Shell Vial Assay for Culture of Vaccinia Virus

Clinical Center

Investigators

Abstract

Diagnosis of smallpox vaccination complications may be difficult in some patients because signs and symptoms may resemble other infectious (e.g. varicella-zoster virus, herpes simplex virus) or noninfectious causes (e.g. allergic dermatitis, drug rash illnesses). To support 2 Clinical Center protocols we developed a rapid shell vial culture assay for detection of vaccinia virus in patient specimens. The Wyeth strain of Vaccinia virus grew equally well at 35C for 24 and 48 hours in HeLa 229, MRC-5, A549, Mink Lung, HEp2, Vero, and RhMK cells, but grew very poorly in CHO cells. Centrifugation (3,500 x g for 15 min at 25C) of the inoculum onto shell vial HeLa 229 cell monolayers increased the sensitivity of the assay by 100 fold compared to uncentrifuged vials. Sonication of the inoculum improved virus recovery compared to an unsonicated virus suspension. We were able to detect infectious virus in the culture medium of shell vials at 24 hours post infection and we detected 100 fold higher virus titer in the culture medium of shell vials at 48 hours post infection. Fixation of cell monolayers before antibody staining did not inactivate the vaccinia virus. We conclude that the shell vial assay for culture of vaccinia virus with specimen sonication and inoculum centrifugation should be useful for rapid and sensitive diagnosis of complications due to smallpox vaccination. This work was presented at the 19th Annual Clinical Virology Symposium, Clearwater, FL, April 27-30, 2003.