Challenges for accurate and prompt molecular diagnosis of clades of highly pathogenic avian influenza H5N1 viruses emerging in Vietnam

Abstract

Forty-six chickens and 48 ducks were sampled from four Vietnamese poultry premises in 2009 infected with H5N1 highly pathogenic avian influenza (HPAI) clade 2.3.2 and 2.3.4 viruses, which also differed by cleavage site (CS) sequences in their haemagglutinin (HA) genes. All clinical specimens (n0282), namely tracheal and cloacal swabs plus feathers, were tested by five Eurasian reverse-transcriptase AI RealTime polymerase chain reaction (RRT-PCR) methods. Bayesian modelling showed similar high sensitivity for the validated H5 HA2 RRT-PCR and a new modified M-gene RRT-PCR that utilizes lyophilized reagents. Both were more sensitive than the validated ''wet'' M-gene RRT-PCR. Another RRT-PCR, which targeted the H5-gene CS region, was effective for clade 2.3.4 detection, but severely compromised for clade 2.3.2 viruses. Reduced sensitivity of the H5 CS and ''wet'' M-gene RRT-PCRs correlated with mismatches between the target and the primer and/or probe sequences. However, the H5 HA2 RRT-PCR sensitively detected both clade 2.3.2 and 2.3.4 viruses, and agreed with N1 RRT-PCR results. Feather testing from diseased chicken and duck flocks by AI RRT-PCRs resulted in the most sensitive identification of H5N1 HPAI-infected birds. Evolution of new H5N1 HPAI clades remains a concern for currently affected Asian countries, but also for more distant regions where it is important to be prepared for new incursions of H5N1 HPAI viruses. Genetic evidence for adamantane resistance and sensitivity was also observed in isolates from both clades.

Genetic Characterization of Highly Pathogenic Avian Influenza (H5N8) Virus from Domestic Ducks, England, November 2014

“…Thirty-seven of 120 individual swab samples and 5 of 12 tissue pools (Technical Appendix) were confirmed positive for avian influenza virus by identification of the matrix (M) gene and H5 real-time reverse transcription PCR (rRT-PCR) as described ( 5 ). A highly pathogenic pathotype was confirmed by RT-PCR, then Sanger sequencing of the hemagglutinin (HA) gene as described ( 5 ).…”

“…A highly pathogenic pathotype was confirmed by RT-PCR, then Sanger sequencing of the hemagglutinin (HA) gene as described ( 5 ). This process showed the polybasic cleavage site PLRERRRKR/GLF from multiple bird swab and tissue samples from each shed.…”

“…EPI547670– EPI547677, EPI550848–EPI550849, and EPI558000–EPI558013. Initial maximum likelihood phylogenetic analysis of the HA gene was conducted as described ( 5 ) and the evolutionary analyses performed by using MEGA6 ( 8 ). …”

Outbreak of Eurasian lineage H5N1 highly pathogenic avian influenza in turkeys in Great Britain in November 2007

“…The possible diagnostic utility of such samples is also noteworthy. A subsequent investigation of H5N1 HPAI infected chickens and ducks in Vietnam showed feathers to increase the sensitivity of AIV RRT-PCR testing and successful virus isolation (Slomka and others 2012). RNA extracted from the 13 feather pools and 32 faecal samples in the present investigation were also tested by a M gene RRT-PCR test which included an internal positive control (Petrauskene and others 2010).…”

A comparative evaluation of feathers, oropharyngeal swabs, and cloacal swabs for the detection of H5N1 highly pathogenic avian influenza virus infection in experimentally infected chickens and ducks

“…Although some field studies have reported the use of feathers for diagnosis of HPAI infection, 19,20 most provide few details on the stage of growth or body location of the feathers used. To our knowledge, there are no studies that have examined in detail the relative diagnostic value of swabs and feathers from different body locations and of varying maturity in individual chickens and ducks.…”