Detection of enteroviruses in cases of neurological disorders in the State of Pará, Brazil.

Eighty-one cerebrospinal fluid (CSF) samples mainly from cases of aseptic meningitis and motor deficiency syndrome were sent to the Virology Section of Evandro Chagas Institute, Belém Pará, in the period of January 1995 to January 1996 in order to isolate viruses. All samples were inoculated onto HEp-2 cell culture and newborn mice, with negative results. The probability of isolating viruses by these methods is reduced because of the low concentration of viral particles in these specimens. In order to obtain more information about the etiology of these cases, a group of 23 samples were selected to be tested by a more sensitive technique than the virus isolation - the reverse transcription polymerase chain reaction (RT-PCR). Specific primers directed to conserved regions in the enterovirus genome were used, considering that this group of viruses is frequently associated with these neurological disorder. The age of the patients ranged from 1 to 55 years and nearly all of them lived in Belém, State of Pará, North of Brazil. Of 15 samples analyzed by RT PCR nine (60%) were positive; of these, 6 (66.6%) had motor deficiency and 3 (33.3%) developed aseptic meningitis. These results show that it is important to investigate enterovirus as cause of these syndromes.

Postpolio syndrome: poliovirus persistence is involved in the pathogenesis.

The pathogenesis of the postpolio syndrome (PPS) remains unclear. In this study we looked for poliovirus (PV) persistence in the CSF of 20 patients with PPS, in a control group including 20 patients with unrelated neurological diseases, and in 7 patients with stable poliomyelitis sequelae. CSF samples and sera were examined using reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of PV or other enterovirus genomes; this assay allows the detection from as little as 1 fg viral RNA. Sequencing of amplified products from 5 patients was performed. PV genomic sequences were detected in the CSF of 11 of 20 patients with PPS and in none of the control group. Sequencing in the 5' untranslated region confirmed the presence of mutated PV sequences. These findings suggest that PPS is related to the persistence of PV in the central nervous system.

Enterovirus genome detection in wastewater: multi centric evaluation of a commercial kit.

A multi-centric study was carried out in three laboratories, to evaluate the efficiency of a standardized kit for the detection of enterovirus genome in wastewater. Twenty one samples of 20 liters of wastewater were analyzed before and after concentration through glass wool. Each sample was analyzed with the Amplicor kit as well as with techniques developed independently in each laboratory. The results show that the Amplicor kit is well suited to the detection of enterovirus genome in treated wastewater. The results may be compared to those obtained with semi-nested RT-PCR techniques used in each laboratory. However, the Amplicor kit technique is more simple and has the advantage of providing a standardized technique useful for comparative studies. During this work it was observed that the sensitivity of the detection of infectious viruses and virus genome was improved when concentrated samples were used for analysis.

Detection of enteroviral sequence in endomyocardial tissues from patients with cardiac diseases in northern Brazil.

In the present report we describe the results from a pilot study aimed at detecting enterovirus sequence in cardiac tissues, obtained through endomyocardial biopsies, from patients suffering from cardiac diseases in the Amazon region. Six samples that were collected from three patients were analysed by RT-PCR showing 3 positive and 3 negative results. These preliminary findings suggest the participation of enteroviruses in the etiology of cardiac diseases, mainly myocarditis, and warrant further and broader local studies on this subject.

Detection of enteroviral sequence in endomyocardial tissues from patients with cardiac diseases in northern Brazil

No presente trabalho, são mostrados resultados de um estudo piloto direcionado à detecção de seqüências de enterovirus em tecido cardíaco obtido a partir de biópsias endomiocárdicas de pacientes com doenças cardíacas na região Amazônica. Seis amostras coletadas de três pacientes foram analisadas por RT-PCR obtendo-se três espécimes positivos e três negativos. Esses achados preliminares sugerem a participação dos enterovirus na etiologia de doenças cardíacas, principalmente miocardites, e justificam estudos mais amplos nesse assunto.

In the present report we describe the results from a pilot study aimed at detecting enterovirus sequence in cardiac tissues, obtained through endomyocardial biopsies, from patients suffering from cardiac diseases in the Amazon region. Six samples that were collected from three patients were analysed by RT-PCR showing 3 positive and 3 negative results. These preliminary findings suggest the participation of enteroviruses in the etiology of cardiac diseases, mainly myocarditis, and warrant further and broader local studies on this subject.

Discovery of ascomycin analogs with potent topical but weak systemic activity for treatment of inflammatory skin diseases.

Drug therapy for the major inflammatory skin diseases, which include atopic dermatitis, psoriasis and allergic contact dermatitis, is often inadequate due to poor efficacy, toxicity, or both. Much research has focused on the macrolactam T cell inhibitors as a promising new class of agents for immunotherapy, and medicinal chemistry efforts to design novel ascomycin analogs have produced clinically promising agents. A synthetic program to modify the ascomycin nucleus to alter its physicochemical properties and promote systemic clearance is described. A biologic screening strategy to identify analogs with reduced systemic activity and rapid pharmacokinetic elimination led to identification of the clinical candidate, ABT-281. A swine contact hypersensitivity model was used as a stringent indicator of skin penetration as human doses of topical corticosteroids produced inhibition only in the 50% range and ED50 values were 100-fold less potent than in rat. Also, cyclosporine was confirmed to be topically inactive in swine, as seen in human. ABT-281 had topical potency equal to tacrolimus (FK506) despite a severalfold lower potency for inhibiting swine T cells in vitro, consistent with superior skin penetration. ABT-281 was found to have a shorter duration of action after i.v. dosing in monkeys using an ex vivo whole blood IL-2 production assay. Systemic potency was reduced by 30-fold or more in rat popliteal lymph node hyperplasia and contact hypersensitivity assays. Following i.v. or i.p. administration in the swine contact hypersensitivity model, ABT-281 was 19- and 61-fold less potent, respectively, than FK506. Pharmacokinetic studies showed that ABT-281 had a shorter half life and higher rate of clearance than FK506 in all three species. The potent topical activity and reduced systemic exposure of ABT-281 may thus provide both efficacy and a greater margin of safety for topical therapy of skin diseases.

Molecular epidemiological survey of rotaviruses in sewage by reverse transcriptase seminested PCR and restriction fragment length polymorphism assay.

Rotavirus double-stranded RNA was detected directly in sewage treatment plant samples over a 1-year period by reverse transcription followed by PCR amplification of the VP7 gene and Southern blot hybridization. The presence of naturally occurring rotaviruses was demonstrated in 42% of raw sewage samples and in 67% of treated effluent samples. Amplified viral sequences were analyzed by restriction enzymes. Ten different restriction profiles were characterized, most of which were found in treated effluent samples. A mixture of restriction profiles was observed in 75% of contaminated effluent samples. The profiles were compared with those obtained from human rotavirus isolates involved in infections in children from the same area (six different profiles were detected). Five identical viral sequences were detected in both environmental and clinical samples. Restriction profiles were also compared to profiles from known genomic sequences of human and animal viruses. Both human and animal origins of rotavirus contamination of water seemed likely.

Minor structural differences in Boc-CCK-4 derivatives dictate affinity and selectivity for CCK-A and CCK-B receptors.

We previously reported novel Boc-CCK-4 (Boc-Trp-Met-Asp-Phe-NH2) derivatives possessing the general structure Boc-Trp-Lys[N epsilon-CO-NH-(R-Ph)]-Asp-Phe-NH2 (Shiosaki et al. J. Med. Chem. 1991, 34, 2837-2842). In contrast to Boc-CCK-4, which is 70-fold selective for the CCK-B receptor, the modified lysine-bearing tetrapeptides were highly potent and selective full agonists at the CCK-A receptor. Further investigation of the structure-activity profile following modification of the substituted phenylurea moiety appended off the lysine revealed that moving certain substituents, e.g. nitro or acetyl, from the 2- or 3-position on the phenyl ring to the 4-position, a relatively minor and subtle structural modification within the tetrapeptide, resulted in loss of CCK-A receptor selectivity and development of a trend toward CCK-B selectivity. These tetrapeptides, e.g. Boc-Trp-Lys[N epsilon-CO-NH-(4-NO2-Ph)]-Asp-Phe-NH2 and Boc-Trp-Lys[N epsilon-CO-NH-(4-Ac-Ph)]-Asp-Phe-NH2, were full agonists relative to CCK-8 in stimulating intracellular calcium mobilization in a cell line that expresses the CCK-B receptor.

[Molecular biology of enteroviruses]. / Molekulární biologie enteroviru.

Molecular biological methods offer new approaches to the study of RNA viruses. On the example of enteroviruses we tried to give an account, of recent progress in the knowledge and understanding of these important human pathogens. Firstly concerning their structural and genomic organisation; secondly as regards understanding of their pathology and pathogenicity; thirdly the impact of such methods on rapid, sensitive and specific diagnosis, treatment, on epidemiology and finally on evolution and classification of the large picornavirus family where the enterovirus genus belongs. We believe that molecular biology will elucidate still unknown features of enteroviruses, which are not yet correctly evaluated human pathogens.

Multicenter evaluating of a commercially available PCR assay for diagnosing enterovirus infection in a panel of cerebrospinal fluid specimens.

Thirteen laboratories participated in blind tests of a panel of 20 coded cerebrospinal fluid specimens (7 uninfected samples, 3 samples infected with 1 50% tissue culture infective dose [TCID50]/0.1 ml [nonenterovirus strains], and 10 samples infected with 10, 1, or 0.1 TCID50/0.1 ml [three different enterovirus serotypes]) on the Amplicor enterovirus PCR assay (Roche Diagnostic Systems). The panel was also evaluated by in-house PCR (two nested-PCR and three one-step PCR assay) or tissue culture (eight laboratories). The viral load was shown to influence greatly the sensitivity of the assay. The average sensitivity of the Amplicor test ranged from 67 to 98% for viral titers of 1 to 10 TCID50/0.1 ml, respectively; titers of 0.1 TCID50/0.1 ml resulted in a sensitivity of only 16%. The overall specificity of the Amplicor test was 98%. The Amplicor assay compared favorably to the five in-house PCR tests (no significant difference in either sensitivity or specificity) and was much more sensitive than tissue culture (P < 0.001), even for high viral loads. It was easy to perform, rapid (about 6 h), well-standardized, and appeared to be suitable for the diagnosis of enterovirus meningitis on a routine basis in laboratories trained in molecular biology techniques.

Evidence of presence of poliovirus genomic sequences in cerebrospinal fluid from patients with postpolio syndrome.

The postpolio syndrome (PPS) is characterized by new neuromuscular symptoms occurring 30 to 40 years after the acute episode of poliomyelitis paralysis. The presence of the poliovirus RNA genome in the cerebrospinal fluid from 10 patients with PPS and from 23 control patients was sought by using reverse transcription and a PCR specific for polioviruses and/or other enteroviruses. Poliovirus-specific genomic sequences in the 5' untranslated region and in the capsid region (VP1) were detected by reverse transcription PCR in 5 of 10 patients with PPS but in none of the control patients. Sequencing confirmed the presence of mutated poliovirus sequences. This finding suggests persistent viral infection in the central nervous system related to the presence of poliovirus genomes.

Genotypic variation in coxsackievirus B5 isolates from three different outbreaks in the United States.

Genomic sequences in VP1/2A and 5'-non-coding region of 10 isolates of Coxsackievirus B5 from three outbreaks were compared with published sequences of another Coxsackievirus B5, swine vesicular disease virus, Coxsackievirus B1, Coxsackievirus B3, and Coxsackievirus B4. Isolates of Coxsackievirus B5 from the same outbreak showed close relations, not exceeding 7.2% in nucleotide differences. Differences were greater between isolates from different outbreaks, varying between 8.4 and 16%. We have also shown that Coxsackie B5 viruses from an outbreak in 1967 are more similar to viruses from an outbreak in 1983 than to the viruses isolated from an intervening outbreak in 1972. The sequence comparison of Coxsackievirus B5 isolates with other Coxsackie B viruses and swine vesicular study, cDNA synthesis, polymerase chain reaction, and sequencing, are suitable for rapid Coxsackie B virus detection and identification of genotypic relations between viruses originating from different outbreaks.

Novel Asp32-replacement tetrapeptide analogues as potent and selective CCK-A agonists.

A series of novel CCK tetrapeptide analogues of the general formula Boc-Trp-Lys(Tac)-N(R)-(CH2)nCON(R')Phe-NH2 (Tac = o-tolylaminocarbonyl), where R,R' = H or Me and n = 1-5, have been synthesized and tested. These analogues, which lack an acidic residue at the penultimate position, demonstrated surprisingly high CCK-A receptor affinity and selectivity. The effect of N-methylation pattern on CCK-A receptor affinity showed consistent trends for analogues in which n = 1, 2, or 3, with the di-N-methylated analogues having the highest affinity in each case. However, none of these analogues had full agonist activity, as measured by percent maximal PI hydrolysis. Two conformationally constrained analogues also demonstrated high CCK-A receptor affinity and selectivity, as well as nearly maximal agonist activity. In addition, one of these conformationally-constrained analogues demonstrated anorectic activity in rats.

Tetrapeptide CCK-A agonists: effect of backbone N-methylations on in vitro and in vivo CCK activity.

N-Methylation of backbone amide bonds was conducted on a tetrapeptide that had been identified previously (Shiosaki, K.; et al. J. Med. Chem. 1991, 34, 2387-2842) as a potent and selective CCK-A agonist. N alpha-Methylation at the position corresponding to Asp32 (CCK-33 numbering) was consistent with high affinity, efficacy, and selectivity for the CCK-A receptor. Combination of this (N-Me)Asp with the (N-Me)Phe modification also provided a highly active analogue. The observation of parallel structure-binding affinity profiles with respect to sites of N-methylation in the C-terminal regions of tetrapeptide vs heptapeptide CCK analogues suggests that the two series interact similarly with the CCK-A receptor.

Detection of enteroviruses in cerebrospinal fluids: enzymatic amplification and hybridization with a biotinylated riboprobe.

Cerebrospinal fluids from 54 children with meningitis syndrome were examined for the presence of enteroviruses by cell culture, by enzymatic amplification of viral cDNA and by hybridization. Viral sequences were found in 37/39 CSF specimens positive by cell culture (95%) and in 6/15 (40%) CSF specimens negative by cell culture. Thus, PCR, as a specific and sensitive technique, may be suitable for rapid diagnosis of enteroviral meningitis in clinical specimens from which enteroviruses are difficult to isolate.

Tetrapeptide CCK agonists: structure-activity studies on modifications at the N-terminus.

We had reported earlier on a novel series of potent and selective tetrapeptide cholecystokinin-A (CCK-A) agonists of the general structure Boc-Trp-Lys[epsilon-Y]-Asp-N(R)PheNH2 [Y = amides, ureas; R = H, Me] that were potent anorectic agents in rats. In an effort to optimize the potency, selectivity, stability, and efficacy of our lead candidate A-71623 [R = Me, Y = o-tolylaminocarbonyl; Tac] toward development of a clinical candidate, we have explored a series of analogues in which the N-terminal Boc functionality was systematically replaced with various amides, ureas, carbamates, and sulfonamides of differing size, hydrophobicity, and stereoelectronic properties. In general, these analogues maintained good potency and selectivity for the CCK-A receptor (guinea pig pancreas), as well as potent anorectic activity in rats. Those analogues exhibiting equal or superior activity compared to A-71623 but differing physicochemical properties may represent superior drug candidates.

Use of the polymerase chain reaction with a murine model of picornavirus-induced myocarditis.

Enteroviruses are common pathogens responsible for a wide spectrum of systemic infections. Conventional diagnosis of these infections relies on the isolation of viruses in cell culture and their identification by seroneutralization with polyclonal or monoclonal antibodies. Among enteroviruses, coxsackieviruses B have been involved as causative agents for viral myocarditis. Most of the time, in the case of cardiac pathologies, viral isolation is negative. Molecular biology techniques appear to be an alternative to conventional diagnosis and could supply evidence for the direct implication of enteroviruses in these severe pathologies. In this paper, we describe a murine experimental model of infection with the presumed highly cardiopathogenic coxsackie-virus B type 3. A kinetics of infection was observed for a period of 31 days, and the classical virological markers (viral isolation from feces and heart biopsies, seroconversion) were monitored and compared by means of molecular techniques (molecular hybridization, polymerase chain reaction [PCR]). In this 31-day period, the detection of coxsackievirus B type 3 RNA in the heart was possible only by using two successive seminested PCRs. After 9 to 11 days of active viral replication, when all other virological markers were negative, positive PCR signals were obtained, which supports the hypothesis of a shift to persistent enteroviral infection.

Double bond isosteres of the peptide bond: synthesis and biological activity of cholecystokinin (CCK) C-terminal hexapeptide analogs.

New and existing methodologies were used to prepare a series of modified CCK analogs in which each amide bond was replaced by a trans-alkene unit. The data indicate that every amide linkage at C-terminal tetrapeptide (CCK-4) region is crucial for biological activity. While the amide bond beyond the Trp residue in the N-terminal direction can be replaced by a trans-alkene and still retain most of the binding potency and functional activity.