Naturally occurring genotype 2b/1a hepatitis C virus in the United States
© Bhattacharya et al; licensee BioMed Central Ltd. 2011
Received: 29 July 2011
Accepted: 3 October 2011
Published: 3 October 2011
Hepatitis C Virus (HCV) infected patients are frequently repeatedly exposed to the virus, but very few recombinants between two genotypes have been reported.
We describe the discovery of an HCV recombinant using a method developed in a United States clinical lab for HCV genotyping that employs sequencing of both 5' and 3' portions of the HCV genome. Over twelve months, 133 consecutive isolates were analyzed, and a virus from one patient was found with discordant 5' and 3' sequences suggesting it was a genotype 2b/1a recombinant. We ruled out a mixed infection and mapped a recombination point near the NS2/3 cleavage site.
This unique HCV recombinant virus described shares some features with other recombinant viruses although it is the only reported recombinant of a genotype 2 with a subtype 1a. This recombinant represents a conundrum for current clinical treatment guidelines, including treatment with protease inhibitors. This recombinant is also challenging to detect by the most commonly employed methods of genotyping that are directed primarily at the 5' structural portion of the HCV genome.
The WHO estimates that 130-170 million people worldwide are infected with HCV . Six major genotypes (lineages) of HCV have spread throughout the world [2, 3]. Viral genotype is well recognized as the most significant prognostic factor in terms of response to therapy, and a characteristic upon which to base the antiviral prescription . Therefore, the best methods and the most definitive viral target(s) for determining the HCV genotype during patient care remains an important area of translational research. Infection with any genotype can lead to liver cirrhosis and liver cancer in a minority of patients. While specific genotypes dominate in certain regions of the world (for example, genotype 4 in the Middle East), many regions have multiple genotypes circulating including Europe and the United States. People with repeated use of intravenous needles and contaminated blood products in these regions likely are exposed to more than one genotype of HCV. Both mixed infections and recombinant viruses have been described. These situations are thought to be uncommon, particularly natural recombination between two genotypes. Genetic incompatibilities between the viral proteins of different genotypes have been suggested as a reason for recombination in HCV to be a rare event .
Here we describe a case report of a patient who was chronically infected over an extended period of time with a recombinant HCV strain. Like all other naturally occurring inter-genotypic recombinants reported to date, this strain has a genotype 2 5' portion of the genome encoding the structural region, while the nonstructural coding region is from a different genotype. The crossover junction was mapped to the NS2/3 region. We also compared the sequence to other recombinants, but could find limited evidence to support the proposed theory that stable RNA hairpin structures can promote recombination and bracket the cross over junction.
Primers used for Amplification and DNA Sequencing of 5' UTR and NS5B regions in the identification of the HCV recombinant isolate (A, B) and subsequently for Amplification of Overlapping Products Across Entire HCV Recombinant Genome (1-7)
Accession no. AF009606)
Forward Primer (5'-3')
Normal: HCV genotyping primer; Bold: 1a primer;Italics: 2b primer
Reverse Primer (5'-3')
Normal: HCV genotyping primer; Bold: 1a primer;Italics: 2b primer
Sequencing of Recombinant
A web-based program http://www.phylogeny.fr/ was used to analyze the recombinant strain relative to other HCV strains . All sequencing data were collected and searched against the NCBI database using a web-based nucleotide BLAST program http://blast.ncbi.nlm.nih.gov/Blast.cgi. For identification of a more specific junction, analyses were performed using the SimPlot program [(Version 3.5.1)  available online from http://sray.med.som.jhmi.edu/SCRoftware/simplot]. The RNA secondary structure was analyzed using the MFOLD program with default parameters. The MFOLD web based program is provided by Michael Zuker, Rensselaer Polytechnic Institute http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form. The stable RNA secondary structure for this chimeric virus was analyzed and compared with the stable hairpin structure 1 as described by . The NS2-NS3 region near the crossover point was also analyzed using the same program.
GenBank nucleotide sequence accession number
The entire nucleotide sequences of this recombinant virus have been submitted to GenBank and assigned accession number is JF779679.
DNA Sequencing clinical isolates at two different regions of the HCV genome produces mostly concordant results
The discordant clinical isolate is a 2b/1a recombinant virus with a crossover at the NS2/NS3 Junction
While HCV recombinants between two different genotypes are rare, closely related putative parental strains and several different isolates of the RF_2k/1b have been sequenced. Based on RNA structural analysis and recombinants seen in the plant virus turnip crinkle virus, mechanisms that might lead to recombination have been proposed including the identification of two stable RNA hairpins upstream and downstream of the crossover site in the parental strains that are putatively destabilized by mutations in the recombinant . For example, the hairpin structure 1 (HS1) observed in the recombinant is present in the parental 2k strain, but is destabilized slightly by the acquisition of two mutations present in the recombinant RF1_2k/1b. Additionally, a hairpin downstream of the crossover site in the 1b parental strain was destabilized by 5 mutations present in the RF_2k/1b recombinant. We examined if this hairpin was also present in the RF8_ 2/1a reported here or in any of the other recombinants with a genotype 2 5' UTR-NS2 region. Although, all recombinants exhibited RNA base pairing in this region, all secondary structures appeared less stable than that of RF_2k/1b (data not shown), with two small stems being predicted rather than one longer stem. Non-recombinant 2b and the RF8_ 2/1a have similarly stable hairpins in this region, however, neither are as stable as the 2k hairpin (recombinant or parental).
For over ten years, HCV genotyping has been the critical parameter to determine both the likelihood of response to therapy, as well as the duration of therapy needed to obtain a Sustained Virologic Response (SVR) [19–21]. While several HCV genotyping methods exist, none are FDA approved in the United States. These methods are based on primarily targeting only the 5'structural regions of virus and thus cannot easily identify recombinant strains. Based on the last Hepatitis Viral Load proficiency survey (HVL-C) administered by the College of American Pathologists in 2010 , the most commonly employed HCV genotyping method, utilized by over 60% (110/177) of participating clinical diagnostic labs in the United States, is the Versant HCV (LiPA) 2.0 assay (manufactured by Innogenetics, distributed by Siemens Healthcare Diagnostics). While CE marked in Europe and approved for research use only in the United States, this assay is based on line probe hybridization targeting DNA sequence information within the 5' UTR and the contiguous structural core (C) region of HCV (Figure 1). While 9% (16/177) of participants did conduct genotyping by DNA sequencing using the Siemens Diagnostics TruGene system, sequencing information is again only obtained from the 5' UTR of HCV. These 5' structural regions have been utilized historically for genotyping because they are adequately conserved such that a limited number of primers or probes can amplify and recognize all isolates, respectively, but have sufficient diversity to distinguish between non-recombinant genotypes 1-6. However, it should be noted that information provided solely by the 5'UTR is insufficient for subtype identification and in some cases for genotype identification.
It remains unclear how much of the genome needs to be genotype 2 in order for the clinical response to justify a 12-24 week treatment course rather than 48 weeks advocated for genotype 1. Both within genotype 1, as well as between genotypes 1 and 2, there are known differences between the ability of NS5A to bind the ds RNA induced PKR . These differences in NS5A binding alter the cellular interferon mediated antiviral response that in turn has been postulated to explain the corresponding clinical response. Clinical response to interferon-based regimens depends upon both viral factors (including NS5A and E2 glycoprotein) as well as host genetic factors, including lambda interferon polymorphisms , but the viral genotype assigned by clinical labs should closely reflect related strains and ideally indicate the historical antiviral response for those strains. Data from a chimeric mouse model, as well as anecdotal clinical data, suggests the RF1_2k/1b strain is more resistant to interferon than some genotype 2 strains . As protease inhibitors and other directly acting antivirals become available, it will become increasingly important to know the genotype of each viral drug target of the isolate infecting the patient in order to determine the most effective therapy for that patient, and minimize the side-effects of therapy. Data from the PROVE 3 Protease Inhibitor trial , among others , suggests that subtyping may be clinically useful. Unfortunately, current methods for HCV genotyping primarily solely targeting the 5'UTR and possibly contiguous core (C) structural regions do not provide sufficient information across the entire genome to detect the possibility of recombinant species which may be critical for the determination for treatment efficacy.
In conclusion, we report here the first naturally occurring HCV recombinant in the United States. While clearly an independent event from other recombinants, this strain shares several characteristics with those previously reported in that it has genotype 2 5' UTR and structural genes, and a crossover point near the NS2/3 junction. At this time we cannot tell whether this recombinant strain is circulating in patients besides the one reported here, but the patient was viremic from this strain for months and likely years. Hybridization probe techniques and DNA sequencing targeting only the 5' UTR/core regions are frequently used to clinically genotype HCV to determine the dose and duration of therapy. One advantage of using direct DNA sequencing to genotype viruses is that the DNA sequence of amplified regions can be aligned with known recombinants, such as the strain reported here, particularly if multiple regions are sequenced. Using this approach, undiscovered recombinants may still be missed depending on the regions amplified, but at least an assessment of whether further testing is needed to rule out known recombinants can be made. The presence of circulating recombinants of HCV may have significant ramifications for the efficacy and selection of therapy. Clearly more comprehensive HCV genotyping is required to ascertain the significance of HCV recombinant isolates in clinical practice.
Hepatitis C Virus
Food and Drug Administration
RS is supported by the American Cancer Society, and a Veteran's Administration Merit Award (5I01CX000117-02)
- Lavanchy D: The global burden of hepatitis C. Liver Int 2009,1(29 Suppl):74-81.View ArticleGoogle Scholar
- Kuiken C, Simmonds P: Nomenclature and numbering of the hepatitis C virus. Methods Mol Biol 2009, 510: 33-53. 10.1007/978-1-59745-394-3_4View ArticlePubMedGoogle Scholar
- Simmonds P, Bukh J, Combet C, Deleage G, Enomoto N, Feinstone S, Halfon P, Inchauspe G, Kuiken C, Maertens G, Mizokami M, Murphy DG, Okamoto H, Pawlotsky JM, Penin F, Sablon E, Shin IT, Stuyver LJ, Thiel HJ, Viazov S, Weiner AJ, Widell A: Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology 2005,42(4):962-973. 10.1002/hep.20819View ArticlePubMedGoogle Scholar
- Seeff LB, Hoofnagle JH: National Institutes of Health Consensus Development Conference: management of hepatitis C: 2002. Hepatology 2002,36(5 Suppl 1):S1-2.View ArticlePubMedGoogle Scholar
- Ramirez S, Perez-del-Pulgar S, Carrion JA, Coto-Llerena M, Mensa L, Dragun J, Garcia-Valdecasas JC, Navasa M, Forns X: Hepatitis C virus superinfection of liver grafts: a detailed analysis of early exclusion of non-dominant virus strains. J Gen Virol 2010,91(Pt 5):1183-1188.View ArticlePubMedGoogle Scholar
- Cook L, Ng KW, Bagabag A, Corey L, Jerome KR: Use of the MagNA Pure LC automated nucleic acid extraction system followed by real-time reverse transcription-PCR for ultrasensitive quantitation of hepatitis C virus RNA. J Clin Microbiol 2004, 42: 4130-4136. 10.1128/JCM.42.9.4130-4136.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Tang YM, Sefers SE, Li H: Primer sequence modification enhances hepatitis C virus genotype coverage. J Clin Microbiol 2005, 43: 3576-3577. 10.1128/JCM.43.7.3576-3577.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Laperche S, Lunel F, Izopet J, Alain S, Deny P, Duverlie G, Gaudy C, Pawlotsky JM, Plantier JC, Pozetto B, Thibault V, Tosetti F, Lefrere JJ: Comparison of hepatitis C virus NS5b and 5' noncoding gene sequencing methods in a multicenter study. J Clin Microbiol 2005, 43: 733-739. 10.1128/JCM.43.2.733-739.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Kuiken C, Yusim K, Boykin L, Richardson R: The Los Alamos hepatitis C sequence database. Bioinformatics 2005,21(3):379-384. 10.1093/bioinformatics/bth485View ArticlePubMedGoogle Scholar
- Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD: Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 2003,31(13):3497-3500. 10.1093/nar/gkg500PubMed CentralView ArticlePubMedGoogle Scholar
- Combet C, Garnier N, Charavay C, Grando D, Crisan D, Lopez J, Dehne-Garcia A, Geourjon C, Bettler E, Hulo C, Le Mercier P, Bartenschlager R, Diepolder H, Moradpour D, Pawlotsky JM, Rice CM, Trepo C, Penin F, Deleage G: euHCVdb: the European hepatitis C virus database. Nucleic Acids Res 2007, (35 Database):D363-6.
- Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, Dufayard JF, Guindon S, Lefort V, Lescot M, Claverie JM, Gascuel O: Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 2008, (36 Web Server):W465-9.
- Lole KS, Bollinger RC, Paranjape RS, Gadkari D, Kulkarni SS, Novak NG, Ingersoll R, Sheppard HW, Ray SC: Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J Virol 1999,73(1):152-160.PubMed CentralPubMedGoogle Scholar
- Kalinina O, Norder H, Magnius LO: Full-length open reading frame of a recombinant hepatitis C virus strain from St Petersburg: proposed mechanism for its formation. J Gen Virol 2004,85(Pt 7):1853-1857.View ArticlePubMedGoogle Scholar
- Chen Z, Weck KE: Hepatitis C virus genotyping: interrogation of the 5' untranslated region cannot accurately distinguish genotypes 1a and 1b. J Clin Microbiol 2002,40(9):3127-3134. 10.1128/JCM.40.9.3127-3134.2002PubMed CentralView ArticlePubMedGoogle Scholar
- Hraber PT, Fischer W, Bruno WJ, Leitner T, Kuiken C: Comparative analysis of hepatitis C virus phylogenies from coding and non-coding regions: the 5' untranslated region (UTR) fails to classify subtypes. Virol J 2006, 3: 103. 10.1186/1743-422X-3-103PubMed CentralView ArticlePubMedGoogle Scholar
- Pickett BE, Striker R, Lefkowitz EJ: Evidence for separation of HCV subtype 1a into two distinct clades. J Viral Hepat 2010,18(9):608-618.PubMed CentralView ArticlePubMedGoogle Scholar
- Kieffer TL, Sarrazin C, Miller JS, Welker MW, Forestier N, Reesink HW, Kwong AD, Zeuzem S: Telaprevir and pegylated interferon-alpha-2a inhibit wild-type and resistant genotype 1 hepatitis C virus replication in patients. Hepatology 2007,46(3):631-639. 10.1002/hep.21781View ArticlePubMedGoogle Scholar
- McHutchison JG, Gordon SC, Schiff ER, Shiffman ML, Lee WM, Rustgi VK, Goodman ZD, Ling MH, Cort S, Albrecht JK: Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis Interventional Therapy Group. N Engl J Med 1998,339(21):1485-1492. 10.1056/NEJM199811193392101View ArticlePubMedGoogle Scholar
- Poynard T, Marcellin P, Lee SS, Niederau C, Minuk GS, Ideo G, Bain V, Heathcote J, Zeuzem S, Trepo C, Albrecht J: Randomised trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon alpha2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. International Hepatitis Interventional Therapy Group (IHIT). Lancet 1998,352(9138):1426-1432. 10.1016/S0140-6736(98)07124-4View ArticlePubMedGoogle Scholar
- Poynard T, McHutchison J, Goodman Z, Ling MH, Albrecht J: Is an "a la carte" combination interferon alfa-2b plus ribavirin regimen possible for the first line treatment in patients with chronic hepatitis C? The ALGOVIRC Project Group. Hepatology 2000,31(1):211-218. 10.1002/hep.510310131View ArticlePubMedGoogle Scholar
- 2010 College of American of Pathologists Hepatitis Viral Load Proficiency (HVC-L) Survey
- Zhou Y, Wang X, Hong G, Tan Z, Zhu Y, Lan L, Mao Q: Natural intragenotypic and intergenotypic HCV recombinants are rare in southwest China even among patients with multiple exposures. J Clin Virol 2010,49(4):272-276. 10.1016/j.jcv.2010.08.007View ArticlePubMedGoogle Scholar
- Sentandreu V, Jimenez-Hernandez N, Torres-Puente M, Bracho MA, Valero A, Gosalbes MJ, Ortega E, Moya A, Gonzalez-Candelas F: Evidence of recombination in intrapatient populations of hepatitis C virus. PLoS One 2008,3(9):e3239. 10.1371/journal.pone.0003239PubMed CentralView ArticlePubMedGoogle Scholar
- Yamamura J, Ichimura H: A natural inter-genotypic (2b/1b) recombinant of hepatitis C virus in the Philippines. J Med Virol 2006, 78: 1423-1428. 10.1002/jmv.20714View ArticlePubMedGoogle Scholar
- Kalinina O, Norder H, Mukomolov S, Magnius LO: A natural intergenotypic recombinant of hepatitis C virus identified in St. Petersburg. J Virol 2002,76(8):4034-4043. 10.1128/JVI.76.8.4034-4043.2002PubMed CentralView ArticlePubMedGoogle Scholar
- Lee YM, Lin HJ, Chen YJ, Lee CM, Wang SF, Chang KY, Chen TL, Liu HF, Chen YM: Molecular epidemiology of HCV genotypes among injection drug users in Taiwan: Full-length sequences of two new subtype 6w strains and a recombinant form_2b6w. J Med Virol 2009, 82: 57-68.View ArticleGoogle Scholar
- Legrand-Abravanel F, Claudinon J, Nicot F, Dubois M, Chapuy-Regaud S, Sandres-Saune K, Pasquier C, Izopet J: New natural intergenotypic (2/5) recombinant of hepatitis C virus. J Virol 2007,81(8):4357-4362. 10.1128/JVI.02639-06PubMed CentralView ArticlePubMedGoogle Scholar
- Noppornpanth S, Lien TX, Poovorawan Y, Smits SL, Osterhaus AD, Haagmans BL: Identification of a naturally occurring recombinant genotype 2/6 hepatitis C virus. J Virol 2006,80(15):7569-7577. 10.1128/JVI.00312-06PubMed CentralView ArticlePubMedGoogle Scholar
- Moreau I, Hegarty S, Levis J, Sheehy P, Crosbie O, Kenny-Walsh E, Fanning LJ: Serendipitous identification of natural intergenotypic recombinants of hepatitis C in Ireland. Virol J 2006, 3: 95. 10.1186/1743-422X-3-95PubMed CentralView ArticlePubMedGoogle Scholar
- Tallo T, Norder H, Tefanova V, Krispin T, Schmidt J, Ilmoja M, Orgulas K, Pruunsild K, Priimagi L, Magnius LO: Genetic characterization of hepatitis C virus strains in Estonia: fluctuations in the predominating subtype with time. J Med Virol 2007,79(4):374-382. 10.1002/jmv.20828View ArticlePubMedGoogle Scholar
- Kurbanov F, Tanaka Y, Chub E, Maruyama I, Azlarova A, Kamitsukasa H, Ohno T, Bonetto S, Moreau I, Fanning LJ, Legrand-Abravanel F, Izopet J, Naoumov N, Shimada T, Netesov S, Mizokami M: Molecular epidemiology and interferon susceptibility of the natural recombinant hepatitis C virus strain RF1_2k/1b. J Infect Dis 2008,198(10):1448-1456. 10.1086/592757View ArticlePubMedGoogle Scholar
- Enomoto N, Sakuma I, Asahina Y, Kurosaki M, Murakami T, Yamamoto C, Ogura Y, Izumi N, Marumo F, Sato C: Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection. N Engl J Med 1996,334(2):77-81. 10.1056/NEJM199601113340203View ArticlePubMedGoogle Scholar
- Ge D, Fellay J, Thompson AJ, Simon JS, Shianna KV, Urban TJ, Heinzen EL, Qiu P, Bertelsen AH, Muir AJ, Sulkowski M, McHutchison JG, Goldstein DB: Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 2009,461(7262):399-401. 10.1038/nature08309View ArticlePubMedGoogle Scholar
- McHutchison JG, Manns MP, Muir AJ, Terrault NA, Jacobson IM, Afdhal NH, Heathcote EJ, Zeuzem S, Reesink HW, Garg J, Bsharat M, George S, Kauffman RS, Adda N, Di Bisceglie AM: Telaprevir for previously treated chronic HCV infection. N Engl J Med 2010,362(14):1292-1303. 10.1056/NEJMoa0908014View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.