PubMed Health⌕ Search

Biomedical subjects

B K Rima

Publications and source records attributed to B K Rima.

At least 19 recordsLinked to original sources
PubMed2006

CD9-dependent regulation of Canine distemper virus-induced cell-cell fusion segregates with the extracellular domain of the haemagglutinin.

Antibodies to CD9, a member of the tetraspan transmembrane-protein family, selectively inhibit Canine distemper virus (CDV)-induced cell-cell fusion. Neither CDV-induced virus-cell fusion nor cell-cell fusion induced by the closely related morbillivirus Measles virus (MV) is affected by anti-CD9 antibodies. As CDV does not bind CD9, an unknown, indirect mechanism is responsible for the observed inhibition of cell-cell fusion. It was investigated whether this effect was restricted to only one viral glycoprotein, either the haemagglutinin (H) or the fusion (F) protein, which form a fusion complex on the surface of virions and infected cells, or whether it is dependent on both in transient co-transfection assays. The susceptibility to CD9 antibodies segregates with the H protein of CDV. By exchanging portions of the H proteins of CDV and MV, it was determined that the complete extracellular domain, including the predicted stem structure (stem 1, barrel strand 1 and stem 2) and globular head domain, of the CDV-H protein mediates the effect. This suggests that interaction of the CDV-H protein with an unknown cellular receptor(s) is regulated by CD9, rather than F protein-mediated membrane fusion.

Amino Acid Sequence↗
PubMed2006

A mouse model of persistent brain infection with recombinant Measles virus.

Measles virus (MV) nucleocapsids are present abundantly in brain cells of patients with subacute sclerosing panencephalitis (SSPE). This invariably lethal brain disease develops years after acute measles as result of a persistent MV infection. Various rodent models for MV infection of the central nervous system (CNS) have been described in the past, in which the detection of viral antigens is based on histological staining procedures of paraffin embedded brains. Here, the usage of a recombinant MV (MV-EGFP-CAMH) expressing the haemagglutinin (H) of the rodent-adapted MV-strain CAM/RB and the enhanced green fluorescent protein (EGFP) is described. In newborn rodents the virus infects neurons and causes an acute lethal encephalitis. From 2 weeks on, when the immune system of the genetically unmodified animal is maturating, intracerebral (i.c.) infection is overcome subclinically, however, a focal persistent infection in groups of neurons remains. The complete brain can be analysed in 50 or 100 microm slices, and infected autofluorescent cells are readily detected. Seven and 28 days post-infection (p.i.) 86 and 81% of mice are infected, respectively, and virus persists for more than 50 days p.i. Intraperitoneal immunization with MV 1 week before infection, but not after infection, protects and prevents persistence. The high percentage of persistence demonstrates that this is a reliable and useful model of a persistent CNS infection in fully immunocompetent mice, which allows the investigation of determinants of the immune system.

Age Factors↗
PubMed2005

Completion of the sequence of a cetacean morbillivirus and comparative analysis of the complete genome sequences of four morbilliviruses.

The gene encoding the large (L) protein and the genome termini of the dolphin strain of cetacean morbillivirus (CeMV) were sequenced. The CeMV genome is 15702 nucleotides long and has been compared with other available morbillivirus genome sequences in regards to the "rule of six" and the "phase" of any particular nucleotide, defined as its position within a given hexamer, which here is defined as a group of six nucleotides starting from the 3' end of the genomic RNA. With exception of the position of the start of the F gene, the phase of the transcription start sites of each gene is strictly conserved between the morbilliviruses, but each gene is in a different phase. The lengths of gene transcripts differ between viruses by multiples of six nucleotides with exception of the M and F transcripts. The differences between the various morbilliviruses result from deletions or insertions of multiples of six nucleotides in the 3' and 5' UTRs of the different viral genes. The four bases were distributed non-randomly over the six positions in the hexamer boxes. However, the distribution patterns of each of the four bases indicated that multiples of three were more prevalent than those of six nucleotides. This reflected the positions of nucleotides in codons and codon usage in the reading frames. The L protein of CeMV was found to be 2183 amino acids in length and similar to that of MV and RPV. The CeMV L protein sequence was found to be equidistant between those of the CDV/PDV and MV/RPV subgroups of the morbilliviruses. This concurs with the analyses carried out on the other structural proteins.

3' Untranslated Regions↗
PubMed2005

'Rescue' of mini-genomic constructs and viruses by combinations of morbillivirus N, P and L proteins.

Chloramphenicol acetyltransferase (CAT)-expressing negative-sense mini-genomic constructs of measles virus (MV) and rinderpest virus (RPV) were rescued by standard technology with helper plasmids expressing the nucleocapsid (N), phospho- (P) and large (L) proteins of MV, canine distemper virus (CDV) or RPV in order to determine whether the proteins of different viruses can function together. Homogeneous sets consisting of N, P and L plasmids derived from one virus were able to generate reporter gene expression from either mini-genomic construct. Heterogeneous sets of proteins from different viruses were not functional, with the exception that a low level of activity was obtained when MV N and P protein were combined with RPV L protein in the rescue of the MV mini-genomic construct, or CDV N was combined with RPV P and L in the rescue of the RPV mini-genome. However, only homogeneous sets of plasmids were able to rescue infectious virus from full-length anti-genome-expressing plasmids.

Amino Acid Sequence↗
PubMed2000

Parvovirus B19 in kidney transplant patients.

Renal transplant patients were screened for the presence of parvovirus B19, before transplantation and monthly for 4 months after transplantation, by means of a sensitive nested PCR assay. Upon screening plasma from 110 patients, we found that two asymptomatic patients were B19 DNA positive. One of these patients was PCR positive in the plasma sample taken 2 months after transplantation; the plasma contained anti-B19 IgG antibodies before transplant and throughout the follow-up period, with an increase in the IgG level in the second posttransplant sample coinciding with the detection of B19 DNA. IgM antibodies to B19 were not detected in this patient. Because, for this patient, the donor's spleen DNA was also B19 DNA positive, we suspect B19 transmission from the donor and limited B19 replication, inasmuch as this patient already had a primed immune response to B19. The other patient was PCR positive in the pretransplant and in the plasma sample taken 1 month after transplant and contained a strong anti-B19 IgG response in the pretransplant sample and throughout the follow-up period-and anti-B19 IgM antibodies were not detected before or after transplantation. By testing samples taken from this patient at 2 weeks, 2 months, and 3 months before transplantation, we were able to determine that the infection occurred shortly before transplantation. Unexpectedly, this graft failed and was removed 2 days after transplantation despite a negative cross-match. A pathological examination of the kidney indicated acute vascular rejection, suggesting a possible role for B19 in this complication.

Antibodies, Viral↗
PubMed2000

The major tegumental antigen of Fasciola hepatica contains repeated elements.

In order to provide a better understanding of the interaction between the liver fluke (Fasciola hepatica) and the immune system of its mammalian host immunoreactive lambda bacteriophage clones containing F. hepatica cDNA have been isolated. Plasmids from these clones were sequenced and found to encode a family of proteins containing certain common elementst. All the clones contained a coding repeating sequence (RRRXCA) which is conserved at the nucleic acid level followed by a non-repeating element coding for the C terminal used by the proteins which shows conservation of amino acids at certain positions. Antisera raised against a beta-galactosidase fusion protein with one of these sequences as a terminal extension was used to localize the immunoreactive antigens. Binding was predominantly in the tegument of the juvenile fluke but was reduced in the adult tegument. The wall of the uterus showed strong reactivity in the adult. Rats immunized with the beta-galactosidase fusion protein showed enhanced resistance to challenge infections. The role of these antigens in the host response to infection by F. hepatica is discussed.

Amino Acid Sequence↗
PubMed2000

In vitro and in vivo infection of neural cells by a recombinant measles virus expressing enhanced green fluorescent protein.

This study focused on the in vitro infection of mouse and human neuroblastoma cells and the in vivo infection of the murine central nervous system with a recombinant measles virus. An undifferentiated mouse neuroblastoma cell line (TMN) was infected with the vaccine strain of measles virus (MVeGFP), which expresses enhanced green fluorescent protein (EGFP). MVeGFP infected the cells, and cell-to-cell spread was studied by virtue of the resulting EGFP autofluorescence, using real-time confocal microscopy. Cells were differentiated to a neuronal phenotype, and extended processes, which interconnected the cells, were observed. It was also possible to infect the differentiated neuroblastoma cells (dTMN) with MVeGFP. Single autofluorescent EGFP-positive cells were selected at the earliest possible point in the infection, and the spread of EGFP autofluorescence was monitored. In this instance the virus used the interconnecting processes to spread from cell to cell. Human neuroblastoma cells (SH-SY-5Y) were also infected with MVeGFP. The virus infected these cells, and existing processes were used to initiate new foci of infection at distinct regions of the monolayer. Transgenic animals expressing CD46, a measles virus receptor, and lacking interferon type 1 receptor gene were infected intracerebrally with MVeGFP. A productive infection ensued, and the mice exhibited clinical signs of infection, such as ataxia and an awkward gait, identical to those previously observed for the parental virus (Edtag). Mice were sacrificed, and brain sections were examined for EGFP autofluorescence by confocal scanning laser microscopy over a period of 6 h. EGFP was detected in discrete focal regions of the brain and in processes, which extended deep into the parenchyma. Collectively, these results indicate (i) that MVeGFP can be used to monitor virus replication sensitively, in real time, in animal tissues, (ii) that infection of ependymal cells and neuroblasts provides a route by which measles virus can enter the central nervous system in mouse models of encephalitis, and (iii) that upon infection, the virus spreads transneuronally.

Animals↗
PubMed2000

Establishment of a rescue system for canine distemper virus.

Canine distemper virus (CDV) has been rescued from a full-length cDNA clone. Besides Measles virus (MV) and Rinderpest virus, a third morbillivirus is now available for genetic analysis using reverse genetics. A plasmid p(+)CDV was constructed by sequential cloning using the Onderstepoort vaccine strain large-plaque-forming variant. The presence of a T7 promoter allowed transcription of full-length antigenomic RNA by a T7 RNA polymerase, which was provided by a host range mutant of vaccinia virus (MVA-T7). Plasmids expressing the nucleocapsid protein, the phosphoprotein, and the viral RNA-dependent RNA polymerase, also under control of a T7 promoter, have been generated. Infection of HeLa cells with MVA-T7 and subsequent transfection of p(+)CDV plus the helper plasmids led to syncytium formation and release of infectious recombinant (r) CDV. Comparison of the rescued virus with the parental virus revealed no major differences in the progression of infection or in the shape and size of syncytia. A genetic tag, consisting of two nucleotide changes within the coding region of the L protein, has been identified in the rCDV genome. Expression by rCDV of all the major viral structural proteins has been demonstrated by immunofluorescence.

Animals↗
PubMed2000

Measles virus-induced disruption of the glial-fibrillary-acidic protein cytoskeleton in an astrocytoma cell line (U-251).

A recombinant measles virus which expresses enhanced green fluorescent protein (MVeGFP) has been used to infect two astrocytoma cell lines (GCCM and U-251) to study the effect of virus infection on the cytoskeleton. Indirect immunocytochemistry was used to demonstrate the cellular localization of the cytoskeletal components. Enhanced green fluorescent protein autofluorescence was used to identify measles virus-infected cells. No alteration of the actin, tubulin, or vimentin components of the cytoskeleton was observed in either cell type, whereas a disruption of the glial-fibrillary-acidic protein filament (GFAP) network was noted in MVeGFP-infected U-251 cells. The relative amounts of GFAP present in infected and uninfected U-251 cells were quantified by image analysis of data sets obtained by confocal microscopy by using vimentin, another intermediate filament on which MVeGFP has no effect, as a control.

Astrocytoma↗
PubMed1999

Aberrant splicing of intron 1 creates a novel null HLA-B*1501 allele.

A comparison of serological and DNA HLA class I typing data identified a serological "blank" HLA-B15 antigen in a volunteer donor on the bone marrow registry. Isoelectric focusing and Western blot analysis of a cell line established from this individual confirmed that the HLA-B15 antigen is not expressed at the cell surface. Nucleotide sequence analysis of the HLA-B*15 null allele revealed a 10-bp deletion near the 3' end of intron 1, when compared to the normal HLA-B*1501 sequence. All of the HLA-B*15 specific cDNA clones examined retained the intron 1 sequence. Reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis demonstrated that the HLA-B*15 mRNA molecule contained the intron 1 sequence, indicating an inability to efficiently splice out intron 1 from the mRNA transcript. The retention of the mutated intron 1 sequence in the mRNA causes a frameshift and premature termination of translation at the start of exon 2, explaining the HLA-B*1501 null phenotype. Our data predicts that the HLA-B*1501 null allele would express a small truncated protein containing the signal sequence fused to an ORF within intron 1 and terminating (out of frame) just within exon 2.

Alleles↗
PubMed1999

Altered virulence of vaccine strains of measles virus after prolonged replication in human tissue.

To understand the molecular determinants of measles virus (MV) virulence, we have used the SCID-hu thymus/liver xenograft model (SCID-hu thy/liv) in which in vivo MV virulence phenotypes are faithfully duplicated. Stromal epithelial and monocytic cells are infected by MV in thymus implants, and virulent strains induce massive thymocyte apoptosis, although thymocytes are not infected. To determine whether passage of an avirulent vaccine strain in human tissue increases virulence, we studied a virus isolated from thymic tissue 90 days after infection with the vaccine strain Moraten (pMor-1) and a virus isolated from an immunodeficient child with progressive vaccine-induced disease (Hu2). These viruses were compared to a minimally passaged wild-type Edmonston strain (Ed-wt) and the vaccine strain Moraten. pMor-1, Hu2, and Ed-wt displayed virulent phenotypes in thymic implants, with high levels of virus being detected by 3 days after infection (10(5.2), 10(2.8), and 10(3. 4), respectively) and maximal levels being detected between 7 and 14 days after infection. In contrast, Moraten required over 14 days to grow to detectable levels. pMor-1 produced the highest levels of virus throughout infection, suggesting thymic adaptation of this strain. Similar to other virulent strains, Ed-wt, Hu2, and pMor-1 caused a decrease in the number of viable thymocytes as assessed by trypan blue exclusion and fluorescence-activated cell sorter analysis. Thymic architecture was also disrupted by these strains. Sequence analysis of the hemagglutinin (H) and matrix (M) genes showed no common changes in Hu2 and pMor-1. M sequences were identical in pMor-1 and Mor and varied in H at amino acid 469 (threonine to alanine), a position near the base of propeller 4 in the propeller blade/stem model of H structure. Further study will provide insights into the determinants of virulence.

Cell Line↗
PubMed1999

Observation of measles virus cell-to-cell spread in astrocytoma cells by using a green fluorescent protein-expressing recombinant virus.

A recombinant measles virus (MV) which expresses enhanced green fluorescent protein (EGFP) has been rescued. This virus, MVeGFP, expresses the reporter gene from an additional transcription unit which is located prior to the gene encoding the measles virus nucleocapsid protein. The recombinant virus was used to infect human astrocytoma cells (GCCM). Immunocytochemistry (ICC) together with EGFP autofluorescence showed that EGFP is both an early and very sensitive indicator of cell infection. Cells that were EGFP-positive and ICC-negative were frequently observed. Confocal microscopy was used to indirectly visualize MV infection of GCCM cells and to subsequently follow cell-to-cell spread in real time. These astrocytoma cells have extended processes, which in many cases are intimately associated. The processes appear to have an important role in cell-to-cell spread, and MVeGFP was observed to utilize them in the infection of surrounding cells. Heterogeneity was seen in cell-to-cell spread in what was expected to be a homogeneous monolayer. In tissue culture, physical constraints govern the integrity of the syncytia which are formed upon extensive cell fusion. When around 50 cells were fused, the syncytia rapidly disintegrated and many of the infected cells detached. Residual adherent EGFP-positive cells were seen to either continue to be involved in the infection of surrounding cells or to remain EGFP positive but no longer participate in the transmission of MV infection to neighboring cells.

Animals↗
PubMed1999

The H gene of rodent brain-adapted measles virus confers neurovirulence to the Edmonston vaccine strain.

Molecular determinants of neuropathogenesis have been shown to be present in the hemagglutinin (H) protein of measles virus (MV). An H gene insertion vector has been generated from the Edmonston B vaccine full-length infectious clone of MV. Using this vector, it is possible to insert complete H open reading frames into the parental (Edtag) background. The H gene from a rodent brain-adapted MV strain (CAM/RB) was inserted into this vector, and a recombinant virus (EdtagCAMH) was rescued by using a modified vaccinia virus which expresses T7 RNA polymerase (MVA-T7). The recombinant virus grew at an equivalent rate and to similar titers as the CAM/RB and Edtag parental viruses. Neurovirulence was assayed in a mouse model for MV encephalitis. Viruses were injected intracerebrally into the right cortex of C57/BL/6 suckling mice. After infection mice inoculated with the CAM/RB strain developed hind limb paralysis and ataxia. Clinical symptoms were never observed with an equivalent dose of Edtag virus or in sham infections. Immunohistochemistry (IHC) was used to detect viral antigen in formalin-fixed brain sections. Measles antigen was observed in neurons and neuronal processes of the hippocampus, frontal, temporal, and olfactory cortices and neostriatum on both sides of symmetrical structures. Viral antigen was not detected in mice infected with Edtag virus. Mice infected with the recombinant virus, EdtagCAMH, became clinically ill, and virus was detected by IHC in regions of the brain similar to those in which it was detected in animals infected with CAM/RB. The EdtagCAMH infection had, however, progressed much less than the CAM/RB virus at 4 days postinfection. It therefore appears that additional determinants are encoded in other regions of the MV genome which are required for full neurovirulence equivalent to CAM/RB. Nevertheless, replacement of the H gene alone is sufficient to cause neuropathology.

Adaptation, Biological↗
PubMed1997

Comparative antibody response in harbour and grey seals naturally infected by a morbillivirus.

The antibody response of free-ranging harbour and grey seals, naturally infected by a morbillivirus, was assessed using a virus neutralizing test and a radio-immunoprecipitation assay. The prevalence of antibody was similar between species, however, grey seals had significantly higher virus neutralizing titers. Serum from clinically healthy grey seals precipitated the nucleocapsid (N) protein along with the hemagglutinin (H) and fusion (F) glycoproteins. By contrast, significantly fewer harbour seal sera precipitated the envelope glycoproteins and responses were weaker than those of grey seals. One harbour seal with acute morbillivirus pneumonia, and two with encephalitis precipitated only the N protein. Serum from four harbour seals with encephalitis weakly recognized the envelope glycoproteins. Thus, the antibody response of grey seals appears more competent than that of harbour seals with respect to morbillivirus antigens. We speculate that this difference between the species may be an important determinant of morbillivirus susceptibility.

Animals↗
PubMed1997

Sequence divergence of measles virus haemagglutinin during natural evolution and adaptation to cell culture.

Phylogenetic analysis of the sequence of the H gene of 75 measles virus (MV) strains (32 published and 43 new sequences) was carried out. The lineage groups described from comparison of the nucleotide sequences encoding the C-terminal regions of the N protein of MV were the same as those derived from the H gene sequences in almost all cases. The databases document a number of distinct genotype switches that have occurred in Madrid (Spain). Well-documented is the complete replacement of lineage group C2, the common European genotype at that time, with that of group D3 around the autumn of 1993. No further isolations of group C2 took place in Madrid after this time. The rate of mutation of the H gene sequences of MV genotype D3 circulating in Madrid from 1993 to 1996 was very low (5 x 10(-4) per annum for a given nucleotide position). This is an order of magnitude lower than the rates of mutation observed in the HN genes of human influenza A viruses. The ratio of expressed over silent mutations indicated that the divergence was not driven by immune selection in this gene. Variations in amino acid 117 of the H protein (F or L) may be related to the ability of some strains to haemagglutinate only in the presence of salt. Adaptation of MV to different primate cell types was associated with very small numbers of mutations in the H gene. The changes could not be predicted when virus previously grown in human B cell lines was adapted to monkey Vero cells. In contrast, rodent brain-adapted viruses displayed a lot of amino acid sequence variation from normal MV strains. There was no convincing evidence for recombination between MV genotypes.

Animals↗
PubMed1997

Dinucleotide and stop codon frequencies in single-stranded RNA viruses.

To identify potential selection pressures which lead to RNA sequence conservation, we examined the occurrence rates of dinucleotides in 64 single-stranded RNA virus genomes. These viruses may offer a particular insight into these pressures since their RNA-dependent RNA polymerases lack proofreading capability. This potentiates introduction of mutations into their genomes, yet unidentified selection processes conserve the genomes to a large degree. We report a strong inverse correlation between the C+G content and the occurrence of the CpG dinucleotide (r=0.71) in the RNA virus genomes, in contrast to earlier reports (Karlin et al., 1994, Journal of Virology 68, 2889-2897). We also detected significant suppression of UpA, correlating inversely with genomic U+A content. These suppressions are coupled with over-representation of the complementary pair of dinucleotides, CpA and UpG. In addition, we highlight the fact that odds ratios for dinucleotides are not independent variables, a situation apparently not widely appreciated in the literature. This led us to view the over-representation of CpA and UpG as a consequential outcome of UpA and CpG suppression in the virus genomes. Potential factors influencing these disturbances are discussed. In addition, higher than random incidence was observed for 'out-of-frame' stop codons in the viral RNA genomes, with some preferences for individual codons being exhibited by certain virus groups. The UAG codon appeared more common in the +1 frame, the UGA in the -1 frame.

Base Sequence↗
PubMed1997

Nucleotide sequence analysis of the large (L) genes of phocine distemper virus and canine distemper virus (corrected sequence).

This paper corrects the previously published sequence of the L gene of canine distemper virus (CDV). Errors in the published sequence (M. S. Sidhu et al., 1993, Virology 193, 50-65) led to frame shifts between residues 1021-1032, 1190-1219 and 1645-1650; a deletion of 21 amino acids between residues 1684-1705, and a single residue deletion at residue 1478. Residue 237 is now found to be glycine rather than tryptophan and residue 1626 proline instead of threonine. The sequence of the L gene of phocine distemper virus (PDV) was also determined. Alignment of the morbillivirus L proteins showed that PDV and CDV are more closely related to each other than to rinderpest virus and measles virus. Two regions of low identity are proposed to function as hinge regions between three highly conserved domains (I-III) in the morbillivirus L proteins. New sequence motifs have been identified on the basis of conservation in the morbilliviruses and the Paramyxovirinae.

Amino Acid Sequence↗