Publications by authors named "Sunando Roy"

36 Publications

Using Whole Genome Sequences to Investigate Adenovirus Outbreaks in a Hematopoietic Stem Cell Transplant Unit.

Front Microbiol 2021 2;12:667790. Epub 2021 Jul 2.

Division of Infection and Immunity, University College London, London, United Kingdom.

A recent surge in human mastadenovirus (HAdV) cases, including five deaths, amongst a haematopoietic stem cell transplant population led us to use whole genome sequencing (WGS) to investigate. We compared sequences from 37 patients collected over a 20-month period with sequences from GenBank and our own database of HAdVs. Maximum likelihood trees and pairwise differences were used to evaluate genotypic relationships, paired with the epidemiological data from routine infection prevention and control (IPC) records and hospital activity data. During this time period, two formal outbreaks had been declared by IPC, while WGS detected nine monophyletic clusters, seven were corroborated by epidemiological evidence and by comparison of single-nucleotide polymorphisms. One of the formal outbreaks was confirmed, and the other was not. Of the five HAdV-associated deaths, three were unlinked and the remaining two considered the source of transmission. Mixed infection was frequent (10%), providing a sentinel source of recombination and superinfection. Immunosuppressed patients harboring a high rate of HAdV positivity require comprehensive surveillance. As a consequence of these findings, HAdV WGS is being incorporated routinely into clinical practice to influence IPC policy contemporaneously.
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http://dx.doi.org/10.3389/fmicb.2021.667790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8284422PMC
July 2021

Mixed cytomegalovirus genotypes in HIV-positive mothers show compartmentalization and distinct patterns of transmission to infants.

Elife 2020 12 31;9. Epub 2020 Dec 31.

Department of Infection, Immunity and Inflammation, UCL Great Ormond Street Institute of Child Health, University College London, London, United Kingdom.

Cytomegalovirus (CMV) is the commonest cause of congenital infection and particularly so among infants born to HIV-infected women. Studies of congenital CMV infection (cCMVi) pathogenesis are complicated by the presence of multiple infecting maternal CMV strains, especially in HIV-positive women, and the large, recombinant CMV genome. Using newly developed tools to reconstruct CMV haplotypes, we demonstrate anatomic CMV compartmentalization in five HIV-infected mothers and identify the possibility of congenitally transmitted genotypes in three of their infants. A single CMV strain was transmitted in each congenitally infected case, and all were closely related to those that predominate in the cognate maternal cervix. Compared to non-transmitted strains, these congenitally transmitted CMV strains showed statistically significant similarities in 19 genes associated with tissue tropism and immunomodulation. In all infants, incident superinfections with distinct strains from breast milk were captured during follow-up. The results represent potentially important new insights into the virologic determinants of early CMV infection.
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http://dx.doi.org/10.7554/eLife.63199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806273PMC
December 2020

Detection of Norovirus Variant GII.4 Hong Kong in Asia and Europe, 2017-2019.

Emerg Infect Dis 2021 01;27(1):289-293

We report a new norovirus GII.4 variant, GII.4 Hong Kong, with low-level circulation in 4 Eurasia countries since mid-2017. Amino acid substitutions in key residues on the virus capsid associated with the emergence of pandemic noroviruses suggest that GII.4 Hong Kong has the potential to become the next pandemic variant.
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http://dx.doi.org/10.3201/eid2701.203351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7774557PMC
January 2021

High prevalence of integrase mutation L74I in West African HIV-1 subtypes prior to integrase inhibitor treatment.

J Antimicrob Chemother 2020 06;75(6):1575-1579

Department of Medicine, University of Cambridge, Cambridge, UK.

Objectives: HIV-1 integrase inhibitors are recommended as first-line therapy by WHO, though efficacy and resistance data for non-B subtypes are limited. Two recent trials have identified the integrase L74I mutation to be associated with integrase inhibitor treatment failure in HIV-1 non-B subtypes. We sought to define the prevalence of integrase resistance mutations, including L74I, in West Africa.

Methods: We studied a Nigerian cohort of recipients prior to and during receipt of second-line PI-based therapy, who were integrase inhibitor-naive. Illumina next-generation sequencing with target enrichment was used on stored plasma samples. Drug resistance was interpreted using the Stanford Resistance Database and the IAS-USA 2019 mutation lists.

Results: Of 115 individuals, 59.1% harboured CRF02_AG HIV-1 and 40.9% harboured subtype G HIV-1. Four participants had major IAS-USA integrase resistance-associated mutations detected at low levels (2%-5% frequency). Two had Q148K minority variants and two had R263K (one of whom also had L74I). L74I was detected in plasma samples at over 2% frequency in 40% (46/115). Twelve (26.1%) had low-level minority variants of between 2% and 20% of the viral population sampled. The remaining 34 (73.9%) had L74I present at >20% frequency. L74I was more common among those with subtype G infection (55.3%, 26/47) than those with CRF02_AG infection (29.4%, 20/68) (P = 0.005).

Conclusions: HIV-1 subtypes circulating in West Africa appear to have very low prevalence of major integrase mutations, but significant prevalence of L74I. A combination of in vitro and clinical studies is warranted to understand the potential implications.
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http://dx.doi.org/10.1093/jac/dkaa033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225870PMC
June 2020

A comprehensive characterization of chronic norovirus infection in immunodeficient hosts.

J Allergy Clin Immunol 2019 11 13;144(5):1450-1453. Epub 2019 Aug 13.

Institute of Immunity and Transplantation, University College London, Royal Free Campus, London, United Kingdom; Department of Clinical Immunology, Royal Free London NHS Foundation Trust, London, United Kingdom. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2019.07.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6843911PMC
November 2019

Whole-genome Sequencing Provides Data for Stratifying Infection Prevention and Control Management of Nosocomial Influenza A.

Clin Infect Dis 2019 10;69(10):1649-1656

Division of Infection and Immunity, University College London, United Kingdom.

Background: Influenza A virus causes annual epidemics in humans and is associated with significant morbidity and mortality. Haemagglutinin (HA) and neuraminidase (NA) gene sequencing have traditionally been used to identify the virus genotype, although their utility in detecting outbreak clusters is still unclear. The objective of this study was to determine the utility, if any, of whole-genome sequencing over HA/NA sequencing for infection prevention and control (IPC) in hospitals.

Methods: We obtained all clinical samples from influenza (H1N1)-positive patients at the Great Ormond Street Hospital between January and March 2016. Samples were sequenced using targeted enrichment on an Illumina MiSeq sequencer. Maximum likelihood trees were computed for both whole genomes and concatenated HA/NA sequences. Epidemiological data was taken from routine IPC team activity during the period.

Results: Complete genomes were obtained for 65/80 samples from 38 patients. Conventional IPC analysis recognized 1 outbreak, involving 3 children, and identified another potential cluster in the haemato-oncology ward. Whole-genome and HA/NA phylogeny both accurately identified the previously known outbreak cluster. However, HA/NA sequencing additionally identified unrelated strains as part of this outbreak cluster. A whole-genome analysis identified a further cluster of 2 infections that had been previously missed and refuted suspicions of transmission in the haemato-oncology wards.

Conclusions: Whole-genome sequencing is better at identifying outbreak clusters in a hospital setting than HA/NA sequencing. Whole-genome sequencing could provide a faster and more reliable method for outbreak monitoring and supplement routine IPC team work to allow the prevention of transmission.
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http://dx.doi.org/10.1093/cid/ciz020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821348PMC
October 2019

Human cytomegalovirus haplotype reconstruction reveals high diversity due to superinfection and evidence of within-host recombination.

Proc Natl Acad Sci U S A 2019 03 28;116(12):5693-5698. Epub 2019 Feb 28.

Division of Infection and Immunity, University College London, London WC1E 6BT, United Kingdom;

Recent sequencing efforts have led to estimates of human cytomegalovirus (HCMV) genome-wide intrahost diversity that rival those of persistent RNA viruses [Renzette N, Bhattacharjee B, Jensen JD, Gibson L, Kowalik TF (2011) 7:e1001344]. Here, we deep sequence HCMV genomes recovered from single and longitudinally collected blood samples from immunocompromised children to show that the observations of high within-host HCMV nucleotide diversity are explained by the frequent occurrence of mixed infections caused by genetically distant strains. To confirm this finding, we reconstructed within-host viral haplotypes from short-read sequence data. We verify that within-host HCMV nucleotide diversity in unmixed infections is no greater than that of other DNA viruses analyzed by the same sequencing and bioinformatic methods and considerably less than that of human immunodeficiency and hepatitis C viruses. By resolving individual viral haplotypes within patients, we reconstruct the timing, likely origins, and natural history of superinfecting strains. We uncover evidence for within-host recombination between genetically distinct HCMV strains, observing the loss of the parental virus containing the nonrecombinant fragment. The data suggest selection for strains containing the recombinant fragment, generating testable hypotheses about HCMV evolution and pathogenesis. These results highlight that high HCMV diversity present in some samples is caused by coinfection with multiple distinct strains and provide reassurance that within the host diversity for single-strain HCMV infections is no greater than for other herpesviruses.
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http://dx.doi.org/10.1073/pnas.1818130116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431178PMC
March 2019

Use of Whole-Genome Sequencing of Adenovirus in Immunocompromised Pediatric Patients to Identify Nosocomial Transmission and Mixed-Genotype Infection.

J Infect Dis 2018 09;218(8):1261-1271

Infection, Immunity and Inflammation Section, UCL Great Ormond Street Institute of Child Health, University College London, United Kingdom.

Background: Adenoviruses are significant pathogens for the immunocompromised, arising from primary infection or reinfection. Serotyping is insufficient to support nosocomial transmission investigations. We investigate whether whole-genome sequencing (WGS) provides clinically relevant information on transmission among patients in a pediatric tertiary hospital.

Methods: We developed a target-enriched adenovirus WGS technique for clinical samples and retrospectively sequenced 107 adenovirus-positive residual diagnostic samples, including viremias (>5 × 104 copies/mL), from 37 patients collected January 2011-March 2016. Whole-genome sequencing was used to determine genotype and for phylogenetic analysis.

Results: Adenovirus sequences were recovered from 105 of 107 samples. Full genome sequences were recovered from all 20 nonspecies C samples and from 36 of 85 species C viruses, with partial genome sequences recovered from the rest. Whole-genome phylogenetic analysis suggested linkage of 3 genotype A31 cases and uncovered an unsuspected epidemiological link to an A31 infection first detected on the same ward 4 years earlier. In 9 samples from 1 patient who died, we identified a mixed genotype adenovirus infection.

Conclusions: Adenovirus WGS from clinical samples is possible and useful for genotyping and molecular epidemiology. Whole-genome sequencing identified likely nosocomial transmission with greater resolution than conventional genotyping and distinguished between adenovirus disease due to single or multiple genotypes.
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http://dx.doi.org/10.1093/infdis/jiy323DOI Listing
September 2018

Norovirus Transmission Dynamics in a Pediatric Hospital Using Full Genome Sequences.

Clin Infect Dis 2019 01;68(2):222-228

Microbiology, Virology and Infection Prevention and Control, Great Ormond Street Hospital National Health Service Foundation Trust.

Background: Norovirus is a leading cause of worldwide and nosocomial gastroenteritis. The study aim was to assess the utility of molecular epidemiology using full genome sequences compared to routine infection prevention and control (IPC) investigations.

Methods: Norovirus genomes were generated from new episodes of norovirus at a pediatric tertiary referral hospital over a 19-month period (n = 182). Phylogeny identified clusters of related sequences that were verified using epidemiological and clinical data.

Results: Twenty-four clusters of related norovirus sequences ("sequence clusters") were observed, including 8 previously identified by IPC investigations ("IPC outbreaks"). Seventeen sequence clusters (involving 77/182 patients) were corroborated by epidemiological data ("epidemiologically supported clusters"), suggesting transmission between patients. Linked infections were identified among 44 patients who were missed by IPC investigations. Thirty-three percent of norovirus sequences were linked, suggesting nosocomial transmission; 24% of patients had nosocomial infections from an unknown source; and 43% were norovirus positive on admission.

Conclusions: We show there are frequent introductions of multiple norovirus strains with extensive onward nosocomial transmission of norovirus in a pediatric hospital with a high proportion of immunosuppressed patients nursed in isolation. Phylogenetic analysis using full genome sequences is more sensitive than classic IPC investigations for identifying linked cases and should be considered when investigating norovirus nosocomial transmission. Sampling of staff, visitors, and the environment may be required for complete understanding of infection sources and transmission routes in patients with nosocomial infections not linked to other patients and among patients with phylogenetically linked cases but no evidence of direct contact.
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http://dx.doi.org/10.1093/cid/ciy438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321856PMC
January 2019

On the effective depth of viral sequence data.

Virus Evol 2017 Jul 14;3(2):vex030. Epub 2017 Nov 14.

Division of Infection and Immunity, University College London, London, UK.

Genome sequence data are of great value in describing evolutionary processes in viral populations. However, in such studies, the extent to which data accurately describes the viral population is a matter of importance. Multiple factors may influence the accuracy of a dataset, including the quantity and nature of the sample collected, and the subsequent steps in viral processing. To investigate this phenomenon, we sequenced replica datasets spanning a range of viruses, and in which the point at which samples were split was different in each case, from a dataset in which independent samples were collected from a single patient to another in which all processing steps up to sequencing were applied to a single sample before splitting the sample and sequencing each replicate. We conclude that neither a high read depth nor a high template number in a sample guarantee the precision of a dataset. Measures of consistency calculated from within a single biological sample may also be insufficient; distortion of the composition of a population by the experimental procedure or genuine within-host diversity between samples may each affect the results. Where it is possible, data from replicate samples should be collected to validate the consistency of short-read sequence data.
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http://dx.doi.org/10.1093/ve/vex030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5724399PMC
July 2017

Characterisation of a rare, reassortant human G10P[14] rotavirus strain detected in Honduras.

Mem Inst Oswaldo Cruz 2018 Jan;113(1):9-16

Centers for Disease Control and Prevention, Gastroenteritis and Respiratory Viruses Laboratory Branch, Atlanta, Georgia, USA.

Background: Although first detected in animals, the rare rotavirus strain G10P[14] has been sporadically detected in humans in Slovenia, Thailand, United Kingdom and Australia among other countries. Earlier studies suggest that the strains found in humans resulted from interspecies transmission and reassortment between human and bovine rotavirus strains.

Objectives: In this study, a G10P[14] rotavirus genotype detected in a human stool sample in Honduras during the 2010-2011 rotavirus season, from an unvaccinated 30-month old boy who reported at the hospital with severe diarrhea and vomiting, was characterised to determine the possible evolutionary origin of the rare strain.

Methods: For the sample detected as G10P[14], 10% suspension was prepared and used for RNA extraction and sequence independent amplification. The amplicons were sequenced by next-generation sequencing using the Illumina MiSeq 150 paired end method. The sequence reads were analysed using CLC Genomics Workbench 6.0 and phylogenetic trees were constructed using PhyML version 3.0.

Findings: The next generation sequencing and phylogenetic analyses of the 11-segmented genome of the G10P[14] strain allowed classification as G10-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3. Six of the genes (VP1, VP2, VP3, VP6, NSP2 and NSP4) were DS-1-like. NSP1 and NSP5 were AU-1-like and NSP3 was T6, which suggests that multiple reassortment events occurred in the evolution of the strain. The phylogenetic analyses and genetic distance calculations showed that the VP7, VP4, VP6, VP1, VP3, NSP1, NSP3 and NSP4 genes clustered predominantly with bovine strains. NSP2 and VP2 genes were most closely related to simian and human strains, respectively, and NSP5 was most closely related to a rhesus strain.

Main Conclusions: The genetic characterisation of the G10P[14] strain from Honduras suggests that its genome resulted from multiple reassortment events which were possibly mediated through interspecies transmissions.
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http://dx.doi.org/10.1590/0074-02760170083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719537PMC
January 2018

Molecular characterization of a human G20P[28] rotavirus a strain with multiple genes related to bat rotaviruses.

Infect Genet Evol 2018 01 26;57:166-170. Epub 2017 Nov 26.

Centers for Disease Control and Prevention, 1600 Clifton Rd. NE, Atlanta, GA 30333, USA.

Group A rotaviruses are the major cause of severe gastroenteritis in the young of mammals and birds. This report describes characterization of an unusual G20P[28] rotavirus strain detected in a 24month old child from Suriname. Genomic sequence analyses revealed that the genotype constellation of the Suriname strain RVA/Human-wt/SUR/2014735512/2013/G20P[28] was G20-P[28]-I13-R13-C13-M12-A23-N13-T15-E20-H15. Genes VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4 and NSP5 were recently assigned novel genotypes by the Rotavirus Classification Working Group (RCWG). Three of the 11 gene segments (VP7, VP4, VP6) were similar to cognate gene sequences of bat-like human rotavirus strain Ecu534 from Ecuador and the VP7, NSP3 and NSP5 gene segments of strain RVA/Human-wt/SUR/2014735512/2013/G20P[28] were found to be closely related to gene sequences of bat rotavirus strain 3081/BRA detected in Brazil. Although distantly related, the VP1 gene of the study strain and bat strain BatLi09 detected in Cameroon in 2014 are monophyletic. The NSP1 gene was found to be most closely related to human strain QUI-35-F5 from Brazil. These findings suggest that strain RVA/Human-wt/SUR/2014735512/2013/G20P[28] represents a zoonotic infection from a bat host.
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http://dx.doi.org/10.1016/j.meegid.2017.11.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818710PMC
January 2018

Super-infections and relapses occur in chronic norovirus infections.

J Clin Virol 2017 11 20;96:44-48. Epub 2017 Sep 20.

Microbiology, Virology and Infection Prevention and Control, Great Ormond Street Hospital for Children NHS Foundation Trust, UK; Infection and Immunity, University College London, UK.

Background: Norovirus causes chronic infections in immunocompromised patients with considerable associated morbidity. It is not known whether chronic infections involve super- or re-infections or relapses.

Objectives: To retrospectively investigate whether longitudinal sampling in chronically infected patients demonstrates persistent infection with the same virus, or super- or re-infection.

Study Design: Norovirus full genomes were generated from 86 longitudinal samples from 25 paediatric patients. Consensus sequences were used for phylogenetic analysis and genotyping.

Results: Super-infections occurred in 17% of chronically infected patients who were continuously PCR positive; including two with mixed norovirus infections. The median duration of infection was 107days longer in those with super-infections; however this was not statistically significant. A third of patients with interrupted norovirus shedding continued to be infected with the same virus despite up to 2 months of PCR negative stools, classified as a relapse. The majority (67%) of patients with interrupted shedding were re-infected with a different genotype.

Conclusions: Chronically infected patients who are continuously PCR positive are most likely to remain infected with the same virus; however super-infections do occur leading to mixed infection. Patients with interrupted shedding are likely to represent re-infection with a different genotype, however relapsing infections also occur. Our findings have implications for infection control as immunosuppressed patients remain susceptible to new norovirus infections despite current or recent infection and may continue to be infectious after norovirus is undetectable in stool. The relevance to children without co-morbidities remains to be determined.
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http://dx.doi.org/10.1016/j.jcv.2017.09.009DOI Listing
November 2017

The emerging GII.P16-GII.4 Sydney 2012 norovirus lineage is circulating worldwide, arose by late-2014 and contains polymerase changes that may increase virus transmission.

PLoS One 2017 29;12(6):e0179572. Epub 2017 Jun 29.

Division of Infection and Immunity, University College London, London, United Kingdom.

Noroviruses are a leading cause of human gastroenteritis worldwide. The norovirus genotype GII.4 is the most prevalent genotype in the human population and has caused six pandemics since 1995. A novel norovirus lineage containing the GII.P16 polymerase and pandemic GII.4 Sydney 2012 capsid was recently detected in Asia and Germany. We demonstrate that this lineage is also circulating within the UK and USA and has been circulating since October 2014 or earlier. While the lineage does not contain unique substitutions in the capsid, it does contain polymerase substitutions close to positions known to influence polymerase function and virus transmission. These polymerase substitutions are shared with a GII.P16-GII.2 virus that dominated outbreaks in Germany in Winter 2016. We suggest that the substitutions in the polymerase may have resulted in a more transmissible virus and the combination of this polymerase and the pandemic GII.4 capsid may result in a highly transmissible virus. Further surveillance efforts will be required to determine whether the GII.P16-GII.4 Sydney 2012 lineage increases in frequency over the coming months.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179572PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5491022PMC
September 2017

Characterization of a triple-recombinant, reassortant rotavirus strain from the Dominican Republic.

J Gen Virol 2017 02 24;98(2):134-142. Epub 2017 Feb 24.

Yale School of Medicine, New Haven, CT, USA.

We report the genome of a novel human triple-recombinant G4P[6-8_R] mono-reassortant strain identified in a stool sample from the Dominican Republic during routine facility-based rotavirus strain surveillance. The strain was designated as RVA/Human-wt/DOM/2013840364/2013/G4P[6-8_R], with a genomic constellation of G4-P[6-8_R]-I1-R1-C1-M1-(A1-A8_R)-N1-(T1-T7_R)-E1-H1. Recombinant gene segments NSP1 and NSP3 were generated as a result of recombination between genogroup 1 rotavirus A1 human strain and a genotype A8 porcine strain and between genogroup 1 rotavirus T1 human strain and a genotype T7 bovine strain, respectively. Analyses of the RNA secondary structures of gene segment VP4, NSP1 and NSP3 showed that all the recombinant regions appear to start in a loop (single-stranded) region and terminate in a stem (double-stranded) structure. Also, the VP7 gene occupied lineage VII within the G4 genotypes consisting of mostly porcine or porcine-like G4 strains, suggesting the occurrence of reassortment. The remaining gene segments clustered phylogenetically with genogroup 1 strains. This exchange of whole or partial genetic materials between rotaviruses by recombination and reassortment contributes directly to their diversification, adaptation and evolution.
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http://dx.doi.org/10.1099/jgv.0.000688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5720362PMC
February 2017

Norovirus Whole-Genome Sequencing by SureSelect Target Enrichment: a Robust and Sensitive Method.

J Clin Microbiol 2016 10 3;54(10):2530-7. Epub 2016 Aug 3.

Microbiology, Virology, and Infection Control, Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom Division of Infection and Immunity, University College London, London, United Kingdom.

Norovirus full-genome sequencing is challenging due to sequence heterogeneity among genomes. Previous methods have relied on PCR amplification, which is problematic due to primer design, and transcriptome sequencing (RNA-Seq), which nonspecifically sequences all RNA, including host and bacterial RNA, in stool specimens. Target enrichment uses a panel of custom-designed 120-mer RNA baits that are complementary to all publicly available norovirus sequences, with multiple baits targeting each position of the genome, which overcomes the challenge of primer design. Norovirus genomes are enriched from stool RNA extracts to minimize the sequencing of nontarget RNA. SureSelect target enrichment and Illumina sequencing were used to sequence full genomes from 507 norovirus-positive stool samples with reverse transcription-real-time PCR cycle threshold (CT) values of 10 to 43. Sequencing on an Illumina MiSeq system in batches of 48 generated, on average, 81% on-target reads per sample and 100% genome coverage with >12,000-fold read depth. Samples included genotypes GI.1, GI.2, GI.3, GI.6, GI.7, GII.1, GII.2, GII.3, GII.4, GII.5, GII.6, GII.7, GII.13, GII.14, and GII.17. When outliers were accounted for, we generated >80% genome coverage for all positive samples, regardless of CT values. A total of 164 samples were tested in parallel with conventional PCR genotyping of the capsid shell domain; 164/164 samples were successfully sequenced, compared to 158/164 samples that were amplified by PCR. Four of the samples that failed capsid PCR analysis had low titers, which suggests that target enrichment is more sensitive than gel-based PCR. Two samples failed PCR due to primer mismatches; target enrichment uses multiple baits targeting each position, thus accommodating sequence heterogeneity among norovirus genomes.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035417PMC
http://dx.doi.org/10.1128/JCM.01052-16DOI Listing
October 2016

Comparison of Next-Generation Sequencing Technologies for Comprehensive Assessment of Full-Length Hepatitis C Viral Genomes.

J Clin Microbiol 2016 10 6;54(10):2470-84. Epub 2016 Jul 6.

Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom Roslin Institute, University of Edinburgh, Edinburgh, United Kingdom.

Affordable next-generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting antiviral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV sequence data sets and evaluated their utility for diagnostics and clinical assessment. NGS methods using (i) unselected HCV RNA (metagenomics), (ii) preenrichment of HCV RNA by probe capture, and (iii) HCV preamplification by PCR implemented in four United Kingdom centers were compared. Metrics of sequence coverage and depth, quasispecies diversity, and detection of DAA resistance-associated variants (RAVs), mixed HCV genotypes, and other coinfections were compared using a panel of samples with different viral loads, genotypes, and mixed HCV genotypes/subtypes [geno(sub)types]. Each NGS method generated near-complete genome sequences from more than 90% of samples. Enrichment methods and PCR preamplification generated greater sequence depth and were more effective for samples with low viral loads. All NGS methodologies accurately identified mixed HCV genotype infections. Consensus sequences generated by different NGS methods were generally concordant, and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. Metagenomic methods identified human pegivirus coinfections. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis, and quasispecies diversity. Enrichment methods are particularly suited for high-throughput analysis while providing the genotype and information on potential DAA resistance.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035407PMC
http://dx.doi.org/10.1128/JCM.00330-16DOI Listing
October 2016

Molecular characterization of the first G24P[14] rotavirus strain detected in humans.

Infect Genet Evol 2016 09 26;43:338-42. Epub 2016 May 26.

Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA. Electronic address:

Here we report the genome of a novel rotavirus A (RVA) strain detected in a stool sample collected during routine surveillance by the Centers for Disease Control and Prevention's New Vaccine Surveillance Network. The strain, RVA/human-wt/USA/2012741499/2012/G24P[14], has a genomic constellation of G24-P[14]-I2-R2-C2-M2-A3-N2-T9-E2-H3. The VP2, VP3, VP7 and NSP3 genes cluster phylogenetically with bovine strains. The other genes occupy mixed clades containing animal and human strains. Strain RVA/human-wt/USA/2012741499/2012/G24P[14] most likely is the product of interspecies transmission and reassortment events. This is the second report of the G24 genotype and the first report of the G24P[14] genotype combination in humans.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5072358PMC
http://dx.doi.org/10.1016/j.meegid.2016.05.033DOI Listing
September 2016

Full genome characterization of the first G3P[24] rotavirus strain detected in humans provides evidence of interspecies reassortment and mutational saturation in the VP7 gene.

J Gen Virol 2016 Feb 20;97(2):389-402. Epub 2015 Nov 20.

Gastroenteritis and Respiratory Viruses Laboratory Branch, Division of Viral Diseases (DVD), National Center for Immunization and Respiratory Diseases (NCIRD), Centers for Disease Control and Prevention, 1600 Clifton Road NE, Atlanta, GA 30329-4027, USA.

During the 2008-2009 rotavirus season of the Centers for Disease Control and Prevention New Vaccine Surveillance Network, one case of paediatric acute gastroenteritis associated with a rotavirus G14P[24] strain was identified. This was the first detection of the genotype G14 and P[24] in humans, and the first detection of the G14P[24] combination. To gain an insight into the origins and the evolution of this strain, we determined the complete ORF sequences of all 11 genes. A majority of the genes identified were similar to the simian strain TUCH, except for the VP1 and VP7 genes that clustered only distantly with the bovine and equine strains, respectively. In addition, this strain carried AU-1-like NSP2 and NSP4 genes. Using codon-partitioning and protein-based phylogenetic approaches, we determined that the VP7 genotype of strain 2009727118 was actually G3; therefore, the proposed full genomic classification of the 2009727118 strain is G3-P[24]-I9-R2-C3-M3-A9-N3-T3-E3-H6. These findings indicate the possibility that the 2009727118 strain originated by interspecies transmission and multiple reassortment events involving human, bovine and equine rotaviruses, resulting in the introduction of some genes into the genome of simian rotaviruses. Additionally, we found evidence of mutational saturation in the third codon position of the VP7 ORF which presented an issue with homoplasy in phylogenetic analyses.
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http://dx.doi.org/10.1099/jgv.0.000349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4827097PMC
February 2016

Full-Genome Sequence of the First G8P[14] Rotavirus Strain Detected in the United States.

Genome Announc 2015 Jun 18;3(3). Epub 2015 Jun 18.

Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA

This is a report of the complete genomic sequence of a rare rotavirus group A G8-P[14]-I2-R3-C2-M2-A3-N2-T6-E2-H3 strain detected in a stool sample from a 57-year-old subject.
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http://dx.doi.org/10.1128/genomeA.00677-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472909PMC
June 2015

Full genomic characterization and phylogenetic analysis of a zoonotic human G8P[14] rotavirus strain detected in a sample from Guatemala.

Infect Genet Evol 2015 Jul 5;33:206-11. Epub 2015 May 5.

Division of Viral Diseases, Gastroenteritis and Respiratory Viruses Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA, USA.

We report the genomic characterization of a rare human G8P[14] rotavirus strain, identified in a stool sample from Guatemala (GTM) during routine rotavirus surveillance. This strain was designated as RVA/Human-wt/GTM/2009726790/2009/G8P[14], with a genomic constellation of G8-P[14]-I2-R2-C2-M2-A13-N2-T6-E2-H3. The VP4 gene occupied lineage VII within the P[14] genotype. Phylogenetic analysis of each genome segment revealed close relatedness to several zoonotic simian, guanaco and bovine strains. Our findings suggest that strain RVA/Human-wt/GTM/2009726790/2009/G8P[14] is an example of a direct zoonotic transmission event. The results of this study reinforce the potential role of interspecies transmission and reassortment in generating novel and rare rotavirus strains which infect humans.
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http://dx.doi.org/10.1016/j.meegid.2015.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5803796PMC
July 2015

Outbreak of Gastroenteritis in Adults Due to Rotavirus Genotype G12P[8].

Clin Infect Dis 2015 Aug 13;61(4):e20-5. Epub 2015 Apr 13.

Chicago Department of Public Heath, Illinois.

Background: Rotavirus infection in adults is poorly understood and few rotavirus outbreaks among US adults have been reported in the literature. We describe an outbreak due to genotype G12P[8] rotavirus among medical students, faculty, and guests who attended a formal dinner event in April 2013.

Methods: A web-based questionnaire was distributed to event attendees to collect symptom and exposure data. A clinical case was defined as a person who developed diarrhea after attending the formal event. A laboratory-confirmed case was defined as a clinical case who attended the formal event, with rotavirus detected in stool by enzyme immunoassay or reverse transcription-polymerase chain reaction (RT-PCR) assay.

Results: Among 334 dinner attendees, 136 (41%) completed the web-based questionnaire; 58 (43%) respondents reported illness. Symptom onset ranged from 1 to 8 days, with peak onset 3 days after the event. In addition to diarrhea, predominant symptoms included fever (91%), abdominal pain (84%), and vomiting (49%). The median duration of illness was 2.5 days. Thirteen (22%) of 58 cases sought medical attention; none were hospitalized. Analysis of food exposures among questionnaire respondents did not identify significant associations between any specific food or drink item and illness. Stool specimens were negative for bacterial pathogens by culture and negative for norovirus by RT-PCR assay; 4 specimens were positive for rotavirus by enzyme immunoassay or PCR. G12P[8]-R1-C1-M1-A1-N1-T1-E1-H1 was identified as the causative full-genome genotype.

Conclusions: Rotavirus outbreaks can occur among adults, including young adults. Health professionals should consider rotavirus as a cause of acute gastroenteritis in adults.
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http://dx.doi.org/10.1093/cid/civ294DOI Listing
August 2015

Full Genome Sequence of a Reassortant Human G9P[4] Rotavirus Strain.

Genome Announc 2014 Dec 11;2(6). Epub 2014 Dec 11.

Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA

This is a report of the complete genomic sequence of a reassortant rotavirus group A G9-P[4]-I2-R2-C2-M2-A2-N2-T2-E6-H2 strain designated RVA/Human-wt/USA/ LB1562/2010/G9P[4].
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http://dx.doi.org/10.1128/genomeA.01284-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263837PMC
December 2014

Comparative genomic analysis of genogroup 1 (Wa-like) rotaviruses circulating in the USA, 2006-2009.

Infect Genet Evol 2014 Dec 6;28:513-23. Epub 2014 Oct 6.

Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA. Electronic address:

Group A rotaviruses (RVA) are double stranded RNA viruses that are a significant cause of acute pediatric gastroenteritis. Beginning in 2006 and 2008, respectively, two vaccines, Rotarix™ and RotaTeq®, have been approved for use in the USA for prevention of RVA disease. The effects of possible vaccine pressure on currently circulating strains in the USA and their genome constellations are still under investigation. In this study we report 33 complete RVA genomes (ORF regions) collected in multiple cities across USA during 2006-2009, including 8 collected from children with verified receipt of 3 doses of rotavirus vaccine. The strains included 16 G1P[8], 10 G3P[8], and 7 G9P[8]. All 33 strains had a Wa like backbone with the consensus genotype constellation of G(1/3/9)-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. From maximum likelihood based phylogenetic analyses, we identified 3-7 allelic constellations grouped mostly by respective G types, suggesting a possible allelic segregation based on the VP7 gene of RVA, primarily for the G3 and G9 strains. The vaccine failure strains showed similar grouping for all genes in G9 strains and most genes of G3 strains suggesting that these constellations were necessary to evade vaccine-derived immune protection. Substitutions in the antigenic region of VP7 and VP4 genes were also observed for the vaccine failure strains which could possibly explain how these strains escape vaccine induced immune response. This study helps elucidate how RVA strains are currently evolving in the population post vaccine introduction and supports the need for continued RVA surveillance.
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http://dx.doi.org/10.1016/j.meegid.2014.09.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4620586PMC
December 2014

Full genomic characterization of a novel genotype combination, G4P[14], of a human rotavirus strain from Barbados.

Infect Genet Evol 2014 Dec 22;28:524-9. Epub 2014 Sep 22.

Gastroenteritis and Respiratory Viruses Laboratory Branch, Division of Viral Diseases, NCIRD, Centers for Disease Control and Prevention, Atlanta, GA, USA. Electronic address:

Since 2004, the Pan American Health Organization (PAHO) has carried out rotavirus surveillance in Latin America and the Caribbean. Here we report the characterization of human rotavirus with the novel G-P combination of G4P[14], detected through PAHO surveillance in Barbados. Full genome sequencing of strain RVA/Human-wt/BRB/CDC1133/2012/G4P[14] revealed that its genotype is G4-P[14]-I1-R1-C1-M1-A8-N1-T1-E1-H1. The possession of a Genogroup 1 (Wa-like) backbone distinguishes this strain from other P[14] rotavirus strains. Phylogenetic analyses suggested that this strain was likely generated by genetic reassortment between human, porcine and possibly other animal rotavirus strains and identified 7 lineages within the P[14] genotype. The results of this study reinforce the potential role of interspecies transmission in generating human rotavirus diversity through reassortment. Continued surveillance is important to determine if rotavirus vaccines will protect against strains that express the P[14] rotavirus genotype.
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http://dx.doi.org/10.1016/j.meegid.2014.09.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4605422PMC
December 2014

Whole-genome analyses of DS-1-like human G2P[4] and G8P[4] rotavirus strains from Eastern, Western and Southern Africa.

Virus Genes 2014 Oct 22;49(2):196-207. Epub 2014 Jun 22.

South African Medical Research Council/UL Diarrhoeal Pathogens Research Unit (MRC/DPRU), Department of Virology, University of Limpopo (Medunsa Campus) and National Health Laboratory Service, PO Box 173, Medunsa, Pretoria, 0204, South Africa.

Group A rotaviruses (RVAs) with distinct G and P genotype combinations have been reported globally. We report the genome composition and possible origin of seven G8P[4] and five G2P[4] human RVA strains based on the genetic evolution of all 11 genome segments at the nucleotide level. Twelve RVA ELISA positive stool samples collected in the representative countries of Eastern, Southern and West Africa during the 2007-2012 surveillance seasons were subjected to sequencing using the Ion Torrent PGM and Illumina MiSeq platforms. A reference-based assembly was performed using CLC Bio's clc_ref_assemble_long program, and full-genome consensus sequences were obtained. With the exception of the neutralising antigen, VP7, all study strains exhibited the DS-1-like genome constellation (P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2) and clustered phylogenetically with reference strains having a DS-1-like genetic backbone. Comparison of the nucleotide and amino acid sequences with selected global cognate genome segments revealed nucleotide and amino acid sequence identities of 81.7-100 % and 90.6-100 %, respectively, with NSP4 gene segment showing the most diversity among the strains. Bayesian analyses of all gene sequences to estimate the time of divergence of the lineage indicated that divergence times ranged from 16 to 44 years, except for the NSP4 gene where the lineage seemed to arise in the more distant past at an estimated 203 years ago. However, the long-term effects of changes found within the NSP4 genome segment should be further explored, and thus we recommend continued whole-genome analyses from larger sample sets to determine the evolutionary mechanisms of the DS-1-like strains collected in Africa.
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http://dx.doi.org/10.1007/s11262-014-1091-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818712PMC
October 2014

Full-Genome Sequence of a Rare Human G3P[9] Rotavirus Strain.

Genome Announc 2014 Mar 27;2(2). Epub 2014 Mar 27.

Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.

This is a report of the complete genomic sequence of a rare rotavirus group A G3-P[9]-I2-R2-C2-M2-A3-N2-T1-E2-H3 strain designated RVA/Human-wt/USA/12US1134/2012/G3P[9].
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http://dx.doi.org/10.1128/genomeA.00143-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3968326PMC
March 2014

Genetic analysis of G12P[8] rotaviruses detected in the largest U.S. G12 genotype outbreak on record.

Infect Genet Evol 2014 Jan 21;21:214-9. Epub 2013 Nov 21.

Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA. Electronic address:

In 2006-07, 77 cases of gastroenteritis in Rochester, NY, USA were associated with rotavirus genotype G12P[8]. Sequence analysis identified a high degree of genetic relatedness among the VP7 and VP4 genes of the Rochester G12P[8] strains and between these strains and currently circulating human G12P[8] strains. Out of 77 samples, two and seven unique nucleotide sequences were identified for VP7 and VP4 genes, respectively. Rochester strain VP7 genes were found to occupy the G12-III lineage and VP4 genes clustered within the P[8]-3 lineage. Six strains contained non-synonymous nucleotide substitutions that produced amino acid changes at 6 sites in the VP8(∗) region of the VP4 gene. Two sites (amino acids 242 and 246) were located in or near a described trypsin cleavage site. Selection analyses identified one positively selected VP7 site (107) and strong purifying selection at 58 sites within the VP7 gene as well as 2 of the 6 variant sites (79 and 218) in VP4.
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http://dx.doi.org/10.1016/j.meegid.2013.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4612336PMC
January 2014
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