首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
Influenza A viruses (IAVs) encode two critical glycoproteins, hemagglutinin and neuraminidase (NA). Hemagglutinin promotes viral docking onto cells via interactions with IAV's receptor, sialic acid and NA facilitates release of newly synthesized virions by cleaving cellular and viral sialic acid. NA inhibitors, such as oseltamivir, are widely used drugs that work by binding to the active site of NA. Although oseltamivir-resistant viruses were easily generated years ago in laboratory experiments, it was widely believed that these viruses would not be able to circulate in the human population as they did not replicate efficiently. However, oseltamivir-resistant H1N1 viruses rapidly spread during the 2007-2008 IAV season and these viruses contained precisely the same exact drug-resistance mutation identified years prior, a histidine to tyrosine substitution at NA residue 274 (H274Y). Unlike the experimentally derived NA inhibitor-resistant viruses, 2007-2008 H1N1 viruses containing H274Y replicated efficiently. Bloom et al. have solved this riddle by identifying permissive NA mutations that allow viruses to tolerate H274Y. Here, we discuss these important findings and speculate how these studies may facilitate early detection of drug-resistant strains in the future.  相似文献   

2.
The surveillance of seasonal influenza virus susceptibility to neuraminidase (NA) inhibitors was conducted using an NA inhibition assay. The 50% inhibitory concentration values (IC50s) of 4,570 viruses collected globally from October 2004 to March 2008 were determined. Based on mean IC50s, A(H3N2) viruses (0.44 nM) were more sensitive to oseltamivir than A(H1N1) viruses (0.91 nM). The opposite trend was observed with zanamivir: 1.06 nM for A(H1N1) and 2.54 nM for A(H3N2). Influenza B viruses exhibited the least susceptibility to oseltamivir (3.42 nM) and to zanamivir (3.87 nM). To identify potentially resistant viruses (outliers), a threshold of a mean IC50 value + 3 standard deviations was defined for type/subtype and drug. Sequence analysis of outliers was performed to identify NA changes that might be associated with reduced susceptibility. Molecular markers of oseltamivir resistance were found in six A(H1N1) viruses (H274Y) and one A(H3N2) virus (E119V) collected between 2004 and 2007. Some outliers contained previously reported mutations (e.g., I222T in the B viruses), while other mutations [e.g., R371K and H274Y in B viruses and H274N in A(H3N2) viruses) were novel. The R371K B virus outlier exhibited high levels of resistance to both inhibitors (>100 nM). A substantial variance at residue D151 was observed among A(H3N2) zanamivir-resistant outliers. The clinical relevance of newly identified NA mutations is unknown. A rise in the incidence of oseltamivir resistance in A(H1N1) viruses carrying the H274Y mutation was detected in the United States and in other countries in the ongoing 2007 to 2008 season. As of March 2008, the frequency of resistance among A(H1N1) viruses in the United States was 8.6% (50/579 isolates). The recent increase in oseltamivir resistance among A(H1N1) viruses isolated from untreated patients raises public health concerns and necessitates close monitoring of resistance to NA inhibitors.  相似文献   

3.
Abed Y  Baz M  Boivin G 《Antiviral therapy》2006,11(8):971-976
Subtype-specific neuraminidase (NA) mutations conferring resistance to NA inhibitors (NAIs) have been reported during in vitro passages and in clinic. In this study, we evaluated the impact of various NA mutations (E119A/G/V, H274Y, R292K and N294S) on the susceptibility profiles to different NAIs (oseltamivir, zanamivir and peramivir) using recombinant NA proteins of influenza A/WSN/33 (H1N1) and A/Sydney/5/97-like (H3N2) viruses. In the Nl subtype, the E119V mutation conferred cross-resistance to oseltamivir, zanamivir and peramivir [1,727-2,144 and 5,050-fold increase in IC50 values compared with wild-type (WT)] whereas only oseltamivir-resistance (1,028-fold increase in IC50) was conferred by the same mutation in the N2 subtype. The N294S mutation conferred resistance to oseltamivir in both the NI and N2 subtypes (197- and 1,879-fold increase in IC50 values, respectively) whereas the H274Y mutation conferred resistance to oseltamivir (754-fold increase) and peramivir (260-fold increase) in the N1 subtype only. The virulence of reverse genetics-rescued A/WSN/33 viruses harbouring H274Y and N294S NA mutations was investigated in Balb/c mice. The WT and H274Y recombinants had identical LD50 values (103 PFUs) and generated similar viral lung titres, whereas a higher LD50 (10 PFUs) and a 1-log decrease in viral lung titres were obtained with the N294S mutant. This study shows that some NA mutations at framework residues may confer resistance to one or three NAIs depending on the viral subtype. It suggests that certain drug-resistant NA mutants may still be virulent although additional studies using clinical isolates are needed to confirm our results.  相似文献   

4.
To identify mutations that can arise in highly pathogenic A(H5N1) viruses under neuraminidase inhibitor selective pressure, two antigenically different strains were serially passaged with increasing levels of either oseltamivir or zanamivir. Under oseltamivir pressure, both A(H5N1) viruses developed a H274Y neuraminidase mutation, although in one strain the mutation occurred in combination with an I222M neuraminidase mutation. The H274Y neuraminidase mutation reduced oseltamivir susceptibility significantly (900- to 2,500-fold compared to the wild type). However the dual H274Y/I222M neuraminidase mutation had an even greater impact on resistance, with oseltamivir susceptibility reduced significantly further (8,000-fold compared to the wild type). A similar affect on oseltamivir susceptibility was observed when the dual H274Y/I222M mutations were introduced, by reverse genetics, into a recombinant seasonal human A(H1N1) virus and also when an alternative I222 substitution (I222V) was generated in combination with H274Y in A(H5N1) and A(H1N1) viruses. These viruses remained fully susceptible to zanamivir but demonstrated reduced susceptibility to peramivir. Following passage of the A(H5N1) viruses in the presence of zanamivir, the strains developed a D198G neuraminidase mutation, which reduced susceptibility to both zanamivir and oseltamivir, and also an E119G neuraminidase mutation, which demonstrated significantly reduced zanamivir susceptibility (1,400-fold compared to the wild type). Mutations in hemagglutinin residues implicated in receptor binding were also detected in many of the resistant strains. This study identified the mutations that can arise in A(H5N1) under either oseltamivir or zanamivir selective pressure and the potential for dual neuraminidase mutations to result in dramatically reduced drug susceptibility.Large-scale outbreaks of highly pathogenic A(H5N1) avian influenza affecting poultry have occurred throughout many parts of Asia, North Africa, and the Middle East since 2003 (1). The virus, which now appears to be enzootic in many regions, has on occasion caused zoonotic infections in humans (1). Humans who acquire the infection develop severe pneumonia that can progress to acute respiratory distress syndrome with high risk of mortality. For the 6-year period 2003 to 2008, 395 confirmed A(H5N1) virus human infections were reported, and 250 were fatal (a case fatality rate of 63%) (http://www.who.int/csr/disease/avian_influenza/en/index.html). Human-to-human transmission of A(H5N1) virus appears to be rare and has been associated only with very close unprotected contact with severely ill patients (30). Of concern is the potential for the A(H5N1) virus to become easily transmissible between humans, which, because of the lack of prior immunity to this strain in humans, might result in a global influenza pandemic. Based on these theoretical concerns and the experiences of large-scale morbidity and mortality from previous influenza pandemics, many countries have prepared plans to address or mitigate such an occurrence, including the stockpiling of inactivated A(H5N1) influenza vaccines, as well as anti-influenza drugs. Because multiple vaccine doses may be necessary to achieve protection and some time would be required to generate a vaccine with an antigenically matched strain (1), antiviral drugs could play a critical role in the treatment or prophylaxis of influenza, particularly during the early stages of a pandemic. The oral neuraminidase (NA) inhibitor oseltamivir (Tamiflu) has been the most widely used anti-influenza drug for the treatment of A(H5N1) virus -infected patients and has been stockpiled for potential broad use. Results from uncontrolled clinical trials suggest that the use of oseltamivir may increase the survival rate of patients with A(H5N1) virus infection, particularly if administered early in the course of illness (1). However, oseltamivir-resistant A(H5N1) virus variants with an H274Y NA mutation have been isolated from treated patients and may be associated with clinical deterioration and fatal outcomes (9). Viruses with the H274Y NA mutations are susceptible to the NA inhibitor zanamivir, which has led to the inclusion of inhaled zanamivir, together with oseltamivir, in pandemic drug stockpiles. The volume of drug that might be used in the event of a pandemic would be significantly greater than has ever been used previously for treatment of seasonal influenza. There is concern that this may lead to a high frequency of drug resistance. While previous studies have identified a number of NA inhibitor resistance mutations that have arisen in seasonal influenza viruses under drug pressure, little is known about which NA inhibitor resistance mutations might arise in highly pathogenic A(H5N1) viruses. To investigate this question, two A(H5N1) strains from different phylogenetic clades were subjected to serial passage in Madin-Darby canine kidney (MDCK) cells in the presence of increasing levels of either oseltamivir or zanamivir, and the resultant viruses were analyzed functionally and genetically.  相似文献   

5.
The evolution of oseltamivir resistance mutations during selection through serial passages in animals is still poorly described. Herein, we assessed the evolution of neuraminidase (NA) and hemagglutinin (HA) genes of influenza A/WSN/33 (H1N1) and A/Victoria/3/75 (H3N2) viruses recovered from the lungs of experimentally infected BALB/c mice receiving suboptimal doses (0.05 and 1 mg/kg of body weight/day) of oseltamivir over two generations. The traditional phenotypic and genotypic methods as well as deep-sequencing analysis were used to characterize the potential selection of mutations and population dynamics of oseltamivir-resistant variants. No oseltamivir-resistant NA or HA changes were detected in the recovered A/WSN/33 viruses. However, we observed a positive selection of the I222T NA substitution in the recovered A/Victoria/3/75 viruses, with a frequency increasing over time and with an oseltamivir concentration from 4% in the initial pretherapy inoculum up to 28% after two lung passages. Although the presence of mixed I222T viral populations in mouse lungs only led to a minimal increase in oseltamivir 50% enzyme-inhibitory concentrations (IC50s) (by a mean of 5.7-fold) compared to that of the baseline virus, the expressed recombinant A/Victoria/3/75 I222T NA protein displayed a 16-fold increase in the oseltamivir IC50 level compared to that of the recombinant wild type (WT). In conclusion, the combination of serial in vivo passages under neuraminidase inhibitor (NAI) pressure and temporal deep-sequencing analysis enabled, for the first time, the identification and selection of the oseltamivir-resistant I222T NA mutation in an influenza H3N2 virus. Additional in vivo selection experiments with other antivirals and drug combinations might provide important information on the evolution of antiviral resistance in influenza viruses.  相似文献   

6.
The new oral neuraminidase (NA) inhibitor A-322278 was evaluated in mice infected with influenza A/H1N1 wild-type virus or the oseltamivir-resistant (H274Y mutant) virus. A-322278 decreased mortality rates and lung virus titers significantly more than oseltamivir in mice infected with the NA H274Y mutant when therapy was started 4 h before or even 48 h after infection.  相似文献   

7.
Peramivir is a neuraminidase (NA) inhibitor (NAI) under development that must be administered by the systemic route. The prophylactic activity of intramuscular (IM) peramivir was evaluated with mice infected with wild-type (WT) and oseltamivir-resistant (H274Y NA mutant) recombinant influenza A/WSN/33 (H1N1) viruses. Treatment regimens consisted of IM injections starting 1 h before viral challenge that were single (45 mg/kg or 90 mg/kg) or multiple (45 mg/kg daily for 5 days). All peramivir regimens prevented mortality and weight loss while significantly reducing lung viral titers (LVT) in mice infected with the WT virus. For animals infected with the H274Y mutant, the multiple-dose regimen completely prevented mortality and was associated with significant reduction in weight loss and LVT compared to untreated animals. In contrast, both single-treatment regimens reduced mortality and weight loss but did not significantly reduce LVT. Although further experiments using different influenza A/H1N1 virus strains and other animal models are needed, our results suggest that 5-day IM peramivir therapy may be considered a prophylactic alternative to control influenza infections caused by oseltamivir-resistant viruses with the H274Y mutation.Neuraminidase (NA) inhibitors (NAIs) constitute one of the most valuable options for the control of influenza epidemics and pandemics. Two NAIs, inhaled zanamivir and oral oseltamivir, have been approved for the treatment and prevention of influenza infections in many countries (16). In addition, other NAIs are at different stages of development. Peramivir, which is a cyclopentane analogue compound, has shown potent in vitro activity against influenza A and B viruses (4). By the use of NAI assays, we previously demonstrated that peramivir 50% inhibitory concentration (IC50) values for Canadian clinical influenza A/H3N2, A/H1N1, and B viruses were lower than those of zanamivir and oseltamivir (10). In other studies, mean IC50 values of clinical influenza A/H1N1 viruses from untreated individuals against peramivir were also lower than those against oseltamivir and zanamivir (14, 15). Furthermore, on-site dissociation studies demonstrated that peramivir remained tightly bound to the NA enzyme with a half-time for the substrate conversion of >24 h compared to 1.25 h for both zanamivir and oseltamivir (5).In controlled trials of prophylaxis and treatment, oral peramivir was associated with reduced viral titers but no significant decrease in time to relief of symptoms, a feature that could be attributed to a low oral bioavailability in humans (6). The bioavailability of peramivir may be improved by using intravenous (IV) or intramuscular (IM) injections. Indeed, comparison of single IM versus oral peramivir with the same dose (10 mg/kg), administered 4 h prior to a lethal influenza A/WSN/33 (H1N1) virus challenge, demonstrated that the IM route was associated with a higher survival rate in mice than that of the oral route (100% versus 50%) (5). Also, a single IV injection of 3 mg/kg of peramivir provided a significant therapeutic effect that was superior to that of oral oseltamivir in a lethal mouse model of influenza A and B virus infections (18). The emergence and rapid dissemination of the seasonal A/Brisbane/59/2007 (H1N1) virus containing the NA mutation H274Y in N2 numbering (H275Y in N1 numbering), which is associated with a high level of resistance to oseltamivir and moderate cross-resistance to peramivir in vitro (9), are a major clinical concern. The aim of the present study was to evaluate the prophylactic efficacy of IM injections of peramivir in mice infected with a recombinant influenza A/WSN/33 (H1N1) virus containing or not containing the H274Y NA mutation, which has been associated with 427- and 48-fold increases in oseltamivir and peramivir IC50 values, respectively, in NAI assays (1).  相似文献   

8.
Neuraminidase inhibitors (NAIs) play a major role for managing influenza virus infections. The widespread oseltamivir resistance among 2007-2008 seasonal A(H1N1) viruses and community outbreaks of oseltamivir-resistant A(H1N1)pdm09 strains highlights the need for additional anti-influenza virus agents. Laninamivir is a novel long-lasting NAI that has demonstrated in vitro activity against influenza A and B viruses, and its prodrug (laninamivir octanoate) is in phase II clinical trials in the United States and other countries. Currently, little information is available on the mechanisms of resistance to laninamivir. In this study, we first performed neuraminidase (NA) inhibition assays to determine the activity of laninamivir against a set of influenza A viruses containing NA mutations conferring resistance to one or many other NAIs. We also generated drug-resistant A(H1N1) and A(H3N2) viruses under in vitro laninamivir pressure. Laninamivir demonstrated a profile of susceptibility that was similar to that of zanamivir. More specifically, it retained activity against oseltamivir-resistant H275Y and N295S A(H1N1) variants and the E119V A(H3N2) variant. In vitro, laninamivir pressure selected the E119A NA substitution in the A/Solomon Islands/3/2006 A(H1N1) background, whereas E119K and G147E NA changes along with a K133E hemagglutinin (HA) substitution were selected in the A/Quebec/144147/2009 A(H1N1)pdm09 strain. In the A/Brisbane/10/2007 A(H3N2) background, a large NA deletion accompanied by S138A/P194L HA substitutions was selected. This H3N2 variant had altered receptor-binding properties and was highly resistant to laninamivir in plaque reduction assays. Overall, we confirmed the similarity between zanamivir and laninamivir susceptibility profiles and demonstrated that both NA and HA changes can contribute to laninamivir resistance in vitro.  相似文献   

9.
The I222K, I222R, and I222T substitutions in neuraminidase (NA) have been found in clinically derived 2009 pandemic influenza A/H1N1 viruses with altered susceptibilities to NA inhibitors (NAIs). The effects of these substitutions, together with the most frequently observed resistance-related substitution, H274Y, on viral fitness and resistance mechanisms were further investigated in this study. Reduced sensitivities to oseltamivir were observed in all three mutants (I222K, I222R, and I222T). Furthermore, the I222K and I222T substitutions had a combinational effect of further increasing resistance in the presence of H274Y, which might result from a conformational restriction in the NA binding site. Of note, by using molecular dynamics simulations, R152, the neighbor of T222, was observed to translate to a position closer to T222, resulting in the narrowing of the binding pocket, which otherwise only subtends the residue substitution of H274Y. Moreover, significantly attenuated NA function and viral growth abilities were found in the I222K+H274Y double mutant, while the I222T+H274Y double mutant exhibited slightly delayed growth but had a peak viral titer similar to that of the wild-type virus in MDCK cells. The relative growth advantage of the I222T mutant versus the I222K mutant and the higher frequency of I222T emerging in N1 subtype influenza viruses raise concerns necessitating close monitoring of the dual substitutions I222T and H274Y.  相似文献   

10.
BACKGROUND: Viruses resistant to zanamivir have been generated in vitro, but no resistant virus has yet been isolated from a zanamivir-treated immunocompetent patient. In contrast most resistant viruses isolated from oseltamivir-treated patients correspond to those selected in vitro. However, despite mutations being in conserved residues in the neuraminidase (NA) they do not confer resistance in all NA subtypes. OBJECTIVES AND METHODS: We have used reverse genetics and the recombinant baculovirus expression system for investigating reasons for the lack of isolation of zanamivir-resistant H3N2 viruses and for further exploring subtype-specific oseltamivir resistance. RESULTS: H3N2 viruses generated by reverse genetics with H274Y, R292K E119V and E119D mutations were rescued. Those with E119G, E119A or R152K mutations could only be rescued in the presence of exogenous NA and after passage in the absence of exogenous NA only isolates that had reverted to the wild-type NA or, surprisingly, E119G/A to E119V NA were isolated. Mutations conferring zanamivir resistance significantly affected enzyme activity, virus replication or NA thermal stability. E119V viruses were stable and grew to similar titres as wild-type virus, consistent with their isolation from oseltamivir-treated patients. Mutations conferring oseltamivir resistance in N1 (H274Y) and B (R152K) NAs also conferred resistance in recombinant G70C N9 NA expressed in insect cells. CONCLUSIONS: These data suggest that zanamivir-resistant H3N2 viruses may not readily arise in vivo due to their poor viability. The G70C N9 NA may also provide a useful model for understanding the structural basis of subtype-specific drug resistance.  相似文献   

11.
A system of reverse genetics was used to generate influenza A/H1N1 viruses harbouring neuraminidase (NA) mutations previously associated with resistance to NA inhibitors in various viral subtypes. The His274Tyr and Glu119Gln mutants were rescued whereas the Arg292Lys and Glu1l9 --> Gly, Val, Ala or Asp mutants could not be generated. In NA inhibition assays, the His274Tyr mutant was resistant to oseltamivir (430-fold over wild-type) and BCX-1812 (50-fold) but was sensitive to zanamivir. A similar trend was seen when the mutant was evaluated by plaque reduction assay (PRA). The Glu119Gln mutant expressed a low level of resistance to oseltamivir (nine-fold) and zanamivir (fourfold) in NA inhibition assay but was only marginally resistant to oseltamivir (fourfold) in PRA. The replication capacity of both mutants, in particular that of the His274Tyr virus, was impaired when compared with the wild-type virus in vitro.  相似文献   

12.
Comparison of the viral persistence of pandemic H1N1 (H1N1pdm) and seasonal H1N1 with or without H275Y mutation after oseltamivir therapy has not been adequately done. Virus was isolated before and on days 4-6 from the start of oseltamivir treatment for 158 cases of seasonal (2007-2008 and 2008-2009 seasons) or pandemic (2009-2010 season) H1N1 influenza. Sequence analysis was done for each season and NA inhibition assay (IC(50)) was done in the 2009-2010 season. H275Y mutation before therapy was 0% in the 2007-2008 and 2009-2010 seasons, but 100% in the 2008-2009 season. Fever and other symptoms were noticeably prolonged after oseltamivir therapy for children with H275Y mutated seasonal H1N1 (2008-2009 season), but not in patients with seasonal H1N1 without mutation (2007-2008) or H1N1pdm (2009-2010). The viral persistence rate was significantly higher for patients 15 years or younger than for those 16 years and older with H275Y mutated seasonal H1N1 (46.2% and 10.5%, respectively) or with H1N1pdm (43.3% and 11.5%, respectively). The H275Y mutation emerged after oseltamivir treatment in 2.4% (2/82) of all patients with H1N1pdm. In two children, the H275Y mutation emerged after therapy and the IC(50) increased more than 200 fold; however, the prolongation of fever was not so prominent. In conclusion, oseltamivir was effective for fever and other clinical symptoms; however, the virus persisted longer than expected after treatment in H1N1pdm influenza-infected children in the 2009-2010 season, similar to seasonal H1N1 with H275Y mutation in the 2008-2009 season.  相似文献   

13.
14.
Neuraminidase (NA) inhibitors (NIs) are the first line of defense against influenza virus. Reverse genetics experiments allow the study of resistance mechanisms by anticipating the impacts of mutations to the virus. To look at the possibility of an increased effect on the resistance phenotype of a combination of framework mutations, known to confer resistance to oseltamivir or zanamivir, with limited effect on virus fitness, we constructed 4 viruses by reverse genetics in the A/Moscow/10/99 H3N2 background containing double mutations in their neuraminidase genes: E119D+I222L, E119V+I222L, D198N+I222L, and H274Y+I222L (N2 numbering). Among the viruses produced, the E119D+I222L mutant virus was not able to grow without bacterial NA complementation and the D198N+I222L mutant and H274Y+I222L mutant were not stable after passages in MDCK cells. The E119V+I222L mutant was stable after five passages in MDCK cells. This E119V-and-I222L combination had a combinatorial effect on oseltamivir resistance. The total NA activity of the E119V+I222L mutant was low (5% compared to that of the wild-type virus). This drop in NA activity resulted from a decreased NA quantity in the virion in comparison to that of the wild-type virus (1.4% of that of the wild type). In MDCK-SIAT1 cells, the E119V+I222L mutant virus did not present a replicative advantage over the wild-type virus, even in the presence of oseltamivir. Double mutations combining two framework mutations in the NA gene still have to be monitored, as they could induce a high level of resistance to NIs, without impairing the NA affinity. Our study allows a better understanding of the diversity of the mechanisms of resistance to NIs.  相似文献   

15.
To understand the current situation of antiviral-resistance of influenza viruses to neuraminidase inhibitors (NAIs) in Mainland China, The antiviral-resistant surveillance data of the circulating influenza viruses in Mainland China during the 2016–2017 influenza season were analyzed.The total 3215 influenza viruses were studied to determine 50% inhibitory concentration (IC50) for oseltamivir and zanamivir using a fluorescence-based assay.Approximately 0.3% (n = 10) of viruses showed either highly reduced inhibition (HRI) or reduced inhibition (RI) against at least one NAI. The most common neuraminidase (NA) amino acid substitution was H275Y in A (H1N1)pdm09 virus, which confers HRI by oseltamivir. Two A (H1N1)pdm09 viruses contained a new NA amino acid substitution respectively, S110F and D151E, which confers RI by oseltamivir or/and zanamivir. Two B/Victoria-lineage viruses harbored a new NA amino acid substitution respectively, H134Q and S246P, which confers RI by zanamivir. One B/Victoria-lineage virus contained dual amino acid substitution NA P124T and V422I, which confers HRI by zanamivir. One B/Yamagata-lineage virus was a reassortant virus that haemagglutinin (HA) from B/Yamagata-lineage virus and NA from B/Victoria-lineage virus, defined as B/Yamagata-lineage virus confers RI by oseltamivir, but as B/Victoria-lineage virus confers normal inhibition by oseltamivir. All new substitutions that have not been reported before, the correlation of these substitutions and observed changes in IC50 should be further assessed.During the 2016–2017 influenza season in Mainland China the majority tested viruses were susceptible to oseltamivir and zanamivir. Hence, NAIs remain the recommended antiviral for treatment and prophylaxis of influenza virus infections.  相似文献   

16.
The influenza virus neuraminidase (NA) inhibitors zanamivir and oseltamivir were introduced into clinical practice in various parts of the world between 1999 and 2002. In order to monitor the potential development of resistance, the Neuraminidase Inhibitor Susceptibility Network was established to coordinate testing of clinical isolates collected through the World Health Organization influenza surveillance network from different regions of the world (M. Zambon and F. G. Hayden, Antivir. Res. 49:147-156, 2001). The present study establishes the baseline susceptibilities prior to and shortly after the introduction of the NA inhibitors. Over 1000 clinical influenza isolates recovered from 1996 to 1999 were tested. Susceptibilities were determined by enzyme inhibition assays with chemiluminescent or fluorescent substrates with known NA inhibitor-resistant viruses as controls. The 50% inhibitory concentrations (IC(50)s) depended upon the assay method, the drug tested, and the influenza virus subtype. By both assays, the mean zanamivir IC(50)s were 0.76, 1.82, and 2.28 nM for the subtype H1N1 (N1), H3N2 (N2), and B NAs, respectively, and the oseltamivir IC(50)s were 1.2, 0.5, and 8.8 nM for the N1, N2, and B NAs, respectively. The drug susceptibilities of known zanamivir- and oseltamivir-resistant viruses with the NA mutations E119V, R292K, H274Y, and R152K fell well outside the 95% confidence limits of the IC(50)s for all natural isolates. Sequence analysis of the NAs of viruses for which the IC(50)s were above the 95% confidence limits and several control isolates for which the IC(50)s were in the normal range revealed variations in some previously conserved residues, including D151, A203, T225, and E375 (N2 numbering). Known resistance mutations are both influenza virus subtype and drug specific, but there was no evidence of naturally occurring resistance to either drug in any of the isolates.  相似文献   

17.
Two neuraminidase (NA) inhibitors, zanamivir (Relenza) and oseltamivir phosphate (Tamiflu), have been licensed for use for the treatment and prophylaxis of influenza. We have reported on laninamivir (code name, R-125489), a novel neuraminidase inhibitor, and have discovered that the laninamivir prodrug CS-8958 worked as a long-acting neuraminidase inhibitor in a mouse influenza virus infection model when it is intranasally administered. In this study, CS-8958 was administered just once 7 days before infection and showed significant efficacy in vivo. The efficacy of a single administration of CS-8958 after viral infection was then compared with that of repeated administrations of oseltamivir phosphate or zanamivir in mice and ferrets. CS-8958 showed efficacy superior or similar to the efficacies of the two licensed NA inhibitors. CS-8958 also significantly reduced the titers of an oseltamivir-resistant H1N1 virus with a neuraminidase H274Y substitution in a mouse infection model. These results suggest that since CS-8958 is characteristically long lasting in the lungs, it may be ideal for the prophylaxis and treatment of influenza.Influenza is a serious respiratory illness which can be debilitating and which causes complications that lead to hospitalization and death, especially in elderly individuals. This respiratory disease is caused by influenza A and B viruses, which are pathogens that are highly contagious for humans. Influenza A viruses are classified into subtypes on the basis of the antigenicities of hemagglutinin (HA) and neuraminidase (NA) molecules. To date, 16 HA subtypes (H1 to H16) and 9 NA subtypes (N1 to N9) have been reported. Seasonal influenza or influenza epidemics are caused by influenza A virus H1N1 and H3N2 and influenza B virus (22), and every year the global burden of influenza epidemics is believed to be 3.5 million cases of severe illness and 300,000 to 500,000 deaths (6), before the new pandemic in 2009.In the last 100 years, humans have experienced three influenza pandemics: the first in 1918 (H1N1), the second in 1957 (H2N2), and the third in 1968 (H3N2) (22). In 2009, a new swine-origin influenza virus (H1N1) infected humans (20) and caused a pandemic. WHO has reported more than 400,000 confirmed cases worldwide as of 18 October 2009 (http://www.who.int/csr/don/2009_10_23/en/index.html). Another possible concern is a pandemic caused by highly pathogenic avian influenza (HPAI) H5N1 viruses. Since 2003, the number of humans infected with the HPAI H5N1 virus has increased, and the fatality rate is high. More than 444 cases infected with the H5N1 virus and as many as 262 deaths were reported as of 27 November 2009 (http://www.who.int/csr/disease/avian_influenza/country/en/). Thus, there is considerable concern that such highly pathogenic viruses will cause sustained human-to-human transmission and the next global pandemic.Two countermeasures, vaccinations and treatment with antivirals, are available to control human influenza. Although vaccinations play a critical role in influenza prophylaxis, they are an insufficient tool both for prophylaxis and against a pandemic virus. Therefore, antivirals are an important tool that may be used to mitigate influenza pandemics. Currently, two types of anti-influenza virus drugs are available: M2 ion channel blockers (adamantane) (5) and NA inhibitors. However, adamantane-resistant viruses readily emerge and are already prevalent worldwide among the seasonal influenza viruses (both the H1N1 and the H3N2 subtypes) (1, 3). The pandemic 2009 H1N1 viruses are also adamantane resistant (9). Moreover, the emergence of adamantane-resistant HPAI H5N1 viruses has prevented the use of adamantane for the treatment of infections caused by these viruses (4). The adamantane drugs have not been recommended for use for the treatment or chemoprophylaxis of influenza in the United States since the 2005 influenza season (1, 2). The second and most recently developed class of drugs with activities against influenza A and B viruses are the NA inhibitors, which bind to the NA surface glycoprotein of newly formed virus particles and prevent their efficient release from the host cell (8). Two NA inhibitors, zanamivir (inhaled drug, 10 mg/dose; Relenza) and oseltamivir (oral drug, 75 mg/dose; Tamiflu), are currently licensed for use. Both drugs require twice-daily administration for treatment. Oseltamivir is predominant and is used worldwide for the treatment of influenza, and the generation and circulation of oseltamivir-resistant seasonal influenza viruses have become major concerns (10, 11, 15, 17, 18). In particular, the worldwide prevalence of neuraminidase H274Y oseltamivir-resistant mutants of seasonal H1N1 virus have been reported, and 95% of H1N1 isolates tested from the fourth quarter of 2008 to January 2009 (WHO, http://www.who.int/csr/disease/influenza/H1N1webupdate20090318%20ed_ns.pdf) and almost all the H1N1 isolates tested since October 2008 in the United States (CDC, http://www.cdc.gov/flu/weekly/) were reported to be oseltamivir resistant. As well, a number of oseltamivir-resistant pandemic 2009 H1N1 viruses (7) and HPAI H5N1 viruses (18) have already appeared, although their appearance is still sporadic. These epidemics of oseltamivir-resistant influenza viruses therefore necessitate the development of alternative antiviral agents.We found a new strong neuraminidase inhibitor, laninamivir (code name, R-125489), and reported that CS-8958 (laninamivir octanoate or the laninamivir prodrug) worked as a long-acting neuraminidase inhibitor (12, 16, 23). Laninamivir potently inhibited the neuraminidase activities of various influenza A and B viruses, including subtypes N1 to N9 and oseltamivir-resistant viruses (23), as well as pandemic 2009 H1N1 virus (14). Due to the long retention of R-125489 in mouse lungs after the intranasal administration of CS-8958 (16), the intranasal administration of a single dose of CS-8958 showed efficacy superior to the efficacies of zanamivir and oseltamivir in mouse models of infection with influenza A virus and seasonal and current pandemic strains (14, 23).In this report, the in vivo efficacy of a single administration of CS-8958 was compared with the efficacies of repeated administrations of zanamivir (intranasal) and oseltamivir (oral) in mouse or ferret models of influenza A and B virus infection and the administration of oseltamivir in a mouse model of H274Y virus infection. We demonstrate the great potential of the single administration of CS-8958 as an alternative treatment against influenza viruses, including oseltamivir-resistant mutants.  相似文献   

18.
RWJ-270201 is a novel cyclopentane inhibitor of influenza A and B virus neuraminidases (NAs). We compared the ability of RWJ-270201 to inhibit NA activity of clinical influenza isolates and viruses with defined resistance mutations with that of zanamivir and oseltamivir carboxylate. In NA inhibition assays with influenza A viruses, the median 50% inhibitory concentration (IC(50)) of RWJ-270201 (approximately 0.34 nM) was comparable to that of oseltamivir carboxylate (0.45 nM) but lower than that of zanamivir (0.95 nM). For influenza B virus isolates, the IC(50) of RWJ-270201 (1.36 nM) was comparable to that of zanamivir (2.7 nM) and less than that of oseltamivir carboxylate (8.5 nM). A zanamivir-resistant variant bearing a Glu119-to-Gly (Glu119-->Gly) or Glu119-->Ala substitution in an NA (N2) remained susceptible to RWJ-270201 and oseltamivir carboxylate. However, a zanamivir-selected variant with an Arg292-->Lys substitution in an NA (N2) showed a moderate level of resistance to RWJ-270201 (IC(50) = 30 nM) and zanamivir (IC(50) = 20 nM) and a high level of resistance to oseltamivir carboxylate (IC(50) > 3,000 nM). The zanamivir-resistant influenza B virus variant bearing an Arg152-->Lys substitution was resistant to each NA inhibitor (IC(50) = 100 to 750 nM). The oseltamivir-selected variant (N1) with the His274-->Tyr substitution exhibited resistance to oseltamivir carboxylate (IC(50) = 400 nM) and to RWJ-270201 (IC(50) = 40 nM) but retained full susceptibility to zanamivir (IC(50) = 1.5 nM). Thus, drug-resistant variants with substitutions in framework residues 119 or 274 can retain susceptibility to other NA inhibitors, whereas replacement of functional residue 152 or 292 leads to variable levels of cross-resistance. We conclude that RWJ-270201 is a potent inhibitor of NAs of wild-type and some zanamivir-resistant or oseltamivir-resistant influenza A and B virus variants.  相似文献   

19.
While the neuraminidase (NA) inhibitor oseltamivir is currently our first line of defense against a pandemic threat, there is little information about whether in vitro testing can predict the in vivo effectiveness of antiviral treatment. Using a panel of five H5N1 influenza viruses (H5 clades 1 and 2), we determined that four viruses were susceptible to the drug in vitro (mean 50% inhibitory concentration [IC50], 0.1 to 4.9 nM), and A/Turkey/65-1242/06 virus was slightly less susceptible (mean IC50, 10.8 nM). Two avian viruses showed significantly greater NA enzymatic activity (Vmax) than the human viruses, and the five viruses varied in their affinity for the NA substrate MUNANA (Km, 64 to 300 μM) and for oseltamivir carboxylate (Ki, 0.1 to 7.9 nM). The protection of mice provided by a standard oseltamivir regimen (20 mg/kg/day for 5 days) also varied among the viruses used. We observed (i) complete protection against the less virulent A/chicken/Jogjakarta/BBVET/IX/04 virus; (ii) moderate protection (60 to 80% survival) against three viruses, two of which are neurotropic; and (iii) no protection against A/Turkey/65-1242/06 virus, which induced high pulmonary expression of proinflammatory mediators (interleukin-1α [IL-1α], IL-6, alpha interferon, and monocyte chemotactic protein 1) and contained a minor subpopulation of drug-resistant clones (I117V and E119A NA mutations). We found no correlation between in vitro susceptibility and in vivo protection (Spearman rank correlation coefficient ρ = −0.1; P > 0.05). Therefore, the in vivo efficacy of oseltamivir against highly pathogenic H5N1 influenza viruses cannot be reliably predicted by susceptibility testing, and more prognostic ways to evaluate anti-influenza compounds must be developed. Multiple viral and host factors modulate the effectiveness of NA inhibitor regimens against such viruses and new, more consistently effective treatment options, including combination therapies, are needed.Highly pathogenic avian H5N1 influenza viruses have spread intercontinentally and evolved into 10 phylogenetically distinct hemagglutinin (HA) clades; the most diverse, clade 2, comprises five subclades (33). Large outbreaks among poultry continue in far-ranging geographical areas, although human infections remain rare (411 confirmed cases since May 2003) (34). However, the pandemic potential of H5N1 influenza viruses should not be underestimated, and preparedness requires that appropriate prophylactic and therapeutic antiviral regimens be established. Importantly, human H5N1 infection differs markedly from human seasonal influenza (35). Viral pneumonia is considered a primary cause of death from H5N1 infection, but disseminated disease and multiorgan failure with renal and cardiac dysfunction, Reye''s syndrome, and hemorrhage often occur (1, 4, 38). Infectious virus and viral RNA have been isolated from the upper and lower respiratory tract, brain, intestines, feces, blood, cerebrospinal fluid, and even from the placentas and fetuses of pregnant women (9, 35).Antiviral drugs can play an important role in the initial response to pandemic influenza. One of the two classes of anti-influenza drugs, M2-ion channel blockers (amantadine and rimantadine), has limited usefulness, because clade 1 H5N1 viruses are frequently resistant (3, 22), although representatives of clade 2 are susceptible to adamantanes (15, 26). Most H5N1 isolates are susceptible in vitro to the second class of drugs, neuraminidase (NA) inhibitors (oseltamivir and zanamivir) (12). Natural genetic variations in NA were reported to affect the susceptibility of H5N1 viruses to oseltamivir in vitro (23), and some clade 2 viruses were found to be 15 to 30 times less susceptible to oseltamivir than clade 1 viruses, based on their 50% inhibitory concentrations (IC50s) (18). Reduced susceptibility may be caused by NA antigenic mutation(s) and by the emergence of specific NA mutations under drug selection pressure (18, 23). NA mutations at positions 274 (H→Y) and 294 (N→S) are considered markers of the oseltamivir-resistant H5N1 phenotype (6, 17).The NA enzyme inhibition assay measures the decrease in functional NA activity in the presence of the drug. This assay is considered the most reliable in vitro method of quantifying the susceptibility of seasonal influenza viruses to NA inhibitors, and it is well correlated with their susceptibility in animal models (29). However, it is unknown whether in vitro data can accurately predict the effectiveness of antiviral drugs against H5N1 viruses in vivo, since viral and host factors that modulate disease manifestations are incompletely understood (20). Experimental animal models are a logical approach to estimating drug effectiveness in vivo against lethal influenza virus infection. Studies in mice showed that more prolonged oseltamivir treatment is required to inhibit residual replication of a highly virulent representative of clade 1, A/Vietnam/1203/04 (H5N1) virus, than to inhibit a less virulent 1997 isolate (36). In a ferret model, the best antiviral effect against H5N1 virus was achieved by increasing the dose of oseltamivir and initiating treatment early (8). These observations show that the optimal dose and duration of an anti-H5N1 regimen may depend on virus virulence, although other viral factors can play a role. Some characteristics, such as the ability to spread systemically, tissue tropism (including neurotropism), virus fitness, the characteristics of individual virus proteins, and a preference for binding to α2,3- or α2,6-linked sialic acid receptors, clearly differ among H5N1 viruses and may affect the protection offered by antiviral therapy. It is also unknown whether the hypercytokinemia reported in human cases of H5N1 infection (7) represents an appropriate immune response or immune dysregulation that may alter the outcome of drug therapy.In the present study, we compared the in vitro NA inhibitor susceptibility and NA protein properties (enzymatic activity, affinity for substrate, and affinity for NA inhibitors) of five highly pathogenic H5N1 influenza viruses with the efficacy of oseltamivir treatment in a mouse model. Viruses of clade 1 and of the more diverse clade 2 were represented. Virus replication in the lungs and brain and production of proinflammatory cytokines were assessed, and virus clones were sequenced to identify minor subpopulations of variants and determine their effect on antiviral treatment. Here we demonstrate that the in vivo efficacy in mice of NA inhibitors against highly pathogenic H5N1 influenza viruses cannot be reliably predicted by susceptibility testing in vitro.  相似文献   

20.
Neuraminidase inhibitors, including oseltamivir and zanamivir are the recommended licensed anti-influenza drugs. However, the emergence in 2007 of oseltamivir-resistant and in 2009 of pandemic A(H1N1) influenza viruses highlighted the need for more efficient antiviral agents for influenza treatment. In this article, we review the advances in knowledge, based on experience from the last pandemic in particular, for patients admitted to the intensive care unit for severe influenza infection. We also focus on undergoing trials evaluating the efficiency of parenteral administration of oseltamivir, zanamivir or peramivir, the associations of antiviral agents, as well as laninamivir, a new long-acting neuraminidase inhibitor. Favipiravir, a polymerase inhibitor and DAS181, a sialidase removing the sialic acids within the host epithelial respiratory tract, are also undergoing clinical trials. In vitro studies showed that small interfering RNAs are able to inhibit the expression of influenza viral RNA and nitazoxide to block the terminal glycosylation of haemagglutinins. To date, new drugs are being actively developed to improve influenza treatment.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号