Efficient and Informative Laboratory Testing for Rapid Confirmation of H5N1 (Clade 2.3.4.4) High-Pathogenicity Avian Influenza Outbreaks in the United Kingdom.

During the early stages of the UK 2021-2022 H5N1 high-pathogenicity avian influenza virus (HPAIV) epizootic in commercial poultry, 12 infected premises (IPs) were confirmed by four real-time reverse-transcription-polymerase chain reaction (RRT)-PCRs, which identified the viral subtype and pathotype. An assessment was undertaken to evaluate whether a large sample throughput would challenge laboratory capacity during an exceptionally large epizootic; hence, assay performance across our test portfolio was investigated. Statistical analysis of RRT-PCR swab testing supported it to be focused on a three-test approach, featuring the matrix (M)-gene, H5 HPAIV-specific (H5-HP) and N1 RRT-PCRs, which was successfully assessed at 29 subsequent commercial IPs. The absence of nucleotide mismatches in the primer/probe binding regions for the M-gene and limited mismatches for the H5-HP RRT-PCR underlined their high sensitivity. Although less sensitive, the N1 RRT-PCR remained effective at flock level. The analyses also guided successful surveillance testing of apparently healthy commercial ducks from at-risk premises, with pools of five oropharyngeal swabs tested by the H5-HP RRT-PCR to exclude evidence of infection. Serological testing at anseriform H5N1 HPAIV outbreaks, together with quantitative comparisons of oropharyngeal and cloacal shedding, provided epidemiological information concerning the chronology of initial H5N1 HPAIV incursion and onward spread within an IP.

Proceedings Paper-Avian Diseases 10th AI Symposium Issue Development and Application of Real-Time PCR Assays for Specific Detection of Contemporary Avian Influenza Virus Subtypes N5, N6, N7, N8, and N9.

Previously published NA subtype-specific real-time reverse-transcriptase PCRs (RRT-PCRs) were further validated for the detection of five avian influenza virus (AIV) NA subtypes, namely N5, N6, N7, N8, and N9. Testing of 30 AIV isolates of all nine NA subtypes informed the assay assessments, with the N5 and N9 RRT-PCRs retained as the original published assays while the N7 and N8 assays were modified in the primer-probe sequences to optimize detection of current threats. The preferred N6 RRT-PCR was either the original or the modified variant, depending on the specific H5N6 lineage. Clinical specimen (n = 137) testing revealed the ability of selected N5, N6, and N8 RRT-PCRs to sensitively detect clade 2.3.4.4b highly pathogenic AIV (HPAIV) infections due to H5N5, H5N6, and H5N8 subtypes, respectively, all originating from European poultry and wild bird cases during 2016-2018. Similar testing (n = 32 clinical specimens) also showed the ability of N7 and N9 RRT-PCRs to sensitively detect European H7N7 HPAIV and China-origin H7N9 low pathogenicity AIV infections, respectively.

Desarrollo y aplicación de ensayos de PCR en tiempo real para la detección específica de subtipos contemporáneos de influenza aviar Virus N5, N6, N7, N8 y N9. Métodos de transcripción reversa y PCR en tiempo real (RRT) específicos para subtipo específico de NA que fueron publicados anteriormente se validaron completamente para la detección de cinco subtipos de NA del virus de la influenza aviar (AIV), incluyendo N5, N6, N7, N8 y N9. El análisis de 30 aislamientos del virus de la influenza aviar de los nueve subtipos de NA proporcionaron información acerca de las evaluaciones de los ensayos, con los métodos de RRT-PCR N5 y N9 evaluados de acuerdo a los ensayos originales, mientras que los métodos N7 y N8 se modificaron en las secuencias del iniciador y de la sonda para optimizar la detección de los virus que constituyen amenazas actuales. Los métodos de RRT-PCR para el subtipo N6 preferido fueron tanto el dirigido al virus original o el dirigido a la variante modificada, dependiendo del linaje específico de H5N6. Las pruebas de muestras clínicas (n=137) revelaron que los métodos RRT-PCR seleccionados para N5, N6 y N8 detectaron con sensibilidad los subtipos del clado 2.3.4.4b del virus de la influenza aviar altamente patógenos H5N5, H5N6 y H5N8, respectivamente, todos originados en Europa de casos en avicultura comercial y de aves silvestres durante el año 2016 al 2018. Estudios similares (muestras clínicas n = 32) también mostraron que los métodos de RRT-PCR para los subtipos N7 y N9 detectaron con sensibilidad las infecciones por el virus H7N7 de alta patogenicidad europeo y por el subtipo H7N9 de origen chino de baja patogenicidad, respectivamente.