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BACKGROUND The prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) has changed unevenly over time around the world. Although whole genome sequencing is the gold standard for virus characterisation, the discovery of alpha VOC causing spike gene target failure (SGTF) result, when tested using an reverse transcription real-time polymerase chain reaction (RT-qPCR) assay, has provided a simple tool for tracking the frequencies of variants. OBJECTIVES The aim of this study was to investigate if a multiplex RT-qPCR assay (BioM 4Plex VOC) could be used to detect SARS-CoV-2 and to perform a VOC screening test in a single reaction tube. Here, we present the multicentre study evaluating this assay. METHODS Twelve laboratories have participated in the multicentre study. The BioM 4Plex VOC was distributed to them with detailed instructions of how to perform the test. They were asked to test the BioM 4Plex VOC in parallel with their routine Commercial SARS-CoV-2 diagnostic assay. Additionally, they were requested to select SARS-CoV-2-positive samples with genome sequenced and lineage definition according to PANGO lineage classification. FINDINGS The BioM 4Plex VOC and commercial RT-PCR assay are equally effective in detecting SARS-CoV-2. Results revealed a specificity of 96.5-100% [95% confidence interval (CI)], a sensitivity of 99.8-100% (95% CI), and an accuracy of 99.8-100% (95% CI). A 99% concordance rate was found between results from the BioM 4Plex VOC and that from available genome sequencing data. MAIN CONCLUSIONS The BioM 4Plex VOC provides an effective solution to detect SARS-CoV-2 infections and screening for VOCs in a single reaction. It is a straightforward method to help us monitor the frequency and distribution of VOCs and develop strategies to better cope with the pandemics.

The Brazilian strategy to overcome the spread of COVID-19 has been particularly criticized due to the lack of a national coordinating effort and an appropriate testing program. Here, a successful approach to control the spread of COVID-19 transmission is described by the engagement of public (university and governance) and private sectors (hospitals and oil companies) in Macaé, state of Rio de Janeiro, Brazil, a city known as the National Oil Capital. In 2020 between the 17th and 38th epidemiological week, over two percent of the 206,728 citizens were subjected to symptom analysis and RT-qPCR testing by the Federal University of Rio de Janeiro, with positive individuals being notified up to 48 h after swab collection. Geocodification and spatial cluster analysis were used to limit COVID-19 spreading in Macaé. Within the first semester after the outbreak of COVID-19 in Brazil, Macaé recorded 1.8% of fatalities associated with COVID-19 up to the 38th epidemiological week, which was at least five times lower than the state capital (10.6%). Overall, considering the successful experience of this joint effort of private and public engagement in Macaé, our data suggest that the development of a similar strategy countrywise could have contributed to a better control of the COVID-19 spread in Brazil. Quarantine decree by the local administration, comprehensive molecular testing coupled to scientific analysis of COVID-19 spreading, prevented the catastrophic consequences of the pandemic as seen in other populous cities within the state of Rio de Janeiro and elsewhere in Brazil.

The emergence of SARS-CoV-2 variants with multiple shared mutations has been described to be more transmissible and could affect COVID-19 morbidity and mortality. Some of these variants, known as B.1.1.7 (originally described in the United Kingdom), B1.351 (originally described in South Africa), and P.1 (originally described in Brazil) have rapidly become dominant within their countries and require a vigorous public health response. Whole Genomic sequencing remains the gold standard method to identify the SARS-CoV-2 variant, even though this approach is laborious, time-consuming, and expensive. Here, we have developed a fast and simple SYBR green-based real-time RT-PCR assay that identifies a distinct signature affecting the Non-Structural Protein 6 (NSP6), a nine-nucleotides deletion leading to amino acid losses: Δ106S, Δ107G, and Δ108F. This NSP6 signature is present in the variants of concern (VOC) described above (B.1.1.7; B1.351; P.1) and has not been detected in other SARS-CoV-2 lineages. The new version has corrections made on the master mix and authorship.

Background Brazilian strategy to overcome the spread of COVID-19 has been particularly criticized due to the lack of a national coordinating effort and an appropriate testing program. Here, a successful approach to control the spread of COVID-19 transmission is described by the engagement of public (university and governance) and private sectors (hospitals and oil companies) in Macaé, state of Rio de Janeiro, Brazil, a city known as the National Oil Capital. Methods Until the 38th epidemiological week, over two percent of the 206,728 citizens were subjected to symptom analysis and massive RT-qPCR testing by the Federal University of Rio de Janeiro, with positive individuals being notified up to 48 hours after swab collection. Geocodification and spatial cluster analysis were used to limit COVID-19 spreading in Macaé. Findings: Within the first semester after the outbreak of COVID-19 in Brazil, Macaé recorded 1.8% of fatality associated to COVID-19 up to the 38th epidemiological week, which was at least five times lower than the state capital (10.92%). Overall, considering the successful experience of this joint effort of private and public engagement in Macaé, our data suggest that the development of a similar strategy country wise would have saved over 50,000 lives. Interpretation: Quarantine decree by the local government, molecular massive testing coupled to scientific analysis of COVID-19 spreading prevented the catastrophic consequences of the pandemic as seen in other populous cities within the state of Rio de Janeiro and elsewhere in Brazil.

Mayaro virus (MAYV) has historically been associated with sylvatic transmission; however, urban outbreaks have been reported in Brazil, including cases of co-detection with dengue virus (DENV). Therefore, we performed a molecular survey to investigate MAYV circulation and cocirculation with DENV within Goiania, a major city in Central-West Brazil. Among 375 subjects with arbovirus-like symptoms, 259 were positive for DENV and 26 for MAYV. Of these, 17 were coinfected with DENV-2, suggesting co-transmission of the viruses. The most common complaints at the time of inclusion were myalgia, headache, fever, arthralgia, retro-orbital pain, and skin rash. No specific symptoms were associated with MAYV when either detected alone or co-detected with DENV, compared to that when DENV was detected alone. Most MAYV-infected subjects were women with no recent travel history to rural/sylvatic areas. Phylogenetic reconstruction indicated that the MAYV identified in this study is closely related with a lineage observed in Peru, belonging to genotype D. Our results corroborate the growing circulation of MAYV in urban environments in Brazil and reinforce the need to implement laboratory diagnosis in the Unified Health System, considering that the clinical manifestations of Mayaro fever are similar to those of other arboviruses, particularly dengue. Furthermore, most cases occurred in association with DENV-2. Further phylogenetic studies are needed to evaluate MAYV, which has not been widely examined.

In response to the Zika epidemics in Brazil, the ZDC molecular assay (Bio-Manguinhos) was developed and registered at the Brazilian Regulatory Agency of Health Surveillance - ANVISA. The circulation of Zika (ZIKV) Dengue (DENV) and Chikungunya (CHIKV) viruses and their clinical similarities are challenges to correctly diagnose these viruses. The simultaneous detection of ZIKV, DENV and CHIKV is an important tool for diagnosis and surveillance. Here, we present the analytical and clinical performance evaluation of ZDC molecular assay (Bio-Manguinhos) at the public health laboratories three years after its registration at ANVISA. The clinical performance demonstrates the ZDC molecular assay (Bio-Manguinhos) has 100% sensitivity and 100% specificity to detect and discriminate ZIKV, CHIKV, and DENV from clinical plasma samples. The ZDC molecular assay (Bio-Manguinhos) results were highly reproducible and no cross-reactivity was seen during testing with a panel of other infectious agents. In conclusion, the ZDC molecular assay (Bio-Manguinhos) is an accurate and reliable tool to monitor Zika, dengue and chikungunya infections in countries like Brazil with simultaneous circulation of the three viruses.

Recent reports indicate that besides respiratory and systemic symptoms among coronavirus disease (COVID-19) patients, the disease has a wide spectrum of neurological manifestations (encephalitis, meningitis, myelitis, acute disseminated encephalomyelitis, metabolic and acute hemorrhagic necrotizing encephalopathy, cerebrovascular diseases, Guillain-Barré syndrome, polyneuritis cranialis, dysautonomia, and myopathies). The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can spread from the respiratory system to the central nervous system, using transneuronal and hematogenous mechanisms. Although not every COVID-19 patient will test positive for the virus in the cerebrospinal fluid exam, the appearance of neurological symptoms associated with SARS-CoV-2 infection reveals the importance of understanding the neurologic manifestations and capacity for neural invasion associated with the pathogen. These aspects are relevant for correct diagnosis and treatment, and for the potential development of vaccines. This review highlights the latest evidence of SARS-CoV-2 infection with a focus on neurological involvement and potential neuropathogenesis mechanisms.

Malaria can be transmitted by blood transfusion through donations collected from asymptomatic donors. Transfusion-transmitted malaria (TTM) poses a great risk to blood services worldwide. A good screening tool for Plasmodium spp. detection in blood banks must have a high sensitivity for prevention of TTM. However, in Brazilian blood banks, screening for malaria still relies on microscopy.In Brazil, screening for human immunodeficiency virus type 1 (HIV), RNA/DNA for hepatitis C (HCV) and hepatitis B (HBV) viruses is mandatory for every blood donation and uses nucleic acid amplification testing (NAT). The aim of this study was to evaluate the inclusion of an assay for malaria to identify Plasmodium sp. from total nucleic acid (TNA; DNA/RNA) by targeting the 18S rRNA gene of the parasite.Considering the limitations of microscopy and the wide availability of the Brazilian NAT platform in the screening of blood units for HIV, HCV, and HBV, a molecular diagnostic tool was validated for detection of Plasmodium sp. in blood banks; a pilot study showed that using this novel NAT assay could reduce the risk of TTM.The prototype HIV/HCV/HBV/malaria NAT assay was effective in detecting infected candidate donors and has good prospects to be applied in routine screening for preventing TTM.

Background The history of the development and implementation of the Brazilian nucleic acid testing (NAT) platform to detect and discriminate among human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) infections in blood donors is described here. The results for the sensitivity, reproducibility, and NAT yield of the platform since program implementation are provided. Study design and methods The Brazilian NAT HIV, HCV, and HBV kit was developed and evaluated with regard to analytical sensitivity, specificity, intralot and interlot reproducibility, interfering substances, and genotype and diagnostic sensitivity. Additionally, a sample of identified NAT-yield cases was characterized with regard to viral load. Results The 95% limits of detection for HIV, HCV, and HBV were 68.02, 102.35, and 9.08 IU/mL, respectively. All replicates were detected with reproducibility assays between the acceptable values. A total of 13,610,536 blood donors was screened from 2010 to 2016, and 63 HIV-yield cases and 28 HCV-yield cases were detected. Among 5,795,424 blood donors screened for HBV from 2014 to 2016, 42 yield cases were found. Conclusion The Brazilian NAT HIV, HCV, and HBV kit is an automated NAT system suitable for routine blood donor screening in a completely traceable process. The analytical sensitivity as well as the diagnostic sensitivity fulfilled all requirements set by the health ministry for blood donor screening. A significant number of transmission cases were prevented by the implementation of this important program.

Hepatitis C virus (HCV) infection is a worldwide health problem. Nowadays, direct-acting antiviral agents (DAAs) are the main treatment for HCV; however, the high level of virus variability leads to the development of resistance-associated variants (RAVs). Thus, assessing RAVs in infected patients is important for monitoring treatment efficacy. The aim of our study was to investigate the presence of naturally occurring resistance mutations in HCV NS3 and NS5 regions in treatment-naïve patients. Ninety-six anti-HCV positive serum samples from blood donors at the Center of Hematology and Hemotherapy of Santa Catarina State (HEMOSC) were collected retrospectively in 2013 and evaluated in this study. HCV 1a (37.9%), 1b (25.3%), and 3a (36.8%) subtypes were found. The frequency of patients with RAVs in our study was 6.9%. The HCV NS5b sequencing reveled 1 sample with L320F mutation and 4 samples with the C316N/R polymorphism. The analysis of the NS3 region revealed the D168A/G/T (3.45%), S122G (1.15%), and V55A (2.3%) mutations. All samples from genotype 3a (36.8%) presented the V170 I/V non-synonymous mutation. In conclusion, we have shown that mutations in NS3 and NS5b genes are present in Brazilian isolates from therapy-naïve HCV patients.

We describe a case of fetal death associated with a recent infection by Chikungunya virus (CHIKV) in a Brazilian pregnant woman (positive RT-PCR in blood and placenta). Zika virus (ZIKV) infection during pregnancy was also identified, based on a positive RT-PCR in a fetal kidney specimen. The maternal infection caused by the ZIKV was asymptomatic and the CHIKV infection had a classical clinical presentation. The fetus had no apparent anomalies, but her weight was between the 3rd and 10th percentile for the gestational age. This is the second case report of congenital arboviral co-infection and the first followed by antepartum fetal death. Keywords: Zika virus, Chikungunya, Dengue, Stillbirth, Arbovirus

The yellow fever virus (YFV) epidemic in Brazil is the largest in decades. The recent discovery of YFV in Brazilian Aedes species mosquitos highlights a need to monitor the risk of reestablishment of urban YFV transmission in the Americas. We use a suite of epidemiological, spatial, and genomic approaches to characterize YFV transmission. We show that the age and sex distribution of human cases is characteristic of sylvatic transmission. Analysis of YFV cases combined with genomes generated locally reveals an early phase of sylvatic YFV transmission and spatial expansion toward previously YFV-free areas, followed by a rise in viral spillover to humans in late 2016. Our results establish a framework for monitoring YFV transmission in real time that will contribute to a global strategy to eliminate future YFV epidemics., info:eu-repo/semantics/published

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 Keywords: HCV, viral load, Real Time PCR, monitoring patients

Objectives Feline immunodeficiency virus (FIV) is a lentivirus that induces AIDS-like disease in cats. Some of the antiretroviral drugs available to treat patients with HIV type 1 are used to treat FIV-infected cats; however, antiretroviral therapy (ART) is not used in cats as a long-term treatment. In this study, the effects of long-term ART were evaluated in domestic cats treated initially with the nucleoside transcriptase reverse inhibitor (NTRI) zidovudine (AZT) over a period ranging from 5–6 years, followed by a regimen of the NTRI lamivudine (3TC) plus AZT over 3 years. Methods Viral load, sequencing of pol (reverse transcriptase [RT]) region and CD4:CD8 lymphocyte ratio were evaluated during and after treatment. Untreated cats were evaluated as a control group. Results CD4:CD8 ratios were lower, and uncharacterized resistance mutations were found in the RT region in the group of treated cats. A slight increase in viral load was observed in some cats after discontinuing treatment. Conclusions and relevance The data strongly suggest that treated cats were resistant to therapy, and uncharacterized resistance mutations in the RT gene of FIV were selected for by AZT. Few studies have been conducted to evaluate the effect of long-term antiretroviral therapy in cats. To date, resistance mutations have not been described in vivo.

The history of the development and implementation of the Brazilian nucleic acid testing (NAT) platform to detect and discriminate among human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) infections in blood donors is described here. The results for the sensitivity, reproducibility, and NAT yield of the platform since program implementation are provided.The Brazilian NAT HIV, HCV, and HBV kit was developed and evaluated with regard to analytical sensitivity, specificity, intralot and interlot reproducibility, interfering substances, and genotype and diagnostic sensitivity. Additionally, a sample of identified NAT-yield cases was characterized with regard to viral load.The 95% limits of detection for HIV, HCV, and HBV were 68.02, 102.35, and 9.08 IU/mL, respectively. All replicates were detected with reproducibility assays between the acceptable values. A total of 13,610,536 blood donors was screened from 2010 to 2016, and 63 HIV-yield cases and 28 HCV-yield cases were detected. Among 5,795,424 blood donors screened for HBV from 2014 to 2016, 42 yield cases were found.The Brazilian NAT HIV, HCV, and HBV kit is an automated NAT system suitable for routine blood donor screening in a completely traceable process. The analytical sensitivity as well as the diagnostic sensitivity fulfilled all requirements set by the health ministry for blood donor screening. A significant number of transmission cases were prevented by the implementation of this important program.

A major concern associated with ZIKV infection is the increased incidence of microcephaly with frequent calcifications in infants born from infected mothers. To date, postmortem analysis of the central nervous system (CNS) in congenital infection is limited to individual reports or small series. We report a comprehensive neuropathological study in ten newborn babies infected with ZIKV during pregnancy, including the spinal cords and dorsal root ganglia (DRG), and also muscle, pituitaries, eye, systemic organs, and placentas. Using in situ hybridization (ISH) and electron microscopy, we investigated the role of direct viral infection in the pathogenesis of the lesions. Nine women had Zika symptoms between the 4th and 18th and one in the 28th gestational week. Two babies were born at 32, one at 34 and 36 weeks each and six at term. The cephalic perimeter was reduced in four, and normal or enlarged in six patients, although the brain weights were lower than expected. All had arthrogryposis, except the patient infected at 28 weeks gestation. We defined three patterns of CNS lesions, with different patterns of destructive, calcification, hypoplasia, and migration disturbances. Ventriculomegaly was severe in the first pattern due to midbrain damage with aqueduct stenosis/distortion. The second pattern had small brains and mild/moderate (ex-vacuo) ventriculomegaly. The third pattern, a well-formed brain with mild calcification, coincided with late infection. The absence of descending fibres resulted in hypoplastic basis pontis, pyramids, and cortico-spinal tracts. Spinal motor cell loss explained the intrauterine akinesia, arthrogryposis, and neurogenic muscle atrophy. DRG, dorsal nerve roots, and columns were normal. Lympho-histiocytic inflammation was mild. ISH showed meningeal, germinal matrix, and neocortical infection, consistent with neural progenitors death leading to proliferation and migration disorders. A secondary ischemic process may explain the destructive lesions. In conclusion, we characterized the destructive and malformative consequences of ZIKV in the nervous system, as reflected in the topography and severity of lesions, anatomic localization of the virus, and timing of infection during gestation. Our findings indicate a developmental vulnerability of the immature CNS, and shed light on possible mechanisms of brain injury of this newly recognized public health threat.

Zika virus tested in human brain organoids The pernicious and resilient Aedes mosquito is rapidly spreading Zika virus (ZIKV) through the Americas. ZIKV infection mostly causes mild disease, but in some patients, nervous system involvement is indicated. A particular worry is an observed correlation between infection of mothers in the first trimester of pregnancy and microcephaly in newborns. Garcez et al. tested the effects of ZIKV compared with dengue virus infection on human neural stem cells grown as organoids. ZIKV targeted the human brain cells, reduced their size and viability in vitro, and caused programmed cell death responses. Science , this issue p. 816

HIV-1 budding requires short peptide motifs in p6(Gag), known as late domains, that promote the release of infectious virions. The primary late domain of HIV-1 is a Pro-(Thr/Ser)-Ala-Pro (hereafter referred to as a PTAP) motif. This motif may be completely or partially duplicated. In this work we analyzed p6(Gag) sequences from 547 isolates from drug-naive patients and 213 isolates from patients failing HAART therapy. Complete duplications within PTAP were selected during HAART therapy in all HIV-1 subtypes analyzed: B (p = 0.0338), F1 (p = 0.0294), and C (p = 0.0001). Nevertheless, the patterns of these duplications were different; subtype C isolates accumulated longer duplications and displayed a higher frequency of duplications in both treated (54%) and drug-naive isolates (23%). Accumulation of PTAP duplications within subtypes B, F1, and C during therapy suggests a potential role of the duplications in antiretroviral drug resistance.

Summary Human immunodeficiency virus (HIV)-1 protease is a known target of CD8+ T cell responses, but it is the only HIV-1 protein in which no fully characterized HIV-1 protease CD4 epitopes have been identified to date. We investigated the recognition of HIV-1 protease by CD4+ T cells from 75 HIV-1-infected, protease inhibitor (PI)-treated patients, using the 5,6-carboxyfluorescein diacetate succinimidyl ester-based proliferation assay. In order to identify putative promiscuous CD4+ T cell epitopes, we used the TEPITOPE algorithm to scan the sequence of the HXB2 HIV-1 protease. Protease regions 4–23, 45–64 and 73–95 were identified; 32 sequence variants of the mentioned regions, encoding frequent PI-induced mutations and polymorphisms, were also tested. On average, each peptide bound to five of 15 tested common human leucocyte antigen D-related (HLA-DR) molecules. More than 80% of the patients displayed CD4+ as well as CD8+ T cell recognition of at least one of the protease peptides. All 35 peptides were recognized. The response was not associated with particular HLA-DR or -DQ alleles. Our results thus indicate that protease is a frequent target of CD4+ along with CD8+ proliferative T cell responses by the majority of HIV-1-infected patients under PI therapy. The frequent finding of matching CD4+ and CD8+ T cell responses to the same peptides may indicate that CD4+ T cells provide cognate T cell help for the maintenance of long-living protease-specific functional CD8+ T cells.

Our objective was to evaluate the in vitro functional profile of T cells from uninfected neonates born from HIV-1-infected pregnant women who controlled (G1) or not (G2) the virus replication. We demonstrated that the lymphoproliferation of T cell to polyclonal activators was higher in the G2 as compared with G1. Nevertheless, no detectable proliferative response was observed in response to HIV-1 antigens in both neonate groups. Cytokine dosage in the supernatants of these polyclonally activated T cell cultures demonstrated that, while IL-10 was the dominant cytokine produced in G1, Th17-related cytokines were significantly higher in G2 neonates. The higher Th17 phenotype tendency in G2 was related to high production of IL-23 by lipopolysaccharide-activated monocyte-derived dendritic cells from these neonates. Our results demonstrated immunological disorders in uninfected neonates born from viremic HIV-1-infected mothers that can help to explain why some of these children have elevated risk of clinical morbidity and mortality due to pathological hypersensitivity.

Objective: Because epidemiological data on circulating HIV subtypes among HIV-positive patients in the state of Paraná were not known until now, the aims of this study were to describe the genetic diversity profile of HIV-1 in treated patients in Paraná, Brazil, and report the differences in protease (PR) and reverse transcriptase (RT) mutations in HIV-1 subtypes. Patients and methods: A cross-sectional study was conducted from 2003 to 2006. Plasma viral RNA of 389 patients was extracted and PR and RT genes were polymerase chain reaction-amplified and sequenced. Sequences were subtyped and examined for antiretroviral resistance mutations. Data on gender of patient harboring the viruses and past history of antiretroviral treatment were also collected. Results: Most viruses were either subtype B (61.44%) or subtype C (20.57%). Subtype C and F were more frequent in women (p < 0.00). The prevalence of subtypes was similar over the years studied. The most frequent RT mutations in all subtypes were M184V and mutations at codons 215, 41, 103, 67, 219, and 190. Mutations 41L, 210W, 215YF, and 74V were significantly more prevalent on subtype B, and the mutation 106M was significantly more prevalent on subtype C. The most frequent major PI mutations in all subtypes occurred at codons 46, 82, and 90. PR mutations 32I, 46I, and 84V were significantly more prevalent on subtype B. The minor PI mutations on codons 36, 93, and 63 were more prevalent on subtypes F, C, and B, respectively. Conclusion: We concluded that the predominant strain of HIV-1 in Paraná is subtype B, followed by subtype C. Some mutations at PR and TR had subtype predominance in accordance with other authors’ report. Keywords: HIV-1 subtypes, genotype, antiretroviral, drug experienced patients

Hepatitis C (HCV) infection is a cause of chronic liver disease such as cirrhosis, carcinoma, or liver failure, and the current therapy is effective in only 50% of patients. Serine proteases, which are present in HCV, are the most studied class of proteolytic enzymes­, and are a primary target in the drug development field. In this paper, we describe the synthesis and biological studies of a novel­ class of peptide mimetic compounds as potential HCV serine protease inhibitors.

International audience; OBJECTIVE: This study aimed to evaluate the impact of pregnancy-related immune events on the HIV-1 replication and to analyze their relationship with the risk of vertical transmission. METHODS: The peripheral blood from HIV-1-infected pregnant women who controlled (G1) or not controlled (G2) their plasma viral load was drawn, and the plasma and the T cells were obtained. The T-cell cultures were activated in vitro with anti-CD3 and anti-CD28, and the proliferation and cytokine production profile were evaluated after 3 days of incubation. The in-vitro HIV-1 replication was measured in culture supernatants in the seventh day following stimulation. The cytokines were also analyzed in the plasma. RESULTS: Our results demonstrated a lower T-cell proliferation and a lower interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma production in polyclonally activated T-cell cultures from G1 patients, when compared with G2. Furthermore, high levels of interleukin-10 were produced both systemically and by activated T-cell cultures from G1 patients. Interestingly, the neutralization of endogenous interleukin-10 by anti-interleukin-10 monoclonal antibody elevated both the inflammatory cytokines' release and the HIV-1 replication in the polyclonally activated T-cell cultures from G1 patients. Additionally, the maternal antiretroviral treatment significantly enhanced the systemic interleukin-10 production. Finally, the higher systemic interleukin-10 levels were inversely correlated with vertical virus transmission risk. CONCLUSION: These results indicate that a high tendency of pregnant women to produce interleukin-10 can help them control the HIV-1 replication, and this can reduce the risk of vertical transmission. Furthermore, our data suggest a role for maternal antiretroviral treatment in enhancing this phenomenon.

The goal of this work was to compare the differences between human immunodeficiency virus type 1 (HIV-1) of B and F1 subtypes in the acquisition of major and minor protease inhibitor (PI)-associated resistance mutations and of other polymorphisms at the protease (PR) gene, through a cross sectional study. PR sequences from subtypes B and F1 isolates matched according to PI exposure time from Brazilian patients were included in this study. Sequences were separated in four groups: 24 and 90 from children and 141 and 99 from adults infected with isolates of subtypes F1 and B, respectively. For comparison, 211 subtype B and 79 subtype F1 PR sequences from drug-naïve individuals were included. Demographic and clinical data were similar among B- and F1-infected patients. In untreated patients, mutations L10V, K20R, and M36I were more frequent in subtype F1, while L63P, A71T, and V77I were more prevalent in subtype B. In treated patients, K20M, D30N, G73S, I84V, and L90M, were more prevalent in subtype B, and K20T and N88S were more prevalent in subtype F1. A higher proportion of subtype F1 than of subtype B strains containing other polymorphisms was observed. V82L mutation was present with increased frequency in isolates from children compared to isolates from adults infected with both subtypes. We could observe a faster resistance emergence in children than in adults, during treatment with protease inhibitors. This data provided evidence that, although rates of overall drug resistance do not differ between subtypes B and F1, the former accumulates resistance at higher proportion in specific amino acid positions of protease when compared to the latter.

The major human immunodeficiency virus type 1 subtype circulating in Brazil is B, followed by F and C. We have genotyped 882 samples from Brazilian patients for whom highly active antiretroviral therapy failed, and we found subtype B and the unique recombinant B/F1 forms circulating. Due to codon usage variation, there is a significantly lower incidence of the substitutions L210W, Q151M, and F116Y in subtype F1 isolates than in the subtype B counterparts.

This work evaluated HIV-1 subtypes from different geographic regions and phenotypic data from drug-naïve HIV-positive pregnant women from Mozambique. We analyzed 75 pol sequences from patients and the distribution of the subtypes in three regions of Mozambique and found that the majority of samples analyzed clustered with subtype C. In the northern region, multiple variants were found 5 (approximately 18%) subtype A, 3 (approximately 11%) subtype D and 2 (approximately 7.1%) mosaics (A/C/D and C/D), whereas 18 (64.3%) isolates were subtype C, from a total of 28 samples. Already in the southern region, only one (5%) isolate of 20 samples was subtype D, and the other 19 (95%) isolates were subtype C. All 27 (100%) isolates from the central region grouped within clade C. No primary resistance mutations to IP, NNRTI or NRTI were found. There was no evidence of phenotypic resistance in any of the isolates tested, suggesting that neither the polymorphism in the protease, nor the one found at codon 215 of the RT gene caused an increase in phenotypic resistance. This finding suggests that HAART regimens indicated by WHO will probably be successful in Mozambique.

Feline immunodeficiency virus (FIV) is the Lentivirus responsible for an immunodeficiency-like disease in domestic cats ( Felis catus ). FIV is divided into five phylogenetic subtypes (A, B, C, D, and E), based on genetic diversity. Knowledge of the geographical distribution of subtypes is relevant for understanding different disease progressions and for vaccine development. In this study, viral sequences of 26 infected cats from Rio de Janeiro, 8 undergoing treatment with zidovudine (AZT) for at least 5 years, were successfully amplified from blood specimens. gag capsid (CA), pol reverse transcriptase (RT), and env gp120 (V3-V4) regions were analyzed to determine subtypes and to evaluate potential mutations related to antiretroviral drug resistance among treated cats. Subtyping based on phylogenetic analysis was performed by the neighbor-joining and maximum likelihood methods. All of the sequences clustered with subtype B in the three regions, exhibiting low genetic variability. Additionally, we found evidence that the same virus is circulating in animals in close contact. The analysis of FIV RT sequences identified two new putative mutations related to drug resistance located in the RT “finger” domain, which has 60% identity to human immunodeficiency virus (HIV) sequence. Amino acid change K→R at codons 64 and 69 was found in 25% and 37.5% of the treated animals, respectively. These signatures were comparable to K65R and K70R thymidine-associated mutations found in the HIV-1 HXB2 counterpart. This finding strongly suggests a position correlation between the mutations found in FIV and the K65R and K70R substitutions from drug-resistant HIV-1 strains.

This work aimed to evaluate immune events in HIV-1-exposed uninfected neonates born from mothers who control (G1) or not (G2) the plasma viral load, using unexposed neonates as controls. Cord blood from each neonate was collected, plasma and mononuclear cells were separated and the lymphoproliferation and cytokine pattern were evaluated. The results demonstrated that the in vitro lymphoproliferation induced by polyclonal activators was higher in the G2 neonates. Nevertheless, no cell culture responded to poll synthetic HIV-1 envelope peptides. The cytokine dosage in the plasma and supernatants of polyclonally-activated cultures demonstrated that, while IL-4 and IL-10 were the dominant cytokines produced in G1 and control groups, IFN-gamma and TNF-alpha were significantly higher in G2 neonates. Systemic levels of IL-10 observed among the G1 neonates were higher in those born from anti-retroviral treated mothers. In summary, our results indicate an altered immune responsiveness in neonates exposed in utero to HIV and support the role of maternal anti-retroviral treatment to attenuate it.

OBJECTIVE To evaluate the impact of age on the proliferative response, cytokine profile and viral kinetics in AIDS patients treated successfully with antiretroviral drugs. METHODS Peripheral blood mononuclear cells (PBMC), CD4 cell-depleted PBMC or CD4 T cells from young adult and aged HIV-1-infected patients were activated in vitro with anti-CD3 with or without interleukin (IL)-2. Lymphoproliferation and cytokines were measured after 3 days and in-vitro HIV-1 replication after 7 days. RESULTS Both lymphoproliferation and cytokine [IL-1beta, tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma)] secretion were higher in younger than in older AIDS patients. In cultures of cells derived from aged patients and activated by anti-CD3, IFN-gamma production was severely damage and IL-10 production was much higher. Although IL-2 addition to activated PBMC elevated IFN-gamma secretion, IL-10 production remained elevated in the aged group. The depletion of CD4 T lymphocytes from these cultures dramatically reduced released IL-10 in the older group but did not alter significantly IFN-gamma production. Interestingly, higher IL-10 levels produced by CD4 T cells were related to lower in-vitro HIV-1 replication, and the blockade of this cytokine by anti-IL-10 monoclonal antibody enhanced virus replication. This effect may be correlated with elevated TNF-alpha secretion. Finally, impaired IFN-gamma secretion detected in activated CD4 T cells obtained from aged patients was not directly correlated with high IL-10 production. CONCLUSIONS Elevated IL-10 production by aged AIDS patients contributed considerably to control of HIV replication and to inhibition of TNF-alpha secretion but not to the reduced IFN-gamma production.

One of the main structural features of the mature HIV-1 virion is the matrix protein (p17). This partially globular protein presents four helixes centrally organized and a fifth one, H5, projecting away from the packed bundle of helixes. Comparison between solution and crystallographic data of p17 indicates a 6 A displacement of a short 3 10 helix and a partial unfolding of H5 in solution related to crystal. While the behavior of the 3 10 helix has been previously addressed to virion assembly, the relevance and origin of H5 partial unfolding is possibly related to the contacts between p17 and other viral elements, such as p24. In this context, we present a 40 ns conformational sampling of monomeric p17 using molecular dynamics simulations. The performed simulations presented a progressive conversion of the p17 crystallographic structure to the NMR conformation, suggesting that the biological form of this protein may have its C-terminal portion partially unfolded.

Potential topical retrovirucides or vaginal microbicides against human immunodeficiency virus type 1 (HIV-1) include nonnucleoside reverse transcriptase inhibitors (NNRTIs). To be successful, such agents have to be highly active against cell-free virions. In the present study, we developed a new real-time PCR-based assay to measure the natural endogenous reverse transcription (NERT) activity directly on intact HIV-1 particles in the presence of reverse transcriptase (RT) inhibitors. We further evaluated the permeability to nevirapine (NVP) and efavirenz (EFV) and their retention within nascent viral particles. We also demonstrated the NVP and EFV inhibitory effects on NERT activity and the impact of resistance mutations measured directly by this new strategy. Furthermore, the results showed a clear correlation between NERT activity and classical infectivity assays. The 50% inhibitory concentrations (IC 50 s) of NVP and EFV were demonstrated to be up to 100-fold higher for cell-free than for cell-associated virions, suggesting that cell-free virions are less permeable to these drugs. Our results suggest that NVP and EFV penetrate both the envelope and the capsid of HIV-1 particles and readily inactivate cell-free virions. However, the characteristics of these NNRTIs, such as lower permeability and lower retention during washing procedures, in cell-free virions reduce their efficacies as microbicides. Here, we demonstrate the usefulness of the NERT real-time PCR as an assay for screening novel antiretroviral compounds with unique mechanisms of action.

Resistance to antiretroviral drugs has been a major obstacle for long-lasting treatment of HIV-infected patients. The development of models to predict drug resistance is recognized as useful for helping the decision of the best therapy for each HIV+ individual. The aim of this study was to develop classifiers for predicting resistance to the HIV protease inhibitor lopinavir using a probabilistic neural network (PNN). The data were provided by the Molecular Virology Laboratory of the Health Sciences Center, Federal University of Rio de Janeiro (CCS-UFRJ/Brazil). Using bootstrap and stepwise techniques, ten features were selected by logistic regression (LR) to be used as inputs to the network. Bootstrap and cross-validation were used to define the smoothing parameter of the PNN networks. Four balanced models were designed and evaluated using a separate test set. The accuracies of the classifiers with the test set ranged from 0.89 to 0.94, and the area under the receiver operating characteristic (ROC) curve (AUC) ranged from 0.96 to 0.97. The sensitivity ranged from 0.94 to 1.00, and the specificity was between 0.88 and 0.92. Four classifiers showed performances very close to three existing expert-based interpretation systems, the HIVdb, the Rega and the ANRS algorithms, and to a k-Nearest Neighbor.