ABSTRACT
West Nile virus (WNV) infection in humans can cause neurological deficits, including flaccid paralysis, encephalitis, meningitis, and mental status change. To better understand the neuropathogenesis of WNV in the peripheral and the central nervous systems (PNS and CNS), we used a mouse footpad inoculation model to simulate a natural peripheral infection. Localization of WNV in the nervous system using this model has suggested two routes of viral invasion of the CNS: axonal retrograde transport (ART) from the PNS and hematogenous diffusion via a breakdown in the blood-choroid-plexus barrier. C57BL/6J mice were treated with nocodazole, a microtubule inhibitor that blocks ART, prior to infection with WNV. Nocodazole-treated WNV-infected mice developed a viremia 1.5 log(10) greater than untreated WNV-infected control mice at days 3 to 4 post infection (PI). Although viremia was greater in nocodazole-treated mice, detection of virus in brain tissue (spinal cord, cortex, brainstem, and cerebellum), as measured by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), did not occur until day 7. At these later time points (7 and 9 days PI), nocodazole-treated WNV-infected animals attained viral titers in these tissues similar to titers in the untreated WNV-infected control animals. These results demonstrate that a single dose of nocodazole delays, but does not block, WNV infection of the brain.
Subject(s)
Brain/virology , Nocodazole/pharmacology , Tubulin Modulators/pharmacology , Virus Internalization/drug effects , West Nile Fever/virology , West Nile virus/physiology , Animals , Brain/pathology , Mice , Mice, Inbred C57BL , Polymerase Chain Reaction , RNA, Viral/genetics , Time Factors , Viral Load , West Nile Fever/pathology , West Nile virus/genetics , West Nile virus/isolation & purificationABSTRACT
We have generated a single-chain variable fragment (ScFv) antibody, from a previously well-characterized monoclonal antibody (MAb) to Venezuelan equine encephalitis (VEE) virus, 5B4D-6. The variable regions of the heavy (V(H)) and light (V(L)) chain antibody genes, were connected by a DNA linker and cloned in the phagemid vector pCANTAB5E. The ScFv clone in Escherichia coli strain TG-1, 5B4D-6-6, was expressed as a approximately 30 kDa ScFv protein and higher molecular weight fusion products which were functional in recognizing VEE virus by enzyme-linked immunosorbent assay (ELISA). Results were reproduced in Escherichia coli strain HB2151, where clone D66 was expressed mainly as soluble periplasmic protein. The D66 ScFv antibody bound VEE virus strongly as determined by ELISA. Nucleotide sequence analysis of 5B4D-6-6 ScFv indicated that the Vkappa gene belonged to family XVI, subgroup V, while the V(H) gene was unique in its sequence, though its amino acid sequence could be subgrouped as IA. The deduced protein sequence of D66 was highly homologous to published murine ScFv protein sequences. This work demonstrates, for the first time, cloning of a functional ScFv antibody against VEE virus.
Subject(s)
Antibodies, Monoclonal/genetics , Antibodies, Viral/genetics , Encephalitis Virus, Venezuelan Equine/immunology , Immunoglobulin Variable Region/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Gene Expression , Genes, Immunoglobulin , Hybridomas/immunology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Mice , Molecular Sequence Data , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Viral Envelope Proteins/immunologyABSTRACT
This report describes the clinical, laboratory, and epidemiological findings on 27 cases of Mayaro virus (MV) disease, an emerging mosquito-borne viral illness that is endemic in rural areas of tropical South America. MV disease is a nonfatal, dengue-like illness characterized by fever, chills, headache, eye pain, generalized myalgia, arthralgia, diarrhea, vomiting, and rash of 3-5 days' duration. Severe joint pain is a prominent feature of this illness; the arthralgia sometimes persists for months and can be quite incapacitating. Cases of two visitors from the United States, who developed MV disease during visits to eastern Peru, are reported. MV disease and dengue are difficult to differentiate clinically.
Subject(s)
Alphavirus Infections/diagnosis , Alphavirus/isolation & purification , Adult , Age Distribution , Alphavirus/classification , Alphavirus/genetics , Alphavirus/immunology , Alphavirus Infections/epidemiology , Alphavirus Infections/virology , Animals , Antibodies, Viral/blood , Culicidae , DNA, Viral/analysis , Female , Humans , Insect Vectors , Middle Aged , Peru/epidemiology , Seasons , Sequence Analysis, DNA , ZoonosesABSTRACT
We compared the alpha/beta interferon (IFN-alpha/beta) sensitivities of the TC-83 vaccine strain and 24 enzootic and epizootic Venezuelan equine encephalitis (VEE) isolates. The IFN-resistant or -sensitive phenotype correlated well with epizootic or enzootic potential. IFN-alpha/beta resistance of Trinidad donkey (TRD) virus correlated with virulence determinants in the 5' noncoding region and glycoproteins. Infection of mice lacking a functional IFN system with the IFN-sensitive TC-83 virus resulted in disease equivalent to that produced by the virulent, IFN-resistant TRD virus, further demonstrating that IFN resistance contributes to VEE virus virulence and is a biological marker of epizootic potential.
Subject(s)
Encephalitis Virus, Venezuelan Equine/pathogenicity , Encephalomyelitis, Venezuelan Equine/veterinary , Horse Diseases/virology , Interferon Type I/pharmacology , Animals , Cell Line , Cytopathogenic Effect, Viral , Drug Resistance, Microbial/genetics , Encephalitis Virus, Venezuelan Equine/genetics , Encephalitis Virus, Venezuelan Equine/isolation & purification , Encephalomyelitis, Venezuelan Equine/virology , Glycoproteins/genetics , Horses , Humans , Mice , Virulence/genetics , Zoonoses/virologyABSTRACT
Although dengue (DEN) virus is the etiologic agent of dengue fever, the most prevalent vector-borne viral disease in the world, precise information on the antigenic structure of the dengue virion is limited. We have prepared a set of murine monoclonal antibodies (MAbs) specific for the envelope (E) glycoprotein of DEN 2 virus and used these antibodies in a comprehensive biological and biochemical analysis to identify 16 epitopes. Following domain nomenclature developed for the related flavivirus, tick-borne encephalitis, three functional domains were identified. Five epitopes associated with domain A were arranged in three spatially independent regions. These A-domain epitopes were destroyed by reduction, and antibodies reactive with these epitopes were able to block virus hemagglutination, neutralize virus infectivity, and block virus-mediated cell membrane fusion. Domain-A epitopes were present on the full-length E glycoprotein, a 45-kDa tryptic peptide representing its first 400 amino acids (aa) and a 22-kDa tryptic peptide representing at least aa 1-120. Four epitopes mapped into domain B, as determined by their partial resistance to reduction and the localization of these epitopes on a 9-kDa tryptic or chymotryptic peptide fragment (aa 300-400). One domain-B-reactive MAb was also capable of binding to a DEN 2 synthetic peptide corresponding to aa 333-351 of the E glycoprotein, confirming the location of this domain. Domain-B epitopes elicited MAbs that were potent neutralizers of virus infectivity and blocked hemagglutination, but they did not block virus-mediated cell-membrane fusion. Domains A and B were spatially associated. As with tick-borne encephalitis virus, determination of domain C was more problematic; however, at least four epitopes had biochemical characteristics consistent with C-domain epitopes.
Subject(s)
Antibodies, Viral/immunology , Antigens, Viral/immunology , Dengue Virus/immunology , Epitope Mapping , Epitopes, B-Lymphocyte/immunology , Viral Envelope Proteins/immunology , Animals , Antibodies, Monoclonal/immunology , Antigens, Viral/chemistry , Binding Sites , Binding, Competitive , Cell Line , Epitopes, B-Lymphocyte/chemistry , Hemagglutination Inhibition Tests , Humans , Jamaica , Male , Membrane Fusion , Mice , Mice, Inbred BALB C , Models, Molecular , Neutralization Tests , Peptide Fragments/chemical synthesis , Peptide Fragments/immunology , Peptide Mapping , Protein Conformation , Structure-Activity Relationship , Viral Envelope Proteins/chemistryABSTRACT
Although dengue (DEN) virus is the etiologic agent of dengue fever, the most prevalent vector-borne viral disease in the world, precise information on the antigenic structure of the dengue virion is limited. We have prepared a set of murine monoclonal antibodies (Mabs) specific for the envelope (E) glycoprotein of DEN 2 virus and used these antibodies in a comprehensive biological and biochemical analysis to identify 16 epitopes. Following domain nomenclature developed for the related flavivirus, tick-bourne encephalitis, three functional domains were identified. Five epitopes associated with domain A were arranged in three spatially independently regions. These A-domain epitopes were destroyed by reduction, and antibodies reactive with these epitopes were able to block virus hemagglutination, neutralize virus infectivity, and block virus haemagglutination, neutralize virus infectivity, and block virus-mediated cell membrane fusion. Domain-A epitopes were present on the full-length E glycoprotein, a 45-kDa tryptic peptide representing its first 400 amino acids (aa) and a 22-kDA tryptic peptide representing at least aa 1-120. Four epitopes mapped into domain B, as determined by their partial resistance to reduction and the localization of these epitopes on a 9-kDa tryptic or chymotryptic peptide fragment (aa 300-400). One domain-B-reactive MAb was also capable of binding to a DEN 2 synthetic peptide corresponding to aa 333-351 of the E glycoprotein, confirming the location of this domain. Domain-B epitopes elicited MAbs that were potent neutralizers of virus infectivity and blocked hemagglutination, but they did not block virus-mediated cell-membrane fusion. Domains A and B were spatially associated. As with tick-bourne encephalitis virus, determination of domain C was more problematic: however, at least four epitopes and biochemical characteristics consistent with C-domain epitopes(AU)
Subject(s)
21003 , Humans , Antibodies, Viral/immunology , Antigens, Viral/immunology , Dengue Virus/immunology , Epitope Mapping , Viral Envelope Proteins/immunology , Hemagglutination Inhibition Tests , Jamaica , Membrane Fusion , Mice , Mice, Inbred BALB C , Models, Molecular , Neutralization Tests , Peptide Fragments/chemical synthesis , Peptide Fragments/immunology , Peptide Mapping , Protein Conformation , Structure-Activity Relationship , Viral Envelope Proteins/chemistry , Antibodies, Monoclonal/immunology , Antigens, Viral/chemistry , Binding Sites , Binding, Competitive , Cell LineABSTRACT
A survey was conducted from October 1, 1993 to June 30, 1995 to determine the arboviral etiologies of febrile illnesses in the city of Iquitos in the Amazon River Basin of Peru. The study subjects were patients who were enrolled at medical care clinics or in their homes by Peruvian Ministry of Health (MOH) workers as part of the passive and active disease surveillance program of the MOH. The clinical criterion for enrollment was the diagnosis of a suspected viral-associated, acute, undifferentiated febrile illness of < or = 5 days duration. A total of 598 patients were enrolled in the study. Demographic information, medical history, clinical data, and blood samples were obtained from each patient. The more common clinical features were fever, headache, myalgia, arthralgia, retro-ocular pain, and chills. Sera were tested for virus by the newborn mouse and cell culture assays. Viral isolates were identified initially by immunofluorescence using polyclonal antibody. An ELISA using viral-specific monoclonal antibodies and nucleotide sequence analysis were used to determine the specific variety of the viruses. In addition, thin and thick blood smears were observed for malaria parasites. Venezuelan equine encephalitis (VEE) virus subtype I, variety ID virus was isolated from 10 cases, including three cases in October, November, and December 1993, five cases in January and February 1994, and two cases in June 1995. The ELISA for IgM and IgG antibody indicated that VEE virus was the cause of an additional four confirmed and four presumptive cases, including five from January through March 1994 and three in August 1994. Sixteen cases were positive for malaria. The 18 cases of VEE occurred among military recruits (n = 7), agriculture workers (n = 3), students (n = 3), and general laborers (n = 5). These data indicated that an enzootic strain of VEE virus was the cause of at least 3% (18 of 598) of the cases of febrile illnesses studied in the city of Iquitos in the Amazon Basin region of Peru.
Subject(s)
Encephalomyelitis, Venezuelan Equine/diagnosis , Encephalomyelitis, Venezuelan Equine/epidemiology , Adolescent , Adult , Aged , Ambulatory Care Facilities , Antibodies, Viral/analysis , Cells, Cultured , Child , Child, Preschool , Encephalitis Virus, Venezuelan Equine/classification , Encephalitis Virus, Venezuelan Equine/genetics , Encephalitis Virus, Venezuelan Equine/immunology , Encephalomyelitis, Venezuelan Equine/blood , Enzyme-Linked Immunosorbent Assay , Epitope Mapping , Female , Fluorescent Antibody Technique, Indirect , Humans , Infant , Malaria/diagnosis , Male , Middle Aged , Molecular Epidemiology , Peru/epidemiology , Phylogeny , Polymerase Chain Reaction , Population Surveillance , RNA, Viral/analysis , RNA, Viral/genetics , Seroepidemiologic Studies , SerotypingABSTRACT
Genetic relationships among viruses defining the Venezuelan equine encephalitis (VEE) virus antigenic complex were determined by analyzing the 3'-terminal 561 nucleotides of the nonstructural protein 4 gene and the entire 26S RNA region of the genome. New sequence information is reported for VEE 78V-3531 (VEE subtype-variety IF), Mucambo (IIIA), Tonate (IIIB), 71D-1252 (IIIC), Pixuna (IV), Cabassou (V), and AG80-663 (VI) viruses. The results reported here and by previous investigators largely support the current classification scheme of these viruses, while clearly identifying Everglades (II) as a subtype I virus. A genetic relationship between 78V-3531 (IF) and AG80-663 (VI) viruses contradicted previous serologic results. Mutations near the amino terminus of the E2 envelope proteins of Pixuna and AG80-663 viruses probably account for the previously reported low reactivity of the protective monoclonal antibody 1A2B-10 with these two viruses. Variations in the distribution of potential glycosylation sites in the E2 glycoprotein are discussed.
Subject(s)
Antigens, Viral/genetics , Encephalitis Virus, Venezuelan Equine/immunology , RNA, Messenger/chemistry , RNA, Viral/chemistry , Amino Acid Sequence , Base Sequence , Capsid/chemistry , Capsid/genetics , DNA, Complementary/chemistry , Encephalitis Virus, Venezuelan Equine/classification , Encephalitis Virus, Venezuelan Equine/genetics , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/geneticsABSTRACT
We used previously characterized murine monoclonal antibodies to develop a panel useful in subtyping Venezuelan equine encephalitis (VEE) viruses by an indirect fluorescent antibody assay. This panel worked well with either prototype VEE viruses or a series of more recent VEE virus isolates. The panel is particularly useful for rapidly differentiating VEE viruses with epidemic-epizootic potential from other endemic varieties of this virus. Using this panel, we identified an antigenic variant of prototype VEE subtype 1E virus currently present in Mexico. This antigenic change in the E2 glycoprotein was confirmed by enzyme-linked immunosorbent assay. Because VEE virus virulence has been associated in part with the E2 glycoprotein, this observed antigenic change in the 1E virus E2 glycoprotein may explain the apparent equine virulence of this unusual VEE 1E virus.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Encephalitis Virus, Venezuelan Equine/isolation & purification , Encephalomyelitis, Venezuelan Equine/virology , Fluorescent Antibody Technique, Indirect/methods , Animals , Encephalitis Virus, Venezuelan Equine/immunology , Horses , HumansABSTRACT
An outbreak of a febrile illness characterized by headache, ocular pain, myalgia, and arthralgia occurred during June 1994 among Peruvian army troops in Northern Peru. On June 14-16, 1994, clinical data and blood samples were obtained from eight soldiers with a febrile illness, and from 26 others who had a history of febrile illness during the past three months. A follow-up blood sample was obtained 107 days later from four of the febrile and seven of the afebrile soldiers. Serum samples were tested for dengue (DEN), Oropouche (ORO), and Venezuelan equine encephalitis (VEE) IgM and IgG antibodies by an enzyme-linked immunosorbent assay (ELISA). Virus isolation was performed by inoculation of newborn mice and Vero cell cultures. Viral isolates were identified by immunofluorescence, ELISA, and nucleotide sequencing. A VEE virus infection was confirmed in three of the eight febrile soldiers, two by virus isolation, and one by serology. Antigenic analysis indicated that one of the virus isolates was similar to VEE subtype I, variety ID, viruses previously isolated in Colombia and Venezuela. Nucleotide sequence data showed that both viral isolates were identical to one another and closely related to VEE ID viruses previously isolated in Peru, Colombia, and Venezuela. Serologic results showed that two of 26 afebrile soldiers had IgM antibody to VEE and four had IgG antibody to VEE; two febrile soldiers had IgG antibody in their first serum samples. Oropouche-specific IgM antibody was detected in one of the eight febrile and five of the afebrile soldiers, and 18 of the 34 soldiers had low titers of ORO IgG antibody titers, which did not meet the diagnostic criteria for confirmed cases. All soldiers were negative for DEN IgM antibody, and 10 had flavivirus IgG antibody that reacted with DEN antigens. These data indicated that VEE ID virus was one of the causes of illness among Peruvians soldiers and that this was the first association of this VEE subtype with human disease in Peru.
Subject(s)
Bunyaviridae Infections/epidemiology , Disease Outbreaks , Encephalomyelitis, Venezuelan Equine/epidemiology , Adolescent , Adult , Animals , Antibodies, Viral/blood , Bunyaviridae Infections/virology , Encephalitis Virus, Venezuelan Equine/immunology , Encephalitis Virus, Venezuelan Equine/isolation & purification , Encephalomyelitis, Venezuelan Equine/virology , Humans , Immunoglobulin G/blood , Immunoglobulin M/blood , Male , Mice , Orthobunyavirus , Peru/epidemiology , Seroepidemiologic Studies , Simbu virus/immunology , Simbu virus/isolation & purificationABSTRACT
In order to define more precisely the protective epitope encoded within the first 25 amino acids (aa) of the E2 glycoprotein of the Trinidad donkey strain of Venezuelan equine encephalomyelitis (VEE) virus, we examined the immunogenicity of smaller peptides within the first 19 aa. pep1-9 and pep3-10 elicited virus-reactive antibody, but failed to protect mice from virus challenge. Additionally, pep3-10 was identified by a competitive binding assay using overlapping peptide octamers as the putative binding site of the antipeptide monoclonal antibody (mAb) 1A2B-10. Since the E2 amino-terminal sequence for all VEE subtype viruses is conserved, we tested the protective capacity in mice of passively transferred mAb 1A2B-10 and found it to protect from both epizootic and enzootic VEE virus challenge. Since horses are an important natural host for VEE virus, pep1-19 was used to immunize horses and was found to be immunogenic and to elicit virus-reactive antibody.
Subject(s)
Encephalitis Virus, Venezuelan Equine/immunology , Encephalomyelitis, Venezuelan Equine/immunology , Epitopes/analysis , Horse Diseases/immunology , Viral Envelope Proteins/immunology , Viral Vaccines/immunology , Animals , Antibodies, Monoclonal/therapeutic use , Antibodies, Viral/blood , Antigens, Viral/immunology , Encephalomyelitis, Venezuelan Equine/prevention & control , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Female , Horse Diseases/prevention & control , Horses , Immunization, Passive , Mice , Peptide Fragments/immunology , Peptide Fragments/therapeutic use , Viral Envelope Proteins/pharmacologyABSTRACT
The T-helper (Th) cell immune response following immunization of C3H (H-2k) mice with a recombinant vaccinia (VAC) virus (TC-5A) expressing the structural proteins (capsid, E1 and E2) of the attenuated vaccine strain (TC-83) of Venezuelan equine encephalitis (VEE) virus was compared with the immune response induced in mice after immunization with TC-83 virus. TC-5A virus elicited Th cells that strongly recognized both VAC and TC-83 viruses in in vitro lymphoblastogenesis tests. Th-cell activation was associated with elevated levels of interleukin-2. TC-5A virus induced long-term humoral immunity; VEE virus-binding and neutralizing antibodies were detected in mouse sera collected from mice 16 months after a single immunization.
Subject(s)
Encephalitis Virus, Venezuelan Equine/immunology , Sindbis Virus/immunology , T-Lymphocytes, Helper-Inducer/immunology , Vaccines, Synthetic/immunology , Vaccinia virus/immunology , Viral Vaccines/immunology , Animals , Antibody Formation , Antigen-Antibody Reactions , Immunization , Immunophenotyping , Interleukin-2/metabolism , Male , Mice , Mice, Inbred C3H , Vaccines, Attenuated/immunologyABSTRACT
To identify T-helper (Th)-cell epitopes, we analyzed 25 synthetic peptides, which included most of the 495-amino-acid sequence of the envelope (E)-glycoprotein of dengue 2 virus. The peptides were analyzed in three mouse strains, BALB/c (H-2d), C57BL/6 (H-2b), and outbred NIH-Swiss, for their ability to elicit antibody or prime the Th-cell compartment following two inoculations in Freund's incomplete adjuvant. Sixteen peptides were able to elicit an antipeptide antibody response in one or more mouse strain. Eleven antipeptide serum pools were able to bind to virus in ELISA. Fifteen peptides primed one or more haplotype for an in vitro antipeptide Th-cell response as measured by blastogenesis. Th-cell activation was generally confirmed by measurable in vitro production of interleukin (IL)-2/IL-4. Nine peptides that were positive for in vitro blastogenesis, 1-2, 35, 4-6, 79, 142, 208, 06, 16, and 17, elicited virus-reactive Th-cells in vitro in H-2d mice. Two of these peptides (4-6 and 17) were able to prime virus-reactive Th-cells in H-2b mice. Nine peptides primed outbred mice in vitro for an antiviral antibody response significantly greater than that seen in animals primed with an irrelevant peptide. These results correlate with, and expand on, our previous observations based on a smaller set of synthetic peptides derived from the E-glycoprotein of Murray Valley encephalitis virus and suggest that synthetic peptides can function as E-glycoprotein Th-cell epitopes. The similarity of results between two distantly related flaviviruses suggests that E-glycoprotein Th-cell epitopes are consistent in location and activity.
Subject(s)
Antigens, Viral/immunology , Dengue Virus/immunology , Epitopes/immunology , T-Lymphocytes, Helper-Inducer/immunology , Viral Envelope Proteins/immunology , Animals , Antibodies, Viral/biosynthesis , Antibodies, Viral/blood , Antigens, Viral/blood , Glycoproteins/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Peptide Fragments/immunology , Viral Envelope Proteins/bloodABSTRACT
The virulent Trinidad donkey (TRD) strain of Venezuelan equine encephalitis (VEE) virus and its live attenuated vaccine derivative, TC-83 virus, have different neurovirulence characteristics. A full-length cDNA clone of the TC-83 virus genome was constructed behind the bacteriophage T7 promoter in the polylinker of plasmid pUC18. To identify the genomic determinants of TC-83 virus attenuation, TRD virus-specific sequences were inserted into the TC-83 virus clone by in vitro mutagenesis or recombination. Antigenic analysis of recombinant viruses with VEE E2- and E1-specific monoclonal antibodies gave predicted antigenic reactivities. Mouse challenge experiments indicated that genetic markers responsible for the attenuated phenotype of TC-83 virus are composed of genome nucleotide position 3 in the 5'-noncoding region and the E2 envelope glycoprotein. TC-83 virus amino acid position E2-120 appeared to be the major structural determinant of attenuation. Insertion of the TRD virus-specific 5'-noncoding region, by itself, into the TC-83 virus full-length clone did not alter the attenuated phenotype of the virus. However, the TRD virus-specific 5'-noncoding region enhanced the virulence potential of downstream TRD virus amino acid sequences.
Subject(s)
Antigens, Viral/immunology , Encephalitis Virus, Venezuelan Equine/immunology , Encephalomyelitis, Venezuelan Equine/prevention & control , Regulatory Sequences, Nucleic Acid/genetics , Vaccines, Attenuated , Viral Envelope Proteins/immunology , Animals , Antibody Formation , Bacteriophage T7/genetics , Base Sequence , Cloning, Molecular , Encephalitis Virus, Venezuelan Equine/genetics , Encephalitis Virus, Venezuelan Equine/pathogenicity , Encephalomyelitis, Venezuelan Equine/immunology , Epitopes , Genome, Viral , Male , Mice , Mice, Inbred ICR , Molecular Sequence Data , Point Mutation , Survival Analysis , Vero Cells , Viral Envelope Proteins/genetics , Viral Plaque Assay , VirulenceABSTRACT
To complete our analysis of the E2 glycoprotein of Venezuelan equine encephalomyelitis (VEE) virus, we prepared six synthetic peptides corresponding to the extramembranal carboxy-terminal one-third of the protein. NIH-Swiss mice were immunized with the peptides, and antipeptide and antiviral titers were determined by enzyme-linked immunosorbent assay (ELISA). Challenge studies revealed that peptide 13 (amino acids 241-265) protected 60-70% of virus-challenged mice. Although the other peptides generally elicited antipeptide ELISA titers but no or low antiviral titers and did not protect mice, significant E2 reactivity was found in immunoblots. These results provide the first direct evidence that much of the E2 carboxy-terminal domain is cryptic in the VEE virion, even when virus was bound to polystyrene ELISA plates.
Subject(s)
Encephalitis Virus, Venezuelan Equine/immunology , Encephalomyelitis, Venezuelan Equine/prevention & control , Peptide Fragments/immunology , Viral Envelope Proteins/immunology , Viral Vaccines/immunology , Animals , Antibodies, Viral/blood , Enzyme-Linked Immunosorbent Assay , Female , Immunoblotting , Mice , Vaccines, Synthetic/immunology , Viral Envelope Proteins/chemistryABSTRACT
A peptide composed of the amino-terminal 25 amino acids of the E2 glycoprotein of the virulent Trinidad donkey (TRD) strain of Venezuelan equine encephalomyelitis virus was found to protect peptide-immunized mice from lethal TRD virus challenge (Hunt et al., 1990). Viral growth in peptide-immunized animals was found to be limited in comparison to that in nonimmunized controls. Although both treated and control groups of mice responded to virus challenge by producing neutralizing antibody, only immunized mice with preexisting antipeptide antibody survived. Polyclonal antipeptide sera as well as a monoclonal antipeptide antibody were able to passively protect naive mice from TRD virus challenge, despite the fact that these antibodies were nonneutralizing. Passive transfer of antipeptide antibody to immunosuppressed recipients was not protective, thus indicating that survival of TRD virus challenge required an in situ immune response as well as preexisting antipeptide antibody. Binding studies of both polyclonal and monoclonal antipeptide antibodies indicated that they recognize only epitopes present on virus-infected cells or denatured virus.
Subject(s)
Antibodies, Viral , Encephalitis Virus, Venezuelan Equine/physiology , Encephalomyelitis, Venezuelan Equine/immunology , Peptides/chemical synthesis , Virus Replication , Animals , Antibody Formation , Antigen-Antibody Complex/analysis , Cell Line , Encephalitis Virus, Venezuelan Equine/immunology , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Female , Fluorescent Antibody Technique , Hemagglutination Inhibition Tests , Horses , Immunization, Passive , Immunoglobulin G/analysis , Immunoglobulin M/analysis , Mice , Mice, Inbred Strains , Peptides/immunologyABSTRACT
Four monoclonal antibody-resistant variants (MARVs) of Venezuelan equine encephalitis (VEE) virus were used to study mosquito-virus interactions. In vitro experiments using an Aedes albopictus cell line, C6/36, demonstrated that an amino acid change in the glycoprotein E2h epitope (MARV 1A3B-7) decreased virus growth when compared with the wild-type, Trinidad donkey virus, and its vaccine derivative, TC-83. The MARVs replicated as efficiently as the parent virus when inoculated into Aedes aegypti mosquitoes, but MARV 1A3B-7 was restricted in its ability to infect and disseminate from the midgut following oral infection. These results demonstrate that a single amino acid change in the E2 glycoprotein can affect the ability of VEE virus to replicate and disseminate in Ae. aegypti mosquitoes.
Subject(s)
Encephalitis Virus, Venezuelan Equine/genetics , Viral Envelope Proteins/genetics , Virus Replication , Aedes/microbiology , Animals , Antibodies, Monoclonal/immunology , Cell Line , Cricetinae , Encephalitis Virus, Venezuelan Equine/chemistry , Encephalitis Virus, Venezuelan Equine/physiology , Insect Vectors/microbiology , Kinetics , Vero Cells , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/immunologyABSTRACT
We have prepared a murine monoclonal antibody (MAb) capable of distinguishing between wild-type Venezuelan equine encephalomyelitis (VEE) virus and the TC-83 vaccine derivative. This MAb, 1A2B-10, was derived from immunization with a synthetic peptide corresponding to the first 19 amino acids of the E2 glycoprotein of Trinidad donkey VEE virus. The MAb reacts with prototype viruses from all naturally occurring VEE subtypes except subtype 6 in an enzyme-linked immunosorbent assay. It does not react with TC-83 virus or members of the western and eastern equine encephalitis virus complex or with Semliki Forest virus. This antibody will also differentiate between TC-83 and Trinidad donkey VEE virus in indirect immunofluorescence assays with virus-infected Vero cells.
Subject(s)
Antibodies, Monoclonal , Encephalitis Virus, Venezuelan Equine/immunology , Viral Vaccines/immunology , Amino Acid Sequence , Animals , Antibody Specificity , Encephalitis Virus, Venezuelan Equine/classification , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/chemistry , Peptides/immunologyABSTRACT
Fourteen peptides representing 67% of the extramembranal domain of the Venezuelan equine encephalomyelititis (VEE) virus E2 glycoprotein were synthesized and analyzed to determine their antigenic, immunogenic, and protective capacities. Thirteen of 14 peptides elicited antibody for the homologous peptide. Thirteen peptides elicited antiviral antibody that recognized either the Trinidad (TRD) strain of VEE virus or the TC-83 vaccine derivative, or both. Two peptides, VE2pep01(TC-83) and VE2pep01(TRD), protected significant numbers of mice from TRD virus challenge. The majority of the peptides were reactive with antisera from mice immunized with the various subtypes of VEE virus. A competition assay using antipeptide antibodies to block virus binding of anti-VEE virus monoclonal antibodies corroborated previous studies on the spatial relationship of E2 epitopes and provided evidence for a spatial overlap of the E2 amino terminus with a domain composed of residues 180-210.
Subject(s)
Antigens, Viral/immunology , Bacteriophage mu/genetics , Encephalitis Virus, Venezuelan Equine/immunology , Viral Envelope Proteins/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Binding, Competitive , Encephalomyelitis, Venezuelan Equine/immunology , Encephalomyelitis, Venezuelan Equine/prevention & control , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/immunology , Viral Vaccines/immunologyABSTRACT
Stable neutralization (N) escape variants of Venezuelan equine encephalitis (VEE) virus were selected by anti-E2 glycoprotein monoclonal antibodies (MAbs) that neutralize viral infectivity, block viral hemagglutination, and passively protect mice. The nucleotide sequence of the E1, E2, and E3 genes of four variants revealed a clustering of single mutations in a domain spanning E2-182 to E2-207. The conformation of this short linear sequence affects antigenicity in the N domain because reduction and alkylation of virus disrupted binding of some E2 neutralizing MAbs. Serologic evidence for interaction of E2 epitopes also was obtained. Mutations in the N domain of VEE virus did not alter the kinetics of binding to Vero cells. They did, in some cases, produce attenuation of virulence in mice.