Diversity in the Toll-Like Receptor Genes of the African Penguin (Spheniscus demersus)

The African penguin, Spheniscus demersus, is listed as Endangered by the IUCN Red List of Threatened Species due to the drastic reduction in population numbers over the last 20 years. To date, the only studies on immunogenetic variation in penguins have been conducted on the major histocompatibility complex (MHC) genes. It was shown in humans that up to half of the genetic variability in immune responses to pathogens are located in non-MHC genes. Toll-like receptors (TLRs) are now increasingly being studied in a variety of taxa as a broader approach to determine functional genetic diversity. In this study, we confirm low genetic diversity in the innate immune region of African penguins similar to that observed in New Zealand robin that has undergone several severe population bottlenecks. Single nucleotide polymorphism (SNP) diversity across TLRs varied between ex situ and in situ penguins with the number of non-synonymous alterations in ex situ populations (n = 14) being reduced in comparison to in situ populations (n = 16). Maintaining adaptive diversity is of vital importance in the assurance populations as these animals may potentially be used in the future for re-introductions. Therefore, this study provides essential data on immune gene diversity in penguins and will assist in providing an additional monitoring tool for African penguin in the wild, as well as to monitor diversity in ex situ populations and to ensure that diversity found in the in situ populations are captured in the assurance populations

Virus uncoating is required for apoptosis induction in cultured mammalian cells infected with African horse sickness virus

Infection of cultured mammalian cells with African horse sickness virus (AHSV) is known to induce cell death. To date, the trigger(s) of this response, the apoptotic pathways activated during AHSV infection and the functional consequences of apoptosis on the virus replication cycle have yet to be characterized. This study demonstrated that extracellular treatment of BHK- 21 cells with both of the AHSV4 outer capsid proteins, VP2 and VP5, was sufficient to trigger apoptosis. Whether steps in AHSV4 replication subsequent to viral attachment were required for AHSV4-induced apoptosis was also investigated. Apoptosis was induced in BHK-21 cells infected with UV-inactivated AHSV4 and in ribavirin-treated cells infected with AHSV4. However, both AHSV4- and VP2/VP5-stimulated apoptotic responses were inhibited in the presence of the endosomal acidification inhibitors ammonium chloride and chloroquine. These results indicated that uncoating of AHSV4 virions, but not viral transcription or subsequent steps in viral replication, was required for AHSV4 to induce apoptosis in BHK-21 cells. Furthermore, this study showed that both the extrinsic (caspase-8) and intrinsic (caspase-9) apoptotic pathways were induced following AHSV4 infection. The inhibition of caspase activity in AHSV4-infected cells did not diminish AHSV4 replication, but reduced the release and dissemination of progeny viral particles. Taken together, the data indicated that uncoating of AHSV virions was required for apoptosis induction, and that apoptosis enhanced virus spread and release.National Research Foundation and the University of Pretoria’s Institutional Research Theme Programme.http://vir.sgmjournals.orghb201

Lack of diversity at innate immunity Toll-like receptor genes in the Critically Endangered White winged Flufftail (Sarothrura ayresi)

The White-winged Flufftail (Sarothrura ayresi) population is listed as globally Critically Endangered. White-winged Flufftails are only known to occur, with any regularity, in the high-altitude wetlands of South Africa and Ethiopia. Threats to the species include the limited number of suitable breeding sites in Ethiopia and severe habitat degradation and loss both in Ethiopia and South Africa. Toll-like receptors (TLRs) are increasingly being studied in a variety of taxa as a broader approach to determine functional genetic diversity. In this study, we confirm low genetic diversity in the innate immune regions of the White-winged Flufftail similar to that observed in other bird species that have undergone population bottlenecks. Low TLR diversity in White-winged Flufftail indicates that this species is more likely to be threatened by changes to the environment that would potentially expose the species to new diseases. Thus, conservation efforts should be directed towards maintaining pristine habitat for White-winged Flufftail in its current distribution range. To date, no studies on immunogenetic variation in White-winged Flufftail have been conducted and to our knowledge, this is the first study of TLR genetic diversity in a critically endangered species

The Culicoides sonorensis inhibitor of apoptosis 1 protein protects mammalian cells from apoptosis induced by infection with African horse sickness virus and bluetongue virus

African horse sickness virus (AHSV) and bluetongue virus (BTV) are arboviruses of the genus Orbivirus that are transmitted to their vertebrate hosts by Culicoides biting midges. These orbiviruses exhibit lytic infection (apoptosis) in mammalian cells, but cause persistent infection with no cytopathic effects in Culicoides sonorensis cells. Although regulation of apoptosis could thus be integral for establishing persistent virus infection in midge cells, nothing is known about the presence and function of apoptosis pathways in Culicoides midges and their derived cell lines. Here, we report the cloning and functional characterization of an inhibitor of apoptosis protein (IAP), designated CsIAP1, from C. sonorensis cells. The CsIAP1 protein contains two baculoviral IAP repeat (BIR) domains and a RING domain. Silencing of the Cs iap1 gene in C. sonorensis cells caused apoptosis, indicating that CsIAP1 plays a role in cell survival. Stable expression of the CsIAP1 protein in BSR mammalian cells suppressed apoptosis induced by AHSV-4 and BTV-10 infection, and biochemical data indicated that CsIAP1 is an inhibitor of mammalian caspase-9, an initiator caspase in the intrinsic apoptotic pathway. Mutagenesis studies indicated that the BIR2 and RING domains are required for the anti-apoptotic activity of CsIAP1. The results suggest that the mechanism by which CsIAP1 suppresses apoptosis in insect cells may involve inhibition of a Culicoides caspase-9 homologue through a mechanism that requires both the BIR2 and RING domains. This study provides the first evidence that the CsIAP1 protein is a key negative regulator of apoptosis in C. sonorensis cells.The University of Pretoria’s Institutional Research Theme Programme (Grant AOU999) and the National Research Foundation, South Africa (Grant 81068).http://www.elsevier.com/locate/virusres2018-03-31hb2017Microbiology and Plant Patholog

Directed genetic modification of African horse sickness virus by reverse genetics

African horse sickness virus (AHSV), a member of the Orbivirus genus in the family Reoviridae, is an arthropod-transmitted pathogen that causes a devastating disease in horses with a mortality rate greater than 90%. Fundamental research on AHSV and the development of safe, efficacious vaccines could benefit greatly from an uncomplicated genetic modification method to generate recombinant AHSV. We demonstrate that infectious AHSV can be recovered by transfection of permissive mammalian cells with transcripts derived in vitro from purified AHSV core particles. These findings were expanded to establish a genetic modification system for AHSV that is based on transfection of the cells with a mixture of purified core transcripts and a synthetic T7 transcript. This approach was applied successfully to recover a directed cross-serotype reassortant AHSV and to introduce a marker sequence into the viral genome. The ability to manipulate the AHSV genome and engineer specific mutants will increase understanding of AHSV replication and pathogenicity, as well as provide a tool for generating designer vaccine strains.The University of Pretoria’s Institutional Research Theme programme. The National Research Foundation (South Africa) provided E.M., D.J.P. and A.C. with bursaries.http://www.sajs.co.zaam201

African horse sickness virus infects BSR cells through macropinocytosis

Cellular pathways involved in cell entry by African horse sickness virus (AHSV), a member of the Orbivirus genus within the Reoviridae family, have not yet been determined. Here, we show that acidic pH is required for productive infection of BSR cells by AHSV-4, suggesting that the virus is likely internalized by an endocytic pathway. We subsequently analyzed the major endocytic routes using specific inhibitors and determined the consequences for AHSV- 4 entry into BSR cells. The results indicated that virus entry is dynamin dependent, but clathrin- and lipid raft/caveolae-mediated endocytic pathways were not used by AHSV-4 to enter and infect BSR cells. Instead, binding of AHSV-4 to BSR cells stimulated uptake of a macropinocytosis-specific cargo and inhibition of Na+/H+ exchangers, actin polymerization and cellular GTPases and kinases involved in macropinocytosis significantly inhibited AHSV-4 infection. Altogether, the data suggest that AHSV-4 infects BSR cells by utilizing macropinocytosis as the primary entry pathway.http://www.elsevier.com/locate/yviro2017-10-31hb2016Microbiology and Plant Patholog

Pangolin genomes and the evolution of mammalian scales and immunity

Pangolins, unique mammals with scales over most of their body, no teeth, poor vision, and an acute olfactory system, comprise the only placental order (Pholidota) without a whole-genome map. To investigate pangolin biology and evolution, we developed genome assemblies of the Malayan (Manis javanica) and Chinese (M. pentadactyla) pangolins. Strikingly, we found that interferon epsilon (IFNE), exclusively expressed in epithelial cells and important in skin and mucosal immunity, is pseudogenized in all African and Asian pangolin species that we examined, perhaps impacting resistance to infection. We propose that scale development was an innovation that provided protection against injuries or stress and reduced pangolin vulnerability to infection. Further evidence of specialized adaptations was evident from positively selected genes involving immunity-related pathways, inflammation, energy storage and metabolism, muscular and nervous systems, and scale/hair development. Olfactory receptor gene families are significantly expanded in pangolins, reflecting their well-developed olfaction system. This study provides insights into mammalian adaptation and functional diversification, new research tools and questions, and perhaps a new natural IFNE-deficient animal model for studying mammalian immunity.University of Malaya and Ministry of Education, Malaysia [UM.C/HIR/MOHE/08]; UMRG grant from the University of Malaya and Ministry of Education, Malaysia [RG541-13HTM]; Russian Ministry of Science [11.G34.31.0068]; NIH-NHGRI grant [5U54HG00307907]SCI(E)[email protected]

Identification of viral proteins and cellular pathways involved in the induction of apoptosis of African horse sickness virus-infected mammalian cells

African horse sickness virus (AHSV) is transmitted by Culicoides spp. biting midges to horses, causing serious effusion and haemorrhage in various organs and tissues, but is asymptomatic in the insect host. Likewise, AHSV causes dramatic cytopathic effect (CPE) in infected mammalian cells in culture, but no CPE is observed in infected insect cell cultures despite productive virus replication. The basis for this differential host response has not yet been investigated, but is suggestive of the induction of apoptosis in mammalian cells following virus infection. Consequently, the aims of this investigation were essentially to determine whether AHSV infection induces apoptosis in cultured mammalian cells, and to subsequently identify the initiators and effectors of AHSV-induced apoptosis in mammalian cells. To determine whether apoptosis is induced in BHK-21 mammalian cells, the cells were infected with AHSV-4 and the key apoptotic indicators of cell morphology, chromosomal DNA fragmentation and caspase-3 activation were monitored. Results were obtained providing evidence that in vitro infection of BHK-21 cells with AHSV-4 results in apoptosis at 12 h post-infection with maximal levels of apoptosis at 24 h post-infection. By making use of inhibitors of endosomal acidification and UV-inactivated AHSV-4, it was demonstrated that virus disassembly, but not productive virus replication, is necessary for AHSV-4 to trigger apoptosis in BHK-21 cells. Subsequent studies indicated that extracellular coadministration of VP2 and VP5, which likely results in the uptake of VP2-VP5 complexes into the endosomes, induces apoptosis. These findings therefore suggest that the outer capsid proteins are sufficient to trigger apoptosis and that they exert their effect during the early events in AHSV cell entry, where cell binding and endosomal membrane penetration is required. To identify apoptotic pathways triggered during AHSV-4 infection of BHK-21 cells, the enzymatic activity of different cellular caspases in cytoplasmic extracts of infected cells was measured by proteolytic cleavage of caspase-specific chromogenic substrates. Results were obtained indicating the activation of caspases-8 and -9, whereas flow cytometry analyses, following staining of the cells with the lipophilic cation DePsipher™, revealed the loss of mitochondrial membrane potential. This data therefore indicated that both the extrinsic and intrinsic apoptotic pathways are activated in AHSV-infected mammalian cells. Moreover, AHSV-4 infection of BHK-21 cells led to the nuclear translocation of nuclear factor B (NF- B) complexes containing the Rel family members p50 and p65, thus suggesting that NF- B may also play a role in the AHSV apoptotic machinery. Collectively, the results obtained during the course of this investigation provide evidence for apoptosis induction following AHSV infection of mammalian cells, and are the first to delineate the role of early events in the virus replication cycle in the induction of apoptosis and to demonstrate that both the death receptor and mitochondrial pathways can play an essential role in AHSV-induced apoptosis. These results therefore add an important new dimension to AHSV-host cell interactions and provide a foundation for the future study of apoptosis and the role thereof in viral pathogenesis.Dissertation (MSc)--University of Pretoria, 2012.Microbiology and Plant PathologyMScUnrestricte