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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. © 2013 Elsevier B.V.

A highly sensitive and specific one-step multiplex RT-PCR assay has been developed and standardised for the simultaneous and differential detection of the most important vesicular viruses affecting livestock foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), and vesicular stomatitis virus (VSV). The method uses three primer sets, each one specific for the corresponding virus, selected to detect of all serotypes of FMD and VS. The detection range was confirmed by examination of a collection of 31 isolates of the three target viruses. The specificity of the assay was also demonstrated by testing other related viruses, uninfected cell line cultures and healthy pig tissues. The testing of blood and serum samples from animals infected experimentally proved that the method can be useful for early diagnosis of the diseases, even before the first vesicular lesions are visualized in the infected pigs. An assessment of the performance of the multiplex RT-PCR was carried out using a panel of more than 100 samples from animals infected experimentally, showing the suitability of the method for a rapid (less than 6 h), sensitive and specific differential diagnosis in clinical samples. Additionally, a uniplex RT-PCR for VSV, that amplifies the two viral serotypes, was also developed and tested as a rapid tool for the diagnosis of this vesicular disease. © 2007 Elsevier B.V. All rights reserved.