USP researchers cultivate monkeypox virus for testing and research

After completing the first genome sequencing of the monkeypox virus (MPXV) in Brazil, scientists from USP (University of São Paulo) are dedicating themselves to cultivating the causative agent of monkeypox in cell lines. The objective is to distribute samples to public and private laboratories across the country, which can be used both in diagnostic tests and in research aimed at understanding viral evolution and developing new treatments and vaccines.

The work has been carried out at the Virology Laboratory (LIM52) of the Institute of Tropical Medicine (IMT-USP), under the command of virologist Lucy dos Santos Vilas Boas.

“We received the clinical sample from the first patient diagnosed in the country and inoculated it into a vero cell culture. [linhagem oriunda de rim de macaco e usada como modelo para pesquisas com vírus]. After 24 hours, it was already possible to observe morphological changes in the cells that are typical of monkeypox. The confirmation was made by RT-PCR”, says Vilas Boas.

The monkeypox-specific RT-PCR test was developed at Hospital Israelita Albert Einstein by the team of physician João Renato Rebello Pinho, who is also a researcher at IMT-USP. The method is the same used in the diagnosis of Covid-19 and numerous other diseases, but each pathogen requires specific reagents so that the genetic material present in the clinical sample can be amplified and detected in the laboratory.

“Until that day [10/06] no center in the country had the RT-PCR kit for this virus assembled. We had already studied the sequence [de nucleotídeos complementar à do DNA viral, que é necessária para a amplificação do material genético durante o teste] and request for a national company to synthesize. What was missing was the positive control, which came with the first confirmed case in the country. He is the one who guarantees that the test is working”, explains Pinho. “As in the case of Covid-19, there is a great deal of collaboration between public and private institutions, which is fundamental.”

After confirming that the virus that was multiplying in vero cells was in fact the cause of monkeypox, the next steps were to extract it from the culture medium and inactivate it to be sent safely to other centers, explains Vilas Boas. .

“Infected cells die and release virus particles into the supernatant — the nutrient-rich liquid in which the culture is kept. We take this liquid and add a reagent that inactivates the virus. Now we are sending samples of this material to private and public laboratories that requested. In these other centers, the viral DNA can be extracted and used as a positive control in RT-PCR tests, expanding the testing capacity in the country”, reports the virologist.

José Eduardo Levi, a researcher at IMT-USP and the Dasa laboratory network, explains that, in general, plates with 96 small wells are used in RT-PCR tests, in which clinical samples from up to 94 individuals with suspected of the disease. The other two wells are intended for the positive and negative controls, necessary for the validation of the results.

“As a negative control we can use just water or the supernatant collected from cell cultures without any virus. The most difficult thing when we have to deal with a new pathogen is to get the positive control. Without it we cannot be sure that the RT- PCR is actually working,” says Levi.

According to the researcher, despite the advances, the scarcity of specific reagents for MPXV in the country is still a major bottleneck for mass testing of the population, if necessary.

“When a laboratory in the United States places an order for reagents, it receives it the next day, as there are companies specializing in the production of these inputs. Here in Brazil it takes at least a month, as it is necessary to import. June and still nothing”, he says.

large scale production

Some centers equipped with the necessary biosecurity infrastructure are receiving from IMT-USP samples of the virus that is still capable of infecting cells and replicating itself, as is the case with USP’s Institute of Biomedical Sciences (ICB). Like SARS-CoV-2, PMXV can only be handled in biosafety level 3 (NB3) laboratories.

“Our role will be to cultivate the virus on a larger scale and within two or three weeks start distributing aliquots to laboratories across the country. This will be possible thanks to an agreement we made at the beginning of the Covid-19 pandemic with the Ministry of Science, Technology and Innovations [MCTI] and with the Post Office. The company makes the specialized transport of samples. They pick it up here and take it to the recipients’ door”, says ICB-USP professor Edison Luiz Durigon.

The ICB-USP laboratory is part of RedeVírus —created by the MCTI in February 2020 to advise the portfolio on strategies in the area of ​​science, technology and innovation needed in the health area— and was responsible for producing SARS-CoV-2 in scale and distribute samples to diagnostic and research centers across the country.

The team has extensive experience in this type of work and has played a key role in successive health crises.

“The work started around 2003, with the creation of the Genetic Diversity Network of Viruses [VGDN], funded by Fapesp. At the time we cultivated and distributed SARS-CoV-1 [causador da síndrome respiratória aguda grave]. In 2005 we did work with influenza [causador da gripe] and then, in 2015, came the zika virus. We received the Zika sample in December from Instituto Evandro Chagas de Belém [PA] and with resources from Fapesp projects, we cultivate and distribute them to several laboratories. With the arrival of the new coronavirus and the agreement with the MCTI, we managed to expand the work scale”, he says.

Duringon points out that the work was only possible because the laboratory already had the necessary material and human resources. “I already had trained staff, who were FAPESP PhD and Master’s scholarship holders. If we started from scratch, it would take at least a year to start operating. There needs to be a structure in place and continuous funding so that a quick response can be given. to public health problems, such as a pandemic.”

Now, in addition to cultivating MPXV on a large scale, Durigon’s group will also sequence some samples to check for differences in relation to the virus initially isolated in the country.

“The scenario I am anticipating is that, in most cases, we will distribute aliquots of the inactivated virus to be used as a positive control in diagnostic laboratories. Only for some centers with an NB3 laboratory will we send samples of the viable virus for research”, says the professor at the ICB-USP.

In the opinion of USP professor Ester Sabino, who coordinated the first genomic sequencing of MPXV in the country, this is yet another example of how public universities can contribute to their installed capacity in crisis situations. “Viral isolation is important not only for the production of positive controls but also for the evaluation of new drugs”, she says.

Pinho emphasizes the importance of virologists from all over the country working together, in a network, and also the need for more investment in science.