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1.
PLoS Negl Trop Dis ; 13(11): e0007346, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31693659

RESUMO

BACKGROUND: Aedes aegypti is a vector mosquito of major public health importance, transmitting arthropod-borne viruses (arboviruses) such as chikungunya, dengue, yellow fever and Zika viruses. Wild mosquito populations are persistently infected at high prevalence with insect-specific viruses that do not replicate in vertebrate hosts. In experimental settings, acute infections with insect-specific viruses have been shown to modulate arbovirus infection and transmission in Ae. aegypti and other vector mosquitoes. However, the impact of persistent insect-specific virus infections, which arboviruses encounter more commonly in nature, has not been investigated extensively. Cell lines are useful models for studying virus-host interactions, however the available Ae. aegypti cell lines are poorly defined and heterogenous cultures. METHODOLOGY/PRINCIPLE FINDINGS: We generated single cell-derived clonal cell lines from the commonly used Ae. aegypti cell line Aag2. Two of the fourteen Aag2-derived clonal cell lines generated harboured markedly and consistently reduced levels of the insect-specific bunyavirus Phasi Charoen-like virus (PCLV) known to persistently infect Aag2 cells. In contrast to studies with acute insect-specific virus infections in cell culture and in vivo, we found that pre-existing persistent PCLV infection had no major impact on the replication of the flaviviruses dengue virus and Zika virus, the alphavirus Sindbis virus, or the rhabdovirus vesicular stomatitis virus. We also performed a detailed characterisation of the morphology, transfection efficiency and immune status of our Aag2-derived clonal cell lines, and have made a clone that we term Aag2-AF5 available to the research community as a well-defined cell culture model for arbovirus-vector interaction studies. CONCLUSIONS/SIGNIFICANCE: Our findings highlight the need for further in vivo studies that more closely recapitulate natural arbovirus transmission settings in which arboviruses encounter mosquitoes harbouring persistent rather than acute insect-specific virus infections. Furthermore, we provide the well-characterised Aag2-derived clonal cell line as a valuable resource to the arbovirus research community.


Assuntos
Aedes/virologia , Arbovirus/crescimento & desenvolvimento , Coinfecção/virologia , Mosquitos Vetores/virologia , Orthobunyavirus/crescimento & desenvolvimento , Replicação Viral , Alphavirus/crescimento & desenvolvimento , Animais , Arbovirus/genética , Sequência de Bases , Técnicas de Cultura de Células/métodos , Linhagem Celular , Vírus da Dengue/crescimento & desenvolvimento , Flavivirus/genética , Flavivirus/crescimento & desenvolvimento , Genoma Viral , Interações Hospedeiro-Patógeno/fisiologia , Orthobunyavirus/genética , Vírus de RNA/genética , Vírus de RNA/crescimento & desenvolvimento , Rhabdoviridae/crescimento & desenvolvimento , Vírus Sindbis/crescimento & desenvolvimento , Transfecção , Zika virus/crescimento & desenvolvimento
2.
Rev Bras Parasitol Vet ; 28(4): 764-768, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576974

RESUMO

Due to anthropic environmental changes, vector-borne diseases are emerging worldwide. Ticks are known vectors of several pathogens of concern among humans and animals. In recent decades, several examples of tick-borne emerging viral diseases have been reported (Crimean Congo hemorrhagic fever virus, Powassan virus, encephalitis virus, heartland virus, severe fever with thrombocytopenia syndrome virus). Unfortunately, few studies addressing the presence of viruses in wild ticks have been carried out in South America. With the aim of detecting flaviviruses and orthobunyaviruses in ticks, we carried out molecular detection in wild ticks collected in the state of Minas Gerais, Brazil. No Flavivirus-positive ticks were detected; however, we detected activity of Orthobunyavirus in 8 Amblyomma tick specimens. One of those individuals was positive for Bunyamwera orthobunyavirus, which represents the first report of this virus among ticks in South America. Further studies related to the ecology of zoonotic diseases are needed to increase knowledge of this topic, including attempts at viral isolation, full genome sequencing and biological characterization. In this way, we will obtain a better picture of the real risk of ticks as a vector for viral diseases for humans and animals on our continent, where no tick-borne viral disease is known to occur.


Assuntos
Flavivirus/genética , Orthobunyavirus/genética , Carrapatos/virologia , Animais , Feminino , Flavivirus/classificação , Flavivirus/isolamento & purificação , Masculino , Orthobunyavirus/classificação , Orthobunyavirus/isolamento & purificação , Filogenia , Inquéritos e Questionários
3.
Vet Microbiol ; 238: 108425, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31648723

RESUMO

Duck Tembusu virus (DTMUV) has caused enormous economic losses to the poultry industry in China. In the current study, we generated chimeric virus-like particles (VLPs) containing E protein of the DTMUV and HA2 protein of the H3N2 avian influenza virus (AIV). The chimeric VLPs could induce specific antibody responses in both mice (n = 5/group) and ducks (n = 10/group). After immunizing ducklings with the chimeric VLPs, all immunized ducks (n = 10/group) were 100% (10/10) protected against homologous DTMUV strain and virus shedding was not detected on day 5 post-challenge, whereas 60% (6/10) of the ducklings immunized with PBS presented typical symptoms with a virus shedding rate of 90% (9/10). Furthermore, viral loads were significantly decreased in the birds of the chimeric VLPs immunized group, comparing to that of the PBS immunized group. Our data demonstrated that the chimeric VLPs used in the current study could be applied as a potential vaccine candidate to control DTMUV infections in young ducks.


Assuntos
Antígenos Virais/imunologia , Flavivirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Antivirais/sangue , Patos , Flavivirus/genética , Infecções por Flavivirus/imunologia , Imunogenicidade da Vacina/imunologia , Camundongos , Proteínas Virais de Fusão/genética , Carga Viral
4.
BMC Vet Res ; 15(1): 362, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31651323

RESUMO

BACKGROUND: Tembusu virus (TMUV) usually affects adult ducks, causing a severe drop of egg production. It has also been shown to be pathogenic in commercial Pekin ducklings below 7 weeks of age. Here, we report a TMUV-caused neurological disease in young egg-type ducklings and the pathogenicity of the egg-type duck-origin TMUV isolates in meat-type Pekin ducklings. RESULTS: The disease occurred in 25 to 40-day-old Jinding ducklings in China, and was characterized by paralysis. Gross lesions were lacking and microscopic lesions appeared chiefly in brain and spleen. Inoculation in embryonated duck eggs resulted in isolation of TMUV Y and GL. The clinical signs and microscopic lesions observed in the spontaneously infected egg-type ducks were repeated in Pekin ducklings by experimental infection. Notably, both Y and GL strains caused 100% mortality in the case of 2-day-old inoculation by intracerebral route. High mortalities (80 and 70%) also occurred following infection of the Y virus at 2 days of age by intramuscular route and at 9 days of age by intracerebral route. CONCLUSIONS: These findings demonstrate that the egg-type duck-origin TMUVs exhibit high pathogenicity in Pekin ducklings, and that the severity of the disease in ducklings is dependent on the infection route and the age of birds at the time of infection. The availability of the highly pathogenic TMUV strains provides a useful material with which to begin investigations into the molecular basis of TMUV pathogenicity in ducks.


Assuntos
Infecções por Flavivirus/veterinária , Flavivirus/patogenicidade , Doenças das Aves Domésticas/virologia , Fatores Etários , Animais , Linhagem Celular , Cricetinae , Vias de Administração de Medicamentos/veterinária , Patos/virologia , Flavivirus/genética , Infecções por Flavivirus/patologia , Infecções por Flavivirus/virologia , Paralisia/veterinária , Paralisia/virologia , Doenças das Aves Domésticas/patologia
5.
Mol Biotechnol ; 61(10): 783-790, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31482466

RESUMO

In our previous study, a recombinant duck enteritis virus (DEV) delivering codon-optimized E gene (named as E-ch) of duck Tembusu virus (DTMUV) optimized referring to chicken's codon bias has been obtained based on the infectious bacterial artificial chromosome (BAC) clone of duck enteritis virus vaccine strain pDEV-EF1, but the expression level of E-ch in recombinant virus rDEV-E-ch-infected cells was very low. To optimize DTMUV E gene expression delivered by the vectored DEV, different forms of E gene (collectively called EG) including origin E gene (E-ori), truncated E451-ori gene, codon-optimized E-dk gene optimized referring to duck's codon bias, as well as the truncated E451-ch and E451-dk, Etpa-ori and Etpa-451-ori, which contain prefixing chick TPA signal peptide genes, were cloned into transfer vector pEP-BGH-end, and several recombinant plasmids pEP-BGH-EG were constructed. Then the expression cassettes pCMV-EG-polyABGH amplified from pEP-BGH-EG by PCR were inserted into US7/US8 gene intergenic region of pDEV-EF1 by two-step Red/ET recombination, 7 strain recombinant mutated BAC clones pDEV-EG carrying different E genes were constructed. Next, the recombinant viruses rDEV-EG were reconstituted from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Western blot analysis showed that E or E451 protein is expressed in rDEV-E-ori, rDEV-E-ch, rDEV-Etpa-ori, rDEV-E451-ori, rDEV-E451-dk, and rDEV-E451-ch-infected CEFs, and protein expression level in rDEV-E451-dk-infected CEFs is the highest. These studies have laid a foundation for developing bivalent vaccine controlling DEV and DTMUV infection.


Assuntos
Flavivirus/metabolismo , Mardivirus/crescimento & desenvolvimento , Proteínas do Envelope Viral/metabolismo , Animais , Linhagem Celular , Embrião de Galinha , Cromossomos Artificiais Bacterianos/genética , Fibroblastos/citologia , Fibroblastos/virologia , Flavivirus/genética , Mardivirus/genética , Proteínas do Envelope Viral/genética , Vacinas Virais/metabolismo
6.
Nucleic Acids Res ; 47(18): 9789-9802, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31392996

RESUMO

Members of the Flaviviridae family, including dengue virus (DENV) and yellow fever virus, cause serious disease in humans, whilst maternal infection with Zika virus (ZIKV) can induce microcephaly in newborns. Following infection, flaviviral RNA genomes are translated to produce the viral replication machinery but must then serve as a template for the transcription of new genomes. However, the ribosome and viral polymerase proceed in opposite directions along the RNA, risking collisions and abortive replication. Whilst generally linear, flavivirus genomes can adopt a circular conformation facilitated by long-range RNA-RNA interactions, shown to be essential for replication. Using an in vitro reconstitution approach, we demonstrate that circularization inhibits de novo translation initiation on ZIKV and DENV RNA, whilst the linear conformation is translation-competent. Our results provide a mechanism to clear the viral RNA of ribosomes in order to promote efficient replication and, therefore, define opposing roles for linear and circular conformations of the flavivirus genome.


Assuntos
Vírus da Dengue/genética , Flavivirus/genética , Biossíntese de Proteínas , Zika virus/genética , Vírus da Dengue/patogenicidade , Flavivirus/patogenicidade , Genoma Viral/genética , Genômica , Humanos , Recém-Nascido , RNA Viral/genética , Replicação Viral/genética , Vírus da Febre Amarela/genética , Vírus da Febre Amarela/patogenicidade , Zika virus/patogenicidade , Infecção por Zika virus/genética , Infecção por Zika virus/virologia
7.
Int J Mol Sci ; 20(16)2019 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-31398956

RESUMO

Dengue, West Nile and Zika, closely related viruses of the Flaviviridae family, are an increasing global threat, due to the expansion of their mosquito vectors. They present a very similar viral particle with an outer lipid bilayer containing two viral proteins and, within it, the nucleocapsid core. This core is composed by the viral RNA complexed with multiple copies of the capsid protein, a crucial structural protein that mediates not only viral assembly, but also encapsidation, by interacting with host lipid systems. The capsid is a homodimeric protein that contains a disordered N-terminal region, an intermediate flexible fold section and a very stable conserved fold region. Since a better understanding of its structure can give light into its biological activity, here, first, we compared and analyzed relevant mosquito-borne Flavivirus capsid protein sequences and their predicted structures. Then, we studied the alternative conformations enabled by the N-terminal region. Finally, using dengue virus capsid protein as main model, we correlated the protein size, thermal stability and function with its structure/dynamics features. The findings suggest that the capsid protein interaction with host lipid systems leads to minor allosteric changes that may modulate the specific binding of the protein to the viral RNA. Such mechanism can be targeted in future drug development strategies, namely by using improved versions of pep14-23, a dengue virus capsid protein peptide inhibitor, previously developed by us. Such knowledge can yield promising advances against Zika, dengue and closely related Flavivirus.


Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Vírus da Dengue , Flavivirus , Sequência de Aminoácidos , Animais , Proteínas do Capsídeo/genética , Sequência Conservada , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Evolução Molecular , Flavivirus/genética , Flavivirus/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Filogenia , Conformação Proteica , Estabilidade Proteica , Relação Estrutura-Atividade
8.
Microb Pathog ; 132: 362-368, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31054366

RESUMO

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused massive economic losses to the duck industry in China. The cellular factors required for DTMUV replication have been poorly studied. The ubiquitin-proteasome system (UPS), the major intracellular proteolytic pathway, mediates diverse cellular processes, including endocytosis and signal transduction, which may be involved in the entry of virus. In the present study, we explored the interplay between DTMUV replication and the UPS in BHK-21 cells and found that treatment with proteasome inhibitor (MG132 and lactacystin) significantly decreased the DTMUV progency at the early infection stage. We further revealed that inhibition of the UPS mainly occurs on the level of viral protein expression and RNA transcription. In addition, using specific siRNAs targeting ubiquitin reduces the production of viral progeny. In the presence of MG132 the staining for the envelope protein of DTMUV was dramatically reduced in comparison with the untreated control cells. Overall, our observations reveal an important role of the UPS in multiple steps of the DTMUV infection cycle and identify the UPS as a potential drug target to modulate the impact of DTMUV infection.


Assuntos
Flavivirus/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Replicação Viral/fisiologia , Acetilcisteína/análogos & derivados , Acetilcisteína/antagonistas & inibidores , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Patos , Flavivirus/efeitos dos fármacos , Flavivirus/patogenicidade , Técnicas de Silenciamento de Genes , Leupeptinas/antagonistas & inibidores , Doenças das Aves Domésticas/virologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , RNA Interferente Pequeno , Transfecção , Ubiquitina/efeitos dos fármacos , Ubiquitina/genética , Proteínas do Envelope Viral , Internalização do Vírus
9.
PLoS Negl Trop Dis ; 13(5): e0007334, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31059502

RESUMO

BACKGROUND: The recent reports of Aedes aegypti and Ae. albopictus populations in Turkey, in parallel with the territorial expansion identified in several surrounding countries, have raised concerns about the establishment and re-establishment of these invasive Aedes mosquitoes in Turkey. This cross-sectional study was performed to detect Aedes aegypti and Ae. albopictus in regions of recent incursions, and screen for viral pathogens known to be transmitted elsewhere by these species. METHODOLOGY: Mosquitoes were collected at several locations in Artvin, Rize and Trabzon provinces of the Black Sea region during 2016-2017, identified morphologically, pooled and analyzed via generic or specific nucleic acid amplification assays. Viruses in positive pools were identified by product sequencing, cell culture inoculation and next generation sequencing (NGS) in selected specimens. PRINCIPAL FINDINGS: The study group comprised 791 specimens. Aedes albopictus was the most abundant species in all locations (89.6%), followed by Ae. aegypti (7.8%) and Culex pipiens (2.5%). Mosquitoes were screened for viruses in 65 pools where fifteen (23.1%) were reactive. The infecting strains was identified as West Nile virus (WNV) in 5 pools (7.7%) with Ae. albopictus or Cx. pipiens mosquitoes. The obtained WNV sequences phylogenetically grouped with local and global lineage 1 clade 1a viruses. In 4 (6.2%) and 6 (9.2%) pools, respectively, cell fusing agent virus (CFAV) and Aedes flavivirus (AEFV) sequences were characterized. NGS provided a near-complete AEFV genome in a pool of Ae. albopictus. The strain is provisionally called "AEFV-Turkey", and functional analysis of the genome revealed several conserved motifs and regions associated with virus replication. Merida-like virus Turkey (MERDLVT), a recently-described novel rhabdovirus, was also co-detected in a Cx. pipiens pool also positive for WNV. CONCLUSIONS/SIGNIFICANCE: Invasive Aedes mosquitoes are established in certain locations of northeastern Turkey. Herein we conclusively show the role of these species in WNV circulation in the region. Biosurveillance is imperative to monitor the spread of these species further into Asia Minor and to detect possible introduction of pathogens.


Assuntos
Aedes/virologia , Mosquitos Vetores/virologia , Febre do Nilo Ocidental/transmissão , Vírus do Nilo Ocidental/isolamento & purificação , Aedes/classificação , Animais , Estudos Transversais , Feminino , Flavivirus/classificação , Flavivirus/genética , Flavivirus/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mosquitos Vetores/classificação , Filogenia , Especificidade da Espécie , Turquia , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/classificação , Vírus do Nilo Ocidental/genética
10.
Virology ; 533: 86-92, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31136895

RESUMO

Duck Tembusu virus (DTMUV) is a novel flavivirus that has caused an outbreak of severe duck egg-drop syndrome since 2010. It has spread rapidly to other avian species, causing enormous economic loss. In the present study, we generated a reporter virus expressing NanoLuc luciferase, which was stable after 10 rounds of continuous propagation without reporter gene deletion. Moreover, we generated two types of replicons driven by the T7 promoter or CMV promoter, both of which worked well in BHK21 cells. Furthermore, we developed the first packaging system for DTMUV by co-transfection into BHK21 cells of a replicon (containing mature C) and a plasmid encoding C16-prM-E, which resulted in the production of single round infectious particles (SRIPs). We also generated a packaging cell line for DTMUV to produce SRIPs. We believe that these multicomponent platform tools are important for DTMUV pathogenesis research and novel vaccine development.


Assuntos
Infecções por Flavivirus/veterinária , Flavivirus/genética , Doenças das Aves Domésticas/virologia , Replicon , Animais , Patos , Flavivirus/fisiologia , Infecções por Flavivirus/virologia , Genes Reporter , Luciferases/genética , Luciferases/metabolismo , Genética Reversa
11.
Transbound Emerg Dis ; 66(5): 1982-1992, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31090210

RESUMO

Duck Tembusu virus (DTMUV) infected cases have increasingly been observed in several duck farms in Thailand since its first report in 2013. However, information on the genetic characteristic of DTMUVs recently circulating in ducks in Thailand is limited. In this study, we investigated the geographic distribution and genetic characteristic of DTMUVs recently circulating in ducks in Thailand during 2015-2017. Of the 288 clinical samples obtained from 89 ducks farms located in duck raising areas of Thailand, 65 samples (22.57%) of 34 duck farms (38.20%) were DTMUV positive. Our results demonstrated that DTMUV was extensively distributed in duck raising areas of Thailand. Phylogenetic analysis of the E and NS5 genes revealed that DTMUVs circulating in Thailand were divided into three distinct clusters, including cluster 1, subcluster 2.1 and a novel cluster 3. Among these three clusters, subcluster 2.1 was a predominant cluster of DTMUV circulating in duck populations in Thailand during 2015-2017. It is interesting to note that a novel cluster of DTMUV (cluster 3), which was genetically different from any of the previously reported DTMUV clusters, was first identified in this study. In conclusion, our data demonstrated the circulation of different clusters of DTMUV and the presence of a novel DTMUV cluster in ducks in Thailand. This study highlights the high genetic diversity of DTMUVs in Thailand and the necessity of the routine surveillance of DTMUV for early detection, prevention and control of newly emerging DTMUVs.


Assuntos
Patos , Infecções por Flavivirus/veterinária , Flavivirus/genética , Doenças das Aves Domésticas/virologia , Animais , Flavivirus/classificação , Infecções por Flavivirus/virologia , Filogenia , Tailândia
13.
EBioMedicine ; 43: 317-324, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31003930

RESUMO

BACKGROUND: A tick-borne segmented RNA virus called Jingmen tick virus (JMTV) was recently identified, variants of which were detected in a non-human primate host and fatal patients with Crimean-Congo haemorrhagic fever. We investigated its infectivity and pathogenicity for humans. METHODS: We obtained skin-biopsy, blood and serum samples from patients with tick bites, and used high-throughput sequencing, in situ hybridisation, and serologic testing to diagnose and ascertain the cases of JMTV infection. FINDINGS: A JMTV strain was isolated from the tick Amblyomma javanense into an embryo-derived tick cell line. We obtained sustained passage of JMTV, and revealed that it was able to accumulate in salivary glands of experimentally infected ticks. Four JMTV-infected patients were identified by high-throughput sequencing of skin biopsies and blood samples. The virus replication in skin tissue was visualised by in situ hybridisation. The four patients all had an itchy or painful eschar at the site of tick bite, with or without lymphadenopathy. Immunohistochemical examination revealed remarkable local inflammation manifested as infiltration by neutrophils. Eight patients were identified by serological testing and showed more severe clinical manifestations. Two Ixodes persulcatus ticks detached from patients were positive for JMTV. All JMTV strains identified in this study formed a well-supported sub-lineage, distinct from those previously reported in China. Interpretation The public significance of JMTV should be highly concerning due to its potential pathogenicity for humans and efficient transmission by potential ticks. FUND: China Natural Science Foundation, State Key Research Development Programme, and United Kingdom Biotechnology and Biological Sciences Research Council.


Assuntos
Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/virologia , Infecções por Flavivirus/epidemiologia , Infecções por Flavivirus/virologia , Flavivirus , Biomarcadores , China , Doenças Transmissíveis Emergentes/diagnóstico , Doenças Transmissíveis Emergentes/transmissão , Flavivirus/classificação , Flavivirus/genética , Infecções por Flavivirus/diagnóstico , Infecções por Flavivirus/transmissão , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Hibridização In Situ , Filogenia , Vigilância em Saúde Pública , RNA Viral , Estudos Retrospectivos , Testes Sorológicos , Pele/patologia , Picadas de Carrapatos
15.
Med Vet Entomol ; 33(3): 397-406, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30887540

RESUMO

New species of insect-specific viruses (ISV) have been reported worldwide. In the present study, the complete genome of Culex flavivirus (CxFV) and partial sequences of other ISVs in Culex quinquefasciatus Say 1823 females (n = 3425) sampled in 200 urban areas census tracts of Cuiaba, state of Mato Grosso, were identified via reverse transcriptase-polymerase chain reaction for a NS5 region of flaviviruses, nucleotide and high-throughput sequencing, and viral isolation in C6/36 cells. CxFV was detected in 16 of 403 mosquito pools; sequences found in the study presented a high similarity with isolates from São Paulo, Brazil and other countries in Latin American that belong to genotype II, supporting the geographical influence on CxFV evolution. The monthly maximum likelihood estimation for CxFV ranged from 1.81 to 9.94 per 1000 mosquitoes. In addition to the CxFV complete genome, one pool contained an ORF1 sequence (756 bp) that belongs to a novel Negevirus from the Sandewavirus supergroup most similar to the Santana virus (77.1%) and another pool presented an RNA-dependent RNA polymerase sequence (1081 bp) of a novel Rhabdovirus most similar to Wuhan mosquito virus 9 (44%). After three passages in C6/36 cells, only CxFV was isolated from these co-infected pools. The importance of ISVs relies on their possible ability to interfere with arbovirus replication in competent vectors.


Assuntos
Culex/virologia , Flavivirus/genética , Genoma Viral , Animais , Brasil , Feminino , Flavivirus/classificação , Flavivirus/isolamento & purificação , Genótipo , Filogenia
16.
Ticks Tick Borne Dis ; 10(4): 742-748, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30902589

RESUMO

Tick-borne encephalitis virus (TBEV), a flavivirus that causes severe neurological symptoms in humans, has been found in Hokkaido, Japan. In the present study, we detected sequences from a novel tick-borne flavivirus, designated Yamaguchi virus (YGV), in liver and serum samples obtained from a wild boar in the Yamaguchi prefecture, Japan. Phylogenetic analysis revealed that YGV belongs to the TBEV complex and is closely related to Langat virus (LGTV). YGV was also detected by specific RT-PCR from 20 in 378 pools of ticks (2923 ticks) collected in Yamaguchi and Wakayama prefectures and from seven in 46 wild boar captured in Wakayama. The major ticks infected with YGV belong to the genus Haemaphysalis. Unfortunately, YGV could not be isolated from any samples from the RT-PCR positive wild boar or ticks. Therefore, ELISA for detection of antibodies against YGV was established using LGTV, and surveillance was performed among wild boar in 10 different prefectures on Honshu Island, the main island of Japan. The results showed that the seroprevalence of tick-borne flavivirus infection in the Wakayama and Hyogo prefectures of western Japan was significantly higher than that in the other prefectures, while antibodies against tick-borne flavivirus were not detected in any wild boar in the Tochigi prefecture in the eastern part of Japan. In addition, wild raccoons or masked palm civets in the Hyogo prefecture did not possess detectable antibodies against tick-borne flaviviruses. In conclusion, YGV appears to be maintained primarily among wild boar and ticks in the western part of Japan. YGV is the second flavivirus (after Japanese encephalitis virus) shown to be circulating on Honshu Island in Japan.


Assuntos
Animais Selvagens/virologia , Encefalite Transmitida por Carrapatos/veterinária , Infecções por Flavivirus/veterinária , Ixodes/virologia , Filogenia , Sus scrofa/virologia , Animais , Animais Selvagens/sangue , Anticorpos Antivirais/sangue , Vírus da Encefalite Transmitidos por Carrapatos/genética , Encefalite Transmitida por Carrapatos/epidemiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Flavivirus/genética , Flavivirus/isolamento & purificação , Infecções por Flavivirus/epidemiologia , Japão/epidemiologia , Masculino , RNA Viral , Estudos Soroepidemiológicos , Sus scrofa/sangue , Suínos
17.
Viruses ; 11(3)2019 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-30909641

RESUMO

Untranslated regions (UTRs) of flaviviruses contain a large number of RNA structural elements involved in mediating the viral life cycle, including cyclisation, replication, and encapsidation. Here we report on a comparative genomics approach to characterize evolutionarily conserved RNAs in the 3 ' UTR of tick-borne, insect-specific and no-known-vector flaviviruses in silico. Our data support the wide distribution of previously experimentally characterized exoribonuclease resistant RNAs (xrRNAs) within tick-borne and no-known-vector flaviviruses and provide evidence for the existence of a cascade of duplicated RNA structures within insect-specific flaviviruses. On a broader scale, our findings indicate that viral 3 ' UTRs represent a flexible scaffold for evolution to come up with novel xrRNAs.


Assuntos
Regiões 3' não Traduzidas , Flavivirus/genética , Genômica , Insetos/virologia , RNA Viral/química , Animais , Evolução Molecular , Conformação de Ácido Nucleico , RNA não Traduzido/química , RNA não Traduzido/genética , RNA Viral/genética
18.
Vet Microbiol ; 230: 32-44, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30827402

RESUMO

Duck Tembusu virus (DTMUV) is a novel member of flavivirus with the highest viral loads in the spleen. Six-month egg-laying shelducks were intramuscularly injected with DTMUV strain XZ-2012. Morphological analysis revealed the presence of vacuolar degeneration in the periellipsoidal lymphatic sheaths (PELS) of spleen white pulp following infection, especially from 12 hpi to 3 dpi. Ultrastructural images showed an obvious swelling of cells and their mitochondria and endoplasmic reticulum. Using RNA-seq analysis, the expression levels of RIG-I like receptors (RLRs), downstream IRF7 and proinflammatory cytokines IL-6 from RIG-I signaling pathway were non-apparently upregulated at 2 hpi and apparently at 3 dpi, while MHC-II expression was obviously downregulated at 2 hpi. The expression levels of downstream antiviral cytokines type-I IFNs, anti-inflammatory cytokines IL-10, cell adhesion molecules (CAMs), chemokines and their receptors associated with lymphocyte homing were significantly upregulated at 3 dpi. The population of lymphocyte was increased at 6 dpi. The immune function of spleen was recovered starting from 9 dpi. These findings of this study suggest that DTMUV invaded into the spleen via RIG-I signaling pathway and enhanced immune evasion by inhibiting MHC-II expression during the early stage of infection. Additionally, DTMUV induced PELS lesions through activating IL-6 expression. Furthermore, DTMUV increased the expression levels of RLRs, antiviral type-I IFNs, lymphocyte homing-related genes and proteins as well as the number of lymphocytes in the infected duck spleen. Taken altogether, this study provides new insights into the cellular and molecular mechanisms of DTMUV infection in duck spleen.


Assuntos
Infecções por Flavivirus/veterinária , Flavivirus/genética , Baço/patologia , Baço/virologia , Animais , Anticorpos Antivirais/sangue , Patos/virologia , Feminino , Flavivirus/patogenicidade , Interações Hospedeiro-Patógeno , Microscopia Eletrônica de Transmissão , Doenças das Aves Domésticas/virologia , Análise de Sequência de RNA , Testes Sorológicos , Baço/ultraestrutura , Transcriptoma
19.
Biomed Res Int ; 2019: 1582709, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30809531

RESUMO

Outbreaks of duck Tembusu virus (DTMUV) have caused substantial economic losses in the major duck-producing regions of China since 2010. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of DTMUV infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography-tandem mass spectrometry to detect the protein changes in duck embryo fibroblast cells (DEFs) infected and mock-infected with DTMUV. In total, 434 cellular proteins were differentially expressed, among which 116, 76, and 339 proteins were differentially expressed in the DTMUV-infected DEFs at 12, 24, and 42 hours postinfection, respectively. The Gene Ontology analysis indicated that the biological processes of the differentially expressed proteins were primarily related to cellular processes, metabolic processes, biological regulation, response to stimulus, and cellular organismal processes and that the molecular functions in which the differentially expressed proteins were mainly involved were binding and catalytic activity. Some selected proteins that were found to be differentially expressed in DTMUV-infected DEFs were further confirmed by real-time PCR. The results of this study provide valuable insight into DTMUV-host interactions. This could lead to a better understanding of DTMUV infection mechanisms.


Assuntos
Patos/virologia , Flavivirus/genética , Interações Hospedeiro-Patógeno/genética , Proteoma/genética , Animais , China , Cromatografia Líquida , Patos/genética , Embrião não Mamífero/virologia , Fibroblastos/virologia , Flavivirus/patogenicidade , Proteômica/métodos , Espectrometria de Massas em Tandem
20.
Viruses ; 11(2)2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30795524

RESUMO

Usutu virus (USUV) is a Culex-associated mosquito-borne flavivirus of the Flaviviridae family. Since its discovery in 1959, the virus has been isolated from birds, arthropods and humans in Europe and Africa. An increasing number of Usutu virus infections in humans with neurological presentations have been reported. Recently, the virus has been detected in bats and horses, which deviates from the currently proposed enzootic cycle of USUV involving several different avian and mosquito species. Despite this increasing number of viral detections in different mammalian hosts, the existence of a non-avian reservoir remains unresolved. In Kedougou, a tropical region in the southeast corner of Senegal, Usutu virus was detected, isolated and sequenced from five asymptomatic small mammals: Two different rodent species and a single species of shrew. Additional molecular characterization and in vivo growth dynamics showed that these rodents/shrew-derived viruses are closely related to the reference strain (accession number: AF013412) and are as pathogenic as other characterized strains associated with neurological invasions in human. This is the first evidence of Usutu virus isolation from rodents or shrews. Our findings emphasize the need to consider a closer monitoring of terrestrial small mammals in future active surveillance, public health, and epidemiological efforts in response to USUV in both Africa and Europe.


Assuntos
Infecções por Flavivirus/veterinária , Flavivirus/genética , Flavivirus/isolamento & purificação , Filogenia , Roedores/virologia , Animais , Camundongos , Reação em Cadeia da Polimerase , RNA Viral/isolamento & purificação , Senegal
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