Hygiene of Medical Devices and Minimum Inhibitory Concentrations for Alcohol-Based and QAC Disinfectants among Isolates from Physical Therapy Departments.

Disinfectants are used intensively to control and prevent healthcare-associated infections. With continuous use and exposure to disinfectants, bacteria may develop reduced susceptibility. The study aimed to check the hygiene of devices in the physiotherapy department. For isolated bacterial strains, we aimed to determine the minimum inhibitory concentration of five different disinfectant wipe products currently in use. Microbiological environmental sampling in four various institutions in four different cities from two counties was performed, followed by CFU calculation and identification using matrix-assisted laser desorption and ionization with time-of-flight analyzer mass spectrometry (MALDI-TOF). The sampling was performed on three different occasions: before patient use, after patient use, and after disinfection. The susceptibility of isolates to three different alcohol-based and three different quaternary ammonium compounds (QAC) disinfectant wipes was examined by determining the minimal inhibitory concentrations (MIC). We identified 27 different bacterial species from 11 different genera. Gram-positive bacteria predominated. The most abundant genera were Staphylococcus, Micrococcus, and Bacillus. The average MIC values of alcohol-based disinfectants range between 66.61 and 148.82 g/L, and those of QAC-based disinfectants range between 2.4 and 3.5 mg/L. Distinctive strains with four-fold increases in MIC values, compared to average values, were identified. The widespread use of disinfectants can induce a reduction in the susceptibility of bacteria against disinfectants and affect the increase in the proportion of antibiotic-resistant bacteria. Therefore, it is urgent to define clear criteria for defining a microorganism as resistant to disinfectants by setting epidemiological cut-off (ECOFF) values and standardizing protocols for testing the resistance of microorganisms against disinfectants.

Prevalence of sexually transmitted infections with Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis: findings from the National Survey of Sexual Lifestyles, Attitudes and Health, Slovenia, 2016 to 2017.

BackgroundTo inform prevention and control of sexually transmitted infections (STIs), we need reliable prevalence estimates.AimOne objective of the Slovenian National Survey of Sexual Lifestyles, Attitudes and Health was to estimate the prevalence of STIs with Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis.MethodsData were collected between October 2016 and July 2017 in a probability sample of the general population aged 18-49 years. Computer-assisted face-to-face interviewing and self-completion of questionnaires were used. Respondents were invited to provide urine samples to be tested for STIs.ResultsOf 1,929 survey participants, 1,087 individuals provided urine samples which were tested confidentially for C. trachomatis and a subset (n = 1,023) were tested anonymously for the other STIs. The prevalence of C. trachomatis was 0.5% (95% confidence interval (CI): 0.1-1.8) in men and 1.7% (95% CI: 0.9-3.2) in women. Age-specific prevalence was the highest among individuals aged 18-24 years, 2.8% (95% CI: 0.7-10.6) in men and 4.7% (95% CI: 1.7-12.3) in women. N. gonorrhoea was not detected. Prevalence of M. genitalium was 0.5% (95% CI: 0.1-2.2) in men and 0.3% (95% CI: 0.1-1.1) in women; the highest prevalence was among men aged 25-34 years (1.1%; 95% CI: 0.2-7.5) and women aged 35-49 years (0.5%; 95% CI: 0.1-2.0). T. vaginalis was detected in the sample from one woman (0.2%; 95% CI: 0.1-1.2).ConclusionThe substantial prevalence of C. trachomatis among young adults suggests gaps in testing, diagnosis and treatment.

Reduced Susceptibility and Increased Resistance of Bacteria against Disinfectants: A Systematic Review.

Disinfectants are used to reduce the concentration of pathogenic microorganisms to a safe level and help to prevent the transmission of infectious diseases. However, bacteria have a tremendous ability to respond to chemical stress caused by biocides, where overuse and improper use of disinfectants can be reflected in a reduced susceptibility of microorganisms. This review aims to describe whether mutations and thus decreased susceptibility to disinfectants occur in bacteria during disinfectant exposure. A systematic literature review following PRISMA guidelines was conducted with the databases PubMed, Science Direct and Web of Science. For the final analysis, 28 sources that remained of interest were included. Articles describing reduced susceptibility or the resistance of bacteria against seven different disinfectants were identified. The important deviation of the minimum inhibitory concentration was observed in multiple studies for disinfectants based on triclosan and chlorhexidine. A reduced susceptibility to disinfectants and potentially related problems with antibiotic resistance in clinically important bacterial strains are increasing. Since the use of disinfectants in the community is rising, it is clear that reasonable use of available and effective disinfectants is needed. It is necessary to develop and adopt strategies to control disinfectant resistance.

Single mutation in the matrix gene of seasonal influenza A viruses critically affects the performance of diagnostic molecular assay.

Reduced intensity of the fluorescence signal in the amplification curve was observed when using a WHO recommended real time RT-PCR for influenza virus detection. A single mutation, G189T, in the conserved region of influenza virus matrix gene was detected by Sanger sequencing. The mutation is located in the probe binding region, hence we speculated it could be the reason for the atypical shape of amplification curve. The mutation was first noted in Slovenia in 2011 and 2013 for seasonal influenza A virus types A(H1N1)pdm09 and A(H3N2), respectively. In the following years, 2014 and 2015, the majority of influenza A(H3N2) viruses alone carried the mutation. The amplification of matrix gene for these influenza A(H3N2) viruses continuously resulted in the atypically shaped amplification curves. The performance of the particular assay was critically affected; therefore, the assay was no longer usable as diagnostic tool for influenza virus detection. Mutations in the conserved region of influenza virus genome are more common than expected and this would need to be considered when targeting matrix gene.

The impact of illegal waste sites on a transmission of zoonotic viruses.

BACKGROUND: Illegal waste disposal impacts public health and causes aesthetic and environmental pollution. Waste disposed in places without permitted and controlled facilities can provide a ready source of nutrition and shelter for rodents and thus promote the spread of their ecto- and endoparasites. The presence of two distinct zoonotic viruses, lymphocytic choriomeningitis virus (LCMV) and tick-borne encephalitis virus (TBEV), was searched at illegal waste sites. The aim of this study was to determine the prevalence of infection with both viruses in rodents and to discuss the virus-rodent relations in such environments. METHODS: Rodents sampled between October 2011 and April 2013 at 7 locations in the Istrian peninsula, were identified morphologically and genetically to minimize misidentification. Serological and molecular techniques were used to determine seroprevalence of infection in rodents and to detect viral RNAs. Serological testing was performed by immune fluorescence assay for detection of LCMV and TBEV specific antibodies. Real-time RT PCR was used for the detection of LCMV nucleoprotein gene and TBEV 3' non-coding region. Data were statistically analysed using SPSS statistic v2.0. RESULTS: Out of 82 rodent sera tested, the presence of LCMV antibodies was demonstrated in 24.93%. The highest prevalence of LCMV infection was found in commensal Mus musculus (47.37%), followed by 11.53%, 19.04% and 25% prevalence of infection in A. agrarius, A. flavicolis and A. sylvaticus, respectively. The highest prevalence of infection in rodents (53.33%) was found in locations with large waste sites and high anthropogenic influence. LCMV seroprevalence was significantly lower in rodents sampled from natural habitats. Viral nucleic acids were screened in 46 samples but yielded no amplicons of LCMV or TBEV. In addition, TBEV specific antibodies were not detected. CONCLUSIONS: Illegal waste sites have considerable impact on the area where they are located. Results have shown that the transmission of human pathogens can be significantly increased by the presence of waste sites. However, the pathogen must be endemic in the environment where the waste site is located. The introduction of a human pathogen as a consequence of the waste site in the area of interest could not be proven.

Prevalence of Neurotropic Viruses in Malignant Glioma and Their Onco-Modulatory Potential.

BACKGROUND: the association between infectious agents and tumour aetiology is relevant in about 20% of cases. PATIENTS AND METHODS: We tested high-grade glioma tissues from 45 patients for the presence of viral nucleic acids of six herpes viruses, human adenoviruses (A-G), and two neurotropic human viruses (enteroviruses, tick-borne encephalitis virus). Real-time polymerase chain reaction was used with immunolabelling. RESULTS: Three species of herpes viruses were detected: HSV-2, Epstein-Barr virus (EBV), HHV-6, and one human enterovirus. Plasma of these patients was not infected with viruses. In sera of patients, low HSV-1 and HSV-2 immunoreactivity were found in five cases, although these were not detected in their tumour tissue. CONCLUSION: Certain common viruses (HSV-1, HSV-2, EBV, human cytomegalovirus) are chronically present in the sera of patients with glioblastoma, but not necessarily in their tissues. Possibly both are associated with glioma progression, as we only found viruses in glioblastoma multiforme, but not in lower stages of glioma. Low titres of viruses in the blood indicate chronic viral virulence.

Clinical and laboratory characteristics of viral lower respiratory tract infections in preschool children.

BACKGROUND: Viral lower respiratory tract infections are the leading cause of hospitalizations in preschool children. Clinical pictures of different viral causes are not well characterized. The aim of this study was to establish the differences in clinical and laboratory characteristics between the different viral causes of lower respiratory tract infections in preschool children. METHODS: We included 278 preschool children hospitalized because of lower respiratory tract infection. White blood cell count and C-reactive protein values were determined and chest X-ray was performed in most patients. Polymerase chain reaction assay was used for the detection of viral pathogens from nasopharyngeal swab. RESULTS: Pneumonia was present in 71.4 % of all coronavirus infections, 35.1 % of all respiratory syncytial virus infections, and 13.0 % of all rhinovirus infections. Coronavirus (p = 0.03) and respiratory syncytial virus (p < 0.01) were retrospectively shown to be associated with the presence of pneumonia and rhinovirus (p < 0.01) with the absence of pneumonia. Wheezing was present in 81.5 % of all rhinovirus infections and in only 33.3 % of all adenovirus infections. Rhinovirus (p < 0.01) was associated with the presence of wheezing and adenovirus (p = 0.05) with the absence of wheezing. In adenovirus infections mean C-reactive protein value was 72.4 mg/L and white blood cell count 19.000/µl, both significantly higher than in other viruses (p < 0.01). CONCLUSIONS: Clinical and laboratory characteristics of viral lower respiratory tract infections significantly differ. With the advance of viral detection methods and increase of knowledge it becomes possible to characterize different respiratory viral infections and to improve the differential diagnosis.

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.

Prevalence and molecular characterization of tick-borne encephalitis virus in Ixodes ricinus ticks collected in Slovenia.

The hard tick Ixodes ricinus is the principal vector of tick-borne encephalitis virus (TBEV) in Slovenia; but until now, there was no information about the prevalence of TBEV infection in Slovenian ticks. We conducted a 2-year survey in 2005 and 2006, during which we were collecting I. ricinus ticks monthly in eight different locations of Slovenia. A total of 4777 I. ricinus ticks were collected: 1515 in year 2005 and 3262 in year 2006. The collected ticks were pooled into groups from which total RNA was extracted. Viral RNA was detected using real-time RT-polymerase chain reaction (PCR). Ticks infected with TBEV were found in six of eight locations. Viral RNA was detected in 8 of the 230 pools of ticks collected in 2005 and in 14 of the 442 pools collected in 2006. Prevalence of TBEV infection in Slovenian ticks was determined as 0.47%: 0.54% in 2005 and 0.43% in 2006. The detected infection rate in ticks significantly correlates with the TBEV incidence rates in selected areas. Using the method of sequencing, we have confirmed that the TBEV in ticks is genetically related to the TBEV in Slovenian patients.

Anaplasma phagocytophilum in ticks in Slovenia.

Ticks act as vectors of many pathogens of domestic animals and humans. Anaplasma phagocytophilum in Europe is transmitted by the ixodid tick vector Ixodes ricinus. A. phagocytophilum causes a disease with diverse clinical signs in various hosts. A great genetic diversity of the groESL operon of A. phagocytophilum has been found in ticks elsewhere. In Slovenia, the variety of the groESL operon was conducted only on deer samples. In this study, the prevalence of infected ticks was estimated and the diversity of A. phagocytophilum was evaluated. On 8 locations in Slovenia, 1924 and 5049 (6973) I. ricinus ticks were collected from vegetation in the years 2005 and 2006, respectively. All three feeding stages of the tick's life cycle were examined. The prevalence of ticks infected with A. phagocytophilum in the year 2005 and in the year 2006 was 0.31% and 0.63%, respectively, and it did not differ considerably between locations. The similarity among the sequences of groESL ranged from 95.6% to 99.8%. They clustered in two genetic lineages along with A. phagocytophilum from Slovenian deer. One sequence formed a separate cluster. According to our study, the prevalence of A. phagocytophilum in ticks is comparable to the findings in other studies in Europe, and it does not vary considerably between locations and tick stages. According to groESL operon analysis, two genetic lineages have been confirmed and one proposed. Further studies on other genes would be useful to obtain more information on genetic diversity of A. phagocytophilum in ticks in Slovenia.

Interacting roles of immune mechanisms and viral load in the pathogenesis of crimean-congo hemorrhagic fever.

Until now, the pathogenesis of Crimean-Congo hemorrhagic fever (CCHF) has not been well described. However, it has been hypothesized that it could be a result of the direct injury of virus-infected tissues in combination with the indirect effects of host immune responses, including cytokines. To shed more light on the role of viral load and cytokines, differential influences of CCHF virus (CCHFV) RNA load, antibody response, and cytokine production on severity and outcome of the disease were studied in sera of 46 patients with confirmed acute CCHF from Kosovo. In this study, viral load proved to be strongly related to the severity and outcome of the disease, with higher viral loads detected in patients with fatal outcomes than in surviving patients. Also, patients with fatal outcome had on average a weaker antibody response, if one was present at all. High levels of interleukin-10 (IL-10), gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) were associated with poor outcome, since detected concentrations were highest in patients with fatal outcome and lowest in patients with moderate disease course. Additionally, a positive linear dependence between viral load and these cytokines was observed. Interestingly, reduced levels of IL-12 were detected in all CCHF patients. Our study favors the hypothesis that CCHF could be a result of a delayed and downregulated immune response caused by IL-10, which leads to an increased replication and spread of CCHFV throughout the body. This consequently triggers increased production of IFN-gamma and TNF-alpha, cytokines mediating vascular dysfunction, disseminated intravascular coagulation, organ failure, and shock.

Rickettsia hoogstraalii sp. nov., isolated from hard- and soft-bodied ticks.

A novel spotted fever group Rickettsia was found in Haemaphysalis sulcata ticks collected from sheep and goats in Croatia in 2006. At the same time, a genetically identical organism was co-isolated with the embryonic cell line CCE3 obtained from the soft tick Carios capensis in Georgia, USA. In this study, further phenotypic and genotypic characteristics of the novel rickettsial strain present in H. sulcata ticks were investigated. Based on the cultivation of bacteria in mosquito and Vero cell cultures, the presence of rickettsiae in tick tissues and cell cultures [confirmed by transmission electron microscopy (TEM)] and the amplification and sequencing of five rickettsial genes, it was demonstrated that the novel Rickettsia strain fulfils the criteria to be classified as a novel species. The name Rickettsia hoogstraalii sp. nov. is proposed for the new strain. Rickettsia hoogstraalii sp. nov., an obligately intracellular bacterium, was grown in Vero cells and arthropod CCE3, ISE6 and C6/36 cell lines. The morphology of the cells of the novel species was typical of SFG rickettsiae. The small coccobacillary appearance of the bacteria was apparent with light microscopy. A Gram-negative bacterial cell wall and a cytoplasmic membrane separated by a narrow periplasmic space were visible by TEM. To date, Rickettsia hoogstraalii sp. nov. has been isolated from two species of ticks, H. sulcata and C. capensis. The novel species appears to be geographically widely distributed, having been detected in Croatia, Spain and Georgia, USA. Although no information is available regarding the possible pathogenicity of the novel species for vertebrate hosts, R. hoogstraalii sp. nov. has a cytopathic effect in Vero, CCE3 and ISE6 cells. Sequence analyses of the 16S rRNA, 17 kDa, gltA, ompA and ompB genes indicated that even though R. hoogstraalii sp. nov. was closely related to Rickettsia felis, it represents a separate species within the spotted fever group. The type strain of R. hoogstraalii sp. nov. is strain Croatica(T) (=DSM 22243(T)=UTMB 00003(T)).

First molecular evidence of Tula hantavirus in Microtus voles in Slovenia.

Different Microtus species, present in a worldwide range habitat populating North America, Europe, Asia, and few other species have been recognized previously as a hantavirus reservoir. Tula hantavirus was first reported in Microtus arvalis and Microtus rossiaemeridionalis from Central Russia and later discovered in several European countries. Using molecular techniques we have demonstrated the presence of Tula hantavirus in three different Microtus species in Slovenia. Phylogenetic analyses of partial S segment placed Slovenian strains in the same genetic lineage as Austrian and Croatian strains.

The hantaviral load in tissues of naturally infected rodents.

Hantaviruses cause a lifelong and asymptomatic infection in naturally infected hosts as well as in experimentally infected rodents. Understanding the ecology and pathogenesis of hantaviruses requires an interdisciplinary research approach, which links laboratory experiments with results gained from field studies. Although several studies report hantavirus persistence and tissue infection patterns for experimentally infected rodents, field data is very limited. For this reason, the aim of our study was to investigate Puumala, Dobrava and Saaremaa virus RNA loads and tissue infection patterns in their natural reservoirs. Hantavirus RNA was demonstrated in all tested internal organs and blood samples of 14 naturally infected rodent hosts. However, the concentration of a specific virus differs depending on the virus, the host and the organ tested. Above all, the Dobrava virus showed a considerably higher viral load in all internal organs and blood samples of infected Apodemus flavicollis hosts. Results obtained in the study support the thesis that virus RNA load reaches its peak in the first month after infection, presumably after the virus has spread throughout all internal organs. This also implies that recently infected rodents are more important for transmission of the virus in the community.

Native valve endocarditis due to Bartonella henselae in an immunocompetent man.

Culture-negative endocarditis accounts for 2.5-31% of all endocarditis cases and remains a diagnostic and therapeutic challenge. Bartonella spp. has only recently been recognized as an important cause of culture-negative endocarditis. We report a case of Bartonella henselae endocarditis occurring in an immunocompetent man who owned a cat and had previously been diagnosed with valvulopathy. Diagnosis was made only after prolonged diagnostic work-up with serology and with PCR and subsequent sequencing to identify the microorganism in the excised valves. The duration of treatment in patients with bartonella endocarditis is not clearly defined, and we decided to treat our patient with a prolonged course of antibiotic. Surgical treatment is usually necessary and was also successful in our patient. To our knowledge, this is the first case of bartonella endocarditis occurring in our geographic area.

Dobrava virus RNA load in patients who have hemorrhagic fever with renal syndrome.

To asses the role of virus load in the pathogenesis of hemorrhagic fever with renal syndrome, the serum Dobrava virus RNA load in 46 patients was measured with a novel quantitative real-time reverse-transcriptase polymerase chain reaction assay and compared to the disease severity. The level of viremia, detected in 26 patients, ranged from 10(2)-10(8) copies/mL of serum. The patients with severe disease had, on average, higher viral RNA loads than patients with a milder course of disease (6.15 vs. 4.67 log(10) copies/mL; P = .053). These results suggest that the Dobrava virus load might be associated with the severity of disease.

The complete genome sequence of a Crimean-Congo hemorrhagic fever virus isolated from an endemic region in Kosovo.

The Balkan region and Kosovo in particular, is a well-known Crimean-Congo hemorrhagic fever (CCHF) endemic region, with frequent epidemic outbreaks and sporadic cases occurring with a hospitalized case fatality of approximately 30%. Recent analysis of complete genome sequences of diverse CCHF virus strains showed that the genome plasticity of the virus is surprisingly high for an arthropod-borne virus. High levels of nucleotide and amino acid differences, frequent RNA segment reassortment and even RNA recombination have been recently described. This diversity illustrates the need to determine the complete genome sequence of CCHF virus representatives of all geographically distinct endemic areas, particularly in light of the high pathogenicity of the virus and its listing as a potential bioterrorism threat. Here we describe the first complete CCHF virus genome sequence of a virus (strain Kosova Hoti) isolated from a hemorrhagic fever case in the Balkans. This virus strain was isolated from a fatal CCHF case, and passaged only twice on Vero E6 cells prior to sequence analysis. The virus total genome was found to be 19.2 kb in length, consisting of a 1672 nucleotide (nt) S segment, a 5364 nt M segment and a 12150 nt L segment. Phylogenetic analysis of CCHF virus complete genomes placed the Kosova Hoti strain in the Europe/Turkey group, with highest similarity seen with Russian isolates. The virus M segments are the most diverse with up to 31 and 27% differences seen at the nt and amino acid levels, and even 1.9% amino acid difference found between the Kosova Hoti and another strain from Kosovo (9553-01). This suggests that distinct virus strains can coexist in highly endemic areas.

Molecular detection of Theileria sp. in ticks and naturally infected sheep.

Seven healthy sheep and 10 sheep diagnosed with piroplasmosis based on clinical signs were tested for the presence of babesiae and theileriae. Using the molecular techniques, two species of theileriae were detected and characterized. Theileria ovis was present mostly in healthy sheep and in Rhipicephalus ticks collected from infected sheep. Theileria sp. OT3 parasite was detected mostly in ill animals which represent additional evidence to the possible pathogenic nature of Theileria sp. OT3. The presence of babesiae in sheep or in ticks was not determined. The results of this study showed that ovine piroplasmosis due to Theileria is present in Southern Croatia. It was concluded that clinical diagnosis of ovine piroplasmosis should be confirmed by molecular analysis in order to identify the species of piroplasm, to select the appropriate treatment and to exclude the threat for public health.

Molecular detection of Bartonella species infecting rodents in Slovenia.

Rodents, collected in three zoogeographical regions across Slovenia, were tested for the presence of bartonellae using direct PCR-based amplification of 16S/23S rRNA gene intergenic spacer region (ITS) fragments from splenic DNA extracts. Bartonella DNA was detected in four species of rodents, Apodemus flavicollis, Apodemus sylvaticus, Apodemus agrarius and Clethrionomys glareolus, in all three zoogeographic regions at an overall prevalence of 40.4%. The prevalence of infection varied significantly between rodent species and zoogeographical regions. Comparison of ITS sequences obtained from bartonellae revealed six sequence variants. Four of these matched the ITS sequences of the previously recognized species, Bartonella taylorii, Bartonella grahamii, Bartonella doshiae and Bartonella birtlesii, but one was new. The identity of the bartonellae from which the novel ITS sequences was obtained were further assessed by sequence analysis of cell division protein-encoding gene (ftsZ) fragments. This analysis demonstrated that the strain is most likely a representative of possible new species within the genus.

Puumala hantavirus in Slovenia: analyses of S and M segment sequences recovered from patients and rodents.

In Slovenia, the co-existence of Dobrava and Puumala (PUUV) hantaviruses in a single endemic region has been demonstrated. This study presents selected Slovenian HFRS cases caused by PUUV combined with genetic analysis of viral genome sequences recovered from clinical specimens and tissue samples of Clethrionomys glareolus (bank voles). Serum samples from nine HFRS patients were included in the study. Rodents study sites were selected with regard to the HFRS cases. Partial S segment sequences were recovered from all nine patients and partial M segment sequences could be recovered from seven. Partial S and M segments sequences were also recovered from five C. glareouls captured at three different study sites. The sequences from Slovenian clinical specimens and rodent tissue samples belonged to the PUUV genotype and formed a distinct genetic lineage of PUUV. Human and rodent PUUV sequences located in the closest proximity to each other on the phylogenetic trees suggest genetic links between the human cases and the hantaviral strains circulating in natural foci of this zoonotic infection. Analysis of the complete S segment sequences recovered for two wild-type PUUV strains confirmed the existence of a distinct genetic lineage and also indicated a possible quasispecies type of Slovenian PUUV.