Hepatitis C Resistance-Associated Substitutions Among People Who Inject Drugs Treated with Direct-Acting Antiviral-Containing Regimens

Background Resistance-associated substitutions (RASs) to HCV direct-acting antivirals (DAAs) can contribute to virologic failure and limit retreatment options. People who inject drugs (PWID) are at highest risk for transmission of resistant virus. We report on RASs at baseline and following virologic failure in DAA-naive and protease inhibitor-experienced PWID. Methods We sequenced the NS3/4A, NS5A, and NS5B regions from 150 PWID with genotype 1 (GT1) viruses; 128 (85.3%) GT1a, 22 (14.7%) GT1b. Results Among the 139 (92.7%) DAA-naive PWID, 85/139 (61.2%) had baseline RASs – 67/139 (48.2%) in NS3 (predominantly Q80K/L); 25/139 (18.0%) in NS5A; and 8/139 (5.8%) in NS5B. Of the 11 protease inhibitor-experienced participants, 9 had baseline NS3 RASs (V36L N=1, Q80K N=9) and 4 had baseline NS5A RASs (M28V N=2, H58P N=1, A92T N=1). Among the 11 participants who had post-treatment samples with detectable virus (seven treatment failures, one late relapse, three reinfections), one sofosbuvir/ledipasvir failure had a baseline H58P. Two sofosbuvir/ledipasvir-treated participants developed new NS5A mutations (Q30H, Y93H, L31M/V). Otherwise, no RASs were detected. Conclusion Our results demonstrate RAS prevalence among DAA-naive PWID is comparable to that in the general population. Only 2/150 (1.3%) in our longitudinal cohort developed treatment-emergent RASs. Concern for transmission of resistant virus may therefore be minimal.

Favipiravir-resistant influenza A virus shows potential for transmission

Favipiravir is a nucleoside analogue which has been licensed to treat influenza in the event of a new pandemic. We previously described a favipiravir resistant influenza A virus generated by in vitro passage in presence of drug with two mutations: K229R in PB1, which conferred resistance at a cost to polymerase activity, and P653L in PA, which compensated for the cost of polymerase activity. However, the clinical relevance of these mutations is unclear as the mutations have not been found in natural isolates and it is unknown whether viruses harbouring these mutations would replicate or transmit in vivo. Here, we infected ferrets with a mix of wild type p(H1N1) 2009 and corresponding favipiravir-resistant virus and tested for replication and transmission in the absence of drug. Favipiravir-resistant virus successfully infected ferrets and was transmitted by both contact transmission and respiratory droplet routes. However, sequencing revealed the mutation that conferred resistance, K229R, decreased in frequency over time within ferrets. Modelling revealed that due to a fitness advantage for the PA P653L mutant, reassortment with the wild-type virus to gain wild-type PB1 segment in vivo resulted in the loss of the PB1 resistance mutation K229R. We demonstrated that this fitness advantage of PA P653L in the background of our starting virus A/England/195/2009 was due to a maladapted PA in first wave isolates from the 2009 pandemic. We show there is no fitness advantage of P653L in more recent pH1N1 influenza A viruses. Therefore, whilst favipiravir-resistant virus can transmit in vivo, the likelihood that the resistance mutation is retained in the absence of drug pressure may vary depending on the genetic background of the starting viral strain.

Broadening a SARS-CoV-1 neutralizing antibody for potent SARS-CoV-2 neutralization through directed evolution

The emergence of SARS-CoV-2 underscores the need for strategies to rapidly develop neutralizing monoclonal antibodies that can function as prophylactic and therapeutic agents and to help guide vaccine design. Here, we demonstrate that engineering approaches can be used to refocus an existing neutralizing antibody to a related but resistant virus. Using a rapid affinity maturation strategy, we engineered CR3022, a SARS-CoV-1 neutralizing antibody, to bind SARS-CoV-2 receptor binding domain with >1000-fold improved affinity. The engineered CR3022 neutralized SARS-CoV-2 and provided prophylactic protection from viral challenge in a small animal model of SARS-CoV-2 infection. Deep sequencing throughout the engineering process paired with crystallographic analysis of an enhanced antibody elucidated the molecular mechanisms by which engineered CR3022 can accommodate sequence differences in the epitope between SARS-CoV-1 and SARS-CoV-2. The workflow described provides a blueprint for rapid broadening of neutralization of an antibody from one virus to closely related but resistant viruses.

Primary HIV drug resistance among newly HIV type-1 diagnosed patients in St. Petersburg

There is concern that the widespread use of antiretroviral drugs (ARV) to treat human immunodeficiency virus 1 (HIV-1) infection may result in the emergence of transmission of drug-resistant virus among persons newly infected with HIV-1. Russia is one of a growing number of countries in the world where drug-resistant HIV is becoming a serious health problem because it has the potential to compromise the efficacy of antiretroviral therapy (ART) at the population level.Materials and methods. We performed a genetic analysis of the HIV-1 plasma derived pol gene among the newly diagnosed ART-naïve HIV-1 infected patients during the period from November 2018 to October 2019 in the St. Petersburg Clinical Infectious Diseases Hospital named after S.P. Botkin. We used reverse transcriptase polymerase chain reaction (RT-PCR) followed by direct sequencing of PCR products to determine the prevalence of primary drug resistance (PDR) conferring mutations. HIV-1 genotypes were determined by phylogenetic analysis.Results. The predominant HIV-1 subtype was A1 (87.2%), followed by B (11.8%) and CRF06_cpx (1%). The overall prevalence of PDR was 11%. Virus with known resistance-conferring mutations to any nucleoside reverse transcriptase inhibitors (NRTIs) was found in 8 individuals, to any non NRTIs in 5 subjects, and to any protease inhibitors in 1 case. Multidrug-resistant virus was identified in 2 individuals (2%).Conclusion. The distribution of HIV-1 genotypes in St. Petersburg, Russia is diverse. The emerging prevalence of PDR in ART-naïve patients demonstrates the significance of constant monitoring due to the challenges it presents towards treatment.

Using Wolbachia to eliminate dengue – will the virus fight back?

Recent field trials have demonstrated that dengue incidence can be substantially reduced by introgressing strains of the endosymbiotic bacterium, Wolbachia into Aedes aegypti mosquito populations. This strategy relies on Wolbachia reducing the susceptibility of Ae. aegypti to disseminated infection by positive-sense RNA viruses like dengue. However, RNA viruses are well known to adapt to antiviral pressures. Here we review the viral infection stages where selection for Wolbachia-resistant virus variants could occur. We also consider the genetic constraints imposed on viruses that alternate between vertebrate and invertebrate hosts, and the likely selection pressures that dengue virus might adapt to in order to be effectively transmitted by Ae. aegypti that carry Wolbachia. Whilst there are hurdles to dengue viruses developing resistance to Wolbachia, we suggest that long-term surveillance for resistant viruses should be an integral component of Wolbachia-introgression biocontrol programs.

A Comparison of Porphyrin Photosensitizers in Photodynamic Inactivation of RNA and DNA Bacteriophages

Effective broad-spectrum antiviral treatments are in dire need as disinfectants and therapeutic alternatives. One such method of disinfection is photodynamic inactivation, which involves the production of reactive oxygen species from dissolved oxygen in response to light-stimulated photosensitizers. This study evaluated the efficacy of functionalized porphyrin compounds for photodynamic inactivation of bacteriophages as human virus surrogates. A blue-light light emitting diode (LED) lamp was used to activate porphyrin compounds in aqueous solution (phosphate buffer). The DNA bacteriophages ΦX174 and P22 were more resistant to porphyrin TMPyP photodynamic inactivation than RNA bacteriophage fr, with increasing rates of inactivation in the order: ΦX174 << P22 << fr. Bacteriophage ΦX174 was therefore considered a resistant virus suitable for the evaluation of three additional porphyrins. These porphyrins were synthesized from TMPyP by inclusion of a central palladium ion (PdT4) and/or the addition of a hydrophobic C14 chain (PdC14 or C14). While the inactivation rate of bacteriophage ΦX174 via TMPyP was similar to previous reports of resistant viruses, ΦX174 inactivation increased by a factor of approximately 2.5 using the metalloporphyrins PdT4 and PdC14. The order of porphyrin effectiveness was TMPyP < C14 < PdT4 < PdC14, indicating that both Pd2+ ligation and C14 functionalization aided virus inactivation.

Favipiravir-resistant influenza A virus shows potential for transmission

Favipiravir is a nucleoside analogue which has been licensed to treat influenza in the event of a new pandemic. We previously described a favipiravir resistant influenza A virus generated by in vitro passage in presence of drug with two mutations: K229R in PB1, which conferred resistance at a cost to polymerase activity, and P653L in PA, which compensated for the cost of polymerase activity. However, the clinical relevance of these mutations is unclear as the mutations have not been found in natural isolates and it is unknown whether viruses harbouring these mutations would replicate or transmit in vivo. Here, we infected ferrets with a mix of wild type p(H1N1) 2009 and corresponding favipiravir-resistant virus and tested for replication and transmission in the absence of drug. Favipiravir-resistant virus successfully infected ferrets and was transmitted by both contact transmission and respiratory droplet routes. However, sequencing revealed the mutation that conferred resistance, K229R, decreased in frequency over time within ferrets. Modelling revealed that due to a fitness advantage for the PA P653L mutant, reassortment with the wild-type virus to gain wild-type PB1 segment in vivo resulted in the loss of the PB1 resistance mutation K229R. We demonstrated that this fitness advantage of PA P653L in the background of our starting virus A/England/195/2009 was due to a maladapted PA in first wave isolates from the 2009 pandemic. We show there is no fitness advantage of P653L in more recent pH1N1 influenza A viruses. Therefore, whilst favipiravir-resistant virus can transmit in vivo, the likelihood that the resistance mutation is retained in the absence of drug pressure may vary depending on the genetic background of the starting viral strain.Author SummaryIn the event of a new influenza pandemic, drugs will be our first line of defence against the virus. However, drug resistance has proven to be particularly problematic to drugs against influenza. Favipiravir is a novel drug which might be used against influenza virus in the event of a new pandemic. Is resistance likely to be a problem for the use of favipiravir? Our previous work has shown that resistance to favipiravir can be generated in cell culture but we don’t know whether there will be a cost preventing the spread of resistance in whole organisms. Here, we used a mix of wild-type and resistant influenza viruses from early in the 2009 pandemic to test whether viruses resistant to favipiravir could transmit between ferrets. We found that the resistant viruses could transmit but that the resistance mutation was selected against within some ferrets. Using modelling and in vitro experiments, we found that the resistant mutation was selected against in the influenza strain from our experiment but not in more recently evolved strains. Our results show that favipiravir resistant viruses could spread if resistance is generated but the probability will depend on the genetic background of the virus.

An Extended Primer Grip of Picornavirus Polymerase Facilitates Sexual RNA Replication Mechanisms

ABSTRACT Picornaviruses have both asexual and sexual RNA replication mechanisms. Asexual RNA replication mechanisms involve one parental template, whereas sexual RNA replication mechanisms involve two or more parental templates. Because sexual RNA replication mechanisms counteract ribavirin-induced error catastrophe, we selected for ribavirin-resistant poliovirus to identify polymerase residues that facilitate sexual RNA replication mechanisms. We used serial passage in ribavirin, beginning with a variety of ribavirin-sensitive and ribavirin-resistant parental viruses. Ribavirin-sensitive virus contained an L420A polymerase mutation, while ribavirin-resistant virus contained a G64S polymerase mutation. A G64 codon mutation (G64Fix) was used to inhibit emergence of G64S-mediated ribavirin resistance. Revertants (L420) or pseudorevertants (L420V and L420I) were selected from all independent lineages of L420A, G64Fix L420A, and G64S L420A parental viruses. Ribavirin resistance G64S mutations were selected in two independent lineages, and novel ribavirin resistance mutations were selected in the polymerase in other lineages (M299I, M323I, M392V, and T353I). The structural orientation of M392, immediately adjacent to L420 and the polymerase primer grip region, led us to engineer additional polymerase mutations into poliovirus (M392A, M392L, M392V, K375R, and R376K). L420A revertants and pseudorevertants (L420V and L420I) restored efficient viral RNA recombination, confirming that ribavirin-induced error catastrophe coincides with defects in sexual RNA replication mechanisms. Viruses containing M392 mutations (M392A, M392L, and M392V) and primer grip mutations (K375R and R376K) exhibited divergent RNA recombination, ribavirin sensitivity, and biochemical phenotypes, consistent with changes in the fidelity of RNA synthesis. We conclude that an extended primer grip of the polymerase, including L420, M392, K375, and R376, contributes to the fidelity of RNA synthesis and to efficient sexual RNA replication mechanisms. IMPORTANCE Picornaviruses have both asexual and sexual RNA replication mechanisms. Sexual RNA replication shapes picornavirus species groups, contributes to the emergence of vaccine-derived polioviruses, and counteracts error catastrophe. Can viruses distinguish between homologous and nonhomologous partners during sexual RNA replication? We implicate an extended primer grip of the viral polymerase in sexual RNA replication mechanisms. By sensing RNA sequence complementarity near the active site, the extended primer grip of the polymerase has the potential to distinguish between homologous and nonhomologous RNA templates during sexual RNA replication.

Evasion of a Human Cytomegalovirus Entry Inhibitor with Potent Cysteine Reactivity Is Concomitant with the Utilization of a Heparan Sulfate Proteoglycan-Independent Route of Entry

ABSTRACT The dependence of viruses on the host cell to complete their replicative cycle renders cellular functions potential targets for novel antivirals. We screened a panel of broadly acting cellular ion channel inhibitors for activity against human cytomegalovirus (HCMV) and identified the voltage-gated chloride ion channel inhibitor 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) as a potent inhibitor of HCMV replication. Time-of-addition studies demonstrated that DIDS inhibited entry via direct interaction with the virion that impeded binding to the plasma membrane. Synthesis and analysis of pharmacological variants of DIDS suggested that intrinsic cysteine, and not lysine, reactivity was important for activity against HCMV. Although sequencing of DIDS-resistant HCMV revealed enrichment of a mutation within UL100 (encoding glycoprotein M) and a specific truncation of glycoprotein RL13, these did not explain the DIDS resistance phenotype. Specifically, only the introduction of the RL13 mutant partially phenocopied the DIDS resistance phenotype. Serendipitously, the entry of DIDS-resistant HCMV also became independent of heparan sulfate proteoglycans (HSPGs), suggesting that evasion of DIDS lowered dependence on an initial interaction with HSPGs. Intriguingly, the DIDS-resistant virus demonstrated increased sensitivity to antibody neutralization, which mapped, in part, to the presence of the gM mutation. Taken together the data characterize the antiviral activity of a novel HCMV inhibitor that drives HCMV infection to occur independently of HSPGs and the generation of increased sensitivity to humoral immunity. The data also demonstrate that compounds with cysteine reactivity have the potential to act as antiviral compounds against HCMV via direct engagement of virions. IMPORTANCE Human cytomegalovirus (HCMV) is major pathogen of nonimmunocompetent individuals that remains in need of new therapeutic options. Here, we identify a potent antiviral compound (4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid [DIDS]), its mechanism of action, and the chemical properties required for its activity. In doing so, the data argue that cysteine-reactive compounds could have the capacity to be developed for anti-HCMV activity. Importantly, the data show that entry of DIDS-resistant virus became independent of heparan sulfate proteoglycans (HSPGs) but, concomitantly, became more sensitive to neutralizing antibody responses. This serendipitous observation suggests that retention of an interaction with HSPGs during the entry process in vivo may be evolutionarily advantageous through better evasion of humoral responses directed against HCMV virions.