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Baek, Song, Song, Pascua, Lee, Han, Woo, Kim, and Choi: Genetic Characteristics and Phylogenetic Analysis of Influenza Type B Viruses Isolated from Nasopharyngeal Suction Samples of Korean Patients
Original Article

J Bacteriol Virol 2009;39(2):125-136.

Published online: 18 January 2009

DOI: https://doi.org/10.4167/jbv.2009.39.2.125

Genetic Characteristics and Phylogenetic Analysis of Influenza Type B Viruses Isolated from Nasopharyngeal Suction Samples of Korean Patients

Yun Hee Baek, Young Jun Song, Min-Suk Song, Philippe Noriel Q. Pascua, Jun Han Lee, Heon-Seok Han, Sung-Il Woo, Seok-Yong Kim, Young-Ki Choi∗

College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Korea

∗Corresponding author: Young Ki Choi. College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong, Heungduk-Ku, Cheongju 361-763, Korea. Phone: +82-43-261-3384, Fax: +82-43-272-1603 e-mail: choiki55@chungbuk.ac.kr

∗∗ This work was supported by internal fund from Chungbuk National University in 2008.

      Revised 1 May 20093 June 2009       Accepted 5 June 2009

Copyright © 2009 Korean Society for Legal Medicine

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

To investigate the genetic characteristics of human influenza type B viruses circulating in Chungbuk province, Korea, we tested 510 clinical samples of nasopharyngeal suction from pediatric patients diagnosed with respiratory illness between June 2007 and June 2008. Twelve out of thirty-six isolates were identified as type B influenza virus by RT-PCR and sequencing analysis. Interestingly, genetic characterization of type B viruses isolated in this study revealed that all type B influenza viruses were the Yamagata lineages, a vaccine strains of southern hemisphere during 2007~2008, rather than the Victoria lineage of northern hemisphere during 2007~2008. Furthermore, there were a total of twelve unique mutations (HA: H40Y, D/G230S, V252M and K272R and NA: P3H, P/T/S42Q, N59S) occurred in our type B isolates. These results suggest that relative high prevalence of type B viruses in Korea during 2007~2008 season might be due to the wrong vaccine strains selection. Taken together, the results of this study demonstrate continuous evolutions of human type B viruses by antigenic drift and also highlight the need to closely monitoring of influenza viruses to aid the early detection of potentially pandemic strains as well as underscore the need for new therapeutics.

Keywords: Influenza B virus, Antigenic drift, Phylogenetic analysis, Vaccine strain

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Figure 1.
Amplification of the eight viral gene segments of influenza B viruses isolated in this study. Full length segments were amplified by RT-PCR using different combination of primers for each gene (Table 1) and visualized by electrophoresis on 0.8% agarose gel. Two representative viruses (B/Cheongju/411/2008 and B/Cheongju/437/2008 with GenBank accession number FJ461674~FJ461689) are shown (PB2: 2396 bp; PB1: 2369 bp; PA: 2308 bp; HA: 1755 bp; NP: 1683 bp; NA: 1557 bp; M: 1076 bp; NS: 846 bp).
jbv-39-125f1.tif
Figure 2.
Phylogenetic analysis. Phylogenetic trees of the nucleotide sequences for the 8 viral Genes (A~H) including (A) HA and (B) NA genes in this study (in bold) compared with nucleotide sequences from selected human influenza B viruses available in GenBank including the three to five vaccine strains. The vaccine strains are indicated by stars. Phylograms were generated by neighbor-joining analysis with 1000 bootstrapped replicates. Bootstrap values for nodes occurring in 60% or more of multiple bootstrap replicates are given immediately to the left of the node.
jbv-39-125f2a.tifjbv-39-125f2b.tifjbv-39-125f2c.tifjbv-39-125f2d.tif
Table 1.
List of primers used for amplifying and sequencing of the eight viral genes
Oligonucleotide Sequence Position Polarity
aMDV-B 5′ HA TATTCGTCTCAGGGAGCAGAAGCAGAGCATTTTCTAATATC 1~40 Sence
bB HA A GTGACTGGTGTGATACCACT 121~140 Sence
bB HA DII TGTTTTCACCCATATTGGGC 1005~1023 Antisense
aMDV-B 3′ HA ATATCGTCTCGTATTAGTAGTAACAAGAGCATTTTTC 1719~1755 Antisense
aNAb-1 TATTCGTCTCAGGGAGCAGAAGCAGAGCA 1~29 Sence
aNAb-1557R ATATCGTCTCGTATTAGTAGTAACAAGAGCATTTT 1523~1557 Antisense
aMDV-B 5′ M TATTCGTCTCAGGGAGCAGAAGCACGCACTTTCTTAAAATG 1~41 Sence
B M 560R GTTCATAGCTGAGACCATCTG 540~560 Antisense
aMDV-B 3′ M ATATCGTCTCGTATTAGTAGAAACAACGCACTTTTTCCAG 1037~1076 Antisense
aMDV-B 5′ NS TATTCGTCTCAGGGAGCAGAAGCAGAGGATTTGTTTAGTC 1~40 Sence
aMDV-B 3′ NS ATATCGTCTCGTATTAGTAGTAACAAGAGGATTTTTAT 809~846 Antisense
aMDV-B 5′ NP TATTCGTCTCAGGGAGCAGAAGCACAGCATTTTCTTGTG 1~39 Sence
aMDV-B 3′ NP ATATCGTCTCGTATTAGTAGAAACAACAGCATTTTTTAC 1645~1683 Antisense
aPB1b-1 TATTCGTCTCAGGGAGCAGAAGCGGAGCCTTTAAGATG 1~38 Sence
PB1b 890 F GGGATCAGCATGACAGTAACA 890~910 Sence
PB1 1560 R GCTGATTCATTTACTCCAGCA 1540~1560 Antisense
aPB1b-2369R ATATCGTCTCGTATTAGTAGAAACACGAGCCTT 2337~2369 Antisense
aPB2b-1 TATTCGTCTCAGGGAGCAGAAGCGGAGCGTTTTCAAGATG 1~40 Sence
PB2 890 F AATCATGTCTGACAGCCATAG 890~910 Sence
aPB2b-1145R TATTCGTCTCTCTCATTTTGCTCTTTTTTAATATTCCCC 1107~1145 Antisense
aPB2b-2396R ATATCGTCTCGTATTAGTAGAAACACGAGCATT 2364~2396 Antisense
aPAb-1 TATTCGTCTCAGGGAGCAGAAGCGGTGCGTTTGA 1~34 Sence
Pab- 830 F GCTACCAGAAGTTCCATATAA 830~850 Sence
aPAb-1261R TATTCGTCTCCCAGGGCCCTTTTACTTGTCAGAGTGC 1225~1261 Antisense
aPAb-2308R ATATCGTCTCGTATTAGTAGAAACACGTGCATT 2276~2308 Antisense

a References: a Rescue of influenza B virus from eight plasmids (24)

b Multiplex PCR for typing and subtyping influenza and respiratory syncytial viruses (25)

Table 2.
Amino acid variations of HA 1 region from Influenza B isolates in Cheongju, Korea compared with B/Yamagata/16/88
Virus strain(s) Amino acid at position
37 40 48 56 73 76 122 126 129 131 146 149 150
B/Yamagata/16/88 T H K N M I H N R P V R N
B/Shanghai/361/02a I • R D V T Q D K L A K S
B/New york/01/08 • Y R D V T Q D K • A K S
B/Cheongju/411/08 • Y R D V T Q D K • A K S
B/Cheongju/437/08 • Y R D V T Q D K • A K S
B/Malaysia/2506/04b • • • • T • • • N • • G •
Table 2a.
Virus strain(s) Amino acid position Glycosylation
162 165 168 183 197 230 233 252 272 320 196~198
B/Yamagata/16/88 R – T K D D N V K N DKT (–)
B/Shanghai/361/02a K N N E • G D • • H DKT (–)
B/New york/01/08 K N N E N S D M R H NKT (+)
B/Cheongju/411/08 K N N E N S D M R H NKT (+)
B/Cheongju/437/08 K N N E N S D M • H NKT (+)
B/Malaysia/2506/04b K N N E N • N • • • NKT (+)

The strain (B/Yamagata/16/88) was used as a reference for comparison. The amino acid substitution in different sites is shown. Dots represent amino acid similar to the reference strain while the dash denotes deletion. Shaded amino acids indicate identical residues at these sites with B/New york/01/08 compared to the reference isolate and vaccine strains.

a 2004~2006 vaccine strain

b 2007~2008 vaccine strain

Table 3.
Amino acid variations of NA region from Influenza B isolates in Cheongju, Korea compared with B/Yamagata/16/88
Virus strain(s) Amino acid at position
3 42 49 50 59 68 71 76 88 107 125 186 193 202 219
B/Yamagata/16/88 P P K V N T M S Q R K R L I N
B/Shanghai/361/02a • T T M • • V P P K K K V L ▪
B/New york/01/08 • Q I M • A V P P K T K V L K
B/Cheongju/411/08 H Q I M S A V P P K T K V L K
B/Cheongju/437/08 H Q I M S A V P P K T K V L K
B/Malaysia/2506/04b • S T M • • V • P K K • • • •
Virus strain(s) Amino acid at position
235 244 250 272 329 340 342 345 373 378 389 392 396 404 436
B/Yamagata/16/88 D S K E D N D R E E T D P K E
B/Shanghai/361/02a N • E K • N N S K G I ▪ • E T
B/New york/01/08 • P E K N D • S K G • ▪ • E T
B/Cheongju/411/08 • P E K N D • S K G • E • E T
B/Cheongju/437/08 • P E K N D • S K G • E • E T
B/Malaysia/2506/04b N • E K • N • • • G A • F E •

The strain (B/Yamagata/16/88) was used as a reference for comparison. The amino acid substitution in different sites is shown. Dots represent amino acid similar to the reference strain while the dash denotes deletion. Shaded amino acids indicate identical residues at these sites with B/New york/01/08 compared to the reference isolate and vaccine strains.

a 2004~2006 vaccine strain

b 2007~2008 vaccine strain

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