- Volume 88, Issue 5, 2007
Volume 88, Issue 5, 2007
- Animal
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- RNA viruses
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Complete genomes for hepatitis C virus subtypes 6f, 6i, 6j and 6m: viral genetic diversity among Thai blood donors and infected spouses
In this study, the first complete genome sequences for hepatitis C virus (HCV) subtypes 6f, 6i, 6j and 6m, obtained from infected blood donors in Chiang Mai, Thailand, are reported. Pairwise genome-wide nucleotide similarities between some of these isolates were higher than the 75–80 % value used previously to define different HCV subtypes. To investigate further, the entire genomes of four prototype isolates, Th602 (6i), Th553 (6j), B4/92 (6m) and D86/93 (6n), were sequenced. Pairwise comparison of these sequences gave a similar range of nucleotide similarities, thereby providing new information for HCV subtype classification. In order to study the hypothesis of interspousal HCV transmission, four additional complete HCV genome sequences were obtained from two infected Thai blood donors and their spouses, C-0044 and C-0046 (6f), and C-0192 and C-0185 (6m). Pairwise comparison of the sequences revealed that C-0044 and C-0046 share a nucleotide similarity of 98.1 %, whilst C-0185 and C-0192 have a similarity of 97.8 %. Several other studies of partial HCV sequences of different genomic regions from HCV-infected couples have shown nucleotide similarities ranging from 96.3 to 100 %. The similarities of the complete genome sequences from the two couples in the current study are consistent with HCV transmission between spouses.
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Complete genomes of hepatitis C virus (HCV) subtypes 6c, 6l, 6o, 6p and 6q: completion of a full panel of genomes for HCV genotype 6
Five hepatitis C virus (HCV) complete genome sequences (Th846, 537796, QC227, QC216 and QC99) from a blood donor in Thailand and three Asian immigrants and one Caucasian in North America were determined. Phylogenetically, they represent the first complete genomes for subtypes 6c, 6l, 6o, 6p and 6q, respectively. Similarity analysis showed no evidence of inter- or intrasubtype recombination. Further analysis in conjunction with partial sequences from the Los Alamos HCV database led to the identification of other closely related isolates from south-eastern Asia or immigrants from that region. However, Th846 did not cluster with any reference sequence and is the sole isolate of subtype 6c reported so far. This study completes the full genome sequencing of all 17 assigned HCV genotype 6 subtypes (6a–6q). The utility of this panel of complete sequences for accurate detection and classification of infection, and for estimating the origin of this genotype of HCV, is discussed.
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Genetic diversity in hepatitis C virus in Egypt and possible association with hepatocellular carcinoma
Egypt has one of the world’s highest prevalences of hepatitis C virus (HCV) infection, with a majority of genotype 4 infections. To explore the genetic diversity of HCV in Egypt, sera from 131 Egyptians [56 from community studies, 37 chronic hepatitis patients, 28 hepatocellular carcinoma (HCC) patients and 10 patients with non-Hodgkin’s lymphoma] were genotyped by restriction fragment-length polymorphism and phylogenetic analyses of sequences from the mid-core and non-structural 5B regions. The different genotyping methods showed good agreement. The majority of the viruses (83 of 131; 63 %) were of subtype 4a, but five other subtypes within genotype 4 were also observed, as well as three genotype 1b, five genotype 1g and one genotype 3a samples. Interestingly, subtype 4o, which was easily identifiable in all three genomic regions, showed an association with HCC (P=0.017), which merits further investigation.
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Egg whites from eggs of chickens infected experimentally with avian hepatitis E virus contain infectious virus, but evidence of complete vertical transmission is lacking
H. Guo, E. M. Zhou, Z. F. Sun and X.-J. MengAvian hepatitis E virus (HEV) is genetically and antigenically related to human HEV. Vertical transmission of HEV has been reported in humans, but not in other animals. In this study, we showed that avian HEV could be detected in chicken egg-white samples. Subsequently, avian HEV in egg white was found to be infectious, as evidenced by the appearance of viraemia, faecal virus shedding and seroconversion in chickens inoculated with avian HEV-positive egg white, but not in chickens inoculated with HEV-negative egg white. To further assess the possibility of vertical transmission of avian HEV, batches of embryonated eggs from infected hens were hatched, and hatched chicks were monitored for evidence of avian HEV infection. However, no virus was detected in samples collected from the hatched chicks throughout this study, suggesting that avian HEV could not complete the vertical transmission cycle. The possible implications of our findings are also discussed.
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Analysis of the complete genomic sequence of an apparently avirulent strain of avian hepatitis E virus (avian HEV) identified major genetic differences compared with the prototype pathogenic strain of avian HEV
P. Billam, Z. F. Sun and X.-J. MengAvian hepatitis E virus (HEV) was identified from chickens with hepatitis–splenomegaly syndrome. In this study, the complete genomic sequence of an apparently avirulent strain of avian HEV was determined to be 6649 nt in length, excluding the poly(A) tail, which is 5 nt shorter than the prototype avian HEV. Sequence analyses revealed that the ORF1 has 89.6 % nucleotide sequence identity, with numerous non-silent mutations and deletions, compared to the prototype strain. The ORF2 capsid gene showed 90.7 % sequence identity with six non-silent mutations, and ORF3 had four non-silent mutations with 97 % sequence identity. Overall, the apparently avirulent strain shares only 90.1 % nucleotide sequence identity with the prototype strain. The identification of significant non-silent mutations in the capsid gene and other regions suggests that these mutations may play a role in HEV attenuation. This is the first report of the full-length sequence of an apparently avirulent strain of HEV.
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Genetic determinants of Sindbis virus strain TR339 affecting midgut infection in the mosquito Aedes aegypti
More LessMosquito midgut epithelial cells (MEC) play a major role in determining whether an arbovirus can successfully infect and be transmitted by mosquitoes. The Sindbis virus (SINV) strain TR339 efficiently infects Aedes aegypti MEC but the SINV strain TE/5′2J poorly infects MEC. SINV determinants for MEC infection have been localized to the E2 glycoprotein. The E2 amino acid sequences of TR339 and TE/5′2J differ at two sites, E2-55 and E2-70. We have altered the TE/5′2J virus genome by site-directed mutagenesis to contain two TR339 residues, E2-55 H→Q (histidine to glutamine) and E2-70 K→E (lysine to glutamic acid). We have characterized the growth patterns of derived viruses in cell culture and determined the midgut infection rate (MIR) in A. aegypti mosquitoes. Our results clearly show that the E2-55 H→Q and the E2-70 K→E mutations in the TE/5′2J virus increase MIR both independently and in combination. TE/5′2J virus containing both TR339 E2 residues had MIRs similar to the parental TR339 virus. In addition, SINV propagated in a mammalian cell line had a significantly lower A. aegypti midgut 50 % infectious dose than virus propagated in a mosquito cell line.
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Further characterization of a paramyxovirus transcription initiation signal: search for required nucleotides upstream and importance of the N phase context
More LessParamyxovirus genomes contain a linear array of five to ten genes sequentially transcribed by the viral RNA polymerase. mRNA synthesis initiates at a nucleotide signal (gs1) within the genomic promoter located at the genome 3′ end. To gain information about the mechanism involved in transcription initiation, a search was carried out for upstream nucleotides required for gs1 and the effects of the gs1 nucleocapsid protein (N) phase context on transcription regulation were determined. For both purposes, tandem promoter mini-genomes carrying a transcription signal ectopically positioned downstream of a replication-only signal were used. The requirement for hygromycin resistance gene expression was used in an attempt to select essential nucleotides within randomized stretches of nucleotides. Nucleotide insertions or deletions were also made on either side of the transcription signal to change its original N phase context in the five remaining possibilities and GFP expression from these modified signals was assessed. Cell cultures resistant to hygromycin treatment were readily obtained following amplification of mini-genomes harbouring randomized sequences. However, selected nucleotides upstream of gs1 could not be identified under conditions where nucleotides within gs1 were selected. In contrast, it was observed that changing the gs1 N phase context progressively decreased transcription by five- to tenfold. These results are discussed in relation to two different mechanisms of transcription initiation.
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A novel monolayer cell line derived from human umbilical cord blood cells shows high sensitivity to measles virus
Measles virus (MeV) research is largely dependent on the B95a cell line, which is derived from marmoset B lymphocytes. As this cell line is persistently infected with Epstein–Barr virus (EBV), a novel cell line, COBL-a, was established from human umbilical cord blood. COBL-a cells have a significant advantage over B95a cells because they are of human origin, are free from EBV and have higher sensitivity to wild-type MeV. Thus, COBL-a cells should prove very valuable for both epidemiological and basic studies of MeV.
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Analysis of gene expression in Lassa virus-infected HuH-7 cells
More LessThe pathogenesis of Lassa fever is poorly understood. As the liver is a major target organ of Lassa virus, gene expression in Lassa virus-infected HuH-7 cells, a differentiated human hepatoma cell line, was studied. Cellular mRNA levels were measured at the late phase of acute infection, when virtually all cells expressed large amounts of nucleoprotein, and virus RNA concentration had reached >108 copies (ml supernatant)−1. Two types of transcription array were used: cDNA-based macroarrays with a set of 3500 genes (Atlas Human 1.2 arrays; Clontech) and oligonucleotide-based microarrays covering 18 400 transcripts (Human Genome U133A array; Affymetrix). Data analysis was based on statistical frameworks controlling the false-discovery rate. Atlas array data were considered relevant if they could be verified by U133A array or real-time RT-PCR. According to these criteria, there was no evidence for true changes in gene expression. Considering the precision of the U133A array and the number of replicates tested, potential expression changes due to Lassa virus infection are probably smaller than twofold. To substantiate the array data, beta interferon (IFN-β) gene expression was studied longitudinally in Lassa virus-infected HuH-7 and FRhK-4 cells by using real-time RT-PCR. IFN-β mRNA levels increased only twofold upon Lassa virus infection, although there was no evidence that the virus inhibited poly(I : C)-induced IFN-β gene expression. In conclusion, Lassa virus interferes only minimally with gene expression in HuH-7 cells and poorly induces IFN-β gene transcription.
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Induction of apoptosis in Saccharomyces cerevisiae results in the spontaneous maturation of tetravirus procapsids in vivo
The Tetraviridae are a family of small, non-enveloped, insect RNA viruses consisting of one or two single-stranded, positive-sense genomic RNAs encapsidated in an icosahedral capsid with T=4 symmetry. Tetravirus procapsids undergo maturation when exposed to a low pH environment in vitro. While the structural biology of the conformational changes that mediate acid-dependent maturation is well understood, little is known about the significance of acid-dependent maturation in vivo. To address this question, the capsid-coding sequence of the tetravirus Helicoverpa armigera stunt virus was expressed in Saccharomyces cerevisiae cells. Virus-like particles were shown to assemble as procapsids that matured spontaneously in vivo as the cells began to age. Growth in the presence of hydrogen peroxide or acetic acid, which induced apoptosis or programmed cell death in the yeast cells, resulted in virus-like particle maturation. The results demonstrate that assembly-dependent maturation of tetravirus procapsids in vivo is linked to the onset of apoptosis in yeast cells. We propose that the reduction in pH required for tetraviral maturation may be the result of cytosolic acidification, which is associated with the early onset of programmed cell death in infected cells.
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Identification of minimal sequences of the Rhopalosiphum padi virus 5′ untranslated region required for internal initiation of protein synthesis in mammalian, plant and insect translation systems
More LessRhopalosiphum padi virus (RhPV) is a member of the family Dicistroviridae. The genomes of viruses in this family contain two open reading frames, each preceded by distinct internal ribosome entry site (IRES) elements. The RhPV 5′ IRES is functional in mammalian, insect and plant translation systems and can form 48S initiation complexes in vitro with just the mammalian initiation factors eIF2, eIF3 and eIF1. Large regions of the 5′ untranslated region (UTR) can be deleted without affecting initiation-complex formation. The minimal sequences required for directing internal initiation in mammalian (rabbit reticulocyte lysate), plant (wheatgerm extract) and insect (Sf21 cells) translation systems have now been defined. A fragment (nt 426–579) from the 3′ portion of the 5′ UTR can direct translation in each of these translation systems. In addition, a distinct region (nt 300–429) is also active. Thus, unstructured regions within the 5′ UTR seem to be critical for IRES function.
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Vaccination with a T-cell-priming Gag peptide of caprine arthritis encephalitis virus enhances virus replication transiently in vivo
CD4+ T cells are involved in several immune response pathways used to control viral infections. In this study, a group of genetically defined goats was immunized with a synthetic peptide known to encompass an immunodominant helper T-cell epitope of caprine arthritis encephalitis virus (CAEV). Fifty-five days after challenge with the molecularly cloned CAEV strain CO, the vaccinated animals had a higher proviral load than the controls. The measurement of gamma interferon and interleukin-4 gene expression showed that these cytokines were reliable markers of an ongoing immune response but their balance did not account for more or less efficient control of CAEV replication. In contrast, granulocyte–macrophage colony-stimulating factor appeared to be a key cytokine that might support virus replication in the early phase of infection. The observation of a potential T-cell-mediated enhancement of virus replication supports other recent findings showing that lentivirus-specific T cells can be detrimental to the host, suggesting caution in designing vaccine candidates.
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- DNA viruses
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In vivo fitness and virulence of a drug-resistant herpes simplex virus 1 mutant
More LessTwo important issues regarding a virus mutant that is resistant to an antiviral drug are its ability to replicate in animal hosts (in vivo fitness) relative to other genetic variants, including wild type, and its ability to cause disease. These issues have been investigated for a herpes simplex virus 1 mutant that is resistant to thiourea compounds, which inhibit encapsidation of viral DNA. Following corneal inoculation of mice, the mutant virus replicated very similarly to its wild-type parent in the eye, trigeminal ganglion and brain. The mutant virus was as lethal to mice as its wild-type parent following this route of inoculation. Indeed, it exhibited increased virulence. Thus, unlike most drug-resistant virus mutants, this mutant retained in vivo fitness and virulence.
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Human herpesvirus 7 infection increases the expression levels of CD46 and CD59 in target cells
More LessCD46 (membrane cofactor protein; MCP) is a molecule that functions as either a complement-regulatory protein (CRP) or a receptor for some pathogens, including human herpesvirus 6. DNA microarray analysis suggested that the expression of CD46 was upregulated in T cells infected with human herpesvirus 7 (HHV-7). Northen and Western blot analyses supported this result at both the transcriptional and translational levels. Flow-cytometric analysis revealed that upregulation of CD46 occurred at a late stage of infection in both SupT1 cells and primary CD4+ T cells, and also that expression of another CRP, CD59, was increased at a late stage of infection. Whether these CRPs actually function in HHV-7-infected cells was addressed and it was found that HHV-7-infected cells were more resistant to complement-dependent cytotoxicity than were uninfected cells. This study is the first report demonstrating that HHV-7 infection causes elevation of the CRPs CD46 and CD59, which may be a possible mechanism for HHV-7 to evade humoral immunity via complement.
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Reciprocal roles of cellular chemokine receptors and human herpesvirus 7-encoded chemokine receptors, U12 and U51
More LessHuman herpesvirus 7 (HHV-7) is a member of the subfamily Betaherpesvirinae that exhibits a restricted cell tropism, preferentially infecting CD4+ T cells in vitro. HHV-7 encodes two functional chemokine receptors, U12 and U51. The human chemokines that act as ligands for these receptors have been identified as CCL22 (the natural ligand for CCR4) and CCL19 (the natural ligand for CCR7). It was found that murine L1.2 cells co-expressing CCR4 or CCR7 and U12 responded to both CCL22 and CCL19 in calcium-mobilization assays, but migrated in response only to the appropriate ligand for the expressed cellular receptor. Similar results were obtained with L1.2 cells co-expressing CCR4 or CCR7 with U51. These results suggest that the HHV-7 U12 and U51 receptors can function in concert with CCR4 and CCR7 in host-cell signalling pathways.
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Processing and MHC class I presentation of human cytomegalovirus pp65-derived peptides persist despite gpUS2–11-mediated immune evasion
Immune control of human cytomegalovirus (HCMV) infection can be mediated by CD8+ cytolytic T lymphocytes (CTL). Adoptive transfer of antiviral CTL confers protection against HCMV reactivation and disease. The tegument protein pp65 and the immediate-early 1 protein (IE1) are recognized to be major CTL targets, even though during productive infection the viral immunoevasion proteins gpUS2–11 act to suppress major histocompatibility complex (MHC) class I-restricted antigen presentation. Thus it was not clear how infected cells could be labelled with antigenic peptides in the face of immunoevasion. We show here that the immunodominant peptide pp65NLV was presented by MHC class I in cells infected with a gpUS2–11-competent virus. Presentation of pp65NLV was still detectable at 96 h post-infection, although at low levels. Partial suppression of pp65NLV presentation was dependent on the ability of the infecting strain to express gpUS2–11. MHC class I-restricted antigen presentation in HCMV-infected cells (encoding gpUS2–11) exhibited specificity for pp65-derived peptides, as infected fibroblasts did not present the IE1-derived nonapeptide IE1TMY. Remarkably, infected cells could restore pp65NLV peptide presentation after acid removal of MHC class I despite gpUS2–11 expression. This recovery was shown to be dependent on proteasome functionality. In contrast to IE1, pp65 peptides are loaded on MHC class I molecules to be transported to the cell surface at early and late times after infection in the face of gpUS2–11-mediated immunoevasion. pp65 is therefore the first example of an HCMV protein only incompletely subjected to gpUS2–11-mediated immunoevasion.
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Upregulation of CD94/NKG2A receptors and Qa-1b ligand during murine cytomegalovirus infection of salivary glands
More LessFollowing acute infection, murine cytomegalovirus (MCMV) replicates persistently in the salivary glands, despite the vigorous response of activated CD8 T cells that infiltrate this gland. Virus-specific CD8 T lymphocytes isolated from this organ were found to express the inhibitory CD94/NKG2A receptor that, in some virus models, confers an inhibitory response to cytotoxic T lymphocytes (CTLs). In response to MCMV infection, expression of the CD94/NKG2A ligand, Qa-1b, increased dramatically in the submandibular gland (SMG) prior to upregulation of H-2Dd. However, there was no net negative impact on virus-specific T-cell function, as virus titres were similar in CD94− and CD94+ mice. CD94/NKG2A expression, also known to inhibit apoptosis, did not influence the numbers of accumulated T, NK and NK T cells. These data indicate that expression of inhibitory CD94/NKG2A receptors does not account for the failure of MCMV-specific CTLs to clear the SMG of infection.
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Murine gammaherpesvirus 68 ORF20 induces cell-cycle arrest in G2 by inhibiting the Cdc2–cyclin B complex
More LessThe objective of this work was to identify novel viral ‘evasion’ genes without homology in the database through functional assays. Using this approach, the ‘unassigned’, conserved murine gammaherpesvirus ORF20 gene was shown to localize in the nucleus and to induce cell-cycle arrest followed by apoptosis in both mouse and human cells. Such growth-arrested cells did not express phospho-histone H3, demonstrating that the virus protein caused arrest at the G2 stage of the cell cycle. To characterize the mechanism further, Western blots of ORF20-recombinant lentivirus-infected cells were developed with antibodies to cyclin B1, Cdc2 and phospho-Tyr-15-Cdc2. This analysis revealed a relative increase in cyclin B and phospho-Tyr-15-Cdc2, from 24 to 72 h after infection with recombinant lentivirus. The demonstration that Cdc2 is in its inactive phosphorylated form and the clearly increased levels of cyclin B indicated that the virus gene blocks the progression of cells into mitosis by acting at the level of the Cdc2–cyclin B complex. To confirm this result, the Cdc2–cyclin B complex in ORF20-expressing cells was shown to be essentially without kinase activity. As the ORF20 gene is conserved in all herpesvirus, it may be presumed to have evolved to fulfil an important, as yet undefined, biological role in host-cell modification.
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Silencing of latent membrane protein 2B reduces susceptibility to activation of lytic Epstein–Barr virus in Burkitt's lymphoma Akata cells
Epstein–Barr virus (EBV) latent membrane protein 2A (LMP2A) blocks B-cell receptor (BCR) signalling after BCR cross-linking to inhibit activation of lytic EBV, and ectopically expressed LMP2B negatively regulates LMP2A. Here, it is demonstrated that silencing of LMP2B in EBV-harbouring Burkitt's lymphoma Akata cells results in reduced expression of EBV immediate-early lytic BZLF1 gene mRNA and late lytic gp350/220 protein upon BCR cross-linking. Similarly, reduction of lytic EBV activation was observed in Akata cells overexpressing LMP2A. In contrast, silencing of LMP2A expression resulted in higher lytic EBV mRNA and protein expression in BCR cross-linked Akata cells. These observations indicate a role for LMP2B distinct from that of LMP2A in regulation of lytic EBV activation in the host cell and support the hypothesis that LMP2B exhibits a negative-regulatory effect on the ability of LMP2A to maintain EBV latency by preventing the switch to lytic replication.
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Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization
Virus-like particle-based vaccines for high-risk human papillomaviruses (HPVs) appear to have great promise; however, cell culture-derived vaccines will probably be very expensive. The optimization of expression of different codon-optimized versions of the HPV-16 L1 capsid protein gene in plants has been explored by means of transient expression from a novel suite of Agrobacterium tumefaciens binary expression vectors, which allow targeting of recombinant protein to the cytoplasm, endoplasmic reticulum (ER) or chloroplasts. A gene resynthesized to reflect human codon usage expresses better than the native gene, which expresses better than a plant-optimized gene. Moreover, chloroplast localization allows significantly higher levels of accumulation of L1 protein than does cytoplasmic localization, whilst ER retention was least successful. High levels of L1 (>17 % total soluble protein) could be produced via transient expression: the protein assembled into higher-order structures visible by electron microscopy, and a concentrated extract was highly immunogenic in mice after subcutaneous injection and elicited high-titre neutralizing antibodies. Transgenic tobacco plants expressing a human codon-optimized gene linked to a chloroplast-targeting signal expressed L1 at levels up to 11 % of the total soluble protein. These are the highest levels of HPV L1 expression reported for plants: these results, and the excellent immunogenicity of the product, significantly improve the prospects of making a conventional HPV vaccine by this means.
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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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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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