Molecular Characterization and Expression Analysis of NS3 and NS4 Genes of Dengue Virus Serotype-2 from Pakistani Isolates

AbstractBackground Dengue virus (DENV) infection poses a significant public health threat, particularly in tropical and subtropical regions. Current estimates suggest that approximately 390 million cases of dengue occur annually, putting around 3.97 billion people at risk of contracting the infection. Despite global efforts, no antivirals or preventive vaccines are presently available in the market for the clinical treatment of dengue infection due to its ill-defined pathogenesis mechanism and lack of suitablein vitroandininfection models. The aim of this study was to identify the prevalent DENV serotypes circulating in Pakistan and to characterize and express the non-structural genes (NS3 and NS4) of dengue virus to better understand the disease pathogenesis. Methods In the present study, we tested NS1 positive serum samples for the identification of dengue serotypes through nested PCR using serotype-specific primers. The viral RNA was purified from the serum samples and complementary DNA (cDNA) was synthesized. We targeted serotype-2 samples for the amplification of NS3 and NS4A genes through nested PCR using two sets of gene-specific primers. The PCR products were initially verified using gel electrophoresis and subsequently confirmed through Sanger sequencing. Further, we cloned the amplified NS3 and NS4A gene fragments in the pCR 2.1 cloning vector and expressed them in mammalian vector (pET28) for further functional analysis. Results The NS1-positive DENV samples were tested for various dengue serotypes, confirming that serotype-2 continues to be predominant in Pakistan after COVID-19 pandemic. We successfully amplified the NS3 and NS4A genes of dengue virus. The computational analysis revealed that NS3 and NS4A gene sequences were closely related to the DENV-2 strains isolated from other areas of Pakistan like Swat. This analysis also confirmed their homology above 98% with Indian and Saudi Arabian isolates. The amplified NS3 and NS4A genes were then cloned in pCR 2.1 cloning vector and subcloned in mammalian expression vector pET28. Further, the NS3 gene was transfected into mammalian cell line and tests were conducted with siRNA targeting the NS3 protein, resulting in approximately a 50% inhibition. Conclusions Our current study has confirmed the prevalence of DENV serotype-2 following the COVID-19 pandemic and provided a molecular characterization of the genetic landscape of the non-structural genes (NS3 and NS4). This investigation serves as an important underpinning for a comprehensive examination of the DENV non-structural genes, proposing valuable insights that can contribute to the development of early diagnosis and antiviral therapies against DENV infections.