Quantification of individual subgenomic mRNA species during replication of the coronavirus transmissible gastroenteritis virus

Abstract

A biotinylated-oligonucleotide-based method was used to isolate the subgenomic mRNAs of the coronavirus transmissible gastroenteritis virus (TGEV) to investigate the amounts of the mRNAs produced at early, middle and late times in the replication cycle. TGEV mRNA 6, which encodes the N protein, was observed to be the most abundant species throughout the replication cycle. The ratios of mRNA 6 to the other mRNAs were 1:0.11 (mRNA 2), 1:0.16 (mRNAs 3 and 4) and 1:0.37 (mRNA 5) at 12 h post-infection. All the mRNA species were differentially regulated throughout the replication cycle, although the rate of accumulation of mRNAs 4, 5 and 6, but not mRNA 3, increased markedly towards the end of the replication cycle. mRNA 7 was not detected in the system used. There was no observable correlation between the amounts of each mRNA synthesised and the potential degree of base pairing between the 3' end of the leader sequence and the transcription associated sequences on the genomic RNA at any time during the replication cycle. This indicates that the extent of base pairing was not the only factor involved in the control of subgenomic mRNA synthesis.

Diagnostics of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) nucleocapsid antigen using chicken immunoglobulin Y

“…The NP is the most predominant virus derived protein throughout the infection, because the NP mRNA levels are amplified 3 to 10 times higher at 12 h postinfection (Hiscox et al, 1995) compared with other structural genes. This feature makes it a suitable candidate for developing a diagnostic kit in suspected individuals.…”

“…The 3 major diagnostic methods currently available are (i) viral RNA detection using real-time reverse-transcription PCR (Yam et al, 2003;Jiang et al, 2004;Poon et al, 2004), (ii) antibody detection Li et al, 2005), and (iii) antigen detection (Che et al, 2004;Lau et al, 2004;Li et al, 2005). The most predominant SARS-CoV-derived protein throughout the infection is the nucleocapsid protein (NP) due to the comparatively high levels of mRNA expressed upon infection (Hiscox et al, 1995). The NP is also reported to be high in humans infected with SARS-CoV.…”

The Identification and Characterization of Two Novel Epitopes on the Nucleocapsid Protein of the Porcine Epidemic Diarrhea Virus

“…The N protein of PEDV consists of 441 amino acids and represents an essential agent in the formation of the virus nucleocapsid structure. The abundant expression of N protein in PEDV-infected cells and the high conservation among different PEDV strains make it fairly suitable for use as a target for PEDV diagnosis3068. However, the epitopes on PEDV N protein have been poorly characterized.…”

Bispecific Antibodies for Diagnostic Applications

“…The assay is designed to detect the most abundant and conserved viral antigen nucleocapsid protein NP (Di et al 2005;Hiscox et al 1995;Lau et al 2004;Rota et al 2003;Suresh et al 2008) because its presence in different body fluids (serum, urine, nasopharyngeal aspirate, throat wash samples, and saliva) is suggestive of current infection. Three different monoclonal antibodies that recognize different epitopes on NP antigen and the anti-HRPO monoclonal antibody were employed to generate the BsMAb using hybrid-hybridoma technology.…”