Gert Bolt

Genetic diversity of the attachment (H) protein gene of current field isolates of canine distemper virus

To characterize the variability of recent field isolates of canine distemper virus (CDV) from different hosts and geographical areas, we conducted nucleotide sequence analysis of the gene encoding the haemagglutinin (H), the attachment protein of this virus. Pronounced differences between field isolates were revealed in comparison to the Convac and Onderstepoort vaccine strains. The diversity of CDV appeared to exceed that determined for measles virus. Phylogenetic analysis also separated the field isolates of CDV from the vaccine strains and provided evidence for the existence of different contemporary genotypes of CDV. Isolates from a Greenlandic sledge dog and a Siberian seal formed a distinct lineage. The remaining isolates formed a group. This group contained two European isolates from mink and ferret, a single lineage comprising three European dog isolates, and another separate lineage of North American isolates from dog, javelina, raccoon and captive leopards.

Measles virus-induced modulation of host-cell gene expression

The influence of measles virus (MV) infection on gene expression by human peripheral blood mononuclear cells (PBMCs) was examined with cDNA microarrays. The mRNA levels of more than 3000 cellular genes were compared between uninfected PBMCs and cells infected with either the Edmonston MV strain or a wild-type MV isolate. The MV-induced upregulation of individual genes identified by microarray analyses was confirmed by RT-PCR. In the present study, a total of 17 genes was found to be upregulated by MV infection. The Edmonston strain grew better in the PBMC cultures than the wild-type MV, and the Edmonston strain was a stronger inducer of the upregulated host cell genes than the wild-type virus. The anti-apoptotic B cell lymphoma 3 (Bcl-3) protein and the transcription factor NF-kappaB p52 subunit were upregulated in infected PBMCs both at the mRNA and at the protein level. Several genes of the interferon system including that for interferon regulatory factor 7 were upregulated by MV. The genes for a number of chaperones, transcription factors and other proteins of the endoplasmic reticulum stress response were also upregulated. These included the gene for the pro-apoptotic and growth arrest-inducing CHOP/GADD153 protein. Thus, the present study demonstrated the activation by MV of cellular mechanisms and pathways that may play a role in the pathogenesis of measles.

Adaptation of wild-type measles virus to CD46 receptor usage

Summary. Vaccine strains of measles virus (MV) use CD46 as receptor and downregulate CD46 from the surface of infected cells. MVs isolated and passaged on B-lymphoid cells (wild-type MVs) seem to use another receptor and do not downregulate CD46. In the present study, we found that isolation of MV on human or marmoset B-lymphoid cells did not alter the MV haemagglutinin (H) protein relative to that in the patient. The wild-type isolates were adapted to the human epithelial HEp-2 cell line or the monkey fibroblast Vero cell line. All HEp-2 cell adapted viruses and 1 out of 4 Vero cell adapted viruses acquired the capacity to use CD46 as receptor, as measured by their ability to infect murine cells expressing human CD46. Adaptation to CD46 receptor usage was coupled to substitution of amino acid 481 of the MV H protein from asparagine to tyrosine but not to CD46 downregulation. The present study demonstrates that CD46 receptor usage can be induced by adaptation of wild-type MV to cells that do not express a wild-type receptor and suggests that a similar mechanism acted on the progenitor viruses of the present MV vaccine strains during their isolation and attenuation.

Cleavage of the respiratory syncytial virus fusion protein is required for its surface expression: role of furin

The fusion (F) glycoprotein of respiratory syncytial virus (RSV) is synthesized as a nonfusogenic precursor protein (F(0)), which during its migration to the cell surface is activated by cleavage into the disulfide-linked F(1) and F(2) subunits. In the present study, soluble secreted human furin produced by a recombinant baculovirus cleaved RSV F(0) into proteins the size of F(1) and F(2). Furthermore, cleavage of F(0) was partially inhibited in the furin defective LoVo cell line, in calcium depleted HEp-2 cells, and in HEp-2 cells treated with the furin inhibitor decanoyl-R-V-K-R-chloromethylketon. These findings strongly suggest an important role for furin in activation of the RSV F protein. The F(0) protein could not be detected on the surface of cells, in which F protein activation was inhibited, and RSV particles did not appear to be released from these cells. It thus seems that in contrast to the F proteins of most other paramyxoviruses, the RSV F(0) protein is very inefficient in reaching the cell surface or is unable to reach the cell surface and therefore cannot be incorporated into virus particles.

Nucleotide and deduced amino acid sequences of the matrix (M) and fusion (F) protein genes of cetacean morbilliviruses isolated from a porpoise and a dolphin

Morbilliviruses have been isolated from stranded dolphins and porpoises. The present paper describes the cloning and sequencing of the porpoise morbillivirus (PMV) F gene and of the dolphin morbillivirus (DMV) M and F genes and their flanking regions. The gene order of the DMV genome appeared to be identical to that of other morbilliviruses. A genomic untranslated region of 837 nucleotides was found between the translated DMV M and F gene regions. The predicted DMV M protein were highly conserved with those of other morbilliviruses. Both the deduced PMV and DMV F0 proteins exhibited three major hydrophobic regions as well as a cysteine rich region, a leucine zipper motif and a cleavage motif allowing cleavage of the F0 protein into F1 and F2 subunits. Apparently the DMV F0 cleavage motif was not modified by adaptation of DMV to Vero cells. The predicted PMV and DMV F proteins were 94% identical. Comparisons with the corresponding sequences of other morbilliviruses demonstrated that the cetacean morbillivirus does not derive from any known morbillivirus but represents an independent morbillivirus lineage.

Comparative analysis of the gene encoding the nucleocapsid protein of dolphin morbillivirus reveals its distant evolutionary relationship to measles virus and ruminant morbilliviruses

A morbillivirus of uncertain origin recently killed hundreds of Mediterranean dolphins. This is the first report of the nucleotide and deduced amino acid sequence of a dolphin morbillivirus (DMV) gene. The sequence of the nucleocapsid (N) gene including boundaries was determined. When the DMV N gene coding region was compared with the corresponding sequences of other morbilliviruses a distant evolutionary relationship between these viruses and DMV was apparent. Phylogenetic analysis of the sequence data provided further evidence that DMV is not closely related to any known morbillivirus, whereas phocine distemper virus exhibits a relatively close relationship to canine distemper virus.

COMPARATIVE ANALYSIS OF THE ATTACHMENT PROTEIN GENE (H) OF DOLPHIN MORBILLIVIRUS

DMV, dolphin morbillivirus, a paramyxovirus of uncertain origin recently emerged in Mediterranean dolphins. This study presents the complete nucleotide sequence of the hemagglutinin (H) gene including the gene boundaries. The single open reading frame of the DMV H gene encodes a protein of 604 residues which exhibits overall sequence characteristics similar to the H genes of other morbilliviruses. When compared to its closest homologues, measles virus (MV) and rinderpest virus (RPV), DMV has, respectively, 44 and 46% of amino acid residues in identical positions. The primary sequence of the DMV H protein is markedly less conserved than that of the fusion protein. The comparative data at the genomic level correspond with cross-neutralization studies with different morbilliviruses. Retrospective serogical studies dating back to 1983 indicate DMV-like infections in whales of the eastern Atlantic. The presented data support and extend previous studies suggesting that this novel morbillivirus is one of the phylogenetically oldest morbilliviruses known to circulate today. The relationship of DMV and established morbilliviruses to the newly emerged candidate morbillivirus infecting horse and man is discussed.

Processing of N-linked oligosaccharides on the measles virus glycoproteins: importance for antigenicity and for production of infectious virus particles.

The envelope of measles virus (MV) particles contains two viral glycoproteins, the haemagglutinin (H) and the fusion (F) protein, which together induce the entry of MV into cells. In the present study, we investigated the role of oligosaccharide processing for the function and antigenicity of the MV glycoproteins by means of glycosidase inhibitors. Golgi alpha-mannosidase inhibitors (1-deoxymannojirimycin and swainsonine) prevented the oligosaccharides on the MV glycoproteins from obtaining Endo H resistance, but that did not appear to influence in vitro MV infections, indicating that conversion of oligosaccharide chains into the complex form was not required for the function of the MV glycoproteins. The alpha-glucosidase inhibitor castanospermine (CSP) quantitatively reduced the production of infectious MV particles in cells infected with both vaccine strain and wild-type MV. CSP reduced the detection of the MV F protein by certain monoclonal antibodies (MAbs) that appeared to recognize nonlinear epitopes. CSP also inhibited syncytium formation in MV infected cells, but did not affect MV induced CD46 downregulation, suggesting that CSP primarily influenced the F protein. We propose that CSP induces aberrant folding of MV glycoproteins in a manner that influences their function and antigenicity.

The phosphoprotein gene of a dolphin morbillivirus isolate exhibits genomic variation at the editing site

The nucleotide sequence of the phosphoprotein (P) gene of a dolphin morbillivirus (DMV) isolate was determined. Like those of other morbilliviruses the DMV P gene encoded P and C proteins in overlapping open reading frames and V protein by editing the P gene transcript. Among P mRNA based clones the editing site variants GGGC, GGGG, GAGC and GGGGGGC predicting a P protein, and the variants GGGGC and GGGGGG predicting a V protein, were found. Surprisingly, the three variants GGGC, GGGG and GAGC were also found among clones generated from genomic RNA of the DMV isolate. Thus, more than one viral genome type appeared to be present in cells infected with the DMV isolate. By a similar analysis of the virus genomes in the tissue from which the DMV isolate was obtained, only the GGGC type was found, indicating that the GGGG and GAGC types arose during adaptation of the virus to growth in cell cultures. No editing site variants likely to have arisen by editing the GAGC type were encountered, and it remains ot be determined whether mRNA encoding V protein can be transcribed from genomes with this editing site. Using antisera raised against the common N terminus and unique C termini of the predicted P and V proteins, the in vivo expression of these proteins was demonstrated.

Changes in the Receptorbinding Haemagglutinin Protein of Wild-type Morbilliviruses are not Required for Adaptation to Vero Cells

We examined the consequences of isolation and adaptation to Vero cells for the receptorbinding haemagglutinin (H) gene of four syncytia-forming isolates of canine distemper virus (CDV) and of a dolphin morbillivirus isolate. A Vero-adapted CDV isolate exhibited biased hypermutation, since 11 out of 12 nucleotide differences to other isolates from the same epidemic were U–C transitions. Most of these transitions appeared to have taken place during in vitro cultivation. Previously, biased hypermutation in morbilliviruses has almost exclusively been described for subacute sclerosing panencephalitis and measles inclusion body encephalitis, which are rare measles virus brain infections. Amino acid changes in the H proteins were not required for Vero cell adaptation, suggesting that Vero cells express receptors for wild-type morbilliviruses. This strongly indicate the existence of other morbillivirus receptors than CD46 and CDw150.