Comparative sequence analysis of the P-, M- and L-coding region of the measles virus CAM-70 live attenuated vaccine strain

Measles virus is a highly contagious agent which causes a major health problem in developing countries. The viral genomic RNA is single-stranded, nonsegmented and of negative polarity. Many live attenuated vaccines for measles virus have been developed using either the prototype Edmonston strain or other locally isolated measles strains. Despite the diverse geographic origins of the vaccine viruses and the different attenuation methods used, there was remarkable sequence similarity of H, F and N genes among all vaccine strains. CAM-70 is a Japanese measles attenuated vaccine strain widely used in Brazilian children and produced by Bio-Manguinhos since 1982. Previous studies have characterized this vaccine biologically and genomically. Nevertheless, only the F, H and N genes have been sequenced. In the present study we have sequenced the remaining P, M and L genes (approximately 1.6, 1.4 and 6.5 kb, respectively) to complete the genomic characterization of CAM-70 and to assess the extent of genetic relationship between CAM-70 and other current vaccines. These genes were amplified using long-range or standard RT-PCR techniques, and the cDNA was cloned and automatically sequenced using the dideoxy chain-termination method. The sequence analysis comparing previously sequenced genotype A strains with the CAM-70 Bio-Manguinhos strain showed a low divergence among them. However, the CAM-70 strains (CAM-70 Bio-Manguinhos and a recently sequenced CAM-70 submaster seed strain) were assigned to a specific group by phylogenetic analysis using the neighbor-joining method. Information about our product at the genomic level is important for monitoring vaccination campaigns and for future studies of measles virus attenuation.

Comparative sequence analysis of the P-, M- and L-coding region of the measles virus CAM-70 live attenuated vaccine strain.

Measles virus is a highly contagious agent which causes a major health problem in developing countries. The viral genomic RNA is single-stranded, nonsegmented and of negative polarity. Many live attenuated vaccines for measles virus have been developed using either the prototype Edmonston strain or other locally isolated measles strains. Despite the diverse geographic origins of the vaccine viruses and the different attenuation methods used, there was remarkable sequence similarity of H, F and N genes among all vaccine strains. CAM-70 is a Japanese measles attenuated vaccine strain widely used in Brazilian children and produced by Bio-Manguinhos since 1982. Previous studies have characterized this vaccine biologically and genomically. Nevertheless, only the F, H and N genes have been sequenced. In the present study we have sequenced the remaining P, M and L genes (approximately 1.6, 1.4 and 6.5 kb, respectively) to complete the genomic characterization of CAM-70 and to assess the extent of genetic relationship between CAM-70 and other current vaccines. These genes were amplified using long-range or standard RT-PCR techniques, and the cDNA was cloned and automatically sequenced using the dideoxy chain-termination method. The sequence analysis comparing previously sequenced genotype A strains with the CAM-70 Bio-Manguinhos strain showed a low divergence among them. However, the CAM-70 strains (CAM-70 Bio-Manguinhos and a recently sequenced CAM-70 submaster seed strain) were assigned to a specific group by phylogenetic analysis using the neighbor-joining method. Information about our product at the genomic level is important for monitoring vaccination campaigns and for future studies of measles virus attenuation.

A polymerase chain reaction-based assay to identify genotype F of hepatitis B virus.

We have developed a polymerase chain reaction (PCR) assay which distinguishes genotype F from the other genotypes of hepatitis B virus (HBV). The method was used to characterize HBV strains isolated in urban areas of the Brazilian Amazon. DNA was amplified in 54 of a total of 78 HBsAg-positive serum samples, using universal, non-genotype-specific primers. Only 4 (7.4%) were identified as genotype F by our genotype-specific PCR assay. This proportion is notably lower than that previously reported in Argentina, Venezuela, Peru, and Central America.

A polymerase chain reaction-based assay to identify genotype F of hepatitis B virus

We have developed a polymerase chain reaction (PCR) assay which distinguishes genotype F from the other genotypes of hepatitis B virus (HBV). The method was used to characterize HBV strains isolated in urban areas of the Brazilian Amazon. DNA was amplified in 54 of a total of 78 HBsAg-positive serum samples, using universal, non-genotype-specific primers. Only 4 (7.4 percent) were identified as genotype F by our genotype-specific PCR assay. This proportion is notably lower than that previously reported in Argentina, Venezuela, Peru, and Central America

Genetic an antigenic analysis of adenovirus type 3 strains showing intermediate behavior in standard seroneutralization test.

During an epidemiological survey of acute respiratory infection in Rio de Janeiro, among 208 adenovirus isolates, we found two strains that we were not able, by a standard neutralization procedure, to distinguish between type 3 or 7. However, DNA restriction pattern for the two strains with different enzymes were analyzed and showed a typical Ad3h profile. Using a cross-neutralization test in which both Ad3p and Ad7p antisera were used in different concentration against 100 TCID50 of each adenovirus standard and both isolates, we were able to confirm that the two isolates belong to serotype 3. An hemagglutination inhibition test also corroborated the identification of both strains as adenovirus type 3. Comparing Ad3h and Ad3p genome, we observed 16 different restriction enzyme sites, three of which were located in genomic regions encoding polypeptides involved in neutralization sites.

Genomic characterization of adenovirus serotype 7 isolated in Brazil from acute respiratory disease patients during the period from 1980 to 1991.

Forty isolates of adenovirus type 7 were analized by restriction enzyme digestion with BamHI, SmaI, EcoRI and HindIII. These isolates were obtained from acute respiratory disease patients during the years 1980 to 1991. Only two genomic types were found: Ad7b and Ad7e, with Ad7b (87.5%) being more frequent than Ad7e (12.5%). The genomic type Ad7e appeared in the years 1980, 1981 and 1983. Ad7b appeared in 1982 and it was the only genomic type found from 1984 to 1991. Both genomic types were responsible for lower (LRTI) and upper (URTI) respiratory tract infection, but the proportion LRTI/URTI is higher for Ad7b (25/6) than for Ad7e (1/4).

Downregulation of major histocompatibility complex class I expression and susceptibility to natural killer cells in cells transformed with the oncogenic adenovirus 12 are regulated by different E1A domains.

All adenoviruses transform rodent cells in vitro, but only cells transformed by serotypes belonging to subgroups A (Ad12) and B (Ad3) are tumorigenic for immunocompetent animals. In these cells, the expression of major histocompatibility complex (MHC) class I antigens is repressed and might allow them to escape from recognition by cytotoxic T lymphocytes and to develop in tumor. Furthermore, these cell lines appear resistant to lysis by natural killer (NK) cells. To determine the E1A domain(s) responsible for these properties several cell lines were created by transforming baby rat kidney cells with a set of plasmids expressing different Ad2/Ad12 hybrid E1A gene products. The class I gene expression was inhibited in cells expressing the Ad12 13S mRNA product and in cells transformed with Ad2/Ad12 hybrid E1A gene product harboring the C-terminal part of the conserved region (CR) 3 of Ad12. Susceptibility of these transformed cell lines to NK cells was determined by cytolytic assays. The results obtained suggest that two of Ad12 E1A domains are required to induce resistance of the cell lines to NK cells.

Sequence analysis of pre-S/S gene of hepatitis B virus strains of genotypes A, D, and F isolated in Brazil.

The nucleotide sequences of pre-S/S gene of nine hepatitis B virus strains (3 adw2, 3 ayw2, and 3 ayw 3) and of pre-S region of two adw4 isolates from Rio de Janeiro, Brazil, were determined. Phylogenetic analysis allowed to classify these strains into three genotypes, A, D and F, reflecting the diverse origin of the population. However, strains belonging to a same genotype were separated by a short evolutionary distance. The presence of aminoacid mutations into Pre-S region not found in hepatitis B viruses isolated in other parts of the world is described.

Genetic diversity of hepatitis B virus strains isolated in Rio de Janeiro, Brazil.

Hepatitis B virus (HBV) genomes have been classified into six groups, A-F, group F being the most divergent. South America has provided the smallest number of strains studied at the genome level. The only HBV strain from this region sequenced completely has been classified in group F, and contains the most divergent of the HBV genomes presently known [Naumann et al. (1983): Journal of General Virology 74:1627-1632]. To evaluate genetic relatedness between strains isolated in South America and in the rest of the world, a restriction endonuclease analysis was carried out on 14 HBV strains (4 adw2, 3 adw4, 3 ayw2, and 4 ayw3) isolated in Rio de Janeiro, Brazil. C, pre-S, and X genes along with the 5' part of the P gene from these strains were amplified by the polymerase chain reaction. The DNA fragments were digested by BamHI, BstEII, EcoRI, HhaI, and TaqI endonucleases. The restriction patterns obtained were compared with those deduced from the nucleotide sequence of 26 HBV strains isolated in other continents. The results showed a large genetic variability of Brazilian strains. Taking into account that all the samples examined possessed the w antigenic subdeterminant, the number of different restriction patterns of strains isolated in Rio de Janeiro was at least as large as that of the reference strains isolated in the rest of the world. Some original restriction patterns were found in adw4 and ayw2 HBV strains.

Restriction site mapping of four genome types of adenovirus types 3 and 7 isolated in South America.

New genome types of Ad3 and Ad7 were found among adenovirus (Ad) strains isolated from stools of children during epidemiological surveys made in São Paulo, Brazil, and Buenos Aires, Argentina. These were characterized by DNA analysis with 11 restriction endonucleases and showed a number of new restriction patterns, notably for BamHI, BcII, BgIII, HindIII, KpnI, and SmaI. Restriction maps of the genome types, named Ad3e1, Ad3e2, Ad3h, and Ad7h, were constructed and compared with those of Ad3p and Ad7p.

Avaliaçäo da açäo protetora do misoprostol sobre a mucosa gastrintestinal em pacientes artrósicos em uso de naproxen / Evaluation of the protective effect of misoprostol in the gastrointestinal mucosa in patients with osteo-arthritis using naproxen

Foi realizado um estudo aberto, näo comparativo, com o Misoprostol, prostaglandina sintética no tipo E1, na terapêutica de 30 pacientes portadores de osteoartrose em uso de Naproxen e que, concomitantemente, exibiram manifestaçöes clínicas relativas ao trato gastrintestinal superior. Esses pacientes receberam Misoprostol na dose de 200 mcg, três vezes ao dia, administrados simultanemante com sua dose diária do antiinflamatório näo hormonal. O tempo de observaçäo máxima seria de oito semanas, com avaliaçöes clínicas intermediárias sendo efetuadas no final das 2ª, 4ª e 8ª semanas. Dezenove (19) pacientes (63,3%), já ao final da 2ª semana, relataram acentuadas diminuiçöes digestivas enquanto que, mais 8 pacientes (26,7%) totalizando 27 (90%) ao final da 4ª semana, exibiam tal melhora. Apenas dois pacientes acusaram efeitos colaterais significativos, porém näo foram demonstradas alteraçöes de monta nos dados vitais nem nos exames laboratoriais executados. Os autores concluem pela validade do uso de Misoprostol na atenuaçäo dos sintomas gastrintestinais superiores de pacientes artrósicos em uso simultâneo de Naproxen