Journal of Veterinary Science

The Korean Society of Veterinary Science (대한수의학회)
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Transcriptome profiling identifies immune response genes against porcine reproductive and respiratory syndrome virus and Haemophilus parasuis co-infection in the lungs of piglets

  • Zhang, Jing (Key Laboratory of Livestock and Poultry Major Epidemic Disease Monitoring and Prevention, Institute of Animal Husbandry and Veterinary Science, Guizhou Academy of Agricultural Sciences) ;
  • Wang, Jing (Key Laboratory of Livestock and Poultry Major Epidemic Disease Monitoring and Prevention, Institute of Animal Husbandry and Veterinary Science, Guizhou Academy of Agricultural Sciences) ;
  • Zhang, Xiong (Key Laboratory of Livestock and Poultry Major Epidemic Disease Monitoring and Prevention, Institute of Animal Husbandry and Veterinary Science, Guizhou Academy of Agricultural Sciences) ;
  • Zhao, Chunping (Key Laboratory of Livestock and Poultry Major Epidemic Disease Monitoring and Prevention, Institute of Animal Husbandry and Veterinary Science, Guizhou Academy of Agricultural Sciences) ;
  • Zhou, Sixuan (Key Laboratory of Livestock and Poultry Major Epidemic Disease Monitoring and Prevention, Institute of Animal Husbandry and Veterinary Science, Guizhou Academy of Agricultural Sciences) ;
  • Du, Chunlin (Key Laboratory of Livestock and Poultry Major Epidemic Disease Monitoring and Prevention, Institute of Animal Husbandry and Veterinary Science, Guizhou Academy of Agricultural Sciences) ;
  • Tan, Ya (Key Laboratory of Livestock and Poultry Major Epidemic Disease Monitoring and Prevention, Institute of Animal Husbandry and Veterinary Science, Guizhou Academy of Agricultural Sciences) ;
  • Zhang, Yu (College of Animal Science, Guizhou University) ;
  • Shi, Kaizhi (Key Laboratory of Livestock and Poultry Major Epidemic Disease Monitoring and Prevention, Institute of Animal Husbandry and Veterinary Science, Guizhou Academy of Agricultural Sciences)
  • Received : 2021.05.10
  • Accepted : 2021.10.20
  • Published : 2022.01.31
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Abstract

Background: Co-infections of the porcine reproductive and respiratory syndrome virus (PRRSV) and the Haemophilus parasuis (HPS) are severe in Chinese pigs, but the immune response genes against co-infected with 2 pathogens in the lungs have not been reported. Objectives: To understand the effect of PRRSV and/or HPS infection on the genes expression associated with lung immune function. Methods: The expression of the immune-related genes was analyzed using RNA-sequencing and bioinformatics. Differentially expressed genes (DEGs) were detected and identified by quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC) and western blotting assays. Results: All experimental pigs showed clinical symptoms and lung lesions. RNA-seq analysis showed that 922 DEGs in co-challenged pigs were more than in the HPS group (709 DEGs) and the PRRSV group (676 DEGs). Eleven DEGs validated by qRT-PCR were consistent with the RNA sequencing results. Eleven common Kyoto Encyclopedia of Genes and Genomes pathways related to infection and immune were found in single-infected and co-challenged pigs, including autophagy, cytokine-cytokine receptor interaction, and antigen processing and presentation, involving different DEGs. A model of immune response to infection with PRRSV and HPS was predicted among the DEGs in the co-challenged pigs. Dual oxidase 1 (DUOX1) and interleukin-21 (IL21) were detected by IHC and western blot and showed significant differences between the co-challenged pigs and the controls. Conclusions: These findings elucidated the transcriptome changes in the lungs after PRRSV and/or HPS infections, providing ideas for further study to inhibit ROS production and promote pulmonary fibrosis caused by co-challenging with PRRSV and HPS.

Keywords

Acknowledgement

We thank Dr. Chang Liu (Guizhou University of Traditional Chinese Medicine) for providing guidance on data analysis.

References

  1. Shi C, Liu Y, Ding Y, Zhang Y, Zhang J. PRRSV receptors and their roles in virus infection. Arch Microbiol. 2015;197(4):503-512. https://doi.org/10.1007/s00203-015-1088-1
  2. Thanawongnuwech R, Thacker B, Halbur P, Thacker EL. Increased production of proinflammatory cytokines following infection with porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae. Clin Diagn Lab Immunol. 2004;11(5):901-908. https://doi.org/10.1128/CDLI.11.5.901-908.2004
  3. Solano GI, Bautista E, Molitor TW, Segales J, Pijoan C. Effect of porcine reproductive and respiratory syndrome virus infection on the clearance of Haemophilus parasuis by porcine alveolar macrophages. Can J Vet Res. 1998;62(4):251-256.
  4. Thanawongnuwech R, Brown GB, Halbur PG, Roth JA, Royer RL, Thacker BJ. Pathogenesis of porcine reproductive and respiratory syndrome virus-induced increase in susceptibility to Streptococcus suis infection. Vet Pathol. 2000;37(2):143-152. https://doi.org/10.1354/vp.37-2-143
  5. Hernandez Reyes Y, Provost C, Traesel CK, Jacques M, Gagnon CA. Actinobacillus pleuropneumoniae culture supernatant antiviral effect against porcine reproductive and respiratory syndrome virus occurs prior to the viral genome replication and transcription through actin depolymerization. J Med Microbiol. 2018;67(2):249-264. https://doi.org/10.1099/jmm.0.000659
  6. Powell LF, Cheney TE, Williamson S, Guy E, Smith RP, Davies RH. A prevalence study of Salmonella spp., Yersinia spp., Toxoplasma gondii and porcine reproductive and respiratory syndrome virus in UK pigs at slaughter. Epidemiol Infect. 2016;144(7):1538-1549. https://doi.org/10.1017/S0950268815002794
  7. Zhang P, Zhang C, Aragon V, Zhou X, Zou M, Wu C, et al. Investigation of Haemophilus parasuis from healthy pigs in China. Vet Microbiol. 2019;231:40-44. https://doi.org/10.1016/j.vetmic.2019.02.034
  8. Zhao Z, Qin Y, Lai Z, Peng L, Cai X, Wang L, et al. Microbial ecology of swine farms and PRRS vaccine vaccination strategies. Vet Microbiol. 2012;155(2-4):247-256. https://doi.org/10.1016/j.vetmic.2011.09.028
  9. Macedo N, Rovira A, Oliveira S, Holtcamp A, Torremorell M. Effect of enrofloxacin in the carrier stage of Haemophilus parasuis in naturally colonized pigs. Can J Vet Res. 2014;78(1):17-22.
  10. Lunney JK, Fang Y, Ladinig A, Chen N, Li Y, Rowland B, et al. Porcine reproductive and respiratory syndrome virus (PRRSV): pathogenesis and interaction with the immune system. Annu Rev Anim Biosci. 2016;4(1):129-154. https://doi.org/10.1146/annurev-animal-022114-111025
  11. Zhang B, Tang C, Liao M, Yue H. Update on the pathogenesis of Haemophilus parasuis infection and virulence factors. Vet Microbiol. 2014;168(1):1-7. https://doi.org/10.1016/j.vetmic.2013.07.027
  12. Liang W, Li Z, Wang P, Fan P, Zhang Y, Zhang Q, et al. Differences of immune responses between Tongcheng (Chinese local breed) and large white pigs after artificial infection with highly pathogenic porcine reproductive and respiratory syndrome virus. Virus Res. 2016;215:84-93. https://doi.org/10.1016/j.virusres.2016.02.004
  13. de la Fuente AJ, Ferri EF, Tejerina F, Frandoloso R, Martinez SM, Martin CB. Cytokine expression in colostrum-deprived pigs immunized and challenged with Haemophilus parasuis. Res Vet Sci. 2009;87(1):47-52. https://doi.org/10.1016/j.rvsc.2008.12.012
  14. Fu S, Guo J, Li R, Qiu Y, Ye C, Liu Y, et al. Transcriptional profiling of host cell responses to virulent Haemophilus parasuis: new insights into pathogenesis. Int J Mol Sci. 2018;19(5):1320. https://doi.org/10.3390/ijms19051320
  15. Liang W, Ji L, Zhang Y, Zhen Y, Zhang Q, Xu X, et al. Transcriptome differences in porcine alveolar macrophages from Tongcheng and large white pigs in response to highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) infection. Int J Mol Sci. 2017;18(7):1475. https://doi.org/10.3390/ijms18071475
  16. Kavanova L, Matiaskova K, Leva L, Stepanova H, Nedbalcova K, Matiasovic J, et al. Concurrent infection with porcine reproductive and respiratory syndrome virus and Haemophilus parasuis in two types of porcine macrophages: apoptosis, production of ROS and formation of multinucleated giant cells. Vet Res (Faisalabad). 2017;48(1):28. https://doi.org/10.1186/s13567-017-0433-6
  17. Li J, Wang S, Li C, Wang C, Liu Y, Wang G, et al. Secondary Haemophilus parasuis infection enhances highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) infection-mediated inflammatory responses. Vet Microbiol. 2017;204:35-42. https://doi.org/10.1016/j.vetmic.2017.03.035
  18. Geissmann F, Gordon S, Hume DA, Mowat AM, Randolph GJ. Unravelling mononuclear phagocyte heterogeneity. Nat Rev Immunol. 2010;10(6):453-460. https://doi.org/10.1038/nri2784
  19. Costa V, Angelini C, De Feis I, Ciccodicola A. Uncovering the complexity of transcriptomes with RNASeq. J Biomed Biotechnol. 2010;2010:853916.
  20. Jia A, Zhou R, Fan H, Yang K, Zhang J, Xu Y, et al. Development of serotype-specific PCR assays for typing of Haemophilus parasuis isolates circulating in Southern China. J Clin Microbiol. 2017;55(11):3249-3257. https://doi.org/10.1128/jcm.00688-17
  21. Liu S, Li W, Wang Y, Gu C, Liu X, Charreyre C, et al. Coinfection with Haemophilus parasuis serovar 4 increases the virulence of porcine circovirus type 2 in piglets. Virol J. 2017;14(1):227. https://doi.org/10.1186/s12985-017-0890-6
  22. Yu J, Wu J, Zhang Y, Guo L, Cong X, Du Y, et al. Concurrent highly pathogenic porcine reproductive and respiratory syndrome virus infection accelerates Haemophilus parasuis infection in conventional pigs. Vet Microbiol. 2012;158(3-4):316-321. https://doi.org/10.1016/j.vetmic.2012.03.001
  23. Pertea M, Kim D, Pertea GM, Leek JT, Salzberg SL. Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nat Protoc. 2016;11(9):1650-1667. https://doi.org/10.1038/nprot.2016.095
  24. Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SL. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol. 2015;33(3):290-295. https://doi.org/10.1038/nbt.3122
  25. Varet H, Brillet-Gueguen L, Coppee JY, Dillies MA. SARTools: a DESeq2- and EdgeR-based R pipeline for comprehensive differential analysis of RNA-Seq data. PLoS One. 2016;11(6):e0157022. https://doi.org/10.1371/journal.pone.0157022
  26. Feiden S, Wolfrum U, Wegener G, Kamp G. Expression and compartmentalisation of the glycolytic enzymes GAPDH and pyruvate kinase in boar spermatogenesis. Reprod Fertil Dev. 2008;20(6):713-723. https://doi.org/10.1071/RD08004
  27. Wu J, Mao X, Cai T, Luo J, Wei L. KOBAS server: a web-based platform for automated annotation and pathway identification. Nucleic Acids Res. 2006;34(Web Server issue):W720-W724. https://doi.org/10.1093/nar/gkl167
  28. Szklarczyk D, Gable AL, Lyon D, Junge A, Wyder S, Huerta-Cepas J, et al. STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res. 2019;47(D1):D607-D613. https://doi.org/10.1093/nar/gky1131
  29. Su G, Morris JH, Demchak B, Bader GD. Biological network exploration with Cytoscape 3. Curr Protoc Bioinformatics. 2014;47(1):8.13.1-8.13.24.
  30. Wang CJ, Zhou ZG, Holmqvist A, Zhang H, Li Y, Adell G, et al. Survivin expression quantified by Image Pro-Plus compared with visual assessment. Appl Immunohistochem Mol Morphol. 2009;17(6):530-535. https://doi.org/10.1097/PAI.0b013e3181a13bf2
  31. Gallo-Oller G, Ordonez R, Dotor J. A new background subtraction method for Western blot densitometry band quantification through image analysis software. J Immunol Methods. 2018;457:1-5. https://doi.org/10.1016/j.jim.2018.03.004
  32. Kavanova L, Prodelalova J, Nedbalcova K, Matiasovic J, Volf J, Faldyna M, et al. Immune response of porcine alveolar macrophages to a concurrent infection with porcine reproductive and respiratory syndrome virus and Haemophilus parasuis in vitro. Vet Microbiol. 2015;180(1-2):28-35. https://doi.org/10.1016/j.vetmic.2015.08.026
  33. Kavanova L, Matiaskova K, Leva L, Nedbalcova K, Matiasovic J, Faldyna M, et al. Concurrent infection of monocyte-derived macrophages with porcine reproductive and respiratory syndrome virus and Haemophilus parasuis: a role of IFNα in pathogenesis of co-infections. Vet Microbiol. 2018;225:64-71. https://doi.org/10.1016/j.vetmic.2018.09.016
  34. Pomorska-Mol M, Dors A, Kwit K, Czyzewska-Dors E, Pejsak Z. Coinfection modulates inflammatory responses, clinical outcome and pathogen load of H1N1 swine influenza virus and Haemophilus parasuis infections in pigs. BMC Vet Res. 2017;13(1):376. https://doi.org/10.1186/s12917-017-1298-7
  35. Binjawadagi B, Dwivedi V, Manickam C, Torrelles JB, Renukaradhya GJ. Intranasal delivery of an adjuvanted modified live porcine reproductive and respiratory syndrome virus vaccine reduces ROS production. Viral Immunol. 2011;24(6):475-482. https://doi.org/10.1089/vim.2011.0040
  36. Eason MM, Fan X. The role and regulation of catalase in respiratory tract opportunistic bacterial pathogens. Microb Pathog. 2014;74:50-58. https://doi.org/10.1016/j.micpath.2014.07.002
  37. Liu JC, Li L, Yan HC, Zhang T, Zhang P, Sun ZY, et al. Identification of oxidative stress-related Xdh gene as a di(2-ethylhexyl)phthalate (DEHP) target and the use of melatonin to alleviate the DEHP-induced impairments in newborn mouse ovaries. J Pineal Res. 2019;67(1):e12577. https://doi.org/10.1111/jpi.12577
  38. Konate MM, Antony S, Doroshow JH. Inhibiting the activity of NADPH oxidase in cancer. Antioxid Redox Signal. 2020;33(6):435-454. https://doi.org/10.1089/ars.2020.8046
  39. Xiao Y, An TQ, Tian ZJ, Wei TC, Jiang YF, Peng JM, et al. The gene expression profile of porcine alveolar macrophages infected with a highly pathogenic porcine reproductive and respiratory syndrome virus indicates overstimulation of the innate immune system by the virus. Arch Virol. 2015;160(3):649-662. https://doi.org/10.1007/s00705-014-2309-7
  40. Zhen Y, Wang F, Liang W, Liu J, Gao G, Wang Y, et al. Identification of differentially expressed non-coding RNA in porcine alveolar macrophages from Tongcheng and large white pigs responded to PRRSV. Sci Rep. 2018;8(1):15621. https://doi.org/10.1038/s41598-018-33891-0
  41. Wang Y, Liu C, Fang Y, Liu X, Li W, Liu S, et al. Transcription analysis on response of porcine alveolar macrophages to Haemophilus parasuis. BMC Genomics. 2012;13(1):68. https://doi.org/10.1186/1471-2164-13-68
  42. Parrish-Novak J, Foster DC, Holly RD, Clegg CH. Interleukin-21 and the IL-21 receptor: novel effectors of NK and T cell responses. J Leukoc Biol. 2002;72(5):856-863. https://doi.org/10.1189/jlb.72.5.856
  43. Odaka C, Tanioka M, Itoh T. Matrix metalloproteinase-9 in macrophages induces thymic neovascularization following thymocyte apoptosis. J Immunol. 2005;174(2):846-853. https://doi.org/10.4049/jimmunol.174.2.846
  44. Liu Y, Du Y, Wang H, Du L, Feng WH. Porcine reproductive and respiratory syndrome virus (PRRSV) upregulates IL-8 expression through TAK-1/JNK/AP-1 pathways. Virology. 2017;506:64-72. https://doi.org/10.1016/j.virol.2017.03.009
  45. Martinez-Martinez S, Rodriguez-Ferri EF, Frandoloso R, Garrido-Pavon JJ, Zaldivar-Lopez S, Barreiro C, et al. Molecular analysis of lungs from pigs immunized with a mutant transferrin binding protein B-based vaccine and challenged with Haemophilus parasuis. Comp Immunol Microbiol Infect Dis. 2016;48:69-78. https://doi.org/10.1016/j.cimid.2016.08.005