Kunjin Research Articles

Complex transmission epidemiology of neglected Australian arboviruses: diverse non-human vertebrate hosts and competent arthropod invertebrate vectors.

More than 75 arboviruses are indigenous to Australia, of which at least 13 are known to cause disease in humans. Alphaviruses are the most common arboviruses, notably including Ross River and Barmah Forest viruses, which contribute a significant public health and economic burden in Australia. Both can cause febrile illness with arthritic symptoms. Each circulates nationally across diverse climates and environments, and has multi-host, multi-vector dynamics. Several medically important flaviviruses also circulate in Australia. Infection with Murray Valley encephalitis or Kunjin viruses is less common but is associated with brain inflammation. Key research priorities for Australian arboviruses aim to understand clinical manifestations, develop timely diagnostics, and identify transmission cycles that permit the maintenance of arboviruses. While these can now be answered for a handful of notifiable alpha- and flaviviruses there are others for which non-human vertebrate hosts and competent arthropod invertebrate vectors are still to be identified and/or whose role in transmission is not well understood. One or more of these 'neglected' arboviruses may be the causative agent of a proportion of the many thousands of fever-related illnesses reported annually in Australia that at present remain undiagnosed. Here, what is known about enzootic cycling of viruses between arthropod vectors and mammalian and avian reservoir hosts is summarised. How and to what extent these interactions influence the epidemiology of arbovirus transmission and infection is discussed.

Amino Acids at Positions 156 and 332 in the E Protein of the West Nile Virus Subtype Kunjin Virus Classical Strain OR393 Are Involved in Plaque Size, Growth, and Pathogenicity in Mice.

The West Nile virus (WNV) subtype Kunjin virus (WNVKUN) is endemic to Australia. Here, we characterized the classical WNVKUN strain, OR393. The original OR393 strain contained two types of viruses: small plaque-forming virus (SP) and large plaque-forming virus (LP). The amino acid residues at positions 156 and 332 in the E protein (E156 and E332) of SP were Ser and Lys (E156S/332K), respectively, whereas those in LP were Phe and Thr (E156F/332T). SP grew slightly faster than LP in vitro. The E protein of SP was N-glycosylated, whereas that of LP was not. Analysis using two recombinant single-mutant LP viruses, rKUNV-LP-EF156S and rKUNV-LP-ET332K, indicated that E156S enlarged plaques formed by LP, but E332K potently reduced them, regardless of the amino acid at E156. rKUNV-LP-EF156S showed significantly higher neuroinvasive ability than LP, SP, and rKUNV-LP-ET332K. Our results indicate that the low-pathogenic classical WNVKUN can easily change its pathogenicity through only a few amino acid substitutions in the E protein. It was also found that Phe at E156 of the rKUNV-LP-ET332K was easily changed to Ser during replication in vitro and in vivo, suggesting that E156S is advantageous for the propagation of WNVKUN in mammalian cells.

Validation of flavivirus infectious clones carrying fluorescent markers for antiviral drug screening and replication studies.

Flaviviruses have emerged as major arthropod-transmitted pathogens and represent an increasing public health problem worldwide. High-throughput screening can be facilitated using viruses that easily express detectable marker proteins. Therefore, developing molecular tools, such as reporter-carrying versions of flaviviruses, for studying viral replication and screening antiviral compounds represents a top priority. However, the engineering of flaviviruses carrying either fluorescent or luminescent reporters remains challenging due to the genetic instability caused by marker insertion; therefore, new approaches to overcome these limitations are needed. Here, we describe reverse genetic methods that include the design and validation of infectious clones of Zika, Kunjin, and Dengue viruses harboring different reporter genes for infection, rescue, imaging, and morphology using super-resolution microscopy. It was observed that different flavivirus constructs with identical designs displayed strikingly different genetic stabilities, and corresponding virions resembled wild-type virus particles in shape and size. A successful strategy was assessed to increase the stability of rescued reporter virus and permit antiviral drug screening based on quantitative automated fluorescence microscopy and replication studies.

Kunjin Virus, Zika Virus, and Yellow Fever Virus Infections Have Distinct Effects on the Coding Transcriptome and Proteome of Brain-Derived U87 Cells.

As obligate intracellular parasites, viruses rely heavily on host cells for replication, and therefore dysregulate several cellular processes for their benefit. In return, host cells activate multiple signaling pathways to limit viral replication and eradicate viruses. The present study explores the complex interplay between viruses and host cells through next generation RNA sequencing as well as mass spectrometry (SILAC). Both the coding transcriptome and the proteome of human brain-derived U87 cells infected with Kunjin virus, Zika virus, or Yellow Fever virus were compared to the transcriptome and the proteome of mock-infected cells. Changes in the abundance of several hundred mRNAs and proteins were found in each infection. Moreover, the alternative splicing of hundreds of mRNAs was found to be modulated upon viral infection. Interestingly, a significant disconnect between the changes in the transcriptome and those in the proteome of infected cells was observed. These findings provide a global view of the coding transcriptome and the proteome of Flavivirus-infected cells, leading to a better comprehension of Flavivirus-host interactions.

CMPK2 restricts Zika virus replication by inhibiting viral translation.

Flaviviruses continue to emerge as global health threats. There are currently no Food and Drug Administration (FDA) approved antiviral treatments for flaviviral infections. Therefore, there is a pressing need to identify host and viral factors that can be targeted for effective therapeutic intervention. Type I interferon (IFN-I) production in response to microbial products is one of the host's first line of defense against invading pathogens. Cytidine/uridine monophosphate kinase 2 (CMPK2) is a type I interferon-stimulated gene (ISG) that exerts antiviral effects. However, the molecular mechanism by which CMPK2 inhibits viral replication is unclear. Here, we report that CMPK2 expression restricts Zika virus (ZIKV) replication by specifically inhibiting viral translation and that IFN-I- induced CMPK2 contributes significantly to the overall antiviral response against ZIKV. We demonstrate that expression of CMPK2 results in a significant decrease in the replication of other pathogenic flaviviruses including dengue virus (DENV-2), Kunjin virus (KUNV) and yellow fever virus (YFV). Importantly, we determine that the N-terminal domain (NTD) of CMPK2, which lacks kinase activity, is sufficient to restrict viral translation. Thus, its kinase function is not required for CMPK2's antiviral activity. Furthermore, we identify seven conserved cysteine residues within the NTD as critical for CMPK2 antiviral activity. Thus, these residues may form an unknown functional site in the NTD of CMPK2 contributing to its antiviral function. Finally, we show that mitochondrial localization of CMPK2 is required for its antiviral effects. Given its broad antiviral activity against flaviviruses, CMPK2 is a promising potential pan-flavivirus inhibitor.

Arthropod-Borne Virus Surveillance as a Tool to Study the Australian Mosquito Virome

Mosquitoes (n = 4381 in 198 pools) were collected in March and April 2018 to survey the presence of West Nile virus Kunjin strain in mosquito populations around crocodile farms in the Darwin region of the Northern Territory (NT) of Australia. While no Kunjin virus was detected in these mosquitoes, we applied our viral replicative intermediates screening system termed monoclonal antibodies to viral RNA intermediates in cells or MAVRIC to this set of samples. This resulted in the detection of 28 pools with virus replicating in C6/36 mosquito cells and the identification of three insect viruses from three distinct virus classes. We demonstrate the persistence of the insect-specific flavivirus Palm Creek virus in Coquillettidia xanthogaster mosquitoes from Darwin over almost a decade, with limited genetic drift. We also detected a novel Hubei macula-like virus 3 strain in samples from two mosquito genera, suggesting the virus, for which the sequence was originally detected in spiders and soybean thrips, might be involved in a horizontal transmission cycle between arthropods and plants. Overall, these data demonstrate the strength of the optimized MAVRIC system and contribute to our general knowledge of the mosquito virome and insect viruses.

Kunjin virus

This datasheet on Kunjin virus covers Identity, Distribution, Hosts/Species Affected, Vectors & Intermediate Hosts.

Flavivirus replication kinetics in early-term placental cell lines with different differentiation pathways

BackgroundThe uncontrollable spread of Zika virus (ZIKV) in the Americas during 2015–2017, and its causal link to microcephaly in newborns and Guillain-Barré syndrome in adults, led the World Health Organisation to declare it a global public health emergency. One of the most notable features of ZIKV pathogenesis was the ability of the virus to pass the placental barrier to infect the growing foetus. This pathogenic trait had not been observed previously for medically important flaviviruses, including dengue and yellow fever viruses.MethodsIn this study we evaluated the replication kinetics of ZIKV and the related encephalitic flavivirus West Nile strain Kunjin virus (WNVKUN) in early-term placental cell lines.ResultsWe have observed that WNVKUN in fact replicates with a greater rate and to higher titres that ZIKV in these cell lines.ConclusionsThese results would indicate the potential for all flaviviruses to replicate in placental tissue but it is the ability to cross the placenta itself that is the restrictive factor in the clinical progression and presentation of congenital Zika syndrome.

Zika virus employs the host antiviral RNase L protein to support replication factory assembly

Infection with the flavivirus Zika virus (ZIKV) can result in tissue tropism, disease outcome, and route of transmission distinct from those of other flaviviruses; therefore, we aimed to identify host machinery that exclusively promotes the ZIKV replication cycle, which can inform on differences at the organismal level. We previously reported that deletion of the host antiviral ribonuclease L (RNase L) protein decreases ZIKV production. Canonical RNase L catalytic activity typically restricts viral infection, including that of the flavivirus dengue virus (DENV), suggesting an unconventional, proviral RNase L function during ZIKV infection. In this study, we reveal that an inactive form of RNase L supports assembly of ZIKV replication factories (RFs) to enhance infectious virus production. Compared with the densely concentrated ZIKV RFs generated with RNase L present, deletion of RNase L induced broader subcellular distribution of ZIKV replication intermediate double-stranded RNA (dsRNA) and NS3 protease, two constituents of ZIKV RFs. An inactive form of RNase L was sufficient to contain ZIKV genome and dsRNA within a smaller RF area, which subsequently increased infectious ZIKV release from the cell. Inactive RNase L can interact with cytoskeleton, and flaviviruses remodel cytoskeleton to construct RFs. Thus, we used the microtubule-stabilization drug paclitaxel to demonstrate that ZIKV repurposes RNase L to facilitate the cytoskeleton rearrangements required for proper generation of RFs. During infection with flaviviruses DENV or West Nile Kunjin virus, inactive RNase L did not improve virus production, suggesting that a proviral RNase L role is not a general feature of all flavivirus infections.

A Putative Lipid-Associating Motif in the West Nile Virus NS4A Protein Is Required for Efficient Virus Replication.

Flavivirus replication is intimately associated with re-organized cellular membranes. These virus-induced changes in membrane architecture form three distinct membranous “organelles” that have specific functions during the flavivirus life cycle. One of these structures is the replication complex in which the flaviviral RNA is replicated to produce progeny genomes. We have previously observed that this process is strictly dependent on cellular cholesterol. In this study we have identified a putative cholesterol recognition/interaction amino acid consensus (CRAC) motif within the West Nile virus strain Kunjin virus (WNVKUN) NS4A protein. Site-directed mutagenesis of this motif within a WNVKUN infectious clone severely attenuated virus replication and the capacity of the mutant viruses to form the replication complex. Replication of the mutant viruses also displayed reduced co-localization with cellular markers recruited to replication sites during wild-type virus replication. In addition, we observed that the mutant viruses were significantly impaired in their ability to remodel cytoplasmic membranes. However, after extensive analysis we are unable to conclusively reveal a role for the CRAC motif in direct cholesterol binding to NS4A, suggesting additional complex lipid-protein and protein-protein interactions. We believe this study highlights the crucial role for this region within NS4A protein in recruitment of cellular and viral proteins to specialized subdomains on membrane platforms to promote efficient virus replication.

Development of a Multivalent Kunjin Virus Reporter Virus-Like Particle System Inducing Seroconversion for Ebola and West Nile Virus Proteins in Mice.

Kunjin virus (KUNV) is an attenuated strain of the severe neurotropic West Nile virus (WNV). The virus has a single-strand positive-sense RNA genome that encodes a polyprotein. Following gene expression, the polyprotein is cleaved into structural proteins for viral packaging and nonstructural proteins for viral replication and expression. Removal of the structural genes generate subgenomic replicons that maintain replication capacity. Co-expression of these replicons with the viral structural genes produces reporter virus-like particles (RVPs) which infect cells in a single round. In this study, we aimed to develop a system to generate multivalent RVPs based on KUNV to elicit an immune response against different viruses. We selected the Ebola virus (EBOV) glycoprotein (GP) and the matrix protein (VP40) genes, as candidates to be delivered by KUNV RVPs. Initially, we enhanced the production of KUNV RVPs by generating a stable cell line expressing the KUNV packaging system comprising capsid, precursor membrane, and envelope. Transfection of the DNA-based KUNV replicon into this cell line resulted in an enhanced RVP production. The replicon was expressed in the stable cell line to produce the RVPs that allowed the delivery of EBOV GP and VP40 genes into other cells. Finally, we immunized BALB/cN mice with RVPs, resulting in seroconversion for EBOV GP, EBOV VP40, WNV nonstructural protein 1, and WNV E protein. Thus, our study shows that KUNV RVPs may function as a WNV vaccine candidate and RVPs can be used as a gene delivery system in the development of future EBOV vaccines.

A Hyperactive Kunjin Virus NS3 Helicase Mutant Demonstrates Increased Dissemination and Mortality in Mosquitoes.

The unwinding of double-stranded RNA intermediates is critical for the replication and packaging of flavivirus RNA genomes. This unwinding activity is achieved by the ATP-dependent nonstructural protein 3 (NS3) helicase. In previous studies, we investigated the mechanism of energy transduction between the ATP and RNA binding pockets using molecular dynamics simulations and enzymatic characterization. Our data corroborated the hypothesis that motif V is a communication hub for this energy transduction. More specifically, mutations T407A and S411A in motif V exhibit a hyperactive helicase phenotype, leading to the regulation of translocation and unwinding during replication. However, the effect of these mutations on viral infection in cell culture and in vivo is not well understood. Here, we investigated the role of motif V in viral replication using West Nile virus (Kunjin subtype) T407A and S411A mutants (T407A and S411A Kunjin, respectively) in cell culture and in vivo We were able to recover S411A Kunjin but unable to recover T407A Kunjin. Our results indicated that S411A Kunjin decreased viral infection and increased cytopathogenicity in cell culture compared to wild-type (WT) Kunjin. Similarly, decreased infection rates in surviving S411A Kunjin-infected Culex quinquefasciatus mosquitoes were observed, but S411A Kunjin infection resulted in increased mortality compared to WT Kunjin infection. Additionally, S411A Kunjin infection increased viral dissemination and saliva positivity rates in surviving mosquitoes compared to WT Kunjin infection. These data suggest that S411A Kunjin increases viral pathogenesis in mosquitoes. Overall, these data indicate that NS3 motif V may play a role in the pathogenesis, dissemination, and transmission efficiency of Kunjin virus.IMPORTANCE Kunjin and West Nile viruses belong to the arthropod-borne flaviviruses, which can result in severe symptoms, including encephalitis, meningitis, and death. Flaviviruses have expanded into new populations and emerged as novel pathogens repeatedly in recent years, demonstrating that they remain a global threat. Currently, there are no approved antiviral therapeutics against either Kunjin or West Nile viruses. Thus, there is a pressing need for understanding the pathogenesis of these viruses in humans. In this study, we investigated the role of the Kunjin virus helicase on infection in cell culture and in vivo This work provides new insight into how flaviviruses control pathogenesis and mosquito transmission through the nonstructural protein 3 helicase.

Flavivirus Cross-Reactivity to Dengue Nonstructural Protein 1 Antigen Detection Assays.

Dengue virus (DENV) and Zika virus (ZIKV) are flaviviruses of public health relevance. Both viruses circulate in the same endemic settings and acute infections generally manifest similar symptoms. This highlights the importance of accurate diagnosis for clinical management and outbreak control. One of the commonly used acute diagnostic markers for flaviviruses is nonstructural protein 1 (NS1). However, false positives due to antigenic cross-reactivity have been reported between DENV and ZIKV infections when using DENV NS1 antigen (NS1 Ag) detection assays in acute cases. Therefore, we investigated the lowest detectable virus titres and cross-reactivity of three commercial dengue NS1 Ag rapid assays and two ELISAs for different flaviviruses. Our results showed that substantially high viral titres of ZIKV, Kunjin virus (KUNV) and yellow fever virus (YFV) are required to give false-positive results when using DENV NS1 rapid detection assays. Commercial DENV NS1 ELISAs did not react with ZIKV and YFV. In comparison, tested assays detected DENV at a significantly low virus titre. Given the relatively low viral loads reported in clinical samples, our findings suggest that commercially available dengue NS1 Ag detection assays are less likely to generate false-positive results among clinical samples in areas where multiple flaviviruses cocirculate.

Neglected Australian Arboviruses Associated With Undifferentiated Febrile Illnesses.

Infections with commonly occurring Australian arthropod-borne arboviruses such as Ross River virus (RRV) and Barmah Forest virus (BFV) are diagnosed routinely by pathology laboratories in Australia. Others, such as Murray Valley encephalitis (MVEV) and Kunjin (KUNV) virus infections may be diagnosed by specialist reference laboratories. Although Alfuy (ALFV), Edge Hill (EHV), Kokobera (KOKV), Sindbis (SINV), and Stratford (STRV) viruses are known to infect humans in Australia, all are considered ‘neglected.’ The aetiologies of approximately half of all cases of undifferentiated febrile illnesses (UFI) in Australia are unknown and it is possible that some of these are caused by the neglected arboviruses. The aims of this study were to determine the seroprevalence of antibodies against several neglected Australian arboviruses among residents of Queensland, north-east Australia, and to ascertain whether any are associated with UFI. One hundred age- and sex-stratified human plasma samples from blood donors in Queensland were tested to determine the prevalence of neutralising antibodies against ALFV, BFV, EHV, KOKV, KUNV, MVEV, RRV, SINV, and STRV. The seroconversion rates for RRV and BFV infections were 1.3 and 0.3% per annum, respectively. The prevalence of antibodies against ALFV was too low to enable estimates of annual infection rates to be determined, but the values obtained for other neglected viruses, EHV (0.1%), KOKV (0.05%), and STRV (0.05%), indicated that the numbers of clinical infections occurring with these agents are likely to be extremely small. This was borne out by the observation that only 5.7% of a panel of 492 acute phase sera from UFI patients contained IgM against any of these arboviruses, as detected by an indirect immunofluorescence assay. While none of these neglected arboviruses appear to be a cause of a significant number of UFIs in Australia at this time, each has the potential to emerge as a significant human pathogen if there are changes to their ecological niches.

Budding induces symmetry defects in flavivirus virions

Flaviviruses assemble at the endoplasmic reticulum in an immature, non-infectious state that is designed to inhibit premature fusion with the host. Maturation of the particle occurs when it enters the trans-Golgi network, where low pH induces a conformational rearrangement of the virus glycoproteins. This transition causes 60 prM-E glycoprotein trimers in the immature virus to rearrange into 90 M-E dimers in the mature virus. We hypothesize that flaviviruses do not have exact icosahedral symmetry, thus providing flexibility for the large conformational rearrangements that are required during the virus life cycle. When icosahedral symmetry constraints were excluded in calculating the cryo-EM reconstruction of immature Kunjin virus (KUNV), a low virulent strain of West Nile virus, the nucleocapsid core touched the inside of the viral lipid membrane at the “proximal pole” and was asymmetrically positioned within the lipid bilayer envelope. The outer glycoprotein spikes on the “distal pole” were either distorted or missing. In the asymmetric reconstruction of mature KUNV, the core was re-positioned, as expected, concentric with the glycoprotein shell and there remained a distortion of the glycoproteins on one pole of the virion. This suggests that the interactions between the core and glycoproteins are altered during viral assembly and budding from the ER. In addition, it implies that the glycoproteins have a geometric defect that perhaps facilitates the transitions that occur during maturation. This defect in number and arrangement of the glycoproteins may reflect the consequence of membrane budding.

Flavivirus Species Recognition Based On the Polyprotein Coding Sequences

Method recognizing the flavivirus species, including a subtype recognition, that based on the genome sequence analysis, is proposed. This method takes into consideration frequency characteristics of amino acid codons in the coding sequences of full-length polyprotein of flavivirus genomes. High reliability of the method is proved in recognizing flavivirus genomes from 15 groups of different species and sub-types, that are sufficiently represented in the GenBank database. Ten various species of the flaviviruses, four sub-types of Dengue virus and Kunjin virus, that is suggested to be a sub-type of West Nile virus, are considered in the work.

Inflammatory responses to a pathogenic West Nile virus strain

BackgroundWest Nile virus (WNV) circulates across Australia and was referred to historically as Kunjin virus (WNVKUN). WNVKUN has been considered more benign than other WNV strains circulating globally. In 2011, a more virulent form of the virus emerged during an outbreak of equine arboviral disease in Australia.MethodsTo better understand the emergence of this virulent phenotype and the mechanism by which pathogenicity is manifested in its host, cells were infected with either the virulent strain (NSW2012), or less pathogenic historical isolates, and their innate immune responses compared by digital immune gene expression profiling. Two different cell systems were used: a neuroblastoma cell line (SK-N-SH cells) and neuronal cells derived from induced pluripotent stem cells (iPSCs).ResultsSignificant innate immune gene induction was observed in both systems. The NSW2012 isolate induced higher gene expression of two genes (IL-8 and CCL2) when compared with cells infected with less pathogenic isolates. Pathway analysis of induced inflammation-associated genes also indicated generally higher activation in infected NSW2012 cells. However, this differential response was not paralleled in the neuronal cultures.ConclusionNSW2012 may have unique genetic characteristics which contributed to the outbreak. The data herein is consistent with the possibility that the virulence of NSW2012 is underpinned by increased induction of inflammatory genes.

Antiviral activity of the FDA-approved drug candesartan cilexetil against Zika virus infection

Zika virus (ZIKV) is a mosquito-borne virus that has risen to prominence as a significant threat to public health in the recent decade. Since its re-emergence in 2007, ZIKV has spread at an alarming rate and has since become endemic to multiple regions around the world. Infections are primarily asymptomatic, however the virus has become associated with the development of severe neurological complications such as Guillain–Barré syndrome (GBS) and congenital microcephaly. At present, there are currently no approved antivirals for ZIKV infections. In this study, we utilised a phenotype-based screening platform to perform a high-throughput screen on a 1172-compound US FDA-approved drug library to identify potential novel inhibitors against ZIKV. Candesartan cilexetil, an angiotensin II receptor inhibitor, displayed potent inhibition effects against ZIKV and subsequent downstream time-course studies revealed that it targets a post-entry stage(s) of the ZIKV replication cycle. Moreover, candesartan cilexetil also inhibited viral RNA production and viral protein synthesis. Candesartan cilexetil also exhibited antiviral effects against Dengue virus serotype-2 (DENV2), Kunjin virus (KUNV) and Chikungunya virus (CHIKV), indicating that its antiviral properties may not be restricted to ZIKV. Our study has demonstrated for the first time the potential application of candesartan cilexetil as an antiviral.

Arbovirus surveillance using FTATM cards in modified CO2 -baited encephalitis virus surveillance traps in the Northern Territory, Australia.

In 2016, modified CO2 -baited encephalitis virus surveillance (EVS) traps were evaluated for flavivirus surveillance in the Northern Territory, Australia. The traps were fitted with honey-soaked nucleic acid preservation cards (FTATM ) for mosquitoes to expectorate virus while feeding on the cards. Cards were tested for the presence of selected arboviruses, with two cards testing positive for Kunjin virus and Alfuy, while sentinel chickens tested in parallel also showed Kunjin virus activity at the same time. The results from the cards and vector mosquito feeding rates indicate that CO2 -baited EVS traps coupled with honey-baited FTATM cards are an effective tool for broad-scale arbovirus surveillance.

Abstract B163: Regulation of translation by the interferon-induced antiviral protein viperin

Abstract The innate immune response involves the induction of hundreds of interferon-stimulated genes (ISGs), many of which play a role in cancer immunosurveillance and antiviral immunity. Therefore, understanding how ISGs function and coordinate with the cellular network is of critical importance. We focus on one such ISG, viperin (virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible). Viperin has been reported to inhibit a broad spectrum of DNA and RNA viruses, including many flaviviruses, human cytomegalovirus (HCMV), Chikungunya virus, influenza A virus, Sindbis virus, vesicular stomatitis virus, tick-borne encephalitis virus, HIV-1 and more. It is highly conserved in evolution, from protozoans to humans. Viperin is a peripheral membrane protein, associating on the cytosolic face of the ER via its N-terminal amphipathic helix. The cytosolic domain contains a radical SAM domain with a [4Fe-4S] cluster coordinated by three cysteine residues in the active site. Although the enzymatic function and substrate of viperin is still under debate, a recent study reveals that viperin catalyzes the conversion of cytidine triphosphate to 3′-deoxy-3′,4′-didehydro-CTP (ddhCTP) via its radical SAM activity. However, a general mechanism for viperin action and its potential roles within the innate immune response remain to be defined. Here, we demonstrate that viperin inhibits global protein translation via its radical SAM-dependent enzymatic activity. Using immortalized mouse macrophages from wild-type and viperin knockout mice we show that, although other ISGs are induced, type I IFN treatment of WT but not viperin KO mouse microphages leads to a global change in polysome profile with an increase in the ribosome fraction and a decrease in actively translating ribosomes. In parallel, the translational regulation function was evaluated in 293T-cells with transient transfection and doxycycline-inducible system. Despite no IFN stimulation and therefore no other ISG expression, viperin effectively represses translation. Furthermore, we find that viperin reduces global protein translation by ~30% in live cells using [35S]-methionine metabolic pulse-labeling or O-propargyl-puromycin (OPP) labelling. The radical SAM enzymatic activity is required for translational inhibition: site-directed mutagenesis of amino residues involved in iron-sulfur cluster and substrate binding abolishes the translational inhibition activity. We also demonstrate that ddhCTP, a recently identified vipern product, inhibits global protein translation in live cells. Viral replication requires the host translation machinery to make viral proteins, and our results suggest that much of its antiviral activity may be attributable to translational regulation. We show that viperin inhibits viral protein synthesis and viral replication of the Kunjin virus, a West Nile Virus variant, in tissue culture cells. Our study suggests a partially unifying mechanism for the broad antiviral function of viperin based on translational regulation. Citation Format: Chun-Chieh Hsu, Maudry Laurent-Rolle, Peter Cresswell. Regulation of translation by the interferon-induced antiviral protein viperin [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B163.