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Rift Valley fever virus (RVFV) is a mosquito-borne virus which has the ability to infect a large variety of animals including humans in Africa and Arabian Peninsula. The abortion rate among these animals are close to 100%, and young animals develop severe disease which often are lethal.

In humans, Rift Valley fever (RVF) presents in most cases as a mild illness with influenza-like symptoms. However, in about 8% of the cases it progresses into a more severe disease with a high case fatality rate. Since there is such a high abortion rate among infected animals, a link between human miscarriage and RVFV has been suggested, but never proven.

We could in paper I for the first time show an association between acute RVFV infection and miscarriage in humans. We observed an increase in pregnant women arriving at the Port Sudan Hospital with fever of unknown origin, and several of the patients experienced miscarriage. When we analysed their blood samples for several viral diseases we found that many had an acute RVFV infection and of these, 54% experienced a miscarriage. The odds of having a miscarriage was 7 times higher for RVFV patients compared to the RVFV negative women of which only 12% miscarried. These results indicated that RVFV infection could be a contributing factor to miscarriage.

RVFV is an enveloped virus containing the viral glycoproteins n and c (Gn and Gc respectively), where Gn most likely is responsible for the initial cellular contact. The protein DC-SIGN on dendritic cells and the glycosaminoglycan heparan sulfate has been suggested as cellular receptors for RVFV, however other mechanisms are probably also involved in binding and entry. Charge is a driving force for molecular interaction and has been shown to be important for cellular attachment of several viruses, and in paper II we could show that when the charge around the cells was altered, the infection was affected. We also showed that Gn most likely has a positive charge at a physiological pH.

When we added negatively charged molecules to the viral particles before infection, we observed a decreased infection efficiency, which we also observed after removal of carbohydrate structures from the cell surface.

Our results suggested that the cellular interaction partner for initial attachment is a negatively charged carbohydrate. Further investigations into the mechanisms of RVFV cellular interactions has to be undertaken in order to understand, and ultimately prevent, infection and disease.

There is currently no vaccine approved for human use and no specific treatments for RVF, so there is a great need for developing safe effective drugs targeting this virus. We designed a whole-cell based high-throughput screen (HTS) assay which we used to screen libraries of small molecular compounds for anti-RVFV properties. After dose-response and toxicity analysis of the initial hits, we identified six safe and effective inhibitors of RVFV infection that with further testing could become drug candidates for treatment of RVF. This study demonstrated the application of HTS using a whole-cell virus replication reporter gene assay as an effective method to identify novel compounds with potential antiviral activity against RVFV.

Rift Valley fever virus (RVFV), a negative-stranded RNA virus, is the etiological agent of the vector-borne zoonotic disease Rift Valley fever (RVF). RVFV causes significant morbidity and mortality in humans and livestock throughout Africa and the Arabian Peninsula. RVFV is an emerging virus and is capable of infecting a broad range of mosquito species distributed around the world, so it poses a potential threat globally. A wide range of livestock animals (e.g. sheep, goats, cows, and camels) and some wild animals become highly affected by RVFV. In humans, RVFV infection presents as an acute self-limiting febrile illness that may lead to more severe hemorrhagic fever and encephalitis. The severity of the disease is mostly dependent on age and the species of mammal, but other factors are also important.

There are no licensed RVFV vaccines for humans, and there is a lack of effective antiviral drugs. Moreover, due to the severe pathogenicity, higher-level facilities are needed―biosafety level 3 (BSL-3) or more―to work with RVFV, which makes antiviral drug development more challenging. Because RVFV causes severe disease in Africa and the Arabian Peninsula, and has the potential to spread globally, it is essential that safe, efficient antiviral drugs against this virus are developed.

The previously reported antiviral compound benzavir-2 inhibits the replication of several DNA viruses, i.e. human adenoviruses, herpes simplex virus (HSV) type 1, and HSV type 2, indicating a broadranging activity. We wanted to evaluate whether benzavir-2 had an effect against the RNA virus RVFV. For these and subsequent studies, we used a recombinant, modified RVFV strain with a deleted NSs gene, which was replaced by a reporter gene (rRVFVΔNSs::Katushka), enabling the studies to be conducted under BSL-2 conditions. The NSs gene is the main virulence factor for RVFV and without it, RVFV become less pathogenic. The reporter gene made it possible for us to quantify infection with the help of the red fluorescent protein. We found that benzavir-2 effectively inhibited RVFV infection in cell culture at an effective concentration showing 50% inhibition (EC50) of 0.6 μM. Benzavir-2 also inhibited the production of progeny virus. When we studied the pharmacokinetic properties, we found that benzavir-2 had good in vitro solubility, permeability, and metabolic stability. When we investigated the oral bioavailability in mice by administering benzavir-2 in peanut butter pellets, high systemic distribution was observed without any adverse toxic effects. Benzavir-2 thus inhibited RVFV infection in cell culture and showed excellent pharmacokinetic properties, suggesting the possibility of evaluating its effectiveness in an animal model. Since benzavir-2 has a broad effect against both RNA and DNA viruses, we speculated that the antiviral mechanism affects cellular targets.

We also wanted to explore a large number of small chemical compounds with unknown properties and identify any anti-RVFV activities. Thus, we developed a whole-cell-based high-throughput reporter-based assay, and screened 28,437 small chemical compounds. The assay was established after optimization of several parameters. After primary and secondary screening, we identified 63 compounds that inhibited RVFV infection by 60% at a concentration of 3.12 μM and showed ≥ 50% cell viability at 25 μM. After a dose-dependent screening of these 63 compounds, several compounds were identified with highly efficient anti-RVFV properties. Finally, N1-(2-(biphenyl-4-yloxy)ethyl)propane-1,3-diamine (compound 1) was selected as the lead compound. We performed a structure-activity relationship (SAR) analysis of compound 1 by replacing and changing component after component of the chemical compound to see how this affected the antiviral activity. After the SAR analysis, the antiviral activity did not change, but we could improve the cytotoxicity profile. Our studies suggested that the improved compound, 13a, might be targeting the early phase of the RVFV lifecycle.

In conclusion, we developed an efficient and reliable screening method that creates possibilities for discovering and developing antivirals against RVFV under BSL-2 conditions. We also identified several chemical compounds with anti-RVFV activities, which might lead to development of therapies for RVFV infection.

Rift Valley fever virus (RVFV) är ett vektorburet RNA-virus som orsakar Rift Valley fever (RVF). RVF virus orsakar betydande sjukdom och död hos människor och boskap i hela Afrika och på den Arabiska halvön. Viruset kan orsaka stora, plötsliga utbrott och eftersom det kan infektera många olika myggarter som finns runt om i världen så utgör RVF ett potentiellt globalt hot. Ett stort antal olika boskapsdjur och några vilda djur drabbas hårt under RVF utbrott. Människor får en sjukdom som kan vara mild, men kan utvecklas till allvarligare symptom som blödarfeber och hjärninflammation (encefalit) och under RVF-utbrott är antalet döda högt. Hur allvarlig sjukdomens blir är mest beroende av ålder, bakomliggande symptom och för djur, vilken djurart som infekteras.

Frånvaron av licensierade vacciner för människor och brist på effektiva antivirala läkemedel gör behandling och prevention av denna sjukdom utmanande. Dessutom, på grund av att den kan orsaka allvarlig sjukdom och död behövs höga nivåer av biosäkerhet (biosäkerhetsnivå-3, BSL-3) för att arbeta med RVFV vilket försvårar utvecklingen av läkemedel. Eftersom RVFV orsakar allvarlig sjukdom i Afrika och Arabiska halvön och har potential att spridas globalt är det viktigt att utveckla säkra effektiva antivirala medel för detta virus.

Den tidigare rapporterade antivirala föreningen benzavir-2 hämmar flera DNA-virus, dvs humant adenovirus, herpes simplexvirus typ-1 och typ-2 infektion, vilket indikerar en bredverkande aktivitet. Vi ville utvärdera om benzavir-2 också hade en effekt mot RNA-viruset RVFV. För dessa och följande studier använde vi en rekombinant, modifierad RVFV med en borttagen NSs-gen, ersatt av en reportergen, som gjorde det möjligt att utföra studierna i BSL-2-förhållanden. NSsgenen är den huvudsakliga virulensfaktorn för RVFV och med den borttagen är RVFV mindre sjukdomsframkallande. Reportergenen gjorde det möjligt att detektera rött fluoroscerande ljus efter virusinfektion och använda det som ett mått på infektion.

Vi observerade att benzavir-2 effektivt inhiberade RVFVinfektion i cellkultur med 50% inhibering vid en koncentration av 0,6μM. Benzavir-2 hämmade också produktion av nya viruspartiklar. När vi studerade dess farmakokinetiska egenskaper fann vi att benzavir-2 hade bra löslighet, permeabilitet och metabolisk stabilitet i cellkultur. När vi undersökte hur bra möss kunde ta upp benzavir-2 genom att äta föreningen blandad med jordnötssmör (den orala biotillgängligheten) observerades en hög serumkoncentration av benzavir-2 utan skadliga effekter. Benzavir-2 hämmade alltså RVFV-infektion i cellkultur och visade på utmärkta farmakokinetiska egenskaper med potential att senare kunna utvärdera effektiviteten i en djurmodell. Eftersom benzavir-2 har en bredverkande effekt mot både RNA och DNA-virus, spekulerar vi att den antivirala mekanismen påverkar cellulära mål.

I den andra studien ville vi undersöka ett stort antal små kemiska föreningar med okända egenskaper och identifiera anti-RVFV aktivitet hos dessa. För att genomföra detta utvecklade vi en helcellsbaserad ”high-throughput-screening” analys då vi använde den rekombinanta, modifierade RVFV beskriven ovan och screenade 28,437 små kemiska föreningar. Till att börja med optimerades analysmetoden med avseende på flera olika parametrar. Därefter utfördes primär och sekundär screening och 63 föreningar som inhiberade RVFV-infektion identifierades. De inhiberade RVFV-infektion med 60% vid 3,12μM och uppvisade ≥50% cellöverlevnad vid 25μM. Efter en dosberoende analys av dessa 63 föreningar kunde vi fokusera på några av föreningarna med mycket effektiva anti-RVFV egenskaper. Slutligen valdes N1-(2-(biphenyl-4-yloxy)ethyl)propane-1,3-diamine (förening 1) som vår ledande förening. Vi utförde därefter en strukturaktivitetsrelationsanalys (SAR) för förening 1. SAR-analys innebär att man byter ut och förändrar komponent efter komponent i den kemiska föreningen för att se hur det påverkar den antivirala aktiviteten. Efter SAR-analysen förändrades inte den antivirala egenskapen, men vi kunde förbättra cytotoxicitetsprofilen så att föreningen hade en låg toxisk effekt på värdcellen. Den förbättrade föreningen benämns 13a, och vår studie föreslog också att förening 13a kan vara aktiv under den tidiga fasen av RVFV-livscykel.

Sammanfattningsvis har vi identifierat flera kemiska föreningar med anti-RVFV aktiviteter som kan leda till utvecklande av terapi mot RVFV. Denna studie öppnar också möjligheter att upptäcka och utveckla antivirala medel mot RVFV i biosäkerhets-2 nivå.