Nucleotide sequence analysis of the Newcastle disease virus nucleocapsid protein gene and phylogenetic relationships among the Paramyxoviridae

The nucleocapsid (N) protein genes from 24 Newcastle disease virus (NDV) isolates representing various pathotypes with different geographical and chronological origins were cloned and sequenced. The N-terminal region of the N protein to residue 401 was highly conserved among isolates with several conservative substitutions occurring that correlated with phylogenetic relationships. Variability of the N protein was detected in the C-terminal portion similar to what has been reported for other members of the Paramyxovirinae. Amino acids previously identified as invariant or highly conserved in N proteins of other paramyxoviruses were also present in the NDV protein. Phylogenetic analysis of N gene coding sequences among NDV isolates again demonstrated the existence of two major groups. One clade contained viruses that included vaccine and virulent strains isolated in the USA prior to 1970 while a second clade included vaccine and virulent viruses isolated worldwide. Comparison of N protein amino acid sequences among members of the Paramyxoviridae resulted in NDV and avian paramyxovirus 6 separating as a cluster distinct from the Rubulavirus genus. This provides further support for avian paramyxoviruses being considered for their own genus among the Paramyxovirinae.     

Conserved structures among the nucleocapsid proteins of the paramyxoviridae: complete nucleotide sequence of human parainfluenza virus type 3 NP mRNA

The nucleotide sequence of the mRNA coding for the nucleocapsid protein (NP) of the paramyxovirus, human parainfluenza virus type 3 (PIV-3), has been determined. The NP mRNA was found to contain 1642 bases, excluding poly(A), and encode a protein of 515 amino acids, with a molecular weight of 57,823. Amino acid residues 1 through 420 of PIV-3 NP protein showed extensive sequence homology with the corresponding amino acids of Sendai virus nucleocapsid protein. There was virtually no homology between the last 95 amino acids. Comparison of the NP proteins of PIV-3, Sendai virus, measles virus, and canine distemper virus revealed, from amino acid residues 160 through 390, some conserved areas between the corresponding proteins of these paramyxoviruses. The 5' terminal sequence of PIV-3 NP mRNA (5'-AGGATTAAAG-3') was similar to the conserved sequence (formula; see text) found at the 5' termini of Sendai virus mRNAs. Both PIV-3 NP and Sendai virus mRNAs had a common 3' terminal tetranucleotide (5'-TAAG-3') preceding the poly (A) tail.     

Complete nucleotide sequence of the matrix protein mRNA of mumps virus

The complete nucleotide sequence of the mumps virus membrane protein or matrix protein (M) has been determined by sequencing cDNA clones and confirmed by partially sequencing the M mRNA and the genome. The mRNA is 1248 nucleotides long excluding the poly(A) and encodes a protein of 375 amino acids. The molecular weight (38,670), deduced from the amino acid sequence, is in agreement with the molecular weight of the viral M protein estimated by polyacrylamide gel electrophoresis (39-40 kDa). The mumps virus M protein shows 23-27% homology with M proteins of Newcastle disease virus (NDV), measles virus, canine distemper virus (CDV), parainfluenza virus type 3, and Sendai virus, respectively. A comparison of the M protein sequences of the above six paramyxoviruses did not reveal any conserved area of homology common among all paramyxovirus M proteins.     

Complete nucleotide sequence of the hemagglutinin-neuraminidase (HN) mRNA of mumps virus and comparison of paramyxovirus HN proteins

The complete nucleotide sequence of the hemagglutinin-neuraminidase protein (HN) mRNA of the virulent SBL-1 strain of mumps virus has been determined. The mRNA contains 1887 nucleotides excluding the poly(A). The protein encoded by the mRNA has 582 amino acids and a membrane anchorage domain near the amino terminus. The calculated molecular mass (64 kDa) of the protein is in good agreement with that of the unglycosylated HN protein (63 kDa) identified in tunicamycin treated mumps virus infected cells (Herrler and Compans, 1983). The predicted sequence has nine potential N-glycosylation sites out of which two contain a cysteine residue and one has a proline residue as the variable amino acid X in the glycosylation site (Asn-X-Ser or Asn-X-Thr) and therefore, may not be utilized. One potential glycosylation site is in the cytoplasmic region which may not also be glycosylated. Comparison of the mumps HN protein sequence with the HN protein sequences of Sendai virus, simian-virus 5 (SV5), parainfluenza virus type 3 and Newcastle disease virus (NDV) shows two major homology regions, one region near the middle of the protein and the other in the second half of the molecule. In terms of percentage amino acid homology, the HN proteins of mumps virus, SV5 and NDV are closely related to each other but distinct from Sendai virus and parainfluenza virus type 3 HN proteins.     

Nucleotide sequence of the leader and nucleocapsid protein gene of mumps virus and epitope mapping with the in vitro expressed nucleocapsid protein.

The nucleotide sequence of the leader and the gene encoding the nucleocapsid protein (NP) of mumps virus Miyahara strain have been determined. The leader sequence is 55 nucleotides in length and the NP gene is 1845 nucleotides in length, exclusive of poly(A). The NP gene codes for a protein of 549 amino acids, with a calculated molecular weight of 61,365. For epitope mapping, a series of NPs from which C-termini were serially deleted were expressed in vitro from five mRNA constructs and were examined by radioimmunoprecipitation assay (RIPA) with eight nonoverlapping monoclonal antibodies (MoAbs) against the mumps virus NP. It was found that seven out of eight MoAbs reacted with the NP synthesized in vitro. Five recognized the epitopes located within the C-terminal 74 amino acids region and one within the adjacent 64 amino acids upstream. The epitope of the remaining one was in the N-terminal half of the NP.     

Nucleotide sequence of cDNA to the rinderpest virus mRNA encoding the nucleocapsid protein

The full-length cDNA corresponding to the mRNA encoding the nucleocapsid protein (NP) of rinderpest virus (RV) was cloned and its complete nucleotide sequence was determined. The gene of RV-NP was composed of 1683 nucleotides and contained a single large open reading frame, which is capable of encoding a protein of 525 amino acids with a molecular weight of 58,241 Da. The nucleotide sequence and predicted amino acid sequence were compared with those of measles virus (MV) and canine distemper virus (CDV). The nucleotide sequence of the coding region of RV-NP (53–1630) revealed a homology of 68.1% and 63.0% with MV and CDV-NP, respectively. Relatively moderate homologies of 68.7% (MV) and 64.3% (CDV) were found at nucleotides 53–592. The highest homology of 75.3–74.3% was equally present between RV and both MV and CDV in the middle region at nucleotides 593–1312. The homologies of the predicted amino acids in this region were 88.3% (MV) and 86.3% (CDV). Relatively low (MV) or little (CDV) homology was detected in the last 318 nucleotides toward the 3 terminus (1313–1630). The predicted secondary structures of amino acids at the C terminus differed between the three viruses.     

Molecular cloning and sequence analysis of bovine respiratory syncytial virus mRNA encoding the major nucleocapsid protein

The nucleotide sequence of the gene encoding the major nucleocapsid (N) protein of bovine respiratory syncytial virus (BRSV) has been determined. The N mRNA is 1196 nucleotides long with a single, large open reading frame. The derived polypeptide has 391 amino acids corresponding to a calculated molecular weight of 42,600 Da. This is in agreement with the molecular weight of 43,000 Da determined for the BRSV N protein by SDS-polyacrylamide gel electrophoresis (PAGE). Comparison of the nucleotide sequence of BRSV N gene with the sequence of the N gene of human respiratory syncytial virus (HRSV) revealed a homology of 80.7%. There is a 93.3% homology at the amino acid level between the N proteins of BRSV and HRSV. The 5'- and 3'-terminal untranslated sequences that are conserved among HRSV mRNAs were also identified in the N mRNA of BRSV. The results indicate that the N genes are highly conserved in the bovine and human strains of respiratory syncytial virus.     

The genomic sequence of a contemporary wild-type mumps virus strain

Vaccination has the potential to eradicate mumps, and 82 countries now include a live attenuated mumps vaccine as part of their childhood vaccination programme. Although, monotypic, genetic variants of mumps virus (MuV) have been described based on comparison of the SH gene sequences, and at least seven genotypes have been identified. We now report the entire sequence of a recently isolated wild type MuV strain, Glouc1/UK96 (Glouc1) by direct sequencing of the cDNA obtained from cell culture fluid. The genome of this recent isolate was 15 384 nucleotides in length. There were 579 nucleotide differences (3.8%) and 71 amino acid differences (1.5%) between Glouc1, a genotype G strain and Ur-AM9, a genotype B strain. Other MuV strains with available sequences were also compared with this pathological strain. The sequence of the contemporary strain reported here provides a picture of the variability of MuV over its entire genome (GenBank accession no. AF280799).     

Identification of the mRNA of the nucleocapsid proteins of pathogenic arenaviruses

Total RNA from cells infected with Machupo and Lassa viruses as well as individual sedimentation classes of these RNAs were translated in the cell-free protein-synthesizing system from rabbit reticulocytes. The translation products were precipitated either with anti-Machupo immune gamma-globulin or monoclonal antibodies to nucleocapsid protein (NP) of Lassa virus. Both total RNA and RNA fraction with the sedimentation coefficient 15-16S promoted the synthesis of protein which comigrated in gel with NP protein of purified virions. It is concluded that monocistron mRNA for NP protein of arenaviruses has the sedimentation coefficient 15-16 S.     

Antibody responses to mumps virus proteins in natural mumps infection and after vaccination with live and inactivated mumps virus vaccines

Paired sera from 20 patients with acute mumps infection, 16 from persons vaccinated with live attenuated mumps virus vaccine, and 12 from persons vaccinated with formalin-inactivated virus vaccine were studied for mumps antibodies by single radial hemolysis (SRH), hemagglutination inhibition (HI), and by enzyme immunoassays (EIA) specific for whole virus, envelope glycoprotein, and nucleocapsid antibodies. Mumps patients had diagnostic rises in serum mumps antibodies in 90–100% of the cases depending on the method of assay. Vaccination resulted in seroconversion in 75–88% (live vaccine) and in 92% (inactivated vaccine) of the cases as detected by SRH or EIAs, whereas HI detected seroconversion only in 38% and 58% of the cases, respectively. Immunoprecipitation analyses revealed that all sera from mumps patients and nearly all postvaccination sera had antibodies against the main structural proteins of mumps virus. By immunoblotting, antibodies against denatured hemagglutinin-neuraminidase (HN) and fusion protein (F) were detected in 15–25 % of mumps patients and persons vaccinated with live vaccine, whereas most postvaccination sera from those vaccinated with inactivated vaccine had HN (92%) and F (83%) protein antibodies, suggesting that antibodies against the denatured form of proteins are formed.     

Molecular homology among the structural proteins of densonucleosis virus from silkworm,Bombyx mori

Summary Similarities among the four structural proteins ofBombyx densonucleosis virus (DNV) were examined by four independent techniques, peptide mapping, immunodiffusion tests, amino acid analysis and enzyme linked immunosorbent (ELISA) technique. The peptide maps produced byStaphylococcus aureus V8 protease, or chymotrypsin indicated the existence of sequence homology among the proteins. The results of immunodiffusion tests revealed that these four structural proteins share common antigens. The amino acid compositions of these proteins were also very similar to each other. However, the amount of amino acid residues (e.g., serine) in VP3 was not always sufficient to account for the amount found in the smaller structural protein, VP2. These results indicate that the four structural proteins ofBombyx DNV probably originate, at least partially, from a common DNA sequence, and that VP2 is not a direct cleavage product of VP3.With 3 Figures     

Sequence analysis of the mumps virus mRNA encoding the P protein

The nucleotide sequence of the mumps virus phospho- or polymerase-associated (P) protein mRNA has been determined by sequencing a full-length cDNA clone and confirmed by partially sequencing the mRNA and the genome. The mRNA contains 1311 nucleotides excluding the poly(A) and encodes a protein of 390 amino acids with a calculated molecular weight of 41,574. Three small polypeptides were seen in in vitro translation of viral mRNA and hybrid-selected P mRNA, possibly representing internal initiation in the same reading frame of the P protein. A second overlapping reading frame is predicted from the sequence which has a capacity to code for two polypeptides of 56 and 34 amino acids, respectively. Whether these two polypeptides are expressed in infected cells is not known. Comparison of the P protein sequence with that of Sendai virus, measles virus, parainfluenza virus type 3, and canine distemper virus (CDV) showed no distinct homology but comparison with the P protein of Newcastle disease virus (NDV) showed 25.6% homology.     

Characterisation of mumps virus genotype C among patients with mumps in India

Measles, mumps and rubella (MMR) vaccine failure had been reported globally and here, we report that it occurs in India now. MMR vaccinated people have developed acute mumps accompanied by anti-mumps immunoglobulin M. Genotypic characterisation revealed that the circulating mumps strain was genotype C, which is distinct from the vaccine strain of genotype N (L-Zagreb). This is the first report in India to suggest that genotype C is responsible for the present mumps infection. Thus, the present MMR vaccine must be revamped and optimised for its efficacy to prevent any future mumps epidemics.     

Purification and amino-terminal protein sequence analysis of the mumps virus fusion protein

The fusion (F) protein of mumps virus was purified by immunoaffinity chromatography using an anti-F monoclonal antibody. The F protein was reduced and alkylated, and the F1 and F2 chains were isolated by high-pressure size exclusion chromatography. Twenty-three amino acid residues from the amino terminus of each chain were identified following automated Edman degradation. The amino-terminal sequence of the F1 chain was homologous to previously reported F1 sequences from three other paramyxoviruses (simian virus 5, Newcastle disease virus, and Sendai virus). Secondary structure predictions suggest an alpha-helical conformation for the mumps virus F1 amino-terminal sequence. A helical wheel model of the paramyxovirus F1 NH2 terminus is presented which defines conserved and variable arcs of the helix and provides a spatial representation of this critical functional domain of the paramyxovirus fusion protein.     

Binding of the V proteins to the nucleocapsid proteins of human parainfluenza type 2 virus

Interaction of the nucleocapsid (NP) and V proteins of human parainfluenza type 2 virus (HPIV-2) was investigated using a transient expression system. When the NP proteins were co-expressed with the V proteins, some of the NP proteins were translocated into the nuclei. These findings suggest that the NP protein interact with the V proteins. We examined the interaction of the NP proteins and the P, V proteins or deletion mutants of V protein using immunofluorescence and co-immunoprecipitation plus Western blotting analyses, and showed that the V proteins of HPIV-2 bind to the NP proteins and that the N-terminal domain of V protein interacts directly with the NP proteins. When the NP proteins were co-expressed with the V proteins or the N-terminal fragments (aa 1–46), the NP proteins were detected diffusely in the nuclei of the transfected cells, and were also detected in cytoplasmic inclusions. The NP and V proteins were co-localized in the nuclei or cytoplasm. Futhermore, the NP proteins were co-precipitated with the P, V, and V(1–164) proteins by a specific antibody. The P proteins interact more closely with the NP proteins than do the V proteins. These findings indicate that the V proteins have the ability to bind the NP proteins. Received: 4 January 1996     

Demonstration of antigenic variation among rabies virus isolates by using monoclonal antibodies to nucleocapsid proteins.

Rabies virus isolates from terrestrial animals in six areas of the United States were examined with a panel of monoclonal antibodies to nucleocapsid proteins. Characteristic differences in immunofluorescence reactions permitted the formation of four antigenically distinct reaction groups from the 231 isolates tested. The geographic distribution of these groups corresponded well with separate rabies enzootic areas recognized by surveillance of sylvatic rabies in the United States. Distinctive reaction patterns were also identified for viral proteins from four infected bat species, and identical patterns were found in eight isolated cases of rabies in terrestrial animals. These findings suggest that monoclonal antibodies can be used to study the prevalence, distribution, and transmission of rabies among wildlife species.     

Sequence variation of the P gene among mumps virus strains

We have determined nucleotide sequences of a 183-nucleotide long region of the P gene of 10 mumps virus strains after gene amplification mediated by DNA polymerase catalyzed chain reaction (PCR) and have compared them with those of two strains which had been reported earlier (K. Takeuchi et al., J. Gen. Virol., 69, 2043-2049 (1988]. It was shown that mutation is generally noncumulative, i.e., most nucleotide substitutions in earlier strains do not appear in later strains. Viruses of different lineages appeared to cocirculate, but the comparison of American and Japanese strains suggested that those isolated in one country are more closely related to each other than to those isolated in the other country.