A novel ViewRNA in situ hybridization method for the detection of the dynamic distribution of Classical Swine Fever Virus RNA in PK15 cells
Abstract Background Classical swine fever (CSF) is a highly contagious fatal infectious disease caused by classical swine fever virus (CSFV). A better understanding of CSFV replication is important for the study of pathogenic mechanism of CSF. With the development of novel RNA in situ Hybridization...
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| Format: | Article |
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BMC
2017-04-01
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| Series: | Virology Journal |
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| Online Access: | http://link.springer.com/article/10.1186/s12985-017-0734-4 |
| _version_ | 1818552654806122496 |
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| author | Qianyi Zhang Lu Xu Yujie Zhang Tuanjie Wang Xingqi Zou Yuanyuan Zhu Yan Zhao Cui Li Kai Chen Yongfang Sun Junxiang Sun Qizu Zhao Qin Wang |
| author_facet | Qianyi Zhang Lu Xu Yujie Zhang Tuanjie Wang Xingqi Zou Yuanyuan Zhu Yan Zhao Cui Li Kai Chen Yongfang Sun Junxiang Sun Qizu Zhao Qin Wang |
| author_sort | Qianyi Zhang |
| collection | DOAJ |
| description | Abstract Background Classical swine fever (CSF) is a highly contagious fatal infectious disease caused by classical swine fever virus (CSFV). A better understanding of CSFV replication is important for the study of pathogenic mechanism of CSF. With the development of novel RNA in situ Hybridization method, quantitatively localization and visualization of the virus RNA molecular in cultured cell or tissue section becomes very important tool to address these pivotal pathogenic questions. In this study, we established ViewRNA ISH method to reveal the dynamic distribution of CSFV RNA in PK15 cells. Methods We designed several specific probes of CSFV RNA and reference gene β-actin for host PK15 cells to monitor the relative location of CSFV RNA and house-keeping gene in the infected cells. After determining the titer of reference strain CSFV (HeBHH1/95) with the 50% tissue culture infective dose (TCID50), we optimized the protease K concentration and formalin fixation time to analyze the hybridization efficiency, fluorescence intensity and repeatability. In order to measure the sensitivity of this assay, we compared it with the fluorescent antibody test (FAT) and immunohistochemical(IHC) method. Specificity of the ViewRNA ISH was tested by detecting several sub genotypes of CSFV (sub genotype 1.1, 2.1, 2.2 and 2.3) which are present in China and other normal pig infectious virus (bovine viral diarrhea virus (BVDV), porcine parvovirus (PPV), porcine pseudorabies virus (PRV) and porcine circovirusII(PCV-2). Results The lowest detection threshold of the ViewRNA ISH method was 10−8, while the sensitivity of FAT and IHC were 10−5 and 10−4, respectively. The ViewRNA ISH was specific for CSFV RNA including 1.1, 2.1, 2.2 and 2.3 subtypes, meanwhile, there was no cross-reaction with negative control and other viruses including BVDV, PPV, PRV and PCV-2. Our results showed that after infection at 0.5 hpi (hours post inoculation, hpi), the CSFV RNA can be detected in nucleus and cytoplasm; during 3–9 hpi, RNA was mainly distributed in nucleus and reached a maximum at 12hpi, then RNA copy number was gradually increased around the cell nucleus during 24–48 hpi and reached the peak at 72hpi. Conclusions To our knowledge, this is the first to reveal the dynamic distribution of medium virulence CSFV RNA in PK15 cells using the ViewRNA ISH method. The sensitivity of the ViewRNA ISH was three to four orders of magnitude higher than that of FAT and IHC methods. The specificity experiment showed that the ViewRNA ISH was highly specific for CSFV and no cross-reaction occurred to negative control and other pig infectious virus. This assay is more suitable for studying the CSFV RNA life cycle in cell nucleus. The results proved that CSFV RNA enters into PK15 cells earlier than 0.5hpi, relative to the eclipse period of cytoplasm is 6–9 hpi and CSFV RNA has ever existed in nucleus. |
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| format | Article |
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| institution | Directory Open Access Journal |
| issn | 1743-422X |
| language | English |
| last_indexed | 2024-12-12T09:16:00Z |
| publishDate | 2017-04-01 |
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| series | Virology Journal |
| spelling | doaj.art-1ab4b34a0f0c45bbaf8075080a60b6022022-12-22T00:29:23ZengBMCVirology Journal1743-422X2017-04-011411810.1186/s12985-017-0734-4A novel ViewRNA in situ hybridization method for the detection of the dynamic distribution of Classical Swine Fever Virus RNA in PK15 cellsQianyi Zhang0Lu Xu1Yujie Zhang2Tuanjie Wang3Xingqi Zou4Yuanyuan Zhu5Yan Zhao6Cui Li7Kai Chen8Yongfang Sun9Junxiang Sun10Qizu Zhao11Qin Wang12National Classical Swine Fever Reference Laboratory, China Institute of Veterinary Drug ControlNational Classical Swine Fever Reference Laboratory, China Institute of Veterinary Drug ControlNational Classical Swine Fever Reference Laboratory, China Institute of Veterinary Drug ControlNational Classical Swine Fever Reference Laboratory, China Institute of Veterinary Drug ControlNational Classical Swine Fever Reference Laboratory, China Institute of Veterinary Drug ControlNational Classical Swine Fever Reference Laboratory, China Institute of Veterinary Drug ControlNational Classical Swine Fever Reference Laboratory, China Institute of Veterinary Drug ControlNational Classical Swine Fever Reference Laboratory, China Institute of Veterinary Drug ControlNational Classical Swine Fever Reference Laboratory, China Institute of Veterinary Drug ControlNational Classical Swine Fever Reference Laboratory, China Institute of Veterinary Drug ControlNational Classical Swine Fever Reference Laboratory, China Institute of Veterinary Drug ControlNational Classical Swine Fever Reference Laboratory, China Institute of Veterinary Drug ControlNational Classical Swine Fever Reference Laboratory, China Institute of Veterinary Drug ControlAbstract Background Classical swine fever (CSF) is a highly contagious fatal infectious disease caused by classical swine fever virus (CSFV). A better understanding of CSFV replication is important for the study of pathogenic mechanism of CSF. With the development of novel RNA in situ Hybridization method, quantitatively localization and visualization of the virus RNA molecular in cultured cell or tissue section becomes very important tool to address these pivotal pathogenic questions. In this study, we established ViewRNA ISH method to reveal the dynamic distribution of CSFV RNA in PK15 cells. Methods We designed several specific probes of CSFV RNA and reference gene β-actin for host PK15 cells to monitor the relative location of CSFV RNA and house-keeping gene in the infected cells. After determining the titer of reference strain CSFV (HeBHH1/95) with the 50% tissue culture infective dose (TCID50), we optimized the protease K concentration and formalin fixation time to analyze the hybridization efficiency, fluorescence intensity and repeatability. In order to measure the sensitivity of this assay, we compared it with the fluorescent antibody test (FAT) and immunohistochemical(IHC) method. Specificity of the ViewRNA ISH was tested by detecting several sub genotypes of CSFV (sub genotype 1.1, 2.1, 2.2 and 2.3) which are present in China and other normal pig infectious virus (bovine viral diarrhea virus (BVDV), porcine parvovirus (PPV), porcine pseudorabies virus (PRV) and porcine circovirusII(PCV-2). Results The lowest detection threshold of the ViewRNA ISH method was 10−8, while the sensitivity of FAT and IHC were 10−5 and 10−4, respectively. The ViewRNA ISH was specific for CSFV RNA including 1.1, 2.1, 2.2 and 2.3 subtypes, meanwhile, there was no cross-reaction with negative control and other viruses including BVDV, PPV, PRV and PCV-2. Our results showed that after infection at 0.5 hpi (hours post inoculation, hpi), the CSFV RNA can be detected in nucleus and cytoplasm; during 3–9 hpi, RNA was mainly distributed in nucleus and reached a maximum at 12hpi, then RNA copy number was gradually increased around the cell nucleus during 24–48 hpi and reached the peak at 72hpi. Conclusions To our knowledge, this is the first to reveal the dynamic distribution of medium virulence CSFV RNA in PK15 cells using the ViewRNA ISH method. The sensitivity of the ViewRNA ISH was three to four orders of magnitude higher than that of FAT and IHC methods. The specificity experiment showed that the ViewRNA ISH was highly specific for CSFV and no cross-reaction occurred to negative control and other pig infectious virus. This assay is more suitable for studying the CSFV RNA life cycle in cell nucleus. The results proved that CSFV RNA enters into PK15 cells earlier than 0.5hpi, relative to the eclipse period of cytoplasm is 6–9 hpi and CSFV RNA has ever existed in nucleus.http://link.springer.com/article/10.1186/s12985-017-0734-4Bovine Viral Diarrhea VirusClassical Swine Fever VirusPK15 CellClassical Swine FeverDynamic Distribution |
| spellingShingle | Qianyi Zhang Lu Xu Yujie Zhang Tuanjie Wang Xingqi Zou Yuanyuan Zhu Yan Zhao Cui Li Kai Chen Yongfang Sun Junxiang Sun Qizu Zhao Qin Wang A novel ViewRNA in situ hybridization method for the detection of the dynamic distribution of Classical Swine Fever Virus RNA in PK15 cells Virology Journal Bovine Viral Diarrhea Virus Classical Swine Fever Virus PK15 Cell Classical Swine Fever Dynamic Distribution |
| title | A novel ViewRNA in situ hybridization method for the detection of the dynamic distribution of Classical Swine Fever Virus RNA in PK15 cells |
| title_full | A novel ViewRNA in situ hybridization method for the detection of the dynamic distribution of Classical Swine Fever Virus RNA in PK15 cells |
| title_fullStr | A novel ViewRNA in situ hybridization method for the detection of the dynamic distribution of Classical Swine Fever Virus RNA in PK15 cells |
| title_full_unstemmed | A novel ViewRNA in situ hybridization method for the detection of the dynamic distribution of Classical Swine Fever Virus RNA in PK15 cells |
| title_short | A novel ViewRNA in situ hybridization method for the detection of the dynamic distribution of Classical Swine Fever Virus RNA in PK15 cells |
| title_sort | novel viewrna in situ hybridization method for the detection of the dynamic distribution of classical swine fever virus rna in pk15 cells |
| topic | Bovine Viral Diarrhea Virus Classical Swine Fever Virus PK15 Cell Classical Swine Fever Dynamic Distribution |
| url | http://link.springer.com/article/10.1186/s12985-017-0734-4 |
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