West Nile virus (WNV) belongs to the Flaviviridae family, a virus family of which many members are known as human pathogens. WNV has a worldwide distribution and strains that cluster in lineage II is endemic to sub-Saharan Africa. The complete nucleotide sequence of four lineage II West Nile virus strains, isolated in South Africa from patients with mild or severe WNV infections, were determined. Using a murine model, these strains had been shown to produce either highly or less neuroinvasive infections and induced similar genes to corresponding highly or less neuroinvasive lineage I strains. Nucleotide and amino acid sequence comparison between highly and less pathogenic lineage II strains demonstrated that the non-structural genes and in particular the gene coding for the NS5 proteins were the most variable. All the lineage II strains sequenced in this study were found to possess the E-protein glycosylation site previously postulated to be associated with virulence. Comparison of the signalase cleavage sites suggested that lineage II strains may be cleaved slightly more efficiently than lineage I strains in the C-prM junction, but less efficiently between prM and E genes. Relative to the highly neuroinvasive strains sequenced in this study major deletions were found in the 3’ noncoding region of 2 lineage II strains shown in previous studies to be either less- or not at all neuroinvasive. This is the first report of full genome sequences of highly neuroinvasive lineage II WNV strains. Currently available commercial WNV ELISA kits were developed with lineage I WNV strains and are expensive to use. For these reasons the development of a potential ELISA diagnostic assay based on the South African lineage II strain, H442, was envisaged. Such assay, if reliable and efficacious would be a useful tool towards WNV surveillance. The prM and E genes were selected to be expressed as recombinant antigens because of their co-expression nature and because the envelope protein is the principal target for neutralization. After cloning of the respective genes and verification of integrity, a mammalian expression system was utilized. Different mammalian cells and transfection media were tested and BHK 21 cells with SuperFect transfection medium were found to be best. Attempted expression of proteins was tested with immunofluorescent antibody testing as well as SDS-PAGE and Western blot analysis. Expression of recombinant WNV antigens were also tested in indirect and sandwich ELISA’s systems. It was however not possible to perform these two ELISA systems at a satisfactory level or clearly indicated if expression of proteins was successful. / Dissertation (MSc (Microbiology))--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

Cytotoxic T Lymphocytes (CTL) are known to play an important role in clearing infectious virus from infected hosts in a variety of viral infections. Depending on the type of virus and mode of virus entry both class I and class II restricted CTL can contribute to protection from virus-induced disease. Although CD8 positive CTL are associated with virus elimination and control in many viral infections, elimination of neurotropic viruses from the Central Nervous system (CNS) is more complex due to the lowered expression of MHC antigens on neuronal cells. This failure to constitutively express high levels of MHC antigens by neurons could serve as an advantage to avoid damage to this differentiated and non-renewable tissue. However, abnormal induction of MHC antigens in the CNS mediated by CD4 positive lymphocytes or by astrocytes have also been shown to cause destructive inflammation in the CNS. The present study deals with CTL responses against one such neurotropic virus called Japanese Encephalitis Virus (JEV). JEV is a positive-stranded RNA virus that belongs to the flavivirus group, a group that is among the most important agents causing human encephalitis worldwide. Although passive transfer of monoclonal antibodies against this virus has been shown to confer protection of mice from lethal challenge with virus, neither the presence of CTL against this virus nor its role in conferring protection has been reported so far. Understanding the CTL responses against these viruses acquired importance in light of recent reports that neurovirulence of JEV and yellow fever viruses can be enhanced by the administration of virus specific antibodies. Hence this study was undertaken to examine the possibility of raising CTL specific to JEV. The specificity of the CTL raised, their therapeutic value and the ability of different lymphocyte subsets to mediate protection in vivo are dealt with in this study. Generation of CTL against JEV The generation of CTL against JEV in BALB/c mice, requires MHC defined cell lines that not only support virus infection but are also histocompatible. Several cell lines were initially examined for their ability to support JEV infection as a prc-rcquisitc before their utilization in in vivo and in vitro stimulation protocols aimed at generating JEV-specific CTL. Virus infection was monitored by immunofluorescence using JEV envelope-specific monoclonal antibodies as well as by titration of virus produced from infected cells by plaque assays. These different cell lines that were characterised for their ability to support JEV infection were then utilised to generate and monitor antiviral CTL. Several in vivo immunisation protocols were examined initially find out which of these infected cells prime BALB/c mice efficiently for generation of virus-specific CTL upon secondary stimulation in vitro with infected syngeneic cells. Immunisation of mice with infected cells per se was preferred over free virus since this was thought to facilitate priming against some viral non-structural proteins preferentially found on infected cells in addition to other viral structural proteins. It was observed that not only infected syngeneic and allogeneic cells but also infected xenogeneic cells prime BALB/c mice for the generation of JEV- specific CTL upon secondary restimulation in vitro. An optimal protocol was standardised for the generation of CTL against JEV. This included primary in vivo immunisation of mice followed by secondary in vitro restimulation of splenocytes with infected syngeneic cells. Either immunisation alone or in vitro stimulation of naive splenocytes alone was unsuccessful. The effector cells generated specifically lysed JEV-infccted P388D1 targets but not uninfected P388D1 or YAC-1 targets suggesting that the lysis on infected targets is not mediated by Natural Killer activity. Specificity and MHC restriction of anti JEV Effectors Cell depletion studies using complement mediated lysis were performed to examine the phenotype of the cells mediating virus specific lysis of infected targets. Depletion of Lyt 2.2+ or Thy 1+ but not L3T4+ sub-populations of effector cells inhibited lysis of infected targets showing that the effectors mediating virus-specific lysis were Lyt-2+ T cells. Examination of target specificities and MHC restriction of the antiviral CTL generated showed that although infected xenogeneic cells were used for immunisation, the effector cells recognised only infected syngeneic (P388D1, Sp2/0) and semisyngeneic (Neuro 2a, YAC-1) cells. Virus-specific recognition was found to be class I Kd and class I Dd restricted. These effector cells were also found to recognise cells infected with a closely related flavivirus, West Nile Virus (WNV) suggesting that they were crossreactive to some degree. Based on the consensus motif that has been established for H-2Kd associated peptides, several nonamers were predicted as possible CTL epitopes by scanning the deduced amino acid sequences of three strains of JEV and WNV. Among several predicted nonamers, three peptides were examined for their ability to reconstitute lysis of uninfected targets by polyclonal anti JEV CTL populations. Results demonstrate that peptides derived from NS1 and NS3 but not NS5 protein of JEV were able to partially reconstitute lysis of uninfected targets by effectors when pulsed with the appropriate peptide. Protective ability of the CTL raised against JEV To examine whether anti-JEV effectors raised in vitro could confer protection from intracerebral challenge with JEV, these effectors were adoptively transferred into adult BALB/c mice intracerebrally along with 10 x LDJ0 dose of JEV. More than 55% of these animals were protected from death and survived beyond 100 days after JEV challenge demonstrating that adoptively transferred anti-JEV effectors could indeed confer protection from lethal challenge with JEV. However, adoptive transfer of effectors by either intravenous or intraperitoneal routes did not protect adult mice from the lethal effects of intracerebral challenge with JEV. In contrast to adult mice, newborn mice were not protected from death by the adoptively transferred effector cells. This was also supported by experiments where a correlation was observed with the increasing age of mice and the success of protection conferred by the adoptively transferred effector cells. To establish the identity of cell subsets responsible for protection, Lyt 2, L3T4 or Thy 1 positive cells were specifically depleted from the polyclonal CTL by multiple cycles of complement mediated lysis and the remaining cells were adoptively transferred intracerebrally along with 10 x LD of JEV. These results demonstrate that both Lyt 2 and L3T4 positive T cells present in the effector population were necessary to confer protection of adult mice. Examination of virus-specific neutralising antibodies in the sera of protected and unprotected mice revealed that presence of L3T4 positive cells in the adoptively transferred population increases virus-specific neutralising antibodies. However presence of neutralising antibodies alone was not sufficient to confer protection. The protection required both Lyt-2 and L3T4 positive cells together. These studies could in the long term throw some light on similar observations about age dependant susceptibility to JEV in humans.

West Nile virus (WNV) is a widespread emerging zoonotic neurotropic flavivirus cycling naturally between mosquitos and birds. WNV causes disease in 20% of infections in the most susceptible incidental hosts which are horses and humans. Up to 40% of affected horses and 1- to approximately 50% of affected humans develop neurological signs and/or flaccid paralysis, in some cases fatal or severely debilitating, due to variable encephalitis, meningitis and poliomyelitis. Two predominant genetic lineages exist, 1 and 2, with neurovirulent lineage 1 strains recorded in the northern and western hemispheres, the milder lineage 1 Kunjin strain in Australia, and the lineage 2 strain endemic to southern Africa and Madagascar and considered, until recently, to have mainly mildly pathogenic strains. Since 2002 investigations into South African lineage 2 WNV strains showed that they resulted in severe neurological disease in horses and humans. From 2004 lineage 2 strains were recorded for the first time in southern Europe as a cause of neurological signs and death in birds, and increasingly, in horses and humans. In 2011 the mild lineage 1 Kunjin strain mutated to an equine neurovirulent strain in New South Wales, Australia, and in 2010 the first South African case of lineage 1 WNV was reported from the western Cape in a mare which showed severe neurological signs, abortion and death. Laboratory strains of mice are extremely susceptible to WNV and have been mostly used in experimental studies since the 1937 discovery of the virus in Uganda. In the early 2000s studies in mice showed that field strains of lineage 1 and 2 WNV ranged from mildly pathogenic to highly neurovirulent, however, the associated pathology of the lineage 2 infections was not studied. In the current study, the macroscopic and microscopic pathology of a South African human-neurovirulent field strain of lineage 2 WNV (SPU93/01) and the neurovirulent lineage 1 (NY99/385) strain were investigated and compared in mice used as controls in 2 WNV vaccine studies. The clinical signs, CNS and extra-CNS pathology were indistinguishable between the lineages and some lesions were comparable to those previously reported. Lineage 1 WNV equine pathology has been well described but that of lineage 2 only briefly previously described. The pathology in 6 naturally-occurring fatal lineage 2 WNV-infected horses with severe neurological signs, was investigated and compared with that of the single South African lineage 1 WNV field infection. Diagnoses were confirmed by real-time RT-PCR. Similarities and some slight differences in lesions were found in both mouse and horse studies when compared with lineage 1 pathology cases and with previous reports, and the neurovirulence of the lineage 2 field strains was confirmed. WNV immunohistochemistry (IHC) of all mouse tissues allowed speculation as to pathogenesis of intestinal lesions, but in equine CNS lesions was mostly negative. Ultrastructure of IHC positive cells showed rare WNV particles. In the horse cases rabies, equine herpes virus, and other arboviral co-infections were excluded and similarities and implications of gross lesions of African horsesickness to those often seen in WNV infections were discussed. / Dissertation (MSc)--University of Pretoria, 2014. / gm2014 / Medical Virology / unrestricted

In South Africa (SA), the arboviruses West Nile virus (WNV), Wesselsbron virus (WSLV), Sindbis virus (SINV) and Middelburg virus (MIDV) are considered the most important flavi- and alphaviruses. Clinical presentation and importance of these viruses as animal pathogens in SA remains ambiguous. Although widely endemic in SA, lineage 2 (L2) WNV has rarely been associated with cases of neurological disease and was therefore assumed to be non-pathogenic. However, fatal encephalitis in a foal was diagnosed as L2 WNV in SA, 1996, leading to the thought that L2 cases were possibly being missed. As the above-mentioned arboviruses have the same transmission vectors, Culex mosquitoes for WNV and SINV and Aedes mosquitoes for WSLV and MIDV, co-screening for these viruses is important. We hypothesise that horses could be used as sentinels for virus activity in SA and cases of unexplained neurological disease or fever in animals overlooked, rather than being non-existent. To this end, the study aimed to screen horses displaying unexplained neurological disease or fever with Flavivirus family-specific RT-PCR. Additionally, samples were screened with an Alphavirus family-specific RT-PCR to determine whether co-circulating viruses could be responsible for neurological symptoms in horses. The results would aid in establishing the molecular epidemiology and disease description of each virus, virus distribution and disease seasonality in SA. In total 261 clinical specimens were collected from horses displaying these symptoms (2008 - 2010). Samples were screened with Flavi- and Alphavirus differential diagnostic RT-PCR and acute serum was screened for WNV-IgM and neutralizing antibodies. Serological screening (WNV haemagglutination inhibition, WNV IgG and/or WNV neutralization) identified 62 suspected WNV cases while 34 cases could be confirmed by RT-PCR (16/34), WNV IgM and neutralization assays (18/34) and virus isolation. Neurological disease made up 91% (31/34) of the cases, mortality was calculated at 44% (15/34). Phylogenetically 12/16 RT-PCR positives grouped with L2 SA strains. The first detection of L1 WNV and horse-associated abortion in SA was reported when a pregnant mare aborted her foetus in Ceres, Western Cape. The first cases of WSLV-associated disease in horses were identified by sequencing Flavivirus RT-PCR positive products from 2 horses displaying severe neurological disease; one being fatal. This suggests missed cases in the past. To elucidate virulence factors of WSLV, a human encephalitic strain AV259, was subjected to Roche FLX454 full-genome sequencing and compared to a previously sequenced febrile strain (H177). Several structural amino acid changes occurred in proteins NS2A, NS4B and NS5 of AV259; necessary for Flavivirus replication. Phylogenetically AV259, clinical horse strains and WSLV strains previously isolated from animals, humans and arthropods were similar. Additionally and in concurrence with other studies, WSLV clusters with Sepik virus (SEPV) within the YFV group of the Flaviviridae family. Alphavirus screening identified 17 cases; 6/17 SINV and 11/17 MIDV. SINV-WNV co-infections resulted in fatal neurological disease; remaining SINV cases recovered after displaying fever and/or mild neurological disease. MIDV symptoms varied from “three-day-stiffness” to severe neurological symptoms, with 2 fatalities. Co-infections with equine encephalosis and Shuni virus were identified. MIDV strains identified in this study were phylogenetically distinct from older strains. Results highlight the use of horses as sentinels for virus activity and suggest that these arboviruses may have been previously missed as horse pathogens in Africa. These viruses should be considered as the aetiological agents in animals displaying unexplained neurological or hepatic disease, fevers or abortions. Awareness of flavi- and alphaviruses and the disease manifestation they may have in horses was illustrated. These findings suggest that a WNV vaccine may be beneficial for horses in SA. / Dissertation (MSc)--University of Pretoria, 2011. / Medical Virology / Unrestricted

Recently, non-cellular viral agents became the focus of a large number of scientific groups. A prominent and widespread group of these viruses are flaviviruses, which include, for example, Zika virus, Dengue fever virus, tick-borne encephalitis virus and West Nile virus. There is a considerable diversity among these viruses, however, highly conserved proteins can be found throughout this viral genus. The largest and most conserved protein encoded by flaviviruses is the nonstructural NS5 protein. Its N-terminal domain bears the methyltransferase (MTase) activity. Thanks to the methylation of its genome, it allows the virus to initiate translation and at the same time mask it from the host's immune system. By blocking the active site of this enzyme with a small molecule, viral infection could be stopped not only in one flavivirus, but, due to the high conservation of MTases, in all other flaviviruses. This diploma thesis deals with the aforementioned MTase domain of the NS5 protein, specifically of the West Nile virus (WNV). After designing an insert encoding the WNV MTase domain, amplifying it and ligating it into the vector, the MTase domain was prepared by a recombinant expression, followed by purification. Subsequently, complexes of the protein with small molecules (MTase ligands) were formed, in...