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Volume 86,
Issue 4,
2005
Volume 86, Issue 4, 2005
- Animal
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- DNA viruses
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Immunization with a bovine herpesvirus 1 glycoprotein B DNA vaccine induces cytotoxic T-lymphocyte responses in mice and cattle
More LessVirus-specific cytotoxic T lymphocytes (CTLs) are considered to be important in protection against and recovery from viral infections. In this study, several approaches to induce cytotoxicity against bovine herpesvirus 1 (BHV-1) were evaluated. Vaccination of C57BL/6 mice with BHV-1 induced a strong humoral, but no CTL, response, which may be due to downregulation of major histocompatibility complex class I molecules. In contrast, vaccinia virus expressing glycoprotein B (gB) elicited a weaker antibody response, but strong cytotoxicity, in mice. As an approach to inducing both strong humoral and cellular immune responses, a plasmid vector was then used to express gB. Both antibody and CTL responses were induced by the plasmid encoding gB in C57BL/6 and C3H mice, regardless of the type of vector backbone. This demonstrated that DNA immunization induces a broad-based immune response to BHV-1 gB. Interestingly, removal of the membrane anchor, which resulted in secretion of gB from transfected cells, did not result in reduced cytotoxicity. Here, it is shown that, compared with the cell-associated counterpart, plasmid-encoded secreted protein may induce enhanced immune responses in cattle. Therefore, calves were immunized intradermally with pMASIAtgB, a plasmid encoding the secreted form of gB (tgB), using a needle-free injection system. This demonstrated that pMASIAtgB elicited both humoral responses and activated gamma interferon-secreting CD8+ CTLs, suggesting that a DNA vaccine expressing tgB induces a CTL response in the natural host of BHV-1.
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Discovery of Epstein–Barr virus (EBV)-encoded RNA signal and EBV nuclear antigen leader protein DNA sequence in pet dogs
More LessThe aim of this study was to investigate Epstein–Barr virus (EBV)-related virus infection in pet dogs. The presence of antibodies to EBV antigens and EBV-related DNA was determined by Western blot analysis and PCR, respectively. Among 36 pet dogs examined for serum antibodies, 32 (88·9 %) were positive for EBV-specific thymidine kinase, 15 (41·7 %) for EBV-encoded DNA-binding protein and 10 (27·8 %) for EBV-specific DNA polymerase. A BamHI W fragment sequence encoding part of the EBV nuclear antigen leader protein was detected by PCR in corresponding leukocyte DNA samples. Among 21 dogs tested, 15 (71·4 %) were positive for the BamHI W fragment sequence. The specificity of the amplified DNA fragments was confirmed by DNA sequencing. Within the amplified region of the BamHI W fragment (241 bp), DNA sequences detected in 10 dogs had 99·2 % (two nucleotide variations), 99·6 % (one nucleotide variation) or 100 % identity to that of EBV. Furthermore, an EBV-encoded RNA signal was detected by in situ hybridization in dog lymphocytes, as well as in bone-marrow sections, indicating a latent infection with EBV or an EBV-like virus. In conclusion, although the sample size was small, these results showed that a widespread EBV-related gammaherpesvirus could be detected in the peripheral blood and bone marrow of pet dogs. Although no evident zoonotic transmission was detected, further studies are imperative for disclosing the biological significance of this canine EBV-like virus, which may correlate with human disorders.
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Development of bovine herpesvirus 4 as an expression vector using bacterial artificial chromosome cloning
Several features make bovine herpesvirus 4 (BoHV-4) attractive as a backbone for use as a viral expression vector and/or as a model to study gammaherpesvirus biology. However, these developments have been impeded by the difficulty in manipulating its large genome using classical homologous recombination in eukaryotic cells. In the present study, the feasibility of exploiting bacterial artificial chromosome (BAC) cloning and prokaryotic recombination technology for production of BoHV-4 recombinants was explored. Firstly, the BoHV-4 genome was BAC cloned using two potential insertion sites. Both sites of insertion gave rise to BoHV-4 BAC clones stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. Reconstituted virus replicated comparably to wild-type parental virus and the loxP-flanked BAC cassette was excised by growing them on permissive cells stably expressing Cre recombinase. Secondly, BoHV-4 recombinants expressing Ixodes ricinus anti-complement protein I or II (IRAC I/II) were produced using a two-step mutagenesis procedure in Escherichia coli. Both recombinants induced expression of high levels of functional IRAC molecules in the supernatant of infected cells. This study demonstrates that BAC cloning and prokaryotic recombination technology are powerful tools for the development of BoHV-4 as an expression vector and for further fundamental studies of this gammaherpesvirus.
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Murine gammaherpesvirus-68 ORF28 encodes a non-essential virion glycoprotein
More LessMurine gammaherpesvirus-68 (MHV-68) ORF28 is a gammaherpesvirus-specific gene of unknown function. Analysis of epitope-tagged ORF28 protein indicated that it was membrane-associated and incorporated into virions in N-glycosylated, O-glycosylated and unglycosylated forms. The extensive glycosylation of the small ORF28 extracellular domain – most forms of the protein appeared to be mainly carbohydrate by weight – suggested that a major function of ORF28 is to attach a variety of glycans to the virion surface. MHV-68 lacking ORF28 showed normal lytic replication in vitro and in vivo and normal latency establishment. MHV-68 ORF28 therefore encodes a small, membrane-bound and extensively glycosylated virion protein, whose function is entirely dispensable for normal, single-cycle host colonization.
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Analysis of the Choristoneura fumiferana nucleopolyhedrovirus genome
The double-stranded DNA genome of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) was sequenced and analysed in the context of other group I nucleopolyhedroviruses (NPVs). The genome consists of 129 593 bp with a G+C content of 50·1 mol%. A total of 146 open reading frames (ORFs) of greater than 150 bp, and with no or minimal overlap were identified. In addition, five homologous regions were identified containing 7–10 repeats of a 36 bp imperfect palindromic core. Comparison with other completely sequenced baculovirus genomes revealed that 139 of the CfMNPV ORFs have homologues in at least one other baculovirus and seven ORFs are unique to CfMNPV. Of the 117 CfMNPV ORFs common to all group I NPVs, 12 are exclusive to group I NPVs. Overall, CfMNPV is most similar to Orgyia pseudotsugata MNPV based on gene content, arrangement and overall amino acid identity. Unlike other group I baculoviruses, however, CfMNPV encodes a viral enhancing factor (vef) and has two copies of p26.
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Gene organization and sequencing of the Choristoneura fumiferana defective nucleopolyhedrovirus genome
More LessTwo distinct nucleopolyhedrovirus species of the eastern spruce budworm, Choristoneura fumiferana, exist in a symbiont-like relationship. C. fumiferana defective nucleopolyhedrovirus (CfDEFNPV) only infects C. fumiferana larvae per os in the presence of C. fumiferana nucleopolyhedrovirus Ireland strain (CfMNPV), but is infective when injected into the haemolymph. CfDEFNPV synergizes CfMNPV in per os infections and CfMNPV is always the predominant progeny. This study was undertaken to report the genomic makeup and organization of CfDEFNPV in an attempt to identify its defect and understand its synergistic role. The genome was mapped, sequenced, characterized and compared to other baculoviruses. The CfDEFNPV genome was 131 160 nt long with 149 putative open reading frames (ORFs) and a G+C content of 45·8 mol%. Homologues of all 62 conserved lepidopteran baculovirus genes were found including those implicated in per os infectivity, p74, per os infectivity factor (pif) and pif-2. Although no obvious deletions were observed to explain the defect, two ORFs, Cfdef79 and Cfdef99 (inhibitor of apoptosis-4), contained potential deletions. Cfdef50 (late expression factor-10)/Cfdef51 (vp1054) and Cfdef76/Cfdef77 (telokin-like protein) had large overlaps and a potential homologue to ac105/he65 was split. Four baculovirus repeat ORFs were present, as were two unique genes, but no enhancins were identified. CfDEFNPV contained 13 homologous regions, each with one to five palindromes. Comparison with fully sequenced baculovirus genomes identified CfDEFNPV as a group I NPV with the closest average amino acid identity to Epiphyas postvittana NPV, followed by Orgyia pseudotsugata MNPV and CfMNPV, with its closest matches being to individual Anticarsia gemmatalis MNPV gene sequences.
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Expression of a Toxoneuron nigriceps polydnavirus-encoded protein causes apoptosis-like programmed cell death in lepidopteran insect cells
The polydnavirus Toxoneuron nigriceps bracovirus (TnBV) is an obligate symbiont associated with the braconid wasp T. nigriceps, a parasitoid of Heliothis virescens larvae. Previously, to identify polydnavirus genes that allow parasitization by altering the host immune and endocrine systems, expression patterns of TnBV genes from parasitized H. virescens larvae were analysed and cDNAs were obtained. To study the function of the protein from one such cDNA, TnBV1, overexpression of the protein was attempted by using the baculovirus Autographa californica multicapsid nucleopolyhedrovirus. Recovery of stable recombinant virus was unsuccessful, with the exception of recombinants with deletions/mutations within the TnBV1 gene. It was hypothesized that TnBV1 expression was cytotoxic to the Spodoptera frugiperda (Sf21) insect cells that were used to produce the recombinants. Therefore, the Bac-to-Bac system was used to create recombinant baculoviruses maintained in Escherichia coli expressing either TnBV1 (Ac-TnBV1) or an initiator-methionine mutant [Ac-TnBV1(ATG−)]. Microscopy revealed substantial cell death of Sf21 and High Five cells from 48 h post-infection with Ac-TnBV1, but not with the Ac-TnBV1(ATG−) recombinant virus. Ac-TnBV1-infected Sf21 cells, but not those with parental virus infection, showed an increased caspase-3-like protease activity, as well as increased terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) for breaks in host genomic DNA. Although indicative of apoptosis, blebbing and apoptotic bodies were not observed in infected cells. Transiently expressing TnBV1 alone caused TUNEL staining in High Five cells. These data suggest that TnBV1 expression alone can induce apoptosis-like programmed cell death in two insect cell lines. Injection of Ac-TnBV1 budded virus, compared with parental virus, did not result in an alteration of virulence in H. virescens larvae.
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Cloning, characterization and analysis by RNA interference of various genes of the Chelonus inanitus polydnavirus
More LessSuccessful parasitism of some endoparasitic wasps depends on an obligately symbiotic association with polydnaviruses. These unique viruses have a segmented genome consisting of circles of double-stranded (ds) DNA and do not replicate in the parasitized host. They are produced in the wasp's ovary and injected into the host along with the egg. Chelonus inanitus is an egg–larval parasitoid; its polydnavirus (CiV) has been shown to protect the parasitoid larva from the host's immune system and to induce developmental arrest in the prepupal stage. The genome of CiV consists of at least 10–12 segments and five have been sequenced up to now. Here, the complete (CiV12g2) or partial (CiV12g1, CiV16.8g1) cloning of three new CiV genes is reported. All three occur only on one viral segment and have no similarity to other known polydnavirus genes, with the exception of a high similarity of CiV12g1 to CiV14g1 and CiV12g2 to CiV14g2. Furthermore, the first attempt of in vivo application of RNA interference to study the function of polydnavirus genes is shown. Injection of dsRNA of two late- and one early- and late-expressed CiV genes into CiV/venom-containing host eggs partially rescued last-instar larvae from developmental arrest. Injection of the same dsRNAs into parasitized eggs partially reduced parasitoid survival, mainly by preventing the successful emergence of the parasitoid from the host. These viral genes thus seem to be involved in inducing developmental arrest and in keeping the cuticle soft, which appears to be necessary for parasitoid emergence and host feeding.
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A new intertype recombinant between genotypes C and D of hepatitis B virus identified in China
Hepatitis B virus (HBV) genotypes have a characteristic geographical distribution. More than 90 % of chronic HBV patients in China are infected with genotypes B or C. Here, eight HBV isolates that were initially classified as genotype D by PCR-restriction fragment length polymorphism analysis were analysed in detail. The complete HBV genome was sequenced and compared with 32 sequences retrieved from GenBank, representing HBV genotypes A–G. Phylogenetic analysis of the S gene (nt 10–800) classified all eight isolates as genotype D. However, phylogenetic analyses of nt 800–10 and the open reading frames (ORFs) of the precore/core and X genes classified all eight isolates as genotype C. This discordance between phylogenetic trees reconstructed on different ORFs suggested that intertype recombination has occurred in all eight isolates. By using the simplot program, the site of recombination with genotype D was located in the preS2/S region, spanning nt 10–799 in seven of eight isolates and nt 10–1499 in the other isolate. These results demonstrate that intertype recombination should be considered as a type of variation that increases the genetic diversity of HBV. Hybrids of different HBV genotypes might exhibit specific virological properties and their significance in the diagnosis and management of chronic hepatitis B deserves further investigation.
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An investigation of the therapeutic value of vaccinia-immune IgG in a mouse pneumonia model
More LessVaccinia-immune globulin (VIG) was used to treat severe complications of smallpox vaccination, but its use was controversial because it resolved disease in only some clinical cases. VIG is a pool of hyperimmune sera collected from individuals with a high neutralizing titre against the intracellular mature form (IMV) of vaccinia virus (VACV), but activity against the extracellular enveloped form (EEV) was often not considered. Here, the efficacy of anti-VACV antibodies (Abs) in protecting mice from intranasal infection with the VACV strain Western Reserve (WR) was evaluated. Mice were immunized passively with hyperimmune rabbit Abs (IgG) generated against inactivated IMV or produced following infection by VACV; subsequently, animals were challenged with VACV WR. The results demonstrated that: (i) good protection requires Abs to EEV in addition to IMV; (ii) Abs were effective when given before or up to 4 days after infection; and (iii) protection of mice from VACV WR correlated with a reduction of virus replication in lungs, but not in brain. In agreement with studies conducted before smallpox was eradicated and recent studies using EEV antigens for immunization, this study reiterates the importance of anti-EEV Abs in protecting against orthopoxvirus infection and illustrates the need to evaluate both anti-IMV and anti-EEV neutralizing Abs in VIG.
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Binding of human papillomavirus 16 E6 to p53 and E6AP is impaired by monoclonal antibodies directed against the second zinc-binding domain of E6
The E6 protein of cancer-associated human papillomavirus type 16 (16E6) binds to p53 and, in association with E6AP, promotes its degradation through the ubiquitin–proteasome pathway. The aim of this work was to develop monoclonal antibodies against 16E6 and to test their effect on the binding of 16E6 to p53 and E6AP, and on the degradation of p53. It was shown that an antibody directed against the N terminus of 16E6 inhibited E6AP-dependent binding to p53 and degradation of p53, whereas two different antibodies directed to the second zinc-binding domain of 16E6 reduced 16E6 E6AP-independent binding to p53 and binding to E6AP but not degradation of p53.
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Minute virus of mice small non-structural protein NS2 localizes within, but is not required for the formation of, Smn-associated autonomous parvovirus-associated replication bodies
The non-structural proteins NS1 and NS2 of the parvovirus minute virus of mice (MVM) are required for efficient virus replication. It has previously been shown that NS1 and NS2 interact and colocalize with the survival motor neuron (Smn) gene product in novel nuclear structures that are formed late in infection, termed Smn-associated APAR (autonomous parvovirus-associated replication) bodies (SAABs). It is not clear what molecular viral intermediate(s) contribute to SAAB formation. The current results address the role of NS2 in SAAB formation. In highly synchronized wild-type MVM infection of murine A92L cells, NS2 colocalizes with Smn and other SAAB constituents. An MVM mutant that does not produce NS2 still generates SAABS, albeit with a temporal delay. The lag in SAAB formation seen in the absence of NS2 is probably related to the temporal delay in virus replication, suggesting that, whilst NS2 is required for efficient viral infection, it is dispensable for SAAB formation.
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- Plant Viruses
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Natural isolates of Brome mosaic virus with the ability to move from cell to cell independently of coat protein
Brome mosaic virus (BMV) requires encapsidation-competent coat protein (CP) for cell-to-cell movement and the 3a movement protein (MP) is involved in determining the CP requirement for BMV movement. However, these conclusions have been drawn by using BMV strain M1 (BMV-M1) and a related strain. Here, the ability of the MPs of five other natural BMV strains to mediate the movement of BMV-M1 in the absence of CP was tested. The MP of BMV M2 strain (BMV-M2) efficiently mediated the movement of CP-deficient BMV-M1 and the MPs of two other strains functioned similarly to some extent. Furthermore, BMV-M2 itself moved between cells independently of CP, demonstrating that BMV-M1 and -M2 use different movement modes. Reassortment between CP-deficient BMV-M1 and -M2 showed the involvement of RNA3 in determining the CP requirement for cell-to-cell movement and the involvement of RNAs 1 and 2 in movement efficiency and symptom induction in the absence of CP. Spontaneous BMV MP mutants generated in planta that exhibited CP-independent movement were also isolated and analysed. Comparison of the nucleotide differences of the MP genes of BMV-M1, the natural strains and mutants capable of CP-independent movement, together with further mutational analysis of BMV-M1 MP, revealed that single amino acid differences at the C terminus of MP are sufficient to alter the requirement for CP in the movement of BMV-M1. Based on these findings, a possible virus strategy in which a movement mode is selected in plant viruses to optimize viral infectivity in plants is discussed.
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Cucumber mosaic virus 2a polymerase and 3a movement proteins independently affect both virus movement and the timing of symptom development in zucchini squash
More LessThe basis for differences in the timing of systemic symptom elicitation in zucchini squash between a pepper strain of Cucumber mosaic virus (Pf-CMV) and a cucurbit strain (Fny-CMV) was analysed. The difference in timing of appearance of systemic symptoms was shown to map to both RNA 2 and RNA 3 of Pf-CMV, with pseudorecombinant viruses containing either RNA 2 or RNA 3 from Pf-CMV showing an intermediate rate of systemic symptom development compared with those containing both or neither Pf-CMV RNAs. Symptom phenotype was shown to map to two single-nucleotide changes, both in codons for Ile at aa 267 and 168 (in Fny-CMV RNAs 2 and 3, respectively) to Thr (in Pf-CMV RNAs 2 and 3). The differential rate of symptom development was shown to be due to differences in the rates of cell-to-cell movement in the inoculated cotyledons, as well as differences in the rate of egress of the virus from the inoculated leaves. These data indicate that both the CMV 3a movement protein and the CMV 2a polymerase protein affect the rate of movement of CMV in zucchini squash and that these two proteins function independently of each other in their interactions with the host, facilitating virus movement.
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Subcellular distribution of mutant movement proteins of Cucumber mosaic virus fused to green fluorescent proteins
More LessThe subcellular distribution of the movement proteins (MPs) of nine alanine-scanning mutants of Cucumber mosaic virus (CMV), fused to the green fluorescent protein (GFP) and expressed from CMV, was determined by confocal microscopy of infected epidermal cells of Nicotiana tabacum and Nicotiana benthamiana, as well as infected N. benthamiana protoplasts. Only those mutant MPs that were functional for movement in all host species tested localized to plasmodesmata of infected epidermal cells and to tubules extending from the surface of infected protoplasts, as for wild-type CMV 3a MP. Various mutant MPs that were either conditionally functional for movement or dysfunctional for movement did not localize to plasmodesmata and did not form tubules on the surface of infected protoplasts. Rather, they showed distribution to different extents throughout the infected cells, including the cytoplasm, nucleus or the plasma membrane. The CMV 3a MP also did not associate with microtubles.
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- Other Agents
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Genotype-level variation in lifetime breeding success, litter size and survival of sheep in scrapie-affected flocks
Five different sheep flocks with natural outbreaks of scrapie were examined to determine associations between individual performance (lifetime breeding success, litter size and survival) and scrapie infection or PrP genotype. Despite different breed composition and forces of infection, consistent patterns were found among the flocks. Regardless of the flock, scrapie-infected sheep produced on average 34 % fewer offspring than non-scrapie-infected sheep. The effect of scrapie on lifetime breeding success appears to be a function of lifespan as opposed to fecundity. Analysis of litter size revealed no overall or genotype differences among the five sheep flocks. Survival, however, depends on the individual's scrapie status (infected or not) and its PrP genotype. Susceptible genotypes appear to perform less well in lifetime breeding success and life expectancy even if they are never affected with clinical scrapie. One possible explanation for these results is the effect of pre-clinical scrapie. Additional evidence supporting this hypothesis is discussed.
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Volumes and issues
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Volume 106 (2025)
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