RESUMO
Comparative analysis of amino acid and nucleotides sequences of ORFs located in extended segments of the terminal variable regions in variola virus genome detected a promising locus for viral genotyping according to the geographic origin. This is ORF O1L of VARV. The primers were calculated for synthesis of this ORF fragment by PCR, which makes it possible to distinguish South America-Western Africa genotype from other VARV strains. Subsequent RFLP analysis reliably differentiated Asian strains from African strains (except Western Africa isolates). This method has been tested using 16 VARV strains from various geographic regions. The developed approach is simple, fast and reliable.
Assuntos
Reação em Cadeia da Polimerase/métodos , Varíola/virologia , Vírus da Varíola/classificação , Vírus da Varíola/isolamento & purificação , África , Sequência de Aminoácidos , Repetição de Anquirina/genética , Ásia , Primers do DNA/genética , Variação Genética , Genoma Viral/genética , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Polimorfismo de Fragmento de Restrição , Sensibilidade e Especificidade , Alinhamento de Sequência , América do Sul , Vírus da Varíola/genéticaRESUMO
The development of a method in macroarray format for the identification of alphaviruses and orthopoxviruses in samples of concern in biodefense is reported. Capture oligonucleotides designed to bind generic members of the orthopox- or alphavirus families and a collection of additional oligonucleotides to bind specifically nucleic acids from five individual alphaviruses, including Venezuelan equine encephalitis, or DNA from each of four orthopoxviruses, including variola virus (VAR) were deposited onto nylon membranes. Hybridization of digoxigenin labeled PCR products to the macroarray produced results easily observable to the naked eye. Multiplex RT-PCR utilizing both orthopox- and alphavirus-generic primers yielded amplification of DNA corresponding to the expected sizes of the orthopoxvirus and alphavirus fragments, respectively. Hybridization of samples to capture oligonucleotides in the macroarray membranes identified correctly generic orthopox- or alphaviral sequences. The hybridizations correctly identified each of the three alphaviruses and two orthopoxviruses tested. We observed cross-hybridization only once (between two alphaviruses) that was less intense than the spots formed by correct hybridization. The macroarray test described below is easy to perform, inexpensive, relatively fast, uncomplicated to interpret, and its end point is read visually without the need of additional equipment. This nucleic acid hybridization assay onto nylon membranes in macroarray format can help in detecting or excluding the presence of threat viruses in environmental samples and appears promising for a variety of biodefense applications.
Assuntos
Alphavirus/classificação , Alphavirus/isolamento & purificação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Orthopoxvirus/classificação , Orthopoxvirus/isolamento & purificação , Alphavirus/genética , DNA Viral/análise , Digoxigenina , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/isolamento & purificação , Hibridização de Ácido Nucleico , Orthopoxvirus/genética , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Coloração e Rotulagem , Vírus da Varíola/genética , Vírus da Varíola/isolamento & purificaçãoAssuntos
Guerra Biológica/história , Surtos de Doenças/história , Varíola/história , Vírus da Varíola/fisiologia , Feminino , História do Século XX , Humanos , Cazaquistão/epidemiologia , Masculino , Varíola/epidemiologia , Varíola/virologia , U.R.S.S. , Vírus da Varíola/isolamento & purificação , VirulênciaRESUMO
Genome DNA terminal region sequences were determined for a Brazilian alastrim variola minor virus strain Garcia-1966 that was associated with an 0.8% case-fatality rate and African smallpox strains Congo-1970 and Somalia-1977 associated with variola major (9.6%) and minor (0.4%) mortality rates, respectively. A base sequence identity of > or = 98.8% was determined after aligning 30 kb of the left- or right-end region sequences with cognate sequences previously determined for Asian variola major strains India-1967 (31% death rate) and Bangladesh-1975 (18.5% death rate). The deduced amino acid sequences of putative proteins of > or = 65 amino acids also showed relatively high identity, although the Asian and African viruses were clearly more related to each other than to alastrim virus. Alastrim virus contained only 10 of 70 proteins that were 100% identical to homologs in Asian strains, and 7 alastrim-specific proteins were noted.
Assuntos
DNA Viral , Variação Genética , Vírus da Varíola/genética , África , Ásia , Sequência de Bases , Brasil , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , Vírus da Varíola/isolamento & purificação , Proteínas Virais/genéticaRESUMO
We examined the nucleotide sequences of the inverted terminal repeat (ITR) regions adjacent to the covalently closed hairpin end sequences of three variola major and four minor strains from smallpox outbreaks in Europe, Asia, Africa, and South America. The ITR regions ranged in size from 581 to 1051 base pairs (bp) and contained no apparent open reading frames. Two nonrepetitive sequence elements, NR1 and NR2, were conserved and resembled nonrepetitive elements in the ITRs of other orthopoxviruses. Depending on strain, the terminally positioned NR1 and the more internal NR2 flanked a direct repeat region containing from none to four copies of a 69-bp sequence and one copy of a 54-bp related sequence partial repeat. A distinctive pattern of ITR topography of NR1 and NR2 flanking a single copy of the 69-bp unit characterized each of three examined alastrim variola minor strains. A nonalastrim African minor strain from the last natural case of smallpox in Somalia in 1977 showed the largest ITR region of the examined viruses because of a second direct repeat cluster following NR2.
Assuntos
Sequências Repetitivas de Ácido Nucleico , Varíola/virologia , Vírus da Varíola/genética , África/epidemiologia , Ásia/epidemiologia , Sequência de Bases , DNA Viral/química , DNA Viral/genética , DNA Viral/isolamento & purificação , Surtos de Doenças , Europa (Continente)/epidemiologia , Genoma Viral , Humanos , Dados de Sequência Molecular , Varíola/epidemiologia , América do Sul/epidemiologia , Vírus da Varíola/isolamento & purificaçãoRESUMO
La eliminación mundial del virus de la viruela con el virus de la vaccinia fue un proceso que duró poco más de siglo y medio, que se desarrolló en varias etapas según la aceptación del procedimiento, de los adelantos técnicos y del esfuerzo colaborativo de las autoridades internacionales de salud con los representantes en cada uno de los países del mundo. El virus de la vaccinia es asimétrico y está recubierto por una membrana obtenida cuando el virus maduro sale de la célula infectada. Tiene la forma de un ladrillo y es muy resistente a los efectos de los inactivadores físicos y químicos. La certificación definitiva de la erradicación mundial de la viruela fue hecha por la Asamblea Mundial de la Salud en mayo de 1980; con esto el virus de la vaccinia parecía también haber quedado condenado a la extinción. Sin embargo, la comunidad científica se volvió a fijar en él a consecuencia del desarrollo de la tecnología del DNA recombinante, cuando surgió la posibilidad de romper la barrera genética de los organismos y se logró la recombinación de genomas de cualquier especie. Si el agente infeccioso no es patogénico y el gene proviene de otro agente que si lo es, entonces ese vector de expresión se puede utilizar con una vacuna prácticamente ideal, ya que puede inmunizar al sujeto contra ese patógeno, sin peligro para la salud. Uno de los más lógicos candidatos a servir como vector fue el virus de la vaccinia, debido a que sus cualidades inmunogénicas y de seguridad son excelentes ya que su genoma puede incorporar una cantidad importante de DNA, por lo menos 25,000 pares de bases, sin por ello perder su inefectividad. El virus de la vaccinia cuenta ahora con enormes posibilidades para continuarse usando, ya no como medida profiláctica, específica o no, sino como un vehículo seguro y eficiente de material genético de otros agentes infecciosos
Assuntos
México , Varíola/diagnóstico , Varíola/epidemiologia , Varíola/história , Varíola/imunologia , Vaccinia virus/análise , Vaccinia virus/efeitos dos fármacos , Vaccinia virus/genética , Vaccinia virus/imunologia , Vaccinia virus/isolamento & purificação , Vacinação/classificação , Vacinação/história , Vacinação/instrumentação , Vacinação/métodos , Vacinação/tendências , Vírus da Varíola/análise , Vírus da Varíola/imunologia , Vírus da Varíola/isolamento & purificação , Vírus da Varíola/fisiologiaRESUMO
Several groups of variola isolates were compared in DNA structure, and by four independent biologic markers. Isolates of variola minor from Europe and South America (alastrim virus) could be distinguished from African isolates of variola minor by DNA structure and by two of the four biologic markers. Taken as a group, the properties of African isolates, in general, differed from those of variola major, but this difference was confined to properties which depended (in the laboratory) on the recent history of the virus concerned. The suggestion made previously that there was an "intermediate" or "African" variety of variola virus is discounted. Laboratory tests did not distinguish any individual African isolate from variola major virus. It is concluded that a virus which may be called "alastrim" represents a "fixed" variant of variola virus, whose distribution is consistent with the dramatic spread of variola minor through the Americas and Europe in the early part of this century, and that variola minor in Africa in recent years was due to variola virus which was not alastrim and which laboratory evidence fails to identify as an entity distinguishable from variola major virus.