jority of concurrent infection samples were co-infected by DENV-1 and DENV-2 (n=26). Ten samples were co-infected by DENV-2 and DENV-3 and one sample was co-infected by DENV-1 and DENV-3. Singleplex nested PCR reactions with D1 and one of the type specific primers (as applicable) were carried out for all the concurrent infection samples and single specific bands were visualized on agarose gel.
Molecular characterization of DENV strains detected in the 2013 outbreak was also carried out by sequencing domain III of the envelope protein gene. E gene fragments of 10 DENV-1, 12 DENV-2 and 7 DENV-3 strains were sequenced in the present study. The sequences were deposited in the GenBank database with following accession numbers; GenBank: KJ729163-KJ729172 (DENV-1 strains) GenBank: KJ729151-KJ729162 (DENV-2 strains) and GenBank: KJ729173-KJ729179 (DENV-3 strains). Phylogenetic analysis of the DENV strains was carried out based upon the sequenced region. A 353 bp (117 amino acid) region of envelope genes of 46 DENV -1 strains including 10 DENV-1 strains sequenced in the present study were aligned. The aligned region spanned 1921-2273 bp of DENV-1 genome, 993-1345 bp of the E protein gene and 332-448 amino acid of the E protein (numbering based on the prototype). These strains grouped within the American African Genotype (Fig.1). Six mutations i.e. PheE339Ile, SerE341Thr, AlaE371Thr, Val E382Ile, Ile E438Val, Ile E441Val were detected in the study strains in comparison to the prototype strain. All these mutations have been previously reported. These study sequences showed nucleotide distance of up to 0.3% amongst themselves while amino acid sequences were identical. The study sequences showed nucleotide distance of 11.2-11.6% and amino acid distance of 5.3 % when compared to the prototype.
For DENV-2 strains, a 449bp (149aa) region of E gene of 45 strains including 12 strains sequenced in the present study was aligned. The region corresponded to 1801-2249 bp of the DENV-2 genome, 865-1313 bp of the E protein gene and 289-437 amino acid of the E protein (numbering based on the prototype). The DENV- 2 strains of the present study clustered within the Cosmopolitan genotype (Fig. 2). The study strains showed nucleotide distance of up to 3% whereas amino acid sequences showed distances of up to 0.7%. Nucleotide distance of 5.8 to 6.3% and amino acid distance of 1.4-2% were identified when compared to the prototype strain. Two mutations, AsnE390Ser and IleE402Phe were detected in the study strains as compared to the prototype strain which are all previously reported.
Phylogenetic analysis of DENV-3 strains was carried out by aligning 485 bp (161 amino acid) region of E gene of 27 strains including 7 DENV-3 strains sequenced in the present study. The aligned region was from 1600 to 2084 bp of the DENV-3 genome, 666-1150 bp of the E protein gene and 223-383 amino acid of the E protein (numbering based on the prototype). The DENV- 3 strains of the present study grouped within the genotype III (Fig. 3). The DENV-3 strains showed nucleotide distances of up to 1.3% and amino acid distances of up to 0.6% among themselves. Nucleotide distance of 5.6 to 6.1% and amino acid distance of 3.2-3.8% were detected when compared to the prototype strain. Five mutations, all previously reported, were detected in the sequenced strains, LysE225Glu, ThrE270Asn, LysE291Glu, LeuE301Thr and LysE383Asn with reference to the prototype.
Both serotypes detected in co-infected samples could not be sequenced for the E gene. Therefore, three co-infected samples were sequenced for C-prM gene junction. Two of such samples were co-infected by DENV-1 and 2 and one sample was co-infected by DENV-2 and 3. The sequences were confirmed by BLAST. These sequences were submitted to the GenBank with accession numbers KM114683- KM114685. The DENV-2 sequences were <200 bp and hence were not accepted by the GenBank (available upon request). As described by Chinnawirotpisan et al.16, the sequences were subjected to a preliminary phylogenetic analysis including prototype strain of all