Dengue infection is an important mosquito-borne viral infection in areas where mosquitoes breed under optimal conditions. As a member of the family Falviviridae, the dengue virus is transmitted to human via Aedes genus, especially Aedes
agypti. This family also includes Hepatitis C Virus, West Nile Virus and Yellow Fever Virus. Dengue virus has four serotypes DEN 1-4. Sequencing of dengue viral RNA has further verified strain variation within a serotype allowing viruses to be classified into genetically distinct groups within serotypes called genotypes. This virus is prevalent in areas of Asia, Africa, Central and South America [1, 2]
. Dengue viral infection can either cause dengue fever (DF), dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). The classical dengue fever is mild, febrile illness which usually results after primary infection with dengue virus. In other cases DF can lead to DHF or DSS which can be life threatening [3, 4]. Infection with a different serotype can show severe outcome due to antibody dependent enhancement [2, 5] and can be a risk factor for DHF and DSS [2, 6–8]. Though dual infection with dengue virus is attributed to cause onset of severe disease [9–11] but a case of mild disease due to dual infection was documented in Brazil in 2003 . Outcome of disease may also depend upon the genotype involved. Some genotypes induce greater viremia and are transmitted more readily, thereby having a higher potential to cause large epidemic [12, 13].
Timely and correct diagnosis is very critical for patient management as no definitive vaccine has been developed against all dengue virus serotypes. Methods are being employed for diagnosing the dengue virus infection like viral isolation techniques, serological methods and molecular methods. Viral isolation methods are time consuming and usually take a week [2, 14]. Use of serological methods by detecting viral anti-IgM anti-IgG can give false positive results due to extensive antigenic cross-reactivity among flavivirus as well as between different dengue virus serotypes [2, 15–17]. Different types of polymerase chain reactions (PCR) like reverse -transcription PCR (RT-PCR), real-time PCR and nested or hemi-nested PCR are used for detecting genomic sequence for serotyping. Use of PCR techniques is a quick and sensitive method for detecting dengue virus and has replaced viral isolation techniques [2, 18].
Several outbreaks due to the dengue virus infection have been reported from Pakistan [19–26]. Dengue infection was first documented in Pakistan in year 1982 from Punjab in which 12 patients out of total 174 were found positive for dengue virus; all these samples were collected in1968 and 1978 . The first outbreak of DHF was documented in 1994 by Chan and colleagues  who observed DEN-1 and DEN-2 in three out of ten tested patients for dengue virus. In the following year, DEN-2 infection was reported from the province of Balochistan [22, 23]. Through serological studies, dengue type 1 and type 2 were found in sera of children in Karachi [24, 25]. Jamil and colleagues  had previously been reported DEN-3 infection in 2005 outbreak of DHF in Karachi. Kan and colleagues  reported co-circulation of dengue virus type 2 and type 3 in 2006 outbreak in Karachi. More recently, Hamayoun and colleagues  reported cases with dengue infection in the 2008 outbreak in Lahore. Out of 17 samples checked via real-time PCR, ten of their patients had DEN-4, five had DEN-2 and two had DEN-3 infection .
Pakistan has a history of outbreaks of dengue viral infection however, the responsible serotype/s is not well known. Therefore, the current study was initiated to determine the circulating serotype/s of dengue virus in Pakistan using molecular based techniques in patients' sera. Samples were selected from stored repository from three most recent outbreaks of dengue virus (2007-2009) and the obtained sequences were compared to other dengue virus sequences reported from other geographical regions of the world to deduce a phylogenetic relationship.