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ABSTRACT: Highly pathogenic (HP) H5N1 influenza viruses are evolving pathogens with the potential to cause sustained human-to-human transmission and pandemic virus spread. Specific antiviral drugs can play an important role in the early stages of a pandemic, but the emergence of drug-resistant variants can limit control options. The available data on the susceptibility of HP H5N1 influenza viruses to neuraminidase (NA) inhibitors and adamantanes is scarce, and there is no extensive analysis. Here, we systematically examined the prevalence of NA inhibitor and adamantane resistance among HP H5N1 influenza viruses that circulated worldwide during 2002-2012. The phenotypic fluorescence-based assay showed that both human and avian HP H5N1 viruses are susceptible to NA inhibitors oseltamivir and zanamivir with little variability over time and ∼5.5-fold less susceptibility to oseltamivir of viruses of hemagglutinin (HA) clade 2 than of clade 1. Analysis of available sequence data revealed a low incidence of NA inhibitor-resistant variants. The established markers of NA inhibitor resistance (E119A, H274Y, and N294S, N2 numbering) were found in 2.4% of human and 0.8% of avian isolates, and the markers of reduced susceptibility (I117V, K150N, I222V/T/K, and S246N) were found in 0.8% of human and 2.9% of avian isolates. The frequency of amantadine-resistant variants was higher among human (62.2%) than avian (31.6%) viruses with disproportionate distribution among different HA clades. As in human isolates, avian H5N1 viruses carry double L26I and S31N M2 mutations more often than a single S31N mutation. Overall, both human and avian HP H5N1 influenza viruses are susceptible to NA inhibitors; some proportion is still susceptible to amantadine in contrast to ∼100% amantadine resistance among currently circulating seasonal human H1N1 and H3N2 viruses. Continued antiviral susceptibility monitoring of H5N1 viruses is needed to maintain therapeutic approaches for control of disease.
Antiviral research 02/2013; · 3.61 Impact Factor
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ABSTRACT: Several novel anti-influenza compounds are in various phases of clinical development. One of these, T-705, has a mechanism of action that is not fully understood but is suggested to target influenza virus RNA-dependent RNA polymerase. We investigated the mechanism of T-705 activity against influenza A (H1N1) viruses by applying selective drug pressure over multiple sequential passages in MDCK cells. We found that T-705 treatment did not select specific mutations in potential target proteins, including PB1, PB2, PA, and NP. Phenotypic assays based on cell viability confirmed that no T-705-resistant variants were selected. In the presence of T-705, titers of infectious virus decreased significantly (P<0.0001) during serial passage in MDCK cells inoculated with seasonal influenza A (H1N1) viruses at a low multiplicity of infection (MOI=0.0001 PFU/cell) or with 2009 pandemic H1N1 viruses at a high MOI (10 PFU/cell). There was no corresponding decrease in the number of viral RNA copies; therefore, specific virus infectivity (the ratio of infectious virus yield to viral RNA copy number) was reduced. Sequence analysis showed enrichment of G-to-A and C-to-T transversion mutations, increased mutation frequency, and a shift of the nucleotide profiles of individual NP gene clones under drug selection pressure. Our results demonstrate that T-705 induces a rapid rate of mutation that generates a non-viable viral phenotype and that lethal mutagenesis is a key antiviral mechanism of T-705. Our findings also explain the broad spectrum of activity of T-705 against viruses of multiple families.
Journal of Virology 01/2013; · 5.40 Impact Factor
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ABSTRACT: The rarely identified influenza A viruses of H15 hemagglutinin subtype have been isolated exclusively in Australia. Here we report the isolation of an H15N4 influenza A virus (A/teal/Chany/7119/2008) in Western Siberia, Russia. Phylogenetic analysis demonstrated that the internal genes of the A/teal/Chany/7119/2008 belong to the Eurasian clade, and that the H15 and N4 genes were introduced into the gene pool of circulating endemic avian influenza viruses through reassortment events.
Journal of Virology 01/2013; · 5.40 Impact Factor
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ABSTRACT: Despite 75 years of research into prevention and treatment of influenza, the viruses that cause this disease continue to rank as some of the most important pathogens afflicting humans today. Progress in development of therapeutics for influenza has been slow for much of that time, but has accelerated in pace over the last two decades. Two classes of antiviral medications are used in humans at present, but each has limitations in scope and effectiveness of use. New strategies involving these licensed agents, including alternate forms of delivery and combination therapy with other drugs, are currently being explored. In addition, several novel antiviral compounds are in various clinical phases of development. Together with strategies designed to target the virus itself, new approaches to interrupt host-pathogen interactions or modulate detrimental aspects of the immune response have been proposed. Therapy for influenza will likely undergo substantial changes in the decades to come, evolving with our knowledge of pathogenesis as new approaches become viable and are validated clinically.
Current topics in microbiology and immunology 01/2012; · 4.93 Impact Factor
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ABSTRACT: Avian influenza viruses are a source of genetic material that can be transmitted to humans through direct introduction or reassortment. Although there is a wealth of information concerning global monitoring for antiviral resistance among human viruses of the N1 and N2 neuraminidase (NA) subtypes, information concerning avian viruses of these and other NA subtypes is limited. We undertook a surveillance study to investigate the antiviral susceptibility of avian influenza N6 NA viruses, the predominant subtype among wild waterfowl. We evaluated 73 viruses from North American ducks and shorebirds for susceptibility to the NA inhibitor oseltamivir in a fluorescence-based NA enzyme inhibition assay. Most (90%) had mean IC(50) values ranging from <0.01 to 5.0nM; 10% were from 5.1 to 50.0nM; and none were >50.0nM. Susceptibility to oseltamivir remained stable among all isolates collected over approximately three decades (P⩽0.74). Two isolates with I222V NA substitution had moderately reduced susceptibility to oseltamivir in vitro (IC(50), 30.0 and 40.0nM). One field sample was a mixed population containing an avian paramyxovirus (APMV) and H4N6 influenza virus, as revealed by electron microscopy and hemagglutination inhibition assays with a panel of anti-APMV antisera. This highlights the importance of awareness and careful examination of non-influenza pathogens in field samples from avian sources. This study showed that oseltamivir-resistant N6 NA avian influenza viruses are rare, and must be tested both phenotypically and genotypically to confirm resistance.
Antiviral research 01/2012; 93(3):322-9. · 3.61 Impact Factor
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ABSTRACT: For the first time, obesity appeared as a risk factor for developing severe 2009 pandemic influenza infection. Given the increase in obesity, there is a need to understand the mechanisms underlying poor outcomes in this population. In these studies, we examined the severity of pandemic influenza virus in obese mice and evaluated antiviral effectiveness. We found that genetically and diet-induced obese mice challenged with either 2009 influenza A virus subtype H1N1 or 1968 subtype H3N2 strains were more likely to have increased mortality and lung pathology associated with impaired wound repair and subsequent pulmonary edema. Antiviral treatment with oseltamivir enhanced survival of obese mice. Overall, these studies demonstrate that impaired wound lung repair in the lungs of obese animals may result in severe influenza virus infection. Alternative approaches to prevention and control of influenza may be needed in the setting of obesity.
The Journal of Infectious Diseases 12/2011; 205(2):252-61. · 6.41 Impact Factor
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Ghazi Kayali,
Rabeh El-Shesheny,
Mohamed A Kutkat,
Ahmed M Kandeil,
Ahmed Mostafa,
Mariette F Ducatez,
Pamela P McKenzie, Elena A Govorkova,
Mohamed H Nasraa,
Robert G Webster,
Richard J Webby,
Mohamed A Ali
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ABSTRACT: Reservoirs for the continuing influenza (H5N1) outbreaks in Egypt are ill-defined. Through active surveillance, we detected highly pathogenic influenza subtype H5 viruses in all poultry sectors; incidence was 5%. No other subtypes were found. Continued circulation of influenza (H5N1) viruses in various regions and poultry sectors perpetuates human exposure in Egypt.
Emerging Infectious Diseases 12/2011; 17(12):2306-8. · 6.79 Impact Factor
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ABSTRACT: Antiviral drugs are important components for the control of influenza. The key question is whether antiviral use or natural virus evolution will lead to the emergence of drug-resistant virus with comparable or superior fitness to drug-susceptible counterpart. Currently, neuraminidase (NA) inhibitors (NAIs) are the first choice for influenza prevention and treatment. In this article we will review complex process of the risk assessment for the fitness of NAIs-resistant seasonal H1N1 and H3N2, pandemic 2009 H1N1, and highly pathogenic H5N1 influenza A viruses: identification of antiviral susceptibility, degree of functional NA loss, molecular markers of resistance, and evaluation of replicative ability in vivo, virulence and transmissibility in animal studies (mouse, ferret, and guinea pig models).
Current opinion in virology. 12/2011; 1(6):574-81.
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ABSTRACT: Neuraminidase (NA) inhibitors are among the first line of defense against influenza virus infection. With the increased worldwide use of the drugs, antiviral susceptibility surveillance is increasingly important for effective clinical management and for public health epidemiology. Effective monitoring requires effective resistance detection methods. We have developed and validated a novel genotyping method for rapid detection of established NA inhibitor resistance markers in influenza viruses by single nucleotide polymorphism (SNP) analysis. The multi- or monoplex SNP analysis based on single nucleotide extension assays was developed to detect NA mutations H275Y and I223R/V in pandemic H1N1 viruses, H275Y in seasonal H1N1 viruses, E119V and R292K in seasonal H3N2 viruses, and H275Y and N295S in H5N1 viruses. The SNP analysis demonstrated high sensitivity for low-content NA amplicons (0.1 to 1 ng/μl) and showed 100% accordant results against a panel of defined clinical isolates. The monoplex assays for the H275Y NA mutation allowed precise and accurate quantification of the proportions of wild-type and mutant genotypes in virus mixtures (5% to 10% discrimination), with results comparable to those of pyrosequencing. The SNP analysis revealed the lower growth fitness of an H275Y mutant compared to the wild-type pandemic H1N1 virus by quantitatively genotyping progeny viruses grown in normal human bronchial epithelial cells. This novel method offers high-throughput screening capacity, relatively low costs, and the wide availability of the necessary equipment, and thus it could provide a much-needed approach for genotypic screening of NA inhibitor resistance in influenza viruses.
Antimicrobial Agents and Chemotherapy 07/2011; 55(10):4718-27. · 4.84 Impact Factor
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ABSTRACT: Pandemic 2009 influenza A (H1N1) virus (H1N1pdm) is different from contemporary seasonal human viruses in that it can cause infection deep in the lungs of critical care patients. Here we establish a mammalian animal model and assessed the efficacy of the neuraminidase (NA) inhibitor oseltamivir treatment against H1N1pdm virus infection. Oseltamivir (25 mg/kg/day twice daily for 5 days) was orally administered to groups of ferrets, starting either 2 or 24 h after inoculation with 10(6)PFU of A/California/04/2009 (H1N1) influenza virus. We determined that virus replication was restricted to 1 or 2 of 4 lung lobes in oseltamivir-treated animals, while virus was consistently isolated from 4 of 4 lung lobes in control animals (1.5-3.8log(10)PFU/g). Analysis of arterial blood oxygenation revealed less pronounced changes in partial oxygen and carbon dioxide pressure in oseltamivir-treated ferrets, and histologic examination confirmed reduced pneumonia. Treated animals had significantly decreased inflammatory responses in the upper respiratory tract (P < 0.05), less fever and weight loss, and less reduction of activity. Virus titers in the nasal washes of treated and control ferrets did not differ significantly. NA sequencing and fluorescence-based phenotypic assays identified no oseltamivir-resistant variants. Overall, oseltamivir treatment decreases the signs of infection and reduced the spread of H1N1pdm influenza virus in the lungs of ferrets and therefore impeded the development of viral pneumonia.
Antiviral research 05/2011; 91(2):81-8. · 3.61 Impact Factor
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Influenza and Other Respiratory Viruses 05/2011; 5 Suppl 1:79-82. · 4.16 Impact Factor
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Influenza and Other Respiratory Viruses 05/2011; 5 Suppl 1:90-3. · 4.16 Impact Factor
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ABSTRACT: With 119 confirmed cases between March 2006 and December 2010, Egypt ranks second among countries reporting human H5N1 influenza virus infections. In 2009-2010, Egypt reported 68 new human cases and became the new epicenter for H5N1 infections. We conducted an epidemiological and molecular analysis in order to better understand the situation in Egypt. The onset of new cases peaked annually during the winter and spring months, with majority of cases reported in the Nile Delta region. Most cases were less than 18 years old (62%) and females (60%). The overall case-fatality rate was 34% and significantly increased by age. There was a significant difference between the case-fatality rates among females and males. We observed a significant drop (p = 0.004) in case fatality rate in 2009 (10%) as compared to higher rates (36%-56%) in other years. Hospitalization within 2 or 3 days after onset of symptoms significantly decreased mortality. Molecular analysis showed that variations do occur among viruses isolated from birds as well as from humans in Egypt, and these mutations were especially noted in 2009 viruses. As the epidemiological profile of Egyptian cases differs from other countries, there is an urgent need to conduct prospective studies to enhance our understanding of incidence, prevalence, and determinants of virulence of human infections with avian H5N1 influenza viruses.
PLoS ONE 01/2011; 6(3):e17730. · 4.09 Impact Factor
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ABSTRACT: Immunocompromised patients are highly susceptible to influenza infection and can have prolonged viral shedding, which is a risk factor for the development of antiviral resistance.
We investigated the emergence of oseltamivir-resistant influenza variants in children and young adults with cancer during the 2002-2008 influenza seasons. The demographic and clinical features of influenza infections in 12 patients who had viral isolates obtained before and after oseltamivir therapy was initiated were studied. Antiviral susceptibilities were determined by the fluorescence-based neuraminidase (NA) enzyme inhibition assay and by sequencing genes encoding NA and matrix M2 proteins.
The mean age of patients was 10.5 (range, 1.1-23.0) years. Ten patients had hematologic malignancies, 4 were recipients of hematopoietic stem cell transplants, and all patients were receiving immunosuppressive therapy. Eleven patients had prolonged respiratory symptoms and 8 had prolonged viral shedding. Serial viral isolates were available for 8 of 12 patients. Oseltamivir-resistant influenza viruses were isolated from 4 children (3 influenza A [H3N2] and 1 influenza B virus): before the initiation of antiviral therapy in 2 patients and during therapy in the other 2 patients. Three resistant influenza A (H3N2) viruses shared a common E119V NA mutation. One patient was infected with oseltamivir-resistant influenza B virus (IC50, 731.86 ± 155.12 nM) that harbored a N294S NA mutation, the first report of this mutation in influenza B viruses.
Oseltamivir-resistant influenza viruses can exist before or rapidly emerge during antiviral therapy in immunocompromised individuals, and this has important implications for therapy and infection control.
The Pediatric Infectious Disease Journal 11/2010; 30(4):284-8. · 3.58 Impact Factor
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Terri D Stoner,
Scott Krauss,
Rebecca M DuBois,
Nicholas J Negovetich,
David E Stallknecht,
Dennis A Senne,
Marie R Gramer,
Seth Swafford,
Tom DeLiberto, Elena A Govorkova,
Robert G Webster
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ABSTRACT: Influenza viruses of the N1 neuraminidase (NA) subtype affecting both animals and humans caused the 2009 pandemic. Anti-influenza virus NA inhibitors are crucial early in a pandemic, when specific influenza vaccines are unavailable. Thus, it is urgent to confirm the antiviral susceptibility of the avian viruses, a potential source of a pandemic virus. We evaluated the NA inhibitor susceptibilities of viruses of the N1 subtype isolated from wild waterbirds, swine, and humans. Most avian viruses were highly or moderately susceptible to oseltamivir (50% inhibitory concentration [IC(50)], <5.1 to 50 nM). Of 91 avian isolates, 7 (7.7%) had reduced susceptibility (IC(50), >50 nM) but were sensitive to the NA inhibitors zanamivir and peramivir. Oseltamivir susceptibility ranged more widely among the waterbird viruses (IC(50), 0.5 to 154.43 nM) than among swine and human viruses (IC(50), 0.33 to 2.56 nM). Swine viruses were sensitive to oseltamivir, compared to human seasonal H1N1 isolated before 2007 (mean IC(50), 1.4 nM). Avian viruses from 2007 to 2008 were sensitive to oseltamivir, in contrast to the emergence of resistant H1N1 in humans. Susceptibility remained high to moderate over time among influenza viruses. Sequence analysis of the outliers did not detect molecular markers of drug-resistance (e.g., H275Y NA mutation [N1 numbering]) but revealed mutations outside the NA active site. In particular, V267I, N307D, and V321I residue changes were found, and structural analyses suggest that these mutations distort hydrophobic pockets and affect residues in the NA active site. We determined that natural oseltamivir resistance among swine and wild waterbirds is rare. Minor naturally occurring variants in NA can affect antiviral susceptibility.
Journal of Virology 10/2010; 84(19):9800-9. · 5.40 Impact Factor
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ABSTRACT: The fitness of oseltamivir-resistant highly pathogenic H5N1 influenza viruses has important clinical implications. We generated recombinant human A/Vietnam/1203/04 (VN; clade 1) and A/Turkey/15/06 (TK; clade 2.2) influenza viruses containing the H274Y neuraminidase (NA) mutation, which confers resistance to NA inhibitors, and compared the fitness levels of the wild-type (WT) and resistant virus pairs in ferrets. The VN-H274Y and VN-WT viruses replicated to similar titers in the upper respiratory tract (URT) and caused comparable disease signs, and none of the animals survived. On days 1 to 3 postinoculation, disease signs caused by oseltamivir-resistant TK-H274Y virus were milder than those caused by TK-WT virus, and all animals survived. We then studied fitness by using a novel approach. We coinoculated ferrets with different ratios of oseltamivir-resistant and -sensitive H5N1 viruses and measured the proportion of clones in day-6 nasal washes that contained the H274Y NA mutation. Although the proportion of VN-H274Y clones increased consistently, that of TK-H274Y virus decreased. Mutations within NA catalytic (R292K) and framework (E119A/K, I222L, H274L, and N294S) sites or near the NA enzyme active site (V116I, I117T/V, Q136H, K150N, and A250T) emerged spontaneously (without drug pressure) in both pairs of viruses. The NA substitutions I254V and E276A could exert a compensatory effect on the fitness of VN-H274Y and TK-H274Y viruses. NA enzymatic function was reduced in both drug-resistant H5N1 viruses. These results show that the H274Y NA mutation affects the fitness of two H5N1 influenza viruses differently. Our novel method of assessing viral fitness accounts for both virus-host interactions and virus-virus interactions within the host.
Journal of Virology 08/2010; 84(16):8042-50. · 5.40 Impact Factor
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ABSTRACT: The emergence of pandemic H1N1 influenza viruses in April 2009 and the continuous evolution of highly pathogenic H5N1 influenza viruses underscore the urgency of novel approaches to chemotherapy for human influenza infection. Anti-influenza drugs are currently limited to the neuraminidase inhibitors (oseltamivir and zanamivir) and to M2 ion channel blockers (amantadine and rimantadine), although resistance to the latter class develops rapidly. Potential targets for the development of new anti-influenza agents include the viral polymerase (and endonuclease), the hemagglutinin, and the non-structural protein NS1. The limitations of monotherapy and the emergence of drug-resistant variants make combination chemotherapy the logical therapeutic option. Here we review the experimental data on combination chemotherapy with currently available agents and the development of new agents and therapy targets.
Viruses 08/2010; 2(8):1510-29. · 1.50 Impact Factor
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ABSTRACT: The emergence and global spread of the 2009 pandemic H1N1 influenza virus reminds us that we are limited in the strategies available to control influenza infection. Vaccines are the best option for the prophylaxis and control of a pandemic; however, the lag time between virus identification and vaccine distribution exceeds 6 months and concerns regarding vaccine safety are a growing issue leading to vaccination refusal. In the short-term, antiviral therapy is vital to control the spread of influenza. However, we are currently limited to four licensed anti-influenza drugs: the neuraminidase inhibitors oseltamivir and zanamivir, and the M2 ion-channel inhibitors amantadine and rimantadine. The value of neuraminidase inhibitors was clearly established during the initial phases of the 2009 pandemic when vaccines were not available, i.e. stockpiles of antivirals are valuable. Unfortunately, as drug-resistant variants continue to emerge naturally and through selective pressure applied by use of antiviral drugs, the efficacy of these drugs declines. Because we cannot predict the strain of influenza virus that will cause the next epidemic or pandemic, it is important that we develop novel anti-influenza drugs with broad reactivity against all strains and subtypes, and consider moving to multiple drug therapy in the future. In this article we review the experimental data on investigational antiviral agents undergoing clinical trials (parenteral zanamivir and peramivir, long-acting neuraminidase inhibitors and the polymerase inhibitor favipiravir [T-705]) and experimental antiviral agents that target either the virus (the haemagglutinin inhibitor cyanovirin-N and thiazolides) or the host (fusion protein inhibitors [DAS181], cyclo-oxygenase-2 inhibitors and peroxisome proliferator-activated receptor agonists).
Drugs 07/2010; 70(11):1349-62. · 4.23 Impact Factor
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ABSTRACT: The acquisition of neuraminidase (NA) inhibitor resistance by H5N1 influenza viruses has serious clinical implications, as this class of drugs can be an essential component of pandemic control measures. The continuous evolution of the highly pathogenic H5N1 influenza viruses results in the emergence of natural NA gene variations whose impact on viral fitness and NA inhibitor susceptibility are poorly defined. We generated seven genetically stable recombinant clade 2.2 A/Turkey/15/06-like (H5N1) influenza viruses carrying NA mutations located either in the framework residues (E119A, H274Y, N294S) or in close proximity to the NA enzyme active site (V116A, I117V, K150N, Y252H). NA enzyme inhibition assays showed that NA mutations at positions 116, 117, 274, and 294 reduced susceptibility to oseltamivir carboxylate (IC(50)s increased 5- to 940-fold). Importantly, the E119A NA mutation (previously reported to confer resistance in the N2 NA subtype) was stable in the clade 2.2 H5N1 virus background and induced cross-resistance to oseltamivir carboxylate and zanamivir. We demonstrated that Y252H NA mutation contributed for decreased susceptibility of clade 2.2 H5N1 viruses to oseltamivir carboxylate as compared to clade 1 viruses. The enzyme kinetic parameters (V(max), K(m) and K(i)) of the avian-like N1 NA glycoproteins were highly consistent with their IC(50) values. None of the recombinant H5N1 viruses had attenuated virulence in ferrets inoculated with 10(6) EID(50) dose. Most infected ferrets showed mild clinical disease signs that differed in duration. However, H5N1 viruses carrying the E119A or the N294S NA mutation were lethal to 1 of 3 inoculated animals and were associated with significantly higher virus titers (P<0.01) and inflammation in the lungs compared to the wild-type virus. Our results suggest that highly pathogenic H5N1 variants carrying mutations within the NA active site that decrease susceptibility to NA inhibitors may possess increased virulence in mammalian hosts compared to drug-sensitive viruses. There is a need for novel anti-influenza drugs that target different virus/host factors and can limit the emergence of resistance.
PLoS Pathogens 05/2010; 6(5):e1000933. · 9.13 Impact Factor
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ABSTRACT: The neuraminidase (NA) inhibitor oseltamivir offers an important immediate option for the control of influenza, and its clinical use has increased substantially during the recent H1N1 pandemic. In view of the high prevalence of oseltamivir-resistant seasonal H1N1 influenza viruses in 2007-2008, there is an urgent need to characterize the transmissibility and fitness of oseltamivir-resistant H1N1/2009 viruses, although resistant variants have been isolated at a low rate. Here we studied the transmissibility of a closely matched pair of pandemic H1N1/2009 clinical isolates, one oseltamivir-sensitive and one resistant, in the ferret model. The resistant H275Y mutant was derived from a patient on oseltamivir prophylaxis and was the first oseltamivir-resistant isolate of the pandemic virus. Full genome sequencing revealed that the pair of viruses differed only at NA amino acid position 275. We found that the oseltamivir-resistant H1N1/2009 virus was not transmitted efficiently in ferrets via respiratory droplets (0/2), while it retained efficient transmission via direct contact (2/2). The sensitive H1N1/2009 virus was efficiently transmitted via both routes (2/2 and 1/2, respectively). The wild-type H1N1/2009 and the resistant mutant appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. We compared viral fitness within the host by co-infecting a ferret with oseltamivir-sensitive and -resistant H1N1/2009 viruses and found that the resistant virus showed less growth capability (fitness). The NA of the resistant virus showed reduced substrate-binding affinity and catalytic activity in vitro and delayed initial growth in MDCK and MDCK-SIAT1 cells. These findings may in part explain its less efficient transmission. The fact that the oseltamivir-resistant H1N1/2009 virus retained efficient transmission through direct contact underlines the necessity of continuous monitoring of drug resistance and characterization of possible evolving viral proteins during the pandemic.
PLoS Pathogens 01/2010; 6(7):e1001022. · 9.13 Impact Factor
