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Why Don’t Mozzies Get a Fever?

mosquito

Viral diseases transmitted by mosquito bite are a major public health problem worldwide.

By Prasad Paradkar, Jean-Bernard Duchemin & Peter Walker

Understanding how mosquitoes protect themselves from the viruses they carry could lead to new ways of controlling the spread of viral diseases like dengue or yellow fever.

Why are some mosquitoes so effective in transmitting disease-causing viruses such as dengue, yellow fever and West Nile? And why do other mosquitoes transmit viruses poorly? Why do viruses that cause devastating diseases in humans and animals cause no disease in the mosquitoes that carry them?

There are lots of unanswered questions in mosquito-borne viral disease transmission, and many of the answers may lie in understanding the mosquito’s own defensive response. In a recent paper published in the Proceedings of National Academy of Sciences USA, we have reported that viral infection in mosquitoes produces a secreted protein called Vago that can alert other mosquito cells to defend against the invading virus. This finding provides an understanding of how mosquitoes respond to viral infections and may provide insights into the processes controlling the efficiency of virus transmission.

Viral diseases transmitted by mosquito bite are a major public health problem worldwide. The World Health Organization (WHO) estimates that between 50 and 100 million dengue virus infections occur annually, causing more than 20,000 deaths – mostly in children. Yellow fever is estimated to affect 200,000 people each year in Africa and Latin America, causing more than 30,000 deaths. Mosquito-borne inflammatory conditions of the brain, caused by viruses such as West Nile and Japanese encephalitis, are an increasing public health problem in Africa, Europe, North America and Asia, while Ross River and chikungunya viruses cause a debilitating arthritis that has affected many thousands of people from Africa through South Asia to Australia and the Pacific Islands.

Many of these diseases were originally localised in the warmer tropics. Through increased travel, trade and global warming, however, they are on the move, spreading geographically to new regions and adapting to infect new mosquito species. Growing urbanisation in combination with poor sanitation have also led to increased risk of viral epidemics.

The dynamics of mosquito-borne disease are complex. They involve a multitude of factors associated with the mosquito transmitter or vector, the virus itself, the vertebrate host (human and other) and the environment.

For efficient transmission, the virus must multiply through alternate cycles in the vertebrate host and a blood-feeding mosquito. When a mosquito bites an infected person or animal, the virus is taken along with the blood meal into the gut and passes through intestinal cells into haemolymph, the circulatory fluid of mosquito. The virus then travels to the salivary gland where it multiplies, and is transmitted to a new host via saliva during a subsequent mosquito bite.

Interestingly, different mosquito species are responsible for the transmission of different viruses. Viruses such as dengue and chikungunya are transmitted by Aedes mosquito species, whilst viruses such as West Nile and Japanese encephalitis are transmitted by Culex mosquitoes.

Different species of mosquito, and even different subpopulations, transmit viruses with different efficiencies. The reasons for these differences, also known as vector competence, remain largely unknown. Variations in mosquito genetics and behaviour are likely to play a role, and there may also be environmental influences. How mosquitoes respond to and defend against viral infection may also be a key factor in understanding vector competence.

The classical immune response to infection in vertebrates, such as birds, fish and mammals, consists of two parts. Invading pathogens first encounter the innate immune system, which comprises cells and proteins that defend against them in a largely non-specific manner. This response occurs in all classes of animals, including insects and other primitive organisms.

The adaptive or acquired immune system consists of highly specialised cells and soluble factors that can recognise specific pathogens and process them for elimination. This system, which occurs only in vertebrate animals, comprises antibodies, B and T lymphocytes, and a complex array of soluble proteins called cytokines that provide not only exquisite specificity but also allow the organism to retain a memory of an initial exposure to a pathogen, ensuring long-term defence against any subsequent infection.

Some of the most important fundamental studies of immune-related genes have been performed using the fruit fly, Drosophila melanogaster. Due to their ease of genetic manipulation and high reproductive frequency, fruit flies have provided a robust genetic model to screen for genes involved in immune recognition and response.

Until recently, however, immunity in flies was studied primarily using bacteria, parasites and other large complex pathogens. So, despite the importance of mosquitoes as vectors of many human and animal viral diseases, virtually nothing was known about the defensive response of insects to viruses.

Indeed, for many years it had been known that insects lacked key components of the vertebrate antiviral immune response, such as antibodies and proteins like interferon, so it had been assumed that they probably lacked effective antiviral immunity. This, it seems, is far from correct.

During the past 10 years or so, a series of discoveries has revealed that fruit flies do indeed have antiviral immunity in the form of RNA interference (RNAi). RNAi is an intracellular mechanism that allows the recognition and degradation of foreign RNA – genetic material akin to DNA but built on a slightly different molecular backbone. While the genome of most organisms is comprised of DNA, many viruses have a single-stranded RNA genome that is converted into a double-stranded (ds) form during replication.

Research originally conducted in fruit flies showed that viral dsRNA is recognised as foreign by insect cells, activating the RNAi pathway and leading to the eventual slicing up of the invading RNA by a protein called Dicer-2. It has now been confirmed that this sort of immunity is involved in the natural defence of mosquitoes against a range of viruses including important pathogens such as O’nyong-nyong, dengue, West Nile and vesicular stomatitis viruses. The role of RNAi in the natural antiviral defensive response of mammalian cells is still a matter of debate.

Following the demonstration that RNAi plays a key role in the invertebrate antiviral immunity, attention turned to the involvement of other genes. French researchers in Strasbourg reported that viral infection in fruit flies leads to the activation of an antiviral protein called Vago. Intriguingly, activation of Vago occurs via Dicer-2, the protein involved in breaking down foreign RNA. Mutant flies that are deficient in Dicer-2 are unable to mount a Vago response, but Vago activation is not dependent on any other known components of the RNAi pathway. This significant discovery suggests the existence of a novel mechanism of immune signalling in insects.

Based on this research, we asked whether a similar antiviral mechanism occurs in mosquitoes and, if so, what the function of Vago might be.

By infecting a cell line derived from Culex mosquitoes with West Nile virus, we demonstrated that Dicer-2 recognises the virus’ genome, leading to activation of Vago. We also showed that Vago is secreted from the infected cells and binds to neighbouring uninfected cells, alerting them to the presence of the invading virus.

Further investigations revealed that binding of Vago to un­infected mosquito cells activates a range of antiviral genes, preventing viral transmission. Some of these results were then confirmed in live mosquitoes using CSIRO’s biosecure insectary, where mosquitoes can be safely housed for virus infection studies.

This research suggests that mosquitoes (and other insects) have effective and complex antiviral defences. Many unanswered questions remain, and have opened up new avenues of research. For instance, the receptor to which Vago binds on the surface of uninfected mosquito cells is unknown. We are also yet to identify the antiviral genes that are activated by Vago. What factors do these genes encode and how do they block the virus?

We believe that, in order to transmit effectively, viruses must have evolved their own mechanisms to overcome the antiviral action of Vago. Viral strategies to overcome the action of interferon in mammalian cells occur commonly, so it is likely that viruses have also learned to disarm the Vago response. These areas of research are currently being pursued and are expected to shed more light on the interaction between viruses and their mosquito carriers.

There is another important question we should ask. Is it possible to exploit or enhance the antiviral activity of Vago (or the proteins it induces) to boost mosquito resistance to infection? Currently, there is no effective treatment for many of these deadly mosquito-borne viruses and, apart from yellow fever virus, there are no effective vaccines. If we could breed mosquitoes with heightened levels of natural resistance to infection, perhaps we could prevent or restrict the transmission of disease.

These are challenging problems and it is still very early days, but the previous work of others has shown that, using appropriate approaches, it is possible to generate a mosquito population that is less efficient in disease transmission. As this technology advances, such interventions may well become feasible.

Australia‘s proximity to South-East Asia, increasing international travel and trade, and environmental instability associated with climate change present particular risks of mosquito-borne viruses entering our borders. Dengue virus is introduced to Australia regularly by travellers but epidemics to date have been relatively rare and restricted by effective containment. As we have seen in other countries, there is clearly potential for diseases such as dengue, Japanese encephalitis and chikungunya to establish in our tropical North and spread over much of the continent.

We believe our research will not only reduce the risk of disease for millions of people worldwide but also better prepare Australia to deal with future biosecurity threats.

Prasad Paradkar is an Australian Research Council DECRA Fellow, Jean-Bernard Duchemin a Senior Research Scientist and Peter Walker Chief Research Scientist at the CSIRO Australian Animal Health Laboratory.