Mechanism by Which Mutations at His274 Alter Sensitivity of Influenza A Virus N1 Neuraminidase to Oseltamivir Carboxylate and Zanamivir

Human infections with avian influenza viruses are a serious public health concern. The neuraminidase (NA) inhibitors (NAIs) are the frontline anti-influenza drugs and are the major option for treatment of newly emerging influenza. Therefore, it is essential to identify the molecular markers of NAI resistance among specific NA subtypes of avian influenza viruses to help guide clinical management. NAI-resistant substitutions in NA subtypes other than N1 and N2 have been poorly studied. Here, we identified NA amino acid substitutions associated with NAI resistance among influenza viruses of N3, N7, and N9 subtypes which have been associated with zoonotic transmission. We applied random mutagenesis and generated recombinant influenza viruses carrying single or double NA substitution(s) with seven internal genes from A/Puerto Rico/8/1934 (H1N1) virus. In a fluorescencebased NA inhibition assay, we identified three categories of NA substitutions associated with reduced inhibition by NAIs (oseltamivir, zanamivir, and peramivir): (i) novel subtype-specific substitutions in or near the enzyme catalytic site (R152W, A246T, and D293N, N2 numbering), (ii) subtype-independent substitutions (E119G/V and/or D and R292K), and (iii) substitutions previously reported in other subtypes (Q136K, I222M, and E276D). Our data show that although some markers of resistance are present across NA subtypes, other subtype-specific markers can only be determined empirically. IMPORTANCE The number of humans infected with avian influenza viruses is increasing, raising concerns of the emergence of avian influenza viruses resistant to neuraminidase (NA) inhibitors (NAIs).Since most studies have focused on NAI-resistance in human influenza viruses, we investigated the molecular changes in NA that could confer NAI resistance in avian viruses grown in immortalized monolayer cells, especially those of the N3, N7, and N9 subtypes, which have caused human infections. We identified not only numerous NAI-resistant substitutions previously reported in other NA subtypes but also several novel changes conferring reduced susceptibility to NAIs, which are subtype specific. The findings indicate that some resistance markers are common across NA subtypes, but other markers need to be determined empirically for each subtype. The study also implies that antiviral surveillance monitoring could play a critical role in the clinical management of influenza virus infection and an essential component of pandemic preparedness.

Section: Discussionsupporting

confidence: 82%

Unique Determinants of Neuraminidase Inhibitor Resistance among N3, N7, and N9 Avian Influenza Viruses

Song

Marathe

Kumar

et al. 2015

“…The neuraminidases of the MZ and MZ-delNA viruses were transiently expressed in 293T cells, and their enzymatic parameters were determined on cell extracts using the MUNANA fluorogenic substrate, as previously described (52). The Michaelis-Menten constant (K m ), which reflects the affinity for the substrate, was very similar for the wild-type and the short-stalk NA (23.9 Ϯ 1.3 and 22.3 Ϯ 1.5 M, respectively) ( Table 2) and in the same range as K m values obtained with other neuraminidases of the N1 subtype (29,52,73). The maximal velocity of the reaction (V max ), which depends on both the specific activity and the amount of enzyme in the reaction, was 2.8-fold higher for the short-stalk NA than for the wild-type NA (P Ͻ 0.001) ( Table 2).…”

Section: Resultsmentioning

confidence: 48%

A Genetically Engineered Waterfowl Influenza Virus with a Deletion in the Stalk of the Neuraminidase Has Increased Virulence for Chickens

Munier

Larcher

Cormier-Aline

et al. 2010

132

126

A deletion of about 20 amino acids in the stalk of the neuraminidase (NA) is frequently detected upon transmission of influenza A viruses from waterfowl to domestic poultry. Using reverse genetics, a recombinant virus derived from a wild duck influenza virus isolate, A/Mallard/Marquenterre/Z237/83 (MZ), and an NA stalk deletion variant (MZ-delNA) were produced. Compared to the wild type, the MZ-delNA virus showed a moderate growth advantage on avian cultured cells. In 4-week-old chickens inoculated intratracheally with the MZ-delNA virus, viral replication in the lungs, liver, and kidneys was enhanced and interstitial pneumonia lesions were more severe than with the wild-type virus. The MZ-delNA-inoculated chickens showed significantly increased levels of mRNAs encoding interleukin-6 (IL-6), transforming growth factor-␤4 (TGF-␤4), and CCL5 in the lungs and a higher frequency of apoptotic cells in the liver than did their MZ-inoculated counterparts. Molecular mechanisms possibly underlying the growth advantage of the MZ-delNA virus were explored. The measured enzymatic activities toward a small substrate were similar for the wild-type and deleted NA, but the MZ-delNA virus eluted from chicken erythrocytes at reduced rates. Pseudoviral particles expressing the MZ hemagglutinin in combination with the MZ-NA or MZ-delNA protein were produced from avian cultured cells with similar efficiencies, suggesting that the deletion in the NA stalk does not enhance the release of progeny virions and probably affects an earlier step of the viral cycle. Overall, our data indicate that a shortened NA stalk is a strong determinant of adaptation and virulence of waterfowl influenza viruses in chickens.

“…The K m provides an approximation of the substrate concentration required for significant catalysis to occur, while the V max provides an approximation for the NA enzyme activity at the given standardized virus dose. The K m values determined with MUNANA substrate concentrations of 0 to 3333.3 M were in general higher than the K m values reported for expressed H1N1 (37,47) or H5N1 (37) virus NA protein determined at a substrate concentration of 5 to 100 M. The K m values of the PR8-H274Y and PR8-N294S NA were significantly higher (P Ͻ 0.05) than that of PR8 NA, suggesting that activation of catalysis by the mutant NA requires a higher substrate concentration (Table 3). The N294S mutation increased the K m of VN1203 NA, but the difference was not statistically significant (Table 3).…”

Section: H274y and Pr8-n294s Viruses (Survival Rate 0% [Data Not Shmentioning

confidence: 44%

Neuraminidase Inhibitor-Resistant Recombinant A/Vietnam/1203/04 (H5N1) Influenza Viruses Retain Their Replication Efficiency and Pathogenicity In Vitro and In Vivo

Yen

Ilyushina

Salomon

et al. 2007

154

124

Effective antiviral drugs are essential for early control of an influenza pandemic. It is therefore crucial to evaluate the possible threat posed by neuraminidase (NA) inhibitor-resistant influenza viruses with pandemic potential. Four NA mutations (E119G, H274Y, R292K, and N294S) that have been reported to confer resistance to NA inhibitors were each introduced into recombinant A/Vietnam/1203/04 (VN1203) H5N1 influenza virus. For comparison, the same mutations were introduced into recombinant A/Puerto Rico/8/34 (PR8) H1N1 influenza virus. The E119G and R292K mutations significantly compromised viral growth in vitro, but the H274Y and N294S mutations were stably maintained in VN1203 and PR8 viruses. In both backgrounds, the H274Y and N294S mutations conferred resistance to oseltamivir carboxylate (50% inhibitory concentration [IC 50 ] increases, >250-fold and >20-fold, respectively), and the N294S mutation reduced susceptibility to zanamivir (IC 50 increase, >3.0-fold). Although the H274Y and N294S mutations did not compromise the replication efficiency of VN1203 or PR8 viruses in vitro, these mutations slightly reduced the lethality of PR8 virus in mice. However, the VN1203 virus carrying either the H274Y or N294S mutation exhibited lethality similar to that of the wild-type VN1203 virus. The different enzyme kinetic parameters (V max and K m ) of avian-like VN1203 NA and human-like PR8 NA suggest that resistance-associated NA mutations can cause different levels of functional loss in NA glycoproteins of the same subtype. Our results suggest that NA inhibitor-resistant H5N1 variants may retain the high pathogenicity of the wild-type virus in mammalian species. Patients receiving NA inhibitors for H5N1 influenza virus infection should be closely monitored for the emergence of resistant variants.The highly pathogenic H5N1 influenza viruses have severely affected the poultry industry and posed a serious threat to human health, resulting in Ͼ50% fatality among more than 300 confirmed human cases (49). Although these viruses have yet to acquire efficient transmissibility between humans, their wide geographical dissemination, broad host range, and high pathogenicity raise concern about the severity of a possible pandemic. In the early stage of an influenza pandemic, unless antigenically matched vaccines are available, antivirals offer the best hope of preventing the spread of infection. The two classes of antivirals available for influenza prophylaxis and treatment are the adamantanes (amantadine and rimantadine), which target the M2 ion channel of influenza A virus, and neuraminidase (NA) inhibitors (NAIs) (oseltamivir and zanamivir), which target the NA glycoproteins of influenza A and B viruses.One of the disadvantages of antiviral therapy is the emergence of drug-resistant variants. Fully pathogenic and transmissible adamantane-resistant H1N1 and H3N2 influenza virus variants emerge rapidly posttreatment (15,17). Further, a high percentage of the recently isolated human H3N2 (4) and avian highly pathogenic H5N1 ...