RESUMO
The gold-standard method to evaluate a functional antiviral immune response is to titer neutralizing antibodies (NAbs) against a viral pathogen. This is historically performed using an in vitro assay of virus-mediated infection, which requires BSL-3 facilities. As these are insufficient in Latin American countries, including Mexico, scant information is obtained locally about viral pathogens NAb, using a functional assay. An alternative solution to using a BSL-3 assay with live virus is to use a BSL-2-safe assay with a non-replicative pseudovirus. Pseudoviral particles can be engineered to display a selected pathogen's entry protein on their surface, and to deliver a reporter gene into target cells upon transduction. Here we comprehensively describe the first development of a BSL-2 safe NAbs-measuring functional assay in Mexico, based on the production of pseudotyped lentiviral particles. As proof-of-concept, the assay is based on Nanoluc luciferase-mediated luminescence measurements from target cells transduced with SARS-CoV-2 Spike-pseudotyped lentiviral particles. We applied the optimized assay in a BSL-2 facility to measure NAbs in 65 serum samples, which evidenced the assay with 100% sensitivity, 86.6% specificity and 96% accuracy. Overall, this is the first report of a BSL-2 safe pseudovirus-based functional assay developed in Mexico to measure NAbs, and a cornerstone methodology necessary to measure NAbs with a functional assay in limited resources settings.
Assuntos
Anticorpos Neutralizantes , COVID-19 , Humanos , SARS-CoV-2 , Testes de Neutralização/métodos , Glicoproteína da Espícula de Coronavírus/metabolismo , Anticorpos Antivirais , México , Luciferases/genética , AntiviraisRESUMO
The causes of the broad spectrum of severity in COVID-19 are unknown. A protective effect through humoral immunity from previous infections by viruses of the SARS-CoV-2 family could explain a mild form of this disease. This study aimed to address whether the presence of antibodies against human seasonal coronaviruses (HCoVs) could prevent severe manifestations of COVID-19. A cross-sectional study was carried out in 165 participants. The presence of pre-existent antibodies against the seasonal HCoV-OC43, HCoV-HKU1, HCoV-229E, and HCoV-NL63 were detected. From all of the seasonal HCoVs studied, it was only found that being seropositive to HCoV-229E presented an association (p = 0.012) with developing mild clinical symptoms of COVID-19 or being asymptomatic. Multinomial regression analysis showed that being seropositive to HCoV-229E is associated with mild or moderate clinical symptoms for COVID-19. Statistical analysis also showed that being female is associated with being asymptomatic for SARS-CoV-2 infection or developing mild COVID-19. A subgroup analysis taking only seropositive to HCoV-229E revealed that females are more likely to develop asymptomatic SARS-CoV-2 infection (OR = 27.242, 95% CI 2.092-354.706, p = 0.012). Our results suggest that previous infections by HCoV-229E could prevent more serious clinical manifestations of COVID-19, but these are not the only variables that influence this event.
Assuntos
COVID-19 , Coronavirus Humano 229E , Anticorpos Antivirais , Estudos Transversais , Feminino , Humanos , SARS-CoV-2RESUMO
BACKGROUND: Interactions between humans and fauna lay in the heart of the history of human subsistence. In Mesoamerica, the Tehuacán-Cuicatlán Valley (TCV) harbours a high biodiversity with archaeological and ethnoecological evidence of its use by people inhabiting the area since at least 12,000 B.P. It is recognized as one of the most ancient areas of agriculture in the Americas, and a broad spectrum of management practices aimed to ensure the availability of desirable plants has been documented, but it has not been analysed for animals. This study aimed to investigate the use and management practices directed to wild animals along current settlements within the TCV and neighbouring areas. METHODS: We conducted an extensive search, review and analysis of documental sources for the period between 1967 and 2018. We found 38 documents providing information about the presence of animal species and 15 describing their use and/or management. We included our own observations from four case studies among the Ixcatec, Cuicatec, Nahua and Mestizo people, as well as from regional studies of biodiversity. We used unconstrained multivariate data analysis to describe the management typology of the animals in the region. RESULTS: Hitherto, 652 vertebrate species and 765 species of insects have been recorded in this area; and until present, 107 wild animal species have been reported to be used in 11 use-type categories, mostly for food (65.42%), ornamental (27.52%) and medicinal (21.10%) purposes by the Nahua, Cuicatec, Popolocan, Ixcatec, Mazatec and Mestizo people. Their extraction entails manual capture and gathering as well as hunting and trapping strategies, some of them involving planning in time or space and communitarian regulations; in addition, relocation actions and care in captivity were recorded. Nearly 178 of the species distributed in the region with no reports of local use are used in other localities of Mesoamerica. Ethnozoological information is still lacking for the Mixtec, Chinantec and Chocholtec people in the area. CONCLUSIONS: Wild fauna is still a valuable resource for the inhabitants of the TCV. Animals are obtained through extractive practices, which vary from one another in their qualitative attributes. With this work, we provide a context for further research priorities on fauna management in a region of high biocultural significance.
Assuntos
Criação de Animais Domésticos/métodos , Animais Selvagens , Animais , Antropologia Cultural , Conservação dos Recursos Naturais , Domesticação , Humanos , Medicina Tradicional , MéxicoRESUMO
Zika virus (ZV) is an arbovirus transmitted by Aedes aegypti and A. albopictus. The neurotropic profile of this virus is known since 1952. The main finding related to ZV in America is microcephaly. Two hypotheses are tested on its involvement in the central nervous system: its neurotropic feature and the direct effect of ZV on the placenta. Malformations and clinical findings on fetal development comprise congenital Zika syndrome. RT-PCR and serology (IgM) are useful for definitive diagnosis. However, we should keep in mind first that the viremia in pregnant women can stay for a longer period of time, and second, a positive IgM for Zika should be properly interpreted in an endemic area to other flavivirus. It is suggested to be part of TORCHS-Z complex the ZV infection in endemic areas.
El virus del Zika (VZ), arbovirus, es transmitido por Aedes aegypti y A. albopictus. Desde 1952 se conoce su perfil neurotrópico. El principal hallazgo relacionado con la infección en las Américas, es la microcefalia. Dos hipótesis se plantean sobre su afectación en el sistema nervioso central: su característica neurotrópica per se, y el efecto directo del virus sobre la placenta. Las malformaciones y hallazgos clínicos sobre el desarrollo fetal conforman el síndrome de Zika congénito. La reacción de polimerasa en cadena-transcriptasa reversa (RPC-TR) y serología (IgM) son útiles para el diagnóstico definitivo; sin embargo, debe tenerse en cuenta, primero, que la viremia en las mujeres embarazadas puede permanecer por un período más prolongado y segundo, que una IgM positiva para Zika, debe ser adecuadamente interpretada en un medio endémico para otros flavivirus. Se propone a la infección por el VZ, en zonas endémicas, como parte del complejo TORCHS-Z.
Assuntos
Humanos , Animais , Feminino , Gravidez , Complicações Infecciosas na Gravidez/virologia , Infecção por Zika virus/complicações , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/virologia , Microcefalia/virologia , Complicações Infecciosas na Gravidez/diagnósticoRESUMO
Zika virus (ZV) is an arbovirus transmitted by Aedes aegypti and A. albopictus. The neurotropic profile of this virus is known since 1952. The main finding related to ZV in America is microcephaly. Two hypotheses are tested on its involvement in the central nervous system: its neurotropic feature and the direct effect of ZV on the placenta. Malformations and clinical findings on fetal development comprise congenital Zika syndrome. RT-PCR and serology (IgM) are useful for definitive diagnosis. However, we should keep in mind first that the viremia in pregnant women can stay for a longer period of time, and second, a positive IgM for Zika should be properly interpreted in an endemic area to other flavivirus. It is suggested to be part of TORCHS-Z complex the ZV infection in endemic areas.
Assuntos
Microcefalia/virologia , Complicações Infecciosas na Gravidez/virologia , Infecção por Zika virus , Animais , Feminino , Humanos , Gravidez , Complicações Infecciosas na Gravidez/diagnóstico , Infecção por Zika virus/complicações , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/virologiaRESUMO
Human astroviruses (HAstV) are a frequent cause of gastroenteritis in young children and immunocompromised patients. To understand the early steps of HAstV infection in the highly permissive Caco-2 cell line, the binding and entry processes of the virus were characterized. The half-time of virus binding to the cell surface was about 10 min, while virus decapsidation took around 130 min. Drugs affecting clathrin-mediated endocytosis, endosome acidification, and actin filament polymerization, as well as those that reduce the presence of cholesterol in the cell membrane, decreased the infectivity of the virus. The infection was also reduced by silencing the expression of the clathrin heavy chain (CHC) by RNA interference or by overexpression of dominant-negative mutants of dynamin 2 and Eps15. Furthermore, the entry of HAstV apparently depends on the maturation of endosomes, since the infection was reduced by silencing the expression of Rab7, a small GTPase involved in the early- to late-endosome maturation. Altogether, our results suggest that HAstV enters Caco-2 cells using a clathrin-dependent pathway and reaches late endosomes to enter cells. Here, we have characterized the mechanism used by human astroviruses, important agents of gastroenteritis in children, to gain entry into their host cells. Using a combination of biochemical and genetic tools, we found that these viruses enter Caco-2 cells using a clathrin-dependent endocytic pathway, where they most likely need to travel to late endosomes to reach the cytoplasm and begin their replication cycle.
Assuntos
Mamastrovirus/fisiologia , Internalização do Vírus , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Antivirais/farmacologia , Infecções por Astroviridae/genética , Infecções por Astroviridae/metabolismo , Infecções por Astroviridae/virologia , Linhagem Celular , Clatrina/genética , Clatrina/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Endorribonucleases/metabolismo , Proteínas Fúngicas/metabolismo , Inativação Gênica , Humanos , Mamastrovirus/efeitos dos fármacos , Mutação , Ligação Viral , Liberação de Vírus , Replicação Viral/efeitos dos fármacos , Desenvelopamento do Vírus , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Rotavirus (RV) is the major cause of childhood gastroenteritis worldwide. This study presents a functional genome-scale analysis of cellular proteins and pathways relevant for RV infection using RNAi. Among the 522 proteins selected in the screen for their ability to affect viral infectivity, an enriched group that participates in endocytic processes was identified. Within these proteins, subunits of the vacuolar ATPase, small GTPases, actinin 4, and, of special interest, components of the endosomal sorting complex required for transport (ESCRT) machinery were found. Here we provide evidence for a role of the ESCRT complex in the entry of simian and human RV strains in both monkey and human epithelial cells. In addition, the ESCRT-associated ATPase VPS4A and phospholipid lysobisphosphatidic acid, both crucial for the formation of intralumenal vesicles in multivesicular bodies, were also found to be required for cell entry. Interestingly, it seems that regardless of the molecules that rhesus RV and human RV strains use for cell-surface attachment and the distinct endocytic pathway used, all these viruses converge in early endosomes and use multivesicular bodies for cell entry. Furthermore, the small GTPases RHOA and CDC42, which regulate different types of clathrin-independent endocytosis, as well as early endosomal antigen 1 (EEA1), were found to be involved in this process. This work reports the direct involvement of the ESCRT machinery in the life cycle of a nonenveloped virus and highlights the complex mechanism that these viruses use to enter cells. It also illustrates the efficiency of high-throughput RNAi screenings as genetic tools for comprehensively studying the interaction between viruses and their host cells.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Infecções por Rotavirus/metabolismo , Rotavirus/metabolismo , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/virologia , ATPases Associadas a Diversas Atividades Celulares , Animais , Células CACO-2 , Chlorocebus aethiops , Estudo de Associação Genômica Ampla , Humanos , Transporte Proteico/fisiologia , Interferência de RNA , Infecções por Rotavirus/virologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Células Vero , Proteínas de Transporte Vesicular/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7 , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
This study examined the placentation in the degu, the origin of the extrasubplacental trophoblast (EST) (extravillous trophoblast in human), and the activity of Na+/K+ ATPase in the placental barrier during different gestational ages, as part of a wider effort to understand the reproductive biology of this species. Fifteen degus at the first stage of gestation, midgestation and at term of pregnancy were studied. At day 27 of gestation, the subplacenta is formed under the wall of the central excavation. Simultaneously, the outermost trophoblast of the ectoplacental cone differentiated into secondary trophoblast giant cells that lie on the outside of the placenta, forming an interface with the maternal cells in the decidua. These giant cells immunostained positive for cytokeratin (CK) and placental lactogen (hPL) until term. During this period, the EST merged from the subplacenta to the decidua and immunostained negative for CK, but at term, immunostained for CK and hPL in the maternal vessels. The vascular mesenchyme of the central excavation invaded the chorioallantoic placenta during this period, forming two fetal lobules of labyrinthine-fine syncytium, the zone of the placental barrier. The activity of Na+/K+ ATPase in the placental barrier was constant during the gestational period. The residual syncytium at the periphery of the placental disc and between the lobules was not invaded by fetal mesenchyme and formed the marginal and interlobular labyrinthine syncytium that immunostained first for CK, and later for hPL, as in the labyrinthine fine syncytium. The presence of intracytoplasmic electron-dense material in the interlobular labyrinthine syncytium suggested a secretory process in these cells that are bathed in maternal blood. Placentas obtained from vaginal births presented a large, single lobe, absence of the subplacenta, and a reduced interlobular labyrinthine syncytium. At day 27, the inverted visceral yolk sac is observed and its columnar epithelium immunostained for CK and hPL. This suggests that the yolk sac is an early secretory organ. The epithelium of the parietal yolk sac covers the placenta. The origin of the EST in the degu placenta and its migration to maternal vessels allows us to present this animal model for the study of pregnancy pathologies related to alterations in the migration of the extravillous trophoblast.