A Zika virus-specific IgM elicited in pregnancy exhibits ultrapotent neutralization.

Congenital Zika virus (ZIKV) infection results in neurodevelopmental deficits in up to 14% of infants born to ZIKV-infected mothers. Neutralizing antibodies are a critical component of protective immunity. Here, we demonstrate that plasma IgM contributes to ZIKV immunity in pregnancy, mediating neutralization up to 3 months post-symptoms. From a ZIKV-infected pregnant woman, we isolated a pentameric ZIKV-specific IgM (DH1017.IgM) that exhibited ultrapotent ZIKV neutralization dependent on the IgM isotype. DH1017.IgM targets an envelope dimer epitope within domain II. The epitope arrangement on the virion is compatible with concurrent engagement of all ten antigen-binding sites of DH1017.IgM, a solution not available to IgG. DH1017.IgM protected mice against viremia upon lethal ZIKV challenge more efficiently than when expressed as an IgG. Our findings identify a role for antibodies of the IgM isotype in protection against ZIKV and posit DH1017.IgM as a safe and effective candidate immunotherapeutic, particularly during pregnancy.

Antiviral Effector RTP4 Bats against Flaviviruses.

Bats harbor diverse viruses and manifest distinct antiviral immune responses. Recently in Cell Host & Microbe, Boys et al. demonstrated that bat receptor transporter protein 4 (RTP4) is an innate antiviral effector that inhibits flavivirus replication, revealing an evolutionary arms race between flaviviruses and their hosts.

Why Is IFN-λ Less Inflammatory? One IRF Decides.

Type I and type III interferons (IFNs) activate similar antiviral transcriptional programs, but the type I IFN response is more inflammatory. In this issue of Immunity, Forero et al. find that selective induction of the transcription factor IRF1 promotes proinflammatory chemokine expression downstream of type I IFN signaling.

Shared and Distinct Functions of Type I and Type III Interferons.

Type I interferons (IFNs) (IFN-α, IFN-ß) and type III IFNs (IFN-λ) share many properties, including induction by viral infection, activation of shared signaling pathways, and transcriptional programs. However, recent discoveries have revealed context-specific functional differences. Here, we provide a comprehensive review of type I and type III IFN activities, highlighting shared and distinct features from molecular mechanisms through physiological responses. Beyond discussing canonical antiviral functions, we consider the adaptive immune priming, anti-tumor, and autoimmune functions of IFNs. We discuss a model wherein type III IFNs serve as a front-line defense that controls infection at epithelial barriers while minimizing damaging inflammatory responses, reserving the more potent type I IFN response for when local responses are insufficient. In this context, we discuss current therapeutic applications targeting these cytokine pathways and highlight gaps in understanding of the biology of type I and type III IFNs in health and disease.

Altered m6A Modification of Specific Cellular Transcripts Affects Flaviviridae Infection.

The RNA modification N6-methyladenosine (m6A) modulates mRNA fate and thus affects many biological processes. We analyzed m6A across the transcriptome following infection by dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), and hepatitis C virus (HCV). We found that infection by these viruses in the Flaviviridae family alters m6A modification of specific cellular transcripts, including RIOK3 and CIRBP. During viral infection, the addition of m6A to RIOK3 promotes its translation, while loss of m6A in CIRBP promotes alternative splicing. Importantly, viral activation of innate immune sensing or the endoplasmic reticulum (ER) stress response contributes to the changes in m6A in RIOK3 or CIRBP, respectively. Further, several transcripts with infection-altered m6A profiles, including RIOK3 and CIRBP, encode proteins that influence DENV, ZIKV, and HCV infection. Overall, this work reveals that cellular signaling pathways activated during viral infection lead to alterations in m6A modification of host mRNAs to regulate infection.

K63-linked polyubiquitination of transcription factor IRF1 is essential for IL-1-induced production of chemokines CXCL10 and CCL5.

Although interleukin 1 (IL-1) induces expression of the transcription factor IRF1 (interferon-regulatory factor 1), the roles of IRF1 in immune and inflammatory responses and mechanisms of its activation remain elusive. Here we found that IRF1 was essential for IL-1-induced expression of the chemokines CXCL10 and CCL5, which recruit mononuclear cells into sites of sterile inflammation. Newly synthesized IRF1 acquired Lys63 (K63)-linked polyubiquitination mediated by the apoptosis inhibitor cIAP2 that was enhanced by the bioactive lipid S1P. In response to IL-1, cIAP2 and the sphingosine kinase SphK1 (the enzyme that generates S1P) formed a complex with IRF1, which led to its activation. Thus, IL-1 triggered a hitherto unknown signaling cascade that controlled the induction of IRF1-dependent genes that encode molecules important for sterile inflammation.

Structure and neutralization mechanism of a human antibody targeting a complex Epitope on Zika virus.

We currently have an incomplete understanding of why only a fraction of human antibodies that bind to flaviviruses block infection of cells. Here we define the footprint of a strongly neutralizing human monoclonal antibody (mAb G9E) with Zika virus (ZIKV) by both X-ray crystallography and cryo-electron microscopy. Flavivirus envelope (E) glycoproteins are present as homodimers on the virion surface, and G9E bound to a quaternary structure epitope spanning both E protomers forming a homodimer. As G9E mainly neutralized ZIKV by blocking a step after viral attachment to cells, we tested if the neutralization mechanism of G9E was dependent on the mAb cross-linking E molecules and blocking low-pH triggered conformational changes required for viral membrane fusion. We introduced targeted mutations to the G9E paratope to create recombinant antibodies that bound to the ZIKV envelope without cross-linking E protomers. The G9E paratope mutants that bound to a restricted epitope on one protomer poorly neutralized ZIKV compared to the wild-type mAb, demonstrating that the neutralization mechanism depended on the ability of G9E to cross-link E proteins. In cell-free low pH triggered viral fusion assay, both wild-type G9E, and epitope restricted paratope mutant G9E bound to ZIKV but only the wild-type G9E blocked fusion. We propose that, beyond antibody binding strength, the ability of human antibodies to cross-link E-proteins is a critical determinant of flavivirus neutralization potency.

Neuroinvasive Flavivirus Pathogenesis Is Restricted by Host Genetic Factors in Collaborative Cross Mice, Independently of Oas1b.

Powassan virus (POWV) is an emerging tick-borne flavivirus that causes neuroinvasive diseases, including encephalitis, meningitis, and paralysis. Similar to other neuroinvasive flaviviruses, such as West Nile virus (WNV) and Japanese encephalitis virus (JEV), POWV disease presentation is heterogeneous, and the factors influencing disease outcome are not fully understood. We used Collaborative Cross (CC) mice to assess the impact of host genetic factors on POWV pathogenesis. We infected a panel of Oas1b-null CC lines with POWV and observed a range of susceptibility, indicating that host factors other than the well-characterized flavivirus restriction factor Oas1b modulate POWV pathogenesis in CC mice. Among the Oas1b-null CC lines, we identified multiple highly susceptible lines (0% survival), including CC071 and CC015, and two resistant lines, CC045 and CC057 (>75% survival). The susceptibility phenotypes generally were concordant among neuroinvasive flaviviruses, although we did identify one line, CC006, that was specifically resistant to JEV, suggesting that both pan-flavivirus and virus-specific mechanisms contribute to susceptibility phenotypes in CC mice. We found that POWV replication was restricted in bone marrow-derived macrophages from CC045 and CC057 mice, suggesting that resistance could result from cell-intrinsic restriction of viral replication. Although serum viral loads at 2 days postinfection were equivalent between resistant and susceptible CC lines, clearance of POWV from the serum was significantly enhanced in CC045 mice. Furthermore, CC045 mice had significantly lower viral loads in the brain at 7 days postinfection than did CC071 mice, suggesting that reduced central nervous system (CNS) infection contributes to the resistant phenotype of CC045 mice. IMPORTANCE Neuroinvasive flaviviruses, such as WNV, JEV, and POWV, are transmitted to humans by mosquitoes or ticks and can cause neurologic diseases, such as encephalitis, meningitis, and paralysis, and they can result in death or long-term sequelae. Although potentially severe, neuroinvasive disease is a rare outcome of flavivirus infection. The factors that determine whether someone develops severe disease after a flavivirus infection are not fully understood, but host genetic differences in polymorphic antiviral response genes likely contribute to the outcome of infection. We evaluated a panel of genetically diverse mice and identified lines with distinct outcomes following infection with POWV. We found that resistance to POWV pathogenesis corresponded to reduced viral replication in macrophages, more rapid clearance of virus in peripheral tissues, and reduced viral infection in the brain. These susceptible and resistant mouse lines will provide a system for investigating the pathogenic mechanisms of POWV and identifying polymorphic host genes that contribute to resistance.

Interferon-λ: Immune Functions at Barrier Surfaces and Beyond.

When type III interferon (IFN-λ; also known as interleukin-28 [IL-28] and IL-29) was discovered in 2003, its antiviral function was expected to be analogous to that of type I IFNs (IFN-α and IFN-ß) via the induction of IFN-stimulated genes (ISGs). Although IFN-λ stimulates expression of antiviral ISGs preferentially in cells of epithelial origin, recent studies have defined additional antiviral mechanisms in other cell types and tissues. Viral infection models using mice lacking IFN-λ signaling and SNP associations with human disease have expanded our understanding of the contribution of IFN-λ to the antiviral response at anatomic barriers and the immune response beyond these barriers. In this review, we highlight recent insights into IFN-λ functions, including its ability to restrict virus spread into the brain and to clear chronic viral infections in the gastrointestinal tract. We also discuss how IFN-λ modulates innate and adaptive immunity, autoimmunity, and tumor progression and its possible therapeutic applications in human disease.

Zika Virus Replicates in the Vagina of Mice with Intact Interferon Signaling.

Zika virus (ZIKV) is unusual among flaviviruses in its ability to spread between humans through sexual contact, as well as by mosquitoes. Sexual transmission has the potential to change the epidemiology and geographic range of ZIKV compared to mosquito-borne transmission and potentially could produce distinct clinical manifestations, so it is important to understand the host mechanisms that control susceptibility to sexually transmitted ZIKV. ZIKV replicates poorly in wild-type mice following subcutaneous inoculation, so most ZIKV pathogenesis studies use mice lacking type I interferon (IFN-αß) signaling (e.g., Ifnar1-/-). We found that wild-type mice support ZIKV replication following intravaginal infection, consistent with prior studies, although the infection remained localized to the lower female reproductive tract. Vaginal ZIKV infection required a high-progesterone state (pregnancy or pretreatment with depot medroxyprogesterone acetate [DMPA]) even in Ifnar1-/- mice that otherwise are highly susceptible to ZIKV infection. Progesterone-mediated susceptibility did not appear to result from a compromised epithelial barrier, blunted antiviral gene induction, or changes in vaginal leukocyte populations, leaving open the mechanism by which progesterone confers susceptibility to vaginal ZIKV infection. DMPA treatment is a key component of mouse vaginal infection models for herpes simplex virus and Chlamydia, but the mechanisms by which DMPA increases susceptibility to those pathogens also remain poorly defined. Understanding how progesterone mediates susceptibility to ZIKV vaginal infection may provide insights into host mechanisms influencing susceptibility to diverse sexually transmitted pathogens. IMPORTANCE Zika virus (ZIKV) is transmitted by mosquitoes, similar to other flaviviruses. However, ZIKV is unusual among flaviviruses in its ability also to spread through sexual transmission. We found that ZIKV was able to replicate in the vaginas of wild-type mice, even though these mice do not support ZIKV replication by other routes, suggesting that the vagina is particularly susceptible to ZIKV infection. Vaginal susceptibility was dependent on a high-progesterone state, which is a common feature of mouse vaginal infection models for other pathogens, through mechanisms that have remained poorly defined. Understanding how progesterone mediates susceptibility to ZIKV vaginal infection may provide insights into host mechanisms that influence susceptibility to diverse sexually transmitted pathogens.

Structurally Conserved Domains between Flavivirus and Alphavirus Fusion Glycoproteins Contribute to Replication and Infectious-Virion Production.

Alphaviruses and flaviviruses have class II fusion glycoproteins that are essential for virion assembly and infectivity. Importantly, the tip of domain II is structurally conserved between the alphavirus and flavivirus fusion proteins, yet whether these structural similarities between virus families translate to functional similarities is unclear. Using in vivo evolution of Zika virus (ZIKV), we identified several novel emerging variants, including an envelope glycoprotein variant in ß-strand c (V114M) of domain II. We have previously shown that the analogous ß-strand c and the ij loop, located in the tip of domain II of the alphavirus E1 glycoprotein, are important for infectivity. This led us to hypothesize that flavivirus E ß-strand c also contributes to flavivirus infection. We generated this ZIKV glycoprotein variant and found that while it had little impact on infection in mosquitoes, it reduced replication in human cells and mice and increased virus sensitivity to ammonium chloride, as seen for alphaviruses. In light of these results and given our alphavirus ij loop studies, we mutated a conserved alanine at the tip of the flavivirus ij loop to valine to test its effect on ZIKV infectivity. Interestingly, this mutation inhibited infectious virion production of ZIKV and yellow fever virus, but not West Nile virus. Together, these studies show that shared domains of the alphavirus and flavivirus class II fusion glycoproteins harbor structurally analogous residues that are functionally important and contribute to virus infection in vivo.IMPORTANCE Arboviruses are a significant global public health threat, yet there are no antivirals targeting these viruses. This problem is in part due to our lack of knowledge of the molecular mechanisms involved in the arbovirus life cycle. In particular, virus entry and assembly are essential processes in the virus life cycle and steps that can be targeted for the development of antiviral therapies. Therefore, understanding common, fundamental mechanisms used by different arboviruses for entry and assembly is essential. In this study, we show that flavivirus and alphavirus residues located in structurally conserved and analogous regions of the class II fusion proteins contribute to common mechanisms of entry, dissemination, and infectious-virion production. These studies highlight how class II fusion proteins function and provide novel targets for development of antivirals.

Flavivirus Envelope Protein Glycosylation: Impacts on Viral Infection and Pathogenesis.

Flaviviruses encode one, two, or no N-linked glycosylation sites on their envelope proteins. Glycosylation can impact virus interactions with cell surface attachment factors and also may impact virion stability and virus replication. Envelope protein glycosylation has been identified as a virulence determinant for multiple flaviviruses, but the mechanisms by which glycosylation mediates pathogenesis remain unclear. In this Gem, we summarize current knowledge on flavivirus envelope protein glycosylation and its impact on viral infection and pathogenesis.

Two Genetic Differences between Closely Related Zika Virus Strains Determine Pathogenic Outcome in Mice.

Recent Zika virus (ZIKV) outbreaks and unexpected clinical manifestations of ZIKV infection have prompted an increase in ZIKV-related research. Here, we identify two strain-specific determinants of ZIKV virulence in mice. We found that strain H/PF/2013 caused 100% lethality in Ifnar1-/- mice, whereas PRVABC59 caused no lethality; both strains caused 100% lethality in Ifnar1-/-Ifngr1-/- double-knockout (DKO) mice. Deep sequencing revealed a high-frequency variant in PRVABC59 not present in H/PF/2013: a G-to-T change at nucleotide 1965 producing a Val-to-Leu substitution at position 330 of the viral envelope (E) protein. We show that the V330 variant is lethal on both virus strain backgrounds, whereas the L330 variant is attenuating only on the PRVABC59 background. These results identify a balanced polymorphism in the E protein that is sufficient to attenuate the PRVABC59 strain but not H/PF/2013. The consensus sequences of H/PF/2013 and PRVABC59 differ by 3 amino acids, but these were not responsible for the difference in virulence between the two strains. H/PF/2013 and PRVABC59 differ by an additional 31 noncoding or silent nucleotide changes. We made a panel of chimeric viruses with identical amino acid sequences but nucleotide sequences derived from H/PF/2013 or PRVABC59. We found that 6 nucleotide differences in the 3' quarter of the H/PF/2013 genome were sufficient to confer virulence in Ifnar1-/- mice. Altogether, our work identifies a large and previously unreported difference in virulence between two commonly used ZIKV strains, in two widely used mouse models of ZIKV pathogenesis (Ifnar1-/- and Ifnar1-/- Ifngr1-/- DKO mice).IMPORTANCE Contemporary ZIKV strains are closely related and often used interchangeably in laboratory research. Here, we identify two strain-specific determinants of ZIKV virulence that are evident in only Ifnar1-/- mice but not Ifnar1-/-Ifngr1-/- DKO mice. These results identify a balanced polymorphism in the E protein that is sufficient to attenuate the PRVABC59 strain but not H/PF/2013. We further identify a second virulence determinant in the H/PF/2013 strain, which is driven by the viral nucleotide sequence but not the amino acid sequence. Altogether, our work identifies a large and previously unreported difference in virulence between two commonly used ZIKV strains, in two widely used mouse models of ZIKV pathogenesis. Our results highlight that even very closely related virus strains can produce significantly different pathogenic phenotypes in common laboratory models.

Envelope Protein Glycosylation Mediates Zika Virus Pathogenesis.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus. Recent ZIKV outbreaks have produced serious human disease, including neurodevelopmental malformations (congenital Zika syndrome) and Guillain-Barré syndrome. These outcomes were not associated with ZIKV infection prior to 2013, raising the possibility that viral genetic changes could contribute to new clinical manifestations. All contemporary ZIKV isolates encode an N-linked glycosylation site in the envelope (E) protein (N154), but this glycosylation site is absent in many historical ZIKV isolates. Here, we investigated the role of E protein glycosylation in ZIKV pathogenesis using two contemporary Asian-lineage strains (H/PF/2013 and PRVABC59) and the historical African-lineage strain (MR766). We found that glycosylated viruses were highly pathogenic in Ifnar1-/- mice. In contrast, nonglycosylated viruses were attenuated, producing lower viral loads in the serum and brain when inoculated subcutaneously but remaining neurovirulent when inoculated intracranially. These results suggest that E glycosylation is advantageous in the periphery but not within the brain. Accordingly, we found that glycosylation facilitated infection of cells expressing the lectins dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) or DC-SIGN-related (DC-SIGNR), suggesting that inefficient infection of lectin-expressing leukocytes could contribute to the attenuation of nonglycosylated ZIKV in mice.IMPORTANCE It is unclear why the ability of Zika virus (ZIKV) to cause serious disease, including Guillain-Barré syndrome and birth defects, was not recognized until recent outbreaks. One contributing factor could be genetic differences between contemporary ZIKV strains and historical ZIKV strains. All isolates from recent outbreaks encode a viral envelope protein that is glycosylated, whereas many historical ZIKV strains lack this glycosylation. We generated nonglycosylated ZIKV mutants from contemporary and historical strains and evaluated their virulence in mice. We found that nonglycosylated viruses were attenuated and produced lower viral loads in serum and brains. Our studies suggest that envelope protein glycosylation contributes to ZIKV pathogenesis, possibly by facilitating attachment to and infection of lectin-expressing leukocytes.

Development of Envelope Protein Antigens To Serologically Differentiate Zika Virus Infection from Dengue Virus Infection.

Zika virus (ZIKV) is an emerging flavivirus that can cause birth defects and neurologic complications. Molecular tests are effective for diagnosing acute ZIKV infection, although the majority of infections produce no symptoms at all or present after the narrow window in which molecular diagnostics are dependable. Serology is a reliable method for detecting infections after the viremic period; however, most serological assays have limited specificity due to cross-reactive antibodies elicited by flavivirus infections. Since ZIKV and dengue virus (DENV) widely cocirculate, distinguishing ZIKV infection from DENV infection is particularly important for diagnosing individual cases or for surveillance to coordinate public health responses. Flaviviruses also elicit type-specific antibodies directed to non-cross-reactive epitopes of the infecting virus; such epitopes are attractive targets for the design of antigens for development of serological tests with greater specificity. Guided by comparative epitope modeling of the ZIKV envelope protein, we designed two recombinant antigens displaying unique antigenic regions on domain I (Z-EDI) and domain III (Z-EDIII) of the ZIKV envelope protein. Both the Z-EDI and Z-EDIII antigens consistently detected ZIKV-specific IgG in ZIKV-immune sera but not cross-reactive IgG in DENV-immune sera in late convalescence (>12 weeks postinfection). In contrast, during early convalescence (2 to 12 weeks postinfection), secondary DENV-immune sera and some primary DENV-immune sera cross-reacted with the Z-EDI and Z-EDIII antigens. Analysis of sequential samples from DENV-immune individuals demonstrated that Z-EDIII cross-reactivity peaked in early convalescence and declined steeply over time. The Z-EDIII antigen has much potential as a diagnostic antigen for population-level surveillance and for detecting past infections in patients.

MAVS Is Essential for Primary CD4+ T Cell Immunity but Not for Recall T Cell Responses following an Attenuated West Nile Virus Infection.

The use of pathogen recognition receptor (PRR) agonists and the molecular mechanisms involved have been the major focus of research in individual vaccine development. West Nile virus (WNV) nonstructural (NS) 4B-P38G mutant has several features for an ideal vaccine candidate, including significantly reduced neuroinvasiveness, induction of strong adaptive immunity, and protection of mice from wild-type (WT) WNV infection. Here, we determined the role of mitochondrial antiviral signaling protein (MAVS), the adaptor protein for RIG-I-like receptor in regulating host immunity against the NS4B-P38G vaccine. We found that Mavs-/- mice were more susceptible to NS4B-P38G priming than WT mice. Mavs-/- mice had a transiently reduced production of antiviral cytokines and an impaired CD4+ T cell response in peripheral organs. However, antibody and CD8+ T cell responses were minimally affected. NS4B-P38G induced lower type I interferon (IFN), IFN-stimulating gene, and proinflammatory cytokine responses in Mavs-/- dendritic cells and subsequently compromised the antigen-presenting capacity for CD4+ T cells. Interestingly, Mavs-/- mice surviving NS4B-P38G priming were all protected from a lethal WT WNV challenge. NS4B-P38G-primed Mavs-/- mice exhibited equivalent levels of protective CD4+ T cell recall response, a modestly reduced WNV-specific IgM production, but more robust CD8+ T cell recall response. Taken together, our results suggest that MAVS is essential for boosting optimal primary CD4+ T cell responses upon NS4B-P38G vaccination and yet is dispensable for host protection and recall T cell responses during secondary WT WNV infection.IMPORTANCE The production of innate cytokines induced by the recognition of pathogen recognition receptors (PRRs) via their cognate ligands are critical for enhancing antigen-presenting cell functions and influencing T cell responses during microbial infection. The use of PRR agonists and the underlying molecular mechanisms have been the major focus in individual vaccine development. Here, we determined the role of mitochondrial antiviral-signaling protein (MAVS), the adaptor protein for RIG-I like receptor in regulating host immunity against the live attenuated West Nile virus (WNV) vaccine strain, the nonstructural (NS) 4B-P38G mutant. We found that MAVS is important for boosting optimal primary CD4+ T cell response during NS4B-P38G vaccination. However, MAVS is dispensable for memory T cell development and host protection during secondary wild-type WNV infection. Overall, these results may be utilized as a paradigm to aid in the rational development of other efficacious live attenuated flavivirus vaccines.

Lack of Durable Cross-Neutralizing Antibodies Against Zika Virus from Dengue Virus Infection.

Cross-reactive antibodies elicited by dengue virus (DENV) infection might affect Zika virus infection and confound serologic tests. Recent data demonstrate neutralization of Zika virus by monoclonal antibodies or human serum collected early after DENV infection. Whether this finding is true in late DENV convalescence (>6 months after infection) is unknown. We studied late convalescent serum samples from persons with prior DENV or Zika virus exposure. Despite extensive cross-reactivity in IgG binding, Zika virus neutralization was not observed among primary DENV infections. We observed low-frequency (23%) Zika virus cross-neutralization in repeat DENV infections. DENV-immune persons who had Zika virus as a secondary infection had distinct populations of antibodies that neutralized DENVs and Zika virus, as shown by DENV-reactive antibody depletion experiments. These data suggest that most DENV infections do not induce durable, high-level Zika virus cross-neutralizing antibodies. Zika virus-specific antibody populations develop after Zika virus infection irrespective of prior DENV immunity.

Zika Virus: New Clinical Syndromes and Its Emergence in the Western Hemisphere.

Zika virus (ZIKV) had remained a relatively obscure flavivirus until a recent series of outbreaks accompanied by unexpectedly severe clinical complications brought this virus into the spotlight as causing an infection of global public health concern. In this review, we discuss the history and epidemiology of ZIKV infection, recent outbreaks in Oceania and the emergence of ZIKV in the Western Hemisphere, newly ascribed complications of ZIKV infection, including Guillain-Barré syndrome and microcephaly, potential interactions between ZIKV and dengue virus, and the prospects for the development of antiviral agents and vaccines.

Interferon-Regulatory Factor 5-Dependent Signaling Restricts Orthobunyavirus Dissemination to the Central Nervous System.

UNLABELLED: Interferon (IFN)-regulatory factor 5 (IRF-5) is a transcription factor that induces inflammatory responses after engagement and signaling by pattern recognition receptors. To define the role of IRF-5 during bunyavirus infection, we evaluated Oropouche virus (OROV) and La Crosse virus (LACV) pathogenesis and immune responses in primary cells and in mice with gene deletions in Irf3, Irf5, and Irf7 or in Irf5 alone. Deletion of Irf3, Irf5, and Irf7 together resulted in uncontrolled viral replication in the liver and spleen, hypercytokinemia, extensive liver injury, and an early-death phenotype. Remarkably, deletion of Irf5 alone resulted in meningoencephalitis and death on a more protracted timeline, 1 to 2 weeks after initial OROV or LACV infection. The clinical signs in OROV-infected Irf5(-/-) mice were associated with abundant viral antigen and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells in several regions of the brain. Circulating dendritic cell (DC) subsets in Irf5(-/-) mice had higher levels of OROV RNA in vivo yet produced lower levels of type I IFN than wild-type (WT) cells. This result was supported by data obtained in vitro, since a deficiency of IRF-5 resulted in enhanced OROV infection and diminished type I IFN production in bone marrow-derived DCs. Collectively, these results indicate a key role for IRF-5 in modulating the host antiviral response in peripheral organs that controls bunyavirus neuroinvasion in mice. IMPORTANCE: Oropouche virus (OROV) and La Crosse virus (LACV) are orthobunyaviruses that are transmitted by insects and cause meningitis and encephalitis in subsets of individuals in the Americas. Recently, we demonstrated that components of the type I interferon (IFN) induction pathway, particularly the regulatory transcription factors IRF-3 and IRF-7, have key protective roles during OROV infection. However, the lethality in Irf3(-/-) Irf7(-/-) (DKO) mice infected with OROV was not as rapid or complete as observed in Ifnar(-/-) mice, indicating that other transcriptional factors associated with an IFN response contribute to antiviral immunity against OROV. Here, we evaluated bunyavirus replication, tissue tropism, and cytokine production in primary cells and mice lacking IRF-5. We demonstrate an important role for IRF-5 in preventing neuroinvasion and the ensuing encephalitis caused by OROV and LACV.

Oropouche virus infection and pathogenesis are restricted by MAVS, IRF-3, IRF-7, and type I interferon signaling pathways in nonmyeloid cells.

UNLABELLED: Oropouche virus (OROV) is a member of the Orthobunyavirus genus in the Bunyaviridae family and a prominent cause of insect-transmitted viral disease in Central and South America. Despite its clinical relevance, little is known about OROV pathogenesis. To define the host defense pathways that control OROV infection and disease, we evaluated OROV pathogenesis and immune responses in primary cells and mice that were deficient in the RIG-I-like receptor signaling pathway (MDA5, RIG-I, or MAVS), downstream regulatory transcription factors (IRF-3 or IRF-7), beta interferon (IFN-ß), or the receptor for type I IFN signaling (IFNAR). OROV replicated to higher levels in primary fibroblasts and dendritic cells lacking MAVS signaling, the transcription factors IRF-3 and IRF-7, or IFNAR than in wild-type (WT) cells. In mice, deletion of IFNAR, MAVS, or IRF-3 and IRF-7 resulted in uncontrolled OROV replication, hypercytokinemia, extensive liver damage, and death, whereas WT congenic animals failed to develop disease. Unexpectedly, mice with a selective deletion of IFNAR on myeloid cells (CD11c Cre(+) Ifnar(f/f) or LysM Cre(+) Ifnar(f/f)) did not sustain enhanced disease with OROV or a selective (flox/flox) deletion La Crosse virus, a closely related encephalitic orthobunyavirus. In bone marrow chimera studies, recipient irradiated Ifnar(-/-) mice reconstituted with WT hematopoietic cells sustained high levels of OROV replication and liver damage, whereas WT mice reconstituted with Ifnar(-/-) bone marrow were resistant to disease. Collectively, these results establish a dominant protective role for MAVS, IRF-3 and IRF-7, and IFNAR in restricting OROV infection and tissue injury and suggest that IFN signaling in nonmyeloid cells contributes to the host defense against orthobunyaviruses. IMPORTANCE: Oropouche virus (OROV) is an emerging arthropod-transmitted orthobunyavirus that causes episodic outbreaks of a debilitating febrile illness in humans in countries of South and Central America. The continued expansion of the range and number of its arthropod vectors increases the likelihood that OROV will spread into new regions. At present, the pathogenesis of OROV in humans or other vertebrate animals remains poorly understood. To define cellular mechanisms of control of OROV infection, we performed infection studies in a series of primary cells and mice that were deficient in key innate immune genes involved in pathogen recognition and control. Our results establish that a MAVS-dependent type I IFN signaling pathway has a dominant role in restricting OROV infection and pathogenesis in vivo.