**5.1 Mutations observed**

100 Current Topics in Tropical Medicine

Eight studies were conducted in the hospital settings (Zhang, C. et al 2006, Rico-Hesse R. et al. 1998, Wittke,V.et al.,2002, Kukreti, H. et al.,2008;Dash, P.K.et al.,2006, Islam, M.A.et al.,2006, al,Kanakaratne, N. et al.,2009), three in the community (Zhou Y, et al 2006, Dash PK, et al 2006, Kanakaratne, N. et al.,2009), where as data was extracted from laboratory records in two studies (Jamil B, et al, 2007, King, C.C. et al.,2008) and dengue virus strain

Age ranges for dengue patients in these studies varied from 1 year to 70 years. The total numbers of dengue patients were 7663 in these studies. Characteristics of the studies included in this review have been summarized in table 2. A total of 285 virus isolates were subjected to genotyping/ sequence analysis in these studies. All four genotypes were studied in three studies (Zhou Y, et al 2006,Jarman RG, et al 2008, Rico-Hesse R. et al. 1998); only DEN 3 in five studies (Wittke,V.et al.,2002, Kukreti, H. et al.,2008; Islam, M.A.et al.,2006), only DEN 2 in three studies (Zhou Y, et al 2006,Mangada MNM et al 1997, Zhang, C. et al 2006), only DEN 4 in one studies (KlungthongC, et al, 2004), whereas DEN 1 and DEN 3 in one study (Kukerti H, et al 2008) and DEN 1, DEN 2 and DEN 3 studied in one

Dengue case was defined on the bases of presence of IgM, IgG, or fourfold or greater rise in hemagglutination inhibiting (HI) antibody titer against dengue virus, and presence of dengue virus specific nucleic acids in RT-PCR. Clinical severity was defined as presence of hemorrhagic manifestation and DHF related symptoms such as thrombocytopenia, skin rash, gum bleeding, gastrointestinal bleeding, hemorrhagic sclera, epistaxis, edema and ascitis. Where as other studies simply defined as presence of DF, DHF grade I, II and III and

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'

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

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

were taken from frozen stock of clinical serum samples in two (18, 21).

**5. Nucleotide sequencing and phylogenetic analysis** 

UTR and complete genomic sequences were studied in one each

severity of these two serotypes will be discussed here in detail.

likely hood methods available e.g. PAUP PROGRM

**4.1.8 Study sample characteristics** 

study (Jarman RG et al 2008).

**4.1.9 Clinical definition** 

DSS as per WHO criteria.

#### **5.1.1 E-gene mutations**

In case of DEN-2 virus, maximum numbers of viral isolates have been analyzed in studies from Thailand. The E-NS1 region of 77 different variants of DEN-2 studied using Maximum Parsimony analysis of 240 nucleotide sequence, showed 11 of 240 nucleotide variation; 4.6% divergence but did not reveal significant segregation of virus according to geographic location (Rico-Hesse R. et al. 1998). Similarly, Phylogenetic analysis of 120 E gene of DEN-2 by another group from Thailand has confirmed existence of six genotypes of this virus; however evolutionary relationships among the genotypes is difficult to determine (Zhang, C. et al 2006). In terms of dengue pathogenesis these studies failed to show segregation of DF versus DHF-associated viruses on the evolutionary tree. There are no clear-cut evolutionary divergence or branching of DF versus DHF isolates, suggesting that nucleotides from this region of the genome encode amino acids that are apparently not under immune selection (Rico-Hesse R. et al. 1998 and Zhang, C. et al 2006, ).

DEN-3 has replaced DEN-2 as most frequently isolated virus in Thailand since late 1980's (Wittke,V.et al.,2002). The evolutionary history of Thai DEN-3 viruses, has been studied by comparative analysis of the nucleotide sequence of E protein genes of currently prevailing isolates with those from all previously published E gene sequences of DEN-3 virus available in Gen Bank (Wittke,V et al. 200218), this study has shown E-gene of DEN-3 to be relatively conserved at amino acid level, however, four amino acid changes have been identified within genotype II of Thai strains. The amino acid changes observed at positions (E172 I-V) and (E479 A-V) are the only difference found between pre and post-1992 viruses. Similarly, there is little evidence to support in-situ evolution among the virus samples that were studied over prolong period ranging from days to months in a selected community in Thailand(Jarman, R.G.et al.,2008) very few mutational changes were noted, and association of these mutations with disease severity could not be delineated either. Analysis by E-sequence of eight DEN-3 strains from Bangladesh (2002 out-break strains) were found to be very closely related to Thai isolates that caused out-break in 1998 in Thailand. The multiple alignment of amino acid (aa) sequence revealed that Bangladeshi isolates and Thai isolates shared common aa changes at position E127 (I-V), suggesting that 2002 outbreak in Bangladesh was due to introduction of Thai isolates (Islam, M.A.et al.,2006), however the statistical association of aa changes with disease severity could not be delineated. In case of Sri Lankan DEN-3, type III is the most frequent strain with two distinct clades IIIA and IIIB linked to mild and severe disease epidemics on the island respectively (Kanakaratne, N. et al.,2009). Phylogenetic studies of E-NS1 junction of DEN-2 isolates from Sri Lanka has categorized the isolates into 4 genotypes designated as Malaysian/Indian subcontinent, Southeast Asian, American, and West African (Sylvatic) and Sri Lankan isolates are closely related to Indian / Malaysian genotype.
