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1.
Front Immunol ; 11: 1450, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32733480

RESUMO

The complement system is a key component of innate immunity which readily responds to invading microorganisms. Activation of the complement system typically occurs via three main pathways and can induce various antimicrobial effects, including: neutralization of pathogens, regulation of inflammatory responses, promotion of chemotaxis, and enhancement of the adaptive immune response. These can be vital host responses to protect against acute, chronic, and recurrent viral infections. Consequently, many viruses (including dengue virus, West Nile virus and Nipah virus) have evolved mechanisms for evasion or dysregulation of the complement system to enhance viral infectivity and even exacerbate disease symptoms. The complement system has multifaceted roles in both innate and adaptive immunity, with both intracellular and extracellular functions, that can be relevant to all stages of viral infection. A better understanding of this virus-host interplay and its contribution to pathogenesis has previously led to: the identification of genetic factors which influence viral infection and disease outcome, the development of novel antivirals, and the production of safer, more effective vaccines. This review will discuss the antiviral effects of the complement system against numerous viruses, the mechanisms employed by these viruses to then evade or manipulate this system, and how these interactions have informed vaccine/therapeutic development. Where relevant, conflicting findings and current research gaps are highlighted to aid future developments in virology and immunology, with potential applications to the current COVID-19 pandemic.


Assuntos
Betacoronavirus/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Evasão da Resposta Imune , Pandemias/prevenção & controle , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Vacinas Virais/imunologia , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Ativação do Complemento/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Flavivirus/imunologia , Infecções por Flavivirus/imunologia , Infecções por Flavivirus/virologia , Humanos , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Internalização do Vírus
2.
PLoS Pathog ; 16(6): e1008513, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32555677

RESUMO

The ability of the endosymbiont Wolbachia pipientis to restrict RNA viruses is presently being leveraged to curb global transmission of arbovirus-induced diseases. Past studies have shown that virus replication is limited early in arthropod cells colonized by the bacterium, although it is unclear if this phenomenon is replicated in mosquito cells that first encounter viruses obtained through a vertebrate blood meal. Furthermore, these cellular events neither explain how Wolbachia limits dissemination of viruses between mosquito tissues, nor how it prevents transmission of infectious viruses from mosquitoes to vertebrate host. In this study, we try to address these issues using an array of mosquito cell culture models, with an additional goal being to identify a common viral target for pathogen blocking. Our results establish the viral RNA as a cellular target for Wolbachia-mediated inhibition, with the incoming viral RNA experiencing rapid turnover following internalization in cells. This early block in replication in mosquito cells initially infected by the virus thus consequently reduces the production of progeny viruses from these same cells. However, this is not the only contributor to pathogen blocking. We show that the presence of Wolbachia reduces the per-particle infectivity of progeny viruses on naïve mosquito and vertebrate cells, consequently limiting virus dissemination and transmission, respectively. Importantly, we demonstrate that this aspect of pathogen blocking is independent of any particular Wolbachia-host association and affects viruses belonging to Togaviridae and Flaviviridae families of RNA viruses. Finally, consistent with the idea of the viral RNA as a target, we find that the encapsidated virion RNA is less infectious for viruses produced from Wolbachia-colonized cells. Collectively, our findings present a common mechanism of pathogen blocking in mosquitoes that establish a link between virus inhibition in the cell to virus dissemination and transmission.


Assuntos
Flavivirus/metabolismo , RNA Viral/metabolismo , Togaviridae/metabolismo , Wolbachia/metabolismo , Aedes , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Drosophila melanogaster , Flavivirus/genética , RNA Viral/genética , Togaviridae/genética , Células Vero , Wolbachia/genética
3.
Mem Inst Oswaldo Cruz ; 115: e200012, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32520074

RESUMO

In Argentina, many Flavivirus were recognised including West Nile virus (WNV). During 2009 several strains of Culex Flavivirus (CxFV), an insect-specific flavivirus, were isolated in the same region where circulation of WNV was detected. Hence, the objective of this study was to analyse the effect of co-infection in vitro assays using CxFV and WNV Argentinean strains in order to evaluate if CxFV could affect WNV replication. Our results showed that WNV replication was suppressed when multiplicity of infection (MOI) for CxFV was 10 or 100 times higher than WNV. Nevertheless, in vivo assays are necessary in order to evaluate the superinfection exclusion potential.


Assuntos
Aedes/virologia , Culex/virologia , Flavivirus/fisiologia , Insetos Vetores/virologia , Superinfecção/virologia , Vírus do Nilo Ocidental/patogenicidade , Animais , Argentina , Linhagem Celular , Ensaio de Placa Viral
4.
Arch Virol ; 165(8): 1863-1868, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32474687

RESUMO

The aim of this study was to improve flavivirus field monitoring in Brazil using a reliable probe-based RT-qPCR assay. Standard flavivirus strains were employed to evaluate the performance of the assay, and its applicability was evaluated using 235 stored pools of Culicidae samples collected between 1993 and 1997 and in 2016. Flavivirus species were identified by sequencing. Sixteen (6.8%) samples tested positive: Ilheus virus, Iguape virus, and Saint Louis encephalitis virus were identified in historical specimens from 1993-1994, while insect-specific flaviviruses were detected in the samples from 2016. This approach was demonstrated to be accurate for flavivirus detection and characterization, and it can be successfully applied for vector surveillance and for monitoring and discovery of insect specific flaviviruses.


Assuntos
Flavivirus/genética , Vigilância em Saúde Pública/métodos , Animais , Brasil , Filogenia , Reação em Cadeia da Polimerase em Tempo Real/métodos
6.
Arch Virol ; 165(8): 1769-1776, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32440701

RESUMO

South Texas has experienced local transmission of Zika virus and of other mosquito-borne viruses such as chikungunya virus and dengue virus in the last decades. Using a mosquito surveillance program in the Lower Rio Grande Valley (LRGV) and San Antonio, TX, from 2016 to 2018, we detected the presence of an insect-specific virus, cell fusing agent virus (CFAV), in the Aedes aegypti mosquito population. We tested 6,326 females and 1,249 males from the LRGV and 659 females from San Antonio for CFAV by RT-PCR using specific primers. Infection rates varied from 0 to 261 per 1,000 mosquitoes in the LRGV and 115 to 208 per 1,000 in San Antonio depending on the month of collection. Infection rates per 1,000 individuals appeared higher in females collected from BG Sentinel 2 traps compared to Autocidal Gravid Ovitraps, but the ratio of the percentage of infected pools did not differ by trap type. The natural viral load in individual males ranged from 1.25 x 102 to 5.50 x 106 RNA copies and in unfed females from 5.42 x 103 to 8.70 x 106 RNA copies. Gravid females were found to harbor fewer viral particles than males and unfed females.


Assuntos
Aedes/virologia , Flavivirus/genética , Animais , Feminino , Vírus de Insetos/genética , Masculino , Mosquitos Vetores/genética , RNA Viral/genética , Texas , Carga Viral/genética
8.
PLoS Negl Trop Dis ; 14(4): e0008223, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32324736

RESUMO

Usutu virus (USUV), an African mosquito-borne flavivirus closely related to West Nile virus, was first isolated in South Africa in 1959. USUV emerged in Europe two decades ago, causing notably massive mortality in Eurasian blackbirds. USUV is attracting increasing attention due to its potential for emergence and its rapid spread in Europe in recent years. Although mainly asymptomatic or responsible for mild clinical signs, USUV was recently described as being associated with neurological disorders in humans such as encephalitis and meningoencephalitis, highlighting the potential health threat posed by the virus. Despite this, USUV pathogenesis remains largely unexplored. The aim of this study was to evaluate USUV neuropathogenicity using in vivo and in vitro approaches. Our results indicate that USUV efficiently replicates in the murine central nervous system. Replication in the spinal cord and brain is associated with recruitment of inflammatory cells and the release of inflammatory molecules as well as induction of antiviral-responses without major modulation of blood-brain barrier integrity. Endothelial cells integrity is also maintained in a human model of the blood-brain barrier despite USUV replication and release of pro-inflammatory cytokines. Furthermore, USUV-inoculated mice developed major ocular defects associated with inflammation. Moreover, USUV efficiently replicates in human retinal pigment epithelium. Our results will help to better characterize the physiopathology related to USUV infection in order to anticipate the potential threat of USUV emergence.


Assuntos
Flavivirus/patogenicidade , Modelos Biológicos , Sistema Nervoso/virologia , Animais , Encéfalo/virologia , Modelos Animais de Doenças , Células Endoteliais/virologia , Células Epiteliais/virologia , Flavivirus/crescimento & desenvolvimento , Humanos , Camundongos , Epitélio Pigmentado Ocular/virologia , Medula Espinal/virologia
9.
PLoS Negl Trop Dis ; 14(3): e0008156, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32226028

RESUMO

Surveillance of Usutu virus is crucial to prevent future outbreaks both in Europe and in other countries currently naïve to the infection, such as the Americas. This goal remains difficult to achieve, notably because of the lack of large-scale cohort studies and the absence of commercially available diagnostic reagents for USUV. This work started with the first identification of USUV in a blood donor in the Friuli Venezia Giulia (FVG) Region in Northern-Eastern Italy, which is endemic for West Nile virus. Considering that only one IgG ELISA is commercially available, but none for IgM, a novel NS1 antigen based IgG/M ELISA has been developed. This assay tested successfully for the detection of Usutu virus in blood donors with the identification of a second case of transmission and high levels of exposure. Furthermore, two pan-flavivirus antiviral drugs, that we previously characterized to be inhibitors of other flavivirus infectivity, were successfully tested for inhibition of Usutu virus with inhibitory concentrations in the low micromolar range. To conclude, this work identifies North-Eastern Italy as endemic for Usutu virus with implications for the screening of transfusion blood. A novel NS1-based ELISA test has been implemented for the detection of IgM/G that will be of importance as a tool for the diagnosis and surveillance of Usutu virus infection. Finally, Usutu virus is shown to be sensitive to a class of promising pan-flavivirus drugs.


Assuntos
Anticorpos Antivirais/sangue , Antivirais/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Infecções por Flavivirus/diagnóstico , Flavivirus/isolamento & purificação , Testes Sorológicos/métodos , Proteínas não Estruturais Virais/imunologia , Sangue/virologia , Doadores de Sangue , Feminino , Flavivirus/efeitos dos fármacos , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Itália , Testes de Sensibilidade Microbiana , Testes de Neutralização/métodos
10.
PLoS Negl Trop Dis ; 14(4): e0008217, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32282830

RESUMO

BACKGROUND: The Asian bush mosquito Aedes japonicus is invading Europe and was first discovered in Lelystad, the Netherlands in 2013, where it has established a permanent population. In this study, we investigated the vector competence of Ae. japonicus from the Netherlands for the emerging Zika virus (ZIKV) and zoonotic Usutu virus (USUV). ZIKV causes severe congenital microcephaly and Guillain-Barré syndrome in humans. USUV is closely related to West Nile virus, has recently spread throughout Europe and is causing mass mortality of birds. USUV infection in humans can result in clinical manifestations ranging from mild disease to severe neurological impairments. METHODOLOGY/PRINCIPAL FINDINGS: In our study, field-collected Ae. japonicus females received an infectious blood meal with ZIKV or USUV by droplet feeding. After 14 days at 28°C, 3% of the ZIKV-blood fed mosquitoes and 13% of the USUV-blood fed mosquitoes showed virus-positive saliva, indicating that Ae. japonicus can transmit both viruses. To investigate the effect of the mosquito midgut barrier on virus transmission, female mosquitoes were intrathoracically injected with ZIKV or USUV. Of the injected mosquitoes, 96% (ZIKV) and 88% (USUV) showed virus-positive saliva after 14 days at 28°C. This indicates that ZIKV and USUV can efficiently replicate in Ae. japonicus but that a strong midgut barrier is normally restricting virus dissemination. Small RNA deep sequencing of orally infected mosquitoes confirmed active replication of ZIKV and USUV, as demonstrated by potent small interfering RNA responses against both viruses. Additionally, de novo small RNA assembly revealed the presence of a novel narnavirus in Ae. japonicus. CONCLUSIONS/SIGNIFICANCE: Given that Ae. japonicus can experimentally transmit arthropod-borne viruses (arboviruses) like ZIKV and USUV and is currently expanding its territories, we should consider this mosquito as a potential vector for arboviral diseases in Europe.


Assuntos
Aedes/virologia , Infecções por Flavivirus/transmissão , Mosquitos Vetores/virologia , Infecção por Zika virus/transmissão , Animais , Feminino , Flavivirus , Humanos , Microcefalia/virologia , Países Baixos , Saliva/virologia , Temperatura , Zika virus
11.
J Vis Exp ; (157)2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32225162

RESUMO

Whole genome sequencing can be used to characterize and to trace viral outbreaks. Nanopore-based whole genome sequencing protocols have been described for several different viruses. These approaches utilize an overlapping amplicon-based approach which can be used to target a specific virus or group of genetically related viruses. In addition to confirmation of the virus presence, sequencing can be used for genomic epidemiology studies, to track viruses and unravel origins, reservoirs and modes of transmission. For such applications, it is crucial to understand possible effects of the error rate associated with the platform used. Routine application in clinical and public health settings require that this is documented with every important change in the protocol. Previously, a protocol for whole genome Usutu virus sequencing on the nanopore sequencing platform was validated (R9.4 flowcell) by direct comparison to Illumina sequencing. Here, we describe the method used to determine the required read coverage, using the comparison between the R10 flow cell and Illumina sequencing as an example.


Assuntos
Flavivirus/genética , Genoma Viral , Sequenciamento por Nanoporos , Sequenciamento Completo do Genoma , Sequência Consenso/genética , Primers do DNA/metabolismo , Análise de Dados , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Reação em Cadeia da Polimerase , Padrões de Referência
12.
Artigo em Inglês | MEDLINE | ID: mdl-32284379

RESUMO

Bunyaviruses are significant human pathogens, causing diseases ranging from hemorrhagic fevers to encephalitis. Among these viruses, La Crosse virus (LACV), a member of the California serogroup, circulates in the eastern and midwestern United States. While LACV infection is often asymptomatic, dozens of cases of encephalitis are reported yearly. Unfortunately, no antivirals have been approved to treat LACV infection. Here, we developed a method to rapidly test potential antivirals against LACV infection. From this screen, we identified several potential antiviral molecules, including known antivirals. Additionally, we identified many novel antivirals that exhibited antiviral activity without affecting cellular viability. Valinomycin, a potassium ionophore, was among our top targets. We found that valinomycin exhibited potent anti-LACV activity in multiple cell types in a dose-dependent manner. Valinomycin did not affect particle stability or infectivity, suggesting that it may preclude virus replication by altering cellular potassium ions, a known determinant of LACV entry. We extended these results to other ionophores and found that the antiviral activity of valinomycin extended to other viral families, including bunyaviruses (Rift Valley fever virus, Keystone virus), enteroviruses (coxsackievirus, rhinovirus), flavirivuses (Zika virus), and coronaviruses (human coronavirus 229E [HCoV-229E] and Middle East respiratory syndrome CoV [MERS-CoV]). In all viral infections, we observed significant reductions in virus titer in valinomycin-treated cells. In sum, we demonstrate the importance of potassium ions to virus infection, suggesting a potential therapeutic target to disrupt virus replication.


Assuntos
Antivirais/farmacologia , Encefalite da Califórnia/tratamento farmacológico , Ionóforos/farmacologia , Vírus La Crosse/efeitos dos fármacos , Potássio/metabolismo , Valinomicina/farmacologia , Replicação Viral/efeitos dos fármacos , Coronavirus/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Encefalite da Califórnia/virologia , Enterovirus/efeitos dos fármacos , Flavivirus/efeitos dos fármacos , Humanos , Orthobunyavirus/efeitos dos fármacos , Estados Unidos
13.
PLoS One ; 15(4): e0232274, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32330205

RESUMO

The Flaviviridae is a family of enveloped viruses with a positive-sense single-stranded RNA genome. It contains many viruses that threaten human health, such as Japanese encephalitis virus (JEV) and yellow fever virus (YFV) of the genus Flavivirus as well as hepatitis C virus of the genus Hepacivirus. Cell culture systems highly permissive for the Flaviviridae viruses are very useful for their isolation, propagation, and diagnosis, an understanding of their biology, and the development of vaccines and antiviral agents. Previously, we isolated a human hepatoma HuH-7-derived cell clone, Huh7.5.1-8, which is highly permissive to hepatitis C virus infection. Here, we have characterized flavivirus infection in the Huh7.5.1-8 cell line by comparing with that in the African green monkey kidney-derived Vero cell line, which is permissive for a wide spectrum of viruses. Upon infection with JEV, Huh7.5.1-8 cells produced a higher amount of virus particles early in infection and were more susceptible to virus-induced cell death than Vero cells. Similar outcomes were obtained when the cells were infected with another flavivirus, YFV (17D-204 strain). Quantification of cellular and extracellular viral RNA revealed that high JEV production in Huh7.5.1-8 cells can be attributed to rapid viral replication kinetics and efficient virus release early in infection. In a plaque assay, Huh7.5.1-8 cells developed JEV plaques more rapidly than Vero cells. Although this was not the case with YFV plaques, Huh7.5.1-8 cells developed higher numbers of YFV plaques than Vero cells. Sequence analysis of cDNA encoding an antiviral RNA helicase, RIG-I, showed that Huh7.5.1-8 cells expressed not only a full-length RIG-I mRNA with a known dominant-negative missense mutation but also variants without the mutation. However, the latter mRNAs lacked exon 5/6-12, indicating functional loss of RIG-I in the cells. These characteristics of the Huh7.5.1-8 cell line are helpful for flavivirus detection, titration, and propagation.


Assuntos
Carcinoma Hepatocelular/virologia , Chlorocebus aethiops/virologia , Flavivirus/crescimento & desenvolvimento , Animais , Linhagem Celular , Linhagem Celular Tumoral , Flavivirus/genética , Infecções por Flavivirus/virologia , Hepacivirus/genética , Humanos , RNA Viral/genética , Células Vero , Replicação Viral/genética
14.
Adv Exp Med Biol ; 1204: 57-73, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32152943

RESUMO

CLEC5A is a spleen tyrosine kinase (Syk)-coupled C-type lectin that is highly expressed by monocytes, macrophages, neutrophils, and dendritic cells and interacts with virions directly, via terminal fucose and mannose moieties of viral glycans. CLEC5A also binds to N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) disaccharides of bacterial cell walls. Compared to other C-type lectins (DC-SIGN and DC-SIGNR) and TLRs, CLEC5A binds its ligands with relatively low affinities. However, CLEC5A forms a multivalent hetero-complex with DC-SIGN and other C-type lectins upon engagement with ligands, and thereby mediates microbe-induced inflammatory responses via activation of Syk. For example, in vivo studies in mouse models have demonstrated that CLEC5A is responsible for flaviviruses-induced hemorrhagic shock and neuroinflammation, and a CLEC5A polymorphism in humans is associated with disease severity following infection with dengue virus. In addition, CLEC5A is co-activated with TLR2 by Listeria and Staphylococcus. Furthermore, CLEC5A-postive myeloid cells are responsible for Concanavilin A-induced aseptic inflammatory reactions. Thus, CLEC5A is a promiscuous pattern recognition receptor in myeloid cells and is a potential therapeutic target for attenuation of both septic and aseptic inflammatory reactions.


Assuntos
Lectinas Tipo C/imunologia , Receptores de Superfície Celular/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Animais , Vírus da Dengue/imunologia , Flavivirus/imunologia , Humanos , Inflamação/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Neutrófilos/imunologia
15.
PLoS Negl Trop Dis ; 14(3): e0008166, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32203536

RESUMO

Flaviviruses such as yellow fever, dengue or Zika viruses are responsible for significant human and veterinary diseases worldwide. These viruses contain an RNA genome, prone to mutations, which enhances their potential to emerge as pathogens. Bamaga virus (BgV) is a mosquito-borne flavivirus in the yellow fever virus group that we have previously shown to be host-restricted in vertebrates and horizontally transmissible by Culex mosquitoes. Here, we aimed to characterise BgV host-restriction and to investigate the mechanisms involved. We showed that BgV could not replicate in a wide range of vertebrate cell lines and animal species. We determined that the mechanisms involved in BgV host-restriction were independent of the type-1 interferon response and RNAse L activity. Using a BgV infectious clone and two chimeric viruses generated as hybrids between BgV and West Nile virus, we demonstrated that BgV host-restriction occurred post-cell entry. Notably, BgV host-restriction was shown to be temperature-dependent, as BgV replicated in all vertebrate cell lines at 34°C but only in a subset at 37°C. Serial passaging of BgV in Vero cells resulted in adaptive mutants capable of efficient replication at 37°C. The identified mutations resulted in amino acid substitutions in NS4A-S124F, NS4B-N244K and NS5-G2C, all occurring close to a viral protease cleavage site (NS4A/2K and NS4B/NS5). These mutations were reverse engineered into infectious clones of BgV, which revealed that NS4B-N244K and NS5-G2C were sufficient to restore BgV replication in vertebrate cells at 37°C, while NS4A-S124F further increased replication efficiency. When these mutant viruses were injected into immunocompetent mice, alongside BgV and West Nile virus chimeras, infection and neurovirulence were enhanced as determined by clinical scores, seroconversion, micro-neutralisation, viremia, histopathology and immunohistochemistry, confirming the involvement of these residues in the attenuation of BgV. Our studies identify a new mechanism of host-restriction and attenuation of a mosquito-borne flavivirus.


Assuntos
Infecções por Flavivirus/virologia , Flavivirus/genética , Flavivirus/patogenicidade , Mutação , Proteínas não Estruturais Virais/genética , Animais , Encéfalo/patologia , Encéfalo/virologia , Linhagem Celular , Chlorocebus aethiops , Culicidae/virologia , Modelos Animais de Doenças , Endorribonucleases/metabolismo , Feminino , Flavivirus/fisiologia , Infecções por Flavivirus/metabolismo , Infecções por Flavivirus/patologia , Células HEK293 , Humanos , Masculino , Camundongos , Mosquitos Vetores/virologia , Células Vero , Virulência/genética , Replicação Viral , Vírus do Nilo Ocidental/genética
16.
Parasit Vectors ; 13(1): 54, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-32041638

RESUMO

BACKGROUND: Wolbachia pipientis are bacterial endosymbionts of arthropods currently being implemented as biocontrol agents to reduce the global burden of arboviral diseases. Some strains of Wolbachia, when introduced into Aedes aegypti mosquitoes, reduce or block the replication of RNA viruses pathogenic to humans. The wAlbB strain of Wolbachia was originally isolated from Aedes albopictus, and when transinfected into Ae. aegypti, persists in mosquitoes under high temperature conditions longer than other strains. The utility of wAlbB to block a broad spectrum of RNA viruses has received limited attention. Here we test the ability of wAlbB to reduce or block the replication of a range of Flavivirus and Alphavirus species in cell culture. METHODS: The C6/36 mosquito cell line was stably infected with the wAlbB strain using the shell-vial technique. The replication of dengue, West Nile and three strains of Zika (genus Flavivirus), and Ross River, Barmah Forest and Sindbis (genus Alphavirus) viruses was compared in wAlbB-infected cells with Wolbachia-free controls. Infectious virus titres were determined using either immunofocus or plaque assays. A general linear model was used to test for significant differences in replication between flaviviruses and alphaviruses. RESULTS: Titres of all viruses were significantly reduced in cell cultures infected with wAlbB versus Wolbachia-free controls. The magnitude of reduction in virus yields varied among virus species and, within species, also among the strains utilized. CONCLUSION: Our results suggest that wAlbB infection of arthropods could be used to reduce transmission of a wide range of pathogenic RNA viruses.


Assuntos
Alphavirus/crescimento & desenvolvimento , Flavivirus/crescimento & desenvolvimento , Interações Microbianas , Replicação Viral , Wolbachia , Aedes/microbiologia , Aedes/virologia , Infecções por Alphavirus/prevenção & controle , Animais , Linhagem Celular/microbiologia , Linhagem Celular/virologia , Dengue/prevenção & controle , Humanos , Insetos Vetores/microbiologia , Insetos Vetores/virologia , Controle Biológico de Vetores , Viroses/prevenção & controle , Viroses/transmissão , Febre do Nilo Ocidental/prevenção & controle , Infecção por Zika virus/prevenção & controle
17.
Nat Med ; 26(2): 228-235, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32015557

RESUMO

Zika virus (ZIKV) has caused significant disease, with widespread cases of neurological pathology and congenital neurologic defects. Rapid vaccine development has led to a number of candidates capable of eliciting potent ZIKV-neutralizing antibodies (reviewed in refs. 1-3). Despite advances in vaccine development, it remains unclear how ZIKV vaccination affects immune responses in humans with prior flavivirus immunity. Here we show that a single-dose immunization of ZIKV purified inactivated vaccine (ZPIV)4-7 in a dengue virus (DENV)-experienced human elicited potent cross-neutralizing antibodies to both ZIKV and DENV. Using a unique ZIKV virion-based sorting strategy, we isolated and characterized multiple antibodies, including one termed MZ4, which targets a novel site of vulnerability centered on the Envelope (E) domain I/III linker region and protects mice from viremia and viral dissemination following ZIKV or DENV-2 challenge. These data demonstrate that Zika vaccination in a DENV-experienced individual can boost pre-existing flavivirus immunity and elicit protective responses against both ZIKV and DENV. ZPIV vaccination in Puerto Rican individuals with prior flavivirus experience yielded similar cross-neutralizing potency after a single vaccination, highlighting the potential benefit of ZIKV vaccination in flavivirus-endemic areas.


Assuntos
Dengue/imunologia , Doadores de Tecidos , Vacinas Virais/uso terapêutico , Infecção por Zika virus/imunologia , Infecção por Zika virus/prevenção & controle , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Reações Cruzadas , Vírus da Dengue , Mapeamento de Epitopos , Feminino , Flavivirus/metabolismo , Humanos , Imunoglobulina G/química , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ligação Proteica , Domínios Proteicos , Vacinação , Vacinas de Produtos Inativados/uso terapêutico , Células Vero , Viremia , Zika virus
18.
Acta Trop ; 205: 105401, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32081658

RESUMO

In Brazil, flaviviruses have caused massive outbreaks. Surveillance programs designed to monitor virus activity in vectors provides a system for mapping disease distribution and for identifying specific vector species for targeted control. The present study aimed to describe the detection, whole genome characterization and phylogenetic analysis of Ilheus virus (ILHV) and Iguape virus (IGUV) strains obtained from historical mosquito's samples. Twelve isolates of pooled mosquito specimens (inoculated in neonate mouse brain) collected in the state of São Paulo, Brazil, in 1993, 1994 and 1997 were investigated. Viral RNA was extracted and analyzed by qRT-PCR using Flavivirus genus-specific primers. Positive samples were sequenced and underwent phylogenetic analyses. Flavivirus was detected in 50% of the specimens. Positive samples were successfully Sanger sequenced. Three Anopholes cruzii pools collected in 1994 were positive for IGUV. One Culex sp. pool, one Anopheles triannulatus pool, and one Coquillettidia juxtamansonia pool, collected in 1994, were positive for ILHV. Metagenomic sequencing successfully characterize one ILHV and four IGUV full genomes, and revealed a high degree of homology between the Brazilian ILHV and IGUV strains and isolates available in GenBank. Phylogenetic analysis of partial ILHV NS5 gene revealed three distinct lineages (clades), an indication of genetic heterogeneity in strains circulating in Brazil. Nucleotide insertions and a high-level of nucleotide diversity were observed in the NS1 protein and capsid region of IGUV strains, respectively. Detection of ILHV and IGUV in mosquitoes from Southeastern Brazil confirms the historical circulation of these viruses in this area. Furthermore, this first evidence of ILHV in Anopheles triannulatus suggests the potential importance of Anopheles mosquitoes in the IGUV transmission cycle. Genomic and phylogenetic analysis of these viruses provided insights into their diversity and evolution, which are important for the emergence patterns of flaviviruses and their evolutionary trends in Brazil, an endemic country for several arbovirus. in In-depth studies of ILHV and IGUV including vector competence and molecular studies are needed to shed light on their epidemiology and potential risk of future emergence.


Assuntos
Culicidae/virologia , Flavivirus/genética , Flavivirus/isolamento & purificação , Genoma Viral , Animais , Sequência de Bases , Brasil/epidemiologia , Infecções por Flaviviridae/epidemiologia , Infecções por Flaviviridae/virologia , Camundongos , Mosquitos Vetores/virologia , Filogenia , RNA Viral/genética
19.
Vet Res ; 51(1): 12, 2020 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-32070432

RESUMO

High-mobility group box 1 protein (HMGB1) shows endogenous damage-associated molecular patterns (DAMPs) and is also an early warning protein that activates the body's innate immune system. Here, the full-length coding sequence of HMGB1 was cloned from the spleen of Cherry Valley duck and analyzed. We find that duck HMGB1(duHMGB1) is mostly located in the nucleus of duck embryo fibroblast (DEF) cells under normal conditions but released into the cytoplasm after lipopolysaccharide (LPS) stimulation. Knocking-down or overexpressing duHMGB1 had no effect on the baseline apoptosis rate of DEF cells. However, overexpression increased weakly apoptosis after LPS activation. In addition, overexpression strongly activated the IFN-I/IRF7 signaling pathway in DEF cells and significantly increased the transcriptional level of numerous pattern recognition receptors (PRRs), pro-inflammatory cytokines (IL-6, TNF-α), IFNs and antiviral molecules (OAS, PKR, Mx) starting from 48 h post-transfection. Overexpression of duHMGB1 strongly impacted duck virus replication, either by inhibiting it from the first stage of infection for novel duck reovirus (NDRV) and at late stage for duck Tembusu virus (DTMUV) or duck plague virus (DPV), or promoting replication at early stage for DTMUV and DPV infection. Importantly, data from duHMGB1 overexpression and knockdown experiments, time-dependent DEF cells transcriptional immune responses suggest that duHMGB1 and RIG-I receptor might cooperate to promote the expression of antiviral proteins after NDRV infection, as a potential mechanism of duHMGB1-mediated antiviral activity.


Assuntos
Proteínas Aviárias/genética , Patos/genética , Infecções por Flavivirus/veterinária , Proteína HMGB1/genética , Infecções por Herpesviridae/veterinária , Imunidade Inata/genética , Doenças das Aves Domésticas/prevenção & controle , Transdução de Sinais/genética , Sequência de Aminoácidos , Animais , Antivirais , Proteínas Aviárias/química , Proteínas Aviárias/metabolismo , Patos/metabolismo , Flavivirus , Infecções por Flavivirus/prevenção & controle , Infecções por Flavivirus/virologia , Perfilação da Expressão Gênica/veterinária , Proteína HMGB1/química , Proteína HMGB1/metabolismo , Infecções por Herpesviridae/prevenção & controle , Infecções por Herpesviridae/virologia , Mardivirus , Filogenia , Doenças das Aves Domésticas/virologia , Alinhamento de Sequência/veterinária
20.
Vet Microbiol ; 240: 108508, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31902493

RESUMO

Duck Tembusu virus (DTMUV) is a major pathogen of duck industry in China. In the current study, we generated different constructs containing envelope (E) protein, pre-membrane-envelope (prM-E) protein, and C-terminally truncated E protein of the DTMUV. The constructed proteins could induce specific antibody responses in young ducks. When ducklings were immunized with the constructed proteins, they were 100% protected against DTMUV infection. Furthermore, the fluorescent signal of the truncated E protein was stronger than other constructed proteins, when Bac-to-Bac baculovirus expression system was applied. Our data demonstrated that the truncated E protein used in the current study could be applied as a potential vaccine candidate to control DTMUV infection in young ducks.


Assuntos
Infecções por Flavivirus/veterinária , Flavivirus/imunologia , Doenças das Aves Domésticas/prevenção & controle , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Fatores Etários , Oxirredutases do Álcool/genética , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Baculoviridae/genética , China , Proteínas de Ligação a DNA/genética , Patos/virologia , Flavivirus/química , Flavivirus/genética , Infecções por Flavivirus/imunologia , Infecções por Flavivirus/prevenção & controle , Doenças das Aves Domésticas/imunologia , Organismos Livres de Patógenos Específicos , Vacinação , Vacinas de Subunidades/imunologia , Proteínas do Envelope Viral/genética
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