Meccanismi evolutivi dei parapoxvirus: caratterizzazione genomica di pseudocowpoxvirus e messa a punto di sistemi per lo studio delle ricombinazioni

The Poxviruses are a family of double stranded DNA (dsDNA) viruses that cause disease in many species, both vertebrate and invertebrate. Their genomes range in size from 135 to 365 kbp and show conservation in both organization and content. In particular, the central genomic regions of the chordopoxvirus subfamily (those capable of infecting vertebrates) contain 88 genes which are present in all the virus species characterised to date and which mostly occur in the same order and orientation. In contrast, however, the terminal regions of the genomes frequently contain genes that are species or genera-specific and that are not essential for the growth of the virus in vitro but instead often encode factors with important roles in vivo including modulation of the host immune response to infection and determination of the host range of the virus. The Parapoxviruses (PPV), of which Orf virus is the prototypic species, represent a genus within the chordopoxvirus subfamily of Poxviridae and are characterised by their ability to infect ruminants and humans. The genus currently contains four recognised species of virus, bovine papular stomatitis virus (BPSV) and pseudocowpox virus (PCPV) both of which infect cattle, orf virus (OV) that infects sheep and goats, and parapoxvirus of red deer in New Zealand (PVNZ). The ORFV genome has been fully sequenced, as has that of BPSV, and is ~138 kb in length encoding ~132 genes. The vast majority of these genes allow the virus to replicate in the cytoplasm of the infected host cell and therefore encode proteins involved in replication, transcription and metabolism of nucleic acids. These genes are well conserved between all known genera of poxviruses. There is however another class of genes, located at either end of the linear dsDNA genome, that encode proteins which are non-essential for replication and generally dictate host range and virulence of the virus. The non-essential genes are often the most variable within and between species of virus and therefore are potentially useful for diagnostic purposes. Given their role in subverting the host-immune response to infection they are also targets for novel therapeutics. The function of only a relatively small number of these proteins has been elucidated and there are several genes whose function still remains obscure principally because there is little similarity between them and proteins of known function in current sequence databases. It is thought that by selectively removing some of the virulence genes, or at least neutralising the proteins in some way, current vaccines could be improved. The evolution of poxviruses has been proposed to be an adaptive process involving frequent events of gene gain and loss, such that the virus co-evolves with its specific host. Gene capture or horizontal gene transfer from the host to the virus is considered an important source of new viral genes including those likely to be involved in host range and those enabling the virus to interfere with the host immune response to infection. Given the low rate of nucleotide substitution, recombination can be seen as an essential evolutionary driving force although it is likely underestimated. Recombination in poxviruses is intimately linked to DNA replication with both viral and cellular proteins participate in this recombination-dependent replication. It has been shown, in other poxvirus genera, that recombination between isolates and perhaps even between species does occur, thereby providing another mechanism for the acquisition of new genes and for the rapid evolution of viruses. Such events may result in viruses that have a selective advantage over others, for example in re-infections (a characteristic of the PPV), or in viruses that are able to jump the species barrier and infect new hosts. Sequence data related to viral strains isolated from goats suggest that possible recombination events may have occurred between OV and PCPV (Ueda et al. 2003). The recombination events are frequent during poxvirus replication and comparative genomic analysis of several poxvirus species has revealed that recombinations occur frequently on the right terminal region. Intraspecific recombination can occur between strains of the same PPV species, but also interspecific recombination can happen depending on enough sequence similarity to enable recombination between distinct PPV species. The most important pre-requisite for a successful recombination is the coinfection of the individual host by different virus strains or species. Consequently, the following factors affecting the distribution of different viruses to shared target cells need to be considered: dose of inoculated virus, time interval between inoculation of the first and the second virus, distance between the marker mutations, genetic homology. At present there are no available data on the replication dynamics of PPV in permissive and non permissive hosts and reguarding co-infetions there are no information on the interference mechanisms occurring during the simultaneous replication of viruses of different species. This work has been carried out to set up permissive substrates allowing the replication of different PPV species, in particular keratinocytes monolayers and organotypic skin cultures. Furthermore a method to isolate and expand ovine skin stem cells was has been set up to indeep further aspects of viral cellular tropism during natural infection. The study produced important data to elucidate the replication dynamics of OV and PCPV virus in vitro as well as the mechanisms of interference that can arise during co-infection with different viral species. Moreover, the analysis carried on the genomic right terminal region of PCPV 1303/05 contributed to a better knowledge of the viral genes involved in host interaction and pathogenesis as well as to locate recombination breakpoints and genetic homologies between PPV species. Taken together these data filled several crucial gaps for the study of interspecific recombinations of PPVs which are thought to be important for a better understanding of the viral evolution and to improve the biosafety of antiviral therapy and PPV-based vectors.

MetaVaccinology: a new vaccine discovery tool

In the last decade, the reverse vaccinology approach shifted the paradigm of vaccine discovery from conventional culture-based methods to high-throughput genome-based approaches for the development of recombinant protein-based vaccines against pathogenic bacteria. Besides reaching its main goal of identifying new vaccine candidates, this new procedure produced also a huge amount of molecular knowledge related to them. In the present work, we explored this knowledge in a species-independent way and we performed a systematic in silico molecular analysis of more than 100 protective antigens, looking at their sequence similarity, domain composition and protein architecture in order to identify possible common molecular features. This meta-analysis revealed that, beside a low sequence similarity, most of the known bacterial protective antigens shared structural/functional Pfam domains as well as specific protein architectures. Based on this, we formulated the hypothesis that the occurrence of these molecular signatures can be predictive of possible protective properties of other proteins in different bacterial species. We tested this hypothesis in Streptococcus agalactiae and identified four new protective antigens. Moreover, in order to provide a second proof of the concept for our approach, we used Staphyloccus aureus as a second pathogen and identified five new protective antigens. This new knowledge-driven selection process, named MetaVaccinology, represents the first in silico vaccine discovery tool based on conserved and predictive molecular and structural features of bacterial protective antigens and not dependent upon the prediction of their sub-cellular localization.

Functional and molecular characterization of ethylene responsive factor genes involved in grape ripening

Grape berry is considered a non climacteric fruit, but there are some evidences that ethylene plays a role in the control of berry ripening. This PhD thesis aimed to give insights in the role of ethylene and ethylene-related genes in the regulation of grape berry ripening. During this study a small increase in ethylene concentration one week before véraison has been measured in Vitis vinifera L. ‘Pinot Noir’ grapes confirming previous findings in ‘Cabernet Sauvignon’. In addition, ethylene-related genes have been identified in the grapevine genome sequence. Similarly to other species, biosynthesis and ethylene receptor genes are present in grapevine as multi-gene families and their expression appeared tissue or developmental specific. All the other elements of the ethylene signal transduction cascade were also identified in the grape genome. Among them, there were ethylene response factors (ERF) which modulate the transcription of many effector genes in response to ethylene. In this study seven grapevine ERFs have been characterized and they showed tissue and berry development specific expression profiles. Two sequences, VvERF045 and VvERF063, seemed likely involved in berry ripening control due to their expression profiles and their sequence annotation. VvERF045 was induced before véraison and was specific of the ripe berry, by sequence similarity it was likely a transcription activator. VvERF063 displayed high sequence similarity to repressors of transcription and its expression, very high in green berries, was lowest at véraison and during ripening. To functionally characterize VvERF045 and VvERF063, a stable transformation strategy was chosen. Both sequences were cloned in vectors for over-expression and silencing and transferred in grape by Agrobacterium-mediated or biolistic-mediated gene transfer. In vitro, transgenic VvERF045 over-expressing plants displayed an epinastic phenotype whose extent was correlated to the transgene expression level. Four pathogen stress response genes were significantly induced in the transgenic plants, suggesting a putative function of VvERF045 in biotic stress defense during berry ripening. Further molecular analysis on the transgenic plants will help in identifying the actual VvERF045 target genes and together with the phenotypic characterization of the adult transgenic plants, will allow to extensively define the role of VvERF045 in berry ripening.