Monday, October 3, 2011

Naturally Occurring Genotype 2b/1a Hepatitis C Virus in the United States

Naturally Occurring Genotype 2b/1a Hepatitis C Virus in the United States

Dipankar Bhattacharya, Molly A Accola, Israr H Ansari, Rob Striker and William M Rehrauer
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Virology Journal 2011, 8:458 doi:10.1186/1743-422X-8-458Published: 3 October 2011
Abstract (provisional)

Hepatitis C Virus (HCV) infected patients are frequently repeatedly exposed to the virus, but very few recombinants between two genotypes have been reported. Findings: 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.

Discussion Only
Full Text
For over ten years, HCV genotyping has been the critical parameter to determine both thelikelihood of response to therapy, as well as the duration of therapy needed obtain a Sustained Virologic Response (SVR) [19,20,21]. 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 can not easily identify recombinant strains. Based on the last Hepatitis Viral Load proficiency survey (HVL-C) administered by the College of American Pathologists in 2010 [22], the most commonly employed HCV genotyping method, utilized by over 60% (110/177) of participating clinical diagnostic labs in the United States, is the VersantHCV (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 ofHCV. 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. Several studies looking for HCV recombinants in intravenous drug users and other populations where multiple exposures are likely have been performed and in general recombination does seem to be a rare event [23].

While other reports of recombination between different genotypes exist [24], the DNA sequence of entire recombinant genomes and site of recombination remains to be determined. Interestingly, all full-length recombinants described todate between two genotypes have included a 5’ portion of genotype 2 [25,26,27,28,29]. So faronly RF1_2k/1b has been shown to be circulating in multiple patients described in Russia [26],Ireland [30], Estonia [31] and Uzbekistan [32].

As shown in Figure 4, all reported HCVrecombinants have similar, but non-identical cross over points to the RF8_2/1a (reported here) orRF1_2k/1b.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 genotype1.

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 [33]. 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 [34], 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 [32].

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 [35], among others [18],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 structura lgenes, 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 prove techniques and DNA sequencing targeting only the 5’ UTR/core regions are frequently used to clinically genotypeHCV to determine the dose and duration of therapy.

One advantage of using direct DNAsequencing 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 comprehensiveHCV genotyping is required to as certain the significance of HCV recombinant isolates in clinical practice.

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