Phylogeographic analysis of Tula hantavirus highlights a single introduction to central Europe.

Orthohantaviruses are zoonotic pathogens of humans, unique among the bunyaviruses in not being transmitted by an arthropod vector. Tula orthohantavirus (TULV) is an old-world hantavirus, of yet unclear human pathogenicity, with few reported cases of clinically relevant human infection. So far, phylogeographic studies exploring the global pathways of hantaviral migration are scarce and generally do not focus on a specific hantavirus species. The aim of the present study was to reconstruct the dispersal history of TULV lineages across Eurasia based on S segment sequences sampled from different geographic areas. Maximum-likelihood and Bayesian inference methods were used to perform the phylogenetic analysis and phylogeographic reconstructions. Sampling time and trapping localities were obtained for a total of 735 TULV S segment sequences available in public databases at the time of the study. The estimated substitution rate of the analyzed partial S segment alignment was 2.26 × 10-3 substitutions/site/year (95 per cent highest posterior density interval: 1.79 × 10-3 to 2.75 × 10-3). Continuous phylogeography of TULV S segment sequences placed the potential root and origin of TULV spread in the Black Sea region. In our study, we detect a single-lineage introduction of TULV to Europe, followed by local viral circulation further on.

West Nile virus in the Republic of Serbia-Diagnostic performance of five serological tests in dog and horse sera.

West Nile virus (WNV) is a zoonotic mosquito-borne virus classified as family Flaviviridae and genus Flavivirus. The first WNV outbreak in humans in the Republic of Serbia was recorded in 2012. Equids and dogs can show clinical symptoms after WNV infection and are often used as sentinels. This study aimed to (i) give insight into seropositivity for WNV in clinically healthy dog and horse sera in different regions of Serbia and (ii) compare diagnostic value of 'in-house' and commercially available indirect immunofluorescence (IFA) and enzyme-linked immunoassay (ELISA) tests to 'gold standard' virus neutralization test (VNT). Due to cross-reactivity, sera were tested for Usutu virus and tick-borne encephalitis virus in VNT based on the epidemiological data of field presence. Blood sera of dogs (n = 184) and horses (n = 232) were collected from 2011 to 2013. The seropositivity was confirmed by VNT in 36.9 % tested dog sera and 34.9% tested horse sera with highest positivity in regions near two big rivers, while in four dog and seven horse sera, positivity resulted from Usutu virus infection. Comparative results of diagnostic tests in dogs ranged from 18.7 % seropositivity by 'in-house' ELISA to 31.9% by commercially available ELISA. In horses, seropositivity ranged from 36.2% by 'in-house' IFA to 32.5% by commercially available IFA and from 26.3% by 'in-house' IgG ELISA to 20.9% by commercially available ELISA. There were no statistically significant differences according to the McNemar test between 'in-house' and commercially available IFA and ELISA test in horse sera, while the same was not true for two ELISAs used in dog sera (χ2  = 8.647, p = .003). Established seropositivity in dogs and horses was in accordance with the epidemiological situation and WNV spread in the Republic of Serbia and proven Usutu virus co-circulation. 'In-house' tests remain a valuable tool in early diagnostics of WNV.

A Forgotten Episode of Marburg Virus Disease: Belgrade, Yugoslavia, 1967.

In 1967, several workers involved in poliomyelitis vaccine development and production fell ill at three different locations in Europe with a severe and often lethal novel disease associated with grivets (Chlorocebus aethiops) imported from Uganda. This disease was named Marburg virus disease (MVD) after the West German town of Marburg an der Lahn, where most human infections and deaths had been recorded. Consequently, the Marburg episode received the most scientific and media attention. Cases that occurred in Frankfurt am Main, West Germany, were also described in commonly accessible scientific literature, although they were less frequently cited than those pertaining to the Marburg infections. However, two infections occurring in a third location, in Belgrade, Yugoslavia, have seemingly been all but forgotten. Due in part to their absence in commonly used databases and in part to the fact that they were written in languages other than English, the important articles describing this part of the outbreak are very rarely cited. Here, we summarize this literature and correct published inaccuracies to remind a younger generation of scientists focusing on Marburg virus and its closest filoviral relatives of this important historical context. Importantly, and unfortunately, the three episodes of infection of 1967 still represent the best in-depth clinical look at MVD in general and in the context of "modern" medicine (fully resourced versus less-resourced capacity) in particular. Hence, each individual case of these episodes holds crucial information for health care providers who may be confronted with MVD today.

Molecular characterization of Dobrava-Belgrade hantavirus in Serbia, 2007-2011.

BACKGROUND: Hantaviruses are etiological agents of emerging zoonotic diseases worldwide, including hemorrhagic fever with renal syndrome (HFRS). A number of hantavirus species is known to be present in Europe. In Serbia, existing data on hantavirus presence and prevalence rely in serological findings. In this study, molecular analysis was performed in order to characterize HFRS causing hantaviruses in Serbia. METHODS: Sixty four serum samples of HFRS cases, previously found seropositive to anti-hantaviral antibodies, were included in the study. Partial hantaviral L and S segments were PCR amplified producing 390nt and 598nt amplicons, respectively, in parallel with human beta-actin mRNA as external reverse transcription positive control. Hantavirus specific PCR products were DNA sequenced in both direction and the obtained sequences phylogenetically confirmed and analyzed. RESULTS: PCR detection of hantavirus L and S genome segments was positive in 18/64 and 11/64 tested samples, respectively. Positive PCR results involved samples obtained from different locations, mostly from central and southern parts of Serbia. All the obtained sequences were identified as Dobrava-Belgrade virus (DOBV). In the phylogenetic analysis sequences from Serbia tended to cluster in distinctive, geographically related clusters. CONCLUSIONS: Our findings indicate DOBV as the main HFRS causing hantavirus in Serbia, the site of its initial isolation.

Prevalence of antibodies to Rickettsiae in different regions of Serbia.

We assayed the presence of antibodies specific for Rickettsia typhi, R. akari, and R. conorii in sera of persons from several localities in Serbia with different geographic, climatic, and lifestyle characteristics. Sera from 140 patients with unclear clinical symptoms and 273 healthy persons were tested for the presence of rickettsiae-specific antibodies by indirect immunofluorescence assay. In this study, for the first time we detected the presence of rickettsiae from the spotted fever group in Serbia. We detected the presence of antibodies against R. conorii in the samples from all tested localities. The proportion of positive cases was low in the plain agricultural areas but reached up to 23% in the mountain areas. We also observed a significant number of cases positive for antibodies against R. akari. Antibodies specific for the antigens of R. typhi were detected in only 2 samples from the municipality of Pec (Kosovo region). These findings contribute to the prevalence of Rickettsia species in Southeast Europe. Our study also revealed a dramatic lack of awareness of rickettsioses among medical personnel and pointed to the need for urgent measures that would help improve the current situation in the region.

Smallpox as an actual biothreat: lessons learned from its outbreak in ex-Yugoslavia in 1972.

Variola (smallpox) virus is classified as class A of potential biological weapons, due to its microbiological, genetic, antigenic and epidemiological characteristics. The potential danger is more real because vaccination against smallpox has stopped since disease eradication in 1979. That is why we want to share our unique, rich experience and acquired knowledge in the fight against this highly contagious and deadly disease during the smallpox outbreak in ex-Yugoslavia in 1972. It was the largest postwar outbreak in Europe when there were officially registered 175 ill patients, 35 of them with lethal outcome. This outbreak was specific by the time of its occurrence, the affected territory, dimensions and some epidemiological characteristics, but also by the well-organized, synchronized and efficient reaction of the competent state services in the fight against it.

Identification of Tula hantavirus in Pitymys subterraneus captured in the Cacak region of Serbia-Yugoslavia.

BACKGROUND: Atypical serum neutralizing antibody responses to prototype strains of Puumala viruses in some patients with hemorrhagic fever with renal syndrome (HFRS) have long suggested the existence of other hantaviruses in the Balkans. OBJECTIVE: To determine the presence of arvicolid rodent-borne Puumala-like hantaviruses in Yugoslavia. MATERIALS AND METHODS: Using reverse transcript-polymerase chain reaction, Tula virus RNA was amplified from lung tissues of a European pine vole (Pitymys subterraneus) captured in 1987, following an outbreak of HFRS in the Cacak region of Serbia-Yugoslavia. RESULTS: Sequence analysis of the entire coding region of the S segment and a 948-nucleotide region of the G2 glycoprotein-encoding M segment revealed divergence of approximately 14% from Tula virus strains harbored by European common voles (Microtus arvalis) captured in Central Russia and the Czech Republic. However, nearly complete identity was found in the corresponding deduced amino acid sequences. Moreover, phylogenetic trees constructed by the maximum parsimony and neighbor-joining methods indicated that this Pitymys-borne hantavirus shared a common ancestry with other Tula virus strains. CONCLUSIONS: The data demonstrate that Pitymys subterraneus also serves as a rodent reservoir of Tula virus in Serbia-Yugoslavia. To what extent this represents virus spillover from Microtus arvalis warrants further investigation.

Evidence of recombination in Tula virus strains from Serbia.

Tula hantavirus (TULV) belongs to Bunyaviridae family, with negative sense RNA genome. Segmented nature of hantaviral genome allows for genetic reassortment, but the evidence of homologous recombination also exists. In this study we analyzed TULV sequences isolated in Serbia on different occasions and from different rodent hosts: 1987 strain from Microtus subterraneus and 2007 strain from Microtus arvalis. Phylogenetic analysis of both L and S segment sequences is suggestive of geographically related clustering, as previously shown for majority of hantaviruses. Reconstruction of phylogenetic tree for TULV S segment showed that both sequences from Serbia clustered together with sequences from East Slovakia, which had previously been shown to be recombinants (Kosice strain). Exploratory recombination analysis, supported by phylogenetic and amino acid pattern analysis, revealed the presence of recombination in the S segment sequences from Serbia, resulting in mosaic-like structure of TULV S segment similar to the one of Kosice strain. Although recombination is considered a rare event in molecular evolution of negative strand RNA viruses, obtained molecular data in this study support evidence of recombination in TULV, in geographically distant regions of Europe.

A case of human monocytic ehrlichiosis in Serbia.

INTRODUCTION: Ehrlichiosis is a bacterial zoonosis transmitted by hematophagous arthropods--ticks. In humans, it occurs as monocytic, granulocytic, and ewingii ehrlichiosis. Pathological process is based on parasitic presence of Ehrlichia organisms within peripheral blood cells--monocytes and granulocytes. CASE OUTLINE: Fifty-two year old patient was admitted to hospital due to high fever of over 40 degrees C that lasted two days, accompanied with chills, muscle aches, malaise, loss of appetite, headache, confusion, breathing difficulties, and mild dry cough. The history suggested tick bite that occurred seven days before the onset of disease. Doxycycline was introduced and administered for 14 days, causing the disease to subside. Indirect immunofluorescence assay was used to analyze three serum samples obtained from this patient for Ehrlichia chaffeensis antibodies, and peripheral blood smear was evaluated for the presence of Ehrlichia and Ehrlichia aggregation into morulae. CONCLUSION: Ehrlichiosis should be considered in each case where there is a history of tick bite together with the clinical picture (high fever, chills, muscle aches, headache, generalized weakness and malaise, and possible maculopapular rash). The presence of Ehrlichia chaffeensis antibodies was confirmed in a patient with the history of tick bite, appropriate clinical picture and indirect immunofluorescence assay. This confirmed the presence of human monocytotropic ehrlichiosis, a disease that is uncommonly identified in our country.

Balloon valvuloplasty as a treatment of congenital aortic stenosis in children and adolescents.

INTRODUCTION: Balloon valvuloplasty (BVP) is one of the primary therapies for congenital aortic stenosis in children and adolescents. The aim of this interventional procedure is to gain time before possible surgical therapy (aortic valve replacement) until adulthood. OBJECTIVE: The aim of this study was to evaluate the efficacy, safety and mid-term results oftranscatheter BVP in children and adolescent in our Center. METHODS: From 2004 to 2011, 50 patients, aged 18 days to 18 years (mean 6.3 years) underwent BVP. Retrospective analysis of the echocardiographic and hemodynamic parameters were performed before and after procedure, especially peak pressure gradient (PG) across the aortic valve, semiquantification of the aortic regurgitation (AR) after the BVP as well as the left ventricle dimensions and functions. RESULTS: The mean peak PG in the whole group decreased from 74.80 +/- 27.72 mm Hg to 27.86 +/- 3.04 mm Hg (p < 0.001) after BVP. In 39 patients (78%), residual PG was lower than 30 mm Hg just after dilation. At the end of follow-up period, 25 patients (50%) had PG above 50 mm Hg, measured by Doppler technique, and four of them underwent re-dilation. Eight patients (16%) had severe AR. During the follow-up period (12-80 months, mean 51 months), six patients (12%) were referred to cardiac surgeons for aortic valve replacement or Ross procedure. CONCLUSIONS: This retrospective study analyzes our first experience of BVP as primary therapy of the congenital aortic stenosis. The results confirmed that BVP effectively postponed the need for surgery in children and adolescents toward the adulthood.