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Biomedical subjects

M J Grubman

Publications and source records attributed to M J Grubman.

At least 37 records · Page 2Linked to original sources
PubMed1996

Pathogenesis of wild-type and leaderless foot-and-mouth disease virus in cattle.

Four calves were experimentally infected via aerosol with foot-and-mouth disease virus. Two were infected with a wild-type virus derived from a full-length infectious clone (A12-IC), and two were infected with a clone-derived virus lacking the leader gene (A12-LLV2), with euthanasia and tissue collection at 24 and 72 h postexposure (hpe). Clinical disease was apparent only in the animal given A12-IC and euthanized at 72 hpe. In situ hybridization revealed that the animal infected with A12-IC and euthanized at 24 hpe had abundant viral nucleic acid in the lung, present in clusters of positive cells in the respiratory bronchiolar epithelium and associated subepithelial regions. At 72 hpe in the A12-IC-infected calf, viral nucleic acid in the lung was present in interstitial areas, and in addition, viral nucleic acid was detectable in epithelial tissues around histologically apparent vesicles. In animals infected with A12-LLV2, viral nucleic acid was detectable in the lung at both 24 and 72 hpe, but staining revealed a more localized distribution with less nucleic acid than was found in animals given A12-IC. Therefore, it appears that after aerosol exposure to A12-IC, early replication is in the region of the lung, with subsequent dissemination to distal sites. In comparison, the A12-LLV2 virus is much less widely disseminated in the lung at 24 hpe, with no lesions or virus detectable in secondary sites at 72 hpe. The greatly reduced pathogenicity of A12-LLV2 may make it an excellent candidate for a modified live viral vaccine.

Animals↗
PubMed1996

Distribution of bluetongue virus in tissues of experimentally infected pregnant dogs as determined by in situ hybridization.

Six female dogs (four pregnant and two nonpregnant) were inoculated with bluetongue virus (BTV), serotype 11. Pregnant animals and one nonpregnant dog received 5.5-6.3 log10 of cell culture-adapted virus. The other nonpregnant dog received a modified live vaccine contaminated with bluetongue virus. The non-pregnant animals never became clinically ill and were euthanatized 35 days post-inoculation. Three of the four pregnant dogs aborted, and all four died or were euthanatized 5-10 days post-inoculation. The predominant pathologic feature in the adults was severe pulmonary edema. Various tissues from the bitches and fetuses were examined by in situ hybridization using a digoxigenin-labeled probe corresponding to the nonstructural protein-1 gene of BTV-17. By this technique, viral nucleic acid was detected predominantly in endothelial cells of lung of all four dogs, with lesser amounts in capillaries of uterus, spleen, and kidney in some of the dogs. In two adult dogs, bluetongue viral nucleic acid was detected in mononuclear cells of the periarteriolar lymphoid sheaths of spleen. There was minimal staining of capillaries in placenta in three of the five fetuses examined. There was no viral nucleic acid detected in any of the other fetal tissues.

Animals↗
PubMed1995

Identification of the active-site residues of the 3C proteinase of foot-and-mouth disease virus.

To identify the active-site residues of the 3C proteinase of foot-and-mouth disease virus (FMDV), we introduced mutations into the 3C coding region and examined the activity of mutant enzymes on various substrates. Based on alignment of FMDV 3C with other picornavirus 3C proteinases and with the trypsin family of serine proteinases, mutations were introduced at residues presumed to be part of the catalytic triad, involved in substrate binding, or present in nonconserved regions. Wild-type and mutant 3C proteins were expressed in Escherichia coli and tested for their ability to cleave synthetic substrates corresponding to different portions of the viral genome. Substitutions at His-46 (catalytic triad), Asp-84 (catalytic triad), or His-181 (substrate binding) produced enzymes unable to process P1, P2, or P3 substrates in trans, whereas a change in the conserved Asp-98 had no effect on enzyme activity. Substitution of Ser for Cys-163 (catalytic triad) yielded an enzyme that retained activity on some substrates, while a substitution of Gly at this position resulted in a completely inactive enzyme. The kinetics of trans processing of translation products from a transcript encoding the P1 and P2 coding regions and the 2C/3A cleavage site with wild-type 3C or a transcript encoding P1 with 3C mutants revealed that the order of cleavage was VP3-VP1, VP0-VP3, VP1-2A, 2C-3A, and 2B-2C. Mutations in 3C that resulted in a partially active enzyme were individually introduced into full-length FMDV cDNA and RNA transcripts were translated in a cell-free system and used to transfect cells. In all cases the virus that was rescued had reverted to the wild-type 3C codon.

3C Viral Proteases↗
PubMed1995

Expression in Escherichia coli and purification of biologically active L proteinase of foot-and-mouth disease virus.

The foot-and-mouth disease virus (FMDV) Lb gene was cloned into bacterial expression vectors under the control of a T7 RNA polymerase promoter. The Lb protein was expressed in both an in vitro transcription-translation system and in Escherichia coli. In vitro expression of a construct containing the Lb gene fused to a portion of the VP4 and 3D genes demonstrated cis cleavage activity that could be blocked by the thiol protease inhibitor E-64. Lb expressed in E. coli was purified from the soluble fraction by metal chelation chromatography. Purified Lb had trans cleavage activity at the L/P1 junction and cleaved the p220 component of the cap-binding protein complex.

Aphthovirus↗
PubMed1995

Identification of the active-site residues of the L proteinase of foot-and-mouth disease virus.

The foot-and-mouth disease virus (FMDV) leader (L) protein is involved in autocatalytic cleavage at the L/P1 junction and in the cleavage of translation initiation factor p220, a subunit of the cap-binding protein complex. It has been suggested that this proteinase has homology to the papain-like family of cysteine proteinases, and from this information, we have investigated the active-site residues by introducing specific mutations into the L gene. Mutations of Cys-23 to Ala or His-120 to Leu resulted in enzymes that lacked cis activity at the L/VP4 cleavage site, trans activity on a truncated L-P1 substrate, and p220 cleavage activity. Mutations of Cys-23 to ser or His-110 to Leu resulted in enzymes that retained some or all cis activity and had reduced p220 cleavage. These mutations were introduced separately into a full-length FMDV cDNA, and RNA transcripts derived from these cDNAs were translated in a cell-free system and transfected into cells. The C23S mutant inefficiently cleaved at the L/P1 junction and within P1, and virus obtained from transfected cells reverted to wild type. The H110L mutant cleaved the L/P1 junction almost as well as the wild-type enzyme, and virus recovered from transfected cells retained the mutation and displayed wild-type viral protein synthesis and host shut-off kinetics.

Animals↗
PubMed1995

The foot-and-mouth disease virus leader proteinase gene is not required for viral replication.

The foot-and-mouth disease virus (FMDV) leader (L) proteinase has only two known functions: (i) autocatalytic removal from the N terminus of the viral polyprotein and (ii) cleavage of the p220 subunit of the eukaryotic initiation factor 4F complex, which helps to shut off host protein synthesis. Cleavage of p220 appears to be important for picornavirus replication, since rhinoviruses and enteroviruses utilize a different proteinase (2A) to cleave p220. To explore the role of L in FMDV replication, we generated synthetic FMDV genomes lacking the L gene and tested their viability in cells. Genomes were constructed with the N-terminal Gly codon of VP4 positioned directly following either the first (Lab) or second (Lb) Met codon of the L protein. Cells transfected with synthetic RNAs lacking L and initiating with the Lab Met codon failed to produce viable virus, but cells transfected with RNAs that utilized the second AUG to drive translation of the viral polyprotein produced viable viruses. These leader-deleted viruses produced plaques on BHK cells that were slightly smaller than those produced by wild-type (WT) virus, grew to slightly lower titers than WT virus in BHK cells, shut off host protein synthesis more slowly than WT virus, and were slightly attenuated in mice. These studies indicate that the L proteinase is not essential for FMDV replication and show that in the cells and animals tested the L gene has a limited effect on virus replication.

Amino Acid Sequence↗
PubMed1994

Phylogenetic analysis of segment 10 from African horsesickness virus and cognate genes from other orbiviruses.

Utilizing the reverse transcriptase-polymerase chain reaction (RT-PCR) procedure, we have synthesized full-length copies of segment 10 from African horsesickness virus (AHSV) serotypes 1, 4 and 8. The genes were cloned, sequenced and compared with the sequence of the cognate gene from AHSV serotypes 3 and 9. Sequences were analyzed to assess evolutionary relationships among serotypes using cladistics. Based on this analysis the data support a close relationship between serotypes 4 and 9 and between serotypes 1 and 8 and a closer relationship of serotype 3 to the 4 and 9 group.

African Horse Sickness Virus↗
PubMed1994

Presence of African horse sickness virus in equine tissues, as determined by in situ hybridization.

In a retrospective study, a negative-sense digoxigenin-labeled RNA probe, corresponding to the gene encoding nonstructural protein-1 of African horse sickness virus (AHSV) serotype 4, was applied to formalin-fixed, paraffin-embedded tissue taken from horses in the terminal stages of infection with AHSV. Fifteen infected ponies and one noninfected control were studied. Ponies exhibited a range of clinical signs and lesions. Thirteen ponies were infected with serotype 4, one with serotype 1, and one with serotype 2. Ponies were monitored clinically and euthanatized when severely clinically ill. The following tissues were available for study by in situ hybridization and histopathology: lung, heart, spleen, neck muscle, and supraorbital fat. Histologically, the most striking changes were pulmonary edema and, in some, acute myocardial necrosis. In situ hybridization revealed virus distributed widely in sections of lung and heart examined, with relatively less in spleen, neck muscle, or supraorbital fat. Virus was localized to target cells with morphologic features compatible with endothelium in all organs except spleen, where it was found in both endotheliumlike cells and large mononuclear cells.

African Horse Sickness↗
PubMed1994

Identification of African horse sickness virus in cell culture using a digoxigenin-labeled RNA probe.

A digoxigenin-labeled RNA probe was synthesized from a plasmid containing a portion of the African horse sickness virus (AHSV) serotype 4 genome segment coding for nonstructural protein 1. In an in situ hybridization procedure, this probe hybridized successfully to Vero cells infected with each of the 9 serotypes of AHSV. There was no hybridization with noninfected cell cultures or cell cultures infected with bluetongue virus.

African Horse Sickness Virus↗
PubMed1993

Characterization of the foot-and-mouth disease virus 3C protease expressed in Escherichia coli.

We have constructed a clone encoding the foot-and-mouth disease virus (FMDV) 3C protease gene (p3C) using the polymerase chain reaction. The construct was engineered to contain initiation and termination codons and cloned into a plasmid under the control of the bacteriophage T7 promoter. The p3C gene was expressed both in an in vitro transcription-translation system and in vivo in an Escherichia coli system containing an inducible T7 RNA polymerase gene. In both systems the expressed products were of the appropriate molecular weight and immunologically reactive with bovine convalescent serum. E. coli-expressed 3C protein was mainly found in the insoluble fraction of cell lysates. The E. coli-expressed protease was assayed in an in vitro system with radiolabeled P1 capsid precursor protein and P2 precursor protein as substrates. E. coli-expressed 3C completely processed the P1 and P2 precursors into mature capsid and nonstructural proteins, respectively. The kinetics of processing of P1 by E. coli-expressed 3C revealed the following order of cleavage: VP3-VP1, VP0-VP3, VP1-2A.

3C Viral Proteases↗
PubMed1993

Protection of swine against foot-and-mouth disease with viral capsid proteins expressed in heterologous systems.

Three groups of swine were each inoculated with a different antigen preparation of foot-and-mouth disease virus (FMDV) capsid proteins and challenged by contact exposure to animals infected with FMDV. One group of four animals was inoculated with an extract from cells infected with a recombinant baculovirus containing the FMDV P1-2A structural protein precursor gene and a portion of the P2 gene. Two out of four animals were protected from clinical disease, but not from virus replication. A second group of animals was inoculated with an extract from Escherichia coli that expressed FMDV proteins from a construct containing the P1-2A gene, a portion of the P2 gene and the 3C protease gene. Three out of four animals in this group did not develop clinical signs of FMD upon challenge and two of four were protected against virus replication. In contrast, inoculation of a third group of swine with an extract from E. coli expressing the same FMDV construct as present in the recombinant baculovirus failed to protect any of the four animals from generalized FMD.

Animals↗
PubMed1993

Use of a digoxigenin-labeled RNA probe to detect all 24 serotypes of bluetongue virus in cell culture.

A digoxigenin-labeled RNA probe, corresponding to the section of the bluetongue virus (BTV) serotype 17 genome coding for nonstructural protein-1 (NS1), was applied to noninfected cell cultures and cell cultures infected with 24 different serotypes of BTV, 2 serotypes of epizootic hemorrhagic disease virus, and African horse sickness virus type 4. The probe hybridized to all cell cultures infected with the various BTV serotypes but did not hybridize to noninfected cell cultures or cell cultures infected with any of the other orbiviruses.

Animals↗
PubMed1992

Identification and characterization of the structural and nonstructural proteins of African horsesickness virus and determination of the genome coding assignments.

Proteins present in purified African horsesickness virus (AHSV) and in infected cells were analyzed by SDS-polyacrylamide gel electrophoresis. Twelve viral proteins were identified, one minor and four major structural proteins, three major and two minor nonstructural proteins, as well as variable amounts of two additional structural proteins. Cell-free translation of total AHS virion RNA in a rabbit reticulocyte system resulted in the synthesis of proteins which were qualitatively and quantitatively similar to those found in infected cells. The in vivo and in vitro synthesized proteins were viral specific as demonstrated by immunoprecipitation. The coding assignments of all the purified genome segments were determined by in vitro translation and confirmed by immunoprecipitation.

African Horse Sickness Virus↗
PubMed1992

Antiviral effects of a thiol protease inhibitor on foot-and-mouth disease virus.

The thiol protease inhibitor E-64 specifically blocks autocatalytic activity of the leader protease of foot-and-mouth disease virus (FMDV) and interferes with cleavage of the structural protein precursor in an in vitro translation assay programmed with virion RNA. Experiments with FMDV-infected cells and E-64 or a membrane-permeable analog, E-64d, have confirmed these results and demonstrated interference in virus assembly, causing a reduction in virus yield. In addition, there is a lag in the appearance of virus-induced cellular morphologic alterations, a delay in cleavage of host cell protein p220 and in shutoff of host protein synthesis, and a decrease in viral protein and RNA synthesis. The implications of using E-64-based compounds as potential antiviral agents for FMDV are discussed.

Animals↗
PubMed1991

Expression, processing, and assembly of foot-and-mouth disease virus capsid structures in heterologous systems: induction of a neutralizing antibody response in guinea pigs.

Plasmids containing the foot-and-mouth disease virus structural protein precursor (P1) and 3C protease genes or the P1 gene alone were expressed in Escherichia coli. A recombinant baculovirus containing the P1 gene was also generated and expressed in Spodoptera frugiperda cells. Expression of the P1 and 3C genes in E. coli resulted in efficient synthesis and processing of the structural protein precursor and assembly into 70S empty capsids. This material reacted with neutralizing monoclonal antibodies which recognize only conformational epitopes and elicited a significant neutralizing antibody response in vaccinated guinea pigs. Expression of the P1 gene in E. coli resulted in synthesis of an insoluble product, whereas in insect cells infected with the recombinant baculovirus a soluble product was synthesized. Both soluble and insoluble P1 reacted with a 12S-specific monoclonal antibody, but only soluble P1 elicited a neutralizing antibody response in guinea pigs.

Animals↗
PubMed1990

Nucleotide and deduced amino acid sequence of the nonstructural phosphoprotein, NS2, of bluetongue virus serotype 17: comparison to two isolates of serotype 10.

The nucleotide sequence of bluetongue virus (BTV) serotype 17 segment 8 from North America (NA) coding for the nonstructural phosphoprotein, NS2, was determined. This segment contains 1125 base pairs and codes for a protein of 40,581 daltons containing 354 amino acids with a net charge of -8.5 at pH 7.0. The carboxyl terminal portion of the protein is very hydrophilic and has a high degree of potential alpha-helix. Serine is the major, if not the exclusive, phosphorylated amino acid residue and ten of the twenty serine residues present in NS2 are found in consensus phosphorylation sites. Comparison of the nucleotide sequence of BTV-17NA segment 8 with the sequence of BTV-10NA and BTV-10 South Africa (SA) revealed a greater degree of homology between different serotypes within the same geographical area, i.e., 17NA and 10NA, than between isolates of the same serotype located in different areas, i.e., 10NA and 10SA. The same homology relationship as above was found at the amino acid level.

Amino Acid Sequence↗
PubMed1990

Bluetongue virus: surface exposure of VP7.

The exposed proteins of bluetongue virus serotype 17 were determined using surface labeling and reactivity with monoclonal antibodies. Iodination of amino groups predominantly labeled VP2; however, iodination of tyrosine residues labeled both VP2 and VP5, with VP7 labeled to a significantly lesser degree. To investigate the exposure of VP7 on the intact virion further, monoclonal antibodies that reacted with this protein were used. At least two antibodies, reacting with different epitopes on VP7, bound to intact virions, as determined by adsorption of infectious particles, electron microscopic observation of antibody-bound virus, and co-sedimentation of antibody and virus. Surface iodination of viral cores was used to show that VP7 and VP3 are major exposed proteins on these particles. We conclude that a major core protein, VP7, has at least two epitopes exposed on the virus surface.

Antibodies, Monoclonal↗