The equine herpesvirus 1 (EHV-1) UL3 gene, an ICP27 homolog, is necessary for full activation of gene expression directed by an EHV-1 late promoter

We have previously reported that the equine herpesvirus 1 (EHV-1) XbaI G restriction fragment (nucleotides 1436 to 7943 relative to the left terminus of the EHV-1 genome [Kentucky A strain]) is required in combination with the EHV-1 immediate-early (IE) gene to achieve significant activation of two representative EHV-1 late promoter-chloramphenicol acetyltransferase (CAT) recombinants in transient expression assays. In this report, we demonstrate that the XbaI G-encoded UL3 gene (an ICP27 homolog) provides a trans-acting factor which acts (in combination with the EHV-1 IE gene product) to increase reporter gene expression directed by an EHV-1 late promoter-CAT recombinant plasmid. We show that cloned copies of UL3 can successfully substitute for the XbaI G fragment in CAT assays and that stop codon insertion within the UL3 open reading frame inhibits the ability of UL3 to activate reporter gene expression in trans.     

Expression and cellular distribution of baculovirus-expressed bovine herpesvirus 1 (BHV-1) glycoprotein D (gD) sequences

Glycoprotein D (gD) of bovine herpesvirus 1 (BHV-1), a homolog of herpes simplex virus gD, represents a major component of the viral envelope and is a dominant immunogen. To study the antigenic properties of the different regions of gD, we have expressed the full-length gD encoding gene and overlapping fragments spanning various regions of the gD open reading frame in a baculovirus (Autographa californica nuclear polyhedrosis virus)--insect cell (Spodoptera frugiperda, SF-9) system. Maximum levels of expression for all proteins were obtained 48 to 72 h post infection of SF-9 cells by recombinant viruses. Full-length and truncated recombinant gD proteins reacted specifically with anti-gD monospecific serum as determined by immunoprecipitation and immunoblotting, indicating that the proteins retained their antigenicity. However, based on the reactivity with a panel of gD-specific monoclonal antibodies (Mabs), the full-length recombinant gD lacked proper expression for two highly neutralizing linear epitopes identified by Mabs R54 and 9D6. The rest of the epitopes appeared to be preserved and antigenically unaltered. Immunofluorescence studies of recombinant baculovirus infected SF-9 cells using gD monospecific serum, revealed no direct correlation between cellular localization of the expressed proteins and their amino acid sequences.     

Application of polymerase chain reaction (PCR) for diagnosis of equine herpes virus-1 (EHV-1)

Fifty aborted foetus samples were diagnosed for the presence of equine herpes virus-1 (EHV-1) by polymerase chain reaction (PCR) technique. Specific primer pair for amplification of a particular segment of EHV-1 DNA in gc region having 3 Hae III restriction endonuclease sites was used. A 409 base pair segment obtained as PCR amplification product in 9 samples was digested with Hae III to confirm the presence of EHV-1 as the infectious agent in aborted tissues. It was observed that PCR technique was more sensitive, specific and rapid than the conventional virological diagnostic methods.     

Genetic immunization with glycoprotein 63 cDNA results in a helper T cell type 1 immune response and protection in a murine model of leishmaniasis

Genetic immunization is a promising gene therapy approach for the prevention and treatment of infectious disease. Plasmid DNA expressing genes of pathogens is directly introduced into host cells and specific cell-mediated and/or humoral immune responses are elicited against the encoded protein. Leishmaniasis is a significant world-wide health problem for which no vaccine exists. In susceptible animals, such as BALB/c mice, protection from leishmaniasis requires induction of a Thl immune response. In this study, cell-mediated immunity to Leishmania major (L. major) was induced by injecting BALB/c mice intradermally with plasmid DNA expressing the conserved L. major cell surface glycoprotein gp63 (gp63-pcDNA-3). CD4 T lymphocytes from gp63-pcDNA-3-immunized mice proliferated and produced IFN-gamma (but not IL-4) when stimulated in vitro with freeze-thawed parasites, consistent with a Th1 immune response. In contrast, lymphocyte proliferation in animals immunized with freeze-thawed parasites was associated with IL-4 (but not IFN-gamma) production, suggesting a nonprotective Th2 response. Challenge studies revealed that gp63-pcDNA-3 vaccination protected 30% of susceptible mice (21 of 70) from Leishmania infection while neither gp63 protein (0 of 20) nor freeze-thawed parasite vaccines (0 of 50) were efficacious. Dendritic cells derived from skin of gp63-pcDNA-3-injected mice also immunized naive recipients and protected them from leishmaniasis. We conclude that gp63-pcDNA-3 genetic vaccination results in a CD4-dependent Th1 immune response that correlates with protection from disease, and suggest that skin-derived dendritic cells are involved in priming this response.     

Immune responses and protective efficacy of recombinant baculovirus-expressed glycoproteins of equine herpesvirus 1 (EHV-1) gB, gC and gD alone or in combinations in BALB/c mice

Baculovirus-expressed glycoproteins of EHV-1 gB, gC and gD alone or in combination evoked antibody responses and protected vaccinated mice against a challenge with EHV-1. gB, gD, gB + gC, gB + gD and gC + gD elicited very high levels of ELISA antibodies while gC and gC + gD elicited high levels of virus neutralising antibodies. Western blotting demonstrated that the antibodies produced were not only specific for the baculovirus-expressed glycoproteins gB, gC and gD, but also highly specific for each EHV-1 glycoprotein. Vaccination of mice with gB or gD prevented clinical signs of infection in mice challenged with EHV-1 and all vaccinated groups of mice except controls showed a rapid clearance of virus from the lungs and a reduction in lesions characteristic of herpesviruses in the lungs post-challenge. Notably, the lungs of mice vaccinated with gB, gD or gB + gD and challenged with EHV-1 showed prominent peribronchiolar and perivascular aggregations of mononuclear cells, predominantly lymphocytes. Immunocytochemical staining of these sections showed large numbers of T cells, suggesting an active role for these cells at the site of virus replication post-challenge.     

Induction of immunity in the respiratory tract and protection from bovine herpesvirus type 1 infection by different routes of immunization with recombinant adenovirus

As part of a larger study, the Lehman Quality of Life Interview (QOLI) was conducted a total of 85 times with 55 clients with serious mental illness. Results revealed widespread adverse objective circumstances (unemployment, poverty and social isolation) despite which most clients rated their satisfaction levels about average (about equally satisfied and dissatisfied). As expected, subjective quality of life indicators were generally better predictors of global well-being (GWB) (itself based on subjective ratings) than were objective indicators. Correlations between objective and subjective indicators were very low and insignificant. Moderate relationships were found between GWB and levels of personal functioning, and changes in levels of personal functioning, as rated by mental health workers. Retests showed that subjective quality of life was relatively stable over intervals of several months. The findings suggest that leisure and social relations would be suitable areas for interventions that might improve clients' quality of life.     

Modification of experimental Porphyromonas gingivalis murine infection by immunization with a polysaccharide-protein conjugate

To better understand the role of the capsular polysaccharide in the virulence of Porphyromonas gingivalis, the effect of immunization with a polysaccharide-protein conjugate on experimental murine infection was evaluated. The conjugate was prepared using polysaccharide isolated from P. gingivalis strain ATCC 53977 and bovine serum albumin. One group of 22 mice was immunized by intraperitoneal injection with the conjugate and a control group of 25 mice was similarly immunized with bovine serum albumin. Serum antibody reactive to the polysaccharide, as determined by enzyme-linked immunosorbent assay, was elevated in the group of mice immunized with the polysaccharide-protein conjugate but not in the mice immunized with bovine serum albumin. Both groups of mice were challenged with P. gingivalis strain ATCC 53977 (10(10) cells) administered subcutaneously on the dorsal surface. Following challenge, the mice immunized with the polysaccharide-protein conjugate appeared healthier and demonstrated less weight loss than did the control group of mice. Ulcerative lesions at secondary locations were smaller in mice immunized with the polysaccharide-protein conjugate. Thus, immunization of mice with a conjugate containing P. gingivalis polysaccharide could reduce the severity of but not prevent an invasive infection with P. gingivalis.     

The V domain of herpesvirus Ig-like receptor (HIgR) contains a major functional region in herpes simplex virus-1 entry into cells and interacts physically with the viral glycoprotein D

The herpesvirus entry mediator C (HveC), previously known as poliovirus receptor-related protein 1 (PRR1), and the herpesvirus Ig-like receptor (HIgR) are the bona fide receptors employed by herpes simplex virus-1 and -2 (HSV-1 and -2) for entry into the human cell lines most frequently used in HSV studies. They share an identical ectodomain made of one V and two C2 domains and differ in transmembrane and cytoplasmic regions. Expression of their mRNA in the human nervous system suggests possible usage of these receptors in humans in the path of neuron infection by HSV. Glycoprotein D (gD) is the virion component that mediates HSV-1 entry into cells by interaction with cellular receptors. We report on the identification of the V domain of HIgR/PRR1 as a major functional region in HSV-1 entry by several approaches. First, the epitope recognized by mAb R1. 302 to HIgR/PRR1, capable of inhibiting infection, was mapped to the V domain. Second, a soluble form of HIgR/PRR1 consisting of the single V domain competed with cell-bound full-length receptor and blocked virion infectivity. Third, the V domain was sufficient to mediate HSV entry, as an engineered form of PRR1 in which the two C2 domains were deleted and the V domain was retained and fused to its transmembrane and cytoplasmic regions was still able to confer susceptibility, although at reduced efficiency relative to full-length receptor. Consistently, transfer of the V domain of HIgR/PRR1 to a functionally inactive structural homologue generated a chimeric receptor with virus-entry activity. Finally, the single V domain was sufficient for in vitro physical interaction with gD. The in vitro binding was specific as it was competed both by antibodies to the receptor and by a mAb to gD with potent neutralizing activity for HSV-1 infectivity.     

Fatal disseminated cercopithecine herpesvirus 1 (herpes B infection in cynomolgus monkeys (Macaca fascicularis)

Two adult female cynomolgus monkeys (Macaca fascicularis) that had been housed together for 4 months died within 2 weeks of each other after brief illnesses. Monkey No. 1 presented with collapse, watery stool, and hypothermia and died overnight. Monkey No. 2 presented with dyspnea, nasal discharge, leukopenia, and hypoproteinemia and was euthanized after 2 days. Both animals had peritoneal effusions, massive necrosis of pharyngeal, esophageal, and gastric mucosa, and multifocal hepatic and pancreatic necrosis. Monkey No. 2 also had lingual ulcers and locally extensive necrosis of spleen, adrenal glands, and lymph nodes. Large numbers of eosinophilic intranuclear inclusion bodies were present in epithelial and syncytial cells adjoining the necrotic foci in Monkey No. 2 but were absent in Monkey No. 1. Monkey No. 1 seroconverted to cercopithecine herpesvirus 1 (CHV-1, commonly known as herpes B) in the month before death. CHV-1 was isolated from a sample of stomach from Monkey No. 2, and electron microscopy of liver from this animal demonstrated herpesvirus particles within hepatocytes. Both animals were seropositive for simian type D retrovirus, and the virus was cultured from the liver of Monkey No. 2. A diagnosis of disseminated CHV-1 infection was made, possibly occurring secondary to immunosuppression due to infection with simian type D retrovirus. Although a high percentage of cynomolgus monkeys are apparently infected with CHV-1, disseminated disease is rare. Because infection with CHV-1 in humans is associated with a high fatality rate, familiarity with the lesions of disseminated infection with this virus is important.     

Antigenic structure of soluble herpes simplex virus (HSV) glycoprotein D correlates with inhibition of HSV infection

Soluble forms of herpes simplex virus (HSV) glycoprotein D (gD) block viral penetration. Likewise, most HSV strains are sensitive to gD-mediated interference by cells expressing gD. The mechanism of both forms of gD-mediated inhibition is thought to be at the receptor level. We analyzed the ability of different forms of soluble, truncated gD (gDt) to inhibit infection by different strains of HSV-1 and HSV-2. Strains that were resistant to gD-mediated interference were also resistant to inhibition by gDt, thereby suggesting a link between these two phenomena. Virion gD was the major viral determinant for resistance to inhibition by gDt. An insertion-deletion mutant, gD-1(delta 290-299t), had an enhanced inhibitory activity against most strains tested. The structure and function of gDt proteins derived from the inhibition-resistant viruses rid1 and ANG were analyzed. gD-1(ridlt) and gD-1(ANGt) had a potent inhibitory effect on plaque formation by wild-type strains of HSV but, surprisingly, little or no effect on their parental strains. As measured by quantitative enzyme-linked immunosorbent assay with a diverse panel of monoclonal antibodies, the antigenic structures of gD-1(rid1t) and gD-1(ANGt) were divergent from that of the wild type yet were similar to each other and to that of gD-1 (delta 290-299t). Thus, three different forms of gD have common antigenic changes that correlate with enhanced inhibitory activity against HSV. We conclude that inhibition of HSV infectivity by soluble gD is influenced by the antigenic conformation of the blocking gDt as well as the form of gD in the target virus.     

Activity of pentamidine-loaded poly (D,L-lactide) nanoparticles against Leishmania infantum in a murine model

Tumors of craniocervical junction (ccjct) may cause a variety of non specific signs and symptoms. Before the advent of computed tomography (CT) diagnosis was sometimes possible only by surgical exploration, as the available radiologic methods--plain film radiography, angiography, and myelocisternography--essentially provided only indirect information about the neural structures. Because of its beam hardening artifacts, CT remained unsatisfactory as well. These diagnostic problems have practically disappeared with the availability of magnetic resonance imaging (MRI), which is now considered the method of choice if a space-occupying lesion is suspected at the ccjct. In some cases invasive angiography may still be necessary for surgical planning, and angiography may also be needed, whenever CT or MRI suggest the presence of an aneurysm. In the first part of this overview we present strategies for the radiological examination of the ccjct.     

Protective activity of a murine monoclonal antibody against European bat lyssavirus 1 (EBL1) infection in mice

A mouse model was designed to test in vivo the efficacy of rabies immune globulins and specific neutralizing monoclonal antibodies to prevent European bat lyssavirus 1 infection. Human or equine rabies immune globulins previously found to contain variable amounts of neutralizing bat lyssavirus crossreactive antibodies were passively transferred to mice receiving intramuscularly a lethal dose of bat lyssavirus type 1. Immune globulins did not protect mice well against bat lyssavirus 1 whereas they reduced the mortality caused by rabies virus. In contrast, mice inoculated with bat lyssavirus 1 or rabies virus survived when passively immunized with bat lyssavirus 1 specific monoclonal antibody (mAb 8-2). This monoclonal antibody, an IgG2 alpha, recognized an epitope located in the antigenic site IIa of rabies glycoprotein. A mutation replacing the lysine 198 by glutamate in a rabies variant abrogated sensitivity to this neutralizing antibody. Because of its broad neutralizing spectrum against wild virus isolates, including European bat lyssaviruses, this monoclonal antibody should be a good candidate for rabies immune globulin replacement. It could improve efficacy of rabies vaccination, used either alone or in conjunction with human rabies immune globulins or monoclonal antibody cocktail to supplement their lack of crossreactivity to European bat lyssavirus 1.     

Detection of antibodies against equine herpesvirus types 1 and 4 by using recombinant protein derived from an immunodominant region of glycoprotein B

The N-terminal fragment comprising residues +1 to +50 (gB1-50) of equine herpesvirus type 1 (EHV-1) glycoprotein B was expressed as a glutathione S-transferase fusion protein in Escherichia coli. Recombinant gB1-50 (rgB1-50) was recognized in immunoblots by sera from rabbits immunized with EHV-1 and by convalescent-phase sera from horses with natural EHV-1 infections. An enzyme-linked immunosorbent assay (ELISA) for monitoring antibody levels against EHV-1 was developed by using rgB1-50, and its specificity was assessed with a panel of reference antisera against other equine viruses. A specific cross-reaction was detected with EHV-4, which was confirmed by inhibition ELISA. Convalescent-phase sera from horses with natural EHV-1 or EHV-4 infections possessed antibody titers against rgB1-50 ranging from 1:2,000 to 1:64,000, indicating the presence of an immunodominant antigenic site. The study demonstrated the potential application of rgB1-50 as a diagnostic antigen and highlights the glutathione S-transferase fusion system as a simple and effective method of producing purified milligram quantities of antigen.     

Proteolytic cleavage of bovine herpesvirus 1 (BHV-1) glycoprotein gB is not necessary for its function in BHV-1 or pseudorabies virus

Glycoprotein B homologs represent the most highly conserved group of herpesvirus glycoproteins. They exist in oligomeric forms based on a dimeric structure. Despite the high degree of sequence and structural conservation, differences in posttranslational processing are observed. Whereas gB of herpes simplex virus is not proteolytically processed after oligomerization, most other gB homologs are cleaved by a cellular protease into subunits that remain linked via disulfide bonds. Proteolytic cleavage is common for activation of viral fusion proteins, and it has been shown that herpesvirus gB homologs are essential for membrane fusion events during infection, e.g., virus penetration and direct viral cell-to-cell spread. To analyze the importance of proteolytic cleavage for the function of gB homologs, we isolated a mutant bovine herpesvirus 1 (BHV-1) expressing a BHV-1 gB that is no longer proteolytically processed because of a deletion of the proteolytic cleavage site and analyzed its phenotype in cell culture. We showed previously that BHV-1 gB can functionally substitute for the homologous glycoprotein in pseudorabies virus (PrV), based on the isolation of a PrV gB-negative PrV recombinant that expresses BHV-1 gB (A. Kopp and T. C. Mettenleiter, J. Virol, 66:2754-2762, 1992). Therefore, we also isolated a mutant PrV lacking PrV gB but expressing a noncleavable BHV-1 gB. Our results show that cleavage of BHV-1 gB is not essential for its function in either a BHV-1 or a PrV background. Compared with the PrV recombinant expressing cleavable BHV-1 gB, deletion of the cleavage site in the recombinant PrV did not detectably alter the viral phenotype, as analyzed by plaque assays, one-step growth kinetics, and penetration kinetics. In the BHV-1 mutant, the uncleaved BHV-1 gB was functionally equivalent to the wild-type protein with regard to penetration and showed only slightly delayed one-step growth kinetics compared with parental wild-type BHV-1. However, the resulting plaques were significantly smaller, indicating a role for proteolytic cleavage of BHV-1 gB in cell-to-cell spread of BHV-1.     

Development of a murine model of chronic Salmonella infection

The invasive disease caused by Salmonella typhimurium in mice resembles the acute phase of human typhoid fever caused by Salmonella typhi, and experimental murine salmonellosis is a widely used experimental model for systemic salmonellosis. In this paper we demonstrate that murine S. typhimurium infection can also be used to model the development of the chronic carrier state that develops in humans after infection with S. typhi. We describe a virulent variant of S. typhimurium that has decreased expression of AgfA fibers under all environmental conditions studied and that causes a chronic carrier state in BALB/c mice after peroral inoculation. The chronic carrier state is associated with persistence of bacteria in the small intestine, spleen, and liver, and chronic infection continues despite the development of protective immunity to challenge with virulent Salmonella.     

Chronic infection of Balb/c mice with murine herpesvirus 72 is associated with neoplasm development

One hundred Balb/c mice were infected with murine herpesvirus strain 72 (MHV-72) and observed for 2.5 years for neoplasm development and virus presence in tumour as well as non-tumour tissues. Out of 13 neoplasm-bearing mice the virus was recovered from solid tumours (one lymphoma, two non-differentiated lymphoblastomas and two fibrosarcomas) of five mice and from the spleen of one mouse with lymphatic leukemia. The virus persisted frequently also in various organs of the neoplasm-bearing mice.     

The equine herpesvirus 1 glycoprotein gp21/22a, the herpes simplex virus type 1 gM homolog, is involved in virus penetration and cell-to-cell spread of virions

Experiments to analyze the function of the equine herpesvirus 1 (EHV-1) glycoprotein gM homolog were conducted. To this end, an Rk13 cell line (TCgM) that stably expressed EHV-1 gM was constructed. Proteins with apparent M(r)s of 46,000 to 48,000 and 50,000 to 55,000 were detected in TCgM cells with specific anti-gM antibodies, and the gM protein pattern was indistinguishable from that in cells infected with EHV-1 strain RacL11. A viral mutant (L11deltagM) bearing an Escherichia coli lacZ gene inserted into the EHV-1 strain RacL11 gM gene (open reading frame 52) was purified, and cells infected with L11deltagM did not contain detectable gM. L11deltagM exhibited approximately 100-fold lower titers and a more than 2-fold reduction in plaque size relative to wild-type EHV-1 when grown and titrated on noncomplementing cells. Viral titers were reduced only 10-fold when L11deltagM was grown on the complementing cell line TCgM and titrated on noncomplementing cells. L11deltagM also exhibited slower penetration kinetics compared with those of the parental EHV-1 RacL11. It is concluded that EHV-1 gM plays important roles in the penetration of virus into the target cell and in spread of EHV-1 from cell to cell.     

Safety evaluation of a polymerized hemoglobin solution in a murine infection model

Several investigators have observed that free hemoglobin may increase the mortality rate in experimental Escherichia coli peritonitis in animals. This effect is probably mediated by the heme moiety of hemoglobin, but the mechanism remains controversial. Free hemoglobin might impair neutrophil function, and it might serve as a source of iron, which is necessary for bacterial replication. Several modified hemoglobin solutions, developed as blood substitutes, are currently being tested in clinical studies, but concern exists that these solutions may have the potential to exacerbate a bacterial infection. At the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, a blood substitute based on modified hemoglobin (PolyHbXl) has been developed that has improved oxygen affinity and prolonged vascular retention. In the present study the potential risk of this solution on the promotion of infections has been evaluated. PolyHbXl was intravenously injected into mice in a clinically relevant dose of 1.5 gm/kg body weight 1 hour before intravenous administration of a sublethal number of Listeria monocytogenes, Salmonella typhimurium, E. coli, or Candida albicans organisms. PolyHbXl did not promote the proliferation of any of these microorganisms in the liver and spleen, nor did it lead to an increased mortality rate in the mice. Also, the in vitro proliferation of L. monocytogenes, S. typhimurium, and E. coli was not increased by PolyHbXl. In conclusion, PolyHbXl does not affect the course of infection with various microorganisms in mice, and no indication was found that this new blood substitute compromises the host defense against infections.     

Detection of Marek's disease virus serotype 1 (MDV1) glycoprotein D in MDV1-infected chick embryo fibroblasts

Chick embryo fibroblasts (CEFs) infected with three strains of Marek's disease virus serotype 1 (MDV1), GA, Md5 and JM, were subjected to indirect immunofluorescence assay with monoclonal antibodies (MAbs) against MDV1 homolog of glycoprotein D (MDV1 gD) of herpes simplex virus. By the MAbs, a number of MDV1 gD-positive cells were detected in CEFs infected with GA, whereas only a few and no positive cells were detected in CEFs infected with Md5 and JM, respectively. The MDV1 gD in GA-infected CEFs was recognized as the band of 64 kDa in immunoblot analysis using one of the MAbs. This is the first report that the MDV1 gD was detected in MDV1-infected cell cultures.