Padronização de um ELISA indireto a partir da utilização de uma proteína de superfície do vírus da anemia infecciosa equina como antígeno para o diagnóstico dessa enfermidade / Standardization of an indirect ELISA using a surface protein of the equine infectious anemia virus as an antigen for the diagnosis of this disease

A anemia infecciosa equina é uma importante enfermidade que acomete os equídeos em todo o mundo, se apresentando de forma aguda, crônica e assintomática causando grandes prejuízos para a economia tanto para criadores que vivem do trabalho desses animais quantos aos criadores que investem no melhoramento das raças, impedindo o acesso ao mercado tanto nacional quanto internacional. O Ministério da Agricultura, Pecuária e Abastecimento considera o IDGA como teste oficial para diagnóstico dessa enfermidade, porém essa técnica é demorada e muita vez acaba sendo subjetiva, dependendo da experiencia particular de cada Laboratorista. Além de não conseguir detectar animais no início da infecção. Logo, a necessidade de se buscar novas técnicas como o ELISA indireto que aperfeiçoem o tempo de análise dos resultados, facilita a automação e obtém resultados confiáveis. O estudo realizado teve como objetivo padronizar uma técnica de ELISA indireto utilizando uma proteína de envelope viral GP90 como antígeno para diagnóstico da anemia infecciosa equina. Avaliando o desempenho do teste a partir da sensibilidade, especificidade e valores preditivos positivo e negativo. Os valores obtidos foram: 91,11%, 93,33%, 91,11% e 93,33% respectivamente. Concluiu-se que o teste apresenta bom desempenho, além da possibilidade de detectar amimais positivos no início da infecção.

Equine infectious anemia is an important disease that affects horses all over the world, presenting in an acute, chronic and asymptomatic way, causing great damage to the economy, both for breeders who live off the work of these animals and for breeders who invest in the improvement of breeds, preventing access to both national and international markets. The Ministry of Agriculture, Livestock and Food Supply considers AGID to be the official test for diagnosing this disease, but this technique takes time and often ends up being subjective, depending on the particular experience of each laboratory worker. In addition to not being able to detect animals at the beginning of the infection. Therefore, the need to seek new techniques such as indirect ELISA that improve the time of analysis of results, facilitate automation and obtain reliable results. The aim of this study was to standardize an indirect ELISA technique using a GP90 viral envelope protein as an antigen for the diagnosis of equine infectious anemia. Evaluating test performance based on sensitivity, specificity and positive and negative predictive values. The values obtained were 91.11%, 93.33%, 91.11 and 93.33 respectively. It was concluded that the test performs well, in addition to the possibility of detecting positive animals at the beginning of the infection.

Nuclei ultrastructural changes of C6/36 cells infected with virus dengue type 2 / Cambios ultraestructurales en núcleos de células C6/36 infectadas con virus dengue de tipo 2

ABSTRACT Introduction: Dengue virus replication has been considered mainly cytoplasmic, however, studies indicate that some flaviviruses may use the intranuclear pathway as part of the machinery that the virus uses to increase infection capacity in the host cell. This paper describes alterations at nuclear level in the cell infected with dengue, which are likely involved in the virus replication processes. Objective: This paper addresses the ultrastructural observations of C6/36 cells of the Aedes albopictus mosquito infected with dengue virus type 2. Materials and methods: C6/36 cells were infected in culture medium with the serum of a patient positively diagnosed for dengue 2. Subsequently, the cells were incubated for 10 days and the cytopathic effect was assessed. The cells were processed for immunofluorescence assays and transmission electron microscopy. Results: The immunofluorescence assays confirmed the presence of viral protein E associated with cellular syncytia in the culture. In the ultrastructural study, the infected cells showed vesicular-tubular structures and dilated cisterns of the endoplasmic reticulum at the cytoplasmic level. Viral particles were found exclusively in cytoplasm localized within the vacuoles. Nuclei of cellular syncytia showed membrane structures arranged in a circular shape and, in some cases, these syncytia displayed lysis; in no case viral particles were observed at the nuclear level. Conclusions: The ultrastructural alterations of nuclei in cells infected with the dengue virus using electron microscopy techniques had not been reported before, as far as we know. It is likely that such modifications are associated with replicative processes at an intranuclear level as an alternate replication mechanism.

RESUMEN Introducción. La replicación del virus del dengue se ha considerado principalmente citoplásmica; sin embargo, en diversos estudios se ha informado que algunos flavivirus pueden utilizar factores intranucleares como parte de la maquinaria que utilizan para aumentar la capacidad de infección en la célula huésped. En este trabajo se describen las alteraciones a nivel nuclear en células infectadas con dengue, probablemente involucradas en procesos de replicación viral. Objetivo. Presentar las observaciones ultraestructurales de células C6/36 de Aedes albopictus infectadas con el virus del dengue de tipo 2. Materiales y métodos. Se infectaron células C6/36 con suero de un paciente con diagnóstico de dengue 2; posteriormente, se mantuvieron en medio de cultivo durante 10 días y se evaluó el efecto citopático. Las células se procesaron para los ensayos de inmunofluorescencia y microscopía electrónica de transmisión, con el fin de hacer el estudio ultraestructural. Resultados. Los ensayos de inmunofluorescencia confirmaron la presencia de la proteína E viral asociada con sincitios celulares en el cultivo. En el estudio ultraestructural, las células infectadas tenían estructuras vesiculares y tubulares, y cisternas dilatadas del retículo endoplásmico en el citoplasma. Las partículas virales se encontraron exclusivamente en vacuolas localizadas en el citoplasma. Los núcleos de los sincitios celulares contenían estructuras de membrana dispuestas en forma circular y, en algunos casos, dichos sincitios presentaban lisis. En ningún caso se observaron partículas virales en el núcleo. Conclusiones. No se habían reportado alteraciones ultraestructurales en los núcleos de células infectadas con el virus del dengue detectadas mediante técnicas de microscopia electrónica. Es probable que tales modificaciones estén asociadas con procesos intranucleares de replicación como un mecanismo alternativo.

Distribution of hepatitis B virus genotypes in Korea / 대한간학회지

BACKGROUND/AIMS: Considering the incidence of prevailing hepatitis B virus (HBV) genotypes in neighboring nations, the predominance of genotype C in Korea is exceptional and needs to be confirmed by nationwide investigation. METHODS: A total of 510 HBsAg (+) or HBeAg (+) serum samples was collected from subjects in several cities and harbors throughout the Korean peninsula for genotype (A-G)-specific multiplex PCR analysis. Another 40 serum samples from chronic HBV carriers from Iksan city were selected for sequencing of the entire HBV genome. Phylogenetic analysis was performed with 22 whole genomic sequences of Korean HBV strains enrolled in GenBank. RESULTS: An amplicon was found in 377 specimens and genotype C occupied 98.1% (370 cases); none of the other genotypes were found. A mixed pattern of genotypes B and C was seen in seven specimens (1.9%), of which five were tested using PCR targeting the X fragment; no genotype B bands were found. With the exception of 1 case, which was subgenotype A2, whole sequences of Korean HBV strains (n=62) belonged to subgenotype C2. CONCLUSIONS: The prevailing HBV genotype in Korea is C2; the other genotypes occur only rarely. Future studies should include confirmation of the detection of genotypes other than C.

Comparison of antigenic sites of the envelope glycoprotein of the Iranian isolate of human T-cell leukemia virus type 1 with different subtypes of the virus

Human T-cell leukemia virus type 1 [HTLV-1] is an enveloped retrovirus, which is associated with a T-cell malignancy known as adult T-cell leukemia [ATL]. Variation in the HTLV-1 envelope nucleotide sequence has been extensively documented and has been used to classify HTLV-1 isolates into different subtypes. The virus occurs in at least 3 subtypes, which have been named A, B, and C. We conducted this study to compare the antigenic proprieties of the Iranian isolate of HTLV-1 with the homologous region of different subtypes of the virus. This study took place in the Department of Biology, College of Sciences, Shiraz University, Iran in 2005. The predicted antigenic sites and secondary structure of the envelope glycoprotein of HTLV-1, present in Iran, have been compared with the antigenic sites and secondary structure of the homologous domains in subtypes A, B, C of the virus. To predict the epitopes of glycoproteins, 21 different scales were used. The number of helices in the Iranian isolate was equal to the number of these regions in all 3 subtypes, but the number of beta-sheets was more than other viruses. One potential glycosylation site, on all these studied envelope glycoproteins, was predicted. Antigenic sites in the Iranian isolate were almost similar to subtype A of the virus and the Iranian isolate of HTLV-1 may be belongs to subtype A. Our results indicate the similarities and differences between the Iranian and other subtypes of HTLV-1. Antigenic sites represent potential candidates for use in a peptide vaccine against HTLV-1 glycoproteins and since most of the properties of a particular protein depend on its structural properties, this type of study can help in better understanding of HTLV-1 isolates present in Iran

Application of polymerase chain reaction to differentiate herpes simplex virus 1 and 2 serotypes in culture negative intraocular aspirates.

PURPOSE: To standardize and apply a polymerase chain reaction (PCR) on the glycoprotein D gene to differentiate Herpes simplex virus (HSV) 1 & 2 serotypes in culture negative intraocular specimens. METHODS: Twenty-one intraocular fluids collected from 19 patients were subjected to cultures for HSV and uniplex PCR (uPCR) for DNA polymerase gene. To differentiate HSV serotypes, as 1 & 2, a seminested PCR (snPCR) targeting the glycoprotein D gene was standardised and applied onto 21 intraocular fluids. The specificity of the snPCR was verified by application onto ATCC strains of HSV 1 and 2, clinical isolates and DNA sequencing of the amplified products. All specimens were also tested for the presence of cytomegalovirus (CMV) and varicella zoster virus (VZV) by nucleic acid amplification methods. RESULTS: Four of the 21 intraocular fluids were positive for HSV by uPCR. snPCR detected HSV in three additional specimens (total of seven specimens), and identified three as HSV 1 and four as HSV 2. DNA sequencing of PCR products showed 100% homology with the standard strains of HSV 1 and 2 respectively. None of the samples were positive in culture. Among the other patients, CMV DNA was detected in two and VZV DNA in five others. CONCLUSIONS: The standardized snPCR can be applied directly onto the culture negative specimens for rapid differentiation of HSV serotypes.

Molecular analysis of yellow fever virus 17DD vaccine strain

The Oswaldo Cruz Foundation produces most of the yellow fever (YF) vaccine prepared world wide. As part of a broader approach to determine the genetic variability in YF l7D seeds and vaccines and its relevance to viral attenuation the 17DD virus was purifed directly from chick embryo homogenates which is the source of virus used for vaccination of millions of people in Brazil and other countries for half a century. Neutralization and hemagglutination tests showed that the purified virus is similar to the original stock. Furthermore, radioimmune precipitation of 35S-methionine-labeled viral proteins using mouse hyperimmune ascitic fluid revealed identical patterns for the purified 17DD virus and the YF l7D-204 strain except for the 17DD E protein which migrated slower on SDS-PAGE. This difference is likely to be due to N-linked glycosylation. Finally, comparison by northern blot nybridization of virion RNAs of purified 17DD with two other strains of YF virus only fenome-sized molecules for all three viruses. These observations suggest that vaccine phenotype is primarily associated with the accumulation of mutations