No abstract available

For decades, human infections with Zika virus (ZIKV), a mosquito-transmitted flavivirus, were sporadic, associated with mild disease, and went underreported since symptoms were similar to other acute febrile diseases. Recent reports of severe disease associated with ZIKV have greatly heightened awareness. It is anticipated that ZIKV will continue to spread in the Americas and globally where competent Aedes mosquito vectors are found. Dengue virus (DENV), the most common mosquito-transmitted human flavivirus, is both well-established and the source of outbreaks in areas of recent ZIKV introduction. DENV and ZIKV are closely related, resulting in substantial antigenic overlap. Through antibody-dependent enhancement (ADE), anti-DENV antibodies can enhance the infectivity of DENV for certain classes of immune cells, causing increased viral production that correlates with severe disease outcomes. Similarly, ZIKV has been shown to undergo ADE in response to antibodies generated by other flaviviruses. We tested the neutralizing and enhancing potential of well-characterized broadly neutralizing human anti-DENV monoclonal antibodies (HMAbs) and human DENV immune sera against ZIKV using neutralization and ADE assays. We show that anti-DENV HMAbs, cross-react, do not neutralize, and greatly enhance ZIKV infection in vitro. DENV immune sera had varying degrees of neutralization against ZIKV and similarly enhanced ZIKV infection. Our results suggest that pre-existing DENV immunity may enhance ZIKV infection in vivo and may lead to increased disease severity. Understanding the interplay between ZIKV and DENV will be critical in informing public health responses and will be particularly valuable for ZIKV and DENV vaccine design and implementation strategies.

Zika virus (ZIKV) infection is typically characterized by a mild self-limiting disease presenting with fever, rash, myalgia and arthralgia and severe fetal complications during pregnancy such as microcephaly, subcortical calcifications and arthrogyropsis. Virus-induced arthralgia due to perturbed osteoblast function has been described for other arboviruses. In case of ZIKV infection, the role of osteoblasts in ZIKV pathogenesis and bone related pathology remains unknown. Here, we study the effect of ZIKV infection on osteoblast differentiation, maturation and function by quantifying activity and gene expression of key biomarkers, using human bone marrow-derived mesenchymal stromal cells (MSCs, osteoblast precursors). MSCs were induced to differentiate into osteoblasts and we found that osteoblasts were highly susceptible to ZIKV infection. While infection did not cause a cytopathic effect, a significant reduction of key osteogenic markers such as ALP, RUNX2, calcium contents and increased expression of IL6 in ZIKV-infected MSCs implicated a delay in osteoblast development and maturation, as compared to uninfected controls. In conclusion, we have developed and characterized a new in vitro model to study the role of bone development in ZIKV pathogenesis, which will help to identify possible new targets for developing therapeutic and preventive measures.

Zika virus (ZIKV) is primarily transmitted through mosquito bites and sexual contact, and vertical transmission of ZIKV has also been observed in humans. In addition, ZIKV infection via unknown transmission routes has been frequently reported in clinical settings. However, whether ZIKV can be transmitted via aerosol routes remains unknown. In this study, we demonstrated that aerosolized ZIKV is fully infectious in vitro and in vivo. Remarkably, intratracheal (i.t.) inoculation with aerosolized ZIKV led to rapid viremia and viral secretion in saliva, as well as robust humoral and innate immune responses in guinea pigs. Transcriptome analysis further revealed that the expression of genes related to viral processes, biological regulation and the immune response was significantly changed. Together, our results confirm that aerosolized ZIKV can result in systemic infection and induce both innate and adaptive immune responses in guinea pigs, highlighting the possibility of ZIKV transmission via aerosols.

Zika-infected patients can have eye involvement ranging from mild conjunctivitis to severe chorioretinal lesions, however the possible long-term sequelae of infection and timeline to recovery remain unknown. Here we describe the partial recovery of chorioretinal lesions in an immunocompetent patient diagnosed with bilateral posterior uveitis associated with Zika infection and show that some lesions resolved with focal atrophy evident as pigmentary changes on funduscopy. To better understand the progression of the lesions and correlate the changes in fundus imaging with local viral load, immune responses, and retinal damage, we developed a symptomatic mouse model of ocular Zika virus infection. Imaging of the fundus revealed multiple hypopigmentary patches indicative of chorioretinal degeneration as well as thinning of the retina that mirror the lesions in patients. Microscopically, the virus primarily infected the optic nerve, retinal ganglion cells, and inner nuclear layer cells, showing thinning of the outer plexiform layer. During acute infection, the eyes showed retinal layer disorganization, retinitis, vitritis, and focal choroiditis, with mild cellular infiltration and increased expression of tumor necrosis factor, interferon-γ, granzyme B, and perforin. Focal areas of gliosis and retinal degeneration persisted 60 dpi. The model recapitulates features of ZIKA infections in patients and should help elucidate the mechanisms underlying the damage to the eyes and aid in the development of effective therapeutics.

In urban settings, chikungunya, Zika, and dengue viruses are transmitted by Aedes aegypti mosquitoes. Since these viruses co-circulate in several regions, coinfection in humans and vectors may occur, and human coinfections have been frequently reported. Yet, little is known about the molecular aspects of virus interactions within hosts and how they contribute to arbovirus transmission dynamics. We have previously shown that Aedes aegypti exposed to chikungunya and Zika viruses in the same blood meal can become coinfected and transmit both viruses simultaneously. However, mosquitoes may also become coinfected by multiple, sequential feeds on single infected hosts. Therefore, we tested whether sequential infection with chikungunya and Zika viruses impacts mosquito vector competence. We exposed Ae. aegypti mosquitoes first to one virus and 7 days later to the other virus and compared infection, dissemination, and transmission rates between sequentially and single infected groups. We found that coinfection rates were high after sequential exposure and that mosquitoes were able to co-transmit both viruses. Surprisingly, chikungunya virus coinfection enhanced Zika virus transmission 7 days after the second blood meal. Our data demonstrate heterologous arbovirus synergism within mosquitoes, by unknown mechanisms, leading to enhancement of transmission under certain conditions.

Currently, Zika virus (ZIKV) is spreading across the world and no ZIKV infection cases have ever been reported in China. Here, we aimed to determine whether ZIKV infection exists in China. Blood samples of 273 healthy individuals were collected from Nanning City, Guangxi Province, China in March 2019. We found that 9.5% (26/273) and 1.8% (5/273) of healthy persons were positive to ZIKV total antibody (IgG and/or IgM) IgM antibody, respectively. All ZIKV positive plasma samples were negative to Dengue virus and West Nile virus. Among the ZIKV antibody positive plasma samples, 65.4% (17/26) exhibited neutralizing activity to ZIKV. Followed up studies showed that none had clinical symptoms of ZIKV infection and oversea experience. Together, our study indicates that endemic ZIKV infections emerge in China, which not only suggested that ZIKV posed a potential threat to public health in China, but also expand the ZIKV epidemic areas in East and Southeast Asia.

The establishment of a sylvatic reservoir of Zika virus (ZIKV) in the Americas is dependent on the susceptibility of primates of sufficient population density, the duration and magnitude of viremia, and their exposure to the human mosquito-borne transmission cycle. To assess the susceptibility of squirrel (Saimiri sp.) and owl monkeys (Aotus sp.) to infection, we inoculated four animals of each species with ZIKV from the current epidemic. Viremia in the absence of detectible disease was observed in both species and seroconversion occurred by day 28. ZIKV was detected in the spleen of three owl monkeys: one at 7 days postinoculation (dpi) and two at 14 dpi. This study confirms the susceptibility to ZIKV infection of two Neotropical primate species that live in close proximity to humans in South America, suggesting that they could support a widespread sylvatic ZIKV cycle there. Collectively, establishment of a ZIKV sylvatic transmission cycle in South America would imperil eradication efforts and could provide a mechanism for continued exposure of humans to ZIKV infection and disease.

The high levels of dengue-virus (DENV) seroprevalence in areas where the Zika virus (ZIKV) is circulating and the cross-reactivity between these two viruses have raised concerns on the risk of increased ZIKV disease severity for patients with a history of previous DENV infections. To determine the role of DENV preimmunity in ZIKV infection, we analyzed the T- and B-cell responses against ZIKV in donors with or without previous DENV infection. Using peripheral blood mononuclear cells (PBMCs) from donors living in an endemic area in Colombia, we have identified, by interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assay, most of the immunodominant ZIKV T-cell epitopes in the nonstructural (NS) proteins NS1, NS3, and NS5. Analyses of the T- and B-cell responses in the same donors revealed a stronger T-cell response against peptides conserved between DENV and ZIKV, with a higher level of ZIKV-neutralizing antibodies in DENV-immune donors in comparison with DENV-naïve donors. Strikingly, the potential for antibody-mediated enhancement of ZIKV infection was reduced in donors with sequential DENV and ZIKV infection in comparison with donors with DENV infection only. Altogether, these data suggest that individuals with DENV immunity present improved immune responses against ZIKV.

Zika virus (ZIKV) is an emergent member of the Flaviviridae family which causes severe congenital defects and other major sequelae, but the cellular processes that support ZIKV replication are incompletely understood. Related flaviviruses use the endoplasmic reticulum (ER) as a membranous platform for viral replication and induce ER stress during infection. Our data suggest that ZIKV activates IRE1α, a component of the cellular response to ER stress. IRE1α is an ER-resident transmembrane protein that possesses a cytosolic RNase domain. Upon activation, IRE1α initiates nonconventional cytoplasmic splicing of XBP1 mRNA. Spliced XBP1 encodes a transcription factor, which upregulates ER-related targets. We find that ZIKV infection induces XBP1 mRNA splicing and induction of XBP1 target genes. Small molecule inhibitors of IRE1α, including those specific for the nuclease function, prevent ZIKV-induced cytotoxicity, as does genetic disruption of IRE1α. Optimal ZIKV RNA replication requires both IRE1α and XBP1. Spliced XBP1 has been described to cause ER expansion and remodeling and we find that ER redistribution during ZIKV infection requires IRE1α nuclease activity. Finally, we demonstrate that inducible genetic disruption of IRE1α and XBP1 impairs ZIKV replication in a mouse model of infection. Together, our data indicate that the ER stress response component IRE1α promotes ZIKV infection via XBP1 and may represent a potential therapeutic target.

Zika virus (ZIKV) is a major human pathogen. ZIKV can replicate in female and male reproductive organs, thus facilitating the human-human transmission cycle. Viral shedding in the semen can increase the risk of ZIKV transmission through sexual mode. Therefore, the vaginal and anorectal mucosa are relevant sites for ZIKV infection. However, the pathobiology of ZIKV transmission through the rectal route is not well understood. Here, we utilize a mouse model system to investigate the immunopathological consequences following ZIKV infection of the rectal mucosa compared to a subcutaneous route of infection. We show that ZIKV-rectal inoculation results in viremia with subclinical infection. ZIKV infects the mucosal epithelium and submucosal dendritic cells, inducing immune and inflammatory cell infiltration. Rectal transmission of ZIKV resulted in the generation of serum-neutralizing antibody responses. Mass cytometry analyses of splenocytes showed a significantly reduced level of inflammatory monocyte and neutrophil cellular responses in the rectal route group. Furthermore, immunological priming through the rectal mucosa with an attenuated ZIKV strain resulted in significant protection from lethal subcutaneous ZIKV challenge, further eliciting robust memory CD4-positive (CD4+) and CD8+ T-cell and ZIKV-specific serum-neutralizing antibody responses. Thus, our study provides deeper immunopathobiological insights on rectal transmission and highlights a rational strategy for mucosal immunization. This model system recapitulates clinical aspects of human ZIKV disease outcome, where most infections are well controlled and result in subclinical and asymptomatic outcomes.IMPORTANCE Zika virus is a clinically significant human pathogen that is primarily transmitted and spread by Aedes species mosquitoes but is also sexually transmissible. The recent pandemic in the Americas led to an unprecedented increase of newborn babies with developmental brain and eye abnormalities. To date, there is no licensed vaccine or therapeutic intervention available for the fight against ZIKV. Understanding the sexual transmission of ZIKV through vaginal and rectal routes is necessary to restrict virus transmission and spread. This study examines the early immunological and pathological consequences of rectal and subcutaneous routes of ZIKV infection using a mouse model. We characterized the primary target cells of ZIKV infection and the subsequent mucosal immune responses to infection, and we demonstrate the protective effect of mucosal rectal immunization using an attenuated ZIKV strain. This mucosal vaccination approach can be further developed to prevent future ZIKV outbreaks.

Pregnant women, and their fetal offspring, are uniquely susceptible to Zika virus and other microbial pathogens capable of congenital fetal infection. Unavoidable exposure to Zika virus in endemic areas underscores the need for identifying at-risk individuals, and protecting expecting mothers and their fetal offspring against prenatal infection. Here we show that primary Zika virus asymptomatic infection in mice confers protection against re-infection, and that these protective benefits are maintained during pregnancy. Zika virus recovery was sharply reduced in maternal tissues and amongst fetal concepti after prenatal challenge in mothers with resolved subclinical infection prior to pregnancy compared with mice undergoing primary prenatal infection. These benefits coincide with expanded accumulation of viral-specific antibodies in maternal serum and fetal tissues that protect against infection by the identical or heterologous Zika virus genotype strains. Thus, preconceptual infection primes Zika virus-specific antibodies that confer cross-genotype protection against re-infection during pregnancy.

Zika virus (ZIKV) is transmitted to people by bite of an infected mosquito and by sexual contact. ZIKV infects primary genital epithelial cells, the same cells targeted by herpes simplex virus 2 (HSV-2). HSV-2 seroprevalence is high in areas where ZIKV is endemic, but it is unknown whether HSV-2 increases the risk for ZIKV infection. Here, we found that pre-infecting female genital tract epithelial cells with HSV-2 leads to enhanced binding of ZIKV virions. This effect did not require active replication by HSV-2, implying that the effect results from the immune response to HSV-2 exposure or to viral genes expressed early in the HSV-2 lifecycle. Treating cells with toll-like receptor-3 ligand poly-I:C also lead to enhanced binding by ZIKV, which was inhibited by the JAK-STAT pathway inhibitor ruxolitinib. Blocking or knocking down the well-studied ZIKV receptor AXL did not prevent binding of ZIKV to epithelial cells, nor prevent enhanced binding in the presence of HSV-2 infection. Blocking the α5 integrin receptor did not prevent ZIKV binding to cells either. Overall, our results indicate that ZIKV binding to genital epithelial cells is not mediated entirely by a canonical receptor, but likely occurs through redundant pathways that may involve lectin receptors and glycosaminoglycans. Our studies may pave the way to new interventions that interrupt the synergism between herpes and Zika viruses.

Zika virus (ZIKV) is a mosquito-borne pathogen which has recently spread beyond Africa and into Pacific and South American regions. Despite first being detected in 1947, very little information is known about the virus, and its spread has been associated with increases in Guillain-Barre syndrome and microcephaly. There are currently no known vaccines or antivirals against ZIKV infection. Progress in assessing interventions will require the development of animal models to test efficacies; however, there are only limited reports on in vivo studies. The only susceptible murine models have involved intracerebral inoculations or juvenile animals, which do not replicate natural infection. Our report has studied the effect of ZIKV infection in type-I interferon receptor deficient (A129) mice and the parent strain (129Sv/Ev) after subcutaneous challenge in the lower leg to mimic a mosquito bite. A129 mice developed severe symptoms with widespread viral RNA detection in the blood, brain, spleen, liver and ovaries. Histological changes were also striking in these animals. 129Sv/Ev mice developed no clinical symptoms or histological changes, despite viral RNA being detectable in the blood, spleen and ovaries, albeit at lower levels than those seen in A129 mice. Our results identify A129 mice as being highly susceptible to ZIKV and thus A129 mice represent a suitable, and urgently required, small animal model for the testing of vaccines and antivirals.

Astrocytes are an early and important target of Zika virus (ZIKV) infection in the developing brain, but the impacts of infection on astrocyte function remain controversial. Given that nonhuman primate (NHP) models of ZIKV infection replicate aspects of neurologic disease seen in human infections, we cultured primary astrocytes from the brain tissue of infant rhesus macaques and then infected the cells with Asian or African lineage ZIKV to identify transcriptional patterns associated with infection in these cells. The African lineage virus appeared to have greater infectivity and promote stronger antiviral signaling, but infection by either strain ultimately produced typical virus response patterns. Both viruses induced hypoxic stress, but the Asian lineage strain additionally had an effect on metabolic and lipid biosynthesis pathways. Together, these findings describe an NHP astrocyte model that may be used to assess transcriptional signatures following ZIKV infection.

The emergence of ZIKV infection has prompted a global effort to develop safe and effective vaccines. We engineered a lipid nanoparticle (LNP) encapsulated modified mRNA vaccine encoding wild-type or variant ZIKV structural genes and tested immunogenicity and protection in mice. Two doses of modified mRNA LNPs encoding prM-E genes that produced virus-like particles resulted in high neutralizing antibody titers (∼1/100,000) that protected against ZIKV infection and conferred sterilizing immunity. To offset a theoretical concern of ZIKV vaccines inducing antibodies that cross-react with the related dengue virus (DENV), we designed modified prM-E RNA encoding mutations destroying the conserved fusion-loop epitope in the E protein. This variant protected against ZIKV and diminished production of antibodies enhancing DENV infection in cells or mice. A modified mRNA vaccine can prevent ZIKV disease and be adapted to reduce the risk of sensitizing individuals to subsequent exposure to DENV, should this become a clinically relevant concern.

Congenital Zika syndrome (CZS) is a set of birth defects caused by Zika virus (ZIKV) infection during pregnancy. Microcephaly is its main feature, but other brain abnormalities are found in CZS patients, such as ventriculomegaly, brain calcifications, and dysgenesis of the corpus callosum. Many studies have focused on microcephaly, but it remains unknown how ZIKV infection leads to callosal malformation. To tackle this issue, we infected mouse embryos in utero with a Brazilian ZIKV isolate and found that they were born with a reduction in callosal area and density of callosal neurons. ZIKV infection also causes a density reduction in PH3+ cells, intermediate progenitor cells, and SATB2+ neurons. Moreover, axonal tracing revealed that callosal axons are reduced and misrouted. Also, ZIKV-infected cultures show a reduction in callosal axon length. GFAP labeling showed that an in utero infection compromises glial cells responsible for midline axon guidance. In sum, we showed that ZIKV infection impairs critical steps of corpus callosum formation by disrupting not only neurogenesis, but also axon guidance and growth across the midline.

Zika virus (ZIKV) is a re-emerging flavivirus. Similar to other flaviviruses, ZIKV antagonizes the host interferon (IFN) signaling pathway to establish infection. Understanding the molecular mechanism by which ZIKV antagonizes IFN-induced antiviral signaling may lead to a new antiviral strategy by cracking the IFN antagonism. Flaviviruses have been reported to employ NS5-dependent and -independent mechanisms to block STAT2-mediated signaling, whereas whether flaviviruses target STAT1 remains controversial. Herein, we found that ZIKV infection triggered caspase-dependent cleavage of STAT1 at the aspartic acid 694 during late infection, whereas murine STAT1 (mSTAT1) was resistant to cleavage. Intriguingly, ectopically expressed cleavage-resistant human STAT1.D694A or complementation of cleavable mSTAT1.D695G exerted comparable anti-ZIKV activity with their counterparts, challenging the role of caspase-mediated STAT1 cleavage in the IFN antagonism in ZIKV-infected cells. These data may also imply a dominant role of the antagonism of STAT2 but not STAT1 in ZIKV-infected cells.

Progress toward understanding Zika virus (ZIKV) pathogenesis is hindered by lack of immunocompetent small animal models, in part because ZIKV fails to effectively antagonize Stat2-dependent interferon (IFN) responses in mice. To address this limitation, we first passaged an African ZIKV strain (ZIKV-Dak-41525) through Rag1-/- mice to obtain a mouse-adapted virus (ZIKV-Dak-MA) that was more virulent than ZIKV-Dak-41525 in mice treated with an anti-Ifnar1 antibody. A G18R substitution in NS4B was the genetic basis for the increased replication, and resulted in decreased IFN-β production, diminished IFN-stimulated gene expression, and the greater brain infection observed with ZIKV-Dak-MA. To generate a fully immunocompetent mouse model of ZIKV infection, human STAT2 was introduced into the mouse Stat2 locus (hSTAT2 KI). Subcutaneous inoculation of pregnant hSTAT2 KI mice with ZIKV-Dak-MA resulted in spread to the placenta and fetal brain. An immunocompetent mouse model of ZIKV infection may prove valuable for evaluating countermeasures to limit disease.

Zika virus (ZIKV) has emerged as an important human pathogen that can cause congenital defects in the fetus and neurological conditions in adults. The interferon (IFN) system has proven crucial in restricting ZIKV replication and pathogenesis. The canonical IFN response is triggered by the detection of viral RNA through RIG-I like receptors followed by activation of the adaptor protein MAVS on mitochondrial membranes. Recent studies have shown that a second organelle, peroxisomes, also function as a signaling platforms for the IFN response. Here, we investigated how ZIKV infection affects peroxisome biogenesis and antiviral signaling. We show that ZIKV infection depletes peroxisomes in human fetal astrocytes, a brain cell type that can support persistent infection. The peroxisome biogenesis factor PEX11B was shown to inhibit ZIKV replication, likely by increasing peroxisome numbers and enhancing downstream IFN-dependent antiviral signaling. Given that peroxisomes play critical roles in brain development and nerve function, our studies provide important insights into the roles of peroxisomes in regulating ZIKV infection and potentially neuropathogenesis.