Dataset Information

Simultaneous Differentiation of the N1 to N9 Neuraminidase Subtypes of Avian Influenza Virus by a GeXP Analyzer-Based Multiplex Reverse Transcription PCR Assay.

ABSTRACT: To date, nine neuraminidase (NA) subtypes of avian influenza virus (AIV) have been identified in poultry and wild birds. Rapid and effective methods for differentiating these nine NA subtypes are needed. We developed and validated a rapid, sensitive, and robust method utilizing a GeXP analyzer-based multiplex RT-PCR assay and capillary electrophoresis for the simultaneous differentiation of the N1 to N9 subtypes in a single-tube reaction. Ten pairs of primers-nine subtype-specific pairs and one pan-AIV pair-were screened and used to establish the GeXP multiplex RT-PCR assay. A single subtype was detected using the developed GeXP assay; the N1 to N9 AIV subtypes individually generated two target peaks: the NA subtype-specific peak and the general AIV peak. Different concentrations of multiplexed subtypes were tested with this GeXP assay and the peaks of the corresponding NA subtypes were generated, suggesting that this GeXP assay is useful for identifying NA subtypes in mixed samples. Moreover, no peaks were generated for other important avian viruses, indicating negative results and validating the lack of cross-reactions between AIV subtypes and other avian pathogens. RNA templates synthesized through in vitro transcription were used to analyze the sensitivity of the assay; the limit of detection was 100 copies per reaction mixture. The results obtained from clinical samples using this GeXP method were consistent with the results of the neuraminidase inhibition (NI) test, and the ability of the GeXP assay to identify mixed infections was superior to amplicon sequencing of isolated viruses. In conclusion, this GeXP assay is proposed as a specific, sensitive, rapid, high-throughput, and versatile diagnostic tool for nine NA subtypes of AIV.

SUBMITTER: Luo S

PROVIDER: S-EPMC6568037 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Simultaneous Differentiation of the N1 to N9 Neuraminidase Subtypes of Avian Influenza Virus by a GeXP Analyzer-Based Multiplex Reverse Transcription PCR Assay.

Luo Sisi S Xie Zhixun Z Huang Jiaoling J Xie Zhiqin Z Xie Liji L Zhang Minxiu M Li Meng M Wang Sheng S Li Dan D Zeng Tingting T Zhang Yanfang Y Fan Qing Q Deng Xianwen X

Frontiers in microbiology 20190607

To date, nine neuraminidase (NA) subtypes of avian influenza virus (AIV) have been identified in poultry and wild birds. Rapid and effective methods for differentiating these nine NA subtypes are needed. We developed and validated a rapid, sensitive, and robust method utilizing a GeXP analyzer-based multiplex RT-PCR assay and capillary electrophoresis for the simultaneous differentiation of the N1 to N9 subtypes in a single-tube reaction. Ten pairs of primers-nine subtype-specific pairs and one ...[more]

PMID: 31231349

Similar Datasets

Project description:UnlabelledHuman infections with avian influenza viruses are a serious public health concern. The neuraminidase (NA) inhibitors (NAIs) are the frontline anti-influenza drugs and are the major option for treatment of newly emerging influenza. Therefore, it is essential to identify the molecular markers of NAI resistance among specific NA subtypes of avian influenza viruses to help guide clinical management. NAI-resistant substitutions in NA subtypes other than N1 and N2 have been poorly studied. Here, we identified NA amino acid substitutions associated with NAI resistance among influenza viruses of N3, N7, and N9 subtypes which have been associated with zoonotic transmission. We applied random mutagenesis and generated recombinant influenza viruses carrying single or double NA substitution(s) with seven internal genes from A/Puerto Rico/8/1934 (H1N1) virus. In a fluorescence-based NA inhibition assay, we identified three categories of NA substitutions associated with reduced inhibition by NAIs (oseltamivir, zanamivir, and peramivir): (i) novel subtype-specific substitutions in or near the enzyme catalytic site (R152W, A246T, and D293N, N2 numbering), (ii) subtype-independent substitutions (E119G/V and/or D and R292K), and (iii) substitutions previously reported in other subtypes (Q136K, I222M, and E276D). Our data show that although some markers of resistance are present across NA subtypes, other subtype-specific markers can only be determined empirically.ImportanceThe number of humans infected with avian influenza viruses is increasing, raising concerns of the emergence of avian influenza viruses resistant to neuraminidase (NA) inhibitors (NAIs). Since most studies have focused on NAI-resistance in human influenza viruses, we investigated the molecular changes in NA that could confer NAI resistance in avian viruses grown in immortalized monolayer cells, especially those of the N3, N7, and N9 subtypes, which have caused human infections. We identified not only numerous NAI-resistant substitutions previously reported in other NA subtypes but also several novel changes conferring reduced susceptibility to NAIs, which are subtype specific. The findings indicate that some resistance markers are common across NA subtypes, but other markers need to be determined empirically for each subtype. The study also implies that antiviral surveillance monitoring could play a critical role in the clinical management of influenza virus infection and an essential component of pandemic preparedness.

Dataset Information

Simultaneous Differentiation of the N1 to N9 Neuraminidase Subtypes of Avian Influenza Virus by a GeXP Analyzer-Based Multiplex Reverse Transcription PCR Assay.

Publications

Simultaneous Differentiation of the N1 to N9 Neuraminidase Subtypes of Avian Influenza Virus by a GeXP Analyzer-Based Multiplex Reverse Transcription PCR Assay.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets