Abstract
Using recombination analysis, we identified a recombinant dengue virus type 1 strain, namely, GD23/95, with three recombination regions, located within the sequences of the prM/E junction, NS1, and NS3, respectively. The recombinant dengue virus was further confirmed by phylogenetic analysis based on its recombination and non-recombination regions. This appears to be the first study to confirm the existence of three recombination regions in a single dengue virus isolate and to report recombination between parent virus strains isolated from the same geographic area (Guangdong province, China). It is also the first to report breakpoints within the NS3 gene of dengue viruses.
Both dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are severe arboviral diseases caused by dengue viruses (DENV), with more than 100 million cases of infection and estimated 25,000 deaths recorded annually around the world [5]. DENV belongs to the genus Flavivirus of the family Flaviviridae. There are four distinct serotypes of DENV—DENV1, DENV2, DENV3, and DENV4. The dengue virus genome is a single-stranded, positive-sense RNA virus of approximately 11 kb that encodes three structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [13]. Recombination events have been proven to occur in RNA viruses such as polioviruses [3], feline calicivirus [4], Western equine encephalitis virus [6], severe acute respiratory syndrome coronavirus (SARS-CoV) [20, 21, 25], and hepatitis C virus (HCV) [2, 9, 10, 12, 17]. Recombination events also occur in DENV, which are one of the most important mosquito-borne RNA viruses in tropical and subtropical areas. In defined recombinant DENV, all confirmed recombination breakpoints (“hot spots”) are located just within the C, prM, E, and NS1 sequences [7, 22–24] rather than in other regions of the dengue genome.
Guangdong province is the most severely affected epidemiological area for dengue in China. Between 1991 and 2007, there were ten major dengue epidemics (1991, 1993, 1995, 1997, 1998, 1999, 2000, 2001, 2002, and 2006) mainly caused by DENV1 in Guangdong province. Among them, the most severe outbreak occurred in 1995, when 6,812 people were infected. In this study, DENV1 isolates from Guangdong province were analyzed to investigate the existence of a recombinant virus in the area.
The DENV strains used in this study are as follows: GZ01/04, 71/02GZ, GD05/99, GD14/97, GZ01/95, GD23/95, GZ/80, Mochizuki, 16007, A88, 98901530, ZJ01/2004, NB04, Fj231/04, 98901518, WestPac 74, ARG9920, BR90, FGA/89, DENV2-43, DENV3-80-2, and DENV4-B5. The first seven strains were isolated from Guangdong province. GD23/95 was recovered from C6/36 cells (that showed distinct cytopathic effect) infected by the acute serum of dengue patients in Guangdong province in 1995. It was identified by serotype assays, nested reverse transcription-polymerase chain reaction (RT-PCR) [15], and sequence analysis. All the data demonstrated that GD23/95 was a dengue virus type 1 strain.
The sequences of the DENV1 isolates from Guangdong province (GD23/95, GZ01/95, 71/02GZ, GD05/99, GD14/97, GZ/80, and GZ01/04) and those of other reference isolates (98901530, ZJ01/2004, NB04, and Fj231/04) were firstly aligned using ClustalW as implemented in the MEGA3.1 (Molecular Evolutionary Genetics Analysis, Pennsylvania State University, PA, USA) [11] software with default parameters. The aligned sequences were then analyzed by RDP as implemented in Recombinant Detection Program version 2 (RDP2, http://darwin.uvigo.es/rdp/rdp.html) [14] with default parameters. The results showed that GD23/95 was a recombinant (daughter) virus and that the major and minor parent viruses were GZ01/95 and GD14/97, respectively. The three regions of recombination (termed regions A, B, and C) between GZ01/95 and GD14/97 were distributed in the structural and nonstructural-coding regions. Region A (between nucleotides 851 and 1,241 in GD23/95), region B (between nucleotides 2,672 and 2,975 in GD23/95), and region C (between nucleotides 5,264 and 5,555 in GD23/95) were located within the sequences of the prM/E junction, NS1, and NS3, respectively. The P value for each recombination region was significantly lower than 0.01 (the P values were 1.625 × 10−27, 1.352 × 10−18, and 1.064 × 10−16 for regions A, B, and C, respectively) (Fig. 1a). The recombination events detected by RDP were further investigated by bootscan [19] as implemented in RDP2. The bootscan analysis also confirmed the three recombination regions between GZ01/95 and GD14/97 (the P value was 4.442 × 10−12, 3.874 × 10−9, and 2.924 × 10−8 for regions A, B, and C, respectively) (Fig. 1b), which were consistent with the RDP results. This is the first study to confirm recombination breakpoints within the NS3 gene in DENV, although they have been confirmed in HCV [9, 10, 17].
To determine the phylogenetic relationships between GD23/95, GZ01/95, GD14/97, and other defined reference isolates (GZ/80, Mochizuki, 16007, A88, 98901518, WestPac 74, ARG9920, BR90, and FGA/89) [18], phylogenetic trees, based on the putative recombination and non-recombination regions described above, were generated and visualized by the neighbor-joining method using MEGA3.1. Bootstrap analysis was performed using 1,000 replicates. As seen in Fig. 2, the left panels represent non-recombination regions and the right ones, recombination regions. We compared these phylogenetic trees and found that the topology of the phylogenetic trees in the left panels (non-recombination regions) was very different from that of the trees in the right panels (recombination regions). For example, with regard to the non-recombination regions (the region upstream of A, the region between A and B, the region between B and C, and the region downstream of C), GD23/95 and GZ01/95 were clustered into the same group as WestPac74, 98901518, and A88, but GD14/97 fell into the same group as GZ80. On the contrary, with regard to the recombination regions (regions A, B, and C), GD23/95 and GD14/97 fell into the same group as GZ80, and GZ01/95 were clustered into the same group as WestPac 74, 98901518, and A88 (Fig. 2). Thus, phylogenetic analysis further confirmed that GD23/95 was a recombinant virus, and that the regions A, B, and C in GD23/95 were inherited from the minor parent virus GD14/97, while the other regions were inherited from the major parent virus GZ01/95.
The recombination events observed in this study are unusual in natural populations. It is unclear whether the recombination events took place in a human host or a mosquito vector co-infected by multiple virus strains. However, when two different virus strains simultaneously infect a single cell, it is theoretically possible for recombination to occur through a copy-choice mechanism [1] wherein recombination results from template switches during viral genome replication. The existence of six breakpoints (two breakpoints per recombination region) in GD23/95 implies six template switches. Recombination might occur during synthesis of the positive strand of the viral genome as observed in the case of polioviruses [8] and pestiviruses [16]. During the recombination processes observed in this study, no insertions, deletions, or duplications occurred. However, the precise mechanism underlying the recombination events observed in the present study is unknown. A better understanding of the recombination process requires the development of experimental models for co-infection and the generation of recombinant DENV.
In conclusion, we confirm a recombinant DENV1 strain, namely, GD23/95, with three regions of recombination between the parent virus strains GZ01/95 and GD14/97. This appears to be the first study that confirms the existence of three recombination regions in a single dengue virus isolate, that identifies breakpoints within the NS3 gene, and that reports recombination between parent virus strains isolated from the same geographic area (Guangdong province). The present study also contributes toward the understanding of the pathogenesis, evolution, vaccine development, treatment, and diagnosis of DENV.
References
Chungue E, Cassar O, Drouet MT, Guzman MG, Laille M, Rosen L, Deubel V (1995) Molecular epidemiology of dengue–1 and dengue-4 viruses. J Gen Virol 7:1877–1884
Colina R, Casane D, Vasquez S, García-Aguirre L, Chunga A, Romero H, Khan B, Cristina J (2004) Evidence of intratypic recombination in natural populations of hepatitis C virus. J Gen Virol 85:31–37
Cooper PD, Steiner-Pryor A, Scotti PD, Delong D (1974) On the nature of poliovirus genetic recombinants. J Gen Virol 23:41–49
Coyne KP, Reed FC, Porter CJ, Dawson S, Gaskell RM, Radford AD (2007) Recombination of Feline calicivirus within an endemically infected cat colony. J Gen Virol 87:921–926
Gubler DJ (1998) Resurgent vector-borne diseases as a global health problem. Emerg Infect Dis 4:442–450
Hahn CS, Lustig S, Strauss EG, Strauss JH (1988) Western equine encephalitis virus is a recombinant virus. Proc Natl Acad Sci USA 85:5997–6001
Holmes EC, Worobey M, Rambaut A (1999) Phylogenetic evidence for recombination in dengue virus. Mol Biol Evol 16:405–409
Jarvis TC, Kirkegaard K (1992) Poliovirus RNA recombination: mechanistic studies in the absence of selection. EMBO J 11:3135–3145
Kageyama S, Agdamag DM, Alesna ET, Leaño PS, Heredia AML, Abellanosa-Tac-An IP, Jereza LD, Tanimoto T, Yamamura J, Ichimura H (2006) A natural inter-genotypic (2b/1b) recombinant of hepatitis C virus in the Philippines. J Med Virol 78:1423–1428
Kalinina O, Norder H, Mukomolov S, Magnius LO (2002) A natural intergenotypic recombinant of hepatitis C virus identified in St. Petersburg. J Virol 76:4034–4043
Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163
Legrand-Abravanel F, Claudinon J, Nicot F, Dubois M, Chapuy-Regaud S, Sandres-Saune K, Pasquier C, Izopet J (2007) New natural intergenotypic (2/5) recombinant of hepatitis C virus. J Virol 81:4357–4362
Lindenbach BD, Rice CM (2003) Molecular biology of flaviviruses. Adv Virus Res 59:23–61
Martin DP, Williamson C, Posada D (2005) RDP2: recombination detection and analysis from sequence alignments. Bioinformatics 21:260–262
Meiyu F, Huosheng C, Cuihua C et al (1997) Detection of flaviviruses by reverse transcriptase-polymerase chain reaction with the universal primer set. Microbiol Immunol 41:209–213
Meyers G, Thiel HJ (1996) Molecular characterization of pestiviruses. Adv Virus Res 47:53–118
Noppornpanth S, Lien TX, Poovorawan Y, Smits SL, Osterhaus ADME, Haagmans BL (2006) Identification of a naturally occurring recombinant genotype 2/6 hepatitis C virus. J Virol 80:7569–7577
Nukui Y, Tajima S, Kotaki A, Ito M, Takasaki T, Koike K, Kurane I (2006) Novel dengue virus type 1 from travelers to Yap state, Micronesia. Emerg Infect Dis 12:343–346
Salminen MO, Carr JK, Burke DS, McCutchan FE (1995) Identification of breakpoints in intergenotypic recombinants of HIV type 1 by bootscanning. AIDS Res Hum Retroviruses 11:1423–1425
Stanhope MJ, Brown JR, Amrine-Madsen H (2004) Evidence from the evolutionary analysis of nucleotide sequences for a recombinant history of SARS-CoV. Infect Genet Evol 4:15–19
Stavrinides J, Guttman DS (2004) Mosaic evolution of the severe acute respiratory syndrome coronavirus. J Virol 78:76–82
Tolou HJG, Couissinier-Paris P, Durand JP, Mercier V, de Pina JJ, de Micco P, Billoir F, Charrel RN, de Lamballerie X (2001) Evidence for recombination in natural populations of dengue virus type 1 based on the analysis of complete genome sequences. J Gen Virol 82:1283–1290
Uzcategui NY, Camacho D, Comach G, de Uzcategui RC, Holmes EC, Gould EA (2001) Molecular epidemiology of dengue type 2 virus in Venezuela: evidence for in situ virus evolution and recombination. J Gen Virol 82:2945–2953
Worobey M, Rambaut A, Holmes EC (1999) Widespread intraserotype recombination in natural populations of dengue virus. Proc Natl Acad Sci USA 6:7352–7357
Zhang XW, Yap YL, Danchin A (2005) Testing the hypothesis of a recombinant origin of the SARS-associated coronavirus. Arch Virol 150:1–20
Acknowledgments
We thank Wei Zhao for providing some DF epidemiological data of Guangdong province.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Chen, SP., Yu, M., Jiang, T. et al. Identification of a recombinant dengue virus type 1 with 3 recombination regions in natural populations in Guangdong province, China. Arch Virol 153, 1175–1179 (2008). https://doi.org/10.1007/s00705-008-0090-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00705-008-0090-1