Researchers complete monkey pox virus sequencing in 18 hours

High-speed treatment of viral genetic material from the first confirmed case in Brazil was possible thanks to the adaptation of an existing technique

Researchers at the Joint UK-Brazil Center for Arbovirus Discovery, Diagnosis, Genomics and Epidemiology (CADDE) concluded in only 18 hours the complete genome sequencing of monkeypox virus (MPXV)isolated from the first patient diagnosed with monkey pox confirmed in Brazil.

The high speed of treatment of viral genetic material was possible thanks to the adaptation to the virus by a rapid metagenomic technique developed during the doctoral degree by researcher Ingra Morales Claro, a fellow at the Fundação de Amparo à Pesquisa do Estado de São Paulo ( Fapesp).

The work was coordinated by Professor of the University of São Paulo (USP) Ester Sabino, who was also responsible for the first sequencing of SARS-CoV-2, the virus that causes Covid-19, in the country, in March 2020, and by the first case of the Gamma variant, which appeared in Manaus about a year later.

The team published the findings Thursday (9) on the virology platform, a website where scientists around the world share real-time disease-causing information.

“We received a sample from a patient admitted to Hospital Emílio Ribas at 4 pm on Tuesday (7) and 10 am the following morning, the genome of the virus, which has almost 200,000 base pairs [bem mais que as 30 mil do SARS-CoV-2], was sequenced and analyzed. The method we developed is on average 45% faster than conventional metagenomic techniques. And the cost is also lower and reaches 30 USD per. try ”, says Ingra.

According to Ester, researchers often resort to analyzes of this type when identifying a newly emerged 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 at. hand. necessary, as is now the case with the monkey pox virus.

The performance of molecular diagnostic tests (RT PCR) requires called “primers” (primers), which are sequences complementary to the viral sequences that initiate the replication of the genetic material. After processing, the result should be compared to negative and positive “controls”. RT PCR is considered the gold standard for the diagnosis of Covid-19 and several other diseases.

“When an epidemic starts with a new infectious agent, one of the biggest bottlenecks for diagnosing cases is lack of specific primers and positive controls. This technique can be useful in these situations, as it allows the identification of pathogens that are still unknown and for which there are no reagents, ”explains Ester.

The earlier the “index” case is detected, the first case, the more likely it is to contain a new virus, Ingra adds.

In the case of metagenomics, random primers (not specific to a particular virus or bacterium) are used, which make it possible to sequence all the genetic material contained in a biological sample, including the host (human, in this case) and other agents that cause diseases. , which he 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 data obtained were mapped on a sequence of the virus already available for studies. And that allowed us to prove that they were monkey cups, ”says Ingra.

shortening of paths

The official confirmation of the first Brazilian case of monkey poop was made on Thursday (9) by the Instituto Adolfo Lutz in São Paulo.

The São Paulo Reference Laboratory performed the metagenomic analysis on a platform known as Illumina, one of the technologies that has been used to detect monkeypox virus in European and North American centers and is considered the gold standard. Sequencing with this method takes an average of 48 hours to complete.

The research team at the Joint Brazil-United Kingdom Center used a portable sequencer known as MinION, from Oxford Nanopore Technologies, and made adjustments to the protocol used to sequence Zika virus and SARS-CoV-2, making it faster.

“One of the benefits of this new protocol are the reduction in time sample preparation for sequencing, which runs from 14 hours to 5:40 minutes, ”reports Ingra.

Since the error rate is slightly higher than for the Illumina platform, the 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 it is not a misreading,” says the researcher.

genetic traits

The next step was to assemble the phylogenetic tree of the smallpox virus isolated in Brazil. For this, the researchers compared the sequence obtained at USP with a further 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, giving clues as to how the virus may develop.

“We downloaded all complete genomes sequenced in 2022 [até 09/06], adjust the sequences and set up the phylogenetic tree. We saw that the MPXV found here fits into a large draft [grupo], the same 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 observed 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 (in 2018, in Africa).

‘What these mutations represent, and whether they have in some way contributed to the increase in the number of cases, is something that is still being investigated by other research groups. We here at CADDE are keeping an eye on the next cases. The idea is to continue sequencing to monitor the development of the virus, ”reveals Ingra.

Although known to cause monkey pox, MPXV is a virus that mainly infects rodents in Africa. The pathogen is part of the Orthopoxvirus family, the same as the human smallpox virus that was eradicated in 1980.

The disease usually starts with fever, fatigue, headache, muscle aches, that is, nonspecific, cold or flu-like symptoms. A few days after the onset of the fever, skin lesions occur, which contain a high viral load.

Spread occurs through direct contact with the lesions or through clothing, sheets and towels used by a person with skin ulcers. It can also occur by coughing or sneezing of infected people.

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

Source: CNN Brazil; Karina Toledo – FAPESP Agency

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