RESUMEN
AIM: Study of circulating 02_AG recombinant form HIV-1 isolates that have been rapidly spreading in Novosibirsk region during 3 recent years. MATERIALS AND METHODS: WHO protocol for primary HIV isolation was used, automatic sequencer was used for genetic characterization of isolates. Virus specific RNA were isolated and env HIV-1 region DNA fragments were processed. Phylogenetic analysis was also performed. RESULTS: CRF_02AG HIV-1 isolated from peripheral blood of HIV-1 positive patients belonged to CCR5 tropic viruses and had various reproduction characteristics. Most of the HIV isolated were rapidly replicating virus variants characterized by an ability to accumulate high levels of virus protein p24 in cultural fluid. Infectivity and reproductive properties of HIV isolates were confirmed in experimental infection by using clarified cultural liquid of mononuclear cells from healthy donors. Phylogenetic analysis of CRF02_AG HIV-1 variants isolated in Novosibirsk region in 2007 - 2010 showed the formation of a separate outbreak in the area caused by emergence of CRF02_AG HIV-1 in human population. CONCLUSION: A collection of genetically and biologically characterized CRF02_AG HIV-1 isolates that has not been spreading previously in Russia.
Asunto(s)
Genes env/genética , Genes pol/genética , Infecciones por VIH/epidemiología , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/genética , Adolescente , Adulto , Preescolar , Femenino , Genotipo , VIH-1/aislamiento & purificación , Humanos , Masculino , Filogenia , Receptores CCR5/inmunología , Recombinación Genética , Análisis de Secuencia de ADN , Siberia/epidemiologíaRESUMEN
Libraries of hybrid plasmids carrying DNA fragments of complete genomes of 8 variola virus strain from the Russian Collection belonging to 2 epidemical types and isolated in various geographic regions of the world were obtained. Genomic sequences of variola virus can be thus preserved for a long time in a biologically safe form and provide the research work on studying the genetic organization of this unique virus and on developing modern methods for rapid detection of variola virus and other orthopoxviruses.
Asunto(s)
Genoma Viral , Virus de la Viruela/genética , ADN Viral/análisis , ADN Viral/genética , Salud Global , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Mapeo RestrictivoAsunto(s)
Evolución Molecular , Monkeypox virus/genética , Infecciones por Poxviridae/virología , Virus de la Viruela/genética , Animales , República Democrática del Congo , Genes Virales/genética , Humanos , Monkeypox virus/aislamiento & purificación , Monkeypox virus/patogenicidad , Sistemas de Lectura Abierta/genética , Infecciones por Poxviridae/epidemiología , Infecciones por Poxviridae/transmisión , Viruela/epidemiología , Viruela/transmisión , Viruela/virología , Virus de la Viruela/patogenicidad , Proteínas Virales/genética , Virulencia/genéticaRESUMEN
Monkeypox virus (MPV) belongs to the orthopoxvirus genus of the family Poxviridae, is endemic in parts of Africa, and causes a human disease that resembles smallpox. The 196,858-bp MPV genome was analyzed with regard to structural features and open reading frames. Each end of the genome contains an identical but oppositely oriented 6379-bp terminal inverted repetition, which similar to that of other orthopoxviruses, includes a putative telomere resolution sequence and short tandem repeats. Computer-assisted analysis was used to identify 190 open reading frames containing >/=60 amino acid residues. Of these, four were present within the inverted terminal repetition. MPV contained the known essential orthopoxvirus genes but only a subset of the putative immunomodulatory and host range genes. Sequence comparisons confirmed the assignment of MPV as a distinct species of orthopoxvirus that is not a direct ancestor or a direct descendent of variola virus, the causative agent of smallpox.
Asunto(s)
Genoma Viral , Monkeypox virus/genética , Sistemas de Lectura Abierta , Análisis de Secuencia de ADN , Animales , Secuencia de Bases , ADN Viral/química , ADN Viral/genética , Humanos , Datos de Secuencia Molecular , Monkeypox virus/química , Filogenia , Telómero/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Monkeypox virus (MPV) causes a human disease which resembles smallpox but with a lower person-to-person transmission rate. To determine the genetic relationship between the orthopoxviruses causing these two diseases, we sequenced the 197-kb genome of MPV isolated from a patient during a large human monkeypox outbreak in Zaire in 1996. The nucleotide sequence within the central region of the MPV genome, which encodes essential enzymes and structural proteins, was 96.3% identical with that of variola (smallpox) virus (VAR). In contrast, there were considerable differences between MPV and VAR in the regions encoding virulence and host-range factors near the ends of the genome. Our data indicate that MPV is not the direct ancestor of VAR and is unlikely to naturally acquire all properties of VAR.
Asunto(s)
Genoma Viral , Monkeypox virus/genética , Monkeypox virus/patogenicidad , Virus de la Viruela/genética , Virus de la Viruela/patogenicidad , Secuencia de Aminoácidos , Ancirinas/química , Evolución Molecular , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , VirulenciaAsunto(s)
Monkeypox virus/química , Orthopoxvirus/química , Proteínas Virales/química , Secuencia de Aminoácidos , Aminoácidos/química , Secuencia de Bases , Sitios de Unión , Proteínas Inactivadoras de Complemento/química , Datos de Secuencia Molecular , Monkeypox virus/genética , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Proteínas Virales/genéticaRESUMEN
The most potent antigen among HSV-1 proteins are glycoproteins gB(UL27) and gD(US6). Multiple amino acid sequence alignment of these proteins shows that gD protein is the most specific for HSV-1. Analysis of gD protein epitopes detected the main antigenic determinants not cross-reactive with antigens of other viruses. Virus was isolated and genome DNA was prepared from morphological elements of a patient with herpes simplex infection. US6 gene fragment was cloned in pUC19 vector. Cloning in bacterial expression vectors helped obtain beta-galactosidase-fused recombinant HSV-1 gD protein with 6-histidines affine target for high-performance chromatography purification. ELISA with a set of HSV-1-positive and negative donor sera and a commercial panel of HSV-1 sera (Vektor-Best) showed that recombinant gD can be used as an antigen to HSV-1-specific IgG.
Asunto(s)
Epítopos/química , Proteínas del Envoltorio Viral/química , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Epítopos/genética , Epítopos/aislamiento & purificación , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Homología de Secuencia de Aminoácido , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/aislamiento & purificaciónRESUMEN
To identify the hantaviruses causing hemorrhagic fever with renal syndrome (HFRS) in the Far East of Russia, blood samples collected from HFRS patients in 1994-1998, were examined by reverse transcription-polymerase chain reaction. In addition, 36 sera were tested by an immunofluorescence assay for antibodies against Hantaan, Seoul, Puumala, and Khabarovsk viruses, and 54 samples were tested by plaque reduction neutralization test. With both serological assays, the highest antibody titers were to Hantaan and/or Seoul viruses. Of 110 blood samples 36 were found RT-PCR positive. Phylogenetic analysis the sequences of a 256-nucleotide (nt) fragment of the hantavirus M genome segment revealed at least 3 genetically distinct hantavirus lineages. Nucleotide sequence comparison showed that two of the lineages, designated as FE and Amur (AMR), differed from one another by 15.9-21.2% and from Hantaan virus by 9.8-17.5%. The third lineage, VDV, differed from Seoul virus by 2.6-5.1%. All S segment sequences were from FE lineage, and differed from Hantaan virus by 10.7-12.6%. Thirty of the 36 (83%) analyzed sequences were found to be the FE genotype, which is very similar to that of Hantaan virus, strain 76-118. Of the remaining hantaviruses, 11% were the AMR genotype, and 6% the VDV genotype, which are genetically novel genotypes of Hantaan or Seoul viruses, respectively.
Asunto(s)
Variación Genética , Virus Hantaan/genética , Fiebre Hemorrágica con Síndrome Renal/virología , Secuencia de Aminoácidos , ADN Viral/análisis , ADN Viral/sangre , Virus Hantaan/aislamiento & purificación , Fiebre Hemorrágica con Síndrome Renal/epidemiología , Humanos , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Federación de Rusia/epidemiología , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
Alastrim variola minor virus, which causes mild smallpox, was first recognized in Florida and South America in the late 19th century. Genome linear double-stranded DNA sequences (186,986 bp) of the alastrim virus Garcia-1966, a laboratory reference strain from an outbreak associated with 0.8% case fatalities in Brazil in 1966, were determined except for a 530-bp fragment of hairpin-loop sequences at each terminus. The DNA sequences (EMBL Accession No. Y16780) showed 206 potential open reading frames for proteins containing >/=60 amino acids. The amino acid sequences of the putative proteins were compared with those reported for vaccinia virus strain Copenhagen and the Asian variola major strains India-1967 and Bangladesh-1975. About one-third of the alastrim viral proteins were 100% identical to correlates in the variola major strains and the remainder were >/=95% identical. Compared with variola major virus DNA, alastrim virus DNA has additional segments of 898 and 627 bp, respectively, within the left and right terminal regions. The former segment aligns well with sequences in other orthopoxviruses, particularly cowpox and vaccinia viruses, and the latter is apparently alastrim-specific.
Asunto(s)
ADN Viral/genética , Genoma Viral , Virus de la Viruela/genética , 3-Hidroxiesteroide Deshidrogenasas/genética , Secuencia de Aminoácidos , Repetición de Anquirina , Secuencia de Bases , Línea Celular , Virus de la Viruela Vacuna/genética , Proteínas de Unión al ADN/genética , Humanos , Recién Nacido , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Orthopoxvirus/genética , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Virus Vaccinia/genética , Proteínas Virales/genéticaRESUMEN
Analysis of published reports helped us single out the most potent antigens among HCMV proteins: phosphoproteins pp150(UL32) and p52(UL44). Theoretical computer analysis of p52 epitopes showed the main antigenic determinants not cross-reacting with antigens of other viruses. Virus-containing (strain AD169) material was obtained and genome DNA was isolated. Amplification of a site of gene UL44 coding for unique determinants detected a PCR fragment of required electrophoretic mobility. The fragment was cloned in vector pLBE. The specificity of cloning was confirmed by restriction analysis of theoretical sites. Nucleotide sequence of cloned fragment of UL44 gene was studied by Maxam-Gilbert's method. Cloning in expressing bacterial vectors helped obtain HCMV recombinant protein p52 in the pure form and fused with beta-galactosidase. Enzyme immunoassay with HCMV-positive and negative donor sera and ABBOTT HCMV sera showed that recombinant p52 increased the sensitivity and specificity of a previously obtained recombinant pp150 as an antigen to HCMV-IgG and HCMV-IgM. The sensitivity and specificity is 100% with 98-99% reliability.
Asunto(s)
Antígenos Virales/aislamiento & purificación , Citomegalovirus/inmunología , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas Virales/aislamiento & purificación , Secuencia de Aminoácidos , Antígenos Virales/química , Antígenos Virales/genética , Secuencia de Bases , Clonación Molecular , Reacciones Cruzadas , ADN Viral , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Técnicas para Inmunoenzimas , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Sequencing and computer analysis of the left (52,283 bp) and right (49,649 bp) variable DNA regions of the cowpox virus strain GRI-90 (CPV-GRI) has revealed 51 and 37 potential open reading frames (ORFs), respectively. Comparison of the structure-function organization of these DNA regions of CPV-GRI with those previously published for corresponding regions of genomes of vaccinia virus, strains Copenhagen (VAC-COP) and Western Reserve (VAC-WR); and variola major virus, strains India-1967 (VAR-IND), Bangladesh-1975 (VAR-BSH); and alastrim variola minor virus, strain Garcia-1966 (VAR-GAR), was performed. Within the left terminal region under study, an extended DNA sequence (14,171 bp), unique to CPV, has been found. Within the right region of the CPV-GRI genome two segments, which are unique to CPV DNA (1579 and 3585 bp) have been found. Numerous differences have been revealed in the genetic structure of CPV-GRI DNA regions, homologous to fragments of the genomes of the above-mentioned orthopoxvirus strains. A cluster of ORFs with structural similarity ot immunomodulatory and host range function of other poxviruses have also been detected. A comparison of the sequences of ORF B, crmA, crmB, crmC, IMP, and CHO hr genes of CPV Brighton strain (CPV-BRI) with the corresponding genes in strain GRI-90 have revealed an identity at the amino acid level ranging from 82 to 96% between the two strains. The findings are significant in light of the recent demonstration of CPV as an important poxvirus model system to probe the precise in vivo role(s) of the unique virally encoded immunomodulatory proteins. Also, the presence of a complete and intact repertoire of immunomodulatory proteins, ring canal proteins family, and host range genes indicates that CPV may have been the most ancient of all studied orthopoxviruses.
Asunto(s)
Virus de la Viruela Vacuna/genética , Genoma Viral , Sistemas de Lectura Abierta , Análisis de Secuencia de ADN , Secuencia de Aminoácidos , Animales , Femenino , Humanos , Datos de Secuencia Molecular , Topos , Orthopoxvirus/genética , Receptores del Factor de Necrosis Tumoral/genética , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Aminoácido , Proteínas Virales/genéticaRESUMEN
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.
Asunto(s)
ADN Viral , Variación Genética , Virus de la Viruela/genética , África , Asia , Secuencia de Bases , Brasil , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , Virus de la Viruela/aislamiento & purificación , Proteínas Virales/genéticaAsunto(s)
Genoma Viral , Virus de la Viruela/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , ADN Viral , Datos de Secuencia Molecular , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Relación Estructura-Actividad , Proteínas Virales/genéticaAsunto(s)
Cisteína Endopeptidasas/genética , Virus de la Encefalitis Equina del Oeste/genética , Recombinación Genética , Proteínas no Estructurales Virales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cisteína Endopeptidasas/metabolismo , Datos de Secuencia Molecular , Filogenia , ARN Viral , Homología de Secuencia de Aminoácido , Proteínas no Estructurales Virales/metabolismoRESUMEN
Sequencing of variola virus (VAR) genome region of 43069 bp was carried out. This area contains 42 potential genes. Computer analysis of proteins coding for these viral genes was done. We compared VAR proteins with the those of vaccinia virus. The region studied is conservative for orthopoxviruses.