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W J Bellini

Publications and source records attributed to W J Bellini.

At least 73 records · Page 4Linked to original sources
PubMed1992

Genetic variability of the glycoprotein genes of current wild-type measles isolates.

The glycoprotein coding sequences from three wild-type measles viruses isolated in the United States during 1988-1989 were examined by mRNA templated sequencing to determine whether contemporary strains have undergone genetic changes relative to the vaccine strain, Moraten. These studies revealed variation in the hemagglutinin (HA) gene and, to a far lesser degree, the fusion (F) gene. The F protein coding region was highly conserved with only three predicted amino acid changes. Among the predicted amino acid changes identified in the HA was a new potential glycosylation site at residue 416, located toward the carboxy-terminal end of the HA peptide. Eighty percent of the predicted amino acid changes in the HA shared by the three wild-type isolates were clustered near the five previously identified potential glycosylation sites. A linear pattern of evolutionary change was observed after comparing the predicted amino acid HA changes from the 1988-1989 viruses to those predicted in the HA protein from U.S. wild types isolated in 1977 and 1983.

Animals↗
PubMed1992

Baculovirus expression of the nucleoprotein gene of measles virus and utility of the recombinant protein in diagnostic enzyme immunoassays.

A recombinant baculovirus that expresses the nucleoprotein gene of measles virus (Edmonston vaccine strain) under the transcriptional control of the polyhedrin promoter was generated. The expressed protein (B-MVN) comigrated with the authentic viral nucleoprotein as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it was phosphorylated. The B-MVN protein proved to be reactive with monoclonal antibodies in radioimmunoprecipitations, and it was immunogenic, eliciting in mice antisera that recognized the native nucleoprotein. In addition, the B-MVN protein was evaluated as a replacement source of antigen for whole virus in enzyme immunoassays (EIAs) for detection of measles virus-specific immunoglobulin M (IgM) and IgG antibodies. A capture IgM EIA with the B-MVN protein as antigen detected specific IgM antibodies in 18 (72%) acute- and all convalescent-phase specimens from 25 clinical measles cases and exceeded 99% specificity with 120 control specimens. An indirect IgG EIA with the B-MVN protein detected specific IgG antibodies in 129 of 131 (98%) serum specimens with antibodies to measles virus, and results obtained from testing 268 additional serum specimens were better correlated with measles virus-neutralizing antibodies than those obtained with a commercial EIA.

Antibodies, Viral↗
PubMed1991

Protection of mice with vaccinia virus recombinants that express the rabies nucleoprotein.

The role of rabies virus nucleoprotein (N) in protection against rabies was examined with recombinant vaccinia viruses expressing the N of the Challenge Virus Standard strain. Two chimeric plasmids were constructed with the open reading frame of the N gene placed downstream of the vaccinia P7.5 promoter (early/late class) or the vaccinia P11 promoter (late class), with each expression cassette flanked by vaccinia thymidine kinase (TK) sequences to enable marker rescue by TK insertional inactivation. Two recombinants were isolated that expressed the rabies N in infected cells as determined by radioimmunoprecipitation and immunofluoresence microscopy with an anti-N monoclonal antibody. Two groups of 25 ICR mice inoculated intradermally with the recombinants and challenged with 75 MFPLD50 of street rabies virus showed high survival ratios (22/25 and 21/25). Intramuscular inoculation, however, was not protective against 25 MFPLD50. The intradermally vaccinated mice developed non-neutralizing antibodies against rabies N.

Animals↗
PubMed1991

Evaluation of monoclonal antibody-based capture enzyme immunoassays for detection of specific antibodies to measles virus.

Monoclonal antibodies to the hemagglutinin protein, fusion protein, phosphoprotein, matrix protein, and nucleoprotein of measles virus were evaluated as detector antibodies in capture enzyme immunoassays (EIAs) for the detection of specific serum immunoglobulin G (IgG), IgA, and IgM antibodies to measles virus. A pool of monoclonal antibodies to hemagglutinin protein and nucleoprotein proved optimal and was further evaluated. Specific IgM was detected in 97% of adolescents with clinical measles, 97% of infants 3 weeks postvaccination, and less than 1% of normal serum specimens. Specific IgA antibodies were found in 97% of adolescents with clinical measles, 97% of infants 3 weeks postvaccination, and less than 1% of normal serum specimens. Specific IgA antibodies were found in 97% of clinical measles cases and vaccinees, in 26% of healthy persons, and in 36% of infants 8 months postvaccination; consequently, IgA antibodies were not a useful indicator of recent measles infection. A significant increase in IgG antibodies between paired specimens was detected in 92% of clinical cases and all vaccinees. Only 59% of infant specimens had persistent IgG antibodies as detected by capture EIA at 8 months postvaccination, whereas all specimens had antibodies as detected by hemagglutination inhibition and plaque neutralization. An alternative indirect EIA, in which antigen was directly absorbed to the solid phase, was more sensitive than the capture design, detecting IgG antibodies in all infants postvaccination. When standardized with a microneutralization assay for the detection of persistent antibodies, the indirect IgG EIA gave predictive values for positive and negative tests exceeding 90%. Our capture IgM and indirect IgG EIAs provide a practical combination of serologic tests for the determination of acute measles virus infection and past exposure to measles virus or vaccine, respectively.

Antibodies, Monoclonal↗
PubMed1991

Raccoon poxvirus recombinants expressing the rabies virus nucleoprotein protect mice against lethal rabies virus infection.

Raccoon poxvirus (RCN) recombinants expressing the rabies virus internal structural nucleoprotein (RCN-N) protected A/WySnJ mice against a lethal challenge with street rabies virus (SRV). Maximum survival was achieved following vaccination by tail scratch and footpad (FP) SRV challenge. RCN-N-vaccinated mice inoculated in the FP with SRV were resistant to infection for at least 54 weeks postvaccination. Protection was also elicited by RCN recombinants expressing the rabies virus glycoprotein (RCN-G). Vaccination with RCN-G evoked rabies virus neutralizing antibody. Rabies virus neutralizing antibody was not detected in RCN-N-vaccinated mice prior to or following SRV infection. Radioimmunoprecipitation assays showed that sera from RCN-N-vaccinated mice which survived SRV infection did not contain antibody to SRV structural protein G, M, or NS. The mechanism(s) of N-induced resistance appears to correlate with the failure of peripherally inoculated SRV to enter the central nervous system (CNS). Support for this correlation with resistance was documented by the observations that SRV-inoculated RCN-N-vaccinated mice did not develop clinical signs of CNS rabies virus infection, infectious SRV was not detected in the spinal cord or brain following FP challenge, and all RCN-N-vaccinated mice died following direct intracranial infection of the CNS with SRV. These results suggest that factors other than anti-G neutralizing antibody are important in resistance to rabies virus and that the N protein should be considered for incorporation with the G protein in recombinant vaccines.

Animals↗
PubMed1990

Rabies diagnostic reagents prepared from a rabies N gene recombinant expressed in baculovirus.

A gene encoding the nucleoprotein (N) of rabies virus was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus. Recombinant gene expression was controlled by the strong polyhedrin gene promoter. Insect cells (Spodoptera frugiperda) infected by a baculovirus recombinant containing the rabies virus N gene produced abundant amounts of a novel 55-kilodalton protein of a size comparable to that of the rabies virus N protein, as demonstrated by polyacrylamide gel electrophoresis. This new gene product possessed the antigenic and immunogenic properties of native viral N protein, as shown by the ability of the new protein to react in immunoprecipitation and immunofluorescence assays with antirabies antibodies, to serve as a substitute for infectious rabies virus in adsorbing suspensions for diagnostic tests, and to induce high-titered antiserum. The baculovirus expression system provides a safe, convenient, and inexpensive source of rabies virus N protein for the production of both antiserum and adsorbing suspensions for use in rabies diagnoses.

Animals↗
PubMed1988

Expression of the gene encoding the 17-kilodalton antigen from Rickettsia rickettsii: transcription and posttranslational modification.

Recently, we reported the molecular cloning and nucleotide sequence analysis of a gene from Rickettsia rickettsii that codes for a 17-kilodalton antigen (17K antigen) and is preceded by sequences closely resembling the -10 and -35 consensus sequences for recognition by Escherichia coli RNA polymerase (Anderson et al., J. Bacteriol. 169:2385-2390, 1987). Experiments described in this report indicate that the start sites for initiating transcription of the 17K antigen gene are identical in the E. coli clone and in intact R. rickettsii. In each case, initiation was shown to begin 9 bases downstream of the presumed Pribnow box sequence (TATACT). A 169-base-pair fragment containing the promoter sequence initiated transcription in both directions when cloned into an E. coli promoter probe vector. The rickettsial fragment was found to contain sequences identical to the -10 region (but not the -35 region) of the E. coli promoter consensus sequence directed away from the 17K antigen gene. The amino-terminal portion (residues 17 to 20) of the deduced amino acid sequence for the 17K antigen contained the tetrapeptide Leu-Gln-Ala-Cys, a sequence that conforms favorably to those described for lipid modification and cleavage by lipoprotein signal peptidase II. The 17K antigen produced by the E. coli clone was shown to be labeled with [3H]palmitate and [3H]glycerol, indicative of lipid modification. In vitro mutagenesis designed to alter the cysteine at residue 20 to a glycine abolished incorporation of [3H]palmitate, suggesting that posttranslational modification occurs via a mechanism similar to that described for other gram-negative bacterial lipoproteins.

Amino Acid Sequence↗
PubMed1987

Sequence analysis of the 17-kilodalton-antigen gene from Rickettsia rickettsii.

DNA obtained from the Sheila Smith strain of Rickettsia rickettsii was digested to completion with the restriction endonucleases BamHI and SalI and ligated with the plasmid vector pUC19. The ligation mixture was used to transform Escherichia coli. A total of 465 bacterial clones were screened for antigen production with hyperimmune rabbit serum. One of the reactive clones, containing a recombinant plasmid designated pSS124, was solubilized and subjected to immunoblot analysis and revealed expression of a 17-kilodalton protein reactive with anti-R. rickettsii serum that comigrated with an antigen from R. rickettsii. A 1.6-kilobase PstI-BamHI fragment from pSS124 was subcloned and continued to direct synthesis of the 17-kilodalton antigen. The nucleotide sequence was determined for this 1.6-kilobase subclone, which encompassed the gene encoding the polypeptide as well as flanking regions containing potential regulatory sequences. The open reading frame consisted of 477 nucleotides that specified a 159-amino-acid protein with a calculated molecular weight of 16,840. The deduced amino acid sequence contained a hydrophobic sequence near the amino terminus that resembled signal peptides described for E. coli. The carboxy terminus was hydrophilic in nature and probably contained the exposed epitopes.

Amino Acid Sequence↗
PubMed1987

Characterization of cloned measles virus mRNAs by in vitro transcription, translation, and immunoprecipitation.

A cDNA library was constructed from poly(A)+ RNA prepared from VERO cells infected with the Edmonston strain of measles virus. Clones corresponding to five major viral-specific transcripts were identified by northern blot hybridization analysis. Probes prepared from these five clones detected an additional five minor viral RNA transcripts. The sizes and hydridization patterns of these minor RNA species are consistent with their being bicistronic transcripts arising from a viral genomic template with the gene order 3'..NP-9/C-M-F-HA-(L)..5'. To assess the coding capacity of these cDNA clones they were inserted into pSP64, transcribed in vitro, the RNA was translated in reticulocyte lysates, and the protein was immunoprecipitated with specific antisera. From this analysis the genes for NP, P/C, M, F, and HA were identified. In vitro translation of natural mRNAs and SP6 transcripts of cDNAs consistently produced smaller polypeptides that appear to be initiated at internal AUGs. The relative abundance of these various cell-free translation products reflects the probability of translational initiation at the various in-frame AUGs. The patterns observed suggest that other factors besides sequences immediately flanking the AUGs have a significant effect on the selection of translational initiation sites. An increase in translational efficiency of the F transcript was achieved by removing 450 bases of the G-C-rich 5' noncoding region.

Animals↗
PubMed1986

Accumulated measles virus mutations in a case of subacute sclerosing panencephalitis: interrupted matrix protein reading frame and transcription alteration.

Subacute sclerosing panencephalitis (SSPE) is a fatal disease affecting the human central nervous system several years after acute measles infection. Measles virus (MV) genomes replicating in SSPE brains do not give rise to budding particles and present various defects in gene expression, mostly concerning the matrix (M) protein. For one SSPE case (K), shown previously to be devoid of M protein expression, we examined here in detail the features involved in this defect. In the brain of patient K the normal, monocistronic MV M mRNA was completely substituted by a bicistronic RNA containing the coding sequence of the preceding phosphoprotein (P) gene in addition to the M coding sequence. Analysis of the P-M intercistronic region by direct cDNA sequencing showed that the consensus sequence at this RNA processing site was unaltered but revealed several distant point mutations. cDNA cloning and sequencing of the entire M coding region established that one of the point mutations leads to a stop codon at triplet 12 of the M reading frame. It is unknown whether this defect, explaining by itself the lack of M protein, is related also to the block of M mRNA formation. In addition we note that as much as 1% of the nucleotides differed between two overlapping clones from the same brain. This high sequence variability could possibly account for the diversity of defects observed in MV gene expression in SSPE brains and may be a general phenomenon associated with RNA virus persistence.

Amino Acid Sequence↗
PubMed1986

Matrix genes of measles virus and canine distemper virus: cloning, nucleotide sequences, and deduced amino acid sequences.

The nucleotide sequences encoding the matrix (M) proteins of measles virus (MV) and canine distemper virus (CDV) were determined from cDNA clones containing these genes in their entirety. In both cases, single open reading frames specifying basic proteins of 335 amino acid residues were predicted from the nucleotide sequences. Both viral messages were composed of approximately 1,450 nucleotides and contained 400 nucleotides of presumptive noncoding sequences at their respective 3' ends. MV and CDV M-protein-coding regions were 67% homologous at the nucleotide level and 76% homologous at the amino acid level. Only chance homology was observed in the 400-nucleotide trailer sequences. Comparisons of the M protein sequences of MV and CDV with the sequence reported for Sendai virus (B. M. Blumberg, K. Rose, M. G. Simona, L. Roux, C. Giorgi, and D. Kolakofsky, J. Virol. 52:656-663; Y. Hidaka, T. Kanda, K. Iwasaki, A. Nomoto, T. Shioda, and H. Shibuta, Nucleic Acids Res. 12:7965-7973) indicated the greatest homology among these M proteins in the carboxyterminal third of the molecule. Secondary-structure analyses of this shared region indicated a structurally conserved, hydrophobic sequence which possibly interacted with the lipid bilayer.

Amino Acid Sequence↗
PubMed1985

Sequence homology within the morbilliviruses.

Double-stranded cDNA synthesized from total polyadenylate-containing mRNA extracted from monkey kidney cells infected with canine distemper virus (CDV) was cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing CDV DNA were identified by hybridization to a CDV-specific 32P-labeled cDNA. A cDNA clone containing an insert 1,700 base pairs (CDV 364) has been identified as the reverse transcript of the mRNA coding for the nucleocapsid protein. The size of the mRNA species complementary to this insert is 1,850 nucleotides, as determined by the Northern technique. Hybridization experiments and heteroduplex mapping indicated homology between the central region of the CDV and measles virus nucleocapsid gene. The completion of the nucleotide sequence analysis of the measles virus gene allowed the reconstruction of the entire coding region of the measles virus gene and a comparison with the counterpart sequence of CDV. This comparison delineated three regions: (i) a region of high homology (nucleotides 501 to 1215), in which 77% of the nucleotides and 88% of the encoded amino acids are identical; (ii) a region of moderate homology at the 5' end of the message (nucleotides 1 to 500), in which 59% of the nucleotides and 66% of the encoded amino acids are identical; (iii) a region of little or no homology (nucleotides 1216 to 1625) near the 3' end of the message.

Amino Acid Sequence↗
PubMed1985

Cloning and characterization of DNA complementary to the canine distemper virus mRNA encoding matrix, phosphoprotein, and nucleocapsid protein.

Double-stranded cDNA synthesized from total polyadenylate-containing mRNA, extracted from monkey kidney cells infected with canine distemper virus (CDV), has been cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing canine distemper virus DNA were identified by hybridization to a canine distemper virus-specific, 32P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-80), 960 (clone 74-16), 1,700 (clone 364), and 950 (clone 40-9) base pairs. The sizes of the mRNA species complementary to these inserts are 1,500, 1,850, 1,850 and 2,500 nucleotides, respectively, as determined by the Northern technique. Three of the cloned DNA fragments were further identified as the reverse transcripts of the mRNA coding for the matrix, phosphoprotein, and nucleocapsid protein of CDV.

Base Sequence↗
PubMed1985

Measles virus P gene codes for two proteins.

The entirety of the phosphoprotein gene of measles virus has been sequenced. The gene is composed of 1,657 nucleotides and specifies a 507-amino-acid protein (P). A second overlapping reading frame was predicted from the sequence and specifies a 186-amino-acid protein (C). Through the use of antisynthetic peptide antibodies, we show that both proteins are expressed in virally infected cells. Both proteins are expressed from a functionally bicistronic mRNA through independent initiation of ribosomes at the respective AUG codons. Using immunofluorescent microscopy, we localized the C protein in the nucleus and in cytoplasmic inclusions within the infected cells.

Animals↗
PubMed1985

Use of antibodies directed against synthetic peptides for identifying cDNA clones, establishing reading frames, and deducing the gene order of measles virus.

A number of cDNA clones complementary to measles virus mRNA and 50S genome RNA have been generated. These clones have been mapped by restriction enzyme analysis and were subsequently sequenced by the method of Maxam and Gilbert (A. M. Maxam and W. Gilbert, Methods Enzymol. 65:499-560, 1980). Computer analysis of these DNA sequences revealed open reading frames which potentially could code for a number of gene products. Portions of these putative polypeptides were synthesized, and rabbit antibodies directed against peptide-hemocyanin conjugates were produced. These antibodies were used to immunoprecipitate virus-specific polypeptides which were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For each of the antisera tested, a unique protein was precipitated whose migration on polyacrylamide gels corresponded to standard gene products identified by monoclonal antibodies and antisera against measles virus. By using this method, we were able to assign the coding regions of cDNA clones to specific protein products and, subsequently, to order the genes of the 3'-terminal third of measles genome RNA.

Antibodies, Monoclonal↗
PubMed1984

Human cellular immune response to measles virus polypeptides.

Measles virus polypeptides were separated by polyacrylamide gel electrophoresis and electroeluted from gel sections. The antigenicity of the polypeptides was determined by enzyme-linked immunosorbent assays. The ability of these measles virus antigens to stimulate lymphoproliferation was measured in both high- and low-responder individuals. In contrast to the low-responder lymphocytes which did not proliferate when stimulated with measles virus antigens, the high-responder lymphocytes proliferated when challenged with hemagglutinin, nucleocapsid-associated phosphoprotein, nucleocapsid protein, matrix protein, and fusion protein.

Electrophoresis, Polyacrylamide Gel↗