Newcastle Disease Virus Genome Research Articles

Sequence variation in the Newcastle disease virus genome

Full-length genome sequences of five virulent and five avirulent strains of Newcastle disease virus isolated between 1998 and 2002 in Victoria and New South Wales, Australia were determined. Comparisons between these strains revealed that coding sequence variability in the haemagglutinin-neuraminidase (HN), matrix (M) and phosphoprotein (P) gene sequences appeared to be more variable than in the fusion (F), nucleocapsid (N) and RNA dependent-RNA replicase (L) genes. Sequence analysis of a number of other isolates made during the recent virulent NDV outbreaks, also identified the presence of a number of variants with altered F gene cleavage sites, which resulted in altered biological properties of those viruses. Quasispecies analysis of a number of field isolates indicated the presence of virulent virus in one particular isolate. Gene sequence analysis of the progenitor virus isolated in 1998 showed very little sequence variation when compared to that of a progenitor-like virus isolated in 2001, demonstrating that in the field, viral genome sequence variation appears to be biologically restricted to that of a consensus sequence.

Detection of Newcastle Disease Virus Genome from the Field Outbreaks in Poultry by Reverse Transcription – Polymerase Chain Reaction

A total of 30 field samples (27 tissue samples comprising of either trachea, lungs or brain; and 3 allantoic fluid samples) were collected from broiler chickens for detection of Newcastle disease virus (NDV) by RT-PCR. In addition, vaccine strains (F, LaSota, R B) were taken as positive control and trachea 2 from unvaccinated healthy bird as negative control. Newcastle disease virus (NDV) was detected in five field samples as well as in all the vaccine strains by RT-PCR. All these samples as well as vaccine strains yielded a band of 356 bp on amplification of F region of NDV. Three more field samples yielded a band of 216 bp with nested PCR, thus making total eight field samples positive for NDV. The results of the present study indicated that RT-PCR followed by nested PCR can be used to detect NDV directly from tissue samples in poultry.

Mapping of the RNA promoter of Newcastle disease virus

The RNA promoters of the genome and antigenome of Newcastle disease virus (NDV) were studied by mutational analysis of their 3′ terminal ends. Similarly to other paramyxoviruses, NDV RNA replication follows the rule of six, and the genomic and antigenomic promoters require two discontinuous regions: conserved region I (first 18 nucleotides) and conserved region II (nucleotides 73–90). Proper spacing between those regions and the phase of six in region II is critical for efficient RNA promoter activity. As expected, the gene start signal at the 3′ end of the NDV genome was required for mRNA transcription, but not for RNA replication. Surprisingly, mutation of the polyadenylation signal in the 5′ end did not affect gene expression transcription. Although the conserved region I of NDV (avulavirus) promoter appears to be more similar to that of Sendai virus (SeV) (respirovirus), conserved region II is analogous to that of rubulaviruses.

RNA-dependent RNA polymerase gene analysis of worldwide Newcastle disease virus isolates representing different virulence types and their phylogenetic relationship with other members of the paramyxoviridae

Nucleotide sequence was determined for the RNA-dependent RNA polymerase (L) gene of 16 Newcastle disease virus (NDV) isolates from diverse geographic and chronological origins. The observed consensus amino acid sequence conformed to the six domains previously identified among paramyxovirus L proteins, and the putative 749QGDNQ753 active site was strictly conserved among all isolates. Analysis of predicted amino acid sequences allowed us to identify a sequencing error in the previously reported L genes for NDV. The correct sequences reported herein provided a more accurate alignment with predicted l-amino acid sequences of other paramyxoviruses. Comparison of L gene coding sequences among isolates revealed that synonymous substitutions dominated non-synonymous substitutions, as observed previously with other NDV genes. However, the overall substitution rate was lower than other genes examined making the L gene the most conserved of the NDV genome. Phylogenetic analysis of L genes among NDV isolates was consistent with previous results that suggested the existence of two major lineages. One group contained strains isolated in North America prior to 1970 and included virulent and vaccine strains, while the second group included virulent viruses isolated worldwide. A comparison of the NDV L coding sequences to other Paramyxoviridae illustrated the unique clustering of the avian-specific paramyxoviruses, further justifying the newly created Avulavirus genus.

Recombinant Newcastle disease virus as a viral vector: effect of genomic location of foreign gene on gene expression and virus replication

Newcastle disease virus (NDV) was examined for its suitability as a vector for the expression and delivery of foreign genes for vaccination and gene therapy. A reporter gene encoding human secreted alkaline phosphatase (SEAP) was inserted as an additional transcription unit at four different positions in the NDV genome, between the NP and P, M and F, and HN and L genes and behind the L gene. Eight infectious recombinant NDV (rNDV) viruses, four in the non-virulent strain NDFL and four in the virulent derivative NDFLtag, were generated by reverse genetics. SEAP expression levels, replication kinetics and virus yield were examined. Replication kinetics of the rNDV viruses in primary chicken embryo fibroblasts showed that the insertion of an additional gene resulted in a delay in the onset of replication. This effect was most prominent when the gene was inserted between the NP and P genes. With the exception of the strain that carried the SEAP gene behind the L gene, all recombinant strains expressed high levels of SEAP, both in cell culture and in embryonated chicken eggs. In embryonated eggs, the rNDV viruses showed a 2.6- to 5.6-fold (NDFL) or 2.1- to 8.1-fold (NDFLtag) reduction in yield compared with the parent strains. These results show that foreign genes can be inserted at different positions in the NDV genome without severely affecting replication efficiency or virus yield.

Epidemiological study of Newcastle disease in backyard poultry and wild bird populations in Switzerland

Blood samples and cloacal swabs from poultry were collected in 107 small chicken flocks and 62 pure-bred poultry flocks to determine their status regarding Newcastle disease virus (NDV) infection. A questionnaire emphasizing potential contacts of poultry with wild birds and management practices associated with NDV infection was completed for each flock. Additionally, 1576 wild bird carcasses of 115 different bird species were collected from hunters and taxidermists. Poultry sera and tissue fluids of wild birds were tested for NDV antibodies using a blocking ELISA. Cloacal swabs were subjected to reverse transcription polymerase chain reaction (RT-PCR) for NDV genome detection. In-herd NDV seroprevalences between 5 and 29% were found in one small chicken flock, as well as in four pure-bred poultry flocks. NDV antibody positive wild birds were found in 10.2% of all wild birds examined. Highest proportions (i.e. 15%) of positive birds per species were found among sparrowhawks, kites, tawny owls, eagle owls, barn owls, cuckoos, swifts, cormorants and grebes. No NDV genome was detected in cloacal swabs. This study suggests that buying eggs or poultry abroad and exchanging poultry within the country were factors, more important than wild birds, to explain the higher NDV seropositivity in pure-bred poultry flocks.

Detection of polycistronic transcripts in Newcastle disease virus infected cells and identification of their sequence content.

The synthesis of six to seven polycistronic transcripts of Newcastle disease virus (NDV) in BHK cells was detected by Northern hybridization using cDNA clones generated by reverse transcription of five NDV mRNAs. Within the molecular weight range resolved by the gel electrophoresis system employed, four of the transcripts were suggested to be distronic, containing sequences of two genes, NP-P, P-M, M-F0 and F0-HN, respectively. In addition, tricistronic molecules of M-F0-HN and possibly of NP-P-M as well as P-M-F0 appeared to develop, although they were very low in amount. These data suggest a gene order of NP-P-M-F0-HN on the NDV genome. The polycistronic as well as monocistronic transcripts were generated with an almost constant proportion in amount throughout the virus replication. Further, at least several of them were also generated under the conditions where only the primary transcription was allowed by inhibiting de novo protein synthesis. Therefore, it appears likely that there is no distinct temporal control in NDV genome expression.

Cloning and nucleotide sequence of newcastle disease virus hemagglutinin-neuraminidase mRNA: Identification of a putative sialic acid binding site

The avian pathogen Newcastle disease virus (NDV) is a member of the Paramyxoviridae, a family which includes Sendai virus, simian virus 5 (SV5) and the human virus parainfluenza 3 (para-3). These enveloped viruses contain a single-stranded RNA genome of negative sense, 15-18 kb in length, encased in a helical nucleocapsid. Six major messenger RNA species are transcribed sequentially by the virion-associated RNA polymerase. They are capped, methylated, and polydenylated by virion proteins (Huang et a/., 1971; Colonno and Stone, 1976). Organization of the NDV genome has been investigated indirectly by both uv-inactivation mapping and by the analysis of polycistronic transcripts (Collins et a/., 1980; Wilde and Morrison, 1984). Both studies indicate that the gene order is identical to that of Sendai virus, which has been determined to be 3’-NP-P-M-F-HN-L-5’ by nucleotide sequence analysis of genomic clones (Blumberg et a/., 1984, 1985a,b). NP and the phosphoprotein P are components of the viral nucleocapsid, L is an RNAdependent RNA polymerase, and M is a protein of undetermined function associated with the inner surface of the viral membrane (reviewed in Kingsbury, 1985). Paramyxovirus glycoproteins are functionally similar to the well-studied surface glycoproteins of influenza (Ward, 1981; Colman and Ward, 1985). They possess cell fusion, hemagglutination and neuraminidase activities (Hsu et a/., 1979; Scheid and Choppin, 1974; Merz et a/., 1981), and constitute the spikes which protrude from the viral envelope (Choppin and Compans, 1975). The F glycoprotein, which is responsible for the paramyxovirus’ ability to fuse with the host cell membrane, is activated by a proteolytic cleavage event (Scheid and

Sequence of 2,617 nucleotides from the 3' end of Newcastle disease virus genome RNA and the predicted amino acid sequence of viral NP protein.

DNA fragments complementary to the Newcastle disease virus genome (strain D26) were cloned and sequenced. The sequence of 2,617 nucleotides from the 3' end of the genome was determined and an open reading frame (OP-1) consisting of 1,467 nucleotides, most likely encoding NP protein, was found in this region. This was followed by a second unfinished open reading frame (OP-2) of at least 729 nucleotides which continued beyond the 2,617th nucleotide. Another relatively short (312 nucleotides long) open reading frame (OP-2') was found overlapping with OP-2, but its significance is still unclear. The amino acid sequence deduced from the nucleotide sequence of OP-1 showed a moderate homology to that of the NP protein of Sendai virus in the central portion of the peptide. The leader sequence of 53 nucleotides was also identified. The 5' end of mRNAs synthesized in the infected cells was analyzed and found to be m7GpppA, suggesting that the transcription of viral mRNAs starts with A, but not with G residue.

RNA sequence and transcriptional properties of the 3′ end of the Newcastle disease virus genome

The 3′ end of the genomic RNA of Newcastle disease virus (NDV) has been sequenced and the leader RNA defined. Using hybridization to a 3′-end-labeled genome, leader RNA species from in vitro transcription reactions and from infected cell extracts were found to be 47 and 53 nucleotides long. In addition, the start site of the 3′-proximal mRNA was determined by sequence analysis of in vitro [β- 32P]GTP-labeled transcription products. The genomic sequence extending beyond the leader region demonstrated an open reading frame for at least 42 amino acids and probably represents the amino terminus of the nucleocapsid protein (NP). The terminal 8 nucleotides of the NDV genome were identical to those of measles virus and Sendai virus while the sequence of the distal half of the leader region was more similar to that of vesicular stomatitis virus. These data argue for strong evolutionary relatedness between the paramyxovirus and rhabdovirus groups.

Cell-free coupling of Newcastle disease virus RNA transcription, translation and Co-translational processing.

A cell-free coupled system for transcription, translation and glycoprotein processing of the Newcastle disease virus genome is described. The system consists of a rabbit reticulocyte lysate preincubated with micrococcal nuclease and of detergent-disrupted purified Newcastle disease virions. [35S]methionine incorporation was linear for 2 h. Polypeptides NP and M, the presumably unglycosylated analogues of glycoproteins HN and possibly F, were identified as translation products. When in vitro synthesis was carried out in the presence of dog pancreas microsomes the HN analogue (pre-HN) was converted to an 80K (approx.) protein which comigrated on polyacrylamide gels with HN synthesized in vivo and which, except for a small fragment, was protected from proteolytic degradation. In immunoprecipitation studies, antiserum against HN purified from virions reacted with both the processed and the unprocessed form of HN synthesized in vitro.

Absence of nucleotide sequence complementarity between Sindbis virus and Newcastle disease virus genomes.

SummaryThe RNA isolated from Sindbis virus was annealed with RNA isoated from Newcastle disease virus (NDV). No ribonuclease-resistant hybrid was formed, indicating that these viral RNA's do not have complementary nucleotide sequences, despite complementary base compositions.

Newcastle disease virus complementary RNA: Its relationship to the viral genome and its accumulation in the presence or absence of actinomycin D

Tritium-labeled RNA isolated from Newcastle disease virus (NDV) was annealed with RNA extracted from chick embryo cells incubated with or without 10 μg/ml actinomycin D for 7 hours after infection by NDV. By measurement of acid-precipitable radioactivity after ribonuclease treatment of annealed materials, it was found that at least 80% of the NDV genome is represented by complementary counterparts in infected cells whether or not the cells are incubated with actinomycin D. Specific dilution assays showed that actinomycin D enhanced accumulation of both NDV parental-type (+) RNA and NDV complementary (−) RNA in cells, but the drug did not lower the ratio of (+) to (−) RNA.