Dataset Information

In situ hybridization detection and subtyping of rotaviruses in swine samples.

ABSTRACT: Rotavirus groups A, B, and C (RVA, RVB, and RVC, respectively) have been the most prevalent and pathogenic in pigs. To date, immunohistochemistry is only available for RVA because of the lack of commercial antibodies for RVB and RVC. We developed a novel in situ hybridization RNA-based chromogenic technique (ISH-RNA) to detect and subtype RVA, RVB, and RVC. We evaluated 33 samples that were reverse-transcription PCR positive for RVA, RVB, and/or RVC. ISH-RNA was able to detect as few as 103 RV RNA copies/mL. The new ISH-RNA test can be useful for routine investigation of rotavirus enteritis in order to guide strategies for control of the infection in pigs, but a full validation study needs to be completed. Pathogenesis studies may be conducted using ISH-RNA based on the identification of replicating virus.

SUBMITTER: Resende TP

PROVIDER: S-EPMC6505766 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Publications

In situ hybridization detection and subtyping of rotaviruses in swine samples.

Resende Talita P TP Marthaler Douglas D Vannucci Fabio A FA

Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 20181212 1

Rotavirus groups A, B, and C (RVA, RVB, and RVC, respectively) have been the most prevalent and pathogenic in pigs. To date, immunohistochemistry is only available for RVA because of the lack of commercial antibodies for RVB and RVC. We developed a novel in situ hybridization RNA-based chromogenic technique (ISH-RNA) to detect and subtype RVA, RVB, and RVC. We evaluated 33 samples that were reverse-transcription PCR positive for RVA, RVB, and/or RVC. ISH-RNA was able to detect as few as 10<sup>3 ...[more]

PMID: 30541408

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Project description:BACKGROUND: Dengue fever (DF) and dengue hemorrhagic fever (DHF) represent a global challenge in public health. It is estimated that 50 to 100 million infections occur each year causing approximately 20,000 deaths that are usually linked to severe cases like DHF and dengue shock syndrome. The causative agent of DF is dengue virus (genus Flavivirus) that comprises four distinct serotypes (DENV-1 to DENV-4). Fluorescence in situ hybridization (FISH) has been used successfully to detect pathogenic agents, but has not been implemented in detecting DENV. To improve our understanding of DENV infection and dissemination in host tissues, we designed specific probes to detect DENV in FISH assays. METHODS: Oligonucleotide probes were designed to hybridize with RNA from the broadest range of DENV isolates belonging to the four serotypes, but not to the closest Flavivirus genomes. Three probes that fit the criteria defined for FISH experiments were selected, targeting both coding and non-coding regions of the DENV genome. These probes were tested in FISH assays against the dengue vector Aedes albopictus (Diptera: Culicidae). The FISH experiments were led in vitro using the C6/36 cell line, and in vivo against dissected salivary glands, with epifluorescence and confocal microscopy. RESULTS: The three 60-nt oligonucleotides probes DENV-Probe A, B and C cover a broad range of DENV isolates from the four serotypes. When the three probes were used together, specific fluorescent signals were observed in C6/36 infected with each DENV serotypes. No signal was detected in either cells infected with close Flavivirus members West Nile virus or yellow fever virus. The same protocol was used on salivary glands of Ae. albopictus fed with a DENV-2 infectious blood-meal which showed positive signals in the lateral lobes of infected samples, with no significant signal in uninfected mosquitoes. CONCLUSION: Based on the FISH technique, we propose a way to design and use oligonucleotide probes to detect arboviruses. Results showed that this method was successfully implemented to specifically detect DENV in a mosquito cell line, as well as in mosquito salivary glands for the DENV-2 serotype. In addition, we emphasize that FISH could be an alternative method to detect arboviruses in host tissues, also offering to circumvent the discontinuity of antibodies used in immunofluorescent assays.

Project description: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.We designed several specific probes of CSFV RNA and reference gene ?-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).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.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.

Dataset Information

In situ hybridization detection and subtyping of rotaviruses in swine samples.

Publications

In situ hybridization detection and subtyping of rotaviruses in swine samples.

Similar Datasets

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