Shell Vial Cultures (Centrifugation-Enhanced Culture)

This method allows for rapid detection viruses in clinical specimens. It has been adapted
for saveral viruses, including cytomegalovirus (CMV) can bee detected in 18-24 hours,
compared with 2-4 weeks for classic cell culture; the sensitivities of shell vial and classic
cell cultures for CMV are comparable. Monolayers of the appropriate cell line (eg. MRC-
5 cells for CMV) are grown on converslips in 15 X 45-mm 1-dram shell vials. After
inoculation with the specimen, the vials are centrifuged at 700 X g for 40 minutes at
room temperature. The vials are incubated at 37 C for 16-24 hours, fixed, and reacted
with a monoclonal antibody specific for a CMV nuclear protein that is present very carly
in the culture; several such antibodies are commercially avaible. Direct or indirect
antibody staining methods and fluorescence microscopy are used to determine positive
shell vial cultures. Positive and negative control vials technique has been developed to
allow the simultaneous recovery and detection of multiple respiratory viruses using R-
Mix cells. This method typically is available through a commercial company
Quided/Diagnostic Hybrids Inc. (Athens, OH) one vial contain (mixes) two cell lines such
as human lung carcinoma A549 and mink lung fibroblast MviLu cells. The laboratory will
typically inoculate two such vials. After 18-24 hours incubation, one vial is stained using
a pooled immunofluorescent antibody reagent that detects all of the common
respiratory viruses. If the stain is positive, then the cells on the coverslip of second vial
are sraped, inoculated onto an eight-well slide, and thenstained with individual
monoclonal antibody reagents that detect the specific virus. Isolates are not obtained
using the shell vial technique. If isolates are needed for susceptibility testing for antiviral
drugs, the classic cell culture technique should be used.

Enzyme-Linked Virus-Inducible System (ELVIS)

This proprictary cell line (Quided/Diagnotic Hybrids, Inc.) is a novel system used to detect
herpes simplex viruses (HSV) in culture. A baby hamster kidney cell line was genetically
engineeral using the promoter sequence of the HSV UL.97 geneand the E.coli lacz gene.
When herpes viruses arepresent in clinical samples, they activate the UL97 promooter,
which antivates the lacZ gene to produce the enzyme -galactosidase. When a subtrate
for the enzyme is added, a blue color is produced indicating the presence of virus. HSV
virus typing can be perfomed on the positive cultures by adding monoclonal antibodies
that detect HSV-2 for example.