Application of genome sequence information to the classification of bovine enteroviruses: the importance of 5'- and 3'-nontranslated regions.

Comparative genomics of viruses in evolutionary and phylogenetic studies is well established. Previous nucleic acid sequence analyses have demonstrated that enteroviruses and rhinoviruses of the family Picornaviridae exhibit a similar structure of the 5'-nontranslated region (NTR) differing significantly from the 5'-NTR of cardiovirus, aphthovirus, hepatovirus, and echovirus 22 (provisionally parechovirus 1). Available nucleotide sequence information of the 5'- and 3'-nontranslated regions of more than 70 serotypes of enteroviruses, bovine enteroviruses and rhinoviruses has been compared and correlated with previous findings obtained after analysis of the coding and noncoding genome regions. As a result, the 5'- and 3'-NTRs of all three virus groups are characterized by group-specific nucleotide sequences. Focusing on bovine enterovirus (BEV) serotypes, unique characteristics in all secondary structures of the NTRs were observed. These features clearly separate the BEVs from the human enteroviruses and rhinoviruses. Concerning the 5'-NTR, the most remarkable property is an insertion of about 110 nucleotides between the putative cloverleaf structure at the very 5'-end of the viral genome and the IRES element. This insertion was demonstrated for BEV 1 and 2 and has a predicted folding pattern which is very similar to the 5'-cloverleaf structure. One stem-loop of this second cloverleaf is almost identical to the 3CDpro-binding domain of rhinoviral 5'-cloverleafs. It was also demonstrated that the IRES elements and the 3'-NTRs of both, enteroviruses and rhinoviruses, have group-specific features which differ significantly from the corresponding genome regions of BEV. These results suggest that bovine enteroviruses hold an exceptional taxonomic position besides the established genera Enterovirus and Rhinovirus. Within the Enterovirus and Rhinovirus genera, the existence of virus clusters representing subgenera was previously proposed. Whereas the 5'-NTRs of the four human enterovirus clusters fall into two groups, all four clusters have characteristic secondary structures at the 3'-NTR supporting the concept of enterovirus clusters. For rhinoviruses, the existence of two virus clusters was confirmed.

DNA vaccine-mediated immune responses in Coxsackie virus B3-infected mice.

DNA immunizations with the major structural protein VP1 of Coxsackie virus B3 (CVB3) have been previously found to protect BALB/c mice from lethal challenge. Here we report that the other CVB3 capsid proteins, VP2, VP3, and VP4, were less effective at preventing CVB3-caused disease. The application of pCMV/VP1 as a vaccine caused decreased myocyte destruction, reduced viral load in the heart tissue, accelerated antibody induction, and an early cytokine expression in heart tissue. In summary, our results indicate that the induction of B cell and/or T cell memory in vaccinated mice prior to challenge is responsible for the protection observed.

Synthesis, cytotoxicity and antiviral activity of N,N'-bis-5-nitropyrimidyl derivatives of dispirotripiperazine.

During the search for new antivirals, various N,N'-bis-5-pyrimidyl derivatives of 3,12-diaza-6,9-diazonia(5,2,5,2)dispirohexadecane dichloride (dispirotripiperazine) were synthesized. To reveal relationships between chemical structure and antiviral activity, the compounds were characterized by fast atom bombardment mass, nuclear magnetic resonance, infra red spectroscopy, and elemental analysis and examined for cytotoxicity, inhibition of cell growth and antiviral activity under in vitro conditions. The results of this study demonstrate an excellent compatibility of the test compounds for confluent as well as proliferating cells and a potent structure-dependent inhibition of herpes simplex virus type 1 replication when added during viral adsorption. Functional group analysis revealed that both the dispirotripiperazine as well as the pyrimidine ring with a nitro group in the 5 position are necessary for activity. A reduction of electron density in the terminal pyrimidine rings enhanced the antiviral activity whereas electron donor substitutions reduced it. Introduction of a methyl group in position 2 of the pyrimidine had no influence on cytotoxicity or antiviral activity.

A rapid assay for evaluation of antiviral activity against coxsackie virus B3, influenza virus A, and herpes simplex virus type 1.

In order to identify new potential antiviral drugs, small amounts of extracts or compounds have to be examined for cytotoxicity and antiviral activity in primary screening using a rapid, easy, inexpensive, and highly standardised test system. In this study, high-throughput cytopathic effect (CPE) inhibitory assays were established for coxsackie virus B3 on HeLa Ohio cells, influenza virus A on Madin-Darby canine kidney cells, and herpes simplex virus type 1 (HSV-1) on green monkey kidney cells that meet these requirements. The cytotoxic and the antiviral effects were quantified using a crystal violet uptake assay allowing automated handling of large numbers of candidate agents. To ensure comparable results with plaque reduction assays, the 50 and 90% plaque inhibitory concentrations of guanidine, amantadine, and phosphonoformic acid were used to standardise the anti-coxsackie virus B3, anti-influenza virus A, and anti-HSV-1 tests, respectively. The strong correlation between the antiviral activity determined by CPE-inhibitory assays and plaque reduction assay was further proved for other antivirals. In summary, low amounts of large numbers of compounds may be tested inexpensively and standardised within 24 h (coxsackie virus B3 and influenza virus A) or 48 h (herpes simplex virus type 1) post-infection using CPE inhibitory assays.

Detection of porcine enteroviruses by nRT-PCR: differentiation of CPE groups I-III with specific primer sets.

Porcine enteroviruses (PEV) comprising at least 13 serotypes grouped into three species are described as causative agents of neurological disorders, fertility disorders, and dermal lesions of swine. Despite their well-documented acid stability, enteric infection route, and similarity of clinical symptoms, most of the porcine enterovirus (PEV) serotypes are set apart from the genus Enterovirus of the Picornaviridae. Hence, PCR procedures used commonly to detect enteroviruses are not applicable to epizootic relevant PEV serotypes. A nested RT-PCR protocol is described now suited to detect all known porcine enterovirus serotypes using three sets of primer pairs. These primer pairs were designed to amplify either highly conserved sequences of the 5'-nontranslated region (5'-NTR) or the polymerase gene region of the relevant virus species. All 13 acknowledged serotypes of three PEV species and several field isolates of clinical specimens were detectable. The specificity of the PCR procedure is supported by the observation that RT-PCR-positive field isolates coincide with serological PEV classification. PEV PCR is more rapid and less laborious than the time-consuming virus isolation by tissue culture techniques over several passages and serotyping. Because other viruses such as classical swine fever virus, pseudorabies virus, porcine parvovirus, swine vesicular disease virus, and foot-and-mouth disease virus may cause diseases with similar clinical symptoms, PCR detection of all PEVs closes a diagnostic gap and offers the opportunity to use comprehensive PCR procedures for the diagnosis of all relevant viruses causing such symptoms.

Matrix remodelling in dilated cardiomyopathy entails the occurrence of oncofetal fibronectin molecular variants.

OBJECTIVES: To investigate whether disturbance of the cellular homoeostasis and integrity of cardiomyocytes in dilated cardiomyopathy (DCM) is accompanied by alterations in cell-matrix relations as indicated by changes in the deposition of fibronectin (FN) isoforms. DESIGN: Tissue from a case series of patients with DCM was investigated by immunohistochemistry with antibodies against FN (all variants, clone IST4), ED-A+ FN (clone IST9), ED-B+ FN (clone BC1), and oncofetal glycosylated FN (clone 5C10). The sites of de novo synthesis of FN were demonstrated by means of non-radioactive RNA in situ hybridisation (ISH) with biotinylated FN cDNA fragments as the probe. SETTING: University hospital. PATIENTS: Samples from 10 patients with clinical criteria and histological diagnosis of DCM and from 3 individuals with normal hearts. INTERVENTIONS: Samples were obtained by right ventricular endomyocardial biopsy. MAIN OUTCOME MEASURE: Distribution of oncofetal FN variants in DCM hearts. RESULTS: Immunostaining of FN (IST4, all variants) showed a coarse interstitial network in normal and diseased myocardium. ED-A+ FN was deposited as fine interstitial spots in normal myocardium and in DCM samples. Immunostaining for oncofetal glycosylated FN and ED-B+ FN was not seen in normal adult myocardium, whereas myocardium from DCM patients showed focal and delicate staining in the interstitium. RNA ISH showed that these deposits resulted from local FN synthesis. CONCLUSION: The results accord with de novo expression of oncofetal FN variants in hearts from patients with DCM. The oncofetal FN variants may serve as disease markers in myocardium affected by DCM.

Measurements of nonspecific humoral immune parameters in patients with chronic pyelonephritis.

The levels of autoantibody classes IgG, IgM, IgA, IgD against renal antigens together with CH50, C3, C4, lysozyme, and phagocytic activity against Staphylococcus aureus SG 511 and E. coli 014 were determined in serum samples from 3 groups of patients with chronic pyelonephritis. Increased levels of IgG and IgD were found in patients with severely impaired renal function while increased levels of IgM and IgD correlated with disease activity. C3 levels were increased with enhanced activity and C4 and lysozyme levels with reduced renal function and distinct activity. The value of these findings is discussed and the clinical usefulness of the determination of levels of IgM, IgD, C3, C4, and lysozyme is pointed out.

Influence of hemoperfusion with XAD-4 resin on the concentration of biologic constituents in the blood.

13 patients, who had been subjected to hemoperfusion with Amberlite XAD-4 resin (Haemoresin, Fa. Braun), because of acute intoxications, were examined for the adsorption of bioactive material from the blood. A mean treatment period of four hours at a blood flow rate of 200 ml/min. resulted in a marked thrombocyte decrease by 43% and changes in the concentrations of proteins, complement components C3 and C4 (16-26%) triglycerides (33%), aldosterone (33%) and testosterone (20%). Low molecular weight substances and electrolytes, however, were only slightly or not affected. Thus hemoperfusion system with its poor biocompatibility and inability to remove BUN, electrolytes and water is only suitable for short-term treatment and cannot be used without detailed control of possible side effects and perhaps substitution for treatment of chronic renal or hepatic failure. Hemoperfusion has proved an efficient method of treating various exogenous intoxications. The adsorption of biologically active material from the blood constitutes several side effects which contraindicate long-term treatment, above all of endogenous intoxications such as chronic renal failure and hepatic failure. With the type of adsorber used - various charcoal preparations, resins etc. such as uncoated material or coated with different substances - these side effects vary and have not yet been investigated in a systematic, comparative and comprehensive way. The present investigations are meant to check the adsorption behaviour of Amberlite XAD-4 Resin in vivo towards some selected blood constituents and the influence of hemoperfusion on the concentrations.

The in vitro and in vivo effects of carrageenin on humoral and cellular factors of natural resistance.

The inhibitory effect of carrageenin, a sulfated algal polygalactose, on humoral factors of natural resistance is discussed. In dependence on dose and time, the influence of non-specific and specific bacteriolysis, on the in vitro and in vivo opsonic activity and on the course of infection was studied.

Preselection of Potential Cancerostatics by Automatic Analysis of Suspended and Adherent Cells Incubated in Microplates.

Alternative toxicological screening programmes, without the use of animal experiments, are intended to eliminate dangerous substances and to find new pharmacologically active agents in cell cultures. They can also provide information on the cytostatic activities of the agents. Intercalating cytostatics which bind DNA were selected by measuring the statistical distributions of the cell diameters of K-562 and L-929 cells by using an electronic cell analyser (CASY1). These compounds were identified by cell enlargement or from flat concentration-activity curves created with the cell analyser system. Incubation for 72 hours with DNA-binding agents, such as doxorubicin, daunorubicin and Mitoxantron®, resulted in enlargement of cell diameter and cell volume. The antineoplastic agents actinomycin D and ambazone had no comparable effect. Comparisons of the different parameters obtained with CASY1 measurement were performed with Microsoft EXCEL.

Abnormal macrophages and NK cell cytotoxicity in human systemic lupus erythematosus and the role of interferon and serum factors.

Macrophage (MO) and natural killer (NK) cell mediated cytotoxicity to K562 target cells were strikingly decreased in patients with systemic lupus erythematosus (SLE). SLE NK cells failed to release soluble factor(s) for lysing the targets. IFN-induced enhancement of both types of cytotoxicity was impaired. NK cells from healthy subjects kept their activity in culture with or without IFN for more than six days whereas SLE NK cell activity declined to zero at day 3. So, the increased IFN level of many SLE patients and a possible prior IFN priming effect seemed unrelated to the insensitivity to exogenous IFN in vitro. Inhibition factor(s) of SLE serum suppressed NK cytotoxicity in the presence of IFN whereas IFN sensitivity of MO remained unaffected indicating the complex regulation by serum components of immune reactions.

[The effect of potential antineoplastic antibiotics and the metal complex compound cisplatin on in vitro phagocytosis]. / Der Einfluss potentiell antineoplastisch wirksamer Antibiotika und der Metallkomplexverbindung Cisplatin auf die in vitro-Phagozytose.

Under standardized conditions the influence of the drugs lambdamycin, violamycin, granatomycin C, daunorubicin and cisplatin on the phagocytosis has been tested. Therapeutical doses of lambdamycin, daunorubicin and cisplatin did not have any obvious impairment on the phagocytosis. Contrarily to this granatomycin C (an isochromanchinon antibiotic) and violamycin (an anthracyclin in high concentrations) significantly depressed the phagocytosis of PMN.

[The clinical value of unspecific humoral immune parameters. Attempt at an immunogram. 1. Quantitative determination of IgG, IgM, IgA and IgD and demonstration of antibodies against cell and tissue bound antigens in patients with chronic pyelonephritis]. / Untersuchungen zur klinischen Wertigkeit von unspezifischen humoralen Immunparametern. Versuch eines Immunogramms. 1. Mitteilung: Die quantitative Bestimmung von IgG, IgM, IgA und IgD sowie der Nachweis von Antikörpern gegen zell- und gewebsgebundene Antigene bei Patienten mit chronischer Pyelonephritis.

In the serum of patients with chronic pyelonephritis IgG, IgM, IgA, IgD and autoantibodies against organ- and species-specific renal antigens were determined. Clinical questions concerned relations to the activity of the disease and the renal function. The Ig-mean-values lying within the biological norm could be precised by taking into consideration individual biological boundary titres and relative ratio values. Pictures of tendency led to the estimation that increases of IgG and IgD appear in severely restricted renal function in normal or decreased IgM. Increases may be a criterion of activity in diseases with normal or slightly restricted renal function. IgA determinations remained without any evidence.-- Autoantibodies were found in 32,5%, increased titres were a sign of deteriorated renal function. The apparently given possibility of a humoral immunogramme for the chronic pyelonephritis is discussed, in which case a combination with other test parameters seems to be necessary.