Cellular transcripts of chicken brain tissues in response to H5N1 and Newcastle disease virus infection

Abstract

Background: Highly-pathogenic avian influenza (HPAI) H5N1 and Newcastle disease (ND) viruses are the two most important poultry viruses in the world, with the ability to cause classic central nervous system dysfunction in poultry and migratory birds. To elucidate the mechanisms of neurovirulence caused by these viruses, a preliminary study was design to analyze host's cellular responses during infections of these viruses.

Methods: An improved mRNA differential display technique (Gene Fishing™) was undertaken to analyze differentially expressed transcripts regulated during HPAI H5N1 and velogenic neurotropic NDV infections of whole brain of chickens. The identification of differentially expressed genes (DEGs) was made possible as this technique uses annealing control primers that generate reproducible, authentic and long PCR products that are detectable on agarose gels.

Results: Twenty-three genes were identified to be significantly regulated during infections with both viruses, where ten of the genes have been selected for validation using a TaqMan® based real time quantitative PCR assay. Some of the identified genes demonstrated to be key factors involving the cytoskeletal system, neural signal transduction and protein folding during stress. Interestingly, Septin 5, one of the genes isolated from HPAI H5N1-infected brain tissues has been reported to participate in the pathogenic process of Parkinson's disease.

Conclusions: In this limited study, the differentially expressed genes of infected brain tissues regulated by the viruses were found not to be identical, thus suggesting that their neurovirulence and neuropathogenesis may not share similar mechanisms and pathways.

Figure 1

A schematic diagram of the experimental procedure.

Figure 2

Gel picture showing DEGs obtained from control and HPAI H5N1 infected brain tissues. There are 12 bands, of which 6 of them are up regulated and 6 down regulated. The identities of these bands are presented in Table 3; C: Control brain tissues; I: Infected with H5N1. Each number indicates the DEG number.

Figure 3

Gel picture showing DEGs obtained from control and velogenic AF2240 NDV infected brain tissues. There are 11 bands, of which 6 of them are up regulated and 5 down regulated. The identities of these bands are presented in Table 4; C: Control brain tissues; I: Infected with NDV. Each number indicates the DEG number.

Figure 4

Relative quantitation of down regulated and up regulated genes. A. Relative quantitation of selected genes from control and infected with HPAI H5N1 brain tissues was determined by real time RT-PCR and the fold difference (2^-ΔΔCT) between the study groups were calculated: Uninfected cells (Control); Infected cells (Infected). B. Relative quantitation of selected genes from control and infected with NDV brain tissues was determined by real time RT-PCR and the fold difference (2^-ΔΔCT) between the study groups were calculated: Uninfected cells (Control); Infected cells (Infected). The ** (P value) for both sets of data is < 0.05, indicating there are significant down regulation and up regulation in the infected cells compared to control.