Development of a RT-nested PCR test detecting pigeon Paramyxovirus-1 directly from organs of infected animals

A RT-nested PCR that amplifies part of the conserved nucleoprotein gene of avian Paramyxovirus type 1 is described. The technique allowed the detection of pigeon Paramyxovirus type 1 (pPMV-1) virus directly from a wide range of infected chicken and pigeon organs, and should be able to detect typical Newcastle disease viruses too. Compared with the reference method, the developed RT-nested PCR was found more sensitive, as it was able to detect virus genome in infected pigeon organs at late stage of infection, when virus isolation failed. Such a molecular technique represents an alternative method of diagnosis for research purposes on pPMV-1 variants, for example to study pathogenesis aspects of the infection or to assess the efficacy of vaccines.     

Quasispecies Nature of an Unusual Avian Paramyxovirus Type-1 Isolated from Pigeons

An avian paramyxovirus type-1 (APMV-1) was classified as virulent according to its Intra Cerebral Pathogenicity Index (ICPI), but as avirulent according to the motif of its F protein cleavage site. Although this atypical APMV-1 was isolated from sick, unvaccinated pigeons, it was not grouped with pigeon variants regarding its antigenic and genetic characterisation. We analysed its quasispecies nature by cloning and sequencing parts of the genome in three different genes to evaluate if heterogeneity might explain the difference observed between the ICPI and the F protein cleavage site motif. Two distinct sub-populations were detected in the phosphoprotein gene. In the fusion protein gene, two clones were found to be related to typical pigeon variants in the hypervariable domain.     

Proteins of newcastle disease virus. a comparison by partial protease digestion among the strains of different pathogenicity

To investigate the relationship between the pathogenicity of Newcastle disease virus and the structure of viral proteins, two typical strains were sampled from each of three groups of different pathogenicities and these six strains were compared for protein structure by sizing peptides generated by partial digestion with Staphylococcus aureus V8 protease and chymotrypsin. These digests yielded closely similar peptide patterns for the internal polypeptides L and NP, whereas those of the glycoproteins HN and F showed apparent variations which appeared to be specific for the individual groups. Although not as significant as in the glycoproteins, group-dependent variations were also detectable with the M protein. These results suggest that the external proteins might undergo considerable changes whereas the internal proteins would be highly stable and that there is a definite correlation between such changes in the external proteins and the pathogenicity of the virus.     

Virus-specific polypeptides of human parainfluenza virus type 4 and their synthesis in infected cells

We have studied the structural components of human parainfluenza virus type 4A (PIV-4A) and identified some virus-specific polypeptides by immunoprecipitation with polyclonal and monoclonal antibodies followed by one- or two-dimensional SDS-PAGE. HN polypeptides existed as monomer, disulfide-linked dimer, and disulfide-linked larger oligomer in cells infected with PIV-4A. Interestingly, the nonreduced NP, the nonreduced fusion, and the reduced F1 proteins migrated as doublets. Two F1 polypeptides were derived from different F1 + 2 proteins which migrated separately under nonreducing condition. In Vero cells infected with two strains of PIV-4A, two lower-molecular-weight proteins related to NP were detected. Oligopeptide patterns of the lower-molecular-weight protein were similar to those of NP protein synthesized in primary monkey kidney cells. The NP-related low-molecular-weight protein(s) was immunoprecipitated by 1 of 11 monoclonal antibodies against mumps virus NP protein. The MAb also reacted with NP proteins of PIV-2 and SV5. Thus, the epitope recognized by the MAb was common among PIV-2, PIV-4, mumps virus, and SV5, suggesting that the epitope might have an important biological function. However, the MAb did not react with the intact NP protein from cells infected with PIV-4, indicating that the epitope of PIV-4A was presented only when NP was cleaved. Phosphorylation was demonstrated for NP and P proteins.     

The polypeptides of human parainfluenza type 2 virus and their synthesis in infected cells

Summary We have studied the structural components of three strains of human parainfluenza virus type 2 (HPI-2) and identified the virus-specific polypeptides. Molecular weight of P and F1 polypeptides determined by us, when compared with that reported previously, was in reversed order. HN polypeptide existed chiefly as disulfide-linked dimers in cells infected with Toshiba strain, while as monomers and dimers in nearly equal proportion in cells infected with two other strains. Similar disulfide-linked NP oligomers were found in cells infected with all three strains. F1 and F2, cleaved forms of F protein, could be detected in cells infected with all three strains, but the ratio of cleaved (F1 and F2) to uncleaved (F₀) forms was markedly lower in 62-M 786- and 63-M 1-infected than in Toshiba strain-infected cells. However, there was no difference of oligopeptide mapping patterns and isoelectric point of F polypeptide between Toshiba and 62-M 786 strains. By contrast, oligopeptide mapping patterns of HN protein of Toshiba strain differed from those of the two other strains. Furthermore, the HN polypeptide of Toshiba strain was phosphorylated in the infected Vero cells, but that of the other two strains was not.     

Human parainfluenza virus 3: Purification and characterization of subviral components,viral proteins and viral RNA

A simple method was established that allowed large quantities of human parainfluenza 3 (PF3) virions to be isolated from tissue culture cells. The purity of the virus was sufficient for biochemical analysis of virion proteins. The density of PF3 virions was 1.18–1.20. Purified virions contained seven viral proteins with estimated molecular weights of: L, 180000; P, 83000; HN, 69000; NP, 66000; f₀, 60000; F₁, 51000; and M, 38000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. There were three phosphoproteins, P, NP and M, and two glycoproteins, HN and F (includes f₀ and f₁). F_1,2, the activated, cleaved, fusion glycoprotein (60000 Da), consisting of two disulfide-linked subunits, f₁ and F₂, was seen only under nonreducing conditions. Because of its small size (approximately 9000 Da) F₂ could be seen only on gels with high acrylamide concentrations. As in other enveloped viruses, cellular actin (43 000 Da) was present in purified virions. Several minor bands migrating between NP and M represented breakdown products of NP.Solubilization of the virion membrane in low salt buffer with non-ionic detergent resulted in the loss of HN and F. In high salt buffer, the M protein was also removed. Nucleocapsids isolated by CsCl centrifugation contained L, P, NP and small amounts of M. Nucleocapsids isolated in the presence of the ionic detergent, sarcosyl, contained only the NP protein. The density of nucleocapsids was 1.29–1.30. Genomic 50S RNA isolated from nucleocapsids had an estimated molecular weight of 5 × 10⁶.     

Evaluation of the molecular basis of pathogenicity of the variant Newcastle disease viruses termed “pigeon PMV-1 viruses”

Summary The amino acid sequence at the F2/F1 cleavage site was determined for 15 strains of the so-called pigeon PMV-1 (PPMV-1) variant of Newcastle disease virus (NDV) which showed close antigenic identity, determined by their reactions with a panel of 28 monoclonal antibodies, but considerable variation in their pathogenicity for chickens. Thirteen of the isolates possessed the motif¹¹²G-R-Q-K-R-F¹¹⁷. This motif was seen for one virus which had initially low pathogenicity and remained unaltered when virulence of the virus for chickens was increased by bird to bird passage. The two other viruses had the sequence¹¹²R-R-Q-K-R-F¹¹⁷ at the cleavage site which is more typical of virulent viruses, however, pathogenicity index tests indicated that these isolates were of moderate and low pathogenicity. The nucleotide sequence coding for the HN/HN0 extension region was determined for two of the PPMV-1 isolates. In both cases a stop codon was present indicating that the product for these viruses would be HN₅₇₁. We conclude that the wide variation in pathogenicity of the variant PPMV-1 for chickens is not related to variation in the amino acid motif at the F2/F1 cleavage site nor due to production of HN0 which may also influence pathogenicity. The high virulence of some of the viruses examined confirms that a double pair of basic amino acids in the region of the F2/F1 cleavage site is not necessary for the full expression of virulence.     

Analysis and gene assignment of mRNAs of a paramyxovirus, simian virus 5

Polypeptides synthesized by the paramyxovirus SV5 in infected CV-1 cells were readily identified when the host cell was treated with actinomycin D. The unglycosylated forms of HN and Fo synthesized in infected cells in the presence of tunicamycin and HN and Fo synthesized in vitro were identified by immunoprecipitation with specific antibodies. Separation of SV5-specific poly(A)-containing RNAs on methyl-mercury agarose gels and in vitro translation of fractions, indicated that the viral polypeptides were translated from individual mRNAs except P (Mr approximately 44K) and the nonstructural polypeptide V (Mr approximately 24K) for which the mRNAs could not be separated. cDNA copies of SV5-specific mRNAs were synthesized and cloned in plasmid pBR322. Clones to NP, P + V, M, F, and HN were identified by hybrid-arrest and hybrid-selection translation of SV5 mRNAs. Tryptic peptide mapping of polypeptides P and V indicated that the peptides of V were a subset of those of P. Hybridization of cDNA probes to infected cell mRNAs separated on agarose gels permitted identification of the NP, P + V, M, F, and HN mRNAs and presumptive polycistronic mRNAs. The sizes and sequence homologies of these polycistronic mRNAs were used to derive a likely gene order on the SV5 50 S genome RNA.     

Completion of the full-length genome sequence of human parainfluenza virus types 4A and 4B: sequence analysis of the large protein genes and gene start, intergenic and end sequences

We have already reported the nucleotide sequences of the NP, P/V, M, F and HN genes of human parainfluenza virus type 4A (hPIV-4A) and type 4B (hPIV-4B). Here, we have determined the sequences of the L protein genes as well as the gene start, intergenic and end sequences, thereby completing the full-length genome sequence of hPIV-4A and 4B. hPIV-4A and 4B have 17,052 and 17,304 nucleotides, respectively. The end sequence of hPIV-4, especially 4B, was extraordinarily long. In a comparison with members of the genus Rubulavirus, the hPIV-4 L proteins were closely related to those of mumps virus (MUV) and hPIV-2, less closely related to those of Menangle virus and Tioman virus, and more distantly related to those of Mapuera virus and porcine rubulavirus.     

Fusion protein of the paramyxovirus SV5: destabilizing and stabilizing mutants of fusion activation

The fusion (F) protein of the paramyxovirus SV5 strain W3A causes syncytium formation without coexpression of the SV5 hemagglutinin-neuraminidase (HN) glycoprotein, whereas the F protein of the SV5 strain WR requires coexpression of HN for fusion activity. SV5 strains W3A and WR differ by three amino acid residues at positions 22, 443, and 516. The W3A F protein residues P22, S443, and V516 were changed to amino acids found in the WR F protein (L22, P443, and A516, respectively). Three single-mutants, three double-mutants, and the triple-mutant were constructed, expressed, and assayed for fusion using three different assays. Mutant P22L did not cause fusion under physiological conditions, but fusion was activated at elevated temperatures. Compared with the W3A F protein, mutant S443P enhanced the fusion kinetics with a faster rate and greater extent, and had a lower activation temperature. Mutant V516A had little effect on F protein-mediated fusion. The double-mutant P22L,S443P was capable of causing fusion, suggesting that the two mutations have opposing effects on fusion activation. The WR F protein requires coexpression of HN to cause fusion at 37 degrees C, and does not cause fusion at 37 degrees C when coexpressed with influenza virus hemagglutinin (HA); however, at elevated temperatures coexpression of WR F protein with HA resulted in fusion activation. In the crystal structure of the core trimer of the SV5 F protein (Baker, K. A., Dutch, R. E., Lamb, R.A., and Jardetzky, T. S. (1999). Mol. Cell 3, 309-319), S443 is the last residue (with interpretable electron density) in an extended chain region and the temperature factor for S443 is high, suggesting conformational flexibility at this point. Thus, the presence of prolines at residues 22 and 443 may destabilize the F protein and thereby decrease the energy required to trigger the presumptive conformational change to the fusion-active state.     

Extensive antigenic diversity among human parainfluenza type 2 virus isolates and immunological relationships among paramyxoviruses revealed by monoclonal antibodies

A panel of 128 monoclonal antibodies (MAbs) directed against hemagglutinin-neuraminidase (HN), fusion (F), matrix (M), and polymerase (P) proteins, and nucleoprotein (NP) of the Toshiba strain of human parainfluenza type 2 virus (PIV2) was prepared to examine the antigenic relationships among clinical isolates of PIV2 and among paramyxoviruses by indirect enzyme-linked immunosorbent assays. The HN proteins of 18 clinical isolates of PIV2 showed extensive antigenic diversity: 23 of 33 anti-HN MAbs showed no or limited reactivity to many isolates, while other structural proteins were antigenically well conserved. Some anti-HN MAbs recognizing conserved epitopes of the isolates exhibited two types of neutralizing activity, that is, these antibodies inhibited viral infectivity through attachment inhibition or fusion inhibition. This result also showed the presence of a potential third function of the HN protein which might affect the fusing activity of the F protein besides the hemagglutinating and neuraminidase activities. Many of the anti-NP and anti-P MAbs reacted with simian virus 41 (SV41) and simian virus 5 (SV5), whereas a few reacted with mumps virus or PIV4. Two of 6 anti-F MAbs reacted with SV41. None of the 128 MAbs showed reactivity with PIV1, PIV3, Newcastle disease virus (NDV), and measles virus. This result confirmed antigenic proximity of SV5 and SV41 to PIV2 and revealed comparatively restricted immunological relatedness among PIV2, PIV4, and mumps virus.     

Antigenic and biological characterization of avian paramyxovirus type 1 isolates from pigeons

Summary 21 A/PMV-1 viruses were isolated from pigeons and characterized using polyclonal and monoclonal antisera in hemagglutination inhibition, sero-neutralization and immunoprecipitation studies.Polyclonal and monoclonal antibodies directed against the HN and F proteins of Italien virus reacted with all pigeon isolates showing a close relationship between chicken velogenic and pigeon viruses. Differences in the M.W. of F₀, P and M proteins were however observed between pigeon and chicken Italien virus.Marked differences in virulence were recorded among pigeon isolates; these were reflected by great variation in the IVPI of the different strains.With 3 Figures     

An improved method for recovery of F-defective Sendai virus expressing foreign genes from cloned cDNA

An improved system is described to recover non-transmissible Sendai virus that lack the envelope fusion (F) gene from cloned cDNA. The system (1) used plasmids that expressed the F and the HN viral envelope proteins, as well as the plasmids that expressed the viral NP, P, and L proteins as helper plasmids for recovery, and (2) overlaid packaging cells that expressed the F protein. With this improved system, we have succeeded in recovery of F-defective Sendai virus expressing two foreign proteins, and expression vectors that do not contain the EGFP reporter gene. This system may provide the basis for constructing recombinant F-defective Sendai virus for preventing and treating human diseases in the form of vaccines and vectors for gene therapy.     

Host-dependent temperature-sensitive growth of HVJ (Sendai virus) wild-type in rat glioma C 6 cells

Summary At non-permissive temperature viral specific RNA synthesis was not restricted in rat glioma (C 6) cells infected with HVJ (Sendai virus) wild-type. However, as has previously been shown (J Gen Virol [1984] 65: 639–643), the synthesis of M protein was reduced at non-permissive temperature, in contrast to the L, P, HN, F₀ and NP proteins which were synthesized in comparable amounts at permissive and non-permissive temperatures. In this report we show additionally that viral nucleocapsids (NC), which consist of L, P and NP proteins, were formed within the infected cells at both temperatures. Hemagglutinin and neuraminidase activities were also detected in samples incubated at non-permissive temperature. By membrane immunofluorescence and cell-surface immunoprecipitation it was shown that migration of HN and F₀ proteins to the cell surface occurred normally at non-permissive temperature. Additionally, the L, P and NP proteins, which were associated with the plasma membrane isolated from the infected cells maintained at permissive temperature, were absent from the membrane of cells incubated at non-permissive temperature. These results suggest that NC and glycoproteins synthesized at non-permissive temperature could not assemble effectively at the plasma membrane because of a lack of M protein. Thus, the host-dependentts lesion of HVJ in C 6 cells was considered to be mainly in M protein synthesis.With 3 Figures     

Association of the Sendai virus C protein with nucleocapsids

Summary The subcellular localization of the nonstructural protein C of Sendai virus was investigated by means of indirect immunofluorescence microscopy of Sendai virus-infected cells, using an antiserum specific for C protein. In infected cells, C protein was detected exclusively in the cytoplasm as granular fluorescence, which coincided very well with the distribution of nucleocapsid protein NP and phosphoprotein P, which were also detected with specific antisera. This suggested that these proteins are present together in inclusions, probably forming nucleocapsids. In contrast, when the NP and C proteins were individually expressed in COS cells by transfection with expression plasmids containing cDNA for these proteins, their distribution patterns in the cytoplasm were found to be quite different from each other. Protein-blot analyses of purified virions revealed the presence of a significant amount of the C protein in virions, which indicated that C protein is integrated into virions. Under conditions in which most of the envelope-associated proteins, such as HN, F, and M, were removed from the virions by a detergent, the C protein remained tightly associated with the nucleocapsids — about 40 molecules per nucleocapsid.     

Molecular characterization of a Class I Newcastle disease virus strain isolated from a pigeon in China

Constant monitoring is performed to elucidate the role of natural hosts in the ecology of Newcastle disease virus (NDV). In this study, an NDV strain isolated from an asymptomatic pigeon was sequenced and analysed. Results showed that the full-length genomes of this isolate were 15,198 nucleotides with the gene order of 3′-NP-P-M-F-HN-L-5′. This NDV isolate was lentogenic, with an intracerebral pathogenicity index of 0.00 and a mean time of death more than 148?h. The isolate possessed a motif of -¹¹²E-R-Q-E-R-L¹¹⁷- at the F protein cleavage site. In addition, 7 and 13 amino acid substitutions were identified in the functional domains of fusion protein (F) and haemagglutinin-neuraminidase protein (HN) proteins, respectively. Analysis of the amino acids of neutralizing epitopes of F and HN proteins showed 3 and 10 amino acid substitutions, respectively, in the isolate. Phylogenetic analysis classified the isolate into genotype Ib in Class I. This isolate shared high homologies with the NDV strains isolated from wild birds and waterfowl in southern and eastern parts of China from 2005 to 2013. To our knowledge, this study is the first to report a NDV strain isolated from pigeon that belongs to genotype Ib in Class I, rather than to the traditional genotype VI or other sub-genotypes in Class II. This study provides information to elucidate the distribution and evolution of Class I viruses for further NDV prevention.     

Evolution of pigeon Newcastle disease virus strains

Twenty-seven Newcastle disease virus isolates obtained during the years 1998 and 1999 from racing pigeons were shown to be antigenically indistinguishable from the pigeon paramyxovirus type 1 (PPMV-1) viruses isolated in the years 1983 and 1984. Partial sequencing of 240 base pairs of the F gene demonstrated at least 94.7% identity at the nucleotide level between isolates from 1983 and 1984, and more recent viruses isolated in 1998 and 1999. Most of the nucleotide changes observed were silent mutations as only six amino acid changes were observed. Three amino acid substitutions were observed in the F2/F1 cleavage site. The sequence of the F2/F1 cleavage site of all isolates was typical for pathogenic paramyxovirus 1 viruses. Amino acids at the F2/F1 cleavage site changed from 112 GRQKRF 117 to 112 RRQKRF 117 , 112 RRKKRF 117 or 112 RRRKRF 117 . The motif 112 RRQKRF 117 was present in the majority of the isolates but the intracerebral pathogenicity indexes of PPMV-1 isolates having this motif was highly variable but largely lower (mean, 0.69) than that reported for PPMV-1 viruses isolated in the years 1983 and 1984 (mean, 1.44).