Transmembrane communication in cells chronically infected with measles virus.

The transmembrane association of the measles virus hemagglutinin and hemolysin surface proteins with intracellular viral antigens was studied. Rabbit antisera monospecific for measles virus matrix and nucleocapsid proteins and a human antiserum containing specificities for both the hemagglutinin and hemolysin proteins were used to study the co-capping of these proteins in human Lu 106 cell-line, chronically infected with measles virus. Capping of the surface-associated envelope components was accompanied by co-capping of the matrix and nucleocapsid proteins, the latter being localized mainly within the inclusions. This demonstrated transmembrane communication between surface-associated envelope components and the intracellular measles virus matrix and nucleocapsid proteins. The results demonstrated the existence of a linkage between viral inclusions and viral proteins associated with cell membranes. In the presence of cytochalasin B (1--2 micrograms/ml), co-capping of the matrix protein was unchanged or slightly enhanced, whereas co-capping of the nucleocapsid protein decreased, indicating that actin filaments may mediate the communication between viral nucleocapsids and the cell membrane.

Impairment of short-term memory and Korsakoff syndrome are common in AIDS patients with cytomegalovirus encephalitis.

BACKGROUND AND PURPOSE: The diagnosis of cytomegalovirus encephalitis (CMV-E) in AIDS patients is challenging as other illnesses may obscure the symptoms. Here, we characterize the clinical symptoms of CMV-E and link them to post-mortem findings. Patients and methods In 254 homosexual men with AIDS, followed from HIV diagnosis to death before the antiretroviral combination therapy era, CMV-E was suspected in 93 cases. All were CMV-positive in blood. Neurological examination, including cognitive testing was performed in 34 of them within 6 months before death. CMV-E was diagnosed by CMV-PCR in cerebrospinal fluid (n = 24) or by post-mortem (n = 24). RESULTS: The majority complained of forgetfulness (91%), balance difficulties (85%) and impotence (85%). Impaired short-term memory was present in 29 patients. It was extreme in 17, justifying the diagnosis of Korsakoff's syndrome. This was often associated with infectious CMV in blood (P = 0.01). Brainstem symptoms were found in 19 patients. Post-mortem examination often revealed ventriculoencephalitis. CMV was found primarily around the ventricles and in other structures, described in Korsakoff's syndrome. CONCLUSION: The location of CMV in the brain corresponded well to the clinical findings, demonstrating the close relationship between the neurological symptoms and the neuroanatomical lesions.

The X4 phenotype of HIV type 1 evolves from R5 in two children of mothers, carrying X4, and is not linked to transmission.

Previously, we found that emergence of the X4 viral phenotype in HIV-1-infected children was related to the presence of X4 in their mothers (C.H. Casper et al., J Infect Dis 2002; 186:914-921). Here, we investigated the origin of the X4 phenotype in the child, analyzing two mother-child pairs (Ma-Ca, Mb-Cb) where the mothers carried X4 and their children developed X4 after an initial presence of R5. We used nested polymerase chain reaction of the env V3 region to generate 203 HIV-1 clones for sequencing (Ma, n = 44; Ca, n = 73; Mb, n = 61; Cb, n = 25) from DNA of peripheral blood mononuclear cell (PBMC) lysates, altogether 167 clones, or from cDNA of plasma RNA, 36 clones. PBMC and plasma isolate sequences from each time point enabled us to assign the probable phenotype to clone sequences in a phylogenetic tree. The transmission and evolution were reconstructed using the maximum likelihood method. In mother-child pair Ma-Ca, one maternal R5 isolate clustered with the child's R5 sequences, at the earliest time when R5 was isolated in the child, confirming this as a likely source of the transmitted R5 phenotype. At age 3, an X4 population was present in the child that had evolved from the child's own R5-associated population, clearly distinct from the maternal X4 sequences. The second mother-child pair (Mb-Cb) displayed a similar pattern. Amino acid substitution patterns corroborated the conclusions from the phylogenetic tree. Thus, in both children, the X4 virus developed from their own R5 population, and was not caused by transmission of X4.

Effect of heat on extracted HIV viral infectivity and antibody activity using the filter paper technique of blood sampling.

The diagnostic potential of the standardized filter paper technique at an outpatient clinic was further evaluated for individuals infected with HIV; the inactivation of HIV in dried blood from filter paper was also investigated. Heat treatment at 56 degrees C or 70 degrees C for 1 h did not demonstrably affect antibody activity as measured by enzyme-linked immunosorbent assay and Western blot. Prolonged treatment up to 18 h reduced the activity to some extent. Drying at room temperature of filter papers which had been soaked with HIV was sufficient to inactivate low, but not high, amounts of infectious virus. After exposure of the filter paper discs to 56 degrees C or 70 degrees C for 1 h, HIV could not be isolated from the extracts. The obvious advantage of blood collection on filter paper for serological studies makes it an alternative to venipuncture, especially when the blood might be contagious for HIV.

The sensitivity of HIV-1 DNA polymerase chain reaction in the neonatal period and the relative contributions of intra-uterine and intra-partum transmission.

OBJECTIVE: To derive reliable estimates of the sensitivity of HIV-1 DNA polymerase chain reaction (PCR) in the neonatal period and to quantify the relative contributions of intra-uterine and intra-partum transmission. METHODS: After reviewing studies on the early diagnosis of HIV-1 infection, investigators were asked to provide published and unpublished PCR test results on prospectively followed, non-breastfed, vertically infected children. Age-specific estimates of the sensitivity of PCR were derived using distribution-free methods for interval-censored data. RESULTS: Data on 271 infected children were combined for analysis. PCR detected HIV-1 DNA in an estimated 38% [90% confidence interval (CI), 29-46] of HIV-infected children tested on the day of, or day after, birth. Sensitivity was observed to rise rapidly in the second week of life, reaching 93% (90% CI, 76-97) by 14 days of age. CONCLUSION: The sensitivity of PCR in the neonatal period is higher than previously reported. This affects the clinical interpretation of an early negative test result and encourages the use of PCR as an endpoint for trials to evaluate interventions to reduce vertical transmission in non-breastfed populations. Approximately one-third of vertically acquired HIV-1 infection could be attributable to intra-uterine transmission.

HIV isolation from cerebrospinal fluid in relation to immunological deficiency and neurological symptoms.

Human immunodeficiency virus (HIV) could be isolated from the cerebrospinal fluid (CSF) of the majority (62%) of 72 patients in various stages of HIV infection. This high rate of successful virus isolation was achieved only when the time from lumbar puncture to initiation of the cell cultures was short, i.e. not exceeding 5 h. The HIV isolation rates were equally high in patients with persistent generalized lymphadenopathy (PGL), AIDS-related complex (ARC) and AIDS. Although the HIV recovery rate was low in patients with normal immunological parameters it was not correlated with the degree of severity of the immunodeficiency in the other patients. Furthermore, the recovery rates were not significantly correlated to the duration of the infection. HIV was recovered as often from patients with neurological symptoms as from patients without such symptoms. These findings suggest that in the majority of patients there is central nervous system (CNS) involvement early in the course of HIV infection and that HIV replication in the CNS may occur in the absence of a pronounced systemic cellular immunodeficiency and frequently without causing overt neurological symptoms.

Rapid detection of cytomegalovirus DNA and RNA in blood of renal transplant patients by in vitro enzymatic amplification.

Cytomegalovirus infection causes significant morbidity and mortality in renal transplant patients. The only marker of CMV infection that appears to correlate with the development of symptomatic illness is viremia. However, CMV grows slowly in tissue culture, requiring 2-6 weeks of incubation for detection of characteristic cytopathic effect. The efficacy of antiviral therapy for CMV may be improved by earlier detection of viremia and institution of antiviral therapy. We performed amplification of CMV DNA and RNA from peripheral blood of renal transplant patients using the polymerase chain reaction (PCR) technique. We consistently detected CMV DNA by PCR earlier than CMV was detected by culture. Detection of CMV RNA in one patient confirmed the presence of actively replicating virus in peripheral blood. Amplification of peripheral blood from healthy CMV-seropositive and seronegative individuals, and from seropositive renal transplant patients without evidence of active CMV disease, was consistently negative. These preliminary data indicate that PCR may provide a means for earlier diagnosis of CMV viremia. Future prospective studies should determine if early detection of CMV DNA by PCR in peripheral blood does predict viremia and symptomatic illness, and if earlier institution of antiviral therapy based on PCR results improves outcome for the CMV-infected transplanted patient.

Coreceptor change appears after immune deficiency is established in children infected with different HIV-1 subtypes.

Change of HIV-1 coreceptor use has been connected to progression of disease in children infected with HIV-1, presumably subtype B. It has not been possible to discern whether the appearance of new viral phenotypes precedes disease development or comes as a consequence of it. We studied the evolution of coreceptor use in HIV-1 isolates from 24 vertically infected children. Their clinical, virological, and immunological status was recorded and the env V3 subtype was determined by DNA sequencing. Coreceptor use was tested on human cell lines, expressing CD4 together with CCR5, CXCR4, and other chemokine receptors. The children carried five different env subtypes (nine A, five B, four C, three D, and one G) and one circulating recombinant form, CRF01_AE (n = 2). Of the 143 isolates, 86 originated from peripheral blood mononuclear cells (PBMCs) and 57 originated from plasma, received at 90 time points. In 52 of 54 paired plasma and PBMC isolates the coreceptor use was concordant. All 74 isolates obtained at 41 time points during the first year of life used CCR5. A change from use of CCR5 to use of CXCR4 occurred in four children infected with subtype A, D, or CRF01_AE after they had reached 1.5 to 5.8 years of age. There was a significant association with decreased CD4+ cell levels and severity of disease but, interestingly, the coreceptor change appeared months or even years after the beginning of the immunological deterioration. Thus CXCR4-using virus may emerge as a possible consequence of immune deficiency. The results provide new insights into AIDS development in children.

Nosocomial calicivirus gastroenteritis in a pediatric hospital.

At St. Göran's Children's Hospital (a tertiary center), we perform electron microscopy of feces in most cases of nosocomial gastroenteritis. From September 1987 through April 1992 we identified 32 episodes of calicivirus infection, 25 of which were nosocomial and, except for one outbreak, sporadic. Systematic study of the nosocomial outbreak of calicivirus gastroenteritis from November 1991 to January 1992, revealed calicivirus in the stools of 8 of 23 children with diarrhea and 0 of 10 without diarrhea. In 3 of 7 sampled after cessation of diarrhea, calicivirus excretion continued for 3 to 6 days. We found no calicivirus in 42 staff members or 9 members of infected patients' families. Nosocomial transmission of calicivirus can occur among infants.

Astroviruses as a cause of nosocomial outbreaks of infant diarrhea.

During a 16-month study period at a children's hospital, 32 children developed nosocomial gastroenteritis caused by astroviruses. Twenty-five of these occurred during 2 epidemic outbreaks in medical and surgical infants' wards. From the community, 13 confirmed cases were admitted during the study period. Both community-acquired and nosocomial cases occurred during autumn, winter and early spring. The attack rates during outbreaks ranged between 7 and 62% and were highest among children with underlying gastrointestinal diseases. Diarrhea and vomiting were the most common clinical manifestations. The median duration of symptoms was 4 days and that of virus excretion was 5 days. Hospital infection with astroviruses is common and usually affects children less than 2 years of age. The probable mode of transmission is spread via contaminated hands.

Foscarnet for pre-emptive therapy of CMV infection detected by a leukocyte-based nested PCR in allogeneic bone marrow transplant patients.

Fifteen allogeneic BMT patients in a phase II study were given foscarnet 60 mg/kg twice daily for 14 days as pre-emptive therapy against CMV disease. CMV infection was diagnosed by a leukocyte-based nested PCR. All 15 patients were evaluable for toxicity. One patient did not fulfill the inclusion criteria of two consecutively positive CMV PCR tests and therefore was not evaluable for efficacy. Thus, 14 of 15 patients were evaluable for development of CMV disease. None of the patients developed CMV disease and all 14 assessable patients had a negative CMV isolation at the end of therapy. None of the 15 patients had to discontinue therapy due to toxicity. Six patients reported mild gastrointestinal disturbances, three patients headaches, and three patients mild urethritis or hemorrhagic cystitis. Serum-electrolyte disturbances were common including abnormal magnesium, potassium and calcium levels. Two patients developed mild serum-creatinine increases requiring adjustment of the foscarnet dosage according to protocol. We conclude that a dosage of foscarnet of 60 mg/kg given twice daily seems to be safe and effective in preventing CMV disease in allogeneic BMT recipients. A study comparing foscarnet and ganciclovir is indicated.

Use of a semi-quantitative PCR for cytomegalovirus DNA as a basis for pre-emptive antiviral therapy in allogeneic bone marrow transplant patients.

The aim of this study was to evaluate the efficacy of pre-emptive antiviral therapy, based on a semi-quantitative nested PCR for cytomegalovirus (CMV) DNA in leukocytes, for the prevention of CMV disease after allogeneic BMT. Fifty-eight patients were prospectively followed with PCR for CMV DNA and antiviral therapy with ganciclovir was initiated after two consecutive positive tests. The levels of CMV DNA were determined by serial dilutions of the positive samples. The probability of detection of CMV DNA was 48.3% and the probability of CMV disease 6% at 100 days after BMT. Patients with CMV disease had higher CMV DNA levels compared with patients without CMV disease (P = 0.001). In comparison to 58 matched historical controls detection of CMV DNA was 5 days earlier (NS) and antiviral therapy could be initiated 10 days earlier in patients followed by PCR (P = 0.05). Pre-emptive antiviral therapy was given to 28 patients in a total of 36 courses. Patients became negative in PCR after 28 of 36 courses (77%). We conclude that PCR for CMV DNA can be used for early detection of CMV infection and as the basis of initiation of pre-emptive antiviral therapy in BMT patients.

Respiratory infection and iatrogenic diarrhea in Honduras and El Salvador during the 1991-1992 season.

The etiology of acute respiratory tract infection and its association with diarrhea was analyzed in 135 hospitalized children less than three years of age with mainly respiratory symptoms in two pediatric hospitals in Honduras and El Salvador. Etiologic diagnoses were performed on nasopharyngeal samples by tissue culture and immunofluorescence, including a search for the presence of respiratory virus-specific immunoglobulin A antibodies. Fecal samples were subjected to electron microscopy and tissue culture. The majority (83%) of the children (65% boys and 35% girls) were less than 12 months old and 45% were less than four months old. In 63 (47%) patients, at least one viral infection of the respiratory tract was identified. Respiratory syncytial virus (RSV) or the respective IgA antibodies were found in 43 (32%) patients., influenza A and B viruses in 29 (21%) patients, adenovirus in four patients, and enterovirus in two patients. Twenty (15%) patients had two or more viral agents. An association between RSV cases and environmental temperature was established. In 124 fecal samples, we identified four cases with astrovirus, seven with nonpolio enterovirus, and eight with adenovirus, but only three cases with rotavirus. Diarrhea was present in 55 (45%) of the patients. There was a statistically significant association between diarrhea and cases with RSV. This was shown to be associated with antibiotic treatment.

Enterovirus IgM detection: specificity of mu-antibody-capture radioimmunoassays using virions and procapsids of Coxsackie B virus.

A predominantly type-specific mu-capture radioimmunoassay (RIA) of IgM antibodies to Coxsackie B1-B5 (CB1-CB5) viruses was previously described (Frisk et al., 1984). The present study is concerned with the specificity of this assay, using as antigen different strains of one serotype (CB5) and procapsids of two serotypes (CB3 and CB5). Eight strains of CB5 virions were tested against acute and/or convalescent sera from 10 patients from whom CB5 had been isolated. Seven patients' sera were tested against their own strain. The frequency of IgM-positive patients varied from 9 of 10 (90%) to 5 of 10 (50%). In three cases the highest titres were obtained with the patients' own strain. When sera from patients with other enterovirus infections were tested against the CB5 strains, heterotypic titres were obtained to a certain extent (0-15.6%). The strains giving a high frequency of homotypic titres varied concerning heterotypic reactions. It is concluded that the choice of strain is important if a high frequency of homotypic titres with no or only a few heterotypic reactions is to be obtained. When procapsids were used as antigen, both homotypic and heterotypic titres were seen to a large extent. All patients with homotypic IgM against CB3 or CB5 virions showed IgM against the CB3 or the CB5 procapsids, respectively. When sera from patients with other enterovirus infections were tested, IgM was found in 54 of 93 patients (58%) with use of the CB3 procapsid and in 52 of 87 patients (60%) with the CB5 procapsid. It was often not the same patients who showed IgM against the two different procapsids. When both procapsids were used, IgM positivity was found in 62 of 81 patients (77%) with other enterovirus infections. It is concluded that the use of two or more procapsids in combination is of value for the diagnosis of a recent or current enterovirus infection.

Rapid detection of cytomegalovirus using immune scanning electron microscopy.

Monoclonal antibodies, specific for human cytomegalovirus, were conjugated to latex microspheres that were already labelled with rabbit anti-mouse immunoglobulin. The beads were then incubated with serum or urine from patients, and then collected on a filter surface, which was analyzed in a scanning electron microscope. Size, immunological specificity, and relative quantity of virus particles were determined within 2 h after serum or urine collection by the visualization of virus particles specifically bound to the latex-bead surface. No such binding of virus particles was detected in the various controls. This method was compared with conventional virus isolation by tissue culture. It enables identification of viruses within a few hours in different body fluids. Even without specific antibodies, the filtration method may permit the rapid detection of particles and the determination of their size in various body fluids.

Rapid detection of cytomegalovirus using immune scanning electron microscopy.

Monoclonal antibodies specific for human cytomegalovirus were conjugated to latex microspheres that were already labelled with rabbit anti-mouse immunoglobulin. The beads were incubated with serum or urine from patients, and then collected on a filter surface, which was analyzed in a scanning electron microscope. Size, immunological specificity, and relative quantity of virus particles were determined within 2 h after serum or urine collection by the visualization of virus particles specifically bound to the latex bead surface. No such binding of virus particles were detected in the various controls. This method was compared with conventional virus isolation by tissue culture. It enables identification of viruses within a few hours in different body fluids. Even without specific antibodies, the filtration method may permit the rapid detection of particles and the determination of their size in various body fluids.

Immune responses and resistance to viral-induced myocarditis in mice exposed to cadmium.

The effects of 10 weeks of treatment with cadmium (Cd) on the immune function and resistance to coxsackievirus B3 (CB3)-induced myocarditis in female Balb/c mice were investigated. A 2mM dose of Cd in the drinking water did not influence mortality due to the CB3 infection. The inflammatory and necrotic lesions in the ventricular myocardium seven days after inoculation (2.94% of tissue section area) were not increased by Cd (2.82% of tissue section area). The response pattern of lymphocyte subsets in situ in myocardial inflammatory lesions was elucidated by an immune histochemical staining technique. With Cd treatment the number of cytotoxic T cells and B cells in these lesions decreased by 22% (n.s.) and 21% (p < 0.05), respectively. Spleen weight and the lymphoproliferative response to the B-lymphocyte mitogen increased by 19% (p < 0.05) and 23% (n.s.), respectively. The titers of neutralizing antibodies increased by 22% (n.s.) with Cd treatment. However, the activity of spleen T lymphocytes and spontaneous cell-mediated cytotoxicity (NK-cell) was unchanged. Thymus weight and WBC count in peripheral blood tended to decrease. Thus, Cd exposure seems to result in a decreased maturation and mobilization of T and B lymphocytes, but increased humoral immune host responses.

[Reduced mother-to-child transmission of HIV-1 infection. Exclusive breast feeding and weaning--a possible model in developing countries?]. / Minskning av HIV-1-smitta från mor till barn. Exklusiv amning och tidig avvänjning--möjlig modell i u-länder?

Antiretroviral therapy with zidovudine during pregnancy, delivery, and to the newborn in combination with Cesarean section has markedly reduced the rate of mother-to-child transmission of HIV-1 in industrialized countries. These strategies are not realistic in the less developed world. Nevertheless, short-term antiretroviral therapy during delivery and shortly after birth has proven feasible as a means of reducing the rate of vertical transmission in less developed countries, in some studies even in combination with exclusive breast-feeding. The risks and benefits of breast-feeding among HIV-1 infected mothers are discussed.