Evolutionary patterns and heterogeneity of Dengue Virus serotypes in Pakistan

Comprehensive and systematic examination of Dengue virus (DENV) evolution is essential in the context of Pakistan as the virus presents a significant public health challenge with the ability to adapt and evolve. To shed light on intricate evolutionary patterns of all four DENV serotypes, we analyzed complete genome sequences (n=43) and envelope (E) gene sequences (n=44) of all four DENV serotypes collected in Pakistan from 1994 to 2023 providing a holistic view of their genetic evolution. Our findings revealed that all four serotypes of DENV co-circulate in Pakistan with a close evolutionary relationship between DENV-1 and DENV-3. Genetically distinct serotypes DENV-2 and DENV-4 indicate that DENV–4 stands out as the most genetically different, while DENV-2 exhibits greater complexity due to the presence of multiple genotypes and the possibility of temporal fluctuations in genotype prevalence. Selective pressure analysis in Envelope (E) gene revealed heterogeneity among sequences (n=44) highlighting 46 codons in the genome experiencing selective pressure, characterized by a bias towards balancing selection indicating genetic stability of the virus. Furthermore, our study suggested an intriguing evolutionary shift of DENV-4 towards the DENV-2 clade, potentially influenced by antibodies with cross-reactivity to multiple serotypes providing a critical insight into the complex factors shaping DENV evolution and contributing to the emergence of new serotype. Author Summary The emergence of the fifth serotype of dengue virus heightened our interest in investigating its presence in Pakistan. In our quest to understand the evolving landscape of dengue in Pakistan, we conducted a comprehensive analysis, comparing whole genome sequences and E gene sequences. Notably, we focused on the E gene recognized as the most mutable component and a key determinant of dengue’s virulence. The phylogenetic analysis unveiled fascinating findings, demonstrating a strong genetic affinity between serotypes 1 and 3. Substantially signifying its implications for vaccine development and understanding of cross-immunity dynamics within serotypes. We delved into the genetic dynamics of dengue by subjecting the whole genome of DENV and E gene to neutrality tests. The outcomes of these tests unveiled a critical aspect of dengue virus evolution: the genome is not evolving neutrally. Instead, the E gene experiences selective pressure, indicating a bias towards balancing selection. The finding underscores the complex interplay of factors shaping the genetic diversity of dengue in Pakistan and provides valuable insights into the virus’s adaptive strategies. ### Competing Interest Statement The authors have declared no competing interest.