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Adeno-associated viruses (AAVs) are key vectors for gene therapy; thus, many aspects of their cell transduction pathway have been revealed in detail. However, the specific mechanisms AAV virions use to enter the host nucleus remain largely unresolved. We therefore aimed to reveal the structural interactions between the AAV capsid (Cap) protein and the nuclear transport protein importin alpha (IMPα). A putative nuclear localization sequence (NLS) in the virion protein 1 capsid protein of the porcine AAV Po1 was identified. This region was complexed with IMPα and a structure solved at 2.26 Å. This is the first time that an NLS of AAV Cap complexed with IMPα has been determined structurally. Our results support the findings that AAV capsids enter the nucleus through binding the nuclear import adapter IMPα.

Wet markets are important for food security in many regions worldwide but have come under scrutiny due to their potential role in the emergence of infectious diseases. The sale of live wildlife has been highlighted as a particular risk, and the World Health Organisation has called for the banning of live, wild-caught mammalian species in markets unless risk assessment and effective regulations are in place. Following PRISMA guidelines, we conducted a global scoping review of peer-reviewed information about the sale of live, terrestrial wildlife in markets that are likely to sell fresh food, and collated data about the characteristics of such markets, activities involving live wildlife, the species sold, their purpose, and animal, human, and environmental health risks that were identified. Of the 59 peer-reviewed records within scope, only 25% (n = 14) focussed on disease risks; the rest focused on the impact of wildlife sale on conservation. Although there were some global patterns (for example, the types of markets and purpose of sale of wildlife), there was wide diversity and huge epistemic uncertainty in all aspects associated with live, terrestrial wildlife sale in markets such that the feasibility of accurate assessment of the risk of emerging infectious disease associated with live wildlife trade in markets is limited. Given the value of both wet markets and wildlife trade and the need to support food affordability and accessibility, conservation, public health, and the social and economic aspects of livelihoods of often vulnerable people, there are major information gaps that need to be addressed to develop evidence-based policy in this environment. This review identifies these gaps and provides a foundation from which information for risk assessments can be collected.

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.

Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from cellular innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flaviviruses, picornaviruses, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform correlates with protection from severe COVID-19. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests that early control of SARS-CoV-2 replication through OAS1 p46 is an important determinant of COVID-19 severity.

Since 2007, African swine fever virus (ASFV) has spread to countries in Europe, Asia and Oceania and has caused devastating impacts on pigs and the pork industry. Transmission can be direct or indirect, and epidemiologic scenarios have been described in which spread occurs between free-living and domestic pigs. The purpose of this scoping review was to identify primary research in which authors made statements to support ASFV transmission between free-living and domestic pigs and assess the circumstances in which transmission events occurred. A search was conducted in five bibliographic databases and the grey literature. Two reviewers (from a team of ten) independently screened each record and charted data (demographics of the pig populations, their husbandry [domestic pigs] and habitat [free-living pigs], the spatial and temporal distribution of ASF, the occurrence or burden of ASF in the populations, and whether ticks were present in the geographic range of the pig populations). Data synthesis included statistics and a narrative summary. From 1,349 records screened, data were charted from 46 individual studies published from 1985 to 2020. Outbreak investigations revealed that whilst poor biosecurity of domestic pig operations was often reported, direct contact resulting in transmission between free-living and domestic pigs was rarely reported. Studies in which quantitative associations were made generally found that spread within populations was more important than spread between populations, although this was not always the case, particularly when domestic pigs were free-ranging. We conclude that there is limited evidence that transmission of ASFV between free-living and domestic pigs is an important feature of ASF epidemiology, especially in the current ASF epidemic in Europe and the Russian Federation. If ASFV elimination cannot be achieved in free-living pigs, compartmentalization of domestic pig populations from free-living populations via biosecurity strategies could be used to support trade of domestic pigs.

Nipah and Hendra viruses are highly pathogenic, zoonotic henipaviruses that encode proteins that inhibit the host’s innate immune response. The W protein is one of four products encoded from the P gene and binds a number of host proteins to regulate signalling pathways. The W protein is intrinsically disordered, a structural attribute that contributes to its diverse host protein interactions. Here, we review the role of W in innate immune suppression through inhibition of both pattern recognition receptor (PRR) pathways and interferon (IFN)-responsive signalling. PRR stimulation leading to activation of IRF-3 and IFN release is blocked by henipavirus W, and unphosphorylated STAT proteins are sequestered within the nucleus of host cells by W, thereby inhibiting the induction of IFN stimulated genes. We examine the critical role of nuclear transport in multiple functions of W and how specific binding of importin-alpha (Impα) isoforms, and the 14-3-3 group of regulatory proteins suggests further modulation of these processes. Overall, the disordered nature and multiple functions of W warrant further investigation to understand henipavirus pathogenesis and may reveal insights aiding the development of novel therapeutics.

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.

Since 2007, African swine fever virus (ASFV) has spread to countries in Europe, Asia and Oceania, and has caused devastating impacts on pigs and the pork industry. Transmission can be direct or indirect, and epidemiologic scenarios have been described in which spread occurs between free-living and domestic pigs. The purpose of this scoping review was to identify primary research in which authors made statements to support ASFV transmission between free-living and domestic pigs and assess the circumstances in which transmission events occurred. A search was conducted in five bibliographic databases and the grey literature. Two reviewers (from a team of ten) independently screened each record and charted data (demographics of the pig populations, their husbandry [domestic pigs] and habitat [free-living pigs], the spatial and temporal distribution of ASF, the occurrence or burden of ASF in the populations, and whether ticks were present in the geographic range of the pig populations). Data synthesis included statistics and a narrative summary. From 1,349 records screened, data were charted from 46 individual studies published from 1985 to 2020. Outbreak investigations revealed that whilst poor biosecurity of domestic pig operations was often reported, direct contact resulting in transmission between free-living and domestic pigs was rarely reported. Studies in which quantitative associations were made generally found that spread within populations was more important than spread between populations, although this was not always the case, particularly when domestic pigs were free-ranging. We conclude that there is limited evidence that transmission of ASFV between free-living and domestic pigs is an important feature of ASF epidemiology, especially in the current ASF epidemic in Europe and the Russian Federation. If ASFV elimination cannot be achieved in free-living pigs, compartmentalisation of free-living and domestic pig populations via biosecurity strategies could be used to support trade of domestic pigs.

Pathogenic flaviviruses antagonize host cell Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling downstream of interferons &alpha, /&beta, Here, we show that flaviviruses inhibit JAK/STAT signaling induced by a wide range of cytokines beyond interferon, including interleukins. This broad inhibition was mapped to viral nonstructural protein 5 (NS5) binding to cellular heat shock protein 90 (HSP90), resulting in reduced Janus kinase&ndash, HSP90 interaction and thus destabilization of unchaperoned JAKs (and other kinase clients) of HSP90 during infection by Zika virus, West Nile virus, and Japanese encephalitis virus. Our studies implicate viral dysregulation of HSP90 and the JAK/STAT pathway as a critical determinant of cytokine signaling control during flavivirus infection.

Interleukin-1 beta (IL-1β) is a pleiotropic mediator of inflammation and is produced in response to a wide range of stimuli. During infection, IL-1β production occurs in parallel with the onset of innate antimicrobial defenses, but the contribution of IL-1β signaling to cell-intrinsic immunity is not defined. Here, we report that exogenous IL-1β induces interferon regulatory factor 3 (IRF3) activation in human myeloid, fibroblast, and epithelial cells. IRF3 activation by IL-1β is dependent upon the DNA-sensing pathway adaptor, stimulator of interferon genes (STING), through the recognition of cytosolic mtDNA by cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS). IL-1β treatment results in interferon (IFN) production and activation of IFN signaling to direct a potent innate immune response that restricts dengue virus infection. This study identifies a new function for IL-1β in the onset or enhancement of cell-intrinsic immunity, with important implications for cGAS-STING in integrating inflammatory and microbial cues for host defense.

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.

Flaviviruses are single-stranded positive sense RNA enveloped viruses. The flavivirus genus includes important human pathogens such as dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), and Murray Valley encephalitis virus (MVEV). In addition to the viral proteins and viral genomic RNA, flaviviruses produce at least two functional non-coding RNAs derived from the 3' untranslated region (3'UTR), the subgenomic flavivirus RNA (sfRNA) and a putative WNV miRNA (KUN-miR-1). In this review we summarize published data from studies with WNV, YFV, DENV, JEV, and MVEV on sfRNA production following incomplete degradation of the viral genomic RNA by the cellular 5'-3' exoribonuclease 1 (XRN1), RNA structural elements involved in stalling XRN1 to generate sfRNA, and functions of sfRNA in modulating cellular mRNA decay and RNAi pathways as well as in modulating anti-viral type I interferon response. In addition, we also summarize data on the mechanisms of biogenesis of 3'UTR-derived KUN-miR-1 and its function in WNV replication in mosquito host, along with recent findings on a discovery of a second potential flaviviral miRNA vsRNA5, derived from the 3'UTR of DENV. This review thus summarizes the known mechanisms of generation and the functions of flaviviral 3'UTR-derived non-coding RNAs.

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.

Flaviviruses are a large group of positive strand RNA viruses transmitted by arthropods that include many human pathogens such as West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus, dengue virus, and tick-borne encephalitis virus. All members in this genus tested so far are shown to produce a unique subgenomic flavivirus RNA (sfRNA) derived from the 3' untranslated region (UTR). sfRNA is a product of incomplete degradation of genomic RNA by the cell 5'-3' exoribonuclease XRN1 which stalls at highly ordered secondary RNA structures at the beginning of the 3'UTR. Generation of sfRNA results in inhibition of XRN1 activity leading to an increase in stability of many cellular mRNAs. Mutant WNV deficient in sfRNA generation was highly attenuated displaying a marked decrease in cytopathicity in cells and pathogenicity in mice. sfRNA has also been shown to inhibit the antiviral activity of IFN-α/β by yet unknown mechanism and of the RNAi pathway by likely serving as a decoy substrate for Dicer. Thus, sfRNA is involved in modulating multiple cellular pathways to facilitate viral pathogenicity; however the overlying mechanism linking all these multiple functions of sfRNA remains to be elucidated.