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Find video protocols related to scientific articles indexed in Pubmed.
Update: influenza activity -- United States and worldwide, May 18-September 20, 2014.
MMWR Morb. Mortal. Wkly. Rep.
PUBLISHED: 10-03-2014
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During May 18-September 20, 2014, the United States experienced low levels of seasonal influenza activity overall. Influenza A (H1N1)pdm09 (pH1N1), influenza A (H3N2), and influenza B viruses were detected worldwide and were identified sporadically in the United States. In August, two influenza A (H3N2) variant viruses (H3N2v) were detected in Ohio. This report summarizes influenza activity in the United States and worldwide during May 18-September 20, 2014.
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Characterization of Drug-Resistant Influenza A(H7N9) Variants Isolated From an Oseltamivir-Treated Patient in Taiwan.
J. Infect. Dis.
PUBLISHED: 08-14-2014
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?Patients contracting influenza A(H7N9) infection often developed severe disease causing respiratory failure. Neuraminidase (NA) inhibitors (NAIs) are the primary option for treatment, but information on drug-resistance markers for influenza A(H7N9) is limited.
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Influenza activity - United States, 2013-14 season and composition of the 2014-15 influenza vaccines.
MMWR Morb. Mortal. Wkly. Rep.
PUBLISHED: 06-06-2014
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During the 2013-14 influenza season in the United States, influenza activity increased through November and December before peaking in late December. Influenza A (H1N1)pdm09 (pH1N1) viruses predominated overall, but influenza B viruses and, to a lesser extent, influenza A (H3N2) viruses also were reported in the United States. This influenza season was the first since the 2009 pH1N1 pandemic in which pH1N1 viruses predominated and was characterized overall by lower levels of outpatient illness and mortality than influenza A (H3N2)-predominant seasons, but higher rates of hospitalization among adults aged 50-64 years compared with recent years. This report summarizes influenza activity in the United States for the 2013-14 influenza season (September 29, 2013-May 17, 2014†) and reports recommendations for the components of the 2014-15 Northern Hemisphere influenza vaccines.
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Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2012-2013.
Antiviral Res.
PUBLISHED: 05-21-2014
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Emergence of influenza viruses with reduced susceptibility to neuraminidase inhibitors (NAIs) is sporadic, often follows exposure to NAIs, but occasionally occurs in the absence of NAI pressure. The emergence and global spread in 2007/2008 of A(H1N1) influenza viruses showing clinical resistance to oseltamivir due to neuraminidase (NA) H275Y substitution, in the absence of drug pressure, warrants continued vigilance and monitoring for similar viruses. Four World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza (WHO CCs) tested 11,387 viruses collected by WHO-recognized National Influenza Centres (NIC) between May 2012 and May 2013 to determine 50% inhibitory concentration (IC50) data for oseltamivir, zanamivir, peramivir and laninamivir. The data were evaluated using normalized IC50 fold-changes rather than raw IC50 data. Nearly 90% of the 11,387 viruses were from three WHO regions: Western Pacific, the Americas and Europe. Only 0.2% (n=27) showed highly reduced inhibition (HRI) against at least one of the four NAIs, usually oseltamivir, while 0.3% (n=39) showed reduced inhibition (RI). NA sequence data, available from the WHO CCs and from sequence databases (n=3661), were screened for amino acid substitutions associated with reduced NAI susceptibility. Those showing HRI were A(H1N1)pdm09 with NA H275Y (n=18), A(H3N2) with NA E119V (n=3) or NA R292K (n=1) and B/Victoria-lineage with NA H273Y (n=2); amino acid position numbering is A subtype and B type specific. Overall, approximately 99% of circulating viruses tested during the 2012-2013 period were sensitive to all four NAIs. Consequently, these drugs remain an appropriate choice for the treatment and prophylaxis of influenza virus infections.
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An investigational antiviral drug, DAS181, effectively inhibits replication of zoonotic influenza A virus subtype H7N9 and protects mice from lethality.
J. Infect. Dis.
PUBLISHED: 02-25-2014
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Human infections caused by avian influenza A virus type subtype H7N9 have been associated with substantial morbidity and mortality. Emergence of virus variants carrying markers of decreased susceptibility to neuraminidase inhibitors was reported. Here we show that DAS181 (Fludase), an antiviral drug with sialidase activity, potently inhibited replication of wild-type influenza A(H7N9) and its oseltamivir-resistant R292K variants in mice. A once-daily administration initiated early after lethal infection hampered body weight loss and completely protected mice from lethality. We observed a time-dependent effect for 24-72-hour delayed DAS181 treatments on morbidity and mortality. The results warrant further investigation of DAS181 for influenza treatment.
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Update: influenza activity - United States, September 29, 2013-February 8, 2014.
MMWR Morb. Mortal. Wkly. Rep.
PUBLISHED: 02-21-2014
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Influenza activity in the United States began to increase in mid-November and remained elevated through February 8, 2014. During that time, influenza A (H1N1)pdm09 (pH1N1) viruses predominated overall, while few B and A (H3N2) viruses were detected. This report summarizes U.S. influenza activity* during September 29, 2013-February 8, 2014, and updates the previous summary.
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Evaluation of the antigenic relatedness and cross-protective immunity of the neuraminidase between human influenza A (H1N1) virus and highly pathogenic avian influenza A (H5N1) virus.
Virology
PUBLISHED: 01-29-2014
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To determine the genetic and antigenic relatedness as well as the cross-protective immunity of human H1N1 and avian H5N1 influenza virus neuraminidase (NA), we immunized rabbits with either a baculovirus-expressed recombinant NA from A/Beijing/262/95 (BJ/262) H1N1 or A/Hong Kong/483/97 (HK/483) H5N1 virus. Cross-reactive antibody responses were evaluated by multiple serological assays and cross-protection against H5N1 virus challenge was evaluated in mice. In a neuraminidase inhibition (NI) test, the antisera exhibited substantial inhibition of NA activity of the homologous virus, but failed to inhibit the NA activity of heterologous virus. However, these antisera exhibited low levels of cross-reactivity measured by plaque size reduction, replication inhibition, single radial hemolysis, and ELISA assays. Passive immunization with HK/483 NA-specific antisera significantly reduced virus replication and disease, and afforded almost complete protection against lethal homologous virus challenge in mice. However, passive immunization with BJ/262 (H1N1) NA-specific antisera was ineffective at providing cross-protection against lethal H5N1 virus challenge and only slightly reduced weight loss. Substantial amino acid variation among the NA antigenic sites was observed between BJ/262 and HK/483 virus, which was consistent with the lack of cross-reactive NI activity by the antibody and limited cross-protective immunity in mice. These results show a strong correlation between the lack of cross-protective immunity and low structural similarities of NA from a human seasonal H1N1 virus and an avian H5N1 influenza virus.
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Antiviral Susceptibility of Highly Pathogenic Avian Influenza A(H5N1) Viruses Isolated from Poultry, Vietnam, 2009-2011.
Emerging Infect. Dis.
PUBLISHED: 11-27-2013
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We assessed drug susceptibilities of 125 avian influenza A(H5N1) viruses isolated from poultry in Vietnam during 2009-2011. Of 25 clade 1.1 viruses, all possessed a marker of resistance to M2 blockers amantadine and rimantadine; 24 were inhibited by neuraminidase inhibitors. One clade 1.1 virus contained the R430W neuraminidase gene and reduced inhibition by oseltamivir, zanamivir, and laninamivir 12-, 73-, and 29-fold, respectively. Three of 30 clade 2.3.4 viruses contained a I223T mutation and showed 7-fold reduced inhibition by oseltamivir. One of 70 clade 2.3.2.1 viruses had the H275Y marker of oseltamivir resistance and exhibited highly reduced inhibition by oseltamivir and peramivir; antiviral agents DAS181 and favipiravir inhibited H275Y mutant virus replication in MDCK-SIAT1 cells. Replicative fitness of the H275Y mutant virus was comparable to that of wildtype virus. These findings highlight the role of drug susceptibility monitoring of H5N1 subtype viruses circulating among birds to inform antiviral stockpiling decisions for pandemic preparedness.
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Efficacy of oseltamivir treatment started within 5 days of symptom onset to reduce influenza illness duration and virus shedding in an urban setting in Bangladesh: a randomised placebo-controlled trial.
Lancet Infect Dis
PUBLISHED: 11-22-2013
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Influenza causes substantial morbidity and mortality worldwide. Few data exist for the efficacy of neuraminidase inhibitors, which are the only readily available influenza treatment options, especially in low-income settings. We assessed the efficacy of treatment with the neuraminidase inhibitor oseltamivir to reduce patient illness and viral shedding in people with influenza, in whom treatment was started within 5 days of symptom onset, in an urban setting in Bangladesh.
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Drug susceptibility surveillance of influenza viruses circulating in the United States in 2011-2012: application of the WHO antiviral working group criteria.
Influenza Other Respir Viruses
PUBLISHED: 10-26-2013
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Assessing susceptibility of influenza viruses to neuraminidase (NA) inhibitors (NAIs) is primarily done in NA inhibition (NI) assays, supplemented by NA sequence analysis. However, two factors present challenges for NI assay data interpretation: lack of established IC50 values indicative of clinically relevant resistance and insufficient harmonization of NI testing methodologies among surveillance laboratories. In 2012, the WHO working group on influenza antiviral susceptibility (WHO-AVWG) developed criteria to facilitate consistent interpretation and reporting of NI assay data.
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Cell culture-selected substitutions in influenza A(H3N2) neuraminidase affect drug susceptibility assessment.
Antimicrob. Agents Chemother.
PUBLISHED: 09-30-2013
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Assessment of drug susceptibility has become an integral part of influenza virus surveillance. In this study, we describe the drug resistance profile of influenza A(H3N2) virus, A/Mississippi/05/2011, collected from a patient treated with oseltamivir and detected via surveillance. An MDCK cell-grown isolate of this virus exhibited highly reduced inhibition by the neuraminidase (NA) inhibitors (NAIs) oseltamivir (8,005-fold), zanamivir (813-fold), peramivir (116-fold), and laninamivir (257-fold) in the NA inhibition assay. Sequence analysis of its NA gene revealed a known oseltamivir-resistance marker, the glutamic acid-to-valine substitution at position 119 (E119V), and an additional change, threonine to isoleucine at position 148 (T148I). Unlike E119V, T148I was not detected in the clinical sample but acquired during viral propagation in MDCK cells. Using recombinant proteins, T148I by itself was shown to cause only a 6-fold increase in the zanamivir 50% inhibitory concentration (IC50) and had no effect on inhibition by other drugs. The T148I substitution reduced NA activity by 50%, most likely by affecting the positioning of the 150 loop at the NA catalytic site. Using pyrosequencing, changes at T148 were detected in 35 (23%) of 150 MDCK cell-grown A(H3N2) viruses tested, which was lower than the frequency of changes at D151 (85%), an NA residue previously implicated in cell selection. We demonstrate that culturing of the A(H3N2) viruses (n = 11) at a low multiplicity of infection delayed the emergence of the NA variants with changes at position 148 and/or 151, especially when conducted in MDCK-SIAT1 cells. Our findings highlight the current challenges in monitoring susceptibility of influenza A(H3N2) viruses to the NAI class of antiviral drugs.
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Outbreak of Variant Influenza A(H3N2) Virus in the United States.
Clin. Infect. Dis.
PUBLISHED: 09-24-2013
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Background.?Variant influenza virus infections are rare but may have pandemic potential if person-to-person transmission is efficient. We describe the epidemiology of a multistate outbreak of an influenza A(H3N2) variant virus (H3N2v) first identified in 2011. Methods.?We identified laboratory-confirmed cases of H3N2v and used a standard case report form to characterize illness and exposures. We considered illness to result from person-to-person H3N2v transmission if swine contact was not identified within 4 days prior to illness onset. Results.?From 9 July to 7 September 2012, we identified 306 cases of H3N2v in 10 states. The median age of all patients was 7 years. Commonly reported signs and symptoms included fever (98%), cough (85%), and fatigue (83%). Sixteen patients (5.2%) were hospitalized, and 1 fatal case was identified. The majority of those infected reported agricultural fair attendance (93%) and/or contact with swine (95%) prior to illness. We identified 15 cases of possible person-to-person transmission of H3N2v. Viruses recovered from patients were 93%-100% identical and similar to viruses recovered from previous cases of H3N2v. All H3N2v viruses examined were susceptible to oseltamivir and zanamivir and resistant to adamantane antiviral medications. Conclusions.?In a large outbreak of variant influenza, the majority of infected persons reported exposures, suggesting that swine contact at an agricultural fair was a risk for H3N2v infection. We identified limited person-to-person H3N2v virus transmission, but found no evidence of efficient or sustained person-to-person transmission. Fair managers and attendees should be aware of the risk of swine-to-human transmission of influenza viruses in these settings.
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R292K substitution and drug susceptibility of influenza A(H7N9) viruses.
Emerging Infect. Dis.
PUBLISHED: 08-23-2013
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Neuraminidase inhibitors are the only licensed antiviral medications available to treat avian influenza A(H7N9) virus infections in humans. According to a neuraminidase inhibition assay, an R292K substitution reduced antiviral efficacy of inhibitors, especially oseltamivir, and decreased viral fitness in cell culture. Monitoring emergence of R292K-carrying viruses using a pH-modified neuraminidase inhibition assay should be considered.
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Bioluminescence-based neuraminidase inhibition assay for monitoring influenza virus drug susceptibility in clinical specimens.
Antimicrob. Agents Chemother.
PUBLISHED: 08-05-2013
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The QFlu prototype bioluminescence-based neuraminidase (NA) inhibition (NI) assay kit was designed to detect NA inhibitor (NAI)-resistant influenza viruses at point of care. Here, we evaluated its suitability for drug susceptibility assessment at a surveillance laboratory. A comprehensive panel of reference viruses (n = 14) and a set of 90 seasonal influenza virus A and B isolates were included for testing with oseltamivir and/or zanamivir in the QFlu assay using the manufacturer-recommended protocol and a modified version attuned to surveillance requirements. The 50% inhibitory concentrations (IC50s) generated were compared with those of NI assays currently used for monitoring influenza drug susceptibility, the fluorescent (FL) and chemiluminescent (CL) assays. To provide proof of principle, clinical specimens (n = 235) confirmed by real-time reverse transcription (RT)-PCR to contain influenza virus A(H1N1)pdm09 and prescreened for the oseltamivir resistance marker H275Y using pyrosequencing were subsequently tested in the QFlu assay. All three NI assays were able to discriminate the reference NA variants and their matching wild-type viruses based on the difference in their IC50s. Unless the antigenic types were first identified, certain NA variants (e.g., H3N2 with E119V) could be detected among seasonal viruses using the FL assays only. Notably, the QFlu assay identified oseltamivir-resistant A(H1N1)pdm09 viruses carrying the H275Y marker directly in clinical specimens, which is not feasible with the other two phenotypic assays, which required prior virus culturing in cells. Furthermore, The QFlu assay allows detection of the influenza virus A and B isolates carrying established and potential NA inhibitor resistance markers and may become a useful tool for monitoring drug resistance in clinical specimens.
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Microevolution of highly pathogenic avian influenza A(H5N1) viruses isolated from humans, Egypt, 2007-2011.
Emerging Infect. Dis.
PUBLISHED: 06-05-2013
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We analyzed highly pathogenic avian influenza A(H5N1) viruses isolated from humans infected in Egypt during 2007-2011. All analyzed viruses evolved from the lineage of subtype H5N1 viruses introduced into Egypt in 2006; we found minimal evidence of reassortment and no exotic introductions. The hemagglutinin genes of the viruses from 2011 formed a monophyletic group within clade 2.2.1 that also included human viruses from 2009 and 2010 and contemporary viruses from poultry; this finding is consistent with zoonotic transmission. Although molecular markers suggestive of decreased susceptibility to antiviral drugs were detected sporadically in the neuraminidase and matrix 2 proteins, functional neuraminidase inhibition assays did not identify resistant viruses. No other mutations suggesting a change in the threat to public health were detected in the viral proteomes. However, a comparison of representative subtype H5N1 viruses from 2011 with older subtype H5N1 viruses from Egypt revealed substantial antigenic drift.
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The effect of the MDCK cell selected neuraminidase D151G mutation on the drug susceptibility assessment of influenza A(H3N2) viruses.
Antiviral Res.
PUBLISHED: 05-05-2013
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Propagation of influenza A(H3N2) viruses in MDCK cells has been associated with the emergence of neuraminidase (NA) variants carrying a change at residue 151. In this study, the pyrosequencing assay revealed that ?90% of A(H3N2) virus isolates analyzed (n=150) contained more than one amino acid variant (D/G/N) at position 151. Susceptibilities of the virus isolates to zanamivir and oseltamivir were assessed using the chemiluminescent and fluorescent NA inhibition (NI) assays. In the chemiluminescent assay, which utilizes NA-Star® substrate, up to 13-fold increase in zanamivir-IC50 was detected for isolates containing a high proportion (>50%) of the G151 NA variant. However, an increase in zanamivir-IC50s was not seen in the fluorescent assay, which uses MUNANA as substrate. To investigate this discrepancy, recombinant NAs (rNAs) were prepared and tested in both NI assays. Regardless of the assay used, the zanamivir-IC50 for the rNA G151 was much greater (>1500-fold) than that for rNA D151 wild-type. However, zanamivir resistance conferred by the G151 substitution was masked in preparations containing the D151 NA which had much greater activity, especially against MUNANA. In conclusion, the presence of NA D151G variants in cell culture-grown viruses interferes with drug susceptibility assessment and therefore measures need to be implemented to prevent their emergence.
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Neuraminidase inhibitor susceptibility surveillance of influenza viruses circulating worldwide during the 2011 Southern Hemisphere season.
Influenza Other Respir Viruses
PUBLISHED: 03-12-2013
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Neuraminidase (NA) inhibitors (NAIs) are currently the only antivirals effective against influenza infections due to widespread resistance to M2 inhibitors.
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Assays for monitoring susceptibility of influenza viruses to neuraminidase inhibitors.
Influenza Other Respir Viruses
PUBLISHED: 01-03-2013
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Close monitoring of drug susceptibility among human influenza viruses was necessitated by widespread resistance to M2 inhibitors in influenza H1N1 (pre-pandemic and 2009 pandemic) and H3N2 viruses, and of oseltamivir resistance in pre-pandemic H1N1 viruses. The FDA-approved neuraminidase (NA) inhibitors (NAIs), oseltamivir and zanamivir, as well as investigational NAIs, peramivir and laninamivir, are currently the principal treatment options for managing influenza infection. However, there are challenges associated with assessing virus susceptibility to this class of drugs. Traditional cell culture-based assays are not reliable for phenotypic testing of NAI susceptibility due to complexity in interpretation. Two types of laboratory assays are currently available for monitoring NAI susceptibility, phenotypic such as the neuraminidase inhibition (NI) assay and genotypic. The NI assays requirement for propagated virus lengthens testing turnaround; therefore, the need for timely detection of molecular markers associated with NAI resistance (e.g., H275Y in H1N1) has spurred the development of rapid, high-throughput assays, such as real-time RT-PCR and pyrosequencing. The high sensitivity of genotypic assays allows testing of clinical specimens thus eliminating the need for virus propagation in cell culture. The NI assays are especially valuable when a novel virus emerges or a new NAI becomes available. Modifications continue to be introduced into NI assays, including optimization and data analysis criteria. The optimal assay of choice for monitoring influenza drug susceptibility varies widely depending on the needs of laboratories (e.g., surveillance purposes, clinical settings). Optimally, it is desirable to combine functional and genetic analyses of virus isolates and, when possible, the respective clinical specimens.
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Antiviral resistance during the 2009 influenza A H1N1 pandemic: public health, laboratory, and clinical perspectives.
Lancet Infect Dis
PUBLISHED: 12-18-2011
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Influenza A H1N1 2009 virus caused the first pandemic in an era when neuraminidase inhibitor antiviral drugs were available in many countries. The experiences of detecting and responding to resistance during the pandemic provided important lessons for public health, laboratory testing, and clinical management. We propose recommendations for antiviral susceptibility testing, reporting results, and management of patients infected with 2009 pandemic influenza A H1N1. Sustained global monitoring for antiviral resistance among circulating influenza viruses is crucial to inform public health and clinical recommendations for antiviral use, especially since community spread of oseltamivir-resistant A H1N1 2009 virus remains a concern. Further studies are needed to better understand influenza management in specific patient groups, such as severely immunocompromised hosts, including optimisation of antiviral treatment, rapid sample testing, and timely reporting of susceptibility results.
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Oseltamivir inhibits H7 influenza virus replication in mice inoculated by the ocular route.
Antimicrob. Agents Chemother.
PUBLISHED: 12-12-2011
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The majority of human infections associated with H7 influenza viruses have resulted in ocular and not respiratory disease. While oseltamivir has been prescribed to individuals presenting with conjunctivitis following H7 virus exposure, it is unknown if oseltamivir inhibits virus replication in ocular tissue. We demonstrate that H7 viruses possess sensitivity to neuraminidase inhibitors and that administration of oseltamivir before ocular virus challenge in mice inhibits H7N7 and H7N3 virus replication in ocular and respiratory tissues.
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Clinical and virologic outcomes in patients with oseltamivir-resistant seasonal influenza A (H1N1) infections: results from a clinical trial.
Influenza Other Respir Viruses
PUBLISHED: 11-26-2011
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Nineteen patients with oseltamivir-resistant seasonal influenza A (H1N1) infections were randomized to receive oseltamivir or placebo. Nasopharyngeal swabs were obtained, and clinical and virologic outcomes were compared, stratified by early or late treatment. Neuraminidase inhibition assay and pyrosequencing for H275Y confirmed resistance. Twelve (63%) patients received oseltamivir; 8 (67%) received late treatment. Seven (37%) patients received placebo; 6 (86%) presented >48 hours after onset. Time to 50% decrease in symptom severity, complete symptom resolution, and first negative culture were shortest among the early treatment group. While sample size prohibits a strong conclusion, future studies should evaluate for similar trends.
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Influenza B viruses with mutation in the neuraminidase active site, North Carolina, USA, 2010-11.
Emerging Infect. Dis.
PUBLISHED: 11-22-2011
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Oseltamivir is 1 of 2 antiviral medications available for the treatment of influenza B virus infections. We describe and characterize a cluster of influenza B viruses circulating in North Carolina with a mutation in the neuraminidase active site that may reduce susceptibility to oseltamivir and the investigational drug peramivir but not to zanamivir.
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An influenza N1 neuraminidase-specific monoclonal antibody with broad neuraminidase inhibition activity against H5N1 HPAI viruses.
Hum Vaccin
PUBLISHED: 09-20-2011
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H5N1 avian influenza continues to be a potential pandemic threat. Several vaccine candidates based on potentially pandemic influenza strains and antiviral drugs have been tested in preclinical and clinical studies. The data obtained so far have shown some promise, but have also revealed some shortcomings with both of these approaches. We have identified and characterized an H5N1 neuraminidasespecific monoclonal antibody which specifically inhibits N1 neuraminidase activity of highly pathogenic avian influenza (HPAI) strains from clades 1 and 2. We have also shown the protective efficacy of this antibody in animal challenge models using homologous virus. Specific and effective inhibition of N1 NA could make this mAb a useful therapeutic tool in the treatment of human infection, in particular with oseltamivirand zanamivir-resistant strains of HPAI.
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A human monoclonal antibody with neutralizing activity against highly divergent influenza subtypes.
PLoS ONE
PUBLISHED: 08-01-2011
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The interest in broad-range anti-influenza A monoclonal antibodies (mAbs) has recently been strengthened by the identification of anti-hemagglutinin (HA) mAbs endowed with heterosubtypic neutralizing activity to be used in the design of "universal" prophylactic or therapeutic tools. However, the majority of the single mAbs described to date do not bind and neutralize viral isolates belonging to highly divergent subtypes clustering into the two different HA-based influenza phylogenetic groups: the group 1 including, among others, subtypes H1, H2, H5 and H9 and the group 2 including, among others, H3 subtype. Here, we describe a human mAb, named PN-SIA28, capable of binding and neutralizing all tested isolates belonging to phylogenetic group 1, including H1N1, H2N2, H5N1 and H9N2 subtypes and several isolates belonging to group 2, including H3N2 isolates from the first period of the 1968 pandemic. Therefore, PN-SIA28 is capable of neutralizing isolates belonging to subtypes responsible of all the reported pandemics, as well as other subtypes with pandemic potential. The region recognized by PN-SIA28 has been identified on the stem region of HA and includes residues highly conserved among the different influenza subtypes. A deep characterization of PN-SIA28 features may represent a useful help in the improvement of available anti-influenza therapeutic strategies and can provide new tools for the development of universal vaccinal strategies.
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RT-PCR/electrospray ionization mass spectrometry approach in detection and characterization of influenza viruses.
Expert Rev. Mol. Diagn.
PUBLISHED: 03-31-2011
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Reverse-transcription PCR (RT-PCR) coupled with electrospray ionization mass spectrometry (ESI-MS) is a high-throughput nucleic acid-based technology that relies on the accurate measurement of the molecular weight of PCR amplicons that can be used to deduce the base counts (number of As, Gs, Cs and Ts) of DNA. These amplicons represent highly variable regions with information-rich sequences, which are flanked by broad-range primers designed based on highly conserved loci. This technology was first introduced in 2005 for microbial identification and subtyping, and was later applied to influenza virus detection and identification. The influenza RT-PCR/ESI-MS assay allows analysis of approximately 300 samples per 24 h, and aids in the characterization of influenza viruses based on their core gene signatures. Notably, this assay was used to identify one of the first cases of the 2009 H1N1 pandemic viruses. One of the main advantages of the RT-PCR/ESI-MS technology is its universality and adaptability for pathogen characterization. Efforts are being made to customize the currently used influenza surveillance assay for use in the diagnosis of the H1N1 pandemic virus. In this article, we provide a summary of known applications of the RT-PCR/ESI-MS assay in the field of influenza.
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Cluster of oseltamivir-resistant 2009 pandemic influenza A (H1N1) virus infections on a hospital ward among immunocompromised patients--North Carolina, 2009.
J. Infect. Dis.
PUBLISHED: 02-24-2011
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Oseltamivir resistance among 2009 pandemic influenza A (H1N1) viruses (pH1N1) is rare. We investigated a cluster of oseltamivir-resistant pH1N1 infections in a hospital ward.
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Detection of hemagglutinin variants of the pandemic influenza A (H1N1) 2009 virus by pyrosequencing.
J. Clin. Microbiol.
PUBLISHED: 02-09-2011
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For influenza viruses, pyrosequencing has been successfully applied to the high-throughput detection of resistance markers in genes encoding the drug-targeted M2 protein and neuraminidase. In this study, we expanded the utility of this assay to the detection of multiple receptor binding variants of the hemagglutinin protein of influenza viruses directly in clinical specimens. Specifically, a customized pyrosequencing protocol that permits detection of virus variants with the D, G, N, or E amino acid at position 222 in the hemagglutinin of the 2009 pandemic influenza A (H1N1) virus was developed. This customized pyrosequencing protocol was applied to the analysis of 241 clinical specimens. The use of the optimized nucleotide dispensation order allowed detection of mixtures of variants in 10 samples (4.1%) which the standard cyclic nucleotide dispensation protocol failed to detect. The optimized pyrosequencing protocol is expected to provide a more accurate tool in the analysis of virus variant composition.
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Oseltamivir-resistant pandemic (H1N1) 2009 virus, Mexico.
Emerging Infect. Dis.
PUBLISHED: 02-05-2011
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During May 2009-April 2010, we analyzed 692 samples of pandemic (H1N1) 2009 virus from patients in Mexico. We detected the H275Y substitution of the neuraminidase gene in a specimen from an infant with pandemic (H1N1) 2009 who was treated with oseltamivir. This virus was susceptible to zanamivir and resistant to adamantanes and oseltamivir.
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Characteristics of patients with oseltamivir-resistant pandemic (H1N1) 2009, United States.
Emerging Infect. Dis.
PUBLISHED: 02-05-2011
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During April 2009-June 2010, thirty-seven (0.5%) of 6,740 pandemic (H1N1) 2009 viruses submitted to a US surveillance system were oseltamivir resistant. Most patients with oseltamivir-resistant infections were severely immunocompromised (76%) and had received oseltamivir before specimen collection (89%). No evidence was found for community circulation of resistant viruses; only 4 (unlinked) patients had no oseltamivir exposure.
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Antiviral agents for the treatment and chemoprophylaxis of influenza --- recommendations of the Advisory Committee on Immunization Practices (ACIP).
MMWR Recomm Rep
PUBLISHED: 01-21-2011
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This report updates previous recommendations by CDCs Advisory Committee on Immunization Practices (ACIP) regarding the use of antiviral agents for the prevention and treatment of influenza (CDC. Prevention and control of influenza: recommendations of the Advisory Committee on Immunization Practices [ACIP]. MMWR 2008;57[No. RR-7]).This report contains information on treatment and chemoprophylaxis of influenza virus infection and provides a summary of the effectiveness and safety of antiviral treatment medications. Highlights include recommendations for use of 1) early antiviral treatment of suspected or confirmed influenza among persons with severe influenza (e.g., those who have severe, complicated, or progressive illness or who require hospitalization); 2) early antiviral treatment of suspected or confirmed influenza among persons at higher risk for influenza complications; and 3) either oseltamivir or zanamivir for persons with influenza caused by 2009 H1N1 virus, influenza A (H3N2) virus, or influenza B virus or when the influenza virus type or influenza A virus subtype is unknown; 4) antiviral medications among children aged <1 year; 5) local influenza testing and influenza surveillance data, when available, to help guide treatment decisions; and 6) consideration of antiviral treatment for outpatients with confirmed or suspected influenza who do not have known risk factors for severe illness, if treatment can be initiated within 48 hours of illness onset. Additional information is available from CDCs influenza website at http://www.cdc.gov/flu, including any updates or supplements to these recommendations that might be required during the 2010-11 influenza season. Health-care providers should be alert to announcements of recommendation updates and should check the CDC influenza website periodically for additional information. Recommendations related to the use of vaccines for the prevention of influenza during the 2010-11 influenza season have been published previously (CDC. Prevention and control of influenza with vaccines: recommendations of the Advisory Committee on Immunization Practices [ACIP], 2010. MMWR 2010;59[No. RR-8]).
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Dual resistance to adamantanes and oseltamivir among seasonal influenza A(H1N1) viruses: 2008-2010.
J. Infect. Dis.
PUBLISHED: 01-12-2011
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Two distinct genetic clades of seasonal influenza A(H1N1) viruses have cocirculated in the recent seasons: clade 2B oseltamivir-resistant and adamantane-susceptible viruses, and clade 2C viruses that are resistant to adamantanes and susceptible to oseltamivir. We tested seasonal influenza A(H1N1) viruses collected in 2008-2010 from the United States and globally for resistance to antivirals approved by the Food and Drug Administration. We report 28 viruses with both adamantane and oseltamivir (dual) resistance from 5 countries belonging to 4 distinct genotypes. Because of limited options for antiviral treatment, emergence of dual-resistant influenza viruses poses a public health concern, and their circulation needs to be closely monitored.
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Comprehensive assessment of 2009 pandemic influenza A (H1N1) virus drug susceptibility in vitro.
Antivir. Ther. (Lond.)
PUBLISHED: 12-15-2010
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Antiviral drugs are an important option for managing infections caused by influenza viruses. This study assessed the drug susceptibility of 2009 pandemic influenza A (H1N1) viruses collected globally between April 2009 and January 2010.
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Neuraminidase inhibitor susceptibility testing in human influenza viruses: a laboratory surveillance perspective.
Viruses
PUBLISHED: 08-30-2010
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Neuraminidase inhibitors (NAIs) are vital in managing seasonal and pandemic influenza infections. NAI susceptibilities of virus isolates (n = 5540) collected during the 2008-2009 influenza season were assessed in the chemiluminescent neuraminidase inhibition (NI) assay. Box-and-whisker plot analyses of log-transformed IC(50)s were performed for each virus type/subtype and NAI to identify outliers which were characterized based on a statistical cutoff of IC(50) >3 interquartile ranges (IQR) from the 75(th) percentile. Among 1533 seasonal H1N1 viruses tested, 1431 (93.3%) were outliers for oseltamivir; they all harbored the H275Y mutation in the neuraminidase (NA) and were reported as oseltamivir-resistant. Only 15 (0.7%) of pandemic 2009 H1N1 viruses tested (n = 2259) were resistant to oseltamivir. All influenza A(H3N2) (n = 834) and B (n = 914) viruses were sensitive to oseltamivir, except for one A(H3N2) and one B virus, with D151V and D197E (D198E in N2 numbering) mutations in the NA, respectively. All viruses tested were sensitive to zanamivir, except for six seasonal A(H1N1) and several A(H3N2) outliers (n = 22) which exhibited cell culture induced mutations at residue D151 of the NA. A subset of viruses (n = 1058) tested for peramivir were sensitive to the drug, with exception of H275Y variants that exhibited reduced susceptibility to this NAI. This study summarizes baseline susceptibility patterns of seasonal and pandemic influenza viruses, and seeks to contribute towards criteria for defining NAI resistance.
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Assessment of pandemic and seasonal influenza A (H1N1) virus susceptibility to neuraminidase inhibitors in three enzyme activity inhibition assays.
Antimicrob. Agents Chemother.
PUBLISHED: 06-28-2010
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The neuraminidase inhibitors (NAIs) zanamivir and oseltamivir are currently the only antiviral drugs effective for the treatment and prophylaxis of 2009 pandemic influenza A (H1N1) virus infections. The proven potential of these viruses to acquire NAI resistance during treatment emphasizes the need to assess their NAI susceptibility. The 50% inhibitory concentrations (IC(50)s) are known to vary depending on the neuraminidase inhibition (NI) test used; however, few side-by-side comparisons of different NI assays have been done. In the present study, a panel of 11 isolates representing 2009 seasonal and pandemic influenza H1N1 viruses, including oseltamivir-resistant H275Y variants, were tested in three functional NI assays: chemiluminescent (CL), fluorescent (FL), and colorimetric (CM). The sensitivities of the viruses to zanamivir, oseltamivir, and three investigational NAIs (peramivir, R-125489, and A-315675) were assessed. All isolates with the exception of H275Y variants were sensitive to all five NAIs by all three NI assays. The H275Y variants showed substantially elevated IC(50)s against oseltamivir and peramivir. The three NI assays generally yielded consistent results; thus, the choice of NI assay does not appear to affect conclusions based on drug susceptibility surveillance. Each assay, however, offers certain advantages compared to the others: the CL assay required less virus volume and the FL assay provided the greatest difference in the IC(50)s between the wild type and the variants, whereas the IC(50)s obtained from the CM assay may be the most predictive of the drug concentrations needed to inhibit enzyme activity in humans. It would be desirable to develop an NI assay which combines the advantages of all three currently available assays but which lacks their shortcomings.
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Genomic signature-based identification of influenza A viruses using RT-PCR/electro-spray ionization mass spectrometry (ESI-MS) technology.
PLoS ONE
PUBLISHED: 06-11-2010
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The emergence and rapid spread of the 2009 H1N1 pandemic influenza A virus (H1N1pdm) in humans highlights the importance of enhancing the capability of existing influenza surveillance systems with tools for rapid identification of emerging and re-emerging viruses. One of the new approaches is the RT-PCR electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology, which is based on analysis of base composition (BC) of RT-PCR amplicons from influenza "core" genes. Combination of the BC signatures represents a "genomic print" of an influenza A virus.
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In vitro antiviral activity of favipiravir (T-705) against drug-resistant influenza and 2009 A(H1N1) viruses.
Antimicrob. Agents Chemother.
PUBLISHED: 03-29-2010
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Favipiravir (T-705) has previously been shown to have a potent antiviral effect against influenza virus and some other RNA viruses in both cell culture and in animal models. Currently, favipiravir is undergoing clinical evaluation for the treatment of influenza A and B virus infections. In this study, favipiravir was evaluated in vitro for its ability to inhibit the replication of a representative panel of seasonal influenza viruses, the 2009 A(H1N1) strains, and animal viruses with pandemic (pdm) potential (swine triple reassortants, H2N2, H4N2, avian H7N2, and avian H5N1), including viruses which are resistant to the currently licensed anti-influenza drugs. All viruses were tested in a plaque reduction assay with MDCK cells, and a subset was also tested in both yield reduction and focus inhibition (FI) assays. For the majority of viruses tested, favipiravir significantly inhibited plaque formation at 3.2 muM (0.5 microg/ml) (50% effective concentrations [EC(50)s] of 0.19 to 22.48 muM and 0.03 to 3.53 microg/ml), and for all viruses, with the exception of a single dually resistant 2009 A(H1N1) virus, complete inhibition of plaque formation was seen at 3.2 muM (0.5 microg/ml). Due to the 2009 pandemic and increased drug resistance in circulating seasonal influenza viruses, there is an urgent need for new drugs which target influenza. This study demonstrates that favipiravir inhibits in vitro replication of a wide range of influenza viruses, including those resistant to currently available drugs.
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Attaching zanamivir to a polymer markedly enhances its activity against drug-resistant strains of influenza a virus.
J Pharm Sci
PUBLISHED: 03-25-2010
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Effects of the commercial drug zanamivir (Relenza) covalently attached to poly-l-glutamine on the infectivity of influenza A viruses are examined using the plaque reduction assay and binding affinity to viral neuraminidase (NA). These multivalent drug conjugates exhibit (i) up to a 20,000-fold improvement in anti-influenza potency compared with the zanamivir parent against human and avian viral strains, including both wild-type and drug-resistant mutants, and (ii) superior neuraminidase (NA) inhibition constants, especially for the mutants. These findings provide a basis for exploring polymer-attached inhibitors as more efficacious therapeutics, particularly against drug-resistant influenza strains.
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5PPP-RNA induced RIG-I activation inhibits drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza virus replication.
Virol. J.
PUBLISHED: 03-25-2010
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Emergence of drug-resistant strains of influenza viruses, including avian H5N1 with pandemic potential, 1918 and 2009 A/H1N1 pandemic viruses to currently used antiviral agents, neuraminidase inhibitors and M2 Ion channel blockers, underscores the importance of developing novel antiviral strategies. Activation of innate immune pathogen sensor Retinoic Acid Inducible Gene-I (RIG-I) has recently been shown to induce antiviral state.
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Detection of E119V and E119I mutations in influenza A (H3N2) viruses isolated from an immunocompromised patient: challenges in diagnosis of oseltamivir resistance.
Antimicrob. Agents Chemother.
PUBLISHED: 03-01-2010
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The clinical use of the neuraminidase inhibitor (NAI) oseltamivir is associated with the emergence of drug resistance resulting from subtype-specific neuraminidase (NA) mutations. The influenza A/Texas/12/2007 (H3N2) virus isolated from an oseltamivir-treated immunocompromised patient exhibited reduced susceptibility to oseltamivir in the chemiluminescent neuraminidase inhibition (NI) assay (approximately 60-fold increase in its 50% inhibitory concentration [IC(50)] compared to that for a control virus). When further propagated in cell culture, the isolate maintained reduced susceptibility to oseltamivir in both chemiluminescent and fluorescent NI assays (approximately 50- and 350-fold increases in IC(50), respectively). Sequencing analysis of the isolate revealed a mix of nucleotides coding for amino acids at position 119 of the NA [E119(V/I)]. Plaque purification of the isolate yielded E119V and E119I variants, both exhibiting reduced susceptibility to oseltamivir. The E119I variant also showed decreased susceptibility to zanamivir and the investigational NAIs peramivir and A-315675. The emergence of E119V variants in oseltamivir-treated patients has been previously reported; however, the E119I mutation detected here is a novel one which reduces susceptibility to several NAIs. Both mutations were not detected in unpropagated original clinical specimens using either conventional sequencing or pyrosequencing, suggesting that these variants were present in very low proportions (<10%) in clinical specimens and gained dominance after virus propagation in MDCK cells. All virus isolates recovered from the patient were resistant to adamantanes. Our findings highlight the potential for emergence and persistence of multidrug-resistant influenza viruses in oseltamivir-treated immunocompromised subjects and also highlight challenges for drug resistance diagnosis due to the genetic instability of the virus population upon propagation in cell culture.
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Detection of molecular markers of drug resistance in 2009 pandemic influenza A (H1N1) viruses by pyrosequencing.
Antimicrob. Agents Chemother.
PUBLISHED: 12-22-2009
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The M2 blockers amantadine and rimantadine and the neuraminidase (NA) inhibitors (NAIs) oseltamivir and zanamivir are approved by the FDA for use for the control of influenza A virus infections. The 2009 pandemic influenza A (H1N1) viruses (H1N1pdm) are reassortants that acquired M and NA gene segments from a Eurasian adamantane-resistant swine influenza virus. NAI resistance in the H1N1pdm viruses has been rare, and its occurrence is mainly limited to oseltamivir-exposed patients. The pyrosequencing assay has been proven to be a useful tool in surveillance for drug resistance in seasonal influenza A viruses. We provide a protocol which allows the detection of adamantane resistance markers as well as the I43T change, which is unique to the H1N1pdm M2 protein. The protocol also allows the detection of changes at residues V116, I117, E119, Q136, K150, D151, D199, I223, H275, and N295 in the NA, known to alter NAI drug susceptibility. We report on the detection of the first cases of the oseltamivir resistance-conferring mutation H275Y and the I223V change in viruses from the United States using the approach described in this study. Moreover, the assay permits the quick identification of the major NA group (V106/N248, I106/D248, or I106/N248) to which a pandemic virus belongs. Pyrosequencing is well suited for the detection of drug resistance markers and signature mutations in the M and NA gene segments of the pandemic H1N1 influenza viruses.
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Outbreak of antiviral drug-resistant influenza a in long-term care facility, Illinois, USA, 2008.
Emerging Infect. Dis.
PUBLISHED: 12-08-2009
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An outbreak of oseltamivir-resistant influenza A (H1N1) occurred in a long-term care facility. Eight (47%) of 17 and 1 (6%) of 16 residents in 2 wards had oseltamivir-resistant influenza A virus (H1N1) infections. Initial outbreak response included treatment and prophylaxis with oseltamivir. The outbreak abated, likely because of infection control measures.
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Genomic events underlying the changes in adamantane resistance among influenza A(H3N2) viruses during 2006-2008.
Influenza Other Respir Viruses
PUBLISHED: 11-12-2009
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Adamantanes resistance in H3N2 viruses has been increasing since 2000, and in 2005-2006 reached nearly 100% in most countries, with the circulation of the N-lineage. In 2006-2007, however, a significant decrease in resistance was observed in many regions.
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Bifunctional polymeric inhibitors of human influenza A viruses.
Pharm. Res.
PUBLISHED: 09-09-2009
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New antiviral agents were prepared by attaching derivatives of sialic acid (1) and of the drug zanamivir (2) to poly(isobutylene-alt-maleic anhydride) (poly-(1 + 2)) or by mixing poly-1 and poly-2, followed by assaying them against wild-type and drug-resistant influenza A Wuhan viruses.
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Host cell selection of influenza neuraminidase variants: implications for drug resistance monitoring in A(H1N1) viruses.
Antiviral Res.
PUBLISHED: 08-04-2009
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The neuraminidase inhibitors (NAIs), oseltamivir and zanamivir, are essential for treatment and prevention of influenza A and B infections. Oseltamivir resistance among influenza A (H1N1) viruses rapidly emerged and spread globally during the 2007-2008 and 2008-2009 influenza seasons. Approximately 20% and 90% of viruses tested for NAI susceptibility at CDC during these seasons, respectively, were resistant to oseltamivir (IC(50) approximately 100-3000 time>those of sensitive viruses), based on the chemiluminescent NA inhibition assay. Pyrosequencing analysis confirmed H274Y mutation (H275Y in N1 numbering) in the neuraminidase (NA) gene of oseltamivir-resistant viruses. Full NA sequence analysis of a subset of oseltamivir-resistant and sensitive virus isolates from both seasons (n=725) showed that 53 (7.3%) had mutations at residue D151 (D-->E/G/N), while 9 (1.2%) had mutations at Q136 (Q-->K) and 2 (0.3%) had mutations at both residues. Viruses with very high IC(50) for oseltamivir and peramivir, and elevated IC(50) for zanamivir, had H274Y in addition to mutations at D151 and/or Q136, residues which can potentially confer NAI resistance based on recent N1 NA crystal structure data. Mutations at D151 without H274Y, did not elevate IC(50) for any tested NAI, however, Q136K alone significantly reduced susceptibility to zanamivir (36-fold), peramivir (80-fold) and A-315675 (114-fold) but not oseltamivir. Mutations at D151 and Q136 were present only in MDCK grown viruses but not in matching original clinical specimens (n=33) which were available for testing, suggesting that these variants were the result of cell culture selection or they were present in very low proportions. Our findings provide evidence that propagation of influenza virus outside its natural host may lead to selection of virus variants with mutations in the NA that affect sensitivity to NAIs and thus poses implications for drug resistance monitoring and diagnostics.
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Influenza in outpatient ILI case-patients in national hospital-based surveillance, Bangladesh, 2007-2008.
PLoS ONE
PUBLISHED: 08-03-2009
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Recent population-based estimates in a Dhaka low-income community suggest that influenza was prevalent among children. To explore the epidemiology and seasonality of influenza throughout the country and among all age groups, we established nationally representative hospital-based surveillance necessary to guide influenza prevention and control efforts.
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Detection of antiviral resistance and genetic lineage markers in influenza B virus neuraminidase using pyrosequencing.
Antiviral Res.
PUBLISHED: 07-30-2009
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We report here the design of a pyrosequencing approach for the detection of molecular markers of resistance to the neuraminidase inhibitors zanamivir and oseltamivir in influenza viruses of type B. Primers were designed to analyze the sequences at eight amino acid positions E119, R152, D198, I222, S250, H274, R371, and G402 (universal A/N2 numbering) in the neuraminidase (NA) which have been previously found to be associated with resistance or reduced susceptibility to oseltamivir and/or zanamivir in the NA inhibition assay. In addition, the designed primers could be utilized to the distinguish between the NAs of influenza B viruses from the two major lineages (Victoria and Yamagata) that have co-circulated globally in recent years, thus providing a valuable tool for virus strain surveillance.
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Inhibition of neuraminidase inhibitor-resistant influenza virus by DAS181, a novel sialidase fusion protein.
PLoS ONE
PUBLISHED: 07-22-2009
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Antiviral drug resistance for influenza therapies remains a concern due to the high prevalence of H1N1 2009 seasonal influenza isolates which display H274Y associated oseltamivir-resistance. Furthermore, the emergence of novel H1N1 raises the potential that additional reassortments can occur, resulting in drug resistant virus. Thus, additional antiviral approaches are urgently needed. DAS181 (Fludase), a sialidase fusion protein, has been shown to have inhibitory activity against a large number of seasonal influenza strains and a highly pathogenic avian influenza (HPAI) strain (H5N1). Here, we examine the in vitro activity of DAS181 against a panel of 2009 oseltamivir-resistant seasonal H1N1 clinical isolates. The activity of DAS181 against nine 2009, two 2007, and two 2004 clinical isolates of seasonal IFV H1N1 was examined using plaque number reduction assay on MDCK cells. DAS181 strongly inhibited all tested isolates. EC50 values remained constant against isolates from 2004, 2007, and 2009, suggesting that there was no change in DAS181 sensitivity over time. As expected, all 2007 and 2009 isolates were resistant to oseltamivir, consistent with the identification of the H274Y mutation in the NA gene of all these isolates. Interestingly, several of the 2007 and 2009 isolates also exhibited reduced sensitivity to zanamivir, and accompanying HA mutations near the sialic acid binding site were observed. DAS181 inhibits IFV that is resistant to NAIs. Thus, DAS181 may offer an alternative therapeutic option for seasonal or pandemic IFVs that become resistant to currently available antiviral drugs.
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Influenza genome analysis using pyrosequencing method: current applications for a moving target.
Expert Rev. Mol. Diagn.
PUBLISHED: 07-08-2009
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Pyrosequencing is a high-throughput non-gel-based DNA sequencing method that was introduced in the late 1990s. It employs a DNA sequencing-by-synthesis approach based on real-time measurement of pyrophosphate released from incorporation of dNTPs. A cascade of enzymatic reactions proportionally converts the pyrophosphate to a light signal recorded in a form of peaks, known as pyrograms. Routinely, a 45-60-nucleotide sequence is obtained per reaction. Recent improvements introduced in the assay chemistry have extended the read to approximately 100 nucleotides. Since its advent, pyrosequencing has been applied in the fields of microbiology, molecular biology and pharmacogenomics. The pyrosequencing approach was first applied to analysis of influenza genome in 2005, when it played a critical role in the timely detection of an unprecedented rise in resistance to the adamantane class of anti-influenza drugs. More recently, pyrosequencing was successfully applied for monitoring the emergence and spread of influenza A (H1N1) virus resistance to oseltamivir, a newer anti-influenza drug. The present report summarizes known applications of the pyrosequencing approach for influenza genome analysis with an emphasis on drug-resistance detection.
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Novel pandemic influenza A(H1N1) viruses are potently inhibited by DAS181, a sialidase fusion protein.
PLoS ONE
PUBLISHED: 06-10-2009
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The recent emergence of a novel pandemic influenza A(H1N1) strain in humans exemplifies the rapid and unpredictable nature of influenza virus evolution and the need for effective therapeutics and vaccines to control such outbreaks. However, resistance to antivirals can be a formidable problem as evidenced by the currently widespread oseltamivir- and adamantane-resistant seasonal influenza A viruses (IFV). Additional antiviral approaches with novel mechanisms of action are needed to combat novel and resistant influenza strains. DAS181 (Fludase) is a sialidase fusion protein in early clinical development with in vitro and in vivo preclinical activity against a variety of seasonal influenza strains and highly pathogenic avian influenza strains (A/H5N1). Here, we use in vitro, ex vivo, and in vivo models to evaluate the activity of DAS181 against several pandemic influenza A(H1N1) viruses.
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Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans.
Science
PUBLISHED: 05-22-2009
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Since its identification in April 2009, an A(H1N1) virus containing a unique combination of gene segments from both North American and Eurasian swine lineages has continued to circulate in humans. The lack of similarity between the 2009 A(H1N1) virus and its nearest relatives indicates that its gene segments have been circulating undetected for an extended period. Its low genetic diversity suggests that the introduction into humans was a single event or multiple events of similar viruses. Molecular markers predictive of adaptation to humans are not currently present in 2009 A(H1N1) viruses, suggesting that previously unrecognized molecular determinants could be responsible for the transmission among humans. Antigenically the viruses are homogeneous and similar to North American swine A(H1N1) viruses but distinct from seasonal human A(H1N1).
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Triple-reassortant swine influenza A (H1) in humans in the United States, 2005-2009.
N. Engl. J. Med.
PUBLISHED: 05-07-2009
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Triple-reassortant swine influenza A (H1) viruses--containing genes from avian, human, and swine influenza viruses--emerged and became enzootic among pig herds in North America during the late 1990s.
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Emergence of a novel swine-origin influenza A (H1N1) virus in humans.
N. Engl. J. Med.
PUBLISHED: 05-07-2009
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On April 15 and April 17, 2009, novel swine-origin influenza A (H1N1) virus (S-OIV) was identified in specimens obtained from two epidemiologically unlinked patients in the United States. The same strain of the virus was identified in Mexico, Canada, and elsewhere. We describe 642 confirmed cases of human S-OIV infection identified from the rapidly evolving U.S. outbreak.
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Infections with oseltamivir-resistant influenza A(H1N1) virus in the United States.
JAMA
PUBLISHED: 03-02-2009
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During the 2007-2008 influenza season, oseltamivir resistance among influenza A(H1N1) viruses increased significantly for the first time worldwide. Early surveillance data suggest that the prevalence of oseltamivir resistance among A(H1N1) viruses will most likely be higher during the 2008-2009 season.
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Oseltamivir resistance mutation N294S in human influenza A(H5N1) virus in Egypt.
J Infect Public Health
PUBLISHED: 02-23-2009
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In December 2006, three human specimens were received that were suspected positive for influenza A(H5N1). The specimens were tested using real time PCR. And the presence of A(H5N1) virus was confirmed in 2 patients (16F and 26M), The NA sequence from A(H5N1) positive specimens collected before and after antiviral therapy revealed a mutation (N294S) (N295S according to N1 numbering), previously associated with resistance to oseltamivir. When tested with NA inhibition assays, the two N294S viruses from Egypt exhibited from 57 to 138-fold reduction in susceptibility to oseltamivir, depending on the assay. To our knowledge, this is the first time oseltamivir resistance has been detected in A(H5N1) infecting a human prior to treatment.
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Pyrosequencing as a tool to detect molecular markers of resistance to neuraminidase inhibitors in seasonal influenza A viruses.
Antiviral Res.
PUBLISHED: 01-16-2009
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Pyrosequencing has been successfully used to monitor resistance in influenza A viruses to the first class of anti-influenza drugs, M2 blockers (adamantanes). In contrast to M2 blockers, resistance to neuraminidase (NA) inhibitors (NAIs) is subtype- and drug-specific. Here, we designed a pyrosequencing assay for detection of the most commonly reported mutations associated with resistance to NAIs, a newer class of anti-influenza drugs. These common mutations occur at residues: H274 (N1), E119 (N2), R292 (N2), and N294 (N2) in seasonal influenza A viruses. Additionally, we designed primers to detect substitutions at D151 in NAs of N1 and N2 subtypes. This assay allows detection of mutations associated with resistance not only in grown viruses but also in clinical specimens, thus reducing the time needed for testing and providing an advantage for disease outbreak investigation and management. The pyrosequencing approach also allows the detection of mixed populations of virus variants at positions of interest. Analysis of viruses in the original clinical specimens reduces the potential for introducing genetic variance in the virus population due to selection by cell culture. Our results showed that, in at least one instance, a D151E change seen in N1NA after virus propagation in cell culture was not detected in the original clinical specimen. Although the pyrosequencing assay allows high throughput screening for established genetic markers of antiviral resistance, it is not a replacement for the NA inhibition assays due to insufficient knowledge of the molecular mechanisms of the NAI-resistance.
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Rapid identification of oseltamivir-resistant influenza A(H1N1) viruses with H274Y mutation by RT-PCR/restriction fragment length polymorphism assay.
Antiviral Res.
PUBLISHED: 01-15-2009
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In the beginning of 2007-2008 Northern Hemisphere influenza season, the frequency of influenza A(H1N1) viruses bearing a previously defined oseltamivir resistance conferring amino acid change of Histidine to Tyrosine at position 274 (H274Y) of the neuraminidase (NA) increased dramatically. In order to rapidly detect such resistant viruses, an RT-PCR/restriction fragment length polymorphism (RT-PCR/RFLP) assay targeting amino acid 274 of the N1 NA molecule was developed to investigate the presence or absence of the H274Y mutation. The reverse primer was engineered to produce a BspHI site in the amplicon for oseltamivir-sensitive viruses with Histidine at position 274 (274H). A total of 50 influenza A(H1N1) specimens including 30 oseltamivir-sensitive and 20 oseltamivir-resistant ones submitted to the Centers for Disease Control and Prevention (CDC) during the 2007-2008 influenza season were successfully characterized by this assay. The assay was specific for grown A(H1N1) viruses and original clinical specimens, with a lower limit of detection of approximately 10 RNA transcript copies per reaction. Our RT-PCR/RFLP assay provides a simple, rapid and sensitive tool to monitor the emergence and spread of H274Y oseltamivir-resistant influenza A(H1N1) viruses.
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Detection of molecular markers of antiviral resistance in influenza A (H5N1) viruses using a pyrosequencing method.
Antimicrob. Agents Chemother.
PUBLISHED: 01-05-2009
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Resistance of influenza viruses to antiviral drugs can emerge following medication or may result from natural variation. Two classes of anti-influenza virus drugs targeting either the M2 protein (amantadine and rimantadine) or neuraminidase (NA; oseltamivir and zanamivir) are currently licensed. These drugs are expected to be important in controlling the early stages of a potential pandemic. In the present study, we describe how a pyrosequencing method can be used to rapidly detect established molecular markers of resistance to M2 blockers and NA inhibitors in influenza A (H5N1) viruses. The residues L26, V27, A30, S31, and G34 in the M2 protein were targeted for pyrosequencing. The NA residues for pyrosequencing analysis included the established markers of drug resistance (H274 and N294), as well as residues of less certain relevance (V116, I117, Q136, K150, and I222). A single pair of pyro-reverse transcription (RT)-PCR primers was designed to allow amplification of an approximately 600-nucleotide-long amplicon of the NA genes of H5N1 viruses from various clades/subclades associated with infections in humans. The sensitivity of the assay was demonstrated by the successful pyrosequencing of RNA extracted from samples of serially diluted (10(-5) to 10(-7)) virus stocks with initial concentrations ranging from 10(5) to 10(8) PFU/ml. The markers of resistance were detected in samples with threshold cycle values ranging from 32 to 37, as determined by real-time RT-PCR. The pyrosequencing approach may provide a valuable tool for rapid detection of markers of drug resistance in H5N1 viruses and facilitate the elucidation of the role of such changes in natural and acquired drug resistance.
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Neuraminidase inhibitor susceptibility testing of influenza type B viruses in China during 2010 and 2011 identifies viruses with reduced susceptibility to oseltamivir and zanamivir.
Antiviral Res.
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Influenza type B viruses are responsible for substantial morbidity and mortality in humans. Antiviral drugs are an important supplement to vaccination for reducing the public health impact of influenza virus infections. Influenza B viruses are not sensitive to M2 inhibitors which limit the current therapeutic options to two neuraminidase inhibitors (NAIs), oseltamivir and zanamivir, which are licensed in many countries. Drug resistance is a public health concern which has necessitated monitoring of influenza virus drug susceptibilities through active global surveillance. Here, we report the results of drug susceptibility surveillance of influenza type B viruses (n=680) collected in mainland China during two calendar years, 2010 and 2011, assessed using functional neuraminidase (NA) inhibition (NI) assays. Four influenza B viruses exhibited reduced susceptibilities to oseltamivir, but not zanamivir, and shared the amino acid substitution I221T (ATC?ACC), at this conserved residue in the NA active site (I222T in N2 numbering). Additionally, a single virus with reduced susceptibility to both oseltamivir and zanamivir was identified and contained an amino acid substitution D197N (GAC?AAC) at another conserved residue in the NA active site (D198N in N2 numbering). This report underlies the importance of continued influenza antiviral susceptibility surveillance globally, even in countries where the use of NAIs has been low or non-existing.
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A cluster of patients infected with I221V influenza b virus variants with reduced oseltamivir susceptibility--North Carolina and South Carolina, 2010-2011.
J. Infect. Dis.
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During 2010-2011, influenza B viruses with a novel neuraminidase substitution, denoted I221V (B/I221V), associated with reduced in vitro oseltamivir susceptibility were detected in North Carolina.
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Molecular epidemiology of influenza A(H1N1)pdm09 viruses from Pakistan in 2009-2010.
PLoS ONE
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In early 2009, a novel influenza A(H1N1) virus that emerged in Mexico and United States rapidly disseminated worldwide. The spread of this virus caused considerable morbidity with over 18000 recorded deaths. The new virus was found to be a reassortant containing gene segments from human, avian and swine influenza viruses.
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Monitoring seasonal influenza A evolution: rapid 2009 pandemic H1N1 surveillance with an reverse transcription-polymerase chain reaction/electro-spray ionization mass spectrometry assay.
J. Clin. Virol.
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The emergence of the pandemic H1N1 influenza strain in 2009 reinforced the need for improved influenza surveillance efforts. A previously described influenza typing assay that utilizes RT-PCR coupled to electro-spray ionization mass spectrometry (ESI-MS) played an early role in the discovery of the pandemic H1N1 influenza strain, and has potential application for monitoring viral genetic diversity in ongoing influenza surveillance efforts.
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The fluorescence neuraminidase inhibition assay: a functional method for detection of influenza virus resistance to the neuraminidase inhibitors.
Methods Mol. Biol.
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Neuraminidase inhibitors (NAIs) are presently the only effective antiviral drugs for treatment and chemoprophylaxis of influenza A and B infections, due to the high prevalence of resistance to the adamantane class of drugs among influenza A(H3N2) and A(H1N1) viruses, including the 2009 pandemic H1N1 strain. The limited pharmaceutical options currently available for control of influenza infections underscore the critical need for surveillance on NAI susceptibility of influenza viruses circulating globally. This chapter describes the fluorescent neuraminidase (NA) inhibition (NI) assay, a functional method used for assessing influenza virus susceptibility to NAIs. The IC(50) (drug concentration needed to reduce the NA enzymatic activity by 50%) values generated in this assay are used to evaluate the NAI-susceptibility of test viruses relative to those of sensitive reference viruses of the same antigenic type and subtype. Test viruses with significantly elevated IC(50)s are further analyzed by pyrosequencing or conventional sequencing to identify known markers of NAI resistance or novel changes in the NA. The harmonization of NI assay conditions and interpretation of results across surveillance laboratories is necessary to improve NAI susceptibility testing and analysis.
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The chemiluminescent neuraminidase inhibition assay: a functional method for detection of influenza virus resistance to the neuraminidase inhibitors.
Methods Mol. Biol.
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Neuraminidase inhibitors (NAIs) represent a newer class of anti-influenza drugs. Widespread natural or acquired resistance to NAIs is a major public health concern as it limits pharmaceutical options available for managing seasonal and pandemic influenza virus infections. Molecular-based methods, such as pyrosequencing, sequencing, and PCR are rapid techniques for detecting known genetic markers of resistance, but they are unable to identify novel mutations that may confer resistance, or subtle differences in the susceptibility of viruses to the NAIs. This chapter describes the chemiluminescent neuraminidase (NA) inhibition (NI) assay, a functional method used for assessing influenza virus susceptibility to NAIs. The assay generates IC(50) values (drug concentration needed to reduce the NA enzymatic activity by 50%) which are determined by curve-fitting analysis. Test viruses showing elevated IC(50) values relative to those of NAI-sensitive reference viruses of the same antigenic type and subtype are further analyzed by pyrosequencing or conventional sequencing to identify known markers of NAI resistance or new changes in the NA. The criteria for NAI resistance are currently not well defined and tend to vary by laboratory and NI assay, therefore harmonization of NI assay conditions and interpretation of results across surveillance laboratories is necessary to improve the NAI susceptibility testing and analysis.
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Human infections with novel reassortant influenza A(H3N2)v viruses, United States, 2011.
Emerging Infect. Dis.
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During July-December 2011, a variant virus, influenza A(H3N2)v, caused 12 human cases of influenza. The virus contained genes originating from swine, avian, and human viruses, including the M gene from influenza A(H1N1)pdm09 virus. Influenza A(H3N2)v viruses were antigenically distinct from seasonal influenza viruses and similar to proposed vaccine virus A/Minnesota/11/2010.
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A non-VH1-69 heterosubtypic neutralizing human monoclonal antibody protects mice against H1N1 and H5N1 viruses.
PLoS ONE
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Influenza viruses are among the most important human pathogens and are responsible for annual epidemics and sporadic, potentially devastating pandemics. The humoral immune response plays an important role in the defense against these viruses, providing protection mainly by producing antibodies directed against the hemagglutinin (HA) glycoprotein. However, their high genetic variability allows the virus to evade the host immune response and the potential protection offered by seasonal vaccines. The emergence of resistance to antiviral drugs in recent years further limits the options available for the control of influenza. The development of alternative strategies for influenza prophylaxis and therapy is therefore urgently needed. In this study, we describe a human monoclonal antibody (PN-SIA49) that recognizes a highly conserved epitope located on the stem region of the HA and able to neutralize a broad spectrum of influenza viruses belonging to different subtypes (H1, H2 and H5). Furthermore, we describe its protective activity in mice after lethal challenge with H1N1 and H5N1 viruses suggesting a potential application in the treatment of influenza virus infections.
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Analysis of influenza viruses from patients clinically suspected of infection with an oseltamivir resistant virus during the 2009 pandemic in the United States.
Antiviral Res.
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During the 2009 influenza pandemic, the Centers for Disease Control and Prevention provided antiviral susceptibility testing for patients infected with suspected drug-resistant viruses. Specimens from 72 patients admitted to an intensive care unit or with a severe immunocompromising condition, who failed to clinically improve after oseltamivir treatment, were accepted for testing. Respiratory specimens were tested for the presence of the oseltamivir resistance-conferring H275Y substitution in the neuraminidase (NA) by pyrosequencing. Virus isolates propagated in MDCK cells were tested in phenotypic NA inhibition (NI) assays using licensed NA inhibitors (NAIs), zanamivir and oseltamivir, and investigational NAIs, peramivir and laninamivir. Conventional sequencing and plaque purification were conducted on a subset of viruses. Pyrosequencing data were obtained for 87 specimens collected from 58 of the 72 (81%) patients. Of all patients, 27 (38%) had at least one specimen in which H275Y was detected. Analysis of sequential samples from nine patients revealed intra-treatment emergence of H275Y variant and a shift from wildtype-to-H275Y in quasispecies during oseltamivir therapy. A shift in the H275Y proportion was observed as a result of virus propagation in MDCK cells. Overall, the NI method was less sensitive than pyrosequencing in detecting the presence of H275Y variants in virus isolates. Using the NI method, isolates containing H275Y variant at?50% exhibited resistance to oseltamivir and peramivir, but retained full susceptibility to zanamivir. H275Y viruses recovered from two patients had an additional substitution I223K or I223R that conferred a 38-52- and 33-97-fold enhancement in oseltamivir- and peramivir-resistance, respectively. These viruses also showed decreased susceptibility to zanamivir and laninamivir. These data suggest that pyrosequencing is a powerful tool for timely detection of NAI resistant viruses and that NI assays are needed for comprehensive testing to detect novel resistance substitutions.
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Neuraminidase inhibitor resistance in influenza viruses and laboratory testing methods.
Antivir. Ther. (Lond.)
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Infection with influenza viruses, including seasonal, avian and pandemic viruses, remains a worldwide public health problem. Although influenza virus infection is both vaccine preventable and drug treatable, high rates of mutation and reassortment of viruses can result in reduced effectiveness of vaccines or drugs. Currently, two classes of drugs, adamantanes (M2 blockers) and neuraminidase (NA) inhibitors (NAIs), are available for treatment and chemoprophylaxis of influenza infections. Given these limited antiviral therapy options, resistance to anti-influenza drugs is a constant concern. The emergence and global spread of adamantane-resistant H3N2 viruses in 2003-2004 and oseltamivir-resistant seasonal H1N1 viruses in 2007-2009 demonstrated the ability of drug-resistant variants to rapidly become predominant worldwide. Since the 2009 H1N1 pandemic, all influenza viruses circulating in humans are M2-blocker-resistant and, in general, NAI-susceptible. However, pandemic H1N1 viruses with resistance to the NAI oseltamivir have been reported. Permissive drift mutations and reassortment of viral gene segments have been proposed as mechanisms underlying the retained replicative fitness of resistant viruses. Nevertheless, the precise role of these genetic changes in the efficient transmission and maintenance of resistant viruses in the absence of drug pressure remains poorly understood. In this review, we summarize NAI resistance in influenza viruses and discuss recent challenges in laboratory testing methods. Close monitoring of antiviral resistance among all influenza viruses, both locally and globally, are essential to inform public health strategies for the control of influenza infections.
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Oseltamivir-resistant pandemic (H1N1) 2009 virus infections, United States, 2010-11.
Emerging Infect. Dis.
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During October 2010-July 2011, 1.0% of pandemic (H1N1) 2009 viruses in the United States were oseltamivir resistant, compared with 0.5% during the 2009-10 influenza season. Of resistant viruses from 2010-11 and 2009-10, 26% and 89%, respectively, were from persons exposed to oseltamivir before specimen collection. Findings suggest limited community transmission of oseltamivir-resistant virus.
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