- Volume 85, Issue 8, 2004
Volume 85, Issue 8, 2004
- Animal
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- RNA viruses
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VP1 of infectious bursal disease virus is an RNA-dependent RNA polymerase
Segment B of the bisegmented, double-stranded RNA genome of infectious bursal disease virus (IBDV) encodes the viral protein VP1. This has been presumed to represent the RNA-dependent RNA polymerase (RdRp) as it contains motifs that are typical for the RdRp of plus-strand RNA viruses. Here it is demonstrated that baculovirus-expressed wild-type but not motif A mutated VP1 acts as an RdRp on IBDV-specific RNA templates. Thus, on a plus-strand IBDV segment A cRNA template, minus-strand synthesis occurred in such a way that a covalently linked double-stranded RNA product was generated (by a ‘copy-back’ mechanism). Importantly, enzyme activity was observed only with templates that comprised the 3′ non-coding region of plus-strand RNAs transcribed from IBDV segments A and B, indicating template specificity. RdRp activity was shown to have a temperature optimum of 37 °C and required magnesium ions for enzyme activity. Thus, it has been demonstrated unequivocally that VP1 represents the RdRp of IBDV.
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Peptides resulting from the pVP2 C-terminal processing are present in infectious pancreatic necrosis virus particles
The capsid of birnaviruses contains two proteins, VP2 and VP3, which derive from the processing of a large polyprotein, NH2–pVP2–VP4–VP3–COOH. The proteolytic cascade involved in processing the polyprotein, and in the final maturation of pVP2 (the precursor of VP2), has recently been shown to generate VP2 and four structural peptides in infectious bursal disease virus and blotched snakehead virus. The presence of peptides in infectious pancreatic necrosis virus particles was investigated using mass spectrometry and N-terminal sequencing of virus particles. Three peptides deriving from the C terminus of pVP2 (residues 443–486, 487–495 and 496–508 of the polyprotein) and 14 additional peptides produced by further processing of peptides [443–486] and [496–508] were identified. These results indicate that the presence of several virus-encoded peptides in the virions is a hallmark of birnaviruses.
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Termination and read-through proteins encoded by genome segment 9 of Colorado tick fever virus
More LessGenome segment 9 (Seg-9) of Colorado tick fever virus (CTFV) is 1884 bp long and contains a large open reading frame (ORF; 1845 nt in length overall), although a single in-frame stop codon (at nt 1052–1054) reduces the ORF coding capacity by approximately 40 %. However, analyses of highly conserved RNA sequences in the vicinity of the stop codon indicate that it belongs to a class of ‘leaky terminators’. The third nucleotide positions in codons situated both before and after the stop codon, shows the highest variability, suggesting that both regions are translated during virus replication. This also suggests that the stop signal is functionally leaky, allowing read-through translation to occur. Indeed, both the truncated ‘termination’ protein and the full-length ‘read-through’ protein (VP9 and VP9′, respectively) were detected in CTFV-infected cells, in cells transfected with a plasmid expressing only Seg-9 protein products, and in the in vitro translation products from undenatured Seg-9 ssRNA. The ratios of full-length and truncated proteins generated suggest that read-through may be down-regulated by other viral proteins. Western blot analysis of infected cells and purified CTFV showed that VP9 is a structural component of the virion, while VP9′ is a non-structural protein.
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A comparison of the effects of oral inoculation with Rotashield and pentavalent reassortant rotavirus vaccine (WC3-PV) on suckling CB17scid mice
More LessThe effects of oral inoculation into infant CB17scid mice of two reassortant rotavirus vaccines were compared. The vaccines were Rotashield and WC3-PV, a mixture of five reassortants (G1, G2, G3, G4 and P1; pentavalent reassortant vaccine). Control mice were inoculated with a placebo. At 6 days post-inoculation (p.i.), 8 of 13 (62 %; P<0·005) Rotashield-inoculated mice developed hepatitis and/or bile-duct obstruction compared with none of 11 mice given WC3-PV and none of 14 given placebo. In the Rotashield-inoculated mice, only serotype G3 rhesus rotavirus (RRV) was isolated from multiple sites, including intestine, liver, pancreas, spleen, blood and mesenteric lymph nodes. Recovery of RRV from Rotashield-inoculated mice followed a biphasic pattern. The two peaks of RRV recovery appeared to coincide firstly with replication in the intestine during days 1–3 p.i., and secondly with virus infection of the liver from days 10 to 15 p.i. WC3 reassortants of four different serotypes were detected only at day 1 p.i. in the intestine, liver, pancreas and blood cells from three WC3-PV-inoculated mouse pups. However, WC3-PV did not produce any hepatopathology. Rotashield and WC3-PV appeared to exhibit different biological activity in infant CB17scid mouse pups.
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Potent virucidal activity in larval Heliothis virescens plasma against Helicoverpa zea single capsid nucleopolyhedrovirus
More LessLepidopteran larvae resist baculovirus infection by selective apoptosis of infected midgut epithelial cells and by sloughing off infected cells from the midgut. Once the infection breaches the midgut epithelial barrier and propagates from infective foci to the haemocoel, however, there are few mechanisms known to account for the resistance and clearance of infection observed in some virus–host combinations. The hypothesis that factors present in the plasma of infected pest larvae act to limit the spread of virus from initial infective foci within the haemocoel was tested. An in vitro bioassay was developed in which Helicoverpa zea single capsid nucleopolyhedrovirus (HzSNPV) was incubated with plasma collected from uninfected Heliothis virescens larvae. Infectious HzSNPV particles were then titrated on HzAM1 cells. Diluted plasma from larval Heliothis virescens exhibited a virucidal effect against HzSNPV in vitro, reducing the TCID50 ml−1 by more than 64-fold (from 4·3±3·6×105 to 6·7±0·6×103). The antiviral activity was heat-labile but was unaffected by freezing. In addition, protease inhibitors and specific chemical inhibitors of phenol oxidase or prophenol oxidase activation added to diluted plasma eliminated the virucidal activity. Thus, in the plasma of larval lepidopterans, the enzyme phenol oxidase may act as a constitutive, humoral innate antiviral immune response.
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Complete nucleotide sequence of Kashmir bee virus and comparison with acute bee paralysis virus
More LessThe complete nucleotide sequence of a novel virus is presented here together with serological evidence that it belongs to Kashmir bee virus (KBV). Analysis reveals that KBV is a cricket paralysis-like virus (family Dicistroviridae: genus Cripavirus), with a non-structural polyprotein open reading frame in the 5′ portion of the genome separated by an intergenic region from a structural polyprotein open reading frame in the 3′ part of the genome. The genome also has a polyadenylated tail at the 3′ terminus. KBV is one of several related viruses that also includes acute bee paralysis virus (ABPV). Although KBV and ABPV are about 70 % identical over the entire genome, there are considerable differences between them in significant areas of the genome, such as the 5′ non-translated region (42 % nucleotide identity), between the helicase and 3C-protease domains of the non-structural polyprotein (57 % amino acid identity) and in a 90 aa stretch of the structural polyprotein (33 % amino acid identity). Phylogenetic analyses show that KBV and ABPV isolates fall into clearly separated clades with moderate evolutionary distance between them. Whether these genomic and evolutionary differences are sufficient to classify KBV and ABPV as separate species remains to be determined.
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Sequence analysis of human rhinoviruses in the RNA-dependent RNA polymerase coding region reveals large within-species variation
More LessHuman rhinoviruses (HRVs; family Picornaviridae), the most frequent causative agents of respiratory infections, comprise more than 100 distinct serotypes. According to previous phylogenetic analysis of the VP4/VP2-coding sequences, all but one of the HRV prototype strains distribute between the two established species, Human rhinovirus A (HRV-A) and Human rhinovirus B (HRV-B). Here, partial sequences of the RNA-dependent RNA polymerase (3D polymerase)-coding gene of 48 HRV prototype strains and 12 field isolates were analysed. The designated division of the HRV strains into the species HRV-A and HRV-B was also seen in the 3D-coding region. Phylogenetically, HRV-B clustered closer to human enterovirus (HEV) species HEV-B, HEV-C and poliovirus than to HRV-A. Intraspecies variation within both HRV-A and HRV-B was greater in the 3D-coding region than in the VP4/VP2-coding region, with the difference maxima reaching 48 % at the nucleotide level and 36 % at the amino acid level in HRV-A and 53 and 35 %, respectively, in HRV-B. Within both species, a few strains formed a separate cluster differing from the majority of strains as much as HEV-B from HEV-C. Furthermore, the tree topology within HRV-A differed from that for VP4/VP2, suggesting possible recombination events in the evolutionary history of the strains. However, all 12 field isolates clustered similarly, as in the capsid region. These results showed that the within-species variation in the 3D region is greater in HRV than in HEV. Furthermore, HRV variation in the 3D region exceeds that in the capsid-coding region.
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All five cold-shock domains of unr (upstream of N- ras) are required for stimulation of human rhinovirus RNA translation
More LessEfficient translation of human rhinovirus-2 (HRV-2) RNA from its internal ribosome entry site (IRES) depends on the presence of cellular trans-acting factors upstream of N- ras (unr) and polypyrimidine-tract-binding protein. unr contains five cold-shock domains (CSDs) and is predicted to act as an RNA chaperone, allowing the HRV-2 IRES to attain the correct conformation for ribosome binding. To investigate the role of each of the CSDs in IRES-dependent translation, five unr mutants, each harbouring a point mutation in a different CSD, were generated. All five mutants were severely impaired in their ability to bind to the IRES and to stimulate translation from it. This showed that the ability of unr to function as an activator of HRV-2 RNA translation requires the RNA-binding activity of all five CSDs.
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Expansion of host-cell tropism of foot-and-mouth disease virus despite replication in a constant environment
Foot-and-mouth disease virus (FMDV) variants adapted to BHK-21 cells showed an expanded host-cell tropism that extended to primate and human cell lines. Virus replication in human HeLa and Jurkat cells has been documented by titration of virus infectivity, quantification of virus RNA, expression of a virus-specific non-structural antigen, and serial passage of virus in the cells. Parallel serial infections of human Jurkat cells with the same variant FMDVs indicates a strong stochastic component in the progression of infection. Chimeric viruses identified the capsid as a genomic region involved in tropism expansion. These results indicate that, contrary to theoretical predictions, replication of an RNA virus in a constant cellular environment may lead to expansion of cellular tropism, rather than to a more specialized infection of the cellular type to which the virus has been adapted.
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Functional properties of a 16 kDa protein translated from an alternative open reading frame of the core-encoding genomic region of hepatitis C virus
More LessHepatitis C virus (HCV) often causes persistent infection in humans. This could be due in part to the effect of viral proteins on cellular gene expression. Earlier observations suggest that the HCV core protein expressed from genotype 1a modulates important cellular genes at the transcriptional level, affects programmed cell death (apoptosis) and promotes cell growth. Recently, different groups of investigators have reported the translation of an ∼16 kDa protein (named F/ARFP/core+1 ORF) from an alternate open reading frame of the HCV core-encoding genomic region. The functional significance of this F protein is presently unknown. Thus, whether the F and core proteins have both shared and distinct functions was investigated here. The experimental observations suggested that the F protein does not significantly modulate c-myc, hTERT and p53 promoter activities, unlike the HCV core protein. Interestingly, the F protein repressed p21 expression. Further studies indicated that the F protein does not inhibit tumour necrosis factor alpha-mediated apoptosis of HepG2 cells or promote rat embryo fibroblast growth. Taken together, these results suggest that the F protein does not share major properties identified previously for the HCV core protein, other than regulating p21 expression.
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Identification of the homotypic interaction domain of the core protein of dengue virus type 2
More LessDengue virus causes dengue haemorrhagic fever or dengue shock syndrome with a high mortality rate. The genome of dengue virus is a positive-sense, single-stranded RNA encoding three structural and seven non-structural proteins. The core protein is one of the three structural proteins and is the building block of the nucleocapsid of dengue virus. The core protein of dengue virus type 2 (DEN2) is composed of 100 aa with four α-helix domains. An internal hydrophobic domain located at aa 44–60 was identified. The DEN2 core protein was shown to form homodimers. Deletion of aa 1–36 or 73–100 decreased but did not completely abolish the core-to-core homotypic interaction, whereas deletion of a portion (aa 44–60) within aa 37–72 completely abolished the ability of the DEN2 core proteins to interact with each other. A recombinant DEN2 core protein corresponding to aa 37–72 was able to undergo homotypic interaction and bound to a native DEN2 core protein. The results of this study indicated that the homotypic interaction domain of the DEN2 core protein is located at aa 37–72 and that the internal hydrophobic domain located at aa 44–60 plays a pivotal role in core-to-core homotypic interaction.
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Mx1 GTPase accumulates in distinct nuclear domains and inhibits influenza A virus in cells that lack promyelocytic leukaemia protein nuclear bodies
More LessThe interferon-induced murine Mx1 GTPase is a nuclear protein. It specifically inhibits influenza A viruses at the step of primary transcription, a process known to occur in the nucleus of infected cells. However, the exact mechanism of inhibition is still poorly understood. The Mx1 GTPase has previously been shown to accumulate in distinct nuclear dots that are spatially associated with promyelocytic leukaemia protein (PML) nuclear bodies (NBs), but the significance of this association is not known. Here it is reported that, in cells lacking PML and, as a consequence, PML NBs, Mx1 still formed nuclear dots. These dots were indistinguishable from the dots observed in wild-type cells, indicating that intact PML NBs are not required for Mx1 dot formation. Furthermore, Mx1 retained its antiviral activity against influenza A virus in these PML-deficient cells, which were fully permissive for influenza A virus. Nuclear Mx proteins from other species showed a similar subnuclear distribution. This was also the case for the human MxA GTPase when this otherwise cytoplasmic protein was translocated into the nucleus by virtue of a foreign nuclear localization signal. Human MxA and mouse Mx1 do not interact or form heterooligomers. Yet, they co-localized to a large degree when co-expressed in the nucleus. Taken together, these findings suggest that Mx1 dots represent distinct nuclear domains (‘Mx nuclear domains’) that are frequently associated with, but functionally independent of, PML NBs.
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Influenza A viruses in feral Canadian ducks: extensive reassortment in nature
The current dogma of influenza accepts that feral aquatic birds are the reservoir for influenza A viruses. Although the genomic information of human influenza A viruses is increasing, little of this type of data is available for viruses circulating in feral waterfowl. This study presents the genetic characterization of 35 viruses isolated from wild Canadian ducks from 1983 to 2000, as the first attempt at a comprehensive genotypic analysis of influenza viruses isolated from feral ducks. This study demonstrates that influenza virus genes circulating in Canadian ducks have achieved evolutionary stasis. The majority of these duck virus genes are clustered in distinct North American clades; however, some H6 and H9 genes are clustered with those from Eurasian viruses. Genes appeared to reassort in a random fashion. None of the genotypes identified remained present throughout all of the years examined and most PA and PB2 genes that crossed over into swine were clustered in one phylogenetic grouping. Additionally, matrix genes were identified that branch very early in the evolutionary tree. These findings demonstrate the diversity of the influenza virus gene pool in Canadian ducks, and suggest that genes which cluster in specific phylogenetic groupings in the PB2 and PA genes can be used for markers of viruses with the potential for crossing the species barrier. A more comprehensive study of this important reservoir is needed to provide further insight into the genomic composition of viruses that crossover the species barrier, which would be a useful component to pandemic planning.
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Total viral genome copies and virus–Ig complexes after infection with influenza virus in the nasal secretions of immunized mice
The kinetics of infectious virus (p.f.u.), total virus and virus–Ig complex formation following influenza A/PR8 (H1N1) viral infection was examined in the nasal secretions of naive mice and mice immunized with A/PR8, A/Yamagata (H1N1), A/Guizhou (H3N2) and B/Ibaraki influenza viruses. The total number of virus particles and the number within virus–Ig complexes, captured in advance using an anti-mouse Ig-coated plate, were determined on the basis of viral genome copy number using quantitative RT-PCR. The kinetics of infectious and total virus particle formation, the latter of which increased by 103–104-fold above infectious virus numbers, showed that virus elimination from the nasal area was earlier in A/PR8, A/Yamagata and A/Guizhou-X virus-immunized mice, in decreasing order, compared with naive mice. Early virus elimination correlated with the level of A/PR8 virus-reactive antibodies in immunized mice. Virus elimination coincided with the appearance of virus–Ig complexes shortly after infection. This result suggested that antibodies led to the formation of immune complexes in a dose-dependent manner together with a reduction in number of infectious virus particles. The fact that a large number of virus particles was observed in immune complexes for a wide range antibody levels made it difficult to detect slight differences in virus number within the immune complexes, depending on antibody level. These results suggested that the formation of virus–Ig complexes in virus-immunized mice shortly after infection is involved in early virus elimination, which is determined by the strength of protective immunity against challenge viruses.
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Active NF-κB signalling is a prerequisite for influenza virus infection
Influenza virus still poses a major threat to human health. Despite widespread vaccination programmes and the development of drugs targeting essential viral proteins, the extremely high mutation rate of influenza virus still leads to the emergence of new pathogenic virus strains. Therefore, it has been suggested that cellular cofactors that are essential for influenza virus infection might be better targets for antiviral therapy. It has previously been reported that influenza virus efficiently infects Epstein–Barr virus-immortalized B cells, whereas Burkitt's lymphoma cells are virtually resistant to infection. Using this cellular system, it has been shown here that an active NF-κB signalling pathway is a general prerequisite for influenza virus infection of human cells. Cells with low NF-κB activity were resistant to influenza virus infection, but became susceptible upon activation of NF-κB. In addition, blocking of NF-κB activation severely impaired influenza virus infection of otherwise highly susceptible cells, including the human lung carcinoma cell lines A549 and U1752 and primary human cells. On the other hand, infection with vaccinia virus was not dependent on an active NF-κB signalling pathway, demonstrating the specificity of this pathway for influenza virus infection. These results might be of major importance for both the development of new antiviral therapies and the understanding of influenza virus biology.
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Cytokine and contact-dependent activation of natural killer cells by influenza A or Sendai virus-infected macrophages
NK cells participate in innate immune responses by secreting gamma interferon (IFN-γ) and by destroying virus-infected cells. Here the interaction between influenza A or Sendai virus-infected macrophages and NK cells has been studied. A rapid, cell–cell contact-dependent production of IFN-γ from NK cells cultured with virus-infected macrophages was observed. Expression of the MHC class I-related chain B (MICB) gene, a ligand for NK cell-activating receptor NKG2D, was upregulated in virus-infected macrophages suggesting a role for MICB in the activation of the IFN-γ gene in NK cells. IL12Rβ2, IL18R and T-bet mRNA synthesis was enhanced in NK cells cultured with virus-infected macrophages. Upregulation of these genes was dependent on macrophage-derived IFN-α. In contrast to IL12Rβ2, expression of WSX-1/TCCR, a receptor for IL27, was reduced in NK cells in response to virus-induced IFN-α. In conclusion, these results show that virus-infected macrophages activate NK cells via cytokines and direct cellular interactions and further emphasize the role of IFN-α in the activation of innate immunity.
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Human CD8+ T cell responses against five newly identified respiratory syncytial virus-derived epitopes
CD8+ T lymphocytes play a major role in the clearance of respiratory syncytial virus (RSV) infections. To be able to study the primary CTL response in RSV-infected children, epitopes presented by a set of commonly used HLA alleles (HLA-A1, -A3, -B44 and -B51) were searched for. Five epitopes were characterized derived from the matrix (M), non-structural (NS2) and second matrix (M2) proteins of RSV. All epitopes were shown to be processed and presented by RSV-infected antigen-presenting cells. HLA-A1 tetramers for one of these epitopes derived from the M protein were constructed and used to quantify and phenotype the memory CD8+ T cell pool in a panel of healthy adult donors. In about 60 % of the donors, CD8+ T cells specific for the M protein could be identified. These cells belonged to the memory T cell subset characterized by expression of CD27 and CD28, and down-regulation of CCR7 and CD45RA. The frequency of tetramer-positive cells varied between 0·4 and 3 per 104 CD8+ T cells in PBMC of healthy asymptomatic adult donors.
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The P gene of Newcastle disease virus does not encode an accessory X protein
More LessMany paramyxoviruses encode non-essential accessory proteins that are involved in the regulation of virus replication and inhibition of cellular antiviral responses. It has been suggested that the P gene mRNA of Newcastle disease virus (NDV) encodes an accessory protein – the so-called X protein – by translation initiation at a conserved in-frame AUG codon at position 120. Using a monoclonal antibody that specifically detected the P and X proteins, it was shown that an accessory X protein was not expressed in NDV-infected cells. Recombinant NDV strains in which the AUG was changed into a GCC (Ala) or GUC (Val) codon were viable but showed a reduction in virulence, probably because the amino acid change affected the function of the P and/or V protein.
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Immunization with dendritic cells can break immunological ignorance toward a persisting virus in the central nervous system and induce partial protection against intracerebral viral challenge
More LessDendritic cells (DCs) have been used successfully to induce CD8 T cells that control virus infections and growth of tumours. The efficacy of DC-mediated immunization for the control of neurotropic Borna disease virus (BDV) in mice was evaluated. Certain strains of mice only rarely develop spontaneous neurological disease, despite massive BDV replication in the brain. Resistance to disease is due to immunological ignorance toward BDV antigen in the central nervous system. Ignorance in mice can be broken by immunization with DCs coated with TELEISSI, a peptide derived from the N protein of BDV, which represents the immunodominant cytotoxic T lymphocyte epitope in H-2k mice. Immunization with TELEISSI-coated DCs further induced solid protective immunity against intravenous challenge with a recombinant vaccinia virus expressing BDV-N. Interestingly, however, this immunization scheme induced only moderate protection against intracerebral challenge with BDV, suggesting that immune memory raised against a shared antigen may be sufficient to control a peripherally replicating virus, but not a highly neurotropic virus that is able to avoid activation of T cells. This difference might be due to the lack of BDV-specific CD4 T cells and/or inefficient reactivation of DC-primed, BDV-specific CD8 T cells by the locally restricted BDV infection. Thus, a successful vaccine against persistent viruses with strong neurotropism should probably induce antiviral CD8 (as well as CD4) T-cell responses and should favour the accumulation of virus-specific memory T cells in cervical lymph nodes.
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Host RNA polymerase II makes minimal contributions to retroviral frame-shift mutations
More LessThe rate of mutation during retrovirus replication is high. Mutations can occur during transcription of the viral genomic RNA from the integrated provirus or during reverse transcription from viral RNA to form viral DNA or during replication of the proviral DNA as the host cell is dividing. Therefore, three polymerases may all contribute to retroviral evolution: host RNA polymerase II, viral reverse transcriptases and host DNA polymerases, respectively. Since the rate of mutation for host DNA polymerase is very low, mutations are more likely to be caused by the host RNA polymerase II and/or the viral reverse transcriptase. A system was established to detect the frequency of frame-shift mutations caused by cellular RNA polymerase II, as well as the rate of retroviral mutation during a single cycle of replication in vivo. In this study, it was determined that RNA polymerase II contributes less than 3 % to frame-shift mutations that occur during retrovirus replication. Therefore, the majority of frame-shift mutations detected within the viral genome are the result of errors during reverse transcription.
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Human T-cell leukaemia virus type I is highly sensitive to UV-C light
The biological characteristics of human T-cell leukaemia virus type I (HTLV-I) are not yet well understood. UV light C (UV-C) sensitivity of HTLV-I was studied using a newly established infectivity assay: infection with cell-free HTLV-I dose-dependently induced syncytial plaques in cat cells transduced with the tax1 gene of HTLV-I. HTLV-I was inactivated by a much lower UV dose than bovine leukaemia virus (BLV). The D10 (10 % survival dose) of HTLV-I was about 20 J m−2, while that of BLV was about 180 J m−2, which was similar to the reported D10 of BLV. The UV sensitivity of HTLV-I and BLV was also examined by detecting viral DNA synthesis 24 h after infection. The D10 values determined by PCR using the gag primers for HTLV-I and BLV were close to those determined by the infectivity assays. Further PCR analyses were then performed to determine D10 values using several different primers located between the 5′-long terminal repeat (5′-LTR) and the tax1 gene. The difference in UV sensitivity between HTLV-I and BLV was detected very early during replication, even during reverse transcription of the 5′-LTR of irradiated viruses, and became more prominent as reverse transcription proceeded towards the tax1 gene. Chimeric mouse retroviruses that contain the LTR-tax1 fragments of HTLV-I and BLV were made and hardly any difference in UV sensitivity was detected between them, suggesting that the difference was not determined by the linear RNA sequences of HTLV-I and BLV. HTLV-I was found to be much more sensitive than other retroviruses to UV.
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Enhanced cellular immunity and systemic control of SHIV infection by combined parenteral and mucosal administration of a DNA prime MVA boost vaccine regimen
The immunogenicity and protective efficacy of a DNA and recombinant modified vaccinia Ankara (MVA) vaccine administered by two different routes were investigated. DNA expressing HIV-1 IIIB env, gag, RT, rev, tat and nef, and MVA expressing HIV-1 IIIB nef, tat and rev and simian immunodeficiency virus (SIV) macJ5 gag/pol and vaccinia HIV-1 env, were used as immunogens. Four cynomolgus macaques received DNA intramuscularly (i.m.) at month 0 and intrarectally (i.r.) and intra-orally (i.o.) at 2 months, followed by MVA i.m. at 4 months and i.r. and i.o. at 8 months. Another group of four monkeys received the same immunogens but only i.m.. Overall, stronger cellular immune responses measured by ELISPOT and T-cell proliferation assay were detected in the group primed i.m. and boosted mucosally. Following homologous intravenous simian-human immunodeficiency virus (SHIV) challenge, one of eight vaccinated animals was completely protected. This monkey, immunized i.m. and i.r.+i.o., exhibited the highest levels of HIV Env, Nef and Tat antibodies, high HIV Tat cytotoxic T-lymphocyte activity and T-lymphocyte proliferative responses to HIV Env. Four weeks post-challenge none of the monkeys immunized i.m. and i.r.+i.o., and only two out of four animals immunized i.m., demonstrated detectable plasma viral RNA levels. In contrast, all eight control animals had demonstrable plasma viral RNA levels 4 weeks post-challenge. Thus, stronger cellular immune responses and reduction of challenge virus burden were demonstrated in animals immunized i.m. as well as mucosally, compared with animals immunized i.m. only. The breadth and magnitude of the induced immune responses correlated with protective efficacy.
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Characterization of germline porcine endogenous retroviruses from Large White pig
Porcine endogenous retroviruses (PERV) are of concern when the microbiological safety aspects of xenotransplantation are considered. Four unique isolates of PERV B have been identified previously from a lambda library constructed from genomic DNA from a Large White pig. This study shows that none of these isolates are replication competent when transfected into permissive human or pig cells in vitro, and the removal of flanking genomic sequences does not confer a human tropic replication competent (HTRC) phenotype on these PERV proviruses. Analysis of the envelope sequences revealed that PERV B demonstrated high similarity to the envelope sequences derived from replication-competent PERV, indicating that lack of replication competence does not appear to be attributable to this region of the provirus. These data complement recent findings that HTRC PERV are recombinants between the PERV A and PERV C subgroups, and that these recombinants are not present in the germline of miniature swine. Together, these results indicate that these individual PERV B proviruses are unlikely to give rise to HTRC PERV.
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- DNA viruses
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Negative regulation of herpes simplex virus type 1 ICP4 promoter by IE180 protein of pseudorabies virus
More LessRecombinant pseudorabies viruses (PRVs) gIS8 and N1aHTK were constructed by the insertion of a chimeric gene (α4–TK) from herpes simplex virus type 1 (HSV-1) into wild-type PRV. HSV-1 TK expression by these recombinant viruses resulted in enhanced sensitivity to ganciclovir, compared to that of the wild-type PRV, and was similar to the sensitivity shown by HSV-1. Infection with gIS8 or N1aHTK recombinant viruses led to expression of HSV-1 TK mRNA as an immediate–early (IE) gene, observed by downregulation of the HSV-1 α4 promoter. This negative regulation was due to a PRV IE protein, IE180. IE180, however, does not have all the regulatory functions of the infected-cell protein ICP4, as it does not restore the growth of ICP4-deficient HSV-1 mutants.
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A synthetic peptide from a heptad repeat region of herpesvirus glycoprotein B inhibits virus replication
More LessGlycoprotein B (gB) is the most conserved glycoprotein of herpesviruses and plays important roles in virus infectivity. Two intervening heptad repeat (HR) sequences were found in the C-terminal half of all herpesvirus gBs analysed. A synthetic peptide derived from the HR region (aa 477–510) of bovine herpesvirus type 1 (BoHV-1) gB was studied for its ability to inhibit virus replication. The peptide interfered with cell-to-cell spread and consistently inhibited replication of BoHV-1, with a 50 % effective concentration value (EC50) of 5 μM. Inhibition of replication was obtained not only with herpesviruses including pseudorabies virus and herpes simplex virus type 1 but also partly with Newcastle disease virus. Possible mechanisms of membrane fusion inhibition by the peptide are discussed.
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CD4+ T-cell responses to herpes simplex virus type 2 (HSV-2) glycoprotein G are type specific and differ in symptomatic and asymptomatic HSV-2-infected individuals
T-cell recognition of the secreted and membrane-bound portions of the herpes simplex virus type 2 (HSV-2) glycoprotein G (sgG-2 and mgG-2, respectively) was compared in symptomatic and asymptomatic HSV-2-infected individuals and in HSV-2-seronegative controls and the responses with HSV-1 glycoproteins C and E (gC-1 and gE-1) were compared. CD4+ T cells from HSV-2-infected individuals specifically recognized both sgG-2 and mgG-2, whereas HSV-1-infected and HSV-seronegative controls did not respond to these glycoproteins. The responses to gC-1 and gE-1, on the other hand, were not type specific, as blood mononuclear cells from both HSV-1- and HSV-2-infected individuals responded in vitro. There was an association between the status of the infection (symptomatic versus asymptomatic) and the CD4+ T-cell responsiveness. Symptomatic HSV-2-seropositive individuals responded with significantly lower Th1 cytokine production to sgG-2 and mgG-2 than did asymptomatic HSV-2-infected carriers, especially within the HSV-1-negative cohort. No differences in T-cell proliferation were observed between asymptomatic and symptomatic individuals. The results have implications for studies of HSV-2-specific CD4+ T-cell reactivity in general and for analysis of immunological differences between asymptomatic and symptomatic individuals in particular.
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Sumoylation of the major immediate-early IE2 protein of human cytomegalovirus Towne strain is not required for virus growth in cultured human fibroblasts
More LessSumoylation of the major immediate-early IE2 protein of human cytomegalovirus has been shown to increase transactivation activity in target reporter gene assays. This study examined the role of IE2 sumoylation in viral infection. A Towne strain-based bacterial artificial chromosome clone was generated encoding a mutated form of the IE2 protein with Lys→Arg substitutions at positions 175 and 180, the two major sumoylation sites. When human fibroblast (HF) cells were infected with the reconstituted mutant virus, (i) viral growth kinetics, (ii) the accumulation of IE1 (UL123), IE2 (UL122), p52 (UL44) and pp65 (UL83) proteins and (iii) the relocalization of the cellular small ubiquitin-like modifier (SUMO)-1, p53 and proliferating cell nuclear antigen proteins into viral DNA replication compartments were comparable with those of the wild-type and the revertant virus. The data demonstrate that sumoylation of IE2 is not essential for virus growth in cultured HF cells.
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Functional co-operation between the Kaposi's sarcoma-associated herpesvirus ORF57 and ORF50 regulatory proteins
More LessKaposi's sarcoma (KS)-associated herpesvirus (KSHV) proteins ORF57 (also known as MTA) and ORF50 (also known as RTA) act post-transcriptionally and transcriptionally to regulate viral lytic gene expression and synergistically activate certain early and late KSHV promoters. When ORF57 and ORF50 were co-expressed, they co-operatively stimulated expression from the promoter of the immediate-early ORF50 gene itself. Co-immunoprecipitations with extracts of KSHV-infected cells showed that ORF57 and ORF50 proteins were present in the same complex. Using the pull-down assay with extracts of KSHV-infected cells, ORF50 protein was shown to interact with a glutathione S-transferase–ORF57 fusion protein. A chromatin immunoprecipitation assay showed that ORF50 promoter sequences were preferentially associated with immunoprecipitated chromatin using both anti-ORF50 and anti-ORF57 antibodies consistent with both an in vivo physical association between ORF57 and ORF50 and a potential role for ORF57 at the transcriptional level. This is the first demonstration of an interaction between these two lytic regulatory proteins in a gammaherpesvirus. Expression of ORF50 protein is sufficient to induce lytic replication in latently infected cells and may determine viral host range, spread and KS pathogenesis in vivo. A new insight into the co-ordinated activities of these two key regulatory proteins is provided in which upregulation of the ORF50 promoter with augmentation of ORF50 activity by ORF57 protein, and vice versa, would facilitate the cascade of lytic viral gene expression, thereby breaking latency. A functional and physical interaction between these two gammaherpesvirus regulatory protein counterparts could be a general feature of the herpesviruses.
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Modified vaccinia virus Ankara induces moderate activation of human dendritic cells
More LessModified vaccinia virus Ankara (MVA) is a highly attenuated strain known to be an effective vaccine vector. Here it is demonstrated that MVA, unlike standard vaccinia virus (VACV) strains, activates monocyte-derived human dendritic cells (DCs) as testified by an increase in surface co-stimulatory molecules and the secretion of pro-inflammatory cytokines. Inhibition of virus gene expression by subjecting MVA to UV light or heat treatment did not alter its ability to activate DCs. On the other hand, standard VACV strains activated DCs if virus gene expression was prevented by prior UV light or heat treatment. These results suggest that MVA or standard VACV particles are responsible for DC activation but, in the case of standard VACV strains, virus gene expression prevents activation. Additional experiments showed that DCs were activated by MVA-infected HeLa cells and, under these conditions, could induce secretion of gamma interferon from T lymphocytes more efficiently than if a replication-competent VACV strain was employed. These data provide one explanation for the remarkable immune-stimulating capacity of MVA in the absence of virus multiplication.
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Characterization of pathogenic and non-pathogenic African swine fever virus isolates from Ornithodoros erraticus inhabiting pig premises in Portugal
More LessTen African swine fever virus isolates from the soft tick Ornithodoros erraticus collected on three farms in the province of Alentejo in Portugal were characterized by their ability to cause haemadsorption (HAD) of red blood cells to infected pig macrophages, using restriction enzyme site mapping of the virus genomes and by experimental infection of pigs. Six virus isolates induced haemadsorption and four were non-haemadsorbing (non-HAD) in pig macrophage cell cultures. The restriction enzyme site maps of two non-HAD viruses, when compared with a virulent HAD isolate, showed a deletion of 9·6 kbp in the fragment adjacent to the left terminal fragment and of 1·6 kbp in the right terminal fragment and an insertion of 0·2 kbp in the central region. The six HAD viruses isolated were pathogenic and produced typical acute African swine fever in pigs and the four non-HAD isolates were non-pathogenic. Pigs that were infected with non-HAD viruses were fully resistant or had a delay of up to 14 days in the onset of disease, after challenge with pathogenic Portuguese viruses. Non-HAD viruses could be transmitted by contact but with a lower efficiency (42–50 %) compared with HAD viruses (100 %). The clinical differences found between the virus isolates from the ticks could have implications for the long-term persistence of virus in the field because of the cross-protection produced by the non-pathogenic isolates. This may also explain the presence of seropositive pigs in herds in Alentejo where no clinical disease had been reported.
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Human papillomavirus genotypes in cervical cancers in Mozambique
More LessThe distribution of human papillomavirus (HPV) types in cervical cancers is essential for design and evaluation of HPV type-specific vaccines. To follow up on a previous report that HPV types 35 and 58 were the dominant HPV types in cervical neoplasia in Mozambique, the HPV types in a consecutive case series of 74 invasive cervical cancers in Mozambique were determined. The most common worldwide major oncogenic HPV types 16 and 18 were present in 69 % of cervical cancers, suggesting that a vaccine targeting HPV-16 and -18 would have a substantial impact on cervical cancer also in Mozambique.
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Broad-spectrum detection of papillomaviruses in bovine teat papillomas and healthy teat skin
To investigate the prevalence of bovine papillomavirus (BPV) in bovine papilloma and healthy skin, DNA extracted from teat papillomas and healthy teat skin swabs was analysed by PCR using the primer pairs FAP59/FAP64 and MY09/MY11. Papillomavirus (PV) DNA was detected in all 15 papilloma specimens using FAP59/FAP64 and in 8 of the 15 papilloma specimens using MY09/MY11. In swab samples, 21 and 8 of the 122 samples were PV DNA positive using FAP59/FAP64 and MY09/MY11, respectively. Four BPV types (BPV-1, -3, -5 and -6), two previously identified putative BPV types (BAA1 and -5) and 11 putative new PV types (designated BAPV1 to -10 and BAPV11MY) were found in the 39 PV DNA-positive samples. Amino acid sequence alignments of the putative new PV types with reported BPVs and phylogenetic analyses of the putative new PV types with human and animal PV types showed that BAPV1 to -10 and BAPV11MY are putative new BPV types. These results also showed the genomic diversity and extent of subclinical infection of BPV.
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Attachment of bovine parvovirus to sialic acids on bovine cell membranes
More LessAlthough it has previously been shown that bovine parvovirus (BPV) attaches to the sialated glycoprotein glycophorin A on erythrocytes, the nature of virus-binding moieties on mammalian nucleated cells is less clear. Buffalo lung fibroblasts (Bu), primary bovine embryonic kidney cells, Madin–Darby bovine kidney cells and bovine embryonic trachea (EBTr) cells were assessed for molecules capable of binding BPV. Competition studies were carried out on both erythrocyte and nucleated cell targets using a variety of sialated compounds and sialic acid-negative compounds. Glycophorin A was found to inhibit BPV binding, while mucin exhibited low-level inhibition. These two sialated compounds also blocked attachment of BPV-modified microsphere carriers to the Bu cell membrane. Influenza A virus was used as a sialic acid competitor and interfered with BPV attachment to erythrocytes and replication in Bu cells. Significantly, the enzyme sialidase removed BPV-binding sites from Bu and EBTr cells. The binding sites could be reconstituted on sialidase-treated cells by the enzymes α-2,3-O-sialyltransferase and α-2,3-N-sialyltransferase. These results indicated that BPV can attach to sialic acid on cell membranes and that the sialylglycoproteins available for virus attachment appear to contain both N- and O-linked carbohydrate moieties, but that not all members of the sialic acid family can bind BPV. Moreover, there may be other moieties that can bind BPV, which may act as either primary or secondary receptors.
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The adenovirus E1A and E1B19K genes provide a helper function for transfection-based adeno-associated virus vector production
More LessAlthough the adenoviral E1, E2A, E4 and VA RNA regions are required for efficient adeno-associated virus (AAV) vector production, the role that the individual E1 genes (E1A, E1B19K, E1B55K and protein IX) play in AAV vector production has not been clearly determined. E1 mutants were analysed for their ability to mediate AAV vector production in HeLa or KB cells, when cotransfected with plasmids encoding all other packaging functions. Disruption of E1A and E1B19K genes resulted in vector yield reduction by up to 10- and 100-fold, respectively, relative to the wild-type E1. Interruption of the E1B55K and protein IX genes had a modest effect on vector production. Interestingly, expression of anti-apoptotic E1B19K cellular homologues such as Bcl-2 or Bcl-xL fully complemented E1B19K mutants for AAV vector production. These findings may be valuable for the future development of packaging cell lines for AAV vector production.
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Phylogenetic evidence of widespread distribution of genotype 3 JC virus in Africa and identification of a type 7 isolate in an African AIDS patient
More LessJC virus (JCV) is the cause of progressive multifocal leukoencephalophathy (PML) in immunocompromised patients. The paucity of reports from Africa has led to the hypothesis that PML is rare because of an absence of virus genotypes associated with the condition. Genotypes 3 and 6 have been identified in East and West Africa but the distribution of types across the rest of Africa is unknown. Full-length sequences of five JCV cerebrospinal fluid samples from PML patients in South Africa are reported here. Three isolates from African AIDS patients grouped with type 3A or 3B, and one with type 7, while one from a Caucasian leukaemia patient grouped with type 2D. Widespread distribution of type 3 on the continent may reflect migration patterns in antiquity, but this is the first report of type 7 in an African individual. Type 2D has only been isolated previously in South Asia, although transmission of this genotype to Europeans who later settled in South Africa is not unlikely.
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- Plant Viruses
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The p36 and p95 replicase proteins of Carnation Italian ringspot virus cooperate in stabilizing defective interfering RNA
More LessThe p36 and p95 proteins of Carnation Italian ringspot virus (CIRV), when expressed in Saccharomyces cerevisiae, supported the replication of defective interfering (DI) RNA. Double-label confocal immunofluorescence showed that both proteins localized to mitochondria, independently of each other. DI RNA progeny was localized by in situ hybridization both to mitochondria and to their proximity. Fractionation of cell extracts showed that replicase proteins associated with membranes with a consistent portion of DI RNA. DI RNA transcripts were stabilized more efficiently when co-expressed with both p36 and p95 than with either protein alone. By using the copper-inducible CUP1 promoter, p36 was shown to have an effect on DI RNA stability only above a threshold concentration, suggesting an ‘all-or-none’ behaviour. Conversely, the stabilizing activity of p95 was proportional to protein concentration in the range examined. Similarly, DI RNA replication level was proportional to p95 concentration and depended on a threshold concentration of p36.
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Population structure and genetic variability within isolates of Grapevine fanleaf virus from a naturally infected vineyard in France: evidence for mixed infection and recombination
More LessThe nematode-borne Grapevine fanleaf virus, from the genus Nepovirus in the family Comoviridae, causes severe degeneration of grapevines in most vineyards worldwide. We characterized 347 isolates from transgenic and conventional grapevines from two vineyard sites in the Champagne region of France for their molecular variant composition. The population structure and genetic diversity were examined in the coat protein gene by IC-RT-PCR-RFLP analysis with EcoRI and StyI, and nucleotide sequencing, respectively. RFLP data suggested that 55 % (191 of 347) of the isolates had a population structure consisting of one predominant variant. Sequencing data of 51 isolates representing the different restrictotypes confirmed the existence of mixed infection with a frequency of 33 % (17 of 51) and showed two major predominant haplotypes representing 71 % (60 of 85) of the sequence variants. Comparative nucleotide diversity among population subsets implied a lack of genetic differentiation according to host (transgenic vs conventional) or field site for most restrictotypes (17 of 18 and 13 of 18) and for haplotypes in most phylogenetic groups (seven of eight and six of eight), respectively. Interestingly, five of the 85 haplotypes sequenced had an intermediate divergence (0·036–0·066) between the lower (0·005–0·028) and upper range (0·083–0·138) of nucleotide variability, suggesting the occurrence of homologous RNA recombination. Sequence alignments clearly indicated a mosaic structure for four of these five variants, for which recombination sites were identified and parental lineages proposed. This is the first in-depth characterization of the population structure and genetic diversity in a nepovirus.
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Mutation of Phe50 to Ser50 in the 126/183-kDa proteins of Odontoglossum ringspot virus abolishes virus replication but can be complemented and restored by exact reversion
More LessSequence comparison of a non-biologically active full-length cDNA clone of Odontoglossum ringspot virus (ORSV) pOT1 with a biologically active ORSV cDNA clone pOT2 revealed a single nucleotide change of T→C at position 211. This resulted in the change of Phe50 in OT2 to Ser50 in OT1. It was not the nucleotide but the amino acid change of Phe50 that was responsible for the inability of OT1 to replicate. Time-course experiments showed that no minus-strand RNA synthesis was detected in mutants with a Phe50 substitution. Corresponding mutants in Tobacco mosaic virus (TMV) showed identical results, suggesting that Phe50 may play an important role in replication in all tobamoviruses. Complementation of a full-length mutant OT1 was demonstrated in a co-infected local-lesion host, a systemic host and protoplasts by replication-competent mutants tORSV.GFP or tORSV.GFPm, and further confirmed by co-inoculation using tOT1.GFP+tORSV (TTC), suggesting that ORSV contains no RNA sequence inhibitory to replication in trans. Surprisingly, a small number of exact revertants were detected in plants inoculated with tOT1+tORSV.GFPm or tOT1.GFP+tORSV (TTC). No recombination was detected after screening of silent markers in virus progeny extracted from total RNA or viral RNA from inoculated and upper non-inoculated leaves as well as from transfected protoplasts. Exact reversion from TCT (OT1) to TTT (OT2), rather than recombination, restored its replication function in co-inoculated leaves of Nicotiana benthamiana.
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Nucleo-cytoplasmic shuttling of the beet necrotic yellow vein virus RNA-3-encoded p25 protein
The protein p25 encoded by beet necrotic yellow vein virus (BNYVV) RNA-3 is involved in symptom expression of infected plants. Confocal microscopy analysis of wild-type and mutated p25 fused to GFP and transiently expressed in BY-2 tobacco suspension cells identified a nuclear localization signal (NLS) in the N-terminal part of the protein. Functionality of the NLS was confirmed by pull-down assays using rice and pepper importin-α. Furthermore, it was demonstrated that p25 contains a nuclear export sequence sensitive to leptomycin B. The nuclear export signal (NES) was characterized by mutagenesis. A GFP–p25 fusion protein expressed during a BNYVV infection of Chenopodium quinoa leaves had the same subcellular localization as observed during transient expression in BY-2 cells. The symptom phenotype induced by expression of GFP–p25 during infection was similar to that induced by wild-type virus. Studies with mutated derivatives of GFP–p25 revealed that symptom phenotype was altered when the subcellular localization of GFP–p25 was modified.
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- Other Agents
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Characterization of two distinct prion strains derived from bovine spongiform encephalopathy transmissions to inbred mice
Distinct prion strains can be distinguished by differences in incubation period, neuropathology and biochemical properties of disease-associated prion protein (PrPSc) in inoculated mice. Reliable comparisons of mouse prion strain properties can only be achieved after passage in genetically identical mice, as host prion protein sequence and genetic background are known to modulate prion disease phenotypes. While multiple prion strains have been identified in sheep scrapie and Creutzfeldt–Jakob disease, bovine spongiform encephalopathy (BSE) is thought to be caused by a single prion strain. Primary passage of BSE prions to different lines of inbred mice resulted in the propagation of two distinct PrPSc types, suggesting that two prion strains may have been isolated. To investigate this further, these isolates were subpassaged in a single line of inbred mice (SJL) and it was confirmed that two distinct prion strains had been identified. MRC1 was characterized by a short incubation time (110±3 days), a mono-glycosylated-dominant PrPSc type and a generalized diffuse pattern of PrP-immunoreactive deposits, while MRC2 displayed a much longer incubation time (155±1 days), a di-glycosylated-dominant PrPSc type and a distinct pattern of PrP-immunoreactive deposits and neuronal loss. These data indicate a crucial involvement of the host genome in modulating prion strain selection and propagation in mice. It is possible that multiple disease phenotypes may also be possible in BSE prion infection in humans and other animals.
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Evaluation of new cell culture inhibitors of protease-resistant prion protein against scrapie infection in mice
More LessIn vitro inhibitors of the accumulation of abnormal (protease-resistant) prion protein (PrP-res) can sometimes prolong the lives of scrapie-infected rodents. Here, transgenic mice were used to test the in vivo anti-scrapie activities of new PrP-res inhibitors, which, because they are approved drugs or edible natural products, might be considered for clinical trials in humans or livestock with transmissible spongiform encephalopathies (TSEs). These inhibitors were amodiaquine, thioridazine, thiothixene, trifluoperazine, tetrandrine, tannic acid and polyphenolic extracts of tea, grape seed and pine bark. Test compounds were administered for several weeks beginning 1–2 weeks prior to, or 2 weeks after, intracerebral or intraperitoneal 263K scrapie challenge. Tannic acid was also tested by direct preincubation with inoculum. None of the compounds significantly prolonged the scrapie incubation periods. These results highlight the need to assess TSE inhibitors active in cell culture against TSE infections in vivo prior to testing these compounds in humans and livestock.
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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 56 (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 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)