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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.

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.

The MERS coronavirus (MERS-CoV) is a highly pathogenic, emerging virus that produces accessory proteins to antagonize the host innate immune response. The MERS-CoV ORF4b protein has been shown to bind preferentially to the nuclear import adapter IMPα3 in infected cells, thereby inhibiting NF-κB-dependent innate immune responses. Here, we report high-resolution structures of ORF4b bound to two distinct IMPα family members. Each exhibit highly similar binding mechanisms that, in both cases, lack a prototypical Lys bound at their P2 site. Mutations within the NLS region dramatically alter the mechanism of binding, which reverts to the canonical P2 Lys binding mechanism. Mutational studies confirm that the novel binding mechanism is important for its nuclear import, IMPα interaction, and inhibition of innate immune signaling pathways. In parallel, we determined structures of the nuclear binding domain of NF-κB component p50 bound to both IMPα2 and α3, demonstrating that p50 overlaps with the ORF4b binding sites, suggesting a basis for inhibition. Our results provide a detailed structural basis that explains how a virus can target the IMPα nuclear import adapter to impair immunity, and illustrate how small mutations in ORF4b, like those found in closely related coronaviruses such as HKU5, change the IMPα binding mechanism.

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.

SUMMARYMany 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 host cell innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense viral 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 flavivirus, picornavirus, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform strongly associates with COVID-19 severity. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests 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.

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

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.

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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.

West Nile virus(WNV; genusFlavivirus, familyFlaviviridae) is an emerging pathogenic arbovirus responsible for outbreaks of encephalitis around the world. Whilst no vaccines are currently available to prevent WNV infection of humans, the use of cDNA copies of flavivirus RNA genomes with large internal deletions within the capsid (C) appears promising. C-deleted vaccines are able to replicate and secrete large amounts of non-infectious immunogenic subviral particles (SVPs) from transfected cells. We have previously generated a WNV DNA vaccine candidate pKUNdC/C where C-deleted WNV cDNA was placed under the control of one copy of the cytomegalovirus (CMV) promoter and the C gene was placed under the control of a second copy of the CMV promoter in the same plasmid DNA. This DNA was shown to generate single-round infectious particles (SRIPs) capable of delivering self-replicating C-deleted RNA producing SVPs to surrounding cells, thus enhancing the vaccine potential. However, the amounts of both SRIPs and SVPs produced from pKUNdC/C DNA were relatively low. In this investigation, we aimed at increasing SRIP production by optimizingtrans-C expression via incorporating different forms of C and the use of a more powerful promoter. The construct containing an elongation factor EF1α promoter encoding an extended form of C was demonstrated to produce the highest titres of SRIPs and was immunogenic in mice. Additionally, SRIP and SVP titres were further improved via incorporation of a glycosylation motif in the envelope protein. The optimized DNA yielded ~100-fold greater titres of SRIPs than the original construct, thus providing a promising candidate for further vaccine evaluation.

Although interferon (IFN) signaling induces genes that limit viral infection, many pathogenic viruses overcome this host response. As an example, 2′-O methylation of the 5′ cap of viral RNA subverts mammalian antiviral responses by evading restriction of Ifit1, an IFN-stimulated gene that regulates protein synthesis. However, alphaviruses replicate efficiently in cells expressing Ifit1 even though their genomic RNA has a 5′ cap lacking 2′-O methylation. We show that pathogenic alphaviruses use secondary structural motifs within the 5′-untranslated region (UTR) of their RNA to alter Ifit1 binding and function. Mutations within the 5′-UTR affecting RNA structural elements enabled restriction by or antagonism of Ifit1 in vitro and in vivo. These results identify an evasion mechanism by which viruses use RNA structural motifs to avoid immune restriction.

Flaviviruses are a group of single-stranded, positive-sense RNA viruses that generally circulate between arthropod vectors and susceptible vertebrate hosts, producing significant human and veterinary disease burdens. Intensive research efforts have broadened our scientific understanding of the replication cycles of these viruses and have revealed several elegant and tightly co-ordinated post-translational modifications that regulate the activity of viral proteins. The three structural proteins in particular – capsid (C), pre-membrane (prM) and envelope (E) – are subjected to strict regulatory modifications as they progress from translation through virus particle assembly and egress. The timing of proteolytic cleavage events at the C–prM junction directly influences the degree of genomic RNA packaging into nascent virions. Proteolytic maturation of prM by host furin during Golgi transit facilitates rearrangement of the E proteins at the virion surface, exposing the fusion loop and thus increasing particle infectivity. Specific interactions between the prM and E proteins are also important for particle assembly, as prM acts as a chaperone, facilitating correct conformational folding of E. It is only once prM/E heterodimers form that these proteins can be secreted efficiently. The addition of branched glycans to the prM and E proteins during virion transit also plays a key role in modulating the rate of secretion, pH sensitivity and infectivity of flavivirus particles. The insights gained from research into post-translational regulation of structural proteins are beginning to be applied in the rational design of improved flavivirus vaccine candidates and make attractive targets for the development of novel therapeutics.

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.