Peter Wildy

Effect of acycloguanosine treatment of acute and latent herpes simplex infections in mice

Systemic treatment of mice with the nucleoside analog 9-(2-hydroxyethoxymethyl)guanine (acycloguanosine [aciclovir]) was found to be highly effective against acute type 1 herpes simplex virus infection of the pinna. The drug ablated clinical signs and reduced virus replication both in tissue local to the inoculation site and within the nervous system. Provided that moderate-sized virus inocula were used, acycloguanosine treatment reduced or prevented the establishment of a latent infection in the dorsal root ganglia relating to the sensory nerve supply of the ear. However, although it aborted artificially produced infections in dorsal root ganglia, acycloguanosine was found not to be effective against the latent infection once established. This finding strongly indicated that latent herpes simplex virus in mice can exist in a nonreplicating form.

Isolation and characterization of acyclovir-resistant mutants of herpes simplex virus

Mutants of HSV which are resistant to acyclovir (acycloguanosine) have been isolated following serial passages of several herpes simplex virus (HSV) strains in the presence of the drug. The majority of the mutants isolated are defective in induction of thymidine kinase (TK) and this is consistent with the observation that independently isolated TK- viruses are naturally resistant to ACV. One mutant is described (SC16 R9C2) which is resistant in biochemically transformed cells which express HSV TK. This suggests that its resistance resides at a level other than TK. It is also resistant to phosphonoacetic acid, suggesting that the DNA polymerase locus may be involved. A further mutant is described [Cl (101) P2C5] which induces normal levels of TK, although the nature of resistance of this virus is not yet elucidated.

The fine structure of polyoma virus.

Abstract Preparations of polyoma virus were examined by electron microscopy, using the negative contrast method. Spherical particles identified as viruses were found to have a mean diameter of 453 A. All the particles showed a shell (capsid) of elongated hollow subunits (capsomeres). In the majority, these surrounded a central core; but some particles appeared to be empty. Though there is no evidence of icosahedral shape, the capsomere arrangement showed 5:3:2 axial symmetry. It has been deduced that the particle contains 42 capsomeres, each aligned radially along an axis of symmetry.

Pathogenesis of Herpes Simplex Virus in Congenitally Athymic Mice: the Relative Roles of Cell-mediated and Humoral Immunity

Athymic nude (nu/nu) mice were inoculated in the ear pinna with 10(4) p.f.u. herpes simplex virus type 1 (strain SC 16). Initially, the virus was observed to replicate in the pinna, spreading via a neurological route to the dorsal root ganglia, spinal cord, brain and adrenal glands. Following the transfer of lymphoid cells from day 7 herpesvirus-infected hairy immunocompetent donors into infected nude mice, virus was not isolated from the pinna and nervous system of the majority of the mice. The passive transfer of neutralizing polyclonal anti-herpesvirus serum or neutralizing monoclonal anti-gp D serum did not reduce infectivity in the pinna, but markedly reduced the amount of virus in the ganglia and spinal cord. These data suggest that neutralizing antibodies play an important role in restricting the movement of virus to the nervous system, whereas cell-mediated immune (CMI) mechanisms are essential for eliminating virus from the pinna.

Cell-mediated immunity in herpes simplex virus-infected mice: functional analysis of lymph node cells during periods of acute and latent infection, with reference to cytotoxic and memory cells

The functional characteristics of lymphoid cells were investigated during acute and latent infection of mice with herpes simplex virus (HSV). Cytotoxic T cells were found in the draining lymph node (DLN) 4 days p.i. and had reached maximum activity between 6 and 9 days. After the 12th day and during the period of latent infection (> 20 days) no cytotoxic cell activity was observed. Cytotoxic activity could only be detected when the lymphoid cells had been cultured for a period of 3 days. In general, the cell killing was specific for syngeneic infected target cells, although some killing of uninfected targets was observed. In contrast to the cytotoxic response, DLN cells responding to HSV in a proliferation assay were detected towards the end of the acute phase and at lease up to 9 months thereafter. The significance of these observations for the pathogenesis of HSV is discussed.

Pathogenesis of herpes simplex virus in B cell-suppressed mice: the relative roles of cell-mediated and humoral immunity.

B cell responses of Balb/c mice were suppressed using sheep anti-mouse IgM serum. At 4 weeks, both B cell-suppressed and normal littermates were infected in the ear pinna with herpes simplex virus type 1 (HSV-1). The B cell-suppressed mice failed to produce neutralizing herpes antibodies in their sera but had a normal cell-mediated immunity (CMI) response as measured by a delayed hypersensitivity skin test. Although the infection was eliminated from the ear in both B cell-suppressed and normal mice by day 10 after infection, there was an indication that B cell-suppressed mice had a more florid primary infection of the peripheral and central nervous system and also a higher incidence of a latent infection. These results support the hypothesis that antibody is important in restricting the spread of virus to the central nervous system, whereas CMI is important in clearing the primary infection in the ear pinna.

The T-Cell-Mediated Immune Response of Mice to Herpes Simplex Virus

The host response to infection with herpes simplex virus (HSV) in which both natural resistance and specific immunological responses become marshaled is clearly very complex. Natural resistance mechanisms, mediated for example by macrophages, NK cells, and interferon, represent a formidable early barrier to the virus (Lopez, Chapter 2, this volume), although the eventual resolution of the infectious process and the establishment of long-lasting antiviral defense is the property of the immune system. Whereas both humoral and cell-mediated responses are readily induced by the virus, it is clear that the action of T cells is central for recovery from the primary infection and in the control of recrudescent lesions. Much of this evidence comes from studies carried out in the mouse in which the precise nature of the T-cell response to herpes can readily be studied. In this respect the variety of inbred and congenic mouse strains has been invaluable in studying MHC restriction of T-cell responses; and the availability of monoclonal antibodies to specific T-cell subsets has enabled a detailed analysis of the antiviral T-cell response to be carried out.

Quantitative cytological studies on HeLa cells infected with herpes virus.

Abstract A cytological study has been made on HeLa cells infected with herpes virus. Two types of specific cellular lesion were recognized: (1) the classic lesions of interphase cells and (2) a lesion of mitosis which rapidly replaces normal mitotic cells and which has a transitory existence only. The results suggest that inhibition of cell division by this virus is characterized either (a) by failure to enter mitosis or (b) by an abnormal mitosis. Microspectrophotometry of Feulgen-stained preparations confirmed that, after a lag period, there is a net increase of the DNA per nucleus in infected cells. At the same time autoradiography has shown that the rate of incorporation of tritiated thymidine is depressed. This paradox may be partly explained if the intracellular deoxyribonucleotide concentration is increased after infection. One factor contributing to such an increase could be breakdown of host nucleic acid as a result of infection. Studies on the loss of label from cells preincubated with tritiated thymidine suggest that such a breakdown may occur.

Atypical Patterns of Neural Infection Produced in Mice by Drugresistant Strains of Herpes Simplex Virus

Mice inoculated intracerebrally (i.c.) with a mutant strain of HSV were found to develop cataracts 1 to 2 months after inoculation. Cataract formation was subsequently shown to follow an acute retinitis which commenced within 1 week of inoculation. The mutant had been selected for high resistance to the nucleoside analogue acyclovir and has been shown previously to be defective in the induction of thymidine kinase and also to express an altered DNA polymerase. The LD50 for mice inoculated i.c. was greater than 10(5) p.f.u. compared with approx 7 p.f.u. for the parental strain. Studies of virus replication following i.c. inoculation with a sublethal dose of the mutant revealed that only small amounts of infectious virus were produced in the brain, but during a period from 6 to 12 days after inoculation vigorous replication occurred in retinal tissue, producing very high titres of virus.

T cell-macrophage interaction in arginase-mediated resistance to herpes simplex virus

Peritoneal macrophages activated by-products derived from a herpes simplex virus-specific helper T cell clone were used to investigate intrinsic and extrinsic resistance mechanisms to herpes simplex virus type 1 infection in vitro. T cell-activated macrophages produced fewer infective centres, indicating enhanced intrinsic resistance, and markedly reduced the growth of virus in a permissive cell line. The reduction in virus growth correlated with the depletion of arginine in the support medium, presumably resulting from increased arginase production by activated macrophages. The significance of these findings for antiviral immunity in vivo is discussed.