Multiple recombinants in two dengue virus, serotype-2 isolates from patients from Oaxaca, Mexico

Abstract

Background: Dengue (DEN) is a serious cause of mortality and morbidity in the world including Mexico, where the infection is endemic. One of the states with the highest rate of dengue cases is Oaxaca. The cause of DEN is a positive-sense RNA virus, the dengue virus (DENV) that evolves rapidly increasing its variability due to the absence of a repair mechanism that leads to approximately one mutational event per genome replication; which results in enhancement of viral adaptation, including the escape from host immune responses. Additionally, recombination may play a role in driving the evolution of DENV, which may potentially affect virulence and cause host tropism changes. Recombination in DENV has not been described in Mexican strains, neither has been described the relevance in virus evolution in an endemic state such as Oaxaca where the four serotypes of DENV are circulating.

Results: To study whether there are isolates from Oaxaca having recombination, we obtained the sequence of 6 different isolates of DENV-2 Asian/American genotype from the outbreak 2005-6, one clone of the C(91)-prM-E-NS1(2400) structural genes, and 10 clones of the E gene from the isolate MEX_OAX_1656_05. Evidence of recombination was found by using different methods along with two softwares: RDP3 and GARD. The Oaxaca MEX_OAX_1656_05 and MEX_OAX_1038_05 isolates sequenced in this study were recombinant viruses that incorporate the genome sequence from the Cosmopolitan genotype. Furthermore, the clone of the E gene namely MEX_OAX_165607_05 from this study was also recombinant, incorporating genome sequence from the American genotype.

Conclusions: This is the first report of recombination in DENV-2 in Mexico. Given such a recombinant activity new genomic combinations were produced, this could play a significant role in the DENV evolution and must be considered as a potentially important mechanism generating genetic variation in this virus with serious implications for the vaccines and drugs formulation as occurs for other viruses like poliovirus, influenza and HIV.

Figure 1

Experimental strategy. A) The flow chart shows the experimental strategy that we followed to detect the recombinants in DENV-2 isolates. The C₍₉₁₎-prM-E-NS1₍₂₄₀₀₎ region from the MEX_OAX_14946_06; MEX_OAX_1020_06; MEX_OAX_739_05; MEX_OAX_1733_05; MEX_OAX_1038_05 and MEX_OAX_1656_05 isolates and the clone MEX_OAX_1656_05 were amplified and sequenced. All sequences were analyzed with RDP3 and GARD software to detect the recombinants. The analysis in silico displayed the recombinants and one parental strain. B) The E protein gene from MEX_OAX_1656_05 was cloned in TOPO TAV4 to detect possible recombinants and/or the parental sequences. One parental sequence was detected in addition to one recombinant.

Figure 2

Recombination plots of structural gene regions from MEX_OAX_1038_05 and MEX_OAX_1656_05 sequences. A) BOOTSCAN plot analysis of the C₍₉₁₎-prM-E-NS1₍₂₄₀₀₎ gene sequences from the MEX_OAX_1038_05 isolate and the parental strains INDI_GWL102_01 and MEX_OAX_1656_05_C241. The first breakpoint is located in the nucleotide 499, the second breakpoint is located in the nucleotide 868 and the third breakpoint is located in the nucleotide 2239; B) BOOTSCAN plot analysis of the C₍₉₁₎-prM-E-NS1₍₂₄₀₀₎ gene sequences from the MEX_OAX_1656_05 isolate and the parental strains INDI_GWL102_01 and MEX_OAX_1656_05_C241. The first breakpoint is located in the nucleotide 512; the second breakpoint is located in the nucleotide 826 and the third breakpoint is located in the nucleotide 2239; C) The breakpoint plots of sequences of isolates MEX_OAX_1038_05 and MEX_OAX_1656_05 determined by GARD displayed the first breakpoint in the nucleotide 498, the second breakpoint in the nucleotide 828nt and the third breakpoint in the nucleotide 2226; D) Representation of recombinant regions in the genome of DENV. The nucleotide number is determined for the first nucleotide of our sequence corresponding to the nucleotide 91 starting with the coding region in the C gene.

Figure 3

Phylogenetic trees of MEX_OAX_1038_05 and MEX_OAX_1656_05 based on putative recombination regions. Maximum Likelihood trees of the putative recombination regions and non-recombination regions of the structural genes C₍₉₁₎-prM-E-NS1₍₂₄₀₀₎ of MEX_OAX_1038_05 and MEX_OAX_1656_05 isolates. Nucleotides (nt) 1-497, nt 498-828, nt 829-2222 and 2223-2310 are displayed in A, B, C and D respectively.

Figure 4

Nucleotide alignment of C(91)-prM-E-NS1(2400) sequence of MEX_OAX_1038_05 and MEX_OAX_1656_05 putative recombinant isolates with the parental strains. The number of nucleotide is determined by the position in our sequences of DENV as described in Methods; the location of the breakpoints of MEX_OAX_1038_05 sequence determined for BOOTSCAN is highlighted by (†); the breakpoints of MEX_OAX_1656_05 sequence determined for BOOTSCAN are indicated by (*); the breakpoints of MEX_OAX_1038_05 and MEX_OAX_1656_05 sequences, determined for GARD are labeled by (•). MEX_OAX_1656241_05 clone is the putative mayor parent and INDI_GWI_102_01 is the putative minor parents.

Figure 5

Recombination plots of clone MEX_OAX_165607_05 of E protein gene. A) BOOTSCAN plot resulted from the analysis of the clone MEX_OAX_165607_05 sequence with 1000 bootstrap, the putative mayor parent MEX_OAX_165617_05, and the putative minor parent MEX_95; B) Breakpoints plot obtained with GARD algorithm by using the sequences as above; C) Phylogenetic trees (E gene) based on putative recombination and non-recombination regions by maximum likelihood methods.

Figure 6

Alignment of recombinant E protein gene sequence MEX_OAX_165607_05 with parental sequences. Location of the breakpoints of MEX_OAX_165607_05 sequence determined by BOOTSCAN is highlighted by (*); and the one determined by GARD is labeled by (•). The number of nucleotide is determined by the position in the sequence of E gene.