Human and animal infections by vaccinia-like viruses in the state of Rio de Janeiro: a novel expanding zoonosis.

Since 1999, vesicular infections caused by Orthopoxvirus in humans and animals, mainly in dairy cattle, have been identified in 20 municipalities in the Rio de Janeiro state of Brazil. This paper describes studies conducted in counties of the northwestern, middle-Paraíba Valley and southern regions of the Rio de Janeiro state where 77 human, 346 bovine and 78 rodent samples were collected over the past ten years. Laboratory investigations using virus isolation, electron microscopy, molecular biology (PCR) and serological analysis confirmed Orthopoxvirus infections in 77.9% of human, 49.2% of dairy cattle and 17.9% of rodent samples. The characterisation of the Cantagalo/IOC strain reconfirmed that this virus was a vaccinia-like virus. In other regions of the Rio de Janeiro state, vesicular/pustular infections in animals and humans are suspected but these have not yet been confirmed. A continuous surveillance system has been established to monitor these regions in addition to several other states of the Brazilian Federation.

Liver injury and viremia in mice infected with dengue-2 virus.

The goal of this study was to test the feasibility of BALB/c mice as an experimental model in the study of dengue disease. BALB/c mice were intraperitoneal infected with DENV-2 obtained from a human patient. Histopathological analysis of infected animals revealed liver injury with viral antigens detection. In initial stages, the most prominent lesions were vacuolization and diffuse steatosis in hepatocytes. Serum levels of ALT and AST increased progressively, reaching the highest values 7 days p.i. and decreasing at the 14th day. Since levels of circulating virus were very low, viremia was analyzed in C6/36 cells. Virus presence was detected by ultrastructural analysis, confirmed by RT-PCR assays. Period of viremia was analyzed by flow cytometry with cells incubated with mouse-infected sera collected in different days, revealing peak virus levels at the 7th day p.i. All such data correlate to the development of the disease described in humans.

Study of hantavirus infection in captive breed colonies of wild rodents.

Wild sigmondontine rodents are known to be the reservoir of several serotypes of New World hantaviruses. The mechanism of viral transmission is by aerosol inhalation of the excreta from infected rodents. Considering that the captive breed colonies of various wild mammals may present a potential risk for hantaviral transmission, we examined 85 specimens of Thrichomys spp. (Echimyidae) and 17 speciemens of Nectomys squamipes (Sigmodontinae) from our colony for the presence of hantavirus infections. Blood samples were assayed for the presence of antibodies to Andes nucleocapsid antigen using enzyme-linked immunosorbent assay (ELISA). Additionally, serum samples from workers previously exposed to wild rodents, in the laboratories where the study was conducted, were also tested by ELISA to investigate prevalence of anti-hantavirus IgG antibodies. All blood samples were negative for hantavirus antibodies. Although these results suggest that those rodent's colonies are hantavirus free, the work emphasizes the need for hantavirus serological monitoring in wild colonized rodents and secure handling potentially infected rodents as important biosafety measures.

Study of hantavirus infection in captive breed colonies of wild rodents

Wild sigmondontine rodents are known to be the reservoir of several serotypes of New World hantaviruses. The mechanism of viral transmission is by aerosol inhalation of the excreta from infected rodents. Considering that the captive breed colonies of various wild mammals may present a potencial risk for hantaviral transmission, we examined 85 speciemens of Thrichomys spp. (Echimyidae) and 17 speciemens of Nectomys squamipes (Sigmodontinae) from our colony for the presence of hantavirus infections. Blood samples were assayed for the presence of antibodies to Andes nucleocapsid antigen using enzyme-linked immunosorbent assay (ELISA). Additionally, serum samples from workers previously exposed to wild rodents, in the laboratories where the study was conducted, were also tested by ELISA to investigate prevalence of anti-hantavirus IgG antibodies. All blood samples were negative for hantavirus antibodies. Although these results suggest that those rodent's colonies are hantavirus free, the work emphasizes the need for hantavirus serological monitoring in wild colonized rodents and secure handling potentially infected rodents as important biosafety measures.

Dengue virus surveillance: the co-circulation of DENV-1, DENV-2 and DENV-3 in the State of Rio de Janeiro, Brazil.

This study presents the results obtained in the monitoring of dengue virus (DENV) transmission in the Greater Metropolitan Region of the State of Rio de Janeiro, in the period 2000-2001. A total of 5324 serum samples from suspected cases of dengue were analysed in order to confirm dengue infection. The introduction of DENV-3 to the region in December 2000 resulted in the co-circulation of three serotypes: DENV-1, DENV-2 and DENV-3. In this study, virus isolation and/or reverse transcriptase PCR (RT-PCR) confirmed 52.3% (42/79) of DENV-3 cases, showing the importance of acute serum samples in the virological surveillance of the disease. Despite the introduction of a new serotype, an outbreak due to DENV-1 was observed in the municipality of Niteroi. The restriction site-specific PCR (RSS-PCR) patterns obtained for DENV-1 and DENV-2 isolated in that period showed that those strains belonged to the subtypes previously circulating in the state. DENV-3 RSS-PCR patterns confirmed that these viruses belonged to subtype C (Sri Lanka/India strains), represented by the strain circulating on the American continent. These data showed the importance of an active surveillance programme in countries where dengue is endemic.

Human immunodeficiency virus type 1: drug resistance in treated and untreated Brazilian children.

Twenty-two vertically human immunodeficiency virus type 1 (HIV-1) infected Brazilian children were studied for antiretroviral drug resistance. They were separated into 2 groups according to the administration of antiretroviral therapy into those who presented disease symptoms or without symptoms and no therapy. Viral genome sequencing reactions were loaded on an automated DNA sampler (TruGene, Visible Genetics) and compared to a database of wild type HIV-1. In the former group 8 of 12 children presented isolates with mutations conferring resistance to protease inhibitors (PIs), 7 presented isolates resistant to nucleoside reverse transcriptase inhibitors (NRTIs) and 2 presented isolates resistant to non-nucleoside reverse transcriptase inhibitors (NNRTIs). Ten children were included in the antiretroviral na ve group. Eight were susceptible to NRTIs and all of them were susceptible to PIs; one presented the V108I mutation, which confers low-level resistance to NNRTIs. The data report HIV mutant isolates both in treated and untreated infants. However, the frequency and the level of drug resistance were more frequent in the group receiving antiretroviral therapy, corroborating the concept of selective pressure acting on the emergence of resistant viral strains. The children who presented alterations at polymorphism sites should be monitored for the development of additional mutations occurring at relevant resistance codons.

Human immunodeficiency virus type 1: drug resistance in treated and untreated Brazilian children

Twenty-two vertically human immunodeficiency virus type 1 (HIV-1) infected Brazilian children were studied for antiretroviral drug resistance. They were separated into 2 groups according to the administration of antiretroviral therapy into those who presented disease symptoms or without symptoms and no therapy. Viral genome sequencing reactions were loaded on an automated DNA sampler (TruGene, Visible Genetics) and compared to a database of wild type HIV-1. In the former group 8 of 12 children presented isolates with mutations conferring resistance to protease inhibitors (PIs), 7 presented isolates resistant to nucleoside reverse transcriptase inhibitors (NRTIs) and 2 presented isolates resistant to non-nucleoside reverse transcriptase inhibitors (NNRTIs). Ten children were included in the antiretroviral naﶥ group. Eight were susceptible to NRTIs and all of them were susceptible to PIs; one presented the V108I mutation, which confers low-level resistance to NNRTIs. The data report HIV mutant isolates both in treated and untreated infants. However, the frequency and the level of drug resistance were more frequent in the group receiving antiretroviral therapy, corroborating the concept of selective pressure acting on the emergence of resistant viral strains. The children who presented alterations at polymorphism sites should be monitored for the development of additional mutations occurring at relevant resistance codons

Outbreak of jaundice and hemorrhagic fever in the Southeast of Brazil in 2001: detection and molecular characterization of yellow fever virus.

Between January and March 2001, an outbreak of jaundice and hemorrhagic fever occurred in the state of Minas Gerais, Southeast region of Brazil, in which a mortality rate of 53% was reported. Seroconversion, virus isolation, histopathological and immunohistochemical findings, and reverse transcription-polymerase chain reaction (RT-PCR) identified yellow fever virus (YFV) as the etiological agent responsible for the outbreak. Partial nucleotide sequence analysis from a fragment of the YFV genome spanning parts of nonstructural (NS) 5 gene and 3' noncoding region (3' UTR) showed that the YFV involved in this outbreak belongs to South American genotype I and differs from the Brazilian virus identified in 1996.

Genetic characterization of dengue virus type 3 isolates in the State of Rio de Janeiro, 2001.

The genetic characterization of dengue virus type 3 (DEN-3) strains isolated from autochthonous cases in the State of Rio de Janeiro, Brazil, in 2001 is presented. Restriction site-specific (RSS)-PCR performed on 22 strains classified the Brazilian DEN-3 viruses as subtype C, a subtype that contains viruses from Sri Lanka, India, Africa and recent isolates from Central America. Nucleic acid sequencing (positions 278 to 2550) of one DEN-3 strain confirmed the origin of these strains, since genotype III - classified by sequencing - and RSS-PCR subtype C are correlated. This genetic subtype has been associated with hemorrhagic dengue epidemics and the information provided here could be useful to implement appropriate prevention and control measures.

Genetic characterization of dengue virus type 3 isolates in the State of Rio de Janeiro, 2001

The genetic characterization of dengue virus type 3 (DEN-3) strains isolated from autochthonous cases in the State of Rio de Janeiro, Brazil, in 2001 is presented. Restriction site-specific (RSS)-PCR performed on 22 strains classified the Brazilian DEN-3 viruses as subtype C, a subtype that contains viruses from Sri Lanka, India, Africa and recent isolates from Central America. Nucleic acid sequencing (positions 278 to 2550) of one DEN-3 strain confirmed the origin of these strains, since genotype III - classified by sequencing - and RSS-PCR subtype C are correlated. This genetic subtype has been associated with hemorrhagic dengue epidemics and the information provided here could be useful to implement appropriate prevention and control measures

Dengue virus infection of the central nervous system (CNS): a case report from Brazil.

Dengue infection that is accompanied by unusual complications has been described in Brazil. We report on the presence of dengue virus in the central nervous system (CNS) of a patient who died in 1998 in Rio Grande do Norte, northeast Brazil. DEN-2 viruses were isolated from the brain liver, and lymphnode tissue of a 67-year-old man whose signs and symptoms were those of dengue infection and a secondary immune response. A postmortem revealed nose bleeds a liver that was brownish with yellow areas, and pulmonary and cerebrae congestion. Immunoperoxidase staining showed a dengue antigen-specific positive reaction in the gray matter cells of the cerebrall cortex; a granular citoplasmatic reaction was seen in the neurons. Dengue infection should always be considered as a cause encephalitis in tropical countries, especially in those where the disease is endemic.

Detection of hepatitis B virus antigens in paraffin-embedded liver specimens from the Amazon region, Brazil.

Hepatic viscerotomy of paraffin-preserved old specimens, collected in the period from 1934 to 1967, were analyzed by immunohistochemical assays to detect hepatitis B, hepatitis D, dengue and yellow fever virus antigens. The material belongs to the Yellow Fever Collection, Department of Pathology, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil and the cases were diagnosed at that time according to clinical aspects and histopathological findings reporting viral hepatitis, yellow fever, focal necrosis and hepatic atrophy. From the 79 specimens, 69 were collected at the Labrea Region and the other 10 in difFerent other localities in the Amazon Region. The five micra thick histological slices were analyzed for the presence of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) by immunoperoxidase technique. An immunofluorescence assay was applied to the detection of hepatitis D, yellow fever and dengue virus antigens. Nine (11.4%) histological samples were HBsAg reactive and 5 (6.3%) were HBcAg reactive. The oldest reactive sample was from 1934. Viral antigens related to the other pathologies were not detected in this study. Our results confirm that the methodology described may be used to elucidate the aetiology of hepatitis diseases even after a long time of conservation of the specimens.

Experimental hepatitis A virus (HAV) infection in Callithrix jacchus: early detection of HAV antigen and viral fate.

Common marmosets (Callithrixjacchus) were orally inoculated with a Brazilian strain (HAF-203) of hepatitis A virus (HAy). Three monkeys were euthanized at postinoculation hours 6, 12 and 24 to investigate the early events of HAV infection. Following others three inoculated and one control marmosets remained throughout the 46 day to evaluation of viral excretion. Different samples were collected to detect sequential presence of HAV RNA by nested reverse transcription-polymerase chain reaction (RT-PCR) in liver, saliva, bile and stools at 6 hours to 461h days postinoculation. Liver tissues were examined by immunofluorescence assay in a confocal laser-scanning microscope for the presence of HAV antigen. HAV RNA was detected in saliva during the course of the study, in bile from 24 hours to 46 days. in stools from 7 to 46 days and liver at 12 hours postinfection. In immunofluorescence of liver stained preparations, viral antigen was present at six hours after inoculation throughout the remainder of the 46-day study. The animals developed histological and biochemical acute hepatitis after second week postinoculation. Spleen, duodenum, and mesenteric lymph nodes specimens were negative for HAV antigens. This study supports the possibility that in Callithrixjacchus orally inoculated with hepatitis A virus the saliva route may be additional way of viral elimination. The viral replication in the liver was responsible for biliary HAV presence and latter HAV detection in fecal samples.

Detection of hepatitis B virus antigens in paraffin-embedded liver specimens from the Amazon Region, Brazil

Hepatic viscerotomy of paraffin-preserved old specimens, collected in the period from 1934 to 1967, were analyzed by immunohistochemical assays to detect hepatitis B, hepatitis D, dengue and yellow fever virus antigens. The material belongs to the Yellow Fever Collection, Department of Pathology, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil and the cases were diagnosed at that time according to clinical aspects and histopathological findings reporting viral hepatitis, yellow fever, focal necrosis and hepatic atrophy. From the 79 specimens, 69 were collected at the Labrea Region and the other 10 in different other localities in the Amazon Region. The five micra thick histological slices were analyzed for the presence of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) by immunoperoxidase technique. An immunofluorescence assay was applied to the detection of hepatitis D, yellow fever and dengue virus antigens. Nine (11.4 percent) histological samples were HBsAg reactive and 5 (6.3 percent) were HBcAg reactive. The oldest reactive sample was from 1934. Viral antigens related to the other pathologies were not detected in this study. Our results confirm that the methodology described may be used to elucidate the aetiology of hepatitis diseases even after a long time of conservation of the specimens

Dengue virus type 3 in Rio de Janeiro, Brazil.

Dengue virus type 3 was isolated for the first time in the country as an indigenous case from a 40 year-old woman presenting signs and symptoms of a classical dengue fever in the municipality of Nova Iguaçu, State of Rio de Janeiro. This serotype has been associated with dengue haemorrhagic epidemics and the information could be used to implement appropriate prevention and control measures. Virological surveillance was essential in order to detected this new serotype.

[Emerging and reemerging viral diseases]. / Viroses emergentes e reemergentes.

Recent decades have witnessed previously unknown viruses like HIV, along with other previously controlled viruses like dengue. The most important mechanisms have been the emergence of new viral strains by genetic alterations, the breakdown of species barriers by viruses, and viral spread from ecological niches. The main factors facilitating such mechanisms have been demographic pressure, with the expansion of the agricultural frontier, social behavior patterns, intensive air traffic, transporting both vectors and infected humans, importation of animals carrying the viruses, large-scale ecological alterations like dam- and road-building, and the widespread transformation of health systems, with a reduction in resources and infrastructure for disease control activities. Discussions on an international scale have recommended investments in the areas of Epidemiological Surveillance, Research Applied to Public Health, an emphasis on disease prevention and vector control measures, and infrastructure improvements in the health sector at the local, State, and federal level to reduce the impact of these viral diseases.

[Smallpox, an old foe]. / A varíola, uma antiga inimiga.

Smallpox has accompanied mankind for centuries, causing deaths and permanent lesions. Used in the past as a biological weapon during wars, it has come into focus again precisely because of this renewed possibility, although the disease has been eradicated in the Americas since 1971 and worldwide since 1977. Data gathered during the eradication campaigns show that the disease spread relatively slowly through close contacts between patients and susceptibles. Sub-clinical infection in non-vaccinated individuals was a rare event, and blockade vaccination surrounding new cases (as long as these cases were confirmed early) was able to prevent the disease from spreading in the community. Even with only one dose, vaccinated individuals rarely developed a serious case of the disease upon reinfection. The use of smallpox as a biological weapon should be considered a real possibility, although according to the available data, highly virulent viral suspensions spread very close to the target population would be necessary to infect a large number of persons.

Detection of poxvirus in cattle associated with human cases in the State of Rio de Janeiro: preliminary report.

This preliminary report describes human and cow cases of poxvirus that recently occurred in the State of Rio de Janeiro. The electron microscopic findings were consistent with parapoxviral and orthopoxviral infection. Orthopoxvirus strains were isolated from human and cow cases. Detailed viral characterization by means of genetical techniques is under investigation. Based on these informations, poxviral diseases should be also considered an emerging viral zoonosis that can affect human beings.

Inducible nitric oxide synthase (iNOS) expression in liver and splenic T lymphocyte rise are associated with liver histological damage during experimental hepatitis A virus (HAV) infection in Callithrix jacchus.

Callithrix jacchus is considered a reliable animal model for hepatitis A virus (HAV) infection. All three HAV orally inoculated marmosets developed hepatitis - the infection was monitored by continuous virus shedding, high levels of serum enzyme alanine aminotransferase, specific antibody and seroconversion 3-6 weeks after HAV inoculation. HAV antigen was detected in liver by immunofluorescence 4 days post inoculation (PI) and onwards. To gain insight into the biological role of inducible nitric oxide synthase (iNOS) during immune-related acute liver injury the enzyme was searched in frozen biopsies: immunofluorescent labeling was found in the cytoplasm of liver cells mainly Kupffer's cells and spleen macrophages (CD68+) starting 11 days PI with maximum intensity on the fifth to sixth week PI. Necroinflammatory liver lesions characteristic of viral hepatitis were also observed at 10 days PI with maximum severity at 4 to 6 weeks PI. Furthermore, T lymphocytes (CD2+) were raised at this time point. No difference was evident in the frequency of B lymphocytes (CD20+). Therefore, iNOS expression preceded necroinflammatory liver lesion and maximal immunofluorescence reaction was coincident with tissue injury, supporting the hypothesis that NO contributes to hepatic cytotoxic mechanism but also to virus clearance. The concomitant rise in T-lymphocyte population may suggest a role for these cells in this and/or other independent HAV-induced pathological changes.