Complete nucleotide sequence analysis of a Brazilian dengue virus type 2 strain

In the last decade, dengue fever (DF) in Brazil has been recognized as an important public health problem, and an increasing number of dengue haemorrhagic fever (DHF) cases have been reported since the introduction of dengue virus type 2 (DEN-2) into the country in 1990. In order to analyze the complete genome sequence of a DEN-2 Brazilian strain (BR64022/98), we designed primers to amplify contiguous segments of approximately 500 base pairs across the entire sequence of the viral genome. Twenty fragments amplified by reverse transcriptase-PCR were cloned, and the complete nucleotide and the deduced amino acid sequences were determined. This constitutes the first complete genetic characterization of a DEN-2 strain from Brazil. All amino acid changes differentiating strains related to the Asian/American-Asian genotype were observed in BR64022/98, indicating the Asiatic origin of the strain.

Antagonistic activity expressed by Shigella sonnei: identification of a putative new bacteriocin

Bacteriocins are antibacterial, proteinaceous substances that mediate microbial dynamics. Bacteriocin production is a highly disseminated property among all major lineages of bacteria, including Shigella. In this paper, we addressed the purification and characterisation of a bacteriocin produced by a Shigella sonnei strain (SS9) isolated from a child with acute diarrhoea. The substance was purified through ammonium-sulphate precipitation and sequential steps of chromatography. The intracellular fraction obtained at 75% ammonium sulphate maintained activity following exposure to pH values from 1-11 and storage at -80ºC for more than two years and was inactivated by high temperatures and proteases. The molecular mass of the purified bacteriocin was determined by mass spectrometry to be 18.56 kDa. The N-terminal sequence of the bacteriocin did not match any other antibacterial proteins described. A putative new bacteriocin produced by S. sonnei has been detected. This bacteriocin may represent a newly described protein or a previously described protein with a newly detected function. Considering that SS9 expresses antagonism against other diarrhoeagenic bacteria, the bacteriocin may contribute to S. sonnei virulence and is potentially applicable to either preventing or controlling diarrhoeal disease.

A single-step purification of bothropstoxin-1

Bothrops venoms are complex mixtures of components with a wide range of biological activities. Among these substances, myotoxins have been investigated by several groups. Bothropstoxin-1 (Bthtx-1) is a phospholipase A2-like basic myotoxin from Bothrops jararacussu. The purification of this component involves two chromatographic steps. Although providing a pure material, the association of these two steps is time consuming and a single-step method using high performance chromatography media would be useful. In the present study, we describe a single-step purification method for Bthtx-1. Bothrops jararacussu venom was dissolved in 1 ml buffer. After centrifugation, the supernatant was injected into a Resource-S cation exchange column connected to an FPLC system and eluted with a linear salt gradient. The complete procedure took 20 min, representing a considerable time gain when compared to a previously described method (Homsi-Brandenburgo MI et al. (1988) Toxicon, 26: 615-627). Bthtx-1 purity and identity, assessed by SDS-PAGE and N-terminal sequencing, resulted in a single band with a molecular mass of about 14 kDa and the expected sequence of the first 5 residues, S-L-F-E-L. Although the amount of protein purified after each run is lower than in the previously described method, we believe that this method may be useful for small-scale purifications.

Partial sequence and toxic effects of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera

A neurotoxic peptide, granulitoxin (GRX), was isolated from the sea anemone Bunodosoma granulifera. The N-terminal amino acid sequence of GRX is AKTGILDSDGPTVAGNSLSGT and its molecular mass is 4958 Da by electrospray mass spectrometry. This sequence presents a partial degree of homology with other toxins from sea anemones such as Bunodosoma caissarum, Anthopleura fuscoviridis and Anemonia sulcata. However, important differences were found: the first six amino acids of the sequence are different, Arg-14 was replaced by Ala and no cysteine residues were present in the partial sequence, while two cysteine residues were present in the first 21 amino acids of other toxins described above. Purified GRX injected ip (800 µg/kg) into mice produced severe neurotoxic effects such as circular movements, aggressive behavior, dyspnea, tonic-clonic convulsion and death. The 2-h LD50 of GRX was 400 ± 83 µg/kg.

Identification of Cryptosporidium spp. oocysts in fecal smears stained with Heidenhain's iron hematoxylin

There is no paucity of methods for diagnosing Cryptosporidium spp. infection. The merits of immunoassays notwithstanding, microscopic identification of Cryptosporidium spp. oocysts in fecal samples remains an important diagnostic procedure. It owes the persistence of its use to such characteristics as dispensing with expensive equipment and kits, requiring only basic laboratory facilities, and having a low probability of false positive results when permanent slides are prepared, which can be re-examined in case of doubt. Cryptosporidium spp. oocysts can be readily identified in fecal smears prepared according to a regressive iron hematoxylin staining technique. The number of steps and their duration, as well as costs, were reduced to a minimum without loss of image quality and permanence of the preparations.

Staining of intestinal protozoa with heidenhain's iron Hematoxylin

Due to its unique properties, iron hematoxylin has been traditionally recommended for staining intestinal protozoa. This process can be simplified by reducing the number of steps and periods of permanence of the slides in some of the liquids used, without detriment to the quality of the results. Thus iron hematoxylin becomes adequate for routine use. Hematoxylin is a natural dye extracted from Haematoxylon campechianum, of the family Leguminosae. It must first be 'ripened', i.e. oxidized to hematein, which reacts with ferric ammonium sulphate to produce the ferric lake (iron hematoxylin), a basic dye. Iron hematoxylin most frequently stains regressively, i.e. the slides are first overstained and then differentiated.

Molecular and seroepidemiologic studies of Enterovirus 71 infection in the State of Pará, Brazil

In many countries, the Enterovirus 71 (EV-71) Picornaviridae family is associated to hand, foot and mouth disease in addition to acute neurological diseases while in Brazil these viruses are more closely associated to the latter group. The aim of this research was to use the first EV-71 isolate of the Northern region of Brazil in molecular and seroepidemiologic studies. Two (2.2%) out of 88 stool samples (44 cases of AFP), collected from January 1998 to December 2000 were positive for EV-71 isolation (73442/PA/99). Nucleotide sequence of the gen that codifies the VP1 protein showed that isolate 73442/PA/99 was similar to the EV-71 strains belonging to genotype B - more closely identified with EV-71 from North America. Neutralization test with 389 sera samples collected from January 1998 to November 2001, from individuals ranging from 0 to 15 years of age living in the city of Belém, State of Pará showed the following results in relation to isolate 73442/PA/99 and prototype BrCr: a total of 207 individuals (53.2%) had neutralization antibodies to both viruses, 167 (42.9%) had no antibodies and 15 showed the presence of neutralizing antibodies to one of the two viruses. Only 20.2% of the children aged 0 to 3 had neutralizing antibodies to EV-71, indicating that these children were more susceptible to the infection. Both the seroprevalence study and VP1 sequencing were important to demonstrate the spread and the molecular pattern of the EV-71 circulating in the Northern Region of Brazil.

Characterization of a clone from an adult worm cDNA library selected with anti-Schistosoma mansoni human antibodies dissociated from immune complexes: a preliminary report

Considering the scarcity of defined antigens, actually useful and reliable for use in the field studies, we propose an alternative method for selection of cDNA clones with potential use in the diagnosis of schistosomiasis. Human antibodies specific to a protein fraction of 31/32 kDa (Sm31/32), dissociated from immune complexes, are used for screening of clones from an adult worm cDNA library. Partial sequencing of five clones, selected through this strategy, showed to be related to Schistosoma mansoni: two were identified as homologous to heat shock protein 70, one to glutathione S-transferase, one to homeodomain protein, and one to a previously described EST (expressed sequence tag) of S. mansoni. This last clone was the most consistently reactive during the screening process with the anti-Sm31/32 antibodies dissociated from the immune complexes. The complete sequence of this clone was obtained and the translation data yielded only one ORF (open reading frame) that code for a protein with 57 amino acids. Based on this amino acid sequence two peptides were chemically synthesized and evaluated separately against a pool of serum samples from schistosomiasis patients and non-schistosomiasis individuals. Both peptides showed strong reactivity only against the positive pool, suggesting that these peptides may be useful as antigens for the diagnosis of schistosomiasis mansoni.

Antigenic and genetic characterization of the first rabies virus isolated from the bat Eumops perotis in Brazil

Although the main transmitters of rabies in Brazil are dogs and vampire bats, the role of other species such as insectivorous and frugivorous bats deserves special attention, as the rabies virus has been isolated from 36 bat species. This study describes the first isolation of the rabies virus from the insectivorous bat Eumops perotis. The infected animal was found in the city of Ribeirão Preto, São Paulo. The virus was identified by immunofluorescence antibody test (FAT) in central nervous system (CNS) samples, and the isolation was carried out in N2A cell culture and adult mice. The sample was submitted to antigenic typing using a panel of monoclonal antibodies (CDC/Atlanta/USA). The DNA sequence of the nucleoprotein gene located between nucleotides 102 and 1385 was aligned with homologous sequences from GenBank using the CLUSTAL/W method, and the alignment was used to build a neighbor-joining distance-based phylogenetic tree with the K-2-P model. CNS was negative by FAT, and only one mouse died after inoculation with a suspension from the bat's CNS. Antigenic typing gave a result that was not compatible with the patterns defined by the panel. Phylogenetic analysis showed that the virus isolated segregated into the same cluster related to other viruses isolated from insectivorous bats belonging to genus Nyctinomops ssp. (98.8% nucleotide identity with each other).

Characterization of a Hepatitis B virus strain in southwestern Paraná, Brazil, presenting mutations previously associated with anti-HBs Resistance

The present study investigated if hepatitis B virus (HBV) mutants circulate in the southwestern region of the State of Paraná, Brazil, by analyzing samples from children who received immunoprophylaxis but were born to HBV carrier mothers. Samples from 25 children were screened for HBV serum markers and for HBV DNA by PCR. Only one sample was positive for HBsAg, anti-HBs and HBV DNA, although the child had been vaccinated. Analysis of the S gene sequence of this sample showed the presence of a proline at position 105, a serine at position 114, three threonines at positions 115, 116 and 140, and a glutamine at position 129. The presence of these amino acids, except for serine at position 114, has been related to monoclonal or polyclonal therapy with anti-HBs after liver transplantation, whereas the presence of threonine at position 116 has been described in immunized children from Singapore. This finding demonstrates the possible circulation of HBV strains resistant to hepatitis B immunoprophylaxis in southwestern Paraná, Brazil. The genotype of the sample was identified as genotype D, which is frequently found in the region studied. Since 36% of the children had received incomplete or no immunoprophylaxis, more extensive follow-up of children born to HBsAg-positive mothers is needed.

Smqnr VARIANTS IN CLINICAL ISOLATES OF Stenotrophomonas maltophilia IN BRAZIL

SUMMARYStenotrophomonas maltophilia contains a novel chromosomally-encoded qnr gene named Smqnr that contributes to low intrinsic resistance to quinolone. We described Smqnr in 13 clinical isolates of S. maltophilia from two Brazilian hospitals, over a 2-year period. The strains were identified by API 20 NE (bioMérieux, France). Susceptibility by microdilution method to trimetroprim/sulfamethoxazole, ciprofloxacin, levofloxacin, minocycline, ceftazidime, chloramphenicol and ticarcillin/clavulanate was performed according to CLSI. PCR detection of Smqnr gene was carried out. The sequence of Smqnr was compared with those deposited in GenBank. Pulsed-field gel electrophoresis (PFGE) of all strains was performed. Thirteen Smqnr positives isolates were sequenced and three novel variants of Smqnr were identified. All 13 Smqnr isolates had distinguishable patterns by PFGE. This is the first report of Smqnr in S. maltophilia isolated in Brazil.

MOLECULAR IDENTIFICATION OF Bartonella henselae IN A SERONEGATIVE CAT SCRATCH DISEASE PATIENT WITH AIDS IN RIO DE JANEIRO, BRAZIL

Bartonella henselae is associated with a wide spectrum of clinical manifestations, including cat scratch disease, endocarditis and meningoencephalitis, in immunocompetent and immunocompromised patients. We report the first molecularly confirmed case of B. henselae infection in an AIDS patient in state of Rio de Janeiro, Brazil. Although DNA sequence of B. henselae has been detected by polymerase chain reaction in a lymph node biopsy, acute and convalescent sera were nonreactive.

PERFORMANCE OF CONVENTIONAL PCRs BASED ON PRIMERS DIRECTED TO NUCLEAR AND MITOCHONDRIAL GENES FOR THE DETECTION AND IDENTIFICATION OF Leishmania spp.

In visceral leishmaniasis, the detection of the agent is of paramount importance to identify reservoirs of infection. Here, we evaluated the diagnostic attributes of PCRs based on primers directed to cytochrome-B (cytB), cytochrome-oxidase-subunit II (coxII), cytochrome-C (cytC), and the minicircle-kDNA. Although PCRs directed to cytB, coxII, cytC were able to detect different species of Leishmania, and the nucleotide sequence of their amplicons allowed the unequivocal differentiation of species, the analytical and diagnostic sensitivity of these PCRs were much lower than the analytical and diagnostic sensitivity of the kDNA-PCR. Among the 73 seropositive animals, the asymptomatic dogs had spleen and bone marrow samples collected and tested; only two animals were positive by PCRs based on cytB, coxII, and cytC, whereas 18 were positive by the kDNA-PCR. Considering the kDNA-PCR results, six dogs had positive spleen and bone marrow samples, eight dogs had positive bone marrow results but negative results in spleen samples and, in four dogs, the reverse situation occurred. We concluded that PCRs based on cytB, coxII, and cytC can be useful tools to identify Leishmania species when used in combination with automated sequencing. The discordance between the results of the kDNA-PCR in bone marrow and spleen samples may indicate that conventional PCR lacks sensitivity for the detection of infected dogs. Thus, primers based on the kDNA should be preferred for the screening of infected dogs.

Severe digestive pathology associated with chronic Chaga's disease in Ecuador: report of two cases

DNA extracted from peripheral blood of two Ecuadorian patients showing severe digestive pathology was amplified by the polymerase chain reaction using a Trypanosoma cruzi specific oligonucleotide primers derived from the primary sequence of a cDNA encoding for a 24 kDa excretory/secretory protein. The positive PCR results together with the clinical findings confirmed that both patients had a digestive pathology due to Chagas' disease. This pathology could be more frequent than previously described in the chagasic endemic regions of Andean countries.

Molecular evidence of mother-to-child transmission of HTLV-IIc in the Kararao Village (Kayapo) in the Amazon Region of Brazil

Blood samples from native Indians in the Kararao village (Kayapo), were analysed using serological and molecular methods to characterize infection and analyse transmission of HTLV-II. Specific reactivity was observed in 3/26 individuals, of which two samples were from a mother and child. RFLP analysis of the pX and env regions confirmed HTLV-II infection. Nucleotide sequence of the 5' LTR segment and phylogenetic analysis showed a high similarity (98%) between the three samples and prototype HTLV-IIa (Mot), and confirmed the occurrence of the HTLV-IIc subtype. There was a high genetic similarity (99.9%) between the mother and child samples and the only difference was a deletion of two nucleotides (TC) in the mother sequence. Previous epidemiological studies among native Indians from Brazil have provided evidence of intrafamilial and vertical transmission of HTLV-IIc. The present study now provides molecular evidence of mother-to-child transmission of HTLV-IIc, a mechanism that is in large part responsible for the endemicity of HTLV in these relatively closed populations. Although the actual route of transmission is unknown, breast feeding would appear to be most likely.