HIV-1, the virus that causes AIDS, is spreading at an alarming rate. Subtype C, which accounts for approximately 50% of infections worldwide, and 98% of infections in Southern Africa, is by far the most prevalent form of the virus. Most molecular and biochemical studies have been performed on HIV-1 subtype B isolates and products, however, and there is a relative scarcity of corresponding data on subtype C. It is therefore of crucial importance to study subtype C HIV-1 strains in order to understand their characteristic pathogenic effects and to develop effective treatment strategies. The aim of research in our laboratory is the development of novel treatment strategies, with particular focus on identifying novel Subtype C Env-binding peptide ligands. This necessitates the development of reagents for use in the discovery and testing of these compounds. In line with this, the aim of this project was the production and characterisation of recombinant Subtype C gp120s generated from a recently compiled HIV-1 virus cohort. To this end, the gp160-coding regions of 20 South African Subtype C HIV-1 strains isolated from AIDS patients presenting at the Johannesburg General hospital in 2005 were amplified by PCR and sequenced. The gp160 amplicons were used to amplify and clone the gp120-encoding regions of these isolates. Two clones, pTriEx- FV3 and pTriEx-FV5, originating from CXCR4- and CCR5-utilising strains respectively, were selected for further use. These clones were cotransfected into insect cells together with a baculoviral DNA backbone in order to generate gp120-expressing baculoviruses by homologous recombination. Recombinant baculoviruses were used to infect Sf9 insect cell cultures for expression of recombinant gp120, which was then purified using a combination of lectin affinity chromatography and ion exchange chromatography. In order to determine the functionality and conformational integrity of the recombinant gp120, the ability of these purified gp120s to bind CD4 and a panel of well-characterised monoclonal antibodies against various epitopes on gp120 (F425 A1g8, 2G12, F425 B4a1, F425 B4e8, 48d, 17b, IgG1 b12, 5F7, 4G10, 9301, ID6, Chessie 13-39.1, 654-30D and 670-30D) was assessed. Gp120 from the CXCR4-using isolate, FV3, appeared to have an intact, functional CD4 binding site as measured by its ability to bind to CD4 and the CD4 binding site antibody 654-30D. It showed low binding to the monoclonal antibody 654- 30D, moderate binding to 2G12, Chessie 13-39.1 and 9301, and high binding to ID6, but did not show binding to any of the other antibodies used in the recognition profile. Gp120 from the CCR5-using isolate, FV5, showed low binding to the monoclonal antibodies F425 B4a1 and Chessie 13-39.1, moderate binding to 2G12, and showed good binding to 9301and ID6. FV5 gp120 could not, however, bind to CD4. This is likely to be related to a D368G substitution, a mutation affecting a critical structural determinant of CD4 binding. The lack of CD4-binding activity of this gp120 highlights the importance of Asp368 for CD4 binding and hints at a region vulnerable for therapeutic targeting. Our results also highlight the challenges of developing broadly therapeutic drugs for HIV-1, as well as the importance of investigating the specific biochemical and pathogenic properties associated with subtype C HIV-1.

This thesis presents a novel approach to type theory called “pure subtype systems”, and a core calculus called DEEP which is based on that approach. DEEP is capable of modeling a number of interesting language techniques that have been proposed in the literature, including mixin modules, virtual classes, feature-oriented programming, and partial evaluation. The design of DEEP was motivated by two well-known problems: “the expression problem”, and “the tag elimination problem.” The expression problem is concerned with the design of an interpreter that is extensible, and requires an advanced module system. The tag elimination problem is concerned with the design of an interpreter that is efficient, and requires an advanced partial evaluator. We present a solution in DEEP that solves both problems simultaneously, which has never been done before. These two problems serve as an “acid test” for advanced type theories, because they make heavy demands on the static type system. Our solution in DEEP makes use of the following capabilities. (1) Virtual types are type definitions within a module that can be extended by clients of the module. (2) Type definitions may be mutually recursive. (3) Higher-order subtyping and bounded quantification are used to represent partial information about types. (4) Dependent types and singleton types provide increased type precision. The combination of recursive types, virtual types, dependent types, higher-order subtyping, and bounded quantification is highly non-trivial. We introduce “pure subtype systems” as a way of managing this complexity. Pure subtype systems eliminate the distinction between types and objects; every term can behave as either a type or an object depending on context. A subtype relation is defined over all terms, and subtyping, rather than typing, forms the basis of the theory. We show that higher-order subtyping is strong enough to completely subsume the traditional type relation, and we provide practical algorithms for type checking and for finding minimal types. The cost of using pure subtype systems lies in the complexity of the meta-theory. Unfortunately, we are unable to establish some basic meta-theoretic properties, such as type safety and transitivity elimination, although we have made some progress towards these goals. We formulate the subtype relation as an abstract reduction system, and we show that the type theory is sound if the reduction system is confluent. We can prove that reductions are locally confluent, but a proof of global confluence remains elusive. In summary, pure subtype systems represent a new and interesting approach to type theory. This thesis describes the basic properties of pure subtype systems, and provides concrete examples of how they can be applied. The Deep calculus demonstrates that our approach has a number of real-world practical applications in areas that have proved to be quite difficult for traditional type theories to handle. However, the ultimate soundness of the technique remains an open question.

Student Number : 0114163J - PhD thesis - Faculty of Health Sciences / HIV-1 gains entry into host cells by binding to CD4 and a coreceptor, predominantly CCR5 or CXCR4. Viruses that use CCR5 are termed R5, those able to use CXCR4 are termed X4 while viruses able to use both coreceptors are referred to as R5X4. Accelerated CD4 decline and disease progression within an infected HIV-1 subtype B infected individual is often associated with the emergence of viruses able to use CXCR4. However, CXCR4 coreceptor usage appears to occur less frequently among HIV-1 subtype C viruses, the most predominant strain circulating globally, including South Africa. The aim of this study was to investigate the genetic determinants of CXCR4 usage in HIV-1 subtype C isolates. The V3 region of the envelope glycoprotein is the major determinant of coreceptor usage. In Chapter 2, 32 subtype C isolates with known phenotypes (16 R5, 8 R5X4 and 8 X4 isolates) were assessed using a subtype C specific V3-heteroduplex tracking assay. Results indicated that there were sufficient genetic differences to discriminate between R5 viruses and those able to use CXCR4 (both R5X4 and X4). In general, R5 isolates had a mobility ratio >0.9 whereas CXCR4-using isolates were usually <0.9. Sequence analysis of the V3 region showed that CXCR4-using viruses were often associated with an increased positive amino acid charge, insertions and loss of a glycosylation site, similar to HIV-1 subtype B. In contrast, where subtype B consensus V3 has a GPGR crown motif irrespective of coreceptor usage, all 16 subtype C R5 viruses had a conserved GPGQ sequence at the tip of the loop, while 12 of the 16 (75%) CXCR4-using viruses had substitutions in this motif, most commonly arginine (R). Thus, the rare occurrence of CXCR4-using viruses in subtype C may be due to the highly conserved nature of the GPGQ crown that may limit the potential for the development of X4 viruses. The usefulness of available genotype-based methods for predicting viral phenotypes in subtype C was explored in Chapter 3. Results indicated that commonly used prediction methods could detect R5 viruses, but were not very sensitive at identifying X4 viruses. We therefore developed a subtype C specific predictor based on position specific scoring matrices (PSSM). Similar methodology, as used in developing the subtype B PSSM, was applied on a training set of 280 subtype C sequences of known phenotype (229 NSI/CCR5 and 51 SI/CXCR4). The C-PSSM had a specificity of 94% (C.I. [92%-96%]) and sensitivity of 75% (C.I. [68%-82%]), indicating that the C-PSSM had improved sensitivity in predicting CXCR4 usage. This method also highlighted amino acid positions within V3 that could contribute differentially to phenotype prediction in subtypes B and C. A reliable phenotype prediction method, such as the C-PSSM, could provide a rapid and less expensive approach to identifying CXCR4 variants, and thus increase our knowledge of subtype C coreceptor usage. In Chapter 4 we examined the genetic changes in full-length gp160 envelope genes of 23 sequential isolates from 5 patients followed for two to three years. Three of the patients' isolates used CCR5 at all time points while 2 patients underwent a coreceptor switch with disease progression. The genetic changes observed over time indicated changes in length of variable loops particularly the V1, V4 and V5 and shifting N-glycosylation sites, particularly in the 2 patients that used CXCR4. Changes in the V3 were only noted in the 2 patients’ that used CXCR4 which included substitutions of specific amino acids including those in the crown and increased amino acid charge in the V3 region. Both of these patients were dually infected suggesting that recombination may contribute to the rapid emergence of X4 viruses. The in vitro and in vivo development of CXCR4 usage was analysed in a pediatric patient that experienced a coreceptor switch during disease progression (Chapter 5). Biological and molecular clones were generated and the V1-V5 regions sequenced. Analyses of the V3 region indicated that the evolution to CXCR4 usage happens in a step-wise manner that included increased charge and changes in the crown motif. The intermediate variants with predicted dualtropism were also associated with increased V1-V2 lengths, suggesting that other regions may contribute to coreceptor switching. Furthermore, the development of CXCR4 usage within this patient was due to two mutational pathways, in which one resulted in R5X4 viruses and the other X4 variants. In Chapter 6, the impact and treatment of acute TB on HIV-1 diversity in co-infected patients was investigated, specifically to determine the genetic characteristics of the viral populations present before, during and after TB treatment. Plasma samples from 18 HIV-1 infected patients were analysed using the C2V3 region, six of whom showed a high degree of variation using a V3-HTA and were selected for further analyses. All patients were predicted as R5 with no evidence of coreceptor switching over time. There was no correlation between the degree of genetic diversity and viral load, although both showed fluctuations over time. Phylogenetic and pairwise genetic distance analysis indicated that there was amplification of existing variants in 3 patients while in the other 3 patients there were dramatic shifts in viral populations suggesting selection of viral sub-populations over time. Thus in some co-infected patients, TB can affect HIV-1 genetic heterogeneity although there was no evidence of a shift towards CXCR4 usage despite the presence of an AIDS defining illness. Observations in this study have shown that the V3 region is the major determinant of coreceptor usage within HIV-1 subtype C, similar to HIV-1 subtype B. Characteristics such as increased charge length variability of the V3 region and loss of the glycosylation site within this region are associated with CXCR4 usage. The limited number of X4 viruses in subtype C does suggest some restricting mechanisms for CXCR4 usage. In this study we looked at genetic determinants and found that the rare occurrence of CXCR4-using viruses in subtype C, may be due to the highly conserved nature of the GPGQ crown that may limit the potential for the development of subtype C X4 viruses. Furthermore, the development of CXCR4 usage happened in a step-wise manner, with R5X4 viruses intermediates, in which an increased V1-V2 was observed suggesting that other regions within the envelope protein do contribute to coreceptor usage. Thus, regions such as V1-V2 and V4-V5 did contribute to coreceptor usage, but the V3 region remained the most important determinant of coreceptor usage in HIV-1 subtype C isolates. Collectively these findings have provided important data on the genetic determinants of CXCR4 usage in HIV-1 subtype C and an understanding of how they might evolve within a patient.

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ABSTRACT Since its discovery as the etiological agent of AIDS in 1983, HIV-1 has been the focus of unrelenting research into an effective vaccine to control viral infection. Neutralizing antibodies constitute a correlate of immune protection for most available vaccines, but the induction of these antibodies against HIV-1 has become a major challenge. The HIV-1 envelope glycoprotein has evolved to evade neutralizing antibodies in an extraordinary way, yet a vaccine that can stimulate such antibodies remains the best hope to provide sterilizing immunity. The existence of a group of monoclonal antibodies, such as IgG1b12, 2G12, 2F5 and 4E10, capable of neutralizing a broad range of primary isolates signals vulnerable areas on the envelope glycoprotein. Furthermore, passive transfer of these antibodies can completely protect against viral challenge in animal models. The epitopes recognized by these antibodies are being intensely pursued as vaccine targets, in the hope of inducing such specificities. This thesis encompasses a series of studies on characterizing the epitopes recognized by these broadly cross-reactive monoclonal antibodies in the context of subtype C viruses. HIV-1 subtype C is responsible for the vast majority of infections worldwide, however, until recently, little research has been done on these viruses in contrast to the well characterized subtype B strains. Chapter Two describes the characterization of paediatric subtype C viruses for their sensitivity to IgG1b12, 2G12, 2F5 and 4E10. This study was done because of a planned clinical trial of some of these antibodies as post-exposure prophylaxis to prevent mother-to-child HIV-1 subtype C transmission. Only the MAb 4E10 was able to neutralize all the viruses tested, while IgG1b12 was only partially effective. 2F5 and 2G12 did not neutralize any of the viruses. The conclusion was that only 4E10 and IgG1b12 would be suitable for use as prophylactic agents in a population where HIV-1 subtype C is prevalent. Given that subtype C viruses were found to be largely insensitive to 2G12 neutralization, the commonly absent glycan at iv position 295 was introduced into envelope glycoproteins from this clade. The The work presented in Chapter Three explores the requirements of the 2G12 epitope on the envelopes of subtype C viruses. However, this antibody binding site was not readily reconstituted, suggesting structural differences from other HIV-1 subtypes in which the 2G12 epitope is naturally expressed. Chapter Four describes the study of 4E10 resistant virus quasispecies isolated from a seven year old perinatally HIV-1 infected child, in whom anti-MPER antibodies were found. Determinants of 4E10 neutralization were mapped to the epitope of this antibody in the MPER, as well as to the cytoplasmic tail, in particular, to four amino acids in the LLP-2 region. The role of neutralizing antibodies in natural HIV-1 subtype C infection was examined in Chapter Five by following the development of autologous and heterologous neutralizing antibodies in 14 patients during the first year of infection. Potent but relatively strain-specific neutralizing antibody responses were detected within 3-12 months of infection. The magnitude of the responses was associated with shorter V1-to-V5 envelope length and fewer glycosylation sites, in particular in the V1-V2 region. Furthermore, anti-MPER and anti-CD4i neutralizing antibodies were detected in some individuals; however, they were not associated with neutralization breadth. Finally, in Chapter Six these results are analyzed collectively, in the context of the latest findings in the field, and suggestions for further research are discussed.

During 2010, faeces samples for parasitology research oriented on Blastocystis were collected from an anonymous farm. A total of 55 samples were collected (of them for 10 faeces samples from hens, ducks and geese and 25 from pigeons). The samples were cultured in Dobell-Leidlaw medium and viewed by light microscopy. However, this method led to only two foundings of Blastocystis (one from hen, the other from pigeon). Thus, PCR and nested-PCR with specific primers were later used for better detection of Blastocystis. The samples for PCR detection were not collected from the aforementioned farm, but they originated from a collection of isolated DNA samples that was available on the Parasitology institute of AS CR. The results of PCR were checked after electrophoresis and verified by sequenation. The obtained sequences of bird-isolated Blastocystis were phylogenetically analysed and as described to subtype 7.

Background: Chinese populations have been reported to have a higher stroke incidence as well as different stroke epidemiology compared with white populations. However, reliable comparisons have been precluded by a lack of methodologically robust studies. I aimed to systematically evaluate the incidence of stroke, the distribution of its main types/subtypes, and risk factor distributions among stroke types/subtypes in Chinese, and to compare these with data from white populations. Methods: I performed a series of systematic reviews and meta-analyses of studies conducted since 1990 which had data on (1) incidence of stroke, (2) pathological types of stroke or ischaemic stroke subtypes, and (3) frequency of risk factors among pathological types of stroke or ischaemic stroke (IS) subtypes in Chinese populations, and in white populations for comparison. I calculated age-standardized stroke incidence and the proportions of each pathological type and ischaemic subtype. For each risk factor, I calculated study-specific and pooled odds ratios (ORs) using a random effects model for intracerebral haemorrhage (ICH) versus IS, for each IS subtype versus other subtypes, and for overall IS patients, comparing findings for Chinese versus Whites. In addition, I conducted individual patient analyses of data from the National Taiwan University Hospital (NTUH) Stroke Registry, which consecutively recruited 6675 acute stroke patients from 2006-2011, comparing risk factor profiles among stroke types and subtypes and using logistic regression to adjust for potential confounding factors. Results: From my systematic reviews, I found a younger onset of stroke, a slightly higher overall stroke incidence and higher proportion of ICH in Chinese versus white populations. Although the overall proportion of lacunar infarct appeared higher in Chinese from hospital-based studies than white populations, confirming the different distributions of ischaemic subtypes will need further comparable population-based studies. In my meta-analyses comparing risk factors for ICH versus IS, in Chinese - but not Whites – hypertension (HTN) and alcohol intake were significantly more frequent, while mean age was lower in ICH than IS. In IS, the overall prevalence of hypertension, diabetes, smoking, and alcohol intake were similar between Chinese and white IS patients, whereas hypercholesterolaemia, ischaemic heart disease (IHD) and atrial fibrillation (AF) were less common in Chinese IS patients. As for IS subtypes, the relative frequencies of risk factors were mostly qualitatively similar (although different in size) in Chinese and white populations. Compared with other ischaemic subtypes: large artery atherosclerosis (LAA) strokes were associated with diabetes; cardioembolic (CE) strokes were associated with AF and IHD; small vessel disease (SVD) strokes or lacunar strokes were associated with hypertension and diabetes. Analyses of NTUH individual patient data showed that HTN and alcohol intake were independent risk factors for ICH versus IS in a Chinese population in Taiwan, regardless of age, sex, or other risk factors. The results were consistent with my previous risk factor meta-analyses for ICH versus IS. In IS analyses, the prevalence of hypertension, diabetes, AF, and hyperlipidaemia in overall IS patients based in Taiwan were higher than the pooled results in my risk factor meta-analysis for IS for all Chinese populations including mainland China. In terms of risk factor associations with IS subtypes, the findings after controlling for potential confounders were mostly close to my previous meta-analysis results with the exception of stronger associations of hypertension and diabetes with SVD (lacunar) strokes. Conclusion: I have shown a younger onset of stroke, a higher overall stroke incidence, an around twofold higher proportion of ICH and different distribution of IS subtypes, as well as some differences in risk factor distributions among pathological types of stroke and IS subtypes in Chinese compared with white populations. My results help to inform us of different stroke mechanisms in different populations, to guide further well-designed research in this area, and to direct better strategies for stroke prevention in Chinese populations.

The complex and sophisticated circuitry of the neocortex is assembled from an extraordinarily diverse repertoire of neuronal subtypes that reside in distinct functional areas. In recent years, a number of key regulators over neocortical projection neuron subtype and area specification have been identified. It is becoming increasingly clear that these regulators function within a highly-interconnected network, acting in parallel, synergistically, and cross-repressively to orchestrate cortical development. Moreover, an emerging understanding of cortical development has revealed that subtype and area identity are intimately interrelated, and that specification occurs based on several sequential molecular decision points. Although great strides have been made in recent years toward understanding molecular controls over neocortical projection neuron development, many important controls remain to be discovered, and mechanisms by which recently-identified regulators act to delineate subtype and area identity are not well understood. In this dissertation, I characterize functions of two zinc finger transcription factors, Ctip2 and Ctip1, in postmitotic projection neuron subtype and area identity acquisition, using in vivo gain- and loss-of-function approaches in the mouse. I find that Ctip2, known for several years as a central functional control over corticospinal motor neuron (CSMN) terminal differentiation and connectivity, is required both cell-autonomously (within CSMN) and non-cell-autonomously (within striatal medium-sized spiny neurons that surround CSMN axons traveling in the internal capsule) for CSMN to achieve proper connectivity with the spinal cord. In addition, I find that Ctip1, a transcription factor not previously functionally investigated in neocortical development, is a novel control over 1) corticothalamic and callosal projection neuron development and projection neuron migration; and 2) postmitotic area identity acquisition and the formation of sensory maps. Taken together, these results reveal previously unknown functions of Ctip1 in neocortical development, and novel sites of action for Ctip2 control over CSMN connectivity. Ctip1 and Ctip2 are transcriptional controls over the postmitotic specification of neocortical projection neuron subtype and area identity, and over projection neuron connectivity with distant targets.

Both posttraumatic stress disorder (PTSD) and major depressive disorder (MDD) are related to an increased risk for cardiovascular disease (CVD), with links to ischemic heart disease, myocardial infarction, cardiac mortality, and other cardiac conditions. Given that these disorders are associated with increased medical costs, disability, and employer burden, research has investigated which factors increase the likelihood that one will develop CVD. While one hypothesis suggests that individuals who exhibit exaggerated responses to stress are at greater risk of developing CVD, individuals with PTSD/depression have been shown to exhibit both exaggerated and blunted stress responses. The purpose of the present study was to examine whether differing PTSD symptomatology, depressive presentation, dissociation, or cognitive appraisals of stressful events can predict the nature of one’s cardiovascular response to stress. It was hypothesized that: (1) PTSD arousal would be positively related to cardiac reactivity and PTSD avoidance negatively related to cardiac reactivity; (2) depressive activation would be positively related to cardiovascular reactivity and depressive withdrawal negatively related to cardiovascular reactivity; (3) dissociative symptoms would be negatively related to cardiovascular reactivity; and (4) challenge appraisals would be related to greater cardiac reactivity and threat appraisals to greater peripheral resistance. Female participants (N = 57) were administered the Clinician-Administered PTSD Scale (CAPS) and Structured Clinical Interview for DSM-IV Axis I Disorders (SCID) by graduate research assistants. Participants subsequently completed a demographic questionnaire and the Traumatic Life Events Questionnaire (TLEQ), Beck Depression Inventory-II (BDI-II), Dissociative Experiences Scale II (DES II), and Stress Appraisal Measure (SAM) to assess traumatic life events, depression, dissociation, and cognitive appraisals, respectively. Participants’ cardiovascular reactivity (HR, CO, SBP, DBP, and TPR) in response to two stress tasks‒mental arithmetic and public speaking‒were assessed via impedance cardiography and an automated blood pressure cuff. Multivariate regression revealed support for some hypotheses as significant relationships were found between reactivity and PTSD avoidance, PTSD arousal, and depressive withdrawal. No significant relationships were found between reactivity and dissociation, depressive activation, or threat appraisals. This study provides preliminary support for the idea that differing psychological presentations can predict the type of cardiovascular response one exhibits in response to stress.

MSc (Med) Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand / In recent years there has been much progress in understanding and defining the key protein structure-function relationships that mediate Human Immunodeficiency Virus (HIV-1) entry into host CD4+ cells. This process involves fusion of the virus and host cell membranes, following engagement of corresponding viral (gp120) and target (CD4) receptor proteins. Binding of gp120 to CD4 triggers extensive conformational changes in gp120, exposing binding sites for the co-receptor proteins (CCR5 or CXCR4), and facilitating insertion of gp41, the viral fusion protein, into the target cell membrane. Following insertion of gp41, oligomerisation of fusogenic domains on gp41 is thought to drive the juxtaposition of the virus and host cell and fusion of their membranes. Recent reports suggest that detergent-resistant membrane domains, known as lipid rafts, play a crucial role in orientating the receptor molecules during this step of HIV-1 infection. Lipid rafts are typically rich in cholesterol, sphingolipids and GPI-anchored proteins, and are biophysically distinct from the glycerophosolipid bilayer, which constitutes the bulk of mammalian cell membranes. The role of lipid rafts in virus entry, however, is still controversial, and further experimentation is needed to define their importance in this regard. To provide insight into the role of lipid rafts during HIV-1 entry, we evaluated the natural distribution of the host receptor proteins in HIV-1 target cells (U87.CD4.CCR5/CXCR4). CD4 was detected in membrane samples fractionated by sucrose density gradient centrifugation using immunoblotting techniques, while CCR5 and CXCR4 were detected on whole cells by fluorescence microscopy. We then used a primary CCR5-utilising subtype C HIV-1 isolate (FV5) to characterise dynamic changes in the distribution of these receptors and gp41 during viral entry in real-time. Viral fusion assays were set up by inoculating v target cells with FV5 at 23 ºC, a temperature that allows HIV-1 attachment, but is nonpermissive for advancement of the fusion reaction. This prefusogenic form of the virus-host receptor complex is defined as the temperature-arrested state (TAS). We found that, under normal, uninfected conditions, CD4, CCR5 and CXCR4 are distributed throughout both raft and non-raft microdomains on the U87 cell surface, and there is little evidence for any significant redistribution of these receptors into lipid rafts during the HIV-mediated fusion reaction. Interestingly, we observed a change in the structure of CD4 during the fusion process, which could describe a functionally important event in HIV-1 entry, or be related to compromises in the integrity of the virally-infected membranes. Moreover, we discovered that gp41 is capable of membrane insertion and oligomerisation at TAS, in contrast to previous reports that suggest the fusion peptide is not capable of breaching the membrane at this temperature. Our results provide valuable novel insights into the HIV-1 subtype C entry process, and the involvement of lipid rafts in this stage of the viral lifecycle, that may be relevant to novel therapy and immunogen design.