Avaliação de dois testes sorológicos comerciais para diagnóstico das infecções pelo FIV e pelo FeLV / Evaluation of two point-of-care tests to diagnosis of FIV and FeLV infections

FIV e FeLV são retrovírus associados principalmente com neoplasias. Dois testes rápidos são disponibilizados no Brasil para o diagnóstico dessas infecções: um kit de imunocromatografia de fluxo bidirecional (SNAP® Combo IDEXX) e um kit de imunocromatografia de fluxo lateral unidirecional (ALERE/BIONOTE Anigen Rapid). O objetivo deste estudo foi comparar o teste SNAP® com o teste ALERE. Amostras de sangue de 178 gatos foram testadas utilizando-se ambos os kits. A reação em cadeia de polimerase em tempo real (qPCR) foi empregada como método confirmatório para todos os resultados. O teste SNAP® apresentou sensibilidade e especificidade de 100% para FIV; a sensibilidade e a especificidade do teste ALERE foram de 96,15% e 98,68%, respectivamente. A sensibilidade e a especificidade para o FeLV foram de 93,02% e 96,30% para o teste SNAP® e de 90,70% e 97,78% para o teste ALERE. Ainda em relação ao FeLV, três amostras com resultado positivo na qPCR obtiveram resultado falso-negativo em ambos os testes. Não houve diferença estatisticamente significante entre os métodos. Considerando a qPCR como padrão-ouro, o teste SNAP® apresentou maior sensibilidade e especificidade para o FIV, e o teste ALERE apresentou maior especificidade para o FeLV. Os resultados mostraram uma boa correlação entre os testes.(AU)

FIV and FeLV are Retrovirus associated mainly with feline neoplasms. Two point-of-care tests are commercially available in Brazil for diagnosis of these infections: a bidirectional flow immunochromatography kit (IDEXX SNAP ® Combo) and a lateral unidirectional flow immunochromatography kit (ALERE/BIONOTE Anigen Rapid). The aim of this study was to compare SNAP ® and ALERE tests. Blood samples obtained from 178 cats were evaluated using both tests. Quantitative real-time polymerase chain reaction (qPCR) was used as confirmatory test for all samples. The sensitivity and specificity of SNAP ® test was 100% for FIV, and for ALERE test was 96.15% and 98.68%, respectively. The sensitivity and specificity for FeLV was 93.02% and 96.30% for SNAP ® test and 90.70% and 97.78% for ALERE test. Three samples with a qPCR positive result for FeLV obtained a false negative result in both SNAP ® and ALERE tests. There was no statistically significant difference between the two methods. Considering qPCR as gold standard method, the SNAP® test showed higher sensitivity and specificity for FIV, and the ALERE test presented higher specificity for FeLV. The results showed good agreement among the tests.(AU)

DBCollHIV: a database system for collaborative HIV analysis in Brazil

We developed a database system for collaborative HIV analysis (DBCollHIV) in Brazil. The main purpose of our DBCollHIV project was to develop an HIV-integrated database system with analytical bioinformatics tools that would support the needs of Brazilian research groups for data storage and sequence analysis. Whenever authorized by the principal investigator, this system also allows the integration of data from different studies and/or the release of the data to the general public. The development of a database that combines sequences associated with clinical/epidemiological data is difficult without the active support of interdisciplinary investigators. A functional database that securely stores data and helps the investigator to manipulate their sequences before publication would be an attractive tool for investigators depositing their data and collaborating with other groups. DBCollHIV allows investigators to manipulate their own datasets, as well as integrating molecular and clinical HIV data, in an innovative fashion.

The N-terminal amino acid sequence is essential for foot-and-mouth disease virus replicase activity

Foot-and-mouth disease virus replicase was expressed by fusing its cDNA to the OmpA signal peptide coding sequence present in the pIN-III ompA series vectors. Two constructions were developed to express either a full-lenghtt or truncated enzyme lacking the 20 aminoacids at the N-terminal en. Bacterial extr5acts expressing the recombinant proteins were submitted to SDS-PAGE and the presence of the replicase was revealed by immunoblotting. The truncated form exhibited a higher mobility and the relative positions of the proteins show that the signal peptide was removed. The biological activity of these two molecules was tested using a poly(A)-dep[endent oligo(U)-primed poly(U)-polymer4ase assay. The full-lenght replicase is active. The aminoterminal truncated wnzyme had 0.02% activity o9f the intact5 one. This result indicates the importaqnce of the twenty N-terminal amino acids for the activity of FMDV RNA dependent RNMA polymerase

In vitro activities of a recombinant foot-and-mouth disease virus replicase expressed in Escherichia coli

The replicase gene of foot-and-mouth disease virus (FMDV) was expressed in Escherichia coli under the control of a tac promoter. The recombinant enzyme was purified by inclusion body precipitation, elution, and poly(U) Sepharose chromatography. The enzyme exhibits poly(A)-dependent oligo(U)-primed poly(U) polymerase activity. The specific activity of the purified replicase is 1.3 x 10 5. The recombinant replicase synthesizes RNA using FMDV RNA as template, as well as heterologous RNAs, such as globin RNA and synthetic RNAs, polyadenylated or not. In all polymerization reactions, RNA products twice the size of the template are formed, both in the presence and absence of an oligo(U) primer. The enzyme is also capable of incorporating [alpha32P]UTP in all RNAs tested except the viral template. This activity does not seem to be related to the primer independent polymerization activity. The products from polymerization reactions were characterized by hybridization. In the absence of primer they consist of the template and a complementary strand covalently attached, while in the presence of primer they consist of two complementary strands synthesized de novo. We propose mechanisms of RNA synthesis by the recombinant FMDV replicase in the absence and presence of primer. These mechanisms are discussed in terms of models for in vitro RNA synthesis of other piconaviruses

Expression of an active foot-and-mouth disease virus RNA polymerase in Escherichia coli

The expression of a native form of the foot-and-mouth disease virus RNA polymerase was obtained. Two oligonucleotides of 66 base pairs were used to renuild the 5' end of the gene and to introduce the first methionine codon. The expression of the active polymerase in E. coli was achieved by inserting the gene before the tac promoter of the pKK223-3 plasmid

Direct expression of a modified foot-and mouth-disease virus RNA-polymerase in Escherichia coli

A RNA-polimerase (RNA-pol) do vírus da Febre Aftosa foi expressa em Escherichia coli de uma maneira controlável. Para isso um vetor de expressäo foi construído, clonando-se um cDNA do gene contendo o primeiro codon (AGT) e as sequências de Shine & Delgarno foi adicionada no terminal 5' do gene. Após termoinduçäo da cepa recombinante, a RNA-pol do virus da Febre Aftosa foi expressa, chegando a atingir 10% da proteína total da célula. Esta RNA-pol recombinante apresentava um peso molecular esperado (50Kda) assim como reaçäo cruzada em Wstern blot com soros anti RNA-pol (natural) e de bovinos convalescentes de Febre Aftosa. Um soro policlonal contra a RNA-pol recombinante foi capaz de inibir a atividade enzimática da RNA-pol natural. A expressäo da RNA-pol do vírus da Febre Aftosa em E. coli facilitará a análise da atividade enzimática desta molécula assim como tornará possível o desenvolvimento de um método diagnóstico eficiente e seguro para a Febre Aftosa