Molecular detection and typing of duck hepatitis A virus directly from clinical specimens

To develop a new approach for the detection and typing of duck hepatitis A virus (DHAV), a pair of non-degenerate primers was designed to amplify a ∼250-bp genomic region in the 5′UTR. 3 reference strains and 6 duck embryo-derived isolates from various regions in China, involving 2 serotypes, were successfully amplified with the primer set. By determining the nucleotide sequence of the amplicon, a molecular typing method was developed. If isolate sequences were compared to DHAV 5′UTR sequences available in public databases, nucleotide identity was ≥94% with homologous serotype and ≤73% with heterologous serotypes. Phylogenetic analysis revealed monophyletic clustering of 5′UTR sequences of a homologous serotype, confirming the new classification of DHAV (serotype 1 and the two new serotypes recently described in Taiwan and South Korea, respectively) into three genotypes (A, B and C) defined by the capsid coding region. Analysis of the results showed that the primer pair should aid in the detection of DHAV, and that the amplicon sequence contains type-specific information and can be used for effective and rapid molecular typing. The molecular methods proved their utility through the detection and typing of DHAV directly from 28 liver specimens collected from dead ducklings during duck viral hepatitis outbreaks in different regions of China between 2001 and 2007. The results confirmed the presence of DHAV in all of the 28 samples and demonstrated that genotypes A (13/28) and C (15/28) of DHAV are co-circulating in China.