Showing papers in "Journal of General Virology in 1985"

Two distinct subtypes of human respiratory syncytial virus.

Abstract: Antigenic variation of human respiratory syncytial (RS) virus strains was analysed using a collection of nine, six, six, nine and one monoclonal antibodies respectively directed against the large glycoprotein (G), fusion protein (F), matrix protein (M), nucleoprotein (NP) and phosphoprotein (P) components of the Long strain of RS virus. A comparison was made with seven other strains isolated during different years in radioimmune precipitation analyses and immune fluorescence tests. Two different subtypes of the virus were demonstrable. Subtype A included the prototype strains Long and A2 and virus isolates from 1973, 1983 and 1984; subtype B included four virus strains isolated in successive years from 1979 to 1982. Subtype A viruses reacted with all the antibodies, whereas subtype B viruses showed different epitope characteristics in four structural components. The number of altered epitopes were 5/6, 1/2, 2/6 and 1/6 in the G, F, M and NP components, respectively. It is concluded that the two subtypes have evolved separately. The finding of two subtypes may explain previously observed strain variations in neutralization tests, and gives a new perspective on the immunobiology of RS virus.

The physical state of human papillomavirus type 16 DNA in benign and malignant genital tumours.

Abstract: Summary Cloned DNA from human papillomavirus (HPV) type 16 was subjected to restriction enzyme analysis. A genome size of 7.8 ± 0.1 kb was determined and restriction maps were prepared. Fragments of HPV 16 DNA were nick-translated and hybridized with fragments of HPV 6b DNA. The two genomes appeared to be colinear. The physical state of HPV 16 DNA in genital tumours was analysed. In each of six benign tumours the viral DNA was detected exclusively as 8 kb circles. In four malignant tumours the viral DNA appeared to be integrated within the host genome but one cervical carcinoma and one case of Bowen's disease also contained oligomeric episomal molecules of viral DNA. One cervical carcinoma (WV 2965), containing only integrated viral DNA, was examined in detail. HPV 16 DNA was integrated as head-to-tail tandem repeats at more than one site. Three virus/cell junction fragments from this tumour were cloned. Two contained lengths of repetitive cellular DNA and one a length of apparently single copy cellular DNA.

Mutagen-directed attenuation of Rift Valley fever virus as a method for vaccine development.

Abstract: Serial mutagenesis with 5-fluorouracil has been employed to derive an attenuated strain of Rift Valley fever virus for use as a live virus vaccine

Survival Characteristics of Airborne Human Coronavirus 229E

Abstract: The survival of airborne human coronavirus 229E (HCV/229E) was studied under different conditions of temperature (20 +/- 1 degree C and 6 +/- 1 degree C) and low (30 +/- 5%), medium (50 +/- 5%) or high (80 +/- 5%) relative humidities (RH). At 20 +/- 1 degree C, aerosolized HCV/229E was found to survive best at 50% RH with a half-life of 67.33 +/- 8.24 h while at 30% RH the virus half-life was 26.76 +/- 6.21 h. At 50% RH nearly 20% infectious virus was still detectable at 6 days. High RH at 20 +/- 1 degree C, on the other hand, was found to be the least favourable to the survival of aerosolized virus and under these conditions the virus half-life was only about 3 h; no virus could be detected after 24 h in aerosol. At 6 +/- 1 degree C, in either 50% or 30% RH conditions, the survival of HCV/229E was significantly enhanced, with the decay pattern essentially similar to that seen at 20 +/- 1 degree C. At low temperature and high RH (80%), however, the survival pattern was completely reversed, with the HCV/229E half-life increasing to 86.01 +/- 5.28 h, nearly 30 times that found at 20 +/- 1 degree C and high RH. Although optimal survival at 6 degree C still occurred at 50% RH, the pronounced stabilizing effect of low temperature on the survival of HCV/229E at high RH indicates that the role of the environment on the survival of viruses in air may be more complex and significant than previously thought.

Flavivirus infection enhancement in macrophages: an electron microscopic study of viral cellular entry.

Abstract: Summary The mode of entry of West Nile virus (WNV) into the macrophage-like cell line P388D1 was investigated at the electron microscopical level using synchronized infections. The presence of the antiviral monoclonal antibody F6/16A at a concentration that enhanced viral attachment to P388D1 cells ninefold made no difference to the entry pathway of WNV. In both the absence and presence of F6/16A the initial uptake of single viral particles was mediated by coated pits, and started within 30 s of warming the cells to 37°C. Viral particles later appeared in fully or partially coated vesicles and later in uncoated prelysosomal endocytic vacuoles before degradation in lysosomes. However, aggregates of viral particles (five or more virus particles in cross-section), appeared to be phagocytosed whole by cells in a process which involved aggregates being engulfed by extensions of the plasma membrane. This process exhibited a slower time course than the uptake of single viral particles, becoming prominent 15 to 30 min after warming the cells to 37°C. The involvement of a prelysosomal vacuolar compartment in the entry process was shown by a failure to stain for acid phosphatase. This compartment could be specifically loaded with viral particles when viral internalization occurred at 20°C in the presence of 50 mm-ammonium chloride.

Synthesis of Proteins and Glycoproteins in Dengue Type 2 Virus-infected Vero and Aedes albopictus Cells

Abstract: Summary Fifteen proteins were detected in Vero cells infected by dengue type 2 (DEN-2) virus that were not observed in mock-infected cells, namely P98, p82, P67, GP60, gp54, GP46, p30, p28, gp22, GP20, p18, gp16, p15, p14 and gp13. With the exceptions of gp54 and gp13, polypeptides corresponding to those listed above were also observed in DEN-2 virus-infected Aedes albopictus C6/36 cells. Pulse-chase labelling experiments suggested a possible precursor-product relationship between p30 and p28, and between gp22 and GP20. Peptide mapping and immunoprecipitation experiments showed that the major glycoproteins GP60, GP46 and GP20 were unrelated. Immunoprecipitations of infected cells with antiserum prepared against the DEN-2 soluble complement-fixing (SCF) antigen demonstrated that this antigen is equivalent to the non-structural glycoprotein GP46. The envelope glycoprotein (E) from virus grown in C6/36 cells migrated faster through polyacrylamide gels containing SDS than E from virus grown in Vero cells. [3H]Mannose-labelled glycopeptides of GP60, GP46 and GP20 were separated by gel filtration and by electrophoresis in Tris-borate gels; in addition, the polypeptides synthesized in infected cells in the presence of tunicamycin were analysed. The results revealed heterogeneity among the glycan units of GP60 and GP46.

Purification and characterization of the respiratory syncytial virus fusion protein.

Abstract: Summary The fusion protein of respiratory syncytial virus was purified by affinity chromatography using a monoclonal antibody. Under various conditions the protein was recovered as a 145K dimer or a 70K monomer. The 70K monomer was composed of disulphide-linked fragments of 48K and 23K. Polyclonal rabbit serum produced to the dimerized fusion protein neutralized virus but did not inhibit fusion, while rabbit serum to the 2-mercaptoethanol-treated dimerized protein neutralized virus and inhibited fusion of infected cells. Only the latter serum strongly recognized the 23K fragment when studied by Western blot analysis.

Sequence determination of the Sendai virus fusion protein gene.

Abstract: Summary From a genomic DNA library of Sendai virus, we have identified and sequenced clones corresponding to the F glycoprotein gene. The limits of the F gene region were defined by mapping the 5′ and 3′ ends of the mRNA with S1 nuclease. The Sendai virus F gene is 1821 nucleotides long. The predicted primary translation product of the single long open reading frame would code for a protein of 565 amino acids, containing a putative signal peptide, three carbohydrate addition sites, a hydrophobic region corresponding to the known cleavage/activation site of F0, and a long, very hydrophobic region near the C-terminus which probably represents the transmembrane region of the protein. The signal peptide cleavage site of the mature protein was determined by mass spectrometry. Interestingly, the amino acid sequence surrounding the cleavage/activation site of the Sendai virus F protein shows significant homology to the same region of the influenza B and C virus HA proteins, suggesting that these genes may have evolved from a common ancestor. The ability of the Sendai virus F protein to fuse membranes relative to its primary structure is discussed.

Preparation and Characterization of Neutralizing Monoclonal Antibodies with Different Reactivity Patterns to Human Rotaviruses

Abstract: Summary By employing three strains of cultivable human rotaviruses with different serotype specificity as immunizing antigens, we prepared 11 hybridomas which secreted neutralizing monoclonal antibodies against human rotaviruses. In neutralization tests with four strains of serotype 1, and three each of serotypes 2 and 3, the monoclonal antibodies showed different reactivity patterns: seven monoclonal antibodies reacted specifically with all strains of either serotype 1, 2 or 3 human rotavirus, but two showed strain-specific reactions; the remaining two were commonly reactive to various human rotavirus strains from each serotype but not to two non-human rotaviruses. By immunoprecipitation analysis, it was found that four serotype 2-specific and two commonly reactive antibodies were directed to VP3 (82000 mol. wt. protein) on the outer shell of the virus particles.

Examination of the immunological relationships between flaviviruses using yellow fever virus monoclonal antibodies.

Abstract: Monoclonal antibodies prepared against vaccine strains of yellow fever (YF) virus were initially characterized by fluorescence microscopy of Vero cells infected with YF virus strain 17D. When similarly tested against representatives of all flavivirus subgroups, the antibodies produced a wide spectrum of reactions ranging from the monospecific to the broadly cross-reactive; at least five antigenic domains in the YF virus envelope glycoprotein were identified. Monoclonal antibodies differentiated between YF virus vaccine strains (17D, 17DD, FNV), wild-type viruses and plaque variants selected from a 17D pool. One isolate from a patient with YF was antigenically similar to the Brazilian vaccine strain 17DD. Several of the antibodies reacting with the YF viral envelope glycoprotein in biological tests identified the 54K envelope glycoprotein; 45K and 26K polypeptides in YF 17D virus-infected cells were also identified by radioimmunoprecipitation and polyacrylamide gel electrophoresis. Neither of these polypeptides was found in uninfected cells. They may represent short-lived precursors of the 54K protein, post-translational cleavage or breakdown products. Other antibodies reacted with a 48K polypeptide in virus-infected cell lysates. This may be the non-structural NV3 protein described for YF virus. Its appearance on the surface of unfixed infected cells, but not on released virions, was demonstrated by fluorescence microscopy.

Cloning and sequencing of the gene encoding the spike protein of the coronavirus IBV

Abstract: RNA sequences encoding the surface projection (spike) of the coronavirus infectious bronchitis virus, strain Beaudette, have been cloned into pBR322 using cDNA primed with a specific oligonucleotide. A 5.3 kilobase viral insert in the clone pMB179 has been identified. The region of this clone coding for the spike gene has been sequenced by the chain termination method, and we present here the first report of DNA sequence data for a coronavirus spike protein, the protein which forms the characteristic 'corona' after which the group is named. The amino acid sequence of the primary translation product, deduced from the DNA sequence, predicts a polypeptide of 1162 amino acids with a molecular weight of 127 006. This has many interesting features which confirm and extend our knowledge of this recently characterized membrane glycoprotein. The polypeptide is subsequently cleaved to S1 and S2, and partial amino acid analysis of the amino-terminus of the S1 polypeptide has been employed to locate the position of this terminus of S1 within the large open reading frame. The amino acid analysis also reveals the presence of an 18 amino acid putative signal sequence on the primary translation product which is not present on the mature S1 polypeptide.

125I-labelled Human Interferons Alpha, Beta and Gamma: Comparative Receptor-binding Data

Abstract: Binding of 125I-labelled human recombinant DNA interferons (IFNs) alpha-2, beta and gamma was compared on various human lymphoid cells and embryonic fibroblasts. While binding constants were within an order of magnitude for all three interferons (10(-10) to 10(-9) M), no competition was observed between IFN-gamma on the one hand and IFN-alpha 2 and IFN-beta on the other. However, consistent with previous reports, IFN-alpha 2 and IFN-beta competed for presumably common receptors. Depending on the cell type, binding sites for IFN-gamma were expressed in different numbers compared to those for IFN-alpha 2 and IFN-beta. These direct comparative binding studies support the hypothesis that the receptor system for IFN-gamma is unrelated to the IFN-alpha/beta system.

Biochemical Differences among Scrapie-associated Fibrils Support the Biological Diversity of Scrapie Agents

Abstract: Summary Scrapie-associated fibrils (SAF) were isolated and purified from animals infected with three different scrapie agents: ME7 and 139A in mice, and 263K in hamsters. Mouse ME7 and 139A SAF differed from hamster 263K SAF in morphology, sedimentation rate and protein composition. SAF from the three scrapie agents were distinguishable from each other by their sensitivity to proteinase K digestion. SAF copurified with infectivity in both the hamster and mouse systems. SAF appear to be a unique class of structures which are related but specific for each individual scrapie agent. These properties may correlate with the biological and pathological differences seen among these agents.

Immunological Priming with Synthetic Peptides of Foot-and-Mouth Disease Virus

Abstract: A sub-immunizing dose of a synthetic peptide corresponding to the amino acids 141 to 160 region of protein VP1 from foot-and-mouth disease virus (FMDV), serotype O1, coupled to keyhole limpet haemocyanin (141-160KLH) has been shown to prime the immune system of guinea-pigs for an FMDV serotype-specific neutralizing antibody response to a second sub-immunizing dose of the same peptide. Optimal priming required an interval of 42 days between the priming dose and the booster dose. No priming was observed in the absence of adjuvant. The secondary response was not restricted by the carrier since animals primed with 141-160KLH could be boosted with uncoupled 141-160 or 141-160 coupled to tetanus toxoid. It has also been shown that uncoupled peptide 141-160 will prime for a neutralizing antibody response when it is incorporated into a relatively non-immunogenic carrier such as small unilamellar liposomes. These results indicate that the 141-160 peptide of FMDV, as well as containing an important neutralizing antibody site, can initiate its own T-helper cell response.

The Experimental Infection of Chickens with Mixtures of Infectious Bronchitis Virus and Escherichia coli

Abstract: Summary By inoculating chickens intranasally with a collection of strains of infectious bronchitis virus (IBV) of the Massachusetts serotype and of Escherichia coli of different serotypes, a pool of viral and bacterial strains was selected which, on inoculation, consistently produced a highly lethal disease closely resembling the natural disease produced by these two organisms. The conditions for reproducing the experimental disease were not rigorous in that, within broad limits, the size of the viral and bacterial inocula were not important; neither were the times at which both organisms were administered in relation to each other. The breed or strain of chicken used was important and the resistance of chickens to fatal infection increased with age. When the E. coli strains of the pool were inoculated intranasally without the IBV component, the chickens remained well; bacteriological examination of chickens inoculated with one of the E. coli strains, O18, revealed little evidence of invasion of the tissues or even of persistence of the inoculated E. coli strain in the upper respiratory tract. A minority of the IBV strains examined were lethal for chickens when inoculated without E. coli but many of them only produced a substantial mortality when the E. coli were included in the inoculum; IBV strains in this latter category included the vaccine strains H52 and H120. High concentrations of IBV strain M41 and E. coli O18 persisted in the upper respiratory tract for a number of days after they had been inoculated together. Much lower concentrations of IBV M41 were found in the internal organs, such as the spleen; E. coli O18 was only found in these sites in some of the inoculated chickens. Coliform organisms proliferated in the upper respiratory tract of chickens inoculated with IBV alone; they were rarely found in their internal organs.

Lactate dehydrogenase-elevating virus.

Abstract: Introduction. Lactate dehydrogenase-elevating virus (LDV) was discovered 25 years ago by Dr Vernon Riley † and his colleagues during their work on plasma enzyme levels in tumour-bearing mice (Riley et al., 1960). They found that transplantable tumours of many types caused a five- to ten-fold increase in plasma lactate dehydrogenase (LDH) activity within 3 days of transplantation and before the tumours were clinically obvious. To produce this dramatic increase in plasma enzyme level it was not necessary to transplant cells; cell-free plasma from tumour-bearing mice was equally effective. The raised enzyme level could be serially transmitted from mouse to mouse and proved to be caused by a virus which replicated rapidly in mouse macrophages. Very high titres of viral infectivity (109 ID50/ml) are present in the plasma 24 h after infection, and a stable viraemia at a lower level (104 ID50/ml) is established after 7 to 10 days.

Antigenic cross-reactivity between caprine arthritis-encephalitis, visna and progressive pneumonia viruses involves all virion-associated proteins and glycoproteins.

Abstract: Antigenic relatedness between the virion-associated proteins of caprine arthritis-encephalitis, visna and progressive pneumonia viruses was examined Antigenic cross-reactivity was assessed by immunoprecipitation of disrupted, radiolabelled virus with goat, sheep and rabbit antisera, followed by resolution of the immunoprecipitation products by SDS-polyacrylamide gel electrophoresis The results indicate that antigenic cross-reactivity between the caprine and ovine virus isolates involves all of the major virion-associated proteins and glycoproteins The common antigenic determinants exhibited by virion structural proteins are immunogenic in goats, sheep and rabbits

Naked dsRNA Associated with Hypovirulence of Endothia parasitica Is Packaged in Fungal Vesicles

Abstract: Summary Hypovirulence in Endothia parasitica is known to be associated with dsRNA. The characteristics of this cytoplasmically transmissible element suggest it might be viral in nature. Attempts to isolate viral particles indicate the presence of two particulate fractions in hypovirulent (HV) strains and a similar one in virulent (V) strains. Composition analyses of the fractions show these to be similar except that those from HV strains include dsRNA. Also present in these particles are carbohydrate, protein and chloroform-methanol-extractable substances. The protein composition is too low to indicate the presence of a capsid. The neutral sugars that are present (arabinose, mannose, galactose, glucose) in these particles are also present in the fungal cell wall. Silver- and ethidium bromide-stained polyacrylamide gels resolve several minor as well as the known major dsRNA components. These components are arbitrarily divided into four mol. wt. groups: 4.5 × 106 to 5.0 × 106, 1.2 × 106 to 1.5 × 106, 5.8 × 105 to 6.2 × 105 and < 4.6 × 105. Not all segments are present at all times, although the phenotype of the culture is not affected. Proteins are not detectable in vesicles of HV strains, but six proteins are detectable from V strains by gel electrophoresis. Assays for [32P]UMP incorporation indicate the presence of a RNA polymerase closely associated with the dsRNA. Our results suggest that the transmissible element responsible for hypovirulence is a naked dsRNA genome packaged within vesicles formed by the host.

Production of a monoclonal antibody against an epitope on HeLa cells that is the functional poliovirus binding site.

Abstract: Summary Cell lines of primate origin carry receptors on their plasma membrane which are responsible for the specific binding of poliovirus. This paper describes the isolation and characterization of a monoclonal antibody reacting with the plasma membrane of HeLa cells. The antibody (D171) was selected for its protection of HeLa cells against the cytopathic effect of poliovirus type 1. This protection was found to extend to all three viral serotypes, while the replication of five other viruses in HeLa cells was not affected. The 125I-labelled purified antibody did not react with cell lines derived from pig, dog or rodents but bound specifically to all lines of human or primate origin. Immunoglobulin or Fab fragments of D171 prevented the binding of 35S-labelled poliovirus to HeLa cells. Conversely, nearly all binding sites of 125I-labelled D171 immunoglobulins or Fab fragments could be blocked after preincubation of HeLa cells with poliovirus. These results indicate that D171 recognizes the poliovirus receptor site on different susceptible cells and that practically all D171 binding sites are involved in the specific attachment of poliovirus to the plasma membrane. To determine whether the epitope recognized by D171 could be separated from the receptor for poliovirus, human-mouse cell hybrids were prepared and analysed. In all 40 clones tested, the susceptibility to poliovirus correlated with the binding of D171.

The ribonucleotide reductase induced by herpes simplex virus type 1 involves minimally a complex of two polypeptides (136K and 38K).

Abstract: Summary Herpes simplex virus type 1 (HSV-1) encodes a polypeptide of apparent mol. wt. 136000 (Vmw136) known to be a component of the virus-specified ribonucleotide reductase. Monoclonal antibodies that precipitate this polypeptide also precipitate a polypeptide of mol. wt. 38000 (Vmw38) from extracts of HSV-1-infected cells. The basis for this co-precipitation has been investigated using a monoclonal antibody directed against Vmw136 and an oligopeptide-induced antiserum directed against the carboxy terminus of Vmw38. We have also made use of a temperature-sensitive (ts) mutant of HSV-1 which maps within the sequences encoding Vmw136 and which induces a thermolabile ribonucleotide reductase. Our experiments show (i) Vmw136 and Vmw38 form a complex in infected cells and (ii) the mutation in the ts mutant results in the two polypeptides being unable to form the complex at the non-permissive temperature. We speculate that association of the two polypeptides is necessary for ribonucleotide reductase activity. No evidence was found for involvement of host proteins in the proposed virus-induced ribonucleotide reductase complex. The terms RR1 and RR2 are suggested for the large and small subunits of the HSV-induced enzyme.

Entry of poliovirus type 1 and Mouse Elberfeld (ME) virus into HEp-2 cells: receptor-mediated endocytosis and endosomal or lysosomal uncoating.

Abstract: Poliovirus type 1 appeared from electron microscope studies to enter HEp-2 cells by receptor-mediated endocytosis. On adsorption the virus was evenly distributed over the cell surface, with some preference for the microvilli and their bases. Invagination of the cell surface membrane with the attached virus commenced at coated pits and led to the formation of virus-containing coated vesicles in the cytoplasm. These coated vesicles fused with intracellular vesicles to form endosomes. When cells infected with poliovirus or Mouse Elberfeld virus were treated with the weak bases chloroquine, NH4Cl or the ionophore monensin to raise the intraendosomal and intralysosomal pH above 6, virus-directed macromolecular synthesis and production of progeny were prevented. These results suggest that the virus genomes are released to the cytoplasm via endosomes and/or lysosomes by a pH-dependent process.

Detection of Viroid and Viroid-like RNAs from Grapevine

Abstract: SUMMARY Analysis by polyacrylamide gel electrophoresis of nucleic acid preparations, obtained from several varieties of grapevine by a procedure designed to isolate and purify viroids, revealed the presence of RNA species with some of the characteristic physical properties of viroids. Under non-denaturing conditions, a band with a mobility faster than that of citrus exocortis viroid (CEV) was detected, and under fully denaturing conditions two bands were observed, one co-migrating with the circular forms of CEV and a second migrating faster than the linear forms of this viroid. This RNA species did not hybridize with a cDNA probe to CEV. Some of the grapevine preparations were infective for Gynura aurantiaca, inducing symptoms similar to those caused by CEV, and the appearance of an RNA which had the same mobility as CEV in denaturing and non-denaturing electrophoretic systems and hybridized with cDNA to CEV. These results suggest that viroid-like and viroid RNAs can be recovered from grapevine, the former (with no detectable sequence homology to CEV) at a concentration sufficient to be observed as a physical entity in gels, and the latter (with close sequence homology to CEV) whose presence could only be revealed by bioassay. The possible involvement of these RNAs in some grapevine diseases of unknown aetiology is discussed.

Variation in Cydia pomonella Granulosis Virus Isolates and Physical Maps of the DNA from Three Variants

Abstract: Summary Cydia pomonella granulosis viruses (CpGV) from seven different sources in Europe, America and New Zealand were compared by restriction enzyme analysis Most samples were indistinguishable from the Mexican isolate (CpGV-M) Isolates from Russia (CpGV-R) and England (CpGV-E) showed small genotypic differences CpGV-E was shown to be a mixture of two variants, E1 and E2 CpGV-E1 was indistinguishable from CpGV-M A physical map of CpGV-M was constructed for the enzymes EcoRI, BamHI, HindIII, SmaI and ApaI A comparison of fragment profiles allowed construction of maps for CpGV-R and CpGV-E2 Relative to CpGV-M, CpGV-R had a single deletion of 24 kbp and CpGV-E2 was modified in one area resulting in an additional EcoRI site, a shift in a BamHI site and in total about 1 kbp more DNA The map was orientated by locating the granulin gene using the cloned granulin gene from Trichoplusia ni GV as a probe There was no significant difference between the infectivities of the Mexican, Russian and English isolates for neonate larvae

Preparation and characterization of monoclonal antibodies directed against four structural components of canine distemper virus.

Abstract: Mouse hybridomas producing antibodies against structural proteins of canine distemper virus (CDV) were produced by fusion of Sp2/0 myeloma cells with spleen cells from BALB/c mice immunized with purified preparations of Vero cell-grown CDV. Ascites fluids collected after intraperitoneal inoculation with 149 CDV antibody-producing hybridoma cell lines were characterized by different serological tests. By immune precipitation tests with [35S]methionine-labelled extracellular virions and intracellular virus polypeptides, 57 clones were found to produce antibodies against the nucleocapsid protein (NP), 22 against the polymerase (P) protein, 10 against the fusion (F) protein and nine against the large uncleaved glycoprotein (named H in analogy with measles virus). By competitive binding enzyme-linked immunosorbent assay (ELISA) tests with monoclonal antibodies against each structural component, a minimum of 18, six, three and seven separate antigenic determinants were identified on the NP, P, F and H proteins, respectively. The reactions of clones directed against F and H surface components of the virus were tested for their ability to inhibit the infectivity of both CDV and measles virus in the absence and presence of anti-gamma-globulin. In addition, the inhibitory activity of the clones on measles haemagglutinating (HA) and haemolysis (HL) activity were examined. Monoclonal antibodies against six of the seven antigenic determinants of the H protein could neutralize the infectivity of the virus. After addition of anti-gamma-globulin to the test, increases of titres varying from twofold to several hundredfold were observed with the different clones. None of all the clones against H could block measles virus infectivity, HA or HL activity. The 10 clones directed against the F protein could not neutralize the infectivity of CDV even in the presence of anti-gamma-globulin. Further, the antibodies could not inhibit measles HA and HL activity in the absence of anti-gamma-globulin. However, after the addition of anti-gamma-globulin, antibodies against two of the three sites were found to block measles virus HL activity. The reactions of all clones were tested in immune fluorescence, ELISA and immune precipitation tests with three strains of CDV. Each strain had a few unique antigenic sites. Variation was found in four, one and three different antigenic sites of the NP, P and H proteins, respectively.

Nucleotide Sequence Analysis of RNA-3 and RNA-4 of Beet Necrotic Yellow Vein Virus, Isolates F2 and G1

Abstract: Summary The nucleotide sequences of cDNA clones corresponding to RNA-3 and RNA-4 of beet necrotic yellow vein virus isolates F2 and G1 have been determined. The cDNA of RNA-3 of isolate F2 is 1775 residues in length and contains a coding region of 219 codons. In isolate G1 this coding region has undergone an internal deletion of 354 nucleotides in such a way as to conserve a shortened reading frame. Otherwise, the RNA-3 sequences of the two isolates were closely similar. RNA-4 of isolate F2 has an extrapolated length of 1431 residues and contains an open reading frame of 282 codons. This open reading frame has undergone an internal deletion of 324 nucleotides in one cDNA clone of RNA-4(G1) with conservation of a shortened reading frame. Sequence analysis of other RNA-4(G1) cDNA clones revealed, however, that the exact boundaries of the deletion are not always the same. RNA-3 and RNA-4 of each isolate are more than 90% homologous for the 3′-terminal 200 nucleotides. Short homologous sequences are also present in RNA-3 and RNA-4 of isolate F2 flanking the regions deleted in each of these RNAs in the G1 isolate. These homologous sequences probably play a role in the deletion process.

Paramyxovirus antigens in osteoclasts from Paget's bone tissue detected by monoclonal antibodies.

Abstract: The fluorescent antibody technique using both monoclonal and specific polyclonal virus antibodies was applied to investigate the nature of the inclusions seen in the abnormal osteoclasts associated with Paget's bone disease. The results show that antigens of measles virus, simian virus 5 (SV5) and human parainfluenza virus type 3 (PF3) could be detected in the osteoclasts but not in control bone cells. Measles and SV5 nucleoprotein (NP) and haemagglutinin-neuraminidase (HN) antigens were apparently present in all the cases of Paget's disease examined, whereas PF3 NP and HN antigens were present only in some of the cases. These investigations suggest that paramyxoviruses may play a role in the aetiology of the bone disease.

Sequences of the nucleocapsid genes from two strains of avian infectious bronchitis virus.

Abstract: cDNAs prepared from viral genomic RNA purified from two strains of infectious bronchitis virus (IBV) (Beaudette and M41) have been cloned into pBR322. Three of these clones, which contain the complete sequences of mRNA A for both strains, except for the leader sequences which are only present on the subgenomic messenger RNAs, have been sequenced using the dideoxy method. The sequences are similar for both strains, each containing a single long open reading frame of 1227 bases which predicts a polypeptide of molecular weight approximately 45 000. The genome position and size of this predicted polypeptide are consistent with it being the gene for the nucleocapsid protein. The amino acid sequence shows considerable homology with those of the nucleocapsids of murine hepatitis virus strains A59 and JHM. The major difference between the sequences determined for the two IBV strains is that the 3' non-coding region of the Beaudette strain contains a 184 base segment which is not present in the M41 strain.

Respiratory syncytial virus polypeptides. IV. The oligosaccharides of the glycoproteins.

Abstract: Summary The cell-associated glycoproteins of respiratory syncytial (RS) virus included GP1 (90K), VP70 (70K), VGP48 (48K) and GP26 (26K). Although present in infected cells, there was no VP70 in purified virus. Trypsin treatment of infected cells removed 80 to 90% of VP70 as well as its products VGP48 and GP26. This suggested that most of the VP70 in the cell is located on the plasma membrane. The glycoproteins of purified RS virus (GP1, VGP48 and GP26) contain mannose, galactose and fucose as well as glucosamine, but the quantity of mannose in GP1 is low when compared to that of the other three sugars. The effects that follow the treatment of infected cells with the glycosylation inhibitors tunicamycin and monensin, and the treatment of the immunoprecipitated product of pulse-chase experiments with endonuclease H demonstrated that VP70 and its products contained N-linked oligosaccharides, and that the oligosaccharides of the mature VGP48 subunit were of the complex type, while GP1 contained both N- and O-linked oligosaccharides. The non-glycosylated forms of VP70 and GP1 have estimated mol. wt. of 50K and 33K respectively. Therefore, the carbohydrate contribution to the mol. wt. of VP70 and GP1, as determined by PAGE, was equivalent to 20K for the former and 57K for the latter. The majority of the GP1 oligosaccharides were O-linked, a form of sugar linkage not previously found among paramyxoviruses.

In vitro phosphorylation of NS protein by the L protein of vesicular stomatitis virus.

Abstract: Summary The structural proteins L and NS of vesicular stomatitis virus were obtained from purified viral ribonucleoprotein complex followed by phosphocellulose column chromatography and assayed for protein kinase activity using [γ-32P]ATP as the phosphate donor. The fractions containing purified L protein phosphorylated NS protein in vitro. 8-Azido-ATP, a photoreactive analogue of ATP, was also used as the phosphate donor for phosphorylation of NS protein by the L protein. In the presence of ultraviolet light, only L protein was specifically cross-linked with 8-azido-[γ-32P]ATP. In the absence of u.v. light 8-azido ATP did no inhibit RNA transcription in a reconstituted reaction or substitute ATP for RNA synthesis in vitro. The above results, taken together, suggest that 8-azido-ATP was bound to the kinase site and phosphorylation of NS protein was mediated by th L protein. Exogenous phosphate acceptor proteins such as phosvitin and casein were also phosphorylated by the L protein fraction. However, addition of an excess of phosvitin failed to compete with the phosphorylation of NS by L, indicating that the protein kinase activity possessed higher affinity for NS. The phosphorylation of NS was strongly inhibited by photoreaction of L protein with 8-azido-ATP with concomitant inhibition of transcription in vitro. These results suggest that phosphorylation of NS protein by L may have a role in the regulation of the virus genome transcription in vitro.

Characterization by Western blotting of the immunogens of infectious bursal disease virus.

Abstract: The Australian isolate of infectious bursal disease (IBD) virus (002/73) was purified from infected bursae by rate-zonal and density-equilibrium centrifugation and characterized by polyacrylamide gel electrophoresis. Two major polypeptides having approximate mol. wt. of 32 000 (32K) and 37K and three other polypeptides of approximate mol. wt. 29K, 41.5K and 91.5K were present in all preparations of virus having a buoyant density of 1.33 g/ml. Western blotting of the polypeptides of IBD virus showed that the initial antibody response of chickens infected with live virus or injected with an inactivated oil-emulsion vaccine was directed primarily towards the 32K polypeptide. Only sera obtained late in the response to live virus or following hyperimmunization contained antibodies recognizing the 29K, 37K and 41.5K polypeptides. An antibody response to the 91.5K polypeptide was not detected routinely by this technique. It was concluded that the 32K polypeptide is a major immunogen of IBD virus.