Differentiation of influenza B virus lineages Yamagata and Victoria by real-time PCR.
ABSTRACT 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.
- SourceAvailable from: gmo-qpcr-analysis.com[show abstract] [hide abstract]
ABSTRACT: Use of PCR in the field of molecular diagnostics has increased to the point where it is now accepted as the standard method for detecting nucleic acids from a number of sample and microbial types. However, conventional PCR was already an essential tool in the research laboratory. Real-time PCR has catalysed wider acceptance of PCR because it is more rapid, sensitive and reproducible, while the risk of carryover contamination is minimised. There is an increasing number of chemistries which are used to detect PCR products as they accumulate within a closed reaction vessel during real-time PCR. These include the non-specific DNA-binding fluorophores and the specific, fluorophore-labelled oligonucleotide probes, some of which will be discussed in detail. It is not only the technology that has changed with the introduction of real-time PCR. Accompanying changes have occurred in the traditional terminology of PCR, and these changes will be highlighted as they occur. Factors that have restricted the development of multiplex real-time PCR, as well as the role of real-time PCR in the quantitation and genotyping of the microbial causes of infectious disease, will also be discussed. Because the amplification hardware and the fluorogenic detection chemistries have evolved rapidly, this review aims to update the scientist on the current state of the art. Additionally, the advantages, limitations and general background of real-time PCR technology will be reviewed in the context of the microbiology laboratory.Clinical Microbiology and Infection 04/2004; 10(3):190-212. · 4.58 Impact Factor
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ABSTRACT: A fluorogenic PCR-based method (TaqMan-PCR) was developed for typing and subtyping of influenza virus genomes in clinical specimens. The TaqMan-PCR employs a probe technology that exploits the endogenous 5'-3' nuclease activity of the Taq DNA polymerase to allow direct detection of the amplicon by release of a fluorescent reporter during the PCR. Therefore, post-PCR analysis is avoided since hybridization with the fluorogenic probe and quantification of the amplified product is performed simultaneously during PCR cycling. The specificity of the method was evaluated on 86 influenza A (25 H1N1 and 61 H3N2) and 49 influenza B virus reference strains and isolates. The sensitivity of the technique was found to be at the level of 0.1 50% tissue culture infective dose. This TaqMan-PCR was applied prospectively to surveillance work by community-based sampling in Germany during the last two influenza seasons. Seven hundred five throat swabs were analyzed during the winter of 1997-1998. A total of 195 of 705 samples (28%) were positive by PCR. Influenza viruses could be isolated from 125 specimens (18%). During the 1998-1999 season, 1,840 respiratory samples were received. Influenza viruses were isolated from 281 specimens (15%) out of 525 throat swabs (29%) which were positive for influenza A or B virus by TaqMan-PCR. Further differentiation of influenza A virus-positive swabs revealed an intensive circulation of the subtype H3N2 during both seasons, 1997-1998 and 1998-1999. The TaqMan-PCR was much more sensitive than culture and revealed an excellent correlation for typing and subtyping of influenza viruses when samples were positive by both methods.Journal of Clinical Microbiology 05/2000; 38(4):1552-8. · 4.07 Impact Factor
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ABSTRACT: Adenoviruses (AdVs) can cause serious disease in immunosuppressed patients, particularly those undergoing allogeneic stem cell transplantation. A method for virus quantification in clinical specimens is essential for monitoring patient adenoviral loads and evaluating new therapeutic approaches. We developed a PCR-based assay that combines detection and genotyping of human AdVs, targeting a highly conserved region of the adenoviral genome coding for the DNA polymerase (AdV DPol PCR). We tested the diagnostic applicability of this PCR-based assay by analyzing 159 clinical specimens from children with respiratory disease and comparing the results with those obtained by nested PCR analysis. The PCR assay detected all currently known AdV serotypes, with a detection limit of approximately 10 genome equivalents per reaction for 49 of 51 serotypes. No cross-reactivity to human DNA or other DNA viruses was observed. In addition, genotyping of PCR-positive samples was achieved within minutes by fluorescence curve melting analysis in a LightCycler instrument using 6 pairs of hybridization probes, each specific for a single AdV species. Results for clinical specimens were in good concordance with those obtained by nested PCR. The presented assay is a suitable tool for the detection and genotyping of human AdVs in clinical samples.Clinical Chemistry 09/2005; 51(8):1365-73. · 7.15 Impact Factor
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
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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-assayPlasmids5 ? 105
5 ? 103
5 ? 101
Cultured virus High
InterassayPlasmids5 ? 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.
1426NOTESJ. CLIN. MICROBIOL.
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