Molecular cloning, characterization, and expression analysis of the Muscovy duck Toll-like receptor 3 (MdTLR3) gene.

Toll-like receptor 3 (TLR3) is an important membrane-bound receptor for recognizing double-stranded RNA in innate immunity. In this study, we described the cloning and characterization of the Muscovy duck TLR3 (MdTLR3) gene. The full-length MdTLR3 cDNA (2,836 bp) encoded a polypeptide of 895 amino acids. The deduced amino acid sequence contained 4 main structural domains: a signal peptide, an extracellular leucine rich repeats domain, a transmembrane domain, and a Toll/IL-1 receptor domain. Quantitative real-time PCR analysis indicated that MdTLR3 mRNA was constitutively expressed in all sampled tissues of uninfected Muscovy duck except muscle. Expression of MdTLR3 in brain was significantly upregulated at 24 h (1.94-fold, P < 0.05), reached a peak at 48 h (4.64-fold, P < 0.05), and recovered to normal levels at 72 h postinfection with the H5N1 highly pathogenic avian influenza virus. In contrast, MdTLR3 expression was downregulated during the test period in spleen and lung. These results implicated MdTLR3 was a novel member of the TLR family, which is involved in the early stage of antiviral innate immunity.

Genetic characterization of H5N1 avian influenza viruses isolated in southern China during the 2003-04 avian influenza outbreaks.

The recent H5N1 avian influenza outbreaks in Asia spread over more than 8 countries. It has caused enormous economic loss and grand challenges for the public health. During these breakouts we isolated three strains of H5N1 Avian Influenza Virus (AIV) from chickens and one from duck in different farms of Southern China. We completely sequenced these four AIVs. Molecular characterization demonstrated that these strains retain the reported H5N1 AIV sequence properties relevant to virus virulence and host adaptation. Phylogeny results demonstrated that three of these isolates (except A/Chicken/Guangdong/174/04) were closely linked to other H5N1 AIVs isolated from the recent H5N1 outbreaks in Asia. Six of 8 segments (except PA and M) of A/Chicken/Guangdong/174/04 also shares a close linkage to other H5N1 AIVs isolated from the recent H5N1 outbreaks. However, the PA gene of A/Chicken/Guangdong/174/04 and another H5N1 strain forms a distinct subgroup along with an H6N1 AIV, and the M gene of A/Chicken/Guangdong/174/04 shows a close linkage to some H5N1 AIVs from aquatic species in China. Our findings suggest that a new genotype of AIV (in addition to previous reported ones) was present during the 2003-04 Asian bird flu outbreaks and that continuing virus surveillance of AIVs be conducted to monitor the evolutionary paths of the A/Chicken/Guangdong/174/04-like AIVs.