Epitope of dengue virus E protein detect human antibodies associated with mild disease: a potential peptide for vaccine development

ABSTRACT The antigenic potential of seven immunogenic peptides of the dengue virus was evaluated in the sera of patients with dengue confirmed by IgM/IgG serology. Antibodies IgM and IgG against dengue virus peptides were analyzed by ELISA in 31 dengue sero-positive and 20 sero-negative patients. The P5 peptide showed significant IgG immunoreactivity mostly in the sera of patients with dengue without warning signs in comparison with patients with dengue with warning signs, correlating with mild disease. This finding suggests that the low antibody response against P5 epitope could be a risk factor for higher susceptibility to dengue virus infection with warning signs, and that P5 could be a potential antigen for vaccine development.

Dengue diagnostics: serious inaccuracies are likely to occur if pre-analytical conditions are not strictly followed

BACKGROUND The heat-labile nature of Dengue virus (DENV) in serum samples must be considered when applying routine diagnostic tests to avoid issues that could impact the accuracy of test results with direct implications for case management and disease reporting. OBJECTIVES To check if pre-analytical variables, such as storage time and temperature, have an impact on the accuracy of the main routine diagnostic tests for dengue. METHODS Virus isolation, reverse transcription real-time polymerase chain reaction (RT-PCR) and NS1 enzyme-linked immunosorbent assay (ELISA) were evaluated using 84 samples submitted to different pre-analytical conditions. FINDINGS Sensitivity and negative predictive value were directly affected by sample storage conditions. RT-PCR and virus isolation showed greater dependence on well-conserved samples for an accurate diagnosis. Interestingly, even storage at -30ºC for a relatively short time (15 days) was not adequate for accurate results using virus isolation and RT-PCR tests. On the other hand, NS1 ELISA showed no significant reduction in positivity for aliquots tested under the same conditions as in the previous tests. MAIN CONCLUSIONS Our results support the stability of the NS1 marker in ELISA diagnosis and indicate that the accuracy of routine tests such as virus isolation and RT-PCR is significantly affected by inadequate transport and storage conditions of serum samples.

Polyspecific antibodies without persisting antigen in multiple sclerosis, neurolupus and Guillain-Barré syndrome: immune network connectivity in chronic diseases / Anticorpos poliespecíficos sem antígeno persistente em esclerose múltipla, neurolupus e síndrome de Guillain-Barré: conectividade imunológica em doenças crônicas

ABSTRACT The polyspecific antibody synthesis in multiple sclerosis (MS) gained diagnostic relevance with the frequent combination of measles-, rubella- and varicella zoster antibodies (MRZ-antibody reaction) but their pathophysiological role remains unknown. This review connects the data for intrathecal polyspecific antibody synthesis in MS and neurolupus with observations in the blood of patients with Guillain-Barré syndrome (GBS). Simultaneously increased antibody and autoantibody titers in GBS blood samples indicate that the polyspecific antibodies are based on a general property of an immune network, supported by the deterministic day-to-day concentration variation of antibodies in normal blood. Strongly correlated measles- and rubella- antibody variations point to a particular connectivity between the MRZ antibodies. The immune network, which provides serological memory in the absence of an antigen, implements the continuous change of the MRZ pattern in blood, not followed by the earlier immigrated B cells without corresponding connectivity in the brain. This may explain the different antibody patterns in cerebrospinal fluid, aqueous humor and blood of the individual MS patient. A complexity approach must implement a different view on causation in chronic diseases and causal therapies.

RESUMO A síntese de anticorpos poliespecíficos em esclerose múltipla (EM) ganhou relevância diagnóstica com a combinação frequente de anticorpos contra sarampo, rubéola e varicela-zoster (reação de anticorpos MRZ), mas seu papel fisiopatológico permanece desconhecido. Esta revisão relaciona os dados da síntese intratecal de anticorpos poliespecíficos em EM e Neurolupus com observações no sangue de pacientes com síndrome de Guillain Barré (SGB). Simultaneamente, os títulos aumentados de anticorpos e autoanticorpos em amostras de sangue de SGB indicam que os anticorpos poliespecíficos se baseiam numa propriedade geral de uma rede imunitária, suportada pela variação determinística da concentração diária de anticorpos no sangue normal. As variações fortemente correlacionadas de anticorpos contra sarampo e rubéola apontam para uma conectividade particular entre os anticorpos MRZ. A rede imunitária, que fornece memória sorológica na ausência de um antígeno, implementa a mudança contínua do padrão MRZ no sangue, não seguida pelas células B que imigraram anteriormente sem conectividade no cérebro. Isto pode explicar os diferentes padrões de anticorpos no LCR, humor aquoso e sangue do paciente individual de EM. Uma abordagem complexa deve implementar uma visão diferente sobre a causalidade em doenças crônicas e terapias causais.

Decrease in Dengue virus-2 infection and reduction of cytokine/chemokine production by Uncaria guianensis in human hepatocyte cell line Huh-7

ABSTRACT BACKGROUND Dengue fever may present hemorrhages and cavitary effusions as result of exacerbated immune responses. We investigated hydro-alcoholic extracts from leaves (UGL) and bark (UGB) of the medicinal species Uncaria guinanensis with respect to antiviral effects in Dengue virus (DENV) infection and in immunological parameters associated with in vivo physiopathological features. METHODS Chemical profiles from UGB or UGL were compared in thin layer chromatography and 1H nuclear magnetic resonance using flavonoid compounds and a pentacyclic oxindole alkaloid-enriched fraction as references. DENV-2-infected hepatocytes (Huh-7) were treated with extracts. Cell viability, DENV antigens and immunological factors were detected by enzyme-linked immunosorbent assay (ELISA) or flow cytometry. FINDINGS The UGL mainly differed from UGB by selectively containing the flavonoid kaempferitrin. UGB and UGL improved hepatocyte viability. Both extracts reduced intracellular viral antigen and inhibited the secretion of viral non-structural protein (NS1), which is indicative of viral replication. Reduction in secretion of macrophage migration inhibitory factor was achieved by UGB, of interleukin-6 by UGL, and of interleukin-8 by both UGB and UGL. MAIN CONCLUSIONS The U. guianensis extracts presented, antiviral and immunomodulatory effects for DENV and possibly a hepatocyte-protective activity. Further studies may be performed to consider these products as potential candidates for the development of an herbal product for the future treatment of dengue.

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity.

Antigenemia y reacción de polimerasa en cadena en tiempo real en el diagnóstico de enfermedad por citomegalovirus en adultos con virus de inmunodeficiencia adquirida / Antigenemia and real time polymerase chain reaction for diagnosis of cytomegal ovirus disease in HIV infected adults

Background: Cytomegalovirus (CMV) infection is frequent in HIV adults. It is unknown usefulness of quantitative methods for diagnosing the CMV disease in Chilean patients. Aim: To determine the performance of antigenemia and real time polymerase chain reaction (rtPCR) in the diagnosis of CMV disease in Chilean HIV adults. Method: Detection of CMV by viral isolation (AVR), antigenemia and quantitative rtPCR in HIV adults. Results: The 102 adults with suspected CMV disease had lower LTCD4 count and higher HIV viral load than 77 patients without suspicion (p < 0.05). Antigenemia and PCR were positive in 47 (46.1%) and 37 (36.3%) adults with clinical suspicion and in 2 (2.6%) and 4 (5.2%) of 77 without suspicion. The sensitivity, specificity, positive and negative predictive value of antigenemia and RPCtr were 92%, 80%, 72% and 95% and 72%, 95%, 92% and 80%, respectively. The cutoff values were ≥ lcell (+) and ≥ 5.5 log10 copies/2 x 10(6) cells. CMV was isolated in 6/179 patients (3.4%), all symptomatic. Conclusión: Positivity of antigenemia and rtPCR are similar for diagnosing CMV disease in Chilean HIV adults. AVR is inappropriate as a gold standard for its low performance.

Introducción: La infección por citomegalovirus (CMV) es frecuente en adultos con virus de inmunodeficiencia humana (VIH). No se ha establecido la utilidad de los métodos cuantitativos para diagnosticar enfermedad por CMV en pacientes chilenos. Objetivo: Determinar la positividad de antigenemia y reacción de polimerasa en cadena en tiempo real (RPC-TR) en el diagnóstico de enfermedad por CMV en adultos chilenos con infección por VIH. Metodología: Se detectó CMV mediante aislamiento viral rápido (AVR), antigenemia y reacción de polimerasa en cadena en tiempo real (RPC-TR) cuantitativa en adultos infectados por VIH, con y sin sospecha de enfermedad por CMV. Resultados: El recuento de LT CD4 fue menor y mayor la carga de VIH en 102 sintomáticos respecto a 77 asintomáticos (p < 0,05). La antigenemia y la RPC-TR fueron positivas en 46 y 36% de los enfermos y en 3 y 5% de los asintomáticos respectivamente. La sensibilidad, especificidad, valor predictor positivo y negativo de la antigenemia y la RPC-TR fueron 92%, 80%, 72% y 95% y 72%, 95%, 92% y 80%, respectivamente. Los valores de corte fueron ≥ 1 núcleo (+) y ≥ 5,5 log10 copias/2 x 10(6) céls. Se aisló CMV en 3,4%, todos los sintomáticos. Conclusión: La antigenemia y la RPC-TR tienen una positividad similar para diagnosticar enfermedad por CMV en adultos chilenos con infección por VIH. El AVR es inapropiado como referencia por su baja positividad.

Antigen targeting to M cells for enhancing the efficacy of mucosal vaccines

Vaccination is one of the most successful applications of immunology and for a long time has depended on parenteral administration protocols. However, recent studies have pointed to the promise of mucosal vaccination because of its ease, economy and efficiency in inducing an immune response not only systemically, but also in the mucosal compartment where many pathogenic infections are initiated. However, successful mucosal vaccination requires the help of an adjuvant for the efficient delivery of vaccine material into the mucosa and the breaking of the tolerogenic environment, especially in oral mucosal immunization. Given that M cells are the main gateway to take up luminal antigens and initiate antigen-specific immune responses, understanding the role and characteristics of M cells is crucial for the development of successful mucosal vaccines. Especially, particular interest has been focused on the regulation of the tolerogenic mucosal microenvironment and the introduction of the luminal antigen into the lymphoid organ by exploiting the molecules of M cells. Here, we review the characteristics of M cells and the immune regulatory factors in mucosa that can be exploited for mucosal vaccine delivery and mucosal immune regulation.

Prevalence of antibody to hantaviruses in humans and rodents in the Caribbean region of Colombia determined using Araraquara and Maciel virus antigens

We tested sera from 286 agricultural workers and 322 rodents in the department of Córdoba, northeastern Colombia, for antibodies against two hantaviruses. The sera were analysed by indirect ELISA using the lysate of Vero E6 cells infected with Maciel virus (MACV) or the N protein of Araraquara virus (ARAV) as antigens for the detection of antibodies against hantaviruses. Twenty-four human sera were IgG positive using one or both antigens. We detected anti-MACV IgG antibodies in 10 sera (3.5%) and anti-ARAV antibodies in 21 sera (7.34%). Of the 10 samples that were positive for MACV, seven (70%) were cross-reactive with ARAV; seven of the 21 ARAV-positive samples were cross-reactive with MACV. Using an ARAV IgM ELISA, two of the 24 human sera (8.4%) were positive. We captured 322 rodents, including 210 Cricetidae (181 Zygodontomys brevicauda, 28 Oligoryzomys fulvescens and 1 Oecomys trinitatis), six Heteromys anomalus (Heteromyidae), one Proechimys sp. (Echimyidae) and 105 Muridae (34 Rattus rattus and 71 Mus musculus). All rodent sera were negative for both antigens. The 8.4% detection rate of hantavirus antibodies in humans is much higher than previously found in serosurveys in North America, suggesting that rural agricultural workers in northeastern Colombia are frequently exposed to hantaviruses. Our results also indicate that tests conducted with South American hantavirus antigens could have predictive value and could represent a useful alternative for the diagnosis of hantavirus infection in Colombia.

Cross-Protective Immune Responses Elicited by Live Attenuated Influenza Vaccines

The desired effect of vaccination is to elicit protective immune responses against infection with pathogenic agents. An inactivated influenza vaccine is able to induce the neutralizing antibodies directed primarily against two surface antigens, hemagglutinin and neuraminidase. These two antigens undergo frequent antigenic drift and hence necessitate the annual update of a new vaccine strain. Besides the antigenic drift, the unpredictable emergence of the pandemic influenza strain, as seen in the 2009 pandemic H1N1, underscores the development of a new influenza vaccine that elicits broadly protective immunity against the diverse influenza strains. Cold-adapted live attenuated influenza vaccines (CAIVs) are advocated as a more appropriate strategy for cross-protection than inactivated vaccines and extensive studies have been conducted to address the issues in animal models. Here, we briefly describe experimental and clinical evidence for cross-protection by the CAIVs against antigenically distant strains and discuss possible explanations for cross-protective immune responses afforded by CAIVs. Potential barriers to the achievement of a universal influenza vaccine are also discussed, which will provide useful guidelines for future research on designing an ideal influenza vaccine with broad protection without causing pathogenic effects such as autoimmunity or attrition of protective immunity against homologous infection.

Obtención y evaluación preliminar de un virus canarypox recombinante como candidato a vacuna antirrábica / Development and preliminary assessment of a recombinant canarypox virus as an antirabic vaccine candidate

En la Argentina, la rabia está circunscripta a algunas provincias del norte. La disponibilidad de nuevas vacunas que eliminen la manipulación del virus rábico y que permitan el control de la enfermedad es de importancia estratégica nacional y regional. Las vacunas basadas en poxvirus recombinantes se han utilizado con éxito como vacunas antirrábicas a nivel mundial. SI bien estos sistemas no están disponibles comercialmente, la plataforma de obtención de virus canarypox (CNPV) recombinantes ya ha sido implementada en nuestro laboratorio. El objetivo de este trabajo fue obtener y evaluar un candidato a vacuna antirrábica basado en CNPV recombinantes que expresan la glicoproteína G (RG) del virus rábico (RV). Se construyó un virus recombinante que expresa la secuencia codificante de RG (CNPV-RG). La inoculación de ratones con este virus indujo altos títulos de anticuerpos seroneutralizantes de RV (3,58 y 9,76 Ul/ml después de una o dos inmunizaciones, respectivamente) y protegió al 78 % de los animales desafiados intracerebralmente con RV. Además, se determinó que el CNPV-RG posee una potencia relativa de 3,5 Ul/ml. Los resultados obtenidos constituyen la primera etapa en la evaluación del CNPV-RG como candidato a vacuna antirrábica. Se requerirán nuevos ensayos para confirmar su utilidad en especies de interés veterinario.

In Argentina, rabies is limited to some northern provinces. Availability of new vaccines abolishing the handling of the rabies virus and allowing disease control has regional and national strategic importance. Vaccines based on recombinant poxviruses have been successfully used as antirabic vaccines worldwide. Although these systems are not commercially available, the platform to obtain recombinant canarypox viruses (CNPV) has been previously set up in our laboratory. The aim of this work was the development and evaluation of an antirabic vaccine candidate based on recombinant CNPV expressing the rabies virus (RV) glycoprotein G (RG). A recombinant virus (CNPV-RG) expressing the RG coding sequence was designed. Inoculation of mice with this virus induced high RV seroneutralizing antibodies (3.58 and 9.76 lU/ml after 1 or 2 immunizations, respectively) and protected 78% of intracerebrally RV-challenged animals. In addition, it was determined that CNPV-RG has a relative potency of 3.5 lU/ml. The obtained results constituted the first stage of CNPV-RG evaluation as antirabic vaccine candidate. Further assays will be necessary to confirm its utility in species of veterinary Interest.

Viral markers in patients with hemophagocytosis: A prospective study in a tertiary care hospital.

Background : Hemophagocytic syndrome (HPS) is a rare clinicopathological condition characterized by the activation of macrophages with prominent hemophagocytosis in bone marrow and other reticulo-endothelial systems. HPS can be familial or secondary to infections including viruses. Aim : To study the viral markers in patients with HPS. Materials and Methods : Serum samples of patients with HPS and control group were screened for anti EBV VCA IgM, and IgG, anti-Parvo B19 IgM, and anti-CMV IgM antibodies using commercially available ELISA kits and CMV and ParvoB19 DNA by polymerase chain reaction (PCR). Results and Discussion : The present prospective study reports the profile of viral markers in HPS cases from north India. Among the 14 HPS cases 43% (6/14) were positive for at least one viral marker tested, of which EBV was found to be the most prevalent (3/6: 50%) followed by parvovirus B19(2/6: 33%) and cytomegalovirus (1/6: 17%). Mortality was noted in 33% of virus associated HPS patients. Our study highlights the higher association of Epstein-Barr virus (EBV) with HPS as compared to other viruses along with higher rate of mortality in both parvovirus B 19 and EBV associated HPS.

Os morcegos e a raiva na região oeste do Estado de São Paulo / The bats and rabies in the Western region of the State of São Paulo, Brazil

INTRODUÇÃO: O laboratório do Pólo da Alta Sorocabana de Presidente Prudente, SP, em parceria com outras instituições de pesquisa, realizou estudos pertinentes aos morcegos da região oeste do Estado de São Paulo, Brasil. Para tal, foram pesquisadas algumas situações, tais como: a) isolamento do vírus rábico, no período 2006 a 2008; b) as respectivas variantes antigênicas; c) abrigos diurnos do morcego hematófago Desmodus rotundus. MÉTODOS: As amostras para exame foram provenientes de morcegos não hematófagos encaminhadas ao laboratório sendo submetidas aos testes de imunofluorescência direta e prova biológica. As amostras positivas foram caracterizadas antigenicamente por meio do teste de anticorpos monoclonais. Quanto aos morcegos, foram identificados e classificados, e também foi realizado mapeamento de abrigos dos mesmos. RESULTADOS: O laboratório recebeu 1.113 morcegos não hematófagos para diagnóstico laboratorial, sendo 11 (1 por cento) deles positivos, e dentre as amostras positivas, 5 (45,5 por cento) delas tiveram variante antigênica 3 associada ao morcego D. rotundus e 4 (36,5 por cento) foram compatíveis com amostras de morcegos insetívoros. Foram pesquisados 16 abrigos de morcegos hematófagos e observou-se a presença de outras 3 espécies de morcegos não hematófagos convivendo com eles. CONCLUSÕES: Os experimentos mostraram que o vírus rábico continua circulando na região com pelo menos 3 variantes antigênicas, e que, a coabitação de morcegos hematófagos com não hematófagos pode ter alguma relação com a disseminação do vírus rábico.

INTRODUCTION: The Polo da Alta Sorocabana Laboratory in Presidente Prudente, SP, in partnership with other research institutions, conducted studies related to bats from the western region of the State of Sao Paulo, Brazil. Thus, certain situations were investigated, including: a) isolation of the rabies virus from 2006 to 2008; b) identification of respective antigenic variants; and c) characterization of daytime shelters of Desmodus rotundus vampire bats. METHODS: Samples for examination originated from nonhematophagous bats forwarded to the laboratory and subjected to direct fluorescent antibody test and mouse inoculation test. Positive samples were characterized by the monoclonal antibody test. Regarding the bats, they were identified and classified and mapping of their shelters was also performed. RESULTS: The laboratory received 1,113 nonhematophagous bats for rabies diagnosis, 11 (1 percent) of which were positives, and among the positive samples, 5 (45.5 percent) presented antigenic variant 3 (from the bat Desmodus rotundus) and 4 (36.5 percent) were compatible with samples derived from Brazilian insectivorous bats. Sixteen vampire bat shelters were investigated and observation confirmed the presence of another 3 species of nonhematophagous bats coexisting with them. CONCLUSIONS: The experiments showed that at least 3 antigenic variants of rabies virus are circulating in the region and that the cohabitation of vampire bats with nonhematophagous bats could be related to the dissemination of the rabies virus.

Desempeño de la prueba de inmunofluorescencia directa en el diagnóstico del virus Influenza A(H1N1) / Direct immunofluorescence assay performance in diagnosis of the Influenza A(H1N1) virus

El 25 de abril de 2009, a menos de un mes de la detección en México del primer humano con virus Influenza A(H1N1), la enfermedad ya se había propagado a más de 40 países superando los 10 000 casos notificados. Dada su naturaleza impredecible, este tipo de virus requiere métodos diagnósticos apropiados, confiables y seguros, pero que también estén al alcance de los laboratorios clínicos. Mediante el estudio de 291 muestras de pacientes con sospecha de infección por virus Influenza A(H1N1) en Neuquén, Argentina, el presente trabajo compara los dos métodos de diagnóstico utilizados simultáneamente: la prueba de inmunofluorescencia directa (DFA) y la de reacción en cadena de la polimerasa en tiempo real (RT-PCR). La DFA dio una sensibilidad de 44,4 por ciento, especificidad de 99,6 por ciento, valor predictivo positivo de 95,2 por ciento y valor predictivo negativo de 90,7 por ciento. Los resultados positivos de la metodología pueden considerarse verdaderos positivos. Un resultado negativo no excluye la presencia del virus y la muestra debe examinarse mediante RT-PCR. Del total de 291 muestras, 45 resultaron positivas por RT-PCR y 21 por DFA.

By 25 April 2009, less than one month after the first human with Influenza A(H1N1) virus was detected in Mexico, the disease had already spread to more than 40 countries, with over 10 000 cases reported. Due to its unpredictability, this type of virus requires appropriate, reliable, and safe diagnostic methods that are also accessible to clinical laboratories. Through the analysis of 291 samples taken from patients with suspected Influenza A(H1N1) virus infection in Neuquén, Argentina, this study compares the two diagnostic methods used simultaneously: direct immunofluorescence assay (DFA) and real-time polymerase chain reaction (RT-PCR). DFA had a sensitivity of 44.4 percent, a specificity of 99.6 percent, a positive predictive value of 95.2 percent, and a negative predictive value of 90.7 percent. Positive results obtained with this method can be considered true positives. A negative result does not rule out the presence of the virus. In this case, the sample should be examined by RT-PCR. Out of a total of 291 samples, there were 45 positive results with RT-PCR and 21 positive results with DFA.

Standardization of an ELISA test using a recombinant nucleoprotein from the Junin virus as the antigen and serological screening for arenavirus among the population of Nova Xavantina, State of Mato Grosso / Padronização de um teste de ELISA utilizando a nucleoproteína recombinante de vírus Junin como antígeno e triagem sorológica para Arenavírus na população de Nova Xavantina, Estado do Mato Grosso

INTRODUCTION: Arenavirus hemorrhagic fever is a severe emerging disease. METHODS: Considering that the levels of antibodies against arenavirus in the Brazilian population are completely unknown, we have standardized an ELISA test for detecting IgG antibodies using a recombinant nucleoprotein from the Junin virus as the antigen. This protein was obtained by inserting the gene of the Junin virus nucleoprotein into the genome of Autographa californica nucleopolyhedrovirus, using the Bac-to-Bac baculovirus expression system. This recombinant baculovirus was used to infect S. frugiperda cells (SF9). RESULTS: The infection resulted in synthesis of high concentrations of recombinant protein. This protein was detected on 12.5 percent polyacrylamide gel and by means of Western blot. Using the standardized ELISA test, 343 samples from the population of Nova Xavantina were analyzed. We observed that 1.4 percent of the serum samples (five samples) presented antibody titers against arenavirus. CONCLUSIONS: These results show the population studied may present exposure to arenavirus infection.

INTRODUÇÃO: A febre hemorrágica por Arenavirus é uma severa doença emergente. MÉTODOS: Considerando que os níveis de anticorpos contra Arenavirus na população brasileira é totalmente desconhecido, nos padronizamos um teste de ELISA para detecção de anticorpos IgG usando uma nucleoproteína recombinante do vírus Junin como antígeno. Esta proteína foi obtida pela inserção do gene da nucleoproteína do vírus Junin no genoma do vírus Autographa californica nucleopolyhedrovirus, utilizando o sistema de expressão em Baculovírus, Bac-To-Bac. Este baculovirus recombinante foi utilizado para infecção de células de S. frugiperda (Sf9). RESULTADOS: A infecção resultou na produção de altas concentrações de proteína recombinante. Esta proteína foi detectada em gel de poliacrilamida 12,5 por cento, e em Western blot. Utilizando o teste de ELISA padronizado, foram analizadas 343 amostras provenientes da população de Nova Xavantina. Observamos que 1,4 por cento dos soros (5 amostras) apresentavam títulos de anticorpos contra arenavírus. CONCLUSÕES: Estes resultados sugerem que a população estudada pode estar sendo exposta a infecções por arenavírus.

Padronização e uso de um método imunoenzimático que utiliza células infectadas como antígeno no diagnóstico rotineiro do dengue / Standardization and use of an immunoenzymatic method using infected cells as antigens in routine diagnosing of dengue

INTRODUÇÃO: Este trabalho mostra a padronização e o uso do método imunoenzimático utilizando células infectadas como antígeno (EIA-ICC) no diagnóstico sorológico rotineiro do dengue. MÉTODOS: Na otimização do teste, com a dose de 1.000 TCID50 de vírus do dengue tipo 3 (DENV-3), foram utilizadas 100.000 células C636 infectadas 1000 TCID50 (DENV-3). RESULTADOS: Os resultados obtidos com EIA-ICC foram comparados com o kit comercial de dengue HUMAN. Os resultados foram altamente coincidentes; o EIA-ICC mostrou-se moderadamente sensível e com alta especificidade. O teste foi usado no diagnóstico sorológico de 1.797 amostras sorológicas de casos suspeitos de dengue durante a epidemia de Ribeirão Preto, em 2006. Na avaliação sorológica, 228 amostras foram positivas para IgM contra DENV-3, e 235 amostras foram positivas para IgG contra DEV-3, e em 35 amostras detectou-se positividade para IgM e IgG. CONCLUSÕES: O EIA-ICC mostrou-se confiável e simples sendo adequado ao diagnóstico sorológico do dengue.

INTRODUCTION: This paper show the standardization and use of the immunoenzymatic method using infected cells as antigens (EIA-ICC) for routine serological diagnosing of dengue. METHODS: In optimizing the test, a dose of 1,000 TCID50 of dengue type 3 virus (DENV-3) was used, and 100,000 C636 cells infected with 1,000 TCID50 (DENV-3) were used. RESULTS: The results obtained with EIA-ICC were compared with the HUMAN commercial dengue kit. The results were highly concordant. The EIA-ICC showed moderate sensitivity and high specificity. The test was used for serologically diagnosing 1,797 blood samples from suspected dengue cases during the 2006 epidemic in Ribeirao Preto. From the serological evaluation, 228 samples were positive for IgM against DENV-3; 235 samples were positive for IgG against DENV-3; and 35 samples were positive for both IgG and IgM. CONCLUSIONS: EIA-ICC was shown to be reliable and simple, and suitable for serologically diagnosing dengue.

Evaluation of neutralization patterns of the five unique Argentine equine arteritis virus field strains reported / Evaluación de los patrones de neutralización de las únicas cinco cepas argentinas descritas de arteritis viral equina

Equine viral arteritis (EVA) is a contagious viral disease that frequently causes mild or subclinical infections in adult horses. Only one EAV serotype has been described. However, there are differences in antigenicity, pathogenicity and neutralization characteristics of virus field strains. The interaction of two viral proteins, GP5 and M, is critical for infectivity and amino acid changes in the GP5 sequences have an effect on the neutralizing phenotype, regardless the effects of other viral proteins. The objective of the present study was to evaluate the neutralization phenotypes of the 5 unique Argentine EAV strains reported and to compare them with the neutralization phenotypes of the EAV-UCD reference strain, with special emphasis on the analysis of M and GP5 proteins. The strains had a similar neutralization phenotype pattern when anti-EAV serum, derived from EAV seropositive horses, was used in the analysis. Meanwhile, low titers were observed when equine polyclonal anti-EAV reference sera were used in the assay. Argentine strains have almost the same amino acid substitutions, with the exception of LP01 strain, that mainly involves the first variable region V1, especially in neutralization sites B and C. However, they are fairly different from the EAV-UCD strain. Nevertheless, the nucleotide and amino acid differences observed among the Argentine strains LP02/R, LP02/C, LP02/P and LP-LT-ARG did not show any variations in the neutralization phenotype.

La arteritis viral equina (AVE) ocasiona infecciones, en su mayoría subclínicas, pero puede causar abortos y enfermedad respiratoria. Si bien se ha descrito un solo serotipo de AVE, existen diferencias en cuanto a la antigenicidad, patogenicidad y patrones de neutralización en las cepas de campo. Los ORF5 y ORF6 del virus codifican las proteínas de envoltura GP5 y M; la interacción entre estas proteínas es crítica para la infectividad. Los cambios en las secuencias de aminoácidos en la proteína GP5, especialmente en la región V1, afectan el fenotipo neutralizante, sin tener en cuenta variaciones aminoacídicas de otras proteínas virales. En este estudio evaluamos los fenotipos neutralizantes de las 5 únicas cepas de arteritis viral equina aisladas en Argentina y los comparamos con los de la cepa de referencia EAV-UCD por virus neutralización cruzada y análisis de secuencias aminoacídicas de las proteínas M y GP5. Las cepas argentinas presentaron un patrón de neutralización similar cuando se utilizaron sueros positivos del banco de sueros, mientras que fueron neutralizadas en menor medida por los sueros policlonales de referencia anti-AVE. A excepción de la cepa LP01, las cepas argentinas tienen casi las mismas sustituciones aminoacídicas en la primera región variable V1 de la proteína GP5, específicamente en los sitios neutralizantes B y C, pero difieren en gran medida respecto de la cepa de referencia EAV-UCD. Las diferencias encontradas en los aislamientos LP02/R, LP02/C, LP02/P y LT-LP-ARG no se reflejaron en variaciones en el fenotipo neutralizante.

Renal involvement in patients with hepatitis c virus infection

Hepatitis C virus [HCV] infection is a hepatotropic virus causing a variety of extrahepatic immunological manifestations and is a risk factor of a variety of extrahepatic diseases, such as mixed cryoglobulinemia and membranoproliferative glomerulonephritis [MPGN], which is the most common glomerulonephritis. The aim of this study was to evaluate renal involvement in HCV-infected patients. A total of 300 randomly-selected HCV antibody-positive outpatients at the HCV clinic of Shariati hospital were enrolled. Serum creatinine was measured and glomerular filtration rate was estimated accordingly. Urine proteinuria was measured in 24-hour urine samples. The patients were 249 men [83.2%] and 51 women [16.8%] with a mean age of 37.8 +/- 11.7 years [range, 18 to 70 years]. Proteinuria was found in 12 HCV antibody-positive adults [4%], 1 of whom underwent biopsy. He was a 55-year-old man with a 4-month history of facial and lower extremities edema and 3-g proteinuria with a normal kidney function [glomerular filtration rate, 85 mL/min] and normocomplementemia. Kidney biopsy specimens showed MPGN. The frequency of low glomerular filtration rate was 0.7% [2 patients] in the HCV antibody-positive adults. There was no significant relationship between HCV seropositivity and low glomerular filtration rate. Our observations showed renal involvement in HCV antibody-positive patients. Among immune complex glomerular kidney diseases, MPGN without cryoglobulins is thought to be the most common in these patients

Red baby syndrome a new disease due to parvovirus B-19 observed in Kerala.

Red Baby Syndrome is a new disease seen in infants and young children. Dramatic onset of clinical symptoms with high intensity, short duration and lack of similarity with other cutaneous lesions makes it distinct. Of 50 such patients studied over a period of 5 years, half were below one year of age. Abrupt onset of high fever and generalized erythema involving the entire skin, which is swollen and tender is characteristic. These children were highly irritable and had paradoxical cry when cuddled. Rapid resolution of symptoms occurred in 7-10 days with extensive desquamation. Routine investigations were normal, C-reactive protein was raised only in 10 patients. Human Parvo virus B-19 IgM antibodies were positive in 15 out of 24 patients. Real time polymerase chain reaction was positive for human parvovirus B 19 DNA in one. Histopathological changes in the skin biopsy showed post infectious vascular injury pattern.

Perfil antigênico do vírus da raiva isolado de diferentes espécies de morcegos não hematófagos da Região de Presidente Prudente, Estado de São Paulo / Antigen profile of rabies virus isolated from different species of non-hematophagous bats in the region of Presidente Prudente, State of São Paulo

O Laboratório de Virologia Clínica e Molecular do Instituto de Ciências Biomédicas da Universidade de São Paulo, utilizando-se da técnica de anticorpos monoclonais, tipificou 18 amostras de vírus rábico provenientes de morcegos não hematófagos de várias espécies provenientes da Região de Presidente Prudente, SP, Brasil. Destas amostras, 15 (82,3 por cento) foram definidas como variante 3 (compatível com amostras isoladas de morcegos Desmodus rotundus) e 3 (16,7 por cento) como variante 4 (compatível com amostras isoladas de morcegos Tadarida brasiliensis).

Using the monoclonal antibody technique, the Clinical and Molecular Virology Laboratory of the Institute of Biomedical Sciences of the University of São Paulo typed 18 rabies virus samples from non-hematophagous bats of several species from the region of Presidente Prudente, SP, Brazil. Among these samples, 15 (82.3 percent) were defined as variant 3 (compatible with samples isolated from Desmodus rotundus bats) and three (16.7 percent) as variant 4 (compatible with samples isolated from Tadarida brasiliensis bats).