title: RT-qPCR-based microneutralization assay for human cytomegalovirus using fibroblasts and epithelial cells.
abstract: Human cytomegalovirus (HCMV) is a leading cause of congenital infection that can result in serious disabilities in affected children. To facilitate HCMV vaccine development, a microscale neutralization assay based on reverse transcription quantitative PCR (RT-qPCR) was developed to quantify HCMV-neutralizing antibodies. Our approach relies on the generation of crude lysates from virus-infected cells that are amenable to direct analysis by RT-qPCR, thereby circumventing rate-limiting procedures associated with sample RNA extraction and purification. By serial passaging of the laboratory HCMV strain AD169 in epithelial cells (ARPE-19), a revertant virus with restored epithelial cell tropism, designated AD169(wt131), was obtained. AD169 and AD169(wt131) were evaluated in both epithelial cells (ARPE-19) and fibroblasts (MRC-5) by one-step RT-qPCR targeting the immediate-early gene IE1 transcript of HCMV. Expression kinetics indicated that RT-qPCR assessment could be conducted as early as 6h post-infection. Human serum samples (n=30) from healthy donors were tested for HCMV-specific IgG using a commercially available ELISA and for HCMV-neutralizing activity using our RT-qPCR-based neutralization assay. In agreement with the ELISA results, higher neutralizing activity was observed in the HCMV IgG seropositive group when compared with the HCMV IgG seronegative group. In addition, HCMV IgG seropositive human sera exhibited higher neutralizing titers using epithelial cells compared with using fibroblasts (geometric mean titers of 344 and 8 in ARPE-19 cells and MRC-5 cells, respectively). Our assay was robust to variation in input virus dose. In addition, a simple lysis buffer containing a non-ionic detergent was successfully demonstrated to be a less costly alternative to commercial reagents for cell-lysate preparation. Thus, our rapid HCMV neutralization assay may be a straightforward and flexible high-throughput tool for measuring antibody responses induced by vaccination and natural infection.
authors.affiliation: Laboratory of DNA Viruses, Division of Viral Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA.
authors.forename: Xiao
authors.initials: X
authors.lastname: Wang
authors.affiliation: Laboratory of DNA Viruses, Division of Viral Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA.
authors.forename: Keith
authors.initials: K
authors.lastname: Peden
authors.affiliation: Laboratory of DNA Viruses, Division of Viral Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA. Electronic address: haruhiko.murata@fda.hhs.gov.
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Murata
mesh_terms: D000328:Adult; D057134:Antibodies, Neutralizing / Q000097:blood*; D000914:Antibodies, Viral / Q000097:blood*; D003587:Cytomegalovirus / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000821:virology*; D005347:Fibroblasts / Q000821:virology*; D064368:Healthy Volunteers; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D008297:Male; D008875:Middle Aged; D009500:Neutralization Tests / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors; D014019:Tissue Donors; D055815:Young Adult
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D007074:Immunoglobulin G
keywords: Cell lysate; Epithelial cell; Fibroblast; Human cytomegalovirus; Reverse transcription quantitative PCR; Virus-neutralization assay