Subrata Barman

Intravenous Zanamivir for Oseltamivir-Resistant 2009 H1N1 Influenza

To the Editor: A 10-year-old girl with acute lymphoblastic leukemia was transferred to our hospital on August 31. The patient required mechanical ventilation for worsening lobar pneumonia, despite the administration of broad-spectrum antibiotics (for details, see the Supplementary Appendix, available with the full text of this letter at NEJM.org). Two weeks earlier, she had completed 5 days of oseltamivir for an upper respiratory tract infection caused by influenza A virus (with positive results on rapid antigen testing). On August 27, she had presented with lobar pneumonia (with negative results on rapid antigen testing). On admission to our hospital, infection with .xa0.xa0.

Swine Outbreak of Pandemic Influenza A Virus on a Canadian Research Farm Supports Human-to-Swine Transmission

BACKGROUNDnSwine outbreaks of pandemic influenza A (pH1N1) suggest human introduction of the virus into herds. This study investigates a pH1N1 outbreak occurring on a swine research farm with 37 humans and 1300 swine in Alberta, Canada, from 12 June through 4 July 2009.nnnMETHODSnThe staff was surveyed about symptoms, vaccinations, and livestock exposures. Clinical findings were recorded, and viral testing and molecular characterization of isolates from humans and swine were performed. Human serological testing and performance of the human influenza-like illness (ILI) case definition were also studied.nnnRESULTSnHumans were infected before swine. Seven of 37 humans developed ILI, and 2 (including the index case) were positive for pH1N1 by reverse-transcriptase polymerase chain reaction (RT-PCR). Swine were positive for pH1N1 by RT-PCR 6 days after contact with the human index case and developed symptoms within 24 h of their positive viral test results. Molecular characterization of the entire viral genomes from both species showed minor nucleotide heterogeneity, with 1 amino acid change each in the hemagglutinin and nucleoprotein genes. Sixty-seven percent of humans with positive serological test results and 94% of swine with positive swab specimens had few or no symptoms. Compared with serological testing, the human ILI case definition had a specificity of 100% and sensitivity of 33.3%. The only factor associated with seropositivity was working in the swine nursery.nnnCONCLUSIONSnEpidemiologic data support human-to-swine transmission, and molecular characterization confirms that virtually identical viruses infected humans and swine in this outbreak. Both species had mild illness and recovered without sequelae.

Live Bird Markets of Bangladesh: H9N2 Viruses and the Near Absence of Highly Pathogenic H5N1 Influenza

Avian influenza surveillance in Bangladesh has been passive, relying on poultry farmers to report suspected outbreaks of highly pathogenic H5N1 influenza. Here, the results of an active surveillance effort focusing on the live-bird markets are presented. Prevalence of influenza infection in the birds of the live bird markets is 23.0%, which is similar to that in poultry markets in other countries. Nearly all of the isolates (94%) were of the non-pathogenic H9N2 subtype, but viruses of the H1N2, H1N3, H3N6, H4N2, H5N1, and H10N7 subtypes were also observed. The highly pathogenic H5N1-subtype virus was observed at extremely low prevalence in the surveillance samples (0.08%), and we suggest that the current risk of infection for humans in the retail poultry markets in Bangladesh is negligible. However, the high prevalence of the H9 subtype and its potential for interaction with the highly pathogenic H5N1-subtype, i.e., reassortment and attenuation of host morbidity, highlight the importance of active surveillance of the poultry markets.

Feasibility of reconstructed ancestral H5N1 influenza viruses for cross-clade protective vaccine development

Since the reemergence of highly pathogenic H5N1 influenza viruses in humans in 2003, these viruses have spread throughout avian species in Asia, Europe, and Africa. Their sustained circulation has resulted in the evolution of phylogenetically diverse lineages. Viruses from these lineages show considerable antigenic variation, which has confounded vaccine planning efforts. We reconstructed ancestral protein sequences at several nodes of the hemagglutinin (HA) and neuraminidase (NA) gene phylogenies that represent ancestors to diverse H5N1 virus clades. By using the same methods that have been used to generate currently licensed inactivated H5N1 vaccines, we were able to produce a panel of replication competent influenza viruses containing synthesized HA and NA genes representing the reconstructed ancestral proteins. We identified two of these viruses that showed promising in vitro cross-reactivity with clade 1, 2.1, 2.2, 2.3.4, and 4 viruses. To confirm that vaccine antigens derived from these viruses were able to elicit functional antibodies following immunization, we created whole-virus vaccines and compared their protective efficacy versus that of antigens from positive control, naturally occurring, and broadly reactive H5N1 viruses. The ancestral viruses’ vaccines provided robust protection against morbidity and mortality in ferrets challenged with H5N1 strains from clades 1, 2.1, and 2.2 in a manner similar to those based on the control strains. These findings provide proof of principle that viable, computationally derived vaccine seed viruses can be constructed within the context of currently licensed vaccine platforms. Such technologies should be explored to enhance the cross reactivity and availability of H5N1 influenza vaccines.

Antigenic and Molecular Characterization of Avian Influenza A(H9N2) Viruses, Bangladesh

Human infection with avian influenza A(H9N2) virus was identified in Bangladesh in 2011. Surveillance for influenza viruses in apparently healthy poultry in live-bird markets in Bangladesh during 2008–2011 showed that subtype H9N2 viruses are isolated year-round, whereas highly pathogenic subtype H5N1 viruses are co-isolated with subtype H9N2 primarily during the winter months. Phylogenetic analysis of the subtype H9N2 viruses showed that they are reassortants possessing 3 gene segments related to subtype H7N3; the remaining gene segments were from the subtype H9N2 G1 clade. We detected no reassortment with subtype H5N1 viruses. Serologic analyses of subtype H9N2 viruses from chickens revealed antigenic conservation, whereas analyses of viruses from quail showed antigenic drift. Molecular analysis showed that multiple mammalian-specific mutations have become fixed in the subtype H9N2 viruses, including changes in the hemagglutinin, matrix, and polymerase proteins. Our results indicate that these viruses could mutate to be transmissible from birds to mammals, including humans.

Molecular requirements for a pandemic influenza virus: An acid-stable hemagglutinin protein

Significance Influenza pandemics occur several times per century, causing millions of deaths. For one of the myriad of zoonotic influenza viruses to do so, a virus containing a hemagglutinin (HA) surface antigen previously unseen by most humans must evolve the necessary, albeit largely unknown, properties for sustained respiratory spread between people. During entry, the prototypic viral fusion protein HA binds receptors and is triggered irreversibly by low pH in endosomes to cause membrane fusion. These studies link a fundamental property, activation energy of a fusion protein measured as its pH of activation (acid stability), to the ability of zoonotic influenza viruses to cause a human pandemic. Monitoring HA stability is expected to enhance prepandemic surveillance and control of emerging influenza viruses. Influenza pandemics require that a virus containing a hemagglutinin (HA) surface antigen previously unseen by a majority of the population becomes airborne-transmissible between humans. Although the HA protein is central to the emergence of a pandemic influenza virus, its required molecular properties for sustained transmission between humans are poorly defined. During virus entry, the HA protein binds receptors and is triggered by low pH in the endosome to cause membrane fusion; during egress, HA contributes to virus assembly and morphology. In 2009, a swine influenza virus (pH1N1) jumped to humans and spread globally. Here we link the pandemic potential of pH1N1 to its HA acid stability, or the pH at which this one-time-use nanomachine is either triggered to cause fusion or becomes inactivated in the absence of a target membrane. In surveillance isolates, our data show HA activation pH values decreased during the evolution of H1N1 from precursors in swine (pH 5.5–6.0), to early 2009 human cases (pH 5.5), and then to later human isolates (pH 5.2–5.4). A loss-of-function pH1N1 virus with a destabilizing HA1-Y17H mutation (pH 6.0) was less pathogenic in mice and ferrets, less transmissible by contact, and no longer airborne-transmissible. A ferret-adapted revertant (HA1-H17Y/HA2-R106K) regained airborne transmissibility by stabilizing HA to an activation pH of 5.3, similar to that of human-adapted isolates from late 2009–2014. Overall, these studies reveal that a stable HA (activation pH ≤ 5.5) is necessary for pH1N1 influenza virus pathogenicity and airborne transmissibility in ferrets and is associated with pandemic potential in humans.

Pathogenicity and Transmissibility of North American Triple Reassortant Swine Influenza A Viruses in Ferrets

North American triple reassortant swine (TRS) influenza A viruses have caused sporadic human infections since 2005, but human-to-human transmission has not been documented. These viruses have six gene segments (PB2, PB1, PA, HA, NP, and NS) closely related to those of the 2009 H1N1 pandemic viruses. Therefore, understanding of these viruses pathogenicity and transmissibility may help to identify determinants of virulence of the 2009 H1N1 pandemic viruses and to elucidate potential human health threats posed by the TRS viruses. Here we evaluated in a ferret model the pathogenicity and transmissibility of three groups of North American TRS viruses containing swine-like and/or human-like HA and NA gene segments. The study was designed only to detect informative and significant patterns in the transmissibility and pathogenicity of these three groups of viruses. We observed that irrespective of their HA and NA lineages, the TRS viruses were moderately pathogenic in ferrets and grew efficiently in both the upper and lower respiratory tracts. All North American TRS viruses studied were transmitted between ferrets via direct contact. However, their transmissibility by respiratory droplets was related to their HA and NA lineages: TRS viruses with human-like HA and NA were transmitted most efficiently, those with swine-like HA and NA were transmitted minimally or not transmitted, and those with swine-like HA and human-like NA (N2) showed intermediate transmissibility. We conclude that the lineages of HA and NA may play a crucial role in the respiratory droplet transmissibility of these viruses. These findings have important implications for pandemic planning and warrant confirmation.

Novel Genotyping and Quantitative Analysis of Neuraminidase Inhibitor Resistance-Associated Mutations in Influenza A Viruses by Single-Nucleotide Polymorphism Analysis

ABSTRACT Neuraminidase (NA) inhibitors are among the first line of defense against influenza virus infection. With the increased worldwide use of the drugs, antiviral susceptibility surveillance is increasingly important for effective clinical management and for public health epidemiology. Effective monitoring requires effective resistance detection methods. We have developed and validated a novel genotyping method for rapid detection of established NA inhibitor resistance markers in influenza viruses by single nucleotide polymorphism (SNP) analysis. The multi- or monoplex SNP analysis based on single nucleotide extension assays was developed to detect NA mutations H275Y and I223R/V in pandemic H1N1 viruses, H275Y in seasonal H1N1 viruses, E119V and R292K in seasonal H3N2 viruses, and H275Y and N295S in H5N1 viruses. The SNP analysis demonstrated high sensitivity for low-content NA amplicons (0.1 to 1 ng/μl) and showed 100% accordant results against a panel of defined clinical isolates. The monoplex assays for the H275Y NA mutation allowed precise and accurate quantification of the proportions of wild-type and mutant genotypes in virus mixtures (5% to 10% discrimination), with results comparable to those of pyrosequencing. The SNP analysis revealed the lower growth fitness of an H275Y mutant compared to the wild-type pandemic H1N1 virus by quantitatively genotyping progeny viruses grown in normal human bronchial epithelial cells. This novel method offers high-throughput screening capacity, relatively low costs, and the wide availability of the necessary equipment, and thus it could provide a much-needed approach for genotypic screening of NA inhibitor resistance in influenza viruses.

Insight into live bird markets of Bangladesh: an overview of the dynamics of transmission of H5N1 and H9N2 avian influenza viruses

Highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been recognized as threats to public health in Bangladesh since 2007. Although live bird markets (LBMs) have been implicated in the transmission, dissemination, and circulation of these viruses, an in-depth analysis of the dynamics of avian transmission of H5N1 and H9N2 viruses at the human–animal interface has been lacking. Here we present and evaluate epidemiological findings from active surveillance conducted among poultry in various production sectors in Bangladesh from 2008 to 2016. Overall, the prevalence of avian influenza viruses (AIVs) in collected samples was 24%. Our data show that AIVs are more prevalent in domestic birds within LBMs (30.4%) than in farms (9.6%). Quail, chickens and ducks showed a high prevalence of AIVs (>20%). The vast majority of AIVs detected (99.7%) have come from apparently healthy birds and poultry drinking water served as a reservoir of AIVs with a prevalence of 32.5% in collected samples. HPAI H5N1 was more frequently detected in ducks while H9N2 was more common in chickens and quail. LBMs, particularly wholesale markets, have become a potential reservoir for various types of AIVs, including HPAI H5N1 and LPAI H9N2. The persistence of AIVs in LBMs is of great concern to public health, and this study highlights the importance of regularly reviewing and implementing infection control procedures as a means of reducing the exposure of the general public to AIVs.Emerging Microbes &Infections (2017) 6, e12; doi:10.1038/emi.2016.142; published online 8 March 2017

Genesis of Influenza A(H5N8) Viruses

Highly pathogenic avian influenza A(H5N8) clade 2.3.4.4 virus emerged in 2016 and spread to Russia, Europe, and Africa. Our analysis of viruses from domestic ducks at Tanguar haor, Bangladesh, showed genetic similarities with other viruses from wild birds in central Asia, suggesting their potential role in the genesis of A(H5N8).