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1.
BMC Infect Dis ; 19(1): 977, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31747889

RESUMO

BACKGROUND: Rabies is estimated to cause 59,000 deaths and economic losses of US$8.6 billion every year. Despite several years of rabies surveillance and awareness programmes, increased availability of post-exposure prophylaxis vaccinations and dog population control, the disease still remains prevalent in Sri Lanka. This study reports the roll-out of a high number, high coverage canine rabies vaccination campaign in Sri Lanka, providing estimates for the vaccination coverage achieved, analysing the local dog demographics, and identifying barriers of attendance to static vaccination clinics. METHODS: A mass dog vaccination campaign was undertaken in Negombo, Sri Lanka. The campaign was composed of static point and door-to-door vaccination stages, with a final survey of vaccination coverage. A large volume of data on the distribution, health, and signalment of vaccinated dogs was collected through a mobile phone application. A logistic regression model was developed to investigate which socio-spatial and dog-related factors influenced attendance of owners to static vaccination points. RESULTS: The campaign vaccinated over 7800 dogs achieving a vaccination coverage of 75.8%. A dog:human ratio of 1:17 was estimated. Most dogs were owned, and the dog population was mostly male, adult, and non-sterilized. Unawareness, unavailability and handling problems were the most common reasons given by owners to explain failure to attend a static vaccination point. The regression analysis showed that increasing distance to a static point, in addition to young age and poor health of the dog, were associated with a decrease in the likelihood of attendance to a static vaccination points. CONCLUSION: This study demonstrates the feasibility of high number, high coverage vaccination campaigns in Sri Lanka. The information on dog ecology and barriers of attendance to static point vaccination clinics will facilitate development of future vaccination campaigns.


Assuntos
Doenças do Cão/prevenção & controle , Vacinas Antirrábicas/imunologia , Raiva/prevenção & controle , Cobertura Vacinal/métodos , Animais , Telefone Celular , Doenças do Cão/imunologia , Cães , Feminino , Humanos , Programas de Imunização , Modelos Logísticos , Masculino , Raiva/imunologia , Sri Lanka , Inquéritos e Questionários , Cobertura Vacinal/estatística & dados numéricos
2.
Biologicals ; 60: 8-14, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31255474

RESUMO

This two-day workshop, co-sponsored by NICEATM and IABS-NA, brought together over 60 international scientists from government, academia, and industry to advance alternative methods for human and veterinary Rabies Virus Vaccine (RVV) potency testing. On day one, workshop presentations focused on regulatory perspectives related to in vitro potency testing, including recent additions to the European Pharmacopoeia (5.2.14) that provide a scientific rationale for why in vivo methods may be less suitable for vaccine quality control than appropriately designed in vitro methods. Further presentations reviewed the role of the consistency approach to manufacturing and vaccine batch comparison to provide supportive data for the substitution of existing animal-based methods with in vitro assays. In addition, updates from research programs evaluating and validating RVV glycoprotein (G) quantitation by ELISA as an in vitro potency test were presented. On the second day, RVV stakeholders participated in separate human and veterinary vaccine discussion groups focused on identifying potential obstacles or additional requirements for successful implementation of non-animal alternatives to the in vivo potency test. Workshop outcomes and proposed follow up activities are discussed herein.


Assuntos
Vacinas Antirrábicas/uso terapêutico , Vírus da Raiva/imunologia , Raiva/prevenção & controle , Potência de Vacina , Animais , Disciplinas das Ciências Biológicas , Educação , Humanos , Controle de Qualidade , Raiva/imunologia , Raiva/patologia , Vacinas Antirrábicas/imunologia , Sociedades Científicas
3.
PLoS Pathog ; 15(6): e1007799, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31220188

RESUMO

The development of high-throughput genome sequencing enables accurate measurements of levels of sub-consensus intra-host virus genetic diversity and analysis of the role played by natural selection during cross-species transmission. We analysed the natural and experimental evolution of rabies virus (RABV), an important example of a virus that is able to make multiple host jumps. In particular, we (i) analyzed RABV evolution during experimental host switching with the goal of identifying possible genetic markers of host adaptation, (ii) compared the mutational changes observed during passage with those observed in natura, and (iii) determined whether the colonization of new hosts or tissues requires adaptive evolution in the virus. To address these aims, animal infection models (dog and fox) and primary cell culture models (embryo brain cells of dog and fox) were developed and viral variation was studied in detail through deep genome sequencing. Our analysis revealed a strong unidirectional host evolutionary effect, as dog-adapted rabies virus was able to replicate in fox and fox cells relatively easily, while dogs or neuronal dog cells were not easily susceptible to fox adapted-RABV. This suggests that dog RABV may be able to adapt to some hosts more easily than other host variants, or that when RABV switched from dogs to red foxes it lost its ability to adapt easily to other species. Although no difference in patterns of mutation variation between different host organs was observed, mutations were common following both in vitro and in vivo passage. However, only a small number of these mutations also appeared in natura, suggesting that adaptation during successful cross-species virus transmission is a complex, multifactorial evolutionary process.


Assuntos
Doenças do Cão , Evolução Molecular , Interações Hospedeiro-Parasita/imunologia , Vírus da Raiva/fisiologia , Raiva , Animais , Linhagem Celular , Doenças do Cão/genética , Doenças do Cão/imunologia , Cães , Feminino , Raposas/genética , Raposas/imunologia , Raposas/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Parasita/genética , Masculino , Mutação , Raiva/genética , Raiva/imunologia
4.
Biologicals ; 60: 49-54, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31105021

RESUMO

The NIH assay is used to assess the potency of rabies vaccine and is currently a key measure required for vaccine release. As this test involves immunization of mice and subsequent viral challenge, efforts are being made to develop alternative analytical methods that do not rely on animal testing. Sanofi Pasteur has reported the development of a G-protein specific ELISA assay that has shown agreement with the NIH test. In this study we have generated several non-conform vaccine lots by an excessive inactivation with ß-propiolactone (BPL) and assessed the capacity of both tests to detect the corresponding consequences. Excessive BPL inactivation causes G-protein unfolding, altering in turn viral morphology and the continuity of the G-protein layer in the viral particle. Both the NIH and the ELISA tests were able to monitor the consequences of excessive inactivation in a similar manner. Of note, the experimental error of the ELISA test was well below that of the NIH test. These results increase the prospect that the ELISA test could be considered a suitable candidate for the replacement of the NIH test.


Assuntos
Bioensaio , Vacinas Antirrábicas , Potência de Vacina , Animais , Ensaio de Imunoadsorção Enzimática , Camundongos , Raiva/imunologia , Raiva/patologia , Raiva/prevenção & controle , Vacinas Antirrábicas/química , Vacinas Antirrábicas/imunologia , Vacinação , Vacinas de Produtos Inativados
5.
Biomed Res Int ; 2019: 4518163, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31008105

RESUMO

Inactivation of rabies virus is essential for rabies vaccine preparation where the inactivating compound that is currently recommended for rabies vaccine preparation is ß-propiolactone (ß-PL). This compound is considered better than phenol and formalin but it is expensive and potentially carcinogenic. Data revealed that Ascorbic acid (AA) with cupric ions could yield complete and irreversible inactivation of rabies virus without adversely affecting its antigenicity. Additionally, the results of testing the vaccine potency with the selected inactivating compounds were comparable (P<0.05), and ED50 was higher than the recommended World Health Organization (WHO) limits. The use of HemaGel (plasma substitute) for testing vaccine stabilization was compared with the currently used vaccine stabilizers (human albumin and lactose). HemaGel yielded better stability than the other tested stabilizers. Monitoring of cellular and humoral immune responses indicated that both the total IgG level against rabies vaccine and the IFN and IL5 levels obtained with the HemaGel-stabilized vaccines were higher than those obtained with human albumin- and lactose-stabilized vaccine candidates.


Assuntos
Imunogenicidade da Vacina/efeitos dos fármacos , Propiolactona/farmacologia , Vacinas Antirrábicas/farmacologia , Raiva/prevenção & controle , Albuminas/farmacologia , Animais , Anticorpos Antivirais/efeitos dos fármacos , Anticorpos Antivirais/imunologia , Ácido Ascórbico/farmacologia , Humanos , Imunoglobulina G/imunologia , Interferons/imunologia , Interleucina-5 , Lactose/química , Propiolactona/química , Raiva/imunologia , Raiva/virologia , Vacinas Antirrábicas/química , Vacinas Antirrábicas/genética , Vacinas Antirrábicas/imunologia , Vírus da Raiva/imunologia , Vírus da Raiva/patogenicidade , Potência de Vacina , Células Vero/virologia
6.
Int J Mol Sci ; 20(7)2019 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-30934732

RESUMO

The human diploid cell line Medical Research Council -5 (MRC-5) is commonly utilized for vaccine development. Although a rabies vaccine developed in cultured MRC-5 cells exists, the poor susceptibility of MRC-5 cells to the rabies virus (RABV) infection limits the potential yield of this vaccine. The underlying mechanism of MRC-5 cell resistance to RABV infection remains unknown. In this study, we demonstrate that viral infection increased exosomal release from MRC-5 cells; conversely, blocking exosome release promoted RABV infection in MRC-5 cells. Additionally, RABV infection up-regulated microRNA (miR)-423-5p expression in exosomes, resulting in feedback inhibition of RABV replication by abrogating the inhibitory effect of suppressor of cytokine signaling 3 (SOCS3) on type I interferon (IFN) signaling. Furthermore, intercellular delivery of miR-423-5p by exosomes inhibited RABV replication in MRC-5 cells. We also show that RABV infection increased IFN-ß production in MRC-5 cells and that blocking the type I IFN receptor promoted RABV infection. In conclusion, MRC-5 cells were protected from RABV infection by the intercellular delivery of exosomal miR-423-5p and the up-regulation of IFN-ß. These findings reveal novel antiviral mechanisms in MRC-5 cells against RABV infection. miR-423-5p, exosomes, and IFN signaling pathways may therefore be potential targets for improving MRC-5 cell-based rabies vaccine production.


Assuntos
Resistência à Doença , Exossomos/metabolismo , Técnicas de Transferência de Genes , MicroRNAs/administração & dosagem , Vírus da Raiva/fisiologia , Raiva/genética , Raiva/virologia , Sequência de Bases , Linhagem Celular , Exossomos/ultraestrutura , Retroalimentação Fisiológica , Humanos , Interferon beta/metabolismo , Raiva/imunologia , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Regulação para Cima , Replicação Viral
7.
Biologicals ; 59: 56-61, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30898479

RESUMO

The Rapid Fluorescent Focus Inhibition Test (RFFIT) is a standard assay used to detect and assess the titers of rabies virus neutralizing antibodies (RVNA) in blood sera. To simplify the multistep RFFIT procedure by eliminating the immunostaining step, we generated a new recombinant RV expressing a green fluorescent protein (rRV-GFP) and assess its suitability for quantifying RVNA. We rescued the rRV-GFP virus from plasmid DNA carrying a full-length genome of the CVS-N2c strain of RV in which the eGFP gene was inserted between the glycoprotein and RNA-polymerase genes. The recombinant virus was genetically stable and grew efficiently in appropriate cells expressing sufficient GFP fluorescence to detect directly 20 h post infection (hpi). We evaluated the feasibility of using rRV-GFP in RFFIT by comparing RVNA titers in 27 serum samples measured by conventional RFFIT and RFFIT-GFP. A linear regression analysis of the data demonstrated a good agreement between these two methods (r = 0.9776) including results with samples having RVNA titers close to the minimally acceptable vaccine potency threshold (0.5 IU/ml). Study results showed that the rRV-GFP virus could replace the CVS-11 challenge virus currently used in the conventional RFFIT and enabling more rapid, simpler, and less expensive detection and quantitation of RVNA.


Assuntos
Anticorpos Neutralizantes/imunologia , Vacinas Antirrábicas/imunologia , Vírus da Raiva/imunologia , Raiva/imunologia , Animais , Anticorpos Neutralizantes/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Fluorescência , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cobaias , Humanos , Medições Luminescentes/métodos , Camundongos , Testes de Neutralização , Coelhos , Raiva/prevenção & controle , Raiva/virologia , Vacinas Antirrábicas/administração & dosagem , Vírus da Raiva/genética , Vírus da Raiva/metabolismo , Recombinação Genética
8.
PLoS Negl Trop Dis ; 13(1): e0007128, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30695032

RESUMO

BACKGROUND: Early ante-mortem laboratory confirmation of human rabies is essential to aid patient management and institute public health measures. Few studies have highlighted the diagnostic value of antibody detection in CSF/serum in rabies, and its utility is usually undermined owing to the late seroconversion and short survival in infected patients. This study was undertaken to examine the ante-mortem diagnostic utility and prognostic value of antibody detection by rapid fluorescent focus inhibition test (RFFIT) in cerebrospinal fluid (CSF)/serum samples received from clinically suspected human rabies cases from January 2015 to December 2017. METHODOLOGY/PRINCIPAL FINDINGS: Samples collected ante-mortem and post-mortem from 130 and 6 patients with clinically suspected rabies respectively, were received in the laboratory during the study period. Ante-mortem laboratory confirmation was achieved in 55/130 (42.3%) cases. Real time PCR for detection of viral nucleic acid performed on saliva, nuchal skin, brain tissue and CSF samples could confirm the diagnosis in 15 (27.2%) of the 55 laboratory confirmed cases. Ante-mortem diagnosis could be achieved by RFFIT (in CSF and/or serum) in 45 (34.6%) of the 130 clinically suspected cases, accounting for 81.8% of the total 55 laboratory confirmed cases. The sensitivity of CSF RFFIT increased with the day of sample collection (post-onset of symptoms) and was found to be 100% after 12 days of illness. Patients who had received prior vaccination had an increased probability of a positive RFFIT and negative PCR result. Patients who were positive by RFFIT alone at initial diagnosis had longer survival (albeit with neurological sequelae) than patients who were positive by PCR alone or both RFFIT and PCR. CONCLUSIONS/SIGNIFICANCE: Detection of antibodies in the CSF/serum is a valuable ante-mortem diagnostic tool in human rabies, especially in patients who survive beyond a week. It was also found to have a limited role as a prognostic marker to predict outcomes in patients.


Assuntos
Anticorpos Neutralizantes/análise , Anticorpos Antivirais/análise , RNA Viral/análise , Vírus da Raiva/isolamento & purificação , Raiva/diagnóstico , Adulto , Idoso , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/líquido cefalorraquidiano , Anticorpos Antivirais/sangue , Anticorpos Antivirais/líquido cefalorraquidiano , Autopsia , Criança , Feminino , Humanos , Masculino , Prognóstico , RNA Viral/genética , Raiva/sangue , Raiva/líquido cefalorraquidiano , Raiva/imunologia , Vírus da Raiva/genética , Vírus da Raiva/imunologia , Estudos Retrospectivos , Saliva/virologia , Pele/virologia
9.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30567978

RESUMO

Marburg virus (MARV) is a filovirus related to Ebola virus (EBOV) associated with human hemorrhagic disease. Outbreaks are sporadic and severe, with a reported case mortality rate of upward of 88%. There is currently no antiviral or vaccine available. Given the sporadic nature of outbreaks, vaccines provide the best approach for long-term control of MARV in regions of endemicity. We have developed an inactivated rabies virus-vectored MARV vaccine (FILORAB3) to protect against Marburg virus disease. Immunogenicity studies in our labs have shown that a Th1-biased seroconversion to both rabies virus and MARV glycoproteins (GPs) is beneficial for protection in a preclinical murine model. As such, we adjuvanted FILORAB3 with glucopyranosyl lipid adjuvant (GLA), a Toll-like receptor 4 agonist, in a squalene-in-water emulsion. Across two different BALB/c mouse challenge models, we achieved 92% protection against murine-adapted Marburg virus (ma-MARV). Although our vaccine elicited strong MARV GP antibodies, it did not strongly induce neutralizing antibodies. Through both in vitro and in vivo approaches, we elucidated a critical role for NK cell-dependent antibody-mediated cellular cytotoxicity (ADCC) in vaccine-induced protection. Overall, these findings demonstrate that FILORAB3 is a promising vaccine candidate for Marburg virus disease.IMPORTANCE Marburg virus (MARV) is a virus similar to Ebola virus and also causes a hemorrhagic disease which is highly lethal. In contrast to EBOV, only a few vaccines have been developed against MARV, and researchers do not understand what kind of immune responses are required to protect from MARV. Here we show that antibodies directed against MARV after application of our vaccine protect in an animal system but fail to neutralize the virus in a widely used virus neutralization assay against MARV. This newly discovered activity needs to be considered more when analyzing MARV vaccines or infections.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Glicoproteínas/imunologia , Doença do Vírus de Marburg/imunologia , Marburgvirus/imunologia , Vírus da Raiva/imunologia , Raiva/imunologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vacinas Antirrábicas/imunologia , Vacinação/métodos , Células Vero , Vacinas Virais/imunologia
10.
BMC Res Notes ; 11(1): 920, 2018 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-30577868

RESUMO

OBJECTIVES: Domestic dogs are the main reservoir of rabies virus (RABV) infection in Nigeria, thus surveillance of rabies in dog populations is crucial in order to understand the patterns of spread of infection and ultimately devise an appropriate rabies control strategy. This study determined the presence of lyssavirus antigen in brain tissues and anti-rabies antibodies in sera of apparently healthy and suspected-rabid dogs slaughtered for human consumption at local markets in South-Eastern Nigeria. RESULTS: Our findings demonstrated that 8.3% (n = 23) of brain tissues were lyssavirus positive and 2.5% (n = 25) of sera had rabies antibody levels as percentage blocking of 70% and above correlating with a cut-off value ≥ 0.5 IU/mL in the fluorescent antibody neutralization test. There was an inverse correlation between lyssavirus positivity and rabies antibody levels confirming that infected individuals most often do not develop virus neutralizing antibodies to the disease. The low percentage of rabies antibodies in this dog population suggests a susceptible population at high risk to RABV infection. These findings highlight a huge challenge to national rabies programs and subsequent elimination of the disease from Nigeria, considering that majority of dogs are confined to rural communal areas, where parenteral dog vaccination is not routinely undertaken.


Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Doenças do Cão/imunologia , Lyssavirus/imunologia , Raiva/imunologia , Animais , Encéfalo/imunologia , Doenças do Cão/sangue , Doenças do Cão/epidemiologia , Cães , Nigéria/epidemiologia , Raiva/sangue , Raiva/epidemiologia
11.
PLoS One ; 13(12): e0209373, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30571707

RESUMO

Rabies is an ancient and neglected zoonotic disease caused by the rabies virus, a neurotropic RNA virus that belongs to the Rhabdoviridae family, genus Lyssavirus. It remains an important public health problem as there are cost and health concerns imposed by the current human post exposure prophylaxis therapy. The use of monoclonal antibodies (mAbs) is therefore an attractive alternative. Rabies mostly affects people that reside in resource-limited areas where there are occasional failures in the cold-chain. These environmental changes may upset the stability of the mAbs. This study focused on mAbs 62-71-3 and E559; their structures, responses to freeze/thaw (F/T) and exposure to reactive oxygen species were therefore studied with the aid of a wide range of biophysical and in silico techniques in order to elucidate their stability and identify aggregation prone regions. E559 was found to be less stable than 62-71-3. The complementarity determining regions (CDR) contributed the most to its instability, more specifically: peptides 99EIWD102 and 92ATSPYT97 found in CDR3, Trp33 found in CDR1 and the oxidised Met34. The constant region "158SWNSGALTGHTFPAVL175" was also flagged by the special aggregation propensity (SAP) tool and F/T experiments to be highly prone to aggregation. The E559 peptides "4LQESGSVL11 from the heavy chain and 4LTQSPSSL11 from the light chain, were also highly affected by F/T. These residues may serve as good candidates for mutation, in the aim to bring forward more stable therapeutic antibodies, thus paving a way to a more safe and efficacious antibody-based cocktail treatment against rabies.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Antivirais/química , Vírus da Raiva/imunologia , Raiva/terapia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/uso terapêutico , Temperatura Baixa/efeitos adversos , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Simulação por Computador , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Humanos , Testes de Neutralização , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Engenharia de Proteínas/métodos , Proteólise , Raiva/imunologia , Raiva/virologia , Espécies Reativas de Oxigênio/química , Tabaco/genética , Tabaco/metabolismo
12.
Vopr Virusol ; 63(5): 224-232, 2018.
Artigo em Russo | MEDLINE | ID: mdl-30550099

RESUMO

The molecular and biological characteristics of the vaccine against rabies virus strain ERA-CB 20M obtained by the Russian rabiologist, doctor of medical sciences S.V. Gribencha by adapting and cloning the strain ERA and SAD in a transplantable BHK-21 C13 cell culture are presented. The spectrum of the most sensitive strain of rabies ERA-CB 20M cell lines was determined and the level of glycoprotein was quantitatively determined. Primary nucleotide sequences of fragments of the genome of the strain ERA-CB 20M (genes N and G) were obtained and phylogenetic analysis was carried out. Molecular analysis showed that this strain belongs to the group of vaccine strains SAD1. When compared with the reference strain SAD1, 10% of the nucleotide differences were revealed in the gene fragment N; 15%, in the gene fragment G.


Assuntos
Vacinas Antirrábicas/genética , Vírus da Raiva/genética , Raiva/genética , Vacinas Atenuadas/uso terapêutico , Genoma Viral/genética , Glicoproteínas/genética , Humanos , Raiva/imunologia , Raiva/prevenção & controle , Raiva/virologia , Vacinas Antirrábicas/uso terapêutico , Vírus da Raiva/patogenicidade , Vacinas Atenuadas/imunologia
13.
PLoS Negl Trop Dis ; 12(12): e0007011, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30550592

RESUMO

The effectiveness of rabies vaccination in both humans and animals is determined by the presence of virus neutralizing antibodies (VNAs). The Rapid Fluorescent Focus Inhibition Test (RFFIT) is the method traditionally used for detection and quantification of VNAs. It is a functional in vitro test for assessing the ability of antibodies in serum to bind and prevent infection of cultured cells with rabies virus (RABV). The RFFIT is a labor intensive, low throughput and semi-quantitative assay performed by trained laboratorians. It requires staining of RABV-infected cells by rabies specific fluorescent antibodies and manual quantification of fluorescent fields for titer determination. Although the quantification of fluorescent fields observed in each sample is recorded, the corresponding images are not stored or captured to be used for future analysis. To circumvent several of these disadvantages, we have developed an alternative, automated high throughput neutralization test (HTNT) for determination of rabies VNAs based on green fluorescent protein (GFP) expression by a recombinant RABV and compared with the RFFIT. The HTNT assay utilizes the recombinant RABV ERA variant expressing GFP with a nuclear localization signal (NLS) for efficient quantification. The HTNT is a quantitative method where the number of RABV-infected cells are determined and the images are stored for future analysis. Both RFFIT and HTNT results correlated 100% for a panel of human and animal positive and negative rabies serum samples. Although, the VNA titer values are generally agreeable, HTNT titers tend to be lower than that of RFFIT, probably due to the differences in quantification methods. Our data demonstrates the potential for HTNT assays in determination of rabies VNA titers.


Assuntos
Anticorpos Neutralizantes/análise , Anticorpos Antivirais/análise , Proteínas de Fluorescência Verde/análise , Ensaios de Triagem em Larga Escala/métodos , Testes de Neutralização/métodos , Vírus da Raiva/genética , Raiva/virologia , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Raiva/diagnóstico , Raiva/imunologia , Vírus da Raiva/imunologia , Vírus da Raiva/isolamento & purificação
14.
PLoS One ; 13(11): e0207009, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30403742

RESUMO

Rabies is a fatal encephalitic disease in humans and animals caused by lyssaviruses, most commonly rabies virus (RABV). Human antemortem diagnosis of rabies is a complex process involving multiple sample types and tests for the detection of antibodies, antigen (protein), and nucleic acids (genomic RNA). Serological diagnosis of human rabies includes the detection of either neutralizing or binding antibodies in the cerebrospinal fluid (CSF) or serum samples from unimmunized individuals without prior rabies vaccination or passive immunization with purified immunoglobulins. While neutralizing antibodies are targeted against the surface-expressed glycoprotein (G protein), binding antibodies to viral antigens are predominantly against the nucleoprotein (N protein), although there can be antibodies against all RABV-expressed proteins. To determine N protein-specific antibody responses in the CSF and serum during RABV infection, we developed an enzyme-linked immunosorbent assay (ELISA) with purified recombinant N protein expressed in E. coli. N protein-specific immunoglobulin (Ig) subtypes IgG and IgM were detected in the CSF or serum of previously diagnosed human rabies cases. In addition, anti-N protein seroconversion was demonstrated over the course of illness in individual rabies cases. We compared the N protein ELISA results to those of an indirect fluorescent antibody (IFA) test, the current binding antibody assay used in diagnosis, and show that our ELISA is consistent with the IFA test. Sensitivity and specificity of the N protein ELISA ranged from 78.38-100% and 75.76-96.77% with respect to the IFA results. Our data provide evidence for the use of an N protein ELISA as an additional option for the detection of RABV-specific IgG or IgM antibodies in human CSF or serum specimens.


Assuntos
Anticorpos Antivirais/sangue , Nucleoproteínas/imunologia , Vírus da Raiva/metabolismo , Raiva/diagnóstico , Proteínas Virais/imunologia , Anticorpos Antivirais/líquido cefalorraquidiano , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/líquido cefalorraquidiano , Imunoglobulina M/sangue , Imunoglobulina M/líquido cefalorraquidiano , Nucleoproteínas/metabolismo , Raiva/imunologia , Raiva/virologia , Vírus da Raiva/isolamento & purificação , Sensibilidade e Especificidade , Proteínas Virais/metabolismo
15.
Vet Microbiol ; 226: 59-63, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30389044

RESUMO

Although juvenile red foxes (Vulpes vulpes) are considered a single age group, essential for monitoring the effectiveness of the oral rabies vaccination (ORV), there appear to be significant differences among age subgroups. Herein, a subset of 335 foxes aged 0-1 year that had not consumed bait in previous campaign were collected for monitoring the effectiveness of the first seven ORV campaigns in Greece, carried out from 2013 to 2017. These juveniles were additionally assigned to three individual 4-month age groups, according to the exact date on which they were killed. The aim was to identify differences in seroconversion rate and bait uptake level and determine whether reconsideration is needed in the way that ORV monitoring is being implemented and evaluated. Statistically significant differences were observed following the analysis of mandible bone, teeth and blood samples obtained from 1-4 and 5-8-month old foxes as compared to the respective samples derived from 9-12-month old animals, whereas no differences were revealed in samples between foxes aged 1-4 and 5-8 months. Hunting juveniles during the whole period of spring ORV campaigns monitoring should be reevaluated and even discouraged. On the contrary, juvenile foxes hunted for the evaluation of autumn campaigns, aged > 8 months, had similar assessment rates to adult individuals and are equally helpful for assessing the efficacy of an ORV campaign. Taking the above into consideration and by distinguishing recent and old tetracycline uptake, ORV monitoring and evaluation could be performed in an alternative, more comprehensive way.


Assuntos
Vacinas Antirrábicas/efeitos adversos , Vírus da Raiva/imunologia , Raiva/veterinária , Soroconversão , Vacinação , Administração Oral , Fatores Etários , Animais , Antibacterianos/administração & dosagem , Anticorpos Antivirais/sangue , Raposas , Raiva/sangue , Raiva/imunologia , Raiva/prevenção & controle , Vacinas Antirrábicas/administração & dosagem , Vacinas Antirrábicas/imunologia , Estações do Ano , Testes Sorológicos , Tetraciclina/administração & dosagem , Potência de Vacina
16.
Virol J ; 15(1): 174, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30424815

RESUMO

BACKGROUND: Rabies is a fatal disease that is preventable when post exposure prophylaxis (PEP) is administered in a timely fashion. CpG oligodeoxynucleotides (ODNs) can trigger cells that express Toll-like receptor 9, and their immunopotentiation activity in an inactivated aluminum-adjuvanted rabies vaccine for dogs has been identified using mouse and dog models. METHODS: A human diploid cell rabies vaccine (HDCV) of humans and a CpG ODNs with cross-immunostimulatory activity in humans and mice were used to evaluate the immunogenicity and protective efficacy of CpG ODN in a mouse model that simulates human PEP. RESULTS: HDCV combined with CpG ODN (HDCV-CpG) stimulated mice to produce rabies virus-specific neutralizing antibody (RVNA) earlier and increased the seroconversion rate. Compared with HDCV alone, either HDCV-1.25 µg CpG or HDCV-5 µg CpG increased the levels of RVNA. In particular, 5 µg CpG ODN per mouse significantly boosted the levels of RVNA compared with HDCV alone. IFN-γ producing splenocytes generated in the HDCV-5 µg CpG group were significantly increased compared to the group treated with HDCV alone. When the immunization regimen was reduced to three injections or the dose was reduced to half of the recommended HDCV combined with CpG ODN, the RVNA titers were still higher than those induced by HDCV alone. After viral challenge, 50% of mice immunized with a half-dose HDCV-CpG survived, while the survival rate of mice immunized with HDCV alone was 30%. CONCLUSIONS: The immunopotentiation activity of CpG ODNs for a commercially available human rabies vaccine was first evaluated in a mouse model on the basis of the Essen regimen. Our results suggest that the CpG ODN used in this study is a potential adjuvant to rabies vaccines for human use.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Oligodesoxirribonucleotídeos/imunologia , Profilaxia Pós-Exposição , Vacinas Antirrábicas/imunologia , Vírus da Raiva/imunologia , Raiva/imunologia , Animais , Anticorpos Antivirais/sangue , Modelos Animais de Doenças , Feminino , Humanos , Imunogenicidade da Vacina , Camundongos , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos/administração & dosagem , Raiva/prevenção & controle , Vacinas Antirrábicas/administração & dosagem , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia
18.
J Vet Med Sci ; 80(10): 1596-1603, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30210066

RESUMO

The protective antibody response of Balb/c mice to Bali rabies virus (RABV) in BHK-21 cells was studied. The virus was isolated from a rabid dog and was adapted to replicate in BHK-21 cell culture for seven passages. The BHK-21-adapted Bali RABV (BHK-Bali RABV) was inactivated with binary ethylenimine and 24 mice were immunized twice at 21-days intervals with the inactivated virus and Rabisin® vaccine. Virus replication was detected using indirect immunofluorescence, immunocytochemistry, and western blotting assays. Enzyme-linked immunosorbent assay examination 2 weeks after the first immunization revealed RABV antibody titers that were mostly below the minimum protective level (<0.5 equivalent unit, EU). Antibody titers increased sharply after the second immunization. Antibody titers in serum of mice induced by inactivated BHK-Bali RABV one week after the second immunization were slightly lower (0.8-3.8 EU) than those induced by Rabisin vaccine (0.9-6.3 EU). RABV antibody titers were stable for at least 6 weeks after the second immunization. Both Rabisin vaccine and inactivated BHK-Bali RABV induced neutralizing antibodies with neutralization titers (50% protective dose per ml) of 29.84 for 0.1 ml Rabisin, 211.41 for 0.2 ml Rabisin, 27.41 for 0.1 ml BHK-Bali RABV, and 28.25 for 0.2 ml BHK-Bali RABV. Thus, inactivated BHK-Bali RABV induces a protective immune response in Balb/c mice, but at lower levels compared to induction by Rabisin vaccine.


Assuntos
Anticorpos Antivirais/imunologia , Vacinas Antirrábicas/imunologia , Vírus da Raiva/crescimento & desenvolvimento , Raiva/veterinária , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Linhagem Celular , Doenças do Cão/imunologia , Doenças do Cão/prevenção & controle , Doenças do Cão/virologia , Cães , Feminino , Masculino , Camundongos Endogâmicos BALB C , Raiva/imunologia , Raiva/prevenção & controle , Vírus da Raiva/imunologia , Inoculações Seriadas , Vacinas de Produtos Inativados/imunologia , Cultura de Vírus , Replicação Viral
19.
PLoS One ; 13(9): e0204115, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30235274

RESUMO

Despite the implementation of control measures (preventive dog vaccination), rabies has become endemic in Croatia, with red foxes being the main reservoir species. Oral rabies vaccination (ORV) campaigns supported by the European Commission have been conducted twice a year since the spring of 2011. The first campaigns were limited to the northern and eastern parts of the country, and from the autumn of 2012, the program was extended to the entire country. The Lysvulpen vaccine containing the SAD Bern strain was used for ORV. Following the vaccination campaigns, the number of rabies cases decreased, and the last positive case was recorded in February 2014. The bait uptake ranged from 24.86% to 84.62% and the immunisation rate from 11.24% to 35.64%.


Assuntos
Erradicação de Doenças , Raiva/epidemiologia , Raiva/prevenção & controle , Animais , Croácia/epidemiologia , Raposas/imunologia , Raposas/virologia , Imunidade Humoral , Incidência , Chacais/imunologia , Chacais/virologia , Oxitetraciclina/uso terapêutico , Filogenia , Raiva/tratamento farmacológico , Raiva/imunologia , Vírus da Raiva/fisiologia , Soroconversão
20.
J Virol ; 92(22)2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30158289

RESUMO

Rabies virus is a neurovirulent RNA virus, which causes about 59,000 human deaths each year. Treatment for rabies does not exist due to incomplete understanding of the pathogenesis. MALT1 mediates activation of several immune cell types and is involved in the proliferation and survival of cancer cells. MALT1 acts as a scaffold protein for NF-κB signaling and a cysteine protease that cleaves substrates, leading to the expression of immunoregulatory genes. Here, we examined the impact of genetic or pharmacological MALT1 inhibition in mice on disease development after infection with the virulent rabies virus strain CVS-11. Morbidity and mortality were significantly delayed in Malt1 -/- compared to Malt1 +/+ mice, and this effect was associated with lower viral load, proinflammatory gene expression, and infiltration and activation of immune cells in the brain. Specific deletion of Malt1 in T cells also delayed disease development, while deletion in myeloid cells, neuronal cells, or NK cells had no effect. Disease development was also delayed in mice treated with the MALT1 protease inhibitor mepazine and in knock-in mice expressing a catalytically inactive MALT1 mutant protein, showing an important role of MALT1 proteolytic activity. The described protective effect of MALT1 inhibition against infection with a virulent rabies virus is the precise opposite of the sensitizing effect of MALT1 inhibition that we previously observed in the case of infection with an attenuated rabies virus strain. Together, these data demonstrate that the role of immunoregulatory responses in rabies pathogenicity is dependent on virus virulence and reveal the potential of MALT1 inhibition for therapeutic intervention.IMPORTANCE Rabies virus is a neurotropic RNA virus that causes encephalitis and still poses an enormous challenge to animal and public health. Efforts to establish reliable therapeutic strategies have been unsuccessful and are hampered by gaps in the understanding of virus pathogenicity. MALT1 is an intracellular protease that mediates the activation of several innate and adaptive immune cells in response to multiple receptors, and therapeutic MALT1 targeting is believed to be a valid approach for autoimmunity and MALT1-addicted cancers. Here, we study the impact of MALT1 deficiency on brain inflammation and disease development in response to infection of mice with the highly virulent CVS-11 rabies virus. We demonstrate that pharmacological or genetic MALT1 inhibition decreases neuroinflammation and extends the survival of CVS-11-infected mice, providing new insights in the biology of MALT1 and rabies virus infection.


Assuntos
Encéfalo/imunologia , Inflamação/prevenção & controle , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/fisiologia , Vírus da Raiva/imunologia , Raiva/prevenção & controle , Linfócitos T/imunologia , Animais , Encéfalo/metabolismo , Encéfalo/virologia , Células Cultivadas , Inflamação/imunologia , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Raiva/imunologia , Raiva/metabolismo , Linfócitos T/patologia , Linfócitos T/virologia
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