Abstract
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.
Highlights
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
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]
The first step in the analysis of a clinical specimen or a viral isolate in our assay is the generation of a DNA copy of the viral RNA, which is accomplished by reverse transcription coupled to polymerase chain reaction (PCR) (RT-PCR)
Summary
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. 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]. 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
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