Varicella-zoster virus gE escape mutant VZV-MSP exhibits an accelerated cell-to-cell spread phenotype in both infected cell cultures and SCID-hu mice.

Varicella-zoster virus is considered to have one of the most stable genomes of all human herpesviruses. In 1998, we reported the unanticipated discovery of a wild-type virus that had lost an immunodominant B-cell epitope on the gE ectodomain (VZV-MSP); the gE escape mutant virus exhibited an unusual pattern of egress. Further studies have now documented a markedly enhanced cell-to-cell spread by the mutant virus in cell culture. This property was investigated by laser scanning confocal microscopy combined with a software program that allows the measurement of pixel intensity of the fluorescent signal. For this new application of imaging technology, the VZV immediate early protein 62 (IE 62) was selected as the fluoresceinated marker. By 48 h postinfection, the number of IE 62-positive pixels in the VZV-MSP-infected culture was nearly fourfold greater than the number of pixels in a culture infected with a low-passage laboratory strain. Titrations by infectious center assays supported the above image analysis data. Confirmatory studies in the SCID-hu mouse documented that VZV-MSP spread more rapidly than other VZV strains in human fetal skin implants. Generally, the cytopathology and vesicle formation produced by other strains at 21 days postinfection were demonstrable with VZV-MSP at 14 days. To assess whether additional genes were contributing to the unusual VZV-MSP phenotype, approximately 20 kb of the VZV-MSP genome was sequenced, including ORFs 31 (gB), 37 (gH), 47, 60 (gL), 61, 62 (IE 62), 66, 67 (gI), and 68 (gE). Except for a few polymorphisms, as well as the previously discovered mutation within gE, the nucleotide sequences within most open reading frames were identical to the prototype VZV-Dumas strain. In short, VZV-MSP represents a novel variant virus with a distinguishable phenotype demonstrable in both infected cell cultures and SCID-hu mice.

Temperature sensitivity of herpes simplex virus type 1 is a tissue-dependent phenomenon.

The temperature sensitivity of herpes simplex virus type 1 (HSV-1) was assessed in primary cultures of mouse central nervous system (MNS) cells and mouse embryo cells (MEC). Infectious yields were determined and the ultrastructural morphogenesis of HSV-1 particles was compared following incubation at 37 or 40.5 degrees C. Yields of infectious virus were significantly reduced for both types of cell cultures following incubation at 40.5 degrees C. However, the effect of supraoptimal temperature (40.5 degrees C) on HSV-1 replication in MEC was significantly greater than the effect of supraoptimal temperature on virus replication in MNS cells. With respect to viral morphogenesis, no significant differences were found in either the quantity or the appearance (empty versus electron opaque core) of intranuclear particles present per infected nucleus, regardless of cell type or incubation temperature. However, complete virus particles (enveloped capsids with dense cores) were never observed in MEC at 40.5 degrees C, either intracytoplasmically or extracellularly. In contrast, complete virus particles were observed in MNS cell cultures at 40.5 degrees C, albeit in reduced numbers. At the permissive temperature (37 degrees C), complete intracytoplasmic and/or extracellular virus particles were associated with every infected cell in the MNS cell or MEC cultures. Thus an interactional effect on HSV-1 replication was found between cell culture type and incubation temperature.

Colchicine treatment in the preparation of varicella-zoster virus inocula.

Two methods, ultrasonic and colchicine treatment, for preparing varicella-zoster virus (VZV) inocula were compared. Ultrasonic treatment was unsuccessful in achieving the release of detectable, infectious, cell-free VZV from infected human foreskin fibroblast (SF) cells. Sonication of infected SF cells resulted in losses of virus infectivity in direct correlation with losses in cell viability (r = 0.82). In the second method of generating VZV inocula, VZV-infected SF cells were treated for 15 min with 15-20 ng of colchicine per cell. Colchicine treatment maintained short-term survival of virus infectivity, but resulted in a dramatic decrease in long-term cell viability. In addition, colchicine-treated cells could not support the replication of VZV.

Preparation of cultured mammalian cells for transmission and scanning electron microscopy using Aclar film.

Common methods for the preparation of cultured cells for concurrent light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) are not completely satisfactory. This article describes how we grow mammalian cells on plastic disks made from Aclar film. Aclar is a transparent fluorinated-chlorinated thermoplastic that contains no volatile components and is, for all practical purposes, chemically inert. Cells adhere to it readily and remain attached after fixation, dehydration, and critical-point drying or embedding. The film also accepts heavy metal coating by ionic bombardment and is extremely stable in the vacuum of the SEM. LM observations are unhindered by Aclar, since the film is as transparent as glass. Fluorescence microscopy is possible with this film, since it exhibits no detectable autofluorescence. During SEM observation, the film has great dimensional stability, and the cells and heavy metal coating remain attached to the Aclar even under high-resolution operating conditions. TEM processing of specimens grown on Aclar is simplified by the fact that Aclar does not stick to the epoxy resins used in EM. Furthermore, Aclar is easily sectioned and does not damage knives used in ultramicrotomy. The use of Aclar film considerably simplifies the preparation of cultured cells for all types of microscopy. This method is particularly useful in correlating surface features between SEM and TEM observations.

In vitro susceptibility of cytomegalovirus isolates from immunocompromised patients to acyclovir and ganciclovir.

Fifty-four isolates of cytomegalovirus (CMV) from 25 immunocompromised patients with CMV infections were examined in a plaque reduction assay to determine in vitro susceptibilities to both acyclovir and ganciclovir. Isolates were approximately 25-fold more sensitive to ganciclovir than to acyclovir. The mean +/- SD ID50 for all isolates to acyclovir was 63.1 +/- 30.2 microM, (median, 52.3 microM; range, 16.7-146.4 microM). The mean ID50 for all isolates to ganciclovir was 2.50 +/- 1.27 microM, (median, 2.15 microM; range, 0.65-7.11 microM). Exposure to acyclovir or ganciclovir for periods of 2-5 wk did not alter the mean susceptibility of clinical isolates. However, one CMV strain isolated from a renal transplant patient after 14 days of acyclovir therapy displayed the lowest susceptibility of all strains tested (acyclovir 146.4 microM; ganciclovir 7.11 microM) and may represent selection of a resistant virus population.

Cytomegalovirus: an ultrastructural study of the morphogenesis of nuclear inclusions in human cell culture.

We investigated the ultrastructural development and maturation of cytomegalovirus (CMV) nuclear inclusions (NIs) in human embryo thyroid cells at 1 to 144 h post-infection. At 5 h, most cells had rounded from an initial fibroblastic appearance and contained early NIs. At 24 h, early NIs were larger and better defined. At 48 h, although early NIs were still present, most cells had larger and presumably more mature NIs. These latter NIs consisted of several subunits, each made up of a fibrillar network enclosing an electron-lucent area which contained coarse and delicate granules. Also, at 48 h, virus particles were first seen in the nucleoplasm. At 72 h, in cells with more developed NIs, virus particles were closely associated with the fibrillar network. Between 96 and 144 h, the NIs reached maximum size and were made up of numerous subunits. The results indicate that two types of NIs coexist during CMV infection. The appearance of the early the late NIs coincides with the reported peaks of CMV DNA synthesis and thus may explain the biphasic pattern of DNA synthesis in CMV infection. Morphogenetic features of the NIs conform with the hypothesis that synthesis of CMV DNA may occur in the centre in each NI subunit and that the fibrillar network represents condensing capsid proteins.

Cytomegalovirus: development and progression of cytopathic effects in human cell culture.

Cytopathic effects of cytomegalovirus infection were studied in human cell cultures at various time intervals. Cells derived from human embryonic thyroid, skin-muscle, and lung were infected with five different strains of cytomegalovirus at multiplicities of infection of approximately 5 plaque forming units per cell. Under these conditions, cell rounding and early cytoplasmic inclusions were first apparent at 5 hours postinfection, whereas nuclear inclusions were first observed as a homogenous eosinophilic bead at 24 hours postinfection. Cytoplasmic and nuclear inclusions underwent extensive morphogenesis through 96 to 120 hours postinfection. Development of nuclear inclusions included the formation of distinctive beadlike subunits, which increased in size from their first appearance at 48 to 72 hours postinfection and underwent apparent contraction and breakup after 96 hours postinfection. While the cytopathology induced by various cytomegalovirus strains studied was generally similar, the kinetics of their development was different and independent of both the multiplicities of infection and the source of the fibroblastic cells. Such cytomegalovirus strain-associated differences in cytopathology could result from variances in biologic characteristics of the strains studied.