Evaluation of an automated extraction system in combination with Affigene CMV Trender for CMV DNA quantitative determination: comparison with nested PCR and pp65 antigen test.

CMV viral load quantitation is a powerful tool to assist clinicians in making accurate diagnoses, managing post-transplant CMV disease and monitoring antiviral therapy. The aim of this study was to evaluate the performance of Affigene CMV Trender for CMV viral load determination used in combination with a non-dedicated nucleic acid extraction system (BioRobot MDx) for high-throughput routine. Linearity, reproducibility and sensitivity were examined. Clinical samples were used to compare results obtained with the Affigene CMV Trender, with an "in house" nested PCR used for routine diagnosis and with pp65 antigenemia. The results indicated that the test is linear in the range of 1.81-5.18 Logcopies/ml and that sensitivity is 77 copies/ml. The concordance of the Affigene CMV Trender with nested PCR was high, (k=0.91, IC 95%=0.82-1.00), whereas a substantial concordance with pp65 antigenemia was observed (k=0.64, IC 95%=0.54-0.73). In conclusion, combined use of a non-dedicated automated nucleic acid extraction method with the Affigene CMV Trender results in an accurate high throughput system, suitable for routine laboratory monitoring of CMV infection.

Markers of human papillomavirus infection and their correlation with cervical dysplasia in human immunodeficiency virus-positive women.

Human papillomavirus (HPV) genotypes and HPV DNA load were analysed in cervical smears from 76 human immunodeficiency virus (HIV)-positive and 54 HIV-negative women. The prevalence of genotypes was similar for all women, with the exception of HPV62, which was over-represented in HIV-positive samples. HIV-positive women showed a higher prevalence of multiple genotypes that correlated neither with CD4(+) T-cell counts nor with cervical dysplasia. No significant differences were observed in terms of total or single-type HPV DNA load. The HPV DNA load in both HIV-positive and HIV-negative women was significantly higher in squamous intra-epithelial lesions than in negative Pap smears.

Bcl-2 inhibits the caspase-dependent apoptosis induced by SARS-CoV without affecting virus replication kinetics.

Vero cells transfected with either neo- or bcl-2-plasmid were infected with SARS-CoV at a high multiplicity of infection. Apoptosis appeared after the onset of CPE and completion of virus replication, and could be prevented by Bcl-2 expression. Apoptosis is likely mediated by the mitochondrial pathway, as demonstrated by its inhibition using Bcl-2, and by the activation of the caspase cascade, resulting in PARP cleavage. Prevention of apoptosis did not affect susceptibility to infection, kinetics and extent of viral replication and release, thus implying that apoptosis is not involved in facilitating release and/or dissemination of SARS-CoV in Vero cells.

A new RT-PCR method for the identification of reoviruses in seawater samples.

The frequent occurrence of reoviruses in environmental samples could be a potential source of interference with enterovirus detection, especially when enterovirus isolation on cell culture is required. In order to evaluate new virus-based criteria for enforcing recreational water quality standards, a new method based on a broad reverse transcribed polymerase chain reaction (RT-PCR) was set up to detect reoviruses. Two primers were engineered to amplify a 538 base pair fragment of the Sigma 2 gene. Reovirus strains obtained from ATCC (Jones, Lang, Dearing, Abney, NC-TEV, SV59 and SV12) were used as references. Twenty-four samples of 101 were collected from two beaches of the Adriatic sea and 12 from the neighbourhood of Fano Harbour Channel. The presence of environmental reoviruses was tested on both concentrated seawater samples and lysates of BGM cells infected with the concentrated seawater samples. The new method was used in parallel with the detection of a 3:3:4 electrophoretic pattern of reovirus RNA in polyacrylamide gel electrophoresis (PAGE). Enterovirus and bacteria were also screened in compliance with EEC directives. No enteroviruses were isolated, and it was not attributable to reovirus interference. All the reovirus found by PAGE (8/72) were confirmed by RT-PCR, while several genomes (14/72) were detected only by RT-PCR. Presumptive methods of virus identification, that is CPE on BGM cells and haemagglutination test, were not able to detect them. The specificity of RT-PCR products was checked by direct nucleotide sequence analyses of the amplicons. The phylogenetic analyses showed heterogeneous taxa including human and animal reoviruses, with strong evidence that they were spreading consistently from the Harbour-Channel. This novel approach for reovirus detection will be very useful as a trace route of faecal pollution; more importantly, it could be very useful in contributing to the creation of a databank of circulating enteric viruses.

[The seroprevalence of HIV, HBV and HCV infections in patients coming to the departments of general surgery of a public hospital (S. Camillo, Rome)]. / Sieroprevalenza di infezione da HIV, HBV e HCV in pazienti afferenti ai reparti di chirurgia generale di un ospedale pubblico (S. Camillo, Roma).

An anonymous unlinked seroprevalence study of HIV, HBV and HCV infections has been conducted on 485 sera consecutively submitted to the virology laboratory to be tested for HBsAg; sera came from patients attending general surgery at San Camillo Hospital in Rome. Carriers of HBsAg were 12 (2.5%); antibodies against HIV have been identified in 4 sera (0.8%) and against HCV in 35 (7.2) by ELISA (first generation assay); 25/35 of anti HCV ELISA positive sera were reactive also by RIBA (first generation assay). The observed prevalences of potentially infectious patients represent a risk of occupational transmission for health care workers. Vaccination against HBV, adherence with universal precautions and development of safer surgical devices and techniques are needed to prevent the risk of exposure and, consequently, of occupational acquired infections.

Prevalence of hepatitis B and C viruses and human immunodeficiency virus infections in women of reproductive age.

OBJECTIVE: To determine the prevalence of hepatitis B and C viruses, and human immunodeficiency virus infections in women of reproductive age attending a health care system. DESIGN: Prospective cross-sectional study. SETTING: Public Obstetric Clinic and Service for Pre- and Perinatal Prevention of Infectious Diseases, Rome, Latium Region, Italy. SUBJECTS: 1142 women attending our centres consecutively for delivery, miscarriage, voluntary interruption of pregnancy or screening for pre- and perinatal prevention of infectious diseases. INTERVENTIONS: Serum samples, collected after informed consent over a period of 2 months, were tested for hepatitis B virus markers (anti-HBc and HBsAg) by enzyme linked immunosorbent assay (ELISA), for antibodies against hepatitis C virus (by ELISA and, if positive, by RIBA) and for human immunodeficiency virus antibodies (by ELISA and, if positive by Western blot). RESULTS: The seroprevalence of hepatitis B virus was 14.4% (95% CI Poisson distribution 12.2-16.5) for anti-HBc and 1.6% (95% CI, 0.9-2.5) for HBsAg. Antibodies against hepatitis C virus were detected by ELISA in 2.4% (CI 1.6-3.5) and by first generation RIBA in 0.9% (CI 0.4-1.6). Human immunodeficiency virus seroprevalence was 1.0% (CI 0.5-1.7). No significant differences were observed by age or by reason for attending. CONCLUSIONS: Women attending our centres have a higher prevalence of hepatitis B virus, hepatitis C virus and human immunodeficiency virus infection than those observed in our country in larger national surveys of newborn babies, in reproductive-aged women or in other selected low-risk groups such as blood donors. This could be due to the attendance of women at increased risk such as drug addicts. The information has the additional value of emphasizing the need for adherence by health care personnel, to the recommendations issued for the prevention of occupational infections.

Identification of a CD21 receptor-deficient, non-Ig-secreting peripheral B lymphocyte subset in HIV-seropositive drug abusers.

We have studied 61 HIV-seropositive heroin addicts (18 asymptomatic, 20 ARC, and 23 AIDS cases), 26 HIV-seronegative heroin addicts, and 45 healthy blood donors, matching the groups each other for age and sex. We have focused on the phenotypic characteristics of B subpopulations in the peripheral blood of HIV-seropositive and -seronegative drug abusers, paying particular attention to the consistence of the "CD20+" B cell subset, which poorly expresses the CD21 membrane receptor for the C3d and Epstein-Barr virus (EBV) (referred to as "CD20 + CD21-" subset). In healthy blood donors, the ratio CD20 + CD21-/CD20+ x 100 is extremely low (mean +/- SEM = 8.1 +/- 0.9) and rarely exceeds the value of 20. On the contrary, in HIV seropositives, the values are much more dispersed, with higher mean values (mean +/- SEM = 25.8 +/- 1.8) ranging from 50 to 60. An intermediate situation characterizes the class of HIV-seronegative heroin addicts, whose values are slightly higher and more dispersed than that of normal controls (mean +/- SEM = 11.6 +/- 1.3). The extent of the amplification of the CD20 + CD21- subset in HIV-seropositive individuals does not apparently correlate with the progression of the disease and represents an early event in the clinical course of HIV infection. For each subject of the study group, the number of CD20 + CD21- B lymphocytes is not correlated to other early markers of HIV infection, as the T4 lymphocyte number, or total Ig levels in sera. A functional characterization of the CD20 + CD21- B cell subset indicates that, in HIV-seropositive patients, these cells are unable to produce specific and nonspecific immunoglobulins (Ig's), either spontaneously or after pokeweed mitogen stimulation. Furthermore, this cell subset is characterized by poor expression of surface Ig's. The data reported suggest that this cell subset can be regarded as situated at an early level of B cell lineage differentiation.

Sendai virus replication in Friend erythroleukemia cells. I. Acutely and persistently infected cells become resistant to virus-induced lysis.

Friend leukemia cells (FLC) are susceptible to infection by Sendai virus, a member of the paramyxovirus group. FLC constitute a most suitable model to study virus-host cell interactions, because they grow in suspension (thus avoiding the use of trypsin), and provide an easy way of deriving single-cell clones. When FLC are infected with Sendai virus at high m.o.i., a direct, extensive lysis of the cells ensues, whereas lower doses of virus result in a cytocidal infection whose lethality depends mainly on the virus used, standard or defective interfering egg-grown Sendai virus (EGSV), and on the multiplicity of infection (m.o.i). At later times after infection, FLC become resistant to the Sendai induced lysis (SIL). The SIL resistance can be maintained in single-cell clones that had survived the first infection. The maintenance of the resistant phenotype of the clones requires the serial subcultivation of the cells in the presence of activated EGSV. The mechanisms that presumably regulate the appearance of SIL resistance in Sendai infected FLC are discussed.

A nonneutralizing human IgM monoclonal antibody inhibiting hemagglutination of H3N2 influenza A strains.

A mouse-human hybridoma has been produced by fusing human splenocytes from a Cooley's anemia patient with the murine myeloma P3-NS1/1-Ag 4-1. The hybridoma is stable after 18 months and secretes human IgM. The antibody reacts with some H3N2 influenza A strains and detects an epitope that is part of the hemagglutinin antigen, but does not affect virus infectivity.

Induction and maintenance of flattened morphology in highly adhesive Friend leukemia clones depends on the time- and space-specific assembly of microtubular networks.

Indirect immunofluorescent staining with anti-tubulin antibodies, SEM and TEM were applied to study microtubule (MT) assembly in clones isolated from Friend leukemia cells (FLC, 745 A strain) on the basis of their sensitivity to exogenous fibronectin (FN). Kinetics of cell spreading and elongation were studied using computerized image analysis and SEM. In contrast to 745 A cells, FN-sensitive clones (referred to as FF clones) showed elaborate MT networks when observed by immunofluorescent staining as well as by TEM. A good correlation was found between the degree of spreading and elongation of FF cells and the degree and cellular distribution of their MT. The highest concentration of MT networks oriented parallel to the main cellular axis was observed in very elongated FF cells. The majority of MT in interphase FF cells radiated from the centrosomes; some MT apparently originated from the nuclear membranes. TEM showed the existence of morphological differences between centrosomes of 745 A and FF cells. The characteristic ultrastructure of the centrosomes of FF cells was maintained in trypsinized cells, even if such FF cells lost MT's and acquired a spherical morphology. FF cells, treated with a wide spectrum of MT-disrupting agents, promptly acquired a rounded morphology with rapid dissolution of polymerized tubulin. Removal of MT-disrupting agents from the culture medium rapidly restored a flattened morphology with concurrent regeneration of MT's. During recovery from MT-disrupting agents, FF cells showed increased numbers of centrosomes per cell. We conclude that MT networks cooperate in the attachment, spreading and elongation of FF cells isolated from FLC. Moreover, we hypothesize the existence in FF cells of a variant form of centrosomes as compared with those of 745 A cells.

SV40 immortalization of adult human mesenchymal cells from neuroretina. Biological, functional and molecular characterization.

Human adult mesenchymal cells from neuroretina (human choroid cells, HC) have acquired an infinite lifespan, following phenotypic transformation with a wild-type SV40. Immortalized cells (HC/SV40) contain high numbers of free circular viral DNA, and integrated molecules in a head-to-tail array in the cellular DNA. HC/SV40 cells express both the virus-coded "T" antigens and the cell-coded p53 transformation-associated protein. The transformed phenotype was further characterized by loss of contact inhibition of cell division, inability to induce the retraction of a fibrin clot and to spread within fibrin, and the existence of an altered distribution of actin cables. For the first time we also describe a coupling of the immunofluorescence and the quantitative cytofluorometric analyses, a new transformation parameter, since we show that SV40 transformation causes reorganization of the cell membrane by inducing the unmasking of the antigen recognized by the 4F2 monoclonal antibody, which is present in a "cryptic" form in the untransformed cells. Though the HC/SV40 cells have been continuously passaged over a 3-year period, they have not yet achieved a fully malignant phenotype, since they retain serum-dependency and the presence of a well developed fibronectin pericellular network, and they are not tumorigenic in nude mice. Thus this human immortal cell line constitutes a very useful tool for studying the progression toward full malignancy and the relationships between evolution of transformation parameters and changes in the viral and cellular genome interplay.

Can SV40 transformation reverse commitment to terminal division in human adult cells in vitro?

Adult Human Eye diploid Fibroblasts (HEF) had an extremely low growth potential in vitro (12 duplication cycles), that was identical in all the isolated clones. HEF cells were transformed with SV40 virus at the 10th duplication cycle, at which stage they were already approaching the crisis phase as demonstrated by the slowing down in the proliferation rate and the inability to produce clones. After SV40-transformation the proliferation rate rapidly increased, reaching a maximal level in a few days, and the transformed cells recovered the ability to produce clones. All the SV40-transformed isolated clones underwent 37 duplication cycles and did not show any slowing down in the proliferation rate or any sign of aging to date. We suggest that SV40 virus was able to "rescue" the cells from an irreversible commitment to cease dividing.

The relationship between prostaglandins and virus replication: endogenous prostaglandin synthesis during infection and the effect of exogenous PGA on virus production in different cell lines and in persistently infected cells.

African Green Monkey Kidney cells were shown to normally synthesize immunoreactive PGE1. Infection of these cells with Sendai virus did not alter rates of PGE1 synthesis, while it stimulated interferon production. PGAs, that we have previously shown to be potent inhibitors of Sendai virus replication in this system, at the same dose (4 micrograms/ml), also strongly inhibited the replication of this virus in HEp-2 cells and in VERO cells, a monkey kidney cell line that does not produce interferon. PGA1 was found to be effective in several cell and virus models, suggesting a broad spectrum of antiviral actions. Finally, we confirmed the observation that PGA1-treatment prevents the establishment of a "carrier state" by Sendai virus, and PGA1-cured cells did not show any sign of persistent infection for periods as long as 110 days after Sendai infection. Attempts to cure already established persistently infected cells were only partially successful.

Acquisition of sensitivity to exogenous fibronectin by Friend leukemia cells correlates with reduction of their tumorigenic potential.

Strongly adhesive, highly flattened clones (FF clones) can be selected from Friend leukemia cells (FLC) cultivated on top of monolayers of human embryonic fibroblasts (HEF). The flattened phenotype of FF clones is stable during cell replication either in soft agar or in vivo. With the number of passages of FLC on HEF the fibronectin (FN) sensitivity of FF clones increases with a proportional reduction of their tumorigenicity. The FN-sensitivity was defined as the ability of a certain dose of FN to flatten 50% of cells of a given FF clone. Tumorigenicity was defined as the number of FF cells able to give palpable tumors, in 50% of inoculated mice. Exogenous FN does not modify the duplication time of FF clones but strongly influences their growth behavior. In the presence of FN, the growth rate of FF cells is controlled by the size of the growth are available. Highly FN-sensitive FF cells grown on FN-coated substrata die at confluency while FF cells not adherent to substrata escape cell death and grow in suspension. We conclude that the high intrinsic FN-sensitivity of FF cells and FN availability at the site of tumor inoculation could be responsible for the reduced tumorigenicity of highly flattened FF clones.

Electron spin resonance of growing normal and virus-transformed cells.

A g = 2.003 ESR signal, attributed to a free radical localized in HeLa cell nuclei and mitochondria but absent in membranes and cytoplasm, has been studied as a function of the culture growth cycle in normal (NRK and 3T3) and virus transformed (NRK/RSV and 3T3/SV40) cells. For both these cell pairs, the signal is higher during the "lag" stage and lower during the "growth" stage. The average specific intensity of the signal in normal cells is about twice that in virus-transformed cells. However, the maximal point of resonance during the lag state is higher in transformed cells than in normal ones. The lag stage in NRK and NRK/RSV cells is much longer than in 3T3 and 3T3/SV40 cells, while the maximal value of the g = 2.003 ESR signal occurs, early in the lag stage of 3T3 and 3T3/SV40 cells and late in the lag stage of NRK and NRK/RSV cells.