Charles Buck

APPLICATION OF PCR FOR DETECTION AND IDENTIFICATION OF MYCOPLASMA CONTAMINATION IN VIRUS STOCKS

SummaryA nested polymerase chain reaction (PCR) was used to detect and identify mycoplasma contaminants in viral stocks. The results of the PCR assay proved to be a sensitive and accurate indicator of the true status of the stock tested. Those samples positive by agar culture or Hoechst stain were also positive by PCR. Those samples that were inconclusive by Hoechst stain (10.05%) could be clearly determined to be mycoplasma positive or negative by PCR. The PCR assay also detected those fastidious species of mycoplasma that gave false negative results by the direct culture method. In many respects the PCR-based mycoplasma detection method described is superior to the agar culture and Hoechst staining detection methods. In this study, the PCR assay detected substantially more mycoplasma-positive viral stocks than did the agar culture assay. Due to its speed, sensitivity, and reliability, the PCR assay is of particular value in monitoring the process of removing mycoplasma from contaminated stocks. Furthermore, the PCR amplification products can be analyzed by restriction analysis to rapidly identify the species of the mycoplasma contaminating the stock tested.

Isolation of St. Louis encephalitis virus from a killer whale.

We report the isolation of St. Louis Encephalitis (SLE) virus from a mature male killer whale (Orcinus orca). This represents the first isolation of SLE virus from a marine mammal. The animal presented with reduced appetite, rapidly became lethargic and subsequently died. Virus-induced CPE was observed in a dolphin cell line, SP-1K (ATCC CCL 78), inoculated with brain, kidney, and lung tissues obtained at necropsy. Electron microscopy of infected SP-1K cells revealed the presence of virions having morphology and size resembling members of the Flaviviridae. Final identification as SLE virus was made by neutralization and immunofluorescence staining tests.

Detection of mycoplasma contamination in viral stocks by a polymerase chain reaction technique

A polymerase chain reaction (PCR) amplification procedure is described for the detection of mycoplasma contaminants in virus stocks. The method uses primers based upon the DNA coding sequence for mycoplasma 16S and 23S ribosomal RNA. The primers evaluated amplified DNA from commonly encountered mycoplasma contaminants to levels readily detectable on ethidium-stained electrophoresis gels. The same primers did not yield detectable DNA bands when human DNA, mouse DNA, and DNA from related genera of bacteria were amplified. The assay is specific and sensitive and useful in testing a wide range of viral stocks for the presence of mycoplasma contaminants. This PCR-based mycoplasma detection test has proven capable of quickly clarifying the status of viral stocks that occasionally yield indeterminate results when tested for mycoplasma by the Hoechst-staining method.

Cell Cultures and Retroviral Particles From a Tumor of a Moray Eel

Abstract: Until recently, fish cell culture primarily has been useful only in the propagation and study of epidemic viruses significant to the fishing industry. Such fish cell lines derived were developed by appropriating classical techniques of mammalian cell culture, with serum as the major growth supplement. Using an approach in which culture medium is formulated in a cell-type-specific manner with minimal serum and a variety of synergistic supplements, several fish cell lines have been derived that may serve multiple uses. We established cell lines from a potentially tumorous skin lesion of a green moray eel (Gymnothorax funebris) and control tissues, and identified putative retroviral particles in the medium from the tumor cells that are not present in medium from cultures of normal cells from the same eel. The relationship between the virus and the cause of the tumor is not clear, but the genomic structure of this virus should provide useful information in understanding the evolution of retroviruses in general.

Cell Cultures and Reverse Transcriptase Activity From a Tumor of a Moray Eel

This work arose from observations of scientists at SeaWorld, Orlando, of nodular, granulomatous tumor-like skin lesions on a number of captive green moray eels Gymnothorax funebris), as well as eels freshly caught from the reef areas around the Florida coast. Homogenates of the lesions were capable of passing the disease when injected into green morays and other anguilliformes. Cultures of tumor-derived and normal moray eel cells were derived and have been continually passaged for more than a year. Stable expression of reverse transcriptase activity was detected in conditioned culture medium from the tumor-derived cells, with no activity detected in controls. Assays of extracts from frozen tissue samples taken at the time of culture initiation showed concentration dependent activity in the tumor, with little or no activity in extracts of ovary, liver and head kidney from the same animal. Conditioned medium from the tumor cells was centrifuged at 27,000 X g (2 hr), and reverse transcriptase activity was found in the pelleted material. When this material was transferred to a 25%–50% sucrose gradient and centrifuged, particles banded at a density of 1.17–1.19 g/ml.