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Bovine Herpesvirus Type 1 (BoHV-1) infection causes infectious bovine rhinotracheitis and genital disease in cattle, with significant economic and welfare impacts. However, the role of cellular host factors during viral replication remains poorly characterised. A previously performed genome-wide CRISPR knockout screen identified pro- and antiviral host factors acting during BoHV-1 replication. Herein we validate a pro-viral role for a candidate from this screen: the cellular protein tetracopeptide repeat protein 4 (TTC4). We show that TTC4 transcript production is upregulated during BoHV-1 infection. Depletion of TTC4 protein impairs BoHV-1 protein production but does not reduce production of infectious virions, whereas overexpression of exogenous TTC4 results in a significant increase in production of infectious BoHV-1 virions. TTC4 itself is poorly characterized (especially in the context of virus infection), but is a known co-chaperone of heat shock protein 90 (HSP90). HSP90 has a well-characterized pro-viral role during the replication of diverse herpesviruses, and we therefore hypothesized that HSP90 is also pro-viral for BoHV-1. Drug-mediated inhibition of HSP90 using geldanamycin at sub-cytotoxic concentrations inhibited both BoHV-1 protein production and viral genome replication, indicating a pro-viral role for HSP90 during BoHV-1 infection. Our data demonstrates pro-viral roles for both TTC4 and HSP90 during BoHV-1 replication; possibly, interactions between these two proteins are required for optimal BoHV-1 replication, or the two proteins may have independent pro-viral roles.

Herpesviruses encode miRNAs that target both virus and host genes; however their role in herpesvirus biology is still poorly understood. We previously identified thirty five miRNAs encoded by OvHV-2; the causative agent of malignant catarrhal fever (MCF) and are investigating the role of these miRNAs in regulating expression of OvHV-2 genes that play important roles in virus biology. Analysis, using RNAHybrid predicted that two OvHV-2 encoded miRNAs, ovhv2-miR-17-10 and ovhv2-miR-61-1, target transcripts coding for the OvHV-2 bZIP protein Ov2. In other herpesvirus bZIP proteins are known to play important roles in lytic virus replication. Here we show by Flow cytometry and western blotting that ovhv2-miR-17-10 and ovhv2-miR-61-1, reduce the expression of Ov2 protein. The predicted target sites for both miRNAs within the Ov2 gene were disrupted whilst retaining the Ov2 coding sequence. Mutation of the ovhv2-miR-61-1 target sequence restored Ov2 protein expression levels to control levels confirming the identity of its target site. However, it was not possible to determine the binding site of ovhv2-miR-17-10 possibly due to potential G:U pairing introduced during the mutation process. The targeting of Ov2 by two virus-encoded miRNAs suggests an important regulatory role for Ov2 in OvHV-2 replication or reactivation.

BACKGROUND: Malignant catarrhal fever (MCF) is a highly fatal lymphoproliferative disease of cattle, deer, bison, water buffalo, and pigs caused by the gamma-herpesviruses alcelaphine herpesvirus-1 (AlHV-1) and ovine herpesvirus-2 (OvHV-2).OBJECTIVES: This study aimed to determine the prevalence of OvHV-2 in sheep, goats, cattle, and buffalo in Rawalpindi and Islamabad, Pakistan, by applying molecular and phylogenetic methods.METHODS: Blood samples were aspirated from sheep (n = 54), goat (n = 50), cattle (n = 46) and buffalo (n= 50) at a slaughterhouse and several farms. The samples were subjected to heminested polymerase chain reaction (PCR), followed by sequencing and phylogenetic analysis of the OvHV-2 POL gene and the OvHV-2 ORF75 tegument protein gene.RESULTS: The highest percentage of MCF positive samples was in sheep (13%), whereas goat, cattle, and buffalo had lower positive percentages, 11%, 9%, and 6.5%, respectively. Four OvHV-2-positive PCR products obtained from sheep samples were sequenced. The sequences obtained were submitted to the NCBI GenBank database (MK852173 for the POL gene; MK840962, MK852171, and MK852172 for the ORF75 tegument protein gene). Phylogenetic analysis revealed a close similarity of study sequences with those of worldwide samples.CONCLUSIONS: This study is the first cross-sectional study on the prevalence and molecular detection of OvHV-2 in apparently healthy cattle and buffalo that could be carrying OvHV-2 acquired from OvHV-2-positive sheep and goats. The results indicate that OvHV-2 is circulating in Pakistan. Further studies are needed to characterize OvHV-2 and elucidate further its prevalence.

In order to identify host factors that impact Bovine Herpes Virus Type 1 (BHV-1) infection we previously applied a genome wide CRISPR knockout screen with a library covering all bovine protein coding genes. We compiled a list of both pro-viral and anti-viral proteins involved in BHV-1 replication; here we provide further analysis of those that are potentially involved in viral entry into the host cell. These entry related factors include the cell surface proteins PVR and PVRL2, a group of enzymes directly or indirectly associated with the biosynthesis of Heparan Sulfate Proteoglycans (HSPG), and proteins that reside in the Golgi apparatus engaging in intra-Golgi trafficking. For the first time, we provide evidence that PVRL2 serves a receptor for BHV-1, mediating more efficient entry than the previously identified PVR. By knocking out two enzymes that catalyze HSPG chain elongation, HST2ST1 and GLCE, we demonstrated the significance of HSPG in BHV-1 entry. Another intriguing cluster of genes, COG1, COG2 and COG4-7 encodes for six subunits of the conserved oligomeric Golgi (COG) complex. MDBK cells lacking COG6 were less infectable by BHV-1 but release newly produced virions more efficiently as evidenced by fewer but bigger plaques compared to control cells, suggesting impaired HSPG biosynthesis. To facilitate candidate validation, we devised a one-step multiplex CRISPR interference (CRISPRi) system named CRISPR3i that enables quick and simultaneous deployment of three CRISPRs for efficient gene inactivation. Using CRISPR3i, we verified an additional 23 candidates, with many implicated in cellular entry.

We produced a genome wide CRISPR knockout library, btCRISPRko.v1, targeting all protein coding genes in the cattle genome and used it to identify host genes important for Bovine Herpes Virus Type 1 (BHV-1) replication. By infecting library transduced MDBK cells with a GFP tagged BHV-1 virus and FACS sorting them based on their GFP intensity, we identified a list of pro-viral and anti-viral candidate host genes that might affect various aspects of the virus biology, such as cell entry, RNA transcription and viral protein trafficking. Among them were VPS51, VPS52 and VPS53 that encode for subunits of two membrane tethering complexes EARP and GARP. Simultaneous loss of both complexes in MDBKs resulted in a significant reduction in the production of infectious cell free BHV-1 virions, suggesting the vital roles they play during capsid re-envelopment with endocytosed membrane tubules prior to endosomal recycling mediated cellular egress. We also observed potential capsid retention and aggregation in the nuclei of these cells, indicating that they might also indirectly affect capsid egress from the nucleus. The btCRISPRko.v1 library generated here greatly expanded our capability in BHV-1 related host gene discovery; we hope it will facilitate efforts intended to study interactions between the host and other pathogens in cattle and also basic host cell biology.

Background Malignant catarrhal fever (MCF) is a highly fatal lymphoproliferative disease of cattle, deer, bison, water buffalo and pigs caused by the gamma herpesviruses; alcelaphine herpesvirus 1 (AlHV-1) and ovine herpesvirus 2 (OvHV-2). AlHV-1 is known to cause wildebeest-associated MCF whereas OvHV-2 is known to cause sheep-associated MCF. AlHV-1 and OvHV-2 cause inapparent infection in their reservoir hosts (wildebeest for AlHV-1 and sheep for OvHV-2) but fatal lymphoproliferative disease in susceptible species. Methods This study was aimed to find prevalence of OvHV-2 in sheep, goat, cattle and buffalo in Rawalpindi and Islamabad cities of Pakistan, using molecular and phylogenetic methods. Blood samples were collected consisting of sheep (n = 54), goat (n = 50), cattle (n = 46) and buffaloes (n = 50) from slaughter houses and farms. The samples were subjected to hemi-nested PCR, sequencing, and phylogenetic analysis of OvHV-2 POL gene and OvHV-2 tegument protein gene. Results The highest percentage of positive samples was found to be in sheep (13%) whereas positive samples from goat, cattle and buffaloes had percentage of 11%, 9% and 6.5% respectively. Four positive PCR products from sheep samples were also sequenced. The sequences obtained were submitted to NCBI GenBank database under the accession numbers of MK852173 for POL gene, and MK840962, MK852171 and MK852172 for ORF75 tegument protein gene. Phylogenetic analysis revealed close identities with some sequences of this virus from worldwide including neighbouring countries including China, India and Iran. Conclusion This study is the first study in Pakistan on the prevalence of OvHV-2 in carrier and susceptible species. This report provides important data about the presence of OvHV-2 in apparently healthy cattle and buffaloes, which might transmitted to them from OvHV-2 positive sheep or goats. It is concluded that a countrywide, large scale OvHV-2 prevalence study should be conducted to avoid economic losses due to any outbreak of MCF.

Ovine herpesvirus-2 (OvHV-2) is the causative agent of the sheep-associated form of malignant catarrhal fever, a usually fatal lymphoproliferative disease of bison, deer and cattle. Malignant catarrhal fever is a major cause of cattle loss in Africa with approximately 24 7% affected annually; and in North America has significant impact on bison farming. Research into the mechanisms by which OvHV-2 induces disease in susceptible species has been hampered by a lack of a cell culture system for the virus. Ov2 is a bZIP protein encoded by OvHV-2. Proteins with bZIP domains in other herpesviruses, such as the Kaposi'ssarcoma-associated herpesvirus K8 protein and the BZLF1 protein of Epstein-Barr virus are known to play important roles in lytic virus replication. Using a reporter based system, we demonstrate that Ov2 can modulate the activity of the major virus transactivator (Replication and Transcriptional Activator protein, RTA) to 1) drive expression of viral genes predicted to be required for efficient reactivation of the virus, including ORF49; and 2) differentially regulate the expression of the two virus encoded Bcl-2 homologues Ov4.5 and Ov9.

Marek’s disease virus (MDV) is an alphaherpesvirus that induces T-cell lymphomas in chickens. Natural infections in vivo are caused by the inhalation of infected poultry house dust and it is presumed that MDV infection is initiated in the macrophages from where the infection is passed to B cells and activated T cells. Virus can be detected in B and T cells and macrophages in vivo, and both B and T cells can be infected in vitro. However, attempts to infect macrophages in vitro have not been successful. The aim of this study was to develop a model for infecting phagocytes [macrophages and dendritic cells (DCs)] with MDV in vitro and to characterize the infected cells. Chicken bone marrow cells were cultured with chicken CSF-1 or chicken IL-4 and chicken CSF-2 for 4 days to produce macrophages and DCs, respectively, and then co-cultured with FACS-sorted chicken embryo fibroblasts (CEFs) infected with recombinant MDV expressing EGFP. Infected phagocytes were identified and sorted by FACS using EGFP expression and phagocyte-specific mAbs. Detection of MDV-specific transcripts of ICP4 (immediate early), pp38 (early), gB (late) and Meq by RT-PCR provided evidence for MDV replication in the infected phagocytes. Time-lapse confocal microscopy was also used to demonstrate MDV spread in these cells. Subsequent co-culture of infected macrophages with CEFs suggests that productive virus infection may occur in these cell types. This is the first report of in vitro infection of phagocytic cells by MDV.

A number of herpesviruses have now been shown to encode microRNAs (miRNAs) that have roles in control of both viral and cellular gene expression. Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever, a fatal lymphoproliferative disease of cattle. Using massively parallel sequencing and Northern hybridization we have identified eight putative miRNAs encoded by OvHV-2 expressed in an OvHV-2-immortalized bovine lymphocyte cell line. These eight miRNAs are encoded in two areas of the OvHV-2 genome that contain no predicted protein coding regions and show no sequence similarity with other herpesvirus or cellular miRNAs. This represents the first report of the expression of virally encoded miRNAs in the genus Macavirus of herpesviruses.

In this report we demonstrate that the herpes simplex virus type 1 reiteration element 1 (RE1) (nt: 117158–117353) in concert with its flanking sequences is both a cell specific and stimulus inducible regulatory domain. This region of the virus genome and specifically the RE1 supports differential reporter gene expression in both baby hamster kidney cells and disassociated rat trigeminal ganglia and is present within a region that is implicated in regulating latency of the virus in neuronal cells. Further we demonstrate that this locus is a transcriptional regulatory domain and a target for the transcription factor CCCTC binding protein.

Alcelaphine herpesvirus-1 (AlHV-1) is the causative agent of Malignant Catarrhal fever, a lymphoproliferative and degenerative disease of large ruminants and ungulate species. The Alcelaphine Herpesvirus-1 gene product encoded by open reading frame 57 (ORF 57) is the positional homologue of the ORF 57 of Herpes Virus Saimiri (HVS), Kaposi's Sarcoma associated herpesvirus (KSHV) and Murine Gammaherpesvirus 68 (MHV 68), the Epstein-Barr virus BMLF1 gene, the herpes simplex virus (HSV-1) ICP 27 and the IE 4 gene of Varicella Zoster virus (VZV). In these viruses the ORF 57 gene product is expressed very early and encodes a regulatory protein, which is essential for viral replication acting both at the transcriptional and post-transcriptional levels. The function of ORF 57 gene product in the life cycle of AlHV-1 however remains unknown. Here we examined the expression of this gene and the function of its product. We have demonstrated that it is expressed very early in infection and have shown that the ORF57 gene product activates the promoter of another classical transactivator gene ORF50. It activates ORF50 promoter driving expression of an intron-less reporter gene to 50 fold and does not have any effect on an intron-containing reporter gene driven by the ORF 50 promoter. The 50 fold increase in the luciferase activity was not correlated with a similar fold increase in the luciferase RNA levels indicating that ORF 57 protein acts at a post-transcriptional level to regulate gene expression.

Alcelaphine herpesvirus-1 (AlHV-1) is a gamma(2) rhadinovirus associated with Malignant Catarrhal Fever (MCF) in cattle. ORF 57 is well conserved among gammaherpesviruses and it has been shown that the ORF 57 gene products of Herpesvirus Saimiri (HVS), Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) play an important role in regulating viral gene expression. The AlHV-1 ORF 57 gene product has not been characterized. In the accompanying paper we have demonstrated that AlHV-1 ORF 57 encodes an immediate early protein that acts as a regulator of gene expression. The ORF 57 gene product has an up-regulatory effect only on another immediate early gene product encoded by ORF 50. Here we show that the ORF 57 gene product is a nuclear protein. When ORF 57 was fused to the gene encoding Enhanced Green Fluorescent Protein (EGFP), the fusion protein exhibited a punctate nuclear distribution that co-localized with the nucleolar phosphoprotein C23. The nuclear localisation signal of ORF 57 gene product was located at the N-terminus. The ORF 57 gene product travels from nucleus to the cytoplasm, where it accumulates during Actinomycin D treatment. The domain involved in nuclear shuttling was also localised at the N-terminal region of the protein. Thus in common with homologues in other herpesviruses the AlHV-1 ORF 57 gene product is a nuclear cytoplasmic shuttling protein which may play a role in export of viral mRNAs from the nucleus of infected cells.

The various members of the different pattern recognition receptor families are now recognized as playing a crucial role in the initial interactions between a pathogen and the host. This paper identifies all 10 members of the TLR family in sheep as well as key members of the C-type lectin and NLR families. Our data show that sheep possess the ‘human’ and not the ‘mouse’ pattern of TLRs and confirm the high degree of sequence identity between orthologous genes in the different species. In the absence of definitive antibodies, qRT-PCR assays were developed to quantify PRR transcript expression patterns in a range of normal sheep tissues as well as isolated dendritic cell (DC) and leukocyte subsets. These data show that the lymphoid organs (spleen and lymph nodes) express the widest range of PRRs and that organs such as the lung and kidney have distinctive arrays of PRRs that reflect their potential risk of pathogen exposure. In addition we show that the two DC subsets, defined by the differential expression of CD172a/CD45RA and their cytokine expression profiles, have different and characteristic PRR complements again possibly reflecting their distinctive function. These data are important for future studies on the role of PRRs in disease pathogenesis and control.

The aim of this study was to investigate the skin immunopathology of gene gun-delivered plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) and hence explore the possible mechanisms of its adjuvant activity. Using sheep as the experimental model, expressible pGM-CSF was administered to the epidermis and the dermal/epidermal junction and its effects on the skin were assessed by histopathology, immunohistology and quantitative RT-PCR for a range of pro-inflammatory and immune response-polarizing cytokines. Both functional and non-functional plasmids caused an acute inflammatory response with the infiltration of neutrophils and micro-abscess formation; however, the response to pGM-CSF was more severe and was also associated with the accumulation of eosinophils, immature (CD1b(-)/CD172a(-)) dendritic cells and B cells. In terms of cytokine expression, an early TNF-alpha response was stimulated by gene gun delivery of plasmid-associated gold beads, which coincided with an immediate infiltration of neutrophils. However, only pGM-CSF triggered the short-lived expression of GM-CSF (peaking at 4 h) and significant long-term increases in both TNF-alpha and IL-1beta. pGM-CSF did not affect the expression of the immune response-polarizing cytokines, IL-10 and IL-12.

Antibody-dependent cell-mediated cytotoxicity (ADCC) specific for maedi visna virus (MVV) has never been described. The IgG antibody response to MVV is restricted to an IgG1 response whilst MVV specific IgG2 is never seen in persistently infected sheep. To determine whether the isotypic restriction of the antibody response is responsible for the lack of ADCC, an ADCC assay was developed using polyclonal serum raised to recombinant MVV ENV protein. Sheep immunised with a recombinant GST:SUenv fusion protein in complete Freund's adjuvant produced an antibody response which contained IgG1 and IgG2 antibodies. The activity of this serum in an ADCC assay was compared to serum from persistently infected sheep. Serum from immunised sheep mediated ADCC reactions whilst no activity was ever seen in persistently infected sheep serum. IgG2 may therefore be the possible effector isotype for ADCC reactions against MVV. Failure of the IgG2 dependent ADCC system in vivo may contribute to the persistence of MVV-infected macrophages in vivo.

The most common complication of herpes zoster is post-herpetic neuralgia (PHN), which has been defined as severe pain occurring 1 month after rash onset or persisting for greater than 3 months. PHN is classed as a neuropathic pain that is associated with mechanical allodynia where normally innocuous tactile stimuli are perceived as painful. The development of therapies to treat PHN has been hampered by the lack of animal models, which mimic the clinical situation. We have previously reported that varicella zoster virus (VZV) infection in the rat results in mechanical allodynia and thermal hyperalgesia. Here, we report that following VZV infection of the left footpad rats develop a chronic mechanical allodynia, which is present for longer than 60 days post-infection and which resolves by 100 days PI. The model is robust and reproducible with animals consistently developing allodynia by 3 days PI and continuing to present with symptoms for at least 30 days. The reproducible nature of the induction and course of the allodynia allows the use of this model to determine the effect of various compounds on, and to investigate the pathogenic mechanisms underlying the development of VZV-induced allodynia. Comparative studies using HSV-1 show that the induction of the chronic allodynia is VZV-specific and is not a result is of virus replication-induced tissue damage or accompanying inflammation. Therefore, we propose that the rat VZV infection model could prove useful in studying the mechanisms underlying post-herpetic neuralgia.

This chapter discusses the various factors that contribute to the failure of host defense mechanism in eliminating microbes. One way of looking at persistent infections is to regard them as failures of the host defense mechanisms which are designed to eliminate invading microorganisms from tissues. Persistent infections usually represent a secondary event, following on from an initial acute infection. In certain acute infections the patient appears to recover, but there is later a relapse. Many of the severe infections causing illness and death in communities (poliomyelitis, plague, yellow fever, cholera) are not persistent and the microorganisms are eliminated from the body after recovery. Persistent infections are often important from the microbe's point of view, enabling it to be maintained in small or isolated host communities. Persistent infections also generally present problems in the development of vaccines. They are becoming relatively more important, both for the individual and for the community, as the nonpersistent infections are eliminated by public health measures and by vaccination. Not only may they reactivate and cause troublesome infections in immunocompromised or immunosuppressed patients, but some of them can cause malignant tumors.

In the opening chapter, we provide an introduction to infectious diseases and some of the key basic concepts required for a deeper understanding of the subject area. Importantly, we discuss the fact that only a very small proportion of microbes have the capacity to cause disease, and that many bacteria are commensal and can play important roles in essential physiological processes in their hosts. We also discuss the impact of new technological advances in our understanding of infectious diseases and the pathogen and host determinants involved. The reader is also introduced to the different stages of the pathogenic cycle which underpin infectious diseases and form the basis for the main chapters of the book to come.

This chapter describes the role of the innate and adaptive immune responses during infection. It highlights the importance of Pattern Recognition Receptor (PRR) in signalling cytokine production and the activation and proliferation of the macrophages, NK cells and dendritic cells (DCs). DCs provide the key link between the innate and adaptive immune systems by processing and presenting microbial antigens via the MHC system to CD8+ T cytotoxic and T helper lymphocytes. CD4+ T helper (Th) cells are defined by the cytokines they produce and their effector and regulatory function in vivo. An important function of Th cells is to provide help for B cell proliferation and maturation, for the proliferation of cytotoxic T cells and for activating macrophages at sites of infection. The significance of antibody and T cells in the immune response to selected pathogens is presented. In addition, sections on NK cells and the complement system have been updated.

This chapter focuses on some of the host and microbial factors influencing susceptibility to disease. These factors include genetic factors in the microorganism, genetic factors in the host, hormonal factors, and stress. The microorganism's ability to infect a given host is genetically determined and many microorganisms infect only one particular host species. Pathogenicity or virulence is also a function of the microbial genome. Virulence is being increasingly recognized to depend on coordinated expression of numerous genes, whose products mediate adherence, antiphagocytic activity, immune evasion, production of toxins, etc. Susceptibility to infectious disease is always influenced and is sometimes determined by the genetic constitution of the host. Inflammation makes an important contribution to tissue damage and pathology in infectious disease and injected corticosteroids (or ACTH) have a pronounced anti-inflammatory effect; their therapeutic use in infectious diseases depending on a reduction in the inflammatory pathological components at sites of infection. Increased circulating levels of corticosteroid hormones are necessary for a successful host response to infectious disease. A host of miscellaneous factors, such as smoking, seasonal changes mental state, influence the course of infectious diseases.

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This chapter addresses the mechanisms by which pathogens evade the host immune responses. The concepts of induced tolerance and energy are discussed and this continues into coverage of the induction of a generalised immunosuppression by pathogens. Strategies used by the pathogen to evade an immune response, e.g. restricted expression of antigens, the structure of the bacterial cell wall, infection of sites where the immune response is ineffficient, the avoidance of the interferon response and the induction of inappropriate immune responses, e.g. non-neutralising antibodies and the Th1/TH2 balance are discussed. Finally antigenic variation both within the host (e.g. HIV, Trypanosomes) and the population (influenza) are discussed.

This chapter describes several attachment and entry procedures applied by the microorganisms to enter the body of the host. The conjunctiva and the alimentary, respiratory and urinogenital tracts offer pathways for infection by microorganisms. Penetration of these surfaces is more easily accomplished than in the case of the intact outer skin. A number of antimicrobial devices have been developed in evolution to deal with this danger and also special cleansing systems to keep the conjunctiva and respiratory tract clean enough to carry out their particular function. In order to colonize or penetrate these bodily surfaces, microorganisms must first become attached to these surfaces. The microorganisms enter the host body through four ways: by specific mechanisms of attachment or simply by penetrating the body surface, by a biting arthropod, through wounds cuts or damage and impairment of defenses at the body surface, and finally by a local or general defect in body defenses.

In this chapter we describe the key events associated with phagocytosis, a critical immune activity required for host innate resistance to infection. In response to phagocytosis, microbial pathogens have evolved a myriad of ways to interfere with this process and avoid killing. In fact, some bacteria have evolved the capacity to survive and proliferate within phagocytic cells. We provide selected examples of the different mechanisms involved in pathogen avoidance of phagocytosis, and also discuss how defects in phagocytic cells can predispose to infection.

This chapter discusses the general principles of vaccines, its complications and side effects, and the development of new vaccines. Effective resistance to infection or disease depends on the vaccine having certain properties – such as inducing the right type of immune resistance, inducing an immune response in the right place, inducing an immune response to the right antigens, and immunity to toxins. Virulent microorganisms are inactivated to make killed vaccine. If inactivation has been incompletely carried out, vaccination introduces infection. Nonmicrobial antigens in vaccines can cause allergic responses. Large numbers of killed salmonellas are present in the vaccine for typhoid, and this vaccine therefore contains large amounts of endotoxin, which can present toxicity. Live vaccines often cause illness in children with immunodeficiencies. Several procedures for the development of synthetic peptides as vaccines also have been described. Successful new vaccines will come from encouraging laboratory advances in immunology and biotechnology and at the same time attending to practical problems, such as vaccine supply, quality testing, licensing, and financing.

This chapter addresses the mechanisms by which pathogens spread through the body from the initial site of infection and the host and pathogen factors which influence that spread. Localised spread across mucosal or epithelial surfaces is considered and factors which allow spread beyond this initial event briefly introduced. The two main routes of spread throughout the body, via the lymphatic system and the blood are discussed. The factors which influence recognition of the pathogen by antigen-presenting cells and uptake into the lymphatic system, and the outcome of this interaction, i.e. clearance of the pathogen or further spread, are discussed in relation to the cell type encountered and pathogen factors. Once the pathogen enters the blood and spreads throughout the body, the interactions with two key cell types, vascular endothelial cells and reticuloendothelial cells are discussed. Spread via nerves, infection of tissues and pleura and the generation of rashes are also discussed.

This chapter addresses the factors involved in recovery from infection. Antibody and cellular responses (CMI) are considered. The mechanisms by which antibodies neutralise viruses/prevent infection are described, as is the involvement of antibody in bacterial and protozoal diseases. The role of CMI in recovery from specific bacterial, protozoal and viral infections is considered. The mechanisms of cell killing by cytotoxic T-cells is described and the role of NK cells and macrophages in pathogen clearance is discussed, as is the role of inflammation. Mechanisms by which the complement system acts and its role in vivo is discussed, as is the interferon response. Recovery from S. typhimurium is used to illustrate the multimechanistic nature of recovery. The role of temperature in recovery and pathogen factors that induce fever are considered. Tissue repair mechanisms are discussed, as is finally resistance to infection.

In this chapter, we describe the early stages of the pathogenic cycle involving entry into the host. For example, some bacteria may enter into epithelial cells, proliferate and then spread at the epithelial surface before being transmitted to another host. Some pathogens then migrate past the epithelial layer to deeper tissues causing invasive infections. Examples of some of the major bacterial, viral and fungal pathogens affecting the epithelial surfaces in the body are provided. The body produces a strong inflammatory immune response to pathogens at the epithelial surface. An introduction to the components of the inflammatory response including the phagocytic cells and the lymphatic system is provided. In addition, the nutritional requirements for invading bacteria including the importance of iron is discussed.

Many infections are associated with damage inflicted either directly or indirectly by invading pathogens. Although some infections do not result in host damage, it is often a natural consequence of the activities of virulence factors produced by the pathogens in order to facilitate survival, and proliferation in the host or onward transmission to another host. The damage often manifests itself as the symptoms of disease which can be useful for diagnosis and for informing appropriate treatments. A wide array of different types of toxins which cause damage to the host are produced by different bacterial pathogens. Here we provide examples of well-characterised toxins and describe their mechanisms of action, and potential function with regard to pathogenesis. In addition we describe indirect damage to the host in the form of inflammation or immunopathology, typically the result of the host's own immune response. Finally, we discuss diarrhoea as a special case and list some of the major pathogens and the toxins associated with this devastating disease.

Ovine herpesvirus 2 (OvHV-2) causes malignant catarrhal fever in cattle, pigs and deer. We have observed intact circular and linear OvHV-2 genomes in infected T cell lines derived from cows and rabbits. Bovine T cell lines were predominantly latently infected but rabbit T cell lines supported OvHV-2 productive cycle gene expression and virus capsids were demonstrated for the first time.

Central to infection by a majority of DNA viruses is the expression of encoded proteins that modify cell cycle. Viruses such as SV40 and Adenovirus viruses encode proteins that interact directly, or indirectly, with key cell cycle proteins such as CBP300 and the retinoblastoma gene product. However, neurons do not have a cell cycle as we generally describe it and this is also reflected in the difficulty in obtaining immortalised neuronal cultures. The replication strategies of viruses that infect post-mitotic cells such as neurons may be different from infection of other somatic cells. The life cycle for viral latency or slow infection of neurons appears to involve silencing or restricting expression of the viral genome until such times as dictated by the environment. These signals from the environment usually reflect cell stress, otherwise the cell appears to tolerate the existence of the virus genome. We will review the genomic structure of alphaherpesviruses in neurons and transcriptional control mechanisms that may regulate expression. Where appropriate we will contrast and compare virus and endogenous neuronal gene expression.

A domain, previously termed RE1, exists within the herpes simplex virus type 1 genome potentially influencing expression of immediate early genes and the latency associated transcripts. This domain consists of 10 tandem copies of a CT-rich sequence. We demonstrate that this domain binds multiple host-cell factors that may allow RE1 to act either as a transcriptional regulator and/or to affect nucleosomal and DNA structure in the latent genome.

This report describes two subpopulations of B cells in sheep. These subpopulations have distinct recirculation characteristics and tissue distributions. Phenotypically the populations are distinguished by their differential expression of the complement receptors, CD21 (CR2) and CD11b/CD18 (CR3). CD11b+ B cells are surface (s)IgMhi, co-express CD11c but are L-selectin negative. They populate the splenic marginal zone but are absent from splenic and ileal Peyer's patch (IPP) follicles and both afferent and efferent lymph compartments. Fluorescent tracing experiments showed that the CD11b+ B cells are non-recirculating as they did not appear in lymph after intravenous inoculation but are restricted to the blood and spleen. The CD11b-negative population expresses a conformational determinant of CD21 that is recognized by the monoclonal antibody Du 2-74. These cells are sIgMlo and co-express L-selectin. They populate the splenic and IPP follicles, are absent from the splenic marginal zone and are the only B cells in afferent lymph, efferent lymph and all lymph nodes. Fluorescence tracing experiments showed that the CD21 B cells are recirculating cells with their entry into efferent lymph being detectable by 16 h and peaking at 24-30 h. These data suggest that there are at least two lineages of B cells in the sheep with different phenotypic, functional and recirculation characteristics.

Herpesviruses encode microRNAs (miRNAs) that target both virus and host genes; however, their role in herpesvirus biology is understood poorly. We identified previously eight miRNAs encoded by ovine herpesvirus-2 (OvHV-2), the causative agent of malignant catarrhal fever (MCF), and have now investigated the role of these miRNAs in regulating expression of OvHV-2 genes that play important roles in virus biology. ORF20 (cell cycle inhibition), ORF50 (reactivation) and ORF73 (latency maintenance) each contain predicted targets for several OvHV-2 miRNAs. Co-transfection of miRNA mimics with luciferase reporter constructs containing the predicted targets showed the 5′ UTRs of ORF20 and ORF73 contain functional targets for ovhv-miR-2 and ovhv2-miR-8, respectively, and the 3′ UTR of ORF50 contains a functional target for ovhv2-miR-5. Transfection of BJ1035 cells (an OvHV-2-infected bovine T-cell line) with the relevant miRNA mimic resulted in a significant decrease in ORF50 and a smaller but non-significant decrease in ORF20. However, we were unable to demonstrate a decrease in ORF73. MCF is a disease of dysregulated lymphocyte proliferation; miRNA inhibition of ORF20 expression may play a role in this aberrant lymphocyte proliferation. The proteins encoded by ORF50 and ORF73 play opposing roles in latency. It has been hypothesized that miRNA-induced inhibition of virus genes acts to ensure that fluctuations in virus mRNA levels do not result in reactivation under conditions that are unfavourable for viral replication and our data supported this hypothesis.

Jaagsiekte retrovirus (JSRV) is an exogenous type D-related retrovirus specifically associated with a contagious lung cancer of sheep (sheep pulmonary adenomatosis; SPA). Recently, epithelial tumour cells in the lungs of SPA-affected sheep were identified as major sites of JSRV replication by immunological techniques and RT-PCR amplification of part of JSRV gag. JSRV was not detected outside the lungs and their draining lymph nodes. However, low levels of JSRV expression in non-respiratory tissues could have been masked by co-amplification of endogenous JSRV-related sequences, which were differentiated from JSRV by the lack of a Scal restriction site in the PCR product. To further investigate the pathogenesis of SPA, an exogenous virus-specific hemi-nested PCR was developed utilizing primers in the U3 region of JSRV LTR, where major differences between endogenous and exogenous sequences exist. This technique was shown to beor = 10(5)-fold more sensitive than the previous gag PCR/ScaI digestion method. Using this new assay the tissue distribution of JSRV in sheep with natural and experimentally induced SPA was analysed. Proviral DNA and JSRV transcripts were found in all tumours and lung secretions of SPA-affected sheep (n = 22) and in several lymphoid tissues. The mediastinal lymph nodes draining the lungs were consistently demonstrated to be infected by JSRV (10/10). JSRV transcripts were also detected in spleen (7/9), thymus (2/4), bone marrow (4/8) and peripheral blood mononuclear cells (3/7). Proviral DNA was also detected in these tissues although in a much lower proportion of cases. JSRV was not detected in 27 samples from unaffected control animals (n = 15).

Sheep pulmonary adenomatosis (SPA) is a naturally occurring contagious lung tumour of sheep which has been associated aetiologically with a type D- and B-related retrovirus (Jaagsiekte retrovirus; JSRV). To improve understanding of the aetio-pathogenesis of SPA, the distribution and the sites of JSRV replication in sheep with naturally or experimentally induced SPA or in unaffected controls were identified. New immunological reagents were produced and a blocking enzyme-linked immunosorbent assay (B-ELISA) and an immunohistochemical technique for the detection of JSRV major capsid protein at the tissue and cellular levels were developed. JSRV was detected only in the respiratory tract of sheep affected by pulmonary adenomatosis and specifically in the transformed epithelial cells of the alveoli of SPA-affected sheep.

We have used the PCR and HIRT DNA obtained from MVV-infected tissue culture cells as a template to generate a number of independently derived clones representing overlapping fragments of the gp135 region (env) of Maedi visna virus (MVV) strain EV1. Sequencing these clones revealed that homology between selected regions of gp135 ranged from 93.2% to 99.8%. Four hypervariable regions and one large highly conserved region have been identified. These data provide information on the variability of EV1 env, which extends and complements the data previously available on env variability between geographically distinct isolates of MVV.

The envelope glycoprotein gp 135 of the ovine lentivirus maedi visna virus (MVV) is the main target for neutralising antibody in vivo, however little is known about the specific regions of gp135 which elicit this neutralising response. We have used the polymerase chain reaction (PCR) to generate overlapping fragments of the gp135 gene which have been expressed as fusion proteins in the yeast Ty-VLP system. These fusion proteins have been used to analyse the antibody response to gp135 in MVV infected sheep and we are able to identify at least three distinct regions of gp135 to which antibodies are directed. The approach described in this paper provides a rapid and simple method of generating overlapping fusion proteins with which to carry out epitope mapping studies.

Summary A 36-year-old donkey developed dyspnoea, pyrexia, hypoalbuminaemia and oedema. Following continued clinical deterioration the donkey was humanely destroyed. Grossly, there were numerous nodules (5–10 mm) scattered throughout the lung. Microscopically, the lung was infiltrated by an angiocentric and bronchocentric to diffuse mixed population of small mature and atypical lymphocytes, histiocytes, plasma cells and fewer eosinophils. The infiltrate was composed of numerous small mature and fewer atypical CD3+ T lymphocytes. Low numbers of CD20+ and CD79a+ B cells, some atypical, accompanied the T cells. These infiltrates were consistent with an angiocentric lymphoma and resembled lymphomatoid granulomatosis, an Epstein-Barr virus (EBV)-associated human tumour. Immunohistochemistry for EBV latent membrane protein and polymerase chain reaction analysis for equine gamma herpesvirus DNA were negative. To the authors’ knowledge this is the first case of angiocentric lymphoma reported in a donkey and the first case of lymphomatoid granulomatosis-type disease in an animal in which possible concurrent infection with a gamma herpesvirus has been investigated.

Postherpetic neuralgia (PHN), a common complication of herpes zoster, which results from reactivation of varicella zoster virus, is a challenging neuropathic pain syndrome. The incidence and severity of herpes zoster and PHN increases with immune impairment or age and may become a greater burden both in terms of health economics and individual suffering. A clearer understanding of the underlying mechanisms of this disease and translation of preclinical outcomes to the clinic may lead to more efficacious treatment options. Here we give an overview of recent findings from preclinical models and clinical research on PHN.

We have isolated a maedi-visna-like virus from the peripheral blood mononuclear cells of a British sheep displaying symptoms of arthritis and pneumonia. After brief passage in fibroblasts this virus (designated EV1) was used to infect choroid plexus cells. cDNA clones of the virus were prepared from these cells and sequenced. Gaps between non-overlapping clones were filled using gene amplification by the polymerase chain reaction. The genome structure is similar to that described for visna virus strain 1514, and differs from that described for visna virus strain SA-OMVV in not having a W reading frame. Overall the genome differs by about 20% between each of these strains, but there is fivefold variation in the amount of divergence of derived amino acid sequences of different open reading frames. Two sequenced EV1 clones each contain only one copy of the 43 bp repeat, with paired AP-1 sites, which is a feature of other ruminant lentiviral long terminal repeats (LTRs). However, analysis of viral DNA in infected cells by gene amplification shows that LTRs with two repeats do occur, albeit at a relatively low frequency.

Pattern recognition receptors (PRRs) play a crucial role in the initiation of the adaptive immune response. Immunological competence of foetal lambs occurs progressively throughout gestation, and in order to understand the role played by PRRs in foetal immunological competence, we quantified transcript expression, in the skin and spleen, of the TLRs, key C-type lectins and CARD15 during the critical second trimester. These data show that lambs express the same spectrum of PRRs as the adult but that the level of expression for most is dependent on developmental age. Key findings include: TLR1 and TLR5 are expressed at high levels in the foetus but are low in the adult; in contrast, TLR4, CD14 and CARD15 increase with age. In addition, TLR9 and TLR10 are expressed by the spleen and not the skin, while CARD15 is low in the spleen and high in the skin. (c) 2008 Elsevier Ltd. Ail rights reserved.

Several studies have implicated a potential role for histamine H 3 receptors in pain processing, although the data are somewhat conflicting. In the present study we investigated the effects of the novel potent and highly selective H 3 receptor antagonists GSK189254 (6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1 H -3-benzazepin-7-yl)oxy]- N -methyl-3-pyridinecarboxamide hydrochloride) and GSK334429 (1-(1-methylethyl)-4-({1-[6-(trifluoromethyl)-3-pyridinyl]-4-piperidinyl}carbonyl)hexahydro-1 H -1,4-diazepine) in two rat models of neuropathic pain, namely the chronic constriction injury (CCI) model and the varicella-zoster virus (VZV) model. Both GSK189254 (0.3, 3 and/or 10 mg/kg p.o.) and GSK334429 (1, 3 and 10 mg/kg p.o.) significantly reversed the CCI-induced decrease in paw withdrawal threshold (PWT) measured using an analgesymeter and/or von Frey hairs. In addition, GSK189254 (3 mg/kg p.o.) and GSK334429 (10 mg/kg p.o.) both reversed the VZV-induced decrease in PWT using von Frey hairs. We also investigated the potential site of action of this analgesic effect of H 3 antagonists using autoradiography. Specific binding to H 3 receptors was demonstrated with [ 3 H]-GSK189254 in the dorsal horn of the human and rat spinal cord, and in human dorsal root ganglion (DRG), consistent with the potential involvement of H 3 receptors in pain processing. In conclusion, we have shown for the first time that chronic oral administration of selective H 3 antagonists is effective in reversing neuropathic hypersensitivity in disease-related models, and that specific H 3 receptor binding sites are present in the human DRG and dorsal horn of the spinal cord. These data suggest that H 3 antagonists such as GSK189254 and GSK334429 may be useful for the treatment of neuropathic pain.

The aim of this study was to investigate skin immunopathology following gene gun delivery of plasmid-encoding interleukin 3 (pIL-3) and hence explore the possible mechanisms of its adjuvant activity. Using the sheep as the experimental model, expressible pIL-3 was administered to the epidermis and the dermal/epidermal junction and its effects on the skin were assessed by histopathology, immunohistology and quantitative RT-PCR for a range of pro-inflammatory and immune response polarizing cytokines. Delivery of both functional and non-functional plasmids caused an acute inflammatory response with the infiltration of neutrophils and micro-abscess formation; however, the response to pIL-3 was more severe and was also associated with an early (24 h) infiltration of B cells and a later accumulation of CD172a-/CD45RA+ dendritic cells (DC). In terms of cytokine transcript expression, an early TNFalpha response was stimulated by gene gun delivery of plasmid-associated gold beads, which coincided with an immediate infiltration of neutrophils. However, only pIL-3 triggered the short-lived expression of IL-3 (peaking at 6 h) and significant long-term increases in both TNFalpha and IL-1beta. pIL-3 did not affect the expression of the immune response polarizing cytokines, IL-10 and IL-12.