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carcinoma-HNSCC) ukazuje se na ulogu onkogenih virusa i to humanih papiloma virusa (HPV) Epštajn-Bar virusa (EBV) i BK i JC poliomavirusa, kao i značaju HLA-G polimorfizama. Cilj istraživanja. Cilj studije je bio određivanje učestalosti infekcija i koinfekcija onkogenim virusima i HLA-G polimorfizama i njihova korelacija sa demografskim i bihevioralnim karakteristikama pacijenata i kliničkim statusom HNSCC. Materijal i metod. Ispitivano je 100 uzoraka tkiva HNSCC. Single PCR, Nested PCR i Real-time PCR su rađeni za dokazivanje HPV i EBV, a seminested PCR za poliomaviruse. Genotipizacija HPV je rađena metodom sekvenciranja po Sangeru, a EBV genotipizacija Nested PCR metodom. Određivanje HLA-G tipova rađeno je metodom sekvenciranja po Sangeru. Rezultati. Učestalost HPV je bila 27%, EBV 20%, BKV 3% i JCV 0%. Dokazano je 5 HPV genotipova sa značajnom učestalošću visoko onkogenih tipova (84%) i to tipa HPV 16 (57,1%). Utvrđena su oba EBV tipa sa dominacijom EBV-1 (93,75%). HPV i EBV su značajno češći kod muškaraca od 50 do 69 godina sa karcinomom larinksa patohistološkog gradusa 2. Pojedinačna infekcija je utvrđena kod 28% uzoraka, a HPV infekcija je značajno najčešća. Koinfekcije su potvrđene kod 11% uzoraka, HPV/EBV koinfekcija je najzastupljenija ali bez razlike u učestalosti vrste koinfekcija. Utvrđeno je 6 HLA-G tipova, gde je *01:01:01 najučestaliji (56,66%), ali bez razlike u zastupljenosti HLA-G tipova i prisustva pojedinačnih infekcija i koinfekcija onkogenim virusima. Zaključak. Pokazana je povezanost prisustva infekcije onkogenim virusima sa karcinomima glave i vrata, ali nije utvrđena korelacija HLA-G polimorfizama sa prisustvom infekcija i koinfekcija onkogenim virusima u HNSCC. Introduction. The oncogenic viruses, namely Human papillomaviruses (HPV), Epstein-Barr virus (EBV) and BK and JC polyomaviruses, as well as HLA-G polymorphisms may play significant role in the etiology of Head and Neck squamous cell carcinoma (HNSCC). Aim. Determination of the frequency of oncogenic virus infections and coinfections and HLA-G polymorphisms and their correlation with the demographic and behavioral characteristics of patients and clinical status of HNSCC. Material and method. 100 HNSCC tissue samples were examined. Single, Nested and Real-time PCR were used to detect HPV and EBV, and seminested PCR for polyomaviruses. HPV and EBV genotyping were done by Sanger sequencing method, and Nested PCR, respectively. Determination of HLA-G types was done by Sanger sequencing method. Results. The prevalence of HPV was 27%, EBV 20%, BKV 3% and JCV 0%. Five HPV genotypes were identified with a significant frequency of high-risk types (84%), namely HPV type 16 (57.1%). Both EBV types were detected with a predominance of EBV-1 (93.75%). HPV and EBV are significantly more common in men aged 50 to 69 with laryngeal cancer of pathohistological grade 2. Single infection was found in 28% of samples with a significant prevalence of HPV infection. Co-infections were confirmed in 11% of samples. HPV/EBV co-infection is the most common but without a difference in the frequency of co-infection type. Six HLA-G types were identified, where *01:01:01 is the most common (56.66%), with no difference between HLA-G types and the presence of single infections and co-infections. Conclusion. The association of the presence of oncogenic virus infection with HNSCC has been shown, but without the correlation between HLA-G polymorphisms and presence of oncogenic virus infections and coinfections in HNSCC.

Karcinom grlića materice predstavlja četvrti po učestalosti maligni tumor kod žena. Infekcija visoko onkogenim tipovima HPV igra centralnu ulogu u razvoju cervikalne intraepitelijalne neoplazije i cervikalnog karcinoma. Pored HR-HPV, brojni kofaktori igraju značajnu ulogu u malignoj transformaciji ćelija. Najznačajniji među njima su faktori domaćina (genetski faktori i imunski status), faktori spoljašnje sredine i bihejvioralni faktori (multiparitet, upotreba oralnih kontraceptiva, pušenje, infekcija drugim seksualno prenosivim patogenim uzročnicima, ranije stupanje u seksualne odnose, promiskuitet, loši socioekonomski uslovi i nutritivni faktori). Ciljevi istraživanja: Cilj ove studije bio je da se kod ispitanica, koje su sa prethodno definisanim kolposkopskim i citološkim nalazom bile grupisane u četiri kategorije: PAP II, ASCUS, LSIL, HSIL, utvrdi prisustvoHR-HPV. Takođe je kod svih HR-HPV pozitivnih žena trebalo ispitati distribuciju genotipova i alela proinflamatornih citokina TNF-α,IFN-γ i IL-12 i njihov uticaj na nastanak cervikalne bolesti udružene sa HR-HPV i utvrditi koji genotipovi imaju protektivni, odnosno favorizujući, značaj za nastanak karcinoma grlića materice. Takođe, cilj istraživanja bio je da se kod svih HR-HPV pozitivnih pacijentkinja na osnovu upitnika utvrde kofaktori rizika za nastanak cervikalne bolesti i ispita povezanost između kofaktora i konstitucionalnih genskih polimorfizama proinflamatornih citokina, kao i njihov zajednički uticaj na nastanak cervikalnog karcinoma. Materijal i metodologija: Istraživanje dizajnirano kao studija preseka sprovedeno je tokom četvorogodišnjeg perioda, od 2014. do 2018. godine i obuhvatilo je 541 pacijentkinju. Svim pacijentkinjama je tokom ginekološkog pregleda utvrđen kolposkopski i citološki status na grliću materice. Na osnovu citološkog nalaza sve pacijentkinje podeljene su u četiri grupe: PAP II, ASCUS, LSIL, HSIL... Cervical cancer is the fourth most common malignant tumor found in women worldwide. Infection with high risk onconegic HPV types plays a central role in the development of cervical precursor lesion and cervical cancer. In addition to HR-HPV infection, numerous cofactors are important for malignant cell transformation. The most important among theme are: host factors (genetic and immune factors), environmental and behavioral factors (multiparity, use of oral contraceptives, smoking, sexually transmitted infections, early sexual intercourse, promiscuity, poor socio-economic conditions and nutritional factors). Aims: The aim of this study was to determinate the presence of HR-HPV infection in women that were previously divided into four groups (PAP II, ASCUS, LSIL, HSIL) based on cytological and colposcopic findings. In all HR-HPV positive women, it was also necessary to investigate the distribution of genotypes and allele of proinflammatory cytokines TNF-α, IFN-γ and IL-12 and their effect on the development of a cervical illness associated with HR-HPV and also to determine which genotypes possese a protective or favoring significance for the development of cervical cancer. Further aims were to establish risk factors for the occurrence of cervical disease in all HR-HPV positive patients and to examine the association between this cofactors and constitutional gene polymorphisms of proinflammatory cytokines, as well as their common influence on the development of cervical carcinoma. Material and methodology: This four year cross sectional study was conduced from 2014 to 2018 and included 541 patients. Colposcopic and cytological status was determinated for each patients during the gynecological examination. Based on the cytological findings, all patients were classifeid into four groups: PAP II, ASCUS, LSIL, HSIL. To determinate the presence of HPV infection and single nucleotide polymorphism of TNF-α, IFN-γ and IL-12, cervical swab were taken from visible lesions on the ectocervix using cervical cytobras. Further procedure involved the isolation of the total DNA using a commercial DNA extraction kit...

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...

Uvod: Virusni gastrointestinalni sindrom je aktuelni zdravstveni problem u celom svetu. To važi kako u razvijenim zemljama, tako i u zemljama u razvoju, a posebno u nerazvijenim zemljama, gde je drugi po redu uzrok mortaliteta. Nagli početak bolesti, praćen pojavom velikog broja tečnih stolica, mukom, povraćanjem, bolovima u stomaku, temperaturom, malaksalošću, ima za posledicu dehidrataciju. U svim starosnim grupama obolelih, a naročito kod sasvim male dece, starih i imunodeficitarnih osoba može da dođe do smrtnog ishoda, ukoliko se brzo ne postavi tačna etiološka dijagnoza bolesti i ne pristupi se odmah nadoknadi vode i elektrolita, kao i primeni svih ostalih mera simptomatske terapije. Brzo postavljena tačna dijagnoza, što se najbolje postiže real-time PCR testom, sprečava pojavu komplikacija, pa i fatalnog ishoda bolesti. Istovremeno, omogućava primenu odgovarajućih epidemioloških mera da se spreči nastanak epidemija i njihovo širenje. Cilj ovog istraživanja bio je da se tačno utvrdi incidenca virusnog gastrointestinalnog sindroma u Vojvodini i učestalost pojave epidemijskog i sporadičnog javljanja ove bolesti. Cilj je bio i postavljanje algoritma za primenu real-time PCR testa u dijagnostici virusnog gastrointestinalnog sindroma u budućem radu. Isto tako, cilj je bio da se molekularnom analizom, sekvenciranjem delova genoma pozitivnih uzoraka stolice, izvrši genetska tipizacija i odredi filogenetska pripadnost virusa. Materijal i metode: Tokom petogodišnjeg istraživanja molekularnim real-time PCR testom pregledane su 1003 obolele osobe sa simptomima virusnog dijarealnog sindroma, starosti od mesec dana do preko 90 godina. Pregledani su na rota, noro, astro i enterične adenoviruse. Na osnovu podataka iz anketnih upitnika i istorija bolesti, detaljno su analizirani svi klinički pokazatelji (javljanje bolesti tokom godine, trajanje bolesti, simptomi). Procena težine kliničke slike vršena je prema Vesikari skali. Svi podaci su upoređivani prema vrsti virusnog uzročnika, prema starosti obolelih, godinama trajanja istraživanja i epidemijskom i sporadičnom javljanju oboljenja. Dobijeni podaci su statistički obrađeni, tabelarno i grafički prikazani. Rezultati: U petogodišnjem periodu real-time PCR testom pregledan je uzorak od 1003 obolele osobe različite starosti na 4 virusna uzročnika dijarealnog sindroma (rota, noro, astro i enterične adenoviruse). Virusni dijarealni sindrom dokazan je kod 709 obolelih (70,69%). Najčešće su dokazane rotavirusne infekcije u 28,81%. Statistički značajno najčešće rotavirusi su bili utvrđeni kod dece do 5 godina (38,90%), ali u visokom procentu i kod dece uzrasta 6 do 14 godina (24,83%). Deca mlađa od 5 godina imala su statistički značajno najtežu kliničku sliku, bila su češće hospitalizovana i imala su statistički značajno višu temperaturu. Pored više temperature kod obolelih od rotavirusa, klinička slika je kod ovih bolesnika bila teža i bolest je duže trajala nego kod obolelih od drugih virusa. Norovirusna infekcija je dokazana u 23,03% obolelih i to statistički značajno češće kod odraslih osoba, starijih od 20 godina. Od kliničkih simptoma kod ovih bolesnika statistički značajno češće su dokazani muka, povraćanje i bolovi u stomaku, nego kod obolelih od drugih virusa. Norovirusi su značajno češće bili uzročnici epidemijskog javljanja bolesti. Astrovirus je dokazan kod znatno manjeg broja obolelih (u 2,29%) i to samo kod dece do 5 godina i dece uzrasta 6 do 14 godina. Infekcija izazvana enteričnim adenovirusima dokazana je kod 13,36% bolesnika. Njačešće je utvrđena kod dece uzrsta do 5 godina i 6 do 14 godina. Oboleli od adenovirusa imali su statistički značajno blažu kliničku sliku bolesti. Dva virusna uzročnika u uzorku stolice dokazana su u 3,19% osoba, obično u toku epidemijskog javljanja bolesti. Ovi bolesnici su imali bitno težu kliničku sliku. Najviše obolelih od dijarealnog sindroma bilo je u hladnim mesecima, mada su dijagnostikovani i tokom cele godine. U petogodišnjem periodu utvrđene su 22 epidemije u kolektivima i 9 porodičnih epidemija. Epidemijsko javljanje bolesti bilo je statistički značajno najčešće kod najstarijih bolesnika (starijih od 50 godina), a sporadično javljanje bilo je statističko značajno najčešće kod dece. U cilju potvrde tačnosti dijagnostike virusa u ispitivanim uzorcima real-time PCR testom, genotipizacije, kao i detaljnije molekularne analize, izabrani su reprezentativni uzorci pozitivni na rota, noro, astro ili adenoviruse. Delovi genoma ovih uzoraka su amplifikovani, a zatim sekvencirani. Sekvencirani izolati rotavirusa pripadali su grupi A i tipovima G1P[8], G2P[4], G3P[8] i G9P[8]. Sekvencirani izolati norovirusa pripadali su genogrupi I tipu 2, zatim genogrupi II tipovima 1, 2, 4 i 17. Sekvencirani izolati astrovirusa pripadali su grupi klasičnih astrovirusa i tipovima 1, 4 i 5. Sekvencirani izolati adenovirusa pripadali su grupi F i tipovima 40 i 41, kao i grupi C tipu 2. Pripadnost dobijenih sekvenci u ovom istraživanju, dodatno je potvrđena izradom filogenetskog stabla za sekvence pozitivne na rota, noro, astro ili adenoviruse. Zaključak: Incidenca virusnog dijarealnog sindroma u Vojvodini (70,69%) vrlo je visoka i viša je nego što je bilo pretpostavljeno prilikom prijave teze (u hipotezi). Real-time PCR test treba da bude redovno korišćen u budućem dijagnostičkom radu, jer dovodi do brze dijagnostike, čak i ako su virusi prisutni u malom broju u uzorcima tečnih stolica, što je utvrđeno tokom ovog dijagnostičkog rada. Ispitivani virusi treba da budu redovno dijagnostikovani kod obolelih od dijarealnog sindroma i to u svim starosnim grupama, tokom epidemijskog i sporadičnog javljanja oboljenja., Introduction: Viral gastrointestinal syndrome is a current ongoing health problem worldwide. This is true of both developed and developing countries, especially underdeveloped ones where it is the second leading cause of mortality. Sudden onset of the disease—accompanied by the occurrence of large numbers of liquid stools, nausea, vomiting, abdominal pain, fever, and exhaustion—leads to dehydration. A fatal outcome can occur in all age groups of patients, especially very young children, the elderly, and the immuno-deficient, unless an accurate etiological diagnosis of the disease is quickly established, followed by a prompt institution of fluid and electrolyte placement, and implementation of other symptomatic therapy measures. Quick establishment of an accurate diagnosis, which is best achieved using the real-time PCR test, prevents the onset of complications, including a potentially fatal outcome of the disease. Simultaneously, it enables the implementation of appropriate epidemiological measures to prevent epidemic outbreaks and their spread. The aim of this study was to accurately determine the incidence of viral gastrointestinal syndrome in Vojvodina and the frequency of epidemic and sporadic occurrence of this disease. The aim was also to set up an algorithm for the application of the real-time PCR test in diagnostics of viral gastrointestinal syndrome in future work. Likewise, the aim was to carry out genetic typing and determine phylogenetic affiliation of the virus using molecular analysis and sequencing of parts of genomes from positive stool samples. Material and Methods: During a five-year study, 1003 patients with symptoms of viral diarrheal syndrome, aged from one month to more than 90 years old, were examined using molecular real-time PCR test. They were screened for rota, noro, astro, and enteric adenoviruses. Based on the data from survey questionnaires and medical case history, all clinical indicators were meticulously analyzed (disease occurrence during the year, disease duration, symptoms). The assessment of the clinical severity was carried out according to the Vesikari Clinical Severity Scoring scale. All data were compared according to the type of the viral causing agent, age of the patients, duration of research in years, and epidemic and sporadic occurrence of the disease. Obtained data were statistically analyzed, tabulated, and graphically displayed. Results: In a five-year period, a sample of 1003 patients of different ages was screened for four different viral causing agents of diarrheal syndrome (rota, noro, astro, and enteric adenoviruses) using the real-time PCR test. Viral diarrheal syndrome was confirmed in 709 patients (70.69%). The most commonly found were rotavirus infections in 28.81% of the cases. Rotaviruses were statistically significantly most common in children younger than 5 years old (38.90%), but were also found in high percentage in children aged 6-14 years old (24.83%). Children under 5 years of age had statistically significantly highest clinical severity and fever, and were more frequently hospitalized. In addition to higher fever in patients with rotavirus, clinical severity in these patients was also higher, and the disease lasted longer than in patients with other viruses. Norovirus infections were reported in 23.03% of the subjects, statistically significantly more frequently in adults over 20 years of age. Regarding the clinical symptoms in these patients, nausea, vomiting, and abdominal pain were statistically significantly more common than in patients with other viruses. Noroviruses were significantly more common as causing agents of epidemic disease outbreaks. Astrovirus was found in a significantly smaller number of patients (in 2.29%), and only in children under 5 years of age and children aged 6-14 years old. Enteric adenovirus infections were reported in 13.36% of the subjects. They were most commonly found in children younger than 5, and those aged 6- 14 years old. Adenovirus sufferers had statistically significantly milder clinical disease. Two viral causing agents in the stool sample were found in 3.19% of the subjects, usually during an epidemic disease outbreak. These patients had a significantly more severe clinical disease. Highest numbers of sufferers from diarrheal syndrome occurred during the cold months, although they were diagnosed throughout the year. In a five-year period, 22epidemics in collective groups and 9 family epidemics were identified. Epidemic outbreaks of the disease were statistically significantly most frequent in the elderly patients (older than 50), while sporadic occurrences were statistically significantly most frequent in children. Representative samples positive for rota, noro, astro, or adenoviruses were selected in order to confirm the accuracy of virus diagnostics in samples tested by the real-time PCR test, and perform genotyping as well as more detailed molecular analyses. Parts of the genomes of these samples were amplified and then sequenced. Sequenced rotavirus isolates belonged to group A and types G1P[8], G2P[4], G3P[8], and G9P[8]. Sequenced norovirus isolates belonged to genogroup I type 2, and genogroup II types 1, 2, 4, and 17. Sequenced astrovirus isolates belonged to the group of classical astroviruses and types 1, 4, and 5. Sequenced adenovirus isolates belonged to group F and types 40 and 41, as well as group C type 2. The affiliation of the obtained sequences in this study was further confirmed by creating a phylogenetic tree for sequences positive for rota, noro, astro, or adenoviruses. Conclusion: The incidence of viral diarrheal syndrome in Vojvodina (70.69%) is very high—higher than what was assumed at the time of the thesis submission (in the hypothesis). The real-time PCR test should be regularly used in future diagnostic work, since it leads to rapid diagnostics even if viruses are present in small numbers in liquid stool samples, as determined in the course of this diagnostic study. The investigated viruses should be regularly tested in patients with diarrheal syndrome belonging to all age groups during both epidemic and sporadic occurrences of the disease.

Human immunodeficiency virus (HIV) is a retrovirus, the causative agent of Acquired immunodeficiency syndrome (AIDS). Since the beginning of the epidemic over 35 years ago, more than 78 million people have been infected so far and over 30 million have died. The high genetic variability and rapid evolution of HIV have been critical to its persistence and spread throughout the world. HIV-1 and HIV-2 comprise two distinct types of HIV. HIV-1 has diversified extensively into numerous genetic forms, including four groups (M, N, O, P), of which group M is causing the pandemic of HIV infection and AIDS. Group M viruses are further classified in multiple phylogenetically distinct subtypes (A-D, F, G, H, J and K), sub-subtypes (A1, A2, F1 and F2) and numerous recombinant forms. The global distribution of HIV-1 is complex and dynamic with regional epidemics representing only a subset of the global diversity. Molecular phylogenetic analysis, a method of reconstructing evolutionary relationships between nucleotide sequences, is one of the strategies for studying viral diversity and transmission dynamics. It is estimated that around half of HIV infected people are undiagnosed, making identification of transmission networks important for targeted public health intervention programs... Virus humane imunodeficijencije (HIV) je retrovirus koji uzrokuje sindrom stečene imunodeficijencije. Od početka epidemije pre 35 godina, ovim virusom je inficirano više od 78 miliona ljudi a preko 30 miliona je umrlo. Visoka genetička varijabilnost i brza evolucija HIV-a su ključni uzroci opstanka i globalnog širenjaepidemije. HIV je filogenetski klasifikovan u dva tipa: HIV-1 i HIV-2. Visoki diverzitet HIV-1 ogleda u postojanju četiri grupe (M, N, O, P) od kojih su virusi grupe M uzročnici globalne HIV-1 pandemije. Grupa M virusa je podeljena u više filogenetski različitih podtipova (A-D, F-H, J i K), pod-podtipove (A1, A2, F1 i F2) i cirkulišuće rekombinantne forme. Distribucija podtipova u svetu je složena i dinamična sa regionalnim HIV-1 epidemijama unutar globalnog diverziteta. Molekularna filogenetska analiza, metod za rekonstrukciju evolutivnih odnosa između nukleotidnih sekvenci, je tehnika za proučavanje varijabilnosti virusa i dinamike transmisije unutar regionalnih populacija. Procenjuje se da kod blizu polovine inficiranih osoba HIV infekcija nije dijagnostikovana, zbog čega je identifikacija puteva transmisije izuzetno značajna u cilju javno zdravstvenog nadzora. U ovom istraživanju primenjene su savremene filogenetske metode u analizi HIV-1 sekvenci izolata iz Srbije u cilju karakterizacije molekularne epidemiologije i dinamike transmisije, što je ključno za bolje razumevanje karakteristika aktuelne HIV-1 epidemije u Srbiji...

Primenom metode PCR u ispitanim uzorcima svinja ustanovljeno je prisustvo virusa Aujeckijeve bolesti, parvovirusa svinja i svinjskog cirkovirusa tip 2. Metodom direktnog sekvenciranja po Sanger-u određen je redosled nukleotida dela gB gena virusa Aujeckijeve bolesti. Tri nukleotidne sekvence virusa Aujeckijeve bolesti uključene u filogenetsku analizu su pokazale visok stepen sličnosti sa sekvencama navedenog virusa izolovanim u u Mađarskoj, Grčkoj i SAD. Na osnovu rezultata filogenetske analize ustanovljeno je da sva tri virusa Aujeckijeve bolesti identifikovana kod svinja u Crnoj Gori pripadaju genotipu I navedenog virusa. Metodom direktnog sekvenciranja po Sanger-u određen je redosled pet odabranih parvovirusa svinja identifikovana u Crnoj Gori. Na osnovu rezultata filogenetske analize parvovirusi svinja identifikovani u Crnoj Gori grupisani su zajedno sa sojevima navedenog virusa poreklom Velike Britanije, Kine, Brazila i SAD. Dobijeni rezultati filogenetske analize su pokazali da svinjski parvovirusi poreklom od svinja sa teritorije Crne Gore pripadaju klasteru E navedenog virusa. Primenom metode direktnog sekvenciranja po Sanger-u određen je redosled nukleotida dela ORF1 regiona genoma virusa PCV2. Nukleotidne sekvence identifikovanih PCV2 virusa imale su visok stepen sličnosti sa sojevima navedenog virusa izolovanim u Italiji, Austriji, Srbiji, Nemačkoj i Kini. Dobijeni rezultati filogenetske analize šest identifikovanih virusa PCV2 su potvrdili da njih pet pripada genotipu PCV2b, a jedan genotipu PCV2c. The presence of Aujeszky's disease virus, porcine parvovirus and porcine circovirus type 2 were detected by PCR method. Nucleotide sequences of Aujeszky's disease virus identified in swine in Montenegro were determined by direct sequencing method according to Sanger. Three nucelotide sequences included in phylogenetic analysis were highly similar with nucleotide sequences of Aujeszky's disease virus strains identified in Hungary, Greece and USA. According to results of phylogenetic analyisis, it was concluded that all three viruses belongs to genotype I. Nucleotide sequences of five porcine parvoviruses identified in Montenegro were determined by direct sequencing method according to Sanger and were highly similar with nucleotide sequences of porcine parvovirus strains isolated in United Kingdom, China, Brasil and USA. Phylogenetic analysis of five porcine parvoviruses revealed that all of them belongs to cluster E. The nucleotide sequences of six porcine circovirus type 2 identified in Montenegro were determined by direct sequencing method according to Sanger and were highly similar with nucleotide sequences of PCV2 strains isolated in Italy, Austria, Serbia, Germany and China. The phylogenetic analysis of six porcine circovirus type 2 showed that five of them belongs to genotype PCV2b, and one to genotype PCV2c.

Periapikalne lezije karakteriše hronična zapaljenska reakcija koja uzrokuje razgradnju alveolarnog koštanog tkiva u predelu vrha korena avitalnih zuba. Dosadašnja istraživanja pokazuju da su bakterijski mikroorganizmi i njihovi štetni produkti najčešći uzročnici nastanka zapaljenske reakcije u apeksnom parodoncijumu. Početkom 21. veka dokazano je prisustvo herpesvirusa u tkivu periapikalnih lezija i postavljena je hipoteza da pomenuti virusi učestvuju u procesu njihovog nastanka po modelu kojim doprinose razgradnji alveolarnog koštanog tkiva, kao i u marginalnom parodontitisu. Do danas je publikovano 20 istraživanja koja su ispitivala učestalost herpesvirusne infekcije u tkivu periapikalnih lezija. Rezultati istraživanja ukazuju da su Epštajn-Bar virus (EBV) i Humani citomegalovirus (HCMV) najčešće detektovani pripadnici porodice Herpesviridae u periapikalnim lezijama. Pojedina istraživanja su pokazala da su herpesvirusi češće detektovani u simptomatskim i velikim u odnosu na asimptomatske i male lezije. Međutim, njihova uloga u patogenezi periapikalnih lezija još uvek nije u potpunosti razjašnjena. Iako se smatra da genetička varijabilnost herpesvirusa može uticati na odnos virusa i imunskog odgovor domaćina, u naučnoj literaturi još uvek nema podataka o učestalosti pojedinih genotipova EBV i HCMV u periapikalnim lezijama. Takođe, nedovoljno je ispitan odnos povećanog broja genskih kopija herpesvirusa i nivoa proinflamatornih citokina u patogenezi periapikalnih lezija. CILJ ISTRAŽIVANJA. Ovo istraživanje je imalo za cilj da odredi učestalost prisustva, broja genskih kopija i vrste genotipova EBV i HCMV u tkivu periapikalnih lezija i kontrolnog pulpnog tkiva, kao i da uporedi dobijeni nalaz među periapikalnim lezijama sa različitim kliničkim, radiološkim i patohistološkim karakteristikama. Takođe, cilj ovog istraživanja bio je i da se odredi vrednost proinflamatornih citokina (TNF-α, IL-1β i IL-6) u tkivu periapikalnih lezija i kontrolnog pulpnog tkiva, te da se dobijene vrednosti uporede sa brojem genskih kopija EBV i HCMV u ispitivanom uzorku. MATERIJAL I METOD. U istraživanju je analizirano 100 uzoraka hroničnih periapikalnih lezija i 25 uzoraka kontrolnog pulpnog tkiva. Uzorci su prikupljani od zdravih pacijenata kod kojih je indikovana resekcija vrha korena zuba ili hirurško vađenje imapaktiranih umnjaka... Periapical lesions are characterized by chronic inflammation and destruction of tooth-supporting tissues in the periapical area of non-vital teeth. Previous studies confirmed that polymicrobial infection of endodontic origin is essential for its development. At the beginning of 21st century herpesviruses have been detected in tissue samples of periapical lesions. It has been hypothesized that concomitant herpesviral infection may contribute to the pathogenesis of apical periodontitis in the same way as it has been proposed for marginal periodontitis. Up to now, 20 studies investigated the occurrence of herpesviral infection in apical periodontitis. Their results indicate that Epstein-Barr virus (EBV) and Human cytomegalovirus (HCMV) are the most frequently detected species of Herpesviridae family in apical periodontitis. Previous studies showed that herpesviruses occurred more frequently in symptomatic and large compared to asymptomatic and small periapical lesions. However, their exact role in the pathogenesis of apical periodontitis has not been fully elucidated. Although it is considered that a great diversity of viral strains may influence the course and severity of disease there are no scientific data about the occurrence of different EBV and HCMV genotypes in apical periodontitis samples. Furthermore, there is a lack of data about the relationship between levels of pro-inflammatory cytokines and herpesviral copy number in the pathogenesis of apical periodontitis. AIMS. This study aimed to detect the occurrence of HCMV and EBV, to assess their copy number and the distribution of their genotypes in periapical lesions and healthy control tissues. Additionally, this study aimed to compare these findings between symptomatic and asymptomatic, large and small periapical lesions, and periapical granulomas and radicular cysts. The goal of the study was also to determine the levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) in periapical lesions and healthy control tissues, and to correlate these data with the copy number of investigated herpesviruses...

UVOD: Značaj infekcije humanim papiloma virusima (HPV) u razvoju različitih karcinoma kod čoveka, a naročito karcinoma grlića materice dobro je dokumentovan. Karcinom grlića materice jedan je od najznačajnijih uzroka smrtnosti žena u svetu. Genotipovi HPV udruženi sa karcinomom grlića materice označavaju se kao visoko onkogeni, a među njima su najučestaliji HPV 16 i HPV 18. U novije vreme, sve je više dokaza da pojedine linije i podlinije u okviru više genotipova HPV igraju značajnu ulogu u razvoju ovog karcinoma. CILJEVI: U ovom istraživanju identifikuju se genotipovi HPV, određuje njihova učestalost u tkivima karcinoma grlića materice, kao i povezanost sa patohistološkim tipom i stadijumom karcinoma grlića materice. Takođe, ciljevi ovog istraživanja odnose se na utvrđivanje i analizu genotipske i intragenotipske varijabilnosti dobijenih HPV izolata analizom E1, L1, E6/E7 (HPV 16) gena, uz poređenje dobijene učestalosti i distribucije HPV genotipova sa učestalošću i distribucijom u drugim zemljama. MATERIJAL I METODOLOGIJA: Studija je uključivala uzorke tkiva karcinoma grlića materice, fiksiranih u formalinu i ukalupljenih u parafin, uzeti od 101 pacijentkinje starosti od 24 do 78 godina. Nakon deparafinizacije i ekstrakcije virusne DNK, primenjivan je PCR metod uz korišćenje odgovarajućih prajmera za dokazivanje L1, E1 i HPV 16 E6/7 gena. Uzorci kod kojih je detektovana HPV DNK odgovarajućih gena, sekvencirani su metodom završetka lanca. Detekcija prisustva HPV DNK i genotipizacija izvedena je i metodom protočne (engl."Flow through") hibridizacije. Identifikovane sekvence, analizirane su potom filogenetskim metodama. Obrađene sekvence upotrebljavane su za utvrđivanje i poređenje genotipske i intragenotipske varijabilnosti HPV. REZULTATI: Od ukupno 101 uzorka tkiva karcinoma grlića materice, devedeset jedan uzorak pripadao je patohistološkom tipu planocelularnog karcinoma (90,09%)...

Cilj rada: analiza kliničkih i laboratorijskih karakteristika novorođenčadi sa infekcijom izazvanom Herpes simpleks virusom (HSV). Procena učestalosti neonatalnog herpesa (NH) u našoj sredini. Ispitivanje korelacije seroloških i molekularnih metoda dijagnostike. Definisanje kriterijuma za dijagnostičke i terapijske postupke. Materijal i metode: Analizirani su podaci o novorođenčadi i odojčadi uzrasta ≤45 dana sa sumnjom na infekciju HSV u periodu od januara 2003. do maja 2015. godine. Dokazana HSV infekcija je podrazumevala ispoljenu kliničku sliku NH i pozitivan PCR i/ili serologiju na HSV. Za ispitivanje incidencije NH korišćeni su podaci Republičkog zavoda za statistiku Srbije. Analizirane su kliničke manifestacije, laboratorijske analize, ishod lečenja, kao i kvatnifikovanje težine bolesti korišćenjem skorova SNAP II i SNAPPE II. Za statističku analizu korišćene su deskriptivne i analitičke metode. Rezultati: Studija ima 168 ispitanika, 29 sa dokazanom, 133 sa suspektnom i 6 sa asimptomskom HSV infekcijom. Incidencija NH tokom ispitivanog perioda je 3,01 na 100.000 živorođenih. U periodu 2010-2015. godine, sa uvođenjem rutinske PCR dijagnostike zabeležen je porast incidencije na 4,78 na 100 000 živorođenih. Najveći broj novorođenčadi sa dokazanom infekcijom, 72,45%, imao je herpesni encephalitis, 24,1% je imalo diseminovani oblik i 3,5% lokalizovani oblik bolesti. Većina infekcija uzrokovana je HSV-2 tipom (55,2%). Teži oblik bolesti imala su novorođenčad sa HSV-1 infekcijom. Ukupna smrtnost iznosila je 13,8%. Zaključak: Učestalost neonatalnog herpes u našoj populaciji odgovara učestalosti u ostalim evropskim zemljama. Sa unapređenjem dijagnostičkih metoda očekujemo dalji porast incidencije. Osobenosti NH u našoj sredini su veći broj HSV-2 infekcija i češća pojava herpes encefalitisa. Smrtni ishod je češći kod HSV-1 infekcije. Aim of study: Analysis of clinical and laboratory characteristics of newborns with Herpes simplex virus infection (HSV). Estimation of neonatal herpes incidence (NH) in our population. Correlation of serologycal and PCR tests. Comparison between suspected and confirmed cases of NH in order to define diagnostic and therapeutic protocol. Material and methods: Medical records of infants age ≤ 45 days with suspected NH, from January 2003 to May 2015. A confirmed case of NH was defined in an infant with clinical signs compatible with HSV disease and positive serology or PCR for HSV. Incidence was calculated by using Statistical Office of Republic of Serbia’s data of live births. We analyzed clinical manifestations, laboratory analysis, treatment outcome and predicting scores (SNAP II and SNAPPE II). Descriptive and analytic statistical methods were used for analysis of study results. Results: There were 168 infants, 133 with suspected, 29 with confirmed and 6 with asymptomatic HSV infection. Incidence of NH during the study period was 3.01 per 100.000 live births. Rise of incidence to 4.78 per live births 100.000 was noted after 2010. Majority of infants, 72.45%, presented with encephalitis, 24.1% had disseminated and 3,5% localized disease. Majority of infections were caused by HSV-2 (55.2%). Mortality was 13.8%, mostly accompanied with HSV-1 disease. Conclusion: Incidence of NH in our population is similar to other European countries. The rise of incidence was observed with improvement of HSV diagnostics. Characteristics of NH in our population are predominance of HSV-2 infections and herpes encephalitis. Mortality in our group is in accordance with other reports of NH.

Uvod: U novije vreme sve veći broj studija sugeriše da se genotipske varijante humanih papiloma virusa (HPV), uprkos filogentskoj srodnosti, mogu razlikovati u patogenosti i različito doprineti razvoju cervikalnih neoplazija. Cilj ovog rada je bio da se definiše zastupljenost različitih onkogenih tipova HPV kod ispitanica sa područja Južnobačkog okruga (AP Vojvodina) i odredi genotipska varijabilnost najprevalentnijih genotipova HPV, analizom DNK sekvenci i RFLP metodom.Materijal i metode: Istraživanje je sprovedeno u periodu od januara 2014. do novembra 2015.godine. Studija je obuhvatila 564 osobe ženskog pola, starosti od 18 do 69 godina. Genotipizacija 12 visokorizičnih tipova HPV izvršena je upotrebom komercijalnog kita HPV High Risk Typing Real-TM (Sacace Biotechnologies, Italy, CE, IVD). Automatskosekvenciranje amplifikovanih fragmenata L1 gena HPV rađeno je na automatskom sekvenatoru ABI Prism BigDye 3.1 (PE Applied Biosystems, Foster, CA, SAD). Bioinformatičkom analizom utvrđene su genetičke distance i filogenetski odnosi HPV izolata dobijenih u AP Vojvodini u odnosu na izolate iz drugih geografskih područja za tipove: 16, 18, 31 i 33. PCR-RFLP metodom ispitivana je heterogenost unutar HPV tipa 51, na nivou E1, L1 i L2 gena 11 odabranih HPV DNK pozitivnih izolata, primenom restrikcionih enima DraI, TaqI i PstI.Rezultati: Prevalencija HPV infekcije kod testiranih žena je iznosila 51,8%. Učestalost različitih tipova vema je varirala, pri čemu su najprevalentniji bili: HPV16 (34,5%); HPV 31 (20,5%); HPV 51 (13%); HPV33 (11,6%); HPV52 (11%) i HPV18 (11%). HPV tipovi 16 i 33 su najčešće dijagnostikovani u starosnoj grupi od 31-40 godina (25,5% i 6,21%), dok su HPV18 (8,16%), HPV31 (7,04% ) i HPV51 (12,3%) najčešće dokazani kod žena mlađih od 30 godina. Određene su genetičke distance i filogenetski odnosi u okviru populacije izolata iz Južnobačkog okruga, AP Vojvodine, u odnosu na druge izolate čije su genomske sekvence dostupneu GenBank bazi podataka. Filogenetska analiza nukleotidnih sekvenci HPV tipa 16 je potvrdila da se najveći broj izolata iz područja AP Vojvodine grupiše u liniju A, (podlinije A3 i A4 ) dok se jedan izolat izdvojio u liniju D (podlinija D1). Tri izolata HPV tipa 18 su grupisana u liniju A, a dva izolata kao posebna grupa. Utvrđeno je da genotipske varijante HPV 31 iz našeg područja pripadaju linijama A, B i C. Izolati HPV tipa 33 pripadali su liniji A (podlinije A1 i A2). Dobijene nukleotidne distance su pokazale da je razlika između analiziranih i referentnih sekvenci manja od 2%, štopotvrđuje sličnost na nivou varijante. Na osnovu PCR-RFLP analize u odabranim DNK pozitivnim na HPV tip 51, restrikcionom analizom PCR produkata na nivou L1, L2 i E1 gena, utvrđeno je da su prisutni samo genotipovi koji odgovaraju liniji A sa podlinijom A1.Zaključak: Rezultati studije predstavljaju prve dostupne podatke o rasprostranjenosti 12 visokorizičnih tipova HPV, kao i podatke o genomskoj arijabilnosti i filogenetskoj srodnosti najprevalentnijih HPV tipova kod žena sa područja Južnobačkog okruga. Najprevalentniji HPV tipovi našeg regiona su pokazali usklađenost sa evropskim izolatima, ali su nađene i ne-evropske varijante.Nizak procenat genetičkih distanci u okviru istog HPV tipa je u skladu sa niskom stopom mutacija kod ovih virusa. Iako se u današnje vreme RFLP metoda primenjuje u manjoj meri zbog uspona tehnologija sekvenciranja DNK, ova tehnika se može koristiti kao brza i jeftinija metoda za određivanje pojedinih linija ovih virusa. S obzirom da je u većini kliničkih laboratorija sekvenciranje DNK nedostupno,razvijena metoda se može koristiti za diferenciranje dve glavne linije HPV tipa 51, koje imaju različit onkogeni potencijal. Rezultati predstavljaju izuzetan doprinos epidemiološkoj proceni realnih potreba uvođenja rutinske imunizacije protiv visokoprevalentnih genotipova prisutnih u prekanceroznim promena na grliću materice inficiranih žena sa našeg područja. Tipizacija HPV virusa primenjenim metodama može biti od velike koristi u ranom otkrivanju maligne transformacijeinficiranih ćelija i prevenciji karcinoma grlića materice., Introduction:In recent times increasing number of researches suggests that genotypic variants of human papillomavirus (HPV), in spite of phylogenic relations, could differ in virulence and contribute the development of cervical neoplasia. The aim of this work was to define the partition of different oncogenic HPV types in examined women from the area of Juznobacka district (AP Vojvodina) and to determine genotypic variability of the most prevalent HPV genotypes, by DNA sequences’ analysis and RFLP method.Material and methods:The research wasconducted in period from January 2014 till November 2015. Study included 564 female persons, age from 18 to 69. The genotypization of 12 high-risk HPV types was performed with use of commercial kit HPV High Risk Typing Real-TM (Sacace Biotechnologies, Italy, CE, IVD). The automatic sequencing of amplified fragments of L1 HPV gene was performed on automatic sequencer ABI Prism BigDye 3.1 (PE Applied Biosystems,Foster, CA, USA). By bioinformatic analysis genetic distances, as well as phylogenic relations of HPV isolates in AP Vojvodina were determined in relation with isolates from other regions for types 16,18, 31 and 33. By PCR-RFLP, the heterogeneity within HPV type 51, at the level of E1, L1 and L2 genes 11 selected HPV DNA positive isolates, by use of restriction enzymes DraI, TaqI and PstI.Results:The prevalence of HPV infection in tested women was 51,8%. The frequency of different types varied considerably, where the most prevalent types were: HPV16 (34,5%); HPV 31 (20,5%); HPV 51 (13%); HPV33 (11,6%); HPV52 (11%) and HPV18 (11%). The HPV types 16 and 33 are diagnosed the most in age group from 31-40 (25,5% and 6,21%), while HPV 18 (8,16%), HPV31 (7,04% ) and HPV51 (12,3%) are the most diagnosed in women younger than 30. The genetic distances and phylogenic relations within samples from population of Južnobacka region, AP Vojvodina, are determined in relation to other samples whose genomic sequences are available in GenBank data base. Phylogenetic analysis of nucleotide sequences of HPV 16 confirmed that the most of isolates from the area of AP Vojvodina are grouped in line A (sublines A3 and A4), while one isolate separated to the line D (subline D1). Three isolates of HPV type 18 are grouped in line A, and two isolates grouped as separate group. It was confirmed that genotypic variants of HPV 31 from our region belong to lines A, B and C. Isolates of HPV type 33 belonged to line A (sublines A1 and A2). Gained nucleotide distances showed that difference between analyzed and referent sequences is lower than 2%, which confirme the similarity at the level of variant. Based on PCR-RFLP analysis in selected DNA of persons positive on HPV type 51, by restriction analysis of PCR products on the level of L1, L2 and E1 genes, it is confirmed that only genotypes that correspond to line A, subline A1 are present .Conclusions: The results of study present the first available data on abundance of 12 high-risk types, as well as data on genomic variability and phylogenic relations of the most prevalent HPV types in females from the Juznobacka region. The most prevalent HPV types of our region showed concordance with European isolates, but non-european variants were also found. Low percentage of genetic distances within the same HPV type is in concordance with low mutation rate of these viruses. Although today RFLP method is applied at lower scale because of rise of DNA technologies,this technique could be used as fast and efficient method for determination of particular lines of these viruses. Regarding that DNA sequencing is unavailable to the most of clinical laboratories,developed technique could be used for discrimination of two main lines of HPV type 51 that have different oncogenic potential. The results present considerable contribution to the epidemiological assessment of real needs for implementation of routine immunization against the high prevalent genotypes, present in precancerous alterations on the cervix of infected women from our area. Typization of HPV viruses by applied methods could be of great benefit in early detection of malignant transformations of infected cells and prevention of cancer of cervix.

Prisustvo svinjskog cirkovirusa 2 i parvovirusa svinja ispitano je u osamdeset zbirnih uzoraka (slezina, limfni čvorovi, pluća) poreklom od nevakcinisanih svinja primenom lančane reakcije polimeraze. Sve životinje su bile iz ekstenzivnog načina gajenja i iz različitih regiona Republike Srpske, BiH. Četiri uzorka limfnih čvorova i dva uzorka slezine su bili pozitivni na prisustvo DNK svinjskog cirkovirusa 2 (7,5%), dok je kod pet uzoraka limfnih čvorova utvrđeno prisustvo DNK parvovirusa svinja (6,25%). U uzorcima poreklom od tri svinje utvrđeno je prisustvo nukleinske kiseline oba prethodno navedena virusa (3,75%). Metodom sekvenciranja određena je nukleotidna sekvenca dela ORF1 regiona genoma svih izolata svinjskog cirkovirusa 2 i dela VP2 gena identifikovanih sojeva parvovirusa svinja. Nukleotidne sekvence virusa PCV2 utvrđene u uzorcima svinja poreklom iz RSBiH uključene u filogenetsku analizu su pokazale visok stepen sličnosti sa nukleotidnim sekvencama soja Mantova izolovanog kod svinja u Italiji, zatim sojeva DE00614 i DE22213 izolovanih kod svinja u Nemačkoj kao i sa sojem Jvnan izolovanog kod svinja u Kini. Istovremeno, virusi PCV2 utvrđeni kod svinja u RSBiH su bili delimično slični sa sojem NIVC SRB virusa PCV2 izolovanim kod svinja u Srbiji. Nukleotidnesekvence svinjskih parvovirusa identifikovanih kod svinja u Republici Srpskoj uključene u filogenetsku analizu su pokazale visok stepen sličnosti sa sojem analognim sekvencama sojeva Challenge izolovanim kod svinja u UK, Kresse izolovanim kod svinja u SADu kao i sa sojevima 77 i LZ izolovanim kod svinja u Kini... The presence of porcine circovirus 2 and porcine parvovirus was examined in eighty clinical samples of spleen, lymph nodes and lungs originating from nonvaccinated swine by polymerase chain reaction. All animals were reared in extensive livestock farming systems in different geographical districts of Republic of Srpska, Bosnia and Herzegovina. Porcine circovirus 2 DNA was detected in four lymph node and two spleen samples (7.5%), while porcine parvovirus DNA was identified in five lymph node samples (6.25%). The presence of both viruses was detected in three lymph node samples (3.75%). Partial nucleotide sequence of ORF1 gene of porcine circovirus 2 and VP2 gene of porcine parvovirus isolates was determined. The nucleotide sequences of identified PCV2 viruses from RSBIH included in phylogenetic typing are similar and cluster together with strain Mantova isolated from domestic pigs in Italy, strains DE00614 and DE22213 isolated from pigs in Germany as well as with the strain Jvnan isolated from pigs in China. Also, analyzed PCV2 isolates were partially similar with the strain NIVC SRB isolated from pigs in Serbia. The nucleotide sequences of identified PPV viruses that were included in phylogenetic typing showed a high level of similarity with the strain Challenge isolated from pigs in UK, strain Kresse isolated from pigs in USA and strains 77 and LZ isolated from pigs in China.

Glavni cilj ove doktorske disertacije je da se izvrši identifikacija i molekularna karakterizacija izolovanih sojeva virusa parainfluence 3 (PI3) goveda sa teritorije Republike Srbije. Izvršena ispitivanja su obuhvatila izolaciju virusa parainfluence 3 iz nosnih briseva goveda na kulturi tkiva i identifikaciju izolovanih sojeva virusa metodom virusneutralizacije, direktne imunofluorescencije i njihovu molekularnu karakterizaciju metodom lanĉane reakcije polimeraze (RT PCR) uz korišćenje prajmera za konzervisane regione HN i F gena virusa PI3 goveda sa sekvenciranjem radi njihove genotipizacije i svrstavanja na filogenetsko stablo izolovanih sojeva pomenutog patogena. Filogenetska analiza izolovanih sojeva izvršena je na osnovu utvrĊivanja homologije i poreĊenja pozicije njihovih nukleotidnih sekvenci sa sekvencama registrovanih sojeva virusa u bazi gena. Na osnovu nukleotidne homologije i pozicije domaćih izolovanih sojeva virusa na filogenetskom stablu, izvršena je njihova genotipizacija. U navedenim ispitivanjima je korišćen referentni soj SF4 virusa PI3 goveda (izolovan u drţavi Tenesi ,SAD). Nazalni brisevi poreklom od 119 goveda razliĉitih rasnih i starosnih kategorija sa ispoljenim simptomima infekcije gornjih partija respiratornog trakta su ispitivani na prisustvo virusa PI3 metodom izolacije na ćelijskoj liniji MDBK (Madin Darby Bovine Kidney). Izolacija virusa vršena je posle najviše tri pasaţe suspektnog materijala na navedenoj ćelijskoj liniji do pojave citopatogenog efekta. Citopatogeni efekat je ustanovljen u ćelijskim linijama posle inokulacije 11 uzoraka ispitivanog materijala, dok je primenom metode virusneutralizacije i direktne imunofluorescencije ustanovljeno prisustvo virusa parainfluece 3 kod 6 inokulisanih ćelijskih linija. U ostalim ćelijskim linijama, pojedinaĉno inokulisanim sa po 5 uzoraka poreklom od ispitivanih nosnih briseva goveda nije dokazano prisustvo virusa parainfluence 3 primenom prethodno navedenih metoda... The main objective of this doctoral thesis was the identification and molecular characterization of isolated strains of parainfluenza 3 (PI3) from cattle on the territory of the Republic of Serbia. The investigations included isolation of parainfluenza 3 virus from nasal swabs from cattle in tissue culture and identification of isolated strains of the virus by method of virusneutralisation, direct imunoflurescense and their molecular caracterisation by method of polymerase chain reaction (RT-PCR) using primers from conserved regions of the HN and F genes of the virus PI3 cattle, with sequencing and genotyping of domestic strains by help of their inclusion on the phylogenetic tree of mentioned pathogens. Phylogenetic analysis of the isolated strains was carried out on the basis of determination of the homology of the nucleotide sequences with the sequences of strains of the virus registered in gene base NCBI . Genotyping of domestic strains was done on the basis of nucleotide homology and position of local isolated virus strains on the phylogenetic tree, of PI3 virus. In these experiments, reference strain SF4 of virus PI3 of cattle was used (isolated in Tennessee, USA). Nasal swabs originating from 119 cattle with symptoms of infection in upper parts of the respiratory tract were examined for the presence of the virus PI3 by method of isolation of virus on cell line MDBK (Madin Darby Bovine Kidney). Virus isolation was done after three passages of suspected material on cell line till the appearance of cytopathic effect. Cytopathic effect was detected in cell lines after inoculation of 11 tested samples, while applying the method of virusneutralisation and direct immunofluorescense, identification of the virus parainfluece 3 were conformed in 6 inoculated cell lines, but not confirmed in other 5 tested samples from nasal swabs of cattle...

Uvod: Infekcija parvoB19 virusom koja se prenosi respiratornim i vertikalnim putem, ali i preko zaražene krvi i krvnih derivata, može predstavljati ozbiljan zdravstveni problem za rizične grupe pacijenata. Ovaj, po dimenzijama mali DNK virus bez omotača je vrlo otporan na standardne tehnike inaktivacije virusa u proizvodnji derivata krvi, a pouzdanu dijagnostiku otežava varijabilnost genomske sekvence virusa. Određivanje karakteristika nukleotidnih sekvenci izolata B19 virusa od značaja je za razvoj dijagnostičkih metoda i daje uvid u puteve nastanka i održavanja genomske varijabilnosti B19 virusa. Cilj ovog rada bio je da se odredi učestalost B19 virusa kod dobrovoljnih davalaca krvi i kod pacijenata sa sumnjom na B19 infekciju i da se na osnovu molekularno genetskih karakteristika dobijenih genomskih sekvenci, a u poređenju sa genomskim sekvencama dostupnim u bazama podataka, odredi evolutivna dinamika varijabilnosti parvoB19 virusa i filogenetski i filogeografski odnosi ispitivanih B19 izolata. Metode: Uzorci plazme dobrovoljnih davalaca i plazme i krvi pacijenata sa sumnjom na B19 infekciju analizirani su na prisustvo B19 DNK primenom lančane reakcije polimeraze (PCR), kako metodama iz literature tako i razvijenom internom metodom. Za uzorke sa detektovanim B19 genomom, direktnim nukleotidnim sekvenciranjem određena je genomska sekvenca. Bioinformatičkom analizom u odnosu na genomske sekvence B19 virusa dostupne u bazama podataka određen je genotip izolovanih virusnih genoma, evolutivni pritisci na osnovu odnosa nesinonimnih i sinonimnih zamena (dN/dS) za nukleotidne sekvence koje kodiraju virusne proteine, kao i genetičke distance i filogenetski odnosi B19 izolata dobijenih u Srbiji u odnosu na izolate iz drugih geografskih regija. Rezultati: Za 5 izolata iz Srbije, utvrđena je sekvenca B19 genoma u regiji kompletne cDNK. Iz baze podataka izdvojeni su i analizirani izolati sa poznatom sekvencom u regiji kompletne ili skoro kompletne cDNK, kojih je do 2012. godine bilo ukupno 146, od toga 117 genotipa 1. Analizom odnosa dN/dS za virusne proteine na osnovu većeg broja nukleotidnih sekvenci u odnosu na dosadašnje literaturne podatke procenjen je uticaj pozitivne i negativne selekcije na varijabilnost virusnog genoma... Abstract: Background: Parvovirus B19 infection, being transmitted via the respiratory route, vertically from mother to fetus as well as through blood-derived products administered parenterally, can represent a serious health problem for risk patients groups. This small non-enveloped DNA virus is extremely resistant to standard viral inactivation techniques, while a reliable diagnosis is challenged by the virus genome sequence variability. Characterization of B19 isolates nucleotide sequences is valuable for development of diagnostic methods, and it gives an insight into the genesis and maintenance of the B19 virus genome variability. Aim of this thesis was to determine the frequency of B19 viremia in voluntary blood donors and in patients with the B19 infection symptoms, investigate the level and evolutionary dynamics of the B19 genome variability as well as to examine phylogenetic and phylogeographic relations of the B19 genomes obtained in Serbia with the isolates from the other parts of the world on the bases of the almost complete B19 cDNA sequences. Methods: Plasma samples of the voluntary blood donors, and plasma and blood samples of the B19 infection suspected patients were analysed for the B19 DNA presence by the polymerase chain reaction (PCR), using the methods described in the literature as well as internally developed method. For the B19 genome positive samples genome sequences were determined by direct sequencing. Bioinformatic analysis was performed by using the generated data as well as the database information in order to determine a genotype of the isolated genomes, evolutionary pressures (based on dN/dS ratio) for virus protein coding sequences, and genetic distances and phylogenetic relations of the B19 isolates from Serbia compared to the other geographic regions. Results: For 5 isolates from Serbia, the B19 genome sequence was determined for the complete cDNA. Database isolates with the known, complete or almost complete cDNA, available until the end of 2012 (146 isolates in total, out of which 117 were genotype 1) were chosen and used for the analysis. Positive and negative selection influence for the virus protein coding sequences was determined by the dN/dS ratio analysis by using larger data set than described in the literature so far...