Rocio virus (ROCV) was the causative agent of an unprecedented outbreak of encephalitis during the 1970s in the Vale do Ribeira, Sao Paulo State, in the Southeast region of Brazil. Surprisingly, no ...further cases of ROCV infection were identified after this outbreak; however, serological surveys have suggested the circulation of ROCV among humans and animals in different regions of Brazil. Cross-protective immunity among flaviviruses is well documented; consequently, immunity induced by infections with other flaviviruses endemic to Brazil could potentially be responsible for the lack of ROCV infections. Herein, we evaluated the cross-protection mediated by other flaviviruses against ROCV infection using an experimental C57BL/6 mouse model. Cross-protection against ROCV infection was observed when animals had prior exposure to Ilheus virus or Saint Louis encephalitis virus, suggesting that cross-reactive anti-flavivirus antibodies may limit ROCV disease outbreaks.
Rocio virus (ROCV) is a highly neuropathogenic mosquito-transmitted flavivirus responsible for an unprecedented outbreak of human encephalitis during 1975-1976 in Sao Paulo State, Brazil. Previous ...studies have shown an increased number of inflammatory macrophages in the central nervous system (CNS) of ROCV-infected mice, implying a role for macrophages in the pathogenesis of ROCV. Here, we show that ROCV infection results in increased expression of CCL2 in the blood and in infiltration of macrophages into the brain. Moreover, we show, using CCR2 knockout mice, that CCR2 expression is essential for macrophage infiltration in the brain during ROCV infection and that the lack of CCR2 results in increased disease severity and mortality. Thus, our findings show the protective role of CCR2-mediated infiltration of macrophages in the brain during ROCV infection.
Dengue, a disease caused by any of the four serotypes of dengue viruses, is the most important arthropod-borne viral disease in the world in terms of both morbidity and mortality. The infection by ...these viruses induces a plethora of clinical manifestations ranging from asymptomatic infections to severe diseases with involvement of several organs. Severe forms of the disease are more frequent in secondary infections by distinct serotypes and, consequently, a dengue vaccine must be tetravalent. Although several approaches have been used on the vaccine development, no vaccine is available against these viruses, especially because of problems on the development of a tetravalent vaccine. Here, we describe briefly the vaccine candidates available and their ability to elicit a protective immune response. We also discuss the problems and possibilities of any of the vaccines in final development stage reaching the market for human use.
In the Americas, hantaviruses cause severe cardiopulmonary syndrome (HCPS) with a high fatality rate. Hantavirus infection is commonly diagnosed using serologic techniques and reverse ...transcription-polymerase chain reaction. This paper presents a novel plaque reduction neutralisation test (PRNT) for detecting antibodies to Brazilian hantavirus. Using PRNT, plaque detection was enhanced by adding 0.6% of dimethyl sulfoxide into the overlay culture medium of the infected cells. This procedure facilitated clear visualisation of small plaques under the microscope and provided for easy and accurate plaque counting. The sera from 37 HCPS patients from the city of Ribeirão Preto, Brazil was evaluated for the Rio Mamoré virus (RIOMV) using PRNT. Six samples exhibited neutralising antibodies; these antibodies exhibited a low titre. The low level of seropositive samples may be due to fewer cross-reactions between two different hantavirus species; the patients were likely infected by Araraquara virus (a virus that has not been isolated) and RIOMV was used for the test. This assay offers a new approach to evaluating and measuring neutralising antibodies produced during hantavirus infections and it can be adapted to other hantaviruses, including viruses that will be isolated in the future.
Development of an enzyme immunoassay for poliovirus antigens Hashimoto, Newton; Benati, Fabrício José; Lauretti, Flávio ...
Brazilian archives of biology and technology,
2007, 2007-01-00, 20070101, 2007-01-01, Letnik:
50, Številka:
1
Journal Article
Recenzirano
Odprti dostop
An indirect solid-phase enzyme immunoassay (EIA) was developed for the detection of poliovirus antigen. Virus antigen was obtained in LLC-MK2 cell cultures and used to prepare antibodies in rabbit ...and guinea pig. Antibodies were evaluated by double immunodiffusion and neutralization test. Optimal concentrations of guinea pig and rabbit immunoglobulins were determined by checkerboard titration. Microtitre plates were coated with 15.0 µg/ml guinea pig anti-polio immunoglobulin and rabbit anti-polio immunoglobulin at the concentration of 7.94 µg/ml was used as detecting antibody. The standard curve with eight different antigen concentrations in eight replicates resulted in a coefficient of variation (CV) between 2.1% to 7.8%. The dose-response relationship was determined by simple linear regression with a coefficient of correlation (R²) equal to 96.4%. The assay detected a minimum of 2.3 µg/ml poliovirus antigen.
O trabalho apresenta o desenvolvimento de um ensaio imunoenzimático indireto para a detecção de antígeno de poliovírus. O antígeno viral foi obtido em cultura de células LLC-MK2 e usado para imunização de coelho e cobaia. Os soros hiperimunes foram avaliados por imunodifusão dupla e teste de neutralização. Após padronização, o soro de captura, produzido em cobaia, foi usado na concentração protéica de 15.0 µg/ml para sensibilizar microplacas de poliestireno e o soro de coelho (detector) foi usado na concentração de 7.94 µg/ml. A curva padrão resultante da utilização de oito diferentes concentrações do antígeno padrão definiu um coeficiente de variação de 2.1% a 7.8%. A relação dose-resposta foi determinada por regressão linear simples com o estabelecimento do coeficiente de correlação (R²) igual a 96.4%. O ensaio possibilitou a detecção mínima de 2.3 µg/ml de antígeno de poliovírus.
Dengue is the most important arbovirus disease throughout the world and it is responsible for more than 500,000 dengue hemorrhagic cases and 22,000 deaths every year. One vaccine was recently ...licensed for human use in Brazil, Mexico and Philippines and although at least seven candidates have been in clinical trials the results of the most developed CYD vaccine have demonstrated immunization problems, such as uneven protection and interference between serotypes. We constructed a vaccine candidate based on vesicular stomatitis virus (VSV) expression of pre-membrane (prM) and envelope (E) proteins of dengue-2 virus (DENV-2) and tested it in mice to evaluate immunogenicity and protection against DENV-2 infection. VSV has been successfully used as vaccine vectors for several viruses to induce strong humoral and cellular immune responses. The VSV-DENV-2 recombinant was constructed by inserting the DENV-2 structural proteins into a VSV plasmid DNA for recombinant VSV-DENV-2 recovery. Infectious recombinant VSV viruses were plaque purified and prM and E expression were confirmed by immunofluorescence and radiolabeling of proteins of infected cells. Forty Balb/C mice were inoculated through subcutaneous (s.c.) route with VSV-DENV-2 vaccine in a two doses schedule 15 d apart and 29 d after first inoculation, sera were collected and the mice were challenged with 50 lethal doses (LD
50
) of a neurovirulent DENV-2. The VSV-DENV-2 induced anti-DENV-2 antibodies and protected animals in the challenge experiment comparable to DENV-2 immunization control group. We conclude that VSV is a promising platform to test as a DENV vaccine and perhaps against others Flaviviridae.
Acridine orange is a metachromatic intercalator used extensively in histochemistry to differentiate double- from single-stranded (ds, ss) nucleic acid by the emission of green and red fluorescence, ...respectively, under ultraviolet light. In the present study we standardised a protocol in order to use acridine orange to detect rotavirus ds RNA in polyacrylamide gels and compared it to silver and ethidium bromide staining. We demonstrated that the simplest and best condition was attained when gels containing rotavirus ds RNA bands, stained in green, were treated with 4.3
μM acridine orange after electrophoresis and destained with distilled water pH 6 at 37
°C. Under this protocol, rotavirus RNA concentration was calculated and the mean minimum amounts of nucleic acid detected by acridine orange, ethidium bromide, and silver staining were 26.0±4.29, 15.6±1.48 and 1.06±0.11
ng, respectively. The comparison of acridine orange sensitivity with ethidium bromide and silver staining, for 25 field strains of rotavirus and one cell-adapted strain (SA11), demonstrated concurrent results in 80% of the specimens. Red colour emission resulting from the interaction of acridine orange with ss nucleic acid was also shown by testing denatured 0.5
kb
HindIII digest of lambda phage DNA. Furthermore, it was demonstrated that rotavirus ds RNA could be used for reverse transcription activity, followed by PCR amplification, after acridine orange staining. In conclusion, although acridine orange is less sensitive than ethidium bromide and silver staining, its practicality, low cost, metachromatic properties, and its non-interference on RT-PCR should be considered. It is suggested the use of acridine orange as an appropriate stain for various purposes in virology, as well as for the molecular biology of nucleic acid.
Rotaviruses have been implicated as the major causal agents of acute
diarrhoea in mammals and fowls. Experimental rotavirus infection have
been associated to a series of sub-cellular pathologic ...alterations
leading to cell lysis which may represent key functions in the
pathogenesis of the diarrhoeic disease. The current work describes the
cytopathic changes in cultured MA-104 cells infected by a simian
(SA-11) and a porcine (1154) rotavirus strains. Trypan blue exclusion
staining showed increased cell permeability after infection by both
strains, as demonstrated by cell viability. This effect was confirmed
by the leakage of infected cells evaluated by chromium release. Nuclear
fragmentation was observed by acridine orange and Wright staining but
specific DNA cleavage was not detected. Ultrastructural changes, such
as chromatin condensation, cytoplasm vacuolisation, and loss of
intercellular contact were shown in infected cells for both strains. In
situ terminal deoxynucleotidyl transferase (Tunel) assay did not show
positive result. In conclusion, we demonstrated that both strains of
rotavirus induced necrosis as the major degenerative effect.
Rotavirus (RV) strain SA-11 was studied with respect to its infectivity in MA-104 cell cultures and the effect of concanavalin A (ConA). Receptors for ConA at the surface of MA-104 cell were ...determined by fluorescence assay and specifically inhibited by D-mannose. The kinetics of virus growth was carried out by plaque assay. Electron microscopy and polyacrylamide gel electrophoresis were used for monitoring the experiments. It was concluded that RV replication was not affected consistently by ConA, however it interfered with the development of cytopathic effect (CPE) without altering virus yields.
A infecciosidade do rotavírus cepa SA-11 foi estudada em cultura de células MA-104 em presença de concanavalina A (ConA). Foi demonstrada a presença de receptores para a ConA nestas células através de ensaio de fluorescência e bloqueio específico dos mesmos através de D-manose. A cinética de replicação viral foi realizada pelo método de plaque e o vírus monitorado, pontualmente, pela microscopia eletrônica de transmissão e eletroforese em gel de poliacrilamida. Concluímos que a ConA não afetou consistentemente a replicação viral, entretanto interferiu no efeito citopático.
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