- Volume 83, Issue 2, 2002
Volume 83, Issue 2, 2002
- Animal: RNA Viruses
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Genetic clustering of all 102 human rhinovirus prototype strains: serotype 87 is close to human enterovirus 70
More LessHuman rhinoviruses (HRV), common agents of respiratory infections, comprise 102 designated serotypes. The genetic relationships of HRV prototype strains and the possibility of using genetic identification of a given HRV field strain were studied. Genomic sequences in the VP4/VP2 region were obtained from all 102 prototype strains. Phylogenetic analysis included 61 recently isolated Finnish field strains. Seventy-six out of the 102 prototype strains clustered in the HRV genetic group A and 25 in group B. Serotype 87 clustered separately and together with human enterovirus 70. The ‘percentage’ interserotypic differences were generally similar to those between different enterovirus serotypes, but for six pairs of HRV serotypes they were less than 10%. The maximum variation in genetic group A was 41% at the nucleotide level and 28% at the amino acid level, and in genetic group B 34% and 20%, respectively. Judging from the observed interserotypic differences, the 61 Finnish field isolates might represent as many as 19 different serotypes. One cluster of the field strains did not directly associate with any of the prototype strains and might represent a new serotype. However, larger numbers of field isolates of known serotype need to be characterized, possibly also in the VP1 region, to evaluate the feasibility of genetic typing of HRV strains.
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Trans-complementation of a genetic defect in the coxsackie B3 virus 2B protein
The enterovirus 2B protein contains a putative amphipathic α-helix that includes three positively charged and one negatively charged residue. Previously, we observed that replacement of the glutamic acid-40 residue with a lysine residue (mutation 2B-E[40]K) in the amphipathic α-helix of the coxsackie B3 virus 2B protein resulted in a quasi-infectious phenotype. On one occasion, however, transfection of 2B-E[40]K RNA transcripts gave rise to a virus stock in which the mutation was retained. This study was aimed at elucidating the molecular mechanism underlying this observation. Sequence analysis of the viral RNA provided no evidence for a second-site suppression mutation that rescued the defect of the 2B-E[40]K mutation in cis. Therefore, the possibility was considered that the defect caused by the 2B-E[40]K mutation was complemented in trans by viable revertants that had emerged in the virus population. The transfection-derived virus stock indeed contained a small fraction of (pseudo)revertant viruses, carrying the original glutamic acid-40, threonine-40 or asparagine-40, rather than the introduced lysine-40. Consistent with the idea that the 2B-E[40]K virus is unable to grow without the aid of trans-acting wild-type(-like) proteins, only the (pseudo)revertant viruses were able to produce individual plaques. Further support for the idea of trans-rescue was obtained using a genetic complementation assay, which revealed the occurrence of a low level of trans-complementation of the 2B-E[40]K mutation by wild-type virus. This is the first report that provides evidence that a genetic defect in the enterovirus 2B protein can be complemented in trans.
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Enhancement of coxsackievirus B3 infection by antibody to a different coxsackievirus strain
More LessGroup B coxsackieviruses (CVBs) are a major cause of viral myocarditis and pancreatitis in humans and produce a similar pattern of disease in inbred strains of mice. As there are six strains of CVBs, individuals can be infected with multiple serotypes. This raises the possibility of antibody enhancement of infectivity (AEI) by cross-reactive but non-neutralizing antibody to a different strain from a prior infection. To determine whether AEI plays a role in coxsackievirus pathogenesis, an in vitro system using the murine macrophage cell line J774.1 was tested for enhanced infection when incubated with CVB3 plus anti-CVB2 antibody. Yields of virus were found to increase by 10–50-fold and the percentage of infected cells increased proportionately. The effect was Fc-mediated as F(ab′)2 fragments of the antibody could not mediate the effect. To determine whether AEI could also be demonstrated in vivo CVB3 was injected into 5-week-old mice together with mouse polyclonal anti-CVB2. Controls included mice injected with PBS or CVB3 alone. Results showed that the titres of virus in tissues of animals injected with virus plus antibody were 1–2 logs higher than when virus was injected alone. This was accompanied by greater histopathological damage, particularly in the heart. These results have implications for human disease as infection with multiple strains likely occurs during the lifetime of an individual.
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Hepatitis A virus polyprotein processing by Escherichia coli proteases
Hepatitis A virus (HAV) encodes a single polyprotein, which is post-translationally processed. This processing represents an essential step in capsid formation. The virus possesses only one protease, 3C, responsible for all cleavages, except for that at the VP1/2A junction region, which is processed by cellular proteases. In this study, data demonstrates that HAV polyprotein processing by Escherichia coli protease(s) leads to the formation of particulate structures. P3 polyprotein processing in E. coli is not dependent on an active 3C protease: the same processing pattern is observed with wild-type 3C or with several 3C mutants. However, this processing pattern is temperature-dependant, since it differs at 37 or 42 °C. The bacterial protease(s) cleave scissile bonds other than those of HAV; this contributes to the low efficiency of particle formation.
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Hepatitis C virus non-structural protein 3-specific cellular immune responses following single or combined immunization with DNA or recombinant Semliki Forest virus particles
The capacity of recombinant Semliki Forest virus particles (rSFV) expressing the hepatitis C virus non-structural protein 3 (NS3) to induce, in comparison or in combination with an NS3-expressing plasmid, specific cellular and humoral immune responses in murine models was evaluated. In vitro studies indicated that both types of vaccine expressed the expected size protein, albeit with different efficacies. The use of mice transgenic for the human HLA-A2.1 molecule indicated that the rSFV-expressed NS3 protein induces, as shown previously for an NS3 DNA vaccine, NS3-specific cytotoxic lymphocytes (CTLs) targeted at one dominant HLA-A2 epitope described in infected patients. All DNA/rSFV vaccine combinations evaluated induced specific CTLs, which were detectable for up to 31 weeks after the first injection. Overall, less than 1 log difference was observed in terms of the vigour of the bulk CTL response induced and the CTL precursor frequency between all vaccines (ranging from 1:2·6×105 to 1:1×106). Anti-NS3 antibodies could only be detected following a combined vaccine regimen in non-transgenic BALB/c mice. In conclusion, rSFV particles expressing NS3 are capable of inducing NS3-specific cellular immune responses targeted at a major HLA-A2 epitope. Such responses were comparable to those obtained with a DNA-based NS3 vaccine, whether in the context of single or combined regimens.
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Efficient delivery and regulable expression of hepatitis C virus full-length and minigenome constructs in hepatocyte-derived cell lines using baculovirus vectors
More LessBaculovirus vectors have been used as efficient delivery vehicles for constitutive gene expression in a variety of mammalian cells. We have further developed the system to allow for regulable expression by placing the gene of interest under the control of an inducible promoter, and complementing it with a second baculovirus vector providing the control elements necessary for promoter activity. We have used this system to express (a) the lacZ gene, (b) a ‘minigenome’ derived from hepatitis C virus (HCV) and carrying lacZ or (c) the full-length HCV viral genome, in human hepatocyte cell lines in an inducible fashion. Control systems that rely on either the absence of tetracycline or presence of ponasterone to induce gene expression were tested. Expression of lacZ was controlled by ponasterone, but β-galactosidase activity was limited to 10–20% of cells. In contrast, the tetracycline-controlled expression system gave a low basal activity and was highly inducible in almost 100% of cells. Inducible expression was also obtained in almost 100% of cells infected with baculoviruses in which an HCV minigenome was placed downstream of the tetracycline-inducible promoter and upstream of either a hammerhead or hepatitis δ virus ribozyme. Northern blot analysis was consistent with accurate cleavage of the minigenome transcript by the hepatitis δ virus ribozyme. Finally, regulable transcript production and viral polypeptide processing could be demonstrated in HepG2 cells infected with baculoviruses bearing the full-length HCV genome. This system thus provides a novel tool for the analysis of HCV replication and host–cell interactions.
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The sialate-4-O-acetylesterases of coronaviruses related to mouse hepatitis virus: a proposal to reorganize group 2 Coronaviridae
More LessGroup 2 coronaviruses are characterized within the order Nidovirales by a unique genome organization. A characteristic feature of group 2 coronaviruses is the presence of a gene encoding the haemagglutinin–esterase (HE) protein, which is absent in coronaviruses of groups 1 and 3. At least three coronavirus strains within group 2 expressed a structural protein with sialate-4-O-acetylesterase activity, distinguishing them from other members of group 2, which encode an enzyme specific for 5-N-acetyl-9-O-acetylneuraminic acid. The esterases of mouse hepatitis virus (MHV) strains S and JHM and puffinosis virus (PV) specifically hydrolysed 5-N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2) as well as the synthetic substrates p-nitrophenyl acetate, 4-methylumbelliferyl acetate and fluorescein diacetate. The K m values of the MHV-like esterases for the latter substrates were two- to tenfold lower than those of the sialate-9-O-acetylesterases of influenza C viruses. Another unspecific esterase substrate, α-naphthyl acetate, was used for the in situ detection of the dimeric HE proteins in SDS–polyacrylamide gels. MHV-S, MHV-JHM and PV bound to horse serum glycoproteins containing Neu4,5Ac2. De-O-acetylation of the glycoproteins by alkaline treatment or incubation with the viral esterases resulted in a complete loss of recognition, indicating a specific interaction of MHV-like coronaviruses with Neu4,5Ac2. Combined with evidence for distinct phylogenetic lineages of group 2 coronaviruses, subdivision into subgroups 2a (MHV-like viruses) and 2b (bovine coronavirus-like viruses) is suggested.
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Defective segment 1 RNAs that interfere with production of infectious influenza A virus require at least 150 nucleotides of 5′ sequence: evidence from a plasmid-driven system
More LessThe presence of at least 80–90 and more typically around 200 nucleotides (nt) at the 5′ end of the virion-sense RNA in all naturally occurring defective influenza A virus RNAs suggests that this is essential sequence, whereas the 3′-end sequence may be as short as 25 nt. The stability of defective RNA on serial passage with infectious helper virus also depends on the length of 5′-end sequence. Here, we have studied the influence of 5′-end sequences of a panel of six defective segment 1 RNAs from H3N8 and H7N7 viruses on their ability to interfere with the multiplication of plasmid-produced infectious A/WSN virus (H1N1). Four of the H3N8 defective RNAs are identical in overall length but vary in the length of 5′ sequence. Transfected defective RNAs interfered with infectious virus production in a concentration-dependent manner. The extent of interference also depended on the length of 5′-end sequence in the defective genome. This required at least 150 nt and was maximal with 220 nt of 5′ end sequence. The reduction in virus multiplication was highly significant and correlated with the presence of detectable intracellular defective RNA. Packaging of full-length segment 1 RNA by progeny virus was inversely proportional to the packaging of defective segment 1 RNA and may explain the reduction in infectivity. In summary, a critical length of 5′-end sequence is essential for the interfering properties of defective influenza virus RNAs, which indicates that this plays some vital role in the virus life cycle.
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H3N2 influenza viruses from domestic chickens in Italy: an increasing role for chickens in the ecology of influenza?
In Italy, multiple H3N2 influenza viruses were isolated from chickens with mild respiratory disease and were shown to replicate in the respiratory tracts of experimentally infected chickens; this finding is the first to show that H3N2 influenza viruses can replicate and cause disease in chickens. H3N2 influenza viruses in pigs on nearby farms seemed a likely source of the virus; however, antigenic and molecular analyses revealed that the gene segments of the viruses in chickens were mainly of Eurasian avian origin and were distinguishable from those isolated from pigs and wild aquatic birds in Italy. Thus, several different H3 influenza viruses were circulating in Italy, but we failed to identify the source of the chicken H3N2 influenza viruses that have disappeared subsequently from Italian poultry. Until recently, the transmission of influenza viruses (other than the H5 and H7 subtypes) from their reservoir in aquatic birds to chickens was rarely detected and highly pathogenic and non-pathogenic viruses were considered to be restricted to poultry species. However, the recent reports of the transmission of H9N2 and H5N1 influenza viruses to chickens in Hong Kong and, subsequently, to humans and our findings of the transmission of H3N2 influenza viruses to domestic chickens in Italy suggest an increased role for chickens as an intermediate host in the ecology of influenza.
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Genomic organization of infectious salmon anaemia virus
More LessThe RNA genome segment order, nucleotide sequence and the putative encoded proteins were determined for infectious salmon anaemia virus (ISAV). Eight segments of genomic viral RNA between 1·0 and 2·4 kb in length were identified. RNA segments 1–6 each had a predicted single open reading frame encoding the P1, PB1, NP, P2, P3 and HA proteins, respectively. Segment 7 encoded the P4/P5 proteins and segment 8 encoded the P6/P7 proteins. Seven virion proteins with molecular masses between 25 and 72 kDa were found by SDS–PAGE analysis. The 72 and 42 kDa proteins were immunoreactive with ISAV antiserum from Atlantic salmon. The molecular mass of the 72 kDa virion protein suggested that it was the NP protein encoded by segment 3. The amino acid sequence was conserved, sharing 96·6% identity with the NP protein of a Scottish ISAV isolate. Comparison of the amino acid sequences obtained by N-terminal analyses and cDNA nucleotide translation revealed that the 42 and 47 kDa proteins were the HA and P3 proteins encoded by segments 6 and 5, respectively. In addition, analysis provided evidence for their protein synthesis initiation sites. Like the HA protein, the signal sequence and potential glycosylation sites of P3 suggested that it was a surface glycoprotein. The predicted amino acid sequence shared 83·1, 84·0 and 99·6% identity to the predicted P3 protein sequences for ISAV isolates from Norway, Scotland and Maine, respectively. These results establish the specificity, migration, number and nucleotide sequence of the eight RNA segments of the ISAV genome.
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Virus-specific CTL responses induced by an H-2Kd-restricted, motif-negative 15-mer peptide from the fusion protein of respiratory syncytial virus
More LessWe describe 15-mer peptide P8:F92–106 from the F protein of respiratory syncytial virus (RSV) that can act as an MHC class I-restricted (H-2Kd) epitope for RSV-specific CD8+ CTL. This peptide is interesting because not only is it the first murine CTL epitope to be identified in the F protein but also because it does not contain a known allele-specific motif, as all 15 amino acids appear to be required for effective presentation to CTL. In in vitro MHC class I refolding experiments, peptide P8:F92–106 induced complex formation with H-2Kd heavy chains and β2-microglobulin. Immunization of BALB/c mice with P8:F92–106 resulted in the induction of peptide and RSV-specific CTL responses as well as peptide-specific proliferative responses. Following intranasal challenge with RSV, P8:F92–106-immunized mice showed a significant reduction in viral load in the lungs compared to that seen in unimmunized mice. Furthermore, passive transfer of purified CD8+ lymphocytes into BALB/c scid mice prior to challenge with RSV also resulted in a reduction in the virus load in lungs of challenged mice. These results indicate the potential of synthetic peptide epitopes for the induction of protective immune responses against RSV infection.
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Fv1-like restriction of N-tropic replication-competent murine leukaemia viruses in mCAT-1-expressing human cells
More LessTo study the replication of murine leukaemia viruses in human cells we have used full-length as well as EGFP-tagged ecotropic viruses in combination with mCAT-1-expressing human cells. We present results showing that N-tropic murine leukaemia viruses are restricted in both infection and replication in such cells while B-tropic viruses, modified at capsid position 110, escape restriction. These results support a recently reported Fv1-like restriction in mammalian cells. We extend the analysis of Fv1-like restriction by demonstrating that NB-tropic viruses also escape restriction and human mCAT-1-expressing cells are thus similar to murine Fv1 b cells with respect to infection though the ecotropic receptor pathway.
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- Animal: DNA Viruses
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A transgenic mouse model for non-immune hydrops fetalis induced by the NS1 gene of human parvovirus B19
Human parvovirus B19 (B19) infection during pregnancy is associated with the adverse foetal outcome known as non-immune hydrops fetalis (NIHF). Although B19 is known to infect erythroid-lineage cells in vivo as well as in vitro, the mechanism leading to the occurrence of NIHF is not clear. To investigate the possible involvement of the B19 non-structural protein NS1 in NIHF, three independent lines of transgenic mice were generated that expressed NS1 under the control of the Cre-loxP system and the GATA1 promoter. Two of the three lines expressed NS1 in erythroid-lineage cells. Most of the transgenic mice died at the embryonic stage, some of which developed hydropic changes caused by severe anaemia at embryonic day 15·5 (E15·5). Histological examination of embryos at E15·5 showed significantly fewer erythropoietic islands in the liver parenchyma, whereas their hearts showed no abnormal signs, such as cardiomegaly and apoptotic cells. The NS1-transgenic mouse lines established here provide an animal model for human NIHF and suggest that NS1 plays a crucial role in the adverse outcome associated with intrauterine B19 infection in humans.
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Mutational analysis of the discs large tumour suppressor identifies domains responsible for human papillomavirus type 18 E6-mediated degradation
More LessThe discs large (Dlg) tumour suppressor protein is targeted for ubiquitin-mediated degradation by the high-risk human papillomavirus E6 proteins. To understand further the mechanisms behind this, a mutational analysis of Dlg was undertaken. This study demonstrates that an intact PDZ domain 2 (PDZ2) on Dlg is necessary for the ability of E6 to bind and degrade Dlg. However, additional residues within the amino-terminal portion of Dlg are also required for optimal E6 activity. Stable cell lines expressing different Dlg mutants were also established and these confirm that Dlg is regulated intrinsically by the proteasome in the absence of E6; however, in this case, the sequences responsible for regulating Dlg stability lie predominantly within PDZ2. These results suggest that there are at least two mechanisms for regulating Dlg protein stability and that the pathways used by E6 are not necessarily the same as those used in the cell in its absence.
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Herpes simplex virus type 1 glycoprotein C is necessary for efficient infection of chondroitin sulfate-expressing gro2C cells
More LessThe role of glycoprotein C (gC) for binding of herpes simplex virus type 1 (HSV-1) to cell surface chondroitin sulfate (CS) and the consequences of this interaction for virus attachment and infectivity were studied. To this end, a panel of HSV-1 gC mutants, including a gC-negative (gC−) variant, and mouse fibroblasts expressing either cell surface CS or heparan sulfate (HS) were used. Comparing gC-positive (gC+) and gC− viruses in terms of their attachment to and infection of CS-expressing cells indicated that gC was essential for both functions. Furthermore, purified gC bound efficiently to isolated CS chains. However, hypertonic NaCl disrupted this interaction more easily as compared to the binding of gC to HS. Also, native and selectively desulfated heparins were approximately 10 times more efficient at inhibiting gC binding to CS-expressing cells than binding to HS-expressing cells. Experiments with the HSV-1 gC mutants revealed that specific, positively charged and hydrophobic amino acids within the N-terminal part of the protein were responsible for efficient binding as well as infectivity in both CS- and HS-expressing cells. When the infectivity of the gC mutants in the two cell types was compared, it appeared that more residues contributed to the infection of CS-expressing cells than to infection of HS-expressing cells. Taken together, analysis of gC function in cell systems with limited expression of glycosaminoglycans revealed that gC could interact with either CS or HS and that these interactions exhibited subtle but definite differences as regards to the involved structural features of gC, ionic strength dependency as well as sensitivity to specifically desulfated heparin compounds.
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Characterization of pseudorabies virus glycoprotein C attachment to heparan sulfate proteoglycans
More LessPseudorabies virus first attaches to cells through an interaction between the envelope glycoprotein C (gC) and the cell surface heparan sulfate (HS) that is linked to proteoglycans (HSPGs). The HS-binding domain of gC is composed of three discrete heparin-binding domains (HBDs), designated HBD1, -2 and -3 for their proximity to the amino terminus of gC. Each HBD can independently mediate virus attachment to HS, yet each also exhibits a distinct binding preference for differentially sulfated derivatives of heparin. To demonstrate this, affinity columns composed of wild-type gC or mutant gC retaining a single HBD to capture several HSPGs from cultured pig and bovine kidney cells were used. The wild-type gC column bound all of the HSPGs well and, overall, bound more than 90% of the total sample applied to the column. Columns composed of either HBD2 or -3 bound intermediate amounts (40%) of the total sample applied, while the HBD1 column bound low amounts of HSPGs. HBD2 and -3 columns did not uniformly bind all of the HSPGs from bovine kidney cells, but the same HSPGs were bound with equal efficiency on each column. Thus, despite their different preferences for sulfation patterns on HS side-chains, HBD2 and -3 appear to bind the same proteoglycan cores. These results established a hierarchy of HBD2=HBD3>HBD1 in importance for HSPG binding. These in vitro-binding results correlated with the attachment phenotype of virus strains expressing gC with a single HBD in their envelopes.
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Early gene m18, a novel player in the immune response to murine cytomegalovirus
More LessThe identification of all antigenic peptides encoded by a pathogen, its T cell ‘immunome’, is a research aim for rational vaccine design. Screening of proteome-spanning peptide libraries or computational prediction is used to identify antigenic peptides recognized by CD8 T cells. Based on their high coding capacity, cytomegaloviruses (CMVs) could specify numerous antigenic peptides. Yet, current evidence indicates that the memory CD8 T cell response in a given haplotype is actually focused on a few viral proteins. CMVs actively interfere with antigen processing and presentation by the expression of immune evasion proteins. In the case of murine CMV (mCMV), these proteins are effectual in the early (E) phase of the virus replication cycle and should thus preclude the presentation of peptides derived from E proteins. Notably, the m18 gene is here added to a growing list of mCMV E genes that encode antigenic peptides in spite of the E phase immune evasion strategies of the virus.
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Evidence for trafficking of Epstein–Barr virus strains between hairy leukoplakia and peripheral blood lymphocytes
More LessHairy leukoplakia (HL), an epithelial lesion found on the side of the tongue in immunocompromised individuals, is characterized by high-level replication of Epstein–Barr virus (EBV) and multiple EBV strains. The source of these strains and their relationship to peripheral blood lymphocyte (PBL) strains has not previously been characterized. Using matched pairs of HL scrapings and PBL from 16 HIV-positive men, variation in EBV strain identity was characterized by detection of a 30 nucleotide deletion of the EBV latent membrane protein (LMP)-1 gene, variation in the LMP-1 repeat region and typing for Epstein–Barr nuclear antigen (EBNA)-2. Multiple EBV strains were found in both the HL and PBL specimens, but 13 of 16 (81%) patients showed evidence of strain identity for at least one strain and analysis of two patients suggested that EBV strains from HL could infect the PBL. Our data are consistent with active trafficking of EBV between these two compartments.
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Replacing the SCR domains of vaccinia virus protein B5R with EGFP causes a reduction in plaque size and actin tail formation but enveloped virions are still transported to the cell surface
More LessA vaccinia virus (VV) recombinant is described in which the outer envelope of extracellular enveloped virus (EEV), cell-associated enveloped virus (CEV) and intracellular enveloped virus (IEV) is labelled with the enhanced green fluorescent protein (EGFP) derived from Aequorea victoria. To construct this virus, EGFP was fused to the VV B5R protein from which the four short consensus repeats (SCRs) of the extracellular domain had been deleted. Cells infected with the recombinant virus expressed a B5R–EGFP fusion protein of 40 kDa that was present on IEV, CEV and EEV, but was absent from IMV. The recombinant virus produced 2- and 3-fold reduced levels of IMV and EEV, respectively. Analysis of infected cells by confocal microscopy showed that actin tail formation by the mutant virus was reduced by 86% compared to wild-type (WT). The virus formed a small plaque compared to WT, consistent with a role for actin tails in promoting cell-to-cell spread of virus. However, the enveloped virions were still transported to the cell surface, confirming that this process is independent of actin tail formation. Lastly, we compared the mutant virus with a recombinant VV in which the B5R SCR domains were deleted and show that, contrary to a previous report, the plaque size of the latter virus was reduced compared to WT. This observation reconciles an inconsistency in the field and confirms that viruses deficient in formation of actin tails form small plaques.
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- Insect
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Assessment of foreign protein production by recombinant Heliothis (Helicoverpa) armigera entomopoxviruses in Spodoptera frugiperda cells
More LessThis report describes the first production of recombinant forms of Heliothis (Helicoverpa) armigera entomopoxvirus (HaEPV). These HaEPVs are engineered at either the spheroidin or fusolin locus, to produce the green fluorescent marker protein (GFP). The growth properties of these recombinant HaEPVs, in comparison to the parental HaEPV, were assessed in cultured Spodoptera frugiperda Sf9 cells. Additionally, GFP production by these recombinant HaEPVs was compared to that of a GFP-expressing recombinant of the baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) in the same in vitro system, at various multiplicities of infection. Expression of GFP from the HaEPV spheroidin locus produced up to 60% of that generated from the AcNPV polyhedrin locus, albeit over a longer period of infection. A considerably lower yield was recorded from the HaEPV fusolin locus, a result that contrasted markedly with the apparent activity of this promoter in caterpillar infections in vivo. The potential applications for further development of HaEPV expression systems are discussed.
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Comparative analysis of the genome and host range characteristics of two insect iridoviruses: Chilo iridescent virus and a cricket iridovirus isolate
More LessThe iridovirus isolate termed cricket iridovirus (CrIV) was isolated in 1996 from Gryllus campestris L. and Acheta domesticus L. (both Orthoptera, Gryllidae). CrIV DNA shows distinct DNA restriction patterns different from those known for Insect iridescent virus type 6 (IIV-6). This observation led to the assumption that CrIV might be a new species within the family Iridoviridae. CrIV can be transmitted perorally to orthopteran species, resulting in specific, fatal diseases. These species include Gryllus bimaculatus L. and the African migratory locust Locusta migratoria migratorioides (Orthoptera, Acrididae). Analysis of genomic and host range properties of this isolate was carried out in comparison to those known for IIV-6. Host range studies of CrIV and IIV-6 revealed no differences in the peroral susceptibility in all insect species and developmental stages tested to date. Different gene loci of the IIV-6 genome were analyzed, including the major capsid protein (274L), thymidylate synthase (225R), an exonuclease (012L), DNA polymerase (037L), ATPase (075L), DNA ligase (205R) and the open reading frame 339L, which is homologous to the immediate-early protein ICP-46 of frog virus 3. The average identity of the selected viral genes and their gene products was found to be 95·98 and 95·18% at the nucleotide and amino acid level, respectively. These data led to the conclusion that CrIV and IIV-6 are not different species within the Iridoviridae family and that CrIV must be considered to be a variant and/or a novel strain of IIV-6.
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Transcription and identification of an envelope protein gene (p22) from shrimp white spot syndrome virus
More LessWhite spot syndrome virus (WSSV) is one of the most virulent pathogens causing high mortality in shrimp. In the present study, an open reading frame (termed the p22 gene) was revealed from a WSSV cDNA library. The gene was expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli and purified. Specific antibody was raised using the purified fusion protein (GST–P22). Temporal analysis showed that the p22 gene was a late gene. After binding between purified WSSV virions and anti-GST–P22 IgG followed by labelling with gold-labelled secondary antibody, the gold particles, under a transmission electron microscope, could be found along the outer envelope of WSSV virions. This experiment suggests that the p22 gene encodes an envelope protein of the virus.
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- Plant
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Mapping of the P1 proteinase cleavage site in the polyprotein of Wheat streak mosaic virus (genus Tritimovirus)
More LessMonopartite members of the family Potyviridae utilize three virus-encoded proteinases to cleave the viral polyprotein into mature proteins. The amino-terminal region of the viral polyprotein is autolytically cleaved by the P1 proteinase. A domain required for P1 proteinase activity of Wheat streak mosaic virus (WSMV) was mapped using a series of templates with nested 3′-truncations or 5′-deletions to program in vitro transcription–translation reactions. The WSMV P1 proteinase cleavage site was mapped to a position downstream of amino acid residue 348 and upstream of amino acid residue 353, with the peptide bond between amino acid residues Y352 and G353 the most probable site of hydrolysis. An alignment of potyvirus polyprotein sequences in the carboxy-terminal region of the P1 domain revealed WSMV P1 contained conserved H257, D267, S303 and FIVXG325–329 residues upstream of the cleavage site that are typical of serine proteinases and shown by others to be required for P1 proteolysis in Tobacco etch virus. Insertion of the GUS reporter gene immediately downstream of the P1 cleavage site in a full-length clone of WSMV resulted in systemic infection and GUS expression upon inoculation of plants with in vitro transcripts. When cleaved by P1 at the amino terminus and NIa proteinase at a site engineered in the carboxy-terminus, active GUS protein expressed by WSMV in infected wheat had electrophoretic mobility similar to wild-type GUS protein.
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- Other Agents
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Distribution and accumulation of PrP in gut-associated and peripheral lymphoid tissue of scrapie-affected Suffolk sheep
More LessThe distribution of disease-associated prion protein (PrP) was investigated in eight animals (20–24 months of age) from a flock of Suffolk sheep that had experienced frequent cases of natural scrapie over a period of several years. Tissue from the central nervous system (CNS), alimentary tract, peripheral nervous system and lymphoreticular system was examined by histopathology and immunohistochemistry. The lymphoid tissues were subjected further to histoblot and immunofluorescence examination. The four clinically affected PrPARQ/ARQ sheep had widespread accumulations of disease-associated PrP in the CNS, lymphoreticular system and peripheral ganglia. In the two PrPARQ/ARQ sheep that did not show clinical signs of scrapie, only limited vacuolation and PrP accumulation were detected in the brain, but the results from the lymphoreticular system and peripheral nervous system were comparable with the clinically affected animals. The remaining PrPARR/ARR and PrPARR/ARQ sheep did not show proteinase K-resistant PrP accumulations in the lymphoid tissues examined and immunohistochemistry did not reveal the presence of disease-associated PrP. In lymphoid tissues of the PrPARQ/ARQ sheep, the dominant localization of disease-associated PrP was in lymphoid nodules and double immunofluorescence labelling for PrP and CD21 provided further support for the role of follicular dendritic cells in scrapie in sheep. A striking finding in the present study was the large accumulations of disease-associated PrP in the lymphoid nodules of the alimentary tract at the late sub-clinical and clinical stage of the infection. The study also identified disease-associated PrP in extra-nodular sites of lymphoid tissues, such as the marginal zone of the spleen, and these observations were used to argue that cells of the mononuclear phagocyte system of sheep may be involved in the uptake, transport, elimination and shedding of the scrapie agent.
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Volumes and issues
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Volume 105 (2024)
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Volume 104 (2023)
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Volume 103 (2022)
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Volume 102 (2021)
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Volume 101 (2020)
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Volume 100 (2019)
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Volume 99 (2018)
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Volume 98 (2017)
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Volume 97 (2016)
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Volume 96 (2015)
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Volume 95 (2014)
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Volume 94 (2013)
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Volume 93 (2012)
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Volume 92 (2011)
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Volume 91 (2010)
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Volume 90 (2009)
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Volume 89 (2008)
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Volume 88 (2007)
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Volume 87 (2006)
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Volume 86 (2005)
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Volume 85 (2004)
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Volume 84 (2003)
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Volume 83 (2002)
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Volume 82 (2001)
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Volume 81 (2000)
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Volume 80 (1999)
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Volume 79 (1998)
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Volume 78 (1997)
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Volume 77 (1996)
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Volume 76 (1995)
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Volume 75 (1994)
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Volume 74 (1993)
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Volume 73 (1992)
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Volume 72 (1991)
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Volume 71 (1990)
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Volume 70 (1989)
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Volume 69 (1988)
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Volume 68 (1987)
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Volume 67 (1986)
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Volume 66 (1985)
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Volume 65 (1984)
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Volume 64 (1983)
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Volume 63 (1982)
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Volume 62 (1982)
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Volume 61 (1982)
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Volume 60 (1982)
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Volume 59 (1982)
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Volume 58 (1982)
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Volume 57 (1981)
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Volume 56 (1981)
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Volume 55 (1981)
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Volume 54 (1981)
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Volume 53 (1981)
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Volume 52 (1981)
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Volume 51 (1980)
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Volume 50 (1980)
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Volume 49 (1980)
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Volume 48 (1980)
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Volume 47 (1980)
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Volume 46 (1980)
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Volume 45 (1979)
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Volume 44 (1979)
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Volume 43 (1979)
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Volume 42 (1979)
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Volume 41 (1978)
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Volume 40 (1978)
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Volume 39 (1978)
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Volume 38 (1978)
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Volume 37 (1977)
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Volume 36 (1977)
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Volume 35 (1977)
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Volume 34 (1977)
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Volume 33 (1976)
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Volume 32 (1976)
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Volume 31 (1976)
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Volume 30 (1976)
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Volume 29 (1975)
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Volume 28 (1975)
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Volume 27 (1975)
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Volume 26 (1975)
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Volume 25 (1974)
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Volume 24 (1974)
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Volume 23 (1974)
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Volume 22 (1974)
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Volume 21 (1973)
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Volume 20 (1973)
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Volume 19 (1973)
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Volume 18 (1973)
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Volume 17 (1972)
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Volume 16 (1972)
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Volume 15 (1972)
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Volume 14 (1972)
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Volume 13 (1971)
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Volume 12 (1971)
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Volume 11 (1971)
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Volume 10 (1971)
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Volume 9 (1970)
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Volume 8 (1970)
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Volume 7 (1970)
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Volume 6 (1970)
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Volume 5 (1969)
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Volume 4 (1969)
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Volume 3 (1968)
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Volume 2 (1968)
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Volume 1 (1967)