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1.
Actual. Sida Infectol. (En linea) ; 32(114): 63-78, 20240000. fig, graf
Artigo em Espanhol | LILACS, BINACIS | ID: biblio-1552316

RESUMO

La encefalitis equina del oeste (WEEV, por su sigla en inglés, Western Equine Encephalitis) es una enfermedad reemergente en Argentina a partir del año 2023. La co-municación inicial fue en 1933, las últimas epizootias ocurrieron en 1983 y el último caso humano se registró en 1996. Se revisan las características del agente causal, la ecología con especial referencia a los vectores iden-tificados en el país, su competencia en la transmisión y el ciclo así como los factores de riesgo para adquirir la enfermedad. La situación epidemiológica en equinos y humanos desde noviembre 2023 hasta marzo 2024 es analizada. Se describen las formas clínicas de presen-tación de la enfermedad humana, las posibilidades evo-lutivas, los datos disponibles en los casos confirmados y el tratamiento. La metodología y algoritmo empleados para el diagnóstico etiológico en el Centro Nacional de Referencia son detallados. Las estrategias para la pre-vención y el control se basan en la vacunación de los equinos, el saneamiento ambiental y el control del foco ante la presentación de la enfermedad animal (vigilancia epidemiológica activa)


Western equine encephalitis (WEE) is a re-emerging dis-ease in Argentina starting in 2023. Since the initial notifi-cation in 1933, the last epizootics occurred in 1983, and the last human case was recorded in 1996.The charac-teristics of the causative agent, the ecology with special reference to vectors identified in the country, their compe-tence in transmission, and the cycle as well as the risks factors for acquiring the disease, are reviewed.The epidemiological situation in horses and humans from November 2023 to March 2024 is analyzed. The clinical presentation of the human disease, its evolutionary po-tential, available data in confirmed cases, and the treat-ment are described.The methodology and algorithm used for the etiological diagnosis at the National Reference Center are detailed. Strategies for prevention and control are based on vaccination of horses, environmental sani-tation and outbreak control in the presence of the animal disease (active epidemiological surveillance)


Assuntos
Humanos , Animais , Masculino , Feminino , Saneamento/legislação & jurisprudência , Fatores de Risco , Encefalomielite Equina do Oeste/epidemiologia , Vírus da Encefalite Equina do Oeste/imunologia , Monitoramento Epidemiológico/veterinária
3.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 40(1): 62-68, 2024 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-38246178

RESUMO

Objective To prepare mouse monoclonal antibodies against the ectodomain of E2 (E2ecto) glycoprotein of Western equine encephalitis virus (WEEV). Methods A prokaryotic expression plasmid pET-28a-WEEV E2ecto was constructed and transformed into BL21 (DE3) competent cells. E2ecto protein was expressed by IPTG induction and presented mainly as inclusion bodies. Then the purified E2ecto protein was prepared by denaturation, renaturation and ultrafiltration. BALB/c mice were immunized with the formulated E2ecto protein using QuickAntibody-Mouse5W as an adjuvant via intramuscular route, boosted once at an interval of 21 days. At 35 days post-immunization, mice with antibody titer above 1×104 were inoculated with E2ecto intraperitoneally, and spleen cells were fused with SP2/0 cells three days later. Hybridoma cells secreting specific monoclonal antibodies were screened by the limited dilution method, and ascites were prepared after intraperitoneal inoculation of hybridoma cells. The subtypes and titers of the antibodies in ascites were assayed by ELISA. The biological activity of the mAb was identified by immunofluorescence assay(IFA) on BHK-21 cells which were transfected with eukaryotic expression plasmid pCAGGS-WEEV-CE3E2E1. The specificity of the antibodies were evaluated with E2ecto proteins from EEEV and VEEV. Results Purified WEEV E2ecto protein was successfully expressed and obtained. Four monoclonal antibodies, 3G6G10, 3D7G2, 3B9E8 and 3D5B7, were prepared, and their subtypes were IgG2c(κ), IgM(κ), IgM(κ) and IgG1(κ), respectively. The titers of ascites antibodies 3G6G10, 3B9E8 and 3D7G2 were 105, and 3D5B7 reached 107. None of the four antibody strains cross-reacted with other encephalitis alphavirus such as VEEV and EEEV. Conclusion Four strains of mouse mAb specifically binding WEEV E2ecto are successfully prepared.


Assuntos
Ascite , Vírus da Encefalite Equina do Oeste , Cavalos , Animais , Camundongos , Imunossupressores , Anticorpos Monoclonais , Imunoglobulina M
4.
Artigo em Chinês | WPRIM (Pacífico Ocidental) | ID: wpr-1009476

RESUMO

Objective To prepare mouse monoclonal antibodies against the ectodomain of E2 (E2ecto) glycoprotein of Western equine encephalitis virus (WEEV). Methods A prokaryotic expression plasmid pET-28a-WEEV E2ecto was constructed and transformed into BL21 (DE3) competent cells. E2ecto protein was expressed by IPTG induction and presented mainly as inclusion bodies. Then the purified E2ecto protein was prepared by denaturation, renaturation and ultrafiltration. BALB/c mice were immunized with the formulated E2ecto protein using QuickAntibody-Mouse5W as an adjuvant via intramuscular route, boosted once at an interval of 21 days. At 35 days post-immunization, mice with antibody titer above 1×104 were inoculated with E2ecto intraperitoneally, and spleen cells were fused with SP2/0 cells three days later. Hybridoma cells secreting specific monoclonal antibodies were screened by the limited dilution method, and ascites were prepared after intraperitoneal inoculation of hybridoma cells. The subtypes and titers of the antibodies in ascites were assayed by ELISA. The biological activity of the mAb was identified by immunofluorescence assay(IFA) on BHK-21 cells which were transfected with eukaryotic expression plasmid pCAGGS-WEEV-CE3E2E1. The specificity of the antibodies were evaluated with E2ecto proteins from EEEV and VEEV. Results Purified WEEV E2ecto protein was successfully expressed and obtained. Four monoclonal antibodies, 3G6G10, 3D7G2, 3B9E8 and 3D5B7, were prepared, and their subtypes were IgG2c(κ), IgM(κ), IgM(κ) and IgG1(κ), respectively. The titers of ascites antibodies 3G6G10, 3B9E8 and 3D7G2 were 105, and 3D5B7 reached 107. None of the four antibody strains cross-reacted with other encephalitis alphavirus such as VEEV and EEEV. Conclusion Four strains of mouse mAb specifically binding WEEV E2ecto are successfully prepared.


Assuntos
Animais , Camundongos , Cavalos , Vírus da Encefalite Equina do Oeste , Ascite , Imunossupressores , Anticorpos Monoclonais , Imunoglobulina M
5.
Viruses ; 15(2)2023 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-36851596

RESUMO

Eastern (EEEV), Venezuelan (VEEV), and western equine encephalitis viruses (WEEV) are members of the genus Alphavirus, family Togaviridae. Typically spread by mosquitoes, EEEV, VEEV, and WEEV induce febrile illness that may develop into more severe encephalitic disease, resulting in myriad severe neurologic sequelae for which there are no vaccines or therapeutics. Here, we summarize the clinical neurologic findings and sequelae induced by these three encephalitic viruses and describe the various animal models available to study them. We emphasize the crucial need for the development of advanced animal modeling combined with the use of telemetry, behavioral testing, and neuroimaging to facilitate a detailed mechanistic understanding of these encephalitic signs and sequelae. Through the use of these systems, much-needed therapeutics and vaccines can be developed.


Assuntos
Infecções por Alphavirus , Alphavirus , Encefalite , Animais , Cavalos , Vírus da Encefalite Equina do Oeste , Progressão da Doença , Modelos Animais
6.
Expert Rev Anti Infect Ther ; 20(12): 1551-1566, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36305549

RESUMO

INTRODUCTION: Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV) are mosquito-borne New World alphaviruses that cause encephalitis in equids and humans. These viruses can cause severe disease and death, as well as long-term severe neurological symptoms in survivors. Despite the pathogenesis and weaponization of these viruses, there are no approved therapeutics for treating infection. AREAS COVERED: In this review, we describe the molecular pathogenesis of these viruses, discuss host-pathogen interactions needed for viral replication, and highlight new avenues for drug development with a focus on host-targeted approaches. EXPERT OPINION: Current approaches have yielded some promising therapeutics, but additional emphasis should be placed on advanced development of existing small molecules and pursuit of pan-encephalitic alphavirus drugs. More research should be conducted on EEEV and WEEV, given their high lethality rates.


Assuntos
Alphavirus , Vírus da Encefalite Equina Venezuelana , Encefalomielite Equina , Viroses , Animais , Humanos , Cavalos , Vírus da Encefalite Equina Venezuelana/fisiologia , Vírus da Encefalite Equina do Oeste/fisiologia , Encefalomielite Equina/tratamento farmacológico
7.
PLoS One ; 17(3): e0262967, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35298486

RESUMO

Herein we describe a previously uninvestigated salivary gland escape barrier (SEB) in Culex tarsalis mosquitoes infected with two different strains of Western equine encephalitis virus (WEEV). The WEEV strains were originally isolated either from mosquitoes (IMP181) or a human patient (McMillan). Both IMP181 and McMillan viruses were fully able to infect the salivary glands of Culex tarsalis after intrathoracic injection as determined by expression of mCherry fluorescent protein. IMP181, however, was better adapted to transmission as measured by virus titer in saliva as well as transmission rates in infected mosquitoes. We used chimeric recombinant WEEV strains to show that inclusion of IMP181-derived structural genes partially circumvents the SEB.


Assuntos
Culex , Encefalomielite Equina , Animais , Vetores de Doenças , Vírus da Encefalite Equina do Oeste/genética , Humanos , Mosquitos Vetores , Glândulas Salivares
8.
Viruses ; 14(2)2022 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-35215864

RESUMO

Venezuelan equine encephalitis virus (VEEV) is an Alphavirus in the Togaviridae family of positive-strand RNA viruses. The viral genome of positive-strand RNA viruses is infectious, as it produces infectious virus upon introduction into a cell. VEEV is a select agent and samples containing viral RNA are subject to additional regulations due to their infectious nature. Therefore, RNA isolated from cells infected with BSL-3 select agent strains of VEEV or other positive-strand viruses must be inactivated before removal from high-containment laboratories. In this study, we tested the inactivation of the viral genome after RNA fragmentation or cDNA synthesis, using the Trinidad Donkey and TC-83 strains of VEEV. We successfully inactivated VEEV genomic RNA utilizing these two protocols. Our cDNA synthesis method also inactivated the genomic RNA of eastern and western equine encephalitis viruses (EEEV and WEEV). We also tested whether the purified VEEV genomic RNA can produce infectious virions in the absence of transfection. Our result showed the inability of the viral genome to cause infection without being transfected into the cells. Overall, this work introduces RNA fragmentation and cDNA synthesis as reliable methods for the inactivation of samples containing the genomes of positive-strand RNA viruses.


Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Genoma Viral , RNA Viral , Inativação de Vírus , Animais , Células Cultivadas , Chlorocebus aethiops , Efeito Citopatogênico Viral , DNA Complementar/biossíntese , Vírus da Encefalite Equina do Leste/genética , Vírus da Encefalite Equina do Leste/fisiologia , Vírus da Encefalite Equina Venezuelana/fisiologia , Vírus da Encefalite Equina do Oeste/genética , Vírus da Encefalite Equina do Oeste/fisiologia , RNA Viral/química , RNA Viral/fisiologia , Ribonucleases/metabolismo , Células Vero
9.
Viruses ; 15(1)2022 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-36680046

RESUMO

The Department of Defense recently began an effort to improve and standardize virus challenge materials and efficacy determination strategies for testing therapeutics and vaccines. This includes stabilization of virus genome sequences in cDNA form where appropriate, use of human-derived virus isolates, and noninvasive strategies for determination of challenge virus replication. Eventually, it is desired that these approaches will satisfy the FDA "Animal Rule" for licensure, which substitutes animal efficacy data when human data are unlikely to be available. To this end, we created and examined the virulence phenotype of cDNA clones of prototypic human infection-derived strains of the alphaviruses, Venezuelan (VEEV INH9813), eastern (EEEV V105) and western (WEEV Fleming) equine encephalitis viruses, and created fluorescent and luminescent reporter expression vectors for evaluation of replication characteristics in vitro and in vivo. Sequences of minimally passaged isolates of each virus were used to synthesize full-length cDNA clones along with a T7 transcription promoter-based bacterial propagation vector. Viruses generated from the cDNA clones were compared with other "wild type" strains derived from cDNA clones and GenBank sequences to identify and eliminate putative tissue culture artifacts accumulated in the cell passaged biological stocks. This was followed by examination of aerosol and subcutaneous infection and disease in mouse models. A mutation that increased heparan sulfate binding was identified in the VEEV INH9813 biological isolate sequence and eliminated from the cDNA clone. Viruses derived from the new human isolate cDNA clones showed similar mouse virulence to existing clone-derived viruses after aerosol or subcutaneous inoculation.


Assuntos
Vírus da Encefalite Equina Venezuelana , Vírus da Encefalite Equina do Oeste , Estados Unidos , Humanos , Animais , Cavalos , Camundongos , DNA Complementar/genética , Fenótipo , Células Clonais
10.
Viruses ; 13(11)2021 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-34835037

RESUMO

The three encephalitic alphaviruses, namely, the Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV), are classified by the Centers for Disease Control and Prevention (CDC) as biothreat agents. Currently, no licensed medical countermeasures (MCMs) against these viruses are available for humans. Neutralizing antibodies (NAbs) are fast-acting and highly effective MCMs for use in both pre- and post-exposure settings against biothreat agents. While significant work has been done to identify anti-VEEV NAbs, less has been done to identify NAbs against EEEV and WEEV. In order to develop anti-EEEV or -WEEV NAbs, mice were immunized using complementary strategies with a variety of different EEEV or WEEV immunogens to maximize the generation of NAbs to each of these viruses. Of the hybridomas generated, three anti-EEEV and seven anti-WEEV monoclonal antibodies were identified with in vitro neutralization activity. The most potent neutralizers (two anti-EEEV NAbs and three anti-WEEV NAbs) were further evaluated for neutralization activity against additional strains of EEEV, a single strain of Madariaga virus (formerly South American EEEV), or WEEV. Of these, G1-2-H4 and G1-4-C3 neutralized all three EEEV strains and the Madariaga virus strain, whereas G8-2-H9 and 12 WA neutralized six out of eight WEEV strains. To determine the protective efficacy of these NAbs, the five most potent neutralizers were evaluated in respective mouse aerosol challenge models. All five NAbs demonstrated various levels of protection when administered at doses of 2.5 mg/kg or 10 mg/kg 24 h before the respective virus exposure via the aerosol route. Of these, anti-EEEV NAb G1-4-C3 and anti-WEEV NAb 8C2 provided 100% protection at both doses and all surviving mice were free of clinical signs throughout the study. Additionally, no virus was detected in the brain 14 days post virus exposure. Taken together, efficacious NAbs were developed that demonstrate the potential for the development of cross-strain antibody-based MCMs against EEEV and WEEV infections.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Vírus da Encefalite Equina do Leste/imunologia , Vírus da Encefalite Equina do Oeste/imunologia , Encefalomielite Equina/prevenção & controle , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/imunologia , Proteção Cruzada , Modelos Animais de Doenças , Imunização , Camundongos , Testes de Neutralização
11.
Exp Neurol ; 346: 113845, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34454938

RESUMO

Viral infection of the central nervous system (CNS) can cause lasting neurological decline in surviving patients and can present with symptoms resembling Parkinson's disease (PD). The mechanisms underlying postencephalitic parkinsonism remain unclear but are thought to involve increased innate inflammatory signaling in glial cells, resulting in persistent neuroinflammation. We therefore studied the role of glial cells in regulating neuropathology in postencephalitic parkinsonism by studying the involvement of astrocytes in loss of dopaminergic neurons and aggregation of α-synuclein protein following infection with western equine encephalitis virus (WEEV). Infections were conducted in both wildtype mice and in transgenic mice lacking NFκB inflammatory signaling in astrocytes. For 2 months following WEEV infection, we analyzed glial activation, neuronal loss and protein aggregation across multiple brain regions, including the substantia nigra pars compacta (SNpc). These data revealed that WEEV induces loss of SNpc dopaminergic neurons, persistent activation of microglia and astrocytes that precipitates widespread aggregation of α-synuclein in the brain of C57BL/6 mice. Microgliosis and macrophage infiltration occurred prior to activation of astrocytes and was followed by opsonization of ⍺-synuclein protein aggregates in the cortex, hippocampus and midbrain by the complement protein, C3. Astrocyte-specific NFκB knockout mice had reduced gliosis, α-synuclein aggregate formation and neuronal loss. These data suggest that astrocytes play a critical role in initiating PD-like pathology following encephalitic infection with WEEV through innate immune inflammatory pathways that damage dopaminergic neurons, possibly by hindering clearance of ⍺-synuclein aggregates. Inhibiting glial inflammatory responses could therefore represent a potential therapy strategy for viral parkinsonism.


Assuntos
Astrócitos/metabolismo , Neurônios Dopaminérgicos/metabolismo , Encefalite Viral/metabolismo , Mediadores da Inflamação/metabolismo , Agregados Proteicos/fisiologia , alfa-Sinucleína/metabolismo , Animais , Astrócitos/imunologia , Neurônios Dopaminérgicos/imunologia , Vírus da Encefalite Equina do Oeste/imunologia , Vírus da Encefalite Equina do Oeste/metabolismo , Encefalite Viral/imunologia , Feminino , Humanos , Mediadores da Inflamação/imunologia , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais/fisiologia
12.
Biotechnol J ; 16(8): e2100008, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34176228

RESUMO

Western equine encephalitis virus (WEEV) causes lethal encephalitis in humans and equines, and it poses a serious public health threat in many countries. Therefore, the development of an efficient vaccine remains an important challenge for the prevention of WEEV infection. This study presents the first description of WEEV virus-like particles (VLPs) generated from insect cells using recombinant baculoviruses. WEEV VLPs with 206 adjuvant could trigger a strong cellular immune response; increase the levels of IL-2, IL-4 and IFN-γ; and induce a high level of neutralizing antibodies against WEEV in mice. These data showed that the insect cell-baculovirus system is suitable for the production of WEEV VLPs and that these VLPs could elicit the strong immunogenicity in mice. These results suggest a new, nonreplicating, and effective vaccine candidate against WEEV infection.


Assuntos
Baculoviridae , Vírus da Encefalite Equina do Oeste , Animais , Anticorpos Antivirais , Baculoviridae/genética , Vírus da Encefalite Equina do Oeste/genética , Cavalos , Imunidade , Imunização , Insetos , Camundongos
13.
Semin Cell Dev Biol ; 111: 148-155, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32665176

RESUMO

Alphaviruses such as chikungunya and western equine encephalitis viruses are important human pathogens transmitted by mosquitoes that have recently caused large epidemic and epizootic outbreaks. The epidemic potential of alphaviruses is often related to enhanced mosquito transmission. Tissue barriers and antiviral responses impose bottlenecks to viral populations in mosquitoes. Substitutions in the envelope proteins and the presence of repeated sequence elements (RSEs) in the 3'UTR of epidemic viruses were proposed to be specifically associated to efficient replication in mosquito vectors. Here, we discuss the molecular mechanisms that originated RSEs, the evolutionary forces that shape the 3'UTR of alphaviruses, and the significance of RSEs for mosquito transmission. Finally, the presence of RSEs in the 3'UTR of viral genomes appears as evolutionary trait associated to mosquito adaptation and emerges as a common feature among viruses from the alphavirus and flavivirus genera.


Assuntos
Infecções por Alphavirus/transmissão , Vírus Chikungunya/genética , Vírus da Encefalite Equina do Oeste/genética , Infecções por Flavivirus/transmissão , Flavivirus/genética , Genoma Viral , Proteínas do Envelope Viral/genética , Regiões 3' não Traduzidas , Infecções por Alphavirus/virologia , Animais , Vírus Chikungunya/classificação , Vírus Chikungunya/patogenicidade , Culicidae/virologia , Vírus da Encefalite Equina do Oeste/classificação , Vírus da Encefalite Equina do Oeste/patogenicidade , Flavivirus/classificação , Flavivirus/patogenicidade , Infecções por Flavivirus/virologia , Regulação da Expressão Gênica , Humanos , Repetições de Microssatélites , Mosquitos Vetores/virologia , Filogenia , Transdução de Sinais , Proteínas do Envelope Viral/metabolismo , Replicação Viral
14.
Antiviral Res ; 182: 104875, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32755661

RESUMO

Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV) are mosquito-borne viruses in the Americas that cause central nervous system (CNS) disease in humans and equids. In this study, we directly characterized the pathogenesis of VEEV, EEEV, and WEEV in cynomolgus macaques following subcutaneous exposure because this route more closely mimics natural infection via mosquito transmission or by an accidental needle stick. Our results highlight how EEEV is significantly more pathogenic compared to VEEV similarly to what is observed in humans. Interestingly, EEEV appears to be just as neuropathogenic by subcutaneous exposure as it was in previously completed aerosol exposure studies. In contrast, subcutaneous exposure of cynomolgus macaques with WEEV caused limited disease and is contradictory to what has been reported for aerosol exposure. Several differences in viremia, hematology, or tissue tropism were noted when animals were exposed subcutaneously compared to prior aerosol exposure studies. This study provides a more complete picture of the pathogenesis of the encephalitic alphaviruses and highlights how further defining the neuropathology of these viruses could have important implications for the development of medical countermeasures for the neurovirulent alphaviruses.


Assuntos
Vírus da Encefalite Equina do Leste/patogenicidade , Vírus da Encefalite Equina Venezuelana/patogenicidade , Vírus da Encefalite Equina do Oeste/patogenicidade , Encefalomielite Equina/patologia , Encefalomielite Equina Venezuelana/patologia , Macaca fascicularis/virologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Feminino , Masculino , Replicação Viral
15.
Vector Borne Zoonotic Dis ; 20(11): 868-871, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32644885

RESUMO

A total of 102 free-range wild boars, 170 hunting dogs, and 49 hunters from 3 Brazilian regions were sampled and tested for antibodies to eastern equine encephalitis virus (EEEV), western equine encephalitis virus, and Venezuelan equine encephalitis virus. Three of the 102 (2.9%) wild boars were positive for antibodies against EEEV by microplate serum neutralization test. Based on our data, free-range wild boars from central-western Brazil may be exposed to EEEV, and further studies are needed to evaluate the potential of incorporating serosurveys in routine arbovirus activity surveillance specifically to identify arbovirus activity foci and to help establish thresholds for epidemic transmission.


Assuntos
Doenças do Cão/virologia , Vírus da Encefalite Equina do Leste , Vírus da Encefalite Equina Venezuelana , Vírus da Encefalite Equina do Oeste , Encefalomielite Equina/veterinária , Doenças dos Suínos/virologia , Animais , Anticorpos Antivirais/sangue , Brasil/epidemiologia , Doenças do Cão/epidemiologia , Cães , Encefalomielite Equina/epidemiologia , Encefalomielite Equina/virologia , Humanos , Estudos Soroepidemiológicos , Sus scrofa , Suínos , Doenças dos Suínos/epidemiologia , Cães Trabalhadores
17.
mBio ; 11(1)2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-32047126

RESUMO

Venezuelan and western equine encephalitis viruses (VEEV and WEEV, respectively) invade the central nervous system (CNS) early during infection, via neuronal and hematogenous routes. While viral replication mediates host shutoff, including expression of type I interferons (IFN), few studies have addressed how alphaviruses gain access to the CNS during established infection or the mechanisms of viral crossing at the blood-brain barrier (BBB). Here, we show that hematogenous dissemination of VEEV and WEEV into the CNS occurs via caveolin-1 (Cav-1)-mediated transcytosis (Cav-MT) across an intact BBB, which is impeded by IFN and inhibitors of RhoA GTPase. Use of reporter and nonreplicative strains also demonstrates that IFN signaling mediates viral restriction within cells comprising the neurovascular unit (NVU), differentially rendering brain endothelial cells, pericytes, and astrocytes permissive to viral replication. Transmission and immunoelectron microscopy revealed early events in virus internalization and Cav-1 association within brain endothelial cells. Cav-1-deficient mice exhibit diminished CNS VEEV and WEEV titers during early infection, whereas viral burdens in peripheral tissues remained unchanged. Our findings show that alphaviruses exploit Cav-MT to enter the CNS and that IFN differentially restricts this process at the BBB.IMPORTANCE VEEV, WEEV, and eastern equine encephalitis virus (EEEV) are emerging infectious diseases in the Americas, and they have caused several major outbreaks in the human and horse population during the past few decades. Shortly after infection, these viruses can infect the CNS, resulting in severe long-term neurological deficits or death. Neuroinvasion has been associated with virus entry into the CNS directly from the bloodstream; however, the underlying molecular mechanisms have remained largely unknown. Here, we demonstrate that following peripheral infection alphavirus augments vesicular formation/trafficking at the BBB and utilizes Cav-MT to cross an intact BBB, a process regulated by activators of Rho GTPases within brain endothelium. In vivo examination of early viral entry in Cav-1-deficient mice revealed significantly lower viral burdens in the brain than in similarly infected wild-type animals. These studies identify a potentially targetable pathway to limit neuroinvasion by alphaviruses.


Assuntos
Barreira Hematoencefálica/virologia , Cavéolas/virologia , Vírus da Encefalite Equina Venezuelana/fisiologia , Vírus da Encefalite Equina do Oeste/fisiologia , Transcitose , Internalização do Vírus , Animais , Caveolina 1/genética , Linhagem Celular , Sistema Nervoso Central/virologia , Células Endoteliais/virologia , Masculino , Camundongos Endogâmicos C57BL , Replicação Viral
18.
PLoS Pathog ; 16(2): e1008102, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32027727

RESUMO

Understanding the circumstances under which arboviruses emerge is critical for the development of targeted control and prevention strategies. This is highlighted by the emergence of chikungunya and Zika viruses in the New World. However, to comprehensively understand the ways in which viruses emerge and persist, factors influencing reductions in virus activity must also be understood. Western equine encephalitis virus (WEEV), which declined during the late 20th century in apparent enzootic circulation as well as equine and human disease incidence, provides a unique case study on how reductions in virus activity can be understood by studying evolutionary trends and mechanisms. Previously, we showed using phylogenetics that during this period of decline, six amino acid residues appeared to be positively selected. To assess more directly the effect of these mutations, we utilized reverse genetics and competition fitness assays in the enzootic host and vector (house sparrows and Culex tarsalis mosquitoes). We observed that the mutations contemporary with reductions in WEEV circulation and disease that were non-conserved with respect to amino acid properties had a positive effect on enzootic fitness. We also assessed the effects of these mutations on virulence in the Syrian-Golden hamster model in relation to a general trend of increased virulence in older isolates. However, no change effect on virulence was observed based on these mutations. Thus, while WEEV apparently underwent positive selection for infection of enzootic hosts, residues associated with mammalian virulence were likely eliminated from the population by genetic drift or negative selection. These findings suggest that ecologic factors rather than fitness for natural transmission likely caused decreased levels of enzootic WEEV circulation during the late 20th century.


Assuntos
Vírus da Encefalite Equina do Oeste/genética , Encefalomielite Equina/genética , Deriva Genética , Seleção Genética , Animais , Culex/imunologia , Culex/virologia , Vírus da Encefalite Equina do Oeste/imunologia , Vírus da Encefalite Equina do Oeste/patogenicidade , Encefalomielite Equina/imunologia , Encefalomielite Equina/patologia , Encefalomielite Equina/transmissão , Humanos , Mesocricetus , Mosquitos Vetores/imunologia , Mosquitos Vetores/virologia , Pardais/imunologia , Pardais/virologia
19.
Front Immunol ; 11: 598847, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33542715

RESUMO

Venezuelan, eastern and western equine encephalitis viruses (EEV) can cause severe disease of the central nervous system in humans, potentially leading to permanent damage or death. Yet, no licensed vaccine for human use is available to protect against these mosquito-borne pathogens, which can be aerosolized and therefore pose a bioterror threat in addition to the risk of natural outbreaks. Using the mouse aerosol challenge model, we evaluated the immunogenicity and efficacy of EEV vaccines that are based on the modified vaccinia Ankara-Bavarian Nordic (MVA-BN®) vaccine platform: three monovalent vaccines expressing the envelope polyproteins E3-E2-6K-E1 of the respective EEV virus, a mixture of these three monovalent EEV vaccines (Triple-Mix) as a first approach to generate a multivalent vaccine, and a true multivalent alphavirus vaccine (MVA-WEV, Trivalent) encoding the polyproteins of all three EEVs in a single non-replicating MVA viral vector. BALB/c mice were vaccinated twice in a four-week interval and samples were assessed for humoral and cellular immunogenicity. Two weeks after the second immunization, animals were exposed to aerosolized EEV. The majority of vaccinated animals exhibited VEEV, WEEV, and EEEV neutralizing antibodies two weeks post-second administration, whereby the average VEEV neutralizing antibodies induced by the monovalent and Trivalent vaccine were significantly higher compared to the Triple-Mix vaccine. The same statistical difference was observed for VEEV E1 specific T cell responses. However, all vaccinated mice developed comparable interferon gamma T cell responses to the VEEV E2 peptide pools. Complete protective efficacy as evaluated by the prevention of mortality and morbidity, lack of clinical signs and viremia, was demonstrated for the respective monovalent MVA-EEV vaccines, the Triple-Mix and the Trivalent single vector vaccine not only in the homologous VEEV Trinidad Donkey challenge model, but also against heterologous VEEV INH-9813, WEEV Fleming, and EEEV V105-00210 inhalational exposures. These EEV vaccines, based on the safe MVA vector platform, therefore represent promising human vaccine candidates. The trivalent MVA-WEV construct, which encodes antigens of all three EEVs in a single vector and can potentially protect against all three encephalitic viruses, is currently being evaluated in a human Phase 1 trial.


Assuntos
Vírus da Encefalite Equina do Leste/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Vírus da Encefalite Equina do Oeste/imunologia , Encefalomielite Equina/prevenção & controle , Vacinas Virais/imunologia , Aerossóis , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteção Cruzada/imunologia , Modelos Animais de Doenças , Encefalomielite Equina/imunologia , Encefalomielite Equina/mortalidade , Feminino , Imunização , Camundongos , Mortalidade , Testes de Neutralização , Vacinas de DNA , Vacinas Virais/administração & dosagem
20.
J Virol ; 94(3)2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31694940

RESUMO

RNA interference (RNAi) is a conserved antiviral immune defense in eukaryotes, and numerous viruses have been found to encode viral suppressors of RNAi (VSRs) to counteract antiviral RNAi. Alphaviruses are a large group of positive-stranded RNA viruses that maintain their transmission and life cycles in both mosquitoes and mammals. However, there is little knowledge about how alphaviruses antagonize RNAi in both host organisms. In this study, we identified that Semliki Forest virus (SFV) capsid protein can efficiently suppress RNAi in both insect and mammalian cells by sequestrating double-stranded RNA and small interfering RNA. More importantly, when the VSR activity of SFV capsid was inactivated by reverse genetics, the resulting VSR-deficient SFV mutant showed severe replication defects in mammalian cells, which could be rescued by blocking the RNAi pathway. Besides, capsid protein of Sindbis virus also inhibited RNAi in cells. Together, our findings show that SFV uses capsid protein as VSR to antagonize RNAi in infected mammalian cells, and this mechanism is probably used by other alphaviruses, which shed new light on the knowledge of SFV and alphavirus.IMPORTANCE Alphaviruses are a genus of positive-stranded RNA viruses and include numerous important human pathogens, such as Chikungunya virus, Ross River virus, Western equine encephalitis virus, etc., which create the emerging and reemerging public health threat worldwide. RNA interference (RNAi) is one of the most important antiviral mechanisms in plants and insects. Accumulating evidence has provided strong support for the existence of antiviral RNAi in mammals. In response to antiviral RNAi, viruses have evolved to encode viral suppressors of RNAi (VSRs) to antagonize the RNAi pathway. It is unclear whether alphaviruses encode VSRs that can suppress antiviral RNAi during their infection in mammals. In this study, we first uncovered that capsid protein encoded by Semliki Forest virus (SFV), a prototypic alphavirus, had a potent VSR activity that can antagonize antiviral RNAi in the context of SFV infection in mammalian cells, and this mechanism is probably used by other alphaviruses.


Assuntos
Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Interferência de RNA/fisiologia , Vírus da Floresta de Semliki/genética , Vírus da Floresta de Semliki/metabolismo , Animais , Capsídeo , Linhagem Celular , Vírus Chikungunya/fisiologia , Drosophila , Vírus da Encefalite Equina do Oeste/fisiologia , Células HEK293 , Humanos , RNA Interferente Pequeno , RNA Viral , Sindbis virus/fisiologia , Vírion , Replicação Viral
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