Mary Pudney

Cell lines from larvae of Aedes (Stegomyia) malayensis Colless and Aedes (S) pseudoscutellaris (Theobald) and their infection with some arboviruses.

Abstract The establishment of 2 cell lines from first stage larvae of the mosquitoes, Aedes malayensis and Aedes pseudoscutellaris is described. The cells of the A. malayensis line are diploid while those of A. pseudoscutellaris are predominantly polyploid. West Nile and Japanese B encephalitis viruses produced a cytopathic effect (CPE) in the A. malayensis cells. Both these viruses and Dengue-2 virus also produced a cytopathic effect in the A. pseudoscutellaris cells. There was some evidence that the CPE was influenced by the nature of the container. A. malayensis cells of early subcultures grown in Falcon plastic flasks formed large syncytia while those of later subcultures infected under identical conditions produced only small syncytia. Cells of the A. malayensis line grown on glass did not show any cytopathic response in early subcultures, but in cells of higher subcultures grown on glass, the viruses produced a definite CPE presenting as degenerate clumps of cells and floating single cells. It is suggested that the different result is due to continual adaptation of the cells to the culture conditions in the early stages. Cells of the A. pseudoscutellaris line, following infection with viruses formed syncytia, clearly visible on plastic surfaces, but less obvious on glass. An unadapted strain of Dengue-2 in infected human serum produced syncytia in the cells, but did not produce any plaques in a stable line in pig kidney cells. The infected cultures recovered after some days and formed healthy monolayers. Such cells were subcultured without showing any cytopathic response, but continued to produce virus.

Anopheles stephensi var. mysorensis: Establishment of a larval cell line (Mos. 43)

Abstract The establishment of a cell line, mosquito tissue culture No. 43 (Mos. 43), derived from first stage larvae of Anopheles stephensi var. mysorensis is described. In the primary culture, cells from the cut ends of the larval pieces suspended in the medium grew out on to the surface of the glass container. The larval pieces continued to produce new viable cells for at least 14 months after they were set up in culture. Although the primary culture contained epithelial-type, fibroblast-type, and occasional giant cells, the proportions of these types altered during serial subcultures until by about the 15th subculture, the majority of the cells were of the fibroblasttype and the giant cells had been greatly reduced in numbers. During the period of active growth, the cells had an estimated population-doubling time of 16 hr and a monolayer was usually formed in 2–4 days. With further increase in cell numbers as the cultures grew older, cells piled up in places producing a network type of layer. The majority of cells retained the normal diploid (2 n = 6) number of chromosomes. The cells have undergone 52 serial subcultures.

Cytopathic effect and plaque formation by arboviruses in a continuous cell line (XTC-2) from the toad Xenopus laevis.

Forty-six arboviruses were tested for c.p.e. and/or plaque formation in an amphibian cell line. C.p.e. was observed with a high proportion of the viruses tested. Comparative plaque assay, in the XTC-2 cells at 28 degrees C and Vero cells at 37 degrees C, suggests that these systems are comparable in sensitivity and susceptibility to infection. Practical uses of this cell line are discussed.

Isolations in a Mosquito (Aedes pseudoscutellaris) Cell Line (Mos. 61) of Yellow Fever Virus Strains from Original Field Material

A simple, rapid and inexpensive method of isolating yellow fever (YF) virus from naturally infected mosquitoes, human liver and the serum of a sentinel monkey by inoculation of a continuous line of mosquito cells is described. The mosquito cells were more sensitive than suckling mice and marginally better than Vero cells for primary isolation. This is the first time that mosquito cells have been successfully used for primary isolation of YF virus from field material.

Establishment and characterization of a cell line (BTC-32) from the triatomine bug, Triatoma infestans (Klug) (Hemiptera: Reduviidae).

The establishment of a cell line from embryonic tissues of the triatomine bug, Triatoma infestans is described. The cell line consists of three cell types which are described, and has a population doubling time of 48 hours during the logarithmic phase of growth. A proportion of the cells remain in the medium as floaters. Of the chromosome preparations that could be counted, the majority contained the diploid (2n = 22) number. The cell line has undergone 129 subcultures and has been maintained for over three years. The potential use of these cells in the study of Trypanosoma cruzi is described.

The culture of embryonic cells from the bug Triatoma maculata (Erichson) (Hemiptera: Reduviidae)

Abstract The culture of embryonic cells of a blood-sucking bug, Triatoma maculata, is described. The culture medium was a modification of one used for cultivating tissues from the culicine mosquito, Culex pipiens var. molestus. Good dissociation of the embryonic cells was obtained by using a 0.1 per cent solution of the protease, Pronase. Frequent mitoses were observed at least up to 40 days. Cultures were maintained in a healthy condition up to this period after which they started degenerating. Attempts at subculturing the cells were unsuccessful.

Characterization of Kawino Virus, an Entero-like Virus Isolated from the Mosquito Mansonia uniformis (Diptera: Culicidae)

Summary Some properties of a small RNA virus isolated from Mansonia uniformis are described. The virus particles have a sedimentation coefficient of about 165S, buoyant density in CsCl of 1.33 g/ml and diameter of 28 nm. They contain a single-stranded RNA which probably lacks poly (A) but is infective. Four polypeptides with mol. wt. of 33, 30, 27 and about 7 × 103 are present in the virus particle.

The growth of some tick-borne arboviruses in cell cultures derived from tadpoles of the common frog, Rana temporaria.

Summary Primary cell cultures and a line consisting of fibroblast-type cells were obtained from tadpoles of the common frog, Rana temporaria. One cell line was taken through 21 subcultures during 5 months, when it was abandoned due to contamination by an anonymous Mycobacterium which was completely resistant to antibiotics. Cells from the primary cultures supported the growth of three tick-borne arboviruses without any cytopathic effect; Quaranfil, louping ill (two strains), and Langat. One of the virus strains, louping ill 369t2, was serially passaged in the cell cultures 11 times in 11 weeks, during which time there was considerable multiplication of virus.