Platelets interact with Coxsackieviruses B and have a critical role in the pathogenesis of virus-induced myocarditis.

BACKGROUND: To further understand the role of platelets in the pathogenesis of viral infections we explored platelet interaction with Coxsackieviruses B (CVB) 1 and 3. CVB is a group of viruses that cause the majority of human enterovirus-related viral myocarditis; their receptor (CAR) is expressed on the platelet surface and there is a well-characterized CVB3-induced myocarditis murine model. METHODS: Human platelets were infected with CVB1 and 3 and viruses were detected in pellets and in supernatants. C57BL/6J mice with or without platelet depletion were inoculated with CVB3 and peripheral blood and heart samples collected at different times post-infection. RESULTS: CVB1 and 3 RNA and a capsid protein were detected in infected platelets. Despite the fact that titration assays in Vero cells showed increasing infectivity titers over time, supernatants and pellets from infected platelets showed similar levels, suggesting that platelets were not susceptible to a replicative infectivity cycle. CVB binding was CAR-independent and resulted in P-selectin and phosphatidylserine (PS) exposure. CVB3-infected mice showed a rapid thrombocytopenia that correlated with an increase in platelet PS exposure and platelet-leukocyte aggregates without modification of platelet P-selectin expression or von Willebrand factor levels. Mortality, viremia, heart viral titers and myocarditis were significantly higher in platelet-depleted than normal animals. Type I IFN levels were not changed but IgG levels were lower in infected and platelet-depleted mice. CONCLUSIONS: Our data reveal that platelets play a critical role in host survival and immune response against CVB3 infection.

Expression of innate immunity genes and damage of primary human pancreatic islets by epidemic strains of Echovirus: implication for post-virus islet autoimmunity.

Three large-scale Echovirus (E) epidemics (E4,E16,E30), each differently associated to the acute development of diabetes related autoantibodies, have been documented in Cuba. The prevalence of islet cell autoantibodies was moderate during the E4 epidemic but high in the E16 and E30 epidemic. The aim of this study was to evaluate the effect of epidemic strains of echovirus on beta-cell lysis, beta-cell function and innate immunity gene expression in primary human pancreatic islets. Human islets from non-diabetic donors (n = 7) were infected with the virus strains E4, E16 and E30, all isolated from patients with aseptic meningitis who seroconverted to islet cell antibody positivity. Viral replication, degree of cytolysis, insulin release in response to high glucose as well as mRNA expression of innate immunity genes (IFN-b, RANTES, RIG-I, MDA5, TLR3 and OAS) were measured. The strains of E16 and E30 did replicate well in all islets examined, resulting in marked cytotoxic effects. E4 did not cause any effects on cell lysis, however it was able to replicate in 2 out of 7 islet donors. Beta-cell function was hampered in all infected islets (P<0.05); however the effect of E16 and E30 on insulin secretion appeared to be higher than the strain of E4. TLR3 and IFN-beta mRNA expression increased significantly following infection with E16 and E30 (P<0.033 and P<0.039 respectively). In contrast, the expression of none of the innate immunity genes studied was altered in E4-infected islets. These findings suggest that the extent of the epidemic-associated islet autoimmunity may depend on the ability of the viral strains to damage islet cells and induce pro-inflammatory innate immune responses within the infected islets.

Echovirus 4 associated to hand, foot and mouth disease / Echovirus 4 associado à doença de mão, pé e boca

Hand, foot and mouth disease (HFMD) is a contagious enteroviral infection occurring primarily in children and characterized by vesicular palmoplantar eruptions and erosive stomatitis. Echovirus 4 (EV-4) has been commonly associated with aseptic meningitis. The association of HFMD with EV-4 has not been reported previously. Two samples of a 14-month child who presented mild fever, sores in the mouth, rash with blisters on the palm of hands and soles of feet were sent to Enteric Viruses Laboratory of Adolfo Lutz Institute. Clinical samples were inoculated in three different cell lines, and those which presented cytopathic effect (CPE), were submitted to Indirect Immunofluorescence Assay (IFA) and "one step" RT-PCR. Agarose gel electrophoresis from RT-PCR product, showed a product with 437 bp, which is characteristic of Enterovirus group. Echovirus 4 was identified by IFA. Although HFMD is a viral infection associated mainly with Enterovirus 71 (HEV-71) and Coxsackievirus A16 (CV-A16), our results demonstrate a diversity of serotype related to HFMD and stress the importance of epidemiological surveillance to this disease and its complications.

A Doença de Mão, Pé e Boca (DMPB) é uma infecção enteroviral contagiosa que ocorre principalmente em crianças sendo caracterizada por erupções palmoplantares vesiculares e estomatite. Echovirus 4 (EV-4) é comumente associado a meningite asséptica. A associação de DMPB por EV-4 não foi descrita anteriormente. Duas amostras provenientes de uma criança de 14 meses apresentando febre, secreções na garganta e exantemas nas palmas das mãos e dos pés, foram enviadas para o Laboratório de Vírus Entéricos do Instituto Adolfo Lutz. As amostras foram inoculadas em três diferentes linhagens celulares; aquelas que apresentaram efeito citopático (ECP), foram submetidas a ensaio de imunofluorescência indireta (IFI) e "one step" RT-PCR. A eletroforese em gel de agarose realizada com o produto de PCR apresentou um produto de 437pb, característico de grupo Enterovirus. O sorotipo EV-4 foi identificado por IFI. Apesar da DMPB ser uma infecção viral associada principalmente com Enterovirus 71 (HEV-71) e Coxsackievirus A16 (CV-A16), nossos resultados enfatizam a necessidade de estudos epidemiológicos e laboratoriais direcionados ao EV-4 como agente causador de DMPB.

Relationship between HLA antigens and infectious agents in contributing towards the development of Graves' disease.

Graves' disease (GD) is an autoimmune thyroid disorder which is associated with the human leucocyte antigens HLA-DR3 and DQA1* O501 in Caucasians. We have explored the possibility that some patients with certain HLA specificities develop anti-HLA antibodies which are correlated with environmental factors that may contribute to the development of GD. We studied 40 GD patients and 157 healthy individuals (controls). Serology was used to type HLA-A, -B, -Cw, and -DR antigens. The frequencies of these antigens in relation to lymphocytotoxic anti-HLA-A-B-Cw-DR antibodies and two environmental factors (Yersinia enterocolitica and Coxsackie B virus) were determined. The frequencies of HLA-B15, -B21 and DR3 antigens were increased, whereas HLA-DR5 antigen was decreased in GD patients. A significant association between HLA-DR3 antigen and lymphocytotoxic antibodies was observed, i.e., IgGs from GD patients were cytotoxic to HLA-DR3+ normal B cells. Following absorption with Yersinia enterocolitica or Coxsackie-B-virus, only Coxsackie-B virus completely inhibited the lymphocytotoxic reactions against HLA-DR3+ B cells. Besides confirming the association of HLA-DR3 with GD, this study also suggests the role of Coxsackie-reactive HLA-DR3 antibodies as contributing factors to the pathogenesis of the disease.

[Outbreak of Coxsackievirus B5 meningoencephalitis in Cuba, 1995]. / Brote de meningoencefalitis por coxsackievirus B5 en Cuba, 1995.

An increase in the reported cases of viral meningoencephalitis (VME) was detected in October and November, 1995, compared with the same period of 1994. 43 stock specimens from children with this diagnosis were received at the Laboratory of Enterovirus from the "Pedro Kourí" Institute of Tropical Medicine. 23 isolations (53.4%) were obtained and identified as Coxsackievirus B5. Besides, in 43 matched sera investigated by the neutralization test against some Enteroviruses, 21 proved to be positive (48.8%) to the isolated agent. This allowed us to affirm, supported by the clinical picture and by epidemiology, that we are in the presence of an VME outbreak produced by Coxsackievirus B5.

An exanthematic disease epidemic associated with coxsackievirus B3 infection in a day care center.

An epidemic of exanthematic illness in a day care center is described. Ten children aged 7 to 13 months were affected by the illness. The exanthem was characterized by nonconfluent macular or maculopapular lesions that appeared on the face, body and limbs. Fifty percent of the infected children had fever of up to 39 degrees C at the beginning of the disease. Coxsackievirus B3 (CB3) was isolated from the stool of one ill child. Paired serum samples were obtained from eight ill children and six of them presented seroconversion to CB3. Antibodies to CB3 were detected at titers higher than 16 in a single serum sample collected from the other two patients. Neutralizing antibodies to CB3 were detected in 71.0% of the contact children.

[Cuban epidemic neuropathy. III. Neutralizing antibodies to the strains isolated and to other enteroviruses in patients and healthy subjects]. / Neuropatía epidémica cubana. Parte III. Anticuerpos neutralizantes a cepas aisladas y otros Enterovirus en pacientes y personas sanas.

Determinations of neutralizing antibodies to the strain 47/93 IPK (CA9) and to the strain 590 were performed in serum samples from patients presenting with epidemic neuropathy and from a group of seemingly healthy subjects. Determinations were also done in the reference strains CA9 and CB1-6 by the microneutralization technique. Patients and their contacts showed significantly higher percentages of neutralizing antibodies to the strain 47/93 than the control group and residents of municipalities with a low rate of the disease. This difference was also confirmed regarding the geometric mean titres with the use of the reference strains CA9 and CB2-4. An increased circulation of the strain 47/93 within the infantile population from 1981 to 1993 was evidenced. Patients exhibited significantly lower percentages and geometric mean titres of neutralizing antibodies to the strain 590 than the control group, despite the fact that in 25/28 certain agents having a mild cytopathogenic effect had been isolated. The possibility of two mechanism of neutralization is stated and an hypothesis on the mechanism by which these viruses may be involved in the pathophysiology of the disease is formulated.

[Cuban epidemic neuropathy. II. The antigenic characteristics of the viral isolates]. / Neuropatía epidémica cubana. Parte II. Algunas características antigénicas de los aislamientos virales.

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.

[Cuban epidemic neuropathy. IV. The antigenic relationship of the viral isolates to the structures of the central nervous system. A possible etiopathogenic mechanism?]. / Neuropatía epidémica cubana. Parte IV. Relación antigénica de los aislamientos virales con estructuras del sistema nervioso central. Posible mecanismo etiopatogénico?

Results which allow to state the existence of antigenic relationships between viruses isolated from the cerebrospinal fluid of patients presenting with epidemic neuropathy and structures of the human central nervous system are reported. These evidences were obtained by 2 different and independent ways: 1) by the double diffusion method in agarose, immunoblot analysis, and immunohistochemical analysis it was confirmed that antibodies induced by isolated viruses react with antigens of the central and peripheral nervous system, 2) serum obtained by the immunization of a rabbit with human brain extract neutralizes the same viruses as those neutralized by hyperimmune sera obtained by isolations. The possible role of viruses as mediators of an autoimmune process in the pathogenesis of the disease is discussed.

Normalización de un ELISA de inhibición para el diagnóstico serológico de las infecciones por virus Coxsackie del grupo B / Standardization of an inhibition ELISA for serological diagnosis of Coxsackie B virus infections

El presente trabajo normalizamos una técnica de ElISA de inhibición para detectar anticuerpos dirigidos contra los virus Coxsackie del grupo B. El método resultó ser tipo específico, ya que fue capaz de detectar anticuerpos a 4 serotipos del B2 y B4 porque no contábamos con las cepas). La comparación con la técnica de neutralización arrojó una coincidencia del 85

, una sensibilidad del 91

y una especificidad del 82

. Todos los reactivos utilizados enel ensayo se produjo en nuestro laboratorio.

Receptor presente en eritrocitos humanos para la variante RD del virus coxsackie B3 / Present receptor in human erythrocytes for the coxsackie virus B3 variant RD

Se efectuó un estudio comparativo de los receptores para la variante RD del virus coxsacki B3 presentes en la membrana plamática de eritrocitos humanos y de células de rhabdomiosarcoma (RD). Para ello se usó un anticuerpo monoclonal, obtenido a partir del receptor presente en la célula RD, el cual fue marcado con 125l. El ligando marcado se unió a los receptores de los eritrocitos humanos e impidió que el virus se uniera a ellos. El anticuerpo monoclonal saturó los receptores, lo que permitió calcular el número de sitios de unión, que fue de 1.95x10 3 por eritrocito, número aproximadamente 10 veces menor que los calculados para las células RD. Los receptores de ambas células fueron bloqueados en su unión al ligando, cuando dos proteasas usadas, la quimotripsina y la pronasa, ejercieron su acción proteolítica, modificando la estructura de los receptores. Los presentes en la membrana plasmática de las células RD fueron más sensibles a la acción proteolítica que los presentes en los eritrocitos. Otra proteasa usada, la tripsina, bloqueó los receptores sobre los eritrocitos en un porcentaje similar a como lo hicieron las otras dos enzimas. Por el contrario, los receptores presentes en las células RD fueron casi insensibles al tratamiento con la tripsina.Las glicoforinas A, tipos MN y MM, fueron capaces de competir por ambos receptores, no así la tipo NN. Se concluye de esto que los receptores presentes en la membrana plasmática de los eritrocitos humanos y de las células RD, son de naturaleza proteica y comparten epítopes. El receptor presente en los eritrocitos humanos para el virus coxsackie B3, variante RD, que es bloqueado por el anticuerpo monoclonal, podría ser la glicoforina A, tipo MN y/o MM

Receptor presente en eritrocitos humanos para la variante RD del virus coxsackie B3 / Present receptor in human erythrocytes for the coxsackie virus B3 variant RD

Se efectuó un estudio comparativo de los receptores para la variante RD del virus coxsacki B3 presentes en la membrana plamática de eritrocitos humanos y de células de rhabdomiosarcoma (RD). Para ello se usó un anticuerpo monoclonal, obtenido a partir del receptor presente en la célula RD, el cual fue marcado con 125l. El ligando marcado se unió a los receptores de los eritrocitos humanos e impidió que el virus se uniera a ellos. El anticuerpo monoclonal saturó los receptores, lo que permitió calcular el número de sitios de unión, que fue de 1.95x10 3 por eritrocito, número aproximadamente 10 veces menor que los calculados para las células RD. Los receptores de ambas células fueron bloqueados en su unión al ligando, cuando dos proteasas usadas, la quimotripsina y la pronasa, ejercieron su acción proteolítica, modificando la estructura de los receptores. Los presentes en la membrana plasmática de las células RD fueron más sensibles a la acción proteolítica que los presentes en los eritrocitos. Otra proteasa usada, la tripsina, bloqueó los receptores sobre los eritrocitos en un porcentaje similar a como lo hicieron las otras dos enzimas. Por el contrario, los receptores presentes en las células RD fueron casi insensibles al tratamiento con la tripsina.Las glicoforinas A, tipos MN y MM, fueron capaces de competir por ambos receptores, no así la tipo NN. Se concluye de esto que los receptores presentes en la membrana plasmática de los eritrocitos humanos y de las células RD, son de naturaleza proteica y comparten epítopes. El receptor presente en los eritrocitos humanos para el virus coxsackie B3, variante RD, que es bloqueado por el anticuerpo monoclonal, podría ser la glicoforina A, tipo MN y/o MM

[Coxsackie B virus in children with acute myocarditis]. / Virus Coxsackie B em crianças com miocardite aguda.

PURPOSE: To study etiopathogenic and follow-up aspects of patients with myocarditis. PATIENTS AND METHODS: Among 44 cases of acute myocarditis in children studied from clinical and virological point of view, it was selected 16 which were positive to coxsackie B virus. The clinical investigation included blood test to enzyme dosage, chest X-ray, electro and echocardiogram. The virological work up to coxsackie B1, B3, B4, B5 and B6 included culture neutralization test and IgM (indirect immunofluorescence). RESULTS: The positive results were: B4 in 9 (57%); B5 in 4 (25%), B1 in 2 (12%) and B3 in 1 (6%). It was not used immunosuppressive treatment. The follow-up (at least one year it was: 7 patients became free of symptoms (43%); 4 (25%) turned to be chronic dilated myocardiopathy 1 (6%) died and 4 (25%) were discharged from hospital but did not return to out-patient clinic. One of patients of chronic group during 4 years of follow-up had ventricular extrasystoles, second degree heart block and now is asymptomatic. CONCLUSION: We did not observe significative differences among the several types of coxsackie B related to clinical course.

Virus Coxsackie B em crianças com miocardite aguda / Coxsackie B virus in children with acute myocarditis

Estudar aspectos etiopatológicos e de evoluçäo em portadores de miocardite. Dentre 44 crianças com miocardite aguda estudadas clínica e virologicamente, foram selecionados 16 casos positivos para Coxasackie B. O protocolo clínico incluiu dosagens enzímicas, radiografia de tórax, eletro e ecocardiograma. A investigaçäo virológica para Coxsacke B1, B3, B4, B5 e B6 se baseou em cultura, teste de neutralizaçäo e pesquisa de IgM por imunofluorescência indireta. Obtivemos vírus B4 em 9 (57%), B5 em 4 (25%), B1 em 2 (12%) e B3 em 1 (6%). Nenhum paciente foi submetido à terapêutica imunossupressora. A evoluçäo de pelo menos 1 ano foi; 7 (43%) permaneceram com miocardiopatia dilatada, 1 (6%) faleceu e 4 (25%) tiveram alta hospitalar, mas näo seguiram o acompanhamento. Uma das pacientes que teve evoluçäo crônica (extra-sístoles ventriculares, BAV do 2§ grau) está agora assintomática. Näo observamos diferença significativa entre os vários tipos de Coxsackie B em relaçäo à evoluçäo clínica

Enterovirus isolation from foetal and placental tissues.

Four spontaneous abortions and two stillbirth occurred during a prospective survey following the teratogenicity of echoviruses in 80 pregnant women selected at random from the Antenatal Care Service. Echovirus types 19, 27, and 33. Coxsackie B2 and B6 were isolated from placental and foetal tissues (brain, liver, kidney, heart, and spleen). The mothers also excreted the virus by faeces at least twenty days before abortion and responded serologically, indicating active virus infection. Almost all aborted children were anomalous with signs of viral infection.

An outbreak of acute flaccid paralysis in Jamaica associated with echovirus type 22.

An outbreak of acute flaccid paralysis in Jamaica in 1986 associated with echovirus type 22 is described. Six patients aged 1 to 27 years developed acute onset of severe flaccid paralysis, with inability to walk. Three cases had facial weakness, four required intensive care with assisted ventilation, and two died. Echovirus type 22 was isolated from the stool of two patients who showed a significant increase in antibody titre. Echovirus type 22 was also isolated from the stool of another patient who had aseptic meningitis without any neurological deficit. There was no evidence of poliovirus infection in any of these patients, most of whom were fully immunized. Of the four surviving cases with flaccid paralysis, three had residual weakness in their lower limbs and walked with an abnormal gait 3 years after the acute paralytic attack. This is the first report in the literature of acute flaccid paralysis associated with type 22 echovirus.

An outbreak of acute flaccid paralysis in Jamaica associated with echovirus

An outbreak of acute flaccid paralysis in Jamaica in 1986 associated with echovirus type 22 is described. Six patients aged 1 to 27 years developed acute onset of severe flaccid paralysis, with inability to walk. Three cases had facial weakness, four required intensive care with assisted ventilation and two died. Echovirus type 22 was isolated from the stool of two patients who showed a significant increase in antibody titre. Echovirus type 22 was also isolated from the stool of another patient who had aseptic meningitis without any neurological deficit. There was no evidence of poliovirus infection in any of these patients, most of whom were fully immunized. Of the four surviving cases with flaccid paralysis, three had residual weakness in their lower limbs and walked with an abnormal gait 3 years after the acute paralytic attack. This is the first report in the literature of acute flaccid paralysis associated with type 22 echovirus. (AU)