Maria Papanastassopoulou

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.

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.

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.

Assessment of bluetongue viraemia in sheep by real-time PCR and correlation with viral infectivity.

Inoculation of embryonated chicken eggs is the standard method for the titration of infectious Bluetongue virus (BTV). Here, six RNA extraction methods coupled with optimised dsRNA denaturation and real-time RT-PCR were evaluated for the quantitation of BTV in blood samples from experimentally infected sheep and results were correlated to infectious virus titres. An exogenous dsRNA internal control (IC) from the closely related Epizootic hemorrhagic disease virus (EHDV) was used to assess the efficiency of BTV genome extraction, dsRNA denaturation, RT, and PCR amplification. Recovery rates of IC and BTV dsRNA copies from extracted blood samples were highly correlated. Adjustment of BTV concentrations according to the IC recovery reduced variation in sample analyses among the different extraction methods and improved the accuracy of BTV quantitation. The EID(50)/ml titre, determined in blood samples from sheep infected experimentally with BTV-1 or BTV-9, correlated highly with the assessed concentration of BTV dsRNA copies. However, this correlation was consistent only during the first 28 days post-infection. The optimised extraction methods and quantitative RT-PCR could be useful for experimental studies of BTV transmission, pathogenesis and vaccine efficacy, or adapted further for the detection and quantitation of EHDV, African horse sickness virus and other dsRNA viruses.

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.

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.

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.

Isolation, tissue distribution and molecular characterization of two recombinant canine coronavirus strains.

Abstract Canine coronavirus (CCoV) is an enveloped RNA virus, responsible for gastrointestinal infection in dogs. To date, two different CCoV genotypes have been recognized, CCoV type I and CCoV type II. Recently, CCoV type II strains of potential recombinant origin with transmissible gastroenteritis virus (TGEV) were detected and characterized as a new subtype (CCoV-IIb) of canine coronavirus, in order to be differentiated from the “classical” CCoV type II strains (CCoV-IIa). In the present study, two CCoV-IIb strains were detected in the faeces and internal organs of two puppies, which died after presenting gastrointestinal symptoms. Mixed infection of both subtypes (CCoV-IIa/IIb) was detected in the faeces, while only CCoV-IIb was detected in the organs. Puppies were also infected by canine parvovirus type 2 (CPV-2). Both CCoV-IIb strains were isolated on cell cultures and subjected to sequence analysis and phylogeny. By means of RT-PCR and real time RT-PCR assays, tissue distribution and quantitation of viral loads took place. These cases represent the first description of tissue distribution and quantitation of CCoV-IIb strains, detected in the organs. The detection of CCoV-IIa strains, which is restricted to the faeces, suggests that CCoV-IIb strains may have an advantage in disseminating throughout a dog with CPV-2 coinfection, in contrast to common enteric CCoV-IIa strains.

Characterization of the PRNP gene locus in Chios dairy sheep and its association with milk production and reproduction traits

The objective of this study was to examine the prion protein gene locus (PRNP) in Chios sheep. PRNP is linked with scrapie resistance in small ruminants. Here, its impact on milk production (test-day and total lactation yield) and reproduction (age at first lambing, conception rate at first service, and prolificacy) was assessed. Genotyping at codons 136, 154 and 171 (classical scrapie) and 141 (atypical scrapie) was performed using DNA from milk somatic cells and PCR-RFLP analysis. A total of 1013 Chios ewes raised in 23 flocks were used. This constituted a random sample of the national breeding population. A total of 15 genotypes and 6 alleles linked to codons 136, 154 and 171 were detected. All animals were homozygous for the leucine allele at codon 141. Linear mixed models were used to assess the impact of PRNP genotypes and alleles on milk production and reproduction traits. The TRQ allele, whose association with such traits was assessed for the first time, had an adverse effect on age at first lambing. All other PRNP alleles, including ARR, which is associated with increased resistance to classical scrapie, had no significant effect on the traits studied. No significant associations of the PRNP genotypes with production and reproduction traits were observed. It was concluded that selection for scrapie-resistant sheep is not expected to affect the ongoing breeding programme that aims to enhance the milk yield and reproduction of the Chios breed.

Canine coronavirus, Greece. Molecular analysis and genetic diversity characterization

Abstract 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 3months 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.