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Journal of Virology logoLink to Journal of Virology
. 2014 Aug;88(16):9060–9071. doi: 10.1128/JVI.01417-14

Absence of Genetic Differences among G10P[11] Rotaviruses Associated with Asymptomatic and Symptomatic Neonatal Infections in Vellore, India

Margaret H Libonati a,*, Allison F Dennis a, Sasirekha Ramani b,*, Sarah M McDonald a,*, Asmik Akopov c, Ewen F Kirkness c, Gagandeep Kang b, John T Patton a,✉
Editor: T S Dermody
PMCID: PMC4136299  PMID: 24899175

ABSTRACT

Rotaviruses (RVs) are leading causes of severe diarrhea and vomiting in infants and young children. RVs with G10P[11] genotype specificity have been associated with symptomatic and asymptomatic neonatal infections in Vellore, India. To identify possible viral genetic determinants responsible for differences in symptomology, the genome sequences of G10P[11] RVs in stool samples of 19 neonates with symptomatic infections and 20 neonates with asymptomatic infections were determined by Sanger and next-generation sequencing. The data showed that all 39 viruses had identical genotype constellations (G10-P[11]-I2-R2-C2-M2-A1-N1-T1-E2-H3), the same as those of the previously characterized symptomatic N155 Vellore isolate. The data also showed that the RNA and deduced protein sequences of all the Vellore G10P[11] viruses were nearly identical; no nucleotide or amino acid differences were found that correlated with symptomatic versus asymptomatic infection. Next-generation sequencing data revealed that some stool samples, both from neonates with symptomatic infections and from neonates with asymptomatic infections, also contained one or more positive-strand RNA viruses (Aichi virus, astrovirus, or salivirus/klassevirus) suspected of being potential causes of pediatric gastroenteritis. However, none of the positive-strand RNA viruses could be causally associated with the development of symptoms. These results indicate that the diversity of clinical symptoms in Vellore neonates does not result from genetic differences among G10P[11] RVs; instead, other undefined factors appear to influence whether neonates develop gastrointestinal disease symptoms.

IMPORTANCE Rotavirus (RV) strains have been identified that preferentially replicate in neonates, in some cases, without causing gastrointestinal disease. Surveillance studies have established that G10P[11] RVs are a major cause of neonatal infection in Vellore, India, with half of infected neonates exhibiting symptoms. We used Sanger and next-generation sequencing technologies to contrast G10P[11] RVs recovered from symptomatic and asymptomatic neonates. Remarkably, the data showed that the RNA genomes of the viruses were virtually indistinguishable and lacked any differences that could explain the diversity of clinical outcomes among infected Vellore neonates. The sequencing results also indicated that some symptomatic and some asymptomatic Vellore neonates were infected with other enteric viruses (Aichi virus, astrovirus, salvirus/klassevirus); however, none could be correlated with the presence of symptoms in neonates. Together, our findings suggest that other poorly defined factors, not connected to the genetic makeup of the Vellore G10P[11] viruses, influence whether neonates develop gastrointestinal disease symptoms.

INTRODUCTION

Group A rotaviruses (RVs) are a major cause of acute dehydrating diarrhea in infants and children under 5 years of age (1, 2). An assessment of the global impact of RV disease estimated that the virus caused 453,000 deaths in 2008, mostly of children living in sub-Saharan Africa and southeast Asia (3, 4). In India alone, RV infections were estimated to cause 122,000 to 153,000 deaths annually (4). Over the last 40 years, several RV strains have been identified that have a predisposition for replication in neonates, frequently without causing gastrointestinal (GI) disease (5–10). In some cases, the asymptomatic phenotype may reflect the unusual genome constellations that can comprise neonatal virus strains (11–14).

The infectious RV particle is a nonenveloped triple-layered icosahedron that contains 11 segments of double-stranded RNA (dsRNA) (1, 15). The genome directs the expression of 6 structural (VP1 to VP4 and VP6 and VP7) and 6 nonstructural (NSP1 to NSP6) proteins (1). Two of these proteins, the VP7 glycoprotein and the VP4 protease-activated spike protein, form the outer capsid shell of the RV particle and induce neutralizing antibody responses in the infected host (2). A classification system has been developed that allows assignment of a genotype to each of the 11 RV genome segments (16, 17). The genotype of segments encoding the viral proteins VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/NSP6 is represented by the acronym Gx-Px-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx, where x is an integer. Whole-genome sequencing has shown that human RVs with the genotype constellation of G1/G3/G4/G9/G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 or G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2 are globally dominant (18–25). Several neonatal RV strains have been identified with overall genotype constellations that are similar to those of the globally dominant strains, with the exception of their P genotypes. For instance, the Australian RV3 neonatal virus has a G3-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1 constellation while the New Delhi neonatal virus 116E has a G9-P[11]-I1-R1-C1-M1-A1-N1-T1-E1-H1 constellation (11, 14, 26). Because such strains are associated with asymptomatic infections and induce immunological protective responses that can ameliorate severe RV diarrhea episodes later in life (8, 11), the RV3 and 116E strains are being pursued as possible vaccine candidates (27–31).

Surveillance studies have determined that G10P[11] RVs are a major cause of neonatal infection in Vellore, India, where the viruses have circulated for more than a decade (32, 33). The persistence of G10P[11] RVs in Vellore may be due, in part, to their widespread contamination of the environment in neonatal nurseries, thus leading to nosocomial infections (33). Unlike the asymptomatic infections caused by some neonatal RVs (e.g., RV3 and 116E), the Vellore viruses have been associated with GI disease in approximately half of the infected neonates (33). The Vellore G10P[11] viruses may not be entirely exclusive to neonates as G10P[11] strains have also been recovered, albeit sporadically, from older children with diarrheal illness (32). In contrast to RV infections in older infants and children, where clinical manifestations typically include watery diarrhea and vomiting, infections by neonatal strains, such as the Vellore G10P[11] virus, can lead to a wide spectrum of other symptoms: abdominal distention, necrotizing enterocolitis, bloody mucoid stools, and intestinal dilation (32–34). It is also noteworthy that while viral load and disease severity can be correlated in older infants and children infected with RVs, viral loads are not significantly different between symptomatic and asymptomatic RV-infected Vellore neonates (35).

Whole-genome sequencing of a G10P[11] RV isolate (N155) recovered in 2003 from a Vellore neonate with GI symptoms revealed that the virus has a genotype constellation of G10-P[11]-I2-R2-C2-M2-A1-N1-T1-E2-H3 (36). Interestingly, G10P[11] RVs are also known to commonly infect calves in India (37, 38). This fact, combined with sequence analysis showing that multiple genome segments of the N155 isolate are highly related to cognate segments of bovine RVs, suggests that the N155 isolate may have originated by reassortment of a bovine G10P[11] RV and a human RV (36, 38).

The discovery that some, but not all, Vellore neonates infected with G10P[11] RVs had GI disease raised the issue of whether multiple, genetically distinct strains of the virus were cocirculating, some which caused illness and some that did not. To address this issue and identify potential virulence determinants (39–42), we used traditional Sanger dideoxynucleotide and next-generation sequencing (NGS) technologies to analyze RNAs contained in stool samples collected from symptomatic and asymptomatic neonates infected with G10P[11] viruses. The results showed that the G10P[11] viruses were nearly indistinguishable, lacking any differences that could be correlated with symptom status. Although additional enteric positive-strand RNA viruses were detected in some stool samples, none could be causally linked to symptoms. Thus, other poorly defined factors, not connected to the genetic makeup of the G10P[11] viruses or to the presence of other RNA viruses, appear to have a significant role in the development of pathology in infected Vellore neonates.

MATERIALS AND METHODS

Sample collection.

Stool samples were collected from neonates admitted in the neonatal nurseries of Christian Medical College (CMC), Vellore, India, during 2003 to 2004 (33). Neonates remaining in the nursery for over 48 h with symptoms of RV-associated GI disease (diarrhea, vomiting, gastroesophageal reflux, GI bleeding, necrotizing enterocolitis, pneumatosis intestinalis, abdominal distension, and/or feed intolerance) were enrolled in the study (33). For each symptomatic neonate enrolled, at least one other neonate was enrolled that had been admitted for 48 h and lacked GI disease symptoms. RV-positive stool samples were identified by enzyme immunoassay (Rota IDEIA; Dako Ltd., United Kingdom). Samples containing G10P[11] RVs were identified by reverse transcription-PCR (RT-PCR) (36). This study was approved by the Institutional Review Board of the CMC, Vellore, India.

Sequencing.

Total RNA was prepared from neonatal G10P[11]-positive stool samples with TRIzol (Invitrogen). The RNA was randomly amplified and prepared for NGS using a sequence-independent single-primer amplification (SISPA) method as described elsewhere (43). Briefly, 50 to 200 ng of RNA was combined with dimethyl sulfoxide and a chimeric oligonucleotide containing a 22-nucleotide (nt) barcode sequence with a downstream random hexamer, incubated at 65°C for 5 min, and immediately placed on ice. Reverse transcription was performed by adding the RNA sample to reaction mixtures containing SuperScript III and First Strand buffer (both from Life Technologies), 100 mM dithiothreitol, 10 mM deoxynucleoside triphosphates (dNTPs), and RNase Out (Life Technologies) and incubating at 25°C for 10 min, 50°C for 50 min, and 85°C for 10 min. The cDNA products were denatured at 95°C for 10 min, mixed with (3′→5′ exo-) Klenow fragment, and incubated at 37°C for 60 min and then at 75°C for 10 min. Subsequently, the cDNA products were amplified by PCR using Taq Gold (Life Technologies) for 35 cycles (denaturation, 30 s at 94°C; annealing, 30 s at 55°C; extension, 48 s at 68°C). PCR mixtures contained primers corresponding to the 22-nucleotide barcode sequence (43). The cDNA products were treated with exonuclease I at 37°C for 60 min, followed by incubation at 72°C for 15 min. Quantities of SISPA products were normalized and pooled into a single reaction mixture that was purified using a PCR purification kit (Qiagen). The purified cDNA products were then gel purified to select for SISPA products that were 300 to 500 bp in size and submitted for sequencing on Roche 454 GS FLX Titanium and Illumina Genome Analyzer GSIIx platforms. The sequence reads were sorted by barcode, trimmed, and de novo assembled into contigs using CLC bio's de_novo_assembler program (Qiagen). To fill in sequence gaps for G10P[11] genome segments, data from NGS were used to design primers that were then used to process viral RNA using Sanger sequencing. The final full-length contigs for G10P[11] viruses were assembled using a combination of data from Sanger, 454, and Illumina sequencing results.

Sequence data analysis.

The numbers of individual sequence reads obtained by 454 and Illumina technologies are given in Table 1. The overall project is described elsewhere (http://www.ncbi.nlm.nih.gov/bioproject/PRJNA220986), and NGS sequence information is available in the SRA bioinformatics database under GenBank accession numbers SRX504629 (454) and SRX504694 (Illumina). Individuals reads were mapped against databases for group A RV (http://www.ncbi.nlm.nih.gov/nuccore/?term=txid28875[Organism:exp]), Achi virus and kobuvirus (http://www.ncbi.nlm.nih.gov/nuccore/?term=txid72149[Organism:exp]), Astroviridae (http://www.ncbi.nlm.nih.gov/nuccore/?term=txid39733[Organism:exp]), salivirus/klassevirus (http://www.ncbi.nlm.nih.gov/nuccore/?term=txid688449[Organism:exp]), enterovirus (http://www.ncbi.nlm.nih.gov/nuccore/?term?txid138950[Organism%3Aexp] with “poliovirus” entered into the definition line), other viruses (http://www.ncbi.nlm.nih.gov/nuccore/?term=txid10239[Organism%3Aexp] RefSeq), bacteria (ftp://ftp.ncbi.nlm.nih.gov/GenBank/genomes/Bacteria/), and the human genome (http://hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips/chromFa.tar.gz). Only reads of greater than 50 nucleotides (after quality and bar-code checks) with identity matches of greater than 80% over at least 90% of the full read were enumerated. Sequence contigs subjected to analysis were, at minimum, 200 nucleotides in length and included two individual reads. Contigs, and unassembled 454 reads, were mapped against the GenBank nonredundant database (http://www.ncbi.nlm.nih.gov/GenBank) using the NCBI's blastn and blastx tools (http://www.ncbi.nlm.nih.gov/staff/tao/URLAPI/blastall).

TABLE 1.

NGS reads for stool specimens recovered from Vellore neonates infected with G10P[11] RVsa

Neonate GI symptoms No. of 454 reads No. of Illumina reads Total no. of reads No. of reads in read class:
Group A RV Aichi virus Astroviridae Salivirus/klassevirus Poliovirus Other virus Bacteria Human Unmapped
N36 No 805 400,528 401,333 384,376 3,339 1,472 0 0 49 9,960 633 1,504
N39 No 620 65,489 66,109 48,334 0 13 0 0 9 1,479 11,022 5,252
N62 No 1,027 84,470 85,497 82,493 5 0 0 0 16 304 1,912 767
N74 No 9,292 177,240 186,532 64,397 8 0 0 0 14,645 72,108 6,667 28,707
N121 No 33,411 163,585 196,996 85,509 0 0 0 0 4,043 69,911 7,603 29,930
N138 No 5,200 221,804 227,004 103,392 0 0 0 0 663 12,175 108,294 2,480
N190 No 5,517 214,282 219,799 188,717 8 0 0 4 69 22,418 682 7,901
N191 No 33,193 191,125 224,318 36,329 14 0 0 0 268 131,049 3,850 52,808
N192 No 14,558 223,896 238,454 27,757 9 0 0 0 291 178,840 14,764 16,793
N203 No 45,486 346,875 392,361 153,093 19 0 0 0 657 135,050 674 102,868
N210 No 7,640 218,292 225,932 38,011 3 0 4 0 706 181,406 2,295 3,507
N215 No 13,655 139,552 153,207 73,965 0 0 0 0 184 55,635 97 23,326
N223 No 2,488 197,936 200,424 159,418 0 0 0 83 90 35,943 279 4,611
N240 No 223 60,796 61,019 58,563 0 0 0 0 7 1,276 232 941
N273 No 4,360 331,278 335,638 178,459 111 0 0 0 1,104 117,351 2,377 36,236
N284 No 885 175,791 176,676 169,877 0 0 349 0 3 5,689 30 728
N291 No 15,219 251,104 266,323 180,311 0 1 2 0 151 74,180 3,253 8,425
N329 No 9,003 42,066 51,069 8,918 0 0 0 0 18 39,965 667 1,501
N330 No 13,992 83,130 97,122 8,130 6 0 132 0 270 81,420 1,278 5,886
N375 No 3,354 96,962 100,316 9,950 1 0 0 0 135 87,762 735 1,733
N37 Yes 7,807 252,890 260,697 127,988 4 128,208 0 0 7 644 2,505 1,341
N83 Yes 2,260 198,636 200,896 185,465 0 0 0 0 337 7,819 2,611 4,664
N137 Yes 3,205 384,902 388,107 368,168 10,526 0 0 0 117 838 175 8,283
N160 Yes 13,012 185,181 198,193 115,819 1 0 0 0 325 48,237 3,972 29,839
N184 Yes 1,276 398,344 399,620 390,606 244 0 0 0 20 4,545 866 3,339
N187 Yes 22,058 150,714 172,772 58,433 1 0 0 0 828 65,950 17,918 29,642
N188 Yes 1,989 119,592 121,581 114,841 0 0 0 0 33 3,845 268 2,594
N196 Yes 20,364 464,194 484,558 1,837 106 0 0 0 778 287,926 136,744 57,167
N197 Yes 34,114 196,142 230,256 2,281 517 0 0 0 310 217,705 5,014 4,429
N198 Yes 2,042 500,616 502,658 492,493 17 1 0 0 24 2,062 2,826 5,235
N212 Yes 11,498 281,050 292,548 184,488 7 0 335 0 291 67,589 14,280 25,558
N214 Yes 15,461 394,557 410,018 242,927 18 0 0 0 4,443 48,312 18,715 95,603
N228 Yes 561 437,349 437,910 436,917 73 0 0 0 8 577 38 297
N232 Yes 229 272,737 272,966 272,803 1 0 0 0 1 47 6 108
N243 Yes 5,512 679,841 685,353 464,457 3 0 0 0 248 203,517 2,843 14,285
N247 Yes 19,053 109,2319 111,1372 259,285 742,512 1 4 0 369 68,425 12,928 27,848
N259 Yes 8,912 213,353 222,265 14,674 44 0 0 0 264 203,244 130 3,909
N292 Yes 773 346,491 347,264 331,950 0 0 230 0 3,415 3,871 350 7,448
N313 Yes 1,872 548,839 550,711 528,014 1 0 0 0 6,856 770 148 14,922
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a

Reads were analyzed by mapping against reference databases, as indicated in Materials and Methods.

The genotypes of RV RNAs were determined using RotaC, version (http://rotac.regatools.be) (44).

Sequence alignments.

Nucleotide and amino acid sequence alignments and identity values were generated using the ClustalW application of the Geneious Pro (version 6.0) software suite. RV strains with nucleotide and amino acid sequences with close similarity to those of G10P[11] viruses were identified using blastn and blastp (http://blast.ncbi.nlm.nih.gov/). JProfileGrid (version 2) (http://www.profilegrid.org/) was used to produce a matrix relating amino acid position, residue identity, and residue frequency (see Dataset S1 in the supplemental material). Identical nucleotide and amino acid sequences were identified using the ElimDupes duplicate sequence removal tool (http://www.hiv.lanl.gov/content/sequence/ELIMDUPES/elimdupes/html).

Nucleotide sequence accession numbers.

Accession numbers for the Vellore G10P[11] RVs are given in Table 2.

TABLE 2.

Neonate parameters and other viruses detected in specimens recovered from Vellore neonates infected with G10P[11] RV

RV strain Specimen recovery (yr/mo) Neonate characteristica
 Other virus in stool specimenb
 GenBank accession numbers for RV sequences
Sex Birthplace Birth stage GI symptoms Aichi virus Astrovirus Salivirus/klassevirus Poliovirus
RVA/Human-wt/IND/N36/2003/G10P[11] 2003-01 Male CMC Term No X X KC174861–KC174871
RVA/Human-wt/IND/N39/2003/G10P[11] 2003-01 Male CMC Preterm No KC174872–KC174882
RVA/Human-wt/IND/N62/2003/G10P[11] 2003-02 Male CMC Preterm No KC174883–KC174893
RVA/Human-wt/IND/N74/2003/G10P[11] 2003-03 Female CMC Term No KC174894–KC174904
RVA/Human-wt/IND/N121/2003/G10P[11] 2003-10 Male CMC Term No KC174905–KC174915
RVA/Human-wt/IND/N138/2003/G10P[11] 2003-11 Female CMC Term No KC174916–KC174926
RVA/Human-wt/IND/N190/2004/G10P[11] 2004-01 Male CMC Term No KC174927–KC174937
RVA/Human-wt/IND/N191/2004/G10P[11] 2004-01 Male CMC Preterm No KC174938–KC174948
RVA/Human-wt/IND/N192/2004/G10P[11] 2004-01 Female CMC Borderline term No KC174949–KC174959
RVA/Human-wt/IND/N203/2004/G10P[11] 2004-01 Male CMC Term No KC174960–KC174969, KJ638481
RVA/Human-wt/IND/N210/2004/G10P[11] 2004-02 Male Outside CMC Term No KC174970–KC174979, KJ638480
RVA/Human-wt/IND/N215/2004/G10P[11] 2004-02 Male Outside CMC Term No KC174980–KC174990
RVA/Human-wt/IND/N223/2004/G10P[11] 2004-03 Female CMC Term No X KC174991–KC175000, KJ638484
RVA/Human-wt/IND/N240/2004/G10P[11] 2004-03 Female CMC Preterm No KC175001–KC175011
RVA/Human-wt/IND/N273/2004/G10P[11] 2004-05 Female CMC Term No X KC175012–KC175022
RVA/Human-wt/IND/N284/2004/G10P[11] 2004-05 No data No data No data No X KC175023–KC175033, KJ638483
RVA/Human-wt/IND/N291/2004/G10P[11] 2004-05 Female CMC Term No KC175034–KC175044, KJ638482
RVA/Human-wt/IND/N329/2004/G10P[11] 2004-06 Female CMC Term No KC175045–KC175055
RVA/Human-wt/IND/N330/2004/G10P[11] 2004-06 Male Outside CMC Preterm No X KC175056–KC175066
RVA/Human-wt/IND/N375/2004/G10P[11] 2004-07 Female CMC Preterm No KC175067–KC175077
RVA/Human-wt/IND/N37/2003/G10P[11] 2003-01 Female CMC Preterm Yes X KC175078–KC175088
RVA/Human-wt/IND/N83/2003/G10P[11] 2003-03 Male Outside CMC Term Yes KC175089–KC175099, KJ638479
RVA/Human-wt/IND/N137/2003/G10P[11] 2003-11 Male CMC Term Yes X KC175100–KC175109, KJ638478
RVA/Human-wt/IND/N160/2003/G10P[11] 2003-12 Female CMC Preterm Yes KC175110–KC175120
RVA/Human-wt/IND/N184/2004/G10P[11] 2004-01 Male CMC Preterm Yes X KC175121–KC175131
RVA/Human-wt/IND/N187/2004/G10P[11] 2004-01 Male CMC Preterm Yes KC175132–KC175142
RVA/Human-wt/IND/N188/2004/G10P[11] 2004-01 Male CMC Term Yes KC175143–KC175153
RVA/Human-wt/IND/N196/2004/G10P[11] 2004-01 Male CMC Term Yes KC175165–KC175175
RVA/Human-wt/IND/N197/2004/G10P[11] 2004-01 Female CMC Preterm Yes X KC175165–KC175175, KJ638477
RVA/Human-wt/IND/N198/2004/G10P[11] 2004-01 Male CMC Preterm Yes KC175176–KC175186
RVA/Human-wt/IND/N212/2004/G10P[11] 2004-02 Female Outside CMC Preterm Yes X KC175187–KC175197
RVA/Human-wt/IND/N214/2004/G10P[11] 2004-02 Male CMC Preterm Yes KC175198–KC175208
RVA/Human-wt/IND/N228/2004/G10P[11] 2004-03 Male CMC Preterm Yes X KC175209–KC175219
RVA/Human-wt/IND/N232/2004/G10P[11] 2004-03 Female CMC Preterm Yes KC175220–KC175230
RVA/Human-wt/IND/N243/2004/G10P[11] 2004-03 Female CMC Preterm Yes KC175231–KC175241
RVA/Human-wt/IND/N247/2004/G10P[11] 2004-03 Male CMC Term Yes X KC175242–KC175252
RVA/Human-wt/IND/N259/2004/G10P[11] 2004-04 Male CMC Preterm Yes KC175253–KC175263
RVA/Human-wt/IND/N292/2004/G10P[11] 2004-05 Male CMC Preterm Yes X KC175264–KC175274
RVA/Human-wt/IND/N313/2004/G10P[11] 2004-05 Female CMC Term Yes KC175275–KC175285
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a

CMC, Christian Medical College hospital; GI, gastrointestinal.

b

Other viruses were detected by BLAST analysis of NGS contigs against the GenBank database. X, stool specimen containing multiple RNA sequences that assembled into a contig matching the genome of the indicated virus.

RESULTS

Sequencing of RNA in stool samples from neonates infected with G10P[11] RVs.

Stool samples were collected from 19 symptomatic and 20 asymptomatic neonates infected with G10P[11] RVs. The neonates included both males and females, most were born at CMC hospital, and some were preterm (Table 2). None of these factors (sex, birthplace, and gestational age) is linked to symptom status, confirming results obtained with larger data sets previously (33). RNAs isolated from neonate stool samples were converted to cDNAs and analyzed by both 454 and Illumina NGS technologies, producing more than 12 million reads (Table 1). Due to the large number of reads, it was not feasible to search all reads individually against the complete GenBank database. Instead, reads were mapped against reference databases that included group A rotavirus, Aichi virus, astrovirus, salivirus/klassevirus, other viruses, bacteria, and the human genome (see Materials and Methods). As represented in Fig. 1, 58% of the NGS reads mapped to group A RV, 24% to bacterial, 7% to Aichi virus, and 1% to astrovirus sequences. One percent or less of the sequences reads mapped to salivirus/klassevirus, poliovirus, or other viruses. Of the bacterial reads, approximately 5% mapped to 16S ribosomal sequences (data not shown). Reads mapping to bacteriophage and the Herpesviridae were common in the “other” virus class; none of the reads in this class correlated with neonate symptom status (data not shown). The reads obtained for each stool sample were assembled de novo into sequence contigs; these were searched against the complete GenBank database. Missing sequence information in RV contigs was filled in using Sanger sequencing and primers designed from NGS data. This approach allowed identification of the complete, or nearly complete, open reading frame (ORF) sequences for all 11 genome segments of each of the 39 Vellore G10P[11] RVs (accession numbers are given in Table 2).

FIG 1.

FIG 1

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Classes of independent sequence reads generated by NGS analysis of neonatal stool samples. Sequence reads were mapped against the databases indicated in Materials and Methods. The “Other virus” category includes salivirus/klassevirus and poliovirus.

Vellore G10P[11] RVs share a genotype constellation.

Evaluation of genome sequences using the RotaC website (44) indicated that all the Vellore G10P[11] RVs had the same genotype constellation (G10-P[11]-I2-R2-C2-M2-A1-N1-T1-E2-H3), regardless of whether the viruses were isolated from symptomatic or asymptomatic neonates. This genotype constellation was identical to that of the Vellore G10P[11] N155 isolate (36) (Table 3). Another neonatal G10P[11] RV, the I321 strain, was isolated during 1988 to 1991 in Banglalore, India, which is approximately 200 km distant from Vellore (45). Although the genotype constellation of I321 is known only partially, it cannot be same as the Vellore viruses examined in this study, as their NSP2 genotypes differ (N1 and N2, respectively) (Table 3). The complete genomes of other human G10 RVs have been described, but these lack a P[11] VP4 or have a genotype constellation that otherwise differs from that of the Vellore G10P[11] viruses (Table 3): G10P[6]/mani-265/07/IND (46), G10P[8]/163/Vietnam (47), G10P[8]/6717/2002/ARN/NGA (48), G10P[8]/6748/2002/ARN/CIV (48), and G10P[14]/V585/AUS (49). Human RVs have also been described that, although lacking a G10 VP7, have a P[11] VP4 (G6P[11]/SI-B17/SVN) (50) and G9P[11]/116E/IND (14) (Table 3); the genotype constellations of these viruses have additional differences that distinguish them from the Vellore G10P[11] viruses. Despite the numerous reports of human RVs with G10 and/or P[11] genotype specificity, such viruses remain relatively rare compared to viruses with the globally dominant G/P-type combinations: G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8] (18, 22).

TABLE 3.

Genotype constellation of Vellore G10P[11] and related rotavirusesb

graphic file with name zjv9990993440004.jpg

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a

Genotype for all Vellore neonatal G10P[11] viruses examined in this study.

b

n.r., not reported.

Vellore G10P[11] RVs likely derived from reassortment of human and bovine RVs.

Comparison of the sequences of the Vellore G10P[11] viruses examined in this study with those of the Vellore G10P[11] N155 isolate (36) showed that they were nearly identical, both at the nucleotide and amino acid levels (Table 4). Thus, all these viruses appear to be derived from the same lineage. Like the results of an earlier study (36), BLAST analysis indicated that several segments (VP7, VP4, VP1, VP2, VP3, VP6, NSP4, and NSP5) of the Vellore strains were closely related to cognate segments of bovine RVs; the sequences of the remaining segments (NSP1, NSP2, and NSP3) were closely related to those of human genotype 1 viruses (Table 4). Consistent with previous proposals (36, 38), these results suggest that the neonatal G10P[11] RVs may have emerged through reassortment of a bovine G10P[11] RV with a human genogroup 1 RV.

TABLE 4.

Comparison of the genome segments of Vellore G10P[11] RVs with the cognate segments of N155 and other related strainsa

Genome segment Genotype G10P[11] N155 strain
 Other strain
Nucleotide identity (%) Amino acid identity (%) GenBank no. Strain name Nucleotide identity (%) Amino acid identity (%) GenBank accession no.
VP1 R2 99 99 EU200793 RVA/bovine-tc/USA/NCDV/1971/G6P[1] 94 99 JF693026
VP2 C2 99 100 EU200794 RVA/bovine-tc/USA/NCDV/1971/G6P[1] 94 99 JF693027
VP3 M3 98 99 EU200795 RVA/bovine-tc/USA/NCDV/1971/G6P[1] 92 95 JF693028
VP4 P[11] 99 99 EU200796 RVA/bovine-wt/CHN/DQ-75/2008/G10P[11] 92 93 GU181281
VP6 I2 99 100 EU200797 RVA/bovine-wt/KOR/KJ9-1/G6P[7] 95 99 HM988974
VP7 G10 99 100 EU200798 RVA/bovine-wt/ARG/B3326_D_BA/2007/G10P[11] 90 92 KC895795
NSP1 A1 99 99 EU200799 RVA/human-wt/USA/DC4092/1988/G1P[8] 90 88 KC579470
NSP2 N1 98 99 EU200800 RVA/human-wt/USA/VU06-07-21/2006/G3P[8] 96 98 JF490909
NSP3 T1 99 99 EU200801 RVA/human-wt/USA/DC1007/1978/G1P[8] 96 97 KC579594
NSP4 E2 100 99 EU200802 RVA/bovine-wt/KOR/CBNU-1 95 98 AF166353
NSP5 H3 98 100 EU200803 RVA/bovine-tc/USA/NCDV/1971/G6P[1] 93 94 JF693036
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a

G10P[11] isolate N184 was used as the reference strain for the Vellore viruses examined in this study.

G10P[11] RVs associated with asymptomatic and symptomatic infections are genetically indistinguishable.

Comparison of the genome segments and encoded proteins of the 39 Vellore G10P[11] viruses showed that they were identical in sequence, or nearly so (96% to 100% identity values). The sequence identity values for viruses that caused asymptomatic or symptomatic infections were not substantially different from the values obtained for all viruses.

Sequence alignments failed to identify any nucleotide in the genome segments of the Vellore G10P[11] viruses that correlated with the presence or absence of disease symptoms (data not shown). Likewise, the proteins of the Vellore viruses lacked any amino acid differences that could be correlated with GI disease symptoms (see Dataset S1 in the supplemental material). Indeed, the protein sequences of G10P[11] viruses recovered from symptomatic neonates were frequently identical to those of viruses recovered from asymptomatic neonates (Table 5). Taken together, these data indicate that the presence or absence of symptoms among the Vellore neonates does not stem from a virulence determinant that differs among the viruses. Thus, differences in the activities of putative RV proteins affecting pathogenesis, including the NSP1 interferon antagonist (51–53), the NSP4 enterotoxin (40, 54, 55), and VP3—an inhibitor of the host oligoadenylate synthetase-RNase L pathway (39)—are not responsible for differences in neonatal symptoms. Rather, our analysis suggests that only a single, genetically nearly identical lineage of the G10P[11] virus circulated in Vellore during 2003 to 2004 and was associated with both symptomatic and asymptomatic infections.

TABLE 5.

Common protein sequences among G10P[11] strains recovered from asymptomatic and symptomatic neonatesa

graphic file with name zjv9990993440005.jpg

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a

Common protein sequences were identified using ElimDupes (http://www.hiv.lanl.gov/). Boxes of the same color represent indistinguishable sequences. A white box represents a unique sequence, with at least one amino acid different from any other.

Detection of other enteric viruses in the stool samples of RV-infected neonates.

To explore the possibility that disease symptoms among RV-infected Vellore neonates were due to coinfection by other viruses, contigs generated from NGS reads were mapped by BLAST against all sequences in the GenBank database. The results showed that of the 39 stool samples analyzed, 12 contained RNAs mapping to enteric positive-strand RNA viruses, suggesting that some neonates were coinfected with RV and at least one other virus (Table 2). The mapping results indicated that the coinfecting virus was most commonly Aichi virus, a member of the Picornaviridae (56), and, less commonly, salivirus/klassevirus, also a member of the Picornaviridae (57), or astrovirus, a member of the Astroviridae (58, 59). The location of the contigs relative to the genomes of reference strains is indicated in Fig. 2. The presence of Aichi virus, salivirus/klassevirus, or astrovirus in stool samples may be of importance in defining factors affecting symptoms among the neonates, as surveillance studies have indicated that these viruses can lead to pediatric gastroenteritis (60–64). However, based on evaluation of the data presented in Table 2 using Fisher's exact test (e.g., P = 0.2351 for Aichi virus), no statistically significant link existed between any of the coinfecting viruses and symptom status. Thus, other factors are likely responsible for the development of clinical disease among the Vellore neonates. Notably, stool samples that contained RV and Aichi virus RNAs were recoverable over several months in 2003 to 2004 (Fig. 3), suggesting that these viruses cocirculate in Vellore and that coinfections may be relatively frequent.

FIG 2.

FIG 2

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Sequences of other RNA viruses detected in stool samples from Vellore neonates infected with G10P[11] RVs. Sequence contigs generated from NGS reads were used in BLAST searches of the GenBank database. Contigs with matches to Aichi virus (A), astrovirus (B), and salivirus/klassevirus (C) are shown mapped (gray lines) relative to the full-length genome of these viruses. The ORFs of the genome sequences are shown in yellow. The neonate number and category (in parentheses) are indicated to the left as follows: A, asymptomatic, S, symptomatic. The accession numbers of the viruses used to prepare the illustration are GQ927704 (Aichi virus), JQ898343 (salivirus), and FJ375759 (astrovirus). CDS, coding DNA sequence.

FIG 3.

FIG 3

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Temporal origin of G10P[11] RVs recovered from Vellore neonates. Blocks represent stool samples containing G10P[11] RV that were sequenced in this study and are colored differently to indicate samples collected from neonates with asymptomatic and symptomatic infections. Samples containing other viral RNAs are coded as indicated.

NGS reads for neonate N223 produced a 236-nt contig that, with the exception of a single residue, perfectly matched that of a sequence shared among the Sabin 1, 2, and 3 strains of oral poliovirus vaccine (e.g., residues 6023 to 6258 of Sabin 2; accession number FJ898290) (Fig. 1). Vellore newborns are given oral poliovirus vaccine at day 0; this may represent the source of poliovirus RNA in this neonate's stool sample.

DISCUSSION

By a combination of next-generation and Sanger sequencing technologies, we have determined that the sequences of G10P[11] RVs infecting symptomatic and asymptomatic Vellore neonates are virtually identical. This result indicates that factors outside the genetic material of the virus determine whether neonates infected with G10P[11] RVs develop symptoms. The nature of these factors is unclear, but their elucidation is important for developing effective antiviral therapies, particularly in neonates which are too young to be afforded protection through vaccination.

Although nearly half of neonatal infections by the Vellore G10P[11] viruses result in GI disease, infections by the Bangalore G10P[11] I321 virus are asymptomatic (45). Because the Vellore and Bangalore viruses have the same G/P-type specificity but differ in their potential to cause GI disease, genome segments of these viruses—other than those encoding VP4 and VP7—may contain differences that affect pathogenesis. Indeed, the I321 genome contains a genotype N1 NSP2 gene while Vellore strains contain a genotype N2 NSP2 gene, hinting at one possible determinant affecting symptom status (Table 3). Previous studies of neonates infected with the Bangalore and Vellore G10P[11] strains produced contrasting results when the subjects were evaluated for protection against subsequent RV infection and disease (65, 66). Specifically, neonates infected with I321-like strains in Bangalore showed evidence of protection, while neonates infected with N155-like strains in Vellore did not (65, 66). Understanding the molecular basis for the differences in the Bangalore and Vellore G10P[11] viruses in inducing neonatal protective responses awaits further sequencing of I321-like strains and should provide information that is important for engineering asymptomatic vaccine candidates.

NGS analysis indicated that some symptomatic neonates infected with G10P[11] RVs were coinfected with Aichi virus, astrovirus, and/or salivirus/klassevirus. These viruses commonly infect young children, often without signs of clinical disease (67–71). Coinfections involving RV and other enteric viral or bacterial pathogens have been frequently reported, and there have been a few studies suggesting that coinfections with certain pathogens can alter GI disease severity (63, 72–76). However, little information is available concerning whether coinfection of young children, and particularly neonates, with RV and Aichi virus, astrovirus, or salivirus/klassevirus has an impact on disease symptoms. Nonetheless, our data failed to establish a causal relationship between neonatal symptom status and the presence of a coinfecting positive-strand RNA virus.

Our NGS data did not reveal the presence of norovirus, a leading cause of pediatric gastroenteritis (69, 70, 77, 78), in any of the neonatal stool samples analyzed in this study. This finding was unexpected given an earlier surveillance study indicating that a third of all norovirus-infected Vellore neonates with GI disease were coinfected with RV (79). Similarly, our NGS data indicated that the Vellore neonates were not infected with orthoreovirus, reovirus, or saffold virus, all RNA viruses that frequently infect children (67, 70, 76, 80–83). At its simplest, the absence of NGS reads for these RNA viruses suggests a possible lack of neonatal exposure or susceptibility to these viruses or a lack of circulation of such viruses in the Vellore area or CMC nurseries. Alternatively, instability or low copy numbers of viral RNAs in stool samples may have also precluded detection by NGS. It is also possible that the cutoff values used in mapping NGS reads against databases were too stringent to identify viruses whose sequences had highly diverged from reference sequences. Because our protocols were directed at detecting RNAs in stool samples, we could have failed to detect enteric DNA viruses or pathogenic bacteria that affect the symptom status of Vellore neonates.

Whether Vellore neonates develop symptoms is not linked to variations in the genetic material of G10P[11] viruses or, apparently, to how well the virus grows (35). Other factors that may impact disease progression include the level and specificity of maternal anti-RV antibodies acquired by the neonate (84, 85, 86, 87). Alternatively, variability in the physiological maturity and microbiome composition of the neonatal gut may lead to differences in host recognition and response to viral infection (88, 89). It is possible that some aspects of GI disease in RV-infected Vellore neonates may reflect immunopathology triggered by misdirected and/or exaggerated inflammatory responses (90, 91). Such responses have been tied to the development of asthma in respiratory virus infections (92, 93), hemorrhagic fevers in infections with hantavirus (94), yellow fever virus (95), or dengue virus (96), and lung pathology in influenza patients (97, 98). The possibility that some GI disease symptoms in Vellore neonates stem from inappropriate inflammatory responses will require an analysis of the types and levels of inflammatory cytokines and chemokines triggered by RV infections.

Supplementary Material

Supplemental material
supp_88_16_9060__index.html (1.2KB, html)

ACKNOWLEDGMENT

M.H.L., A.F.D., S.M.M., and J.T.P. were supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH). This project was also supported by funds provided by NIAID, NIH, contract number HHSN272200900007C.

We thank Kristen Ogden and Natalie Leach for their comments and suggestions.

Footnotes

Published ahead of print 4 June 2014

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JVI.01417-14.

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