ABSTRACT
Four closely related chymotrypsin genes were identified in Anopheles aquasalis and Anopheles darlingi (Anachy1, Anachy2, Andchy1 and Andchy2). The deduced amino-acid sequences were compared to other chymotrypsin sequences. These sequences were used to infer phylogenetic relationships among the different species. Genomic cloning revealed that, in contrast to An. aquasalis and A. gambiae, the chymotrypsin genomic locus in An. darlingi had a short intergenic region that accompanied the inverted position of the genes, suggesting inversion mechanisms in this species related to transposable elements. Alignments of the sequences upstream of the transcription start sites of Anachy1, Anachy2, Andchy1 and Andchy2 revealed areas with high similarity containing palindromic sequences. Northern analysis from An. aquasalis indicated that the transcription of chy 1 and 2 are induced by blood feeding.
Subject(s)
Anopheles/genetics , Chymotrypsin/genetics , Amino Acid Sequence , Animals , Anopheles/enzymology , Anopheles/parasitology , Base Sequence , Cloning, Molecular , DNA Transposable Elements , Molecular Sequence Data , Phylogeny , Sequence Homology, Amino Acid , Sequence Homology, Nucleic AcidABSTRACT
Chimeric yellow fever (YF)-dengue type 2 (Den 2) viruses were constructed by replacing the premembrane (prM) and envelope (E) genes of YF 17D virus with those from Den 2 virus strains of south-east Asian genotype. Whereas viable chimeric viruses were successfully recovered when the YF 17D C gene and the Den 2 prM gene were fused at the signalase cleavage site, no virus could be rescued from the constructions fused at the viral protease cleavage site. Unlike YF virus that replicated in all the cell lines tested and similar to the Den 2 virus, the recombinant viruses did not replicate in vaccine-production certified CEF and MRC5 cells. Besides, chimeric 17D/Den 2 viruses and their parental viruses reached similar growth titers in Vero and C6/36 cell cultures. Analysis of mouse neurovirulence, performed by intracerebral inoculation, demonstrated that the 17D/Den 2 chimera is more attenuated in this system than the YF 17DD virus. Immunization of mice with this chimera induced a neutralizing antibody response associated with a partial protection against an otherwise lethal dose of mouse neurovirulent Den 2 NGC virus. Overall, these results provide further support for the use of chimeric viruses as an attractive methodology for the development of new live flavivirus vaccines.
Subject(s)
Dengue Virus/genetics , Yellow fever virus/genetics , Amino Acid Sequence , Animals , Chlorocebus aethiops , Dengue Virus/growth & development , Dengue Virus/immunology , Dengue Virus/pathogenicity , Electrophoresis, Polyacrylamide Gel/methods , Mice , Molecular Sequence Data , Recombination, Genetic , Sequence Analysis, DNA , Vero Cells , Viral Proteins/analysis , Yellow fever virus/growth & development , Yellow fever virus/immunology , Yellow fever virus/pathogenicityABSTRACT
Outbreaks of an epidermic disease suggesting parapox virus infections have been observed in all major herds of sheep and goats from different geographical areas of Brazil. Clinical samples (dried scabs) were collected and orf virus was isolated and characterized by electron microscopy in previous work. In order to characterize these viruses at the molecular level, a modified methodology for genomic DNA extraction directly from scabs was used and such DNA was used to derive the restriction enzyme digestion patterns for clinical samples from three distinct geographic origins. Pulsed field gel electrophoresis was used to separate restriction enzyme DNA fragments and heterogeneity among isolates from different geographic areas could be observed on stained gels. The HindIII-G DNA fragment from orf-A virus genome was cloned and hybridized to DNA of other orf virus isolates. Further heterogeneity was confirmed by these hybridizations.
Subject(s)
Ecthyma, Contagious/virology , Goat Diseases/virology , Orf virus/genetics , Animals , Brazil/epidemiology , Cloning, Molecular , DNA Probes/chemistry , DNA, Viral/chemistry , DNA, Viral/isolation & purification , Deoxyribonuclease HindIII/chemistry , Disease Outbreaks/veterinary , Ecthyma, Contagious/epidemiology , Electrophoresis, Agar Gel/veterinary , Electrophoresis, Gel, Pulsed-Field/veterinary , Goat Diseases/epidemiology , Goats , Microscopy, Electron/veterinary , Nucleic Acid Hybridization , Orf virus/chemistry , Orf virus/classification , SheepABSTRACT
The complete nucleotide sequence of the genome from two yellow fever (YF) virus strains, 17DD and 17D-213 was determined. Comparison of these sequences with those of other YF viruses, including the parental virulent Asibi strain, allowed the identification of 48 nucleotide sequence differences which are 17D strain-specific and potentially related to viral attenuation. Another 43 nucleotide sequence differences were not common to all 17D substrains and are therefore substrain specific. Of the 21 changes between 17DD and Asibi 15 only five led to amino acid substitutions whereas 13 substrain differences common to all 17D-204 substrains produced six amino acid substitutions. Since the exact passage histories of these viruses is known it was possible to calculate, for each strain, the number of accumulated changes per passage. Based on these data the 17DD strain was the most genetically stable virus.
Subject(s)
Genetic Variation , Yellow fever virus/classification , Yellow fever virus/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary/genetics , DNA, Viral/genetics , Genome, Viral , Humans , Phenotype , Species Specificity , Vaccines, Attenuated/genetics , Viral Proteins/genetics , Viral Vaccines/genetics , Virulence/genetics , Virulence/immunology , Yellow fever virus/immunologyABSTRACT
The Ilheus (ILH) virus has long been known to belong to group B of the arboviruses. Previous attempts to relate it to existing serogroups within the Flavivirus genus using conventional serological techniques such as hemagglutination inhibition, neutralization and complement fixation tests have been inconclusive. We have first used denaturing gel electrophoresis to estimate the molecular weight of immunoprecipitated radiolabeled viral proteins and the cross-reactivity among ILH proteins and hyperimmune sera to several flaviviruses only from the mosquito-borne encephalitis virus serogroups. The estimated molecular weight for the three proteins was in the same order of magnitude, as previously established, for mosquito-borne flaviviruses. Cross-immunoprecipitation tests showed that NS3 protein is the most cross-reactive. Partial nucleotide sequence analyses of the NS3 gene, corresponding to an area linking the helicase and the RNA triphosphatase domains, revealed that ILH virus is very closely related to the Japanese encephalitis virus complex confirming earlier serological data.
Subject(s)
Encephalitis Virus, Japanese/classification , Flaviviridae/classification , Viral Nonstructural Proteins/genetics , Animals , Arboviruses/classification , Arboviruses/genetics , Chlorocebus aethiops , Cross Reactions , Encephalitis Virus, Japanese/genetics , Flaviviridae/genetics , Genome, Viral , RNA Helicases , Serine Endopeptidases , Vero Cells , Viral Proteins/analysis , Viral Proteins/immunologyABSTRACT
The complete nucleotide sequence of the genome from two yellow fever (YF) virus vaccine strains, 17DD and 17D-213, has been determined. Comparison of these sequences with those of other YF viruses including the parental virulent Asibi strain allowed the identification of 48 nucleotide sequence differences which are common to all 17D substrains. This is a significant reduction from the 67 nucleotide changes originally reported as being 17D-specific and potentially related to viral attenuation. The 48 changes are scattered throughout the genome, 26 of which are silent and 22 led to amino acid substitutions. These 22 changes are bona fide candidates to test by mutating the infectious YF cDNA to investigate their role in viral attenuation.
