RNA viruses can hijack vertebrate microRNAs to suppress innate immunity.

Currently, there is little evidence for a notable role of the vertebrate microRNA (miRNA) system in the pathogenesis of RNA viruses. This is primarily attributed to the ease with which these viruses mutate to disrupt recognition and growth suppression by host miRNAs. Here we report that the haematopoietic-cell-specific miRNA miR-142-3p potently restricts the replication of the mosquito-borne North American eastern equine encephalitis virus in myeloid-lineage cells by binding to sites in the 3' non-translated region of its RNA genome. However, by limiting myeloid cell tropism and consequent innate immunity induction, this restriction directly promotes neurologic disease manifestations characteristic of eastern equine encephalitis virus infection in humans. Furthermore, the region containing the miR-142-3p binding sites is essential for efficient virus infection of mosquito vectors. We propose that RNA viruses can adapt to use antiviral properties of vertebrate miRNAs to limit replication in particular cell types and that this restriction can lead to exacerbation of disease severity.

Antibody Preparations from Human Transchromosomic Cows Exhibit Prophylactic and Therapeutic Efficacy against Venezuelan Equine Encephalitis Virus.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne RNA virus that causes low mortality but high morbidity rates in humans. In addition to natural outbreaks, there is the potential for exposure to VEEV via aerosolized virus particles. There are currently no FDA-licensed vaccines or antiviral therapies for VEEV. Passive immunotherapy is an approved method used to protect individuals against several pathogens and toxins. Human polyclonal antibodies (PAbs) are ideal, but this is dependent upon serum from convalescent human donors, which is in limited supply. Non-human-derived PAbs can have serious immunoreactivity complications, and when "humanized," these antibodies may exhibit reduced neutralization efficiency. To address these issues, transchromosomic (Tc) bovines have been created, which can produce potent neutralizing human antibodies in response to hyperimmunization. In these studies, we have immunized these bovines with different VEEV immunogens and evaluated the protective efficacy of purified preparations of the resultant human polyclonal antisera against low- and high-dose VEEV challenges. These studies demonstrate that prophylactic or therapeutic administration of the polyclonal antibody preparations (TcPAbs) can protect mice against lethal subcutaneous or aerosol challenge with VEEV. Furthermore, significant protection against unrelated coinfecting viral pathogens can be conferred by combining individual virus-specific TcPAb preparations.IMPORTANCE With the globalization and spread or potential aerosol release of emerging infectious diseases, it will be critical to develop platforms that are able to produce therapeutics in a short time frame. By using a transchromosomic (Tc) bovine platform, it is theoretically possible to produce antigen-specific highly neutralizing therapeutic polyclonal human antibody (TcPAb) preparations in 6 months or less. In this study, we demonstrate that Tc bovine-derived Venezuelan equine encephalitis virus (VEEV)-specific TcPAbs are highly effective against VEEV infection that mimics not only the natural route of infection but also infection via aerosol exposure. Additionally, we show that combinatorial TcPAb preparations can be used to treat coinfections with divergent pathogens, demonstrating that the Tc bovine platform could be beneficial in areas where multiple infectious diseases occur contemporaneously or in the case of multipathogen release.

The 17D-204 Vaccine Strain-Induced Protection against Virulent Yellow Fever Virus Is Mediated by Humoral Immunity and CD4+ but not CD8+ T Cells.

A gold standard of antiviral vaccination has been the safe and effective live-attenuated 17D-based yellow fever virus (YFV) vaccines. Among more than 500 million vaccinees, only a handful of cases have been reported in which vaccinees developed a virulent wild type YFV infection. This efficacy is presumed to be the result of both neutralizing antibodies and a robust T cell response. However, the particular immune components required for protection against YFV have never been evaluated. An understanding of the immune mechanisms that underlie 17D-based vaccine efficacy is critical to the development of next-generation vaccines against flaviviruses and other pathogens. Here we have addressed this question for the first time using a murine model of disease. Similar to humans, vaccination elicited long-term protection against challenge, characterized by high neutralizing antibody titers and a robust T cell response that formed long-lived memory. Both CD4+ and CD8+ T cells were polyfunctional and cytolytic. Adoptive transfer of immune sera or CD4+ T cells provided partial protection against YFV, but complete protection was achieved by transfer of both immune sera and CD4+ T cells. Thus, robust CD4+ T cell activity may be a critical contributor to protective immunity elicited by highly effective live attenuated vaccines.

Stable, high-level expression of reporter proteins from improved alphavirus expression vectors to track replication and dissemination during encephalitic and arthritogenic disease.

Engineered alphavirus vectors expressing reporters of infection have been used for a number of years due to their relatively low costs for analysis of virus replication and the capacity to utilize imaging systems for longitudinal measurements of growth within single animals. In general, these vectors have been derived from Old World alphaviruses using a second viral subgenomic promoter to express the transgenes, placed either immediately after the nonstructural proteins or at the 3' end of the viral coding sequences. However, the relevance of these vectors to natural infections is questionable, as they have not been rigorously tested for virulence in vivo in comparison with parental viruses or for the retention of the reporter during replication. Here, we report construction of new expression vectors for two Old World arthritogenic alphaviruses (Sindbis and Chikungunya viruses) and two New World encephalitic alphaviruses (eastern and Venezuelan equine encephalitis viruses) based upon either fusion of the reporter protein in frame within nonstructural protein 3 (nsP3) or insertion of the reporter as a cleavable element between the capsid and PE2 structural proteins. We have compared these with a traditional 3' double subgenomic promoter virus expressing either a large, firefly luciferase (fLuc; 1,650 nucleotides), or small, NanoLuc (nLuc; 513 nucleotides), luminescent reporter protein. Results indicate that the nLuc is substantially more stable than fLuc during repeated rounds of infection regardless of the transgene location. However, the capsid-PE2 insertion and nsP3 fusion viruses exhibit the most authentic mimicking of parental virus infection regardless of expressed protein. IMPORTANCE As more antiviral therapeutics and vaccines are developed, rapid and accurate in vivo modeling of their efficacy will be required. However, current alphavirus vectors expressing reporters of infection have not been extensively tested for accurate mimicking of the infection characteristics of unmodified parental viruses. Additionally, use of in vivo imaging systems detecting light emitted from luciferase reporters can significantly decrease costs associated with efficacy studies by minimizing numbers of animals. Herein we report development and testing of new expression vectors for Sindbis, Chikungunya, and eastern and Venezuelan equine encephalitis viruses and demonstrate that a small (∼500-nucleotide) reporter gene (NanoLuc; Promega) is very stable and causes a disease severity similar to that caused by unmodified parental viruses. In contrast, expression of larger reporters is very rapidly lost with virus replication and can be significantly attenuating. The utility of NanoLuc for in vivo imaging is also demonstrated.

Mosquito saliva serine protease enhances dissemination of dengue virus into the mammalian host.

Dengue virus (DENV), a flavivirus of global importance, is transmitted to humans by mosquitoes. In this study, we developed in vitro and in vivo models of saliva-mediated enhancement of DENV infectivity. Serine protease activity in Aedes aegypti saliva augmented virus infectivity in vitro by proteolyzing extracellular matrix proteins, thereby increasing viral attachment to heparan sulfate proteoglycans and inducing cell migration. A serine protease inhibitor reduced saliva-mediated enhancement of DENV in vitro and in vivo, marked by a 100-fold reduction in DENV load in murine lymph nodes. A saliva-mediated infectivity enhancement screen of fractionated salivary gland extracts identified serine protease CLIPA3 as a putative cofactor, and short interfering RNA knockdown of CLIPA3 in mosquitoes demonstrated its role in influencing DENV infectivity. Molecules in mosquito saliva that facilitate viral infectivity in the vertebrate host provide novel targets that may aid in the prevention of disease.

Comparison of the live attenuated yellow fever vaccine 17D-204 strain to its virulent parental strain Asibi by deep sequencing.

BACKGROUND: The first comparison of a live RNA viral vaccine strain to its wild-type parental strain by deep sequencing is presented using as a model the yellow fever virus (YFV) live vaccine strain 17D-204 and its wild-type parental strain, Asibi. METHODS: The YFV 17D-204 vaccine genome was compared to that of the parental strain Asibi by massively parallel methods. Variability was compared on multiple scales of the viral genomes. A modeled exploration of small-frequency variants was performed to reconstruct plausible regions of mutational plasticity. RESULTS: Overt quasispecies diversity is a feature of the parental strain, whereas the live vaccine strain lacks diversity according to multiple independent measurements. A lack of attenuating mutations in the Asibi population relative to that of 17D-204 was observed, demonstrating that the vaccine strain was derived by discrete mutation of Asibi and not by selection of genomes in the wild-type population. CONCLUSIONS: Relative quasispecies structure is a plausible correlate of attenuation for live viral vaccines. Analyses such as these of attenuated viruses improve our understanding of the molecular basis of vaccine attenuation and provide critical information on the stability of live vaccines and the risk of reversion to virulence.

Natural variation in the heparan sulfate binding domain of the eastern equine encephalitis virus E2 glycoprotein alters interactions with cell surfaces and virulence in mice.

Recently, we compared amino acid sequences of the E2 glycoprotein of natural North American eastern equine encephalitis virus (NA-EEEV) isolates and demonstrated that naturally circulating viruses interact with heparan sulfate (HS) and that this interaction contributes to the extreme neurovirulence of EEEV (C. L. Gardner, G. D. Ebel, K. D. Ryman, and W. B. Klimstra, Proc. Natl. Acad. Sci. U. S. A., 108:16026-16031, 2011). In the current study, we have examined the contribution to HS binding of each of three lysine residues in the E2 71-to-77 region that comprise the primary HS binding site of wild-type (WT) NA-EEEV viruses. We also report that the original sequence comparison identified five virus isolates, each with one of three amino acid differences in the E2 71-to-77 region, including mutations in residues critical for HS binding by the WT virus. The natural variant viruses, which possessed either a mutation from lysine to glutamine at E2 71, a mutation from lysine to threonine at E2 71, or a mutation from threonine to lysine at E2 72, exhibited altered interactions with heparan sulfate and cell surfaces and altered virulence in a mouse model of EEEV disease. An electrostatic map of the EEEV E1/E2 heterotrimer based upon the recent Chikungunya virus crystal structure (J. E. Voss, M. C. Vaney, S. Duquerroy, C. Vonrhein, C. Girard-Blanc, E. Crublet, A. Thompson, G. Bricogne, and F. A. Rey, Nature, 468:709-712, 2010) showed the HS binding site to be at the apical surface of E2, with variants affecting the electrochemical nature of the binding site. Together, these results suggest that natural variation in the EEEV HS binding domain may arise during EEEV sylvatic cycles and that this variation may influence receptor interaction and the severity of EEEV disease.

Heparan sulfate binding by natural eastern equine encephalitis viruses promotes neurovirulence.

The Alphavirus genus of the family Togaviridae contains mosquito-vectored viruses that primarily cause either arthritogenic disease or acute encephalitis. North American eastern equine encephalitis virus (NA-EEEV) is uniquely neurovirulent among encephalitic alphaviruses, causing mortality in a majority of symptomatic cases and neurological sequelae in many survivors. Unlike many alphaviruses, NA-EEEV infection of mice yields limited signs of febrile illness typically associated with lymphoid tissue replication. Rather, signs of brain infection, including seizures, are prominent. Use of heparan sulfate (HS) as an attachment receptor increases the neurovirulence of cell culture-adapted strains of Sindbis virus, an arthritogenic alphavirus. However, this receptor is not known to be used by naturally circulating alphaviruses. We demonstrate that wild-type NA-EEEV strain FL91-4679 uses HS as an attachment receptor and that the amino acid sequence of its E2 attachment protein is identical to those of natural isolates sequenced by RT-PCR amplification of field samples. This finding unequivocally confirms the use of HS receptors by naturally circulating NA-EEEV strains. Inactivation of the major HS binding domain in NA-EEEV E2 demonstrated that the HS binding increased brain replication and neurologic disease but reduced lymphoid tissue replication, febrile illness signs, and cytokine/chemokine induction in mice. We propose that HS binding by natural NA-EEEV strains alters tropism in vivo to antagonize/evade immune responses, and the extreme neurovirulence of wild-type NA-EEEV may be a consequence. Therefore, reinvestigation of HS binding by this and other arboviruses is warranted.

Host responses to alphavirus infection.

SUMMARY: Members of the Alphavirus genus are classified into New World and Old World groups based upon their disease characteristics and primary areas of endemicity. The two groups exhibit noteworthy differences in pathogenesis during human infection, with Old World viruses primarily causing febrile and arthritogenic diseases and the New World viruses causing encephalitis. In this review, we summarize the major factors contributing to disease manifestations observed in murine models of alphavirus infection. We concentrate upon differences between particular viruses as they relate to interaction with myeloid lineage cells (particularly dendritic cells and macrophages), both in terms of virus replication efficiency and host cell responses to infection. In addition, we discuss the effects of mutations acquired during cell culture-adaptation of alphaviruses upon our understanding of important factors in pathogenesis. Finally, we focus on the role of host innate immune responses, in particular the type I interferon (IFN-alpha/beta) system, in determining the permissivity of myeloid and other cell types. Recent contributions to the current understanding of identities and mechanisms of action of IFN-alpha/beta-induced antiviral effectors in vitro and in vivo are also discussed.

A mouse model for studying viscerotropic disease caused by yellow fever virus infection.

Mosquito-borne yellow fever virus (YFV) causes highly lethal, viscerotropic disease in humans and non-human primates. Despite the availability of efficacious live-attenuated vaccine strains, 17D-204 and 17DD, derived by serial passage of pathogenic YFV strain Asibi, YFV continues to pose a significant threat to human health. Neither the disease caused by wild-type YFV, nor the molecular determinants of vaccine attenuation and immunogenicity, have been well characterized, in large part due to the lack of a small animal model for viscerotropic YFV infection. Here, we describe a small animal model for wild-type YFV that manifests clinical disease representative of that seen in primates without adaptation of the virus to the host, which was required for the current hamster YF model. Investigation of the role of type I interferon (IFN-alpha/beta) in protection of mice from viscerotropic YFV infection revealed that mice deficient in the IFN-alpha/beta receptor (A129) or the STAT1 signaling molecule (STAT129) were highly susceptible to infection and disease, succumbing within 6-7 days. Importantly, these animals developed viscerotropic disease reminiscent of human YF, instead of the encephalitic signs typically observed in mice. Rapid viremic dissemination and extensive replication in visceral organs, spleen and liver, was associated with severe pathologies in these tissues and dramatically elevated MCP-1 and IL-6 levels, suggestive of a cytokine storm. In striking contrast, infection of A129 and STAT129 mice with the 17D-204 vaccine virus was subclinical, similar to immunization in humans. Although, like wild-type YFV, 17D-204 virus amplified within regional lymph nodes and seeded a serum viremia in A129 mice, infection of visceral organs was rarely established and rapidly cleared, possibly by type II IFN-dependent mechanisms. The ability to establish systemic infection and cause viscerotropic disease in A129 mice correlated with infectivity for A129-derived, but not WT129-derived, macrophages and dendritic cells in vitro, suggesting a role for these cells in YFV pathogenesis. We conclude that the ability of wild-type YFV to evade and/or disable components of the IFN-alpha/beta response may be primate-specific such that infection of mice with a functional IFN-alpha/beta antiviral response is attenuated. Consequently, subcutaneous YFV infection of A129 mice represents a biologically relevant model for studying viscerotropic infection and disease development following wild-type virus inoculation, as well as mechanisms of 17D-204 vaccine attenuation, without a requirement for adaptation of the virus.

Similarities and differences in antagonism of neuron alpha/beta interferon responses by Venezuelan equine encephalitis and Sindbis alphaviruses.

Venezuelan equine encephalitis virus (VEEV) is highly virulent in adult laboratory mice, while Sindbis virus (SINV) is avirulent regardless of dose or inoculation route, dependent upon functioning alpha/beta interferon (IFN-alpha/beta) responses. We have examined each virus' resistance to and/or antagonism of IFN-alpha/beta responses in neurons, a cell type targeted by both viruses in mice, by infecting IFN-alpha/beta-treated or untreated primary cultures with viruses or virus-derived replicons that lacked the structural proteins. Priming with IFN-alpha/beta prior to infection revealed that VEEV replication and progeny virion production were resistant to an established antiviral state while those of SINV were more sensitive. Postinfection IFN-alpha/beta treatment revealed that phosphorylation of STAT1 and STAT2 was partially blocked by infection with either virus, dependent upon expression of nonstructural proteins (nsP), but not structural proteins (sP). However, inhibition of STAT phosphorylation by VEEV replicons was not correlated with inhibition of IFN-stimulated gene (ISG) mRNA induction, yet ISG induction was inhibited when sP were present. Host translation was inhibited by VEEV nsP even when cells were pretreated with IFN-alpha/beta. SINV blocked ISG induction and translation, associated with nsP-mediated shutoff of macromolecular synthesis, but both activities were sensitive to IFN-alpha/beta pretreatment. We conclude that both VEEV and SINV limit ISG induction in infected neurons through shutoff of host transcription and translation but that inhibition by VEEV is more resistant to IFN-alpha/beta priming. Likewise, both viruses inhibit IFN receptor-initiated signaling, although the effect upon host responses is not clear. Finally, VEEV appears to be more resistant to effectors of the preestablished antiviral state.

Eastern and Venezuelan equine encephalitis viruses differ in their ability to infect dendritic cells and macrophages: impact of altered cell tropism on pathogenesis.

Eastern and Venezuelan equine encephalitis viruses (EEEV and VEEV, respectively) cause severe morbidity and mortality in equines and humans. Like other mosquito-borne viruses, VEEV infects dendritic cells (DCs) and macrophages in lymphoid tissues, fueling a serum viremia and facilitating neuroinvasion. In contrast, EEEV replicates poorly in lymphoid tissues, preferentially infecting osteoblasts. Here, we demonstrate that infectivity of EEEV for myeloid lineage cells including DCs and macrophages was dramatically reduced compared to that of VEEV, whereas both viruses replicated efficiently in mesenchymal lineage cells such as osteoblasts and fibroblasts. We determined that EEEV infection of myeloid lineage cells was restricted after attachment, entry, and uncoating of the genome. Using replicon particles and translation reporter RNAs, we found that translation of incoming EEEV genomes was almost completely inhibited in myeloid, but not mesenchymal, lineage cells. Alpha/beta interferon (IFN-alpha/beta) responses did not mediate the restriction, as infectivity was not restored in the absence of double-stranded RNA-dependent protein kinase, RNase L, or IFN-alpha/beta receptor-mediated signaling. We confirmed these observations in vivo, demonstrating that EEEV is compromised in its ability to replicate within lymphoid tissues, whereas VEEV does so efficiently. The altered tropism of EEEV correlated with an almost complete avoidance of serum IFN-alpha/beta induction in vivo, which may allow EEEV to evade the host's innate immune responses and thereby enhance neurovirulence. Taken together, our data indicate that inhibition of genome translation restricts EEEV infectivity for myeloid but not mesenchymal lineage cells in vitro and in vivo. In this regard, the tropisms of EEEV and VEEV differ dramatically, likely contributing to observed differences in disease etiology.

Alpha/beta interferon inhibits cap-dependent translation of viral but not cellular mRNA by a PKR-independent mechanism.

The alpha/beta interferon (IFN-alpha/beta) response is critical for host protection against disseminated replication of many viruses, primarily due to the transcriptional upregulation of genes encoding antiviral proteins. Previously, we determined that infection of mice with Sindbis virus (SB) could be converted from asymptomatic to rapidly fatal by elimination of this response (K. D. Ryman et al., J. Virol. 74:3366-3378, 2000). Probing of the specific antiviral proteins important for IFN-mediated control of virus replication indicated that the double-stranded RNA-dependent protein kinase, PKR, exerted some early antiviral effects prior to IFN-alpha/beta signaling; however, the ability of IFN-alpha/beta to inhibit SB and protect mice from clinical disease was essentially undiminished in the absence of PKR, RNase L, and Mx proteins (K. D. Ryman et al., Viral Immunol. 15:53-76, 2002). One characteristic of the PKR/RNase L/Mx-independent antiviral effect was a blockage of viral protein accumulation early after infection (K. D. Ryman et al., J. Virol. 79:1487-1499, 2005). We show here that IFN-alpha/beta priming induces a PKR-independent activity that inhibits m(7)G cap-dependent translation at a step after association of cap-binding factors and the small ribosome subunit but before formation of the 80S ribosome. Furthermore, the activity targets mRNAs that enter across the cytoplasmic membrane, but nucleus-transcribed RNAs are relatively unaffected. Therefore, this IFN-alpha/beta-induced antiviral activity represents a mechanism through which IFN-alpha/beta-exposed cells are defended against viruses that enter the cytoplasm, while preserving essential host activities, including the expression of antiviral and stress-responsive genes.

Gamma-interferon exerts a critical early restriction on replication and dissemination of yellow fever virus vaccine strain 17D-204.

Live attenuated viruses are historically among the most effective viral vaccines. Development of a safe vaccine requires the virus to be less virulent, a phenotype that is historically arrived by empirical evaluation often leaving the mechanisms of attenuation unknown. The yellow fever virus 17D live attenuated vaccine strain has been developed as a delivery vector for heterologous antigens; however, the mechanisms of attenuation remain elusive. The successful and safe progress of 17D as a vaccine vector and the development of live attenuated vaccines (LAVs) to related flaviviruses requires an understanding of the molecular mechanisms leading to attenuation. Using subcutaneous infection of interferon-deficient mouse models of wild type yellow fever virus (WT YFV) pathogenesis and 17D-mediated immunity, we found that, in the absence of type I IFN (IFN-α/ß), type II interferon (IFN-γ) restricted 17D replication, but not that of WT YFV, by 1-2 days post-infection. In this context, IFN-γ responses protected 17D-infected animals from mortality, largely restricted the virus to lymphoid organs, and eliminated viscerotropic disease signs such as steatosis in the liver and inflammatory cell infiltration into the spleen. However, WT YFV caused a disseminated infection, gross liver pathology, and rapid death of the animals. In vitro, IFN-γ treatment of myeloid cells suppressed the replication of 17D significantly more than that of WT YFV, suggesting a direct differential effect on 17D virus replication. Together these data indicate that an important mechanism of 17D attenuation in vivo is increased sensitivity to IFN-γ stimulated responses elicited early after infection.

The Interferon-Induced Exonuclease ISG20 Exerts Antiviral Activity through Upregulation of Type I Interferon Response Proteins.

Type I interferon (IFN)-stimulated genes (ISGs) have critical roles in inhibiting virus replication and dissemination. Despite advances in understanding the molecular basis of ISG restriction, the antiviral mechanisms of many remain unclear. The 20-kDa ISG ISG20 is a nuclear 3'-5' exonuclease with preference for single-stranded RNA (ssRNA) and has been implicated in the IFN-mediated restriction of several RNA viruses. Although the exonuclease activity of ISG20 has been shown to degrade viral RNA in vitro, evidence has yet to be presented that virus inhibition in cells requires this activity. Here, we utilized a combination of an inducible, ectopic expression system and newly generated Isg20-/- mice to investigate mechanisms and consequences of ISG20-mediated restriction. Ectopically expressed ISG20 localized primarily to Cajal bodies in the nucleus and restricted replication of chikungunya and Venezuelan equine encephalitis viruses. Although restriction by ISG20 was associated with inhibition of translation of infecting genomic RNA, degradation of viral RNAs was not observed. Instead, translation inhibition of viral RNA was associated with ISG20-induced upregulation of over 100 other genes, many of which encode known antiviral effectors. ISG20 modulated the production of IFIT1, an ISG that suppresses translation of alphavirus RNAs. Consistent with this observation, the pathogenicity of IFIT1-sensitive alphaviruses was increased in Isg20-/- mice compared to that of wild-type viruses but not in cells ectopically expressing ISG20. Our findings establish an indirect role for ISG20 in the early restriction of RNA virus replication by regulating expression of other ISGs that inhibit translation and possibly other activities in the replication cycle.IMPORTANCE The host immune responses to infection lead to the production of type I interferon (IFN), and the upregulation of interferon-stimulated genes (ISGs) reduces virus replication and virus dissemination within a host. Ectopic expression of the interferon-induced 20-kDa exonuclease ISG20 suppressed replication of chikungunya virus and Venezuelan equine encephalitis virus, two mosquito-vectored RNA alphaviruses. Since the replication of alphavirus genomes occurs exclusively in the cytoplasm, the mechanism of nucleus-localized ISG20 inhibition of replication is unclear. In this study, we determined that ISG20 acts as a master regulator of over 100 genes, many of which are ISGs. Specifically, ISG20 upregulated IFIT1 genes and inhibited translation of the alphavirus genome. Furthermore, IFIT1-sensitive alphavirus replication was increased in Isg20-/- mice compared to the replication of wild-type viruses but not in cells ectopically expressing ISG20. We propose that ISG20 acts as an indirect regulator of RNA virus replication in the cytoplasm through the upregulation of many other ISGs.

Interplay between Keratinocytes and Myeloid Cells Drives Dengue Virus Spread in Human Skin.

The skin is the site of dengue virus (DENV) transmission following the bite of an infected mosquito, but the contribution of individual cell types within skin to infection is unknown. We studied the dynamics of DENV infection in human skin explants using quantitative in situ imaging. DENV replicated primarily in the epidermis and induced a transient IFN-α response. DENV infected a wide range of cells, including Langerhans cells, macrophages, dermal dendritic cells, mast cells, fibroblasts, and lymphatic endothelium, but keratinocytes were the earliest targets of infection and made up 60% of infected cells over time. Virus inoculation led to recruitment and infection of Langerhans cells, macrophages, and dermal dendritic cells, and these cells emigrated from skin in increased numbers as a result of infection. DENV induced expression of proinflammatory cytokines and chemokines by infected keratinocytes. Blocking keratinocyte-derived IL-1ß alone reduced infection of Langerhans cells, macrophages, and dermal dendritic cells by 75-90% and reduced the overall number of infected cells in dermis by 65%. These data show that the innate response of infected keratinocytes attracts virus-permissive myeloid cells that inadvertently spread DENV infection. Our findings highlight a role for keratinocytes and their interplay with myeloid cells in dengue.

The Efficacy of the Interferon Alpha/Beta Response versus Arboviruses Is Temperature Dependent.

Interferon alpha/beta (IFN-α/ß) is a critical mediator of protection against most viruses, with host survival frequently impossible in its absence. Many studies have investigated the pathways involved in the induction of IFN-α/ß after virus infection and the resultant upregulation of antiviral IFN-stimulated genes (ISGs) through IFN-α/ß receptor complex signaling. However, other than examining the effects of genetic deletion of induction or effector pathway components, little is known regarding the functionality of these responses in intact hosts and whether host genetic or environmental factors might influence their potency. Here, we demonstrate that the IFN-α/ß response against multiple arthropod-vectored viruses, which replicate over a wide temperature range, is extremely sensitive to fluctuations in temperature, exhibiting reduced antiviral efficacy at subnormal cellular temperatures and increased efficacy at supranormal temperatures. The effect involves both IFN-α/ß and ISG upregulation pathways with a major aspect of altered potency reflecting highly temperature-dependent transcription of IFN response genes that leads to altered IFN-α/ß and ISG protein levels. Discordantly, signaling steps prior to transcription that were examined showed the opposite effect from gene transcription, with potentiation at low temperature and inhibition at high temperature. Finally, we demonstrate that by lowering the temperature of mice, chikungunya arbovirus replication and disease are exacerbated in an IFN-α/ß-dependent manner. This finding raises the potential for use of hyperthermia as a therapeutic modality for viral infections and in other contexts such as antitumor therapy. The increased IFN-α/ß efficacy at high temperatures may also reflect an innate immune-relevant aspect of the febrile response.IMPORTANCE The interferon alpha/beta (IFN-α/ß) response is a first-line innate defense against arthropod-borne viruses (arboviruses). Arboviruses, such as chikungunya virus (CHIKV), can infect cells and replicate across a wide temperature range due to their replication in both mammalian/avian and arthropod hosts. Accordingly, these viruses can cause human disease in tissues regularly exposed to temperatures below the normal mammalian core temperature, 37°C. We questioned whether temperature variation could affect the efficacy of IFN-α/ß responses against these viruses and help to explain some aspects of human disease manifestations. We observed that IFN-α/ß efficacy was dramatically lower at subnormal temperatures and modestly enhanced at febrile temperatures, with the effects involving altered IFN-α/ß response gene transcription but not IFN-α/ß pathway signaling. These results provide insight into the functioning of the IFN-α/ß response in vivo and suggest that temperature elevation may represent an immune-enhancing therapeutic modality for a wide variety of IFN-α/ß-sensitive infections and pathologies.

Electroporation of Alphavirus RNA Translational Reporters into Fibroblastic and Myeloid Cells as a Tool to Study the Innate Immune System.

The ability to transfect synthetic mRNAs into cells to measure processes such as translation efficiency or mRNA decay has been an invaluable tool in cell biology. The use of electroporation over other methods of transfection is an easy, inexpensive, highly efficient, and scalable method to introduce synthetic mRNA into a wide range of cell types. More recently, coupling of noncoding RNA sequences or protein coding regions from viral pathogens to fluorescent or bioluminescence proteins in RNA "reporters" has permitted study of host-pathogen interactions. These can range from virus infection of cells to translation of the viral genome, replication and stability of viral RNAs, or the efficacy of host antiviral responses. In this chapter, we describe a method for electroporating viral RNA reporters into both fibroblastic and myeloid cells that encode firefly or Renilla luciferase, whose reaction with specific substrates and light emitting activity is a measure of viral RNA translation efficiency. We have used this method to examine host interferon-dependent responses that inhibit viral translation along with identifying secondary structures in the 5' nontranslated region (NTR) and microRNA binding sites in the 3' NTR that are responsible for antagonizing the host innate immune responses and restricting viral cell tropism.

Host translation shutoff mediated by non-structural protein 2 is a critical factor in the antiviral state resistance of Venezuelan equine encephalitis virus.

Most previous studies of interferon-alpha/beta (IFN-α/ß) response antagonism by alphaviruses have focused upon interruption of IFN-α/ß induction and/or receptor signaling cascades. Infection of mice with Venezuelan equine encephalitis alphavirus (VEEV) or Sindbis virus (SINV) induces serum IFN-α/ß, that elicits a systemic antiviral state in uninfected cells successfully controlling SINV but not VEEV replication. Furthermore, VEEV replication is more resistant than that of SINV to a pre-existing antiviral state in vitro. While host macromolecular shutoff is proposed as a major antagonist of IFN-α/ß induction, the underlying mechanisms of alphavirus resistance to a pre-existing antiviral state are not fully defined, nor is the mechanism for the greater resistance of VEEV. Here, we have separated viral transcription and translation shutoff with multiple alphaviruses, identified the viral proteins that induce each activity, and demonstrated that VEEV nonstructural protein 2-induced translation shutoff is likely a critical factor in enhanced antiviral state resistance of this alphavirus.