Fast processing of viral genetic material from the first confirmed case in Brazil was possible thanks to the adaptation of existing techniques.
Researchers at the United Kingdom-Brazil Joint Center for Arbovirus Discovery, Diagnosis, Genomics and Epidemiology (CADDE) Complete genomic sequencing of monkeypox virus (MPXV) takes only 18 hoursWas isolated from the first patient diagnosed with monkeypox, confirmed in Brazil.
Fundação de Amparoà Pesquisado Estadode São Paulo (Fapesp).
This work was coordinated by Professor Esther Sabino of the University of Sao Paulo (USP). He was also responsible for the initial sequencing of SARS-CoV-2, the virus that causes Covid-19 in the country in March 2020. And the first case of a gamma mutant that appeared in Manaus about a year later.
The team announced results on Thursday (9) on the Virological platform, a site where scientists around the world share real-time information about disease-causing substances.
“I received a sample from a patient admitted to the hospital Emily Olives at 4 pm on Tuesday (7) and received a nearly 200,000 base pair virus genome at 10 am the next morning. [bem mais que as 30 mil do SARS-CoV-2], Sequenced and analyzed. The methodology we have developed is, on average, 45% faster than traditional metagenomics techniques. It’s also low cost, reaching $ 30 per sample, “says Ingra.
According to Ester, scientists detect known viruses in patient samples when they need to identify emerging viruses, as in the case of SARS-CoV-2 in 2019, or without the use of reagents. You often rely on this type of analysis when you need to. As with the Monkey Poxvirus, you need a hand.
To perform a molecular diagnostic test (RT PCR), Called a “primer” (Primer), which is a sequence complementary to the viral sequence that initiates 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 diagnosing Covid-19 and several other diseases.
“If the epidemic begins with a new infectious pathogen, one of the major bottlenecks for diagnosing cases is Lack of specific primers and positive controls.. This technique can be useful in this situation because it can identify pathogens that are not yet known without reagents, “Ester explains.
If the “index” case is detected early, the first case is more likely to contain a new virus and Ingra will be added.
For metagenomics, random primers are used (not specific to a particular virus or bacterium), thereby causing the host (in this case a human) and other drugs that cause the disease he ultimately causes.
This information is then analyzed by bioinformatics technology and compared to the bibliography panel. “It’s exactly the same as it was done with MPXV. The data obtained was mapped to a sequence of viruses already available for study, and it proves that it was monkeypox. Has made it possible, “says Ingra.
Shorten the path
The official confirmation of the first Brazilian case of monkeypox was made on Thursday (9) by the Adolfo Lutz Institute in São Paulo.
The São Paulo Reference Lab conducted metagenomic analysis on a platform called Illumina, one of the techniques used to detect monkeypox virus in centers in Europe and North America and is considered the gold standard. Sequences made this way take an average of 48 hours to complete.
A research group at a joint center in Brazil and the United Kingdom used a portable sequencer called MinION from Oxford Nanopore Technologies to adapt and accelerate the protocol used to sequence Zika virus and SARS-CoV-2.
“one of The advantage of this new protocol is the time savings. Sample preparation for the sequence from 14 hours to 5:40 minutes, “Ingra reports.
The error rate is slightly higher than the Illumina platform error rate, so the team sought to generate up to 300 redundant reads for each region of the viral genome. “When you cover the same area several times and find the same results, you can be confident that it’s not a misreading,” says the researchers.
Genetic characteristics
The next step was to build a phylogenetic tree of the monkeypox virus isolated in Brazil. To this end, 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 purpose was to assess the degree of similarity between sequences, which provided clues as to how the virus evolved.
“Downloaded all complete genomes sequenced in 2022 [até 09/06], Align the sequence and set the phylogenetic tree.It turns out that the MPXV detected here fits into a large branch group [grupo], Same as the virus 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, and only three mutations were observed, “says Ingra.
For comparison, the first MPXV genome sequenced in 2022 showed 47 mutations compared to the last case described so far (2018, Africa).
“What these mutations represent and whether they have contributed to an increase in the number of cases in any way is still being studied by other research groups. We, CADDE, are: Keep an eye on the case. The idea is to continue the sequence to monitor the evolution of the virus, “Ingra reveals.
Although known to cause monkeypox, MPXV is a virus that mainly infects rodents in Africa. The pathogen is part of the same orthopoxvirus family as the human smallpox virus, which was eradicated in 1980.
The illness usually begins with fever, malaise, headaches, and muscle aches, that is, symptoms like a nonspecific cold or flu. A few days after the fever, skin lesions with high viral load appear.
Spreading is done through direct contact with the lesion or through clothing, sheets, or towels used by people with skin scars. It can also result from an infected person’s coughing or sneezing.
Until the beginning of this year, the infection was common only in Central Africa. However, new cases have already been detected in 33 countries, most of which have no previous history of the disease.
Source: CNN Brazil; Karina Toledo – FAPESP Agency