Prevalence of Virus Infections and GLRaV-3 Genetic Diversity in Selected Clones of Croatian Indigenous Grapevine Cultivar Plavac Mali.

The cultivar Plavac Mali (Vitis vinifera L.), the most important indigenous red grapevine cultivar in Croatia, was tested for the presence of 16 grapevine viruses. Thirty-five samples from the collection vineyard were tested for the presence of grapevine leafroll-associated viruses-1, -2, and -3 (GLRaV-1, GLRaV-2 and GLRaV-3, respectively), grapevine fanleaf virus (GFLV), arabis mosaic virus (ArMV), grapevine virus-A (GVA), -B (GVB), -G (GVG), -H (GVH), -I (GVI), -J (GVJ), grapevine fleck virus (GFkV), grapevine rupestris stem pitting associated virus (GRSPaV), and grapevine pinot gris virus (GPGV) by reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, standard PCR was conducted for grapevine badnavirus 1 (GBV-1) and grapevine red blotch virus (GRBV). Mixed infections were most common and GLRaV-3, the most abundant virus found in 85.71% of the vines tested, was further molecularly characterised. Different genomic variants of the heat shock protein homologue (HSP70h) were separated by cloning, detected by single-strand conformation polymorphism (SSCP) analysis, sequenced, and phylogenetically analysed. The presence of phylogenetic groups I and II was only confirmed. This study demonstrates the high virus infection rate of Plavac Mali vines and the heterogeneity of GLRaV-3 present nowadays in a collection vineyard.

Grapevine Leafroll-Associated Virus 3 Replication in Grapevine Hosts Changes through the Dormancy Stage.

Grapevine leafroll-associated virus 3 (GLRaV-3) is a graft-transmissible virus present in every viticultural region of the world and poses a large threat to grapevine production. Frequent coinfections with other viruses, the large number of grapevine varieties, the complexity of processes involved in plant response to virus infection, and the lack of studies on GLRaV-3 replication limit our knowledge of GLRaV-3 damaging effects and their background. In this study, five different inocula, one containing GLRaV-3 and others containing GLRaV-3 in combination with different grapevine viruses were green grafted to 52 different grapevine plants of four varieties to analyze the influence of the phenological stage and virus composition on GLRaV-3 replication. Relative concentration analysis by quantitative PCR conducted over a 16-month period revealed that other viruses as well as plant stage had a significant effect on GLRaV-3 replication and symptoms expression. The replication was most pronounced in the deep dormancy stage at the beginning of the infection, and the least at the exit of the dormancy stage. This study brings new insight into GLRaV-3 replication and discusses about viral interactions in one of the most economically important perennial plants, the grapevine.

Virome Analysis of Signal Crayfish (Pacifastacus leniusculus) along Its Invasion Range Reveals Diverse and Divergent RNA Viruses.

Crayfish are a keystone species of freshwater ecosystems and a successful invasive species. However, their pathogens, including viruses, remain understudied. The aim of this study was to analyze the virome of the invasive signal crayfish (Pacifastacus leniusculus) and to elucidate the potential differences in viral composition and abundance along its invasion range in the Korana River, Croatia. By the high-throughput sequencing of ribosomal RNA, depleted total RNA isolated from the crayfish hepatopancreas, and subsequent sequence data analysis, we identified novel and divergent RNA viruses, including signal crayfish-associated reo-like, hepe-like, toti-like, and picorna-like viruses, phylogenetically related to viruses previously associated with crustacean hosts. The patterns of reads abundance and calculated nucleotide diversities of the detected viral sequences varied along the invasion range. This could indicate the possible influence of different factors and processes on signal crayfish virome composition: e.g., the differences in signal crayfish population density, the non-random dispersal of host individuals from the core to the invasion fronts, and the transfer of viruses from the native co-occurring and phylogenetically related crayfish species. The study reveals a high, previously undiscovered diversity of divergent RNA viruses associated with signal crayfish, and sets foundations for understanding the potential risk of virus transmissions as a result of this invader's dispersal.

Legacy of Plant Virology in Croatia-From Virus Identification to Molecular Epidemiology, Evolution, Genomics and Beyond.

This paper showcases the development of plant virology in Croatia at the University of Zagreb, Faculty of Science, from its beginning in the 1950s until today, more than 70 years later. The main achievements of the previous and current group members are highlighted according to various research topics and fields. Expectedly, some of those accomplishments remained within the field of plant virology, but others make part of a much-extended research spectrum exploring subviral pathogens, prokaryotic plant pathogens, fungi and their viruses, as well as their interactions within ecosystems. Thus, the legacy of plant virology in Croatia continues to contribute to the state of the art of microbiology far beyond virology. Research problems pertinent for directing the future research endeavors are also proposed in this review.

Predominance and Diversity of GLRaV-3 in Native Vines of Mediterranean Croatia.

Sixteen grapevine cultivars from Mediterranean Croatia were surveyed for the presence of 10 of the most economically important grapevine viruses. The presence of Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Grapevine leafroll associated virus-1, -2, and -3 (GLRaV-1; GLRaV-2 and GLRaV-3), Grapevine virus A (GVA) and B (GVB), Grapevine fleck virus (GFkV), Grapevine rupestris stem pitting associated virus (GRSPaV), and Grapevine Pinot gris virus (GPGV) were tested by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). All 71 analyzed clones were positive for the presence of one or more viruses. The most abundant one, detected in almost 95% of samples was GLRaV-3. In most of cases it was reported in mixed infections with GVA, GRSPaV, and GPGV. Virus genomes of GLRaV-3 infected vines were further characterized molecularly in order to determine their genetic diversity. Different genomic variants of heat shock 70 protein homologue (HSP70h) were identified by single-strand conformation polymorphism (SSCP) and sequenced. Sequence analysis confirmed their clustering into phylogenetic group I and/or phylogenetic group II. This study emphasizes the wide virus heterogenicity in Mediterranean vines and the predominant presence of GLRaV-3 phylogenetic groups I and II, either individually or in combination.

Distribution and Molecular Characterization of Hepatitis E virus in Domestic Animals and Wildlife in Croatia.

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.

Stem pitting and seedling yellows symptoms of Citrus tristeza virus infection may be determined by minor sequence variants.

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.