The effective and efficient management of cancer patients relies upon early diagnosis and/or the monitoring of treatment, something that is often difficult to achieve using standard tissue biopsy techniques. Biological fluids such as blood hold great possibilities as a source of non-invasive cancer biomarkers that can act as surrogate markers to biopsy-based sampling. The non-invasive nature of these “liquid biopsies” ultimately means that cancer detection may be earlier and that the ability to monitor disease progression and/or treatment response represents a paradigm shift in the treatment of cancer patients. Below, we review one of the most promising classes of circulating cancer biomarkers: microRNAs (miRNAs). In particular, we will consider their history, the controversy surrounding their origin and biology, and, most importantly, the hurdles that remain to be overcome if they are really to become part of future clinical practice.

A strain ofTick-borne encephalitis virusdesignated Zausaev (Za) was isolated in Siberia from a patient who died of a progressive (2-year) form of tick-borne encephalitis 10 years after being bitten by a tick. The complete genomic sequence of this virus was determined, and an attempt was made to correlate the sequence with the biological characteristics of the virus. Phylogenetic analysis demonstrated that this virus belongs to the Siberian subtype ofTick-borne encephalitis virus. Comparison of Za virus with two related viruses, a Far Eastern isolate, Sofjin, and a Siberian isolate, Vasilchenko, revealed differences among the three viruses in pathogenicity for Syrian hamsters, cytopathogenicity for PS cells, plaque morphology, and the electrophoretic profiles of virus-specific nonstructural proteins. Comparative amino acid alignments revealed 10 individual amino acid substitutions in the Za virus polyprotein sequence that were different from those of other tick-borne flaviviruses. Notably, the dimeric form of the Za virus NS1 protein migrated in polyacrylamide gels as a heterogeneous group of molecules with a significantly higher electrophoretic mobility than those of the Sofjin and Vasilchenko viruses. Two amino acid substitutions, T₂₇₇→V and E₂₇₉→G, within the NS1 dimerization domain are probably responsible for the altered oligomerization of Za virus NS1. These studies suggest that the patient from whom Za virus was isolated died due to increased pathogenicity of the latent virus following spontaneous mutagenesis.

We have previously demonstrated that the replacement of the S gene from an avirulent strain (Beaudette) of infectious bronchitis virus (IBV) with an S gene from a virulent strain (M41) resulted in a recombinant virus (BeauR-M41(S)) with the in vitro cell tropism of the virulent virus but that was still avirulent. In order to investigate whether any of the other structural or accessory genes played a role in pathogenicity we have now replaced these from the Beaudette strain with those from M41. The recombinant IBV was in effect a chimaeric virus with the replicase gene derived from Beaudette and the rest of the genome from M41. This demonstrated that it is possible to exchange a large region of the IBV genome, approximately 8.4 kb, using our transient dominant selection method. Recovery of a viable recombinant IBV also demonstrated that it is possible to interchange a complete replicase gene as we had in effect replaced the M41 replicase gene with the Beaudette derived gene. Analysis of the chimaeric virus showed that it was avirulent indicating that none of the structural or accessory genes derived from a virulent isolate of IBV were able to restore virulence and that therefore, the loss of virulence associated with the Beaudette strain resides in the replicase gene.

Millions of domestic and wild European rabbits (Oryctolagus cuniculus) have died in Europe, Asia, Australia and New Zealand during the past 17 years following infection by Rabbit haemorrhagic disease virus (RHDV). This highly contagious and deadly disease was first identified in China in 1984. Epidemics of RHDV then radiated across Europe until the virus apparently appeared in Britain in 1992. However, this concept of radiation of a new and virulent virus from China is not entirely consistent with serological and molecular evidence. This study shows, using RT–PCR and nucleotide sequencing of RNA obtained from the serum of healthy rabbits stored at 4 °C for nearly 50 years, that, contrary to previous opinions, RHDV circulated as an apparently avirulent virus throughout Britain more than 50 years ago and more than 30 years before the disease itself was identified. Based on molecular phylogenetic analysis of British and European RHDV sequences, it is concluded that RHDV has almost certainly circulated harmlessly in Britain and Europe for centuries rather than decades. Moreover, analysis of partial capsid sequences did not reveal significant differences between RHDV isolates that came from either healthy rabbits or animals that had died with typical haemorrhagic disease. The high stability of RHDV RNA is also demonstrated by showing that it can be amplified and sequenced from rabbit bone marrow samples collected at least 7 weeks after the animal has died.

The gold standard for cancer diagnosis remains the histological examination of affected tissue, obtained either by surgical excision, or radiologically guided biopsy. Such procedures however are expensive, not without risk to the patient, and require consistent evaluation by expert pathologists. Consequently, the search for non‐invasive tools for the diagnosis and management of cancer has led to great interest in the field of circulating nucleic acids in plasma and serum. An additional benefit of blood‐based testing is the ability to carry out screening and repeat sampling on patients undergoing therapy, or monitoring disease progression allowing for the development of a personalized approach to cancer patient management. Despite having been discovered over 60 years ago, the clear clinical potential of circulating nucleic acids, with the notable exception of prenatal diagnostic testing, has yet to translate into the clinic. The recent discovery of non‐coding (nc)RNA(in particular micro(mi)RNAs) in the blood has provided fresh impetuous for the field. In this review, we discuss the potential of thecirculating transcriptome(coding and ncRNA), as novel cancer biomarkers, the controversy surrounding their origin and biology, and most importantly the hurdles that remain to be overcome if they arereallyto become part of future clinical practice.

The spike (S) glycoprotein of the avian gammacoronavirus infectious bronchitis virus (IBV) is comprised of two subunits (S1 and S2), has a role in virulence in vivo, and is responsible for cellular tropism in vitro. We have previously demonstrated that replacement of the S glycoprotein ectodomain from the avirulent Beaudette strain of IBV with the corresponding region from the virulent M41-CK strain resulted in a recombinant virus, BeauR-M41(S), with the in vitro cell tropism of M41-CK. The IBV Beaudette strain is able to replicate in both primary chick kidney cells and Vero cells, whereas the IBV M41-CK strain replicates in primary cells only. In order to investigate the region of the IBV S responsible for growth in Vero cells, we generated a series of recombinant IBVs expressing chimeric S glycoproteins, consisting of regions from the Beaudette and M41-CK S gene sequences, within the genomic background of Beaudette. The S2, but not the S1, subunit of the Beaudette S was found to confer the ability to grow in Vero cells. Various combinations of Beaudette-specific amino acids were introduced into the S2 subunit of M41 to determine the minimum requirement to confer tropism for growth in Vero cells. The ability of IBV to grow and produce infectious progeny virus in Vero cells was subsequently narrowed down to just 3 amino acids surrounding the S2′ cleavage site. Conversely, swapping of the 3 Beaudette-associated amino acids with the corresponding ones from M41 was sufficient to abolish Beaudette growth in Vero cells. IMPORTANCE Infectious bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines. IBV vaccines, both live attenuated and inactivated, are currently grown on embryonated hen9s eggs, a cumbersome and expensive process due to the fact that most IBV strains do not grow in cultured cells. The reverse genetics system for IBV creates the opportunity for generating rationally designed and more effective vaccines. The observation that IBV Beaudette has the additional tropism for growth on Vero cells also invokes the possibility of generating IBV vaccines produced from cultured cells rather than by the use of embryonated eggs. The regions of the IBV Beaudette S glycoprotein involved in the determination of extended cellular tropism were identified in this study. This information will enable the rational design of a future generation of IBV vaccines that may be grown on Vero cells.