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

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• 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

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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"

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