Human vaccinia-like virus outbreaks in São Paulo and Goiás States, Brazil: virus detection, isolation and identification

Since October 2001, the Adolfo Lutz Institute has been receiving vesicular fluids and scab specimens of patients from Paraíba Valley region in the São Paulo and Minas Gerais States and from São Patricio Valley, in the Goiás State. Epidemiological data suggested that the outbreaks were caused by Cowpox virus or Vaccinia virus. Most of the patients are dairy milkers that had vesiculo-pustular lesions on the hands, arms, forearms, and some of them, on the face. Virus particles with orthopoxvirus morphology were detected by direct electron microscopy (DEM) in samples of 49 (66.21%) patients of a total of 74 analyzed. Viruses were isolated in Vero cell culture and on chorioallantoic membrane (CAM) of embryonated chicken eggs. Among 21 samples submitted to PCR using primers for hemagglutinin (HA) gene, 19 were positive. Restriction digestion with TaqI resulted in four characteristic Vaccinia virus fragments. HA nucleotide sequences showed 99.9% similarity with Cantagalo virus, described as a strain of Vaccinia virus. The only difference observed was the substitution of one nucleotide in the position 616 leading to change in one amino acid of the protein in the position 206. The phylogenetic analysis showed that the isolates clustered together with Cantagalo virus, other Vaccinia strains and Rabbitpox virus.

Notification of the first isolation of Cacipacore virus in a human in the State of Rondônia, Brazil

Flavivirus is a genus of arthropod-transmitted viruses of the family Flaviviridae, and in Brazil, up to eleven different Flavivirus have been isolated. We collected blood from farmers in the municipality of Theobroma, which is located 320km from the City of Porto Velho, the former capital of the Brazilian State of Rondônia. For viral isolation, we used newborn mouse brain, followed by RT-PCR with specific universal Flavivirus primers. We obtained fragments 958bp and 800bp in length. Based on BLAST, these sequences were 91% similar to a sequence of Cacipacore virus.

Primate populations in continuous forest and forest fragments in Central Amazonia

Population densities of six primate species (Saguinus midas, Pithecia pithecia, Cebus apella, Chiropotes satanas, Alouatta seniculus and Ateles paniscus) were estimated in continuous forest and in isolated reserves (one of 100 ha and four of 10 ha). Saguinusdensities in the continuous forest were found to be low, probably due to the lack of edge habitat and second growth favoured by them; Pithecia, Cebus and Ateles populations are also low, possibly because of more widely distributed and/or less abundant food sources than is true for other Amazonian regions, although hunting in the past, particularly of Ateles may also be a contributing factor; and Chiropotes and Alouatta densities were found to be similar to those observed in other areas of Amazonas forests. Ateles and Chiropotes, which occupy ranges on the order of three km2 were excluded from the 100-ka reserve at the time of its isolation. Unfortunately populations were not known prior to isolation of this reserve but during isolation there remained four groups of Saguinus, two Pitheciagroups, one Cebus groups and five Alouatta groups. One Saguinus group disappeared two months later, and one year post-isolation the Cebus group also left the reserve. Single Alouatta groups survive in the isolated 10-ha reserves. Saguinus, present in the four 10-ha reserves following isolation, have disappeared from two of them. One 10-ha reserve retains a group of Pithecia.

The persistence of ant-following birds in small amazonian forest fragments

SUMMARYMist-net captures of army ant-following bird were monitored during the isolation of central Amazonian forest fragments of 1 ha (n = 5), 10 ha (n = 4) and 100 ha (n = 1). Post-isolation captures of the three obligate ant-following birds Dendrocinela merula, Pithys albifrons, and Gymnopithys rufigula decreased significantly in all fragments. post-isolation captures of four facultative ant-following species were not significantly different in forest fragments of 1 ha and 10 ha, although two species decreased significantly in the 100 ha fragment. Experimental introductions of obligate species into small forest fragments in the absence and presence of introduced Eciton burchelli army ant colonies resulted in significantly greater recaptures of introduced birds when active army ant colonies were present. Of the 105 birds introduced, 58 ' 55%) crossed 100-320 m od dedorested area an were recaptured in continuous forest.

RS virus diagnosis: comparison of isolation, immunofluorescence and enzyme immunoassay

Two techniques for rapid diagnosis, immunofluorescence (IFAT) and enzyme immunoassay (EIA), have been compared with virus isolaion in tissue culture for the detection of respiratory syncytial virus (RSV) in specimens of nasopharyngeal secretions. The specimens were obtained from children under five years of age suffering from acute respiratory iliness, during a period of six months from January to June 1982. Of 471 specimens examined 54 (11.5%) were positive by virus isolation and 180 (38.2%) were positive by immunofluorescence. The bacterial contamination of inoculated tissue cultures unfortunately prevented the isolation of virus from many samples. Specimens from 216 children were tested to compare enzyme immunoassay and immunofluorescence. Of these 60 (27%) were positive by EIA and 121 (56%) were positive by IFAT. Our results suggest that the EIA technique although highly specific is rather insensitive. This may be because by the time these tests were done the originl nasopharyngeal secretions were considerably diluted and contained more mucus fragments than the call suspension used for IFAT. Of the three techniques, IFAT gives the best results although EIA may be useful where IFAT is not possible.

Isolation of a distally located gene possibly correlated with gametocyte production ability

Previous studies were focussed on the attempt to correlate observable variations in the size of Plasmodium berghei chromosomes with the loss of ability to produce viable gametocytes. A temporal coincidence between the appearance of a subtelomeric deletion on P. berghei chromosome 5 and the loss of the ability to produce viable gametocytes was observed in a clone (HPE) directly derived from the high gametocyte-producer clone 8417 during mechanical passages. Interestingly enough, three P. berghei sexual-specific genes have already been mapped on internal fragments of this chromosome. A novel gene, clone 150, isolated from a genomic library of clone 8417 using a probe enriched for sexual-specific transcripts, maps on chromosome 5 within 100kb from the telomere. Subtelomeric deletions of chromosome 5 affecting two non-producer clones involve part of the transcribed region of this gene.

Human scFv antibody fragments specific for the epithelial tumour marker MUC-1, selected by phage display on living cells.

New anti-cancer agents are being developed that specifically recognise tumour cells. Recognition is dependent upon the enhanced expression of antigenic determinants on the surface of tumour cells. The tumour exposure and the extracellular accessibility of the mucin MUC-1 make this marker a suitable target for tumour diagnosis and therapy. We isolated and characterised six human scFv antibody fragments that bound to the MUC-1 core protein, by selecting a large naive human phage display library directly on a MUC-1-expressing breast carcinoma cell line. Their binding characteristics have been studied by ELISA, FACS and indirect immunofluorescence. The human scFv antibody fragments were specific for the tandem repeat region of MUC-1 and their binding is inhibited by soluble antigen. Four human scFv antibody fragments (M2, M3, M8, M12) recognised the hydrophilic PDTRP region of the MUC-1 core protein, which is thought to be an immunodominant region. The human scFv antibody fragments were stable in human serum at 37 degrees C and retained their binding specificity. For imaging or targeting to tumours over-expressing MUC-1, it might be feasible to use these human scFv, or multivalent derivatives, as vehicles to deliver anti-cancer agents.

Chronic delivery of antibody fragments using immunoisolated cell implants as a passive vaccination tool.

BACKGROUND: Monoclonal antibodies and antibody fragments are powerful biotherapeutics for various debilitating diseases. However, high production costs, functional limitations such as inadequate pharmacokinetics and tissue accessibility are the current principal disadvantages for broadening their use in clinic. METHODOLOGY AND PRINCIPAL FINDINGS: We report a novel method for the long-term delivery of antibody fragments. We designed an allogenous immunoisolated implant consisting of polymer encapsulated myoblasts engineered to chronically release scFv antibodies targeted against the N-terminus of the Aβ peptide. Following a 6-month intracerebral therapy we observed a significant reduction of the production and aggregation of the Aβ peptide in the APP23 transgenic mouse model of Alzheimer's disease. In addition, functional assessment showed prevention of behavioral deficits related to anxiety and memory traits. CONCLUSIONS AND SIGNIFICANCE: The chronic local release of antibodies using immunoisolated polymer cell implants represents an alternative passive vaccination strategy in Alzheimer's disease. This novel technique could potentially benefit other diseases presently treated by local and systemic antibody administration.

Isolation of a fragment homologous to the rp49 constitutive gene of Drosophila in the Neotropical malaria vector Anopheles aquasalis (Diptera: Culicidae)

The constitutive ribosomal gene rp49 is frequently used as an endogenous control in Drosophila gene expression experiments. Using the degenerate primer PCR technique we have cloned a fragment homologous to this gene in Anopheles aquasalis Curry, a Neotropical vector of malaria. In addition, based on this first sequence, a new primer was designed, which allowed the isolation of fragments of rp49 in two other species, Aedes aegypti (Linnaeus) and Culex quinquefasciatus Say, suggesting that it could be used to clone fragments of this gene in a number of other mosquito species. Primers were also designed to specifically amplify rp49 cDNA fragments in An. aquasalis and Ae. aegypti, showing that rp49 could be used as a good constitutive control in gene expression studies of these and other vectorially important mosquito species.

Lesion aspirate culture for the diagnosis and isolation of Leishmania spp. from patients with cutaneous leishmaniasis

The detection of Leishmania spp. in skin lesion aspirates, using a puncture technique, was evaluated in 76 patients with cutaneous leishmaniasis (CL) who were referred to a Leishmaniasis Reference Centre in Brazil. CL was defined based on skin lesions suggestive of the disease and on a positive result of the Montenegro skin test or Giemsa-stained imprints of biopsy fragments. The aspirates were cultured using a vacuum tube device containing culture medium and evaluated for the presence of Leishmania spp. The biphasic medium culture was examined once a week for three weeks. Promastigotes were observed in 53/76 (69.7%) cultures. Stained smears from 60 of the 76 patients were evaluated using PCR-RFLP to detect the conserved minicircle region of Leishmania spp. and to classify the parasite. Of these patients, 45 (75%) showed positive results in aspirate culture and 15 presented negative results. The PCR was positive in 80% (53/60) samples. The PCR-RFLP profile was determined in 49 samples, of which 45 (92%) showed a pattern compatible with Leishmania (Viannia) braziliensis. The aspirate culture is a sensitive and feasible method for diagnosing CL and may be routinely adopted by health services for L. (V.) braziliensis isolation and identification.

Alpha 3 domain mutants of peptide/MHC class I multimers allow the selective isolation of high avidity tumor-reactive CD8 T cells.

The goal of adoptive T cell therapy in cancer is to provide effective antitumor immunity by transfer of selected populations of tumor Ag-specific T cells. Transfer of T cells with high TCR avidity is critical for in vivo efficacy. In this study, we demonstrate that fluorescent peptide/MHC class I multimeric complexes incorporating mutations in the alpha3 domain (D227K/T228A) that abrogate binding to the CD8 coreceptor can be used to selectively isolate tumor Ag-specific T cells of high functional avidity from both in vitro expanded and ex vivo T cell populations. Sorting, cloning, and expansion of alpha3 domain mutant multimer-positive CD8 T cells enabled rapid selection of high avidity tumor-reactive T cell clones. Our results are relevant for ex vivo identification and isolation of T cells with potent antitumor activity for adoptive T cell therapy.

Gene transfer into the airway epithelium of animals by targeting the polymeric immunoglobulin receptor.

Genes of interest can be targeted specifically to respiratory epithelial cells in intact animals with high efficiency by exploiting the receptor-mediated endocytosis of the polymeric immunoglobulin receptor. A DNA carrier, consisting of the Fab portion of polyclonal antibodies raised against rat secretory component covalently linked to poly-L-lysine, was used to introduce plasmids containing different reporter genes into airway epithelial cells in vivo. We observed significant levels of luciferase enzyme activity in protein extracts from the liver and lung, achieving maximum values of 13,795 +/- 4,431 and 346,954 +/- 199,120 integrated light units (ILU) per milligram of protein extract, respectively. No luciferase activity was detected in spleen or heart, which do not express the receptor. Transfections using complexes consisting of an irrelevant plasmid (pCMV lacZ) bound to the bona fide carrier or the expression plasmid (pGEMluc) bound to a carrier based on an irrelevant Fab fragment resulted in background levels of luciferase activity in all tissues examined. Thus, only tissues that contain cells bearing the polymeric immunoglobulin receptor are transfected, and transfection cannot be attributed to the nonspecific uptake of an irrelevant carrier-DNA complex. Specific mRNA from the luciferase gene was also detected in the lungs of transfected animals. To determine which cells in the lungs are transfected by this method, DNA complexes were prepared containing expression plasmids with genes encoding the bacterial beta-galactosidase or the human interleukin 2 receptor. Expression of these genes was localized to the surface epithelium of the airways and the submucosal glands, and not the bronchioles and alveoli. Receptor-mediated endocytosis can be used to introduce functional genes into the respiratory epithelium of rats, and may be a useful technique for gene therapy targeting the lung.

Identification and isolation of two ascomycete fungi from spores of the arbuscular mycorrhizal fungus Scutellospora castanea.

Two filamentous fungi with different phenotypes were isolated from crushed healthy spores or perforated dead spores of the arbuscular mycorrhizal fungus (AMF) Scutellospora castanea. Based on comparative sequence analysis of 5.8S ribosomal DNA and internal transcribed spacer fragments, one isolate, obtained from perforated dead spores only, was assigned to the genus Nectria, and the second, obtained from both healthy and dead spores, was assigned to Leptosphaeria, a genus that also contains pathogens of plants in the Brassicaceae. PCR and randomly amplified polymorphic DNA-PCR analyses, however, did not indicate similarities between pathogens and the isolate. The presence of the two isolates in both healthy spores and perforated dead spores of S. castanea was finally confirmed by transmission electron microscopy by using distinctive characteristics of the isolates and S. castanea. The role of this fungus in S. castanea spores remains unclear, but the results serve as a strong warning that sequences obtained from apparently healthy AMF spores cannot be presumed to be of glomalean origin and that this could present problems for studies on AMF genes.

Vitellogenin B2 gene in Xenopus laevis: isolation, in vitro transcription and relation to other vitellogenin genes.

The isolation of the four Xenopus laevis vitellogenin genes has been completed by the purification from a DNA library of the B2 gene together with its flanking sequences. The overlapping DNA fragments analyzed cover 34 kilobases. The B2 gene which has a length of 17.5 kilobases was characterized by heteroduplex and R-loop mapping in the electron microscope and by in vitro transcription in a HeLa whole-cell extract. Its structural organization is compared with that of the closely related B1 gene. The mRNA-coding sequence of about 6 kilobases is interrupted 34 times in the B1 gene and 33 times in the B2 gene. Sequence homology between the two genes was not only found in exons. In addition, 54% of the intron sequences as well as 63% and 48.5% respectively of the 5' and 3' flanking sequences, show enough homology to form stable duplexes. These findings are compared with earlier results obtained with the two other closely related members of the vitellogenin gene family, the A1 and the A2 genes.

INOCULATION AND ISOLATION OF PLANT GROWTH-PROMOTING BACTERIA IN MAIZE GROWN IN VITÓRIA DA CONQUISTA, BAHIA, BRAZIL

Maize is among the most important crops in the world. This plant species can be colonized by diazotrophic bacteria able to convert atmospheric N into ammonium under natural conditions. This study aimed to investigate the effect of inoculation of the diazotrophic bacterium Herbaspirillum seropedicae (ZAE94) and isolate new strains of plant growth-promoting bacteria in maize grown in Vitória da Conquista, Bahia, Brazil. The study was conducted in a greenhouse at the Experimental Area of the Universidade Estadual do Sudoeste da Bahia. Inoculation was performed with peat substrate, with and without inoculation containing strain ZAE94 of H. seropedicae and four rates of N, in the form of ammonium sulfate (0, 60, 100, and 140 kg ha-1 N). After 45 days, plant height, dry matter accumulation in shoots, percentage of N, and total N (NTotal) were evaluated. The bacteria were isolated from root and shoot fragments of the absolute control; the technique of the most probable number and identification of bacteria were used. The new isolates were physiologically characterized for production of indole acetic acid (IAA) and nitrogenase activity. We obtained 30 isolates from maize plants. Inoculation with strain ZAE94 promoted an increase of 14.3 % in shoot dry mass and of 44.3 % in NTotal when associated with the rate 60 kg ha-1 N. The strains N11 and N13 performed best with regard to IAA production and J06, J08, J10, and N15 stood out in acetylene reduction activity, demonstrating potential for inoculation of maize.