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 anti-bodies, 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. (C) 2019 Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Hepatitis C virus as cause of fulminant hepatitis-sequence analysis of the 5’ nontranslated region

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Detection of the Xanthi Chryso-like Virus in New Geographical Area and a Novel Arthropod Carrier

Here, we report on a serendipitous finding of a chryso-like virus associated with Culex pipiens mosquitos in the course of study aimed to detect and characterize West Nile virus (WNV) circulating in mosquitos in Serbia, Southern Europe. Upon initial detection of unexpected product in a PCR protocol for partial WNV NS5 gene amplification, further confirmation and identification was obtained through additional PCR and Sanger sequencing experiments. Bioinformatic and phylogenetic analysis identified the obtained sequences as Xanthi chryso-like virus (XCLV). The finding is particular for the fact that it associates XCLV with a new potential vector species and documents a novel geographical area of its distribution

ABO histo-blood groups and Rh systems in relation to malignant tumors of the digestive tract in Bosnia and Herzegovina

The distribution of ABO blood groups and the Rhesus factor was analyzed in 279 patients who suffered from malignant tumors of the digestive system. Patients were registered retrospectively in the Gastroenterohepatology Clinic, Clinical Center, University of Sarajevo over a discontinuous period of 88 months. From the results obtained, it was concluded that: (a) men became ill from gastric cancer significantly more frequently than women; (b) the frequency of liver carcinoma was three times higher than the global frequency and the frequency neighboring ethnic groups; and (c) patients with blood group B and patients with RhD(-) exhibited a significantly higher proportion of disease.Analizirana je distribucija krvnih grupa ABO sistema i Rezus faktora kod 279 pacijenata obolelih od malignih tumora digestivnog sistema. Pacijenti su registrovani retrospektivno u Gastroenterohepatološkoj klinici Kliničkog centra Univerziteta u Sarajevu u diskontinuitetu tokom 88 meseci (1987-1998). Na osnovu analizirane populacije pacijenata zaključeno je da: (a) muškarci značajno češće oboljevaju od kancera želuca u odnosu na žene; (b) učestalost karcinoma jetre je tri puta veća upoređenju sa učestalošću ovog oboljenja u svetu i susednim zemljama; (c) pacijenti sa V krvnom grupom i pacijenti sa RhD(-) su u značajnom stepenu češći u ispitivanoj populaciji obolelih od očekivanog.Projekat ministarstva br. 143010 i 14301

Should MTHFR 1298 A>C be tested together with MTHFR 677 C>T polymorphism in women with reproductive challenges?

Methylenetetrahydrofolate reductase (MTHFR) plays a critical role in the folate metabolism. The polymorphism 677C > T of the MTHFR gene, producing thermolabile enzyme with decreased function, is widely studied and associated with many conditions. Additionally, it has been shown that another polymorphism, 1298A > C, also reduces the activity of this enzyme, although to a lesser extent. The aim of this study is to evaluate the clinical informativeness of testing both MTHFR polymorphisms. Genomic DNA, were extracted from peripheral blood of 180 female patients with pregnancy complications and 183 healthy female controls, and genotyped for MTHFR 677C > T and 1298A > C loci, using TaqMan assays. Our study found similar frequency of alleles and genotypes between two groups. Based on MTHFR 677C > T genotype, 11.7% of patients homozygous for this mutation were under the possible risk. When the position 1298 was included in the testing, 22.8% of the patients were heterozygous for both polymorphisms. Additionally, 8.9% of the patients were homozygous only for the MTHFR 1298 mutation. Although, there was no differences compared to healthy control (p > 0.05), 43% of patients were found to have elevated risk which is about four time higher than results with only MTHFR 677C > T genotyping. After obtaining information for the 677 position, testing for the second polymorphism (1298A > C) should be considered, since we have shown that it dramatically increases the rate of detection of patients who are potentially at risk for MTHFR associated conditions

Evolutionary dynamics of Usutu virus: Worldwide dispersal patterns and transmission dynamics in Europe

Background: Usutu virus (USUV) is an emerging mosquito-borne Flavivirus, with birds as the main zoonotic reservoir. Humans are accidental hosts and mostly develop mild or even asymptomatic infections, although severe complications such as encephalitis can also arise. Detailed characterization of the pathogen's phylogenetics may offer valuable insights into the prediction and prevention of potential epidemics; however, lack of uniformity and the number of available USUV sequences worldwide hamper comprehensive investigation. Aim: The study aimed to investigate USUV spatio-temporal dispersal inter- and intracontinentally and to estimate the dynamics of viral spread within Europe. Methods: Phylogeographic and phylodynamic analyses were done using advanced phylogenetic methods implemented in Beast 1.10.4 and Beast 2.6.4 software packages. Results: Herein, we report on a new USUV isolate from Culex pipiens collected in 2019 from Serbia. The results of this research revealed two newly described intercontinental migration events of USUV from Africa to Germany in the 1970s and from Africa to the Middle East (Israel) in the late 90s. Finally, phylodynamic analysis substantiated the ongoing active expansion of USUV in Europe. Conclusion: The data would imply a high potential for further USUV expansion in Europe. Detailed phylogenetic characterization of the pathogen may offer valuable insights into prediction and prevention of potential epidemics; however, lack of uniformity and number of available USUV sequences worldwide hampers comprehensive investigation. This study draws attention to the need for upscaling USUV surveillance

Sequence variability at the internal ribosome entry site of the HCV genome in relation to therapy outcome

Different types of interferon are widely used to treat hepatitis C virus (HCV) infection. Results obtained in vitro suggest that interferon inhibits internal ribosome entry site (IRES)-mediated translation of the HCV genome. To elucidate the possible effect of the nucleotide sequence of IRES on therapy outcome, we compared HCV isolates from patients with sustained response and non-response to interferon/ribavirin combination therapy. In 56 analyzed HCV isolates, nucleotide changes appeared strictly in the stem-loop IIIb region, the stem part from 243 nt to 248 nt, and the polypyrimidine-II region. The natural sequence variability of IRES in isolates of genotype 3a was significantly higher than in isolates of genotype 1b (p < 0.05). The average number of nucleotide changes in genotype 3a correlated with response to therapy (p < 0.05).Interferonska terapija se danas najčešće koristi u lečenju infekcije virusom hepatitisa tipa C (HCV). In vitro rezultati su pokazali da interferoninhibira translaciju kod ovog virusa preko interakcije sa delom genoma koji učestvuje u inicijaciji translacije tzv. 'unutrašnje ribozomalno ulazno mesto' (IRES). U ovom radu smo ispitivali nukleotidne izmene u IRES-u kod izolata HCV-a dobijenih iz seruma osoba koje su primale kombinovanu terapiju interferon/ribavirin. U analiziranoj grupi od 56 HCV izolata, nukleotidne izmene su utvrđene u: IIIb petlji, regiji između 243 nt i 248 nt i polipirimidin-II regiji. Utvrđena varijabilnost IRES-a kod izolata genotipa 3a značajno je veća u poređenju sa izolatima genotipa 1b (p < 0.05). Prosečan broj nukleotidnih izmena kod izolata genotipa 3a je u korelaciji sa odgovorom na primenjenu terapiju (p < 0.05).Projekat ministarstva br. 143010 i 14301

Principles of NAT technology in view of the results obtained at the Institute of Transfusiology of MMA from 2007 till 2011

Nucleic-acid Amplification Testing (NAT) consist of extraction, amplification, hybridization and detection. Polymerase Chain Reaction (PCR) is method for amplification of viral genetic material (DNA/RNA) in enough number of copies, so we can detect them. Since year 1943, when transmission of hepatitis via transfusion was reported till beginning of 21st century, when NAT was implemented, large number of tests for detection of hepatitis B and C virus (HBV, HCV) and human immunodeficiency virus (HIV) were developed. In this article, we describe basic princples of detection of these viruses by NAT. We also show results from testing samples of blood donors in Institute of Transfusiology from April 2007 till June 2011. From 50 369 donors testing in Mini Pools (MP) of 24, we found two HCV RNA positive samples, one HBV DNA positive sample and all pools were HIV RNA negative. From 2 689 samples which were testing by Individual Donation (ID) NAT, 135 were HBV DNA positive, 108 were HCV RNA positive and 7 were HIV RNA positive. All samples also were tested by Enzyme Link Immunosorbent Assay (ELISA) and Confirmatory tests. Comparison of our results show validity of NAT testing, especially when ELISA tests of less specificity and sensitivity were used.Da bi testiranje nukleinskih kiselina bilo moguće, neophodno je izdvojiti ih iz virusa (ekstrakcija), zatim umnožiti (amplifikacija), obeležiti specifičnim probama (hibridizacija) i detektovati. To se obavlja korišćenjem metode 'Polymerase Chain Reaction' (PCR) ili lančane reakcije polimeraze koja nam omogućava da umnožimo deo virusnog genoma do količine koju je moguće detektovati. Od 1943. godine, kada je transmisija hepatitisa preko transfuzije prvi put prijavljena, do početka 21. veka, kada je masovno uvedena NAT tehnologija (eng. Nucleic-acid Amplification Testing - testiranje amplifikovanih nukleinskih kiselina), došlo je do značajnog poboljšanja u otkrivanju virusa uzročnika hematogenih transmisivnih bolesti. U ovom radu dati su osnovni principi NAT-a, odnosno detekcije genetskog materijala virusa uzročnika hepatitisa tipa B, C i virusa humane imunodeficijencije (HIV-a). Takođe, opisani su NAT rezultati dobijeni kod uzoraka krvi davalaca u Institutu za transfuziologiju VMA u periodu od aprila 2007. do juna 2011. godine. Od 50.369 testiranih davalaca u pulu, detektovana su dva HCV RNK pozitivna uzorka, jedan HBV DNK pozitivan uzorak, a za HIV RNK svi pulovi su bili negativni. Od 2.689 pojedinačno obavljenih NAT testiranja, najviše pozitivnih bilo je za HBV DNK (135 uzoraka), zatim za HCV RNK (108 pozitivnih) i 7 pozitivnih za HIV RNK. Za sve navedene uzorke paralelno su rađeni određeni enzimoimunski i potvrdni testovi. Komparacija rezultata primenjenih testova na našim ispitanim uzorcima potvrdila je opravdanost primene NAT-a, posebno prilikom korišćenja manje specifičnih i osetljivih enzimoimunskih testova.nul

ABO histo-blood groups and Rh systems in relation to malignant tumors of the digestive tract in Bosnia and Herzegovina

The distribution of ABO blood groups and the Rhesus factor was analyzed in 279 patients who suffered from malignant tumors of the digestive system. Patients were registered retrospectively in the Gastroenterohepatology Clinic, Clinical Center, University of Sarajevo over a discontinuous period of 88 months. From the results obtained, it was concluded that: (a) men became ill from gastric cancer significantly more frequently than women; (b) the frequency of liver carcinoma was three times higher than the global frequency and the frequency neighboring ethnic groups; and (c) patients with blood group B and patients with RhD(-) exhibited a significantly higher proportion of disease.Analizirana je distribucija krvnih grupa ABO sistema i Rezus faktora kod 279 pacijenata obolelih od malignih tumora digestivnog sistema. Pacijenti su registrovani retrospektivno u Gastroenterohepatološkoj klinici Kliničkog centra Univerziteta u Sarajevu u diskontinuitetu tokom 88 meseci (1987-1998). Na osnovu analizirane populacije pacijenata zaključeno je da: (a) muškarci značajno češće oboljevaju od kancera želuca u odnosu na žene; (b) učestalost karcinoma jetre je tri puta veća upoređenju sa učestalošću ovog oboljenja u svetu i susednim zemljama; (c) pacijenti sa V krvnom grupom i pacijenti sa RhD(-) su u značajnom stepenu češći u ispitivanoj populaciji obolelih od očekivanog.Projekat ministarstva br. 143010 i 14301