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.

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

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 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

Prevalence and risk factors for HBV, HCV and HDV infections among injecting drug users from Rio de Janeiro, Brazil

Viral hepatitis constitutes a major health issue, with high prevalence among injecting drug users (IDUs). The present study assessed the prevalence and risk determinants for hepatitis B, C and D viruses (HBV, HCV and HDV) infections among 102 IDUs from Rio de Janeiro, Brazil. Serological markers and HCV-RNA were detected by enzyme immunoassay and nested PCR, respectively. HCV genotyping was determined by restriction fragment length polymorphism analysis (RFLP). HBsAg, anti-HBc and anti-HBs were found in 7.8, 55.8 and 24.7 percent of IDUs, respectively. In the final logistic regression, HBV infection was independently associated with male homosexual intercourse within the last 5 years (odds ratio (OR) 3.1; 95 percent confidence interval (CI) 1.1-8.8). No subject presented anti-delta (anti-HD). Anti-HCV was detected in 69.6 percent of subjects, and was found to be independently associated with needle sharing in the last 6 months (OR 3.4; 95 percent CI 1.3-9.2) and with longer duration of iv drug use (OR 3.1; 95 percent CI 1.1-8.7). These data demonstrate that this population is at high risk for both HBV and HCV infection. Among IDUs from Rio de Janeiro, unprotected sexual intercourse seems to be more closely associated with HBV infection, whereas HCV is positively correlated with high risk injecting behavior. Comprehensive public health interventions targeting this population and their sexual partners must be encouraged

Retrospective study on dengue in Fortaleza, State of Ceará, Brazil

A retrospective serologic study was carried out in Fortaleza, State of Ceará, Brazil, in order to detect the dengue virus activity before recognizing the epidemic of 1994. Mac-Elisa was performed by using a mixture of specific DEN-1 and DEN-2 antigens on serum samples from the Emilio Ribas Laboratory collection. Samples were obtained from 1,224 patients with exanthematic febrile disease and negative serological results for rubella. All specimens were taken during November 1993 to May 1994. The results confirmed dengue infections in Fortaleza by November 1993, approximately six months before the beginning of the epidemic, proving how misleading diagnosis of dengue infection are still troublesome, in spite of the strong dengue activity in Ceará. The authors stress the urgent necessity to implement the active surveillance system in order to prevent another extensive fever epidemics in the state. Epidemiological background of the dengue activity in the State of Ceará is also described.

Dengue infection in Paracambi, State of Rio de Janeiro, 1990-1995

Um inquérito soroepidemiológico foi realizado em uma amostra de escolares, em 1994, no município de Paracambi, Estado do Rio de Janeiro. Positividade do teste de Inibição da Hemaglutinação foi detectada em 39,2% (145/370) dos escolares pesquisados. A freqüência de positividade foi de 53,8% (78/145) para o sexo feminino e de 46,2% (67/145) para o sexo masculino. A distribuição por faixa etária mostrou uma positividade crescente com o aumento da idade. Cepas do vírus dengue tipo 1 e vírus dengue tipo 2 foram isoladas anteriormente (1990), mostrando a co-circulação de ambos os sorotipos na área. Os índices de infestação predial pelo Aedes aegypti e pelo Aedes albopictus foram determinados.

A seroepidemiological survey was carried out during 1994 in the municipality of Paracambi, state of Rio de Janeiro. Haemagglutination inhibition test positivity was detected in 145 out of 370 (39.2%) schoolchildren. The frequency of positive test by sex was 53.8% (78/145) female and 46.2% (67/145) male. Distribution by age showed the increasing of antibody positivity in older children. Strains of dengue virus type 1 and dengue virus type 2 were isolated before (1990) showing the co-circulation of both serotypes in that area. The house index infestation of Aedes albopictus and Aedes aegypti has been determined.

Temporal association between the isolation of Sabin-related poliovirus vaccine strains and the Guillain-Barre syndrome

Thirty eigth paralysis classified as Guillain-Barre syndrome (GBS) in Brazil were analysed. In all these cases Sabin-related poliovirus vaccine strains were isolated. In most of the cases the last vaccine dose was given months or years before the onset of GBS, suggesting a persistent infection or transmission of the Sabin-related strains to the patients...

Poliovirus type 1 isolated from a vaccine-associated case of paralytic poliomyelitis in Brazil

This study reports a type 1 poliovirus strain isolated in Brazil from a case classified as vaccine-associated paralytic poliomyelitis (VAPP). After serotyping of the viral isolate with hyperimmune equine sera, PCR and molecular hybridization techniques characterized the strain as P1/Sabin-derived. The isolate was partially sequenced to identify mutations at nucleotides 480, 525 and 6203, which are important for reversion of the P1/Sabin strain to neurovirulence. In a recent study, a P1/ Sabin-derived strain isolated from the central nervous system of a VAPP case did not mutate at these positions, but maintained 480-G and 525-U (and 6203-C), suggesting that these mutations are not essential for the occurrence of disease (Georgescu et al., (1994), Journal of Virology, 68: 8089-8101). Although the Brazilian strain also maintained 480-G and 525-U (and 6203-C) and was isolated from the stool, the possibility that this isolate invaded the central nervous system after replicating in the gut, causing the paralysis, cannot be ruled out. This is the first report of a type I VAPP case in Brazil, although some cases caused by type 2 and type 3 strains have been described.

Genomic characterization of type 2 polioviruses isolated from vaccine-associated cases in Brazil

Twenty strains of P2/Sabin-related polioviruses isolated in Brazil were analyzed; ten from persistent paralytic poliomyelitis cases, three from suspected polio cases with transient paralysis, and seven from healthy contacts. The serotypes of the viral isolates were identified by the neutralization test with hyperimmune equine sera. The relationship of the isolates to the P2/Sabin strain was demonstrated by molecular hydridization and polymerase chain reaction (PCR). Partial sequencing demonstrated mutations at nucleotide 481 in the 5' noncoding region and at amino acid 143 of the capsid protein VP1 in most of these isolates from accine-associated cases in Brazil. These data support previous studies on the importance of mutations at these attenuated determinants in the establishment of the disease. However, the existence of isolates without mutations at these positions suggests that they are not essential. The results also strengthen the possibility of the participation of a mutation at nucleotide 398 in the establishment of the disease, and suggest that a mutation at nucleotide 491 or 500 may also be involved in this process. The isolates from healthy contacts presented the same mutations as the isolates from vaccine-associated cases with they were in contact. This strengthens the observation that, although mutations in the genome of the P2/Sabin strain are important for the establishment of the disease, host factors are also involved

Intragastric infection induced in marmosets (Callithrix jacchus) by a Brazilian hepatitis A virus (HAF-203)

Several specied of non-human primates have been used in studies on experimental infection with hepatitis A virus (HAV). Attempts to infect a South-American marmoset (Callithrix jacchus) with a Brazilian HAV isolate (HAF-203) are described here. Four seronegative animals were inoculated intragastrically and one was sacrificed on day 11,20,47 and 62 after infection. One uninfected animal was included as control. Liver, small intestine, lymph node, spleen and kidney samples were collected for histological diagnosis and immunocytochemistry studies. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum enzymes and anti-HAV antibodies were monitored by a colorimetric procedure (Abbott) and an enzyme immunoassay (ELISA), respectively. Feces were collected daily for HAV antigen (HAVAg) detection by ELISA. Increased levels of HAVAg were detected in hepatocytes 11 days after infection, with a gradual decrease during the course of infection. Shedding of HAVAg in feces was observed from the late incubation to the early acute phase (20th day to 47th day after infection). The end of the incubation period was indicated by the initial increases in serum ALT and AST. Severe hepatic lesions such as piecemeal necrosis and bridging necrosis were detected during the acute phase, coinciding with the maximum transaminase levels and the appearance of anti-HAV antibodies. On the 62nd day (convalescent phase), the hepatic tissue showed evidence of regeneration and the transaminase values had returned to baselines. The serological, biochemical, antigenic and histological evidence of hepatitis A was similar to that observed in several primate models inoculated with other HAV isolates. The data suggest that C. jacchus can be a valuable model for the study of hepatitis A and for the evaluation of HAV vaccines

Genomic characterization of type 3 polioviruses isolated from vaccine-associated poliomyelitis cases in Brazil

Eight strains of P3/Sabin-related polioviruses were analyzed; four from persistent paralytic poliomyelitis cases classified as vaccine associated, one from a transient paralysis case classified as transverse myelitis, one from a transient paralysis case classified as Guillain-Barr'e syndrome, one from a transient facial paralysis case, and one from a healthy vaccine. The serotypes of the viral isolates were identified by the neutralization test with hyperimmune equine sera and the relationship of the isolates with the P3/Sabin strain was demonstrated by molecular hybridization of the viral RNA of the isolates with a P3/Sabin-specific probe. The P3/Sabin relationship was confirmed by PCR, using a pair of specific primers for P3/Sabin-related isolates. The available data indicate that a U C mutation at nucleotide 472 in the 5' noncoding region of the genome of the type 3 Sabin strain increases the neurovirulence of this strain and this mutation was observed in all type 3 isolates from vaccine-associated cases. These eight P3/Sabin-related isolates were partially sequenced in the 5" noncoding region and seven presented a U C mutation at nucleotide 472, except the isolate from a transient paralysis case classfied as transverse myelitis, that maintained a U at nucleotide 472. Although this virus maintaining U at nucleotide 472 may not be the etiological agent of the disease, the possibility that virus was the causative agent of the disease could not be ruled out

Detection and isolation of human T-cell leukemia/lymphoma virus type I (HTLV-I) from cultured lymphocytes of a Brazilian TSP/HAM patient

Some Brazilian regions are considered to be endemic for human T-cell leukemia/lymphoma virus type I (HTLV-I) infection. Several studies have shown a high prevalence of HTLV-I infection among different groups such as blood donors, hemophiliacs and patients suffering from hematological and neurological diseases. Cases of adult T -cell leukemia/lymphoma as well as tropical spastic paraparesis/HTLV-i-infected T -cell line (ROB) expressing viral antigens was established and reverse transcriptase activity could be detected in the culture supernatant. Ultrastructural analysis showed immature and mature HTLV retrovirus particles. Finally, HTLV-I provirus type I was demonstrated by the plymerase chain reaction. This is the first isolation completely carried out in Latin America. The molecular analysis of viral strains, now in progress, should clarify the molecular epidemiology of HTLV-I in Brazil

Intratypic differentiation of polioviruses isolated from suspected cases of poliomyelitis in Brazil during the period of 1990 to 1993

This study analyzed 3129 fecal samples derived from 1626 patients with sudden onset acute flaccid paralysis clinically compatible with poliomyelitis. The samples were collected in the period ranging from January 1990 to September 1993 in all regions of Brazil. Among the 1626 cases studied, 196 had isolation of poliovirus. Nevertheless, it was observed that some factors influenced the isolation rate and the intratypic characterization of these polioviruses. No cases of acute flaccid paralysis has been found to be etiologically related with wild polioviruses.

Hepatitis B and C prevalences among blood donors in the south region of Brazil

The prevalence of hepatitis B and C infection has been determined in a seroepidemiological survey among blood donors from the south of Brazil (Florianópolis, State of Santa Catarina). These markers has also been correlated with the levels of alanine aminotransferase (ALT), a surrogate marker to prevent post-transfusion hepatitis. Sera from 5000 donors were randomly collected in the period of April to November 1991. The prevalences of HBsAg, anti-HBs and anti-HBc were respectively 0.78, 7.02 and 13.98. The anti-HCV prevalence after confirmation testing with line immunoassay (LIA), was 1.14. Normal values of ALT ( < = 32 U/ml) were found in 59.78, values slightly above the mean (ALT between 32-70 U/ml) in 37.74 and high values of ALT ( > = 70 U/ml) in 2.48. The positivity of anti-HCV antibodies increased with the elevation of ALT levels. This correlation was not observed in relation to HBsAg. There exists a diversity in the recognition of HCV epitopes among HCV positive donors. Via the confirmation test used, we could observe that 94.7 of donors recognize the structural core antigen. Besides that, we observed that 5.26 of the HCV reactive sera recognized only epitopes located in the NS4 and/or NS5 region, indicating the importance of these epitopes for the improvement of assays.

Fast growth of a Brazilian hepatitis A virus (HAF-203) in a primate cell line

A hepatitis A virus (HAV, HAF-203) isolated in Brazil was submitted to 8 serial passages through fetal Rhesus kidney cells (FRhK-4). The kinetics of replication were monitored by enzyme immunoassay (EIA-HAVAg) and cDNA-RNA dot blot hybridization. The maximum level of RNA, which was observed 21 days post-infection (p.i.) during the 3rd passage, when HAVAg was still undetectable by EIA, served as a basis to establish subsequent passages every 21 days p.i. This schedule of passage resulted in a progressive reduction of time between culture infection and HAVAg and RNA production, together with an enhancement in antigen titer content of cell lysates. During the 7th passage, maximum HAVAg and RNA levels were detected at 7 days. Fourteen days after the 8th passage, clear morphological modifications appeared, suggesting a good adaptation of HAF-203 to FRhK-4 cells. Obtaining a fast-growing Brazilian HAV is very important for the development of vaccines

Human antibodies to dengue and yellow fever do not react in diagnostic assays for hepatitis C virus

Hepatitis C virus (HCV) is a recently described causative agent of the great majority of post-transfusion non A-non B hepatitis and is classified within the Flaviviridae family. Due to a high prevalence of anti-HCV and other flaviviruses circulating in Brazil, such as dengue and yellow fever, we investigated the possibility of serological cross-reactivity between these viruses. Different panels of human sera positive for dengue type 1 (9 cases) and type 2 (7 cases) from 6 patients naturally infected with yellow fever and from 94 adults vaccinated against the 17D strain of yellow fever were tested against HCV antigens used in diagnostic assays. Two enzyme immunoassay systems were tested: one, an in-house test using recombinant antigens from core, NS3 and NS5 regions of the HCV genome (Research Foundation for Microbial Disease of Osaka University, Japan); and another, using synthetic peptides representing immunodominant epitopes of structural core and non-structural NS4 and NS5 HCV regions (INNOTEST HCV Ab, Innogenetics, Belgium). A line immunoassay (INNO-LIA HCV Ab, Innogenetics, Belgium) was used as a confirmatory test. In this, HCV antigens are coated as discrete lines on a nylon strip with plastic backing. Besides 4 control lines on each strip, a total of 6 HCV lines are present: line A consists of several NS4 epitopes, line B consists of several NS5 epitopes and lines C-F contain several core epitopes. This test not only confirms but differentiates antibodies to hepatitis C virus. No positive results were detected with these tests, indicating that hepatitis C infection can be evaluated by current assays in regions where flaviviruses are endemic

Primary isolation of a brazilian strai9n of hepatitis A virus (HAF-203) and growth in a primate cell line (FRhK-4)

Although hepatitis A is endemic in Brazil, this is the first report describing the isolation of a Brazilian strain of hepatitis A virus (HAV). Fecal specimens obtained from patients in the acute phase of hepatitis A were iunoculated into fetal Rhesus kidney cell cultures (FRhK-4). Only one inoculum, denoted HAF-203, could be propagated serially. Both cell lysates and tissue culture fluids of infected cells were used as inocula and evaluated for viral antigen and RNA content by enzyme immunoassay and cDNA-RNA hybridization, respectively. Cell lysates gave better yields when used as viral inocula. After three passages, viral RNA and antigen were detected in cell lysates 4 and 14 days post-infection, respectively. Using tissue culture fluid as inoculum, the incubation period was decreased from 49 to 7 days after 4 serial passages, reflecting the adaptation of HAF-203 to growth in FRhK-4 cells. FRhK-4 cells can now be used for HAV antigen production for diagnostic assays and molecular characterization