Please cite this paper as: Govorkova EA. (2012) Consequences of resistance: in vitro fitness, in vivo infectivity, and transmissibility of oseltamivir‐resistant influenza A viruses. Influenza and ...Other Respiratory Viruses 7(Suppl. 1), 50–57.
The development of drug resistance is a major drawback to any antiviral therapy, and the specific anti‐influenza drugs, the neuraminidase (NA) inhibitors (NAIs), are not excluded from this rule. The impact of drug resistance depends on the degree of reduction in fitness of the particular drug‐resistant virus. If the resistance mutations lead to only a modest biological fitness cost and the virus remains highly transmissible, the effectiveness of antiviral use is likely to be reduced. This review focuses on the fitness of oseltamivir‐resistant seasonal H1N1 and H3N2, 2009 pandemic H1N1 (H1N1pdm09), and highly pathogenic H5N1 influenza A viruses carrying clinically derived NAI resistance‐associated NA mutations.
H5N1 influenza viruses transmitted from poultry to humans in Asia cause high mortality and pose a pandemic threat. Viral genes important for cell tropism and replication efficiency must be identified ...to elucidate and target virulence factors. We applied reverse genetics to generate H5N1 reassortants combining genes of lethal A/Vietnam/1203/04 (VN1203), a fatal human case isolate, and nonlethal A/chicken/Vietnam/C58/04 (CH58) and tested their pathogenicity in ferrets and mice. The viruses' hemagglutinins have six amino acids differences, identical cleavage sites, and avian-like alpha-(2,3)-linked receptor specificity. Surprisingly, exchanging hemagglutinin and neuraminidase genes did not alter pathogenicity, but substituting CH58 polymerase genes completely attenuated VN1203 virulence and reduced viral polymerase activity. CH58's NS gene partially attenuated VN1203 in ferrets but not in mice. Our findings suggest that for high virulence in mammalian species an avian H5N1 virus with a cleavable hemagglutinin requires adaptive changes in polymerase genes to overcome the species barrier. Thus, novel antivirals targeting polymerase proteins should be developed.
Neuraminidase (NA) inhibitors (NAIs) are the only antiviral drugs recommended for influenza treatment and prophylaxis. Although NAI-resistant influenza B viruses that could pose a threat to public ...health have been reported in the field, their fitness is poorly understood. We evaluated in ferrets the pathogenicity and relative fitness of reverse genetics (rg)-generated influenza B/Yamanashi/166/1998-like viruses containing E119A or H274Y NA substitutions (N2 numbering). Ferrets inoculated with NAI-susceptible rg-wild-type (rg-WT) or NAI-resistant (rg-E119A or rg-H274Y) viruses developed mild infections. Growth of rg-E119A virus in the nasal cavities was delayed, but the high titers at 3 days post-inoculation (dpi) were comparable to those of the rg-WT and rg-H274Y viruses (3.6-4.1 log10TCID50/mL). No virus persisted beyond 5 dpi and replication did not extend to the trachea or lungs. Positive virus antigen-staining of the nasal turbinate epithelium was intermittent with the rg-WT and rg-H274Y viruses; whereas antigen-staining for the rg-E119A virus was more diffuse. Virus populations in ferrets coinoculated with NAI-susceptible and -resistant viruses (1:1 mixture) remained heterogeneous at 5 dpi but were predominantly rg-WT (>70%). Although the E119A substitution was associated with delayed replication in ferrets, the H274Y substitution did not measurably affect viral growth properties. These data suggest that rg-H274Y has undiminished fitness in single virus inoculations, but neither rg-E119A nor rg-H274Y gained a fitness advantage over rg-WT in direct competition experiments without antiviral drug pressure. Taken together, our data suggest the following order of relative fitness in a ferret animal model: rg-WT > rg-H274Y > rg-E119A.
Laninamivir (LAN) is a long-acting neuraminidase (NA) inhibitor (NAI) with a similar binding profile in the influenza NA enzyme active site as those of other NAIs, oseltamivir (OS), zanamivir (ZAN), ...and peramivir, and may share common resistance markers with these NAIs. We screened viruses with NA substitutions previously found during OS and ZAN selection in avian influenza viruses (AIVs) of the N3 to N9 subtypes for LAN susceptibility. Of the 72 NA substitutions, 19 conferred resistance to LAN, which ranged from 11.2- to 549.8-fold-decreased inhibitory activity over that of their parental viruses. Ten NA substitutions reduced the susceptibility to all four NAIs, whereas the remaining 26 substitutions yielded susceptibility to one or more NAIs. To determine whether the
susceptibility of multi-NAI-resistant AIVs is associated with
susceptibility, we infected BALB/c mice with recombinant AIVs with R292K (ma81K-N3
) or Q136K (ma81K-N8
) NA substitutions, which impart
susceptibility only to LAN or OS, respectively. Both ma81K-N3
and ma81K-N8
virus-infected mice exhibited reduced weight loss, mortality, and lung viral titers when treated with their susceptible NAIs, confirming the
susceptibility of these substitutions. Together, LAN resistance profiling of AIVs of a range of NA subtypes improves the understanding of NAI resistance mechanisms. Furthermore, the association of
and
NAI susceptibility indicates that our models are useful tools for monitoring NAI susceptibility of AIVs.
The chemical structures of neuraminidase inhibitors (NAIs) possess similarities, but slight differences can result in variable susceptibility of avian influenza viruses (AIVs) carrying resistance-associated NA substitutions. Therefore, comprehensive susceptibility profiling of these substitutions in AIVs is critical for understanding the mechanism of antiviral resistance. In this study, we profiled resistance to the anti-influenza drug laninamivir in AIVs with substitutions known to impart resistance to other NAIs. We found 10 substitutions that conferred resistance to all four NAIs tested. On the other hand, we found that the remaining 26 NA substitutions were susceptible to at least one or more NAIs and showed for a small selection that
data predicted
behavior. Therefore, our findings highlight the usefulness of screening resistance markers in NA enzyme inhibition assays and animal models of AIV infections.
Influenza B virus infections remain insufficiently studied and antiviral management in immunocompromised patients is not well defined. The treatment regimens for these high-risk patients, which have ...elevated risk of severe disease-associated complications, require optimization and can be partly addressed via animal models.
We examined the efficacy of monotherapy with the RNA-dependent RNA polymerase inhibitor T-705 (favipiravir) in protecting genetically modified, permanently immunocompromised BALB scid mice against lethal infection with B/Brisbane/60/2008 (BR/08) virus. Beginning at 24 h post-infection, BALB scid mice received oral T-705 twice daily (10, 50 or 250 mg/kg/day) for 5 or 10 days.
T-705 had a dose-dependent effect on survival after BR/08 challenge, resulting in 100% protection at the highest dosages. With the 5 day regimens, dosages of 50 or 250 mg/kg/day reduced the peak lung viral titres within the treatment window, but could not efficiently clear the virus after completion of treatment. With the 10 day regimens, dosages of 50 or 250 mg/kg/day significantly suppressed virus replication in the lungs, particularly at 45 days post-infection, limiting viral spread and pulmonary pathology. No T-705 regimen decreased virus growth in the nasal turbinates of mice, which potentially contributed to the viral dynamics in the lungs. The susceptibility of influenza B viruses isolated from T-705-treated mice remained comparable to that of viruses from untreated control animals.
T-705 treatment is efficacious against lethal challenge with BR/08 virus in immunocompromised mice. The antiviral benefit was greatest when longer T-705 treatment was combined with higher dosages.
Current anti-influenza therapy depends on administering drugs soon after infection, which is often impractical. We assessed whether combinations of oseltamivir (a neuraminidase inhibitor) and T-705 ...(a nonspecific inhibitor of viral polymerases) could extend the window for treating lethal infection with highly pathogenic A(H5N1) influenza virus in mice. Combination therapy protected 100% of mice, even when delayed until 96 h postinoculation. Compared to animals receiving monotherapy, mice receiving combination therapy had reduced viral loads and restricted viral spread in lung tissues, limited lung damage, and decreased inflammatory cytokine production. Next-generation sequencing showed that virus populations in T-705-treated mice had greater genetic variability, with more frequent transversion events, than did populations in control and oseltamivir-treated mice, but no substitutions associated with resistance to oseltamivir or T-705 were detected. Thus, combination therapy extended the treatment window for A(H5N1) influenza infection in mice and should be considered for evaluation in a clinical setting.
Current influenza treatment relies on a single class of antiviral drugs, the neuraminidase inhibitors (NAIs), raising concern over the potential emergence of resistant variants and necessitating the ...development of novel drugs. In recent years, investigational inhibitors targeting the endonuclease activity of the influenza acidic polymerase (PA) protein have yielded encouraging results, although there are only limited data on their
efficacy. Here, we examined the antiviral potential of the PA endonuclease inhibitor RO-7 in prophylactic and therapeutic regimens in BALB/c mice inoculated with influenza A/California/04/2009 (H1N1)pdm09 or B/Brisbane/60/2008 viruses, which represent currently circulating antigenic variants. RO-7 was administered to mice intraperitoneally twice daily at dosages of 6, 15, or 30 mg/kg/day for 5 days, starting 4 h before or 24 or 48 h after virus inoculation, and showed no adverse effects. Prophylactic administration completely protected mice from lethal infection by influenza A or B virus. The level of therapeutic protection conferred depended upon the time of treatment initiation and RO-7 dosage, resulting in 60 to 100% and 80 to 100% survival with influenza A and B viruses, respectively. RO-7 treatment significantly decreased virus titers in the lung and lessened the extent and severity of lung damage. No PA endonuclease-inhibitor resistance was observed in viruses isolated from lungs of RO-7-treated mice, and the viruses remained susceptible to the drug at nanomolar concentrations in phenotypic assays. These
efficacy results further highlight the potential of RO-7 for development as antiviral therapy for influenza A and B virus infections.
Influenza B viruses cause annual outbreaks of respiratory illness in humans and are increasingly recognized as a major cause of influenza-associated pediatric mortality. Neuraminidase (NA) inhibitors ...(NAIs) are the only available therapy for patients infected with influenza B viruses, and the potential emergence of NAI-resistant viruses is a public health concern. The NA substitutions located within the enzyme active site could not only reduce NAI susceptibility of influenza B virus but also affect virus fitness. In this study, we investigated the effect of single NA substitutions on the fitness of influenza B/Yamanashi/166/1998 viruses (Yamagata lineage). We generated recombinant viruses containing either wild-type (WT) NA or NA with a substitution in the catalytic (R371K) or framework (E119A, D198E, D198Y, I222T, H274Y, and N294S) residues. We assessed NAI susceptibility, NA biochemical properties, NA protein expression, and virus replication in vitro and in differentiated normal human bronchial epithelial (NHBE) cells. Our results showed that four NA substitutions (D198E, I222T, H274Y, and N294S) conferred reduced inhibition by oseltamivir and three (E119A, D198Y, and R371K) conferred highly reduced inhibition by oseltamivir, zanamivir, and peramivir. All NA substitutions, except for D198Y and R371K, were genetically stable after seven passages in MDCK cells. Cell surface NA protein expression was significantly increased by H274Y and N294S substitutions. Viruses with the E119A, I222T, H274Y, or N294S substitution were not attenuated in replication efficiency in vitro or in NHBE cells. Overall, viruses with the E119A or H274Y NA substitution possess fitness comparable to NAI-susceptible virus, and the acquisition of these substitutions by influenza B viruses should be closely monitored.
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