Development of an in-situ hybridization assay using riboprobes for detection of viral haemorrhagic septicemia virus (VHSV) mRNAs in a cell culture model.

Autor: Qadiri SSN; Department of Aqualife Medicine, College of Fisheries and Ocean Science, Chonnam National University, Yeosu, 59626, Republic of Korea., Kim SJ; Department of Aqualife Medicine, College of Fisheries and Ocean Science, Chonnam National University, Yeosu, 59626, Republic of Korea., Krishnan R; Department of Aqualife Medicine, College of Fisheries and Ocean Science, Chonnam National University, Yeosu, 59626, Republic of Korea., Kim JO; Department of Aqualife Medicine, College of Fisheries and Ocean Science, Chonnam National University, Yeosu, 59626, Republic of Korea., Kim WS; Department of Aqualife Medicine, College of Fisheries and Ocean Science, Chonnam National University, Yeosu, 59626, Republic of Korea., Oh MJ; Department of Aqualife Medicine, College of Fisheries and Ocean Science, Chonnam National University, Yeosu, 59626, Republic of Korea. Electronic address: ohmj@jnu.ac.kr.
Jazyk: angličtina
Zdroj: Journal of virological methods [J Virol Methods] 2019 Feb; Vol. 264, pp. 1-10. Date of Electronic Publication: 2018 Nov 08.
DOI: 10.1016/j.jviromet.2018.11.003
Abstrakt: An in situ hybridization (RNA-ISH) assay has been developed and optimized to detect viral haemorrhagic septicemia virus (VHSV), an OIE listed piscine rhabdovirus, in infected fish cells using fathead minnow (FHM) as a model cell line. Two antisense riboprobes (RNA probes) targeting viral transcripts from a fragment of nucleoprotein (N) and glycoprotein (G) genes were generated by reverse transcription polymerase chain reaction (RT-PCR) using VHSV specific primers followed by a transcription reaction in the presence of digoxigenin dUTP. The synthesized RNA probes were able to detect viral mRNAs in formalin fixed VHSV infected FHM cells at different time points post inoculation (pi). To correlate the signal intensity, a time dependent quantitation of the viral mRNA transcript and infectivity titer was done by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and 50% tissue culture infectivity dose (TCID 50 ), respectively, from the infected cells and culture supernatants. Further, we compared the diagnostic sensitivity of ISH assay with immunocytochemistry (ICC). Both the riboprobes used in the ISH assay detected VHSV as early as 6 hpi in the FHM cells inoculated with a multiplicity of infection (moi) of 2. Also, the signal detection in ISH was at an early stage in comparison to ICC, wherein, signal was first detected at 12 hpi. Our results clearly highlight that current ISH assay can be of value as a diagnostic tool to localize and detect VHSV in conjunction with conventional virus isolation in cell culture.
(Copyright © 2018 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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