Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (21): 4146-4156.doi: 10.3864/j.issn.0578-1752.2018.21.013

;

• SPECIAL FOCUS: SWINE FEVER AND AFRICAN SWINE FEVER • Previous Articles     Next Articles

Dynamic Distribution of Classical Swine Fever Virus in vivo After Infection by Intermediate Virulent Strains

JunXiang SUN1,QianYi ZHANG1,HeMin XU2,TuanJie WANG1,Lu XU1,XingQi ZOU1,YuanYuan ZHU1,Cui LI1,YingJu XIA1,Yuan XU1,Kai CHEN1,YuJie ZHANG1,QiZu ZHAO1,Qin WANG1   

  1. 1National/OIE Reference Laboratory for Classical Swine Fever, China Institute of Veterinary Drug Control, Beijing 100081
    2China Animal Husbandry Industry Co., LTD, Beijing 100070
  • Received:2018-04-18 Accepted:2018-05-28 Online:2018-11-01 Published:2018-11-01
  • Contact: JunXiang SUN,QianYi ZHANG,QiZu ZHAO,Qin WANG

RichHTML

8

PDF

546

Abstract:

【Objective】At present, the infection type of classical swine fever in China is mainly subacute or chronic infection. The aim of this study was to investigate the differences and distribution of RNA and protein expressions in pigs infected with the intermediate virulent strain of classical swine fever virus. The results could help to elucidate the replication and distribution of the subacute disease virus and to provide technical support for the early diagnosis and prevention of classical swine fever.【Method】Using the medium virulence strain (HEBHH1/95), we successfully established a subacute CSF infection animal model. Duodenum, spleen, kidney, lung, pancreas and ileocecal samples were collected from pigs for viewing in situ hybridization (ISH), immunohistochemistry (IHC) and hematoxylin-eosin staining (HE) at 1 day post infection (dpi), 3 dpi, 6 dpi, 10 dpi, 13 dpi, 20 dpi, 24 dpi and 28 dpi. The ViewRNA ISH was used to study the dynamic distribution of viral RNA in infected tissues. Immunohistochemistry (IHC) and HE staining were used to detect the distribution of viral proteins in infected tissues and their contribution to tissue damage.【Result】The clinical score increased rapidly from 6 dpi to 10 dpi, then from 11 dpi to 26 dpi the score remained at approximately 15, until at 28 dpi clinical score peaked at 20 points. The body temperature showed an upward trend from 8 dpi to 10 dpi, and then livestock suffered from continuous fever which persisted at about 40℃ from 13 dpi to 24 dpi, thereafter, the body temperature began to fall back to about 39.5℃ before the animal died. Viral RNA were detected in duodenum, pancreas, ileocecal valve and kidney at 1 dpi and in the lungs of the bronchioles, spleen oval body at 3 dpi; Viral RNAs widely distributed in each tissue at 28 dpi, and were mainly observed in the spleen artery around the lymphatic sheath, pancreatic acinar, renal tubular with secretion function. The IHC and HE staining were used to verify the results of ViewRNA ISH in similar fields of vision. The positive signals of viral proteins and the corresponding histopathological changes of duodenum, pancreas and kidney were also detected at 1 dpi, but the viral protein and tissue pathological changes were detected in the ileocecal valve, spleen and lung at 3 dpi. Viral RNA and protein localization tended to be the same in each tissue after 3 dpi.【Conclusion】All the results showed that CSFV had an increasing virus load from 1 dpi to 28 dpi detected by ViewRNA ISH, which was in consistent with the result of IHC. Moreover, CSFV was firstly tropism to secretory cells such as pancreatic acinar, renal tubular and spleen artery at the beginning of infection (1 dpi-3 dpi), and then showed pantropically infectious to all the tissues during the infection period (6 dpi-13 dpi), and during the final stage CSFV was accumulated both around lymphocytes and secretory cells (20 dpi-28 dpi).

Key words: CSFV, medium virulent, RNA, ViewRNA ISH, distribution

Fig. 1

Clinical scores after infection of CSFV HeBHH1/95 strain"

Fig. 2

Change of body temperatures after infection of CSFV HeBHH1/95 strain"

Table 1

The optimization of boiling and proteinase digestion time for pre-treated tissue samples"

组织名称
Tissue		 预处理液处理时间
Preconditioning liquids work time (min)		 蛋白酶消化时间
Proteinase K work time (min)
肺脏Lung 10 20
十二指肠
Duodenum		 10 10
回盲瓣
Ileocecal valve		 10 10
胰腺Pancreas 10 10
脾脏Spleen 20 10
肾脏Kidney 20 20

Fig. 3

Duodenum ViewRNA ISH, IHC and H.E staining results after infection of CSFV HeBHH1/95 strain (200×) “a” represents the ViewRNA ISH test result, where the RNA target sequence of CSFV is dyed red and the nucleus is stained blue. “b” represents the IHC test results, where the target protein of CSFV is dyed red. “c” denotes the result of H.E staining. Negative control was done by injection of saline pig. IV: intestinal villus; GC: goblet cell"

Fig. 4

Pancreas ViewRNA ISH, IHC and H.E staining results after infection of CSFV HeBHH1/95 strain (200×) “a” represents the ViewRNA ISH test result, where the RNA target sequence of CSFV is dyed red and the nucleus is stained blue. “b” represents the IHC test results, where the target protein of CSFV is dyed red. “c” denotes the result of H.E staining. Negative control was done by injection of saline pig. PA: Pancreas acinar; PI: Pancreatic islet"

Fig. 5

Ileocecal valve ViewRNA ISH, IHC and H.E staining results after infection of CSFV HeBHH1/95 strain (200×) “a” represents the ViewRNA ISH test result, where the RNA target sequence of CSFV is dyed red and the nucleus is stained blue. “b” represents the IHC test results, where the target protein of CSFV is dyed red. “c” denotes the result of H.E staining. Negative control was done by injection of saline pig. LP: Lamina propria; LC: Crypt of Lieberkuhn"

Fig. 6

Spleen ViewRNA ISH, IHC and H.E staining results after infection of CSFV HeBHH1/95 strain (200×) “a” represents the ViewRNA ISH test result, where the RNA target sequence of CSFV is dyed red and the nucleus is stained blue. “b” represents the IHC test results, where the target protein of CSFV is dyed red. “c” denotes the result of H.E staining. Negative control was done by injection of saline pig. E: Ellipsoid; PALS: Periarterial lymphatic sheath; MZ: Marginal zone"

Fig. 7

Kidney ViewRNA ISH, IHC and H.E staining results after infection of CSFV HeBHH1/95 strain (200×) “a” represents the ViewRNA ISH test result, where the RNA target sequence of CSFV is dyed red and the nucleus is stained blue. “b” represents the IHC test results, where the target protein of CSFV is dyed red. “c” denotes the result of H.E staining. Negative control was done by injection of saline pig. G: glomerulus; RT: renal tubular"

Fig. 8

Lung ViewRNA ISH, IHC and H.E staining results after infection of CSFV HeBHH1/95 strain (200×) “a” represents the ViewRNA ISH test result, where the RNA target sequence of CSFV is dyed red and the nucleus is stained blue. “b” represents the IHC test results, where the target protein of CSFV is dyed red. “c” denotes the result of H.E staining.Negative control was done by injection of saline pig. B: Bronchiole; LI: Lung interstitium"

[1] SAMBANDAM R, ANGAMUTHU R, KANAGARAJ V, KATHAPERUMAL K, SHUBHADA K C, RUTH H N, RHIANNON M B, BHUSHAN M J, SURESH V K . An immuno-chromatographic lateral flow assay (LFA) for rapid on-the-farm detection of classical swine fever virus (CSFV). Archives of Virology, 2017,162(3/4):1-6.
doi: 10.1007/s00705-016-3065-7 pmid: 27695950
[2] 王琴, 涂长春. 猪瘟. 北京:中国农业出版社, 2015: 15.
WANG Q, TU C C . Classical Swine Fever. Beijing: China Agriculture Press, 2015: 15. (in Chinese)
[3] EDWARDS S, FUKUSHO A, LEFEVRE P C, LIPOWSKI A, PEJSAK Z, ROEHE P . Classical swine fever: The global situation. Veterinary Microbiology, 2000,73(2):103-119.
doi: 10.1016/S0378-1135(00)00138-3 pmid: 10785321
[4] CHEVILLE N F, MENGELING W L . The pathogenesis of chronic hog cholera (swine fever). Histologic, immunofluorescent, and electron microscopic studies. Laboratory investigation. Journal of Technical Methods and Pathology, 1969,20:261-274.
pmid: 5773219
[5] LIU J, FAN X Z, WANG Q, XU L, ZHAO Q Z, HUANG W, ZHOU Y C, TANG B, CHEN L, ZOU X Q, SHA S, ZHU Y Y . Dynamic distribution and tissue tropism of classical swine fever virus in experimentally infected pigs. Journal of Virology, 2011,8(1):201-211.
doi: 10.1186/1743-422X-8-201 pmid: 3107811
[6] 陈锴 . 猪瘟慢性感染对猪免疫功能影响的细胞与分子机制研究[D]. 雅安:四川农业大学, 2012.
CHEN K . Research on the immune and cellular mechanism after classical swine fever virus chronic infection[D]. Yaan: Sichuan Agricultural University, 2012. ( in Chinese)
[7] TAYLOR A M, BERCHTOLD N C, PERREAU V M, TU C H, LI J N, COTMAN C W . Axonal mRNA in uninjured and regenerating cortical mammalian axons. The Journal of Neuroscience, 2009,29(15):4697-4707.
doi: 10.1523/JNEUROSCI.6130-08.2009 pmid: 3632375
[8] YU M, DAVID T T, STOTT S L, WINTER B S, OZSOK L F, PAUL S, CICILIANO J C, SMAS M E, WINOKUR D, GILMAN A J, ULMAN M J, XEGA K, CONTIO G, ALAGESN B, BRANNGIN B W, MILOS P M, RYAN D P, SEQUIST L V, BARDEESY N, RAMASWAMY S, TONER M, MAHESWARAN S, HABER D A . RNA sequencing of pancreatic circulating tumour cells implicates WNT signaling in metastasis. Nature, 2012,487(7408) : 510-513.
doi: 10.1038/nature11217 pmid: 3408856
[9] ZHANG Q Y, XU L, ZHANG Y J, WANG T J, ZOU X Q, ZHU Y Y, ZHAO Y, LI C, CHEN K, SUN Y F, SUN J X, ZHAO Q Z, WANG Q . A novel View RNA in situ hybridization method for the detection of the dynamic distribution of Classical Swine Fever Virus RNA in PK15 cells. Virology Journal, 2017,14(1):81-89.
doi: 10.1186/s12985-017-0734-4 pmid: 5395781
[10] MITTELHOLZER C, MOSER C, TRATHSIN J D, HOFMANN M A . Porcine cells persistently infected with classical swine fever virus protected from pestivirus-induced cytopathic effect. Journal of General Virology, 1998,79:2981-2987.
doi: 10.1099/0022-1317-79-12-2981 pmid: 9880012
[11] 朱长康 . 猪瘟病毒人工慢性感染的组织病理学和病毒核酸载量动态分布研究[D]. 雅安: 四川农业大学, 2012.
ZHU C K . Histopathology and dynamic distribution of classical swine fever virus in chronically infected pigs[D]. Yaan: Sichuan Agricultural University, 2012. ( in Chinese)
[12] 刘同慎, 李冰 . HE染色程序的使用和维护. 生物学通报, 2013 , 48(4) : 55-57.
LIU T S, LI B . Use and maintenance of HE staining program. Bulletin of Biology, 2013 , 48(4):55-57. (in Chinese)
[13] 赵燕 . 猪瘟病毒QuantiGene ViewRNA原位杂交方法的建立及初步应用[D]. 北京:中国兽医药品监察所, 2014.
ZHAO Y . Establishment and rreliminary application of CSFV QuantiGene ViewRNA in situ hybridization technique[D]. Beijing:China Institute of Veterinary Drugs Control, 2014. ( in Chinese)
[14] LAIHO J E, OIKARINEN S, OIKARINEN M, LARSSON P G, STONE V M, HOBER D, OBSERTE S, FLODSTROM T M, ISOLA J, HYOTY H . Application of bioinformatics in probe design enables detection of enteroviruses on different taxonomic levels by advanced in situ hybridization technology. Journal of Clinical Virology, 2015,69 : 165-171.
doi: 10.1016/j.jcv.2015.06.085
[15] 王珊珊, 马建, 史楠, 刘强韦, 华冕, 周建华 . 应用ViewRNA技术特异性检测感染细胞中的不同马传染性贫血病毒株. 中国预防兽医学报, 2011,33(10) : 800-803.
doi: 10.3969/j.issn.1008-0589.2011.10.11
WANG S S, MA J, SHI N, LIU Q W, HUA M, ZHOU J H . In situ detection of co-infected equine infectious anemia virus heterologous strains in fetal donkey dermal cells using a View RNA assay. Chinese Journal of Preventive Veterinary Medicine, 2011, 33(10):800-803. (in Chinese)
doi: 10.3969/j.issn.1008-0589.2011.10.11
[16] 张玉杰, 赵燕, 徐璐, 张乾义, 陈锴, 孙永芳, 邹兴启, 朱元源, 赵启祖, 宁宜宝, 王琴 . RNA可视化原位杂交技术对感染细胞中猪瘟病毒RNA定位与分布. 中国农业科学, 2016 , 49(12):2397-2407.
doi: 10.3864/j.issn.0578-1752.2016.12.015
ZHANG Y J, ZHAO Y, XU L, ZHANG Q Y, CHEN K, SUN Y F, ZOU X Q, ZHU Y Y, ZHAO Q Z, NING Y B, WANG Q . Study of location and distribution of classical swine fever virus RNA in PK15 cells by visualization in situ hybridization technology. Scientia Agricultura Sinica, 2016,49(12) : 2397-2407. (in Chinese)
doi: 10.3864/j.issn.0578-1752.2016.12.015
[17] 孙佳鑫, 侯明星 . 促吞噬肽免疫学机制的研究进展. 世界最新医学信息文摘, 2018,18(16):53-54.
SUN J X, HOU M X . The advances of Tuftsin on immunological mechanisms. World Latest Medicine Information, 2018, 18(16):53-54. (in Chinese)
[18] SONG H, CHEN L, SUN F . Expansion of spleen myeloid suppressor cells represses NK cell cytotoxicity in tumor-bearing host. Blood, 2007, 109(10):4336-4342.
doi: 10.1182/blood-2006-09-046201 pmid: 17244679
[19] BUCKLEY P J, SMITH M R, BRAVERMAN M F, DICKSON S A . Human spleen contains phenotypic subsets of macrophages and dendritic cells that occupy discrete microanatomic locations. American Journal of Pathology, 1987,128(3):505-520.
pmid: 3307443
[20] MARVEL D M, FINN O J . Global inhibition of DC priming capacity in the spleen of self-antigen vaccinated mice requires IL-10. Frontiers in Immunology, 2014,5:59-83.
doi: 10.3389/fimmu.2014.00059 pmid: 3925839
[21] SUMMERFIELD A, KNOETIG S M, TSCHUDIN R, MCULLOUGH K C . Pathogenesis of granulocytopenia and bone marrow atrophy during classical swine fever involves apoptosis and necrosis of uninfected cells. Virology, 2000,272(1):50-60.
doi: 10.1006/viro.2000.0361 pmid: 10873748
[22] LAMBRECHT B N . Alveolar macrophage in the driver's seat. The Journal of Immunology, 2006,24(4):366-368.
doi: 10.1016/j.immuni.2006.03.008 pmid: 16618595
[23] JAKUBZICK C, TACKE F, LODRA J, VAN R N, RANODPH G J . Modulation of dendritic cell trafficking to and from the airways. The Journal of Immunology, 2006,176(6):3578-3584.
doi: 10.4049/jimmunol.176.6.3578 pmid: 16517726
[24] NING P B, ZHANG Y M, GUO K K, CHEN R, LANG W L, LIN Z, LI H L . Discovering up-regulated VEGF-C expression in swine umbilical vein endothelial cells by classical swine fever virus Shimen. Veterinary Research, 2014,45(1):48-53.
doi: 10.1186/1297-9716-45-48 pmid: 24758593
[25] GOMEZ-VILLAMANDOS J C, SALGUERO F J, RUIZ- VILLAMOR E, SANCHEZ P J, BAUTISTA M J, SIERRA M A . Classical swine fever: Pathology of bone marrow. Veterinary Pathology, 2003,40(2):157-163.
doi: 10.1354/vp.40-2-157 pmid: 12637755
[26] ALBERT Q L, BENJAMIN S F, RYUJI M, PAUL H L, VALERIUS M T, BONVENTRE J V . Rapid and efficient differentiation of human pluripotent stem cells into intermediate mesoderm that forms tubules expressing kidney proximal tubular markers. Journal of the American Society of Nephrology, 2014,25(6):1211-1225.
doi: 10.1681/ASN.2013080831 pmid: 24357672
[27] XU L, FAN X Z, ZHAO Q Z, ZHANG Z X, CHEN K, NING Y B, ZHANG Q Y, ZOU X Q, ZHU Y Y, LI C, ZHANG Y J, WANG Q . Effects of vaccination with the C-Strain vaccine on immune cells and cytokines of pigs against classical swine fever virus. Viral Immunology, 2017,31(1):34-39.
doi: 10.1089/vim.2017.0010 pmid: 28514189
[28] 林鸷 . 白介素-1β和胆固醇在猪瘟病毒感染中的作用[D]. 杨凌: 西北农林科技大学, 2015.
LIN Z . The role of IL-1β and cholesterol in classical swine fever virus infection [D] . Yangling: North-West Agriculture and Forestry University, 2015. ( in Chinese)
[29] SCHMITA J, OWYANG A, OLDHAM E, SONG Y, MURPHY E, MCCLANAHAN T K, ZURASWKI G, MOSHREFI M, QIN J, LI X, GORMAN D M, BAZAN J F, KASTELEIN R A . IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity, 2005s,23(5):479-490.
doi: 10.1016/j.immuni.2005.09.015
[30] 李素, 王惟, 张淑琴, 董鹏, 朱妍, 郭焕成, 涂长春 . 猪瘟病毒感染猪细胞因子和趋化因子的动态变化. 中国生物制品学志, 2010,23(10):1038-1042.
LI S, WANG W, ZHANG S Q, DONG P, ZHU Y, GUO H C, TU C C . Kinetics of cytokines and chemokines of pigs infected with classical swine fever virus. Chinese Journal of Biologicals, 2010,23(10):1038-1042. (in Chinese)
[1] LI YuanJing, YUAN RuiXiang, LI YongTai, SUN TianGe, LIU Feng, LI YanJun, ZHANG XinYu. Identification and Functional Characterization of β-Glucosidase Genes in Verticillium dahliae for Pathogenicity on Cotton [J]. Scientia Agricultura Sinica, 2026, 59(7): 1380-1399.
[2] ZHANG Qi, CHEN ErHu, SUN DeHong, TANG PeiAn. Relationship Between Glutathione S-Transferase Genes CfGSTe1 and CfGSTd1 and Ethyl Formate Tolerance in Cryptolestes ferrugineus [J]. Scientia Agricultura Sinica, 2026, 59(5): 1008-1019.
[3] GUO FuCheng, TANG HaiJiang, HAO XinYi, MA GuoLin, YANG JiuJu, HUANG LinFeng, TIAN Lei, WANG Bin, LUO ChengKe. Effects of Different Irrigation Methods on Water-Salt Transport, Rice Yield, and Water Use Efficiency in Saline Soil in Ningxia [J]. Scientia Agricultura Sinica, 2026, 59(4): 750-764.
[4] LUO Wei, YU Hong, YUAN LiXin, WANG LingLing, ZHAO JinPeng, YIN Wei, WANG MingTian, WANG RuLin. Change of Geographic Distributions of Ratoon Rice in Sichuan- Chongqing Under Global Climate Change [J]. Scientia Agricultura Sinica, 2026, 59(4): 765-780.
[5] KONG Yuan, CUI ShaSha, LI Mei, LI Jian, YANG SiYu, FANG Feng, LIU ShuaiShuai, LIU MingPing, ZENG Yan, GAO XingXiang, BAI LianYang. Spatiotemporal Distribution Dynamics of Five Grass Weed Species Including Lolium multiflorum in Winter Wheat Fields of the Huang- Huai-Hai Region [J]. Scientia Agricultura Sinica, 2026, 59(4): 807-823.
[6] YAN WenYing, ZHANG YuanZhen, WU HongXin, PANG Rui, CHEN ZePeng, JIN FengLiang, XU XiaoXia. Construction of an RNAi-Enhanced Metarhizium anisopliae Targeting PxGNBP3 and Its Immunoregulatory Mechanism in Plutella xylostella [J]. Scientia Agricultura Sinica, 2026, 59(3): 556-574.
[7] WANG JiaMei, XU FengQi, ZHOU Hui, HU XiangDong. The Current Situation, Regional Characteristics and International Experience of Agricultural Socialized Services in China [J]. Scientia Agricultura Sinica, 2026, 59(2): 459-474.
[8] LÜ XuDong, SUN ShiYuan, LI YaNan, LIU YuLong, WANG YanQun, FU Xin, ZHANG JiaYing, NING Peng, PENG ZhengPing. Effects of Intelligent Mechanized Layered Fertilization on Root-Soil Nutrient Distribution and Yield in Wheat Fields [J]. Scientia Agricultura Sinica, 2026, 59(1): 129-146.
[9] ZHOU Qi, ZHANG ShiHao, ZHANG Liang, PAN Yu, ZHANG LiJuan, TU Zhi, PAN HongMei, LONG Xi. Prediction of the Potential Habitat Suitability of Luopanshan Pigs in Chongqing Based on the Optimized MaxEnt Model [J]. Scientia Agricultura Sinica, 2026, 59(1): 205-219.
[10] WANG BingJie, QIN ShiHan, LI DeCheng, HU WenYou, JIANG Jun, CHI FengQin, ZHANG Chao, ZHANG JiuMing, XU YingDe, WANG JingKuan. Spatial Distribution Pattern and Transfer Function Construction of Soil Bulk Density in Nenjiang City, Heilongjiang Province [J]. Scientia Agricultura Sinica, 2025, 58(9): 1791-1803.
[11] WANG JiYing, LI JingXuan, WANG YanPing, GUO JianFeng, LIN HaiChao, ZHAO XueYan. Weighted Gene Co-Expression Network Analysis Reveals Potential Candidate Genes Affecting Fat Deposition in Pigs [J]. Scientia Agricultura Sinica, 2025, 58(9): 1845-1855.
[12] LI Lin, ZHANG YuanZhen, YAN WenYing, ZENG Lu, PANG Rui, XU XiaoXia, JIN FengLiang. The Role of miR-6497-x in Regulating the Reaction of Plutella xylostella to Fungal Infection [J]. Scientia Agricultura Sinica, 2025, 58(8): 1550-1563.
[13] JU XiaoJun, ZHANG Ming, LIU YiFan, JI GaiGe, SHAN YanJu, TU YunJie, ZOU JianMin, ZHANG HaiTao, BIAN LiangYong, SHU JingTing. Integration of Intestinal Flora and Small Molecule Metabolite to Analyze the Role of Factors Regulating Feed Conversion in Broiler Chickens [J]. Scientia Agricultura Sinica, 2025, 58(6): 1223-1238.
[14] CHEN ErHu, TANG JingJie, HU ShunJie, TANG PeiAn. The Roles of Heat Shock Protein Genes CfHsp70-1 and CfHsp70-2 in Enhancing the High-Temperature Tolerance after Heat Acclimation in Cryptolestes ferrugineus [J]. Scientia Agricultura Sinica, 2025, 58(5): 918-928.
[15] CONG QiQi, ZHANG JingYi, MENG XiangLong, DAI PengBo, LI Bo, HU TongLe, WANG ShuTong, CAO KeQiang, WANG YaNan. Identification of Hypovirus in Apple Ring Rot Fungus Botryosphaeria dothidea and Detection of Virus-Carrying Status in China [J]. Scientia Agricultura Sinica, 2025, 58(3): 478-492.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd