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.

Establishment of a T-Ag and NF-kappaB binding elements gene co-transfected stable HUVECs cell line.

To set up a T-Ag gene transfected stable human umbilical venous endothelial cells (HUVECs) cell line and a T-Ag, nuclear transcriptional factor kappa B (NF-kappaB) binding elements linked with luciferase reporter gene co-transfected stable HUVECs cell line. Cultured HUVECs were transfected with pCI-neo-T-Ag and pRSV-luc-3XkappaB by lipofectin. The G418 selected monoclones were subcultured. The expression of marker protein, vWF and the characteristic of uptake of lipids were compared by Western blotting and immunocytochemistry in non-transfected and transfected HUVECs. The reporter gene assay was done in the presence of TNF-alpha. A T-Ag gene transfected stable HUVECs cell line and a T-Ag and NF-kappaB binding elements linked with luciferase reporter gene co-transfected stable HUVECs cell lines were set up. The expression of vWF of these cell lines was similar with those in non-transformed HUVECs. The function of uptaking of lipids was preserved as well in transfected cell lines. Furthermore, TNF-alpha, a typical cytokine increasing the activity of NF-kappaB was used to treat the transfected cells O/N. The higher luciferase reporter gene activity was seen. A pCI-neo-T-Ag and pRSV-luc-3X kappaB co-transfected stable HUVECs cell line might be used to check reporter gene activity directly. It might be a useful tool to screen drugs acting on transcription level.