Potato leafroll luteovirus is an aphid-transmissible virus which has isometric particles and is confined to the phloem tissue of infected plants. Its multiplication was investigated by using plant protoplasts as a model system. In protoplasts, net accumulation of PLRV ceased at approximately 48 his post-inoculation. Virus-specific products were detectable 15 hrs or more post-inoculation and remained detectable at approximately 100 hrs postinoculation. The amount of PLRV accumulated depended on the conditions in which protoplasts were incubated. Incubation at 25°C rather than 20°C and incubation in the dark for a period rather than continuous light resulted in more PLRV accumulation. RNA extracted from PLRV-infected protoplasts was identical on northern blots to that extracted from leaf tissue of PLRV-infected Maris Piper potato plants. Northern blots of RNA from other plants, some resistant, some susceptible to PLRV multiplication, were very similar. Resistant plants appeared to contain smaller quantities of subgenomic RNA. The genes at the 3'-end of the genome are expressed by translation of a subgenomic RNA. This was mapped to position 3376 on the PLRV genome and is therefore 2505 nucleotides long. The untranslated leader sequence of 212 nucleotides contains some putative promoter sequences although not in the same order as described for other viruses. A sequence of 8 nucleotides at the 5'-end of the genomic RNA was found to be repeated at the 5'-end of subgenomic RNA. The complement of this sequence may form part of an internal initiation site for the viral replicase complex in the minus strand RNA. The possibility of the untranslated leader sequence containing several promoters for both subgenomic RNA synthesis and ORF expression is discussed. Protoplast lysates contained a component that sedimented nearer the top of a sucrose gradient than virus particles. This contained subgenomic RNA and was detectable by ELISA but not by electron microscopy. It was not present in extracts of PLRV-infected plant tissue or in preparations of purified virus particles and may therefore be an unstable structure possibly - playing a role in particle assembly.

The technique of in situ hybridisation was optimised for the detection of viral RNA using radioactively-labelled single-stranded DNA and RNA probes, and applied to three areas of interest. Optimum hybridisation conditions were determined in vitro using cells infected with the single-stranded negative sense RNA paramyxoviruses. Transcription of RNA probes was the most rapid and efficient method of probe labelling, since electrophoretic purification was not required and large amounts of RNA were produced. However, their use for in situ hybridisation was problematic due to RNase contamination and low sensitivity. In contrast, DNA probes produced from M13 clones and oligonucleotide probes gave consistent hybridisation results and were preferred in subsequent studies for their ease of use, stability and sensitivity. The effect of virus-host interactions on the clearance of the paramyxovirus, SV5, in a mouse model was investigated by detection of viral RNA and protein in lung sections. Immunisation with purified SV5 proteins prior to infection provided protection against infection, indicated by a reduction in the level of viral RNA and protein, due to enhanced clearance of virus by primed T cells. X-irradiation of the host prior to infection resulted in prolonged or persistent infection in which RNA was detected up to 19 days post-infection. The potential of in situ hybridisation for detection of aetiological agents was demonstrated by investigation of the presence of measles virus in two chronic human diseases. Thus, measles virus RNA was detected in brain sections from a patient with subacute sclerosing panencephalitis and in the osteoclasts of bone sections from a patient with Paget's disease of bone. In situ hybridisation was used to analyse expression of the two immediate-early genes of herpesvirus saimiri, the 52K gene and the hinG gene. Differential expression was detected by hybridisation to mRNA using oligonucleotide probes, in productively-infected cells. The 52K gene was expressed asynchronously throughout the population in agreement with immunocytochemical detection of the 52K protein. In contrast, the hinG gene was expressed synchronously, with all cells showing similar levels of hybridisation, indicating a specific control mechanism for expression of the 52K gene, which differs from that of the hinG gene in requiring or being inhibited by additional factors. This may have relevance to the mechanism of establishment of latency in this virus.

La résistance au séchage des virus dépend de plusieurs facteurs : le type de support, la température, l'humidité et la composition de l'enveloppe ou de la capside. Un virus déposé sur une surface peut conserver son pouvoir infectieux pendant plusieurs jours voire plusieurs mois si les conditions sont réunies. Inactiver des virus présents sur différentes surfaces est un enjeu majeur dans la lutte contre la propagation des maladies nosocomiales. Cette problématique est importante et elle nécessite de réaliser des travaux pour connaître davantage la sensibilité des virus aux substances et produits utilisés dans ce but. [...] Dans le cadre de cette thèse des méthodes ont été développées pour comparer l'activité virucide de désinfectants sur des virus en suspension ou sur une surface (acier inoxydable ou support non tissé-fonctionnalisé). Deux approches ont été mises en oeuvre : la première afin de tester l'activité virucide de supports préalablement fonctionnalisés, la seconde afin de comparer l'activité virucide de produits sous forme liquide ou diffusés par voie aérienne à l'encontre de virus en suspension ou séchés sur un support solide. [...] / The resistance of viruses to drying relies on several factors : the type of surface, temperature, humidity and the compositon of the viral envelope or capsid. A virus adsorbed on a surface can remain infectious for several days or months if the conditions are met. [...] In this thesis, methods were developed to compare the virucidal activity of disinfectants towards viruses in suspension and on surfaces (stainless steel or non-woven fuctionalized textiles). Two approaches were implemented : the first to test the virudical activity of functionalized surfaces

Reciprocal transplant experiments have been made to investigate the .intra-specific variation in two clonal species, Primula vulgaris and RBDUDculus repens. Primula transplants performed best when returned to their native populations, indicating that they were differentia~ed in response to local conditions. There was marked variation in the degree of local specialisation of plants in different primrose populations and possible causes of this variation are discussed. Although buttercup transplants also showed great variability, there was no evidence that they were specialised, either between, or within, local populations. The lack of genetic specialisation in RBDUDculus repens may be due to its spreading growth form, widespread distribution and low level of seedling recruitment. In glasshouse experiments, the presence or absence of neighbours affected many parameters of buttercup growth. Within a genet the effect of edaphic and biotic heterogeneity was integrated, so that ramets in favourable conditions supported interconnected ramets in less favourable sites. Plants of R. repens vary phenotypically in different environments but appear to respond to heterogeneous local conditions by phenotypic plasticity of individual ramets rather than genetic specialisation. The assumption that differences between transplants are solely indicative of genetic specialisation has been questioned. Virus infection was detected in 7 of 14 primrose populations surveyed. Infected plants showed no symptoms of disease, yet they produced significantly fewer but larger leaves than uninfected plants. Differences between transplants which could easily be attributed to genetic variation may be due to differential virus infection. Furthermore, viruses may ultimately contribute to genetic differentiation and have a role as selective forces in the environment. Phenotypic differences between ramets of the same genet of R. repens were maintained and even increased after 26 week's growth in a cammon environment. It is clearly imPortant in transplant experiments to use comparable phenotypes and virus-free plants when determining the role of genotype in the match between organism and environment.

No description available.

Thesis (Masters Diploma (Information Technology))--Cape Technikon, Cape Town, 1994 / Computer virus programs are generally perceived to be a threat to the information stored by computer users. This research evaluated the impact computer viruses have on information stored by computer users. The emphasis was on the effects of computer viruses rather than on the detail of their operation. The main hypotheses involved the question of whether or not computer viruses do pose a threat to the information stored by computer users. The effect of computer viruses on the information of users in industry was measured by sending a questionnaire to 388 companies country-wide. &~ average of 2l,5% of the respondents claimed detrimental effects to information stored on disk due to computer viruses. This and other data was used to guide laboratory experiments on the actual damage done by computer viruses to stored information. A set of test disks was prepared to represent programs and data of a typical PC user in industry. Fifteen different virus programs were used individually to infect the test disks. After each infection, all the test disks were inspected to ascertain damage to data, system and program files as well as to separate disk sectors. The research established that: The damage done by computer viruses to stored information is generally limited to one file or disk area. Where damage to stored information did occur, it was often reversible. Irrational user responses to virus symptoms provide a large potential source for damage to stored information. The availability of master program disks (for program file restoration) and recent, validated data backup is essential to recovery from a computer virus infection. A user can solve most problems caused by virus infections if he has a basic understanding of disk structure, i.e. tracks, sectors, sides, the FAT, etc, and of the use of disk utility programs like Norton Utilities or PCTools. The fact that some of the findings of prominent virus researchers could not be verified, suggests that virus programs could be unstable. Claims regarding the damage inflicted by viruses must be considered to be valid only for a specific copy of the virus under discussion. The importance of using original application software (to minimize the transfer of viruses and to enable program file restoration) , regular back-ups (to enable data file restoration) and basic user awareness (infection prevention, symptoms, the use of anti-viral and utility programs, etc.) was emphasized. The average PC user should be able to clear up a virus infection without assistance by following the given disinfection procedure. Suggestions for further study include virus origins, generations, mutations, multiple infections, and the effect of viruses on computer networks.

M.Sc. / It is the widely accepted view that the human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS) and that South Africa harbors mainly HIV type 1 subtype C (HIV-1 C). Extensively characterized biological isolates (especially of HIV-1 subtype C) for use in HIV/AIDS vaccine and drug development are not readily available. This study evaluated three different protocols for the expansion of virus from infected PBMC's of 68 HIV/AIDS patients (designated HJ1 — 22 and INN1 — 97). Factors influencing the success of a protocol for the expansion of HIV-1 were 1) the amount and time of addition of IL-2 and PHA to the culture media; 2) the fact that freshly isolated clean PBMC's (treated with PHA prior to co-cultivation) was necessary while infected PBMC's could be used fresh or frozen; 3) whether the absence or presence of polybrene as a tissue culture additive had any effect. The I-11V-status of patients could be confirmed with rapid tests and/or NASBA assays, while successful expansion of the virus could be confirmed or refuted by determining p24 levels of sera or culture supematant (with values ranging from <7.8pg/ml to about 280pg/ml). Less sensitive assays like the reverse transcriptase (RT) and gpl 20 ELISA's give much lower absorbance values when compared to the p24 ELISA. Using expanded virus to infect PBMC's and T-cell lines (PM1, U87.CD4-CCR5, U87.CD4-CXCR4 and CEM.NKR-CCR5) and then measuring p24 levels showed that the final protocol chosen was capable of producing high titre, biologically active virus. To further test the biological activity of the isolates, the virus was used in assays evaluating the potential inhibitory ability of natural products and neutralizing antibodies. PCR using universal primers (SK22/SK38/SK39) was not consistently successful in amplifying out the correct sized region of gag (for SK22/SK39 a fragment of 600bp and a 115bp fragment for SK38/SK39 was obtained but not for all the samples). Primers (Cgag189(+/-)) were designed during this project to specifically amplify an 189bp region of gag from subtype C, which proved (in some instances) to be more successful. PCR amplification of the proviral DNA fragments was confirmed by sequencing of selected PCR products. Virus titre was determined by calculating TCID50 (login: 1.054). By modifying expansion and detection protocols it is possible to standardize the process to suit a particular isolate and/or circumstance. This production of large volumes of high titre, biologically active isolates has filled a desperate need for reagents to aid HIV researchers in the development of an effective vaccine or other drug therapy.

No description available.

Black beetle virus (BBV) is a bipartite single-stranded RNA virus belonging to the family Nodaviridae. Its host range has been found to be limited to insects. RNA 1, the larger of the two RNA molecules, with a MW of 1,15 x 10⁶ and the smaller RNA 2 with a MW of 0,46 x 10⁶, are both packaged in the same virus particle. The two RNA molecules are translated separately, with RNA 1 coding for protein A of MW 105 x 10³ and RNA 2 coding for protein α of MW 47 x 10³. Protein α is the major capsid protein precursor, which during in vivo maturation is cleaved to form the coat protein β of MW 43 x 10³, and protein γ of MW 5 x 10³. Cell-free translation of BBV (strain W17) mRNA was carried out in rabbit reticulocyte lysates. Protein α was detectable between 0 and 30 minutes after RNA addition. A protein 'β', which was found to co-electrophorese on polyacrylamide gels with authentic β and which was immunoprecipitated by anti-BBV antiserum, was detectable after 30 minutes. Results of this work show that the formation of 'β' could be prevented by the addition of RNase to the lysate, indicating that intact RNA is necessary for α to β cleavage. Arresting protein synthesis by the addition of cycloheximide to the lysate mix did not inhibit the cleavage. The formation of β could also be prevented by cooling the lysate mix to 1°C. Cleavage of α to β still occurred when RNA 2, without the presence of RNA 1, was translated. Therefore the cleavage is not dependent on a translation product of RNA 1. Sedimentation of lysate on sucrose density gradients showed that α to β cleavage was not accompanied by assembly of BBV RNA and protein lnto a viral substructure as has been shown to occur with some viruses, for example certain picornaviruses. Serial dilution of lysate containing α showed that the level of β decreased with increasing dilution, indicating that the cleavage is not mediated by autocatalysis, but by some other unknown factor. Although much work has been carried out on black beetle virus, no work has been published to date concerning α to β cleavage as an indication of assembly in rabbit reticulocyte lysates. Results of these cell-free translation experiments thus indicate that BBV coat protein precursor α, in association with its messenger RNA 2, undergoes a maturation cleavage in the lysate to produce BBV coat protein β. In addition, this cleavage seems to occur without assembly into any intermediate viral structure

The Tetraviridae are a family of ss (+) RNA viruses that specifically infect lepidopteran insects. Their icosahedral capsids are non-enveloped and approximately 40 nm in diameter with T=4 quasi-equivalent symmetry. The omegatetraviruses, which are structurally the best characterised in the family, include Helicoverpa armigera stunt virus (HaSV) and Nudaurelia capensis omega virus (NwV). The omegatetravirus procapsid is composed of 240 identical copies of the capsid precursor proteins, which undergo autoproteolytic cleavage at its carboxyl-terminus generating the mature capsid protein (b) and γ-peptide. This process occurs in vitro following a shift from pH 7.6 to pH 6.0. The viral capsid encapsidates two ss genomic RNAs: The larger RNA1 encodes the viral replicase as well as three small ORFs while RNA2 encodes the capsid precursor protein together with an overlapping ORF designated P17. While a wealth of structural data pertaining to the assembly and maturation of omegatetraviruses is available, little is known about how this relates to their lifecycle. The principle aim of the research described in this thesis was to use an experimental system developed in the yeast, Saccharomyces cerevisiae, to investigate the assembly of HaSV and NwV virus-like particles (VLPs) in terms of maturation and encapsidation of viral RNAs, in vivo. The yeast expression system used two promoter systems for expression of capsid precursor protein: in the first, a hybrid promoter (PGADH) was used for high-level expression, while the second, PGAL1, produced substantially lower levels of the virus capsid protein precursors. An increase in the level of HaSV capsid protein precursor (p71) via the PGADH promoter resulted in a dramatic increase in VLP assembly as compared with the PGAL system. A protein equivalent to the mature capsid protein (p64) appeared at later time intervals following induction of transcription. Transmission electron microscopic studies showed that p64 correlated with the presence of mature VLPs as opposed to procapsids in cells containing p71. This confirmed that the presence of p64 denoted maturation of VLPs in vivo. Further investigation indicated that maturation correlated with cell aging and the onset of apoptosis. It was shown that induction of apoptosis resulted in VLP maturation while inhibition of apoptosis prevented maturation. These results suggested that the process of apoptosis might be the trigger for maturation of virus procapsids in their host cells. The increase in the efficiency of VLP assembly observed in the high-level expression system was proposed to be due to an increase in the cellular concentrations of viral RNA. To test this hypothesis, HaSV P71 was co-expressed with either P71 mRNA or full length RNA2. An increase in the solubility of p71 was observed in cells expressing increased levels of both RNAs, but there was no increase in the efficiency of VLP assembly. Northern analysis of encapsidated RNAs revealed that there was no selective encapsidation of either P71 mRNA or viral RNA2. This data indicated that the increase in viral RNA was not the reason for increased efficiency of VLP assembly, but most likely resulted from higher concentrations of p71 itself. It was decided to determine whether a highly efficient nodavirus replication system developed in yeast for heterologous production of proteins, could be used as a method for expressing the capsid protein precursor. The aim of using this system was to determine if VLPs assembled in a replication system specifically encapsidated viral RNA. Transcripts encoding the NwV capsid protein precursor (p70) were generated in yeast cells by replication of a hybrid RNA template by the Nodamura virus (NoV) replicase. Western analysis confirmed the presence of p70 as well as a protein of 62 kDa corresponding to the mature NwV capsid protein. Northern analysis of purified VLPs showed that NoV RNA1 and RNA3 were encapsidated, but no RNA2 was detected. Taken together, the data lead to the conclusion that specific encapsidation of tetraviral RNAs required more than close proximity of the viral RNAs and assembling virus-like particles. Encapsidation specificity in the omegatetraviruses may require additional viral proteins such as p17 during encapsidation or specific viral RNA encapsidation was replication-dependent. Replication-dependent assembly has been shown in the nodaviruses.