Pneumovirus-like characteristics of the mRNA and proteins of turkey rhinotracheitis virus.

Electronmicroscopy has indicated that turkey rhinotracheitis virus (TRTV), the causative agent of an acute respiratory disease in turkeys, is a member of the Paramyxoviridae family. To determine if TRTV belongs to one of the three defined genera of this family (Paramyxovirus, Morbillivirus and Pneumovirus) we have analysed the RNA and proteins induced during replication of TRTV in Vero cells. Following replication in the presence of actinomycin D 10 polyadenylated RNA bands, ranging in Mr from 0.22 to 2.0 X 10(6), were detected in infected cells; some bands probably contained 2 or more RNA species. Viral proteins were studied after radiolabelling in the presence of [35S]methionine and [3H]glucosamine. Comparison of the polypeptides in mock-infected and infected cells, virions and nucleocapsids and after lentil-lectin chromatography and immunoprecipitation revealed seven virus-specific polypeptides (p), some of which were glycosylated (gp): gp82 (Mr 82K), gp68, gp53, gp15, p43, p40 and p35. These are considered to be analogous to the large glycopolypeptide (HN, H and G), fusion protein precursor F0, the F protein cleavage products F1 and F2, nucleocapsid (N), phosphorylated (P) and matrix (M) polypeptides, respectively, of the Paramyxoviridae. Two other polypeptides (Mr 200K and 22K) were also detected, as was a glycopolypeptide of Mr 97K, probably related to gp82. Tunicamycin inhibited glycosylation of gp53 and gp15 but gp82 was little affected, most glycans still being present on a glycopolypeptide of approximately 79K. This finding, indicating that gp82 has mostly O-linked glycans, considered with the mRNA profile and the molecular weight of the N protein shows that of the three genera in this family, TRTV most closely resembles the Pneumovirus genus.

Morbillivirus group: genome organisation and proteins.

The Paramyxoviridae family is divided into three genera: Paramyxovirus, Pneumovirus and Morbillivirus. In the last group, there are four closely related viruses which are seriously pathogenic for man and animals, and usually cause acute diseases. At least two of them (measles and canine distemper viruses) can cause a persistent infection which leads to a chronic disease of the nervous system that, in the end, is fatal. For a long time, the biochemical analysis of morbilliviruses was hampered by the high susceptibility of some of their proteins to proteolysis. With cloning and sequencing technology, more data on the biology of those viruses are now available.

A review of the 1988 European seal morbillivirus epizootic.

An epizootic of morbillivirus infection killed thousands of common seals (Phoca vitulina) in European seas in 1988. Most of the affected seals had respiratory signs and the main post mortem finding was acute pneumonia. The histopathological changes were similar to those of canine distemper. Six common porpoises (Phocoena phocoena) found stranded on the coast of Northern Ireland in late 1988 had similar lesions. Morbillivirus infection also killed several thousand Siberian seals (Phoca siberica) in Lake Baikal in 1987 and 1988. A morbillivirus (phocine distemper virus) has been isolated from affected seals in several European countries and studies of the antigenicity of the virus indicate that it has several unique epitopes that distinguish it from the other known morbilliviruses. Biochemical studies of the viral proteins, RNA and nucleotide sequence confirm that it is a new morbillivirus. There is seroepizootiological evidence of morbillivirus infection in Greenland harp seals (Pagophilus groenlandicus), ringed seals (Phoca hispida) and Dutch common seals several years before the 1988 epizootic. Antibodies to a morbillivirus have also been found in bottlenose dolphins (Tursiops truncatus) from the eastern coast of the USA. Further studies are required to determine whether these sea mammal populations have been infected with phocine distemper virus.

Biochemical characterization of a Yucaipa-like virus.

A Yucaipa-like virus (PLOC/Senegal/273/77) was grown in embryonated chicken eggs and used for biochemical investigations after purification. The genome of the virus is composed of one fragment of single-stranded (ss)RNA with an estimated mol. wt. of 5.6 X 10(6). There are six virus structural polypeptides with mol. wt. of 126 000, 68 000 (major), 60 000, 52 000 (major), 44 000 and 39 000 (major). The fatty acid composition of the virus envelope seems to be very selective since we found only fatty acids containing 14 and 16 carbon atoms.

The structural polypeptides of avian paramyxoviruses.

The structural polypeptides of twenty-three avian paramyxoviruses from five serotypes were examined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate under reducing and non-reducing conditions. All virus polypeptide profiles consisted of 7--10 polypeptides of which two were glycosylated. Some variation was seen in the profiles of viruses from the same serotype, but groups formed on the basis of their serological relationships in haemagglutination inhibition tests were identical to those formed on the basis of similarities in their polypeptide profiles.

Comparison of 6-94 virus and Sendai virus RNA by RNA-RNA hybridization.

The genomic RNA of 6/94 virus, an agent isolated from the brains of multiple sclerosis patients, was studied for sequence homology by RNA-RNA hybridization with closely related Sendai virus and another paramyxovirus virus, Newcastle disease virus. It was found that the genomic RNA of 6/94 virus hybridizes equally as well to the virus-specific 18S RNA found in Sendai-infected cells as that of Sendai virus.

Fusion glycoprotein of measles virus: nucleotide sequence of the gene and comparison with other paramyxoviruses.

The sequence of the fusion (F) glycoprotein mRNA of the Hallé strain of measles virus was determined from a cDNA clone representing the entire length of the mRNA. It contained 2384 nucleotides, excluding poly(A), with a 5' consensus sequence typical of paramyxoviruses and a 3' terminus found in measles virus mRNAs. The coding sequence was preceded by an unusually long (580 nucleotide) 5' non-translated region, which contained 44% cytosine. The longest open reading frame coded for a polypeptide of 553 amino acids with a predicted molecular weight of 59.84 K. Comparison of the sequence with that of the Edmonston strain of measles virus showed that the gene is highly conserved. No amino acid differences were observed between the two strains. The F polypeptide had three regions of high hydrophobicity: an N-terminal signal peptide, the N-terminus of F1 and a C-terminal membrane-spanning region. The four potential asparagine-linked glycosylation sites (one in the signal peptide) were all in the F2 subunit. Comparison of the measles virus F amino acid sequence with other paramyxoviruses revealed homologies with these viruses. Certain regions such as the N terminus of F1 and ten cysteine residues which probably impose structural restraints were highly conserved.

Antigenic and structural relationships between avian paramyxoviruses isolated from ducks in Hong Kong and Mississippi, U.S.A.

Representative isolates of the paramyxoviruses duck/Hong Kong/75 and duck/Mississippi/75 were shown to be serologically closely related by haemagglutination and neuraminidase inhibition tests. The structural polypeptides of these viruses were also shown to be similar. For each of the isolates tested, polyacrylamide gel electrophoresis in the presence of SDS revealed a similar polypeptide pattern consisting, under reducing conditions, of seven polypeptides with apparent mol. wt. ranging from 46000 to 190000. Each virus had two glycosylated polypeptides with apparent mol. wt. of 56000 and 71000 to 72000 under reducing conditions and 62000 to 63000 and 135000 to 142000 under non-reducing conditions.

Comparison of proteins induced in cells infected with rinderpest and peste des petits ruminants viruses.

The two morbilliviruses rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) are closely related and cause severe disease in large and small ruminants, respectively. They show distinct epidemiological patterns and are distinguishable by reciprocal cross-neutralization tests. We have analysed the proteins induced by these viruses in infected cells and have shown that they can be distinguishable by a very marked difference in the apparent mol. wt. of the nucleocapsid (N) protein. The N protein of PPRV is almost identical in mobility on polyacrylamide gels to the N proteins of measles virus and canine distemper virus (60K). Several strains of RPV and PPRV from widespread geographical locations were studied and found to show this difference in the N protein.

Immunological relationships of simian virus 41 (SV41) to other paramyxoviruses and serological evidence of SV41 infection in human populations.

Antigenic relationships of simian virus 41 (SV41) to other paramyxoviruses were examined by immunoprecipitation of isotope-labelled SV41-infected cell lysates with specific antisera. SV41 is closely related to the group comprising human parainfluenza virus 2 (HPIV-2), simian virus 5 (SV5), parainfluenza virus 4 and mumps virus. Slight cross-neutralization was detected between SV41, HPIV-2 and SV5. Anti-SV41 activities were detected in 21 of 1116 human serum specimens, indicating that a proportion of the human population is infected with SV41. The haemagglutinin-neuraminidase of SV41 was preferentially immunoprecipitated by anti-SV41 positive sera.

Study of a new strain of paramyxoviruses isolated from wild ducks: structural polypeptides.

By polyacrylamide gel electrophoresis, Duck/Mississippi/75 virus was shown to contain five different types of polypeptides of mol. wt. ranging from 76 X 1O(3) to 43 X 10(3), two of which were glycosylated (mol. wt. 76 X 10(3) and 57 X 10(3). A comparison of this data with similar results obtained using Yucaipa and Newcastle disease virus (NDV) revealed a similarity with NDV, concerning the number, position and mol. wt. of the polypeptides. Haemagglutinating and neuraminidase properties are associated with surface glycoproteins which represent at least 40% of the virus protein. After KCl and Triton X-100 treatment and centrifugation on linear sucrose density gradients (10 to 25%, w/w) it was possible to isolate glycoprotein VP1 of mol. wt 76 X 10(3) with which haemagglutinating and neuraminidase activities were associated.

Intrinsic fluorescence of some myxo- and paramyxoviruses and of their subviral fractions.

Fluorescence spectra of Sendai and influenza A(H1N1) viruses have different emission maxima; their quantum yield is much lower than that of tobacco mosaic virus. Solubilized envelopes and nucleoproteins of Sendai, influenza and mumps virus have different half-bandwidth and relative quantum yield values of emission, according to the agent used for disruption. Thus emission (as well as excitation and absorption) spectra of Triton X-100-solubilized envelopes show a marked hypsochromic shift as compared with the envelopes obtained by Tween20-disruption. The results are correlated with the different disruption extent achieved with the two agents.

The structural proteins of a porcine paramyxovirus (LPMV).

The porcine paramyxovirus is a newly identified agent of a fatal disease in piglets, endemic in Mexico since 1980, where it was seen around the town of La Piedad, Michoacan, Mexico (hence LPM virus). At least six [35S]methionine-labelled proteins could be resolved by SDS-PAGE and five of them were clearly immunoprecipitated. Selective labelling of LPMV-infected cells with [3H]glucosamine revealed two bands with an Mr of about 66K and 59K, corresponding to the two viral glycoproteins, the haemagglutinin-neuraminidase protein and the fusion protein. Labelling of virus with [32P]orthophosphate disclosed one band with an Mr of 52K, corresponding to the phosphoprotein. Analysis of nucleocapsids obtained from purified virus or from a permanently infected cell line revealed one major band with an Mr of 68K, the nucleoprotein. Two other proteins were also identified, the large protein and the matrix protein, with apparent Mr of about 200K and 40K, respectively. The protein migration pattern of LPMV was compared, by SDS-PAGE, with that of Newcastle disease virus, bovine parainfluenza 3 virus and Sendai virus. Differences in the Mr of LPMV proteins and the proteins of these paramyxoviruses were observed. We propose that LPMV should be classified as a novel member of the genus Paramyxovirus.

Sulfated components of enveloped viruses.

The glycoproteins of several enveloped viruses, grown in a variety of cell types, are labeled with 35SO4(-2), whereas the nonglycosylated proteins are not. This was shown for the HN and F glycoproteins of SV5 and Sendai virus, the E1 and E2 glycoproteins of Sindbis virus, and for the major glycoprotein, gp69, as well as for a minor glycoprotein, gp52, of Rauscher leukemia virus. The minor glycoprotein of Rauscher leukemia virus is more highly sulfated, with a ratio of 35SO4- [3H]glucosamine about threefold greater than that of gp69. The G protein of vesicular stomatitis virus was labeled when virions were grown in the MDBK line of bovine kidney cells, although no significant incorporation of 35SO4(-2) into this protein was observed in virions grown in BHK21-F line of baby hamster kidney cells. In addition to the viral glycoproteins, sulfate was also incorporated into a heterogenous component with an electrophoretic mobility lower than that of any labeled with 35SO4(-2) and [3H]leucine, this component had a much greater 35S-3H ratio than any of the viral polypeptides and thus could not represent aggregated viral proteins. This material is believed to be a cell-derived mucopolysaccharide and can be removed from virions by treatment with hyaluronidase without affecting the amount of sulfate present on the glycoproteins.