Replication of herpes simplex virus and cytomegalovirus in human leukocytes.

Human peripheral blood leukocytes, lymphocyte subpopulations, and hemic cell lines were examined for their ability to supprot HSV and CMV replication. Mitogen-stimulated mononuclear leukocytes, B lymphocytes, and T lymphcytes supported the replication of HSV to high titers over 3 to 5 days of infection. HSV replicated in unstimulated mononuclear leukocyte cultures of one of five donors, and to a limited degree in untreated B lymphocytes of three of five donors; HSV replication was not detected in unstimulated T lymphocytes (five donors). There was no evidence of enhanced uptake of 3H-thymidine in the untreated donor cells that replicated HSV. CMV replication was not detected during 9 to 10 days of infection in untreated or mitogen-treated mononuclear leukocytes and lymphocyte subpopulations from the same adult donors or in neonatal cord blood leukocytes. The ability of the cells to support HSV or CMV replication did not correlate with the presence of specific antiviral antibodies in the donor serum. HSV replication in B, T, and myeloid cell lines to high titers over 5 days of infection, whereas CMV failed to replicate in any of the hemic cell lines. A persistent HSV infection has been established in a T cell line (CEM) with high titers of infectious virus being produced concurrently with growth of the cells over the first 11 weeks of infection.     

Humoral and cellular CMV responses in healthy donors; identification of a frequent population of CMV-specific, CD4+ T cells in seronegative donors

CMV status is an important risk factor in immune compromised patients. In hematopoeitic cell transplantations (HCT), both donor and recipient are tested routinely for CMV status by serological assays; however, one might argue that it might also be of relevance to examine CMV status by cellular (i.e., T lymphocyte) assays. Here, we have analyzed the CMV status of 100 healthy blood bank donors using both serology and cellular assays. About half (56%) were found to be CMV seropositive, and they all mounted strong CD8+ and/or moderate CD4+ T cell responses ex vivo against the immunodominant CMV protein, pp65. Of the 44 seronegative donors, only five (11%) mounted ex vivo T cell responses; surprisingly, 33 (75%) mounted strong CD4+ T cell responses after a brief in vitro peptide stimulation culture. This may have significant implications for the analysis and selection of HCT donors.     

Direct diagnostic methods for the demonstration of cytomegaloviruses in the blood

CMV can be detected in blood with two techniques: isolation of CMV from the buffy-coat on human embryonic fibroblasts; CMV genome detection by DNA hybridization technique. Isolation of CMV from blood plated on embryonic fibroblasts necessitates a delay of 10-30 days. Using monoclonal antibodies we have developed a 96 hours test to diagnose viremia. Fibroblasts grown on coverslip in tubes are inoculated with buffy-coat. Indirect immunofluorescence test is performed using the monoclonal antibodies E 13, 48 and 96 hours post-infection. DNA-DNA hybridization technique necessitates purification of CMV DNA. CMV DNA is then labelled with 32 P by nick-translation. This labelled DNA is used as probe for detection of CMV genome in DNA extracted from leukocytes. The hybridization is virus-specific. Cytomegaloviremia correlates with active infection and is not indicate of the carrier state of blood donors. CMV genome research in blood can potentially identify infective donors. Further studies are needed to correlate CMV genome detection in blood with presence of CMV antibodies.     

HLA-DR-restricted cytotoxicity of cytomegalovirus-infected monocytes mediated by Leu-3-positive T cells

Mononuclear leukocytes from 14 cytomegalovirus (CMV)-seropositive and six CMV-seronegative normal healthy donors were treated with soluble CMV antigen for 5 days to generate cytotoxic T lymphocyte (CTL) activity. CMV-antigen-stimulated lymphocytes from CMV-seropositive but not CMV-seronegative donors lysed autologous peripheral blood monocyte targets infected with CMV in 13 of 14 donors (mean percentage of virus-specific lysis = 19.0 +/- 4.5%, effector to target ratio of 50:1). Freshly donated, unstimulated lymphocytes displayed little or no lysis of CMV-infected monocytes. Lysis was virus specific in that CMV-stimulated CTL did not kill herpes simplex virus-infected monocytes. The mean level of lysis of CMV-infected autologous targets was equivalent to that of HLA-DR-matched targets (20.0 +/- 8.0%), and was significantly greater than that of HLA-A/B-matched targets (6.3 +/- 2.5%, p less than 0.035) and HLA-mismatched targets (3.3 +/- 2.5%, p less than 0.01). Enrichment for T cell subsets with the use of selective depletion methods with monoclonal antibodies showed that CTL activity against autologous and HLA-DR-matched allogeneic targets was present predominantly in Leu-3-positive T lymphocytes. These results show for the first time that short term stimulation of heterogeneous lymphocytes from CMV-seropositive donors with CMV antigen can generate CMV-specific, Leu-3-positive CTL that are primarily restricted in their activity to autologous and class II, HLA-DR-matched targets. Our findings suggest a role for Leu-3-phenotypic CTL in immunity to CMV, and provide a model for analysis of this antiviral effector function during immunodeficient states.     

T and B cell responses to cytomegalovirus antigens in healthy blood donors and bone marrow transplant recipients

Abstract We measured the production of interferon-gamma (IFN-γ) from single T cells and the T cell proliferative response to different cytomegalovirus (CMV) antigens in healthy blood donors and bone marrow transplant recipients. The antigens consisted of a CMV nuclear antigen (CMV na) containing the pp65-kDa matrix protein and the immediate early antigens but lacking CMV glycoproteins, and an antigen comprising native CMV glycoproteins (CMV gp). We also measured the IgG antibodies to CMV na and CMV gp. The T cells reacted to CMV na in CMV seropositive blood donors both with the production of IFN-γ and with proliferation, while bone marrow transplant recipients had a deficient T cell response. After stimulation with CMV gp, no T cell response could be observed in CMV seropositive subjects. IgG antibodies to CMV na coexisted in plasma with similar levels of antibodies to CMV gp.     

Infectious Mononucleosis and Mononucleosis Syndromes: Clinical,Virological and Immunological Features

Infectious mononucleosis (IM) and cytomegalovirus (CMV) mononucleosis are caused by a primary infection with related viruses, Epstein-Barr virus (EBV) and CMV. Despite the similarity of clinical manifestations, basic differences exist: (1) The heterophil antibody (HA) response is absent in CMV mononucleosis, whereas it is present in IM. (2) In IM atypical lymphocytosis reflects proliferation of B cells early and of T cells later in the disease course; in CMV mononucleosis the situation appears complex. (3) In blood, EBV is restricted to B lymphocytes, whereas CMV is found in polymorphonuclear and mononuclear leukocytes. (4) Complications of CMV mononucleosis such as hepatitis and pneumonitis may be due to virus cytopathic effect in target organs. Prominent tonsillopharyngitis with adenopathy, and visceral complications of IM are related to lymphoproliferation which is self-limited except in males with a rare familial defect in defense against EBV. Immune complex-mediated pathology may occur in both diseases. (5) CMV is frequently transmitted to a fetus in utero or to an infant during or after birth, and this occasionally leads to severe cytomegalic inclusion disease; vertical transmission of EBV appears to be exceptional. (6) Secondary EBV infections are associated with certain malignancies whereas such an association has not been recognized in the case of CMV.Toxoplasma gondii is another cause of HA-negative mononucleosis. Its complications in the heart, in skeletal muscle and in the central nervous system are related to direct invasion by the parasite. Cellular immunity plays an important role in defense against all three agents.     

Problems posed by the preparation of specific anti-cytomegalovirus immunoglobulins

The authors describe the preparation of a first batch of intravenous cytomegalovirus (CMV) immune globulin at the Nancy Regional blood transfusion centre. Immune plasmas were selected from 3 640 healthy volunteer blood donors on the basis of CF antibody titers to CMV (Kolmer's method modified) of, at least, 1:8; plasmas from approximately 10% of the donors were therefore selected. The 68 liters of pooled immune plasma had à CF antibody titer of 1:16 (CMV antibody titers of 1: 10 000 and 1: 640 when tested in the ELISA assay and passive hemagglutination assay respectively). Intravenous immune globulin was produced from pooled plasma by Cohn fractionation and treatment with pepsin at pH 4; 4.8 liters of immune globulin were prepared and divided in 96 doses of 50 ml each. The final product was found to have a CMV antibody titer of 1: 32 (CF) 1: 50 000 (ELISA) or 1: 2 560 (passive hemagglutination). Recent reports on the preparation of CMV immune globulin are briefly reviewed.     

Human cytomegalovirus genome sequences in lymph nodes

Human cytomegalovirus (CMV) is a major cause of morbidity in heart and lung transplant patients, resulting from immunosuppression-mediated reactivation of latent CMV originating either from the transplanted tissue, or the recipient. We showed that out of eight donor/recipient pairs, the lymph nodes (LNs) of three donors and four recipients, all CMV seropositive, harboured CMV DNA at exceeding levels compared with those of matched blood samples, as well as CMV RNA otherwise undetectable in patients' blood. On follow-up, patients positive for CMV DNA and RNA in LNs developed viraemia 4 to 5 weeks earlier than those initially polymerase chain reaction-negative for CMV. Our results indicate that LN are a significant site for sequestration and persistence of CMV and that LN may be important in seeding of CMV-infected cells into the circulation.     

A cost-effective strategy for selection of third-party donors for a virus-specific T-cell bank for an Asian patient population

Background aimsThird party virus-specific T cells (VST) has shown efficacy for opportunistic virus infection which do not have effective treatment or are drug-refractory. We describe our preparatory work in setting up a third-party VST bank for a multi-ethnic Asian population.MethodsDiscarded white cells from regular blood bank plateletpheresis donors with known locally prevalent HLA antigens were cultured in small scale to generate VST against Adenovirus, BK virus, Cytomegalovirus, Epstein–Barr virus, and Human Herpes Virus 6. Multi-virus specific T cells (multi-VST) were also generated against all 5 viruses in single cultures. A strategy of allelic typing for donors with good and broad-spectrum cytotoxicity together with consideration on HLA restriction for the virus epitope was used to select combinations of VST lines for a hypothetical third party VST bank. The breadth of coverage based on these selection criteria was validated using our database of 100 post haematopoietic stem cell transplant patients.ResultsWe show that 50%, 42%, 56%, 56% and 42% of single VST cultures demonstrated specific cytotoxicity against AdV, BKV, CMV, EBV and HHV6 respectively. Twenty four of the 36 multi-VST lines showed activity against at least 2 of the 5 viruses studied. A carefully selected combination of just 6 VST lines can offer VST with at least 1 allelic match to 99% of potential recipients, while 92% can find 2 allelic matches and 79% can find 3 allelic matches.ConclusionsThis preparatory work confirms that a cost-effective strategy recruiting a small number of pre-characterized donors can generate VST lines with broad coverage for a multi-ethnic Asian patient population, thereby laying the foundation for setting up of a third party VST bank for Asian patients.     

Adherence of Peripheral Blood Leukocytes to Cytomegalovirus-Infected Fibroblasts

Peripheral blood monocytes and B cells adhered to cytomegalovirus (CMV)-infected fibroblasts, whereas T cells and polymorphonuclear leukocytes did not adhere to either CMV-infected or uninfected fibroblasts. When T cells were activated with anti-CD3 antibody, activated T cells demonstrated adherence and cytotoxicity to both CMV-infected and uninfected fibroblasts. Adherence of peripheral blood mononuclear cells (PBMC) and cytotoxicity mediated by adherent activated T cells were blocked by treatment of CMV-infected fibroblasts with anti-ICAM-1 antibody and by treatment of leukocytes with anti-LFA-1 antibody. These data suggest that an interaction of ICAM-1 and LFA-1 is responsible for the adherence of leukocytes and for adherent activated T cell-mediated cytotoxicity against CMV-infected fibroblasts.     

Prevalence of anti-R-13 antibodies in human Trypanosoma cruzi infection

Abstract Infection with Trypanosoma cruzi develops in three phases: acute, indeterminate or asymptomatic, and chronic phase (with cardiac or digestive manifestations). Moreover, transmission may occur from infected mothers to newborn, the so-called congenital form. In the present study, humoral responses against T. cruzi total extract and against the 13 amino acid peptide named R-13 derived from the parasite ribosomal P protein, previously described as a possible marker of chronic Chagas heart disease, were determined pateints and in blood bank donors from endemic areas. While in sera from acute phase, only IgM anti- T.cruzi response was observed, both IgM and IgG anti- T. cruzi antibodies were detected in sera from congenitally infected newborns. The percentage of positive response in sera from blood bank donors was relatively high in endemic regions. Antibodies against the R-13 peptide were present in a large proportion of cardiac chagasic patients but were totally lacking in patients with digestive form of Chagas disease. Furthermore, anti-R-13 positive responses were detected in congenitally infected newborns.     

PCR-RFLP analysis of cytomegalovirus infections associated with bone marrow transplantation in Japanese children.

In order to investigate the longitudinal molecular epidemiology of cytomegalovirus (CMV) infections associated with bone marrow transplantation (BMT) in Japanese children, we analyzed 36 CMV strains from 11 cases. Three regions (DNA polymerase, glycoprotein H, and immediate-early regions) of CMV DNA were amplified by polymerase chain reaction (PCR), and amplified products were each digested with two restriction enzymes, followed by electrophoresis. These restriction fragment length polymorphism (RFLP) analyses allowed the differentiation of 36 strains into 13 genotypes. Each patient excreted his or her own CMV with distinct genotype over the study period of up to one year. CMVs of two different genotypes were recovered during a one-month study from one recipient, who received a peripheral blood stem cell transplantation. Although the majority of patients and donors were CMV-seropositive before BMT, multiple CMV infections might not be common and the reactivation of latently infected CMV might be prominent in Japanese children receiving transplants.     

CMV infections in bone marrow transplanted patients--evaluation of prophylaxis with Cytotect (CMV hyperimmuneglobulin)

Cytomegalovirus (CMV) infection is a frequent and clinically important infection following bone marrow transplantation. Candidates for this study were patients admitted for transplantation: 22 patients received bone marrow from a HLA-identical, MCR-nonreactive sibling, in 9 patients an autologous BMT was performed. The anti-CMV IgG (Cytotect) was administered at a dosage of 1 ml/kg on days -7, 13, 33, 53, 73 and 93 after BMT. 5 patients in the very beginning of our BMT program did not receive Cytotect. Patients were given random blood products from the bloodbank not tested for CMV positivity. Active CMV infection or seroconversion in our patients was defined as a rise in IgG titer against the late antigen of fourfold or more or an IgM increase. In the allogeneic BMT group the pretransplant serological status was in 6 cases negative in recipients and donor, in 7 patients positive in recipients and negative in donors, and in 4 patients positive in recipients and donors. Of the 6 patients seronegative in recipients and donors, 3 developed active infection and of the 7 patients pretransplant positive with seronegative donors 3 developed active infection and 4 latent infections during the period from 2 to 100 days following grafting. 1 patient out of the group transplanted in third partial remission of AML developed interstitial pneumonia and died on day +30.4 of the 4 cases with seropositivity of recipients and donors developed active CMV infection. Of 9 patients with autologous transplantation 6 patients were pretransplant seropositive. 3 of these 6 developed active infection and 2 latent infection 30 to 180 days after grafting.(ABSTRACT TRUNCATED AT 250 WORDS)     

建立海南黎族永生细胞库

采用ＥＢ病毒转化外周血Ｂ淋巴细胞加环胞霉素Ａ 法,建立了含５４ 株海南黎族永生细胞库（男性５６％ ,女性４４％ ）,有亲缘关系者占１３％ 。供血者身体健康,三代无与其他民族或支系通婚史。     

Systemic immune deficiency necessary for cytomegalovirus invasion of the mature brain

Cytomegalovirus (CMV) is a significant opportunistic pathogen associated with AIDS and immunosuppressive therapy. Infection of the mature central nervous system (CNS) can cause significant pathology with associated neurological deficits, mental disorders, and cognitive impairment and may have potentially fatal consequences. Using genetically immunocompromised mice, we studied mechanisms of CMV invasion into, and behavior within, the CNS. Adult immunodeficient (nude and SCID) and control mice were peripherally infected with recombinant mouse CMV expressing a green fluorescent protein reporter gene. Control mice actively eliminated acute peripheral infection and were resistant to invasion of CMV into the brain. In contrast, virus infected brains of immunodeficient mice but only after a minimum of 21 days postinoculation. After inoculation, CMV was found in circulating leukocytes (MAC-3/CD45⁺) and in leukocytes within the brain, suggesting these cells as a possible source of CMV entry into the CNS. CNS infection was observed in many different cell types, including neurons, glial cells, meninges, ependymal cells, and cells of cerebral vessels. Infection foci progressively expanded locally to adjacent cells, resulting in meningitis, choroiditis, encephalitis, vasculitis, and necrosis; clear indication of axonal transport of CMV was not found. Regional distribution of CMV was unique in each brain, consisting of randomly distributed, unilateral foci. Testing whether CMV gained access to brain through nonspecific vascular disruption, vascular injections of a tracer molecule revealed no obvious disruption of the blood brain barrier in mice with CMV in the brain. Results indicate the importance of host adaptive immunity (particularly T cells) in controlling entry and dissemination of CMV into the brain and are consistent with the view that virus may be carried into the brain by circulating mononuclear cells that traffic through the blood brain barrier.     

Absence of detectable XMRV and other MLV-related viruses in healthy blood donors in the United States

Background

Preliminary studies in chronic fatigue syndrome (CFS) patients and XMRV infected animals demonstrated plasma viremia and infection of blood cells with XMRV, indicating the potential risk for transfusion transmission. XMRV and MLV-related virus gene sequences have also been detected in 4–6% of healthy individuals including blood donors in the U.S. These results imply that millions of persons in the U.S. may be carrying the nucleic acid sequences of XMRV and/or MLV-related viruses, which is a serious public health and blood safety concern.

Methodology/Principal Findings

To gain evidence of XMRV or MLV-related virus infection in the U.S. blood donors, 110 plasma samples and 71 PBMC samples from blood donors at the NIH blood bank were screened for XMRV and MLV-related virus infection. We employed highly sensitive assays, including nested PCR and real-time PCR, as well as co-culture of plasma with highly sensitive indicator DERSE cells. Using these assays, none of the samples were positive for XMRV or MLV-related virus.

Conclusions/Significance

Our results are consistent with those from several other studies, and demonstrate the absence of XMRV or MLV-related viruses in the U.S. blood donors that we studied.     

Endemic impact of human T cell leukemia virus type 1 screening in bone allografts

Allograft bone is a widely used as a convenient tool for reconstructing massive bone defects in orthopedic surgery. However, allografts are associated with the risk of viral disease transmission. One of the viruses transmitted in this manner is human T-lymphotropic virus type 1 (HTLV-1), which is found worldwide but is unevenly distributed. The southwestern parts of Japan are a highly endemic for HTLV-1. We investigated the HTLV-1 seroprevalence in candidate allograft donors at the regional bone bank in Kagoshima, Japan during its first 5 years of service. Between 2008 and 2012, we collected 282 femoral heads at the Kagoshima regional bone bank from living donors with osteoarthritis of the hip joint. Among the 282 candidate donors, 32 donors (11.3 %) were seropositive for anti-HTLV-1 antibody; notably, this prevalence is higher than that reported for blood donors in this area. Additionally, to determine if HTLV-1 genes are detectable after processing, we examined the bone marrow of the femoral heads from seropositive donors by conducting PCR assays. Our results confirm the existence of viral genes following the heat treatment processing of the femoral heads. Therefore, it is important to inactivate a virus completely by heat-treatment. Together, our findings highlight the importance of HTLV-1 screening at bone banks, particularly in HTLV-1-endemic areas such as southwest Japan.     

Antibody-dependent cellular cytotoxicity (AAZZ) against leukemia cells--K-cell activity of human leukocytes

The activity of human leukocytes from the peripheral blood (PB) and bone-marrow (BM) to take the function as K-cells in the antibody dependent cellular cytotoxicity (ADCC) was tested in a 51Cr-test against mouse leukaemic cells and ALL cells covered with specific heterologous antibodies. Mononuclear PB-leukocytes and granulocytes of healthy donors and patients with leukemia and lymphoma in remission lysed murine and human leukaemic cells in the presence of specific antibodies. There was no lowering of K-cell activity of mononuclear PB-leukocytes of patients with leukaemia and lymphoma in remission under chemotherapy as compared with healthy donors and patients in remission without chemotherapy. There was a good correlation between the percentage of K-cell active mononuclear leukocytes in PB and BM. Attempts of fractionation with peripheral blood leukocytes of healthy donors resulted in the non-adherent mononuclear PB-leukocytes (lymphocytes) and granulocytes being effector cells in ADCC. To a high degree K-cell active lymphocytes could be identified in the non-B-fraction and only slightly in the fraction forming E rosettes.     

Enzyme linked immunosorbent assay (ELISA) for determination of IgG antibodies to human cytomegalovirus

A solid-phase enzyme linked immunosorbent assay (ELISA) for determination of IgG antibodies to cytomegalovirus (CMV) is described. The assay used purified CMV and extracts of CMV infected cells as antigen. Antigens were desiccated onto the bottom surface of polystyrene microcuvettes. The antibodies bound to the antigens were assayed by anti-IgG-alkaline phosphate conjugate followed by addition of the enzyme substrate. Titration curves have been obtained from the sera of 35 blood donors and of 23 patients. Comparison of results obtained by ELISA with those obtained by complement fixation (CF) shows that there is agreement between the tests. Both purified CMV and extracts of CMV infected cells were found to be suitable antigens. Purified CMV was of value particularly in those sera which show high reactivity against control antigen. The ELISA technique described is approximately 412 to 548 times more sensitive than the CF test when purified CMV or extracts of CMV infected cells, respectively, are used as antigens. No significant heterotypic rise to CMV was observed by ELISA in three sets of sera with seroconversion to herpes simplex virus. The ELISA technique gives objective results, is easily performed, and may be adaptable as a routine test both for serological diagnosis of CMV infection and for screening of the general population.     

Screening for arboviruses in healthy blood donors: Experience from Karachi,Pakistan

Highlights:
1 Potential transfusion-related transmission of DENV, WNV and JEV was investigated in healthy blood donors from the blood bank of Aga Khan University during July to December 2018.
2 ELISA for DENV, WNV and JEV IgM antibodies and RT-PCR for viral RNA detection were performed.
3 Of the 360 blood donors screened, IgM antibodies for DENV and WNV were positive in 3.9% and JEV in 0.28% respectively while none of the blood donors tested positive for RT-PCR.
4 Majority of the seropositive donors were between 19 to 30 years of age and residents of urban areas, mainly from Karachi City.
5 DENV and WNV seropositivity were significantly associated with residence in Malir District of Karachi.