title: Detection of D151G/N mutations in the neuraminidase gene of influenza A (H3N2) viruses by real-time RT-PCR allelic discrimination assay.
abstract: Single nucleotide polymorphisms (SNPs) at D151 position of neuraminidase (NA) gene of influenza A (H3N2) virus has been associated with drug resistance and increased binding affinity. NA-D151G/N-substitutions of influenza A (H3N2) viruses are frequently induced and selected by culturing in Madin-Darby canine kidney (MDCK) cell lines. It is important to consider and exclude D151G/N mutants after isolation of influenza virus in MDCK cell line; since, the substitutions can highly influence the results of experimental research. The study aims to develop an allelic discrimination real-time reverse transcriptase polymerase chain reaction (RT-PCR) for the screening of D151G/N mutants. Thirty-six influenza A (H3N2) virus isolates were included and screened for D151G/N mutants using allelic discrimination assay. Out of the 36 isolates, 11 isolates (30.5%) were detected as heterozygous for D and G/N substitutions. Twenty-one (58.3%) isolates were identified as homozygous wild type and four isolates (11.1%) were undetermined. Isolates with substitutions at D151 position were sequenced by Sanger sequencing method. The present study demonstrates a rapid and convenient method for primary screening of the mutation after culturing of the influenza virus in MDCK cell lines in order to avoid potential misinterpretations of results and improve the quality of experimental research.
authors.affiliation: Manipal Centre for Virus Research, Regional Reference Laboratory for Influenza Virus and ICMR Grade-I Virus Diagnostic Laboratory, Manipal University, Manipal, Karnataka, India.
authors.forename: Vahid Rajabali
authors.initials: VR
authors.lastname: Zadeh
authors.affiliation: Manipal Centre for Virus Research, Regional Reference Laboratory for Influenza Virus and ICMR Grade-I Virus Diagnostic Laboratory, Manipal University, Manipal, Karnataka, India.
authors.forename: Anitha
authors.initials: A
authors.lastname: Jagadesh
authors.affiliation: Manipal Centre for Virus Research, Regional Reference Laboratory for Influenza Virus and ICMR Grade-I Virus Diagnostic Laboratory, Manipal University, Manipal, Karnataka, India.
authors.forename: Anjana
authors.initials: A
authors.lastname: Krishnan
authors.affiliation: Manipal Centre for Virus Research, Regional Reference Laboratory for Influenza Virus and ICMR Grade-I Virus Diagnostic Laboratory, Manipal University, Manipal, Karnataka, India.
authors.forename: Govindakarnavar
authors.initials: G
authors.lastname: Arunkumar
mesh_terms: D000483:Alleles; D000818:Animals; D004285:Dogs; D024882:Drug Resistance, Viral; D006801:Humans; D053122:Influenza A Virus, H3N2 Subtype / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D007251:Influenza, Human / Q000821:virology; D061985:Madin Darby Canine Kidney Cells; D009154:Mutation; D009439:Neuraminidase / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA
chemical_list: D009439:Neuraminidase
keywords: MDCK; SNP; allelic discrimination; influenza virus; neuraminidase gene; real-time PCR