Researchers from the British-Brazilian Joint Center for Arbovirus Discovery, Diagnosis, Genomics and Epidemiology (CADDE) completed in just 18 hours sequencing the complete genome of Monkeypox virus (MPXV) isolated from one patient. First confirmed diagnosis of smallpox in Brazil .
This achievement was made possible by the rapid adaptation to MPXV of a metagenomics technique developed during the Ph.D. of Ingra Morales Claro, a FAPESP Fellowship holder. This work was coordinated by Professor Ester Sabino of The University of Sao Paulo (USP), which was responsible for the first SARS-CoV-2 sequencing in the country in March 2020 and in the first case of the new gamma variant, which appeared in Manaus about a year later.
The CADDE team released the results yesterday (09/06) at virological.org. This is a website where virologists from around the world share real-time information about pathogens of interest.
“We received a sample from a patient admitted to Emílio Ribas Hospital at 4 p.m. on Tuesday. [07/06] and at 10:00 a.m. the next morning The viral genome contains almost 200,000 base pairs. [bem mais que as 30 mil do SARS-CoV-2]were sequenced and analyzed. The way we developed it On average, it is 45% faster than conventional metagenomics techniques, and the cost is also lower, reaching $30 per sample,” Claro told Agência FAPESP.
As Sabino explains, scientists often use metagenomic analysis when they want to identify emerging viruses. (as was the case with SARS-CoV-2 in 2019) or detect known viruses in patient samples without having the necessary specialized reagents on hand (eg now with MPXV).
This is because the RT-PCR test, the gold standard for diagnosing COVID-19 and many other diseases, requires what is known as a primer. which is the nucleotide sequence coupled with the viral sequence that initiates the replication of the genetic material. and the results had to be compared with the negative and positive controls.
“When an epidemic starts with a new infection One of the major bottlenecks for diagnosing cases is the lack of specific primers and positive controls. This technique can be helpful in these situations. This is because it allows identification of unknown pathogens. which has no reagents,” explains Sabino.
And the sooner an “index” case (the first case) is detected, the more likely it is to contract the emerging virus, Claro added.
know more
In the case of metagenomics, random primers are used. (not specific to some viruses or bacteria), which makes it possible to sequence all the genetic material contained in a biological sample. including those of the host (human, in this case) and other pathogens. This data is then analyzed by bioinformatics techniques and compared with reference groups.
“Just as was done with MPXV, the data obtained were mapped to an existing viral sequence for the study. And that allowed us to prove it was smallpox,” Claro said.
shorter path
Instituto Adolfo Lutz officially confirmed the first case of chickenpox in Brazil yesterday (06/09). A reference laboratory in Sao Paulo has carried out a methanomic analysis on a platform called Illumina, which is one of In the technology used to detect MPXV in European and North American centers and is considered the gold standard This sequence takes approximately 48 hours to complete.
The CADDE group used a portable sequencer called MinION from Oxford Nanopore Technologies and modified the protocols used in sequencing the Zika virus (since 2015) and SARS-CoV-2 (since 2020). the most
know more
“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,” Claro said.
Because the error rate was slightly higher than that of the Illumina platform, the CADDE team attempted to generate a maximum of 300 redundant reads for each region of the viral genome. The researchers said “When we covered the same area multiple times and found the same results, We can be sure it’s not a misread,” the researchers said.
The next step is to assemble the isolated MPXV phylogenetic tree in Brazil. For this, the CADDE team compared the sequence obtained from the USP with 102 others published this year by scientists from countries such as Belgium, Portugal, the United Kingdom, Germany, Spain and the United States. Its purpose is to measure the degree of similarity between the sequences. which gives clues about evolutionary relationships
“We downloaded the entire genome sequentially in 2022. [até 09/06]Sorting and setting up a phylogenetic tree, we see that the MPXV detected here fit a large clad. [grupo]The same is true for viruses that rank in Europe and the United States. Compared to the CDC reference genome [o Centro de Controle de Doenças norte-americano]which updated in May We only observed three mutations,” Claro said.
By comparison, the first MPXV genome sequenced in 2022 had 47 mutations compared to the last case described so far (in 2018 in Africa).
“What shows these mutations and whether they contribute to the increase in case numbers are what other research groups have. Still studying, we at CADDE will keep an eye on the case. The idea is to continue sequencing to track the evolution of the virus,” Claro revealed.
Although it is known to cause chickenpox or smallpox, MPXV is the virus that infects most of the African rodents. Pathogens are part of the family. Orthopoxvirus Like the human smallpox virus, which was eradicated in 1980.
Illness often begins with fever, fatigue, headache, muscle aches, ie, non-specific symptoms. have a cold or flu-like illness A few days after the onset of fever, skin lesions appear, which have a high viral load. Transmission is through direct contact with the wound or to the clothing, bedding, and towels used by people with skin wounds. It can also occur from the coughing or sneezing of an infected person.
until earlier this year The infection is common only in Central Africa. But new cases have been found in 33 countries, most of them have no history of the disease.
– Revista Galileu