The arterivirus family (order Nidovirales) of single-stranded, positive-sense RNA viruses includes porcine respiratory and reproductive syndrome virus and equine arteritis virus (EAV). Their replicative enzymes are translated from their genomic RNA, while their seven structural proteins are encoded by a set of small, partially overlapping genes in the genomic 3'-proximal region. The latter are expressed via synthesis of a set of subgenomic mRNAs that, in general, are functionally monocistronic (except for a bicistronic mRNA encoding the E and GP2 proteins). ORF5, which encodes the major glycoprotein GP5, has been used extensively for phylogenetic analyses. However, an in-depth computational analysis now reveals the arterivirus-wide conservation of an additional AUG-initiated ORF, here termed ORF5a, that overlaps the 5' end of ORF5. The pattern of substitutions across sequence alignments indicated that ORF5a is subject to functional constraints at the amino acid level, while an analysis of substitutions at synonymous sites in ORF5 revealed a greatly reduced frequency of substitution in the portion of ORF5 that is overlapped by ORF5a. The 43-64 aa ORF5a protein and GP5 are probably expressed from the same subgenomic mRNA, via a translation initiation mechanism involving leaky ribosomal scanning. Inactivation of ORF5a expression by reverse genetics yielded a severely crippled EAV mutant, which displayed lower titres and a tiny plaque phenotype. These defects, which could be partially complemented in ORF5a-expressing cells, indicate that the novel protein, which may be the eighth structural protein of arteriviruses, is expressed and important for arterivirus infection.

Discovery of a small arterivirus gene that overlaps the GP5 coding sequence and is important for virus production

Substantial variation in the virulence of Newcastle disease virus (NDV) isolates means that the detection of NDV or evidence of infection is insufficient for an adequate diagnosis, as control measures for avirulent viruses are very different to those for virulent viruses. Diagnosis therefore requires further characterization, at least as to whether an isolate is virulent or avirulent. Conventional detection and differentiation of ND viruses is perceived as slow, laborious and requiring an undesirable use of in vivo techniques. In addition, further characterization is needed to give greater information on origin and spread. This review concentrates on the application of monoclonal antibody and molecular biological approaches. Panels of monoclonal antibodies were a major advance for the characterization of NDV isolates, although confirmation of virulence for poultry still required in vivo testing. As molecular-based techniques become easier and more reliable, they are likely to supersede the use of monoclonal antibodies, especially for characterizing viruses for epidemiological purposes. The attraction of molecular-based techniques is that they may be able to cover all three aspects of Newcastle disease diagnosis (detection of virus, characterization, including inference of virulence, and epidemiology) quickly, accurately and definitively in a single test. A number of approaches based on the reverse transcriptase polymerase chain reaction have been developed, with subsequent analysis of the product by restriction enzyme analysis, probe hybridization and nucleotide sequencing. Although extensive variation among NDVs still poses technical problems, the real and potential advantages of a molecular biological approach to Newcastle disease diagnosis appear to be overwhelming.

https://www.tandfonline.com/doi/pdf/10.1080/03079450120044515

Detection and differentiation of Newcastle disease virus (avian paramyxovirus type 1).

PRRSV structure, replication and recombination: Origin of phenotype and genotype diversity.

A review of RT-PCR technologies used in veterinary virology and disease control: sensitive and specific diagnosis of five livestock diseases notifiable to the World Organisation for Animal Health.

Over the past two decades, enormous advances have occurred in the structural and biological characterization of Newcastle disease virus (NDV). As a result, not only the complete sequence of the viral genome has been fully determined, but also a clearer understanding of the viral proteins and their respective roles in the life cycle has been achieved. This article reviews the progress in the molecular biology of NDV with emphasis on the new technologies. It also identifies the fundamental problems that need to be addressed and attempts to predict some research opportunities in NDV that can be realized in the near future for the diagnosis, prevention and treatment of disease(s).

https://www.tandfonline.com/doi/pdf/10.1080/03079450120078626

Newcastle disease virus: macromolecules and opportunities.

To provide information on the epidemiology of Newcastle disease (ND) of poultry in Taiwan, ND virus isolates from chickens and an owl were investigated by restriction site analysis and sequencing of their gene. A 1, 349 base fragment of the F (fusion protein) gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The PCR products were analyzed using restriction endonucleases, HinfI, BstOI, and RsaI. Three strains isolated from chickens during the 1995 epidemic outbreak had the same restriction sites as that of a 1994 isolate; the number of the restriction sites of HinfI, BstOI, and RsaI were 4, 2, and 4, respectively. In the F gene of the strain isolated from an owl during the same outbreak an additional restriction site of HinfI was found. The 1991 isolate had only 3 restriction sites. The F gene of the owl isolate was amplified by RT-PCR and followed by direct sequencing. The deduced amino acid sequence at the cleavage site of the F protein was of virulent strains, 112R-R-Q-K-R-F117. The F gene of Ow/Tw/2209/95 was phylogenetically most closely related to that of Ck/Tw/2137/95 isolated from the same outbreak. The present results indicate that the causative virus of the 1995 ND outbreak had already been present in Taiwan.

Restriction Fragment Length Polymorphism Analysis of the F Gene of Newcastle Disease Viruses Isolated from Chickens and an Owl in Taiwan

In combination of utilizing a leader specific primer and primers complementary to porcine reproductive and respiratory syndrome virus (PRRSV) genome through RT-PCR, the leader junction sequences of subgenomic mRNA (sg mRNA) was identified from a Taiwanese isolate of PRRSV. Thirty-six cDNA clones derived from sg mRNAs 2, 3, 4, 5, 6 and 7 were determined. The junction sequences analyzed from different sg mRNA were found to contain a similar 5 nucleotide sequence motif, (U/G)(C/A)(A/G)CC. The distance between the junction site and the translation initiation codon of the down stream open reading frame varied from 4 to 226 nucleotides. Minor heterogenecity was observed in the nucleotide sequence surrounding the junctions from all six sg mRNA analyzed. However, for sg mRNA 7, two junction sites approximately 103 nucleotides apart from each other were identified. The additional site is a new junction not previously reported in sg mRNA 7 from other PRRSV strains.

Ling-Ling Chueh

Papers

Restriction Fragment Length Polymorphism Analysis of the F Gene of Newcastle Disease Viruses Isolated from Chickens and an Owl in Taiwan

Leader-body junction sequence of the viral subgenomic mRNAs of porcine reproductive and respiratory syndrome virus isolated in Taiwan.