Singh Sneha, Anupriya M G, Sreekumar Easwaran
Molecular Virology Laboratory, Rajiv Gandhi Centre for Biotechnology (RGCB), Thycaud P.O., Thiruvananthapuram 695014, Kerala, India.
Molecular Virology Laboratory, Rajiv Gandhi Centre for Biotechnology (RGCB), Thycaud P.O., Thiruvananthapuram 695014, Kerala, India.
Infect Genet Evol. 2017 Aug;52:34-43. doi: 10.1016/j.meegid.2017.04.022. Epub 2017 Apr 27.
The role of genetic differences among dengue virus (DENV) in causing increased microvascular permeability is less explored. In the present study, we compared two closely related DENV serotype-2 strains of Cosmopolitan genotype for their in vitro infectivity phenotype and ability to induce trans-endothelial leakage. We found that these laboratory strains differed significantly in infecting human microvascular endothelial cells (HMEC-1) and hepatocytes (Huh7), two major target cells of DENV in in vivo infections. There was a reciprocal correlation in infectivity and vascular leakage induced by these strains, with the less infective strain inducing more trans-endothelial cell leakage in HMEC-1 monolayer upon infection. The cells infected with the strain capable of inducing more permeability were found to secrete more Non-Structural protein (sNS1) into the culture supernatant. A whole genome analysis revealed 37 predicted amino acid changes and changes in the secondary structure of 3' non-translated region between the strains. But none of these changes involved the signal sequence coded by the C-terminal of the Envelope protein and the two glycosylation sites within the NS1 protein critical for its secretion, and the N-terminal NS2A sequence important for surface targeting of NS1. The strain that secreted lower levels of NS1 and caused less leakage had two mutations within the NS1 protein coding region, F103S and T146I that significantly changed amino acid properties. A comparison of the sequences of the two strains with published sequences of various DENV strains known to cause clinically severe dengue identified a number of amino acid changes which could be implicated as possible key genetic differences. Our data supports the earlier observations that the vascular leakage induction potential of DENV strains is linked to the sNS1 levels. The results also indicate that viral genetic determinants, especially the mutations within the NS1 coding region, could affect this critical phenotype of DENV strains.
登革病毒（DENV）基因差异在导致微血管通透性增加方面的作用鲜少被研究。在本研究中，我们比较了两个密切相关的泛在基因型DENV血清型2毒株的体外感染表型以及诱导跨内皮渗漏的能力。我们发现，这些实验室毒株在感染人微血管内皮细胞（HMEC-1）和肝细胞（Huh7）方面存在显著差异，这两种细胞是DENV体内感染的两个主要靶细胞。这些毒株的感染性与诱导的血管渗漏之间存在相互关联，感染性较低的毒株在感染后会在HMEC-1单层中诱导更多的跨内皮细胞渗漏。感染能够诱导更高通透性的毒株的细胞被发现会向培养上清液中分泌更多的非结构蛋白（sNS1）。全基因组分析揭示了毒株之间37个预测的氨基酸变化以及3'非翻译区二级结构的变化。但这些变化均未涉及包膜蛋白C末端编码的信号序列以及NS1蛋白分泌关键的两个糖基化位点，也未涉及NS1表面靶向重要的N末端NS2A序列。分泌较低水平NS1且引起较少渗漏的毒株在NS1蛋白编码区内有两个突变，即F103S和T146I，这显著改变了氨基酸特性。将这两个毒株的序列与已知会导致临床严重登革热的各种DENV毒株的已发表序列进行比较，发现了一些可能作为关键基因差异的氨基酸变化。我们的数据支持了早期的观察结果，即DENV毒株诱导血管渗漏的潜力与sNS1水平相关。结果还表明，病毒遗传决定因素，尤其是NS1编码区内的突变，可能会影响DENV毒株的这一关键表型。
