The role of redox-dependent CD4 isomerization and membrane re-distribution in HIV-1 infection

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand in fulfilment of the requirement for the degree of Doctor of Philosophy Johannesburg, 2016CD4, a key molecule of the immune system, is expressed on the surface of certain T lymphocytes (T cells) and participates in MHC class II driven lymphocyte activation. It is also the essential primary receptor for Human Immunodeficiency Virus (HIV) cell entry. Reactive oxygen species (ROS) and other redox-active molecules are important components of the immunological response. They initiate cytocidal responses of the pathogen defence scheme, and redox-activated signalling events ensure appropriate induction of adaptive immunological responses. A redox imbalance can result in failure of essential regulatory mechanisms and the development of pathological immune conditions. An increasing amount of evidence suggests that redox active enzymes such as thioredoxin (Trx) are implicated in CD4 immunological function and in HIV entry at the cell surface, and the dynamic localization of CD4 in specific plasma membrane microdomains, like detergent resistant membrane microdomains (DRM) or lipid rafts, has been shown to play a key role in these regards. However, the biological utility of both the microdomain distribution and the disulphide reduction of CD4, together with the interplay between these processes and the role of cellular oxidoreductases therein remain poorly understood. In this study, we investigated a cell surface-based Trx redox system, and asked whether a relationship exists between these two fundamental aspects of CD4 function by analysing how manipulating cell surface redox conditions affects CD4 membrane domain localization and HIV entry into host CD4-positive (CD4 +) cells. Our investigation of the role of a cell surface redox system in regulating CD4 function was prefaced by research into the membrane association of a variant of Thioredoxin reductase 1 (TrxR1), the enzyme responsible for reducing (and thereby recharging) the active site cysteines of Trx. These studies, carried out in the laboratory of Prof. E Arner (Medical Biochemistry and Biophysics, Karolinska Institutet), were the first to show that a TrxR1 variant called TXNRD1_v3 (henceforth v3) is targeted to DRM domains via N-terminal acylation. Although the role(s) of v3 in this context remains poorly understood, the evidence suggesting that TrxR can associate with the plasma membrane under certain circumstances alludes to the importance of redox capacity at the cell surface, which increasingly suggests it is essential for the function of CD4. To this end, using a transgenic cell line that has been extensively used to model HIV entry and various HIV pseudoviruses, we then analysed the effects of manipulating cell surface redox conditions on CD4 membrane domain distribution and HIV entry. Our results showed that under normal cell growth conditions, the majority of CD4 is associated with detergent soluble regions of the plasma membrane (non-raft regions). Intriguingly, we found that the inhibition of cellular oxidoreductases, and specifically Trx1, results in a redistribution of CD4 into DRMs. CD4 DRM redistribution appears to be targeted, as other cell surface molecules (such as the HIV co-receptor, CCR5) remain unaffected. Furthermore, the redistribution of CD4 to the DRM’s correlates with reduced CD4-dependent HIV infection. Overall, these findings provide evidence for the presence of cell surface-acting redox systems and demonstrate how redox exchanges influence CD4 localization and function. In the context of HIV, our data support previous findings that the thioredoxin system plays an important role in regulating viral entry, which may be related to uncoupled trafficking of CD4 and the HIV co-receptor. Trx-mediated regulation of CD4 membrane domain trafficking may represent a redox switch for functional CD4 clustering during T cell activation.MT201

Development of a Pan-Filoviridae SYBR green qPCR assay for biosurveillance studies in bats

DATA AVAILABILITY: The data are contained within the article or supplementary materials.SUPPLEMENTARY MATERIALS : TABLE S1: Publically available sequences used for primer design; SUPPLEMENTARY FILE S1: Synthetic constructs sequences, TABLE S2: SYBR Green qPCR results for field samples.Recent studies have indicated that bats are hosts to diverse filoviruses. Currently, no panfilovirus molecular assays are available that have been evaluated for the detection of all mammalian filoviruses. In this study, a two-step pan-filovirus SYBR Green real-time PCR assay targeting the nucleoprotein gene was developed for filovirus surveillance in bats. Synthetic constructs were designed as representatives of nine filovirus species and used to evaluate the assay. This assay detected all synthetic constructs included with an analytical sensitivity of 3–31.7 copies/reaction and was evaluated against the field collected samples. The assay’s performance was similar to a previously published probe based assay for detecting Ebola- and Marburgvirus. The developed pan-filovirus SYBR Green assay will allow for more affordable and sensitive detection of mammalian filoviruses in bat samples.National Research Foundation (NRF) of South Africa.https://www.mdpi.com/journal/virusesMedical Virolog

Human rabies associated with domestic cat exposures in South Africa, 1983–2018

Rabies is a fatal encephalitic disease caused by lyssaviruses belonging to the family Rhabdoviridae. At the time of this report, a total of 16 species of lyssaviruses, which included the prototype rabies virus (RABV), and 2 related but unclassified bat lyssaviruses, Taiwan and Kothalati, had been recognised by the International Committee on Taxonomy of Viruses (ICTV 2019). Globally RABV, also referred to as ‘classic rabies’, circulates in natural transmission cycles involving domestic dogs and various wildlife species. In the Americas, RABV is found in certain insectivorous and haematophagous bat species (Banyard et al. 2013). The public health burden of rabies is, however, very closely related to the occurrence of the disease in domestic dogs; thus, human cases of rabies are mostly reported from areas where dog rabies is uncontrolled (Hampson et al. 2015). An annual estimation of 59 000 human deaths occur worldwide with 95% of rabies cases occurring in Africa and Asia (Hampson et al. 2015). In South Africa, RABV circulates both in domestic animals and wildlife cycles, involving the canid and mongoose variants of the virus (Nel, Thomson & Von Teichman 1993). The urban cycle involves domestic dogs reported from various locations in the country, but particularly from the KwaZulu-Natal, Eastern Cape, Limpopo and Mpumalanga provinces (Cohen et al. 2007; Zulu, Sabeta & Nel 2009). Sylvatic cycles of the canid variant RABV in bat-eared foxes and black-backed jackal (Zulu et al. 2009) and the mongoose variant RABV in certain species of mongoose occur in South Africa (Van Zyl, Markotter & Nel 2010). Apart from the reservoir species, canid and mongoose RABV infections are reported in an array of domestic and wildlife species in the country, with these animals primarily serving as dead-end hosts (Sabeta et al. 2018). Laboratory-confirmed human rabies cases in South Africa are predominantly dogmediated, and seven cases of rabies linked to other domestic species and wildlife have been reported (Weyer et al. 2011).http://www.jsava.co.zaam2020Medical VirologyVeterinary Tropical Disease

First report of an imported case of haemorrhagic fever with renal syndrome in South Africa

Haemorrhagic fever with renal syndrome (HFRS) is caused by hantavirus infection. Hantaviruses are not endemic to South Africa, and we report the first detection of an imported case of HFRS in the country. The case involved a traveller from Croatia who presented to a Johannesburg hospital with an acute febrile illness with renal dysfunction. The patient reported visiting rurally located horse stables in Croatia before falling ill, and that a worker in the stables with similar illness was diagnosed with HFRS. Given the exposure history and clinical findings of the case, a clinical diagnosis of HFRS was made and confirmed by laboratory testing.The NICD, a division of the National Health Laboratory Service.http://www.samj.org.zadm2022Medical Virolog

Coding-complete genome sequences for two confirmed monkeypox cases in South Africa 2022

DATA AVAILABILITY : The genome sequences for NICD-SVPL223 and NICD-SVPL232 were submitted to NCBI GenBank (accession numbers ON918611 and ON927248). Raw sequence data have also been deposited in NCBI (BioProject accession number PRJNA856120 and SRA accession number SRR19995508 and SRR19995509).The coding-complete genome sequences of monkeypox virus (MPXV) were obtained from skin lesion swabs from two human cases detected in South Africa in June 2022. Sequence analyses indicated that the genetic sequences of the viruses associated with these two cases were related most closely to the genetic sequences of other MPXVs reported during the 2022 multicountry outbreak and belong to the monkeypox hMPXV-1 clade (previously West Africa clade) and B.1 lineage.https://journals.asm.org/journal/mraam2023BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMedical VirologyMicrobiology and Plant Patholog

Lack of Marburg virus transmission from experimentally infected to susceptible in-contact Egyptian fruit bats

Egyptian fruit bats (Rousettus aegyptiacus) were inoculated subcutaneously (n = 22) with Marburg virus (MARV). No deaths, overt signs of morbidity, or gross lesions was identified, but microscopic pathological changes were seen in the liver of infected bats. The virus was detected in 15 different tissues and plasma but only sporadically in mucosal swab samples, urine, and fecal samples. Neither seroconversion nor viremia could be demonstrated in any of the in-contact susceptible bats (n = 14) up to 42 days after exposure to infected bats. In bats rechallenged (n = 4) on day 48 after infection, there was no viremia, and the virus could not be isolated from any of the tissues tested. This study confirmed that infection profiles are consistent with MARV replication in a reservoir host but failed to demonstrate MARV transmission through direct physical contact or indirectly via air. Bats develop strong protective immunity after infection with MARV.This work was supported by the National Institute for Communicable Diseases.http://jid.oxfordjournals.orgam2016Paraclinical Science

Development of a Pan-<i>Filoviridae</i> SYBR Green qPCR Assay for Biosurveillance Studies in Bats

Recent studies have indicated that bats are hosts to diverse filoviruses. Currently, no pan-filovirus molecular assays are available that have been evaluated for the detection of all mammalian filoviruses. In this study, a two-step pan-filovirus SYBR Green real-time PCR assay targeting the nucleoprotein gene was developed for filovirus surveillance in bats. Synthetic constructs were designed as representatives of nine filovirus species and used to evaluate the assay. This assay detected all synthetic constructs included with an analytical sensitivity of 3–31.7 copies/reaction and was evaluated against the field collected samples. The assay’s performance was similar to a previously published probe based assay for detecting Ebola- and Marburgvirus. The developed pan-filovirus SYBR Green assay will allow for more affordable and sensitive detection of mammalian filoviruses in bat samples

Near-complete genome sequence of Ndumu virus from Garissa, Kenya, 1997

We report a nearly complete genome sequence of Ndumu virus (NDUV) identified using a metagenomics approach. The sequence was derived from a viral isolate obtained from a bovine calf following a diagnostic investigation of the 1997 to 1998 Rift Valley fever (RVF) outbreak in the Garissa District of northeastern Kenya.https://mra.asm.orgam2022Medical Virolog

Epidemiology of human rabies in South Africa, 2008-2018

BACKGROUND. Human rabies cases continue to be reported annually in South Africa (SA). Previous investigations have shown the association between the occurrence of human rabies cases and dog rabies cases in the country. OBJECTIVES. To describe the epidemiology of laboratory-confirmed human rabies cases in SA for the period 2008 - 2018. METHODS. A retrospective document review of laboratory-confirmed human rabies cases for the period 2008 - 2018 was performed using a case register and related documentation available from the National Institute for Communicable Diseases. RESULTS. A total of 105 human rabies cases were laboratory confirmed from 2008 to 2018, with cases reported from all the provinces of SA except the Western Cape. Children and adolescents were most affected by the disease during the study period. In almost half of the cases, medical intervention was not sought after exposure. When victims did seek healthcare, deviations from post-exposure prophylaxis protocols were reported in some cases. CONCLUSIONS. The epidemiological trends of human rabies cases reported in SA for the period 2008 - 2018 remained largely the same as in previous reports. Dog-mediated rabies remains the main source of human rabies in SA.The NICD of the National Health Laboratory Service.http://www.samj.org.zaam2021Medical VirologyVeterinary Tropical Disease