CD1- and CD1+ porcine blood dendritic cells are enriched for the orthologues of the two major mammalian conventional subsets.

Conventional dendritic cells (cDC) are professional antigen-presenting cells that induce immune activation or tolerance. Two functionally specialised populations, termed cDC1 and cDC2, have been described in humans, mice, ruminants and recently in pigs. Pigs are an important biomedical model species and a key source of animal protein; therefore further understanding of their immune system will help underpin the development of disease prevention strategies. To characterise cDC populations in porcine blood, DC were enriched from PBMC by CD14 depletion and CD172a enrichment then stained with lineage mAbs (Lin; CD3, CD8α, CD14 and CD21) and mAbs specific for CD172a, CD1 and CD4. Two distinct porcine cDC subpopulations were FACSorted CD1- cDC (Lin-CD172+ CD1-CD4-) and CD1+ cDC (Lin-CD172a+ CD1+ CD4-), and characterised by phenotypic and functional analyses. CD1+ cDC were distinct from CD1- cDC, expressing higher levels of CD172a, MHC class II and CD11b. Following TLR stimulation, CD1+ cDC produced IL-8 and IL-10 while CD1- cDC secreted IFN-α, IL-12 and TNF-α. CD1- cDC were superior in stimulating allogeneic T cell responses and in cross-presenting viral antigens to CD8 T cells. Comparison of transcriptional profiles further suggested that the CD1- and CD1+ populations were enriched for the orthologues of cDC1 and cDC2 subsets respectively.

Association between complete response and outcomes in transplant-eligible myeloma patients in the era of novel agents.

OBJECTIVES: Achieving complete response (CR) has been linked to improved progression-free (PFS) and overall (OS) survival in myeloma. A meta-analysis was conducted to investigate whether this holds true in the era of novel agents (bortezomib, lenalidomide, thalidomide). METHODS: A total of 24 studies in newly diagnosed patients undergoing autologous stem cell transplantation (ASCT) that reported associations between responses and long-term outcomes (PFS/OS rates post-ASCT by response, or hazard ratios with 95% confidence intervals from Cox models) were identified and analyzed. RESULTS: Achievement of CR vs.

Factors affecting the infectivity of tissues from pigs with classical swine fever: thermal inactivation rates and oral infectious dose.

Outbreaks of classical swine fever are often associated with ingestion of pig meat or products derived from infected pigs. Assessment of the disease risks associated with material of porcine origin requires knowledge on the likely amount of virus in the original material, how long the virus may remain viable within the resulting product and how much of that product would need to be ingested to result in infection. Using material from pigs infected with CSFV, we determined the viable virus concentrations in tissues that comprise the majority of pork products. Decimal reduction values (D values), the time required to reduce the viable virus load by 90% (or 1 log10), were determined at temperatures of relevance for chilling, cooking, composting and ambient storage. The rate of CSFV inactivation varied in different tissues. At lower temperatures, virus remained viable for substantially longer in muscle and serum compared to lymphoid and fat tissues. To enable estimation of the temperature dependence of inactivation, the temperature change required to change the D values by 90% (Z values) were determined as 13 °C, 14 °C, 12 °C and 10 °C for lymph node, fat, muscle and serum, respectively. The amount of virus required to infect 50% of pigs by ingestion was determined by feeding groups of animals with moderately and highly virulent CSFV. Interestingly, the virulent virus did not initiate infection at a lower dose than the moderately virulent strain. Although higher than for intranasal inoculation, the amount of virus required for infection via ingestion is present in only a few grams of tissue from infected animals.

Rab6 dependent post-Golgi trafficking of HSV1 envelope proteins to sites of virus envelopment.

Herpes simplex virus 1 (HSV1) is an enveloped virus that uses undefined transport carriers for trafficking of its glycoproteins to envelopment sites. Screening of an siRNA library against 60 Rab GTPases revealed Rab6 as the principal Rab involved in HSV1 infection, with its depletion preventing Golgi-to-plasma membrane transport of HSV1 glycoproteins in a pathway used by several integral membrane proteins but not the luminal secreted protein Gaussia luciferase. Knockdown of Rab6 reduced virus yield to 1% and inhibited capsid envelopment, revealing glycoprotein exocytosis as a prerequisite for morphogenesis. Rab6-dependent virus production did not require the effectors myosin-II, bicaudal-D, dynactin-1 or rabkinesin-6, but was facilitated by ERC1, a factor involved in linking microtubules to the cell cortex. Tubulation and exocytosis of Rab6-positive, glycoprotein-containing membranes from the Golgi was substantially augmented by infection, resulting in enhanced and targeted delivery to cell tips. This reveals HSV1 morphogenesis as one of the first biological processes shown to be dependent on the exocytic activity of Rab6.

Endocytic tubules regulated by Rab GTPases 5 and 11 are used for envelopment of herpes simplex virus.

Enveloped viruses employ diverse and complex strategies for wrapping at cellular membranes, many of which are poorly understood. Here, an ultrastructural study of herpes simplex virus 1 (HSV1)-infected cells revealed envelopment in tubular membranes. These tubules were labelled by the fluid phase marker horseradish peroxidase (HRP), and were observed to wrap capsids as early as 2 min after HRP addition, indicating that the envelope had recently cycled from the cell surface. Consistent with this, capsids did not colocalise with either the trans-Golgi network marker TGN46 or late endosomal markers, but showed coincidence with the transferrin receptor. Virus glycoproteins were retrieved from the plasma membrane (PM) to label wrapping capsids, a process that was dependent on both dynamin and Rab5. Combined depletion of Rab5 and Rab11 reduced virus yield to <1%, resulting in aberrant localisation of capsids. These results suggest that endocytosis from the PM into endocytic tubules provides the main source of membrane for HSV1, and reveal a new mechanism for virus exploitation of the endocytic pathway.

Characterisation of vaccine-induced, broadly cross-reactive IFN-γ secreting T cell responses that correlate with rapid protection against classical swine fever virus.

Live attenuated C-strain classical swine fever viruses (CSFV) provide a rapid onset of protection, but the lack of a serological test that can differentiate vaccinated from infected animals limits their application in CSF outbreaks. Since immunity may precede antibody responses, we examined the kinetics and specificity of peripheral blood T cell responses from pigs vaccinated with a C-strain vaccine and challenged after five days with a genotypically divergent CSFV isolate. Vaccinated animals displayed virus-specific IFN-γ responses from day 3 post-challenge, whereas, unvaccinated challenge control animals failed to mount a detectable response. Both CD4(+) and cytotoxic CD8(+) T cells were identified as the cellular source of IFN-γ. IFN-γ responses showed extensive cross-reactivity when T cells were stimulated with CSFV isolates spanning the major genotypes. To determine the specificity of these responses, T cells were stimulated with recombinant CSFV proteins and a proteome-wide peptide library from a related virus, BVDV. Major cross-reactive peptides were mapped on the E2 and NS3 proteins. Finally, IFN-γ was shown to exert potent antiviral effects on CSFV in vitro. These data support the involvement of broadly cross-reactive T cell IFN-γ responses in the rapid protection conferred by the C-strain vaccine and this information should aid the development of the next generation of CSFV vaccines.

Challenge of pigs with classical swine fever viruses after C-strain vaccination reveals remarkably rapid protection and insights into early immunity.

Pre-emptive culling is becoming increasingly questioned as a means of controlling animal diseases, including classical swine fever (CSF). This has prompted discussions on the use of emergency vaccination to control future CSF outbreaks in domestic pigs. Despite a long history of safe use in endemic areas, there is a paucity of data on aspects important to emergency strategies, such as how rapidly CSFV vaccines would protect against transmission, and if this protection is equivalent for all viral genotypes, including highly divergent genotype 3 strains. To evaluate these questions, pigs were vaccinated with the Riemser® C-strain vaccine at 1, 3 and 5 days prior to challenge with genotype 2.1 and 3.3 challenge strains. The vaccine provided equivalent protection against clinical disease caused by for the two challenge strains and, as expected, protection was complete at 5 days post-vaccination. Substantial protection was achieved after 3 days, which was sufficient to prevent transmission of the 3.3 strain to animals in direct contact. Even by one day post-vaccination approximately half the animals were partially protected, and were able to control the infection, indicating that a reduction of the infectious potential is achieved very rapidly after vaccination. There was a close temporal correlation between T cell IFN-γ responses and protection. Interestingly, compared to responses of animals challenged 5 days after vaccination, challenge of animals 3 or 1 days post-vaccination resulted in impaired vaccine-induced T cell responses. This, together with the failure to detect a T cell IFN-γ response in unprotected and unvaccinated animals, indicates that virulent CSFV can inhibit the potent antiviral host defences primed by C-strain in the early period post vaccination.

The classical swine fever virus N-terminal protease N(pro) binds to cellular HAX-1.

The positive-stranded RNA genome of classical swine fever virus (CSFV) encodes 12 known proteins. The first protein to be translated is the N-terminal protease (N(pro)). N(pro) helps evade the innate interferon response by targeting interferon regulatory factor-3 for proteasomal degradation and also participates in the evasion of dsRNA-induced apoptosis. To elucidate the mechanisms by which N(pro) functions, we performed a yeast two-hybrid screen in which the anti-apoptotic protein HAX-1 was identified. The N(pro)-HAX-1 interaction was confirmed using co-precipitation assays. A dramatic redistribution of both N(pro) and HAX-1 was observed in co-transfected cells, as well as in transfected cells infected with wild-type CSFV, but not in cells infected with an N(pro)-deleted CSFV strain.

Characterisation of virus-specific peripheral blood cell cytokine responses following vaccination or infection with classical swine fever viruses.

Existing live attenuated classical swine fever virus (CSFV) vaccines provide a rapid onset of complete protection but pose problems in discriminating infected amongst vaccinated animals. With a view to providing additional information on the cellular mechanisms that may contribute to protection, which in turn may aid the development of the next generation of CSFV vaccines, we explored the kinetics of the cytokine responses from peripheral blood cells of pigs vaccinated with an attenuated C-strain vaccine strain and/or infected with a recent CSFV isolate. Peripheral blood cells were isolated over the course of vaccination/infection and stimulated in vitro with C-strain or UK2000/7.1 viruses. Virus-specific responses of peripheral blood cells isolated from C-strain vaccinated pigs were dominated by the production of IFN-gamma. IFN-gamma production in response to the C-strain virus was first detected in vaccinates 9 days post-vaccination and was sustained over the period of observation. In contrast, cells from challenge control animals did not secrete IFN-gamma in response to stimulation with C-strain or UK2000/7.1 viruses. Supernatants from UK2000/7.1 infected animals contained significant levels of pro-inflammatory cytokines from day 8 post-infection and these cytokines were present in both virus and mock stimulated cultures. The results suggest that the C-strain virus is a potent inducer of a type-1 T cell response, which may play a role in the protection afforded by such vaccines, whereas the pro-inflammatory cytokine responses observed in cultures from infected pigs may reflect a pathological pro-inflammatory cascade initiated in vivo following the replication and spread of CSFV.

A dominant-negative mutant of rab5 inhibits infection of cells by foot-and-mouth disease virus: implications for virus entry.

Foot-and-mouth disease virus (FMDV) can use a number of different integrins (alphavbeta1, alphavbeta3, alphavbeta6, and alphavbeta8) as receptors to initiate infection. Infection mediated by alphavbeta6 is known to occur by clathrin-mediated endocytosis and is dependent on the acidic pH within endosomes. On internalization, virus is detected rapidly in early endosomes (EE) and subsequently in perinuclear recycling endosomes (PNRE), but not in late endosomal compartments. Due to the extreme sensitivity of FMDV to acidic pH, it is thought that EE can provide a pH low enough for infection to occur; however, definitive proof that infection takes place from within these compartments is still lacking. Here we have investigated the intracellular transport steps required for FMDV infection of IBRS-2 cells, which express alphavbeta8 as their FMDV receptor. These experiments confirmed that FMDV infection mediated by alphavbeta8 is also dependent on clathrin-mediate endocytosis and an acidic pH within endosomes. Also, the effect on FMDV infection of dominant-negative (DN) mutants of cellular rab proteins that regulate endosomal traffic was examined. Expression of DN rab5 reduced the number of FMDV-infected cells by 80%, while expression of DN rab4 or DN rab7 had virtually no effect on infection. Expression of DN rab11 inhibited infection by FMDV, albeit to a small extent ( approximately 35%). These results demonstrate that FMDV infection takes place predominantly from within EE and does not require virus trafficking to the late endosomal compartments. However, our results suggest that infection may not be exclusive to EE and that a small amount of infection could occur from within PNRE.

Importance of arginine 20 of the swine vesicular disease virus 2A protease for activity and virulence.

A major virulence determinant of swine vesicular disease virus (SVDV), an Enterovirus that causes an acute vesicular disease, has been mapped to residue 20 of the 2A protease. The SVDV 2A protease cleaves the 1D-2A junction in the viral polyprotein, induces cleavage of translation initiation factor eIF4GI, and stimulates the activity of enterovirus internal ribosome entry sites (IRESs). The 2A protease from an attenuated strain of SVDV (Ile at residue 20) is significantly defective at inducing cleavage of eIF4GI and the activation of IRES-dependent translation compared to the 2A protease from a pathogenic strain (J1/73, Arg at residue 20), but the two proteases have similar 1D-2A cleavage activities (Y. Sakoda, N. Ross-Smith, T. Inoue, and G. J. Belsham, J. Virol. 75:10643-10650, 2001). Residue 20 has now been modified to every possible amino acid, and the activities of each mutant 2A protease has been analyzed. Selected mutants were reconstructed into full-length SVDV cDNA, and viruses were rescued. The rate of virus growth in cultured swine kidney cells reflected the efficiency of 2A protease activity. In experimentally infected pigs, all four of the mutant viruses tested displayed much-reduced virulence compared to the J1/73 virus but a significant, albeit reduced, level of viral replication and excretion was detected. Direct sequencing of cDNA derived from samples taken early and late in infection indicated that a gradual selection-reversion to a more efficient protease occurred. The data indicated that extensive sequence change and selection may introduce a severe bottleneck in virus replication, leading to a decreased viral load and reduced or no clinical disease.

Functional and structural similarities between the internal ribosome entry sites of hepatitis C virus and porcine teschovirus, a picornavirus.

Initiation of protein synthesis on picornavirus RNA requires an internal ribosome entry site (IRES). Typically, picornavirus IRES elements contain about 450 nucleotides (nt) and use most of the cellular translation initiation factors. However, it is now shown that just 280 nt of the porcine teschovirus type 1 Talfan (PTV-1) 5' untranslated region direct the efficient internal initiation of translation in vitro and within cells. In toeprinting assays, assembly of 48S preinitiation complexes from purified components on the PTV-1 IRES was achieved with just 40S ribosomal subunits plus eIF2 and Met-tRNA(i)(Met). Indeed, a binary complex between 40S subunits and the PTV-1 IRES is formed. Thus, the PTV-1 IRES has properties that are entirely different from other picornavirus IRES elements but highly reminiscent of the hepatitis C virus (HCV) IRES. Comparison between the PTV-1 IRES and HCV IRES elements revealed islands of high sequence identity that occur in regions critical for the interactions of the HCV IRES with the 40S ribosomal subunit and eIF3. Thus, there is significant functional and structural similarity between the IRES elements from the picornavirus PTV-1 and HCV, a flavivirus.