Serotyping of Dengue Virus from Deadly Outbreaks of Pakistan

Dengue virus (DENV) belonging to the family Flaviviridae consists of 4 serotypes (DEN-1, 2, 3 & 4) causing severe illnesses like dengue fever (DF), Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS). Dengue fever has become an emerging disease in Pakistan during the past 5-7 years with an increase in the number of cases every year since 2006. The current study aimed at identifying the serotypes of dengue virus involved in 2011 outbreak of Pakistan. Samples were collected from all provinces of Pakistan. Patients were confirmed in the hospital by antibody captured Enzyme linked immunosorbent assay (ELISA). These ELISA positive serum samples were inoculated on HeLa cell line to raise the viral titer of dengue virus and then RT-PCR was done for serotyping of dengue virus. Initially D1, D2 primers were tried for serotyping of dengue virus but poor results were obtained. Then serotype specific primers were designed having equal fragment length targeting NS1 gene. This approach was successful in serotyping of dengue virus from Pakistani population. On Real time RT-PCR, Dengue virus (DENV) was detected in 43.33% (n=26) patientsout of 60 analyzed samples. Among 26 positive cases, 76.9% (n=20) were children and 23.1% (n=6) were adultsand 46.1% (n=12) were male and 53.8 % (n=14) were females. ELISA results of26 PCR positive cases show that 53.8% (n=14) were positive for IgM, 15.4 % (n=4) were positive for IgG and 30.8 % (n=8) were positive for both IgG and IgM. In this study DEN-2 was detected in 13.3% (n=8) patients, DEN-3 was detected in 16.67% (n=10) patients and DEN-2 & DEN-3 coinfection was detected in 13.3% (n=8) patients. We did not detected DEN-1 & DEN-4 in 2011 outbreak of Pakistan in 60 analyzed samples. By this study we can conclude that DEN-2 and DEN-3 were the main serotypes involved in 2011 outbreaks in Pakistan.