BACKGROUND
the arbovirus, o'nyong-nyong virus  belongs to genus alphavirus, and is an enveloped, single stranded,  rna virus with a genome of approximately  <dig> kb  <cit> .
unlike other arboviruses, onnv is primarily transmitted by anopheline mosquitoes such as anopheles gambiae and an.
funestus  <cit> .
onnv was first identified during an epidemic in uganda in  <dig>  which ultimately infected over  <dig> million people across east africa from  <dig> to  <dig>  <cit> .
recently it has reemerged in  <dig> and  <dig> sporadically in africa  <cit> .
although mosquitoes are critical vectors in many arboviral transmission cycles, there is limited information on how arboviruses influence mosquito gene expression and how mosquito immune systems defend arthropod vectors from deleterious consequences of viral infection.
the recent completion of the sequencing of the an.
gambiae genome has allowed us to investigate modulation of mosquito gene expression resulting from arbovirus infection.
genome-wide screening of differentially expressed transcripts of onnv-infected female an.
gambiae relative to naïve females was conducted at  <dig> day p.i.
<cit> .
seven genes were identified for their differential expression in onnv-infected an.
gambiae compared with controls by cdna microarrays followed by paired t-test and quantitative real time pcr  analysis.
the products of the seven genes are seemingly involved in protein translation, dna replication, or intracellular transport pathways  <cit> .
among the seven candidates, hsc70b, ef-1α and agglutinin were chosen for further functional studies because of their roles in protein folding, protein elongation, cell adhesion, and cytoprotection, which are all important molecular processes for viral replication.
we hypothesize that elevated gene expression of hsc70b for example, may protect the mosquito cells from onnv-induced molecular damage  <cit> .
since molecular chaperons including heat shock protein  <dig> families regulate protein folding and degradation, it is possible that hsc70b may suppress the non-native viral structural or non-structural protein synthesis in mosquito cells.
it was also reported that mammalian and mosquito ef-1α binds to the 3' utrs of west nile virus  and a range of rna viruses  <cit> .
thus, anopheles ef-1α may have a similar role for onnv replication in an.
gambiae.
lastly, agglutinin is a membrane attachment subunit that may interact with onnv on the membranes of endosomes and lysosomes.
because non-structural proteins and rnas of alphaviruses including onnv are associated with the membranes of modified endosomes and lysosomes in the replication complex  <cit> , agglutinin may therefore be involve in a membrane attachment of the replication complex of onnv.
based on these assumptions, hsc70b, ef-1α and agglutinin genes were subjected to a detailed functional analysis for their potential involvement in onnv replication.
using rnai, we post-transcriptionally silenced target transcripts of the three genes, hsc70b, ef-1α and agglutinin by co-injecting dsrnas of each target transcript with onnv into female an.
gambiae.
the result showed that silencing the hsc70b transcript caused significant increase of onnv titers in female mosquitoes whilst the remaining two genes had no noticeable effects.
herein, we discuss potential antiviral activity of hsc70b in an.
gambiae.
RESULTS
phylogenetic analysis of  <dig> kda heat shock protein family from an.
gambiae and d. melanogaster
phylogenetic analysis of hsp <dig> genes from an.
gambiae and d. melanogaster indicate that anopheles hsc70b gene is evolutionarily more conserved with drosophila hsc70- <dig> and hsc70- <dig> genes than other anopheles homologues .
for example, anopheles hsc70b is most tightly liked to drosophila hsc70- <dig> .
multiple sequence alignments indicated that there are high polymorphisms near the 5' and 3'-end coding and non-coding regions of the anopheles hsc <dig> family .
indeed, based on these sequence polymorphisms among the anopheles hsc <dig> family, we were able to detect unique transcripts of the anopheles hsc70b gene among the anopheles hsp <dig> genes by using the rt-pcr and qrt-pcr primers based on the 3'and 5'-end regions, respectively .
in addition, the dsrna of anopheles hsc70b was designed based on the 5' end that was specific to the hsc70b gene, which enabled hsc70b specific knockdown in anopheles gambiae.
aq represent the primer pairs for quantitative rt-pcr, bds represents the primer pairs of the templates for dsrna includes t <dig> promoter for in vitro transcription.
camplification efficiencies were calculated from the slope of standard curves as e = 10- <dig>  100% pcr efficiency corresponds to an amplification efficiency of 1; dregression coefficient of linear standard curve.
the effect of hsc70b on onnv replication
first, efficiency of dsrnai was assessed by using semi-quantitative rt-pcr and qrt-pcr analyses.
in contrast to high induction of hsc70b in dsβ-gal and onnv/onnv-egfp coinjected mosquitoes, only traces of hsc70b mrna were detected in dshsc70b mosquitoes using semi-quantitative rt-pcr and primers corresponding to the 3' end of the hsc70b gene .
this result shows that the injection of specific dshsc70b successfully reduced endogenous target transcripts of hsc70b gene after onnv injection.
similarly, rt-pcr results also showed the successful post-transcriptional inhibition in agglutinin and ef-1α genes .
mosquitoes were coinjected with ~ <dig>  ×  <dig> pfu of virus and  <dig> ng of the respective dsrnas targeting the hsc70b, agglutinin and ef-1α gene or the β-gal gene as an internal control .
to quantify onnv in the respective dsrnai-treated mosquitoes, each an.
gambiae was triturated and onnv titer was quantified by plaque assay from the treatment groups.
mosquitoes coinjected with onnv and dshsc70b had significantly more plaques than those mosquitoes coinjected with onnv and dsβ-gal  at  <dig> d.p.i.
.
no significant differences in the number of plaques were observed among the mosquitoes injected with onnv and each of ds-agglutinin, dsef-1α, and dsβ-gal  .
to investigate whether up-regulation of hsc70b gene impedes onnv replication, an.
gambiae was infected with egfp-tagged onnv whilst hsc70b transcripts were silenced by dsrnai.
onnv titers were then indirectly estimated by visually comparing egfp expression among three mosquito groups that were coinoculated with dshsc70b, or dsβ-gal or buffer.
in control mosquitoes injected with dsβ-gal or buffer, expression of egfp was usually weak in head, thorax and abdomen tissues .
mosquitoes coinjected with onnv-egfp and dshsc70b typically had stronger expression of egfp in all three tissues .
at  <dig> dpi, 47%  of mosquitoes receiving dsβ-gal expressed egfp in thoracic tissues.
however, 87%  expressed egfp in thoracic tissues when dshsc70b was silenced .
in a similar way, the mosquitoes receiving dsβ-gal showed 38%  and 22%  of egfp expression in head and abdomen, respectively .
in contrast, mosquitoes receiving dshsc70b showed 70%  and 65%  of egfp expression in head and abdomen, respectively .
the ribosomal proteins s <dig>  and s <dig>  were used as internal controls for infection studies of onnv and the malaria parasite, plasmodium berghei, respectively  <cit> .
when rps <dig> was analyzed by qrt-pcr and cdna microarray studies, student's t test determined that the mrna expression levels of rps <dig> in uninfected and onnv-infected mosquitoes collected at  <dig> h,  <dig> h and  <dig> days p.i.
were not significantly different .
as shown in figure  <dig>  rt-pcr analysis of rps <dig> and qrt-pcr analysis of rps <dig> transcript levels at  <dig> days p.i., detected no significant difference between the relative mrna levels derived from treated mosquitoes.
the results indicate that the low expression levels observed for candidate genes were related to the observed knockdown effect by dsrnai, and not attributed to variation in sample loading.
suppression of hsc70b expression by rnai and higher onnv infection level decreases the survival rate of an.
gambiae
co-injection of onnv and the dshsc70b significantly shortened the lifespan of adult mosquitoes compared with the co-injected mosquitoes of onnv and dsβ-gal, causing a significant reduction of survival rates from  <dig> days p.i.
.
in addition, an.
gambiae mosquitoes with down-regulated hsc70b alone lead a reduced survival rate  at  <dig> days post injection, though it is much less harmful than co-injection of onnv and dshsc70b.
this suggests that both reduced expression levels of hsc70b gene and increased onnv infection level synergistically shorten the lifespan of an.
gambiae .
discussion
in general, the hsp <dig> family including hsc70b is one of the best studied among the heat shock protein families.
the structures of hsp70s and hsc70s are highly conserved in all organisms, from prokaryotes to eukaryotes  <cit> .
the conserved region of the molecule consists of an n-terminal adenosine triphosphatase domain , a substrate-binding domain , and a carboxyl-terminal domain of variable length and polymorphism  <cit> .
furthermore, hsc70b, a constitutively induced form of the hsp <dig> family, was inferred to be located in the cytoplasm  <cit> , and so therefore has may interact with structural and non-structural gene products of onnv.
in fact, from the in vitro study with another alphavirus sindbis, hsc <dig> purified from bovine brain inhibits the autoprotease activity of sindbis capsid protein present in the cytoplasm  <cit> .
interestingly, the prominent antiviral drug cyclopentenone prostanoids and its derivatives, specifically induced hsp70s and hsc70s in treated vero and mosquito, aedes albopictus, cells  <cit> .
induction of hsp70s has a cytoprotective effect, interrupting viral replication against a wide range of dna and rna viruses including sindbis  <cit> .
this antiviral response seems to depend on synthesis of molecular chaperons, which are involved in controlling virus replication in various ways  <cit> .
in addition, recent studies suggested that hsp <dig> and hsc <dig> chaperones play a key role in protein quality control, being involved in either folding or degrading of non-native proteins by the ubiquitin-proteasome pathway  <cit> .
alternatively, hsc70b may impede viral replication by modulating other an.
gambiae proteins that are critical for viral replication in mosquito cells.
since one of the key roles of hsc70b is to regulate nascent unfolded protein as a chaperone  <cit> , the overexpression of hsc70b may shift metabolism of mosquito cells to suppress viral replication.
thus, hsc70b may indirectly repress nascent protein folding of host factors required for onnv replication in the mosquito.
in contrast, a cdna microarray study in ae.
aegypti, which is the principal vector of yellow fever and dengue viruses, reported that sindbis infection significantly down-regulates the mosquito chaperones, hsp <dig> and hsp <dig>  <cit> .
perhaps, viral suppression of host chaperones may be important for propagation of the virus.
previous studies of the gene products of alphaviruses have shown that these gene products suppress and maneuver host gene expression to favor the viral gene expression system  <cit> .
with respect to the regulation/repression of host cell gene expression, there may thus be competition between viral and host factors.
furthermore, the mosquito and virus may compete for the ef-1α and agglutinin, and this could explain why these proteins are induced upon infection.
however, it cannot be ruled out that these knockdown effects may not be sufficient to mimic loss-of-function phenotypes to impair or enhance onnv replication in an.
gambiae, because the rnai technique cannot completely abolish target transcripts.
this lack of complete knockout is expected to allow residual expression of target genes, which could result in protein expression that cannot be phenotypically distinguished from wild type or control groups.
therefore proteomics assays measuring viral protein expression in conjunction with ef-1α or agglutinin knockdown will provide more definitive pictures for these proteins' role in onnv replication in an.
gambiae.
suppression of hsc70b by dsrnai substantially reduced the survival rate of onnv-infected an.
gambiae .
in d. melanogaster, it has been reported that apoptosis affects primarily cells with the lowest level of hsc70s during embryogenesis, suggesting a role for hsc70s in the control of apoptosis  <cit> .
several studies have shown that hsp70s and hsc70s binding to the anti-apoptotic protein bag- <dig>  <cit> .
it has also been reported that hsc70s interacts with the ecdysone receptor and ultraspiracle   <cit> , and that high induction of the chaperones extends longevity in d. melanogaster and in c. elegans  <cit> .
our data indicates that an.
gambiae hsc70b is also important for the survival of mosquitoes during viral infection .
CONCLUSIONS
in summary, our results demonstrate that hsc70b impede onnv replication in an.
gambiae.
the balance between onnv propagation and hsc70b abundance may determine, at least in part, the level of onnv replication and cytotoxicity.
understanding the molecular details of hsc70b interaction with structural and non-structural gene products of onnv will lead to the elucidation of new approaches to controlling this pathogenic virus.
furthermore, this may suggest new control strategies relying upon the development of transgenic mosquitoes that selectively turn on chaperone genes during arbovirus infection.
while molecular mechanisms regarding the regulation of expression of hsp <dig> genes are relatively well characterized  <cit> , the regulation of hsc <dig> genes remains to be determined  <cit> .
our study, in conjunction with the previous microarray experiments, have proven to be effective to identify candidate genes involved in an.
gambiae-onnv interactions at the genome level  <cit> .
in addition to genome-wide gene expression studies, an analysis of tissue-specific gene expression is also important, e.g., midgut, fat body, and salivary glands.
the discovery of genes that can provide barriers of onnv transmission in those mosquito tissues will be beneficial to control the viral infection in humans.

 the arbovirus, o'nyong-nyong virus belongs to genus alphavirus, and is an enveloped, single stranded, rna virus with a genome of approximately kb. unlike other arboviruses, onnv is primarily transmitted by anopheline mosquitoes such as anopheles gambiae and an. funestus. onnv was first identified during an epidemic in uganda in which ultimately infected over million people across east africa from to. recently it has reemerged in and sporadically in africa. although mosquitoes are critical vectors in many arboviral transmission cycles, there is limited information on how arboviruses influence mosquito gene expression and how mosquito immune systems defend arthropod vectors from deleterious consequences of viral infection. the recent completion of the sequencing of the an. gambiae genome has allowed us to investigate modulation of mosquito gene expression resulting from arbovirus infection. genome-wide screening of differentially expressed transcripts of onnv-infected female an. gambiae relative to naïve females was conducted at day p.i.. seven genes were identified for their differential expression in onnv-infected an. gambiae compared with controls by cdna microarrays followed by paired t-test and quantitative real time pcr analysis. the products of the seven genes are seemingly involved in protein translation, dna replication, or intracellular transport pathways. among the seven candidates, hsc70b, ef-1α and agglutinin were chosen for further functional studies because of their roles in protein folding, protein elongation, cell adhesion, and cytoprotection, which are all important molecular processes for viral replication. we hypothesize that elevated gene expression of hsc70b for example, may protect the mosquito cells from onnv-induced molecular damage. since molecular chaperons including heat shock protein families regulate protein folding and degradation, it is possible that hsc70b may suppress the non-native viral structural or non-structural protein synthesis in mosquito cells. it was also reported that mammalian and mosquito ef-1α binds to the 3' utrs of west nile virus and a range of rna viruses. thus, anopheles ef-1α may have a similar role for onnv replication in an. gambiae. lastly, agglutinin is a membrane attachment subunit that may interact with onnv on the membranes of endosomes and lysosomes. because non-structural proteins and rnas of alphaviruses including onnv are associated with the membranes of modified endosomes and lysosomes in the replication complex, agglutinin may therefore be involve in a membrane attachment of the replication complex of onnv. based on these assumptions, hsc70b, ef-1α and agglutinin genes were subjected to a detailed functional analysis for their potential involvement in onnv replication. using rnai, we post-transcriptionally silenced target transcripts of the three genes, hsc70b, ef-1α and agglutinin by co-injecting dsrnas of each target transcript with onnv into female an. gambiae. the result showed that silencing the hsc70b transcript caused significant increase of onnv titers in female mosquitoes whilst the remaining two genes had no noticeable effects. herein, we discuss potential antiviral activity of hsc70b in an. gambiae.  phylogenetic analysis of kda heat shock protein family from an. gambiae and d. melanogaster phylogenetic analysis of hsp genes from an. gambiae and d. melanogaster indicate that anopheles hsc70b gene is evolutionarily more conserved with drosophila hsc70- and hsc70- genes than other anopheles homologues. for example, anopheles hsc70b is most tightly liked to drosophila hsc70-. multiple sequence alignments indicated that there are high polymorphisms near the 5' and 3'-end coding and non-coding regions of the anopheles hsc family. indeed, based on these sequence polymorphisms among the anopheles hsc family, we were able to detect unique transcripts of the anopheles hsc70b gene among the anopheles hsp genes by using the rt-pcr and qrt-pcr primers based on the 3'and 5'-end regions, respectively. in addition, the dsrna of anopheles hsc70b was designed based on the 5' end that was specific to the hsc70b gene, which enabled hsc70b specific knockdown in anopheles gambiae. aq represent the primer pairs for quantitative rt-pcr, bds represents the primer pairs of the templates for dsrna includes t promoter for in vitro transcription. camplification efficiencies were calculated from the slope of standard curves as e = 10- 100% pcr efficiency corresponds to an amplification efficiency of 1; dregression coefficient of linear standard curve. the effect of hsc70b on onnv replication first, efficiency of dsrnai was assessed by using semi-quantitative rt-pcr and qrt-pcr analyses. in contrast to high induction of hsc70b in dsβ-gal and onnv/onnv-egfp coinjected mosquitoes, only traces of hsc70b mrna were detected in dshsc70b mosquitoes using semi-quantitative rt-pcr and primers corresponding to the 3' end of the hsc70b gene. this result shows that the injection of specific dshsc70b successfully reduced endogenous target transcripts of hsc70b gene after onnv injection. similarly, rt-pcr results also showed the successful post-transcriptional inhibition in agglutinin and ef-1α genes. mosquitoes were coinjected with ~ × pfu of virus and ng of the respective dsrnas targeting the hsc70b, agglutinin and ef-1α gene or the β-gal gene as an internal control. to quantify onnv in the respective dsrnai-treated mosquitoes, each an. gambiae was triturated and onnv titer was quantified by plaque assay from the treatment groups. mosquitoes coinjected with onnv and dshsc70b had significantly more plaques than those mosquitoes coinjected with onnv and dsβ-gal at d.p.i.. no significant differences in the number of plaques were observed among the mosquitoes injected with onnv and each of ds-agglutinin, dsef-1α, and dsβ-gal. to investigate whether up-regulation of hsc70b gene impedes onnv replication, an. gambiae was infected with egfp-tagged onnv whilst hsc70b transcripts were silenced by dsrnai. onnv titers were then indirectly estimated by visually comparing egfp expression among three mosquito groups that were coinoculated with dshsc70b, or dsβ-gal or buffer. in control mosquitoes injected with dsβ-gal or buffer, expression of egfp was usually weak in head, thorax and abdomen tissues. mosquitoes coinjected with onnv-egfp and dshsc70b typically had stronger expression of egfp in all three tissues. at dpi, 47% of mosquitoes receiving dsβ-gal expressed egfp in thoracic tissues. however, 87% expressed egfp in thoracic tissues when dshsc70b was silenced. in a similar way, the mosquitoes receiving dsβ-gal showed 38% and 22% of egfp expression in head and abdomen, respectively. in contrast, mosquitoes receiving dshsc70b showed 70% and 65% of egfp expression in head and abdomen, respectively. the ribosomal proteins s and s were used as internal controls for infection studies of onnv and the malaria parasite, plasmodium berghei, respectively. when rps was analyzed by qrt-pcr and cdna microarray studies, student's t test determined that the mrna expression levels of rps in uninfected and onnv-infected mosquitoes collected at h, h and days p.i. were not significantly different. as shown in figure rt-pcr analysis of rps and qrt-pcr analysis of rps transcript levels at days p.i., detected no significant difference between the relative mrna levels derived from treated mosquitoes. the results indicate that the low expression levels observed for candidate genes were related to the observed knockdown effect by dsrnai, and not attributed to variation in sample loading. suppression of hsc70b expression by rnai and higher onnv infection level decreases the survival rate of an. gambiae co-injection of onnv and the dshsc70b significantly shortened the lifespan of adult mosquitoes compared with the co-injected mosquitoes of onnv and dsβ-gal, causing a significant reduction of survival rates from days p.i.. in addition, an. gambiae mosquitoes with down-regulated hsc70b alone lead a reduced survival rate at days post injection, though it is much less harmful than co-injection of onnv and dshsc70b. this suggests that both reduced expression levels of hsc70b gene and increased onnv infection level synergistically shorten the lifespan of an. gambiae. discussion in general, the hsp family including hsc70b is one of the best studied among the heat shock protein families. the structures of hsp70s and hsc70s are highly conserved in all organisms, from prokaryotes to eukaryotes. the conserved region of the molecule consists of an n-terminal adenosine triphosphatase domain, a substrate-binding domain, and a carboxyl-terminal domain of variable length and polymorphism. furthermore, hsc70b, a constitutively induced form of the hsp family, was inferred to be located in the cytoplasm, and so therefore has may interact with structural and non-structural gene products of onnv. in fact, from the in vitro study with another alphavirus sindbis, hsc purified from bovine brain inhibits the autoprotease activity of sindbis capsid protein present in the cytoplasm. interestingly, the prominent antiviral drug cyclopentenone prostanoids and its derivatives, specifically induced hsp70s and hsc70s in treated vero and mosquito, aedes albopictus, cells. induction of hsp70s has a cytoprotective effect, interrupting viral replication against a wide range of dna and rna viruses including sindbis. this antiviral response seems to depend on synthesis of molecular chaperons, which are involved in controlling virus replication in various ways. in addition, recent studies suggested that hsp and hsc chaperones play a key role in protein quality control, being involved in either folding or degrading of non-native proteins by the ubiquitin-proteasome pathway. alternatively, hsc70b may impede viral replication by modulating other an. gambiae proteins that are critical for viral replication in mosquito cells. since one of the key roles of hsc70b is to regulate nascent unfolded protein as a chaperone, the overexpression of hsc70b may shift metabolism of mosquito cells to suppress viral replication. thus, hsc70b may indirectly repress nascent protein folding of host factors required for onnv replication in the mosquito. in contrast, a cdna microarray study in ae. aegypti, which is the principal vector of yellow fever and dengue viruses, reported that sindbis infection significantly down-regulates the mosquito chaperones, hsp and hsp. perhaps, viral suppression of host chaperones may be important for propagation of the virus. previous studies of the gene products of alphaviruses have shown that these gene products suppress and maneuver host gene expression to favor the viral gene expression system. with respect to the regulation/repression of host cell gene expression, there may thus be competition between viral and host factors. furthermore, the mosquito and virus may compete for the ef-1α and agglutinin, and this could explain why these proteins are induced upon infection. however, it cannot be ruled out that these knockdown effects may not be sufficient to mimic loss-of-function phenotypes to impair or enhance onnv replication in an. gambiae, because the rnai technique cannot completely abolish target transcripts. this lack of complete knockout is expected to allow residual expression of target genes, which could result in protein expression that cannot be phenotypically distinguished from wild type or control groups. therefore proteomics assays measuring viral protein expression in conjunction with ef-1α or agglutinin knockdown will provide more definitive pictures for these proteins' role in onnv replication in an. gambiae. suppression of hsc70b by dsrnai substantially reduced the survival rate of onnv-infected an. gambiae. in d. melanogaster, it has been reported that apoptosis affects primarily cells with the lowest level of hsc70s during embryogenesis, suggesting a role for hsc70s in the control of apoptosis. several studies have shown that hsp70s and hsc70s binding to the anti-apoptotic protein bag-. it has also been reported that hsc70s interacts with the ecdysone receptor and ultraspiracle, and that high induction of the chaperones extends longevity in d. melanogaster and in c. elegans. our data indicates that an. gambiae hsc70b is also important for the survival of mosquitoes during viral infection.  in summary, our results demonstrate that hsc70b impede onnv replication in an. gambiae. the balance between onnv propagation and hsc70b abundance may determine, at least in part, the level of onnv replication and cytotoxicity. understanding the molecular details of hsc70b interaction with structural and non-structural gene products of onnv will lead to the elucidation of new approaches to controlling this pathogenic virus. furthermore, this may suggest new control strategies relying upon the development of transgenic mosquitoes that selectively turn on chaperone genes during arbovirus infection. while molecular mechanisms regarding the regulation of expression of hsp genes are relatively well characterized, the regulation of hsc genes remains to be determined. our study, in conjunction with the previous microarray experiments, have proven to be effective to identify candidate genes involved in an. gambiae-onnv interactions at the genome level. in addition to genome-wide gene expression studies, an analysis of tissue-specific gene expression is also important, e.g., midgut, fat body, and salivary glands. the discovery of genes that can provide barriers of onnv transmission in those mosquito tissues will be beneficial to control the viral infection in humans. 
furthermore, co-injection of onnv with dshsc70b significantly reduced the lifespan of adult mosquitoes as compared with the control, co-injection of onnv with dsβ-gal treated mosquitoes. onnv titers from mosquitoes co-injected with dshsc70b were about 9-fold higher at days post-injection as compared to the control mosquitoes. we previously reported that expression of hsc70b and other genes including elongation factor-1α and the agglutinin attachment subunit were up-regulated in o'nyong-nyong virus -infected female an. therefore, mosquitoes can function as vectors of onnv transmission to humans while onnv infection in an. these results indicate that hsc70b plays important roles in homeostasis and suppression of onnv replication in the vector, an. double-stranded rna interferences have been applied to further investigate hsc70b, ef-1α and the agglutinin functions in onnv replication in an. among these three rnai silenced genes, only dsrnas of hsc70b promoted onnv replication in adult an. phylogenetic and functional analysis was conducted on an anopheles gambiae gene, ensangg based on phylogenetic analysis, this gene belongs to the same lineage as heat shock protein cognate 70- in drosophila. gambiae compared to the control mosquitoes that were co-injected with onnv and dsrna of β-galactosidase.
 phylogenetic and functional analysis was conducted on an anopheles gambiae gene, ensangg based on phylogenetic analysis, this gene belongs to the same lineage as heat shock protein cognate 70- in drosophila. accordingly, we propose to name this gene heat shock protein cognate 70b. we previously reported that expression of hsc70b and other genes including elongation factor-1α and the agglutinin attachment subunit were up-regulated in o'nyong-nyong virus -infected female an. gambiae. double-stranded rna interferences have been applied to further investigate hsc70b, ef-1α and the agglutinin functions in onnv replication in an. gambiae.  among these three rnai silenced genes, only dsrnas of hsc70b promoted onnv replication in adult an. gambiae compared to the control mosquitoes that were co-injected with onnv and dsrna of β-galactosidase. onnv titers from mosquitoes co-injected with dshsc70b were about 9-fold higher at days post-injection as compared to the control mosquitoes. by using onnv tagged with enhanced green fluorescent protein, co-injection of onnv-egfp with dshsc70b also showed approximately ~ 3-fold higher gfp expression rates than the controls in the head, thorax, and abdomen of the mosquito. furthermore, co-injection of onnv with dshsc70b significantly reduced the lifespan of adult mosquitoes as compared with the control, co-injection of onnv with dsβ-gal treated mosquitoes.  these results indicate that hsc70b plays important roles in homeostasis and suppression of onnv replication in the vector, an. gambiae. biological implications of these findings are that while mosquitoes allow onnv to replicate in them, they also check viral titers so that onnv infection will result in no harmful effect on mosquitoes. therefore, mosquitoes can function as vectors of onnv transmission to humans while onnv infection in an. gambiae remains asymptomatic. 