title: 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 (n=282), 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.
authors.affiliation: Animal Health and Veterinary Laboratories Agency (AHVLA-Weybridge), Woodham Lane, Addlestone, Surrey, UK. Marek.Slomka@ahvla.gsi.gov.uk
authors.forename: Marek J
authors.initials: MJ
authors.lastname: Slomka
authors.forename: Thanh L
authors.initials: TL
authors.lastname: To
authors.forename: Hien H
authors.initials: HH
authors.lastname: Tong
authors.forename: Vivien J
authors.initials: VJ
authors.lastname: Coward
authors.forename: Amanda
authors.initials: A
authors.lastname: Hanna
authors.forename: Wendy
authors.initials: W
authors.lastname: Shell
authors.forename: Theo
authors.initials: T
authors.lastname: Pavlidis
authors.forename: Anstice L E
authors.initials: AL
authors.lastname: Densham
authors.forename: Georgios
authors.initials: G
authors.lastname: Kargiolakis
authors.forename: Mark E
authors.initials: ME
authors.lastname: Arnold
authors.forename: Jill
authors.initials: J
authors.lastname: Banks
authors.forename: Ian H
authors.initials: IH
authors.lastname: Brown
mesh_terms: D000818:Animals; D001483:Base Sequence; D001499:Bayes Theorem; D002645:Chickens; D016000:Cluster Analysis; D004372:Ducks; D005241:Feathers / Q000821:virology; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D053124:Influenza A Virus, H5N1 Subtype / Q000145:classification / Q000235:genetics* / Q000472:pathogenicity; D005585:Influenza in Birds / Q000175:diagnosis / Q000453:epidemiology*; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000235:genetics; D010802:Phylogeny; D011201:Poultry Diseases / Q000175:diagnosis / Q000453:epidemiology* / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014744:Vietnam / Q000453:epidemiology
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; C109963:hemagglutinin, avian influenza A virus; D009439:Neuraminidase