Immunosuppression in sheep induced by cyclophosphamide, bluetongue virus and their combination: Effect on clinical reaction and viremia.

The main purpose of this work was to establish an experimental model for immunosuppression in sheep, and evaluate its possible effects on bluetongue viremia. Animals were allocated in 4 groups: Cy (cyclophosphamide), BT (bluetongue), CyBT (combined Cy and BT) and Co (control), and underwent clinical evaluations, virological testing, peripheral blood immunophenotyping and determination of antiviral humoral immune responses. Intravenous administration of cyclophosphamide (37.5 mg/kg body weight) resulted in immunosuppresion induction, as significant drops were observed in blood leukocytes and lymphocyte subset counts (CD2+, CD4+, CD8+, CD19+), lasting 3-10 days after its administration. Reduction in B-cell (CD19+) counts was more pronounced than in T-/NK-cell (CD2+) counts (92% and 59%, respectively). BTV-9 inoculation resulted in pronounced lymphocytopenia observed from day 1 post-inoculation. Their combined administration resulted in a more intense immunosuppressive effect, as indicated by the greater reduction in lymphocyte, granulocyte, CD4+ and CD8+ cell counts. In group CyBT, earlier initiation of fever by one day (day 6 p.i.) compared to group BT (day 7 p.i.), and delay in antibody responses by one day was observed, compared to group BT. Neutralizing antibodies in both groups (BT, CyBT) were detectable from day 10 p.i., but no significant titer differences were observed. Infectious virus titers were detected from day 4 p.i. in group BT and from day 3 in group CyBT. Statistical significances in virus titers were also observed (greatest mean titer difference: 1.4 log10 CEID50/ml RBCs at day 5 p.i., P < 0.001), indicating possible impact of immunosuppression on virus transmission and epidemiology of bluetongue.

Evaluation of Cross-Protection of a Lineage 1 West Nile Virus Inactivated Vaccine against Natural Infections from a Virulent Lineage 2 Strain in Horses, under Field Conditions.

Although experimental data regarding cross-protection of horse West Nile virus (WNV) vaccines against lineage 2 infections exist, the cross-protective efficacy of these vaccines under field conditions has not been demonstrated. This study was conducted to evaluate the capability of an inactivated lineage 1 vaccine (Equip WNV) to protect against natural infections from the Nea Santa-Greece-2010 lineage 2 strain. In total, 185 WNV-seronegative horses in Thessaloniki, Greece, were selected during 2 consecutive years (2011 and 2012); 140 were immunized, and 45 were used as controls. Horses were examined for signs compatible with WNV infection. Neutralizing antibody titers against the Greek strain and the PaAn001/France lineage 1 strain were determined in immunized horses. WNV circulation was detected during both years in the study area. It was estimated that 37% and 27% of the horses were infected during 2011 and 2012, respectively. Three control animals developed clinical signs, and the WNV diagnosis was confirmed. Signs related to WNV infection were not observed in the vaccinated animals. The nonvaccinated animals had a 7.58% ± 1.82% higher chance of exhibiting signs than immunized animals (P < 0.05). Neutralizing antibodies raised against both strains in all immunized horses were detectable 1 month after the initial vaccination course. The cross-protective capacity of the lowest titer (1:40) was evident in 19 animals which were subsequently infected and did not exhibit signs. Neutralizing antibodies were detectable until the annual booster, when strong anamnestic responses were observed (geometrical mean titer ratio [GMTR] for lineage 1 of 30.2; GMTR for lineage 2 of 27.5). The results indicate that Equip WNV is capable of inducing cross-protection against natural infections from a virulent lineage 2 WNV strain in horses.

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.

Evidence of Schmallenberg virus circulation in ruminants in Greece.

During March 2013, we investigated the presence and the levels of Schmallenberg virus (SBV) circulation in three dairy cow herds and three sheep flocks in Central Macedonia, Greece. In two cow herds, a high number of abortions had been observed during the winter. Six bulk-tank milk samples and 147 individual sera were screened for SBV-specific antibodies by ELISA. Positive reactions were obtained from 5 out of 6 bulk-tank milk samples, 58 out of 90 sera from the 3 cow herds, and 2 sera from 2 of the 3 sheep flocks. Twenty-two ELISA-positive sera were tested by serum neutralization test (SNT). SNT confirmed the presence of neutralizing antibodies against SBV in all samples tested, with titers ranging between 1:32 and ≥1:256. No neutralizing antibodies against Akabane virus (AKAV) or Shamonda virus (SHAV) were detected, indicating that neutralizing antibodies against SBV do not cross react with AKAV or SHAV in SNT. ELISA testing of bulk-tank milk samples proved to be convenient and reliable. None of the tested sera was found positive for SBV by real-time RT-PCR, indicating that the sampling was conducted past the viremia stage. This is the first report of SBV circulation in Greece.

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.

Detection and early warning of West Nile Virus circulation in Central Macedonia, Greece, using sentinel chickens and mosquitoes.

Following the West Nile Virus (WNV) epidemic in 2010 in Central Macedonia, Greece, which resulted in 197 human neuroinvasive disease cases, we determined the seasonal appearance and prevalence of the virus in 2011 by testing weekly for WNV genomic RNA in mosquitoes collected in carbon dioxide-baited traps, and for anti-WNV antibodies in sentinel chickens. Preliminary findings of the surveillance program regarding the circulation of "Nea Santa-Greece-2010" in sentinel chickens were rapidly communicated to public health authorities. In the present article, the full 2011 data produced by this surveillance program are presented. We detected enzootic circulation of WNV in chickens 1 month prior to the onset of the first human cases in 2011. Culex pipiens and Cx. modestus were abundant throughout the sampling period and at all sites of increased transmission. Molecular identification and phylogenetic analysis of WNV isolates from two chickens and one Cx. pipiens mosquito pool suggested that: (1) the virulent "Nea Santa-Greece-2010" WNV lineage 2 strain responsible for the 2010 epidemic was actively circulating in 2011, and (2) all Greek isolates belong to a distinct recent evolutionary clade. In Europe, where numerous strains of different virulence coexist, sequencing information for WNV is important for phylogeography and identification of virulent strains for human health risk assessment.

Canine coronavirus, Greece. Molecular analysis and genetic diversity characterization.

Canine coronavirus (CCoV) is an etiologic agent of diarrhea in dogs and is known to have spread worldwide. Mild disease or asymptomatic carriage are probably in many cases common outcomes of infection. To date, two different genotypes of CCoV are known, CCoV type I (CCoV-I) and CCoV type II (CCoV-II). CCoV type II is divided in two subtypes, CCoV-IIa (classical strains) and CCoV-IIb, with CCoV-IIb emerging as a result of a putative recombination between CCoV-IIa and transmissible gastroenteritis virus (TGEV). The aim of the present study was to investigate the presence of CCoV in Greece and to genetically analyze the circulating strains. Between December 2007 and December 2009, 206 fecal samples were collected from dogs with diarrhea from kennels, pet shops and veterinary clinics of different country regions. RT-PCR and real time RT-PCR assays were used for CCoV detection and characterization. CCoV was identified in 65.1% of the dogs presenting diarrhea, being more frequently detected in animals younger than 3 months old and in animals housed in groups. In 47% of the positive samples more than one CCoV genotype/subtype were detected, with triple CCoV-I/CCoV-IIa/CCoV-IIb infections being identified for the first time. Molecular and phylogenetic analysis revealed that CCoV-I Greek strains share low genetic relatedness to each other and to the prototype CCoV-I strains in the 5' end of the S gene. Moreover, a divergent CCoV-IIa strain was identified. The circulation of highly variable CCoV-I and CCoV-IIb emerging strains, as well as the detection of the divergent strain, raise concerns on the importance of these new strains as primary pathogens of diarrhoeic syndromes diagnosed in dogs.

Molecular characterization of a canine coronavirus NA/09 strain detected in a dog's organs.

In the present study, the detection of a pantropic canine coronavirus (CCoV) strain in a dog with lethal diarrhoea is reported. RT-PCR and real-time RT-PCR assays were used for the detection, characterization and quantitation of CCoV. Sequence and phylogenetic analysis of the CCoV NA/09 revealed a high degree of sequence identity with the pantropic strain CB/05, indicating the presence of CB/05-like pantropic strains in Greece. The absence of the 38-nucleotide deletion in ORF3b, which is characteristic of CB/05, indicates the need to identify new genetic markers for pantropic variants of CCoV, probably in the spike-protein gene region.

Distinct proteinase K-resistant prion protein fragment in goats with no signs of disease in a classical scrapie outbreak.

Considerable efforts have been directed toward the identification of small-ruminant prion diseases, i.e., classical and atypical scrapie as well as bovine spongiform encephalopathy (BSE). Here we report the in-depth molecular analysis of the proteinase K-resistant prion protein core fragment (PrP(res)) in a highly scrapie-affected goat flock in Greece. The PrP(res) profile by Western immunoblotting in most animals was that of classical scrapie in sheep. However, in a series of clinically healthy goats we identified a unique C- and N-terminally truncated PrP(res) fragment, which is akin but not identical to that observed for atypical scrapie. These findings reveal novel aspects of the nature and diversity of the molecular PrP(res) phenotypes in goats and suggest that these animals display a previously unrecognized prion protein disorder.

Evolution and phylogenetic analysis of full-length VP3 genes of Eastern Mediterranean bluetongue virus isolates.

Bluetongue virus (BTV) is the 'type' species of the genus Orbivirus within the family Reoviridae. The BTV genome is composed of ten linear segments of double-stranded RNA (dsRNA), each of which codes for one of ten distinct viral proteins. Previous phylogenetic comparisons have evaluated variations in genome segment 3 (Seg-3) nucleotide sequence as way to identify the geographical origin (different topotypes) of BTV isolates. The full-length nucleotide sequence of genome Seg-3 was determined for thirty BTV isolates recovered in the eastern Mediterranean region, the Balkans and other geographic areas (Spain, India, Malaysia and Africa). These data were compared, based on molecular variability, positive-selection-analysis and maximum-likelihood phylogenetic reconstructions (using appropriate substitution models) to 24 previously published sequences, revealing their evolutionary relationships. These analyses indicate that negative selection is a major force in the evolution of BTV, restricting nucleotide variability, reducing the evolutionary rate of Seg-3 and potentially of other regions of the BTV genome. Phylogenetic analysis of the BTV-4 strains isolated over a relatively long time interval (1979-2000), in a single geographic area (Greece), showed a low level of nucleotide diversity, indicating that the virus can circulate almost unchanged for many years. These analyses also show that the recent incursions into south-eastern Europe were caused by BTV strains belonging to two different major-lineages: representing an 'eastern' (BTV-9, -16 and -1) and a 'western' (BTV-4) group/topotype. Epidemiological and phylogenetic analyses indicate that these viruses originated from a geographic area to the east and southeast of Greece (including Cyprus and the Middle East), which appears to represent an important ecological niche for the virus that is likely to represent a continuing source of future BTV incursions into Europe.

Molecular analysis of the NS3/NS3A gene of Bluetongue virus isolates from the 1979 and 1998-2001 epizootics in Greece and their segregation into two distinct groups.

The sequence of the genome segment 10 (Seg-10) encoding NS3/NS3A was determined for 19 field isolates of Bluetongue virus (BTV) of serotypes BTV-1, BTV-4, BTV-9 and BTV-16, derived from epizootics in Greece in the years 1979 and 1998-2001. The aim of the study was to define the molecular epidemiology of the virus in this part of the Mediterranean basin. On the basis of the Seg-10 sequences, the isolates grouped into two distinct phylogenetic clusters. These were Greek group I of solely serotype BTV-4 viruses, and Greek group II of serotypes BTV-1, BTV-9 and BTV-16 viruses. The isolates in Greek group I clustered with the Corsican and Tunisian BTV-2 serotypes and US group II strains of BTV-10 and BTV-13 serotypes, while those in Greek group II with Chinese, Indian and Australian viruses of different serotypes suggesting that viruses derived from two distinct ecosystems have caused BT incursions in Greece over the last 25 years. The NS3/NS3A sequences of most of the BTV-4 isolates were identical, irrespective of the year of isolation, geographical location and host species or tissue origin. Maximum of 15-16% nucleic acid sequence variation, but only 4% deduced amino acid substitution, were observed between groups I and II. Furthermore, the clustering of the NS3/NS3A sequences was independent of the viral serotype, indicating the occurrence of genome segment reassortment during the course of evolution of the viruses.

Molecular analysis of the proviral DNA of equine infectious anemia virus in mules in Greece.

Molecular analysis of the regulatory and structurally important genetic segments of equine infectious anemia virus (EIAV) in mules is presented. We have previously reported clinicopathological and laboratory findings in mules infected with EIAV, both naturally and after experimental inoculation. In this study the fragment coding for integrase, gp90, tat and the fusion domain of gp45 of the proviral genome from these animals was sequenced and compared with one another and with that of EIAV strains already published in the literature. Significant variations were observed mainly in the sequences of the gp90 surface protein. In the two wild type sequences, there were substitutions in the V5 hypervariable domain of this protein. In the sequences of the experimentally inoculated animals and the donor strain, variations were due to insertions/duplications in the V3 principal neutralizing domain (PND) and substitutions in the V5 hypervariable domain. Finally, when compared with the already published strains, the wild type sequences had single amino acid substitutions across the whole protein and multiple substitutions in the V4-V6 variable domains. In general, the two Greek wild type sequences were closer to two of the American strains (WSU5 and Massachusetts), than to the two Japanese (V26 and V70) or the third American strain (Wyoming_wi) used in this study.

Transmission and pathogenicity of encephalomyocarditis virus (EMCV) among rats.

Due to the probable role played by rodents as a reservoir for the transmission of the EMC virus to pigs, the experiment reported here was performed in order to assess the transmission rate of EMCV within a rat population. Twenty-five eight-week-old Wistar rats housed in individual plastic cages were experimentally infected either with a Greek myocardial EMCV strain (5 rats with a 0.2 x 10(6) TCID50 dose per rat and 10 rats with a 0.5 x 10(4.5) TCID50 dose per rat, oronasally) or a Belgian myocardial EMCV strain (10 rats with a 0.5 x 10(4.5) TCID50 dose per rat, oronasally). Two to five days later, each inoculated rat was moved to a new clean cage and coupled with a contact rat to compare the pathogenicity of the two strains and to estimate the basic reproduction ratio R0, indicating the level of EMCV transmission. During the experiments, faecal virus excretion was measured as well as the serological response against EMCV. After euthanasia, virus isolation was attempted from different rat tissues. Neither strains produced mortality, nor clinical signs and only low titres of neutralising antibodies were found. All contact rats, however, were infected and the virus was isolated from their faeces and from various tissues. Both 10-pair experiments revealed a point estimate for the R0 of infinity (95%-CI for both the Greek and Belgian EMCV strains = 4.48 - infinity), as did the 5-pair experiment with a higher dose of the Greek strain (95%-CI = 1.83 - infinity). Combining the results from the two 10-pair experiments resulted in an estimate for R0 of infinity (95%-CI: 9.87 - infinity). These results indicate that the EMC virus can spread very easily within a rat population by horizontal rat-to-rat transmission (R0 >> 1).