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A system for purification and concentration of Venezuelan equine encephalomyelitis (VEE) virus omitting large-scale ultracentrifugation was developed. The first step consists in prefiltration through large pore nuclear filters (NF) to remove large particle admixtures from the virus-containing fluid. In the second step, the resulting filtrage undergoes concentrating microfiltration through small pore NF. In this way the virus, but not total protein, is concentrated approximately 30-fold without any loss of biological activity. For VEE virus purification the next step uses gel filtration chromatography in a column with chemically modified macropore silica. In this stage, the purification factor by protein is approximately 100-fold and virus yield reaches 40%. It is suggested that the procedure used be applied for purification and concentration of a wide range of enveloped viruses.

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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.

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The equine-virulent Venezuelan encephalitis virus, Trinidad donkey (TRD), was compared to its vaccine derivative, TC-83 virus, by examining the glycosylation of the two structural envelope glycoproteins (E1 and E2). The number of size classes of glycopeptides on the glycoproteins was determined by P-6 column chromatography following Pronase digestion. The E1 glycoprotein had three glycopeptide size species and the E2 glycoprotein contained four size species ranging in mol. wt. from 1900 to 2700. Both viruses contained similar glycopeptide size species, although the relative amounts on the E2 glycoproteins appeared to be somewhat different. All of the glycopeptide species appeared to be complex, since all were labelled with glucosamine, mannose, galactose and fucose. No mannose-rich species could be detected. The different glycopeptide species appeared to be sialylation isomers of a smaller core glycopeptide with an apparent mol. wt. of 1800 which was the sole product following desialylation of the larger glycopeptides. The number of oligosaccharide attachment sites present on both E1 and E2 of each virus was determined using reverse-phase high pressure liquid chromatography. This analysis indicated that the E1 glycoprotein of both viruses had six or seven similar sugar-labelled peptide fragments following trypsin digestion. However, the E2 glycoprotein of TRD virus contained three oligosaccharide attachment sites, whereas TC-83 E2 glycoprotein had only two.

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The method of electron paramagnetic resonance and spin labels was used to study the structural characteristics of Venezuelan equine encephalomyelitis virus envelope and cell membranes of chick embryo fibroblasts. The virus envelope lipids form a bilayer structural typical of biological membranes. The lipid bilayer of virions is more rigid than the cytoplasmic membrane of chick embryo fibroblasts. Investigation of interaction of a small hydrophilic molecule with Venezuelan equine encephalomyelitis virus particles showed free water to be absent in the virions. A structural translation was detected at 25 degrees-30 degrees C in the virion membrane. The role of membrane structures in virus reproduction is discussed.

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Virion polypeptide compositions of 26 isolates of Venezuelan encephalitis virus were analyzed by a reproducible and comparative technique of discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Although the molecular weight of the core polypeptide for each isolate was 36,000, numbers and molecular weights of envelope glycoproteins were heterogeneous. Isolates associated with human, but not equine, disease usually had two glycoproteins of 50,000 to 51,000 and 51,000 to 55,000 molecular weight, whereas isolates associated with both human and equine disease usually had an additional, third polypeptide band of either 45,000 to 46,000 or 56,000 to 58,000 molecular weight. The former isolates were in hemagglutination inhibition subtypes I-D, I-E, III, or IV, and the latter were in subtypes I-A, I-B, I-C, or II. Thus virion envelope glycoproteins should be useful markers of Venezuelan encephalitis virus isolates in epidemiological investigations.

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An electronmicroscopic study of a suspension of Venezuelan equine encephalomyelitis (VEE) virus by freeze-drying and freeze-etching methods showed that glycoprotein peplomers are located on the surface of the lipoprotein shell. These peplomers are organized with trimer clustering in a T = 4 icosahedral surface lattice. The mode of glycoprotein clustering for the two clones of VEE are different.

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Pulse-chase experiments after synchronous initiation of translation indicate that the larger Venzuelan equine encephalomyelitis (VEE) virus membrane glycoprotein E2, is derived by proteolytic cleavage of the precursor, PE2. The structural proteins of VEE virus strains representing each of the antigenic subtypes and varieties have been compared by discontinuous SDS-polyacrylamide gel electrophoresis. Nucleocapsid proteins of all isolates were similar in size (mol. wt. 35 to 36 X 10(3). The mol. wt. of E1 varied from 48 to 51 X 10(3) and the mol. wt. of E2 glycoproteins ranged from 53 to 59 X 10(3). Pixuna virus contained a third envelope glycoprotein of 59 X 10(3) mol. wt. in addition to the two major glycoproteins of mol. wt. 53 X 10(3) and 48 X 10(3) respectively. The isoelectric points (pI) of E1 and E2 for all VEE strains studied were approx. 7 and 9 respectively. Both glycoproteins of TC-83 virus induced precipitating antibodies which reacted only with the homologous purified E1 and E2 glycoproteins. Antibodies to E2 protein of each virus neutralized virus infectivity and inhibited the agglutination of goose erythrocytes by virions. Haemagglutination-inhibition tests using antisera to E2 glycoproteins of prototype viruses, representing each of the antigenic subtypes and varieties, differentiated the viruses into subtypes I, II, III and IV with subtype I divided into variants 1AB, 1C, 1D and 1E.

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RNA oligonucleotide fingerprint analyses indicate that the genome RNA obtained from Trinidad donkey (TRD) Venezuelan equine encephalomyelitis (VEE) virus serotype I A, its vaccine strain derivative TC-83, and the VEE I B virus isolate PTF-39, have almost identical patterns of characteristic ribonuclease T1 resistant oligonucleotides. The TC-83 strain and the I B isolate can, on the basis of these analyses, be considered as variants of the TRD virus and categorized as I AB serotypes. Comparisons made by single and co-electrophoreses of the ribonuclease T1 digests of the RNA species of TC-83 and a VEE I C isolate P676 indicate that 16 of 37 large oligonucleotides of the TC-83 virus co-migrate with the oligonucleotides obtained from the I C isolate. Similar single and co-electrophoreses of ribonuclease T1 digests of the RNA species of TC-83 and a VEE I D isolate 3880 indicate that 18 of 41 TC-83 large oligonucleotides co-migrate with the oligonucleotides obtained from the I D virus isolate. At least nine of the TC-83 large oligonucleotides appear on the basis of these analyses, to be present in the digests of the genome RNA obtained from these selected I B, I C and I D virus isolates. The ribonucleast T1 digests of three I E virus isolates (Mina II, 63U2 and 71U388) give oligonucleotide fingerprints which, although comparable to each other, are more distinct from the I A and I B RNA fingerprints than are those of the I C and I D RNA species. The ribonuclease T1 resistant oligonucleotide fingerprints of VEE virus isolates belonging to serotypes (VEE subtypes) II, III and IV show little similarity to each other or to those of the serotype I virus isolates we have studied. The results obtained here agree with the reported close antigenic relationships of VEE, I A, I B, I C and I D virus isolates, and our studies suggest that these viruses have conserved nucleotide sequences. The I E virus isolates appear to have more distinct nucleotide sequences than do the other serotype 1 viruses. The results also agree with the serological differentiation of VEE, I, II, III and IV subtypes in that the oligonucleotide fingerprints of subtypes II to IV are different from each other and from those of the different serotype I virus isolates. On the basis of antigenic and genome relationships, VEE isolates can be classified as serotypes I to IV with serotype I viruses differentiated into the categories I AB, I C, I D and I E.

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