Performance of Tsol-p27 antigen for the serological diagnosis of cysticercosis in Mozambique.

The diagnosis of neurocysticercosis (NCC) requires expensive neuroimaging techniques that are seldom affordable for people in endemic countries. Accordingly, there is a need for new low-cost diagnostic methods that offer high sensitivity and specificity. In this study, we evaluated Western blot analysis of the previously described recombinant antigen Tsol-p27 in relation to a commercial or in-house enzyme-linked immunosorbent assay (ELISA) for NCC, and compared the results with those provided by a commercial enzyme-linked immunoelectrotransfer blot (EITB) assay, which was regarded as the reference standard method. The analysed serum samples were obtained from 165 people, 18 of whom were confirmed to be NCC positive by EITB. Comparing our Western blot analysis of Tsol-p27 with a previous evaluation performed in Central America showed similar specificity (96.69% versus 97.8%) and sensitivity (85.71% versus 86.7%). The present results indicate that the recombinant Tsol-p27 antigen provides good sensitivity and specificity, and might be preferable as a diagnostic antigen in poorly equipped laboratories in endemic countries.

Stability of a formalin-inactivated Rift Valley fever vaccine: evaluation of a vaccination campaign for cattle in Mozambique.

In Africa and the Arabian Peninsula, outbreaks of Rift Valley fever (RVF) are characterized by abortions in gestating animals and high mortality rates among domestic ruminants. An immunization program using a formalin-inactivated vaccine was initiated in Mozambique in 2002 to control RVF in cattle. In this intervention, the vaccine must be transported for more than a week within the country before it can be administered to the animals, and it is practically impossible to maintain low storage temperatures during that time. Here, we evaluated the influence of transportation conditions on the efficacy of the vaccine. Sixty-three previously unvaccinated and RVF virus seronegative cattle were divided into four groups, which were given vaccine that had been stored for 1 week at 4°C (n=9, group A), at 25°C (n=8, group B), or alternating between 4 and 25°C (n=8, group C), or under the temperature conditions ordinarily occurring during transportation within Mozambique (n=38, group D). The antibody responses induced were monitored for 6-9 months and in some animals up to 21 months. Two immunizations (3 weeks apart) with the formalin-inactivated vaccine induced a long-lasting neutralizing antibody response that was still detectable up to 21 months later. The antibody titers in the animals did not differ significantly between the temperature-assigned vaccine groups A, B, and C, whereas they were significantly higher in group D. These results show that the formalin-inactivated RVF virus vaccine is stable, and, importantly, it is not adversely affected by the variation in temperature that ordinarily occurs during transport within Mozambique.

Anti-Epstein-Barr virus antibodies as serological markers of multiple sclerosis: a prospective study among United States military personnel.

BACKGROUND: Elevated Epstein-Barr virus (EBV) antibody titers are risk factors for multiple sclerosis (MS), but the strength and consistency of this association are not well characterized. OBJECTIVES: The objectives of this study were to determine whether this association is confounded by vitamin D or modified by gender or race, and the usefulness of EBV nuclear antigen (EBNA) antibodies as a marker for MS. METHODS: We conducted a prospective study among US military personnel. Antibody titers against EBV antigens were measured in serum samples from 222 individuals who developed MS and 444 age, sex, and race/ethnicity matched controls. Conditional logistic regression was used to estimate relative risks. RESULTS: MS risk increased with increasing titers of anti-EBNA complex (p < 10(-9)) and anti-EBNA-1 (p = 5.8 × 10(-9)) titers. MS risk was 36-fold higher among individuals with anti-EBNA complex IgG titers ≥320 than among those with titers <20 (95% confidence interval [CI] 9.6-136), and 8-fold higher among those with anti-EBNA-1 ≥320 than among those with anti-EBNA-1 <20 (95% CI 2.6-23). These associations were consistent across gender and race/ethnicity groups and independent from 25-hydroxyvitamin D levels. Areas under the receiver operating characteristic (ROC) curves were 0.67 for EBNA complex and 0.65 for EBNA-1. CONCLUSIONS: Serum titers of pre-onset anti-EBNA antibodies are strong, robust markers of MS risk and could be useful in an MS risk score.

Gene segment reassortment between American and Asian lineages of avian influenza virus from waterfowl in the Beringia area.

Since prehistoric times, the Bering Strait area (Beringia) has served as an avenue of dispersal between the Old and the New Worlds. On a field expedition to this area, we collected fecal samples from dabbling ducks, geese, shorebirds, and gulls on the Chukchi Peninsula, Siberia, and Pt. Barrow, Alaska, and characterized the subtypes of avian influenza virus present in them. Four of 202 samples (2%) from Alaska were positive for influenza A virus RNA in two independent polymerase chain reaction (PCR)-based screening assays, while all shorebird samples from the Chukchi Peninsula were negative. Subtypes H3N8 and H6N1 were recorded once, while subtype H8N4 was found in two samples. Full-length sequences were obtained from the three unique isolates, and phylogenetic analysis with representative sequences for the Eurasian and North American lineages of influenza A virus showed that one HA gene clustered with the Eurasian rather than the North American lineage. However, the closest relative to this sequence was a North American isolate from Delaware described in 2002, indicating that a H6 spillover from Asia has established itself in North America.

Plasma titers of antibodies against Epstein-Barr virus BZLF1 and risk of multiple sclerosis.

BACKGROUND/AIMS: Results of recently conducted prospective studies have demonstrated that the presence of high titers of anti-EBNA-1 or anti-EBNA complex IgG antibodies in healthy individuals is a strong risk factor for multiple sclerosis (MS). Antibodies to BZLF1, the product of the homonymous early lytic gene, have been found to be related to risk of nasopharyngeal carcinoma, but have not been previously measured in MS studies. METHODS: We examined whether high levels of anti-BZLF1 IgG antibodies also predict MS risk in a nested case-control study among women in the Nurses Health Study and Nurses Health Study II cohorts. RESULTS: Results of this prospective study suggest that antibody titers to EBNAs are the strongest predictor of MS risk. CONCLUSION: Little further contribution may be provided by measuring anti-BZLF1 antibodies in regard to MS risk.

Exogenous nitric oxide inhibits Crimean Congo hemorrhagic fever virus.

Crimean Congo hemorrhagic fever virus (CCHFV) is a geographically widespread pathogen that causes severe hemorrhagic fever with high mortality. Even though one of the main objectives focuses on the progress of antiviral agents, the research on CCHFV is strongly hampered due to its BSL-4 classification. Nitric oxide (NO), a mediator with broad biological effects, has been shown to possess inhibitory properties against various pathogens. The molecule constitutes a component of the innate immunity and serves to assist in the early immunological events where it contributes to clearance of microorganisms. In this study, we investigated the inhibitory properties of exogenous NO on CCHFV. We found that NO had a significant antiviral activity against CCHFV replication. By using the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) we were able to show up to 99% reduction in virion progeny yield. In contrast, 3-morpholinosydnonimine hydrochloride (SIN-1), a peroxynitrite donor, had no significant antiviral activity against CCHFV. Furthermore the expression of viral proteins; the nucleocapsid protein and the glycoprotein, were clearly reduced with increasing concentrations of SNAP. We have also shown that the amount of total vRNA in SNAP-treated cells was reduced by about 50% compared to the controls.

Circulating epstein-barr virus in children living in malaria-endemic areas.

Children living in malaria-endemic regions have high incidence of Burkitt's lymphoma (BL), the aetiology of which involves Plasmodium falciparum malaria and Epstein-Barr virus (EBV) infections. Acute malarial infection impairs the EBV-specific immune responses with the consequent increase in the number of EBV-carrying B cells in the circulation. To further understand the potential influence of malarial infection on the EBV persistence in children living in malaria-endemic areas, we studied the occurrence and quantified cell-free EBV-DNA in plasma from 73 Ghanaian children with and without acute malarial infection. Viral DNA was detected in 40% of the samples (47% in the malaria-infected and 34% in the nonmalaria group) but was absent in plasma from Ghanaian adults and healthy Italian children. These findings provide evidence that viral reactivation is common among children living in malaria-endemic areas, and may contribute to the increased risk for endemic BL. The data also suggest that the epidemiology of EBV infection and persistence varies in different areas of the world.

Delayed viremia and antibody responses in Puumala hantavirus challenged passively immunized cynomolgus macaques.

No specific therapy is currently available against hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome. In order to study if passive immunization could inhibit hantavirus infection and/or symptoms, we inoculated two cynomolgus macaques with neutralizing monoclonal antibodies and subsequently challenged them with wild-type Puumala virus (PUUV), recently shown to induce typical signs of milder HFRS in cynomolgus macaques. Although viral load and antibody titers did not differ substantially as compared to the two control monkeys, a delayed onset of viremia and seroconversion was observed in the immunized monkeys. Interestingly, one of the immunized monkeys showed no symptoms, nor elevated of levels of IL-6, IL-10, and TNF-alpha, while the other developed severe symptoms and elevated levels of those cytokines, believed to be involved in PUUV-pathogenesis.

Detection of Epstein-Barr virus, but not human herpesvirus 8, DNA in cervical secretions from Swedish women by real-time polymerase chain reaction.

BACKGROUND: Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV-8) are two related herpesviruses that may be sexually transmitted. GOAL: To examine the presence of HHV-8 and EBV DNA in the female genital tract. STUDY DESIGN: Real-time polymerase chain reaction systems for quantification of DNA from HHV-8, EBV, and herpes simplex virus type 2 were developed and used for examination of cervical secretions from 112 Swedish women. HHV-8, EBV, and herpes simplex virus type 2 serology was also performed on samples from all subjects. RESULTS: EBV DNA was found in 10 cervical secretion samples, sometimes in high amounts. No cervical secretion or leukocyte sample contained detectable HHV-8 DNA. Antibodies to HHV-8-latent and -lytic antigens were found in 2.7 % and 24% of serum samples, respectively. CONCLUSION: This study supports a possible sexual route of transmission for EBV but not for HHV-8. The new real-time polymerase chain reaction systems could be valuable in future studies of relations between virus load and disease.

A repetitive sequence of Epstein-Barr virus nuclear antigen 6 comprises overlapping T cell epitopes which induce HLA-DR-restricted CD4(+) T lymphocytes.

Most human adults carry the Epstein-Barr virus (EBV) and develop immunological memory against the structural and the virus-encoded cellular proteins. The EBV nuclear antigen 6 (EBNA6) elicits cytotoxic T cell responses and it also maintains a persistent antibody response. The majority of sera from EBV-seropositive individuals reacts with a synthetic peptide, p63, comprising 21 amino acids of a repetitive region of EBNA6. CD4(+) T lymphocytes, with specificity for p63, could be recalled from the T cell repertoire of EBV carriers that expressed certain HLA-DR allotypes which were identified as good binders of p63 by an in vitro flow cytometric assay. Analysis of the HLA-DR/p63 interaction by molecular mechanics calculations indicated the presence of multiple overlapping epitopes which were predicted to bind in a HLA-DRB1 allo- and subtype-specific manner. Specific activation of p63-selected long-term CD4(+) T cell cultures resulted in a proliferative response, in the production of IL-2 and in the secretion of high levels of tumor necrosis factor as measured by bioassays. Proliferation and cytokine production of p63-specific T cells could be induced by p63-loaded HLA-DR-matched antigen-presenting cells and by B cells co-expressing relevant HLA-DR molecules and EBNA6. Our results show that peptides of an EBNA6 repeat region induce CD4(+) T cells which can react with EBNA6-carrying cells in many individuals. We suggest that these T(h) cells may be important in conditioning dendritic cells for initiation potent virus-specific immune responses, provide help for EBV-specific B cells, drive IgG isotype switch and support the sustained effector function of memory cytotoxic T lymphocytes.

Demethylation of the Epstein-barr virus origin of lytic replication and of the immediate early gene BZLF1 is DNA replication independent. Brief report.

Epstein-Barr virus (EBV) episomal DNA is extensively methylated in Burkitt lymphoma derived cell lines. In this study we examined whether lytic viral cycle reactivation is dependent on demethylation of critical viral genes. Viral replication was induced in the Burkitt's lymphoma cell line Daudi by the combination of 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium-butyrate. Two regions necessary for EBV replication, the BZLF1 immediate early region and the origin of lytic cycle replication (ori Lyt) were demethylated during the early phase of the lytic virus cycle. Demethylation was observed while production of new (unmethylated) viral DNA was blocked by phosphonoformic acid (PFA). This suggests that demethylation, which may be instrumental for the onset of the lytic cycle, is an active process independent of viral DNA replication

Horizontal transfer of DNA by the uptake of apoptotic bodies.

In this study we have raised the question of whether DNA can be transferred from one cell to another by phagocytosis of apoptotic bodies. We have used integrated copies of the Epstein-Barr virus (EBV) as a marker to follow the fate and expression pattern of apoptotic DNA in the phagocytotic host. Apoptosis was induced in EBV-carrying cell lines by irradiation before cultivation with either human fibroblasts, macrophages, or bovine aortic endothelial cells. Analysis of the expression pattern of EBV-encoded genes was performed by immunofluorescent staining as well as in situ hybridization. Cocultivation of apoptotic bodies from lymphoid cell lines containing integrated but not episomal copies of EBV resulted in expression of the EBV-encoded genes EBER and EBNA1 in the recipient cells at a high frequency. Fluorescence in situ hybridization analysis showed uptake of human chromatin as well as integrated EBV-DNA into the nuclei of bovine aortic endothelial cells. These data show that DNA may be rescued and reused from apoptotic bodies by somatic cells. In addition, our findings suggest that apoptotic bodies derived from EBV-carrying B lymphocytes may serve as the source of viral transfer to cells that lack receptors for the EBV virus in vivo.

Visualization of alternative Epstein-Barr virus expression programs by fluorescent in situ hybridization at the cell level.

Epstein-Barr virus (EBV) transforms human B lymphocytes into immortalized lymphoblastoid cell lines (LCLs). They regularly express six virally encoded nuclear proteins (EBNA1 to EBNA6) and three membrane proteins (LMP1, LMP2A, and LMP2B). In contrast, EBV-carrying Burkitt lymphoma (BL) cells in vivo and derived type I cell lines that maintain the BL phenotype express only EBNA1. During prolonged in vitro culturing, most EBV-carrying BL lines drift toward a more immunoblastic (type II or III) phenotype. Their viral antigen expression is upregulated in parallel. We have used fluorescent in situ hybridization to visualize viral transcripts in type I and III BL lines and LCLs. In type I cells, EBNA1 is encoded by a monocistronic message that originates from the Qp promoter. In type III cells, the EBNA1 transcript is spliced from a giant polycistronic message that originates from one of several alternative Wp or Cp promoters and encodes all six EBNAs. We have obtained a "track" signal with a BamHI W DNA probe that could hybridize with the polycistronic but not with the monocistronic message in two type III BL lines (Namalwa-Cl8 and MUTU III) and three LCLs (LCL IB4-D, LCL-970402, and IARC-171). A BamHI K probe that can hybridize to both the monocistronic and the polycistronic message visualized the same pattern in the type III BLs and the LCLs as the BamHI W probe. A positive signal was obtained with the BamHI K but not the BamHI W probe in the type I BL lines MUTU I and Rael. The RNA track method can thus distinguish between cells that use a type III and those that use a type I program. The former cells hybridize with both the W and the K probes, but the latter cells hybridize with only the K probe. Our findings may open the way for studies of the important but still unanswered question of whether cells with type I latency arise from immunoblasts with a full type III program or are generated by a separate pathway during primary infection.

Specific methylation patterns in two control regions of Epstein-Barr virus latency: the LMP-1-coding upstream regulatory region and an origin of DNA replication (oriP).

The Epstein-Barr virus (EBV) can establish at least four different forms of latent infection. Previously, we have shown that the level of methylation of the EBV genome varies, depending on the form of latency. The methylation status of CpGs was analyzed by the bisulfite genomic sequencing technique in four different cell types representing different forms of latency. The dyad symmetry element of the origin of replication (oriP) region and the latent membrane protein 1 (LMP-1) regulatory sequence (LRS) were studied. The dyad symmetry element has four binding sites for EBNA-1. In a cell with type I latency, a region upstream of the dyad symmetry element was highly methylated, whereas the dyad symmetry element was unmethylated in the EBNA-1-binding region. The LRS was extensively methylated in the LMP-1-negative cell line Rael, in contrast to a LMP-1-expressing nasopharyngeal carcinoma tumor (NPC C15), which was almost completely unmethylated. The methylation pattern of LRS in type I and type III Burkitt lymphoma cells of similar parental origins confirmed that demethylation of some regions takes place upon phenotypic drift.

Direct identification by PCR of EBV types and variants in clinical samples.

Both Epstein-Barr virus (EBV) type A and type B, and variants of type A, were identified simultaneously by polymerase chain reaction (PCR) amplification of a DNA region coding for a 13 amino acid repeat in the Epstein-Barr virus nuclear antigen (EBNA) 6. Whereas this region varies extensively in type A isolates, no variation was seen in type B isolates. When a repetitive region in the LMP1-coding region was amplified by PCR, it was possible to distinguish individual variants of type B isolates from each other. Forty-two saliva samples from HIV-1-carrying individuals were examined for the presence of type A and type B virus. Both types and multiple variants of each type were found with a much higher frequency than in the saliva samples from healthy individuals. Type A EBV alone was detected in mouthwash samples from 6 infectious mononucleosis (IM) patients. Both type A and B were detected in the peripheral blood B-lymphocytes (PBL) from 1 healthy individual. The same type A variant was demonstrated both in PBL and in the mouthwash sample from another healthy individual. In this study it was shown that a combination of the EBNA 6- and LMP 1-specific PCRs followed by Southern hybridisation can be used to identify both type A and type B virus, as well as to distinguish between multiple variants of the same strain, in saliva and B-cells from both healthy and immunosuppressed individuals.

Rapid occurrence of lymphoproliferative disease after pancreas-kidney transplantation performed during acute primary Epstein-Barr virus infection.

Generalized lymphoproliferative disease occurred in a 30-year-old woman 15 days after she underwent simultaneous pancreas-kidney transplantation. Because of the rapid progression of this disorder, it was necessary to remove the grafts and discontinue immunosuppression 19 days after transplantation. Serological analysis demonstrated that the patient, who was Epstein-Barr virus (EBV)-seronegative 3 months before, was seroconverting at the time of the transplantation. EBV therefore was acquired just before the transplantation, either by a blood transfusion 4 months earlier or from the patient's EBV-positive boyfriend. The latter source appeared most likely, as concluded from the investigation of the EBV strains from the patient's boyfriend and from the blood and organ donors. Donor origin of lymphoblastoid cells was excluded by sex chromosomal analysis. Initiation of immunosuppression during a primary EBV infection carries the risk of very rapid development of B-cell lymphoproliferative disease. This emphasizes the need for active monitoring of EBV infections in transplant recipients and for the development of preventive strategies.

Transmission of donor Epstein-Barr virus (EBV) in transplanted organs causes lymphoproliferative disease in EBV-seronegative recipients.

Epstein-Barr virus (EBV) is associated with post-transplant lymphoproliferative disease (PTLD). To determine whether the donor EBV isolate is transmitted to the recipient via the allograft and causes PTLD, EBV isolates from four cases of PTLD in cadaveric heart and/or lung transplant recipients were compared with the donor isolates by PCR and DNA sequence analysis. Two recipients who were EBV seronegative at transplantation acquired an EBV isolate indistinguishable from that of the donor and developed PTLD. In contrast, in two patients who were seropositive before transplantation, the donor isolate differed from that present in PTLD of the recipient. The results suggest that the acquisition of donor EBV is a risk factor for PTLD development in a previously seronegative transplant recipient.