BACKGROUND
den is a serious cause of mortality and morbidity in the tropical and subtropical regions that infects fifty million people every year; approximately  <dig>  of them are hospitalized and 5% to 15% of them die, which is a dramatic data  <cit> .
positive-sense rna viruses evolve rapidly,  <cit>  allowing the virus population to quickly adapt to new environments and escape from host anti-viral responses.
one of the principal causes of genetic diversity in denv is the error-prone replication with rna-dependent rna polymerase ,  <cit>  so that one genomic mutation occurs in nearly every cycle of virus replication.
rna virus, such as denv populations at a particular region, may also rapidly change due to periodic selective sweeps <cit> , by the introduction of foreign strains of virus  <cit> , and due to intra-serotypic recombination  <cit> .
however, there has been considerable debate about whether recombination occurs in denv  <cit>  and the relevance of any recombination to the development of live-attenuated flavivirus vaccines  <cit> .
besides, there is a number of reasons for believing that recombination can occur in denv and this process is being described with increasing frequency in denv- <dig>  <cit>  and other members of the family flaviviridae  <cit> .
the recombination in denv was reported in the structural genes region and particularly in e gene sequence through the use of the bootscan, diverse plots, and lard software  <cit> .
the co-circulation of multiple denv populations increases the opportunities for a mosquito vector to ingest several variants by feeding on a number of diverse infected hosts, or for a host to be infected by vectors infected with distinct denv variants.
these conditions exist in mexico, the caribbean area and south-east asia  <cit> .
this is supported by the fact that there are many reports of multiple serotypes of denv from single hosts  <cit> .
furthermore, it is likely that mixed infections with different genotypes of the same serotype may also occur where they co-circulate  <cit> .
oaxaca, mexico is one of the states where denv is endemic and serotypes - <dig>  - <dig> and - <dig> of denv are co-circulating  <cit> .
denv- <dig> was reported as the serotype with higher frequency compared with denv- <dig>  - <dig> or - <dig>  six partial sequences of the genes encoding proteins: capsid , pre-membrane-membrane , envelop , and non-structural  <dig>  represented as c-prm-e-ns <dig> from six different isolates of denv- <dig> from the oaxaca outbreaks 2005- <dig> were obtained.
in addition, the rt-pcr products of c-prm-e-ns <dig> and e genes obtained from the mex_oax_1656_ <dig> isolate were cloned and sequenced.
mex_oax_1656_ <dig> and mex_oax_1038_ <dig> isolates displayed recombination in the prm-e and e-ns <dig> genes and the parental strains were the asian/american and cosmopolitan genotypes.
in addition, the e gene sequences from the clone  <dig>  showed recombination between the nucleotides  <dig> to  <dig> and the parental strains were asian/american and american genotypes.
RESULTS
to determine recombinant sequences in denv- <dig>  the nucleotide sequences of the partial c-prm-e-ns <dig> genome from six isolates and  <dig> representative sequences of the different genotypes were aligned and analyzed by rdp <dig> and gard.
in addition, the rt-pcr product of the partial c-prm-e-ns <dig> genome from the mex_oax_1656_ <dig> isolate was cloned in pgem-3z.
the sequences of  <dig> clones were aligned with all of the above sequences .
we also sequenced  <dig> clones of the e structural gene from the isolate mex_oax_1656_ <dig> and aligned with  <dig> representative sequences containing different genotypes by the programs mentioned above .
the first task in this phylogenetic analysis was to determine the best model of nucleotide substitution for denv- <dig> virus sequence evolution.
this assignment was undertaken using the model selection test from datamonkey online server  <cit> , which compares  <dig> models of dna substitution.
our results demonstrated that the best model was trn <dig>  <cit> .
accordingly, the most complex general time-reversible value was the best fit to the data ; the ln likelihood = - <dig> ; parameter count = 38; and aic =  <dig> .
finally, the estimated base composition was a =  <dig> , c =  <dig> , g =  <dig> , and t =  <dig> .
our analysis with rdp <dig> showed that the sequences of isolate mex_oax_1038_ <dig> and mex_oax_1656_ <dig> present statistical evidence of recombinants for genecov , bootscan , maxchi , chimera , siscan , and 3seq , in two regions : the first breakpoints were located in 499nt and 512nt respectively; the second breakpoints were located in 868nt and 826nt respectively, and the third breakpoint was located in 2239nt in both recombinants .
in addition, the analysis with gard confirmed the breakpoints and recombination data for maximum likelihood.
this analysis displayed the same site for the three breakpoints in both isolates: the first, second and third breakpoints were located in the nucleotides  <dig>   <dig> and  <dig>  respectively .
the recombinant regions were the intersection of prm-m structural gene to intersection of m-e structural genes and the second recombinant region started in the intersection of e-ns <dig> genes .
interestingly, we found that the parental major strain was the non-recombinant clone mex_oax_1656_05_ c <dig>  and the minor parental strain was the cosmopolitan genotype strain indi_gwl_102_ <dig> .
the ml tree constructed with our sequence of structural gene c-prm from nucleotide 1- <dig> from the mex_oax_1038_ <dig> and mex_oax_1656_ <dig> isolates clustered with the asian/american genotype ; the analysis of the region from nucleotides 498- <dig> of the isolates mex_oax_1038_ <dig> and mex_oax_1656_ <dig> moved to the cosmopolitan genotype  and when the region from the nucleotides 828- <dig> was analyzed the two strains clustered again with the asian/american genotype .
finally, when the region corresponding to nucleotides 2223- <dig> was analyzed the isolates clustered with the cosmopolitan genotype .
to determine the nucleotides involved in these recombinants, the c-prm-e-ns <dig> sequences of the clone mex_oax_1656_05_c <dig>  recombinants sequences mex_oax_1038_ <dig>  mex_oax_1656_ <dig> and the cosmopolitan strain indi_gwl_102_ <dig> were analyzed.
the changes in the recombinant isolates are labeled with a black dot .
this analysis showed no evidence of recombination in the recombinant strain mex_oax_1656_ <dig>
like other rna viruses, denv undergoes low fidelity replication  <cit> , resulting in virus pools of mutants.
therefore, to determine if one of the parental strains and/or a recombinant sequence is present in these pools, the rt-pcr product of the e protein gene from the recombinant strain, mex_oax_1656_ <dig> was cloned and analyzed .
we obtained  <dig> e protein gene clones that were studied using the rdp <dig> software and it was determined that the sequence of clone mex_oax_1656_05_c <dig> presents statistical evidence of recombination by genecov , bootscan , maxchi , chimera  and 3seq .
the e protein gene of said clones contains two breakpoints.
the first breakpoint was located in the nucleotide  <dig> of the coding region for protein e; the second breakpoint was located in the nucleotide  <dig> of the same gene .
gard analysis confirmed that this clone is recombinant displaying the first breakpoint in the nucleotide  <dig> and the second breakpoint in the nucleotide <dig> .
the constructed ml trees showed that the mex_oax_1656_05_c <dig> clone clustered in the asian/american genotype branch when the 1- <dig> e gene region was examined, and clustered in the american genotype when the e gene region from nucleotide  <dig> to <dig> was analyzed .
finally, when region 1048- <dig> was analyzed, the clone clustered again with the asian/american strains.
the nucleotides involved in this recombinant are displayed in the alignment of the e gene region sequences of the recombinant mex_oax_1656_05_c <dig> clone, the parental clone mex_oax_1656_05_c <dig> and the strain mex_ <dig> .
discussion
mutation rate studies indicate that denv genome averages  <dig> nucleotide change per cycle of virus replication  <cit>  because of the lack of proofreading activity.
another means to generate genetic changes is through recombination that has been reported in different flaviviruses, including hepatitis c virus , diarrhea bovine virus , denv, japanese encephalitis virus , and saint louis encephalitis virus   <cit> .
recently, some evidences have showed recombination in natural populations of denv- <dig> - <dig>  - <dig> and - <dig>  <cit>  and the incorporation of this recombination as a probable mechanism contributing to genetic variation in denv.
furthermore, the circulation of different serotypes and genotypes of denv in a particular geographical region has been documented  <cit> , as well as the coexistence of two different serotypes or genotypes in a given mosquito or patient  <cit> , which makes feasible the recombination in denv.
from the first identification of an intergenotypic denv recombinant  <cit> , several denv- <dig>  - <dig>  - <dig> and - <dig> recombinant strains have been identified  <cit> .
more importantly, the identification of this recombinant strains demonstrates that denv is capable of successfully completing all the simultaneous stages of the infection in the same cell: the simultaneous replication of both viral genomes and the template shift by the viral rna polymerase, while keeping the correct reading frame, encapsidation and release of the recombinant genomes in the process.
the products will be subjected to the population processes guiding the maintenance, expansion or disappearance of new variants in the heterogeneous viral population.
all these reports focused on denv- <dig>  <cit>  recombination, and to date, there are a few reports of den- <dig> recombinant strains detected by analysis of protein e sequences  <cit> .
besides, protein e gene of clones or c-prm-e-ns <dig> region from human serum isolates have not been reported.
there is only one single report of putative denv- <dig> recombinant clone isolated from mosquitoes in the coding region for protein e  <cit> .
in this report, the isolates mex_oax1656_ <dig> and mex_oax1038_ <dig> showed recombination within the c-prm-e-ns <dig> region.
in addition, there was recombination clearly identified within the e protein gene of the clone mex_oax1656_05_c <dig>  furthermore, the parental strains from the recombinants were identified.
these results are a strong evidence of the creation of new variants in a heterogeneous viral population.
furthermore, this is the first report of denv- <dig> recombination in mexico.
we detected two isolates containing recombination highly similar to the one obtained from different cities in the state of oaxaca, which is an evidence of the maintenance and expansion of new variants.
these two recombinants in the c-prm-e-ns <dig> region contained  <dig> breakpoints non-previously reported: one in the prm and two in the e protein .
we are showing denv- <dig> recombination between different genotypes in the isolates and clones analyzed with high frequency of approximately 30% and 10%, respectively.
the detection of the denv recombinants supports a potentially significant role for recombination in the evolution of denv by creating genetic variation.
this result is very important since recombination may shift the virulence of denv.
one could speculate if this shift may increase or diminish the virulence, like has happened in other rna viruses, such as poliovirus  <cit>  influenza virus  <cit>  and the hiv virus  <cit> .
a dramatic example is the loss of the attenuated phenotype of the poliovirus vaccine by recombination, resulting in the generation of new phenotypes that produce the acute paralytic disease.
consequently, recombinants have the potential to generate strains with a higher or lower virulence.
to test this issue for denv recombinants will be necessary to have an animal model to study the virulence of these recombinants.
the two points in our experimental procedure that have been instrumental in obtaining the reported result and to build confidence are: first, we analyzed  <dig> isolates and one clone in the coding region c-prm-e-ns <dig> from oaxaca and concentrated our efforts in sequencing the e gene of  <dig> clones from one isolate.
these regions were chosen based on its biological relevance and on the location of breakpoints identified in previous reports of recombination in denv  <cit> ; secondly, we minimized the chance of detecting false, artifactual recombination by using long extension times  <cit>  and a proofreading dna polymerase   <cit> .
moreover, the breakpoints tested by rdp <dig> resulted significant by  <dig> statistical methods; besides, the gard software displayed the same breakpoints as the rdp <dig> software package.
the analysis of  <dig> clones obtained from the isolate mex_oax_1656_ <dig> showed one clone  containing recombination in the e gene .
interestingly, the parental strains for this recombinant were the asian/american and the american genotypes.
this result is very important because the american genotype has the highest divergence among all the genotypes for denv- <dig>  furthermore, this is the first report on recombination between the asian/american  and american genotypes , which is supported by the analysis with rdp <dig> and gard .
this recombinant displays the breakpoints between the nucleotides  <dig> and  <dig>  these results suggest that the frequency of recombination in denv is higher than thought earlier, and the process will remain fundamentally hidden until more studies of clonal diversity to be undertaken.
nevertheless, the precise mechanism underlying the recombination events for denv is unknown.
to understand the mechanism of recombination the development of experimental models for co-infection to generate denv recombinants is required.
the second breakpoint in the c-prm-e-ns <dig> region  for the mex_oax_1038_ <dig> and mex_oax_1656_ <dig> isolates was different for  <dig> nucleotides when determined by bootscan, but it was the same when gard was used .
this was not associated with a sequence that permits the inference of a hot-spot of recombination as previously reported  <cit>  and does not permit the deduction of the mechanism of recombination as has been described for other flavivirus  <cit>  <cit> .
recombination may be a consequence of circulation of several genotypes at the same time in the same site and probably inside the same cell in the mosquito or human patient; in addition to the high density of different viruses circulating in the geographic area of oaxaca.
this is supported by a previous work that suggests that density of geographical and temporal sampling increases the probability for identifying recombinant sequences  <cit> .
phylogenetic studies have shown the circulation of the american  <cit> , american/asian  <cit> , and cosmopolitan  <cit>  genotypes in mexico, which makes feasible their recombination and explains the fact of the cosmopolitan and american genotypes to recombine with the asian/american genotype spread more broadly.
our results in combination with previous reports  <cit>  on denv- <dig> recombination suggest that the different genotypes of denv- <dig> are circulating in the virus pool infecting the mosquitoes or the human cells around the world.
until now, it remains unclear whether the frequency of recombination seen in this and previous studies is driving an increasing virulence of denv strains.
however, the recombinant strains of this study were obtained from the outbreak 2005- <dig> where the frequency of dhf cases was higher than the df cases in comparing to previous epidemics  <cit> .
to elucidate the role of recombination in denv virulence will be necessary to follow the generation of recombinants in outbreaks from other mexican states.
CONCLUSIONS
it is unclear whether the recombination events took place in a human host or a mosquito vector co-infected by multiple denv genotypes.
in this study, we detected two recombinant isolates of denv- <dig> from human hosts namely mex_oax_1038_ <dig> and mex_oax_1656_ <dig>  which identify  <dig> breakpoints within the prm-e-ns <dig> genome.
particularly the recombination appeared to have involved two genotypes of denv- <dig>  the asian/american clone  from the same strain and the cosmopolitan strain .
it is remarkable that parental and recombinant viral sequences of protein e were observed in an isolate from a single patient, particularly when the recombination appeared to have involved two genotypes of denv- <dig>  from the same geographic area .
this is only the second observation of one parental and recombinant of denv- <dig> in a population within a single host  <cit> .
there are two more studies where both parental and recombinant viral genomes were observed in a denv- <dig> isolate from a single patient.
denv recombination mechanism will be clarified by undertaking more studies of clonal diversity in both human and mosquito vector in mexico.

 den is a serious cause of mortality and morbidity in the tropical and subtropical regions that infects fifty million people every year; approximately of them are hospitalized and 5% to 15% of them die, which is a dramatic data. positive-sense rna viruses evolve rapidly, allowing the virus population to quickly adapt to new environments and escape from host anti-viral responses. one of the principal causes of genetic diversity in denv is the error-prone replication with rna-dependent rna polymerase, so that one genomic mutation occurs in nearly every cycle of virus replication. rna virus, such as denv populations at a particular region, may also rapidly change due to periodic selective sweeps, by the introduction of foreign strains of virus, and due to intra-serotypic recombination. however, there has been considerable debate about whether recombination occurs in denv and the relevance of any recombination to the development of live-attenuated flavivirus vaccines. besides, there is a number of reasons for believing that recombination can occur in denv and this process is being described with increasing frequency in denv- and other members of the family flaviviridae. the recombination in denv was reported in the structural genes region and particularly in e gene sequence through the use of the bootscan, diverse plots, and lard software. the co-circulation of multiple denv populations increases the opportunities for a mosquito vector to ingest several variants by feeding on a number of diverse infected hosts, or for a host to be infected by vectors infected with distinct denv variants. these conditions exist in mexico, the caribbean area and south-east asia. this is supported by the fact that there are many reports of multiple serotypes of denv from single hosts. furthermore, it is likely that mixed infections with different genotypes of the same serotype may also occur where they co-circulate. oaxaca, mexico is one of the states where denv is endemic and serotypes - - and - of denv are co-circulating. denv- was reported as the serotype with higher frequency compared with denv- - or - six partial sequences of the genes encoding proteins: capsid, pre-membrane-membrane, envelop, and non-structural represented as c-prm-e-ns from six different isolates of denv- from the oaxaca outbreaks 2005- were obtained. in addition, the rt-pcr products of c-prm-e-ns and e genes obtained from the mex_oax_1656_ isolate were cloned and sequenced. mex_oax_1656_ and mex_oax_1038_ isolates displayed recombination in the prm-e and e-ns genes and the parental strains were the asian/american and cosmopolitan genotypes. in addition, the e gene sequences from the clone showed recombination between the nucleotides to and the parental strains were asian/american and american genotypes.  to determine recombinant sequences in denv- the nucleotide sequences of the partial c-prm-e-ns genome from six isolates and representative sequences of the different genotypes were aligned and analyzed by rdp and gard. in addition, the rt-pcr product of the partial c-prm-e-ns genome from the mex_oax_1656_ isolate was cloned in pgem-3z. the sequences of clones were aligned with all of the above sequences. we also sequenced clones of the e structural gene from the isolate mex_oax_1656_ and aligned with representative sequences containing different genotypes by the programs mentioned above. the first task in this phylogenetic analysis was to determine the best model of nucleotide substitution for denv- virus sequence evolution. this assignment was undertaken using the model selection test from datamonkey online server, which compares models of dna substitution. our results demonstrated that the best model was trn. accordingly, the most complex general time-reversible value was the best fit to the data; the ln likelihood = -; parameter count = 38; and aic =. finally, the estimated base composition was a =, c =, g =, and t =. our analysis with rdp showed that the sequences of isolate mex_oax_1038_ and mex_oax_1656_ present statistical evidence of recombinants for genecov, bootscan, maxchi, chimera, siscan, and 3seq, in two regions: the first breakpoints were located in 499nt and 512nt respectively; the second breakpoints were located in 868nt and 826nt respectively, and the third breakpoint was located in 2239nt in both recombinants. in addition, the analysis with gard confirmed the breakpoints and recombination data for maximum likelihood. this analysis displayed the same site for the three breakpoints in both isolates: the first, second and third breakpoints were located in the nucleotides and respectively. the recombinant regions were the intersection of prm-m structural gene to intersection of m-e structural genes and the second recombinant region started in the intersection of e-ns genes. interestingly, we found that the parental major strain was the non-recombinant clone mex_oax_1656_05_ c and the minor parental strain was the cosmopolitan genotype strain indi_gwl_102_. the ml tree constructed with our sequence of structural gene c-prm from nucleotide 1- from the mex_oax_1038_ and mex_oax_1656_ isolates clustered with the asian/american genotype; the analysis of the region from nucleotides 498- of the isolates mex_oax_1038_ and mex_oax_1656_ moved to the cosmopolitan genotype and when the region from the nucleotides 828- was analyzed the two strains clustered again with the asian/american genotype. finally, when the region corresponding to nucleotides 2223- was analyzed the isolates clustered with the cosmopolitan genotype. to determine the nucleotides involved in these recombinants, the c-prm-e-ns sequences of the clone mex_oax_1656_05_c recombinants sequences mex_oax_1038_ mex_oax_1656_ and the cosmopolitan strain indi_gwl_102_ were analyzed. the changes in the recombinant isolates are labeled with a black dot. this analysis showed no evidence of recombination in the recombinant strain mex_oax_1656_ like other rna viruses, denv undergoes low fidelity replication, resulting in virus pools of mutants. therefore, to determine if one of the parental strains and/or a recombinant sequence is present in these pools, the rt-pcr product of the e protein gene from the recombinant strain, mex_oax_1656_ was cloned and analyzed. we obtained e protein gene clones that were studied using the rdp software and it was determined that the sequence of clone mex_oax_1656_05_c presents statistical evidence of recombination by genecov, bootscan, maxchi, chimera and 3seq. the e protein gene of said clones contains two breakpoints. the first breakpoint was located in the nucleotide of the coding region for protein e; the second breakpoint was located in the nucleotide of the same gene. gard analysis confirmed that this clone is recombinant displaying the first breakpoint in the nucleotide and the second breakpoint in the nucleotide. the constructed ml trees showed that the mex_oax_1656_05_c clone clustered in the asian/american genotype branch when the 1- e gene region was examined, and clustered in the american genotype when the e gene region from nucleotide to was analyzed. finally, when region 1048- was analyzed, the clone clustered again with the asian/american strains. the nucleotides involved in this recombinant are displayed in the alignment of the e gene region sequences of the recombinant mex_oax_1656_05_c clone, the parental clone mex_oax_1656_05_c and the strain mex_. discussion mutation rate studies indicate that denv genome averages nucleotide change per cycle of virus replication because of the lack of proofreading activity. another means to generate genetic changes is through recombination that has been reported in different flaviviruses, including hepatitis c virus, diarrhea bovine virus, denv, japanese encephalitis virus, and saint louis encephalitis virus. recently, some evidences have showed recombination in natural populations of denv- - - and - and the incorporation of this recombination as a probable mechanism contributing to genetic variation in denv. furthermore, the circulation of different serotypes and genotypes of denv in a particular geographical region has been documented, as well as the coexistence of two different serotypes or genotypes in a given mosquito or patient, which makes feasible the recombination in denv. from the first identification of an intergenotypic denv recombinant, several denv- - - and - recombinant strains have been identified. more importantly, the identification of this recombinant strains demonstrates that denv is capable of successfully completing all the simultaneous stages of the infection in the same cell: the simultaneous replication of both viral genomes and the template shift by the viral rna polymerase, while keeping the correct reading frame, encapsidation and release of the recombinant genomes in the process. the products will be subjected to the population processes guiding the maintenance, expansion or disappearance of new variants in the heterogeneous viral population. all these reports focused on denv- recombination, and to date, there are a few reports of den- recombinant strains detected by analysis of protein e sequences. besides, protein e gene of clones or c-prm-e-ns region from human serum isolates have not been reported. there is only one single report of putative denv- recombinant clone isolated from mosquitoes in the coding region for protein e. in this report, the isolates mex_oax1656_ and mex_oax1038_ showed recombination within the c-prm-e-ns region. in addition, there was recombination clearly identified within the e protein gene of the clone mex_oax1656_05_c furthermore, the parental strains from the recombinants were identified. these results are a strong evidence of the creation of new variants in a heterogeneous viral population. furthermore, this is the first report of denv- recombination in mexico. we detected two isolates containing recombination highly similar to the one obtained from different cities in the state of oaxaca, which is an evidence of the maintenance and expansion of new variants. these two recombinants in the c-prm-e-ns region contained breakpoints non-previously reported: one in the prm and two in the e protein. we are showing denv- recombination between different genotypes in the isolates and clones analyzed with high frequency of approximately 30% and 10%, respectively. the detection of the denv recombinants supports a potentially significant role for recombination in the evolution of denv by creating genetic variation. this result is very important since recombination may shift the virulence of denv. one could speculate if this shift may increase or diminish the virulence, like has happened in other rna viruses, such as poliovirus influenza virus and the hiv virus. a dramatic example is the loss of the attenuated phenotype of the poliovirus vaccine by recombination, resulting in the generation of new phenotypes that produce the acute paralytic disease. consequently, recombinants have the potential to generate strains with a higher or lower virulence. to test this issue for denv recombinants will be necessary to have an animal model to study the virulence of these recombinants. the two points in our experimental procedure that have been instrumental in obtaining the reported result and to build confidence are: first, we analyzed isolates and one clone in the coding region c-prm-e-ns from oaxaca and concentrated our efforts in sequencing the e gene of clones from one isolate. these regions were chosen based on its biological relevance and on the location of breakpoints identified in previous reports of recombination in denv; secondly, we minimized the chance of detecting false, artifactual recombination by using long extension times and a proofreading dna polymerase. moreover, the breakpoints tested by rdp resulted significant by statistical methods; besides, the gard software displayed the same breakpoints as the rdp software package. the analysis of clones obtained from the isolate mex_oax_1656_ showed one clone containing recombination in the e gene. interestingly, the parental strains for this recombinant were the asian/american and the american genotypes. this result is very important because the american genotype has the highest divergence among all the genotypes for denv- furthermore, this is the first report on recombination between the asian/american and american genotypes, which is supported by the analysis with rdp and gard. this recombinant displays the breakpoints between the nucleotides and these results suggest that the frequency of recombination in denv is higher than thought earlier, and the process will remain fundamentally hidden until more studies of clonal diversity to be undertaken. nevertheless, the precise mechanism underlying the recombination events for denv is unknown. to understand the mechanism of recombination the development of experimental models for co-infection to generate denv recombinants is required. the second breakpoint in the c-prm-e-ns region for the mex_oax_1038_ and mex_oax_1656_ isolates was different for nucleotides when determined by bootscan, but it was the same when gard was used. this was not associated with a sequence that permits the inference of a hot-spot of recombination as previously reported and does not permit the deduction of the mechanism of recombination as has been described for other flavivirus. recombination may be a consequence of circulation of several genotypes at the same time in the same site and probably inside the same cell in the mosquito or human patient; in addition to the high density of different viruses circulating in the geographic area of oaxaca. this is supported by a previous work that suggests that density of geographical and temporal sampling increases the probability for identifying recombinant sequences. phylogenetic studies have shown the circulation of the american, american/asian, and cosmopolitan genotypes in mexico, which makes feasible their recombination and explains the fact of the cosmopolitan and american genotypes to recombine with the asian/american genotype spread more broadly. our results in combination with previous reports on denv- recombination suggest that the different genotypes of denv- are circulating in the virus pool infecting the mosquitoes or the human cells around the world. until now, it remains unclear whether the frequency of recombination seen in this and previous studies is driving an increasing virulence of denv strains. however, the recombinant strains of this study were obtained from the outbreak 2005- where the frequency of dhf cases was higher than the df cases in comparing to previous epidemics. to elucidate the role of recombination in denv virulence will be necessary to follow the generation of recombinants in outbreaks from other mexican states.  it is unclear whether the recombination events took place in a human host or a mosquito vector co-infected by multiple denv genotypes. in this study, we detected two recombinant isolates of denv- from human hosts namely mex_oax_1038_ and mex_oax_1656_ which identify breakpoints within the prm-e-ns genome. particularly the recombination appeared to have involved two genotypes of denv- the asian/american clone from the same strain and the cosmopolitan strain. it is remarkable that parental and recombinant viral sequences of protein e were observed in an isolate from a single patient, particularly when the recombination appeared to have involved two genotypes of denv- from the same geographic area. this is only the second observation of one parental and recombinant of denv- in a population within a single host. there are two more studies where both parental and recombinant viral genomes were observed in a denv- isolate from a single patient. denv recombination mechanism will be clarified by undertaking more studies of clonal diversity in both human and mosquito vector in mexico. 
the oaxaca mex_oax_1656_ and mex_oax_1038_ isolates sequenced in this study were recombinant viruses that incorporate the genome sequence from the cosmopolitan genotype. 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. to study whether there are isolates from oaxaca having recombination, we obtained the sequence of different isolates of denv- asian/american genotype from the outbreak 2005- one clone of the c-prm-e-ns structural genes, and clones of the e gene from the isolate mex_oax_1656_ evidence of recombination was found by using different methods along with two softwares: rdp and gard. the cause of den is a positive-sense rna virus, the dengue virus 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. furthermore, the clone of the e gene namely mex_oax_165607_ from this study was also recombinant, incorporating genome sequence from the american genotype.
 dengue 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 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.  to study whether there are isolates from oaxaca having recombination, we obtained the sequence of different isolates of denv- asian/american genotype from the outbreak 2005- one clone of the c-prm-e-ns structural genes, and clones of the e gene from the isolate mex_oax_1656_ evidence of recombination was found by using different methods along with two softwares: rdp and gard. the oaxaca mex_oax_1656_ and mex_oax_1038_ 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_ from this study was also recombinant, incorporating genome sequence from the american genotype.  this is the first report of recombination in denv- 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. 