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A A Nash

Publications and source records attributed to A A Nash.

At least 19 recordsLinked to original sources

The m4 gene of murine gammaherpesvirus modulates productive and latent infection in vivo.

Murine gammaherpesvirus 68 (MHV-68) infection of mice represents a viable small-animal model for the study of gammaherpesvirus pathogenesis. MHV-76 is a deletion mutant of MHV-68, which lacks four MHV-68-specific genes (M1 to M4) and eight viral tRNA-like sequences at the 5' end of the genome. These genes are implicated in latency and/or immune evasion. Consequently, MHV-76 is attenuated in the acute phase of in vivo infection with respect to MHV-68. Little is known about the role of M4 in viral infection, except that it is expressed as an immediate-early/early transcript during lytic replication of MHV-68 in vitro. To elucidate the contribution M4 makes to in vivo pathogenesis, we created a novel MHV-76 mutant (MHV-76inM4), in which the region of MHV-68 coding for M4 and accompanying putative promoter elements were inserted into the 5' region of the MHV-76 genome. The growth of MHV-76inM4 in vitro was indistinguishable from that of MHV-76 and MHV-68. However, virus titers from MHV-76inM4-infected BALB/c mice were significantly increased with respect to MHV-76 at early times in the lung. In addition, at days 17 and 21 postinfection, there was a significant elevation in latent viral load in splenocytes of MHV-76inM4-infected mice compared to MHV-76. Like MHV-76-infected mice, MHV-76inM4-infected mice display no evidence of overt splenomegaly, a finding characteristic of MHV-68 infection. M4 expression in vivo was detectable during productive infection in the lung and during the establishment of latency in the spleen, but in general M4 was not detectable during long-term latency (day 100 postinfection).

Animals↗

Natural history of murine gamma-herpesvirus infection.

Murine gamma-herpesvirus 68 (MHV-68) is a natural pathogen of small rodents and insectivores (mice, voles and shrews). The primary infection is characterized by virus replication in lung epithelial cells and the establishment of a latent infection in B lymphocytes. The virus is also observed to persist in lung epithelial cells, dendritic cells and macrophages. Splenomegaly is observed two weeks after infection, in which there is a CD4+ T-cell-mediated expansion of B and T cells in the spleen. At three weeks post-infection an infectious mononucleosis-like syndrome is observed involving a major expansion of Vbeta4+CD8+ T cells. Later in the course of persistent infection, ca. 10% of mice develop lymphoproliferative disease characterized as lymphomas of B-cell origin. The genome from MHV-68 strain g2.4 has been sequenced and contains ca. 73 genes, the majority of which are collinear and homologous to other gamma-herpesviruses. The genome includes cellular homologues for a complement-regulatory protein, Bcl-2, cyclin D and interleukin-8 receptor and a set of novel genes M1 to M4. The function of these genes in the context of latent infections, evasion of immune responses and virus-mediated pathologies is discussed. Both innate and adaptive immune responses play an active role in limiting virus infection. The absence of type I interferon (IFN) results in a lethal MHV-68 infection, emphasizing the central role of these cytokines at the initial stages of infection. In contrast, type II IFN is not essential for the recovery from infection in the lung, but a failure of type II IFN receptor signalling results in the atrophy of lymphoid tissue associated with virus persistence. Splenic atrophy appears to be the result of immunopathology, since in the absence of CD8+ T cells no pathology occurs. CD8+ T cells play a major role in recovery from the primary infection, and also in regulating latently infected cells expressing the M2 gene product. CD4+ T cells have a key role in surveillance against virus recurrences in the lung, in part mediated through 'help' in the genesis of neutralizing antibodies. In the absence of CD4+ T cells, virus-specific CD8+ T cells are able to control the primary infection in the respiratory tract, yet surprisingly the memory CD8+ T cells generated are unable to inhibit virus recurrences in the lung. This could be explained in part by the observations that this virus can downregulate major histocompatibility complex class I expression and also restrict inflammatory cell responses by producing a chemokine-binding protein (M3 gene product). MHV-68 provides an excellent model to explore methods for controlling gamma-herpesvirus infection through vaccination and chemotherapy. Vaccination with gp150 (a homologue of gp350 of Epstein-Barr virus) results in a reduction in splenomegaly and virus latency but does not block replication in the lung, nor the establishment of a latent infection. Even when lung virus infection is greatly reduced following the action of CD8+ T cells, induced via a prime-boost vaccination strategy, a latent infection is established. Potent antiviral compounds such as the nucleoside analogue 2'deoxy-5-ethyl-beta-4'-thiouridine, which disrupts virus replication in vivo, cannot inhibit the establishment of a latent infection. Clearly, devising strategies to interrupt the establishment of latent virus infections may well prove impossible with existing methods.

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Murine gammaherpesvirus-68 infection causes multi-organ fibrosis and alters leukocyte trafficking in interferon-gamma receptor knockout mice.

Murine gammaherpesvirus-68 (MHV-68) infection in interferon-gamma receptor knockout mice (IFN-gammaR(-)/(-)) results in splenic fibrosis and excessive loss of splenocytes. In our present study we found that MHV-68 infection in IFN-gammaR(-)/(-) mice also resulted in fibrosis and atrophy of the mediastinal lymph nodes, interstitial pulmonary fibrosis and fibrotic changes in the liver. Atrophy and cellular depletion of the spleen in IFN-gammaR(-)/(-) was not the result of increased cell death. The loss of splenocytes in IFN-gammaR(-)/(-) mice, which was most evident on day 23 after infection, correlated with an increase in the number of leukocytes in peripheral blood. At the peak of leukocytosis, on day 23 after infection, peripheral blood cells from infected IFN-gammaR(-)/(-) mice were unable to traffic through the fibrosed spleens of IFN-gammaR(-)/(-) mice but were able to enter the spleens of wild-type mice. This indicates that leukocytosis was in part the result of emigration of cells from the spleen and their subsequent exclusion of re-entry at the height of fibrosis. Significant cytokine and chemokine changes were observed in spleens of IFN-gammaR(-)/(-) mice. IFN-gamma, tumor necrosis factor-alpha (TNF-alpha ), TNF-beta, interleukin-1beta (IL-1beta), transforming growth factor-beta1 (TGF-beta1), lymphotactin, and MIP-1beta were elevated on day 14 after infection whereas chemokines IP-10 and MIG were significantly reduced. These changes suggest a role for dysregulated cytokines and chemokines in severe organ-specific fibrosis with implications for immune-mediated fibrotic disorders.

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Analysis of a novel strain of murine gammaherpesvirus reveals a genomic locus important for acute pathogenesis.

Infection of mice by murine gammaherpesvirus 68 (MHV-68) is an excellent small-animal model of gammaherpesvirus pathogenesis in a natural host. We have carried out comparative studies of another herpesvirus, murine herpesvirus 76 (MHV-76), which was isolated at the same time as MHV-68 but from a different murid host, the yellow-necked mouse (Apodemus flavicollis). Molecular analyses revealed that the MHV-76 genome is essentially identical to that of MHV-68, except for deletion of 9,538 bp at the left end of the unique region. MHV-76 is therefore a deletion mutant that lacks four genes unique to MHV-68 (M1, M2, M3, and M4) as well as the eight viral tRNA-like genes. Replication of MHV-76 in cell culture was identical to that of MHV-68. However, following infection of mice, MHV-76 was cleared more rapidly from the lungs. In line with this, there was an increased inflammatory response in lungs with MHV-76. Splenomegaly was also significantly reduced following MHV-76 infection, and much less latent MHV-76 was detected in the spleen. Nevertheless, MHV-76 maintained long-term latency in the lungs and spleen. We utilized a cosmid containing the left end of the MHV-68 genome to reinsert the deleted sequence into MHV-76 by recombination in infected cells, and we isolated a rescuant virus designated MHV-76(cA8+)4 which was ostensibly genetically identical to MHV-68. The growth properties of the rescuant in infected mice were identical to those of MHV-68. These results demonstrate that genetic elements at the left end of the unique region of the MHV-68 genome play vital roles in host evasion and are critical to the development of splenic pathology.

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Murine gammaherpesvirus M11 gene product inhibits apoptosis and is expressed during virus persistence.

The murine gammaherpesvirus (MHV-68) M11 gene encodes a protein with BH1 domain homology to Bcl-2. We found that the M11 gene product (MHVBcl-2) protected murine epithelial cells from TNF-alpha induced apoptosis. M11 was transcribed during early lytic infection in vitro. During early infection of mice, M11 message was detected in spleen and lung along with lytic cycle messages. During persistence, lytic cycle gene expression was undetectable but M11 RNA was still present. This suggests that MHVBcl-2 promotes virus survival by protecting not only productively infected but also persistently infected cells from apoptotic death.

Amino Acid Sequence↗

Herpes virus latency in sensory ganglia--a comparison with endogenous neuronal gene expression.

Central to infection by a majority of DNA viruses is the expression of encoded proteins that modify cell cycle. Viruses such as SV40 and Adenovirus viruses encode proteins that interact directly, or indirectly, with key cell cycle proteins such as CBP300 and the retinoblastoma gene product. However, neurons do not have a cell cycle as we generally describe it and this is also reflected in the difficulty in obtaining immortalised neuronal cultures. The replication strategies of viruses that infect post-mitotic cells such as neurons may be different from infection of other somatic cells. The life cycle for viral latency or slow infection of neurons appears to involve silencing or restricting expression of the viral genome until such times as dictated by the environment. These signals from the environment usually reflect cell stress, otherwise the cell appears to tolerate the existence of the virus genome. We will review the genomic structure of alphaherpesviruses in neurons and transcriptional control mechanisms that may regulate expression. Where appropriate we will contrast and compare virus and endogenous neuronal gene expression.

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Rta of murine gammaherpesvirus 68 reactivates the complete lytic cycle from latency.

Herpesviruses are characterized as having two distinct life cycle phases: lytic replication and latency. The mechanisms of latency establishment and maintenance, as well as the switch from latency to lytic replication, are poorly understood. Human gammaherpesviruses, including Epstein-Barr virus (EBV) and human herpesvirus-8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), are associated with lymphoproliferative diseases and several human tumors. Unfortunately, the lack of cell lines to support efficient de novo productive infection and restricted host ranges of EBV and HHV-8 make it difficult to explore certain important biological questions. Murine gammaherpesvirus 68 (MHV-68, or gammaHV68) can establish de novo lytic infection in a variety of cell lines and is also able to infect laboratory mice, offering an ideal model with which to study various aspects of gammaherpesvirus infection. Here we describe in vitro studies of the mechanisms of the switch from latency to lytic replication of MHV-68. An MHV-68 gene, rta (replication and transcription activator), encoded primarily by open reading frame 50 (ORF50), is homologous to the rta genes of other gammaherpesviruses, including HHV-8 and EBV. HHV-8 and EBV Rta have been shown to play central roles in viral reactivation from latency. We first studied the kinetics of MHV-68 rta gene transcription during de novo lytic infection. MHV-68 rta was predominantly expressed as a 2-kb immediate-early transcript. Sequence analysis of MHV-68 rta cDNA revealed that an 866-nucleotide intron 5' of ORF50 was removed to create the Rta ORF of 583 amino acids. To test the functions of MHV-68 Rta in reactivation, a plasmid expressing Rta was transfected into a latently infected cell line, S11E, which was established from a B-cell lymphoma in an MHV-68-infected mouse. Rta induced expression of viral early and late genes, lytic replication of viral DNA, and production of infectious viral particles. We conclude that Rta alone is able to disrupt latency, activate viral lytic replication, and drive the lytic cycle to completion. This study indicates that MHV-68 provides a valuable model for investigating regulation of the balance between latency and lytic replication in vitro and in vivo.

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Type I interferons and IRF-1 play a critical role in the control of a gammaherpesvirus infection.

The murine gammaherpesvirus 68 (MHV-68) is an ideal model system for the study of interactions between gammaherpesviruses and their hosts. Intranasal infection of mice with MHV-68 results in replication of the virus in the lung epithelium followed by latent infection of B cells. Resolution of productive MHV-68 infection depends on the adaptive immune system, but little is known about the role of innate immune mechanisms and the early interaction between the host and the virus. In this report, we have used mice that are deficient in components of the early defence system, the common type I interferon (IFN) receptor (IFN R), the transcriptional activator IRF-1, and the inducible nitric oxide synthase, to investigate the contribution of these mechanisms to control of MHV-68 infection. We show that while wild-type mice are highly resistant to infection with MHV-68, mice unresponsive to type I IFNs (IFN-alpha/beta R(-/-) ) are highly susceptible to the virus. At high multiplicities of infection (m.o.i. ; 4 x 10(6) PFU), 80-90% of IFN-alpha/beta R(-/-) mice succumb to infection, and at low m.o.i. (4 x 10(3) PFU), 50% mortality rates occur. Both high and low doses of virus lead to 100- to 1000-fold higher lung virus titres in IFN-alpha/beta R(-/-) mice than are found in wild-type mice and result in systemic dissemination of the virus. Latently infected cells are detectable in the spleens of IFN-alpha/beta R(-/-) mice earlier than in wild-type mice, and the numbers of latently infected cells are 10-fold higher in the IFN-alpha/beta R(-/-) mice during the acute phase of infection. We find IRF-1 has a critical role in protection from fatal disease, whereas inducible nitric oxide synthase does not appear to be important. The results indicate that innate immune mechanisms are critical for the early control of MHV-68 and may play a role in the establishment of latency.

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Murine gamma-herpesvirus 68 glycoprotein 150 protects against virus-induced mononucleosis: a model system for gamma-herpesvirus vaccination.

Murine gamma-herpesvirus 68 (MHV-68) is a model for the study of the pathogenesis of gamma-herpesviruses. Epstein-Barr virus (EBV) is a highly related gamma-herpesvirus that causes significant disease in humans. The major membrane antigen gp350 of EBV is a candidate vaccine antigen for protection against EBV-related disease. An MHV-68 glycoprotein, gp150, has significant homology to EBV gp350. We have therefore used the MHV-68 gp150 to model the potential efficacy of EBV gp350 in protecting from virus-associated disease. A recombinant vaccinia virus expressing MHV-68 gp150 was constructed. This recombinant vaccinia virus was used to infect mice via the subcutaneous route. This vaccination resulted in production of MHV-68-neutralising antibodies. Mice were then challenged intra-nasally with MHV-68. MHV-68-associated mononucleosis was virtually abrogated in immunised mice. However, mice did establish MHV-68 latency. The results suggest that gp350 may be effective as an immunogen to prevent EBV-associated infectious mononucleosis in humans that are EBV-seronegative.

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The role of endogenous interleukin-12 in resistance to murine cytomegalovirus (MCMV) infection and a novel action for endogenous IL-12 p40.

Biologically active interleukin-12 (IL-12), comprising a 40 kDa subunit (p40) covalently linked to a 35 kDa subunit (p35), is produced in response to a range of infectious stimuli. Here, we demonstrate that mice deficient in either IL-12 p40 (p40-/-) or IL-12 p35 (p35-/-) are susceptible to murine cytomegalovirus (MCMV) infection in terms of survival (Balb/c p35-/-) and viral clearance (Balb/c p35-/- and Balb/c p40-/-), and this susceptibility may be correlated to a deficiency in serum interferon-gamma (IFN-gamma) levels. These data support a role for endogenous IL-12 in controlling MCMV infection. The IL-12 p40 subunit is produced in excess of IL-12 p35, and to date the function of the excess endogenous p40 has been assumed to be one of IL-12 antagonism, as demonstrated by experiments with exogenous p40 both in vivo and in vitro. We show that Balb/c p35-/- alone are significantly compromised in survival of a sublethal infection and in clearance of virus from the spleen. These mice produce a very early IFN-gamma spike (8 h after infection) and an aberrant tumor necrosis factor-alpha (TNF-alpha) spike (day 2 after infection). MCMV infection has revealed an altered Balb/c p35-/- phenotype compared with Balb/c p40-/-, and this indicates that endogenous p40 may have an activity independent of and additional to IL-12 antagonism in vivo.

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Behavioural changes in the rat following infection with varicella-zoster virus.

Following the establishment of a chronic varicella-zoster virus infection in the rat, behavioural allodynia and hyperalgesia were observed in the injected, but not the contralateral hind limb up to 33 days post-infection. This model may prove useful in investigating mechanisms involved in the establishment of post-herpetic neuralgia.

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2'-Deoxy-5-ethyl-beta-4'-thiouridine inhibits replication of murine gammaherpesvirus and delays the onset of virus latency.

The antiviral thionucleoside analogue 2'-deoxy-5-ethyl-beta-4'-thiouridine (4'-S-EtdU) was shown to be a more potent inhibitor of gammaherpesvirus infection than acyclovir. This compound inhibits replication of murine herpesvirus (MHV)-68 in the lungs of mice when given 3 days post-infection. However, as with other nucleoside analogues, it was unable to prevent the establishment of latency, despite delaying the onset of latent infection in the spleen. In contrast, virus persistence in the lung was inhibited following drug treatment, although persistence was re-established in mice when treatment was suspended after 12 days. These data suggest that 4'-S-EtdU is a highly effective inhibitor of murine gammaherpesvirus replication and as such provides a powerful tool to study the pathogenesis of this virus in vivo.

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Substance use among adolescents with juvenile rheumatoid arthritis.

OBJECTIVE: To determine the prevalence of substance use among adolescents with juvenile rheumatoid arthritis and to assess available opportunities for rheumatologists to identify high risk teens. METHODS: Fifty-two teens (mean age 13.9 years, 86% female) completed questionnaires regarding substance use (alcohol, tobacco, marijuana, and other illicit substances), functional disability, and frequency of health care contacts. RESULTS: Alcohol use was reported by 30.7% of teens, including 23.5% of those for whom methotrexate was prescribed; 15.4% reported tobacco use in the last year, and 13.4% reported other illicit substance use in their lifetime, although most use was experimental. No teen reported marijuana use. The majority reported regular contact with their rheumatologist but only 26.9% were ever interviewed alone. CONCLUSION: Many teens with juvenile rheumatoid arthritis, including those prescribed methotrexate, used substances, especially alcohol. When rheumatologists see adolescents, particularly in situations where methotrexate may be prescribed, a clinical setting conductive to confidentially, physician comfort in asking about sensitive topics such as substance abuse, and referral relationships with skilled adolescent health and substance abuse counseling providers are essential.

Activities of Daily Living↗

Investigation of the role of delayed-type-hypersensitivity responses to myelin in the pathogenesis of Theiler's virus-induced demyelinating disease.

The contribution of autoimmune responses to the pathogenesis of Theiler's virus-induced demyelinating disease was investigated. Delayed-type hypersensitivity responses to myelin were examined in both symptomatic and asymptomatic mice at different times post-infection, in order to determine whether autoreactivity correlates with the development of demyelination. The results indicate that although autoimmune responses probably do not play a major role in the initiation of demyelination at early times post-infection, autoreactivity to myelin antigens dose eventually develop in symptomatic animals, perhaps through the mechanism of epitope spreading. Autoimmunity to myelin components is therefore an additional factor that may contribute to lesion progression in chronically diseased animals.

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The PI capsid region of Theiler's virus controls replication in mouse glial cell cultures.

The GDVII strain of Theiler's virus is virulent. The DA strain is avirulent and can persist and initiate lesions of inflammatory demyelination in the CNS of susceptible strains of mice. Other, resistant strains of mice clear the infection. Replication of the GDVII and DA strains of Theiler's virus and their genetic recombinants R2, R3 and R4 were compared in mixed glial cell cultures derived from the mouse CNS. Differences were observed in the early rate of viral production. These mapped to the P1 capsid region of the viral genome. Viruses with GDVII P1 sequences produced virus and spread more rapidly than viruses with DA P1 sequences. GDVII virus infected greater numbers of cells than DA virus. Both strains of virus rapidly replicated at least to the level of translation in astrocytes (GFAP+), macrophage/microglial cells (F4/80+), oligodendrocytes (O4+) and bipotential precursor (A2B5+) cells. Early in infection many A2B5+ and GFAP+ cells were infected and destroyed. In contrast, O4+ cells were relatively resistant to cell-death. The cultures survived and produced virus over 14 days of study, at which time all 4 cell-type were present in the culture but < 1% of all the cells, the majority of which were O4+, expressed viral protein. Most of these infected O4+ cells retained a healthy morphology with extensive sheets of cytoplasm, suggesting that Theiler's virus infection of mature oligodendrocytes was non-destructive.

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Characterisation of the immune response in a neural xenograft rejection paradigm.

We have looked at both donor and host MHC expression in a neural xenograft rejection paradigm. Grafts of either mouse corpus callosum or an SV40 large T transformed astrocytic cell line were placed in the mid-brain of neonatal rats. Three weeks later graft rejection was induced by the application of a skin graft of the same donor origin. MHC expression in the neural graft and the host brain was examined histologically four and ten days after the animals had received a skin graft. Donor MHC expression was detected in the corpus callosal grafts at both time points and preceded host MHC expression and the lymphocytic infiltrate. The grafts of the transformed cell line could not be induced to express MHC antigens under the experimental protocol used nor were they rejected. The migratory patterns of the transformed cells were compared to the well characterised migration patterns of astrocytes from the corpus callosal grafts.

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