Giuseppe
Emeritus
Antimicrob Agents Chemother. 2009 Jan 5. [Epub ahead of print]
Detection of molecular markers of antiviral resistance in influenza A (H5N1) viruses using pyrosequencing method.
Deyde VM, Nguyen T, Bright RA, Balish A, Shu B, Lindstrom S, Klimov AI, Gubareva LV. - Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta GA, USA; National Centre for Veterinary Diagnostics, DAH11-78th lane - Giai Phong str Phuong Mai - Dong Da - Hanoi, Vietnam.
Resistance to antiviral drugs in influenza viruses can emerge following medication or may result from natural variation.
Two classes of anti-influenza 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 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 to NA inhibitors in A(H5N1) viruses. The residues L26, V27, A30, S31, and G34 in the M2 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 RT-PCR primers was designed to allow amplification of an approximately 600nt-long amplicon of the NA gene 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 Ct values ranging from 32 to 37 determined by real-time RT-PCR.
The pyosequencing approach may provide a valuable tool in 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.
PMID: 19124660 [PubMed - as supplied by publisher
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Detection of molecular markers of antiviral resistance in influenza A (H5N1) viruses using pyrosequencing method.
Deyde VM, Nguyen T, Bright RA, Balish A, Shu B, Lindstrom S, Klimov AI, Gubareva LV. - Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta GA, USA; National Centre for Veterinary Diagnostics, DAH11-78th lane - Giai Phong str Phuong Mai - Dong Da - Hanoi, Vietnam.
Resistance to antiviral drugs in influenza viruses can emerge following medication or may result from natural variation.
Two classes of anti-influenza 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 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 to NA inhibitors in A(H5N1) viruses. The residues L26, V27, A30, S31, and G34 in the M2 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 RT-PCR primers was designed to allow amplification of an approximately 600nt-long amplicon of the NA gene 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 Ct values ranging from 32 to 37 determined by real-time RT-PCR.
The pyosequencing approach may provide a valuable tool in 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.
PMID: 19124660 [PubMed - as supplied by publisher
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