Rapid processing of viral genetic material from the first confirmed case in Brazil was possible thanks to the adaptation of the existing technique
Researchers at the Joint British-Brazilian Center for Arbovirus Detection, Diagnosis, Genomics and Epidemiology (CADDE) have concluded only 18 hours complete monkey virus virus genome sequencing (MPXV) isolated from the first patient diagnosed with monkeypox confirmed in Brazil.
High speed in the processing of viral genetic material was possible thanks to the adaptation of the virus by the technique of rapid metagenomy developed during the doctorate of researcher Ingra Morales Claro, associate at the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).
The work was coordinated by Ester Sabino, a professor at the University of Sao Paulo (USP), who was also in charge of the first sequencing of SARS-CoV-2, the virus that causes Covid-19, in the country, in March 2020. appeared in Manaus about a year later.
The team released the results Thursday (9) on the Virology Platform, a page where scientists around the world share real-time information on the causes of disease.
“We received a sample from a patient admitted to Emílio Ribas Hospital on Tuesday (7) at 4pm and, at 10am the next morning, the genome of the virus, which has nearly 200,000 base pairs. [bem mais que as 30 mil do SARS-CoV-2], was sequenced and analyzed. The methodology we have developed is on average 45% faster than conventional metagenomic techniques. And the price is also lower, reaching $ 30 per sample, ”says Ingra.
According to Esther, scientists often resort to this type of analysis when they need to identify a new emerging virus, as was the case with SARS-CoV-2 in 2019, or to detect an already known virus in patient samples without having the reagents in hand. , as is now the case with the monkeypox virus.
Performing molecular diagnostic tests (RT PCR) requires called “primers” (primers), which are sequences complementary to viral sequences that initiate the replication of genetic material. After processing, the result must be compared to negative and positive “controls”. RT PCR is considered the gold standard for diagnosing Covid-19 and several other diseases.
“When an epidemic starts with a new infectious agent, one of the main bottlenecks in diagnosing cases is lack of specific primers and positive controls. This technique can be useful in such situations, because it enables the identification of as yet unknown pathogens for which there are no reagents “, explains Esther.
The earlier the “index” case is detected, the first case, the higher the probability that it contains a developing virus, adds Ingra.
In the case of metagenomy, random primers (not specific for a particular virus or bacterium) are used, which allow sequencing of all genetic material contained in the biological sample, including the host (in this case human) and other agents that cause diseases that it eventually brings.
This information is then analyzed using bioinformatics techniques and compared with a panel of references. “Exactly as it was done with MPXV. The obtained data were mapped to a virus sequence that is already available for studies. And that allowed us to prove that they were monkey goddesses, ”says Ingra.
shortening of roads
The official confirmation of the first Brazilian case of monkeypox was given on Thursday (9) by the Instituto Adolfo Lutz, in Sao Paulo.
A reference laboratory in Sao Paulo performed a metagenomic analysis on a platform known as Illumina, one of the technologies used to detect monkeypox viruses in European and North American centers and which is considered the gold standard. Sequencing with this method takes an average of 48 hours.
A research group at the Joint Center of Brazil and the United Kingdom used a portable sequencer known as MinION, from Oxford Nanopore Technologies, and adapted the protocol used to sequence Zika and SARS-CoV-2 viruses, making it faster.
“One of The advantage of this new protocol is the reduction of time preparation of the sample for sequencing, which lasts from 14 hours to 5:40 minutes “, reports Ingra.
As the error rate is slightly higher than that on the Illumina platform, the team sought to generate up to 300 redundant readings for each region of the viral genome. “When we cover the same region several times and find the same result, we can be sure that it is not a misreading,” says the researcher.
genetic characteristics
The next step was to assemble a phylogenetic tree of the monkeypox virus isolated in Brazil. For this, the scientists compared the sequence obtained at the USP with another 102 published this year by experts from countries such as Belgium, Portugal, the United Kingdom, Germany, Spain and the United States. The aim was to assess the degree of similarity between the sequences, which gives indications of how the virus could evolve.
“We have taken over all the complete genomes sequenced in 2022 [até 09/06], align the sequences and place the phylogenetic tree. We have seen that the MPXV discovered here fits into a big log [grupo], as well as viruses sequenced in Europe and the United States. Compared to the CDC reference genome [o Centro de Controle de Doenças norte-americano]updated in May, we detected only three mutations, ”says Ingra.
By comparison, the first MPXV genome sequenced in 2022 showed 47 mutations compared to the last case described so far (2018 in Africa).
“What these mutations represent and whether they have contributed to the increase in the number of cases is something that is still being studied by other research groups.” We here at CADDE will look out for the following cases. The idea is to continue sequencing to monitor the evolution of the virus, ”reveals Ingra.
Although known to cause monkeypox, MPXV is a virus that mainly infects rodents in Africa. The pathogen is part of the Orthopoxvirus family, as well as the smallpox virus in humans, eradicated in 1980.
The disease usually begins with fever, fatigue, headache, muscle aches, or nonspecific symptoms of colds or flu. A few days after the onset of fever, skin lesions appear that contain a high viral load.
It is spread by direct contact with lesions or through clothing, bedding and towels used by someone with skin wounds. It can also be caused by coughing or sneezing of infected people.
Until the beginning of this year, the infection was common only in Central Africa. But new cases have already been detected in 33 countries, most without a previous medical history.
Source: CNN Brazil; Karina Toledo – FAPESP Agency