Virus RNA Load in Patients with Tick-Borne Encephalitis, Slovenia.

We determined levels of tick-borne encephalitis (TBE) virus (TBEV) RNA in serum samples obtained from 80 patients during the initial phase of TBE in Slovenia. For most samples, levels were within the range of 3-6 log10 copies RNA/mL. Levels were higher in female patients than in male patients, but we found no association between virus load and several laboratory and clinical parameters, including severity of TBE. However, a weak humoral immune response was associated with a more severe disease course, suggesting that inefficient clearance of virus results in a more serious illness. To determine whether a certain genetic lineage of TBEV had a higher virulence potential, we obtained 56 partial envelope protein gene sequences by directly sequencing reverse transcription PCR products from clinical samples of patients. This method provided a large set of patient-derived TBEV sequences. We observed no association between phylogenetic clades and virus load or disease severity.

Fiber-optic immunosensor for detection of Crimean-Congo hemorrhagic fever IgG antibodies in patients.

Crimean-Congo hemorrhagic fever (CCHF) is a severe viral disease with high fatality rate. CCHF virus is endemic in parts of Africa, Asia, the Middle East, and southeastern Europe. Rapid diagnostics of CCHF is vital for appropriate clinical management and prevention of secondary spread from human-to-human. Currently, diagnostics relies on real-time RT-PCR and antibody or antigen detection using ELISA. These methods require trained personnel and expensive equipment and are not appropriate for point-of-care (POC) diagnostics. Furthermore, there are no POC assays available for CCHF. We developed a fiber-optic biosensor for the detection of CCHF IgG antibodies. In order to improve sensitivity, we optimized both the bioreceptor immobilization protocol and the chemiluminescence substrate formulation. The resulting protocol showed a 100-fold greater sensitivity for detection of CCHF antibodies. Finally, we evaluated the fiber-optic biosensor with two CCHF patient sera. We showed that the fiber-optic biosensor is 10-times more sensitive than colorimetric ELISA and is able to detect both patients with high and low levels of IgG antibodies. We believe that the fiber-optic biosensor is a suitable alternative to ELISA as it is much more sensitive and makes it possible to detect a small amount of antibodies at an early stage of infection and can be integrated as a point-of-care diagnostic system of CCHF.

Crimean-Congo hemorrhagic fever virus nucleoprotein suppresses IFN-beta-promoter-mediated gene expression.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a member of the family Bunyaviridae and is a causative agent of severe hemorrhagic disease. Knowledge regarding the pathogenesis of CCHFV is limited due to the requirement for high-containment laboratories and the lack of an immunocompetent animal host. Previous studies have shown that CCHFV delays the activation of the human innate immune response, specifically, the type I interferon response. Our study results show that antagonism of the interferon-beta promoter is mediated by the nucleoprotein of CCHFV strain Hoti, while strains IbAr10200 and AP92 do not suppress the activity of the IFN-beta promoter. Our results also suggest that several viral factors may provide antagonistic action against the type I interferon response.

HMGB1 Is a Potential Biomarker for Severe Viral Hemorrhagic Fevers.

Hemorrhagic fever with renal syndrome (HFRS) and Crimean-Congo hemorrhagic fever (CCHF) are common representatives of viral hemorrhagic fevers still often neglected in some parts of the world. Infection with Dobrava or Puumala virus (HFRS) and Crimean-Congo hemorrhagic fever virus (CCHFV) can result in a mild, nonspecific febrile illness or as a severe disease with hemorrhaging and high fatality rate. An important factor in optimizing survival rate in patients with VHF is instant recognition of the severe form of the disease for which significant biomarkers need to be elucidated. To determine the prognostic value of High Mobility Group Box 1 (HMGB1) as a biomarker for disease severity, we tested acute serum samples of patients with HFRS or CCHF. Our results showed that HMGB1 levels are increased in patients with CCHFV, DOBV or PUUV infection. Above that, concentration of HMGB1 is higher in patients with severe disease progression when compared to the mild clinical course of the disease. Our results indicate that HMGB1 could be a useful prognostic biomarker for disease severity in PUUV and CCHFV infection, where the difference between the mild and severe patients group was highly significant. Even in patients with severe DOBV infection concentrations of HMGB1 were 2.8-times higher than in the mild group, but the difference was not statistically significant. Our results indicated HMGB1 as a potential biomarker for severe hemorrhagic fevers.

Molecular epidemiology of Crimean-Congo hemorrhagic fever virus in Kosovo.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a zoonotic agent that causes severe, life-threatening disease, with a case fatality rate of 10-50%. It is the most widespread tick-borne virus in the world, with cases reported in Africa, Asia and Eastern Europe. CCHFV is a genetically diverse virus. Its genetic diversity is often correlated to its geographical origin. Genetic variability of CCHFV was determined within few endemic areas, however limited data is available for Kosovo. Furthermore, there is little information about the spatiotemporal genetic changes of CCHFV in endemic areas. Kosovo is an important endemic area for CCHFV. Cases were reported each year and the case-fatality rate is significantly higher compared to nearby regions. In this study, we wanted to examine the genetic variability of CCHFV obtained directly from CCHF-confirmed patients, hospitalized in Kosovo from 1991 to 2013. We sequenced partial S segment CCHFV nucleotide sequences from 89 patients. Our results show that several viral variants are present in Kosovo and that the genetic diversity is high in relation to the studied area. We also show that variants are mostly uniformly distributed throughout Kosovo and that limited evolutionary changes have occurred in 22 years. Our results also suggest the presence of a new distinct lineage within the European CCHF phylogenetic clade. Our study provide the largest number of CCHFV nucleotide sequences from patients in 22 year span in one endemic area.

Prevalence of Crimean-Congo hemorrhagic fever virus in healthy population, livestock and ticks in Kosovo.

Crimean-Congo hemorrhagic fever (CCHF) is an acute, tick borne disease often associated with hemorrhagic presentations and high case fatality rate. Kosovo is a highly endemic area for CCHF, with a significant case fatality rate. The aim of our study was to determine the prevalence of CCHF in Kosovo. We tested 1105 serum samples from healthy population in both endemic and non-endemic areas in the country. Our results revealed a seroprevalence of 4.0% (range 0-9.3%) which is comparable to the seroprevalence in other countries. We show that seroprevalence is correlated to the disease incidence in each studied municipality. We also tested 401 animal sera (353 cow, 30 sheep, 10 goat and 8 chicken) in four endemic municipalities in Kosovo. We detected specific antibodies in all animals except in chicken. Seroprevalence in cows is comparable to other endemic areas and correlates to the seroprevalence in humans. No CCHF RNA could be detected in 105 tick samples obtained in 2012 and 2013. Sequencing of CCHFV positive ticks from 2001 revealed that the virus is most closely related to viral strains that were detected in CCHF patients from Kosovo. Results suggest that mild CCHF cases are most probably underdiagnosed and consequently that the burden of disease is higher than reported. Our study provides key information for CCHF surveillance and raises awareness for possible imported cases in CCHF non-endemic countries.

Molecular evidence and high genetic diversity of shrew-borne Seewis virus in Slovenia.

Seewis virus, the shrew-borne hantavirus from Sorex araneus, has been molecularly detected in reservoir hosts in many different central European countries and Russia. Slovenia is a known endemic country for rodent-borne hantaviruses, therefore the aim of the study was to investigate the presence of shrew-borne hantaviruses in insectivores. Viral L, S and M segment have been recovered only from tissue samples of 7 S. araneus, despite several shrew species were tested. Phylogenetic analysis showed high genetic diversity of SWSV in Slovenia, ranging from 3 to 19.4% for different viral segments. The most divergent were M segment sequences, with 19.4% nucleotide divergence among Slovenian strains. Above that, different SWSV strains from Slovenia do not group into separate geographic clusters. While three separate genetic clades were determined, two of them were simultaneously present in one location at the same time.

Phylogeographic diversity of pathogenic and non-pathogenic hantaviruses in slovenia.

Slovenia is a very diverse country from a natural geography point of view, with many different habitats within a relatively small area, in addition to major geological and climatic differences. It is therefore not surprising that several small mammal species have been confirmed to harbour hantaviruses: A. flavicollis (Dobrava virus), A. agrarius (Dobrava virus-Kurkino), M. glareolus (Puumala virus), S. areanus (Seewis virus),M. agrestis, M. arvalis and M. subterraneus (Tula virus). Three of the viruses, namely the Dobrava, Dobrava-Kurkino and Puumala viruses, cause disease in humans, with significant differences in the severity of symptoms. Due to changes in haemorrhagic fever with renal syndrome cases (HFRS) epidemiology, a detailed study on phylogenetic diversity and molecular epidemiology of pathogenic and non-pathogenic hantaviruses circulating in ecologically diverse endemic regions was performed. The study presents one of the largest collections of hantavirus L, M and S sequences obtained from hosts and patients within a single country. Several genetic lineages were determined for each hantavirus species, with higher diversity among non-pathogenic compared to pathogenic viruses. For pathogenic hantaviruses, a significant geographic clustering of human- and rodent-derived sequences was confirmed. Several geographic and ecological factors were recognized as influencing and limiting the formation of endemic areas.

Phylogeographic characterization of tick-borne encephalitis virus from patients, rodents and ticks in Slovenia.

Tick-borne encephalitis virus (TBEV) is the most important arboviral agent causing infections of the central nervous system in central Europe. Previous studies have shown that TBEV exhibits pronounced genetic variability, which is often correlated to the geographical origin of TBEV. Genetic variability of TBEV has previously been studied predominantly in rodents and ticks, while information about the variability in patients is scarce. In order to understand the molecular relationships of TBEV between natural hosts, vectors and humans, as well as correlation between phylogenetic and geographical clustering, sequences of TBEV E and NS5 protein genes, were obtained by direct sequencing of RT-PCR products from TBE-confirmed patients as well as from rodents and ticks collected from TBE-endemic regions in Slovenia. A total of 27 partial E protein gene sequences representing 15 human, 4 rodent and 8 tick samples and 30 partial NS5 protein gene sequences representing 17 human, 5 rodent and 8 tick samples were obtained. The complete genome sequence of TBEV strain Ljubljana I was simultaneously obtained. Phylogenetic analysis of the E and NS5 protein gene sequences revealed a high degree of TBEV variability in patients, ticks and rodents. Furthermore, an evident correlation between geographical and phylogenetic clustering was shown that was independent of the TBEV host. Moreover, we show the presence of a possible recombination event in the TBEV genome obtained from a patient sample, which was supported with multiple recombination event detection methods. This is the first study that simultaneously analyzed the genetic relationships of directly sequenced TBEV samples from patients, ticks and rodents and provides the largest set of patient-derived TBEV sequences up to date. In addition, we have confirmed the geographical clustering of TBEV sequences in Slovenia and have provided evidence of a possible recombination event in the TBEV genome, obtained from a patient.

Evidence of an autochthonous Toscana virus strain in Croatia.

BACKGROUND: Phleboviruses are large and widespread group of viruses that are transmitted by arthropods and they have been reported to circulate in endemic regions of Mediterranean Basin, including Croatia. OBJECTIVES: To investigate the role of Toscana virus, as a cause of the aseptic meningitis, in summer months in Croatia. STUDY DESIGN: Samples from 30 patients with aseptic meningitis were retrospectively tested by serology and RT-PCR for TOSV. RESULTS: TOSV RNA was detected in 2/30 and TOSV IgM antibodies were found in 4/30 of patients. Phylogenetic analysis of partial L and S segments suggests that TOSV from Croatia represents an autochthonous strain. CONCLUSIONS: The study has confirmed the role of TOSV as an agent that causes aseptic meningitis in Croatia, therefore it should be considered by physicians when encountering meningitis or febrile illness among indigenous population or travellers during the summer months.