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.
Hepatitis C Virus
Food and Drug Administration
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.
RS is supported by the American Cancer Society, and a Veteran's Administration Merit Award (5I01CX000117-02)
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