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.
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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)