A remarkable feature of herpes simplex virus type 1 (HSV-1) is its ability to persist in the natural host (human) after primary infection in a latent or subdetectable form. Persistence of HSV-1 has been documented in patients with recurrent HSV-1 infections, herpes keratitis, and urogenital tumors. Nonetheless, the sites where HSV-1 maintains itself in the human organism vary with the clinical manifestations. Until now, persistence of the virus has been reported in neurons, corneal epithelium, and lymphocytes, although other cell types such as macrophages should also be considered as hosts for HSV-1 persistence.

Macrophages are widely distributed in the body, are important effector cells in host responses to virus infection, and participate in both innate and acquired immunity. Their presence in major organs, tissues, serous spaces and sites of inflammation suggests that they are the first immune cells encountered by the invading virus. In addition, microphages are among the cell types selected for the persistence of DNA and RNA; therefore, it is likely that HSV-1 can remain in these cells.

Persistence of HSV-1 in human mononuclear cells has not been documented, although circumstantial evidence suggests that the virus can maintain itself in these cells. HSV-1 persistence has been reported ex vivo in a primary murine spleen macrophage culture obtained from mice resistant to infection and in vitro in a promonocyte cell line, and in a macrophage-like cell line. In cells from patients with recurrent HSV-1 infections, higher concentration of HSV-1 DNA are present in the recrudescent phase than in the convalescent phase, although it is not clear if the increase in DNA results from replication of latent viruses or by exogenous infection. Moreover, mononuclear phagocytes (human and murine) are ex vivo–susceptible to HSV-1 infection with varied permissivity, which seems to be related to the particular differentiation state in which monocytes are restrictive.

In this report, we readdress the issue of HSV-1 persistence in macrophages. Due to the intrinsic heterogeneity of macrophages from primary cultures as hosts for virus persistence, we selected a well-defined macrophage-like cell line, P388D1, which displays a number of macrophage characteristics and is considered as a mature macrophage.

We describe here the establishment and characterization of a stable, productive, persistently infected macrophage culture that has been maintained for more than 2 years. Viral persistence was confirmed and monitored by measuring extracellular virus titer, presence of cell-viral antigen, and macrophage production of infectious viruses. In addition, the phenotype of viruses obtained from the persistently infected culture was determined together with the presence of viral and host proteins in the persistent culture and in HSV-1-infected macrophages.

Herpes simplex virus type 1 (HSV-1) macroplaque strain (MP), originally obtained from Dr. B. Roizman, was used because of its large plaque size. Viruses obtained from passages 47 and 60 of the persistently infected culture were named HSV_A and HSV_B, respectively. Viral propagation and titration were carried out by standard techniques in Vero cells.

P388D1 cells, a continuous DBA/2 mouse macrophage-like cell line (ATCC TIB 63), courtesy of Dr. A. Tingle, University of British Columbia, Vancouver, Canada, were grown as a monolayer in RPMI 1640 medium (Gibco BRL, Inc., Gaithersburg, MD, USA) supplemented with 10% heat-inactivated (56°C/30 min) fetal bovine serum (FBS) (Bioexport, México, D.F., México), antibiotics (100 ui/mL penicillin, 100 ?g/mL streptomycin) (Sigma Chemical Co., St. Louis, MO, USA), and 0.00001% ?-mercaptoethanol (Sigma Chemical Co., complete medium). Vero cells originally from ATCC (CCL 81) were used and maintained in MEM medium (Gibco BRL) supplemented with antibiotics, 10 ?g/mL amphotericin B (Sigma Chemical) and 5% heat-inactivated FBS.

Persistently infected cultures were obtained by the infection of macrophage monolayers using HSV-1 at multiplicity of infection (MOI) 0.001–0.1 after being washed with PBS. Non-adsorbed virus was removed after 1 h of adsorption at 37°C in a 5%-CO₂ humid atmosphere. Subsequently, RPMI 1640 medium was added, with antibiotics but without serum. The cultures were maintained for 7 days with a daily change of the medium. Thereafter, the cells were subcultured every 2–3 days at a split ratio of 1:2 with a daily change of complete medium. At each passage, the supernatant was collected for virus titration.

Cell-viral antigens were detected by direct immunofluorescence (IF). The Syva Microtrak HSV1/HSV2 Culture Identification/Typing Test Kit (Syva Co., Palo Alto, CA, USA) was used using the technique recommended by the manufacturer. The percentage of fluorescent cells was determined by observing three separate slides of the same passage. A minimum of 100 cells was observed and an average was calculated.

Cells releasing infectious virus were detected by the infectious center assay. This was carried out on a monolayer of Vero cells grown on 24-well microtiter plates (Costar, Cambridge, MA, USA). Persistently infected cells were extensively washed (>20 times) with PBS to eliminate extracellular virus, and were later detached from the surface of the Petri dish with a pipette. Subsequently, a serial three-fold limiting dilution of the cell suspension was made with RPMI 1640 medium containing 1% FBS. One hundred microliters of each dilution were delivered per well. Thereafter, 200 ?L of MEM medium with 1% FBS and 0.7% agar (Difco Laboratories, Detroit, MI, USA) were added and incubated for 5 days at 37°C. The cultures were MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide: thiazolyl blue (Sigma Chemical) stained or fixed with 3% formaldehyde in PBS and stained with crystal violet. The plaques were counted and the percentage of cells releasing infectious virus was calculated from an average of three determinations of the same passage.

Vero cell monolayers were infected with ten-fold serial dilutions of HSV-1, HSV_A, or HSV_B. Non-adsorbed virus was removed after 1 h of adsorption at 37°C, and medium with 0.7% agar was added. Cultures were incubated for 5 days and fixed with 3% formaldehyde in PBS and stained with crystal violet. The cytopathic effect (CPE) in Vero and P388D1 cells infected at a MOI of 1.0 was examined after 48 h of infection under the microscope.

Persistent acute infected (MOI 1 at 48 h post-infection) or non-infected cells were extensively washed with PBS and then resuspended in 100 ?L of lysis buffer (10 mM Tris-HCl pH 7.5; 140 mM NaCl; 5 mM EDTA; 1% Triton X-100 and 1% sodium deoxycholate) and centrifuged at 12,000 × g for 5 min. The supernatants were kept at ?20°C until used. Protein content was measured by the method of Lowry.

Radioactively labeled cellular extract analysis was performed essentially as described elsewhere. Control cells, acute infected cells (MOI of 1.0, 24 h post-infection), and persistent cell cultures were incubated for 2 h at 37°C in a methionine-free MEM medium (Gibco BRL). Then, 100 ?Ci/mL of Tran³⁵SLabel (ICN Pharmaceuticals, Inc., Costa Mesa, CA, USA) was added. After 4 h, the cellular extracts were prepared and separated by 10% SDS-PAGE electrophoresis (40 ?g/well). The radioactive proteins were detected by autoradiography.