Simple protocol for population (Sanger) sequencing for Zika virus genomic regions

BACKGROUND A number of Zika virus (ZIKV) sequences were obtained using Next-generation sequencing (NGS), a methodology widely applied in genetic diversity studies and virome discovery. However Sanger method is still a robust, affordable, rapid and specific tool to obtain valuable sequences. OBJECTIVE The aim of this study was to develop a simple and robust Sanger sequencing protocol targeting ZIKV relevant genetic regions, as envelope protein and nonstructural protein 5 (NS5). In addition, phylogenetic analysis of the ZIKV strains obtained using the present protocol and their comparison with previously published NGS sequences were also carried out. METHODS Six Vero cells isolates from serum and one urine sample were available to develop the procedure. Primer sets were designed in order to conduct a nested RT-PCR and a Sanger sequencing protocols. Bayesian analysis was used to infer phylogenetic relationships. FINDINGS Seven complete ZIKV envelope protein (1,571 kb) and six partial NS5 (0,798 Kb) were obtained using the protocol, with no amplification of NS5 gene from urine sample. Two NS5 sequences presented ambiguities at positions 495 and 196. Nucleotide analysis of a Sanger sequence and consensus sequence of previously NGS study revealed 100% identity. ZIKV strains described here clustered within the Asian lineage. MAIN CONCLUSIONS The present study provided a simple and low-cost Sanger protocol to sequence relevant genes of the ZIKV genome. The identity of Sanger generated sequences with published consensus NGS support the use of Sanger method for ZIKV population studies. The regions evaluated were able to provide robust phylogenetic signals and may be used to conduct molecular epidemiological studies and monitor viral evolution.

Avaliação da resposta à terapia antirretroviral de resgate contendo antagonista do correceptor CCR5 em pessoas vivendo com HIV/AIDS / Assessment of antiretroviral salvage therapy response to CCR5 co-receptor antagonist containing regimen in people living with HIV/AIDS

O presente estudo apresenta um novo algoritmo de ferramentas de bioinformática capaz de determinar de forma robusta o tropismo do HIV analisando sequências da região V3 do envelope viral. O critério ...A análise do tropismo viral usando a genotipagem da V3 e o Critério IAL foi empregada em 157 pacientes do Programa Estadual de Aids de São Paulo, que necessitavam de terapia de resgate, dos anos de 2008 a 2012. Foram detectados, 89 pacientes com tropismo HIV para o correceptor CCR5 (R5) e 68 CXCR4 (X4). Com base nestes resultados foi estabelecido o esquema terapêutico pelo médico e comissão estadual, e indicado antagonista do CCR5 (maraviroque–MQV) para alguns pacientes.Em 73 casos (41R5 e 32X4) foi possível obter o desfecho clínico, levando em consideração dados virológicos e imunológicos. Foi calculada a pontuação.....Houve aumento de células T CD4+ de mais de 100 células/mm3 de sangue quando comparado ao baseline em 76% dos pacientes: 86% do Grupo 1, 75% do Grupo 2 e 69% do Grupo 3. O grupo 1 apresentou o menor nadir e o maior aumento de células T CD4, embora semsignificância estatística. Não houve diferença na pontuação de drogas entre os Grupos 1 e 2 (2,75 – 2,38), mas quando foi retirado o MQV da análise, houve queda da pontuação do Grupo 1 com valores semelhantes ao Grupo 3 (1,75–2). Concluindo, o Critério IAL para determinar o tropismo HIV se mostrou mais sensível e específico que as ferramentas de bioinformática isoladamente e com sensibilidade e especificidade semelhantes ao ensaio de imunofenotipagem, sendo considerado útil na indicação do melhor esquema terapêutico de resgate ao paciente. O estudo longitudinal com MQV mostrou ser esta uma boa opção terapêutica em pacientes com vírus R5.

HI-1 viral tropism is an important phenotypic characteristic may be classified as CCR5 tropic, CXCR4 tropic or dual tropic. As phenotypic assays are complex and expensive, genotypic prediction has been largely used. This study presents a new bioinformatics algorithm capable of robustly determining the tropism of HIV analyzing sequences of V3 region of the viral envelope tools. The criterion, .... The analysis of viral tropism using the V3 genotyping and IAL Criterion was used in 157 patients with advance disease in Sao Paulo from 2008 to 2012. CCR5 HIV tropism (R5) was predicted for 89 patients and X4 in 68. Based on these results, the therapeutic regimen was established by the physician and state commission. In 73 cases (41R5 and 32X4) it was possible to obtain the clinical outcome, including virological and immunological data. Scores of salvage ...There was an increase of more than 100 cells/mm3 of blood CD4cells when compared to baseline in 76% of patients: 86% of group 1, 75% in group 2 and 69 % of Group 3. Group 1 had the lowest nadir and the highest increase of CD4 + cells, although without statistical significance. There was no difference in the scores of drugs between Groups 1 and 2 (2.25 - 3), but when MQV was removed from the analysis, there was a drop in the scores of Group 1 with values similar to Group 3 (1.25-2). In conclusion, the IAL criterion to determine HIV tropism was more sensitive and specific than the tools of bioinformatics alone and showing comparable results to that of the phenotype, and its use to predict viral tropism allowed the use MQV for some R5 patients. Its use as part of salvage therapy showed a good therapeutic response suggesting that the test was useful in predicting CCR5 antagonist drug activity.