Correction to: International prospective observational cohort study of Zika in infants and pregnancy (ZIP study): study protocol.

Following publication of the original article [1], the author mentioned that two additional NIH staff were involved in the development of the protocol who did not receive recognition in the Acknowledgments section in their published article.

International prospective observational cohort study of Zika in infants and pregnancy (ZIP study): study protocol.

BACKGROUND: Until recently, Zika virus (ZIKV) infections were considered mild and self-limiting. Since 2015, they have been associated with an increase in microcephaly and other birth defects in newborns. While this association has been observed in case reports and epidemiological studies, the nature and extent of the relationship between ZIKV and adverse pregnancy and pediatric health outcomes is not well understood. With the unique opportunity to prospectively explore the full spectrum of issues related to ZIKV exposure during pregnancy, we undertook a multi-country, prospective cohort study to evaluate the association between ZIKV and pregnancy, neonatal, and infant outcomes. METHODS: At research sites in ZIKV endemic regions of Brazil (4 sites), Colombia, Guatemala, Nicaragua, Puerto Rico (2 sites), and Peru, up to 10,000 pregnant women will be recruited and consented in the first and early second trimesters of pregnancy and then followed through delivery up to 6 weeks post-partum; their infants will be followed until at least 1 year of age. Pregnant women with symptomatic ZIKV infection confirmed by presence of ZIKV RNA and/or IgM for ZIKV will also be enrolled, regardless of gestational age. Participants will be tested monthly for ZIKV infection; additional demographic, physical, laboratory and environmental data will be collected to assess the potential interaction of these variables with ZIKV infection. Delivery outcomes and detailed infant assessments, including physical and neurological outcomes, will be obtained. DISCUSSION: With the emergence of ZIKV in the Americas and its association with adverse pregnancy outcomes in this region, a much better understanding of the spectrum of clinical outcomes associated with exposure to ZIKV during pregnancy is needed. This cohort study will provide information about maternal, fetal, and infant outcomes related to ZIKV infection, including congenital ZIKV syndrome, and manifestations that are not detectable at birth but may appear during the first year of life. In addition, the flexibility of the study design has provided an opportunity to modify study parameters in real time to provide rigorous research data to answer the most critical questions about the impact of congenital ZIKV exposure. TRIAL REGISTRATION: NCT02856984 . Registered August 5, 2016. Retrospectively registered.

Tetherin Antagonism by HIV-1 Group M Nef Proteins.

Although Nef is the viral gene product used by most simian immunodeficiency viruses to overcome restriction by tetherin, this activity was acquired by the Vpu protein of HIV-1 group M due to the absence of sequences in human tetherin that confer susceptibility to Nef. Thus, it is widely accepted that HIV-1 group M uses Vpu instead of Nef to counteract tetherin. Challenging this paradigm, we identified Nef alleles of HIV-1 group M isolates with significant activity against human tetherin. These Nef proteins promoted virus release and tetherin downmodulation from the cell surface and, in the context of vpu-deleted HIV-1 recombinants, enhanced virus replication and resistance to antibody-dependent cell-mediated cytotoxicity (ADCC). Further analysis revealed that the Vpu proteins from several of these viruses lack antitetherin activity, suggesting that under certain circumstances, HIV-1 group M Nef may acquire the ability to counteract tetherin to compensate for the loss of this function by Vpu. These observations illustrate the remarkable plasticity of HIV-1 in overcoming restriction by tetherin and challenge the prevailing view that all HIV-1 group M isolates use Vpu to counteract tetherin. IMPORTANCE Most viruses of HIV-1 group M, the main group of HIV-1 responsible for the global AIDS pandemic, use their Vpu proteins to overcome restriction by tetherin (BST-2 or CD317), which is a transmembrane protein that inhibits virus release from infected cells. Here we show that the Nef proteins of certain HIV-1 group M isolates can acquire the ability to counteract tetherin. These results challenge the current paradigm that HIV-1 group M exclusively uses Vpu to counteract tetherin and underscore the importance of tetherin antagonism for efficient viral replication.

Comparison of Antibody-Dependent Cell-Mediated Cytotoxicity and Virus Neutralization by HIV-1 Env-Specific Monoclonal Antibodies.

UNLABELLED: Although antibodies to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein have been studied extensively for their ability to block viral infectivity, little data are currently available on nonneutralizing functions of these antibodies, such as their ability to eliminate virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 Env-specific antibodies of diverse specificities, including potent broadly neutralizing and nonneutralizing antibodies, were therefore tested for ADCC against cells infected with a lab-adapted HIV-1 isolate (HIV-1NL4-3), a primary HIV-1 isolate (HIV-1JR-FL), and a simian-human immunodeficiency virus (SHIV) adapted for pathogenic infection of rhesus macaques (SHIVAD8-EO). In accordance with the sensitivity of these viruses to neutralization, HIV-1NL4-3-infected cells were considerably more sensitive to ADCC, both in terms of the number of antibodies and magnitude of responses, than cells infected with HIV-1JR-FL or SHIVAD8-EO ADCC activity generally correlated with antibody binding to Env on the surfaces of virus-infected cells and with viral neutralization; however, neutralization was not always predictive of ADCC, as instances of ADCC in the absence of detectable neutralization, and vice versa, were observed. These results reveal incomplete overlap in the specificities of antibodies that mediate these antiviral activities and provide insights into the relationship between ADCC and neutralization important for the development of antibody-based vaccines and therapies for combating HIV-1 infection. IMPORTANCE: This study provides fundamental insights into the relationship between antibody-dependent cell-mediated cytotoxicity (ADCC) and virus neutralization that may help to guide the development of antibody-based vaccines and immunotherapies for the prevention and treatment of HIV-1 infection.

Envelope Glycoprotein Internalization Protects Human and Simian Immunodeficiency Virus-Infected Cells from Antibody-Dependent Cell-Mediated Cytotoxicity.

UNLABELLED: The cytoplasmic tails of human and simian immunodeficiency virus (HIV and SIV, respectively) envelope glycoproteins contain a highly conserved, membrane-proximal endocytosis motif that prevents the accumulation of Env on the surface of infected cells prior to virus assembly. Using an assay designed to measure the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC), we show that substitutions in this motif increase the susceptibility of HIV-1- and SIV-infected cells to ADCC in a manner that directly correlates with elevated Env levels on the surface of virus-infected cells. In the case of HIV-1, this effect is additive with a deletion in vpu recently shown to enhance the susceptibility of HIV-1-infected cells to ADCC as a result of tetherin-mediated retention of budding virions on the cell surface. These results reveal a previously unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from antibody responses by regulating the amount of Env present on the cell surface. IMPORTANCE: This study reveals an unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from elimination by Env-specific antibodies. Thus, strategies designed to interfere with this mechanism of Env internalization may improve the efficacy of antibody-based vaccines and antiretroviral therapies designed to enhance the immunological control of HIV-1 replication in chronically infected individuals.

Tethering viral restriction to signal transduction.

Tetherin serves as an innate sensor of viral infection in addition to its role in inhibiting virus release from infected cells. In this issue, Galão et al. (2014) provide important insights into the mechanism of virus-induced signal transduction by tetherin.

Tetherin antagonism by Vpu protects HIV-infected cells from antibody-dependent cell-mediated cytotoxicity.

Tetherin is an IFN-inducible transmembrane protein that inhibits the detachment of enveloped viruses from infected cells. HIV-1 overcomes this restriction factor by expressing HIV-1 viral protein U (Vpu), which down-regulates and degrades tetherin. We report that mutations in Vpu that impair tetherin antagonism increase the susceptibility of HIV-infected cells to antibody-dependent cell-mediated cytotoxicity (ADCC), and conversely that RNAi knockdown of tetherin, but not other cellular proteins down-modulated by Vpu, decreases the susceptibility of HIV-infected cells to ADCC. These results reveal that Vpu protects HIV-infected cells from ADCC as a function of its ability to counteract tetherin. By serving as link between innate and adaptive immunity, the antiviral activity of tetherin may be augmented by virus-specific antibodies, and hence much greater than previously appreciated.

Retroelements versus APOBEC3 family members: No great escape from the magnificent seven.

Retroelements comprise a large and successful family of transposable genetic elements that, through intensive infiltration, have shaped the genomes of humans and other mammals over millions of years. In fact, retrotransposons now account for approximately 45% of the human genome. Because of their genomic mobility called retrotransposition, some retroelements can cause genetic diseases; such retrotransposition events occur not only in germ cells but also in somatic cells, posing a threat to genomic stability throughout all cellular populations. In response, mammals have developed intrinsic immunity mechanisms that provide resistance against the deleterious effects of retrotransposition. Among these, seven members of the APOBEC3 (A3) family of cytidine deaminases serve as highly active, intrinsic, antiretroviral host factors. Certain A3 proteins effectively counteract infections of retroviruses such as HIV-1, as well as those of other virus families, while also blocking the transposition of retroelements. Based on their preferential expression in the germ cells, in which retrotransposons may be active, it is likely that A3 proteins were acquired through mammalian evolution primarily to inhibit retrotransposition and thereby maintain genomic stability in these cells. This review summarizes the recent advances in our understanding of the interplay between the retroelements currently active in the human genome and the anti-retroelement A3 proteins.

Sites of action of HIV-1 Vpu in BST-2/tetherin downregulation.

The interferon-inducible host restriction factor bone marrow stromal antigen 2 (BST-2/tetherin) blocks the release of human immunodeficiency virus type 1 (HIV-1) by directly cross-linking virions to the membrane of infected cells. This antiviral effect is counteracted by the HIV-1 accessory protein viral protein U (Vpu) through mechanisms that remain unclear. Accumulating evidence suggests that Vpu antagonizes BST-2 by removing it from the plasma membrane; however, neither the cellular sites of interaction nor the effector mechanisms that result in the downregulation of BST-2 cell-surface expression have been fully determined. Based on current evidence regarding the subcellular localization of Vpu and BST-2 and the latter's trafficking defects induced by their interaction, three models have been proposed. In the first, Vpu is hypothesized to block the traffic of newly synthesized BST-2 towards the cell surface by retaining it in the biosynthetic/secretory compartment. The second model suggests that Vpu sequesters BST-2 within intracellular compartments corresponding to recycling endosomes and the trans-Golgi network by blocking its recycling after endocytosis. In the third model, we and others have proposed that Vpu directly internalizes BST-2 from the plasma membrane and induces its enhanced endolysosomal trafficking and degradation. As for its intracellular fate, the viral antagonism of BST-2 is likely dependent on the intracellular sequestration, or the proteasomal/lysosomal degradation of the restriction factor. This review summarizes the current advances in our understanding of the cellular pathways and sites of action of Vpu in the downregulation of cell-surface BST-2.

Structural Basis for the Antiviral Activity of BST-2/Tetherin and Its Viral Antagonism.

The interferon-inducible host restriction factor bone marrow stromal antigen 2 (BST-2/tetherin) blocks the release of HIV-1 and other enveloped viruses. In turn, these viruses have evolved specific antagonists to counteract this host antiviral molecule, such as the HIV-1 protein Vpu. BST-2 is a type II transmembrane protein with an unusual topology consisting of an N-terminal cytoplasmic tail (CT) followed by a single transmembrane (TM) domain, a coiled-coil extracellular (EC) domain, and a glycosylphosphatidylinositol (GPI) anchor at the C terminus. We and others showed that BST-2 restricts enveloped virus release by bridging the host and virion membranes with its two opposing membrane anchors and that deletion of either one completely abrogates antiviral activity. The EC domain also shows conserved structural properties that are required for antiviral function. It contains several destabilizing amino acids that confer the molecule with conformational flexibility to sustain the protein's function as a virion tether, and three conserved cysteine residues that mediate homodimerization of BST-2, as well as acting as a molecular ruler that separates the membrane anchors. Conversely, the efficient release of virions is promoted by the HIV-1 Vpu protein and other viral antagonists. Our group and others provided evidence from mutational analyses indicating that Vpu antagonism of BST-2-mediated viral restriction requires a highly specific interaction of their mutual TM domains. This interpretation is further supported and expanded by the findings of the latest structural modeling studies showing that critical amino acids in a conserved helical face of these TM domains are required for Vpu-BST-2 interaction and antagonism. In this review, we summarize the current advances in our understanding of the structural basis for BST-2 antiviral function as well as BST-2-specific viral antagonism.

Immunofluorescence assay in India for confirmation of HIV-1 infection using a T-cell line infected with defective HIV-1.

BACKGROUND: In India, the enzyme immunoassay (EIA)/rapid test is used for screening and confirmatory antibody testing of HIV infection, and all HIV reactive samples are further confirmed by two other rapid tests working on different principles; however, Western blotting (WB) and immunofluorescence (IF) assays are not routinely performed in this country. METHODS: A total of 2104 sera from Indian subjects were tested for the presence of HIV-1 antibody using EIA/rapid tests, according to the guidelines of the National AIDS Control Organization of India, and were also subjected to IF test using L-2 cells persistently infected with defective HIV-1. WB and a nested reverse transcriptase polymerase chain reaction (RT-PCR) were performed on discrepant samples. RESULTS: IF results were 100% concordant with EIA/rapid tests for 212 HIV-1-positive samples and 1889 HIV-1-negative samples. Interestingly, three (0.14%) samples negative by EIA/rapid tests were weakly or moderately positive (1+/2+) by IF test. All three of these samples were confirmed to be negative by WB (reactive with Gag/Pol, but not with Env), but positive by RT-PCR with primers targeting the C2-V5 fragment of the env gene. These three samples were from individuals who voluntarily reported for HIV testing because of high-risk practices, and they may have been at an early stage of HIV infection. CONCLUSIONS: These results confirm that the IF test using L-2 cells is a sensitive and specific alternative method for confirmation of HIV-1 infection and could be included in the diagnostic algorithm in reference laboratories in developing countries.

High systemic levels of interleukin-10, interleukin-22 and C-reactive protein in Indian patients are associated with low in vitro replication of HIV-1 subtype C viruses.

BACKGROUND: HIV-1 subtype C (HIV-1C) accounts for almost 50% of all HIV-1 infections worldwide and predominates in countries with the highest case-loads globally. Functional studies suggest that HIV-1C is unique in its biological properties, and there are contradicting reports about its replicative characteristics. The present study was conducted to evaluate whether the host cytokine environment modulates the in vitro replication capacity of HIV-1C viruses. METHODS: A small subset of HIV-1C isolates showing efficient replication in peripheral blood mononuclear cells (PBMC) is described, and the association of in vitro replication capacity with disease progression markers and the host cytokine response was evaluated. Viruses were isolated from patient samples, and the corresponding in vitro growth kinetics were determined by monitoring for p24 production. Genotype, phenotype and co-receptor usage were determined for all isolates, while clinical category, CD4 cell counts and viral loads were recorded for all patients. Plasmatic concentrations of cytokines and, acute-phase response, and microbial translocation markers were determined; and the effect of cytokine treatment on in vitro replication rates was also measured. RESULTS: We identified a small number of viral isolates showing high in vitro replication capacity in healthy-donor PBMC. HIV-1C usage of CXCR4 co-receptor was rare; therefore, it did not account for the differences in replication potential observed. There was also no correlation between the in vitro replication capacity of HIV-1C isolates and patients' disease status. Efficient virus growth was significantly associated with low interleukin-10 (IL-10), interleukin-22 (IL-22), and C-reactive protein (CRP) levels in plasma (p < .0001). In vitro, pretreatment of virus cultures with IL-10 and CRP resulted in a significant reduction of virus production, whereas IL-22, which lacks action on immune cells appears to mediate its anti-HIV effect through interaction with both IL-10 and CRP, and its own protective effect on mucosal membranes. CONCLUSIONS: These results indicate that high systemic levels of IL-10, CRP and IL-22 in HIV-1C-infected Indian patients are associated with low viral replication in vitro, and that the former two have direct inhibitory effects whereas the latter acts through downstream mechanisms that remain uncertain.

Human and mosquito infections by dengue viruses during and after epidemics in a dengue-endemic region of Colombia.

We conducted a study in a dengue-endemic area of Colombia to evaluate the dynamics of transmission of dengue viruses during and after epidemics. Information was simultaneously gathered about occurrence of infection in humans and mosquitoes every three months in four cities with endemic transmission. Viral isolation was confirmed in 6.7% of the persons and most were asymptomatic. Adult mosquito and larvae house indexes were not found associated with increased burden of disease. The only entomologic indicator related to dengue infection in humans was the pooled infection rate of mosquitoes. Aedes aegypti infection rates showed significant differences between the epidemic (10.68, 95% confidence interval [CI] = 7.04-15.62) and after epidemic periods of the study (6.15, 95% CI = 3.46-10.19). In addition, Ae. albopictus were also infected with dengue viruses. Increases in mosquito infection rates were associated with increases in human infection rates in the following trimester.