Publications (13) View all
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Article: Molecular Assays for Quantitative and Qualitative Detection of Influenza Virus and Oseltamivir Resistance Mutations.
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ABSTRACT: Sensitive and reproducible molecular assays are essential for influenza virus diagnostics. This manuscript describes the design, validation, and evaluation of a set of real-time RT-PCR assays for quantification and subtyping of human influenza viruses from patient respiratory material. Four assays are included for detection of oseltamivir resistance mutations H275Y in prepandemic and pandemic influenza A/H1N1 and E119V and R292K in influenza A/H3N2 neuraminidase. The lower limits of detection of the quantification assay were determined to be 1.7 log10 virus particles per milliliter (vp/mL) for influenza A and 2.2 log10 vp/mL for influenza B virus. The lower limits of quantification were 2.1 and 2.3 log10 vp/mL, respectively. The RT-PCR efficiencies and lower limits of detection of the quantification assays were only marginally affected when tested on the most dissimilar target sequences found in the GenBank database. Finally, the resistance RT-PCR assays detected at least 5% mutant viruses present in mixtures containing both wild-type and mutant viruses with approximated limits of detection of 2.4 log10 vp/mL. Overall, this set of RT-PCR assays is a powerful tool for enhanced influenza virus surveillance.The Journal of molecular diagnostics: JMD 04/2013; · 3.48 Impact Factor -
Article: Reply to Chan-Tack: Treatment of severely ill influenza infected patients. Please notice the immune compromised.
The Journal of Infectious Diseases 10/2012; · 6.41 Impact Factor -
Article: H1N1 2009 Pandemic Influenza Virus: Resistance of the I223R Neuraminidase Mutant Explained by Kinetic and Structural Analysis.
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ABSTRACT: Two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that antiviral resistant viruses emerge and spread in the human population. The 2009 pandemic H1N1 virus is already resistant to adamantanes. Recently, a novel neuraminidase inhibitor resistance mutation I223R was identified in the neuraminidase of this subtype. To understand the resistance mechanism of this mutation, the enzymatic properties of the I223R mutant, together with the most frequently observed resistance mutation, H275Y, and the double mutant I223R/H275Y were compared. Relative to wild type, K(M) values for MUNANA increased only 2-fold for the single I223R mutant and up to 8-fold for the double mutant. Oseltamivir inhibition constants (K(I)) increased 48-fold in the single I223R mutant and 7500-fold in the double mutant. In both cases the change was largely accounted for by an increased dissociation rate constant for oseltamivir, but the inhibition constants for zanamivir were less increased. We have used X-ray crystallography to better understand the effect of mutation I223R on drug binding. We find that there is shrinkage of a hydrophobic pocket in the active site as a result of the I223R change. Furthermore, R223 interacts with S247 which changes the rotamer it adopts and, consequently, binding of the pentoxyl substituent of oseltamivir is not as favorable as in the wild type. However, the polar glycerol substituent present in zanamivir, which mimics the natural substrate, is accommodated in the I223R mutant structure in a similar way to wild type, thus explaining the kinetic data. Our structural data also show that, in contrast to a recently reported structure, the active site of 2009 pandemic neuraminidase can adopt an open conformation.PLoS Pathogens 09/2012; 8(9):e1002914. · 9.13 Impact Factor -
Article: The potential for multidrug-resistant influenza.
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ABSTRACT: The 2009 influenza pandemic introduced a new influenza A/H1N1 subtype in the human population. This pandemic 2009 influenza A/H1N1 virus has natural resistance to the adamantanes class and has a low threshold to become resistant to the neuraminidase class of antiviral drugs. This review describes recent findings on influenza antiviral resistance in pandemic 2009 influenza A/H1N1 virus. Pandemic 2009 viruses have emerged with novel resistance patterns to the neuraminidase inhibitors. In addition, the identification of mutations that facilitated oseltamivir resistance in prepandemic influenza emphasizes the ability of influenza to become resistant to antiviral drugs without significant loss of fitness. Novel initiatives are required to find and develop high genetic barrier influenza therapeutic regimens for effective treatment of severe influenza virus infections.Current Opinion in Infectious Diseases 12/2011; 24(6):599-604. · 4.93 Impact Factor -
Article: Multidrug resistant 2009 A/H1N1 influenza clinical isolate with a neuraminidase I223R mutation retains its virulence and transmissibility in ferrets.
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ABSTRACT: Only two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that influenza virus becomes resistant to these antiviral drugs and spreads in the human population. The 2009 pandemic A/H1N1 influenza virus is naturally resistant to adamantanes. Recently a novel neuraminidase I223R mutation was identified in an A/H1N1 virus showing cross-resistance to the neuraminidase inhibitors oseltamivir, zanamivir and peramivir. However, the ability of this virus to cause disease and spread in the human population is unknown. Therefore, this clinical isolate (NL/2631-R223) was compared with a well-characterized reference virus (NL/602). In vitro experiments showed that NL/2631-I223R replicated as well as NL/602 in MDCK cells. In a ferret pathogenesis model, body weight loss was similar in animals inoculated with NL/2631-R223 or NL/602. In addition, pulmonary lesions were similar at day 4 post inoculation. However, at day 7 post inoculation, NL/2631-R223 caused milder pulmonary lesions and degree of alveolitis than NL/602. This indicated that the mutant virus was less pathogenic. Both NL/2631-R223 and a recombinant virus with a single I223R change (recNL/602-I223R), transmitted among ferrets by aerosols, despite observed attenuation of recNL/602-I223R in vitro. In conclusion, the I223R mutated virus isolate has comparable replicative ability and transmissibility, but lower pathogenicity than the reference virus based on these in vivo studies. This implies that the 2009 pandemic influenza A/H1N1 virus subtype with an isoleucine to arginine change at position 223 in the neuraminidase has the potential to spread in the human population. It is important to be vigilant for this mutation in influenza surveillance and to continue efforts to increase the arsenal of antiviral drugs to combat influenza.PLoS Pathogens 09/2011; 7(9):e1002276. · 9.13 Impact Factor
