Tetraviruses are entomopathogenic viruses that propagate solely in lepidopteran hosts. Viruses of this group possess non-enveloped 38- to 40-nm capsids arranged in T = 4 surface symmetry. The viral genome consists of one or two single stranded positive sense RNA strands, which define the two genera of this family, the monopartite betatetraviruses and the bipartite omegatetraviruses. Two extensively studied members of the tetraviruses are the omegatetraviruses, Helicoverpa armigera stunt virus (HaSV) and the closely related Nudaurelia capensis ω virus (NωV). The larger genomic strand of HaSV (RNA1) encodes the viral replicase, while the other (RNA2) encodes the 71-kDa capsid precursor protein (p71). The pro-capsid is assembled from 240 copies of p71, which undergo a maturation auto-catalytic cleavage into the 64-kDa (p64) capsid protein and a 7-kDa peptide (p7) forming the capsid shell. The mechanism for the recognition and packaging of the viral genome is poorly understood for these viruses. The principle objective of the research described in this study was to develop in vitro and in vivo experimental systems to investigate interactions between the N terminal domain of HaSV p71 and viral RNAs. More specifically, the two positively charged clusters of predominantly arginine residues that are conserved amongst tetraviruses and the structurally analologous nodaviruses capsid protomers’ N terminal domains were investigated. An in vitro RNA-protein “pull down” system was developed using the rapid protein purification technique of the IMPACTTM-CN system. The coding sequence of the N terminal domain of p71 was fused to that of a chitin binding affinity tag (intein). This fusion protein was used as protein bait for the viral RNA. It was proposed that if RNA interacted with the fusion protein, it would be pulled down by the mass of affinity matrix and be precipitated and fluoresce when analysed by agarose gel electrophoresis using ethidium bromide. Despite optimisation of the in vitro assay, results were affected by the interaction between the intein-tag and nucleic acids, the state of the expressed fusion protein (in particular self-cleavage) and the excessive fluorescence present on the gels. The ADH2-GAPDH yeast expression system was used to investigate the in vivo assembly of p71 containing deletions of either one or both clusters within N terminal domain. It was found that all p71 mutants were expressed with the exception of the mutant containing a deletion of the second cluster. The reasons for this still require further investigation. The expressed p71 mutants were not processed into p64 and were degraded in vivo. In addition, an experimental attempt to purify assembled p71 mutant VLPs was unsuccessful. The assembly defect of p71 mutants emphasised the significance of the clusters, which are possibly required for interaction with viral RNAs for efficient VLP assembly. The results of this study suggest that an alternative tag or in vitro RNA-protein interaction assay be used. In addition, further experiments are required to investigate whether the co-expression of full length viral RNAs are required to rescue the in vivo assembly defect of p71 mutants into VLPs.

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.

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).

After the discovery that Zika virus (ZIKV) infection in pregnant women may result in severe adverse outcomes such as fetal microcephaly, ZIKV must be added to an ever-expanding list of teratogenic viruses. As only a small minority of newborns will display congenital abnormalities after maternal primary infection, innate immune mechanisms must exist in the placenta to prevent viral transmission to the fetus. Understanding the innate antiviral defenses of the placenta is critical to improving diagnosis, treatment, and prevention of adverse pregnancy outcomes stemming from viral infections. We hypothesized that RLRs are expressed in either one or both of the outer cell layers of the chorionic villi, either the syncytiotrophoblast (STB, outermost layer) or in the villous cytotrophoblast (CTB, inner layer), and that expression of these receptors will increase with advancing gestational age.  In order to determine the expression of RLRs in placental tissue (6-32 weeks and full-term), we used immunohistochemistry to stain for the RIG-I-like receptors (RLRs) RIG-I, DHX58/LGP2, and MDA5, as well as the endosomal Toll-like receptor TLR7, that serve as antiviral innate immune receptors involved in detecting microbial ligands and cytoplasmic viral nucleic acids. Hofbauer (HB) cells stained positive for all receptors and served as a positive internal control. TLR7 was not present in either the STB or CTB throughout gestation. MDA5 was localized to the STB cytoplasm up to 13 weeks. After 13 weeks, MDA5 was localized to the CTB cytoplasm. DHX58/LGP2 was localized to the STB cytoplasm at 6 weeks of gestation, the STB apical and basal membranes in addition to cytoplasm at 7 weeks of gestation, and also CTB cytoplasm after 7 weeks of gestation. Lastly, RIG-I was localized to the CTB cytoplasm throughout gestation. The differential expression of these RLRs suggest an innate immune defense system unique to the placenta that is responsible for protecting the conceptus from viral attack.  These findings will complement ongoing work in characterizing replication of teratogenic viruses in the placenta.

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