Virus isolation by cell culture

Samples are typically added to susceptible cell lines, such as EPC, CHSE-214 or FHM. Methods used for detecting and determining the quantity of IHNV in the sample are end-point titrations and plaque assays. The plaque assay is more sensitive than end-point titrations (Fendrick et al., 1982; Leong et al., 1983a). The American Fisheries Society 'Blue Book' (Amos, 1985; Ganzhorn and LaPatra, 1994) and the Manual of Compliance of the Department of Fisheries and Oceans (1984) in Canada recommend that two cell lines (pH 7.6) are inoculated when 80-90% confluent and incubated at 15°C for a minimum of 14 days for end point dilutions or 7 days for plaque assays.

For plaque assays, the inoculum volume should be adjusted to reflect the size of the cell-culture well, since using too high a volume can reduce the efficiency of plaquing (Burke and Mulcahy, 1980; Batts et al., 1991). Viral adsorption is optimal after 1 h; shorter times reduce the detectable viral titre and longer times have no effect on the number of plaques (Burke and Mulcahy, 1980; Brunson et al., 1988; Batts and Winton, 1989a,b; Batts et al., 1991). Plaques are larger when cells are overlaid with methyl cellulose, but gum tragacanth or agarose at a pH of 7.2-8.0 can be used (Wolf and Quimby, 1973; Burke and Mulcahy, 1980; Okamoto et al, 1985; Batts et al, 1991).

Cells should be examined daily for cytopathic effect (CPE). If no CPE occurs, then the sample is virus negative, especially if the sample has been passed in tissue culture several times (blind passaged). If there is CPE characteristic of IHNV, then a presumptive diagnosis of IHNV is made. Typical CPE in cell cultures consists of grape-like clusters of rounded cells, with margination of the chromatin of the nuclear membrane (Wolf, 1988). Typical IHNV plaques consist of a cell sheet that retracts or piles up at the inner margins of the opening and the centre contains granular debris (Wolf and Quimby, 1973).

Attempts have been made to enhance the sensitivity of viral detection in cell cultures. Pretreatment of cell monolayers with polybrene, a polycation that enhances the ionic attraction of viruses to cells, increased titres of a laboratory strain of IHNV by five- to tenfold (Leong et al., 1981b) but did not affect titres of field isolates of IHNV assayed by plaque assays or end-point dilution (Fendrick et al., 1982). Pretreatment of cell monolayers with 7% polyethylene glycol (PEG, 20,000 Mr) improved the speed and sensitivity of plaque assays for the quantification of low levels of IHNV and resulted in the production of larger plaques (Batts and Winton, 1989a,b; Batts et al., 1991). Polyethylene glycol can also be added directly to the antibiotic solution that samples are incubated in, prior to inoculation on to cells (Brunson et al., 1988). The discovery of the effectiveness of PEG pretreatment is an important advance in the detection of IHNV in cell culture. Once a virus is detected in cell culture, its identity must be confirmed.

Was this article helpful?

0 0

Post a comment