RESUMEN
Mosquitoes transmit many disease-relevant flaviviruses. Efficient viral transmission to mammalian hosts requires mosquito salivary factors. However, the specific salivary components facilitating viral transmission and their mechanisms of action remain largely unknown. Here, we show that a female mosquito salivary gland-specific protein, here named A. aegypti Neutrophil Recruitment Protein (AaNRP), facilitates the transmission of Zika and dengue viruses. AaNRP promotes a rapid influx of neutrophils, followed by virus-susceptible myeloid cells toward mosquito bite sites, which facilitates establishment of local infection and systemic dissemination. Mechanistically, AaNRP engages TLR1 and TLR4 of skin-resident macrophages and activates MyD88-dependent NF-κB signaling to induce the expression of neutrophil chemoattractants. Inhibition of MyD88-NF-κB signaling with the dietary phytochemical resveratrol reduces AaNRP-mediated enhancement of flavivirus transmission by mosquitoes. These findings exemplify how salivary components can aid viral transmission, and suggest a potential prophylactic target.
Asunto(s)
Aedes , Virus Zika , Animales , Aedes/virología , Aedes/metabolismo , Femenino , Virus Zika/fisiología , Ratones , Virus del Dengue/fisiología , Proteínas y Péptidos Salivales/metabolismo , Mosquitos Vectores/virología , Proteínas de Insectos/metabolismo , Células Mieloides/virología , Células Mieloides/metabolismo , Infección por el Virus Zika/transmisión , Infección por el Virus Zika/virología , Infección por el Virus Zika/metabolismo , Dengue/transmisión , Dengue/virología , Dengue/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Factor 88 de Diferenciación Mieloide/metabolismo , Factor 88 de Diferenciación Mieloide/genéticaRESUMEN
Antigenic similarities between Zika virus (ZIKV) and other flaviviruses pose challenges to the development of virus-specific diagnostic tools and effective vaccines. Starting with a DNA-encoded one-bead-one-compound combinatorial library of 508,032 synthetic, non-natural oligomers, we selected and characterized small molecules that mimic ZIKV epitopes. High-throughput fluorescence-activated cell sorter-based bead screening was used to select molecules that bound IgG from ZIKV-immune but not from dengue-immune sera. Deep sequencing of the DNA from the "Zika-only" beads identified 40 candidate molecular structures. A lead candidate small molecule "CZV1-1" was selected that correctly identifies serum specimens from Zika-experienced patients with good sensitivity and specificity (85.3% and 98.4%, respectively). Binding competition studies of purified anti-CZV1-1 IgG against known ZIKV-specific monoclonal antibodies (mAbs) showed that CZV1-1 mimics a nonlinear, neutralizing conformational epitope in the domain III of the ZIKV envelope. Purified anti-CZV1-1 IgG neutralized infection of ZIKV in cell cultures with potencies comparable to highly specific ZIKV-neutralizing mAbs. This study demonstrates an innovative approach for identification of synthetic non-natural molecular mimics of conformational virus epitopes. Such molecular mimics may have value in the development of accurate diagnostic assays for Zika, as well as for other viruses.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Epítopos , Infección por el Virus Zika , Virus Zika , Virus Zika/inmunología , Epítopos/inmunología , Humanos , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/virología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Inmunoglobulina G/inmunología , Anticuerpos Monoclonales/inmunología , Imitación Molecular/inmunologíaRESUMEN
During their blood-feeding process, ticks are known to transmit various viruses to vertebrates, including humans. Recent viral metagenomic analyses using next-generation sequencing (NGS) have revealed that blood-feeding arthropods like ticks harbor a large diversity of viruses. However, many of these viruses have not been isolated or cultured, and their basic characteristics remain unknown. This study aimed to present the identification of a difficult-to-culture virus in ticks using NGS and to understand its epidemic dynamics using molecular biology techniques. During routine tick-borne virus surveillance in Japan, an unknown flaviviral sequence was detected via virome analysis of host-questing ticks. Similar viral sequences have been detected in the sera of sika deer and wild boars in Japan, and this virus was tentatively named the Saruyama virus (SAYAV). Because SAYAV did not propagate in any cultured cells tested, single-round infectious virus particles (SRIP) were generated based on its structural protein gene sequence utilizing a yellow fever virus-based replicon system to understand its nationwide endemic status. Seroepidemiological studies using SRIP as antigens have demonstrated the presence of neutralizing antibodies against SAYAV in sika deer and wild boar captured at several locations in Japan, suggesting that SAYAV is endemic throughout Japan. Phylogenetic analyses have revealed that SAYAV forms a sister clade with the Orthoflavivirus genus, which includes important mosquito- and tick-borne pathogenic viruses. This shows that SAYAV evolved into a lineage independent of the known orthoflaviviruses. This study demonstrates a unique approach for understanding the epidemiology of uncultured viruses by combining viral metagenomics and pseudoinfectious viral particles.
Asunto(s)
Ciervos , Flavivirus , Metagenómica , Garrapatas , Animales , Metagenómica/métodos , Japón/epidemiología , Ciervos/virología , Flavivirus/genética , Flavivirus/aislamiento & purificación , Flavivirus/clasificación , Garrapatas/virología , Filogenia , Viroma/genética , Virión/genética , Sus scrofa/virología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estudios Seroepidemiológicos , Genoma ViralRESUMEN
Mosquito-borne flaviviruses such as dengue (DENV) and Zika (ZIKV) cause hundreds of millions of infections annually. The single-stranded RNA genome of flaviviruses is translated into a polyprotein, which is cleaved equally into individual functional proteins. While structural proteins are packaged into progeny virions and released, most of the nonstructural proteins remain intracellular and could become cytotoxic if accumulated over time. However, the mechanism by which nonstructural proteins are maintained at the levels optimal for cellular fitness and viral replication remains unknown. Here, we identified that the ubiquitin E3 ligase HRD1 is essential for flaviviruses infections in both mammalian hosts and mosquitoes. HRD1 directly interacts with flavivirus NS4A and ubiquitylates a conserved lysine residue for ER-associated degradation. This mechanism avoids excessive accumulation of NS4A, which otherwise interrupts the expression of processed flavivirus proteins in the ER. Furthermore, a small-molecule inhibitor of HRD1 named LS-102 effectively interrupts DENV2 infection in both mice and Aedes aegypti mosquitoes, and significantly disturbs DENV transmission from the infected hosts to mosquitoes owing to reduced viremia. Taken together, this study demonstrates that flaviviruses have evolved a sophisticated mechanism to exploit the ubiquitination system to balance the homeostasis of viral proteins for their own advantage and provides a potential therapeutic target to interrupt flavivirus infection and transmission.
Asunto(s)
Aedes , Infecciones por Flavivirus , Flavivirus , Infección por el Virus Zika , Virus Zika , Animales , Ratones , Flavivirus/genética , Virus Zika/genética , Ubiquitina/metabolismo , Ligasas/metabolismo , Proteínas Virales/metabolismo , MamíferosRESUMEN
The ZIKA virus (ZIKV) evades the host immune response by degrading STAT2 through its NS5 protein, thereby inhibiting type I interferon (IFN)-mediated antiviral immunity. However, the molecular mechanism underlying this process has remained elusive. In this study, we performed a genome-wide CRISPR/Cas9 screen, revealing that ZSWIM8 as the substrate receptor of Cullin3-RING E3 ligase is required for NS5-mediated STAT2 degradation. Genetic depletion of ZSWIM8 and CUL3 substantially impeded NS5-mediated STAT2 degradation. Biochemical analysis illuminated that NS5 enhances the interaction between STAT2 and the ZSWIM8-CUL3 E3 ligase complex, thereby facilitating STAT2 ubiquitination. Moreover, ZSWIM8 knockout endowed A549 and Huh7 cells with partial resistance to ZIKV infection and protected cells from the cytopathic effects induced by ZIKV, which was attributed to the restoration of STAT2 levels and the activation of IFN signaling. Subsequent studies in a physiologically relevant model, utilizing human neural progenitor cells, demonstrated that ZSWIM8 depletion reduced ZIKV infection, resulting from enhanced IFN signaling attributed to the sustained levels of STAT2. Our findings shed light on the role of ZIKV NS5, serving as the scaffold protein, reprograms the ZSWIM8-CUL3 E3 ligase complex to orchestrate STAT2 proteasome-dependent degradation, thereby facilitating evasion of IFN antiviral signaling. Our study provides unique insights into ZIKV-host interactions and holds promise for the development of antivirals and prophylactic vaccines.
Asunto(s)
Proteínas Cullin , Interferón Tipo I , Proteolisis , Factor de Transcripción STAT2 , Transducción de Señal , Ubiquitina-Proteína Ligasas , Ubiquitinación , Proteínas no Estructurales Virales , Infección por el Virus Zika , Virus Zika , Humanos , Factor de Transcripción STAT2/metabolismo , Virus Zika/inmunología , Virus Zika/fisiología , Virus Zika/metabolismo , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Interferón Tipo I/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Infección por el Virus Zika/metabolismo , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/virología , Proteínas Cullin/metabolismo , Células A549 , Células HEK293 , Sistemas CRISPR-CasRESUMEN
West Nile virus (WNV) is an arthropod-borne, positive-sense RNA virus that poses an increasing global threat due to warming climates and lack of effective therapeutics. Like other enzootic viruses, little is known about how host context affects the structure of the full-length RNA genome. Here, we report a complete secondary structure of the entire WNV genome within infected mammalian and arthropod cell lines. Our analysis affords structural insights into multiple, conserved aspects of flaviviral biology. We show that the WNV genome folds with minimal host dependence, and we prioritize well-folded regions for functional validation using structural homology between hosts as a guide. Using structure-disrupting, antisense locked nucleic acids, we then demonstrate that the WNV genome contains riboregulatory structures with conserved and host-specific functional roles. These results reveal promising RNA drug targets within flaviviral genomes, and they highlight the therapeutic potential of ASO-LNAs as both WNV-specific and pan-flaviviral therapeutic agents.
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Genoma Viral , ARN Viral , Virus del Nilo Occidental , Virus del Nilo Occidental/genética , Animales , ARN Viral/genética , ARN Viral/metabolismo , Humanos , Línea Celular , Conformación de Ácido Nucleico , Fiebre del Nilo Occidental/virología , Especificidad del Huésped/genética , Interacciones Huésped-Patógeno/genéticaRESUMEN
Flaviviruses such as Zika (ZIKV) and dengue virus (DENV) are positive-sense RNA viruses belonging to Flaviviridae The flavivirus genome contains a 5' end stem-loop promoter sequence known as stem-loop A (SLA) that is recognized by the flavivirus polymerase NS5 during viral RNA synthesis and 5' guanosine cap methylation. The crystal structures of ZIKV and DENV SLAs show a well-defined fold, consisting of a bottom stem, side loop, and top stem-loop, providing unique interaction sites for small molecule inhibitors to disrupt the promoter function. To facilitate the identification of small molecule binding sites in flavivirus SLA, we determined high-resolution structures of the bottom and top stems of ZIKV SLA, which contain a single U- or G-bulge, respectively. Both bulge nucleotides exhibit multiple orientations, from folded back on the adjacent nucleotide to flipped out of the helix, and are stabilized by stacking or base triple interactions. These structures suggest that even a single unpaired nucleotide can provide flexibility to RNA structures, and its conformation is mainly determined by the stabilizing chemical environment. To facilitate discovery of small molecule inhibitors that interfere with the functions of ZIKV SLA, we screened and identified compounds that bind to the bottom and top stems of ZIKV SLA.
Asunto(s)
Conformación de Ácido Nucleico , ARN Viral , Bibliotecas de Moléculas Pequeñas , Virus Zika , Virus Zika/genética , Virus Zika/efectos de los fármacos , ARN Viral/química , ARN Viral/genética , ARN Viral/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Sitios de Unión , Antivirales/farmacología , Antivirales/química , Cristalografía por Rayos X , Modelos Moleculares , Regiones Promotoras GenéticasRESUMEN
Many viruses have evolved structured RNA elements that can influence transcript abundance and translational efficiency, and help evade host immune factors by hijacking cellular machinery during replication. Here, we evaluated the functional impact of sub-genomic flaviviral RNAs (sfRNAs) known to stall exoribonuclease activity, by incorporating these elements into recombinant adeno-associated viral (AAV) genome cassettes. Specifically, sfRNAs from Dengue, Zika, Japanese Encephalitis, Yellow Fever, Murray Valley Encephalitis, and West Nile viruses increased transcript stability and transgene expression compared to a conventional woodchuck hepatitis virus element (WPRE). Further dissection of engineered transcripts revealed that sfRNA elements (i) require incorporation in cis within the 3' untranslated region (UTR) of AAV genomes, (ii) require minimal dumbbell structures to exert the observed effects, and (iii) can stabilize AAV transcripts independent of 5'-3' exoribonuclease 1 (XRN1)-mediated decay. Additionally, preliminary in vivo assessment of AAV vectors bearing sfRNA elements in mice achieved increased transcript abundance and expression in cardiac tissue. Leveraging the functional versatility of engineered viral RNA elements may help improve the potency of AAV vector-based gene therapies. IMPORTANCE: Viral RNA elements can hijack host cell machinery to control stability of transcripts and consequently, infection. Studies that help better understand such viral elements can provide insights into antiviral strategies and also potentially leverage these features for therapeutic applications. In this study, by incorporating structured flaviviral RNA elements into recombinant adeno-associated viral (AAV) vector genomes, we show improved AAV transcript stability and transgene expression can be achieved, with implications for gene transfer.
Asunto(s)
Dependovirus , Vectores Genéticos , ARN Viral , Dependovirus/genética , Animales , ARN Viral/genética , ARN Viral/metabolismo , Vectores Genéticos/genética , Ratones , Humanos , Estabilidad del ARN , Flaviviridae/genética , Transgenes , Células HEK293 , Genoma Viral , Regiones no Traducidas 3'/genética , Exorribonucleasas/metabolismo , Exorribonucleasas/genéticaRESUMEN
Japanese encephalitis virus (JEV) is an arthropod-borne, plus-strand flavivirus causing viral encephalitis in humans with a high case fatality rate. The JEV non-structural protein 5 (NS5) with the RNA-dependent RNA polymerase activity interacts with the viral and host proteins to constitute the replication complex. We have identified the multifunctional protein Nucleolin (NCL) as one of the several NS5-interacting host proteins. We demonstrate the interaction and colocalization of JEV NS5 with NCL in the virus-infected HeLa cells. The siRNA-mediated knockdown of NCL indicated that it was required for efficient viral replication. Importantly, JEV grew to higher titers in cells over-expressing exogenous NCL, demonstrating its pro-viral role. We demonstrated that NS5 interacted with the RRM and GAR domains of NCL. We show that the NCL-binding aptamer AS1411 containing the G-quadruplex (GQ) structure and the GQ ligand BRACO-19 caused significant inhibition of JEV replication. The antiviral effect of AS1411 and BRACO-19 could be overcome in HeLa cells by the overexpression of exogenous NCL. We demonstrated that the synthetic RNAs derived from the 3'-NCR of JEV genomic RNA containing the GQ sequence could bind NCL in vitro. The replication complex binding to the 3'-NCR is required for the viral RNA synthesis. It is likely that NCL present in the replication complex destabilizes the GQ structures in the genomic RNA, thus facilitating the movement of the replication complex resulting in efficient virus replication.IMPORTANCEJapanese encephalitis virus (JEV) is endemic in most parts of South-East Asia and the Western Pacific region, causing epidemics of encephalitis with a high case fatality rate. While a tissue culture-derived JEV vaccine is available, no antiviral therapy exists. The JEV NS5 protein has RNA-dependent RNA polymerase activity. Together with several host and viral proteins, it constitutes the replication complex necessary for virus replication. Understanding the interaction of NS5 with the host proteins could help design novel antivirals. We identified Nucleolin (NCL) as a crucial host protein interactor of JEV NS5 having a pro-viral role in virus replication. The NS5-interacting NCL binds to the G-quadruplex (GQ) structure sequence in the 3'-NCR of JEV RNA. This may smoothen the movement of the replication complex along the genomic RNA, thereby facilitating the virus replication. This study is the first report on how NCL, a host protein, helps in JEV replication through GQ-binding.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie) , Nucleolina , Fosfoproteínas , Proteínas de Unión al ARN , Proteínas no Estructurales Virales , Replicación Viral , Humanos , Virus de la Encefalitis Japonesa (Especie)/fisiología , Virus de la Encefalitis Japonesa (Especie)/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Células HeLa , Unión Proteica , Encefalitis Japonesa/virología , Encefalitis Japonesa/metabolismo , Interacciones Huésped-Patógeno , G-Cuádruplex , AnimalesRESUMEN
Flavivirus infection capitalizes on cellular lipid metabolism to remodel the cellular intima, creating a specialized lipid environment conducive to viral replication, assembly, and release. The Japanese encephalitis virus (JEV), a member of the Flavivirus genus, is responsible for significant morbidity and mortality in both humans and animals. Currently, there are no effective antiviral drugs available to combat JEV infection. In this study, we embarked on a quest to identify anti-JEV compounds within a lipid compound library. Our research led to the discovery of two novel compounds, isobavachalcone (IBC) and corosolic acid (CA), which exhibit dose-dependent inhibition of JEV proliferation. Time-of-addition assays indicated that IBC and CA predominantly target the late stage of the viral replication cycle. Mechanistically, JEV nonstructural proteins 1 and 2A (NS1 and NS2A) impede 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) activation by obstructing the liver kinase B1 (LKB1)-AMPK interaction, resulting in decreased p-AMPK expression and a consequent upsurge in lipid synthesis. In contrast, IBC and CA may stimulate AMPK by binding to its active allosteric site, thereby inhibiting lipid synthesis essential for JEV replication and ultimately curtailing viral infection. Most importantly, in vivo experiments demonstrated that IBC and CA protected mice from JEV-induced mortality, significantly reducing viral loads in the brain and mitigating histopathological alterations. Overall, IBC and CA demonstrate significant potential as effective anti-JEV agents by precisely targeting AMPK-associated signaling pathways. These findings open new therapeutic avenues for addressing infections caused by Flaviviruses. IMPORTANCE: This study is the inaugural utilization of a lipid compound library in antiviral drug screening. Two lipid compounds, isobavachalcone (IBC) and corosolic acid (CA), emerged from the screening, exhibiting substantial inhibitory effects on the Japanese encephalitis virus (JEV) proliferation in vitro. In vivo experiments underscored their efficacy, with IBC and CA reducing viral loads in the brain and mitigating JEV-induced histopathological changes, effectively shielding mice from fatal JEV infection. Intriguingly, IBC and CA may activate 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) by binding to its active site, curtailing the synthesis of lipid substances, and thus suppressing JEV proliferation. This indicates AMPK as a potential antiviral target. Remarkably, IBC and CA demonstrated suppression of multiple viruses, including Flaviviruses (JEV and Zika virus), porcine herpesvirus (pseudorabies virus), and coronaviruses (porcine deltacoronavirus and porcine epidemic diarrhea virus), suggesting their potential as broad-spectrum antiviral agents. These findings shed new light on the potential applications of these compounds in antiviral research.
RESUMEN
Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that has been associated with congenital neurological defects in fetuses born to infected mothers. At present, no vaccine or antiviral therapy is available to combat this devastating disease. Repurposing drugs that target essential host factors exploited by viruses is an attractive therapeutic approach. Here, we screened a panel of clinically approved small-molecule kinase inhibitors for their antiviral effects against a clinical isolate of ZIKV and thoroughly characterized their mechanisms of action. We found that the Raf kinase inhibitors Dabrafenib and Regorafenib potently impair the replication of ZIKV, but not that of its close relative dengue virus. Time-of-addition experiments showed that both inhibitors target ZIKV infection at post-entry steps. We found that Dabrafenib, but not Regorafenib, interfered with ZIKV genome replication by impairing both negative- and positive-strand RNA synthesis. Regorafenib, on the other hand, altered steady-state viral protein levels, viral egress, and blocked NS1 secretion. We also observed Regorafenib-induced ER fragmentation in ZIKV-infected cells, which might contribute to its antiviral effects. Because these inhibitors target different steps of the ZIKV infection cycle, their use in combination therapy may amplify their antiviral effects which could be further explored for future therapeutic strategies against ZIKV and possibly other flaviviruses. IMPORTANCE: There is an urgent need to develop effective therapeutics against re-emerging arboviruses associated with neurological disorders like Zika virus (ZIKV). We identified two FDA-approved kinase inhibitors, Dabrafenib and Regorafenib, as potent inhibitors of contemporary ZIKV strains at distinct stages of infection despite overlapping host targets. Both inhibitors reduced viral titers by ~1 to 2 log10 (~10-fold to 100-fold) with minimal cytotoxicity. Furthermore, we show that Dabrafenib inhibits ZIKV RNA replication whereas Regorafenib inhibits ZIKV translation and egress. Regorafenib has the added benefit of limiting NS1 secretion, which contributes to the pathogenesis and disease progression of several flaviviruses. Because these inhibitors affect distinct post-entry steps of ZIKV infection, their therapeutic potential may be amplified by combination therapy and likely does not require prophylactic administration. This study provides further insight into ZIKV-host interactions and has implications for the development of novel antivirals against ZIKV and possibly other flaviviruses.
Asunto(s)
Antivirales , Imidazoles , Oximas , Compuestos de Fenilurea , Inhibidores de Proteínas Quinasas , Piridinas , Replicación Viral , Infección por el Virus Zika , Virus Zika , Replicación Viral/efectos de los fármacos , Oximas/farmacología , Virus Zika/efectos de los fármacos , Piridinas/farmacología , Humanos , Imidazoles/farmacología , Infección por el Virus Zika/virología , Infección por el Virus Zika/tratamiento farmacológico , Chlorocebus aethiops , Animales , Compuestos de Fenilurea/farmacología , Células Vero , Antivirales/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Línea Celular , Virus del Dengue/efectos de los fármacosRESUMEN
Tick-borne flaviviruses (TBFs) are transmitted to humans through milk and tick bites. Although a case of possible mother-to-child transmission of tick-borne encephalitis virus (TBEV) through breast milk has been reported, this route has not been confirmed in experimental models. Therefore, in this study, using type I interferon receptor-deficient A129 mice infected with Langat virus (LGTV), we aimed to demonstrate the presence of infectious virus in the milk and mammary glands of infected mice. Our results showed viral RNA of LGTV in the pup's stomach milk clots (SMCs) and blood, indicating that the virus can be transmitted from dam to pup through breast milk. In addition, we observed that LGTV infection causes tissue lesions in the mammary gland, and viral particles were present in mammary gland epithelial cells. Furthermore, we found that milk from infected mice could infect adult mice via the intragastric route, which has a milder infection process, longer infection time, and a lower rate of weight loss than other modes of infection. Specifically, we developed a nano-luciferase-LGTV reporter virus system to monitor the dynamics of different infection routes and observed dam-to-pup infection using in vivo bioluminescence imaging. This study provides comprehensive evidence to support breast milk transmission of TBF in mice and has helped provide useful data for studying TBF transmission routes.IMPORTANCETo date, no experimental models have confirmed mother-to-child transmission of tick-borne flavivirus (TBF) through breastfeeding. In this study, we used a mouse model to demonstrate the presence of infectious viruses in mouse breast milk and mammary gland epithelial cells. Our results showed that pups could become infected through the gastrointestinal route by suckling milk, and the infection dynamics could be monitored using a reporter virus system during breastfeeding in vivo. We believe our findings have provided substantial evidence to understand the underlying mechanism of breast milk transmission of TBF in mice, which has important implications for understanding and preventing TBF transmission in humans.
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Virus de la Encefalitis Transmitidos por Garrapatas , Encefalitis Transmitida por Garrapatas , Transmisión Vertical de Enfermedad Infecciosa , Glándulas Mamarias Animales , Leche , Animales , Femenino , Ratones , Virus de la Encefalitis Transmitidos por Garrapatas/crecimiento & desarrollo , Virus de la Encefalitis Transmitidos por Garrapatas/fisiología , Encefalitis Transmitida por Garrapatas/transmisión , Encefalitis Transmitida por Garrapatas/virología , Glándulas Mamarias Animales/virología , Leche/virología , Animales Recién Nacidos/virologíaRESUMEN
Powassan virus (POWV) is an emergent tick-borne flavivirus that causes fatal encephalitis in the elderly and long-term neurologic sequelae in survivors. How age contributes to severe POWV encephalitis remains an enigma, and no animal models have assessed age-dependent POWV neuropathology. Inoculating C57BL/6 mice with a POWV strain (LI9) currently circulating in Ixodes ticks resulted in age-dependent POWV lethality 10-20 dpi. POWV infection of 50-week-old mice was 82% fatal with lethality sequentially reduced by age to 7.1% in 10-week-old mice. POWV LI9 was neuroinvasive in mice of all ages, causing acute spongiform CNS pathology and reactive gliosis 5-15 dpi that persisted in survivors 30 dpi. High CNS viral loads were found in all mice 10 dpi. However, by 15 dpi, viral loads decreased by 2-4 logs in 10- to 40-week-old mice, while remaining at high levels in 50-week-old mice. Age-dependent differences in CNS viral loads 15 dpi occurred concomitantly with striking changes in CNS cytokine responses. In the CNS of 50-week-old mice, POWV induced Th1-type cytokines (IFNγ, IL-2, IL-12, IL-4, TNFα, IL-6), suggesting a neurodegenerative pro-inflammatory M1 microglial program. By contrast, in 10-week-old mice, POWV-induced Th2-type cytokines (IL-10, TGFß, IL-4) were consistent with a neuroprotective M2 microglial phenotype. These findings correlate age-dependent CNS cytokine responses and viral loads with POWV lethality and suggest potential neuroinflammatory therapeutic targets. Our results establish the age-dependent lethality of POWV in a murine model that mirrors human POWV severity and long-term CNS pathology in the elderly. IMPORTANCE: Powassan virus is an emerging tick-borne flavivirus causing lethal encephalitis in aged individuals. We reveal an age-dependent POWV murine model that mirrors human POWV encephalitis and long-term CNS damage in the elderly. We found that POWV is neuroinvasive and directs reactive gliosis in all age mice, but at acute stages selectively induces pro-inflammatory Th1 cytokine responses in 50-week-old mice and neuroprotective Th2 cytokine responses in 10-week-old mice. Our findings associate CNS viral loads and divergent cytokine responses with age-dependent POWV lethality and survival outcomes. Responses of young mice suggest potential therapeutic targets and approaches for preventing severe POWV encephalitis that may be broadly applicable to other neurodegenerative diseases. Our age-dependent murine POWV model permits analysis of vaccines that prevent POWV lethality, and therapeutics that resolve severe POWV encephalitis.
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Citocinas , Modelos Animales de Enfermedad , Virus de la Encefalitis Transmitidos por Garrapatas , Encefalitis Transmitida por Garrapatas , Ratones Endogámicos C57BL , Neuroglía , Carga Viral , Animales , Ratones , Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Encefalitis Transmitida por Garrapatas/inmunología , Encefalitis Transmitida por Garrapatas/virología , Encefalitis Transmitida por Garrapatas/mortalidad , Encefalitis Transmitida por Garrapatas/patología , Citocinas/metabolismo , Citocinas/inmunología , Neuroglía/virología , Neuroglía/inmunología , Neuroglía/patología , Femenino , Factores de Edad , Ixodes/virología , Ixodes/inmunología , Sistema Nervioso Central/virología , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/patología , Encéfalo/virología , Encéfalo/patología , Encéfalo/inmunologíaRESUMEN
Live-attenuated flavivirus vaccines confer long-term protection against disease, but the design of attenuated flaviviruses does not follow a general approach. The non-coding, subgenomic flavivirus RNA (sfRNA) is produced by all flaviviruses and is an essential factor in viral pathogenesis and transmission. We argue that modulating sfRNA expression is a promising, universal strategy to finetune flavivirus attenuation for developing effective flavivirus vaccines of the future.
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Infecciones por Flavivirus , Flavivirus , ARN Viral , Vacunas Atenuadas , Vacunas Virales , Vacunas Atenuadas/inmunología , Flavivirus/inmunología , Flavivirus/genética , ARN Viral/genética , Humanos , Vacunas Virales/inmunología , Infecciones por Flavivirus/prevención & control , Infecciones por Flavivirus/virología , Animales , Desarrollo de VacunasRESUMEN
We report fatal West Nile virus (WNV) infection in a 7-year-old mare returning to the United Kingdom from Spain. Case timeline and clustering of virus sequence with recent WNV isolates suggest that transmission occurred in Andalusía, Spain. Our findings highlight the importance of vaccination for horses traveling to WNV-endemic regions.
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Fiebre del Nilo Occidental , Animales , Femenino , Análisis por Conglomerados , Caballos , España/epidemiología , Reino Unido/epidemiología , Fiebre del Nilo Occidental/diagnóstico , Fiebre del Nilo Occidental/veterinariaRESUMEN
Flaviviruses target their replication on membranous structures derived from the ER, where both viral and host proteins play crucial structural and functional roles. Here, we have characterized the involvement of the ER-associated degradation (ERAD) pathway core E3 ligase complex (SEL1L-HRD1) regulator proteins in the replication of Japanese encephalitis virus (JEV). Through high-resolution immunofluorescence imaging of JEV-infected HeLa cells, we observe that the virus replication complexes marked by NS1 strongly colocalize with the ERAD adapter SEL1L, lectin OS9, ER-membrane shuttle factor HERPUD1, E3 ubiquitin ligase HRD1 and rhomboid superfamily member DERLIN1. NS5 positive structures also show strong overlap with SEL1L. While these effectors show significant transcriptional upregulation, their protein levels remain largely stable in infected cells. siRNA mediated depletion of OS9, SEL1L, HERPUD1 and HRD1 significantly inhibit viral RNA replication and titres, with SEL1L depletion showing the maximum attenuation of replication. By performing protein translation arrest experiments, we show that SEL1L, and OS9 are stabilised upon JEV infection. Overall results from this study suggest that these ERAD effector proteins are crucial host-factors for JEV replication.
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Virus de la Encefalitis Japonesa (Especie) , Degradación Asociada con el Retículo Endoplásmico , Proteínas de la Membrana , Ubiquitina-Proteína Ligasas , Replicación Viral , Humanos , Virus de la Encefalitis Japonesa (Especie)/fisiología , Virus de la Encefalitis Japonesa (Especie)/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Células HeLa , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Interacciones Huésped-Patógeno , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Proteínas/metabolismo , Proteínas/genética , Antígenos de DiferenciaciónRESUMEN
Tick-borne orthoflaviviruses (TBFs) are classified into three conventional groups based on genetics and ecology: mammalian, seabird and probable-TBF group. Recently, a fourth basal group has been identified in Rhipicephalus ticks from Africa: Mpulungu flavivirus (MPFV) in Zambia and Ngoye virus (NGOV) in Senegal. Despite attempts, isolating these viruses in vertebrate and invertebrate cell lines or intracerebral injection of newborn mice with virus-containing homogenates has remained unsuccessful. In this study, we report the discovery of Xinyang flavivirus (XiFV) in Haemaphysalis flava ticks from Xìnyáng, Henan Province, China. Phylogenetic analysis shows that XiFV was most closely related to MPFV and NGOV, marking the first identification of this tick orthoflavivirus group in Asia. We developed a reverse transcriptase quantitative PCR assay to screen wild-collected ticks and egg clutches, with absolute infection rates of 20.75â% in adult females and 15.19â% in egg clutches, suggesting that XiFV could be potentially spread through transovarial transmission. To examine potential host range, dinucleotide composition analyses revealed that XiFV, MPFV and NGOV share a closer composition to classical insect-specific orthoflaviviruses than to vertebrate-infecting TBFs, suggesting that XiFV could be a tick-only orthoflavivirus. Additionally, both XiFV and MPFV lack a furin cleavage site in the prM protein, unlike other TBFs, suggesting these viruses might exist towards a biased immature particle state. To examine this, chimeric Binjari virus with XIFV-prME (bXiFV) was generated, purified and analysed by SDS-PAGE and negative-stain transmission electron microscopy, suggesting prototypical orthoflavivirus size (~50 nm) and bias towards uncleaved prM. In silico structural analyses of the 3'-untranslated regions show that XiFV forms up to five pseudo-knot-containing stem-loops and a prototypical orthoflavivirus dumbbell element, suggesting the potential for multiple exoribonuclease-resistant RNA structures.
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Flavivirus , Ixodidae , Filogenia , Animales , Flavivirus/genética , Flavivirus/clasificación , Flavivirus/aislamiento & purificación , China , Ixodidae/virología , FemeninoRESUMEN
BACKGROUND: Flavivirus is a challenge all over the world. The replication of flavivirus takes place within membranous replication compartments (RCs) derived from endoplasmic reticulum (ER). Flavivirus NS1 proteins have been proven essential for the formation of viral RCs by remodeling the ER. The glycosylation of flavivirus NS1 proteins is important for viral replication, yet the underlying mechanism remains unclear. METHODS: HeLa cells were used to visualize the ER remodeling effects induced by NS1 expression. ZIKV replicon luciferase assay was performed with BHK-21 cells. rZIKV was generated from BHK-21 cells and the plaque assay was done with Vero Cells. Liposome co-floating assay was performed with purified NS1 proteins from 293T cells. RESULTS: We found that the glycosylation of flavivirus NS1 contributes to its ER remodeling activity. Glycosylation deficiency of NS1, either through N-glycosylation sites mutations or tunicamycin treatment, compromises its ER remodeling activity and interferes with viral RCs formation. Disruption of NS1 glycosylation results in abnormal aggregation of NS1, rather than reducing its membrane-binding activity. Consequently, deficiency in NS1 glycosylation impairs virus replication. CONCLUSIONS: In summary, our results highlight the significance of NS1 glycosylation in flavivirus replication and elucidate the underlying mechanism. This provides a new strategy for combating flavivirus infections.
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Proteínas no Estructurales Virales , Replicación Viral , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Glicosilación , Humanos , Animales , Compartimentos de Replicación Viral/metabolismo , Células HeLa , Chlorocebus aethiops , Flavivirus/fisiología , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Células VeroRESUMEN
Outbreaks of West Nile virus (WNV) occur periodically, affecting both human and equine populations. There are no vaccines for humans, and those commercialised for horses do not have sufficient coverage. Specific antiviral treatments do not exist. Many drug discovery studies have been conducted, but since rodent or primate cell lines are normally used, results cannot always be transposed to horses. There is thus a need to develop relevant equine cellular models. Here, we used induced pluripotent stem cells to develop a new in vitro model of WNV-infected equine brain cells suitable for microplate assay, and assessed the cytotoxicity and antiviral activity of forty-one chemical compounds. We found that one nucleoside analog, 2'C-methylcytidine, blocked WNV infection in equine brain cells, whereas other compounds were either toxic or ineffective, despite some displaying anti-viral activity in human cell lines. We also revealed an unexpected proviral effect of statins in WNV-infected equine brain cells. Our results thus identify a potential lead for future drug development and underscore the importance of using a tissue- and species-relevant cellular model for assessing the activity of antiviral compounds.
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Enfermedades de los Caballos , Células Madre Pluripotentes Inducidas , Fiebre del Nilo Occidental , Virus del Nilo Occidental , Animales , Caballos , Humanos , Fiebre del Nilo Occidental/veterinaria , Fiebre del Nilo Occidental/epidemiología , Encéfalo , Antivirales/farmacología , Antivirales/uso terapéutico , Enfermedades de los Caballos/tratamiento farmacológicoRESUMEN
OBJECTIVES: Tick-borne encephalitis (TBE) is an infection caused by the tick-borne encephalitis virus (TBEV) that can lead to symptoms of central nervous system inflammation. There are five subtypes of TBEV, three of which - European, Siberian and Far Eastern - occur in Europe. As it is thought that different subtype infections exhibit varying clinical courses and outcomes, serological differentiation of the virus subtypes is clearly important. However, to date, this has proved difficult to achieve. METHODS: An ELISA format was developed based on TBE virus NS1 antigen against the European, Siberian and Far Eastern subtype. The three NS1 antigens were biotechnologically produced in a human cell line and used for ELISA coating. Sera from German (European subtype) and Russian (Siberian and/or Far Eastern subtypes) TBE patients with positive TBEV IgG were used to test the reactivity against these three NS1 antigens. RESULTS: Testing of 23 German and 32 Russian TBEV IgG-positive sera showed that the ELISA was able to differentiate between TBEV European subtype and TBEV Siberian and Far Eastern subtype infections. CONCLUSIONS: In geographical areas where two or more TBEV subtype infections can occur, the NS1-IgG ELISA developed here constitutes an important diagnostic tool to differentiate between European subtype infections and Siberian/Far Eastern subtype infections and to use the new assay for epidemiological studies to clarify the importance of particular subtype infections in an area. Consequently, it may help to better describe and anticipate the clinical courses and outcomes of particular TBEV subtype infections.