_Archives
Vi rology
Arch Virol (1992) 127:49-63
© Springer-Verlag 1992 Printed in Austria
Temperature sensitivity of herpes simplex virus type 1 is a tissue-dependent phenomenon Nancy L. Cole
Indiana University School of Medicine, South Bend Center for Medical Education, Notre Dame, Indiana, U.S.A.
Accepted April 25, 1992
Summary. 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 °C. Yields of infectious virus were significantly reduced for both types of cell cultures following incubation at 40.5 °C. However, the effect of supraoptimal temperature (40.5 °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 °C, either intracytoplasmicallyor extracellularly. In contrast, complete virus particles were observed in MNS cell cultures at 40.5 °C, albeit in reduced numbers. At the permissive temperature (37 °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.
Introduction The effect of supraoptimal temperature on the permissive replication of viruses is a useful means of further assessing the complex interaction of viruses with host cells. Cell- or tissue-dependent effects of supraoptimal temperature may indicate important differences in the cellular components utilized by the virus under various cellular or enviromental circumstances, or important differences in tissue susceptibilities to virus infection given certain environmental conditions
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Nancy L. Cole
(e.g., fever). Earlier studies reported cell-type dependent differences in HSV temperature sensitivity in vitro between rabbit kidney and hamster e m b r y o cells [2] and between H e L a cells and h u m a n skin fibroblasts [19]. However, the temperature sensitivity of HSV- 1 replication has n o t been c o m p a r e d in nervous system versus non-nervous system cells from a single species. In this study, two aspects of HSV-1 replication were examined following incubation at 37 and 40.5 °C. First, the temperature sensitivity o f HSV-1 replication in mouse central nervous system a n d m o u s e embryo cells was determined by infectivity studies. Second, the ultrastructural morphogenesis o f HSV-1 particles in the two types of cell cultures was c o m p a r e d to begin to explore the m e c h a n i s m of the observed tissue-dependent effect of supraoptimal temperature on virus replication. Materials and methods Cells and cell viabitities Primary cultures of mouse central nervous system (MNS) cells, mouse embryo cells (MEC) and human foreskin fibroblast (SF) cells were prepared as follows. The cerebral contents of 2-3 days old Swiss ICR mice were aspirated and trypsin dissociated (0.05% trypsinEDTA; Gibco Laboratories, Grand Island, NY) to yield single cell suspensions. These suspensions of MNS cells were seeded into 8-well plates (Nunc, Naperville, IL) in HL-1 media (Ventrex Laboratories, Portland, ME) supplemented with 1% (v/v) fetal bovine serum (FBS; Gibco), 0.06% (w/v) glutamine and 0.4% (w/v) dextrose. Suspensions of M EC were obtained by trypsin dissociation of minced 15-18 day Swiss ICR embryos from which the head and legs had been surgically removed. The MEC were seeded into 8-well plates or 75 cm2 tissue culture flasks (Coming, Corning, NY) in 50/50 media [-equal quantities of Opti-MEM (Gibco) and MEM with Earle's salts (Gibco)] supplemented with 5% FBS and 0.03% glutamine. Confluent cultures of MEC were maintained with 50/50 media supplemented with 2% FBS and 0.03% glutamine. Primary cultures of human SF cells were prepared by trypsin dissociation of minced human foreskin tissues, and initiated and maintained in 50/50 media supplemented with 0.03% glutamine and 5% or 2% Calf Supreme (Gibco), respectively. Cell viabilities were determined by trypan blue exclusion. Suspensions of trypsinized cells were diluted in 0.25% (w/v) trypan blue in complete PBS. Following a 20 rain incubation period, viable cells were counted using a hemacytometer. Virus Stocks of HSV- 1 strain KOS were produced from infected human foreskin fibroblasts (SF) cells. HSV-infected SF cells were frozen and thawed, and physically disrupted using a Fisher Scientific model 300 probe sonic dismembrator (Fisher Scientific, Lexington, MA) at 50% output. The supernatants (1000 rpm, 10 rain) were stored at - 85 °C. Plaque assays were performed on thawed supernatants to establish the plaque forming units of virus per ml (PFU/ml) of each stock. Clinical strains of HSV-1 were obtained from Dr. Kenneth Fife (Indiana University School of Medicine); stocks of the clinical strains were produced and assayed as described for HSV-1 KOS. Infectivity studies Confluent monolayers of MNS cells or MEC were inoculated with 10-15 PFU per ceU and maintained in a humidified 5% CO2 atmosphere at the desired temperature following a
HSV-1 replication in mouse CNS versus mouse embryo cells
51
45 rain adsorption period (37 °C). PFU per cell were estimated based on cell viability counts taken at the beginning of each experiment and the previously determined PFU per ml of the particular virus stock employed. At appropriate times post-infection, individual wells of infected cells were scraped and frozen at - 85 °C until assayed. Plaque assays of infectious virus were performed by inoculating confluent monolayers of SF cells with serial 10-fold dilutions of the thawed, sonicated (50% output, 20 s) samples. Inoculated monolayers were incubated at 37 °C for 45 min to allow virus adsorption, after which the inoculum fluids were removed and replaced with 1.5 ml of a methylcellulose overlay [equal volumes of 1% (w/v) methylcellulose and 2 x MEM with Earle's salts, supplemented with 10% FBS and 0.03% glutamine] per well. After 4 days incubation at 37 °C, assay monolayers were fixed (10% formalin), stained (0.03% methylene blue), and the numbers of plaques counted with a stereomicroscope.
Experimental protocol and electron microscopy methods Confluent cultures of MNS cells or MEC in 8-well plates were inoculated with 10-15 P F U per cell and maintained in a humidified, 5% CO2 atmosphere at either 37 °C or 40.5 °C following a 45 rain adsorption period (37 °C). At appropriate times post-infection, the cells in individual wells were scraped into complete PBS (274 mM NaC1, 537 mM KC1, 1.8 mM CaC12, 1.0 mM MgC12, 21 mM Na2 HPO4, 2.9 mM KH2 PO4) and pelleted (200 x g, 10 min) directly in Beem capsules (Electron Microscopy Sciences, Fort Washington PA). Cell pellets were fixed (30 min) in cold 0.1 M sodium cacodylate buffer, pH 7.4, containing 2% glutaraldehyde and 1% formaldehyde. After fixation, the cells were rinsed with three changes of the same buffer, followed by post-fixation for 1 h in cold 0.1 M sodium cacodylate buffer containing 1% osmimn tetraoxide and 1.5% potassium ferricyanide [6]. After osmication, the infected cell pellets were rinsed three times in buffer, dehydrated in graded ethanols, and embedded in Spurr's resin (Polysciences, Inc., Warrington, PA). Ultrathin sections were cut in a Sorvall MT-2 ultramicrotome (RMC, Inc., Tucson, AZ) with a Diatome diamond knife (Electron Microscopy Sciences, Fort Washington, PA) and stained with uranyl acetate and lead citrate [18] (Reynolds, 1963) for 10 min and 5 rain, respectively. The sections were observed using an Hitachi H-600 electron microscope (Hitachi Scientific Instruments, Mountain View, CA) operating at 75 kV. The images were recorded on Kodak electron microscope 4489 film, and developed in D-19 for 4.5 rain at 23 °C.
Statistical analyses In order to determine whether the supraoptimal temperature differentially affected the replication of HSV in mouse central nervous system versus mouse embryo cells, two approaches were taken. The first was a descriptive approach, in which means and standard deviations were determined for various parameters of virus replication. These parameters included the number of plaque-forming units per ml detectable at 24-30 h postinfection, the number of infected cells per grid hole (as evidenced by presence of intranuclear capsids), the number of intranuclear capsids, the percent empty out of total intranuclear capsids, and the number of infected cells with complete virus particles. The second approach was statistical, and was conducted to determine the probability that the differences in the parameter means calculated above could have occurred by chance alone (i.e., no interactional effect of cell type and temperature). Thus the parameters of virus replication were analyzed using a two-way analysis of variance (ANOVA), as this allows one to compare the means of several groups within a matrix (cell type x temperature).
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Nancy L. Cole Results
Effect oj' temperature on replication of HSV-1 KOS The effect of temperature on the productive replication of HSV-1 in MNS cells and MEC is shown in Fig. 1 A and B, respectively. The mean PFU/ml (4- SEM) for each time point was calculated based on data from nine separate experiments. At 37 °C, the replication of HSV-1 was productive in both MNS cells and MEC. At 40.5 °C, however, a significant restriction of virus replication was observed in both MNS cells (p < 0.05) and MEC (p < 0.01), based on Student's t-test. Unfortunately, these analyses could not answer the question whether virus replication was restricted by supraoptimal temperature to a greater extent in one type of cell culture as opposed to the other. To address this question, data from the plaque assays were examined by a two-way analysis of variance, which allows one to determine the reliability of the mean differences in PFU per ml produced in MNS cells versus MEC, at 37 versus 40.5 °C. These analyses confirmed that the restrictive effect of supraoptimal temperature was significantly stronger in MEC compared to MNS cells (p < 0.05). Figure 1 also suggests that larger amounts of virus were obtained from MNS ceils as opposed to MEC following incubation at 37 °C. However, the cultures 9
9
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Fig. 1. Kinetics of HSV-1 replication A in MNS cells and B in MEC. Infected cells were scraped, frozen and thawed, sonicated, and assayed for infectivityin SF cells. 0, • 37 °C; O, [] 40.5 °C
HSV-1 replication in mouse CNS versus mouse embryo cells
53
of MNS cells characteristically had significantly greater cell densities compared to cultures of MEC (p < 0.01). The difference in virus yields at 37 °C for the two types of cell cultures was not significant when the estimates of infectious virus were statistically corrected for the density of the cell cultures (p > 0.10). The ability of HSV-1 KOS to replicate at intermediate temperatures (38.5 and 39.5 °C) was also determined (Fig. 2 A and B). As illustrated by the data from one of duplicate experiments, virus replication was minimally affected by either of the two intermediate temperatures, regardless of the type of cell culture.
Effect of temperature on replication of clinical HSV strains Low passage clinical isolates of HSV- 1 were evaluated with respect to the effect of temperature on virus replication. Four clinical isolates were evaluated, three of which were isolated from the CNS of patients with HSV encephalitis. Representative results from one of duplicate experiments performed with each of
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Fig.2. Kinetics of HSV-1 replication A in MNS cells and B in MEC. Infected cells were scraped, frozen and thawed, sonicated, and assayed for infectivity in SF cells. • 37 °C, ¢r 38.5 °C, [] 39.5 °C, O 40 °C
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Nancy L. Cole
two encephalitis isolates are shown in Fig. 3 A and B. Comparable findings to those described for the laboratory strain, KOS, were obtained for all of the clinical isolates.
Effect of temperature on cell viability The effect of temperature on the viability of uninfected MNS cells or MEC was examined to determine whether losses in cell viability might account for the apparent restriction of virus replication at the supraoptimal temperature. Representative results from one of triplicate experimental determinations are shown in Fig. 4. As described above, a significant difference existed between the characteristic density of each of the two types of cell cultures (p < 0.01). However, supraoptimal temperature did not have a significant effect on cell viability within each type of culture ~ > 0.10).
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Fig. 3. Kinetics of replication of clinical HSV-1 isolates in MNS cells (O 37°C, O 40.5 °C) and in MEC (11 37 °C, [] 40.5 °C). (A strain WG; B strain Hemme). Infected cells were scraped, frozen and thawed, sonicated, and assayed for infectivity in SF cells
HSV-1 replication in mouse CNS versus mouse embryo cells
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co
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0.1) or the proportion of empty capsids to capsids with cores (nucleocapsids) per 40,000 x field (p > 0.05) (Table 1). Although the density of nucleocapsid cores varied considerably, no convincing differences were observed in the relative frequency of faint, homogeneous versus granular cores at either temperature or in either type of cell culture. With respect to the different cell types within the MNS cell or MEC cultures, all cell types were able to support the development of intranuclear particles. Minor cell type-specific differences were observed, particularly with respect to the formation of crystalline arrays of intranuclear particles. The largest, most well-developed crystalline arrays were typically seen in MNS-B (Fig. 6 c) and C cells and MEC-B cells (Fig. 7 c), but were also occassionally observed in MNS-D and MEC-C (Fig. 7 e) cells. Small crystalline arrays were only rarely observed within nuclei of infected MNS-F or MEC-A cells, despite the presence of large numbers of intranuclear particles.
Effect of temperature on the development of complete virus particles in infected M N S cells and MEC The development of complete virus particles was significantly affected by incubation at the supraoptimal temperature. Complete virus particles were defined as nucleocapsids containing a dense granular core and surrounded by an electron-opaque envelope. Abundant complete virus particles were observed intracellularly and extracellularly in cultures of both MNS cells (Fig. 6 a, c, e, g, and i) and MEC (Fig. 7 a, c, and e) incubated at 37 °C. These particles were observed within all cell types present within the respective cultures. Complete virus particles were also observed within all cell types present in MNS cell cultures incubated at 40.5 °C (Fig. 6 b, d, f, h, and j). However, the frequency of infected cells (based on presence of intranuclear particles) containing complete particles was significantly reduced (p < 0.001), from 100% in MNS cells at 37 °C to 15% in MNS cells at 40.5 °C (Table 1). The greatest effect of supraoptimal temperature was observed in cultures of
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Fig. 6. Comparison of virus particles observed at 24-30 h postinfection in HSV-infected MNS cell at 37 °C (a, e, e, g, and i) or 40.5 °C (b, d, f, h, and j). Intranuclear particles included empty nucleocapsids (short arrows) and nucleocapsids with partial cores of varying density (long arrows). Complete, enveloped particles with a dense granular core (curved arrows) were observed intracytoplasmicatly and extracellularly at both 37 and 40.5 °C. a, b MNS-A cells (x 22,800 and x 22,100, respectively), e, d MNS-B cells (x 19,200), e, f MNS-C cells (x 19,200 and x 28,000, respectively), g, h MNS-D cells (x 19,040 and x 22,500, respectively), i, j MNS-E cells (x 18,000)
HSV-1 replication in mouse CNS versus mouse embryo cells
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MEC incubated at 40.5 °C (Table t; Fig. 7 b, d, and f). Complete virus particles, either intra- or extracellular, were never observed in infected MEC at 40.5 °C. This total absence of observed complete particles was irrespective of cell type, and despite the presence of comparable numbers of intranuclear particles relative to infected MNS cells incubated at 37 or 40.5 °C or MEC incubated at 37 °C. Discussion
A tissue-specific temperature sensitivity of productive HSV-1 replication was observed in the current study. By infectivity studies, supraoptimal[ temperature (40.5 °C) had a significantly greater effect on the productive replication of HSV1 within infected mouse embryo cells as opposed to mouse central nervous system cells. This differential effect could not be accounted for by differences in the cell density of the two types of cell cultures. Furthermore, this effect could not be accounted for by a differential effect of supraoptimal temperature on cell viability of the two types of cell cultures. The tissue-specific temperature sensitivity of productive HSV-1 replication appears to be a consistent phenom-
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Fig. 7. Comparison of virus particles observed at 24-30 h postinfection in HSV-infected MEC at 37 °C (a, e, and e) or 40.5 °C (b, d, and f). Intranuclear particles include empty nucleocapsids (short arrows) and nucleocapsids with partial cores of varying density (long arrows). Complete intracytoplasmic or extracellular virus particles (curved arrows) were only observed at 37 °C. a, b MEC-A cells ( x 16,800 and x 19,200, respectively), e, d MECB cells ( x 19,200), e, f MEC-C cells ( x 16,800 and x 19,200, respectively)
HSV-1 replication in mouse CNS versus mouse embryo cells
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Table 1. Quantitative assessment of HSV-1 replication in mouse central nervous system and mouse embryo cells MNS
37 °C % infected cellsc (mean 4- SEM)
94.4 4-2.0 N. capsids per 40,000 x field 26.4 (mean + SEM) 4- 5.4 % empty capsidsa (mean + SEM) 21.3 4-4.6 % infected cells with complete particles 100
MEC b
a
40.5 °C
37 "C
40.5 °C
40.3 4- 5.4 23.4 4- 3.8 10.7 4-2.0 14.9 4-2.6
100
18.4 4-3.2 24.6 4- 3.2 17.3 4-4.4 0
49.1 4- 10.7 14.0 4-2.2 100
a Mouse central nervous system cells b Mouse embryo cells ° Per grid hole, 200 mesh copper grids, 24-30 h postinfection d Percent empty capsids out of total capsids
enon of this virus, as both clinical and laboratory strains exhibited the same results. Supraoptimal temperature also profoundly restricted the morphogenesis of herpes simplex virus particles within cultures of mouse embryo cells, but not cultures of mouse central nervous system cells. Complete enveloped virus particles could not be detected in infected mouse embryo cells incubated at 40.5 °C, despite the presence of abundant intranuclear capsids. The ultrastructural development of herpes simplex virus type 1 (HSV-1) particles under permissive conditions is thoroughly described in the literature I-5, 8, 9, 13, 15]. Electron microscopic comparisons of particle development have been useful in evaluating the ability of various cell types to support HSV replication I-3, 4, 7, 16, 17] and in evaluating the effect of various conditions or treatments on HSV replication [1, 10, 12, 14-1. The conclusion from these studies is that restrictive cellular environments, treatments or physical conditions often do not completely block expression of the viral genome, but do result in a block in the formation of complete, enveloped virus particles in HSV-infected cells. One question that arises is whether the observed differential temperature sensitivity has significance to the in vivo interactions of HSV-1 with different tissues within the host animal. The differential temperature sensitivity observed in this study may offer a partial explanation for the fulminating nature of HSV encephalitis. HSV encephalitis is a rapidly progressive infection associated with high morbidity and mortality [20]. In addition to the rapid replication that is characteristic of this virus, the fulminant nature of this infection suggests that either the specific or the nonspecific host defenses, or both, are ineffective in
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controlling the replication and spread of this virus within the central nervous system. Consistent with the data presented in this report, the ability of HSV-1 to replicate productively in nervous system cells, regardless of the presence or absence of fever (supraoptimal temperature), may contribute significantly to the overall progression of the virus once it has gained access to the CNS. As one of the nonspecific defense mechanisms, fever may restrict virus replication via temperature-sensitive viral proteins that are nonfunctional at the increased temperature. Alternatively, fever (supraoptimal temperature) may restrict virus replication via temperature-sensitive host cell proteins that are crucial to the synthesis of virus-specified RNA or DNA or the production of functional viral proteins. The data presented in this report suggest the latter role for fever with respect to restriction of HSV- 1 replication. Rather than a virus-specified feature of the interaction with host cells, the temperature sensitivity described appears to be a feature of the specific host cells, defined by their tissue of origin. This conclusion that a cellular phenomenon is involved is further confirmed by consistent observation of the temperature sensitivity, regardless of whether a laboratory or clinical isolates of HSV-1 were examined, and regardless of the site from which the clinical strains were isolated. The current study suggests that temperature sensitive host factors may play a crucial role in determining the outcome of HSV-1 infection, and would predict the route of exposure or invasion to be a significant factor due to differences in the extent to which host tissues are protected from the effects of naturally occurring supraoptimal temperature (fever). Fibroblasts and epithelial cells, which are likely components of the mouse embryo cell cultures, are relatively expendable, all-purpose cells that can be readily replenished within tissues. In contrast, the components of the mouse nervous system cell cultures have much more specialized functions in vivo. These cells, due to their natural importance to the functioning of the host nervous system, may possess variants of cellular factors that are more resistant to the effects of supraoptimal temperature. Additional studies are needed to assess the specific host factors (e.g., cellular enzymes) that may be involved in the tissue-specific temperature sensitivity described in this report.
Acknowledgements I am indebted to Dr. Robert Kingsley, for sharing his invaluable electron microscopy expertise with a novice, and to William Archer, for his excellent technical assistance. I would also like to acknowledge the diligent work and valuable contributions of Kerry Sieger and Lorrie Miller-Rice, student researchers in my laboratory. This work was supported in part by grant 5 SO7RR 5371, awarded by the Biomedical Research Support Grant program, Division of Research Resources, National Institutes of Health.
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References 1. Chatterjee S, Hunter E, Whitley R (1985) Effect of cloned human interferons on protein synthesis and morphogenesis of herpes simplex virus. J Virol 56:419-425 2. Crouch NA, Rapp F (1972) Cell-dependent differences in the production of infectious herpes simplex virus at a supraoptimal temperature. J Virol 9:223-230 3. Dupuy-Coin AM, Arnoult J, Bouteilte M (1978) Quantitative correlation of morphological alterations of the nucleus with functional events during in vitro infection of glial cells with herpes simplex hominis (HSV2). J Ultrastruct Res 65:60-72 4. Hill TJ, Field HJ (1973) The interaction of herpes simplex virus with cultures of peripheral nervous tissue: an electron microscopic study. J Gen Virol 21:123-133 5. Katsumoto T, Hirano A, Kurimoto T, Takagi A (1981) In situ electron microscopical observations of cells infected with herpes simplex virus. J Gen Virol 52:267-278 6. Langford LA, Coggeshall RE (1980) The use of potassium ferricyanide in neural fixation. Anat Rec 197:297-303 7. Leestma JE, Bornstein MB, Sheppard RD, Feldman LA (1969) Ultrastructural aspects of herpes simplex virus infection in organized cultures of mammalian nervous tissue. Lab Invest 20:70-77 8. Morgan C, Rose HM, Holden M, Jones EP (1959) Electron microscopic observations on the development of herpes simplex virus. J Exp Med 110:643-656 9. Nii S, Morgan C, Rose HM (1968) Electron microscopy of herpes simplex virus. II. Sequence of development. J Virol 2:517-536 10. Nii S, Rosenkranz S, Morgan C, Rose HM (1968) Electron microscopy of herpes simplex virus. III. Effect of hydroxyurea. J Virol 2:1163-1171 11. Reynolds ES (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17:208-212 12. Schaffer PA, Brunschwig JP, McCombs RM, Benyesh-Melnick M (1974) Electron microscopic studies of temperature-sensitive mutants of herpes simplex virus type 1. Virology 62:444-457 13. Schwartz J, Roizman B (1969) Similarities and differences in the development of laboratory strains and freshly isolated strains of herpes simplex virus in HEp-2 cells: electron microscopy. J Virol 4:879-889 14. Siminoff P, Menefee MG (1966) Normal and 5-bromodeoxyuridine-inhibited development of herpes simplex virus. An electron microscope study. Exp Ceil Res 44: 241255 15. Smith JD, de Harven E (1973) Herpes simplex virus and human cytomegalovirus replication in WI-38 cells. I. Sequence of viral replication. J Virol 12:919-930 16. Spring SB, Roizman B, Schwartz J (1968) Herpes simplex virus products in productive and abortive infection. II. Electron microscopic and immunological evidence for failure of virus envelopment as a cause of abortive infection. J Virol 2:384-392 17. Stevens JG, Cook ML (1971) Restriction of herpes simplex virus by macrophages. An analysis of the cell-virus interaction. J Exp Med 133:19-38 18. Watson ML (1958) Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem Cytol 4:475-478 19. Wheeler CE (1958) The effect of temperature upon the production of herpes simplex virus in tissue culture. J Immunol 81:98-106 20. Whitley RJ, Alford CA, Hirsch MS (1986) Vidarabine versus acyclovir therapy in herpes simplex encephalitis. N Eng J Med 314:144.149 Author's address: Nancy L. Cole, South Bend Center for Medical Education, B 22 Haggar Hall, University of Notre Dame, Notre Dame, IN46556, U.S.A. Received February 4, 1992
Vi rology
Arch Virol (1992) 127:49-63
© Springer-Verlag 1992 Printed in Austria
Temperature sensitivity of herpes simplex virus type 1 is a tissue-dependent phenomenon Nancy L. Cole
Indiana University School of Medicine, South Bend Center for Medical Education, Notre Dame, Indiana, U.S.A.
Accepted April 25, 1992
Summary. 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 °C. Yields of infectious virus were significantly reduced for both types of cell cultures following incubation at 40.5 °C. However, the effect of supraoptimal temperature (40.5 °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 °C, either intracytoplasmicallyor extracellularly. In contrast, complete virus particles were observed in MNS cell cultures at 40.5 °C, albeit in reduced numbers. At the permissive temperature (37 °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.
Introduction The effect of supraoptimal temperature on the permissive replication of viruses is a useful means of further assessing the complex interaction of viruses with host cells. Cell- or tissue-dependent effects of supraoptimal temperature may indicate important differences in the cellular components utilized by the virus under various cellular or enviromental circumstances, or important differences in tissue susceptibilities to virus infection given certain environmental conditions
50
Nancy L. Cole
(e.g., fever). Earlier studies reported cell-type dependent differences in HSV temperature sensitivity in vitro between rabbit kidney and hamster e m b r y o cells [2] and between H e L a cells and h u m a n skin fibroblasts [19]. However, the temperature sensitivity of HSV- 1 replication has n o t been c o m p a r e d in nervous system versus non-nervous system cells from a single species. In this study, two aspects of HSV-1 replication were examined following incubation at 37 and 40.5 °C. First, the temperature sensitivity o f HSV-1 replication in mouse central nervous system a n d m o u s e embryo cells was determined by infectivity studies. Second, the ultrastructural morphogenesis o f HSV-1 particles in the two types of cell cultures was c o m p a r e d to begin to explore the m e c h a n i s m of the observed tissue-dependent effect of supraoptimal temperature on virus replication. Materials and methods Cells and cell viabitities Primary cultures of mouse central nervous system (MNS) cells, mouse embryo cells (MEC) and human foreskin fibroblast (SF) cells were prepared as follows. The cerebral contents of 2-3 days old Swiss ICR mice were aspirated and trypsin dissociated (0.05% trypsinEDTA; Gibco Laboratories, Grand Island, NY) to yield single cell suspensions. These suspensions of MNS cells were seeded into 8-well plates (Nunc, Naperville, IL) in HL-1 media (Ventrex Laboratories, Portland, ME) supplemented with 1% (v/v) fetal bovine serum (FBS; Gibco), 0.06% (w/v) glutamine and 0.4% (w/v) dextrose. Suspensions of M EC were obtained by trypsin dissociation of minced 15-18 day Swiss ICR embryos from which the head and legs had been surgically removed. The MEC were seeded into 8-well plates or 75 cm2 tissue culture flasks (Coming, Corning, NY) in 50/50 media [-equal quantities of Opti-MEM (Gibco) and MEM with Earle's salts (Gibco)] supplemented with 5% FBS and 0.03% glutamine. Confluent cultures of MEC were maintained with 50/50 media supplemented with 2% FBS and 0.03% glutamine. Primary cultures of human SF cells were prepared by trypsin dissociation of minced human foreskin tissues, and initiated and maintained in 50/50 media supplemented with 0.03% glutamine and 5% or 2% Calf Supreme (Gibco), respectively. Cell viabilities were determined by trypan blue exclusion. Suspensions of trypsinized cells were diluted in 0.25% (w/v) trypan blue in complete PBS. Following a 20 rain incubation period, viable cells were counted using a hemacytometer. Virus Stocks of HSV- 1 strain KOS were produced from infected human foreskin fibroblasts (SF) cells. HSV-infected SF cells were frozen and thawed, and physically disrupted using a Fisher Scientific model 300 probe sonic dismembrator (Fisher Scientific, Lexington, MA) at 50% output. The supernatants (1000 rpm, 10 rain) were stored at - 85 °C. Plaque assays were performed on thawed supernatants to establish the plaque forming units of virus per ml (PFU/ml) of each stock. Clinical strains of HSV-1 were obtained from Dr. Kenneth Fife (Indiana University School of Medicine); stocks of the clinical strains were produced and assayed as described for HSV-1 KOS. Infectivity studies Confluent monolayers of MNS cells or MEC were inoculated with 10-15 PFU per ceU and maintained in a humidified 5% CO2 atmosphere at the desired temperature following a
HSV-1 replication in mouse CNS versus mouse embryo cells
51
45 rain adsorption period (37 °C). PFU per cell were estimated based on cell viability counts taken at the beginning of each experiment and the previously determined PFU per ml of the particular virus stock employed. At appropriate times post-infection, individual wells of infected cells were scraped and frozen at - 85 °C until assayed. Plaque assays of infectious virus were performed by inoculating confluent monolayers of SF cells with serial 10-fold dilutions of the thawed, sonicated (50% output, 20 s) samples. Inoculated monolayers were incubated at 37 °C for 45 min to allow virus adsorption, after which the inoculum fluids were removed and replaced with 1.5 ml of a methylcellulose overlay [equal volumes of 1% (w/v) methylcellulose and 2 x MEM with Earle's salts, supplemented with 10% FBS and 0.03% glutamine] per well. After 4 days incubation at 37 °C, assay monolayers were fixed (10% formalin), stained (0.03% methylene blue), and the numbers of plaques counted with a stereomicroscope.
Experimental protocol and electron microscopy methods Confluent cultures of MNS cells or MEC in 8-well plates were inoculated with 10-15 P F U per cell and maintained in a humidified, 5% CO2 atmosphere at either 37 °C or 40.5 °C following a 45 rain adsorption period (37 °C). At appropriate times post-infection, the cells in individual wells were scraped into complete PBS (274 mM NaC1, 537 mM KC1, 1.8 mM CaC12, 1.0 mM MgC12, 21 mM Na2 HPO4, 2.9 mM KH2 PO4) and pelleted (200 x g, 10 min) directly in Beem capsules (Electron Microscopy Sciences, Fort Washington PA). Cell pellets were fixed (30 min) in cold 0.1 M sodium cacodylate buffer, pH 7.4, containing 2% glutaraldehyde and 1% formaldehyde. After fixation, the cells were rinsed with three changes of the same buffer, followed by post-fixation for 1 h in cold 0.1 M sodium cacodylate buffer containing 1% osmimn tetraoxide and 1.5% potassium ferricyanide [6]. After osmication, the infected cell pellets were rinsed three times in buffer, dehydrated in graded ethanols, and embedded in Spurr's resin (Polysciences, Inc., Warrington, PA). Ultrathin sections were cut in a Sorvall MT-2 ultramicrotome (RMC, Inc., Tucson, AZ) with a Diatome diamond knife (Electron Microscopy Sciences, Fort Washington, PA) and stained with uranyl acetate and lead citrate [18] (Reynolds, 1963) for 10 min and 5 rain, respectively. The sections were observed using an Hitachi H-600 electron microscope (Hitachi Scientific Instruments, Mountain View, CA) operating at 75 kV. The images were recorded on Kodak electron microscope 4489 film, and developed in D-19 for 4.5 rain at 23 °C.
Statistical analyses In order to determine whether the supraoptimal temperature differentially affected the replication of HSV in mouse central nervous system versus mouse embryo cells, two approaches were taken. The first was a descriptive approach, in which means and standard deviations were determined for various parameters of virus replication. These parameters included the number of plaque-forming units per ml detectable at 24-30 h postinfection, the number of infected cells per grid hole (as evidenced by presence of intranuclear capsids), the number of intranuclear capsids, the percent empty out of total intranuclear capsids, and the number of infected cells with complete virus particles. The second approach was statistical, and was conducted to determine the probability that the differences in the parameter means calculated above could have occurred by chance alone (i.e., no interactional effect of cell type and temperature). Thus the parameters of virus replication were analyzed using a two-way analysis of variance (ANOVA), as this allows one to compare the means of several groups within a matrix (cell type x temperature).
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Nancy L. Cole Results
Effect oj' temperature on replication of HSV-1 KOS The effect of temperature on the productive replication of HSV-1 in MNS cells and MEC is shown in Fig. 1 A and B, respectively. The mean PFU/ml (4- SEM) for each time point was calculated based on data from nine separate experiments. At 37 °C, the replication of HSV-1 was productive in both MNS cells and MEC. At 40.5 °C, however, a significant restriction of virus replication was observed in both MNS cells (p < 0.05) and MEC (p < 0.01), based on Student's t-test. Unfortunately, these analyses could not answer the question whether virus replication was restricted by supraoptimal temperature to a greater extent in one type of cell culture as opposed to the other. To address this question, data from the plaque assays were examined by a two-way analysis of variance, which allows one to determine the reliability of the mean differences in PFU per ml produced in MNS cells versus MEC, at 37 versus 40.5 °C. These analyses confirmed that the restrictive effect of supraoptimal temperature was significantly stronger in MEC compared to MNS cells (p < 0.05). Figure 1 also suggests that larger amounts of virus were obtained from MNS ceils as opposed to MEC following incubation at 37 °C. However, the cultures 9
9
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I
I
I
I
I
3
6
12
18
24
30
0
3
6
12
18
24
30
HOURS POST-INFECTION
Fig. 1. Kinetics of HSV-1 replication A in MNS cells and B in MEC. Infected cells were scraped, frozen and thawed, sonicated, and assayed for infectivityin SF cells. 0, • 37 °C; O, [] 40.5 °C
HSV-1 replication in mouse CNS versus mouse embryo cells
53
of MNS cells characteristically had significantly greater cell densities compared to cultures of MEC (p < 0.01). The difference in virus yields at 37 °C for the two types of cell cultures was not significant when the estimates of infectious virus were statistically corrected for the density of the cell cultures (p > 0.10). The ability of HSV-1 KOS to replicate at intermediate temperatures (38.5 and 39.5 °C) was also determined (Fig. 2 A and B). As illustrated by the data from one of duplicate experiments, virus replication was minimally affected by either of the two intermediate temperatures, regardless of the type of cell culture.
Effect of temperature on replication of clinical HSV strains Low passage clinical isolates of HSV- 1 were evaluated with respect to the effect of temperature on virus replication. Four clinical isolates were evaluated, three of which were isolated from the CNS of patients with HSV encephalitis. Representative results from one of duplicate experiments performed with each of
A
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o _J
t 0
6
12
18
24
30
0
t
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6
12
18
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HOURS POST-INFECTION
Fig.2. Kinetics of HSV-1 replication A in MNS cells and B in MEC. Infected cells were scraped, frozen and thawed, sonicated, and assayed for infectivity in SF cells. • 37 °C, ¢r 38.5 °C, [] 39.5 °C, O 40 °C
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Nancy L. Cole
two encephalitis isolates are shown in Fig. 3 A and B. Comparable findings to those described for the laboratory strain, KOS, were obtained for all of the clinical isolates.
Effect of temperature on cell viability The effect of temperature on the viability of uninfected MNS cells or MEC was examined to determine whether losses in cell viability might account for the apparent restriction of virus replication at the supraoptimal temperature. Representative results from one of triplicate experimental determinations are shown in Fig. 4. As described above, a significant difference existed between the characteristic density of each of the two types of cell cultures (p < 0.01). However, supraoptimal temperature did not have a significant effect on cell viability within each type of culture ~ > 0.10).
A
B
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12
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24
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O
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12
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30
HOURS POST--INFECTION
Fig. 3. Kinetics of replication of clinical HSV-1 isolates in MNS cells (O 37°C, O 40.5 °C) and in MEC (11 37 °C, [] 40.5 °C). (A strain WG; B strain Hemme). Infected cells were scraped, frozen and thawed, sonicated, and assayed for infectivity in SF cells
HSV-1 replication in mouse CNS versus mouse embryo cells
55
co
/
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7
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0.1) or the proportion of empty capsids to capsids with cores (nucleocapsids) per 40,000 x field (p > 0.05) (Table 1). Although the density of nucleocapsid cores varied considerably, no convincing differences were observed in the relative frequency of faint, homogeneous versus granular cores at either temperature or in either type of cell culture. With respect to the different cell types within the MNS cell or MEC cultures, all cell types were able to support the development of intranuclear particles. Minor cell type-specific differences were observed, particularly with respect to the formation of crystalline arrays of intranuclear particles. The largest, most well-developed crystalline arrays were typically seen in MNS-B (Fig. 6 c) and C cells and MEC-B cells (Fig. 7 c), but were also occassionally observed in MNS-D and MEC-C (Fig. 7 e) cells. Small crystalline arrays were only rarely observed within nuclei of infected MNS-F or MEC-A cells, despite the presence of large numbers of intranuclear particles.
Effect of temperature on the development of complete virus particles in infected M N S cells and MEC The development of complete virus particles was significantly affected by incubation at the supraoptimal temperature. Complete virus particles were defined as nucleocapsids containing a dense granular core and surrounded by an electron-opaque envelope. Abundant complete virus particles were observed intracellularly and extracellularly in cultures of both MNS cells (Fig. 6 a, c, e, g, and i) and MEC (Fig. 7 a, c, and e) incubated at 37 °C. These particles were observed within all cell types present within the respective cultures. Complete virus particles were also observed within all cell types present in MNS cell cultures incubated at 40.5 °C (Fig. 6 b, d, f, h, and j). However, the frequency of infected cells (based on presence of intranuclear particles) containing complete particles was significantly reduced (p < 0.001), from 100% in MNS cells at 37 °C to 15% in MNS cells at 40.5 °C (Table 1). The greatest effect of supraoptimal temperature was observed in cultures of
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Nancy L. Cole
Fig. 6. Comparison of virus particles observed at 24-30 h postinfection in HSV-infected MNS cell at 37 °C (a, e, e, g, and i) or 40.5 °C (b, d, f, h, and j). Intranuclear particles included empty nucleocapsids (short arrows) and nucleocapsids with partial cores of varying density (long arrows). Complete, enveloped particles with a dense granular core (curved arrows) were observed intracytoplasmicatly and extracellularly at both 37 and 40.5 °C. a, b MNS-A cells (x 22,800 and x 22,100, respectively), e, d MNS-B cells (x 19,200), e, f MNS-C cells (x 19,200 and x 28,000, respectively), g, h MNS-D cells (x 19,040 and x 22,500, respectively), i, j MNS-E cells (x 18,000)
HSV-1 replication in mouse CNS versus mouse embryo cells
59
MEC incubated at 40.5 °C (Table t; Fig. 7 b, d, and f). Complete virus particles, either intra- or extracellular, were never observed in infected MEC at 40.5 °C. This total absence of observed complete particles was irrespective of cell type, and despite the presence of comparable numbers of intranuclear particles relative to infected MNS cells incubated at 37 or 40.5 °C or MEC incubated at 37 °C. Discussion
A tissue-specific temperature sensitivity of productive HSV-1 replication was observed in the current study. By infectivity studies, supraoptimal[ temperature (40.5 °C) had a significantly greater effect on the productive replication of HSV1 within infected mouse embryo cells as opposed to mouse central nervous system cells. This differential effect could not be accounted for by differences in the cell density of the two types of cell cultures. Furthermore, this effect could not be accounted for by a differential effect of supraoptimal temperature on cell viability of the two types of cell cultures. The tissue-specific temperature sensitivity of productive HSV-1 replication appears to be a consistent phenom-
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Nancy L. Cole
Fig. 7. Comparison of virus particles observed at 24-30 h postinfection in HSV-infected MEC at 37 °C (a, e, and e) or 40.5 °C (b, d, and f). Intranuclear particles include empty nucleocapsids (short arrows) and nucleocapsids with partial cores of varying density (long arrows). Complete intracytoplasmic or extracellular virus particles (curved arrows) were only observed at 37 °C. a, b MEC-A cells ( x 16,800 and x 19,200, respectively), e, d MECB cells ( x 19,200), e, f MEC-C cells ( x 16,800 and x 19,200, respectively)
HSV-1 replication in mouse CNS versus mouse embryo cells
61
Table 1. Quantitative assessment of HSV-1 replication in mouse central nervous system and mouse embryo cells MNS
37 °C % infected cellsc (mean 4- SEM)
94.4 4-2.0 N. capsids per 40,000 x field 26.4 (mean + SEM) 4- 5.4 % empty capsidsa (mean + SEM) 21.3 4-4.6 % infected cells with complete particles 100
MEC b
a
40.5 °C
37 "C
40.5 °C
40.3 4- 5.4 23.4 4- 3.8 10.7 4-2.0 14.9 4-2.6
100
18.4 4-3.2 24.6 4- 3.2 17.3 4-4.4 0
49.1 4- 10.7 14.0 4-2.2 100
a Mouse central nervous system cells b Mouse embryo cells ° Per grid hole, 200 mesh copper grids, 24-30 h postinfection d Percent empty capsids out of total capsids
enon of this virus, as both clinical and laboratory strains exhibited the same results. Supraoptimal temperature also profoundly restricted the morphogenesis of herpes simplex virus particles within cultures of mouse embryo cells, but not cultures of mouse central nervous system cells. Complete enveloped virus particles could not be detected in infected mouse embryo cells incubated at 40.5 °C, despite the presence of abundant intranuclear capsids. The ultrastructural development of herpes simplex virus type 1 (HSV-1) particles under permissive conditions is thoroughly described in the literature I-5, 8, 9, 13, 15]. Electron microscopic comparisons of particle development have been useful in evaluating the ability of various cell types to support HSV replication I-3, 4, 7, 16, 17] and in evaluating the effect of various conditions or treatments on HSV replication [1, 10, 12, 14-1. The conclusion from these studies is that restrictive cellular environments, treatments or physical conditions often do not completely block expression of the viral genome, but do result in a block in the formation of complete, enveloped virus particles in HSV-infected cells. One question that arises is whether the observed differential temperature sensitivity has significance to the in vivo interactions of HSV-1 with different tissues within the host animal. The differential temperature sensitivity observed in this study may offer a partial explanation for the fulminating nature of HSV encephalitis. HSV encephalitis is a rapidly progressive infection associated with high morbidity and mortality [20]. In addition to the rapid replication that is characteristic of this virus, the fulminant nature of this infection suggests that either the specific or the nonspecific host defenses, or both, are ineffective in
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controlling the replication and spread of this virus within the central nervous system. Consistent with the data presented in this report, the ability of HSV-1 to replicate productively in nervous system cells, regardless of the presence or absence of fever (supraoptimal temperature), may contribute significantly to the overall progression of the virus once it has gained access to the CNS. As one of the nonspecific defense mechanisms, fever may restrict virus replication via temperature-sensitive viral proteins that are nonfunctional at the increased temperature. Alternatively, fever (supraoptimal temperature) may restrict virus replication via temperature-sensitive host cell proteins that are crucial to the synthesis of virus-specified RNA or DNA or the production of functional viral proteins. The data presented in this report suggest the latter role for fever with respect to restriction of HSV- 1 replication. Rather than a virus-specified feature of the interaction with host cells, the temperature sensitivity described appears to be a feature of the specific host cells, defined by their tissue of origin. This conclusion that a cellular phenomenon is involved is further confirmed by consistent observation of the temperature sensitivity, regardless of whether a laboratory or clinical isolates of HSV-1 were examined, and regardless of the site from which the clinical strains were isolated. The current study suggests that temperature sensitive host factors may play a crucial role in determining the outcome of HSV-1 infection, and would predict the route of exposure or invasion to be a significant factor due to differences in the extent to which host tissues are protected from the effects of naturally occurring supraoptimal temperature (fever). Fibroblasts and epithelial cells, which are likely components of the mouse embryo cell cultures, are relatively expendable, all-purpose cells that can be readily replenished within tissues. In contrast, the components of the mouse nervous system cell cultures have much more specialized functions in vivo. These cells, due to their natural importance to the functioning of the host nervous system, may possess variants of cellular factors that are more resistant to the effects of supraoptimal temperature. Additional studies are needed to assess the specific host factors (e.g., cellular enzymes) that may be involved in the tissue-specific temperature sensitivity described in this report.
Acknowledgements I am indebted to Dr. Robert Kingsley, for sharing his invaluable electron microscopy expertise with a novice, and to William Archer, for his excellent technical assistance. I would also like to acknowledge the diligent work and valuable contributions of Kerry Sieger and Lorrie Miller-Rice, student researchers in my laboratory. This work was supported in part by grant 5 SO7RR 5371, awarded by the Biomedical Research Support Grant program, Division of Research Resources, National Institutes of Health.
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References 1. Chatterjee S, Hunter E, Whitley R (1985) Effect of cloned human interferons on protein synthesis and morphogenesis of herpes simplex virus. J Virol 56:419-425 2. Crouch NA, Rapp F (1972) Cell-dependent differences in the production of infectious herpes simplex virus at a supraoptimal temperature. J Virol 9:223-230 3. Dupuy-Coin AM, Arnoult J, Bouteilte M (1978) Quantitative correlation of morphological alterations of the nucleus with functional events during in vitro infection of glial cells with herpes simplex hominis (HSV2). J Ultrastruct Res 65:60-72 4. Hill TJ, Field HJ (1973) The interaction of herpes simplex virus with cultures of peripheral nervous tissue: an electron microscopic study. J Gen Virol 21:123-133 5. Katsumoto T, Hirano A, Kurimoto T, Takagi A (1981) In situ electron microscopical observations of cells infected with herpes simplex virus. J Gen Virol 52:267-278 6. Langford LA, Coggeshall RE (1980) The use of potassium ferricyanide in neural fixation. Anat Rec 197:297-303 7. Leestma JE, Bornstein MB, Sheppard RD, Feldman LA (1969) Ultrastructural aspects of herpes simplex virus infection in organized cultures of mammalian nervous tissue. Lab Invest 20:70-77 8. Morgan C, Rose HM, Holden M, Jones EP (1959) Electron microscopic observations on the development of herpes simplex virus. J Exp Med 110:643-656 9. Nii S, Morgan C, Rose HM (1968) Electron microscopy of herpes simplex virus. II. Sequence of development. J Virol 2:517-536 10. Nii S, Rosenkranz S, Morgan C, Rose HM (1968) Electron microscopy of herpes simplex virus. III. Effect of hydroxyurea. J Virol 2:1163-1171 11. Reynolds ES (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17:208-212 12. Schaffer PA, Brunschwig JP, McCombs RM, Benyesh-Melnick M (1974) Electron microscopic studies of temperature-sensitive mutants of herpes simplex virus type 1. Virology 62:444-457 13. Schwartz J, Roizman B (1969) Similarities and differences in the development of laboratory strains and freshly isolated strains of herpes simplex virus in HEp-2 cells: electron microscopy. J Virol 4:879-889 14. Siminoff P, Menefee MG (1966) Normal and 5-bromodeoxyuridine-inhibited development of herpes simplex virus. An electron microscope study. Exp Ceil Res 44: 241255 15. Smith JD, de Harven E (1973) Herpes simplex virus and human cytomegalovirus replication in WI-38 cells. I. Sequence of viral replication. J Virol 12:919-930 16. Spring SB, Roizman B, Schwartz J (1968) Herpes simplex virus products in productive and abortive infection. II. Electron microscopic and immunological evidence for failure of virus envelopment as a cause of abortive infection. J Virol 2:384-392 17. Stevens JG, Cook ML (1971) Restriction of herpes simplex virus by macrophages. An analysis of the cell-virus interaction. J Exp Med 133:19-38 18. Watson ML (1958) Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem Cytol 4:475-478 19. Wheeler CE (1958) The effect of temperature upon the production of herpes simplex virus in tissue culture. J Immunol 81:98-106 20. Whitley RJ, Alford CA, Hirsch MS (1986) Vidarabine versus acyclovir therapy in herpes simplex encephalitis. N Eng J Med 314:144.149 Author's address: Nancy L. Cole, South Bend Center for Medical Education, B 22 Haggar Hall, University of Notre Dame, Notre Dame, IN46556, U.S.A. Received February 4, 1992
