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Antivirus Fortresses

High security laboratories give the country autonomy in research into emergent diseases

EDUARDO CESARRigor: instruments of work and personal objects are sterilized with detergent and ultraviolet radiation when they go into the laboratory and when they come outEDUARDO CESAR

There are new guests in the Fernando de Noronha archipelago, 545 kilometers from Recife. They are the egrets that began to arrive from Africa a few years ago, and sometimes appear, flying in flocks, in the environs of the airport and the houses. In other countries, in these two species of egrets – the cattle egret, more common, and the great egret – the virus of the West Nile Virus (WNV) has been found, which has worried specialists in public health, as it is spreading over the world and causing an emergent disease with a high mortality rate. Since it was identified, in 1937, in the blood of a woman from Uganda, the West Nile Virus has spread over Africa, moved into the Middle East and Europe, spread over the former Soviet Union, reached India, and for three years now has been terrifying the United States. It has now spread over 44 American states and, in a rare demonstration of aggressiveness, infected 7,000 victims, children and elderly in the main, and caused 145 deaths. Transmitted to human beings by mosquitoes that feed on the birds contaminated blood, WNV causes severe inflammations in the brain or in neighboring organs, like the cerebellum.

“There is no reason to suppose that this virus will not arrive in Brazil”, says virologist Edison Luiz Durigon, from the Biomedical Sciences Institute (ICB) of the University of São Paulo (USP). Epidemiologist Eduardo Massad, from USP’s College of Medicine, has also reached this conclusion, on the basis of the same arguments: the country is on the route of the migratory birds that have spread this virus over the world, and we have a lot of vectors – the mosquitoes of the Culex genus, common domestic insects. But, they recall, it is necessary to know basic information about the transmission rate in a group of a thousand, is it only one or a hundred birds that carry the virus? – before defining precisely the seriousness of the situation.

Up until now, Brazilian scientists did not have any laboratories suitable for working in safety with viruses like the West Nile Virus or of the Severe Acute Respiratory Syndrome (Sars), the most recent representatives of the so-called emergent diseases, which cause severe damage, because the organism is not yet used to dealing with them. The situation is starting to change this month, with the inauguration, on the 11th , of the Klaus Eberhard Stewien Laboratory, so named in homage to a German virologist, naturalized Brazilian, now at the age of 65, who helped to contain polio in Brazil.Built in the course of a year and a half on the second floor of one of the buildings of USP’s Biomedical Sciences Institute, the new laboratory is the first in the country with BSL 3 (biosafety level 3).

It is almost the maximum possible for civilian research, there are more sophisticated installation only in the Centers for Disease Control and Prevention (CDC), of Atlanta, United States, a world-wide benchmark in emergent diseases. “This laboratory at USP is serving as a benchmark for the development of projects for the network of 12 BSL 3 level laboratories that the Ministry of Health began building this year”, says Mário Althoff, the coordinator general of the network of public health laboratories of the Secretariat for Surveillance in Health of the Ministry of Health. According to him, eight of these new areas are now under construction. They are to come into operation by the end of 2004 in Brasília, Fortaleza, Manaus, Salvador, Recife, Porto Alegre and São Paulo, with the purpose of dealing with viruses and bacteria that are important for public health, with minimum risks for the life of the researchers or of the population. “With this network”, says Althoff, “research with tuberculosis bacilli will be carried out for the first time under proper conditions in Brazil.”

Overalls, mask and boots
It is not at all easy or comfortable to enter these fortresses. For the time being, Edison Durigon, the coordinator of the ICB’s laboratory, is asking guests just to cover their shoes with white slippers, before getting to know the area of almost 50 square meters, entirely sealed and surrounded by wall half a meter thick. But from January onwards, when the laboratory is working normally, only six researchers will carry the electronic card that unlocks the entry door and they will have to follow a series of procedures that will take them at least ten minutes to get in and another ten to get out. In a room 1 meter by 2, they are going to undress and put on waterproof overalls, boots, masks and glasses. Only then, passing through doors that are opened only if others are locked, will they be able to reach the main room, with the freezers, incubators for cultivating viruses, a space for experiments with cells or animals, and genome sequencing equipment. The ICB’s laboratory will be the first of the four BSL 3s to be built in São Paulo.By the end of next year, if the work proceeds as expected, another unit should start to work at USP in Ribeirão Preto, a third one at the São Paulo State University (Unesp) in São José do Rio Preto, and the fourth in the Adolfo Lutz Institute, in the capital. The construction of the four laboratories is part of a larger project, the Viral Genetic Diversity Network, or VGDN, created at the end of 2000 with about R$ 12 million in finance from FAPESP.

Today, almost 150 researchers from 22 São Paulo research groups – including organs of the State Secretariat for Health and a private hospital, the Albert Einstein – are looking into the diversity and the peculiarities of four viruses: the strain of the Aids virus that is most common in Brazil, the HIV-1, whose genome should be sequenced by the end of next year; the HCV, which causes hepatitis C, to be sequenced straight afterwards; the Respiratory syncytial virus or SRV, which causes serious infections in the lungs and is the main cause of death of children up to the age of 18 months; and hantavirus, which causes grave forms of pneumonia, lethal in up to 40% of the cases. The ICB’s laboratory will house studies into hantavirus, arboviruses and the West Nile Virus, on the basis of blood samples from rodents and birds already collected in the Amazon and in the Atlantic Rain Forest. “For the sake of security, we are not going to keep exotic viruses in the laboratory before they appear in Brazil, Durigonreassures. From 1990 to 1994, he worked in Atlanta, at the Centers for Disease Control and Prevention (CDC), in a laboratory similar to the one he is now coordinating.In the Network, specialists in insects work with physicians, virologists, geneticists and molecular biologists. The conclusions which they reach should explain – or explain better – the origin, the forms of transmission and propagation, and the best techniques for treating the diseases caused by viruses. What is wanted is to find molecular markers of virulence (genes, stretches of genes or proteins) that may, for example, reduce the resistance of the Aids virus to medicines.

With the data in hand, it becomes easier to give more consistency to public health measures and to give guidance on vaccination campaigns. For Massad, at least, it will not be the first time. A rare example of a physician skilled in equations (he was in the fifth year of medicine when he joined the physics course), he creates mathematical models that indicate the behavior of epidemics and that have been useful, for example, in the German measles epidemic in 1992: the recommendation was to vaccinate all the children and youngsters aged between 9 months and 15 years, but Massad showed that it would suffice to vaccinate children in the 1 to 10 year age group, with just 7 million doses of vaccine. This made it possible to reduce costs by almost half (see Pesquisa FAPESP no 61, of January 2001). Once again called in when there was an outbreak of measles in 1997, the team from the College of Medicine helped to create a vaccination strategy that made it possible to limit the impact of the virus to 20,000 cases, far fewer than the 300,000 originally forecast.

Support for doctors
The information that emerges from the laboratories of the Network is now reaching the doctors who are attending to the victims of the viruses. Two biologists from the ICB, Danila Vedovello and Larissa do Amaral, are receiving samples of nasal secretions from children in-patients, suspected to be infected with the syncytial virus, at the seven hospitals connected with the project. At the laboratory, at the same time that they gather the material together for research, they identify the virus that causes the symptoms, and in a day or two send the result to the doctors, who accordingly choose the most suitable treatment, since even bacteria and funguses can lead to very similar symptoms. In Ribeirão Preto, the team led by Luiz Tadeu Moraes Figueiredo has drawn up a table of symptoms making it possible for the doctors to reinforce their suspicions of infection caused by hantavirus, transmitted through the air in places where their natural reservoirs, hairy-tailed bolo mice (Bolomys lasiurus) have been. “It is now enough to suspect contamination by hantavirus to send the individuals affected to hospitals that have ICUs (intensive care units)”, says Figueiredo.

At the end of the 90’s, when cases of victims of hantavirus in the Ribeirão Preto region intensified, Figueiredo succeeded in being sufficiently agile to develop a method of diagnosis that recognizes the viruses of this family in four hours. Paolo Zanotto, from USP’s ICB, was just as quick with Sars virus, responsible for an atypical form of pneumonia that killed 800 people in over 30 countries, since it was identified in February this year. In July, when he worked at the University of Freiburg, in Germany, he developed a method for detecting the virus that increases 100,000 times the efficiency of the test traditionally used. He embarked for Germany with just the idea for a new test, suggested by Carlos Augusto Pereira, a researcher at the Butantan Institute: the terminal region of the genome of the coronavirus, of the family to which the Sars virus belongs, induces an intense production of molecules of ribonucleic acid (RNA). In his baggage on return, Zanotto brought RNA, cloned material and fragments ofgenes that made a more precise diagnosis possible.

Zanotto is only going to touch this material – and even the virus, should Sars appear in Brazil – inside the new laboratory. The space at the ICB is kept under negative pressure (lower than the pressure outside) precisely to prevent any microorganism from getting out of there. Inside there, the temperature remains at 20° Celsius – even so, the researchers are expected to feel hot when they wear the complete working uniform – and the air is filtered and entirely changed 12 times every hour. The instruments of work must be sterilized, and even personal objects like glasses are passed through a bath with detergent, under ultraviolet radiation, before being put inside or outside. When the work is done, the researchers themselves leave by a route that forces them to have a bath with 50 liters of water, and the overalls they wore go into an autoclave – a closed tank – where they are washed under pressure and with steam. Anyone who suffers a bit from claustrophobia will certainly be content to observe thework only through the double-glazed window by the side of the entry door.

Sabia set free
As it becomes consolidated, the Network is creating more suitable conditions for facing viruses like the Sabia, that arose, out of the blue, in 1990. Its story began when a land surveyor who lived in Sabiá Garden, in Cotia, in Greater São Paulo, was taken, seriously ill, to the hospital of the Emílio Ribas Institute. The doctors were not able to do much: a victim of intensive internal bleeding, she died on the same day. Researchers from the Adolpho Lutz received the blood samples containing the virus, and sent them to the Evandro Chagas Institute, in Belém, another referral center for virology in Brazil, which over the last 50 years has characterized about 11,000 strains of almost 200 kinds of arbovirus. There, a technician contaminated himself when inoculating newborn mice with this virus, which spreads in the air. The material with the virus to be identified then went on to the unit at Yale University, in the United States, accredited by the World Health Organization (WHO) to deal with exotic viruses. Not even there did the Sabia keep quiet. A French trainee contaminated himself, when a test tube broke in his hand, when being taken out of the centrifuge. Instead of communicating the fact to the team, he cleaned the centrifuge, closed the laboratory, and went away. One week later, as had happened with the technician in Belém, he was admitted to hospital with intense fevers. They both survived, but to this day nobody knows how the virus arose, whether it really has disappeared, and where it went; it was only identified three months after being found (today, one week would be sufficient).

Made up by just one molecule of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) wrapped in a protein shell, viruses are extremely simple structures, so simple that there is still some doubt as to whether they really are living beings, as they need other organisms to reproduce. But they persecute human beings from infancy to old age. Vaccines now exist to check the viruses that cause measles, German measles, polio (infantile paralysis), varicella (chicken pox) and rabies, but can still do little against others than torment the mothers of small children, such as those that cause mumps, viral meningitis and hepatitis. And adults have to take precautions against the Aids virus and try and dribble past those that cause dengue, yellow fever and the strains that cause some kinds of cancer, and there are still the painful eruptions that appear because of the herpes virus. Later on, particularly for those past the age of 65, the recommendation is to take the vaccine against the influenza virus, which in earlier decades has not been much of a worry. There have been advances, of course. Today, the diarrheas caused by virus, which in the 70’s used to be the main cause of death in Brazil, are not so worrying.

In the 70’s, the Rocio virus
Another one transmitted by the Aedes mosquito caused an epidemic of encephalitis in the Paraíba Valley, between the cities of São Paulo and Rio de Janeiro. Rocio has apparently disappeared, but there are still children with antibodies against it, a sign that it may still be in circulation, even though in a less dangerous version. Durigon observes that, besides the possibility of old viruses reappearing, new varieties may always arise, as the forests are destroyed. Pedro Vasconcelos, a virologist from the Evandro Chagas, adds another risk factor: the need to produce food in larger quantities favors the growth of the population of wild rodents and mosquitoes, which facilitates the transmission of microorganisms with which the human organism has not yet been in contact.

One of the targets of the coordinators of the Virus Network is to anticipate problems – through predictive virology. Or, in practical terms, discovering viruses before they show up in people. This is the purpose for which biologist Luiz Francisco Sanfilippo spent two months camping on the banks of the Machado river in Acre, collecting blood and samples of secretion from the trachea and cloaca of the birds that land there. He does not lack practice: for 12 years, Sanfilippo was head of the bird section of a zoo, until last year, when he began his studies for a doctorate, in Durigon’s team. This month, Sanfilippo is to go on to Fernando de Noronha and collect material from the two species of African egrets, the cattle egret (Bubucus ibis) and the great egret (Casmerodius albus), reinforcing the survey by the team from the Brazilian Institute for the Environment and Renewable Natural Resources (Ibama), which up until now has found no signs of the West Nile Virus in Brazil. However, the list of the 110 species of birds that can be infected includes the domestic chicken (Gallus gallus), pigeons (Columba livia), quails (Coturnix coturnix) and mallards (Anas platyrhynchos), besides other migratory species, such as the gray heron (Ardea cinerea) and the little stint (Calidris minuta).

It is also to discover problems before they appear that the team from Ribeirão Preto is going into the field at least once a month, in a trailer coupled to a light truck and adapted to be a mobile laboratory – the hantamobile is how it is called -, to hunt for rodents than may be hosts for hantavirus. In the next few weeks, as soon as the laboratory at USP starts to operate, analysis will begin of the 300 blood samples from birds and another 600 from rodents, brought from Rondônia, and from the Mar mountains or hinterland in São Paulo to be analyzed. The researchers want to know how they live and whether they spread viruses that are already known, or if there are new species arriving.

The Project
Viral Genetic Diversity Network; Modality Special Programs; Coordinators Eduardo Massad – USP’s College of Medicine, and Edison Durigon and Paolo Zanotto – ICB at USP; Investment
R$ 6,687,937.23 and US$ 1,674,373.74