A phylogenetic analysis of Bovine Viral Diarrhoea Virus (BVDV) isolates from six different regions of the UK and links to animal movement data.

Bovine Viral Diarrhoea Virus (BVDV) is a pestivirus which infects cattle populations worldwide and is recognised as a significant source of economic loss through its impact on health and productivity. Studies investigating the molecular epidemiology of BVDV can give invaluable information about the diversity of viral strains present in a population and this, in turn, can inform control programs, drive vaccine development and determine likely infection sources. The current study investigated 104 viral isolates from forty farms across the UK. Through phylogenetic and nucleotide sequence analysis of the 5'UTR and Npro regions of the isolates investigated, it was determined that BVDV 1a was the predominant sub-genotype. However, BVDV 1b, 1e and 1i were also identified and, for the first time in the UK, BVDV 1d. Through analysis of animal movement data alongside the phylogenetic analysis of these BVD isolates, it was possible to link animal movements to the viral isolates present on several premises and, for the first time, begin to elucidate the routes of viral transmission. With further work, this type of analysis would enable accurate determination and quantification of the true biosecurity risk factors associated with BVDV transmission.

Validation of endogenous reference genes for RT-qPCR normalisation in bovine lymphoid cells (BL-3) infected with Bovine Viral Diarrhoea Virus (BVDV).

Reverse transcription quantitative PCR (RT-qPCR) is a highly sensitive tool that can be used for accurate and reliable gene expression analysis; however, careful normalisation to a set of stably expressed endogenous reference genes is essential. Expression levels of many reference genes in RT-qPCR analyses can be extremely variable under different experimental conditions, producing potentially erroneous results (Bustin, 2002). This limitation can be overcome with a systematic evaluation of candidate reference genes to determine the most stable. In the present study eight candidate reference genes were evaluated in a bovine lymphoid (BL-3) cell culture system over seven different time points in response to three different Bovine Viral Diarrhoea Virus (BVDV) strains. Data were analysed using BestKeeper (Pfaffl et al., 2004), geNorm (Vandesompele et al., 2002), and NormFinder (Andersen et al., 2004) validation programs and results enable the candidate reference genes to be ranked from most to least stable. Quantification cycle (C(q)) variability was determined between samples, i.e. between treatment groups and time points, and variability was also observed between the three validation programs. The reference gene combination of beta-actin and hypoxanthine-guanine phosphoribosyl transferase (HPRT) was found to be the most stable in Norm Finder. BestKeeper and geNorm both demonstrated beta-microglobulin and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase (YWHAZ) as the most stable. The determination of a stable set of reference genes in the BL-3 cell culture system facilitates analysis of expression levels for appropriate genes of interest. This study further emphasises the need to accurately validate candidate reference genes before use in gene expression RT-qPCR studies.

Infectivity of pestivirus following persistence of acute infection.

Bovine viral diarrhoea virus (BVDV) is an endemic pathogen worldwide and eradication strategies focus on the identification and removal of persistently infected (PI) animals arising after in utero infection. Despite this, acute infections with BVDV can persist for months or years after the removal of the PI source despite repeated screening for PIs and tight biosecurity measures. Recent evidence for a prolonged duration of viraemia in the testicles of bulls following acute BVDV infection suggests the possibility of a form of chronic persistence that may more closely resemble the persistence strategies of hepatitis C virus (HCV). To investigate the potential for virus transmission from infected and recovered cattle to virus naïve hosts we established an acute infection of 5 BVDV-naïve calves and monitored animals over 129 days. Infectious BVDV was detected in white blood cells between days 3 and 7 post-challenge. The animals seroconverted by day 21 post-infection and subsequently were apparently immune and free from infectious virus and viral antigen. Animals were further monitored and purified white blood cells were stimulated in vitro with phytohaemagglutinin A (PHA) during which time BVDV RNA was detected intermittently. Ninety-eight days following challenge, blood was transferred from these apparently virus-free and actively immune animals to a further group of 5 BVDV-naïve calves and transmission of infection was achieved. This indicates that BVDV-infected, recovered and immune animals have the potential to remain infectious for BVDV-naïve cohorts for longer than previously demonstrated.

Partial redistribution of the Autographa californica nucleopolyhedrovirus chitinase in virus-infected cells accompanies mutation of the carboxy-terminal KDEL ER-retention motif.

During virus infection of insect cells, the Autographa californica nucleopolyhedrovirus chitinase is localized primarily within the endoplasmic reticulum (ER), which is consistent with the presence of a carboxy-terminal ER retention motif (KDEL). Release of chitinase into the extracellular medium appears to be concomitant with terminal cell lysis, rather than by active secretion. In this study, we have shown that mutation of the KDEL motif induces a partial redistribution of the chitinase at both early and late times post-infection. Deletion of the KDEL motif or substitution with glycine residues allowed chitinase to move through the secretory pathway, accumulating to detectable levels in the extracellular medium by 24 h post-infection; more than 48 h prior to cell lysis. Deletion of the KDEL motif did not compromise enzyme activity, with the modified enzyme exhibiting characteristic endo- and exo-chitinolytic activity. Trichoplusia ni larvae infected with the modified virus were found to liquefy approximately 24 h earlier than larvae infected with a control virus in which the chitinase KDEL motif had not been deleted.