Helena Kopecka

Postpolio syndrome: poliovirus persistence is involved in the pathogenesis.

Abstract 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.

In vivo incorporation of cytosine arabinoside into Simian virus 40 DNA

Abstract Inhibition of Simian virus 40 DNA replication by cytosine arabinoside was found to be essentially irreversible. At high concentration of the inhibitor (20 μg/ml), cytosine arabinoside was incorporated into the growing viral DNA chains in internucleotide linkage. Use of lower concentration of the same inhibitor (2 μg/ml) allowed its recovery into supercoiled viral DNA component I.

Intérêt des sondes ARNc (ribosondes) synthétisés in vitro dans la détection des entérovirus par hybridation moléculaire

Summary Radioactively labelled RNA transcripts made in vitro of various fragments from cDNA clones of poliovirus type 1 and of hepatitis A virus under the control of bacteriophage T7 or SP6 promoters have been evaluated for diagnostic purposes. The RNA transcripts were 2 orders of magnitude more sensitive as hybridization probes than corresponding cDNA preparations labelled by the nick translation procedure. A combination of hybridization analysis and sequence comparison showed that some regions of the genome of a number of enteroviruses are highly conserved, while others show very little homology; the general order of conservation is: 5′-non-coding>3′-terminal>central (2C)>VP3>VP1. The 350 bases of the poliovirus VP1 region were highly specific for that virus, while the 450-bases of the 5′NC region showed extensive cross-reaction with other enteroviruses. However, these probes did not hybridize with HAV, which was detected only by HAV-specific riboprobes. The transcripts have been successfully applied as hybridization probes in diagnostic tests on supernatants of infected cell culture lysates and in clinical samples, mainly in stool extracts.

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.

Poliovirus cDNA Cloned in Bacterial Plasmids

The genome of poliovirus is a 7440 nucleotide long, infectious, single-stranded RNA molecule of “plus” strand polarity (1). Its 5′ end is covalently attached to a small MW protein termed VPg (for a review see reference 2), whereas its 3′ end is polyadenylated (Fig. 1). Most of the progress in our understanding of the organization of the viral genome comes from the recent determination of the complete nucleotide sequence of the RNAs of both the Mahoney strain (wild type) and the attenuated Sabin strain (Lsc2ab strain) of type 1 virus (3–5).

Analysis by electron microscopy of recombinant plasmids carrying poliovirus type-1 cDNA inserts

Summary The genome of poliovirus type 1 was reverse transcribed into cDNA, inserted into the DNA of plasmid pBR322 and cloned in Escherichia coli . DNA molecules from cloned recombinant plasmids were examined by electron microscopy after hybridization with viral RNA molecules under conditions favoring RNA-DNA hybridization. Resulting R-loops were used to measure the size of the cDNA inserts. Examination under the electron microscope of heteroduplex molecules formed by annealing of the same recombinant plasmid DNA molecules together, allowed to determine the extent of homology between the cDNA inserts, and the relative location of each insert on the map of the viral genome. A simple procedure which can be used for both RNA-DNA and DNA-DNA hybridization experiments is described.

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.