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We have followed throughout time the development of allergic airway disease (AAD) in both uninfected mice and mice infected intranasally with murine cytomegalovirus (MCMV). Histological evaluation of lung tissue from uninfected mice with AAD demonstrated mucus plugging after 14 and 21 days of ovalbumin-aerosol challenge, with resolution of mucus plugging occurring by 42 days. In MCMV/AAD mice, mucus plugging was observed after 7 days of ovalbumin-aerosol challenge and remained present at 42 days. The level of interleukin-13 in bronchoalveolar lavage fluid from MCMV/AAD mice was decreased compared with AAD mice and was accompanied by increased levels of interferon-gamma. Levels of Muc5A/C, Muc5B, or Muc2 mucin mRNA in the lungs of MCMV/AAD mice were not elevated compared with AAD mice. MCMV was able to infect the airway epithelium, resulting in decreased expression of Foxj1, a transcription factor critical for ciliogenesis, and a loss of ciliated epithelial cells. In addition, an increase in the number of epithelial cells staining positive for periodic acid-Schiff was observed in MCMV/AAD airways. Together, these findings suggest that MCMV infection of the airway epithelium enhances goblet cell metaplasia and diminishes efficient mucociliary clearance in mice with AAD, resulting in increased mucus plugging.

Routine immunization against mumps has substantially reduced the number of cases annually, yet recent outbreaks such as the one in Iowa and Great Britain remind us that we must suspect it and be able to recognize both its typical and its less common signs and symptoms. The Iowa outbreak from December 2005 through April 2006 affected nearly 2600 people in 11 states and prompted the US Centers for Disease Control and Prevention (CDC) to update its recommendations for mumps vaccination, which are discussed here.

Several studies have demonstrated that antiretroviral therapy diminishes the adverse effects of HIV/AIDS on brain function. Yet, few studies have examined the role of comorbid psychiatric disorders in limiting the magnitude of recovery. The present study examined the effects of the presence versus absence of one such disorder--antisocial personality disorder (ASPD)--on brain function in an HIV-1 seronegative control group (n=68) and in HIV-1 seropositive patients receiving (n=26), versus not receiving (n=71), antiretroviral treatment. The primary dependent measures of brain function were the amplitude, latency, and topography of the P300 event-related potential. ANCOVA revealed a significant main effect of ASPD as well as an interaction between ASPD and HIV-1/treatment status. Participants with ASPD exhibited a significant decrement in frontal P300 amplitude in comparison to the ASPD-negative groups. More importantly, further analyses showed that ASPD blunted the decrement in P300 accompanying untreated HIV/AIDS as well as the improvement in P300 accompanying antiretroviral treatment. The results suggest that P300 is minimally improved by antiretroviral therapy among HIV-1 seropositive patients with comorbid ASPD. The lack of antiretroviral efficacy is not easily explained by poor medication compliance. The diminished response to treatment is more likely related to a pre-existing neurophysiological decrement localized to the same brain regions where HIV/AIDS and antiretroviral treatment exert their maximal effect., (Copyright (c) 2006 S. Karger AG, Basel.)

Objectives: Balance and gait problems have repeatedly been mentioned in case descriptions of patients infected with Human Immunodeficiency Virus (HIV-1). Objective evidence of these problems has rarely been reported, however. Furthermore, the extent to which balance and gait are influenced by antiretroviral medications or comorbid psychiatric disorders has rarely been examined., Design: The study compared 78 HIV-1 seronegative volunteers to 28 HIV/AIDS patients receiving no antiretroviral therapy, 25 patients receiving only nucleoside analogue therapy, and 37 patients receiving Highly Active Antiretroviral Therapy (HAART)., Methods: The dependent measures included Equilibrium Quotient scores recorded during 3 subtests of the Sensory Organization Test (SOT), the number of falls during each subtest, the functional base of support, gait speed and cadence, single leg balance time, proximal strength, and vibrotactile threshold of the foot. The analysis employed the number of alcohol and drug abuse problems, depression severity, and body mass index as covariates., Results: ANCOVAs revealed significant decrements in the 3 HIV-1 seropositive groups relative to the control group on Equilibrium Quotient scores during the most difficult of the SOT subtests (sway-referenced support surface with eyes-closed). HIV/AIDS patients also exhibited a smaller functional base of support and greater vibrotactile thresholds. Antiretroviral treatment did not affect balance; but, it did alter sensory threshold in a complex manner., Conclusions: HIV/AIDS is associated with reliable decrements in balance and peripheral sensory function which are variably sensitive to antiretroviral treatment. The implications of these findings for mobility, and workplace or operator safety, should be contemplated.

A vaccine vector, designated AdV-gH, was constructed by inserting the complete open reading frame of MCMV gH under control of the human CMV IE-1 promoter into the E-1 region of a replication-deficient adenovirus 5. In vitro infection of QB1-293 cells and mouse embryo cells with AdV-gH resulted in expression of MCMV gH detected by IFA. Immunization of BALB/c mice with AdV-gH (1 x 10(7) PFU) given by the intranasal route induced a humoral response with antibody detected in serum of 100% of vaccines. Antibody to MCMV gH was also detected in the bronchoalveolar lavage, fecal suspensions and vaginal washings. The viral titer of lung and salivary gland of immunized mice 10 days after intranasal challenge with MCMV (1 x 10(5) PFU) were significantly reduced compared to controls, but virus infection was not prevented. Re-exposure of mice to AdV-gH 30 days after primary immunization induced a significant boost of serum antibody response. When rechallenged with MCMV intranasally, these mice had further reduction of MCMV titers in the lung and salivary glands. Such a strategy may be important in reducing horizontal transmission of CMV infections across mucosal surfaces and in altering host immunity to CMV.

The murine cytomegalovirus (MCMV) glycoprotein B (gB) gene was expressed in an adenovirus replication-deficient vector. This virus, designated Ad-gB, was used to immunize BALB/c and B6 mice by the intranasal (i.n.) route to induce an immune response. Following primary immunization, antibody was detected in serum of 100% of vaccinees, as well as the bronchoalveolar lavage, fecal suspensions and vaginal washings. The viral titer of lung and salivary gland of vaccinees 10 days after intranasal challenge with MCMV at 10(5) or 10(3)plaque forming units (PFU) were significantly reduced compared to controls. Re-exposure of vaccinees to Ad-gB 30 days after primary immunization induced a remarkable boost of serum and mucosal antibody responses and further reduction of MCMV titers in the lung and salivary glands. The ability to induce both a systemic and mucosal immune response to a specific gene product may be important in reducing horizontal transmission of CMV infections across mucosal surfaces and in altering host immunity to CMV.

These studies demonstrate that in vitro stimulation of spleen cells from murine cytomegalovirus (MCMV) immune mice with MCMV-infected fibroblasts induces production of interferon (IFN)-gamma. This response is specific to MCMV, is not generalized to heterologous viruses, and also is not H-2 restricted. Both early and late CMV antigens induce IFN-gamma. In in vitro cell depletion and direct cell selection experiments, T lymphocytes were responsible for IFN-gamma production. Although both CD4 and CD8 cells appear to be required to induce the response, the cell subset that releases the IFN-gamma is not yet undefined. In vivo, this IFN-gamma response appears early after acute infection and persists > or =1 year. The response is not seen in T cell-deficient mice. Thus, previous MCMV infection results in a virus-specific IFN-gamma response in spleen cells exposed to MCMV antigens. The pathophysiologic significance of these observations is now under study.

The murine cytomegalovirus (MCMV) monoclonal antibody 5C7:6 was used in Western analysis to probe MCMV infected murine embryo cells (MEC). This antibody recognizes three virus specific polypeptides of 130, 105, and 95 kDa and pulse-chase experiments demonstrated that these three proteins, although antigenically related, are distinct. The 105- and 95-kDa species were expressed with early kinetics, whereas the 130-kDa protein was synthesized as a true late. By screening a lambdagt11 MCMV cDNA library, the gene encoding these proteins was identified as the M25 open reading frame previously reported by Dallas et al. (Dallas, P. B., Lyons, P. A., Hudson, J. B., Scalzo, A. A., and Shellam, G. R., 1994, Virology 200, 643-650). Immunofluorescent studies monitored the location of pM25, present in the nucleus at 15 h after infection, condensing around the periphery of the nucleus at 18 h, before finally accumulating in the cytoplasm. Immunoelectron microscopy detected gold particles associated with the viral tegument of enveloped virions located in the cytoplasm and extracellular space but not with naked nucleocapsids. Western analysis of MCMV purified virions depicted the presence of the 130-kDa protein, the predominant M25 species, in mature virus particles. Together these findings provide compelling evidence that the 130-kDa M25 polypeptide is a component of the viral tegument.

The murine cytomegalovirus (MCMV) M44 gene product pp50 is normally present in the nuclei of virus-infected cells. During transient expression of pp50 in COS-1 cells, the phosphoprotein was readily detectable in the nuclei, indicating that it possesses a nuclear localization signal (NLS). Studies on the subcellular locations of N- and C-terminal deletion mutants of pp50 suggested that alterations in both the C terminus and the highly conserved N-terminal domains of pp50 affect nuclear localization. In particular, the C-terminal 11 amino acids of pp50, which includes a "KKQK" motif, were able to mediate the import of a beta-galactosidase fusion protein into the nucleus. The pair of lysine residues in this motif constitutes an essential element of the C-terminal NLS as mutation of this motif to AAQK directly affected the nuclear localization of either pp50 or beta-galactosidase fusion proteins containing the C-terminal portion of pp50. Furthermore our results indicated that the functionality of the C-terminal NLS is dependent on the structural integrity of the highly conserved N-terminal portion of the molecule, as deletion of amino acids 157-201 alone adversely affected nuclear localization. In the absence of a functional C-terminal NLS, the subcellular localization of pp50 is sensitive to potential conformational changes induced by mutations within the N-terminal half of the molecule. Under those circumstances, mutation of the YK residues at position 22-23 or deletion of amino acids 267-283 was sufficient to produce a protein that was impaired in nuclear import or retention., (Copyright 1999 Academic Press.)

Human herpesvirus-6 (HHV-6) exhibits a predominant tropism for CD4+ T-lymphocytes, but can infect other components of the blood as well as surrounding tissue and organs. To understand the role of the endothelium in the transmission and haematogenous spread of this virus, human umbilical vein endothelial cells (HUVEC) were infected with HHV-6 and monitored for viral gene expression. The presence of both early and late viral antigens was demonstrated by indirect immunofluorescence in 37.6 and 6.5%, respectively, of HUVEC. However, attempts to detect the release of infectious virus were not successful, indicating infection is semipermissive in nature. Upon continued passage of infected HUVEC monolayers, HHV-6 antigen-positive cells persisted up to 27 days post-infection. Furthermore, the virus could be recovered from HUVEC monolayers that contained fewer than 1% antigen-positive cells by co-cultivation with peripheral blood mononuclear cells. Together, these findings suggest that endothelial cells may serve as a reservoir for harbouring HHV-6.

The human cytomegaloviruses (HCMVs) appear to have the potential to disrupt production of hematopoietic cytokines. We examined the production of granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-8 by cultured and CMV-infected human umbilical vein endothelial cells (HUVECs) and compared this production with that of uninfected cells. Endothelial cells are, among other things, an integral component of human bone marrow stroma, and are responsible for production of factors that modulate the proliferation and differentiation of human hematopoietic progenitors. HCMV infection increased the production of GM-CSF in IL-1-primed HUVECs without altering GM-CSF levels in infected but unprimed HUVECs. However, this same virus was capable of causing increased production of the inhibitory cytokine IL-8. Both the viral pellet and the cleared viral supernatant appeared to contribute equally to the increased IL-8 and GM-CSF production, because each of these preparations alone was capable of exerting only half the effect seen with whole virus preparations. That both live virus and soluble protein factors within the viral stock contributed to the enhancement in GM-CSF and IL-8 production was further confirmed by inactivation with either ultraviolet or heat treatment of the viral stocks. Although the identity of the factor within the HCMV stock that contributes to this effect remains unknown, studies conducted in the presence of neutralizing antibodies or polymyxin B ruled out a role for tumor necrosis factor-alpha, IL-6, or endotoxin, all known inducers of GM-CSF. These studies indicate that HCMVs can exert both direct and indirect effects on the production of the hematopoietic factor GM-CSF and the inflammatory/inhibitory cytokine IL-8.

Murine cytomegalovirus (MCMV) infection in the lungs of T cell-deficient athymic BALB/c (Nu/ Nu) mice and their immunocompetent heterozygous (Nu/+) littermates was examined. Following intranasal inoculation, MCMV replicated in the lungs of both Nu/Nu and Nu/+ mice, but virus titers were significantly higher in T cell-deficient mice. After subcutaneous inoculation, virus disseminated to lung tissue of athymic mice, leading to progressive MCMV replication in lungs that was not seen in the immunocompetent mice. Athymic mice failed to develop an antibody response to MCMV. Histologically, athymic mice uniformly developed focal interstitial cellular aggregates adjacent to blood vessels or airways, which progressively enlarged and coalesced. Pneumonitis was not seen in the lungs of any Nu/+ mice. Thus, MCMV can replicate in the lungs without pneumonitis in immunocompetent mice, but MCMV produces a progressive focal pneumonitis during deficiency of T cell-mediated immunity.

UL9 is the origin binding protein of herpes simplex virus type-1 (HSV-1). A UL9-specific monoclonal antibody (17B) whose epitope maps to the N-terminal 33 amino acids was used to study the localization of UL9 in infected and transfected cells. We demonstrate the colocalization of UL9 and the HSV-1 single-strand DNA binding protein (ICP8 or UL29) in replication compartments, sites of viral DNA synthesis. On the other hand, UL9 does not completely colocalize with ICP8 in prereplicative sites, structures observed under conditions that inhibit viral DNA polymerase. Cells transfected with various deletion or pyruvate kinase fusion constructs were analyzed by indirect immunofluorescence assay to define the nuclear localization signal (NLS) of UL9. Deletion analysis showed that the region required for nuclear localization lies within the C-terminal DNA binding domian (amino acids 535-851). Various regions of UL9 were tested in fusion constructs for their ability to direct the normally cytoplasmic chicken pyruvate kinase protein to the nucleus. A fusion construct containing the carboxy-terminal 107 residues (amino acids 745-851) localized efficiently to the nucleus, whereas a fusion construct containing the N-terminal 660 amino acids of UL9 was unable to do so. Mutations designed to alter a potential NLS sequence (793-KREFAGARFKLR-804) within the C-terminal 107 residues result in a mutant UL9 protein which falls to localize efficiently to the nucleus. These results suggest that the major NLS of UL9 maps within the C-terminal 107 amino acids.

The detection of hepatitis C virus (HCV) RNA by nested polymerase chain reaction (PCR) is believed to be the most reliable method to diagnose HCV infections. A pitfall of nested PCR is that it is prone to contamination. Single step reverse transcription-PCR (RT-PCR) was performed, prospectively, on 80 sera from 59 patients with a set of primers that amplified a 273 bp sequence unique to the 5' noncoding (NC) region of the HCV genome. Nested PCR, was performed on all PCR negative specimens with a set of primers that amplified a 255 bp internal to the original primers. Single step RT-PCR was positive on 45 sera from 35 patients following gel electrophoresis and on two additional sera from two patients following Southern blot hybridization. Nested PCR was positive on two more sera following gel electrophoresis of the nested PCR products. These two patients were seropositive and subsequent serum from one patient was positive by single step PCR. Three additional sera were positive following Southern blot analysis of the nested PCR products. Two patients were seropositive and had elevated serum alanine aminotransferase (ALT) levels. The third patient was seronegative with normal ALT level and was considered a false positive. The remaining seronegative control specimens were PCR negative by both methods. The majority of PCR positive patients (82%) had elevated ALT levels, while the majority of PCR negative seropositive patients had normal ALT levels. We conclude that single step PCR is a sensitive test for the laboratory diagnoses of the majority of the HCV infections.

A rapid and sensitive radioimmunoassay for the quantitation of HCMV binding and infection of human fibroblasts (HFF) was developed. The protocol involves the use of a monoclonal antibody (27-156) reactive with HCMV gB (alpha-gB), followed by an 125I-labeled second antibody to mouse IgG. Antibody to gB bound specifically to HFF inoculated with HCMV when compared to sham inoculated cells or cells inoculated with HSV (strain KOS). Antibody to gB also bound to HFF infected with HCMV 48 h prior to assay. The binding of antibody to HFF inoculated with HCMV was found to be dependent on antibody concentration and to demonstrate saturable kinetics. Moreover, antibody binding was directly dependent on the concentration of the virus inoculum, using either conventional viral preparations or gradient purified HCMV. The binding of antibody to HFF inoculated with HCMV at 4 degrees C was found to be dependent on antibody concentration and to demonstrate saturable kinetics. Displacement of HCMV binding to HFF with the proteoglycan heparin sulfate could be detected, thus allowing for competitive binding studies. This binding assay allows for the relative quantitation of HCMV binding to cells and will be useful for examining the early events of cell-viral interactions.

To study the roles of beta 2-microglobulin (beta 2-m) and major histocompatibility complex (MHC) class I expression in human cytomegalovirus (HCMV) infection, the ability of HCMV strain AD-169 to infect and replicate in a human melanoma cell line (FO-1), which is beta 2-m-deficient and cannot express MHC class I on its cell surface, was examined. Susceptibility of FO-1 cells was compared with human foreskin fibroblasts (HFF) and FO-1H cells (FO-1 cells that have been transfected with the human beta 2-m gene, restoring MHC I expression on the cell surface). As judged by the HCMV immediate early 1 (IE-1) antigen expression, HCMV was able to infect FO-1 cells, although somewhat less efficiently than HFF. However, the expression of HCMV late (L) antigen and the production of virus was significantly less for FO-1 cells than for HFF. Analysis of the FO-1H transfectants revealed that expression of IE-1 and L HCMV antigens was comparable to FO-1 cells, which lack MHC I. Treatment of FO-1 and FO-1H cells with sodium butyrate prior to inoculation did not alter the expression of MHC I in either cell type, but did increase susceptibility of both cell types to HCMV infection, as well as the expression of L antigens and production of virus. These studies indicate that HCMV infection of FO-1 cells is independent of beta 2-m and MHC class I expression.

The role of tumor necrosis factor-alpha (TNF alpha) in acute lethal and sublethal murine cytomegalovirus (MCMV) infection in BALB/c mice was examined. During the course of acute infection, TNF alpha was not detectable in the serum or bronchoalveolar lavage (BAL) fluids, while TNF alpha was uniformly detected in both serum and BAL following intravenous administration of lipopolysaccharide (LPS). Administration of recombinant murine (rMu) TNF alpha did not consistently alter the virus content of tissues during acute infection. Passive transfer of purified polyclonal immunoglobulin containing neutralizing antibody to TNF alpha did not alter mortality or MCMV replication in tissues during acute infection but did block the TNF alpha response when LPS was administered to BALB/c mice. Thus, TNF alpha appears to play little role in the course and outcome of acute MCMV infection.

We have compared the replication of three strains of human cytomegalovirus (HCMV), HCMV AD-169, HCMV Towne, or HCMV RC-256, an insertional mutant of Towne containing the LacZ gene of E. coli, in human umbilical vein endothelial cells (HUVEC) and human forskin fibroblasts (HFF). We also examine the effects of salts of short-chain fatty acids on the susceptibility of HUVEC to infection by HCMV. All three virus strains replicated in both cell types, but 10-to 100-fold less virus was produced in HUVEC cells than HFF. For all virus strains, expression of HCMV IE-1 antigen in HFF was > 70% 24 h after inoculation. In contrast, the number of HUVEC exhibiting IE-1 antigen at 24 h was < 15%. Treatment of HUVEC with sodium butyrate, sodium hexanoate, or sodium propionate prior to virus inoculation increased the IE-1 and late HCMV antigen expression in a dose- and time-dependent manner. Virus yield was also increased. This increased susceptibility was inhibited by cycloheximide and tunicamycin, indicating a requirement for new cellular protein synthesis. Treatment with both sodium hexanoate and propionate after virus inoculation increased HUVEC susceptibility to HCMV infection. Treatment of HUVEC with sodium butyrate after virus inoculation also increased HCMV IE-1 antigen expression, but only after removal of the drug. These studies demonstrate that the susceptibility of HUVEC to HCMV infection can be increased by the treatment of the host cell with salts of short-chain fatty acids, such as sodium butyrate, before or after virus inoculation.

Events were examined that might contribute to mortality in acute murine cytomegalovirus (MCMV) infection after intraperitoneal inoculation. Specifically, viral replication in the liver, spleen, and pancreas and the concomitant biochemical abnormalities induced by MCMV during lethal and nonlethal acute viral infection were compared. Mortality was limited to susceptible strains of mice infected by the intraperitoneal (ip) route. In addition, the virus content of the lung, liver, spleen, and pancreas was 100- to 1000-fold greater with lethal infection in the ip-infected group than in those with nonlethal infection. Serum transaminase and lipase levels were markedly elevated in susceptible mice inoculated with MCMV ip. Histopathologic and immunocytochemical changes in the liver, coupled with elevated serum transaminase levels indicating severe hepatitis, appear sufficient to explain the early mortality seen with the ip route of infection.

Data derived from the murine models described in this report now serve as the cornerstone for our understanding of at least some of the mechanisms by which MCMV in combination with other cofactors can trigger the series of events that result in the development of interstitial pneumonia in the MCMV-infected mouse. What is clear from the studies completed to date is that MCMV by itself is not particularly pathogenic when it replicates in the lung, but in the presence of a perturbation in the immune system of the host, can produce serious pulmonary disease. It is hoped that the knowledge gained from these studies in mice will someday be shown to be relevant to humans.

We have developed a hybridoma, designated 25G11, which produced a monoclonal antibody (MAb) reactive with a 52K protein of murine cytomegalovirus (MCMV). This MAb, 25G11, was reactive with a protein band of 52K in MCMV-infected cell lysates and with a protein of 49K in human CMV (HCMV)-infected cell lysates as detected by immunoblot analysis. With purified MCMV virions, 25G11 gave a faintly immunoreactive band of 52K. However, no immunoreactive protein band was detected with purified HCMV virions, nor with purified HCMV or MCMV envelope preparations. By immunocytochemistry, 25G11 detected viral antigen primarily in the nucleus of HCMV- or MCMV-infected cells. The antibody 25G11 was used to screen a lambda gt11 library of HCMV DNA fragments. One of the isolated clones (lambda 32323B) was employed for gene mapping on the HCMV genome, which suggested that the immunoreactive HCMV protein was the DNA-binding protein (ICP36). Analysis of the recombinant fusion protein with antibody 25G11 and with an MAb (CH16) specific for an HCMV DNA-binding protein confirmed the identity of the cross-reacting protein as ICP36. Furthermore, we found that whereas the epitope recognized by 25G11 was conserved between HCMV and MCMV proteins, the epitope recognized by CH16 was unique to HCMV and thus represents a variable region in the protein.

The development of acute graft-vs-host disease (GVHD) is a common outcome after the injection of fully MHC disparate parental T cells into unirradiated F1 mice. Murine cytomegalovirus (MCMV) infection has been previously shown to augment the development of acute GVHD in the parent-into-F1 (P----F1) model, such that 10-fold fewer parental cells are required. In the present study, we have investigated the effect of MCMV infection on the induction of non-lethal GVHD that occurs in P----F1 combinations involving MHC class I only or class II only differences. Using P----F1 combinations involving either an H-2K only difference or an H-2D only difference, MCMV infection of F1 mice 3 days before the injection of parental spleen cells led to a profound T cell immunodeficiency that strongly resembled that observed in acute GVHD. Further studies examining the H-2K disparate P----F1 combination, C57Bl/6---- (C57Bl/6xB6.C-H-2bm1) F1 and combined MCMV infection showed that the immunodeficiency is characterized by a profound loss of in vitro Th cell production of IL-2 and an intrinsic defect in T effector function as shown by an inability of rIL-2 to restore defective CTL responses. Additional experiments in these mice revealed the presence of suppressor cells as well as significant parent-anti-F1 CTL activity possibly accounting for the suppressor effect. This pattern of immunodeficiency was not seen after the administration of either MCMV or MHC class I disparate parental cells alone. MCMV infection did not detectably alter the immunodeficiency observed in a P----F1 combination involving a MHC class II difference only. These results indicate that MCMV infection can alter the pattern of GVHD in the setting of an MHC class I disparity, but not in the setting of class II disparity, such that it resembles acute GVHD. These results may have relevance to the human transplant setting where intercurrent CMV infection has been associated with an adverse clinical outcome.

Reovirus type 3 interfered with the binding of beta-adrenergic antagonist ligands to receptors on Y1 adrenal, C6 glioma, and mouse L cells. This inhibition of beta-adrenergic binding was dose related. Reovirus did not interfere with dopaminergic binding or isoproterenol-induced activation of adenylate cyclase. In addition, reovirus infected Y1 cells, which bind beta-adrenergic antagonist ligands but lack agonist-induced activity. These results suggest that reovirus infection is initiated by binding to antagonist (nonfunctional) domains of the adrenergic receptor complex.

Monoclonal antibody to the NK 1.1 antigen, found on the natural killer cells of a number of strains of mice, specifically suppresses NK cell function when given in vivo. Using this monoclonal antibody, we have examined the effects of specific suppression of natural killer (NK) cells in vivo on acute murine cytomegalovirus (MCMV) infection in C57BL/10ScN mice. Administration of antibody to NK 1.1 substantially lowered the resistance of C57BL/10ScN mice to lethal virus challenge. In addition, antibody administration prior to intraperitoneal infection significantly increased MCMV replication in salivary glands, lungs, and spleens. In C3H/HeN mice, a strain that lacks the NK 1.1 antigen, antibody to NK 1.1 had no effect on virus replication or lethal infection. Thus, in vivo administration of monoclonal antibody to NK 1.1 alters the course of acute MCMV infection. These findings further substantiate the role of NK cells in defense against acute MCMV infection.

We examined the role of T lymphocytes bearing the L3T4 phenotype in acute murine cytomegalovirus (MCMV) infection. In vivo administration of rat IgG2b monoclonal antibody (MAb) GK 1.5 was used to deplete mice of L3T4+ lymphocytes during acute MCMV infection. Unlike the saline-treated controls that resolved their infections, mice receiving the MAb developed persistent and high levels of virus in the salivary gland, lung and spleen. The production of antibody to MCMV was delayed and the titres achieved were markedly less than in the controls. Despite the higher levels of virus replication, there was no increase in mortality seen in animals treated with the MAb. Following intraperitoneal challenge with MCMV, depletion of L3T4+ lymphocytes was protective, increasing the dose of MCMV required to produce death. These data indicate that T lymphocytes of the L3T4 phenotype influence the degree of MCMV replication during acute infection and may contribute to mortality following intraperitoneal virus challenge.

An enzyme-linked immunosorbent assay for immunoglobulin G antibody to encephalomyocarditis virus was developed. This assay was comparable to antibody assay by neutralization. Its adaptability should be useful for laboratory and epidemiological studies of infections due to encephalomyocarditis virus.

We have compared murine cytomegalovirus (MCMV) antibody determination by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay. A comparison of antibody detection with 146 serum samples at a 1:20 dilution showed 100% agreement (60 negatives and 86 positives) between the assays. There was close agreement of endpoint determinations of sera by both methods. After experimental MCMV infection, antibody to MCMV was detected by both assays as early as day 7, and high titers persisted as late as 6 months. In contrast to immunocompetent littermates, athymic nude mice did not develop antibody after infection. Mice lacking antibody detectable by ELISA were susceptible to lethal MCMV challenge. In a survey of animals from five commercial sources, MCMV antibody was not detected unless mice were experimentally infected. MCMV antibody determination by ELISA is a convenient method, comparable to the indirect immunofluorescence assay in sensitivity and specificity.

Sixteen weeks after inoculation, murine cytomegalovirus (MCMV) can no longer be detected in the tissues of mice. However, a 2-week course of immunosuppression with antilymphocyte serum and cortisone acetate results in reactivation and dissemination of the latent virus in all animals. In this study of reactivation, MCMV was first detected in the liver, usually during the first week of immunosuppression, and virus replication was shown to be restricted to hepatocytes. Subsequently, a viremia occurred, with spread of infection to other organs. The highest titers of virus were reached in salivary glands in which replication occurred in serous acinar cells. In the lung, virus-specific abnormalities were difficult to detect because of superimposed bacterial and fungal infections. However, interstitial pneumonitis could be produced when cortisone acetate was deleted from the immunosuppressive regimen. Although the site of virus latency has not been defined, this model system will be useful for study of reactivation of latent cytomegalovirus infection.

Acute murine cytomegalovirus (MCMV) infection alters the course of graft-vs-host (GVH) disease involving major histocompatibility (MHC) antigens and induces interstitial pneumonitis. F1 (B10 x B10.BR) mice given 20 x 10(6) B10.BR spleen cells and MCMV (1 x 10(5) plaque-forming units [PFU]) develop severe, diffuse pneumonitis not seen with either MCMV or GVH alone. As one index of the host immune processes operating in the lungs during MCMV/GVH pneumonitis, we examined the types of cells recovered from the lung by bronchoalveolar lavage (BAL) during pneumonitis. During MCMV/GVH pneumonitis, the total cells recovered significantly increased, due primarily to an influx of Thy 1.2 lymphocytes. Characterization of cells using multiparameter flow cytometric analysis revealed that greater than 80% of all BAL cells were Thy 1.2-positive lymphocytes of donor origin. In addition, donor Thy 1.2-positive cells were of both the L3T4+ (43% of BAL cells) and Lyt 2+ (38% of BAL cells) phenotype. Thus, MCMV infection during GVH to MHC antigens induces interstitial pneumonitis, characterized by an influx of T lymphocytes (both helper and suppressor/cytotoxic) from the donor. The antigenic specificity of these cells is not known.

Glycoproteins are integral components of cell-surface structure and participate in adherence of pathogenic microbes to host cells. We have initiated studies of the glycoproteins of Pneumocystis carinii. Biotin-conjugated lectins, followed by reaction with avidin-peroxidase, were used to detect glycoproteins in electrophoretically separated proteins of P. carinii and on whole organisms when using light microscopy. Glycoproteins of P. carinii were clearly different from rat cell glycoproteins. Multiple glycoproteins were present in P. carinii and exhibited intense reactivity to both concanavalin A and wheat-germ agglutinin. Those lectins that reacted with the electrophoretically separated proteins also stained both alcohol-fixed P. carinii and the extracellular granular material present only in P. carinii preparations. In electron micrographs of P. carinii, which were stained with colloidal-gold-labeled concanavalin A, we found that the lectin bound to the outer surface of the organisms and to the tubular extensions emanating from the exterior surface.

Cytomegalovirus (CMV) is a major source of morbidity for immunocompromised patients, such as AIDS patients. The folic acid antagonists have not been explored as potential antiviral agents against CMV. We examined the effects of methotrexate, compared to acyclovir and ganciclovir, on both murine CMV (MCMV) and human CMV (HCMV) in vitro. Using a plaque assay in mouse embryo cells or human foreskin fibroblasts for MCMV and HCMV respectively, we found that methotrexate, in micromolar concentrations, was a potent inhibitor of both viruses. This effect was due to folic acid antagonism since folinic acid abrogated the antiviral effect of methotrexate, but not ganciclovir. Cellular toxicity due to methotrexate appeared insufficient to account for the antiviral effects. The ability of methotrexate to inhibit CMV in vivo merits exploration.

The severity of the graft-versus-host (GVH) reaction, judged by splenomegaly and immunosuppression, was augmented by murine cytomegalovirus (MCMV) infection. Profound GVH-induced immunosuppression was seen in adult unirradiated MCMV-infected F1, mice even after challenge with extremely low doses of parental spleen cells. Mice receiving MCMV+GVH challenge died from days 16-21, with interstitial pneumonia being the most prominent pathological lesion. Pulmonary disease was unrelated to levels of viral replication in the lung. These results suggest that in human marrow recipients, cytomegalovirus infection may play a primary role both in provoking or accentuating GVH disease, as well as in the development of interstitial pneumonia.

In BALB/c mice, murine cytomegalovirus (MCMV) in conjunction with a single dose of cyclophosphamide (CP) induces a diffuse interstitial pneumonitis not seen with either virus or CP alone. To gain insight into the host immune mechanisms operating in the lung during interstitial pneumonitis, we examined the cells recovered in bronchoalveolar lavage (BAL) fluids of mice with MCMV with and without CP. During MCMV interstitial pneumonitis, there was a significant increase in the total BAL cells recovered, primarily because of an influx of lymphocytes bearing the Thy 1.2 marker. Although the number of cells with Lyt 1 and Lyt 2 markers increased, the most significant increase was in the proportion of lymphocytes with surface asialo-GM1. Delineating the roles of these various cell populations may provide insight into the pathogenetic mechanisms leading to CMV interstitial pneumonitis.

During studies of the pathogenesis of murine cytomegalovirus (MCMV) infection in athymic nude mice, we noted striking virus involvement of the adrenal glands. Because patients with the acquired immunodeficiency syndrome (AIDS) have recently been reported to have adrenal necrosis and evidence of infection of the adrenal gland with human cytomegalovirus (HCMV), we have further evaluated adrenal gland involvement during MCMV infection. Following virus inoculation, MCMV replicated to high titer in the adrenal glands of T-cell deficient, homozygous nude mice, but not heterozygous littermates with intact T-cell function. Concomitant with the high titers of virus, there appeared overt histological evidence of herpes-virus virus infection accompanied by patchy necrosis of adrenal cortical and medullary tissues. Acyclovir, which inhibits growth of MCMV, reduced virus replication in the adrenal gland. Similarly, virus replication was diminished in homozygous nude mice immunologically reconstituted by infusion of normal spleen cells three weeks prior to infection. Thus, in the absence of functioning T lymphocytes, MCMV can infect and replicate in adrenal tissues causing a progressive destructive adrenalitis.

Recently, the biological and biochemical characteristics of EMC virus have received considerable attention, but little is known concerning the relative susceptibility of various mammalian cells to EMC virus. In this study, a variety of mammalian cell types was evaluated for susceptibility to the M strain of EMC virus by comparing peak viral titers, time course of infection, plaquing efficiency, and formation of viral antigens. Infection of all murine and guinea pig cell types resulted in substantial virus replication and induction of viral antigens in greater than 95% of cells. Human cell lines supported replication with a slower time course, but significant increases in viral yield and plaquing efficiency occurred with viral adaptation following successive replication cycles in human cells. All rat cell lines were relatively resistant to EMC virus infection and replication, and less than 1% of rat cells expressed viral antigens. Attempts were unsuccessful to adapt virus by successive replication cycles in rat cells. The possible reasons for the relative resistance of rat cells are currently under investigation.

Murine Y-1 cells, a continuous epithelial cell line derived from an adrenal adenocarcinoma, are highly susceptible to murine cytomegalovirus (MCMV) infection. Inoculation of these cells with MCMV results in production of infectious virus and is associated with development of both cytopathic changes and the expression of viral-specific nuclear and cytoplasmic antigens. Examination of the ultrastructure of infected Y-1 cells reveals MCMV in various stages of replication. Cytomegalovirus replication in this hormone-responsive epithelial cell line should provide a model system in which virus--host cell interactions and their effects on cellular metabolism can be studied.

We reviewed the charts of 59 pediatric and adult patients hospitalized because of animal bites (46 dog bites, 10 cat bites, 3 monkey bites). The bites of 40 of the 59 patients were infected at the time of admission. Gram-stained specimens correctly predicted the infecting bacteria in only 5 of 20 cases. Eighty-three percent of the bacterial isolates were penicillin-susceptible. Before admission 14 patients had received outpatient antibiotic prophylaxis and the infections in 11 of these 14 patients were caused by bacteria susceptible to the prophylactic antibiotic. Complications were more common if antimicrobial therapy had not been altered according to susceptibility testing results. Of the 59 patients 19 were admitted immediately after being bitten because of severe uninfected bites. Of these 19 patients 18 received prophylactic antibiotics and none developed a serious complication.

Acute murine cytomegalovirus (MCMV) infection enhances the ability of parental spleen cells to induce graft-vs.-host immunodeficiency (GVHID) in F1 hybrid mice when the two processes occur simultaneously in the recipient. The present study assessed GVHID as the ability of spleen cells to generate in vitro cytotoxic T lymphocyte responses to trinitrophenyl-modified syngeneic cells. The results indicate that MCMV infection not only reduces the number of parental spleen cells required to induce GVHID, but accelerates the onset of GVHID, which occurs as early as 3 days after cell and virus challenge. To determine whether MCMV infection exerts this synergistic effect primarily through the donor or the host component, we examined the effect of MCMV infection of either donor mice or recipient mice at 3, 10, and 17 days prior to spleen cell transfer. Two weeks after cell transfer, splenocytes were tested for their ability to generate CTL. When donor mice were infected with MCMV three days prior to cell transfer, the ability of donor cells to induce GVHID was reduced. In contrast, MCMV infection of the recipients three days prior to cell transfer increased their susceptibility to GVHID induction. Infection of either donor or host mice 10 days or 17 days prior to parental spleen cell transfer had little effect on the ability to induce or resist GVHID when compared with sham-infected mice. Thus, acute MCMV infection can modulate the severity of GVHID depending on whether it is the donor or the host that is infected. The ability of acute MCMV to alter the course and severity of GVHID may be relevant for human bone marrow transplants in which preceding CMV infection has been associated with chronic GVH. In this setting, CMV may lower the threshold necessary to induce a GVH reaction.

Macrophages infected in vitro with murine cytomegalovirus (MCMV) manifest depressed phagocytic uptake of a variety of particles within hours after the initiation of infection. Analysis of kinetics of uptake of radiolabeled Staphylococcus aureus by MCMV-infected macrophages indicates that the diminished uptake results from a depression in the calculated maximum velocity of uptake (Vmax) with the apparent Michaelis constant (KM) remaining unaltered. This pattern of altered uptake is typical of that seen after manipulations that affect the surface interactions of macrophages with ingestible particles. Coincubation of macrophages and radiolabeled Staphylococcus with opsonizing antibody resulted in normalization of the phagocytic rates. The surface localization of the defective phagocytosis was further confirmed by light and scanning electron microscopy of the macrophages incubated with Staphylococcus or latex spherules. These data indicate that defective macrophage surface that interferes with the initial macrophage-particle interactions that initiate nonimmune phagocytosis.