Rapid and highly informative diagnostic assay for H5N1 influenza viruses

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.

Figure 1

Primers, markers and key sites on H5N1 influenza A hemagglutinin gene. A region of 768 bases of cDNA of the hemagglutinin gene of nine strains of H5N1 was assayed to obtain sequence information for three key biologically significant sites (glycosylation, receptor specificity, and HA1/HA2 cleavage; (sites indicated in red)). Two markers (green) distinguish clades of H5N1. Three additional polymorphic sites (yellow) provide unambiguous strain identification. The PCR primers used to bracket this region are indicated in purple; internal primers are blue.

Figure 2

Phylogenetic relationships of H5N1 hemagglutinin (HA) genes from highly pathogenic H5N1 avian influenza viruses used in this study. Phylogenetic trees were inferred from nucleotide sequences by the neighbor joining method in the MEGA program. Horizontal distances are proportional to the number of nucleotide changes between the viruses. HA clade determinations are shown on the right.

Figure 3

Amplicon DNA yield from PCR with different combinations of biotinylated primers. All PCR products were Sanger-sequenced to confirm the identity of the sample. A) The nine H5N1 samples amplified with a biotinylated forward primer (B-F-H5N1-1) and nonbiotinylated reverse primer (R-H5N1-2). From left to right, the samples are A/goose/Guangdong/1/96 (AF144305), A/Hong Kong/156/97 (AF036356), A/Hong Kong/483/97 (AF046097), A/Hong Kong/213/2003 (AY575869), A/chicken/Korea/ES/03 (AY676035), A/Vietnam/1203/2004 vaccine candidate with deletion of polybasic cleavage site (AY651334), A/Vietnam/JP14/2005 (ISDN117778), A/Vietnam/HN30408/2005 (ISDN119678), A/Indonesia/5/05 (ISDN125873). B) The H5N1 PCR products amplified with a biotinylated reverse primer (B-R-H5N1-3) and a nonbiotinylated forward primer (F-H5N1-4). Samples are in the order stated for part (A).

Figure 4

Panels A–I: Pyrograms obtained by pyrosequencing of sample A/Vietnam/HN30408/2005 with the HS 96 system. Peaks above a given nucleotide sample indicate nucleotide incorporation (with height proportional to number of consecutive incorporation events). Initial pyrosequencing was performed with systematic nucleotide dispensation for de novo sequencing (pre-programmed dispensation runs can also be performed). Panel J: Map of the HA sequence of A/Vietnam/HN30408/2005 obtained by Sanger sequencing; pyrosequencing data is overlaid and color-coded to show correlation between the sequence data obtained by the two methods. Reverse-primed pyrosequencing results should be read as reverse complements. For clarity, primers and biologically significant sequence segments are colored to correspond to the sequence map presented in Figure 1.

Figure 5

A representative pyrogram illustration. Comparison of pyrosequencing results obtained using the de novo (top) and consensus pre-programmed (bottom) sequencing methods for site 3 (a clade marker) in sample A1 (goose/Guangdong/1/96). Both methods give the sequence GACAAAGCTCTATCAAAAC for our laboratory stock. In contrast the GenBank sequence (accession # AF144305) reads GACAAAGCTATATCAAAAC. Database information was found to be based off wild-type sequence information, while the sample we analyzed was obtained via reverse genetics. A longer read length is obtained via the consensus pre-programmed dispensation method (22 versus 12 nucleotides).