Brazilians sequence the genome of the monkeypox virus in 18 hours – Revista Galileu

Monkeypox virus genome sequencing technique in 18 hours (Photo: CADDE / outreach)

Researchers at the United Kingdom-Brazil Center for the Discovery, Diagnosis, Genomics and Epidemiology of Drugs (CADDE) completed the 18-hour complete sequencing of the genus of the monkey virus (MPXV) in the first patient to confirm the diagnosis of the monkey in Brazil. .

This feat was made possible by the adaptation of the rapid metagenomics technique developed in the PhD by Ingra Morales Clara of FAPESP Fellow to MPXV. The work was coordinated by Professor Ester Sabino of 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 the first cases of the new gamma variant. He was born in Manaus about a year later.

The CADDE team released the results yesterday (09/06) on virological.org, a website where virologists from around the world share information about pathogens of interest in real time.

“We received a sample of a patient admitted to Emílio Ribas Hospital on Tuesday at 4pm [07/06] and at 10 a.m. the genome of the virus, which contains nearly 200 thousand 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 metagenomics techniques. And the cost is even lower, reaching $ 30 per sample, ”Claro told Agência FAPESP.

Sabino explained that scientists often resort to metagenomic analysis when they need to identify a new virus that is emerging (as was the case with SARS-CoV-2 in 2019) or when they need to detect an already known virus in patient samples without the necessary specific reagents available. (as is now the case with MPXV).

That is, the gold standard for diagnosing RT-PCR, Covid-19 and many other diseases, which requires so-called primers, are nucleotide sequences that complement the viral sequence that triggers the replication of genetic material. And the result must be compared with negative and positive controls.

“When an epidemic starts with a new infectious agent, one of the main bottlenecks in diagnosing cases is the lack of specific triggers and positive controls. This technique can be useful in these situations, as it allows the identification of pathogens that are still unknown to those who do not have reagents, ”explained Sabino.

And the earlier the “index” case is detected (the first case), the greater the likelihood of developing a virus that is emerging, Claro added.

In the case of metagenomics, random raw materials are used (not specific to a particular virus or bacterium), which allow all genetic material in a biological sample to be sequenced, including that of the host (human, in this case) and other pathogens that it eventually adopts. this is analyzed using bioinformatics techniques and compared to a reference panel.

“Just like with MPXV. The data obtained were mapped to a sequence of viruses available for research. And we proved that he was a monkey, ”says Clara.

shortening paths

The official confirmation of the first case of the Brazilian monkey was made yesterday (06/09) by the Adolfo Lutz Institute. The São Paulo Reference Laboratory performed 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 a gold standard. Sequencing using this method takes an average of 48 hours to complete.

The CADDE team used a portable sequencer called MinION, from Oxford Nanopore Technologies, and made adjustments to the protocol used for sequencing the Zika virus (2015) and SARS-CoV-2 (2020), making it the fastest.

“One of the advantages of this new protocol is that it reduces the time it takes to prepare samples for sequencing, 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 has sought to create 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.

The next step was to assemble an isolated phylogenetic MPXV tree in Brazil. To this end, the sequence obtained by the CADDE team at USP has been compared with the 102 released this year by scientists from Belgium, Portugal, the United Kingdom, Germany, Spain and the United States. The aim was to measure the degree of similarity between the sequences, which provides guidance on evolutionary relationships.

“In 2022, we downloaded all the genomes sequenced [até 09/06], align the sequences and set the phylogenetic tree. We saw that the MPXV detected here fits into a large clade [grupo], The same as the 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 only saw three mutations, ”says Claro.

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 somehow helped to increase the number of cases is something that is still being studied by other research groups. Here at CADDE we will keep an eye on the following cases. The aim is to continue monitoring the evolution of the virus, ”explained Claro.

Although it causes monkeypox or monkeypox, MPXV is a virus that mainly infects rodents in Africa. The pathogen is part of the Orthopoxvirus family, identical to the human smallpox virus, which became extinct in the 1980s.

The disease usually includes fever, fatigue, headaches, muscle aches, which are non-specific symptoms, similar to cold or flu. A few days after the onset of the fever, skin lesions appear with a high viral load. Spread occurs through direct contact with wounds or clothing, sheets, and towels worn by someone with skin lesions. It can also be caused by coughing or sneezing of infected people.

Until the beginning of the year, the infection was only common in Central Africa. But new cases have already been detected in 33 countries, most with no history of the disease.

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