Non-permissive C6/36 cell culture for the Australian isolate of Macrobrachium rosenbergii nodavirus

Hayakijkosol, O., and Owens, L. (2013) Non-permissive C6/36 cell culture for the Australian isolate of Macrobrachium rosenbergii nodavirus. Journal of Fish Diseases, 36 (4). pp. 401-409.

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Abstract

Macrobrachium rosenbergii nodavirus (MrNV) that causes white tail disease (WTD) is an emerging disease that contributes to serious production losses in Macrobrachium hatcheries worldwide. Mosquito cell lines (C6/36) have been reported to support the growth of MrNV and used to observe the cytopathic effects (CPE) in infected cells. This study determined the susceptibility of C6/36 mosquito cells to the Australian isolate of MrNV in order to use fewer animals in further investigations. Different staining methods were used to observe MrNV viral activity in C6/36 cells. Typical cytopathic effects such as vacuolation and viral inclusion bodies were observed in infected C6/36 cells with H&E and Giemsa staining. With acridine orange, it was easier to detect presumptive MrNV messenger ribonucleic acid in the infected cells. Using neutral red staining to measure mitochondrial activity showed light absorption of infected cells maximized at day 4 (O.D. = 0.6) but was significantly lower (chi-square = 41.265, df = 1, P < 0.05) than control groups (O.D. = 2) which maximized at day 12. Using trypan blue staining to count the number of cells with disrupted cell membranes, the maximum number of presumptively dead cells at day 8 (4 × 10⁵ cells) in infected treatments was higher than the control treatment at day 10 (1.8 × 10⁵ cells). However, TaqMan real-time PCR did not confirm the replication of MrNV in the cells over 14 days. The mean viral copies and mean cycle times of positive samples were stable at 2.07 × 10⁴ and 24.12, respectively. Limited evidence of viral replication was observed during four serial passages. This study determined the mortality of the C6/36 cell line to the Australian isolate of MrNV but suggests limited patent replication was occurring. Trying different cell lines or adapting the virus to the C6/36 cells may be necessary to successfully replicate Australian MrNV in cell lines.

Item ID: 26098
Item Type: Article (Research - C1)
ISSN: 1365-2761
Keywords: C6/36 cell culture; Macrobrachium rosenbergii nodavirus; white tail disease
Date Deposited: 03 Apr 2013 23:07
FoR Codes: 06 BIOLOGICAL SCIENCES > 0605 Microbiology > 060506 Virology @ 50%
07 AGRICULTURAL AND VETERINARY SCIENCES > 0704 Fisheries Sciences > 070404 Fish Pests and Diseases @ 50%
SEO Codes: 83 ANIMAL PRODUCTION AND ANIMAL PRIMARY PRODUCTS > 8301 Fisheries - Aquaculture > 830101 Aquaculture Crustaceans (excl. Rock Lobster and Prawns) @ 100%
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