RESUMO
Usutu virus (USUV) and West Nile virus (WNV) are two closely related emerging mosquito-borne flaviviruses. Their natural hosts are wild birds, but they can also cause severe neurological disorders in humans. Both viruses are efficiently suppressed by type I interferon (IFN), which interferes with viral replication, dissemination, pathogenesis and transmission. Here, we show that the replication of USUV and WNV are inhibited through a common set of IFN-induced genes (ISGs), with the notable exception of ISG20, which USUV is resistant to. Strikingly, USUV was the only virus among all the other tested mosquito-borne flaviviruses that demonstrated resistance to the 3'-5' exonuclease activity of ISG20. Our findings highlight that the intrinsic resistance of the USUV genome, irrespective of the presence of cellular or viral proteins or protective post-transcriptional modifications, relies on a unique sequence present in its 3' untranslated region. Importantly, this genomic region alone can confer ISG20 resistance to a susceptible flavivirus, without compromising its infectivity, suggesting that it could be acquired by other flaviviruses. This study provides new insights into the strategy employed by emerging flaviviruses to overcome host defense mechanisms.
Assuntos
Regiões 3' não Traduzidas , Flavivirus , Replicação Viral , Vírus do Nilo Ocidental , Regiões 3' não Traduzidas/genética , Flavivirus/genética , Flavivirus/fisiologia , Humanos , Animais , Replicação Viral/genética , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/fisiologia , Infecções por Flavivirus/virologia , Exonucleases/metabolismo , Exonucleases/genética , Chlorocebus aethiops , Exorribonucleases/metabolismo , Exorribonucleases/genética , Células HEK293 , Células Vero , Linhagem Celular , Interferon Tipo I/metabolismo , Genoma ViralRESUMO
The ongoing epidemic of flaviviruses worldwide has underscored the importance of studying flavivirus vector competence, considering their close association with mosquito vectors. Tembusu virus is an avian-related mosquito-borne flavivirus that has been an epidemic in China and Southeast Asia since 2010. However, the reason for the outbreak of Tembusu virus in 2010 remains unclear, and it is unknown whether changes in vector transmission played an essential role in this process. To address these questions, we conducted a study using Culex quinquefasciatus as a model for Tembusu virus infection, employing both oral infection and microinjection methods. Our findings confirmed that both vertical and venereal transmission collectively contribute to the cycle of Tembusu virus within the mosquito population, with persistent infections observed. Importantly, our data revealed that the prototypical Tembusu virus MM_1775 strain exhibited significantly greater infectivity and transmission rates in mosquitoes than did the duck Tembusu virus (CQW1 strain). Furthermore, we revealed that the viral E protein and 3' untranslated region are key elements responsible for these differences. In conclusion, our study sheds light on mosquito transmission of Tembusu virus and provides valuable insights into the factors influencing its infectivity and transmission rates. These findings contribute to a better understanding of Tembusu virus epidemiology and can potentially aid in the development of strategies to control its spread.
Assuntos
Culex , Infecções por Flavivirus , Flavivirus , Mosquitos Vetores , Animais , Culex/virologia , Flavivirus/fisiologia , Infecções por Flavivirus/veterinária , Infecções por Flavivirus/transmissão , Infecções por Flavivirus/virologia , Mosquitos Vetores/virologia , FemininoRESUMO
West Nile virus (WNV) and Usutu virus (USUV) are closely related flaviviruses with differing capacities to cause neurological disease in humans. WNV is thought to use a transneural route of neuroinvasion along motor neurons and causes severe motor deficits. The potential for use of transneural routes of neuroinvasion by USUV has not been investigated experimentally, and evidence from the few clinical case reports of USUV-associated neuroinvasive disease is lacking. We hypothesised that, compared with WNV, USUV is less able to infect motor neurons, and therefore determined the susceptibility of human induced pluripotent stem cell (iPSC)-derived spinal cord motor neurons to infection. Both viruses could grow to high titres in iPSC-derived neural cultures. However, USUV could not productively infect motor neurons due to restriction by the antiviral response, which was not induced upon WNV infection. Inhibition of the antiviral response allowed for widespread infection and transportation of USUV along motor neurons within a compartmented culture system. These results show a stark difference in the ability of these two viruses to evade initiation of intrinsic antiviral immunity. Our data suggests that USUV cannot infect motor neurons in healthy individuals but in case of immunodeficiency may pose a risk for motor-related neurological disease and transneural invasion.
West Nile virus, but not Usutu virus, can productively infect human motor neurons as a possible route of neuroinvasion.
Assuntos
Flavivirus , Células-Tronco Pluripotentes Induzidas , Neurônios Motores , Vírus do Nilo Ocidental , Humanos , Vírus do Nilo Ocidental/fisiologia , Vírus do Nilo Ocidental/patogenicidade , Neurônios Motores/virologia , Células Cultivadas , Flavivirus/fisiologia , Células-Tronco Pluripotentes Induzidas/virologia , Infecções por Flavivirus/virologia , Febre do Nilo Ocidental/virologia , Animais , Medula Espinal/virologiaRESUMO
Insect-specific viruses (ISVs) include viruses that are restricted to the infection of mosquitoes and are spread mostly through transovarial transmission. Despite using a distinct mode of transmission, ISVs are often phylogenetically related to arthropod-borne viruses (arboviruses) that are responsible for human diseases and able to infect both mosquitoes and vertebrates. ISVs can also induce a phenomenon called "superinfection exclusion", whereby a primary ISV infection in an insect inhibits subsequent viral infections of the insect. This has sparked interest in the use of ISVs for the control of pathogenic arboviruses transmitted by mosquitoes. In particular, insect-specific flaviviruses (ISFs) have been shown to inhibit infection of vertebrate-infecting flaviviruses (VIFs) both in vitro and in vivo. This has shown potential as a new and ecologically friendly biological approach to the control of arboviral disease. For this intervention to have lasting impacts for biological control, it is imperative that ISFs are maintained in mosquito populations with high rates of vertical transmission. Therefore, these strategies will need to optimise vertical transmission of ISFs in order to establish persistently infected mosquito lines for sustainable arbovirus control. This review compares recent observations of vertical transmission of arboviral and insect-specific flaviviruses and potential determinants of transovarial transmission rates to understand how the vertical transmission of ISFs may be optimised for effective arboviral control.
Assuntos
Culicidae , Infecções por Flavivirus , Flavivirus , Transmissão Vertical de Doenças Infecciosas , Mosquitos Vetores , Vertebrados , Animais , Flavivirus/fisiologia , Flavivirus/genética , Flavivirus/classificação , Mosquitos Vetores/virologia , Culicidae/virologia , Infecções por Flavivirus/transmissão , Infecções por Flavivirus/virologia , Humanos , Vertebrados/virologia , Arbovírus/fisiologia , Arbovírus/classificaçãoRESUMO
West Nile virus (WNV) was first detected in the Netherlands in 2020, with circulation observed in birds, mosquitoes, and humans in two geographical areas. Usutu virus (USUV) has been circulating in the Netherlands since 2016. Following the detection of WNV in the Netherlands, we investigated the possible use of petting zoos as urban sentinel sites to examine the extent of WNV and USUV circulation around the two WNV outbreak locations. Chickens at petting zoos and in backyards were sampled within a 15-kilometer radius of the confirmed WNV circulation areas at three timepoints over one year (2021-2022). Sera were analysed using a protein microarray for binding antibodies to orthoflavivirus NS1 antigens and reactive samples were confirmed through micro-focus reduction neutralization tests (mFRNT). Furthermore, mosquitoes at sampling locations were collected to assess their blood feeding behaviour. This serosurvey detected the circulation of USUV and WNV in petting zoo and backyard chickens in 2021, both within and outside the 2020 outbreak areas. The WNV circulation was not detected by other existing surveillance schemes in mosquitoes, wild birds, horses and humans. In addition, the results show rapid decay of USUV antibodies in approximately 20 weeks. Our findings support the utility and the added value of petting zoo chickens as sentinels for monitoring USUV and WNV circulation compared to other available methods. Seroconversions observed in petting zoos and backyard chickens living in or near densely populated urban areas further highlighted potential public health risks that went undetected.
Assuntos
Anticorpos Antivirais , Galinhas , Doenças das Aves Domésticas , Vigilância de Evento Sentinela , Febre do Nilo Ocidental , Vírus do Nilo Ocidental , Animais , Vírus do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/isolamento & purificação , Países Baixos/epidemiologia , Galinhas/virologia , Febre do Nilo Ocidental/veterinária , Febre do Nilo Ocidental/epidemiologia , Febre do Nilo Ocidental/virologia , Anticorpos Antivirais/sangue , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/epidemiologia , Vigilância de Evento Sentinela/veterinária , Flavivirus/imunologia , Flavivirus/isolamento & purificação , Infecções por Flavivirus/epidemiologia , Infecções por Flavivirus/veterinária , Infecções por Flavivirus/virologia , Animais de Zoológico/virologia , Culicidae/virologia , Surtos de Doenças/veterináriaRESUMO
MicroRNAs (miRNAs) are molecules that influence messenger RNA (mRNA) expression levels by binding to the 3' untranslated region (3' UTR) of target genes. Host miRNAs can influence flavivirus replication, either by inducing changes in the host transcriptome or by directly binding to viral genomes. The 3' UTR of the flavivirus genome is a conserved region crucial for viral replication. Cells might exploit this well-preserved region by generating miRNAs that interact with it, ultimately impacting viral replication. Despite significant efforts to identify miRNAs capable of arresting viral replication, the potential of all these miRNAs to interact with the flavivirus 3' UTR is still poorly characterised. In this context, bioinformatic tools have been proposed as a fundamental part of accelerating the discovery of interactions between miRNAs and the 3' UTR of viral genomes. In this study, we performed a computational analysis to reveal potential miRNAs from human and mosquito species that bind to the 3' UTR of flaviviruses. In humans, miR-6842 and miR-661 were found, while in mosquitoes, miR-9-C, miR-2945-5p, miR-11924, miR-282-5p, and miR-79 were identified. These findings open new avenues for studying these miRNAs as antivirals against flavivirus infections.
Assuntos
Regiões 3' não Traduzidas , Biologia Computacional , Flavivirus , Genoma Viral , MicroRNAs , MicroRNAs/genética , MicroRNAs/metabolismo , Regiões 3' não Traduzidas/genética , Flavivirus/genética , Humanos , Animais , Biologia Computacional/métodos , Replicação Viral/genética , Antivirais/farmacologia , Infecções por Flavivirus/virologia , Infecções por Flavivirus/genética , Culicidae/virologia , Culicidae/genéticaRESUMO
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.
Assuntos
Proteínas Quinases Ativadas por AMP , Antivirais , Vírus da Encefalite Japonesa (Espécie) , Encefalite Japonesa , Metabolismo dos Lipídeos , Replicação Viral , Animais , Metabolismo dos Lipídeos/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Vírus da Encefalite Japonesa (Espécie)/efeitos dos fármacos , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Camundongos , Antivirais/farmacologia , Humanos , Encefalite Japonesa/tratamento farmacológico , Encefalite Japonesa/virologia , Proteínas Quinases Ativadas por AMP/metabolismo , Chalconas/farmacologia , Triterpenos/farmacologia , Proteínas não Estruturais Virais/metabolismo , Infecções por Flavivirus/tratamento farmacológico , Infecções por Flavivirus/virologia , Infecções por Flavivirus/metabolismo , Flavivirus/efeitos dos fármacos , Linhagem CelularRESUMO
Flaviviruses comprise a large number of arthropod-borne viruses, some of which are associated with life-threatening diseases. Flavivirus infections are rising worldwide, mainly due to the proliferation and geographical expansion of their vectors. The main human pathogens are mosquito-borne flaviviruses, including dengue virus, Zika virus, and West Nile virus, but tick-borne flaviviruses are also emerging. As with any viral infection, the body's first line of defense against flavivirus infections is the innate immune defense, of which type I interferon is the armed wing. This cytokine exerts its antiviral activity by triggering the synthesis of hundreds of interferon-induced genes (ISGs), whose products can prevent infection. Among the ISGs that inhibit flavivirus replication, certain tripartite motif (TRIM) proteins have been identified. Although involved in other biological processes, TRIMs constitute a large family of antiviral proteins active on a wide range of viruses. Furthermore, whereas some TRIM proteins directly block viral replication, others are positive regulators of the IFN response. Therefore, viruses have developed strategies to evade or counteract TRIM proteins, and some even hijack certain TRIM proteins to their advantage. In this review, we summarize the current state of knowledge on the interactions between flaviviruses and TRIM proteins, covering both direct and indirect antiviral mechanisms.
Assuntos
Infecções por Flavivirus , Flavivirus , Replicação Viral , Humanos , Infecções por Flavivirus/virologia , Infecções por Flavivirus/imunologia , Flavivirus/fisiologia , Flavivirus/imunologia , Animais , Imunidade Inata , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Interações Hospedeiro-Patógeno/imunologia , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismoRESUMO
Bagaza virus (BAGV) is a mosquito-borne flavivirus of the family Flaviviridae, genus Orthoflavivirus, Ntaya serocomplex. Like other viruses of the Ntaya and Japanese encephalitis serocomplexes, it is maintained in nature in transmission cycles involving viremic wild bird reservoirs and Culex spp. mosquitoes. The susceptibility of red-legged partridge, ring-necked pheasant, Himalayan monal and common wood pigeon is well known. Determining whether other species are susceptible to BAGV infection is fundamental to understanding the dynamics of disease transmission and maintenance. In September 2023, seven Eurasian magpies were found dead in a rural area in the Mértola district (southern Portugal) where a BAGV-positive cachectic red-legged partridge had been found two weeks earlier. BAGV had also been detected in several red-legged partridges in the same area in September 2021. Three of the magpies were tested for Bagaza virus, Usutu virus, West Nile virus, Avian influenza virus and Avian paramyxovirus serotype 1, and were positive for BAGV only. Sequencing data confirmed the specificity of the molecular detection. Our results indicate that BAGV is circulating in southern Portugal and confirm that Eurasian magpie is potential susceptible to BAGV infection. The inclusion of the abundant Eurasian magpie in the list of BAGV hosts raises awareness of the potential role of this species as as an amplifying host.
Assuntos
Flavivirus , Animais , Portugal , Flavivirus/genética , Flavivirus/isolamento & purificação , Filogenia , Doenças das Aves/virologia , Doenças das Aves/epidemiologia , Infecções por Flavivirus/virologia , Infecções por Flavivirus/veterinária , Infecções por Flavivirus/transmissão , Infecções por Flavivirus/epidemiologiaRESUMO
USUV in Europe is detected in vectors (mosquitoes) and has a reservoir in vertebrates. There are known fatal epidemics among birds, especially blackbirds. Currently, USUV also causes rare infections in humans. However, the emergence of clinical cases, including severe neurological symptoms, and the finding of seroprevalence in asymptomatic people (e.g. blood donors, forest workers), indicate that USUV, due to its neurotropism, may become a potential public health problem. Therefore, it is very important to monitor cases infections in humans, migratory and resident birds and other animals that may constitute a reservoir of the virus, but also detection of the virus in mosquitoes (vectors), including alien and invasive species, as well as the impact of climatic factors on the ability to spread the virus in the Europe. There is currently no evidence of virus transmission during transfusion or transplantation, but the potential risk of virus transmission from an asymptomatic blood donor to an mmunocompromised recipient must be considered. Although the occurrence of USUV in European countries is currently not a significant threat, surveillance and screening of blood donors for USUV should be carried out during the period of vector activity and during WNV epidemics, as well as in patients with symptoms of meningitis and encephalitis.
Assuntos
Flavivirus , Animais , Humanos , Culicidae/virologia , Infecções por Flavivirus/virologia , Mosquitos Vetores/virologiaRESUMO
Tick-borne encephalitis virus (TBEV) is a neurotropic member of the genus Orthoflavivirus (former Flavivirus) and is of significant health concern in Europe and Asia. TBEV pathogenesis may occur directly via virus-induced damage to neurons or through immunopathology due to excessive inflammation. While primary cells isolated from the host can be used to study the immune response to TBEV, it is still unclear how well these reflect the immune response elicited in vivo. Here, we compared the transcriptional response to TBEV and the less pathogenic tick-borne flavivirus, Langat virus (LGTV), in primary monocultures of neurons, astrocytes and microglia in vitro, with the transcriptional response in vivo captured by single-nuclei RNA sequencing (snRNA-seq) of a whole mouse cortex. We detected similar transcriptional changes induced by both LGTV and TBEV infection in vitro, with the lower response to LGTV likely resulting from slower viral kinetics. Gene set enrichment analysis showed a stronger transcriptional response in vivo than in vitro for astrocytes and microglia, with a limited overlap mainly dominated by interferon signaling. Together, this adds to our understanding of neurotropic flavivirus pathogenesis and the strengths and limitations of available model systems.
Assuntos
Astrócitos , Vírus da Encefalite Transmitidos por Carrapatos , Encefalite Transmitida por Carrapatos , Microglia , Neurônios , Animais , Astrócitos/virologia , Microglia/virologia , Vírus da Encefalite Transmitidos por Carrapatos/genética , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Camundongos , Neurônios/virologia , Encefalite Transmitida por Carrapatos/virologia , Infecções por Flavivirus/virologia , Infecções por Flavivirus/imunologia , Células Cultivadas , Flavivirus/fisiologia , Flavivirus/genética , Camundongos Endogâmicos C57BL , Transcrição GênicaRESUMO
Usutu (USUV), West Nile (WNV), and Zika virus (ZIKV) are neurotropic arthropod-borne viruses (arboviruses) that cause severe neurological disease in humans. However, USUV-associated neurological disease is rare, suggesting a block in entry to or infection of the brain. We determined the replication, cell tropism and neurovirulence of these arboviruses in human brain tissue using a well-characterized human fetal organotypic brain slice culture model. Furthermore, we assessed the efficacy of interferon-ß and 2'C-methyl-cytidine, a synthetic nucleoside analogue, in restricting viral replication. All three arboviruses replicated within the brain slices, with WNV reaching the highest titers, and all primarily infected neuronal cells. USUV- and WNV-infected cells exhibited a shrunken morphology, not associated with detectable cell death. Pre-treatment with interferon-ß inhibited replication of all arboviruses, while 2'C-methyl-cytidine reduced only USUV and ZIKV titers. Collectively, USUV can infect human brain tissue, showing similarities in tropism and neurovirulence as WNV and ZIKV. These data suggest that a blockade to infection of the human brain may not be the explanation for the low clinical incidence of USUV-associated neurological disease. However, USUV replicated more slowly and to lower titers than WNV, which could help to explain the reduced severity of neurological disease resulting from USUV infection.
Assuntos
Encéfalo , Flavivirus , Replicação Viral , Vírus do Nilo Ocidental , Zika virus , Humanos , Vírus do Nilo Ocidental/patogenicidade , Vírus do Nilo Ocidental/fisiologia , Zika virus/patogenicidade , Zika virus/fisiologia , Encéfalo/virologia , Replicação Viral/efeitos dos fármacos , Flavivirus/patogenicidade , Flavivirus/fisiologia , Flavivirus/efeitos dos fármacos , Feto/virologia , Interferon beta/farmacologia , Animais , Virulência , Técnicas de Cultura de Órgãos , Tropismo Viral , Neurônios/virologia , Infecções por Flavivirus/virologia , Infecção por Zika virus/virologia , Chlorocebus aethiops , Células VeroRESUMO
One third of all emerging infectious diseases are vector-borne, with no licensed antiviral therapies available against any vector-borne viruses. Zika virus and Usutu virus are two emerging flaviviruses transmitted primarily by mosquitoes. These viruses modulate different host pathways, including the PI3K/AKT/mTOR pathway. Here, we report the effect on ZIKV and USUV replication of two AKT inhibitors, Miransertib (ARQ-092, allosteric inhibitor) and Capivasertib (AZD5363, competitive inhibitor) in different mammalian and mosquito cell lines. Miransertib showed a stronger inhibitory effect against ZIKV and USUV than Capivasertib in mammalian cells, while Capivasertib showed a stronger effect in mosquito cells. These findings indicate that AKT plays a conserved role in flavivirus infection, in both the vertebrate host and invertebrate vector. Nevertheless, the specific function of AKT may vary depending on the host species. These findings indicate that AKT may be playing a conserved role in flavivirus infection in both, the vertebrate host and the invertebrate vector. However, the specific function of AKT may vary depending on the host species. A better understanding of virus-host interactions is therefore required to develop new treatments to prevent human disease and new approaches to control transmission by insect vectors.
Assuntos
Infecções por Flavivirus , Flavivirus , Proteínas Proto-Oncogênicas c-akt , Replicação Viral , Zika virus , Animais , Flavivirus/fisiologia , Flavivirus/efeitos dos fármacos , Flavivirus/genética , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Linhagem Celular , Zika virus/fisiologia , Zika virus/efeitos dos fármacos , Infecções por Flavivirus/virologia , Infecções por Flavivirus/transmissão , Vertebrados/virologia , Antivirais/farmacologia , Mosquitos Vetores/virologia , Chlorocebus aethiops , Culicidae/virologia , Interações Hospedeiro-PatógenoRESUMO
Flaviviridae is a family of positive-stranded RNA viruses, including human pathogens, such as Japanese encephalitis virus (JEV), dengue virus (DENV), Zika virus (ZIKV), and West Nile virus (WNV). Nuclear localization of the viral core protein is conserved among Flaviviridae, and this feature may be targeted for developing broad-ranging anti-flavivirus drugs. However, the mechanism of core protein translocation to the nucleus and the importance of nuclear translocation in the viral life cycle remain unknown. We aimed to identify the molecular mechanism underlying core protein nuclear translocation. We identified importin-7 (IPO7), an importin-ß family protein, as a nuclear carrier for Flaviviridae core proteins. Nuclear import assays revealed that core protein was transported into the nucleus via IPO7, whereas IPO7 deletion by CRISPR/Cas9 impaired their nuclear translocation. To understand the importance of core protein nuclear translocation, we evaluated the production of infectious virus or single-round-infectious-particles in wild-type or IPO7-deficient cells; both processes were significantly impaired in IPO7-deficient cells, whereas intracellular infectious virus levels were equivalent in wild-type and IPO7-deficient cells. These results suggest that IPO7-mediated nuclear translocation of core proteins is involved in the release of infectious virus particles of flaviviruses.
Assuntos
Transporte Ativo do Núcleo Celular , Núcleo Celular , Flavivirus , Humanos , Flavivirus/metabolismo , Flavivirus/fisiologia , Animais , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Replicação Viral/fisiologia , Proteínas do Core Viral/metabolismo , Proteínas do Core Viral/genética , Carioferinas/metabolismo , Carioferinas/genética , Infecções por Flavivirus/metabolismo , Infecções por Flavivirus/virologia , Chlorocebus aethiops , Células HEK293RESUMO
Usutu virus (USUV) is an emerging flavivirus that can infect birds and mammals. In humans, in severe cases, it may cause neuroinvasive disease. The innate immune system, and in particular the interferon response, functions as the important first line of defense against invading pathogens such as USUV. Many, if not all, viruses have developed mechanisms to suppress and/or evade the interferon response in order to facilitate their replication. The ability of USUV to antagonize the interferon response has so far remained largely unexplored. Using dual-luciferase reporter assays we observed that multiple of the USUV nonstructural (NS) proteins were involved in suppressing IFN-ß production and signaling. In particular NS4A was very effective at suppressing IFN-ß production. We found that NS4A interacted with the mitochondrial antiviral signaling protein (MAVS) and thereby blocked its interaction with melanoma differentiation-associated protein 5 (MDA5), resulting in reduced IFN-ß production. The TM1 domain of NS4A was found to be essential for binding to MAVS. By screening a panel of flavivirus NS4A proteins we found that the interaction of NS4A with MAVS is conserved among flaviviruses. The increased understanding of the role of NS4A in flavivirus immune evasion could aid the development of vaccines and therapeutic strategies.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Flavivirus , Helicase IFIH1 Induzida por Interferon , Interferon beta , Transdução de Sinais , Proteínas não Estruturais Virais , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Flavivirus/imunologia , Flavivirus/genética , Flavivirus/fisiologia , Interferon beta/genética , Interferon beta/imunologia , Interferon beta/metabolismo , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon/imunologia , Células HEK293 , Evasão da Resposta Imune , Infecções por Flavivirus/imunologia , Infecções por Flavivirus/virologia , Interações Hospedeiro-Patógeno/imunologia , Ligação Proteica , Imunidade Inata , AnimaisRESUMO
Usutu virus (USUV) is a mosquito-borne flavivirus originating from Africa, that belongs to the Japanese encephalitis virus (JEV) complex. In nature, USUV involves Culex spp. mosquitoes acting as vectors and birds as amplifying hosts. The virus has recently spread in Europe and is considered an emerging human pathogen. This is the first research study performed in Greece revealing the presence and circulation of USUV in Culex spp. mosquito populations. Out of the 1,500 mosquito pools tested with real-time RT-PCR, four (Roesch et al., 2019) were positive for USUV. All four pools were collected from the region of Central Macedonia, Northern Greece.
Assuntos
Culex , Flavivirus , Mosquitos Vetores , Animais , Culex/virologia , Grécia , Flavivirus/genética , Flavivirus/isolamento & purificação , Flavivirus/classificação , Mosquitos Vetores/virologia , Reação em Cadeia da Polimerase em Tempo Real , Infecções por Flavivirus/virologia , Infecções por Flavivirus/veterinária , Infecções por Flavivirus/transmissão , RNA Viral/genética , RNA Viral/isolamento & purificaçãoRESUMO
Annexin A2 (ANXA2) is a multifaceted protein implicated in various stages of viral infections, particularly in envelope virus replication through mechanisms such as endocytosis and exocytosis. This study delves into the characterization and functional dynamics of duck ANXA2 (duANXA2). We successfully cloned the full-length coding sequence of duANXA2 and conducted a detailed structural analysis. The open reading frame (ORF) of duANXA2 is 1020 bp, encoding 339 amino acids and featuring 4 conserved domains. Phylogenetic tree analysis indicates that duANXA2 is most closely related to Gallus gallus, with significantly lesser homology to fish species. We evaluated the tissue-specific expression of duANXA2 in healthy ducks, noting its ubiquitous presence but varying expression levels across different organs, with notably high expression in the esophagus and immune organs. Upon infecting duck embryo fibroblast (DEF) cells with the duck Tembusu virus (DTMUV), a flavivirus causing ducks substantial mortality and a dramatic decline in egg production, we observed a pronounced upregulation of duANXA2. Functional assays demonstrated that overexpression of duANXA2 in DEF cells augments DTMUV replication, while its interference markedly reduces DTMUV replication. These findings underscore the role of duANXA2 as a facilitator of DTMUV replication, presenting it as a potential target for therapeutic intervention in managing DTMUV infections.
Assuntos
Anexina A2 , Proteínas Aviárias , Patos , Flavivirus , Filogenia , Doenças das Aves Domésticas , Replicação Viral , Animais , Patos/genética , Anexina A2/genética , Anexina A2/metabolismo , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/genética , Flavivirus/fisiologia , Flavivirus/genética , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Proteínas Aviárias/química , Clonagem Molecular , Infecções por Flavivirus/veterinária , Infecções por Flavivirus/virologia , Infecções por Flavivirus/genética , Sequência de Aminoácidos , Alinhamento de Sequência/veterináriaRESUMO
Usutu virus (USUV) is an emerging flavivirus that is maintained in an enzootic cycle with mosquitoes as vectors and birds as amplifying hosts. In Europe, the virus has caused mass mortality of wild birds, mainly among Common Blackbird (Turdus merula) populations. While mosquitoes are the primary vectors for USUV, Common Blackbirds and other avian species are exposed to other arthropod ectoparasites, such as ticks. It is unknown, however, if ticks can maintain and transmit USUV. We addressed this question using in vitro and in vivo experiments and field collected data. USUV replicated in IRE/CTVM19 Ixodes ricinus tick cells and in injected ticks. Moreover, I. ricinus nymphs acquired the virus via artificial membrane blood-feeding and maintained the virus for at least 70 days. Transstadial transmission of USUV from nymphs to adults was confirmed in 4.9% of the ticks. USUV disseminated from the midgut to the haemocoel, and was transmitted via the saliva of the tick during artificial membrane blood-feeding. We further explored the role of ticks by monitoring USUV in questing ticks and in ticks feeding on wild birds in the Netherlands between 2016 and 2019. In total, 622 wild birds and the Ixodes ticks they carried were tested for USUV RNA. Of these birds, 48 (7.7%) carried USUV-positive ticks. The presence of negative-sense USUV RNA in ticks, as confirmed via small RNA-sequencing, showed active virus replication. In contrast, we did not detect USUV in 15,381 questing ticks collected in 2017 and 2019. We conclude that I. ricinus can be infected with USUV and can transstadially and horizontally transmit USUV. However, in comparison to mosquito-borne transmission, the role of I. ricinus ticks in the epidemiology of USUV is expected to be minor.
Assuntos
Doenças das Aves , Infecções por Flavivirus , Flavivirus , Ixodes , Ninfa , Animais , Ixodes/virologia , Ixodes/fisiologia , Flavivirus/fisiologia , Flavivirus/genética , Infecções por Flavivirus/transmissão , Infecções por Flavivirus/veterinária , Infecções por Flavivirus/virologia , Ninfa/virologia , Doenças das Aves/virologia , Doenças das Aves/transmissão , Aves/virologia , Vetores Aracnídeos/virologia , Vetores Aracnídeos/fisiologia , Países Baixos , FemininoRESUMO
Usutu virus is an emerging pathogen transmitted by mosquitoes. Culex modestus mosquitoes are widespread in Europe, but their role in disease transmission is poorly understood. Recent data from a single infectious mosquito suggested that Culex modestus could be an unrecognized vector for Usutu virus. In this study, our aim was to corroborate this finding using a larger sample size. We collected immature Culex modestus from a reedbed pond in Flemish Brabant, Belgium, and reared them in the laboratory until the third generation. Adult females were then experimentally infected with Usutu virus in a blood meal and incubated at 25 °C for 14 days. The presence of Usutu virus in the saliva, head and body of each female was determined by plaque assay and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). The transmission efficiency was 54% (n = 15/28), confirming that Belgian Culex modestus can experimentally transmit Usutu virus.
Assuntos
Culex , Infecções por Flavivirus , Flavivirus , Mosquitos Vetores , Animais , Culex/virologia , Feminino , Mosquitos Vetores/virologia , Flavivirus/genética , Flavivirus/fisiologia , Bélgica , Infecções por Flavivirus/transmissão , Infecções por Flavivirus/virologia , Saliva/virologiaRESUMO
Orthoflaviviruses are enveloped positive-sense RNA viruses comprising numerous human pathogens transmitted by hematophagous arthropods. This includes viruses such as dengue virus, Zika virus, and yellow fever virus. The viral nonstructural protein NS1 plays a central role in the pathogenesis and cycle of these viruses by acting in two different forms: associated with the plasma membrane (NS1m) or secreted outside the cell (NS1s). The versatility of NS1 is evident in its ability to modulate various aspects of the infectious process, from immune evasion to pathogenesis. As an intracellular protein, it disrupts many processes, interfering with signaling pathways and facilitating viral replication in concert with other viral proteins. As a secreted protein, NS1 actively participates in immune evasion, interfering with the host immune system, inhibiting the complement system, facilitating viral dissemination, and disrupting the integrity of endothelial barriers. This review primarily aims to address the role of NS1 in viral pathogenesis associated with orthoflaviviruses.