-
Volume 88,
Issue 9,
2007
Volume 88, Issue 9, 2007
- Animal
-
- DNA viruses
-
-
A comprehensive library of mutations of Epstein–Barr virus
A mutant library of 249 mutants with mutations that span the entire Epstein–Barr virus (EBV) genome was generated by transposition with EZ : : TN
and insertion with an apramycin resistance gene by a PCR-targeting method. This study also demonstrates the feasibility of generating deletions and site-specific mutations in the BRLF1 promoter on the EBV genome to determine the regions in the promoter that are crucial to transcription. Analysing BZLF1 and BRLF1 mutants by microarray analysis revealed that these two genes regulate the transcription of EBV lytic genes differently. A BZLF1 mutation affects global expression of EBV lytic genes; almost no lytic gene is expressed by the mutant after lytic induction. However, although a BRLF1 mutant still transcribes most lytic genes, the expression of these lytic genes is inefficient. Furthermore, this study shows that the proximal Zta-response element in the BRLF1 promoter is crucial to BRLF1 transcription from the EBV genome, despite the fact that another work demonstrated that this site was unimportant in transient transfection analysis. Furthermore, mutants with a mutation in BDLF1 and BORF1 cannot assemble viral capsids. Results of this study demonstrate the usefulness of a comprehensive mutant library in genetic analyses of EBV.
-
-
-
Virus distribution of the attenuated MVA and NYVAC poxvirus strains in mice
Recombinant vaccinia viruses based on the attenuated NYVAC and MVA strains are promising vaccine candidates against a broad spectrum of diseases. Whilst these vectors are safe and immunogenic in animals and humans, little is known about their comparative behaviour in vivo. In this investigation, a head-to-head analysis was carried out of virus dissemination in mice inoculated by the mucosal or systemic route with replication-competent (WRluc) and attenuated recombinant (MVAluc and NYVACluc) viruses expressing the luciferase gene. Bioluminescence imaging showed that, in contrast to WRluc, the attenuated recombinants expressed the reporter gene transiently, with MVAluc expression limited to the first 24 h and NYVACluc giving a longer signal, up to 72 h post-infection, for most of the routes assayed. Moreover, luciferase levels in MVAluc-infected tissues peaked earlier than those in tissues infected by NYVACluc. These findings may be of immunological relevance when these vectors are used as recombinant vaccines.
-
-
-
Reprogramming the chiA expression profile of Autographa californica multiple nucleopolyhedrovirus
More LessExpression of chiA and v-cath RNA and enzyme activity in wild-type Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was compared with that of recombinant AcMNPV viruses reprogrammed for expression of the endogenous chiA. To establish a baseline for our recombinant AcMNPV studies, we compared, for the first time, the temporal expression profiles of both AcMNPV chiA transcription and translation simultaneously. The rate of intracellular chitinase accumulation during AcMNPV infection followed the same pattern observed for chiA transcription but was delayed by about 6 h. Replacement of 21 nucleotides containing the native late chiA and v-cath promoters with a selectable polh–EGFP cassette was sufficient to eliminate expression of both chiA and v-cath. Viruses were generated that express chiA from either the late p6.9 or very late polh promoters of AcMNPV, replacing the native chiA promoter. There was a marked difference in the temporal chiA transcription profiles from the native, p6.9 and polh promoters, resulting in respective specific activities of chitinase at 48 h p.i. of 62, 160 and 219 mU (mg lysate total protein)−1. Based on temporal analysis of v-cath transcription by Northern blot, AcMNPV v-cath was transcribed from 9 h p.i. in Sf21 cells. However, expression of v-cath RNA or enzyme from a reconstructed v-cath promoter in the chiA-reprogrammed viruses was not detected at 48 h of virus replication. Reprogramming for increased chitinase (and putatively cathepsin) expression with native baculovirus promoters might provide a means for designing environmentally benign biological insecticides.
-
-
-
The Chilo iridescent virus DNA polymerase promoter contains an essential AAAAT motif
More LessThe delayed-early DNA polymerase promoter of Chilo iridescent virus (CIV), officially known as Invertebrate iridescent virus, was fine mapped by constructing a series of increasing deletions and by introducing point mutations. The effects of these mutations were examined in a luciferase reporter gene system using Bombyx mori cells transfected with promoter constructs and infected with CIV. When the size of the upstream element was reduced from position −19 to −15, relative to the transcriptional start site, the luciferase activity was reduced to almost zero. Point mutations showed that each of the 5 nt (AAAAT) located between –19 and –15 were equally essential for promoter activity. Mutations at individual bases around the transcription initiation site showed that the promoter extended until position −2 upstream of the transcription start site. South-Western analysis showed that a protein of approximately 100 kDa interacted with the −19 nt promoter fragment in CIV-infected cells. This binding did not occur with a point mutant that lacked promoter activity. The AAAAT motif was also found in the DNA polymerase promoter region of other iridoviruses and in other putative CIV delayed-early genes.
-
- Plant
-
-
-
2b ORFs encoded by subgroup IB strains of cucumber mosaic virus induce differential virulence on Nicotiana species
More LessCucumber mosaic virus (CMV)-encoded 2b protein from subgroup IA or subgroup II was shown to be a determinant of virulence in many solanaceous hosts. In this study, the virulence of 2b proteins from subgroup IB strains was analysed using four intraspecies hybrid viruses, which were generated by precise replacement of the 2b open reading frame (ORF) in subgroup IA strain Fny-CMV with the 2b ORFs of four subgroup IB strains, Cb7-CMV, PGs-CMV, Rad35-CMV and Na-CMV, generating FCb72b-CMV, FPGs2b-CMV, FRad352b-CMV and FNa2b-CMV, respectively. FCb72b-CMV was more virulent than Fny-CMV, and was similar in phenotype to its parental virus Cb7-CMV on the three Nicotiana species tested. FNa2b-CMV also was virulent on these host species, equivalent to Fny-CMV or Na-CMV. However, FRad352b-CMV only caused mild mosaic or undetectable symptoms on all the host species tested, and was less virulent than Fny-CMV or Rad35-CMV. FPGs2b-CMV infected all the host species systemically, and induced either mosaic or barely visible symptoms, demonstrating that the inability of PGs-CMV to infect these three Nicotiana species was not due to its 2b protein. The diverse virulence was shown to be mediated by the 2b proteins rather than the C-terminal overlapping parts of the 2a proteins, and was associated with the level of viral progeny RNA accumulation in systemically infected leaves, but not with the rate of long-distance viral movement in host plants. Through analysis of encapsidation of viral RNAs, there was an apparent correlation between the virulence and the high level of encapsidated RNA 2 in virions of Fny-CMV, FCb72b-CMV and FNa2b-CMV.
-
-
-
-
Analysis of the serological variability of Lettuce mosaic virus using monoclonal antibodies and surface plasmon resonance technology
A panel of 19 monoclonal antibodies (mAbs) was used to study the immunological variability of Lettuce mosaic virus (LMV), a member of the genus Potyvirus, and to perform a first epitope characterization of this virus. Based on their specificity of recognition against a panel of 15 LMV isolates, the mAbs could be clustered in seven reactivity groups. Surface plasmon resonance analysis indicated the presence, on the LMV particles, of at least five independent recognition/binding regions, correlating with the seven mAbs reactivity groups. The results demonstrate that LMV shows significant serological variability and shed light on the LMV epitope structure. The various mAbs should prove a new and efficient tool for LMV diagnostic and field epidemiology studies.
-
-
-
Combinations of two amino acids (Ala40 and Phe75 or Ser40 and Tyr75) in the coat protein of apple chlorotic leaf spot virus are crucial for infectivity
More LessAmino acid sequences of apple chlorotic leaf spot virus (ACLSV) coat protein (CP) were compared between 12 isolates from apple, plum and cherry, and 109 cDNA clones that were amplified directly from infected apple tissues. Phylogenetic analysis based on the amino acid sequences of CP showed that the isolates and cDNA clones were separated into two major clusters in which the combinations of the five amino acids at positions 40, 59, 75, 130 and 184 (Ala40-Val59-Phe75-Ser130-Met184 or Ser40-Leu59-Tyr75-Thr130-Leu184) were highly conserved within each cluster. Site-directed mutagenesis using an infectious cDNA clone of ACLSV indicated that the combinations of two amino acids (Ala40 and Phe75 or Ser40 and Tyr75) are necessary for infectivity to Chenopodium quinoa plants by mechanical inoculation. Moreover, an agroinoculation assay indicated that the substitution of a single amino acid (Ala40 to Ser40 or Phe75 to Tyr75) resulted in extreme reduction in the accumulation of viral genomic RNA, double-stranded RNAs and viral proteins (movement protein and CP) in infiltrated tissues, suggesting that the combinations of the two amino acids at positions 40 and 75 are important for effective replication in host plant cells.
-
- Other Agents
-
-
-
Amyloid-specific fluorophores for the rapid, sensitive in situ detection of prion contamination on surgical instruments
More LessPrion diseases or transmissible spongiform encephalopathies (TSEs) are a group of rare, transmissible and fatal neurodegenerative diseases associated with the protein agent (PrPSc). As such, the sensitive and rapid detection of prion PrPSc amyloid on the surface of suspect surgical instruments is of great importance and may even allow remedial action to be taken prior to any further operative intervention and possible iatrogenic transmission. However, conventional PrPSc detection methodologies tend to rely on the inefficient and unreliable removal of suspect material from a surface using swabs or wipes prior to antibody analysis. Here we show how the combination of an advanced light microscope technique, episcopic differential interference contrast/epifluorescence (EDIC/EF) microscopy, and the application of β-amyloid fluorescent thiazole markers (thioflavin T, thioflavin S) can be used to detect, in situ, submicron (attomole) levels of prion protein amyloid contamination in brain and spleen sections, smears and homogenate on surgical stainless steel surfaces and surgical instruments. This technique, although not specific to an amyloid type, can be used to verify that surgical instruments are substantially free from prion amyloid protein soiling and hence reduce the risk of iatrogenic transmission.
-
-
- Jgv Direct
-
-
-
Reduction of vector gene expression increases foreign antigen-specific CD8+ T-cell priming
Viral vectors have been shown to induce protective CD8+ T-cell populations in animal models, but significant obstacles remain to their widespread use for human vaccination. One such obstacle is immunodominance, where the CD8+ T-cell response to a vector can suppress the desired CD8+ T-cell response to a recombinantly encoded antigen. To overcome this hurdle, we broadly reduced vector-specific gene expression. We treated a recombinant vaccinia virus, encoding antigen as a minimal peptide determinant (8–10 aa), with psoralen and short-wave UV light. The resulting virus induced 66 % fewer vector-specific immunodominant CD8+ T cells, allowing the in vivo induction of an increased number of CD8+ T cells specific for the recombinant antigen.
-
-
-
-
Mapping to completeness and transplantation of a group-specific, discontinuous, neutralizing epitope in the envelope protein of dengue virus
More LessDengue is caused by a taxonomic group of four viruses, dengue virus types 1–4 (DENV1–DENV4). A molecular understanding of the antibody-mediated protection against this disease is critical to design safe vaccines and therapeutics. Here, the energetic epitope of antibody mAb4E11, which neutralizes the four serotypes of DENV but no other flavivirus, and binds domain 3 (ED3) of their envelope glycoprotein, was characterized. Alanine-scanning mutagenesis of the ED3 domain from serotype DENV1 was performed and the affinities between the mutant domains and the Fab fragment of mAb4E11 were measured. The epitope residues (307–312, 387, 389 and 391) were at the edges of two distinct β-sheets. Four residues constituted hot spots of binding energy. They were aliphatic and contributed to form a hydrophobic pocket (Leu308, Leu389), or were positively charged (Lys307, Lys310). They may bind the diversity residues of mAb4E11, H-Trp96-Glu97. Remarkably, cyclic residues occupy and block the hydrophobic pocket in all unrelated flaviviruses. Transplanting the epitope from the ED3 domain of DENV into those of other flaviviruses restored affinity. The epitope straddles residues of ED3 that are involved in virulence, e.g. Asn/Asp390. These results define the epitope of mAb4E11 as an antigenic signature of the DENV group and suggest mechanisms for its neutralization potency.
-
-
-
Adaptation of two flaviviruses results in differences in genetic heterogeneity and virus adaptability
More LessWest Nile virus (WNV) is a mosquito-borne flavivirus that was first introduced into the USA in the New York City area in 1999. Since its introduction, WNV has steadily increased both its host and geographical ranges. Outbreaks of the closely related flavivirus, St. Louis encephalitis virus (SLEV), occur in the USA periodically, but levels of activity and host range are more restricted than those of WNV. Understanding the selective pressures that drive arbovirus adaptation and evolution in their disparate mosquito and avian hosts is crucial to predicting their ability to persist and re-emerge. Here, we evaluated the in vivo phenotypes of mosquito cell-adapted WNV and SLEV. Results indicated that in vitro adaptations did not translate to in vivo adaptations for either virus, yet SLEV displayed attenuated growth in both mosquitoes and chickens, while WNV generally did not. In vitro growth analyses also indicated that WNV adaptations could be generalized to cell cultures derived from other mosquito species, while SLEV could not. Analysis of genetic diversity for passaged SLEV revealed a highly homogeneous population that differed significantly from previous results of high levels of diversity in WNV. We hypothesize that this difference in genetic diversity is directly related to the viruses' success in new and changing environments in the laboratory and that differences in a viruses' ability to produce and maintain heterogeneous populations in nature may in some instances explain the variable levels of success seen among arboviruses.
-
Volumes and issues
-
Volume 106 (2025)
-
Volume 105 (2024)
-
Volume 104 (2023)
-
Volume 103 (2022)
-
Volume 102 (2021)
-
Volume 101 (2020)
-
Volume 100 (2019)
-
Volume 99 (2018)
-
Volume 98 (2017)
-
Volume 97 (2016)
-
Volume 96 (2015)
-
Volume 95 (2014)
-
Volume 94 (2013)
-
Volume 93 (2012)
-
Volume 92 (2011)
-
Volume 91 (2010)
-
Volume 90 (2009)
-
Volume 89 (2008)
-
Volume 88 (2007)
-
Volume 87 (2006)
-
Volume 86 (2005)
-
Volume 85 (2004)
-
Volume 84 (2003)
-
Volume 83 (2002)
-
Volume 82 (2001)
-
Volume 81 (2000)
-
Volume 80 (1999)
-
Volume 79 (1998)
-
Volume 78 (1997)
-
Volume 77 (1996)
-
Volume 76 (1995)
-
Volume 75 (1994)
-
Volume 74 (1993)
-
Volume 73 (1992)
-
Volume 72 (1991)
-
Volume 71 (1990)
-
Volume 70 (1989)
-
Volume 69 (1988)
-
Volume 68 (1987)
-
Volume 67 (1986)
-
Volume 66 (1985)
-
Volume 65 (1984)
-
Volume 64 (1983)
-
Volume 63 (1982)
-
Volume 62 (1982)
-
Volume 61 (1982)
-
Volume 60 (1982)
-
Volume 59 (1982)
-
Volume 58 (1982)
-
Volume 57 (1981)
-
Volume 56 (1981)
-
Volume 55 (1981)
-
Volume 54 (1981)
-
Volume 53 (1981)
-
Volume 52 (1981)
-
Volume 51 (1980)
-
Volume 50 (1980)
-
Volume 49 (1980)
-
Volume 48 (1980)
-
Volume 47 (1980)
-
Volume 46 (1980)
-
Volume 45 (1979)
-
Volume 44 (1979)
-
Volume 43 (1979)
-
Volume 42 (1979)
-
Volume 41 (1978)
-
Volume 40 (1978)
-
Volume 39 (1978)
-
Volume 38 (1978)
-
Volume 37 (1977)
-
Volume 36 (1977)
-
Volume 35 (1977)
-
Volume 34 (1977)
-
Volume 33 (1976)
-
Volume 32 (1976)
-
Volume 31 (1976)
-
Volume 30 (1976)
-
Volume 29 (1975)
-
Volume 28 (1975)
-
Volume 27 (1975)
-
Volume 26 (1975)
-
Volume 25 (1974)
-
Volume 24 (1974)
-
Volume 23 (1974)
-
Volume 22 (1974)
-
Volume 21 (1973)
-
Volume 20 (1973)
-
Volume 19 (1973)
-
Volume 18 (1973)
-
Volume 17 (1972)
-
Volume 16 (1972)
-
Volume 15 (1972)
-
Volume 14 (1972)
-
Volume 13 (1971)
-
Volume 12 (1971)
-
Volume 11 (1971)
-
Volume 10 (1971)
-
Volume 9 (1970)
-
Volume 8 (1970)
-
Volume 7 (1970)
-
Volume 6 (1970)
-
Volume 5 (1969)
-
Volume 4 (1969)
-
Volume 3 (1968)
-
Volume 2 (1968)
-
Volume 1 (1967)
Most Read This Month
