Immune control of mammalian gamma-herpesviruses: lessons from murid herpesvirus-4.

Many acute viral infections can be controlled by vaccination; however, vaccinating against persistent infections remains problematic. Herpesviruses are a classic example. Here, we discuss their immune control, particularly that of gamma-herpesviruses, relating the animal model provided by murid herpesvirus-4 (MuHV-4) to human infections. The following points emerge: (i) CD8(+) T-cell evasion by herpesviruses confers a prominent role in host defence on CD4(+) T cells. CD4(+) T cells inhibit MuHV-4 lytic gene expression via gamma-interferon (IFN-gamma). By reducing the lytic secretion of immune evasion proteins, they may also help CD8(+) T cells to control virus-driven lymphoproliferation in mixed lytic/latent lesions. Similarly, CD4(+) T cells specific for Epstein-Barr virus lytic antigens could improve the impact of adoptively transferred, latent antigen-specific CD8(+) T cells. (ii) In general, viral immune evasion necessitates multiple host effectors for optimal control. Thus, subunit vaccines, which tend to prime single effectors, have proved less successful than attenuated virus mutants, which prime multiple effectors. Latency-deficient mutants could make safe and effective gamma-herpesvirus vaccines. (iii) The antibody response to MuHV-4 infection helps to prevent disease but is suboptimal for neutralization. Vaccinating virus carriers with virion fusion complex components improves their neutralization titres. Reducing the infectivity of herpesvirus carriers in this way could be a useful adjunct to vaccinating naive individuals with attenuated mutants.

Parainfluenza virus 5 genomes are located in viral cytoplasmic bodies whilst the virus dismantles the interferon-induced antiviral state of cells.

Although the replication cycle of parainfluenza virus type 5 (PIV5) is initially severely impaired in cells in an interferon (IFN)-induced antiviral state, the virus still targets STAT1 for degradation. As a consequence, the cells can no longer respond to IFN and after 24-48 h, they go out of the antiviral state and normal virus replication is established. Following infection of cells in an IFN-induced antiviral state, viral nucleocapsid proteins are initially localized within small cytoplasmic bodies, and appearance of these cytoplasmic bodies correlates with the loss of STAT1 from infected cells. In situ hybridization, using probes specific for the NP and L genes, demonstrated the presence of virus genomes within these cytoplasmic bodies. These viral cytoplasmic bodies do not co-localize with cellular markers for stress granules, cytoplasmic P-bodies or autophagosomes. Furthermore, they are not large insoluble aggregates of viral proteins and/or nucleocapsids, as they can simply and easily be dispersed by 'cold-shocking' live cells, a process that disrupts the cytoskeleton. Given that during in vivo infections, PIV5 will inevitably infect cells in an IFN-induced antiviral state, we suggest that these cytoplasmic bodies are areas in which PIV5 genomes reside whilst the virus dismantles the antiviral state of the cells. Consequently, viral cytoplasmic bodies may play an important part in the strategy that PIV5 uses to circumvent the IFN system.

K3-mediated evasion of CD8(+) T cells aids amplification of a latent gamma-herpesvirus.

The murine gamma-herpesvirus-68 (MHV-68) K3 protein, like that of the Kaposi's sarcoma associated herpesvirus, down-regulates major histocompatibility complex (MHC) class I expression. However, how this contributes to viral replication in vivo is unclear. After intranasal MHV-68 infection, K3 was transcribed both during acute lytic infection in the lung and during latency establishment in lymphoid tissue. K3-deficient viruses were not cleared more rapidly from the lung, but the number of latently infected spleen cells was reduced and the frequency of virus-specific CD8(+) cytotoxic T lymphocytes (CTLs) was increased. CTL depletion reversed the viral latency deficit. Thus, a major function of K3 appears to be CTL evasion during viral latency expansion.

A secreted chemokine binding protein encoded by murine gammaherpesvirus-68 is necessary for the establishment of a normal latent load.

Herpesviruses encode a variety of proteins with the potential to disrupt chemokine signaling, and hence immune organization. However, little is known of how these might function in vivo. The B cell-tropic murine gammaherpesvirus-68 (MHV-68) is related to the Kaposi's sarcoma-associated herpesvirus (KSHV), but whereas KSHV expresses small chemokine homologues, MHV-68 encodes a broad spectrum chemokine binding protein (M3). Here we have analyzed the effect on viral pathogenesis of a targeted disruption of the M3 gene. After intranasal infection, an M3 deficiency had surprisingly little effect on lytic cycle replication in the respiratory tract or the initial spread of virus to lymphoid tissues. However, the amplification of latently infected B cells in the spleen that normally drives MHV-68-induced infectious mononucleosis failed to occur. Thus, there was a marked reduction in latent virus recoverable by in vitro reactivation, latency-associated viral tRNA transcripts detectable by in situ hybridization, total viral DNA load, and virus-driven B cell activation. In vivo CD8(+) T cell depletion largely reversed this deficiency, suggesting that the chemokine neutralization afforded by M3 may function to block effective CD8(+) T cell recruitment into lymphoid tissue during the expansion of latently infected B cell numbers. In the absence of M3, MHV-68 was unable to establish a normal latent load.

A broad spectrum secreted chemokine binding protein encoded by a herpesvirus.

Chemokines are a family of small proteins that interact with seven-transmembrane domain receptors and modulate the migration of immune cells into sites of inflammation and infection. The murine gammaherpesvirus 68 M3 gene encodes a secreted 44-kD protein with no sequence similarity to known chemokine receptors. We show that M3 binds a broad range of chemokines, including CC, CXC, C, and CX(3)C chemokines, but does not bind human B cell-specific nor mouse neutrophil-specific CXC chemokines. This herpesvirus chemokine binding protein (hvCKBP) blocks the interaction of chemokines with high-affinity cellular receptors and inhibits chemokine-induced elevation of intracellular calcium levels. hvCKBP is the first soluble chemokine receptor identified in herpesviruses; it represents a novel protein structure with the ability to bind all subfamilies of chemokines in solution and has potential therapeutic applications.

Analysis of murine gammaherpesvirus-68 transcription during lytic and latent infection.

Murine gammaherpesvirus-68 (MHV-68) is a gamma2-herpesvirus that upon experimental infection of laboratory mice establishes a latent infection in B lymphocytes. To date, no virus-encoded gene products have been reported to be expressed during latent infection. In this study, viral transcription has been analysed in a persistently infected B-cell line and abundant and preferential transcription of open reading frame M3 has been identified. Significantly, in situ hybridization analysis of latently infected mouse spleens with probes corresponding to 20 MHV-68 ORFs demonstrated active transcription of a single ORF, corresponding to M3. The kinetics and pattern of transcription of M3 were compared with that of the virally encoded tRNAs (vtRNAs), previously demonstrated to constitute a marker for latent infection in the spleen. Transcription of vtRNAs in splenic tissue could be first detected at 7 days post-inoculation (p.i.) in scattered cells in periarteriolar lymphoid sheaths (PALS). At 10 days p.i., vtRNA transcription was widespread and localized not only to cells in PALS but also to cells within developing germinal centres and from 21 days p.i. expression was detected exclusively within lymphoid follicles. Transcription of vtRNAs could be detected as late as 70 days p.i. In contrast, the histological localization of M3 transcription, which was first detected at 7 days p.i. in scattered cells in PALS, never changed and transcription could not be detected beyond 21 days p.i. These results suggest that M3 is an ORF that is expressed early during the establishment of latency in vivo.

BSE in Portugal: anticipating the decline of an epidemic.

BACKGROUND: Attention throughout Europe continues to focus on bovine spongiform encephalopathy (BSE), with increasing evidence linking it to the new variant of Creutzfeldt-Jakob disease in humans. In particular, recent attention has been directed at Portugal, where the incidence of confirmed BSE cases continues to rise. METHODS: We modelled the age-specific incidence of BSE in Portuguese-born cattle by birth cohort as a function of: the survival distribution; the cohort-specific incidence of BSE infection; the age-specific probability, conditional on survival, that an infected animal will experience clinical onset; and the under-reporting rate of BSE cases prior to 1998. RESULTS: We obtained good fits to the age-specific incidence of BSE by birth cohort in Portugal. Under a range of assumptions, the estimated incidence of BSE infection was relatively low initially, except possibly in the 1989 cohort, and then rose gradually between the 1992 and 1994 cohorts. The estimated decrease in infection incidence between the 1994 and 1995 cohorts probably reflects the effectiveness of the ban on the use of mammalian meat and bone-meal introduced in Portugal in mid-1994. Assuming no infections in animals born after June 1995, the models predict that the incidence of BSE cases in Portugal will peak in 1999 with BSE case-incidence declining thereafter. DISCUSSION: Our results illustrate the power of epidemiological analysis to detect decreasing trends in infection incidence prior to the resulting decrease in case incidence. The findings should inform the deliberations of the European Commission, which recently reported concerns about the sharp increase in case incidence from 1997 to 1998.

Murine gammaherpesvirus 68: a model for the study of gammaherpesvirus pathogenesis.

Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring herpesvirus of wild rodents and is genetically related to human herpesvirus 8 and Epstein-Barr virus. The ability of MHV-68 to establish acute and persistent infection within laboratory mice offers a unique opportunity to investigate immunological and virological aspects of gammaherpesvirus pathogenesis.

Four tRNA-like sequences and a serpin homologue encoded by murine gammaherpesvirus 68 are dispensable for lytic replication in vitro and latency in vivo.

Experimental infection of inbred strains of laboratory mice with murine herpesvirus 68 (MHV-68), a natural pathogen of wild rodents, results in acute productive infection of the lung followed by a latent infection of B lymphocytes. We have previously shown that MHV-68 encodes an open reading frame with similarity to poxvirus serpins, designated ORF1, and eight novel tRNA-like sequences. The latter are processed into mature, uncharged tRNAs and are abundantly expressed during both lytic and latent infection. In this study it is demonstrated that deletion of four of the tRNA-like sequences and ORF1 from the virus genome does not affect the ability of MHV-68 to replicate in vitro or to establish, and reactivate from, latency in vivo.

Murine gammaherpesvirus 68 encodes tRNA-like sequences which are expressed during latency.

Murine gammaherpesvirus 68 (MHV-68) is a virus of wild rodents and is a convenient small animal model for studies of gammaherpesvirus pathogenesis. We have sequenced 6162 bp at the left end of the MHV-68 genome and identified two unique open reading frames (ORFs) (ORF2 and ORF3) and an ORF (ORF1) which displays similarity to poxvirus members of the serpin family. Interspersed with the ORFs is a family of eight novel tRNA-like sequences sharing tRNA-like predicted secondary structures and RNA polymerase III promoter elements. These sequences are expressed to high levels during lytic infection and are processed into mature tRNAs with post-transcriptionally added 3' CCA termini, indicating their recognition as tRNAs by cellular machinery. Acidic Northern analysis of four tRNAs tested has demonstrated that they are not aminoacylated by aminoacyl-tRNA synthetases present in the infected cell. Thus, it is currently unclear what biological function these uncharged viral tRNA-like sequences may fulfil. In situ hybridization analysis has shown that in addition to being expressed within productively infected tissues during acute stages of infection, the tRNA-like sequences are abundantly expressed within splenic germinal centres of latently infected mice. Therefore, the MHV-68 viral tRNAs represent a marker for latent infection and constitute the first report of tRNA-like sequences encoded by a virus of eukaryotes.

The course of disease and persistence of virus in the central nervous system varies between individual CBA mice infected with the BeAn strain of Theiler's murine encephalomyelitis virus.

Following intracerebral inoculation of the BeAn strain of Theiler's murine encephalomyelitis virus the course of the acute infection and persistence of virus in the CNS varies between individual CBA mice. On the basis of clinical signs, virus distribution, virus titres, histopathology and Southern blot hybridization analysis of virus specific RT-PCR amplified products from total brain and spinal cord RNA, individual CBA mice could be placed into one of three groups. The first group were those animals which died of acute encephalitis. The second group were animals with or without clinical signs which had early high CNS virus titres, and in addition to scattered foci of infection had spread of virus in specific neuronal nuclei followed by destruction of these areas and thereafter persistence of virus in the CNS. The third group had no clinical signs, low CNS virus titres, small foci of CNS infection and were negative for virus after 28 days. This third pattern of infection was also seen in BALB/c mice. Between 50 and 268 days post-infection 53% of CBA mice were positive for viral RNA in the CNS by RT-PCR. No BALB/c mice were positive. In both the acute and persistent infection there was a correlation between serum neutralizing antibody titre and clinical signs. During the acute infection BeAn RNA could be detected in neurons and astrocytes. Oligodendrocytes were negative. In those CBA mice with persistence, viral RNA was observed in scattered individual or small foci of cells, predominantly oligodendrocytes, in both the brain and spinal cord.

The neurovirulent GDVII strain of Theiler's virus can replicate in glial cells.

The distribution, spread, neuropathology, tropism, and persistence of the neurovirulent GDVII strain of Theiler's virus in the central nervous system (CNS) was investigated in mice susceptible and resistant to chronic demyelinating infection with TO strains. Following intracerebral inoculation, the virus spread rapidly to specific areas of the CNS. There were, however, specific structures in which infection was consistently undetectable. Virus spread both between adjacent cell bodies and along neuronal pathways. The distribution of the infection was dependent on the site of inoculation. The majority of viral RNA-positive cells were neurons. Many astrocytes were also positive. Infection of both of these cell types was lytic. In contrast, viral RNA-positive oligodendrocytes were rare and were observed only in well-established areas of infection. The majority of oligodendrocytes in these areas were viral RNA negative and were often the major cell type remaining; however, occasional destruction of these cells was observed. No differences in any of the above parameters were observed between CBA and BALB/c mice, susceptible and resistant, respectively, to chronic CNS demyelinating infection with TO strains of Theiler's virus. By using Southern blot hybridization to detect reverse-transcribed PCR-amplified viral RNA sequences, no virus persistence could be detected in the CNS of immunized mice surviving infection with GDVII. In conclusion, the GDVII strain of Theiler's murine encephalomyelitis virus cannot persist in the CNS, but this is not consequent upon an inability to infect glial cells, including oligodendrocytes.