Amilcar Tanuri

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.

Protease sequences from HIV-1 group M subtypes A-H reveal distinct amino acid mutation patterns associated with protease resistance in protease inhibitor-naive individuals worldwide.

BackgroundAlthough numerous mutations that confer resistance to protease inhibitors (PRI) have been mapped for HIV-1 subtype B, little is known about such substitutions for the non-B viruses, which globally cause the most infections. ObjectivesTo determine the prevalence of PRI-associated mutations in PRI-naive individuals worldwide. DesignUsing the polymerase chain reaction, protease sequences were amplified from 301 individuals infected with HIV-1subtypes A (79), B (95), B’ (19), C (12), D (26), A/E (23), F (26), A/G (11), and H (3) and unclassifiable HIV-1 (7). Amplified DNA was directly sequenced and translated to amino acids to analyze PRI-associated major and accessory mutations. ResultsOf the 301 sequences, 85% contained at least one codon change giving substitution at 10, 20, 30, 36, 46, 63, 71, 77, or 82 associated with PRI resistance; the frequency of these substitutions was higher among non-B (91%) than B (75%) viruses (P < 0.0005). Of these, 25% carried dual and triple substitutions. Two major drug resistance-conferring mutations, either 20M or 30N, were identified in only three specimens, whereas drug resistance accessory mutations were found in 252 isolates. These mutations gave distinct prevalence patterns for subtype B, 63P (62%) > 77I (19%) > 10I/V/R (6%) = 36I (6%) = 71T/V (6%) > 20R (2%), and non-B strains, 36I (83%) > 63P (17%) > 10I/V/R (13%) > 20R(10%) > 77I (2%), which differed statistically at positions 20, 36, 63, 71, and 77. ConclusionsThe high prevalence of PRI-associated substitutions represent natural polymorphisms occurring in PRI-naive patients infected with HIV-1 strains of subtypes A−H. The significance of distinct mutation patterns identified for subtype B and non-B strains warrants further clinical evaluation. A global HIV-1 protease database is fundamental for the investigation of novel PRI.

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.

Congenital Brain Abnormalities and Zika Virus: What the Radiologist Can Expect to See Prenatally and Postnatally

Purpose To document the imaging findings associated with congenital Zika virus infection as found in the Instituto de Pesquisa in Campina Grande State Paraiba (IPESQ) in northeastern Brazil, where the congenital infection has been particularly severe. Materials and Methods From June 2015 to May 2016, 438 patients were referred to the IPESQ for rash occurring during pregnancy or for suspected fetal central nervous system abnormality. Patients who underwent imaging at IPESQ were included, as well as those with documented Zika virus infection in fluid or tissue (n = 17, confirmed infection cohort) or those with brain findings suspicious for Zika virus infection, with intracranial calcifications (n = 28, presumed infection cohort). Imaging examinations included 12 fetal magnetic resonance (MR) examinations, 42 postnatal brain computed tomographic examinations, and 11 postnatal brain MR examinations. Images were reviewed by four radiologists, with final opinion achieved by means of consensus. Results Brain abnormalities seen in confirmed (n = 17) and presumed (n = 28) congenital Zika virus infections were similar, with ventriculomegaly in 16 of 17 (94%) and 27 of 28 (96%) infections, respectively; abnormalities of the corpus callosum in 16 of 17 (94%) and 22 of 28 (78%) infections, respectively; and cortical migrational abnormalities in 16 of 17 (94%) and 28 of 28 (100%) infections, respectively. Although most fetuses underwent at least one examination that showed head circumference below the 5th percentile, head circumference could be normal in the presence of severe ventriculomegaly (seen in three fetuses). Intracranial calcifications were most commonly seen at the gray matter-white matter junction, in 15 of 17 (88%) and 28 of 28 (100%) confirmed and presumed infections, respectively. The basal ganglia and/or thalamus were also commonly involved with calcifications in 11 of 17 (65%) and 18 of 28 (64%) infections, respectively. The skull frequently had a collapsed appearance with overlapping sutures and redundant skin folds and, occasionally, intracranial herniation of orbital fat and clot in the confluence of sinuses. Conclusion The spectrum of findings associated with congenital Zika virus infection in the IPESQ in northeastern Brazil is illustrated to aid the radiologist in identifying Zika virus infection at imaging. (©) RSNA, 2016 Online supplemental material is available for this article.

Geographic and Temporal Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance: An Individual-Patient- and Sequence-Level Meta-Analysis

Background Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. Methods and Findings We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05–1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06–1.25), North America (OR = 1.19; 95% CI: 1.12–1.26), Europe (OR = 1.07; 95% CI: 1.01–1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12–1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92–1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling. Conclusions Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen.

HIV-1 subtype C dissemination in southern Brazil

Objectives:To describe the molecular and epidemiological profile of HIV-1 in patients followed at the University Hospital of Rio Grande, Brazil. Design and methods:A cross-sectional study was conducted from September to December 2002. Plasma viral RNA of 85 patients was extracted and protease and reverse transcriptase genes were polymerase chain reaction-amplified and sequenced. Sequences were subtyped and examined to antiretroviral resistance mutations. Laboratory data and past history of antiretroviral treatment were also collected. Results:Most viruses were either subtype B (42%) or subtype C (45%). No risk behaviour, sexual orientation or laboratory parameter was associated with any specific subtype, but subtype C tended to be more frequently found in women (P = 0.06). The prevalence of subtype C has increased over the HIV/AIDS epidemic, accounting for almost 60% of cases diagnosed in 2002. Intra-subtype genetic distances were smaller in subtype C than in subtype B, suggesting a more recent introduction of the former in the epidemic. Of patients under treatment, 60% had at least one antiretroviral drug resistance mutation, but no mutation was specifically associated with any HIV-1 subtype. Only one resistance mutation each was found in drug-naive patients with subtypes B and C. Conclusion:Despite the fact that subtype C appeared in southern Brazil more recently than subtype B, it is now the predominant strain in Rio Grande. The epidemic spread of subtype C could be taking place in Brazil, and possibly in south America, a phenomenon similar to that seen in other countries where this subtype is now totally dominant.

Dual and Recombinant Infections: An Integral Part of the HIV-1 Epidemic in Brazil

We systematically evaluated multiple and recombinant infections in an HIV-infected population selected for vaccine trials. Seventy-nine HIV-1 infected persons in a clinical cohort study in Rio de Janeiro, Brazil, were evaluated for 1 year. A combination of molecular screening assays and DNA sequencing showed 3 dual infections (3.8%), 6 recombinant infections (7.6%), and 70 (88.6%) infections involving single viral subtypes. In the three dual infections, we identified HIV-1 subtypes F and B, F and D, and B and D; in contrast, the single and recombinant infections involved only HIV-1 subtypes B and F. The recombinants had five distinct B/F mosaic patterns: Bgag-p17/Bgag -p24/Fpol/Benv , Fgag-p17/Bgag -p24/Fpol/Fenv , Bgag-p17/B-Fgag -p24/Fpol/Fenv , Bgag-p17/B-Fgag -p24/Fpol/Benv , and Fgag-p17/B-Fgag -p24/Fpol/Fenv . No association was found between dual or recombinant infections and demographic or clinical variables. These findings indicate that dual and recombinant infections are emerging as an integral part of the HIV/AIDS epidemic in Brazil and emphasize the heterogenous character of epidemics emerging in countries where multiple viral subtypes coexist.

Early Diagnosis of Human Immunodeficiency Virus in Infants Using Polymerase Chain Reaction on Dried Blood Spots in Botswana's National Program for Prevention of Mother-to-Child Transmission

Background: Botswana has high antenatal human immunodeficiency virus (HIV) prevalence (33.4%). The public health system provides free services for prevention of mother to child transmission of HIV (PMTCT) and antiretroviral therapy, which can reduce vertical HIV transmission from 35% to <5%. Infant HIV diagnosis is challenging in resource-limited settings, and HIV prevalence among HIV-exposed infants in Botswana is unknown. Dried blood spot (DBS) polymerase chain reaction (PCR) provides a feasible method to assess PMTCT programs and identify HIV-infected children. Methods: We trained staff in 15 clinics and a hospital to obtain DBS on HIV-exposed infants age 6 weeks to 17 months receiving routine care. Samples were sent to the national HIV reference laboratory. Roche Amplicor 1.5 DNA PCR testing was performed. Results: Between June–December 2005, 1931 HIV-exposed infants age 6 weeks to 17 months were tested for HIV, of whom 136 (7.0%) were HIV infected. Among infants ≤8 weeks old, 27 of 544 (5.0%) were HIV infected. Among infants tested in clinics (primarily during routine health visits), 65 of 1376 (4.7%) were infected; among infants tested in the hospital, 71 of 555 (12.8%) were infected. Conclusions: Collection and testing of DBS was successfully integrated into routine infant care in the public health system. HIV prevalence among infants in the Botswana PMTCT program is low. National expansion of infant DBS PCR in Botswana is planned.

Identification of Single and Dual Infections with Distinct Subtypes of Human Immunodeficiency Virus Type 1 by Using Restriction Fragment Length Polymorphism Analysis

The simultaneous presence of multiple HIV-1 subtypes has become common in communities with the growth of the pandemic. As a consequence, the potentiality for an increased frequency of HIV-1 mixed infections caused by viruses of distinct subtypes could be expected. Thus, there is a need to estimate the prevalence and geographic distribution of infections caused by viruses of a singular subtype as well as coinfections caused by two or more HIV-1 strains of distinct subtypes. To address this need, we have developed a genetic method based on restriction fragment length polymorphism (RFLP) to screen for these two types of infections within infected populations. In this assay, restriction enzymes may be used to predict the phylogroup of HIV-1 infected samples. A 297 bp pol fragment spanning the entire viral protease gene and a 311 bp fragment of the p24 gag region are used for this analysis. The viral regions are amplified by nested PCR using DNA templates from uncultured peripheral blood mononuclear cells (PBMC) or virus culture. Classification of HIV-1 strains to well defined subtypes B, D, F, and A/C is done by sequential endonuclease restriction analysis of a PCR amplified-protease gene followed by analysis of the p24 gag region. The electrophoretic migration patterns visualized by ethidium bromide staining or by radiolabeled probes are then determined on a 10% polyacrylamide gel. In infections caused by viruses of a singular subtype, a single restriction pattern is detected, whereas in multiple infections caused by two or more viral strains of different subtypes, the combination of different digestion patterns are observed in infected individuals. Using this methodology we have screened for genetic variations in HIV-1 proviral DNA from thirty-three Brazilian samples. Our RFLP procedure classified thirty-two samples as single infections caused by viruses of subtypes B (31) and F (1), and one sample as dual infection caused by distinct viral strains. Subsequent sequence and phylogenetic analysis of the viral protease gene in lymphocytes of all these patients confirmed our RFLP findings in single infections, and demonstrated the existence of two distinct HIV-1 strains of subtypes F and D in a patient which lymphocytes showed the simultaneous presence of two different digestion patterns. As up to now, single infections caused by subtype D variants were not identified in Brazil, our data provide the first evidence of subtype D HIV-1 in this country. Because sequencing of HIV proviral DNA is not particularly practical for large-scale molecular epidemiological studies, the protease/gag-based RFLP screening method will be useful to predict the phylogroup of HIV-1, and to identify multiple infections caused by HIV-1 strains of distinct subtypes. We believe that this information is crucial for both evaluation of the HIV-1/AIDS pandemic and intervention strategies.

Discordances between Interpretation Algorithms for Genotypic Resistance to Protease and Reverse Transcriptase Inhibitors of Human Immunodeficiency Virus Are Subtype Dependent

ABSTRACT The major limitation of drug resistance genotyping for human immunodeficiency virus remains the interpretation of the results. We evaluated the concordance in predicting therapy response between four different interpretation algorithms (Rega 6.3, HIVDB-08/04, ANRS [07/04], and VGI 8.0). Sequences were gathered through a worldwide effort to establish a database of non-B subtype sequences, and demographic and clinical information about the patients was gathered. The most concordant results were found for nonnucleoside reverse transcriptase (RT) inhibitors (93%), followed by protease inhibitors (84%) and nucleoside RT inhibitor (NRTIs) (76%). For therapy-naive patients, for nelfinavir, especially for subtypes C and G, the discordances were driven mainly by the protease (PRO) mutational pattern 82I/V + 63P + 36I/V for subtype C and 82I + 63P + 36I + 20I for subtype G. Subtype F displayed more discordances for ritonavir in untreated patients due to the combined presence of PRO 20R and 10I/V. In therapy-experienced patients, subtype G displayed a lot of discordances for saquinavir and indinavir due to mutational patterns involving PRO 90 M and 82I. Subtype F had more discordance for nelfinavir attributable to the presence of PRO 88S and 82A + 54V. For the NRTIs lamivudine and emtricitabine, CRF01_AE had more discordances than subtype B due to the presence of RT mutational patterns 65R + 115 M and 118I + 215Y, respectively. Overall, the different algorithms agreed well on the level of resistance scored, but some of the discordances could be attributed to specific (subtype-dependent) combinations of mutations. It is not yet known whether therapy response is subtype dependent, but the advice given to clinicians based on a genotypic interpretation algorithm differs according to the subtype.