RESUMO
The Zika virus (ZIKV) was first isolated in Africa in 1947. It was shown to be a mild virus that had limited threat to humans. However, the resurgence of the ZIKV in the most recent Brazil outbreak surprised us because it causes severe human congenital and neurologic disorders including microcephaly in newborns and Guillain-Barré syndrome in adults. Studies showed that the epidemic ZIKV strains are phenotypically different from the historic strains, suggesting that the epidemic ZIKV has acquired mutations associated with the altered viral pathogenicity. However, what genetic changes are responsible for the changed viral pathogenicity remains largely unknown. One of our early studies suggested that the ZIKV structural proteins contribute in part to the observed virologic differences. The objectives of this study were to compare the historic African MR766 ZIKV strain with two epidemic Brazilian strains (BR15 and ICD) for their abilities to initiate viral infection and to confer neurocytopathic effects in the human brain's SNB-19 glial cells, and further to determine which part of the ZIKV structural proteins are responsible for the observed differences. Our results show that the historic African (MR766) and epidemic Brazilian (BR15 and ICD) ZIKV strains are different in viral attachment to host neuronal cells, viral permissiveness and replication, as well as in the induction of cytopathic effects. The analysis of chimeric viruses, generated between the MR766 and BR15 molecular clones, suggests that the ZIKV E protein correlates with the viral attachment, and the C-prM region contributes to the permissiveness and ZIKV-induced cytopathic effects. The expression of adenoviruses, expressing prM and its processed protein products, shows that the prM protein and its cleaved Pr product, but not the mature M protein, induces apoptotic cell death in the SNB-19 cells. We found that the Pr region, which resides on the N-terminal side of prM protein, is responsible for prM-induced apoptotic cell death. Mutational analysis further identified four amino-acid residues that have an impact on the ability of prM to induce apoptosis. Together, the results of this study show that the difference of ZIKV-mediated viral pathogenicity, between the historic and epidemic strains, contributed in part the functions of the structural prM-E proteins.
Assuntos
Neuroglia/virologia , Proteínas do Envelope Viral/genética , Ligação Viral , Infecção por Zika virus/patologia , Zika virus/patogenicidade , África , Apoptose , Encéfalo/citologia , Encéfalo/virologia , Brasil , Surtos de Doenças , Epidemias , Humanos , Mutação , Neuroglia/imunologia , Replicação Viral , Zika virus/classificaçãoRESUMO
A strong correlation between Zika virus (ZIKV) infection and severe neurological disease in newborns and occasionally adults has emerged in the Brazilian outbreak. Efficient human cell-based assays are required to test candidate inhibitors of ZIKV replication. The aim of this work was to investigate ZIKV propagation and quantification in different cell lines. The human (U87, A549, Huh7), mosquito (C6/36) and monkey (VERO E6) cell lines tested were all permissive to ZIKV infection. When assessed by plaque forming units (PFU) in three different target cell lines, the maximal production of ZIKV was achieved in Huh7 at day 3 post-infection (6.38±0.44 log10PFU/ml). The C6/36 cell line showed a low and slow production of virus when compared with other cell lines. A549 readout cells generated a larger number of plaques compared to Huh7 but not to VERO E6 cells. ZIKV PFU and RNA titers showed the highest correlation when Huh7 and A549 were used as the producer and readout cells, respectively. Also, U87 cells produced ZIKV RNA titers which were highly correlated with PFU independently from the readout cell line. Using the best virus-cell system, sofosbuvir and ribavirin EC50 were 1.2µM and 1.1µM when measured through plaque assay, and 4.2µM and 5.2µM when measured by quantitative real time PCR (qRT-PCR), respectively. In summary, ZIKV can efficiently infect different human cell lines and rapidly reach peak viral titers. Overall, A549 cells appear to be as efficient as the VERO E6 gold standard for plaque assay allowing the use of human, rather than simian, cells for evaluating candidate anti-ZIKV compounds by the reference assay. The possibility to replace the labor-intensive plaque assay with the more rapid and easy-to-perform qRT-PCR is appealing and warrants further investigation.
Assuntos
Antivirais/farmacologia , Especificidade de Hospedeiro , RNA Viral/antagonistas & inibidores , Ribavirina/farmacologia , Sofosbuvir/farmacologia , Replicação Viral/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Células A549 , Animais , Brasil , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Culicidae , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Testes de Sensibilidade Microbiana , Neuroglia/efeitos dos fármacos , Neuroglia/virologia , RNA Viral/biossíntese , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Células Vero , Carga Viral/efeitos dos fármacos , Ensaio de Placa Viral , Zika virus/genética , Zika virus/crescimento & desenvolvimento , Zika virus/metabolismoRESUMO
BACKGROUND: Zika virus is an arthropod-borne virus that is a member of the family Flaviviridae transmitted mainly by mosquitoes of the genus Aedes. Although usually asymptomatic, infection can result in a mild and self-limiting illness characterised by fever, rash, arthralgia, and conjunctivitis. An increase in the number of children born with microcephaly was noted in 2015 in regions of Brazil with high transmission of Zika virus. More recently, evidence has been accumulating supporting a link between Zika virus and microcephaly. Here, we describe findings from three fatal cases and two spontaneous abortions associated with Zika virus infection. METHODS: In this case series, formalin-fixed paraffin-embedded tissue samples from five cases, including two newborn babies with microcephaly and severe arthrogryposis who died shortly after birth, one 2-month-old baby, and two placentas from spontaneous abortions, from Brazil were submitted to the Infectious Diseases Pathology Branch at the US Centers for Disease Control and Prevention (Atlanta, GA, USA) between December, 2015, and March, 2016. Specimens were assessed by histopathological examination, immunohistochemical assays using a mouse anti-Zika virus antibody, and RT-PCR assays targeting the NS5 and envelope genes. Amplicons of RT-PCR positive cases were sequenced for characterisation of strains. FINDINGS: Viral antigens were localised to glial cells and neurons and associated with microcalcifications in all three fatal cases with microcephaly. Antigens were also seen in chorionic villi of one of the first trimester placentas. Tissues from all five cases were positive for Zika virus RNA by RT-PCR, and sequence analyses showed highest identities with Zika virus strains isolated from Brazil during 2015. INTERPRETATION: These findings provide strong evidence of a link between Zika virus infection and different congenital central nervous system malformations, including microcephaly as well as arthrogryposis and spontaneous abortions. FUNDING: None.
Assuntos
Encéfalo/patologia , Encéfalo/virologia , Deformidades Congênitas dos Membros/virologia , Microcefalia/virologia , Complicações Infecciosas na Gravidez/patologia , Complicações Infecciosas na Gravidez/virologia , Primeiro Trimestre da Gravidez , Infecção por Zika virus/congênito , Infecção por Zika virus/patologia , Zika virus/isolamento & purificação , Aborto Espontâneo/virologia , Adulto , Antígenos Virais/isolamento & purificação , Autopsia , Brasil , Evolução Fatal , Feminino , Humanos , Imuno-Histoquímica/métodos , Lactente , Deformidades Congênitas dos Membros/diagnóstico por imagem , Masculino , Microcefalia/patologia , Neuroglia/patologia , Neuroglia/virologia , Placenta/patologia , Placenta/virologia , Gravidez , Segundo Trimestre da Gravidez , Terceiro Trimestre da Gravidez , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Síndrome , Ultrassonografia Pré-Natal , Zika virus/imunologiaRESUMO
Oncolytic viruses have the ability to infect tumor cells and leave healthy cells intact. In this study, bovine herpesvirus 1 (BHV1; Los Angeles, Cooper, and SV56/90 strains) and bovine herpesvirus 5 (BHV5; SV507/99 and GU9457818 strains) were used to infect two neuronal tumor cell lineages: neuro2a (mouse neuroblastoma cells) and C6 (rat glial cells). BHV1 and BHV5 strains infected both cell lines and positively correlated with viral antigen detection (p < 0.005). When neuro2a cells were infected by Los Angeles, SV507/99, and GU9457818 strains, 40 % of infected cells were under early apoptosis and necroptosis pathways. Infected C6 cells were >40 % in necroptosis phase when infected by BHV5 (GU9457818 strain). Blocking caspase activation did not interfere with cell death. However, when necroptosis was blocked, 60-80 % of both infected cells with either virus switched to early apoptosis pathway with no interference with virus replication. Moreover, reactive oxygen species production and mitochondrial membrane dysfunction were detected at high levels in both infected cell lines. In spite of apoptosis and necroptosis blockage, tumor necrosis factor alpha (TNFA) and virus transcription were positively correlated for all viral strains studied. Thus, these results contribute to the characterization of BHV1 and BHV5 as potential oncolytic viruses for non-human cells. Nonetheless, the mechanisms underlying their oncolytic activity in human cells are still to be determined.
Assuntos
Apoptose/genética , Herpesvirus Bovino 1/crescimento & desenvolvimento , Herpesvirus Bovino 5/crescimento & desenvolvimento , Necrose/virologia , Neuroglia/virologia , Neurônios/virologia , Animais , Antígenos Virais/genética , Bovinos , Linhagem Celular Tumoral , Expressão Gênica , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 5/genética , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/virologia , Necrose/genética , Necrose/patologia , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Vírus Oncolíticos/genética , Vírus Oncolíticos/crescimento & desenvolvimento , Especificidade de Órgãos , Estresse Oxidativo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Replicação ViralRESUMO
The mechanisms leading to the neurocognitive deficits in humans with immunodeficiency virus type 1 (HIV-1) are not well resolved. A number of cell culture models have demonstrated that the HIV-envelope glycoprotein 120 (gp120) decreases the reuptake of glutamate, which is necessary for learning, memory, and synaptic plasticity. However, the impact of brain HIV-1 gp120 on glutamate uptake systems in vivo remains unknown. Notably, alterations in brain glutamate uptake systems are implicated in a number of neurodegenerative and neurocognitive disorders. We characterized the kinetic properties of system XAG (sodium-dependent) and systems xc- (sodium-independent) [3H]-L-glutamate uptake in the striatum and hippocampus of HIV-1 gp120 transgenic mice, an established model of HIV neuropathology. We determined the kinetic constant Vmax (maximal velocity) and Km (affinity) of both systems XAG and xc- using subcellular preparations derived from neurons and glial cells. We show significant (30-35 %) reductions in the Vmax of systems XAG and xc- in both neuronal and glial preparations derived from the striatum, but not from the hippocampus of gp120 mice relative to wild-type (WT) controls. Moreover, immunoblot analysis showed that the protein expression of glutamate transporter subtype-1 (GLT-1), the predominant brain glutamate transporter, was significantly reduced in the striatum but not in the hippocampus of gp120 mice. These extensive and region-specific deficits of glutamate uptake likely contribute to the development and/or severity of HIV-associated neurocognitive disorders. Understanding the role of striatal glutamate uptake systems in HIV-1 gp120 may advance the development of new therapeutic strategies to prevent neuronal damage and improve cognitive function in HIV patients.
Assuntos
Disfunção Cognitiva/metabolismo , Corpo Estriado/metabolismo , Transportador 2 de Aminoácido Excitatório/genética , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/metabolismo , HIV-1/patogenicidade , Neuroglia/metabolismo , Animais , Disfunção Cognitiva/complicações , Disfunção Cognitiva/genética , Disfunção Cognitiva/virologia , Corpo Estriado/virologia , Modelos Animais de Doenças , Transportador 2 de Aminoácido Excitatório/deficiência , Ácido Glutâmico/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/complicações , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/fisiologia , Hipocampo/metabolismo , Hipocampo/virologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neuroglia/virologia , Neurônios/metabolismo , Neurônios/virologia , Especificidade de Órgãos , Sinapses/metabolismo , Sinapses/virologia , TransgenesRESUMO
Argentine haemorrhagic fever (AHF) is a systemic febrile syndrome characterized by several haematological and neurological alterations caused by Junín virus (JUNV), a member of the Arenaviridae family. Newborn mice are highly susceptible to JUNV and the course of infection has been associated with the viral strain used. Galectin-3 (Gal-3) is an animal lectin that has been proposed to play an important role in some central nervous system (CNS) diseases. In this study, we analysed Gal-3 expression at the transcriptional and translational expression levels during JUNV-induced CNS disease. We found that Candid 1 strain induced, with relatively low mortality, a subacute/chronic CNS disease with significant glia activation and upregulation of Gal-3 in microglia cells as well as in reactive astrocytes that correlated with viral levels. Our results suggest an important role for Gal-3 in viral-induced CNS disease.
Assuntos
Infecções por Arenaviridae/metabolismo , Encefalite Viral/metabolismo , Galectina 3/biossíntese , Vírus Junin/patogenicidade , Neuroglia/metabolismo , Neuroglia/virologia , Regulação para Cima/fisiologia , Animais , Animais Recém-Nascidos , Infecções por Arenaviridae/patologia , Astrócitos/metabolismo , Astrócitos/patologia , Astrócitos/virologia , Modelos Animais de Doenças , Encefalite Viral/patologia , Febre Hemorrágica Americana/metabolismo , Febre Hemorrágica Americana/patologia , Febre Hemorrágica Americana/virologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microglia/patologia , Microglia/virologia , Neuroglia/patologiaRESUMO
In previous reports we proposed a new genus for Rhabdoviridae and described neurotropic preference and gross neuropathology in newborn albino Swiss mice after Curionopolis and Itacaiunas infections. In the present report a time-course study of experimental encephalitis induced by Itacaiunas and Curionopolis virus was conducted both in vivo and in vitro to investigate cellular targets and the sequence of neuroinvasion. We also investigate, after intranasal inoculation, clinical signs, histopathology and apoptosis in correlation with viral immunolabeling at different time points. Curionopolis and Itacaiunas viral antigens were first detected in the parenchyma of olfactory pathways at 2 and 3 days post-inoculation (dpi) and the first clinical signs were observed at 4 and 8 dpi, respectively. After Curionopolis infection, the mortality rate was 100% between 5 and 6 dpi, and 35% between 8 and 15 dpi after Itacaiunas infection. We identified CNS mice cell types both in vivo and in vitro and the temporal sequence of neuroanatomical olfactory areas infected by Itacaiunas and Curionopolis virus. Distinct virulences were reflected in the neuropathological changes including TUNEL immunolabeling and cytopathic effects, more intense and precocious after intracerebral or in vitro inoculations of Curionopolis than after Itacaiunas virus. In vitro studies revealed neuronal but not astrocyte or microglial cytopathic effects at 2 dpi, with monolayer destruction occurring at 5 and 7 dpi with Curionopolis and Itacaiunas virus, respectively. Ultrastructural changes included virus budding associated with interstitial and perivascular edema, endothelial hypertrophy, a reduced and/or collapsed small vessel luminal area, thickening of the capillary basement membrane, and presence of phagocytosed apoptotic bodies. Glial cells with viral budding similar to oligodendrocytes were infected with Itacaiunas virus but not with Curionopolis virus. Thus, Curionopolis and Itacaiunas viruses share many pathological and clinical features present in other rhabdoviruses but distinct virulence and glial targets in newborn albino Swiss mice brain.
Assuntos
Encefalite Viral/patologia , Infecções por Rhabdoviridae/patologia , Rhabdoviridae/classificação , Rhabdoviridae/patogenicidade , Animais , Animais Recém-Nascidos , Apoptose , Encéfalo/embriologia , Encéfalo/patologia , Encéfalo/virologia , Células Cultivadas , Modelos Animais de Doenças , Encefalite Viral/etiologia , Feminino , Imunofluorescência , Técnicas Imunoenzimáticas , Camundongos , Neuroglia/citologia , Neuroglia/metabolismo , Neuroglia/virologia , Neurônios/citologia , Neurônios/metabolismo , Neurônios/virologia , Gravidez , Infecções por Rhabdoviridae/virologiaRESUMO
Pig neural cells express glycoproteins with sialylated N-linked oligosaccharide chains (SNOC) which are used by the porcine rubulavirus (PoRv) as receptors. Pig neuronal or glial cell cultures were employed to investigate (a) whether PoRv infects such cells using a molecule expressing SNOC, and (b) the role of viral envelope glycoproteins in establishing the infection. Enriched neuronal or glial cell cultures were exposed to PoRv and infection was detected immunocytochemically. Neuronal cultures prepared from neonatal pigs were treated enzymatically to eliminate sialic acid or N-linked oligosaccharide chains. Primary neural cultures were exposed to anti-HN or anti-F preincubated with PoRv to study the role of the viral glycoproteins. In enriched cultures, PoRv infected neurons and glial cells, and sialic acid expressed in N-linked oligosaccharide chains appeared to play a central role in infection. It was concluded that HN and F viral glycoproteins are required to infect neurons and glial cells.