Researchers from the United Kingdom-Brazil Center for the Detection, Diagnosis, Genomics and Epidemiology (CADDE) completed complete sequencing of the monkeypox virus (MPXV) genome in just 18 hours, isolated from the first patient diagnosed with monkeypox in Brazil. .
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This feat was made possible by the adaptation to MPXV of a rapid metagenomics technique developed during the FAPESP Fellow’s Ingra Morales Claro PhD. The work was coordinated by Esther Sabino, a professor at the University of São Paulo (USP), who was also responsible for the first sequencing of SARS-CoV-2 in the country in March 2020 and for the first cases of the new gamma variant that appeared in Manaus around a year later.
The CADDE team published the results yesterday (06/09) on virological.org, a website where virologists from around the world share information about pathogens of interest in real time.
“We received a sample from a patient admitted to Emilio Ribas Hospital at 4 pm on Tuesday [07/06] and at 10 a.m. the next morning the genome of the virus, which is 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, “Claro told Agência FAPESP.
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As Sabino explains, scientists often resort to metagenomic analysis when they have to identify a new emerging virus (as was the case with SARS-CoV-2 in 2019) or detect an already known virus in patient samples without the necessary specific reagents on hand. (as is now the case with MPXV).
This is because the RT-PCR test, the gold standard for diagnosing Covid-19 and several other diseases, requires so-called primers, which are nucleotide sequences complementary to viral sequences that initiate the replication of genetic material. The result should then be compared with negative and positive controls.
“When the epidemic starts with a new infectious agent, one of the main obstacles to diagnosing cases is the lack of specific primers and positive controls. This technique can be useful in these situations, as it allows the identification of pathogens that are not yet known, for which there are no reagents, “explains Sabino.
And the earlier the “index” case is detected (the first case), the more likely it is to contain a new virus, Claro added.
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In the case of metagenomics, random primers (non-specific for a particular virus or bacterium) are used, which make it possible to sequence all genetic material contained in a biological sample, including that of the host (human, in this case) and other pathogens that ultimately account contains. This information is then analyzed using bioinformation techniques and compared with a reference panel.
“Just like it was done with MPXV. The data obtained were mapped in sequence from the virus, which is already available for research. And that allowed us to prove that it’s monkeypox, “says Claro.
shortening of roads
The official confirmation of the first Brazilian case of monkeypox was made yesterday (09/06) by the Instituto Adolfo Lutz. The Sao Paulo Reference Laboratory conducted a metagenomic analysis on a platform known as Illumina, one of the technologies used to detect MPXV in European and North American centers and considered the gold standard. The sequence of this method takes an average of 48 hours.
The CADDE Group uses a portable sequencer, known as MinION, from Oxford Nanopore Technologies, and has made adaptations to the protocol used to sequence Zika virus (since 2015) and SARS-CoV-2 (since 2020), making it the most the fastest.
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“One of the advantages of this new protocol is the reduction of sample preparation time for sequencing, which takes from 14 hours to 5:40 minutes,” says Claro.
Because the error rate is slightly higher than that of the Illumina platform, the CADDE team tried 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 this is not a misreading,” said the researcher.
The next step was to collect the MPXV phylogenetic tree isolated in Brazil. Therefore, the CADDE team compares the sequence obtained in the USP with another 102 released this year by scientists from countries such as Belgium, Portugal, the United Kingdom, Germany, Spain and the United States. The aim was to measure the degree of similarity between the sequences, which gives evidence of evolutionary relationships.
“We downloaded all the complete genomes sequenced in 2022 [até 09/06], align the sequences and adjust the phylogenetic tree. We saw that the MPXV detected here fits into a large log [grupo], the same as viruses sequenced in Europe and the United States. Compared to the reference genome of CDC [o Centro de Controle de Doenças norte-americano]updated in May, we observed only three mutations, ”says Claro.
By comparison, the first genome of MPXV, sequenced in 2022, shows 47 mutations compared to the last case described so far (in 2018, in Africa).
“What these mutations are 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 monitor the following cases. The idea is to continue sequencing to monitor the evolution of the virus, “said Claro.
Although known to cause smallpox or smallpox, MPXV is a virus that mainly infects rodents in Africa. The pathogen is part of the Orthopoxvirus family, the same as the human measles virus that was eradicated in 1980.
The disease usually begins with fever, fatigue, headache, muscle aches, ie non-specific, colds or flu-like symptoms. A few days after the onset of fever, skin lesions appear that contain a high viral load. Spread occurs through direct contact with wounds or clothing, sheets, and towels used by someone with skin lesions. It can also occur when infected people cough or sneeze.
– Revista Galileu