Novel Approach to Herpesvirus Infections

Stop Herpes Now

You'll discover: What foods are bad for you, encouraging outbreaks. What foods are good for discouraging outbreaks. The connection between genital herpes and stress. What herbs actually suppress the herpes virus. How to heal your body naturally and safely. How to manage stress in your life.

Stop Herpes Now Summary


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Herpes Life Cycle Exam Short Notes

Undergoing Herpes Simplex Virus Transcriptional Activation in the Nervous System In Vivo Nancy M. 6 Reporter Cell Lines for the Detection of Herpes Simplex Viruses Szu-Hao Kung 73 9 Herpes Simplex Virus-Cell Interactions Studied by Low-Fading 10 Herpes Simplex Virus-Cell Interactions Studied by Immunogold 16 Identification and Characterization of Herpesviral 23 Molecular Genetics of Herpesviruses 25 Genetic Analysis of Cytomegalovirus by Shuttle Mutagenesis Manfred Lee and Fenyong herpesvirus 1 Using Infectious Clone Technology Timothy J. Mahony, Fiona M. McCarthy, Jennifer L. Gravel, 28 Design of a Herpes Simplex Virus Type 2 Long Processive DNA Synthesis of Herpesviruses There are several methods for detecting DNA viruses. One widespread method that has been used in several studies is nested polymerase chain reaction (PCR) analysis of viral DNA. Nested PCR amplification has been used to detect the Epstein-Barr virus latent membrane protein-1 (LMP-1) gene in clinical infections (1)....

Relationship to other herpesviruses

HHV-8 is a y2 herpesvirus (rhadinovirus) and is the first human representative of this group. Several animal rhadinoviruses are known and some of them, herpesvirus saimiri (HVS), alcephaline herpesvirus 1 (AHV-1) and ovine herpesvirus 2 (OHV-2), cause lymphoid malignancies. Among human herpesviruses, HHV-8 is most closely related to Epstein-Barr virus (EBV), a y, herpesvirus, which also causes lymphoid tumors in immunosuppressed individuals and may play a role in other malignant diseases (African Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma). While EBV-mediated cellular transformation has been studied in detail,

Varicella and Herpes Zoster

Varicella zoster virus (VZV) is a double-stranded DNA virus of the Herpesviridae family. Varicella (chickenpox) is the primary manifestation of VZV infection. Once VZV infection has occurred, the virus is permanently established in the dorsal root and trigeminal ganglia, persists in latent form, and recurs when reactivated as herpes zoster (shingles). More than 90 percent of humans become infected with VZV.774 In the United States, there are over 3.5 million cases of chickenpox and 300,000 cases of herpes zoster per year.7576 Chickenpox is highly contagious and poses a serious nosocomial and occupational infection risk. Varicella (chickenpox) transmission occurs by contact, droplet, airborne, and transplacental routes. 74 Transmission associated with herpes zoster (shingles) occurs with direct contact with vesicular lesions. Airborne transmission of herpes zoster may also occur, especially if the source patient is immunosuppressed. 7 78 VZV infections may be prevented or modified via...

Common Viral Infections Influenzaviruses And Herpesviruses

Herpesviruses Herpes, .Simplexviruses, i a,n.d. 2 Herpes, .Zoster , C.hic.k.enpo.x Herpes. .Zoster Shingles Epst.ei.n B.a.r.r V.i, ., . nfectious M.o.non.ucleosis Cytomegalovirus Chapter, References Viral illnesses are among the most common reasons that people come to an emergency department. Although some may be trivial, such as the common cold, others are life threatening, such as viral encephalitis, hemorrhagic fever viruses, and HIV, and still others are capable of causing chronic diseases and may contribute to the development of certain malignancies. Effective therapies for some viruses are now available, with more drugs and vaccines undergoing clinical trials. Influenzaviruses and the herpesvirus family are discussed in this chapter.

Herpes Zoster Shingles

Herpes zoster (shingles) is the reactivation of latent VZV infection. There is a lifetime incidence of almost 20 percent, with the majority of cases being among the elderly. It occurs only in people who have had chicken pox. After a single occurrence in an immunocompetent host, there is a 4 percent likelihood of a second occurrence. The lesions of shingles are identical to those of chickenpox, but are limited to a single dermatome in distribution. Thoracic and lumbar dermatomes are most common. The cranial nerves may be affected as well, with the potential complications of herpes zoster ophthalmicus (HZO) and Ramsay Hunt syndrome. What triggers the reactivation is unknown. The disease begins with a prodrome of pain in the affected area for 1 to 3 days, followed by the outbreak of a maculopapular rash that quickly progresses to a vesicular rash. The course of the disease is usually around 2 weeks, but may persist for a full month. CN VII can also be affected. Involvement of the...

Quantitative Taq Man Assay for the Detection and Monitoring of Cytomegalovirus Infection in Organ Transplant Patients

Quantitative polymerase chain reaction assays have become the most common methods in the determination of viral load during cytomegalovirus (CMV) infection of transplant patients. In recent years, the development of automated nucleic acid extraction devices together with the introduction of real-time technology have been important elements for improvements of these assays. Key Words Automated nucleic acid extraction cytomegalovirus (CMV) MagNA Pure LC organ transplant patient plasma quantitation real-time PCR TaqMan viral load. Over the past recent years, the knowledge of the clinical significance, diagnosis, and management of cytomegalovirus (CMV) infection and disease in transplant patients has increased considerably. Major advances have been achieved through the development of new diagnostic techniques for the detection of the virus and through the use of antiviral agents. Most centers have protocols for the frequent monitoring of transplant recipients for CMV and 1. Human...

Herpes Zoster Ophthalmicus

Herpes zoster ophthalmicus (HZO) represents shingles in the trigeminal nerve distribution with ocular involvement. When the cutaneous lesions include the tip of the nose (Hutchinson sign), the nasociliary nerve is involved and the eye frequently becomes inflamed. An iritis can occur with photophobia and pain. Cutaneous lesions and conjunctival involvement is treated with erythromycin ointment to prevent secondary bacterial infection. The cornea can have a pseudodendrite, which is a poorly staining mucous plaque with no epithelial erosion (unlike HSV, which has a true dendrite with epithelial erosion and staining). The anterior chamber on slit-lamp examination can show manifestations of iritis (cell and flare). Iritis can be treated with topical steroids prednisolone acetate 1 (Pred Forte), one drop four to five times a day, and pain reduction can be achieved with topical cycloplegic agents (scopolamine 0.25 one drop tid or cyclopentolate 1 one drop tid). If HZO is diagnosed, admission...

Infection by herpes simplex virus HSV

There are two related subtypes of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), now called human herpesviruses 1 and 2 respectively. Both are large, double-stranded DNA viruses (ca. 150 kb) The first problem for the host in defending itself against HSV is the site of infection. The skin and nervous system have been called 'immune privileged' sites and certainly the immune surveillance occurring in these tissues is not as great as in highly vascularized tissues such as the liver. To compound this problem, especially in recrudescent lesions, the virus is restricted to the epidermal layer until a well-established infection results in enough cell death to cause inflammation in the dermal layer. The second problem for the host is that once the virus has established a latent infection, no detectable viral products are expressed and so there is apparently no way for the host's immune system to attack latently infected cells. However, the immune system does have an important part to...

Herpes Simplex and Eczema Herpeticum

Penicillin Wright Stain

The vast majority of HSV type 1 or type 2 infections involve only localized areas of skin or mucous membranes. In neonates, and adults with atopic dermatitis, malignancy, immunosuppression, and the acquired immunodeficiency syndrome (AIDS), the HSV infection may disseminate, resulting in widespread vesicles, pustules, and ulcerations, and causing multisystem involvement. The neonate may acquire the infection either in utero due to maternal infection during pregnancy or during delivery. Adults undergoing chemotherapy or transplant recipients who are HSV seropositive may reactivate HSV and develop disseminated disease. Patients with atopic dermatitis are also at risk of disseminated disease, which is called eczema herpeticum.17 FIG. 241-7 (P at.e 3.3). Disseminated HSV infection in an immunocompromised adult patient note the widespread distribution of vesicular lesions on an erythematous base. Eczema herpeticum is an association of two common conditions atopic dermatitis and HSV...

Herpes Simplex Resistance History

Acyclovir (ACV) is the gold standard for the treatment of HSV infections. This drug is a nucleoside analogue of guanosine that has to be phosphorylated three times. The first phosphorylation is achieved by the thymidine kinase (TK) encoded by the virus this step is important as it allows ACV to become active only in infected cells. The second and third phosphorylations are carried out by cellular thymidylate kinases. Acyclovir triphosphate is a competitive inhibitor of viral DNA polymerase and is a DNA chain terminator. 1 Herpes simplex virus strains resistant to ACV have been reported since 1982. 2 They were most often recovered from immunocompromised patients previously treated with ACV. 3 Herpes simplex virus resistance to ACV may be associated with a mutation in one of the two viral enzymes involved in ACV mechanism of action TK and or DNA polymerase. Mutations occurring in the viral gene encoding TK are the most frequent and 95 of ACV-resistant isolates present a TK-deficient...

Herpes Simplex Virus Infections

Herpes simplex virus (HSV) type I most commonly occurs on the face. Initial infection occurs during childhood or adolescence and varies in its presentation. Many individuals experience mild symptoms while a few experience a debilitating eruption. Recurrences tend to be mild and occur primarily on the lips, in the nose, and in the oral cavity. Recurrent HSV is typically seen as herpes labialis (fever blisters or cold sores). The individual often experiences a prodrome of localized tingling or burning several hours before the onset of the eruption. The herpetic lesion usually occurs along the lip margin and completely heals within 10 days. Ultraviolet light, fever, or local trauma can induce these eruptions. The diagnosis is established in the same manner as that for herpes zoster with a positive Tzanck preparation and viral culture. Treatment for primary HSV gingivostomatitis includes symptomatic treatment, as mentioned previously for herpes zoster infections, including compresses and...

Herpes simplex viruses type 1 HSV1 and type 2 HSV2

There are several features which distinguish herpes viruses from other viruses. The genome is a linear double-stranded DNA ranging from 125 000 to 250 000 base pairs. The viron is composed of the DNA-containing core an icosahedral capsid, which is about 100 nm in diameter and exhibits 162 morphological units (capsomeres) on its surface and an outer lipid-containing membrane or envelope. These viruses use both the nucleus and the cytoplasm of the host cell for their replication. The pathology and epidemiology of herpes virus infections depend on the mode of viral replication, associated cytotoxicity as well as their capacity to cause latent infections. Clinically many of the infections caused by HSV-1 and HSV-2 are indistinguishable. Despite the fact that these strains are predominantly isolated from different anatomical sites, it is clear that strains of either type can initiate and establish infection at either genital or extragenital sites. HSV rarely causes severe disseminated...

TABLE 1045 Treatment of Genital Herpes

The safety of acyclovir and valacyclovir during pregnancy has not been established. In pregnant patients with life-threatening disease, such as encephalitis, pneumonitis, or hepatitis, intravenous acyclovir should be used. It should not be used for recurrent episodes or as suppressive therapy. Pregnant women treated with the drug should be reported to the Glaxo-Wellcome registry, which is kept in cooperation with the CDC (1-800-722-9292, extension 38465). Current registry findings do not indicate an increased risk for major birth defects after acyclovir treatment.2,3., 2


The herpesviruses are a ubiquitous class of enveloped DNA viruses that cause an expanding list of human illness. The herpesviruses all have the ability to dwell in the host as a lifelong latent infection and may cause clinical disease or recurrent disease at a time distant from the primary infection. Some have been shown to be carcinogenic. Each herpesvirus has distinguishing clinical characteristics and will be discussed individually. Human herpesviruses 6 and 7, both which cause roseola, and human herpesvirus 8, implicated in Kaposi sarcoma, are not discussed in this chapter. As a class, the herpesviruses are transmitted by close contact, since they are unable to survive in the environment and are unable to penetrate intact skin. The varicella zoster virus (VZV) can be spread via aerosolized particles as well as by close contact. Most transmission of the herpes simplex virus (HSV) and of Epstein-Barr virus (EBV) occurs during asymptomatic shedding. Viruses discussed below are HSV,...

Other herpesviruses

Cytomegalovirus and Epstein-Barr virus can cause acute encephalitis syndromes.77 Varicella-zoster virus (VZV) infection may also be complicated by encephalitis, which usually develops a week after the exanthem begins. Acute cerebellar ataxia is the most common complication of chickenpox.57,61 An eruption of herpes zoster may be complicated by encephalomyelitis and granulomatous arteritis, the latter of which has been associated with zoster ophthalmicus.57

Genital Herpes

Genital herpes is a sexually transmitted infection caused by a DNA-containing virus specific to human beings. Sexual transmission can occur during asymptomatic periods. Usually, genital herpes is the most frequently encountered of the diseases causing genital ulcers and is thought to be associated with an increased risk for HIV infection.3 There are two antigenic groups herpes simplex virus 1 (HSV-1) and herpes simplex virus 2 (HSV-2). Initially, HSV-1 caused oral lesions and HSV-2 genital lesions, but this is no longer the case. Overall, 85 to 90 percent of genital herpes infections are caused by HSV-2, but some studies have found up to 30 percent are caused by HSV-1. Symptomatic genital herpes is the most frequent cause of painful lesions of the lower genital tract in American women. Neonatal Approximately 25 percent of initial presentations occur in women with preexisting antibody to herpes simplex virus. The initial episodes tend to be less severe and resemble recurrent...

Herpes Simplex

Herpes simplex type 1 most commonly affects the oral cavity. Herpes simplex type 2 can occur orally and will be discussed later. The primary infection, herpes gingivostomatitis, causes acute painful ulcerations on the gingiva and mucosal surfaces. Fever and lymphadenopathy are commonly associated findings and may occur up to 3 days prior to the appearance of oral lesions. Vesicular lesions appear and rupture after 1 to 2 days, leaving painful ulcers that heal gradually over 1 to 2 weeks. Secondary infection affects mostly the lips but may affect the hard palate and attached gingiva. By adulthood, most of the population has been exposed to herpes simplex type 1. The virus is harbored in sensory ganglion such as the gasserian ganglion of the trigeminal nerve. Periodic stresses activate the virus from its normally dormant state and allow for infection along the sensory distribution of the affected nerve. A prodrome of burning or tingling frequently occurs 1 to 2 days preceding outbreak...


Cytomegalovirus (CMV, or human herpesvirus 5) is another ubiquitous virus with worldwide distribution. It is, like the other members of the herpesvirus family, capable of causing a primary illness and then existing in a latent state in the human host indefinitely with the ability to reactivate at a later time. It is present in approximately 1 CMV is one of the TORCH agents toxoplasmosis, other (viruses), rubella, CMV, and herpes (simplex viruses) and is capable of causing intrauterine infection. Fewer than 25 percent of neonates with intrauterine CMV will display symptoms. Those at highest risk are those whose mother acquires primary disease during the first half of the pregnancy. A seropositive mother's antibodies to CMV appear to provide the fetus some protection. Classic intrauterine CMV (congenital cytomegalic inclusion disease) involves multiple organs, including jaundice, hepatosplenomegaly, microcephaly, petechiae, and inner ear problems, as well as CNS defects. Children who...

Herpetic Whitlow

Herpetic whitlow is a viral infection of the distal finger caused by the herpes simplex virus. Infection typically occurs when the dermis has come in contact with oral herpetic infections. Herpetic whitlow in children tends to be associated with gingivostomatitis and herpes simplex type 1 (HSV-1), whereas adults most commonly harbor herpes simplex type 2 (HSV-2).13 Health care professionals historically have been at risk for this infection, although the incidence appears to have diminished with the advent of latex gloves. The patient will present with a burning, pruritic sensation similar to all herpes simplex infections. On examination, the lesion is erythematous and tender, with vesicular bullae being the hallmark of herpetic whitlow ( Fig . 277-6). Although indurated, the infection is not tense, as is seen in a felon. It is important that a herpetic whitlow not be misidentified as a felon because incision and drainage may result in increased morbidity and prolonged failure to heal....

Herpes Zoster

Zoster Nose

Varicella zoster infections (VZI), also referred to as shingles or zoster, represent reactivation of the previously dormant virus, Herpesvirus varicellae, in a patient with an altered immune response. At reactivation, the virus travels down specific sensory nerves to the skin resulting in the skin manifestations of shingles. Patients with lymphoma, leukemia, or diabetes mellitus, and who are immunocompromised, are at risk for reactivated or disseminated infection.15 The rash of herpes zoster consists of clusters of vesicles and papules grouped on an erythematous base. Vesicles initially appear clear but become cloudy or FIG. 241-5 (Plate.31). A single dermatomal involvement in a patient with herpes zoster infection in a thoracic distribution. FIG. 241-6 (Plate.32). Herpes zoster ophthalmic infection Facial zoster involving the ophthalmic branch of the fifth cranial nerve with nasal lesions strongly suggestive of corneal infection. Approximately one-half of cases with VZI particularly...

Reporter Cell Based Antiviral Susceptibility Testing

To ensure consistency of experiments, the stable transfected cells should be assessed for their responsiveness to HSV infection from time to time (say every 3 mo). If the stable clones lose their sensitivity to viral infection, performing limiting dilution is recommended to gain an appropriate clone. 1. Whitley, R. J. and Roizman, B. (2002) Herpes simplex viruses, in Clinical Virology, 2nd ed. (Richman, D. D., Whitley, R. J., and Hayden F. G., eds.), ASM, Washington, DC, pp. 375-401. 4 Goldstein, D. J. and Weller, S. K. (1988) Herpes simplex virus type-1 induced ribonucleotide reductase activity is dispensable for virus growth and DNA synthesis isolation and characterization of an ICP6 lacZ insertion mutant. J. Virol. 62,196-205. 5 Wymer, J. P., Chung, T. D., Chang, Y. N., Hayward, G. S., and Aurelian, L. (1989) Identification of immediate-early-type cis-response elements in the promoter for the ribonucleotide reductase large subunit from herpes simplex type 2. J. Virol. 63,...

Helle Lone Jensen and Bodil Norrild

A technique is presented for high-resolution postembedding immunolocalization of one or two (or several) antigens in the same ultrathin cryosection using primary monoclonal antibodies from the same species. The optimized three-layer indirect immunogold-labeled cryosection electron microscopy described is recommended for studies of virus-cell interactions, because (1) it is a simple and reproducible method (2) colloidal gold markers are electron-dense, stable, and easy to recognize (3) the membraneous ultrastructure and immunolabeling are well preserved (4) immunolabeling is less in the two-layer method (5) silver-enhanced gold particles vary in size and shape (6) it is possible to demonstrate herpes simplex virus type 1 glycoproteins gC-1 and gD-1 in the nuclear membranes and gC-1- and gD-1-labeled viral particles in the perinuclear space and to observe virions in the endoplasmic reticulum and Golgi area. The use of buffered 3 paraformaldehyde plus 2 glutaraldehyde for 2 h at room...

Vitaly Erukhimovitch Marina Talyshinsky Yelena Souprun and Mahmoud Huleihel

Fourier-transform infrared (FTIR) microscopy is considered a comprehensive and sensitive method for detection of molecular changes in cells. The advantage of FTIR microspectroscopy over conventional FTIR spectroscopy is that it facilitates inspection of restricted regions of a cell culture or a tissue. We have shown that it is possible to apply FTIR microscopy as a sensitive and effective assay for the detection of cells infected with various members of the herpes family of viruses and retroviruses. Detectable and significant spectral differences between normal and infected cells were evident at early stages of the infection. Impressive changes in several spec-troscopic parameters were seen in infected compared with uninfected cells. It seems that the change in spectral behavior is specific to the infecting virus, because cells infected with her-pesviruses showed different spectral changes compared with cells infected with retoviruses. Key Words Viruses FTIR microscopy malignant cells...

FTIR Spectra Analysis

This peak is attributed to N-type sugars. Our results showed a gradual shift in the position of this peak from 861 cm-1 in normal samples to about 854 cm-1 in correspondence with the development of herpes viruses infection (29). For retrovirus-transformed cells, there was no shift in this peak. Our data showed a gradual disappearance of the peak at 1023 cm-1 over time with the development of herpesviruses infection (29). This spectral peak can be attributed to carbohydrates, as was previously reported (31). There was also a notable decrease in this peak in cells infected with retroviruses (Fig. 1). This peak is attributed to PO2 symmetric stretching vibration. A significant and detectable shift of the peak at 1080-1081 cm-1 for normal cells to 1086-1087 cm for retrovirus-transformed cells was observed (23), whereas there was no shift in this peak in cells infected with herpesviruses. The peaks in the region 1200-1400 cm-1 represent PO2- asymmetric stretching vibrations. Our results...

Analysis of Fusion Using a Virus Free Cell Fusion Assay Marisa P McShane and Richard Longnecker

For enveloped viruses, such as viruses within the herpesvirus family, of which Epstein-Barr virus (EBV) is a member, infection of target cells includes two distinct steps. The first is characterized by the binding of viral envelope glycoproteins to host cellular receptors. After binding, the viral membrane and the cellular membrane fuse. Without both binding and fusion, the virus is not able to enter the host target cell efficiently. Combined with the specific tropism of EBV for primarily two cell types, B lymphocytes and epithelial cells, and the difficulty in inducing lytic replication of EBV in vitro, there is a lack of a good experimental model to study EBV-induced viral fusion. To study fusion more efficiently and effectively, we have employed a virus-free cell-cell fusion assay. In the effector cell, the viral glycoproteins and a plasmid containing the T7 promoter, driving the luciferase gene, are expressed. In the target cell type, T7 RNA polymerase is transfected. Fusion is...

Transient Replication and Plasmid Maintenance Assays in Mammalian Cells

PcDNA3 (Fig. 1) is a 5.4-kb vector that contains a bacterial origin of replication (colEl) and the ampicillin resistance marker, allowing its propagation in bacteria it also contains the SV40 origin of replication, allowing its propagation in mammalian cells expressing the SV40 T antigen. The plasmid expresses the neomycin resistance gene, which allows selection of cells containing the plasmid by growth in the antibiotic G418 (see Note 1). It also contains the mammalian cytomegalovirus (CMV) promoter, with a downstream multicloning site, allowing cloning expression of a gene of interest in mammalian cells.

Gary D Luker and David A Leib

Herpes simplex virus 1 (HSV-1) is a common and significant neurotropic human pathogen that infects 80 of all persons by adulthood. During acute HSV-1 infection, virus replicates peripherally in epithelia, enters axonal terminals, and is transported retrogradely to sensory nerve ganglia, where HSV-1 may establish latency or progress to life-threatening infection of the central nervous system. Studies of viral and host factors that influence pathogenesis have largely used experimental mouse models that rely on sacrifice of infected mice to determine distribution and titer of virus. Although this experimental paradigm has provided important data, it precludes real-time investigations of the same animal over the entire course of disease progression. This limits potentially significant insights from animal-to-animal variations in host-pathogen relationships. Unexpected sites of infection also may be missed because appropriate tissues are not analyzed for virus. To improve investigations of...

Culturing Primary and Transformed Neuronal Cells for Studying Pseudorabies Virus Infection

This chapter discusses the culture of primary sympathetic neurons (superior cervical ganglia) from rat embryos and PC12 cells differentiated into neurons for use in viral infection experiments. Methods are described for the use of a neurotropic herpesvirus, pseudorabies virus (PRV), to analyze the assembly, egress, and transport of viral antigens in neurons. Key Words Cultured neuron herpesvirus immunofluorescence infection laminin nerve growth factor neuron ornithine PC12 pheochromocytoma pseudorabies virus superior cervical ganglia sympathetic neurons. This chapter focuses on the culture of primary sympathetic neurons (superior cervical ganglia) from rat embryos and PC12 cells differentiated into neurons for use in viral infection experiments. Although methods for culturing these types of neuronal cells are well established (1,2), it is often necessary to modify the cell culture method to suit the requirements of a particular experiment. We use these neuronal cultures to understand...

Recombinant Virus Technology

Key Words Herpesviruses Epstein-Barr virus viral recombinants genetic analysis viral mutants. and ligation (1). With increasing size, virus genomes become more difficult to manipulate and propagate. Herpesviruses, for example, possess large genomes (up to 200 kb) that are not amenable to standard cloning techniques, and modification of these genomes relies on homologous recombination. Classically, homologous recombination was performed in eukaryotic cells carrying the virus to be modified (2-4). A major difficulty of this kind of approach is that the mutant virus coexists with the wild-type virus in the infected cell and therefore must first be purified. More recently, single-copy F-plasmid replicons (also known as bacterial artificial chromosomes BACs ) were used to clone up to 300 kb of genomic DNA (5). Introduction of these replicons into large viral DNAs has allowed cloning and manipulation of these genomes in the context of a prokaryotic host (6-10). Genetic analysis using...

Manfred Lee and Fenyong

The genomes of Herpesviridae family members are among the largest of all viruses and therefore present a formidable challenge in understanding the roles of every gene in replication or pathogenesis. For example, murine cytomegalovirus (MCMV) has a genome of 230 kb that encodes more than 170 genes, many of which have unknown functions. Many techniques for the genetic analysis of a herpesvirus have been developed over the past two decades. One such procedure involves the use of a shuttle mutagenesis system, and it has successfully generated a pool of MCMV mutants that contained an engineered Tn3-type transposon inserted within their genome. The process of shuttle mutagenesis involves the construction of a genomic fragment library, transposon mutagenesis of the library, and generation of virus mutants through homologous recombination. This chapter details the methodologies required for implementing a Tn3-based shuttle mutagenesis system for construction of a mutant virus library. Key...

Timothy J Mahony Fiona M McCarthy Jennifer L Gravel and Peter L Young

The application of infectious clone technology to herpesvirus biology has revolutionized the study of these viruses. Previously the ability to manipulate these large DNA viruses was limited to methods dependent on homologous recombination in mammalian cells. However, the construction of herpesvirus infectious clones using bacterial artificial chromosome vectors has permitted the application of powerful bacterial genetics for the manipulation of these viruses. A method is described for the construction and characterization of a gene inactivation library of Bovine herpesvirus 1 using an infectious clone. The method utilizes transposon-mediated gene inactivation, which permits gene inactivation without any prior knowledge of the viral genomic sequence. Furthermore, as the genetic manipulation is performed in bacteria the inactivation of those viral genes that are essential for viral replication is also possible. The method described here can be readily applied to any herpesvirus clone...

J S Aguilar Peter Ghazal and Edward K Wagner

The design and construction of a long (75-mer) oligonucleotide-based DNA microarray for herpes simplex virus type 2 transcripts is described. This array is utilized to generate an analysis of HSV-2 transcript abundance as a function of conditions of infection of human cells, and global patterns of HSV-2 transcript abundance are compared with those for HSV-1. General similarities in patterns along with notable differences in specific details are noted. These results reveal a marked conservation in the program of gene activity between phenotypically diverged strains. Key Words Herpes simplex virus type 2 human herpesvirus 2 oligonucleotide-based DNA microarrays kinetics of transcript abundance DNA replication inhibitor global profiling of viral gene expression.

HSV2 Compared With HSV1

Full DNA sequences are available for the two closely related herpes simplex viruses of humans, HSV-1 and HSV-2 (HHV1 and HHV2). HSV-1 is normally associated with primary infection of the lip, with latency being established in the trigeminal nerve ganglia, whereas HSV-2 is associated with genital infections and latency in the sciatic nerve ganglia (1). In humans both can be transmitted sexually, although this mode is generally associated with HSV-2. The initial infection normally takes place in epithelial tissue, from which virus can reach the innervating sensory neurons. Here, they can become latent, with their genomes forming an episome in the nuclei of the neurons. In this state, only the Although both viruses are medically important, the vast bulk of basic research on the basic virology and molecular biology of HSV including patterns of viral gene expression during replication and latency has been carried out with HSV-1 (2-4). Structurally, herpes virions consist of a...

Differences Between HSV2 and HSV1

Despite their high degree of genomic identity (> 80 ) (7,8), their significantly different pathology in humans is reflected in differences in the behavior of the virus in animal systems (9,10). An important problem in herpes virology is to understand the molecular basis leading to the different pathologies between HSV-1 and HSV-2. Differences in viral functions as well as in the cellular functions affected by each type of virus could explain these different pathologies. Thus, some viral functions are different in HSV-1 and HSV-2. For instance, whereas in HSV-1 glycoprotein C is the major viral function responsible for virus attachment to cells and glycoprotein B mediates penetration, in HSV-2 glycoprotein B is the major protein involved in both binding and penetration (11). Also, the viral host shutoff activity is much stronger in HSV-2 than in HSV-1 (12). Furthermore, the protein kinase activity associated with ribonucleotide reductase 1 has different ATP requirements in HSV-1 and...

Grouping HSV2 Transcripts Into Kinetic Classes

The ability to group HSV-1 transcripts into immediate-early, early, and late classes is readily accomplished by examination of those transcripts abundantly expressed in the absence of de novo protein synthesis, such as during a cycloheximide blockage during infection, and those abundantly expressed in the absence of viral DNA replication. This latter condition is best accomplished by analysis of RNA levels expressed following infection with tight mutant of one of the genes essential for viral DNA replication at several times, compared with control levels with a wt infection. We have carried out some careful control experiments using the HSV-1 chip to show that generally equivalent results can be attained using a DNA synthesis inhibitor, such as PAA or acyclovir.

James Papin Wolfgang Vahrson Rebecca Hines Boykin and Dirk P Dittmer

Whole-genome profiling using DNA arrays has led to tremendous advances in our understanding of cell biology. It has had similar success when applied to large viral genomes, such as the herpesviruses. Unfortunately, most DNA arrays still require specialized and expensive resources and, generally, large amounts of input RNA. An alternative approach is to query entire viral genomes using real-time quantitative PCR. We have designed such PCR-based arrays for every open reading frame of human herpesvirus 8 and describe here the general design criteria, validation procedures, and detailed application to quantify viral mRNAs. This should provide a useful resource either for whole-genome arrays or just to measure transcription of any one particular mRNA of interest. Because these arrays are RT-PCR-based, they are inherently more sensitive and robust than current hybridization-based approaches and are ideally suited to query viral gene expression in models of pathogenesis. Key Words Real-time...

Induction of Kshv Ie Gene Expression

Theoretically, herpesvirus IE genes can be selectively induced in the presence of inhibition of protein synthesis. Since protein synthesis inhibitors, such as cycloheximide, are in general toxic to host cells and cause apoptosis in these cells, a range of cycloheximide concentrations has to be determined in which protein synthesis is completely inhibited but cell viability rate remains high at least for 8-12 h.

Robert P Ricciardi Kai Lin Xulin Chen Dorjbal Dorjsuren Robert Shoemaker and Shizuko

Processivity factors associate with DNA polymerases, enabling them to incorporate thousands of nucleotides without dissociating from the template. The processivity factors encoded by each of the herpesviruses are ideal targets for specifically blocking viral replication, particularly since they have unique primary amino acid sequences. Here we provide details of a rapid mechanistic plate assay and its potential application to high-throughput screening of libraries of tens of thousands of chemical compounds to identify inhibitors of processive DNA synthesis. Methods of validation testing are presented. Key Words Processivity factor DNA polymerase PF-8 Pol-8 KSHV herpesviruses inhibitor screening mechanistic plate assay high-throughput screening chemical combinatorial library.

Recombinant Technology Approach

Transposon Plasmid Coli

Herpesvirus genetics have long been hindered by the large size of the typical herpesvirus genome and the consequent recalcitrance of these genomes to manipulation by standard molecular genetics techniques. However, two primary strategies have emerged that allow for the generation of targeted viral mutants. With these mutants, investigators can pursue critical questions regarding the relationship between specific viral genetic elements and the viral life cycle. The first strategy of viral genome manipulation utilizes the mammalian homologous recombination machinery to introduce specific changes into the native viral genome. This approach involves construction of a targeting vector containing both the desired mutation and a significant flanking viral sequence to permit efficient recombination. The targeting vector is then introduced into mammalian cells, along with viral DNA, and recombinant virus is subsequently selected, harvested, and purified. The second, and more recent, approach...

Modification of the Rapid Mechanistic Plate Assay for HTS

The original rapid mechanistic plate assay for KSHV Pol-8 PF-8 DNA synthesis assay (9) described above (Subheadings 3.1.-3.9.) can be modified for HTS. The assay is modified so that known herpesvirus polymerase inhibitors cause at least a 50 reduction in DNA synthesis at micromolar concentrations. Lowering the compound test dose to the micromolar range is one of our critical goals for optimization, to avoid selecting promiscuous inhibitors of KSHV Pol-8 PF-8, as it has previously been cautioned that higher doses of test compounds commonly result in many nonspecific screening hits from molecular target-based in vitro HTS assays (13).

Szu Hao Kung

Virus culture has played significant roles in basic and clinical virology, with a number of advantages that cannot be attainable by modern molecular techniques. However, virus culture is generally a slower process, as it inevitably takes the period of a full replication cycle of a given virus. A genetically modified cell culture with a virus-inducible marker is described here, using a frequently isolated DNA virus (herpes simplex virus) as a model. The assay system relies on expression of the reporter gene driven by a specific viral promoter that is triggered early in the course of viral infection. The reporter gene employed was green fluorescent protein (GFP) or secreted alkaline phosphatase (SEAP), whose assays offer real-time detection or quantification, respectively. This cell-based assay is simple, rapid, sensitive, specific, and quantitative and serves as a phenotypic method for determination of antiviral susceptibilities. Key Words Herpes simplex virus reporter cell line...


To establish a reporter cell line that generates the EGFP or SEAP reporter in response to HSV infection, several steps are followed. The HSV-2 ICP10 promoter is isolated by PCR amplification and cloned into the reporter gene-harboring plasmid. Stable transfection with the recombinant plasmid into Vero cells is carried out, and appropriate clones are selected following HSV infection those with the least background expression and the highest inducible expression are chosen. These clones are evaluated for EGFP or SEAP production by fluorescence microscopy or chemoluminescent assay, respectively (see Note 1).


Turnip yellow mosaic virus (TYMV), cucumber mosaic virus (CMV), herpes simplex virus-1 (HSV-1), and intracellular mature vaccinia (IMV) virus were isolated and purified according to procedures described in detail elsewhere (5-8). AFM imaging of intact virions under physiological conditions was conducted in appropriate buffer solutions, which were normally those utilized for resuspension of virus during late stages of purification. Detergents and enzymes typically used for disassembly of virions were utilized in the AFM dissection experiments.

Nancy M Sawtell

Herpes simplex virus (HSV), in contrast to most other members of the herpes virus family, has the ability to infect, enter latency, and reactivate from latency in a number of nonhuman species, including mice. This provides a unique opportunity to study the complex lytic-latent cycle of a human neurotropic virus in a mouse model. This chapter details basic methods for inducing and quantifying reactivation, with emphasis on the first strategy for detecting and quantifying the initiation of HSV reactivation in vivo. Key Words Herpes simplex virus latent infection persistent infection viral reactivation hyperthermic stress initiation of reactivation whole tissue in situ detection viral latency immunohistochemistry.

Yan Yuan

Immediate-early (IE) genes are the first class of viral genes expressed after primary infection or reactivation. As transcription of IE genes does not require prior viral protein synthesis, this class of genes is experimentally defined by their transcription following primary infection or reactivation in the presence of inhibitors of protein synthesis. This chapter describes an approach to identify IE genes in a novel herpesvirus genome. Transcription of IE genes is selectively induced with sodium butyrate in the presence of the protein synthesis inhibitor cycloheximide. The transcripts of the induced genes are identified by using a cDNA subtraction-based method of gene expression screening. Key Words Immediate-early transcripts cDNA subtraction KSHV y-herpesvirus reactivation.

Validation Testing

Which reveals the sizes of the DNA products resulting from processive DNA synthesis on agarose gels. The details of the M13 gel assay performed with herpesvirus polymerases and their cognate processivity factors are presented here, followed by an illustration of the assay using candidate inhibitors from the rapid mechanistic plate assay. Fig. 2. Use of the M13 gel assay to validate putative DNA synthesis inhibitors identified by the rapid mechanistic plate assay. M13 single-stranded DNA with an annealed oligonucleotide primer was incubated with the premix solution containing in vitro translated Pol-8 and PF-8 and 32P dCTP for radiolabeling. The synthesized DNA products were fractionated on a 1.3 alkaline agarose gel and then analyzed by autoradiography. The control reaction (buffer, no compound) generated DNA strands corresponding to the full-length (FL) template (7249 nt) as well as characteristic shorter products (SP) that were several hundred nts in length. Numbers above each lane...

Cluster Analysis

FTIR spectra in the region of 1000-1050 cm-1 of (A) noninfected Vero cells and cells infected with 1 MOI of HSV-1 and (B) noninfected NIH 3T3 cells and cells infected with 1 FFU cell of MuSV. Results are means of five different and separate experiments for each cell culture. The SD for these means was < 0.001. FFU, focus-forming unit HSV, herpes simplex virus MuSV, murine sarcoma virus AU, asymmetric unit. Fig. 1. FTIR spectra in the region of 1000-1050 cm-1 of (A) noninfected Vero cells and cells infected with 1 MOI of HSV-1 and (B) noninfected NIH 3T3 cells and cells infected with 1 FFU cell of MuSV. Results are means of five different and separate experiments for each cell culture. The SD for these means was < 0.001. FFU, focus-forming unit HSV, herpes simplex virus MuSV, murine sarcoma virus AU, asymmetric unit. Fig. 2. FTIR spectra in the region of 1200-1400 cm-1 of (A) noninfected Vero cells and cells infected with 1 MOI of HSV-1 and (B) noninfected NIH 3T3 cells and...

Adenoassociated Virus

Adeno-associated virus is a small, DNA-containing parvovirus that has been isolated from humans and other animal species. 1-4 Several serotypes of AAV have been isolated from humans and nonhuman primates. 1,4 Adeno-associated virus is defective and replicates only in host cell nuclei when certain functions are provided by a coinfecting helper adenovirus (Ad) or herpesvirus.1-1,2-1 The mechanism of the helper function is not clearly defined but only a limited set of adenovirus genes, E1, E2A, E4, and the VA RNA are required. 1 Adeno-associated virus has a broad host range but the efficiency of transduction with AAV vectors of different serotypes varies because of differences in cell receptors utilized for entry, cellular trafficking of AAV particles, and uncoating of the viral genome in the cell nucleus. For AAV to replicate, or to function as a gene delivery vehicle, the uncoated single-stranded DNA genome must be converted to a double-stranded molecule to permit transcription and...

Design and Production of AAV Vectors

In DNA transfection-based systems, human 293 cells are transfected with DNA plasmids containing the AAV vector cassette, the rep and cap genes, and the adenovirus E2A, E4, and VA genes. Alternatively, stable producer cell lines that contain the rep and cap complementing genes and the vector genome are infected with Ad. Producer cell lines are readily scalable AAV, but a new producer cell line must be generated for each individual AAV vector. A third approach uses a packaging cell line containing a rep-cap gene cassette that is then coinfected with an Ad AAV hybrid virus, which is an E1 gene-deleted Ad containing the AAV-ITR vector cassette, and Ad to provide E1. After infection, the rep-cap genes, the AAV-ITR cassette is amplified and packaged into AAV particles. The same packaging cell line can be used for production of different AAV vectors simply by changing the Ad AAV hybrid virus. Variations of these methods use herpes simplex virus (HSV) in the production of AAV vectors by...

Preface to the second edition

Events have moved fast since the first edition of this book. In the United States the Office of Scientific Integrity has been replaced by the Office of Research Integrity (ORI), whose newsletters and annual reports show that the system is now dealing with many cases expeditiously and with due process - while seminars and conferences on the topic have been described as a growth industry. In particular, one fear that the courts may assume a more important role has been realised by a successful qui tam suit in May 1995 (though the decision is now under appeal). Feeling that she would get nowhere through the official channels, Pamela Berge, a former Cornell epidemiologist, filed a suit under the False Claims Act alleging that the University of Alabama, Birmingham, had made false claims in its NIH grant application and had used her work on cytomegalovirus. The jury in the Federal District Court found for Berge, stating that the false claims amounted to 550 000 by statute (the article in...

Vaccination of kittens

In most diseases of cats, MDA declines to non-interfering levels in the majority of animals by 9-12 weeks of age. The standard protocol is therefore to vaccinate kittens at 8 or 9 weeks and then repeat in 3-4 weeks' time. However, the duration of MDA may be variable between different litters of kittens and even between kittens in a litter, depending on the antibody levels in the queen and the intake of colostrum. MDA against feline herpesvirus has been shown to last for 2-10 weeks, and against feline calicivirus for up to 10-14 weeks (Chapter 22). For feline panleucopenia the majority of kittens born to queens with moderate titres will lose their MDA by 8-12 weeks, but for those born to queens with high titres, MDA may persist for 16 weeks or more (Chapters 14 and 21). Serology for antibody level estimation can be carried out where

Vaccination of adutt cats

In previously unvaccinated cats, or animals of unknown vaccination history, a primary vaccination course of two doses 3-4 weeks apart should be undertaken. In cats vaccinated as kittens, the first booster at 1 year of age is important in case the first vaccination course was ineffective (e.g. if MDA was still at interfering levels). The onset of immunity is 7-10 days after systemic vaccination and 2-4 days after an intranasal vaccine. Although annual boosters are generally recommended for all injectable vaccines, there is some evidence that for panleucopenia the duration of immunity may be longer, up to several years (Gaskell et al, 2002a, b). However, clearly a number of variables may influence this, including the potency of the actual product. For feline herpesvirus and feline calicivirus, although immunity may again last for up to several years, it is only partial in most animals (Scott & Geissinger, 1997, 1999). For feline leukaemia, there is limited evidence that, after...

Antimicrobial mechanisms of phagocytic cells

Over, it is likely that the uptake of enzymes and cations from other cells plays a significant role in macrophage antibacterial function in vivo. The defensins will kill organisms as diverse as Staphylococcus aureus, Pseudomonas aeruginosa, E. coli, Cryptococcus neoformans and even the enveloped virus, Herpes simplex.

Vaccination schedules

Because of these concerns, some authorities in the USA have designated certain vaccines as 'core' vaccines, such that all animals should undergo primary vaccination as indicated by the manufacturer, followed by revaccination 1 year later and then boosters every 3 years. A vaccine is designated as core either if the consequences of infection are particularly severe (e.g. feline panleucopenia) or the prevalence of disease is high and the disease is easily transmitted (e.g. feline herpesvirus and feline calicivirus infection). In the USA and some other countries where rabies is endemic and poses a substantial zoonotic risk, rabies vaccines are mandatory and are also considered core vaccines. Other vaccines are designated 'non-core' and it is recommended that they should only be used following a risk benefit assessment.

Pediatric Cardiac Transplantation

Childhood infections are frequently encountered and should be treated according to routine practice. Vaccinations with live attenuated virus are avoided. Exposure to chickenpox (varicella) is avoided if possible. If exposure does occur in a recipient without a history of previous infection, treatment with varicellazoster immune globulin (VZIG) is indicated. Recipients who develop chickenpox are treated with intravenous acyclovir (Zovirax).

Management Of Posttransplant Patients

Posttransplant patients are at risk for several complications related to their underlying disease, medication side effects, and immunocompromised state. Most centers use cyclosporine, azathioprine, and prednisone for maintenance immunosuppression. In addition, prophylaxis against Pneumocystis carinii pneumonia is undertaken with trimethoprim-sulfamethoxazole (TMP-SMX). Prophylaxis against herpes simplex virus (HSV) and CMV is indicated based on the specific immunologic status of the donor and recipient. Patients learn to measure their pulmonary function (FEV1 and FVC), systemic blood pressure, and temperature daily. They carry a diary with daily vital signs, present medications and doses, names of hospital contacts, and guidelines for contacting the nurse coordinator. Bronchoscopy is necessary to diagnose subclinical rejection and infection. Each transplant center has a protocol concerning bronchoscopy indications. Common warning signs of a fever (> 37 C), cough, sputum, or FEV1...

Other Conditions and Causes

The list of other conditions that can cause abdominal pain in the older patient is extensive, highlighting the need for the comprehensive evaluation of such patients. The most important disease to suspect is acute myocardial ischemia. Some 1 to 2 percent of elderly patients with abdominal pain will be having a myocardial infarction.10 Virtually all other chest diseases can cause abdominal pain, including pneumonia, pulmonary embolism, empyema, tuberculosis, congestive heart failure, esophageal rupture, and endocarditis. Genitourinary disease including renal colic, pyelonephritis, epididymitis, and testicular torsion is a possible cause of abdominal pain in the elderly. Diabetic ketoacidosis, herpes zoster, hypercalcemia, addisonian crisis, hemochromatosis, and retroperitoneal or rectus sheath hematomas secondary to anticoagulant therapy are examples of medical causes of abdominal pain in the elderly.

Prophylaxis and treatment of infection

Opportunistic infections after transplantation continue to constitute a challenge for management (table 7.8). Cytomegalovirus (CMV) remains the most important infection affecting heart transplant recipients. In the prevention of CMV disease, those at risk of primary disease (donor seropositive, recipient seronegative) should receive prophylaxis.54 In many units, oral ganciclovir is now the preferred route of prophylactic treatment. To monitor activity of CMV infection, assessment of viral load has become a valuable tool. Legionella pneumophila may cause pneumonia of variable severity after cardiac transplantation. Chlorination and heating of water is an important

Skin Surface Analysis

Microanatomy The Epidermis

Skin surface analysis is often tailored to the goals of restorative surgery or nonsur-gical treatments. Patients undergoing scar revisions require analyses to include the resting skin tension lines. In the patient undergoing nasal reconstructive surgery after Mohs' resection, an analysis of the topographical units of the face is important. If cutaneous resurfacing is planned, an analysis of skin type and reaction to solar damage are also needed. Koebner's phenomenon is also an important consideration. This phenomenon describes the tendency for some skin diseases such as psoriasis, lichen planus, discoid lupus erythematosus, and herpes simplex to localize to areas of recent surgery or scars.

Environmental Factors

Many viruses have been implicated in the patho-genesis of T1D they may have a direct effect on 0 cells by infection and cell lysis, or alternatively they may act as triggers to the autoimmune process. Among the viruses that have been implicated in humans are coxsackie A, coxsackie B, rubella, cytomegalovirus, mumps, and Epstein-Barr viruses. The enteroviruses (Coxsackie A, Coxsackie B, and Echovirus) are the most commonly associated viruses with diabetes and serve as a major trigger for T1D in the young possibly by induction of islet cell antibodies. The evidence for viral involvement in type 1 diabetes came from several sources, including anecdotal case reports, epidemiological studies, seasonal incidence studies, and animal models. There is data to support the theory that enterovirus infection either accompanies or precedes the development of T1D in young people in many instances.

Immunocompetent Patient

Therapy with oral acyclovir is not recommended routinely for the treatment of uncomplicated varicella in the otherwise healthy child < 12 years of age. B. Oral acyclovir may be given within 24 hours of the onset of rash. Administration results in a modest decrease in the duration and magnitude of fever and a decrease in the number and duration of skin lesions. C. Acyclovir (Zovirax) 80 mg kg day PO q6h for five days, max 3200 mg day cap 200 mg susp 200 mg 5 mL tabs 400, 800 mg

Choose The Method Of Overexpression

It is important to determine whether continuous or limited overexpression of a particular factor is required. On the one hand, tissue-specific promoters will allow the factor to be expressed for only a relatively short time upon transdifferentiation, the promoter will no longer be active. Ubiquitous promoters, on the other hand, will express the chosen factor continuously and may produce undesired results. Many tran-scriptional regulators are expressed only transiently and require a strict temporal regulation for proper development to occur. For example, continuous overexpression of Hlxb9 in the normal pancreas interferes with the differentiation of both exocrine and endocrine cells. Thus, the use of the constitutive promoters, such as cytomegalovirus, may not be suitable. We believe that the use of tissue-specific promoters is best suited to the type of experiments described here to prevent the chosen factor from interfering with the proper differentiation of the new phenotype.

Characteristics of the organism and its antigens

Cytomegalovirus is a typical herpesvirus. It contains double-stranded DNA of 2.4 X 105 base pairs which makes it the largest virus known to infect humans in terms of genome size. The proteins are expressed in an infected cell in an ordered cascade sequence termed a (immediate early), (3 (early) or y (late). Typically, they function as regulators of cell metabolism, enzymes and structural proteins of the virus, respectively. Many of the primary translation products are modified by glycosylation, cleavage or phosphorylation. Chee and colleagues have analyzed the complete sequence of one strain of CMV which encodes approximately 202 proteins. Recently, an additional 22 open reading frames have been Approximately 25 of the total open reading frames can be assigned functions, with many more having sequence homologies to known proteins. The surface structures which may be important for immunity exist as high molecular weight glycoprotein complexes which have been termed gCl, gCN and gCIII....

Classificationtype Of Pathogen

The Epstein-Barr virus (EBV) belongs to the family of herpesviruses.1-1-1 With respect to morphology, EBV is hardly distinguishable from other family members. Based on pathogenesis, characteristics of replication, and host cell tropism, herpesviruses can be classified into three subfamilies alpha-, beta-, and gamma-herpesvirus. EBV belongs to the genus Lymphocrypto-virus of gamma-herpesviruses, which are characterized by a narrow host (cell) range and a slow rate of replication in cell culture.

Management Of Ebvassociated Diseases

Most cases of infectious mononucleosis do not require therapeutic intervention. In cases of clinically severe IM and life-threatening forms of SCAEBV, intravenous application of the nucleoside analog acyclovir or gancy-clovir may be necessary to reduce active viral replica-tion. 4 In addition, but not alone, antiphlogistic drugs to reduce the unspecific effects of the cellular immune response may be beneficial. Tonsillectomy is frequently used and was found to reduce symptoms of IM possibly

Evolutionary Conservation Of Bcl2 Family Proteins

Notably, this family of proteins is evolutionarily conserved. A number of viruses encode Bcl-2 homologs, including most, if not all, gamma herpes viruses (7). Most of these viral homologs are anti-apoptotic, probably because viruses need to keep the infected cells alive for latent and persistent infection (7,8). The nematode Caenorhabditis elegans has its own sequence and functional homologs for a death antagonist, CED-9 (9), and a BH3-only death agonist, EGL-1 (10). On the other hand, only prodeath homologs (dBorg-1 Drob-1 Debcl and dBorg-2 Buffy) have been described in the Drosophila (11). These homologs are discussed in details in Chapters 9 and 10, respectively.

Characteristics of EBV and its antigens

EBV is a member of the gamma-herpesvirus family. It is an enveloped virus of about 150-180 nm diameter with an icosahedral nucleocapsid containing a double-stranded DNA viral genome of about 172 000 base pairs. The nucleocapsid is composed of a major 160 kDa nonglycosylated polypeptide and a minor 125 kDa glycoprotein, both of which form part of the viral capsid antigen (VCA) complex. The virus lipoprotein envelope contains at least three virally-encoded glycoproteins, designated the membrane antigen (MA) complex. Two antigenically-related MA glycoproteins, gp340 and gp220, potentiate binding of the virus to a specific cell surface receptor molecule during the infection of target cells. The third MA glycoprotein, gp85, is involved in the fusion of bound virus with the host cell membrane. Following fusion, viral DNA is released into the cell and is transcribed and replicated in the nucleus, where it persists as multiple episomal copies.

Immune responses of the host to EBV infection

An important feature of EBV shared by all herpesviruses is that following primary infection of the host the virus is carried as a persistent infection for life. More than 90 of all adults in all populations worldwide carry EBV as an asymptomatic infection. Infectious virus can be detected in the throat washings of nearly all healthy carriers. In addition, EBV-positive LCLs can be established spontaneously from cultures of circulating B lymphocytes. The virus is transmitted horizontally via salivary secretions, but current opinion is divided as to the target cell of primary infection. Until recently, dogma held that

Limitation Of Clearance The Granuloma

Mechanisms of immune evasion. Pathogens utilise a range of mechanisms to subvert detection by the immune system across the spectra of innate and adaptive responses. Upper panel Residing in immune privilege sites and low antigen production (e.g., herpes simplex virus latency in central nervous system) result in lack of detection by immune recognition mechanisms. Lower panel - left to right (1) Prevent phagocytosis (e.g., Streptococcus pneumoniae polysaccharide). (2) Inhibit pathogen processing and destruction (e.g., Mycobacterium tuberculosis inhibition of phagolysosome fusion. (3) Prevent clearance (e.g., Mycobacterium tuberculosis granuloma induction). (4) Modulate cytokine effects (e.g., Vaccinia virus induced secretion of antagonistic cytokine homologue). (5) Vary antigen expression (e.g., Trypansoma Cruzi antigen switching). (6) Inhibit antigen processing and presentation (e.g., cytomegalovirus inhibition of MHC expression).

Clinical features

Fever, weight loss, diarrhoea, skin changes, CNS manifestations. Haematological abnormalities include thrombocy-topaenia, leucopaenia, neutropaenia, hypergammaglob-ulinaemia and anaemia. Infections may start to occur with organisms such as herpes simplex or zoster, Pneumococcus and Salmonella. When the CD4 count falls below 0.2 X 109 l, the patient becomes susceptible to a wide spectrum of opportunistic infections (Streptococcus pneumoniae, Haemophilus influenzae, Pneumocystis carinii, toxoplasmosis, Mycobacterium tuberculosis, atypical mycobacteria, histoplasmosis, Cryptococcus, cryptosporidiosis, fungal infections, Jamestown Canyon (JC) virus infection and CMV infections), malignancies (Kaposi's sarcoma and non-Hodgkin's lymphoma) and CNS disease such as dementia may develop. This stage of infection is classified by the Centre for Disease Control (CDC) as fully developed acquired immunodeficiency syndrome (AIDS). Many staging systems for HIV have been proposed and exist. Mostly...

Microbial counterparts of CD59

It has been to the advantage of many microbial pathogens to acquire mechanisms for immune evasion. For example, an open reading frame encoding a sequence highly homologous to that of human and various monkey species of CD59 has been identified in the genome of herpesvirus saimiri (HVS), whose natural host is the squirrel monkey. It appears highly likely that the virus has captured this sequence from its host during the course of evolution. Preliminary studies indicate that the sequence does indeed encode a functional protein, so expression of this sequence by HVS could be considered as a virulence factor in aiding the virus to evade the detrimental effects of complement activation. Complement-inhibiting proteins that appear to have some antigenic relationship to CD59 have also been described in Entamoeba histolytica and Schistosoma mansoni. The E. histolytica protein is an integrin-like molecule consisting of two subunits, one of which shares very limited sequence homology with CD59....

Neurologic Complications

OTHER NEUROLOGIC DISORDERS Other, less common CNS infections that should be considered in the presence of neurologic symptoms include bacterial meningitis, histoplasmosis (usually disseminated), CMV, progressive multifocal leukoencephalopathy, herpes simplex virus, neurosyphilis, and TB. Noninfectious CNS processes include CNS lymphoma (occurring in 5 percent of AIDS patients and typically manifested as a subacute neurologic deterioration over several months), cerebrovascular accidents, and metabolic encephalopathies.

Clinical Description And Epidemiology

Among immunocompetent patients, resistance to ACV is rare, with a prevalence below 1 6 and most often detected in the course of recurrent genital herpes. In this situation, the observed prevalence ranged from 3.5 7 to 8.6 , 8 and in most cases, clinical course was unchanged. Prevalence of ACV resistance among immunocompro-mised patients is about 5 according to several published reports recently reviewed by Bacon et al. 6 But this global incidence has to be specified according to the type of immunosuppression as resistance may be detected in up to 25 of allogeneic bone marrow transplant patients presenting with an HSV infection. 9

Management Of Resistant Infections

The first way to manage ACV-resistant HSV infection is, when possible, to decrease immunosuppressive treat-ments. 15 An increase of antiviral dose should also be Several alternative antiviral drugs are also available. Penciclovir is a nucleoside analogue of guanosine and is very similar to ACV. Indeed, most ACV-resistant HSV isolates are also resistant to penciclovir. Foscarnet, a pyrophosphate analogue, and cidofovir, a nucleotide analogue of cytidine, act directly on viral DNA polymerase without previous activation by viral TK and both these molecules are active on viruses resistant to ACV because of a mutation in the TK gene. 17,18 Acyclovir-resistant management strategies usually recommend the use of Cross-resistance between ACV and foscarnet has been documented in immunocompromised patients. 19-21 Nevertheless, foscarnet-resistant strains sensitive to ACV have also been reported. 21 Acyclovir-resistant strains, cross resistant or not to foscarnet, have always been shown to be...

Genetic Characterization

Herpes simplex virus TK is involved in 95 of HSV resistance to ACV. It is a 376-amino acid protein, encoded by a gene of 1128 bp (UL 23). It exhibits an ATP binding site (amino acid 51 to 63) and a nucleoside binding site (amino acid 168 to 176) and six regions conserved among herpesviridae TK (amino acid 50 to 66, 79 to 91,162 to 178, 212 to 226 and 281 to 292). Figure 1 presents the localization of mutations reported in ACV-sensitive and ACV-resistant HSV clinical isolates, in relation to the localization of active and conserved sites of the enzyme. These data were obtained by genetic characterization of about 70 ACV-resistant HSV isolates and 30 ACV-sensitive HSV isolates. 24,25 These studies revealed a large degree of polymorphism in the HSV TK gene. The mutations unrelated to resistance are located throughout the gene but conserved regions among herpesviridae TK Q GC homopolymer repeats b.s. binding site Ns nucleoside conserved regions among herpesviridae TK Q GC homopolymer...

Application Of Dna Microarrays To The Study Of Bladder Cancer

The following study provides an example of the functional classification of genes applied to bladder cancer, relating the expression patterns of p53-mediated apoptosis in resistant tumor cell lines vs sensitive tumor cell lines. The ECV-304 bladder carcinoma cell line was selected for resistance to p53 by repeated infections with a p53 recombinant adenovirus Ad5-cytomegalovirus (CMV)-p53. Its expression pattern using cDNA arrays containing 5730 genes was compared with p53-sensitive ECV-304 cells. A number of potential p53 transcription or related targets were identified playing roles in cell cycle regulation, DNA repair, redox control, cell adhesion, apoptosis, and differentiation. Proline oxidase, a mitochondrial enzyme involved in the proline-pyrroline-5-carboxylate redox cycle, was up-regulated in sensitive, but not in resistant cells. Further experiments with pyrroline-5-carboxylate (P5C), a proline-derived metabolite generated by proline oxidase, inhibited the proliferation and...

Chapter References

Kesson AM Position paper of the Pediatric Special Interest Group of the Australian Society for Infectious Diseases Use of acyclovir in herpes simplex virus infections. J Paediatr Child Health 34 9, 1998. 11. Spruance SL, Rea TL, Thomig C, et al Penciclovir cream for the treatment of herpes simplex labialis A randomized, multicenter, double-blind, placebo-controlled trial. JAMA 277 1374, 1997. 12. Hutto C, Arvin A, Jacobs R, et al Intrauterine herpes simplex virus infections. J Pediatr 110 97, 1987. 13. Gibbs RS, Amstey MS, Sweet RL, et al Management of genital herpes infection in pregnancy. Obstet Gynecol 71 779, 1988. 14. Brown ZA, Bendetti J, Ashley R Neonatal herpes simplex virus infection in relation to asymptomatic shedding at the time of labor. N Engl J Med 324 1227, 1991. 15. Herpetic Eye Disease Study Group Acyclovir for the prevention of recurrent herpes simplex virus eye disease. N Engl J Med 339 300, 1998. 17. Adour KK, Ruboyianes JM, VanDoersten PG, et al Bell's palsy...

Case presentation 2 continued

Proven antiviral therapy is currently limited to HSV. In two separate trials comparing vidarabine to acyclovir in HSE, acyclovir was found to be superior.87,88 The recommended dose is 10 mg kg1 intravenously every 8 hours for 10-14 days.89 The dose should be adjusted in patients with renal Herpes simplex virus type 1 Cytomegalovirus Herpes B virus insufficiency. Both mortality and later sequelae can be substantially reduced if therapy is instituted before there is a major alteration in consciousness.87 Therefore, early treatment is essential and should be initiated as soon as the diagnosis is suspected. Although several new antiviral drugs with activity against HSV are available in oral formulations with good bioavailability, none has been studied for HSV infections of the CNS.

Nonstreptococcal Pharyngitis

ETIOLOGY Most cases of acute pharyngitis in children are caused by viral infections. Examples include adenovirus, Epstein-Barr virus (see below), influenza virus, parainfluenza virus, rhinovirus, herpes simplex virus, and enterovirus. Although many of these viruses cause symptoms in addition to sore throat and fever, such as cough, coryza, conjunctivitis, or mucosal ulcerations, some viral infections can be clinically difficult to distinguish from GABHS. EBV is a herpesvirus that is a common cause of infection in childhood and adolescence. While EBV has been associated with a variety of clinical syndromes, most children infected with EBV are asymptomatic or have only mild nonspecific symptoms. EBV can cause isolated tonsillopharyngitis and pharyngitis as a manifestation of infectious mononucleosis (IM). Clinically, the classic IM syndrome begins with malaise, fatigue, and sore throat. Fever and adenopathy are the most common signs. Splenomegaly and hepatomegaly are also present in the...

In vitro biological activities

IL-8 has profound effects on nonleukocytic cells. IL-8 is haptotactic (induces cell migration to cell surface- or matrix-bound gradient), and promotes adhesion and proliferation of melanoma cclls. It inhibits collagen expression in synovial fibroblasts and enhances cytomegalovirus replication in lung fibroblasts. Furthermore, IL-8 induces the migration of human umbilical vein endothelial cclls in vitro and angiogenesis in rat cornea.

Validating Novel Genes Gene To Phenotype

The development of EGS technology for silencing gene expression stemmed from the characterization of RNase-P-mediated cleavage of pre-tRNA molecules to produce mature tRNAs (85). These studies identified RNA as the catalytic component of the RNase P ribonucleoprotein complex. Moreover, characterization of the substrate showed that the structure recognized by RNase P could be mimicked using antisense RNAs, called EGSs, which hybridize with any target RNA and reproduce a tRNA-like structure. EGSs can be synthetic or gene-expressed and have been used primarily to target mammalian viruses, including herpes simplex virus (HSV), cytomegalovirus (CMV), and human immunodeficiency virus (HIV) (86, 87). As with all gene-silencing technologies operating at the posttranscriptional level, the identification of target RNA sites accessible to EGS binding is essential for efficient control of gene expression. One advantage associated with EGSs for gene silencing is its action through a naturally...

Course of HIV Infection

The collapse of cell-mediated immunity sets the stage for infection by organisms that are normally held in check by these mechanisms. These include various mycobacteria, fungi, yeasts, protozoa, as well as cancers stimulated by proliferation by other viruses (e.g., Kaposi sarcoma, which is linked to herpesvirus infection).

Selection of Gene Targeted Cells

Homologous recombination is a very rare event, and scientists using it to modify or knock out mouse genes must identify the cells in which it has occurred. In addition to injecting the gene they are trying to incorporate, scientists also inject selectable genes whose products permit cells to live or cause them to die in the presence of a particular drug. The two most common selectable genes used in gene targeting are the neomycin resistance (neor) gene, which allows cells to survive in the presence of the antibiotic neomycin, G418, and the thymidine kinase (TK) gene from the herpes virus. Cells with this gene die in the presence of the antiviral agent gancyclovir. The neor and TK genes are generally used together for maximum selection.

Immunogenicity of antigens borne by ISCOMs

Protective immunity has been induced against a variety of microorganisms including viruses, bacteria, mycoplasma and parasites. Of particular interest is that protective immunity was induced by ISCOM-borne antigens to a number of retroviruses, e.g. feline leukemia virus infection in cats, to SIV, HIV-1 and HIV-2 in primates, to a tumor-inducing herpesvirus (e.g. Epstein-Barr virus) in cotton top tamarin monkeys, and against lethal infection of mice with Trypanosoma cruzi causing Chagas' disease in humans. Table 1 lists examples of ISCOM-borne antigens having induced protective immunity.

Gene disruption by homologous recombination

Initially, a targeting construct is assembled that contains part of the target gene, separated by a positive selection marker. Typically, this marker is the neo gene that confers resistance to the aminoglycoside G418 on cells that have taken up the construct. To enrich for integration of the vector into the target gene via homologous recombination, a negative selection step is employed simultaneously, eliminating cell clones that have undergone random integration of the construct. This is achieved by introducing the thymidine kinase gene of herpes simplex virus (HSV-tk) in the targeting vector. HSV-tk converts inactive nucleoside analogus such as l- 2-deoxy, 2-fluoro-j3-D-arabinofuranosyl -5 iodouracil (FIAU) into chain terminators that are incorporated into the DNA of the target cell and block DNA synthesis. In the vector described in Figure 1, the HSV-tk gene flanks the homologous gene region and is Figure 1 Generation of a gene targeting construct. The gene targeting construct is...

Implications for practice

What is the role of anti-herpes virus therapy in The discovery of KSHV (also known as HHV8) has provided another potential target for the prevention and treatment of AIDS-related KS. KSHV replication is inhibited in vitro by cidofovir, ganciclovir and foscarnet, but not by aciclovir.89,90 Cohort studies have reported a decreased risk of developing KS for HIV-positive patients treated with ganciclovir or foscarnet for cytomegalovirus (CMV) infection and there have been occasional case reports of patients with AIDS-related KS having prolonged responses to antiherpetic therapy.91-95 One cohort study of 3688 HIV-positive patients followed up for a median of 4-2 years, during which time 16 (598 patients) developed KS, found a statistically significant reduction in the relative hazard of developing KS for those who received foscarnet or ganciclovir. The relative hazard for foscarnet was 0-38 (95 CI 0-15-0-95 P 0-038) and for ganciclovir 0-39 (95 CI 0-19-0-84 P 0-015).

Latent and slow persistent viral infections

After an infection has passed, a virus may sometimes remain in the body for long periods, causing no harm. It may be reactivated, however, by stress or some change in the individual's health, and initiate a disease state. Well known examples of latent viral infections are cold sores and shingles, both caused by members of the herpesvirus family. A virus of this sort will remain with an individual throughout their lifetime.

T cell targeting and effector function

Mal cell events, such as viral infection, to recirculating CD8 ' virus-immune T cells. These lymphocytes can potentially kill virus-infected target cells and thus terminate the progression of the infectious process. An exception to this rule is found in the brain where some cells, especially sensory neurons, remain MHC class I negative. This may be the reason that herpesviruses can persist, for instance, in the trigeminal ganglia.

Complex Designer Cells in Screens

For historic reasons, viral promoters such as the SV40 early promoter, the Rous sarcoma virus (RSV) long terminal repeat (LTR) and, most frequently, the immediate early promoter from human cytomegalovirus (hCMV), are used to drive the expression of foreign genes. However, despite the 10- to 50-fold different promoter activity in transient assays (expression measured at 2-3 days post introduction of recombinant DNA), stable producer clones containing the strongest promoter (hCMV) have no clear advantage over clones derived with

Structurefunction Relationship Of Chemokine Ligands

The only known natural ligand of CXCR4 is the stromal cell-derived factor (SDF)-1a, a CXC chemokine that plays critical roles in the migration, proliferation, and differentiation of leukocytes (Bleul et al., 1996b Oberlin et al., 1996). CXCR4 can also be recognized by an antagonistic ligand, the viral macrophage inflammatory protein (vMIP)-II encoded by the Kaposi's sarcoma-associated herpes virus (Moore et al., 1996). vMIP-II displays a broader spectrum of receptor activities than any mammalian chemokine, because it binds with high affinity to a number of both CXC and CC chemokine receptors, including CXCR4, CCR5, and CCR2, and inhibits cell entry of HIV-1 mediated by these receptors (Boshoff et al., 1997 Kledal et al., 1997). Studies with knockout mice of CXCR4 and SDF-1a have demonstrated that

Opportunistic Infections

Bacteremias pneumonitis herpesvirus and mycotic infections EBV-induced lymphoma The pattern of infection in the compromised host has been altered somewhat by the availability of effective prophylaxis for some of the commoner opportunistic infections. Cotrimoxazole effectively prevents Pneumocystis pneumonia in patients with hematologic malignancy and in transplant patients. It is less useful in HIV infection as hypersensitivity reactions to the sulfonamide component are common. Inhaled pentamidine is a useful alternative. Herpes simplex virus (HSV) reactivation can be prevented by acyclovir some protection may also extend to other herpesviruses including cytomegalovirus (CMV). Some of the newer quinolone antibiotics such as ciprofloxacin and norfloxacin can reduce the incidence of bacteremia in neutropenic patients.

Infection in transplant recipients

The pattern of infection in BMT recipients changes with time after transplant. In the first 3-4 weeks patients experience severe mucosal ulceration, are profoundly neutropenic, and are prone to bacteremia and reactivation of herpes simplex infections. During the second and third months a major and often fatal problem is interstitial pneumonitis. Total body irradiation is an important factor in the development of pneumonitis, but about one-half are associated with CMV infection or reactivation. Pneumocystis pneumonia is uncommon. Primary CMV infection can be avoided in CMV-negative patients provided their donor is also CMV-negative and only CMV-negative blood products are used. Prevention of reactivation in CMV-positive recipients is difficult, but early detection and therapy with ganciclovir may be effective. Fatal CMV infections are less common following recovery of endogenous antibody production about 2 months after transplantation. In the first month after transplantation bacterial...

TABLE 2273 Viral Pathogens Causing Encephalitis in North America

Signs of meningeal irritation and increased intracranial pressure should be sought. Neurologic findings reflect the areas of involvement. A careful assessment of cognition is crucial. Sensorimotor deficits are not typical. Encephalitides may show special regional tropism. Herpes simplex virus (HSV) involves limbic structures of the temporal and frontal lobes, with prominent psychiatric features, memory disturbance, and aphasia. Some arboviruses predominantly affect the basal ganglia, causing choreoathetosis and parkinsonism. Involvement of the brainstem nuclei that control swallowing leads to the hydrophobic choking response characteristic of rabies encephalitis.1 I5

Photoimmunology in humans

One question of considerable importance concerns the potential of UV-induced immune suppression to alter the immune response to pathogenic microorganisms. While animal studies have clearly shown that UV exposure can suppress the immune response to a variety of infectious agents and increase the incidence, severity and duration of infection, it is not clear whether photoimmunosuppression similarly affects the immune response to infectious agents in humans. Some evidence suggests that the ability of sunlight to trigger recrudescence of herpes simplex virus infection is due to the immunosuppressive activity of UV radiation. In addition, UV irradiation of the site of elicitation of a delayed hypersensitivity reaction to leprosy antigens in healthy, immune subjects reduced the granulomatous reaction and decreased the number of CD4 T cells in the reaction site. Thus, UV radiation seems to have the ability to modify the immune responses of humans to infectious agents, but whether this...

Lymphocyte proliferation as a consequence of molecular mimicry by infectious agents

An increasing number of viruses are known to infect lymphocytes, and directly or indirectly induce lymphocyte proliferation. Both retroviruses and DNA viruses infect lymphocytes. Human GD4 ' T cells are infected by the nontransforming retroviruses human immunodeficiency virus type 1 (HIV-1) and HIV-2 and by the transforming retrovirus human T lympho-tropic virus type I (HTLV-I) while CDS cells are transformed by HTLV-I. Human B cells are infected with two herpesviruses Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV8). Likewise, human T cells are infected with at least three herpesviruses HHV6 and HHV7 and herpesvirus saimiri. Mouse B cells are infected with murine mammary tumor virus (MMTV), resulting in augmented T and B cell proliferation. Other organisms have subverted lymphocyte signal transduction pathways for enhanced rep-licative fitness, to impair the host immune response or both. A list of some of these agents and their targets are found in Table 4. HHV-6 HHV-7 H....

Other reproductive tract pathogens

See also Acquired immune deficiency syndrome (AIDS) Candida, infection and immunity Chlamydia, infection and immunity Cytomegalovirus, infection and immunity Epstein-Barr virus, infection and immunity Escherichia coli, infection and immunity Herpes simplex virus, infection and immunity IgA Immunodeficiency, primary Mucosal immunity Mucosa-associated lymphoid tissue (MALT) Mycoplasma, infection and immunity Neisseria, infection and immunity Opportunistic infections Papillomavirus, infection and immunity Treponema, infection and immunity Urinary tract infections.

Treatment of patients with SCID

Patients with SCID have a poor prognosis and in most cases do not survive more than a few months without appropriate treatment. Supportive treatment with intravenous immunoglobulin infusions at regular intervals, and prophylaxis for Pneumocystis car-inii is indicated. Bacterial or fungal infections are treated with intravenous infusions of antibiotics or antifungal agents. Epstein-Barr virus, herpes virus and cytomegalovirus infection may result in systemic and lethal disorders and should be treated with antiviral reagents. Prophylactic infusions of immunoglobulin with high titer for cytomegalovirus has been advocated. For ADA deficiency, enzyme replacement with polyethylene glycol-modified ADA (PEG-ADA) resulted in limited but significant improvement of the immunodeficiency.

HIVAssociated Peripheral Neurologic Disease

Cytomegalovirus (CMV) Radiculitis In the latter stages of AIDS, patients may suffer from an acute radiculitis caused by CMV infection. These patients almost always have evidence of CMV infection elsewhere in the body and may have ongoing CMV retinitis. Patients become acutely weak, with primarily lower extremity involvement, and may have variable degrees of bowel and bladder dysfunction. The examination shows primarily lower extremity weakness and hyporeflexia, with decreased sensation in the lower extremities and groin. Rectal tone may be impaired. Lumbar puncture reveals a pleocytosis with predominantly polymorphonuclear cells and modestly increased protein viral DNA is detected by polymerase chain reaction in most patients and is highly specific. MRI of the lumbosacral spine demonstrates swelling and clumping of the cauda equina. Imaging of these patients is mandatory to rule out mass lesions of the lower spine or nerve roots. The treatment of CMV radiculitis is intravenous...

The etiological role of viruses

In common with other autoimmune diseases, it is believed that polygenic genetic susceptibility factors interact with environmental factors to induce SS. Potential viral triggers include sialatropic and lym-photropic viruses such as the herpesviruses, in particular Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human herpesvirus type 6 (HHV-6). Evidence for involvement of any of these viruses has been conflicting, with some studies finding elevated titers of antibodies to all three viruses in patients with SS but others finding them normal. EBV has been detected in parotid and labial salivary glands by DNA hybridization techniques. Salivary epithelium can contain up to 50 copies of EBV DNA per cell in healthy people without inducing an immune response, suggesting that the gland is an important site of persistence. The extent of EBV infection load in salivary glands in SS is still controversial with conflicting reports. While these inconsistent data may reflect the different...

Expression in mammalian cells

Most vectors use the enhancer promoters from the human cytomegalovirus (CMV), the SV40 virus, or the herpes simplex virus thymidine kinase (HS-TK) to drive transcription. These give high level, constitutive, expression. As in prokaryotic systems, it is sometimes desirable to control the onset of expression. One way of achieving such regulation is by interposing the operator sequence (tetO) from the bacterial tetracycline resistance operon between the promoter and the cloned gene. If the mammalian cells are cotransfected with a second plasmid containing the tetracycline repressor gene (tetR), also expressed using the CMV promoter, the TetR protein will bind to the tetO site, thus preventing transcription. When tetracycline is added to the culture medium it will bind to the TetR protein, altering its conformation and releasing it from the DNA, thus derepressing transcription of the cloned gene. The advantage of this is that, because this system is of prokaryotic origin, its activation...