Identification and Molecular Characterization of NS1 and NS2 Genes of Dengue Virus from two mini outbreaks (2022-2023) in Pakistan

Abstract

Background Dengue viral infection is a global threat and approximately 400 million infections and 22000 deaths occur due to this virus worldwide annually. The exact mechanism of dengue virus pathogenesis remains unclear due to the absence of suitable in vivo and in vitro models. So far no antiviral medications available for the clinical treatment of Dengue virus (DENV) infection. The non-structural genes that are involved in disease pathogenesis are still not well defined. For that reason, there is a dire need to characterize these non-structural genes for further functional characterization. The main goal of this study is to identify DENV serotypes circulating in Pakistan during the year 2022 to 2023, characterize the non-structural (NS1 and NS2) genes for identification of their sequence variations and further functional analysis. Methods In this study, NS1 positive dengue serum samples were collected from the different molecular diagnostic laboratories and hospitals, in Pakistan. Viral RNA was purified and synthesized complementary DNA (cDNA). Serotype specific primers were used for the identification of dengue virus serotypes (DENV 1–4). Through nested PCR, inner and outer sense primers were used to amplify NS1 and NS2 genes of DENV and confirmed through gel electrophoresis. All purified DNA products were processed for sequencing. For cloning these fragments were then cloned in the TA cloning vector (pcDNA 2.1) and expressed expression vector (pcDNA3.) Results In our study, it was confirmed that the most prevalent serotype of DENV in Pakistan is Serotype-2 during the year 2022-23 and also amplified NS1 and NS2 genes. These amplified products were confirmed through Sanger sequencing. Through phylogenetic analysis of these sequences, it was confirmed that the isolated strains are closely related to the strains from Swat, Jeddah, India etc with homology about 97%. The amplified NS1 and NS2A gene fragments were then cloned in the TA cloning vector (pcDNA 2.1) and expressed in mammalian expression vector (pcDNA3.1) for further functional characterization. Conclusion Our study has shed light on the most prevalent serotype between the year 2022 to 2023 and characterization on the molecular genetic landscape of non-structural genes (NS1 and NS2). This study will give us a prime way for in-depth investigation of non-structural genes in the dengue virus, contributing valuable insights for the development of antiviral therapy against DENV infection.