Serafeim C. Chaintoutis

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.

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.

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.

Lumpy skin disease outbreaks in Greece during 2015–16, implementation of emergency immunization and genetic differentiation between field isolates and vaccine virus strains

The objective of this study is to present epizootiological data from the lumpy skin disease (LSD) outbreaks in Greece during 2015-16, following the implementation of emergency vaccination and total stamping-out, along with laboratory data regarding the genetic differentiation between field isolates and live attenuated vaccine virus strains. Descriptive geographical chronology analysis was conducted to present the progressive shift of the outbreaks westwards, and at the same time, the absence of further outbreaks in previously affected regional units where high vaccination coverage was achieved. Isolation and molecular characterization of LSDV from the first recorded case in Greece (Evros/GR/15 isolate) was performed. The two live attenuated LSD vaccine viruses, currently used for emergency immunization in Greece, were sequenced and compared to the Evros/GR/15 isolate, in 3 genomic regions (GPCR gene, RPO30 gene, and partial LSDV126/LSDV127 genes). Sequence comparisons revealed prominent differences between the Evros/GR/15 isolate and the vaccine strains. Phylogenetic analysis resulted in the classification of the Evros/GR/15 isolate in the same clade with all field LSDV isolates, whereas vaccine strains were grouped in a distinct cluster within the LSDV clade. Additional samples from animals presenting skin nodules (N=13) were characterized by sequencing in the 3 aforementioned genomic regions. Among them, in 5 animals that were vaccinated, the attenuated vaccine virus was identified. A PCR-RFLP method targeting the LSDV127 gene was developed and proved to be able to discriminate between the characterized field and vaccine strains. The findings of the present study substantiate the importance of timely and intensive vaccinations for the control of LSDV epizootic and the genetic differences between the Evros/GR/15 isolate and the vaccine strains. This provides the basis for the development of PCR-based DIVA assays, which would be of major importance for effective disease surveillance and stamping-out during LSD vaccination campaigns.

A Novel Pan-Flavivirus Detection and Identification Assay Based on RT-qPCR and Microarray

The genus Flavivirus includes arthropod-borne viruses responsible for a large number of infections in humans and economically important animals. While RT-PCR protocols for specific detection of most Flavivirus species are available, there has been also a demand for a broad-range Flavivirus assay covering all members of the genus. It is particularly challenging to balance specificity at genus level with equal sensitivity towards each target species. In the present study, a novel assay combining a SYBR Green-based RT-qPCR with a low-density DNA microarray has been developed. Validation experiments confirmed that the RT-qPCR exhibited roughly equal sensitivity of detection and quantification for all flaviviruses tested. These PCR products are subjected to hybridization on a microarray carrying 84 different oligonucleotide probes that represent all known Flavivirus species. This assay has been used as a screening and confirmation tool for Flavivirus presence in laboratory and field samples, and it performed successfully in international External Quality Assessment of NAT studies. Twenty-six Flavivirus strains were tested with the assay, showing equivalent or superior characteristics compared with the original or even with species-specific RT-PCRs. As an example, test results on West Nile virus detection in a panel of 340 mosquito pool samples from Greece are presented.

Development and validation of a TaqMan probe-based real-time PCR method for the differentiation of wild type lumpy skin disease virus from vaccine virus strains

Lumpy skin disease (LSD) is a transboundary viral disease of cattle with severe economic impact. Immunization of cattle with homologous live attenuated vaccines poses a number of diagnostic problems, as it has been associated with adverse reactions resembling disease symptoms. The latter hampers clinical diagnosis and poses challenges in virus identification. To this end, a duplex quantitative real-time PCR method targeting the GPCR gene was developed and validated, for the concurrent detection and differentiation of wild type and vaccine Lumpy skin disease virus (LSDV) strains. The method was evaluated in three laboratories. The evaluation included a panel of 38 poxvirus isolates/strains and the analytical characteristics of the method were determined. Amplification efficiencies were 91.3% and 90.7%, for wild type and vaccine LSDV, respectively; the limit of detection was 8 DNA copies for both targets and the inter-assay CV was 0.30% for wild type and 0.73% for vaccine LSDV. The diagnostic performance was assessed using 163 LSDV-positive samples, including field specimens and samples from experimentally vaccinated/infected animals. The method is able to confirm diagnosis in suspect cases, it differentiates infected from vaccinated animals (DIVA) and can be regarded as an important tool for effective LSD surveillance and eradication during vaccination campaigns.

Surveillance and Early Warning of West Nile Virus Lineage 2 Using Backyard Chickens and Correlation to Human Neuroinvasive Cases

In 2010, a West Nile virus (WNV) epidemic was reported in Central Macedonia, Northern Greece, with 197 neuroinvasive disease (WNND) cases in humans. The following 3 years, WNV spreads to new areas of Greece and human cases reoccurred during the transmission periods. After the initial outbreak, a WNV surveillance system using juvenile backyard chickens was established in Central Macedonia (after the 2011 outbreak) and Eastern Macedonia‐Thrace (after the 2012 outbreak). Sera were screened for the presence of antibodies against WNV using cELISA and serum neutralization test, to monitor the spread of WNV and to assess the correlation between the WNV point seroprevalence in chickens and the incidence rates of human WNND cases in the aforementioned areas. WNV seroprevalence in chickens was 10.4% (95% CI: 7–15) in Central Macedonia (2011) and 18.1% (95% CI: 14–23) in Eastern Macedonia‐Thrace (2012). Seroprevalence in chickens and incidence rates of human WNND cases in Eastern Macedonia‐Thrace were strongly positively correlated (ρ = 0.98, P = 0.005) at the regional unit level, with the incidence of WNND in humans increasing with increasing WNV point seroprevalence in chickens. In Central Macedonia, the correlation was weaker (ρ = 0.68, P = 0.20), apparently due to small number of reported human WNND cases. Another study was also conducted using juvenile backyard chickens in Central Macedonia, aiming to detect early WNV enzootic circulation, before the onset of human cases during 2011 and 2013. The first seroconverted chickens were detected about 1.5 months before the laboratory diagnosis of any human WNND cases in Central Macedonia, for both years. WNV surveillance, using juvenile backyard chickens, was reliable for the identification of areas with WNV enzootic and silent transmission, and for early warning. Timely diffusion of information to public health authorities facilitated the successful implementation of preparedness plans to protect public health.

Serological monitoring of backyard chickens in Central Macedonia-Greece can detect low transmission of West Nile virus in the absence of human neuroinvasive disease cases.

During 2010-13, West Nile virus (WNV) epidemics occurred in Greece with high numbers of human cases. In parallel, WNV serological surveillance utilizing domestic birds was applied mainly in Central Macedonia, as well as in other areas of the country, and allowed efficient detection of WNV activity during this period. The objective of the study was to evaluate the sensitivity of chicken-based WNV surveillance in periods of low-level virus transmission (2014-15) in a well-studied area, i.e. the epicenter of the 2010 WNV epidemic (Central Macedonia), which is considered endemic since then. WNV activity was monitored via determination of antiviral immune responses in juvenile backyard chickens. The birds were sampled twice per transmission period. WNV-specific antibodies were detected by ELISA in 2.8% out of 255 chickens sampled early in the 2014 transmission period (95% CI: 1-6%). Continued virus transmission was detected at the end of the period, as 4.2% out of 240 sampled chickens seroconverted to WNV (95% CI: 2-8%). Although 14 human neuroinvasive cases occurred in Greece during 2014, no such cases were reported in the study area. During the 2015 early warning period, antibodies against WNV were not detected in sampled chickens (n=250, 95% CI: 0-2%). However, humoral immune responses were detected in 6 out of 240 chicken sampled at the end of the transmission period (2.5%; 95% CI: 1-6%), indicating continued WNV activity. No human cases were reported in Greece during 2015. All samples were negative with real-time RT-PCR. Serological surveillance of chickens resulted in identification of areas with low WNV activity levels during 2014-15, and provided indications of its overwintering in Central Macedonia. The findings suggest that surveillance based on serological testing of domestic birds is sensitive and able to detect low-level of WNV enzootic transmission, in the absence of human cases.

Development of one-tube real-time qRT-PCR and evaluation of RNA extraction methods for the detection of Eggplant mottled dwarf virus in different species

A one-tube real-time qRT-PCR assay was developed, for the detection and quantification of Eggplant mottled dwarf virus (EMDV), a pathogen affecting cultivated and ornamental plants. The amplification efficiency of the assay was 98% and the linear range of quantification was from 20 to 2×10(8) RNA transcripts. Total RNA extraction methods (three developed methods and one commercially available RNA extraction kit) were evaluated using tissues from seven different plant species and synthetic EMDV RNA transcripts of known concentration. The recovery rates of RNA and the effect of co-extracted inhibitors revealed that methods involving PVPP and phenol-chloroform extraction were the most efficient. These modifications were necessary for processing samples containing high phenolic and polysaccharide compounds such as woody plants. The developed EMDV detection protocol was successfully applied in forty naturally infected woody and herbaceous plants belonging to six different species. The protocol comprises a useful method for low-cost detection of ssRNA viruses in diverse plant tissues.

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

ABSTRACT 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.