Application of 16S rRNA next generation sequencing in ticks in Greece.

Tick-borne bacteria pose a significant threat to human and veterinary public health. Greece is a Mediterranean country with rich tick fauna and the most commonly detected tick-borne bacterial pathogens are members of the Rickettsia and Anaplasma species. The variable V2-V4 and V6-V9 regions of 16S rRNA gene of seven ticks belonging to four genera representative in Greece (Ixodes, Rhipicephalus, Dermacentor, Haemophyssalis) were analysed using multiple primer pairs by next generation sequencing (NGS). Nine bacterial phyla corresponding to 95 families, 116 genera and 172 species were identified. Proteobacteria was the predominant phylum in five of the seven ticks, followed by Actinobacteria, which predominated in two ticks. The tick-borne bacteria included Rickettsia and Anaplasma species, while "Candidatus Midichloria mitochondrii" were detected in high abundance in I. ricinus ticks and less in Rhipicephalus bursa; Coxiella-like endosymbionts were detected in Rh. sanguineus, H. parva, and less in Rh. bursa ticks. Co-infections with Rickettsia and Anaplasma were also observed. 16S rRNA NGS is a powerful tool to investigate the tick bacteriome and can improve the strategies for prevention and control of tick-borne diseases.

A PCR-based NGS protocol for whole genome sequencing of West Nile virus lineage 2 directly from biological specimens.

Lineage 2 West Nile virus (WNV) strains have been implicated in severe encephalitis outbreaks in humans and equines residing in Europe. WNV molecular characterization is important for the development of diagnostic assays, as well as for obtaining molecular information, which is necessary for epidemiological investigations of virus transmission in areas at risk. For whole genome sequencing of lineage 2 WNV strains, directly from biological specimens, a PCR-based NGS protocol was developed. The method was applied in WNV-positive specimens obtained from animal, human and mosquito hosts in Greece. The results of its application indicate that, even in cases of low virus titers, the developed PCR-based NGS approach is able to provide whole genome sequences of lineage 2 WNV strains.

Emergence of West Nile virus lineage 2 belonging to the Eastern European subclade, Greece.

West Nile virus (WNV) emerged in Greece in 2010 and since then human outbreaks occurred every year except 2015 and 2016. An early start and prolonged WNV transmission season was observed in 2018 with a record number of 316 reported cases and 47 fatalities. The Greek WNV strains detected during 2010-2018 clustered within the central European subclade of lineage 2. A novel WNV genetic variant was detected in August 2018 in one human case in the north-eastern region of Greece, at the land cross-border with Turkey and Bulgaria. The strain belongs to the Eastern European subclade of lineage 2 suggesting a new virus introduction in the country and the continuously changing epidemiology of the disease.

Crimean-Congo haemorrhagic fever in a Greek worker returning from Bulgaria, June 2018.

We report a tick-borne case of severe Crimean-Congo haemorrhagic fever (CCHF) imported into Greece from Bulgaria. The patient presented severe thrombocytopenia, hemophagocytosis, haemodynamic instability, large haematomas and altered mental status. Supportive treatment and ribavirin were administered. Symptoms started one day after the tick was removed; the patient was discharged from the hospital 26 days after symptom onset. No secondary cases were observed. Phylogenetically the CCHF virus strain belongs to clade Europe 1.

Molecular epidemiology of Dobrava-Belgrade virus in Greece.

In order to gain an insight into the genetic relatedness of the Dobrava-Belgrade virus (DOBV) in Greece, a phylogenetic analysis was performed based on all currently available DOBV sequences obtained from hospitalized cases with hemorrhagic fever with renal syndrome (HFRS). Most cases occurred in northwestern and north central part of the country. Two sequence datasets consisted of 41 S and 12 M partial DOBV RNA segment sequences were analyzed. All DOBV strains belong to Dobrava genotype which is associated with the rodent Apodemus flavicollis. In both phylogenetic trees (S and M segments), two main clusters of Greek strains could be distinguished. Phylogenetic analysis showed a spatial rather than temporal relation of the strains, since their genetic clustering was highly associated with the geographic distribution of the cases. Besides previous characterized endemic foci, novel ones have been identified, expanding our knowledge on the epidemiology of HFRS in Greece.

Uukuniemi virus, Czech Republic.

Following the identification of severe fever with thrombocytopenia syndrome and Heartland viruses, the interest on tick-borne phleboviruses has increased rapidly. Uukuniemi virus has been proposed as a model for tick-borne phleboviruses. However, the number of available sequences is limited. In the current study we performed whole-genome sequencing on two Uukuniemi viral strains isolated in 2000 and 2004 from Ixodes ricinus ticks in the Czech Republic. Both strains cluster together with Potepli63 strain isolated in the country in 1963. Although the Czech strains were isolated many years apart, a high identity was seen at the nucleotide and amino acid levels, suggesting that UUKV has a relatively stable genome.

Isolation and whole-genome sequencing of a Crimean-Congo hemorrhagic fever virus strain, Greece.

Crimean-Congo hemorrhagic fever virus (CCHFV) was isolated from a pool of two adult Rhipicephalus bursa ticks removed from a goat in 2015 in Greece. The strain clusters into lineage Europe 2 representing the second available whole-genome sequenced isolate of this lineage. CCHFV IgG antibodies were detected in 8 of 19 goats of the farm. Currently CCHFV is not associated with disease in mammals other than humans. Studies in animal models are needed to investigate the pathogenicity level of lineage Europe 2 and compare it with that of other lineages.

Acute viral infections of the central nervous system, 2014-2016, Greece.

In order to investigate the viral etiology of acute infections of central nervous system (CNS), multiplex and single PCRs combined with serology for arboviruses were applied on samples from 132 hospitalized patients in Greece during May 2014-December 2016. A viral pathogen was detected in 52 of 132 (39.4%) cases with acute CNS infection. Enteroviruses predominated (15/52, 28.8%), followed by West Nile virus (9/52, 17.3%). Phleboviruses, varicella-zoster virus, and Epstein-Barr virus accounted for 15.4%, 13.5%, and 11.5% of the cases, respectively. The study gives an insight into the etiology of viral CNS infections in a Mediterranean country, where arboviruses should be included in the differential diagnosis of acute CNS infections.

Genomic variants in the FTO gene are associated with sporadic amyotrophic lateral sclerosis in Greek patients.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a devastating disease whose complex pathology has been associated with a strong genetic component in the context of both familial and sporadic disease. Herein, we adopted a next-generation sequencing approach to Greek patients suffering from sporadic ALS (together with their healthy counterparts) in order to explore further the genetic basis of sporadic ALS (sALS). RESULTS: Whole-genome sequencing analysis of Greek sALS patients revealed a positive association between FTO and TBC1D1 gene variants and sALS. Further, linkage disequilibrium analyses were suggestive of a specific disease-associated haplotype for FTO gene variants. Genotyping for these variants was performed in Greek, Sardinian, and Turkish sALS patients. A lack of association between FTO and TBC1D1 variants and sALS in patients of Sardinian and Turkish descent may suggest a founder effect in the Greek population. FTO was found to be highly expressed in motor neurons, while in silico analyses predicted an impact on FTO and TBC1D1 mRNA splicing for the genomic variants in question. CONCLUSIONS: To our knowledge, this is the first study to present a possible association between FTO gene variants and the genetic etiology of sALS. In addition, the next-generation sequencing-based genomics approach coupled with the two-step validation strategy described herein has the potential to be applied to other types of human complex genetic disorders in order to identify variants of clinical significance.

Zika virus infection in a newly married Greek couple.

Zika virus (ZIKV) is a member of the Flaviviridae family causing asymptomatic or mildly symptomatic infections with fever, rash, arthralgia and headache. It is transmitted by the Aedes species mosquitoes and also sexually and transplacentally, and has been recently associated with congenital neurologic birth defects in South and Central America. We report the case of a newly married couple from Greece who travelled to Cuba for their honeymoon and developed mild symptoms consistent with arboviral infection. After returning to Greece, they were found to have been infected by Zika virus during their honeymoon. These are the first two cases of Zika virus infection in Greece, the southeastern border of Europe, denoting that Zika virus infection poses a threat for public health worldwide, since returning travelers could be asymptomatic carriers of the virus, not only leading to risk of neurologic birth defects for their offspring but also the real risk of transmission of the virus in their country by local Aedes mosquitoes.

Challenges in laboratory diagnosis of acute viral central nervous system infections in the era of emerging infectious diseases: the syndromic approach.

INTRODUCTION: Many acute viral infections of the central nervous system (CNS) remain without etiological diagnosis. Specific treatment is available for only few of them; however, accurate diagnosis is essential for patient's life and public health. AREAS COVERED: In the current article, the main parameters playing a role for a successful etiological diagnosis of acute CNS infections are analysed and the syndromic approach based on clinical and demographic data combined with surrogated indicators is discussed. For the development of a relevant test panel, knowledge on the microbes causing CNS infections in a particular geographic region is essential. The modern screening strategies covering a large panel of potential causative agents are described. Examples of the successful application of next generation sequencing in the identification of etiological agents, including novel and emerging viruses, are given. Expert commentary: Knowledge on epidemiology of the viruses, expertise on syndromic grouping of the etiological agents and advances in technology enable the laboratory diagnosis of acute CNS infections, and the rapid identification, containment and mitigation of probable outbreaks.

Insect-specific flaviviruses in Aedes mosquitoes in Greece.

Mosquitoes of the genus Aedes are known vectors of pathogenic flaviviruses, and insect-specific flaviviruses (ISFs) have been detected in members of this genus in numerous parts of the world. In order to gain insight into whether Aedes mosquitoes in Greece are infected by flaviviruses, 1173 Aedes spp. mosquitoes collected in 2010 and 2012 were grouped in 53 pools and tested by RT nested PCR using flavivirus generic primers. Eight pools (15.09 %) were found to be PCR positive: five pools (5/53, 9.4 %) contained RNA sequences related to Ochlerotatus caspius flavivirus (OCFV), an ISF previously detected in the Iberian peninsula, two pools (2/53, 3.8 %) contained sequences related to a mosquito flavivirus detected in Aedes vexans (AeveV) in Italy and the Czech Republic, and one pool contained a DNA sequence that was too short to identify accurately. The highest OCFV prevalence (12.9 %) was observed in August 2010 in the regional unit of Thessaloniki. Similar sequences were later obtained from two Culex spp. pools collected in 2013 in the same regions. A genetic difference of 0.2-1.4 % was seen among the Greek OCFV strains, which differed by 2.2-4.1 % from the Iberian strains and by 6.2-11.1 % from the Finnish Hanko virus. The genetic distances among strains varied depending on the genome region (genes for E, NS3 and NS5 proteins), with NS3 being the most variable. The present study shows no evidence of infection of Aedes mosquitoes with known pathogenic flaviviruses, but it expands the geographic distribution of OCFV in the eastern Mediterranean area. Any implication of ISFs for public health (either directly or through interactions with other flaviviruses in the mosquitoes) remains to be elucidated.

Imported Chikungunya fever case in Greece in June 2014 and public health response.

We report about the first imported case of Chikungunya fever in Greece in a Greek traveler returning from the Dominican Republic and the associated public health response. We investigated the case and performed focused epidemiological and entomological investigation in all areas the patient visited during the infectious period, to identify the targeted interventions needed. Entomological investigation revealed the occurrence of the competent vector Aedes albopictus (Diptera: Culicidae) in the environment surrounding the hospital where the patient was admitted and in her workplace. All captured mosquitoes tested negative for Chikungunya virus. We further conducted clinical and laboratory examination of the patient's co-travelers, gave advice on appropriate personal preventive measures against mosquito bites to the patient and co-travelers and on vector control, and raised awareness among health professionals throughout Greece. The risk of introduction and local transmission of Chikungunya and other arboviruses in Greece and other European countries is present, as the competent vector exists in many parts of Europe. Public health professionals, travel medicine specialists and clinicians should maintain awareness regarding this possibility of importation of arbovirus cases in order to provide the appropriate advice, seek the prompt diagnosis, and implement appropriate interventions. Mobilization of various stakeholders will lead to enhanced epidemiological and entomological surveillance that will allow for improved risk assessment in each area.

Molecular epidemiology of Crimean-Congo hemorrhagic fever in Bulgaria--An update.

Crimean-Congo hemorrhagic fever (CCHF) is endemic in Bulgaria. During 2013-2014, 11 confirmed CCHF cases have been reported in the country (seven in 2013 and four in 2014). The present study provides the CCHF molecular epidemiology in Bulgaria based on all currently available S, M, and L RNA segment nucleotide sequences spanning the years 1978-2014. A relatively low genetic difference (0-6%, the maximum seen in the M RNA segment) was seen among the CCHFV sequences suggesting that a slow evolving CCHFV strain belonging to "Europe 1" clade is present in Bulgaria. Although the virus emerged in new foci during the recent years, it is more active in the established endemic foci which seem to offer the most suitable ecosystem and environment. Understanding the CCHF epidemiology and virus evolution is the basis for public health programs and vaccine design.

Detection of West Nile virus and insect-specific flavivirus RNA in Culex mosquitoes, central Macedonia, Greece.

BACKGROUND: West Nile virus (WNV) emerged in central Macedonia, northern Greece, in 2010. The aim of the study was to test Culex mosquitoes for genetic detection of WNV and other flaviviruses, and to check whether the detection of WNV in mosquitoes was correlated with the occurrence of human WNV cases. METHODS: During 2013, 25 780 Culex spp. mosquitoes were collected from central Macedonia, grouped into 295 pools, according to collection site and date, and tested for the presence of flavivirus RNA. RESULTS: Thirteen (4.4%) pools were flavivirus-positive. WNV lineage 2 was detected in nine (3.1%) pools, and insect-specific flaviviruses (ISFV) in four (1.4%). Three ISFV nucleotide sequences were 98% identical to Culex theileri flavivirus, previously detected in the Iberian peninsula, while the fourth sequence differed by >25% from all known flaviviruses. In most units of central Macedonia WNV detection in mosquitoes preceded the occurrence of human cases by approximately one month. CONCLUSIONS: Detection of WNV lineage 2 in mosquitoes for a fourth consecutive year suggests that the virus is established in Greece. Entomological surveillance provides an early warning system for the circulation of the virus. The detection of Culex theileri flavivirus extends the known range of this virus.

Detection of West Nile virus lineage 2 in the urine of acute human infections.

West Nile virus (WNV) lineage 2 emerged in Greece in 2010 and since then outbreaks in humans have been reported for four consecutive years. Laboratory diagnosis is based mainly on serology. A real-time RT-PCR was applied on urine samples obtained from 35 patients with acute WNV infection. WNV RNA was detected in 40% of the samples with cycle threshold (CT) values ranging from 26.95 to 39.89 (mean 33.11). WNV was isolated from two of four urine samples with low CT (<30). Viral load was not associated with patients' age, sex, day of illness, presence of WNV antibodies, and neurological symptoms. However, it seems that sample shipment and storage conditions are very important for virus detection and isolation. The usefulness of the WNV RNA detection in urine as a diagnostic tool of acute WNV infections is discussed.

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 of West Nile virus lineage 2 in Culex mosquitoes, Greece, 2012.

To gain insight into the strain(s) causing human cases of West Nile virus (WNV) infection in Greece in 2012, mosquitoes were collected and tested for probable WNV infection. WNV lineage 2 sequences were obtained from two of 14 Culex spp. pools collected in Xanthi Prefecture, and especially in the municipality with the highest incidence in Greece. As in the 2 previous years, the strain contains the H249P substitution in the NS3 protein. It seems that this specific strain is fully established in Greece and causes large-scale outbreaks. Thus, prevention and control measures have to be taken, while enhanced surveillance is needed.