BioMed Central Page 1 of 9 (page number not for citation purposes) Virology Journal Open Access Research Phylogenetic studies reveal existence of multiple lineages of a single genotype of DENV-1 (genotype III) in India during 1956–2007 Himani Kukreti †1 , Paban Kumar Dash †2 , Manmohan Parida 2 , Artee Chaudhary 1 , Parag Saxena 2 , RS Rautela 1 , Veena Mittal 3 , Mala Chhabra 3 , D Bhattacharya 3 , Shiv Lal 1,3 , PV Lakshmana Rao 2 and Arvind Rai* 1 Address: 1 Division of Biochemistry and Biotechnology, National Institute of Communicable Diseases (NICD), 22 Shamnath Marg, Delhi 110054, India, 2 Division of Virology, Defence Research & Development Establishment, Jhanshi Road, Gwalior-474002, MP, India and 3 Division of Zoonosis, National Institute of Communicable Diseases (NICD), 22 Shamnath Marg, Delhi 110054, India Email: Himani Kukreti - himani_kukreti@yahoo.co.in; Paban Kumar Dash - pabandash@rediffmail.com; Manmohan Parida - paridamm@hotmail.com; Artee Chaudhary - rt_rt2005@yahoo.com; Parag Saxena - paragsaxena@rediffmail.com; RS Rautela - rautelanicd@yahoo.co.in; Veena Mittal - veena_m12@yahoo.com; Mala Chhabra - malachhabra@yahoo.co.in; D Bhattacharya - dipesh.3656@gmail.com; Shiv Lal - dirnicd@bol.net.in; PV Lakshmana Rao - pvlrao@rediffmail.com; Arvind Rai* - arvindrai_16@hotmail.com * Corresponding author †Equal contributors Abstract Background: Dengue virus type 1 (DENV-1) have been mostly circulating silently with dominant serotypes DENV-2 and DENV-3 in India. However recent times have marked an increase in DENV- 1 circulation in yearly outbreaks. Many studies have not been carried out on this virus type, leaving a lacunae pertaining to the circulating genotypes, since its earliest report in India. In the present study, we sequenced CprM gene junction of 13 DENV-1 isolated from Delhi and Gwalior (North India) between 2001–2007 and one 1956 Vellore isolate as reference. For comparison, we retrieved 11 other Indian and 70 global reference sequences from NCBI database, making sure that Indian and global isolates from all decades are available for comparative analysis. Results: The region was found to be AT rich with no insertion or deletion. Majority of the nucleotide substitutions were silent, except 3 non-conservative amino acid changes (I → T, A → T and L → S at amino acid positions 59,114 and 155 respectively) in the Indian DENV-1 sequences, sequenced in this study. Except two 1997–98 Delhi isolates, which group in genotype I; all other Indian isolates group in genotype III. All Indian genotype III DENV-1 exhibited diversity among them, giving rise to at least 4 distinct lineages (India 1–4) showing proximity to isolates from diverse geographic locations. Conclusion: The extensive phylogenetic analysis revealed consistent existence of multiple lineages of DENV-1 genotype III during the last 5 decades in India. Background Dengue fever (DF) is one of the most important arboviral diseases of humans in tropic and sub-tropics [1,2]. In South-East Asia, with a total population of 1.5 billion, approximately 1.3 billion people live at risk of acquiring DF or DHF [3,4]. Its etiological agent, Dengue virus belongs to family Flaviviridae, genus Flavivirus; and exists in 4 antigenically distinct serotypes, Dengue virus type 1– Published: 6 January 2009 Virology Journal 2009, 6:1 doi:10.1186/1743-422X-6-1 Received: 11 August 2008 Accepted: 6 January 2009 This article is available from: http://www.virologyj.com/content/6/1/1 © 2009 Kukreti et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Virology Journal 2009, 6:1 http://www.virologyj.com/content/6/1/1 Page 2 of 9 (page number not for citation purposes) 4 (DENV-1 to 4) [5]. Although history of dengue virus in India dates back to 1946, however the first major out- break was reported in 1963 in Calcutta. Since then many outbreaks have been reported from all over the country [6- 9]. Although all 4 dengue serotypes have been reported to circulate in the country [10] but only DENV-2 and DENV- 3 have been implicated in major DF/dengue hemorrhagic fever (DHF) outbreaks [11-14]. We and some other work- ers have earlier reported CprM gene based genotyping of DENV-2 and DENV-3 which has proved to be useful for carrying out molecular epidemiology of these viruses [12,14-16]. Lately there has been a rise in DENV-1 associ- ated cases, which account for around 30% of the total cases in most recent 2006 DF outbreak, when it co-existed with pre-dominant DENV-3 [17]. Earlier DENV-1 has been reported from south India (Vellore) in 1956 and 1962–64; and from north India (Delhi and Gwalior) in 1970, 1982, 1997–98 and 2002–2006. Inspite of all these reports, no attempt has been made to study the phylogeny of this virus since its emergence. With increase in DENV-1 cases, the need to understand the genetic nature of circu- lating DENV-1 has become even more necessary as it will harbors significant information regarding the genotypes of the DENV-1 circulating in the country for so long. In addition it will also indicate whether these viruses were pre-existing or an importation. With this view, present study was undertaken to understand the genetic nature of circulating DENV-1 in India and to trace their evolution during the last 50 years, by sequencing CprM gene junc- tion of 13 DEN-1 isolated in India during 2001–2007 and comparing 354 bp of this region with 11 other Indian DENV-1 sequences reported till date with at least one sequence from each decade, since it was first reported in the country. Seventy other global reference sequences were also retrieved from NCBI nucleotide database for comparison. This study shall help to fill the lacunae per- taining to knowledge of circulating DENV-1 genotypes in India since its first report. Results Thirteen serum samples found positive for DENV-1 dur- ing dengue outbreaks in northern India during 2001– 2007 were included in this study. For sequence compari- son and phylogenetic analysis we selected a 354 bp sequence (nucleotides 208–561) from CprM gene junc- tion of 13 DENV-1 sequenced in this study and compared them with 11 representatives Indian and 70 other global geographically diverse DENV-1 sequences, spanning last 5 decades. All these sequences were aligned with the proto- type Indian DENV-1 isolate (India-56 (Vellore) that was also sequenced in the study. This region was found to be AT rich and the AT composition of the Indian DENV-1 varied from 52.6–53.7%. The alignment did not reveal any base insertion or deletion, only substitutions which were mostly synonymous in nature. Deduced amino acid alignment of the Indian isolates sequenced in this study revealed that the non-synonymous nucleotide substitu- tions, gave rise to only 3 non-conservative amino acid changes i.e. isoleucine to threonine (Amino acid position 59) and leucine to serine (Amino acid position 155) as seen in some 2006 and 2007 Delhi isolates; and alanine to threonine (Amino acid position 114) as observed in 2001 Delhi and 2002 Gwalior isolates (amino acid align- ment shown as Figure 1 and 2). On comparison of sequences, we observed that one 2002 Gwalior and two 2001 Delhi CprM sequences exhibited close nucleotide identity of 99.7% to a 2004 Reunion Island sequence. It was also found that the most recent Indian isolates from 2006 and 2007 show mean sequence identities of 96.45%, 95.35%, 97.5% and 96.75% with 1956 Vellore, 1970 Delhi, 1982 Delhi and 2004 Gwalior sequences; and 98.6–99.2% (mean 98.9%) nucleotide identity among themselves. On analyzing the Indian ref- erence sequences taken for comparison, it was observed that 2005 Delhi sequences exhibited 95.8–99.2% (mean 97.5%) identity with sequences from South American iso- lates sampled between 1989–2001. Three sequences from south India (Vellore) sampled in 1962–64 show a nucle- otide identity of 96.6–96.9% (mean 96.75%) with a 1956 sequence from the same area. Two Delhi sequences from years 1997 and 1998 show more nucleotide identity i.e. 95.8–96.6% (mean 96.2%) with Japan Mochizuki sequence isolated in 1943; than to the other Indian sequences, with whom theses two sequences show a nucleotide identity of only 93%. On carrying out the phylogenetic analysis of all these DENV-1, on basis of CprM sequences, we observed that they grouped in 3 distinct clusters (Figure 3) or genotypes with a mean inter-genotype sequence divergence ~7.2%. All the Indian isolates, grouped in genotype III except 2 Delhi 1997 and 1998 isolates which grouped in genotype I showing closeness to 1943 Mochizuki strain from Japan. All the Indian Isolates classified in genotype III show intra-genotypic clustering giving rise to 4 distinct Indian lineages (India-1 through 4) and some Indian isolates although clustering together but not giving rise to a sepa- rate lineage. India-1 lineage consists of DENV-1 from Delhi isolated in 2005 and is close to South American viruses isolated from Brail, Argentina, Nicaragua, Para- guay, Peru and Venezuela between 1990–2007. Lying close to this lineage is another lineage, India-4 clustering with a 2004 African isolate from Reunion Island. Delhi 2001 and Gwalior 2002 sequences exhibit 2 C → T (at nucleotide position 274 and 522) and 2 G → A (at nucle- otide position 434 and 436) transitions, which they share with 2004 Reunion island sequence. Alanine to threonine amino acid change at position 114 was also observed exclusively in this lineage and hence considered as a line- Virology Journal 2009, 6:1 http://www.virologyj.com/content/6/1/1 Page 3 of 9 (page number not for citation purposes) Amino acid alignment of CprM gene junction sequences of all Indian and global DENV-1 showing changes in comparison to the consensus sequenceFigure 1 Amino acid alignment of CprM gene junction sequences of all Indian and global DENV-1 showing changes in comparison to the consensus sequence. The Indian sequences that are sequenced in this study are in bold. The number- ing of amino acid position corresponds to the ORF of DEN-1 strain "Singapore 8114/93" (GenBank Acc. No. AY762084 ). Dot (.) indicates amino acid similarities with the consensus. 39 48 58 68 78 88 98 108 118 | | | | | | | | | | | | | | | | Consensus SGQGPMKLVMAFIAFLRFLA*PPTAGILARW*SFKKNGAIKVLRGFKKEIS*MLN*MNRRK*SVTML*ML*PT*L*FHLT India 01 D1/1CprM/Del01 I S S I R L L A.T India 01 D1/2CprM/Del01 I S S I R L L A.T India 02 GWL14 I S S I R L L A.T India 06 07/1/del2006 T S S I R L L A.A India 06 08/1/del2006 T S S I R L L A.A India 06 09/1/del2006 T S S I R L L A.A India 06 10/1/del2006 I S S I R L L A.A India 06 11/1/del2006 I S S I R L L A.A India 06 12/1/del2006 T S S I R L L A.A India 06 13/1/del2006 T S S I R L L A.A India 07 D1/1CprM/Del07 I S S I R L L A.A India 07 D1/2CprM/Del07 I S S I R L L A.A India 05 del/05/1474/D1 I S S I R L L A.A India 05 del/05/902/D1 I S S I R L L A.A India 05 AARS01 I S S I R L L A.A India 05 AARS02-2005 I S S I R L L A.A India 56 vellore I S S I R F L A.A India 70 703180/1970/delhi I S S I R A.FL L A.A India 82 826881/1982/delhi I S S I R P L A.A India 04 GWL19 I S S I R L L A.A India 62 623996/vellore SI S N I R L L A.A India 63 631286/1963/vellore I S A AN I R L L A.A India 64 64411/1964/vellore V I S N I R L L A.A India 97 1021/1997/delhi L I G.L N SI K L L A.A India 98 1412/1998/delhi I G.L N ST R L L A.A Comoros 93 04.329/93 I S S I R L L A.A Singapore 93 8114/93 I S S I R L L A.A Brunei 05 DS06-210505 M I S S I R L L A.A Brazil 97 BR/97-409 I S S I R L L A.A Argentina 00 295arg00 I S S I R L L A.A Paraguay 00 259par00 I S S I R L L A.A Brazil 97 BR/97-233 I S S I R L L A.A Brazil 90 Den1BR/90 I S S I R L L A.A Reunion 04 191/04 I S S I R L L A.T Argentina 00 ARG0028c1 I S S I R L L A.A Peru 01 Sullana 6682-01 I S N.S I R L L A.A Brazil 01 BR/01-MR I S S I R L L A.A Argentina 00 ARG0044c1 I S S I K L L A.A Argentina 00 ARG0044 I S S I K L L A.A Indonesia 04 SC01 I S N I R L L A.A Argentina 00 ARG0048 I S S I K L L A.A Myanmar 98 D1.32514/98 I S S I R L L A.A Myanmar 96 D1.23819/96 I S S I R L L A.A Myanmar 76 D1.40568/76 I S S I R L L A.A Myanmar 71 D1.40553/71 I S N I R L L A.A USA 01 HawO3663 I S N I R L L A.A Indonesia 00 ET243 I G N I R F L A.A Argentina 99 ARG9920c1 I S S I KP L L A.A China 95 GZ01/95 I G N I R L L AMT China 95 GD23/95 I G N I R L L AMT Yap 04 D1/hu/Yap/NIID27/2004 I S N I R L L V.T Singapore 90 S275/90 I G N I R L L A.A USA 01 HawM2540 I G N I R L L V.T Reunion 04 185/04 I G N I R L L A.T China 99 GD05/99 I P G N I R L L AMT Reunion 04 257/04 I G N I R L L A.T China 97 GD14/97 I V G N I R L L A.A USA 01 HawO3758 I G N I R L L V.T Cambodia 01 DENV-1/KHM/2001 I G N I R L M A.A China 02 71/02GZ I G N I R SV.F L A.A Thailand 94 ThD1_0097_94 I G N I R L L A.A Thailand 01 ThD1_0102_01 I G N I R L M A.A Thailand 94 ThD1_0488_94 I S N V R L L A.A China 04 Fj231/04 I G S I R L M A.A Thailand 81 ThD1_0008_81 I G N I R L L A.A Thailand 01 ThD1_0049_01 I G R N I R L M A.A Thailand 82 ThD1_0081_82 I G N I R L L A.A French Guiana 89 FGA/89 I S S I R L L V.A Venezuela 07 BID V1134 I S S I R L L A.A Japan 43 Mochizuki I G N I R L L A.A Thailand 64 16007 I G R N I R L L A.A Thailand 64 16007 (PDK-13) I G R N I R L L A.A Nauru Island-74 clone WestPac I G N I R L L A.A Philippines 84 I G N I R L L A.A Djibouti 98 D1/H/IMTSSA/98/606 T I G N I R L L V.A Vietnam 07 BID V1561 I G S N T R L M A.A Vietnam 06 BID V1504 I G N I R L M A.A Vietnam 07 ID V1543 I G S V.N I R L M A.A Vietnam 06 BID V1499 I G S N T R L M A.A Vietnam 06 BID V1321 I G N I R L M A.A Nicaragua 05 BID V606 I S S I R L L A.A Nicaragua 04 BID V653 I S S I R L L A.A Vietnam BID V931 I G N I R L M A.A China 01 DGVgz01 V I G N I RK LT.M A.A Singapore 06 D1/SG/06K2290DK1/ I G N I R L M A.A Singapore 05 D1/SG/05K4820DK1/ I G N I R L M A.A Seychelles 04 1480/04 I G N I R L L A.T Seychelles 03 D1/hu/NIID41/200 I G N I R L L A.T Thailand 91 ThD1_0336_91 I G N V R L L V.A Thailand 93 2302/1993/thi I V G N I R L L A.A Thailand 99 49/1999/thi I V G R N I R F L A.A Thailand 96 630/1996/thi I G N I R L L A.A Indonesia 98 98901530 DF DV-1 I G N I R F L A.A Indonesia 98 98901518 DHF DV-1 I G N I R L L V.T Combodia 01 DENV-1/KHM/2001 L1 I G N I R L M A.A Virology Journal 2009, 6:1 http://www.virologyj.com/content/6/1/1 Page 4 of 9 (page number not for citation purposes) Amino acid alignment of CprM gene junction sequences of all Indian and global DENV-1 showing changes in comparison to the consensus sequenceFigure 2 Amino acid alignment of CprM gene junction sequences of all Indian and global DENV-1 showing changes in comparison to the consensus sequence. The Indian sequences that are sequenced in this study are in bold. The number- ing of amino acid position corresponds to the ORF of DEN-1 strain "Singapore 8114/93" (GenBank Acc. No. AY762084 ). Dot (.) indicates amino acid similarities with the consensus. 119 128 138 148 155 | | | | | | | Consensus TRGGEPHM*V*KQERGKSLLFKTS*GVNMCTLIAMDL India 01 D1/1CprM/Del01 I.S V India 01 D1/2CprM/Del01 I.S V India 02 GWL14 I.S V India 06 07/1/del2006 I.S A India 06 08/1/del2006 I.S A India 06 09/1/del2006 I.S A India 06 10/1/del2006 I.S A S India 06 11/1/del2006 I.S A S India 06 12/1/del2006 I.S A India 06 13/1/del2006 I.S A India 07 D1/1CprM/Del07 I.S A S India 07 D1/2CprM/Del07 I.S A S India 05 del/05/1474/D1 I.S A India 05 del/05/902/D1 I.S A India 05 AARS01 I.S A India 05 AARS02-2005 I.S A India 56 vellore I.S A India 70 703180/1970/delhi I.S A India 82 826881/1982/delhi I.S A India 04 GWL19 I.S A India 62 623996/vellore I.S A India 63 631286/1963/vellore I.S A India 64 64411/1964/vellore I.S A India 97 1021/1997/delhi I.S F A India 98 1412/1998/delhi I.S R A Comoros 93 04.329/93 I.S A Singapore 93 8114/93 I.S A Brunei 05 DS06-210505 I.S A Brazil 97 BR/97-409 I.S A Argentina 00 295arg00 I.S A Paraguay 00 259par00 I.S A Brazil 97 BR/97-233 I.S A Brazil 90 Den1BR/90 I.S A Reunion 04 191/04 I.S V Argentina 00 ARG0028c1 I.S A Peru 01 Sullana 6682-01 I.S A Brazil 01 BR/01-MR I.S G Argentina 00 ARG0044c1 I.S A.I Argentina 00 ARG0044 I.S A.I Indonesia 04 SC01 I.S A Argentina 00 ARG0048 I.S A.I Myanmar 98 D1.32514/98 V.S A Myanmar 96 D1.23819/96 V.S A Myanmar 76 D1.40568/76 V.S A Myanmar 71 D1.40553/71 V.S A USA 01 HawO3663 I.S A Indonesia 00 ET243 I.S A Argentina 99 ARG9920c1 I.S A.I R China 95 GZ01/95 I.S A China 95 GD23/95 I.S A Yap 04 D1/hu/Yap/NIID27/2004 I.S A Singapore 90 S275/90 I.S E V USA 01 HawM2540 I.S A Reunion 04 185/04 I.G A China 99 GD05/99 R L.I.S L A Reunion 04 257/04 I.G F A China 97 GD14/97 I.S T USA 01 HawO3758 I.S PA Cambodia 01 DENV-1/KHM/2001 I.S A China 02 71/02GZ I.S A Thailand 94 ThD1_0097_94 I.S A Thailand 01 ThD1_0102_01 I.S A Thailand 94 ThD1_0488_94 I.S A China 04 Fj231/04 I.S A Thailand 81 ThD1_0008_81 I.T T Thailand 01 ThD1_0049_01 I.S A Thailand 82 ThD1_0081_82 I.T A French Guiana 89 FGA/89 I.S A Venezuela 07 BID V1134 I.S A Japan 43 Mochizuki M.S A Thailand 64 16007 I.S A Thailand 64 16007 (PDK-13) I.S A Nauru Island-74 clone WestPac I.S A Philippines 84 TLI.S PA Djibouti 98 D1/H/IMTSSA/98/606 I.T T Vietnam 07 BID V1561 I.S A Vietnam 06 BID V1504 I.S A Vietnam 07 ID V1543 I.S A Vietnam 06 BID V1499 I.S A Vietnam 06 BID V1321 I.S A Nicaragua 05 BID V606 I.S A Nicaragua 04 BID V653 I.S A Vietnam BID V931 I.T A China 01 DGVgz01 I.S A Singapore 06 D1/SG/06K2290DK1/ I.S A Singapore 05 D1/SG/05K4820DK1/ I.S A Seychelles 04 1480/04 I.G A Seychelles 03 D1/hu/NIID41/200 I.G A Thailand 91 ThD1_0336_91 I.T A Thailand 93 2302/1993/thi I.S G A Thailand 99 49/1999/thi I.S A Thailand 96 630/1996/thi I.S A Indonesia 98 98901530 DF DV-1 I.S A Indonesia 98 98901518 DHF DV-1 I.S A Combodia 01 DENV-1/KHM/2001 L1 I.S A Virology Journal 2009, 6:1 http://www.virologyj.com/content/6/1/1 Page 5 of 9 (page number not for citation purposes) DENV-1 phylogenetic treeFigure 3 DENV-1 phylogenetic tree. Tree was generated by Neighbor-Joining method based on 354 bp nucleotide sequences of CprM gene region. Each isolate is denoted by country of isolation and last two digits of year of isolation, followed by virus ID. Bootstrap support values (>50%) are shown for major nodes on the tree. All horizontal branch lengths are drawn to scale. DENV-1 sequences that were sequenced in the study are in bold and other Indian sequences taken for comparison are in bold italics. Paraguay 00 259par00 Argentina 00 ARG0028c1 Argentina 00 295arg00 Brazil 97 BR/97- 233 Brazil 01 BR/01-MR Brazil 97 BR/97-409 Peru 01 Sullana 6682- 01 Venezuela 07 BID V1134 Nicaragua 05 BID V606 Nicaragua 04 BID V653 Brazil 90 Den1BR/90 Frenc h Guiana 89 FGA/89 Argentina 00 ARG0044c1 Argentina 00 ARG0044 Argentina 00 ARG0048 Argentina 99 ARG9920c1 Reunion 04 191/04 India 01 D1/2CprM/Del01 India 02 GWL14 India 01 D1/1CprM/Del01 India-4 India 05 AARS02-2005 India 05 del/05/1474/D1 India 05 del/05/902/D1 India 05 AARS01 Ind ia -1 Myanmar 98 D1.32514/98 Myanmar 96 D1.23819/96 Myanmar 76 D1.40568/76 Myanmar 71 D1.40553/71 India 56 vellore India 70 703180/1970/delhi Brunei 05 DS06- 210505 Singapore 93 8114/93 India 82 826881/1982/delhi Comoros 93 04.329/93 India 04 GWL19 India 07 D1/1CprM/Del07 India 07 D1/2CprM/Del07 India 06 10/1/del2006 India 06 11/1/del2006 India 06 12/1/del2006 India 06 08/1/del2006 India 06 07/1/del2006 India 06 09/1/del2006 India 06 13/1/del2006 India-2 India 64 64411/1964/vellore India 63 631286/1963/vellore India 62 623996/vellore India-3 genotype III Seyc helles 04 1480/04 Reunion 04 257/04 Seyc helles 03 D1/hu/NIID41/2003 Reunion 04 185/04 Indonesia 98 98901518 DHF DV-1 USA 01 HawO3758 USA 01 HawM2540 China 99 GD05/99 China 95 GD23/95 China 95 GZ01/95 Micronesia 04 D1/hu/Y ap/NIID27/2004 China 02 71/02GZ Indonesia 00 ET243 Indonesia 98 98901530 DF DV-1 Nauru Island-74 clone WestPac USA 01 HawO3663 Indonesia 04 SC01 Philippines 84 genotype II Viet nam 06 BID V1321 Vietnam 06 BID V1504 Combodia 01 DENV- 1/KHM/2001 L1108177 China 01 DGVgz01 Thailand 01 T hD1 0049 01 Cambodia 01 DENV-1/KHM/2001 Thailand 01 T hD1 0102 01 China 04 Fj231/04 Singapore 05 D1/SG/05K4820DK1/2005 Singapore 06 D1/SG/06K2290DK1/2006 Viet nam 07 ID V1543 Vietnam 06 BID V1499 Viet nam 07 BID V1561 Viet nam BID V931 Thailand 94 T hD1 0488 94 Thailand 96 630/1996/thi Thailand 94 T hD1 0097 94 Thailand 93 2302/1993/t hi Thailand 99 49/1999/thi China 97 GD14/97 Djibouti 98 D1/H/IMTSSA/98/606 Thailand 81 T hD1 0008 81 Thailand 91 ThD1 0336 91 Thailand 82 T hD1 0081 82 Singapore 90 S275/90 Japan 43 Moc hizuki India 98 1412/1998/delhi India 97 1021/1997/delhi Thailand 64 16007 (PDK-13) Thailand 64 16007 genotype I 100 70 100 80 64 99 58 98 97 77 61 98 70 95 98 75 98 97 91 94 92 86 84 80 79 75 75 69 63 58 56 54 80 53 55 79 78 78 62 67 78 54 78 65 0.01 Virology Journal 2009, 6:1 http://www.virologyj.com/content/6/1/1 Page 6 of 9 (page number not for citation purposes) age specific change (Figure 1). Designation of these Indian lineages i.e. India-1–4 do not signify a chronological order, however they were sequentially designated in order of discovery of these lineages. 1962–64 strains from South India (Vellore) also formed a different lineage (India-3) that diverged long ago, with no other Indian and global isolates clustering in this group. There were other Indian sequences which clustered together but did not form a dis- tinct grouping or lineage. These include 1956 Vellore iso- late, 1970 and 1982 Delhi isolates and 2004 Gwalior isolate. These sequences were close to 1993 isolates from Singapore and Comoros and a 2005 isolate form Brunei. All these sequences showed a common T → C transition at nucleotide position 208. These DENV-1 were also close to most recent isolates from Delhi sampled in 2006 and 2007 which clustered together to give rise to another inde- pendent lineage (India-2) that exhibited 3 lineage specific nucleotide changes (A → G, C → T and T → G at nucle- otide position 307, 469 and 520). Amino acid changes Isoleucine to threonine (at position 59 in Figure 1) and leucine to serine (at position 155 in Figure 2) were also observed in some and not all isolates in this lineage and hence could not be designated as lineage specific changes. Genotype I in which two of the Indian isolates from Delhi (1997–98) cluster; consists of viruses from Asia except 1 African isolate from Djibouti isolated in 1998. Although, most Asian strains in this genotype are from South East Asian countries viz. Cambodia, Vietnam, Thailand and Singapore; 2 recent isolates from China and 1 very old strain from Japan (Japan 43 Mochizuki strain) are the only 3 strains from North Asia that lie in this genotype. None of the Indian strains was classified as genotype II. This genotype was more cosmopolitan in nature with strains from East Africa, Asia and South Pacific. East Afri- can strains include recent viruses from East African islands of Seychelles and Reunion isolated during 2003–04; South Pacific strains include viruses from Hawaii, Nauru Island and Micronesia isolated between 74-04; and Asian strains include viruses from China, Philippines and Indo- nesia isolated during 1995–2004. Although African strains are clustering together, making a distinct grouping; strains from South Pacific and Asia do not show any such geographical clustering within this genotype. Discussion The large scale climatic and demographic changes have resulted in distribution of Aedes species in hitherto unknown areas. This has led to appearance of dengue infection in many newer areas with an estimated 3 billion people living at risk of this infection around the world. Considering the high rates of Aedes infestation and the presence of high-risk susceptible population, the proba- bility of dengue epidemic always remain very high in India [18]. The pathogenesis of dengue is not yet fully understood. Though re-infection with a heterologous dengue serotype remain a major risk factor for DHF, how- ever, occurrence of DHF even in primary infection makes the role of virus in pathogenesis all the more important. The incursion of new genotype into an area is also being attributed to the severe form of the disease [19]. All these facts drive the scientific community to pay more attention towards the genetic nature of dengue viruses and their spread in the population. Like other RNA viruses, the dengue viruses also revealed strong genetic diversity. Dengue serotypes are further clas- sified into 4–5 'genotypes' based on their genetic diversity [20]. However in recent reports many researchers have cat- egorized DENV-1 in three distinct genotypes [21-23]. Researchers have also reported clustering of sequences below the genotype level that correlate with the geograph- ical origin and/or time of isolation and defined as line- ages/clades [21,23,24]. Different regions of dengue genome like Envelope, E-NS1, C-prM and complete genome has been utilized for the genotyping [15,20,21,24]. We have utilized the sequence information of CprM gene junction in this study. The genotyping based on the CprM gene junction has been adopted by several researchers including us in the recent past [12,14,15,25]. This results in faster and economical geno- typing due to utilization of a single set of primer pair for both amplification and sequencing [26]. The involvement of DENV-1 in major dengue outbreaks in India was not recorded. Most of the major dengue out- breaks are attributed to DENV-2 and DENV-3 viruses. However, DENV-1 were isolated from different parts of India at regular intervals, since its first isolation from southern India (Vellore) in 1956. It is increasingly impli- cated as a minor serotype during recent outbreaks in India, including the Delhi outbreak in 2006 [17,27]. The lack of sequence analysis of Indian DENV-1 also affects the effective monitoring of circulating genotypes in India. In this study, we determined the nucleotide sequence of 13 recent DENV-1 directly from clinical samples to avoid selection and sequencing of mutant viral RNA which may occur during isolation/propagation in mouse brain/cell culture [28]. The comparison of deduced amino acid sequence revealed 3 important unique substitutions in 2001–02 and some of the 2006–07 Indian isolates. These involve substitutions of hydrophobic Isoleucine and Leu- cine by hydrophilic Threonine and Serine respectively in some 2006–07 isolates; and hydrophobic Alanine by hydrophilic Threonine in Delhi 2001 and Gwalior 2002 isolates. The hydrophilic amino acids are found mostly on the surface of the protein and are involved in the immu- nological interactions. The implications of these unique Virology Journal 2009, 6:1 http://www.virologyj.com/content/6/1/1 Page 7 of 9 (page number not for citation purposes) changes in recent Indian DENV-1 warrants further studies to understand their virulence and epidemic potential. The phylogenetic analysis revealed the Indian DENV-1 collected over last 5 decades fall into two genotypes (I and III). However, majority of the viruses are grouped within genotype III, two isolates sampled from Delhi during 1997–98 group in genotype I. These genotype I viruses from Delhi were found closely related to the 1943 Japa- nese Mochizuki strain. Their proximity was however diffi- cult to explain. Similar type of genotype I viruses were also found circulating predominantly in other Asian countries including Thailand and China around the same time. Presence of an African isolate from Djibouti in this group probably signifies the introduction of these Asian viruses in Djibouti or vice-versa. Thailand strains belonging to this genotype were isolated over four decades (1964– 2001); indicating persistence of this genotype in Thailand for a long time. However, the restricted circulation of gen- otype I viruses in Delhi for just 2 years is very surprising. The phylogenetic analysis clearly revealed continuous cir- culation of genotype III viruses over the last 50 years in India. Interestingly, majority of the Indian genotype III viruses are found to be phylogenetically quite distinct. It is surprising to find that the recent viruses recovered in quick succession in 2001–02, 2004 and 2006–07 from northern India also belong to separate lineages. In con- trast, this type of distinct lineage pattern was not observed among Indian DENV-2 and 3 [12,14]. Domingo et al., [24] has earlier established 2 distinct lin- eages of DENV-1 in India and designated them as India-1 and India-2. While India-1 has shown closeness to South American strains, India-2 exhibited close proximity to a 1993 isolate from Singapore. In our earlier study we have substantiated the presence of India-1 and India-2 lineages during 2005 and 2006 respectively and also reported a third lineage that was present in south India (Vellore) between 1962–64, whose present existence could not be established [17]. In this study we now report another lin- eage, which consists of viruses from Delhi and Gwalior sampled in 2001 and 2002 respectively; that we desig- nated as India-4. During the same period presence of India-2 lineage was also reported [24], there by suggesting the co-circulation of India-2 and India-4 lineage in India during 2001. As evident from the phylogenetic tree (Fig- ure 3) Indian DENV-1 isolated in 1956, 1970, 1982 and 2004 could not be designated in a separate lineage but their relatedness could be deciphered by clustering together of these isolates in the phylogenetic tree. In the cosmopolitan genotype (genotype II) with viruses from East Africa, Asia and South Pacific, only African iso- lates clustered together indicating independent evolution of these viruses in this area. Conclusion Thus we conclude that during the last 5 decades there has been a persistence of genotype III of DENV-1 in India with genotype I being present only during 2 years i.e. 1997–98 in Delhi. Most of the Indian DENV-1, grouping in geno- type III were quite diverse, giving rise to distinct lineages. Thus, there has been persistence of multiple lineages of DENV-1 genotype III during the last 50 years in India. This study clearly identified the genotypes of DENV-1 cir- culating in India since 1956. It also confirmed the utility of CprM gene junction for rapid and economical genotyp- ing in endemic areas. The prevalence of multiple lineages of DENV-1 in India warrants sustained monitoring of the circulating viruses, to implement effective control meas- ures at the earliest. Materials and methods Clinical samples Serum samples from febrile patients suspected for dengue infection were collected from Delhi and Gwalior during 2001–2007. Informed consent from all the patients and/ or their parents (in minors) was obtained, before collec- tion of clinical samples. Approval of the ethical commit- tee of both institutions was obtained to carry out the present study. Thirteen serum samples found positive for DENV-1 RNA were included in this study. Virus Dengue virus serotype 1 (P-23086) obtained from the National Institute of Virology (NIV), Pune, India was used as reference strain in this study. This is the prototype Indian DENV-1 virus isolated from Vellore, India in 1956. Extraction of viral RNA Viral RNA was extracted from 140 μl of serum samples and P-23086 infected C6/36 supernatant by using QIAamp viral RNA mini kit (Qiagen, Germany) in accord- ance with the manufacturer's instructions. Finally RNA was eluted in 50 μl of nuclease free water and stored at - 80°C until use. Reverse transcription- Polymerase chain reaction (RT- PCR) The RT-PCR was carried out in a 25 μl reaction volume using the access quick one-step RT-PCR kit (Promega, USA) containing PCR master mix, AMV-RT, and respective sense and antisense primers [(D1: 5'-TCAATATGCT- GAAACGCGCGAGAAACCG-3') (D2: 5'-TTGCAC- CAACAGTCAATGTCTTCAGGTTC-3')] [26] in a thermal cycler (BioRad, USA). The thermal profile of the RT-PCR reaction was- RT step at 42°C for 45 min, followed by PCR Virology Journal 2009, 6:1 http://www.virologyj.com/content/6/1/1 Page 8 of 9 (page number not for citation purposes) step of initial denaturation at 95°C for 2 min, followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 2 min and final extension at 72°C for 10 min. The PCR products were gel purified from 1.2% agarose gel using the QIAquick PCR purification kit (Qiagen, Germany) and used as template in sequencing reaction. Sequencing reaction Both strands of the purified amplicons were sequenced employing Big dye terminator cycle sequencing ready reaction kit (Applied Biosystems, USA) following the standard protocol described by us earlier [12]. The cycle sequenced product was purified by precipitation with 75% isopropanol and was vaccum dried. The DNA pellet was resuspended in 10 μl of template suppression reagent (TSR), heated at 95°C for 2 min and loaded on the ABI 310 automated DNA sequencer (Applied Biosystems, USA). Sequence alignment and phylogenetic analysis The CprM gene sequences of fourteen Indian DENV-1 obtained in the present study were submitted to GenBank at http://www.ncbi.nlm.nih.gov (accession numbers are mentioned in Table 1). BLAST search was carried-out to confirm the identity of strains. For comparison, we retrieved 11 DENV-1 CprM sequences from India and 70 belonging to diverse geographical locations from the glo- bal data base as shown in phylogenetic tree (Figure 3). Lasergene 5 software package (DNASTAR Inc, USA) was used to examine the percent identity and diversity among sequences. Sequences were translated into amino acid and aligned using BioEdit v7.0.9. Phylogenetic analysis was carried out using MEGA version 3.1 [29]. Phylogenetic tree was constructed employing Neighbor Joining method [30] with bootstrap analysis of 10000 replicates. Competing interests The authors declare that they have no competing interests. Authors' contributions HK and PKD contributed equally to this work and carried out the sequencing experiments and phylogenetic analysis and drafted the manuscript. MMP, AC, PS, RSR carried out the collection of samples and carrying out RNA extraction and RT-PCR experiments. MC, DB coordinated with MCD for collection of clinical samples used in this study. SL provided overall scientific and infrastructural support. PVLR provided scientific support and supervision. VM Table 1: Details of all the Indian DENV-1 isolates included in this study. Isolated Viruses Year of Isolation GenBank Accession No. Genotype India 56 vellore 1956 EU626489 genotype III India 62 623996/vellore 1962 AY593211 genotype III India 63 631286/1963/vellore 1963 AY593212 genotype III India 64 64411/1964/vellore 1964 AY593214 genotype III India 70 703180/1970/delhi 1970 AY593215 genotype III India 82 826891/1982/delhi 1982 AY593217 genotype III India 97 1021/1997/delhi 1997 AY584591 genotype I India 98 1412/1998/delhi 1998 AY584594 genotype I India 01 D1/1CprM/Del01 2001 EU846232 genotype III India 01 D1/2CprM/Del01 2001 EU846233 genotype III India 02 GWL14 2002 EU626490 genotype III India 04 GWL19 2004 EU626491 genotype III India 05 del/05/1474/D1 2005 EF064776 genotype III India 05 del/05/902/D1 2005 EF064774 genotype III India 05 AARS01 2005 EF222443 genotype III India 05 AARS02-2005 2005 EF222444 genotype III India 06 07/1/del2006 2006 EU181194 genotype III India 06 08/1/del2006 2006 EU181195 genotype III India 06 09/1/del2006 2006 EU181196 genotype III India 06 10/1/del2006 2006 EU181197 genotype III India 06 11/1/del2006 2006 EU181198 genotype III India 06 12/1/del2006 2006 EU181199 genotype III India 06 13/1/del2006 2006 EU181200 genotype III India 07 D1/1CprM/Del07 2007 EU846230 genotype III India 07 D1/2CprM/Del07 2007 EU846231 genotype III 'DENV-1 sequenced in the study are written in bold' Indian isolates sequenced in this study are in Bold. Each isolate is denoted by country of isolation and last two digits of year of isolation, followed by virus ID. Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Virology Journal 2009, 6:1 http://www.virologyj.com/content/6/1/1 Page 9 of 9 (page number not for citation purposes) and AR provided overall supervision and critical analysis at every level of scientific work. Acknowledgements The authors are thankful to the Director, DRDE, Gwalior and Director, NICD, Delhi for their keen interest and constant support in this study. The authors are also thankful to the Superintendent, JA Hospital, Gwalior and Dr. Ashok Rawat, Deputy Health Officer, Municipal Corporation of Delhi, for providing the clinical samples. HK also acknowledges partial financial support form Council of Scientific and Industrial Research (CSIR), Delhi and WHO country office, Delhi, India. References 1. Guzmán MG, Kourí G: Dengue: an update. Lancet Infect Dis 2002, 2:33-42. 2. Gubler DJ: Dengue/dengue haemorrhagic fever: history and current status. Novartis Found Symp 2006, 277:3-16. 3. Setiati TE, Wagenaar JFP, de Kruif MD, Mairuhu ATA, Gorp ECM, Soemantri A: Changing epidemiology of changing haemor- rhagic fever in Indonesia. Dengue Bull 2006, 30:1-14. 4. World Health Organization: Dengue and dengue haemorrhagic fever. 2002. Fact sheet 117 5. 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Kumar S, Tamura K, Nei M: MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence align- ment. Brief Bioinform 2004, 5:150-63. 30. Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987, 4:406-25. . [12,14]. Domingo et al., [24] has earlier established 2 distinct lin- eages of DENV-1 in India and designated them as India- 1 and India- 2. While India- 1 has shown closeness to South American strains, India- 2. (genotype III) in India during 1956–2007 Himani Kukreti †1 , Paban Kumar Dash †2 , Manmohan Parida 2 , Artee Chaudhary 1 , Parag Saxena 2 , RS Rautela 1 , Veena Mittal 3 , Mala Chhabra 3 , D Bhattacharya 3 ,. Artee Chaudhary - rt_rt2005@yahoo.com; Parag Saxena - paragsaxena@rediffmail.com; RS Rautela - rautelanicd@yahoo.co .in; Veena Mittal - veena_m12@yahoo.com; Mala Chhabra - malachhabra@yahoo.co .in;