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During the ongoing COVID-19 epidemic many efforts have gone into the investigation of the SARS-CoV-2–specific antibodies as possible therapeutics. Currently, conclusions cannot be drawn due to the lack of standardization in antibody assessments. Here we describe an approach of establishing antibody characterisation in emergent times which would, if followed, enable comparison of results from different studies. The key component is a reliable and reproducible assay of wild-type SARS-CoV-2 neutralisation based on a banking system of its biological components - a challenge virus, cells and an anti-SARS-CoV-2 antibody in-house standard, calibrated to the First WHO International Standard immediately upon its availability. Consequently, all collected serological data were retrospectively expressed in an internationally comparable way. The neutralising antibodies (NAbs) among convalescents ranged from 4 to 2869 IU mL-1 in a significant positive correlation to the disease severity. Their decline in convalescents was on average 1.4-fold in a one-month period. Heat-inactivation resulted in 2.3-fold decrease of NAb titres in comparison to the native sera, implying significant complement activating properties of SARS-CoV-2 specific antibodies. The monitoring of NAb titres in the sera of immunocompromised COVID-19 patients that lacked their own antibodies evidenced the successful transfusion of antibodies by the COVID-19 convalescent plasma units with NAb titres of 35 IU mL-1 or higher.

Objectives: The patients with hematological malignancies are a vulnerable group to COVID-19, due to the immunodeficiency resulting from the underlying disease and oncological treatment that significantly impair cellular and humoral immunity. Here we report on a beneficial impact of a passive immunotherapy with convalescent plasma to treat a prolonged, active COVID-19 infection in a patient with a history of nasopharyngeal diffuse large B-cell lymphoma treated with the therapy inducing substantial impairment of particularly humoral arm of immune system. The specific aim was to quantify SARS-CoV2 neutralizing antibodies in a patient plasma during the course of therapy. Materials and methods: Besides the standard of care treatment and monitoring, neutralizing antibody titers in patient's serum samples, calibrated according to the First WHO International Standard for anti- SARS-CoV-2 immunoglobulin (human), were quantified in a time- dependent manner. During the immunotherapy period peripheral blood flow cytometry immunophenotyping was conducted to characterize lymphocyte subpopulations. Results: The waves of clinical improvements and worsening coincided with transfused neutralizing antibodies rises and drops in the patient's systemic circulation, proving their contribution in controlling the disease progress. Besides the patient's lack of own humoral immune system, immunophenotyping analysis revealed also the reduced level of helper T-lymphocytes and immune exhaustion of monocytes. Conclusion: Therapeutic approach based on convalescent plasma transfusion transformed a prolonged, active COVID-19 infection into a manageable chronic disease.

The SARS-CoV-2 pandemic is a major global threat that sparked global research efforts. Pre-clinical and biochemical SARS-CoV-2 studies firstly rely on cell culture experiments where the importance of choosing an appropriate cell culture model is often underestimated. We here present a bottom-up approach to identify suitable permissive cancer cell lines for drug screening and virus research. Human cancer cell lines were screened for the SARS-CoV-2 cellular entry factors ACE2 and TMPRSS2 based on RNA-seq data of the Cancer Cell Line Encyclopedia (CCLE). However, experimentally testing permissiveness towards SARS-CoV-2 infection, we found limited correlation between receptor expression and permissiveness. This underlines that permissiveness of cells towards viral infection is determined not only by the presence of entry receptors but is defined by the availability of cellular resources, intrinsic immunity, and apoptosis. Aside from established cell culture infection models CACO-2 and CALU-3, three highly permissive human cell lines, colon cancer cell lines CL-14 and CL-40 and the breast cancer cell line CAL-51 and several low permissive cell lines were identified. Cell lines were characterised in more detail offering a broader choice of non-overexpression in vitro infection models to the scientific community. For some cell lines a truncated ACE2 mRNA and missense variants in TMPRSS2 might hint at disturbed host susceptibility towards viral entry.

A large variation in the severity of disease symptoms is one of the key open questions in COVID-19 pandemics. The fact that only a small subset of people infected with SARS-CoV-2 develop severe disease suggests that there have to be some predisposing factors, but biomarkers that reliably predict disease severity have not been found so far. Since overactivation of the immune system is implicated in a severe form of COVID-19 and the IgG glycosylation is known to be involved in the regulation of different immune processes, we evaluated the association of inter-individual variation in IgG N-glycome composition with the severity of COVID-19. The analysis of 166 severe and 167 mild cases from hospitals in Spain, Italy and Portugal revealed statistically significant differences in the composition of the IgG N-glycome. The most notable difference was the decrease in bisecting N-acetylglucosamine (GlcNAc) in severe patients from all three cohorts. IgG galactosylation was also lower in severe cases in all cohorts, but the difference in galactosylation was not statistically significant after correction for multiple testing. To our knowledge, this is the first study exploring IgG N-glycome variability in COVID-19 severity.

According to anti-SARS-CoV-2 seroresponse in patients with COVID-19 from Croatia, we emphasised the issue of different serological tests and need for combining diagnostic methods for COVID-19 diagnosis. Anti-SARS-CoV-2 IgA and IgG ELISA and IgM/IgG immunochromatographic assay (ICA) were used for testing 60 sera from 21 patients (6 with severe, 10 moderate, and 5 with mild disease). The main clinical, demographic, and haemato-biochemical data were analysed. The most common symptoms were cough (95.2%), fever (90.5%), and fatigue and shortness of breath (42.9%). Pulmonary opacities showed 76.2% of patients. Within the first 7 days of illness, seropositivity for ELISA IgA and IgG was 42.9% and 7.1%, and for ICA IgM and IgG 25% and 10.7%, respectively. From day 8 after onset, ELISA IgA and IgG seropositivity was 90.6% and 68.8%, and for ICA IgM and IgG 84.4% and 75%, respectively. In general, sensitivity for ELISA IgA and IgG was 68.3% and 40%, and for ICA IgM and IgG 56.7% and 45.0%, respectively. The anti-SARS-CoV-2 antibody distributions by each method were statistically different (ICA IgM vs. IgG, p = 0.016; ELISA IgG vs. IgA, p

At present, the novel pandemic coronavirus SARS-CoV-2 is a major global threat to human health and hence demands united research activities at different levels. Finding appropriate cell systems for drug screening and testing molecular interactions of the virus with the host cell is mandatory for drug development and understanding the mechanisms of viral entry and replication. For this, we selected human cell lines represented in the Cancer Cell Line Encyclopedia (CCLE) based on RNA-seq data determined transcript levels of ACE2 and TMPRSS2, two membrane proteins that have been identified to aid SARS-CoV-2 entry into the host cell. mRNA and protein expression of these host factors were verified via RQ-PCR and western blot. We then tested permissiveness of these cell lines towards SARS-CoV-2 infection, cytopathic effect, and viral replication finding limited correlation between receptor expression and infectability. One of the candidate cancer cell lines, the human colon cancer cell line CL-14, tested positive for SARS-CoV-2 infection. Our data argue that SARS-CoV-2 in vitro infection models need careful selection and validation since ACE2/TMPRSS2 receptor expression on its own does not guarantee permissiveness to the virus.Author summaryIn the midst of the pandemic outbreak of corona-virus SARS-CoV-2 therapeutics for disease treatment are still to be tested and the virus-host-interactions are to be elucidated. Drug testing and viral studies are commonly conducted with genetically manipulated cells. In order to find a cell model system without genetic modification we screened human cell lines for two proteins known to facilitate entry of SARS-CoV-2. We confirmed and quantified permissiveness of current cell line infection models, but dismissed a number of receptor-positive cell lines that did not support viral replication. Importantly, ACE2/TMPRSS2 co-expression seems to be necessary for viral entry but is not sufficient to predict permissiveness of various cancer cell lines. Moreover, the expression of specific splice variants and the absence of missense mutations of the host factors might hint on successful infection and virus replication of the cell lines.

The purpose of this paper is to present SARS-CoV-2 diagnostic methods and their optimal implementation. The main routine microbiological diagnostic tools are molecular and serological methods. Sensitivity and utilisation of molecular methods is the best at the disease onset, while serological methods are the best if used at least 5-7 days after disease onset. Positive molecular test result (real-time polymerase chain reaction; RT-PCR) must be interpreted in the clinical presentation context. A sampling site for molecular analysis is based on disease severity; upper and lower respiratory tract samples are used for mild and severe presentations, respectively. Virus cultivation is the only method of proving the existence of a viable virus in a tissue sample, but due to the method complexity, it is not a part of a routine process. Ground glass opacities with or without reticular pattern and/or consolidation are typical findings for COVID-19 pneumonia. Multi-slice computerized tomography (MSCT) is a superior radiological method for performing X-ray of the chests., Ovaj članak ima za cilj prikazati dijagnostičke metode SARS-CoV-2 infekcije, kao i njihovu optimalnu primjenu. Glavne rutinske mikrobiološke dijagnostičke postupke predstavljaju molekularne i serološke metode. Osjetljivost i primjena molekularnih metoda najbolja je na početku bolesti, dok je primjena seroloških metoda najprikladnija barem 5-7 dana nakon početka bolesti. Pozitivan rezultat molekularnog testa (lančana reakcija polimeraze u stvarnom vremenu, engl. real-time polymerase chain reaction; RT-PCR) mora biti protumačen u skladu s kliničkom prezentacijom. Mjesto uzorkovanja za molekularnu analizu ovisi o težini bolesti; uzorci iz gornjeg i donjeg respiratornog trakta koriste se pri blagim i teškim prezentacijama, navedenim redoslijedom. Uzgoj virusa na staničnoj kulturi jedina je metoda koja se primjenjuje u svrhu dokazivanja živog virusa u uzorku tkiva, međutim, metoda se ne izvodi rutinski zbog složenosti metodologije. Promjene po tipu mliječnog stakla s ili bez retikularnog uzorka i/ili konsolidacija tipične su pojave kod COVID-19 upale pluća. Višeslojna kompjutorizirana tomografija (engl. multi-slice computerized tomography; MSCT) predstavlja superiornu metodu, u odnosu na klasični RTG srca i pluća.

A large variation in the severity of disease symptoms is one of the key open questions in coronavirus disease 2019 (COVID-19) pandemics. The fact that only a small subset of people infected with severe acute respiratory syndrome coronavirus 2 develops severe disease suggests that there have to be some predisposing factors, but biomarkers that reliably predict disease severity have not been found so far. Since overactivation of the immune system is implicated in a severe form of COVID-19 and the immunoglobulin G (IgG) glycosylation is known to be involved in the regulation of different immune processes, we evaluated the association of interindividual variation in IgG N-glycome composition with the severity of COVID-19. The analysis of 166 severe and 167 mild cases from hospitals in Spain, Italy and Portugal revealed statistically significant differences in the composition of the IgG N-glycome. The most notable difference was the decrease in bisecting N-acetylglucosamine in severe patients from all three cohorts. IgG galactosylation was also lower in severe cases in all cohorts, but the difference in galactosylation was not statistically significant after correction for multiple testing.

Francisella tularensisis is a causative agent of tularemia a zoonotic disease that infects wide range of hosts including arthropods, mammals and birds. In this study, the prevalence of tularemia among small mammals in Croatia was investigated. The 444 samples of small rodents and insectivores were collected in eight different localities in continental Croatia during the 2-year study. Spleen samples of: 197 Apodemus agrarius (striped field mouse), 78 Apodemus sylvaticus (wood mouse), 92 Apodemus flavicollis (yellow-necked mouse), 17 Myodes glareolus (bank vole), 27 Mycrotus agrestis (field vole), 20 Microtus arvalis (common vole) and 13 Sorex araneus (common shrew) were investigated for the presence of DNA of Francisella spp. using qRT-PCR method. Two striped field mouses and one wood mouse, originated from the same area - locality of Lipovljani, were found to be positive on Francisella spp. revealing the presence of bacteria among small mammals population in Croatia., Francisella tularensisis uzročnik je zoonoze tularemije, koja ima široki raspon domaćina, uključujući člankonošce, sisavce i ptice. Dvije podvrste F. tularensis, subsp. tularensis (Tip A) i subsp. holartica (Tip B), opisane su kao najčešći uzročnici bolesti u ljudi (Kingry and Petersen, 2014; Kuehn et al., 2013). Tip A i Tip B razlikuju se po svojim biokemijskim značajkama, patogenosti, ekologiji, epidemiologiji te geografskoj rasprostranjenosti. Rasprostranjenost Tipa A uglavnom je ograničena na sjevernoamerički kontinent, za razliku od Tipa B, koji se javlja duž cijele sjeverne polutke, a ujedno je i najčešći uzročnik tularemije u Europi(Keim et al., 2007; Kuehn et al., 2013). No ekologija i kruženje ove bakterije u prirodi do danas još nisu u potpunosti razjašnjeni. Različiti glodavci (voluharice, miševi, tekunice, bizantski štakori, dabrovi) te dvojezupci (kunić i zec) opisani su kao mogući rezervoari F.tularensis. Zadnja epidemiološka i epizootiološka istraživanja tularemije u Hrvatskoj datiraju iz šezdesetih i sedamdesetih godina prošloga stoljeća(Borcić et al., 1975). Stoga je cilj ovoga rada bio prikupiti nove podatke o prevalenciji tularemije u populaciji sitnih sisavaca u Hrvatskoj. Ukupno 444 uzoraka sitnih glodavaca i kukcojeda sakupljeno je u razdoblju od dvije godine na osam različitih lokaliteta u šumama kontinentalne Hrvatske: Lipovljani, Nova Subocka, Velika Gorica, Stara Gradiška, Županja, Sunja, Koprivnica i Čakovec (Slika 1). Pretraženi su uzorci slezene od: 197 Apodemus agrarius (poljski miš), 78 Apodemus sylvaticus (šumski miš), 92 Apodemus flavicollis (žutogrli miš), 17 Myodes glareolus (šumska voluharica), 27 Mycrotus agrestis (livadna voluharica), 20 Microtus arvalis (poljska voluharica) te 13 Sorex araneus (rovka). Uzorcisu pretraženi na prisutnost DNA Francisella spp. koristeći qRT-PCR metodu. Dobiveni rezultati pokazuju da su od ukupno 444 prikupljene životinje tri uzorka (0.67%), od kojih dva poljska miša te jedan šumski miš, bili pozitivni su na francizelu (Tablica 1). Sva tri pozitivna uzorka prikupljena su na istom području – lokalitetu Lipovljani. Tularemija je bolest koja se primarno povezuje s „nizinskim“ i „vodenim“ ekosustavima (Borcić, 1973). Područje duž toka rijeke Save (Posavina) opisuje se kao prirodno žarište tularemije u Hrvatskoj (Borcic et al., 1976), akao glavni rezervoar tularemije na tom području navodi se poljska voluharica (Borcic et al., 1976). Trimiša pozitivna na F. tularensis upućuju da je ova bakterija i dalje prisutna u populaciji sitnih glodavaca u Hrvatskoj. Nadalje, svi pozitivni uzorci prikupljeni su na lokalitetu Lipovljani, smještenom u srednjem dijelu toka rijeke Save, stoga ova regija zadržava obilježje prirodnog žarišta tularemije u Hrvatskoj.

This is the second subsequent year of West Nile neuroinvasive disease (WNND) outbreak in Croatia. Between July and October 2013, 22 patients presented with symptoms of WNND: all with meningitis and 18 additionally with encephalitis. In contrast to 2012, where six autochthonous infections were confirmed in eastern Croatia, the majority of this year's cases occurred in and around the city of Zagreb, where no West Nile virus infections have been observed before. Viral RNA was recovered from two patients and phylogenetic analyses revealed West Nile virus lineage 2. This represents the first molecular characterization and phylogenetic analysis of the circulating West Nile virus strain in Croatia.

The inability of RNA polymerase to perform proofreading during replication causes high mutation rates in Hantaviruses and other RNA viruses. Our goal was to clone and determine the sequence of the Puumala virus strain Sotkamo from WHO arbovirus collection (Dr. R. Tesh, UTMB) and to compare it to the published sequence. RNA from VeroE6 cells infected with Puumala virus strain Sotkamo was isolated using chloroform-phenol extraction and the cDNA was prepared using random hexamers. Viral sequences were amplified using specific primers and cloned into suitable vectors. These plasmids were sequenced using dye termination sequencing on Applied Biosystems Model 3100 Genetic Analyzer. All detected “mutations” were confirmed by direct sequencing of PCR products. The sequence obtained for the three segments showed more than 99% homology compared to the published genome for the Puumala Sotkamo strain. Sequencing showed seven mutations in the S segment, four in the non-coding region and three silent mutations in the open reading frame for the nucleocapsid protein. M segment contained eleven mutations in the coding region for Puumala glycoprotein, all but two caused amino acid changes. Only five mutations were observed in L segment, causing four amino acid changes in the 6471 nucleotides long open reading frame for the viral polymerase. Although only one clone was sequenced, all mutations were confirmed by direct sequencing. Interestingly, these mutations aren't spread evenly through the genome. The L segment shows proportionally the smallest number of mutations, whereas M and S segments have a higher but proportionally similar amount of changes. However, their potential effects differ within the genome segment. While 80% of mutations cause an amino acid change in the M segment, all mutations are silent in the S segment. The segment's non-coding region probably accumulates more nucleotide changes than the coding region. Acknowledgements: We thank J.W. LeDuc, PhD (University of Texas Medical Branch, Galveston, USA) for his support during this study. This study was prepared as a part of the following projects: 143-1430115-0103 and J-29-2007 (P.I. Dr. A. Markotić) supported by the Croatian Ministry of Sciences, Education and Sports. These materials are based on a thesis development funded by the National Foundation for Science, Higher Education and Technological Development of the Republic of Croatia.

Ebola virusna bolest (EVB) je opasna, karantenska zarazna bolest čija se do sada najveća zabilježena epidemija pojavila u zapadnoj Africi krajem 2013.g. i početkom 2014.g. te se proširila u pet zemalja regije (Gvineja, Liberija, Nigerija, Sierra Leone i Mali). Tjekom kasnog ljeta 2014 je došlo do nepovezane epidemije u Demokratskoj Republici Kongo. Postoji pet vrsta virusa ebole [Zaire (EBOV), Sudan (SUDV), Bundibugyo (BDBV), Taï Forest (TAFV) i Reston (RESTV)] koji su svi endemski u Africi, osim soja Reston, koji je endemski i u dijelovima jugoistočne Azije i Kini. Rezervoarom virusa ebole smatraju se voćni šišmiši (ili letipsi), kod kojih su dokazana ebolavirusna RNA i antitijela, no nisu definitivno potvrđeni kao glavni rezervoari EBOV. Imunopatogeneza ebole je kompleksna i zahtijeva ozbiljna istraživanja. Teški oblici bolesti praćeni su brojnim komplikacijama, multiorganskim zatajenjem i znacima septičkog šoka. Smrtnost se kreće od 55 – 75%, ponekad i više. Preživjeli prolaze dugo razdoblje rekonvalescencije. Za sada nema odobrenog cjepiva, a medicinsko osoblje pri liječenju i zbrinjavanju bolesnika s EVB mora primjenjivati propisane striktne mjere osobne zaštite. Liječenje je simptomatsko uz po potrebi mjere intenzivnog liječenja. Bez obzira na moguću ekonomsku neprofitabilnost, jasno pokazana potreba za razvojem dijagnostika, cjepiva i lijekova za liječenje ebole i drugih hemoragijskih vrućica, koji bi predstavljali garanciju sprječavanja širenja ovih opasnih bolesti., Ebola virus disease (EVD) is a dangerous and highly contagiuos infectious disease. The largest recorded outbreak so far occurred in West Africa in late 2013 and in early 2014 and spread to five countries of the region (Guinea, Liberia, Nigeria, Sierra Leone and Mali). During the late summer of 2014 an unrelated outbreak of Ebola virus disease occured in the Democratic Republic of Congo. There are five types of Ebola virus [Zaire (EBOV), Sudan (SUDV), Bundibugyo (BDBV), Taï Forest (TAFV) i Reston (RESTV)] all of which are endemic in Africa, except Reston ebolavirus, which is also endemic in parts of South East Asia and China. The reservoir of Ebola virus are considered to be fruit bats, in which Ebola virus RNA and antibodies were detected, but they are not definitively confirmed as the main reservoirs. Immunopathogenesis of Ebola virus disease is complex and requires serious research. Severe forms of the disease are accompanied by numerous complications, multiorgan failure and signs of septic shock. Mortality ranges from 55 – 75%, and sometimes more. Survivors undergo a long period of convalescence. So far there is no approved vaccine, and medical personnel who treat and care for patients with EVD must apply strict measures of proposed personal protection. Treatment is symptomatic with, if necessary, measures of intensive treatment. Regardless of the possible economic unprofitability, the need for the development of diagnostics, vaccines and drugs for the treatment of EVD and other hemorrhagic fevers is clearly demonstrated and would represent a mean to prevent future outbreaks of this dangerous disease.

SUMMARYAn outbreak of haemorrhagic fever with renal syndrome (HFRS) started on Medvednica mountain near Zagreb in January 2012. In order to detect the aetiological agent of the disease in small rodents and to make the link with the human outbreak, rodents were trapped at four different altitudes. Using nested RT–PCR, Puumala virus (PUUV) RNA was detected in 41/53 (77·4%) bank voles (Myodes glareolus) and Dobrava virus (DOBV) RNA was found in 6/61 (9·8%) yellow-necked mice (Apodemus flavicollis). Sequence analysis of a 341-nucleotide region of the PUUV S segment, obtained from all infected bank voles and five HFRS patients, showed 98·8–100% sequence similarity, indicating that the patients were probably exposed to PUUV on Medvednica mountain. A very large bank-vole population combined with an extremely high infection rate of PUUV was responsible for this unusual winter outbreak of HFRS in Croatia.

In Croatia, several rodent- and vector-borne agents are endemic and of medical importance. In this study, we investigated hantaviruses and, for the first time, tick-borne encephalitis virus (TBEV) and Rickettsia spp. in small wild rodents from two different sites (mountainous and lowland region) in Croatia. In total, 194 transudate and tissue samples from 170 rodents (A. flavicollis, n=115; A. agrarius, n=2; Myodes glareolus, n=53) were tested for antibodies by indirect immunofluorescence assays (IIFT) and for nucleic acids by conventional (hantaviruses) and real-time RT-/PCRs (TBEV and Rickettsia spp.). A total of 25.5% (24/94) of the rodents from the mountainous area revealed specific antibodies against hantaviruses. In all, 21.3% (20/94) of the samples from the mountainous area and 29.0% (9/31) from the lowland area yielded positive results for either Puumala virus (PUUV) or Dobrava-Belgrade virus (DOBV) using a conventional RT-PCR. All processed samples (n=194) were negative for TBEV by IIFT or real-time RT-PCR. Serological evidence of rickettsial infection was detected in 4.3% (4/94) rodents from the mountainous region. Another 3.2% (3/94) rodents were positive for Rickettsia spp. by real-time PCR. None of the rodents (n=76) from the lowland area were positive for Rickettsia spp. by real-time PCR. Dual infection of PUUV and Rickettsia spp. was found in one M. glareolus from the mountainous area by RT-PCR and real-time PCR, respectively. To our knowledge, this is the first detection of Rickettsia spp. in small rodents from Croatia. Phylogenetic analyses of S- and M-segment sequences obtained from the two study sites revealed well-supported subgroups in Croatian PUUV and DOBV. Although somewhat limited, our data showed occurrence and prevalence of PUUV, DOBV, and rickettsiae in Croatia. Further studies are warranted to confirm these data and to determine the Rickettsia species present in rodents in these areas.

West nile virus (WNV), family Flaviviridae, genus Flavivirus is a mosquito-borne virus that can cause influenza-like illness, termed West nile Fever (WNF) in approximately 20-40% infected patients, or West nile neuroinvasive disease (WNND) in 1% of cases. First human infections in Europe were observed in the 1960s. since the first outbreak in 1996 in Romania, clusters of cases and outbreaks were observed in several Mediterranean countries as well as in Hungary, Bulgaria and Romania. Serological evidence for WNV in Croatia dates back to 1980s, with seroprevalence ranging from 0.28 to 4.9%, and current data for the continental part showed similar seroprevalence. During September 2012, seven human cases of WNND in the eastern parts of Croatia were confirmed by Plaque reduction neutralization test. Between July and October 2013, 22 WNND patients were admitted at the University hospital for Infectious Diseases, Zagreb, all were confirmed or probable cases of WNND as defined by Croatian Public Health Authorities. Except one, all the patients were exposed to WNV in two north-western counties: City of Zagreb and Zagreb County, where until now no WNV infections were observed. Of all WNND cases, 18 (81.8%) presented with encephalitis and four (18.2%) with meningitis alone. Ten (45.5%) patients had tremor and/or ataxia. Ten WNND patients (45.5%) were admitted to the Intensive care unit where three patients were mechanically ventilated due to central respiratory failure. As of 10 October 2013, all patients survived WNND. All samples showed positive IgM and in nine available paired sera, specific IgG was detected. Cross-reactivity for TBEV was seen in five cases. Amplification of WNV RNA was unsuccessful in all available (54.5%) CSF and urine (77.3%) samples but nested RT-PCR amplified a WNV specific sequence from total RNA isolated from the blood of two patients. The sequences of the two obtained amplicons were 100% identical and phylogenetic analyses showed clustering with WNV lineage 2 strains from Austria, Greece, Hungary or Serbia. This represents the first molecular analysis of the circulating WNV strain in Croatia. Virus isolation of RT-PCR positive samples was so far unsuccessful. No evidence of WNF cases was documented, probably due to mild presentation. Considering that only 1% of infected persons develop WNND it seems probable that several hundred undocumented infections occurred this summer.

During August and September 2012, seven cases of West Nile neuroinvasive disease were identified in three north-eastern counties of Croatia. Four cases were reported in Osijek-Baranja County, two in Brod-Posavina County and one in Vukovar-Srijem County. The median age of the patients was 62.7 years. All patients were hospitalized for 2-5 weeks. The patients from Slavonski Brod had more severe clinical presentation of disease with prolonged hospitalization. Medical entomological research was carried out in 64 localities, where 1785 mosquitoes were captured. Among the analyzed mosquitoes, 114 were determined to be Culex pipiens and subjected to molecular characterization for the presence of virus. No viral RNA was detected in mosquitoes. Subsequent public health measures taken include mosquito control in all settlements where disease was detected.

In the summer of 2010, two autochthonous dengue fever cases were detected in Croatia. Here we report the retrospective detection of an additional case of dengue fever, representing the first sustained autochthonous transmission in Europe since 1928. In addition, we present the phylogenetic analyses based on two sequences from the Pelješac peninsula, southern Croatia. The sequences were identified as dengue virus genotype 1 and recovered from two out of the three Pelješac patients in whom infection occurred.

Emerging and re-emerging infectious diseases create constant and serious concerns for public health. The majority of emerging infectious diseases (EID) are wildlife zoonotic diseases and vector-borne diseases. Croatia has a long tradition in the control, management and research of EID zoonotic diseases and vector-borne diseases. There has also been a long and advantageous tradition in the collaboration of different experts and professionals in EID research in Croatia involving physician clinicians in infectious diseases, microbiologists, pathologists, veterinarians and animal scientists, ecologists, forestry experts, wildlife scientists, public health specialists and epidemiologists and laboratory scientists. The University Hospital for Infectious Diseases in Zagreb established the Centre for Emerging and Re-emerging Infectious Diseases in liaison with national and international partners from Europe and the United States. This Centre is working in line with the 'One Health initiative' which recognises the inter-relationships between human, animal and environmental health.

Dugo se smatralo da hantavirusi nisu citopatogeni virusi. Tek smo nedavno dokazali da patogeni hantavirusi izazivaju citopatski učinak i apoptozu u staničnoj liniji humanih epitelnih stanica bubrega embrija (HEK293). Cilj našeg rada je bio na razini ekspresije gena istražiti ulogu signalnog puta dušikovog oksida (NO) pri apoptozi uzrokovanoj patogenim hantavirusima: Hantaan i Andes u HEK293 stanicama. Genska ekspresija je analizirana metodom GEArray (GEArray, Superarray Bioscience, Frederic, MD, SAD). Naši rezultati su pokazali važnost uloge unutarstaničnog puta apoptoze pri infekciji HEK293 stanica patogenim hantavirusima, a virus Andes je imao veći učinak na promjene u genskoj ekspresiji od virusa Hantaan. Promjene u ekpresiji gena: ABCC1, BNIP3, CCL5, GCLC, HSPA4, MYC, PRKDC, PRMT3, p11, SLC6A6, TFRC vjerojatno sudjeluju u složenoj interakciji pro- i anti-apoptotičkih mehanizama tijekom infekcije HEK293 stanica patogenim hantavirusima. U nastavku naših istraživanja potrebno je na razini proteina utvrditi molekule odgovorne za pokretanje unutarnjeg puta apoptoze, te moguću interakciju s vanjskim putem apoptoze., For a long time it was believed that Hantaviruses don't cause cytopathic effect (CPE). Only recently it was proved that pathogenic hantaviruses induce CPE and apoptosis in the human embryonic kidney cells (HEK293). The goal of our paper was to examine the nitric-oxide (NO) signaling pathway on the level of gene expression during apoptosis caused by pathogenic hantaviruses: Hantaan virus and Andes virus in HEK293 cells. Gene expression was analyzed with a commercial DNA microarray. Our results showed the importance of the mitochondrial pathway of apoptosis in HEK293 cells infected with pathogenic hantaviruses, where Andes virus induced more changes in gene expression than Hantaan virus. Changes in gene expression: ABCC1, BNIP3, CCL5, GCLC, HSPA4, MYC, PRKDC, PRMT3, p11, SLC6A6, TFRC probably participate in the complex pro- and anti- apoptotic mechanisms during the infection of HEK293 with pathogenic hantaviruses. Further studies have to determine signal molecules of the mitochondrial pathway on the protein level and possible interactions with the extrinsic pathway.