Detection, Identification and Molecular Characterization of Dengue Virus Variants Prevalent in KPK Province Pakistan

Abstract

Dengue fever is a vector borne infection causing self-limiting to life threatening disease and is a major public health problem in many tropical and subtropical countries across the world including Pakistan. The objectives of this study were to detect and identify the genetic diversity of dengue virus serotypes circulating in Khyber-Pakhtunkhwa (KPK) province of Pakistan during 2013-2015. A total of 1,297 suspected cases were registered in four districts of KPK along with their demographic and clinical data were recorded. Blood samples from study subjects were collected and transported to Department of Virology, National Institute of Health Islamabad-Pakistan for confirmation of dengue infection using ELISA and realtime PCR. Samples positive for viral RNA were further subjected to genetic sequencing of E-NS1and C-prM genes. Dengue infection was confirmed in 63% cases on the basis of NS1 ELISA, while 50.4% were seropositive for IgM and 29% for IgG antibodies. On the basis of real-time PCR, 52.3% cases were confirmed to be infected. The total number of positive cases in 2013, 2014 and 2015 remained 64.7%, 65% and 62% respectively based on at least one of the three markers tested for diagnosis of acute dengue infection. Dengue cases were reported more frequently during the monsoon and post-monsoon season largely during the months of September and October. The highest rate of dengue positive cases was found among patients of age group 15-30 years followed by 31-45 years of age (31% and 16% respectively).Fever, body pain and headache were the most common clinical signs recorded in63%,55% and 45% respectively of the confirmed cases. Comparison of four different commonly used diagnostic assays revealed that NS-1 ELISA is more suitable and sensitive test compared to those targeting anti-IgM and anti- IgG. Genetic characterization of dengue virus strains indicated prevalence of all four dengue serotypes in our study population. DENV-3 (48%) and DENV-2 (32%) were xxi found as the dominant serotypes circulating in KPK province while DENV-1 (5.4%) and DENV-4 (0.1%) were comparatively less prevalent during 2013-2015. The genetic characterization based on partial E-NS1 gene and C-prM gene suggested the circulation of DENV-2 (Cosmopolitan genotype), DENV-3 (genotype-III) and DENV-4 (genotype-I) with closest nucleotide and amino acid homologies to the dengue strains already reported from Pakistan. The phylogenetic analysis showed that DENV-2 serotype showed 91.6-92.8% and 83.1-86.1% nucleotide and amino acid homology with reference strains (AF038403, DENV-2 Prototype strain) and DENV-3 serotype showed 94.8-97% and 89.8-91.5% nucleotide and amino acid similarity to prototype strain (M93130, DENV-3 Reference strain). DENV-4 serotype shared close homology 97.9% to the reference strain (AY947539, DENV-4 Prototype). Our findings affirm the prevalence of multiple dengue virus serotypes infecting local populations. It also warrants that the correct and timely diagnosis along with serotype identification would be helpful in prevention of progression of dengue fever to severe forms of dengue haemorrhagic fever and shock syndrome. The genetic data from this study highlights that the infection due to DENV-2 and DENV-3 in KPK province may have possibility originated from Karachi and Lahore outbreaks through vector transmission as revealed by phylogenetic analysis. This study thus emphasizes for continuous monitoring and surveillance of dengue cases throughout the country to trigger preparedness and preventive measures early on.