- Volume 83, Issue 6, 2002
Volume 83, Issue 6, 2002
- Review Article
-
-
-
Genetic variability in hepatitis B viruses
More LessIn 1988, it was reported that the full nucleotide sequences of 18 hepatitis B virus (HBV) strains clustered into four genetic groups (A to D) with more than 8% divergence between the groups. This classification of strains in terms of genome sequence has since proven to be an important tool in the understanding of HBV epidemiology and evolution and has been expanded to include three more genotypes. In parallel with the HBV genotypes described in humans, HBV strains isolated from different primates and hepadnaviruses found in woodchucks, ground squirrels, ducks and herons have been studied. Sequence differences between HBV genotypes can lead to structural differences at the level of the pregenome and can also lead to dramatic differences at the translational level when specific and commonly occurring mutations occur. There is increasing evidence that the clinical picture, the response to treatment and the long-term prognosis may differ depending on which genotype has infected the patient. The consideration of traditional serological patterns in a patient must therefore take the genotype of the infecting strain into account. Nucleotide variability between HBV strains has been used in several studies to trace routes of transmission and, since it is becoming increasingly clear that the differences between HBV genotypes are important, the need for reliable and easy methods of differentiating HBV genotypes has arisen. This review summarizes the knowledge of HBV genotypes with regard to their genetic, structural and clinically significant differences and their origin and evolution in the context of the hepadnaviruses in general.
-
- Animal: RNA Viruses
-
-
-
Selection following isolation of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and herpesvirus saimiri-transformed T cells is comparable
More LessIn attempts to improve isolation rates and virus yields for human immunodeficiency virus (HIV), the use of herpesvirus saimiri-immortalized T cells (HVS T cells) has been investigated as an alternative to/improvement over peripheral blood mononuclear cells (PBMCs). Here we characterize isolates rescued, in the two cell types, from two asymptomatic, long-term non-progressing HIV-1-infected individuals. All rescued viruses replicated in PBMCs and HVS T cells only, displaying a non-syncytium inducing (NSI) phenotype, and using CCR5 as co-receptor. Furthemore, PBMC/HVS T cell virus pairs displayed similar neutralization profiles. Full-length, expression-competent env genes were rescued from all virus isolates and directly from the patient samples using proviral DNA and viral RNA as templates. Compared with the sequences retrieved directly from the patient samples, both cell types showed similar selection characteristics. Whilst the selections were distinct for individual patient samples, they shared a common characteristic in selecting for viruses with increased negative charge across the V2 domain of the viral glycoproteins. The latter was observed at the env gene sequencing level for three other patients whose HIV strains were isolated in PBMCs only. This further supports a common selection for viral sequences that display a macrophage-tropic/NSI phenotype and shows that HVS T cells are a viable alternative to PBMCs for HIV-1 isolation.
-
-
-
-
Transient immune suppression of inapparent carriers infected with a principal neutralizing domain-deficient equine infectious anaemia virus induces neutralizing antibodies and lowers steady-state virus replication
The genetic variation of equine infectious anaemia virus (EIAV) clearly affects the antigenic properties of the viral envelope; however, effects on immunogenicity remain undefined, although widely assumed. Here, the immunogenicity is reported of a novel, neutralization-resistant, pony-isolate envelope EIAVPV564ΔPND that contains a 14-residue deletion in the designated principal neutralizing domain (PND) of the gp90 protein. Two ponies inoculated with a chimeric virus, EIAVΔPND, containing the EIAVPV564ΔPND envelope in a reference provirus strain, remained asymptomatic through 14 months post-inoculation, producing high steady-state levels of envelope-specific antibodies but no detectable serum-neutralizing antibodies. Consequent dexamethasone-induced immune suppression produced characteristic EIA that resolved concomitantly with the development of high-titre, strain-specific, neutralizing antibodies and a 100-fold reduction in steady-state virus loads. These results demonstrate: natural variations in the EIAV envelope have profound effects on both antigenic and immunogenic properties; the PND is not required for neutralizing antibody responses; and transient immune suppression can enhance established host immunity to achieve more effective control of steady-state lentivirus replication.
-
-
-
Walleye dermal sarcoma virus reverse transcriptase is temperature sensitive
More LessWalleye dermal sarcoma virus (WDSV) is a piscine retrovirus that replicates naturally in fish at temperatures near 4 °C. The reverse transcriptase (RT) protein from virus particles isolated from walleye tumours was purified and biochemically characterized. Like the RT of the distantly related murine leukaemia virus, WDSV RT sediments as a monomer in the absence of template. It exhibits a K m of 22 μM for TTP in an assay with poly(rA) as a template and oligo(dT) as a primer. The enzyme is rapidly inactivated at temperatures greater than 15 °C. The ratio of RT activity at 15 °C to that at 4 °C is similar for WDSV and recombinant human immunodeficiency virus type 1, suggesting that, at least with this template, the fish enzyme is not specially adapted to function more efficiently in the cold.
-
-
-
Infection with enterovirus 71 or expression of its 2A protease induces apoptotic cell death
More LessEnterovirus 71 (EV71) is the causative agent of human diseases with distinct severity, from mild hand-foot-and-mouth disease to severe neurological syndromes, such as encephalitis and meningitis. Infection of several different cell lines with EV71 causes extensive cytopathic effect, leading to destruction of the entire monolayer and the death of infected cells. In this study, cell death processes during EV71 infection and the underlying mechanisms of them were investigated. The hallmarks of apoptosis, nuclear condensation and fragmentation, were observed 24 h after infection. Apoptosis in infected cells was also confirmed by detectable cleavage of cellular DNA and degradation of poly(ADP-ribose) polymerase. Transient expression of EV71 2A protease (2Apro) alone resulted in the induction of apoptotic change. Infection of EV71 or expression of EV71 2Apro leads to cleavage of the eukaryotic initiation factor 4GI, a key factor for host protein synthesis. This study added one more example to the growing list of human viruses that induce apoptosis by a virus-encoded protein.
-
-
-
Primary replication of a recombinant Sendai virus vector in macaques
An efficient antigen expression system using a recombinant Sendai virus (SeV) has been established recently and its potential to induce resistance against immunodeficiency virus infections in macaques has been shown. SeV replication has been well characterized in mice, the natural host, but not in primates, including humans. Here, primary SeV replication was investigated in macaques. After intranasal immunization with a recombinant SeV expressing simian immunodeficiency virus Gag protein, SeV-Gag, robust gag expression was observed in the nasal mucosa and much lower but significant levels of gag expression were observed in the local retropharyngeal and submandibular lymph nodes (LN). Expression peaked within a week and lasted at least up to 13 days after immunization. SeV-Gag was isolated from nasal swabs consistently at day 4 but not at all at day 13. Gag expression was undetectable in the lung as well as in remote lymphoid tissues, such as the thymus, spleen and inguinal LN, indicating that the spread of the virus was more restricted in macaques than in mice. SeV-specific T cells were detectable in SeV-immunized macaques at day 7. Finally, no naive macaques showed significant levels of anti-SeV antibodies in the plasma, even after living in a cage together with an acutely SeV-infected macaque for 5 weeks, indicating that SeV transmission from SeV-infected macaques to naive ones was inefficient. None of the SeV-immunized macaques displayed appreciable clinical manifestations. These results support the idea that this system may be used safely in primates, including humans.
-
-
-
Mapping of linear epitopes on the capsid proteins of swine vesicular disease virus using monoclonal antibodies
More LessThe antigenic linear map of swine vesicular disease virus (SVDV) has been studied using a repertoire of monoclonal antibodies (mAbs) raised against a recombinant SVDV polyprotein, P1. Peptide-scanning analyses, cross-reactivity studies with homologous and heterologous viruses and predicted location on a computer-generated three-dimensional model of the capsid proteins have allowed the identification of five main linear sites. Two sites, the N terminus of VP3 and amino acids 51–60 on VP1, correspond to internal areas, conserved not only between SVDV isolates but also in the related enterovirus coxsackievirus B5. In contrast, three other regions, amino acids 142–161 of VP2, 61–70 of VP3 and the C terminus of VP1, are exposed on the external face of the capsid and subjected to antigenic variation, even among different SVDV isolates. Further minor sites that were antigenically conserved were identified on VP4. In contrast with conformational sites described previously, none of the linear epitopes identified in this work is involved in neutralization of virus infectivity and post-infection swine sera did not inhibit the binding of mAbs with the relevant epitopes. Both of these observations suggest that linear epitopes are poorly immunogenic in pigs. The characterization of linear sites has contributed to a better understanding of the antigenic structure of SVDV and mAbs used to this purpose may provide a useful tool for the improvement of diagnostic methods, such as antigen detection systems, and analyses of the antigenic profile of SVDV isolates.
-
-
-
Evolutionary relationships among Astroviridae
More LessTo study the evolutionary relationships among astroviruses, all available sequences for members of the family Astroviridae were collected. Phylogenetic analysis distinguished two deep-rooted groups: one comprising mammalian astroviruses, with ovine astrovirus being an outlier, and the other comprising avian astroviruses. All virus species as well as serotypes of human astroviruses represented individual lineages within the tree. All human viruses clustered together and separately from non-human viruses, which argue for their common evolutionary origin and against ongoing animal-to-human transmissions. The branching order of mammalian astroviruses was exactly the opposite of that of their host species, suggesting at least two cross-species transmissions involving pigs, cats and humans, possibly through intermediate hosts. Analysis of synonymous (Ds) versus non-synonymous (Da) distances revealed that negative selection is dominating in the evolution of astroviruses, with the Ds:Da ratios being up to 46 for the comparisons of the most closely related viruses. Phylogenetic analyses of all open reading frames (ORFs) based on Ds resulted in the loss of tree structures, with virus species – and in ORF2, even serotypes of human astroviruses – branching out from virtually a single node, suggesting their ancient separation. The strong selection against non-synonymous substitutions, the low number of which is, therefore, not proof of a recent separation between lineages, together with the position of the oldest available human astrovirus strain (1971) far from the common node of its serotype 4, suggest that intraserotype diversification originates from an earlier date.
-
-
-
Porcine B-cells recognize epitopes that are conserved between the structural proteins of American- and European-type porcine reproductive and respiratory syndrome virus
More LessBy selecting phage display libraries with immune sera from experimentally infected pigs, porcine B-cell epitopes in the open reading frame (ORF) 2, 3, 5 and 6 proteins of European-type porcine reproductive and respiratory syndrome virus (PRRSV) were identified. The sequences of all the epitopes were well conserved in European-type PRRSV and even between European- and American-type PRRSV. Accordingly, sera from pigs infected with American-type PRRSV cross-reacted with the European-type epitopes. Thus, this study showed, for the first time, the presence of highly conserved epitopes in the matrix protein and envelope glycoproteins of PRRSV. ORF5 and 6 epitopes localized to protein parts that are predicted to be hidden in PRRSV virions. In contrast, ORF2 and 3 epitopes localized to putative protein ectodomains. Due to the interesting localization, the sequence surrounding the ORF2 and 3 epitopes was subjected to closer scrutiny. A heptad motif, VSRRIYQ, which is present in a single copy in ORF2 and 3 proteins, was identified; this arrangement is completely conserved in all European-type PRRSV sequences available. The VSRRIYQ repeat motif colocalized closely with one of the ORF2 epitopes and secondary structure modelling showed that this segment of the ORF2 protein could form an amphipathic helix. Intriguingly, a mutation associated with virulence/attenuation of an American vaccine strain of PRRSV also localized to this ORF2 protein segment and affected the hydrophobic face of the predicted amphipathic helix. Further work is needed to determine whether these findings delineate a functional domain in the PRRSV ORF2 protein.
-
-
-
Increased positive selection pressure in persistent (SSPE) versus acute measles virus infections
More LessWe compared the extent of positive selection acting on acute and persistent strains of measles virus (MV). Far stronger positive selection was found in the fusion (F) and haemagglutinin (H) genes from subacute sclerosing panencephalitis (SSPE) compared to acute MV cases. Most of the positively selected sites identified in these surface glycoprotein genes from SSPE cases correspond to structural, functional or antigenic areas, and could not be explained by the effects of cell passaging. The correlations between selected sites and functional studies of MV are discussed in detail with reference to the maintenance of persistent infection. No positive selection was found in the matrix (M) gene from acute cases of MV and the effects of including hypermutated SSPE M gene sequences in phylogenetic inference were also explored. Finally, using H gene data, we estimated the rate of molecular evolution for SSPE strains as 3·4×10−4 substitutions/site/year, which is similar to previous estimates obtained for acute strains.
-
-
-
Analysis of receptor (CD46, CD150) usage by measles virus
In order to investigate which measles virus (MV)-strains use CD46 and/or CD150 (signalling lymphocytic activation molecule, SLAM) as receptors, CHO cells expressing either recombinant CD46 or SLAM were infected with a panel of 28 MV-strains including vaccine strains, wild-type strains with various passage histories and recombinant viruses. We found that SLAM served as a common receptor conferring virus uptake and syncytium formation for all MV-strains tested. Predominantly vaccine and laboratory adapted strains, but also a minor fraction of the wild-type strains tested, could utilize both CD46 and SLAM. Using recombinant viruses, we demonstrate that the single amino acid exchange in the haemagglutinin (H) protein at position 481 Asn/Tyr (H481NY) determines whether the virus can utilize CD46. This amino acid alteration has no affect on the usage of SLAM as receptor, and as such demonstrates that the binding sites for SLAM and CD46 are distinct.
-
-
-
Genetic characterization of wild-type measles viruses circulating in suburban Khartoum, 1997–2000
Measles remains endemic in many East African countries, where it is often associated with high morbidity and mortality. We collected clinical specimens from Sudanese measles patients between July 1997 and July 2000. Sequencing of the 3′ 456 nucleotides of the nucleoprotein gene from 33 measles virus (MV) isolates and 8 RNA samples extracted from clinical specimens demonstrated the presence of a single endemic MV strain with little sequence variation over time (overall nucleotide divergence of 0 to 1·3%). This was confirmed by sequencing of the complete H gene of two isolates from 1997 and two from 2000, in which the overall divergence ranged between 0 and 0·5%. Comparison with MV reference strains demonstrated that the viruses belonged to clade B, genotype B3, and were most closely related to a set of viruses recently isolated in Nigeria. Our study demonstrates a remarkable genetic stability of an endemically circulating MV strain.
-
-
-
A model for the generation of multiple A to G transitions in the human respiratory syncytial virus genome: predicted RNA secondary structures as substrates for adenosine deaminases that act on RNA
More LessHuman respiratory syncytial virus (HRSV) escape mutants selected with antibodies specific for the attachment (G) protein contain diverse genetic alterations, including point mutations, premature stop codons, frame shift changes and A to G hypermutations. The latter changes have only been found in mutants selected with antibodies directed against the conserved central region of the G protein. This gene segment fulfils substrate requirements for adenosine deaminases that act on RNA (ADARs): i.e. it is an A+U rich region of 137 residues and 98 or 106 of them – for A/Mon/3/88 or Long HRSV strains, respectively – are predicted to form intramolecular base pairs leading to a stable RNA secondary structure. In addition, when sequences of the G gene from natural isolates are compared in terms of pairwise substitutions, A to G+G to A changes are preferentially observed in regions where stable intramolecular dsRNA secondary structures are predicted to occur. In this study, a model is proposed in which, in addition to nucleotide misincorporations, reiterative A to G changes in HRSV are generated by ADAR activity operating in short segments (100–200 ribonucleotide residues) of the HRSV genome with high tendency for intramolecular base pairing.
-
-
-
Rinderpest virus H protein: role in determining host range in rabbits
A major molecular determinant of virus host-range is thought to be the viral protein required for cell attachment. We used a recombinant strain of Rinderpest virus (RPV) to examine the role of this protein in determining the ability of RPV to replicate in rabbits. The recombinant was based on the RBOK vaccine strain, which is avirulent in rabbits, carrying the haemagglutinin (H) protein gene from the lapinized RPV (RPV-L) strain, which is pathogenic in rabbits. The recombinant virus (rRPV-lapH) was rescued from a cDNA of the RBOK strain in which the H gene was replaced with that from the RPV-L strain. The recombinant grew at a rate equivalent to the RPV-RBOK parental virus in B95a cells but at a lower rate than RPV-L. The H gene swap did not affect the ability of the RBOK virus to act as a vaccine to protect cattle against virulent RPV challenge. Rabbits inoculated with RPV-L became feverish, showed a decrease in body weight gain and leukopenia. High virus titres and histopathological lesions in the lymphoid tissues were also observed. Clinical signs of infection were never observed in rabbits inoculated with either RPV-RBOK or with rRPV-lapH; however, unlike RPV-RBOK, both RPV-L and rRPV-lapH induced a marked antibody response in rabbits. Therefore, the H protein plays an important role in allowing infection to occur in rabbits but other viral proteins are clearly required for full RPV pathogenicity to be manifest in this species.
-
-
-
Rabies virus glycoprotein can fold in two alternative, antigenically distinct conformations depending on membrane-anchor type
More LessRabies virus glycoprotein (G) is a trimeric type I transmembrane glycoprotein that mediates both receptor recognition and low pH-induced membrane fusion. We have previously demonstrated that a soluble form of the ectodomain of G (G1–439), although secreted, is folded in an alternative conformation, which is monomeric and antigenically distinct from the native state of the complete, membrane-anchored glycoprotein. This has raised questions concerning the role of the transmembrane domain (TMD) in the correct native folding of the ectodomain. Here, we show that an ectodomain anchored in the membrane by a glycophosphatidylinositol is also folded in an alternative conformation, whereas replacement of the TMD of G by other peptide TMDs results in correct antigenicity of G. However, mutants with an insertion of a hydrophilic linker between the ectodomain and the TMD also fold in an alternative conformation. The influence of the membrane-anchor type on G ectodomain trimerization and folding is discussed.
-
-
-
Nucleotide sequences of segments 1, 3 and 4 of the genome of Bombyx mori cypovirus 1 encoding putative capsid proteins VP1, VP3 and VP4, respectively
More LessThe complete nucleotide sequences of genomic segments S1, S3 and S4 from Bombyx mori cypovirus 1 (BmCPV-1) have been determined. The segments consisted of 4190, 3846 and 3262 nucleotides encoding putative proteins of 1333, 1239 and 1058 amino acids with molecular masses of approximately 148, 140 and 120 kDa (p148, p140 and p120, respectively). All segments possess a single open reading frame. Homology searches showed that all three proteins have homologies to proteins of Rice ragged stunt virus, a member of the genus Oryzavirus within the family Reoviridae. Partial homologies of p140 to structural proteins in other viruses were also found. The predicted molecular masses and the homologies with structural proteins in other viruses lead us to suggest that S1, S3 and S4 encode the capsid proteins VP1, VP3, and VP4, respectively, of BmCPV-1.
-
-
-
Molecular cloning and characterization of Antheraea mylitta cytoplasmic polyhedrosis virus genome segment 9
More LessGenome segment 9 of the 11-segment RNA genomes of three cytoplasmic polyhedrosis virus (CPV) isolates from Antheraea mylitta (AmCPV), Antheraea assamensis (AaCPV) and Antheraea proylei (ApCPV) were converted to cDNA, cloned and sequenced. In each case, this genome segment consists of 1473 nucleotides with one long ORF of 1035 bp and encodes a protein of 345 amino acids, termed NSP38, with a molecular mass of 38 kDa. Secondary structure prediction showed the presence of nine α-helices in the central and terminal domains with localized similarity to RNA-binding motifs of bluetongue virus and infectious bursal disease virus RNA polymerases. Nucleotide sequences were 99·6% identical between these three strains of CPVs, but no similarity was found to any other nucleotide or protein sequence in public databases. The ORF from AmCPV cDNA was expressed as a His-tagged fusion protein in E. coli and polyclonal antibody was raised against the purified protein. Immunoblot as well as immunofluorescence analysis with anti-NSP38 antibody showed that the protein was not present in polyhedra or uninfected cells but was present in AmCPV-infected host midgut cells. NSP38 was expressed in insect cells as soluble protein via a baculovirus expression vector and shown to possess the ability to bind poly(rI)–(rC) agarose, which was competitively removed by AmCPV viral RNA. These results indicate that NSP38 is expressed in virus-infected cells as a non-structural protein. By binding to viral RNA, it may play a role in the regulation of genomic RNA function and packaging.
-
- Animal: DNA Viruses
-
-
-
Hepatitis B virus surface antigen suppresses the activation of monocytes through interaction with a serum protein and a monocyte-specific receptor
More LessDuring hepatitis B virus (HBV) infection, high numbers of non-infectious HBV surface antigen (HBsAg) particles are present in circulation. It is shown here that recombinant HBsAg (rHBsAg) particles, which contain the S protein only, bind almost exclusively to monocytes. Attachment of rHBsAg to the THP-1 pre-monocytic cell line occurs upon 1,25-dihydroxyvitamin D3-induced differentiation. Binding to monocytes is enhanced by a heat-labile serum protein and is inhibited by Ca2+/Mg2+, low pH and an HBsAg-specific monoclonal antibody. Furthermore, it is shown that rHBsAg suppresses lipopolysaccharide- and IL-2-induced production of cytokines. These results suggest the existence of a monocyte-specific receptor, the engagement of which by HBsAg suppresses the activity of these cells.
-
-
-
-
Genomic characterization of TT viruses (TTVs) in pigs, cats and dogs and their relatedness with species-specific TTVs in primates and tupaias
Using PCR with primers derived from a non-coding region of the human TT virus (TTV) genome, the TTV sequence in serum samples obtained from pigs (Sus domesticus), dogs (Canis familiaris) and cats (Felis catus) was identified and the entire genomic sequence was determined for each representative isolate. Three TTV isolates (Sd-TTV31 from a pig, Cf-TTV10 from a dog and Fc-TTV4 from a cat) comprising 2878, 2797 and 2064 nucleotides, respectively, each had three open reading frames (ORFs) encoding 436–635 (ORF1), 73–105 (ORF2) and 224–243 (ORF3) aa but lacked ORF4, similar to tupaia TTV. ORF3 was presumed to arise from a splicing of TTV mRNA, similar to human prototype TTV. Although the nucleotide sequence of Sd-TTV31, Cf-TTV10 and Fc-TTV4 differed by more than 50% from each other and from previously reported TTVs of 3·4–3·9 kb and TTV-like mini viruses (TLMVs) of 2·8–3·0 kb isolated from humans and non-human primates as well as tupaia TTVs of 2·2 kb, they resembled known TTVs and TLMVs with regard to genomic organization and presumed transcriptional profile rather than animal circoviruses of 1·7–2·3 kb. Phylogenetic analysis revealed that Sd-TTV31, Cf-TTV10 and Fc-TTV4 were closer to TTVs from lower-order primates and tupaias than to TTVs from higher-order primates and TLMVs. These results indicate that domestic pigs, cats and dogs are naturally infected with species-specific TTVs with small genomic size and suggest a wide distribution of TTVs with extremely divergent genomic sequence and length in animals.
-
-
-
Human adenovirus serotypes 4p and 11p are efficiently expressed in cell lines of neural tumour origin
More LessMost currently used adenovirus vectors are based upon adenovirus serotypes 2 and 5 (Ad2 and Ad5), which have limited efficiencies for gene transfer to human neural cells. Both serotypes bind to the known adenovirus receptor, CAR (coxsackievirus and adenovirus receptor), and have restricted cell tropism. The purpose of this study was to find vector candidates that are superior to Ad5 in infecting human neural tumours. Using flow cytometry, the vector candidates Ad4p, Ad11p and Ad17p were compared to the commonly used adenovirus vector Ad5v for their binding capacity to neural cell lines derived from glioblastoma, medulloblastoma and neuroblastoma cell lines. The production of viral structural proteins and the CAR-binding properties of the different serotypes were also assessed in these cells. Computer-based models of the fibre knobs of Ad4p and Ad17 were created based upon the crystallized fibre knob structure of adenoviruses and analysed for putative receptor-interacting regions that differed from the fibre knob of Ad5. The non CAR-binding vector candidate Ad11p showed clearly the best binding capacity to all of the neural cell lines, binding more than 90% of cells of all of the neural cell lines tested, in contrast to 20% or less for the commonly used vector Ad5v. Ad4p and Ad11p were also internalized and produced viral proteins more successfully than Ad5. Ad4p showed a low binding ability but a very efficient capacity for infection in cell culture. Ad17p virions neither bound or efficiently infected any of the neural cell lines studied.
-
-
-
Efficient mobilization of E1-deleted adenovirus type 5 vectors by wild-type adenoviruses of other serotypes
Mobilization of replication-deficient adenovirus vectors can lead to spread and shedding of the vector. Here we show that in cultured HepG2 cells wild-type (wt) adenoviruses of subgroup A (Ad12), B (Ad7, 11 and 16), C (Ad1, 2 and 5) and E (Ad4) can efficiently mobilize Ad5CMVluc, a ΔE1ΔE3-Ad5 vector carrying the firefly luciferase gene as reporter. In addition, we show that Ad5CMVluc can be propagated on Ad12E1-transformed human embryonic retinoblasts. This provides evidence that expression of the E1 region of Ad12 is sufficient for mobilizing ΔE1-Ad5-derived vectors. Thus, in therapeutic applications of replication-defective Ad vectors any active Ad infection is of potential concern, independent of the serotype involved. To prevent vector mobilization by wt Ads, new vectors should be developed in which essential functions such as the initiation of DNA replication and genome packaging are restricted.
-
-
-
The products of human cytomegalovirus genes UL23, UL24, UL43 and US22 are tegument components
We have investigated the human cytomegalovirus (HCMV) US22 gene family members UL23, UL24, UL43 and US22. Specific antibodies were generated to identify pUL23 (33 kDa), pUL24 (40 kDa) and pUL43 (48 kDa), while pUS22 was identified by monoclonal antibody HWLF1. A C-terminally truncated UL43 product (pUL43t; 21 kDa) produced by a deletion mutant was also investigated. The UL24 and UL43 genes were expressed with early-late (γ1) and true-late (γ2) kinetics, respectively. Immunoblot and immuno-EM studies demonstrated that pUL23, pUL24, pUL43 and pUS22 were virion tegument components. Immunofluorescence and immuno-EM studies showed that pUL23, pUL24, pUL43 and pUL43t were located in cytoplasmic protein aggregates, manifesting two forms: complex juxtanuclear structures and smaller, membrane-bound aggregates resembling dense bodies. The complex-type aggregate is a putative site of particle maturation. Because pUL43t was present in protein aggregates, but under-represented in virus particles compared to pUL43, it was concluded that N-terminal sequences target pUL43 to protein aggregates and that C-terminal sequences are important for incorporation into particles. Since three other US22 family products (pUL36, pTRS1 and pIRS1) are documented tegument components, at least seven of the twelve US22 family genes encode tegument proteins, suggesting that the products of the remaining five genes might be similarly located. These findings demonstrate a common biological feature among most, if not all, US22 family proteins and implicate the family in events occurring immediately after virus penetration.
-
-
-
Partial characterization of a novel gammaherpesvirus isolated from a European badger (Meles meles)
More LessA herpesvirus causing a cytopathic effect was isolated from pulmonary fibroblast cultures established from a European badger (Meles meles). A study was undertaken to classify and to assess some in-vitro growth characteristics of this virus. From a panel of 27 mammalian cell lines, in-vitro replication of the badger herpesvirus (BadHV) was only demonstrated with a mink lung cell line, suggesting a high degree of host specificity. Using PCR with degenerate primers, three independent fragments of the BadHV genome were sequenced. The largest of these fragments comprised a 6·2 kb segment including the DNA polymerase and glycoprotein B genes. Phylogenetic analysis of these sequences demonstrated that the BadHV is novel and clearly grouped with members of the Gammaherpesvirinae. In view of the oncogenic and immunosuppressive potential of many related herpesviruses, it is possible that BadHV can impact on existing acute or chronic disease in badgers.
-
-
-
Identification of the principal serological immunodeterminants of African swine fever virus by screening a virus cDNA library with antibody
More LessProtective immunity to African swine fever virus (ASFV) may involve a combination of both serological and cellular mechanisms. This work is focused on the identification of the possible relevant serological immunodeterminants of immunity. Thus, 14 serological immunodeterminants of ASFV have been characterized by exhaustive screening of a representative lambda phage cDNA expression library of the tissue culture-adapted Ba71V strain of ASFV. The library was constructed using RNA extracted from Vero cells infected for 3, 6, 9 and 12 h. A total of 150 clones was selected arbitrarily by antibody screening of the library with a polyclonal antiserum from a domestic pig surviving infection with the virulent Malta isolate of ASFV. Sequencing of these clones permitted identification of 14 independent viral proteins that stimulated an antibody response. These included six proteins encoded by previously unassigned open reading frames (ORFs) (B602L, C44L, CP312R, E184L, K145R and K205R) as well as some of the more well-studied structural (A104R, p10, p32, p54 and p73) and non-structural proteins (RNA reductase, DNA ligase and thymidine kinase). Immunogenicity of these proteins was confirmed by demonstrating the corresponding antibodies in sera from pigs infected either with the Malta isolate or with the OURT88/3–OURT88/1 isolate combination. Furthermore, the majority of these ORFs were also recognized by immune antiserum from the natural host, the bush pig, following secondary challenge with the virulent Malawi (SINT90/1) isolate of ASFV. Thus, it is possible that some of these determinants may be important in protection against virus infection.
-
- Plant
-
-
-
Phloem loading and unloading of Cowpea mosaic virus in Vigna unguiculata
More LessWithin their host plants, viruses spread from the initially infected cell through plasmodesmata to neighbouring cells (cell-to-cell movement), until reaching the phloem for rapid invasion of the younger plant parts (long-distance or vascular movement). Cowpea mosaic virus (CPMV) moves from cell-to-cell as mature virions via tubules constructed of the viral movement protein (MP). The mechanism of vascular movement, however, is not well understood. The characteristics of vascular movement of CPMV in Vigna unguiculata (cowpea) were examined using GFP-expressing recombinant viruses. It was established that CPMV was loaded into both major and minor veins of the inoculated primary leaf, but was unloaded exclusively from major veins, preferably class III, in cowpea trifoliate leaves. Phloem loading and unloading of CPMV was scrutinized at the cellular level in sections of loading and unloading veins. At both loading and unloading sites it was shown that the virus established infection in all vascular cell types with the exception of companion cells (CC) and sieve elements (SE). Furthermore tubular structures, indicative of virion movement, were never found in plasmodesmata connecting phloem parenchyma cells and CC or CC and SE. In cowpea, SE are symplasmically connected only to the CC and these results therefore suggest that CPMV employs a mechanism for phloem loading and unloading that is different from the typical tubule-guided cell-to-cell movement in other cell types.
-
-
-
-
Size-dependent cell-to-cell movement of defective interfering RNAs of Cymbidium ringspot virus
More LessCo-inoculation of Nicotiana benthamiana plants with in vitro transcripts of both genomic and short defective interfering (DI) RNAs of Cymbidium ringspot virus results in an accumulation of de novo generated DI RNA dimers. Time-course analysis of DI RNA accumulation in the inoculated leaves showed early accumulation of DI RNA dimers followed by increased levels of DI RNA monomers. In contrast, DI RNA dimers were barely detectable in systems where cell-to-cell movement does not take place (protoplasts) or is less important (monomeric DI RNA-expressing transgenic plants). Our results also demonstrated that the size of DI RNAs is important in the colonization of inoculated leaves, suggesting that DI RNA dimers are quickly selected for cell-to-cell movement if short DI RNA monomers are used for infection.
-
-
-
Molecular evolution of Turnip mosaic virus: evidence of host adaptation, genetic recombination and geographical spread
Turnip mosaic virus (TuMV), a species of the genus Potyvirus, occurs worldwide. Seventy-six isolates of TuMV were collected from around the world, mostly from Brassica and Raphanus crops, but also from several non-brassica species. Host tests grouped the isolates into one or other of two pathotypes; Brassica (B) and Brassica–Raphanus (BR). The nucleotide sequences of the first protein (P1) and coat protein (CP) genes of the isolates were determined. One-tenth of the isolates were found to have anomalous and variable phylogenetic relationships as a result of recombination. The 5′-terminal 300 nt of the P1 gene of many isolates was also variable and phylogenetically anomalous, whereas the 380 nt 3′ terminus of the CP gene was mostly conserved. Trees calculated from the remaining informative parts of the two genes of the non-recombinant sequences by neighbour-joining, maximum-likelihood and maximum-parsimony methods were closely similar, and so these parts of the sequences were concatenated and trees calculated from the resulting 1150 nt. The isolates fell into four consistent groups; only the relationships of these groups with one another and with the outgroup differed. The ‘basal-B’ cluster of eight B-pathotype isolates was most variable, was not monophyletic, and came from both brassicas and non-brassicas from southwest and central Eurasia. Closest to it, and forming a monophyletic subgroup of it in most trees, and similarly variable, was the ‘basal-BR’ group of eight BR pathotype Eurasian isolates. The third and least variable group, the ‘Asian-BR’ group, was of 22 BR-pathotype isolates, all from brassicas, mostly Raphanus, and all from east Asia mostly Japan. The fourth group of 36 isolates, the ‘world-B’ group, was from all continents, most were isolated from brassicas and most were of the B-pathotype. The simplest of several possible interpretations of the trees is that TuMV originated, like its brassica hosts, in Europe and spread to the other parts of the world, and that the BR pathotype has recently evolved in east Asia.
-
- Phage
-
-
-
Nucleotide sequence of a ssRNA phage from Acinetobacter: kinship to coliphages
More LessThe complete nucleotide sequence of ssRNA phage AP205 propagating in Acinetobacter species is reported. The RNA has three large ORFs, which code for the following homologues of the RNA coliphage proteins: the maturation, coat and replicase proteins. Their gene order is the same as that in coliphages. RNA coliphages or Leviviridae fall into two genera: the alloleviviruses, like Qβ, which have a coat read-through protein, and the leviviruses, like MS2, which do not have this coat protein extension. AP205 has no read-through protein and may therefore be classified as a levivirus. A major digression from the known leviviruses is the apparent absence of a lysis gene in AP205 at the usual position, overlapping the coat and replicase proteins. Instead, two small ORFs are present at the 5′ terminus, preceding the maturation gene. One of these might encode a lysis protein. The other is of unknown function. Other new features concern the 3′-terminal sequence. In all ssRNA coliphages, there are always three cytosine residues at the 3′ end, but in AP205, there is only a single terminal cytosine. Distantly related viruses, like AP205 and the coliphages, do not have significant sequence identity; yet, important secondary structural features of the RNA are conserved. This is shown here for the 3′ UTR and the replicase-operator hairpin. Interestingly, although AP205 has the genetic map of a levivirus, its 3′ UTR has the length and RNA secondary structure of an allolevivirus. Sharing features with both MS2 and Qβ suggests that, in an evolutionary sense, AP205 should be placed between Qβ and MS2. A phylogenetic tree for the ssRNA phages is presented.
-
-
Volumes and issues
-
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)