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Circoviruses represent a rapidly expanding group of viruses that infect both vertebrate and invertebrate hosts. Members are responsible for diseases of veterinary and economic importance, including postweaning multisystemic wasting syndrome in pigs, and beak and feather disease (BFD) in birds. These viruses are associated with lymphoid depletion and immunosuppressive conditions in infected animals leading to systemic illness. Circoviruses are small nonenveloped DNA viruses containing a single-stranded circular genome, encoding two major proteins: the capsid-associated protein (Cap), comprising the entirety of the viral capsid, and the replication-associated protein (Rep). Cap is the only protein component of the virion and plays crucial roles throughout the virus replication cycle, including viral attachment, cell entry, genome uncoating, and packaging of newly formed viral particles. Rep mediates recognition of replication origin motifs in the viral genome sequence and is responsible for endonuclease activity enabling nicking of the circular DNA and initiation of rolling-circle replication (RCR). Porcine circovirus 2 (PCV2) was the first circovirus capsid structure to be solved at atomic resolution using X-ray crystallography. The structure revealed an assembly comprising 60 monomeric subunits to form virus-like particles. Each Cap monomer harbors a canonical viral jelly roll domain composed of two, four-stranded antiparallel β-sheets. Crystal structures of two distinct macromolecular assemblies from BFD virus Cap were also resolved at high resolution. In these structures, the exposure of the N-terminal arginine-rich motif, responsible for DNA binding and nuclear localization is reversed. Additional structural investigations have also elucidated a PCV2 type-specific neutralizing epitope, and interaction between the PCV2 capsid and polymers such as heparin. In this review, we provide a snapshot of the structural and functional aspects of circovirus proteins.

Henipaviruses are single-stranded RNA viruses that have recently emerged as zoonotic pathogens, capable of causing severe acute respiratory disease and encephalitis in humans. The prototypical henipaviruses, Hendra henipavirus and Nipah henipavirus, are a major health concern as they have high mortality rates and no currently approved human vaccine or drug therapy. Understanding the mechanisms of viral replication and pathogenicity is of critical importance for therapeutic developments. A novel target for such therapies is the Henipavirus Matrix (M) protein, a multifunctional protein that drives viral assembly and inhibits the innate immune response. These multifunctional attributes promote a complicated lifecycle: while viral replication occurs in the cytoplasm, M traffics to the nucleus, where it is ubiquitinated, for correct cellular targeting and virion packaging. In this study, we review the relationship between the structure and functions of M. In specific cases, the compatibility between structural accessibility and protein functionality is not always evident, and we highlight areas that require further investigation.

Zika virus (ZIKV) is a mosquito-transmitted flavivirus that caused a public health emergency in the Americas when an outbreak in Brazil became linked to congenital microcephaly. Understanding how ZIKV could evade the innate immune defenses of the mother, placenta, and fetus has become central to determining how the virus can traffic into the fetal brain. ZIKV, like other flaviviruses, evades host innate immune responses by leveraging viral proteins and other processes that occur during viral replication to allow spread to the placenta. Within the placenta, there are diverse cell types with coreceptors for ZIKV entry, creating an opportunity for the virus to establish a reservoir for replication and infect the fetus. The fetal brain is vulnerable to ZIKV, particularly during the first trimester, when it is beginning a dynamic process, to form highly complex and specialized regions orchestrated by neuroprogenitor cells. In this review, we provide a conceptual framework to understand the different routes for viral trafficking into the fetal brain and the eye, which are most likely to occur early and later in pregnancy. Based on the injury profile in human and nonhuman primates, ZIKV entry into the fetal brain likely occurs across both the blood/cerebrospinal fluid barrier in the choroid plexus and the blood/brain barrier. ZIKV can also enter the eye by trafficking across the blood/retinal barrier. Ultimately, the efficient escape of innate immune defenses by ZIKV is a key factor leading to viral infection. However, the host immune response against ZIKV can lead to injury and perturbations in developmental programs that drive cellular division, migration, and brain growth. The combined effect of innate immune evasion to facilitate viral propagation and the maternal/placental/fetal immune response to control the infection will determine the extent to which ZIKV can injure the fetal brain.

Zika virus (ZIKV) is a mosquito-borne pathogen that caused an epidemic in 2015–2016. ZIKV-specific T cell responses are functional in animal infection models, and helper CD4 T cells promote avid Abs in the vaccine context. The small volumes of blood available from field research limit the determination of T cell epitopes for complex microbes such as ZIKV. The goal of this project was efficient determination of human ZIKV CD4 T cell epitopes at the whole proteome scale, including validation of reactivity to whole pathogen, using small blood samples from convalescent time points when T cell response magnitude may have waned. Polyclonal enrichment of candidate ZIKV-specific CD4 T cells used cell-associated virus, documenting that T cells in downstream peptide analyses also recognize whole virus after Ag processing. Sequential query of bulk ZIKV-reactive CD4 T cells with pooled/single ZIKV peptides and molecularly defined APC allowed precision epitope and HLA restriction assignments across the ZIKV proteome and enabled discovery of numerous novel ZIKV CD4 T cell epitopes. The research workflow is useful for the study of emerging infectious diseases with a very limited human blood sample availability.

In recent years, Asian lineage Zika virus (ZIKV) strains emerged to cause pandemic outbreaks associated with a high rate of congenital ZIKV syndrome (CZVS). The reasons for the enhanced spread and severe disease caused by newly emerging strains are not fully understood. Here we compared viral sequences, viral replication, and innate immune signaling induction of three different ZIKV strains derived from African and Asian lineages and West Nile virus, another flavivirus. We found pronounced differences in activation of innate immune signaling and inhibition of viral replication across ZIKV strains. The newly emerged Asian ZIKV strain Brazil Fortaleza 2015, which is associated with a higher rate of neurodevelopmental disorders like microcephaly, induced much weaker and delayed innate immune signaling in infected cells. However, superinfection studies to assess control of innate immune signaling induced by Sendai virus argue against an active block of IRF3 activation by the Brazilian strain of ZIKV and rather suggest an evasion of detection by host cell pattern recognition receptors. Compared to the Asian strain FSS13025 isolated in Cambodia, both ZIKV Uganda MR766 and ZIKV Brazil Fortaleza appear less sensitive to the interferon-induced antiviral response. ZIKV infection studies of cells lacking the different RIG-I-like receptors identified RIG-I as the major cytosolic pattern recognition receptor for detection of ZIKV. IMPORTANCE Zika Virus (ZIKV), discovered in 1947, is divided into African and Asian lineages. Pandemic outbreaks caused by currently emerging Asian lineage strains are accompanied by high rates of neurological disorders and exemplify the global health burden associated with this virus. Here we compared virological and innate immunological aspects of two ZIKV strains from the Asian lineage, an emerging Brazilian strain and a less-pathogenic Cambodian strain, and the prototypic African lineage ZIKV strain from Uganda. Compared to the replication of other ZIKV strains, the replication of ZIKV Brazil was less sensitive to the antiviral actions of interferon (IFN), while infection with this strain induced weaker and delayed innate immune responses in vitro. Our data suggest that ZIKV Brazil directs a passive strategy of innate immune evasion that is reminiscent of a stealth virus. Such strain-specific properties likely contribute to differential pathogenesis and should be taken into consideration when choosing virus strains for future molecular studies.

The initial response to viral infection is anticipatory, with host antiviral restriction factors and pathogen sensors constantly surveying the cell to rapidly mount an antiviral response through the synthesis and downstream activity of interferons. After pathogen clearance, the host's ability to resolve this antiviral response and return to homeostasis is critical. Here, we found that isoforms of the RNA-binding protein ZAP functioned as both a direct antiviral restriction factor and an interferon-resolution factor. The short isoform of ZAP bound to and mediated the degradation of several host interferon messenger RNAs, and thus acted as a negative feedback regulator of the interferon response. In contrast, the long isoform of ZAP had antiviral functions and did not regulate interferon. The two isoforms contained identical RNA-targeting domains, but differences in their intracellular localization modulated specificity for host versus viral RNA, which resulted in disparate effects on viral replication during the innate immune response.

Zika virus (ZIKV) is a flavivirus with teratogenic effects on fetal brain, but the spectrum of ZIKV-induced brain injury is unknown, particularly when ultrasound imaging is normal. In a pregnant pigtail macaque (Macaca nemestrina) model of ZIKV infection, we demonstrate that ZIKV-induced injury to fetal brain is substantial, even in the absence of microcephaly, and may be challenging to detect in a clinical setting. A common and subtle injury pattern was identified, including (i) periventricular T2-hyperintense foci and loss of fetal noncortical brain volume, (ii) injury to the ependymal epithelium with underlying gliosis and (iii) loss of late fetal neuronal progenitor cells in the subventricular zone (temporal cortex) and subgranular zone (dentate gyrus, hippocampus) with dysmorphic granule neuron patterning. Attenuation of fetal neurogenic output demonstrates potentially considerable teratogenic effects of congenital ZIKV infection even without microcephaly. Our findings suggest that all children exposed to ZIKV in utero should receive long-term monitoring for neurocognitive deficits, regardless of head size at birth.

Chronic hepatitis C virus (HCV) infection results in sustained immune activation in both the periphery and hepatic tissue. HCV infection induces innate immune signaling that is responsible for recognition of dsRNA, leading to activation of transcription factors and production of Type I and III IFNs, as well as pro-inflammatory cytokines and chemokines. Continued activation of host-immune mediated inflammation is thought to contribute to pathologic changes that result in progressive hepatic fibrosis. The current standard treatment for chronic HCV infection is directly-acting antivirals (DAAs), which have provided the unique opportunity to determine whether successful, rapid treatment-induced eradication of viral RNA normalizes the dysregulated antiviral innate immune response in patients chronically infected with HCV. Results First, in patients receiving two different combinations of DAAs, we found that DAAs induced not only rapid viral clearance, but also a re-setting of antiviral immune responses in the peripheral blood. Specifically, we see a rapid decline in the expression of genes associated with chronic IFN stimulation (IFIT3, USP18, IFIT1) as well as a rapid decline in genes associated with inflammation (IL1β, CXCL10, CXCL11) in the peripheral blood that precedes the complete removal of virus from the blood. Interestingly, this rapid reversal of innate immune activation was not seen in patients who successfully clear chronic HCV infection using IFN-based therapy. Next, using a novel humanized mouse model (Fah-/-RAG2-/-IL2rgnull—FRG), we assessed the changes that occur in the hepatic tissue following DAA treatment. DAA-mediated rapid HCV clearance resulted in blunting of the expression of proinflammatory responses while functionally restoring the RIG-I/MAVS axis in the liver of humanized mice. Conclusions Collectively, our data demonstrate that the rapid viral clearance following treatment with DAAs results in the rebalancing of innate antiviral response in both the peripheral blood and the liver as well as enhanced antiviral signaling within previously infected hepatocytes.

Flavivirus genomes with deletions in the capsid (C) gene are attractive vaccine candidates, as they secrete highly immunogenic subviral particles (SVPs) without generating infectious virus. Here, we report that cytomegalovirus promoter-driven cDNA of West Nile virus Kunjin (KUNV) containing a glycosylation motif in the envelope (E) gene and a combined deletion of alpha helices 1, 2, and 4 in C produces significantly more SVPs than KUNV cDNAs with nonglycosylated E and various other deletions in C.

The neurotropic arthropod-borne flavivirus West Nile virus (WNV) is responsible for sporadic outbreaks of fever and encephalitis in infected humans, with an almost global distribution of circulating strains. During WNV infection in mammals Interferon-α/β (IFN-α/β) drives signaling through JAK/STAT pathways to enhance the expression of antiviral genes with an overall protective effect against WNV infection. However, WNV strains have been demonstrated to be able to antagonize IFN-α/β signaling within infected cells, interrupting this JAK/STAT cascade and promoting viral replication. We hypothesized that JAK/STAT signaling downstream of multiple cytokines would be broadly inhibited in WNV infected cells as a consequence of viral replication. To assess the degree of cytokine signaling in infected cells, A549 and PMA-differentiated THP-1 cells were infected with WNV for 24 hr prior to acute course treatment with various cytokines. We reveal that STAT3 and STAT5 phosphorylation, in addition to STAT1 and STAT2, is blocked in WNV-infected cells in response to IFN-β. Remarkably, JAK/STAT signaling cascades downstream of IFN-γ, IFN-λ3, IL-4, and IL-6 are also similarly dysregulated upon WNV infection, suggesting that global JAK/STAT signaling may be inhibited independent of the identity of the cytokine receptor. The breadth and mechanisms of the WNV-imposed JAK/STAT signaling blockade will be presented.