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Human gammaherpesviruses such as Epstein-Barr virus (EBV) cause lifelong infections and associated diseases, by virtue of their ability to establish latent infection. Many studies performed in the past years in murine herpesvirus 68 (MHV-68) model of infection suggested that the limited immunity generated against isolated viral components by subunit vaccines cannot counteract the multiple immune evasion strategies operated by gammaherpesviruses. Indeed, a significant inhibition of long-term latency establishment could be observed in mice vaccinated with strains of genetically modified MHV-68 defective in reactivation or establishment of latency. In this study, we focused on the effects of interferon-α (IFN-α) on both the lytic and latent phase of MHV-68 infection, as exerted by the constitutive release of IFN-α1 by a clone of MHV-68 genetically modified to produce this cytokine (MHV-68mIFNα1). Although the MHV-68mIFNα1 recombinant virus exhibited in vitro replication features indistinguishable from those of the wild type MHV-68, its pathological properties were severely attenuated in vivo in immunocompetent mice and not in mice rendered genetically unresponsive to type I IFN, suggesting that a stronger immune response was primed in the presence of the cytokine. Notably, MHV-68mIFNα1 attenuation did not result in a reduced level of long-term spleen latency establishment. These results prompted us to evaluate the efficacy of MHV-68mIFNα1 in a prophylactic vaccination regimen aimed at inhibiting the symptoms of acute virus infection and the establishment of long-term latency after MHV-68 challenge. Our results show that mice vaccinated with MHV-68mIFNα1, administered as a live-attenuated or partially inactivated (by Psoralen and UV treatment) vaccine, were protected against the challenge with wt MHV-68 from all phases of infection. The ability of MHV-68mIFNα1 to produce IFN-α at the site of the infection, thus efficiently stimulating the immune system in case of virus reactivation from latency, makes this recombinant virus a safer live-attenuated vaccine as compared to the previously reported latency-deficient clones.

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.

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.

Theiler's virus strains GDVII and FA cause an acute encephalitis when injected intracerebrally into mice, whereas strains To, BeAn and DA establish a persistent infection and produce a chronic demyelinating disease. The chronic infection is also dependent on the mouse strain used, with susceptibility linked in part to the D locus of the MHC. The region of the virus genome associated with neurovirulence maps to the P1 region, encoding the capsid proteins, and to the 5′ non-coding region. There is evidence that BeAn DA virus persists in oligodendrocytes, where it reactivates to initiate demyelinating disease. Host factors are involved in the development of the lesion, including CD4 + T cell responses. These lymphocytes most probably mediate damage through activation of macrophages leading to local destruction of glial cells. Another possible pathological role for the immune system is the recognition of nerve cell antigens and the initiation of autoimmune disease. Such a virus-triggered phenomenon may well underlie human CNS diseases such as multiple sclerosis.

Mice inoculated intranasally with murine gammaherpesvirus-68 were used to evaluate the efficacy of acyclovir (ACV) in the treatment of acute and latent infections. Effectiveness was measured by infectious virus assay of the lung (site of active replication) and infectious centre assay of spleen cells (site of latency). Intraperitoneal administration of ACV at 6-h intervals starting soon after inoculation was more effective in reducing infectious virus in the lung than was treatment with 12-hourly injections commencing 3 days post-infection. Further, ACV treatment during acute infection resulted in an approximately 10-fold reduction in the number of infectious centres in the spleen as compared to placebo-treated animals. However, once latency was established, ACV treatment was not effective in reducing the number of infectious centres in the spleen. This is the first report demonstrating that ACV can be used to minimize the replication of murine gammaherpesvirus in mice at the site of primary infection, resulting in a reduction in the number of latently infected spleen lymphocytes.

Our understanding of the host response to gammaherpesviruses comes largely from studies on Epstein-Barr virus. A recent addition to this family is murine herpesvirus-68 which, like Epstein-Barr virus, establishes a latent infection in B lymphocytes and is associated with lymphoproliferative disease. This virus provides a unique opportunity to explore the relationship between gammaherpesviruses and the immune system.

The outcome of a viral infection within the nervous system depends on a complex interplay between the virus, its target cell and the immune system. Recent research has elucidated a variety of mechanisms involved in these interactions and their role in the production of disease.

Vaccinia virus recombinants expressing glycoproteins B (vgB11), D (VgD52), E (gE/7.5 and gE/4B), G (gG-vac), H (gH-vac), and I (gI-vac) of HSV-1 were used to compare the protective response to these individual glycoproteins in the mouse. Glycoprotein D induced the best neutralizing antibody titers and the most increased rates of HSV clearance from the ear as well as good protection from the establishment of latent HSV infections in the sensory ganglia. Glycoprotein B also induced good neutralizing antibody titers and as great a protection from the establishment of latency as gD although the rate of virus clearance from the ear was not as great as after immunization with gD. Glycoprotein E induced weak neutralizing antibody but gG, gH, and gI did not show a neutralizing antibody response. At higher challenge doses of virus (10(6) PFU HSV-1 in the ear), gE induced a protective response by increasing the rate of virus clearance and reducing the acute infection of ganglia as compared to negative control immunized mice. However there was no protection from the establishment of latent infections after immunization with gE. No protective response was seen to gG, gH, or gl.

This chapter discusses the various strategies employed by the microbes in response against host defense mechanisms. Phagocyte, the most important part of the host defense system, is equipped with a powerful array of antimicrobial weapons and lysosomal enzymes. A detailed description of the process of phagocytosis is provided in the chapter. Microorganisms that readily attract phagocytes, and are then ingested and killed by them, are by definition unsuccessful as they fail to cause a successful infection. Microbial factors that damage the host or actively promote the spread of infection are often called agressins. Such factors have a 'toxic' activity that is demonstrable in a suitable test system. In many instances, however, microbial factors inhibit the operation of host defense mechanisms without actually doing any damage. Microbial products that kill phagocytes may at lower concentrations interfere with their locomotion or their phagocytic activity, for instance, by inhibiting protein synthesis. Microorganisms can avoid the attentions of phagocytic cells by inhibiting chemotaxis, and as a result of this, the host is less able to focus polymorphs and macrophages into the exact site of infection. In case the particle is enclosed in a phagocytic vacuole, there are other methods of entry such as endocytosis.

This chapter discusses the events and responses that follow immediately after the entry of an organism in a host cell. The first activity that could be observed is the growth of the epithelial cells. At the site of entry into the body, microorganisms that are shed directly to the exterior multiply in the epithelial surface producing a spreading infection in the epithelium. Certain microorganisms spread through the body to reach susceptible target organs after free microorganism particles have entered vessels below the skin or intestinal epithelium. The infected host has a variety of defenses that operate without delay, before the immune response comes into action. However, many microbes have strategies for interfering with these defenses. After traversing the epithelial cell layer, a microorganism encounters the basement membrane. After reaching the subepithelial tissues they are exposed to three important host defense systems: the tissue fluids, the lymphatic system leading to the lymph nodes, and phagocytic cells. In addition to being able to resist host defense mechanisms, a pathogenic organism—be it an obligate intracellular, facultative intracellular, or extracellular pathogen—must also overcome the problem of obtaining essential nutrients if it is to be successful.

This chapter summarizes the various strategies used by the microbes against the immune defenses of the host cell. Many infections that are restricted to body surfaces have short incubation periods, with less than a week between initial infection and disease onset. The microbe thus evades the immune response by completing its growth and shedding within a few days before the host deploys specific antibody and T cells. Immunological tolerance is another factor that reduces the immune response to a given antigen. There is commonly a degree of tolerance to a microorganism when infection occurs during fetal or early postnatal life. Tolerance to a given microorganism can also arise when large amounts of microbial antigen or antigen-antibody complexes are circulating in the body. If a microbial antigen is very similar to normal host antigens, the immune response to this antigen may be weak or absent, giving a degree of tolerance. A large variety of microorganisms causes immunosuppression in the infected host, i.e., the host shows a depressed immune response to antigens unrelated to those of the infecting microorganism. Some of the other strategies include, latent phase of the microbes, microbial presence in bodily sites inaccessible to the immune response, induction of inappropriate antibody and T-cell responses, antibodies mopped up by soluble microbial antigens, and antigenic variations.

This chapter describes several methods through which the microbes spread in the host body. Direct spread occurs to organs and tissues below the body surfaces, extending the focus of infection and sometimes accounting for striking complications. Several microbial factors – such as some soluble substances and enzymes – help in the promotion of direct spread. The spread of the microbes can also occur through lymphatics blood and various other pathways. Microbes in tissue fluids enter lymphatic capillaries rather than blood capillaries, and are transported to the nearest lymph node. A rich lymphatic network lies below the epithelium in the nasopharynx, mouth, and lung; microorganisms traversing these epithelia enter cervical and pulmonary lymph nodes. The blood is the most effective vehicle of all for the spread of microbes through the body, particularly the blood tissue junctions. In small vessels – such as capillaries and sinusoids – where blood flows slowly, there is an opportunity for the microorganism to be arrested and to establish infection in neighboring tissues. Some of the other pathways of microbial spread include cerebrospinal fluid (CSF), pleural and peritoneal cavities, and peripheral nerves.

We have used a mouse model that utilizes the exclusively epithelial nature of human papillomavirus (HPV) infections to investigate the in vivo immune response to the E7 protein of human papillomavirus type-16. A keratinocyte cell line expressing E7 protein has been established and grafted onto syngeneic mice using a transplantation technique that permits the reformation of a differentiated epithelium on a granulation tissue bed. In this way viral antigens may be presented to the immune system in a way comparable to natural infection. A delayed-type hypersensitivity (DTH) response was studied post grafting by intradermal challenge with recombinant E7 protein. A significant response to E7 has been demonstrated in this way; however, priming with a low amount of HPV-16 E7 antigen induces immunological unresponsiveness, as measured by a loss of DTH reactivity to the protein, and persistence of keratinocytes expressing E7. Lymphocytes from mice exhibiting DTH reactivity have been shown to proliferate when stimulated with purified recombinant E7 protein in vitro, while immunoperoxidase staining of tissue from the sites of immunologically-induced inflammation has defined the cell infiltrate to be phenotypically characteristic of DTH. The observations reported here have important implications for vaccine strategy.

We have developed an animal. model which mimics a number of aspects of clinical. infection with HPV. In this system 107 keratinocytes expressing HPV 16 E7 protein are grafted onto syngeneic mice using a transplantation technique which permits the reformation of a differentiated epithelium. By this route viral. antigens may be presented to the immune system in a manner comparable to the natural. infection. Subsequent intradermal. challenge of the recipients at a site distant from the graft with an HPV-16 E7 recombinant vaccinia virus (vacc-E7) results in a delayed type hypersensitivity (DTH) response1.

Murine gammaherpesvirus is a natural pathogen of wild rodents. We have established that in vivo the virus persists in B lymphocytes in a latent form and therefore has similar biological properties to Epstein-Barr virus and related gamma-I-herpesviruses. In this report we have established a persistent infection in mouse myeloma (B) cells (NSO cell line), but not in mouse thymoma (T) cells (BW 5147 cell line). The virus persists indefinitely in myeloma cells, without any apparent cytopathic effect, but with the production of infectious virus. We demonstrate that ACV abolishes the productive infection, but large numbers of cells harbor the virus in a latent form, as determined by an infectious center assay. Analysis of the viral DNA has shown that during a persistent infection linear virus genomes predominated, with low levels of circular DNA also present. Treatment of cells with ACV results in a significant reduction of linear genomes, but has no effect on the level of circular DNA molecules. These data provide further evidence to support our earlier observations on B cells as the site of latency and provides an in vitro model with which to study the molecular basis of MHV-68 latency/persistence.

The herpesviruses are ubiquitous in nature, infecting fish, amphibians, reptiles, birds, and mammals. They are highly successful parasites, requiring only a small host range in which to maintain an infection: typically 102–103 individuals are sufficient to maintain a varicella-zoster virus (VZV) infection in the population, compared with >105 individuals for measles virus.1 The great success of the herpesviruses is attributed to a remarkable strategy for persisting within their host, termed latency. This strategy involves the virus persisting as genetic material, but without expressing any detectable viral proteins and thereby evading host immune defenses. Although the ability to establish a latent infection is a characteristic of all herpesviruses, we consider classical latency to be a property of those viruses involved with infections of the nervous system, notably herpes simplex virus (HSV) types 1 and 2 and VZV.

Congenitally athymic nude mice were infected with 10 4 p.f.u. herpes simplex type I (strain SC16). Following the passive transfer of neutralizing monoclonal antibodies (AP 7 , AP 8 and AP 12 ) it was observed that AP 7 alone reduced the virus infectivity in the nervous system; AP 8 and AP 12 failed to protect mice probably due to poor in vivo binding to the neutralization site on the virus. Latent ganglionic infection could be established in nude mice following adoptive transfer of optimum number (2 × 10 7 cells/mouse) of immune lymph node cells from day 7 herpes virus-infected hairy immunocompetent donor mice. Moreover, in some of the immune lymph node cell protected nudes, latency could be maintained even in complete absence of neutralizing antibodies. Results of ear-ablation experiments revealed that removal of primary source of infection after day 5 of infection reduced the amount of virus in the ganglia and spinal cord. Acute neurological infection was not detected following transfer of protective anti-gp-D neutralizing antibody (LP 2 ) in combination with removal of infected pinna. These data suggest that continuous seeding of virus occurs in related ganglia via the axonal route from infected ear pinna. It appears that local T-cell-mediated immune mechanisms are involved in maintenance of latency.

Peritoneal macrophages activated by-products derived from a herpes simplex virus-specific helper T cell clone were used to investigate intrinsic and extrinsic resistance mechanisms to herpes simplex virus type 1 infection in vitro. T cell-activated macrophages produced fewer infective centres, indicating enhanced intrinsic resistance, and markedly reduced the growth of virus in a permissive cell line. The reduction in virus growth correlated with the depletion of arginine in the support medium, presumably resulting from increased arginase production by activated macrophages. The significance of these findings for antiviral immunity in vivo is discussed.

Summary During the thirty-five year period from 1937 1973, 100 parathyroid tumors were found and pathologically proved at Memorial Hospital, New York. Ninety-one were found at surgery and nine at autopsy. There were eighty-seven functioning adenomas, one case of multiple adenomas or hyperplasia, and four parathyroid carcinomas. Hypercalcemia was documented in ninety-one patients and presented a serious diagnostic problem. One third of the patients had cancer either before, during, or after the discovery of hypercalcemia. Another third had a benign tumor coincidental with a parathyroid tumor. One third had the benign bone tumors and pathologic fractures of primary hyperparathyroidism itself. A survey of the same years at The New York Hospital revealed 180 cases of proved primary hyperparathyroidism wherein 10 per cent had cancer fortuitously. The sites of these tumors, multiplicity, histology, surgical management, and complications of treatment were analogous to the experience in the series at Memorial Hospital. The signs and symptoms of this disease have perceptibly changed over the years as diagnosis and detection have improved. The necessary laboratory studies have been progressively simplified and are more accurate. Surgical exploration is safe and necessary in early diagnosis and treatment.

A HSV-specific, Lyt 1+2− T cell clone (D7.1) was established by repetitive in vitro restimulation of in vivo HSV-1 primed draining lymph node cells with UV-inactivated homologous virus. Earlier work had shown that this clone could provide specific helper activity for HSV-1 primed B cells for antibody production in vivo. In this paper, further functional analysis has demonstrated that this clone and its subclones could also help unprimed T cells in the generation of effector DTH T cells in vitro. The helper activity was found to be antigen-specific and cell dose-dependent. In addition, this helper T cell clone showed little, if any, natural killer-like activity and was non-cytolytic towards both HSV-infected and uninfected syngeneic targets. However, they could exert potent cytotoxic activity towards P815 and EL-4 tumour targets in the presence of lectins.

Systemic treatment of mice with the nucleoside analog 9-(2-hydroxyethoxymethyl)guanine (acycloguanosine [aciclovir]) was found to be highly effective against acute type 1 herpes simplex virus infection of the pinna. The drug ablated clinical signs and reduced virus replication both in tissue local to the inoculation site and within the nervous system. Provided that moderate-sized virus inocula were used, acycloguanosine treatment reduced or prevented the establishment of a latent infection in the dorsal root ganglia relating to the sensory nerve supply of the ear. However, although it aborted artificially produced infections in dorsal root ganglia, acycloguanosine was found not to be effective against the latent infection once established. This finding strongly indicated that latent herpes simplex virus in mice can exist in a nonreplicating form.