Between October 2006 and February 2007, 452 throat swabs from patients with symptoms related to respiratory tract infection were collected at the clinical virology laboratory of the Taipei Veterans General Hospital. This laboratory has a contract with the Taiwan's Centers for Disease Control and is responsible for the virological evaluation of all the specimens sent from different hospitals in the northern region of Taiwan. The epidemiological curve and geographic distribution were shown in Figures 1 and 2. The median age of 145 patients with influenza B infection was 8 years old, with a range from 8 days to 14 years old except that B/Taiwan/3807/06, B/Taiwan/3842/06, B/Taiwan/1575/07 and B/Taiwan/1540/07 were 19, 18, 23 and 43 years old respectively. Seventy-four patients are male and seventy-one are female. To study the hemagglutinin inhibition titers for the influenza virus B isolates, five adult volunteers including four normal individuals and one patient (P139) with impaired immune function were recruited to receive vaccines containing A/New Caledonia/20/99, A/Wisconsin/67/05 and B/Malaysia/2506/04 strains. Informed consent was obtained from all participants. Patient P139 described that he was incapable of generating anti-hepatitis B virus (HBV) surface antibodies after repeatedly inoculated with HBV vaccine. Serum samples were collected prior to and 3 months after vaccination.

Filtrates from transport medium containing throat swabs were inoculated into Madin-Darby Canine Kidney (MDCK) cells. HA tests with guinea pig red blood cells were used to test the cultural supernatant for influenza B viral infection. Infected cells were detected using an IMAGEN™ influenza virus test kits (DaKo Cytomation Ltd, Cambs, UK) contain fluorescein isothiocyanate(FITC) conjugated monoclonal antibodies which bind specifically to influenza virus type B.

Viral RNA was extracted from 140 μL of infected tissue culture fluid using a QIAamp Viral RNA mini Kit (QIAGEN Inc., CA). Real-time PCR was performed with 5 μL RNA, Taqman one-step RT-PCR master mix reagents (Roche, New Jersey USA), 400 nM primer and 100 nM probe. The primers and probe used were according to Garbino et al. 20 and the cycling condition is 30 min at 48°C, 10 min at 95°C, and 40 cycles of 15 seconds at 95°C and 1 min at 60°C. In addition, 5 μL of eluted RNA were used for amplification with a QIAGEN one-step reverse transcription (RT)-PCR reagent. RT-PCR and sequencing for HA and NA were performed according to Tsai et al. 19.

DNA sequence analysis was performed using software from the Wisconsin Genetics Computer Group (GCG) 192122. All nucleotide sequences identified in this study have been deposited in GenBank (accession numbers EU234376 to EU234451).

Alignment of the respective 739 and 251 base pairs of HA and NA gene sequences was performed using the DNASTAR MegAlign clustal method. Phylogenetic trees were constructed using the neighbor-joining method based on Kimura's 2-parameter distance matrix with 1000 bootstrap replicates, using the MEGA (version 3.0) and PHYLIP (version 3.6) software packages 232425. We used MEGA for construction of neighbor-joining trees and PHYLIP for parsimony and maximum-likelihood methods to verify the topology of taxas shown in the trees. In this study, all trees are shown as the neighbor-joining tree from MEGA.

HI testing of 2006–2007 samples was performed using the World Health Organization (WHO) Influenza Reagent Kit for identifying influenza isolates, produced and distributed by WHO collaborating center for surveillance, epidemiology and control of influenza in American Continent (CDC, USA) as described in the kit's manual. Reference strain samples consisted of distinct influenza B anti-sera from four epidemics: B/Malaysia/2506/04 (B reassortant lineage), B/Hong Kong/330/01 (Victoria lineage), B/shanghai/361/02 (Yamagata lineage), and B/Sichuan/379/99 (Yamagata lineage).

Among HA screening tests positive cases from 452 specimens, 145 (32%) were confirmed to have influenza B infection using both real-time PCR and IFA test. The epidemiological curve and geographic distribution analyses demonstrated that the influenza B infection reach the peak in December 2006 and there were four outbreaks in Yilan county located in the northern region of Taiwan: outbreak I, 8 children from Jiaosi town; outbreak II, 7 students from Shai-Jin Elementary School in Wujie town; outbreak III, 5 students from Li-Tse Elementary School in Wujie town; and outbreak IV, 5 children from Toucheng town (Figures 1 and 2). Major symptoms included vomit, rhinorrhea, myalgia, diarrhea, prostration, lethargy, pharynegeal vesicles or ulcers, cough, sore throat, headache, and fever (temperature > 38°C), with a duration of 1–2 weeks. Of the patients from Yilan county, all 25 were in kindergarten or primary school, aged between 6 and 8 years old.

According to results obtained from phylogenetic analyses of nearly full-length HA nucleotide sequences for the HA1 subunit (1100 nt) of influenza B virus isolates from 39 patients including 38 children and 1 adult (B/Taiwan/3842/06), all isolates clustered with B/Malaysia/2506/04-HA with a bootstrap value of 96 (P < 0.01) (Figure 3). B/Malaysia/2506/04 has been identified by the WHO as the primary vaccine strain for the 2006–2007 flu season in the northern hemisphere. Note that 4 variants (cluster I) in outbreak II clustered on a single branch with a bootstrap value of 79 and statistical p value < 0.01 (Figure 3). In terms of NA genes, all 37 Taiwanese isolates were identified as belonging to the B reassortant lineage (P < 0.05) except two isolates were not PCR amplified (Figure 4).

Compared to amino acid residues 22–234 of the B/Malaysia/2506/04 HA protein, most of the Taiwanese isolates had only 2–3 changes. Exceptions included 1 strain from outbreak I (B/Taiwan/3009/06, 9 amino acid changes) and 4 variants (cluster I) from outbreak II (B/Taiwan/3802/06, B/Taiwan/3804/06, B/Taiwan/3805/06, and B/Taiwan/3790/06, with a combined total of 14 amino acid changes) (Table 1). Conserved region results indicate that amino acid residues 16–77 of the B/Malaysia/2506/04 NA protein originated in the previous season's Yamagata lineage strain (data not shown).

We used a panel of 4 reference sheep antisera to test the HI titers of 10 influenza B isolates; results are presented in Table 2. The HI titers of the B/Malaysia/2506/04-sheep antiserum to the Taiwanese influenza virus B strains belonged to outbreaks III, IV and the sporadic influenza B cases ranged from 1,280 to 2,560. In contrast, only 640 to 1,280 HI titers of the B/Malaysia/2506/04-sheep antiserum were identified as belonging to outbreak II. The number of HI titers for the B/Hong Kong/330/01-sheep antiserum ranged from 160 to 640. The smallest numbers of HI titers were observed in the antisera against the two virus isolates belonging to the Yamagata lineage.

Pre- and post-immunized serum samples were collected from 5 volunteers who had received the influenza vaccine for purposes of evaluating HI titers; all 5 generated antibody titers between 160 and 640 to the virus variants in outbreak II (Table 3). Of those 5, 3 showed 4 to 8-fold increases in HI antibody titers. The seroconversion factor was 1 for the patient diagnosed as immunologically incompetent. Another patient who reported to have received very few influenza vaccinations in the preceding ten years had exceptionally low antibody titers in his pre-immunized serum but an acceptable number following immunization.