**5. Nucleotide sequencing and phylogenetic analysis**

Envelope –gene (E-gene) sequence was most frequently investigated loci, nine studies were focused on this region followed by C-prM gene, in three studies both genetic loci studied in one study (9) and one study included NS1 along with PrM and E loci. The 3'-UTR, 5' and 3' UTR and complete genomic sequences were studied in one each

Homology search and comparisons of most obtained sequences were performed using commercially available software systems such as DNASIS, DNAStar, 3' –UTR secondary structures were estimated using MFOLD package, while nucleotide sequence alignments (Phylogenetic analysis) were performed using CLUSTAL X, MEGA version, and maximum likely hood methods available e.g. PAUP PROGRM

The quality of included studies was assessed by using criterion which asses the quality of studies by focusing on study type, sample size calculation, clarity of objective, selection of cases, and internal validity of selected studies. From total of 16 points scale, individual score on quality assessment criteria was as follows 8.5 (Rico-Hesse R. et al. 1998), 7.0 (Zhou Y, et al 2006,Jarman RG, et al 2008, Jamil B, et al, 2007), 10.5 (Dash PK, et al 2006, Kukreti, H. et al.,2008), 5.0 (Wittke,V.et al.,2002), 6.5 (Zhang, C. et al 2006,), 10 (Zhou Y, et al 2006, Mangada MNM et al 1997), 7.5 (Wittke,V.et al.,2002, Jarman RG, et al 2008, King, C.C. et al.,2008), 12(KlungthongC, et al, 2004). Since most of the severe DHF outbreaks in SEA have been associated with DEN-2 and DEN-3, mutational changes and its relation to disease severity of these two serotypes will be discussed here in detail.
