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At least 73 records · Page 4Linked to original sources
PubMed1991

Methods for viral isolation and DNA extraction for a penaeid shrimp baculovirus.

Procedures for the purification of virions and nucleocapsids of Baculovirus penaei (BP) of penaeid shrimp and subsequent extraction of the viral nucleic acid are described. BP-infected hepatopancrata, from two species of shrimp from different geographical locations in the Americas, were removed and homogenized in a solution of TN buffer (0.01 M Tris-HCl, 0.10 M NaCl, pH 8.0). The homogenized mixture was strained through a 100-mesh screen to remove large pieces of tissue and centrifuged to concentrate the remaining material. The pellet was suspended in TN buffer and layered on to a handmade CsCl gradient. Fractions were collected according to the bands observed in the gradient, and the optical density at 254 nm was recorded for each fraction. The resultant data was tabulated and graphed. Additionally, each fraction was examined by transmission electron microscopy to determine relative numbers of viral particles present. Large amounts of virus consistently corresponded to a specific band in the gradient, which produced a peak when the spectrophometric data was graphed. Nucleic acid was then extracted from the purified viral particles. Removal of polysaccharides was accomplished with the addition of CTAB/NaCl. The BP DNA was visualized on an agarose gel with phage lambda DNA markers for size estimation, and a preliminary endonuclease digestion was performed using BamHI.

Animals↗
PubMed2000

Cellular proviral HIV-DNA decline and viral isolation in naïve subjects with <5000 copies/ml of HIV-RNA and >500 x 10(6)/l CD4 cells treated with highly active antiretroviral therapy.

OBJECTIVE: To evaluate the decay rate of cellular proviral HIV-DNA and viral replication in patients receiving highly active antiretroviral therapy (HAART) in the very early phase of infection. METHODS: Thirty-four patients treated with HAART and retrospectively selected for progressive decline of plasma viraemia up to undetectable levels (< 20 copies/ml), were stratified according to CD4+ cell count and plasma viraemia at base line: > 500 x 10(6) cells/l with < 5000 copies/ml (group 1) or with > 5000 copies/ml (group 2), > 5000 copies/ml with 300-500 x 10(6) cells/l (group 3) or with < 300 x 10(6) cells/l (group 4). Plasma HIV-RNA and proviral HIV-DNA were analysed at baseline and after 1, 2, 3, 6, 9 and 12 months of treatment. RESULTS: After 1 year of treatment, a significant decrease of proviral DNA titre was observed in all patients and a decrease > 1 log was achieved in 24 of 29 subjects of the first three groups. The more pronounced decay of HIV-DNA (half-life 28 weeks) up to < 50 HIV-DNA copies/10(6) CD4+ cells was detected in patients of group 1. At the year's endpoint, five patients (four in group 1 and one in group 2) had < 20 HIV-DNA copies. However, HIV strains sensitive to antiretroviral drugs were isolated from peripheral lymphocytes of 16 out of 34 patients. CONCLUSION: In patients with undetectable plasma viraemia after 1 year of HAART, the highest reduction of proviral DNA up to < 50 copies/10(6) CD4+ cells was obtained only in subjects in the early asymptomatic phase of infection. Nevertheless, a replication-competent virus can be detected in all phases of antiretroviral therapy.

Adult↗
PubMed1987

Physiology and metabolism in isolated viral septicemia. Further evidence of an organism-independent, host-dependent response.

The hypothesis has been advanced that the human systemic septic response is a function of the host and not of the type of infecting organism. Metabolic and physiologic data from five immunosuppressed transplant recipients with isolated cytomegaloviral sepsis and viremia were prospectively evaluated. Serial cultures obtained from lung, sputum, urine, wound, blood, and invasive lines were positive for virus and negative for bacterial or fungal pathogens. The results were compared with two data banks derived from either victims of multiple trauma without sepsis or surgical patients with early bacterial or fungal sepsis. Statistically significant differences between the patients and the nonseptic reference group were noted for cardiac index, total peripheral resistance, arteriovenous oxygen content difference, oxygen consumption, and levels of triglycerides, proline, phenylalanine, tyrosine, alpha-aminobutyrate, and alanine. No such differences were present for these data compared with the septic reference group. Physiologic data obtained just before death in three patients indicated a failure of oxygen transport. It appears that the systemic septic response to viral agents is indistinguishable by physiologic and metabolic criteria from that resulting from bacterial or fungal agents.

Adult↗
PubMed1991

Comparison of Directigen FLU-A with viral isolation and direct immunofluorescence for the rapid detection and identification of influenza A virus.

Directigen FLU-A, an enzyme immunoassay membrane test, was compared prospectively to isolation in cell culture and direct immunofluorescence (IF) for the detection of influenza A virus. One hundred ninety specimens were evaluated by Directigen FLU-A and cell culture; 184 of these specimens were also tested by direct IF. The sensitivity of Directigen FLU-A compared to isolation in cell culture and direct IF was 100%. The specificities of Directigen FLU-A compared to isolation and direct IF were identical, 91.6%. Fourteen specimens that were positive by Directigen FLU-A did not yield virus in culture; two of the specimens, however, were positive by direct IF, and four other specimens were not specimens of choice for the test. A positive Directigen result had positive predictive values of 62.6 and 75.0% compared to isolation and direct IF, respectively; a positive Directigen result with an intensity reading of 2+ or greater, however, had positive predictive values of 85 and 100% compared to isolation and direct IF, respectively. In all comparisons, the negative predictive value was 100%. There was no evidence that cross-reactivity occurred with non-influenza A antigens. Directigen FLU-A should serve as a convenient screening test for influenza A and as a rapid test supported by isolation in cell culture during an influenza outbreak.

Antigens, Viral↗
PubMed2003

High prevalence of human metapneumovirus infection in young children and genetic heterogeneity of the viral isolates.

RNA of the newly identified human metapneumovirus (HMPV) was detected in nasopharyngeal aspirates of 11 of 63 (17.5%) young children with respiratory tract disease. Markers of infection caused by another member of the Pneumovirinae subfamily of the family Paramyxoviridae, respiratory syncytial virus (RSV), were identified in 15 of these patients (23.8%). Three patients were simultaneously infected with HMPV and RSV. Studies of the clinical characteristics of HMPV-infected children did not reveal any difference between HMPV-infected patients and a control population of RSV-infected patients with regard to disease severity, but the duration of symptoms was significantly shorter for HMPV-infected patients. Phylogenetic analysis of the amplified viral genome fragments confirmed the existence and simultaneous circulation within one epidemic season of HMPV isolates belonging to two genetic lineages.

Genetic Variation↗
PubMed1989

Genotypic transitions among bluetongue viral isolates from domestic ruminants in Colorado during 1981-1984.

Two predominant electropherotypes of bluetongue virus (BTV) serotype 11 isolates from cattle during a 1981-1984 field study in eastern Colorado were characterized. The genomes of strains isolated from the first 2 years of the study had 1 predominant electropherotype (CO81), with the exception of 1 isolate that differed only in the migration of segment 3. A second electropherotype (CO83), with differences in the migration of 4 segments, coexisted in the same region during 1983 and 1984 with strains having the CO81 RNA profile. The genomes of CO81 and CO83 were also distinguishable from those of the US prototype of BTV 11. Analysis of the polypeptides of representative strains of each electropherotype by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the proteins were very similar. The occurrence of the CO81 electropherotype was apparently the result of multiple viral infections since the positions of 7 segments had faint second bands and single-banded variants were isolated after serial plaque purifications. In addition, protein 7 of 1 of the CO81 isolates and protein 7 of the single-banded variant differed as shown by reverse phase-high performance liquid chromatography of 35S-methionine-labeled tryptic peptides.

Animals↗
PubMed2006

A comparative study between viral isolation and indirect immunofluorescence in the diagnosis of herpes simplex virus.

Fifty patients with oral ulcers were studied clinically and investigated for the detections of Herpes Simplex Virus (HSV) through virus isolation from their lesions (vesicles and ulcers) and detection of the presence of antiviral antibodies (both, IgM and IgG) in their sera using the indirect immunofluorescene (IIF) technique. The results of this study proved that virus isolation is the most reliable method for diagnosis, though the use of antibody serological tests could be a useful adjunct to virus isolation in situations where a rapid laboratory diagnosis is needed. Oral Herpes Simplex virus infection can be viewed, in the main, as a trivial disorder causing patients minor physical discomfort. The prevalence of HSV may be high in innocent infections, as high as 1/3 of the population. However, HSV infection and its complications with troublesome recurrences may make the problem worse. The apparent increase in HSV infection over recent years may be partly due to increased publicity about the disease, the current antiviral treatment, the inclusion of both primary and recurrent cases in clinic follow up and the increased use of viral cultures for diagnosis. The aim of this work is to share in the study of the detection of HSV through virus isolation and detection of antiviral antibodies using IIF technique, as well as the evaluation of the diagnosis by the above mentioned methods.

Adolescent↗
PubMed2001

Concordant utilization of macrophage entry coreceptors by related variants within an HIV type 1 primary isolate viral swarm.

There is considerable diversity among HIV-1 strains in terms of their ability to use entry coreceptors on macrophages, especially CXCR4, but it is not known whether virus-specific differences exist among related members of a viral swarm. Defining how entry coreceptors on primary target cells are utilized by the spectrum of HIV-1 variants that emerge in vivo is important for understanding the relationship between coreceptor selectivity and pathogenesis. HIV-1 89.6(PI) is a dual-tropic primary isolate, and the prototype 89.6-cloned R5X4 Env uses both CXCR4 and CCR5 on macrophages. We generated a panel of env clones from the 89.6(PI) quasispecies and found a mixture of R5, R5X4, and X4 variants on the basis of fusion and infection of coreceptor-transfected cell lines. Here we address the use of macrophage coreceptors by these related Envs by analyzing fusion and infection of primary monocyte-derived macrophages mediated specifically through each coreceptor. All R5X4 Envs utilized both CXCR4 and CCR5 on macrophages, while R5 variants used CCR5 only. One variant characterized in cell lines as X4 used both CXCR4 and CCR5 on macrophages. No Env variant fused with macrophages through alternative coreceptor pathways. Thus, there was heterogeneity in coreceptor use among the related Env variants, but use of each coreceptor specifically in macrophages was consistent among members of the viral swarm. Coreceptor use in transfected cells generally predicted use in primary macrophages, although for some Envs macrophages may be a more sensitive indicator of CCR5 use than transfected cell lines.

Cell Line↗
PubMed1988

Endogenous murine leukemia viruses: frequency of radiation-activation and novel pathogenic effects of viral isolates.

Female C57BL/6 and BALB/c mice were injected i.p. with 0.06 microCi/kg or 0.5 microCi/kg of the short-lived alpha-emitting radionuclide 224radium at 3-day intervals. Infectious N-ecotropic XC+, and xenotropic C-type retroviruses were activated in several tissues in both strains. In C57BL/6 mice the activation of ecotropic and xenotropic virus was dose-dependent as observed 4 weeks after the start of irradiation. In BALB/c mice a few animals showed activation of ecotropic virus after four weeks of irradiation. The expression of xenotropic virus was similar in irradiated mice and controls. Viral antigen, indicative for viraemia, was not detected in irradiated or control animals. Antiviral antibodies were found in both control and irradiated mice but higher titers were found in the irradiated mice. Bone tissue-derived N-tropic XC+ virus isolates were found to be non-oncogenic in newborn mice of the parental strain. In contrast, the same virus isolates induced a novel pattern of disease, such as osteopetrosis and osteomas together with malignant lymphomas in NMRI mice. The data indicate that the pattern of endogenous murine leukemia virus activation by internal alpha-irradiation is dependent on the dose rate, and on the genetics of the mouse strain.

Animals↗
PubMed1995

[Cuban epidemic neuropathy. I. Viral isolation].

From the end of 1991 to June, 1993, an epidemic neuropathy affecting 50,963 persons occurred in Cuba. Two clinical forms of the disease were observed: the optic form (with or without peripheral manifestations, 52% of the cases) and the peripheral form (48%). The epidemiological studies revealed nutritional disorders, mainly a vitamin B complex deficiency due to economic difficulties faced by the country in the "special period". Smoking habit and alcohol consumption were identified as risk factors, particularly for the most severe forms of the disease. In the virological studies of the cerebrospinal fluid (CSF) a Coxsackie 9 virus was isolated in Vero cells in 4% of the cases, as well as another agent with mild cytopathogenic effect in 80% of the samples. Sixty seven percent of the CSF samples inoculated to lactating mice caused the disease or the death of the animals. The viral persistence was confirmed in the cerebrospinal fluid during al least 21 days in a group of patients, and for a year in another group.

Animals↗
PubMed1995

[Cuban epidemic neuropathy. II. The antigenic characteristics of the viral isolates].

With the aim of characterizing antigenically isolations producing mild cytopathogenic effect obtained from the cerebrospinal fluid of patients presenting with epidemic neuropathy, neutralization tests and western blot analysis were performed using hyperimmune sera of patients and hyperimmune sera of rabbits. It was confirmed that isolations with mild cutopathogenic effect studied have the same antigenic characteristics and that they are related to Coxsackie A9 and B4 viruses. Structural proteins were not detected in the strains with mild cytopathogenic effects, only antigens having a high molecular weight which were considered as precursor proteins for viral replication were confirmed.

Animals↗
PubMed1981

Rat sarcoma virus: further analysis of individual viral isolates and the gene product.

Rasheed rat sarcoma virus, derived by in vitro cocultivation of two rat cell lines (Rasheed et al., Proc. Natl. Acad. Sci. U.S.A. 75:2972-2976, 1978), has been reported to code for a protein of 29,000 Mr, immunologically related to the 21,000 Mr src gene product of Harvey and Kirsten sarcoma viruses. Rat sarcoma virus p29 was thought to contain at least part of a rat type C virus structural protein, since antiserum prepared against whole rat virus was able to immunoprecipitate rat sarcoma virus p29 but not Harvey or Kirsten sarcoma virus p21 (Young et al., Proc. Natl. Acad. Sci. U.S.A. 76:3523-3527, 1979). We now report that antiserum directed against rat type C virus p15, but not viral p12, p10, or p27, immunoprecipitated rat sarcoma virus p29. The p15 antiserum was also able to immunoprecipitate both denatured p29 and a peptide derived by V-8 protease cleavage of p29, indicating that this antiserum contains antibodies directed against primary amino acid determinants. Finally, five separate isolates of rat sarcoma virus were found to code for p29, which indicates that a highly specific site of recombination is involved in the generation of sarcoma viruses in rat cells.

Animals↗
PubMed2001

Molecular epidemiology of TT virus in Italy and phylogenesis of viral isolates from subjects at different risk for parenteral exposure.

The prevalence and genotype distribution of human TT virus (TTV) in Italy were analysed in 593 subjects at different risk of parenteral infection who included blood donors, patients with chronic type C hepatitis (HCV), thalassemic patients, patients on haemodialysis, human immunodeficiency virus type 1 (HIV-1)-negative intravenous drug users (IVDUs), and HIV-1-infected subjects (IVDUs, heterosexual contacts and homosexual males). Plasma TTV-DNA was detected using nested PCR with primers deduced from the N22 region of the open reading frame 1 (ORF-1) and from the untranslated region (UTR) of the viral genome. Phylogenetic analysis of the sequences obtained from ORF-1 was also undertaken. A high prevalence of plasma TTV-DNA was observed using the UTR primers, with rates varying from 83-100% in the study groups. Using the N22 primers, HIV-1 positive IVDUs and homosexual males, haemodialysed patients and thalassemic patients had a significantly higher TTV prevalence (range: 23.0-86.1%) than blood donors, who displayed a high frequency of positivity (10.6%). Sequence analysis of 127 N22-positive isolates revealed that 42.5% were of type 1, 53.5% of type 2, 2.4% of type 3, and that two isolates (1.6%) were closely related to genotypes 1-2 but distinct from the other major genotypes. TTV-2 was significantly more prevalent in patients at high risk for parenteral infection and in HIV-1 positive homosexuals. In sequential samples from 15 TTV-infected subjects, N22 sequences were detectable persistently in 12 (80.0%) and UTR sequences persisted in all 15 patients over a mean period of 29.6 months. This data indicates that TTV is widespread in Italy in parenterally exposed subjects, and that the infection frequently persists.

Adult↗