RESUMO
Venezuelan equine encephalitis (VEE) virus antigenic subtypes and varieties are considered either epidemic/epizootic or enzootic. In addition to epidemiological differences between the epidemic and enzootic viruses, several in vitro and in vivo laboratory markers distinguishing the viruses have been identified, including differential plaque size, sensitivity to interferon (IFN), and virulence for guinea pigs. These observations have been shown to be useful predictors of natural, equine virulence and epizootic potential. Chimeric viruses containing variety IAB (epizootic) nonstructural genes with variety IE (enzootic) structural genes (VE/IAB-IE) or IE nonstructural genes and IAB structural genes (IE/IAB) were constructed to systematically analyze and map viral phenotype and virulence determinants. Plaque size analysis showed that both chimeric viruses produced a mean plaque diameter that was intermediate between those of the parental strains. Additionally, both chimeric viruses showed intermediate levels of virus replication and virulence for guinea pigs compared to the parental strains. However, IE/IAB produced a slightly higher viremia and an average survival time 2 days shorter than the VE/IAB-IE virus. Finally, IFN sensitivity assays revealed that only one chimera, VE/IAB-IE, was intermediate between the two parental types. The second chimera, containing the IE nonstructural genes, was at least five times more sensitive to IFN than the IE parental virus and greater than 50 times more sensitive than the IAB parent. These results implicate viral components in both the structural and nonstructural portions of the genome in contributing to the epizootic phenotype and indicate the potential for epidemic emergence from the IE enzootic VEE viruses.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/patogenicidade , Animais , Antivirais/farmacologia , Linhagem Celular , Cricetinae , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Vírus da Encefalite Equina Venezuelana/crescimento & desenvolvimento , Cobaias , Interferon-alfa/farmacologia , Interferon beta/farmacologia , Recombinação Genética , Ensaio de Placa Viral , VirulênciaRESUMO
The 5' nontranslated region (5'NTR) and nonstructural region nucleotide sequences of nine enzootic Venezuelan equine encephalitis (VEE) virus strains were determined, thus completing the genomic RNA sequences of all prototype strains. The full-length genomes, representing VEE virus antigenic subtypes I-VI, range in size from 11.3 to 11.5 kilobases, with 48-53% overall G+C contents. Size disparities result from subtype-related differences in the number and length of direct repeats in the C-terminal nonstructural protein 3 (nsP3) domain coding sequence and the 3'NTR, while G+C content disparities are attributable to strain-specific variations in base composition at the wobble position of the polyprotein codons. Highly-conserved protein components and one nonconserved protein domain constitute the VEE virus replicase polyproteins. Approximately 80% of deduced nsP1 and nsP4 amino acid residues are invariant, compared to less than 20% of C-terminal nsP3 domain residues. In two enzootic strains, C-terminal nsP3 domain sequences degenerate into little more than repetitive serine-rich blocks. Nonstructural region sequence information drawn from a cross-section of VEE virus subtypes clarifies features of alphavirus conserved sequence elements and proteinase recognition signals. As well, whole-genome comparative analysis supports the reclassification of VEE subtype-variety IF and subtype II viruses.
Assuntos
Regiões 5' não Traduzidas/genética , Antígenos Virais/genética , Vírus da Encefalite Equina Venezuelana/genética , RNA Viral/genética , Sequência de Aminoácidos , Animais , Antígenos Virais/química , Sequência de Bases , Sequência Conservada , Equidae , Variação Genética , Genoma Viral , Cavalos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Epizootics of Venezuelan equine encephalitis (VEE) involving subtype IAB viruses occurred sporadically in South, Central and North America from 1938 to 1973. Incompletely inactivated vaccines have long been suspected as a source of the later epizootics. We tested this hypothesis by sequencing the PE2 glycoprotein precursor (1,677 nucleotides) or 26S/nonstructural protein 4 (nsP4) genome regions (4,490 nucleotides) for isolates representing most major outbreaks. Two distinct IAB genotypes were identified: 1) 1940s Peruvian strains and 2) 1938-1973 isolates from South, Central, and North America. Nucleotide sequences of these two genotypes differed by 1.1%, while the latter group showed only 0.6% sequence diversity. Early VEE virus IAB strains that were used for inactivated vaccine preparation had sequences identical to those predicted by phylogenetic analyses to be ancestors of the 1960s-1970s outbreaks. These data support the hypothesis of a vaccine origin for many VEE outbreaks. However, continuous, cryptic circulation of IAB viruses cannot be ruled out as a source of epizootic emergence.
Assuntos
Surtos de Doenças , Vírus da Encefalite Equina Venezuelana/genética , Encefalomielite Equina/epidemiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , América Central/epidemiologia , Primers do DNA/química , Vírus da Encefalite Equina Venezuelana/classificação , Encefalomielite Equina/virologia , Dados de Sequência Molecular , América do Norte/epidemiologia , Filogenia , RNA Viral/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , América do Sul/epidemiologia , Vacinas Virais/efeitos adversosRESUMO
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.
Assuntos
Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/veterinária , Doenças dos Cavalos/virologia , Interferon Tipo I/farmacologia , Animais , Linhagem Celular , Efeito Citopatogênico Viral , Resistência Microbiana a Medicamentos/genética , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/isolamento & purificação , Encefalomielite Equina Venezuelana/virologia , Glicoproteínas/genética , Cavalos , Humanos , Camundongos , Virulência/genética , Zoonoses/virologiaRESUMO
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.
Assuntos
Antígenos Virais/genética , Vírus da Encefalite Equina Venezuelana/imunologia , RNA Mensageiro/química , RNA Viral/química , Sequência de Aminoácidos , Sequência de Bases , Capsídeo/química , Capsídeo/genética , DNA Complementar/química , Vírus da Encefalite Equina Venezuelana/classificação , Vírus da Encefalite Equina Venezuelana/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
The evolution of yellow fever virus over 67 years was investigated by comparing the nucleotide sequences of the envelope (E) protein genes of 20 viruses isolated in Africa, the Caribbean, and South America. Uniformly weighted parsimony algorithm analysis defined two major evolutionary yellow fever virus lineages designated E genotypes I and II. E genotype I contained viruses isolated from East and Central Africa. E genotype II viruses were divided into two sublineages: IIA viruses from West Africa and IIB viruses from America, except for a 1979 virus isolated from Trinidad (TRINID79A). Unique signature patterns were identified at 111 nucleotide and 12 amino acid positions within the yellow fever virus E gene by signature pattern analysis. Yellow fever viruses from East and Central Africa contained unique signatures at 60 nucleotide and five amino acid positions, those from West Africa contained unique signatures at 25 nucleotide and two amino acid positions, and viruses from America contained such signatures at 30 nucleotide and five amino acid positions in the E gene. The dissemination of yellow fever viruses from Africa to the Americas is supported by the close genetic relatedness of genotype IIA and IIB viruses and genetic evidence of a possible second introduction of yellow fever virus from West Africa, as illustrated by the TRINID79A virus isolate. The E protein genes of American IIB yellow fever viruses had higher frequencies of amino acid substitutions than did genes of yellow fever viruses of genotypes I and IIA on the basis of comparisons with a consensus amino acid sequence for the yellow fever E gene. The great variation in the E proteins of American yellow fever virus probably results from positive selection imposed by virus interaction with different species of mosquitoes or nonhuman primates in the Americas.
Assuntos
Evolução Biológica , Produtos do Gene env/genética , Genes env , Variação Genética , Febre Amarela/virologia , Vírus da Febre Amarela/genética , Aedes/virologia , África , Algoritmos , Sequência de Aminoácidos , Animais , Sequência de Bases , Região do Caribe , Sequência Consenso , Primers do DNA , Produtos do Gene env/química , Genótipo , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Primatas/virologia , RNA Viral/genética , Homologia de Sequência de Aminoácidos , América do Sul , Vírus da Febre Amarela/classificação , Vírus da Febre Amarela/isolamento & purificaçãoRESUMO
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.
Assuntos
Vírus da Encefalite Equina Venezuelana/imunologia , Sindbis virus/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas Sintéticas/imunologia , Vaccinia virus/imunologia , Vacinas Virais/imunologia , Animais , Formação de Anticorpos , Reações Antígeno-Anticorpo , Imunização , Imunofenotipagem , Interleucina-2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Vacinas Atenuadas/imunologiaRESUMO
Enzootic strains of Venezuelan equine encephalitis (VEE) virus occur in the United States (Florida), Mexico, Central America and South America. Epizootic VEE first occurred in North and Central America in a widespread outbreak between 1969 and 1972. To investigate the likelihood that this epizootic VEE virus, identified as VEE antigenic subtype I-AB, evolved from enzootic viruses extant in the region, we cloned and sequenced the 26S mRNA region of the genomes of the Florida VEE subtype II virus, strain Everglades Fe3-7c, and the Middle American subtype I-E virus, strain Mena II. This region of the genome encodes the viral structural proteins. The sequences of the 26S mRNA regions of the Everglades and Mena virus genomes differed from that of the reference epizootic VEE subtype I-AB virus, Trinidad donkey strain, by 453 and 887 nucleotides and by 66 and 131 amino acids, respectively. These data confirm previous reports demonstrating significant antigenic and genetic distance between VEE I-AB virus and viruses of subtypes I-E and II. It is unlikely that the epizootic VEE I-AB virus responsible for the 1969 outbreak originated from mutation of enzootic VEE viruses in North or Middle America.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , RNA Mensageiro/genética , RNA Viral/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Evolução Biológica , Vírus da Encefalite Equina Venezuelana/química , Vírus da Encefalite Equina Venezuelana/classificação , Genoma Viral , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Antígenos Virais/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/prevenção & controle , Sequências Reguladoras de Ácido Nucleico/genética , Vacinas Atenuadas , Proteínas do Envelope Viral/imunologia , Animais , Formação de Anticorpos , Bacteriófago T7/genética , Sequência de Bases , Clonagem Molecular , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/imunologia , Epitopos , Genoma Viral , Masculino , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Mutação Puntual , Análise de Sobrevida , Células Vero , Proteínas do Envelope Viral/genética , Ensaio de Placa Viral , VirulênciaRESUMO
An important question pertaining to the natural history of Venezuelan equine encephalitis (VEE) virus concerns the source of epizootic, equine-virulent strains. An endemic source of epizootic virus has not been identified, despite intensive surveillance. One of the theories of epizootic strain origin is that epizootic VEE viruses evolve from enzootic strains. Likely enzootic sources of VEE virus occur in Colombia and Venezuela where many of the epizootic outbreaks of VEE have occurred. We have determined the nucleotide sequences of the entire genomes of epizootic VEE subtype I-C virus, strain P676, isolated in Venezuela, and of enzootic VEE subtype I-D virus, strain 3880, isolated in Panama. VEE subtype I-D viruses are maintained in enzootic foci in Panama, Colombia, and Venezuela. The genomes of P676 and 3880 viruses differ from that of VEE subtype I-AB virus, strain Trinidad donkey (TRD), by 417 (3.6%) and 619 (5.4%) nucleotides, respectively. The translated regions of P676 and 3880 genomes differ from those of TRD virus by 54 (1.4%) and 66 (1.8%) amino acids, respectively. This study and the oligonucleotide fingerprint analyses of South American I-C and I-D viruses (Rico-Hesse, Roehrig, Trent, and Dickerman, 1988, Am. J. Trop. Med. Hyg. 38, 187-194) provide the most conclusive evidence to date suggesting that equine-virulent strains of VEE virus arise naturally from minor variants present in populations of I-D VEE virus maintained in enzootic foci in northern South America.
Assuntos
Evolução Biológica , Vírus da Encefalite Equina Venezuelana/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Impressões Digitais de DNA , Vírus da Encefalite Equina Venezuelana/classificação , Genes Virais/genética , Variação Genética , Genoma Viral , Cavalos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Proteínas Virais/genética , Proteínas Estruturais Virais/genéticaRESUMO
Venezuelan equine encephalitis (VEE) virus is a mosquito-borne pathogen that has caused encephalitis in equine species and humans during sporadic outbreaks in the western hemisphere. The last, and most widespread, VEE outbreak occurred in South America, Central America, Mexico and the U.S.A. (Texas) during 1969 to 1972. We have cloned and sequenced the genome of a virulent VEE subtype I-AB virus, strain 71-180, isolated in Texas in 1971. Thirty-four nucleotide differences were detected between the genome of 71-180 virus and that of the subtype I-AB Trinidad donkey (TRD) virus isolated during the 1943 VEE epizootic in Trinidad. Fifteen nucleotide changes occurred in the non-structural genes, 16 in the structural genes and three in the 3' non-coding region. Only six of the nucleotide differences resulted in amino acid substitutions: one change in each of non-structural proteins nsP1 and nsP3, two in the E2 envelope glycoprotein, one in the 6K polypeptide and one in the E1 envelope glycoprotein. The close genetic relationship between 71-180 virus and TRD virus, commonly used for production of formalin-inactivated VEE vaccines, suggests that incompletely inactivated virulent vaccine virus may have been the source of this and other VEE outbreaks. Use of formalized virulent virus was discontinued during the 1969 to 1972 panzootic. No VEE epizootics have been reported since the introduction of the live attenuated TC-83 vaccine virus.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Encefalite por Arbovirus/microbiologia , Animais , Vírus da Encefalite Equina Venezuelana/patogenicidade , História do Século XX , Humanos , América do Norte , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , América do SulRESUMO
Nucleotide sequence analysis of cDNA clones covering the entire genomes of Trinidad donkey (TRD) Venezuelan equine encephalitis (VEE) virus and its vaccine derivative, TC-83, has revealed 11 differences between the genomes of TC-83 virus and its parent. One nucleotide substitution and a single nucleotide deletion occurred in the 5'- and 3'-noncoding regions of the TC-83 genome, respectively. The deduced amino acid sequences of the nonstructural polypeptides of the two viruses differed only in a conservative Ser(TRD) to Thr(TC-83) substitution in nonstructural protein (nsP) three at amino acid position 260. The two silent mutations (one each in E1 and E2), one amino acid substitution in the E1 glycoprotein, and five substitutions in the E2 envelope glycoprotein of TC-83 virus were reported previously (B.J.B. Johnson, R.M. Kinney, C.L. Kost, and D.W. Trent, 1986, J. Gen. Virol. 67, 1951-1960). The genome of TRD virus was 11,444 nucleotides long with a 5'-noncoding region of 44 nucleotides. The carboxyl terminal portion of VEE nsP3 contained two peptide segments (7 and 34 amino acids long) that were repeated with high fidelity. The open reading frame of the nonstructural polyprotein was interrupted by an in-frame opal termination codon between nsP3 and nsP4, as has been reported for Sindbis, Ross River, and Middelburg viruses. The deduced amino acid sequences of the VEE TRD nsP1, nsP2, nsP3, and nsP4 polypeptides showed 60-66%, 57-58%, 35-44%, and 73-71% identity with the aligned sequences of the cognate polypeptides of Sindbis and Semliki Forest viruses, respectively. The lack of homology in the nsP3 of the viruses is due to sequence variation in the carboxyl terminal half of this polypeptide.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Vacinas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Capsídeo/genética , Genes Virais , Dados de Sequência Molecular , Perissodáctilos/microbiologia , RNA Viral/genética , Proteínas do Core Viral/genética , Proteínas não Estruturais ViraisRESUMO
cDNA molecules encoding the structural proteins of the virulent Trinidad donkey and the TC-83 vaccine strains of Venezuelan equine encephalitis (VEE) virus were inserted under control of the vaccinia virus 7.5K promoter into the thymidine kinase gene of vaccinia virus. Synthesis of the capsid protein and glycoproteins E2 and E1 of VEE virus was demonstrated by immunoblotting of lysates of CV-1 cells infected with recombinant vaccinia/VEE viruses. VEE glycoproteins were detected in recombinant virus-infected cells by fluorescent antibody (FA) analysis performed with a panel of VEE-specific monoclonal antibodies. Seven E2-specific epitopes and two of four E1-specific epitopes were demonstrated by FA.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/biossíntese , Vaccinia virus/genética , Proteínas Virais/biossíntese , Anticorpos Monoclonais , Sequência de Bases , Clonagem Molecular , DNA , Vírus da Encefalite Equina Venezuelana/metabolismo , Epitopos/genética , Imunofluorescência , Immunoblotting , Plasmídeos , RNA Viral , Proteínas Recombinantes de Fusão/genética , Vaccinia virus/metabolismo , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Proteínas Virais/genética , Proteínas Estruturais ViraisRESUMO
Mice immunized with recombinant vaccinia virus (VACC) expressing Venezuelan equine encephalitis (VEE) virus capsid protein and glycoproteins E1 and E2 or with attenuated VEE TC-83 virus vaccine developed VEE-specific neutralizing antibody and survived intraperitoneal challenge with virulent VEE virus strains including Trinidad donkey (subtype 1AB), P676 (subtype 1C), 3880 (subtype 1D), and Everglades (subtype 2). However, unlike immunization with TC-83 virus, immunization with the recombinant VACC/VEE virus did not protect mice from intranasal challenge with VEE Trinidad donkey virus. These results suggest that recombinant VACC/VEE virus is a vaccine candidate for equines and humans at risk of mosquito-transmitted VEE disease but not for laboratory workers at risk of accidental exposure to aerosol infection with VEE virus.
Assuntos
Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina/prevenção & controle , Encefalomielite Equina Venezuelana/prevenção & controle , Vacinas Sintéticas , Vacinas , Vacinas Virais , Animais , Anticorpos Antivirais/biossíntese , Capsídeo/imunologia , Reações Cruzadas , Vírus da Encefalite Equina Venezuelana/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Glicoproteínas/imunologia , Testes de Inibição da Hemaglutinação , Masculino , Camundongos , Camundongos Endogâmicos , Testes de Neutralização , Vacinas/imunologia , Vacinas Atenuadas , Vacinas Sintéticas/imunologia , Vaccinia virus/genética , Vaccinia virus/imunologia , Vacinas Virais/imunologiaRESUMO
The sequence of the 5'-end of the genome of dengue 2 (Jamaica genotype) virus has been previously reported (V. Deubel, R. M. Kinney, and D. W. Trent, 1986, Virology 155, 365-377). We have now cloned and sequenced the remaining 75% of the genomic RNA that encodes the nonstructural proteins. The complete genome is 10,723 bases in length with a single open reading frame extending from nucleotides 97 to 10,269 encoding 3391 amino acids. The 3'-noncoding extremity presents a stem- and loop-structure and contains a repeated oligonucleotide sequence. Comparisons of the nucleotide sequences of the genomes of dengue 2 viruses of different topotypes reveal 90-95% similarity, with 64-66% similarity evident between dengue viruses of different serotypes. The amino acid sequence of the polyprotein of dengue 2 Jamaica virus shows 97, 68, 50, and 44% similarity with those of other dengue 2, dengue 1, or dengue 4, West Nile, and yellow fever viruses, respectively. Despite amino acid sequence divergence, the hydrophobic profile of the flavivirus proteins is highly conserved. Proteins NS1, NS3, and NS5 are the most conserved. Conserved amino acid stretches present in all flavivirus proteins may be involved in common essential biological functions.
Assuntos
Vírus da Dengue/genética , Genes Virais , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Homologia de Sequência do Ácido NucleicoRESUMO
The nucleotide sequence of the 5'-terminal 2469 bases of dengue 2 (Jamaica genotype) virus has been determined and the encoded proteins compared with those of yellow fever and West Nile viruses, which belong to different flavivirus serogroups. The cDNA clone which was sequenced contains a 5'-noncoding region of 96 nucleotides followed by a single open reading frame coding for the structural proteins 5'-C-prM(M)-E-3' and the beginning of the NS1 nonstructural protein. The amino acid sequence homology between the structural polyprotein precursor of dengue 2 virus and those of yellow fever and West Nile viruses is 36.5 and 42%, respectively. The dengue virus structural proteins are similar in size and composition to those of the other flaviviruses. The basic capsid protein and the membrane and envelope proteins have hydrophobic regions at their C termini. The dengue 2 capsid C, membrane M, and envelope E proteins share 13, 36, and 43% homology, respectively, with the cognate proteins of yellow fever virus, and 33, 32, and 47% homology with the cognate proteins of West Nile virus. All 6 cysteine residues in the dengue 2 premembrane protein and all 12 cysteine residues in the dengue 2 envelope protein are conserved in the cognate proteins of yellow fever and West Nile viruses.
Assuntos
Vírus da Dengue/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Códon , DNA/genética , Vírus da Dengue/classificação , Flavivirus/genética , Conformação Proteica , SolubilidadeRESUMO
The nucleotide and deduced amino acid sequences of the structural proteins of the TC-83 vaccine strain of Venezuelan equine encephalitis (VEE) virus have been determined from a cDNA clone containing the 26S mRNA coding region. A cDNA clone encoding the equivalent region of the virulent parent VEE virus [Trinidad donkey strain (TRD)] has been sequenced previously. Comparison of the sequences of the TC-83 and TRD cDNA clones revealed 13 nucleotide differences. Neither the organization of the structural proteins (5'-capsid-E3-E2-6K-E1-3') nor the length (3762 nucleotides) of the open reading frame coding for the viral polyprotein precursor was altered during attenuation. Of the 13 nucleotide differences between the cDNA clones of TC-83 and TRD, nine occurred in the dominant population of the respective genomic RNAs from plaque-purified viruses. Six of the nine mutations were clustered in the E2 surface glycoprotein gene. All five of the nucleotide changes which produced non-conservative amino acid substitutions in the encoded proteins were located in the E2 gene. Two mutations occurred in the E1 glycoprotein gene; one was silent and the other did not alter the chemical character of the E1 protein. One nucleotide difference was found in the non-coding region immediately preceding the 5'-end of the 26S mRNA. The E2 and non-coding region mutations are candidates for the molecular determinants of VEE virus neurovirulence.
Assuntos
Encéfalo/microbiologia , Vírus da Encefalite Equina Venezuelana/patogenicidade , Glicoproteínas/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA , Vírus da Encefalite Equina Venezuelana/genética , Genes Virais , Mutação , RNA Viral/genética , Proteínas Estruturais Virais , VirulênciaRESUMO
A cDNA clone containing all of the 26 S mRNA coding region of the RNA genome of Venezuelan equine encephalitis (VEE) virus, virulent strain Trinidad donkey (TRD), has been constructed and sequenced. The nucleotide and deduced amino acid sequences of the 26 S RNA of VEE virus conform to the general organization of the alphavirus subgenomic mRNA. Excluding the poly(A) tail, the VEE 26 S RNA is 3913 nucleotides long with a protein coding region of 3762 nucleotides. Codon usage in the translated region is nonrandom and correlates well with that reported for Sindbis (SIN), Semliki Forest (SF), and Ross River (RR) alphaviruses. Highly conserved sequences of 19 to 22 nucleotides representing putative replicase recognition sites occur at the 26 S RNA junction region of the 42 S genomic RNA and at the 3' terminus immediately preceding the poly(A) tail. The conserved sequence at the 26 S/42 S junction region of VEE virus differs from that of other alphaviruses in that an ochre termination codon (UAA) is substituted for a GGU (Gly) codon present in the other viruses. The 5' and 3' noncoding regions (30 and 121 nucleotides, respectively) of the VEE 26 S RNA are shorter than has been reported for several other alphaviruses. The approximate transmembrane domains of the VEE E1 and E2 envelope glycoproteins have been identified. VEE E1 contains a single asparagine-linked glycosylation site, whereas E2 has three such sites, all of which are apparently glycosylated. The deduced amino acid sequence of the VEE polyprotein shows an overall homology of 44 to 46% with the precursor polyproteins of SIN, SF, and RR viruses. VEE virus capsid, E1, and E2 structural proteins show 43 to 46%, 50 to 53%, and 36 to 41% homology, respectively, with the cognate proteins of SIN, SF, and RR viruses.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , RNA Mensageiro/análise , Proteínas Virais/análise , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Sequência de Bases , Capsídeo , Linhagem Celular , Clonagem Molecular , DNA/análise , Genes , Haplorrinos , Biossíntese de Proteínas , Proteínas Estruturais ViraisRESUMO
Unclassified Venezuelan equine encephalitis (VEE) viruses Tonate (TON), Bijou Bridge (BB), Paramana (PARA), 71D-1252 and Cabassou (CAB) were characterized serologically and biochemically. The envelope glycoproteins of these and nine other VEE viruses representing VEE subtype variants I-AB, I-C, I-D, I-E, II, III and IV were separated by column isoelectric focusing. The E1 and E2 glycoproteins of all the Zwittergent-dissociated VEE viruses focused at pI 6.3 to 6.9 and pI 8.6 to 9.3 respectively. Haemagglutination-inhibition and neutralization tests using rabbit sera to the E2 glycoprotein of TON, BB and PARA viruses showed them to be indistinguishable from each other and closely related to prototype subtype III virus Mucambo (MUC). VEE strain 71D-1252 was also serologically closely related to prototype MUC virus. We proposed that MUC, TON and 71D-1252 VEE viruses be classified subtype III viruses, designated variants III-A, III-B and III-C respectively. CAB virus, which is not closely related to other VEE isolates, may represent a new VEE subtype (V). SDS-PAGE resolved the capsid protein (35 to 36 kdal) and two major envelope glycoproteins of 50 to 51 kdal (E1) and 51 to 58 kdal (E2) for all VEE viruses except CAB; the two glycoproteins of CAB virus co-migrated by PAGE with apparent identical mol. wt. of 51 kdal. Limited digestion of SDS-dissociated virus proteins with Staphylococcus aureus V8 protease produced identical peptide maps for serologically indistinguishable viruses. Oligonucleotide fingerprinting of virus RNA supported the close serological relationships observed at the genome level.
Assuntos
Vírus da Encefalite Equina Venezuelana/classificação , RNA Viral/análise , Proteínas Virais/análise , Capsídeo/análise , Eletroforese em Gel de Poliacrilamida , Vírus da Encefalite Equina Venezuelana/análise , Glicoproteínas/imunologia , Testes de Inibição da Hemaglutinação , Ponto Isoelétrico , Oligorribonucleotídeos/análise , Peptídeos/análise , Sorotipagem , Proteínas do Envelope Viral , Proteínas Virais/imunologiaRESUMO
Two strains of recently isolated Venezuelan equine encephalitis (VEE) complex virus from southern Brazil, avirulent for 6- to 8-week-old mice and short-haired guinea pigs, were characterized by biologic, serologic, and biochemical means. They were shown serologically to represent a single, newly recognized variant of subtype I. Two-dimensional polyacrylamide gel electrophoresis (PAGE) of ribonuclease T1 digests of viral ribonucleic acid showed considerable homology between the genomes of the new variant prototype and variant IA. Three structural proteins were visualized by discontinuous sodium dodecyl sulfate-PAGE (SDS-PAGE). Although the smallest protein of both recent isolates migrates with the capsid proteins of other subtype I viruses, the larger structural proteins of the new variants differ in molecular weight from the E1 and E2 envelope glycoproteins of the other subtype I variants. The new isolates produced peptide fragment patterns that were identical to each other, but different from the patterns of other subtype I viruses, following SDS-PaGE of dissociated virions digested with Staphylococcus aureus V8 protease. Since these two isolates were from Culex (Melanoconion) species mosquitoes and from a bat (Carollia perspicillata), were postulated that this is an enzootic VEE virus variant for which the classification IF is suggested.