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HEALTH

The spread of Zika

Sequencing confirms that the variety in circulation in Brazil came from Polynesia and is projected to spread to other countries

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Two Brazilian teams in recent weeks have completed sequencing of the genetic material of the Zika virus isolated in the states of São Paulo and Paraíba. The results suggest that the Zika variety circulating in different regions of Brazil is of the same Zika strain as that of French Polynesia, where there was an outbreak in 2013 and 2014. They also indicate that the virus was probably introduced in Brazil in a single event.

At the Adolfo Lutz Institute in São Paulo, virologist Renato Pereira de Souza and his team sequenced Zika’s genetic material extracted from a person who became ill in Campinas. This individual contracted the virus through a blood transfusion. The donor fell ill days later and told the blood center of the University of Campinas (Unicamp) he suspected he had dengue. The analyzes done at Adolfo Lutz eliminated dengue and confirmed the Zika infection. “Blood centers will also have to pay attention to this virus, since many cases are asymptomatic,” says Souza. “In this case, the virus remained viable and infected another person,” he says. He was one of the coordinators of the analysis, carried out in partnership with researchers from Unicamp and the University of São Paulo (USP), in Ribeirão Preto, and accepted for publication by the journal Genome Announcements.

The sequencing of the genetic material of the virus showed a lean genome. There are about 10,600 units (nucleotides) making up a single strand of ribonucleic acid (RNA). This strand is home to a total of only genes capable of producing 10 different proteins—some genes are multipurpose. “The genome analysis indicates that the virus is of the same lineage as the virus that circulated in French Polynesia and Easter Island,” says Souza.

At the Federal University of Rio de Janeiro (UFRJ), virologist Amilcar Tanuri and his team also sequenced the Zika genome extracted from the amniotic fluid of two women who had given birth to babies with microcephaly in Paraíba State in northeastern Brazil. The conclusions are the same. “I am positive it’s the same strain as the one in Polynesia,” says Tanuri. Tanuri notes that the difference between the genetic material of the virus in circulation in Brazil and the genetic material of the virus in Polynesia is small, about 20 nucleotides and only one of 3,500 amino acids (protein-forming units). He says that such a close similarity indicates that the virus is spreading very quickly and was introduced only once in Brazil. Tanuri says that the Zika sequenced in Rio de Janeiro is very similar to the dengue virus, in particular serotype 4, which can hamper the development of a diagnostic kit to specifically identify Zika antibodies. “We will have to circumvent this similarity when producing a test,” he says.

... and zebrafish embryos: models to study the development of the central nervous system

Ângela Costa and Murilo Carvalho / LNBio… and zebrafish embryos: models to study the development of the central nervous systemÂngela Costa and Murilo Carvalho / LNBio

From Brazil to the world
The virus that haunts the world with the threat of microcephaly took almost 70 years to cross half the globe. But in a short time it has gained an explosive potential for dissemination. Its ability to spread seems to have increased in recent times, especially after arriving in Brazil, where, according to government estimates, it has already infected 440,000 to 1.3 million people.

Adaptations undergone by the virus during its journey from Africa apparently facilitated its ability to reproduce in the human body. This characteristic, coupled with the high degree of mobility of today’s population and the fact that the virus usually hitchhikes in human blood without being noticed (in 80% of cases the infection causes no symptoms) is transforming Zika into an international headache. In a brief article published in the January 23, 2016 issue of The Lancet, one of the most important journals in the field of medicine, a group of researchers from Canada, the United States and England provided a scenario of Zika’s rapid expansion by region with a high concentration of people in the Americas and Europe.

The team led by Dr. Kamran Khan, an infectious disease specialist at the University of Toronto who investigates how diseases are spread by travelers, used a mathematical model that reproduces dengue outbreaks to estimate Zika’s ability to spread. The researchers fed the model with information on areas where the Aedes genus of mosquitoes, which in addition to Zika also transmits yellow fever, dengue and chikungunya viruses, is currently found, and regions with a climate favorable to the proliferation of these insects. With this data, they were able to get an idea of where the conditions would be favorable to the spread of Zika once it arrived there.

In the next step, the researchers needed to calculate the probability of the virus reaching the regions where its vector lives—for the Aedes aegypti, the Americas and Africa, and for the Aedes albopictus, Asia and Europe. To do this, they mapped the international destinations of travelers who between September 2014 and August 2015 were in regions of Brazil where there was Zika transmission.

During this period, nearly 10 million people traveled abroad from 146 Brazilian airports located in areas where the virus was circulating. About 6.5 million people (65% of the total) went to countries in North and South America. Another 27% traveled to Europe and 5% to Asia. The United States alone received 2.8 million people coming from Brazil, while Argentina received 1.3 million and Chile, 614,000. The most common destinations in Europe were Italy, Portugal and France, each receiving 400,000 people. Tens of thousands also traveled to Asia, especially to China, and to Africa, mainly to Angola.

This scenario worries health authorities for several reasons. First, some regions receiving travelers are densely populated areas. “More than 60% of the populations of Argentina, Italy and the United States live in regions favorable to seasonal transmission of the virus,” the researchers wrote. Between 23 million and 30 million people in Mexico, Colombia as well as in the United States would be in areas at risk of ongoing transmission, where insects can spread the virus throughout the year.

The second cause for concern is that Zika seems to have acquired the ability to more easily infect the human body during its long and slow path traveled in Asia, after leaving the forests of Uganda around 1945, up to arriving in French Polynesia in 2013, from where it traveled to Brazil. In this passage, recently mapped by Caio Melo Freire, a biomedical specialist at the Federal University of São Carlos (UFSCar), and colleagues from USP and the Pasteur Institute in Senegal, the virus humanized: today some of its genes contain recipes for making proteins more compatible with the human body, which facilitates infection (see Pesquisa FAPESP issue nº 239). “This may have occurred because over the course of this trip the virus circulated among only a few vectors, probably humans and insects,” notes Atila Iamarino, biologist and co-author of the study. Iamarino, a member of the USP team, also disseminates scientific information and, with zoologist Sônia Carvalho Lopes, coordinated the production of material available on the Wikiversity site, which guides elementary and secondary school teachers to assist students in disproving rumors about Zika spread over the Internet.

As the virus advances, researchers from all over Brazil are continuing to study Zika in an attempt to understand what it does in the human body and how the virus could cause the cases of microcephaly attributed to it. Between October 22, 2015 and January 30, 2016, the Ministry of Health reported the births of 4,783 babies suspected of having microcephaly (prior to the Zika epidemic, this notification was not mandatory).

Of the 1,113 cases analyzed so far, 404 were confirmed. The brains of these babies are in fact too small for a newborn and, in addition to the clinical symptoms, show signs of brain damage consistent with an infection acquired during pregnancy (congenital). Thus far, however, Zika infection has only been proven in 17 of the 404 cases of microcephaly—another 387 will depend on the performance of immunoassays, not yet available, to rule out this association.

Colonies of bacteria of the genus Bacillus...

Leticia Gushi/UnespColonies of bacteria of the genus BacillusLeticia Gushi/Unesp

Congenital syndrome
Some groups are trying to better characterize the problems of babies born to mothers possibly infected by the virus. Under the coordination of geneticist Dr. Lavinia Schüler-Faccini of the Federal University of Rio Grande do Sul (UFRGS), experts in congenital anomalies from various Brazilian institutions have conducted clinical, genetic and imaging tests on 35 children with microcephaly related to Zika born in eight Brazilian states, including São Paulo. “The tests allowed us to exclude genetic diseases or infections by other agents known to cause microcephaly,” says Schüler-Faccini.

From this analysis, a pattern typical of infections caused by viruses during pregnancy began to emerge. Although there was no evidence that the mothers had been infected by the virus, 74% showed signs consistent with Zika fever during pregnancy, such as red spots that itched, low-grade fever and joint pain—mostly during the first trimester of pregnancy, when the fetus is in an accelerated phase of development. Of the 35 babies examined, 25 (71% of the total) had severe microcephaly, with a skull circumference of less than 31 centimeters at birth.

Twenty-seven of the infants underwent imaging studies, which showed neurological abnormalities. The most common were calcifications, which are small nodules that act as a sort of scar on brain tissue. One in three of the children had lissencephaly, an absence of the characteristic folds of a healthy brain, or pachygyria, wider folds. More severe, these changes suggest that the infection occurred at an early stage of development.

Four babies developed a severe joint problem know as arthrogryposis. This disease, which hinders movement of the joints, manifests itself primarily in the knees, hips and elbows and, according to Schüler-Faccini, may mean that the baby did not move much during intrauterine development because of neurological damage. “We have already analyzed another 15 cases and the pattern we see is always very similar, which supports the hypothesis that the Zika infection does not just cause microcephaly, but also a new syndrome, as already proposed by some researchers,” says Schüler-Faccini, who presented the results in an article in the January 22, 2016 issue of Morbidity and Mortality Weekly Report.

Characterization of the damage associated with Zika is essential to guiding the work of other groups trying to clarify the biological mechanisms behind the microcephaly. In a new initiative to study the virus organized by Kleber Gomes Franchini of the Brazilian Biosciences National Laboratory (LNBio) at the Center for Research in Energy and Materials (CNPEM), physician and researcher José Xavier Neto and his team in Campinas plan to inoculate the virus into the embryos of mice, chickens and zebrafish of different ages.

In vertebrates, the extensive group of animals that includes everything from fish to mammals (including humans), the formation and development of the central nervous system organs follow a sequence of known and standardized steps. Interference at different stages often leads to distinct changes in brain architecture, which makes it possible to anticipate how the virus acts. “First,” notes Xavier Neto, “we have to find the biological model that is most appropriate for studying the infection.”

Xavier Neto intends to examine alterations at the cellular and genetic level. The pattern observed in cases of microcephaly associated with Zika suggests that there may be interference both in cell proliferation and in the migration phase, in which they move circumferentially to the positions they will occupy in the fetal brain, a phenomenon that occurs during the second trimester of pregnancy. “There is a lot of information coming in, and we still need to determine what is most relevant,” says Xavier Neto. His experiments will use samples of the virus being grown in the laboratory by Lucio Freitas Júnior, also of LNBio. Freitas Júnior, in turn, is developing screening tests for finding new compounds that can be used to treat Zika infections.

... and Microbacterium: tests to assess whether or not they eliminate the Zika virus

Leticia Gushi / Unesp… and Microbacterium: tests to assess whether or not they eliminate the Zika virusLeticia Gushi / Unesp

The virus and the mosquito
While some researchers are mapping the damage caused by the virus in the human body, others are looking for ways to reduce or block transmission of the virus by the mosquito. At São Paulo State University (Unesp) in Botucatu, biologist Jayme Souza Neto has begun to investigate what factors make Aedes more susceptible to Zika infection. His work will involve using what has been learned in recent years about the interaction between the mosquito and the dengue virus.

Years ago, while studying for his postdoctorate at Johns Hopkins University in the United States, Souza Neto noted that bacteria comprising the insect’s gut microbiota, found naturally in its digestive system, protect it from the dengue virus in some cases. This effect was evident when researchers treated a population of mosquitoes with antibiotics—thereby changing their gut microbiota—and found that they are more easily infected with the virus than mosquitoes with intact microbiota.

Expanding upon this work, Souza Neto found that some groups of bacteria seem to provide a more protective effect than others. Aedes females fed a mixture of blood and high concentrations of certain groups of bacteria—for example, bacteria of the genus Paenibacillus and Proteus—had fewer copies of the virus in their gut. Apart from stimulating the insect’s immune system, it is already known that some of these bacteria act directly on the virus by inhibiting its replication. “Since the dengue and Zika viruses are very close from an evolutionary standpoint, it is possible that some varieties of bacteria that act against one also work against the other,” says Souza Neto. If successful, this strategy for fighting the virus could be added to the fight against the mosquito itself—for now, the most efficient way to prevent the spread of Zika.

In addition to seeking ways to prevent mosquitoes from becoming infected with the virus, thereby preventing the insect from passing on the infection, Souza Neto plans to compare the efficiency of Aedes aegypti in transmitting the dengue virus with its ability to propagate the Zika virus. This work will be conducted in partnership with the team led by Margareth Capurro, a USP entomologist, and may help explain why this virus seems to spread faster than the dengue virus. In a project to be developed with researchers at London’s Imperial College, he intends to determine whether Aedes albopictus, commonly found in Europe and forested areas of Brazil, could also be a good propagator of Zika.

While at work on the longer-term studies, Souza Neto will be undertaking a task of immediate impact. Like other teams of the Zika Network, his group in Botucatu will look for mosquito activity in areas with suspected cases of the disease. “This way we expect to know the number of infected mosquitoes in the environment and varieties of the virus in circulation,” says Souza Neto, who will work with Margareth Capurro and Lincoln Suesdek of the Butantan Institute, both members of the research network that began to take shape in São Paulo in December 2015 and that is now expected to increase its efficiency through optimized management.

Original Title: aegypti668sRGB.tifJames Gathany/CDCA more efficient network
At a meeting held in early February 2016 of the Council of SP State University Presidents (Cruesp), the deans of research at USP, Unicamp and Unesp presented a proposal to the leadership of the Zika Network: create a structure to optimize the use of available resources, coordinate the interaction between these universities and other institutions in Brazil and abroad, and accelerate access to more funding for research. This proposed structure would consist of scientific and advisory committees, both comprising members of the three universities, as well as a spokesperson, who would be responsible for communicating with the media.

The goal, according to the research deans, is to design an action plan that will quickly lead to an understanding of how the Zika virus acts in the human body; the development of effective diagnostic tests; and the production of a serum or vaccine against the virus, in addition to vector control. An initial step will be to conduct a survey of all network researchers and their areas of expertise. “So, we want to clarify the scenario with which we are working to determine what resources we have available and what we need to look for, including searching abroad,” says Maria José Giannini, the dean of research at Unesp. “It will be an attempt to make these collaborations achieve results more quickly on an issue that has become a matter of national and international urgency,” she adds.

“Perhaps such coordination was not needed before,” says USP’s dean of research, José Eduardo Krieger. “Now, however, we feel that it has become necessary for the network to achieve greater efficiency.” Krieger believes this structure will allow better use of the resources and infrastructure of the three universities. “If we do not combine the internal capacities of each institution, then more than one group could be working in isolation on the same area of research,” says Glaucia Pastore, research dean at Unicamp. “In this emergency situation, we need a new working model, in which we work in a complementary way to overcome barriers more quickly.”

Carlos Henrique de Brito Cruz, FAPESP’s scientific director, attended the meeting and asked the three universities to prepare a plan with proposals, methodologies and objectives that can be submitted to some funding sources. “Our intention was to have a new program subsidized by FAPESP, aimed at not only a better understanding of the Zika virus in particular, but also dengue and chikungunya,” says Brito Cruz. “We will have more meetings about it in the future.”

Project
Characterization of the microbiota-mediated anti-dengue mechanisms in wild Aedes aegypti populations (nº 2013/11343-6); Grant Mechanism Young Investigators Award; Principal Investigator Jayme Augusto de Souza Neto (IBTeC-Unesp); Investment R$1,843,243.92

Scientific articles
CUNHA, M.S. et al. First complete genome sequence of Zika virus (Flaviviridae, Flavivirus) from an autochthonous transmission in the Americas. Genome Announcements. In press.
SCHÜLER-FACCINI, L. et al. Possible association between zika virus infection and microcephaly – Brazil, 2015. Morbidity and Mortality Weekly Report. January 22, 2016.
BOGOCHI, I.I. et al. Anticipating the international spread of zika virus from Brazil. The Lancet. V. 387, pp. 334-5. January 23, 2016.
OLIVEIRA MELO, A.S. et al. Zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg? Ultrasound Obstetrics and Gynecology. V. 47, pp. 6-7. 2016.
VENTURA, C.V. et al. Zika virus in Brazil and macular atrophy in a child with microcephaly. The Lancet. V. 387, p. 228. January 23, 2016.

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