RESUMO
BACKGROUND: Dengue, chikungunya and Zika viruses (DENV, CHIKV and ZIKV) are transmitted in sylvatic transmission cycles between non-human primates and forest (sylvan) mosquitoes in Africa and Asia. It remains unclear if sylvatic cycles exist or could establish themselves elsewhere and contribute to the epidemiology of these diseases. The Caribbean island of St. Kitts has a large African green monkey (AGM) (Chlorocebus aethiops sabaeus) population and is therefore ideally suited to investigate sylvatic cycles. METHODS: We tested 858 AGM sera by ELISA and PRNT for virus-specific antibodies and collected and identified 9704 potential arbovirus vector mosquitoes. Mosquitoes were homogenized in 513 pools for testing by viral isolation in cell culture and by multiplex RT-qPCR after RNA extraction to detect the presence of DENV, CHIKV and ZIKVs. DNA was extracted from 122 visibly blood-fed individual mosquitoes and a polymorphic region of the hydroxymethylbilane synthase gene (HMBS) was amplified by PCR to determine if mosquitoes had fed on AGMs or humans. RESULTS: All of the AGMs were negative for DENV, CHIKV or ZIKV antibodies. However, one AGM did have evidence of an undifferentiated Flavivirus infection. Similarly, DENV, CHIKV and ZIKV were not detected in any of the mosquito pools by PCR or culture. AGMs were not the source of any of the mosquito blood meals. CONCLUSION: Sylvatic cycles involving AGMs and DENV, CHIKV and ZIKV do not currently exist on St. Kitts.
Assuntos
Febre de Chikungunya/transmissão , Febre de Chikungunya/veterinária , Chlorocebus aethiops/virologia , Dengue/transmissão , Dengue/veterinária , Infecção por Zika virus/transmissão , Infecção por Zika virus/veterinária , Aedes/genética , Aedes/virologia , Animais , Anticorpos Antivirais/sangue , Vírus Chikungunya/genética , Vírus Chikungunya/imunologia , Vírus da Dengue/genética , Vírus da Dengue/imunologia , Feminino , Humanos , Hidroximetilbilano Sintase/genética , Mosquitos Vetores/genética , Mosquitos Vetores/virologia , São Cristóvão e Névis , Zika virus/genética , Zika virus/imunologiaRESUMO
Vaccinia virus (VACV) is a notorious virus for a number of scientific reasons; however, most of its notoriety comes from the fact that it was used as a vaccine against smallpox, being ultimately responsible for the eradication of that disease. Nonetheless, many different vaccinia virus strains have been obtained over the years; some are suitable to be used as vaccines, whereas others are virulent and unsuitable for this purpose. Interestingly, different vaccinia virus strains elicit different immune responses in vivo, and this is a direct result of the genomic differences among strains. In order to evaluate the net result of virus-encoded immune evasion strategies of vaccinia viruses, we compared antiviral immune responses in mice intranasally infected by the highly attenuated and nonreplicative MVA strain, the attenuated and replicative Lister strain, or the virulent WR strain. Overall, cell responses elicited upon WR infections are downmodulated compared to those elicited by MVA and Lister infections, especially in determined cell compartments such as macrophages/monocytes and CD4+ T cells. CD4+ T cells are not only diminished in WR-infected mice but also less activated, as evaluated by the expression of costimulatory molecules such as CD25, CD212, and CD28 and by the production of cytokines, including tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), interleukin-4 (IL-4), and IL-10. On the other hand, MVA infections are able to induce strong T-cell responses in mice, whereas Lister infections consistently induced responses that were intermediary between those induced by WR and MVA. Together, our results support a model in which the virulence of a VACV strain is proportional to its potential to downmodulate the host's immune responses.IMPORTANCE Vaccinia virus was used as vaccine against smallpox and was instrumental in the successful eradication of that disease. Although smallpox vaccination is no longer in place in the overall population, the use of vaccinia virus in the development of viral vector-based vaccines has become popular. Nonetheless, different vaccinia virus strains are known and induce different immune responses. To look into this, we compared immune responses triggered by mouse infections with the nonreplicative MVA strain, the attenuated Lister strain, or the virulent WR strain. We observed that the WR strain was capable of downmodulating mouse cell responses, whereas the highly attenuated MVA strain induced high levels of cell-mediated immunity. Infections by the intermediately attenuated Lister strain induced cell responses that were intermediary between those induced by WR and MVA. We propose that the virulence of a vaccinia virus strain is directly proportional to its ability to downmodulate specific compartments of antiviral cell responses.
Assuntos
Imunidade Celular/imunologia , Vaccinia virus/imunologia , Vacínia/imunologia , Virulência/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Galinhas/imunologia , Galinhas/virologia , Chlorocebus aethiops/imunologia , Chlorocebus aethiops/virologia , Citocinas/imunologia , Vetores Genéticos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Varíola/imunologia , Vacinação/métodos , Vacínia/virologia , Vacinas Virais/imunologiaRESUMO
Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation, but the DNA ladder was detected at different times post-infection depending on the cell line, i.e., 48, 72 and 96 h post-infection in RK-13, Vero and BHK-21 cells, respectively. Measurement of viral titers obtained with each cell line indicated that apoptosis causes interference with viral replication and therefore decreased viral titers. As an unequivocal marker of apoptosis, we measured the expression of caspase-3 and caspases-8 and -9 as extrinsic and intrinsic markers of apoptosis pathways, respectively. Caspase-8 in BHK-21 cells was the only protease that was not detected at any of the times assayed. We found that Bucyrus EAV strain exhibited a distinctive apoptosis pathway depending on the cell line.
Assuntos
Apoptose/fisiologia , Equartevirus/patogenicidade , Células Vero , Replicação Viral/fisiologia , Animais , Linhagem Celular/virologia , Chlorocebus aethiops/virologia , Cricetinae , HaplorrinosRESUMO
The increasingly limited availability and high cost of the hitherto most commonly used monkey species in dengue vaccine research has augmented the importance of identifying alternative suitable models for these studies. In this study we examined the capacity of green monkeys (Chlorocebus aethiops sabaeus) to develop dengue viremia, and thus provide a potential model for dengue vaccine testing. Monkeys were inoculated with two different doses of dengue virus type 2. All animals in both groups became viremic after inoculation of the virus. In the lower dose group, mean viremia duration of 5.66 days was detected, whereas in the group that received the 106 PFU dose, viremia had a mean duration of only 1.66 days. Antibody titers were similar to those obtained in previous experiments with rhesus and cynomolgus macaques. We conclude that green monkeys develop viremia and antibody responses and therefore provide a potential model for the preclinical evaluation of novel candidates for dengue vaccines.
Assuntos
Anticorpos Antivirais/sangue , Chlorocebus aethiops/virologia , Vírus da Dengue/imunologia , Vírus da Dengue/patogenicidade , Dengue/imunologia , Dengue/prevenção & controle , Viremia , Animais , Dengue/virologia , Modelos Animais de Doenças , Vacinas/imunologiaRESUMO
HCV (hepatitis C virus) is a worldwide health problem nowadays. No preventive vaccine is available against this pathogen, and therapeutic treatments currently in use have important drawbacks, including limited efficacy. In the present work a recombinant fowlpox virus, FPCoE1, expressing a truncated HCV core-E1 polyprotein, was generated. FPCoE1 virus generally failed to elicit a humoral immune response against HCV antigens in BALB/c mice. By contrast, mice inoculated with FPCoE1 elicited a positive interferon-gamma secretion response against HCV core in ex-vivo ELISPOT (enzyme-linked immunospot) assays. Remarkably, mice inoculated with FPCoE1 significantly controlled viraemia in a surrogate challenge model with vvRE, a recombinant vaccinia virus expressing HCV structural antigens. In fact, 40% of the mice had no detectable levels of vvRE in their ovaries. Administration of FPCoE1 in vervet monkeys [Chlorocebus (formerly Cercophitecus) aethiops sabaeus] induced lymphoproliferative response against HCV core and E1 proteins in 50% of immunized animals. Monkeys immunized with FPCoE1 had no detectable levels of vvRE in their blood, whereas monkeys inoculated with FP9, the negative control virus, had detectable levels of vvRE in blood up to 7 days after challenge. In conclusion, recombinant fowlpox virus FPCoE1 is able to induce an anti-HCV immune response in mice and monkeys. This ability could be rationally employed to develop effective strategies against HCV infection by using FPCoE1 in combination with other vaccine candidates or antiviral treatments.
Assuntos
Chlorocebus aethiops/imunologia , Vírus da Varíola das Aves Domésticas/genética , Hepatite C/imunologia , Imunização , Polimorfismo de Nucleotídeo Único/imunologia , Vaccinia virus/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Chlorocebus aethiops/virologia , DNA Viral/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Vírus da Varíola das Aves Domésticas/imunologia , Hepatite C/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/genéticaRESUMO
Three fractions of fucoidans isolated from the brown seaweed Leathesia difformis (Ee, Ec and Ea) were found to be selective antiviral agents against herpes simplex virus (HSV) types 1 and 2 and human cytomegalovirus. Fraction Ea was the most active, with IC50 values in the range 0.5-1.9 microg/ml without affecting cell viability at concentrations up to 400 microg/ml. The antiherpetic activity of Ea was assessed by three different methods, plaque reduction, inhibition of virus yield and prevention of HSV-2 induced shut-off of cell protein synthesis, demonstrating that the inhibitory effect was independent of the antiviral assay and the multiplicity of infection. The mode of action of Ea could be ascribed to an inhibitory action on virus adsorption. The fucoidans did not inhibit the blood coagulation process even at concentrations exceeding more than 100 times the IC50 value.