Dengue virus is more stable than originally thought

In a study published in PLoS One, Brazilian scientists present the results of the complete genome sequencing of viral particles found in patient samples.

By Karina Toledo

Agência FAPESP – In an article published this past August in PLoS One, researchers from Universidade de São Paulo (USP) described how the dengue virus (DENV) undergoes mutations at a much slower pace than originally imagined, increasing the chances of finding an effective vaccine against this virus.

The scientists reached this conclusion by sequencing the complete genome of thousands of viral particles that were found in ten blood samples from patients who were diagnosed during the epidemic that hit the Santos Basin in 2010. During the outbreak, the area registered 33,000 cases of type-2 dengue, although specialists estimate that the actual number of infected people was at least five-times higher.

Camila Malta Romano, a researcher at USP’s São Paulo Tropical Medicine Institute and author of the FAPESP-funded study, explained that the genetic variability of the virus found in a single individual (intra-host variability) was approximately 0.002%, which was lower than the rates that were reported in previous studies.

“The earlier studies used much more expensive and laborious traditional sequencing methods. For this reason, only a certain region of the genome was analyzed and not all particles were shown. In our study, thanks to new large scale sequencing techniques, we practically generated a complete sequence for each particle of virus found in the sample, which gives greater depth to the analysis,” she explained.

Romano also added that because the samples were collected at different times throughout the epidemic, from February through June, the scientists were able to observe that the virus remained essentially stable throughout the entire outbreak.

“I believed that the genetic variability of DENV was much higher because it is an RNA virus, much like HIV (which causes Aids) and HCV (which causes hepatitis C). Unlike DNA viruses, which use the cellular machinery of the host to replicate, RNA viruses replicate on their own. This means there are no mechanisms to correct errors in the process, and because of this, at least one mutation is expected in each progeny,” explained Romano.

One of the scientists’ hypotheses explaining the lower genetic variability of DENV in relation to other RNA viruses is based on the fact that it must swap between two very different hosts, i.e., mosquitoes and humans, to complete its transmission cycle.

“From an evolutionary standpoint, this alternation between a vertebrate and invertebrate exerts very strong pressure on DENV not to change very much. If it accumulates many mutations, it could lose the adaptation that allows it to replicate both in humans and mosquitoes. This type of evolutionary mechanism has already been proven in the virus that causes yellow fever,” said Romano.

Another possible explanation for the greater stability of DENV is related to the fact that the dengue virus causes an acute infection that lasts between 5 and 10 days.

“The organism does not have time to raise a specific immune response against the virus. It is different from HIV, for example, which causes a chronic disease, is in a constant battle with the immune system, and needs to modify itself continuously to sidestep the organism’s defenses. In the case of DENV, this pressure is selective and lower,” affirmed Romano.

Implications

According to the infectious diseases specialist and immunologist Esper Georges Kallás, who is a professor at the Immunology and Allergy Clinic at USP’S Medical School (FMUSP) and co-author of the study, the detailed information that the researchers obtained about the DENV genome can help to identify the regions of the virus that are capable of activating an immune response, which will pave the way for the development of a vaccine.

“As the dengue virus is less diverse than originally imagined, the chances of an immunization working increase. Although we have studied only serotype 2, we have a strong suspicion that this low variability occurs in other serotypes as well,” affirmed Kallás.

The major challenge, according to Kallás, is finding the correct combination of antigens that is capable of inducing an efficient immune response against the four serotypes simultaneously.

“We know that the worst manifestations of the disease are associated with a second infection. If you make a vaccine that only partially protects [individuals], it could be like shooting oneself in the foot. If an immunized person is infected by a virus serotype that the vaccine is not effective against, he or she will run a greater risk of developing the hemorrhagic type of dengue fever than will an unvaccinated individual who has never had dengue,” explained Kallás.

Analysis of the samples that were collected during the epidemic in the Santos Basin also resulted in another article that was published in PLoS One, in 2010. On that occasion, the researchers showed that the outbreak on the São Paulo coast was caused by a serotype 2 strain of the virus that originated from the Caribbean.

Through phylogenetic analysis, the group also revealed that the introduction of this strain into São Paulo State occurred well before the epidemic, at some time between 2003 and 2005.

“This result serves as a warning for the need to increase epidemiological vigilance. The virus enters a certain region and could spend some time unnoticed before exploding and causing an epidemic,” evaluated Romano.

Currently, Romano is coordinating another research project that is funded by FAPESP and has the objective of describing how adaptive mutations of DENV occur.

“The virus is adapting, and in many regions, it can infect other mosquitoes of the Aedes genus, in addition to A. aegypti. We want to understand how this happens because we have shown that the virus changes very little. We want to understand what can and cannot change when it jumps from one host to another,” she explained.

To reach this objective, researchers are infecting mosquitoes of the Aedes genus, and as they did with samples of human blood, they are analyzing the genomes of the thousands of viral fractions that are found in the insects. Furthermore, the group has initiated a new research line that has the objective of developing an efficient anti-viral drug against DENV.

The article Inter- and Intra-Host Viral Diversity in a Large Seasonal DENV2 Outbreak (doi: 10.1371/journal.pone.0070318) can be read at www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0070318#abstract0.

The article Characterization of Dengue Virus Type 2: New Insights on the 2010 Brazilian Epidemic (doi: 10.1371/journal.pone.0011811) can be read at www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0011811.