In my doctoral project, I studied the evolution of retroviruses and long-term interactions with their hosts. Retroviruses infect a broad range of species including possibly all vertebrates. They are unique in their ability to efficiently create endogenous retroviruses (ERVs) - viral copies integrated into the host genomes and consequently inherited by successive generations as usual genomic locus. ERVs represent a significant portion of vertebrate genomes and play an important role in a variety of cellular processes and pathologies; however, their sequences are still largely unexplored. The results of my work contributed to the uncovering of ancient evolutionary history of retroviruses. In this regard, I employed the ERV sequences, as they represent "genetic fossils" of viral infections that occurred throughout entire retroviral evolution. By discovery and analysis of ancient ERV lineages, I shed light on the deep history of retroviruses and revealed how the past infections shaped the evolution of vertebrate antiviral defense. In addition to the investigation of retroviral evolution, I also studied process of ongoing endogenization and fixation of newly emerged ERVs in a mammalian host population. In this part of my work, I focused on a unique model of ERV that have been recently invading mule deer genome.

Context: West Nile virus (WNV) is an arthropod-borne virus considered a One Health challenge because of its increasing impact on human and animal health. It is one the most widely distributed viruses of the encephalitic Flaviviruses. It may cause severe neurological symptoms in humans and animals and is recognized as a serious public health problem also because of its impact on blood transfusion and organ transplantation. First identified in Africa in 1937, it was later introduced and spread in Italy, where in many regions it is now endemic, due to the increasingly favorable climatic and environmental conditions. Aim: The main objectives of this study, based on an interdisciplinary One Health approach, were: (1) to characterize the geographical distribution within specific host and vector populations in Africa; (2) to describe its phylogeographical patterns between Africa and Europe; (3) to define the genetic structure and epidemiology of Italian WNV strains, giving an insight of the viral circulation dynamics in the Italian territory. Methodology: Ecological and epidemiological studies were combined with molecular and phylogenetic analyses, carrying out field sampling activities, cellular culture, viral infection, immunofluorescent assay, multiplexed RT-PCR, sequencing, data analysis, and novel technique design. These activities were carried out both in Italy and in Senegal. Results: Our study evidences: (i) the circulation of several WNV lineages [Lineage 1 (L1), 2 (L2), 7 (L7), and 8 (L8)] in the African Continent; (ii) the presence of diverse competent mosquito vectors in Africa, mainly belonging to the Culex genus; (iii) the lack of vector competence studies for several other mosquito species found naturally infected with WNV in Africa; (iv) the need of more vector competence studies on ticks; (v) the circulation of WNV among humans, animals and vectors in at least 28 African countries; (vi) the lack of knowledge on the epidemiological situation of WNV for 19 African countries, and (vii) the importance of carrying out specific serological surveys in order to avoid possible bias on WNV circulation in Africa (objective 1). Furthermore, a new set of WNV L1 and L2 genome-specific primers for tiled-amplicon sequencing have been designed and a consistent dataset of 64 WNV L2 and 31 WNV L1 Italian genomes and of 3 WNV L2 and 7 WNV L1 Senegalese genome sequences from samples collected in Italy and Senegal between 2006 and 2022 has been produced. Twenty more WNV L1 and L2 Senegalese sequences obtained from samples collected in Senegal between 1985 and 2018 have been shared by the Institut Pasteur Dakar of Senegal and added to the dataset. This allowed the conduction of phylogenetic and phylogeographic analyses, evidencing: (viii) the presence of a strong viral connection between Africa and Europe, with intercontinental circulation supported by birds crossing international boundaries while migrating through the African-Eurasian flyways; (ix) the WNV L1 Western-Mediterranean cluster probable spread from Senegal, where the virus was first reported in 1979, to Italy, where the lineage first appeared in Europe in 1998, and to France in 2000, and the presence of back re-introductory events from Italy, Spain, and France to North and West Africa from the 2010s; and (x) the first African introduction of WNV L2 in Europe in Hungary in 2004, possibly from South African countries (objective 2). Our study also gives an insight of the dynamics of the viral circulation in Italy, demonstrating: (xi) the endemic presence of WNV L1 and L2 in part of Italy supported by resident wild birds and vector competent mosquitoes mainly belonging to the Culex genus; (xii) the current existence of two diverse WNV L1 strains circulating in Italy, one in the North-East, and one circulating intra-regionally in the Campania region; (xiii) suggested characteristic silent periods observed for WNV L1 in the country, with unnoticed circulation lasting sometimes for more than 10 years; (xiv) the 2022 WNV L1 increasing incidence of neurological disease cases in humans; (xv) the presence of genetically stable WNV L2 strains in Italy with continuous circulation throughout the time; (xvi) the presence of overwintering mechanisms supported by bird-to bird, rodent-to bird, or mosquito-to bird transmission routes; (xvii) the existence of WNV L1 and L2 co-infections in birds and mosquitoes; (xviii) the existence of a continuous transmission of the two strains between Western Mediterranean countries, supported by short distance migratory birds; and (xix) the crucial importance of the surveillance system other than the strategic role of wildlife rescue centers in monitoring both the introduction and circulation of avian emerging zoonotic diseases in Italy (objective 3). Conclusion: Our work points out the existence of high genetic diversity of WNV strains in Africa, the spread of L1 and L2 strains from Africa to Europe, and the existence of continuous transmission episodes among several Western-Mediterranean countries, with few recently suspected back introductory events from Europe to Africa. The progressive increase of the WNV L2 circulation both temporally and spatially in the Mediterranean countries and the WNV L1 re-appearance in Europe, both associated with a significant impact on humans and animal health, other than the strong WNV incidence in Italy and its endemization in part of its territory, evidence a solid WNV epidemic risk for Italy and a persistent threat for WNV spread into new areas. To predict and control future epidemics, it is crucial to constantly monitor the circulation and evolution of WNV in Europe and Africa, and to implement coordinated surveillance plans in both Continents, even in areas which are not currently affected.

Murine polyomavirus is an important member of Polyomaviridae family offering potential applications in gene therapy and immunotherapy. Viral mutant analysis is crucial for study of the virus, however, commonly used methods of its production are laborious and give low yields. This thesis involves development of the new experimental system that can produce intact viral genome from recombinant plasmid in vivo using Cre/loxP-mediated recombination. One loxP site is unavoidably introduced into newly generated viral genome during recombination. Two variants of production plasmids generating wild type viral genome with incorporation of loxP between the poly(A) signal sites of early and late genes or into the intronic region of early genes were prepared. LoxP insertion between the poly(A) signal sites has a dramatic effect on viral gene expression and leads to complete loss of virus infectivity. Conversely, the infectious virus was obtained from the viral genome containing loxP site in the early intronic region. To ensure expression of Cre recombinase I also prepared stably transfected cell lines which can simplify the virus production. This thesis shows that newly designed system gives satisfactory yield of the virus, solves restrictions connected with commonly used methods and can be used for low infectious viral...

Samples of lucerne plants characteristic with local necrosic lesions, leave malformation and yellow spots on leaves were investigated with transmission electron microscopy. Virus particles observed there were filamentous ones of 600 to 700 nm long. Nucleic acid was isolated, transcribed and amplified using PCR. Genus-specific primers were designed based on reverse genetics from the highly conserved genes for carlaviruses, potexviruses and potyviruses. Successful amplification with carlavirus-specific primers, sequencing and comparison with sequences in GenBank database revealed presence of a carlavirus. This was later identified by nucleotide sequence comparison as a new isolate V4 of Alfalfa latent virus. Specific primers for isolate V4 were designed in a coat protein position. Half of the genom of this virus was obtained with PCR and PCR modified amplifications and compared with sequences of Alfalfa latent virus and Pea streak virus from GenBank.

Viral particles derived from mouse polyomavirus can be potentially used as a delivery system for therapeutic genes and drugs into target cells. This thesis focuses on preparation and characterization of polyomaviral particles that are modified with cell-penetrating peptides in order to increase efficiency of transduction of reporter genes into human cells. Viral particles that are composed of major capsid protein VP1 in combination with minor capsid protein VP2 and minor capsid protein VP3 that is modified with octaarginine, LAH4 peptide or with transduction domain of adenoviral protein VI are analysed in transduction assays. The thesis also provides information about the effect of the modification on encapsidation of heterologous DNA. The results of transduction assays performed with modified particles containing encapsidated luciferase gene revealed that efficiency of transduction did not increase but decreased in comparison with unmodified particles. These findings help to elucidate the role of polyomaviral minor capsid proteins in gene transfer mediated by viral particles and contribute to the design of new strategies for modifications of viral particles derived from mouse polyomavirus for their successful application in nanomedicine. Key words: mouse polyomavirus, pseudovirions, virus-like...

Hyaluronic acid (HA) is widely studied as a targeting moiety to CD44 overexpressing cancer cells. Various types of nanoparticles (NPs) were modified by HA. Virus-like particles (VLPs) derived from mouse polyomavirus are an interesting class of NPs that can be modified by various targeting agents to increase their potential as gene or drug delivery vehicles for e.g. theragnostic purposes. HA has not been tested as a targeting moiety on VLPs, hence this was the focus of the current study. HA (~14 kDa) was attached to the VLPs via a bispecific Bodipy-derived fluorescent probe. To test the targeting potential of HA on comparable non-viral NPs, nanodiamonds were prepared in a similar manner. NPs functionalized with HA, together with Bodipy-labeled control variants, were tested on interactions with MDA-MB-231 cells overexpressing CD44. The NP-cell interaction via CD44 was assessed by a competitive cell-binding assay, where non-labeled HA competed for HA-binding sites at CD44 with the NPs. CD44 specific cell interactions were detected in studies with HA functionalized nanodiamonds, whereas VLP-HA* associated with cells in a less specific manner. Control VLPs with polyethylene glycol (PEG) did not interact with the cells. Results indicate that the HA targeting strategy for the VLPs requires optimization to...

Influenza virus causes severe respiratory infections in birds and mammals and it is responsible for up to half a million deaths of human beings worldwide each year. Two molecular targets in influenza viral life cycle, neuraminidase and M2 proton channel are exploited in treatment. However, the recent emergence of new pandemic type along with increasing resistance against approved drugs has urged the need for a new drug target discovery and potential search of its inhibitor. Recently, an interesting protein-protein interaction between two subunits PA and PB1 of influenza A viral polymerase has been identified by X-ray crystallography as a new promising drug target. The fact that relatively few residues drive the binding and that the binding interface is highly conserved presents an intriguing possibility to identify antiviral lead compounds effective against all subtypes of influenza A virus. In our laboratory, we expressed and purified two fusion tag constructs of the recombinant C-terminal domain of polymerase acidic subunit (CPA) from the pandemic isolate A/California/07/2009 H1N1. First, GST-CPA fusion protein was used for kinetic evaluation of PA-PB1 interaction by surface plasmon resonance. Moreover, this construct was used in the development of high-throughput screening method for search of...

Influenza virus causes severe respiratory infections in birds and mammals and it is responsible for up to half a million deaths of human beings worldwide each year. Two molecular targets in influenza viral life cycle, neuraminidase and M2 proton channel are exploited in treatment. However, the recent emergence of new pandemic type along with increasing resistance against approved drugs has urged the need for a new drug target discovery and potential search of its inhibitor. Recently, an interesting protein-protein interaction between two subunits PA and PB1 of influenza A viral polymerase has been identified by X-ray crystallography as a new promising drug target. The fact that relatively few residues drive the binding and that the binding interface is highly conserved presents an intriguing possibility to identify antiviral lead compounds effective against all subtypes of influenza A virus. In our laboratory, we expressed and purified two fusion tag constructs of the recombinant C-terminal domain of polymerase acidic subunit (CPA) from the pandemic isolate A/California/07/2009 H1N1. First, GST-CPA fusion protein was used for kinetic evaluation of PA-PB1 interaction by surface plasmon resonance. Moreover, this construct was used in the development of high-throughput screening method for search of...

Plasmacytoid dendritic cells (pDC) represent innate immune cells capable to detect viruses in their endosomal environment via Toll-like receptors (TLRs). Viral nuclear acid recognition leads to the massive production of type I interferon (IFN I) and induction of the antiviral state in uninfected cells. Crosslinking of the surface regulatory receptors, such as BDCA-2, with monoclonal antibodies or with some viruses leads to the activation of MEK1/2- ERK signaling pathway and inhibition of IFN I production in pDC. In this study, the role of MEK1/2 kinase has been highlighted. Its inhibition reversed the inhibitory effect of BDCA-2 crosslinking and its direct activation with PMA led to the inhibition of IFN-α production. Yet an unclear role of pDC in sensing of BK polyomavirus virus (BKV) responsible for kidney transplant rejection was investigated as a major topic of this thesis. Experiments with the pDC cell line Gen2.2 and HRPTEC primary cell line showed that pDCs were not able to detect BKV particles, however, exposure of activated Gen2.2 cells to BKV inoculum dramatically upregulated production of IFN-α. Most importantly, coculture of Gen2.2 cells with BKV- infected HRPTEC cells resulted in IFN-α and TNF-α production, which was prevented by Bafilomycin. These results suggest that BKV-infected...

One of the important tasks of virology and immunology is to explore the species- and cell-barriers preventing virus horizontal transmission and reveal the ways how viruses overcome these barriers and "adapt" to different species. This work is based on a well- established retroviral model - avian Rous sarcoma virus (RSV) and studies virus replication blocks in mammalian cells at both pre- and post-integration level. Interaction of the viral envelope glycoprotein (Env) with a specific cellular receptor mediates virus entry into cells. Although mammalian orthologues of specific chicken receptors do not support RSV entry, it was observed that some RSV strains are able to enter mammalian cells. Several RSV-transformed rodent cells lines were described and analysis of provirus H20- RSV in one these cells lines (hamster H-20 tumor cell line) showed multiple mutations including two crucial amino acid substitutions in different regions of Env. Substitutions D32G and L378S confer virus transmission to hamster, human and also chicken cells lacking the appropriate receptor. Altered conformation of H20-RSV Env is similar to a receptor-primed (activated) state of Env. This observation indicates that virus can circumvent the need of original cell receptor because of spontaneous Env activation caused by single...