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Active transmissible spongiform encephalopathy (TSE) surveillance in small ruminants across Europe was implemented in 2002 following the epizootic of bovine spongiform encephalopathy. Here, we report the potential emergence of classical scrapie in Portugal, in a background of enzootic atypical scrapie. Between 2003 and 2008, 375,459 small ruminants were screened in total, with 328 animals confirmed positive for NOR98 atypical scrapie. During this period, the prevalence rate of atypical scrapie for all years combined was 0.0874% across the country. In this scenario, classical scrapie emerged as a single outbreak in 2008, with 12 identified cases. In contrast to other European countries, where classical scrapie has been enzootic for decades, these data indicate that, in Portugal, atypical scrapie is the predominant form of TSE. The findings reported here will have implications for the control of classical scrapie in Portugal, namely in terms of keeping the country free of enzootic classical scrapie.

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.

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.

Luciferase-based imaging allows a global view of microbial pathogenesis. We applied this technique to gammaherpesvirus infection by inserting a luciferase expression cassette into the genome of murine herpesvirus-4 (MuHV-4). The recombinant virus strongly expressed luciferase in lytically infected cells without significant attenuation. We used it to compare different routes of virus inoculation. After intranasal infection of anaesthetized mice, luciferase was expressed in the nose and lungs for 7-10 days and in lymphoid tissue, most consistently the superficial cervical lymph nodes, for up to 30 days. Gastrointestinal infection was not observed. Intraperitoneal infection was very different to intranasal, with strong luciferase expression in the liver, kidneys, intestines, reproductive tract and spleen, but none in the nose or lungs. The nose has not previously been identified as a site of MuHV-4 infection. After intranasal infection of non-anaesthetized mice, it was the only site of non-lymphoid luciferase expression. Nevertheless, lymphoid colonization and persistence were still established, even at low inoculation doses. In contrast, virus delivered orally was very poorly infectious. Inoculation route therefore had a major impact on pathogenesis. Low dose intranasal infection without anaesthesia seems most likely to mimic natural transmission, and may therefore be particularly informative about normal viral gene functions.

The gammaherpesviruses are characteristically latent in lymphocytes and exploit lymphocyte proliferation to establish a large, persistent pool of latent genomes. Murine gammaherpesvirus 68 (MHV-68) allows the in vivo analysis of viral genes that contribute to this and other aspects of host colonization. In this study, the MHV-68 bcl-2 homologue, M11, was disrupted either in its BH1 homology domain or upstream of its membrane-localizing C-terminal domain. Each M11 mutant showed normal lytic replication in vitro and in vivo, but had a reduction in peak splenic latency. Lower infectious-centre titres correlated with lower in vivo B-cell activation, lower viral genome loads and reduced viral tRNA expression. This was therefore a true latency deficit, rather than a deficit in ex vivo reactivation. Stable, long-term levels of splenic latency were normal. M11 function therefore contributed specifically to viral latency amplification in infected lymphoid tissue.

Experimental transmission of bovine spongiform encephalopathy to sheep has prompted the implementation of a surveillance plan of scrapie in small ruminants by the European Union in all member states. Since its start over 30,000 animals have been tested, and the first seven cases of sheep with detectable PrP(res) deposition in the central nervous system have been identified in Portugal. Notably, the pattern of PrP(res) distribution in the brainstem was different from that previously described for scrapie and consistent in all seven animals. Moreover, the profile of the electrophoretic mobility of PrP(res) after proteinase K treatment was equivalent in all cases analysed but distinct from that observed for scrapie. Notably, four animals had genotypes rarely associated with scrapie, including one animal homozygous for A(136)R(154)R(171). There were no cases found to exhibit vacuolation, a pattern of PrP(res) distribution or PrP(res) electrophoretic mobility corresponding to scrapie. These data reveal a putative atypical scrapie strain in Portugal not linked to specific Prnp genotypes.

Infection of mice with murine gammaherpesvirus 68 is characterized by a marked transient expansion of latently infected splenic germinal centre (GC) B cells, which is followed by lower levels of persistent infection in GC and memory B cells. Virus transcription within GC B cells is restricted to a number of latency-associated open reading frames, including M2. This gene encodes a structurally unique protein of unknown function, which has been shown to be essential for the transient peak of virus latency during the establishment of latent infection in the spleen. This study shows that upon infection of mice with M2-defective viruses, at 14 days post-infection during the establishment of latency in the spleen, there was a reduction in the number of latently infected follicles when compared with wild-type virus. However, the mean number of latently infected cells within each follicle was equivalent between wild-type and M2-defective viruses. Late in infection, disruption of M2 resulted in sustained and abnormally high levels of virus persistence in splenic GC B cells but not memory B cells. These data indicate that during the establishment of latency in the spleen, the M2 gene product is required for efficient colonization of splenic follicles but is dispensable for the expansion of latently infected GC B cells and that M2 might be a critical modulator of B-cell function.

In vitro studies have established that the latency-associated nuclear antigen encoded by human Kaposi's sarcoma-associated herpesvirus and the related ORF73 gene product of herpesvirus saimiri interact with virus origins of replication to facilitate maintenance of episomal DNA. Such a function implies a critical role for ORF73 in the establishment and maintenance of latency in vivo. To determine the role of ORF73 in virus pathogenesis, the ORF73 gene product encoded by murine herpesvirus-68 (MHV-68) was disrupted by making an ORF73 deletion mutant, Delta73, and an independent ORF73 frameshift mutant, FS73. The effect of the mutations introduced in ORF73 on MHV-68 pathogenesis was analysed in vivo using a well-characterized murine model system. These studies have revealed that ORF73 is not required for efficient lytic replication either in vitro or in vivo. In contrast, a severe latency deficit is observed in splenocytes of animals infected with an ORF73 mutant, as assessed by infectious centre reactivation assay or by in situ hybridization detection of latent virus. Assessment of viral genome-positive cells in sorted splenocyte populations confirmed the absence of ORF73 mutant virus from splenic latency reservoirs, including germinal centre B cells. These data indicate a crucial role for ORF73 in the establishment of latency and for virus persistence in the host.

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.

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 (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.

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.