Biomedical subjects
Publications and source records attributed to B K Rima.
The SH gene and its flanking sequences have been analysed for 10 strains of mumps virus (MuV) and compared to 5 others. A new lineage has been identified among UK isolates. Changes in the transcription pattern could not be correlated with differences in the sequences of the F-SH and SH-HN intergenic regions of the genome.
The gene encoding the major nucleocapsid, N, protein of measles virus has been inserted into a baculovirus vector under the control of the polyhedrin promoter. Insect cells infected with this recombinant baculovirus synthesize high levels of measles N protein, up to 40% of total soluble cell protein. The recombinant protein is recognized by sera from convalescent patients, vaccinees and patients with subacute sclerosing panencephalitis and thus could form the basis of a simple diagnostic assay. Nucleocapsid-like structures, similar to those found in mammalian cells infected with measles virus, can be observed in both the nucleus and cytoplasm of the infected insect cells. These have many structural features in common with nucleocapsids found in measles virus-infected cells, but are longer (up to 2 microns) and have a lower buoyant density. Measles N protein thus appears to be capable of assembling into nucleocapsid-like structures in the absence of measles virion RNA or other viral proteins.
The biochemical characterisation of phocine distemper virus (PDV) has shown that PDV is related to but clearly distinct from canine distemper virus (CDV) and relative to its relationship with CDV is only remotely related to the other morbilliviruses, namely measles virus (MV) or rinderpest virus (RPV) and peste-des-petits-ruminants virus (PPRV). Comparative studies with monoclonal antibodies indicate that the virus is serologically closely related to CDV with many conserved epitopes, particularly on the internal proteins of the virus, while the external attachment (H) protein shows the greatest level of variability among the distemper virus isolates. The analysis of the viral proteins by electrophoresis indicates molecular weight differences between CDV and PDV in the fusion (F), phosphoprotein (P), H, nucleocapsid (N) and matrix (M) proteins. The RNA profiles of CDV and PDV are indistinguishable and different from those for RPV and MV. Nucleotide sequence analysis of cDNA clones of the virus show approximately 70% homology between CDV and PDV and approximately 48% with MV. These data prove that PDV is a different virus from CDV and co-circulates with it probably primarily in sea mammals.
A virus isolated from a porpoise during the 1988 seal epizootic was shown to be a morbillivirus. In order to determine the relationship of the virus to phocine distemper virus (PDV) a battery of monoclonal antibodies raised against canine distemper virus (CDV), PDV or the porpoise isolate were assessed for their ability to bind to CDV, PDV or porpoise virus epitopes in indirect immunofluorescence assays and ELISAs. The porpoise isolate contained several unique epitopes and several epitopes present on CDV and PDV were absent on the porpoise isolate. The data presented in this study indicate that the porpoise virus is an antigenically distinct morbillivirus and as such has been tentatively named as delphinoid distemper virus (DDV).
The nucleotide sequence of the gene encoding the fusion protein of phocine distemper virus has been determined. The mRNA is 2206 nucleotides in length and contains one major open reading frame (ORF) of 1893 nucleotides encoding a potential protein of 631 amino acid residues. However, analogy with canine distemper virus (CDV) suggests that translation of the F protein starts at the sixth AUG codon in the mRNA sequence which is located at position 461, resulting in an F0 protein of exactly the same size (537 aa) as that of CDV. The overall homology at nucleotide level between the CDV and PDV F genes is 66%. The homology between the two F proteins of these respective viruses is 83%.
The nucleotide sequences of the N and P genes of two wild type measles virus strains JM and CM in two distinct lineages of the virus have been analyzed and compared with those of other MV strains in order to assess which parts of the internal proteins are variable. Most variations in the P protein appear to occur in the N-terminus, while the middle part of the protein (residues 201-350) and the C-terminus are conserved. The C protein varies primarily in its N-terminal amino acids. The C-terminal amino acid residues of the V protein, which are unique to this protein, do not vary significantly between measles virus strains. The data show that evolutionary trees determined on the basis of the N, P, or M genes are the same and that probably no recombination has taken place between these genes in the strains investigated so far. The M protein appears to be less variable than the other genes and thus changes observed in this gene in some SSPE and MIBE viruses may be of greater significance than were assumed earlier.
Generation of a set of cDNA clones covering the N, P/V/C, M, F, H and part of the L gene of phocine distemper virus (PDV) has been described. The gene order of PDV determined from a physical as well as a transcriptional map, was identical to that of the other morbilliviruses so far studied. The H gene sequence (1951 nucleotides) contains one large open reading frame which encodes a protein of 607 amino acids, identical in length to that of the H protein of the Convac strain of canine distemper virus (CDV). Nucleotide and protein sequence comparisons between PDV and the other morbilliviruses provide further evidence in favour of PDV's inclusion in the morbillivirus genus as a distinct species closely related to CDV and more distantly to measles virus and rinderpest virus.
The nucleotide sequences of the phosphoprotein (P)/V/C and matrix (M) protein genes of phocine distemper virus (PDV) have been determined and the deduced amino acid sequences of the proteins derived from these genes compared with those of the other morbilliviruses. The 1655 nucleotides of the P gene encode a phosphoprotein of 507 amino acid residues (from nucleotide numbers 60 to 1583) which is 75% identical to that of canine distemper virus (CDV). The C proteins of the two viruses are 73% identical. The C protein has the same length, 174 amino acid residues, as C of CDV. The nucleotide sequences of the P genes are 78% identical. The editing site in the P gene is present as a stretch of 18 nucleotides, conserved in all other morbilliviruses. A V-like protein can be accessed by insertion of one G residue at this site. The P and M genes are separated from adjacent genes and each other by the CTT trinucleotide sequence which is totally conserved in morbilliviruses. The M gene is 1447 nucleotides long and encodes a typical paramyxovirus M protein of 335 amino acids (identical in length to those of CDV, measles virus and rinderpest virus). The gene is 78% identical to CDV in the coding region (nucleotides 32 to 1039) but only 34% identical to CDV in the 407 nucleotides of the 3' untranslated sequence of the M mRNA. The M protein of PDV is 91% identical to the M protein of CDV. The data demonstrate interesting variations in the level of conservation of various genome areas and prove the distinct nature of PDV.
A morbillivirus was isolated from lung tissue of a porpoise which had lesions similar to those of phocine distemper. Porpoise and seal isolates differed in their reactions to monoclonal antibodies to canine distemper virus.
The role of the humoral immune response in clearance or prevention of canine distemper viral encephalitis of dogs infected with a virulent strain of canine distemper virus has been evaluated. Dogs that have demyelinating lesions, CDV proteins and infectious virus in their brains demonstrate an impaired humoral immune response. In dogs that recover from infection and contain no demyelinating lesions, viral proteins or infectious virus in the brain, antibodies to the internal proteins of CDV are observed early after infection. Later antibodies to primarily the H protein are detectable in sera of these dogs and the appearance of antibodies against the surface glycoprotein (H) correlates with the absence of lesions, CDV antigen and infectious virus in the brains of these dogs. Very late after infection immunoprecipitating antibody to all CDV antigens diminished rapidly so that at about ten weeks post infection antibodies that precipitate CDV antigens are barely detectable.
Inclusion body myositis (IBM) is a distinct type of muscle disease. The characteristic electron microscopic findings, intranuclear or intracytoplasmic inclusions composed of microtubular filaments, morphologically resemble paramyxovirus nucleocapsids. These findings and the reported immunoreactivity of the inclusions with mumps virus antibodies have suggested that inclusion body myositis is a chronic virus infection. We analyzed skeletal muscle specimens from three patients with characteristic light microscopic features and electron microscopically verified inclusions of IBM by immunocytochemistry using antibodies raised against members of the paramyxovirus group, and by in situ hybridization with a cRNA probe representing the mumps virus nucleocapsid gene. The specificity of the reactions was demonstrated with infected and uninfected cultured cells. No immunocytochemical staining or hybridization signal was observed in biopsy specimens from IBM patients. These findings speak against a paramyxovirus etiology of IBM.
The sequences of a region of the nucleocapsid protein gene, between nucleotides 1231 and 1686, encoding the C-terminal 151 amino acid residues of the nucleocapsid protein have been determined for 16 strains of measles virus. Analysis of this region showed that it is highly divergent (up to 7.2% divergence in the nucleotide sequence and 10.6% divergence in the amino acid sequence between most distant strains) and that several lineages of measles virus can be found to co-circulate at a given time. Some of the lineages show geographical restriction. The results for measles virus are similar to those reported for other human paramyxoviruses such as mumps virus, parainfluenza type 3 virus and the avian Newcastle disease virus.
The sequence of the H gene and flanking sequences in the F and L genes of canine distemper virus (CDV) have been determined. The H gene of CDV (1946 nucleotides) contains one large open reading frame starting at position 21 and terminating at position 1835, encoding a protein of 604 amino acid residues. This protein contains three potential glycosylation sites in the extracellular domain and, like all other paramyxoviruses, a N-terminal membrane-spanning hydrophobic anchor domain. The deduced H protein sequence shows an identity of 36% with rinderpest virus (RPV) and measles virus (MV). The identities at the nucleotide level are higher (RPV 52% and MV 53%). The amino acid sequence shows conservation of all the structural determinants with the H proteins of MV and RPV. The data also show that CDV is evolutionarily equidistant to RPV and MV with respect to the H gene.
The nucleotide sequences of the matrix protein (M) genes of two wild-type measles virus (MV) isolates (JM and CM) have been determined and shown to differ in 56 positions; 31 of these differences are located in the non-coding region and 25 in the coding region of the gene. Most (80%) of the mutations in the coding region are changes to the third base of a codon. A maximum parsimony analysis of the available M gene nucleotide sequences allowed the construction of a tree with at least three lineages or subtypes. One wild-type strain (JM) was very similar to a subacute sclerosing panencephalitis virus strain (case B); the second wild-type strain, CM, showed nucleotide sequence similarity with MV from a case of measles inclusion body encephalitis. Both wild-type virus sequences are distinct from those so far determined for vaccine strains.
During 1988 thousands of harbor seals (Phoca vitulina) died in European seas as a result of morbillivirus infection. Six harbor porpoises (Phocoena phocoena) found stranded on the coast of Northern Ireland in late 1988 were submitted to our laboratory for necropsy. Pneumonia was the main necropsy finding in three of these animals. Microscopic lung lesions characterized by necrosis of bronchial and bronchiolar epithelium and infiltration of alveoli with leukocytes, lymphoid cells, macrophages, and multinucleate syncytia were seen in all six porpoises. Cytoplasmic and nuclear acidophilic inclusions characteristic of morbillivirus infection were common in bronchial and bronchiolar epithelial cells and in alveolar macrophages and syncytia. Brain alterations included degeneration and necrosis of neurons, microglial infiltration, and perivascular cuffing. There were cytoplasmic and nuclear acidophilic inclusions in many neurons. Immunoperoxidase staining of morbillivirus antigen was seen in many tissues including lung, brain, spleen, and urinary bladder. Alterations in our porpoises were similar to those seen in distemper in seals and many species of terrestrial mammals. Systemic viral disease has not previously been documented in Cetacea.
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Evidence has been presented that generation of polycistronic readthrough RNAs in mumps virus-infected cells is not a simple stochastic process with strain-dependent variations in the generation of certain readthrough products, but that this process is affected by host as well as viral factors. RNAs extracted from infected Vero cells or chicken embryo fibroblast (CEF) cells have been analysed by Northern blotting with virus-specific probes for the nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), small hydrophobic (SH) and haemagglutinin-neuraminidase (HN) genes. Vero cells infected with tissue culture cell-adapted virus strains generate monocistronic as well as polycistronic RNAs. Transcription analysis of Vero cells infected with an egg-adapted strain reveal the absence of monocistronic M and F transcripts, with a concomitant increase in readthrough transcripts involving these genes. When the same virus infects CEF cells monocistronic RNAs accumulate. The presence of viral proteins in the various virus/host cell combinations assessed by immunofluorescence with mumps virus-specific monoclonal antibodies for the N, P, M, F and HN proteins correlates well with the patterns of transcription.