{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,31]],"date-time":"2025-12-31T11:11:54Z","timestamp":1767179514842,"version":"build-2238731810"},"update-to":[{"DOI":"10.12688\/wellcomeopenres.14438.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2019,1,10]],"date-time":"2019-01-10T00:00:00Z","timestamp":1547078400000}}],"reference-count":54,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2019,1,10]],"date-time":"2019-01-10T00:00:00Z","timestamp":1547078400000},"content-version":"unspecified","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100009053","name":"Wellcome Trust\/DBT India Alliance","doi-asserted-by":"publisher","award":["EarlyCareerFellowship"],"award-info":[{"award-number":["EarlyCareerFellowship"]}],"id":[{"id":"10.13039\/501100009053","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000288","name":"Royal Society, SERB","doi-asserted-by":"publisher","award":["NewtonInternationalFellowship"],"award-info":[{"award-number":["NewtonInternationalFellowship"]}],"id":[{"id":"10.13039\/501100000288","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100007601","name":"Horizon 2020","doi-asserted-by":"publisher","award":["734584"],"award-info":[{"award-number":["734584"]}],"id":[{"id":"10.13039\/501100007601","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100011102","name":"Seventh Framework Programme","doi-asserted-by":"publisher","award":["278976"],"award-info":[{"award-number":["278976"]}],"id":[{"id":"10.13039\/100011102","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000272","name":"National Institute for Health Research","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100000272","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001407","name":"Department of Biotechnology , Ministry of Science and Technology","doi-asserted-by":"publisher","award":["ResearchAssociateship"],"award-info":[{"award-number":["ResearchAssociateship"]}],"id":[{"id":"10.13039\/501100001407","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001407","name":"Department of Biotechnology , Ministry of Science and Technology","doi-asserted-by":"publisher","award":["GlueGrant"],"award-info":[{"award-number":["GlueGrant"]}],"id":[{"id":"10.13039\/501100001407","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["wellcomeopenresearch.org"],"crossmark-restriction":false},"short-container-title":["Wellcome Open Res"],"abstract":"<ns4:p>\n                    <ns4:bold>Background:<\/ns4:bold>\n                    Mosquito-borne flaviviruses, such as dengue and Japanese encephalitis virus (JEV), cause life-threatening diseases, particularly in the tropics.\n                  <\/ns4:p>\n                  <ns4:p>\n                    <ns4:bold>Methods:<\/ns4:bold>\n                    Here we performed unbiased metagenomic sequencing of RNA extracted from the serum of four patients and the plasma of one patient, all hospitalized at a tertiary care centre in South India with severe or prolonged febrile illness, together with the serum from one healthy control, in 2014.\n                  <\/ns4:p>\n                  <ns4:p>\n                    <ns4:bold>Results:<\/ns4:bold>\n                    We identified and assembled a complete dengue virus type 3 sequence from a case of severe dengue fever. We also identified a small number of JEV sequences in the serum of two adults with febrile illness, including one with severe dengue. Phylogenetic analysis revealed that the dengue sequence belonged to genotype III. It has an estimated divergence time of 13.86 years from the most highly related Indian strains. In total, 11 amino acid substitutions were predicted for this strain in the antigenic envelope protein, when compared to the parent strain used for development of the first commercial dengue vaccine.\u00a0\n                  <\/ns4:p>\n                  <ns4:p>\n                    <ns4:bold>Conclusions:<\/ns4:bold>\n                    We demonstrate that both genome assembly and detection of a low number of viral sequences are possible through the unbiased sequencing of clinical material. These methods may help ascertain causal agents for febrile illnesses with no known cause.\n                  <\/ns4:p>","DOI":"10.12688\/wellcomeopenres.14438.2","type":"journal-article","created":{"date-parts":[[2019,1,10]],"date-time":"2019-01-10T10:20:18Z","timestamp":1547115618000},"page":"44","update-policy":"https:\/\/doi.org\/10.12688\/wellcomeopenres.crossmark-policy","source":"Crossref","is-referenced-by-count":5,"title":["Complete assembly of a dengue virus type 3 genome from a recent genotype III clade by metagenomic sequencing of serum"],"prefix":"10.12688","volume":"3","author":[{"given":"Mary","family":"Dias","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9707-3358","authenticated-orcid":false,"given":"Chitra","family":"Pattabiraman","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shilpa","family":"Siddappa","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Malali","family":"Gowda","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anita","family":"Shet","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2393-1890","authenticated-orcid":false,"given":"Derek","family":"Smith","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Barbara","family":"Muehlemann","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Krishnapriya","family":"Tamma","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tom","family":"Solomon","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Terry","family":"Jones","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sudhir","family":"Krishna","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"2560","published-online":{"date-parts":[[2019,1,10]]},"reference":[{"key":"ref-1","first-page":"719-26","article-title":"Acute 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Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/3-44\/v2\/pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/3-44\/v2\/xml","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/3-44\/v2\/iparadigms","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,7,13]],"date-time":"2024-07-13T19:15:43Z","timestamp":1720898143000},"score":1,"resource":{"primary":{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/3-44\/v2"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,1,10]]},"references-count":54,"URL":"https:\/\/doi.org\/10.12688\/wellcomeopenres.14438.2","relation":{},"ISSN":["2398-502X"],"issn-type":[{"value":"2398-502X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,1,10]]},"assertion":[{"value":"Indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/3-44\/v2#article-reports","order":0,"name":"referee-status","label":"Referee status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.21956\/wellcomeopenres.15716.r33079, Easwaran Sreekumar, Shijulal Nelson-Sathi, Molecular Virology Laboratory, Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananthapuram, Kerala, India, 17 May 2018, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/3-44\/v1#referee-response-33079","order":0,"name":"referee-response-33079","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Chitra Pattabiraman<\/b>; <i>Posted: 03 Jan 2019<\/i>; We thank the reviewers for the comments. They are addressed point wise below <i>1. New Generation sequencing approaches are attractive options to identify viral quasispecies in patients. In the present article, the authors have presented a common consensus final sequence of DENV-3 and compared it with reference sequence and the closely related GWL-25 genome. Did the authors had a chance&nbsp; to look at the raw read clusters, and identify possible variations that could represent quasi species by looking at sites\/ sequence regions showing consistent variability?&nbsp;<\/i> <i>A comparison table showing the non-synonymous sites could have been included.<\/i>We have not performed a systematic analysis for viral variants within our sample, however, given that we got greater that 100X coverage of many regions of the genome, we believe this analysis is possible. A comparison table showing the changes between the DENV3 strain reported here and the RefSeq DENV3 strain are included in Supplementary File 5.&nbsp;2.&nbsp; <i>Considering that DENV virus protein translation starts as a polyprotein formation from a single ORF, identifying two internal stop codons at positions 58 &amp; 168 seems to be curious. Have the authors done a manual verification of the corresponding raw read alignments? What could be the implications of this finding<\/i>?We have realigned the sequence and manually verified this. Both changes are well supported. Our experiment cannot distinguish between defective viral particles and viable ones. Also, we are potentially putting together sequences from different viruses to make the consensus. These two factors limit our ability to interpret these findings.&nbsp;3. <i>The authors have identified two missing regions-one each in the 5' and 3' UTR. Are they internal gaps or terminal truncation in the sequence? Could this be a failure to sequence these regions by the technique or is it possible that the DENV-3 strain itself is lacking these regions? Since the UTRs contain major replication regulatory elements, discussing this observation seems to be important.<\/i>Given the importance of the UTRs in viral replication, we strongly suspect that our approach has limitations in capturing the ends of the genome.&nbsp;4. <i>In the result section, the unique substitution is mentioned as D361E, where as in the fig.3A it is shown as E361D. The reader gets confused on the order of the sequences that are being compared.<\/i>The texthas been corrected in line with the figure.&nbsp;Most of the amino acid substitutions were shared among all the Indian strains, while a D361E substitution was unique to the DENV3 strain reported here (Figure 3A).toMost of the amino acid substitutions were shared among all the Indian strains, while a&nbsp;E361Dsubstitution was unique to the DENV3 strain reported here (Figure 3A).5.&nbsp; <i>In Fig.3b(ii), the authors have highlighted the amino acid residues that are important in antibody binding and antigenicity. But how many of these changes are functionally important, based on the chemical nature of the amino acid substitutions observed? Also, the authors should indicate the wild type as well as the mutant amino acids along with the position number in the figure.<\/i>The figure and accompanying legend have been modified.&nbsp;6. <i>Was there any specific reason in using plasma sample from one patient and serum from others?It would be good to indicate, if available, the duration of fever in F1, and treatment and outcome in F4, for reasons of consistency.<\/i>In this study, we used samples remaining after routine testing and plasma sample&nbsp;was available for that patient.","URL":"https:\/\/wellcomeopenresearch.org\/articles\/3-44\/v2#referee-comment-3772","order":1,"name":"referee-comment-3772","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.15716.r33558, John A Lednicky, Department of Environmental and Global Health, College of Public Health and Health Professions, Emerging Pathogens Institute, University of Florida, Gainesville, FL, USA, 08 Aug 2018, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/3-44\/v1#referee-response-33558","order":2,"name":"referee-response-33558","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Chitra Pattabiraman<\/b>; <i>Posted: 13 Jan 2019<\/i>; Response to Reviewer 2 We thank the reviewer for the comments on the manuscript. We address them point wise below&nbsp; 1. OPTIONAL: On page 3, the authors mention Dengvaxia. It will actually strengthen the authors\u2019 projected use of metagenomics sequencing if they mentioned the problems in the Philippines and elsewhere with this vaccine. Will it help to know before vaccines are used which virus strains are in circulation? And will that sequence knowledge be useful in the future for evaluating the possible side-effects of vaccines before the vaccines are administered? Earlier work from Sanofi Pasteur showed that the tetravalent vaccine was able to induce antibodies that neutralized a large number of clades of circulating dengue viruses (Barban et al., Virology, 2012). It is not clear if variation in the wild-type strain could explain the results seen in the clinical trials (Guy et al, Annu Rev Med. 2016).&nbsp; We do believe that knowledge of circulating strain and their variations from the vaccine strain is important for predicting response to a vaccine, however, our work does not directly test this hypothesis. Therefore, we have not commented on that in this work and hope to address it in future studies.&nbsp; 2. Figure 3: As viewed on my computer and a printed copy of the manuscript, neither blue nor violet colors were discernable for FIG. 3b. &nbsp; &nbsp; &nbsp; &nbsp; The figure and legend have been modified for clarity. &nbsp; &nbsp; &nbsp; &nbsp; 3. OPTIONAL: The authors might clarify: On page 7, DISCUSSION, par. 1: It is not clear if the authors agree with the reference cited (35), or whether they have experienced this first hand. In our work, we did not use a viral capture method, but such methods are available and are likely to be more sensitive in picking up viral sequences at low sequencing depths. The text has been modified to reflect this more clearly.&nbsp; Changed text: We believe that, in the future, the sequence-based -enrichment of viral sequences using conserved sequences, will enable the recovery of complete genomes from routine clinical samples even with by lower-depth sequencing [35] &nbsp; &nbsp; &nbsp; &nbsp; 4. Page 8: Isolation of RNA: Why was the All-Prep kit chosen for serum\/plasma instead of a QIAGEN viral RNA kit? The former is typically used for whole blood. How was the eluted RNA concentrated? Why? &nbsp; &nbsp; &nbsp; &nbsp; We have previously had good results with the All-prep Kit for isolation of DNA and RNA from low number of cells, albeit from cell cultures. We also wanted to extract both DNA and RNA from the samples. The eluted RNA was concentrated by freeze drying (Scan speed 40, Labgene) so that we could take all of the RNA into the cDNA preparation steps, which limits the volume of RNA.&nbsp; 5. OPTIONAL: The authors make no mention of the costs\/time commitments\/ personnel\/training\/facilities to accomplish the type of work they performed. Though the approach is very good (and clearly the wave of the future), it is at present very costly and not practical for most medical laboratories, even in developed countries. How can this be remedied? &nbsp; &nbsp; &nbsp; &nbsp; There are two aspects to this questions. 1) Economics of sequencing \u2013 &nbsp;Sequencing is still prohibitively expensive, both in terms of reagents as well as the skilled personnel required to become a part of routine diagnostics. However, it may become necessary for clinical microbiologists to cultivate these skills as sequencing costs drop. 2) Is it practical? We do not envision the use of metagenomics for every sample, we think it will become a part of the discovery phase for the designing of locally suited diagnostic panels.&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; 6. OPTIONAL: The authors might comment further on this: It will be curious to many readers why JEV sequences were also detected. Is this a finding of major importance? Has this been observed in other studies (persistence of JEV sequences)? Could the severity of illness in patient F2 have been affected by a \u201csmoldering\u201d JEV infection? &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;We found sequencing reads mapping uniquely to Japanese encephalitis virus (JEV) in the serum. JEV is known to cause acute febrile illness in South East Asia (Mayxay et al., Lancet Glob Health., 2013). An alternate explanation is that this is a remnant of previous infection. JEV sequences are known to persist, infectious virus has been cultured from cerebrospinal fluid &gt; 3 weeks post onset of illness (Ravi V et al., J Med Virol 1993). With our data, we cannot currently distinguish between the two possibilities. We did not carry out antibody testing for JEV as all of the sample was used in the sequencing reaction.&nbsp; 7. More explanation would be useful regarding how the sequencing libraries were made. For example, flavivirus RNA is not polyadenylated and is capped. Do the authors have a specific approach to capture these types of RNA? &nbsp; &nbsp; &nbsp; &nbsp; As we were performing a metagenomics experiment with low amount of starting material from a clinical sample, we used all of the RNA for cDNA synthesis using random hexamers. This allows us to capture non-polyadenylated viral RNA.&nbsp; *OPTIONAL: Some possible clarifications\/comments from the authors: (1) Next generation sequencing definitely has a role diagnostics, phylogeographic studies, etc. On page 3, the authors state \u201cWe hypothesized that \u2026\u201d. Some readers will interpret that to mean the authors claim they conceived this concept, and this is the first time the approach has been used. The authors might reword that statement. We have reworded this statement We hypothesized that the unbiased sequencing, or metageomic, approach would help us determine both the identity and the sequences of viruses in febrile illness.&nbsp; Here we used an unbiased sequencing\/metagenomic approach, in order to determine both the identity and sequences of viruses associated with febrile illness.&nbsp; (2) The authors did not comment on the specificity of the DENV IgM test. In this study, we used the Panbio dengue IgM capture ELISA. The serological specificity is stated to be 95.7-100% (3) Many NGS-derived virus sequences deposited in data banks have errors. Thus, whereas the NGS-generated data is useful for the diagnosis, indels etc. are not always verified in the submitted sequences, the general understanding among users being that errors may be present. Also, often, the 5\u2019 and 3\u2019 UTRs are not included. The authors might discuss these issues. In particular, there are artefacts associated with Ion Proton sequencing associated with repeats of the same nucleotide (example: TTTTT). Many of the sequencing error related issues can be circumvented by using multiple reads covering the same base. Coverage \/depth of sequencing, can be used as a proxy for how confident we can be about the consensus. &nbsp;However in the case of RNA viruses, the differences from consensus may also be intra-host variants and not necessarily errors from sequencing. We had difficulty in getting the complete UTRs even though we had good read depth.&nbsp;","URL":"https:\/\/wellcomeopenresearch.org\/articles\/3-44\/v2#referee-comment-3784","order":3,"name":"referee-comment-3784","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This study was supported by the Department of Biotechnology, Glue Grant to SK. PC was supported by the Department of Biotechnology Research Associate fellowship, Royal Society, SERB Newton International fellowship and the India Alliance (DBT-Wellcome Trust) Early Career Fellowship. TS is supported by the NIHR Health Protection Research Unit in Emerging and Zoonotic Infections and the European Union's Horizon 2020 research and innovation program ZikaPLAN (Preparedness Latin America Network), grant agreement No. 734584. DS, BM, and TJ were supported by the European Union FP7 programme ANTIGONE (grant agreement No. 278976). The funders had no role in the in any steps of experimental design, data collection or analysis or the decision to publish. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":4,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}