JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2010, p. 1425–1427
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 48, No. 4
Differentiation of Influenza B Virus Lineages Yamagata and
Victoria by Real-Time PCR?
Barbara Biere,* Bettina Bauer, and Brunhilde Schweiger
Robert Koch Institut, FG17 Influenza/Respiratorische Viren, Nordufer 20, 13353 Berlin, Germany
Received 29 October 2009/Returned for modification 8 December 2009/Accepted 20 January 2010
Since the 1970s, influenza B viruses have diverged into two antigenically distinct virus lineages called the
Yamagata and Victoria lineages. We present the first real-time PCR assay for virus lineage differentiation to
supplement classical antigenic analyses. The assay was successfully applied to 310 primary samples collected
in Germany from 2007 to 2009.
Influenza viruses are members of the family Orthomyxoviri-
dae and are divided into three genera, A, B, and C (8). Influ-
enza A and B viruses are most relevant clinically, since they
cause severe respiratory infections in humans (2). While influ-
enza A viruses comprise a large group of different subtypes (8),
influenza B viruses formed a homogenous group and started to
diverge into two antigenically distinguishable lineages only in
the 1970s (3, 4, 6). These virus lineages were named after their
first representatives, B/Victoria/2/87 and B/Yamagata/16/88, as
the Victoria and Yamagata lineages (6). Today, the antigenic
differences between the lineages allow their differentiation by
hemagglutination inhibition testing (HIT) by using specific im-
mune sera raised against contemporary strains of either lin-
eage. However, HIT is a time-consuming and tedious process
and needs virus isolation as a prerequisite. In contrast, PCR is
well known to be a fast, specific, and sensitive diagnostic
method, and furthermore, real-time PCR reduces the risk of
carryover contamination and allows large-scale diagnostics (5).
However, to date, there has been no real-time PCR assay
described that enables the differentiation of influenza B vi-
ruses, which would greatly speed up and thus improve influ-
enza virus surveillance. We therefore present an assay that not
only amplifies viruses of both lineages but also discriminates
between them by the application of two differently labeled
minor-groove binder (MGB) probes, with either one being
specific for one lineage.
The target region of the assay was chosen from an alignment
with recent influenza B virus hemagglutinin (HA) database
sequences (from the years 2000 to 2008). The 81-bp amplicon
comprises a 13-bp stretch that differs in 6 positions between the
two lineages. The stability of the characteristic nucleotide
changes was confirmed by an alignment comprising all avail-
able influenza B virus hemagglutinin database sequences
(1,622 sequences, from the years 1954 to 2008). The distinctive
nucleotides have been stable from the late 1990s until today, so
nucleotide changes are not impossible but are unlikely to occur
in the near future. Thus, an MGB probe was designed for
either lineage targeting this 13-bp stretch. By the application of
both probes with different color labels (6-carboxyfluorescein
[FAM] and VIC) in a single PCR, both virus lineages can be
detected and discriminated simultaneously, as only one of the
two probes will give a fluorescence signal.
Reaction conditions were established for the LightCycler
480 system in a total reaction mixture volume of 25 ?l con-
taining 1? PCR buffer, 5 mM MgCl2, 1.25 ?M deoxynucleo-
side triphosphate (dNTP) (Invitrogen) with dUTP (GE
Healthcare, Great Britain), 0.5 U Platinum Taq polymerase
(Invitrogen), 900 nM forward primer F432 (5?-ACCCTACAR
AMTTGGAACYTCAGG-3?), 600 nM reverse primer R479
(5?-ACAGCCCAAGCCATTGTTG-3?), 150 nM Yamagata
probe MGB437 (5?-FAM–AATCCGMTYTTACTGGTAG–
MGB-3?), 100 nM Victoria probe MGB470 (5?-VIC–ATCCG
TTTCCATTGGTAA–MGB-3?), and 3 ?l of template cDNA.
Cycling conditions were 5 min at 95°C, followed by 45 cycles of
15 s at 95°C and 30 s at 60°C.
The assay was evaluated by using two plasmids that were
cloned according to routine procedures (1) and contained 610
and 613 bp of the hemagglutinin genes of B/Bayern/7/08 (plas-
mid pYam) and B/Berlin/38/08 (plasmid pVic), two contem-
porary German isolates representing the Yamagata and Vic-
toria lineages, respectively. Thus, the complete primer- and
probe-binding regions represent the original sequences of
these two isolates. Amplification of 10-fold serial dilutions of
each plasmid in ? DNA (1 ng/?l) revealed a linear detection
range from 107to 102genome equivalents per reaction with a
correlation (R2) of ?0.998 and slopes of ?3.32 (pYam) and
?3.33 (pVic) (Fig. 1A), resembling a PCR efficiency of 1 (E ?
10?1/slope? 1). We performed a probit analysis as a model of
nonlinear regression that indicated a 95% detection probabil-
ity of 24.4 genome equivalents per reaction for plasmid pYam
and 12.4 genome equivalents per reaction for pVic (Fig. 1B).
Additionally, from virus culture material of the corresponding
virus isolates B/Bayern/7/08 (Yamagata) and B/Berlin/38/08
(Victoria), the 95% detection probabilities were determined to
be 1.3 ? 10?5and 3.8 ? 10?5HA units per reaction, respec-
tively. The overall variability was assessed by the repeated
examination of three different plasmid copy numbers as well as
virus culture material with a high, medium, or low virus load.
The standard deviations of threshold cycle (CT) values were
found to be very low and were comparable for Yamagata and
Victoria viruses and plasmids (Table 1). We found no cross-
* Corresponding author. Mailing address: Nationales Referenzzen-
trum fu ¨r Influenza, FG 17 Influenza/Respiratorische Viren, Robert
Koch Institut, Nordufer 20, 13353 Berlin, Germany. Phone: 49 3018
754 2383. Fax: 49 3018 754 2699. E-mail: email@example.com.
?Published ahead of print on 27 January 2010.
reactivity with DNA/cDNA of isolates from seasonal influenza
A virus subtypes H1N1 and H3N2; pandemic influenza
A/H1N1 virus; respiratory syncytial viruses A and B; adenovi-
rus serotypes 2, 3, and 4; human metapneumovirus; parainflu-
enza viruses 1, 2, and 3; coxsackievirus; and rhinovirus as well
as human DNA from swab samples.
Finally, to confirm the applicability of the assay to clinical
diagnostics, we examined 310 influenza B virus-positive pri-
mary samples from the 2007-2008 and 2008-2009 influenza
seasons. All samples were taken from German patients pre-
senting with influenza-like illness and successfully under-
went HIT after virus isolation on MDCK2 cells. The nasal
and throat swabs were washed in minimal essential medium
(MEM) cell culture medium immediately after arrival. RNA
was extracted by using either the RTP DNA/RNA virus
MiniKit (Invitek) or the MagAttract viral RNA M48 kit
(Qiagen) according to the manufacturer’s suggestions.
cDNA was synthesized from 25 ?l of RNA by applying
Moloney murine leukemia virus (M-MLV) reverse trans-
criptase (Invitrogen) and random hexamer primers as de-
scribed elsewhere previously (7). Residual RNA was stored
at ?80°C until further use.
By applying the presented assay, viruses were amplified from
all 310 primary samples with CTvalues between 22 and 37. All
samples were genetically identified as Yamagata or Victoria
lineage viruses in concordance with HIT results. The 310 pri-
mary samples comprised 185 Yamagata and 3 Victoria lineage
viruses from the 2007-2008 season as well as 120 Victoria and
2 Yamagata lineage viruses from the 2008-2009 season. Since
the assay’s introduction into our diagnostic routine in February
2009, it has been run on approximately 5,000 samples, and to
our knowledge, no false-positive or false-negative results have
In summary, we present the first real-time PCR assay for the
differentiation of influenza B viruses. This assay speeds up
virus lineage identification in clinical specimens considerably
and will therefore help to improve the surveillance of influenza
B viruses. Furthermore, it will enable a timely recognition of
the circulating B virus lineage during influenza seasons and will
thus allow short-term decisions on patient care, e.g., in the case
of a nonmatching vaccine, as well as the early onset of on-time
epidemiological examinations, including WHO decisions on
We thank Julia Hinzmann and Madlen Sohn for excellent technical
1. Chmielewicz, B., A. Nitsche, B. Schweiger, and H. Ellerbrok. 2005. Develop-
ment of a PCR-based assay for detection, quantification, and genotyping of
human adenoviruses. Clin. Chem. 51:1365–1373.
2. Hampson, A. W., and J. S. Mackenzie. 2006. The influenza viruses. Med. J.
3. Hay, A. J., V. Gregory, A. R. Douglas, and Y. P. Lin. 2001. The evolution of
human influenza viruses. Philos. Trans. R. Soc. Lond. B Biol. Sci. 356:1861–1870.
4. Kanegae, Y., S. Sugita, A. Endo, M. Ishida, S. Senya, K. Osako, K. Nerome,
and A. Oya. 1990. Evolutionary pattern of the hemagglutinin gene of influenza
FIG. 1. PCR assay validation. (A) Mean CTvalues (double reactions) of plasmid dilutions containing 107to 102genome equivalents of pYam
and pVic were plotted against the cycle number. The slope and correlation (R2) are indicated. (B) Probit analyses were performed by examination
of plasmid dilutions containing 100 to 0.1 genome equivalents of pYam and pVic in 10-fold reactions. Results were analyzed by using SPSS 17.0
TABLE 1. PCR assay validation: detection variabilitya
(no. of genome
SD of CTvalue
Intra-assay Plasmids5 ? 105
5 ? 103
5 ? 101
Interassay Plasmids5 ? 105
5 ? 103
5 ? 101
aVariability runs were performed by examination of pYam and pVic plasmid
dilutions (5 ? 105, 5 ? 103, and 5 ? 101genome equivalents per reaction) as well
as cultured virus material with a high (6.67 ? 108genome copies/ml), medium
(6.67 ? 106genome copies/ml), or low (6.67 ? 104genome copies/ml) virus load.
Intra-assay variability was tested in sextuplicate reactions. Interassay variability
was determined by 2-fold examinations of duplicate reactions with the inclusion
of data from the intra-assay variability run (total, 3-fold examination). The
standard deviations (SD) of obtained CTvalues are listed.
1426NOTES J. CLIN. MICROBIOL.
B viruses isolated in Japan: cocirculating lineages in the same epidemic Download full-text
season. J. Virol. 64:2860–2865.
5. Mackay, I. M. 2004. Real-time PCR in the microbiology laboratory. Clin.
Microbiol. Infect. 10:190–212.
6. Rota, P. A., T. R. Wallis, M. W. Harmon, J. S. Rota, A. P. Kendal, and K.
Nerome. 1990. Cocirculation of two distinct evolutionary lineages of influenza
type B virus since 1983. Virology 175:59–68.
7. Schweiger, B., I. Zadow, R. Heckler, H. Timm, and G. Pauli. 2000. Application
of a fluorogenic PCR assay for typing and subtyping of influenza viruses in
respiratory samples. J. Clin. Microbiol. 38:1552–1558.
8. Wright, P. F., G. Neumann, and Y. Kawaoka. 2007. Orthomyxoviruses, p.
1691–1740. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A.
Martin, B. Roizman, and S. E. Straus (ed.), Fields virology, 5th ed. Lippincott
Williams & Wilkins, Philadelphia, PA.
VOL. 48, 2010 NOTES1427