Pesquisa de polimorfismos no gene UL23 do herpes simplex v??rus do tipo 2 em amostras de ??lceras genitais

• Autor(es): Almeida, Tatiana Amaral Pires de
• Data: 2015
• Origem: Oasisbr
• Assunto(s): HSV-2; UL23; Polimorfismos; Aciclovir; Resist??ncia; Polymorphisms; Resistance; CI??NCIAS DA SA??DE
• Tipo de Documento: Dissertação de mestrado
• Idioma: Português

Descrição

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FAPEAM - Funda????o de Amparo ?? Pesquisa do Estado do Amazonas

The Herpes Simplex Virus type 2 (HSV-2) is a widespread, incurable agent that establishes latency in nervous cells and is responsible for the majority of genital herpes cases. The UL23 is a polymorphic gene, which encodes the viral thymidine kinase (TK) of the Herpes virus. TK has six conserved regions, including two active sites, which plays an important role in acyclovir (ACV) metabolism. Therefore, some mutations in UL23 are implicated with ACV resistance phenoty pe. In this work, we analyzed 67 samples collected between Ap ril 2008 and September 2009 at the STI/STD clinic of ???Alfredo da Matta??? Foundation, in Manaus, Amazonas, Brazil obtained from HSV-2 genital ulcer patients. In order to characterize polymorphisms, all sp ecimens were processed for total nucleic acid isolation, submitted to PCR amplification targeting the complete ORF of UL23 gene followed by automated nucleotide sequencing with dideoxy chain terminators. Sequences analysis shown that 45 sequences were 100% similar to the reference strain ???HG52???. The remaining 22 sequences (32.8%) showed at least one nucleotide substitution; none insertions or deletions were found. Amino acids substitutions were detected in 19 out of 22. The mutation Gly39Glu was the most frequently detected (16/19), followed by Asn78Asp (5/19); Gly39Glu and Asn78Asp were simultaneously found in four samples. Deduced amino acids sequences were aligned with 66 available on GenBank from African and American continents. A total of 23 variable sites were identified and, once more, Gly39Glu mutation was the most frequent found (74/133). Substitutions previously associated with ACV resistance were not detected, however, Gly39Glu was recently described as a probable resistant mutation. Another four mutations observed: His4Tyr, Met70Arg, Asn245Ser and Ala366Val had not been previously described. The theoretical models of TK???s structure built for seven samples showed that amino acids substitutions were found away from the conserved and/or catalytic sites of TK, with the exception of Arg338Gln which was nearly from ATP-binding site. Mutations associated with ACV resistance were not detected. However one of the mutations found, Gly39Glu, was p reviously associated with this resistance phenoty pe. Another four mutations found in his work: His4Tyr, Met70Arg, Asn245Ser and Ala366Val had not been previously described elsewere. To our knowledge, this is the first study on UL23 polymorphisms on Brazilian HSV-2 samples, a research field considered as a priority by WHO HIV/STI program.

lat??ncia em c??lulas nervosas sendo respons??vel pela maioria dos casos de herpes genital. O UL23 ?? um gene polim??rfico que codifica a timidinaquinase viral (TK) do Herpesv??rus. A TK possui seis regi??es conservadas, incluindo dois s??tios ativos; esta enzima desempenha um papel importante no metabolismo do aciclovir (ACV). Consequentemente, algumas muta????es no gene UL23 est??o relacionadas a fen??tipos de resist??ncia ao ACV. Nesta disserta????o, analisamos 67 amostras coletadas entre Abril de 2008 e Setembro de 2009 na cl??nica de DST/IST da Funda????o ???Alfredo da Matta???, em Manaus, Amazonas, Brasil, obtidas de pacientes com ??lceras genitais por HSV-2. Com o objetivo de se caracterizar polimorfismos, todos os esp??cimes foram processados para extra????o total de ??cido nucleico e submetidos ?? amplifica????o da CDS do gene UL23 pela t??cnica de PCR, seguida de sequenciamento nucleot??dico automatizado utilizando-se terminadores dide??xi. As an??lises de sequ??ncias mostraram que 45 delas foram 100% similares ?? cepa-refer??ncia ???HG52???. As 22 sequ??ncias restantes (32,8%) mostraram pelo menos uma substitui????o de nucleot??deo; nenhuma inser????o ou dele????o foi encontrada. Substitui????es de amino??cidos foram detectadas em 19 dessas 22 sequ??ncias. A muta????o Gly39Glu foi a mais frequente (16/19), seguida pela Asn78Asp (5/19); Gly39Glu e Asn78Asp foram encontradas simultaneamente em quatro amostras. Sequ??ncias deduzidas de amino??cidos foram alinhadas com 66 sequ??ncias Africanas e Americanas dispon??veis no GenBank. Um total de 23 s??tios vari??veis foram identificados e, mais uma vez, a muta????o Gly39Glu foi a mais frequente (74/133). Substitui????es previamente associadas ?? resist??ncia ao ACV n??o foram detectadas, entretanto, a Gly39Glu foi recentemente descrita como uma prov??vel muta????o de resist??ncia. Outras quatro muta????es observadas, His4Tyr, Met70Arg, Asn245Ser e Ala366Val, foram descritas pela primeira vez por este trabalho. Nos modelos te??ricos da estrutura da TK, constru??dos para sete amostras, foi poss??vel visualizar que as substitui????es de amino??cidos estavam situadas longe dos s??tios ativos/conservados desta enzima, com exce????o da muta????o Arg338Gln que apresentou maior proximidade com o s??tio de liga????o de ATP. N??o foram detectadas muta????es sugestivas de resist??ncia ao ACV. Ao nosso conhecimento, este ?? o primeiro estudo de polimorfismos do gene UL23 do HSV-2 em amostras brasileiras, um campo considerado prioridade pelo Programa de HIV/IST da OM S.