Thesis (M.S.)--University of Wisconsin--Madison, 1982. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

The adenovirus type 2 origin of DNA replication is located within the terminal 51 bp of the viral genome and contains three recognisable domains: the minimal origin or 'core' and binding sites for the cellular transcription factors Nuclear Factor I and Nuclear Factor III. Initiation of Ad2 DNA replication is preceded by the assembly of a nucleoprotein complex at the viral origin of DNA replication. Recombinant baculoviruses were previously constructed which express full-length Nuclear Factor I (NFIFL) or its DNA binding domain (NFTDBD) for use in experiments. DNase I footprinting experiments were carried out to examine the cooperative interaction between NFI and DBP. DBP can increase the binding affinity of both NFIFL and NFTDBD for their recognition site in the Ad2 origin of DNA replication. Mini-columns containing NFIDBD covalently linked to CNBr-activated Sepharose were used to demonstrate that the adenovirus DNA polymerase (pol) interacts with NFI on its own and as part of the precursor terminal protein (pTP)-pol heterodimer. Immunofluorescence experiments have shown that NFI is specifically targeted to sites of ongoing viral DNA replication in Ad2 infected cells but not in Ad4 infected cells. This localisation of NFI to discrete subnuclear sites reflects the requirement for NFI for efficient initiation of Ad2 DNA replication. The viral transcriptional activator Ela is also localised to discrete subnuclear sites which are very different from those of DBF and are cell cycle dependant. Antibodies to RNA polymerase II show that although its cellular distribution is also cell cycle dependant it is different from both DBF and Ela. When the sites of active transcription were labelled in Ad2 infected cells they were shown to be distinct from both the Ela and RNA polymerase II nuclear distribution.

Bunyamwera (BUN) virus multiplied readily in mosquitoes following intrathoracic injection and also after imbibing'-an infective blood meal. This agent also multiplied following inoculation of human and avian cells in tissue cultures, with production of cytopathic effects. Both in arthropod and vertebrate tissues, enveloped virions 84 nm diameter were visualized by electron microscopic observation of tissues collected after maximum viral proliferation was attained. Following intrathroacic injection of 10 ²•² mouse LD₅₀ of BUN virus into groups of wild-caught mosquitoes comprising both Aedes canadensis and A. vexans, increments of infectivity were first detected in salivary glands and gut at 3 days, and maximum titres of 10 ⁵•² mouse LD₅₀ per organ were attained in salivary glands at 10 days. However, 50 the virus content of legs, which provided a convenient means of sampling hemocelic fluid, increased at 2 days. Virus transmission by biting mice was demonstrated with mosquitoes injected 10 days previously, but not after shorter intervals. No virus replication was demonstrated following ingestion of 10⁴•⁰ mouse LD₅₀ of BUN virus in a blood meal. Aedes aegypti mosquitoes readily supported the replication of BUN virus following injection with 10 ³•³ mouse LD₅₀ or imbibing of 10 ⁴•⁶ mouse LD₅₀. After injection, virus titres of whole mosquitoes declined to 10 ¹•⁷ mouse LD₅₀ at 12 hours, followed by an increase to a peak amount of 10 ⁵•⁰ mouse LD₅₀ at 2 days. After feeding, virus was first detected in legs and salivary glands at 4 days, and attained maximum titres of 10 ⁵•⁰ mouse LD₅₀ in salivary glands at 10 days. Transmission of virus to mice was effected by A. aegypti following feeding and injection 10 days previously, but not at earlier intervals. Following exposure of whole gut cultures of adult A. aegypti mosquitoes to 10 ³•⁷mouse LD₅₀ maximum yields of 10 ⁶•⁰ mouse LD₅₀ per ml. were observed after k days incubation at 29°C, after an initial decline of infectivity to 10 ¹•⁸ mouse LD₅₀ at 12 hours. Enveloped virions with cores 45 nm diameter and total diameters 80 to 100 nm were observed within vacuoles and lining vacuolar membranes of salivary glands and gut cells of A. aegypti mosquitoes 10 days or more after infection with BUN virus. No particles were observed earlier, despite high virus titres 4 days or more after injection. After inoculation of continuous live tissue cultures of human epidermoid corcinoma cells (H.Ep. 2) with 10 ⁶•⁵ mouse LD₅₀, the highest amount of virus produced was 10 ⁷•⁰ mouse LD₅₀ per ml. cell suspension after 24 hours incubation at 37°C. Maximum yields of BUN virus (10 ⁶•² mouse LD₅₀ per ml. cell suspension) were attained 24 hours after inoculation of primary chick embryo fibroblast monolayers with 10 ⁵•² mouse LD₅₀ following incubation at 37 C . However, a peak titre of 10 ⁵•⁰ mouse LD₅₀ was attained 3 days after inoculation with 10 ³•⁷mouse LD₅₀ in cultures incubated at 29°C. Before an increment of virus titre was observed infectivity declined to zero during the initial 4 hours after inoculation of cultures incubated at 37°C, and a tenfold decline of infectivity was noted in cultures incubated at 29°C. Enveloped virions with total diameter 84 nm which contained electron-dense nucleoids 44 nm diameter were observed extracellularly in thin sections of chick embryo fibroblasts infected 12 hours previously with BUN virus. These particles were released by budding. Precursor particles 41 nm diameter were associated with intracellular membranes in occasional cells sectioned at 4 hours. Extracellular virions released one day after inoculation of H.Ep. 2 cultures were tagged by ferritin-labelled anti-BUN antibody. Enveloped virions with mean diameters 100 nm were observed in suspensions of suckling mouse brain infected with BUN virus and stained negatively with phosphotungstic acid. These results show clearly that BUN virus exhibits the essential biological and morphological characteristics of a mosquito-borne arbovirus. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Emerging and re-emerging diseases have increased worldwide in incidence in the past decades. Of these emerging diseases 60.3% are caused by zoonotic pathogens of which 22.8% are arboviruses or arthropod borne viruses. Arboviruses are transmitted by hematophagous insects, especially moquitoes. Multiple factors such as human population growth, climate change and adaptations of certain Aedes mosquito vector species to urban environments and anthropophilic have been attributed to causing this rise in arboviral infections. In Southern Africa, zoonotic arboviruses belonging to the families Flaviviridae (genus Flavivirus), Togaviridae (genus Alphavirus), and those in the order Bunyavirales, family Phenuiviridae: (genus Phlebovirus) and Peribunyaviridae (genus Orthobunyaviruses), have proven, in the past, to be of both medical and veterinary importance. Recent detection of neurological cases in South Africa, most likely, due to flaviviruses, alphaviruses and orthobunyaviruses in the Simbu serogroup, has rekindled interest in these zoonotic diseases. This interest is also warranted because of lack of recent information on arboviral prevalence in mosquito species, distributions, abundance, and ecology, especially of Aedes species, the likely primary vectors of these arboviruses in Southern Africa. To update this lack of information, this study t reports on zoonotic arboviruses circulating in selected areas in the north-eastern provinces of South Africa in mosquitoes with a focus on Aedes. Many Aedes species are morphologically quite difficult to identify especially when they are old, and scales rubbed off in the process of trapping. To aid in the identification of Aedes in this study we provide molecular barcodes for Aedes species occurring in in South Africa and define their phylogenetic relationship with other mainly Afrotropical Aedes mosquitoes based on the cytochrome oxidase I gene sequences. The first Chapter provides a comprehensive review of the literature and describes the importance of arboviruses worldwide and in South Africa, highlighting the role of Aedes mosquitoes as vectors. In Chapter 2, what is known about the broad patterns of Aedes mosquito species diversity, abundance, and distribution in different habitats across selected sites in five different provinces in South Africa is described. The sites selected were chosen because of evidence of neurological cases in humans and animals in recent years likely due to arboviral infections. In total, 61,737 adult mosquitoes were collected from January 2014 to May 2018, using three kinds of carbon dioxide baited trap types About 16% (11,440) were Aedes species, of which, 14 species were recognised or suspected vectors of mosquito-borne diseases because of positive infections, including Aedes mcintoshi which was the most abundant Aedes species captured. The effect of the climatic conditions on the mosquito population dynamics were also investigated. Aedes species were present in the sites following the peak of the rainfall and were mostly captured in temperatures between 18°C and 27°C. Chapter 3 focuses on determining the blood meal source present in engorged Aedes mosquitoes sampled to give an assessment of blood feeding tendencies that would serve useful to determine their vector status. Aedes species were identified feeding on a broad range of livestock, and wildlife, only two specimens were identified as feeding on avian species.Chapter 4 focuses on interpretations of cytochrome oxidase subunit 1 (COI) gene sequences to identify Aedes species in South Africa and to analyse the relationship among the species. A total of 52 COI sequences were aligned representing 21 Aedes species. In several cases these were the first African aedine species uploaded in NCBI GenBank. Neomelaniconion species clustered together, except for Ae. aurovenatus. Finally, the data also suggested that Ae. cumminsii present in South Africa belongs to the subspecies ssp. mediopunctatus. In Chapter 5 results of arboviral infections in Culicidae mosquitoes captured from the selected sites, particularly Aedes species is provided. Arboviral infection or prevalence screening was performed using multiple genus specific polymerase chain reactions (PCR). Alphavirus and Orthobunyavirus were detected in different Culicidae genera, including Aedes, Culex, Anopheles and Mansonia. There were no isolations of pathogenic flaviviruses in mosquitoes. The only alphaviruses detected in mosquitoes were Middelburg, Sindbis and Ndumu viruses during the period of the study. Shuni virus was the only member of Orthobunyavirus genus, detected. Even though, the main aim was to identify pathogenic viruses, several insect-specific viruses belonging to Alphavirus and Flavivirus genera were also detected and these are described in Chapter 6. The numerous arboviruses detected in Culicine mosquitoes, including Aedes species, demonstrate that some species are likely maintaining natural cycling of these arboviruses. Noteworthy, is that mosquito species positive for arboviruses are often the most abundant in the selected sampling locations and that these species blood feed mostly on the larger vertebrates present in the area. Outbreaks possibly occur when the prevalence of certainmosquito species are high due to favourable climatic conditions. Highest arbovirus detections occurred in peri-urban, rural, and conservation areas, indicating that livestock and wildlife likely play an important role in the amplification of these arboviruses. This study highlights the importance of a continues mosquito-based surveillance for arboviruses in South Africa, and the role that Aedes species might be playing in the circulation of these arboviruses. Surveillance for the species that tested positive for pathogenic arboviruses during the arbovirus season may act as an early warning system and can also help to avoid spill over in animals and humans in the area surveyed. / Thesis (PhD)--University of Pretoria, 2020. / Centres for Disease Control and Prevention National Research Foundation / Medical Virology / PhD / Unrestricted

The family Bunyaviridae is the largest known taxonomic group of arboviruses. Four of the five genera possess members which are responsible for serious human and livestock disease. The worldwide distribution of these viruses justify studies which will allow understanding of the replication and transcription cycles within permissive cells. The bunyaviruses have been shown to possess a tripartite single strand RNA genome of negative polarity. Replication is confined to the cytoplasm and the virion envelope is acquired when the genome ribonucleoproteins bud into the golgi. Virus release is presumed to be through exocytosis and ultimately cell lysis. The messenger RNA species of all five genera do not possess a poly-A tail of sufficient length to bind to an oligo(dT) cellulose column. This has made separation of viral transcripts from replicating RNAs difficult. In an effort to achieve this separation, infected cell extracts were centrifuged over 20-40% CsCl gradients which permitted replicating RNA structures to band at a density of 1.32 while cellular and viral mRNAs pellet. Recovery of viral transcripts from the CsCl pelleted RNA required synthesis of a cDNA copy of the virus genome to use as a probe. This was done by an unusual method which employs both genome and antigenomic RNA as templates for reverse transcriptase in a first strand synthesis reaction. Recombinant viral clones were then used in a hybrid selection scheme to recover virus mRNA from pelleted material. After recovery, the messages were visualized on acid urea agarose gels pH 3.5, or used to program an in vitro translation reaction. Using these methods, it was established that each genome segment codes for a single messenger RNA which is most likely capped, and that for at least the mid sized segment, proteins with molecular weights which exceed the coding capacity of the genome are translated from the single message.

Arthropod-borne viruses, arboviruses, have the ability to replicate in both vertebrates and invertebrates and are transmitted to susceptible vertebrate hosts by vectors such as mosquitoes and ticks. Ticks are important vectors of many highly pathogenic arboviruses, including the flavivirus tick-borne encephalitis virus (TBEV) and the nairovirus Crimean-Congo haemorrhagic fever virus. In contrast, alphaviruses are principally mosquito-borne and have been isolated only rarely from ticks; ticks have not been implicated as their vectors. Nevertheless, the alphavirus Semliki Forest virus (SFV) replicates in cell lines derived from many different tick species, including those of the genus Ixodes, which includes vectors of TBEV and its lesspathogenic relative Langat virus (LGTV). In vertebrate cells, arboviruses generally cause cytopathic effects; however, arbovirus infection of arthropod cells usually results in a persistent low-level infection without cell death. While little is known about antiviral immunity in tick cells, the immune system of other arbovirus vectors such as mosquitoes has been studied extensively over the last decade. In insects, pathways such as RNA interference (RNAi), JAK/STAT, Toll, Imd and melanisation have been implicated in controlling arbovirus infection, with RNAi being considered the most important antiviral mechanism. In tick cells, RNAi has been shown to have an antiviral effect, but current knowledge of other immunity pathways is limited and none have been implicated in the antiviral response. In the present study, SFV and LGTV replication in selected Ixodes spp. tick cell lines was characterised and the Ixodes scapularis-derived cell line IDE8 was identified as a suitable cell line for this project. Potential antiviral innate immunity pathways were investigated; putative components of the tick JAK/STAT, Toll and Imd pathways were identified by BLAST search using available sequences from well-studied arthropods including the fruit fly Drosophila melanogaster. Using gene silencing, an attempt was made to determine whether these pathways play a role in controlling SFV and LGTV infection in tick cell lines. Selected genes were silenced in IDE8 cells using long target-specific dsRNA and cells were subsequently infected with either SFV or LGTV. Effects of gene silencing on virus replication were assessed by quantitative real time PCR (qPCR) or luciferase reporter assay. Effects on infectious virus production were measured by plaque assay. Replication of the orbivirus St Croix River virus (SCRV), which chronically infects IDE8 cells, was also quantified by qPCR after silencing of selected genes. Interestingly, SFV or LGTV infection of IDE8 cells resulted in a significant increase in SCRV replication, possibly as a result of interference with antiviral pathways by SFV and LGTV or possibly due to diversion of cellular responses from sole control of SCRV. No evidence for an antiviral role for the JAK/STAT or Toll pathways was found in IDE8 cells. However, an antiviral effect was observed for protein orthologues putatively involved in the RNAi response. Argonaute proteins play an important role in translation inhibition and target degradation mediated by RNAi, and silencing of selected Argonaute proteins resulted in a significant increase in SFV and SCRV replication. The carboxypeptidase CG4572 is essential for an efficient antiviral response in D. melanogaster, and supposedly involved in the systemic RNAi response. A putative tick orthologue of CG4572 was identified and this appeared to be involved in the antiviral response in IDE8 tick cells. When expression of CG4572 was silenced and cells subsequently infected with SFV or LGTV, replication of both viruses was significantly increased. In addition, it was shown that three mosquito orthologues of CG4572 also had an antiviral role against SFV in Aedes mosquito cells. In conclusion, of the tick cell lines investigated, IDE8 provided a suitable model system for investigating tick cell responses against arboviruses and new insight into the nature of the tick cell antiviral response was gained.

Published work submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, for the degree Doctor of Science in Medicine. / Andrew Chakane 2018

Arthropod-borne viruses (arboviruses) belonging to the Togaviridae, Flaviviridae and Bunyaviridae pose a significant threat to human and animal health worldwide. Many of these (re-)emerging viruses have increased in geographic range and severity. Developing vaccine strains of these viruses that cannot infect, or be transmitted by, mosquito vectors would be useful tools to block virus transmission cycles in their vectors. Exploiting the miRNA pathway to generate viruses that are attenuated, or restricted in their replication, has recently received much attention due to the site-specific expression of miRNAs within a host organism. Recently it has been shown for a number of single-stranded and segmented viruses, that virus spread can be restricted in a cell- or tissue-specific manner by engineering miRNA recognition elements (MREs), which are complementary to cellular or tissue-specific miRNAs, into the virus genome. The aim of this proof-of-principle study was to generate recombinant Semliki Forest viruses (rSFVs) that are unable to replicate in aedine mosquito cell lines. RNA was extracted from uninfected and SFV4-infected Aag2 (Ae. aegypti) and U4.4 (Ae. albopictus) cells and analysed using high-throughput Illumina Solexa sequencing in order to identify mosquito-specific miRNAs. Several of the most highly abundant mosquito-specific miRNAs were selected and MRE cassettes were designed. MRE cassettes were cloned into the SFV4 backbone to produce rSFV, which were able to replicate in mammalian BHK-21 cells but unable to replicate in mosquito-derived cells. Each cassette encoded a Gaussia luciferase (Gluc) reporter gene and four copies of each MRE under the control of a duplicated subgenomic promoter. The resulting viruses were viable, infectious and were used to determine the effect of mosquito-specific MREs on virus replication. A significant reduction was observed in both luciferase (~4 – 5 logs) and virus (~3 logs) production in mosquito cells infected with the rSFVs. Further characterisation of the three most inhibited rSFVs (SFV4-MRE276, SFV4-MRE2940 and SFV4-MRE2945) suggested that the insertion of the MRE cassette into the SFV4 backbone had no significant effect on the growth kinetics of these viruses. Each virus replicated to titres comparable to wildtype (wt) SFV. In stability assays, the rSFVs virus maintained high luciferase expression and high virus titre for 5 low multiplicity passages in mammalian cells. Taken together, these results suggest that the incorporation of MRE cassettes into the SFV4 genome did not affect the stability of, or ability for, these recombinant viruses to replicate, efficiently in a mammalian system. rSFV stability in mosquito cells is questionable as luciferase expression was not maintained over 5 low multiplicity passages in these cells. In order to take this work forward, characterisation of the rSFVs in various mosquito species is required in order to determine whether or not these results are replicated in vivo. In order to apply this technology to a virus that is economically important, the three most effective MREs were cloned into an attenuated Rift Valley Fever virus (RVFV) strain, MP12. The Gluc reporter was removed from the MRE cassettes due to size constraints of the RFVF plasmids. Modified MRE cassettes were cloned into the S and L segments, specifically into the 3’ untranslated regions (UTRs) of the NSs and L genes, to generate single or double rRVFV mutants. All viruses were rescued and were viable. Infected cells displayed cytopathic effect characteristic of RVFV-infected cells. Single S segment mutant viruses reached similar titres to, and took the same amount of time to rescue as, wtRVFV. L segment and double segment mutants (recombinant viruses with MREs in both the S and L segments) reached titres two logs lower and took two days longer to rescue than wtRVFV. This suggests that the incorporation of the MRE cassettes into the L segment UTR is affecting virus transcription or translation in some way. Unfortunately, due to time constraints, further characterisation of these viruses could not be carried out. The technology described here may provide an innovative way to create environmentally contained vaccines that are no longer transmitted by their mosquito vectors.

Thesis (Ph.D. (Microbiology))--Mahidol Uniersity, 1973.

Arboviruses are transmitted between vertebrate hosts by arthropod vectors, such as mosquitoes or ticks. In vertebrates arboviruses cause cytopathic effects and disease, however, arbovirus infection of arthropods usually results in persistence. Control of arboviral infection is mediated by the arthropod’s immune system. Pathways such as RNAi, JAK/STAT, Toll and IMD have previously been implicated in controlling arbovirus infections. In contrast, the antiviral role of other pathways in mosquitoes, such as melanisation, is unknown. Using high through output 454 sequencing the transcriptome of U4.4 cells infected with the model arbovirus Semliki Forest virus (SFV)(Togaviridae, Alphavirus) was generated. This experiment revealed intriguing patterns of differential transcript abundance that suggest a broad impact of SFV infection in U4.4 cells, such as in metabolism, cell structure and nucleic acid processing. SFV infection induces differential expression of genes in pathways such as apoptosis, stress response and cell cycle. Most interestingly, this study indicated that melanisation might have an antiviral role in mosquitoes. In arthropods, melanisation is a process involved in wound healing and antimicrobial defences. Phenoloxidase (PO), a key enzyme involved in melanisation, is cytotoxic and therefore kept in its inactive form, prophenoloxidase (PPO), until activation is triggered. The PPO activation process is tightly regulated by serine protease inhibitors (serpins) which inhibit the proteolytic activation reaction. In this thesis I demonstrate that the supernatant of cultured Aedes albopictus-derived U4.4 cells contains a functional proPO-activating system, which is activated by infection with bacteria and virions of SFV. Activation of this pathway reduces the spread and infectivity of SFV in vitro and in vivo. In order to further characterise the PO cascade and its antiviral role the serpins in Ae. albopictus were also investigated. Using the transcriptome sequencing and bioinformatics we identified and classified 11 serpins. We silenced each of the serpins and monitored PPO levels and antiviral activity showing that homologues to drosophila’s serpin- 27a plays a role in melanisation against SFV in vitro. Collectively, these results characterise the mosquito PO cascade as a novel immune defence against arbovirus infection in mosquitoes.