The Oswaldo Cruz Institute (IOC/Fiocruz) carried out the first complete genome sequencing of yellow fever virus samples from the current outbreak of the disease in Brazil.
Two samples of monkeys from Espírito Santo, which died at the end of February 2017, were investigated. The analysis showed that the microorganisms belong to the genetic subtype known as the South American lineage 1E, which has been predominant in the country since 2008.
However, from the analysis of the complete sequence of the virus genome, it was possible to verify the presence of variations in genetic sequences that are associated with proteins involved in viral replication. There is no previous record of these mutations in the world scientific literature.
The researchers involved in the discovery reinforce that the impacts on public health still need to be investigated and point to the need to evaluate more samples, related to different locations and including cases in humans, monkeys and mosquitoes.
The results of the analyzes were published in the scientific journal 'Memórias do Instituto Oswaldo Cruz'.
The data were communicated by the Presidency of the Fiocroz to the Department of Communicable Disease Surveillance of the Ministry of Health. Additionally, unpublished data point to the same results for the analysis of mosquitoes collected in Espírito Santo and for a monkey that died in the State of Rio de Janeiro.
The study started from a finding that has been gaining more and more space: the current situation of yellow fever in the country has gaps in understanding about its dispersion dynamics.
The outbreak is the most severe in recent decades, and the disease has spread rapidly, with epizootics and human cases diagnosed even in places considered free of the disease for almost 70 years.
The researchers from the Laboratory of Molecular Biology of Flaviviruses and the Laboratory of Mosquitoes Transmitting Hematozoa of the IOC devoted themselves to looking for evidence that could contribute to clarifying an important question: is there anything different about the yellow fever virus that is currently circulating?
"Right now, the commitment of each one of us researchers must be to generate knowledge in their area of expertise and share these discoveries, in an accelerated way, so that we can contribute to filling out a mosaic of evidence that allows us to help explain the scenario current situation", says researcher Myrna Bonaldo, head of the Flavivirus Molecular Biology Laboratory at IOC, who coordinated the study with researcher Ricardo Lourenço, head of the Laboratory of Mosquitoes Transmitting Hematozoa in the IOC. Both are part of the Sylvester Yellow Fever Situation Room created by the Presidency of the Oswaldo Cruz Foundation (Fiocruz).
Led by Myrna Bonaldo and Ricardo Lourenço, at the center, scientists from the Flavivirus Molecular Biology and Hematozoan Transmitting Mosquito Laboratories were responsible for the findings. Photo: Gutemberg Brito
In collaboration with the Department of Health Surveillance of the Ministry of Health (SVS), the Laboratory of Mosquitoes Transmitting Hematozoa has been working to collect samples of primates and mosquitoes in strategic locations to study the risk of transmission and re-emergence of the urban cycle of yellow fever.
It was in this context that, at the end of February 2017, the group collected blood from two howler monkeys (of the species Alouatta clamitans) who fell ill in a forest area in Espírito Santo, confirmed the virus infection and obtained the genetic material for genome sequencing.
"Howler monkeys are especially important in investigations into yellow fever because they are considered 'sentinels': as they are very vulnerable to the virus, they are among the first to die when affected by the disease. In addition, these animals efficiently amplify the virus in their bodies, favoring the infection of mosquitoes that inhabit the forests and the dissemination of sylvatic transmission, in which human beings are accidentally infected. Therefore, their death triggers an alert for the possible presence of the virus in a locality", describes Ricardo, who combines the experiences as a veterinarian and entomologist.
The collections were carried out by Filipe Abreu, a graduate student in Parasitic Biology do IOC, who works on the team led by Ricardo.
"Since yellow fever had not been recorded in the Atlantic Forest for decades, I thought I would not see its consequences in practice. It was a huge learning experience to have the opportunity to visualize and work, in the field, with the object of study of my thesis", the young biologist comments. . [Click here to find out about the activities carried out by the group in Casimiro de Abreu, the municipality where the first death from the disease in the state of Rio de Janeiro was recorded].
The researchers collected blood samples from two howler monkeys that fell ill in Espírito Santo. Animals are considered 'sentinels' for detection of yellow fever because they are very vulnerable to the virus. Photo: Disclosure
After extracting the genetic material (RNA) from the samples, the complete genome sequencing process was carried out, an activity that was supported by the Technological Platform for DNA Sequencing at the IOC. The analyzes point to three main pieces of evidence.
As the first evidence, 100% identity was observed between the genetic sequences of the viruses present in the animals - that is: the viruses had identical genetic sequences.
The second evidence was the verification of the presence of modifications in the genetic code of the viruses. These mutations were identified when the complete genetic sequence obtained was compared to the complete genetic sequence of viruses related to outbreaks that occurred since the 1980s in Brazil and Venezuela, a country where the South American 1E lineage is also predominant.
For the comparison, international databases dedicated to the deposit of genetic sequences were used.
"The changes we observed are unprecedented, they are not described in previous findings", Myrna details.
The third piece of evidence was obtained in the analysis of viral proteins, in a step following the verification of changes in the genetic sequence.
"In a very simplified way, the genome is a code that has the role of guiding the production of proteins. These proteins are the basis of the very structure of the virus, forming its constituent elements, such as the walls of the virus, for example. We can compare the genome to a script: the virus has a repertoire of proteins that are manufactured from information in the genome. Some genetic changes do not impact the proteins of the virus. Therefore, it is important to observe whether genetic variations could modify the repertoire of proteins manufactured" , describes the molecular virologist, who specializes in flaviviruses, the dengue group, Zika and yellow fever.
Alterations were detected after sequencing the complete genome and comparing it with microorganisms related to outbreaks that occurred since the 1980s in Brazil and Venezuela. Photo: Joshua Damacena
It was identified that the changes in the genome were related to eight substitutions of amino acids (the molecules that make up proteins). Seven of these substitutions occurred in the two most important proteins for viral replication, known as NS3 and NS5.
It is the process of virus replication - multiplication by producing copies of itself - which ensures that the microorganism causes the disease. In addition to the impact on proteins related to viral replication, a modification was also observed in protein C, which forms the capsid (envelope that protects the genetic material inside the virus).
Myrna Bonaldo points out that the biological and epidemiological implications of the finding depend on other studies and that more data are needed to clarify the possible role of the genetic alterations detected in the context of the current outbreak of the disease.
"We have evidence that constitutes a new element, something that had not been observed before. However, we still don't know what the impacts of these mutations are. countries that are dedicated to the subject of yellow fever can consider this aspect in their analyses. Science is done in a collaborative way, with results that add up", he evaluates.
The researchers emphasize that genome sequencing of more pathogens circulating in the current outbreak, both in human cases and in infected mosquitoes and monkeys, is critical to complement the evidence obtained in the research.
"This is an initial result. We cannot generalize, as we still don't know if this virus is predominant in the current outbreak", says Ricardo Lourenço.
Considering that there is a limited number of complete genetic sequences of yellow fever viruses from the Americas deposited in international genome banks, the hypothesis that this genetic alteration is older is not ruled out, and that the virus with this characteristic has been circulating for a longer time. and has not been identified before.
According to the researchers, the genetic alterations detected do not affect the virus envelope proteins, which are central to the vaccine's functioning. Photo: Joshua Damacena
Bearing in mind that changes in the composition of proteins important for viral replication were verified, the researchers consider that it is possible that there is a selective advantage, reflected in the capacity for infection and dissemination of the virus.
However, further research is critical to determine whether these genome modifications are specific to the microorganisms involved in the current outbreak.
"Right now, we are looking for yellow fever virus genome samples from different hosts - including humans, monkeys and mosquitoes - and from diverse geographic origins, especially in Southeast Brazil, where the epidemic has been more intense to better understand this phenomenon", informs Ricardo.
Regarding a possible impact on the available vaccine, the researchers explain that the vaccine currently adopted protects against different genotypes of the virus, including South American and African. Furthermore, the changes detected in the study do not affect the virus's envelope proteins, which are central to the vaccine's functioning.
They point out that the complete genetic sequences of the viruses analyzed in the study have already been published in GenBank, so that they are available for comparisons that can be carried out by other scientists from Brazil and the world.
The Oswaldo Cruz Institute (IOC/Fiocruz) carried out the first complete genome sequencing of yellow fever virus samples from the current outbreak of the disease in Brazil.
Two samples of monkeys from Espírito Santo, which died at the end of February 2017, were investigated. The analysis showed that the microorganisms belong to the genetic subtype known as the South American lineage 1E, which has been predominant in the country since 2008.
However, from the analysis of the complete sequence of the virus genome, it was possible to verify the presence of variations in genetic sequences that are associated with proteins involved in viral replication. There is no previous record of these mutations in the world scientific literature.
The researchers involved in the discovery reinforce that the impacts on public health still need to be investigated and point to the need to evaluate more samples, related to different locations and including cases in humans, monkeys and mosquitoes.
The results of the analyzes were published in the scientific journal 'Memórias do Instituto Oswaldo Cruz'.
The data were communicated by the Presidency of the Fiocroz to the Department of Communicable Disease Surveillance of the Ministry of Health. Additionally, unpublished data point to the same results for the analysis of mosquitoes collected in Espírito Santo and for a monkey that died in the State of Rio de Janeiro.
The study started from a finding that has been gaining more and more space: the current situation of yellow fever in the country has gaps in understanding about its dispersion dynamics.
The outbreak is the most severe in recent decades, and the disease has spread rapidly, with epizootics and human cases diagnosed even in places considered free of the disease for almost 70 years.
The researchers from the Laboratory of Molecular Biology of Flaviviruses and the Laboratory of Mosquitoes Transmitting Hematozoa of the IOC devoted themselves to looking for evidence that could contribute to clarifying an important question: is there anything different about the yellow fever virus that is currently circulating?
"Right now, the commitment of each one of us researchers must be to generate knowledge in their area of expertise and share these discoveries, in an accelerated way, so that we can contribute to filling out a mosaic of evidence that allows us to help explain the scenario current situation", says researcher Myrna Bonaldo, head of the Flavivirus Molecular Biology Laboratory at IOC, who coordinated the study with researcher Ricardo Lourenço, head of the Laboratory of Mosquitoes Transmitting Hematozoa in the IOC. Both are part of the Sylvester Yellow Fever Situation Room created by the Presidency of the Oswaldo Cruz Foundation (Fiocruz).
Led by Myrna Bonaldo and Ricardo Lourenço, at the center, scientists from the Flavivirus Molecular Biology and Hematozoan Transmitting Mosquito Laboratories were responsible for the findings. Photo: Gutemberg Brito
In collaboration with the Department of Health Surveillance of the Ministry of Health (SVS), the Laboratory of Mosquitoes Transmitting Hematozoa has been working to collect samples of primates and mosquitoes in strategic locations to study the risk of transmission and re-emergence of the urban cycle of yellow fever.
It was in this context that, at the end of February 2017, the group collected blood from two howler monkeys (of the species Alouatta clamitans) who fell ill in a forest area in Espírito Santo, confirmed the virus infection and obtained the genetic material for genome sequencing.
"Howler monkeys are especially important in investigations into yellow fever because they are considered 'sentinels': as they are very vulnerable to the virus, they are among the first to die when affected by the disease. In addition, these animals efficiently amplify the virus in their bodies, favoring the infection of mosquitoes that inhabit the forests and the dissemination of sylvatic transmission, in which human beings are accidentally infected. Therefore, their death triggers an alert for the possible presence of the virus in a locality", describes Ricardo, who combines the experiences as a veterinarian and entomologist.
The collections were carried out by Filipe Abreu, a graduate student in Parasitic Biology do IOC, who works on the team led by Ricardo.
"Since yellow fever had not been recorded in the Atlantic Forest for decades, I thought I would not see its consequences in practice. It was a huge learning experience to have the opportunity to visualize and work, in the field, with the object of study of my thesis", the young biologist comments. . [Click here to find out about the activities carried out by the group in Casimiro de Abreu, the municipality where the first death from the disease in the state of Rio de Janeiro was recorded].
The researchers collected blood samples from two howler monkeys that fell ill in Espírito Santo. Animals are considered 'sentinels' for detection of yellow fever because they are very vulnerable to the virus. Photo: Disclosure
After extracting the genetic material (RNA) from the samples, the complete genome sequencing process was carried out, an activity that was supported by the Technological Platform for DNA Sequencing at the IOC. The analyzes point to three main pieces of evidence.
As the first evidence, 100% identity was observed between the genetic sequences of the viruses present in the animals - that is: the viruses had identical genetic sequences.
The second evidence was the verification of the presence of modifications in the genetic code of the viruses. These mutations were identified when the complete genetic sequence obtained was compared to the complete genetic sequence of viruses related to outbreaks that occurred since the 1980s in Brazil and Venezuela, a country where the South American 1E lineage is also predominant.
For the comparison, international databases dedicated to the deposit of genetic sequences were used.
"The changes we observed are unprecedented, they are not described in previous findings", Myrna details.
The third piece of evidence was obtained in the analysis of viral proteins, in a step following the verification of changes in the genetic sequence.
"In a very simplified way, the genome is a code that has the role of guiding the production of proteins. These proteins are the basis of the very structure of the virus, forming its constituent elements, such as the walls of the virus, for example. We can compare the genome to a script: the virus has a repertoire of proteins that are manufactured from information in the genome. Some genetic changes do not impact the proteins of the virus. Therefore, it is important to observe whether genetic variations could modify the repertoire of proteins manufactured" , describes the molecular virologist, who specializes in flaviviruses, the dengue group, Zika and yellow fever.
Alterations were detected after sequencing the complete genome and comparing it with microorganisms related to outbreaks that occurred since the 1980s in Brazil and Venezuela. Photo: Joshua Damacena
It was identified that the changes in the genome were related to eight substitutions of amino acids (the molecules that make up proteins). Seven of these substitutions occurred in the two most important proteins for viral replication, known as NS3 and NS5.
It is the process of virus replication - multiplication by producing copies of itself - which ensures that the microorganism causes the disease. In addition to the impact on proteins related to viral replication, a modification was also observed in protein C, which forms the capsid (envelope that protects the genetic material inside the virus).
Myrna Bonaldo points out that the biological and epidemiological implications of the finding depend on other studies and that more data are needed to clarify the possible role of the genetic alterations detected in the context of the current outbreak of the disease.
"We have evidence that constitutes a new element, something that had not been observed before. However, we still don't know what the impacts of these mutations are. countries that are dedicated to the subject of yellow fever can consider this aspect in their analyses. Science is done in a collaborative way, with results that add up", he evaluates.
The researchers emphasize that genome sequencing of more pathogens circulating in the current outbreak, both in human cases and in infected mosquitoes and monkeys, is critical to complement the evidence obtained in the research.
"This is an initial result. We cannot generalize, as we still don't know if this virus is predominant in the current outbreak", says Ricardo Lourenço.
Considering that there is a limited number of complete genetic sequences of yellow fever viruses from the Americas deposited in international genome banks, the hypothesis that this genetic alteration is older is not ruled out, and that the virus with this characteristic has been circulating for a longer time. and has not been identified before.
According to the researchers, the genetic alterations detected do not affect the virus envelope proteins, which are central to the vaccine's functioning. Photo: Joshua Damacena
Bearing in mind that changes in the composition of proteins important for viral replication were verified, the researchers consider that it is possible that there is a selective advantage, reflected in the capacity for infection and dissemination of the virus.
However, further research is critical to determine whether these genome modifications are specific to the microorganisms involved in the current outbreak.
"Right now, we are looking for yellow fever virus genome samples from different hosts - including humans, monkeys and mosquitoes - and from diverse geographic origins, especially in Southeast Brazil, where the epidemic has been more intense to better understand this phenomenon", informs Ricardo.
Regarding a possible impact on the available vaccine, the researchers explain that the vaccine currently adopted protects against different genotypes of the virus, including South American and African. Furthermore, the changes detected in the study do not affect the virus's envelope proteins, which are central to the vaccine's functioning.
They point out that the complete genetic sequences of the viruses analyzed in the study have already been published in GenBank, so that they are available for comparisons that can be carried out by other scientists from Brazil and the world.
The non-profit reproduction of the text is allowed as long as the source is cited (Comunicação / Instituto Oswaldo Cruz)
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