Background Avian influenza (AI) caused by H7 AI viruses (AIVs) of both low pathogenicity (LP) and high pathogenicity (HP) are notifiable poultry diseases.
Objectives Design and validate two RealTime reverse transcriptase polymerase chain reactions (RRT PCRs) for Eurasian H7 AIV detection and pathotyping.
Methods The H7 RRT PCRs amplified within the (i) HA2 and (ii) cleavage site CS regions of the haemagglutinin gene. Both were validated against 65 H7 AIVs, 57 non‐H7 AIVs and 259 poultry swabs in comparison to M gene (AI generic) RRT PCR and virus isolation (VI). An additional 38 swabs and 20 tissue specimens extended validation against M gene RRT PCR.
Results Both H7 RRT PCRs amplified all 61 Eurasian lineage H7 AIVs and none of 57 non‐H7 AIVs. A total of 297 poultry swabs were used to determine diagnostic sensitivity and specificity relative to M gene RRT PCR, sensitivity was 95·4% and 64·6% for the HA2 and CS RRT PCRs respectively, and specificity 97·9% and 99·6% respectively. The H7 HA2 RRT PCR was more sensitive than VI. This was emphasized by analysis of 37 swabs from turkeys infected experimentally with HPAI H7N1 virus sampled at 24 hours post‐inoculation and LPAI H7N1 chicken infections sampled at 40–64 hours. Although less sensitive, usefulness of the H7 CS RRT PCR was confirmed by the correct molecular pathotyping for all 61 Eurasian lineage H7 AIVs tested.
Conclusions The high sensitivity of H7 HA2 RRT PCR confirms its suitability for use in poultry surveillance and disease diagnosis. H7 CS RRT PCR provides an opportunity for rapid pathotyping of H7 AIVs.
Many different polymerase chain reaction (PCR) protocols have been used for detection and characterization of avian influenza (AI) virus isolates, mainly in research settings. Blind ring trials were conducted to determine the most sensitive and specific AI PCR protocols from a group of six European Union (EU) laboratories. In part 1 of the ring trial the laboratories used their own methods to test a panel of 10 reconstituted anonymized clinical specimens, and the best methods were selected as recommended protocols for part 2, in which 16 RNA specimens were tested. Both panels contained H5, H7, other AI subtypes, and non-AI avian pathogens. Outcomes included verification of 1) generic AI identification by highly sensitive and specific M-gene real-time PCR, and 2) conventional PCRs that were effective for detection and identification of H5 and H7 viruses. The latter included virus pathotyping by amplicon sequencing. The use of recommended protocols resulted in improved results among all six laboratories in part 2, reflecting increased sensitivity and specificity. This included improved H5/H7 identification and pathotyping observed among all laboratories in part 2. Details of these PCR methods are provided. In summary, this study has contributed to the harmonization of AI PCR protocols in EU laboratories and influenced AI laboratory contingency planning following the first European reports of H5N1 highly pathogenic AI during autumn 2005.
Porcine reproductive and respiratory virus (PRRSV) primarily infects and destroys alveolar macrophages of the pig. The aim of the present study was to characterize the changes of leukocyte populations in the broncho-alveolar lavage fluid (BALF) of PRRSV-infected pigs. Piglets were inoculated intranasally with PRRSV strain LV ter Huurne. On various days post-infection the piglets were sacrificed and the lungs removed, washed semi-quantitatively and analysed by flow cytometry. The total number of recovered BALF cells increased approximately 10 times between day 10 and day 21 of infection and decreased thereafter. The number of small low-autofluorescent cells (SLAC), i.e. lymphocytic and monocytic cells, increased very strongly from day 2 until day 21 of infection; in contrast, the number of large highly autofluorescent cells (LHAC), i.e. mostly macrophages, remained constant until day 14 of infection, increased slightly on day 21 and then decreased. On day 21 of infection in specific-pathogen-free piglets approximately 60% of the SLAC consisted of CD2(+)CD8(+)CD4(-)gammadeltaTCR(-) cells, which were partly CD8(+)CD6(+) and partly CD8(+)CD6(-). These phenotypes correspond to that of cytotoxic T-cells and natural killer cells respectively. From these results we can conclude that during a PRRSV infection the total number of BALF cells increases mainly due to an influx of lymphocytic cells with a cytolytic phenotype.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.