Šolaja Sofija, Goletić Šejla, Veljović Ljubiša, Glišić Dimitrije
Department of Virology, Institute of Veterinary Medicine of Serbia, Belgrade, Serbia.
Veterinary Faculty, University of Sarajevo, Sarajevo, Bosnia and Herzegovina.
Front Vet Sci. 2024 Nov 20;11:1494746. doi: 10.3389/fvets.2024.1494746. eCollection 2024.
West Nile Virus, an emerging zoonotic pathogen, has been circulating in Serbia for over a decade, with its first detection in mosquitoes in 2010. Since then, the virus has led to increasing cases in both animals and humans, peaking in 2018 with 415 human cases and 36 fatalities. This study aimed to explore the phylogenetic relationships between previously sequenced West Nile virus strains from Serbia and those sequenced in this study, while also identifying possible virulence factors.
Whole genome sequencing was conducted using a targeted approach on the MinION Mk1C platform, following a two-step process involving cDNA synthesis and amplification. Bioinformatics analysis included demultiplexing, primer trimming, and sequence mapping using tools such as iVar, Minimap2, and Samtools. Phylogenetic analysis was performed using MAFFT alignment and the Maximum Likelihood method with the Tamura Nei model in MEGA X software. Virulence factors were assessed in both structural and nonstructural proteins, focusing on key glycosylation motifs and specific mutations. Homology modeling of the E protein was also performed to evaluate potential structural changes due to mutations.
Phylogenetic analysis revealed two major sublineages within the E subclade, representing the majority of strains from Western and Central Europe. These sublineages likely originated from Austria, Serbia, and Hungary between 2008 and 2012. The study also identified three distinct sublineages within the D subclade, which includes more diverse strains from Southern Europe. The E protein exhibited significant variations, particularly at the E159 site, which is crucial for virulence. The EI159T aa change has become dominant in recent years, replacing the previously prevalent EI159M. Additionally, changes in the NS1 glycoprotein and NS3 protein, both of which are involved in immune modulation and viral replication, were identified, with potential implications for the virus's virulence.
The study's findings highlight the Western Balkans and Central Europe as key regions for the mixing and dissemination of West Nile virus strains from both Western-Central and Southern Europe. These results underscore the importance of continuous surveillance and phylogenetic analysis to monitor the evolution and spread of West Nile virus, particularly in light of the frequent mutations observed in virulence-associated sites.
西尼罗河病毒是一种新出现的人畜共患病原体，在塞尔维亚已经传播了十多年，2010年首次在蚊子中被检测到。从那时起，该病毒导致动物和人类病例不断增加，2018年达到峰值，有415例人类病例和36例死亡。本研究旨在探讨塞尔维亚先前测序的西尼罗河病毒株与本研究中测序的病毒株之间的系统发育关系，同时确定可能的毒力因子。
采用靶向方法在MinION Mk1C平台上进行全基因组测序，该过程包括两步，即cDNA合成和扩增。生物信息学分析包括使用iVar、Minimap2和Samtools等工具进行解复用、引物修剪和序列映射。使用MAFFT比对和MEGA X软件中的Tamura Nei模型的最大似然法进行系统发育分析。在结构蛋白和非结构蛋白中评估毒力因子，重点关注关键糖基化基序和特定突变。还对E蛋白进行了同源建模，以评估由于突变导致的潜在结构变化。
系统发育分析揭示了E亚分支内的两个主要亚谱系，代表了来自西欧和中欧的大多数毒株。这些亚谱系可能在2008年至2012年期间起源于奥地利、塞尔维亚和匈牙利。该研究还在D亚分支内确定了三个不同的亚谱系，该亚分支包括来自南欧的更多样化的毒株。E蛋白表现出显著变化，特别是在对毒力至关重要的E159位点。EI159T氨基酸变化近年来已占主导地位，取代了先前流行的EI159M。此外，还发现了参与免疫调节和病毒复制的NS1糖蛋白和NS3蛋白的变化，这可能对病毒的毒力产生影响。
该研究结果突出了西巴尔干地区和中欧是来自西欧-中欧和南欧的西尼罗河病毒株混合和传播的关键地区。这些结果强调了持续监测和系统发育分析对于监测西尼罗河病毒的进化和传播的重要性，特别是鉴于在毒力相关位点观察到的频繁突变。
