Article

Differentiation of influenza B virus lineages Yamagata and Victoria by real-time PCR.

Robert Koch Institut, FG17 Influenza/Respiratorische Viren, Nordufer 20, 13353 Berlin, Germany.
Journal of clinical microbiology (Impact Factor: 4.23). 04/2010; 48(4):1425-7. DOI: 10.1128/JCM.02116-09
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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.

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Available from: Barbara Biere, Sep 23, 2014
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    • "However, rRT-PCR assay for universally detecting influenza B viruses, whose target region was the nonstructural protein (NS) gene (B/NS rRT-PCR; the sequences of the primers and probe are listed in Table 1), was not able to discriminate between the B/Victoria and B/Yamagata lineages. An rRT-PCR assay for discriminating the B/Victoria and B/Yamagata lineages was first reported by Biere et al. (2010). However , the sequences of the forward primer and the two probes used in the study of Biere et al. had low homology with the recent influenza B viruses circulating in Japan. "
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    ABSTRACT: Since the late 1980s, two genetically and antigenically distinct lineages of influenza B virus, namely, B/Victoria/2/87-like (B/Victoria) and B/Yamagata/16/88-like (B/Yamagata), have co-circulated. In this study, one-step real-time reverse transcription-PCR (rRT-PCR) assays were developed to differentiate B/Victoria and B/Yamagata lineages. The assays were evaluated using in vitro transcribed control RNA, isolated viruses, and other respiratory pathogenic viruses, and were shown to have high sensitivity, good linearity (R(2)=0.99), and high specificity. Using the developed rRT-PCR assays, 169 clinical specimens collected between 2010 and 2013 were then tested, resulting in the identification of 20 clinical specimens as positive for influenza B virus. Of these, 14 and 6 samples were identified as positive for the B/Victoria and B/Yamagata lineages, respectively, whereas 149 samples were negative for the influenza B virus. The rRT-PCR assays were also examined using 20 clinical isolates from 20 influenza B virus-positive specimens, revealing that there was no discrepancy between the results from the rRT-PCR assays and the hemagglutination inhibition (HI) test, with the exception that one clinical isolate with different antigenicity could not be discriminated by the HI test. The present results suggest that these highly sensitive and specific assays are useful not only for diagnosing influenza viruses but also for their surveillance.
    Journal of virological methods 05/2014; 205. DOI:10.1016/j.jviromet.2014.04.016 · 1.88 Impact Factor
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    • "Viral genomic RNA was extracted from 400 í µí¼‡L of the original samples using RTP DNA/RNA Virus Mini Kit (Invitek, Germany) according to manufacturers' instructions. Realtime PCR assays were carried out to detect HMPV and other respiratory viruses including influenza A and B viruses [32] [33], respiratory syncytial virus (RSV A and B) [34], and adenovirus (AdV) [35]. A two-step RT-PCR method was used for the detection and amplification of HMPV fusion gene [36]. "
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    ABSTRACT: Introduction. Since 2001, when Human metapneumovirus (HMPV) was isolated in the Netherlands, the virus has been detected in several continents. Although reports have confirmed the prevalence of HMPV worldwide, data from Egypt remain limited. HMPV plays an important role in respiratory tract infections in individuals of all ages particularly in children. This study was aimed at estimating the prevalence of HMPV in patients with community-acquired lower respiratory infection in Upper Egypt and characterizing the circulating Egyptian HMPV strains for the first time. Materials and Methods. From 2005 to 2008, respiratory samples from 520 patients were analyzed for the presence of HMPV by real-time RT-PCR. Molecular and phylogenetic analyses were performed on partial fusion gene sequences of HMPV-positive patients. Results. HMPV-positive patients were detected in 2007-2008. The overall infection rate was 4%, while 57% of the patients were children. Sequence analysis demonstrated circulation of subgroup B viruses with predominance of lineage B2. Nucleotide sequence identity within lineage B1 was 98.8%-99.7% and higher than that in lineage B2 (94.3%-100%). Three new amino acid substitutions (T223N, R229K, and D280N) of lineage B2 were observed. Conclusion. HMPV is a major viral pathogen in the Egyptian population especially in children. During 2007-2008, predominantly HMPV B2 circulated in Upper Egypt.
    International Journal of Microbiology 01/2014; 2014:290793. DOI:10.1155/2014/290793
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    • "RT- PCR and RFLP) for influenza A and B characterization and further sub-typing of influenza A viruses are in use throughout the globe, most of them allowing virus detection directly from clinical specimens (Ellis et al., 1997; Wang and Taubenberger, 2010). However, besides HI and nucleic acid sequencing, only one molecular protocol for influenza B lineage differentiation, based on real time RT-PCR, can be found in the literature at the time of this writing (Biere et al., 2010). In this study, a PCR–DGGE protocol was proposed as an alternative approach for rapid influenza B lineage differentiation, for laboratories without access to real time or nucleic acid sequencing facilities. "
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    ABSTRACT: In this study, a PCR-DGGE protocol was standardized in order to distinguish Victoria and Yamagata influenza B lineages directly from clinical samples. After routine multiplex PCR characterization, amplicons of the haemagglutinin gene bearing a 40bp-length GC clamp were generated by nested-PCR and analyzed by electrophoresis in 6% polyacrylamide gel with a 25-45% urea-formamide gradient. The results showed a perfect correlation between DGGE and phylogenetic analyses for all compared samples, besides some distinct profiles in Victoria and Yamagata groups that could be used to infer variability inside these groups. In summary, this DGGE protocol for the haemagglutinin gene is rapid, useful and efficient, being an alternative for discrimination between the influenza B lineages.
    Journal of virological methods 11/2011; 179(1):212-6. DOI:10.1016/j.jviromet.2011.10.018 · 1.88 Impact Factor
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