Scientist Live spoke with Dr. Rasgon about the discovery and its possible uses.
How did you get involved in the original project?
We originally were working on a project, and still are, completely unrelated to this. We were working with an endosymbiotic bacterium called wolbachia. This bacteria is of interest to control vector and pest insects such as mosquitoes. It is maternally transmitted and it causes reproductive abnormalities that we want to exploit to control the mosquitoes. It is a really common bacteria and found in lots of different insects and mosquitoes but it is not found in any mosquito in the genus Anopheles which is the mosquito that transmits malaria to humans. We were interested to see if we could transfer this bacterium into anopheles mosquitoes and we started out by working not with the mosquito itself but rather with cell cultures made from anopheles mosquito cells. These are cultured insect mosquito cells growing in a dish or a flask in the laboratory. This bacterium grows inside the cells of other organism. It is an intracellular bacteria. We wanted to see first of all could we get this bacterium to grow inside anopheles cells. (As an aside, we were able to accomplish this.) While we were doing this we would normally infect the cells with the bacterium, we would wait a certain period of time and the first thing we would run a PCR test to see whether the cells were infected with the bacterium or not. We were expecting a fragment of amplified DNA of about 600 bp. From one of our cell lines, we kept seeing a band at about 400 bp. These artifacts can be common in the lab, but we kept seeing it over and over. And we finally got smart and we isolated that band and actually sequenced it to see what we were picking up.
I have seen these kinds of artifacts before and they are usually garbage, randomly amplified mosquito DNA or something like that but this time when we got the sequence back and compared it to other sequences in the public genome database it came back very closely 80-90% related to the Aedes aegypti densonucleosis virus. Now I thought this was very interesting. These viruses have been used and studied for a long time in Aedes mosquitoes as a potential control agent. But all of those studies suggested that the virus does not infect anopheles mosquitoes. This was a densovirus that seemed to be a contaminant in our cell line. We thought because this virus has been growing for god knows how long in these cells maybe it actually came from anopheles mosquitoes and was able to infect them. So we took it from there. We isolated the virus.
First we confirmed that the cells were actually infected. We isolated the virus. We infected mosquitoes with that isolated virus and showed that it did infect them. When we were able to isolate the genome clone, we put a marker gene in there. We showed we could actually infect mosquitoes with these recombinant viruses and have the virus express the green fluorescent protein and see it under the microscope. This is a proof of principal study because it suggests that we can now engineer this virus and instead of putting green fluorescent protein in there we could put a gene that would make the mosquito unable to transmit malaria for instance. Or we could put an insect specific toxin gene that would kill the mosquito after a certain number of days. These viruses are interesting because you can infect the larvae. It will express the genome in the adults and then when the female reproduces she transmits the virus to the next generation both directly by transovarial transmission and also indirectly because she will inoculate virus into the larval water and it will go and infect whatever larvae happen to be in there. You can introduce it into nature in theory and the mosquitoes can spread it to the population. You can think of it as gene therapy for mosquitoes.
What made you decide to pursue this artifact further?
We kept seeing it again and again. We saw it from one particular cell line and finally the post-doc who was working on the project just got curious. Generally, if we keep seeing consistent bands in a gel we will generally sequence them just to see what it is just to make sure we do not have a contamination problem or to make sure our primers are not bad. So we generally will sequence them when we see them. 99 times out of a hundred they are nothing. We just got lucky this time so it was a complete accident. Once we realised what was in there it was obvious what direction to go into. There was no way for us to predict that the virus was in there. Sometimes when these things are in cell culture can cause certain characteristic pathologies in cells. We had no evidence that it was in the cell line until we accidentally pulled it out with these primers.
Did you immediately recognise the possibility of using it as a vector?
Yes, I did. As I am fairly familiar with the work that has been done with the aedes aegypti densovirus with aedes aegypti mosquitoes. They tried to infect anopheles with that virus some six or seven years ago. They found the can get point infections with the virus but it never disseminated. When we found this, we thought wow if we can get this to infect anopheles mosquitoes it would be a major thing. Number one for these applied measures of using it as a control strategy but also very useful as a tool in the lab. Anopheles is not the easiest mosquito to do genetic work with in the lab. Your options are limited because it is difficult to make transgenics. Other viral expression systems do not work very well. So this is a very easy system for basic research on the mosquito in the lab.
What is the next step in terms of further research and applications?
From applied standpoint in terms of using it as a control agent, we are taking two different strategies. One would be to engineer this virus to express genes that would make the mosquito unable to transmit malaria. We have already shown that the virus will infect the virus mid-gut and the mosquito fat body which are relevant tissues for this. We can very easily insert genes into the virus that when they are expressed in the mosquito can block malaria invasion or development in the mosquito. So if it becomes infected by the virus it should in theory become unable to transmit the malaria parasites. That is one strategy. The other is using this as a way to kill mosquitoes. We are not so much thinking of it as a larvacide but rather we're working on a strategy dubbed time bomb lethality. This is putting a lethal gene in the virus that would kill the mosquito adults. They would be infected as larvae and die as adults. We want to time the time of death so the mosquitoes are around ten days old. This allows the mosquito to reproduce because they will take a blood meal around day one or two. Then lay a batch of eggs by day five and another set of eggs by day eight or nine. It will allow the mosquitoes to reproduce and allow the virus to get transmitted to the next generation. Because they are reproducing it will severely limit the evolution of resistance in the mosquito to the virus pathogenicity. Mosquitoes have to live about 14 days to transmit malaria, but if we can kill them by day ten we will give them a long enough time to reproduce and pass the virus on and limit resistance evolution but not long enough to transmit the parasites.
What's next for your laboratory?
We are trying to mutate the virus to see if we can alter the infection phenotype to get it to infect earlier or later. We are developing it into just an expression system. There are a lot of genes we would like to test in mosquitoes for various applications. We really just cannot make transgenics to test them because it is very difficult. Now we have a system fairly developed that we can in a couple of weeks engineer a virus and infect mosquitoes and see what it does. That is the basic research applications. In general we are interested in developing this as a tool to manipulate the mosquito in both an applied manner and in a basic manner. We are following several lines of research along that path.
Reporting by Marc Landas
