Brote epidémico de meningitis aséptica: experiencias en un hospital infantil privado / Epidemic of aseptic meningitis experience of private hospital

Se informa acerca de las experiencias obtenidas en 193 niños con meningitis aséptica, estudiados durante un brote epidémico registrado entre febrero y julio de 1992. Los cultivos de líquido cefalorraquídeo hechos en el Instituto de Referencia Epidemiológica permitieron identificar al virus Echo 30 como un agente a la meningitis en 48 niños

Protein analysis of newly isolated variants of echovirus type 18 by electrophoresis and western blotting.

The protein patterns of two newly isolated antigenic variants of echovirus type 18 were compared with those of the prototype strain. Electrophoretic mobilities of VP1 and VP2 of the variants differed from those of the prototype strain. The antiserum against the prototype strain reacted to VP0 and VP2 in the Western blotting experiment. In addition, the variants differed from the prototype strain in growth behavior in the cultured cells.

Analysis of virus protein heterogeneity among group B coxsackie viruses using a "mini" two-dimensional gel electrophoresis system.

The application of a small format two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) system to the study of protein heterogeneity among group B coxsackie virus (CVB) isolates is described. Under the conditions of electrophoresis developed during this study, protein samples could be processed within 7 h and up to 300 intracellular proteins were resolved from uninfected HEp-2 cell lysates. 2D-PAGE was used to characterise the intracellular proteins of clinical CVB isolates of serotypes 4 and 5. Intracellular proteins from virus-infected cells were radiolabelled using a pulse-chase protocol under conditions which promoted inhibition of cellular protein synthesis. Depending on the CVB serotype up to 11 intracellular virus proteins were identified, ranging in molecular weight between 14,000 and 54,000. Although the overall two-dimensional protein profiles were characteristic for the two CVB serotypes, within a CVB serotype there was some heterogeneity of the virus proteins, mainly affecting the proteins' net charge. The sensitivity of 2D-PAGE in detecting subtle differences in virus proteins combined with the convenience of the small gel format makes this a suitable approach for the study of the molecular epidemiology of human virus pathogens.

Characterization of a non-structural 147-kDa precursor polypeptide of echovirus 33 and its immunogenicity in man.

Zinc chloride (0.9 mM)--an inhibitor of the processing of the initial polypeptides of Picornaviridae--was used to accumulate large precursors of echovirus 33 (EV33) and notably two proteins of 147- and 97-kDa. Polyclonal hyperimmune sera were raised in mice against these polypeptides and assayed by immunoblotting against EV33-infected cells blocked or not with ZnCl2, showing that protein 147--2ABC3ABCD according to the L434 convention--can be considered to be the precursor of the two non-structural proteins P2 and P3. Sixty-three serum specimens from subjects exhibiting varying antibody status against EV33 by seroneutralization were investigated by immunoblotting against an EV33 ZnCl2-blocked antigen. Some subjects infected with EV33 were shown to elicit antibodies which recognize the non-structural precursor polypeptides, a fact whose clinical significance needs further evaluation.

A solid-phase reverse immunosorbent test for the detection of enterovirus IgM.

IgM antibodies against enterovirus antigen were determined by solid-phase reverse immunosorbent test (SPRIST). The 145 sera studied were sampled from cases of enterovirus infections diagnosed by virus isolation and/or complement fixation. In 91 sera from enterovirus infections diagnosed by virus isolation 11/40, 16/28 and 9/23 were positive in SPRIST against ECHO 3 and/or Coxsackie B3 antigen during the first, during the second and third, and after 3 weeks of illness, respectively. The corresponding figures for 85 sera from enterovirus infections diagnosed by a greater than or equal to 4-fold rise in complement fixing (CF) antibody titre against antigen from ECHO 18 and/or Coxsackie B5 antigen were 10/39, 12/20 and 9/26. None out of 22 sera with rheumatoid factor reacted in SPRIST, but 1/92 and 1/154 sera from blood donors was positive in SPRIST with Coxsackie B3 and ECHO 3 antigen, respectively. IgM antibodies against ECHO 3 antigen as determined by SPRIST were found to be cross-reactive over a broad range of enterovirus types and positive results were recorded with sera from infections with Coxsackie A9, B3, B4, B5, ECHO 3, 9, 17, 18, 25 and 30. Heterotypic titres in SPRIST were of the same magnitude (up to 25,600) as recorded against the homotypic virus, 12,800 and 1,600, in two sera from one patient with an ECHO 3 infection. SPRIST was found to be a rapid convenient, cross-reactive and a cheap mu-capture assay for enteroviruses with more than 50% sensitivity during the second and third weeks after onset of illness.

[cRNA probes (riboprobes) synthesized in vitro for detecting enterovirus by molecular hybridization]. / Intérêt des sondes ARNc (ribosondes) synthétisés in vitro dans la détection des entérovirus par hybridation moléculaire.

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 greater than 3'-terminal greater than central (2C) greater than VP3 greater than 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.

The relationship between Coxsackie-B-virus-specific IgG responses and genetic factors (HLA-DR, GM, KM) in insulin-dependent diabetes mellitus.

IgG antibody titres to Coxsackie B1-B6 were measured in 113 insulin-dependent diabetes mellitus (IDDM) patients whose mean age was 12.2 years and mean duration of IDDM was 4.6 years, and in 87 normal sibling controls whose mean age was 13.8 years. Compared with their normal siblings, the diabetics had a significantly increased frequency of high response (titre greater than or equal to 320) to Coxsackie B2 (8% versus 1%, p = 0.028), to Coxsackie B4 (15% versus 1%, p = 0.0006), and to Coxsackie B viruses in general (25% versus 5%, p = 0.0001). The frequencies of HLA-DR and immunoglobulin (GM, KM) antigens did not differ between diabetics with and without a high response to Coxsackie B2, B4, or B viruses in general. We conclude that there is an association between IDDM and IgG response to Coxsackie B2, B4, and B viruses in general, a finding which is consistent with the interpretation that infection with Coxsackie B viruses, especially B4, may initiate the development of IDDM in a portion of individuals who have HLA-DR region susceptibility genes.

Hallazgos inmunologicos sen maningoencefalitis por Neisseria meningitidis B125, Echo 4 y Angiostrongylus cantonensis / Immunological findings in meningoencephalitis due to Neisseria menignitidis B15, ECHO 4 and angiostrongylus cantonensis

Las meningoencefalitis infecciosas resultan de interés por su frecuencia en Cuba. Por ello hemos estudiado 370 pacientes con estas patologías en forma longitudinal en 5 ocasiones durante el año posterior al egreso y en la fase aguda. De ellos fueran 47 con Neisseria meningitidis, 945 con ECHO 4 y 3 con Angiostrongylus cantonensis. Se realiza análisis microbiológico y citolquímico del líquido cefalorraquídeo y sangre. Se cuantifica IgA, IgM, IgG, IgE, albúmina, alfa 1 antitripsna, alfafetoproteína, ácido láctico, anticuerpos contra serogrupos de Neisseria meningitidis por radioinmunoensayo, inmunodifusión radial,contrainmunoelectroforesis y ELISA. En la fase aguda se observó síntesis intratecal de IgG, IgA, IgE en Neisseria meningitidis y en ECHO 4. En los casos de angiostrongylus cantonensis, la ruptura de la barrera hematoencefálica impidió determinar síntesis intratecal. En los estudios longitudinales se observó una depresión de IgM inicial, seguida de un incremento de IgG. Hubo punto de inflexión en la curva de los niveles de IgM e IgA a los 60 dias, con un homogéneo comportamiento de los anticuerpos de serogrupos de Neisseria meningitidis. La IgA tuvo un comportamiento variable. Se observa un incremento de alfa I antitripsina en la fase aguda en todas las meningoencefalitis no eosinofílicas y altos valores de ácido láctico en meningoencefalitis por Neisseria meningitidis

Biochemistry and pathogenicity of echovirus 9. II. Mutants of echovirus 9, strain Hill, with altered capsid surface properties.

The two echovirus 9 strains Hill and Barty have been shown previously not only to differ in pathogenicity for newborn mice but also in a number of in vitro characteristics which depend on viral capsid structure. Three spontaneously occurring mutants of the mouse-apathogenic echovirus 9 prototype strain Hill being resistant to an inhibitor of plaque formation present in agar were isolated and compared biochemically and biophysically to their parent strain and to the mouse-pathogenic echovirus 9 strain Barty, which is resistant to this inhibitor. The mutants differ from their apathogenic parent strain Hill in most of the same in vitro characteristics as strain Barty, namely adsorption to cells in culture, sedimentation behavior in low salt sucrose gradients, distribution of mutant virus particles in isoelectric focusing, and antigenic determinants inducing neutralizing antibodies. For two of the three mutants, Ag2 and Ag3, evidence was obtained from fingerprinting that they differ from their parent strain Hill in VP1; thus, the observed in vitro properties may be caused by the change in this capsid protein. All mutants, however, were found to be apathogenic for newborn mice and do not replicate in the tissues of these animals. It is concluded that the observed changes in capsid structure do not covary with virulence.

Membrane-bound virions of coxsackievirus B4: cellular localization, analysis of the genomic RNA, genome-linked protein, and effect on host macromolecular synthesis.

Hela cells infected with several group B coxsackieviruses contain, in addition to standard virions, a population of virus-specific ribonucleoprotein particles which we (5) designated membrane-bound virions (MBV). MBVs differ from standard virions in buoyant density, yield, appearance, protein composition and infectivity. Here we present several new features of MBVs of coxsackievirus B4. The MBVs are lighter (rho about 1.30) and are localized in rough membranes, intermixed with virions. They contain 35S virion RNA covalently linked with a small protein, VPg. The VPg contain two proteins of different charge. MBV VPg is considerably smaller than the 5300-dalton virion VPg. MBV RNA is homologous to the base sequence present in B4 virus double-stranded RNA. The T1 oligonucleotide fingerprint of MBV RNA is distinguishable from that of virion RNA by one oligonucleotide. Several oligonucleotides of virion RNA appear to occur in submolar quantities in MBV RNA. MBVs are 75 to greater than 200 times less infective; they inhibit host cell macromolecular synthesis less efficiently than virions. In coinfected cells, the extent of inhibition of host synthesis is less severe than in cells infected with virions alone, which suggest interference by MBV particles.

Agarose isoelectrofocusing of intact virions.

A convenient and accurate method for determining the isoelectric points of intact virions is described. Tritium-labeled poliovirus 1 (strains Brunhilde and LSc-2) and echovirus 1 (isolates V239, V248, V212, R115 and 4CH-1) were successfully focused into sharp bands at their respective isoelectric points using a thin-layer agarose isoelectric focusing system. In situ detection of labeled virus bands in the agarose was by fluorography. Freezing and thawing of virus samples prior to isoelectric focusing did not alter their respective isoelectric points.

Evidence for secondary structure within the virion RNA of echovirus 22.

Phenol-extracted echovirus 22 virion RNA is infectious, but unlike poliovirus virion RNA, it resists digestion with pancreatic RNase and nuclease P-1, a 3' exonuclease selective for single-stranded RNA. These data indicate the presence of an enzyme-resistant portion somewhere in the RNA molecule and suggest that it is a double-stranded or base-paired region distant from the unblocked 3' terminus. Equilibrium density gradient centrifugation of native echovirus 22 virion RNA results in a single peak with a density of 1.63 g/cm3. When sheared before centrifugation, the molecule is resolved into two RNA species: one with an approximate density of 1.70 to 1.71 g/cm3, as is observed also for single-stranded poliovirus virion RNA, and the other with a density of 1.58 to 1.59 g/cm3. Data obtained from rate zonal centrifugation may be used to calculate an approximate sedimentation coefficient corrected to water at 20 degrees C of 34 and a molecular weight of 2.4 X 10(6) for the virion RNA. We propose a model for echovirus 22 RNA composed of a linear RNA molecule with a 5' hairpin.

Echovirus 11 dense particles: isolation and preliminary characterization.

Dense particles (density 1.44 g/ml) were isolated during purification in CsCl of echovirus 11 produced in HeLa cells. The dense particles had similar antigenic properties and similar RNA and protein composition to standard (density 1.33 g/ml) echovirus 11 particles. They differed from standard particles in their higher buoyant density, lower infectivity and slightly smaller diameter. In contrast to other picornavirus dense particles, echovirus 11 dense particles were present as a major component of virus population. This high ratio of dense particles was found only in virus from HeLa cells which produced non-haemagglutinating echovirus 11. Haemagglutinating echovirus 11 from primary monkey kidney (MK) cells did not contain any detectable levels of dense particles.

Comparison of capsid polypeptides of group B coxsackie-viruses and polypeptide synthesis in infected cells.

Capsid polypeptides of all six types (B1-6) of group B coxsackieviruses were compared by high-resolution gel electrophoresis, and synthesis of protein and RNA in B4- or B5-infected HeLA cells was analyzed. Four polypeptides, VP1-4, were detected in each type. Another polypeptide, VP0, slightly larger than VP1, was also detected in trace amounts in some types. VP1-3 showed different but characteristic molecular weights (VP1, 34,500 to 37,000; VP2, 31,000 to 36,000; VP3, 26,000 to 32,500), and presented well-defined and reproducible differences in electrophoretic mobility. The molecular weight of VP4 ranged from 5,000 to 5,500. VP1 was largest in B2 and B4, smallest in B1, and of intermediate size in the other types. VP2 was largest in B4 and smallest in B2; VP3 was largest in B5 and B6 and smallest in B4. In B4- or B5-infected HeLa cells, host protein synthesis began to decline after 2 hours postinfection and was less than 20 percent of the control by 6 hours postinfection. Actinomycin D-resistant viral RNA synthesis started at about 2 hours postinfection, peaked by 5 hours, and then declined rapidly. Virus-specific protein synthesis began while host protein synthesis was declining, increased during the ensuing period, and declined in late infection. A number of virus-specific proteins with molecular weights from 23,500 to greater than 92,500 were detected in the host cytoplasm. At least three of these proteins were also present in the nucleus. The kinetics pf processing of virus-specific proteins were examined by pulse-chase experiments in B5-infected cells. The relative intensities of [35S]-methionine-labeled polypeptides suggest that a number of smaller, stable chains (MW 23,500 to 38,000) are generated by cleavage of a precursor polypeptide (MW 92,500 to 100,000).

Antigenicity and polypeptide composition of native and heated echovirus type 7 procapsids.

The native procapsid (naturally occurring empty capsid) of echovirus type 7 (E7) possesses N-antigenicity, which is as highly strain-specific as the native virion. When the native procapsid is heated, its antigenicity is converted to H-antigenicity which is common among strains of E7 and thus type-specific. However, no difference was detected in the sedimentation rate (80S) and polypeptide composition (VP0, 1 and 3) of the native and heated procapsids.

Surface charge of dengue virus hemagglutinins prepared from infected mouse brains.

Surface charge of dengue virus hemagglutinin (HA) prepared from infected suckling mouse brains was studied by sucrose gradient electrophoresis. The findings were as follows: (1) Both the slow- and rapid-sedimenting hemagglutinins (SHA and RHA, respectively) prepared after streptomycin treatment of the brain homogenate were distributed broadly within the gradient after electrophoresis, while echovirus type 7 HA which was used as a reference migrated narrowly, suggesting heterogeneity of the surface charge of both dengue HAs. (2) Neuraminidase treatment of dengue HA slightly retarded its migration toward the anode but did not alter the broad distribution. (3) Protamine treatment used for purification of HA from the brain homogenate changed the surface charge from negative to positive.

A high density component in several vertebrate enteroviruses.

In addition to the major infective component, which bands at a density of 1:34 g/ml in caesium chloride ("light component"), a component with a density of 1:44 g/ml ("heavy component") has been found in harvests of poliovirus (type I), Coxsackie B5 virus, a bovine enterovirus (VG-5-27) and swine vesicular disease virus (SVDV). With SVDV about 98% of the infectivity equilibrated at 1 . 34 g/ml but approx. 2% was present as a peak at 1 . 44 g/ml. The morphology of the two forms was similar but the heavy component had a smaller diameter (28 nm) than the light component (30 nm). No inter-conversion of the two forms was observed on re-cycling in fresh caesium chloride gradients and the two components had the same proportions of RNA and protein and the same polypeptide composition. Each component gave a similar proportion of the light and heavy forms on replication, but the light component had a specific infectivity about fourfold higher than that of the heavy component and was also much more efficient in eliciting the formation of neutralizing antibodies in guinea pigs. Although these results suggest that the two particles are alternative stable configurations of the virus, iodination failed to reveal any differences in the extent or pattern of labelling of the polypeptides in the two forms.

Electrophoretic analysis of capsid and non-capsid polypeptides of echovirus 12, and selective inhibtion of the formation of virus particles by actinomycin D.

Electrophoretic analysis of purified echovirus virus particles yielded four polypeptides of mol. wt. 37000, 30000, 25000 and 7600. The 75S empty capsids of echovirus 12 lack the 25000 and 7600 mol. wt. polypeptides, and possess polypeptides of 41000 mol. wt. A total of 14 virus-induced polypeptide species was found in the cytoplasm of infected cells. Actinomycin D reduced the synthesis of virus, virus RNA, and virus polypeptides and also reduced the proportion of virus particles to empty capsids in the virus yields.