PCV2感染猪各组织病毒载量与细胞内质网应激和自噬相关性
|
摘要
通过对PCV2阳性猪剖检,采集了心脏、肝脏、肾脏、肺脏、肾脏、大脑、肠、胃等组织样品,使用定量PCR(qPCR)对PCV2衣壳蛋白Cap基因、ERS和自噬标志基因进行定量分析。结果显示,肺脏中PCV2载量最高,其次为肝脏和肾脏含有较多病毒,小肠、心脏与大脑中病毒载量较少,胃内并未检测到PCV2。ERS和自噬在肺脏中发生最多,其次为小肠,再次为肝脏和肾脏,ERS和自噬水平较高,心脏与大脑中仅发生少量ERS与自噬,而在胃中几乎没有发生。通过透射电子显微镜观察心脏、肝脏、肾脏、肺脏、大脑、肠等组织样品。观察结果显示,所有器官中均可观察到PCV2感染,除肺脏内含量较多外其余各组织中差异不大。各组织中均可见不同数量的自噬小体与内质网应激,其中以肺脏内数量最多,其次为肠道,其余各组织中数量差异不大。电镜观察结果与qPCR检测结果一致,肺脏为病毒载量最大的器官同时也是ERS与自噬发生最多的器官,胃中几乎无病毒感染同时也几乎没有ERS与自噬发生。试验结果表明,除了小肠以外的各组织中ERS与自噬发生情况均与PCV2载量成正相关。为深入研究PCV2诱导细胞ERS和自噬的机制提供了新的理论依据。
Porcine circovirus type 2(PCV2) can cause pathological changes such as lymphocytosis and swollen lymph nodes in infected pigs,eventually becoming immunocompromised.The virus infection is closely related to the occurrence of multiple system failure syndrome(PMWS) in weanling piglets.It has been reported that PCV2 infection induces endoplasmic reticulum stress(ERS) and autophagy in host cells.However,current research almost focus the cellular level in vitro,lacking living animals.Therefore,this study compared the viral load,endoplasmic reticulum stress and autophagy marker genes in different tissues and organs of pigs infected with PCV2,and described their relationship.We collected PCV2 infected pig heart,liver,kidney,lung,kidney,brain,intestine,stomach samples,quantitative PCR(qPCR) was used to quantify the PCV2 capsid protein gene and the key genes of ERS and autophagy.The results showed that PCV2 viral load was the most in lung,followed by the liver and kidney;that of small intestine,heart and brain was less;PCV2 was not detected in gastric.ERS and autophagy occurred most frequently in the lungs,followed by the small intestine,liver and kidney;only a small amount of ERS and autophagy in the heart and brain;almost no ERS and autophagy occurred in the stomach.Transmission electron microscopy observations showed that all organs can be observed virus particle distribution,with the largest number in the lungs.The ERS and autophagy can be observed in all organs,of which the lungs were the most obvious,followed by the small intestine,the rest was not significant differences.Electron microscope observation results was consistent with qPCR.PCV2 viral load was the most in lung where autophagy and ERS were the same,no virus was detected in stomach,In addition to the small intestine,others tissues autophagy and ERS were positively correlated with PCV2 load.
Porcine circovirus type 2(PCV2) can cause pathological changes such as lymphocytosis and swollen lymph nodes in infected pigs,eventually becoming immunocompromised.The virus infection is closely related to the occurrence of multiple system failure syndrome(PMWS) in weanling piglets.It has been reported that PCV2 infection induces endoplasmic reticulum stress(ERS) and autophagy in host cells.However,current research almost focus the cellular level in vitro,lacking living animals.Therefore,this study compared the viral load,endoplasmic reticulum stress and autophagy marker genes in different tissues and organs of pigs infected with PCV2,and described their relationship.We collected PCV2 infected pig heart,liver,kidney,lung,kidney,brain,intestine,stomach samples,quantitative PCR(qPCR) was used to quantify the PCV2 capsid protein gene and the key genes of ERS and autophagy.The results showed that PCV2 viral load was the most in lung,followed by the liver and kidney;that of small intestine,heart and brain was less;PCV2 was not detected in gastric.ERS and autophagy occurred most frequently in the lungs,followed by the small intestine,liver and kidney;only a small amount of ERS and autophagy in the heart and brain;almost no ERS and autophagy occurred in the stomach.Transmission electron microscopy observations showed that all organs can be observed virus particle distribution,with the largest number in the lungs.The ERS and autophagy can be observed in all organs,of which the lungs were the most obvious,followed by the small intestine,the rest was not significant differences.Electron microscope observation results was consistent with qPCR.PCV2 viral load was the most in lung where autophagy and ERS were the same,no virus was detected in stomach,In addition to the small intestine,others tissues autophagy and ERS were positively correlated with PCV2 load.
引文
[1] 夏春香,肖啸,李志敏.猪圆环病毒病研究进展[J].动物医学进展,2005,26(1):35-38.
[2] 周莹珊,谷元兴,齐宝珠,等.猪圆环病毒2型感染细胞的内质网应激-自噬-凋亡:独立事件还是因果关系?[C]// 中国畜牧兽医学会动物传染病学分会第十六次学术研讨会.2015.
[3] 周莹珊.猪圆环病毒2型感染诱导PK-15细胞内质网应激与凋亡的机制研究[D].杭州:浙江大学,2016.
[4] 彭立玮,张晓晓,吴兴安,等.细胞自噬在病毒感染与免疫过程中的作用[J].细胞与分子免疫学杂志,2015,31(4):557-561.
[5] SIR D,OU J H.Autophagy in viral replication and pathogenesis[J].Mol Cells,2010,29(1):1-7.
[6] ZHANG L,WANG A.Virus-induced ER stress and the unfolded protein response[J].Front Plant Sci,2012,3:293.
[7] ZHOU Y,QI B,GU Y,et al.Porcine circovirus 2 deploys PERK pathway and GRP78 for Its enhanced replication in PK-15 cells[J].Viruses,2016,8(2):56.
[8] KOUROKU Y,FUJITA E,TANIDA I,et al.ER stress (PERK/eIF2alpha phosphorylation) mediates the polyglutamine-induced LC3 conversion,an essential step for autophagy formation[J].Cell Death Differ,2007,14(2):230-239.
[9] CHENG X,LIU H,JIANG C C,et al.Connecting endoplasmic reticulum stress to autophagy through IRE1/JNK/beclin-1 in breast cancer cells[J].Int J Mol Med,2014,34(3):772.
[10] GHISLAT G,PATRON M,RIZZUTO R,et al.Withdrawal of essential amino acids increases autophagy by a pathway involving Ca2+/calmodulin-dependent kinase kinase-β (CaMKK-β)[J].J Biol Chem,2012,287(46):38625.
[11] ZHU B,ZHOU Y,XU F,et al.Porcine Circovirus Type 2 Induces Autophagy via the AMPK/ERK/TSC2/mTOR Signaling Pathway in PK-15 Cells[J].J Virol,2012,86(22):12003-12012.
[12] ZHU B,XU F,LI J,et al.Porcine circovirus type 2 explores the autophagic machinery for replication in PK-15 cells[J].Virus Res,2012,163(2):476-485.
[2] 周莹珊,谷元兴,齐宝珠,等.猪圆环病毒2型感染细胞的内质网应激-自噬-凋亡:独立事件还是因果关系?[C]// 中国畜牧兽医学会动物传染病学分会第十六次学术研讨会.2015.
[3] 周莹珊.猪圆环病毒2型感染诱导PK-15细胞内质网应激与凋亡的机制研究[D].杭州:浙江大学,2016.
[4] 彭立玮,张晓晓,吴兴安,等.细胞自噬在病毒感染与免疫过程中的作用[J].细胞与分子免疫学杂志,2015,31(4):557-561.
[5] SIR D,OU J H.Autophagy in viral replication and pathogenesis[J].Mol Cells,2010,29(1):1-7.
[6] ZHANG L,WANG A.Virus-induced ER stress and the unfolded protein response[J].Front Plant Sci,2012,3:293.
[7] ZHOU Y,QI B,GU Y,et al.Porcine circovirus 2 deploys PERK pathway and GRP78 for Its enhanced replication in PK-15 cells[J].Viruses,2016,8(2):56.
[8] KOUROKU Y,FUJITA E,TANIDA I,et al.ER stress (PERK/eIF2alpha phosphorylation) mediates the polyglutamine-induced LC3 conversion,an essential step for autophagy formation[J].Cell Death Differ,2007,14(2):230-239.
[9] CHENG X,LIU H,JIANG C C,et al.Connecting endoplasmic reticulum stress to autophagy through IRE1/JNK/beclin-1 in breast cancer cells[J].Int J Mol Med,2014,34(3):772.
[10] GHISLAT G,PATRON M,RIZZUTO R,et al.Withdrawal of essential amino acids increases autophagy by a pathway involving Ca2+/calmodulin-dependent kinase kinase-β (CaMKK-β)[J].J Biol Chem,2012,287(46):38625.
[11] ZHU B,ZHOU Y,XU F,et al.Porcine Circovirus Type 2 Induces Autophagy via the AMPK/ERK/TSC2/mTOR Signaling Pathway in PK-15 Cells[J].J Virol,2012,86(22):12003-12012.
[12] ZHU B,XU F,LI J,et al.Porcine circovirus type 2 explores the autophagic machinery for replication in PK-15 cells[J].Virus Res,2012,163(2):476-485.