PMID- 19124660
OWN - NLM
STAT- MEDLINE
DCOM- 20090519
LR  - 20211020
IS  - 1098-6596 (Electronic)
IS  - 0066-4804 (Print)
IS  - 0066-4804 (Linking)
VI  - 53
IP  - 3
DP  - 2009 Mar
TI  - Detection of molecular markers of antiviral resistance in influenza A (H5N1) 
      viruses using a pyrosequencing method.
PG  - 1039-47
LID - 10.1128/AAC.01446-08 [doi]
AB  - Resistance of influenza viruses to antiviral drugs can emerge following 
      medication or may result from natural variation. Two classes of anti-influenza 
      virus drugs targeting either the M2 protein (amantadine and rimantadine) or 
      neuraminidase (NA; oseltamivir and zanamivir) are currently licensed. These drugs 
      are expected to be important in controlling the early stages of a potential 
      pandemic. In the present study, we describe how a pyrosequencing method can be 
      used to rapidly detect established molecular markers of resistance to M2 blockers 
      and NA inhibitors in influenza A (H5N1) viruses. The residues L26, V27, A30, S31, 
      and G34 in the M2 protein were targeted for pyrosequencing. The NA residues for 
      pyrosequencing analysis included the established markers of drug resistance (H274 
      and N294), as well as residues of less certain relevance (V116, I117, Q136, K150, 
      and I222). A single pair of pyro-reverse transcription (RT)-PCR primers was 
      designed to allow amplification of an approximately 600-nucleotide-long amplicon 
      of the NA genes of H5N1 viruses from various clades/subclades associated with 
      infections in humans. The sensitivity of the assay was demonstrated by the 
      successful pyrosequencing of RNA extracted from samples of serially diluted 
      (10(-5) to 10(-7)) virus stocks with initial concentrations ranging from 10(5) to 
      10(8) PFU/ml. The markers of resistance were detected in samples with threshold 
      cycle values ranging from 32 to 37, as determined by real-time RT-PCR. The 
      pyrosequencing approach may provide a valuable tool for rapid detection of 
      markers of drug resistance in H5N1 viruses and facilitate the elucidation of the 
      role of such changes in natural and acquired drug resistance.
FAU - Deyde, Varough M
AU  - Deyde VM
AD  - Influenza Division, National Center for Immunization and Respiratory Diseases, 
      Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
FAU - Nguyen, Tung
AU  - Nguyen T
FAU - Bright, Rick A
AU  - Bright RA
FAU - Balish, Amanda
AU  - Balish A
FAU - Shu, Bo
AU  - Shu B
FAU - Lindstrom, Stephen
AU  - Lindstrom S
FAU - Klimov, Alexander I
AU  - Klimov AI
FAU - Gubareva, Larisa V
AU  - Gubareva LV
LA  - eng
PT  - Journal Article
DEP - 20090105
PL  - United States
TA  - Antimicrob Agents Chemother
JT  - Antimicrobial agents and chemotherapy
JID - 0315061
RN  - 0 (Antiviral Agents)
RN  - 0 (Genetic Markers)
RN  - 0 (RNA, Viral)
RN  - 0T2EF4JQTU (Rimantadine)
RN  - 20O93L6F9H (Oseltamivir)
RN  - BF4C9Z1J53 (Amantadine)
RN  - EC 3.2.1.18 (Neuraminidase)
RN  - L6O3XI777I (Zanamivir)
SB  - IM
MH  - Amantadine/pharmacology
MH  - Animals
MH  - Antiviral Agents/pharmacology
MH  - Birds
MH  - Drug Resistance, Viral/drug effects/*genetics
MH  - *Genetic Markers
MH  - Humans
MH  - Influenza A Virus, H5N1 Subtype/*genetics/metabolism
MH  - Influenza in Birds/drug therapy/virology
MH  - Influenza, Human/drug therapy/virology
MH  - Neuraminidase/pharmacology
MH  - Oseltamivir/pharmacology
MH  - RNA, Viral/genetics
MH  - Reverse Transcriptase Polymerase Chain Reaction/methods
MH  - Rimantadine/pharmacology
MH  - Sensitivity and Specificity
MH  - Sequence Analysis, RNA/*methods
MH  - Zanamivir/pharmacology
PMC - PMC2650582
EDAT- 2009/01/07 09:00
MHDA- 2009/05/20 09:00
PMCR- 2009/09/01
CRDT- 2009/01/07 09:00
PHST- 2009/01/07 09:00 [entrez]
PHST- 2009/01/07 09:00 [pubmed]
PHST- 2009/05/20 09:00 [medline]
PHST- 2009/09/01 00:00 [pmc-release]
AID - AAC.01446-08 [pii]
AID - 1446-08 [pii]
AID - 10.1128/AAC.01446-08 [doi]
PST - ppublish
SO  - Antimicrob Agents Chemother. 2009 Mar;53(3):1039-47. doi: 10.1128/AAC.01446-08. 
      Epub 2009 Jan 5.