Heterogeneity within the hemagglutinin genes of canine distemper virus (CDV) strains detected in Italy.

Canine distemper virus (CDV) is a highly contagious viral pathogen causing lethal disease in dogs and other mammalians. A high degree of genetic variation is found between recent CDV strains and the old CDV isolates used in the vaccines and such genetic variation is regarded as a possible cause of the increasing number of CDV-related diseases in dogs. The H gene shows the greatest extent of genetic variation that allows for distinction of various lineages, according to a geographical pattern of distribution and irrespective of the species of identification. In the present study, hemagglutinin (H) genes obtained from field strains detected from clinical specimens of Italian dogs were analyzed genetically. Phylogenetic analysis revealed that a homogeneous group of CDV strains is widespread in Italian dogs, all which are included into the European lineage. Unexpectedly, strains 179/04 and 48/05 clustered along with CDVs of the Arctic lineage, the highest identity being to strain GR88 (98.0 and 98.4%aa, respectively). The full-length sequence of a red fox CDV strain, 207/00 was also determined and analyzed. The H protein of the fox CDV strain was unrelated to strains within the major European lineage. These results suggest that at least three different CDV lineages are present in Italy.

The nucleotide and predicted amino acid sequence of the attachment protein of canine distemper virus.

The nucleotide sequence of the gene coding for the attachment protein of the Convac strain of the canine distemper virus (CDV), corresponding to the haemagglutinin (H) gene of measles virus was determined using a mRNA-derived cDNA clone and genomic viral RNA. The mRNA transcribed from the CDV H gene is 1944 nucleotides long excluding the polyadenylated tail. Only one long open reading frame was found comprising nucleotides 21-1841. The predicted protein has a single hydrophobic region which can serve as a membrane anchoring domain. The deduced 607 amino acids would code for a protein of 68,247 Da, to be compared with an approximate protein molecular weight in SDS-PAGE of the glycosylated protein, which is 85,000 Da. The CDV H protein exhibited seven potential N-linked glycosylation sites. These were concentrated to the carboxyterminal part of the CDV H protein and differed markedly from measles virus (MV) and rinderpest virus (RPV) where the potential sites were mostly conserved and located in the amino-terminal half of the proteins. In spite of the differences in amino acid composition of these three H proteins their hydrophilicity/hydrophobicity plots were closely similar with the major hydrophobic region at an identical location. All the 12 cysteine residues found in the CDV H protein were conserved in MV and RPV. The amino acid homology between CDV and MV H protein was 37% and between CDV and RPV H protein 38%. The fact that the corresponding homology between the MV and RPV proteins is almost 60% shows that the evolutionary separation between CDV and RPV occurred at a much earlier time than the separation between RPV and MV.

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 nucleotide and deduced amino acid sequence of the M gene of phocid distemper virus (PDV). The most conserved protein of morbilliviruses shows a uniquely close relationship between PDV and canine distemper virus.

The nucleotide sequence of the matrix gene (M) of a recently identified morbillivirus, phocid distemper virus (PDV), was determined and the amino acid composition deduced. The M gene of PDV shared many characteristics with the corresponding gene in other morbilliviruses. The nucleotide homology with the closely related canine distemper virus (CDV) was maximum at 67% followed by measles virus (MV) (58%) and rinderpest virus (RPV) (56%). The length of the 5' long untranslated region of PDV (408) was similar to that of CDV (406) but was somewhat shorter than that of MV (425) and RPV (437). The deduced matrix protein of PDV showed structural characteristics similar to the corresponding proteins of other morbilliviruses. PDV and CDV M proteins showed a remarkably high amino acid homology of 90%. The percent amino acid homology among other morbilliviruses was between 73-77%. The M protein was the most highly conserved protein among all morbilliviruses viral components.

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.

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.

Nucleic acid hybridization analyses confirm the presence of a hitherto unknown morbillivirus in Mediterranean dolphins.

In 1990 an epidemic caused by a morbillivirus was noticed among Mediterranean dolphins. RNA was extracted from the tissues of dolphins and from cell cultures infected with a corresponding dolphin morbillivirus isolate. By nucleic acid hybridization this RNA was compared to RNA extracted from animal tissue or cell cultures infected with canine distemper virus (CDV), phocine distemper virus (PDV) or measles virus (MV). The presence of morbillivirus RNA in the dolphin tissue was demonstrated. Morbillivirus N, P, M and F gene mRNAs were detected in the RNA from dolphin morbillivirus infected cells. These mRNA species seemed to be of approximately the same size as the corresponding mRNA species of CDV, PDV and MV. The results of the comparison demonstrated that the dolphin morbillivirus is genetically different from CDV, PDV and MV. No indication of a close relationship between the dolphin isolate and either CDV, PDV or MV was found.

Humanized animal viruses with special reference to the primate adaptation of morbillivirus.

This review article discusses the evolution of human viruses with special reference to paramyxoviruses. This family of viruses causes epidemics representing the dissemination of infection from one acutely infected host to the next. Since there is no repository for human paramyxoviruses in animals or in the form of persistent infections in man, the history of epidemics afflicting human civilization is short, presumably not exceeding 4000-5000 years. Evolutionary relationships can be deduced for comparison of nucleotide sequences of genes or even complete genomes. The present paramyxovirus genus will probably in the future be divided into two separate genera. In the genus morbillivirus, two pairs of more closely related virus types can be distinguished: canine and phocid viruses, and rinder-pest and measles viruses, respectively. It is speculated that recombination events may have occurred in the evolution of the morbillivirus archetype.

Round table on morbilliviruses in marine mammals.

Since 1988 morbilliviruses have been increasingly recognized and held responsible for mass mortality amongst harbour seals (Phoca vitulina) and other seal species. Virus isolations and characterization proved that morbilliviruses from seals in Northwest Europe were genetically distinct from other known members of this group including canine distemper virus (CDV), rinderpest virus, peste des petits ruminants virus and measles virus. An epidemic in Baikal seals in 1987 was apparently caused by a morbillivirus closely related to CDV so that two morbilliviruses have now been identified in two geographically distant seal populations, with only the group of isolates from Northwest Europe forming a new member of the genus morbillivirus: phocid distemper virus (PDV). Because of distemper-like disease, the Baikal seal morbillivirus was tentatively named PDV-2 in spite of its possible identity with CDV. The appearance of morbilliviruses in the Mediterranean Sea causing high mortality amongst dolphins should further increase the research activities on protection strategies for endangered species of marine mammals.

Serological and genetic characterisation of bovine respiratory syncytial virus (BRSV) indicates that Danish isolates belong to the intermediate subgroup: no evidence of a selective effect on the variability of G protein nucleotide sequence by prior cell culture adaption and passages in cell culture or calves.

Danish isolates of bovine respiratory syncytial virus (BRSV) were characterised by nucleotide sequencing of the G glycoprotein and by their reactivity with a panel of monoclonal antibodies (MAbs). Among the six Danish isolates, the overall sequence divergence ranged between 0 and 3% at the nucleotide level and between 0 and 5% at the amino acid level. Sequence divergences of 7-8%, 8-9% and 2-3% (nucleotide) and 9-11%, 12-16% and 4-6% (amino acid) were obtained in the comparison made between the group of Danish isolates and the previously sequenced 391-2USA, 127UK and 220-69Bel isolates, respectively. Phylogenetic analysis showed that the Danish isolates formed three lineages within a separate branch of the phylogenetic tree. Nevertheless, the Danish isolates were closely related to the 220-69Bel isolate, the prototype of the intermediate antigenic subgroup. The sequencing of the extracellular part of the G gene of additional 11 field BRSV viruses, processed directly from lung samples without prior adaption to cell culture growth, revealed sequence variabilities in the range obtained with the propagated virus. In addition, several passages in cell culture and in calves had no major impact on the nucleotide sequence of the G protein. These findings indicated that the previously established variabilities of the G protein of RS virus isolates were not attributable to mutations induced during the propagation of the virus. The reactivity of the Danish isolates with G protein-specific MAbs were similar to that of the 220-69Bel isolate. Furthermore, the sequence of the immunodominant region was completely conserved among the Danish isolates on one side and the 220-69Bel isolate on the other. When combined, these data strongly suggested that the Danish isolates belong to the intermediate subgroup.

Studies on manifestations of canine distemper virus infection in an urban dog population.

An upsurge of canine distemper was recognized at the beginning of 1991 in the urban dog population of the Copenhagen area. The outbreak had the characteristics of a virulent morbillivirus introduction in a partly immune population, where the disease primarily was manifested in young individuals. Testing of single serum samples for the presence of canine distemper virus (CDV) IgM antibodies using an IgM ELISA confirmed current and recent CDV infections in an urban dog population, where the use of attenuated CDV vaccines was widespread. In 49 out of 66 sera from clinical cases suspected of canine distemper we detected CDV IgM antibodies, as compared to the detection of viral antigen by indirect immunofluorescence in 27 of 65 specimens of conjunctival cells. The antigenic make-up of isolates from acute and subacute clinical cases was investigated with a panel of 51 monoclonal antibodies directed against CDV and the related phocine distemper virus. The isolates exhibited an homogeneous reaction pattern and shared overall antigenic characteristics of the CDV prototype. The majority of cases were diagnosed among unvaccinated dogs and individuals with unknown or obscure vaccination record. However, severe clinical cases were also diagnosed in vaccinated individuals.

Morbilliviruses in aquatic mammals: report on round table discussion.

A workshop was organised to ascertain the current situation with regard to morbillivirus infections in aquatic animals. The great interest generated by the discovery of these new virus infections in 1988 has to some extent abated but much high quality research has continued in this field as the workshop showed. There is some serological evidence that the viruses have continued to circulate in most areas since the initial epizootics. As to their origin, it appears that the most likely source of the European seal morbillivirus (PDV-1) is the North Atlantic and Artic seal populations. As to the origin of the Mediterranean dolphin morbillivirus and the morbilliviruses isolated from porpoises, there is serological evidence that the viruses are widespread in many cetacean species in the Atlantic and 93% of long-finned pilot whales (Globicephala melas) which mass stranded between 1982 and 1993 were morbillivirus seropositive. The epizootic in freshwater seals in Lake Baikal was unrelated to events in the European marine mammal populations. The virus which infected these animals (PDV-2) is indistinguishable from canine distemper field strains. Serological and molecular biological studies provided evidence for the presence of the virus in the seals, at least as late as the Summer of 1992 when the animals were last sampled.

Reactivity of eleven anti-human leucocyte monoclonal antibodies with lymphocytes from several domestic animals.

Nine commercially available monoclonal antibodies and two monoclonal antibodies from The American Type Culture Collection, raised against various human leucocyte surface antigens, were tested on lymphocytes from cow, sheep, goat, swine, horse, cat, dog, mink, and rabbit as well as man. Four antibodies bound to lymphocytes from some of the animals. These were the antibodies against CD8 and CD4 antigen, the antibody to C3b-receptor, and the antibody to the HLA-DR antigen. The CD8 antigen-reactive antibody reacted with lymphocytes from mink, cat, dog, and sheep, while the CD4 antigen-reactive antibody reacted with lymphocytes from mink. The anti-C3b-R antibody reacted with lymphocytes from horse, swine, dog, and cat, and the anti-HLA-DR reacted with lymphocytes from cow, goat, sheep, horse, dog, cat, and mink.

Detection of IgM antibodies against canine distemper virus in dog and mink sera employing enzyme-linked immunosorbent assay (ELISA).

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgM antibodies against canine distemper virus (CDV) in canine and mink serum is described. The diagnostic potential of this technique was evaluated by analyzing sera from natural or experimental infections in dog and mink and negative control sera. These results were compared with results obtained in the developed CDV IgG ELISA and in the virus neutralization test. The IgM test, which requires only a single serum specimen, is a useful method for diagnosing current or recent CDV infections in dog and mink.

Detection of intracellular canine distemper virus antigen in mink inoculated with an attenuated or a virulent strain of canine distemper virus.

Using an indirect immunofluorescence technique, the distribution of viral antigen in various tissues and blood mononuclear leukocytes was studied in wild mink, either vaccinated with an attenuated vaccine strain of canine distemper virus (CDV) or experimentally inoculated with the virulent Snyder-Hill strain of CDV. Viral antigen was detected in cells of the lymphoid system 6 to 12 days after vaccination. From 2 to 3 days after inoculation with the virulent strain, CDV antigen was demonstrated in cells of the lymphoid system and, during the incubation period, the antigen had spread to the epithelia and brain at days 6 and 12, respectively. In clinical cases of acute fatal canine distemper, the viral antigen was detected in a wide variety of tissues, including the cells of the lymphoid system, epithelial cells of skin, mucous membranes, lung, kidney, and cells of the CNS. The diagnostic importance of CDV antigen detection is discussed on the basis of these findings.

Lights and shades on an historical vaccine canine distemper virus, the Rockborn strain.

Both egg- and cell-adapted canine distemper virus (CDV) vaccines are suspected to retain residual virulence, especially if administered to immuno-suppressed animals, very young pups or to highly susceptible animal species. In the early 1980s, post-vaccine encephalitis was reported in dogs from various parts of Britain after administration of a particular batch of combined CDV Rockborn strain/canine adenovirus type-1 vaccine, although incrimination of the Rockborn strain was subsequently retracted. Notwithstanding, this, and other reports, led to the view that the Rockborn strain is less attenuated and less safe than other CDV vaccines, and the Rockborn strain was officially withdrawn from the markets in the mid 1990s. By sequencing the H gene of the strain Rockborn from the 46th laboratory passage, and a commercial vaccine (Candur(®) SH+P, Hoechst Rousell Vet GmbH), the virus was found to differ from the commonly used vaccine strain, Onderstepoort (93.0% nt and 91.7% aa), and to resemble more closely (99.6% nt and 99.3% aa) a CDV strain detected in China from a Lesser Panda (Ailurus fulgens). An additional four CDV strains matching (>99% nt identity) the Rockborn virus were identified in the sequence databases. Also, Rockborn-like strains were identified in two vaccines currently in the market. These findings indicate that Rockborn-like viruses may be recovered from dogs or other carnivores with distemper, suggesting cases of residual virulence of vaccines, or circulation of vaccine-derived Rockborn-like viruses in the field.

Biological properties of phocine distemper virus and canine distemper virus.

Morbilliviruses constitute a major threat to the health of animal and man. To date the Morbillivirus genus in the Paramyxoviridae family comprises five established members, namely canine distemper virus (CDV), phocine distemper virus (PDV), measles virus (MV), rinderpest virus (RPV), and peste-des-petits-ruminants virus (PPRV). In addition, morbillivirus candidates infecting aquatic mammals were recently discovered. The present review on the biology of morbilliviruses focuses on knowledge gained by our group in studies on PDV and CDV. The aims of these studies were: i) to investigate the biological properties of the recently recognized PDV, which was found to be the primary etiology of epidemics with high mortality in seals in Western Europe, ii) to extend our knowledge of the biological properties of CDV. The morbillivirus particle is enveloped. The helical nucleocapsid core contains a single-stranded, non-segmented RNA genome of negative sense of 15 to 16 kilobases in length. The genome is organized in six transcriptional units or genes. Overall, the studies of the genome of PDV revealed a genetic map principally fitting with that determined for other morbilliviruses. The nucleotide and deduced amino acid sequences have been determined for five PDV genes named in analogy with the encoded structural proteins of other morbilliviruses in the order: 3'N(1683)-P(1644)-M(1443)-F(2206)-H(1952)-L5' (The figures in brackets denote nucleotide lengths of the genes of the Danish PDV isolate). The L gene (covering approximately 8900 nucleotides) remains to be sequenced. The six genes are likely to code for at least eight distinct proteins. The nucleocapsid (N) protein was found to consist of 523 amino acids in PDV. The following gene of the transcription map encoded the P protein of 507 amino acid residues. Similar to other morbilliviruses, the P gene of PDV was shown to have additional coding capacity for two distinct proteins V (299 amino acids) and C (174 amino acids). The results presented provide evidence for editing at transcript of the PDV P gene by insertion of nontemplated G residues at a specific site. The edited version of the mRNA was found to encode the cystein-rich V protein. The three envelope-associated proteins of PDV were predicted to consist of 335 (M), 537 (F0) and 607 (H) amino acid residues. The nucleotide and deduced amino acid sequences of the N, P, M, F, and H genes of PDV were aligned with corresponding sequences of other established members of the genus Morbillivirus.(ABSTRACT TRUNCATED AT 250 WORDS)

Two cases of torsio testis in dogs.

Two unusual cases of torsio testis in dogs are presented. The dogs were 8 and 24 months old, and the retained testicles were not neoplastic. In the first case there was a foreign body anamnesis and in the second an apparent accident anamnesis.

A serious outbreak of canine distemper among sled-dogs in northern Greenland.

A canine distemper outbreak in a highly susceptible sled dog population of Northern Greenland was recognized in the beginning of January 1988. A high morbidity and mortality in all age groups was a characteristic of the epizootic. The actual canine distemper virus infection was identified by isolation of the virus and demonstration of viral antigens by immunofluorescence and also by demonstration of conventional inclusion bodies. Virus specific IgM antibodies were demonstrated in affected dogs. In places where vaccination was carried out too late to be effective, the losses were up to 80 per cent. In a settlement, which was under rabies quarantine four weeks before the distemper outbreak started in other settlements, no cases occurred before or after vaccination. The spread of canine distemper seems connected with a Canadian outbreak and communicated by foxes. Once established the further spread apparently was by travelling dog teams. Suitable future prophylactic measures are discussed.