No abstract

Varicella zoster virus (VZV) latency is characterized by limited virus gene expression and the absence of virus DNA replication. Investigations of VZV latency and reactivation have been hindered by the lack of an in vitro model of virus latency. Since VZV is an exclusively human pathogen, we used naturally infected human trigeminal ganglia (TG) obtained at autopsy to study virus latency. Herein, we report optimization of medium to maintain TG integrity as determined by histology and immunohisto-chemistry. Using the optimized culture medium, we also found that both herpes simplex virus-1 (HSV-1) and VZV DNA replicated in TG explants after 5 days in culture. The increase in HSV-1 DNA was fourfold greater than the increase in VZV DNA. Overall, we present a model for alphaherpesvirus latency in human neurons in which the key molecular events leading to virus reactivation can be studied.

Varicella-zoster virus (VZV) is a ubiquitous, highly cell-associated, and exclusively human neurotropic alphaherpesvirus. VZV infection is initiated by membrane fusion, an event dependent in part on VZV glycoproteins gH and gL. Consistent with its location on the virus envelope, the gH/gL complex is a target of neutralizing antibodies produced after virus infection. One week after immunizing a 59-year-old VZV-seropositive man with Zostavax, we sorted his circulating blood plasma blasts and amplified expressed immunoglobulin variable domain sequences by single-cell PCR. Sequence analysis identified two plasma blast clones, one of which was used to construct a recombinant monoclonal antibody (rec-RC IgG). V aricella-zoster virus (VZV), the etiologic agent of varicella (chickenpox) and zoster (shingles), is an exclusively human pathogen and a member of the Herpesviridae family of enveloped DNA viruses. Herpesviruses cause both lytic and latent infections. Lytic infection requires membrane fusion, an event governed by a core complex consisting of conserved glycoproteins gB, gH, and gL. Along with membrane fusion, VZV gH and gL are also involved in virus egress and are essential for virus replication (1-4). In addition to mediating virus entry, VZV glycoproteins can traffic from infected cells to uninfected cells (5). VZV glycoproteins induce strong humoral immune responses both in naturally infected individuals and in varicella or zoster vaccine recipients (6-10). While VZV gE is the most immunogenic and predominant glycoprotein in VZV-infected cell membranes, antibodies to VZV gH are the major neutralizing antibodies (11-16). It is likely that neutralizing activity by the gH/gL complex effectively prevents cell-to-cell virus spread (5,(17)(18)(19)(20).Analysis of VZV attachment and membrane fusion requires highly specific neutralizing antibodies. Hybridoma cell lines and phage display libraries produce human anti-VZV gE and gH monoclonal antibodies (MAbs) that neutralize virus infection (21-24). Monoclonal antibodies that recognize the VZV gH/gL protein complex hold promise in therapies involving passive transfer of neutralizing VZV antibodies (15,16,25,26). Here we present a new method for constructing a recombinant human monoclonal anti-VZV antibody and show that this antibody detects a conformational epitope on the gH/gL complex and neutralizes virus. MATERIALS AND METHODS Cells and virus.Human lung fibroblast (HFL) and human embryonic kidney (HEK-EBNA 293) cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with 4 mM L-glutamine (DMEM; Sigma-Aldrich, St. Louis, MO) and 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA). VZV was propagated by cocultivating infected cells with uninfected cells as described previously (27). Infected HFL cultures were harvested at the height of virus-induced cytopathic effect, usually at 3 days postinfection (dpi).Construction of recombinant antibody. Blood was collected 7 days after immunization...