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.

Emergence of a new genetic lineage of Newcastle disease virus in West and Central Africa--implications for diagnosis and control.

Newcastle disease (ND) is an OIE listed disease caused by virulent avian paramyxovirus type 1 (APMV-1) strains, which affect many species of birds and may cause severe economic losses in the poultry sector. The disease has been officially and unofficially reported in many African countries and still remains the main poultry disease in commercial and rural chickens of Africa. Unfortunately, virological and epidemiological information concerning ND strains circulating in the Western and Central regions of Africa is extremely scarce. In the present study, sequence analysis, pathotyping and detailed genetic characterization of virulent ND strains detected in rural poultry in West and Central Africa revealed the circulation of a new genetic lineage, distinguishable from the lineages described in the Eastern and Southern parts of the continent. Several mismatches were observed in the segment of the matrix gene targeted by the primers and probe designed for the molecular detection of APMV-1, which were responsible for the false negative results in the diagnostic test conducted. Furthermore, deduced amino acid sequences of the two major antigens eliciting a protective immune response (F and HN glycoprotein) revealed protein similarities <90% if compared to some common vaccine strains. Distinct mutations located in the neutralizing epitopes were revealed, indicating the need for detailed assessment of the efficacy of the current vaccines and vaccination practices in Africa. The present investigation provides important information on the epidemiology, diagnosis and control of NDV in Africa and highlights the importance of supporting surveillance in developing countries for transboundary animal diseases.

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.

Co-circulation of distinct genetic lineages of astroviruses in turkeys and guinea fowl.

Viruses belonging to the genus Astrovirus have been increasingly associated with enteritis in mammalian and avian species, including turkeys. More recently, astroviruses have also been detected in diseased guinea fowl. In turkeys, two genetically distinct types of astrovirus have been reported, namely turkey astrovirus 1 and 2 (TAstV1 and TAstV2). The prevalence and the pathogenesis of astrovirus infections in this species is currently unknown, with the exception of data generated in the USA. In the present report, we have demonstrated the co-circulation of distinct genetic lineages of astroviruses infecting turkeys and guinea fowl. The predominant lineage infecting turkeys is genetically related to the American TAstV2, but genetic variability within this lineage was demonstrated. Other isolates appeared to be either TastV1-related or unrelated to any other known isolate. Astroviruses infecting guinea fowl were more closely related to TAstV2, and interspecies transmissions between turkeys and guinea fowl is suggested as a possible mechanism by which these viruses may have evolved, based on the genetic data available. This investigation provides genetic and epidemiological information contributing to a better understanding of enteric viral infections in turkeys and guinea fowl.