Biomedical subjects

R M Kinney

Publications and source records attributed to R M Kinney.

At least 37 records · Page 2Linked to original sources

PubMed1993

Phylogenetic relationships of dengue-2 viruses.

RNA oligonucleotide fingerprinting studies on a large number of virus isolates previously demonstrated considerable genetic variation in isolates of dengue (DEN)-2 serotype. We report the entire envelope (E) glycoprotein gene and deduced amino acid sequences of 16 DEN-2 viruses and the phylogenetic relationships of these, plus 17 additional published DEN E gene sequences. Comparison of DEN-2 E glycoprotein gene sequences revealed base substitutions scattered throughout the entire gene with as much as 22% sequence divergence. Aligned E glycoprotein amino acid sequences revealed the viruses differed by as much as 10%. There appeared to be constraints on the overall structure of the E protein to maintain biological function. Clusters of amino acid substitutions were present in the hydrophobic membrane anchor region at the carboxyl terminal end of the protein. Maximum parsimony analysis of the E gene sequences allowed construction of a phylogram indicating evolutionary relationships of the virus isolates within the DEN-2 serotype. Five genetic subtypes were identified. Phylogenetic relationships of the DEN-2 serotype and other flaviviruses based on E protein sequences reflected traditional antigenic and serologic classifications.

Adult↗

PubMed1993

Molecular evidence that epizootic Venezuelan equine encephalitis (VEE) I-AB viruses are not evolutionary derivatives of enzootic VEE subtype I-E or II viruses.

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.

Amino Acid Sequence↗

PubMed1993

Attenuation of Venezuelan equine encephalitis virus strain TC-83 is encoded by the 5'-noncoding region and the E2 envelope glycoprotein.

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.

Animals↗

PubMed1992

Genetic evidence that epizootic Venezuelan equine encephalitis (VEE) viruses may have evolved from enzootic VEE subtype I-D virus.

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.

Amino Acid Sequence↗

PubMed1992

Variation in the biological function of envelope protein epitopes of yellow fever vaccine viruses detected with monoclonal antibodies.

Three different virus strains (17D-204, 17DD and the French neurotropic vaccine) have been used as live attenuated yellow fever (YF) vaccines and are manufactured in different centres around the world. The envelope proteins of these vaccine viruses were examined and compared using mouse monoclonal antibodies (MAbs) in haemagglutination inhibition (HAI) and neutralization (N) tests. The epitopes eliciting HAI and/or N were found to vary depending on the virus examined. Such variation was also found between vaccine viruses of the same strain manufactured in different centres. These data were confirmed by the use of mouse polyclonal antisera. On the basis of the MAb results in HAI tests a dendrogram of the similarity coefficients between the viruses was constructed and showed that the viruses could be placed into three major groups. Thus, it is concluded that YF vaccines manufactured in different centres are antigenically distinct as recognized by the mouse immune system.

Antibodies, Monoclonal↗

PubMed1992

Molecular evidence for the origin of the widespread Venezuelan equine encephalitis epizootic of 1969 to 1972.

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.

Animals↗

PubMed1990

Attenuation of wild-type yellow fever virus by passage in HeLa cells.

During the 1960s three different research groups reported that passage of wild-type yellow fever (YF) virus [strain Asibi (YF-Asibi)] in HeLa cells resulted in attenuation of the virus for monkeys so that the virus no longer caused viscerotropic disease. We have repeated and extended this observation to analyse the process of attenuation of YF virus during cell culture passage. A large plaque (LP) variant of YF-Asibi virus became attenuated for both monkeys and mice following six serial subcultures in HeLa cells (YF-Asibi-LP HeLa p6). Thus, attenuation was probably due to a genetic change in the virus population rather than to selective enrichment of a pre-existing variant of YF-Asibi-LP virus. No evidence was obtained to implicate defective interfering particles in the attenuation process. Comparison of the YF-Asibi-LP viruses before and after passage in HeLa cells, using a panel of envelope protein-reactive monoclonal antibodies (MAbs), showed that MAbs which specifically neutralize YF-Asibi-LP virus, and not YF 17D-204 vaccine virus, also neutralized YF-Asibi-LP HeLa p6. This indicated that the epitopes involved in the biological process of neutralization were not altered during attenuation. However, two MAbs that recognize envelope protein epitopes did distinguish between HeLa- and non-HeLa-passaged YF-Asibi-LP virus. One of these (MAb 117) which is YF wild-type-specific, recognized YF-Asibi-LP virus but not YF-Asibi-LP HeLa p6 virus, whereas the other (MAb411), which is YF vaccine-specific, recognized YF-Asibi-LP HeLa p6 virus but not YF-Asibi-LP virus. These results suggest that antigenic changes in the viral envelope protein may determine the relative virulence or attenuation of YF virus.

Animals↗

PubMed1989

The full-length nucleotide sequences of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and its attenuated vaccine derivative, strain TC-83.

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.

Amino Acid Sequence↗

PubMed1988

In vitro mechanisms of monoclonal antibody neutralization of alphaviruses.

We have previously identified at least eight epitopes on the E2 glycoprotein of Venezuelan equine encephalomyelitis (VEE) virus vaccine strain TC-83 by using monoclonal antibodies (MAbs). Several of these antibodies identified a critical neutralization (N) domain in competitive binding assays. Passive transfer of these MAbs protected animals from a lethal virus challenge. Using radioactive, purified virus as a marker, we have demonstrated that antibody-mediated virus N, preattachment, can be effected by one of three mechanisms. Interaction of antibody can block virus attachment to susceptible Vero or human embryonic lung cells. The MAbs that were most efficient at blocking attachment were those that defined epitopes spatially proximal to the E2c epitope. The E2c MAbs were, however, the most efficient antibodies for neutralizing virus postattachment. Other E2 MAbs were unable to efficiently block virus attachment to cells; however, resulting replication as monitored by plaque assay or intracellular viral RNA synthesis could not be detected. One novel MAb that defined the E2f epitope appeared to enhance virus attachment to Vero cells, but not BHK-21 or LLC-MK2 cells, by stabilizing virus-cell interaction. This antibody did, however, efficiently neutralize virus infectivity. Once virus had attached to cells, the ability of most MAbs to neutralize infectivity was diminished, except for E2c MAbs. On a molar basis antibody Fab fragments were less efficient than intact antibody at blocking virus attachment.

Animals↗

PubMed1988

Nucleotide sequence and deduced amino acid sequence of the nonstructural proteins of dengue type 2 virus, Jamaica genotype: comparative analysis of the full-length genome.

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.

Amino Acid Sequence↗

PubMed1988

Recombinant vaccinia/Venezuelan equine encephalitis (VEE) virus expresses VEE structural proteins.

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.

Antibodies, Monoclonal↗

PubMed1988

Dengue 2 virus envelope protein expressed by a recombinant vaccinia virus fails to protect monkeys against dengue.

A cDNA copy of the dengue (DEN) 2 virus genome region encoding the virion capsid, membrane and envelope structural proteins has been inserted into vaccinia virus (VV) DNA under the control of its 11K late promoter. The DEN-2 envelope protein was expressed and processed in cells infected with the VV recombinant (VV/D2S). No DEN-2 virus antibody response was detected in mice, hamsters or monkeys vaccinated with VV/D2S. Furthermore, a viraemia was observed in recombinant-vaccinated monkeys after challenge with infectious DEN-2 virus.

Animals↗

PubMed1988

Recombinant vaccinia virus/Venezuelan equine encephalitis (VEE) virus protects mice from peripheral VEE virus challenge.

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.

Animals↗

PubMed1987

Partial nucleotide sequence of St. Louis encephalitis virus RNA: structural proteins, NS1, ns2a, and ns2b.

cDNA clones of the St. Louis encephalitis (SLE) virus genome have been obtained and the nucleotide sequence of 4.7 kb corresponding to the 5' terminal half of the genome determined. The genome contains a 5' noncoding region of 98 nucleotides followed by a single continuous open reading frame that encodes three structural proteins in the order capsid (C), membrane precursor (prM)-membrane (M), and envelope (E). Immediately following the C-terminus of E are located nonstructural proteins NS1 through NS3. The SLE amino acid sequence homology with yellow fever (YF), Murray Valley encephalitis (MVE), West Nile (WN), and dengue-2 (DEN) viruses over the sequenced region is 39, 66, 64, and 43%, respectively. The start of each SLE protein has been assigned on the basis of N-terminal sequence data and potential proteolytic cleavage sites homologous with YF and MVE viruses. Flaviviruses have conserved glycosylation sites in prM and NS1 proteins, although only one of the two glycosylation sites in the SLE E protein is conserved in MVE and DEN viruses. An evolutionary tree showing relationships of SLE, MVE, WN, YF, and DEN-2 flaviviruses is proposed on the basis of the amino acid sequences of the C proteins.

Base Sequence↗

PubMed1986

Nucleotide sequence of the 26 S mRNA of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and deduced sequence of the encoded structural proteins.

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.

Amino Acid Sequence↗

PubMed1986

Nucleotide sequence and deduced amino acid sequence of the structural proteins of dengue type 2 virus, Jamaica genotype.

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.

Amino Acid Sequence↗

PubMed1986

Molecular determinants of alphavirus neurovirulence: nucleotide and deduced protein sequence changes during attenuation of Venezuelan equine encephalitis virus.

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.

Amino Acid Sequence↗

PubMed1985

Amino-terminal amino acid sequences of structural proteins of three flaviviruses.

N-terminal amino acid sequences of structural proteins of three flaviviruses, yellow fever, St. Louis encephalitis, and dengue-2 viruses, have been obtained. The glycoproteins of these three viruses are 52-60% conserved in the region sequenced, depending upon which pair of viruses are compared, and 40% of the amino acids are invariant in all three viruses. Thus, flaviviruses are closely related and have in all probability descended from a common ancestor. Furthermore, residues important in the secondary structure of proteins are conserved, suggesting that the overall conformation of the glycoproteins is the same in all three viruses while considerable variation in the primary sequence can be accommodated. The N-terminal regions of the nucleocapsid proteins of yellow fever and St. Louis encephalitis viruses show markedly less homology (25%) and this region is highly basic with one-quarter (yellow fever) or one-third (St. Louis encephalitis) of the residues being lysine or arginine. N-terminal sequences for the M protein of yellow fever and for NV2(GP19) of St. Louis encephalitis viruses are also reported.

Amino Acid Sequence↗