Endemic population dynamics of West Nile virus in mosquitoes from the territory of Serbia

Prvi slučajevi infekcije virusom Zapadnog Nila (VZN) u Srbiji zabeleženi su 2012. godine, dok je 2018., prema izveštaju Evropskog centra za kontrolu i prevenciju bolesti (ECDC), Srbija prijavila najveći broj obolelih (385) od svih zemalja jugoistočne Evrope. Cilj ove studije je bio da se genetički okarakterišu sojevi VZN-a, koji cirkulišu u Srbiji. U studiji je ispitano 200 pulova komaraca, skupljenih u Beogradu i široj okolini, u okviru redovnog nadzora koji sprovodi Zavod za biocide i medicinsku ekologiju u Beogradu u periodu od 2018-2020. Prisustvo VZN-a utvrđeno je reakcijom lančanog umnožavanja (engl. polymerase chain reaction – PCR), za detekciju NS5 gena, sa primenom Sangerovog DNK sekvenciranja dobijenih PCR produkata i njihovom preliminarnom identifikacijom primenom BLAST alata u NCBI bazi podataka. Konstrukcija filogenetskog stabla rađena je primenom metode Bajesove statistike implementirane u MrBayes softverski paket, uz odgovarajući evolutivni model koji je izabran u jModelTest softveru. Iz 200 analiziranih pulova komaraca, koji su skupljeni u periodu od 2018-2020, dobili smo 45 sekvenci NS5 gena VZN-a. Rezultati studije su pokazali da je 22.5% (45/200) pulova komarca bilo pozitivno na prisustvo VZN-a. Najveći procenat pozitivnih pulova je iz 2018. godine i iznosi 57.8% (26/45), zatim iz 2019 26.7% (12/45) i iz 2020 15.5% (7/45). Identifikacija dobijenih sekvenci pokazala je da je u Srbiji trenutno u cirkulaciji samo linija 2 VZN-a. Dalja analiza je potvrdila da su sojevi VZN-a iz Srbije najsličniji izolatima iz Grčke. Dodatno, u jednom pulu komaraca, uzorkovanom u okolini Šapca 2019. godine, identifikovano je prisustvo Usutu virusa. Dobijeni rezultati su potvrdili da u Srbiji cirkuliše linija 2 VZN-a kao i da postoji potreba za kontinuiranim molekularno-bioinformatički nadzorom nad ovim i sličnim zoonotskim virusima.The first cases of West Nile virus (WNV) infection in Serbia recorded in 2012, while in 2018, according to the report of the European Centre for Disease Control and Prevention (ECDC), Serbia reported the highest number of cases (385) in the Southeast Europe. The aim of this study was to genetically characterize strains of WNV circulating in Serbia. The present study included 200 pools of mosquitoes, collected in Belgrade and the surrounding area by the Institute for Biocides and Medical Ecology in Belgrade in the period from 2018-2020, as part of regular surveillance. The presence of WNV was detected by Nested-PCR (Polymerase chain reaction) method together with specific primers for detection of partial NS5 gene. All obtained specific PCR products were directly sequenced in both directions. Preliminary sequence identification was done by BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Bayesian method was employed to infer the evolutionary relationship of studied sequence dataset, using MrBayes software with the best-fitting nucleotide substitution model for the final sequence dataset, as selected by jModelTest 2. In total, 45 partial NS5 gene WNV sequences were obtained from mosquito pools collected in Serbia between 2018 and 2020. The results obtained in this study showed that 22.5% (45/200) of mosquito pools were positive for the presence of WNV. The highest percentage of positive pools was detected in 2018 (57.8% (26/45)), followed by 2019 (26.7% (12/45)) and 2020 (15.5% (7/45)). Phylogenetic analysis conformed that only linage 2 of WNV is circulating in Serbia. Majority of Serbian isolates clustered with Greek strains, thus forming the Balkan cluster. Of note, in one mosquito pool sampled in the Western Serbia in 2019, we detected the presence of Usutu virus. Results obtained in the present study confirmed the presence of WNV linage 2 in Serbia and the necessity for continuous molecular-bioinformatic monitoring of zoonotic viruses

Routes of pathogens spread: phytogeographic analyses

Filogeografija podrazumeva prostorno-vremensku rekonstrukciju evolutivne istorije različitih patogena u cilju predviđanja pojave zaraznih bolesti, detekcijom ključnih vrsta domaćina i geografskih područija sa kojih se patogeni šire, kao i potencijalni uticaj kretanja prirodnih rezervoara u prirodi na dalje širenje zoonotskih bolesti. Filogeografska analiza može se raditi primenom dva različita pristupa: filogeografija sa diskretnim lokacijama i filogeografija u kontinuiranom prostoru. Za rekonstrukciju prostorno-vremenske evolutivne istorije koristi se Bajesova metoda implementirana u BEAST softverski paket. Pored odgovarajućeg evolutivnog modela, izabranog na osnovu vrednosti AIC kriterijuma u jModelTest programu, neophodno je kalibrisati i ostale parametre u BEAST softveru (molekularni sat, demografski model) u cilju dobijanja što pouzdanijeg modela za analizu distribucije patogena. U studiju smo uključili 275 sekvenci NS5 gena linije 2 Virusa Zapadnog Nila (VZN), izolovanih sa različitih geografskih lokacija u cilju analize prostorno-vremenskog širenja pomenutog patogena. Geografska lokacija zajedno sa godinom izolacije su bile dostupne u NCBI (https://www.ncbi.nlm. nih.gov/nucleotide/) bazi podataka. Filogeografska analiza je rađena u BEAST v1.10.4 softverskom paketu primenom relaksiranog molekularnog sata i Gaussian Markov random field (GMRF) Skyride modela. Za procenu konvergencije Markovljevog lanaca Monte Karlo (MCMC) i za utvrđivanje efektivne veličine uzorka za svaki od parametara (≥200), korišćen je program TRACER v1.6. Filogenetsko stablo konstruisano primenom Bajesove metode, analizirano je u programu FigTree v1.4.4. Vremenski kalibrisano filogenetsko stablo NS5 gena VZN-a je pokazalo da se većina sekvenci iz Srbije grupiše zajedno sa sekvencama iz Grčke, formirajući jedan veliki monofiletski klaster. Analizom molekularnog sata je utvrđeno da je VZN-a ušao u Srbiju 2002. godine. Filogeografska analiza je ukazala da je VZN najverovatnije u Srbiju ušao iz Mađarske i da je nastavio lokalno da se širi. Rezultati naše studije su pokazali da je u Srbiji linija 2 VZN-a bila u cirkulaciji čitavu deceniju pre prve epidemije 2012. godine, i time potvrdili značaj molekularno-bioinformatičkog pristupa u sagledavanju evolutivne istorije i puteva širenja ovog patogena.Phylogeographic analysis reconstruction of evolutionary history of different pathogens aimed to predict of emergence of infectious diseases by detecting the key host species and the geographic areas from which pathogens spread and prediction of the impact of movement of natural reservoirs on the spread of viral diseases. Spatiotemporal reconstructions can be assessed by two different phylogeographic approaches; phylogeographic inference in discrete space and phylogeography in continuous space and time. The Bayesian method implemented in the BEAST software package is used to reconstruct the spatio-temporal evolutionary history. In addition to the appropriate evolutionary model, chosen based on the value of the AIC criteria in the jModelTest program, it is necessary to calibrate other parameters in the BEAST software (molecular clock, demographic model) in order to obtain the most reliable model for distribution analysis. In the present study we included 319 partial NS5 segment sequences of WNV, isolated from different geographic areas with the aim to explore spatio-temporal dispersal out of Africa. Sampling time and trapping localities for all isolates were available in NCBI (https://www.ncbi.nlm.nih.gov/nucleotide/ ) public databases at the time of the study. Phylogeographic analysis was done in BEAST v1.10.4 software package using relaxed molecular clock i Gaussian Markov random field (GMRF) Skyride model. The convergence of parameters was assessed through the ESS>200 checked using Tracer v1.6. Phylogenetic tree was visualized using FigTree software v1.4.4. Bayesian clade credibility (MCC) tree of the NS5 gene showed that majority of Serbian isolates was grouped in a single large clade together with sequences from Greece and Romania, thus forming large monophyletic clade. Molecular clock analysis estimated the time of the most recent common ancestor (tMRCA) of the local WNV epidemic in Serbia to around 2002. Phylogeographic analysis implied introduction of WNV-2 to Serbia from Hungary and further local spread. Our results evidence presence and circulation of WNV-2 in Serbia a decade prior to the first evidenced outbreak in 2012, and demonstrate the usefulness of bioinformatic methods providing a comprehensive view of the viral evolutionary history

Filogenetska analiza molekularne evolucije hantavirusa u različitim vrstama glodara

Hantaviruses, members of the order Bunyavirales, family Hantaviridae, are enveloped viruses with a negative sense three-segmented RNA genome, consisting of large – L (6.5–6.6 kb), medium – M (3.6–3.7 kb) and small – S (1.7–2.0 kb) segments, coding for viral polymerase, viral glycoprotein precursor further processed into two separate envelope glycoproteins (Gn and Gc) and viral nucleocapsid protein, respectively. Hantaviruses are unique among the bunyaviruses in not being transmitted by an arthropod vector. These viruses are persistently active in their natural reservoirs including rodents, insectivores and bats. In nature, hantaviruses are circulating via horizontal transmission among infected natural reservoirs. Their primary natural hosts are rodents belonging to four different subfamilies, including Sigmodontinae, Arvicolinae, Murinae and Neotominae. Newly published data have extended hantavirus hosts to insectivores (families Soricidae and Talpidae) and bats (order Chiroptera). Hantaviruses are commonly divided into two groups: old world hantaviruses, present in Europe and Far East, mostly including pathogenic hantavirus species that cause hemorrhagic fever with renal syndrome (HFRS), and new world hantaviruses, present in the Americas, defined as etiological agents of hantavirus pulmonary syndrome (HPS). Mortality rates of these zoonotic diseases, caused by pathogenic hantaviruses, vary with up to 12%-18% for HFRS and 60% for HPS. Human infection occurs through respiratory exposure to contaminated secreta and excreta including urine, saliva, and feces. Humans are not among the natural hosts of hantaviruses and they are thought to be dead-end hosts, without further virus transmission, with the exception of few reported cases of human-to-human transmission of Andes virus (ANDV) in Argentina and Chile...Hantavirusi, taksonomski svrstani u red Bunyavirales, familija Hantaviridae, su virusi sa omotačem i negativnim jednolančanim RNK genomom koji je segmentiran i sastoji se od velikog – L (6.5–6.6 kb), srednjeg – M (3.6–3.7 kb) i malog – S (1.7–2.0 kb) segmenta. L segment kodira virusnu polimerazu, M segment kodira glikoproteinski prekursor od koga nastaju dva glikoproteina (Gn i Gc) i S segment kodira nukleokapsidni protein. Za razliku od ostalih bunjavirusa, transmisija hantavirusa se ne odvija putem artropodnog vektora. Ovi virusi trajno cirkulišu u svojim prirodnim rezervoarima koji obuhvataju glodare, insektivore i slepe miševe. U prirodi, hantavirusi se prenose putem horizontalne i vertikalne transmisije između zaraženih životinja. Najznačajniji rezervoari hantavirusa su glodari koji pripadaju različitim potfamilijama (Sigmodontinae, Arvicolinae, Murinae and Neotominae). Hantavirusi su podeljeni u dve grupe: hantavirusi starog sveta (prisutni u Evropi i na dalekom istoku) koji obuhvataju patogene vrste hantavirusa koji izazivaju hemoragijsku groznicu sa bubrežnim sindromom (HGBS) i hantavirusi novog sveta (prisutni u Americi) koji obuhvataju patogene vrste hantavirusa koji izazivaju hantavirusni pulmonarni sindrom (HPS). Stopa smrtnosti kod ovih zoonoza, uzrokovanih patogenim vrstama hantavirusa, varira od 12-18% za HGBS i do 60% za HPS. Čovek se inficira resporatornim putem u kontaktu sa izlučevinama zaraženih životinja (urin, feces, pljuvačka). S obzirom da čovek nije prirodni domaćin hantavirusa, dalji interhumani prenos hantsvirusa nije moguć, sa izuzetkom nekoliko opisanih slučajeva prenosa Andes virusa (ANDV) u Čileu i Argentini. Hantavirusi, kao virusi sa segmentiranim RNK genomom , spadaju u visoko varijabilne mikroorganizme sa supstitucionom stopom od 10-2 - 10-4 izmena/mestu/godini. Genetički drift, homologa rekombinacija i izmena genskih segmenata su glavni mehanizmi koji leže u osnovi evolucije hantavirusa. U ovom istraživanju smo se fokusirali na analizu evolucije hantavirusa..

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/)

Evolutionary dynamics and geographical dispersal of Borrelia lusitaniae

Background: Borrelia lusitaniae is a species within the complex Borrelia burgdorferi sensu lato, associated with lizards as reservoirs and Ixodes ricinus as its main vector. Borrelia lusitaniae is predominantly distributed in Central and Southeast Europe, and in countries of the Mediterranean basin, such as Portugal, Morocco, Tunisia, and Italy where this spirochete appears to infect vector ticks more frequently than other genospecies. Evolution of this zoonotic tick-borne microparasite is shaped by different environmental factors. Comprehensive phylogenetic analysis may give insight into how B. lusitaniae spreads to new geographic locations. Aim: We applied Bayesian statistical methods to B. lusitaniae multilocus sequence typing (MLST) data to study the migration routes of B. lusitaniae and its potential for further spread. Results: The discrete phylogeographic analysis placed origins of B. lusitaniae in Southeast Europe and identified at least two introductions of B. lusitaniae from Europe to North Africa. Estimated effective reproductive potential (Re), as a key indicator for a pathogen spread, suggested potential for further spread. Conclusion: The results of this study can provide beneficial information about the potential for further spread of B. lusitaniae in Europe and North Africa and estimation of necessity for the development of strategies to monitor and control Lyme borreliosis

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

Variability of the HCV core region and host genetic and epigenetic factors can predict the response to pegylated interferon/ribavirin therapy in genotype 1b hepatitis C patients from Serbia

Variations in the hepatitis C virus (HCV) core sequence have been related to disease progression and response to antiviral therapy. Previously we showed that the methylation status of RASSF1A and p16 genes, and IL28B genotypes affects the response to pegylated interferon/ribavirin (PEG-IFN/RBV) therapy. Herein we investigated whether amino acid (aa) substitutions in the HCV core region alone or in combination with IL28B genotypes and RASSF1A/p16 methylation affect the response to PEG-IFN/RBV therapy and liver disease progression. Among 29 examined patients, we found no association between single aa substitutions and response to therapy. However, we observed that patients with the HCV core aa substitution at position 75 and CT/TT IL28B genotypes were non-responders (NR), (P=0.023). Moreover, these patients had unmethylated RASSF1A. In contrast, most patients (75%) with aa substitutions at position 91 and CC IL28B genotype achieved sustained virologic response (SVR), (P=0.030), and 70% of them had methylated RASSF1A gene. Our results suggest that combined analysis of aa substitutions in the core protein, the IL28B rs12979860 polymorphism, and the methylation status of the RASSF1A gene may help in predicting treatment response to PEG-IFN/RBV in genotype 1b chronic hepatitis C patients

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

Evolutionary dynamics and geographical dispersal of Borrelia lusitaniae

Background: Borrelia lusitaniae is a species within the complex Borrelia burgdorferi sensu lato, associated with lizards as reservoirs and Ixodes ricinus as its main vector. Borrelia lusitaniae is predominantly distributed in Central and Southeast Europe, and in countries of the Mediterranean basin, such as Portugal, Morocco, Tunisia, and Italy where this spirochete appears to infect vector ticks more frequently than other genospecies. Evolution of this zoonotic tick-borne microparasite is shaped by different environmental factors. Comprehensive phylogenetic analysis may give insight into how B. lusitaniae spreads to new geographic locations. Aim: We applied Bayesian statistical methods to B. lusitaniae multilocus sequence typing (MLST) data to study the migration routes of B. lusitaniae and its potential for further spread. Results: The discrete phylogeographic analysis placed origins of B. lusitaniae in Southeast Europe and identified at least two introductions of B. lusitaniae from Europe to North Africa. Estimated effective reproductive potential (Re), as a key indicator for a pathogen spread, suggested potential for further spread. Conclusion: The results of this study can provide beneficial information about the potential for further spread of B. lusitaniae in Europe and North Africa and estimation of necessity for the development of strategies to monitor and control Lyme borreliosis