Isolation of the Human Cytomegalovirus from bodily fluids

Abstract

In vitro studies on the pathogenesis of the human cytomegalovirus (HCMV) are conducted regularly using laboratory adapted strains that lose some characteristics during the adaptation process. Since HCMV is excreted from bodily fluids during infection or reactivation, this work aimed to isolate and culture HCMV from the MRC-5 human cells found in the urine, bronchoalveolar lavage, saliva, and plasma samples of pediatric patients with probable or confirmed infection. The samples were inoculated on cell cultures either for 14 days or until a cytopathic effect (CPE) of 80 % was observed. The cell lysates and supernatants were used to perform successive viral passages. Besides HCMV, the herpes simplex virus was detected from all the saliva samples. Inoculation of the HCMV positive sera induced cell clustering and immediate monolayer damage that restricted their use. One sample of bronchoalveolar lavage induced a CPE after inoculation like that of the HCMV reference strains (Towne and Merlin), which was consequently propagated and titrated. A second viral isolate derived from the urine sample of a patient with congenital infection did not demonstrate a CPE, although presence of the virus had been confirmed using PCR. The viral isolates were examined and found to be negative for adenoviruses or enteroviruses. Despite the evident difficulty encountered for the isolation and harvesting of the HCMV, this work shows that it was possible to obtain a low passage viral strain using a modified shell vial method and inoculation protocol with extended follow-up and confirmation.