RV strains. Analysis of linkage within these genes showed concordance (G2-P[4]-I2-E2) and discordance (G9-P[4]-I2-E6), equally. The sequences of amplified VP6 (n = 20) and NSP4 (n = 2) genes from G and P nontypeable RV strains (80.0%, 28/35) were most homologous to human group-A RV strains. CONCLUSION: The study underscores the significant temporal variations in RV strains, identifies circulation of intergenogroup reassortants among adolescent and adult patients with acute gastroenteritis and emphasizes the need for continued surveillance and whole genome analysis of emerging rotavirus strains.

Sequence analysis of VP7 and VP4 genes of G1P[8] rotaviruses circulating among diarrhoeic children in Pune, India: a comparison with Rotarix and RotaTeq vaccine strains.

BACKGROUND: The G1P[8] rotaviruses are a common cause of rotavirus diarrhoea among children in India. Two rotavirus vaccines licensed in India, Rotarix and RotaTeq, contain strains with G1 and P[8] genotypes. A comparative analysis of these genotypes in the live rotavirus vaccines with circulating rotavirus strains is essential for assessment of rotavirus diversity. METHODS: G1P[8] strains detected during rotavirus surveillance among diarrhoeic children hospitalized in Pune in 1992-1993 and 2006-2008, were included in the study. Amplification, sequencing and phylogenetic analysis of the VP7 and VP4 genes were carried out for identification of the G1 and P[8] lineages, respectively. Antigenic epitopes of VP7 and VP4 encoded proteins were compared to determine the differences between the G1P[8] strains from Pune and the vaccine strains. RESULTS: G1-Lineage 1, P[8]-Lineage 3 strains were predominant in Pune during 1992-1993 and 2006-2008. Strains of G1-Lineage 2, P[8]-Lineage 3 and G1-Lineage 1, P[8]-Lineage 4 were detected at low levels during 2006-2008. The G1-Lineage 1, P[8]-Lineage 3 strains showed up to eight amino acid changes, each in the VP7 and VP4 epitopes, with respect to the Rotarix vaccine strain (G1-Lineage 2, P[8]-Lineage 1) and the G1 (Lineage-3) and P[8] (Lineage 2) components of the RotaTeq vaccine. The G1-Lineage 2 strains were closer to both vaccine strains with no or only two amino acid substitutions in the VP7 epitopes. The divergent P[8]-Lineage 4 (OP354-like) strains showed fourteen and fifteen amino acid differences, with Rotarix and RotaTeq vaccine strains, respectively, in the VP4 epitopes. CONCLUSION: The differences between the G1P[8] strains in Pune and the G1 and P[8] components of the vaccine strains need to be described for appropriate evaluation of vaccine shedding. Continuous monitoring of the G1P[8] subgenotypic lineages would be necessary to study any long term impact of vaccine use on G1P[8] strain evolution.