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Non‐permissive C 6/36 cell culture for the A ustralian isolate of M acrobrachium rosenbergii nodavirus
Author(s) -
Hayakijkosol O,
Owens L
Publication year - 2013
Publication title -
journal of fish diseases
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.819
H-Index - 85
eISSN - 1365-2761
pISSN - 0140-7775
DOI - 10.1111/j.1365-2761.2012.01414.x
Subject(s) - acridine orange , biology , cytopathic effect , staining , microbiology and biotechnology , trypan blue , cell culture , vero cell , virus , virology , cell , biochemistry , genetics
Macrobrachium rosenbergii nodavirus (Mr NV ) that causes white tail disease ( WTD ) is an emerging disease that contributes to serious production losses in M acrobrachium hatcheries worldwide. Mosquito cell lines ( C 6/36) have been reported to support the growth of Mr NV and used to observe the cytopathic effects ( CPE ) in infected cells. This study determined the susceptibility of C 6/36 mosquito cells to the A ustralian isolate of M r NV in order to use fewer animals in further investigations. Different staining methods were used to observe M r NV viral activity in C 6/36 cells. Typical cytopathic effects such as vacuolation and viral inclusion bodies were observed in infected C 6/36 cells with H & E and G iemsa staining. With acridine orange, it was easier to detect presumptive M r NV 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 × 10 5 cells) in infected treatments was higher than the control treatment at day 10 (1.8 × 10 5 cells). However, T aq M an real‐time PCR did not confirm the replication of M r NV in the cells over 14 days. The mean viral copies and mean cycle times of positive samples were stable at 2.07 × 10 4 and 24.12, respectively. Limited evidence of viral replication was observed during four serial passages. This study determined the mortality of the C 6/36 cell line to the Australian isolate of M r NV but suggests limited patent replication was occurring. Trying different cell lines or adapting the virus to the C 6/36 cells may be necessary to successfully replicate A ustralian M r NV in cell lines.