HSV-infected Mice Research Articles

Macrophages and microglia in HSV-1 infected mouse brain

We have examined the macrophage and microglial cell response in mouse brains during the course of acute herpes simplex encephalitis produced by intranasal inoculation of virus. Macrophage infiltration and a microglial cell reaction were detected from 24 h after initial detection of HSV-1 antigen in brain. Macrophage infiltration was confined to focal areas of infection but the microglial cell reaction occurred in a more widespread distribution. Following disappearance of viral antigen, with resolution of the acute infection, macrophage infiltration and a focal microglial cell reaction remained for several weeks, providing a marker of sites of previous infection.

Immunomodulatory effects of HSV2 glycoprotein D in HSV1 infected mice: implications for immunotherapy of recurrent HSV infection

Immunological analyses in this laboratory and others have suggested that a nonrecurrent HSV seropositive immune status is more closely correlated with a type 1 T helper cell (Th1) response characterized by elevated levels of interferon-γ and IL2 rather than high titers of virus-specific antibodies. Effective intervention with an immunotherapeutic vaccine may require modulation of the regulatory network of T helper cells such that there is selective restimulation and expansion of the Th1 response. We have established a murine model for assessing the immunomodulatory capacity of an HSV glycoprotein subunit vaccine in animals with pre-existing herpes immunity. Animals were infected with varying doses of HSV1 and then administered glycoprotein D (gD) vaccine adjuvanted with aluminum phosphate at 3-week intervals. Observed changes in serological and cellular responses indicated that administration of subunit vaccine adjuvanted with aluminum phosphate could shift a dominant Th1 response, induced by sensitization with live HSV, towards a Th2 profile of activity. These data suggest that use of aluminum based adjuvants will not selectively stimulate Th1-associated responses and alternative adjuvants may be required for effective use of subunit vaccine in an immunotherapeutic indication in humans.

Efficacy of A-73209, a potent orally active agent against VZV and HSV infections

A-73209 is a novel oxetanocin derivative with potent in vitro and in vivo activity against VZV, HSV-1, and HSV-2. A-73209 was two logs more potent than acyclovir against five thymidine kinase positive (TK +) strains of VZV in vitro (mean EC 50 0.01 vs. 1.22 μg/ml). The activity of A-73209 was one log more potent than acyclovir against TK + HSV-1 strains in vitro (EC 50 = 0.03 vs. 0.32 μg/ml). A-73209 yielded a mean EC 50 of 2.2 μg/ml compared to a mean EC 50 of 0.37 μg/ml for acyclovir against a panel of TK + HSV-2 strains in vitro. The in vitro activity of A-73209 against thymidine kinase negative or deficient strains of VZV, HSV-1 and HSV-2 was much lower than for the corresponding TK + strains. A-73209 produced efficacy superior to acyclovir against lethal systemic or intracerebral HSV-1 infections in mice. The greater efficacy of A-73209 relative to acyclovir was especially apparent with oral dosing. Against HSV-2 infections in mice, the efficacy of A-73209 ranged from equal to 1.7 times less active relative to acyclovir with oral dosing. A-73209 was orally bioavailable in mice, with maximal serum concentrations well in excess of in vitro inhibitory concentrations. A-73209 appears to be a potent and selective agent against varicella-zoster virus and herpes simplex virus infections.

Cationic lipids direct a viral glycoprotein into the class I major histocompatibility complex antigen-presentation pathway.

Recombinant glycoprotein B (gB) of herpes simplex virus (HSV) was processed and presented by class I major histocompatibility complex (MHC) molecules after delivery into cells by using N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), a commercially available cationic lipid used for DNA transfection. Cells treated with DOTAP-associated gB were susceptible to lysis by class I MHC-restricted, HSV-specific cytotoxic T lymphocytes (CTL), and the treated cells restimulated memory gB-specific CTL activity in spleen cells from HSV-infected mice. gB-specific CTL responses were detected in mice immunized with recombinant gB and DOTAP but not in those receiving gB emulsified in complete Freund's adjuvant. Thus, cationic lipids may facilitate induction of CD8+ T-cell responses in vaccinations with recombinant antigens, and they may serve as readily available reagents for dissecting class I MHC immunity to viruses and other intracellular pathogens.

Spontaneous reactivation of thymidine kinase deficient (TK −) herpes simplex virus (HSV) infection without acyclovir (ACV) pressure in a patient with the acquired immunodeficiency syndrome (AIDS) : J.J. Sasadeusz, B.A. Rennie, S.L. Sacks. University of British Columbia, Vancouver, BC Canada

Alternative therapies are needed for HSV-1 infections in patients refractory to treatment with Acyclovir (ACV) and its derivatives. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded DNA analogues that enter cells readily and reduce target gene expression through steric blockage of complementary RNA. When applied before or soon after infection PPMO targeting the translation-start-site regions of HSV-1 ICP0 or ICP27 mRNA reduced HSV-1 plaque formation by 70–98% in vitro. The ICP0 PPMO also reduced ACV-resistant HSV-1 (strain 615.9) plaque formation by 70–90%, while an equivalent dose of ACV produced only 40–50% inhibition when the treatment was applied between 1 and 3 hpi. Seven daily topical treatments of 100 μg ICP0 PPMO caused no gross or microscopic damage to the corneas of uninfected mice. Topical application of 10 μg ICP0 PPMO to the eyes of HSV-1 infected mice reduced the incidence of eye disease by 37.5–50% compared to controls. This study demonstrates that topically applied PPMO holds promise as an antiviral drug candidate against HSV-1 ocular infection.

A new class of acyclic phosphonate nucleotide analogues: phosphonate isosteres of acyclovir and ganciclovir monophosphates as antiviral agents.

Novel phosphonate isosteres of acyclovir (ACV) and ganciclovir (DHPG) monophosphates were found to be potent and selective antiherpesvirus agents. In the series of phosphonate analogues of ACV monophosphate, only the guanine analogue 20 exhibited activity against herpesviruses, similar to the structure-activity relationship observed for base modification of ACV analogues. The phosphonate isostere of ACV monophosphate (20) was more effective than ACV in the HSV-1 infected mouse model. The 3'-carba analogues of 9-[3-hydroxy-2-(phosphonomethoxy)propyl]purines/pyrimidines (adenine, HPMPA; guanine, HPMPG; cytosine, HPMPC) are devoid of antiherpesvirus activity. This result confirms that the beta-oxygen atom of the phosphonomethyl ether functionality in HPMP-purines/pyrimidines plays a critical role for activity against herpesviruses.

Latent HSV-1 infection in mice immunized with a zwitterionic detergent-extracted HSV-1 antigen preparation

A HSV-1 antigen preparation obtained by zwitterionic detergent-extraction of HSV-1 infected Vero cells was investigated for its ability to protect mice against establishment of latent infection in the trigeminal ganglion following HSV-1 challenge by ear scarification. The antigen preparation was shown to induce antibody production and correlation between pre-challenge antibody level and establishment of latency was found. Although immunization did not reduce the overall incidence of ear erythema following challenge the duration of erythema was significantly decreased. Latency in the trigeminal ganglion was also significantly reduced in vaccinated mice and in addition, a correlation was found between the duration of erythema and latency, there being significantly more latent infections in mice with erythema of three or more days duration, than in those with erythema lasting less than three days.

HSV hepatitis in the mouse: A light and electron microscopic study with immunohistology and in situ hybridization

In order to characterize better the morphology and immune response in acute necrotizing HSV infection, murine HSV hepatitis was examined. BALB/c mice were inoculated intraperitoneally with 10(6) plaque-forming units (PFU) of HSV-1 (Lenette) and HSV-2 (D316). In both groups half the animals were pretreated with silica particles to block macrophage function. Up to 6 days after infection four mice from each group were sacrificed at daily intervals and the livers were examined by light and electron microscopy, immunohistology, in situ hybridization, combined immunohistology/in situ hybridization and titration of viral PFU. HSV-2 infected mice developed severe necrotizing hepatitis with persistence of HSV in the liver tissue until the end of the study. HSV-1 infected mice rapidly eliminated the virus and revealed only small necrotic foci. Early phase alterations and necrotic phase lesions were distinguished and characterized and morphologic evidence of a direct cytopathic effect of HSV was detected. A specific immune reaction in late stages appeared to be mediated by T4-positive T-lymphocytes. In situ hybridization and immunohistochemistry showed a close correlation with virus titration and were valuable in characterizing early phases and in the assessment of prognosis and differential diagnosis.

Die verhinderung der lymphohämatogenen Streuung als pathogenetische voraussetzung für die neurale persistenz des Herpes simplex Virus

In the experimental model of genital herpes simplex virus (HSV) infection of the mouse--in spite of abundant virus replication on the mucous membranes--no infectious virus can be isolated from the inflamed and swollen draining lymph nodes (DLN), contrary to the positive results in the lumbosacral nerves and their associated ganglia. Attempts to reactivate an abortive infection possibly established in lymph node or spleen cells by stimulation with phytohemagglutinin or lipopolysaccharide rendered no positive results, not even when adult (i.e. at least 6-week old), but immunodeficient mice were used as test animals. On the other hand, isolation of infectious virus from lymph node and--at a lesser rate--from spleen cells was successful in immature 4 to 6-week old mice, particularly when these had undergone pretreatment with cyclophosphamide, silica, antimacrophage serum and/or cortisone; 5 days post infectionem being the date of optimum virus yield. HSV-1 infected mice were more frequently positive than those with HSV-2, and genetically sensitive animals more so than resistant mice. The data indicate that the lymphohaematogenous spread of the virus is inhibited by means of an active defence mechanism, and that unspecific defence factors on the cellular level, probably macrophages and NK-cells, are essentially responsible. This reveals that the lethal generalized infection is prevented and the neural spread can gain its essential role in the pathogenesis of the HSV-infection.

Herpes simplex virus infection in capsaicin-treated mice

Following inoculation into the snout herpes simplex virus (HSV) spread to neurons in mouse trigeminal ganglion and subsequently to the brain. Capsaicin treatment of neonatal mice, which causes a loss of unmyelinated sensory neurons, some of which contain substance P, reduced the mortality rate of HSV-infected mice. Moreover, a lower percentage of mice survived the infection with reactivatable virus. There was also an extensive infection of glial cells proximal to the transitional zone in the trigeminal root between the peripheral and central nervous system. Distal to this zone there was an accumulation of substance P immunoreactivity in centrally directed fibres. This amplified degenerative effect on central branches of the substance P containing sensory nerves by glial infection may contribute to the deafferentiation pain syndrome following HSV infection.

Synthesis and antiviral activity of the carbocyclic analogues of (E)-5-(2-halovinyl)-2'-deoxyuridines and (E)-5-(2-halovinyl)-2'-deoxycytidines.

The carbocyclic analogues of the potent and selective antiherpes agents (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU), and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC) were synthesized by conventional methods with use of carbocyclic 2'-deoxyuridine as starting material. C-BVDU, C-IVDU, and C-BVDC were equally selective, albeit slightly less potent, in their antiherpes action than BVDU, IVDU, and BVDC. Although resistant to degradation by pyrimidine nucleoside phosphorylases, C-BVDU did not prove more effective than BVDU in the systemic (oral, intraperitoneal) or topical treatment of HSV-1 infections in mice.

Murine cellular cytotoxicity to syngeneic and xenogeneic herpes simplex virus-infected cells.

Cellular cytotoxicity of C57BL/6 adult mice peritoneal cells to xenogeneic (Chang liver) and syngeneic (BL/6-WT3) herpes simplex virus (HSV)-infected cells was analyzed in a 6-h 51Cr release assay. There was no difference in antibody-dependent cellular cytotoxicity to either target. There was no natural killer cytotoxicity to targets with cells from uninfected mice except at very high effector cell ratios. HSV-infected (2 X 10(4) PFU intraperitoneally 1 day previously) mice mediated significantly higher antibody-dependent cellular cytotoxicity and required less antibody (10(-5) versus 10(-2) dilution), fewer cells, and less time to kill than cells from uninfected mice. HSV-infected mice mediated natural killer cytotoxicity but preferentially killed syngeneic HSV-infected cells. Stimulation of cytotoxicity was not virus specific since influenza-infected mice mediated similar levels of cytotoxicity to HSV-infected targets. There was no difference in morphology (95% macrophage) or in the percentage of FcR-positive cells, but infected mice had more peritoneal cells and generated higher levels of superoxide in response to opsonized zymosan or phorbolmyristate acetate. These data demonstrate nonspecific virus-stimulated metabolic and effector cell function which may enhance clearance of virus in an infected host.

Mechanism of Antibody‐Mediated Protection against Herpes Simplex Virus Infection in Athymic Nude Mice: Requirement of Fc Portion of Antibody

Experiments were performed to investigate the resistance of the host due to antibody-mediated mechanisms to herpes simplex virus (HSV) infection. Transfer of hyperimmune anti-HSV mouse serum inhibited the development of skin lesions and prolonged the survival of lethally HSV-infected nude mice. Relatively high concentrations of antibody were required to achieve this protection. Antisera prepared in heterologous animals were also effective, while administration of anti-cowpox virus serum or interferon provided no protection. This type of protection is therefore due to specific antibody and cannot be attributed to interferon. In order to delineate the requirement for antibody in antibody-mediated protection, human gamma globulin preparations were transferred to lethally HSV-infected nude mice. Transfer of intact human gamma globulin (GG) was effective in controlling infection. S-sulfonation of GG did not diminish the protective ability. However, purified F(ab')2 did not have any protective action even when it was administered frequently to maintain serum neutralizing antibody titer. GG was effective in C5-deficient mice lethally infected with HSV. These results indicate that in vivo antibody-mediated protection to HSV infection requires the Fc region of the intact IgG molecule and suggest that antibody-dependent cell-mediated cytotoxicity may be operative in vivo.

MHC matching shows that at least two T-cell subsets determine resistance to HSV

DISTINCT T-cell subsets recognise antigens in association with different major histocompatibility complex (MHC) products, and this provides a means of identifying the subsets which operate in protective immunity against pathogens. Thus, cytotoxic T cells (Tc) recognise antigen in association with products of the K and D regions of the H–2 complex of the mouse; and because protection against lymphocytic choriomeningitis1, ectromelia2 and influenza3 requires compatibility at K or D in adoptive transfer experiments, it follows that the subset mediates protective immunity against these viruses. I-region compatibility requirements have not hitherto been observed in viral infections, but are necessary for protection against the intracellular parasite Listeria monocytogenes. This suggests that T helper cells (TH) and/or the lymphocytes responsible for delayed hypersensitivity and lymphokine release may be involved4 (these lymphocytes can provisionally be included within the TH subset). Adoptive transfer studies in mice have clearly established a major role for T lymphocytes in recovery from infection with herpes simplex virus (HSV-1)5–7. The T-lymphocyte subclass or classes responsible for this protection are not known. In vitro cytotoxic T cells (Tc)8, and their primed precursors9 can be obtained from HSV-1 infected mice. The present study indicates that immune protection against HSV-1 can be conferred by immune spleen cells possessing either I or K-D compatibility, and that long-lasting protection is conferred only by cells with I-region compatibility.

Latent herpes simplex virus in ganglia of mice after primary infection and reinoculation at a distant site

Herpes simplex virus (HSV)-infected hairless mice with evidence of latent infection in spinal ganglia did not develop latent HSV infections in trigeminal ganglia upon reinfection in the oro-facial area. HSV-infected and PAA-treated mice without evidence of latent HSV infection in spinal ganglia were resistant to reinfection in the lumbar region, but not to that performed in the oro-facial area.

Characteristics of Herpes Simplex Virus Resistance to Disodium Phosphonoacetate

Herpes simplex virus (HSV), which was partially resistant to the inhibitory effect of disodium phosphonoacetate (PAA), could be recovered following four virus passages in the presence of 100 microgram/ml PAA. Resistant strains were isolated from both HSV type 1 and HSV type 2. Virus resistance to PAA was not complete, and in most isolations a significant proportion of the virus stock remained susceptible to the drug. Resistance was shown to be heritable and persisted through virus passage and cloning experiments. PAA inhibited the replication of virus-specific DNA in sensitive strains of HSV but not in resistant strains of HSV. In vitro experiments directly demonstrated that PAA inhibited the activity of the virus-specific DNA polymerase 10 times more effectively in PAA-susceptible HSV than in PAA-resistant HSV. The treatment of HSV-infected mice with high levels of PAA did not induce the formation of resistant virus strains.

Immune response and latent infection after topical treatment of herpes simplex virus infection in hairless mice

Treatment of herpes simplex virus (HSV)-infected hairless mice with a 2% phosphonoacetic acid (PAA) ointment prevented the appearance of virus-induced skin lesions and subsequent central nervous system (CNS) involvement. Treatment started 24 h after infection significantly reduced the intensity of the skin lesions and also prevented CNS involvement. After four to six applications of PAA ointment, a moderate skin erythemia developed, followed by scaling and complete healing 7 days after cessation of the treatment. Mice treated early after HSV infection had low or undetectable levels of virus-specific antibodies but were completely resistant to reinfection. Early treatment prevented the development of a latent ganglionic infection, but treatment initiated 24 h after infection could not prevent the establishment of the latent infection. PAA-treated and HSV-infected mice with nondetectable levels of antibodies did not develop, with a single exception, a latent ganglionic infection unpon reinfection. The cell-mediated immune response determined by levels of [14C]thymidine incorporation in Ficoll-Hypaque-purified spleen lymphocytes cultures was low in PAA-treated mice; it increased slightly after challenge infection but was strong in mice that proved to harbor a latent HSV infection in the ganglia.

T-cell-mediated cytotoxicity against herpes simplex virus-infected target cells

THE control of herpes simplex virus (HSV) infection by immunological mechanisms seems to be complex and is poorly understood. Neutralising antibodies to HSV plus complement seem to have no effect on the propagation of HSV infection, because HSV spreads to adjacent cells by passing through intercellular bridges1–3. Anti-HSV antibodies plus complement, however, destroy virus-infected cells, but cannot prevent the spread of HSV, suggesting that the virus must be transferred to neighbouring cells before immune lysis occurs1,5. Therefore if lymphocyte-mediated cytolytic mechanisms are instrumental in blocking the spread of HSV in vivo, they ought to destroy infected cells at a very early stage in the viral maturation cycle, before infectious viral particles are produced. Indeed, effective antibody dependent cell-mediated cytotoxicity (ADCC) has been demonstrated in vitro using target cells infected with HSV4,5. In this system K-cell-mediated destruction of virus-infected cells takes place very early in the infectious cycle5. As in vivo protection against HSV infection is predominantly T-cell-dependent6,7, the in vivo spread of HSV infections may be controlled by virus-specific cytotoxic T lymphocytes (CTL). But no virus-specific CTL so far have been detected in the spleen of HSV-infected mice. Using a different experimental approach we have now shown that T lymphocytes of mice undergoing a local HSV infection, when transferred in vitro, differentiate into effective CTL. The CTL generated are highly cytotoxic for HSV-infected target cells.

Role of interferon in the pathogenesis of virus diseases in mice as demonstrated by the use of anti-interferon serum. II. Studies with herpes simplex, Moloney sarcoma, vesicular stomatitis, Newcastle disease, and influenza viruses.

The effect of potent sheep anti-mouse interferon globulin was investigated in several different experimental virus diseases of mice. In anti-interferon globulin-treated mice infected intraperitoneally with herpes simplex virus (HSV) type I, the latent period was shortened, and the overall LD50 was increased several hundredfold compared to virus-infected control mice. When HSV was inoculated subcutaneously all anti-interferon globulin-treated mice died, whereas only 5% of virus-infected control mice died. Subsequent treatment with anti-interferon globulin of previously HSV-infected mice did not result in reactivation of HSV. Treatment of adult mice with anti-interferon globulin resulted in an earlier appearance of MSV-induced tumors, a greater number of mice bearing tumors, an increase in tumor size, and an increase in the duration of tumors. All tumors eventually regressed despite reinjection of anti-interferon globulin. Anti-interferon globulin treatment resulted in a rapid onset of disease and death in adult mice inoculated (intranasal) with VSV and in newborn mice infected with NDV. Anti-interferon globulin exerted no effect on the course of influenza virus infection of mice. We conclude that the early production of interferon is an importane element in the response of the mouse to several viruses exhibiting different pathogeneses.

Influence of changes in brain monoamine metabolism on behaviour of herpes simplex-infected mice

In herpes simplex virus (HSV)-infected mice hypermotility was observed associated with an increased release of transmitter from adrenergic neurons. Inhibition of dopamine (DA) metabolism by the administration of α-methyltyrosine to the infected mice decreased motility rate counts while p-chlorophenylalanine, by inhibiting the formation of 5-hydroxytryptamine (5-HT) caused an increase in rate of motility. Pimozide and phenoxybenzamine, used for blocking DA and noradrenaline (NA) receptors, respectively, were both observed to reduce the hypermotility efficiently. Administration of FLA-63 as an inhibitor of NA-metabolism was associated with increased death rates in HSV-infected mice. Changes in behaviour towards aggression were encountered when levodopa was administered to HSV-infected mice and when apomorphine and clonidine were used for stimulation of the receptor functions of adrenergic neurons. The observations reported suggest that hypermotility, excitation and aggression might all be symptoms associated with a pathologically increased activity of adrenergic neurons.