Mutant Ninja viruses
Authors: Graham R. Foster
Journal: Hepatology (2014)

Potential conflict of interest: Prof. Foster consults, advises, and is on the speakers' bureau of Boehringer Ingelheim, Bristol‐Myers Squibb, Roche, Gilead, Merck, Novartis, and Janssen. See Article on Page 471 As the epidemic of morbidity and mortality from end‐stage liver disease resulting from hepatitis C threatens to engulf us,1 new treatment regimes have emerged that seem likely to turn back the tide and allow us to consider a total victory over this virus.2 Some, including myself, believe that global eradication of hepatitis C may now be possible. This issue of Hepatology contains an article from Hongmei Mo's team,5 which suggests that declarations of victory may be premature and the virus may still have a few tricks up its sleeve. The new all‐oral treatments for chronic hepatitis C infection focus on combination regimes with multiple drugs that attack different viral proteins that are critical for viral replication. Two, subtly different, approaches are leading the way—one involves augmented triple therapy with a protease inhibitor (ABT‐450), a nonstructural (NS)5A inhibitor (ombitasvir), and a non‐nucleotide inhibitor (dasabuvir)4 and the other is based around the nucleotide inhibitor, sofosbuvir (SOF),2 either with or without a second direct‐acting antiviral agent. The hepatitis C virus (HCV) is a highly divergent virus that can be classified into seven genotypes,6 which differ from one another in their genomic sequence and their geographical distribution: Genotype 1 is common in the United States, Japan, and Europe, whereas genotype 4 is found in Egypt and genotype 3 dominates in the Indian subcontinent. The all‐oral treatment regimes require different drug combinations for the different viral genotypes, for example, the current triple‐therapy regime based around ABT‐450 is only active against genotype 1,4 with small‐scale studies suggesting activity against genotype 4,7 and SOF‐based regimes require different adjuncts for the different genotypes. For genotype 2 infection, 12 weeks of SOF plus ribavirin (RBV) is required,8 but for genotype 1–infected patients, this is insufficient and an additional drug (either an NS5A inhibitor [such as ledipasvir]2 or a protease inhibitor [simeprevir]9 is required. For genotype 3–infected patients, a further 12 weeks of SOF plus RBV is needed,3 although adding SOF to pegylated interferon (Peg‐IFN) and RBV for 12 weeks may suffice.10 Genotyping the virus to decide the optimal treatment regime is a well‐established technique involving an assessment of a conserved region of the virus that is known to differ among genotypes. The viral sequence can be determined by direct sequencing or monitoring hybridization of viral polymerase chain reaction products to genotype‐specific control DNA sequences. Both approaches are believed to generate identical results, and commercial hybridization assays based on the 5' region of the virus and sequencing assays commonly based on the 3' (NS5B) regions of the virus are often employed. The article by Hedskog et al.5 suggests that the approach used may be very important in determining which drugs to use. Hepatitis C is a flavivirus that replicates through RNA intermediates. It is well established that, for some flaviviridae, infection of a cell with two different viral strains may occur and allow "crossover" of the replicating viral strands with the generation of a hybrid, recombinant virus that contains features of both viruses. These hybrid virions have been described in HCV, but they are believed to be rare and of doubtful clinical significance.11 Hedskog et al.'s article confirms that these hybrid viruses are indeed rare, but they may be of clinical importance. The article examines samples collected as part of the extensive SOF clinical development program. All samples were assigned a genotype using a commercial hybridization assay (InnoLipa), which looks at conserved sequences in the 5' region of the virus. Over 2,000 samples were reexamined and regenotyped using a technique in which the 3' region of the virus was sequenced. In 0.5% of the cases, the two assays gave divergent results, with genotyping of the 5' end indicating infection with genotype 2 HCV and sequencing of the 3' (opposite end) indicating genotype 1. Figure 1 illustrates how the different assays lead to different results. Detailed analysis of these discordant viruses showed that they were hybrid viruses with a crossover point located in the NS2/NS3 region. Essentially, these hybrid/recombinant viruses are trains where the front half has a different destination from the rear, and looking at the sign of the front of the train and then sitting in the rear may lead to the disappointment of ending up in Baltimore when you dreamed of Boston. Virologically, these hybrid virions pose interesting questions about how two different genotypes can infect the same cell and why the recombination site is restricted to the NS2/3 region. Dissecting out these questions may reveal new information about the virology of HCV, and understanding why the NS2/3 region is fluid is likely to provide new insights into the structure‐function relationship of viral RNA. For the clinician, the interesting, and worrying, finding from the article is that the hybrid viruses respond differently to treatment.Figure 1: Schematic illustrating the HCV genome and highlighting the sites at which different genotyping assays assess viral sequence. The hybrid virus detected by Hedskog et al. is illustrated in the lower panel.Genotype 2 HCV is exquisitely sensitive to SOF, and only 12 weeks of therapy with RBV is required. This regime is inadequate for genotype 1 HCV, where longer durations of treatment or additional drugs are needed. In the Gilead trials, patients were allocated treatment regimes based on their genotype as determined by an analysis of their 5' region. However, the site of action of SOF is at the opposite end of the virus (the NS5B, 3' region), and for the 11 patients with the hybrid viral infections whose viral sequence could be studied in the Hepatology article, the response to treatment was suboptimal; only 3 of 11 patients responded to a typical genotype 2 regime, whereas, in general, responses in genotype 2 are well in excess of 90%. Retreatment of some of these patients with "genotype 1 type" SOF regimes (either 24 weeks of SOF or SOF plus Peg‐IFN and RBV) led to viral clearance in most patients, indicating that they were infected with SOF‐sensitive viruses. The implication from these findings is clear—some patients have recombinant viruses with a genotype two 5' region and a genotype one 3' region, and the 3' region determines the outcome of therapy. These novel viruses are true viral Ninjas hiding a challenging array of "difficult‐to‐cure" genotype 1 enzymes under an "easy‐to‐cure" genotype 2 coat. Although the problems with these hybrid virions have only been observed in SOF‐based clinical studies, it is probable that other regimes involving genotype 2 HCV will have similar problems—as seen in Fig. 1, all of the critical viral enzymes are switched in the hybrid virus so that any drug regime that has different efficacy against genotype 2 and other genotypes will be exposed to this problem. Only truly pangenotypic regimes may be immune. It is interesting that Hedskog et al.'s article only identified genotype 2/1 hybrid viruses. It is unclear whether this is a feature of these genotypes, which may combine more readily than other viral strains, or whether the regions involved in this trial program have a high prevalence of these genotypes leading to selection bias. It is not yet clear why the recombinants all had genotype two 5' ends, and it remains to be seen whether hybrids with nongenotype two 5' domains are viable. It will be interesting, and important, to determine whether other genotypes combine, and as therapy moves into areas with different viral strains, we will need to be alert to the possibility of new viral hybrids combining, for example, genotypes 3 and 4. The clinical importance of this new information will depend upon the patient population and the viral genotyping assay. Clinicians treating patient populations where genotype 2 is rare may choose to carry on regardless, and physicians who rely on 3' sequencing assays are unlikely to be troubled by these new data. However, those who regularly treat patients with genotype 2 infection and who rely on 5' genotyping assays may need to reconsider their approach to pretreatment assessment. It is clear that viral hybrids are rare—only 2.5% of patients with genotype 2 had hybrid viruses in the Hedskog et al. study, but, given the costs of the new therapies, patients will not look kindly upon clinicians who give them the wrong treatment based on an inadequate assessment of their viral strain. Many clinicians breathed a sigh of relief when complex virological measurements were dropped from the therapeutic monitoring required for patients with HCV and many will have deleted the phone number of the local virologist. This issue of Hepatology reminds us that we should perhaps reintroduce ourselves to our virology colleagues; we may still need their guidance to manage our patients with hepatitis C.