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Potato virus Y (PVY, genus Potyvirus) is an economically important aphid-transmissible virus with a very wide host range reported in many tomato-growing areas (Rivarez et al. 2021). Potato virus S (PVS, genus Carlavirus) has a limited host range (Lin et al. 2014) and occurs in tomato (Predajňa et al. 2017), mostly in mixed infections with other viruses. In 2021, greenhouse tomatoes from Vidovec (46° 17’ 3.4’’ N, 16° 15’ 37.0’’ E) in the northwestern and Sedlarica (45° 54’ 23.0’’ N, 17° 12’ 0.5’’ E) in the eastern regions of Croatia were surveyed for virus-like diseases. In total, 30 plants were sampled (12 from Vidovec and 18 from Sedlarica) showing symptoms of mild mottling, leaf rugosity and mild bronzing followed by leaf necroses later in the season. Nucleic acids were extracted from leaves by adapted CTAB procedure (Murray and Thompson 1980) and DNase treated. Four representative samples from Vidovec and four from Sedlarica were pooled for high throughput sequencing (HTS). After rRNA depletion (RiboMinus™ Plant Kit for RNA-Seq, Invitrogen) and polyA tailing, two location specific libraries (PCR-cDNA sequencing kit, Oxford Nanopore Technologies) were prepared for nanopore HTS on MinION Mk1C device. From Vidovec samples, 459, 285 raw reads (mean length 354 nt) were obtained and 206, 718 (mean length 446 nt) from Sedlarica and mapped (Minimap2, v.2.17) against Kraken2 viral genome sequences database (https://benlangmead.github.io/aws-indexes/k2).The number of reads mapped to PVS genome was 1004 from Vidovec (coverage depth 1.56) and those mapped to PVY genome were 781 (coverage depth 0.99) and 57 (coverage depth 1), from Vidovec and Sedlarica, respectively. The PVS complete consensus genome from Vidovec (ON468562, 8485 nt) had 99.09% nucleotide identity (BLASTn) to a potato isolate from the Netherlands (MF418030). The PVY consensus genome sequences from Vidovec (ON505007, 9698 nt) and Sedlarica (ON505008, 9698 nt) had respectively 98.37% and 98.48% identities to a tomato isolate from Slovakia (MW685827). Reverse transcription polymerase chain reaction (RT-PCR) was performed for all 30 samples and amplicons were Sanger sequenced, with primers PVS-7773F/PVS-3’endR for a 720 nt PVS genome portion spanning the 3’-part of the CP and a complete 11K gene (Lin et al. 2014) and PVY-2F/2R primers for a 510 nt portion of PVY CP gene (Aramburu et al. 2006). Only one tomato out of 12 (‘Borana’) from Vidovec harbored PVS in the mixed infection with PVY. Two additional tomatoes from Vidovec and two from Sedlarica were infected solely by PVY. Amplicon sequences of PVS (ON651427) and PVY (ON707000-4, ON734067-8) had 100% identity with the HTS assembled sequences. The PVS isolate from Croatia grouped with PVSO (ordinary) strain in phylogenetic analysis and the PVY isolates from both sites grouped with the PVY-NTN strain (Cox and Jones 2010). Although PVY is considered to be widespread in tomato (Nikolić et al. 2018 ; Rivarez et al. 2021), this is its first report from Croatia. PVS, newly reported from Croatia here, is probably not associated with the symptoms recorded because the same symptomatology was observed in the singly and mixed infected ‘Borana’ tomato plants. The occurrence of PVY in the geographically distant (100 km apart) Vidovec and Sedlarica, suggests that Page 1 of 2it is widespread in the continental Croatia where tomatoes are commercially grown in plastic greenhouses. Further analyses are needed to elucidate PVY and PVS epidemiology and impact on the local tomato production.

Asymptomatic Plectranthus scutellarioides (syn. Coleus blumei) plants of four different cultivars were investigated for the presence of Coleus blumei viroid (CbVd) species (genus Coleviroid, family Pospiviroidae). Cloned and sequenced amplicons obtained by generic coleviroid primers were analyzed for their primary and secondary structure. CbVd-1 was found in one of the coleus cultivars in a single infection and in another cultivar in a mixed infection with CbVd-3. Additionally, CbVd-3 was found in a third coleus cultivar in a single infection. In singly and doubly infected cultivars, both CbVd-1 and CbVd-3 variants displayed higher variability regarding both length and nucleotide composition in a doubly infected cultivar. Sequences from different cultivars showed different phylogenetic grouping suggesting distinct infection sources, probably introduced infected seeds. For all phylogenetic subgroups, characteristic nucleotide changes were identified and their influence on the secondary structure was predicted. The most noticeable structural changes were observed in nucleotide positions 25–26 in CbVd-1 and in the position 138 in CbVd-3 variants. This work includes the first report of coleviroids in Croatia, the second reported coleviroids sequences from Europe and the third report of CbVd-3 in the world enlarging the pool of knowledge on the variability and distribution of viroids in horticultural crops., This study was funded by the University of Zagreb grants to D. Škorić and S. Černi numbers: 20281214, 20282308 and 20283119.

Significance Our article presents the most comprehensive reconstruction of the evolutionary and phylogeographic history of a major plant pathogen of brassica vegetables in Eurasia. Sampling across such a large landmass poses considerable challenges, and our study attempts to describe the spatial and temporal patterns of migration for a plant pathogen on a large scale. Our phylogeographic and molecular clock analyses show that the migration pathways of turnip mosaic potyvirus retrace some of the historical trade arteries of the Silk Road. This study demonstrates how a comprehensive genetic analysis can provide a large-scale view of the epidemiology and human-mediated spread of a plant pathogen across centuries of evolutionary history., Plant pathogens have agricultural impacts on a global scale and resolving the timing and route of their spread can aid crop protection and inform control strategies. However, the evolutionary and phylogeographic history of plant pathogens in Eurasia remains largely unknown because of the difficulties in sampling across such a large landmass. Here, we show that turnip mosaic potyvirus (TuMV), a significant pathogen of brassica crops, spread from west to east across Eurasia from about the 17th century CE. We used a Bayesian phylogenetic approach to analyze 579 whole genome sequences and up to 713 partial sequences of TuMV, including 122 previously unknown genome sequences from isolates that we collected over the past five decades. Our phylogeographic and molecular clock analyses showed that TuMV isolates of the Asian-Brassica/Raphanus (BR) and basal-BR groups and world-Brassica3 (B3) subgroup spread from the center of emergence to the rest of Eurasia in relation to the host plants grown in each country. The migration pathways of TuMV have retraced some of the major historical trade arteries in Eurasia, a network that formed the Silk Road, and the regional variation of the virus is partly characterized by different type patterns of recombinants. Our study presents a complex and detailed picture of the timescale and major transmission routes of an important plant pathogen.

Although Croatia is not often perceived as a citrus growing country, citrus species have been planted in the Croatian southern coastal part for centuries. Citrus had always been important as a source of vitamins and micronutrients for local consumption, but with the introduction of Satsuma mandarins (Citrus unshiu Marc.) citrus production started its commercial development. The Croatian coast is probably the northernmost commercial citrus growing area globally with several agroecological constraints influencing citrus production. However, Satsuma mandarins, the most cold-tolerant citrus of economic interest, are successfully cultivated resulting in an excellent quality of fruits that easily finds a market niche. Unfortunately, with the import of plant material in the last century, the simultaneous introduction of viral and subviral pathogens occurred. The most important are Citrus tristeza virus and Citrus exocortis viroid. As certain rootstock-scion combinations are tolerant to virus or viroid infections and display no obvious symptoms, these pathogens have been continuously spreading by plant propagation. Molecular and biological analyses revealed the existence of pathotypes not believed to be endemically present in the Mediterranean region. Their high intra-isolate genetic variability and frequent coinfections suggest that this geographical region presents a sort of a biological enclave of potentially threatening pathogens. Their transmission from this agroecological niche to areas where sensitive rootstock-scion combinations are common presents a serious risk to citrus production, especially when global warming and the resulting migration of efficient vector species are considered. Comprehensive pathogen monitoring is important for improving citrus production and the prevention of pathogen transmission.

Since the first description of patients with pneumonia of unknown origin in Wuhan in December 2019, unprecedented efforts of the international scientific community led to the identification and molecular characterization of its etiological agent, e.g. SARS-CoV-2. The global pandemic of COVID-19 represents an outstanding challenge for the scientists and medical professionals worldwide. In this review, we discuss the most important aspects of SARS-CoV-2 biology and virology including antiviral and immunomodulatory treatment strategies as well as vaccine development.

The propagation materials such as rootstocks, cuttings and other types of grafting materials used as scions play a relevant role in the dissemination of phytoplasma-associated diseases. In particular in the woody plants the propagation material sanitary status plays an important role for both long-distance transmission and disease introduction in the new areas. Since the phytoplasma infection is systemic in the plants, the vegetative propagation of many horticultural crops allows their spread through cuttings, bud wood, tubers, runners and bulbs. It is, therefore, an efficient method of phytoplasma spreading and establishing infection in new plants. Although the phytoplasma spread through vegetative plant propagation occur over short distances by the use of infected propagation materials such as tubers, the worldwide movement of phytoplasmas should be mainly attributed to the man distributing infected propagation materials. The possibility for the phytoplasma vegetative propagation is present in all the shoots and roots comprizing basal shoots, stems, rhizomes, tubers, stolons, corms, buds and bulbs. Some crops like potato, sweet potato, cassava, carrot, onion, garlic, ginger, sugarcane, banana, pineapple, strawberry and many ornamentals like carnations and Chrysanthemum are only vegetatively propagated and hence they have the maximum chances of phytoplasma spread. The fruit tree propagation is usually achieved by grafting or budding of the selected variety onto a suitable rootstock, and this is the main propagation method for the stone and pome fruit trees, grapevine and other fruit trees and shrubs. Also the shoot micropropagation together with grafting, cutting, and other systems to propagate plant germplasm that avoid sexual reproduction is an efficient manner to maintain and transmit the phytoplasma diseases. The importance of phytoplasma infection spread by the vegetatively propagated plants is discussed in this chapter.

The wild boar is an ancestor of the domestic pig and an important game species with the widest geographical range of all ungulates. Although a large amount of data are available on major histocompatibility complex (MHC) variability in domestic pigs, only a few studies have been performed on wild boars. Due to their crucial role in appropriate immune responses and extreme polymorphism, MHC genes represent some of the best candidates for studying the processes of adaptive evolution. Here, we present the results on the variability and evolution of the entire MHC class II SLA- DRB1 locus exon 2 in 133 wild boars from Croatia. Using direct sequencing and cloning methods, we identified 20 SLA-DRB1 alleles, including eight new variants, with notable divergence. In some individuals, we documented functional locus duplication, and SLA- DRB1*04:10 was identified as the allele involved in the duplication. The expression of a duplicated locus was confirmed by cloning and sequencing cDNA- derived amplicons. Based on individual genotypes, we were able to assume that alleles SLA-DRB1*04:10 and SLA- DRB1*06:07 are linked as an allelic combination that co-evolves as a two-locus haplotype. Our investigation of evolutionary processes at the SLA-DRB1 locus confirmed the role of intralocus recombination in generating allelic variability, whereas tests of positive selection based on the dN/dS (nonsynonymous/synonymous substitution rate ratio) test revealed atypically weak and ambiguous signals.

UMR BFP - Equipe Mollicutes; International audience; Flavescence dorée phytoplasma (FDp) is a quarantine pathogen associated with a severe and epidemic grapevine yellows disease representing a great threat for grapevine cultivation in Europe. An increase in disease spread prompted efforts to identify FDp strains in Croatia. Over 800 samples of grapevine together with presumed reservoir plants and almost 400 samples of Scaphoideus titanus and other potential vectors were collected countrywide and analysed. FDp isolates were characterized by multilocus sequence typing (MLST) of map, secY and uvrB‐degV genes in order to determine genetic diversity and structure of FDp populations, and to trace transmission pathways. FD‐related phytoplasmas were found in Croatia for the first time in alder, the invasive tree species Ailanthus altissima and leafhopper Phlogotettix cyclops. Phylogenetic analysis revealed the presence of three mapFD strain clusters: mapFD1, mapFD2 and mapFD3, and for the first time in Croatia a case of Palatinate grapevine yellows strain A (PGY‐A). In total, 7 different map, 10 secY and 11 uvrB‐degV genotypes were detected. The identification of 15 comprehensive FDp genotypes based on MLST suggests separate routes for disease introduction and propagation origins in Croatia. Moreover, high genetic variability of Croatian isolates indicates a complex ecological cycle of FDp involving various hosts.

Summary Background Acinetobacter baumannii is a leading emerging pathogen that is frequently recovered from patients during hospital outbreaks. The role of environmental A. baumannii reservoirs is therefore of great concern worldwide. Aim To investigate the connection between A. baumannii causing hospital outbreaks and environmental isolates from hospital wastewater, urban sewage and river water as the final natural recipient of wastewaters. Methods Clinical isolates from patients with hospital-acquired pneumonia and environmental isolates from water were collected during a two-month monitoring period. Recovery of A. baumannii was performed using CHROMagar Acinetobacter plates, incubated at 42°C for 48 h. Identification was performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and analyses of rpoB gene. The antibiotic resistance profiles were interpreted according to criteria given for clinical isolates of A. baumannii . The sequence types (ST) were retrieved by multi-locus sequence typing. Results Fourteen of 19 isolates recovered from patients, hospital wastewaters, urban sewage and river water belonged to ST-195. The remaining five isolates recovered from patients and river water were assigned to ST-1421. All isolates showed very strong relatedness and clustered into CC92, which corresponds to IC2. All isolates were non-susceptible to at least one agent in all but two or fewer antimicrobial categories, and thus were classified as ‘extensively-drug-resistant' (XDR). Heteroresistance to colistin was found in two isolates from hospital wastewater. Conclusion Close relatedness of clinical and environmental isolates suggests the emission of XDR A. baumannii via the untreated hospital wastewater in the natural environment.

Recent advances in high-throughput sequencing technologies and bioinformatics have generated huge new opportunities for discovering and diagnosing plant viruses and viroids. Plant virology has undoubtedly benefited from these new methodologies, but at the same time, faces now substantial bottlenecks, namely the biological characterization of the newly discovered viruses and the analysis of their impact at the biosecurity, commercial, regulatory, and scientific levels. This paper proposes a scaled and progressive scientific framework for efficient biological characterization and risk assessment when a previously known or a new plant virus is detected by next generation sequencing (NGS) technologies. Four case studies are also presented to illustrate the need for such a framework, and to discuss the scenarios.

The biology of viroids is a wide term encompassing biological phenomena exerted by these noncoding, 246–401 nt long, single-stranded circular RNAs. As viroids interfere with plant transcription and trafficking machinery, the term commonly comprises plant host range, symptomatology, distribution within a host, and dispersal in the environment (transmission modes). A viroid’s capacity to replicate and evolve is affected by diverse environmental factors, including biotic ones, encountered within dissimilar hosts and in simultaneous infections with different viroids, viruses, or other pathogens. The knowledge of viroid biology ultimately enables us to understand their ecology, evolution, pathogenicity, and to control the adverse effects of viroid diseases in host crops.

Potato spindle tuber viroid (PSTVd) is the causal agent of a number of agriculturally important diseases. It is a single-stranded, circular and unencapsidated RNA molecule with only 356-360 nucleotides and no coding capacity. Because of its peculiar structural features, it is very stable ex vivo and it is easily transmitted mechanically by contaminated hands, tools, machinery, etc. In this work, we describe the development and optimization of a method for concentrating PSTVd using Convective Interaction Media (CIM) monolithic columns. The ion-exchange chromatography on diethylamine (DEAE) monolithic analytical column (CIMac DEAE-0.1 mL) resulted in up to 30% PSTVd recovery whilst the hydrophobic interaction chromatography on C4 monolithic analytical column (CIMac C4-0.1 mL) improved it up to 60%. This was due to the fact that the binding of the viroid to the C4 matrix was less strong than to the highly charged anion-exchange matrix and could be easier and more completely eluted under the applied chromatographic conditions. Based on these preliminary results, a C4 HLD-1 (High Ligand Density) 1 mL monolithic tube column was selected for further experiments. One-litre-water samples were mixed with different viroid quantities and loaded onto the column. By using reverse transcription quantitative polymerase chain reaction (RT-qPCR), the viroid RNA was quantified in the elution fraction (≈5 mL) indicating that 70% of the viroid was recovered and concentrated by at least two orders of magnitude. This approach will be helpful in screening irrigation waters and/or hydroponic systems' nutrient solutions for the presence of even extremely low concentrations of PSTVd.

Citrus tristeza virus (CTV) is the most destructive viral pathogen of citrus and displays a high level of genetic and phenotypic diversity. In this study the biological characterization of monophyletic CTV-isolates based on p25 gene (Gp 1, Gp 2, Gp 3a, Gp 4, Gp 5, Gp M) was analyzed for the first time on the set of standard indicator plants and unde r the same environmental conditions, in order to compare the phenotypic characteristics of p25 genomic variants. The results showed that tested CTV-isolates varied in their ability to induced symptoms as well as in severity of symptoms e. g. pathogenicity. (C) 2012 Elsevier Ltd. All rights reserved.

The reaction to Citrus tristeza virus (CTV) infection was investigated by different diagnostic methods in 13 Satsuma (Citrus unshiu (Mak.) Marc.) symptomless clones. Optical density after performed ELISA (Enzyme Linked Immunosorbent Assay), intensity of colouration after DTBIA (Direct Tissue Blot Immunoassay), and the severity of symptoms in biological testing were compared in order to correlate intensities of these three reactions and the reliability of the obtained results, as well as to correlate serological results to tristeza disease symptoms on indicator plants. ELISA and DTBIA were performed directly on collection plants (clones) and on inoculated Mexican lime tissue samples, while biological indexing was performed on four main citrus indicators. Additional goal of this study was to evaluate the efficiency of CTV DTBIA testing using different tissue types, including Satsuma immature ovaries and flower petioles. ELISA performed on spring shoots tissue of Mexican lime and DTBIA performed on mature ovaries of Satsuma field samples, showed high intensity of reactions and detection efficiency. Therefore, if applied accordingly, both tests are equally reliable and make a good choice for fast CTV detection. Expectedly, for further biological characterization of CTV isolates, biological indexing needs to be performed.

A survey started in 2002 (Križanac et al. 2008) was continued to determine the diversity of phytoplasmas infecting fruit trees and their vectors in Croatia. Leaf and twig samples from 32 commercial orchards located in all major fruit growing regions of Croatia, were visually inspected for symptoms and tested for the presence of the phytoplasma 16S rDNA as were adjacent symptomless wild Prunus species, and potential insect vectors from seven orchards. Phytoplasmas belonging to ‘Candidatus Phytoplasma pyri’ and ‘Ca. P. prunorum’ (riboso-mal subgroups 16SrX-C and 16SrX-B, respectively) were the most widespread and had the highest incidence in pears and stone fruits, respectively. ‘Ca. P. asteris’ (16SrI-B) was found sporadically in both fruit trees and vectors, while phytoplas-mas of the stolbur group (16SrXII-A) was frequently found in pear. Mixed infections were detected in one pear and one Japanese plum. The high incidence and wide geographical distribution of stolbur phytoplasma in pears along with its presence in Cacopsyla pyri, the main vector of pear phytoplasma, calls for investigation of the role of this phytoplasma in the development of pear decline in Croatia. Cacopsylla pyrisuga, a suspected ‘Ca. P. pyri’ psyllid vector, was also found to harbour stolbur phytoplasma. This study revealed higher phytoplasma diversity in fruit trees and vectors than anticipated.

In effort to determine the incidence and distribution of plum pox virus (PPV) in stone fruit orchards throughout the country, a survey was carried out in the year 2004. Solitary Prunus trees found as a part of spontaneous vegetation were also included in this study. After visual inspections, samples were taken mostly from trees showing sharka symptoms. Total of 367 samples were ELISA tested by using universal and specific (PPV-D, PPV-M, PPV-EA and PPV-Ch) antibodies. Over fifty percent (54, 49%) of these samples were PPV positive. The incidence of PPV in 253 symptomatic samples was 69, 56% while reaching 21, 05% in symptomless ones. Fifty samples (33 symptomatic and 17 symptomless) were retested using IC/RT-PCR and, in total, 60% was positive (78, 78% symptomatic and 23, 52% symptomless). PPV is present in every Croatian stone fruit growing region, with the exception of some isolated zones in the central part of the country (Lika). However, its incidence varies with respect to tested Prunus species. In continental northeastern part of Croatia, plum is widely infected, while infected peach samples can be found only in very restricted part of this region. Infected sweet cherry trees are mainly present in the northern part of the country. Apricots that harbor PPV, on the other hand, have been found only along the Adriatic cost. The presence of PPV-M strain has been confirmed by using serological and IC/RT-PCR/RFLP assays in all geographical regions. PPV-D strain has been confirmed only in Istra (northern coastal part) and Međimurje (northern continental part). PPV-M strain has been found in plum, peach, nectarine, sweet cherry and in spontaneous P. myrabolana trees. PPV-D strain has been confirmed only in peach and P. myrabolana. These findings, in conjunction with aphid populations monitoring, will be used in devising protection measures and strategies for PPV control.

Replicative double-stranded RNA (dsRNA) is useful in preliminary identification of Cucumber mosaic virus and its satellite RNA (satRNA). This plant pathogen complex yields sufficient quantity of the replicative RNA form that can be isolated by chromatography on chemically unmodified graded cellulose powder (CF-11). In this work, much faster and more efficient procedure using DEAE monoliths was developed in which dsRNA was separated from other species in total nucleic acids extract originating from the infected plant tissue. The developed chromatographic method revealed the pathogens' presence in only 15 min, avoiding nucleic acid precipitation and electrophoretic analysis.

Hepatitis E is becoming a growing health concern in European countries as an increase of sporadic human cases of unknown origin has been recorded lately. Its causative agent, Hepatitis E virus (HEV), is known to have zoonotic potential and thus the role of domestic and wild animals in the chain of viral spread should be considered when investigating risk factors and the epidemiology of the disease. A comprehensive survey based on viral RNA detection was carried out in Croatia including blood, spleen and liver samples originating from 1816 different domestic and wild animals and digestive gland samples from 538 molluscs. A high HEV prevalence was detected in domestic pigs (24.5 %) and wild boars (12.3 %), whereas cattle, molluscs, ruminant and carnivore wildlife samples tested negative. Molecular characterization of both ORF1 and ORF2 genomic regions confirmed the phylogenetic clustering of the obtained sequences into genotype 3, previously reported in Europe. Furthermore, our results proved the presence of identical sequence variants in different samples, regardless of their origin, age or habitat of the host, suggesting transmission events between domestic swine, as well as between domestic swine and wild boars in the country. Moreover, a close genetic relationship of Croatian animal strains and known human HEV strains from GenBank opens the question of possible cross-species HEV transmission in Croatia, especially in the areas with an intensive swine production.

Genetic heterogeneity of RNA populations influences virus pathogenesis, epidemiology and evolution. Therefore, accurate information regarding virus genetic structure is highly important for both diagnostic and scientific purposes. For the Hepatitis E virus (HEV), the causal agent of hepatitis in humans, the intra-host population structure has been poorly investigated, mainly using the less sensitive RFLP-based approach. The objective of this study was to assess the suitability and the accuracy of single strand conformation polymorphism (SSCP) analysis, a well-established tool in genetic variation research, for the characterization of HEV quasispecies. The analysis was conducted on 50 clones of five swine isolates and 30 clones of three human HEV isolates. To identify and quantify the sequence variants present in each HEV isolate, 348 bp long fragments of the amplified conserved ORF2 region were separated by cloning. Ten clones per isolate were subjected to SSCP and sequenced in a parallel experiment. The results show a high correlation of SSCP haplotype profiling with the sequencing results, confirming the sensitivity and reliability of this simple, rapid and low cost approach in the characterization of HEV quasispecies.

Aim Molecular characterization of a pathogenic complex infecting winter oilseed rape (Brassica napus ssp. oleifera (DC.) Metzg.) plants showing typical rape phyllody symptoms along with some atypical changes. Methods and Results Phytoplasma (‘Candidatus Phytoplasma’) presence was confirmed by PCR-RFLP and 16S rRNA gene sequencing. Phylogenetic analyses of phytoplasma amp, tufB, secY, groEL and ribosomal protein genes confirmed its affiliation to the ‘Ca. P. asteris’ species. However, in the amp gene encoding a specific protein crucial for insect transmission specificity, significant SNPs were found. Biological and serological tests revealed the co-infection with Turnip mosaic virus (TuMV). The phylogenetic analysis of full TuMV genome sequence, the first reported from the Balkans, classified it into the world-B phylogenetic lineage. Conclusions A pathogenic complex consisting of ‘Ca. P. asteris’ and TuMV found to co-infect oilseed rape plants for the first time was molecularly characterized. Significance and Impact of the Study Rape phyllody is a serious problem in rapeseed production. The molecular information from this first multi-gene analysis of ‘Ca. P. asteris’ strain associated with rape phyllody as well as the first report of the complete sequence of TuMV isolate from the Balkans is a starting point for understanding the disease complexity and management.

Natural selection of two new variants of citrus exocortis viroid (CEVd) was detected by observing tissues displaying both severe and mild symptoms from a single Gynura aurantiaca. The variants CEVd-S (severe) and CEVd-M (mild), differing by only five nucleotides confined to the pathogenic (P) domain, remained stable when propagated by rooted cuttings or from successive plants inoculated with tissue extracts or transcripts from cDNA clones. CEVd-S induces a very severe reaction in Gynura that is consistent throughout a range of environmental conditions. However, symptoms resulting from CEVd-M infection can vary from a nonsymptomatic condition to a severe reaction when grown at 40°C. This differential response was confined to a single host, Gynura aurantiaca, and expressed under standard growing conditions. The distinct host responses induced by these variants could not be correlated with any changes in sequence or conformation of the dominant viroid variant, as predicted by molecular modeling. Therefore, the variable symptom expression appears to be associated with a specific temperature-sensitive response of Gynura aurantiaca.

Visual symptom assesment, polymerase chain reaction amplification and restriction fragment length polymorphism analyses were used to detect and identify phytoplasmas infecting grapevines in Croatia. Samples were collected from different viticultural areas in order to examine geographic distribution of phytoplasmas throughout the country. Only phytoplasmas belonging to Bois Noir (subgroup 16SrXII-A or stolbur) were found in vineyards of continental Croatia. The fact that no phytoplasmas were detected in Dalmatia and Istria was in accordance with the absence of grapevine yellows symptoms in these regions.

Single-strand conformation polymorphism (SSCP) analysis is a sensitive and rapid technique for detecting DNA polymorphisms and mutations in PCR-amplified fragments. Due to its technical simplicity, it is widely used as a screening tool in various investigations, ranging from clinical diagnosis of human hereditary diseases to the characterization of microbial communities. This method can also be used successfully on phytoplasmas as a tool for the detection of molecular variability in conserved housekeeping genes such as 16S rRNA and tuf, as well as in more variable genes, revealing the presence of polymorphisms undetected by routine RFLP analyses. The reliability of SSCP has been confirmed by multiple alignments and phylogenetic analyses of representative sequences showing different SSCP profiles. However, it is not broadly applied in phytoplasma research yet. The technique provides an inexpensive, convenient, and sensitive method for determining sequence variation and to differentiate phytoplasma strains, and is particularly suitable for epidemiological studies or as a fast screening, typing tool when dealing with a large number of field samples.

Citrus exocortis viroid (CEVd) is an economically important plant pathogen, which infects a broad range of hosts. In order to investigate complex interactions among viroid, host-growing conditions, and plant secondary metabolism, we setup an in vitro system for the cultivation of CEVd-infected and viroid-free Gynura aurantiaca (Blume) DC. shoots. Both basal Murashige and Skoog (MS) medium and MS medium supplemented with cytokinines supported the growth of shoots, but viroid-free shoots performed better than infected ones. The addition of cytokinines induced an increase in the concentration of purple-reddish pigment, cyanidin tetra-glucoside (GAA), which did not depend on the presence of viroid. The differences in single strand conformation polymorphism patterns between CEVd sequence variants obtained from plants grown on medium supplemented with cytokinines and those obtained from plants grown on basal MS medium or in the greenhouse, indicated that cytokinines may influence not only the plant metabolism, but also the viroid population structure.

Leaf and branch samples collected from 10 Populus nigra L. &#8216 ; Italica&#8217 ; trees were taken from the urban Zagreb area in late summer/autumn 2001. One of the trees exhibited leaf yellowing, overall sparse foliage, stunting and decline. Phytoplasma 16S rDNA was amplified in direct and nested PCR assays using universal and specific phytoplasma primer pairs, from nucleic acids extracted by two different procedures. Strong amplification signals were observed in sample from symptomatic Lombardy poplar as well as in samples from 4 of the asymptomatic trees. RFLP analyses of amplicons showed patterns characteristic of the phytoplasmas belonging to the Aster yellows group (16SrI). This is the first report of a phytoplasma naturally infecting poplar in Croatia.

The genetic diversity of three temperate fruit tree phytoplasmas ‘Candidatus Phytoplasma prunorum’, ‘Ca. P. mali’ and ‘Ca. P. pyri’ has been established by multilocus sequence analysis. Among the four genetic loci used, the genes imp and aceF distinguished 30 and 24 genotypes, respectively, and showed the highest variability. Percentage of substitution for imp ranged from 50 to 68 % according to species. Percentage of substitution varied between 9 and 12 % for aceF, whereas it was between 5 and 6 % for pnp and secY. In the case of ‘Ca P. prunorum’ the three most prevalent aceF genotypes were detected in both plants and insect vectors, confirming that the prevalent isolates are propagated by insects. The four isolates known to be hypo-virulent had the same aceF sequence, indicating a possible monophyletic origin. Haplotype network reconstructed by eBURST revealed that among the 34 haplotypes of ‘Ca. P. prunorum’, the four hypo-virulent isolates also grouped together in the same clade. Genotyping of some Spanish and Azerbaijanese ‘Ca. P. pyri’ isolates showed that they shared some alleles with ‘Ca. P. prunorum’, supporting for the first time to our knowledge, the existence of inter-species recombination between these two species.

Preliminary surveys of citrus field trees in Croatia showed their unsatisfactory sanitary status especially with regard to the presence of Citrus tristeza virus (CTV) even though symptoms in the field are mostly absent. The highest CTV incidence was recorded in the Satsuma mandarin (Citrus unshiu Marc), commercially the most important citrus in the country. The Satsuma collection orchard was founded in the year 2005 at the Institute for Adriatic Crops and Karst Reclamation. It encompasses 12 mandarin cultivars and 51 Satsuma clones from different sources. With the aim of assessing the phytosanitary status of Satsuma collection, biological indexing of all 12 cultivars for principal virus and virus-like diseases was performed. Seven citrus bioindicators were used ; Mexican lime, Madame Vinous and Pineapple sweet oranges, Sicilian sour orange, CRC grapefruit, Troyer and Carrizo citranges. Herbaceous hosts (white sesame, red kidney bean and Chenopodium quinoa) were also used for cool-climate pathogen detection. Serological tests (ELISA and DTBIA) were performed for CTV and Citrus psorosis virus (CPsV) directly on 18 representative clones and on Mexican lime and Madame Vinous indicators. CPsV was not detected by serology while only 2 clones were CTV-negative, both in biological and laboratory tests. Furthermore, the most severe CTV strains displaying stem-pitting and seedling yellows in grapefruit and sweet oranges were found in 4 tested clones. No biological indication of other disease presence in the collection has been found so far. Further testing of the collection Satsumas is under way, as well as the sanitation of the most important cultivars.

Citrus tristeza virus (CTV) represents one of the major threats to citrus production worldwide. In the East Adriatic region, CTV symptoms are mostly absent due to traditional citrus grafting on trifoliate orange (Poncirus trifoliata), a CTV-tolerant rootstock. Therefore, the virus has been continuously spreading by the propagation of infected material. The genetic variability of CTV was studied on nineteen citrus samples, collected from orchards in the coastal region of Croatia, Montenegro and Albania, that previously tested positive by ELISA and immunocapture RT-PCR. Single-strand conformation polymorphism of the amplified coat protein gene demonstrated the presence of different CTV variants in each amplicon, while sequence analysis of cloned CP gene variants confirmed their clustering into six out of the seven phylogenetic groups so far delineated. Four of these groups include sequences of severe quick decline, seedling yellows and stem-pitting (SP) isolates, thought to be found only rarely in the Mediterranean region. Regardless of the lack of symptoms in the field, CTV isolates from the East Adriatic displayed high genetic variability and pathogenic potential, additionally confirmed by biological characterisation. The high percentage of mixed infections suggest the potential for further diversification and a greater risk of severe variants spreading into new areas.

The isolates of Citrus tristeza virus (CTV), the most destructive viral pathogen of citrus, display a high level of variability. As a result of genetic bottleneck induced by the bud-inoculation of CTV-infected material, inoculated seedlings of Citrus wilsonii Tanaka displayed different symptoms. All successfully grafted plants showed severe symptoms of stem pitting and seedling yellows, while plants in which inoculated buds died displayed mild symptoms. Since complex CTV population structure was detected in the parental host, the aim of this work was to investigate how it changed after the virus transmission, and to correlate it with observed symptoms. The coat protein gene sequence of the predominant genotype was identical in parental and grafted plants and clustered to the phylogenetic group 5 encompassing severe reference isolates. In seedlings displaying severe symptoms, the low-frequency variants clustering to other phylogenetic groups were detected, as well. Indicator plants were inoculated with buds taken from unsuccessfully grafted C. wilsonii seedlings. Surprisingly, they displayed no severe symptoms despite the presence of phylogenetic group 5 genomic variants. The results suggest that the appearance of severe symptoms in this case is probably induced by a complex CTV population structure found in seedlings displaying severe symptoms, and not directly by the predominant genomic variant.

About 30 citrus field samples (Satsuma mandarins, oranges, lemons) were collected from different orchards in the Croatian coastal region (Dalmatia), including one remote island. The majority of these trees was grafted on Poncirus trifoliata and displayed no symptoms of Citrus tristeza virus (CTV) infection. However, about 50% of tested trees were found CTV infected. The genetic variability and population structure of CTV from these samples were studied by single-strand conformation polymorphism (SSCP) and sequence analysis of the cloned coat protein (p25) and RNA-binding protein (p23) genes. In the majority of isolates, CTV populations consisted of a swarm of genomic variants with the one being predominant. Surprisingly, the results of phylogenetic analysis, corroborated by a biological characterization, showed that both CP and p23 sequences clustered into five out of seven phylogenetic groups defined by Zemzami et al. (Proc. 15th IOCV, 8-12, 2002). Three of these phylogenetic groups harbor sequences of severe quick decline, seedling yellows and stem pitting isolates. It has been generally believed that some of these strains do not exist in the Mediterranean region, or if found, they are immediately eradicated. It is possible that these CTV strains were introduced into the country with the infected Satsuma material 40-20 years ago and has been continuously spreading by grafting. We also found evidence of mixed infections and recombination events in our isolates. High genetic diversity of Croatian CTV isolates and their potential for further diversification should not be overlooked in the CTV epidemiological studies of the Mediterranean basin.

Surveys of pomaceous fruit trees were carried out in the years 2003 and 2004 in order to determine the current status of 'Candidatus Phytoplasma pyri' (pear decline, PD) and 'Ca. P. mali' (apple proliferation, AP) in Croatia. PD and AP were in the focus of our interest because they are on the Croatian A2 quarantine list of pests and diseases. Eleven orchards in continental and coastal fruit growing regions were visually inspected for the symptom presence during summer and autumn. Leaf and twig samples were collected from symptomatic pear and apple trees. In 2004, phytoplasma vectors Cacopsylla pyri, C. pyrisuga and C. pyricola were collected from 5 orchards in continental and coastal Croatia by using entomological nets and yellow sticky traps. Nucleic acids extracted from insects and pome fruit trees were PCR-tested using phytoplasma universal primers R16F1/R0 for direct and R16F2n/R2 for the first nested PCR. Amplicons digested with Tru9I (MseI) showed profiles consistent with the presence of phytoplasmas belonging to one of the ribosomal groups (16SrX, 16SrXII, 16SrI), except in two cases where mixed infections were detected. For samples showing profiles typical of 16SrX group, additional digestion with RsaI and SspI was done to determine the ribosomal subgroup lately designated as 'Ca. P. pyri', 'Ca. P. mali' and 'Ca. P. prunorum'. Second nested PCR assays by using group specific primer pairs R16(X)F1/R1 and R16(I)F1/R1 were also performed. So far, the presence of following 'Candidatus Phytoplasma' species has been confirmed: -'Ca. P. pyri' in pear, apple, Cacopsylla pyri and C. pyrisuga, -stolbur (16SrXII-A) in pear, apple, Cacopsylla pyri, C. pyrisuga and C. pyricola, as well as -'Ca. P. asteris' (16SrI-B) in pear and Cacopsylla pyri. In this research, we have found no molecular evidence for the presence of 'Ca. P. mali' in Croatia.

The beginning of the science of virology was marked by the discovery of the causal agent of tobacco mosaic disease at the end of the 19th century. Besides the virus concept, many fundamental concepts of molecular biology had the tobacco mosaic virus as the main research object. It was the first virus to be purified, the first to be reconstituted from its protein and RNA components, its coat protein sequence was the first viral protein sequence to be determined. Our understanding of molecular biology (overlapping and silent genes, subgenomic RNAs, RNA-dependent RNA-polymerase) gained much through the use of TMV. The Croatian virologists through their work on TMV also gave a contribution to this pool of knowledge. As one of the best known and the most thoroughly researched viruses, TMV will continue to serve as a research model giving answers to the pertaining questions considering molecular mechanisms of plant-pathogen interactions, cell signaling and RNA-trafficking in plants.

Citrus tristeza Closterovirus is widespread in major citrus-growing regions of the world often causing destructive diseases. Citrus samples were taken from orchards in the Croatian coastal region. CTV was detected in two symptomless field trees of Satsuma mandarins and one diseased lemon tree. Double-stranded RNA was isolated from the field trees and the dsRNA patterns were compared in polyacrylamide gels. The same dsRNA extracts were used as templates in RT-PCR experiments amplifying the CTV coat protein sequence. Amplicons were subjected to SSCP and RFLP analyses. The results indicate greater similarity between CTV isolates from Satsuma mandarins than between these two and the lemon isolate.

To elucidate the potential role of weed species in an epidemic of tomato lethal necrosis, that has been going on for years in the southern coastal Croatia, 71 weed species were collected in a commercial tomato field with a high incidence of tomato lethal necrosis (about 80%), throughout 1995 and 1996. Bioassays, serological tests and dsRNA analyses were used to investigate the presence of CMV and the associated satRNA in all samples. CMV was found in eight weed species. In three species (Anagallis arvensis, Solanum nigrum and Sonchus sp.), CMV was associated with CrFG-satRNA, the same that was previously reported to codetermine tomato lethal necrosis. Since Solanum nigrum is bi-annual and Sonchus sp. a perennial plant species, they could easily enable overwintering of the virus and associated necrogenic satellite. The pathogen complex was also found in two cultivated species: squash (Lagenaria vulgaris) and pumpkin (Cucurbita pepo). Among 13 aphid species collected in the field, 7 are known CMV-vectors. Inoculation of the necrogenic CMV+satRNA complex on young tomato plants caused lethal necrosis in all of them.

Citrus is grown in Croatia (approximately 1,500 ha of citrus groves) on the Dalmatian Coast and Islands between 42 and 43°30′N. The major species, Citrus unshiu Marc. (Satsuma mandarin), is grafted on trifoliate rootstock. The presence of Citrus tristeza virus (CTV) in Satsumas in the Neretva Valley Region was previously reported (3). During the course of a biomolecular characterization of isolates from Croatia, 15 budsticks were collected from field-infected, enzyme-linked immunosorbent assay (ELISA)-positive sources during the autumn of 2003 near Kaštela, Split, Metković (Neretva Valley), and on the island of Vis. Isolates were propagated by graft transmission to Madam Vinous sweet orange (SwO) and maintained in an insect-proof greenhouse at 21 to 33°C. Eight months later, the bark of terminal twigs was peeled off, and the wood was examined for the occurrence of pits. Typical tristeza stem-pitting (SP) was observed in four isolates originating from cvs. Fukumoto navel, Washington navel, and Ichimaru Satsuma and C. wilsonii. The bark from the infected sources was analyzed using immunocapture reverse transcription-polymerase chain reaction (RT-PCR) with primers CTV1 and CTV10 (1), targeting the whole coat protein (CP) gene. The PCR products of the expected size (669 nucleotides) were obtained and TA cloned (pGEM-T Easy Vector; Promega, Madison, WI) in E. coli cells. Thirty-two clones harboring the CTV CP gene were sequenced. Two of the SP isolates contained four genomic variants that differed an average of 2.0% from the severe SwO SP strains SY568 and Nuaga (4) from California and Japan, respectively. The other two SP isolates contained four variants that differed as little as 1.6% from the severe SwO SP from India, CTV-Puna, and CTV-Bangalore (2). The net average distance between these two clusters of sequences is 5.2%. One sequence from each of the four SP isolates was deposited in GenBank (Accession Nos. AY791841 to AY791844). These findings were confirmed by direct observation of SP symptoms in a Satsuma orchard in the Neretva Valley during the spring of 2004. No other conspicuous symptoms that could be attributed to CTV were observed in the field. Most Satsumas were introduced to the Neretva Region from Japan between 1964 and 1984. Together with the fact that the related Nuaga strain was also isolated from Satsumas in Japan (4), our results suggest that SwO SP strains were introduced into Croatia at the same time and have been spreading for several decades. It has been generally believed that this kind of CTV strains either do not exist in the Mediterranean basin or, when found (e.g., Spain), are immediately eradicated. The findings reported here suggest that the epidemiological scenario for the Mediterranean Basin requires revision. References: (1) G. Nolasco et al. Eur. J. Plant Pathol. 108:293, 2002. (2) A. Roy et al. Arch. Virol. 148:707, 2003. (3) A. Šarić and I. Dulić. Agric. Conspectus Sci. 55:171, 1990. (4) G. Suastika et al. J. Gen. Plant Pathol. 67:73, 2001.

Background and purpose: Naturally occurring cucumber mosaic Cucumovirus (CMV) and peanut stunt Cucumovirus (PSV) occasionally encapsidate satellite RNA (satRNA) along with genomic and subgenomic RNAs. CMV satRNA variants reported so far range in size from 330-405 nucleotides. PSV satRNA variants are, in average, longer (391-393 nt). To our knowledge there have been no reports of robinia mosaic Cucumovirus (RbMV) satRNAs. Materials and methods: A cucumovirus from Robinia pseudacacia was studied by the reaction of several ordinary test-plants, by means of biological separation of the virus from its satellite, and by double-stranded RNA analysis. Results: The type of symptoms in naturally infected Robinia and experimental hosts together with distinctive electrophoretic pattern leave no doubt about a cucumoviral, highly le likely RbMV, infection. The dsRNA patterns from originally infected Robinia leaf tissue or Nicotiana megalosiphon infected with original Robinia isolate showed also the existence of a satellite-like dsRNA band. It was slower compared to the CMV satellite dsRNA used as a marker. This satellite-like molecule was eliminated by serial inoculations through Chenopodium quinoa. Conclusions: The characteristics of investigated cucumovirus from symptomatic leaves of Robinia closely resemble RbMV. Its satellite-like RNA is longer than most CMV satellites and accordingly similar to PSV satRNA variants reported so far.

Phytoplasmas are uncultivable prokaryotic wall-less pathogens belonging to the class Mollicutes that inhabit plant phloem and insects. Their identification and classification is difficult and mainly based on the polymorphism of their 16S rRNA gene sequences. Aster yellows (AY ; "Candidatus Phytoplasma asteris") phytoplasmas (ribosomal subgroup 16SrI-P) had been previously identified in several Populus nigra L. "Italica" (Lombardy poplar) trees from the urban area of Zagreb. The aim of this research was to examine the phytoplasmosis distribution in the poplar tree population of wider Zagreb urban area. Total nucleic acids were extracted from leaf samples of 30 symptomatic and 4 asymptomatic trees. Phytoplasma 16S rDNA was amplified in direct and nested PCRs by using universal and group-specific primers. The pathogens were classified on the basis of 16S rDNA amplicon RFLP analyses. Phytoplasmas belonging to the 16SrI ribosomal group (AY) were detected in 12 out of 34 examined trees. In addition, phytoplasma putative gene for aa kinase was analyzed for the positive samples. RFLP profiles from 10 samples were referable to the phytoplasma 16SrI-P ribosomal subgroup previously found only in poplar from Zagreb area. In two samples, unique restriction patterns were found, showing the presence of molecular variability within these conserved gene region. In northern and north-western part of the area the infection was equally distributed, while in the southern part of the city the presence of phytoplasmas was not detected in sampled trees. Further research, including a search for potential insect vectors, is needed in order to investigate ecological and epidemiological implications of these diseases and their impact on the sanitary status of ornamental trees in the city of Zagreb.

Citrus is commercially grown in Croatia between the 42° and 44° of northern latitudes. This is one of the northernmost commercial citrus growing areas in the world. Due to their relative frost tolerance, cultivars of the Satsuma mandarin (Citrus unshiu (Mak.) Marc.) are commercially the most important citrus crop in this agroecological niche and potentially an important export fruit product. The main objective of this work was to eliminate the Citrus tristeza virus (CTV) from clones of extra-early Satsuma Zorica Rana (Croatian selection). The presence of CTV was confirmed in all Zorica Rana mother plants. Following the thermotherapy of Zorica Rana plants, shoot-tip grafting was performed on in vitro grown virus free Troyer citrange rootstock. The efficiency of CTV elimination was confirmed by means of DAS-ELISA and IC/RT-PCR in all tested clones. This is the first report on CTV elimination procedure in Zorica Rana Satsuma selection and the first step towards the production of virus-free citrus planting material in the region.

Seven Croatian grapevine samples found positive for the presence of 'flavescence dorée'(FD) phytoplasmas in 2010 survey confirm the first findings of FD phytoplasma from previous year (ŠERUGA MUSIĆ et al., 2010), as well as the existence of new GY heavily affected narrow area west and southwest of Zagreb along the border with Slovenia. Two new locations 100 km east and 60 km north-east of Zagreb, respectively, were found to be affected, widening the potential FD-risk zone and placing the capital in its centre. Grapevine varieties infected with FD-related phytoplasmas encompassed widely known Pinot Noir and Riesling, but also indigenous red Plemenka Crvena and Ružica Crvena, as well as white variety Škrlet. Preliminary molecular characterization results suggest the variability of 16SrV-C phytoplasmas, especially from the locations west and southwest of Zagreb

Phytoplasmas from the ribosomal subgroup 16SrXII-A (stolbur) were identified by PCR and RFLP analyses of 16S rRNA gene and elongation factor-Tu phytoplasma gene (tuf) in symptomatic grapevines from the Veles and Skopje areas in Macedonia. Two methods were used for nucleic acid extraction. Amplification and RFLP results of 16S rDNA were not influenced by the method used, but RFLP patterns of tuf gene revealed additional bands if a less time consuming method was applied. The possible origin of these bands is discussed. This is the first report of the presence of the stolbur phytoplasma in cvs Chardonnay and Vranac in Macedonia., VITIS - Journal of Grapevine Research, Vol. 42 No. 4 (2003): Vitis

In order to investigate the genetic variability among selected grapevine strains belonging to 16SrXII-A subgroup (stolbur) as a case study, multilocus sequence typing (MLST) approach was used. Three non-ribosomal house-keeping genes were analyzed: tufB, secY and the recently characterized variable vmp1 gene, encoding a putative membrane protein of stolbur phytoplasma. Restriction analysis of tufB gene amplicons revealed the presence of both tuf-a (VK-I) and tuf-b (VK-II) types, with the latter being more prevalent among stolbur strains from Croatian grapevine analyzed in this study. In genotyping of vpm1 gene, five different RFLP profiles were obtained including a mixed pattern observed in one sample. The vmp1 diversity was therefore important among selected strains. Phylogenetic analyses of secY sequences were in accordance with the results of tufB and vmp1 typing ; however in some of the analyzed strains incongruence was observed. Nevertheless, the results presented have shown the presence of considerable genetic variability among relatively small number of 10 selected stolbur isolates from grapevine and proved the importance of MLST application in differentiation of closely related strains.

Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS). Infections with hantaviruses are not lytic, and it is currently unknown why infections in humans cause the disease. It is considered that pathogenesis of HFRS is mainly mediated by immune response. The aim of this study was to explore the components of innate and adaptive immunity important in the early peripheral immune response as well as the possible regulatory effect of miRNA on early immunoreactions during the hantaviral infection. Based on this, biological pathways relevant for the immunopathogenesis of HFRS have also been defined. Using real-time PCR array technology, the relative expression of immune response genes and miRNA was measured in peripheral blood mononuclear cells of patients infected with Puumala virus (PUUV). The results showed suppression of the early immune response to PUUV i.e. genes coding the synthesis of pattern recognition receptors, chemokines and their receptors, cytokines, transcription factors, as well as some signalling molecules. Functional analysis showed that down regulation of the expression of described set of genes modulates inflammatory response by interfering with various cell-signalling pathways. For the first time, the biological importance of miRNA in the regulation of immune response during HFRS was shown. Changes detected at the immune response level have been associated with disease severity but not with the viral load in the blood.

Apoptin, a protein from chicken anemia virus, has the ability to kill human tumor cells. It accumulates in the cytoplasm of normal cells, where it will be subsequently degraded by proteasomal activity. In tumor cells, apoptin enters the nucleus and redirects cell signaling toward apoptosis. It has been found that apoptin senses early stages of malignant transformation, but it is not clear what follows these early changes in cells that would be recognized by the apoptin. In the study presented here, antiproliferative activities of apoptin toward cancer stem cells were compared to its effects toward other tumor cell lines. Transformed primary mammary epithelial cells (HMLE) with silenced gene for E cadherin (HMLE- shEcad) were used as a cancer stem cells (CSC) model. These cells were experimentally induced to pass through epithelial-mesenchymal transition, what was sufficient to acquire stem cells properties. An adenoviral vector that expresses Flag-tagged apoptin gene and a control vector that expresses lacZ gene were used to transfect cells. Localization within the cells, antiproliferative activity, different modes of programmed cell death, and cell cycle disturbances upon Ad-Ap infection were investigated. Although efficiency of transduction was low in the HMLE-shEcad cells, modest growth inhibition was measured. Apoptin localised in the nucleus of HMLE-shEcad cells, but it was not sufficient to induce apoptosis. Apoptin induced cell cycle G2 arrest 24 hours after infection, which delayed cell’s growth, but cells overcame this effect and recovered later. Apoptin showed distinct levels of cytotoxicity towards various tumor cell lines, identifying NCI-H1299 (non-small cell lung cancer) as the most sensitive. In these cells apoptin induced apoptosis, and also modulated autophagy. Our study confirmed apoptin as a potent tumor-cell killer, able to modulate different modes of programmed cell’s death, and moreover, to interfere with CSC. Mechanisms of CSC resistance to apoptin should be further analyzed and evaluated in more details.

Many zoonotic viruses are transmitted by arthropode vectors (e.g. mosquitoes and ticks) between their reservoir hosts and humans and are therefore called arboviruses. This non- systemic classification comprises approximatley 250 different virus species, 80 of them being pathogenic for humans, for example: Dengue virus or Chikungunya virus. Similarly the term roboviruses describes zoonotic viruses that are being transmitted by rodents. Human pathogenic roboviruses belong to two genera, Hantavirus and Arenavirus. The newly coined term ArboRoboviruses includes all viruses from both groups that are mostly emerging or re-emerging pathogens. We will focus here on molecular epidemiology of several emerging and re- emerging ArboRoboviruses in Croatia. Hantaviruses cause hemorrhagic fever with renal syndrome, which was described in Croatia in the 1960’s. Since then several epidemics occurred caused by Puumala and Dobrava viruses. Additionally were Tula and Saarema viruses in small rodents detected. The percentage of infected small rodents varies between different hantaviruses and regions and can reach up to 77 %. Molecular epidemiology showed that Puumala virus at higher altitudes of Gorski Kotar represent a phylogenetically distinct clade in comparison to low-land Puumala viruses. In 2010 three cases of autochthonous Dengue virus infection were discovered at the Pelješac peninsula. Molecular characterization discovered Dengue virus genotype 1 as the causing pathogen, imported by an unrecognized index case, probably from the Indian subcontinent. Retrospective analysis showed this to be the first autochthonous infection of Dengue fever since the 1930’s in Europe. West Nile neuroinvasive disease (WNND), is a serious illness caused by West Nile virus and presents with one of three major clinical syndromes: meningitis, encephalitis or flaccid paralysis. In 2012 the first cases of WNND have been recorded in Croatia. In the following year three times as many cases were hospitalized. In contrast to 2012, where all patients contracted WNND in the eastern parts of Croatia, in 2013 the patients got infected in and around the City of Zagreb. Phylogenetic analysis of parts of the genome showed close relation to strains circulating in neighbouring countries. A limited molecular analysis of Aedes albopictus and ¬Culex pipiens, from various parts of Croatia, did not show any traces of human pathogenic flavi- or alphaviruses.