Bibliography on inactivation of viruses and rickettsiae by heat

Inactivation of viruses and rickettsiae by heat - bibliograph

Molecular studies on the sweet potato virus disease and its two causal agents

The studies presented in this thesis contribute to an increased understanding of the molecular aspects, variability and interaction of the two most important viral pathogens of sweet potato (Ipomoea batatas L): Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV), which cause the severe sweet potato virus disease (SPVD) when co-infecting sweet potato plants. SPVD is the most important disease affecting sweet potato in Africa, and may be the most important virus disease of sweet potato globally. The coat protein gene sequences of several African SPFMV isolates were determined and compared by phylogenetic analyses. Results showed that East African SPFMV isolates were genetically distinct. They could furthermore be divided into two serotypes which differed in their ability to systemically infect the sweet potato cultivar Tanzania. The aetiology of SPVD was studied in sweet potato plants co-infected with SPFMV and SPCSV using nucleic acid hybridisation, bioassays, tissue printing and thin section immunohistochemistry. Resistance to SPFMV in East African sweet potato cultivars was found to be due to inhibition of virus replication rather than movement and resistance was suppressed by infection with SPCSV, resulting in a ca. 600-fold increase in titres of SPFMV. Furthermore, in SPVD affected plants SPFMV is detected outside of the phloem, whereas SPCSV is detected only inside the phloem, which suggests novel as yet unknown mechanisms how SPCSV synergises SPFMV. The genomic sequence of SPCSV was determined. It was composed of two RNA molecules (9407 and 8223 nucleotides), representing the second largest (+)ssRNA genome of plant viruses. The genomic organization of SPCSV revealed novel features for the genus Crinivirus, such as i) the presence of a gene putatively encoding an ribonuclease III-like protein, ii) near-identical, 208 nucleotides long 3’-sequences on both viral RNAs, and iii) the placement of the SHP gene at a new position on the genome of SPCSV relative to other closteroviridae. Northern analyses showed the presence of several sub-genomic RNAs, of which the accumulation was temporally regulated in infected tissues. The 5’-ends of seven sub-genomic RNAs were determined using a PCR based method, which indicated that the sgRNAs were capped

Field and laboratory studies on Rhabdoviruses associated with Epizootic Ulcerative Syndrome (EUS) of fishes.

Epizootic Ulcerative Syndrome (EUS) is a seasonal and widely spread ulcerative disease condition of fresh and brackishwater fishes in Asia caused by a complex of etiological agents. Viral agents have been found to be associated with EUS but the role of viruses in the complex etiology has still to be identified. Further virological examinations were, therefore, conducted in this study. Two warm-water fish cell lines were established from hybrid catfish, male Clarias gariepinus x female C. macrocephalus. The HCK line was derived from head kidney and the HCT line was derived from tail tissue. Both lines were susceptible to 3 birnaviruses, sand goby virus (SGV) and infectious pancreatic necrosis virus (IPNV) serotypes Ab and Sp, 2 reoviruses, golden shiner virus (GSV) and catfish reovirus (CRV), but refractory to all 6 strains of ulcerative disease rhabdovirus (UDRV), channel catfish herpesvirus (CCV) and 1 EUS-associated reovirus (T9231). Only the HCK line was susceptible to recent EUS-associated rhabdovirus strain T9204 and tench and chub reoviruses. Using HCK, BF-2 and SSN-1 fish cell lines, 9 rhabdoviruses were successfully recovered from EUS-diseased fishes during the first 2 weeks of a 1993- 1994 epizootic. Rhabdovirus strain T9412 caused death in fry and skin damage in juvenile striped snakehead fish. A combination of this rhabdovirus and pathogenic Aphanomyces fungus appeared to induce more severe EUS disease in snakehead fish than a single infection with the fungus. EUS transmission was also experimentally achieved by co-habitation of healthy and diseased fish. Three characterised virus strains T9415, T9416 and T9429 possessed a typical bullet- or bacillus-shaped morphology and also exhibited a lyssavirus-like electrophoreotype of structural proteins similar to snakehead rhabdovirus (SHRV) and rhabdovirus strain T9204, while UDRV strains SL11, BP and 20E possessed vesiculovirus-like eletrophoreotypes. The lyssavirus-like EUS-associated rhabdoviruses, except strain T9416, were structurally and serologically similar for which the ‘serotype Sh’ is proposed while the UDRV strains are grouped as a proposed ‘serotype Ud\ Strain T9416 could not be grouped in either serotype as the homologous antiserum was capable of neutralising viruses of both serotypes. The results of this study suggest that the rhabdovirus is one of a complex of etiological agents for EUS and that at least 2 serotypes of EUS-associated rhabdoviruses are identified

Evaluation of transgenic cassava expressing mismatch and non-mismatch hpRNA constructs derived from African cassava mosaic virus and South African cassava mosaic virus open reading frames

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy in the School of Molecular and Cell Biology. Johannesburg, 2015.With rising global food prices, growing populations, climate change and future demand for tuber crops for feed and potential energy source, cassava is well positioned to meet the needs of many countries in the SADC region, including South Africa. However a major constraint to cassava cultivation is cassava infecting begomoviruses (CBVs), including African cassava mosaic virus (ACMV) and South African cassava mosaic virus (SACMV). ACMV and SACMV belong to the family Geminiviridae, comprising of circular single-stranded bipartite. Symptoms associated with CBVs infection include yellow and/or green mosaic, leaf deformation, leaf curling and stunted plant growth. Since no chemical control of virus diseases of plants is possible, one approach to develop virus resistance is via biotechnology, through genetic engineering (GE) of cassava to express hairpin RNA (hpRNA) silencing constructs against CBV. However cassava is recalcitrant and difficult to transform and regenerate. The aim of this study was to produce hpRNA/inverted repeat (IR) hpRNA constructs targeting ACMV AC1/4:AC2/3 open reading frames (ORF) and hpRNA targeting SACMV BC1 ORF to engineer hpRNA expressing transgenic cassava resistant to ACMV and SACMV. Furthermore, the approach was to stack two ACMV contiguous overlapping reading frames (AC1/4) and (AC2/3) in an attempt to improve resistance to CBV. However IR sequences are prone to unfavourable tight secondary structure formation known as cruciform structures. To circumvent this, one set of constructs (mutated sense-arm: mismatch constructs) were designed to contain sodium bisulfite deamination-induced mutations in the hairpin sense-arm making it less complementary to the antisense arm and therefore enhancing IR stability and cruciform junction formation. MM2hp (mismatch construct targeting ACMV AC1/4:AC2/3) and MM4hp (mismatch construct targeting SACMV BC1) were generated. The second construct set, non-mismatch: gateway, was designed based on the most currently used Gateway construct system. Gateway constructs contained an intron positioned between the IR fragments. MM6hp (non-mismatch construct targeting ACMV AC1/4:AC2/3) and MM6hp (non-mismatch construct targeting SACMV BC1) were generated. Similar to the deamination-induced mutations, the intron assisted with IR stability. ACMV- or SACMV-derived hpRNA constructs were transformed into model cassava cultivar cv.60444. Additionally, since few farmer-preferred cultivars or landraces have been transformed for resistance, South African high starch landrace T200 was also transformed with the hpRNA constructs. Agrobacterium-mediated transformation of friable embryogenic callus (FEC) was used and plants regenerated. Several transgenic cv.60444 and T200 lines were regenerated. Cassava landraces are generally less amenable to transformation however were able to report 79 % and 76 % for model cv.60444 and landrace T200, respectively. T200 transformation efficiency reported in this study is 43% higher than previously reported. This is also the first report of South African cassava landrace T200 transformation with ACMV and SACMV-derived hpRNA constructs. Transgenic lines were selected and infected with ACMV and SACMV infectious virus clones. Lines were then monitored at 12, 32 and 67 days post infection (dpi) for symptom development, plant growth and SACMV and ACMV viral load. At 67 dpi, a more significant difference between transgenic lines and untransformed infected cv.60444 was observed. At 67 dpi, 69 % and 75% of ACMV AC1/4:AC2/3 and SACMV BC1 transgenic lines, respectively, showed lower symptoms and reduced viral load compared to control susceptible wild-type cv.60444, but comparable to virus-challenged non-transgenic tolerant landrace control TME3. Notably, a lack of correlation between viral load and symptoms was not always observed. Plant to plant variation was observed between individual transgenic lines generated from each construct (MM2hp; MM4hp; MM6hp and MM8hp) transformation events (A-MM2, A-MM4, C-MM6 and C-MM8). However, overall a positive correlation between symptoms and viral load was observed for virus challenge trials of transgenic lines generated from A-MM4, C-MM6 and C-MM8 transformation events, this overall positive correlation was observed at all 3 dpi (12, 32 and 67 dpi). A number of ACMV and SACMV tolerant transgenic lines were obtained for both mismatch and non-mismatch hpRNA expressing transgenic lines, where virus replication persisted, but symptoms were lower at 67 dpi compared to non-transgenic plants. CBV tolerance levels observed in transgenic lines expressing mismatch technology hpRNA was not significantly different to CBV tolerance levels observed in transgenic lines expressing non-mismatch hpRNA. Expression of ACMV and SACMV- derived constructs generated tolerant cassava lines, where tolerance is defined as plants displaying virus replication but lower to no symptoms. In addition to this, a recovery phenotype was observed in five MM2hp (ACMV AC1/4:AC2/4)- derived hp expressing transgenic lines at 365 dpi, where recovery is defined as no to mild symptoms after an initial period of symptoms, and a reduction in or no viral load. In five MM4hp (SACMV BC1)-derived hpRNA expressing transgenic lines, complete recovery was observed at 365 dpi; no symptoms and no detectable virus. From this study we propose that expression of CBV- derived hpRNA targeting ACMV AC1/4:AC2/4 and SACMV BC1 in CBV susceptible cv.60444 enhances cv.60444 ACMV and SACMV tolerance. Mismatch (mutated sense-arm) construct technology offered tolerance levels comparable to the more conventional and more expensive non-mismatch (Gateway) technology. We therefore also propose that the use of mismatch hpRNA technology in cassava genetic engineering can be used as an alternative approach to transgenic crop production. Promising transgenic lines, showing moderate SACMV and ACMV resistance, were identified and these will be used in further trials as they could be considered favourable to farmers

A Genetic-Proteomic Approach to Identify Cellular Components that Interact with HIV-1

Given the limited genetic coding capacity of HIV-1, it is reasonable to expect that the virus must interact with an extensive set of cellular factors and their complexes to complete its passage through the cell. Indeed, it is remarkable that the viral genome, comprising only about 0.0003% of the entire genetic capacity of the cell, commandeers the cellular environment to its own advantage. However, to date, only a small group of cellular proteins have been shown to be required for viral propagation. In an effort to recover and identify those host proteins that interact in complex with the viral machinery, we have developed a systematic genetic method to select derivatives that can encode a small, but potent, foreign epitope tag yet remain fully replication-competent in culture. In conjunction with a novel cryogenic methodology to capture and preserve viralhost interactions usually lost when more conventional isolation techniques are employed, we have recovered host complexes that interact specifically with each of three independently tagged HIV-1 proteins during progressive infection. Thus, the quantitative purification of the tagged viral proteins has allowed the identification of both described factors already known to interact with each of the targeted viral proteins and as well, unanticipated sets of new host proteins in complex with the virus and previously obscured from investigation. Identification and characterization of protein-protein interactions between the host and the virus will provide insight into the cellular processes expropriated by the virus to complete its life cycle

Bornavirus closely associates and segregates with host chromosomes to ensure persistent intranuclear infection.

Bornaviruses are nonsegmented negative-strand RNA viruses that establish a persistent infection in the nucleus and occasionally integrate a DNA genome copy into the host chromosomal DNA. However, how these viruses achieve intranuclear infection remains unclear. We show that Borna disease virus (BDV), a mammalian bornavirus, closely associates with the cellular chromosome to ensure intranuclear infection. BDV generates viral factories within the nucleus using host chromatin as a scaffold. In addition, the viral ribonucleoprotein (RNP) interacts directly with the host chromosome throughout the cell cycle, using core histones as a docking platform. HMGB1, a host chromatin-remodeling DNA architectural protein, is required to stabilize RNP on chromosomes and for efficient BDV RNA transcription in the nucleus. During metaphase, the association of RNP with mitotic chromosomes allows the viral RNA to segregate into daughter cells and ensure persistent infection. Thus, bornaviruses likely evolved a chromosome-dependent life cycle to achieve stable intranuclear infection