Molecular studies on the densovirus of Mytbimna loreyi, MIDNV a candidate for biological control
Québec, Université du Québec, Institut National de la Recherche Scientifique, Doctorat en virologie et immunologie, 229 p.
Densoviruses of arthropods belong with the parvoviruses of vertebrates to the Parvoviridae family. These isometric viroses, with a diameter between 22 to 25 nm, contain genomes of 4-6 kb of linear, single-stranded DNA. The limited number of coding genes implies a high dependence on a coding complement from their hosts and may explain their narrow host range. Densoviruses are potential biological control agents due to their specifie and acute effect. Yet, their use in biological control has been delayed for a long time since early unconfirmed reports claimed that these insect viroses infect vertebrate cells in culture. However, the advance in molecular biology research makes it increasingly clear that many differences exist between vertebrate and invertebrate biology. In tum, viroses that are obligatory parasites of invertebrates are adapted to the distinct biology of their hosts, thus fitness of these insect viroses to vertebrates is not anticipated. On the other band, sorne vertebrate viroses such as bunyaviruses and flaviruses, that are transmitted to their hosts through an insect vector, can readily replicate and survive for long periods in their intermediate insect hosts without damage. However, these viroses have RNA genomes that carry several viral accessories, which makes the virus Jess dependent on the cell machinery.
Little is known about the biology of densoviruses, although in recent years several densovirus genomes have been cloned. In the current work, two new densoviruses have been characterized and their expression and tropism mechanisms have been explored. The first objective of this thesis was to study the molecular biology of three densoviruses; Mythimna loreyi, Galleria mel/one/la and Helicoverpa armigera densoviruses (MlDNV, GmDNV and HaDNV), respectively. These viroses were isolated from lepidopteran pests that are economically important in agriculture. Their genomes were cloned and the complete sequence and genomic organization were determined. They have genomes of about 6 kb and ITRs of around 550 nts which are the longest among all parvoviruses. A high level of sequence identity (90%) was found among them. The cloned genomes were shown to be infectious both in vitro and in vivo by initiating infections typical to that of the wild-type virus, after transfection. Studying their expression showed that they employ alternative
splicing and leak.y scanning mechanisms to produce three nonstructural proteins (NS1-3) from one viral transcript. These viruses use a leak.y scanning mechanism to produce a set of four structural proteins (VPs) from one transcript produced from the complementary strand without employing splicing or internai ribosome entry sites.
The second objective was to localize the allotropie determinants that control the tropism of GmDNV and MIDNV. The two viruses share over 90% sequence identity but differ in their host preference. MIDNV is polyspecific, infecting severallepidopteran species, whereas GmDNV is restricted toits host Galleria mel/one/la. The two infectious clones, pMI28 and pGml ofMIDNV and GmDNV, respectively, were used to swap a number of domains that covered the whole genome to obtain chimeric genomes. We established a cellular system, consisting oftwo cell types, LD652 thatis permissive for both viruses and T-ni03 that is permissive for MIDNV only, as markers to distinguish these phenotypes. The data obtained after transfection of the different viral chimerae in these cells showed that neither the coding sequences nor the viral origin of replication were responsible in controlling the virus tropism. The transfer of the MIDNV VP promoter into GmDNV background extended the tropism ofGmDNV to T-ni03 cells. However, the full expression ofGmDNV in these cells needed also the trans-activation ofthe viral NS proteins. These data showed that the in vitro tropism ofthese densoviruses is controlled bythe two viral promoters ofNS and VP, respectively, in contrast to the allotropie determinants ofvertebrate parvoviruses.
The third objective was to study in vitro the safety of densoviruses MIDNV and GmDNV as a first step before proceeding towards homologation (regulatory standards for biological control agents). Both infection by virus preparations and transfection by the viral infectious clone were carried out on severa!mammalian cell types, COS-7, 293 T and PT as weil as L929. Using indirect immunofluorescence assays, no virus replication could be detected in mammalian cells. However, in insect cells, virus antigens were readily detected after infection and transfection. Molecular studies were also carried out to define the level at which the virus replication was blocked. These tests were performed specifically with
L929 cells since they were previously reported to be permissive for densovirus infection. Virus transcription in these mammalian cells was not detected even after the transfer of the virus genome into the cells by transfection. Viral DNA could integrate into L cell chromosomes after transfection but not after infection, which indicates a blockage in the virus DNA transfer to the nucleus. However, after integration no viral transcription could be detected. Thus, our results contradict the previous reports about the permissiveness of mammalian cells to densoviruses.
The collective data presented in the current thesis contribute to the understanding of the biology of densoviruses. These viruses use unique expression strategies compared to other members of Parvoviridae. They do not infect mammalian cells in culture, and hence, in vivo homologation tests that are required before proceeding to field applications are
expected to be successful.
|Type de document:
|Directeur de mémoire/thèse:
|Co-directeurs de mémoire/thèse:
||Fédière, Gilles (Faculté d'agriculture Université du Caire)
||densovirus ; virus
||Centre INRS-Institut Armand Frappier
|Date de dépôt:
||27 sept. 2013 14:11
||08 déc. 2015 15:11
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