Isolation of Inoue-Melnick virus from human colorectal carcinoma cell lines

Inoue-Melnick virus (IMV) was isolated from five of six human colorectal carcinoma cell lines, whereas the virus was not isolated from three normal colon-derived cell lines, three pancreas carcinoma cell lines, two bladder carcinoma cell lines, one kidney adenocarcinoma cell line, and one cervical carcinoma cell line. IMV synthesis was remarkably enhanced in the colorectal carcinoma cell lines by treatment with 5-bromodeoxyuridine (BUDR). However, virus was not detected in any of the other cell lines in spite of BUDR treatment. Five strains of virus isolated from colorectal carcinoma cell lines were subsequently identified by the neutralization test as intermediate-type IMV.     

Direct isolation of Inoue-Melnick virus from human colorectal carcinoma tissues but not from stomach carcinoma tissues.

Inoue-Melnick virus (IMV) was isolated directly from all of the seven human colorectal adenocarcinoma tissues studied, whereas IMV was not isolated from five human stomach carcinoma tissues. Seven isolates from the Japanese colorectal carcinoma tissues were identified as type 1 IMV by the neutralization test and were sensitive to phosphonoacetic acid, a growth inhibitor of IMV. Furthermore, type 1 IMV-antibody was detected in six of seven sera of the patients whose colorectal carcinoma tissues were positive for virus isolation. In contrast, IMV-antibody was not detected in the five sera of the patients whose stomach carcinoma tissues were tested and found negative for IMV.     

Enhancement of Inoue-Melnick virus synthesis by 5-bromodeoxyuridine in human meningioma (MG-1) cells

The authors found remarkable enhancement of Inoue-Melnick virus (IMV) synthesis by 5-bromodeoxyuridine (BUDR) in human meningioma (MG-1) cells, a virus-producer line of type 1 IMV. Treatment with BUDR resulted in rapid and abundant synthesis of infectious IMV in MG-1 cells. The titer of the cell-associated virus in treated cells increased approximately 6.0 log 10 compared with that in untreated cells. Immunofluorescent antibody tests revealed that IMV-associated late antigen was induced by BUDR in the cytoplasm of approximately 50% of treated cells. A clonal difference was also found in the enhancement of BUDR on the IMV synthesis in MG-1 cells. The most remarkable enhancing effect of BUDR was observed in the clone C line, and a herpes-type virus was detected by negative-staining electron microscopy in the culture fluid of the clone C treated with BUDR.     

Isolation of Inoue-Melnick virus from human meningioma-derived cell cultures and detection of antibody in patients with meningioma

Inoue-Melnick virus (IMV) was isolated from six of seven human meningioma-derived cell cultures, while the virus was not isolated from six other brain tumor cell cultures. Sera of 145 consecutive neurosurgical inpatients were tested for IMV-neutralizing antibody. Of 26 patients with meningioma, 22 were positive for IMV antibody (84.6%). Of the remaining 119 patients, 16 were positive.     

Endogenous production of infectious Inoue-Melnick virus in a human meningioma cell line

We investigated continuous production of Inoue-Melnick virus (IMV) in the MG-1 cell line, established from human meningioma. The infectious virus, identified as a type 1 virus, was mostly recovered extracellularly. Assay of MG-1 cells as infective centers indicated that most of the cells were capable of producing infectious virus. By immunofluorescence, more than 90% of the cells were found to have IMV-associated cytoplasmic antigen(s) (IMCA).     

Growth of tick-borne encephalitis virus (European subtype) in cell lines from vector and non-vector ticks

We undertook a comparative study of the susceptibility of different tick cell lines to infection with the European subtype of tick-borne encephalitis virus (TBEV), prototype strain Neudoerfl. The growth of TBEV was investigated in lines derived from vector Ixodes ricinus L. ticks (IRE/CTVM18, 19, and 20), as well as non-vector ticks, namely Ixodes scapularis Say (IDE2), Boophilus microplus Canestrini (BME/CTVM2), Hyalomma anatolicum anatolicum Koch (HAE/CTVM9), Rhipicephalus appendiculatus Neumann (RA-257) and recently established and herein described lines from the argasid tick Ornithodoros moubata Murray (OME/CTVM21 and 22). All the tick cell lines tested were susceptible to infection by TBEV and the virus caused productive infection without any cytopathic effect. However, there was a clear difference between the TBEV growth in vector and non-vector cell lines, since I. ricinus cell lines produced 100-1000-fold higher virus yield than the non-vector cell lines. The lowest virus production was observed in O. moubata and R. appendiculatus cell lines.     

Seroepidemiology of inoue-melnick virus in the general population of buffalo,New York

The prevalence of antibodies to Inoue-Melnick virus (IMV) types 1 and 2 in the general population of Buffalo, New York, was studied. Serum specimens were collected from blood donors, and pediatric sera were provided from the Buffalo Children's Hospital. Neutralizing antibody titers against IMV were measured with established procedures. Very high prevalence of antibodies to both IMV types 1 and 2 were found in sera from children of Buffalo, especially in the age group 15-19 years (nearly 100%). The antibody positivity gradually decreased with advancing age, except for the groups of age 60 and older. This is in sharp contrast to a previous report examining sera from the people of Osaka, Japan, in which IMV antibody was not found in children under 10 years of age. In the group of teenagers in Buffalo, with high geometric mean antibody titers, an association with any disease was not found. The peculiar distribution of antibodies to IMV was considered to be possibly related to the high incidence of multiple sclerosis (MS) in the Buffalo area, in contrast to the data from Osaka, Japan, which is a low incidence area for MS. © 1995 Wiley-Liss, Inc.     

Effects of phosphonoacetic acid on subacute myeloopticoneuropathy virus in vitro and in vivo.

The effect of phosphonoacetic acid (PAA) in vitro and in vivo on subacute myeloopticoneuropathy (SMON) virus isolated from the spinal fluid of SMON patients was studied. PAA inhibited multiplication of SMON virus in cultures, but it did not show a direct effect on the virus. The drug did not influence the disease when the medication was started from 10 days after infection of suckling mice. However, the drug did elicit a delay in the incubation period.     

逆转录-聚合酶链反应方法检测乙型脑炎病人标本

目的 逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）检测乙型脑炎（乙脑）病人标本方法的建立和评估。方法 建立ＲＴ－ＰＣＲ法,了解该方法用于乙脑病毒检测的敏感性,特异性,并用于临床疑似乙脑病人血清及脑脊液（ＣＳＦ）标本的检测,并与反向被动血凝抑制实验（ＲＰＨＩ）方法进行比较分析。结果 用该ＲＴ－ＰＣＲ法检测高顺生株（高株）敏感性可达６４ＰＦＵ。共检测临床疑似乙脑病人标本３８份,对ＣＳＦ中乙型脑炎病毒（ＪＥＶ）     

Malignant lymphoma induction of rabbits with oral spray of Epstein-Barr virus-related herpesvirus from Si-IIA cells (HTLV-II-transformed Cynomolgus cell line): A possible animal model for Epstein-Barr virus infection and subsequent virus-related tumors in humans

Malignant lymphoma (ML) was Induced in eight of nine rabbb inoculated by oral spray of the cell-free pellets from SI-IIA culture (MLV-ll-transformed leukocyte cell line of the Cynomolgus-producing Epsteln-Barr virus (EBV)-related herpesvirus) after 64–141 days. None of the rabbits inoculated with EBV from B-95–8 cells or HTLV-II from MOT cells developed ML. Malignant lymphomas were usually of diffuse, large-cell or mixed type. HTLV-II infection was excluded by the polymerase chain reaction (PCR) and the particle agglutination test. EBV-encoded RNA-1 and EBV-related DNA were detected in the tumor tissues by In situ hybridization and PCR, respectively. Anti-viral capsid antigen of EBV antibody (anti-VCA) was observed 3 weeks after oral inoculation of Si-IIA cell-free pellets. Polymerase chain reaction revealed continuous detection of EBV-related virus DNA in the peripheral blood leukocytes from 3 days after oral inoculation. These results show that ML induced orally wtth SI-IIA cell-free pellets was caused by EBV-related herpesvirus harbored by Si-IIA cells. Oral spray of EBV from B-95–8 also induced EBV Infection in rabbits, which was confirmed both by the presence of anti-VCA and by PCR. These oral infection and mallgnant lymphoma induction systems of rabbit using EBV-related virus from Si-IIA or human EBV are useful animal models for the study of EBV infection and EBV-related lymphomas in humans.     

Characterization of fresh (uncultured) tumour-infiltrating lymphocytes (TIL) and TIL-derived T cell lines from patients with renal cell carcinoma.

Fresh (uncultured) TIL from 12 untreated patients with primary renal cell carcinoma were prepared from tumour specimens by enzymatic digestion, and were characterized by immunofluorescence using MoAbs recognizing leucocyte differentiation antigens or particular V alpha or V beta segments of the T cell receptor (TCR). These fresh TIL comprised CD3+ (20-84%); CD4+ (3-15%); CD8+ (13-35%); alpha beta TCR+ (20-50%); gamma delta TCR+ (3-17%); CD16+ (1-18%) and CD56+ (3-10%) cells. Significant proportions of V alpha 2+, V beta 5.1+ and V beta 6+ cells were found in TIL of certain patients with renal cell carcinoma, suggesting that they comprised oligoclonal T cells. T cell lines were developed in low concentrations of rIL-2 (200 U/ml) from TIL from 11 patients with renal cell carcinoma, and were characterized by immunofluorescence and cell-mediated cytotoxicity. These T cell lines consisted primarily of CD3+ (51-94%); CD4+ (1-80%); CD8+ (0-84%); alpha beta TCR+ (65-87%); gamma delta TCR+ (0-25%); CD16+ (0-16%) and CD56+ (2-57%) cells. These T cell lines exhibited non-specific cytotoxicity against autologous and allogeneic renal tumour cells, with the exception of one T cell line that exhibited preferential cytotoxicity against autologous renal tumour cells. These results suggest that fresh TIL from patients with renal cell carcinoma contain significant proportions of oligoclonal T cells that may have accumulated at the tumour site as a result of a clonal expansion.     

稳定表达鼻咽癌来源潜伏膜蛋白1基因的鼻咽癌细胞系的建立

目的:建立稳定表达鼻咽癌(NPC)来源潜伏膜蛋白1(LMP1)的鼻咽癌细胞系。方法:利用基因重组技术构建NPC来源LMP1的一般性真核表达载体及上皮特异性表达载体,并将其转染鼻咽癌细胞系CNE-2,用PCR、RT-PCR及蛋白印迹等检测N-LMP1在CNE-2中的整合和表达。结果:①成功构建了N-LMP1的一般性和上皮特异性表达载体。②N-LMP1基因在CNE-2细胞中获得了正确表达。结论:成功建立了稳定表达NPC来源LMP1的鼻咽癌细胞系,为进一步研究LMP1在鼻咽癌细胞中的作用机制奠定基础。     

Detection of herpesvirus DNA in the large intestine of patients with ulcerative colitis and Crohn's disease using the nested polymerase chain reaction.

The prevalence of herpesvirus DNA was examined in inflammatory bowel disease tissue. DNA was extracted from resection and biopsy specimens of the large intestine from patients with ulcerative colitis (n = 21), patients with Crohn's disease (n = 29), and patients with noninflammatory bowel disease (controls) (n = 21). The nested polymerase chain reaction was used to detect viral DNA using primer pairs specific for either cytomegalovirus (CMV), herpes simplex virus 1 (HSV1), human herpesvirus 6 (HHV6), varicella zoster virus (VZV), or Epstein Barr virus (EBV). HSV1 and VZV DNA were not detected in any of tissue samples. There was a high prevalence of CMV (81%), HHV6 (76%), and EBV (76%) DNA in ulcerative colitis tissue compared to Crohn's disease tissues (CMV 66%, HHV6 45%, EBV 55%). Control tissue had a relatively low frequency of CMV (29%) and EBV (19%) DNA but a prevalence of HHV6 DNA similar to that of ulcerative colitis (86%). However, the simultaneous presence of HHV6 and CMV and/or EBV DNA in ulcerative colitis tissue (76%) was much greater than in either Crohn's disease tissues (38%) or control tissue (29%) (P < 0.05). There was a low prevalence of CMV, HHV6, and EBV DNA in peripheral blood mononuclear cells from all patient groups. CMV and EBV are capable of reactivating HHV6: the high prevalence of coexistent HHV6 infection with either or both of these two viruses in ulcerative colitis tissue suggests that they may play a synergistic role in the pathogenesis of this disease.     

Serological survey of human immunodeficiency virus (HIV) in Ethiopia

The presence of anti-human immunodeficiency virus 1 antibodies was tested in 5,565 serum samples from Ethiopia of which 5,265 were collected from military recruits in the framework of a hepatitis B (HBV) seroepidemiological study performed on a national scale in 1985-1986; the remaining were 300 sera from a population of outpatients belonging to the Arsi region. Of the 5,565 sera, 121 (2.1%) were found to be repeatedly reactive by enzyme-linked immunosorbent assay (ELISA) test for HIV-1 antibodies, but these reactivities were confirmed by Western Blot (WB) assay in only four cases (0.07%) and by ENVACOR (confirmatory competitive ELISA) in three samples. Twenty-three sera were positive by WB to one or two bands related to core proteins but were all negative by ENVACOR. However, according to accepted criteria for positivity, these sera must be regarded as indeterminant reactors. A sample of 409 sera, both reactive and nonreactive by HIV-1 ELISA, were further tested for antibodies to HIV-2 by ELISA. Reactive sera were analysed by WB and by radioimmunoprecipitation assay (RIPA) using 35S-cysteine metabolically labelled SIVmac (HTLV-IV) infected cell lysates. Only 11 sera were found to be slightly reactive in ELISA, but this was not confirmed by WB or RIPA. Data indicate that HIV infection was not widespread in the general population of Ethiopia up to 1986.     

Detection of herpes simplex virus (1 and 2), varicella-zoster virus, cytomegalovirus, human herpesvirus 6 and enterovirus in immunocompetent Tunisian patients with acute neuromeningeal disorder

Enteroviruses (EVs) and human herpesviruses (HHVs) are involved frequently in acute neurological disorders of viral etiology. This study aimed to investigate the incidence of herpes simplex virus types-1 (HSV-1) and 2 (HSV-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6) and human enteroviruses (EVs) in cerebrospinal fluid (CSF) samples of Tunisian immunocompetent patients with neuromeningeal disorders. The patients had been hospitalized at the Fattouma Bourguiba University Hospital (Monastir, Tunisia) between September 2007 and June 2009. At least one viral genome was detected in 58 (46%) out of 126 CSF samples collected. Enterovirus was detected in 31 of the positive samples (53.4%), CMV in 20 (34.5%), HSV-1 in 3 (5.2%), HSV-2 in 6 (10.3%), VZV in 4 (6.9%), HHV-6 in 2 (3.4%). More than one viral genome was detected in seven CSF samples, including CMV DNA in six of the samples. The high frequency of enteroviral infections in aseptic meningitis was confirmed. The detection of CMV DNA only suggests a direct role of this virus in the etiology of acute neuromeningeal disorder.     

Generation of human monoclonal antibody-producing cell lines by Epstein-Barr virus (EBV)-transformation of B lymphocytes and somatic cell hybridization techniques

Summary The use of Epstein-Barr virus to immortalize human B cells and generate monoclonal antibody-producting B cell lines is described.     

Growth of brown bullhead (BB) and other fish cell lines on microcarriers and the production of channel catfish virus (CCV)

The growth of brown bullhead (BB) fish cells and other poikilothermic cell lines on four commercially available brands of microcarrier beads was evaluated. Three of the microcarriers carry a positive surface charge, while the fourth carries a negative surface charge. The four microcarriers vary in their capacity to support the growth of the BB cells and other poikilothermic cell lines tested. The polyacrylamide beads by Bio-Rad and the polystyrene beads by Nunc were found to best support the growth of the anchorage-dependent BB cells. Although the DEAE-dextran beads by Flow Laboratories were also found to be satisfactory, similar Cytodex 1 beads by Pharmacia, were found to support the growth of BB cells poorly. Both the polyacrylamide and polystyrene beads were found to be satisfactory for reuse after resterilization. In contrast, the DEAE-dextran beads were unsatisfactory since they sustained obvious mechanical damage. The BB growth rate and final cell density in microcarrier culture were dependent on the concentration of beads in culture and of the size of the initial cell inoculum. In terms of cell yield per milliliter of culture medium the microcarrier culture was superior to conventional stationary cultures. Microcarrier spinner cultures of BB cells consistently produced higher yields of channel catfish virus than conventional monolayers of the same cells. Two important advantages of the microcarrier system are its suitability for large scale as well as small scale production of both cells and virus.     

Reduced E-cadherin expression correlates with increased invasiveness in colorectal carcinoma cell lines

A reduction in cell adhesiveness and cell invasion are essential steps in tumour progression to metastasis. In the present study two out of seven colorectal carcinoma cell lines exhibited reduced expression of the cell-cell adhesion molecule E-cadherin as assessed by immunofluorescence. The same two cell lines were invasive in the collagen gel and membrane invasion culture system invasion assays. Addition of anti-E-cadherin antibody to a non-invasive carcinoma cell line caused the cells to assume a dissociated morphology on plastic and to become invasive in collagen gels. This demonstrates a causal role for E-cadherin in the maintenance of intercellular adhesion and the suppression of tumour cell invasion and possibly metastasis in colorectal tumour cells.     

Characterization of cell lines stably transfected with rubella virus replicons

Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was ∼9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.