Characterization of a trypsin-dependent avian influenza H5N1-pseudotyped HIV vector system for high throughput screening of inhibitory molecules.
ABSTRACT: In this study, we have generated and characterized an avian influenza H5N1 hemagglutinin (HA), neuraminidase (NA) and M2 ion channel pseudotyped HIV-based vector system (HaNaM-pseudotyped HIV vector). The cleavage site of the HA protein was modified to necessitate trypsin-dependent maturation of the glycoprotein. HA, NA and M2 were efficiently incorporated in HIV vector particles which could transduce different cell lines in a trypsin-dependent manner. Results also showed that the presence of avian influenza M2 and NA proteins maximized both vector production and transduction and that transduction was highly sensitive to the specific NA inhibitor oseltamivir (Tamiflu). H5N1 HaNaM-pseudotyped HIV vector system was also adapted for cell-based high throughput screening of drug candidates against influenza virus infection, and its high sensitivity to the specific oseltamivir validates its potential utility in the identification of new influenza inhibitors. Overall, the trypsin-dependent H5N1-pseudotyped HIV vector can mimic avian influenza virus infection processes with sufficient precision to allow for the identification of new antivirals and to study avian influenza virus biology in a lower biosafety level laboratory environment.
Project description:If highly pathogenic H5N1 influenza viruses acquire affinity for human rather than avian respiratory epithelium, will their susceptibility to neuraminidase (NA) inhibitors (the likely first line of defense against an influenza pandemic) change as well? Adequate pandemic preparedness requires that this question be answered. We generated and tested 31 recombinants of A/Vietnam/1203/04 (H5N1) influenza virus carrying single, double, or triple mutations located within or near the receptor binding site in the hemagglutinin (HA) glycoprotein that alter H5 HA binding affinity or specificity. To gain insight into how combinations of HA and NA mutations can affect the sensitivity of H5N1 virus to NA inhibitors, we also rescued viruses carrying the HA changes together with the H274Y NA substitution, which was reported to confer resistance to the NA inhibitor oseltamivir. Twenty viruses were genetically stable. The triple N158S/Q226L/N248D HA mutation (which eliminates a glycosylation site at position 158) caused a switch from avian to human receptor specificity. In cultures of differentiated human airway epithelial (NHBE) cells, which provide an ex vivo model that recapitulates the receptors in the human respiratory tract, none of the HA-mutant recombinants showed reduced susceptibility to antiviral drugs (oseltamivir or zanamivir). This finding was consistent with the results of NA enzyme inhibition assay, which appears to predict influenza virus susceptibility in vivo. Therefore, acquisition of human-like receptor specificity does not affect susceptibility to NA inhibitors. Sequence analysis of the NA gene alone, rather than analysis of both the NA and HA genes, and phenotypic assays in NHBE cells are likely to adequately identify drug-resistant H5N1 variants isolated from humans during an outbreak.
Project description: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.
Project description:Highly pathogenic avian H5N1 influenza viruses remain a pandemic threat. Antiviral drugs such as neuraminidase (NA) inhibitors will be crucial for disease control in the event of a pandemic. Should drug-resistant H5N1 viruses develop, all defense strategies will be compromised. To determine the likelihood and mechanisms of emergence of NA inhibitor-resistant H5N1 variants in humans, we serially passaged two H5N1 viruses, A/Hong Kong/213/03 and A/Turkey/65-1242/06, in normal human bronchial epithelial (NHBE) cells in the presence of oseltamivir, zanamivir, or peramivir. To monitor the emergence of changes associated with the adaptation of H5N1 viruses to humans, we passaged the strains in the absence of drugs. Under pressure of each NA inhibitor, A/Turkey/65-1242/06 developed mutations in the hemagglutinin (HA) (H28R and P194L/T215I) and NA (E119A) proteins that reduced virus binding to ?2,3-sialyl receptor and NA activity. Oseltamivir pressure selected a variant of A/Hong Kong/213/03 virus with HA P194S mutation that decreased viral binding to ?2,6 receptor. Under peramivir pressure, A/Hong Kong/213/03 virus developed a novel NA mutation, R156K, that reduced binding to all three drugs, caused about 90% loss of NA activity, and compromised replication in NHBE cells. Both strains were eliminated in NHBE cells when they were cultivated in the absence of drugs. Here, we show for the first time that decreased NA activity mediated through NA inhibitors is essential for the adaptation of pandemic H5N1 influenza virus to humans. This ability of decreased NA activity to promote H5N1 infection underlines the necessity to optimize management strategies for a plausible H5N1 pandemic.
Project description:Antibodies to the hemagglutinin (HA) and neuraminidase (NA) glycoproteins are the major mediators of protection against influenza virus infection. Here, we report that current influenza vaccines poorly display key NA epitopes and rarely induce NA-reactive B cells. Conversely, influenza virus infection induces NA-reactive B cells at a frequency that approaches (H1N1) or exceeds (H3N2) that of HA-reactive B cells. NA-reactive antibodies display broad binding activity spanning the entire history of influenza A virus circulation in humans, including the original pandemic strains of both H1N1 and H3N2 subtypes. The antibodies robustly inhibit the enzymatic activity of NA, including oseltamivir-resistant variants, and provide robust prophylactic protection, including against avian H5N1 viruses, in vivo. When used therapeutically, NA-reactive antibodies protected mice from lethal influenza virus challenge even 48 hr post infection. These findings strongly suggest that influenza vaccines should be optimized to improve targeting of NA for durable and broad protection against divergent influenza strains.
Project description:The use of antiviral drugs such as influenza neuraminidase (NA) inhibitors is a critical strategy to prevent and control flu pandemic, but this strategy faces the challenge of emerging drug-resistant strains. For a highly pathogenic avian influenza (HPAI) H5N1 virus, biosafety restrictions have significantly limited the efforts to monitor its drug responses and mechanisms involved. In this study, a rapid and biosafe assay based on NA pseudovirus was developed to study the resistance of HPAI H5N1 virus to NA inhibitor drugs. The H5N1 NA pseudovirus was comprehensively tested using oseltamivir-sensitive strains and their resistant mutants. Results were consistent with those in previous studies, in which live H5N1 viruses were used. Several oseltamivir-resistant mutations reported in human H1N1 were also identified to cause decreased oseltamivir sensitivity in H5N1 NA by using the H5N1 NA pseudovirus. Thus, H5N1 NA pseudoviruses could be used to monitor HPAI H5N1 drug resistance rapidly and safely.
Project description:Currently, two neuraminidase (NA) inhibitors, oseltamivir and zanamivir, which must be administrated twice daily for 5 days for maximum therapeutic effect, are licensed for the treatment of influenza. However, oseltamivir-resistant mutants of seasonal H1N1 and highly pathogenic H5N1 avian influenza A viruses have emerged. Therefore, alternative antiviral agents are needed. Recently, a new neuraminidase inhibitor, R-125489, and its prodrug, CS-8958, have been developed. CS-8958 functions as a long-acting NA inhibitor in vivo (mice) and is efficacious against seasonal influenza strains following a single intranasal dose. Here, we tested the efficacy of this compound against H5N1 influenza viruses, which have spread across several continents and caused epidemics with high morbidity and mortality. We demonstrated that R-125489 interferes with the NA activity of H5N1 viruses, including oseltamivir-resistant and different clade strains. A single dose of CS-8958 (1,500 microg/kg) given to mice 2 h post-infection with H5N1 influenza viruses produced a higher survival rate than did continuous five-day administration of oseltamivir (50 mg/kg twice daily). Virus titers in lungs and brain were substantially lower in infected mice treated with a single dose of CS-8958 than in those treated with the five-day course of oseltamivir. CS-8958 was also highly efficacious against highly pathogenic H5N1 influenza virus and oseltamivir-resistant variants. A single dose of CS-8958 given seven days prior to virus infection also protected mice against H5N1 virus lethal infection. To evaluate the improved efficacy of CS-8958 over oseltamivir, the binding stability of R-125489 to various subtypes of influenza virus was assessed and compared with that of other NA inhibitors. We found that R-125489 bound to NA more tightly than did any other NA inhibitor tested. Our results indicate that CS-8958 is highly effective for the treatment and prophylaxis of infection with H5N1 influenza viruses, including oseltamivir-resistant mutants.
Project description:To study the precise role of the neuraminidase (NA), and its stalk region in particular, in the assembly, release, and entry of influenza virus, we deleted the 20-aa stalk segment from 2009 pandemic H1N1 NA (09N1) and inserted this segment, now designated 09s60, into the stalk region of a highly pathogenic avian influenza (HPAI) virus H5N1 NA (AH N1). The biological characterization of these wild-type and mutant NAs was analyzed by pseudotyped particles (pseudoparticles) system. Compared with the wild-type AH N1, the wild-type 09N1 exhibited higher NA activity and released more pseudoparticles. Deletion/insertion of the 09s60 segment did not alter this relationship. The infectivity of pseudoparticles harboring NA in combination with the hemagglutinin from HPAI H5N1 (AH H5) was decreased by insertion of 09s60 into AH N1 and was increased by deletion of 09s60 from 09N1. When isolated from the wild-type 2009H1N1 virus, 09N1 existed in the forms (in order of abundance) dimer>>tetramer>monomer, but when isolated from pseudoparticles, 09N1 existed in the forms dimer>monomer>>>tetramer. After deletion of 09s60, 09N1 existed in the forms monomer>>>dimer. AH N1 from pseudoparticles existed in the forms monomer>>dimer, but after insertion of 09s60, it existed in the forms dimer>>monomer. Deletion/insertion of 09s60 did not alter the NA glycosylation pattern of 09N1 or AH N1. The 09N1 was more sensitive than the AH N1 to the NA inhibitor oseltamivir, suggesting that the infectivity-enhancing effect of oseltamivir correlates with robust NA activity.
Project description:BACKGROUND: The neuraminidase (NA) inhibitor oseltamivir is widely used to treat patients infected with influenza viruses. An Ile-to-Val change at position 117 in influenza A virus subtype H5N1 NA (NA-I117V) confers a reduction in susceptibility to oseltamivir carboxylate. However, the in vivo relevance and molecular basis of the decreased sensitivity mediated by this mutation are poorly understood. METHODS: We created single-point-mutant viruses with 3 genetically different backgrounds (ie, 1 belonging to clade 1 and 2 belonging to clade 2.3.4) and evaluated the effects of the I117V mutation on oseltamivir susceptibility in vitro, in vivo, and in silico. RESULTS: The NA-I117V mutation conferred a slight reduction in susceptibility to oseltamivir in vitro (1.3- to 6.3-fold changes), although it did not substantially compromise NA enzymatic activity. Mice infected with I117V virus exhibited reduced susceptibility to oseltamivir and decreased survival in 2 of 3 virus pairs tested. Molecular dynamics simulations revealed that I117V caused the loss of hydrogen bonds between an arginine at position 118 and the carboxyl group of oseltamivir, leading to a lower binding affinity for oseltamivir. CONCLUSIONS: Our findings provide new insight into the mechanism of NA-I117V-mediated oseltamivir resistance in highly pathogenic H5N1 avian influenza viruses.
Project description:Influenza neuraminidase (NA) is essential for virus release from its host cells and it is one of the targets for structure-based antiviral drug design.In this report, we established a pseudoviral particle release assay to study NA function, which is based on lentiviral particles pseudotyped with influenza glycoproteins HA and NA as a surrogate system. Through an extensive molecular analysis, we sought to characterize important residues governing NA function. We identified five residues of NA, 234, 241, 257, 286 and 345, four of which (except 345) map away from the active site of NA when projected onto the three-dimensional structure of avian influenza H5N1 NA, and substitutions of these residues adversely affected the NA-mediated viral particle release, suggesting that these residues are critical for NA enzymatic activity.Through extensive chimeric and mutational analyses, we have identified several residues, which map away from the active site and are critical for NA function. These findings provide new insights into NA-mediated pseudoviral particle release and may have important implications in drug design and therapeutics against influenza infection.
Project description:The emergence of novel influenza viruses that cause devastating human disease is an ongoing threat and serves as an impetus for the continued development of novel approaches to influenza vaccines. Influenza vaccine development has traditionally focused on producing humoral and/or cell-mediated immunity, often against the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Here, we describe a new vaccine candidate that utilizes a replication-defective vesicular stomatitis virus (VSV) vector backbone that lacks the native G surface glycoprotein gene (VSV?G). The expression of the H5 HA of an H5N1 highly pathogenic avian influenza virus (HPAIV), A/Vietnam/1203/04 (VN1203), and the NA of the mouse-adapted H1N1 influenza virus A/Puerto Rico/8/34 (PR8) in the VSV?G vector restored the ability of the recombinant virus to replicate in cell culture, without the requirement for the addition of trypsin. We show here that this recombinant virus vaccine candidate was nonpathogenic in mice when given by either the intramuscular or intranasal route of immunization and that the in vivo replication of VSV?G-H5N1 is profoundly attenuated. This recombinant virus also provided protection against lethal H5N1 infection after a single dose. This novel approach to vaccination against HPAIVs may be widely applicable to other emerging strains of influenza virus.Preparation for a potentially catastrophic influenza pandemic requires novel influenza vaccines that are safe, can be produced and administered quickly, and are effective, both soon after administration and for a long duration. We have created a new influenza vaccine that utilizes an attenuated vesicular stomatitis virus (VSV) vector, to deliver and express influenza virus proteins against which vaccinated animals develop potent antibody responses. The influenza virus hemagglutinin and neuraminidase proteins, expressed on the surface of VSV particles, allowed this vaccine to grow in cell culture and induced a potent antibody response in mice that was effective against infection with a lethal influenza virus. The mice showed no adverse reactions to the vaccine, and they were protected against an otherwise lethal influenza infection after only 14 days postvaccination and after as many as 140 days postvaccination. The ability to rapidly produce this safe and effective vaccine in cell culture is additionally advantageous.