Researchers from the UK-Brazil Joint Center for Arbovirus Discovery, Diagnosis, Genomics and Epidemiology (CADDE) completed in just 18 hours the complete genome sequencing of the monkeypox virus (MPXV) isolated from the first patient with a confirmed diagnosis of monkeypox in Brazil. .
This feat was made possible thanks to the adaptation to MPXV of a rapid metagenomics technique developed during the doctorate of Ingra Morales Claro, a FAPESP scholarship holder. The work was coordinated by University of São Paulo (USP) professor Ester Sabino, who was also in charge of the first SARS-CoV-2 sequencing in the country, in March 2020, and of the first cases of the new gamma variant, which emerged in Manaus about a year later.
The CADDE team released the results yesterday (09/06) on virological.org, a site where virologists from around the world share information on pathogens of interest in real time.
“We received a sample from a patient admitted to Hospital Emílio Ribas at 4 pm on Tuesday [07/06] and at 10 am the next morning the genome of the virus, which is almost 200 thousand base pairs [bem mais que as 30 mil do SARS-CoV-2], was sequenced and analyzed. The methodology we developed is, on average, 45% faster than conventional metagenomics techniques. And the cost is also lower, reaching US$ 30 per sample”, Claro tells Agência FAPESP.
As Sabino explains, scientists often resort to metagenomic analyzes when they need 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 having the necessary specific reagents at 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 the viral sequences that initiate the replication of genetic material. And the result then needs to be compared with negative and positive controls.
“When an epidemic starts with a new infectious agent, one of the major bottlenecks for 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 still unknown, for which there are no reagents”, explains Sabino.
And the earlier the detection of the “index” case (the first case) occurs, the greater the probability of containing an emerging virus, adds Claro.
In the case of metagenomics, random primers are used (not specific for a particular virus or bacterium), which make it possible to sequence all the genetic material contained in a biological sample, including that of the host (human, in this case) and other pathogens that it eventually harbors. . Then, this information is analyzed by bioinformatics techniques and compared with a panel of references.
“Exactly as was done with the MPXV. The data obtained were mapped onto a sequence of the virus already available for studies. And that allowed us to prove that it was monkeypox”, says Claro.
shortening paths
The official confirmation of the first Brazilian case of monkeypox was made yesterday (09/06) by the Instituto Adolfo Lutz. The São Paulo reference laboratory conducted the metagenomic analysis on a platform known as Illumina, one of the technologies that has been used to detect MPXV in European and North American centers and considered the gold standard. Sequencing by this method takes an average of 48 hours to complete.
The CADDE group used a portable sequencer known as MinION, from Oxford Nanopore Technologies, and made adaptations to the protocol used to sequence the Zika virus (from 2015) and SARS-CoV-2 (from 2020), making -the fastest.
“One of the advantages of this new protocol is the reduction in sample preparation time for sequencing, which goes from 14 hours to 5:40 minutes”, says Claro.
As the error rate is slightly higher than that of the Illumina platform, the CADDE team sought to generate up to 300 redundant reads 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 the phylogenetic tree of MPXV isolated in Brazil. For this, the CADDE team compared the sequence obtained at USP with another 102 released this year by scientists from countries such as Belgium, Portugal, United Kingdom, Germany, Spain and the United States. The objective was to measure the degree of similarity between the sequences, which gives clues about evolutionary relationships.
“We downloaded all complete genomes sequenced in 2022 [até 09/06], align the sequences and set up 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. When compared to the CDC reference genome [o Centro de Controle de Doenças norte-americano]updated in May, we observed only three mutations”, says Claro.
By way of 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 somehow 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 keep an eye on the next cases. The idea is to continue sequencing to monitor the evolution of the virus”, reveals Claro.
Although it is known to cause monkeypox or monkeypox, 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, eradicated in 1980.
The illness usually starts with fever, fatigue, headache, muscle aches, that is, non-specific, cold or flu-like symptoms. A few days after the onset of fever, skin lesions appear, which contain a high viral load. Spread is through direct contact with the wounds or with clothing, sheets and towels used by someone with the skin lesions. It can also occur from the coughing or sneezing of infected people.
Until earlier this year, the infection was common only in Central Africa. But new cases have already been detected in 33 countries, most with no previous history of the disease.
– Revista Galileu