Molecular epidemiology of human respiratory syncytial virus in Uruguay: 1985-2001 - A review

The variability of the G glycoprotein from human respiratory syncytial viruses (HRSV) (groups A and B) isolated during 17 consecutive epidemics in Montevideo, Uruguay have been analyzed. Several annual epidemics were studied, where strains from groups A and B circulated together throughout the epidemics with predominance of one of them. Usually, group A predominates, but in some epidemics group B is more frequently detected. To analyse the antigenic diversity of the strains, extracts of cells infected with different viruses of group A were tested with a panel of anti-G monoclonal antibodies (MAbs). The genetic variability of both groups was analyzed by sequencing the C-terminal third of the G protein gene. The sequences obtained together with previously published sequences were used to perform phylogenetic analyses. The data from Uruguayan isolates, together with those from the rest of the world provide information regarding worldwide strain circulation. Phylogenetic analyses of HRSV from groups A and B show a model of evolution analogous to the one proposed for influenza B viruses providing information that would be beneficial for future immunization programs and to design safe vaccines.

Re-mapping the molecular features of the human immunodeficiency virus type 1 and human T-cell lymphotropic virus type 1 Brazilian sequences using a bioinformatics unit established in Salvador, Bahia, Brazil, to give support to the viral epidemiology studies

The analysis of genetic data for human immunodeficiency virus type 1 (HIV-1) and human T-cell lymphotropic virus type 1 (HTLV-1) is essential to improve treatment and public health strategies as well as to select strains for vaccine programs. However, the analysis of large quantities of genetic data requires collaborative efforts in bioinformatics, computer biology, molecular biology, evolution, and medical science. The objective of this study was to review and improve the molecular epidemiology of HIV-1 and HTLV-1 viruses isolated in Brazil using bioinformatic tools available in the Laboratório Avançado de Sáude Pública (Lasp) bioinformatics unit. The analysis of HIV-1 isolates confirmed a heterogeneous distribution of the viral genotypes circulating in the country. The Brazilian HIV-1 epidemic is characterized by the presence of multiple subtypes (B, F1, C) and B/F1 recombinant virus while, on the other hand, most of the HTLV-1 sequences were classified as Transcontinental subgroup of the Cosmopolitan subtype. Despite the high variation among HIV-1 subtypes, protein glycosylation and phosphorylation domains were conserved in the pol, gag, and env genes of the Brazilian HIV-1 strains suggesting constraints in the HIV-1 evolution process. As expected, the functional protein sites were highly conservative in the HTLV-1 env gene sequences. Furthermore, the presence of these functional sites in HIV-1 and HTLV-1 strains could help in the development of vaccines that pre-empt the viral escape process.

New species emergence via recombination among isolates of the Brazilian tomato infecting Begomovirus complex

Partial nucleotide sequences of five tomato infecting Begomovirus isolates were determined from DNA-A fragments, corresponding to the 5' region of the replication associated protein gene, the intergenic region and the 5' region of the coat protein gene. Isolate DFM shared 95% identity with Tomato mottle leaf curl virus (TMoLCV), isolates 34, PA-05, and Ta4 were 88% identical to Tomato yellow vein streak virus and isolate DF-BR3 shared 77% identity with TMoLCV. Recombination analysis indicated that isolate DF-BR3 was a chimaera, and it provided evidence that there is a complex and actively recombining population of tomato infecting begomoviruses in Brazil.

Sequence analysis of the capsid protein gene of an isolate of Apple stem grooving virus, and its survey in Southern Brazil

Apple stem grooving virus (ASGV) is one of the most important viruses infecting fruit trees. This study aimed at the molecular characterization of ASGV infecting apple (Malus domestica) plants in Santa Catarina (SC). RNA extracted from plants infected with isolate UV01 was used as a template for RT-PCR using specific primers. An amplified DNA fragment of 755 bp was sequenced. The coat protein gene of ASGV isolate UV01 contains 714 nucleotides, coding for a protein of 237 amino acids with a predicted Mr of approximately 27 kDa. The nucleotide and the deduced amino acid sequences of the coat protein gene showed identities of 90.9% and 97.9%, respectively, with a Japanese isolate of ASGV. Very high amino acid homologies (98.7%) were also found with Citrus tatter leaf capillovirus (CTLV), a very close relative of ASGV. These results indicate low coat protein gene variability among Capillovirus isolates from distinct regions. In a restricted survey, mother stocks in orchards and plants introduced into the country for large scale fruit production were indexed and shown to be infected by ASGV (20%), usually in a complex with other (latent) apple viruses (80%).

Genetic variation among pathogens causing "Helminthosporium" diseases of rice, maize and wheat

Twenty isolates of four fungal species, agents of "Helminthosporium" diseases in cereals, were collected from different regions: nine Bipolarisoryzae isolated from rice (Oryza sativa), seven B.sorokiniana from wheat (Triticum aestivum), two B. maydis, and two Exserohilumturcicum from maize (Zea mays). The strains were compared by PCR-RFLP and RAPD analysis. Size polymorphism among the isolates in the ITS region comprising the 5.8 S rDNA indicated genetic differences among the isolates, while a UPGMA phenogram constructed after the digestion of this region with restriction enzymes showed inter- and intra-specific polymorphism. The RAPD profiles indicated an expressive level of polymorphism among different species, compared with a low level of polymorphism among isolates of the same species. A UPGMA phenogram grouped the isolates according to the species and their host plant. RAPD profiles did not reveal polymorphism that directly correlated climatic factors with geographic source of the isolates of B. sorokiniana, and B. oryzae. Teleomorphic species revealed high similarity with their correspondent anamorphs.

Variability of the coat protein gene of Grapevine leafroll-associated virus 3 in Brazil

Leafroll is an economically important disease affecting grapevines (Vitis spp.). Nine serologically distinct viruses, Grapevine leafroll-associated virus-1 through 9, are associated with this disease. The present study describes the coat protein gene sequence of four GLRaV-3 isolates occurring in the São Francisco River basin, Northeastern Brazil. The viral RNA was extracted from GLRaV-3 ELISA-positive plants and the complete coat protein gene was amplified by RT-PCR. Sequences were generated automatically and compared to the complete coat protein sequence from North American (NY1) and Chinese (Dawanhong Nº2 and SL10) GLRaV-3 isolates. The four studied isolates, named Pet-1 through 4, showed deduced amino acid identities of 98-100% (Pet-1 through 3) and 95% (Pet-4) with North American and Chinese isolates. A total of seventeen amino acid substitutions was detected among the four characterized isolates in comparison to the NY1, Dawanhong No.2 and SL10 sequences. The results indicated the existence of natural variation among GLRaV-3 isolates from grapevines, also demonstrating a lack of correlation between sequence data and geographic origin. This variability should be considered when selecting regions of the viral genome targeted for reliable and consistent virus molecular detection.

Identification and characterization of a Hydrangea ringspot virus isolate infecting hydrangea in São Paulo State

Hydrangea plants showing leaves with chlorotic and necrotic rings from Arujá Municipality, São Paulo State, were analyzed for the identification of the viral species. Elongated filamentous particles of 490 nm were visualized under transmission electron microscope. Oligonucleotides for Hydrangea ringspot virus (HdRSV), a potexvirus commonly found in Europe and in the United States, were tested using total RNA from hydrangea plants, amplifying two fragments, one around 550 and another one of 250 nucleotides. Nucleotide identity with HdRSV (accession number AJ 707100.1) was 96% and 88% for the longest and shortest fragment, respectively, indicating the presence of this virus. To evaluate its dissemination in the matrices of hydrangea used in the commercial production, 17 samples were collected in the region of Arujá, and eight were infected by HdRSV. For the analyzed viral replicase portion, the isolates from the varieties 'Azul LZR', 'Rosita', 'Renat Blue' and 'Vermelho Comum' did not differ in their amino acid sequences from isolates with sequences deposited in the GenBank (accession numbers AY 707100 and NC_006943). The isolates from 'Azul Rendado' and "Rosa Japonesa' showed few differences but were related to the remaining isolates. An antiserum was obtained for HdRSV and can be efficiently used to detect such virus in hydrangea and Primula malacoides, another ornamental plant also infected by HdRSV.

Genetic grouping of avian infectious bronchitis virus isolated in Brazil based on RT-PCR/RFLP analysis of the S1 gene

Twelve Brazilian isolates and one reference vaccine strain of avian infectious bronchitis virus (IBV) were propagated in embryonating chicken eggs. The entire S1 glycoprotein gene of these viruses was analysed by reverse-transcriptase-polymerase chain reaction and restriction fragment length polymorphism (RT-PCR-RFLP), using the restriction enzymes HaeIII, XcmI and BstyI. The RFLP patterns led to the classification of these isolates into five distinct genotypes: A, B, C, D and Massachusetts. Five of twelve isolates were grouped in Massachusetts genotype and the remaining seven viruses were classified into four distinct genotypes: A (2), B (2), C (2) or D (1). Such genotyping classification agreed with previous immunological analysis for most of these viruses, highlighting the occurrence of a relevant variability among the IBV strains that are circulating in Brazilian commercial poultry flocks.

Genomic characterization of Brazilian hepatitis C virus genotypes 1a and 1b

Parts of 5' non-coding (5' NC) and of E1 envelope regions of the hepatitis C virus (HCV) genome were amplified from sera of 26 Brazilian anti-HCV antibody-positive patients using the reverse transcription-polymerase chain reaction (RT-PCR). Fourteen samples were PCR positive with primers from the 5' NC region and 8 of them were also positive with primers from the E1 region. A genomic segment of 176 bp from the E1 region of 7 isolates was directly sequenced from PCR products. The sequences were compared with those of HCV strains isolated in other countries and the Brazilian isolates were classified by phylogenetic analysis into genotypes 1a and 1b. This could have a clinical importance since it has been shown that individuals infected with type 1 viruses are less likely to respond to treatment with interferon than individuals infected with types 2 and 3 viruses. Two quasispecies isolated from the same patient with an interval of 13 months differed by two base substitutions (1.1%). The sequence of another isolate presented a three-nucleotide deletion at codon 329

Low occurrence of occult hepatitis B virus infection and high frequency of hepatitis C virus genotype 3 in hepatocellular carcinoma in Brazil

Occult hepatitis B virus (HBV) infection has been reported among patients with hepatitis C virus (HCV) infection and hepatocellular carcinoma (HCC). Our aim was to evaluate the presence of occult HBV infection in patients with HCV-related liver cirrhosis (LC) with or without HCC in São Paulo, Brazil. Serum and liver tissue samples from 50 hepatitis B surface antigen-negative patients with HCV-related LC who underwent liver transplantation at the University of São Paulo School of Medicine Hospital from 1993 to 2004 were divided into groups with LC only (N = 33) and with LC plus HCC (N = 17). HBV DNA was assayed for serum and paraffin-embedded liver tissue (tumoral and non-tumoral) using real time PCR and only 1 case with HCC had HBV DNA-positive serum. All liver samples were negative. HCV genotype 3 was detected in 17/39 (43.7%) cases. In conclusion, using a sensitive real time PCR directed to detect HBV variants circulating in Brazil, occult hepatitis B infection was not found among HCV-positive cirrhotic patients and was rarely found among HCV-positive HCC patients. These results are probably related to the low prevalence of HBV infection in our population. Furthermore, we have also shown that HCV genotype 3 is frequently found in Brazilian cirrhotic patients, particularly when they also have HCC. More studies involving a large number of cases should be carried out to confirm these data and to further characterize Brazilian HCV genotype isolates to elucidate genetic features that might be related to its carcinogenic potential.

Response of rice cultivars to nitrogen in upland conditions

Against the background of a growing world population, rice (Oryza sativa L.) consumption is expected to grow faster than its production. Therefore, an appropriate question would be: how to increase productivity in the short-term? In this respect, it becomes important the implementation of modern agricultural production systems, such as upland rice with supplemental sprinkler irrigation. Additional information is needed to maximize the available resources, with special attention given to research on the use of nitrogen. This study aimed to evaluate the agronomic performance of commercial rice cultivars with different plant characteristics in upland conditions with supplemental sprinkler irrigation, when subjected to nitrogen in topdress application at the R1 stage (panicle differentiation). The experiment was arranged in a randomized block with split plot design, with 65 treatments, consisting of the combination of 13 cultivars in the plots, and five nitrogen levels in the subplots (0, 40, 80, 120 and 160 kg ha-1), with four replications. Genetic variability was detected among rice cultivars and the agronomic performance in response to the applied nitrogen. The topdressing application of nitrogen increases, in general, the production components and grain yield in rice. Cultivars BRS Primavera, Caiapó and IAC 202 stood out for grain yield, followed by Baldo, Carnaroli, BRS Curinga and IAC 500 with lower yields.

Studies on mosquitoes (Diptera: Culicidae) and anthropic environment: 6 - Breeding in empty conditions of rice fields in South-Eastern Brazil

Studies on culicid breeding in empty rice fields were carried out during the cultivation cycle from May to November 1993. This period corresponded to stages 1 and 2, when empty conditions prevailed. Breeding occurred in stage 1 and the first part of stage 2, corresponding respectively to fallow uncultivated and ploughing situations. No breeding was found to take place during the second part of stage 2 when transient floods and harrowing occurred. The predominant species were Aedes scapularis, Culex nigripalpus and Cx. mollis. The Pilosus Group of Culex (Melanoconion) was found at lower densities. Some epidemiological considerations are presented.

Comparative study of the biological behaviour in hamster of two isolates of leishmania characterized respectively as L. major-like and L. donovani

Hamster inoculated intraperitoneally with 1 x 10(7) parasites of L. donovani and L. major-like of the New World were studied in groups of 15, 30, 60 and 90 days of infection. The parasite load and density showed progressive increase with the evolution of the infection and was higher in the L. donovani groups than in the L. major-like groups. The L. major-like groups showed parasite density higher in the spleen than in the liver and was similar in both organs in L. donovani groups. The histopathology showed a diffuse marked hyperplasia and hypertrophy of the reticuloendothelial system with high parasitism in the L. donovani groups while there was focal involvement of these organs in L. major-like groups, forming nodules of macrophages that were scantly parasitised. The biological behaviour could be useful in the preliminary studies of Leishmania strain in regional laboratories and understanding the histopathology of lesions caused by different leishmania species.

Comparison of serum hepatitis B virus replication markers in patients with chronic hepatitis B: studies on HBeAg/Anti-HBe system, viral dna polymerase and HBV-DNA

The detection of HBV-DNA in serum by molecular hybridization is the most sensitive and specific marker of replication and infectivity of hepatitis B virus and currently is proposed as a routine diagnostic technique in the follow-up of HBV - related diseases. Comparing different techniques already described, we found that direct spotting of serum samples on nitrocellulose membranes under vacuum filtration, followed by denaturing and neutralizing washes is more practical, simple, sensible and reproducible. DNA polymerase assay using phosphonoformic acid as specific viral inhibitor has shown 86.8% of concordance with HBV-DNA detection, and so, it is an useful alternative in the follow-up of hepatitis B chronic patients. We found 19.2% HBeAg positive samples with no other markers of viral replication and no anti-HBe positive sample had detectable HBV-DNA. Discordance between the 2 systems have been extensively described, and we confirm this for the first time in our country. Molecular biological techniques are essential to determine the replication status of chronic hepatitis B patients.

Susceptibility of clinical isolates of Bacteroides fragilis group strains to cefoxitin, cefoperazone and ticarcillin/clavulanate

A total of 40 strains of the B. fragilis group was isolated from clinical specimens in two hospital centers in Fortaleza from 1993 to 1997. The most frequently isolated species was Bacteroides fragilis (19 strains) and most isolates came from intra-abdominal and wound infections. The susceptibility profile was traced for cefoxitin, cefoperazone and ticarcillin-clavulanate by using the agar dilution reference method. All isolates were susceptible to ticarcillin-clavulanate (128/2mug/ml). Resistance rates of 15 and 70% were detected to cefoxitin (64mug/ml) and cefoperazone (64mug/ml), respectively. Such regional results permit a better orientation in choosing this group of antibiotics for prophylaxis and therapy especially in relation to cefoxitin, which is frequently used in the hospital centers studied.