Browsing by Subject "Viruses"

Now showing 1 - 4 of 4
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    Open Access
    Biochemical characterization of the nucleic acids of some human and animal viruses
    (1982) Mew, Ronald Terence
    In Part I, the isolation and partial characterization of human polyomaviruses from a number of renal transplant patients is described. These isolates proved refractory to cell culture propagation by the methods used, and were thus extracted directly from large volumes of patient's urine. This approach has the advantage that the virus cannot undergo any possible genomic modification, as tends to occur during adaptation to cell culture. Human polyomavirus DNA is very susceptible to mutation during cell passage. Four isolates from different patients yielded sufficient DNA for limited restriction endonuclease characterization. Surprisingly, all four gave the same patterns with EcoRI, BamHI and HindIII. Two isolates that were also digested with PstI gave an identical pattern. These patterns are similar to, but distinct from, other strains of the human polyomavirus BK that have been described. Our isolates had a similar-sized genome to BK, but only 3 HindIII sites compared with 4 in the prototype, and 2 PstI sites compared with only 1 in the prototype. The quantity of DNA obtained directly from urine was usually very limited. In order to produce adequate DNA for complete analysis, viral DNA was recombined with· a bacterial vector (pBR322) and cloned into Escherichia coli strains HB101 and C600. Initially, the well-studied strain BK(MM) was successfully cloned. This clone was used to prepare radioactively-labelled DNA probes for the detection of BK-specific sequences in urine isolates and in subsequent recombinants with patient material. Such cloned material is easier to prepare in bulk than DNA from virus passaged in cell culture. Early attempts to clone DNA from clinical isolates failed, but BK-specific DNA from a patient (P.R.) has recently been cloned successfully. These clones are presently being used to investigate the differences in sequence between our isolates and the known strains of BK. It is hoped that this will shed light on the mechanisms of gene expression of these potentially oncogenic viruses. In Part II, the genomes of four rotaviruses were studied. "Simian agent 11" (SAll) and "offal agent" (OA) were cell culture-adapted strains, whereas "epizootic diarrhoea of infant mice virus" (EDIM) and "infantile gastroenteritis virus" (IGV) were isolated from stool specimens. Experiments were performed to confirm the double-stranded RNA (dsRNA) nature of the SAll genome. It ran at a characteristic density of l.595g/ml in caesium sulphate density gradients, and was resistant to DNase and RNase at high ionic strengths, but susceptible to RNase at low ionic strength. At the start of the project few or no polyacrylamide gel pictures of the nucleic acids of these viruses had been published, although it was known they resembled reovirus in consisting of segmented double-stranded RNA. Such pictures were obtained, and molecular weight estimations made by comparison with dsRNA markers of known MW from a cytoplasmic polyhedrosis virus (CPV) from Heliothis armigera (Harley, Rubenstein, Losman and Lutton, 1977. Virology 76: 210-216). The difficulties in obtaining precise MW values for rotavirus genome segments are discussed. All four genomes consist of 11 dsRNA segments. The pattern of bands produced by PAGE is very similar, and high-resolution gels are required to detect the small mobility differences between some segments. Gel systems were developed to improve on the resolution obtained in co-electrophoresis experiments. During attempts to culture SAll and OA viruses in cell culture, it was observed that treatment of the cells and/or virus with versene-trypsin solution during infection gave a marked increase in virus yield. While this effect was being investigated, reports appeared on the potentiating effect of trypsin on the cell culture of previously refractory rotaviruses. We confirmed that trypsin, when present in the culture medium, greatly increased the yield of progeny SAll virus.
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    Open Access
    Characterisation of two aphid picorna-like viruses
    (1988) Williamson, Carolyn; Rybicki, Edward P; Von Wechmar, M Barbara
    A new aphid virus, aphid lethal paralysis virus (ALPV), was isolated from laboratory-propagated Rhopalosiphum padi aphids co-infected with R. padi virus (RhPV). ALPV and RhPV were separated and ALPV was characterised in detail. Virions are isometric with a diameter of 26 nm, a sedimentation coefficient of 164 Sand a density in CsCl of 1.34 g/ml. Virions contain a 9.7 kb polyadenylated, singlestranded RNA and three major proteins with molecular weights of approximately 30 kilodaltons. By characterising RhPV further, two additional putative capsid proteins were found, an RNA poly(A) tract was detected and an RNA size of 10 kb was determined. A South African isolate of RhPV (RhPVoFs) was found to be serologically identical but physically distinct from a USA isolate. Complementary DNA was synthesized from RhPVOFS RNA and cloned into the plasmid vector, pBR322. This clone was used for the detettion of virus in aphids. ALPV and RhPV are serologically unrelated. ALPV is serologically distantly related to two insect picornaviruses, cricket paralysis virus (CrPV) and Drosophila C virus. No nucleic acid homology was detected between ALPV cDNA and CrPV by dot-blot hybridization. ALPV is serologically unrelated to seven other insect picornalike viruses. RhPV is serologically unrelated to any of the above mentioned viruses. ALPV and RhPV RNAs were efficiently translated in rabbit reticulocyte lysate into high molecular weight polypeptides, the sum of which exceeded the coding capacity of the genomes. Putative capsid precursor proteins of ALPV and RhPV were identified by immunoprecipitation. ALPV translation products were post-translationally cleaved as demonstrated in pulse-chase experiments and in experiments using a translation inhibitor. The efficiency of cleavage was concentration-dependent indicating the action of a protease. In parallel experiments with RhPV RNA, no evidence of post-translational cleavage was observed. In a survey of aphids collected in South Africa, ALPV and RhPV were detected in aphids from two major small-grain producing areas. Both viruses were found to naturally infect most of the cereal aphid species found in this country. ALPV and RhPV infections of R. padi resulted in a marked reduction in longevity and fecundity relative to uninfected aphids. Both viruses were found to be horizontally and vertically transmitted through aphid populations, and aphid host plants and aphid predators could be implicated in virus dissemination. ALPV and RhPV have many properties in common with each other as well as with insect and mammalian picornaviruses. Based on this data, it is proposed that ALPV and RhPV be classified into the picornavirus group (family Picornaviridae).
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    Open Access
    Structure and Recognition of a Novel HIV-1 gp120-gp41 Interface Antibody that Caused MPER Exposure through Viral Escape
    (2017) Wibmer, Constantinos Kurt; Gorman, Jason; Ozorowski, Gabriel; Bhiman, Jinal N; Sheward, Daniel J; Joyce, M Gordon; Asokan, Mangai; Burton, Dennis R; Connors, Mark; Abdool Karim, Salim S; Mascola, John R; Robinson, James E; Ward, Andrew B; Kwong, Peter D; Morris, Lynn; Moore, Penny L
    A comprehensive understanding of the regions on HIV-1 envelope trimers targeted by broadly neutralizing antibodies may contribute to rational design of an HIV-1 vaccine. We previously identified a participant in the CAPRISA cohort, CAP248, who developed trimer-specific antibodies capable of neutralizing 60% of heterologous viruses at three years post-infection. Here, we report the isolation by B cell culture of monoclonal antibody CAP248-2B, which targets a novel membrane proximal epitope including elements of gp120 and gp41. Despite low maximum inhibition plateaus, often below 50% inhibitory concentrations, the breadth of CAP248-2B significantly correlated with donor plasma. Site-directed mutagenesis, X-ray crystallography, and negative-stain electron microscopy 3D reconstructions revealed how CAP248-2B recognizes a cleavage-dependent epitope that includes the gp120 C terminus. While this epitope is distinct, it overlapped in parts of gp41 with the epitopes of broadly neutralizing antibodies PGT151, VRC34, 35O22, 3BC315, and 10E8. CAP248-2B has a conformationally variable paratope with an unusually long 19 amino acid light chain third complementarity determining region. Two phenylalanines at the loop apex were predicted by docking and mutagenesis data to interact with the viral membrane. Neutralization by CAP248-2B is not dependent on any single glycan proximal to its epitope, and low neutralization plateaus could not be completely explained by N- or O-linked glycosylation pathway inhibitors, furin co-transfection, or pre-incubation with soluble CD4. Viral escape from CAP248-2B involved a cluster of rare mutations in the gp120-gp41 cleavage sites. Simultaneous introduction of these mutations into heterologous viruses abrogated neutralization by CAP248-2B, but enhanced neutralization sensitivity to 35O22, 4E10, and 10E8 by 10-100-fold. Altogether, this study expands the region of the HIV-1 gp120-gp41 quaternary interface that is a target for broadly neutralizing antibodies and identifies a set of mutations in the gp120 C terminus that exposes the membrane-proximal external region of gp41, with potential utility in HIV vaccine design.
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    Treatment guidelines and early loss from care for people living with HIV in Cape Town, South Africa: A retrospective cohort study
    (2017) Katz, Ingrid T; Kaplan, Richard; Fitzmaurice, Garrett; Leone, Dominick; Bangsberg, David R; Bekker, Linda‐Gail; Orrell, Catherine
    South Africa has undergone multiple expansions in antiretroviral therapy (ART) eligibility from an initial CD4+ threshold of ≤200 cells/μl to providing ART for all people living with HIV (PLWH) as of September 2016. We evaluated the association of programmatic changes in ART eligibility with loss from care, both prior to ART initiation and within the first 16 weeks of starting treatment, during a period of programmatic expansion to ART treatment at CD4+ ≤ 350 cells/μl.