Autophagy impairment by African swine fever virus.

African swine fever is a devastating disease of domestic swine and wild boar caused by a large double-stranded DNA virus that encodes for more than 150 open reading frames. There is no licensed vaccine for the disease and the most promising current candidates are modified live viruses that have been attenuated by deletion of virulence factors. Like many viruses African swine fever virus significantly alters the host cell machinery to benefit its replication and viral genes that modify host pathways represent promising targets for development of gene deleted vaccines. Autophagy is an important cellular pathway that is involved in cellular homeostasis, innate and adaptive immunity and therefore is manipulated by a number of different viruses. Autophagy is regulated by a complex protein cascade and here we show that African swine fever virus can block formation of autophagosomes, a critical functional step of the autophagy pathway through at least two different mechanisms. Interestingly this does not require the A179L gene that has been shown to interact with Beclin-1, an important autophagy regulator.

Inhibition of a large double-stranded DNA virus by MxA protein.

Increasing evidence points to the importance of the interferon (IFN) response in determining the host range and virulence of African swine fever virus (ASFV). Infection with attenuated strains of ASFV leads to the upregulation of genes controlled by IFN pathways, including myxovirus resistance (Mx) genes that are potent effectors of the antiviral state. Mx gene products are known to inhibit the replication of many negative-sense single-stranded RNA viruses, as well as double-stranded RNA viruses, positive-sense single-stranded RNA viruses, and the reverse-transcribing DNA virus hepatitis B virus. Here, we provide data that extend the known range of viruses inhibited by Mx to include the large double-stranded DNA viruses. Stably transfected Vero cells expressing human MxA protein did not support ASFV plaque formation, and virus replication in these cells was reduced 100-fold compared with that in control cells. In contrast, ASFV replication in cells expressing MxB protein or a mutant MxA protein was similar to that in control Vero cells. There was a drastic reduction in ASFV late protein synthesis in MxA-expressing cells, correlating with the results of previous work on the effect of IFN on viral replication. Strikingly, the inhibition of ASFV replication was linked to the recruitment of MxA protein to perinuclear viral assembly sites, where the protein surrounded the virus factories. Interactions between ASFV and MxA were similar to those seen between MxA and different RNA viruses, suggesting a common inhibitory mechanism.

The envelope of intracellular African swine fever virus is composed of a single lipid bilayer.

African swine fever virus (ASFV) is a member of a family of large nucleocytoplasmic DNA viruses that include poxviruses, iridoviruses, and phycodnaviruses. Previous ultrastructural studies of ASFV using chemical fixation and cryosectioning for electron microscopy (EM) have produced uncertainty over whether the inner viral envelope is composed of a single or double lipid bilayer. In this study we prepared ASFV-infected cells for EM using chemical fixation, cryosectioning, and high-pressure freezing. The appearance of the intracellular viral envelope was determined and compared to that of mitochondrial membranes in each sample. The best resolution of membrane structure was obtained with samples prepared by high-pressure freezing, and images suggested that the envelope of ASFV consisted of a single lipid membrane. It was less easy to interpret virus structure in chemically fixed or cryosectioned material, and in the latter case the virus envelope could be interpreted as having two membranes. Comparison of membrane widths in all three preparations indicated that the intracellular viral envelope of ASFV was not significantly different from the outer mitochondrial membrane (P < 0.05). The results support the hypothesis that the intracellular ASFV viral envelope is composed of a single lipid bilayer.

Identification of novel foot-and-mouth disease virus specific T-cell epitopes in c/c and d/d haplotype miniature swine.

To identify foot-and-mouth disease virus (FMDV) specific T-cell epitopes within the non-structural protein 3D in swine, pentadecapeptides were tested in proliferation and Interferon-gamma ELISPOT assays using lymphocytes from two strains of inbred miniature pigs (c/c and d/d haplotype) experimentally infected with FMDV. Lymphocytes of c/c pigs recognized peptides from three different regions in 3D, d/d lymphocytes recognized peptides from two regions, one of them being adjacent to an epitope of c/c pigs and comprising amino acid residues 346-370. Analyses of the response of d/d lymphocytes against peptides representing the structural protein 1A revealed another novel T-cell epitope. Investigation of the phenotype of responding lymphocytes showed a response of CD4(+)CD8(+)MHC-class-II(+) cells, identifying them as activated T-helper cells. This is the first report on FMDV specific T-cell epitopes recognized by swine leukocyte antigen (SLA) inbred swine and provides information useful for the design of novel vaccines against FMDV.

Perforin expression can define CD8 positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic T, natural killer T and MHC un-restricted cytotoxic T-cells.

In this study we have used the expression of perforin to characterize subsets of porcine cytotoxic lymphocytes. Perforin positive lymphocytes expressed both CD2 and CD8alpha, most were small dense lymphocytes (SDL) and up to 90% were CD3 negative. However, the numbers of perforin positive T-cells increased with the age of the animal and their populations increased after specific antigen stimulation in vitro. The remaining perforin positive lymphocytes were large and granular and contained more CD3+CD5+CD6+ T-cells (-40%) of which a substantial proportion also co-expressed CD4. Perforin was expressed in subpopulations of both CD8alphaalpha and CD8alphabeta lymphocytes, but was not expressed in gammadelta T-cells or monocyte/macrophages. The perforin positive CD3- subset was phenotypically homogeneous and defined as CD5-CD6-CD8beta-CD16+CD11b+. This population had NK activity and expressed mRNA for the NK receptor NKG2D, and adaptors DAP10 and DAP12. Perforin positive T-cells (CD3+) could be divided into at least three subsets. The first subset was CD4-CD5+CD6+CD11b-CD16- most were small dense lymphocytes with cytotoxic T-cell activity but not all expressed CD8beta. The second subset was mainly observed in the large granular lymphocytes. Their phenotype was CD4+CD5+CD6+CD8beta+CD16-CD11b- and also showed functional CTL activity. Thus not all of double positive T-cells are memory helper T-cells. The third subset did not express the T-cell co-receptor CD6, but up to half of them expressed another T-cell co-receptor CD5. The majority of this subset expressed CD11b and CD16, thus the third perforin positive T-cell subset was CD3+CD4-CD5+CD6-CD8beta+/-CD11b+CD16+, and possessed MHC-unrestricted cytotoxicity and LAK activity.

African swine fever virus organelle rearrangements.

Like most viruses African swine fever virus (ASFV) subsumes the host cell apparatus in order to facilitate its replication. ASFV replication is a highly orchestrated process with a least four stages of transcription, immediate-early, early, intermediate and late. As the infective cycle progresses through these stages most if not all of the organelles that comprise a nucleated cell are modified, adapted or in some cases destroyed. The entry of the virus is receptor-mediated, but the precise mechanism of endocytosis is a matter of keen, current debate. Once ASFV has exited from the endosomal-lysosomal complex the virus life-cycle enters into an intimate relationship with the microtubular network. Genome replication is believed to be initiated within the nucleus and ASFV infection completely reorders the structure of this organelle. The majority of replication and assembly occurs in discrete, perinuclear regions of the cell called virus factories and finally progeny virions are transported to the plasma membrane along microtubules where they bud out or are propelled away along actin projections to infect new cells. The generation of ASFV replication sites induces profound reorganisation of the organelles that comprise the secretory pathway and may contribute to the induction of cellular stress responses that ASFV modulates. The level of organisation and complexity of virus factories are not dissimilar to those seen in cellular organelles. Like their cellular counterparts the formation of virus factories, as well as virus entry and exit, are dependent on the various components of the cytoskeleton. This review will summarise these rearrangements, the viral proteins involved and their functional consequences.

African swine fever virus causes microtubule-dependent dispersal of the trans-golgi network and slows delivery of membrane protein to the plasma membrane.

Viral interference with secretory cargo is a common mechanism for pathogen immune evasion. Selective down regulation of critical immune system molecules such as major histocompatibility complex (MHC) proteins enables pathogens to mask themselves from their host. African swine fever virus (ASFV) disrupts the trans-Golgi network (TGN) by altering the localization of TGN46, an organelle marker for the distal secretory pathway. Reorganization of membrane transport components may provide a mechanism whereby ASFV can disrupt the correct secretion and/or cell surface expression of host proteins. In the study reported here, we used the tsO45 temperature-sensitive mutant of the G protein of vesicular stomatitis virus to show that ASFV significantly reduces the rate at which the protein is delivered to the plasma membrane. This is linked to a general reorganization of the secretory pathway during infection and a specific, microtubule-dependent disruption of structural components of the TGN. Golgin p230 and TGN46 are separated into distinct vesicles, whereupon TGN46 is depleted. These data suggest that disruption of the TGN by ASFV can slow membrane traffic during viral infection. This may be functionally important because infection of macrophages with virulent isolates of ASFV increased the expression of MHC class I genes, but there was no parallel increase in MHC class I molecule delivery to the plasma membrane.

Loss of interferon regulatory factor 3 in cells infected with classical swine fever virus involves the N-terminal protease, Npro.

We show that cells infected with the pestivirus classical swine fever virus (CSFV) fail to produce alpha/beta interferon not only following treatment with double-stranded RNA but also after superinfection with a heterologous virus, the alphavirus Sindbis virus, a virus shown to normally induce interferon. We investigated whether the inhibition of interferon synthesis by CSFV involved a block in interferon regulatory factor 3 (IRF3) activity. Cells infected with CSFV exhibited a lack of translocation of green fluorescent protein-IRF3 to the nucleus; however, constitutive shuttling of IRF3 was not blocked, since it could still accumulate in the nucleus in the presence of leptomycin B. Interestingly subcellular fractionation analysis showed that IRF3 was lost from the cytoplasm of infected cells from 18 h postinfection onwards. Using IRF3 promoter-luciferase reporter constructs, we demonstrate that loss of IRF3 was due to an inhibition of transcription of the IRF3 gene in CSFV-infected cells. Further, we investigated which viral protein may be responsible for the inhibition of interferon and loss of IRF3. We used cell lines expressing the CSFV N-terminal protease (Npro) to show that this single viral protein, unique to pestiviruses, inhibited interferon production in response to Sindbis virus. In addition to being lost from CSFV-infected cells, IRF3 was lost from Npro-expressing cells. The results demonstrate a novel viral evasion of innate host defenses, where interferon synthesis is prevented by inhibiting transcription of IRF3 in CSFV-infected cells.

Characterization of the porcine neonatal Fc receptor--potential use for trans-epithelial protein delivery.

The neonatal Fc receptor transports maternal immunoglobulin across the gut wall and has the potential to deliver genetically engineered proteins bearing immunoglobulin Fc domains across the gut to the mucosal immune system. Here we have characterized the porcine neonatal Fc receptor and tested its utility as a model system to study this kind of protein delivery. The complete DNA sequence obtained from an EST revealed 70-80% homology to mouse and human receptors, respectively, and tyrptophan and di-leucine endocytosis motifs were identified in the cytoplasmic tail. Reverse transcription-polymerase chain reaction analysis showed expression of the receptor mRNA in gut, liver, kidney and spleen tissue, aortic endothelial cells and monocytes. Pig kidney cell lines showed saturable pH-dependent binding and uptake of porcine immunoglobulin G (IgG) and also bovine, mouse and human IgG. Polyclonal antibodies raised against the receptor immunoprecipitated a protein of 40,000 MW when the cDNA was expressed in cells and the receptor required assembly with porcine beta2-microglobulin for transport from the endoplasmic reticulum to recycling and early endosomes. Immunohistochemical analysis showed the receptor expressed in epithelial cells of the gut of young and adult animals. The ability of the receptor to deliver immunoglobulin across the gut was demonstrated by feeding piglets bovine colostrum as a source of bovine IgG. Bovine IgG was delivered into the pig circulation. Pigs express the neonatal Fc receptor and the receptor has the potential to deliver protein antigens to the pig immune system.