Biomedical subjects

D R Kilpatrick

Publications and source records attributed to D R Kilpatrick.

16 recordsLinked to original sources

PubMed2001

Type-specific detection of echovirus 30 isolates using degenerate reverse transcriptase PCR primers.

Following an approach used to specifically identify polioviruses and enterovirus 71, we have developed reverse transcriptase (RT) PCR primers containing mixed-base residues or deoxyinosine at positions of codon degeneracy. These primers permit specific RT-PCR amplification of echovirus 30 (E30) sequences by targeting sites that encode conserved amino acid motifs within the major capsid protein, VP1. All 221 E30 strains tested, isolated in 16 countries over a 44-year period, yielded the predicted 158-bp PCR product. No specific products were obtained by PCR assays containing templates from any of the other 63 EV serotypes. Inosine-containing degenerate primers may be widely applicable to the identification of echovirus serotypes by PCR.

Amino Acid Sequence↗

PubMed2000

Serotype-specific identification of enterovirus 71 by PCR.

BACKGROUND: Enterovirus 71 and coxsackievirus A16 are closely related genetically and are causative agents of hand foot and mouth disease. Because enterovirus 71 is more often associated with severe neurological disease, there is a need to rapidly discriminate between enterovirus 71 and coxsackievirus A16 during hand, foot, and mouth disease outbreaks. OBJECTIVES: Our goal was to develop and evaluate a serotype-specific reverse transcription-polymerase chain reaction (RT/PCR)-based typing method for enterovirus 71. STUDY DESIGN: Two sets of PCR primers were designed to match conserved amino acid intervals of enterovirus 71. One diagnostic primer pair contains deoxyinosine at sites of 4-fold codon degeneracy. A second primer pair was designed for use in sequencing and molecular epidemiology studies. Primer pairs were tested on strains encountered in routine diagnostic samples. RESULTS: Using both sets of primers on a panel of 61 prototype enteroviral strains, both primer pairs gave strong positive signals for only enterovirus 71. These primers amplified all enterovirus 71 isolates tested and discriminated between enterovirus 71 and the most closely related enterovirus, coxsackievirus A16. CONCLUSIONS: Our RT-PCR assay can be used for specific identification of enterovirus 71 clinical isolates. Furthermore, the 484-bp product of one primer pair has proven useful in sequencing studies to identify distinct genotypes of enterovirus 71.

Amino Acid Sequence↗

PubMed2000

Comparison of classic and molecular approaches for the identification of untypeable enteroviruses.

Members of the family Picornaviridae are the most common viruses infecting humans, and species in several genera also infect a wide variety of other mammals. Picornaviruses have traditionally been classified by antigenic type, based on a serum neutralization assay. However, this method is time-consuming and labor-intensive, is sensitive to virus aggregation and antigenic variation, and requires a large number of antisera to identify all serotypes, even when antiserum pools are used. We developed generic reverse transcription (RT)-PCR primers that will amplify all human enterovirus serotypes, as well as many rhinoviruses and other picornaviruses, and used RT-PCR amplification of the VP1 gene and amplicon sequencing to identify enteroviruses that were refractory to typing by neutralization with pooled antisera. Enterovirus serotypes determined by sequencing were confirmed by neutralization with monospecific antisera. Of 55 isolates tested, 49 were of known enterovirus serotypes, two were rhinoviruses, and four were clearly picornaviruses but did not match any known picornavirus sequence. All four untyped picornaviruses were closely related to one another in sequence, suggesting that they are of the same serotype. RT-PCR, coupled with amplicon sequencing, is a simple and rapid method for the typing and classification of picornaviruses and may lead to the identification of many new picornavirus serotypes.

Animals↗

PubMed1999

Typing of human enteroviruses by partial sequencing of VP1.

Human enteroviruses (family Picornaviridae) are the major cause of aseptic meningitis and also cause a wide range of other acute illnesses, including neonatal sepsis-like disease, acute flaccid paralysis, and acute hemorrhagic conjunctivitis. The neutralization assay is usually used for enterovirus typing, but it is labor-intensive and time-consuming and standardized antisera are in limited supply. We have developed a molecular typing system based on reverse transcription-PCR and nucleotide sequencing of the 3' half of the genomic region encoding VP1. The standard PCR primers amplify approximately 450 bp of VP1 for most known human enterovirus serotypes. The serotype of an "unknown" may be inferred by comparison of the partial VP1 sequence to those in a database containing VP1 sequences for the prototype strains of all 66 human enterovirus serotypes. Fifty-one clinical isolates of known serotypes from the years 1991 to 1998 were amplified and sequenced, and the antigenic and molecular typing results agreed for all isolates. With one exception, the nucleotide sequences of homologous strains were at least 75% identical to one another (>88% amino acid identity). Strains with homologous serotypes were easily discriminated from those with heterologous serotypes by using these criteria for identification. This method can greatly reduce the time required to type an enterovirus isolate and can be used to type isolates that are difficult or impossible to type with standard immunological reagents. The technique may also be useful for the rapid determination of whether viruses isolated during an outbreak are epidemiologically related.

5' Untranslated Regions↗

PubMed1999

Molecular evolution of the human enteroviruses: correlation of serotype with VP1 sequence and application to picornavirus classification.

Sixty-six human enterovirus serotypes have been identified by serum neutralization, but the molecular determinants of the serotypes are unknown. Since the picornavirus VP1 protein contains a number of neutralization domains, we hypothesized that the VP1 sequence should correspond with neutralization (serotype) and, hence, with phylogenetic lineage. To test this hypothesis and to analyze the phylogenetic relationships among the human enteroviruses, we determined the complete VP1 sequences of the prototype strains of 47 human enterovirus serotypes and 10 antigenic variants. Our sequences, together with those available from GenBank, comprise a database of complete VP1 sequences for all 66 human enterovirus serotypes plus additional strains of seven serotypes. Phylogenetic trees constructed from complete VP1 sequences produced the same four major clusters as published trees based on partial VP2 sequences; in contrast to the VP2 trees, however, in the VP1 trees strains of the same serotype were always monophyletic. In pairwise comparisons of complete VP1 sequences, enteroviruses of the same serotype were clearly distinguished from those of heterologous serotypes, and the limits of intraserotypic divergence appeared to be about 25% nucleotide sequence difference or 12% amino acid sequence difference. Pairwise comparisons suggested that coxsackie A11 and A15 viruses should be classified as strains of the same serotype, as should coxsackie A13 and A18 viruses. Pairwise identity scores also distinguished between enteroviruses of different clusters and enteroviruses from picornaviruses of different genera. The data suggest that VP1 sequence comparisons may be valuable in enterovirus typing and in picornavirus taxonomy by assisting in the genus assignment of unclassified picornaviruses.

Amino Acid Sequence↗

PubMed1998

Serotype-specific identification of polioviruses by PCR using primers containing mixed-base or deoxyinosine residues at positions of codon degeneracy.

We have developed a method for determining the serotypes of poliovirus isolates by PCR. Three sets of serotype-specific antisense PCR-initiating primers (primers seroPV1A, seroPV2A, and seroPV3A) were designed to pair with codons of VP1 amino acid sequences that are conserved within but that differ across serotypes. The sense polarity primers (primers seroPV1S, seroPV2S, and seroPV3S) matched codons of more conserved capsid sequences. The primers contain mixed-base and deoxyinosine residues to compensate for the high rate of degeneracy of the targeted codons. The serotypes of all polioviruses tested (48 vaccine-related isolates and 110 diverse wild isolates) were correctly identified by PCR with the serotype-specific primers. None of the genomic sequences of 49 nonpolio enterovirus reference strains were amplified under equivalent reaction conditions with any of the three primer sets. These primers are useful for the rapid screening of poliovirus isolates and for determining the compositions of cultures containing mixtures of poliovirus serotypes.

Amino Acid Sequence↗

PubMed1996

Group-specific identification of polioviruses by PCR using primers containing mixed-base or deoxyinosine residue at positions of codon degeneracy.

We have developed a method for differentiating polioviruses from nonpolio enteroviruses using PCR. A pair of panpoliovirus PCR primers were designed to match intervals encoding amino acid sequences within VP1 that are strongly conserved among polioviruses. The initiating primer hybridizes with codons of a 7-amino-acid sequence that has been found only in polioviruses; the second primer matches codons of a domain thought to interact with the cell receptor. The panpoliovirus PCR primers contain mixed-base and deoxyinosine residues to compensate for the high degeneracy of the targeted codons. All RNAs from 48 vaccine-related and 110 wild poliovirus isolates of all three serotypes served as efficient templates for amplification of 79-bp product. None of the genomic sequences of 49 nonpolio enterovirus reference strains were amplified under equivalent reaction conditions. Sensitivities of poliovirus detection were as low as 100 fg (equivalent to approximately 25,000 genomic copies or 25 to 250 PFU) when the amplified products were visualized by ethidium bromide fluorescence. These degenerate PCR primers should aid in the detection of all polioviruses, including those wild poliovirus isolates for which genotype-specific reagents are unavailable.

Amino Acid Sequence↗

PubMed1992

A 585-bp deletion found in the spleen focus-forming virus (SFFV) env gene is responsible for the defective intracellular transport of SFFV gp52.

The Friend spleen focus-forming virus (F-SFFV) codes for a transport defective, leukemogenic envelope glycoprotein designated as gp52. Gp52 closely resembles the envelope glycoproteins (gp70-p15E) encoded by the mink cell focus-forming viruses (MCFV). The major differences between SFFV and MCFV include a 585-bp deletion and a frame-shift mutation near the 3' end of the SFFV env gene. We have constructed a mutant MCFV env gene, which contains a 585-bp deletion like that found in the SFFV env gene, and expressed this gene using recombinant vaccinia vectors or retroviral vectors. The mutant MCFV env gene expressed a truncated, transport defective glycoprotein (gp57). Only a small proportion of gp57 underwent further oligosaccharide processing. Intracellular gp57 remained predominantly monomeric and only a small proportion of gp57 (and its processed forms) formed disulfide-linked dimers and trimers which resembled those formed by SFFV gp52. Processed forms of gp57 were found on the cell surfaces and in culture fluids. The extracellular forms had a faster electrophoretic mobility than the intracellular-processed forms of gp57. These results indicate that the 585-bp deletion found in SFFV env gene is responsible for the folding, transport, and secretion of gp52. Retroviral vectors carrying the mutant MCFV env gene were nonpathogenic (or weakly pathogenic) in adult mice. The results indicate that the 585-bp deletion, although essential, is not the sole determinant of SFFV-induced disease in adult mice.

3T3 Cells↗

PubMed1991

Comparison of the binding characteristics to BHK-21 cells of viruses representing the two Theiler's virus neurovirulence groups.

The binding characteristics of the highly virulent GDVII and less virulent BeAn strains of Theiler's murine encephalomyelitis viruses (TMEV) to whole BHK-21 cells were determined using a direct viral binding assay. The overall rates of association and dissociation of BeAn and GDVII viruses were similar. Using a saturation binding assay intended for multivalent ligands, such as picornaviruses, the number of binding sites per cell was calculated as 1.6 x 10(5). Competitive binding assays with both viruses showed one-way blocking. In addition, treatment of cell monolayers with neuraminidase reduced binding of BeAn virus by 90% but did not affect GDVII binding. Wheat germ agglutinin, a lectin which blocks binding to sialic acid and N-acetylglucosamine residues, substantially reduced binding of radiolabeled GDVII and BeAn viruses. Treatment of asialylated cells with O-glycanase further reduced the binding of BeAn virus, suggesting that O-linked oligosaccharides are involved in viral binding. These results suggest members of the two TMEV virulence groups share a common receptor but bind it differently.

Animals↗

PubMed1991

Predominant binding of Theiler's viruses to a 34-kilodalton receptor protein on susceptible cell lines.

Western immunoblots of BHK-21 cell lysates probed with the highly virulent GDVII and the less virulent BeAn strains of Theiler's murine encephalomyelitis virus (TMEV) revealed predominant binding to a 34-kDa membrane protein and much lower levels of binding to 100- and 18-kDa membrane proteins. Complete inhibition of virus binding to both the 34- and 18-kDa membrane species by excess unlabeled TMEV demonstrated specificity of binding. Virus binding was also blocked by wheat germ agglutinin, which specifically binds to sialic acid residues and blocks TMEV binding to whole BHK-21 cells. Radiolabeled TMEV also bound to 100-, 34-, and 18-kDa membrane proteins expressed on other TMEV permissive cell lines but not on the nonpermissive cell lines tested. These data suggest that a 34-kDa cellular protein may be the primary determinant of susceptibility to TMEV infection by mediating the binding of GDVII and BeAn viruses to susceptible cells.

Animals↗

PubMed1991

The hydrophobic membrane-spanning sequences of the gp52 glycoprotein are required for the pathogenicity of Friend spleen focus-forming virus.

Friend spleen focus-forming virus (SFFV) codes for a transport-defective envelope glycoprotein designated gp52, which is responsible for the leukemogenic properties of the virus. gp52 is a monotopic integral membrane protein anchored in the membrane by a stretch of hydrophobic amino acid residues located near the carboxy terminus of the molecule. We have constructed a mutant SFFV envelope gene in which the sequences that code for the hydrophobic membrane-spanning domain have been deleted, and we expressed this gene by using recombinant vaccinia virus vectors or retroviral vectors. The mutant SFFV envelope gene was found to encode a truncated glycoprotein (gp52t) which was also transport defective; a majority of gp52t remained cell associated, while a small proportion of the molecules underwent oligosaccharide processing. The processed form of gp52t was secreted from the cells. Retroviral vectors carrying the mutant SFFV envelope gene were found to be nonpathogenic in adult mice. These results indicate that the hydrophobic membrane-spanning region of gp52 is required for pathogenicity of SFFV and suggest that these sequences may play a role in signal transduction. The results also indicate that the transport defect of SFFV gp52 is due to structural features of the ectodomain of the molecule.

Amino Acid Sequence↗

PubMed1989

The spleen focus-forming virus envelope glycoprotein is defective in oligomerization.

The gp52 envelope glycoprotein of Friend spleen focus-forming virus (SFFV) is a recombinant molecule derived from Friend murine leukemia virus (MuLV) by various deletions, insertions, and substitutions. The SFFV gp52 glycoprotein, unlike MuLV envelope glycoproteins, is defective in transport to the cell surface. Only 3-5% of gp52 eventually reaches the cell surface as a processed form (gp65). Although gp52 lacks cytoplasmic tail residues found in MuLV glycoproteins, we have previously shown that this deletion is not responsible for its defective transport. In order to investigate the basis for the defective transport of gp52, we have examined the folding and assembly of gp52 molecules into oligomeric molecules. CV-1 cells infected with vaccinia virus recombinants expressing SFFV gp52 were pulse labeled and the cell extracts were fractionated by velocity centrifugation through sucrose gradients. Immediately after a 10-min pulse, gp52 was detected as a monomer in the upper part of the sucrose gradient (fractions 12 and 14) and it remained as such after a 2-h chase period. However, the processed form, gp65, was found in a lower part of the gradient (fraction 8) after a 2-h chase. The position of gp65 was found to correspond to the position of trimeric influenza hemagglutinin which was analyzed on a parallel sucrose gradient, suggesting that gp65 also exists as a trimer in this fraction. These results indicate that changes in the external domain of gp52 result in improper folding of the glycoprotein molecule, and suggest that this lack of oligomerization is responsible for the defective transport of the molecules. Only those molecules that do form oligomeric structures are transported to the Golgi complex and undergo further oligosaccharide processing, and transport to the cell surface.

Animals↗

PubMed1988

Expression of the spleen focus-forming virus envelope gene in a polarized epithelial cell line.

Friend spleen focus-forming virus (F-SFFV) encodes a glycoprotein designated gp52, which is defective in its intracellular transport and accumulates in the rough endoplasmic reticulum. Only 3-5% of the mature form of gp52 eventually reaches the cell surface. Compared to transport-competent murine leukemia virus (MuLV) glycoproteins, the gp52 molecule exhibits several structural differences which may have resulted in the possible loss of signals required for transport to the cell surface. To determine the effect of these alterations on the specific sites of surface expression of the molecule, the SFFV env gene was expressed from a vaccinia virus recombinant in a polarized epithelial cell line in which retrovirus glycoproteins are expressed exclusively on basolateral surfaces. We also determined the site of expression of a chimeric env protein which contains the external domain of SFFV gp52 the transmembrane, and the cytoplasmic tail residues of Friend MuLV. The wild-type and chimeric env gene products were defective in transport, and remained primarily in an unprocessed form in MDCK cells or CV-1 cells. However, both glycoproteins were detected at low levels on the basolateral surfaces of MDCK cells, a line of polarized epithelial cells. These results indicate that the presence or absence of a cytoplasmic tail as well as a 585-base deletion in the external domain has no affect on the site of polarized expression of a murine retrovirus glycoprotein.

Biological Transport↗

PubMed1987

Effects of deletion of the cytoplasmic domain upon surface expression and membrane stability of a viral envelope glycoprotein.

The envelope protein (gp52) of Friend spleen focus-forming virus (F-SFFV) is defective in its intracellular transport and accumulates in the rough endoplasmic reticulum of F-SFFV-infected cells. This defect in transport has been attributed to the lack of a cytoplasmic domain, and possible loss of signals required for transport to the cell surface. The mature form of gp52, designated gp65, is also reported to be secreted from SFFV-infected cells. To determine the specific changes in the envelope protein which may lead to its lack of transport and to its lack of stability in associating with membranes, the 3' end of the F-SFFV envelope gene, which encodes the transmembrane domain, was inserted in place of the normal 3' end of the Friend murine leukemia virus genome. This chimeric envelope gene was expressed using the vaccinia virus expression system. The chimeric gp70/p15E glycoprotein molecule lacks the cytoplasmic tail residues and as a consequence is about 3300 daltons smaller. The chimeric PrEnv molecule was found to be cleaved efficiently as indicated by pulse-chase experiments. Immunofluorescence studies demonstrate that the chimeric molecule is efficiently transported to the surface of cells, unlike the SFFV gp52 glycoprotein. The chimeric molecule was found to be unstable in its membrane association and is released into the culture medium. These results indicate that the changes in the membrane spanning region and the lack of a cytoplasmic tail do not determine the defective transport of gp52, but may determine the stability of its association with membranes.

Amino Acid Sequence↗

PubMed1987

Intracellular transport and leukemogenicity of spleen focus-forming virus envelope glycoproteins with altered transmembrane domains.

Friend murine spleen focus-forming virus (SFFV) encodes a glycoprotein designated gp52, which is responsible for the leukemogenic properties of the virus. gp52 lacks a cytoplasmic domain and is defective in its transport to the cell surface. We constructed a chimeric envelope gene which codes for a molecule with an external domain derived from the SFFV envelope gene and membrane-spanning and cytoplasmic domains derived from the Friend murine leukemia virus envelope gene. Like gp52, the chimeric protein was defective in its transport to the cell surface, indicating that the absence of a cytoplasmic tail is not responsible for the defective intracellular transport of SFFV gp52. However, unlike wild-type SFFV, the chimeric SFFV genome failed to induce erythroleukemia in adult mice. The results indicate that the altered membrane-spanning domain, lack of a detectable cytoplasmic tail in gp52, or both factors are prerequisites for the erythroleukemia-inducing properties of SFFV but are not responsible for the block in intracellular transport of the glycoprotein.

Amino Acid Sequence↗

PubMed1985

Cloning and physical mapping of Yaba monkey tumor virus DNA.

The physical map positions for the BamHI, EcoRI, and SalI restriction fragments of Yaba monkey tumor pox virus DNA were determined using cloned virus DNA fragments as probes for hybridization as well as analyzing the secondary digests of larger DNA restriction fragments. Digests of EcoRI A and B fragments and SalI A and B fragments with BamHI allowed for the orientation of most of the BamHI restriction map. These secondary digest products were confirmed and the map positions for the EcoRI fragments were established using cloned BamHI fragments. Yaba monkey tumor virus DNA was cloned using the plasmid vector pBR322.

Animals↗