RESUMO
Bluetongue (BT) is a severe and economically important disease of ruminants that is widely distributed around the world, caused by the bluetongue virus (BTV). More than 28 different BTV serotypes have been identified in serum neutralisation tests (SNT), which, along with geographic variants (topotypes) within each serotype, reflect differences in BTV outer-capsid protein VP2. VP2 is the primary target for neutralising antibodies, although the basis for cross-reactions and serological variations between and within BTV serotypes is poorly understood. Recombinant BTV VP2 proteins (rVP2) were expressed in Nicotiana benthamiana, based on sequence data for isolates of thirteen BTV serotypes (primarily from Europe), including three 'novel' serotypes (BTV-25, -26 and -27) and alternative topotypes of four serotypes. Cross-reactions within and between these viruses were explored using rabbit anti-rVP2 sera and post BTV-infection sheep reference-antisera, in I-ELISA (with rVP2 target antigens) and SNT (with reference strains of BTV-1 to -24, -26 and -27). Strong reactions were generally detected with homologous rVP2 proteins or virus strains/serotypes. The sheep antisera were largely serotype-specific in SNT, but more cross-reactive by ELISA. Rabbit antisera were more cross-reactive in SNT, and showed widespread, high titre cross-reactions against homologous and heterologous rVP2 proteins in ELISA. Results were analysed and visualised by antigenic cartography, showing closer relationships in some, but not all cases, between VP2 topotypes within the same serotype, and between serotypes belonging to the same 'VP2 nucleotype'.
Assuntos
Vírus Bluetongue/classificação , Vírus Bluetongue/genética , Proteínas do Capsídeo/classificação , Proteínas do Capsídeo/genética , Reações Cruzadas/imunologia , Sorogrupo , Animais , Antígenos Virais/imunologia , Bluetongue/imunologia , Bluetongue/virologia , Vírus Bluetongue/imunologia , Proteínas do Capsídeo/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Coelhos/imunologia , Ruminantes/imunologia , Sorotipagem , Ovinos/imunologia , Nicotiana/genéticaRESUMO
BACKGROUND: Louping ill virus (LIV) and tick-borne encephalitis virus (TBEV) are tick-borne flaviviruses that are both transmitted by the major European tick, Ixodes ricinus. Despite the importance of I. ricinus as an arthropod vector, its capacity to acquire and subsequently transmit viruses, known as vector competence, is poorly understood. At the molecular scale, vector competence is governed in part by binary interactions established between viral and cellular proteins within infected tick cells. METHODS: To investigate virus-vector protein-protein interactions (PPIs), the entire set of open reading frames for LIV and TBEV was screened against an I. ricinus cDNA library established from three embryonic tick cell lines using yeast two-hybrid methodology (Y2H). PPIs revealed for each viral bait were retested in yeast by applying a gap repair (GR) strategy, and notably against the cognate protein of both viruses, to determine whether the PPIs were specific for a single virus or common to both. The interacting tick proteins were identified by automatic BLASTX, and in silico analyses were performed to expose the biological processes targeted by LIV and TBEV. RESULTS: For each virus, we identified 24 different PPIs involving six viral proteins and 22 unique tick proteins, with all PPIs being common to both viruses. According to our data, several viral proteins (pM, M, NS2A, NS4A, 2K and NS5) target multiple tick protein modules implicated in critical biological pathways. Of note, the NS5 and pM viral proteins establish PPI with several tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins, which are essential adaptor proteins at the nexus of multiple signal transduction pathways. CONCLUSION: We provide the first description of the TBEV/LIV-I. ricinus PPI network, and indeed of any PPI network involving a tick-borne virus and its tick vector. While further investigation will be needed to elucidate the role of each tick protein in the replication cycle of tick-borne flaviviruses, our study provides a foundation for understanding the vector competence of I. ricinus at the molecular level. Indeed, certain PPIs may represent molecular determinants of vector competence of I. ricinus for TBEV and LIV, and potentially for other tick-borne flaviviruses.
Assuntos
Proteínas de Artrópodes/metabolismo , Vírus da Encefalite Transmitidos por Carrapatos/genética , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Interações entre Hospedeiro e Microrganismos , Ixodes/genética , Ixodes/virologia , Proteínas Virais/metabolismo , Animais , Proteínas de Artrópodes/genética , Feminino , Biblioteca Gênica , Fases de Leitura Aberta , Domínios e Motivos de Interação entre Proteínas , Proteínas Virais/genéticaRESUMO
Bluetongue is a severe, economically important disease of ruminants that is widely distributed in tropical and temperate regions around the world. It is associated with major production losses, restrictions of animal movements and trade, as well as costs associated with developing and implementing effective surveillance and control measures. Mammalian hosts infected with bluetongue virus (BTV) generate a protective neutralising antibody response targeting the major BTV outer-capsid protein and serotype-specific antigen, VP2. BTV VP2 proteins that have been expressed in plants are soluble, with a native conformation displaying neutralising epitopes and can assemble with other BTV structural proteins to form virus-like particles (VLPs). His-tagged VP2 proteins of BTV serotypes 4 and 8 were transiently expressed in Nicotiana benthamiana then purified by immobilised metal affinity chromatography (IMAC). Antisera from IFNAR -/- mice prime/boost vaccinated with the purified proteins, were shown to contain VP2-specific antibodies by Indirect ELISA (I-ELISA), western blotting and serum neutralisation tests (SNT). Vaccinated mice, subsequently challenged with either the homologous or heterologous BTV serotype, developed viraemia by day 3 post-infection. However, no clinical signs were observed in mice challenged with the homologous serotype (either prime-boost or single-shot vaccinated), all of which survived for the duration of the study. In contrast, all of the vaccinated mice challenged with a heterologous serotype, died, showing no evidence of cross-protection or suppression of viraemia, as detected by real-time RT-qPCR or virus isolation. The induction of protective, serotype-specific neutralising antibodies in IFNAR -/- mice, indicates potential for the use of plant-expressed BTV VP2s as subunit vaccine components, or as a basis for serotype-specific serological assays.
RESUMO
Viruses belonging to the species Wallal virus and Warrego virus of the genus Orbivirus were identified as causative agents of blindness in marsupials in Australia during 1994/5. Recent comparisons of nucleotide (nt) and amino acid (aa) sequences have provided a basis for the grouping and classification of orbivirus isolates. However, full-genome sequence data are not available for representatives of all Orbivirus species. We report full-genome sequence data for three additional orbiviruses: Wallal virus (WALV); Mudjinabarry virus (MUDV) and Warrego virus (WARV). Comparisons of conserved polymerase (Pol), sub-core-shell 'T2' and core-surface 'T13' proteins show that these viruses group with other Culicoides borne orbiviruses, clustering with Eubenangee virus (EUBV), another orbivirus infecting marsupials. WARV shares <70% aa identity in all three conserved proteins (Pol, T2 and T13) with other orbiviruses, consistent with its classification within a distinct Orbivirus species. Although WALV and MUDV share <72.86%/67.93% aa/nt identity with other orbiviruses in Pol, T2 and T13, they share >99%/90% aa/nt identities with each other (consistent with membership of the same virus species - Wallal virus). However, WALV and MUDV share <68% aa identity in their larger outer capsid protein VP2(OC1), consistent with membership of different serotypes within the species - WALV-1 and WALV-2 respectively.
Assuntos
Ceratopogonidae/virologia , Genoma Viral/genética , Marsupiais/virologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Dados de Sequência Molecular , Orbivirus/genética , Filogenia , Análise de Sequência/métodos , Proteínas Estruturais Virais/genéticaRESUMO
The species Corriparta virus (CORV), within the genus Orbivirus, family Reoviridae, currently contains six virus strains: corriparta virus MRM1 (CORV-MRM1); CS0109; V654; V370; Acado virus and Jacareacanga virus. However, lack of neutralization assays, or reference genome sequence data has prevented further analysis of their intra-serogroup/species relationships and identification of individual serotypes. We report whole-genome sequence data for CORV-MRM1, which was isolated in 1960 in Australia. Comparisons of the conserved, polymerase (VP1), sub-core-shell 'T2' and core-surface 'T13' proteins encoded by genome segments 1, 2 and 8 (Seg-1, Seg-2 and Seg-8) respectively, show that this virus groups with the other mosquito borne orbiviruses. However, highest levels of nt/aa sequence identity (75.9%/91.6% in Seg-2/T2: 77.6%/91.7% in Seg-8/T13, respectively) were detected between CORV-MRM1 and California mosquito pool virus (CMPV), an orbivirus isolated in the USA in 1974, showing that they belong to the same virus species. The data presented here identify CMPV as a member of the Corriparta virus species and will facilitate identification of additional CORV isolates, diagnostic assay design and epidemiological studies.
Assuntos
Genoma Viral , Orbivirus/genética , DNA Intergênico/genética , Tipagem de Sequências Multilocus , Orbivirus/classificação , Filogenia , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Proteínas Estruturais Virais/genéticaRESUMO
Eubenangee virus has previously been identified as the cause of Tammar sudden death syndrome (TSDS). Eubenangee virus (EUBV), Tilligery virus (TILV), Pata virus (PATAV) and Ngoupe virus (NGOV) are currently all classified within the Eubenangee virus species of the genus Orbivirus, family Reoviridae. Full genome sequencing confirmed that EUBV and TILV (both of which are from Australia) show high levels of aa sequence identity (>92%) in the conserved polymerase VP1(Pol), sub-core VP3(T2) and outer core VP7(T13) proteins, and are therefore appropriately classified within the same virus species. However, they show much lower amino acid (aa) identity levels in their larger outer-capsid protein VP2 (<53%), consistent with membership of two different serotypes - EUBV-1 and EUBV-2 (respectively). In contrast PATAV showed significantly lower levels of aa sequence identity with either EUBV or TILV (with <71% in VP1(Pol) and VP3(T2), and <57% aa identity in VP7(T13)) consistent with membership of a distinct virus species. A proposal has therefore been sent to the Reoviridae Study Group of ICTV to recognise 'Pata virus' as a new Orbivirus species, with the PATAV isolate as serotype 1 (PATAV-1). Amongst the other orbiviruses, PATAV shows closest relationships to Epizootic Haemorrhagic Disease virus (EHDV), with 80.7%, 72.4% and 66.9% aa identity in VP3(T2), VP1(Pol), and VP7(T13) respectively. Although Ngoupe virus was not available for these studies, like PATAV it was isolated in Central Africa, and therefore seems likely to also belong to the new species, possibly as a distinct 'type'. The data presented will facilitate diagnostic assay design and the identification of additional isolates of these viruses.
Assuntos
Orbivirus/classificação , Orbivirus/genética , África Central , Animais , Austrália , Sequência de Bases , Sequência Conservada , Genoma Viral , Vírus da Doença Hemorrágica Epizoótica/classificação , Vírus da Doença Hemorrágica Epizoótica/genética , Macropodidae/virologia , Orbivirus/isolamento & purificação , Orbivirus/patogenicidade , Filogeografia , RNA Viral/genética , Infecções por Reoviridae/veterinária , Infecções por Reoviridae/virologia , Especificidade da Espécie , Proteínas do Core Viral/genética , Proteínas Estruturais Virais/genéticaRESUMO
Bluetongue virus is the "type" species of the genus Orbivirus, family Reoviridae. Twenty four distinct bluetongue virus (BTV) serotypes have been recognized for decades, any of which is thought to be capable of causing "bluetongue" (BT), an insect-borne disease of ruminants. However, two further BTV serotypes, BTV-25 (Toggenburg orbivirus, from Switzerland) and BTV-26 (from Kuwait) have recently been identified in goats and sheep, respectively. The BTV genome is composed of ten segments of linear dsRNA, encoding 7 virus-structural proteins (VP1 to VP7) and four distinct non-structural (NS) proteins (NS1 to NS4). We report the entire BTV-26 genome sequence (isolate KUW2010/02) and comparisons to other orbiviruses. Highest identity levels were consistently detected with other BTV strains, identifying KUW2010/02 as BTV. The outer-core protein and major BTV serogroup-specific antigen "VP7" showed 98% aa sequence identity with BTV-25, indicating a common ancestry. However, higher level of variation in the nucleotide sequence of Seg-7 (81.2% identity) suggests strong conservation pressures on the protein of these two strains, and that they diverged a long time ago. Comparisons of Seg-2, encoding major outer-capsid component and cell-attachment protein "VP2" identified KUW2010/02 as 26th BTV, within a 12th Seg-2 nucleotype [nucleotype L]. Comparisons of Seg-6, encoding the smaller outer capsid protein VP5, also showed levels of nt/aa variation consistent with identification of KUW2010/02 as BTV-26 (within a 9th Seg-6 nucleotype - nucleotype I). Sequence data for Seg-2 of KUW2010/02 were used to design four sets of oligonucleotide primers for use in BTV-26, type-specific RT-PCR assays. Analyses of other more conserved genome segments placed KUW2010/02 and BTV-25/SWI2008/01 closer to each other than to other "eastern" or "western" BTV strains, but as representatives of two novel and distinct geographic groups (topotypes). Our analyses indicate that all of the BTV genome segments have evolved under strong purifying selection.
Assuntos
Vírus Bluetongue/genética , Genoma Viral/genética , Animais , Vírus Bluetongue/classificação , Cabras , Kuweit , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ovinos , Proteínas não Estruturais Virais/genética , Proteínas Estruturais Virais/genéticaRESUMO
The subtype distribution of 142 genotype 2 and 97 genotype 4 hepatitis C virus (HCV) isolates from the sera of 1,319 volunteer blood donors in France was determined by gene sequencing and by phylogenetic analysis of the NS5B region and E1 envelope. Findings underlined a wide range of subtypes in both genotypes, that is, 20 in HCV-2 and 11 in HCV-4. Eighteen of these 31 subtypes had not been defined previously. Some subtypes, that is, 2a, 2b, 2c, 2i, 2k, 4a, and 4d, showed numerous strains while subtypes in donors from West Africa or Central Africa showed an endemic profile with only a few strains. A Bayesian coalescence approach was used to estimate the demographic history of each HCV subtype. The estimated mean dates of the most recent common ancestors (MRCA) were 1,889 (confidence interval (CI), 1,842-1,930) for HCV-2a, 1,886 (CI, 1,843-1,921) for HCV-2b, 1,791 (CI, 1,699-1,848) for HCV-2c, 1,846 (CI, 1,803-1,878) for HCV-2i, 1,911 (CI, 1,879-1,937) for HCV-4a, and 1,957 (CI, 1,943-1,967) for HCV-4d. The period of spread for subtype 2b, 2c, and 2i was between 1900 and 1960 whereas rapid exponential spread for subtype 2a, 4a, and 4d occurred in the 1960s. The inferred histories of population growth indicated that transmission rates differed according to HCV subtype. These results may help to predict the future burden of HCV in France.
Assuntos
Doadores de Sangue , Hepacivirus/classificação , Hepacivirus/genética , Hepatite C/epidemiologia , Hepatite C/virologia , Teorema de Bayes , França/epidemiologia , Hepacivirus/isolamento & purificação , Humanos , Epidemiologia Molecular , Filogenia , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genéticaRESUMO
Ten virus isolates belonging to species Getah virus (GETV) have been obtained during surveys for arboviruses in China since 1964. Seven of these isolates (YN0540, YN0542, SH05-6, SH05-15, SH05-16, SH05-17 and GS10-2) were obtained during the current study. The full-length sequences of three Chinese isolates (M1, isolated in 1964; HB0234, isolated in 2002; YN0540, isolated in 2005) were determined. The full-length sequences of these isolates were respectively 11 696, 11 686 and 11 690 nt, and showed more than 97 % intraspecies identity. Deletions were found in the capsid protein of strain M1 and non-structural protein nsP3 of strain HB0234. The E2 gene and 3' UTR of all ten isolates were also characterized. The E2 gene of the Chinese GETV isolates showed nucleotide sequence identities of 98-100 % when compared with other GETV isolates. In the 3' UTR of the Chinese isolates, an insertion of 10 consecutive adenine residues (nt 189-198) appeared in strain M1, and 9 or 3 consecutive adenines were found towards the 3' end of the third RES in strains SH05-6 and SH05-15, respectively. The 3' UTRs of the Chinese isolates showed a deletion between positions 45 and 54 and nucleotide transitions at positions 43, 64 and 148. Sequence and phylogenetic analyses showed that there was a relatively high degree of conservation among GETV isolates. The isolation of GETV from various provinces in China and also in Russia and Mongolia (including regions of the northern tundra) are an indication of changes in the world distribution of this re-emerging virus.
Assuntos
Alphavirus/genética , Genoma Viral , Regiões 3' não Traduzidas/genética , Alphavirus/classificação , Animais , Proteínas do Capsídeo/genética , Linhagem Celular , China , Cricetinae , Culicidae/classificação , Culicidae/virologia , Deleção de Genes , Dados de Sequência Molecular , Filogenia , Análise de Sequência , Homologia de Sequência de Aminoácidos , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genéticaRESUMO
The complete nucleotide sequence of Middelburg virus (MIDV) was determined for strain MIDV-857 from Zimbabwe. The isolation of this virus in 1993 from a horse that died showing severe clinical signs represents the first indication that MIDV can cause severe disease in equids. Full-length cDNA copies of the viral genome were successfully synthesized by an innovative RT-PCR amplification approach using an 'anchor primer' combined with the SMART methodology described previously for the synthesis of full-length cDNA copies from genome segments of dsRNA viruses. The MIDV-857 genome is 11,674 nt, excluding the 5'-terminal cap structure and poly(A) tail (which varies in length from approximately 180 to approximately 220 residues). The organization of the genome is like that of other alphaviruses, including a read-through stop codon between the nsP3 and nsP4 genes. However, phylogenetic analyses of the structural protein amino acid sequences suggested that the MIDV E1 gene was generated by recombination with a Semliki Forest virus-like virus. This hypothesis was supported by bootscanning analysis using a recombination-detection program. The 3' untranslated region of MIDV-857 also contains a 112 nt duplication. This study reports the first full-length sequence of MIDV, which was obtained from a single RT-PCR product.
Assuntos
Alphavirus/genética , Genoma Viral , Regiões 3' não Traduzidas/genética , Alphavirus/isolamento & purificação , Infecções por Alphavirus/veterinária , Animais , Sequência de Bases , Códon de Terminação/genética , Doenças dos Cavalos/virologia , Cavalos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , Capuzes de RNA/genética , RNA Mensageiro/genética , Recombinação Genética , Vírus da Floresta de Semliki/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Baço/virologia , Proteínas não Estruturais Virais/genética , Proteínas Estruturais Virais/genética , ZimbábueRESUMO
Banna virus (BAV) particles contain seven structural proteins: VP4 and VP9 form an outer-capsid layer, whilst the virus core contains three major proteins (VP2, VP8 and VP10) and two minor proteins (VP1 and VP3). Sequence analysis showed that VP3 contains motifs [Kx(I/V/L)S] and (Hx(n)H) that have previously been identified in the guanylyltransferases of other reoviruses. Incubation of purified BAV-Ch core particles with [alpha-32P]GTP resulted in exclusive covalent labelling of VP3, demonstrating autoguanylation activity (which is considered indicative of guanylyltransferase activity). Recombinant VP3 prepared in a cell-free expression system was also guanylated under similar reaction conditions, and products were synthesized (in the presence of non-radiolabelled GDP) that co-migrated with GMP, GDP and GpppG during TLC. This reaction, which required magnesium ions for optimum activity, demonstrates that VP3 possesses nucleoside triphosphatase (GTPase) activity and is the BAV guanylyltransferase (RNA 'capping' enzyme).
Assuntos
Coltivirus/enzimologia , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Proteínas do Core Viral/química , Proteínas do Core Viral/metabolismo , Sequência de Aminoácidos , GTP Fosfo-Hidrolases/metabolismo , Metiltransferases , Dados de Sequência Molecular , Complexos Multienzimáticos , Nucleotidiltransferases/genética , Monoéster Fosfórico Hidrolases , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Proteínas do Core Viral/genética , Proteínas ViraisRESUMO
Banna virus (BAV) is the type species of the genus Seadornavirus within the family Reoviridae. The Chinese BAV isolate (BAV-Ch), which causes encephalitis in humans, was shown to have a structural organization and particle morphology reminiscent of that of rotaviruses, with fibre proteins projecting from the surface of the particle. Intact BAV-Ch virus particles contain seven structural proteins, two of which (VP4 and VP9) form the outer coat. The inner (core) particles contain five additional proteins (VP1, VP2, VP3, VP8 and VP10) and are 'non-turreted', with a relatively smooth surface appearance. VP2 is the 'T = 2' protein that forms the innermost 'subcore' layer, whilst VP8 is the 'T = 13' protein forming the core-surface layer. Sequence comparisons indicate that BAV VP9 and VP10 are equivalent to the VP8* and VP5* domains, respectively, of rotavirus outer-coat protein VP4 (GenBank accession no. P12976). VP9 has also been shown to be responsible for virus attachment to the host-cell surface and may be involved in internalization. These similarities reveal a previously unreported genetic link between the genera Rotavirus and Seadornavirus, although the expression of BAV VP9 and VP10 from two separate genome segments, rather than by the proteolytic cleavage of a single gene product (as seen in rotavirus VP4), suggests a significant evolutionary jump between the members of these two genera.
Assuntos
Coltivirus/genética , Encefalite Viral/virologia , Genoma Viral , Infecções por Reoviridae/virologia , Proteínas do Core Viral/química , Proteínas Estruturais Virais/química , Sequência de Aminoácidos , Animais , Coltivirus/classificação , Coltivirus/isolamento & purificação , Coltivirus/ultraestrutura , Eletroforese , Humanos , Camundongos , Microscopia Eletrônica , Dados de Sequência Molecular , Filogenia , Rotavirus/genética , Análise de Sequência de DNA , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo , Vírion/ultraestruturaRESUMO
Banna virus (BAV: genus Seadornavirus, family Reoviridae) has a double-shelled morphology similar to rotavirus and bluetongue virus. The structure of BAV outer-capsid protein VP9 was determined by X-ray crystallography at 2.6 A resolution, revealing a trimeric molecule, held together by an N-terminal helical bundle, reminiscent of coiled-coil structures found in fusion-active proteins such as HIV gp41. The major domain of VP9 contains stacked beta sheets with marked structural similarities to the receptor binding protein VP8 of rotavirus. Anti-VP9 antibodies neutralize viral infectivity, and, remarkably, pretreatment of cells with trimeric VP9 increased viral infectivity, indicating that VP9 is involved in virus attachment to cell surface and subsequent internalization. Sequence similarities were also detected between BAV VP10 and VP5 portion of rotavirus VP4, suggesting that the receptor binding and internalization apparatus, which is a single gene product activated by proteoloysis in rotavirus, is the product of two separate genome segments in BAV.
Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Coltivirus/genética , Proteínas do Core Viral/química , Proteínas do Core Viral/metabolismo , Sequência de Aminoácidos , Western Blotting , Proteínas do Capsídeo/genética , Coltivirus/classificação , Cristalografia por Raios X , Imuno-Histoquímica , Modelos Moleculares , Dados de Sequência Molecular , Testes de Neutralização , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rotavirus/química , Homologia de Sequência de Aminoácidos , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologiaRESUMO
Flavivirus-related sequences have been discovered in the dsDNA genome of Aedes albopictus and Aedes aegypti mosquitoes, demonstrating for the first time an integration into a eukaryotic genome of a multigenic sequence from an RNA virus that replicates without a recognized DNA intermediate. In the Aedes albopictus C6/36 cell line, an open reading frame (ORF) of 1557 aa with protease/helicase and polyprotein processing domains characteristic of flaviviruses was identified. It is closely related to NS1-NS4A genes of the Cell Fusing Agent and Kamiti River virus and the corresponding mRNAs were detected. Integrated sequences homologous to the envelope, NS4B and polymerase genes of flaviviruses were identified. Overall, approximately two-thirds of a flavivirus-like genome were characterized. In the Aedes aegypti A20 cell line, a 492 aa ORF related to the polymerase of the Cell Fusing Agent and Kamiti River virus was identified. These flavivirus-related integrated DNA sequences were detected in laboratory-bred and wild Aedes albopictus and Aedes aegypti mosquitoes, demonstrating that their discovery is not an artefact resulting from the manipulation of mosquito cell lines, since they exist under natural conditions. This finding has major implications regarding evolution, as it represents an entirely different mechanism by which genetic diversity may be generated in eukaryotic cells distinct from accepted processes.
Assuntos
Aedes/virologia , DNA Viral/genética , Flavivirus/classificação , Flavivirus/genética , Genoma Viral , Integração Viral/genética , Aedes/classificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , DNA Viral/isolamento & purificação , Dados de Sequência Molecular , Filogenia , Vírus de RNA/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
This paper describes the study of hepatitis C virus (HCV) evolution in the largest cohort of HCV-infected blood donors (BDs)/blood recipients (BRs) reported to date (25 pairs). A molecular analysis of partial sequences in the E1 (envelope) and NS5-B (polymerase) genes was performed. Phylogenetic reconstruction showed that the evolution of dominant strains was qualitatively and quantitatively different in BDs and BRs. The evolutionary rate was significantly higher in BRs, in which, in addition, most substitutions observed were antonymous. These findings corroborate the hypothesis that a large part of virus evolution - which was evaluated to be equivalent to approximately 20 years of chronic evolution - is acquired during the early phase of infection. These findings should be taken into account for the modelling of the long-term evolution of HCV and their possible contribution to improve our understanding of HCV natural history is discussed.
Assuntos
Doadores de Sangue , Transfusão de Sangue , Evolução Molecular , Hepacivirus/genética , Estudos de Coortes , Hepatite C/virologia , Humanos , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Proteínas não Estruturais Virais/genética , Proteínas Estruturais Virais/genéticaRESUMO
TT virus-like minivirus (TLMV) was recently discovered as a human circovirus. Little is known about its natural history and molecular epidemiology. A study of TLMV infection is described in a population of French hemodialysis patients. TLMV DNA was tested by seminested PCR system located in the noncoding region in 81 patients divided into seven groups according to the origin of their renal disease. Quantitation of TLMV DNA in serum was carried out. Sequences from 28 patients were compared with 40 sequences retrieved from databases and 53 TLMV sequences cloned from the serum of a single patient. The prevalence of TLMV DNA in hemodialysis patients was 95.1%. In this study, 24 samples (29.6%) presented viral loads of > 125 equivalents of plasmid (Ep)/ml, and only 6 (7.8%) had viral loads of > 125 x 10(2) Ep/ml. A significant correlation (P < 0.029) was found between viral loads of > 125 x 10(2) Ep/ml and the neoplastic origin of end-stage renal disease. Analysis of 53 sequences cloned from a single individual demonstrated high sequence variability, as shown by the genetic distance of 40.2%. This genetic distance is comparable to that between the most divergent sequences of TLMV reported to date (43.5%). These data suggest that TLMV viral load is possibly related to the level of immunocompetence of hemodialysis patients; the genetic diversity of TLMV is extremely high; and co-infection by different strains is possible.