| Porcine Reproductive and Respiratory Syndrome, caused by porcine reproductive and respiratory syndrome virus (PRRSV) is an infectious disease, characterized by severe reproductive deficiency in pregnant sows, respiratory symptoms in piglets and high lethal rate. The genome of PRRSV are highly variable, therefore, there is no an effective vaccine against PRRS infection in pigs. Part of vaccinated pigs still become infected with PRRSV, the cultivation of disease resistance pigs against PRRSV is an practical and feasible way to solve this difficult problem. In this study, we used a Affymetrix porcine microarray to investigate changes in gene transcription in lungs of Dapulian pigs and Duroc×Yorkshire×Landrace commercial pigs that occur during in vitro infeetion with PRRSV. We cloned the porcine interferon receptors(IFNGR1, IFNAR2)cDNA and analyzed the mRNA expression of IFNGR1and IFNAR2 genes in different tissues of different pigs, the influences of PRRSV infection to mRNA expression of IFNGR1and IFNAR2 gene in tissues of Dapulian pigs and Duroc×Yorkshire×Landrace commercial pigs were also analyzed.1. 6 - weak - old Dapulian pigs and Duroc×Yorkshire×Landrace commercial pigs were inoculated intranasally with porcine reproductive and respiratory syndrome virus (PRRSV), the uninfected control were inoculated intranasally with PBS, rectal temperature were recorded every day after inoculation, body weight were recorded and blood samples were drawn from each pig on day 0 before inoculation and on day 4, 7, 14, 21, 28 after inoculation. Cell factors ( IL -1β, IL - 2, IL - 10, IFN–γand TNF - a), immune globulin IgG and blood count were detected. The nucleic acid for PRRSV in serum was detected by RT- PCR and real- time quantitive PCR on day 4, 7, 14, 21, 28 after inoculation. The clinical signs were different between the two pigs after PRRSV infection. The clinical signs of Dapulian pigs infected with PRRSV included mild fever, lethargy, the clinical signs were not apparent. The clinical signs of Duroc×Yorkshire×Landrace commercial pigs infected with PRRSV included fever, the body temperature was as high as 42℃, the red body skin, periocular edema, nasal discharge, dyspnea, lethargy, sometimes with nervous symptom, peribronchial lymphoid hyperplasia in the peribronchial area, interstitial pneumonia and so on. The histological lesions were characterized by infiltration of macrophages and lymphocytes in alveolar septum, pulmonary edema. Tracheal and bronchial bleeding, lymph nodes especially inguinal lymph nodes and hilar lymph node edema, hemorrhage and so on. On day 0-14 after PRRSV infection, Serum TNF-αwas elevated in both Dapulian pigs and Duroc×Yorkshire×Landrace commercial pigs, on day 14 after PRRSV infection, serum IL - 10 in Dapulian pigs maintained high level, while serum IL– 10 in Duroc×Yorkshire×Landrace commercial pigs was decreased. Dynamic features of serum IFN–γexhibit a first drop and subsequent rise. On day 0-14 after PRRSV infection, the level of serum IgG showed a rising trend and peaked on day 14. Leukocyte count in Dapulian pigs and Duroc×Yorkshire×Landrace commercial pigs is reduced or increased after PRRSV infection. Other index basically had no change. The result of RT-PCR and real- time quantitive PCR showed that the content of the nucleic acid for PRRSV in Dapulian pigs infected with PRRSV was lower than that of Duroc×Yorkshire×Landrace commercial pigs infected with PRRSV.2. There were 259 differentially expressed genes in lung tissues of the two pigs after infection with PRRSV, bioinformatics analysis were performed for these differentially expressed genes.Cluster analysis showed that Dapulian pigs were initially clustered together because their expression profiles were similar, Duroc×Yorkshire×Landrace commercial pigs were clustered in another class. Go analysis indicated that the protein encoded by these differently expressed genes are associated with cellular process, physiological process, developmental process, biological regulation process and so on.By real - time PCR, we analysis the mRNA expression of twelve differentially expressed genes, the results showed that eleven of the genes are consistent with microarray, including six up-regulated genes, they are ENPEP, TF, USP18, Scarb2, CACNA2D1and CYP3A29, five down- regulated genes, they are GSTA2, CYP3A88, ATP1B1, LPL and MRC1. The mRNA expression level of CYP3A29, USP18, IF and ENPEP in Dapulian pigs were 9.09, 7.10, 6.24 and 5.43 times of that in Duroc×Yorkshire×Landrace commercial pigs, individually. The mRNA expression level of CYP3A88 and GSTA2 in Dapulian pigs only 1 % and 4 % of that in Duroc×Yorkshire×Landrace commercial pigs. By comparativing analysis of gene expression between Dapulian pigs and Duroc×Yorkshire×Landrace commercial pigs, We preliminarily determined that ENPEP, TF, USP18 and CYP3A29 were the genes underlying PRRSV resistance in pigs, we also preliminarily determined CYP3A88 and GSTA2 were the genes underlying PRRSV susceptibility in pigs.3. Porcine IFNGR1 cDNA was cloned by RT-PCR, 5′- RACE and 3′-RACE, two transcripts were identified. Porcine IFNGR1 cDNA shares 62.95 %, 63.73 %, 72.90 % and 81.10 % identity in nucleotide sequence; and 45.64 %, 46.69 %, 58.04 % and 72.55 % homology in amino acid sequence to those of rat, mouse, human and cattle, respectively. The porcine IFNGR1 genomic structure consists of seven exons and six introns and is located on porcine chromosome 1. The mRNA expression of porcine IFNGR1 gene is detected in all tissues examined, with higher expression in spleen and liver tissues and lower expression in cerebrum, cerebellum and uterus tissues, respectively. A different developmental pattern in IFNGR1 mRNA expression between Laiwu and Duroc breeds was revealed by real-time quantitative PCR: in Duroc pigs, a significantly higher expression was found in the tissues of heart (P<0.05), liver (P<0.01), kidney (P<0.01) and skeletal muscle (P<0.05) of adult pigs compared to piglets. In PRRSV- infected Dapulian pigs, compared to the uninfected ones, the expression level of IFNGR1 mRNA in spleen was significantly up-regulated (P<0.05), whereas its expression in the lymph node was significantly down-regulated (P<0.05); in PRRSV-infected Duroc×Yorkshire×Landrace commercial pigs, however, the differences both in spleen and lymph node tissues were not significant(P>0.05).4. Porcine IFNAR2 cDNA was cloned by using RT-PCR and 5′RACE, the deduced protein of which shares 38.18 %, 55.29 %, 62.01 % and 63.39 % identity to those of mouse, human, sheep and cattle, respectively. The genomic structure of porcine IFNAR2 consists of nine exons and eight introns. The expression of porcine IFNAR2 mRNA was detected in all tissues examined, with stong expression being detected in spleen, small intestine and uterus and relatively weak expression in stomach tissues. Developmental dynamics in IFNAR2 mRNA expression was revealed in adult pigs compared to piglets by real-time quantitative PCR: In Laiwu pigs, IFNAR2 mRNA expression in both liver and spleen was significantly higher (P<0.01); in Duroc pigs, however, significantly higher IFNAR2 mRNA expression was only found in liver (P<0.05). In Duroc×Yorkshire×Landrace commercial pigs infected with PRRSV, the expression of IFNAR2 mRNA in lung tissue was significantly down-regulated as compared to the uninfected ones (P<0.05).To sum up, we discussed the PRRSV resistance mechanism of Dapulian pigs, the results showed the biological responses to PRRSV were different in Dapulian pigs and Duroc×Yorkshire×Landrace commercial pigs. There were lots of differentially expressed genes in lung tissues of the two pigs after infection with PRRSV. We firstly cloned the IFNGR1 and IFNAR2 gene of pigs, and analyzed their expression profiles in tissues. The expression level of IFNGR1 gene in lung tissue and lymph nodes were different in Dapulian pigs and Duroc×Yorkshire×Landrace commercial pigs, we found some genes,expressed in lungs, underlying PRRSV resistance or susceptibility in pigs. The study laid the foundation for further studying the pathogenesis of PRRS and finding the molecular markers associated PRRSV resistance. |
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