Whole-genome characterization of common rotavirus strains circulating in Vellore, India from 2002 to 2017: emergence of non-classical genomic constellations

Rotaviruses (RVs) are the most common etiological agent of acute gastroenteritis among young children, even after vaccine introduction in low-income countries. A whole-genome classification representing the 11 RV genes, was introduced for surveillance and characterization of RVs. This study characterized the common circulating strains in Vellore, India from 2002 to 2017 to understand rotavirus strain diversity and evolution using Whole genome sequencing (WGS) carried out on Illumina MiSeq. The 89% (92% of Wa-like, 86% of DS-1-like) of strains had classical constellations, while reassortant constellations were seen in 11% (8% of Wa-like, 14% of DS-1-like) of the strains. The rare E6-NSP4 in combination with DS-1 like G1P[8] and the emergence of the OP-354 subtype of P[8] were identified. Phylogenetics of RV strains revealed multiple subtypes circulating in the past 15 years, with strong evidence of animal to human gene transmission among several strains. Supplementary Information The online version contains supplementary material available at 10.1186/s13099-023-00569-6.


Introduction
Rotavirus A (RVA) is the most common cause of acute gastroenteritis in children less than five years of age and is responsible for approximately 128,500 annual deaths worldwide, mostly in low-middle income countries.India accounted for 21,357 deaths from the rotaviral disease in 2015, accounting for 20% of all global rotaviral deaths in children under five years of age [1,2].Rotavirus still remains the leading cause of diarrheal hospitalizations in low and middle income countries despite the large-scale use of rotavirus vaccines globally [3].
The whole genome characterisation of all 11 genes of RVA has provided valuable insights into RVA diversity that results from the accumulation of point mutations, genetic reassortment, and intragenic recombination [9][10][11][12][13].In order to trace the evolutionary pattern of various strains, full genome characterization is required for interpretation of the origin of each segment of the RV genome [6].Global studies on RVA strain surveillance and characterization have revealed high diversity of human RVA strains, including the possibility of gene reassortment between Wa-like and DS-1-like strains [14][15][16][17][18][19].
Surveillance from a single location over time can be valuable in understanding viral diversity and evolution.G1P [8] was the most predominant strain in India prior to vaccination (2012-2016), followed by G2P [4], G9 and G12 [20].Vellore reported 32% of diarrheal hospitalizations due to RVA from 2005 to 2016.G2P [4] was the predominant strain in the initial years and was gradually replaced by G1P [8].The emergence of G9P [4] replacing G9P [8] and emergence of G12 strains was also documented [21].Molecular characterization of Indian strains has been performed mainly for the VP7 and VP4 genes, and data on the remaining nine genes are limited.In this study we report the whole genome analysis of common RVA strains circulating in Vellore, India, for the years 2002-2017.

Diversity of Wa-like/DS-1-like strains
All the gene segments showed considerable diversity among Wa-like strains.Among the structural genes of Wa-like strains, the G12 and G1-VP7 gene segments showed the least diversity and among the non-structural genes, H1-NSP5 gene showed the least nucleotide diversity.Highest diversity was observed in P [8]-VP4 gene and A1-NSP1 gene (Table S5).On comparing the different genotypes of the same gene of Wa-like strains, it was observed that among the VP4 gene, the P[8]-genotype shows higher diversity than P [6]-genotype and among the VP7 gene, the G1-genotype shows higher diversity than the G12-genotype.
Among the structural genes of DS-1-like strains, VP4-P [4] showed the least diversity and VP3 gene showed the highest diversity.The NSP5 gene was the most conserved gene amongst the non-structural proteins and E2-NSP4 gene was the most diverse gene (Table S6).Overall, the G2P [4] showed higher diversity than G9P [4] strains across all the gene segments except for the VP7 gene which was more diverse among G9 than G2 strains.

Divergence of Wa-like/DS-1-like strains
Comparison of the study sequences with other RV strains sequences from GenBank clearly shows the divergence of gene sequences and continuous diversification over years.All the gene segments of the Wa-like strains from Vellore shared the least similarity at both NT and AA level with the human classical RV strains like Wa, KU, P, DC-2241, WI61 and ST3 which were circulating and characterized in 1970s (Table S7-S8).On the other hand, the study sequences shared more similarity with the classical RV strains like Dhaka-16, AM06-1 (Indian classical strain), Dhaka-12, Matlab-13, GER-172, Dhaka-25, B4633 and GER-172, which were isolated and characterized in the early 2000s.The highest similarities of the study strains were found with the strains from India reported previously (year 2000-2016).The study sequences shared very less similarity with the vaccine strain sequences like Rotarix, RotaTeq and 116E.Similarly, the DS-1-like study strains showed the least similarity with the DS-1 strain which is the primitive RV strain from 1970s and the strain 116E3D, which is a classical strain from India isolated in 1993 (Table S9).The study sequences shared a little higher similarity to the recently reported DS-1-like strains from Benin.The highest similarity was observed with the DS-1-like strains from India reported in past few years.Therefore, the majority of our study strains are human RV strains, which have diverged from the primitive RV strains circulating 50 years back around 1970s and have diversified its genome accumulating mutations over the years.

Analysis of VP7 gene
GTR + F + I + I + R4 substitution model was used to infer the VP7 gene tree (Fig. 1(A)).The G1 genotype sequences clustered into four different human clusters (HC-1 to HC-4) which represent the sub genotypes of the G1 genotype circulating in the study population (Fig. 2).Of these four different clusters, the human cluster 1(HC-1 N = 76) represents the major subtype which circulated in the past 15 years.This cluster share a close genetic similarity with  The G2 genotype sequences formed only one human cluster representing single sub genotype circulating in the study population over the past 15 years.This is also supported by the high NT sequence similarity (96.75-100%) observed between G2 strains in the distance analysis.
Two G4 strains share a close genetic similarity with the porcine RVA from India indicating animal to human transmission of the gene segment.These strains also clusters away from classical G4 strains (ST3 and DC2241).
G12 strains forms two distinct human clusters.The major cluster (HC-1) includes twenty-one G12 study strains which persisted over past 10 years.The minor cluster (HC-2) includes eight G12 strains circulating between 2005 and 2012 representing the sub-genotype that circulated in the early 2000s and diminished later.

Analysis of VP4 gene
Maximum Likelihood (ML) tree was built for VP4 gene sequences using the GTR + F + I + I + R4 substitution model (Fig. 1(B)).P [8] study sequences clustered into four human clusters (HC-1 to HC-4, Refer Fig. 2).The major cluster (HC-2) represents the sub-genotype that circulated from 2002 to 2013 and shared a close similarity with early 2000s RV classical strains.Reassortant G2P [8] ) also clustered together in this branch putting forward evidence that the P [8] VP4 gene was probably introduced from the human Wa-like virus into the DS-1 backbone.The minor cluster HC-1 represent the current circulating sub-genotype of P [8] sequences that persistently circulated from 2005 to 2017.The other two clusters of P [8] were observed occasionally in which the HC-4 cluster includes six study strains identified between 2003 and 2017 clustering closely with OP-354 like P [8] sequences which has been reported for rapid spread across the globe [22].
All the P [4] sequences clustered into one cluster (HC-1) and show a separate branch away from the early classical RV strains like DS-1 and 116E3D.A single strain (RVA/Human-wt/IND/RO1-2748/2010/G12P [4]) with a Wa-like backbone lies as a singlet branch.
Two P [14] sequences clustered with other two Indian human strains reported from 2014 (G10P [14]) and share close branches with animal sequences.This indicates P [14] had originally evolved from animals and is now reported from humans across globe in combination with different G types.

Analysis of VP6, VP1-VP3 genes
A maximum likelihood tree for VP6, VP1, VP2 and VP3 gene (Fig. 1(C-F)) were built using GTR + F + I + I + R4/R5 substitution model.These viral protein genes sequences showed multiple sub genotype clusters for genogroup1 (I1, R1, C1 and M1) and genogroup 2 (I2, R2, C2 and M2) as shown in Fig. 2. Three major human clusters were observed in VP6-I1 of which HC-2 shared close similarity with 2000s classical RV strains and persisted in our setting from 2002 to 2013.HC-1 and HC-4 represent the recent circulating strains which has diverged considerably from the 1970 and 2000 s classical RV strains.The VP1-R1 sequences showed one major cluster (HC-3) that persisted throughout the study period and shared close similarity with 2000s classical RV strains.The other two sub-genotype clusters were found occasionally.The VP2-C1 sequences showed one major (HC-1), two minor (HC-2 & HC-3) and one occasionally occurring subgenotypic clusters.The major cluster persisted throughout the study period sharing close similarity with classical 2000s RVs strains.The minor clusters declined towards 2013 to 2015 and it is important to note that the minor cluster (HC-3) includes gene sequences specifically from only G1P [8] specificity.VP3-M1 sequences clustered into one major cluster which persisted throughout and one minor cluster which persisted till 2013.
Looking at the structural genes of DS-1 like viruses, the VP6-I2 sequences showed one major sub-genotypic cluster and two occasionally occurring clusters.One minor cluster includes two study strains (one G10P [11] and a reassortant G12P [6]) which clustered with neonatal I2 human sequences reported previously from India [23].This emphasizes that neonatal I2-VP6 sequences vary significantly from other human I2-VP6 sequences.The VP1-R2 gene sequences resolve into two major sub-genotypic cluster (HC-1 & HC-2), which share close similarity with previously reported Indian sequences and persists till date.One rarely occurring sub-genotype cluster was

Analysis of non-structural proteins (NSPs) genes
Diversity of NSP gene sequences varied with the genogroup (Refer Fig. 1(G-K) and Fig. 3).Among the Wa-like viruses (Genogroup I), NSP1-A1 gene appeared conserved while the other NSPs were diverse showing multiple sub-genotypic clusters of which the NSP4-E1 gene showed the highest diversity.On the other hand, among the DS-1 like viruses (Genogroup II), NSP3 and NSP5 genes were conserved, while the NSP1, NSP2 and NSP4 gene had multiple sub genotype clusters.NSP1-A2 gene showed the highest diversity among the Wa -like viruses.

Inter-species transmission
Animal derived RV backbone and genes were seen in a few of our study strains details of which is shown in Table 2. Six of the study strains bearing atypical G and P type showed multiple genes that clustered closely to the animal RV sequences.Of these, one strain (G4P [6] genotype) was a porcine like strain circulating in human hosts with most of the genes of porcine origin.Another six strains bearing common genotypes showed single-reassortant gene of animal origin in their genetic backbone.Genome constellation of these reassortants is mentioned in Table 2. Pigs and cattle RV were the most common source of animal reassortant strains in our setting.The genotypes G6, P [14], A11, T6 and H3 were associated specifically with animal-human reassortant RVs.Among the common genotypes (G1P [8] and G2P [4]) only single gene reassortment was reported.Though the genotype constellation of these common strains appeared like a stable backbone, analysis of single gene sequences by the in depth molecular phylogenetics revealed the presence of rare animal-human reassortant genes.One G1P [8] strain(RVA/Human-wt/IND/CM-0059/2012/G1P [8]) carried VP6 gene of a bovine origin.One G2P [4] strain (RVA/Human-wt/IND/C-65/2006/G2P [4]) carried single NSP4 gene of bovine source and four G2P [4] strains carried NSP2 gene which had originated from cattle strains.Presence of such population of reassortant strains reflects the potency of these strains to expand its host and cause symptomatic disease warranting regular monitoring of circulating RVA strains.

Vaccine reassortants
In-depth molecular phylogenetics of genes revealed that few RV vaccine reassortant strains circulated in our setting (Table 2).Two RotaTeq (RV5) vaccine reassortant strains were detected with single reassortant gene.Reassortment was seen in VP3 gene of a G4P [8] strain and VP6 gene of a G1P [8] strain.A G12P [6] strain RVA/ Human-wt/IND/RVGE-505/2012/G12P [6] was detected with three reassortant genes-VP6, NSP3 and NSP5 from 116E natural reassortant neonatal vaccine strain.The rest of its gene shared high similarity with neonatal strains from India.Detection of vaccine reassortants in our setting is an indication of multiple complex reassortment events happening between vaccine strains and human strains during mixed infections and the ability of these new reassortants to persist and circulate through natural selection.
Few vaccine reassortants were also detected in our setting.A RotaTeq (RV5) vaccine VP6 reassortant strain-RVA/Human-wt/IND/IRID-2631/2009/G1P [8] was identified with constellation G1(WT)P [8](WT)-I2(RV5)-R1(WT)-C1(WT)-Mx-A1(WT)-NI(WT)-T1(WT)-E1(WT)-H1(WT).VP6 gene from RV5 strain could have reassorted into Wa-like strain.Another RotaTeq derived human strain (RVA/Human-wt/ IND/C-1351/2010/G4P [8]) was identified with the constellation G4(WT)P [8] (WT)-I1(WT)-R1(WT)-C1( W T )-M1(RV5)-A1( W T )-N1( W T )-T1( W T )-E1(WT)-H1(WT), where M1-VP3 gene appear to be of vaccine origin which reassorted with Wa-like strain.RotaTeq was not included in the national immunization program at this time but was licensed for private use.It is quite possible that shed vaccine strains could circulate and reassort.It is important to note that such strain appear like a classical Wa-like RV from its constellations, but the in-depth whole genome analysis reveals the true origin of the strain.Hence whole genome analysis becomes an important tool to fully characterize and understand the true origin of the circulating strains.This observation is consistent with the findings of another research group from Brazil, Nicaragua and United States who have reported that such reassortment events are expected considering attenuation of RotaTeq vaccine and the segmented nature of RVA genome [50][51][52][53].Vaccine reassortant strain with partial Rotarix and partial RotaTeq genes in backbone was previously reported from USA [39].Triple reassortant strain RVA/Humanwt/IND/RVGE-505/2012/G12P [6] was identified with constellation G12P [6]-I1-R1-C1-M1-A1-N1-T1-E1-H1 where the VP6, NSP3 and NSP5 gene clustered closely to the 116E strain and rest of the genes clustered with other Indian neonatal strains.116E is a natural reassortant neonatal RV strain that was identified in 1985 in New Delhi, India, and was later developed into Rotavac vaccine strain [54].It was licensed in 2014 and included in National Immunization programme in 2016.Our strain was isolated in 2012 prior to licensure and introduction of 116E vaccine.Therefore, this strain does not appear to be of vaccine origin instead it could have originated from 116E like wildtype strains which could have been circulating at that time.
Gene specific analyses of the sequenced strains clearly reflects the heterogeneity and diversity among each genotype as indicated by multiple clusters of sequences for each gene.Among the Wa like strains the VP7, VP4, VP1, VP3, NSP1 and NSP5/6 genes appear less diverse than VP6, VP2, NSP2, NSP3 and NSP4 genes.While among the DS-1 like strains the VP4, VP7, VP1, VP3, NSP3 and NSP5 genes appear less diverse than VP6, VP2, NSP1, NSP2 and NSP4 genes.DS-1 like strain's G2-VP7, P[4]-VP4, T2-NSP3 and H2-NSP5 genotypes showed only one sub-genotype circulating in the past 15 years suggesting these genotypes to be highly conserved.While genes of other genotypes showed at least two different circulating sub-genotypes.Amongst the Wa-like strain genotypes, only A1-NSP1 gene appeared to be highly conserved with only one human sub-genotype circulating in the past 15 years.Previous comparative studies have reported circulation of multiple sub-genotypes referred as alleles for each RVA gene which also differ at different geographical settings [38][39][40][41][42].Each RV gene and genotype has varying mutation/substitution rates, as reported by some studies [12,[61][62][63][64][65].This could be one the reasons for varying diversity observed for different RV gene or genotype.It is important to note that six strains with OP-354 like P[8]-VP7 sequences were circulating in our setting.OP-354 like P [8], the new divergent form of P [8] strains, originated in south east Asia and has rapidly spread across the continent [22].OP-354 like P [8] are known to exist with various G types.In our study, OP-354 like P [8] were seen in combination with G1 and G9 strains.There have been few reports which suggest that OP-354 P [8] are associated with severe forms of diarrhea [66,67] and in future it would be interesting to analyse such association in our setting.Equine like rotavirus strains are now being reported from various studies where equine like DS-1 backbone has been observed in combination with G3P [8], G3P [6] and G3P [4] [68][69][70].In our study, five equine like R2-VP1 sequences and 22 equine-like N2-NSP2 sequences were detected in G2P [4] strains.This result is quite unusual and new G2P [4] strains with equine like genes have not been reported earlier.There could be a possibility that these VP1 and NSP2 genes could have reassorted from equine like human strains however, more analysis would be required to support the data.
Our study has a few limitations since we were not able to sequence all the strains isolated during 2002-2017 and only few strains from each year were randomly selected for characterization.Other important prevalent strains like G3P [8], G1P [6] and G9P [8]/P [4] were not included whose genetic surveillance is equally important.Very few unusual strains (where evidence of reassortments and zoonotic transmission are more likely) were sequenced and there are chances that characterizing more of such strains will add to the knowledge on rotavirus diversity in Indian settings.For some of the study strains, few genes failed to be sequenced (specially for the unusual strains) and better sequencing protocols are required to meet such challenges.

Conclusion
To summarize, this study found that majority of Wa and DS-1 like strains in our setting carry a stable genetic constellation but the individual genes keep evolving into new sub-genotypes.All the genes are prone to occasional reassortment events and emergence of mixed genetic constellation, though the chances are less than 10%.Some genes have proved to be very conserved in the past years, while few other genes have diverged significantly to create new sub-genotypes.Our study provides the baseline whole-genome sequence data on the common circulating strains in India from the pre-vaccine period which will be crucial to study the evolution of RVA strains since large scale use of rotavirus vaccines especially in the context that India uses indigenously developed vaccines (Rotavac and Rotasiil) which is different from the rest of the world [71].Whole genome characterization also helped detect the vaccine reassortant strains.Detection of novel genotypes like E6-NSP4, T6-NSP3 and H3-NSP5, unusual strains like OP-354, DS-1 like G1P [8], equine like strains is a constant reminder to continue genome level surveillance of RV strains for tracking virus evolution in India especially in the post vaccine era.

Ethics statement and sample collection
This study was approved by the Institutional Review Board of Christian Medical College (CMC), Vellore.In this study samples were selected from the biorepository of stool samples already collected and stored for different community-and hospital-based rotavirus studies at the Wellcome Trust Research Laboratory, at CMC, Vellore.The rotavirus positive stool samples were obtained from our laboratory biorepository.These diarrheal samples were collected from children under 5 years of age as a part of rotavirus surveillance studies conducted both in the hospital or community settings.The stool samples were first screened for rotavirus particles by ELISA and the rotavirus positive samples were genotyped by RT-PCR or sanger sequencing.

Selection of samples for whole genome sequencing
The VP7 and VP4 genotype and Sanger sequence data from the laboratory database were used to select a subset of stool samples for next generation sequencing (NGS).At least 3 strains each of G1P [8], G2P [4] and G12 genotypes were randomly selected from each year from 2002 to 2017.A small number of strains of G4, G6, G8 and G9 and uncharacterised genotypes were also included.A total of 127 Wa-like (88 G1P [8] X], 1G9P[X] and 1 GXP [14]) were sequenced by NGS.

Viral dsRNA extraction for NGS
Viral nucleic acid was extracted from 20%(v/v) fecal extract using QIAmp 96 Virus QIAcube HT kit on the QIAcube HT automated platform (Qiagen, Germany) according to manufacturer's instructions.

Genotype confirmation
Complementary DNA (cDNA) synthesized by reverse transcription using Moloney murine reverse transcriptase enzyme (superscript II MMLV-RT, Invitrogen, United States) and random primers (Invitrogen, United States) were used as templates for VP7 and VP4 typing by a hemi-nested multiplex PCR using published primers [72,73].The untyped samples and unusual strains were sequenced by Sanger sequencing.

Amplification of 11 segments
The protocol followed in this study for amplification of RVA 11 genes segment was standardized and developed through a collaboration with the J. Craig Venter Institute, La Jolla, CA, USA.Briefly, a one-step reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify 11 genes from extracted RNA in four multiplex reactions using Superscript IV One-step RT-PCR kit (Invitrogen, United States) and gene specific primers (see Table S1).The Superscript IV PCR reaction uses Superscript IV reverse transcriptase enzyme for cDNA synthesis and a super fidelity Taq polymerase enzyme from amplification of DNA in consecutive steps.The gene segments amplified were confirmed by running the amplified product on 1% agarose gel.The PCR products were purified using AMPure XP magnetic beads (Beckman Coulter, United States) according to manufacturer's specifications.

Next generation sequencing
Sequencing libraries were prepared using Illumina Nextera DNA flex library preparation kit following manufacturer's guidelines (Illumina, USA).The quality of libraries was assessed using Tapestation D1000 screen tape auto electrophoresis system (Agilent, USA) and Qubit fluorometric assay (Invitrogen, USA).Libraries were diluted to 12 picomoles and spiked with 5% PhiX sequencing control (Illumina, USA).NGS was carried out on an Illumina MiSeq sequencing platform (Illumina, USA) using MiSeq Reagent Kit v3 for 600 cycles with 300 bp paired end reads method.

Accession numbers
The sequences for all the study strains were deposited to GenBank under accession numbers listed in the Supplementary File 10.

Genotype assignment
Genotypes to each gene sequence was assigned using ViPR (Virus Pathogen Resource) Rotavirus A genotype determination tool (This web tool has now been upgraded as RIVM Rotavirus A genotyping tool version 1.0, manuscript is yet in preparation) [76].After the genotype assignment, the strains were classified into genogroups Wa-like (genogroup 1) or DS-1-like (genogroup 2) for further analysis.

Bioinformatics pipeline
Sequence alignment was carried out in Aliview software using MUSCLE algorithm [77].Nucleotide and deduced amino acid sequence identities among strains were calculated for each gene using p-distance algorithm in MEGA 7 software [78].The Model Finder program was used to identify the optimal substitution model that best fit sequence datasets using Bayesian Information Criterion (BIC) [79].Maximum likelihood trees were constructed using IQTree-2 software with 1000 bootstrap replicates [80,81].Maximum likelihood trees for each gene were built using the best substitution model according to the Bayesian criterion.The trees were visualized using iTOL online server and the sub-genotype clusters were defined based on branching patterns and bootstrap value [82].Where sub-genotype cluster is defined as a distinct phylogenetic branch observed within a genotype containing the sequences sharing high similarity (> 95%) and supported by a bootstrap value of > 70%.Branches with bootstrap higher than 70 were considered for inference.The clusters with large number of leaves were collapsed for easy visualization.The human sub genotypic clusters of each genotype were read as HC-1 to HC-N, in the order of its appearance in the tree from top to bottom.The sequences that do not group with any reference other study strain sequences exist as a singlet leaf.For comparison, various classical strains, vaccine strains, reassortant wild-type human strains and wild-type animal strains were included (Refer Table S4).

Fig. 1
Fig. 1 Maximum likelihood trees with bootstrap values for RVA VP7(A), VP4(B), VP6(C), VP1(D), VP2(E), VP3(F), NSP1(G), NSP2(H), NSP3(I), NSP4(J) and, NSP5/6(K) genes.The study sequences are coloured red, the reference classical human, equine-like, DS-1-like G1P[8] and vaccine sequences from Gen-Bank are coloured blue, the animal sequences from GenBank are in green and the recent human sequences from India are in black.Phylogenetic clusters were collapsed for easy visualization and to make comprehensive trees.Clusters in shades of red represent RV gene sequences isolated from human hosts bearing Wa-like (genogroup 1) virus genotype, while clusters in shades of green represent RV gene sequences isolated from animal source bearing Walike virus genotype.Clusters in shades of blue represent RV gene sequences isolated from human hosts bearing DS-1-like (genogroup 2) virus genotype, while clusters in shades of purple represent RV gene sequences isolated from animal source bearing DS-1-like virus genotype.Vaccine and tissue culture reassortant clusters are highlighted in orange.Trees were drawn to scale as indicated by the scale bar which represents substitution per nucleotide site Only one major sub-genotypic cluster and one neonatal cluster was observed in NSP1-A1 gene sequences, where the major cluster persisted throughout the study period.NSP2-N1 gene sequences were diverse and segregated into two major (HC-1 & HC-4), one minor (HC-3), two occasional (HC-2 & HC-6), and one neonatal (HC-5) sub-genotypic clusters.HC-4 and HC-3 represent the sub-genotype which circulated until 2013 while, HC-1 represent the current circulating sub-genotype.NSP3-T1 gene sequences resolved into one major (HC-2), three occasional and one neonatal (HC-5) sub-genotype clusters.NSP4-E1 gene sequences showed the maximum sub-genotypic clusters with one major (HC-5), two minor (HC-3 & HC-6) and four occasional clusters.None of the sub-genotype clusters seem to persist throughout the study period.The NSP5-H1 gene sequences show one major, one minor and one occasional sub-genotypic clusters.The major cluster persisted throughout the past 15 years while the minor cluster persisted from 2005 to 2014.

Fig. 2
Fig. 2 Different sub-genotype clusters observed within each RV genotype's viral protein genes (HC-Human cluster, N-Number of strains in the cluster)

Fig. 3
Fig. 3 Different sub-genotype clusters observed within each RV genotype's non-structural protein genes (HC-Human cluster, N-Number of strains in the cluster)

Table 1
Summary of the RVA strains sequenced from Vellore from year 2002 to 2017.Reassortant genes are marked in bold