http://repub.eur.nl/res/aut/17988/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/39240/ 2013-01-01T00:00:00Z http://repub.eur.nl/res/pub/39044/ 2012-09-01T00:00:00Z 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, KMvalues for MUNANA increased only 2-fold for the single I223R mutant and up to 8-fold for the double mutant. Oseltamivir inhibition constants (KI) 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. http://repub.eur.nl/res/pub/33990/ 2011-12-01T00:00:00Z PURPOSE OF REVIEW: 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. RECENT FINDINGS: 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. SUMMARY: Novel initiatives are required to find and develop high genetic barrier influenza therapeutic regimens for effective treatment of severe influenza virus infections. http://repub.eur.nl/res/pub/30849/ 2011-10-01T00:00:00Z Enhanced surveillance of infections due to the pandemic A(H1N1) influenza virus, which included monitoring for antiviral resistance, was carried out in the Netherlands from late April 2009 through late May 2010. More than 1100 instances of infection with the pandemic A(H1N1) influenza virus from 2009 and 2010 [A(H1N1) 2009] distributed across this period were analyzed. Of these, 19 cases of oseltamivir-resistant virus harboring the H275Y mutation in the neuraminidase (NA) were detected. The mean 50% inhibitory concentration (IC50) levels for oseltamivir- and zanamivir-susceptible A(H1N1) 2009 viruses were 1.4-fold and 2-fold, respectively, lower than for the seasonal A(H1N1) influenza viruses from 2007/2008; for oseltamivir-resistant A(H1N1) 2009 virus the IC50was 2.9-fold lower. Eighteen of the 19 patients with oseltamivir-resistant virus showed prolonged shedding of the virus and developed resistance while on oseltamivir therapy. Sixteen of these 18 patients had an immunodeficiency, of whom 11 had a hematologic disorder. The two other patients had another underlying disease. Six of the patients who had an underlying disease died; of these, five had received cytostatic or immunosuppressive therapy. No indications for onward transmission of resistant viruses were found. This study showed that the main association for the emergence of cases of oseltamivir-resistant A(H1N1) 2009 virus was receiving antiviral therapy and having drug-induced immunosuppression or an hematologic disorder. Except for a single case of a resistant virus not linked to oseltamivir therapy, the absence of detection of resistant variants in community specimens and in specimens from contacts of cases with resistant virus suggested that the spread of resistant A(H1N1) 2009 virus was limited. Containment may have been the cumulative result of impaired NA function, successful isolation of the patients, and prophylactic measures to limit exposure. http://repub.eur.nl/res/pub/33307/ 2011-09-01T00:00:00Z A retrospective nationwide study on the use of intravenous (IV) zanamivir in patients receiving intensive care who were pretreated with oseltamivir in the Netherlands was performed. In 6 of 13 patients with a sustained reduction of the viral load, the median time to start IV zanamivir was 9 days (range, 4-11 days) compared with 14 days (range, 6-21 days) in 7 patients without viral load reduction (P = .052). Viral load response did not influence mortality. We conclude that IV zanamivir as late add-on therapy has limited effectiveness. The effect of an immediate start with IV zanamivir monotherapy or in combination with other drugs need to be evaluated. http://repub.eur.nl/res/pub/34463/ 2011-09-01T00:00:00Z 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. http://repub.eur.nl/res/pub/21311/ 2010-09-30T00:00:00Z http://repub.eur.nl/res/pub/33080/ 2010-04-01T00:00:00Z http://repub.eur.nl/res/pub/27548/ 2010-03-01T00:00:00Z Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to determine the impact on public health and to develop protective measures for the human population. At present, however, the necessity of executing pandemic influenza virus research under biosafety level 3 (BSL-3) high-containment conditions severely hampers timely characterization of such viruses. We tested heat, formalin, Triton X-100, and β-propiolactone treatments for their potencies in inactivating human influenza A(H3N2) and avian A(H7N3) viruses, as well as seasonal and pandemic A(H1N1) virus isolates, while allowing the specimens to retain their virological and immunological properties. Successful heat inactivation coincided with the loss of hemagglutinin (HA) and neuraminidase (NA) characteristics, and β-propiolactone inactivation reduced the hemagglutination titer and NA activity of the human influenza virus 10-fold or more. Although Triton X-100 treatment resulted in inconsistent HA activity, the NA activities in culture supernatants were enhanced consistently. Nonetheless, formalin treatment permitted the best retention of HA and NA properties. Triton X-100 treatment proved to be the easiest-to-use influenza virus inactivation protocol for application in combination with phenotypic NA inhibitor susceptibility assays, while formalin treatment preserved B-cell and T-cell epitope antigenicity, allowing the detection of both humoral and cellular immune responses. In conclusion, we demonstrated successful influenza virus characterization using formalin- and Triton X-100-inactivated virus samples. Application of these inactivation protocols limits work under BSL-3 conditions to virus culture, thus enabling more timely determination of public health impact and development of protective measures when a new influenza virus, e.g., pandemic A(H1N1)v virus, is introduced in humans. Copyright http://repub.eur.nl/res/pub/28404/ 2010-01-01T00:00:00Z Background: Rapid and specific molecular tests for identification of the recently identified pandemic influenza A/H1N1 2009 virus as well as rapid molecular tests to identify antiviral resistant strains are urgently needed. Objectives: We have evaluated the performance of two novel reverse transcriptase polymerase chain reactions (RT-PCRs) targeting specifically hemagglutinin and neuraminidase of pandemic influenza A/H1N1 virus in combination with a conserved matrix PCR. In addition, we investigated the performance of a novel discrimination RT-PCR for detection of the H275Y resistance mutation in the neuraminidase gene. Study design: Clinical performance of both subtype specific RT-PCR assays was evaluated through analysis of 684 throat swaps collected from individuals meeting the WHO case definition for the novel pandemic influenza virus. Analytical performance was analyzed through testing of 10-fold serial dilutions of RNA derived from the first Dutch sequenced and cultured confirmed case of novel pandemic influenza infection. Specificity and discriminative capacities of the H275Y discrimination assay were performed by testing wild type and recombinant H275Y pandemic influenza. Results: 121 throat swaps collected from April 2009 to July 2009 were positive by at least two out of three RT-PCRs, and negative for the seasonal H3/H1 subtype specific RT-PCR assays. 117 of these were tested positive for all three (Ct-values from 15.1 to 36.8). No oseltamivir resistance was detected. Conclusions: We present a sensitive and specific approach for detection of pandemic influenza A/H1N1 2009 and a rapid RT-PCR assay detecting a primary oseltamivir resistance mutation which can be incorporated easily into clinical virology algorithms. http://repub.eur.nl/res/pub/15200/ 2008-09-04T00:00:00Z