Periodic Reporting for period 1 - ZIKVMosTransmit (Molecular mechanisms underlying Zika virus transmission by Aedes aegypti mosquitoes)
Reporting period: 2023-01-16 to 2025-01-15
Project summary:
The public health response to emerging mosquito-borne viral diseases is often inefficient, because the causative agents are recognized and the prevention measures are deployed, only after the disease has spread.
To predict which viruses and mosquitoes have the highest potential to cause future outbreaks, understanding the mechanisms determining the virus susceptibility of mosquitoes and the mosquito transmissibility of viruses is needed.
In this project, I proposed a twopronged approach to identify both the viral and mosquito factors involved in molecular compatibility between the human pathogenic Zika virus (ZIKV) and its main vector Aedes aegypti
by combining my skills in molecular virology with the host lab’s expertise in mosquito experimental infections in vivo.
First, I used an innovative reverse genetics system to create chimeric ZIKV strains from two parental virus strains with different levels of mosquito transmissibility.
The viral genes required for efficient transmission by mosquitoes were identified by comparing transmissibility between chimeric constructs.
Second, I examined the mosquito-virus protein interactome by a proteomics strategy based on cross-linking, immunoaffinity purification and mass spectrometry.
Mosquito factors regulating virus susceptibility will be identified by subtractive comparison of virus-binding mosquito proteins between two mosquito strains displaying different levels of Zika virus susceptibility.
The correlation between virus susceptibility and the identified proteins will be validated by gene-knockdown assays and expression surveys using a collection of mosquito strains.
Together, this project provides important new insights into vector-virus specificity and significantly improve our understanding of mosquito-borne virus emergence.
The public health response to emerging mosquito-borne viral diseases is often inefficient, because the causative agents are recognized and the prevention measures are deployed, only after the disease has spread.
To predict which viruses and mosquitoes have the highest potential to cause future outbreaks, understanding the mechanisms determining the virus susceptibility of mosquitoes and the mosquito transmissibility of viruses is needed.
In this project, I proposed a twopronged approach to identify both the viral and mosquito factors involved in molecular compatibility between the human pathogenic Zika virus (ZIKV) and its main vector Aedes aegypti
by combining my skills in molecular virology with the host lab’s expertise in mosquito experimental infections in vivo.
First, I used an innovative reverse genetics system to create chimeric ZIKV strains from two parental virus strains with different levels of mosquito transmissibility.
The viral genes required for efficient transmission by mosquitoes were identified by comparing transmissibility between chimeric constructs.
Second, I examined the mosquito-virus protein interactome by a proteomics strategy based on cross-linking, immunoaffinity purification and mass spectrometry.
Mosquito factors regulating virus susceptibility will be identified by subtractive comparison of virus-binding mosquito proteins between two mosquito strains displaying different levels of Zika virus susceptibility.
The correlation between virus susceptibility and the identified proteins will be validated by gene-knockdown assays and expression surveys using a collection of mosquito strains.
Together, this project provides important new insights into vector-virus specificity and significantly improve our understanding of mosquito-borne virus emergence.
I investigated the viral genetic determinants underlying the different mosquito-borne transmissibility between Asian and African lineage ZIKV strains by comparing an African strain and an Asian strain using chimeric viruses, in which segments of the parental genomes are swapped.
My results show that the structural genes from the African strain enhance viral internalization, while the non-structural genes improve genome replication and infectious particle production in mosquito cells.
I also performed the artificial blood meal feeding using mosquitoes in vivo. In vivo mosquito transmission is most significantly influenced by the structural genes, although no single viral gene is solely responsible for this effect.
Additionally, I develop a stochastic model of in vivo viral dynamics in mosquitoes that mirrors the observed patterns, suggesting that the primary difference between the African and Asian strains lies in their ability to traverse the mosquito salivary glands.
I conclude that natural ZIKV variation in the efficiency of mosquito-borne transmission is determined by multiple viral genetic factors, predominantly affecting the final stage of propagation within mosquitoes.
I also investigated the Ae. aegypti factors responsible for the susceptibility of ZIKV by comparison of virus-binding mosquito proteins between two mosquito strains displaying different levels of ZIKV susceptibility.
I conducted pilot studies to explore the method for isolating virus-binding mosquito proteins. After mosquito midguts were sonicated by using COVARIS and centrifuged, supernatants were collected as midgut protein lysates.
Midgut protein lysates were incubated with ZIKV, fixed and reacted with anti-ZIKV primary antibodies and secondary fluorescent antibodies.
I then investigated whether ZIKV-midgut protein complex was able to be detected by flow cytometry. Notably, ZIKV particle was able to be detected using anti-E protein monoclonal antibody and some mosquito proteins could be identified by mass spectrometry.
My results show that the structural genes from the African strain enhance viral internalization, while the non-structural genes improve genome replication and infectious particle production in mosquito cells.
I also performed the artificial blood meal feeding using mosquitoes in vivo. In vivo mosquito transmission is most significantly influenced by the structural genes, although no single viral gene is solely responsible for this effect.
Additionally, I develop a stochastic model of in vivo viral dynamics in mosquitoes that mirrors the observed patterns, suggesting that the primary difference between the African and Asian strains lies in their ability to traverse the mosquito salivary glands.
I conclude that natural ZIKV variation in the efficiency of mosquito-borne transmission is determined by multiple viral genetic factors, predominantly affecting the final stage of propagation within mosquitoes.
I also investigated the Ae. aegypti factors responsible for the susceptibility of ZIKV by comparison of virus-binding mosquito proteins between two mosquito strains displaying different levels of ZIKV susceptibility.
I conducted pilot studies to explore the method for isolating virus-binding mosquito proteins. After mosquito midguts were sonicated by using COVARIS and centrifuged, supernatants were collected as midgut protein lysates.
Midgut protein lysates were incubated with ZIKV, fixed and reacted with anti-ZIKV primary antibodies and secondary fluorescent antibodies.
I then investigated whether ZIKV-midgut protein complex was able to be detected by flow cytometry. Notably, ZIKV particle was able to be detected using anti-E protein monoclonal antibody and some mosquito proteins could be identified by mass spectrometry.
By using the chimeric viruses from African and Asian ZIKV genomes, I found that structural proteins from African strain enhanced in vivo mosquito transmissibility.
To further understand ZIKV genetic basis for the potential of the epidemic transmission, I will investigate which ZIKV genome regions modulate efficient ZIKV vertebrate infection and host-vector transmission by using the set of chimeric viruses.
I've already started collaboration with research teams inside and outside Pasteur on these future studies. I expect that these studies will provide new insights into how ZIKV evolved and emerged in the human population.
I established the methods to identify the ZIKV-binding mosquito proteins by using anti-E protein monoclonal antibody and mass spectrometry.
In future, I plan to apply this method to mosquito midgut proteins extracted from mosquito species with different susceptibilities to ZIKV infection to compare the ZIKV-interacting proteins.
I wish identifying mosquito factors underlying virus susceptibility would allow active surveillance to predict if field-derived mosquitoes can transmit pathogenic viruses in advance.
To further understand ZIKV genetic basis for the potential of the epidemic transmission, I will investigate which ZIKV genome regions modulate efficient ZIKV vertebrate infection and host-vector transmission by using the set of chimeric viruses.
I've already started collaboration with research teams inside and outside Pasteur on these future studies. I expect that these studies will provide new insights into how ZIKV evolved and emerged in the human population.
I established the methods to identify the ZIKV-binding mosquito proteins by using anti-E protein monoclonal antibody and mass spectrometry.
In future, I plan to apply this method to mosquito midgut proteins extracted from mosquito species with different susceptibilities to ZIKV infection to compare the ZIKV-interacting proteins.
I wish identifying mosquito factors underlying virus susceptibility would allow active surveillance to predict if field-derived mosquitoes can transmit pathogenic viruses in advance.