RESUMO
Hepatitis A virus (HAV) is closely related to the genus enterovirus. HAV is very stable and resistant to acid pH and elevated temperature, as well as to chemicals and environmental influences. Human poliovirus is still one of the model viruses for testing disinfectants but there are discussions about changing to hepatitis A virus. The purpose of this study was to develop a method for using adapted hepatitis A virus to test hand disinfectants. Using HAV strains HM175/24a and FRhK-4 cytopathic effects were visible rarely, and not before 14 days. To verify virus growth in cells a RT-PCR was developed. Two disinfectants tested did not show the required virucidal activity to satisfy current German guidelines.
Assuntos
2-Propanol/farmacologia , Anti-Infecciosos Locais/farmacologia , Desinfetantes/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Etanol/farmacologia , Desinfecção das Mãos/métodos , Vírus da Hepatite A/efeitos dos fármacos , Cultura de Vírus/métodos , Animais , Efeito Citopatogênico Viral/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/normas , Ensaio de Imunoadsorção Enzimática , Guias como Assunto , Desinfecção das Mãos/normas , Vírus da Hepatite A/classificação , Vírus da Hepatite A/patogenicidade , Humanos , Concentração de Íons de Hidrogênio , Macaca mulatta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Temperatura , Cultura de Vírus/normasRESUMO
Glycoproteins I (gI) and E (gE) of the Varicella-zoster virus are encoded by the neighbouring open reading frames 67 and 68. From earlier work it is known that both genes are transcribed into several transcript species which differ in size. From gI, three transcripts of 1.65, 2.7 and 3.6 kb and from gE, two transcripts of 2.15 and 3.6 kb in size are known. We present further northern analysis and show that these various transcript species appear in different amounts at different times after infection. 12 h after infection, transcripts of 1.65 kb (gI) and 2.15 kb (gE) were clearly detectable, whereas the other transcripts appeared later on. RT-PCR experiments using a set of seven different primers provided clear evidence that gI and gE are transcribed both, mono- and bicistronically with predominance on the respective monocistronic transcript.
Assuntos
Herpesvirus Humano 3/genética , RNA Viral/análise , Proteínas do Envelope Viral/genética , Northern Blotting , Células Cultivadas , Herpesvirus Humano 3/metabolismo , Humanos , Fases de Leitura Aberta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Proteínas do Envelope Viral/metabolismoRESUMO
Varicella-zoster virus glycoprotein E (ORF 68) belongs to the group of late genes. It is a major component of the virion envelope and can be found complexed with glycoprotein I on the infected host cell surface. Glycoprotein E (gE) expression is activated by IE4 and IE62. Also, cellular transcription factors, like Sp1, are able to influence the gE expression. Performing quantitative reverse transcription-polymerase chain reaction, we found no decrease in Sp1 mRNA levels at different times post-infection, indicating that Sp1 mRNA evade virion host shutoff effects. In addition, the Sp1 protein was detectable in highly infected cells. Electrophoretic mobility shift assays have shown a binding of Sp1 to both GC elements within the gE-5'untranslated region (5'UTR). Additional shift assays have notified a binding of TATA box binding protein to the TATA box of the gE promoter, which is characterized by an untypical TATACA motif. Promoter-reporter constructs have been made using mutated variants of the gE-5'UTR as promoters. In transfection studies, we found that the TATA deletion, as well as inactivations of both GC boxes, reduced the basal activity of the promoter. A complete loss of activity did not become measurable until eliminating both GC elements and the TATA box, indicating that these cis-elements substitute for each other in initiation of transcription of the gE-5'UTR.
Assuntos
Herpesvirus Humano 3/metabolismo , Fator de Transcrição Sp1/metabolismo , Proteínas do Envelope Viral/metabolismo , Regiões 5' não Traduzidas , Sequência de Bases , Sítios de Ligação/genética , Células Cultivadas , Primers do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Herpesvirus Humano 3/genética , Humanos , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Viral/análise , RNA Viral/genética , TATA Box , Proteína de Ligação a TATA-Box , Fatores de Transcrição/metabolismo , Proteínas do Envelope Viral/genéticaRESUMO
Varicella-zoster virus (VZV) glycoproteins E (ORF 68) and I (ORF 67) are members of late genes. They belong to the major components of the virion envelope and can be found on the host cell surface as well. To get further insights in the regulation of gE and gI expression, which are known to be activated by IE4 and IE62, we analysed the intergenic regions of ORF 66/67 and ORF 67/68, containing the putative promoters of gI and gE. We have mapped the transcriptional start site of gE and have identified an extensive set of eucaryotic cis-elements: typical TATA- and CAAT-motifs and further regulatory sequences to facilitate interaction with eucaryotic transcription factors. Reporter constructs have been made using the intergenic regions of ORF 66/67 and ORF 67/68 as promoter elements. In cis-trans interaction studies, an influence on the regulation of transcription and reporter gene expression of overexpressed transfactors, LAP/LIP, Sp1, YY1 and NF-E2 has become measurable. In addition, protein-DNA binding assays using both gE- and gI-intergenic regions and cellular extracts from different VZV-permissive cells have suggested a binding of a 32 and 18 kD protein. In conclusion, these data indicate an involvement of common cellular transcription factors in the regulation of VZV late gene expression.
