Serotyping of Dangue Virus and Estimation of T cells in Circulating Peripheral Blood of Dengue Patients

Abstract

Dengue is a mosquito borne viral disease mostly transmitted through bites of infected Aedes aegypti. Epidemiological studies suggest that dengue has been rapidly spreading in tropical and sub-tropical countries imposing a significant health, economic and social burden. The geographical expansion of four dengue virus serotypes (DENV1-4) signifies the need for serotyping of dengue virus for proper surveillance and epidemiological studies. Dengue virus serotypes share 65% similarities in their genome but genetic variation can be observed even within single serotype. The understanding of changing pattern of dengue virus serotypes is important. Cellular immunity plays a vital role during dengue infection. CD8 T cells have been responsible for both protection and immunopathology in dengue patients. Though various epidemiological studies regarding dengue has been carried out but the study about immunological response in dengue patients is not done in Nepal. In this study, serotyping of dengue virus was performed and the change in pattern of serotype was observed. We used selective clinical samples available from two different hospitals of Nepal in 2016 and 2017. RNA was extracted from serum samples and Real-Time PCR was performed. The serum samples were collected from confirmed dengue cases. The samples labeled with the antibodies lysed with FACS lysing solution after incubation , were stained and run in a flowcytometer. Then the data was obtained for further analysis in Flowjo. The flow cytometer analysis of dengue positive samples from 2017 was carried out for the study of immunological response in dengue patients in BD FACS Calibur. The percentage of CD4 and CD8 T cells in dengue patient was estimated. Change in pattern of dengue serotype was observed between two different years. DENV1 was found prevalent in 2016 whereas DENV2 was found prevalent in 2017. The percentages of CD4 cell of samples were similar to that of control whereas the percentage of CD8 cells in dengue positive samples were higher than that of control. Hence, Real-Time PCR is rapid and effective method for serotyping of dengue virus. There was a pattern change observed in dengue virus serotype in the samples of two consecutive years. The increase in percentage of CD8 cells was due to dengue infection. Keywords: Dengue, Serotyping, Real Time PCR, FACS, T cells