Early and late pathologic changes in the adrenal glands of mice after infection with herpes simplex virus type 1

Intranasal infection of CH/HeN mice with herpes simplex virus Type 1 (HSV-1), VR3 strain, caused a high death rate in the 3-12 days following inoculation. Acute interstitial pneumonia and focal adrenal necrosis developed in almost all the animals. About 20% of the mice showed meningoencephalitis and myocarditis. Viral antigen was demonstrated by immunoperoxidase staining in the alveolar walls, around bronchi and blood vessels, and in focal areas of the adrenal cortex as early as 24-48 hours after infection. The virus disseminated probably hematogenously from the lungs and seemed to localize preferentially in the adrenals. In mice surviving the acute stage of infection a subcapsular cell reaction developed in the adrenal cortex. The extensive pneumonia and adrenal necrosis contributed to the high mortality rate of weanling mice infected with HSV-1. The route of viral inoculation and the age of the host animal seemed to influence the localization and outcome of the pathologic process.     

Efficacy of herpes simplex virus type 1 immunization in protecting against acute and latent infection by herpes simplex virus type 2 in mice.

ICR mice were immunized with herpes simplex virus type 1 (HSV-1) and later challenged with HSV-2 by footpad inoculation. Both immunized animals and age-matched, nonimmunized controls were observed for ascending neurological disease and latent infection of spinal ganglia resulting from the HSV-2 challenge. Control animals had a 78% incidence of acute and latent infection compared with a 1.7% incidence in immunized mice. The data show immunity to HSV-1 is protective against both acute and latent infection by HSV-2.     

Hemagglutination by herpes simplex virus type 1

Summary The McIntyre and HSZP strains as well as clinical isolate of herpes simplex virus type 1 were found to agglutinate C57B1/10su and CBA mouse red blood cells. The hemagglutinating activity was inhibited by antisera that neutralized the infectivity of the virus.     

5-Methoxymethyldeoxyuridine-resistant mutants of herpes simplex virus type 1

5-Methoxymethyldeoxyuridine (MMdU)-resistant (MMdU^r) mutants of herpes simplex virus type 1 (HSV-1) were isolated by a single passage of the virus in MMdU at 100 μg/ml (3.7 × 10^?4M). The isolates were cloned and passed in the presence of MMdU at 5 μg/ml (0.18 × 10^?4M). All MMdU^r mutants showed considerable cross-resistance to the nucleoside analogs acycloguanosine (ACG), (E)-5-bromovinyldeoxyuridine (BVdU), and arabinosylthymine (araT), but were as sensitive as the parental strain to phosphonoacetate (PAA). One mutant, MMdU^r-20, induced significant deoxythymidine kinase (dTK) activity. Because it was only 30 times more resistant to BVdU than the parental wild-type virus, while mutants MMdU^r-2 and -12-3 were about 10,000 times more resistant, it was suspected that the mutation was in the dTK locus. All three mutants, however, showed a similar pattern of resistance to the other nucleoside analogs ACG, MMdU, and araT. These results suggest differences in the active sites for PAA and nucleoside analogs (with respect to the viral DNA polymerase), and also among the nucleoside analogs (with respect to the viral dTK).     

Replication of herpes simplex virus type 1 in macrophages from resistant and susceptible mice.

Studies were carried out to determine whether the in vitro capacity of adherent peritoneal cells to replicate herpes simplex virus type 1 (HSV-1) might correlate with the in vivo susceptibility of mice genetically resistant, moderately susceptible, or very susceptible to HSV-1 infection. Unstimulated and proteose peptone-stimulated monolayers restricted viral replication when infected immediately, but replicated HSV-1 when infected after 3 to 7 days of culture. Macrophages from resistant C57Bl/6 mice restricted HSV-1 replication significantly better than cells from susceptible mice. This function did not segregate with resistance, since macrophages from resistant F1 mice failed to restrict HSV-1 replication. Induction of peritoneal exudate cells with thioglycolate yielded cells capable of replicating HSV-1 when infected immediately after plating and after 4 days of culture.     

Heterozygosis and genetic recombination in herpes simplex type 1 virus

Two- and three-factor crosses with temperature-sensitive (ts) and syncytial plaque morphology (syn) mutants of herpes simplex type 1 virus have been used to study the possible role of syn-syn⁺ mixed plaque-forming virus in genetic recombination. Under the conditions of a standard genetic cross, recombinants first appear about 6 hr after infection, the time of formation of the first infectious progeny virus, and their frequency progressively rises until about 20 hr postinfection. During this period the frequency of mixed plaques remained constant at approximately 5%. The frequency of mixed plaques and recombinants were both increased several-fold when crosses were made in the presence of the DNA synthesis inhibitor 5-fluorodeoxyuridine (FUdR). In view of the genetic instability of the partially heterozygous genomes of mixed plaque forming virus this result is interpreted to mean that mixed plaques identify virus which is an intermediate in recombinant formation and that the molecular structure of their genome is probably that of a partial heteroduplex. Measurements of deoxynucleoside triphosphate pools showed that FUdR inhibited the large increase in the dTTP pool size which normally accompanies HSV-1 infection of BHK cells.     

Gastrointestinal invasion by herpes simplex virus type 1 inoculated cutaneously into the immunosuppressed mice

Summary The pathogenesis of infection in mice with herpes simplex virus type 1 (HSV-1) strain 7401H was studied. Mice immunosuppressed by intraperitoneal injection of cyclophosphamide were inoculated cutaneously into the flank with the virus and developed severe zosteriform skin lesions. All of them died within 2 weeks after the infection, while most of the normal mice survived the viral infection with healing of the lesions. In the gastrointestinal tract of the immunosuppressed mice, macroscopic abnormalities were frequently observed, and infectious viruses were detected on days 7 to 9. The viruses were also detectable in the dorsal root ganglia and the spinal cord of thoracolumbar area on days 5 to 7, and in the celiac plexus on day 7. However, no viruses were detected in the blood. Immunohistological examination of the gastrointestinal tract revealed that the viral antigens were localized in Auerbach's myenteric plexus. These results suggest that HSV-1 inoculated into the flank skin invaded the gastrointestinal tract via the nervous system, which gastrointestinal involvement might possibly have caused the death of the mice.     

Experimental myelitis in BALB/cN and C57BL/6N mice caused by herpes simplex virus type 1 compared with herpes simplex virus type 2

Intraperitoneal and footpad inoculations of herpes simplex virus type 1 (HSV) into BALB/cN (HSV-susceptible) and C57BL/6N (HSV-resistant) mice were carried out to induce experimental myelitis. Standard laboratory strains (McIntyre, F, RK, and recently Okinawa strain R1) were inoculated in mice. As a control, the HSV 2 standard laboratory strain SAV was also inoculated. The McIntyre strain was the most virulent, while the F strain was the least. RK and R1 were both moderately virulent. Myelitis was induced in BALB/cN mice after intraperitoneal and footpad inoculations of low to high doses of the McIntyre strain, and intraperitoneal inoculation of moderate and high doses of the RK and R1 strains. Symptoms of paraplegia of the hind legs and rectal and urinary incontinence were observed, but not until 3-5 hours before death. The symptoms caused by footpad inoculation were slightly different from those following intraperitoneal inoculation; rectal incontinence, in particular, was inconspicuous in the former. In the case of footpad inoculation of RK and R1, only one mouse inoculated with R1 showed symptoms and histology of myelitis. The F strain caused no symptoms. In the case of C57BL/6N mice, high dose intraperitoneal and footpad inoculations of the McIntyre strain also caused myelitis, and the symptoms were observed about 6-7 hours before death. In only one C57BL/6N mouse intraperitoneally inoculated with a high dose of R1 did symptoms appear about 6 hours before death. The same symptoms caused by intraperitoneal and footpad inoculations of HSV 2 (SAV) were observed more clearly and for a longer period (half to one day) than those caused by HSV 1 inoculation. Spinal cord necrosis was noted with McIntyre, RK and R1 inoculations, but it was not marked with randomly located foci, when compared with that caused by SAV. Further, the foci of necrosis in C57BL/6N mice were smaller than in BALB/cN mice, even when high dose McIntyre strain was used. Nuclear pyknosis and edema of the brain in the dead mice following HSV 1 inoculation were more marked than in those killed by SAV.     

The herpes simplex virus type 2 equivalent of the herpes simplex virus type 1 US7 gene and its flanking sequences

Nucleotide sequencing studies (D. J. McGeoch, A. Dolan, S. Donald, and F. Rixon, 1985, J. Mol. Biol. 181, 1-14) have indicated that herpes simplex virus type 1 (HSV-1) has a coding sequence, referred to as US7, between the genes for the glycoproteins D and E (gD and gE). Northern blot analysis and nucleotide sequencing have been carried out to show that the type 2 virus (HSV-2) has an equivalent to the US7 gene. A comparison with the HSV-1 sequence has revealed some surprising similarities and differences. At the nucleotide level, HSV-2 has inserted a large sequence into the gE promoter, retained a large palindrome present in the coding sequence but not some tandem repeats, and deleted a region beside those repeats. At the amino acid level, the putative transmembrane sequence has been remarkably well conserved, and hydrophobic moment analysis indicates that it could be interacting with polar species within the plane of the membrane. Immediately after the deletion in the HSV-2 sequence, there is an N-glycosylation signal, and HSV-2 has one more such signal than HSV-1. The longest conserved sequence at the nucleotide level codes for a region of polypeptide that is strongly predicted to fold into alpha-helix. Implications of these analyses to the structure and possible function of these molecules are discussed.     

Blocked herpes simplex virus type 2-specific DNA synthesis in simian virus 40-transformed hamster cells permissive for herpes simplex virus type 1.

Simian virus 40-transformed hamster cells (LL-1) permissive to herpes simplex virus type 1 (HSV-1) were shown to be relatively nonpermissive to HSV-2. When LL-1 cells were infected with HSV-2, there was a 3- to 4-log reduction in infectious viral progeny at 24 h postinfection as compared with HSV-1 under identical cultured conditions. HSV-2 could be carried in the LL-1 cell line for up to 12 passages without any appreciable cytopathology. Various early functions of the replicative cycle of HSV-2 appeared to be normal. Experiments demonstrated that early enzyme activity, HSV-2 thymidine kinase, and DNA polymerase appeared at permissive levels in extracts of HSV-2-infected LL-1 cells. However, DNA analysis of HSV-2 infected LL-1 cells demonstrated a block in HSV-2-specific DNA synthesis, although HSV-2 was capable of inhibiting DNA synthesis in LL-1 cells. Furthermore, indirect immunofluorescence studies indicate that late HSV-2 structural protein synthesis was inhibited in infected LL-1 cells. Thus, the inability of HSV-2 to replicate in LL-1 cells is due to a block at or before HSV-specific DNA synthesis, resulting in a reduction of the structural protein synthesis required for viral maturation.     

Recombination between temperature-sensitive mutants of herpes simplex virus type 1

Fifteen temperature-sensitive (ts) mutants of herpes simplex virus type 1 representing 10 complementation groups were examined for their ability to recombine. Efficient recombination was demonstrated between most mutant pairs in standard two-factor crosses. Mutants which failed to complement each other also failed to recombine or recombined with low frequency. Progeny analysis of putative ts⁺ recombinants demonstrated that complementing clumps and/or multiploid particles did not contribute significantly to recombinant yields. Using recombination data from crosses between 11 mutants representing 7 complementation groups, a provisional linkage map has been constructed. The map spans approximately 38 recombination units and is linear.     

Heterotypic and homotypic re-inoculation of mice already latently infected with herpes simplex virus type 1

Summary Mice were infected at 4 weeks of age with a type 1 strain of herpes simplex virus (HSV) and re-infected 4 weeks later with either a type 1 or a type 2 strain of HSV. The virus used for first infection could be distinguished from that used later since it was resistant to phosphonoformic acid and formed syncytial plaques. Sites used for the second inoculation were as follows: at the site of primary infection, at a different site within the same dermatome or in the equivalent dermatome on the opposite side (also called remote site).Re-infection caused no detectable reactivation of the latent PFA resistant virus. After re-infection with a homotypic virus replication of the re-infecting virus was limited to the inoculation site. However after heterotypic re-infection the type 2 strain was occasionally isolated from the ganglia. Previous infection with the PFA resistant type 1 strain clearly reduced the ability of the homotypic or heterotypic strains to establish a latent infection. However, in a few animals ganglia were found to be latently infected with virus from both the first and second inoculations. Analysis of the results suggests that resistance to the establishment of a second latent infection in a ganglion is determined by the general immunity of the animal rather than immunity of the latently infected ganglion itself.     

Some structural antigens of herpes simplex virus type 1.

Several of the major structural polypeptides of herpes simplex virus were obtained in purified form by polyacrylamide gel electrophoresis of purified virus particle polypeptides. Antisera made by footpad inoculation of these polypeptides into rabbits were used to study the antigenic properties of two envelope glycoproteins and of the major capsid protein.     

Inhibitory effect of herpes simplex virus type 1 on type 2 virus replication.

Simultaneous infection with herpes simplex type I and type 2 viruses of chick embryo fibroblasts (CEF), which are only permissive for type 2 virus, or rabbit embryo fibroblasts (REF), which are permissive for both virus types, resulted in a marked reduction of type 2 virus production. This effect was dependent on the m.o.i. of type I, being expressed at a high rather than a low m.o.i. The rate of interference decreased with the prolongation of the interval between infection with type 2 and type I viruses. No evidence suggestive of interferon involvement was obtained. Partial inactivation of type 2 virus by ultraviolet irradiation enhanced the inhibitory effect of type I virus. On the other hand, u.v. irradiation of type I virus resulted in a progressive loss of inhibitory activity. The results of the present experiments suggest that a type I genome function is responsible for the interfering effect, and that an early step in the growth of type 2 virus is sensitive to the particular type I virus product involved.     

Immunogenicity of herpes simplex virus type 1 glycoproteins expressed in vaccinia virus recombinants

Vaccinia virus recombinants expressing glycoproteins B (vgB11), D (VgD52), E (gE/7.5 and gE/4B), G (gG-vac), H (gH-vac), and I (gI-vac) of HSV-1 were used to compare the protective response to these individual glycoproteins in the mouse. Glycoprotein D induced the best neutralizing antibody titers and the most increased rates of HSV clearance from the ear as well as good protection from the establishment of latent HSV infections in the sensory ganglia. Glycoprotein B also induced good neutralizing antibody titers and as great a protection from the establishment of latency as gD although the rate of virus clearance from the ear was not as great as after immunization with gD. Glycoprotein E induced weak neutralizing antibody but gG, gH, and gI did not show a neutralizing antibody response. At higher challenge doses of virus (10(6) PFU HSV-1 in the ear), gE induced a protective response by increasing the rate of virus clearance and reducing the acute infection of ganglia as compared to negative control immunized mice. However there was no protection from the establishment of latent infections after immunization with gE. No protective response was seen to gG, gH, or gl.     

Neurovirulence of herpes simplex virus type 1 depends on age in mice and thymidine kinase expression

The susceptibility of mice of different ages (from four to 28 days) to infection with herpes simplex virus type 1 (HSV-1) mutants inoculated onto scarified corneas was studied. The TK+ isolate from wild type virus was pathogenic in mice of all age groups. An HSV-1 mutant (designated TK1/4) with a less active thymidine kinase (TK) gene expressing 25 per cent of the TK activity of the TK+ isolate was pathogenic for mice up to 10 days of age. In older mice, virus pathogenicity was dependent on the inoculum dose: increasing the TK1/4 virus dose tenfold raised the level of TK activity and thus the virulence of the virus. A TK- mutant with no TK activity was pathogenic for four to eight day old mice that have TK activity in the brain, but not in older mice. Thus, resistance to HSV-1 that is age-dependent in mice can be determined by the extent to which the virus strain is liable to express its TK gene and by the amount of TK activity present in the brain.     

Roles for herpes simplex virus type 1 UL34 and US3 proteins in disrupting the nuclear lamina during herpes simplex virus type 1 egress

Cells infected with wild type HSV-1 showed significant lamin A/C and lamin B rearrangement, while UL34-null virus-infected cells exhibited few changes in lamin localization, indicating that UL34 is necessary for lamin disruption. During HSV infection, US3 limited the development of disruptions in the lamina, since cells infected with a US3-null virus developed large perforations in the lamin layer. US3 regulation of lamin disruption does not correlate with the induction of apoptosis. Expression of either UL34 or US3 proteins alone disrupted lamin A/C and lamin B localization. Expression of UL34 and US3 together had little effect on lamin A/C localization, suggesting a regulatory interaction between the two proteins. The data presented in this paper argue for crucial roles for both UL34 and US3 in regulating the state of the nuclear lamina during viral infection.