Investigating the host range of Neoromicia capensis coronavirus in vitro

Kotze, Andrea (2019-04)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Coronaviruses are known to cause disease in humans and animals. Two important human coronaviruses that have caused epidemics are severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). These coronaviruses originated in animals and were introduced into the human population through zoonotic transmission. Neoromicia capensis coronavirus (NeoCoV), a bat coronavirus that was discovered in the South African bat species Neoromicia capensis, is 85.5% genetically identical to MERS-CoV. It is believed that NeoCoV is an ancestor of MERS-CoV; however, the potential for NeoCoV to emerge as a potential zoonotic agent has not yet been investigated. This study investigated the host range of NeoCoV in order to assess its potential to cross the species barrier from bats to other mammals. This study attempted to isolate NeoCoV in cell culture to investigate its behaviour in vitro. The host range of NeoCoV was further explored by developing viral pseudoparticles that expressed the spike proteins of NeoCoV, MERS-CoV or SARS-CoV. These pseudoparticles were used to infect various cell lines of mammalian origin to determine which animal species NeoCoV may be able to infect and if its host range has any similarities to that of MERS- and/or SARS-CoV. Attempts were made to isolate NeoCoV in cell culture by inoculating host-derived cells with NeoCoV-positive bat faecal homogenate. Attempts were proven unsuccessful by a highly sensitive quantitative reverse transcription polymerase chain reaction assay. Infecting cell lines with pseudoparticles bearing either the NeoCoV, MERS- or SARS-CoV spike protein revealed that NeoCoV could possibly utilise N. capensis kidney cells for replication, and not the lungs or trachea. Infection of Pipistrellus pipistrellus kidney cells with the three different pseudotypes yielded low levels of infection, suggesting that this cell line is less susceptible to infection by the three viruses. None of the pseudotypes generated were able to infect a kidney cell line derived from Camelus dromedarius, a known host of MERS-CoV, indicating that camel kidney cells are likely not the site of MERS-CoV replication. Results from pseudoparticle infection experiments suggest that NeoCoV would have the ability to infect Vero cells, which originate from African green monkey kidneys, with the same efficiency as MERS- and SARS-CoV. Since pseudoparticles bearing the spike protein of NeoCoV have the ability to infect Vero cells, NeoCoV might have the ability to cross the species barrier from its natural host to non-human primates such as Cercopithecus aethiops. As the human population encroaches on wildlife habitats, the transmission of viruses capable of crossing the species barrier becomes an increasing risk to public health.

AFRIKAANSE OPSOMMING: Coronavirusse veroorsaak siektes in mense en diere. Twee prominente menslike coronavirusse, erge akute respiratoriese sindroomcoronavirus (SARS-CoV) en Midde-Ooste respiratoriese sindroomcoronavirus (MERS-CoV), het epidemies in mense veroorsaak. Hierdie virusse is van diergashere afkomstig en het die menslike bevolking deur zoönotiese oordrag binnegedring. Neoromicia capensis-coronavirus (NeoCoV), „n vlermuiscoronavirus wat in die Suid-Afrikaanse vlermuisspesie Neoromicia capensis ontdek is, is 85.5% geneties identies aan MERS-CoV. Daar word vermoed dat NeoCoV „n voorouer van MERS-CoV is; NeoCoV se potensiaal om as zoönotiese agent op te tree, is egter nog nie ondersoek nie. Hierdie studie het die gasheeromvang van NeoCoV ondersoek in „n poging om die virus se potensiaal om die grens van vlermuise na ander soogdiere oor te steek, te evalueer. Die studie het gepoog om NeoCoV in selkultuur te isoleer om die gedrag van die virus in vitro te bestudeer. Die gasheeromvang van NeoCoV is verder ondersoek deur virale pseudopartikels wat die uitsteekselproteïene van NeoCoV, MERS-CoV of SARS-CoV op hul oppervlak uitdruk, te ontwikkel. Hierdie pseudopartikels is gebruik om verskeie sellyne van soogdieroorsprong te infekteer om vas te stel of NeoCoV oor die vermoë om hierdie selle binne te gaan, beskik, en of die virus se gasheeromvang enige ooreenkomste met dié van MERS- en/of SARS-CoV toon. Daar is gepoog om NeoCoV in selkultuur te isoleer deur selle afkomstig van die gasheer met NeoCoV-positiewe vlermuisontlastingshomogenaat te inokuleer. Pogings is deur „n hoogs sensitiewe kwantitatiewe trutranskripsie-polimerasekettingreaksie-toets onsuksesvol bewys. Die infektering van sellyne met pseudopartikels wat die NeoCoV-, MERS-CoV of SARS-CoV-uitsteekselproteïen uitdruk, het geopenbaar dat NeoCoV moontlik N. capensis-nierselle, en nie die longe of trachea nie, vir replisering gebruik. Infektering van Pipistrellus pipistrellus-nierselle met die drie pseudotipes het lae vlakke van infeksie gelewer, wat suggereer dat die sellyn minder vatbaar vir infeksie deur die drie virusse is. Geeneen van die gegenereerde pseudotipes kon „n sellyn wat afkomstig is van Camelus dromedarius, „n bevestigde MERS-CoV-gasheer, infekteer nie, wat aandui dat kameelnierselle waarskynlik nie die repliseringsetel van MERS-CoV is nie. Pseudopartikelinfekteringsresultate dui daarop dat NeoCoV die vermoë het om Vero-selle, wat van Afrikaanse groenaapnier afkomstig is, met dieselfde doeltreffendheid as MERS- en SARS-CoV te infekteer. Aangesien pseudopartikels wat die uitsteekselproteïene van NeoCoV uitdruk die vermoë het om Vero-selle te infekteer, mag NeoCoV moontlik oor die vermoë om die spesiegrens van sy natuurlike gasheer na nie-menslike primate soos Cercopithecus aethiops oor te steek, beskik. Soos die menslike bevolking inbreuk maak op diere se habitat, word die oordrag van virusse wat die spesiegrens kan oorsteek „n groter risiko vir openbare gesondheid.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/105756
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