Influenza AH1N2 Viruses, United Kingdom, 2001-02 Influenza Season

Joanna S. Ellis, Adriana Alvarez-Aguero, Vicky Gregory, Yi Pu Lin, A. Hay, Maria C. Zambon

Disclosures

Emerging Infectious Diseases. 2003;9(3) 

In This Article

Materials and Methods

Data on the rates of community cases of influenza and influenzalike illness were collected by the Birmingham Unit of the Royal College of General Practitioners and are based on weekly returns from approximately 69 sentinel practices throughout England and Wales. The Communicable Disease Surveillance Centres in Wales and Northern Ireland and the Scottish Centre for Infection and Environmental Health provided additional data. These three mechanisms for monitoring clinical incidence of influenzalike illness cover a total population of 800,000 persons. Incidence data were recorded as new physician consultations per 100,000 persons.

Influenza viruses isolated in hospital laboratories throughout the U.K. were sent to the Enteric, Respiratory and Neurological Laboratory at the Central Public Health Laboratory for antigenic characterization. Throughout the influenza season (October-March), a subset of the clinical practices group in the Birmingham Unit of the Royal College of General Practitioners network obtained nose and throat swabs from patients who had influenzalike illness,[17] which were sent directly by mail to Enteric, Respiratory and Neurological Laboratory for virus isolation and subsequent antigenic and genetic characterization. All specimens were given a laboratory sample number upon arrival.

Combined nose and throat swab specimens in virus transport medium were treated as previously described and inoculated onto confluent Madin-Darby canine kidney cells.[18] The cells were incubated at 33°C, and the medium was tested at day 7 for HA of turkey red cells.

Influenza viruses were typed by using ferret antisera in hemagglutination inhibition tests as described.[19] All HI tests were carried out by using 8 HA U of virus and 0.5% (v/v) turkey red blood cells. All ferret sera were treated with receptor-destroying enzyme. After typing, virus isolates were given a unique strain designation number in strict chronologic order.

Susceptibility of influenza virus replication to inhibition by amantadine was determined by virus infectivity assays.[20] Madin-Darby canine kidney cells were overlaid with medium containing amantadine (SigmaAldrich, Poole, Dorset, England) at a concentration of 0.1, 1.0, or 10.0 µg/mL.

Viral RNA was extracted from 150-µL samples by using the MagNA Pure LC total nucleic acid isolation kit, on a MagNA Pure LC extraction robot (Roche Molecular Biochemicals, Mannheim, Germany).[21] Reverse transcription was performed as previously described.[20] PCR to detect N1 and N2 neuraminidase genes was performed with nested primer sets (primer sequences available on request), modified from previously described assays.[22]

         Viruses selected for genetic analysis are shown in          Table 1          . Amplification of the HA1 domain of the HA gene, the complete coding region of the NA gene and portions of the PB2, PB1, PA, NP, NS, and M genes required primers specific for the eight genes (primer sequences available by request). The regions amplified for sequence analysis were PB2, 46-487; PB1, 970-1427; PA, 499-817; HA, 84-1058; NP, 1045-1428; NA, 20-1426; M, 249-612; and NS, 146-843. PCR products were purified by using agarose gel electrophoresis and a QIAquick gel extraction kit (Qiagen Ltd, Crawley, West Sussex, England) and sequenced by using a Beckman Coulter CEQ 2000 capillary sequencer and CEQ 2000 Dye Terminator cycle sequencing Quick Start kit (Beckman Coulter, Fullerton, CA). The fragment lengths compared were PB2, 442; PB1, 458; PA, 319; HA, 975; NP, 384; NA, 1407; M, 364; and NS, 698 nucleotides. Nucleotide sequences determined in this study are available from the European Molecular Biology Laboratory database (accession nos. AJ489485-AJ489502, AJ489530-AJ489559, and AJ489846-AJ489862). The nucleotide sequences for the NA, M1, and NP of A/Panama/2007/99 and A/Moscow/10/99 viruses are available in the European Molecular Biology Laboratory database under accession numbers AJ457937, AJ457966, AJ458298, AJ458297, AJ458268, and AJ458267.[23]

Sequences were analyzed by neighbor joining with the Phylip (version 3.57) suite of programs (DNADist and Fitch) and bootstrapping by Seqboot.[24] Bootstrap values >70 were regarded as statistically significant.

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