High-speed processing 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 just 18 hours the complete genome sequencing of the monkeypox virus (MPXV) isolated from the first patient diagnosed with monkeypox confirmed in Brazil.
The high speed in the processing of viral genetic material was possible thanks to the adaptation for the virus of a rapid metagenomics technique developed during the doctorate of 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 at the University of São Paulo (USP) Ester Sabino, who was also in charge of the first sequencing of SARS-CoV-2, the virus that causes Covid-19, in the country, in March 2020, and of the first cases of the Gamma variant, which appeared in Manaus about a year later.
The team released the results on Thursday (9) on the Virological platform, a site where scientists around the world share information about disease-causing agents in real time.
“We received a sample from a patient admitted to Hospital Emílio Ribas at 4 pm on Tuesday (7) and, at 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 methodology we developed is, on average, 45% faster than conventional metagenomics techniques. And the cost is also lower, reaching US$ 30 per sample”, says Ingra.
According to Ester, scientists often resort to analyzes of this type when they need to identify a new emerging 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. needed, as is now the case with the monkeypox virus.
The performance of molecular diagnostic tests (RT PCR) requires the called “primers” (primers), which are sequences complementary to the viral sequences that initiate the replication of the genetic material. After processing, the result needs to be compared to negative and positive “controls”. RT PCR is considered the gold standard for diagnosing Covid-19 and several other diseases.
“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 Ester.
The earlier the “index” case is detected, the first case, the greater the likelihood of containing an emerging virus, adds Ingra.
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 agents that cause diseases that he eventually brings.
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 Ingra.
shortening paths
The official confirmation of the first Brazilian case of monkeypox was made on Thursday (9) by the Instituto Adolfo Lutz, in São Paulo.
The São Paulo reference laboratory conducted the metagenomic analysis on a platform known as Illumina, one of the technologies that have been used to detect the monkeypox virus in European and North American centers and considered the gold standard. Sequencing by this method takes an average of 48 hours to complete.
The research group at the Joint Brazil-United Kingdom Center used a portable sequencer known as MinION, from Oxford Nanopore Technologies, and made adaptations to the protocol used to sequence the Zika virus and SARS-CoV-2, making it faster.
“One of advantages of this new protocol is the reduction in time sample preparation for sequencing, which goes from 14 hours to 5:40 minutes”, reports Ingra.
As the error rate is slightly higher than that of the Illumina platform, the 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.
genetic characteristics
The next step was to assemble the phylogenetic tree of the monkeypox virus isolated in Brazil. For this, the scientists compared the sequence obtained at USP with another 102 released 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, which gives clues about how the virus might evolve.
“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 Ingra.
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 Ingra.
Although known to cause 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 lesions or through clothing, sheets and towels used by someone with the skin wounds. It can also occur from the coughing or sneezing of infected people.
Until the beginning of 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.
Source: CNN Brasil; Karina Toledo – FAPESP Agency