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Research

Novel Avian Influenza H7N3 Strain Outbreak, British Columbia

Martin Hirst,* Caroline R. Astell,* Malachi Griffith,* Shaun M. Coughlin,* Michelle Moksa,* Thomas Zeng,* Duane E. Smailus,* Robert A. Holt,* Steven Jones,* Marco A. Marra,* Martin Petric,† Mel Krajden,† David Lawrence,† Annie Mak,† Ron Chow,† Danuta M. Skowronski,† S. Aleina Tweed,† SweeHan Goh,† Robert C. Brunham,† John Robinson,‡ Victoria Bowes,‡Ken Sojonky,‡ Sean K. Byrne,‡ Yan Li,§ Darwyn Kobasa,§ Tim Booth,§ and Mark Paetzel¶
*British Columbia Cancer Agency (BCCA) Genome Sciences Centre, Vancouver, British Columbia, Canada; †British Columbia Centre for Disease Control and University of British Columbia Centre for Disease Control, Vancouver, British Columbia, Canada; ‡Ministry of Agriculture, Abbotsford, British Columbia, Canada; §Canadian Centre for Human and Animal Health, Winnipeg, Manitoba, Canada; and ¶Simon Fraser University, Burnaby, British Columbia, Canada

Appendix

Materials and Methods

Growth of Avian Influenza in Embryonated Chicken Eggs and Cell Culture

	Appendix Figure 1
	Click to view enlarged image Appendix Figure 1. Complete nucleotide (A) and protein (B) alignments from the 5 isolates sequenced in this study...
	Appendix Figure 2
	Click to view enlarged image Appendix Figure 2. Phylogenetic tree of isolates described in the text with 65 full-length H7 HA gene segments present in GenBank...
	Appendix Figure 3
	Click to view enlarged image Appendix Figure 3. A structure-based sequence alignment of the influenza A virus hemagglutinin precursor protein...

The tissue samples were homogenized in 10 volumes of virus isolation diluent (peptone 10 g/L, gelatin 2.5 g/L, phosphate-buffered saline pH 7.4, gentamicin 100 mg/L, amphotericin B 5 mg/L, cyclohexamide 10 mg/L ). The homogenate was centrifuged twice at 3,000 x g for 30 min to remove debris, and 0.2 mL of supernatant was injected into four 9- to 11-day-old embryonated eggs obtained from a cloistered nonvaccinated flock. Controls were injected with virus isolation fluid. Eggs were incubated at 37°C and assessed for viability at 24 h. At 48 h, the eggs were cooled, and the allantoic fluid was harvested. A hemagglutination test was run using 0.5% chicken red blood cells, and hemagglutinin-positive samples were confirmed by polymerase chain reaction (PCR). Nasopharyngeal and conjunctival swab specimens from affected patients were injected into primary rhesus monkey kidney cells and passaged further in Madin Darby canine kidney cells (Diagnostic Hybrids, Athens, OH). The isolated viruses were shown to be influenza A H7 by RT (reverse transcription)-PCR of the RNA (1) and subsequently typed as H7N3 (National Microbiology Laboratory, Winnipeg, MB, Canada).

RNA Extraction and RT-PCR

Trizol (Invitrogen, Burlington, ON, Canada) was used to extract vRNA from 100 μL of allantoic fluid from infected embryonated eggs or 1 mL of infected Madin-Darby kidney cell culture. The vRNA was isolated by using Phase Lock Gel, heavy 2 mL Eppendorf tubes (Brinkmann, Mississauga, ON, Canada) according to the manufacturer's recommended protocol. RNA was precipitated in 0.5 volumes of isopropanol in the presence of 0.2 mg of mussel glycogen (Invitrogen, Burlington, ON, Canada). The resulting pellet was washed in 75% ethanol and resuspended in 40 μL of diethylpyrocarbonate-treated dH₂0. A two step RT-PCR was used to amplify each of the viral gene segments. The RNA (6 μL) was synthesized into cDNA by using 500 ng of Uni12 primer (Appendix Table) and the Powerscript Reverse Transcriptase (BD Biosciences, Mississauga, ON), according to the manufacturer's recommended protocol. The RT reaction was performed at 42°C for 60 min. The cDNA was amplified by using the Expand Long Template PCR System (Roche Diagnostics, Laval, QB, Canada), following the protocol provided. The Mg²⁺ concentration was 3.5 mM, the primer concentration was 0.4 mmol/L, and betaine was added to a final concentration of 1 mol/L. Segment-specific primers were based on the universal influenza A primer set designed by Hoffman et al. (2). The cycling conditions consisted of an initial denaturation at 95^oC for 5 min followed by 10 touchdown PCR cycles starting with 95°C for 15 s, 68^oC (decreased by 1^oC in each subsequent cycle) for 15 s, 68°C for 3 min; then 20 cycles of 95°C for 15 s, 62°C for 15 s, 68°C for 3 min; followed by an extension at 68°C for 10 min.

Isolation and Cloning of vRNA Gene Segments

Ten microliters of the PCR reaction for each sample was loaded on an 1% agarose gel. The gel was stained with >SYBR Green (Mandel, Guelph, ON, Canada) and visualized using a Typhoon 9400 Variable Mode Imager (Amersham, Baie d'Urfe, QB, Canada). Visible bands of expected size were excised from the agarose gel, and purified using the MinElute Gel Extraction Kit (Qiagen, Mississauga, ON) following the manufacturer's protocol. Purified amplicons were cloned into the pCR4-TOPO vector by using the TOPO TA Cloning Kit for Sequencing (Invitrogen), according to the manufacturer's protocol.

vRNA gene Segment Sequencing

Eight clone inserts for each genomic segment were randomly selected and sequenced on an ABI PRISM 3730 XL DNA Analyzer with BigDye v3.1 primer cycle sequencing reagents (Applied Biosystems Canada, Streetsville, ON, Canada). End reads were obtained by using T3 and T7 reverse primers. Sequence reads were processed, and their quality was assessed by using Phred and assembled by using Phrap (3,4). When needed, specific sequencing primers were designed by manual selection from primer sequences generated by Consed (5), and full-length sequences were obtained by primer walking.

The Appendix Table gives a comparison of individual gene segments of the Fraser Valley H7N3 outbreak with influenza genes from GenBank showing the highest similarity. Appendix Figure 1 provides complete nucleotide and protein alignments for the HA gene for the five isolates sequences in this study, and Appendix Figure 2 is a phylogenetic tree of isolates described in the text.

Homology Modeling

The 0.28-nm resolution crystal structure of the hemagglutinin precursor protein from influenza A virus, human H3, strain CV-1 (PDB code HA01) was used as the template (6). An initial sequence alignment was performed with the program ClustalW (7) and then modified by hand, resulting in 49.9 % sequence identity (Appendix Figure 3). The initial model was made by using the program O (8) and then energy minimized with the program CNS (9). The monomer model was superimposed onto the trimeric 1HA0 template structure by using the program superimpose (10) and then further energy minimized by using CNS. The model includes residues 21 through 519 (lacking the N-terminal 20 residues and the C-terminal 48 residues) of the human A/Canada/504/04 (Hu504) hemagglutinin precursor. The figures were prepared by using the program PyMol (11).

Appendix References

1. Spackman E, Senne DA, Myers TJ, Bulaga LL, Garber LP, et al. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J Clin Microbiol. 2002;40:3256–60.
2. Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR. Universal primer set for the full-length amplification of all influenza viruses. Arch Virol. 2001;146:2275–80.
3. Ewing B, Green P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 1998;8:186–94.
4. Ewing B, Hillier L, Wendl MC, Green P. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 1998;8:175–85.
5. Gordon D, Abajian C, Green P. Consed: a graphical tool for sequence finishing. Genome Res. 1998;8:195–202.
6. Chen J, Lee KH, Steinhauer DA, Stevens DJ, Skehel JJ, Wiley DC. Structure of the hemagglutinin precursor cleavage site, a determinant of influenza pathogenicity and the origin of the labile conformation. Cell. 1998;95:409–17.
7. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673–80.
8. Jones TA, Zou JY, Cowan SW, Kjelgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991;47:110–9.
9. Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, et al. Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr. 1998;54:905–21.
10. Diederichs K. Structural superposition of proteins with unknown alignment and detection of topological similarity using a six-dimensional search algorithm. Proteins. 1995;23:187–95.
11. DeLano WL. The PyMOL user's manual. San Carlos (CA): DeLano Scientific, USA; 2002. Available from http://www.pymol.org

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This page posted November 8, 2004
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Emerging Infectious Diseases Journal
National Center for Infectious Diseases
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