- Volume 88, Issue 5, 2007
Volume 88, Issue 5, 2007
- Animal
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- DNA viruses
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Identification of HLA-DR1- and HLA-DR15-restricted human papillomavirus type 16 (HPV16) and HPV18 E6 epitopes recognized by CD4+ T cells from healthy young women
More LessHuman papillomavirus (HPV) infection, particularly with types 16 and 18, is causally associated with the development of cervical cancer. Prophylactic vaccines against HPV have recently been licensed and have the primary aim of protecting children against future HPV infection and cervical cancer. However, these vaccines are unlikely to be effective in women with pre-existing HPV infection and disease. Previous studies have suggested that HPV16 E6-specific CD4+ T cells play a role in controlling viral infection; however, the epitopes recognized by such T-cells have not been defined. In this study, we analysed T-cell responses against HPV16 and 18 in ten healthy young women in an age group (21–31) with a high prevalence of HPV infection and clearance. Five individuals made HPV E6 responses, from which five candidate T-cell epitopes (three HPV16 E6 and two HPV18 E6) were identified. More detailed characterization of epitopes from HPV16 E6(127–141) and HPV18 E6(43–57) revealed HLA-DRB1*01 and HLA-DRB1*15 restriction, respectively. Furthermore, generation of a T-cell line against HPV16 E6(127–141) demonstrated that this epitope could be presented after endogenous processing of soluble HPV16 E6 protein. Overall we demonstrate a powerful approach for defining novel CD4+ T-cell epitopes from two oncogenic HPV types. This approach could be applied to study populations in developing countries with a high incidence of cervical cancer. Such epitopes could provide a more precise way of investigating the role of natural and vaccine-induced T-cell responses against HPV in blood and at sites of disease.
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Characterization of two novel cutaneous human papillomaviruses, HPV93 and HPV96
Two novel human papillomaviruses (HPVs), HPV93 and HPV96, with genomes of 7450 and 7438 bp, respectively, are described. The L1 open reading frame of HPV93 showed highest identity to HPV24 (79 %) and that of HPV96 had highest identity to HPV92 (71 %). Real-time PCR for HPV92, 93 and 96 on stripped biopsies from tumours and healthy skin from 269 immunocompetent patients found HPV DNA in 2.6 % of tumours and in 0.4 % of healthy skin samples. Double infections were observed in two tumours. HPV92 was detected in four, HPV93 in two and HPV96 in three tumours. The range of viral loads spanned from one copy per 45 cells to one copy per 10 000 cells. The E7 proteins of HPV92, 93 and 96 were found to bind the retinoblastoma protein (pRb). These results suggest a possible role for these HPV types in skin carcinogenesis that deserves further study.
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Complete genomic characterization of a murine papillomavirus isolated from papillomatous lesions of a European harvest mouse (Micromys minutus)
The papillomaviruses form a large group of species-specific pathogens that cause epithelial proliferations in a wide spectrum of animal hosts. Previous reports demonstrated a relatively high frequency of a variety of skin lesions in captive European harvest mice. The Micromys minutus papillomavirus (MmPV) was isolated from one of these lesions found on a captive European harvest mouse in a regional zoo in Chicago. In this study we present the entire genomic sequence of MmPV. The MmPV genome is organized into the seven classical papillomaviral open reading frames. Phylogenetic analysis places MmPV together with a papillomavirus (PV) isolated from a Syrian golden Hamster (HaOPV) in the genus Pipapillomavirus. The similar clustering pattern of the MmPV–HaOPV pair and their rodent hosts support the hypothesis of papillomaviral and host co-phylogenetic descent. The availability of the complete genomic sequence of a mouse PV should allow researchers to use MmPV as a model for PV carcinogenesis.
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Betapapillomaviruses frequently persist in the skin of healthy individuals
Infections with human papillomaviruses (HPVs) belonging to the genus Betapapillomavirus have been linked to the development of non-melanoma skin cancer. Although persistence is expected, systematic investigation of this aspect of betapapillomavirus (β-PV) infection has not been conducted. This study investigated the prevalence and persistence of 25 known β-PV types in the skin of immunocompetent individuals. Over a 2 year period, eight consecutive plucked eyebrow hair samples taken from 23 healthy individuals were analysed for the presence of β-PV DNA. Using a recently published general β-PV PCR and genotyping method, 61 % of the individuals were β-PV DNA positive for one or more types at intake, whereas during follow-up this percentage rose to 96 %. HPV23 was the most frequently detected β-PV type. Type-specific β-PV DNA was detected over 6 months or longer in 74 % of the individuals. In 57 % of the individuals, DNA from multiple β-PV types was detected simultaneously for 6 months or longer. When the detection intervals of all β-PV type-specific infections in the study population were considered, a substantial proportion, 48 %, lasted at least half a year. The consistent β-PV patterns found over time in most individuals strongly suggested that β-PV DNA detection in plucked eyebrow hairs reveals true β-PV infection. If the minimum interval of detection was set at 6 months, persistent β-PV infections were found in the majority of the study population (74 %).
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Identification and functional analysis of the origins of DNA replication in the Cydia pomonella granulovirus genome
More LessThe entire genome of Cydia pomonella granulovirus (CpGV) was systematically screened for origins of DNA replication, using an infection-dependent DNA replication assay in the granulovirus-permissive Cydia pomonella cell line, Cp14R. All seven cosmids in an overlapping library that covered the CpGV genome were found to replicate in the assay. A genomic library of 32 overlapping plasmids was subsequently screened. Plasmids that replicated were in turn subcloned into 1–2 kbp overlapping fragments. Eleven subclones replicated, each containing at least one of the 13 single-copy 74–76 bp imperfect palindromes, previously identified in the CpGV genome as possible origins of replication. Genome fragments of 156 bp, each containing one of the 13 palindromes, were cloned to verify replication and provided confirmation that these 13 palindromes are the only origins of replication in the genome. A real-time PCR method was developed for the quantification of DNA replication, which eliminated the need for Southern blotting and hybridization. A set of deletion clones allowed further quantitative characterization of one of the palindromes. The previously proposed non-homologous region origin of replication did not replicate in the assay.
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- Plant
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Molecular dissection of the potato virus Y VPg virulence factor reveals complex adaptations to the pvr2 resistance allelic series in pepper
More LessThe virulence properties of potato virus Y (PVY) towards an allelic series at the pvr2 locus in pepper genotypes are related to variations in the genome-linked viral protein (VPg). Eleven amino acid substitutions in the central part of the VPg were identified in strains differing by their virulenceproperties and were introduced, either singly or in combination, in an infectious PVY clone to get an in-depth genetic analysis of the virulence determinant. The virulence spectrum of these mutants was evaluated by inoculation of four pepper genotypes carrying different alleles at the pvr2 locus. The mutations introduced had complex effects on virulence, including antagonisticepistasis and trade-offs for virulence towards different pvr2 alleles. In addition, several mutants showed new virulence properties that were unknown in the natural environment. Such complex effects of mutations on plant virus virulence are unprecedented. They provide a better understanding of the variable levels of durability of the resistance conferred by the different pvr2 alleles, and have important consequences for a durable management of the resistances.
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In vitro association between the helper component–proteinase of zucchini yellow mosaic virus and cuticle proteins of Myzus persicae
More LessPotyviruses, as typical non-persistently transmitted viruses, are carried within the stylets of aphids. Cuticle proteins (CuPs), which are a major component of the insect cuticle, were examined for in vitro binding to the potyviral helper component–proteinase (HC–Pro). Proteins in 8 M urea extracts from Myzus persicae were separated by SDS-PAGE, electroblotted onto membranes and identified as CuPs by using specific antibodies to M. persicae CuP. Blotted M. persicae protein extracts were overlaid with two HC–Pros, differing by the presence of K or E in the KLSC domain. The HC–Pro with KLSC, known to assist transmission, was found to bind M. persicae proteins, whereas the HC–Pro with ELSC, being deficient in assisting transmission, did not. To identify CuPs that react with HC–Pro, protein extracts were separated by two-dimensional gel electrophoresis. Nine proteins reacting with HC–Pro were sequenced by mass spectrometry. Sequences of peptides in four proteins, of molecular masses between 22 and 31 kDa, were identified as CuPs according to comparison with sequences in GenBank. The putative CuPs from M. persicae that bind HC–Pro are potentially of interest in locating receptors for virions bound to HC–Pro in aphids’ stylets.
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RNA4-encoded p31 of beet necrotic yellow vein virus is involved in efficient vector transmission, symptom severity and silencing suppression in roots
More LessRNA3 and RNA4 of beet necrotic yellow vein virus (BNYVV) are not essential for virus multiplication, but are associated with vector-mediated infection and disease development in sugar beet roots. Here, a unique role for RNA4 in virus transmission, virulence and RNA silencing suppression was demonstrated. Mutagenic analysis revealed that the RNA4-encoded p31 open reading frame (ORF) was involved in efficient vector transmission and slight enhancement of symptom expression in some Beta species. No effects of RNA4 on virus accumulation in infected tissue were observed. Furthermore, the p31 ORF was involved in the induction of severe symptoms by BNYVV in Nicotiana benthamiana plants without affecting viral RNA accumulation. In contrast, RNA3-encoded p25, previously identified as a major contributor to symptom induction in sugar beet, had no such effect on N. benthamiana. In two different silencing suppression assays, neither p31 nor p25 was able to suppress RNA silencing in leaves, but the presence of p31 enhanced a silencing suppressor activity in roots without alteration in viral RNA accumulation. Thus, BNYVV p31 plays a multifunctional role in efficient vector transmission, enhanced symptom expression and root-specific silencing suppression.
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A novel cleavage site within the potato leafroll virus P1 polyprotein
More LessTo study the proteolytic processing of the potato leafroll virus replicase proteins, the multidomain P1 protein with a c-myc epitope tag attached at the N terminus was expressed in insect cells by using the baculovirus system. Western blotting showed that P1 was cleaved at a site upstream of the serine protease domain, in addition to the cleavage site downstream of the protease domain. Mutational analysis showed that the serine protease domain within P1 was responsible for this cleavage. To characterize this novel cleavage site further, a portion of the P1 protein comprising the protease domain and the two cleavage sites was expressed in Escherichia coli. A similar cleavage event was observed in bacteria and was abolished when the P1 protease was inactivated by mutation. Peptide-sequencing studies indicated that this cleavage occurred at a Glu/Arg junction, separating the N-terminal 204 residues from the serine protease domain of P1.
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Infectivity, pseudorecombination and mutagenesis of Kenyan cassava mosaic begomoviruses
Cloned DNA-A and DNA-B components of Kenyan isolates of East African cassava mosaic virus (EACMV, EACMV-UG and EACMV-KE2), East African cassava mosaic Kenya virus (EACMKV) and East African cassava mosaic Zanzibar virus (EACMZV) are shown to be infectious in cassava. EACMV and EACMKV genomic components have the same iteron sequence (GGGGG) and can form viable pseudorecombinants, while EACMZV components have a different sequence (GGAGA) and are incompatible with EACMV and EACMKV. Mutagenesis of EACMZV has demonstrated that open reading frames (ORFs) AV1 (encoding the coat protein), AV2 and AC4 are not essential for a symptomatic infection of cassava, although mutants of both ORF AV1 and AV2 produce attenuated symptoms in this host. Furthermore, ORF AV1 and AV2 mutants were compromised for coat protein production, suggesting a close structural and/or functional relationship between these coding regions or their protein products.
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Cymbidium ringspot virus defective interfering RNA replication in yeast cells occurs on endoplasmic reticulum-derived membranes in the absence of peroxisomes
More LessThe replication of Cymbidium ringspot virus (CymRSV) defective interfering (DI) RNA in cells of the yeast Saccharomyces cerevisiae normally takes place in association with the peroxisomal membrane, thus paralleling the replication events in infected plant cells. However, previous results with a peroxisome-deficient mutant strain of yeast had suggested that the presence of peroxisomes is not a strict requirement for CymRSV DI RNA replication. Thus, a novel approach was used to study the putative alternative sites of replication by using S. cerevisiae strain YPH499 which does not contain normal peroxisomes. In this strain, CymRSV p33 and p92 accumulated over portions of the nuclear membrane and on membranous overgrowths which were identified as endoplasmic reticulum (ER) strands, following immunofluorescence and immunoelectron microscope observations. The proteins were not released by high-pH treatment, but were susceptible to proteolytic digestion, thus indicating peripheral and not integrated association. ER-associated p33 and p92 proteins supported in trans the replication of DI RNA. The capacity of plus-strand RNA viruses to replicate in association with different types of cell membranes was thus confirmed.
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- Jgv Direct
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Properties of H7N7 influenza A virus strain SC35M lacking interferon antagonist NS1 in mice and chickens
Non-structural protein NS1 of influenza A virus counteracts the host immune response by blocking the synthesis of type I interferon (IFN). As deletion of the complete NS1 gene has to date been reported only in the human H1N1 strain A/PR/8/34, it remained unclear whether NS1 is a non-essential virulence factor in other influenza A virus strains as well. In this report, the properties of NS1-deficient mutants derived from strain SC35M (H7N7) are described. A mutant of SC35M that completely lacks the NS1 gene was an excellent inducer of IFN in mammalian and avian cells in culture and, consequently, was able to multiply efficiently only in cell lines with defects in the type I IFN system. Virus mutants carrying C-terminally truncated versions of NS1 were less powerful inducers of IFN and were attenuated less strongly in human A549 cells. Although attenuated in wild-type mice, these mutants remained highly pathogenic for mice lacking the IFN-regulated antiviral factor Mx1. In contrast, the NS1-deficient SC35M mutant was completely non-pathogenic for wild-type mice, but remained pathogenic for mice lacking Mx1 and double-stranded RNA-activated protein kinase (PKR). Wild-type SC35M, but not the NS1-deficient mutant virus, was able to replicate in the upper respiratory tract of birds, but neither virus induced severe disease in adult chickens. Altogether, this study supports the view that NS1 represents a non-essential virulence factor of different influenza A viruses.
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Volumes and issues
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Volume 105 (2024)
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Volume 73 (1992 - 2024)
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Volume 2 (1968)
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Volume 1 (1967)