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After demonstrating that the Brazilian Zika virus causes microcephaly, researchers are beginning to investigate other factors that may contribute to neurological damage

Brains in miniature: organoids cultured in USP’s Stem Cell Laboratory and used in tests of the Zika virus

Léo RamosBrains in miniature: organoids cultured in USP’s Stem Cell Laboratory and used in tests of the Zika virusLéo Ramos

Researchers from São Paulo and other Brazilian states have ended their initial investigation into how the Zika virus influences cases of microcephaly. Six months after the appearance of the initial evidence that this infectious agent was behind the birth of infants with brains too small in relation to the gestational age, Brazilian teams who have studied this connection believe there is already enough information to say that Zika causes microcephaly and neurological damage.

Several conditions necessary to establish a cause-and-effect relationship have been met. During this period, there have been cases of women infected during pregnancy who gave birth to babies with microcephaly and the virus’s ability to cross the placenta has been determined. The researchers identified peculiarities that differentiate microcephaly associated with Zika from other forms of the problem, and they confirmed Zika’s preference for cells of the nervous system. In May 2016, the evidence that had been missing appeared: a São Paulo team presented an animal model of microcephaly.

The researchers used the type of virus circulating in Brazil, and demonstrated that it is more aggressive than the African strain, which was isolated from a monkey in 1947. At the University of São Paulo (USP), the group led by the neuroimmunologist Jean Pierre Peron inoculated pregnant female mice with the virus and monitored their pregnancies. The Brazilian strain, the ZIKVBR, crosses the placentas of a variety of female mice more susceptible to infection by the virus and compromises the development of their offspring.

The mice were born at less than half the normal weight, had smaller brains and exhibited damage to brain tissue similar to that caused by Zika in humans. Like the African virus, the Brazilian Zika virus invades and preferentially damages the neural progenitor cells that produce different types of brain cells and are abundant in the early development of the fetus. But the Brazilian variety causes more severe cell death.

Presented on May 11, 2016, in the journal Nature, this model will allow researchers to investigate Zika’s mechanism of injury in detail and do initial testing of candidate compounds for a vaccine and drug against the virus. “Before, it was impossible to know if it was really Zika or another associated factor causing the malformation cases in Brazil,” says Brazilian neuroscientist Alysson Muotri, a researcher at the University of California at San Diego and co-author of the study. “Our work shows that Brazilian Zika is enough to cause microcephaly and other birth defects,” she says.

“We addressed a major problem,” says virologist Paolo Zanotto, of USP, one of the authors of the study. Zanotto coordinates the Zika Network, a consortium of nearly 50 São Paulo laboratories investigating the virus with support from FAPESP. He knows that not all has been solved. “Now,” he says, “we need to understand the complexity of the epidemic and monitor the cognitive development of children with microcephaly.”

Immune weakness
The more aggressive strain of the virus, in effect, only caused microcephaly in a strain of mice offspring less resistant to viral infections. At USP’s Neuroimmune Interactions Laboratory, Peron and his team injected the virus into the bloodstreams of pregnant female mice of two strains—the C57BL/6, with a more robust defense system, and SLJ, whose cells produce less interferon, a chemical marker that protects them from viral invasion. Only the offspring of the SLJ strain were smaller at birth, a sign that they had experienced growth restriction in the womb, and had suffered brain damage. “This model seems to simulate well what happens during pregnancy, a period in which the immune system undergoes some suppression and becomes more susceptible to infections,” says neuroscientist Patrícia Beltrão Braga, head of USP’s Stem Cell Laboratory and one of the coordinators of the study.

According to Peron, these results may explain why not every woman infected by Zika during pregnancy will have a child with microcephaly. “The mother’s genetic makeup seems to be important in preventing the virus from reaching the fetus,” he says. One of his hypotheses is that women with certain gene variations containing the recipe for producing interferon or regulating its synthesis are more susceptible to infection and having a baby with microcephaly.

Zika01_244More aggressive
The strongest confirmation that the Brazilian Zika virus is more aggressive than the African strain came from experiments conducted in Braga’s laboratory. She and her team extracted stem cells from the baby teeth of healthy children and chemically reprogrammed them to become more versatile cells, neural progenitors. When cultured in suspension in a liquid nutrient, the progenitors form microscopic spheres (neurospheres). Over time, the neurosphere cells produce different cell types that organize themselves into layers like mini-brains.

In Braga’s laboratory, biologists Fernanda Cugola, Isabella Fernandes and Fabiele Russo infected the neurospheres and mini-brains with the Brazilian and African Zika strains. Already on day one, the two types of virus invaded the neural progenitors and began to multiply. On the fourth day, the neurospheres infected by ZIKVBR were one-fourth the size of those infected by the African virus and nearly one-tenth the size of those not infected by the virus. Zika also deformed their structure. And, the greater the amount of virus, the more severe the damage.

In addition to deforming the neurospheres, the virus prevented their cells from migrating, a phenomenon in which they move and populate different brain regions. Mini-brains with Brazilian Zika decreased the thickness of the layer that generates the cortex, the outermost layer of the brain and the one most affected in babies with microcephaly caused by Zika.

Changes in the size and structure of the neurospheres and mini-brains are the result of cell death, which appears to occur in two ways: apoptosis or programmed death, in which cells whither due to signals indicating that recovery of their normal operation is impossible; and autophagy, in which sacs containing acids and enzymes break apart and digest cellular content.

In the case of Zika, death by apoptosis is preceded by disorders identified by the group led by Patricia Garcez and Stevens Rehen, neuroscientists at the Federal University of Rio de Janeiro (UFRJ) and the D’Or Institute for Research and Education (IDOR). The Rio de Janeiro team infected neural progenitors with Zika and, three days later, asked Janaina Vasconcelos and João Vianez Júnior of the Evandro Chagas Institute in Belém (Pará State, northern Brazil) to analyze the gene activation pattern, and Juliana Nascimento, Juliana Cassoli and Daniel Martins de Souza of the University of Campinas (Unicamp) to identify the proteins that were being produced.

Combined, these strategies have revealed that, once inside the cells, the Zika virus begins to control cellular functioning. It prevents neural progenitors from multiplying and blocks orders to repair damage from being executed. It also forces cellular machinery to produce copies of the virus. Unable to return to its normal routine, the cell activates its self-destruct mechanisms.

The death of neural progenitors, however, explains only part of the reduction in the number of neurons. The virus also disables the programming that directs these cells to turn into neurons. “We already knew that cells were dying, but cell death can affect neuron production in different ways,” says Garcez. “Identifying these molecular pathways may perhaps lead us to find ways to block the infection,” she says. She plans to investigate factors that may favor the passage of the virus from the mother to the fetus.

This, incidentally, is one of Zanotto’s current interests. He and his colleagues are trying to find out if and how other infections that the mother had before or during her pregnancy could facilitate access of the virus to the fetus. In May 2016, Zanotto and Dr. Mauro Hanaoka described one of the first cases of microcephaly caused by the Zika virus reported in the state of São Paulo. The baby is a girl born in November of 2015, in the 38th week of pregnancy. She is the daughter of a 32-year-old woman who lives in Santos and had dengue in 2013, in addition to symptoms of Zika infection early in her pregnancy. In July 2015, when the woman was treated for a respiratory infection, doctors noticed that the baby had microcephaly and sent the case to São Paulo. A blood test performed on the mother showed the presence of antibodies against dengue fever and Zika. And even against cytomegalovirus, herpes virus and the parasite toxoplasmosis—infectious agents that can cause microcephaly and are on what is referred to as the STORCH list (acronym for syphilis, toxoplasmosis, rubella, cytomegalovirus and herpes).

As the pregnancy advanced, Zanotto and his colleagues noticed that the concentration of antibodies against Toxoplasma gondii reached levels of a new infection. Researchers do not know whether the increase represented a reaction from the mother to the resurgence of the parasites, which can occur with a weakened immune system, or the proliferation of antibody-producing cells (polyclonal expansion) against Toxoplasma. But they believe it was not a good sign. There may have been damage to the placenta, making it easier for Zika to invade the tissues of the fetus,” says Zanotto.

Zika02_244Other infections
The Santos case reinforces the suspicion that the occurrence of other infections helps explain the concentration of microcephaly in some regions of Brazil and among the poorest people. Toxoplasmosis seems to be one of them. Among 13 common risk factors during pregnancy, it was the only one that increased the likelihood of microcephaly caused by Zika, according to a study published in the Bulletin of the World Health Organization. It is estimated that, in certain regions of Brazil, up to 70% of the population has already been in contact with the parasite. “The Health Ministry recently reported that 77% of the cases of microcephaly in the Northeast occurred in families with the lowest HDI [Human Development Index]” says Zanotto. “This population is more susceptible to these infectious agents.”

Dengue fever is also a concern. It is estimated that between 50% and 80% of the Brazilian population has already been infected by the virus and has antibodies against dengue. A U.S. study indicates that when there are antibodies against dengue the infectivity of Zika increases up to 200 times.

“The full context cannot be ignored,” says Zanotto, who plans to test cases of microcephaly to which he has access for STORCH agents. “The mother who lives in Santos lives in a low HDI region,” he says. He notes: “We will only know whether these factors exert a real influence if we compare the occurrence of congenital manifestations in children of infected mothers with different HDIs.”

1. The role of tryptophan-kinureninas axis in the regulation
of the immune response through NMDA glutamate receptors in experimental autoimmune encephalomyelitis and in brain ischemia-reperfusion injury (nº 2011/18703-2); Grant Mechanism Young Investigators Award; Principal Investigator Jean Pierre Schatzmann Peron (ICB-USP); Investment R$1,077,384.82.
2. A systemic approach to study permissivity on the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) (nº 2014/17766-9); Grant Mechanism Regular Research Grant; Principal Investigator Paolo Marinho Zanotto (ICB-USP), Investment R$500,009.45.
3. Developing a predictive test for a successful medication [response] and understanding the molecular bases of schizophrenia through proteomics (nº 2013/08711-3); Grant Mechanism Young Investigators Award; Principal Investigator Daniel Martins de Souza (IB-Unicamp); Investment R$1,379,511.67.

Scientific articles
CUGOLA, F.R. et al. The Brazilian zika virus strain causes
birth defects in experimental models. Nature. Online. May 11, 2016.
GARCEZ, P.P. et al. Combined proteome and transcriptome analyzes reveal that [the] Zika virus circulating in Brazil alters cell cycle and neurogenic programs in human neurospheres. Peer J Preprints. May 9, 2016.
HANAOKA, M.M. et al. The Zika virus-associated microcephaly case with background exposure to STORCH agents. bioRxiv, May 10, 2016.