TRANSCRIPTOME ANALYSIS OF LUNGS AND BRAINS OF CHICKENS DURING INFECTION WITH A LOW PATHOGENIC (LP) H5N2 AND HIGH PATHOGENIC (HP) H5N1 AVIAN INFLUENZA VIRUSES Sharifah SH1, Vinod B1, Maizan M2, Suriani MN2, Ramlan M2, Othman I1 and Omar AR3 1School of Medicine and Health Sciences, Monash University Sunway Campus, Selangor, Malaysia. 2Veterinary Research Institute, Ipoh, Perak, Malaysia. 3Institute of Bioscience, UPM, Serdang, Selangor, Malaysia. ABSTRACT A preliminary study to compare the cellular gene expression patterns induced by infections of LPAI and HPAI virus in the lungs and brains of chickens was undertaken. An improved mRNA differential display technique using annealing control primers that generates reproducible, authentic and long PCR products that are detectable on agarose gels was used for the identification of differentially expressed genes (DEGs). These full length transcriptomes detected, were cloned into TOPO vector and sequenced. Up regulated cellular transcripts screened using only 5 out of the 120 annealing control primers ACP’s have resulted in the identification of at least 4 authentic DEGs from lungs and 4 DEGs from brains of chickens infected with HPAI virus. For LPAI infected chickens, only 10 DEGs were detected and isolated from the lungs . The BLAST analysis and tentative functions of the genes or proteins isolated and identified are presented. INTRODUCTION RESULTS Avian Influenza (AI) virus infection, polygenic virus factors contribute to the virulence of the virus, resulting in variations in the pathogenicity, severity of the disease and the distributions of lesions. Generally, in chickens, high pathogenic (HP) AI viruses have a common capability to spread beyond the respiratory tract, which is the common route of infection. However for low pathogenic (LP) AI viruses, although they have the ability to replicate in the respiratory tract, they however exhibited a restricted ability to replicate or produce lesions or both in the other organs. In nearly all HPAI infections reported, pathological lesions are observed in the brain, compared to LPAI infections where the brain do not seem to be infected or affected. The interaction of viral genes with host gene products were also known to affect the virulence or pathogenicity of viruses, as this depends greatly on the balance between negative and positive regulators of gene expressions. Studies which analyze virus interference usually focus on the effect of one viral product to one cellular gene. In order to identify a group of up-regulated cellular genes associated with the highly pathogenic H5N1 Avian Influenza Virus infection and also low pathogenic H5N2 Avian Influenza Virus, a study involving mRNA differential display technique was carried out in Monash University, Sunway campus. The main objectives of this study is (i) to identify the expression pattern of regulated genes during infection at various time kinetics, (ii) to isolate unique genes regulated in infected and non-infected chickens and (iii) to deduce their putative functions using bioinformatics tools .   Fig 1: Cytopathic effect (CPE) of highly pathogenic avian influenza virus (H5N1) in mammalian cell line (Madine Darby Canine Kidney) 400x Control uninfected cells Advanced CPE causing the total destruction of cell morphology Cells showing early CPE Cells without CPE CPE of virus infected cells Fig. 1c: 36 hour pi Fig. 1d: 72 hour pi Fig.1a: Control Fig. 1b: 24 hour pi *pi – post infection METHODOLOGY Infection of HPAI and LPAI to chickens (1 week old) in BSL 3 facility, VRI Harvesting the infected (HPAI & LPAI) lungs and brains according to a time kinetic of 10h, 24h, 48h and 72h. Fig. 2: Experion Automated Gel Electrophoresis picture showing various up regulated and down regulated genes present in both HPAI and LPAI infected organs at different time frames. Extraction of mRNA from the lungs and brains at various time frames and first round of RT-PCR using dt-ACP1 as a tag to the poly A tail of the mRNA from the infected organs resulting in 1ST strand cDNA synthesis. Sequence analysis(BLAST) TOPO 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 500bp The identified bands are run in 2% agarose gel electrophoresis. Bands are then harvested and purified using Promega Wizard® SV gel cleanup and cloned into Invitrogen® TOPO 2.1 cloning vector and sequenced. 500bp M 1 2 3 4 5 6 7 8 9 10 11 M C I C I C I C I C I C I C I C I C I Fig. 3: 2% Gel electrophoresis showing up regulated and down regulated genes both in control and infected organs. Fig. 4: 2% Gel electrophoresis showing restriction digest of some of the genes that are cloned in TOPO Table 1: Putative genes /proteins identified by sequence analysis using NCBI BLAST Gene NCBI BLAST Accession Description A Brain (10h, H5N1, ACP2), 250bp - Unknown sequence (no significant similarity found) B Lung (48h, H5N1, ACP2), 518bp EF112270.1 Part of Influenza A viral polymerase complex C Lung (48h, H5N1, ACP2), 284bp XM 419804.2 Prenyl (decaprenyl) diphosphate synthase, subunit 2 mRNA D Lung (72h, H5N2, ACP2), 429bp AC187512.2 Gallus gallus BAC clone CH261-111G7 E Lung (72h, H5N2, ACP2), 193bp EU309724.1 Dendrobium crumenatum putative pectin methylesterase mRNA F Lung (72h, H5N2, ACP2), 154bp FJ268731.1 Sus scrofa calcium transporter 1 (TRPV6) mRNA G Lung (72h, H5N2, ACP2), 93bp H Brain (10h, H5N1, ACP3), 243bp V00401.1 Messenger fragment for chicken pro-alpha-1 (I) collagen J Lung (72h, H5N2, ACP3), 736bp CP000948.1 Escherichia coli str. K12 substr. DH10B, complete genome K Lung (72h, H5N2, ACP3), 982bp DQ073476.1 Chloroplast transformation vector pRvdB1, complete sequence M Brain (10h, H5N1, ACP4), 973bp EU919404.1 Himar1-delivery and mutagenesis vector pHBurk5, complete sequence N Brain (10h, H5N1, ACP4), 181bp O Lung (24h, H5N1, ACP6), 169bp NM_031080.1 Rattus norvegicus phosphodiesterase mRNA P Lung (24h, H5N1, ACP6), 214bp AJ719809.1 Gallus gallus mRNA for hypothetical protein Q Lung (72h, H5N2, ACP6), 224bp R Lung (72h, H5N2, ACP6), 166bp Sus scrofa calcium transporter 1 (TRPV6) Mrna S Lung (72h, H5N2, ACP7), 403bp AY013304.1 Avian leukosis virus strain ev-3, complete genome T Lung (72h, H5N2, ACP7), 207bp The PCR products are run in BIO-RAD Experion GeneChip to identify unique bands The 1st strand cDNA is subjected to 2nd round PCR using various ACP primers (1-5) together with dt-ACP2 as a complementary sequence which binds only to the dt-ACP1 tag at the poly A tail. DISCUSSION & CONCLUSION The application of mRNA differential display technique using annealing control primers by RT-PCR/PCR in this study has resulted in the successful identification of novel authentic DEG’s of host (cell lines and chickens) infected with HP and LP avian influenza viruses at various time kinetics. The use of regulator sequences ensures that there are no false positives thus generating authentic, reproducible and long PCR products. In addition, this system does not require expensive detection methods as the DEGs can be detected on normal agarose gels thus enabling easy profiling of DEGs. Up-regulated cellular transcripts during Avian Influenza Virus (AIV) infection screened using this technique had resulted in the identification of at least 18 authentic differentially expressed genes (DEGs) at the mRNA level in virus-infected and non-infected cells and chickens. This study has resulted in the identification of novel DEGs of the hosts that have been up-regulated only during infection and not found in control or non-infected hosts. The genes identified in hosts infected with HP and LP AIVs have open up avenues of research especially into exploration and elucidation of the functions of these genes and any role it might play in the virulence or pathogenicity of the virus. These novel genes might be utilized as markers or inhibitors for the development of novel biologics and reagents for diagnostic and anti-viral therapies. Apart from that, the functions of these genes can be used as an indicator in gene knock-out or siRNA studies. Silencing host cellular genes without causing disruption to host cellular genetic and phenotypic functions might lead to the development of Avian Influenza Virus, AIV resistant cells or chickens that are resistant to AIV infection. Reference 1. Liang, P. and Pardee, A.B. 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 257:967-971.  2. McClelland, M., Mathieu-Daude, M. and Welsh, J. 1995. RNA fingerprinting and differential display using arbitrarily primed PCR. TIG 11:242-246. Acknowledgement The authors thank Dr.Ramlan, Director of VRI and also Dr.Satoshi Ogawa from Brain Research Institute, Monash for their collaboration with us.