Immunogenicity and serological applications of flavivirus ED III proteins and multiplex RT-PCR for detecting novel Southern African viruses - Mathengtheng, Lehlohonolo (University of the Free State, 2015)
English: West Nile virus (WNV) is endemic to southern Africa but the true burden of disease associated with WNV infection remains unknown in this region. The presence of the mosquito-borne Wesselsbron virus (WESSV) has also been established in southern Africa. Although not considered a serious human pathogen, WESSV has been associated with encephalitis in humans. No routine testing is performed for WESSV diagnosis in South African patients and hence, similar to WNV infections, the virus remains unreported and overlooked. The presence of tick-borne flaviviruses in southern Africa on the other hand, has not been established despite the presence of suitable vectors. A challenge associated with serological identification of flaviruses is the high level of cross-reactivity between members of flaviviruses and the impracticality of using neutralization assays. Serological assays using reagents that can be handled in a biosafety level 2, or lower facility, were developed and evaluated for the detection and differentiation of tick- and mosquito-borne flaviviruses in the Free State province of South Africa. A total of 2393 serum samples from a variety of species including humans, cattle and sheep were tested using Kunjin virus (KUNV) cell lysate antigen for the detection of anti-flavivirus antibodies in an indirect IgG enzyme-linked immonosorbent assay (ELISA). To further differentiate positive reactors on KUNV assay for antibodies against tick- or mosquito-borne flaviviruses, recombinant envelope domain III (r-EDIII) proteins of Langat virus (LGTV), WNV and WESSV were expressed in a bacterial expression system and used in ELISA. A total of 722 samples were positive on the KUNV assay of which 71, 457 and 431 were positive on the r-LGTVEDIII, r-WNVEDIII and r-WESSVEDIII assays, respectively. A total of 70 samples were reactive on the KUNV assay but not on any of the other assays, suggesting that there are other flaviviruses circulating in the Free State province for which specific r-EDIII assays were not available. Collectively, the results suggest a strong presence of flaviviruses co-circulating in the Free State province with an abundance of mosquito-borne flaviviruses. There is evidence suggesting the presence of tick-borne flaviviruses but it has yet to be confirmed. The EDIII protein is a useful tool that can be utilized in the detection and differentiation of flaviviruses in resource-limited laboratories. Vertebrate hosts play a role in the maintenance and circulation of flaviviruses and, although not involved in the direct transmission of tick- and mosquito-borne flaviviruses, form a link for virus transmission between vectors. In addition to rodent involvement in maintenance of flaviviruses, rodents have also been implicated in the transmission of other medically significant viruses such as arenaviruses, lyssaviruses and hantaviruses. Arboviruses and viral heamorrhagic fevers are among the most pathogenic and devastating disease agents in many parts of the world. It is therefore important for surveillance of such pathogens to be conducted as they may result in considerable public health implications. Molecular assays were developed for the detection of a selected number of arboviruses and viral heamorrhagic fevers, specifically Crimean-Congo haemorrhgaic fever virus (CCHFV), mosquito-borne and tick-borne flaviviruses, as well as hantaviruses. To date, the presence of hantaviruses have not been confirmed in southern Africa despite their emergence in the western and eastern parts of Africa in recent years. In our study, serum samples of patients presenting with a tick-bite and febrile illness without diagnosis were screened for hantavirus IgG antibodies using commercial assays that represent the American and Eurasian hantavirus species. The overall seropositivity rate obtained was 10% and 6% for assays representing the Eurasia and America hantavirus species, respectively. The emergence of hantaviruses in Africa and their seroprevalence in the Cape region of South Africa as well as in our study warranted the development of a molecular assay to further investigate the presence of these viruses in southern Africa. In order to achieve this, a real-time RT-PCR was designed and optimized. The assay was designed by identifying in-house primers targeting the partial region of the S-segment of hantaviruses and hydrolysis probes targeting the inner region of the amplicon. The probes were based on nucleotide sequences targeting the Murinae-associated hantaviruses for the HNLS probe, Sigmodontinae- and Arvicolinae-associated hantaviruses for the ASPRB probe, as well as the SANGV probe for the African hantavirus Sangassou virus. The flavivirus RT-PCR targeted the NS5 region with a probe shown to successfully detect RNA samples that represent eight different flavivirus species. The hantavirus primers and probes were evaluated using RNA transcribed from synthetic genes representing the different hantaviral genotypes and subsequently reverse transcribed cDNA. The limit of detection was determined to range from ~160 to ~17 copies of DNA for the various hantaviral probes and flavivirus probe. In addition, a conventional multiplex PCR assay aimed at detecting CCHFV and flavivirus RNA in samples collected from undiagnosed patients presenting with a tick-bite and febrile illness was developed by using nested primers targeting the partial region of the genome of the S-segment of CCHFV and hemi-nested primers targeting the partial region of the NS5 gene of flaviviruses. When clinical samples from patients with known tick-bites, mild disease and no diagnosis were screened, a patient was restrospectively diagnosesd as having a CCHFV infection. This result highlights the need for awareness to arboviruses and viral hemorrhagic fevers in mild cases that may easy be overlooked but constitute a significant public health risk. Similarly, there needs to be an increase in awareness for travelers to South Africa at risk of returning to their country with an exotic viral haemorrhagic fever, highlighting the need for increased awareness and increased diagnostic capacity for arboviruses. Finally the current lack of registered human vaccines warrants continued investigation of the immunogenicity of selected viral proteins. The recombinant antigens developed for serological purposes were further employed in this study to determine the immunogenicity of the envelope domain III proteins of WNV and LGTV in a mouse model. Small molecule antigens or weakly immunogenic antigens frequently require an adjuvant to stimulate a stronger immune response. In addition, adjuvants can shift an immune response towards a Th1 or Th2 response as required based on immune correlates of protection. Groups of mice were immunized with purified r-WNVEDIII or r-LGTVEDIII protein alone, r-WNVEDIII or r-LGTVEDIII protein in combination with one of three adjuvants, including saponin, Titermax® gold and Alhydrogel® or one of the three adjuvants without a flavivirus protein. In the absence of any adjuvant the results from WNV protein alone were inconclusive whereas a strong IgG1 response was induced by LGTV EDIII. Briefly, protein alone or mixed with alum elicited a predominantly Th2 response whereas protein in combination with saponin or Titermax® gold induced a mixed Th1 and Th2 response. Mice immunized with r-WNVEDIII reacted against KUNV native antigen indicating that the protein was expressed in conformation exposing epitopes that are required to induce a detectable antibody response. The formulation of the WNV and LGTV proteins with different adjuvants produced similar results with a shift in response depending on the adjuvant. Despite an absence of being able to assess cell mediated responses using antigen stimulated splenocytes and profiling cytokine production as initially planned, the results do confirm that r-WNVEDIII and r-LGTVEDIII proteins are immunogenic in the absence of complete E protein, with ability to induce detactable antibody when formulated with adjuvant and that different adjuvants are able to have an immunomodulatory influence on the type of response induced.

Afrikaans: Wes-Nyl-virus (WNV) is endemies in suider-Afrika, maar die werklike siektelas wat met WNV-infeksie geassosieer word, is steeds onbekend in hierdie streek. Die teenwoordigheid van die muskiet-gedraagde Wesselsbron-virus (WESSV) is ook in suider-Afrika aangetoon. Alhoewel dit nie as 'n ernstige menspatogeen beskou word nie, word WESSV geassosieer met enkefalitis in mense. Geen roetinetoetsing word gedoen vir die diagnose van WESSV in Suid-Afrikaanse pasiënte nie en daarom, soortgelyk aan WNV-infeksies, bly die virus onaangemeld en oor die hoof gesien. Aan die anderkant is die teenwoordigheid van bosluis-gedraagde flavivirusse in suider-Afrika nog nie vasgestel nie, ten spyte van die teenwoordigheid van geskikte vektore. Die hoë vlak van kruisreaktiwiteit tussen flavivirusse en die gebruik van neutraliserende toetse wat onprakties is, is uitdagings geassosieer met die serologiese identifikasie van flavivirusse. Serologiese toetse wat reagense gebruik wat in 'n bioveilligheidvslak 2 of 'n laer fasiliteit gebruik kan word, was ontwikkel en geëvalueer vir die waarneming en differensiasie van bosluis- en muskiet-gedraagde flavivirusse in die Vrystaat provinsie in Suid-Afrika. 'n Totaal van 2393 serummonsters van verskeie spesies, insluitend mense, beeste en skape, was getoets met behulp van die Kunjin-virus (KUNV) sel lisaat-antigeen vir die aantoning van anti-flavivirus teenliggame in 'n indirekte IgG ensiem-gekoppelde immunosorbent-toets (ELISA). Ten einde verdere positiewe reageerders op KUNV toetsing vir teenliggame teen bosluis- en muskiet-gedraagde flavivirusse te onderskei, was rekombinante omhulsel domein III (r-EDIII) proteïene van Langat-virus (LGTV), WNV en WESSV in 'n bakterieë uitgedruk en in die ELISA gebruik. 'n Totaal van 722 monsters het positief getoets met die KUNV toets, waarvan 71, 457 en 431 positief getoets het vir die r-LGTVEDIII, r-WNVEDIII en r-WESSVEDIII toetse, onderskeidelik. 'n Totaal van 70 monsters het reageer op die KUNV toets, maar nie met enige van die ander toetse nie, wat ‘n aanduiding is dat daar ander flavivirusse waarvoor daar nie spesifieke r-EDIII toetse beskikbaar is nie, in die Vrystaat provinsie sirkuleer. Gesamentlik dui die resultate op 'n sterk teenwoordigheid van flavivirusse wat in die Vrystaat provinsie sirkuleer met verskeie muskiet-gedraagde flavivirusse wat ko-sirkluleer. Daar is bewyse wat op die teenwoordigheid van bosluis-gedraagde flavivirusse dui, alhoewel dit nog nie bevestig is nie. Die EDIII proteïen is 'n nuttige middel wat vir die waarneming en differensiasie van flavivirusse in hulpbron-beperkte laboratoriums gebruik kan word. Gewerwelde gashere speel 'n rol in die onderhoud en sirkulasie van flavivirusse en, hoewel nie betrokke by die direkte oordrag van bosluis- en muskiet-gedraagde flavivirusse nie, vorm 'n skakel vir virusoordrag tussen vektore. Bykomend tot knaagdierbetrokkenheid in die handhawing van flavivirusse, word knaagdiere ook geïmpliseer in die oordrag van ander medies-belangrike virusse soos arenavirusse, lyssavirusse en hantavirusse. Arbovirusse en virale hemorragiese koors virusse is van die mees patogeniese en vernietigende siekte-agente in baie dele van die wêreld. Dit is daarom belangrik dat waarneming van sulke patogene uitgevoer word, omdat hierdie agent tot omvangryke openbare gesondheidsimplikasies kan lei. Molekulêre toetse was ontwikkel vir 'n geselekteerde aantal arbovirusse en virale hemorragiese koors agente, spesifiek Krimeaanse-Kongo hemorragiese koors virus (KKHKV), muskiet- en bosluis-gedraagde flavivirusse, asook hantavirusse. Huidiglik is die teenwoordigheid van hantavirusse in suidelike Afrika nog nie bevestig nie, ten spyte van hulle onlangse verskyning in die westelike en oorstelike dele van Afrika. In hierdie studie was die serummonsters van pasiënte wat met 'n bosluisbyt en koorsagtige siektebeeld sonder 'n diagnose presenteer het, getoets vir hantavirus IgG teenliggame met behulp van kommersiële toetse wat die Amerikaanse en Eurasiese hantavirus spesies verteenwoordig. Die algehele seropositiewe vlak wat gevind was, was 10% en 6% vir toetse wat die Amerikaanse en Eurasiese hantavirus spesies verteenwoordig, onderskeidelik. Die verskyning van hantavirusse in Afrika en die seroprevalensie van hierdie virusse in die Kaapse streek van Suid-Afrika en in ons studie, het die ontwikkeling van 'n molekulêre toets regverdig om die teenwoordigheid van hierdie virusse in suider-Afrika verder te ondersoek. Ten einde dit te bereik, was 'n reële tyd trutranskriptase polimerase ketting reaksie (RT-PKR) ontwerp en geöptimiseer. Die toets was ontwerp deur in-huis peilstukke ‘n deel van die S-segment van hantavirusse teiken en hidrolise probes wat die binneste streek van die amplikon teiken, te identifiseer. Die probes is gebaseer op die nukleotiedopeenvolgings wat die Murinae-geassosieerde hantavirus teiken vir die HNLS probe, Sigmodontinae- en Arvicolinae-geassosieerde hantavirusse vir die ASPRB probe, asook die SANGV probe vir die Afrika hantavirus Sangassou-virus. Die flavivirus RT-PKR teiken die NS5 streek met 'n probe wat suksesvol RNS in monsters op spoor van agt verskillende flavivirus spesies. Die hantavirus peilstukke en probes is geëvalueer met behulp van getranskribeerde RNS van sintetiese gene wat verteenwoordigend is van die verskillende hantavirale genotipes en tru-getranskribeerde kDNS. Die limiet van opsporing was gevind om van ~160 tot ~17 kopieë van DNS vir die verskillende hantavirus probes en die flavivirus probe te wees. 'n Gewone multipleks PKR toets wat gerig was op die opsporing van KKHKV en flavivirus RNS in monsters versamel vanaf ongediagnoseerde pasiënte met 'n bosluisbyt en koorsagtige siektebeeld, is verder ontwikkel deur gebruik te maak van geneste peilstukke gerig teen ‘n deel van die S-segment van KKHKV, en hemi-geneste peilstukke gerig teen ‘n deel van die NS5 geen van flavivirusse. Wanneer kliniese monsters van pasiënte met bosluisbyte, 'n matige siektebeeld en geen diagnose ondersoek is, is 'n pasiënt retrospektief gediagnoseer met KKHKV. Die resultate beklemtoon die belang van die bewustheid van arbovirusse en virale hemorragiese koors agente in matige gevalle wat maklik oor die hoof gesien kan word, maar 'n beduidende openbare gesondheidsrisiko verteenwoordig. Soortgelyk behoort die bewustheid opgeskerp te word vir reisigers na Suid-Afrika wat die risiko loop om na hulle land terug te keer met 'n eksotiese virale hemorragiese koors, wat verder die behoefte beklemtoon vir toenemende bewustheid en diagnostiese kapasiteit vir arbovirusse. Laastens regverdig die huidige gebrek aan geregistreerde menslike entstowwe voortgesette ondersoek na die immunogenisiteit van geselekteerde virale proteïene. Die rekombinante antigene wat ontwikkel is vir serologiese doeleindes, was verder in hierdie studie aangewend om die immunogenisiteit van die omhulsel domein III proteïene van WNV en LGTV in 'n muismodel te bepaal. Klein-molekule antigene of swak immunogeniese antigene vereis dikwels 'n versterkingsmiddel om 'n sterker immuunrespons te stimuleer. Versterkingsmiddels kan verder 'n immuunrespons verskuif na 'n Th1 of Th2 respons soos nodig, gebaseer op die immuunkorrelate van beskerming. Groepe muise was geïmmuniseer met gesuiwerde r-WNVEDIII of r-LGTVEDIII proteïne alleenlik, r-WNVEDIII of r-LGTVEDIII proteïne in kombinasie met een van drie versterkingsmiddels, naamlik saponien, Titermax® gold en Alhydrogel®, of een van die drie versterkingsmiddels sonder 'n flavivirus proteïen. In die afwesigheid van enige versterkingsmiddel was die resultate van die WNV proteïen alleenlik twyfelagtig, terwyl 'n sterk IgG1 respons deur LGTV EDIII geïnduseer is. In kort, proteïen alleenlik of gemeng met alum het 'n oorwegend Th2 respons uitgelok, terwyl proteïen in kombinasie met saponien of Titermax® gold 'n gemengde Th1 en Th2 reaksie geïnduseer het. Muise wat met r-WNVEDIII geïmmuniseer was, het teen natuurlike KUNV antigeen gereageer, wat daarop dui dat die proteïen uitgedruk is in konformasie met blootstelling van die epitope wat vereis word om 'n waarneembare teenliggaamrespons te induseer. Dir formulasie van die WNV en LGTV proteïene met verskillende adjuvante het soortgelyke resultate opgelewer, met 'n verskuiwing in respons afhangend van die versterkingsmiddel. Ten spyte van die onvermoë om selbemiddelde reaksies te assesseer met behulp van antigeen-gestimuleerde splenosiete en karakterisering van sitokienproduksie soos aanvanklik beplan, het die resultate bevestig dat r-WNVEDIII en r-LGTVEDIII proteïne immunogenies was in die afwesigheid van volledige E proteïen, met die vermoë om waarneembare teenliggame te induseer wanneer geformuleer met versterkingsmiddel, en dat verskillende versterkingsmiddels die vermoë het om 'n immuunmodulerende invloed op die tipe respons wat geïndiseer word, uit te oefen.
Keywords: Immunogenetics; Serology; Flaviviral diseases; Viral genetics; West Nile virus -- Diagnosis; Virusses -- Diagnosis; Arbovirus infections; Antigens; Thesis (Ph.D. (Medical Microbiology and Virology))--University of the Free State, 2015