Dynamic Distribution of Classical Swine Fever Virus in vivo After Infection by Intermediate Virulent Strains
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| JunXiang SUN,QianYi ZHANG,HeMin XU,TuanJie WANG,Lu XU,XingQi ZOU,YuanYuan ZHU,Cui LI,YingJu XIA,Yuan XU,Kai CHEN,YuJie ZHANG,QiZu ZHAO,Qin WANG |
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Scientia Agricultura Sinica
2018,51(21
):4146
-4156. DOI:10.3864/j.issn.0578-1752.2018.21.013
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【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).
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Screen of MicroRNAs in Classical Swine Fever Virus-Infected PK-15 Cells and the Regulation of Virus Replication by miR-214
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| ShaoFeng DENG,ZuoDong YE,ShuangQi FAN,JinDing CHEN,JingYuan ZHANG,MengJiao ZHU,MingQiu ZHAO |
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Scientia Agricultura Sinica
2018,51(21
):4157
-4168. DOI:10.3864/j.issn.0578-1752.2018.21.014
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【Objective】 In this study, differential expression of miRNAs in CSFV-infected PK-15 cells were determined by miRNA expression array, and further explore the function of miRNAs in the pathogenic of Classical swine fever virus (CSFV), and provide some new basis for the prevention and control of Classical swine fever (CSF) .【Method】 In order to investigate the changes of the miRNAs expression in CSFV-infected PK-15 cells, we synthesized probes of 326 miRNAs of pig according to the miRBase database version 19.0, and screening of differential expression of miRNAs in CSFV-infected PK-15 cells using miRNA expression array. Then, miR-214, the most obvious difference in expression of CSFV-infected PK-15 cells, was selected as the further study object to investigate the function of miR-214 in the infection process of CSFV. We detected mRNA expression of miR-214 in CSFV-infected PK-15 cells using qRT-PCR. In order to further study the effect of miR-214 of CSFV infection, we synthesized miR-214 analog and inhibitor and transfected into PK-15 cells respectively, follow with CSFV infection at 24 h post-transfection, and then detected CSFV titers and quantity of CSFV genomic copies. In order to further explore the mechanism of miR-214 participate in the regulation of CSFV replication, we predicted the target protein of miR-214 using bioinformatics software and confirmed it by luciferase reporter gene system. Given TRADD can specific interacts with TNFR1 intracellular dead zones and participate in the programmed cell death, we assume that miR-214 influencing apoptosis of PK 15 cells by influencing expression level of target protein TRADD. PK 15 cells transfected with miR-214 and inhibitor respectively, follow with CSFV infection at 24 h post-transfection. At 48 h post-infection, the expression levels of TRADD were detected, and the effect of miR-214 on the apoptosis of CSFV-infected PK-15 cells was detected by flow cytometry. 【Result】 69 miRNAs with different expressions were screened by miRNA expression array in CSFV-infected PK-15 cells. Among which the expression changes of miR-214 were most obvious and up-regulated, and confirmed it by qRT-PCR. After transfected with miR-214 to PK-15 cells, CSFV titers and quantity of CSFV genomic copies decreased significantly, while transfected with miR-214 inhibitor, CSFV titers and quantity of CSFV genomic copies were increased significantly, which suggested that miR-214 promoted the replication of CSFV. In order to further explore the mechanism of miR-214 promoting CSFV replication, we confirmed TRADD is the target protein of miR-214. After transfected miR-214 to PK-15 cells, mRNA and protein expression of TRADD were increased significantly, while transfected with miR-214 inhibitor, that were decreased significantly, suggesting that miR-214.inhibits the expression of TRADD. And then, we verified that CSFV infection inhibits apoptosis of PK-15 cells, and miR-214 inhibits apoptosis of CSFV-infected PK-15 cells. 【Conclusion】 The expression of miR-214 in cells was up-regulated after CSFV infected PK-15. miR-214 inhibits the apoptosis of PK-15 cells and promote the replication of CSFV in cells by targeting inhibits the expression of TRADD protein.
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Eradication of Classical Swine Fever in China: Is It Far Away?
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| Yuan SUN,HuaJi QIU |
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Scientia Agricultura Sinica
2018,51(21
):4169
-4176. DOI:10.3864/j.issn.0578-1752.2018.21.015
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Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is one of the most devastating diseases to the pig industry worldwide from both economic and sanitary points of view. CSF is notifiable to the World Organization for Animal Health (OIE) and classified as a List-I animal infectious disease in China. The National Middle- to Long-Term Plan for the Prevention and Control of Animal Infectious Diseases (2012-2020) has listed CSF as one of five priority animal diseases to be prevented. C-strain, a lapinized attenuated live vaccine developed by Chinese scientists in the 1950s, is highly safe and efficacious, which plays an important role in the control and eradication of CSF all over the world. Up to now, CSF has been eradicated in more than 30 countries and regions in the world. CSF has a high consequence on the pork industry in China. Therefore, we must be committed to eradicate CSF in China. In the middle of the last century, China had put forward a strategy to eradicate CSF. Now sixty years have passed, however, CSF is still endemic in most parts of China. Some factors seriously restrict the eradication of CSF in China, such as vast territory, complex pig-raising environment, diversified pig-raising modes, various animal diseases, and low-level biosecurity measures, etc. At present, China has entered a new era, the overall national strength has grown considerably, the breeding industry is developing towards a scientific, large-scale and environment-friendly direction, and the scientific research is being gradually improved. The current prevalence of CSF is pretty low and the vaccine manufacturing technologies are being improved. New CSF marker vaccines and companion differential diagnosis techniques have been developed. Some pig-raising enterprises in China are also aware of the importance and significance of the eradication of CSF. The government is also actively taking actions to formulate corresponding regulations and policies and to organize farmers, pig-raising enterprises and veterinary administrative departments to carry out local CSF eradication. All these make the eradication of CSF possible and practical. This review summarized the recent situation concerning CSF in China and the world. The importance of the eradication of CSF in China and the favorable and unfavorable conditions for the eradication of CSF were discussed. The cost and benefit of the eradication of CSF in China was also analyzed. This review also summarized the eradication experiences of CSF in Europe and USA and discussed the ideas and plans for the eradication of CSF in China. Considering its historical inevitability, strategic necessity, realistic possibility, and technical feasibility, we believe that the eventual eradication of CSF in China is never so far away from us.
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African Swine Fever: A Major Threat to the Chinese Swine Industry
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| YuZi LUO,Yuan SUN,Tao WANG,HuaJi QIU |
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Scientia Agricultura Sinica
2018,51(21
):4177
-4187. DOI:10.3864/j.issn.0578-1752.2018.21.016
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African swine fever (ASF) is a devastating disease of domestic and wild pigs caused by African swine fever virus (ASFV), and causes signi?cant economic losses to the pig industry in affected countries. Acute disease is characterized by high fever, hemorrhages in the skin and internal organs, and a high mortality rate up to 100%. The continuous spread of the disease through Africa, Europe and Russian Federation keeps the neighboring countries on heightened alert. In August 2018, ASF emerged in China for the first time and subsequently rapidly spread across many regions of China, posing a major threat to the Chinese pig industry. Considering unavailability of ASF vaccines, rapid and early diagnostic assays are urgently needed for on-site field testing. Developing an effective ASF vaccine continues to be challenging due to incomplete understanding of the virus. Recent attempts on attenuated live vaccines and gene-deleted vaccines have been reported with promising efficacy, which have been demonstrated to provide effective homologous protection and partial heterologous protection. This review summarizes the epidemiology, diagnostics, vaccines and control strategies and challenges of ASF.
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