BACKGROUND
denv is a flavivirus within the arboviruses class, more than  <dig> of which have so far been identified.
denv is distributed worldwide in tropical and subtropical countries in association with its mosquito vector ae.
aegypti.
dengue infection ranges from self-limited asymptomatic or mild illness  to a severe hemorrhagic disease  that can progress to dengue shock syndrome  characterized by circulatory failure  <cit> .
more than fifty million dengue infection cases occur every year  <cit> , resulting in approximately  <dig>  deaths due to dss.
in mexico  <cit> ,  <dig>  cases of df and  <dig>  of dhf were reported from  <dig> to  <dig>  although den is the most common vector-borne viral disease, few studies have investigated the complex relationship between denv and ae.
aegypti through their genetic characteristics.
transmission of infection depends on denv virulence, host immunity and the susceptibility of the mosquito to infection.
susceptibility will depend on the interaction between the mosquito and denv: the interaction between midgut  cell membrane receptors and the virus envelope glycoprotein is the initial step  <cit>  in receptor-mediated endocytosis  <cit> .
this essential step determines cell permissiveness to infection.
furthermore, it is known that populations of ae.
aegypti differ greatly in their susceptibility to denv  <cit>  and this variability is determined by the effects of several genes  <cit> .
it has also been proposed  <cit>  that the infection barriers in the mosquitoes are the mg infection barrier , which prevents denv infection of mg epithelial cells, and the mg escape barrier , which prevents denv from leaving the mg and infecting peripheral tissues, limiting the infection to mg cells.
three ae.
aegypti strains differing in denv vector competence were selected: the susceptible ds <dig> strain lacks mib and meb, so 95–100% of these mosquitoes have disseminated infection  <cit> ; the dmeb strain has meb, and although 85% of them are infected in the mg epithelial cells, 27% develop a disseminated infection  <cit> ; and the refractory strain, ibo- <dig>  has mib  <cit> .
differences in vector competence may, at least in part, be due to the presence of specific mg epithelial receptors and their identification would be a significant step forward in understanding the interaction of the virus with the mosquito vector with possible implications for vector surveillance and control of virus transmission.
fc gamma receptor-mediated entry of infectious denv immune complexes into human monocytes/macrophages is hypothesized to be a key event in the pathogenesis of dhf  <cit> .
however, this mechanism does not explain virus entry in primary infections or in cells with non-fc receptors such as those of mosquitoes  <cit> .
putative non-fc gamma receptors differ in chemical structure  <cit> ; proteins  <cit>  or glycoproteins  <cit> ; heparan sulfates  <cit>  and lps/cd14-associated binding proteins  <cit>  have been proposed as cellular receptors for denv.
we have previously shown that the four serotypes of denv mainly recognize two proteins with apparent molecular weights of  <dig> and  <dig> kda  <cit>  from ae.
albopictus c6/ <dig> cells and ae.
aegypti mg. although a variety of receptors have been described in many studies, the mechanism by which the virus enters the cell is unknown.
a study of receptors in the ds <dig>  dmeb and ibo- <dig> strains will allow us to determine whether they are related to the susceptibility of ae.
aegypti to denv infection and transmission, and consequently whether they may serve as genetic markers of vector competence.
to date, none have been described.
knowledge of one or more genes responsible for susceptibility will help in designing new control strategies that may prevent denv infection and dissemination in the mosquito vector  <cit> .
therefore, the objective of this work was to determine whether denv receptors from ae.
aegypti mg are related to vector competence.
the aim was to identify the proteins recognized by denv in three ae.
aegypti strains that differ in their susceptibility to infection.
RESULTS
identification of denv binding proteins in mg from ds <dig>  dmeb and ibo- <dig> by vopba
to determine the apparent molecular weights of denv-binding mg proteins from each ae.
aegypti strain, equivalent amounts of mg protein extracts  were separated by sds-page, blotted on to pvdf membranes and analyzed by vopba  using the same specific antibodies against dengue virus used in our previous study  <cit> .
in the ibo- <dig> strain, denv bound mainly to a protein of apparent molecular weight about  <dig> kda, whereas in the ds <dig> and dmeb strains the apparent molecular mass of this protein was  <dig> kda.
proteins with molecular masses of  <dig> and  <dig> kda were also visualized, as previously described  <cit> , though these bands were not very strong.
because the ibo- <dig> strain showed a very faint band, we repeated the experiment, increasing the protein concentration approximately 3-fold compared to dmeb .
the masses of protein loaded on to the gel for each strain were:  <dig> μg for ds <dig>   <dig> μg for dmeb and  <dig> μg for ibo- <dig>  denv recognized the protein with apparent molecular mass of  <dig> kda with approximately the same intensity in all three strains; the  <dig> kda protein had the same molecular weight in the ds <dig> and dmeb strains, but in the ibo- <dig> strain the apparent molecular mass was  <dig> kda.
as a control for gel loading accuracy, blots were probed with anti-actin antibody; each specific band on the membranes was quantified by densitometry and normalized to actin.
the amount of denv bound to the 67/ <dig> kda proteins in each strain varied in the proportions dmeb: ds3: ibo- <dig> = 77: 56:  <dig>  these differences may be due to differences in the amount of mg protein from each mosquito strain or in their affinity for denv.
to determine whether the proteins identified by vopba represent one or more different molecules or whether there are differences among the strains,  <dig> mgs were subjected to two-dimensional gel electrophoresis  and a vopba was performed on the gel .
in the ds <dig> and dmeb strains, denv mainly recognized two protein spots with almost the same apparent molecular mass of  <dig> kda .
the ibo- <dig> strain displayed the same spots, although the lower one, which was more evident, probably corresponds to the protein with apparent molecular mass about  <dig> kda.
the faint upper spot may correspond to the protein with molecular mass of  <dig> kda.
the isoelectric point of both proteins in the three strains was about  <dig> .
identification of r <dig> and r <dig> in ds <dig>  dmeb and ibo- <dig> by immunoblotting
to determine whether the  <dig> and  <dig> kda mg proteins from the three ae.
aegypti strains are related, we obtained specific polyclonal antibodies against those proteins from the ds <dig> and ibo- <dig> strains and used them for immunoblotting of sds-page.
both the  <dig> and  <dig> kda proteins were recognized by the antibodies.
figure  <dig> shows an immunoblot with the specific antibody against r <dig> from the ibo- <dig> strain; equivalent results were observed when we used the other antibodies.
the main protein recognized by the antibodies in ds <dig> and dmeb had an apparent molecular weight of  <dig> kda.
the ibo- <dig> strain displayed a protein with apparent molecular weight of  <dig> kda, although the band was very faint, probably because its relative concentration was lower than in the other two strains.
thus, we found that the differences observed by vopba  were not due to differences in the affinity of r <dig> and r <dig> for denv, but may have been due to differences in the amounts of r <dig> and r <dig> in mg from each strain.
each specific band in the membranes was analyzed by densitometry and normalized to actin as control of the quantity of protein loaded on to each lane.
distribution of r67/r <dig> in aedes aegypti midgut
to determine the distribution of r <dig> and r <dig> along the mg in the ds <dig>  dmeb and ibo- <dig> strains, we performed confocal microscopy on dissected mg, immunofluorescently labeled with the specific antibody against those proteins .
this revealed the distribution of the proteins between the cells facing the lumen and the basal lamina  of the epithelium, and from the cardia through the anterior mg  and posterior mg .
the proteins were detected in the bl all along the mg in all three ae.
aegypti strains .
in addition, the specific antibody bound to epithelial cells beside the cell membrane in all strains from the middle to the end of the pmg ; in this region the fluorescence was more intense in ds <dig> and dmeb mgs  than in ibo- <dig> mg .
these results suggest a higher density of r <dig> and r <dig> in ds <dig> and dmeb mgs.
control mgs stained with pre-immune serum showed no-fluorescence .
time-course of denv infection in aedes aegypti midgut
we analyzed the time-course of infection all along the mg in order to determine the density and distribution of denv and its binding proteins at the outset of the infective process.
mosquitoes were infected via an artificial membrane feeder and the entire mg was dissected at  <dig>   <dig>   <dig> h and  <dig> days of cultivation after infection.
the mg was examined from the cardia through the amg and pmg by confocal microscopy after immunofluorescent labeling with anti-den- <dig> protein e. figures  <dig>   <dig> and  <dig> correspond to the ds <dig>  dmeb and ibo- <dig> strains respectively.
denv reached the epithelial cells in all strains  <dig> h after infection, before the peritrophic matrix  was formed.
the distribution of denv in the ibo- <dig> strain was similar to the other strains until  <dig> h, but virus density decreased with incubation time; the fluorescence had very low intensity at  <dig> h after infection; and at  <dig> h and  <dig> days after infection it had completely disappeared in 80% of the analyzed mgs.
in the ds <dig> and dmeb strains the fluorescence was very similar in each region analyzed at all times after infection; the infection increased with time and viral envelope antigen was apparent in the bl from  <dig> h until  <dig> days after infection irrespective of the distribution in epithelial cells .
we quantified the fluorescence  as mentioned in methods and noticed that the initial level was very similar in all three strains.
in ds <dig> and dmeb the maximum level was very alike, containing no statistically significant difference after  <dig> h of infection at ppmg; after  <dig> days, this region showed less viral envelope antigen and virus was observed at the anterior pmg.
virus infection was higher at this time compare to infection after  <dig> h .
if we compare virus infection of ibo- <dig> strain with ds <dig> or dmeb at  <dig>   <dig> and  <dig> h the difference is very evident ; contrary to infection at  <dig> h. furthermore, if we compare, infection at  <dig> h with all times in each strain, we observe that ds <dig> and dmeb showed an increase ; opposite to the ibo- <dig> strain where there is an infection decrease .
importantly, the highest fluorescence intensity observed in the mg at the beginning of infection  was very similar to that observed by immunofluorescence assays using anti-r67/r <dig> antibody.
all infected or non-infected mgs stained with pre-immune serum showed no-fluorescence .
discussion
the relationships between denv and its arthropod vector ae.
aegypti are crucial, and analysis of host cell responses to flavivirus infection of mosquito vectors is particularly important for understanding the maintenance and transmission of the disease.
mosquito populations differ in their susceptibility to flavivirus development, termed "vector competence", reflecting the different barriers encountered by the virus from its entry into the mosquito to its release in the saliva.
factors such as specific mosquito receptors on the epithelial cells as well as differential viral replication in the mosquito are critical for vector competence in addition to other genes as has been exhibited by the qtl studies  <cit> .
in the laboratory we have three mosquito strains with different susceptibilities to denv infection  and these have allowed us to determine whether mg cell receptors for denv may be markers of vector competence.
a possible explanation for the wide spectrum of denv receptors in host cells may be that different cell types have been used; alternatively, the reported proteins may not all be cell membrane components.
increasing evidence  <cit>  suggests that denv interacts differently with mammalian and mosquito cells; accordingly, we have studied the molecules that serve as true denv receptors in the mg of ae.
aegypti.
we previously showed by vopba  <cit>  that the four serotypes of denv mainly recognized two membrane proteins with apparent molecular weights of  <dig>  and  <dig>  kda in ae.
albopictus c6/ <dig> cells and ae.
aegypti mg.
both of these receptors were detected in the mg of strains ds <dig>  dmeb and ibo- <dig> in this study, although ibo- <dig> displayed a protein with a slightly lower apparent molecular weight  in addition to the  <dig> kda protein, and the quantity of r <dig> was much lower than in the other two strains.
r <dig> and r <dig> seem to be the same protein, because both are recognized by polyclonal anti-r <dig> and anti-r <dig> antibodies from the ds <dig> and ibo- <dig> strains.
the size difference was also detected by two dimensional gels .
munoz et al.
<cit>  and mercado-curiel et al.
<cit>  showed that anti-r <dig> antibody inhibited denv infection more strongly than anti-r <dig> in c6/ <dig> cells.
this is in agreement with the present results, since r <dig> was recognized in all three strains with no apparent change, suggesting that although this protein participates in denv binding; its role may be different.
it will be necessary future studies to determine r <dig> function during mosquito virus infection.
difference between r <dig> and r <dig> will be the objective of future studies, since they may be attributable to many facts such as post-translational modifications, different splicing or a deletion in the coding region.
studies of the vector competence of ae.
aegypti for denv are typically qualitative, involving the detection of viral antigens in the mg and the head.
this is the first study to scan denv infection and r <dig> protein distribution thoroughly along the entire mg, taking into account that mg contains morphologically different epithelial cells  <cit>  with different biochemical characteristics  <cit> .
previous studies have not determined denv distribution in detail; most of them have concerned virus titer  in the whole mosquito mg  <cit> .
r67/r <dig> were not homogeneously distributed over the epithelial cells along the mg in the three strains; they were mainly present in the cell membrane and behind the intercellular spaces in the region named ppmg, which extends from middle to the end of the posterior mg in the neighborhood of the hindgut.
the quantity of r67/r <dig> in the ppmg region was noticeably higher in ds <dig> and dmeb than in the ibo- <dig> strain .
the receptor distribution was similar to the distribution of infection at the outset.
moreover, we observed a correlation among receptor abundance, presence of denv and mosquito strain susceptibility .
analysis of the time course of denv infection from the beginning to  <dig> days gave insight into the earliest events in denv infection of the mg in the three strains.
mg infection diminished by  <dig> days post-infection, as described previously  <cit> .
we also observed that mgs from the different strains showed a difference in the degree of infection, depending on the susceptibility of the strain.
for example, the ibo- <dig> strain showed almost no fluorescence after  <dig> days.
interestingly, dmeb strain showed increase in infection up to the  <dig> h in all the three mg regions infection, having the maximal virus accumulation in the ppmg and then diminished by  <dig> h post-infection compare to the susceptible strain ds <dig> that have the maximal virus accumulation in apmg at  <dig> days post-infection.
these results also display a statistically significative mg infection increase from the first  <dig> h post-infection to  <dig> h in ds <dig> and dmeb strains .
moreover, ibo- <dig> strain exhibited a significative decrease  of mg infection from  <dig> to  <dig> h post-infection.
furthermore, virus infection of ibo- <dig> strain was almost completely abolish  compared to ds <dig> and dmeb strains from  <dig> to  <dig> h post-infection.
recently, the ae.
aegypti genome has been sequenced  <cit> .
this will facilitate the identification of genes encoding the r67/r <dig> denv receptors, which could be important for influencing the mg infection barrier .
this is in agreement with miller and mitchell  <cit> , who showed that susceptibility, depends on multiple genes.
they selected refractory or highly susceptible strains and obtained progeny with intermediate susceptibility, which suggests that alleles at vector competence loci act additively.
bosio et al.
<cit>  proposed a significant additive genetic effect in mib and demonstrated that the denv titer in the mg and head did not correlate with the rate of infection.
they also showed that the heritability for virus titer in tissues  were almost identical in different strains of ae.
aegypti formosus; and showed that the amount of virus in the mg did not determine if virus was disseminated, which hypothetical may be due to the presence or absence of denv receptors.
in the present study we also observed similar maximum levels of denv in the ds <dig> and dmeb strains, suggesting that genes that influence the virus titer have minimal impact on overall vector competence.
further studies are needed to explain the fact that the dmeb strain, which has meb, showed the presence of denv in the bl all along the mg throughout the time course examined.
the lack of infection in peripheral tissues may have been mainly due to the lack of cell receptors, or to additional factors specific to the kind of cell that allows denv to infect those organs.
ae.
aegypti seems to be the most vulnerable link in the transmission chain; at present there is no genetic marker of vector competence for denv.
such a marker would be very important in the design of new mosquito control strategies, such as campaigns focused on natural populations that could be easily identified by their high denv susceptibility, in order to prevent dengue epidemics.
CONCLUSIONS
our results may suggest that r67/r <dig> from the ae.
aegypti mg could be used as a vector competence marker, since those proteins are the main ones involved in the recognition of denv by mg cells.
the amount of these proteins in the mg varied proportionally to mosquito vector competence and their distribution along the mg correlated with the distribution of denv infection.

 denv is a flavivirus within the arboviruses class, more than of which have so far been identified. denv is distributed worldwide in tropical and subtropical countries in association with its mosquito vector ae. aegypti. dengue infection ranges from self-limited asymptomatic or mild illness to a severe hemorrhagic disease that can progress to dengue shock syndrome characterized by circulatory failure. more than fifty million dengue infection cases occur every year, resulting in approximately deaths due to dss. in mexico, cases of df and of dhf were reported from to although den is the most common vector-borne viral disease, few studies have investigated the complex relationship between denv and ae. aegypti through their genetic characteristics. transmission of infection depends on denv virulence, host immunity and the susceptibility of the mosquito to infection. susceptibility will depend on the interaction between the mosquito and denv: the interaction between midgut cell membrane receptors and the virus envelope glycoprotein is the initial step in receptor-mediated endocytosis. this essential step determines cell permissiveness to infection. furthermore, it is known that populations of ae. aegypti differ greatly in their susceptibility to denv and this variability is determined by the effects of several genes. it has also been proposed that the infection barriers in the mosquitoes are the mg infection barrier, which prevents denv infection of mg epithelial cells, and the mg escape barrier, which prevents denv from leaving the mg and infecting peripheral tissues, limiting the infection to mg cells. three ae. aegypti strains differing in denv vector competence were selected: the susceptible ds strain lacks mib and meb, so 95–100% of these mosquitoes have disseminated infection; the dmeb strain has meb, and although 85% of them are infected in the mg epithelial cells, 27% develop a disseminated infection; and the refractory strain, ibo- has mib. differences in vector competence may, at least in part, be due to the presence of specific mg epithelial receptors and their identification would be a significant step forward in understanding the interaction of the virus with the mosquito vector with possible implications for vector surveillance and control of virus transmission. fc gamma receptor-mediated entry of infectious denv immune complexes into human monocytes/macrophages is hypothesized to be a key event in the pathogenesis of dhf. however, this mechanism does not explain virus entry in primary infections or in cells with non-fc receptors such as those of mosquitoes. putative non-fc gamma receptors differ in chemical structure; proteins or glycoproteins; heparan sulfates and lps/cd14-associated binding proteins have been proposed as cellular receptors for denv. we have previously shown that the four serotypes of denv mainly recognize two proteins with apparent molecular weights of and kda from ae. albopictus c6/ cells and ae. aegypti mg. although a variety of receptors have been described in many studies, the mechanism by which the virus enters the cell is unknown. a study of receptors in the ds dmeb and ibo- strains will allow us to determine whether they are related to the susceptibility of ae. aegypti to denv infection and transmission, and consequently whether they may serve as genetic markers of vector competence. to date, none have been described. knowledge of one or more genes responsible for susceptibility will help in designing new control strategies that may prevent denv infection and dissemination in the mosquito vector. therefore, the objective of this work was to determine whether denv receptors from ae. aegypti mg are related to vector competence. the aim was to identify the proteins recognized by denv in three ae. aegypti strains that differ in their susceptibility to infection.  identification of denv binding proteins in mg from ds dmeb and ibo- by vopba to determine the apparent molecular weights of denv-binding mg proteins from each ae. aegypti strain, equivalent amounts of mg protein extracts were separated by sds-page, blotted on to pvdf membranes and analyzed by vopba using the same specific antibodies against dengue virus used in our previous study. in the ibo- strain, denv bound mainly to a protein of apparent molecular weight about kda, whereas in the ds and dmeb strains the apparent molecular mass of this protein was kda. proteins with molecular masses of and kda were also visualized, as previously described, though these bands were not very strong. because the ibo- strain showed a very faint band, we repeated the experiment, increasing the protein concentration approximately 3-fold compared to dmeb. the masses of protein loaded on to the gel for each strain were: μg for ds μg for dmeb and μg for ibo- denv recognized the protein with apparent molecular mass of kda with approximately the same intensity in all three strains; the kda protein had the same molecular weight in the ds and dmeb strains, but in the ibo- strain the apparent molecular mass was kda. as a control for gel loading accuracy, blots were probed with anti-actin antibody; each specific band on the membranes was quantified by densitometry and normalized to actin. the amount of denv bound to the 67/ kda proteins in each strain varied in the proportions dmeb: ds3: ibo- = 77: 56: these differences may be due to differences in the amount of mg protein from each mosquito strain or in their affinity for denv. to determine whether the proteins identified by vopba represent one or more different molecules or whether there are differences among the strains, mgs were subjected to two-dimensional gel electrophoresis and a vopba was performed on the gel. in the ds and dmeb strains, denv mainly recognized two protein spots with almost the same apparent molecular mass of kda. the ibo- strain displayed the same spots, although the lower one, which was more evident, probably corresponds to the protein with apparent molecular mass about kda. the faint upper spot may correspond to the protein with molecular mass of kda. the isoelectric point of both proteins in the three strains was about. identification of r and r in ds dmeb and ibo- by immunoblotting to determine whether the and kda mg proteins from the three ae. aegypti strains are related, we obtained specific polyclonal antibodies against those proteins from the ds and ibo- strains and used them for immunoblotting of sds-page. both the and kda proteins were recognized by the antibodies. figure shows an immunoblot with the specific antibody against r from the ibo- strain; equivalent results were observed when we used the other antibodies. the main protein recognized by the antibodies in ds and dmeb had an apparent molecular weight of kda. the ibo- strain displayed a protein with apparent molecular weight of kda, although the band was very faint, probably because its relative concentration was lower than in the other two strains. thus, we found that the differences observed by vopba were not due to differences in the affinity of r and r for denv, but may have been due to differences in the amounts of r and r in mg from each strain. each specific band in the membranes was analyzed by densitometry and normalized to actin as control of the quantity of protein loaded on to each lane. distribution of r67/r in aedes aegypti midgut to determine the distribution of r and r along the mg in the ds dmeb and ibo- strains, we performed confocal microscopy on dissected mg, immunofluorescently labeled with the specific antibody against those proteins. this revealed the distribution of the proteins between the cells facing the lumen and the basal lamina of the epithelium, and from the cardia through the anterior mg and posterior mg. the proteins were detected in the bl all along the mg in all three ae. aegypti strains. in addition, the specific antibody bound to epithelial cells beside the cell membrane in all strains from the middle to the end of the pmg; in this region the fluorescence was more intense in ds and dmeb mgs than in ibo- mg. these results suggest a higher density of r and r in ds and dmeb mgs. control mgs stained with pre-immune serum showed no-fluorescence. time-course of denv infection in aedes aegypti midgut we analyzed the time-course of infection all along the mg in order to determine the density and distribution of denv and its binding proteins at the outset of the infective process. mosquitoes were infected via an artificial membrane feeder and the entire mg was dissected at h and days of cultivation after infection. the mg was examined from the cardia through the amg and pmg by confocal microscopy after immunofluorescent labeling with anti-den- protein e. figures and correspond to the ds dmeb and ibo- strains respectively. denv reached the epithelial cells in all strains h after infection, before the peritrophic matrix was formed. the distribution of denv in the ibo- strain was similar to the other strains until h, but virus density decreased with incubation time; the fluorescence had very low intensity at h after infection; and at h and days after infection it had completely disappeared in 80% of the analyzed mgs. in the ds and dmeb strains the fluorescence was very similar in each region analyzed at all times after infection; the infection increased with time and viral envelope antigen was apparent in the bl from h until days after infection irrespective of the distribution in epithelial cells. we quantified the fluorescence as mentioned in methods and noticed that the initial level was very similar in all three strains. in ds and dmeb the maximum level was very alike, containing no statistically significant difference after h of infection at ppmg; after days, this region showed less viral envelope antigen and virus was observed at the anterior pmg. virus infection was higher at this time compare to infection after h. if we compare virus infection of ibo- strain with ds or dmeb at and h the difference is very evident; contrary to infection at h. furthermore, if we compare, infection at h with all times in each strain, we observe that ds and dmeb showed an increase; opposite to the ibo- strain where there is an infection decrease. importantly, the highest fluorescence intensity observed in the mg at the beginning of infection was very similar to that observed by immunofluorescence assays using anti-r67/r antibody. all infected or non-infected mgs stained with pre-immune serum showed no-fluorescence. discussion the relationships between denv and its arthropod vector ae. aegypti are crucial, and analysis of host cell responses to flavivirus infection of mosquito vectors is particularly important for understanding the maintenance and transmission of the disease. mosquito populations differ in their susceptibility to flavivirus development, termed "vector competence", reflecting the different barriers encountered by the virus from its entry into the mosquito to its release in the saliva. factors such as specific mosquito receptors on the epithelial cells as well as differential viral replication in the mosquito are critical for vector competence in addition to other genes as has been exhibited by the qtl studies. in the laboratory we have three mosquito strains with different susceptibilities to denv infection and these have allowed us to determine whether mg cell receptors for denv may be markers of vector competence. a possible explanation for the wide spectrum of denv receptors in host cells may be that different cell types have been used; alternatively, the reported proteins may not all be cell membrane components. increasing evidence suggests that denv interacts differently with mammalian and mosquito cells; accordingly, we have studied the molecules that serve as true denv receptors in the mg of ae. aegypti. we previously showed by vopba that the four serotypes of denv mainly recognized two membrane proteins with apparent molecular weights of and kda in ae. albopictus c6/ cells and ae. aegypti mg. both of these receptors were detected in the mg of strains ds dmeb and ibo- in this study, although ibo- displayed a protein with a slightly lower apparent molecular weight in addition to the kda protein, and the quantity of r was much lower than in the other two strains. r and r seem to be the same protein, because both are recognized by polyclonal anti-r and anti-r antibodies from the ds and ibo- strains. the size difference was also detected by two dimensional gels. munoz et al. and mercado-curiel et al. showed that anti-r antibody inhibited denv infection more strongly than anti-r in c6/ cells. this is in agreement with the present results, since r was recognized in all three strains with no apparent change, suggesting that although this protein participates in denv binding; its role may be different. it will be necessary future studies to determine r function during mosquito virus infection. difference between r and r will be the objective of future studies, since they may be attributable to many facts such as post-translational modifications, different splicing or a deletion in the coding region. studies of the vector competence of ae. aegypti for denv are typically qualitative, involving the detection of viral antigens in the mg and the head. this is the first study to scan denv infection and r protein distribution thoroughly along the entire mg, taking into account that mg contains morphologically different epithelial cells with different biochemical characteristics. previous studies have not determined denv distribution in detail; most of them have concerned virus titer in the whole mosquito mg. r67/r were not homogeneously distributed over the epithelial cells along the mg in the three strains; they were mainly present in the cell membrane and behind the intercellular spaces in the region named ppmg, which extends from middle to the end of the posterior mg in the neighborhood of the hindgut. the quantity of r67/r in the ppmg region was noticeably higher in ds and dmeb than in the ibo- strain. the receptor distribution was similar to the distribution of infection at the outset. moreover, we observed a correlation among receptor abundance, presence of denv and mosquito strain susceptibility. analysis of the time course of denv infection from the beginning to days gave insight into the earliest events in denv infection of the mg in the three strains. mg infection diminished by days post-infection, as described previously. we also observed that mgs from the different strains showed a difference in the degree of infection, depending on the susceptibility of the strain. for example, the ibo- strain showed almost no fluorescence after days. interestingly, dmeb strain showed increase in infection up to the h in all the three mg regions infection, having the maximal virus accumulation in the ppmg and then diminished by h post-infection compare to the susceptible strain ds that have the maximal virus accumulation in apmg at days post-infection. these results also display a statistically significative mg infection increase from the first h post-infection to h in ds and dmeb strains. moreover, ibo- strain exhibited a significative decrease of mg infection from to h post-infection. furthermore, virus infection of ibo- strain was almost completely abolish compared to ds and dmeb strains from to h post-infection. recently, the ae. aegypti genome has been sequenced. this will facilitate the identification of genes encoding the r67/r denv receptors, which could be important for influencing the mg infection barrier. this is in agreement with miller and mitchell, who showed that susceptibility, depends on multiple genes. they selected refractory or highly susceptible strains and obtained progeny with intermediate susceptibility, which suggests that alleles at vector competence loci act additively. bosio et al. proposed a significant additive genetic effect in mib and demonstrated that the denv titer in the mg and head did not correlate with the rate of infection. they also showed that the heritability for virus titer in tissues were almost identical in different strains of ae. aegypti formosus; and showed that the amount of virus in the mg did not determine if virus was disseminated, which hypothetical may be due to the presence or absence of denv receptors. in the present study we also observed similar maximum levels of denv in the ds and dmeb strains, suggesting that genes that influence the virus titer have minimal impact on overall vector competence. further studies are needed to explain the fact that the dmeb strain, which has meb, showed the presence of denv in the bl all along the mg throughout the time course examined. the lack of infection in peripheral tissues may have been mainly due to the lack of cell receptors, or to additional factors specific to the kind of cell that allows denv to infect those organs. ae. aegypti seems to be the most vulnerable link in the transmission chain; at present there is no genetic marker of vector competence for denv. such a marker would be very important in the design of new mosquito control strategies, such as campaigns focused on natural populations that could be easily identified by their high denv susceptibility, in order to prevent dengue epidemics.  our results may suggest that r67/r from the ae. aegypti mg could be used as a vector competence marker, since those proteins are the main ones involved in the recognition of denv by mg cells. the amount of these proteins in the mg varied proportionally to mosquito vector competence and their distribution along the mg correlated with the distribution of denv infection. 
notwithstanding studies of quantitative trait loci that influence the ability of aedes aegypti midgut to become infected with dengue virus, no study to date has been undertaken to identify genetic markers of vector competence. to determine whether the same proteins were identified in all three strains, we obtained polyclonal antibodies against r and r and tested them against the three strains by immunoblotting; both antibodies recognized the and kda proteins, corroborating the vopba results. consequently, our results strongly suggest that this protein may be a marker of vector competence for denv in ae. specific antibodies against both proteins were used for immunofluorescent location by confocal microscopy; the antibodies recognized the basal lamina all along the mg, and cell membranes and intercellular spaces from the middle to the end of the posterior midgut in the neighborhood of the hindgut. the main protein identified had an apparent molecular weight of kda, although the protein identified in the ibo- strain showed a lower mass. furthermore, it is known that mosquito populations differ greatly in their susceptibility to flaviviruses. vector competence refers to the intrinsic permissiveness of an arthropod vector for infection, replication and transmission of a virus. differences in vector competence may, at least in part, be due to the presence of specific midgut epithelial receptors and their identification would be a significant step forward in understanding the interaction of the virus with the mosquito. aegypti strains that differ in their vector competence for denv, we investigated the ds strain, the ibo- strain and the membrane escape barrier strain, dmeb, which is infected exclusively in the midgut epithelial cells. in this paper we provide evidence that the kda protein, described previously as a denv receptor, is related to vector competence in ae. the midgut proteins recognized by denv were also determined by vopba after two-dimensional gel electrophoresis. quantitative analysis showed more intense fluorescence in ds and dmeb than in ibo- the viral envelope antigen was not homogeneously distributed during mg infection but correlated with mg density and the distribution of r67/r we determined the mg proteins that bind denv by an overlay protein binding assay in ae.
 vector competence refers to the intrinsic permissiveness of an arthropod vector for infection, replication and transmission of a virus. notwithstanding studies of quantitative trait loci that influence the ability of aedes aegypti midgut to become infected with dengue virus, no study to date has been undertaken to identify genetic markers of vector competence. furthermore, it is known that mosquito populations differ greatly in their susceptibility to flaviviruses. differences in vector competence may, at least in part, be due to the presence of specific midgut epithelial receptors and their identification would be a significant step forward in understanding the interaction of the virus with the mosquito. the first interaction of denv with the insect is through proteins in the apical membrane of the midgut epithelium resulting in binding and receptor-mediated endocytosis of the virus, and this determines cell permissiveness to infection. the susceptibility of mosquitoes to infection may therefore depend on their specific virus receptors. to study this interaction in ae. aegypti strains that differ in their vector competence for denv, we investigated the ds strain, the ibo- strain and the membrane escape barrier strain, dmeb, which is infected exclusively in the midgut epithelial cells.  we determined the mg proteins that bind denv by an overlay protein binding assay in ae. aegypti mosquitoes of the ds dmeb and ibo- strains. the main protein identified had an apparent molecular weight of kda, although the protein identified in the ibo- strain showed a lower mass. the midgut proteins recognized by denv were also determined by vopba after two-dimensional gel electrophoresis. to determine whether the same proteins were identified in all three strains, we obtained polyclonal antibodies against r and r and tested them against the three strains by immunoblotting; both antibodies recognized the and kda proteins, corroborating the vopba results. specific antibodies against both proteins were used for immunofluorescent location by confocal microscopy; the antibodies recognized the basal lamina all along the mg, and cell membranes and intercellular spaces from the middle to the end of the posterior midgut in the neighborhood of the hindgut. quantitative analysis showed more intense fluorescence in ds and dmeb than in ibo- the viral envelope antigen was not homogeneously distributed during mg infection but correlated with mg density and the distribution of r67/r  in this paper we provide evidence that the kda protein, described previously as a denv receptor, is related to vector competence in ae. aegypti. consequently, our results strongly suggest that this protein may be a marker of vector competence for denv in ae. aegypti mosquitoes. 