Abstract
Infection by human cytomegalovirus (CMV) elicits a strong humoral immune
response and robust anti-CMV antibody production. Diagnosis of virus
infection can be carried out by using a variety of serological assays;
however, quantification of serum antibodies against CMV may not present
an accurate measure of a patient's ability to control a virus infection.
CMV strains that express green fluorescent protein (GFP) fusion proteins
can be used as screening tools for evaluating characteristics of CMV
infection in vitro. In this study, we employed a CMV virus strain,
AD169, that ectopically expresses a yellow fluorescent protein (YFP)
fused to the immediate-early 2 (IE2) protein product (AD169(IE2-YFP)) to
quantify a CMV infection in human cells. We created a high-throughput
cell-based assay that requires minimal amounts of material and provides
a platform for rapid analysis of the initial phase of virus infection,
including virus attachment, fusion, and immediate-early viral gene
expression. The AD169(IE2-YFP) cell infection system was utilized to
develop a neutralization assay with a monoclonal antibody against the
viral surface glycoprotein gH. The high-throughput assay was extended to
measure the neutralization capacity of serum from CMV-positive subjects.
These findings describe a sensitive and specific assay for the
quantification of a key immunological response that plays a role in
limiting CMV dissemination and transmission. Collectively, we have
demonstrated that a robust high-throughput infection assay can analyze
the early steps of the CMV life cycle and quantify the potency of
biological reagents to attenuate a virus infection.
