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.

Use of Sanger protocols to identify variants of concern, key mutations and track evolution of SARS-CoV-2.

Vaccination and the emergence of SARS-CoV-2 variants mark the second year of the pandemic. Variants have amino acid mutations at the spike region, a viral protein central in the understanding of COVID-19 pathogenesis and vaccine response. Variants may dominate local epidemics, as Gamma (P.1) in Brazil, emerging in 2020 and prevailing until mid-2021. Different obstacles hinder a wider use of Next-Generation Sequencing for genomic surveillance. We describe Sanger based sequencing protocols: i) Semi-nested RT-PCR covering up to 3.684 kb (>96 %) spike gene; ii) One-Step RT-PCR for key Receptor Binding Domain (RBD) mutations (codons 417-501); iii) One-Step RT-PCR of partial N region to improve genomic capability. Protocols use leftovers of RNA extracted from nasopharyngeal swabs for quantitative RT-PCR diagnosis; with retro-transcribed DNA sequenced at ABI 3500 using dye termination chemistry. Analyses of sequences from 95 individuals (late 2020/early 2021) identified extensive amino acid variation, 57 % with at least one key mutation at the Receptor Binding Domain, with B.1.1.28 lineage most prevalent, followed by Gamma and Zeta variants, with no Delta variant observed. The relatively low cost and simplicity may provide an accessible tool to improve surveillance of SARS-CoV-2 evolution, monitor new variants and vaccinated breakthroughs.

Major drug resistance mutations to HIV-1 protease inhibitors (PI) among patients exposed to PI class failing antiretroviral therapy in São Paulo State, Brazil.

BACKGROUND: Protease inhibitors (PI) are especially important in salvage therapy. Previous treatment failure with a PI containing regimen may elicit resistance mutations, reducing PI susceptibility and limiting treatment options. The aim of this study was to describe major PI mutations among patients exposed to at least one PI to evaluate predictors of mutation emergence and the impact of subtypes on resistance. METHODOLOGY: Partial HIV-1 pol sequences (Sanger Sequencing) from patients exposed to PI with virological failure were genotyped from January 2014 to December 2017. Drug resistance mutations (DRM), antiretroviral susceptibility (GSS) and subtypes, along clinical and laboratory parameters, were evaluated using logistic regression to access the predictors of mutation emergence. RESULTS: In 27.5% (466/1696) of the cases at least one major PI mutations was identified, most commonly M46 (14.7%), V82 (13.8%) and I54 (13.3%). Mutations to NRTI and NNRTI were observed in 69.6% and 59.9%, respectively, of the 1696 sequences. Full activity to darunavir was predicted in 88% (1496/1696), but was only 57% among those with at least one PI-DRM. Subtype C sequences had less major PI-DRMs (10%, 9/87) compared to B (28%, 338/1216) or F (35%, 58/168) (p <0.001) but adjusted analysis suggested that this association is not independent from a shorter treatment time and fewer regimens (OR 0.59, Confidence Interval 95: 0.2-2.5, p = 0.48). Subtype F, together with NRTI mutations and longer time on treatment was associated to presence of PI-DRM, to a lower darunavir GSS and to mutations at codon I50. CONCLUSIONS: Among patients with PI-DRM, full activity to darunavir was compromised in almost half of the cases and efforts to detect failure at earlier time are warranted, particularly for HIV-1 subtype F that showed association to the emergence of resistance, with potential impact in protease inhibitors sequencing. Furthermore, NRTI mutations may serve as an indicative of sufficient adherence to allow PI-DRM emergence.

Isolation of infectious Zika virus from a urine sample cultured in SIRC cells from a patient suspected of having rubella virus.

A great variety of viruses which cause exanthema share other clinical manifestations, making the etiologic identification a very difficult task, relying exclusively on the clinical examination. Rubella virus (RV) infection during the early stages of pregnancy can lead to serious birth defects, known as congenital rubella syndrome (CRS). In the present report, we described the presence of Zika virus (ZIKV) particles in urine samples and also ZIKV isolation in SIRC cells from the urine of a patient in acute phase of suspected rubella disease. The 50-year-old unvaccinated woman living in Sao Paulo, Brazil, was admitted to the emergency room with fever, headache, rash, arthralgia and prostration. Urine samples were collected for virus isolation and RT-qPCR. SIRC and Vero cells were inoculated with urine samples during 7 days. RT-qPCR was performed using measles virus (MV) and RV primers and both were found to be negative. After this result, RT-qPCR was performed for parvovirus B19, herpes virus 6 and ZIKV. The urine sample and the isolate were positive by Real Time PCR for ZIKV and negative for all other viruses tested. The sequences isolated are from the Asiatic lineage.

High Prevalence of Drug Resistance Mutations Among Patients Failing First-Line Antiretroviral Therapy and Predictors of Virological Response 24 Weeks After Switch to Second-Line Therapy in São Paulo State, Brazil.

Universal antiretroviral treatment with sustained viral suppression benefits patients and reduces HIV transmission. Effectiveness of therapy may be limited by antiretroviral drug resistance. Information on the resistance profile at treatment failure and its impact on antiretroviral drugs may subsidize subsequent treatment strategies. Partial pol sequences from 319 patients failing first-line therapy were analyzed for resistance associated mutations (RAMs) and HIV subtype. Demographic data, CD4 T cell count, viral load, and antiretroviral regimens and mutational profile at first-line failure were also investigated for associations to the response to second-line regimens. RAMs at the reverse transcriptase gene were frequent. Most sequences (88%) showed at least one mutation. A higher number of reverse transcriptase RAMs were associated to lower CD4 T cell counts and the use of tenofovir/lamivudine in first line. Among 205 with follow-up data, 76.6% were virally suppressed (below 200 copies/ml) after 24 weeks of second-line therapy. Most cases initiated second line with a regimen genotypic susceptibility score ≥2, but it did not predict viral suppression, that was independently associated with higher CD4 T cell counts and with the presence of nucleos(t)ide analog reverse transcriptase inhibitor (NRTI) RAMs. This study documented extensive resistance at first-line failure in this area in Brazil, highlights the risks of low CD4 T cell counts to second-line therapy, and supports the notion that recycled NRTIs may contribute to viral suppression even when genotypic resistance is present.

Antiretroviral adherence, elevated viral load, and drug resistance mutations in human immunodeficiency virus - infected women initiating treatment in pregnancy: a nested case-control study

Background Elevated viral load (VL) early after antiretroviral therapy (ART) initiation appears frequently in pregnant and postpartum women living with human immunodeficiency virus; however the relative contributions of pre-ART drug resistance mutations (DRMs) vs nonadherence in the etiology of elevated VL are unknown. Methods Within a cohort of women initiating ART during pregnancy in Cape Town, South Africa, we compared women with elevated VL after initial suppression (cases, n = 80) incidence-density matched to women who maintained suppression over time (controls, n = 87). Groups were compared on pre-ART DRMs and detection of antiretrovirals in stored plasma. Results The prevalence of pre-ART DRMs was 10% in cases and 5% in controls (adjusted odds ratio [aOR], 1.53 [95% confidence interval {CI}, .4­5.9]); all mutations were to nonnucleoside reverse transcriptase inhibitors. At the time of elevated VL, 19% of cases had antiretrovirals detected in plasma, compared with 87% of controls who were suppressed at a matched time point (aOR, 131.43 [95% CI, 32.8­527.4]). Based on these findings, we estimate that <10% of all elevated VL in the cohort may be attributable to pre-ART DRMs vs >90% attributable to ART nonadherence...

Genotypic tropism prediction from paired cell and plasma using single and replicate sequences.

HIV-1 tropism determination is necessary prior to CCR5 antagonist use as antiretroviral therapy. Genotypic prediction of coreceptor use is a practical alternative to phenotypic tests. Cell DNA and plasma RNA-based prediction has shown discordance in many studies. We evaluate paired cell and plasma either as single or replicate V3 sequences to assess prediction comparability. The HIV-1 partial env region was sequenced and tropism was predicted using geno2pheno and position-specific scoring matrices (PSSM). Nucleotide ambiguities at V3 were quantified and genetic distance (Protdist) was determined using BioEdit. Wilcoxon signed-rank test, t tests, and Spearman correlation were performed with Prism GraphPad5.0. Results are expressed as medians, with a level of significance of p<0.05, two tailed. Single (n=28) or replicate (n=26) paired cell/plasma sequences were obtained from 54 patients. Although the clonalfalse-positive rate (FPR) value from both compartments strongly correlated (r=0.86 p<0.0001), discordance in tropism prediction was observed in both singles and replicates using geno2pheno or PSSM. Applying clonalFPR(10%) 46% (25/54) were X4 tropic, with a plasma/cell discordance of 11% in singles and 23% in replicates. Genetic distance (p<0.0001) and clonalFPR value dispersion (p=0.003) were significantly higher among replicate sequences from cells. Discordance of viral tropism prediction is not uncommon and the use of replicates does not decrease its occurrence, but improves X4 sensitivity. Sequences from provirus had greater genetic distance and dispersion of clonalFPR values. This may suggest that DNA replicate assays may better represent the diversity of HIV-1 variants, but the clinical significance of these findings needs further evaluation.

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.

Concordance of HIV type 1 tropism phenotype to predictions using web-based analysis of V3 sequences: composite algorithms may be needed to properly assess viral tropism.

Genotypic prediction of HIV-1 tropism has been considered a practical surrogate for phenotypic tests and recently an European Consensus has set up recommendations for its use in clinical practice. Twenty-five antiretroviral-experienced patients, all heavily treated cases with a median of 16 years of antiretroviral therapy, had viral tropism determined by the Trofile assay and predicted by HIV-1 sequencing of partial env, followed by interpretation using web-based tools. Trofile determined 17/24 (71%) as X4 tropic or dual/mixed viruses, with one nonreportable result. The use of European consensus recommendations for single sequences (geno2pheno false-positive rates 20% cutoff) would lead to 4/24 (16.7%) misclassifications, whereas a composite algorithm misclassified 1/24 (4%). The use of the geno2pheno clinical option using CD4 T cell counts at collection was useful in resolving some discrepancies. Applying the European recommendations followed by additional web-based tools for cases around the recommended cutoff would resolve most misclassifications.

Evaluation of genotypic prediction of HIV-1 tropism using population sequencing of replicates.

Determination of human immunodeficiency virus tropism has contributed to the understanding of the pathogenesis of HIV and is necessary prior to the use of CCR5 antagonists. Replicate V3 sequences may generate different sequences and improve viral tropism prediction. The diversity of HIV was evaluated to access its influence on prediction. Plasma RNA was retro-transcribed and amplified using a one-step protocol, followed by nested PCR and sequencing using an ABI3130XL. Eighty-one patients, 74% male and 26% female, with a median age of 44 years had either a single sequence (n=50) or 2-4 replicates (n=31) evaluated. Most patients (92%) had used multiple anti-retroviral regimens. Tropism prediction was performed using the Geno2pheno clonal option. The number of ambiguous nucleotides, the deduced non-synonymous amino acids at V3 and the genetic distance were quantified. Using a 20% false positive rate (FPR) cut-off, 41/81 (50.6%) was predicted as X4. TCD4 was lower, 226 cells/mm(3) (IQR 82-378), in patients infected with X4; TCD4 for R5 was 324 cells/mm(3) (IQR 200-538, p<0.05). The number of ambiguous nucleotides correlated with a lower FPR value (p<0.0027). Although different sequences may be generated, the number of replicates was not associated to a lower FPR or X4 assignment, and may allow a better prediction of this biological characteristic. Ambiguous nucleotides correlate inversely to a lower FPR.

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.