Quantification of viral infectious units is traditionally measured by methods based on forming plaques in semisolid media (PFU) or endpoint dilution of a virus-containing solution (TCID(50)), methods that are laborious, time-consuming and take on average 3-7 days to carry out. Quantitative real-time PCR is an established method to quantify nucleic acids at high accuracy and reproducibility, routinely used for virus detection and identification. In the present study, a procedure was developed using a two-step real-time PCR and the SYBR Green detection method to study whether there are correlations between TCID(50)/ml, PFU/ml and Ct values generated by real-time PCR enabling rapid and efficient calculation of titer equivalents when working with viruses in the research laboratory. In addition, an external standard with known concentrations was included using in vitro transcribed viral RNA, thus allowing the calculation of the amount of RNA copies needed for various applications (i.e. per plaque or TCID(50)).The results show that there is a correlation between the three quantification methods covering a wide range of concentration of viruses. Furthermore, a general regression line between TCID(50) and Ct values was obtained for all viruses included in the study, which enabled recording titer equivalents using real-time PCR. Finally, by including an external standard, the amount of RNA genomes generating one TCID(50) or PFU for each enterovirus serotype included was determined.

Background: Measuring virus attachment to host cells is of great importance when trying to identify novel receptors. The presence of a usable receptor is a major determinant of viral host range and cell tropism. Furthermore, identification of appropriate receptors is central for the understanding of viral pathogenesis and gives possibilities to develop antiviral drugs. Attachment is presently measured using radiolabeled and subsequently gradient purified viruses. Traditional methods are expensive and time-consuming and not all viruses are stable during a purification procedure; hence there is room for improvement. Real-time PCR (RT-PCR) has become the standard method to detect and quantify virus infections, including enteroviruses, in clinical samples. For instance, primers directed to the highly conserved 5' untranslated region (5'UTR) of the enterovirus genome enable detection of a wide spectrum of enteroviruses. Here, we evaluate the capacity of the RT-PCR technology to study enterovirus host cell interactions at the cell surface and compare this novel implementation with an established assay using radiolabeled viruses. Results: Both purified and crude viral extracts of CVB5 generated comparable results in attachment studies when analyzed with RT-PCR. In addition, receptor binding studies regarding viruses with coxsackie- nd adenovirus receptor (CAR) and/or decay accelerating factor (DAF) affinity, further demonstrated the possibility to use RT-PCR to measure virus attachment to host cells. Furthermore, the RT-PCR technology and crude viral extracts was used to study attachment with low multiplicity of infection (0.05 x 10(-4)TCID(50)/cell) and low cell numbers (250), which implies the range of potential implementations of the presented technique. Conclusion: We have implemented the well-established RT-PCR technique to measure viral attachment to host cells with high accuracy and reproducibility, at low cost and with less effort than traditional methods. Furthermore, replacing traditional methods with RT-PCR offers the opportunity to use crude virus containing extracts to investigate attachment, which could be considered as a step towards viral attachment studies in a more natural state.

Like other RNA viruses, coxsackievirus B5 (CVB5) exists as circulating heterogeneous populations of genetic variants. In this study, we present the reconstruction and characterization of a probable ancestral virion of CVB5. Phylogenetic analyses based on capsid protein encoding regions (the VP1 gene of 41 clinical isolates and the entire P1 region of eight clinical isolates) of CVB5 revealed two major co-circulating lineages. Ancestral capsid sequences were inferred from sequences of these contemporary CVB5 isolates using maximum likelihood methods. By using Bayesian phylodynamic analysis, the inferred VP1 ancestral sequence was dated back to 1854 (1807-1898). In order to study the properties of the putative ancestral capsid, the entire ancestral P1 sequence was synthesized de novo and inserted into the replicative backbone of an infectious CVB5 cDNA clone. Characterization of the recombinant virus in cell culture showed that fully functional infectious virus particles were assembled and that these viruses displayed properties similar to those of modern isolates, in terms of receptor preferences, plaque phenotype, growth characteristics and cell tropism. This is the first report describing resurrection and characterization of a picornavirus with a putative ancestral capsid. Our approach, including phylogenetics-based reconstruction of viral predecessors, could serve as a starting point for experimental studies of viral evolution and might also provide an alternative strategy in the development of vaccines.

Aichi virus (AiV), genus Kobuvirus, family Picornaviridae, is associated with gastroenteritis in humans. Previous studies have shown high seroprevalence but low incidence (0.9-4.1%) in clinical samples. We report here the first detection of AiV in Sweden. Two hundred twenty-one specimens from hospitalized patients with diarrhea, who were negative for other enteric viruses, were included in the study. AiV were detected in three specimens, all from elderly patients. Phylogenetic analysis revealed that the three Swedish isolates belonged to genotype A and were genetically closest to European and Asian strains of AiV.