Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated, not contaminated genomic DNA is prerequisite for successful and reliable large scale genotyping analysis. High quantities of pure DNA are also required for the creation of DNA-banks. In the present study, eleven different DNA extraction procedures, including phenol-chloroform, silica and magnetic beads based extractions, were examined to ascertain their relative effectiveness for extracting DNA from ovine blood samples. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements, Qubit measurements, real-time PCR amplifications and gel electrophoresis. Processing time, intensity of labor and cost for each method were also evaluated. Results revealed significant differences among the eleven procedures and only four of the methods yielded satisfactory outputs. These four methods, comprising three modified silica based commercial kits (Modified Blood, Modified Tissue, Modified Dx kits) and an in-house developed magnetic beads based protocol, were most appropriate for extracting high quality and quantity DNA suitable for large-scale microarray genotyping and also for long-term DNA storage as demonstrated by their successful application to 600 individuals.

Since 1997, a yellowing disease has been observed in greenhouse tomato (Lycopersicon escu-lentum). By 2001, the disease was widespread, including open field tomato crops, and in most cases its incidence was 80 to 90% or even 100%. Epidemics in glasshouses were mainly associated with high populations of the whitefly Trialeurodes vaporariorum and Bemisia tabaci, the major whitefly pests in vegetable crops in Greece. The main leaf symptoms were severe yellowing, rolling, and brittleness. Samples from symptomatic plants were analyzed by polymerase chain reaction (PCR) and shown to be infected with Tomato infectious chlorosis virus (TICV) and Tomato chlorosis virus (ToCV) (family Closteroviridae, genus Crinivirus). TICV was found in 164 of 183 symptomatic samples, while ToCV was less representative (25/183). Sequence comparisons of the amplified 229-bp and 466-bp products revealed 99 and 100% identity with the reported sequences of TICV and ToCV, respectively. A reverse transcription (RT) multiplex PCR assay using a simple sample preparation procedure was developed to allow rapid, specific, and simultaneous detection of both ToCV and TICV sequences in two steps. The method involves a one-tube RT-PCR step in which the combination of primers amplifies part of the heat shock protein to homologue gene of both ToCV and TICV, followed by a multiplex nested PCR amplification. This is the first report of TICV and ToCV in Greece and, as far as we know, the first report of TICV in Europe.

In the summer of 2010 an epidemic of West Nile virus (WNV) occurred in Central Macedonia, Greece, with 197 human neuroinvasive disease (WNND) cases. In the following years the virus spread to new areas, with a total of 76 WNND cases in 2011, and 109 WNND cases in 2012 (14 and 12 WNND cases, respectively, in Central Macedonia). We established a surveillance system based on serological testing of domestic pigeons, using cELISA confirmed by serum neutralization test. In Central Macedonia, pigeon seroprevalence was 54% (95% CI: 49-59%) and 31% (95% CI: 24-37%) at the end of the 2010 and 2011 epidemic seasons, respectively. One serum was positive for neutralizing antibodies directed against Usutu virus. Pigeon WNV seroprevalence and incidence rates of human WNND after the 2010 epidemic were positively correlated (ρ=0.94, at the regional unit level), while in 2011 the correlation (ρ=0.56) was not statistically significant, possibly due to small number of human WNND cases recorded. To evaluate the efficacy of the system at alerting upon WNV enzootic circulation before the onset of human cases, we tested 270 pigeons in 2011 and 240 pigeons in 2012. In Central Macedonia, the first seroconversions in pigeons were recorded 44 and 47 days, respectively, before the first human WNND cases. Pigeon surveillance was used successfully for identification of areas with WNV enzootic transmission and for early warning. Timely diffusion of information to health authorities facilitated the implementation of preparedness plans to protect public health.