Characterization of Oseltamivir-Resistant 2009 H1N1 Pandemic Influenza A Viruses

Influenza viruses resistant to antiviral drugs emerge frequently. Not surprisingly, the widespread treatment in many countries of patients infected with 2009 pandemic influenza A (H1N1) viruses with the neuraminidase (NA) inhibitors oseltamivir and zanamivir has led to the emergence of pandemic strains resistant to these drugs. Sporadic cases of pandemic influenza have been associated with mutant viruses possessing a histidine-to-tyrosine substitution at position 274 (H274Y) in the NA, a mutation known to be responsible for oseltamivir resistance. Here, we characterized in vitro and in vivo properties of two pairs of oseltaimivir-sensitive and -resistant (possessing the NA H274Y substitution) 2009 H1N1 pandemic viruses isolated in different parts of the world. An in vitro NA inhibition assay confirmed that the NA H274Y substitution confers oseltamivir resistance to 2009 H1N1 pandemic viruses. In mouse lungs, we found no significant difference in replication between oseltamivir-sensitive and -resistant viruses. In the lungs of mice treated with oseltamivir or even zanamivir, 2009 H1N1 pandemic viruses with the NA H274Y substitution replicated efficiently. Pathological analysis revealed that the pathogenicities of the oseltamivir-resistant viruses were comparable to those of their oseltamivir-sensitive counterparts in ferrets. Further, the oseltamivir-resistant viruses transmitted between ferrets as efficiently as their oseltamivir-sensitive counterparts. Collectively, these data indicate that oseltamivir-resistant 2009 H1N1 pandemic viruses with the NA H274Y substitution were comparable to their oseltamivir-sensitive counterparts in their pathogenicity and transmissibility in animal models. Our findings highlight the possibility that NA H274Y-possessing oseltamivir-resistant 2009 H1N1 pandemic viruses could supersede oseltamivir-sensitive viruses, as occurred with seasonal H1N1 viruses.     

Oseltamivir boosts 2009 H1N1 virus infectivity in vitro

The neuraminidase inhibitor oseltamivir has been identified to have significant anti-influenza activity in clinical practice. However, its efficacy has not been verified in enough subtypes of influenza A virus, particularly, the current pandemic virus, H1N1. In vitro, using our influenza pseudotyped particle system, oseltamivir displayed significant inhibitory effects on viral NA activity and pp release. Conversely, a boosting effect on viral infection was observed, particularly with the 2009 H1N1 pp at oseltamivir concentrations above 0.025 μM. Further testing on two wild 2009 H1N1 virus strains, A/California/07/09 and A/Sichuan/1/09, as well as a seasonal flu virus, A/Baoan/51/2008, confirmed these findings.     

Induction of Long-Term Protective Immune Responses by Influenza H5N1 Virus-Like Particles

Background

Recurrent outbreaks of highly pathogenic H5N1 avian influenza virus pose a threat of eventually causing a pandemic. Early vaccination of the population would be the single most effective measure for the control of an emerging influenza pandemic.

Methodology/Principal Findings

Influenza virus-like particles (VLPs) produced in insect cell-culture substrates do not depend on the availability of fertile eggs for vaccine manufacturing. We produced VLPs containing influenza A/Viet Nam1203/04 (H5N1) hemagglutinin, neuraminidase, and matrix proteins, and investigated their preclinical immunogenicity and protective efficacy. Mice immunized intranasally with H5N1 VLPs developed high levels of H5N1 specific antibodies and were 100% protected against a high dose of homologous H5N1 virus infection at 30 weeks after immunization. Protection is likely to be correlated with humoral and cellular immunologic memory at systemic and mucosal sites as evidenced by rapid anamnestic responses to re-stimulation with viral antigen in vivo and in vitro.

Conclusions/Significance

These results provide support for clinical evaluation of H5N1 VLP vaccination as a public health intervention to mitigate a possible pandemic of H5N1 influenza.     

Oseltamivir-Resistant Pandemic A/H1N1 Virus Is as Virulent as Its Wild-Type Counterpart in Mice and Ferrets

The neuraminidase inhibitor oseltamivir is currently used for treatment of patients infected with the pandemic A/H1N1 (pH1N1) influenza virus, although drug-resistant mutants can emerge rapidly and possibly be transmitted. We describe the characteristics of a pair of oseltamivir-resistant and oseltamivir-susceptible pH1N1 clinical isolates that differed by a single change (H274Y) in the neuraminidase protein. Viral fitness of pH1N1 isolates was assessed in vitro by determining replication kinetics in MDCK α2,6 cells and in vivo by performing experimental infections of BALB/c mice and ferrets. Despite slightly reduced propagation of the mutant isolate in vitro during the first 24 h, the wild-type (WT) and mutant resistant viruses induced similar maximum weight loss in mice and ferrets with an identical pyrexic response in ferrets (AUC of 233.9 and 233.2, P = 0.5156). Similarly, comparable titers were obtained for the WT and the mutant strains on days 1, 3, 6 and 9 post-infection in mouse lungs and on days 1–7 in ferret nasal washes. A more important perivascular (day 6) and pleural (days 6 and 12) inflammation was noted in the lungs of mice infected with the H274Y mutant, which correlated with increased pulmonary levels of IL-6 and KC. Such increased levels of IL-6 were also observed in lymph nodes of ferrets infected with the mutant strain. Furthermore, the H274Y mutant strain was transmitted to ferrets. In conclusion, viral fitness of the H274Y pH1N1 isolate is not substantially altered and has the potential to induce severe disease and to disseminate.     

The binding properties of the H5N1 influenza virus neuraminidase as inferred from molecular modeling

The avian influenza H5N1 virus has emerged as an important pathogen, causing severe disease in humans and posing a pandemic threat. Substrate specificity is crucial for the virus to obtain the ability to spread from avian to human. Therefore, an investigation of the binding properties of ligands at the molecular level is important for understanding the catalytic mechanism of the avian influenza virus neuraminidase and for designing novel and specific inhibitors of H5N1 neuraminidase. Based on the available crystal structure of H5N1, we have characterized the binding properties between sialic acid, methyl 3’sialyllactoside, methyl 6’sialyllactoside and the H5N1 influenza virus neuraminidase using molecular docking and molecular dynamics simulations. Obtained molecular dynamics trajectories were analyzed in terms of ligand conformations, N1-ligand interactions, and in terms of loop flexibility. It was found that in the N1-SA complex the sialic acid ring undergoes a transition from the B _2,5 to the ² C ₅ conformation. However, in the N1-3SL and N1-6SL complexes sialic acid remained in the distorted boat conformation. The obtained results indicate that 3SL has only weak interactions with the 150-loop, whereas the N1-6SL complex shows strong interactions. Most of the differences arise from the various conformations around the glycosidic linkage, between the sialic acid and galactose, which facilitate the above interactions of 6SL with the enzyme, and as a consequence the interactions between the 150- and 430- loops. This finding suggests that the altered flexibility of loops in and around the active site is one of the reasons why the avian N1 preferentially cleaves sialic acid from α-(2-3)-Gal glycoconjugates over α-(2-6)-Gal. These molecular modeling results are consistent with available experimental results on the specificity of N1.     

Growth and Pathogenic Potential of Naturally Selected Reassortants after Coinfection with Pandemic H1N1 and Highly Pathogenic Avian Influenza H5N1 Viruses

Coinfection of ferrets with H5N1 and pH1N1 viruses resulted in two predominate genotypes in the lungs containing surface genes of highly pathogenic avian influenza H5N1 virus in the backbone of pandemic H1N1 2009 (pH1N1). Compared to parental strains, these reassortants exhibited increased growth and virulence in vitro and in mice but failed to be transmitted indirectly to naive contact ferrets. Thus, this demonstrates a possible natural reassortment following coinfection as well as the pathogenicity of the potential reassortants.     

Design,synthesis and biological evaluation of substituted flavones and aurones as potential anti-influenza agents

We designed a series of substituted flavones and aurones as non-competitive H1N1 neuraminidase (NA) inhibitors and anti-influenza agents. The molecular docking studies showed that the designed flavones and aurones occupied 150-cavity and 430-cavity of H1N1-NA. We then synthesized these compounds and evaluated these for cytotoxicity, reduction in H1N1 virus yield, H1N1-NA inhibition and kinetics of inhibition. The virus yield reduction assay and H1N1-NA inhibition assay demonstrated that the compound 1f (4-methoxyflavone) had the lowest EC₅₀ of 9.36 nM and IC₅₀ of 8.74 μM respectively. Moreover, kinetic studies illustrated that compounds 1f and 2f had non-competitive inhibition mechanism.     

Aspulvinones from a mangrove rhizosphere soil-derived fungus Aspergillus terreus Gwq-48 with anti-influenza A viral (H1N1) activity

A new butenolide isoaspulvinone E (1), together with two known butenolides aspulvinone E (2) and pulvic acid (3) were isolated from the marine-derived fungus, Aspergillus terreus Gwq-48. They showed significant anti-influenza A H1N1 virus activities, with IC₅₀ values of 32.3, 56.9, and 29.1 μg/mL, respectively. Moreover, only compound 1 exhibited effective inhibitory activity against H1N1 viral neuraminidase (NA), and docking of two isomers (1–2) into the active sites of NA showed that the E double bond Δ⁵⁽¹⁰⁾ was essential to achieve activity.     

Construction and comparison of different source neuraminidase candidate vaccine strains for human infection with Eurasian avian-like influenza H1N1 virus

Human infections with Eurasian avian-like swine influenza H1N1 viruses have been reported in China in past years. One case resulted in death and others were mild case. In 2016, the World Health Organization recommended the use of A/Hunan/42443/2015(H1N1) virus to construct the first candidate vaccine strain for Eurasian avian-like swine influenza H1N1 viruses. Previous reports showed that the neuraminidase of A/Puerto Rico/8/34(H1N1) might improve the viral yield of reassortant viruses. Therefore, we constructed two reassortant candidate vaccine viruses of A/Hunan/42443/2015(H1N1) by reverse genetic technology, with (6+2) and (7+1) gene constitution, respectively. The (6+2) virus had hemagglutinin and neuraminidase from A/Hunan/42443/2015, and the (7+1) one had hemagglutinin from A/Hunan/42443/2015, while all the other genes were from A/Puerto Rico/8/34. Our data revealed that although the neuraminidase of the (7+1) virus was from high yield A/Puerto Rico/8/34, the hemagglutination titer and the hemagglutinin protein content of the (7+1) virus was not higher than that of the (6+2) virus. Both of the (7+1) and (6+2) viruses reached a similar level to that of A/Puerto Rico/8/34 at the usual harvest time in vitro. Therefore, both reassortant viruses are potential candidate vaccine viruses, which could contribute to pandemic preparedness.     

Genetic variability of isolates of pandemic influenza A virus H1N1 isolated in Russia in 2009

Complete nucleotide sequence of the genome segments encoding the surface glycoproteins, hemagglutinin, and neuraminidase of influenza A virus H1N1 derived from the patients with influenza in the context of pandemic (H1N1) 2009 was determined out of 14 isolates of pandemic influenza. The philogenetic analysis of these sequences demonstrated their genetic similarity to the corresponding genes of the pandemic influenza virus A (H1N1) 2009 isolates obtained in other countries; each gene homology was greater than 99%. Neuraminidase mutations causing virus resistance to oseltamivir and other neuraminidase inhibitors, known from the literature, were not detected. The hemagglutinin gene mutation D222G was found in 4 isolates from autopsy material. In the hemagglutinin of pandemic A/Salekhard/01/2009(H1N1) isolate a mutation G155E leading to the increase in viral replication in developing chick embryos was detected. The nature and frequency of nucleotides substitutions within HA and NA genes were determined in the current research.     

A contributing role for anti-neuraminidase antibodies on immunity to pandemic H1N1 2009 influenza A virus

Background

Exposure to contemporary seasonal influenza A viruses affords partial immunity to pandemic H1N1 2009 influenza A virus (pH1N1) infection. The impact of antibodies to the neuraminidase (NA) of seasonal influenza A viruses to cross-immunity against pH1N1 infection is unknown.

Methods and Results

Antibodies to the NA of different seasonal H1N1 influenza strains were tested for cross-reactivity against A/California/04/09 (pH1N1). A panel of reverse genetic (rg) recombinant viruses was generated containing 7 genes of the H1N1 influenza strain A/Puerto Rico/08/34 (PR8) and the NA gene of either the pandemic H1N1 2009 strain (pH1N1) or one of the following contemporary seasonal H1N1 strains: A/Solomon/03/06 (rg Solomon) or A/Brisbane/59/07 (rg Brisbane). Convalescent sera collected from mice infected with recombinant viruses were measured for cross-reactive antibodies to pH1N1 via Hemagglutinin Inhibition (HI) or Enzyme-Linked Immunosorbent Assay (ELISA). The ectodomain of a recombinant NA protein from the pH1N1 strain (pNA-ecto) was expressed, purified and used in ELISA to measure cross-reactive antibodies. Analysis of sera from elderly humans immunized with trivalent split-inactivated influenza (TIV) seasonal vaccines prior to 2009 revealed considerable cross-reactivity to pNA-ecto. High titers of cross-reactive antibodies were detected in mice inoculated with either rg Solomon or rg Brisbane. Convalescent sera from mice inoculated with recombinant viruses were used to immunize naïve recipient Balb/c mice by passive transfer prior to challenge with pH1N1. Mice receiving rg California sera were better protected than animals receiving rg Solomon or rg Brisbane sera.

Conclusions

The NA of contemporary seasonal H1N1 influenza strains induces a cross-reactive antibody response to pH1N1 that correlates with reduced lethality from pH1N1 challenge, albeit less efficiently than anti-pH1N1 NA antibodies. These findings demonstrate that seasonal NA antibodies contribute to but are not sufficient for cross-reactive immunity to pH1N1.     

Genetic Characterization of H1N1 and H1N2 Influenza A Viruses Circulating in Ontario Pigs in 2012

The objective of this study was to characterize H1N1 and H1N2 influenza A virus isolates detected during outbreaks of respiratory disease in pig herds in Ontario (Canada) in 2012. Six influenza viruses were included in analysis using full genome sequencing based on the 454 platform. In five H1N1 isolates, all eight segments were genetically related to 2009 pandemic virus (A(H1N1)pdm09). One H1N2 isolate had hemagglutinin (HA), polymerase A (PA) and non-structural (NS) genes closely related to A(H1N1)pdm09, and neuraminidase (NA), matrix (M), polymerase B1 (PB1), polymerase B2 (PB2), and nucleoprotein (NP) genes originating from a triple-reassortant H3N2 virus (tr H3N2). The HA gene of five Ontario H1 isolates exhibited high identity of 99% with the human A(H1N1)pdm09 [A/Mexico/InDRE4487/09] from Mexico, while one Ontario H1N1 isolate had only 96.9% identity with this Mexican virus. Each of the five Ontario H1N1 viruses had between one and four amino acid (aa) changes within five antigenic sites, while one Ontario H1N2 virus had two aa changes within two antigenic sites. Such aa changes in antigenic sites could have an effect on antibody recognition and ultimately have implications for immunization practices. According to aa sequence analysis of the M2 protein, Ontario H1N1 and H1N2 viruses can be expected to offer resistance to adamantane derivatives, but not to neuraminidase inhibitors.     

Anti N1 Cross-Protecting Antibodies Against H5N1 Detected in H1N1 Infected People

The A(H5N1) influenza virus pandemic may be the result of avian H5N1 adapting to humans, leading to massive human to human transmission in a context of a lack of pre-existing immunity. As A(H1N1) and A(H5N1) share the same neuraminidase subtype, anti-N1 antibodies subsequent to H1N1 infections or vaccinations may confer some protection against A(H5N1). We analysed, by microneutralization assay, the A/Vietnam/1194/04 (H5N1) anti-N1 cross-protection acquired either during A/NewCaledonia/20/99 (H1N1) infection or vaccination. In cases with documented H1N1 infection, H5N1 cross-protection could be observed only in patients born between 1930 and 1950. No such protection was detected in the sera of vaccinated individuals.     

An Eight-Segment Swine Influenza Virus Harboring H1 and H3 Hemagglutinins Is Attenuated and Protective against H1N1 and H3N2 Subtypes in Pigs

Swine influenza virus (SIV) infections continue to cause production losses in the agricultural industry in addition to being a human public health concern. The primary method of controlling SIV is through vaccination. The killed SIV vaccines currently in use must be closely matched to the challenge virus, and their protective efficacy is limited. Live attenuated influenza vaccines (LAIV) provide strong, long-lived cell-mediated and humoral immunity against different influenza virus subtypes with no need for antigen matching. Here we report the generation of a new potential LAIV, an eight-segment SIV harboring two different SIV hemagglutinins (HAs), H1 and H3, in the genetic background of H1N1 SIV. This mutant SIV was generated by fusing the H3 HA ectodomain from A/Swine/Texas/4199-2/98 (H3N2) to the cytoplasmic tail, transmembrane domain, and stalk region of neuraminidase (NA) from A/Swine/Saskatchewan/18789/02 (H1N1) SIV. While this H1-H3 chimeric SIV, when propagated in vitro in the presence of exogenous neuraminidase, showed kinetics and growth properties similar to those of the parental wild-type virus, in vivo it was highly attenuated in pigs, demonstrating a great potential for serving as a dual LAIV. Furthermore, vaccination with the H1-H3 virus elicited robust immune responses, which conferred complete protection against infections with both H1 and H3 SIV subtypes in pigs.     

Estimated epidemiologic parameters and morbidity associated with pandemic H1N1 influenza

Background

In the face of an influenza pandemic, accurate estimates of epidemiologic parameters are required to help guide decision-making. We sought to estimate epidemiologic parameters for pandemic H1N1 influenza using data from initial reports of laboratory-confirmed cases.

Methods

We obtained data on laboratory-confirmed cases of pandemic H1N1 influenza reported in the province of Ontario, Canada, with dates of symptom onset between Apr. 13 and June 20, 2009. Incubation periods and duration of symptoms were estimated and fit to parametric distributions. We used competing-risk models to estimate risk of hospital admission and case-fatality rates. We used a Markov Chain Monte Carlo model to simulate disease transmission.

Results

The median incubation period was 4 days and the duration of symptoms was 7 days. Recovery was faster among patients less than 18 years old than among older patients (hazard ratio 1.23, 95% confidence interval 1.06–1.44). The risk of hospital admission was 4.5% (95% CI 3.8%–5.2%) and the case-fatality rate was 0.3% (95% CI 0.1%–0.5%). The risk of hospital admission was highest among patients less than 1 year old and those 65 years or older. Adults more than 50 years old comprised 7% of cases but accounted for 7 of 10 initial deaths (odds ratio 28.6, 95% confidence interval 7.3–111.2). From the simulation models, we estimated the following values (and 95% credible intervals): a mean basic reproductive number (R₀, the number of new cases created by a single primary case in a susceptible population) of 1.31 (1.25–1.38), a mean latent period of 2.62 (2.28–3.12) days and a mean duration of infectiousness of 3.38 (2.06–4.69) days. From these values we estimated a serial interval (the average time from onset of infectiousness in a case to the onset of infectiousness in a person infected by that case) of 4–5 days.

Interpretation

The low estimates for R₀ indicate that effective mitigation strategies may reduce the final epidemic impact of pandemic H1N1 influenza.The emergence and global spread of pandemic H1N1 influenza led the World Health Organization to declare a pandemic on June 11, 2009. As the pandemic spreads, countries will need to make decisions about strategies to mitigate and control disease in the face of uncertainty.For novel infectious diseases, accurate estimates of epidemiologic parameters can help guide decision-making. A key parameter for any new disease is the basic reproductive number (R₀), defined as the average number of new cases created by a single primary case in a susceptible population. R₀ affects the growth rate of an epidemic and the final number of infected people. It also informs the optimal choice of control strategies. Other key parameters that affect use of resources, disease burden and societal costs during a pandemic are duration of illness, rate of hospital admission and case-fatality rate. Early in an epidemic, the case-fatality rate may be underestimated because of the temporal lag between onset of infection and death; the delay between initial identification of a new case and death may lead to an apparent increase in deaths several weeks into an epidemic that is an artifact of the natural history of the disease.We used data from initial reports of laboratory-confirmed pandemic H1N1 influenza to estimate epidemiologic parameters for pandemic H1N1 influenza. The parameters included R₀, incubation period and duration of illness. We also estimated risk of hospital admission and case-fatality rates, which can be used to estimate the burden of illness likely to be associated with this disease.     

Enteric absorption and pharmacokinetics of oseltamivir in critically ill patients with pandemic (H1N1) influenza

Background

Whether the enteric absorption of the neuraminidase inhibitor oseltamivir is impaired in critically ill patients is unknown. We documented the pharmacokinetic profile of oseltamivir in patients admitted to intensive care units (ICUs) with suspected or confirmed pandemic (H1N1) influenza.

Methods

We included 41 patients 18 years of age and older with suspected or confirmed pandemic (H1N1) influenza who were admitted for ventilatory support to nine ICUs in three cities in Canada and Spain. Using tandem mass spectrometry, we assessed plasma levels of oseltamivir free base and its active metabolite carboxylate at baseline (before gastric administration of the drug) and at 2, 4, 6, 9 and 12 hours after the fourth or later dose.

Results

Among the 36 patients who did not require dialysis, the median concentration of oseltamivir free base was 10.4 (interquartile range [IQR] 4.8–14.9) μg/L; the median concentration of the carboxylate metabolite was 404 (IQR 257–900) μg/L. The volume of distribution of the carboxylate metabolite did not increase with increasing body weight (R² = 0.00, p = 0.87). The rate of elimination of oseltamivir carboxylate was modestly correlated with estimations of creatinine clearance (R² = 0.27, p < 0.001). Drug clearance in the five patients who required continuous renal replacement therapy was about one-sixth that in the 36 patients with relatively normal renal function.

Interpretation

Oseltamivir was well absorbed enterically in critically ill patients admitted to the ICU with suspected or confirmed pandemic (H1N1) influenza. The dosage of 75 mg twice daily achieved plasma levels that were comparable to those in ambulatory patients and were far in excess of concentrations required to maximally inhibit neuraminidase activity of the virus. Adjustment of the dosage in patients with renal dysfunction requiring continuous renal replacement therapy is appropriate; adjustment for obesity does not appear to be necessary.A substantial number of cases of pandemic (H1N1) influenza have involved young adults and adolescents without serious comorbidities who present with severe viral pneumonia complicated by acute respiratory distress syndrome, rhabdomyolysis, renal failure and, occasionally, shock.^1,2 Antiviral therapy in such critically ill patients typically requires oral or nasogastric administration of the neuraminidase inhibitor oseltamivir. Current guidelines from the World Health Organization for the pharmacologic management of progressive or severe pandemic (H1N1) influenza recommend the consideration of high-dose therapy (≥ 150 mg twice daily).^3,4 Critically ill patients exhibit defects in gastrointestinal absorption because of impaired gut perfusion, edema of the bowel wall and ileus as a consequence of critical illness and shock.⁵ Whether the enteric absorption of oseltamivir is impaired in such patients is unknown.We undertook this study to document the pharmacokinetic profile of oseltamivir administered orally or by nasogastric tube in patients admitted to intensive care units (ICUs) with respiratory failure due to suspected or confirmed pandemic (H1N1) influenza.     

Analogs of zanamivir with modified C4-substituents as the inhibitors against the group-1 neuraminidases of influenza viruses

Unlike the group-2 neuraminidase, the group-1 neuraminidase of influenza virus possesses a flexible loop (the 150-loop) and a cavity (the 150-cavity) adjacent to the active site, and renders a conformational change from the ‘open’ form to the ‘closed’ form on binding with substrate (sialo-glycoprotein) or inhibitor (e.g., zanamivir). Zanamivir derivative 8a having an extended (piperazinocarbonyl)propyl substituent at the internal N-position of the guanidino group is designed as a possible inhibitor on the basis of computer docking to the open form of N1 subtype neuraminidase. Indeed, compound 8a exhibits strong neuraminidase inhibition and good anti-influenza activity against H1N1 virus with IC₅₀ = 2.15 μM and EC₅₀ = 0.77 μM, respectively. This study may provide a clue to future design of better group-1 neuraminidase inhibitors.     

Seroprevalence of pandemic (H1N1) 2009 in pregnant women in China: an observational study

Background

We investigated the seropositive rates and persistence of antibody against pandemic (H1N1) 2009 virus (pH1N1) in pregnant women and voluntary blood donors after the second wave of the pandemic in Nanjing, China.

Methodology/Principal Findings

Serum samples of unvaccinated pregnant women (n = 720) and voluntary blood donors (n = 320) were collected after the second wave of 2009 pandemic in Nanjing. All samples were tested against pH1N1 strain (A/California/7/2009) with hemagglutination inhibition assay. A significant decline in seropositive rates, from above 50% to about 20%, was observed in pregnant women and voluntary blood donors fifteen weeks after the second wave of the pandemic. A quarter of the samples were tested against a seasonal H1N1 strain (A/Brisbane/59/2007). The antibody titers against pH1N1 strain were found to correlate positively with those against seasonal H1N1 strain. The correlation was modest but statistically significant.

Conclusions and Significance

The high seropositive rates in both pregnant women and voluntary blood donors suggested that the pH1N1 virus had widely spread in these two populations. Immunity derived from natural infection seemed not to be persistent well.     

Human-like receptor specificity does not affect the neuraminidase-inhibitor susceptibility of H5N1 influenza viruses

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.