Differential Gene Expression Of Porcine Alveolar Macrophage Associated With Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus Infection

Porcine reproductive and respiratory syndrome(PRRS) is a severe swine disease that was first identified in North America in 1987,which causes reproductive failure in late term gestation in sows and respiratory disease in pigs of all ages,and is one of the most economically devastating diseases in pig-producing countries.In May 2006,a highly pathogenic PRRS(HP-PRRS) was emerged in South China and spread quickly to most of the provinces of China,which was characterized by high fever, high morbidity,and high mortality in pigs of all ages,and has caused more than one million pigs death. In comparison with the classical PRRSV isolated in China before 2006,the pathogenicity of HP-PRRSV was significantly enhanced,but the reasons of it are still unclear and there is no comprehensive report about it at present.The viral pathogenicity mainly depends on two aspects:one is the enchange of virulence due to change of viral structure,another is the alteration of cellular response to the virus.In particular,the differential gene expression of host cells during viral infection would be the molecular basis of the occurrence of disease.In vivo,PRRSV has a strict tropism for cells of the monocyte/macrophage lineage,and the porcine alveolar macrophages(PAM) are the primary target cells for PRRSV infection. Macrophages,one of the important component elements of immunological system,play a key role in the process of clearing the extraneous micro-organism and keeping the self-balance of body since the macrophages shares the ability of scavenger.Due to the notable role of PAM,the interaction of PAM-PRRSV maybe contributes to discover the relationships between HP-PRRSV infection and pathogenesis.In the present study,a PRRSV specific TaqMan FQ-PCR method was established and employed for detecting the replicative curve of PRRSV in PAMs.At the time of 40h.p.i.(just before the replication of PRRSV rearches the peak),the total RNA of PRRSV-infected-PAM cells was extracted and used for construction of cDNA libraries(HN library and NH library) by suppression subtractive hybridization.HN library,HP-PRRSV-infected PAM/normal PAM library,contains the genes which were up-regulated by HP-PRRSV infection;NH library,normal PAM/HP-PRRSV-infected PAM library, contains the genes which were down-regulated by HP-PRRSV infection.Two hundreds of different clones from each library were randomly picked,followed by PCR amplification of the inserted up/down-regulated expressed DNA fragments,the individual DNA fragments were dotted on membranes and examined by Southern-blot.After DNA sequencing of the positive clones,the inserted up/down-regulated expressed DNA fragments were blasted by homological searching.As a result,56 genes were found to be altered(including 34 up-regulated and 22down-regulated genes).From the functional point of view,those genes were mainly classified into 3 types:receptors,cytokines,and metabolizing enzymes.Such as:TCR-α/β,MHC classⅠ,IL-8,IL-16,TLR6,TLR1,TLR10,and AMCF-Ⅰwas up-regulated expressed;cytoskeletal-βand MHC classⅡwas down-regulated expressed.The expression of genes mentioned above was also investigated in vivo.PRRS antibody and virus negative pigs were randomly divided into two groups,pigs in group 1 were inoculated with HUN4,and pigs in group 2 were inoculated with DMEM.At different time course,pigs were euthanized and the PAM cells of each pig were collected for detecting the up-regulated or down-regulated genes(total of 28 putative up-regulated genes and 16 down-regulated genes were selected).The RNA of PAM was extracted and reverse transcripted into cDNA,then was examined by Real Time quantitative RT-PCR. For each detected gene,double standard curves were established,one curve was set up with gene-specific primers,and other curve was built on the house-keeping gene,GAPDH,which was used to normalize the amount of cDNA present in each PCR experiment.In order to find out the interaction among the differential expressed genes,the genes finely confirmed by comparative real-time fluorescent quantitative RT-PCR,were analyzed by bio-software. Among the complex and intricate pathways analyzed by bio-software,in combination with the currently experiment-provided data,the pathway of Toll-like receptor in which the NF-κB involved may be a valuable clue for further study.And the roles of these differentially expressed genes play in the pathogenesis of PRRSV,is also needed to be confirmed in the next sTaqe of research.