Resistance of high fitness hepatitis C virus to lethal mutagenesis

Abstract

Viral fitness quantifies the degree of virus adaptation to a given environment. How viral fitness can influence the mutant spectrum complexity of a viral quasispecies subjected to lethal mutagenesis has not been investigated. Here we document that two high fitness hepatitis C virus populations display higher resistance to the mutagenic nucleoside analogues favipiravir and ribavirin than their parental, low fitness HCV. All populations, however, exhibited a mutation transition bias indicative of active mutagenesis. Resistance to the analogues was associated with a limited expansion of mutant spectrum complexity, as evidenced by several diversity indices used to characterize mutant spectra. The results are consistent with a replicative site-drug competition mechanism that was previously proposed for HCV fitness-associated resistance to non-mutagenic inhibitors. Other alternative, non-mutually exclusive mechanisms are considered. The results introduce viral fitness as a relevant parameter to evaluate the response of viruses to lethal mutagenesis, with implications for antiviral designs.

Guanosine inhibits hepatitis C virus replication and increases indel frequencies, associated with altered intracellular nucleotide pools

“…Here, we document inhibition of HCV replication by Gua in single and serial infections of Huh-7.5 cells that led to loss of infectivity without significant toxicity for the host cells. The antiviral action of Gua was also exerted on high fitness HCV, albeit without loss of infectivity after 5 passages in the presence of Gua, in agreement with the drug resistance phenotype displayed by high fitness HCV (Fig 1) [14][15][16][17]. The antiviral effect of Gua was not observed for FMDV in BHK-21 cells or for LCMV and VSV in Huh-7.5 cells (Fig 3).…”

“…The HCV p100 virus [HCV p0 passaged 100 times in Huh-7.5 reporter cells], shows a relative fitness that is 2.2 times higher than that of the HCV p0 parental population [14]. Since viral fitness can influence the response of the virus to antiviral agents [15][16][17], HCV p100 was used to study the response of a high fitness HCV to Gua. For this, HCV p100 was subjected to 5 serial passages in Huh-7.5 reporter cells using an initial m.o.i.…”

Synergistic Lethal Mutagenesis of Hepatitis C Virus

“…Two hundred serial passages of HCV p0 in Huh-7.5 cells resulted in population HCV p200, which displayed a 2-to 3-fold increase in replicative fitness, as calculated from progeny production in single and serial infections, as well as from growth competition experiments (72,73). The high-fitness intermediate-passage HCV p100 and HCV p200 displayed a lower sensitivity to the anti-HCV agents than their parental virus, HCV p0, including to favipiravir and ribavirin (72,74,75), thus providing HCV populations for a stringent evaluation of synergistic activities. The infectious progeny production upon single infections of Huh-7.5 cells by HCV p0, HCV p100, and HCV p200 was 10-to 100-fold lower with favipiravir-ribavirin combinations than with the individual analogues ( Fig.…”

“…Cells, viruses, and infections. Huh-7.5 cells and Huh-7.5 reporter cells were grown in Dulbecco's modification of Eagle's medium (DMEM) at 37°C in 5% CO 2 as previously described (74,90,91). Huh-7.5 reporter cells were used for all infections in the absence and the presence of drugs, while Huh-7.5 cells were used for titration of infectivity.…”

“…At 3 days postinfection, the monolayers were washed with phosphate-buffered saline (PBS), fixed with cold methanol, and stained using anti-NS5A monoclonal antibody 9E10 (92). Virus titers (expressed as the 50% tissue culture infective dose [TCID 50 ] per milliliter) were calculated as previously described (67,74).…”