ÖZET

Amaç:

Kırım-Kongo Kanamalı Ateşi (KKKA), Kırım Kongo Kanamalı Ateşi Virüsü’nün (KKKAV) neden olduğu, kenelerle bulaşan ciddi bir viral hastalıktır. KKKA’nın küresel yayılımı ve yüksek ölüm oranları, etkili tedavilerin ve aşıların araştırılmasına ve geliştirilmesine yönelik kritik ihtiyacın önemini vurgulamaktadır. Bununla birlikte, yüksek bulaşma riski ve yüksek biyogüvenlikli tesislere duyulan gereksinim, canlı virüsle yapılan araştırmaları engellemektedir. Bu çalışmada, KKKAV’nin biyolojisini ve immünolojisini araştırmak için plazmid tabanlı virüs benzeri partikül (VLP) sistemi ve minigenom modeli kullanarak bu zorlukların üstesinden gelmeyi amaçladık.

Yöntemler:

KKKAV yapısal genlerini kodlayan plazmitler, Huh- 7 hücrelerine transfekte edildi. Viral protein ekspresyonu, floresan görüntüleme, immünolojik ve moleküler yöntemler kullanılarak doğrulandı. T7 polimeraz, T7 ifade eden hücresel hatlar veya viral ribonükleer protein komplekslerine olan ihtiyacı ortadan kaldıran ve bir yardımcı virüs olmadan otonom virüs replikasyonuna izin veren bir minigenom sistemi geliştirildi.

Bulgular:

Floresan mikroskopisi, NP-EGFP ve GC-EGFP proteinlerinin çeşitli hücre altı lokalizasyonlarda başarılı bir şekilde üretildiğini gösterdi. Western blot analizi ile pre-Gc, Gc, pre-Gn, Gn ve Np proteinlerinin varlığı hücre lizatlarında ve süpernatanlarında gösterildi. ELISA analizi, yalnızca Np’nin, Np ile Gc’nin birlikte veya üç proteinin aynı anda transfeksiyonunun farklı VLP oluşumlarına neden olabileceğini göstermektedir. Ayrıca, minigenom RNA transkriptlerinin transfeksiyonu sonrasında Huh- 7 hücreleri raportör genleri başarıyla açıklamıştır.

Sonuç:

Bu çalışma, virüs biyolojisi ve immunolojisi için yeni araçların kullanılmasını sağlayarak KKKAV araştırmalarına katkı sağlayacaktır. Elde edilen bulgular kullanılarak, bu ihmal edilmiş viral hastalığa karşı halk sağlığı alanında daha hazırlıklı olma imkanları araştırılacaktır.

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Kırım-Kongo Kanamalı Ateşi Virüsünü Araştırmak için Plazmit Tabanlı Yeni Bir Ökaryotik Modelin Geliştirilmesi

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References