|
1
|
Garbuglia AR, Pauciullo S, Zulian V, Del Porto P. Update on Hepatitis C Vaccine: Results and Challenges. Viruses 2024; 16:1337. [PMID: 39205311 PMCID: PMC11359353 DOI: 10.3390/v16081337] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2024] [Revised: 08/15/2024] [Accepted: 08/20/2024] [Indexed: 09/04/2024] Open
Abstract
Therapy against the Hepatitis C virus (HCV) has significantly improved with the introduction of direct-acting antiviral drugs (DAAs), achieving over 95% sustained virological response (SVR). Despite this, the development of an effective anti-HCV vaccine remains a critical challenge due to the low number of patients treated with DAAs and the occurrence of HCV reinfections in high-risk groups. Current vaccine strategies aim to stimulate either B-cell or T-cell responses. Vaccines based on E1 and E2 proteins can elicit broad cross-neutralizing antibodies against all major HCV genotypes, though with varying efficiencies and without full protection against infection. In humans, the neutralizing antibodies induced by such vaccines mainly target the AR3 region, but their levels are generally insufficient for broad neutralization. Various HCV proteins expressed through different viral vectors have been utilized to elicit T cell immune responses, showing sustained expansion of HCV-specific effector memory T cells and improved proliferation and polyfunctionality of memory T cells over time. However, despite these advancements, the frequency and effectiveness of T-cell responses remain limited.
Collapse
Affiliation(s)
- Anna Rosa Garbuglia
- Laboratory of Virology, National Institute for Infectious Diseases “Lazzaro Spallanzani” (IRCCS), 00149 Rome, Italy; (S.P.); (V.Z.)
| | - Silvia Pauciullo
- Laboratory of Virology, National Institute for Infectious Diseases “Lazzaro Spallanzani” (IRCCS), 00149 Rome, Italy; (S.P.); (V.Z.)
| | - Verdiana Zulian
- Laboratory of Virology, National Institute for Infectious Diseases “Lazzaro Spallanzani” (IRCCS), 00149 Rome, Italy; (S.P.); (V.Z.)
| | - Paola Del Porto
- Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, 00100 Rome, Italy;
| |
Collapse
|
|
2
|
Roddur MS, Snir S, El-Kebir M. Enforcing Temporal Consistency in Migration History Inference. J Comput Biol 2024; 31:396-415. [PMID: 38754138 DOI: 10.1089/cmb.2023.0352] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/18/2024] Open
Abstract
In addition to undergoing evolution, members of biological populations may also migrate between locations. Examples include the spread of tumor cells from the primary tumor to distant metastases or the spread of pathogens from one host to another. One may represent migration histories by assigning a location label to each vertex of a given phylogenetic tree such that an edge connecting vertices with distinct locations represents a migration. Some biological populations undergo comigration, a phenomenon where multiple taxa from distinct lineages simultaneously comigrate from one location to another. In this work, we show that a previous problem statement for inferring migration histories that are parsimonious in terms of migrations and comigrations may lead to temporally inconsistent solutions. To remedy this deficiency, we introduce precise definitions of temporal consistency of comigrations in a phylogenetic tree, leading to three successive problems. First, we formulate the temporally consistent comigration problem to check if a set of comigrations is temporally consistent and provide a linear time algorithm for solving this problem. Second, we formulate the parsimonious consistent comigrations (PCC) problem, which aims to find comigrations given a location labeling of a phylogenetic tree. We show that PCC is NP-hard. Third, we formulate the parsimonious consistent comigration history (PCCH) problem, which infers the migration history given a phylogenetic tree and locations of its extant vertices only. We show that PCCH is NP-hard as well. On the positive side, we propose integer linear programming models to solve the PCC and PCCH problems. We demonstrate our algorithms on simulated and real data.
Collapse
Affiliation(s)
- Mrinmoy Saha Roddur
- Department of Computer Science, University of Illinois Urbana-Champaign, Urbana, Illinois, USA
| | - Sagi Snir
- Department of Evolutionary Biology, University of Haifa, Haifa, Israel
| | - Mohammed El-Kebir
- Department of Computer Science, University of Illinois Urbana-Champaign, Urbana, Illinois, USA
- Cancer Center at Illinois, University of Illinois Urbana-Champaign, Urbana, Illinois, USA
| |
Collapse
|
|
3
|
Holmes KE, VanInsberghe D, Ferreri LM, Elie B, Ganti K, Lee CY, Lowen AC. Viral expansion after transfer is a primary driver of influenza A virus transmission bottlenecks. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.11.19.567585. [PMID: 38014182 PMCID: PMC10680852 DOI: 10.1101/2023.11.19.567585] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/29/2023]
Abstract
For many viruses, narrow bottlenecks acting during transmission sharply reduce genetic diversity in a recipient host relative to the donor. Since genetic diversity represents adaptive potential, such losses of diversity are though to limit the opportunity for viral populations to undergo antigenic change and other adaptive processes. Thus, a detailed picture of evolutionary dynamics during transmission is critical to understanding the forces driving viral evolution at an epidemiologic scale. To advance this understanding, we used a novel barcoded virus library and a guinea pig model of transmission to decipher where in the transmission process diversity is lost for influenza A viruses. In inoculated guinea pigs, we show that a high level of viral genetic diversity is maintained across time. Continuity in the barcodes detected furthermore indicates that stochastic effects are not pronounced within inoculated hosts. Importantly, in both aerosol-exposed and direct contact-exposed animals, we observed many barcodes at the earliest time point(s) positive for infectious virus, indicating robust transfer of diversity through the environment. This high viral diversity is short-lived, however, with a sharp decline seen 1-2 days after initiation of infection. Although major losses of diversity at transmission are well described for influenza A virus, our data indicate that events that occur following viral transfer and during the earliest stages of natural infection have a predominant role in this process. This finding suggests that immune selection may have greater opportunity to operate during influenza A transmission than previously recognized.
Collapse
Affiliation(s)
- Katie E. Holmes
- Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA
| | - David VanInsberghe
- Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA
| | - Lucas M. Ferreri
- Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA
| | - Baptiste Elie
- MIVEGEC, CNRS, IRD, Université de Montpellier, Montpellier, France
| | - Ketaki Ganti
- Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA
| | - Chung-Young Lee
- Department of Microbiology, School of Medicine, Kyungpook National University, Jung-gu, Daegu, Republic of Korea
| | - Anice C. Lowen
- Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA
- Emory Center of Excellence for Influenza Research and Response (CEIRR), Atlanta, GA, USA
| |
Collapse
|
|
4
|
Lapointe HR, Dong W, Dong WWY, Kirkby D, Woods C, Poon AFY, Howe AYM, Harrigan PR, Brumme CJ. Validation of a Genotype-Independent Hepatitis C Virus Near-Whole Genome Sequencing Assay. Viruses 2021; 13:v13091721. [PMID: 34578305 PMCID: PMC8473162 DOI: 10.3390/v13091721] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2021] [Revised: 08/17/2021] [Accepted: 08/26/2021] [Indexed: 11/16/2022] Open
Abstract
Despite the effectiveness of direct-acting antiviral agents in treating hepatitis C virus (HCV), cases of treatment failure have been associated with the emergence of resistance-associated substitutions. To better guide clinical decision-making, we developed and validated a near-whole-genome HCV genotype-independent next-generation sequencing strategy. HCV genotype 1-6 samples from direct-acting antiviral agent treatment-naïve and -treated HCV-infected individuals were included. Viral RNA was extracted using a NucliSens easyMAG and amplified using nested reverse transcription-polymerase chain reaction. Libraries were prepared using Nextera XT and sequenced on the Illumina MiSeq sequencing platform. Data were processed by an in-house pipeline (MiCall). Nucleotide consensus sequences were aligned to reference strain sequences for resistance-associated substitution identification and compared to NS3, NS5a, and NS5b sequence data obtained from a validated in-house assay optimized for HCV genotype 1. Sequencing success rates (defined as achieving >100-fold read coverage) approaching 90% were observed for most genotypes in samples with a viral load >5 log10 IU/mL. This genotype-independent sequencing method resulted in >99.8% nucleotide concordance with the genotype 1-optimized method, and 100% agreement in genotype assignment with paired line probe assay-based genotypes. The assay demonstrated high intra-run repeatability and inter-run reproducibility at detecting substitutions above 2% prevalence. This study highlights the performance of a freely available laboratory and bioinformatic approach for reliable HCV genotyping and resistance-associated substitution detection regardless of genotype.
Collapse
Affiliation(s)
- Hope R. Lapointe
- Department of Medicine, Division of Social Medicine, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; (H.R.L.); (P.R.H.)
- BC Centre for Excellence in HIV/AIDS, Vancouver, BC V6Z 1Y6, Canada; (W.D.); (W.W.Y.D.); (D.K.); (C.W.)
| | - Weiyan Dong
- BC Centre for Excellence in HIV/AIDS, Vancouver, BC V6Z 1Y6, Canada; (W.D.); (W.W.Y.D.); (D.K.); (C.W.)
| | - Winnie W. Y. Dong
- BC Centre for Excellence in HIV/AIDS, Vancouver, BC V6Z 1Y6, Canada; (W.D.); (W.W.Y.D.); (D.K.); (C.W.)
| | - Don Kirkby
- BC Centre for Excellence in HIV/AIDS, Vancouver, BC V6Z 1Y6, Canada; (W.D.); (W.W.Y.D.); (D.K.); (C.W.)
| | - Conan Woods
- BC Centre for Excellence in HIV/AIDS, Vancouver, BC V6Z 1Y6, Canada; (W.D.); (W.W.Y.D.); (D.K.); (C.W.)
| | - Art F. Y. Poon
- Department of Pathology and Laboratory Medicine, Western University, London, ON N6A 3K7, Canada;
| | - Anita Y. M. Howe
- British Columbia Centre for Disease Control, Vancouver, BC V5Z 4R4, Canada;
| | - P. Richard Harrigan
- Department of Medicine, Division of Social Medicine, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; (H.R.L.); (P.R.H.)
| | - Chanson J. Brumme
- BC Centre for Excellence in HIV/AIDS, Vancouver, BC V6Z 1Y6, Canada; (W.D.); (W.W.Y.D.); (D.K.); (C.W.)
- Department of Medicine, Division of Infectious Diseases, University of British Columbia, Vancouver, BC V6T 1Z4, Canada
- Correspondence:
| |
Collapse
|
|
5
|
Intra-host evolutionary dynamics of the hepatitis C virus among people who inject drugs. Sci Rep 2021; 11:9986. [PMID: 33976241 PMCID: PMC8113533 DOI: 10.1038/s41598-021-88132-8] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2020] [Accepted: 03/31/2021] [Indexed: 02/03/2023] Open
Abstract
Most individuals chronically infected with hepatitis C virus (HCV) are asymptomatic during the initial stages of infection and therefore the precise timing of infection is often unknown. Retrospective estimation of infection duration would improve existing surveillance data and help guide treatment. While intra-host viral diversity quantifications such as Shannon entropy have previously been utilized for estimating duration of infection, these studies characterize the viral population from only a relatively short segment of the HCV genome. In this study intra-host diversities were examined across the HCV genome in order to identify the region most reflective of time and the degree to which these estimates are influenced by high-risk activities including those associated with HCV acquisition. Shannon diversities were calculated for all regions of HCV from 78 longitudinally sampled individuals with known seroconversion timeframes. While the region of the HCV genome most accurately reflecting time resided within the NS3 gene, the gene region with the highest capacity to differentiate acute from chronic infections was identified within the NS5b region. Multivariate models predicting duration of infection from viral diversity significantly improved upon incorporation of variables associated with recent public, unsupervised drug use. These results could assist the development of strategic population treatment guidelines for high-risk individuals infected with HCV and offer insights into variables associated with a likelihood of transmission.
Collapse
|
|
6
|
Schreiber SJ, Ke R, Loverdo C, Park M, Ahsan P, Lloyd-Smith JO. Cross-scale dynamics and the evolutionary emergence of infectious diseases. Virus Evol 2021; 7:veaa105. [PMID: 35186322 PMCID: PMC8087961 DOI: 10.1093/ve/veaa105] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2023] Open
Abstract
When emerging pathogens encounter new host species for which they are poorly adapted, they must evolve to escape extinction. Pathogens experience selection on traits at multiple scales, including replication rates within host individuals and transmissibility between hosts. We analyze a stochastic model linking pathogen growth and competition within individuals to transmission between individuals. Our analysis reveals a new factor, the cross-scale reproductive number of a mutant virion, that quantifies how quickly mutant strains increase in frequency when they initially appear in the infected host population. This cross-scale reproductive number combines with viral mutation rates, single-strain reproductive numbers, and transmission bottleneck width to determine the likelihood of evolutionary emergence, and whether evolution occurs swiftly or gradually within chains of transmission. We find that wider transmission bottlenecks facilitate emergence of pathogens with short-term infections, but hinder emergence of pathogens exhibiting cross-scale selective conflict and long-term infections. Our results provide a framework to advance the integration of laboratory, clinical, and field data in the context of evolutionary theory, laying the foundation for a new generation of evidence-based risk assessment of emergence threats.
Collapse
Affiliation(s)
| | - Ruian Ke
- T-6: Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, NM 87545, USA
| | - Claude Loverdo
- Laboratoire Jean Perrin, Sorbonne Université, CNRS, Paris 75005, France
| | - Miran Park
- Department of Ecology & Evolution, University of California, Los Angeles, CA 90095, USA
| | - Prianna Ahsan
- Department of Ecology & Evolution, University of California, Los Angeles, CA 90095, USA
| | - James O Lloyd-Smith
- Department of Ecology & Evolution, University of California, Los Angeles, CA 90095, USA
| |
Collapse
|
|
7
|
Johnson KEE, Ghedin E. Quantifying between-Host Transmission in Influenza Virus Infections. Cold Spring Harb Perspect Med 2020; 10:cshperspect.a038422. [PMID: 31871239 DOI: 10.1101/cshperspect.a038422] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The error-prone replication and life cycle of influenza virus generate a diverse set of genetic variants. Transmission between hosts strictly limits both the number of virus particles and the genetic diversity of virus variants that reach a new host and establish an infection. This sharp reduction in the virus population at transmission--the transmission bottleneck--is significant to the evolution of influenza virus and to its epidemic and pandemic potential. This review describes transmission bottlenecks and their effect on the diversity and evolution of influenza virus. It also reviews the methods for calculating and predicting bottleneck sizes and highlights the host and viral determinants of influenza transmissibility.
Collapse
Affiliation(s)
- Katherine E E Johnson
- Center for Genomics and Systems Biology, Department of Biology, New York University, New York, New York 10003, USA
| | - Elodie Ghedin
- Center for Genomics and Systems Biology, Department of Biology, and Department of Epidemiology, College of Global Public Health, New York University, New York, New York 10003, USA
| |
Collapse
|
|
8
|
Caudill VR, Qin S, Winstead R, Kaur J, Tisthammer K, Pineda EG, Solis C, Cobey S, Bedford T, Carja O, Eggo RM, Koelle K, Lythgoe K, Regoes R, Roy S, Allen N, Aviles M, Baker BA, Bauer W, Bermudez S, Carlson C, Castellanos E, Catalan FL, Chemel AK, Elliot J, Evans D, Fiutek N, Fryer E, Goodfellow SM, Hecht M, Hopp K, Hopson ED, Jaberi A, Kinney C, Lao D, Le A, Lo J, Lopez AG, López A, Lorenzo FG, Luu GT, Mahoney AR, Melton RL, Nascimento GD, Pradhananga A, Rodrigues NS, Shieh A, Sims J, Singh R, Sulaeman H, Thu R, Tran K, Tran L, Winters EJ, Wong A, Pennings PS. CpG-creating mutations are costly in many human viruses. Evol Ecol 2020; 34:339-359. [PMID: 32508375 PMCID: PMC7245597 DOI: 10.1007/s10682-020-10039-z] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2019] [Accepted: 03/11/2020] [Indexed: 01/26/2023]
Abstract
Mutations can occur throughout the virus genome and may be beneficial, neutral or deleterious. We are interested in mutations that yield a C next to a G, producing CpG sites. CpG sites are rare in eukaryotic and viral genomes. For the eukaryotes, it is thought that CpG sites are rare because they are prone to mutation when methylated. In viruses, we know less about why CpG sites are rare. A previous study in HIV suggested that CpG-creating transition mutations are more costly than similar non-CpG-creating mutations. To determine if this is the case in other viruses, we analyzed the allele frequencies of CpG-creating and non-CpG-creating mutations across various strains, subtypes, and genes of viruses using existing data obtained from Genbank, HIV Databases, and Virus Pathogen Resource. Our results suggest that CpG sites are indeed costly for most viruses. By understanding the cost of CpG sites, we can obtain further insights into the evolution and adaptation of viruses.
Collapse
Affiliation(s)
- Victoria R. Caudill
- Department of Biology, San Francisco State University, San Francisco, CA USA
- Department of Biology, University of Oregon, Eugene, OR USA
| | - Sarina Qin
- Department of Biology, San Francisco State University, San Francisco, CA USA
- Quantitative Systems Biology, Univeristy of California, Merced, CA USA
| | - Ryan Winstead
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Jasmeen Kaur
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Kaho Tisthammer
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - E. Geo Pineda
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Caroline Solis
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Sarah Cobey
- Department of Ecology and Evolution, University of Chicago, Chicago, IL USA
| | - Trevor Bedford
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA USA
| | - Oana Carja
- Department of Computational Biology, School of Computer Science, Carnegie Mellon University, Pittsburgh, USA
| | - Rosalind M. Eggo
- Department of Infectious Disease Epidemiology, London School of Hygiene & Tropical Medicine, London, UK
| | - Katia Koelle
- Department of Biology, Emory University, Atlanta, GA USA
| | | | - Roland Regoes
- Department of Environmental Systems Science, ETH Zurich, Zurich, Switzerland
| | - Scott Roy
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Nicole Allen
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Milo Aviles
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Brittany A. Baker
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - William Bauer
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Shannel Bermudez
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Corey Carlson
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Edgar Castellanos
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Francisca L. Catalan
- Department of Biology, San Francisco State University, San Francisco, CA USA
- Department of Neurological Surgery, University of California, San Francisco, CA USA
| | | | - Jacob Elliot
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Dwayne Evans
- Department of Biology, San Francisco State University, San Francisco, CA USA
- Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA USA
| | - Natalie Fiutek
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Emily Fryer
- Department of Biology, San Francisco State University, San Francisco, CA USA
- Department of Plant Biology, Carnegie Institution for Science, Stanford, CA USA
| | - Samuel Melvin Goodfellow
- Department of Biology, San Francisco State University, San Francisco, CA USA
- Health Sciences Center, University of New Mexico, Albuquerque, NM USA
| | - Mordecai Hecht
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Kellen Hopp
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - E. Deshawn Hopson
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Amirhossein Jaberi
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Christen Kinney
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Derek Lao
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Adrienne Le
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Jacky Lo
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Alejandro G. Lopez
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Andrea López
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Fernando G. Lorenzo
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Gordon T. Luu
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Andrew R. Mahoney
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Rebecca L. Melton
- Department of Biology, San Francisco State University, San Francisco, CA USA
- UCSD Biomed Sciences PhD Program, University of California, San Diego, CA USA
| | | | - Anjani Pradhananga
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Nicole S. Rodrigues
- Department of Biology, San Francisco State University, San Francisco, CA USA
- Biochemistry, Molecular, Cellular and Developmental Biology Graduate Group, University of California, Davis, CA USA
| | - Annie Shieh
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Jasmine Sims
- Department of Biology, San Francisco State University, San Francisco, CA USA
- UCSF Tetrad Graduate Program, University of California, San Francisco, CA USA
| | - Rima Singh
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Hasan Sulaeman
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Ricky Thu
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Krystal Tran
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Livia Tran
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | | | - Albert Wong
- Department of Biology, San Francisco State University, San Francisco, CA USA
| | - Pleuni S. Pennings
- Department of Biology, San Francisco State University, San Francisco, CA USA
| |
Collapse
|
|
9
|
Shared HCV Transmission Networks Among HIV-1-Positive and HIV-1-Negative Men Having Sex With Men by Ultradeep Sequencing. J Acquir Immune Defic Syndr 2020; 82:105-110. [PMID: 31169768 DOI: 10.1097/qai.0000000000002099] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
OBJECTIVE Several studies reported hepatitis C virus (HCV) transmission networks among men having sex with men (MSM) in Europe and the spread of HCV strains from HIV-HCV coinfected toward HCV monoinfected MSM. We aimed to investigate HCV transmission dynamics among HIV-positive and HIV-negative MSM by ultradeep sequencing (UDS). DESIGN AND METHODS NS5B fragment (388 bp) was sequenced from virus of 50 HIV-positive and 18 HIV-negative patients diagnosed with recent HCV infection. UDS data were analyzed by Geneious (version 10.3.2). Phylogenetic trees were constructed by FastTree (version 2.1) and submitted to ClusterPicker (version 1.2.3) for transmission chain detection at different thresholds of maximum genetic distance (MGD) (3% for Sanger, 3% and 4.5% for UDS). RESULTS Ten, 17, and 18 HCV transmission chains were identified by Sanger at 3%, UDS at 3% and at 4.5% of MGD, respectively. Of 68 subjects enrolled, 38 (55.9%), 38 (55.9%), and 43 (65.3%) individuals were involved in transmission networks found by Sanger at 3%, UDS at 3%, and at 4.5% of MGD, respectively. Mixed transmission chains including HIV-positive and HIV-negative subjects were detected for 8/10 chains by Sanger at 3%, for 9/17 by UDS at 3%, and for 10/18 by UDS at 4.5% of MGD. Overall, the number of HIV-negative individuals clustering with HIV-positive ones was 9/18 by Sanger, 9/18 by UDS at 3%, and 10/18 by UDS at 4.5% of MGD. CONCLUSIONS HIV-positive and HIV-negative MSM shared HCV transmission networks, which emphasizes the need for HCV surveillance and prevention measures in these communities regardless of the HIV status.
Collapse
|
|
10
|
Dennin RH, Wo JE. DNA sequences homologous to hepatitis C virus (HCV) in the extrachromosomal circular DNA in peripheral blood mononuclear cells of HCV-negative subjects. J Zhejiang Univ Sci B 2020; 20:637-646. [PMID: 31273961 DOI: 10.1631/jzus.b1800453] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
OBJECTIVE This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (HCV). These DNA sequences are present in the whole DNA extracted from peripheral blood mononuclear cells (PBMCs) of HCV-negative subjects. We presumed that these experimentally proven 5'-noncoding region (5'-NCR) homologous DNA sequences could be contained in the extrachromosomal circular DNA (eccDNA) fraction as part of the whole cellular DNA. METHODS Home-made polymerase chain reaction (PCR) with whole cellular and isolated eccDNA, nucleotide basic local alignment search tool (BLASTn) alignments, and tests for patterns of methylation in selected sequence sections were performed. RESULTS The PCR tests revealed DNA sequences of up to 320 bp that broadly matched the corresponding sequence sections of known HCV genotypes. In contrast, BLASTn alignment searches of published HCV 5'-NCR sequences with human genome databases revealed only sequence segments of up to 36 bp of the 5'-NCR. The composition of these sequences shows missing base pairs, base pair mismatches as well as complete homology with HCV reference sequences. These short sequence sections are present in numerous copies on both the same and different chromosomes. The selected sequence region within the DNA sequences of the 5'-NCR revealed a broad diversity of individual patterns of methylation. CONCLUSIONS The experimental results confirm our assumption that parts of the HCV 5'-NCR genomic RNA sequences are present at the DNA level in the eccDNA fraction of PBMCs. The tests for methylation patterns therein revealed individual methylomes which could represent an epigenetic feature. The respective sequence section might be subject to genetic regulation.
Collapse
Affiliation(s)
- Reinhard H Dennin
- Formerly Department of Infectious Diseases and Microbiology, University of Lübeck, University Hospital Schleswig-Holstein, 23538 Lübeck, Germany
| | - Jian-Er Wo
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Institute of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China
| |
Collapse
|
|
11
|
Orton RJ, Wright CF, King DP, Haydon DT. Estimating viral bottleneck sizes for FMDV transmission within and between hosts and implications for the rate of viral evolution. Interface Focus 2019; 10:20190066. [PMID: 31897294 DOI: 10.1098/rsfs.2019.0066] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/30/2019] [Indexed: 01/01/2023] Open
Abstract
RNA viruses exist as populations of closely related genomes, characterized by a high diversity of low-frequency variants. As viral genomes from one population disperse to establish new sites of replication, the fate of these low-frequency variants depends to a large extent on the size of the founding population. Focusing on foot-and-mouth disease virus (FMDV) we conjecture that variants are more likely to be transmitted through wide bottlenecks, but more likely to approach fixation in new populations following narrow bottlenecks; therefore, the longer-term rate of accumulation of 'nearly neutral' variants at high frequencies is likely to be inversely related to the bottleneck size. We examine this conjecture in vivo by estimating bottleneck sizes relating 'parent' and 'daughter' populations observed at different scales ranging from within host to between host (within the same herd, and in different herds) using a previously established method. Within hosts, we find bottleneck sizes to range from 5 to 20 viral genomes between populations transmitted from the pharynx to the serum, and from 4 to 54 between serum and lesion populations. Between hosts, we find bottleneck sizes to range from 2 to 39, suggesting inter-host bottlenecks are of a similar size to intra-host bottlenecks. We establish a statistically significant negative relationship between the probability of genomic consensus level change and bottleneck size, and present a simple sampling model that captures this empirical relationship. We also present a novel in vitro experiment to investigate the impact of bottleneck size on the frequency of mutations within FMDV populations, demonstrate that variant frequency in a population increases more rapidly during small population passages, and provide evidence for positive selection during the passage of large populations.
Collapse
Affiliation(s)
- Richard J Orton
- Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.,MRC-University of Glasgow Centre for Virus Research, University of Glasgow, Sir Michael Stoker Building, 464 Bearsden Road, Glasgow G61 1QH, UK
| | | | - Donald P King
- The Pirbright Institute, Ash Road, Pirbright GU24 0NF, UK
| | - Daniel T Haydon
- Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK
| |
Collapse
|
|
12
|
Ghorbani A, Ngunjiri JM, Lee CW. Influenza A Virus Subpopulations and Their Implication in Pathogenesis and Vaccine Development. Annu Rev Anim Biosci 2019; 8:247-267. [PMID: 31479617 DOI: 10.1146/annurev-animal-021419-083756] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The concept of influenza A virus (IAV) subpopulations emerged approximately 75 years ago, when Preben von Magnus described "incomplete" virus particles that interfere with the replication of infectious virus. It is now widely accepted that infectious particles constitute only a minor portion of biologically active IAV subpopulations. The IAV quasispecies is an extremely diverse swarm of biologically and genetically heterogeneous particle subpopulations that collectively influence the evolutionary fitness of the virus. This review summarizes the current knowledge of IAV subpopulations, focusing on their biologic and genomic diversity. It also discusses the potential roles IAV subpopulations play in virus pathogenesis and live attenuated influenza vaccine development.
Collapse
Affiliation(s)
- Amir Ghorbani
- Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio 44691, USA; , , .,Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210, USA
| | - John M Ngunjiri
- Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio 44691, USA; , ,
| | - Chang-Won Lee
- Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio 44691, USA; , , .,Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210, USA
| |
Collapse
|
|
13
|
Zahid MN, Wang S, Learn GH, Abt PL, Blumberg EA, Reese PP, Goldberg DS, Shaw GM, Bar KJ. High multiplicity infection following transplantation of hepatitis C virus-positive organs. J Clin Invest 2019; 129:3134-3139. [PMID: 31112523 PMCID: PMC6668813 DOI: 10.1172/jci127203] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2019] [Accepted: 05/10/2019] [Indexed: 02/06/2023] Open
Abstract
Highly effective direct-acting antivirals against Hepatitis C virus (HCV) have created an opportunity to transplant organs from HCV-positive individuals into HCV-negative recipients, since de novo infection can be routinely cured. As this procedure is performed more widely, it becomes increasingly important to understand the biological underpinnings of virus transmission, especially the multiplicity of infection. Here, we used single genome sequencing of plasma virus in four genotype 1a HCV-positive organ donors and their seven organ recipients to assess the genetic bottleneck associated with HCV transmission following renal and cardiac transplantation. In all recipients, de novo infection was established by multiple genetically distinct viruses that reflect the full phylogenetic spectrum of replication-competent virus circulating in donor plasma. This was true in renal and cardiac transplantation and in recipients with peak viral loads ranging between 2.9 and 6.6 log10 IU/mL. The permissive transmission process characterized here contrasts sharply with sexual or injection-related transmission, which occurs less frequently per exposure and is generally associated with a stringent genetic bottleneck. These findings highlight the effectiveness of current anti-HCV regimens, while raising caution regarding the substantially higher multiplicity of infection seen in organ transplantation-associated HCV acquisition.
Collapse
|
|
14
|
Hepatitis C Virus Genetic Variability, Human Immune Response, and Genome Polymorphisms: Which Is the Interplay? Cells 2019; 8:cells8040305. [PMID: 30987134 PMCID: PMC6523096 DOI: 10.3390/cells8040305] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2019] [Revised: 03/26/2019] [Accepted: 03/30/2019] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) infection is the main cause of chronic hepatitis, affecting an estimated 150 million people worldwide. Initial exposure to HCV is most often followed by chronic hepatitis, with only a minority of individuals spontaneously clearing the virus. The induction of sustained and broadly directed HCV-specific CD4+ and CD8+ T cell responses, together with neutralizing antibodies (nAb), and specific genetic polymorphism have been associated with spontaneous resolution of the infection. However, due to its high variability, HCV is able to overwhelm the host immune response through the rapid acquisition of mutations in the epitopes targeted by T cells and neutralizing antibodies. In this context, immune-mediated pressure represents the main force in driving HCV evolution. This review summarizes the data on HCV diversity and the current state of knowledge about the contributions of antibodies, T cells, and host genetic polymorphism in driving HCV evolution in vivo.
Collapse
|
|
15
|
Kalemera M, Mincheva D, Grove J, Illingworth CJR. Building a mechanistic mathematical model of hepatitis C virus entry. PLoS Comput Biol 2019; 15:e1006905. [PMID: 30883541 PMCID: PMC6445459 DOI: 10.1371/journal.pcbi.1006905] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2018] [Revised: 04/02/2019] [Accepted: 02/28/2019] [Indexed: 12/12/2022] Open
Abstract
The mechanism by which hepatitis C virus (HCV) gains entry into cells is a complex one, involving a broad range of host proteins. Entry is a critical phase of the viral lifecycle, and a potential target for therapeutic or vaccine-mediated intervention. However, the mechanics of HCV entry remain poorly understood. Here we describe a novel computational model of viral entry, encompassing the relationship between HCV and the key host receptors CD81 and SR-B1. We conduct experiments to thoroughly quantify the influence of an increase or decrease in receptor availability upon the extent of viral entry. We use these data to build and parameterise a mathematical model, which we then validate by further experiments. Our results are consistent with sequential HCV-receptor interactions, whereby initial interaction between the HCV E2 glycoprotein and SR-B1 facilitates the accumulation CD81 receptors, leading to viral entry. However, we also demonstrate that a small minority of viruses can achieve entry in the absence of SR-B1. Our model estimates the impact of the different obstacles that viruses must surmount to achieve entry; among virus particles attaching to the cell surface, around one third of viruses accumulate sufficient CD81 receptors, of which 4-8% then complete the subsequent steps to achieve productive infection. Furthermore, we make estimates of receptor stoichiometry; in excess of 10 receptors are likely to be required to achieve viral entry. Our model provides a tool to investigate the entry characteristics of HCV variants and outlines a framework for future quantitative studies of the multi-receptor dynamics of HCV entry.
Collapse
Affiliation(s)
- Mphatso Kalemera
- Institute of Immunity and Transplantation, Division of Infection and Immunity, University College London, United Kingdom
| | - Dilyana Mincheva
- Department of Genetics, University of Cambridge, Cambridge, United Kingdom
| | - Joe Grove
- Institute of Immunity and Transplantation, Division of Infection and Immunity, University College London, United Kingdom
| | | |
Collapse
|
|
16
|
Huang SW, Hung SJ, Wang JR. Application of deep sequencing methods for inferring viral population diversity. J Virol Methods 2019; 266:95-102. [PMID: 30690049 DOI: 10.1016/j.jviromet.2019.01.013] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2018] [Revised: 01/11/2019] [Accepted: 01/24/2019] [Indexed: 12/13/2022]
Abstract
The first deep sequencing method was announced in 2005. Due to an increasing number of sequencing data and a reduction in the costs of each sequencing dataset, this innovative technique was soon applied to genetic investigations of viral genome diversity in various viruses, particularly RNA viruses. These deep sequencing findings documented viral epidemiology and evolution and provided high-resolution data on the genetic changes in viral populations. Here, we review deep sequencing platforms that have been applied in viral quasispecies studies. Further, we discuss recent deep sequencing studies on viral inter- and intrahost evolution, drug resistance, and humoral immune selection, especially in emerging and re-emerging viruses. Deep sequencing methods are becoming the standard for providing comprehensive results of viral population diversity, and their applications are discussed.
Collapse
Affiliation(s)
- Sheng-Wen Huang
- National Mosquito-Borne Diseases Control Research Center, National Health Research Institutes, Tainan, Taiwan
| | - Su-Jhen Hung
- Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, Tainan, Taiwan
| | - Jen-Ren Wang
- Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, Tainan, Taiwan; Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan; Department of Pathology, National Cheng Kung University Hospital, Tainan, Taiwan; National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Tainan, Taiwan.
| |
Collapse
|
|
17
|
Molecular Identification of Transmitted/Founder Hepatitis C Viruses and Their Progeny by Single Genome Sequencing. Methods Mol Biol 2019; 1911:139-155. [PMID: 30593623 DOI: 10.1007/978-1-4939-8976-8_9] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Chronic hepatitis C virus (HCV) infection exists as a complex mixture of genetically distinct viruses, commonly referred to as a "quasispecies." Quasispecies complexity can vary substantially during the course of natural infection as a consequence of viral population "bottlenecking." This occurs at the time of transmission from one individual to the next and during the course of chronic infection of an individual when adaptive immune responses eliminate certain viruses but allow others to escape and expand. Antiviral treatment with drugs that fail to eradicate virus can also lead to virus population bottlenecks and emergence of drug-resistant variants. Single genome sequencing (SGS) combined with mathematical modeling and phylogenetic inference is a recently described approach for characterizing the HCV quasispecies in unprecedented detail, allowing for the first time the retention of genetic linkage across genes and near full-length genomes and precise identification of transmitted/founder (T/F) genomes. Here, we describe the methodological approach to SGS and show how this strategy allows for the precise and unambiguous molecular identification of transmitted viruses as well as those that repopulate the body after drug or immune-mediated selective sweeps. This is an enabling experimental strategy that allows for a precise genetic, biologic, and antigenic characterization of HCV viruses that are responsible for transmission and persistence. Such an approach can be particularly valuable to future HCV vaccine design efforts, as it has been for human immunodeficiency virus type 1 (HIV-1).
Collapse
|
|
18
|
Full-Length Envelope Analyzer (FLEA): A tool for longitudinal analysis of viral amplicons. PLoS Comput Biol 2018; 14:e1006498. [PMID: 30543621 PMCID: PMC6314628 DOI: 10.1371/journal.pcbi.1006498] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2018] [Revised: 01/02/2019] [Accepted: 09/10/2018] [Indexed: 01/07/2023] Open
Abstract
Next generation sequencing of viral populations has advanced our understanding of viral population dynamics, the development of drug resistance, and escape from host immune responses. Many applications require complete gene sequences, which can be impossible to reconstruct from short reads. HIV env, the protein of interest for HIV vaccine studies, is exceptionally challenging for long-read sequencing and analysis due to its length, high substitution rate, and extensive indel variation. While long-read sequencing is attractive in this setting, the analysis of such data is not well handled by existing methods. To address this, we introduce FLEA (Full-Length Envelope Analyzer), which performs end-to-end analysis and visualization of long-read sequencing data. FLEA consists of both a pipeline (optionally run on a high-performance cluster), and a client-side web application that provides interactive results. The pipeline transforms FASTQ reads into high-quality consensus sequences (HQCSs) and uses them to build a codon-aware multiple sequence alignment. The resulting alignment is then used to infer phylogenies, selection pressure, and evolutionary dynamics. The web application provides publication-quality plots and interactive visualizations, including an annotated viral alignment browser, time series plots of evolutionary dynamics, visualizations of gene-wide selective pressures (such as dN/dS) across time and across protein structure, and a phylogenetic tree browser. We demonstrate how FLEA may be used to process Pacific Biosciences HIV env data and describe recent examples of its use. Simulations show how FLEA dramatically reduces the error rate of this sequencing platform, providing an accurate portrait of complex and variable HIV env populations. A public instance of FLEA is hosted at http://flea.datamonkey.org. The Python source code for the FLEA pipeline can be found at https://github.com/veg/flea-pipeline. The client-side application is available at https://github.com/veg/flea-web-app. A live demo of the P018 results can be found at http://flea.murrell.group/view/P018. Viral populations constantly evolve and diversify. In this article we introduce a method, FLEA, for reconstructing and visualizing the details of evolutionary changes. FLEA specifically processes data from sequencing platforms that generate reads that are long, but error-prone. To study the evolutionary dynamics of entire genes during viral infection, data is collected via long-read sequencing at discrete time points, allowing us to understand how the virus changes over time. However, the experimental and sequencing process is imperfect, so the resulting data contain not only real evolutionary changes, but also mutations and other genetic artifacts caused by sequencing errors. Our method corrects most of these errors by combining thousands of erroneous sequences into a much smaller number of unique consensus sequences that represent biologically meaningful variation. The resulting high-quality sequences are used for further analysis, such as building an evolutionary tree that tracks and interprets the genetic changes in the viral population over time. FLEA is open source, and is freely available online.
Collapse
|
|
19
|
Boddicker AM, Mosier AC. Genomic profiling of four cultivated Candidatus Nitrotoga spp. predicts broad metabolic potential and environmental distribution. THE ISME JOURNAL 2018; 12:2864-2882. [PMID: 30050164 PMCID: PMC6246548 DOI: 10.1038/s41396-018-0240-8] [Citation(s) in RCA: 36] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/05/2018] [Revised: 06/28/2018] [Accepted: 07/03/2018] [Indexed: 12/21/2022]
Abstract
Nitrite-oxidizing bacteria (NOB) play a critical role in the mitigation of nitrogen pollution by metabolizing nitrite to nitrate, which is removed via assimilation, denitrification, or anammox. Recent studies showed that NOB are phylogenetically and metabolically diverse, yet most of our knowledge of NOB comes from only a few cultured representatives. Using cultivation and genomic sequencing, we identified four putative Candidatus Nitrotoga NOB species from freshwater sediments and water column samples in Colorado, USA. Genome analyses indicated highly conserved 16S rRNA gene sequences, but broad metabolic potential including genes for nitrogen, sulfur, hydrogen, and organic carbon metabolism. Genomic predictions suggested that Ca. Nitrotoga can metabolize in low oxygen or anoxic conditions, which may support an expanded environmental niche for Ca. Nitrotoga similar to other NOB. An array of antibiotic and metal resistance genes likely allows Ca. Nitrotoga to withstand environmental pressures in impacted systems. Phylogenetic analyses highlighted a deeply divergent nitrite oxidoreductase alpha subunit (NxrA), suggesting a novel evolutionary trajectory for Ca. Nitrotoga separate from any other NOB and further revealing the complex evolutionary history of nitrite oxidation in the bacterial domain. Ca. Nitrotoga-like 16S rRNA gene sequences were prevalent in globally distributed environments over a range of reported temperatures. This work considerably expands our knowledge of the Ca. Nitrotoga genus and suggests that their contribution to nitrogen cycling should be considered alongside other NOB in wide variety of habitats.
Collapse
Affiliation(s)
- Andrew M Boddicker
- Department of Integrative Biology, University of Colorado Denver, Campus Box 171, Denver, CO, USA
| | - Annika C Mosier
- Department of Integrative Biology, University of Colorado Denver, Campus Box 171, Denver, CO, USA.
| |
Collapse
|
|
20
|
Using high-throughput sequencing for investigating intra-host hepatitis C evolution over long retrospective periods. INFECTION GENETICS AND EVOLUTION 2018; 67:136-144. [PMID: 30395998 DOI: 10.1016/j.meegid.2018.11.004] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/26/2018] [Revised: 09/11/2018] [Accepted: 11/02/2018] [Indexed: 12/13/2022]
Abstract
Collections of biological samples held by hospitals represent invaluable resources for conducting retrospective evolutionary studies of chronic infections. Using high-throughput sequencing, those collections permit analysis of within-host genetic diversity over long follow-up periods, and allow a better understanding of resistance to treatment regimes during disease evolution. Here, we studied the evolution of hepatitis C virus (HCV) populations in two patients with an absence of response to dual therapies. We implemented amplicon sequencing to survey genomic variation at the Core and NS5B regions of HCV over a period of 13 years from blood samples obtained at multiple time points. We observed mixed infection by multiple HCV genotypes in both patients. Genetic heterogeneity and sample composition analysis provided information about the changes in viral population over the course of clinical treatment, with NS5B experiencing an increase in diversity after treatment initiation. Secondary infections were estimated to predate treatment year, and our results pointed towards diversifying selection occurring post-treatment, acting on standing genomic variation and maintaining high genetic heterogeneity during infection. For these two patients infected with multiple HCV genotypes, the maintenance of viral diversity was explained with the hypothesis of soft selective sweep started at the same time as antiviral treatment was initiated.
Collapse
|
|
21
|
Parreira R. Laboratory Methods in Molecular Epidemiology: Viral Infections. Microbiol Spectr 2018; 6:10.1128/microbiolspec.ame-0003-2018. [PMID: 30387412 PMCID: PMC11633636 DOI: 10.1128/microbiolspec.ame-0003-2018] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2018] [Indexed: 01/05/2023] Open
Abstract
Viruses, which are the most abundant biological entities on the planet, have been regarded as the "dark matter" of biology in the sense that despite their ubiquity and frequent presence in large numbers, their detection and analysis are not always straightforward. The majority of them are very small (falling under the limit of 0.5 μm), and collectively, they are extraordinarily diverse. In fact, the majority of the genetic diversity on the planet is found in the so-called virosphere, or the world of viruses. Furthermore, the most frequent viral agents of disease in humans display an RNA genome, and frequently evolve very fast, due to the fact that most of their polymerases are devoid of proofreading activity. Therefore, their detection, genetic characterization, and epidemiological surveillance are rather challenging. This review (part of the Curated Collection on Advances in Molecular Epidemiology of Infectious Diseases) describes many of the methods that, throughout the last few decades, have been used for viral detection and analysis. Despite the challenge of having to deal with high genetic diversity, the majority of these methods still depend on the amplification of viral genomic sequences, using sequence-specific or sequence-independent approaches, exploring thermal profiles or a single nucleic acid amplification temperature. Furthermore, viral populations, and especially those with RNA genomes, are not usually genetically uniform but encompass swarms of genetically related, though distinct, viral genomes known as viral quasispecies. Therefore, sequence analysis of viral amplicons needs to take this fact into consideration, as it constitutes a potential analytic problem. Possible technical approaches to deal with it are also described here. *This article is part of a curated collection.
Collapse
Affiliation(s)
- Ricardo Parreira
- Unidade de Microbiologia Médica/Global Health and Tropical Medicine (GHTM) Research Centre, Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), 1349-008 Lisboa, Portugal
| |
Collapse
|
|
22
|
Next-generation sequencing analysis of a cluster of hepatitis C virus infections in a haematology and oncology center. PLoS One 2018; 13:e0194816. [PMID: 29566084 PMCID: PMC5864040 DOI: 10.1371/journal.pone.0194816] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2017] [Accepted: 03/10/2018] [Indexed: 01/17/2023] Open
Abstract
Molecular characterization of early hepatitis C virus (HCV) infection remains rare. Ten out of 78 patients of a hematology/oncology center were found to be HCV RNA positive two to four months after hospitalization. Only two of the ten patients were anti-HCV positive. HCV hypervariable region 1 (HVR1) was amplified in seven patients (including one anti-HCV positive) and analyzed by next generation sequencing (NGS). Genetic variants were reconstructed by Shorah and an empirically established 0.5% variant frequency cut-off was implemented. These sequences were compared by phylogenetic and diversity analyses. Ten unrelated blood donors with newly acquired HCV infection detected at the time of donation (HCV RNA positive and anti-HCV negative) served as controls. One to seven HVR1 variants were found in each patient. Sequences intermixed phylogenetically with no evidence of clustering in individual patients. These sequences were more similar to each other (similarity 95.4% to 100.0%) than to those of controls (similarity 64.8% to 82.6%). An identical predominant variant was present in four patients, whereas other closely related variants dominated in the remaining three patients. In five patients the HCV population was limited to a single variant or one predominant variant and minor variants of less than 10% frequency. In conclusion, NGS analysis of a cluster of HCV infections acquired in the hospital setting revealed the presence of low diversity, very closely related variants in all patients, suggesting an early-stage infection with the same virus. NGS combined with phylogenetic analysis and classical epidemiological analysis could help in tracking of HCV outbreaks.
Collapse
|
|
23
|
Patterson EI, Khanipov K, Rojas MM, Kautz TF, Rockx-Brouwer D, Golovko G, Albayrak L, Fofanov Y, Forrester NL. Mosquito bottlenecks alter viral mutant swarm in a tissue and time-dependent manner with contraction and expansion of variant positions and diversity. Virus Evol 2018; 4:vey001. [PMID: 29479479 PMCID: PMC5814806 DOI: 10.1093/ve/vey001] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Viral diversity is theorized to play a significant role during virus infections, particularly for arthropod-borne viruses (arboviruses) that must infect both vertebrate and invertebrate hosts. To determine how viral diversity influences mosquito infection and dissemination Culex taeniopus mosquitoes were infected with the Venezuelan equine encephalitis virus endemic strain 68U201. Bodies and legs/wings of the mosquitoes were collected individually and subjected to multi-parallel sequencing. Virus sequence diversity was calculated for each tissue. Greater diversity was seen in mosquitoes with successful dissemination versus those with no dissemination. Diversity across time revealed that bottlenecks influence diversity following dissemination to the legs/wings, but levels of diversity are restored by Day 12 post-dissemination. Specific minority variants were repeatedly identified across the mosquito cohort, some in nearly every tissue and time point, suggesting that certain variants are important in mosquito infection and dissemination. This study demonstrates that the interaction between the mosquito and the virus results in changes in diversity and the mutational spectrum and may be essential for successful transition of the bottlenecks associated with arbovirus infection.
Collapse
Affiliation(s)
- Edward I Patterson
- Department of Pathology, Institute for Human Infections and Immunity, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0610, USA
| | - Kamil Khanipov
- Department of Pharmacology and Toxicology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555-0617, USA
| | - Mark M Rojas
- Department of Pharmacology and Toxicology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555-0617, USA
| | - Tiffany F Kautz
- Department of Pathology, Institute for Human Infections and Immunity, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0610, USA
| | - Dedeke Rockx-Brouwer
- Department of Pathology, Institute for Human Infections and Immunity, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0610, USA
| | - Georgiy Golovko
- Department of Pharmacology and Toxicology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555-0617, USA
| | - Levent Albayrak
- Department of Pharmacology and Toxicology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555-0617, USA
| | - Yuriy Fofanov
- Department of Pharmacology and Toxicology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555-0617, USA
| | - Naomi L Forrester
- Department of Pathology, Institute for Human Infections and Immunity, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0610, USA
| |
Collapse
|
|
24
|
Li F, Ma L, Feng Y, Ruan Y, Hu J, Song H, Liu P, Ma J, Rui B, Kerpen K, Scheinfeld B, Srivastava T, Metzger D, Li H, Bar KJ, Shao Y. HIV-1 and hepatitis C virus selection bottleneck in Chinese people who inject drugs. AIDS 2018; 32:309-320. [PMID: 29194114 PMCID: PMC5765877 DOI: 10.1097/qad.0000000000001702] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
OBJECTIVES For both HIV-1 and hepatitis C virus (HCV), assessing the stringency of the transmission process is a scientific priority. Enumerations of transmitted/founder (TF) viruses have shown a strict transmission bottleneck in sexual transmission of HIV-1 and a wide range in the multiplicity of infection in HCV. Here, we aim to determine the stringency of parenteral transmission for HIV-1 and HCV in people who inject drugs (PWID). DESIGN We used molecular sequencing and several complementary analyses to enumerate the TF HIV-1 and HCV variants in a well described cohort of PWID in Xinjiang, China. METHODS We performed single genome sequencing of HIV-1 env and 5' half HCV genomes, then applied phylogenetic analysis and validated models of early virus diversification to enumerate TF viruses in 60 PWID. We used multivariate analysis to determine correlates of multivariant transmission (MVT). RESULTS We generated 1070 env region sequences from 33 HIV-1 early infected individuals and 773 5' half region sequences from 27 HCV early infected individuals. We found rates of MVT of 39 and 54%, respectively, for HIV-1 and HCV, with a limited range in the number of TF viruses in both infections. Behavioural characteristics suggested high-risk injection practices and lower risk sexual practices; we did not find an association between any specific behaviours and MVT. CONCLUSION MVT is frequent in parenteral transmission of both HIV-1 and HCV in Xinjiang PWID, indicating a less stringent transmission process than sexual transmission.
Collapse
Affiliation(s)
- Fan Li
- State Key Laboratory for Infectious Disease Prevention and Control, National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Beijing
| | - Liying Ma
- State Key Laboratory for Infectious Disease Prevention and Control, National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Beijing
| | - Yi Feng
- State Key Laboratory for Infectious Disease Prevention and Control, National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Beijing
| | - Yuhua Ruan
- State Key Laboratory for Infectious Disease Prevention and Control, National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Beijing
| | - Jing Hu
- State Key Laboratory for Infectious Disease Prevention and Control, National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Beijing
| | - Hongshuo Song
- State Key Laboratory for Infectious Disease Prevention and Control, National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Beijing
| | - Pengtao Liu
- State Key Laboratory for Infectious Disease Prevention and Control, National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Beijing
| | - Jun Ma
- Xinjiang Center for Disease Control and Prevention, Urumqi, China
| | - Baolin Rui
- Urumqi Center for Disease Control and Prevention, Urumqi, China
| | - Kate Kerpen
- Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Benjamin Scheinfeld
- Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Tuhina Srivastava
- Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - David Metzger
- Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
- The Treatment Research Institute, Philadelphia PA 19104
| | - Hui Li
- Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Katharine J. Bar
- Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Yiming Shao
- State Key Laboratory for Infectious Disease Prevention and Control, National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Beijing
- Center of Infectious Diseases, Peking University, Beijing, China
- Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China
| |
Collapse
|
|
25
|
Campo DS, Zhang J, Ramachandran S, Khudyakov Y. Transmissibility of intra-host hepatitis C virus variants. BMC Genomics 2017; 18:881. [PMID: 29244001 PMCID: PMC5731494 DOI: 10.1186/s12864-017-4267-4] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Background Intra-host hepatitis C virus (HCV) populations are genetically heterogeneous and organized in subpopulations. With the exception of blood transfusions, transmission of HCV occurs via a small number of genetic variants, the effect of which is frequently described as a bottleneck. Stochasticity of transmission associated with the bottleneck is usually used to explain genetic differences among HCV populations identified in the source and recipient cases, which may be further exacerbated by intra-host HCV evolution and differential biological capacity of HCV variants to successfully establish a population in a new host. Results Transmissibility was formulated as a property that can be measured from experimental Ultra-Deep Sequencing (UDS) data. The UDS data were obtained from one large hepatitis C outbreak involving an epidemiologically defined source and 18 recipient cases. k-Step networks of HCV variants were constructed and used to identify a potential association between transmissibility and network centrality of individual HCV variants from the source. An additional dataset obtained from nine other HCV outbreaks with known directionality of transmission was used for validation. Transmissibility was not found to be dependent on high frequency of variants in the source, supporting the earlier observations of transmission of minority variants. Among all tested measures of centrality, the highest correlation of transmissibility was found with Hamming centrality (r = 0.720; p = 1.57 E-71). Correlation between genetic distances and differences in transmissibility among HCV variants from the source was found to be 0.3276 (Mantel Test, p = 9.99 E-5), indicating association between genetic proximity and transmissibility. A strong correlation ranging from 0.565–0.947 was observed between Hamming centrality and transmissibility in 7 of the 9 additional transmission clusters (p < 0.05). Conclusions Transmission is not an exclusively stochastic process. Transmissibility, as formally measured in this study, is associated with certain biological properties that also define location of variants in the genetic space occupied by the HCV strain from the source. The measure may also be applicable to other highly heterogeneous viruses. Besides improving accuracy of outbreak investigations, this finding helps with the understanding of molecular mechanisms contributing to establishment of chronic HCV infection.
Collapse
Affiliation(s)
- David S Campo
- Division of Viral Hepatitis, Molecular Epidemiology and Bioinformatics, Centers for Disease Control and Prevention, Atlanta, GA, USA.
| | - June Zhang
- Division of Viral Hepatitis, Molecular Epidemiology and Bioinformatics, Centers for Disease Control and Prevention, Atlanta, GA, USA.,Department of Electrical Engineering, University of Hawaii, Manoa, HI, USA
| | - Sumathi Ramachandran
- Division of Viral Hepatitis, Molecular Epidemiology and Bioinformatics, Centers for Disease Control and Prevention, Atlanta, GA, USA
| | - Yury Khudyakov
- Division of Viral Hepatitis, Molecular Epidemiology and Bioinformatics, Centers for Disease Control and Prevention, Atlanta, GA, USA
| |
Collapse
|
|
26
|
Vertical Transmission of Hepatitis C Virus: Variable Transmission Bottleneck and Evidence of Midgestation In Utero Infection. J Virol 2017; 91:JVI.01372-17. [PMID: 28931691 DOI: 10.1128/jvi.01372-17] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2017] [Accepted: 09/15/2017] [Indexed: 12/14/2022] Open
Abstract
Hepatitis C virus (HCV) can be transmitted from mother to child during pregnancy and childbirth. However, the timing and precise biological mechanisms that are involved in this process are incompletely understood, as are the determinants that influence transmission of particular HCV variants. Here we report results of a longitudinal assessment of HCV quasispecies diversity and composition in 5 cases of vertical HCV transmission, including 3 women coinfected with human immunodeficiency virus type 1 (HIV-1). The population structure of HCV variant spectra based on E2 envelope gene sequences (nucleotide positions 1491 to 1787), including hypervariable regions 1 and 2, was characterized using next-generation sequencing and median-joining network analysis. Compatible with a loose transmission bottleneck, larger numbers of shared HCV variants were observed in the presence of maternal coinfection. Coalescent Bayesian Markov chain Monte Carlo simulations revealed median times of transmission between 24.9 weeks and 36.1 weeks of gestation, with some confidence intervals ranging into the 1st trimester, considerably earlier than previously thought. Using recombinant autologous HCV pseudoparticles, differences were uncovered in HCV-specific antibody responses between coinfected mothers and mothers infected with HCV alone, in whom generalized absence of neutralization was observed. Finally, shifts in HCV quasispecies composition were seen in children around 1 year of age, compatible with the disappearance of passively transferred maternal immunoglobulins and/or the development of HCV-specific humoral immunity. Taken together, these results provide insights into the timing, dynamics, and biologic mechanisms involved in vertical HCV transmission and inform preventative strategies.IMPORTANCE Although it is well established that hepatitis C virus (HCV) can be transmitted from mother to child, the manner and the moment at which transmission operates have been the subject of conjecture. By carrying out a detailed examination of viral sequences, we showed that transmission could take place comparatively early in pregnancy. In addition, we showed that when the mother also carried human immunodeficiency virus type 1 (HIV-1), many more HCV variants were shared between her and her child, suggesting that the mechanism and/or the route of transmission of HCV differed in the presence of coinfection with HIV-1. These results could explain why cesarean section is ineffective in preventing vertical HCV transmission and guide the development of interventions to avert pediatric HCV infection.
Collapse
|
|
27
|
Pandey U, Renner DW, Thompson RL, Szpara ML, Sawtell NM. Inferred father-to-son transmission of herpes simplex virus results in near-perfect preservation of viral genome identity and in vivo phenotypes. Sci Rep 2017; 7:13666. [PMID: 29057909 PMCID: PMC5654476 DOI: 10.1038/s41598-017-13936-6] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2017] [Accepted: 10/02/2017] [Indexed: 12/23/2022] Open
Abstract
High throughout sequencing has provided an unprecedented view of the circulating diversity of all classes of human herpesviruses. For herpes simplex virus 1 (HSV-1), we and others have previously published data demonstrating sequence diversity between hosts. However the extent of variation during transmission events, or in one host over years of chronic infection, remain unknown. Here we present an initial example of full characterization of viruses isolated from a father to son transmission event. The likely occasion of transmission occurred 17 years before the strains were isolated, enabling a first view of the degree of virus conservation after decades of recurrences, including transmission and adaptation to a new host. We have characterized the pathogenicity of these strains in a mouse ocular model of infection, and sequenced the full viral genomes. Surprisingly, we find that these two viruses have preserved their phenotype and genotype nearly perfectly during inferred transmission from father to son, and during nearly two decades of episodes of recurrent disease in each human host. Given the close genetic relationship of these two hosts, it remains to be seen whether or not this conservation of sequence will occur during non-familial transmission events.
Collapse
Affiliation(s)
- Utsav Pandey
- Department of Biochemistry and Molecular Biology, Center for Infectious Disease Dynamics, and the Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania, 16802, USA
| | - Daniel W Renner
- Department of Biochemistry and Molecular Biology, Center for Infectious Disease Dynamics, and the Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania, 16802, USA
| | - Richard L Thompson
- Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio, 45229, USA
| | - Moriah L Szpara
- Department of Biochemistry and Molecular Biology, Center for Infectious Disease Dynamics, and the Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania, 16802, USA.
| | - Nancy M Sawtell
- Division of Infectious Diseases, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, 45229, USA
| |
Collapse
|
|
28
|
Quantifying perinatal transmission of Hepatitis B viral quasispecies by tag linkage deep sequencing. Sci Rep 2017; 7:10168. [PMID: 28860476 PMCID: PMC5578979 DOI: 10.1038/s41598-017-10591-9] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2017] [Accepted: 08/11/2017] [Indexed: 02/07/2023] Open
Abstract
Despite full immunoprophylaxis, mother-to-child transmission (MTCT) of Hepatitis B Virus still occurs in approximately 2–5% of HBsAg positive mothers. Little is known about the bottleneck of HBV transmission and the evolution of viral quasispecies in the context of MTCT. Here we adopted a newly developed tag linkage deep sequencing method and analyzed the quasispecies of four MTCT pairs that broke through immunoprophylaxis. By assigning unique tags to individual viral sequences, we accurately reconstructed HBV haplotypes in a region of 836 bp, which contains the major immune epitopes and drug resistance mutations. The detection limit of minor viral haplotypes reached 0.1% for individual patient sample. Dominance of “a determinant” polymorphisms were observed in two children, which pre-existed as minor quasispecies in maternal samples. In all four pairs of MTCT samples, we consistently observed a significant overlap of viral haplotypes shared between mother and child. We also demonstrate that the data can be potentially useful to estimate the bottleneck effect during HBV MTCT, which provides information to optimize treatment for reducing the frequency of MTCT.
Collapse
|
|
29
|
The Number of Target Molecules of the Amplification Step Limits Accuracy and Sensitivity in Ultradeep-Sequencing Viral Population Studies. J Virol 2017; 91:JVI.00561-17. [PMID: 28566384 DOI: 10.1128/jvi.00561-17] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2017] [Accepted: 05/25/2017] [Indexed: 11/20/2022] Open
Abstract
The invention of next-generation sequencing (NGS) techniques marked the coming of a new era in the detection of the genetic diversity of intrahost viral populations. A good understanding of the genetic structure of these populations requires, first, the ability to identify the different isolates or variants and, second, the ability to accurately quantify them. However, the initial amplification step of NGS studies can impose potential quantitative biases, modifying the variant relative frequencies. In particular, the number of target molecules (NTM) used during the amplification step is vastly overlooked although of primary importance, as it sets the limit of the accuracy and sensitivity of the sequencing procedure. In the present article, we investigated quantitative biases in an NGS study of populations of a multipartite single-stranded DNA (ssDNA) virus at different steps of the procedure. We studied 20 independent populations of the ssDNA virus faba bean necrotic stunt virus (FBNSV) in two host plants, Vicia faba and Medicago truncatula FBNSV is a multipartite virus composed of eight genomic segments, whose specific and host-dependent relative frequencies are defined as the "genome formula." Our results show a significant distortion of the FBNSV genome formula after the amplification and sequencing steps. We also quantified the genetic bottleneck occurring at the amplification step by documenting the NTM of two genomic segments of FBNSV. We argue that the NTM must be documented and carefully considered when determining the sensitivity and accuracy of data from NGS studies.IMPORTANCE The advent of next-generation sequencing (NGS) techniques now enables study of the genetic diversity of viral populations. A good understanding of the genetic structure of these populations first requires the ability to identify the different isolates or variants and second requires the ability to accurately quantify them. Prior to sequencing, viral genomes need to be amplified, a step that potentially imposes quantitative biases and modifies the viral population structure. In particular, the number of target molecules (NTM) used during the amplification step is of primary importance, as it sets the limit of the accuracy and sensitivity of the sequencing procedure. In this work, we used 20 replicated populations of the multipartite faba bean necrotic stunt virus (FBNSV) to estimate the various limitations of ultradeep-sequencing studies performed on intrahost viral populations. We report quantitative biases during rolling-circle amplification and the NTM of two genomic segments of FBNSV.
Collapse
|
|
30
|
Shao X, Levin B, Nemenman I. Single variant bottleneck in the early dynamics ofH. influenzaebacteremia in neonatal rats questions the theory of independent action. Phys Biol 2017; 14:045004. [DOI: 10.1088/1478-3975/aa731b] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
|
|
31
|
Merani S, Lucas M, Deshpande P, Pfafferott K, Chopra A, Cooper D, Leary S, Luciani F, Gaudieri S. Influence of Transmitted Virus on the Host's Immune Response: A Case Study. Viral Immunol 2017; 30:533-541. [PMID: 28530508 DOI: 10.1089/vim.2017.0001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Host hepatitis C virus (HCV)-specific T cell responses and the ability of the virus to escape this response are important correlates of infection outcome. Understanding this host-viral interplay has been difficult given the often asymptomatic nature of acute HCV infection. We studied a recent transmission case to determine whether adapted viral strains can be transmitted and influence the recipient's anti-HCV T cell response. The diversity of viral populations was examined using next-generation sequencing, and HCV-specific T cell interferon (IFN)-γ responses were assessed using a peptide panel representing the autologous viruses. HCV-specific T cell responses in the source were directed against peptides that did not match the dominant autologous virus but rather low-frequency variants, implying existing viral adaptation in the source strain. Most HCV T cell epitopes that elicited an IFN-γ response in the source did not in the recipient, despite the pair sharing human leukocyte antigen alleles that govern antigen presentation and similar autologous viruses. Intrahost HCV variation in the recipient fell within predicted T cell epitopes, suggesting alternative targets of the immune response. These data suggest that transmission of adapted viral species can direct the host's HCV-specific immune response profile during acute infection.
Collapse
Affiliation(s)
- Shahzma Merani
- 1 School of Human Sciences, University of Western Australia , Crawley, Australia
| | - Michaela Lucas
- 2 Institute of Immunology and Infectious Diseases, Murdoch University , Murdoch, Australia .,3 School of Medicine and Pharmacology, Harry Perkins Institute, University of Western Australia , Crawley, Australia .,4 School of Pathology and Laboratory Medicine, University of Western Australia , Crawley, Australia
| | - Pooja Deshpande
- 1 School of Human Sciences, University of Western Australia , Crawley, Australia
| | - Katja Pfafferott
- 2 Institute of Immunology and Infectious Diseases, Murdoch University , Murdoch, Australia
| | - Abha Chopra
- 2 Institute of Immunology and Infectious Diseases, Murdoch University , Murdoch, Australia
| | - Don Cooper
- 2 Institute of Immunology and Infectious Diseases, Murdoch University , Murdoch, Australia
| | - Shay Leary
- 2 Institute of Immunology and Infectious Diseases, Murdoch University , Murdoch, Australia
| | - Fabio Luciani
- 5 Systems Immunology, School of Medical Sciences, University of New South Wales , Sydney, Australia
| | - Silvana Gaudieri
- 1 School of Human Sciences, University of Western Australia , Crawley, Australia .,2 Institute of Immunology and Infectious Diseases, Murdoch University , Murdoch, Australia .,6 Division of Infectious Disease, Department of Medicine, Vanderbilt University Medical Centre , Nashville, Tennessee
| |
Collapse
|
|
32
|
Caro-Pérez N, Martínez-Rebollar M, Gregori J, Quer J, González P, Gambato M, García-González N, González-Candelas F, Costa J, Esteban JI, Mallolas J, Forns X, Laguno M, Pérez-Del-Pulgar S. Phylogenetic analysis of an epidemic outbreak of acute hepatitis C in HIV-infected patients by ultra-deep pyrosequencing. J Clin Virol 2017; 92:42-47. [PMID: 28521213 DOI: 10.1016/j.jcv.2017.05.008] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2017] [Revised: 04/29/2017] [Accepted: 05/06/2017] [Indexed: 01/18/2023]
Abstract
BACKGROUND The incidence of acute hepatitis C (AHC) among HIV-infected men who have sex with men (MSM) has increased significantly in the last 10 years. Several studies point to a social and sexual network of HIV-positive MSM that extends internationally. OBJECTIVES The aim of our study was to investigate the dynamics of HCV transmission in an outbreak of AHC in HIV-infected MSM in Barcelona by ultra-deep pyrosequencing. STUDY DESIGN Between 2008 and 2013, 113 cases of AHC in HIV-infected MSM were diagnosed in the Infectious Diseases Unit, Hospital Clínic, Barcelona. Massive sequencing was performed using the Roche 454 GS Junior platform. To define possible transmission networks, maximum likelihood phylogenetic trees were constructed, and levels of genetic diversity within and among patients were compared. RESULTS Among the 70 cases analyzed, we have identified 16 potential clusters of transmission: 8 for genotype 1a (23 cases involved), 1 for genotype 1b (3 cases) and 7 for genotype 4d (27 cases). Although the initial phylogenetic reconstruction suggested a local transmission cluster of HCV gt4d, our approach based on low genetic differentiation did not corroborate it. Indeed, gt4d strains formed 4 independent groups related to patients from other countries. CONCLUSIONS Frequent clustering of HIV-positive MSM shows that HCV infection has spread through a local network in Barcelona. This outbreak is related to a large international HCV transmission network among MSM. Public health efforts are needed to reduce HCV transmission among this high-risk group.
Collapse
Affiliation(s)
- Noelia Caro-Pérez
- Liver Unit, Hospital Clínic, IDIBAPS, CIBERehd, Universitat de Barcelona, Barcelona, Spain.
| | | | - Josep Gregori
- Liver Unit, Vall d'Hebron Institut de Recerca-Hospital Universitari Vall d'Hebron, CIBERehd, Barcelona, Spain; Roche Diagnostics SL, Sant Cugat del Vallès, Barcelona, Spain.
| | - Josep Quer
- Liver Unit, Vall d'Hebron Institut de Recerca-Hospital Universitari Vall d'Hebron, CIBERehd, Barcelona, Spain.
| | - Patricia González
- Liver Unit, Hospital Clínic, IDIBAPS, CIBERehd, Universitat de Barcelona, Barcelona, Spain.
| | - Martina Gambato
- Liver Unit, Hospital Clínic, IDIBAPS, CIBERehd, Universitat de Barcelona, Barcelona, Spain.
| | - Neris García-González
- Joint Research Unit Infección y Salud Pública, FISABIO-Universitat de València, I2SysBio, CIBERESP, Valencia, Spain.
| | - Fernando González-Candelas
- Joint Research Unit Infección y Salud Pública, FISABIO-Universitat de València, I2SysBio, CIBERESP, Valencia, Spain.
| | - Josep Costa
- Microbiology Department, Centre Diagnòstic Biomèdic, Hospital Clínic, CIBERehd, Barcelona, Spain.
| | - Juan Ignacio Esteban
- Liver Unit, Vall d'Hebron Institut de Recerca-Hospital Universitari Vall d'Hebron, CIBERehd, Barcelona, Spain.
| | - Josep Mallolas
- Infectious Diseases Unit, Hospital Clínic, IDIBAPS, Barcelona, Spain.
| | - Xavier Forns
- Liver Unit, Hospital Clínic, IDIBAPS, CIBERehd, Universitat de Barcelona, Barcelona, Spain.
| | - Montse Laguno
- Infectious Diseases Unit, Hospital Clínic, IDIBAPS, Barcelona, Spain.
| | - Sofía Pérez-Del-Pulgar
- Liver Unit, Hospital Clínic, IDIBAPS, CIBERehd, Universitat de Barcelona, Barcelona, Spain.
| |
Collapse
|
|
33
|
Poirier EZ, Vignuzzi M. Virus population dynamics during infection. Curr Opin Virol 2017; 23:82-87. [PMID: 28456056 DOI: 10.1016/j.coviro.2017.03.013] [Citation(s) in RCA: 51] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2016] [Revised: 03/23/2017] [Accepted: 03/24/2017] [Indexed: 12/14/2022]
Abstract
During RNA virus infection of a host, error-prone viral replication will give rise to a cloud of genetically-linked mutants, as well as truncated, defective genomes. In this review, we describe the dynamics of viral diversity during infection, illustrating that the viral population fluctuates greatly in number of genomes and composition of mutants, in relation with the existence of physical barriers or immune pressures. We illustrate the importance of generating diversity by analyzing the case of fidelity variants, largely attenuated in vivo. Recombination is also considered in its various roles: redistribution of mutations on full-length genomes, and production of highly-immunostimulatory defective genomes. We cover these notions by underlining, when they exist, the differences between acute and persistent infections.
Collapse
Affiliation(s)
- Enzo Z Poirier
- Institut Pasteur, Centre National de la Recherche Scientifique, UMR 3569, Paris, France; University of Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France
| | - Marco Vignuzzi
- Institut Pasteur, Centre National de la Recherche Scientifique, UMR 3569, Paris, France.
| |
Collapse
|
|
34
|
Characterization of Hepatitis C Virus (HCV) Envelope Diversification from Acute to Chronic Infection within a Sexually Transmitted HCV Cluster by Using Single-Molecule, Real-Time Sequencing. J Virol 2017; 91:JVI.02262-16. [PMID: 28077634 DOI: 10.1128/jvi.02262-16] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2016] [Accepted: 12/29/2016] [Indexed: 12/18/2022] Open
Abstract
In contrast to other available next-generation sequencing platforms, PacBio single-molecule, real-time (SMRT) sequencing has the advantage of generating long reads albeit with a relatively higher error rate in unprocessed data. Using this platform, we longitudinally sampled and sequenced the hepatitis C virus (HCV) envelope genome region (1,680 nucleotides [nt]) from individuals belonging to a cluster of sexually transmitted cases. All five subjects were coinfected with HIV-1 and a closely related strain of HCV genotype 4d. In total, 50 samples were analyzed by using SMRT sequencing. By using 7 passes of circular consensus sequencing, the error rate was reduced to 0.37%, and the median number of sequences was 612 per sample. A further reduction of insertions was achieved by alignment against a sample-specific reference sequence. However, in vitro recombination during PCR amplification could not be excluded. Phylogenetic analysis supported close relationships among HCV sequences from the four male subjects and subsequent transmission from one subject to his female partner. Transmission was characterized by a strong genetic bottleneck. Viral genetic diversity was low during acute infection and increased upon progression to chronicity but subsequently fluctuated during chronic infection, caused by the alternate detection of distinct coexisting lineages. SMRT sequencing combines long reads with sufficient depth for many phylogenetic analyses and can therefore provide insights into within-host HCV evolutionary dynamics without the need for haplotype reconstruction using statistical algorithms.IMPORTANCE Next-generation sequencing has revolutionized the study of genetically variable RNA virus populations, but for phylogenetic and evolutionary analyses, longer sequences than those generated by most available platforms, while minimizing the intrinsic error rate, are desired. Here, we demonstrate for the first time that PacBio SMRT sequencing technology can be used to generate full-length HCV envelope sequences at the single-molecule level, providing a data set with large sequencing depth for the characterization of intrahost viral dynamics. The selection of consensus reads derived from at least 7 full circular consensus sequencing rounds significantly reduced the intrinsic high error rate of this method. We used this method to genetically characterize a unique transmission cluster of sexually transmitted HCV infections, providing insight into the distinct evolutionary pathways in each patient over time and identifying the transmission-associated genetic bottleneck as well as fluctuations in viral genetic diversity over time, accompanied by dynamic shifts in viral subpopulations.
Collapse
|
|
35
|
Tracking HCV protease population diversity during transmission and susceptibility of founder populations to antiviral therapy. Antiviral Res 2017; 139:129-137. [PMID: 28062191 DOI: 10.1016/j.antiviral.2017.01.001] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2016] [Revised: 12/22/2016] [Accepted: 01/02/2017] [Indexed: 12/16/2022]
Abstract
Due to the highly restricted species-tropism of Hepatitis C virus (HCV) a limited number of animal models exist for pre-clinical evaluation of vaccines and antiviral compounds. The human-liver chimeric mouse model allows heterologous challenge with clinically relevant strains derived from patients. However, to date, the transmission and longitudinal evolution of founder viral populations in this model have not been characterized in-depth using state-of-the-art sequencing technologies. Focusing on NS3 protease encoding region of the viral genome, mutant spectra in a donor inoculum and individual recipient mice were determined via Illumina sequencing and compared, to determine the effects of transmission on founder viral population complexity. In all transmissions, a genetic bottleneck was observed, although diverse viral populations were transmitted in each case. A low frequency cloud of mutations (<1%) was detectable in the donor inoculum and recipient mice, with single nucleotide variants (SNVs) > 1% restricted to a subset of nucleotides. The population of SNVs >1% was reduced upon transmission while the low frequency SNV cloud remained stable. Fixation of multiple identical synonymous substitutions was apparent in independent transmissions, and no evidence for reversion of T-cell epitopes was observed. In addition, susceptibility of founder populations to antiviral therapy was assessed. Animals were treated with protease inhibitor (PI) monotherapy to track resistance associated substitution (RAS) emergence. Longitudinal analyses revealed a decline in population diversity under therapy, with no detectable RAS >1% prior to therapy commencement. Despite inoculation from a common source and identical therapeutic regimens, unique RAS emergence profiles were identified in different hosts prior to and during therapeutic failure, with complex mutational signatures at protease residues 155, 156 and 168 detected. Together these analyses track viral population complexity at high-resolution in the human-liver chimeric mouse model post-transmission and under therapeutic intervention, revealing novel insights into the evolutionary processes which shape viral protease population composition at various critical stages of the viral life-cycle.
Collapse
|
|
36
|
Spontaneous Elimination of Hepatitis C Virus Infection. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2017; 1039:45-54. [PMID: 29164488 DOI: 10.1007/5584_2017_76] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Hepatitis C virus (HCV) is the etiological agent of chronic hepatitis C and a major cause of liver cirrhosis and hepatocellular carcinoma. Only a minority of infected individuals can clear the virus spontaneously. The knowledge of the determinants of virus clearance would allow the development of effective methods preventing its further spread and optimizing treatment regimens. Viral factors associated with spontaneous virus clearance in the acute phase of infection, such as HCV genotype, virus heterogeneity, and the impact of viral proteins on the immune system have been characterized. Likewise, host genetic markers, such as the interleukin genotypes, HLA alleles, and factors affecting the T lymphocyte response appear to play an important role. Studies have revealed that natural clearance of HCV infection in the chronic phase is rare and its mechanisms are not well understood. In this review, we present the state-of-the art knowledge on the viral and host factors affecting the spontaneous elimination of HCV infection.
Collapse
|
|
37
|
Leung P, Eltahla AA, Lloyd AR, Bull RA, Luciani F. Understanding the complex evolution of rapidly mutating viruses with deep sequencing: Beyond the analysis of viral diversity. Virus Res 2016; 239:43-54. [PMID: 27888126 DOI: 10.1016/j.virusres.2016.10.014] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2016] [Revised: 10/24/2016] [Accepted: 10/25/2016] [Indexed: 12/24/2022]
Abstract
With the advent of affordable deep sequencing technologies, detection of low frequency variants within genetically diverse viral populations can now be achieved with unprecedented depth and efficiency. The high-resolution data provided by next generation sequencing technologies is currently recognised as the gold standard in estimation of viral diversity. In the analysis of rapidly mutating viruses, longitudinal deep sequencing datasets from viral genomes during individual infection episodes, as well as at the epidemiological level during outbreaks, now allow for more sophisticated analyses such as statistical estimates of the impact of complex mutation patterns on the evolution of the viral populations both within and between hosts. These analyses are revealing more accurate descriptions of the evolutionary dynamics that underpin the rapid adaptation of these viruses to the host response, and to drug therapies. This review assesses recent developments in methods and provide informative research examples using deep sequencing data generated from rapidly mutating viruses infecting humans, particularly hepatitis C virus (HCV), human immunodeficiency virus (HIV), Ebola virus and influenza virus, to understand the evolution of viral genomes and to explore the relationship between viral mutations and the host adaptive immune response. Finally, we discuss limitations in current technologies, and future directions that take advantage of publically available large deep sequencing datasets.
Collapse
Affiliation(s)
- Preston Leung
- School of Medical Sciences, Faculty of Medicine, UNSW Australia, Sydney, NSW 2052, Australia; The Kirby Institute, UNSW Australia, Sydney, NSW 2052, Australia
| | - Auda A Eltahla
- School of Medical Sciences, Faculty of Medicine, UNSW Australia, Sydney, NSW 2052, Australia; The Kirby Institute, UNSW Australia, Sydney, NSW 2052, Australia
| | - Andrew R Lloyd
- The Kirby Institute, UNSW Australia, Sydney, NSW 2052, Australia
| | - Rowena A Bull
- School of Medical Sciences, Faculty of Medicine, UNSW Australia, Sydney, NSW 2052, Australia; The Kirby Institute, UNSW Australia, Sydney, NSW 2052, Australia
| | - Fabio Luciani
- School of Medical Sciences, Faculty of Medicine, UNSW Australia, Sydney, NSW 2052, Australia; The Kirby Institute, UNSW Australia, Sydney, NSW 2052, Australia.
| |
Collapse
|
|
38
|
Grubaugh ND, Rückert C, Armstrong PM, Bransfield A, Anderson JF, Ebel GD, Brackney DE. Transmission bottlenecks and RNAi collectively influence tick-borne flavivirus evolution. Virus Evol 2016; 2:vew033. [PMID: 28058113 PMCID: PMC5210029 DOI: 10.1093/ve/vew033] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Arthropod-borne RNA viruses exist within hosts as heterogeneous populations of viral variants and, as a result, possess great genetic plasticity. Understanding the micro-evolutionary forces shaping these viruses can provide insights into how they emerge, adapt, and persist in new and changing ecological niches. While considerable attention has been directed toward studying the population dynamics of mosquito-borne viruses, little is known about tick-borne virus populations. Therefore, using a mouse and Ixodes scapularis tick transmission model, we examined Powassan virus (POWV; Flaviviridae, Flavivirus) populations in and between both the vertebrate host and arthropod vector. We found that genetic bottlenecks, RNAi-mediated diversification, and selective constraints collectively influence POWV evolution. Together, our data provide a mechanistic explanation for the slow, long-term evolutionary trends of POWV, and suggest that all arthropod-borne viruses encounter similar selective pressures at the molecular level (i.e. RNAi), yet evolve much differently due to their unique rates and modes of transmission.
Collapse
Affiliation(s)
- Nathan D Grubaugh
- Department of Microbiology Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Fort Collins, CO, USA
| | - Claudia Rückert
- Department of Microbiology Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Fort Collins, CO, USA
| | - Philip M Armstrong
- The Connecticut Agricultural Experiment Station, Center for Vector Biology and Zoonotic Diseases, New Haven, CT, USA
| | - Angela Bransfield
- The Connecticut Agricultural Experiment Station, Center for Vector Biology and Zoonotic Diseases, New Haven, CT, USA
| | - John F Anderson
- The Connecticut Agricultural Experiment Station, Center for Vector Biology and Zoonotic Diseases, New Haven, CT, USA
| | - Gregory D Ebel
- Department of Microbiology Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Fort Collins, CO, USA
| | - Doug E Brackney
- The Connecticut Agricultural Experiment Station, Center for Vector Biology and Zoonotic Diseases, New Haven, CT, USA
| |
Collapse
|
|
39
|
Milazzo L, Lai A, Calvi E, Ronzi P, Micheli V, Binda F, Ridolfo AL, Gervasoni C, Galli M, Antinori S, Sollima S. Direct-acting antivirals in hepatitis C virus (HCV)-infected and HCV/HIV-coinfected patients: real-life safety and efficacy. HIV Med 2016; 18:284-291. [DOI: 10.1111/hiv.12429] [Citation(s) in RCA: 64] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/19/2016] [Indexed: 12/19/2022]
Affiliation(s)
- L Milazzo
- Department of Biomedical and Clinical Sciences L. Sacco; University of Milan; Milan Italy
| | - A Lai
- Department of Biomedical and Clinical Sciences L. Sacco; University of Milan; Milan Italy
| | - E Calvi
- Department of Biomedical and Clinical Sciences L. Sacco; University of Milan; Milan Italy
| | - P Ronzi
- Department of Biomedical and Clinical Sciences L. Sacco; University of Milan; Milan Italy
| | - V Micheli
- Clinical Microbiology Virology and Diagnosis of Bioemergency; L. Sacco University Hospital; Milan Italy
| | - F Binda
- Department of Biomedical and Clinical Sciences L. Sacco; University of Milan; Milan Italy
| | - AL Ridolfo
- Department of Biomedical and Clinical Sciences L. Sacco; University of Milan; Milan Italy
| | - C Gervasoni
- Department of Biomedical and Clinical Sciences L. Sacco; University of Milan; Milan Italy
| | - M Galli
- Department of Biomedical and Clinical Sciences L. Sacco; University of Milan; Milan Italy
| | - S Antinori
- Department of Biomedical and Clinical Sciences L. Sacco; University of Milan; Milan Italy
| | - S Sollima
- Department of Biomedical and Clinical Sciences L. Sacco; University of Milan; Milan Italy
| |
Collapse
|
|
40
|
Montoya V, Olmstead A, Tang P, Cook D, Janjua N, Grebely J, Jacka B, Poon AFY, Krajden M. Deep sequencing increases hepatitis C virus phylogenetic cluster detection compared to Sanger sequencing. INFECTION GENETICS AND EVOLUTION 2016; 43:329-37. [PMID: 27282472 DOI: 10.1016/j.meegid.2016.06.015] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/23/2016] [Revised: 06/03/2016] [Accepted: 06/04/2016] [Indexed: 01/17/2023]
Abstract
Effective surveillance and treatment strategies are required to control the hepatitis C virus (HCV) epidemic. Phylogenetic analyses are powerful tools for reconstructing the evolutionary history of viral outbreaks and identifying transmission clusters. These studies often rely on Sanger sequencing which typically generates a single consensus sequence for each infected individual. For rapidly mutating viruses such as HCV, consensus sequencing underestimates the complexity of the viral quasispecies population and could therefore generate different phylogenetic tree topologies. Although deep sequencing provides a more detailed quasispecies characterization, in-depth phylogenetic analyses are challenging due to dataset complexity and computational limitations. Here, we apply deep sequencing to a characterized population to assess its ability to identify phylogenetic clusters compared with consensus Sanger sequencing. For deep sequencing, a sample specific threshold determined by the 50th percentile of the patristic distance distribution for all variants within each individual was used to identify clusters. Among seven patristic distance thresholds tested for the Sanger sequence phylogeny ranging from 0.005-0.06, a threshold of 0.03 was found to provide the maximum balance between positive agreement (samples in a cluster) and negative agreement (samples not in a cluster) relative to the deep sequencing dataset. From 77 HCV seroconverters, 10 individuals were identified in phylogenetic clusters using both methods. Deep sequencing analysis identified an additional 4 individuals and excluded 8 other individuals relative to Sanger sequencing. The application of this deep sequencing approach could be a more effective tool to understand onward HCV transmission dynamics compared with Sanger sequencing, since the incorporation of minority sequence variants improves the discrimination of phylogenetically linked clusters.
Collapse
Affiliation(s)
- Vincent Montoya
- BC Centre for Disease Control, Vancouver, BC, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada
| | - Andrea Olmstead
- BC Centre for Disease Control, Vancouver, BC, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada
| | | | - Darrel Cook
- BC Centre for Disease Control, Vancouver, BC, Canada
| | - Naveed Janjua
- BC Centre for Disease Control, Vancouver, BC, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada
| | - Jason Grebely
- The Kirby Institute, UNSW Australia, Sydney, NSW, Australia
| | - Brendan Jacka
- The Kirby Institute, UNSW Australia, Sydney, NSW, Australia
| | - Art F Y Poon
- BC Centre for Excellence in HIV/AIDS, St Paul's Hospital, Vancouver, BC, Canada
| | - Mel Krajden
- BC Centre for Disease Control, Vancouver, BC, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.
| |
Collapse
|
|
41
|
Trémeaux P, Caporossi A, Thélu MA, Blum M, Leroy V, Morand P, Larrat S. Hepatitis C virus whole genome sequencing: Current methods/issues and future challenges. Crit Rev Clin Lab Sci 2016; 53:341-51. [PMID: 27068766 DOI: 10.3109/10408363.2016.1163663] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Therapy for hepatitis C is currently undergoing a revolution. The arrival of new antiviral agents targeting viral proteins reinforces the need for a better knowledge of the viral strains infecting each patient. Hepatitis C virus (HCV) whole genome sequencing provides essential information for precise typing, study of the viral natural history or identification of resistance-associated variants. First performed with Sanger sequencing, the arrival of next-generation sequencing (NGS) has simplified the technical process and provided more detailed data on the nature and evolution of viral quasi-species. We will review the different techniques used for HCV complete genome sequencing and their applications, both before and after the apparition of NGS. The progress brought by new and future technologies will also be discussed, as well as the remaining difficulties, largely due to the genomic variability.
Collapse
Affiliation(s)
- Pauline Trémeaux
- a Laboratoire de Virologie , Institut de Biologie et Pathologie, CHU Grenoble-Alpes , Grenoble , France .,b Institut de Biologie Structurale (IBS), UMR 5075 CEA-CNRS-UGA , Grenoble , France
| | - Alban Caporossi
- c Centre d'investigation clinique, Santé publique, CHU Grenoble-Alpes , Grenoble , France .,d Laboratoire TIMC-IMAG , Université de Grenoble Alpes , Grenoble , France , and
| | - Marie-Ange Thélu
- e Clinique d'Hépato-gastroentérologie, Pôle Digidune, CHU Grenoble-Alpes , Grenoble , France
| | - Michael Blum
- d Laboratoire TIMC-IMAG , Université de Grenoble Alpes , Grenoble , France , and
| | - Vincent Leroy
- e Clinique d'Hépato-gastroentérologie, Pôle Digidune, CHU Grenoble-Alpes , Grenoble , France
| | - Patrice Morand
- a Laboratoire de Virologie , Institut de Biologie et Pathologie, CHU Grenoble-Alpes , Grenoble , France .,b Institut de Biologie Structurale (IBS), UMR 5075 CEA-CNRS-UGA , Grenoble , France
| | - Sylvie Larrat
- a Laboratoire de Virologie , Institut de Biologie et Pathologie, CHU Grenoble-Alpes , Grenoble , France .,b Institut de Biologie Structurale (IBS), UMR 5075 CEA-CNRS-UGA , Grenoble , France
| |
Collapse
|
|
42
|
Ferns RB, Tarr AW, Hue S, Urbanowicz RA, McClure CP, Gilson R, Ball JK, Nastouli E, Garson JA, Pillay D. Hepatitis C virus quasispecies and pseudotype analysis from acute infection to chronicity in HIV-1 co-infected individuals. Virology 2016; 492:213-24. [PMID: 26971243 DOI: 10.1016/j.virol.2016.02.003] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2015] [Revised: 01/14/2016] [Accepted: 02/05/2016] [Indexed: 01/01/2023]
Abstract
HIV-1 infected patients who acquire HCV infection have higher rates of chronicity and liver disease progression than patients with HCV mono-infection. Understanding early events in this pathogenic process is important. We applied single genome sequencing of the E1 to NS3 regions and viral pseudotype neutralization assays to explore the consequences of viral quasispecies evolution from pre-seroconversion to chronicity in four co-infected individuals (mean follow up 566 days). We observed that one to three founder viruses were transmitted. Relatively low viral sequence diversity, possibly related to an impaired immune response, due to HIV infection was observed in three patients. However, the fourth patient, after an early purifying selection displayed increasing E2 sequence evolution, possibly related to being on suppressive antiretroviral therapy. Viral pseudotypes generated from HCV variants showed relative resistance to neutralization by autologous plasma but not to plasma collected from later time points, confirming ongoing virus escape from antibody neutralization.
Collapse
Affiliation(s)
- R Bridget Ferns
- Division of Infection & Immunity, Faculty of Medical Sciences, University College London, United Kingdom; Clinical Microbiology & Virology, UCL Hospital NHS Foundation Trust, United Kingdom.
| | - Alexander W Tarr
- School of Life Sciences & NIHR Biomedical Research Unit in Gastrointestinal & Liver Diseases, Faculty of Medicine and Health Sciences, University of Nottingham, United Kingdom
| | - Stephane Hue
- Division of Infection & Immunity, Faculty of Medical Sciences, University College London, United Kingdom
| | - Richard A Urbanowicz
- School of Life Sciences & NIHR Biomedical Research Unit in Gastrointestinal & Liver Diseases, Faculty of Medicine and Health Sciences, University of Nottingham, United Kingdom
| | - C Patrick McClure
- School of Life Sciences & NIHR Biomedical Research Unit in Gastrointestinal & Liver Diseases, Faculty of Medicine and Health Sciences, University of Nottingham, United Kingdom
| | - Richard Gilson
- Research Department of Infection and Population Health, University College London, United Kingdom
| | - Jonathan K Ball
- School of Life Sciences & NIHR Biomedical Research Unit in Gastrointestinal & Liver Diseases, Faculty of Medicine and Health Sciences, University of Nottingham, United Kingdom
| | - Eleni Nastouli
- Clinical Microbiology & Virology, UCL Hospital NHS Foundation Trust, United Kingdom
| | - Jeremy A Garson
- Division of Infection & Immunity, Faculty of Medical Sciences, University College London, United Kingdom
| | - Deenan Pillay
- Division of Infection & Immunity, Faculty of Medical Sciences, University College London, United Kingdom; Wellcome Trust Africa Centre for Health and Population Sciences, University of KwaZulu, Natal, South Africa
| |
Collapse
|
|
43
|
Campo DS, Xia GL, Dimitrova Z, Lin Y, Forbi JC, Ganova-Raeva L, Punkova L, Ramachandran S, Thai H, Skums P, Sims S, Rytsareva I, Vaughan G, Roh HJ, Purdy MA, Sue A, Khudyakov Y. Accurate Genetic Detection of Hepatitis C Virus Transmissions in Outbreak Settings. J Infect Dis 2016; 213:957-965. [PMID: 26582955 PMCID: PMC5119477 DOI: 10.1093/infdis/jiv542] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2015] [Accepted: 10/08/2015] [Indexed: 12/18/2022] Open
Abstract
Hepatitis C is a major public health problem in the United States and worldwide. Outbreaks of hepatitis C virus (HCV) infections are associated with unsafe injection practices, drug diversion, and other exposures to blood and are difficult to detect and investigate. Here, we developed and validated a simple approach for molecular detection of HCV transmissions in outbreak settings. We obtained sequences from the HCV hypervariable region 1 (HVR1), using end-point limiting-dilution (EPLD) technique, from 127 cases involved in 32 epidemiologically defined HCV outbreaks and 193 individuals with unrelated HCV strains. We compared several types of genetic distances and calculated a threshold, using minimal Hamming distances, that identifies transmission clusters in all tested outbreaks with 100% accuracy. The approach was also validated on sequences obtained using next-generation sequencing from HCV strains recovered from 239 individuals, and findings showed the same accuracy as that for EPLD. On average, the nucleotide diversity of the intrahost population was 6.2 times greater in the source case than in any incident case, allowing the correct detection of transmission direction in 8 outbreaks for which source cases were known. A simple and accurate distance-based approach developed here for detecting HCV transmissions streamlines molecular investigation of outbreaks, thus improving the public health capacity for rapid and effective control of hepatitis C.
Collapse
Affiliation(s)
- DS Campo
- Molecular Epidemiology and Bioinformatics Laboratory, Division of Viral hepatitis, Centers for Disease Control and Prevention, Atlanta, 30333, USA
| | - GL Xia
- Molecular Epidemiology and Bioinformatics Laboratory, Division of Viral hepatitis, Centers for Disease Control and Prevention, Atlanta, 30333, USA
| | - Z Dimitrova
- Molecular Epidemiology and Bioinformatics Laboratory, Division of Viral hepatitis, Centers for Disease Control and Prevention, Atlanta, 30333, USA
| | - Y Lin
- Molecular Epidemiology and Bioinformatics Laboratory, Division of Viral hepatitis, Centers for Disease Control and Prevention, Atlanta, 30333, USA
| | - JC Forbi
- Molecular Epidemiology and Bioinformatics Laboratory, Division of Viral hepatitis, Centers for Disease Control and Prevention, Atlanta, 30333, USA
| | - L Ganova-Raeva
- Molecular Epidemiology and Bioinformatics Laboratory, Division of Viral hepatitis, Centers for Disease Control and Prevention, Atlanta, 30333, USA
| | - L Punkova
- Molecular Epidemiology and Bioinformatics Laboratory, Division of Viral hepatitis, Centers for Disease Control and Prevention, Atlanta, 30333, USA
| | - S Ramachandran
- Molecular Epidemiology and Bioinformatics Laboratory, Division of Viral hepatitis, Centers for Disease Control and Prevention, Atlanta, 30333, USA
| | - H Thai
- Molecular Epidemiology and Bioinformatics Laboratory, Division of Viral hepatitis, Centers for Disease Control and Prevention, Atlanta, 30333, USA
| | - P Skums
- Molecular Epidemiology and Bioinformatics Laboratory, Division of Viral hepatitis, Centers for Disease Control and Prevention, Atlanta, 30333, USA
| | - S Sims
- Molecular Epidemiology and Bioinformatics Laboratory, Division of Viral hepatitis, Centers for Disease Control and Prevention, Atlanta, 30333, USA
| | - I Rytsareva
- Molecular Epidemiology and Bioinformatics Laboratory, Division of Viral hepatitis, Centers for Disease Control and Prevention, Atlanta, 30333, USA
| | - G Vaughan
- Molecular Epidemiology and Bioinformatics Laboratory, Division of Viral hepatitis, Centers for Disease Control and Prevention, Atlanta, 30333, USA
| | - HJ Roh
- Molecular Epidemiology and Bioinformatics Laboratory, Division of Viral hepatitis, Centers for Disease Control and Prevention, Atlanta, 30333, USA
| | - MA Purdy
- Molecular Epidemiology and Bioinformatics Laboratory, Division of Viral hepatitis, Centers for Disease Control and Prevention, Atlanta, 30333, USA
| | - A Sue
- Molecular Epidemiology and Bioinformatics Laboratory, Division of Viral hepatitis, Centers for Disease Control and Prevention, Atlanta, 30333, USA
| | - Y Khudyakov
- Molecular Epidemiology and Bioinformatics Laboratory, Division of Viral hepatitis, Centers for Disease Control and Prevention, Atlanta, 30333, USA
| |
Collapse
|
|
44
|
Liu L, Nardo D, Li E, Wang GP. CD4+ T-cell recovery with suppressive ART-induced rapid sequence evolution in hepatitis C virus envelope but not NS3. AIDS 2016; 30:691-700. [PMID: 26645605 DOI: 10.1097/qad.0000000000000997] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
OBJECTIVES CD4 T-cell depletion from HIV infection leads to a global decline in anti-hepatitis C virus (HCV) envelope neutralizing antibody (nAb) response, which may play a role in accelerating liver fibrosis. An increase in anti-HCV nAb titers has been reported during antiretroviral therapy (ART) but its impact on HCV remains poorly understood. The objective of this study is to determine the effects of ART on long-term HCV evolution. DESIGN AND METHODS We examined HCV quasispecies structure and long-term evolution in HIV/HCV coinfected patients with ART-induced CD4 T-cell recovery, and compared with patients with CD4 T-cell depletion from delayed ART. We applied a single-variant sequencing (SVS) method to construct authentic viral quasispecies and compared sequence evolution in HCV envelope, the primary target for humoral immune responses, and NS3, a target for cellular immunity, between the two cohorts. RESULTS The SVS method corrected biases known to skew the proportions of viral variants, revealing authentic HCV quasispeices structures. We observed higher rates of HCV envelope sequence evolution in patients with ART-induced CD4 T-cell recovery, compared with patients with CD4 T-cell depletion from delayed ART (P = 0.03). Evolutionary rates for NS3 were considerably lower than the rates for envelope (P < 0.01), with no significant difference observed between the two groups. CONCLUSION ART-induced CD4 T-cell recovery results in rapid sequence evolution in HCV envelope, but not in NS3. These results suggest that suppressive ART disproportionally enhances HCV-specific humoral responses more than cellular responses, resulting in rapid sequence evolution in HCV envelope but not NS3.
Collapse
|
|
45
|
Caraballo Cortes K, Zagordi O, Jabłońska J, Pawełczyk A, Kubisa N, Perlejewski K, Bukowska-Ośko I, Płoski R, Radkowski M, Laskus T. Spouse-to-Spouse Transmission and Evolution of Hypervariable Region 1 and 5' Untranslated Region of Hepatitis C Virus Analyzed by Next-Generation Sequencing. PLoS One 2016; 11:e0150311. [PMID: 26918636 PMCID: PMC4769329 DOI: 10.1371/journal.pone.0150311] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2015] [Accepted: 02/11/2016] [Indexed: 12/16/2022] Open
Abstract
Hepatitis C virus (HCV) transmission between spouses remains poorly characterized, largely due to the limited availability of samples from the early stage of infection, as well as methodological constraints. A fifty-eight year-old male developed acute hepatitis C infection and his 53-year old spouse has been HCV-positive for over 10 years. Serum samples were collected from both at the time of acute hepatitis C diagnosis in male (baseline) and then at 9 and 13 months. Hypervariable region 1 (HVR1) and 5’ untranslated region (5’UTR) sequences were amplified and subjected to next generation sequencing (NGS) using a pyrosequencing platform. Genetic variants were inferred by Shorah reconstruction method and compared by phylogenetic and sequence diversity analysis. As the sequencing error of the procedure was previously determined to be ≤ 1.5%, the analysis was conducted with and without the 1.5% cut-off with regard to the frequency of variants. No identical HVR1 variants were identified in spouses at baseline and follow-up samples regardless whether the cut-off was applied or not. However, there was high similarity (98.3%) between a minor baseline donor variant (1.7% frequency) and the most abundant baseline recipient variant (62.5% frequency). Furthermore, donor and recipient strains clustered together when compared to 10 control subjects from the same area and infected with the same HCV subtype. There was an increase in HVR1 complexity (number of genetic variants) over time in both spouses. In contrast, the 5'UTR region was stable and of low complexity throughout the study. In conclusion, intrafamilial HCV transmission may be established by a very minor variant and investigation of this phenomenon requires high-sensitivity assays, such as NGS.
Collapse
Affiliation(s)
- Kamila Caraballo Cortes
- Department of Immunopathology of Infectious and Parasitic Diseases, Medical University of Warsaw, Warsaw, Poland
- * E-mail:
| | - Osvaldo Zagordi
- Institute of Medical Virology, University of Zurich, Zurich, Switzerland
| | - Joanna Jabłońska
- Clinics of Infectious, Tropical Diseases and Hepatology, Medical University of Warsaw, Warsaw, Poland
| | - Agnieszka Pawełczyk
- Department of Immunopathology of Infectious and Parasitic Diseases, Medical University of Warsaw, Warsaw, Poland
| | - Natalia Kubisa
- Department of Immunopathology of Infectious and Parasitic Diseases, Medical University of Warsaw, Warsaw, Poland
| | - Karol Perlejewski
- Department of Immunopathology of Infectious and Parasitic Diseases, Medical University of Warsaw, Warsaw, Poland
| | - Iwona Bukowska-Ośko
- Department of Immunopathology of Infectious and Parasitic Diseases, Medical University of Warsaw, Warsaw, Poland
| | - Rafał Płoski
- Department of Medical Genetics, Medical University of Warsaw, Warsaw, Poland
| | - Marek Radkowski
- Department of Immunopathology of Infectious and Parasitic Diseases, Medical University of Warsaw, Warsaw, Poland
| | - Tomasz Laskus
- Department of Immunopathology of Infectious and Parasitic Diseases, Medical University of Warsaw, Warsaw, Poland
| |
Collapse
|
|
46
|
Li H, Stoddard MB, Wang S, Giorgi EE, Blair LM, Learn GH, Hahn BH, Alter HJ, Busch MP, Fierer DS, Ribeiro RM, Perelson AS, Bhattacharya T, Shaw GM. Single-Genome Sequencing of Hepatitis C Virus in Donor-Recipient Pairs Distinguishes Modes and Models of Virus Transmission and Early Diversification. J Virol 2016; 90:152-66. [PMID: 26468546 PMCID: PMC4702571 DOI: 10.1128/jvi.02156-15] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2015] [Accepted: 10/02/2015] [Indexed: 01/06/2023] Open
Abstract
UNLABELLED Despite the recent development of highly effective anti-hepatitis C virus (HCV) drugs, the global burden of this pathogen remains immense. Control or eradication of HCV will likely require the broad application of antiviral drugs and development of an effective vaccine. A precise molecular identification of transmitted/founder (T/F) HCV genomes that lead to productive clinical infection could play a critical role in vaccine research, as it has for HIV-1. However, the replication schema of these two RNA viruses differ substantially, as do viral responses to innate and adaptive host defenses. These differences raise questions as to the certainty of T/F HCV genome inferences, particularly in cases where multiple closely related sequence lineages have been observed. To clarify these issues and distinguish between competing models of early HCV diversification, we examined seven cases of acute HCV infection in humans and chimpanzees, including three examples of virus transmission between linked donors and recipients. Using single-genome sequencing (SGS) of plasma vRNA, we found that inferred T/F sequences in recipients were identical to viral sequences in their respective donors. Early in infection, HCV genomes generally evolved according to a simple model of random evolution where the coalescent corresponded to the T/F sequence. Closely related sequence lineages could be explained by high multiplicity infection from a donor whose viral sequences had undergone a pretransmission bottleneck due to treatment, immune selection, or recent infection. These findings validate SGS, together with mathematical modeling and phylogenetic analysis, as a novel strategy to infer T/F HCV genome sequences. IMPORTANCE Despite the recent development of highly effective, interferon-sparing anti-hepatitis C virus (HCV) drugs, the global burden of this pathogen remains immense. Control or eradication of HCV will likely require the broad application of antiviral drugs and the development of an effective vaccine, which could be facilitated by a precise molecular identification of transmitted/founder (T/F) viral genomes and their progeny. We used single-genome sequencing to show that inferred HCV T/F sequences in recipients were identical to viral sequences in their respective donors and that viral genomes generally evolved early in infection according to a simple model of random sequence evolution. Altogether, the findings validate T/F genome inferences and illustrate how T/F sequence identification can illuminate studies of HCV transmission, immunopathogenesis, drug resistance development, and vaccine protection, including sieving effects on breakthrough virus strains.
Collapse
Affiliation(s)
- Hui Li
- Departments of Medicine and Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Mark B Stoddard
- Departments of Medicine and Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Shuyi Wang
- Departments of Medicine and Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Elena E Giorgi
- T-Division, Los Alamos National Laboratory, Los Alamos, New Mexico, USA
| | - Lily M Blair
- T-Division, Los Alamos National Laboratory, Los Alamos, New Mexico, USA Department of Biology, Stanford University, Stanford, California, USA
| | - Gerald H Learn
- Departments of Medicine and Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Beatrice H Hahn
- Departments of Medicine and Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Harvey J Alter
- Department of Transfusion Medicine, National Institutes of Health, Bethesda, Maryland, USA
| | - Michael P Busch
- Blood Systems Research Institute, University of California San Francisco, San Francisco, California, USA
| | - Daniel S Fierer
- Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, New York, USA
| | - Ruy M Ribeiro
- T-Division, Los Alamos National Laboratory, Los Alamos, New Mexico, USA
| | - Alan S Perelson
- Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, New Mexico, USA
| | - Tanmoy Bhattacharya
- T-Division, Los Alamos National Laboratory, Los Alamos, New Mexico, USA Santa Fe Institute, Santa Fe, New Mexico, USA
| | - George M Shaw
- Departments of Medicine and Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| |
Collapse
|
|
47
|
Gonçalves Rossi LM, Escobar-Gutierrez A, Rahal P. Multiregion deep sequencing of hepatitis C virus: An improved approach for genetic relatedness studies. INFECTION GENETICS AND EVOLUTION 2015; 38:138-145. [PMID: 26733442 DOI: 10.1016/j.meegid.2015.12.020] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/22/2015] [Revised: 12/23/2015] [Accepted: 12/24/2015] [Indexed: 12/19/2022]
Abstract
Hepatitis C virus (HCV) is a major public health problem that affects more than 180 million people worldwide. Identification of HCV transmission networks is of critical importance for disease control. HCV related cases are often difficult to identify due to the characteristic long incubation period and lack of symptoms during the acute phase of the disease, making it challenging to link related cases to a common source of infection. Additionally, HCV transmission chains are difficult to trace back since viral variants from epidemiologically linked cases are genetically related but rarely identical. Genetic relatedness studies primarily rely on information obtained from the rapidly evolving HCV hypervariable region 1 (HVR1). However, in some instances, the rapid divergence of this region can lead to loss of genetic links between related isolates, which represents an important challenge for outbreak investigations and genetic relatedness studies. Sequencing of multiple and longer sub-genomic regions has been proposed as an alternative to overcome the limitations imposed by the rapid molecular evolution of the HCV HVR1. Additionally, conventional molecular approaches required to characterize the HCV intra-host genetic variation are laborious, time-consuming, and expensive while providing limited information about the composition of the viral population. Next generation sequencing (NGS) approaches enormously facilitate the characterization of the HCV intra-host population by detecting rare variants at much lower frequencies. Thus, NGS approaches using multiple sub-genomic regions should improve the characterization of the HCV intra-host population. Here, we explore the usefulness of multiregion sequencing using a NGS platform for genetic relatedness studies among HCV cases.
Collapse
Affiliation(s)
- Livia Maria Gonçalves Rossi
- Department of Biology, Institute of Bioscience, Language and Exact Science, São Paulo State University, São José do Rio Preto, Sao Paulo, Brazil; Instituto de Diagnóstico y Referencia Epidemiológicos, Mexico City, Mexico.
| | | | - Paula Rahal
- Department of Biology, Institute of Bioscience, Language and Exact Science, São Paulo State University, São José do Rio Preto, Sao Paulo, Brazil
| |
Collapse
|
|
48
|
Fourati S, Pawlotsky JM. Virologic Tools for HCV Drug Resistance Testing. Viruses 2015; 7:6346-59. [PMID: 26690198 PMCID: PMC4690865 DOI: 10.3390/v7122941] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2015] [Revised: 11/28/2015] [Accepted: 11/30/2015] [Indexed: 12/22/2022] Open
Abstract
Recent advances in molecular biology have led to the development of new antiviral drugs that target specific steps of the Hepatitis C Virus (HCV) lifecycle. These drugs, collectively termed direct-acting antivirals (DAAs), include non-structural (NS) HCV protein inhibitors, NS3/4A protease inhibitors, NS5B RNA-dependent RNA polymerase inhibitors (nucleotide analogues and non-nucleoside inhibitors), and NS5A inhibitors. Due to the high genetic variability of HCV, the outcome of DAA-based therapies may be altered by the selection of amino-acid substitutions located within the targeted proteins, which affect viral susceptibility to the administered compounds. At the drug developmental stage, preclinical and clinical characterization of HCV resistance to new drugs in development is mandatory. In the clinical setting, accurate diagnostic tools have become available to monitor drug resistance in patients who receive treatment with DAAs. In this review, we describe tools available to investigate drug resistance in preclinical studies, clinical trials and clinical practice.
Collapse
Affiliation(s)
- Slim Fourati
- National Reference Center for Viral Hepatitis B, C, and D; Department of Virology, Hôpital Henri Mondor, Université Paris-Est and INSERM U955, Créteil 94010, France.
| | - Jean-Michel Pawlotsky
- National Reference Center for Viral Hepatitis B, C, and D; Department of Virology, Hôpital Henri Mondor, Université Paris-Est and INSERM U955, Créteil 94010, France.
| |
Collapse
|
|
49
|
Investigation of a Case of Genotype 5a Hepatitis C Virus Transmission in a French Hemodialysis Unit Using Epidemiologic Data and Deep Sequencing. Infect Control Hosp Epidemiol 2015; 37:134-9. [PMID: 26510471 DOI: 10.1017/ice.2015.263] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
BACKGROUND Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. A patient was recently found to be HCV seropositive during hemodialysis follow-up. OBJECTIVE To determine whether nosocomial transmission had occurred and which viral populations were transmitted. DESIGN HCV transmission case. SETTING A dialysis unit in a French hospital. METHODS Molecular and epidemiologic investigations were conducted to determine whether 2 cases were related. Risk analysis and auditing procedures were performed to determine the transmission pathway(s). RESULTS Sequence analyses of the NS5b region revealed a 5a genotype in the newly infected patient. Epidemiologic investigations suggested that a highly viremic genotype 5a HCV-infected patient who underwent dialysis in the same unit was the source of the infection. Phylogenetic analysis of NS5b and hypervariable region-1 sequences revealed a genetically related virus (>99.9% nucleotide identity). Deep sequencing of hypervariable region-1 indicated that HCV quasispecies were found in the source whereas a single hypervariable region-1 HCV variant was found in the newly infected patient, and that this was identical to the major variant identified in the source patient. Risk analysis and auditing procedures were performed to determine the transmission pathway(s). Nosocomial patient-to-patient transmission via healthcare workers' hands was the most likely explanation. In our dialysis unit, this unique incident led to the adjustment of infection control policy. CONCLUSIONS The data support transmission of a unique variant from a source with a high viral load and genetic diversity. This investigation also underlines the need to periodically evaluate prevention and control practices.
Collapse
|
|
50
|
Tarr AW, Khera T, Hueging K, Sheldon J, Steinmann E, Pietschmann T, Brown RJP. Genetic Diversity Underlying the Envelope Glycoproteins of Hepatitis C Virus: Structural and Functional Consequences and the Implications for Vaccine Design. Viruses 2015; 7:3995-4046. [PMID: 26193307 PMCID: PMC4517138 DOI: 10.3390/v7072809] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2015] [Revised: 06/19/2015] [Accepted: 07/08/2015] [Indexed: 12/13/2022] Open
Abstract
In the 26 years since the discovery of Hepatitis C virus (HCV) a major global research effort has illuminated many aspects of the viral life cycle, facilitating the development of targeted antivirals. Recently, effective direct-acting antiviral (DAA) regimens with >90% cure rates have become available for treatment of chronic HCV infection in developed nations, representing a significant advance towards global eradication. However, the high cost of these treatments results in highly restricted access in developing nations, where the disease burden is greatest. Additionally, the largely asymptomatic nature of infection facilitates continued transmission in at risk groups and resource constrained settings due to limited surveillance. Consequently a prophylactic vaccine is much needed. The HCV envelope glycoproteins E1 and E2 are located on the surface of viral lipid envelope, facilitate viral entry and are the targets for host immunity, in addition to other functions. Unfortunately, the extreme global genetic and antigenic diversity exhibited by the HCV glycoproteins represents a significant obstacle to vaccine development. Here we review current knowledge of HCV envelope protein structure, integrating knowledge of genetic, antigenic and functional diversity to inform rational immunogen design.
Collapse
Affiliation(s)
- Alexander W Tarr
- School of Life Sciences, Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham NG7 2RD, UK.
| | - Tanvi Khera
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, A Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centrefor Infection Research (HZI), Hannover D-30625, Germany.
| | - Kathrin Hueging
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, A Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centrefor Infection Research (HZI), Hannover D-30625, Germany.
| | - Julie Sheldon
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, A Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centrefor Infection Research (HZI), Hannover D-30625, Germany.
| | - Eike Steinmann
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, A Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centrefor Infection Research (HZI), Hannover D-30625, Germany.
| | - Thomas Pietschmann
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, A Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centrefor Infection Research (HZI), Hannover D-30625, Germany.
- German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Braunschweig 38124, Germany.
| | - Richard J P Brown
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, A Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centrefor Infection Research (HZI), Hannover D-30625, Germany.
| |
Collapse
|