key: cord-1004014-sh0v7ec9
authors: Tatte, Vaishali S.; Gentsch, Jon R.; Chitambar, Shobha D.
title: Characterization of group A rotavirus infections in adolescents and adults from Pune, India: 1993–1996 and 2004–2007
date: 2010-01-19
journal: J Med Virol
DOI: 10.1002/jmv.21708
sha: 66b40ec7b8e45df62e778d299ad8b0718736ad00
doc_id: 1004014
cord_uid: sh0v7ec9

A total of 1,591 fecal specimens were collected in 1993–1996 and 2004–2007 from adolescents and adults with acute gastroenteritis in Pune, India for detection and characterization of rotavirus. At the two time points, group A rotavirus was detected in 8.6% and 16.2% of the adolescents and 5.2% and 17.2% of the adults, respectively. Reverse transcription‐PCR with consensus primers followed by multiplex genotyping PCR detected common strains G1P[8], G2P[4], G3P[8], and G4P[8] in a total of 53.1% of the samples from 1993 to 1996, while the only prevalent strain identified in 2004–2007 was G2P[4] (23.5% of total). Uncommon rotavirus strains (G1P[4], G2P[8] G9P[6]/P[4]) increased from 7.8% (1993–1996) to 41.2% (2004–2007), while the prevalence of mixed rotavirus infections was high (39%/35%) at both time points. Mixed infections detected by multiplex PCR were confirmed by sequencing two or more individual genotype‐specific PCR products of the VP7 and VP4 genes from the same sample. Phylogenetic analysis of the sequences showed circulation of a heterogeneous rotavirus strain population comprising genotypes G1 (lineages I and IIb), G2 (lineages I and IIb), G4 (lineage Ia), P[4] (lineages P[4]‐5 and P[4]‐1), P[8] (lineages P[8]‐II and P[8]‐III), and P[6] (M37‐like lineage). The VP6 gene sequences of the nontypeable strains were most homologous to animal strains. This study documents the molecular epidemiology of rotavirus strains in adolescents and adults in India, and suggests that it may be important to monitor these strains over time for the potential impact on rotavirus vaccines under development for use in the Indian population. J. Med. Virol. 82:519–527, 2010. © 2010 Wiley‐Liss, Inc.

Group A rotaviruses are the most important etiologic agents of acute gastroenteritis in infants and young children worldwide. Annually, global mortality from rotavirus gastroenteritis has been estimated to be 611,000 with a majority of deaths occurring in lowincome countries [Parashar et al., 2006] .

Rotaviruses belong to the family Reoviridae, and are classified in seven antigenic groups (A-G), of which groups A, B, and C are known to infect humans. Group A rotaviruses infect all children in their first few years of life and account for the vast majority of rotavirus infections worldwide, while B and C rotaviruses are often found to be associated with outbreaks and sporadic cases. Morphologically, rotavirus is an icosahedral particle consisting of 11 segments of double stranded (ds) RNA encased within a triple layered capsid composed of VP4, VP6, and VP7 proteins. The VP6 protein carries antigens that determine group and subgroup specificities, while VP4 (P-protease sensitive) and VP7 (G-glycoprotein), the outer layer proteins carry epitopes for specific neutralizing antibodies and define different serotypes [Estes and Kapikian, 2007] . The two serotype antigens are encoded by separate genome segments that segregate independently of one another during mixed infections in vivo. As a result, a dual classification system to track both serotypes VP7 (G) and VP4 (P) serotypes has been established for rotaviruses.

To date, 20 G and 28 P genotypes have been reported Solberg et al., 2009] , of which G1-G4, G9, P[4], P[6], and P[8] represent common strains detected among children worldwide [Gentsch et al., 2005; Santos and Hoshino, 2005] . Reassortment in vivo among rotaviruses of the known genotypes has led to the isolation of strains with more than 50 different G-P combinations. However, only the five most common types (G1-G4, P[8]) have been targeted in rotavirus vaccines. Countries considering use of these vaccines conduct surveillance to identify the most common strains in circulation so that subsequent impact of vaccines on circulating strains can be assessed. Virtually, all such studies are conducted in infants.

Group A rotaviruses are also known to cause gastroenteritis among adults in a variety of settings including epidemic outbreaks, travel-related gastroenteritis, infections transmitted from children to adults and endemic disease [Hrdy, 1987] . Only a few studies document the genotypic diversity of rotaviruses and occurrence of unusual rotavirus strains in adults that may pose challenges to new rotavirus vaccine programs [Anderson and Weber, 2004; Fischer et al., 2005; Wang et al., 2007] .

The aim of this study was to investigate the molecular epidemiology of rotaviruses causing sporadic acute gastroenteritis in both adolescents and adults from Pune, western India at two different time points.

A total of 1,591 stool specimens were collected from adolescent (10-18 years, n ¼ 211) and adult (>18 years, n ¼ 1,380) cases admitted to or visiting out-patient departments in local hospitals from the city of Pune for acute gastroenteritis at two time points, 1993-1996 (n ¼ 1,338) and 2004-2007 (n ¼ 253) . The small number of samples analyzed at the later time point was due to less number of admissions in the hospitals that were routinely visited by National Institute of Virology, Pune. The ethical committee of National Institute of Virology approved the methods for specimen collection. Epidemiologic data inclusive of age, date of diarrhea onset, date of specimen collection were available from a majority of the patients. Ten percent (w/v) stool suspension of each of the specimens was prepared in 0.01 M phosphate buffered saline (PBS) pH 7.2 containing 0.01 M CaCl 2. The suspensions were centrifuged at 805g for 15 min to remove debris. The supernatants were stored at À708C until tested for rotavirus antigen and genotype.

All specimens were tested for the presence of rotavirus by an in-house antigen capture ELISA . Stool specimens known to be negative and positive for rotavirus and culture supernatants of normal and human rotavirus serotype G1 infected MA-104 cells were included as controls. Specimens having optical density (OD) values above the cut off value (2.0Â OD of negative control wells) were considered positive for rotavirus antigen.

Rotavirus ds RNA was extracted from stool specimens by using TRIZOL reagent (Invitrogen, Carlsbad, CA) as per the manufacturer's protocol. The VP7 and VP4 genes were genotyped by multiplex reverse transcription (RT)-PCR according to the method described earlier [Gouvea et al., 1990; Gentsch et al., 1992] with a minor modification in the thermal cycling programme [Chitambar et al., 2008] . The rotavirus strains that remained nontypeable (in this protocol) were subjected to one step RT-PCR (Qiagen, Hilden, Germany) using following sets of primers: 9Con1-L/VP7-Rdeg [Freeman et al., 2008] and Con 3 5 0 -TGG CTT CGC TCA TTT ATA GAC A-3 0 /Con 2 provided by Centers for Disease Control and Prevention, Atlanta, USA [Gentsch et al., 1992] . Briefly, 4 ml of extracted rotavirus ds RNA was used for RT by denaturing at 958C for 5 min and then chilling in ice for 2 min. A reaction mix of 46 ml containing 5Â buffer, dNTPs, Rnase-free water, primers 9Con1-L/ Con3 and VP7-Rdeg/Con2 and 2 ml of enzyme mix was added to make a final volume of 50 ml. The reaction tubes were placed in a thermocycler for 30 min at 45 and 958C for 15 min followed by 40 cycles of 948C for 1 min, 508C for 1 min, 708C for 2.5 min with a final extension at 708C for 7 min. One step RT-PCR based on a target region of 220 bp in gene segment 6 was also performed on all nontypeable specimens [Wilde et al., 1990] .

All PCR products, including that from the first-round and multiplex PCRs, were analyzed by electrophoresis using 1Â Tris acetate EDTA (TAE) buffer pH 8.3 on 2% agarose gels, containing ethidium bromide (0.5 mg/ml) and visualized under UV illumination.

The PCR products obtained for gene 6 (220 bp), and multiplex PCR products of genes 7 and 4 were purified on minicolumns (QIAquick; Qiagen, Valencia, CA) and sequencing was carried out using ABI-PRISM Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster city, CA) and a ABI-PRISM 310 Genetic Analyzer (Applied Biosystems).

The sequences of genes 6, 7, and 4 were aligned with the sequences of rotavirus strains available in the GenBank by using Clustal X version 1.83 [Thompson et al., 1997] . The phylogenetic analyzes were carried out in MEGA 3 by using Jukes-Canter (JC) distance and Neighbour Joining (NJ) algorithm [Kumar et al., 2004] . The reliability of different phylogenetic groupings was confirmed by using the bootstrap test (1,000 bootstrap replications) available in MEGA 3.

The sequences of VP7, VP4, and VP6 genes of this study have been submitted to the GenBank under the accession numbers FJ623188-FJ623262.

The proportions across two different periods were compared using the chi-squared test with Yates's correction and the proportions within the same period were compared by Binomial test. P-values <0.05 were considered statistically significant.

In total, rotavirus was detected in 10.0% (21/211) and 7% (97/1,380) of the specimens collected from adolescents and adults, respectively. Overall, the combined prevalence values obtained for the two time points (1993-1996 and 2004-2007) were not different (P > 0.05) in two groups examined in the study (Table I) . Age distribution analysis showed that most rotavirus infections occurred in the 10-14 years age group for adolescents and in the 19-40 years group of adults (Fig. 1) . Significantly higher prevalence of rotavirus was observed during 2007 for adults (P < 0.01) but not for adolescents (P > 0.05; Fig. 2 Genotyping VP7 and VP4 typing was conducted for all 118 rotavirus positive samples from adolescent (n ¼ 21) and adult (n ¼ 97) cases of acute gastroenteritis. Both genes were successfully genotyped in 86.7% and 85% of the specimens, respectively, during 1993-1996 but only 50% and 37.8% of the specimens could be typed in [2004] [2005] [2006] [2007] . Though the number of nontypeable rotavirus strains was higher in 2004-2007 for both groups of patients, it was not statistically different from that of the 1993-1996 (P > 0.05; Table II) .

Among the strains typeable for VP7 or VP7 and VP4 (n ¼ 75) recovered during 1993-1996, G1 and G2 were predominant genotypes at 26.7% of total followed by G3 (20.0%), G4 (13.3%), and G9 (6.7%) in adolescents. However, in adults there was predominance of G1 (30.0%) followed by G2 (28.3%), G4 (10.0%), G9 (8.3%), and G3 (1.7%) in adults. Mixed infections caused by different genotypes were 6.7% and 8.3%, in adolescents and adults, respectively. Nontypeable specimens (10.0%) made up the remainder. Among 43 specimens analyzed in 2004-2007, nontypeable strains were predominant in both groups [50% (3/6) in adolescents and 64.9% (24/37) in adults] followed by mixed infections (33.3%) and G2 (16.7%) strains in adolescents, and by G9 (13.5%), G2 (10.8%), G1 (2.7%), G3 (2.7%), G4 (2.7%), and mixed infections (2.7%) in adults.

At both time points, for rotavirus strains (15 in 1993-1996 and 6 in 2004-2007) 

A total of 32 different G-P combinations were detected in 81 rotavirus strains that were typed for both genes (Table III) and G9P[6] were found at both time points, but only in adults. Twenty-one different mixed infections with diverse combinations of G-P types were detected in adolescents and adults (Table III) . Triple infections with either G or P types were detected in individual patients in 1993-1996.

Analysis of VP7 gene sequences of 20 rotavirus strains detected in 9 of 31 total mixed infections confirmed that the multiplex assay detected true mixed infections. The 20 genes sequenced came from specimens with the following genotypes; 4 G1/G2, 1 G1/G4, 2 G2/G4, and 2 G1/G2/G4. Thus, in total 7 G1, 8 G2, and 5 G4 genotypes were detected in the mixed infections. All G1 strains clustered in lineage I of G1 genotypes with nucleotide divergence of 6.3-8.4% from the prototype strains KU and Wa of lineage III (Fig. 4) . The G2 strains clustered in lineages II b (5/8) and I (3/8) with nucleotide divergence of 5.3-6.1% from the prototype strain DS-1 of lineage I. The G4 strains showed 0.3-1.3% nucleotide divergence from the prototype strain Hochi (Fig. 4) (Fig. 5) .

VP6 genes of rotavirus strains recovered in [2004] [2005] [2006] [2007] , that were nontypeable for both the VP7 and VP4 genes had 99.5-100% nucleotide identity with their counter parts in bovine/simian strains (Table IV) .

In India, extensive studies have been carried out on characterization of the rotavirus strains causing acute gastroenteritis among children [Ramachandran et al., 1996; Jain et al., 2001; Kang et al., 2005; Ramani and Kang, 2007] , while studies in adults with acute gastroenteritis were limited to only serologic aspects of rotavirus infections [Kelkar and Ayachit, 2000; Ray and Kelkar, 2004] . This study reports, for the first time, the epidemiologic and molecular characteristics of rotavirus infections in adolescent and adult cases of acute gastroenteritis from India. The studies conducted in countries from Europe, America, Asia, and Australia have reported 2-40% rates of rotavirus prevalence in adults with gastroenteritis [del Refugio Gonzalez-Losa et al., 2001; Anderson and Weber, 2004; Faruque et al., 2004; Rubliar-Abreu et al., 2005] . However, there is no comparative data available for different time points in the same country or region. In the study presented here, the observed prevalence of rotavirus infection in 2004-2007 (16.2%/17.2%) was higher than that in 1993-1996 (5.2%/8.6%) demonstrating a rise in rotavirus infections in Indian adolescents and adults.

The occurrence of rotavirus infections was noted throughout the year with increased rotavirus activity in the winter (November-February) and rainy (June-August) months (Fig. 3) . This is distinct from seasonal rotavirus infections in temperate climates [Cook et al., 1990] , but similar to previous studies reporting a lack of seasonality with a deviation of infections towards warmer months in children and adults from tropical countries [Maldonado and Yolken, 1990; Carraro et al., 2008] . In the study presented here, the contribution of common G-P types and mixed infections (59/64) to diarrhea was significantly higher than the uncommon types (5/64) in adolescents and adults in 1993-1996 (P < 0.01 by Binomial test, Table III ). Interestingly, this pattern was not retained in 2004-2007 when significantly higher numbers of infections were due to uncommon and mixed types (13/17) (P < 0.05 by Binomial test). While most G-P combinations were of the uncommon variety during 2007, all of the individual G and P genotypes except P[6] were those recognized as globally common. These results are in contrast to those reported for children with rotavirus gastroenteritis from western India where common genotypes were more prevalent than uncommon types [Zade et al., 2009] , but similar to those from southern and eastern India where uncommon G-P combinations were detected frequently among children [Ramchandran et al., 1996; Das et al., 2002] . In this study, uncommon G-P serotype combinations were detected in Indian adults for the first time. Thus, as is the case in children, a wide variety of reassortant rotavirus strains cause diarrhea in adults. In addition, circulation of such strains, for example, G9P[4] which do not share serotype antigens with the vaccine could influence success of vaccination programs, if protective immunity to such strains is lower.

In the present study, the proportion of mixed infections (39.0%/35.3%) detected among adolescents and adults at both time points (Table III) , was relatively high compared to that reported for children from India [Ramchandran et al., 1996; Sharma et al., 2008] , Brazil [Timenetsky et al., 1996] , Bangladesh [Unicomb et al., 1999] , UK [Iturriza-Gomara et al., 2000a] , Tunisia [Chouikha et al., 2007] , and Denmark [Fisher et al., 2005] . In order to confirm that these represented true mixed infections and identify VP7/VP4 lineages present in infected adolescents and adults, the DNA products obtained in multiplex PCR were sequenced. It is of note that the distribution of rotavirus strains (G1, G2, G4, P[4], P[8], and P[6]) in the lineages/sublineages (Figs. 4 and 5) was similar to that reported for children from different countries [Xin et al., 1993; Iturriza-Gomara et al., 2000b; Kudo et al., 2001; Cunliffe et al., 2001; Bok et al., 2002; Banyai et al., 2004; Page and Steele, 2004; Arista et al., 2006; Mascarenhas et al., 2006; Ansaldi et al., 2007; Araujo et al., 2007; Espinola et al., 2008] . Interestingly, cocirculation of two P[4] lineages was observed for the first time. Unlike the VP7 and VP4 genes of adult strains that were homologous to common human strains, some VP6 genes of these strains were most homologous to bovine and simian rotaviruses (Table IV) . This also contrasted with the VP6 genes of strains from children that are typically closely related to other common human rotavirus strains [ Iturriza-Gomara et al., 2002; Kerin et al., 2007] . These findings suggest that strains causing infections in adolescents and adults need to be studied further to examine the extent of reassortment in other genes to determine their origin.

Although rotaviruses have been recognized for several decades as the most common cause of gastroenteritis in infants and children, their role as a pathogen in adults has not been investigated sufficiently. Comparative analysis of rotavirus strains/genotypes in different age groups is needed for better understanding of the spread of rotavirus in the community. It is to be noted that the studies conducted in cattle to determine the prevalence of enteric virus shedding have suggested the possibility of adult animal as a source of rota or coronavirus infection in calves [Crouch and Acres, 1984] . Since natural infections with rotavirus do not confer life-long immunity, adolescents and adults may repeatedly undergo infections, which can vary from asymptomatic status to severe sustained infections depending on the immune status of the host. Under such circumstances, asymptomatic adults infected with rotavirus could potentially serve as a reservoir to maintain rotavirus circulation in the population [Pietruchinski et al., 2006] . Further, adult infections may serve as another source of rotavirus genomic diversity, which may limit the beneficial use of rotavirus vaccines being introduced in children from India. 

Rotavirus infection in adults

Molecular characterization of a new variant of rotavirus P[8]G9 predominant in a sentinal-based survey in central Italy

Nucleotide sequence and phylogenetic analysis

Heterogeneity and temporal dynamics of evolution of G1 human rotaviruses in a settled population

Sequencing and phylogenetic analysis of human genotype P[6] rotavirus strains detected in Hungary provides evidence for genetic heterogeneity within the P[6] VP4 gene

Genetic variation of capsid protein VP7 in genotype G4 human rotavirus strains: Simultaneous emergence and spread of different lineages in Argentina

Rotavirus infections in children and adult patients attending in a tertiary hospital of Sao Paulo, Brazil

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Group A rotavirus strains circulating in the eastern center of Tunisia during a ten year period

Global seasonality of rotavirus infections

Prevalence of rotavirus and coronavirus antigens in the feces of normal cows

Rotavirus strain diversity in

Genomic diversity of group A rotavirus strains infecting humans in eastern India

Acute gastroenteritis associated with rotavirus in adults

Sequence and phylogenetic analysis of the VP4 gene of human rotaviruses in Paraguay

Rotaviruses

Diarrhea in elderly people: Aetiology, and clinical characteristics

Characterization of rotavirus strains in a Danish population: High frequency of mixed infections and diversity within the VP4 gene of P[8] strains

Enhancement of detection and quantification of rotavirus in stool using a modified real time RT-PCR assay

Identification of group A rotavirus gene 4 types by polymerase chain reaction

Serotype diversity and reassortment between human and animal rotavirus strains: Implications for rotavirus vaccine programs

Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens

Epidemiology of rotaviral infection in adults

Molecular epidemiology of human group A rotavirus infections in the United Kingdom between 1995 and 1998

Diversity within the VP4 gene of rotavirus P[8] strains: Implications for the reverse transcription-PCR genotyping

Molecular characterization of VP6 genes of human rotavirus isolates: Correlation of genogroups with subgroups and evidence of independent segregation

Great diversity of group A rotavirus strains and high prevalence of mixed rotavirus infections in India

Epidemiological profile of rotavirus infection in India: Challenges for the 21st century

Circulation of group A rotavirus subgroups and serotypes in Pune, India, 1990-1997

Rapid ELISA for the diagnosis of rotavirus

Characterization of VP6 genes from rotavirus strains collected in the United States from

Molecular characterization in the VP7, VP4 and NSP4 genes of human rotavirus serotype 4 (G4) isolated in Japan and Kenya

MEGA 3: Integrated software for molecular evolutionary genetics analysis and sequence alignment

Molecular analysis of VP4, VP7 and NSP4 genes of P[6]G2 rotavirus genotype strains recovered from neonates admitted to hospital in Belem, Brazil

Antigenic and genetic characterization of serotype G2 human rotavirus strains from the African continent

Rotavirus and severe childhood diarrhea

Rotavirus diarrhea in children and adults in a southern city of Brazil in 2003: Distribution of G-P types and finding of a rare G12 strain

Unusual diversity of human rotavirus G and P genotypes in India

Burden of disease and molecular epidemiology of group A rotavirus infections in India

Measurement of antirotavirus IgM/IgA/IgG responses in the serum samples of Indian children following rotavirus diarrhea and their mothers

Serotype G9 rotavirus infections in adults in Sweden

Global distribution of rotavirus serotypes/ genotypes and its implication for the development and implementation of an effective rotavirus vaccine

Emergence of [12G] rotavirus strains in Dehli

Characterization of novel VP7, VP4, and VP6 genotypes of a previously untypeable group A rotavirus

The Clustal X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools

Outbreak of severe gastroenteritis in adults and children associated with type G2 rotavirus

Evidence of high-frequency genomic reassortment of group A rotavirus strains in Bangladesh: Emergence of type G9 in 1995

Molecular epidemiologic analysis of group A rotaviruses in adults and children with Wuhan city

Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reaction

Genetic variation in VP7 gene of human rotavirus serotype I (G1 type) isolated in Japan and China

Characterization of VP7 and VP4 genes of rotavirus strains

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention