Identification And Characterization Of Genes Related To Prrsv And PCV2 Resistance In Pigs

Disease has become one of the key factors impacting the healthy development of pig industry. The PRRS and PCVAD are two major infectious diseases that affect the pig production throughout the world. PRRSV and PCV2 are two major pathogens that can lead to reproductive disorders of pigs. In addition, these two diseases can not only cause immune suppression of the swine but also cause secondary affection or complicating disease caused by other pathogenic microorganism, which causes huge economic loss to the global swine industry. Swine genetics can affect the host in susceptibility to the virus. Anecdotal information from the field suggests that there are host genetic differences in susceptibility to PRRS and PCV2 associated disease among different breeds. This study first analyzed the promoter region of porcine USP18 and Mx1 gene which are candidate genes related to resistance of PRRSV. The objective of this study is to determine if a difference exists in the susceptibility to PCV2 infection between Laiwu pigs and Yorkshire × Landrace pigs and explore the mechanism underlying such differences and finding genetic markers of pigs associated with PRRSV and PCV2 resistance. The main results were shown as follows:1. We set out to compare the promoter activity of USP18 in Chinese indigenous Dapulian(DPL) pigs and Duroc × Landrace × Yorkshire(DLY) commercial pigs and screen single nucleotide polymorphism(SNP) affecting porcine USP18 transcription. We found that the promoter activity was significantly higher in DPL pigs than DLY commercial pigs( P < 0.05), deletion of the promoter from –1790 to –1314 bp decreased the transcriptional activity by roughly 60%(P < 0.05) and a SNP G–1533A in this region increased the mRNA expression both prior to and post PRRSV infection in MARC-145 cells. Population genetics analysis showed that allele A was only detected in Chinese pig breeds which are generally resistant to PRRSV. These results suggest that the SNP G–1533A polymorphism in the promoter region of porcine USP18 gene is a potential DNA marker for the resistance to PRRSV.2. We demonstrated that positive elements were present in the two promoter regions of-2713 to-2565 and-688 to-431 in porcine Mx1 gene. Sequencing and alignment of the amplified porcine Mx1 gene promoter region identified a short interspersed repetitive element(SINE) insertion of 275 bp at site-547. At this site, allele B(an insertion of 275 bp) is dominant in Chinese indigenous pig breeds but has a workable minor allele frequency in western lean-type pig breeds. Luciferase activity was compared between promoters with and without the insertion of the 275 bp fragment in transiently transfected MARC-145 cells. The insertion of the 275 bp fragment increased luciferase activity significantly(P < 0.05) both prior to and post porcine reproductive and respiratory syndrome(PRRS) virus inoculation. These results suggest that the SINE insertion polymorphism at site-547 of the Mx1 gene promoter region is a potential DNA marker for PRRS resistance in pigs.3. Fifteen purebred LW pigs and 15 YL crossbred weaned pigs of 6-week-old in average, respectively. All pigs were seronegative for PCV1,PCV2, porcine reproductive and respiratory syndrome virus(PRRSV) and porcine parvovirus(PPV) as determined by commercial ELISA assays. Pigs were randomly assigned to four groups and raised in isolation rooms of the same farm, namely LW-i groups(n=10), LW-u groups(n=5), YL-i groups(n=10) and YL-u groups(n=5). Pigs of groups LW-i and YL-i were infected intramuscularly with 3m L(103.8TCID50/mL) of PCV2-SD Strain to ensure infection of each pig with a same dose at the time of infection and the uninfected groups(LW-u and YL-u) were treated similarly with an identical volume of phosphate buffered saline(PBS). This day was designated as 0 d after infection. After PCV2 infection, Rectal temperature and clinical symptoms were recorded daily during the experiment. The pigs were weighed and collected blood samples on the day of PCV2 infection at 0,4,7,10,14, 21, 28 and 35(dpi). Blood samples was collected from the infected and control groups for measuring PCV2 virus copy number, cytokines including GM-CSF/CSF2, IL-1β, IL-6, IL-10, TGF-β1, IFN-γ, IL-4, IL-8, IL-12p40 and TNF-α, and immunoglobulin G(Ig G) levels. All pigs were necropsied at 35 dpi and collected tissue samples including lung, tonsil, thymus, ileum, kidney, spleen, liver and lymph node et al. and processed for 10% hae-matoxylin, eosin(H&E) for subsequent histological examinations.The results showed that, in comparison with PCV2-infected LW pigs, 60%(6\10) of PCV2-infected YL pigs exhibited typical clinical symptoms of PMWS, such as appetite dropping, coughing, diarrhea and progressive weight loss, occasionally, sneezing; one of the most obvious sign is the presence of irregular, red macules and papules in the skin, primarily on the back area and hind limbs from approximately 14 dpi onward. About one-half of PCV2-infected YL pigs experienced shortness of breath from 14 to 28 dpi. LW pigs did not show any obvious clinical symptoms, only mild clinical symptoms such as anorexia, coughing and pyrexia. PCV2-infected YL pigs demonstrated a persistent high average rectal temperature of from 10 dpi onward to 14 dpi( > 40oC) compared to control pigs, however, no significant differences were observed in LW groups. From the day of PCV2 inoculation onward to 35 dpi, the weight gain was not significantly different(P >0.05) among the LW groups,while YL pigs showed lower mean body weight compared to control pigs at 21 dpi until 35 dpi. Regarding pathological studies, PCV2-infected YL pigs showed symptoms ranging from congestion to bleeding in the lungs, severe interstitial pneumonia, characterized by interstitial broadening of lung lobules obviously, serious bleeding, a large number of lymphocyte infiltration in the bronchus. However, these pathological symptoms were not observed in PCV2-infected LW pigs. The concentration of IgG in serum were no significant difference during the test period between LW pigs and YL pigs, But the total content of IgG in LW pigs was higher than YL pigs. by ELISA. The results showed that the protein levels of pro-inflammatory cytokines IL-6(P < 0.01), IL-4, IL-8 and TGF-β1 significantly increased(P < 0.05) in the serum of Laiwu pigs, and of TNF-α from YL pigs increased significantly(P < 0.05) in the early stages after infection; the protein level of IL-8 were restrained persistently, regulatory cytokine IL-10 was expressed strongly in later period( P < 0.05). Together, it is suggested that, PCV2 infection caused YL pigs to display inflammatory response, reduced the early anti-virus and chemotactic fuctions and the decreased functions of induced immune modulation and appeared disorder. The results of PCR and absolute quantitative PCR indicated that the content of PCV2 DNA virus in LW-i pigs was lower than YL pigs after infected with PCV2.4. We compared the changes in gene expression profiles of the lung tissues collected from the LW and Yorkshire × Landrace(YL) pigs by RNA-seq. At 35 dpi, a total of 374 differentially expressed genes between the two breeds were identified in the lung tissues. For LW pigs, 83 genes were up-regulated and 86 genes were down-regulated after PCV2 infection compared to the control group. For YL pigs, the number is 187 and 18,respectively. Among these differentially expressed genes, five were up-regulated and four were down-regulated in both breeds. When annotated to Gene Ontology(GO) database,150,160 and 156 genes were found in biological processes, cellular components and molecular functions items, respectively, in Yorkshire × Landrace pigs; there are 149,134 and 134 genes were found in these three terms, respectively. Mining of the Kyoto Encyclopedia of Genes and Genomes(KEGG) database using the 374 differentially expressed genes retrieved 299 pathways. The number of differentially expressed genes varied from one to 25 per pathway. The most represented pathways were complement and coagulation Cascades, RIG-I-like receptor signaling pathway and B cell receptor signaling pathway. We identified four differentially expressed genes(TFPI, SERPINC1, SERPINA1 and SERPINA5) that were related to complement and coagulation cascades and were associated with lung tissues damage and up-regulation in infected Laiwu pigs compare to uninfected pigs. Through analysis of the expression profile, we preliminary screened differentially expressed genes, which are related to PCV2 infection between the two breeds.In conclusion, in this study, we analyzed polymorphisms of USP18 and Mx1 genes associated with PRRSV infection. The SNP G–1533A polymorphism and the SINE insertion polymorphism at site-547 in the promoter region of porcine USP18 and Mx1 genes are two potential DNA markers for the resistance to PRRSV. We tested if a difference exists in PCV2 susceptibility between Laiwu and Yorkshire × Landrace commercial pigs under experimental conditions and explored the mechanism underlying such differences. The results indicated that different biological responses to PCV2 between Laiwu pigs and Yorkshire ×Landrace commercial pigs exist, and hundreds of differentially expressed genes in lung tissues of these two pigs after inoculation with PCV2 virus are revealed. We further validated four differentially expressed genes(TFPI, SERPINC1,SERPINA1 and SERPINA5) that were related to complement and coagulation cascades and were associated with lung tissues damage by real time PCR, the mRNA expression level of which in lung tissue of LW pigs were different from YL commercial pigs after infection with PCV2 virus.