RESUMO
Equine rhinitis A virus (ERAV) is a picornavirus associated with respiratory disease in horses and is genetically closely related to foot-and-mouth disease virus (FMDV), the prototype aphthovirus. ERAV has recently gained interest as an FMDV alternative for the study of aphthovirus biology, including cell entry and uncoating or antiviral testing. As described for FMDV, current data support that acidic pH inside cellular endosomes triggers ERAV uncoating. In order to provide further insights into aphthovirus uncoating mechanism, we have isolated a panel of ERAV mutants with altered acid sensitivity and that differed on their degree of sensitivity to the inhibition of endosome acidification. These results provide functional evidence of the involvement of acidic pH on ERAV uncoating within endosomes. Remarkably, all amino acid substitutions found in acid-labile or acid-resistant ERAVs were located in the capsid protein VP3, indicating that this protein plays a pivotal role for the control of pH stability of the ERAV capsid. Moreover, all amino acid substitutions mapped at the intraprotomer interface between VP3 and VP2 or between VP3 and the N terminus of VP1. These results expand our knowledge on the regions that regulate the acid stability of aphthovirus capsid and should be taken into account when using ERAV as a surrogate of FMDV. IMPORTANCE: The viral capsid constitutes a sort of dynamic nanomachine that protects the viral genome against environmental assaults while accomplishing important functions such as receptor attachment for viral entry or genome release. We have explored the molecular determinants of aphthovirus capsid stability by isolating and characterizing a panel of equine rhinitis A virus mutants that differed on their acid sensitivity. All the mutations were located within a specific region of the capsid, the intraprotomer interface among capsid proteins, thus providing new insights into the regions that control the acid stability of aphthovirus capsid. These findings could positively contribute to the development of antiviral approaches targeting aphthovirus uncoating or the refinement of vaccine strategies based on capsid stabilization.
Assuntos
Ácidos/metabolismo , Aphthovirus/genética , Proteínas do Capsídeo/genética , Cavalos/virologia , Substituição de Aminoácidos/genética , Animais , Antivirais/farmacologia , Aphthovirus/efeitos dos fármacos , Capsídeo/efeitos dos fármacos , Endossomos/virologia , Vírus da Febre Aftosa/efeitos dos fármacos , Genoma Viral/genética , Concentração de Íons de Hidrogênio , Mutação/genética , Infecções por Picornaviridae/tratamento farmacológico , Infecções por Picornaviridae/virologia , Internalização do Vírus/efeitos dos fármacosRESUMO
Injection of the synthetic lipid amine, Avridine, in the form of liposomes, protected guinea pigs against the development of lesions from foot-and-mouth disease virus (FMDV) inoculated intradermally into the rear footpads. The animals were protected against the development of vesicles at the inoculation site as well as the systemic spread of virus. Maximal protection was obtained after intracardial injection of 5-10 mg doses of liposomal Avridine. Lower doses yielded decreased protection. Subcutaneous or intraperitoneal routes of liposomal Avridine injection were ineffective. Protection was maximal 0-24 h after injection of liposomes. Ethanol and emulsion formulations of Avridine could induce protection when injected intracardially but had toxic side effects. Guinea pigs protected against the first FMDV inoculation by liposomal and ethanol formulations of Avridine continued to be protected against lesions after a second inoculation 15-45 days later. FMDV protective antibody titers of these animals ranged from a low of less than 1:10 to greater than 1:1000.
Assuntos
Aphthovirus/efeitos dos fármacos , Diaminas/uso terapêutico , Febre Aftosa/prevenção & controle , Lipossomos/administração & dosagem , Animais , Anticorpos Antivirais/análise , Aphthovirus/imunologia , Diaminas/administração & dosagem , Relação Dose-Resposta a Droga , Feminino , Febre Aftosa/tratamento farmacológico , Febre Aftosa/imunologia , Cobaias , Poli I-C/uso terapêuticoRESUMO
The effect of various vaccines against foot and mouth disease virus (FMDV) was tested on Langerhans cell density in the footpad epidermis of mice. Injection of monovalent, bivalent and trivalent FMDV vaccines caused a reduction in Langerhans cell density in the murine skin, which was more marked at the center of the footpad, the site of injection, than at the periphery. Testing of the various components of the vaccine showed that saponin caused a marked reduction in Langerhans cells while injection of aluminium hydroxide had a lesser effect and the virus alone had no effect on these cells. Thus Langerhans cell density could serve as an efficient marker to test the safety of vaccines to FMDV since the integrity of Langerhans cells, which are the antigen-presenting cells in the skin epidermis, is needed for an effective immune response to the vaccine.
Assuntos
Anticorpos Antivirais/biossíntese , Aphthovirus/efeitos dos fármacos , Ilhotas Pancreáticas/imunologia , Vacinas Virais/farmacologia , Adenosina Trifosfatases , Animais , Aphthovirus/ultraestrutura , Avaliação de Medicamentos , Epiderme/efeitos dos fármacos , Epiderme/imunologia , Pé , Ilhotas Pancreáticas/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Coloração e RotulagemRESUMO
Crude extracts of leaves and fruits of Guarea guidona were tested for antiviral activity against pseudorabies virus and foot-and-mouth disease virus in the IB-RS-2 pig cell line and against bovine herpesvirus-1 (BHV-1) in the GBK bovine cell line. The highest nontoxic doses of extracts from fruits and leaves were 125 micrograms/ml and 500 micrograms/ml. respectively. Crude extracts presented antiviral activity against pseudorabies virus with a decrease in virus titer of 3.0 log units at 500 micrograms/ml. Virucidal activity was not observed at 62.5 micrograms/ml. Preformed cell monolayers showed no cytotoxic effect after 48 h in the presence of 500 micrograms/ml in pig cells. G. guidona leaves did not induce an antiviral state but exhibited antiviral effects during the early stage of viral infection.
Assuntos
Antivirais/farmacologia , Aphthovirus/efeitos dos fármacos , Herpesvirus Bovino 1/efeitos dos fármacos , Herpesvirus Suídeo 1/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Bovinos , Linhagem Celular/efeitos dos fármacos , Células Cultivadas , Testes de Sensibilidade Microbiana , Extratos Vegetais/toxicidade , SuínosRESUMO
Ethyleneimine (EI) and N-acetylethyleneimine (AEI) have been shown to inactivate viruses belonging to most of the families described by the International Committee for the Taxonomy of Viruses. The mechanism by which they inactivate the viruses has not been established. In this paper, experiments with foot-and-mouth disease virus (FMDV) and poliovirus are described which indicate that the inactivating lesion is on the RNA.
Assuntos
Antivirais/farmacologia , Aphthovirus/efeitos dos fármacos , Aziridinas/farmacologia , Poliovirus/efeitos dos fármacos , Animais , Aphthovirus/genética , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Humanos , Poliovirus/genética , Poliovirus/ultraestrutura , Células VeroRESUMO
In order to protect farm animals from infections such as foot and mouth disease (FMD) and tuberculosis, the pasteurisation of milk and milk products designated for the feeding of animals is compulsory in Switzerland. Nowadays, milk products are often treated chemically with acids or with hydrogen peroxide in order to keep bacterial contamination low. The capacity of these chemical treatments to inactivate FMD virus in skimmed milk within 6 h at 5 degrees C was tested in this study. The results indicated that the addition of 0.1%-0.3% of consumable acids, such as citric acid or propionic acid, could not guarantee the complete inactivation of FMD virus in skimmed milk. Similar results were obtained both with FMD virus deliberately added to skimmed milk and with skimmed milk obtained from naturally infected cows. Hydrogen peroxide in concentrations of 0.1%-0.3% was also an ineffective means of controlling the risk of FMD virus transmission from contaminated milk.
Assuntos
Aphthovirus/efeitos dos fármacos , Citratos/farmacologia , Peróxido de Hidrogênio/farmacologia , Leite/microbiologia , Propionatos/farmacologia , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Ácido Cítrico , Feminino , Febre Aftosa/microbiologia , Concentração de Íons de Hidrogênio , CinéticaRESUMO
The inactivating effect of methyl glyoxal on foot-and-mouth disease (FMD) virus was studied. The rate of inactivation depended upon the drug concentration, incubation temperature and pH of the medium. RNA recovered from the inactivated virus was not infectious. The complement-fixing activity of the virus was not reduced by methyl glyoxal treatment. The antigenicity of inactivated virus preparations determined by levels of virus neutralizing antibody in the blood sera of immunized white rats and rabbits was not inferior to that of the initial uninactivated virus.
Assuntos
Aldeídos/farmacologia , Aphthovirus/efeitos dos fármacos , Aldeído Pirúvico/farmacologia , Animais , Anticorpos Antivirais/biossíntese , Antígenos Virais , Aphthovirus/imunologia , Aphthovirus/patogenicidade , Testes de Fixação de Complemento , Concentração de Íons de Hidrogênio , Camundongos , RNA Viral , Coelhos , Ratos , TemperaturaRESUMO
Growth pattern of foot-and-mouth disease virus types 0 and Asia 1 in BHK-21 Razi cells was compared; while type 0 virus grew in high titre, Asia 1 virus was produced in low titre. Inhibition of host protein synthesis in type 0 virus-infected cells was more pronounced than in Asia 1 virus-infected cells. Foot-and-mouth disease virus type 0 infected cells showed higher lactic dehydrogenase activity when compared to Asia 1 virus. A significant decrease in virus yield was observed when Actinomycin D had been added at 50 micrograms/ml to infected cells.
Assuntos
Aphthovirus/crescimento & desenvolvimento , Animais , Aphthovirus/efeitos dos fármacos , Aphthovirus/genética , Aphthovirus/metabolismo , Linhagem Celular , Cricetinae , Dactinomicina/farmacologia , L-Lactato Desidrogenase/metabolismo , Biossíntese de Proteínas , RNA Viral/biossíntese , RNA Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
RGD-containing peptides from the immunodominant region of VP1 between amino acids 135-160 from foot-and-mouth disease virus (FMDV) type O1 Kaufbeuren (O1K) prevented virus adsorption to piglet kidney (PK) cells. The highly conserved amino acid RGD sequence (Arg.-Gly.-Asp.) was a prerequisite of this effect. To prevent infection with 100-200 TCID50 in 10(6) PK cells, 20-250 micrograms of each peptide should have been added.
Assuntos
Aphthovirus/efeitos dos fármacos , Capsídeo/farmacologia , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptores Virais/efeitos dos fármacos , Adsorção , Sequência de Aminoácidos , Animais , Aphthovirus/química , Aphthovirus/imunologia , Aphthovirus/fisiologia , Capsídeo/imunologia , Proteínas do Capsídeo , Células Cultivadas , Epitopos Imunodominantes/imunologia , Dados de Sequência Molecular , Oligopeptídeos/imunologia , SuínosRESUMO
Foot and mouth disease virus (FMDV) type 'O' was inactivated either with formaldehyde or binaryethyleneimine (BEI). Vaccines were prepared with inactivated virus incorporating aluminum hydroxide gel or mineral oil as an adjuvant. The antibody response in sheep was monitored by serum neutralization and ELISA test for a period of six months. Significant difference in antibody response was not observed between vaccines inactivated with formaldehyde or BEI. On the other hand significant difference in the antibody response was noticed between alhydrogel and oil vaccines. The high titer of antibodies stimulated by oil adjuvant vaccines persisted longer than those of alhydrogel vaccines within the period of study.
Assuntos
Adjuvantes Imunológicos , Anticorpos Antivirais/biossíntese , Aphthovirus/imunologia , Febre Aftosa/prevenção & controle , Ovinos/imunologia , Vacinas Virais/imunologia , Hidróxido de Alumínio/farmacologia , Animais , Aphthovirus/efeitos dos fármacos , Aziridinas/farmacologia , Bovinos , Cricetinae , Ensaio de Imunoadsorção Enzimática , Feminino , Formaldeído/farmacologia , Cobaias , Lanolina/farmacologia , Masculino , Óleo Mineral/farmacologia , Testes de Neutralização , Coelhos , Fatores de Tempo , Vacinas de Produtos Inativados/imunologiaRESUMO
A set of five neutralizing monoclonal antibodies (MAbs) to an Indian strain (IND17/77) of type A (subtype A22) foot-and-mouth disease (FMD) virus (FMDV) was used in the study. Four of the MAbs (27S, 37S, 85S, and 143S) identified a trypsin-sensitive (TS) epitope(s) and were specific for VP1, while the remaining MAb (145S) reacted with a trypsin-resistant (TR) epitope and was specific for VP3 in Western blot analysis. Both the epitopes (TS and TR) were conformation-independent in nature. Results obtained in MAb-competition enzyme-linked immunosorbent assay (ELISA), and profiling of the (MAb) neutralization-escape mutants in ELISA and cross-neutralization test revealed two overlapping TS epitopes (27S/37S and 85S/143S) on the virus. Variation at both these epitopes was observed in some field isolates of serotype A. Comparison of deduced amino acid sequence in the VP1 region (aa 140-213) between the parent virus and the mutants identified Gly148 and Arg153 as critical for the formation of both the TS epitopes. Substitution of R153 by Gly or Ser was observed in mutants with no reactivity for the MAbs 85S/143S. However, these mutants maintained partial reactivity with MAbs 27S/37S, and substitution of Gly148 by Glu eliminated both the epitopes. No amino acid substitution was observed in the VP1 region of aa 200-213. Efficient neutralization of the MAb neutralization escape mutants (MAb-resistant (MAR) mutants) by bovine vaccinate serum (BVS) indicated involvement of other epitopes on the virion surface in eliciting neutralizing antibodies following vaccination.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Aphthovirus/imunologia , Capsídeo/imunologia , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Especificidade de Anticorpos , Aphthovirus/classificação , Aphthovirus/efeitos dos fármacos , Aphthovirus/genética , Capsídeo/química , Capsídeo/genética , Proteínas do Capsídeo , Bovinos , Resistência Microbiana a Medicamentos , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/imunologia , Febre Aftosa/virologia , Camundongos , Mutação , Testes de Neutralização , Análise de Sequência de DNA , Tripsina/farmacologia , Vírion/imunologia , Ativação ViralRESUMO
The results obtained studying the genetic markers g, t and rtc of different clones of subtypes of foot-and-mouth disease virus type A are presented in this paper. The subtypes were isolated during outbreaks of foot and mouth disease in Argentine. No significative differences among the subtypes were observed with the t marker. For the other markers, the results seem to indicate a gradual change related with the serological variation. Because of their sensibility to guanidine hidrochloride, it is possible to conclude that the studied clones do not belong to the so called European strains.
Assuntos
Aphthovirus/genética , Marcadores Genéticos , Aphthovirus/classificação , Aphthovirus/efeitos dos fármacos , Células Clonais , Meios de Cultura , Efeito Citopatogênico Viral , Resistência Microbiana a Medicamentos , Guanidinas/farmacologia , Temperatura , Replicação ViralRESUMO
A method of negative staining of foot-and-mouth disease virus preparations permits to obtain separately positive (2% phosphotungstic acid solution, pH 3.0) and negative (2% PTA solution, pH 6.8 +/- 8.0) stainings. When a 3--4% PTA solution, pH 6.8 +/- 8.0 is used, simultaneous positive and negative staining of each virion is possible which characterizes the functional heterogeneity of the virion protein membrane in interaction with PTA anions.
Assuntos
Aphthovirus/efeitos dos fármacos , Microscopia Eletrônica/métodos , Ácido Fosfotúngstico/farmacologia , Aphthovirus/ultraestrutura , Soluções , Coloração e Rotulagem/métodosRESUMO
Foot-and-mouth disease virus (FMDV) is a highly pathogenic member of the genus Aphthovirus (family Picornaviridae) that is only to be manipulated in high-containment facilities, thus complicating research on and discovery of antiviral strategies against the virus. Bovine rhinitis B virus (BRBV) and equine rhinitis A virus (ERAV), phylogenetically most closely related to FMDV, were explored as surrogates for FMDV in antiviral studies. Although no efficient cell culture system has been reported so far for BRBV, we demonstrate that infection of primary bovine kidney cells resulted in an extensive but rather poorly-reproducible induction of cytopathic effect (CPE). Madin-Darby bovine kidney cells on the other hand supported viral replication in the absence of CPE. Antiviral tests were developed for ERAV in Vero A cells employing a viral RNA-reduction assay and CPE-reduction assay; the latter having a Z' factor of 0.83±0.07. The BRBV and ERAV models were next used to assess the anti-aphthovirus activity of two broad-spectrum antiviral agents 2'-C-methylcytidine (2CMC) and ribavirin, as well as of the enterovirus-specific inhibitor enviroxime. The effects of the three compounds in the CPE-reduction (ERAV) and viral RNA-reduction assays (BRBV and ERAV) were comparable. Akin to 2CMC, compound A, a recently-discovered non-nucleoside pan-serotype FMDV inhibitor, also inhibited the replication of both BRBV and ERAV, whereas enviroxime was devoid of activity. The BRBV and ERAV surrogate models reported here can be manipulated in BSL-2 laboratories and may facilitate studies to unravel the mechanism of action of novel FMDV inhibitors.
Assuntos
Antivirais/isolamento & purificação , Antivirais/farmacologia , Aphthovirus/efeitos dos fármacos , Descoberta de Drogas/métodos , Animais , Benzimidazóis/farmacologia , Bovinos , Linhagem Celular , Chlorocebus aethiops , Citidina/análogos & derivados , Citidina/farmacologia , Efeito Citopatogênico Viral/efeitos dos fármacos , Febre Aftosa/tratamento farmacológico , Modelos Teóricos , Oximas , RNA Viral/análise , Ribavirina/farmacologia , Sulfonamidas , Cultura de Vírus/métodos , Replicação Viral/efeitos dos fármacosRESUMO
Recombinant equine interferon-gamma (reIFN-gamma) was prepared using a baculovirus expression system and its antiviral activity was investigated using several equine viruses. The reIFN-gamma suppressed the replication of all equine viruses used in the present experiment in horse cell cultures, but did not affect the growth of host cells at concentrations of less than 1000 u/ml. A strong antiviral effect was observed, especially against RNA viruses. Equine picornavirus, equine rhinovirus and equine arteritis virus could not be propagated at all in 100 u/ml reIFN-gamma when 100 TCID(50) of infective viruses was inoculated to cultivated horse cells. DNA viruses, equine herpesvirus types 1, 2, 3 and 4 and equine adenovirus, were less sensitive to reIFN-gamma but their growth became less than 1/100 in the cells treated with 100 u/ml reIFN-gamma compared to untreated cells. The antiviral effects were decreased in the cells of heterologous species and more than 1000 u/ml reIFN-gamma was required to induce an antiviral effect.