Sequence characteristics of HA gene in influenza type A (H1N1) virus isolated during the 2005-2006 season in Aichi Prefecture, Japan.

In Aichi Prefecture, the first influenza outbreak of the 20052006 influenza season occurred from November to December 2005 in the northeast area (1). As of March 26, 2006, we had obtained a total of 182 influenza virus isolates from the respiratory specimens collected through the surveillance program in Aichi Prefecture. Of these, 70 (39%) were influenza AH1 virus, 106 (58%) were influenza AH3 virus, and 6 (3%) were influenza B virus. The percentage of AH1 virus isolates in Aichi was somewhat higher than that in the entire country (22%, Infectious Disease Surveillance Center, National Institute of Infectious Diseases [NIID], Tokyo, Japan. Available online at .). In the present report, we describe antigenic and genetic aspects of AH1 isolates obtained in the 2005-2006 season in Aichi Prefecture. Throat swab specimens were collected from influenza patients as sentinels in the influenza surveillance, and were inoculated onto Madin-Darby Canine Kidney (MDCK) cells. Influenza virus was identified by hemagglutination inhibition (HI) test using culture supernatant of infected MDCK cells as the hemagglutinin and type-specific sera against influenza viruses (provided from the NIID). Viral RNA was extracted from culture supernatants with High Pure Vial RNA Kit (Roche Applied Science, Penzberg, Germany). The complete coding sequence of the HA1 gene was amplified by One-step RT-PCR Kit (Invitrogen, Carlsbad, Calif., USA). The primers used were 5 ́-gcaaaagcaggggaaaataa-3 ́and 5 ́ttgatctgcagcatagccag-3 ,́ which yield a 1,177-bp amplicon. Direct sequencing of the amplified fragment was performed with a Model-4200 automated DNA sequencer (Li-cor, Lincoln, Nebr., USA). The NA subtype of each isolate was determined using type-specific primers (2). All 14 AH1 isolates were proved to be of the N1 subtype. Out of the 70 AH1 isolates identified so far in the 2005 2006 season, we determined nucleotide sequences of the HA1 gene of 14 isolates. The phylogenetic tree of the isolates is shown in Fig. 1. The isolates formed two clusters in the 20052006 season (Fig. 1). The isolates in the early season (1) are clustered into a group (cluster b in Fig. 1) including Aichi/168/2005, the first isolate of the season in our institute (isolated from a specimen collected on November 17). The identity among the isolates in cluster b was 99.1 100%, without changes in the predicted amino acid sequences. A/ New Caledonia/20/99, one of the 2005-2006 season vaccine strains, also belonged to cluster b, and the Aichi isolates in cluster b exhibited 98.1 -98.4% nucleotide sequence identity to this vaccine strain. In contrast, the isolates in cluster a were genetically more distant from A/New Caledonia than were those in cluster b, showing 97.4 -97.5% identity. A comparison of the predicted amino acid residues between each of the Aichi isolates and A/New Caledonia/20/99 is shown in Fig. 2. Amino acid sequences were 100% identical among isolates within each of two clusters. Aichi/168/2005 and Aichi/21/2006 (isolated from the specimen collected on January 13) are shown as representative strains of clusters b and a, respectively, in Fig. 2. Amino acid numbering corresponds to the number of HA1 in the H3 subtype, as reported previously (3). A total of 5 amino acid changes from A/New Caledonia/20/99 were detected in the HA1 protein of Aichi/ 168/2005-like viruses, including V169A, N190D, W255R, Y256F, and V317A (not shown in Fig. 2). In contrast, Aichi/ 21/2006-like viruses contained 11 predicted amino acid