Background:
Cytomegalovirus (CMV) is an important pathogen in immunocompromised populations. Recent literature has shown that highly purified particles of the lab strain CMV AD169 contain five RNA transcripts. The delivery of these transcripts to cells could be important to CMV pathogenesis immediately following virus adsorption. Before examining this hypothesis directly, we chose to determine whether these transcripts are present in other CMV strains, including a low passage clinical isolate CL203. The objective of this is to detect the UL 21.5 and IRL/TRL 2-5 transcripts in CMV strains AD169, Towne, Davis and a low passage clinical isolate, CL203.

Methods:
The purification process involves ultracentrifugation through three different gradients. First, the clarified CMV-infected cell supernatant is concentrated through 20% D-sorbitol. A glycerol-potassium tartrate gradient is used to separate the virions from the dense bodies. Before the final gradient, particles recovered from the previous gradient are treated with RNase A and DNase I to digest nucleic acids that may be bound to the envelopes. Finally, a cesium chloride (CsCl) step gradient serves to remove the nucleases and further purify the particles. Electron microscopy is used to confirm the identity of the purified particles following each gradient. Endpoint dilution assays are performed to test the infectivity of the purified particles, and RNA is isolated for detection of the transcripts by RT-PCR.

Results:
The results for AD169 have confirmed the published data. The UL21.5 transcript has been consistently detected in CL203. The same transcript has been shown to absent from CMV Towne.

Conclusion:
The presence of the UL21.5 transcript in the clinical isolate shows the packaged transcripts are not unique to lab-adapted strains and suggests that they could be important in CMV pathogenesis.

 

Presented at:

42nd INTERSCIENCE CONFERENCE ON ANTIMICROBIAL AGENTS AND CHEMOTHERAPY (ICAAC), San Diego, California, USA, September 28, 2002. 11:00am to 12:30pm.