Rotavirus isolation and cultivation in the presence of trypsin

Abstract

Rotaviruses are generally difficult to isolate and culture in vitro; therefore, virus isolation has not been used as a method of diagnosing this group of agents. The present report describes a simple procedure for isolating bovine rotaviruses directly from feces after pretreatment of fecal samples with trypsin. This procedure resulted in virus isolation from five of five samples that contained virus particles, as demonstrated by electron microscopy, and four of seven samples where virus particles could not be observed but were considered positive by the presence of immunofluorescent-staining cells in feces. Virus could not be isolated from "normal" feces. If the virus was not passaged in the presence of trypsin, the infectivity was gradually lost, but infectivity could be restored again if trypsin was added, resulting in increased virus spread and concomitant increase in virus yield. The application of this technique as a diagnostic tool for bovine and other rotaviruses is briefly discussed.

Isolation, Propagation and Genotyping of Human Rotaviruses Circulating among Children with Gastroenteritis in Two Egyptian University Hospitals

“…For further propagation, the infected cells were freeze-thawed three times and inoculated onto fresh confluent monolayer cells for another passage after virus activation with treated trypsin as described above. Usually, RV CPE is observed after multiple passages, however, the absence of CPE after six passages, considering RV isolation negative [ 29 , 30 ]. Cell monolayers were photographed using an inverted microscope (Olympus Corporation) equipped with a digital camera.…”