Researchers in São Paulo produce new coronavirus in the laboratory

Samples of the virus will be distributed to public and private clinical laboratories all over Brazil for use as positive controls. The goal is to enhance the labs’ capacity to perform diagnostic tests.

By Elton Alisson | Agência FAPESP – Researchers at the University of São Paulo’s Institute of Biomedical Sciences (ICB-USP) in Brazil have cultured the SARS-CoV-2 coronavirus in a laboratory. The virus was isolated from the first two patients diagnosed with the disease in São Paulo City, at the Albert Einstein Jewish Hospital (HIAE).

The viruses will be distributed to research groups and public and private clinical laboratories throughout Brazil to enhance their capacity to perform diagnostic tests and investigate how the disease spreads.

“This supply of cell-cultured coronavirus samples will enable clinical labs to set up positive controls for validation of diagnostic tests and assurance of their accuracy,” Edison Luiz Durigon, a professor at ICB-USP, told Agência FAPESP. Durigon is principal investigator for the project, which is supported by FAPESP.

A lack of samples of the virus for use as positive controls, he added, was one of the factors limiting diagnosis of the new coronavirus disease in Brazil.

Given its origin abroad, the samples of SARS-CoV-2 used as positive controls in the diagnostic tests by laboratories at the start of the outbreak in Brazil had to be imported from Europe and the United States at a unit cost ranging from 12,000 to 14,000 Brazilian Reais (now about 2,600-3,000 US Dollars).

For this reason, diagnostic tests in Brazil have been performed mainly by private laboratories, as well as public-sector laboratories in the health centers that have treated suspected cases.

Four national reference laboratories currently perform diagnostic tests: Instituto Adolfo Lutz in São Paulo State; Instituto Evandro Chagas in Pará State; Fiocruz in Rio de Janeiro State; and the Goiás State Central Laboratory, which was certified to perform a specific test to find out whether the Brazilians repatriated from China and quarantined in the Anápolis Air Force Base were infected with the new coronavirus.

Tests are performed first by top-tier hospitals in each state. The material collected then has to be sent to one of these four laboratories for a double-check. This lengthy procedure can now be simplified.

“The viruses we’ve succeeded in growing in the lab can be used in a diagnostic kit to be distributed by the Health Ministry to Public Health Central Laboratories [LACENS] nationwide. All states will be equipped to perform diagnostic testing for the new coronavirus disease as a result,” Durigon said.

The viruses will be inactivated before being shipped at room temperature to the LACENS so that they cannot infect cells. The viruses currently imported by Brazilian laboratories must be refrigerated in dry ice during transportation, which is more expensive as a result, he explained.

The laboratories will receive samples containing roughly 1 milliliter (ml) of inactivated viruses. Their nucleic acid will be extracted for use as a positive control in a test based on the technique known as real-time polymerase chain reaction (RT-PCR).

The technique amplifies the viral genome, increasing the number of copies of the coronavirus’s RNA by millions so that it can be detected and quantified in a clinical sample. 

“PCR enables a diagnosis to be completed in up to four hours, but few Brazilian labs have the necessary equipment to use the technique,” Durigon said.

To surmount this limitation the researchers plan to develop other diagnostic tests based on more accessible techniques, such as immunofluorescence, which uses antibodies chemically labeled with fluorescent dyes to visualize molecules.

“If we succeed in validating a test of this specific type for the new coronavirus, other labs and hospitals that don’t have the equipment to perform RT-PCR testing will also be able to diagnose the disease,” Durigon said.

Zika legacy

Isolation of the new coronavirus and its reproduction in the laboratory, Durigon added, were made possible by funding from FAPESP for installation at ICB-USP of two laboratories certified for Biosafety Level III (BSL-3). Facilities with BSL-3 certification must be specially designed to handle substances that can cause serious and potentially lethal diseases (including bioterrorism agents). This infrastructure at ICB-USP was originally built to cultivate zika virus.

Construction of these laboratories in early 2016, when the zika epidemic was at its peak in Brazil, now enables the scientists at ICB-USP to grow not just the new coronavirus but also the human influenza viruses, as well as other viruses for use in diagnosing emerging viral diseases, Durigon stressed.

“When the zika outbreak began in late 2015, we were taken by surprise but thanks to funding from FAPESP we were able to be the first to isolate the virus and culture it in our lab for distribution to other labs and research groups,” he said.

Under the aegis of a project called “Virus Genome”, also supported by FAPESP and begun in 2003, a network of 18 laboratories was set up in São Paulo State and equipped to perform RT-PCR diagnostic tests for respiratory viruses as well as genome sequencing.

When the outbreak of zika virus began, the network played a vital role in diagnosing and understanding the disease, according to Durigon.

“This is why continuous funding for research is so important,” he said. “Thanks to the investment made in the past, we have a research infrastructure in São Paulo that will enable us to respond more speedily to needs as they arise, without having to start from scratch.” 

The researchers at ICB-USP have monitored seasonal circulation of four other coronaviruses in Brazil. The results of their studies show that this circulation occurs mainly in the winter.

“São Paulo State and Rio Grande do Sul State will probably have the largest number of cases of infection in the winter because they’re the coldest states in Brazil,” Durigon said.