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1.
Chinese Medical Journal ; (24): 2048-2053, 2021.
Article in English | WPRIM | ID: wpr-887657

ABSTRACT

BACKGROUND@#With the ongoing worldwide coronavirus disease 2019 (COVID-19) pandemic, an increasing number of viral variants are being identified, which poses a challenge for nucleic acid-based diagnostic tests. Rapid tests, such as real-time reverse transcription-polymerase chain reaction (rRT-PCR), play an important role in monitoring COVID-19 infection and controlling its spread. However, the changes in the genotypes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants may result in decreased sensitivity of the rRT-PCR assay and it is necessary to monitor the mutations in primers and probes of SARS-CoV-2 detection over time.@*METHODS@#We developed two rRT-PCR assays to detect the RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) genes of SARS-CoV-2. We evaluated these assays together with our previously published assays targeting the ORF1ab and N genes for the detection and confirmation of SARS-CoV-2 and its variants of concern (VOCs). In addition, we also developed two rRT-PCR assays (S484K and S501Y) targeting the spike gene, which when combined with the open reading frames (ORF)1ab assay, respectively, to form duplex rRT-PCR assays, were able to detect SARS-CoV-2 VOCs (lineages B.1.351 and B.1.1.7).@*RESULTS@#Using a SARS-CoV-2 stock with predetermined genomic copies as a standard, the detection limit of both assays targeting RdRp and N was five copies/reaction. Furthermore, no cross-reactions with six others human CoVs (229E, OC43, NL63, HKU1, severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus) were observed using these assays. In addition, the S484K and S501Y assays were combined with the ORF1ab assay, respectively.@*CONCLUSIONS@#Four rRT-PCR assays (RdRp, N, S484K, and S501Y) were used to detect SARS-CoV-2 variants, and these assays were shown to be effective in screening for multiple virus strains.


Subject(s)
COVID-19 , Humans , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcription , SARS-CoV-2 , Sensitivity and Specificity
3.
Braz. j. med. biol. res ; 54(5): e10725, 2021. graf
Article in English | LILACS | ID: biblio-1153554

ABSTRACT

Phylogenetic and pathogenesis studies of the severe acute respiratory syndrome-related coronaviruses (SARS-CoVs) strains have highlighted some specific mutations that could confer the RNA genome fitness advantages and immunological resistance for their rapid spread in the human population. The analyses of 30 kb RNA SARS-CoVs genome sequences, protein structures, and functions have provided us a perspective of how host-virus protein-protein complexes act to mediate virus infection. The open reading frame (ORF)1a and ORF1b translation yields 16 non-structural (nsp1-16) and 6 accessory proteins (p6, p7a, p8ab, p9b) with multiple functional domains. Viral proteins recruit over 300 host partners forming hetero-oligomeric complexes enabling the viral RNA synthesis, packing, and virion release. Many cellular host factors and the innate immune cells through pattern-recognition receptors and intracellular RNA sensor molecules act to inhibit virus entry and intracellular replication. However, non-structural ORF proteins hijack them and suppress interferon synthesis and its antiviral effects. Pro-inflammatory chemokines and cytokines storm leads to dysfunctional inflammation, lung injury, and several clinical symptoms in patients. During the global pandemic, COVID-19 patients were identified with non-synonymous substitution of G614D in the spike protein, indicating virus co-evolution in host cells. We review findings that suggest that host RNA editing and DNA repair systems, while carrying on recombination, mutation, and repair of viral RNA intermediates, may facilitate virus evolution. Understanding how the host cell RNA replication process may be driven by SARS-CoV-2 RNA genome fitness will help the testing of vaccines effectiveness to multiple independent mutated coronavirus strains that will emerge.


Subject(s)
Humans , SARS-CoV-2 , COVID-19 , Phylogeny , RNA, Viral/genetics , COVID-19 Vaccines
4.
Braz. j. infect. dis ; 24(1): 13-24, Feb. 2020. tab, graf
Article in English | LILACS | ID: biblio-1089334

ABSTRACT

ABSTRACT Dengue has been a significant public health problem in Colombia since the simultaneous circulation of the four dengue virus serotypes. The replicative fitness of dengue is a biological feature important for virus evolution and contributes to elucidating the behavior of virus populations and viral pathogenesis. However, it has not yet been studied in Colombian isolates. This study aimed to compare the replicative fitness of the four dengue virus serotypes and understand the association between the serotypes, their in vitro infection ability, and their replication in target cells. We used three isolates of each DENV serotype to infect Huh-7 cells at an MOI of 0.5. The percentage of infected cells was evaluated by flow cytometry, cell viability was evaluated by MTT assay, and the pathogenicity index was calculated as a ratio of both parameters. The replicative fitness was measured by the number of viral genome copies produced using quantitative PCR and the production of infectious viral progeny was measured by plaque assay. We showed that Huh-7 cells were susceptible to infection with all the different strain isolates. Nevertheless, the biological characteristics, such as infectious ability and cell viability, were strain-dependent. We also found different degrees of pathogenicity between strains of the four serotypes, representative of the heterogeneity displayed in the circulating population. When we analyzed the replicative fitness using the mean values obtained from RT-qPCR and plaque assay for the different strains, we found serotype-dependent behavior. The highest mean values of replicative fitness were obtained for DENV-1 (log 4.9 PFU/ml) and DENV-4 (log 5.28 PFU/ml), followed by DENV-2 (log 3.9 PFU/ml) and DENV-3 (log 4.31 PFU/ml). The internal heterogeneity of the replicative fitness within each serotype could explain the simultaneous circulation of the four DENV serotypes in Colombia.


Subject(s)
Humans , Virus Replication/genetics , Dengue Virus/genetics , Dengue Virus/pathogenicity , Serogroup , Viral Plaque Assay , Reference Values , Tetrazolium Salts , Time Factors , RNA, Viral/genetics , Cell Line , Cell Survival , Cells, Cultured , Colombia , Reverse Transcriptase Polymerase Chain Reaction , Flow Cytometry , Formazans , Liver/cytology
5.
Mem. Inst. Oswaldo Cruz ; 115: e200009, 2020. tab, graf
Article in English | SES-SP, LILACS, SES-SP | ID: biblio-1135259

ABSTRACT

BACKGROUND Influenza viral load (VL) can be a decisive factor in determining the antiviral efficacy in viral clearance. OBJECTIVE This study aimed to evaluate the rate of infection and the role of influenza VL on the clinical spectrum of illnesses among different patient groups attended at a tertiary hospital in Brazil. METHODS Samples were collected from patients presenting acute respiratory infection from 2009 to 2013. Overall, 2262 samples were analysed and distributed into three groups: (i) asymptomatic (AS); (ii) symptomatic outpatients (OP); and (iii) hospitalised patients (HP). VL (expressed in Log10 RNA copies/mL) was calculated through a quantitative real-time one-step reverse transcription-polymerase chain reaction (RT-PCR) assay aimed at the M gene, with human RNAseP target as internal control and normalising gene of threshold cycle values. FINDINGS A total of 162 (7.16%) H1N1pdm09 positive samples were analysed. Patients aged from 0.08 to 77 years old [median ± standard deviation (SD): 12.5 ± 20.54]. Children with 5 to 11 years old presented the highest detection (p < 0.0001). AS patients had the lowest VL, with a significant difference when compared with symptomatic patients (p = 0.0003). A higher VL was observed within two days of disease onset. Ten patients (HP group) received antiviral treatment and were followed up and presented a mean initial VL of 6.64 ± 1.82. A complete viral clearance for 50% of these patients was reached after 12 days of treatment. MAIN CONCLUSIONS It is important to evaluate AS patients as potential spreaders, as viral shedding was still present, even at lower VL. Our results suggest that patients with underlying diseases and severe clinical symptoms may be considered for prolonged viral treatment.


Subject(s)
Humans , Male , Female , Infant, Newborn , Infant , Child, Preschool , Child , Adolescent , Adult , Aged , Young Adult , Respiratory Tract Infections/virology , Influenza, Human/virology , Influenza A Virus, H1N1 Subtype/genetics , RNA, Viral/genetics , Acute Disease , Viral Load , Influenza A Virus, H1N1 Subtype/classification , Influenza A Virus, H1N1 Subtype/pathogenicity , Real-Time Polymerase Chain Reaction , Middle Aged
6.
Mem. Inst. Oswaldo Cruz ; 115: e190423, 2020. graf
Article in English | SES-SP, LILACS, SES-SP | ID: biblio-1135264

ABSTRACT

BACKGROUND Despite efforts to mitigate the impact of dengue virus (DENV) epidemics, the virus remains a public health problem in tropical and subtropical regions around the world. Most DENV cases in the Americas between January and July 2019 were reported in Brazil. São Paulo State in the southeast of Brazil has reported nearly half of all DENV infections in the country. OBJECTIVES To understand the origin and dynamics of the 2019 DENV outbreak. METHODS Here using portable nanopore sequencing we generated20 new DENV genome sequences from viremic patients with suspected dengue infection residing in two of the most-affected municipalities of São Paulo State, Araraquara and São José do Rio Preto. We conducted a comprehensive phylogenetic analysis with 1,630 global DENV strains to better understand the evolutionary history of the DENV lineages that currently circulate in the region. FINDINGS The new outbreak strains were classified as DENV2 genotype III (American/Asian genotype). Our analysis shows that the 2019 outbreak is the result of a novel DENV lineage that was recently introduced to Brazil from the Caribbean region. Dating phylogeographic analysis suggests that DENV2-III BR-4 was introduced to Brazil in or around early 2014, possibly from the Caribbean region. MAIN CONCLUSIONS Our study describes the early detection of a newly introduced and rapidly-expanding DENV2 virus lineage in Brazil.


Subject(s)
Humans , Genetic Variation , Genomics , Dengue/virology , Dengue Virus/genetics , Phylogeny , Brazil , RNA, Viral/genetics , Genotype
7.
Rev. Soc. Bras. Med. Trop ; 53: e20200657, 2020. tab, graf
Article in English | ColecionaSUS, LILACS, ColecionaSUS, SES-SP | ID: biblio-1143868

ABSTRACT

Abstract INTRODUCTION: Quantitative reverse transcription polymerase chain reaction (RT-qPCR) can detect the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) in a highly specific manner. However, a decrease in the specificity of PCR assays for their targets may lead to false negative results. METHODS: Here, 177 high-coverage complete SARS-CoV-2 genome sequences from 13 Brazilian states were aligned with 15 WHO recommended PCR assays. RESULTS: Only 3 of the 15 completely aligned to all Brazilian sequences. Ten assays had mismatches in up to 3 sequences and two in many sequences. CONCLUSION: These results should be taken into consideration when using PCR-based diagnostics in Brazil.


Subject(s)
Humans , Genome, Viral , Coronavirus Infections/virology , Betacoronavirus/genetics , Computer Simulation , Brazil , RNA, Viral/genetics , Sensitivity and Specificity , Reverse Transcriptase Polymerase Chain Reaction , Pandemics
8.
Rev. Soc. Bras. Med. Trop ; 53: e20190439, 2020. tab, graf
Article in English | LILACS | ID: biblio-1101443

ABSTRACT

Abstract INTRODUCTION: DENV-2 is the cause of most dengue epidemics worldwide and is associated with severe cases. METHODS: We investigated arboviruses in 164 serum samples collected from patients presenting with clinical symptoms of dengue fever and 152 mosquito pools. RESULTS: We detected the Asian II genotype of DENV-2 in humans and mosquitoes. Our results confirmed the circulation of the Asian II genotype in Brazil, in addition to the prevalent Asian/American genotype. CONCLUSIONS: The detection of Asian II genotype of DENV-2 in mosquito pools collected in a forest park may be related to a spillback event of human dengue virus.


Subject(s)
Humans , Animals , Dengue/virology , Dengue Virus/genetics , Culicidae/virology , Phylogeny , Seasons , Brazil , RNA, Viral/genetics , Polymerase Chain Reaction , Genotype , Culicidae/classification
9.
Mem. Inst. Oswaldo Cruz ; 115: e200339, 2020. tab, graf
Article in English | LILACS | ID: biblio-1154865

ABSTRACT

We evaluated sweat, blood and urine specimens obtained from an ongoing cohort study in Brazil. Samples were collected at pre-established intervals after the initial rash presentation and tested for Zika virus (ZIKV) RNA presence by real-time reverse transcriptase polymerase chain reaction (rRT-PCR). From 254 participants with confirmed infection, ZIKV RNA was detected in the sweat of 46 individuals (18.1%). Sweat presented a median cycle threshold (Ct) of 34.74 [interquartile range (IQR) 33.44-36.04], comparable to plasma (Ct 35.96 - IQR 33.29-36.69) and higher than urine (Ct 30.78 - IQR 28.72-33.22). Concomitant detection with other specimens was observed in 33 (72%) of 46 participants who had a positive result in sweat. These findings represent an unusual and not yet investigated virus shedding through eccrine glands.


Subject(s)
Humans , Male , Female , Adult , Sweat/virology , RNA, Viral/genetics , Zika Virus/isolation & purification , Zika Virus Infection/diagnosis , Urine/virology , Blood/virology , Brazil/epidemiology , RNA, Viral/isolation & purification , RNA, Viral/classification , Cohort Studies , Reverse Transcriptase Polymerase Chain Reaction , Real-Time Polymerase Chain Reaction , Zika Virus/genetics , Zika Virus Infection/epidemiology
10.
Mem. Inst. Oswaldo Cruz ; 115: e190338, 2020. tab, graf
Article in English | LILACS | ID: biblio-1091240

ABSTRACT

Oropouche virus (OROV) is an arthropod-borne virus of the Peribunyaviridae family, transmitted to humans primarily by Culicoides paraensis. It is one of the main arboviruses infecting humans in Brazil, primarily in the Amazon Region. Here, we report the detection of OROV in the saliva and urine of a patient whose samples were collected five days after the onset of symptoms. Nucleotide sequencing and phylogenetic analysis further confirmed the results. To our knowledge, this is the first study reporting the detection of OROV in the saliva and urine of an infected patient. In addition, the results of our study expand the current knowledge pertaining to the natural history of Oropouche fever.


Subject(s)
Humans , Female , Saliva/virology , Urine/virology , Orthobunyavirus/isolation & purification , Orthobunyavirus/genetics , Bunyaviridae Infections/diagnosis , Phylogeny , RNA, Viral/genetics , Base Sequence , Amino Acid Sequence , Reverse Transcriptase Polymerase Chain Reaction , Middle Aged
11.
Mem. Inst. Oswaldo Cruz ; 114: e190074, 2019. tab, graf
Article in English | LILACS | ID: biblio-1020080

ABSTRACT

BACKGROUND Hepatitis delta virus (HDV) infections in hepatitis B virus (HBV) carriers are the most severe form of viral hepatitis. HDV prevalence is high in the Brazilian Amazon, but studies in other regions of the country are still scarce and often underestimated its prevalence by including a small numbers of individuals. OBJECTIVE This study aimed to determine the serological prevalence of hepatitis D, the genotypes circulating and to evaluate the associated risk factors for acquisition of HDV in Minas Gerais state, Brazil. METHODS We screened plasma samples (n = 498) from HBV chronic carriers for anti-HD antibodies using a commercial enzyme-linked immunosorbent assay (ELISA) kit. For those samples that were positive for anti-HD antibodies, we performed a reverse transcriptase (RT) nested-polymerase chain reaction (nested-PCR) in order to detect the viral genome and identify the viral genotypes circulating in the state. FINDINGS The prevalence was 6.22% (31/498). Blood transfusion was the only risk factor associated with HDV infection [risk ratio: 3.73; 95% confidence interval (CI): 1.44 to 9.65]. For 26 anti-HD positive patients, HDAg gene sequences were determined and in all patients HDV genotype 1 was found. CONCLUSIONS This study confirmed the circulation of HDV in Minas Gerais, an area previously considered non-endemic for hepatitis D in Brazil. The prevalence found in this study is much higher when compared to other studies performed in Brazil, probably because the population in our study was selected with minimal bias. Furthermore, in 26 anti-HD positive plasma samples, we were also able to detect the viral genome, indicating that these patients were experienced an active infection at the time of sample collection. These findings emphasise the importance of anti-HD testing in HBV infected individuals, which may contribute to this disease control in Brazil.


Subject(s)
Humans , Male , Female , Infant, Newborn , Infant , Child, Preschool , Child , Adolescent , Adult , Middle Aged , Aged , RNA, Viral/genetics , Hepatitis Antibodies/blood , Polymerase Chain Reaction , Hepatitis B, Chronic/epidemiology , Hepatitis B/complications , Brazil , Genotype
12.
Mem. Inst. Oswaldo Cruz ; 114: e190160, 2019. graf
Article in English | LILACS | ID: biblio-1040614

ABSTRACT

Human enteroviruses (EVs) are associated with a wide spectrum of human diseases. Here we report the complete genome sequences of one EV-C99 strain and one E29 strain obtained from children suffering from acute gastroenteritis, without symptoms of enteroviral syndromes. This is the first report of EV-C99 in South America, and the second E29 genome described worldwide. Continuous surveillance on EVs is vital to provide further understanding of the circulation of new or rare EV serotypes in the country. The present study also highlights the capacity of EVs to remain in silent circulation in populations.


Subject(s)
Humans , Male , Child, Preschool , Aged , RNA, Viral/genetics , Enterovirus B, Human/genetics , Enterovirus C, Human/genetics , Enterovirus Infections/virology , Phylogeny , Brazil , Enterovirus B, Human/isolation & purification , Enterovirus C, Human/isolation & purification , Feces/virology
13.
Mem. Inst. Oswaldo Cruz ; 114: e190198, 2019. tab, graf
Article in English | LILACS | ID: biblio-1040605

ABSTRACT

BACKGROUND In Brazil the implementation of the Sentinel Surveillance System of Influenza began in 2000. Central public health laboratories use reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for diagnosis of respiratory viruses, but this protocol identifies only specific targets, resulted in inconclusive diagnosis for many samples. Thus, high-throughput sequencing (HTS) would be complementary method in the identification of pathogens in inconclusive samples for RT-qPCR or other specific detection protocols. OBJECTIVES This study aimed to detect unidentified viruses using HTS approach in negative samples of nasopharynx/tracheal secretions by the standard RT-qPCR collected in the Federal District, Brazil. METHODS Nucleic acids were extracted from samples collected in winter period of 2016 and subjected to HTS. The results were confirmed by the multiplex PR21 RT-qPCR, which identifies 21 respiratory pathogens. FINDINGS The main viruses identified by HTS were of families Herpesviridae, Coronaviridae, Parvoviridae and Picornaviridae, with the emphasis on rhinoviruses. The presence of respiratory viruses in the samples was confirmed by the PR21 multiplex RT-qPCR. Coronavirus, enterovirus, bocavirus and rhinovirus were found by multiplex RT-qPCR as well as by HTS analyses. MAIN CONCLUSIONS Wide virus diversity was found by different methodologies and high frequency of rhinovirus occurrence was confirmed in population in winter, showing its relevance for public health.


Subject(s)
Humans , Parvoviridae/isolation & purification , Picornaviridae/isolation & purification , Trachea/virology , Nasopharynx/virology , Coronaviridae/isolation & purification , Herpesviridae/isolation & purification , Parvoviridae/classification , Parvoviridae/genetics , Picornaviridae/classification , Picornaviridae/genetics , DNA, Viral/genetics , RNA, Viral/genetics , Coronaviridae/classification , Coronaviridae/genetics , Reverse Transcriptase Polymerase Chain Reaction , Real-Time Polymerase Chain Reaction , Herpesviridae/classification , Herpesviridae/genetics
14.
Braz. j. infect. dis ; 22(5): 418-423, Sept.-Oct. 2018. tab, graf
Article in English | LILACS | ID: biblio-974233

ABSTRACT

ABSTRACT The Brazilian Public Health Service provides freely αPEG-IFN to treat patients infected with HCV. The primary goal of HCV therapy is the long-term elimination of HCV from the blood to reduce the risk of HCV associated complications and death. Patient viremia affects the treatment duration and response, thus influencing clinical decisions. We developed a high-throughput method to perform the quantification of RNA hepatitis C virus (HCV) virus load in plasma samples to monitor patients under treatment. The method is based on a duplex detection, in a one-step real-time RT-PCR assay and it has been validated according to the rules established by the official Brazilian regulatory agency (ANVISA). This new method was compared to a commercial kit (Cobas/Taqman HCV Test v2.0 - Roche), showing virus load results with significant correlation between them (p= 0,012) using commercial and clinical panels. In addition, 611 samples from patients treated with peguilated alfa-interferon (αPEG-IFN) from different regions of Brazil were analyzed. Our one-step real-time RT-PCR assay demonstrated good performance in viral load measurement and in treatment course monitoring, with acceptable sensitivity and specificity values.


Subject(s)
Humans , RNA, Viral/isolation & purification , Hepatitis C/virology , Hepacivirus/isolation & purification , Viral Load/methods , Real-Time Polymerase Chain Reaction/methods , Antiviral Agents/therapeutic use , Polyethylene Glycols/therapeutic use , Time Factors , Viremia , Recombinant Proteins/therapeutic use , Brazil , RNA, Viral/genetics , RNA, Viral/blood , Prospective Studies , Reproducibility of Results , Interferon-alpha/therapeutic use , Hepatitis C/drug therapy , Hepatitis C/blood , Hepacivirus/genetics , Genotyping Techniques , Genotype
15.
Rev. Soc. Bras. Med. Trop ; 51(4): 508-512, July-Aug. 2018. tab, graf
Article in English | LILACS | ID: biblio-1041479

ABSTRACT

Abstract INTRODUCTION This study reports the genotype prevalence of hepatitis C virus (HCV) in Pará, Brazil. METHODS: A retrospective cross-sectional study was conducted on 344 plasma samples sent to the Lacen-Pará for diagnostics by molecular techniques. RESULTS: HCV genotypes identified in the different regions of Pará were 1b (47.7%), 3 (23.3%), 1a (18%), and 2 (4.4%). Genotype 1 occurred in 41.6% of men and 30.8% of women in the 18-86-year-old group. CONCLUSIONS: Genotype 1 is the most predominant in Pará, which reinforces the idea of its relationship with late-diagnosed chronic infection.


Subject(s)
Humans , Male , Female , Adolescent , Adult , Aged , Aged, 80 and over , Young Adult , Hepacivirus/genetics , Hepatitis C, Chronic/epidemiology , Brazil/epidemiology , RNA, Viral/genetics , Prevalence , Cross-Sectional Studies , Retrospective Studies , Hepatitis C, Chronic/virology , Genotype , Middle Aged
16.
Braz. j. microbiol ; 49(1): 144-147, Jan.-Mar. 2018. tab, graf
Article in English | LILACS | ID: biblio-889187

ABSTRACT

ABSTRACT Many countries in the Americas have detected local transmission of multiple arboviruses that cause febrile illnesses. Therefore, laboratory testing has become an important tool for confirming the etiology of these diseases. The present study aimed to compare the sensitivity and specificity of three different Zika virus detection assays. One hundred serum samples from patients presenting with acute febrile symptoms were tested using a previously reported TaqMan® RT-qPCR assay. We used a SYBR® Green RT-qPCR and a conventional PCR methodologies to compare the results. Of the samples that were determined to be negative by the TaqMan® RT-qPCR assay, 100% (Kappa = 0.670) were also found to be negative by SYBR® Green RT-qPCR based on Tm comparison; however, 14% (Kappa = 0.035) were found to be positive by conventional PCR followed by agarose gel electrophoresis. The differences between the ZIKV strains circulating worldwide and the low viremia period can compromise diagnostic accuracy and thereby the accuracy of outbreak data. Therefore, improved assays are required to improve the diagnosis and surveillance of arbovirus.


Subject(s)
Humans , Polymerase Chain Reaction/methods , Zika Virus/isolation & purification , Zika Virus Infection/virology , RNA, Viral/genetics , Sensitivity and Specificity , Zika Virus/classification , Zika Virus/genetics , Zika Virus Infection/diagnosis
17.
Säo Paulo med. j ; 136(2): 129-135, Mar.-Apr. 2018. tab, graf
Article in English | LILACS | ID: biblio-904150

ABSTRACT

ABSTRACT BACKGROUND: Increasing genetic diversity of HIV-1 and emergence of drug-resistant mutations may reduce the efficacy of antiretroviral therapy and prophylaxis that are used to prevent mother-to-child transmission. The aim of this study was to assess the genetic diversity and prevalence of drug-resistant mutations among HIV-infected pregnant women. DESIGN AND SETTING: Cross-sectional study at an outpatient clinic for infectious diseases within gynecology and obstetrics. METHODS: This study evaluated the dynamics of HIV-1 subtypes and the prevalence of transmitted and acquired drug-resistant mutations among 38 HIV-infected pregnant women (20 previously exposed to antiretroviral therapy and 18 naive), in Ribeirão Preto (SP), Brazil, between 2010 and 2011. Genotyping was performed by means of molecular sequencing of the protease and reverse transcriptase regions of the HIV-1 pol gene. RESULTS: Subtype B was identified in 84.2% of the samples, recombinant forms between B and F in 7.9%, subtype F1 in 5.3% and the recombinant form K/F in 2.6%. No mutation associated with transmitted drug resistance was detected in the samples from the naive pregnant women, whereas mutations associated with acquired drug resistance were found in 35.0% of the pregnant women previously exposed to antiretroviral therapy. CONCLUSION: The results showed that subtype B predominated, while there was low prevalence of sequences with transmitted drug resistance.


Subject(s)
Humans , Female , Pregnancy , Pregnancy Complications, Infectious/virology , Genetic Variation , HIV Infections/virology , HIV-1/genetics , Anti-HIV Agents/therapeutic use , Drug Resistance, Viral/genetics , Mutation/genetics , Phylogeny , Pregnancy Complications, Infectious/drug therapy , Socioeconomic Factors , RNA, Viral/genetics , HIV Infections/drug therapy , Prevalence , Cross-Sectional Studies , HIV-1/drug effects , Genotype
18.
Mem. Inst. Oswaldo Cruz ; 113(1): 38-44, Jan. 2018. tab, graf
Article in English | LILACS | ID: biblio-894888

ABSTRACT

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.


Subject(s)
RNA, Viral/genetics , Genome, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , Zika Virus/genetics , Phylogeny , Viral Nonstructural Proteins , High-Throughput Nucleotide Sequencing
19.
Braz. j. med. biol. res ; 51(6): e7221, 2018. tab
Article in English | LILACS | ID: biblio-889096

ABSTRACT

Clinical manifestations of Zika, dengue, and chikungunya virus infections are very similar, making it difficult to reach a diagnosis based only on clinical grounds. In addition, there is an intense cross-reactivity between antibodies directed to Zika virus and other flaviviruses, and an accurate Zika diagnosis is best achieved by real-time RT-PCR. However, some real-time RT-PCR show better performance than others. To reach the best possible Zika diagnosis, the analytic sensitivity of some probe-based real-time RT-PCR amplifying Zika virus RNA was evaluated in spiked and clinical samples. We evaluated primers and probes to detect Zika virus, which had been published before, and tested sensitivity using serum spiked and patient samples by real-time RT-PCR. When tested against spiked samples, the previously described primers showed different sensitivity, with very similar results when samples from patients (serum and urine) were analyzed. Real-time RT-PCR designed to amplify Zika virus NS1 showed the best analytical sensitivity for all samples.


Subject(s)
Humans , RNA, Viral/genetics , Dengue/diagnosis , Chikungunya Fever/diagnosis , Zika Virus/genetics , Zika Virus Infection/diagnosis , Clinical Protocols , Sensitivity and Specificity , Reverse Transcriptase Polymerase Chain Reaction , Coinfection , Real-Time Polymerase Chain Reaction
20.
Mem. Inst. Oswaldo Cruz ; 113(4): e170208, 2018. tab, graf
Article in English | LILACS | ID: biblio-1040593

ABSTRACT

The lack of an experimental animal model for the study of dengue pathogenesis is a limiting factor for the development of vaccines and drugs. In previous studies, our group demonstrated the susceptibility of BALB/c mice to infection by dengue virus (DENV) 1 and 2, and the virus was successfully isolated in several organs. In this study, BALB/c mice were experimentally infected intravenously with DENV-4, and samples of their saliva were collected. Viral RNA extracted from the saliva samples was subjected to qRT-PCR, with a detection limit of 0.002 PFU/mL. The presence of DENV-4 viral RNA was detected in the saliva of two mice, presenting viral titers of 109 RNA/mL. The detection of DENV RNA via saliva sampling is not a common practice in dengue diagnosis, due to the lower detection rates in human patients. However, the results observed in this study seem to indicate that, as in humans, detection rates of DENV RNA in mouse saliva are also low, correlating the infection in both cases. This study reports the first DENV detection in the saliva of BALB/c immunocompetent mice experimentally infected with non-neuroadapted DENV-4.


Subject(s)
Humans , Animals , Male , Mice , Saliva/virology , Dengue Virus/isolation & purification , RNA, Viral/isolation & purification , RNA, Viral/genetics , Immunocompromised Host , Viral Load/genetics , Reverse Transcriptase Polymerase Chain Reaction , Dengue Virus/genetics , Disease Models, Animal , Mice, Inbred BALB C
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