title: Rapid molecular haemagglutinin subtyping of avian influenza isolates by specific real-time RT-PCR tests.
abstract: Sixteen haemagglutinin (HA) subtypes of avian influenza viruses (AIV) have been described to date. Rapid subtype identification of any AIV is of major interest because of the possible serious consequences for the poultry industry and even public health. Molecular techniques currently allow immediate accurate subtype characterisation prior to virus isolation. In this study, a set of fourteen specific real-time RT-PCR methods were developed and evaluated for AIV HA subtyping (H1-H4, H6-H8, H10-H16), H5 and H9 being excluded on the basis of the current validity of the European Union (EU) recommended specific assays. Specific primers and probes sets for each HA-subtype were designed to hybridise the largest isolates range within each single subtype, considering the Eurasian lineage as a major target. The robustness and general application of the 14 HA-subtype methods were verified by the analysis of 110 AIV isolates belonging to all 16 HA-subtypes, performed in different laboratories. The developed real-time RT-PCR assays proved to be highly specific and revealed suitable sensitivity, allowing direct HA-subtyping of clinical material. In summary, this study provides for the first time a panel of molecular tests using specific hydrolysis probes for rapid and complete AIV HA-subtype identification.
authors.affiliation: Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos, 28130 Madrid, Spain.
authors.forename: Maia
authors.initials: M
authors.lastname: Elizalde
authors.affiliation: Laboratorio Central de Veterinaria (LCV), Algete, Madrid, Spain.
authors.forename: Montserrat
authors.initials: M
authors.lastname: Agüero
authors.affiliation: Laboratorio Central de Veterinaria (LCV), Algete, Madrid, Spain.
authors.forename: Dolores
authors.initials: D
authors.lastname: Buitrago
authors.affiliation: Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos, 28130 Madrid, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Yuste
authors.affiliation: Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos, 28130 Madrid, Spain.
authors.forename: María Luisa
authors.initials: ML
authors.lastname: Arias
authors.affiliation: Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos, 28130 Madrid, Spain.
authors.forename: María Jesús
authors.initials: MJ
authors.lastname: Muñoz
authors.affiliation: Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (IZSLER), Via Bianchi 9, 25124 Brescia, Italy.
authors.forename: Davide
authors.initials: D
authors.lastname: Lelli
authors.affiliation: Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos, 28130 Madrid, Spain.
authors.forename: Elisa
authors.initials: E
authors.lastname: Pérez-Ramírez
authors.affiliation: Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (IZSLER), Via Bianchi 9, 25124 Brescia, Italy.
authors.forename: Ana María
authors.initials: AM
authors.lastname: Moreno-Martin
authors.affiliation: Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos, 28130 Madrid, Spain. Electronic address: fpinero@inia.es.
authors.forename: Jovita
authors.initials: J
authors.lastname: Fernández-Pinero
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D005062:European Union; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics*; D009980:Influenza A virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000821:virology*; D015345:Oligonucleotide Probes / Q000235:genetics; D011200:Poultry; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*
chemical_list: D017931:DNA Primers; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D015345:Oligonucleotide Probes
keywords: Avian influenza virus; Haemagglutinin subtyping; Real-time RT-PCR