Microscopic detection and molecular characterization of Sarcocystis miescheriana in wild boars (Sus scrofa): first report from Greece.

The genus Sarcocystis includes protozoan parasites with an indirect life cycle. Sarcocystis spp. can infect various animal species and humans, causing sarcocystosis, a parasitosis of economic importance and zoonotic concern. Wild boars can act as intermediate hosts for Sarcocystis miescheriana and the zoonotic Sarcocystis suihominis that infects humans by consumption of raw or undercooked infected swine meat. In the present study, the diaphragmatic muscle tissue of 123 wild boars hunted in Greece was examined to determine the frequency of Sarcocystis spp. The samples were examined by tissue compression and molecular techniques. Under light microscopy, 34 out of 123 (27.6%) wild boars tested positive for Sarcocystis spp., while a higher infection prevalence (75%) was revealed by multiplex PCR performed in 100 of the samples. The partial mtDNA cox1 gene (~ 1100 bp) of 20 samples tested positive for S. miescheriana by multiplex PCR was amplified and sequenced. Sarcocystis miescheriana was identified as the only species involved in these infections. This is the first study on the prevalence of Sarcocystis spp. in wild animals in Greece. Further, large-scale surveys are needed to assess the prevalence and species of this parasite in Greece and to design efficient control and preventive measures in a One Health perspective.

Outbreaks of SARS-CoV-2 in naturally infected mink farms: Impact, transmission dynamics, genetic patterns, and environmental contamination.

SARS-CoV-2 infection outbreaks in minks have serious implications associated with animal health and welfare, and public health. In two naturally infected mink farms (A and B) located in Greece, we investigated the outbreaks and assessed parameters associated with virus transmission, immunity, pathology, and environmental contamination. Symptoms ranged from anorexia and mild depression to respiratory signs of varying intensity. Although the farms were at different breeding stages, mortality was similarly high (8.4% and 10.0%). The viral strains belonged to lineages B.1.1.218 and B.1.1.305, possessing the mink-specific S-Y453F substitution. Lung histopathology identified necrosis of smooth muscle and connective tissue elements of vascular walls, and vasculitis as the main early key events of the acute SARS-CoV-2-induced broncho-interstitial pneumonia. Molecular investigation in two dead minks indicated a consistently higher (0.3-1.3 log10 RNA copies/g) viral load in organs of the male mink compared to the female. In farm A, the infected farmers were responsible for the significant initial infection of 229 out of 1,000 handled minks, suggesting a very efficient human-to-mink transmission. Subsequent infections across the sheds wherein animals were being housed occurred due to airborne transmission. Based on a R0 of 2.90 and a growth rate equal to 0.293, the generation time was estimated to be 3.6 days, indicative of the massive SARS-CoV-2 dispersal among minks. After the end of the outbreaks, a similar percentage of animals were immune in the two farms (93.0% and 93.3%), preventing further virus transmission whereas, viral RNA was detected in samples collected from shed surfaces and air. Consequently, strict biosecurity is imperative during the occurrence of clinical signs. Environmental viral load monitoring, in conjunction with NGS should be adopted in mink farm surveillance. The minimum proportion of minks that need to be immunized to avoid outbreaks in farms was calculated at 65.5%, which is important for future vaccination campaigns.

A highly sensitive semi-nested real-time PCR utilizing oligospermine-conjugated degenerate primers for the detection of diverse strains of small ruminant lentiviruses.

Small ruminant lentiviruses (SRLVs) are highly diverse retroviruses infecting sheep and goats. Although PCR-based testing is being utilized for diagnostics, its application is hampered by various factors. These include, among others, the exceptionally high genetic variability of SRLVs, as well as the low number of infected blood monocytes. For this reason, a highly sensitive and specific semi-nested real-time PCR for proviral DNA detection and quantification was developed. The method is innovative in that a) its design is based on selecting the preferred codon usage in the targeted conserved genomic regions and b) oligospermine-conjugated degenerate primers with increased Tm were utilized. Modifications permitted primer/template duplex formation in the cases of mismatches due to sporadic nucleotide polymorphisms in a number of variant SRLV strains and consequently, the detection of highly diverse SRLV strains. The potential loss of analytical sensitivity and specificity was counterbalanced by including a semi-nested step in combination with LNA probes. An in silico procedure for the evaluation of hybridization efficiency of the designed oligonucleotides to all known targeted variants was also implemented. The method presents a linear range of quantification over a 3-log10 range and a limit of detection of 3.9 proviral dsDNA copies per reaction. Its diagnostic performance was evaluated by testing field samples from seropositive and seronegative animals, followed by phylogenetic analysis of the strains detected. To further increase the diagnostic sensitivity, a DNA extraction protocol for blood leukocytes was developed and evaluated. A minimum of 500 ng input DNA is recommended for PCR-based detection of SRLV proviral DNA, given the low numbers of infected blood monocytes. The developed methodology may serve as a useful tool, which can be adjusted for the quantitative detection of viruses exhibiting high genetic variability.

Development of real-time PCR-based methods for the detection of enzootic nasal tumor virus 2 in goats.

Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of sheep and goats, associated with the oncogenic retroviruses enzootic nasal tumor virus (ENTV) 1 and 2, respectively. It appears to be common in countries with substantial small ruminant-production. ENA diagnosis in goats is based on autopsy and histopathology, and there is no real-time PCR method available for ENTV-2 detection. Here, a novel one-tube real-time RT-PCR (RT-qPCR) method for the detection and quantification of ENTV-2 in nasal swabs is presented. The method targets the env gene/U3 region. For the design of ENTV-2-specific oligonucleotides, molecular characterization of seven Greek ENTV-2 strains was performed. Phylogenetic analysis revealed three distinct phylogenetic clades of ENTV-2 that correlate with the country of sample collection. Evaluation of the analytical performance of the RT-qPCR revealed an amplification efficiency of 92.8% and a linear range of quantification between 2 × 108 and 2 × 102 RNA transcripts. Analysis of nasal swabs from 23 histopathologically confirmed, naturally occurring ENA cases via RT-qPCR yielded positive results. Moreover, modification of the method for use in a real-time PCR (qPCR) assay enables detection of proviral DNA in tumor specimens. Both methods are highly specific and can be used for the confirmation of ENA-suspected cases. Future applications could include ante-mortem diagnosis, verification of the ENTV-2-free status in animal trade, disease surveillance, and control programs.

Occurrence and molecular characterization of betanodaviruses in fish and invertebrates of the Greek territorial waters.

Betanodaviruses are small ssRNA viruses that cause viral encephalopathy and retinopathy, a severe neuropathological infectious disease in marine fish species worldwide. In the present study, the occurrence of betanodaviruses was investigated in wild and cultured populations of fishes and invertebrates of the Greek territorial waters. Betanodaviruses were detected in 35 species belonging to 21 families and 12 orders. To our knowledge, 23 of those are reported for the first time in Greek waters, while 11 of them are reported for the first time globally. The positive samples were subjected to sequencing and phylogenetic analysis of partial segments of RNA1 and RNA2 genes. Almost all the viruses circulating in Greece fell within RGNNV genotype, while reassortant viruses were detected in three samples, namely two inter-RGNNV and one RGNNV/SJNNV. A novel unclassified Betanodavirus sequence was also identified. Most of the Greek sequence types have a restricted geographic distribution except for two RNA1 and one RNA2 sequence types that are widespread throughout the Mediterranean basin. The results of this study indicate the range of reservoirs/hosts of betanodaviruses and also their wide spread in the Greek territorial waters and reinforce the hypothesis that wild fish species transmit the virus to cultured ones and vice versa.

Comparison of 2016-17 and Previous Epizootics of Highly Pathogenic Avian Influenza H5 Guangdong Lineage in Europe.

We analyzed the highly pathogenic avian influenza (HPAI) H5 epizootic of 2016-17 in Europe by epidemiologic and genetic characteristics and compared it with 2 previous epizootics caused by the same H5 Guangdong lineage. The 2016-17 epizootic was the largest in Europe by number of countries and farms affected and greatest diversity of wild birds infected. We observed significant differences among the 3 epizootics regarding region affected, epidemic curve, seasonality, and outbreak duration, making it difficult to predict future HPAI epizootics. However, we know that in 2005-06 and 2016-17 the initial peak of wild bird detections preceded the peak of poultry outbreaks within Europe. Phylogenetic analysis of 2016-17 viruses indicates 2 main pathways into Europe. Our findings highlight the need for global surveillance of viral changes to inform disease preparedness, detection, and control.

A Combined Approach for the Characterization of Small Ruminant Lentivirus Strains Circulating in the Islands and Mainland of Greece.

Small ruminant lentiviruses are a group of viruses infecting goat and sheep worldwide. These viruses exhibit an extraordinary degree of genetic and antigenic variability that severely influence in vivo and in vitro features, as well as diagnostic test results. Small ruminant farming is the most important animal farming business in Greece, with a high impact on the Greek primary economy. Although SRLV infection and its impact on animal production are well established in the country, little is known about the circulating SRLV strains and their prevalence. The aim of this study was to characterize SRLVs circulating in Greece with a combined serological and molecular approach, using the bulk milk matrix collected from 60 farms in different municipalities. This study allowed us to estimate a seroprevalence of around 52% at the herd level. The B1, B2 and A3 subtypes and a novel A viral cluster were identified. Moreover, the amplicon sequencing method allowed us to identify more than one viral subtype in a sample. These results again confirm the high variability of these viruses and highlight the importance of the constant monitoring of viral evolution, in particular in antigens of diagnostic interest.

Circulation of Pestiviruses in Small Ruminants from Greece: First Molecular Identification of Border Disease Virus.

The incidence of small ruminant pestivirus infections in Greece remains unknown as they have not been diagnosed in the country since 1974 when the most recent Border Disease Virus (BDV) outbreak was reported. The objective of our study was to explore the possible occurrence of pestiviral infections among sheep and goat farms in Greece and to further determine the variants of major concern. Thus, serum samples were collected from 470 randomly selected animals belonging to 28 different flocks/herds. ELISA on p80 antibody revealed the existence of seropositive animals in four out of the 24 studied sheep flocks, whereas all the goats in the four studied herds were seronegative. Viral RNA and antigens were detected in two sheep out of the four seropositive flocks by RT-PCR and ELISA, respectively. Sequencing and phylogenetic analysis showed that the newly identified Greek variants were closely related to the strains of the BDV-4 genotype. One of the BDV-positive sheep demonstrated the diagnostic profile of a persistently infected (PI) animal, providing additional information regarding the source of the infection. This is the first molecular identification of BDV isolates in Greece. Our findings indicate that BDV infections are likely to remain undiagnosed, highlighting the need for further epidemiological studies and active surveillance programs to determine the prevalence and impact of BDV infections on a countrywide level.

SARS-CoV-2 wastewater monitoring using a novel PCR-based method rapidly captured the Delta-to-Omicron ΒΑ.1 transition patterns in the absence of conventional surveillance evidence.

Conventional SARS-CoV-2 surveillance based on genotyping of clinical samples is characterized by challenges related to the available sequencing capacity, population sampling methodologies, and is time, labor, and resource-demanding. Wastewater-based variant surveillance constitutes a valuable supplementary practice, since it does not require extensive sampling, and provides information on virus prevalence in a timely and cost-effective manner. Consequently, we developed a sensitive real-time RT-PCR-based approach that exclusively amplifies and quantifies SARS-CoV-2 genomic regions carrying the S:Δ69/70 deletion, indicative of the Omicron BA.1 variant, in wastewater. The method was incorporated in the analysis of composite daily samples taken from the main Wastewater Treatment Plant of Thessaloniki, Greece, from 1 December 2021. The applicability of the methodology is dependent on the epidemiological situation. During Omicron BA.1 global emergence, Thessaloniki was experiencing a massive epidemic wave attributed solely to the Delta variant, according to genomic surveillance data. Since Delta does not possess the S:Δ69/70, the emergence of Omicron BA.1 could be monitored via the described methodology. Omicron BA.1 was detected in sewage samples on 19 December 2021 and a rapid increase of its viral load was observed in the following 10-day period, with an estimated early doubling time of 1.86 days. The proportion of the total SARS-CoV-2 load attributed to BA.1 reached 91.09 % on 7 January, revealing a fast Delta-to-Omicron transition pattern. The detection of Omicron BA.1 subclade in wastewater preceded the outburst of reported (presumable) Omicron cases in the city by approximately 7 days. The proposed wastewater surveillance approach based on selective PCR amplification of a genomic region carrying a deletion signature enabled rapid, real-time data acquisition on Omicron BA.1 prevalence and dynamics during the slow remission of the Delta wave. Timely provision of these results to State authorities readily influences the decision-making process for targeted public health interventions, including control measures, awareness, and preparedness.

Detecting SARS-CoV-2 lineages and mutational load in municipal wastewater and a use-case in the metropolitan area of Thessaloniki, Greece.

The COVID-19 pandemic represents an unprecedented global crisis necessitating novel approaches for, amongst others, early detection of emerging variants relating to the evolution and spread of the virus. Recently, the detection of SARS-CoV-2 RNA in wastewater has emerged as a useful tool to monitor the prevalence of the virus in the community. Here, we propose a novel methodology, called lineagespot, for the monitoring of mutations and the detection of SARS-CoV-2 lineages in wastewater samples using next-generation sequencing (NGS). Our proposed method was tested and evaluated using NGS data produced by the sequencing of 14 wastewater samples from the municipality of Thessaloniki, Greece, covering a 6-month period. The results showed the presence of SARS-CoV-2 variants in wastewater data. lineagespot was able to record the evolution and rapid domination of the Alpha variant (B.1.1.7) in the community, and allowed the correlation between the mutations evident through our approach and the mutations observed in patients from the same area and time periods. lineagespot is an open-source tool, implemented in R, and is freely available on GitHub and registered on bio.tools.

Investigation of the Food-Transmitted Parasites Trichinella spp. and Alaria spp. in Wild Boars in Greece by Classical and Molecular Methods and Development of a Novel Real-Time PCR for Alaria spp. Detection.

Foodborne parasitic diseases represent a major threat to public health. Trichinellosis, caused by the nematode parasite Trichinella spp., is one of the most important foodborne diseases, while alariosis, caused by the trematode parasite Alaria spp., is less common in humans, and rare cases have been reported only in the USA and Canada. Both parasites can infect humans via the consumption of raw or undercooked wild boar meat. In order to investigate the prevalence of these parasites in wild boar meat in Greece, samples from the diaphragm pillars and the region of the mandibular angle from 128 wild boars, hunted in Greece, were collected. The samples were examined by classical parasitological (compression, artificial digestion, and Alaria spp. migration) and by molecular (real-time PCR) methods. For Trichinella spp. an existent real-time PCR detecting all species likely to be present in Greece was applied, while for Alaria spp. a real-time PCR was developed, employing an LNA TaqMan probe targeting the large subunit ribosomal RNA gene. All examined wild boar samples from Greece resulted negative for Trichinella and Alaria species, indicating a low prevalence of infection in the examined population. The novel real-time PCR for Alaria spp. has 81.5% amplification efficiency and is able to detect 0.12 larvae per 50 g of tissue and could be utilized as a complementary to AMT diagnostic tool in surveillance.

A one-step real-time RT-PCR assay for simultaneous typing of SARS-CoV-2 mutations associated with the E484K and N501Y spike protein amino-acid substitutions.

The emergence of SARS-CoV-2 mutations resulting in the S protein amino-acid substitutions N501Y and E484K, which have been associated with enhanced transmissibility and immune escape, respectively, necessitates immediate actions, for which their rapid identification is crucial. For the simultaneous typing of both of these mutations of concern (MOCs), a one-step real-time RT-PCR assay employing four locked nucleic acid (LNA) modified TaqMan probes was developed. The assay is highly sensitive with a LOD of 117 copies/reaction, amplification efficiencies >94 % and a linear range of over 5 log10 copies/reaction. Validation of the assay using known SARS-CoV-2-positive and negative samples from human and animals revealed its ability to correctly identify wild type strains, and strains possessing either one or both targeted amino-acid substitutions, thus comprising a useful pre-screening tool for rapid MOC identification. The basic principles of the methodology for the development of the assay are explained in order to facilitate the rapid design of similar assays able to detect emerging MOCs.

Design and structural bioinformatic analysis of polypeptide antigens useful for the SRLV serodiagnosis.

Due to their intrinsic genetic, structural and phenotypic variability the Lentiviruses, and specifically small ruminant lentiviruses (SRLV), are considered viral quasispecies with a population structure that consists of extremely large numbers of variant genomes, termed mutant spectra or mutant cloud. Immunoenzymatic tests for SRLVs are available but the dynamic heterogeneity of the virus makes the development of a diagnostic "golden standard" extremely difficult. The ELISA reported in the literature have been obtained using proteins derived from a single strain or they are multi-strain based assay that may increase the sensitivity of the serological diagnosis. Hundreds of SRLV protein sequences derived from different viral strains are deposited in GenBank. The aim of this study is to verify if the database can be exploited with the help of bioinformatics in order to have a more systematic approach in the design of a set of representative protein antigens useful in the SRLV serodiagnosis. Clustering, molecular modelling, molecular dynamics, epitope predictions and aggregative/solubility predictions were the main bioinformatic tools used. This approach led to the design of SRLV antigenic proteins that were expressed by recombinant DNA technology using synthetic genes, analyzed by CD spectroscopy, tested by ELISA and preliminarily compared to currently commercially available detection kits.

A Novel Real-Time RT-PCR-Based Methodology for the Preliminary Typing of SARS-CoV-2 Variants, Employing Non-Extendable LNA Oligonucleotides and Three Signature Mutations at the Spike Protein Receptor-Binding Domain.

Mutations resulting in amino-acid substitutions of the SARS-CoV-2 spike protein receptor-binding domain (RBD) have been associated with enhanced transmissibility and immune escape of the respective variants, namely Alpha, Beta, Gamma or Delta. Rapid identification of the aforementioned variants of concern and their discrimination of other variants is thus of importance for public health interventions. For this reason, a one-step real-time RT-PCR assay employing four locked nucleic acid (LNA) modified TaqMan probes was developed, to target signature mutations associated with amino-acid substitutions at positions 478, 484 and 501 present in the receptor-binding motif (RBM) of the spike protein RBD. This region contains most contacting residues of SARS-CoV-2 that bind to ACE2. A novel strategy employing the use of non-extendable LNA oligonucleotide blockers that can reduce non-specific hybridization of probes increased the number of different mutated sites examined in a multiplex PCR. The combinatory analysis of the different fluorescence signals obtained enabled the preliminary differentiation of SARS-CoV-2 variants of concern. The assay is sensitive with a LOD of 263 copies/reaction for the Delta variant, 170 copies/reaction for the Beta variant, amplification efficiencies > 91% and a linear range of >5 log10 copies/reaction against all targets. Validation of the assay using known SARS-CoV-2-positive and negative samples from humans and animals revealed its ability to correctly identify the targeted mutations and preliminary characterize the SARS-CoV-2 variants. The novel approach for mutation typing using LNA oligonucleotide blockers can be modified to target signature mutations at four different sites in the RBM and further expand the range of variants detected.