O uso de microrganismos patogênicos em atos de bioterrorismo é já há algum tempo objeto de grande preocupação em vários países. O presente trabalho apresenta a possível aplicação de vírus e bactérias para fins bélicos e terroristas, bem como o diagnóstico laboratorial para a identificação desses agentes. Foram salientados, entre outros, como agentes de infecções humanas visando o bioterrorismo, os vírus da varíola (ortopoxvírus), os de febres hemorrágicas e os pertencentes aos filovírus. Entre as bactérias foram destacadas as do antrax ( Bacillus anthracis ), da peste ( Yersinia pestis ), do botulismo ( Clostridium botulinum ) e da tularemia ( Francisella tularensis ), incluindo ainda a ricina ( Ricinus communis ) como componente do grupo B de agentes.

In recent years the use of pathogenic microorganisms in acts of bioterrorism has been the subject of major concern in many countries. This paper presents a possible application of viruses and bacteria for warfare and terrorist purposes, as well as a laboratory diagnosis to identify those agents. The viruses of smallpox (orthopoxvirus), of hemorrhagic fever and those belonging to filovirus have been highlighted, inter alia, as agents of human infection with bioterrorist intent. Among the bacteria, the emphasis has been on anthrax (Bacillus anthracis), the plague (Yersinia pestis), botulism (Clostridium botulinum) and tularemia (Francisella tularensis), not to mention ricin (Ricinus communis), as one of the Group B agents.

Despite evidence of West Nile virus (WNV) activity in Colombia, Venezuela and Argentina, this virus has not been reported in most South American countries. In February 2009, we commenced an investigation for WNV in mosquitoes, horses and caimans from the Pantanal, Central-West Brazil. The sera of 168 horses and 30 caimans were initially tested using a flaviviruses-specific epitope-blocking enzyme-linked immunosorbent assay (blocking ELISA) for the detection of flavivirus-reactive antibodies. The seropositive samples were further tested using a plaque-reduction neutralisation test (PRNT90) for WNV and its most closely-related flaviviruses that circulate in Brazil to confirm the detection of specific virus-neutralising antibodies. Of the 93 (55.4%) blocking ELISA-seropositive horse serum samples, five (3%) were seropositive for WNV, nine (5.4%) were seropositive for St. Louis encephalitis virus, 18 (10.7%) were seropositive for Ilheus virus, three (1.8%) were seropositive for Cacipacore virus and none were seropositive for Rocio virus using PRNT90, with a criteria of > four-fold antibody titre difference. All caimans were negative for flaviviruses-specific antibodies using the blocking ELISA. No virus genome was detected from caiman blood or mosquito samples. The present study is the first report of confirmed serological evidence of WNV activity in Brazil.

A partir de 1999, infecções humanas por Orthopoxvirus vem sendo observadas em pelo menos oito estados no país, com a formação de vesículas as quais evoluem para pústulas e crostas, principalmente nos membros superiores e face, após contacto com bovinos apresentando lesões semelhantes no úbere. Alem das lesões na pele, foram descritas nos pacientes reações ganglionares axilares por vezes dolorosas, febre, cefaléia, fadiga, desidratação, anorexia, sudorese, artralgia e mialgia, evoluindo o quadro por três a quatro semanas. Lesão vulvar bem como transmissão intrafamiliar foram igualmente descritas. Estudos moleculares demonstraram que os poxvirus identificados são geneticamente relacionados a amostras do vírus vaccinia utilizadas no passado, nas campanhas de vacinação. Especimens clínicos de 80 infecções humanas foram estudados no laboratório e a infecção por orthopoxvirus confirmada em 68 casos. São apresentadas lesões observadas em pacientes bem como discutidas as implicações desta zoonose no Brasil.

Since 1999, human infection caused by Orthopoxvirus has been observed in at least eight Brazilian states, with the presence of vesicles that evolve to pustules and crusts, especially on the hands, arms and face, after contact with cows showing comparable lesions on the udder. In addition to the skin lesions, there have been descriptions of patients with axillary ganglionic reactions that are sometimes painful, along with fever, headache, fatigue, dehydration, anorexia, sudoresis, arthralgia and muscle pain. The condition evolves over a three to four-week period. Vulvar lesions and transmission within families have also been described. Molecular studies have shown that the poxviruses identified are genetically related to vaccinia virus samples that were used in vaccination campaigns in the past. Clinical specimens from 80 human infections were studied in the laboratory, and orthopoxvirus infections were confirmed in 68 cases. The lesions observed in these patients are presented and the implications of this zoonosis in Brazil are discussed.

Neste estudo avaliou-se a presença de infecções por vírus semelhantes ao vírus vaccínia (VACV) em gado leiteiro em 12 municípios no estado do Rio de Janeiro, ao longo dos últimos nove anos. Amostras clínicas foram coletadas de animais com vesículas, pústulas e crostas no úbere e tetas, e da região do nariz e da cavidade oral de bezerros. Um teste de neutralização viral por redução de placas foi desenvolvido para investigar a presença de anticorpos contra Orthopoxvirus. Os fluidos de vesículas / pústulas e as crostas foram testadas por PCR, microscopia eletrônica (ME) e por inoculação em células VERO para isolamento viral. Anticorpos contra Orthopoxvirus foram detectados na grande maioria dos animais. O teste de PCR demonstrou homologia entre os vírus isolados e amostras de vírus vaccínia usados como controles. Na ME, partículas típicas de Orthopoxvirus foram observadas em vários espécimes analisados. Os vírus isolados em cultivo celular foram confirmados como Orthopoxvirus por PCR e ME. O gene HA da amostra Cantagalo/IOC isolada em nosso laboratório foi seqüenciado e comparado com outras amostras semelhantes ao vaccínia, mostrando uma alta homologia com a amostra original Cantagalo, tendo sido as duas amostras isoladas em 1999 de gado leiteiro. Anticorpos para Orthopoxvirus foram detectados em um roedor silvestre do gênero Akodon sp. coletado na região noroeste do estado, sugerindo uma circulação de poxvírus na natureza. No entanto, os testes de PCR aplicados a tecidos de roedores silvestres foram negativos. Infecções vesiculares / pustulares em humanos que mantinham contato com os animais afetados também foram relatadas. Assim, infecções por amostras semelhantes ao vírus VACV em bovinos e em humanos parecem em expansão no estado, gerando perdas econômicas em animais e quadros de doença incapacitante temporária em pacientes humanos. Dessa forma, a possibilidade da imunização do gado leiteiro no estado deve ser devidamente avaliada.

In the present study we investigated the presence of infections by vaccinia-like viruses in dairy cattle from 12 counties in the state of Rio de Janeiro in the last 9 years. Clinical specimens were collected from adult animals with vesicular/pustular lesions mainly in the udder and teats, and from calves with lesions around the nose and mouth. A plaque reduction neutralization test (PRNT) was applied to search for antibodies to Orthopoxvirus; the vesicular/pustular fluids and scabs were examined by PCR, electron microscopy (EM) and by inoculation in VERO cells for virus isolation. Antibodies to Orthopoxvirus were detected in most cases. The PCR test indicated a high nucleotide homology among the isolates and the vaccinia viruses (VACV) used as controls. By EM, typical orthopoxvirus particles were observed in some specimens. The agents isolated in tissue culture were confirmed as vaccinia-like viruses by EM and PCR. The HA gene of the vaccinia-like Cantagalo/IOC virus isolated in our laboratory was sequenced and compared with other vaccinia-like isolates, showing high homology with the original Cantagalo strain, both strains isolated in 1999 from dairy cattle. Antibodies to Orthopoxvirus were detected in one wild rodent (genus Akodon sp.) collected in the northwestern region of the state, indicating the circulation of poxvirus in this area. Nonetheless, PCR applied to tissue samples collected from the wild rodents were negative. Vesicular/pustular lesions in people in close contact with animals have been also recorded. Thus, the vaccinia-like virus infections in cattle and humans in the state seem to be an expanding condition, resulting in economic losses to dairy herds and leading to transient incapacitating human disease. Therefore, a possible immunization of the dairy cattle in the state should be carefully evaluated.

Circulation of a new dengue virus (DENV)-3 genotype was recently described in Brazil and Colombia, but the precise classification of this genotype has been controversial. Here we perform phylogenetic and nucleotide-distance analyses of the envelope gene, which support the subdivision of DENV-3 strains into five distinct genotypes (GI to GV) and confirm the classification of the new South American genotype as GV. The extremely low genetic distances between Brazilian GV strains and the prototype Philippines/L11423 GV strain isolated in 1956 raise important questions regarding the origin of GV in South America.

Os autores relatam três casos de varíola bovina em humanos, ordenhadores manuais em vacas infectadas, na microrregião de Itajubá, MG. As técnicas diagnósticas foram: isolamento de amostra semelhante ao vírus vaccinia de secreções das lesões cutâneas, reação em cadeia de polimerase, microscopia eletrônica e anticorpos para Orthopoxvirus no sangue dos pacientes.

The authors report three human cases of cowpox infection, among farm workers who were manually milking infected cows, in the microregion of Itajubá, Minas Gerais. The diagnostic techniques used were: isolation of samples similar to the vaccinia virus, from skin lesion secretions; polymerase chain reaction; electronic microscopy; and antibodies for Orthopoxvirus in the patients' blood.

Em Pernambuco, os primeiros casos de dengue ocorreram em 1987. Após intervalo de 7 anos sem casos autóctones, em 1995 ocorreu uma nova epidemia. Foram analisados aspectos relevantes das epidemias (1995-2006), utilizando-se dados epidemiológicos, clínicos e laboratoriais. Um total de 378.374 casos foi notificado, com 612 casos confirmados de febre hemorrágica do dengue e 33 óbitos. A taxa de mortalidade foi de 5.4%. A taxa de incidência aumentou de 134 para 1.438/100.000 habitantes, correspondente às epidemias pelos sorotipos 2 e 3, em 1995 e 2002, respectivamente. O dengue acometeu principalmente pessoas adultas (20-49 anos); 40,7% do sexo masculino e 59,3%, feminino. A partir de 2003, aumentou o número de casos em menores de 15 anos. Entre 225 casos de dengue hemorrágico, foram identificadas 42,7% e 57,3% infecções primárias e secundárias, respectivamente (p = 0.0279). Manifestações neurológicas também foram observadas. A partir de 2002, circularam os sorotipos 1, 2 e 3; predominando o sorotipo 3.

In Pernambuco, the first dengue cases occurred in 1987. After a seven-year interval without autochthonous cases, a new epidemic occurred in 1995. Important aspects of the dengue epidemics during the period 1995-2006 have been analyzed here, using epidemiological, clinical and laboratory data. A total of 378,374 cases were notified, with 612 confirmed cases of dengue hemorrhagic fever and 33 deaths. The mortality rate was 5.4%. The incidence rate increased from 134 to 1,438/100,000 inhabitants, corresponding to the epidemics due to serotypes 2 and 3, in 1995 and 2002, respectively. Dengue mainly affected adults (20-49 years); 40.7% were male and 59.3% were female. From 2003 onwards, the number of cases among individuals younger than 15 years old increased. Out of 225 dengue hemorrhagic fever cases, 42.7% primary and 57.3% secondary infections were identified (p = 0.0279). Neurological manifestations were also observed. From 2002 onwards, serotypes 1, 2 and 3 were circulating; serotype 3 was predominant.

A total of 4 243 049 dengue cases have been reported in Brazil between 1981 and 2006, including 5 817 cases of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) and a total of 338 fatal cases. Although all Brazilian regions have been affected, the Northeast and Southeast regions have registered the highest number of notifications. DENV-1 and DENV-4 were isolated for the first time in the Amazon region of Brazil in 1981 and 1982. The disease became a nationwide public health problem following outbreaks of DENV-1 and DENV-2 in the state of Rio de Janeiro in 1986 and 1990, respectively. The introduction of DENV-3 in 2000, also in the state of Rio de Janeiro, led to a severe epidemic with 288 245 reported dengue cases, including 91 deaths. Virus strains that were typed during the 2002 epidemic show that DENV-3 has displaced other dengue virus serotypes and entered new areas, a finding that warrants closer evaluation. Unusual clinical symptoms, including central nervous system involvement, have been observed in dengue patients in at least three regions of the country.

En Brasil se han notificado 4 243 049 casos de dengue entre 1981 y 2006, de ellos 5 817 casos de dengue hemorrágico/síndrome de choque por dengue (DH/SCD) y un total de 338 casos mortales. A pesar de que la enfermedad ha afectado a todas las regiones brasileñas, el mayor número de casos se ha notificado en las regiones nororiental y suroriental. Los virus del dengue (DENV) 1 y 4 se aislaron por primera vez en la región amazónica de Brasil en 1981 y 1982. La enfermedad se convirtió en un problema nacional de salud pública después de los brotes de DENV-1 y DENV-2 en el Estado de Río de Janeiro en 1986 y 1990, respectivamente. La introducción del DENV-3 en 2000, también en el Estado de Río de Janeiro, llevó a una grave epidemia con 288 245 casos notificados de dengue y 91 muertes. Las cepas del virus identificadas durante la epidemia de 2002 demostraron que el DENV-3 ha desplazado a los otros serotipos y se ha expandido a nuevas zonas, algo que merece una evaluación más profunda. En los pacientes con dengue de al menos tres regiones del país se han observado síntomas clínicos atípicos, entre ellos alteraciones del sistema nervioso central.

Histological and ultrastructural alterations in lung tissue of BALB/c mice infected with dengue virus serotype 2 (non-neuroadapted), by intraperitoneal and intravenous routes were analyzed. Lung tissues were processed following the standard techniques for photonic and electron transmission microscopies. Histopathological and ultrastructural studies showed interstitial pneumonia, characterized by the presence of mononuclear cells. In the mouse model, the dengue virus serotype 2 seems to led to a transient inflammatory process without extensive damage to the interalveolar septa, but caused focal alterations of the blood-exchange barrier. Endothelial cells of blood capillaries exhibited phyllopodia suggesting activation by presence of dengue virus. Morphometrical analysis of mast cells showed an expressive increase of the number of these cells in peribronchiolar spaces and adjacent areas to the interalveolar septa. Alveolar macrophages showed particles dengue virus-like inside rough endoplasmic reticulum and Golgi complex, suggesting viral replication. The tissue alterations observed in our experimental model were similar to the observed in human cases of dengue fever and dengue hemorrhagic fever. Our results show that BALB/c mice are permissive host for dengue virus serotype 2 replication and therefore provides an useful model to study of morphological aspects of dengue virus infection.

One of the main difficulties in studying dengue virus infection in humans and in developing a vaccine is the absence of a suitable animal model which develops the full spectrum of dengue fever, dengue haemorrhagic fever, and dengue shock syndrome. It is our proposal to present morphological aspects of an animal model which shows many similarities with the dengue infection in humans. BALB/c mice were intraperitoneally infected with non-neuroadapted dengue virus serotype 2 (DENV-2). Histopathological and morphometrical analyses of liver tissue revealed focal alterations along the infection, reaching wide-ranging portal and centrolobular veins congestion and sinusoidal cell death. Additional ultrastructural observations demonstrated multifocal endothelial injury, platelet recruitment, and alterated hepatocytes. Dengue virus antigen was detected in hepatocytes and in the capillar endothelium of the central lobular vein area. Liver function tests showed high levels of aspartate transaminase and alanine transaminase enzyme activity. Lung tissue showed interstitial pneumonia and mononuclear cells, interseptal oedema, hyperplasia, and hypertrophy of the bronchiolar epithelial cells. DENV-2 led to a transient inflammatory process, but caused focal alterations of the blood-exchange barrier. Viremia was observed from 2nd to 11th day p.i. by isolation of DENV-2 in C6/36 mosquito cell line inoculated with the supernatant of macerated liver, lung, kidney, and cerebellum tissues of the infected mice.

Oral susceptibility and vertical transmission of dengue virus type 2 (DENV-2) in an Aedes albopictus sample from Rio de Janeiro was estimated. The infection (36.7%) and transmission (83.3%) rates for Ae. albopictus were higher than those of an Ae. aegypti colony used as control, 32.8 and 60%, respectively. Fourth instar larvae and females descendants of 48.5 and 39.1% of experimentally infected Ae. albopictus showed to harbor the virus. The oral susceptibility and the high capacity to assure vertical transmission exhibited by Ae. albopictus from Brazil reinforce that this species may play a role in the maintenance of the virus in nature and be a threat for dengue control in the country.

Hantavírus é o agente etiológico da síndrome cardio-pulmonar por hantavirus (SCPH), uma importante doença transmitida por roedores no Brasil. Com o objetivo de se conhecer a ocorrência de infecção por hantavírus em pequenos roedores silvestres, uma análise sorológica foi conduzida em três diferentes áreas do subúrbio da cidade de Pedreira, São Paulo. Dos 145 animais silvestres capturados, pertecentes a 12 diferentes espécies identificadas por morfologia e por análise cariológica, 107 eram roedores das seguintes espécies: Akodon montensis, Bolomys lasiurus, Calomys tener, Oligoryzomys nigripes, Oligoryzomys flavescens, e Myocastor coypus. Amostras de sangue desses roedores foram testadas para a presença de anticorpos IgG contra o antígeno do nucleocapsídeo do vírus Andes através do teste ELISA. Reatividade sorológica ao vírus Andes foi observada em duas espécies diferentes, O. nigripes and O. flavescens. Estes resultados indicam o potencial risco de transmissão de hantaví-rus nesta região, onde roedores reservatórios freqüentemente são vistos em peridomicílio.

Hantaviruses are the etiological agents of the Hantavirus Cardio-Pulmonary Syndrome, a serious rodent-borne disease in Brazil. In order to investigate the occurrence of hantavirus infection in wild rodents, a survey was conducted in three different suburban areas of the municipality of Pedreira, State of São Paulo, Brazil. Of the 145 wild animals captured belonging to 12 different species identified by morphology and karyological analysis, 107 were rodents of the following species: Akodon montensis, Bolomys lasiurus, Calomys tener, Oligoryzomys nigripes, Oligoryzomys flavescens, and Myocastor coypus. Blood samples from these rodents were assayed for the presence of antibodies against hantavirus by IgG ELISA using Andes recombinant nucleocapsid antigen. Antibody reactive to Andes virus was found in two different species, O. nigripes and O. flavescens. These results indicate a potential risk for hantavirus transmission to humans in this area, where reservoir rodents are present in peridomestic settings.

The present paper reports a laboratory investigation performed between the years of 2000 and 2002 to stydy a virological surveillance program introduced in the state of Piauí to support an epidemiological survey of the disease. Dengue virus type 3 (DENV-3) existence in the state was detected in May 2002 when a high number of dengue cases due to DENV-1 and DENV-2 were reported. An assessment on the population knowledge about the disease and its transmission showed that almost 50% of the population were still unaware of the epidemiological features of dengue.

A tipagem de cepas é uma ferramenta fundamental para a análise epidemiológica molecular e pode fornecer informações importantes sobre a dispersão dos vírus dengue. Neste trabalho, foi realizada a caracterização molecular de amostras de vírus DEN-2 isoladas no Brasil entre 1990-2000, de áreas geograficamente e temporalmente distintas, com o objetivo de investigar a distribuição genética deste sorotipo circulante no país. A reação em cadeia pela polimerase baseada em sítios de restrição específicos (RSS-PCR) apresentou o mesmo padrão para as 52 amostras Brasileiras, mostrando a circulação de apenas uma variante de vírus DEN-2. A análise filogenética utilizando alinhamento progressivo de sequências de 240 nucleotídeos da junção E/NS1 de 15 cepas mostrou que estas pertencem ao genotipo III (genotipo Jamaica).

Strain typing is a critical tool for molecular epidemiological analysis and can provide important information about the spread of dengue viruses. Here, we performed a molecular characterization of DEN-2 viruses isolated in Brazil during 1990-2000 from geographically and temporally distinct areas in order to investigate the genetic distribution of this serotype circulating in the country. Restriction site-specific polymerase chain reaction (RSS)-PCR presented the same pattern for all 52 Brazilian samples, showing the circulation of just one DEN-2 variant. Phylogenetic analysis using progressive pairwise alignments from 240-nucleotide sequences of the E/NS1 junction in 15 isolates showed that they belong to genotype III (Jamaica genotype).

Com base nas pesquisas moleculares sobre o genoma e proteínas, novas vacinas virais deverão ser utilizadas de forma rotineira nas próximas décadas. Por outro lado, espera-se que cada vez mais sejam associados diferentes antígenos imunizantes em uma mesma dose, visando a reduzir o número de aplicações de vacinas nas populações a serem imunizadas. Pela importância de sua estrutura científica e tecnológica, o Brasil deve aumentar a participação nos processos de desenvolvimento de novas vacinas e na avaliação de sua eficácia, envolvendo maior número de pesquisadores e tecnologistas, com o incremento de investimento nessas atividades.

New viral vaccines, based on molecular research into genomes and proteins, should come into routine use in the coming decades. At the same time, use of different immunizing antigens within a single dose is also expected to grow more common, with the purpose of reducing the number of vaccinations administered to target populations. Given Brazil's scientific and technological importance, the country should enhance its participation in the development of new vaccines and in the evaluation of their efficacy by involving a larger number of researchers and technologists and boosting investment in these activities.