Next-Generation Whole Genome Sequencing Identifies the Direction of Norovirus Transmission in Linked Patients

Kundu, Samit; Lockwood, Julianne; Depledge, Daniel P.; Chaudhry, Yasmin; Aston, Antony; Rao, Kanchan; Hartley, John C.; Goodfellow, Ian; Breuer, Judith

doi:10.1093/cid/cit287

Cited by 80 publications

(53 citation statements)

References 40 publications

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“…PCR-based methods yield high read depth values; however, due to sequence heterogeneity among genotypes, primers generally need to be genotype specific (9). Although broad-range primers were reported by Cotten et al (10), the approach retained a limited success rate; full genome sequences were amplified from comparable proportions of samples of GII.13 (83% versus 100% in this study), GII.6 (88% versus 95%), and GII.4 (92% versus 89% or 93% irrespective of read depth).…”

Section: Discussionmentioning

confidence: 99%

“…To date, norovirus sequencing from clinical material has been achieved by two methods, namely, sequencing of overlapping PCR fragments (9)(10)(11)(12) and direct sequencing of total RNA (13)(14)(15)(16). The former generates a pure viral template, which improves the quality of sequencing but requires multiple PCR amplifications; the latter necessitates great depth of sequencing to generate the target norovirus genome.…”

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confidence: 99%

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

et al. 2016

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cNorovirus 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 (C T ) 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 C T 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. N orovirus is a leading cause of outbreaks of acute gastroenteritis (1, 2), with an estimated prevalence of 20% in cases of acute gastroenteritis in developed countries (3) and a large financial burden, associated with ward and hospital closures, in health care settings (4). In countries in which rotavirus vaccine has been introduced, norovirus is now the leading cause of medically attended gastroenteritis in children (5, 6).Norovirus has a 7.5-kb single-stranded RNA genome organized into 3 open reading frames (ORFs), i.e., ORF1, ORF2, and ORF3. ORF1 encodes a nonstructural polyprotein that is cleaved posttranslationally and includes the RNA-dependent RNA polymerase. ORF2 encodes the major structural capsid protein, which is divided into shell (S) and protruding (P) domains. The P domain has two subdomains, P1 and P2. P2 is the most exposed antigenic site and contains immunogenic epitopes; consequently, it has the greatest sequence variation. ORF3 codes for a minor capsid protein.Comparison of viral genetic sequences allows linking of previously unrecognized transmission events or exclusion of cases from an outbreak. Traditionally, norovirus genotyping has involved PCR amplification and capillary sequencing of part...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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

et al. 2016

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“…The data provided in our study will also help to provide enhanced disinfection and prevention strategies, such as focusing on early control and intervention measures in LTCF, the single most important setting for GII.4 norovirus outbreaks. Although it is currently not feasible for routine analysis, future typing methods may include sequencing of the complete genome, which will allow better resolution of outbreaks and detection of emerging variants (44). In addition to tracking of norovirus genotypes based on the viral capsid, the continuous emergence of new recombinant norovirus strains will provide an impetus to identify which nonstructural proteins are important in norovirus virulence and possible immune evasion.…”

Section: Epidemiologic Characteristics Of Norovirus Outbreaks In Thementioning

confidence: 99%

Genotypic and Epidemiologic Trends of Norovirus Outbreaks in the United States, 2009 to 2013

et al. 2014

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In addition, three non-GII.4 viruses, i.e., GII.12, GII.1, and GI.6, caused 528 (13%) of all outbreaks. Several non-GII.4 genotypes (GI.3, GI.6, GI.7, GII.3, GII.6, and GII.12) were significantly more associated with food-borne transmission (odds ratio, 1.9 to 7.1; P < 0.05). Patients in LTCF and people >65 years of age were at higher risk for GII.4 infections than those in other settings and with other genotypes (P < 0.05). Phylogeographic analysis identified three major dispersions from two geographic locations that were responsible for the GI.6 outbreaks from 2011 to 2013. In conclusion, our data demonstrate the cyclic emergence of new (non-GII.4) norovirus strains, and several genotypes are more often associated with food-borne outbreaks. These surveillance data can be used to improve viral food-borne surveillance and to help guide studies to develop and evaluate targeted prevention methods such as norovirus vaccines, antivirals, and environmental decontamination methods.

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“…The accuracy of this strategy could be further improved by increasing the sample size from individual cities and analyzing sequences from recently infected individuals with current residential data. Another tool that could help improve HCV transmission inference studies is next-generation sequencing (NGS) which has recently been investigated for analyzing small-scale transmission events and within-patient connections (43)(44)(45). Minor sequence variants in one individual may predominate in a different individual, as illustrated by studies of liver transplants in patients with HCV (46,47), and NGS may enable the identification of connections involving minority variants which are not apparent using traditional sequencing techniques.…”

Section: Gt3mentioning

confidence: 99%

Spatiotemporal Reconstruction of the Introduction of Hepatitis C Virus into Scotland and Its Subsequent Regional Transmission

McNaughton

Cameron

Wignall-Fleming

et al. 2015

J Virol

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A more comprehensive understanding of hepatitis C virus (HCV) transmission dynamics could facilitate public health initiatives to reduce the prevalence of HCV in people who inject drugs. We aimed to determine how HCV sequences entered and spread throughout Scotland and to identify transmission hot spots. A Scottish data set with embedded demographic data was created by sequencing the NS5B of 125 genotype 1a (Gt1a) samples and 166 Gt3a samples and analyzed alongside sequences from public databases. Applying Bayesian inference methods, we reconstructed the global origin and local spatiotemporal dissemination of HCV in Scotland. Scottish sequences mainly formed discrete clusters interspersed between sequences from the rest of the world; the most recent common ancestors of these clusters dated to 1942 to 1952 (Gt1a) and 1926 to 1942 (Gt3a), coincident with global diversification and distribution. Extant Scottish sequences originated in Edinburgh (Gt1a) and Glasgow (Gt3a) in the 1970s, but both genotypes spread from Glasgow to other regions. The dominant Gt1a strain differed between Edinburgh (cluster 2 [C2]), Glasgow (C3), and Aberdeen (C4), whereas significant Gt3a strain specificity occurred only in Aberdeen. Specific clusters initially formed separate transmission zones in Glasgow that subsequently overlapped, occasioning city-wide cocirculation. Transmission hot spots were detected with 45% of samples from patients residing in just 9 of Glasgow's 57 postcode districts. HCV was introduced into Scotland in the 1940s, concomitant with its worldwide dispersal likely arising from global-scale historical events. Cluster-specific transmission hubs were identified in Glasgow, the key Scottish city implicated in HCV dissemination. This fine-scale spatiotemporal reconstruction improves understanding of HCV transmission dynamics in Scotland. IMPORTANCEHCV is a major health burden and the leading cause of hepatocellular carcinoma. Public health needle exchange and "treatment as prevention" strategies targeting HCV are designed to reduce prevalence of the virus in people who inject drugs (PWID), potentially mitigating the future burden of HCV-associated liver disease. Understanding HCV transmission dynamics could increase the effectiveness of such public health initiatives by identifying and targeting regions playing a central role in virus dispersal. In this study, we examined HCV transmission in Scotland by analyzing the genetic relatedness of strains from PWID alongside data inferring the year individuals became infected and residential information at a geographically finer-scale resolution than in previous studies. Clusters of Scotland-specific strains were identified with regional specificity, and mapping the spread of HCV allowed the identification of key areas central to HCV transmission in Scotland. This research provides a basis for identifying HCV transmission hot spots. H epatitis C virus (HCV) currently infects an estimated 180 million people throughout the world. Following a short acute phase, the virus ...

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Next-Generation Whole Genome Sequencing Identifies the Direction of Norovirus Transmission in Linked Patients

Cited by 80 publications

References 40 publications

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

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

Genotypic and Epidemiologic Trends of Norovirus Outbreaks in the United States, 2009 to 2013

Spatiotemporal Reconstruction of the Introduction of Hepatitis C Virus into Scotland and Its Subsequent Regional Transmission

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