Rapid and Highly Informative Diagnostic Assay for H5N1 Influenza Viruses

A highly discriminative and information-rich diagnostic assay for H5N1 avian influenza would meet immediate patient care needs and provide valuable information for public health interventions, e.g., tracking of new and more dangerous variants by geographic area as well as avian-to-human or human-to-human transmission. In the present study, we have designed a rapid assay based on multilocus nucleic acid sequencing that focuses on the biologically significant regions of the H5N1 hemagglutinin gene. This allows the prediction of viral strain, clade, receptor binding properties, low- or high-pathogenicity cleavage site and glycosylation status. H5 HA genes were selected from nine known high-pathogenicity avian influenza subtype H5N1 viruses, based on their diversity in biologically significant regions of hemagglutinin and/or their ability to cause infection in humans. We devised a consensus pre-programmed pyrosequencing strategy, which may be used as a faster, more accurate alternative to de novo sequencing. The available data suggest that the assay described here is a reliable, rapid, information-rich and cost-effective approach for definitive diagnosis of H5N1 avian influenza. Knowledge of the predicted functional sequences of the HA will enhance H5N1 avian influenza surveillance efforts

Evaluation of the antigenic relatedness and cross-protective immunity of the neuraminidase between human influenza A (H1N1) virus and highly pathogenic avian influenza A (H5N1) virus

AbstractTo determine the genetic and antigenic relatedness as well as the cross-protective immunity of human H1N1 and avian H5N1 influenza virus neuraminidase (NA), we immunized rabbits with either a baculovirus-expressed recombinant NA from A/Beijing/262/95 (BJ/262) H1N1 or A/Hong Kong/483/97 (HK/483) H5N1 virus. Cross-reactive antibody responses were evaluated by multiple serological assays and cross-protection against H5N1 virus challenge was evaluated in mice. In a neuraminidase inhibition (NI) test, the antisera exhibited substantial inhibition of NA activity of the homologous virus, but failed to inhibit the NA activity of heterologous virus. However, these antisera exhibited low levels of cross-reactivity measured by plaque size reduction, replication inhibition, single radial hemolysis, and ELISA assays. Passive immunization with HK/483 NA-specific antisera significantly reduced virus replication and disease, and afforded almost complete protection against lethal homologous virus challenge in mice. However, passive immunization with BJ/262 (H1N1) NA-specific antisera was ineffective at providing cross-protection against lethal H5N1 virus challenge and only slightly reduced weight loss. Substantial amino acid variation among the NA antigenic sites was observed between BJ/262 and HK/483 virus, which was consistent with the lack of cross-reactive NI activity by the antibody and limited cross-protective immunity in mice. These results show a strong correlation between the lack of cross-protective immunity and low structural similarities of NA from a human seasonal H1N1 virus and an avian H5N1 influenza virus

Influenza in Outpatient ILI Case-Patients in National Hospital-Based Surveillance, Bangladesh, 2007–2008

Recent population-based estimates in a Dhaka low-income community suggest that influenza was prevalent among children. To explore the epidemiology and seasonality of influenza throughout the country and among all age groups, we established nationally representative hospital-based surveillance necessary to guide influenza prevention and control efforts.We conducted influenza-like illness and severe acute respiratory illness sentinel surveillance in 12 hospitals across Bangladesh during May 2007–December 2008. We collected specimens from 3,699 patients, 385 (10%) which were influenza positive by real time RT-PCR. Among the sample-positive patients, 192 (51%) were type A and 188 (49%) were type B. Hemagglutinin subtyping of type A viruses detected 137 (71%) A/H1 and 55 (29%) A/H3, but no A/H5 or other novel influenza strains. The frequency of influenza cases was highest among children aged under 5 years (44%), while the proportions of laboratory confirmed cases was highest among participants aged 11–15 (18%). We applied kriging, a geo-statistical technique, to explore the spatial and temporal spread of influenza and found that, during 2008, influenza was first identified in large port cities and then gradually spread to other parts of the country. We identified a distinct influenza peak during the rainy season (May–September).Our surveillance data confirms that influenza is prevalent throughout Bangladesh, affecting a wide range of ages and causing considerable morbidity and hospital care. A unimodal influenza seasonality may allow Bangladesh to time annual influenza prevention messages and vaccination campaigns to reduce the national influenza burden. To scale-up such national interventions, we need to quantify the national rates of influenza and the economic burden associated with this disease through further studies

Genomic Signature-Based Identification of Influenza A Viruses Using RT-PCR/Electro-Spray Ionization Mass Spectrometry (ESI-MS) Technology

BACKGROUND: The emergence and rapid spread of the 2009 H1N1 pandemic influenza A virus (H1N1pdm) in humans highlights the importance of enhancing the capability of existing influenza surveillance systems with tools for rapid identification of emerging and re-emerging viruses. One of the new approaches is the RT-PCR electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology, which is based on analysis of base composition (BC) of RT-PCR amplicons from influenza "core" genes. Combination of the BC signatures represents a "genomic print" of an influenza A virus. METHODOLOGY/PRINCIPAL FINDINGS: Here, 757 samples collected between 2006 and 2009 were tested, including 302 seasonal H1N1, 171 H3N2, 7 swine triple reassortants, and 277 H1N1pdm viruses. Of the 277 H1N1pdm samples, 209 were clinical specimens (throat, nasal and nasopharyngeal swabs, nasal washes, blood and sputum). BC signatures for the clinical specimen from one of the first cases of the 2009 pandemic, A/California/04/2009, confirmed it as an unusual, previously unrecognized influenza A virus, with "core" genes related to viruses of avian, human and swine origins. Subsequent analysis of additional 276 H1N1pdm samples revealed that they shared the genomic print of A/California/04/2009, which differed from those of North American swine triple reassortant viruses, seasonal H1N1 and H3N2 and other viruses tested. Moreover, this assay allowed distinction between "core" genes of co-circulating groups of seasonal H1N1, such as clades 2B, 2C, and their reassortants with dual antiviral resistance to adamantanes and oseltamivir. CONCLUSIONS/SIGNIFICANCE: The RT-PCR/ESI-MS assay is a broad range influenza identification tool that can be used directly on clinical specimens for rapid and accurate detection of influenza virus genes. The assay differentiates the H1N1pdm from seasonal and other nonhuman hosts viruses. Although not a diagnostic tool, this assay demonstrates its usefulness and robustness in influenza virus surveillance and detection of novel and unusual viruses with previously unseen genomic prints

Rapid and Highly Informative Diagnostic Assay for H5N1 Influenza Viruses

A highly discriminative and information-rich diagnostic assay for H5N1 avian influenza would meet immediate patient care needs and provide valuable information for public health interventions, e.g., tracking of new and more dangerous variants by geographic area as well as avian-to-human or human-to-human transmission. In the present study, we have designed a rapid assay based on multilocus nucleic acid sequencing that focuses on the biologically significant regions of the H5N1 hemagglutinin gene. This allows the prediction of viral strain, clade, receptor binding properties, low- or high-pathogenicity cleavage site and glycosylation status. H5 HA genes were selected from nine known high-pathogenicity avian influenza subtype H5N1 viruses, based on their diversity in biologically significant regions of hemagglutinin and/or their ability to cause infection in humans. We devised a consensus pre-programmed pyrosequencing strategy, which may be used as a faster, more accurate alternative to de novo sequencing. The available data suggest that the assay described here is a reliable, rapid, information-rich and cost-effective approach for definitive diagnosis of H5N1 avian influenza. Knowledge of the predicted functional sequences of the HA will enhance H5N1 avian influenza surveillance efforts

Acid Stability of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity

Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulate the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 Å resolution and two structures of HP HA at 2.95 and 3.10 Å resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species

Influenza A Virus Nucleoprotein Exploits Hsp40 to Inhibit PKR Activation

BACKGROUND: Double-stranded RNA dependent protein kinase (PKR) is a key regulator of the anti-viral innate immune response in mammalian cells. PKR activity is regulated by a 58 kilo Dalton cellular inhibitor (P58(IPK)), which is present in inactive state as a complex with Hsp40 under normal conditions. In case of influenza A virus (IAV) infection, P58(IPK) is known to dissociate from Hsp40 and inhibit PKR activation. However the influenza virus component responsible for PKR inhibition through P58(IPK) activation was hitherto unknown. PRINCIPAL FINDINGS: Human heat shock 40 protein (Hsp40) was identified as an interacting partner of Influenza A virus nucleoprotein (IAV NP) using a yeast two-hybrid screen. This interaction was confirmed by co-immunoprecipitation studies from mammalian cells transfected with IAV NP expressing plasmid. Further, the IAV NP-Hsp40 interaction was validated in mammalian cells infected with various seasonal and pandemic strains of influenza viruses. Cellular localization studies showed that NP and Hsp40 co-localize primarily in the nucleus. During IAV infection in mammalian cells, expression of NP coincided with the dissociation of P58(IPK) from Hsp40 and decrease PKR phosphorylation. We observed that, plasmid based expression of NP in mammalian cells leads to decrease in PKR phosphorylation. Furthermore, inhibition of NP expression during influenza virus replication led to PKR activation and concomitant increase in eIF2α phosphorylation. Inhibition of NP expression also led to reduced IRF3 phosphorylation, enhanced IFN β production and concomitant reduction of virus replication. Taken together our data suggest that NP is the viral factor responsible for P58(IPK) activation and subsequent inhibition of PKR-mediated host response during IAV infection. SIGNIFICANCE: Our findings demonstrate a novel role of IAV NP in inhibiting PKR-mediated anti-viral host response and help us understand P58(IPK) mediated inhibition of PKR activity during IAV infection

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery