Virological investigation of four outbreaks of influenza B reassortants in the northern region of Taiwan from October 2006 to February 2007

Background From October 2006 to February 2007, clinical specimens from 452 patients with symptoms related to respiratory tract infection in the northern region of Taiwan were collected. Real-time PCR and direct immunofluorescent antibody tests showed that 145 (32%) patients had influenza B virus infections. Subsequently, nucleotide sequence analyses of both hemagglutinin (HA) and neuraminidase (NA) genes of 39 isolates were performed. Isolated viruses were antigenically characterized using hemagglutinin inhibition (HI) test. Findings Phylogenetic tree analysis showed that all the isolates belonged to the B reassortant lineage with HA gene belonged to the B/Victoria/2/87 lineage and the NA gene belonged to the B/Yamagata/16/88 lineage. In addition, a group of children aged between 6 to 8 years old resided in Yilan county were infected with a variant strain. Hemagglutinin inhibition (HI) tests confirmed that all the reassortant influenza B viruses were B/Malaysia/2506/04-like viruses. Pre- and post-immunized serum samples from 4 normal volunteers inoculated with 2007 influenza vaccine were evaluated for their HI activity on 6 reassortant B isolates including two variants that we found in the Yilan county. The results demonstrated that after vaccination, all four vaccinees had at least 4-fold increases of their HI titers. Conclusion The results indicate that the 2006–2007 seasonal influenza vaccine was effective in stimulating protective immunity against the influenza B variants identified in Yilan county. Continuous surveillance of emerging influenza B variants in the northern region of Taiwan is important for the selection of proper vaccine candidate in the future.


Background
Enveloped orthomyxoviruses have segmented negativesense RNA genomes that facilitate new strain development via mutations and the reassortment of gene segments. This genetic instability is responsible for annual international epidemics and periodic pandemics of influenza infections [1,2]. Influenza viruses are spread by small airborne droplets expelled while talking, breathing, and coughing. The most susceptible population is children, and school-age children are the most common spreaders of infection [3][4][5][6][7]. Vaccines are thought to be the most effective modality for influenza control, and phylogenetic analyses of genes of viruses provide valuable information for vaccine development and prophylaxis.
We conducted phylogenetic analyses of HA and NA genes from influenza B virus isolates collected from a group of children complaining of severe headaches in north Tai

RNA Extraction and RT-PCR
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].

Nucleotide Sequence Analysis
DNA sequence analysis was performed using software from the Wisconsin Genetics Computer Group (GCG) [19,21,22]. All nucleotide sequences identified in this study have been deposited in GenBank (accession numbers EU234376 to EU234451).

Phylogenetic Analysis
Alignment of the respective 739 and 251 base pairs of HA and NA gene sequences was performed using the DNAS-TAR 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 [23][24][25]. We used MEGA for construction of neighbor-joining trees and PHYLIP for parsimony and maximum-likelihood meth-ods to verify the topology of taxas shown in the trees. In this study, all trees are shown as the neighbor-joining tree from MEGA.

Reassortant Influenza B Virus Epidemiology
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 epi-demiological 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.

Phylogenetic Analysis
According to results obtained from phylogenetic analyses of nearly full-length HA nucleotide sequences for the HA1 subunit  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).

HI Titers
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.

Post-Vaccination Serum Antibody Responses
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 preimmunized serum but an acceptable number following immunization.      Table 2). The data also indicate that the lowest HA titers were observed with sheep antisera against the other  two isolates belonging to the Yamagata lineage. We used a panel of serum samples from 5 patients who received influenza vaccinations to test their ability to generate protective immunity against influenza B variants isolated from young children living in Yilan county, using seroconversion factor as our criteria. Of the 5 patients, 4 showed a minimum of 4-fold increases in HI titers postvaccination; the seroconversion factor was 1 for the fifth patient, who was later diagnosed as immunologically incompetent (Table 3). These results suggest that the current influenza vaccine is still effective in stimulating protective immunity against the influenza B variants that we isolated [26][27][28] The gene variable analysis of influenza viruses can provide information for epidemic and pandemic outbreak surveillance and determination of vaccine strain selection. In this study, we have shown data suggesting that the contemporary vaccine was still effective in stimulating protective immunity against the influenza B variants that we isolated. Therefore, continuous monitoring of emerging influenza B virus variants is vital to successful control not only locally, but also internationally.

Discussion
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