Scientists sequence monkeypox virus in 18 hours

Scientists sequence monkeypox virus in 18 hours

High-speed processing of viral genetic material from Brazil’s first confirmed case is possible thanks to existing technology

Researchers from the UK-Brazil Joint Centre for Arbovirus Discovery, Diagnostics, Genomics and Epidemiology (CADDE) Complete genome sequencing of monkeypox virus (MPXV) in as little as 18 hoursisolated from the first patient diagnosed with monkeypox in Brazil.

The high-speed processing of viral genetic material is possible thanks to the adaptation of the virus by rapid metagenomic techniques developed during researcher Ingra Morales Claro, Ph.D., researcher at Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp).

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, the virus that causes Covid-19, in the country in March 2020, and in Manaus about a year later The first instance of the Gamma variant.

The team published the results on Thursday (9) on the Virology Platform, a website where scientists around the world share information about causative agents in real time.

“We received a sample from an inpatient at Emílio Ribas Hospital on Tuesday (7) at 4 pm, and at 10 am 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], were sequenced and analyzed. The method we developed was on average 45% faster than traditional metagenomic techniques. And the cost is lower, at $30 per sample,” Ingra said.

According to Ester, when scientists need to identify an emerging virus (as was the case with SARS-CoV-2 in 2019), or to test for a known virus in patient samples without reagents, they often would resort to this type of analysis. hand. Need, just like the monkeypox virus now.

Molecular Diagnostic Test (RT PCR) Performance Needs called “primers” (primers), which are sequences complementary to viral sequences that initiate replication of genetic material.The processed result needs Compared to negative and positive “controls”. RT PCR is considered the gold standard for diagnosing Covid-19 and several other diseases.

“When an epidemic begins with a new infectious agent, one of the major bottlenecks in diagnosing cases is Lack of specific primers and positive controls. This technique is useful in these cases because it can identify pathogens that are still unknown, without reagents,” Ester explained.

Ingra added that the sooner an “index” case is detected, the more likely the first case, containing the emerging virus, is.

In the case of metagenomics, random primers (not specific to 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 (in this case, humans) and other potions that caused the disease he eventually brought.

This information is then analyzed by bioinformatics techniques and compared with a set of references. “It’s exactly the same as MPXV. The data obtained was mapped onto viral sequences that were already available for research. This allowed us to show that it was monkeypox,” Ingra said.

shorten the path

The Adolph Lutz Institute in São Paulo officially confirmed the first case of monkeypox in Brazil on Thursday.

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

Using a portable sequencer called the MinION from Oxford Nanopore Technologies, the team from the joint Brazil-UK centre adapted the protocol used to sequence Zika and SARS-CoV-2, make it faster.

“one The advantage of this new protocol is the reduced time Sample preparation for sequencing, from 14 hours to 5:40 minutes”, Ingra reports.

With a slightly higher error rate than the Illumina platform, the team attempted to generate up to 300 redundant reads for each region of the viral genome. “When we covered the same area multiple times and found the same result, we could be sure it wasn’t a misreading,” the researchers said.

genetic traits

The next step was to assemble a phylogenetic tree of monkeypox viruses isolated in Brazil. To do this, the scientists compared the sequences obtained at USP with another 102 sequences published this year by experts in countries including Belgium, Portugal, the UK, Germany, Spain and the US. The aim is to assess the degree of similarity between sequences, thereby providing clues to how the virus evolved.

“We downloaded all complete genomes sequenced in 2022 [até 09/06], align the sequences and build a phylogenetic tree.We see that the MPXVs detected here fit into a large clade [grupo], the same virus sequenced in Europe and the United States.Compared to the CDC reference genome [o Centro de Controle de Doenças norte-americano]in the May update, we only observed three mutations,” Ingra said.

In contrast, the first MPXV genome sequenced in 2022 showed 47 mutations compared to the last case described to date (2018, in Africa).

“What these mutations represent and whether they are somehow contributing to the increase in the number of cases is still being studied by other research groups. We at CADDE will be watching the next case closely. Our idea is to continue sequencing to monitor the evolution of the virus”, Ingra disclose.

Although known to cause monkeypox, MPXV is a virus that primarily infects African rodents. The pathogen is part of the orthopoxvirus family, the same as the human smallpox virus, and was eradicated in 1980.

Illness usually begins with fever, fatigue, headache, muscle aches, i.e. nonspecific, cold or flu-like symptoms. A few days after the fever, skin lesions appear, which contain a high viral load.

Transmission is through direct contact with the lesion or through clothing, sheets and towels used by patients with skin wounds. It can also occur when an infected person coughs or sneezes.

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

Source: CNN Brazil; Karina Toledo – FAPESP Agency

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