Chrysostomos I. Dovas

A spot nested RT-PCR method for the simultaneous detection of members of the Vitivirus and Foveavirus genera in grapevine.

A reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to allow rapid, and simultaneous detection of Vitivirus and Foveavirus sequences in two steps. The method involved a one-step RT-PCR, in which the combination of degenerate deoxyinosine-substituted primers amplified part of the polymerase region of both genera, followed by a nested PCR amplification that increased specificity and sensitivity of detection. The increase in sensitivity also permitted the use of a simple and rapid template preparation protocol, involving the spotting of plant sap extract on a nylon membrane. Consistent amplification with infected grapevine plants was possible after inclusion of additives for inhibiting polyphenolic compounds during template preparation. This spot nested RT-PCR method can reliably detect virus species of both genera in grapevine allowing simple, fast, and cost-effective analysis of a large number of samples in certification schemes.

Evaluation of a West Nile virus surveillance and early warning system in Greece, based on domestic pigeons

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.

Caprine PRNP polymorphisms at codons 171, 211, 222 and 240 in a Greek herd and their association with classical scrapie

The association between PRNP variation and scrapie incidence was investigated in a highly affected Greek goat herd. Four mutations were identified at codons 171Q/R, 211R/Q, 222Q/K and 240P/S. Lysine at codon 222 was found to be associated with the protection from natural scrapie (P=0.0111). Glutamine at codon 211 was observed in eight animals, all of them being scrapie-negative, indicating a possible protective role of this polymorphism although statistical analysis failed to support it (P=0.1074). A positive association (P=0.0457) between scrapie-affected goats and the wild-type Q(171)R(211)Q(222)S(240) allele is presented for the first time. In addition, a novel R(171)RQS allele, which is identical to the A(136)R(154)R(171) allele that has been associated with resistance to classical scrapie in sheep, was observed in low frequency. Resistant alleles that include K(222) and Q(211) are absent or rare in sheep and can provide the basis for the development of a feasible breeding programme for scrapie eradication in goats.

Protective efficacy of Bluetongue virus-like and subvirus-like particles in sheep: presence of the serotype-specific VP2, independent of its geographic lineage, is essential for protection.

There have been multiple separate outbreaks of Bluetongue (BT) disease of ruminants in Europe since 1998, often entering via the Mediterranean countries of Italy, Spain and Greece. BT is caused by an orbivirus, Bluetongue virus (BTV), a member of the family Reoviridae. BTV is a non-enveloped double-capsid virus, which encodes 7 structural proteins (VP1-VP7) and several non-structural proteins (NS1, NS2, NS3/3a and NS4) from ten double-stranded RNA segments of the genome. In this report, we have prepared BTV virus-like particles (VLPs, composed of VP2, VP3, VP5 and VP7) and sub-viral, inner core-like particles (CLPs, VP3 and VP7) using a recombinant baculovirus expression system. We compared the protective efficacy of VLPs and CLPs in sheep and investigated the importance of geographical lineages of BTV in the development of vaccines. The Greek crossbred Karagouniko sheep, which display mild to sub-clinical BT, were vaccinated with VLPs or CLPs of BTV-1, derived from western lineage and were challenged with virulent BTV-1 from an eastern lineage. All VLP-vaccinated animals developed a neutralising antibody response to BTV-1 from both lineages prior to challenge. Moreover, post-challenged animals had no clinical manifestation or viraemia and the challenged virus replication was completely inhibited. In contrast, CLP-vaccinated animals did not induce any neutralising antibody response but developed the group specific VP7 antibodies. CLPs also failed to prevent the clinical manifestation and virus replication, but in comparison to controls, the severity of disease manifestation and viraemia was mitigated. The data demonstrated that the outer capsid was essential for complete protection, while the geographical origin of the BTV was not critical for development of a serotype specific vaccine.

A comparison of six methods for genomic DNA extraction suitable for PCR-based genotyping applications using ovine milk samples

Isolation of amplifiable genomic DNA is a prerequisite for the genetic assessment of diseases and disease susceptibility in farm animals. Milk somatic cells are a practical, animal friendly and cost-effective source of genomic DNA in milking ruminants. In this study, six different DNA extraction methods were optimized, evaluated and compared for the isolation of DNA from ovine milk samples. Methods 1 and 2 were direct applications of two commercial kits, Nucleospin((R)) Blood and Nucleospin((R)) Tissue, respectively. Methods 3 and 4 were based on modified protocols of methods 1 and 2, respectively, aiming at increasing DNA recovery and integrity, and eliminating PCR inhibitors. Method 5 was a standard Phenol-Chloroform protocol application and method 6 was based on an in-house developed protocol using silica as the affinity matrix. Spectrophotometer, gel electrophoresis and real-time PCR measurements were used as criteria for evaluating quantity and quality of the extracted DNA. Processing time, intensity of labor and cost for each method were also evaluated. Results suggested that methods 1-4 were considered suitable for molecular downstream applications and performed better than methods 5 and 6. Modifications of protocols 3 and 4 increased the quantity and quality of the extracted DNA from ovine milk samples. Method 3 was proved to be highly efficient and robust for large scale use as demonstrated by its successful application to 1000 individual ovine milk and 50 bulk milk samples.

Evolutionary relationships of virus species belonging to a distinct lineage within the Ampelovirus genus

A study of the evolutionary relationships of GLRaV-4,-5,-6 and -9, and two new Ampelovirus isolates (GLRaV-Pr and -De) related to grapevine leafroll disease was conducted based on molecular variability, positive selection analysis and maximum likelihood phylogenetic reconstructions. Sequences corresponding to the N-terminal HSP70h and full CP encoding genes were determined for these viruses and datasets including homologous genomic regions from different members of the Closteroviridae were analyzed. GLRaV-Pr and -De were further characterised as distinct from the other closely related species after determination of a large genomic region (4319-4358 nts). ML phylogenetic topologies for both genes established the closer phylogenetic relationships of GLRaV-4,-5,-6,-9,-Pr and -De in regard to the other ampeloviruses, revealing very low inter-species evolutionary distances for this multitudinous lineage. The HSP70h segment phylogeny and bootstrap analysis enabled the identification of species within this lineage and provides a useful taxonomic tool for the rapid demarcation of these viruses. Estimations of d(N)/d(S) using the CP and HSP70h datasets revealed that, within the Closteroviridae, these viruses are subjected to the strongest constraints against amino acid substitutions. These estimations demonstrated a distinct evolutionary trait for this lineage probably related to its particular ecological niche that involves successful adaptation to the host, transmission through vegetative propagation and lack of vectors with high transmission efficiency.

Comparison of Eleven Methods for Genomic DNA Extraction Suitable for Large-Scale Whole-Genome Genotyping and Long-Term DNA Banking Using Blood Samples

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.

Detection and Quantification of Infectious Avian Influenza A (H5N1) Virus in Environmental Water by Using Real-Time Reverse Transcription-PCR

ABSTRACT Routes of avian influenza virus (AIV) dispersal among aquatic birds involve direct (bird-to-bird) and indirect (waterborne) transmission. The environmental persistence of H5N1 virus in natural water reservoirs can be assessed by isolation of virus in embryonated chicken eggs. Here we describe development and evaluation of a real-time quantitative reverse transcription (RT)-PCR (qRT-PCR) method for detection of H5N1 AIV in environmental water. This method is based on adsorption of virus particles to formalin-fixed erythrocytes, followed by qRT-PCR detection. The numbers of hemagglutinin RNA copies from H5N1 highly pathogenic AIV particles adsorbed to erythrocytes detected correlated highly with the infectious doses of the virus that were determined for three different types of artificially inoculated environmental water over a 17-day incubation period. The advantages of this method include detection and quantification of infectious H5N1 AIVs with a high level of sensitivity, a wide dynamic range, and reproducibility, as well as increased biosecurity. The lowest concentration of H5N1 virus that could be reproducibly detected was 0.91 50% egg infective dose per ml. In addition, a virus with high virion stability (Tobacco mosaic virus) was used as an internal control to accurately monitor the efficiency of RNA purification, cDNA synthesis, and PCR amplification for each individual sample. This detection system could be useful for rapid high-throughput monitoring for the presence of H5N1 AIVs in environmental water and in studies designed to explore the viability and epidemiology of these viruses in different waterfowl ecosystems. The proposed method may also be adapted for detection of other AIVs and for assessment of their prevalence and distribution in environmental reservoirs.

West Nile Virus State of the Art Report of MALWEST Project

During the last three years Greece is experiencing the emergence of West Nile virus (WNV) epidemics. Within this framework, an integrated surveillance and control programme (MALWEST project) with thirteen associate partners was launched aiming to investigate the disease and suggest appropriate interventions. One out of seven work packages of the project is dedicated to the State of the Art report for WNV. Three expert working groups on humans, animals and mosquitoes were established. Medical databases (PubMed, Scopus) were searched together with websites: e.g., WHO, CDC, ECDC. In total, 1,092 relevant articles were initially identified and 258 of them were finally included as references regarding the current knowledge about WNV, along with 36 additional sources (conference papers, reports, book chapters). The review is divided in three sections according to the fields of interest: (1) WNV in humans (epidemiology, molecular characteristics, transmission, diagnosis, treatment, prevention, surveillance); (2) WNV in animals (epidemiological and transmission characteristics concerning birds, horses, reptiles and other animal species) and (3) WNV in mosquitoes (control, surveillance). Finally, some examples of integrated surveillance programmes are presented. The introduction and establishment of the disease in Greece and other European countries further emphasizes the need for thorough research and broadening of our knowledge on this viral pathogen.

Campylobacter in small ruminants at slaughter: Prevalence, pulsotypes and antibiotic resistance

The present study aimed to address the prevalence, pulsotypes, and antimicrobial susceptibility patterns of Campylobacter species present in sheep and goat carcasses at slaughter. In total, 851 samples were collected (343 meat surfaces, 282 ileum contents, 226 liver surfaces) and 835 Campylobacter isolates were detected in 274 out of 343 carcasses (116 kids, 110 lambs, 63 goats and 54 sheep). The contamination rates per carcass category were 78.4% for kids, 94.5% for lambs, 63.5% for goats, and 72.2% for sheep. On average, 30% of the intestinal content samples and more than 70% of carcass and liver surfaces yielded the presence of campylobacters. Multiplex-PCR and RFLP analysis identified Campylobacter coli as the most prevalent species (76.2%) followed by Campylobacter jejuni (21.4%), albeit 2.4% of selected colonies yielded the concurrent presence of both these species. Macrorestriction profiling by pulsed-field gel electrophoresis (PFGE) was applied in order to characterise a subset of isolates. SmaI-PFGE successfully clustered 222 isolates in 82 SmaI-PFGE types indicating high heterogeneity among the campylobacter isolates (67 types among 174C. coli isolates and 15 types among 48C. jejuni isolates). No carcass-type (lamb, kid, sheep, and goat) specific PFGE clusters were recognised since there was a general overlapping of PFGE patterns regarding ovine and caprine isolates. Multiple pulsotypes were simultaneously present on single carcasses in the majority of tested animals. PFGE provided data regarding the potential routes of meat and liver contamination such as spillage of faecal material and cross-contamination during slaughter. Antimicrobial susceptibility patterns of Campylobacter isolates (n=240), determined by disk diffusion method, revealed resistance to tetracycline (47.9%) followed by streptomycin (22.9%) and ciprofloxacin along with nalidixic acid (18.3%). Isolates exhibited low resistance to erythromycin (2.5%) and were susceptible to gentamicin. The findings of the present study confirm the contamination of sheep and goats at slaughter with thermophilic campylobacters and underline their potential input in the epidemiology of human campylobacteriosis.