**4.1 Methods**

96 Current Topics in Tropical Medicine

DEN-3 viruses have undergone independent evolution which has resulted in emergence of four genetic subtypes of which subtype I-III circulate in the South East Asian Region. Subtype I comprises of viruses from Indonesia, Malaysia and the Philippines; subtype II of viruses from Thailand and subtype III includes viruses from Sri Lanka India and Pakistan. The genetic evolution in these subtypes is primarily reported mutations in the prM/M and E structural protein genes. In spite of these mutations, the genomic region has retained greater than 95% amino acid sequence similarity (Lanciotti, R.S et al.,1994), suggesting that these are highly

Phylogenetic studies suggest that there are regional foci of virus extinction and selection, one such region is Thailand where the indigenous DEN-3 virus circulating up to 1992 disappeared and was replaced by two new lineages perhaps from a common ancestor (Wittke, V. et al. 2002). The sequence of all Thai DEN-3 isolates recovered after 1992 had T at position 2370 in contrast to the C at this site in the pre-1992 samples(Wittke, V. et al. 2002), and nucleotides difference was observed in at least 45 sites of total 96 sites studied. It appears that the post-1992 strains have replaced the pre-1992 strains). These studies point towards potential of regular extinctions of strains of DEN-3 virus and replacement by new variants in the region (Wittke, V. et al. 2002). Natural selection and / or genetic bottle neck could be the plausible causes for this variation. Since the extinction of pre 1992 strains and appearance of new epidemic strain in Thailand occurred during inter-epidemic period it is therefore hypothesized that the genetic bottleneck is perhaps the cause of regional replacement. This is further supported by studies from India reporting shift and dominance of the dengue virus serotype-3 (subtype III) replacing the earlier circulating serotype-2 (subtype IV) with emergence of increased incidence of DHF and DSS in subsequent outbreaks (Dash, P.K.et al. 2006). Strains from the 2005 outbreak in Karachi (Pakistan) were found to be similar to those from Indian strains of dengue serotype 3, and were responsible for deadly outbreak in 2005-06 (Jamil. B. et al. 2007). Thus over the period 1989 and 2000, a new clades of DENV-3 genotype III viruses have replaced older genotype and clades in this region and emergence of new clades coincided with severe epidemics. The epidemiologic data suggests that the DEN-3 virus responsible for recent epidemic outbreaks in Mozambiques, Gutamealla, Pakistan and SriLanka may have been introduced from India, and changing age structure of dengue patients from 1996–2005 may also be indicative of the

The micro evolutionary change within dengue serotypes has resulted in substantial genetic diversity with emergence of endemic and epidemic genotypes. With current advances in the field of genetic and molecular techniques scientists are now trying to decipher relation of changing clades with disease severity and epidemic potential. With the availability of complete genomic sequence of the Dengue virus different genetic loci have been investigated to find this relationship. Envelope –gene (E-gene) sequence is the most frequently investigated locus, (Wittke,V.et al., 2002;-Thu, H.M. et al.,2004;Islam, M.A.et al., 2006,27) followed by capsuler C-prM gene (Kukreti, H. 2008;,Dash, P.K. 2006;,Kanakaratne, N. 2009;Jamil B,2007). In addition non-structural (NS) viral proteins such as NS1 and untranslated genomic region 3'-UTR, 5' UTR along with complete genomic sequences have been investigated to relate the genetic changes with the disease severity (Mangada, M.N. et al., 1997;Zhou,Y.et al.,2006;Islam, M.A.et al.,2006. Despite the wealth of genomic data

conserved regions responsible for protein architecture and / or biological function.

selected virus moving into new areas(Kanakaratne, N. et al.2009 ).

**4. Genetic evolution and disease severity in SEA** 

### **4.1.1 Literature search**

The literature search was performed from February 2010 to June 2010. Data sources include Medline via Pubmed (1950-February 2010), Cochrane data base of systematic reviews, Google scholar and experts in the field. Secondary references and review articles were scanned for thematic review. Hand search of the journal was also carried out. However, unpublished and ongoing studies could not be explored. Terminologies i.e. dengue type 1-4,


Table 1. Features of the excluded studies

Genetic Diversity of Dengue Virus and

PRISMA Flow Diagram

included in this study

Associated Clinical Severity During Periodic Epidemics in South East Asia 99

Fourteen studies were finally selected based on inclusion criteria i.e., association of dengue genotype and clinical severity of the diseases in the patients and were conducted in different countries of South East Asia. Setting of these studies were; Thailand 6 (Zhang, C. et al 2006, Rico-Hesse R. et al. 1998, Wittke,V.et al.,2002), Myanmar (Thu H.M. et al.,2004), India (Kukreti, H. et al.,2008;Dash, P.K.et al.,2006), Bangladesh (Islam, M.A.et al.,2006), Sri Lanka (Kanakaratne, N. et al.,2009), Taiwan (King, C.C. et al.,2008) and Pakistan (Jamil B, et al 2007). These studies were published from 1997 to 2009. Since the focus of our study was on DEN-2 and DEN3 viruses 12 studies out of these 14 were finally

dengue fever, dengue hemorrhagic fever, genetic variation, sequence analysis, south East Asia were used individually as well as in various combinations. Two independent reviewers; reviewed the titles, abstracts and full text articles and selected potentially relevant studies based on inclusion criteria established prior to the literature search. Discrepancy between the reviewers were sought to reach on consensus in consultation with third reviewer. Those potentially irrelevant studies that were ultimately excluded are listed together with the reason for exclusion in Table 1.

### **4.1.2 Inclusion criteria**

Studies which reported dengue virus genotype (mutation / sequencing of viral genetic material) and clinical features of dengue fever patients were included.

#### **4.1.3 Design of the studies**

All type of observational studies i.e. case report, case series, surveys and descriptive crosssectional studies which were focusing on genotype and clinical presentation of dengue patients were included in the review. *Population:* Population includes patients of dengue fever of all age groups. No age and sex restriction were applied. *Outcome of interest:*  Difference in nucleotide and protein sequences were analyzed and compared according to geographical origin, the sampling period and the clinical presentation. Clinical severity of the disease is defined as presence of DF, DHF or DSS. *Language:* Only articles in English language were included in the review.

#### **4.1.4 Exclusion criteria**

All those studies focusing on dengue vector control, clinical trials on vaccines, clinical trials on drugs, pure prevalence or incidence, unusual case report or case series without genotype and studies conducted in countries other than south East Asian region were excluded.

#### **4.1.5 Data extraction**

Data extraction of the included studies was done by using structured data extraction form specifically made for the review. Data was extracted for country of origin, year of publication, clarification of objectives, type of study, its duration and setting, results on both genotype and clinical severity etc.

#### **4.1.6 Data synthesis**

A narrative data synthesis was carried out to show result summary of all included studies which include description of clinical features and genotype of dengue virus. However, meta-analysis could not be performed due to non availability of required data i.e. measure of strength of association. Hence, pooled effect of genetic variation on clinical severities among dengue patients could not be provided.

#### **4.1.7 Quality assessment**

According to Cochrane Collaboration's recommendation, the quality of included studies have been 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.

dengue fever, dengue hemorrhagic fever, genetic variation, sequence analysis, south East Asia were used individually as well as in various combinations. Two independent reviewers; reviewed the titles, abstracts and full text articles and selected potentially relevant studies based on inclusion criteria established prior to the literature search. Discrepancy between the reviewers were sought to reach on consensus in consultation with third reviewer. Those potentially irrelevant studies that were ultimately excluded are listed

Studies which reported dengue virus genotype (mutation / sequencing of viral genetic

All type of observational studies i.e. case report, case series, surveys and descriptive crosssectional studies which were focusing on genotype and clinical presentation of dengue patients were included in the review. *Population:* Population includes patients of dengue fever of all age groups. No age and sex restriction were applied. *Outcome of interest:*  Difference in nucleotide and protein sequences were analyzed and compared according to geographical origin, the sampling period and the clinical presentation. Clinical severity of the disease is defined as presence of DF, DHF or DSS. *Language:* Only articles in English

All those studies focusing on dengue vector control, clinical trials on vaccines, clinical trials on drugs, pure prevalence or incidence, unusual case report or case series without genotype and studies conducted in countries other than south East Asian region were excluded.

Data extraction of the included studies was done by using structured data extraction form specifically made for the review. Data was extracted for country of origin, year of publication, clarification of objectives, type of study, its duration and setting, results on both

A narrative data synthesis was carried out to show result summary of all included studies which include description of clinical features and genotype of dengue virus. However, meta-analysis could not be performed due to non availability of required data i.e. measure of strength of association. Hence, pooled effect of genetic variation on clinical severities

According to Cochrane Collaboration's recommendation, the quality of included studies have been 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

material) and clinical features of dengue fever patients were included.

together with the reason for exclusion in Table 1.

**4.1.2 Inclusion criteria** 

**4.1.3 Design of the studies** 

**4.1.4 Exclusion criteria** 

**4.1.5 Data extraction** 

**4.1.6 Data synthesis** 

**4.1.7 Quality assessment** 

validity of selected studies.

genotype and clinical severity etc.

among dengue patients could not be provided.

language were included in the review.

PRISMA Flow Diagram

Fourteen studies were finally selected based on inclusion criteria i.e., association of dengue genotype and clinical severity of the diseases in the patients and were conducted in different countries of South East Asia. Setting of these studies were; Thailand 6 (Zhang, C. et al 2006, Rico-Hesse R. et al. 1998, Wittke,V.et al.,2002), Myanmar (Thu H.M. et al.,2004), India (Kukreti, H. et al.,2008;Dash, P.K.et al.,2006), Bangladesh (Islam, M.A.et al.,2006), Sri Lanka (Kanakaratne, N. et al.,2009), Taiwan (King, C.C. et al.,2008) and Pakistan (Jamil B, et al 2007). These studies were published from 1997 to 2009. Since the focus of our study was on DEN-2 and DEN3 viruses 12 studies out of these 14 were finally included in this study

Genetic Diversity of Dengue Virus and

**5.1 Mutations observed 5.1.1 E-gene mutations** 

**5.2 C-prM mutations** 

Associated Clinical Severity During Periodic Epidemics in South East Asia 101

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

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

under immune selection (Rico-Hesse R. et al. 1998 and Zhang, C. et al 2006, ).

Sri Lankan isolates are closely related to Indian / Malaysian genotype.

The phylogenetic analysis of 433 base pair region (nucleotides 180—612) of the DEN-3 *CprM*  gene junction showed that sequences of Delhi isolates (2006 outbreak) were closely related to sequences from Guatemala (1998) and presented a nucleotide identity of 95.9—98.2% (mean 97.05%). On comparison of Delhi 2006 sequences with other Indian sequences from years 2003, 2004, and 2005, mean sequence divergence of 2.85%, 2.15%, and 1.6%, respectively, were observed (Kukreti, H. et al.,2008. Common amino acid mutations observed in 2006 DENV-3 sequences are given in table 3. Similar study performed on DEN-3 isolates of 2003-04 outbreaks in New Delhi, found them to be closely related and belonged to subtype III from Sri Lanka (Dash, P.K.et al.,2006). Moreover, Phylogenetic analysis of C/PrM/M region of
