Rodrigo M. Brindeiro

Zika virus impairs growth in human neurospheres and brain organoids.

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 Zika virus infection in cell culture models damages human neural stem cells to limit growth and cause cell death. Since the emergence of Zika virus (ZIKV), reports of microcephaly have increased considerably in Brazil; however, causality between the viral epidemic and malformations in fetal brains needs further confirmation. We examined the effects of ZIKV infection in human neural stem cells growing as neurospheres and brain organoids. Using immunocytochemistry and electron microscopy, we showed that ZIKV targets human brain cells, reducing their viability and growth as neurospheres and brain organoids. These results suggest that ZIKV abrogates neurogenesis during human brain development.

Congenital Zika Virus Infection: Beyond Neonatal Microcephaly

Importance Recent studies have reported an increase in the number of fetuses and neonates with microcephaly whose mothers were infected with the Zika virus (ZIKV) during pregnancy. To our knowledge, most reports to date have focused on select aspects of the maternal or fetal infection and fetal effects. Objective To describe the prenatal evolution and perinatal outcomes of 11 neonates who had developmental abnormalities and neurological damage associated with ZIKV infection in Brazil. Design, Setting, and Participants We observed 11 infants with congenital ZIKV infection from gestation to 6 months in the state of Paraíba, Brazil. Ten of 11 women included in this study presented with symptoms of ZIKV infection during the first half of pregnancy, and all 11 had laboratory evidence of the infection in several tissues by serology or polymerase chain reaction. Brain damage was confirmed through intrauterine ultrasonography and was complemented by magnetic resonance imaging. Histopathological analysis was performed on the placenta and brain tissue from infants who died. The ZIKV genome was investigated in several tissues and sequenced for further phylogenetic analysis. Main Outcomes and Measures Description of the major lesions caused by ZIKV congenital infection. Results Of the 11 infants, 7 (63.6%) were female, and the median (SD) maternal age at delivery was 25 (6) years. Three of 11 neonates died, giving a perinatal mortality rate of 27.3%. The median (SD) cephalic perimeter at birth was 31 (3) cm, a value lower than the limit to consider a microcephaly case. In all patients, neurological impairments were identified, including microcephaly, a reduction in cerebral volume, ventriculomegaly, cerebellar hypoplasia, lissencephaly with hydrocephalus, and fetal akinesia deformation sequence (ie, arthrogryposis). Results of limited testing for other causes of microcephaly, such as genetic disorders and viral and bacterial infections, were negative, and the ZIKV genome was found in both maternal and neonatal tissues (eg, amniotic fluid, cord blood, placenta, and brain). Phylogenetic analyses showed an intrahost virus variation with some polymorphisms in envelope genes associated with different tissues. Conclusions and Relevance Combined findings from clinical, laboratory, imaging, and pathological examinations provided a more complete picture of the severe damage and developmental abnormalities caused by ZIKV infection than has been previously reported. The term congenital Zika syndrome is preferable to refer to these cases, as microcephaly is just one of the clinical signs of this congenital malformation disorder.

HIV-1 subtypes among blood donors from Rio de Janeiro, Brazil.

The prevalence of HIV infection in Brazil is one of the highest in the world. In addition, transfusion-transmitted HIV accounts for 2.3% of all AIDS cases in Brazil. The objective of this study was to evaluate genetic diversity and distribution of HIV-1 strains circulating in the blood-donor population. We characterized 43 seropositive blood units collected from volunteer blood donors residing throughout Rio de Janeiro, Brazil. Viral RNA was extracted from plasma, reverse transcribed, and amplified by nested polymerase chain reaction (PCR) using HIV group M degenerate primers. Genetic heterogeneity was evaluated by direct automated cycle sequencing of the following gene fragments: gag p24 (399 bp), env C2V3 (345 bp), and env gp41 (369 bp). Phylogenetic analysis reflected the complexity of the Brazilian HIV epidemic: the majority of specimens, 33 of 43 (76.7%) were subtype B, and 6 of 43 (14%) were subtype F. The remaining 4 samples (9.3%) involved potential mosaic viruses of subtypes B and F or B and D. This survey is the first to document HIV-1 genetic variation in the Brazilian blood-donor population.

Synonymous Genetic Polymorphisms within Brazilian Human Immunodeficiency Virus Type 1 Subtypes May Influence Mutational Routes to Drug Resistance

BACKGROUND Most published data on antiretroviral-drug resistance is generated from in vitro or in vivo studies of subtype B virus. However, this subtype is associated with <10% of HIV infections worldwide, and it is essential to explore subtype-specific determinants of drug resistance. One potential cause of the differences between subtypes is the synonymous codon usage at key resistance positions. METHODS We investigated the nucleotide sequences at drug resistance-related sites, for all major Brazilian subtypes (B, C, and F1) of human immunodeficiency virus type 1 (HIV-1) group M. RESULTS We identified a change at positions 151 and 210 of the reverse-transcriptase region in subtype F1, such that the emergence of these key nucleoside/nucleotide analogue resistance mutations required an extra nucleotide change in subtype F1, compared with subtypes B and C. The clinical significance of position 210 was confirmed within a large Brazilian database, in which we identified a lower prevalence of the L210W mutation in subtype F1 virus, compared with subtype B virus, in patients matched for thymidine-analogue experience. An inverse relationship between the L210W and K70R mutations was also observed. CONCLUSIONS The findings of the present study illustrate an important mechanism by which a subtype may determine genetic routes to resistance, with implications for treatment strategies for populations infected with HIV-1 subtype F.

Low Accumulation of L90M in Protease from Subtype F HIV-1 with Resistance to Protease Inhibitors Is Caused by the L89M Polymorphism

BACKGROUND This work evaluates the role of subtype F human immunodeficiency virus type 1 (HIV-1) protease (PR) substitutions L89M and L90M in viral replication and resistance to PR inhibitors (PIs). METHODS Subtype B and F PR genes were subjected to site-directed mutagenesis, to create and reverse the methionine at positions 89 and 90. Viruses were re-created in cell culture, and their replicative capacity was assessed by fitness assay. Generated viruses were also phenotyped for PI resistance. RESULTS The subtype F clone (89M90L) showed a replicative capacity comparable to that of the PI-susceptible subtype B clone (89L90L) and was more fit than the L89M mutated subtype B clone (89M90L). Both 89M90M subtype B and F clones presented the lowest fitness s values. The L89M mutation impacted phenotypic resistance to all PIs in half of the subtype F isolates but not in the subtype B isolates. Subtype F isolates presented a phenotypic profile similar to that of subtype B isolates when the M89L mutation was introduced. CONCLUSION The L89M mutation in subtype F viruses is a high genetic barrier to the accumulation of the L90M resistance mutation and can function as a resistance mutation, depending on the presence of other polymorphisms in the subtype F PR backbone.

Impact of nelfinavir resistance mutations on in vitro phenotype, fitness, and replication capacity of human immunodeficiency virus type 1 with subtype B and C proteases.

ABSTRACT Human immunodeficiency virus type 1 subtype B and C proteases were manipulated to contain 90M, 88D, or 89L, and their in vitro biological properties were studied. We showed that D30N has significantly more impact in subtype C than in subtype B counterparts, accounting for the reported low prevalence of this mutation in patients failing nelfinavir-based regimens.

Genotypic and phenotypic evidence of different drug-resistance mutation patterns between B and non-B subtype isolates of human immunodeficiency virus type 1 found in Brazilian patients failing HAART.

We have investigated the phenotypic and genotypic susceptibility of 14 HIV-1 strains isolated from individuals failing HAART therapy to protease inhibitors (PI). Proviral and plasma viral pol gene fragment were amplified, sequenced and subtyped. Nine samples clustered with protease subtype B reference strains and the remaining samples were classified as non-B subtype corresponding to subtype F (n=4) and subtype A (n=1). Although all patients were treated with similar PI drug regimen, the non-B subtype isolates did not present the L90M and I84V mutations and used mainly G48V and V82A/F to achieve drug resistance. A strong cross-resistance phenotype among all four PI was associated with the mutation L90M in the subtype-B isolates, and with G48V and V82A/F in the non-B counterparts. This observation revealed that the non-B viruses tested had specific genotypic characteristics contrasting with the subtype-B isolates.

Prevalence of human immunodeficiency virus drug resistance mutations and subtypes in drug-naive, infected individuals in the army health service of Rio de Janeiro, Brazil.

ABSTRACT The prevalence of mutations that confer resistance to antiretroviral drugs was examined in 56 drug-naive, human immunodeficiency virus type 1 (HIV-1)-infected individuals from the Army Health Service in Rio de Janeiro, Brazil. No primary protease inhibitor mutations were found, but secondary mutations were observed in 51.2% of the samples. Fourteen percent of the viruses had reverse transcriptase inhibitor-associated mutations. Comparative analysis of protease secondary mutations from four different time periods in drug-naive patients in the city of Rio de Janeiro has indicated constant rates for particular mutations. Changes in CD4 cell counts and HIV viral load over time in subtype B- and non-B-infected drug-naive patients were not significantly different.

Testing Genotypic and Phenotypic Resistance in Human Immunodeficiency Virus Type 1 Isolates of Clade B and Other Clades from Children Failing Antiretroviral Therapy

ABSTRACT The emergence of resistance to antiretroviral drugs is a major obstacle to the successful treatment of human immunodeficiency virus type 1 (HIV-1)-infected patients. In this work, we correlate clinical and virological trends such as viral load (VL) and CD4 counts to genotypic and phenotypic antiretroviral (ARV) resistance profiles of HIV-1 isolates from the B and non-B subtypes found in vertically infected children failing ARV therapy. Plasma samples were collected from 52 vertically HIV-1-infected children failing different ARV therapies. Samples underwent HIV-1 pol sequencing and phenotyping and were clustered into subtypes by phylogenetic analysis. Clinical data from each patient were analyzed together with the resistance (genotypic and phenotypic) data obtained. Thirty-five samples were from subtype B, 10 samples were non-B (subtypes A, C, and F), and 7 were mosaic samples. There was no significant difference concerning treatment data between B and non-B clades. Prevalence of known drug resistance mutations revealed slightly significant differences among B and non-B subtypes: L10I, 21 and 64%, K20R, 13 and 43%, M36I, 34 and 100%, L63P, 76 and 36%, A71V/T, 24 and 0%, and V77I, 32 and 0%, respectively, in the protease (0.0001 ≤ P ≤ 0.0886), and D67N, 38 and 8%, K70R, 33 and 0%, R211K, 49 and 85%, and K219Q/E, 31 and 0%, respectively, in the reverse transcriptase (0.0256 ≤ P ≤ 0.0704). Significant differences were found only in secondary resistance mutations and did not reflect significant phenotypic variation between clade B and non-B.

Altered immunological reactivity in HIV-1-exposed uninfected neonates

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.