Drosophila S2 Cells Are Non-Permissive for Vaccinia Virus DNA Replication Following Entry via Low pH-Dependent Endocytosis and Early Transcription

Vaccinia virus (VACV), a member of the chordopox subfamily of the Poxviridae, abortively infects insect cells. We have investigated VACV infection of Drosophila S2 cells, which are useful for protein expression and genome-wide RNAi screening. Biochemical and electron microscopic analyses indicated that VACV entry into Drosophila S2 cells depended on the VACV multiprotein entry-fusion complex but appeared to occur exclusively by a low pH-dependent endocytic mechanism, in contrast to both neutral and low pH entry pathways used in mammalian cells. Deep RNA sequencing revealed that the entire VACV early transcriptome, comprising 118 open reading frames, was robustly expressed but neither intermediate nor late mRNAs were made. Nor was viral late protein synthesis or inhibition of host protein synthesis detected by pulse-labeling with radioactive amino acids. Some reduction in viral early proteins was noted by Western blotting. Nevertheless, synthesis of the multitude of early proteins needed for intermediate gene expression was demonstrated by transfection of a plasmid containing a reporter gene regulated by an intermediate promoter. In addition, expression of a reporter gene with a late promoter was achieved by cotransfection of intermediate genes encoding the late transcription factors. The requirement for transfection of DNA templates for intermediate and late gene expression indicated a defect in viral genome replication in VACV-infected S2 cells, which was confirmed by direct analysis. Furthermore, VACV-infected S2 cells did not support the replication of a transfected plasmid, which occurs in mammalian cells and is dependent on all known viral replication proteins, indicating a primary restriction of DNA synthesis

Reprodutibilidade do diagnóstico histopatológico de dermatoses por fotomicrografias digitais versus microscopia óptica convencional Reproducibility of histopathologic diagnosis of skin diseases by digital photomicrographs versus conventional optical microscopy

FUNDAMENTOS: A telepatologia é considerada boa alternativa para consultas de segunda opinião. Sua implementação é desejável, mas estudos que confirmem sua aplicação prática são necessários. OBJETIVOS: Analisar a concordância entre os diagnósticos histopatológicos de dermatoses feitos com base em microfotografias digitais em comparação com a microscopia convencional. MÉTODOS: As lâminas de 135 pacientes foram avaliadas por dois consultores por meio de microscópio. Após quatro semanas, os mesmos consultores avaliaram 1.738 microfotografias digitais obtidas dessas lâminas. Estimou-se a taxa geral de concordância intraobservadores e interobservadores e obteve-se o índice Kappa com a categorização das dermatoses: dermatoses neoplásicas, dermatoses infectocontagiosas e não infectocontagiosas. RESULTADOS: A concordância do Consultor 1, que analisou lâminas ao microscópio versus imagens, foi de 88,1% (IC 95% = 81,5% - 93,1%). O Consultor 2 obteve concordância de 80,7% (IC 95% = 73,1% - 87%). A concordância interconsultores ao microscópio foi de 81,5% (IC 95% = 73,9% - 87,6%). A mesma análise por meio de microfotografias mostrou concordância de 85,9% (IC 95% = 78,9% - 91,3%). O índice Kappa, que avalia as dermatoses categorizadas, foi de 98,6% para o Consultor 1, de 93,1% para o Consultor 2, de 95,8% para interconsulta por imagens e de 95,9% para interconsultas ao microscópio óptico. Todos esses valores são considerados ótimos. CONCLUSÃO: A avaliação das microfotografias apresentou ótimo nível de reprodutibilidade quando comparada à microscopia tradicional, sendo uma opção viável para consultas de segunda opinião em dermatopatologia<br>BACKGROUND: Telepathology is considered a good alternative for a second opinion consultation. Its implementation is desirable, but studies to confirm its practical application are required. OBJECTIVES: To evaluate the reproducibility of histopathologic diagnoses of skin diseases obtained through digital photomicrographs compared with conventional microscopy. METHODS: We evaluated the surgical specimens of 135 patients using an optical microscope. After 4 weeks, the same consultants independently evaluated a total of 1,738 digital photomicrographs obtained from the histopathological slides. We estimated the overall rate of intra and interobserver agreement, and the Kappa coefficient was obtained with the categorization of the skin diseases: neoplastic skin diseases, infectious-contagious skin diseases and non-infectious-contagious skin diseases. RESULTS: Consultant 1 obtained an agreement of 88.1% (95% CI = 81.5% - 93.1%) between conventional microscopy and images. Consultant 2 obtained an agreement of 80.7% (95% CI = 73.1% - 87%). The interobserver agreement for the microscopy analysis was 81.5% (95% CI = 73.9% - 87.6%). The same analysis using microphotography showed an agreement of 85.9% (95% CI = 78.9% - 91.3%). The Kappa coefficient, which evaluated the categorized skin diseases, was 98.6% for Consultant 1, 93.1% for Consultant 2, 95.8% for interconsultant analyses of images and 95.9% for interconsultant analyses using conventional optical microscopy. All of these values are considered optimal. CONCLUSION: The evaluation of microphotographs shows a good level of reproducibility when compared to traditional microscopy and, therefore, is a viable option for a second opinion consultation in dermatopatholog