RESUMO
Canine distemper virus (CDV, Paramyxoviridae, Morbillivirus) is the causative agent of a severe infectious disease affecting terrestrial and marine carnivores worldwide. Phylogenetic relationships and the genetic variability of the hemagglutinin (H) protein and the fusion protein signal-peptide (Fsp) allow for the classification of field strains into genetic lineages. Currently, there are nine CDV lineages worldwide, two of them co-circulating in South America. Using the Fsp-coding region, we analyzed the genetic variability of strains from Uruguay, Brazil, and Ecuador, and compared them with those described previously in South America and other geographical areas. The results revealed that the Brazilian and Uruguayan strains belong to the already described South America lineage (EU1/SA1), whereas the Ecuadorian strains cluster in a new clade, here named South America 3, which may represent the third CDV lineage described in South America.
Assuntos
Vírus da Cinomose Canina/genética , Vírus da Cinomose Canina/isolamento & purificação , Cinomose/virologia , Variação Genética , Sequência de Aminoácidos , Animais , Vírus da Cinomose Canina/química , Vírus da Cinomose Canina/classificação , Cães , Hemaglutininas Virais/química , Hemaglutininas Virais/genética , Dados de Sequência Molecular , Tipagem Molecular , Filogenia , Alinhamento de Sequência , América do SulRESUMO
RT-PCR was used to detect canine distemper virus (CDV) RNA in clotted blood from Argentine domestic dogs. The NP gene was detected in 73 out of 99 blood samples analyzed. The deduced amino acid sequence of these gene fragments showed 100% identity with the sequence of other wild-type and vaccine strains. A fragment of the hemagglutinin gene was amplified from 24 (32.9%) of the NP-RNA-positive clinical specimens. These H fragments were further analyzed by restriction fragment length polymorphism (RFLP) and sequencing. A single NdeI site was detected in all 24 wild-type strains but was absent in the vaccine strains. Phylogenetic analysis of the partial hemagglutinin amino acid sequences showed close clustering for local strains, clearly distinct from vaccine strains and other wild-type foreign CDV strains. One of the local strains, Arg 23, branched out of the root of the Argentine clade, close to the European strains, suggesting that two different pathogenic CDV genotypes are currently circulating in Argentina, one of them clearly predominant.
Assuntos
Vírus da Cinomose Canina/genética , Vírus da Cinomose Canina/isolamento & purificação , Cinomose/virologia , Sequência de Aminoácidos , Animais , Argentina , Sequência de Bases , Cães , Feminino , Hemaglutininas Virais/química , Hemaglutininas Virais/genética , Masculino , Dados de Sequência Molecular , Filogenia , Polimorfismo de Fragmento de Restrição , RNA Bacteriano/química , RNA Bacteriano/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Alinhamento de SequênciaRESUMO
HLA-DRbeta1*0101-HA and HLA-DRbeta1*0401-HA complexes are studied and compared by means of their computationally derived multipolar moments and electrostatic potentials. Changes in electrostatic potential are associated with definite pocket interaction profiles. Thus, Pocket 1 projects itself as an anchoring pocket for both complexes, in accordance with experimental results. While Pocket 4 has an anchoring profile in the HLA-DRbeta1*0101 allele, it presents itself as modulating pocket-peptide interactions in HLA-DRbeta1*0401. Pockets 6 and 7 both strongly contribute to allele specificity, with Pocket 7 being very important for HLA-DRbeta1*0401-HA. Pocket 9 acts as a "double purpose" interaction site for both alleles. It both projects itself as an anchoring pocket as well as modulating pocket-peptide interactions.
Assuntos
Antígenos HLA-DR/química , Hemaglutininas Virais/química , Modelos Moleculares , Fragmentos de Peptídeos/química , Peptídeos/química , Alelos , Sítios de Ligação , Antígenos HLA-DR/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Humanos , Eletricidade EstáticaRESUMO
Sequence analysis was performed on 50 measles viruses (MV) isolated in Argentina. Forty-six were obtained during the current outbreak (1997-1998), three from the previous outbreak (1991) and one sporadic case (1994). A 377-bp fragment of the hemagglutinin (H) gene was directly amplified by RT-PCR from nasopharyngeal secretions. Nucleotides 8152 to 8417 were sequenced and subjected to phylogenetic analysis. Multiple silent changes and point mutations were found in all MVs. In 1991, substitutions affected the third base in codons resulting in silent changes. In 1994 an A-->C substitution at position 8321 changed amino acids 351 (Leu-->Ile). In 1997-1998, an A-->G substitution at position 8339 changed amino acids 357 (Val-->Ile). In 3/46 viruses, guanine deletion at position 8205 changed the reading frame and insertion of an extra cytosine at nucleotide 8235 shifted it back to the original frame. Phylogenetic analysis revealed that viruses leading to the last two major outbreaks are clustered into two separate branches. MVs that prevailed until 1994 were related to genotype C1 and MVs of the current outbreak to D6. Random drift mutations rendered a 0.5 ratio of nonsilent over silent mutations in most of the MVs analyzed. However, in those showing a reading frame shift, the ratio was greater than 1, suggesting that it was driven by immune selection.
Assuntos
Surtos de Doenças , Hemaglutininas Virais/genética , Vírus do Sarampo/genética , Sarampo/virologia , Análise de Sequência de DNA , Sequência de Aminoácidos , Argentina/epidemiologia , Variação Genética , Hemaglutininas Virais/química , Humanos , Sarampo/epidemiologia , Vírus do Sarampo/isolamento & purificação , Vírus do Sarampo/metabolismo , Dados de Sequência Molecular , Mutação , Filogenia , Mutação Puntual , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
To determine the association between specific structural changes in the hemagglutinin gene and pathogenicity of avian influenza viruses (AIVs), groups of 4-week-old White Plymouth Rock chickens were inoculated intravenously or intranasally with AIVs of varying pathogenicities isolated from chickens in central Mexico during 1994-1995. Mildly pathogenic (MP) viruses had a common hemagglutinin-connecting peptide sequence of Pro-Gln-Arg-Glu-Thr-Arg decreases Gly and had restricted capability for replication and production of lesions in tissues. The principle targets for virus replication or lesion production were the lungs, lymphoid organs, and visceral organs containing epithelial cells, such as kidney and pancreas. Death was associated with respiratory and/or renal failure. By contrast, highly pathogenic (HP) AIVs had one substitution and the addition of two basic amino acids in the hemagglutinin connecting peptide, for a sequence of Pro-Gln-Arg-Lys-Arg-Lys-Thr-Arg decreases Gly. The HP AIVs were pantropic in virus replication and lesion production ability. However, the most severe histologic lesions were produced in the brain, heart, adrenal glands, and pancreas, and failure of multiple critical organs was responsible for disease pathogenesis and death. No differences in lesion distribution patterns or in sites of AIV replication were evident to explain the variation in mortality rates for different HP AIVs, but HP AIVs that produced the highest mortality rates had more severe necrosis in heart and pancreas. The ability of individual HP AIVs to produce low or high mortality rates could not be explained by changes in sequence of the hemagglutinin-connecting peptide alone, but probably required the addition of other undetermined genomic changes.
Assuntos
Galinhas , Vírus da Influenza A Subtipo H5N2 , Vírus da Influenza A , Influenza Aviária/patologia , Glândulas Suprarrenais/química , Glândulas Suprarrenais/patologia , Glândulas Suprarrenais/virologia , Animais , Encéfalo/patologia , Encéfalo/virologia , Química Encefálica , Hemaglutininas Virais/química , Hemaglutininas Virais/genética , Imuno-Histoquímica , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Vírus da Influenza A/fisiologia , Influenza Aviária/epidemiologia , Influenza Aviária/mortalidade , Rim/química , Rim/patologia , Rim/virologia , México/epidemiologia , Miocárdio/química , Miocárdio/patologia , Pâncreas/química , Pâncreas/patologia , Pâncreas/virologia , Organismos Livres de Patógenos Específicos , Baço/química , Baço/patologia , Baço/virologia , Proteínas Virais/análise , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Molecular changes in the haemagglutinin (HA)-coding regions and proteolytic cleavage sites from multiple H5N2 subtype viruses isolated during a recent outbreak of avian influenza (AI) in central Mexico have been characterized. Eighteen isolates, collected during a 15 month period (October 1993 to January 1995) from six central states, were sequenced. None of the 18 predicted HA1 amino acid sequences were identical and changes were not restricted to a specific region of the sequence. Phylogenetic analyses of the HA1 sequences demonstrated two virus lineages, designated Puebla and Jalisco, with sequence variation as high as 10.5 percent for amino acid and 6.2 percent for nucleotide sequences. During the latter months of the surveillance period, highly pathogenic (HP) strains of AI emerged causing lethal disease in commercial poultry flocks. In each of the HP strains isolated, the HA protein was cleaved in chicken embryo fibroblast cells in the absence of trypsin, and two alterations not found in earlier non-HP isolates were detected. In the HA protein, HP strains all had a glutamic acid --> lysine substitution at amino acid position 324 and an insertion of arginine and lysine as new residues 325 and 326. The insertion appears to be due to a duplication of the nucleotide sequence AAAGAA at nucleotide positions 965-970 of the HA1-coding region. Computer-assisted secondary structure analyses place the target for the insertion in a predicted RNA stem-loop structure. A mechanism is suggested by which the polymerase duplicates the sequence.
Assuntos
Galinhas , Hemaglutininas Virais/genética , Vírus da Influenza A Subtipo H5N2 , Vírus da Influenza A/genética , Influenza Aviária/virologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Embrião de Galinha , DNA Viral , Surtos de Doenças , Endopeptidases/metabolismo , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Hemaglutininas Virais/química , Processamento de Imagem Assistida por Computador , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/patogenicidade , Influenza Aviária/epidemiologia , México/epidemiologia , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , RNA Viral/química , Homologia de Sequência de Aminoácidos , VirulênciaRESUMO
In October of 1993, there was decreased egg production and increased mortality among Mexican chickens, in association with serologic evidence of an H5N2 influenza virus. First isolated from chickens in May of 1994, after spreading widely in the country, the virus caused only a mild respiratory syndrome in specific pathogen-free chickens. Because eradication of the virus by destruction of infected birds posed major obstacles to the poultry industry in Mexico, we were able to conduct a "field experiment" to determine the fate of an avirulent virus after repeated cycles of replication in millions of chickens. By the end of 1994, the virus had mutated to contain a highly cleavable hemagglutinin (HA), but remained only mildly pathogenic in chickens. Within months, however, it had become lethal in poultry. Nucleotide sequence analysis of the HA cleavage site of the original avirulent strain revealed R-E-T-R, typical of avirulent viruses and unlike the K-K-K-R sequence characterizing viruses responsible for the 1983 outbreak in poultry in the United States. Both mildly and highly pathogenic isolates contained insertions and a substitution of basic residues in the HA connecting peptide, R-K-R-K-T-R, which made the HA highly cleavable in trypsin-free chicken embryo fibroblasts. Phylogenetic analysis of the HA of H5 avian influenza viruses, including the Mexican isolates, indicated that the epidemic virus had originated from the introduction of a single virus of the North American lineage into Mexican chickens. This sequence of events demonstrates, apparently for the first time, the stepwise acquisition of virulence by an avian influenza virus in nature.