Modern science using ancient pieces of genetic machinery to study, fight disease

By Susan Trulove

Related story: The "interfering scientists" at the Vector-Borne Disease Research Group at Virginia Tech

As life evolved, survival tactics were fierce. One life form would not only invade another’s space, but also their bodies and very cells. Billions of years later, diseases that kill and sicken millions of people and animals are a result of the evolutionary deal made between mosquitoes and the pathogens they harbor and transmit. Scientists are discovering that evolution has also provided tools for interfering with disease transmission.

Members of the Vector-Borne Disease Research Group at Virginia Tech are using ancient pieces of genetic material as tools to explore the mosquito immune system and are among researchers across the world experiment- ing with ways to engineer mosquitoes so they cannot play host to pathogens.

Tool 1: Transposable elements

In the late 1940s, Barbara McClintock discovered jumping genes in the genome of maize (1983 Nobel Prize). In recent decades, with scientists sequencing the genomes of all manner of plants, animals, and bugs, the library of genomic information has grown and the ability to compare its details has advanced. It is now known that jumping, or transposable, gene sequences are common to many kinds of creatures. “If you look at the genome as an ecological system, these transposable elements are different lineages that co-evolved with the rest of the genome,” says Jake Tu, associate professor of biochemistry and a member of the Vector Borne Disease Research Group.

As indicated by their name, transposable elements (TEs) can move from place to place on the genome. Some types of TEs only move copies of themselves. Other types, called DNA transposons, have the ability to relocate. They can drop into line for a different program — from a DNA sequence that initiates hair growth to the sequence that initiates reproduction — and they can bring a date. The relocated TEs then appear in subsequent copies of the DNA.

The ability of TEs to move around on the genome may have contributed to mutations and new species. In self defense and to save energy, genome machinery and their resident TEs have been fighting a war of inactivation and transposition. But TEs are still active in insects, and now they may become tools for quashing the spread of diseases carried to millions of people by mosquitoes.

When an international team sequenced the genome of the mosquito that carries the yellow fever and dengue fever viruses (Aedes aegypti), Tu led the five research labs that annotated the TEs in the Ae. aegypti genome, describing the more than 1,000 transposable elements that occupy half of the entire genome. He reported good news from his studies of the viability of these TEs. “Although the majority of protein coding TE copies in Ae. aegypti appear to be degenerate, a significant number of elements have potentially active TE copies, indicating that they may be developed as tools for genetic studies of mosquitoes,” Tu says.

Even before the Ae. aegypti sequence was reported, scientists were using the TEs of other insects as research tools and in experiments to create disease-free mosquitoes. For example, Zach Adelman, assistant professor of entomology at Virginia Tech, and colleagues at the University of California at Irvine and Colorado State University used a TE isolated from a house fly to introduce an anti-viral gene into the Ae. aegypti mosquito genome.

“While this resulted in these genetically modified mosquitoes being resistant to viral infection, we still know very little about the mosquito’s natural immune responses to viral pathogens,” says Adelman.

Tool 2: RNA interference

So Adelman, who researches the biology of the vectors, and Kevin Myles, an assistant professor of entomology who studies viruses, are collaborating to understand mosquito-virus interaction. They are testing the hypothesis that mosquitoes have an innate immunity mechanism based on RNA interference (RNAi) that is also present in plants, worms, and humans.

RNA is created from a DNA template, then RNA in turn acts as a template for protein synthesis. Different forms of RNA have different functions in the process of transcribing specific gene sequences from DNA to create proteins with specific functions. Normally, RNA is single stranded, but there are a variety of ways in which double-stranded RNA (dsRNA) may form n the cell. In 1998, Andrew Fire and Craig Mello discovered RNAi, a process by which dsRNA silences genes that have similar sequences (2006 Nobel Prize). RNAi is often triggered by foreign dsRNAs.

“Double-stranded RNA is a sign that something is not right,” Adelman says. “The dsRNA will be fed into the RNAi pathway, which will target messenger RNA that has the same nucleic acid sequence as the double-strand and shut it down — stopping protein formation.”

For example, many viruses produce dsRNA while replicating in the infected cell. “The viruses we work with produce double-stranded RNA as part of their replication machinery,” says Myles. “So it is likely that the RNAi pathway is essential to the mosquito’s innate immune response against viruses. This may explain why some mosquitoes become infected with viruses while others do not.”

“To make a mosquito immune to a particular virus, you can expose it to double-stranded RNA from a small part of the virus. Later, if that virus tries to invade, the mosquito immune system will recognize it and be prepared to destroy it,” says Adelman. “In the natural setting, we suspect that the immune response does not completely eradicate the virus. But the virus may be weakened. We want to enhance that ability so all mosquitoes have the ability to eliminate the virus.”

“We may someday be able to manipulate the RNAi pathway of the mosquito in order to reduce disease transmission. However, first we need to understand the immune pathway and how viruses overcome it in order to establish infections in the mosquito host, ” Myles says.

Putting it together: A green-eyed RNAi sensor

Myles and Adelman have created a novel assay system for the study of virus-vector interactions, mosquito genetics, and virus genetics. In an elegant experiment to test the hypothesis about innate immune response in mosquitoes, they have created a genetically-modified mosquito that serves as an RNAi “sensor” — a sequence that will turn a mosquitoe’s eyes fluorescent green if the RNAi immune machinery stops working.

“We used a TE to place three genes into the mosquito genome. One gene produces a red fluorescent protein that shows up in the mosquito’s eyes and lets you know the TE is present. The second gene produces a green fluorescent protein, also visible in the eyes. The third gene activates RNAi, which destroys the messenger RNA for the green fluorescent protein, and prevents the eyes from glowing green.

“If we knock down a gene important in the functioning of RNAi, it will turn on the green gene so the mosquito’s eyes turn green,” says Adelman.

“The red and green eyes research is a way to discover genes involved in immunity,” says Myles.

“Today it is still too early to tell how well we might be able to manipulate the immune system,” he says. “However, as we learn more about this pathway, we validate the immunity hypothesis. In the process, we may also discover other ways to halt the transmission of disease that do not involve genetic engineering of mosquitoes.

“Some mosquitoes are better than others at transmitting disease, perhaps because they have weaker immune systems. If we can understand the reasons, we may be able to predict which populations are more likely to be involved in epidemic transmission,” says Myles. “One day we may have a rapid test to use in the field to determine if a mosquito population has some degree of immunity that makes it less competent. It is difficult to control mosquito populations. If we knew which ones we had to control, it would be easier to sustain control tactics for longer periods of time.”

Focus on the vector

Adelman and Myles are also members of the Vector-Borne Disease Research Group. Other researchers with the large group study other aspects of mosquito-pathogen interaction, and the development, transmission, and nature of vector-borne diseases.

“The ultimate goal is to interrupt transmission of the disease,” says Tu.

“In the past, research on vector-borne diseases looked at two pieces of a three-piece puzzle,” he says. “Scientists looked at us, as the host to the disease organism, and at the disease organism itself — the parasite in the case of malaria and the viruses in the case of encephalitis, dengue fever, and other diseases.”

The disease-carrier, or vector, was missing.

“The mosquito is not a simple syringe that sucks up infected blood from a host and leaks the infection into its next meal,” says Myles.

When does a mosquito become infected? “They can be born with a virus, but it is more common for them to pick it up with their first blood meal of an infected person,” says Adelman.

But it takes time, at least several days, for the virus to traverse the mosquito midgut, replicate to increase its numbers, move into the mosquito's blood, and finally to the salivary glands. Then the mosquito must bite again in order to pass on the infection, Adelman says.

“The virus must replicate in both a vertebrate and intertebrate host,” says Myles. “Continual transmission of the virus is dependent on the establishment of a persistently infected state in the mosquito.”

“There is a complicated biochemistry in the relationship between the mosquito and the virus or parasite. There is a lot of interesting biology to study in how the mosquito deals with the parasite or virus, and the life cycle of the parasite or virus,” says Tu.

How modern genomic tools help find genes involved in immunity

What’s different in the past 10 years is that the genome is available for study. Thanks to the tools of genomics and proteomics — the merger of biology and computation via high-performance computing, “We are able to look at the genome, at genes and their function, in a systematic way. We can look at interactions within a cell and within an organism. We can compare functions of genes and proteins in different organisms. We can look at how function evolved,” says Tu, who studies gene regulatory networks and the use of such modern scientific systems as comparative genomics and bioinformatics to study mosquito and pathogen biochemistry.

“The strength of the Vector-Borne Disease Research Group is our experience and skill in applying these modern techniques to learn about mosquito-pathogen interaction and to use genetics to reduce disease transmission,” he says.

There may be 200 million years of evolution between the mosquito that carries one pathogen and the mosquito that carries another. Meanwhile, in two strains of the same mosquito, one can be a disease carrier while the other is not.

“Thanks to genomics, we can compare the vast amounts of information in the genomes of different species and discover minute differences — such as a gene that might be a key player in whether a mosquito is a good vector,” says Tu.

The researchers infect mosquitoes with a parasite or virus, compare it to uninfected mosquitoes from the same cohort, such as sisters, and use genomics to observe what genes are turned on in the infected mosquito and not in her sister.

“A lot of people would stop there, but a correlation is not sufficient,” says Tu. “You need to challenge the mosquito — to observe the function of the genes that were activated. To be functional, genes have to produce proteins. It is the proteins that perform. Enzymes, antibodies, and structural elements are all proteins. You want to know what proteins are being produced and what they do.”

In order to discover the role of specific genes that become active in an infected mosquito, scientists have created experiments where they use TEs to over-express a gene by copying it and carrying the copies into the mosquito. And they do other experiments where they knock down a gene using RNAi. The finalists become candidates for tomorrow’s anti-viral gene.

Adelman says that the ultimate goal of his research is “to develop genetic control strategies to supplement the currently available methods of containing and eradicating vector-borne diseases, which include source reduction, vaccination, insecticides, and anti-pathogen drug development.

“But we still know very little about how mosquitoes defend themselves against foreign DNA elements,” says Adelman. “What are the effects of inserting transgenic gene constructs? Will the mosquito recognize and shut down such a transgene over time? And what effect will this have on the potential for genetic control? The answers to these questions are of vital importance to the successful and ethical implementation of a genetic control strategy.”

“Meanwhile, even if a modified mosquito is never deployed, a lot of technology is being developed that allows us to understand the biology of the mosquito. Just a basic understanding of how diseases are transmitted can lead to applications,” Myles says.

A new impetus: West Nile virus in the U.S.

Unfortunately, there is a new impetus for stopping vector- borne disease. In 1999, the first case of West Nile virus was reported in the United States, which has been largely spared from the burden of such arboviral diseases so prevalent in other parts of the world.

Few Americans had heard of West Nile virus before it arrived in the United States. “Now, the spreading epidemic illustrates that arboviral diseases represent an emergent and resurgent threat to even the wealthiest countries,” says Myles.

Contributing factors include complex environmental changes resulting from human activity, changes in public health policy and infrastructure, increasing insecticide resistance, and global demographic, economic, and societal changes, as well as the genetic variation of pathogens, he says.

Available options for addressing the problem are limited, he says. “Vector elimination, previously the most successful strategy for controlling arboviral diseases, is no longer sustainable for a variety of reasons. Therefore, it appears that sustainable control of these important pathogens, and the diseases they cause, depends upon the development and application of novel approaches, likely in combination with more traditional strategies. Novel control strategies will be realized only through increases in our understanding of the biology of disease vectors and the pathogen-vector relationship.”

Tu says, “The research community has had significant breakthroughs — sequencing the genomes of two of the mosquitoes that carry deadly diseases to millions of people and development of research tools like RNAi that allow important discoveries about mosquito-pathogen interaction at the molecular level. And there are the experiments that show that we can make disease-resistant mosquitoes in the laboratory.”

Members of the Vector-Borne Disease Research Group at Virginia Tech are also advancing novel insecticides, environmental controls, disease modeling and information systems, anti-parasite immune responses, and various metabolic and biochemical interferences.

Assistant Professor Kevin Myles works to understand at the molecular level the virus-vector interactions in the mosquito. See photo detail for details about Myles's work and about his students.

As part of the work of the Vector-Borne Disease Research Group at Virginia Tech, entomology undergraduate students Elaine Morazzani and Nicole Plaskon examine an agarose gel to identify DNA molecules.

Entomology Assistant Professor Zach Adelman in his lab. See photo detail for information about Adelman's students.

The mosquito is not a simple syringe that sucks up infected blood from a host and leaks the infection into its next meal.

Associate Professor Zhijian (Jake) Tu. See photo detail for details about Tu's work and about his students.

Undergraduate students Jessica Overcash and Mallory Brangan bloodfeeding cages of transgenic mosquitoes. See photo detail for more information.

The eyes of this genetically altered mosquito serve as a "sensor" of the functional status of part of the mosquito’s immune system. Click on the photo for more detail.

Novel control strategies will only be realized through increases in our understanding of the biology of disease vectors and the pathogen-vector relationship.


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