A Novel Strategy for Live Detection of Viral Infection in Drosophila melanogaster

We have created a transgenic reporter for virus infection, and used it to study Nora virus infection in Drosophila melanogaster. The transgenic construct, Munin, expresses the yeast transcription factor Gal4, tethered to a transmembrane anchor via a linker that can be cleaved by a viral protease. In infected cells, liberated Gal4 will then transcribe any gene that is linked to a promoter with a UAS motif, the target for Gal4 transcription. For instance, infected cells will glow red in the offspring of a cross between the Munin stock and flies with a UAS-RFP(nls) transgene (expressing a red fluorescent protein). In such flies we show that after natural infection, via the faecal-oral route, 5-15% of the midgut cells are infected, but there is little if any infection elsewhere. By contrast, we can detect infection in many other tissues after injection of virus into the body cavity. The same principle could be applied for other viruses and it could also be used to express or suppress any gene of interest in infected cells

Utvärdering av vägkonstruktioner med stabilisering

Swedish transport administration has over the past few years changed its way of purchasing contractors. The new way is totalentreprenad, which gives the contractor the opportunity to develop and renew the construction methods. Stabilization is one of the outcomes of that development. It is an unusual method in Sweden and therefore it is important to learn more about its characteristics. Analyses of bearing capacity and deformation have been made in this thesis along with interviews. Knowledge of possible E-modulus on the stabilized layers has been calculated in the program PVD. Experiences and knowledge have been collected and together with the results from the investigation part, given the conclusion that stabilization evolves rut depth and unevenness slower. Furthermore, the investigations have shown that stabilization has good opportunities in future road projects

Nora Virus Persistent Infections Are Not Affected by the RNAi Machinery

Drosophila melanogaster is widely used to decipher the innate immune system in response to various pathogens. The innate immune response towards persistent virus infections is among the least studied in this model system. We recently discovered a picorna-like virus, the Nora virus which gives rise to persistent and essentially symptom-free infections in Drosophila melanogaster. Here, we have used this virus to study the interaction with its host and with some of the known Drosophila antiviral immune pathways. First, we find a striking variability in the course of the infection, even between flies of the same inbred stock. Some flies are able to clear the Nora virus but not others. This phenomenon seems to be threshold-dependent; flies with a high-titer infection establish stable persistent infections, whereas flies with a lower level of infection are able to clear the virus. Surprisingly, we find that both the clearance of low-level Nora virus infections and the stability of persistent infections are unaffected by mutations in the RNAi pathways. Nora virus infections are also unaffected by mutations in the Toll and Jak-Stat pathways. In these respects, the Nora virus differs from other studied Drosophila RNA viruses

Algorithms for Aligning Genetic Sequences to Reference Genomes

The technologies for sequencing genetic materials have improved vastly during the last fifteen years. Sequences can now be determined to affordable costs and therefore are more genetic sequences available than ever before. The bottleneck is no longer to obtain genetic sequences but rather to analyze all the sequence data. A primary step in sequence analysis is to determine a sequence fragment’s position in the genome, a process called aligning. From Computing Science point of view, this is essentially text matching. This is however a much more complex task than searching for strings in ordinary text documents. First, there is large amount of data. An ordinary sequencing experiment could generate more than 100 million sequences. Each sequence should be matched to a reference genome sequence, which is some billions characters in size. Second, the obtained sequences may have differences compared to the reference genome sequence. The algorithms are thus not only searching for exact matches, but for the best approximate matches. In this work I review the algorithms behind modern sequence alignment softwares. I also propose to evaluate to the fast Fourier transform for the task

Ljungan Virus Replication in Cell Culture

Ljungan virus (LV) is a recently identified picornavirus of the genus Parechovirus. LV has been isolated from voles trapped in Sweden and also in the United States. LV infected small rodents may suffer from diabetes type 1 and type 2 like symptoms, myocarditis and encephalitis. LV has been proposed as a human pathogen, with indications of causing diabetes type 1, myocarditis and intrauterine fetal deaths. In this thesis, cell culture adapted LV strains were utilised for development and adaptation of several basic methodological protocols to study the LV biology, e.g. real time PCR, highly specific antibodies and a reverse genetics system. These methods allowed detailed studies of this virus and how it interacts with the host cell. The genomic 5'-end was identified and modelling showed unique secondary structure folding of this region. The LV encodes an aphthovirus-like 2A protein with a DvExNPGP motif. This motif was found to mediate primary cleavage of the LV polyprotein in vitro and is proposed to constitute the carboxy terminus of the structural protein VP1 in LV. Rabbit polyclonal antibodies generated against recombinant structural proteins were used to verify that the LV virion is composed of the structural proteins VP0, VP1 and VP3. Cell culture studies showed that LV replicates to low titer with an absent or delayed cell lysis. LV is proposed to be able to spread by a, for picornaviruses, not previously demonstrated direct cell-to-cell transmission. All results taken together suggest a maintenance strategy of LV including low amounts of the LV genome and persistently infected hosts. Stability studies showed that the LV virion not only maintain activity in acidic and alkaline environments but also exhibit resistance to the commonly used disinfectant Virkon®.The results presented in this thesis show that LV has several unique properties, not previously observed for a picornavirus

Structure of Nora virus at 2.7 Å resolution and implications for receptor binding, capsid stability and taxonomy.

Nora virus, a virus of Drosophila, encapsidates one of the largest single-stranded RNA virus genomes known. Its taxonomic affinity is uncertain as it has a picornavirus-like cassette of enzymes for virus replication, but the capsid structure was at the time for genome publication unknown. By solving the structure of the virus, and through sequence comparison, we clear up this taxonomic ambiguity in the invertebrate RNA virosphere. Despite the lack of detectable similarity in the amino acid sequences, the 2.7 Å resolution cryoEM map showed Nora virus to have T=1 symmetry with the characteristic capsid protein β-barrels found in all the viruses in the Picornavirales order. Strikingly, α-helical bundles formed from the extended C-termini of capsid protein VP4B and VP4C protrude from the capsid surface. They are similar to signalling molecule folds and implicated in virus entry. Unlike other viruses of Picornavirales, no intra-pentamer stabilizing annulus was seen, instead the intra-pentamer stability comes from the interaction of VP4C and VP4B N-termini. Finally, intertwining of the N-termini of two-fold symmetry-related VP4A capsid proteins and RNA, provides inter-pentamer stability. Based on its distinct structural elements and the genetic distance to other picorna-like viruses we propose that Nora virus, and a small group of related viruses, should have its own family within the order Picornavirales.Peer reviewe

Drosophila melanogaster transcriptional response to Nora virus infection

Pathogens induce the activation and suppression of several genes, including potent immune response genes in Drosophila melanogaster. Although several different immune response pathways have been shown to play important roles in the defense against viruses, most of these responses are virus-specific and there appears to be no pathway or set of genes that are induced in response to all viruses. We have here identified genes that are differentially expressed in Drosophila melanogaster in response to Nora virus infection. Our results indicate that none of the known immune response pathways are induced following Nora virus infection, but among the upregulated genes we find three members of the same family as CHKov1 and CHKov2 (ref(3)D), which are involved in resistance to the σ virus. Although Nora virus is a known target of the RNAi pathway, none of the genes belonging to this antiviral system were affected. On the contrary, known immunity responders, such as targets of the Toll and Imd pathways, were downregulated. Several heat-shock proteins, which are usually activated in response to external stimuli, are induced during Nora virus infection. Strikingly, several stress response genes, chaperones and genes required for repair were induced, while digestive enzymes and proteins involved in nutrient uptake and transport were shut down

Drosophila melanogaster transcriptional response to Nora virus infection

Pathogens induce the activation and suppression of several genes, including potent immune response genes in Drosophila melanogaster. Although several different immune response pathways have been shown to play important roles in the defense against viruses, most of these responses are virus-specific and there appears to be no pathway or set of genes that are induced in response to all viruses. We have here identified genes that are differentially expressed in Drosophila melanogaster in response to Nora virus infection. Our results indicate that none of the known immune response pathways are induced following Nora virus infection, but among the upregulated genes we find three members of the same family as CHKov1 and CHKov2 (ref(3)D), which are involved in resistance to the σ virus. Although Nora virus is a known target of the RNAi pathway, none of the genes belonging to this antiviral system were affected. On the contrary, known immunity responders, such as targets of the Toll and Imd pathways, were downregulated. Several heat-shock proteins, which are usually activated in response to external stimuli, are induced during Nora virus infection. Strikingly, several stress response genes, chaperones and genes required for repair were induced, while digestive enzymes and proteins involved in nutrient uptake and transport were shut down

Drosophila melanogaster transcriptional response to Nora virus infection

Pathogens induce the activation and suppression of several genes, including potent immune response genes in Drosophila melanogaster. Although several different immune response pathways have been shown to play important roles in the defense against viruses, most of these responses are virus-specific and there appears to be no pathway or set of genes that are induced in response to all viruses. We have here identified genes that are differentially expressed in Drosophila melanogaster in response to Nora virus infection. Our results indicate that none of the known immune response pathways are induced following Nora virus infection, but among the upregulated genes we find three members of the same family as CHKov1 and CHKov2 (ref(3)D), which are involved in resistance to the σ virus. Although Nora virus is a known target of the RNAi pathway, none of the genes belonging to this antiviral system were affected. On the contrary, known immunity responders, such as targets of the Toll and Imd pathways, were downregulated. Several heat-shock proteins, which are usually activated in response to external stimuli, are induced during Nora virus infection. Strikingly, several stress response genes, chaperones and genes required for repair were induced, while digestive enzymes and proteins involved in nutrient uptake and transport were shut down

Nora virus clearance.

<p>Viral RNA in single flies kept (A) four days on contaminated food (unflip), or serially transferred for (B) four and (C) fourteen days to clean food (flip). Every dot represents the quantification from a single fly. OR: Oregon R, CS. Canton S, n.d: not detected.</p