West Nile Virus-Inclusive Single-Cell RNA Sequencing Reveals Heterogeneity in the Type I Interferon Response within Single Cells.

West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus of global importance. Neuroinvasive WNV infection results in encephalitis and can lead to prolonged neurological impairment or death. Type I interferon (IFN-I) is crucial for promoting antiviral defenses through the induction of antiviral effectors, which function to restrict viral replication and spread. However, our understanding of the antiviral response to WNV infection is mostly derived from analysis of bulk cell populations. It is becoming increasingly apparent that substantial heterogeneity in cellular processes exists among individual cells, even within a seemingly homogenous cell population. Here, we present WNV-inclusive single-cell RNA sequencing (scRNA-seq), an approach to examine the transcriptional variation and viral RNA burden across single cells. We observed that only a few cells within the bulk population displayed robust transcription of IFN-ß mRNA, and this did not appear to depend on viral RNA abundance within the same cell. Furthermore, we observed considerable transcriptional heterogeneity in the IFN-I response, with genes displaying high unimodal and bimodal expression patterns. Broadly, IFN-stimulated genes negatively correlated with viral RNA abundance, corresponding with a precipitous decline in expression in cells with high viral RNA levels. Altogether, we demonstrated the feasibility and utility of WNV-inclusive scRNA-seq as a high-throughput technique for single-cell transcriptomics and WNV RNA detection. This approach can be implemented in other models to provide insights into the cellular features of protective immunity and identify novel therapeutic targets.IMPORTANCE West Nile virus (WNV) is a clinically relevant pathogen responsible for recurrent epidemics of neuroinvasive disease. Type I interferon is essential for promoting an antiviral response against WNV infection; however, it is unclear how heterogeneity in the antiviral response at the single-cell level impacts viral control. Specifically, conventional approaches lack the ability to distinguish differences across cells with varying viral abundance. The significance of our research is to demonstrate a new technique for studying WNV infection at the single-cell level. We discovered extensive variation in antiviral gene expression and viral abundance across cells. This protocol can be applied to primary cells or in vivo models to better understand the underlying cellular heterogeneity following WNV infection for the development of targeted therapeutic strategies.

Zika Virus Antagonizes Type I Interferon Responses during Infection of Human Dendritic Cells.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that is causally linked to severe neonatal birth defects, including microcephaly, and is associated with Guillain-Barre syndrome in adults. Dendritic cells (DCs) are an important cell type during infection by multiple mosquito-borne flaviviruses, including dengue virus, West Nile virus, Japanese encephalitis virus, and yellow fever virus. Despite this, the interplay between ZIKV and DCs remains poorly defined. Here, we found human DCs supported productive infection by a contemporary Puerto Rican isolate with considerable variability in viral replication, but not viral binding, between DCs from different donors. Historic isolates from Africa and Asia also infected DCs with distinct viral replication kinetics between strains. African lineage viruses displayed more rapid replication kinetics and infection magnitude as compared to Asian lineage viruses, and uniquely induced cell death. Infection of DCs with both contemporary and historic ZIKV isolates led to minimal up-regulation of T cell co-stimulatory and MHC molecules, along with limited secretion of inflammatory cytokines. Inhibition of type I interferon (IFN) protein translation was observed during ZIKV infection, despite strong induction at the RNA transcript level and up-regulation of other host antiviral proteins. Treatment of human DCs with RIG-I agonist potently restricted ZIKV replication, while type I IFN had only modest effects. Mechanistically, we found all strains of ZIKV antagonized type I IFN-mediated phosphorylation of STAT1 and STAT2. Combined, our findings show that ZIKV subverts DC immunogenicity during infection, in part through evasion of type I IFN responses, but that the RLR signaling pathway is still capable of inducing an antiviral state, and therefore may serve as an antiviral therapeutic target.

The amphibian peptide Yodha is virucidal for Zika and dengue viruses.

Zika virus (ZIKV) has emerged as a serious health threat in the Americas and the Caribbean. ZIKV is transmitted by the bite of an infected mosquito, sexual contact, and blood transfusion. ZIKV can also be transmitted to the developing fetus in utero, in some cases resulting in spontaneous abortion, fetal brain abnormalities, and microcephaly. In adults, ZIKV infection has been correlated with Guillain-Barre syndrome. Despite the public health threat posed by ZIKV, neither a vaccine nor antiviral drugs for use in humans are currently available. We have identified an amphibian host defense peptide, Yodha, which has potent virucidal activity against ZIKV. It acts directly on the virus and destroys Zika virus particles within 5 min of exposure. The Yodha peptide was effective against the Asian, African, and South American Zika virus strains and has the potential to be developed as an antiviral therapeutic in the fight against Zika virus. The peptide was also effective against all four dengue virus serotypes. Thus, Yodha peptide could potentially be developed as a pan-therapeutic for Zika and dengue viruses.

Cross-Reactive Dengue Virus Antibodies Augment Zika Virus Infection of Human Placental Macrophages.

Zika virus (ZIKV), which emerged in regions endemic to dengue virus (DENV), is vertically transmitted and results in adverse pregnancy outcomes. Antibodies to DENV can cross-react with ZIKV, but whether these antibodies influence ZIKV vertical transmission remains unclear. Here, we find that DENV antibodies increase ZIKV infection of placental macrophages (Hofbauer cells [HCs]) from 10% to over 80% and enhance infection of human placental explants. ZIKV-anti-DENV antibody complexes increase viral binding and entry into HCs but also result in blunted type I interferon, pro-inflammatory cytokine, and antiviral responses. Additionally, ZIKV infection of HCs and human placental explants is enhanced in an immunoglobulin G subclass-dependent manner, and targeting FcRn reduces ZIKV replication in human placental explants. Collectively, these findings support a role for pre-existing DENV antibodies in enhancement of ZIKV infection of select placental cell types and indicate that pre-existing immunity to DENV should be considered when addressing ZIKV vertical transmission.

Postnatal Zika virus infection is associated with persistent abnormalities in brain structure, function, and behavior in infant macaques.

The Zika virus (ZIKV) epidemic is associated with fetal brain lesions and other serious birth defects classified as congenital ZIKV syndrome. Postnatal ZIKV infection in infants and children has been reported; however, data on brain anatomy, function, and behavioral outcomes following infection are absent. We show that postnatal ZIKV infection of infant rhesus macaques (RMs) results in persistent structural and functional alterations of the central nervous system compared to age-matched controls. We demonstrate ZIKV lymphoid tropism and neurotropism in infant RMs and histopathologic abnormalities in the peripheral and central nervous systems including inflammatory infiltrates, astrogliosis, and Wallerian degeneration. Structural and resting-state functional magnetic resonance imaging (MRI/rs-fMRI) show persistent enlargement of lateral ventricles, maturational changes in specific brain regions, and altered functional connectivity (FC) between brain areas involved in emotional behavior and arousal functions, including weakened amygdala-hippocampal connectivity in two of two ZIKV-infected infant RMs several months after clearance of ZIKV RNA from peripheral blood. ZIKV infection also results in distinct alterations in the species-typical emotional reactivity to acute stress, which were predicted by the weak amygdala-hippocampal FC. We demonstrate that postnatal ZIKV infection of infants in this model affects neurodevelopment, suggesting that long-term clinical monitoring of pediatric cases is warranted.

Zika Virus Infects Human Placental Macrophages.

The recent Zika virus (ZIKV) outbreak in Brazil has been directly linked to increased cases of microcephaly in newborns. Current evidence indicates that ZIKV is transmitted vertically from mother to fetus. However, the mechanism of intrauterine transmission and the cell types involved remain unknown. We demonstrate that the contemporary ZIKV strain PRVABC59 (PR 2015) infects and replicates in primary human placental macrophages, called Hofbauer cells, and to a lesser extent in cytotrophoblasts, isolated from villous tissue of full-term placentae. Viral replication coincides with induction of type I interferon (IFN), pro-inflammatory cytokines, and antiviral gene expression, but with minimal cell death. Our results suggest a mechanism for intrauterine transmission in which ZIKV gains access to the fetal compartment by directly infecting placental cells and disrupting the placental barrier.

Single-Cell Analysis of the Plasmablast Response to Vibrio cholerae Demonstrates Expansion of Cross-Reactive Memory B Cells.

We characterized the acute B cell response in adults with cholera by analyzing the repertoire, specificity, and functional characteristics of 138 monoclonal antibodies (MAbs) generated from single-cell-sorted plasmablasts. We found that the cholera-induced responses were characterized by high levels of somatic hypermutation and large clonal expansions. A majority of the expansions targeted cholera toxin (CT) or lipopolysaccharide (LPS). Using a novel proteomics approach, we were able to identify sialidase as another major antigen targeted by the antibody response to Vibrio cholerae infection. Antitoxin MAbs targeted both the A and B subunits, and most were also potent neutralizers of enterotoxigenic Escherichia coli heat-labile toxin. LPS-specific MAbs uniformly targeted the O-specific polysaccharide, with no detectable responses to either the core or the lipid moiety of LPS. Interestingly, the LPS-specific antibodies varied widely in serotype specificity and functional characteristics. One participant infected with the Ogawa serotype produced highly mutated LPS-specific antibodies that preferentially bound the previously circulating Inaba serotype. This demonstrates durable memory against a polysaccharide antigen presented at the mucosal surface and provides a mechanism for the long-term, partial heterotypic immunity seen following cholera. IMPORTANCE: Cholera is a diarrheal disease that results in significant mortality. While oral cholera vaccines are beneficial, they do not achieve equivalent protection compared to infection with Vibrio cholerae Although antibodies likely mediate protection, the mechanisms of immunity following cholera are poorly understood, and a detailed understanding of antibody responses to cholera is of significance for human health. In this study, we characterized the human response to cholera at the single-plasmablast, monoclonal antibody level. Although this approach has not been widely applied to the study of human bacterial infection, we were able to uncover the basis of cross-reactivity between different V. cholerae serotypes and the likely impact of prior enterotoxigenic Escherichia coli exposure on the response to cholera, as well as identify novel antigenic targets. In addition to improving our understanding of the repertoire and function of the antibody response to cholera in humans, this study has implications for future cholera vaccination efforts.