Rapid and Highly Informative Diagnostic Assay for H5N1 Influenza Viruses

PLOS ONE, Dec 2006

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.

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Rapid and Highly Informative Diagnostic Assay for H5N1 Influenza Viruses

et al (2006) Rapid and Highly Informative Diagnostic Assay for H5N1 Influenza Viruses. PLoS ONE 1(1): e95. doi:10.1371/journal.pone.0000095 Rapid and Highly Informative Diagnostic Assay for H5N1 Influenza Viruses Nader Pourmand pourmand@stanford 0 1 Lisa Diamond 0 1 Rebecca Garten 0 1 Julianna P. Erickson 0 1 Jochen Kumm 0 1 Ruben O. Donis 0 1 Ronald W. Davis 0 1 0 Academic Editor: Paul Digard, University of Cambridge , United Kingdom 1 1 Stanford Genome Technology Center, Stanford University , Palo Alto , California, United States of America , 2 Molecular Virology and Vaccines Branch , Influenza Division, Centers for Disease Control and Prevention , Atlanta, Georgia , United States of America 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. - INTRODUCTION The worldwide spread of high-pathogenicity H5N1 avian influenza A virus in poultry and wild birds has resulted in many human infections, with high fatality rates. Although sustained human-to-human transmission has not yet occurred, concern about a potential pandemic continues to mount. The current HA lineage of H5N1 avian influenza was first found among domestic poultry populations in 1996 in southern China [1]. A similar H5N1 influenza virus spread directly from poultry to humans in Hong Kong in 1997, causing death in 6 out of 18 persons diagnosed with infection with this virus [2]. While the massive culling of poultry in 1997 temporarily eradicated the virus in Hong Kong, the virus has continued to spread across Asia, causing human deaths in Thailand, Vietnam, Indonesia, China and elsewhere [2,3]. The rapid spread of H5N1 in birds from Asia into Europe and Africa in recent months has intensified efforts to control the virus and avert a pandemic. To address the recognized need for rapid, low-cost diagnosis, tracking critically important genetic changes in the virus among animal and human host populations, and identifying specific viral clades [4], we have developed high-throughput methods for monitoring viral mutations that may control virulence and transmissibility in humans [5]. Accurate and rapid detection and tracking of H5N1 will be critical to prevent or control a potential pandemic. Diagnosis of influenza type A infections in clinical microbiology laboratories has traditionally been performed using cell culture and/or direct fluorescent antibody assays [57]. These methods are time-consuming and require biosafety level 3 enhanced biocontainment facilities and equipment to protect laboratory personnel from exposure to H5N1 cultured in the laboratory. Because these facilities not widely available, culture-based assays are increasingly being replaced in clinical settings by the various polymerase chain reaction (PCR) methods [811]. PCR is more sensitive than traditional tests and detection does not require viable virus or morphologically intact infected cells in the sample. A PCR-based molecular diagnostic test is currently the most widely used by public health laboratories to diagnose the presence of H5N1 in clinical specimens [12]. We hypothesized that coupling a PCR assay to a rapid sequencing method would further increase the value of molecular techniques for virus identification and characterization, especially if implemented into automated robotic platforms in the near future. Nucleic acid sequencing is considered the most reliable and highest-resolution method for virus identification, but is typically too slow and costly to use as a primary assay. Samples can be prepared sequentially for PCR diagnosis of H5N1 influenza virus, and pyrosequencing, yielding results in approximately 90 minutes, with immediate availability of the viral sequence data. The speed, sensitivity, precision, low cost, and high thr (...truncated)


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Nader Pourmand, Lisa Diamond, Rebecca Garten, Julianna P. Erickson, Jochen Kumm, Ruben O. Donis, Ronald W. Davis. Rapid and Highly Informative Diagnostic Assay for H5N1 Influenza Viruses, PLOS ONE, 2006, 1, DOI: 10.1371/journal.pone.0000095