Simian rotavirus SA-11 plaque formation in the presence of trypsin

Abstract

Incorporation of 5 jig of trypsin per ml of the overlay (Eagle minimal essential medium-0.7% lonagar no. 2) was found to be necessary for plaque formation by simian rotavirus SA-11. Plaques of 3 to 4 mm in diameter were produced in MA-104 cells after 5 days of incubation at 37°C. Plaque size was even larger (5 to 6 mm) in monolayers of African green monkey kidney cells. Addition of diethylaminoethyl-dextran, protamine sulfate, or 5-bromodeoxyuridine to the trypsincontaining overlay did not improve plaque formation by the virus. Incorporation of beef extract or yeast extract to a final concentration of 0.5% in the trypsincontaining overlay inhibited plaque formation. On the other hand, the presence of lactalbumin hydrolysate or peptone at a similar concentration in the overlay did not inhibit plaque formation. When methylcellulose was used instead of the agar as the solidifying agent in the overlay, no plaques were seen. SA-11 is a useful model for the study of human rotaviruses, and this relatively simple plaque assay system should further enhance its usefulness in this regard.

Isolation and characterization of a canine rotavirus

“…Rotaviruses are, in general, difficult to isolate and to grow in cell cultures using conventional techniques. However, certain strains may be serially propagated in cell cultures if the virus inuculum is treated with proteolytic enzymes or if proteolytic enzymes are incorporated into the medium after infection (1,2,3,6,16,40,41,44,45,49,56). ENGI, A~D and POSTON (9) reported that rotavirus observed in feces by E?¢[ from a diarrheal dog grew in cell cultures with the aid of trypsin, but did not grow in cell cultures without trypsin treatment.…”

Development of methods to study the survival of airborne viruses

“…The procedure for fixation and staining of the monolayer before counting pfaques has been described earlier (Ramia and Sattar, 1979). Plaque assays of HCV 229E were performed by standard method in monolayers of L-132 cells in 75 cm' disposable culture flasks (Lux, Scientific CJtrrp.).…”

“…Poliovirus couId readily grow and form countable plaques in MA-104 cek Kotaviruses are also able to form plaques in MA-104 cells but only in the presence of trypsin in the overlay medium (Ramia and Sattar, 1979). Therefore.…”

Plaque formation by avian infectious bronchitis virus in primary chick embryo fibroblast cells in the presence of trypsin

“…We further showed that the addition of trypsin to the overlay medium enables other strains of IBV to produce plaques in CE cells. In many other investigators (1, 6,7,8,11,12,13,14,15), trypsin has been incorporated into the overlay medium at a concentration of 10 9g per ml. The results of the present investigation, however, show that the incorporation of 20--40 y.g of trypsin per ml of overlay is optimal for plaque formation by IBV.…”

“…Trypsin markedly enhances the in vitro infectivity of various viruses, i.e. rotavirus (11,12), influenza viruses (1, 2,8,15), Sendal virus (7), reovirus (13) and vaeeinia virus (6). Quite recently, S:ro~z et al (14) reported that trypsin enhanced plaque formation of an enteropathogenic bovine coronavirus in bovine fetal thyroid and bovine fetal brain cells.…”