Increasing Appearance of Reassortant Influenza B Virus in Taiwan from 2002 to 2005

Viral isolation.Throat and nasal swabs from patients with flu-like syndromes were collected by sentinel and National Cheng Kung University Hospital physicians and immediately placed in 2 ml of a homemade viral transport medium which contains 0.5% gelatin, Earle's minimum essential medium, 200 U/ml penicillin-streptomycin, 0.05 mg/ml gentamicin, and 1.25 μg/ml amphotericin B (Fungizone). Refrigerated samples were inoculated within 24 h into tubes containing Madin-Darby canine kidney (MDCK) cells. Influenza virus infection of cells was confirmed by the use of monoclonal antibodies for influenza A and B viruses (Chemicon, Inc.). Hemagglutinin inhibition (HI) assays for antigenic typing were carried out using the World Health Organization influenza reagent kit provided by the Centers for Disease Control and Prevention, Atlanta, GA.

Nucleotide sequencing analysis.Viral RNA was extracted from 140 μl of infected tissue culture fluid by using the QIAamp viral RNA mini kit. Five microliters of eluted RNA was used for amplification using a QIAGEN one-step reverse transcription (RT)-PCR reagent. Primers used for RT-PCR and sequencing (3) were as follows: B/HA98 (forward, 5′-ATAACATCGTCAAACTCACC-3′), B/HA836 (reverse, 5′-GCACCATGTAATCAACAACA-3′), B/NA1 (forward, 5′-GCTACCTTCAACTATACAAACG-3′), and B/NA2 (reverse, 5′-AACGAGGGTATGTCCACTCC-3′). Cycle conditions for RT-PCR for HA or NA gene amplification were as follows: 30 min at 50°C for reverse transcription, 1 cycle for 5 min at 95°C followed by 35 cycles of 30 s at 94°C, 30 s at 50°C (for HA) or 60°C (for NA), and 1 min at 72°C followed by 1 cycle for 10 min at 72°C, with subsequent holding at 4°C. Prior to sequencing, the PCR products were purified with a QIAquick PCR purification kit (QIAGEN). The purified DNA was sequenced using a BigDye Terminator cycle sequencing kit (Applied Biosystems, California) and a model 373A DNA sequencer (Perkin-Elmer). DNA sequence analysis was performed using software from the Wisconsin Genetics Computer Group (GCG). Phylogenetic comparisons were carried out using version 3.573c of the Phylogeny Inference Package (PHYLIP). Bootstrap values were obtained by performing 1,000 replicates.

Nucleotide sequence accession numbers.Influenza virus sequences reported in this study have been deposited in the GenBank database under accession numbers DQ514388 to DQ514476.

Epidemiology of influenza B viruses in Taiwan.From January 1999 to May 2005, 404 influenza B viruses were isolated from 18,319 throat swab samples collected from patients with influenza-like symptoms. Influenza B virus epidemics occurred annually, with a peak in activity between March and May. Antigenic characterization of HA in circulating influenza B viruses was performed by using HI tests. The HI results showed that the majority of circulating influenza B viruses from 1998 to 2001 belonged to the Yamagata lineage. Most isolates from the 1998-1999 and 1999-2000 seasons were B/Beijing/184/93-like viruses, and most of the 2000-2001 season isolates were B/Yamanashi/166/98-like viruses (Fig. 1). In the 2001-2002 season, B/Hong Kong/22/01-like (Victoria lineage) and B/Sichuan/379/99-like (Yamagata lineage) viruses cocirculated. B/Hong Kong/1434/02-like (Victoria lineage) viruses were identified in the 2002-2003 season. The very mild influenza B activity (B/Shanghai/361/02-like strain, Yamagata lineage) seen during the 2003-2004 season was probably due to increased precautionary measures (such as isolating patients, wearing surgical masks in the hospital and public areas, and decreasing public gatherings) taken during the outbreak of severe acute respiratory syndrome. From December 2004 to April 2005, B/Hong Kong/1434/02-like viruses (Victoria lineage) reappeared, and some B/Shanghai/361/02-like strains (Yamagata lineage) also cocirculated between January and May 2005.

• Open in new tab
• Download powerpoint

FIG. 1.

Monthly distribution of influenza B viruses based on HI tests in Taiwan from 1998 to 2005.

Phylogenetic analysis of influenza B viruses circulating in Taiwan.To determine the molecular evolution of the HA and NA genes, 44 and 45 strains, respectively, isolated in Taiwan from 1999 to 2005 were analyzed. Genetic analyses of the HA genes from 44 local isolates showed that 25 influenza B viruses belonged to the Yamagata lineage and 19 belonged to the Victoria lineage (Fig. 2). There were two clusters in the Yamagata lineage. Cluster 1 could be further divided into two clades, one of which included local isolates from 1999 which were similar to the B/Yamanashi/166/98 vaccine strain. The other clade included isolates from 2000 to 2001, which clustered with B/Sichuan/379/99 and B/Victoria/504/00 vaccine strains. Cluster 2 included isolates from 2002 and 2004 to 2005, which segregated with B/Shanghai/361/02 and B/Jilin/20/03 vaccine strains, respectively. B/Jilin/20/03 and B/Shanghai/361/02 vaccine strains (the vaccines recommended for the 2004-2005 season in the Northern hemisphere) were identified frequently during the 2003-2004 season globally. This B/Shanghai/361/02-like strain had appeared largely in Taiwan since 2002, indicating that this lineage occurred in Taiwan one to two seasons earlier than in other areas of the world. The B/Victoria/2/87 lineage, which cocirculated with the Yamagata lineage, was initially isolated and identified in Taiwan in March 2001. Nineteen local isolates of the Victoria lineage segregated into two clades. One clade contained four local isolates from 2002 and 2003 which clustered with the B/Hong Kong/1434/02 and B/Hong Kong/330/01 vaccine strains, and the second clade, which included two, five, one, one, and six isolates from 2001, 2002, 2003, 2004, and 2005, respectively, clustered with the B/Hong Kong/1351/02 and B/Brisbane/32/02 strains.

• Open in new tab
• Download powerpoint

FIG. 2.

Phylogenetic analysis of HA genes of influenza B virus isolates obtained during the 1999-2005 influenza season. The dendrogram of the 44 influenza B virus outbreak strains, with 28 reference strains from GenBank, is based on 738 nucleotides (nucleotides 98 to 836) of the HA1 gene using the neighbor-joining method with the DNADIST distance measure program (PHYLIP, version 3.573c). The percentage of bootstrap frequency of each branch in the tree is indicated. B/Lee/40 was included as an outgroup.

Sequence analysis of the NA genes from 45 Taiwan isolates showed that 40 influenza B viruses belonged to the Yamagata lineage and 5 belonged to the Victoria lineage (Fig. 3). One cluster among the Yamagata lineage included five, three, one, and five isolates from 2002, 2003, 2004, and 2005, respectively, which clustered with B/Hong Kong/1351/02 and B/Brisbane/32/02 and was recognized as a reassortant strain of viruses with an HA gene from the Victoria lineage but which had acquired an NA gene from a distinctly different lineage, the Yamagata lineage. The other cluster in the Yamagata lineage contained isolates from 1999 to 2005, which were similar to the B/Sichuan/379/99 vaccine strain. Four influenza B viruses of the Victoria lineage segregated into two clades. Two of the influenza B viruses from 2002 were similar to the B/Hong Kong/330/01 strain, and two viruses from 2001 were similar to the B/Shangdong/7/97 and B/Beijing/243/97 vaccine strains. The results showed that the B reassortant viruses were first identified in April 2002 and that there were three groups of influenza B viruses, including those belonging to the Yamagata lineage and the Victoria lineage and the B reassortant viruses which circulated in Taiwan in 2002.

• Open in new tab
• Download powerpoint

FIG. 3.

Phylogenetic analysis of NA genes of influenza B virus isolates obtained during the 1999-2005 influenza season. The dendrogram of the 45 influenza B virus outbreak strains, with 24 reference strains from GenBank, of the NA gene using the neighbor-joining method with the DNADIST distance measure program (PHYLIP, version 3.573c). The percentage of bootstrap frequency of each branch in the tree is indicated. B/Lee/40 was included as an outgroup.

Variations in HA and NA amino acid sequences of influenza B viruses.Regions of the local influenza B virus isolates containing 248 and 66 amino acids, respectively, of HA and NA were compared with those of recent vaccine strains in order to understand the variations between the Yamagata and Victoria lineages of influenza B viruses (Table 1, Table 2, and Table 3). The isolates with HAs clustering within the Yamagata lineage can be segregated into two groups. Taiwan isolates from the 1999 to 2001 Yamagata lineage were similar to B/Sichuan/379/99, and isolates from 2002 to 2005 were similar to B/Shanghai/361/02 or B/Jilin/20/03. Ten signature amino acids in HA, R48, D56, T75, N116, D126, K149, N168, Y179, E183, and G184, of B/Shanghai/361/02, are all conserved in our 2002 to 2005 isolates. However, the HA of all Taiwan isolates from 1999 to 2005 had an asparagine (N) at position 197 instead of an aspartic acid (D), as had B/Sichuan/379/99 and B/Shanghai/361/02. Interestingly, there were two signature amino acids at positions H40Y and L131P of HA found between the B/Shanghai/361/02 strain and our 2004-2005 local isolates which were also found in B/Jilin/20/03 of the Yamagata lineage (Table 1). However, as found in B/Jilin/20/03, two common amino acid changes in HA, I180V and D233A, were seen in some local isolates from 2004 to 2005. The Victoria lineage strains were divided into two distinct groups, B/Hong Kong/330/01 and B/Hong Kong/1351/02 (B reassortant) (Table 2). Local isolates from 2002 to 2005 had an asparagine (N) at position 197 instead of a lysine (K) in HA, as seen in B/Hong Kong/1351/02. The reassortant B viruses from 2004 to 2005 had three notable amino acid changes from the strains of 2002 at K48E, K80R or K80E, and K129N in their HA genes. Notably, B reassortants from 2002 (strains B/Taiwan/S82/02, -H96/02, and -N3849/02) had all the same amino acids in their HA as the isolates from the 2001 Victoria lineage (B/Taiwan/N612/01 and -N701/01). In addition, one 2003 reassortant, B/Taiwan/N384/03, had the same amino acid in its HA as the Victoria isolate from 2002 (B/Taiwan/N1582/02). These results indicated that the HA genes of B reassortants were obtained from the Victoria lineage of the previous year.

Within the 66-amino-acid region of NA, a position 42 substitution (P42T or P42Q) was noticed in nonreassortant strains of the Yamagata lineage from 2001 to 2005 (Table 3). Interestingly, the reassortant B viruses from 2004 to 2005 had two notable amino acid changes from the strains from 2002 to 2003 at S41P and P42S. The Victoria lineage strains from 2002 had three signature amino acid changes from the strains of B/Beijing/243/97, V45I, M50T, E69K (Table 3). It should be noted that B reassortants (B/Taiwan/H96/02, -N253/03, -M53/05, and -N371/05) were found to have the same amino acids in their NA genes as those found in the Yamagata lineage (B/Taiwan/N767/01). In addition, the B reassortant B/Taiwan/N3849/02 had the same amino acids in its NA gene as those in the previous Yamagata lineage strain (B/Taiwan/S76/02). The results indicated that the NA genes of B reassortants were obtained from the Yamagata strains of the previous season.

Comparison of HI test results and phylogenetic analysis of influenza virus.The results of HI testing and phylogenetic analysis were compared (Table 4). The lineage of HI and phylogenetic analysis of the HA gene were very consistent. There were two peaks of reassortant B viruses circulating in Taiwan from April 2002 to April 2005; the first peak was in 2002, and the second was in 2005. Interestingly, these reassorted B viruses accounted for 46% (5/11) of influenza B viruses tested in 2002 and became dominant (73% [19/26]) in January to April 2005.