Identification of infected B-cell populations by using a recombinant murine gammaherpesvirus 68 expressing a fluorescent protein.
Infection of inbred mice with murine gammaherpesvirus 68 (MHV68) has proven to be a powerful tool to study gammaherpesvirus pathogenesis. However, one of the limitations of this system has been the inability to directly detect infected cells harvested from infected animals. To address this issue, we generated a transgenic virus that expresses the enhanced yellow fluorescent protein (YFP), driven by the human cytomegalovirus immediate-early promoter and enhancer, from a neutral locus within the viral genome. This virus, MHV68-YFP, replicated and established latency as efficiently as did the wild-type virus. During the early phase of viral latency, MHV68-YFP efficiently marked latently infected cells in the spleen after intranasal inoculation. Staining splenocytes for expression of various surface markers demonstrated the presence of MHV68 in distinct populations of splenic B cells harboring MHV68. Notably, these analyses also revealed that markers used to discriminate between newly formed, follicular and marginal zone B cells may not be reliable for phenotyping B cells harboring MHV68 since virus infection appears to modulate cell surface expression levels of CD21 and CD23. However, as expected, we observed that the overwhelming majority of latently infected B cells at the peak of latency exhibited a germinal center phenotype. These analyses also demonstrated that a significant percentage of MHV68-infected splenocytes at the peak of viral latency are plasma cells (ca. 15% at day 14 and ca. 8% at day 18). Notably, the frequency of virus-infected plasma cells correlated well with the frequency of splenocytes that spontaneously reactivate virus upon explant. Finally, we observed that the efficiency of marking latently infected B cells with the MHV68-YFP recombinant virus declined at later times postinfection, likely due to shut down of transgene expression, and indicating that the utility of this marking strategy is currently limited to the early stages of virus infection.
Collins
Christopher M
CM
Emory Vaccine Center, Atlanta, Georgia 30322, USA.
Boss
Jeremy M
JM
Speck
Samuel H
SH
D000818:Animals; D001402:B-Lymphocytes / Q000821:virology*; D001426:Bacterial Proteins / Q000378:metabolism*; D002460:Cell Line; D005260:Female; D005434:Flow Cytometry; D018858:Germinal Center / Q000166:cytology / Q000821:virology; D006566:Herpesviridae Infections / Q000821:virology*; D008164:Luminescent Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D016254:Mutagenesis, Insertional; D018144:Rhadinovirus / Q000235:genetics / Q000378:metabolism*; D013154:Spleen / Q000166:cytology / Q000821:virology; D010948:Viral Plaque Assay
D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria