Expansion and activation of NK cell populations in a gammaherpesvirus infection.

NK cells are an important component of the innate immune response to many virus infections. In particular, they play a major role in control of alpha and beta herpesvirus infections in humans and mice and there is evidence for a protective role in Epstein-Barr virus infection. MHV-68 has been widely used to study gammaherpesvirus pathogenesis and provides a tractable means of investigating the role of NK cells in gammaherpesvirus infections. We have shown that, following MHV-68 infection of mice, the NK cell population is expanded and activated and capable of cytotoxic killing in vitro. However, depletion of NK cells prior to MHV-68 infection did not affect viral loads in vivo. To investigate the possibility that MHV-68 was downregulating NK cell activity in vivo and evading the NK cell response, we infected NK cell-depleted mice with the related virus, MHV-76, which lacks a 9.5 kb region of the genome known to be involved in modulating the host immune response. Infection of NK cell-depleted mice with MHV-76 did not result in increased viral loads indicating that genes within this region do not encode products which modulate NK cell activity.

Development of an enzyme-linked immunosorbent assay for the detection of antibodies against malignant catarrhal fever viruses in cattle serum.

Malignant catarrhal fever (MCF) is a sporadic but fatal lymphoproliferative viral disease of cattle, deer and other ruminants. The causative agents are highly-cell-associated herpesviruses of the subfamily gammaherpesvirinae. In this study, an ELISA (WC11-ELISA) was developed to detect antibody to malignant catarrhal fever virus (MCFV) in cattle serum and compared to the commercially produced competitive-inhibition ELISA (CI-ELISA). Crude lysate antigen from alcelaphine herpesvirus-1 strain WC11 was bound to 96-well microplates and used to capture antibodies to MCFV. Dilutions of test sera were added to wells containing bound MCF antigen and control wells containing uninfected cell lysates. A horseradish peroxidase-labelled rabbit-anti-bovine IgG conjugate detected antibodies to MCF, and the results were expressed as absorbance readings at 450 nm. Samples were selected blind from cattle sera which had been sent to the laboratory for diagnostic testing for MCFV antibodies and were tested in both the WC11-ELISA and the CI-ELISA. Good agreement between the WC11-ELISA and CI-ELISA test (k=0.86, n=95) results was found.

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).

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.

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.

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.

Control of gammaherpesvirus latency by latent antigen-specific CD8(+) T cells.

The contribution of the latent antigen-specific CD8(+) T cell response to the control of gammaherpesvirus latency is currently obscure. Some latent antigens induce potent T cell responses, but little is known about their induction or the role they play during the establishment of latency. Here we used the murine gammaherpesvirus system to examine the expression of the latency-associated M2 gene during latency and the induction of the CD8(+) T cell response to this protein. M2, in contrast to the M3 latency-associated antigen, was expressed at day 14 after infection but was undetectable during long-term latency. The induction of the M2(91-99)/K(d) CD8(+) T cell response was B cell dependent, transient, and apparently induced by the rapid increase in latently infected cells around day 14 after intranasal infection. These kinetics were consistent with a role in controlling the initial "burst" of latently infected cells. In support of this hypothesis, adoptive transfer of an M2-specific CD8(+) T cell line reduced the initial load of latently infected cells, although not the long-term load. These data represent the first description of a latent antigen-specific immune response in this model, and suggest that vaccination with latent antigens such as M2 may be capable of modulating latent gammaherpesvirus infection.

Sequence and transfection of BoLA-DRB3 cDNAs.

Bovine MHC (BoLA-) DRB3 alleles encoded by the DH8A, DH22A and DH24A class II haplotypes were cloned from cDNA and characterized by sequence analysis. Comparison with other full-length DRB3 sequences suggested that DRB3 alleles may have evolved through multiple lineages. All three BoLA-DRB3 alleles were shown to express on the surface of transfected cells, and the transfectants were used to define or confirm the class II specificity of a panel of monoclonal antibodies.

Sheep CD1 genes and proteins.

Interest in CD1 genes and proteins was initially stimulated by their close evolutionary and structural relationship to MHC class I molecules. The demonstration that CD1b and c molecules present novel non-peptide antigens to T-cells and play a role in protection against mycobacterial infection then focused attention on the functional role of CD1 proteins. Sheep possess at least seven CD1 genes, including CD1B, D and E, which is the most complex genetic arrangement identified so far in any animal. OvCD1B consists of at least three distinct genes, with the probability of limited polymorphism and the existence of splice variants. Most anti-sheep CD1-specific monoclonal antibodies react with OvCDlb and phenotypic and immunochemical data suggests the existence of two variants. CD1D genes have been identified in all species studied, suggesting a conserved role for CDld proteins across mammalian species. Presumptive evidence for the existence of OvCDIE has been obtained by NH2-terminal sequencing of protein precipitated by the mAb 20.27 (SBU-T6). Confirmatory evidence from gene cloning experiments is currently being sought. Collectively, these factors make the sheep CD1 family a highly relevant model for investigating the in vivo role of CD1 molecules. In this survey, the properties of monoclonal antibodies specific for sheep CD1, the cellular distribution and physicochemical characteristics of sheep CD1 molecules and the current state of knowledge on sheep CD1 genetics are reviewed.

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.

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.

Amino-terminal sequencing of sheep CD1 antigens and identification of a sheep CD1D gene.

The anti-CD1 monoclonal antibodies IAH-CC14 and SBU-T6 were used to immunopurify CD1 antigens from sheep thymocytes. The amino-terminal sequence of IAH-CC14 yielded 13 amino acids, and 29 amino acids were obtained from the SBU-T6 antigen. The sequence of the IAH-CC14 antigen was 100% identical to the predicted sequence of the sheep CD1B clone, SCD1B-42. The 29 amino acid sequence of the SBU-T6 antigen did not match identically with the derived amino acid sequence of any of the previously reported sheep CD1 genes but had closest similarity to the derived sequence of human CD1E. Degenerate polymerase chain reaction primers based on this sequence identified a group 2 sheep CD1 gene. The predicted amino acid sequence of this gene shows that it is not identical to the SBU-T6 peptide, indicating that a different, CD1D-like gene was cloned.

Pathological changes in the spleens of gamma interferon receptor-deficient mice infected with murine gammaherpesvirus: a role for CD8 T cells.

Murine gammaherpesvirus is a natural rodent pathogen which causes a primary infection in the lungs and establishes a persistent infection in B lymphocytes. During the primary infection, large amounts of gamma interferon (IFN-gamma) are produced by spleen, mediastinal, and cervical lymph node cells. To investigate the role of IFN-gamma in control of the virus infection, mice lacking the cellular receptor for IFN-gamma (IFN-gamma R-/- mice) were infected with murine gammaherpesvirus 68 (MHV68). IFN-gamma R-/- mice showed no difference from wild-type mice in the titers of infectious virus in the lungs or in the rate of clearance of the lung infection. In the spleen, however, clear differences were observed. By 14 days postinfection, spleens from IFN-gamma R-/- mice were pale, shrunken, and fibrous. Histological examination showed that there was an early (day 10) infiltration of granulocytes followed by widespread destruction of splenic architecture (days 14 to 17). A marked decrease in the number of splenic B cells and CD4+ and CD8+ T cells occurred. These changes were accompanied by a 10- to 100-fold greater load of latently infected cells in IFN-gamma R-/- mice than in wild-type mice at 14 to 17 days postinfection, but this was reduced to the levels found in wild-type mice by 21 days postinfection. Treatment of the mice with the antiviral drug 2'-deoxyl-5-ethyl-beta-4'-thiouridine from 6 days postinfection did not prevent the occurrence of these changes. The changes were, however, completely reversed by depletion of CD8+ T cells prior to and during the primary infection. Depletion of CD4+ T cells also reversed the major pathological and virological changes, although in this case there was evidence of some histological changes. Thus, the lack of IFN-gamma receptor had profound consequences in spleens of MHV68-infected mice. The possible mechanisms involved in these changes are discussed.

Discrimination of two subsets of CD1 molecules in the sheep.

This paper examines the expression of CD1 in the sheep utilising the monoclonal antibodies (mAbs) which were assigned to OvCD1 in the First and Second Workshops on Ruminant Leukocyte Differentiation Antigens along with those primarily clustered as Bov/OvCD1 in the Third Workshop. Detailed immunohistological studies of both lymphoid and non-lymphoid tissues and flow cytometry of isolated cell populations revealed two distinct patterns of CD1 expression in the sheep. The mAbs assigned to the sub-cluster BovCD1w1 (SBU-T6) and BovCD1w3 (IAH-CC43 and IAH-CC118) were much more widely distributed than those of the sub-cluster BovCD1w2. In addition to cortical thymocytes and dendritic cells (DC) the CD1w1 and w3 molecules are expressed by peripheral blood B lymphocytes, monocytes and many tissue macrophages.

The sheep CD1 gene family contains at least four CD1B homologues.

We identified four cDNA sequences encoding sheep homologues of the CD1 molecule. The sheep sequences were selected from lambdagt11 thymocyte cDNA libraries by hybridization with a human CD1C probe and a homologous sheep probe. The SCD1B-42 and SCD1A25 sequences encode complete CD1 molecules. The third sequence, SCD1B-52, which is closely related to SCD1B-42 and may be an allele, has the sequence encoding the alpha3 region precisely deleted. The fourth sequence, SCD1T10, is truncated at the 5' end. All four sequences are related to the human CD1B and domestic rabbit CD1B-like sequences at both nucleotide and amino acid level. Comparison of the derived CD1 amino acid sequences with the sequence of major histocompatibility complex class I molecules showed that the sheep CD1 molecules, like human CD1 molecules, lack most of the conserved class I residues known to be involved in interaction with beta2-microglobulin and the CD8 molecule. They do not contain the peptide docking residues involved in anchoring peptides in the peptide binding groove of class I molecules. Southern hybridization of sheep DNA with a sheep CD1 exon 4/alpha3 probe showed that the sheep genome encodes at least seven CD1 genes. The implications of these analyses for CD1 function are discussed.

Discrimination between major histocompatibility complex class II DQ and DR locus products in cattle.

We have used a panel of anti-major histocompatibility complex (MHC) class II monoclonal antibodies (mAbs) and have assessed their specificity for the products of the individual bovine MHC (BoLA) class II subregions. The mAbs identified two distinct class II molecules by affinity purification and ELISA. Two-dimensional immunoblotting confirmed these data and NH2-terminal sequencing of the purified class II alpha chains of one member of each group identified the subregion specificity of the mAbs. The mAbs VPM36, TH22A and TH81A are specific for BoLA DQ, whereas VPM54, TH14B and J11 are specific for BoLA DR. SW73.2 reacts with both MHC subgroups of all cattle tested.

Analysis of the fine specificities of sheep major histocompatibility complex class II-specific monoclonal antibodies using mouse L-cell transfectants.

The fine specificities of two panels of monoclonal antibodies (mAbs) for sheep major histocompatibility complex (MHC) class II molecules were determined using five mouse L-cell transfectants, each expressing a defined sheep DQ or DR MHC class II A/B gene pair. Using the transfectants in an indirect fluorescence antibody assay, previous immunochemical characterization of the mAbs was confirmed for 16 of 23 mAbs tested. The MHC class II subtype specificity (DQ or DR) of each mAb was assigned without interference from the products of other expressed class II loci. This allowed the identification of both cross-locus specificities as well as defining fine specificities of mAbs previously only partially characterized by immunochemical techniques.

Sequence of the sheep interleukin-10-encoding cDNA.

The ovine interleukin-10 (oIL-10)-encoding cDNA has been cloned and sequenced using gene amplification by the polymerase chain reaction (PCR). We present the complete coding sequence of the ovine IL-10 gene, as well as the predicted amino acid (aa) sequence. The oIL10 DNA coding sequence is 531 nucleotides long and the mature protein product is predicted to be 18,367 Da, consisting of 158 aa, excluding a 19-aa N-terminal hydrophobic signal peptide. The oIL-10 protein is > 77% identical to pig and human IL-10, > 71% identical to rodent IL-10 and > 68% identical to viral IL-10.

Evidence for the expression of two distinct MHC class II DR beta like molecules in the sheep.

This study used monoclonal antibodies to sheep MHC class II molecules as well as an L cell transfectant (T8.1) which expresses DRA and DRB genes to show that two distinct DR beta chains are expressed in the sheep. Two anti-beta chain specific monoclonal antibodies VPM37 and VPM43 react with DR antigen but not DQ antigen by ELISA. These two antibodies do not react with the DR beta chain expressed in the T8.1 cell line. Two-dimensional immunoblotting shows that these antibodies recognize a subgroup of the spots recognized by the DR-specific monoclonal antibody VPM57 which does react with the T8.1 beta chain. Amino-terminal sequence analysis of the alpha chain associated with VPM37 beta chain shows that this alpha chain is homologous to the human DR alpha chain strongly indicating that the beta chain is DR-like. VPM37 and VPM43 are shown to be directed against different epitopes on sheep MHC class II molecules so it is highly unlikely that the data can be explained by the presence of post-translational modifications or the existence of a very common allele. These data provide clear evidence for the expression of two distinct DR beta chains in the sheep.

Improved discrimination of bovine class II DR beta-chains polymorphisms using immunoblotting.

An immunoblotting technique is reported that reveals electrophoretic variants in the beta-chains of class II antigens of the bovine major histocompatibility complex. One monoclonal antibody, mAb VPM57, reacted on immunoblots with an epitope present in approximately half of the haplotypes investigated. This reagent is especially useful in discriminating electrophoretic variants that have similar isoelectric points.