An SYBR Green-based real-time RT-PCR assay for the detection of H5 hemagglutinin subtype avian influenza virus.

Increasing diversity among H5 hemagglutinin (HA) subtype avian influenza (AI) viruses has resulted in the need of novel sensitive and specific molecular assays. In this study, an SYBR Green-based real-time reverse transcription-PCR (RRT-PCR) assay was developed for the detection of H5 subtype AI virus. Sequence analysis of the Mexican lineage H5N2 isolates (subgroup B) revealed several mismatches in the primer/hydrolysis probe set reported in the commonly used RRT-PCR assay for the detection of H5 North American lineage. The present assay was designed to circumvent the challenge that these viruses represent for the specific detection of H5 subtype AI viruses. This RRT-PCR assay successfully detected a range of different H5 subtype AI strains from both Eurasian and North American lineages representing different avian H5 HA clades from diverse geographical locations. The sensitivity of the present method was determined by using in vitro-transcribed RNA and 10-fold serial dilutions of titrated AI viruses. High sensitivity levels were obtained, with limits of detection of 10(0) 50% egg infectious dose (EID50)/mL and 4.2 gene copies/µl. The linear ranges of the assay span within 10(6)-10(0) EID50/mL and 10(6)-10(0) gene copies/µl. The results obtained from this method were directly compared with those of the H5 RRT-PCR assay recommended by the OIE. The comparison was performed with 110 tracheal and cloacal swabs from various bird species collected during field and laboratory investigations in Eurasia and Africa in 2006 and 2008 and showed 100% agreement. This assay is recommended as an alternative method, also allowing a 'double check' approach detection, to be use mainly in outbreak scenarios with higher risk of poultry infections by Central American/Caribbean H5 AI viruses.

Resistance of turkeys to experimental infection with an early 2009 Italian human influenza A(H1N1)v virus isolate.

We performed an experimental infection of 21- and 70-day-old meat turkeys with an early human isolate of the 2009 pandemic H1N1 influenza virus exhibiting an alpha-2,3 receptor binding profile. Virus was not recovered by molecular or conventional methods from blood, tracheal and cloacal swabs, lungs, intestine or muscle tissue. Seroconversion was detected in a limited number of birds with the homologous antigen only. Our findings suggest that in its present form, the pandemic H1N1 influenza virus is not likely to be transmitted to meat turkeys and does therefore not represent an animal health or food safety issue for this species.