- Volume 88, Issue 3, 2007
Volume 88, Issue 3, 2007
- Animal
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- RNA viruses
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Intracellular localization and effects of individually expressed human parechovirus 1 non-structural proteins
More LessHuman parechovirus 1 (HPEV-1) has many unique features compared with other picornaviruses and it has been shown that the replication complex formed during HPEV-1 infection is different from that of other picornaviruses. Here, the intracellular localization and functional effects of individually expressed HPEV-1 non-structural proteins were studied. The 2A and 3D proteins were found diffusely in the cytoplasm and nucleus of the cell. The 3A and 3AB proteins were observed to co-localize with the markers for the Golgi apparatus, whereas 2B co-localized with markers for the endoplasmic reticulum and the 2C and 2BC proteins were observed mainly on the surface of lipid droplets. The 2C protein, which has been implicated in replication-complex formation in enterovirus-infected cells, was not able to induce vesicles similar to those seen in HPEV-1-infected cells when expressed individually. However, in superinfected cells, the fusion protein was able to relocate to the virus replication complexes. Similar to other picornaviruses, HPEV-1 was found to interfere with cellular secretion, but this function could not be ascribed to any of the individually expressed non-structural proteins.
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Protective immune responses in guinea pigs and swine induced by a suicidal DNA vaccine of the capsid gene of swine vesicular disease virus
A suicidal DNA vaccine based on a Semliki Forest virus (SFV) replicon was evaluated for the development of a vaccine against swine vesicular disease virus (SVDV). The 1BCD gene of SVDV was cloned and inserted into pSCA1, an SFV DNA-based replicon vector. The resultant plasmid, pSCA/1BCD, was transfected into BHK-21 cells and the antigenicity of the expressed protein was confirmed using an indirect immunofluorescence assay. Immunogenicity was studied in guinea pigs and swine. Animals were injected intramuscularly three times with pSCA/1BCD at regular intervals. Anti-SVDV antibodies were detected by ELISA, the lymphocyte proliferation response was tested by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide method and neutralizing antibodies were measured by microneutralization tests. The data showed that SVDV-specific antibodies, neutralizing antibodies and lymphocyte proliferation were induced in both guinea pigs and swine. Furthermore, after three successive vaccinations with pSCA/1BCD, half of the pigs were protected against challenge with SVDV. These results should encourage further work towards the development of a DNA vaccine against SVDV.
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Enterovirus 94, a proposed new serotype in human enterovirus species D
The genus Enterovirus (family Picornaviridae) contains five species with strains isolated from humans: Human enterovirus A (HEV-A), HEV-B, HEV-C, HEV-D and Poliovirus. In this study, a proposed new serotype of HEV-D was characterized. Four virus strains were isolated from sewage in Egypt and one strain from acute flaccid paralysis cases in the Democratic Republic of the Congo. The complete genome of one environmental isolate, the complete coding sequence of one clinical isolate and complete VP1 regions from the other isolates were sequenced. These isolates had 66.6–69.4 % nucleotide similarity and 74.7–76.6 % amino acid sequence similarity in the VP1 region with the closest enterovirus serotype, enterovirus 70 (EV70), suggesting that the isolates form a new enterovirus type, tentatively designated enterovirus 94 (EV94). Phylogenetic analyses including sequences of the 5′ UTR, VP1 and 3D regions demonstrated that EV94 isolates formed a monophyletic group within the species HEV-D. No evidence of recombination was found between EV94 and the other HEV-D serotypes, EV68 and EV70. Further biological characterization showed that EV94 was acid stable and had a wide cell tropism in vitro. Attempts to prevent replication with protective antibodies to known enterovirus receptors (poliovirus receptor, vitronectin α v β 3 receptor and decay accelerating factor) were not successful. Seroprevalence studies in the Finnish population revealed a high prevalence of this virus over the past two decades.
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Thermostable variants are not generally represented in foot-and-mouth disease virus quasispecies
More LessA severe limitation to fully realize the dramatic potential for adaptation of RNA virus quasispecies may occur if mutations in vast regions of the sequence space of virus genomes lead to significant decreases in biological fitness. In this study the detection and selection by heat of thermostable variants from different foot-and-mouth disease virus (FMDV) populations were attempted, in order to explore whether FMDV may generally accept a substantial increase in thermostability without compromising its infectivity. The results obtained with both uncloned and cloned populations of different serotypes, recovered from cytolytic or persistent infections and subjected to either very few passages or extensive passaging in cells, indicate that the presence of thermostable virus variants, even in small proportions, is not a general feature of FMDV quasispecies. This suggests that no substantial increase in the thermostability of FMDV may readily occur without a negative effect on viral function.
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Role of the mutant spectrum in adaptation and replication of West Nile virus
West Nile virus (WNV) has successfully spread throughout the USA, Canada, Mexico, the Caribbean and parts of Central and South America since its 1999 introduction into North America. Despite infecting a broad range of both mosquito and avian species, the virus remains highly genetically conserved. This lack of evolutionary change over space and time is common with many arboviruses and is frequently attributed to the adaptive constraints resulting from the virus cycling between vertebrate hosts and invertebrate vectors. WNV, like most RNA viruses studied thus far, has been shown in nature to exist as a highly genetically diverse population of genotypes. Few studies have directly evaluated the role of these mutant spectra in viral fitness and adaptation. Using clonal analysis and reverse genetics experiments, this study evaluated genotype diversity and the importance of consensus change in producing the adaptive phenotype of WNV following sequential mosquito cell passage. The results indicated that increases in the replicative ability of WNV in mosquito cells correlate with increases in the size of the mutant spectrum, and that consensus change is not solely responsible for alterations in viral fitness and adaptation of WNV. These data provide evidence of the importance of quasispecies dynamics in the adaptation of a flavivirus to new and changing environments and hosts, with little evidence of significant genetic change.
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West Nile virus isolates from India: evidence for a distinct genetic lineage
More LessThe complete genomic sequence of one human isolate of West Nile virus (WNV) and the partial genomic sequences of 14 other strains from India isolated in the period 1955–1982 from different hosts and geographical areas were determined. Phylogenetic analyses based on complete and partial genomic sequences (921 nt of the C–prM–E region) revealed that WNV could be classified into five distinct groups that differed from each other by 20–25 % at the complete genome level and by 20–26 % using partial sequences. Of the Indian isolates, 13 formed a distinct genetic lineage, lineage 5, whereas two isolates, one from a human patient (1967) and another from a bat (1968), were related closely to lineage 1 strains. The complete genomic sequence of the Indian isolate, 804994, showed 20–22 % genetic divergence from the previously proposed lineage 1 and 2 strains and 24–25 % divergence from isolates of the newly proposed lineages 3 (Rabensburg isolate 97-103 of 1997) and 4 (Russian isolate LEIV-Krnd88-190 of 1998). Similarly, the partial genomic sequences of the Indian isolates showed 21–26 % divergence from lineage 1 and 2 strains and from the Rabensburg (97-103) and Russian (LEIV-Krnd88-190) isolates. Cross-neutralization using strain-specific polyclonal antibodies against lineage 1 strain Eg-101 and representative Indian strains suggests substantial antigenic variation. This study documents circulation of WNV strains typical to India for 27 years and the introduction of lineage 1 strains during 1967–1968. These results indicate strongly that WNV should be classified into five genetic lineages, with Indian viruses constituting the distinct genetic lineage 5.
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Molecular epidemiological analysis of Japanese encephalitis virus in China
Sixty-two new Japanese encephalitis virus (JEV) isolates were obtained from mosquitoes, biting midges, human cerebrospinal fluid and human blood samples in China during 2002–2005. The E and prM genes were sequenced and phylogenetic analyses were performed with 38 JEV other isolates from China and 36 JEV strains from other countries. Phylogenetic trees based on the E and prM gene sequences were similar. The results indicate that: (i) recent JEV isolates from China are divided into two genotypes, genotype 1 and genotype 3; (ii) recent JEV isolates from China are grouped into the same clusters within genotypes 1 and 3; and (iii) genotype 1 JEV strains have been isolated in China since 1979, whilst genotype 3 JEV strains were isolated before the 1970s. The results suggest that genotype 1 JEV was introduced to China around 1979 and that JEV strains belonging to genotypes 1 and 3 circulate in China.
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A chimeric GB virus B encoding the hepatitis C virus hypervariable region 1 is infectious in vivo
More LessTwo GB virus B (GBV-B) chimeric genomes, GBV-HVR and GBV-HVRh (with a hinge), containing the coding region of the immunodominant hypervariable region 1 (HVR1) of the E2 envelope protein of Hepatitis C virus (HCV) were constructed. Immunoblot analysis confirmed that HVR1 was anchored to the GBV-B E2 protein. To investigate the replication competence and in vivo stability of in vitro-generated chimeric RNA transcripts, two naïve marmosets were inoculated intrahepatically with the transcripts. The GBV-HVR chimeric genome was detectable for 2 weeks post-inoculation (p.i.), whereas GBV-HVRh reverted to wild type 1 week p.i. Sequencing analysis of the HVR1 and flanking regions from GBV-HVR RNA isolated from marmoset serum demonstrated that the HVR1 insert remained unaltered in the GBV-HVR chimera for 2 weeks. Inoculation of a naïve marmoset with serum collected at 1 week p.i. also resulted in viraemia and confirmed that the serum contained infectious particles. All animals cleared the infection by 3 weeks p.i. and remained negative for the remaining weeks. The chimera may prove useful for the in vivo examination of any HCV HVR1-based vaccine candidates.
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Development and evaluation of an efficient cell-culture system for Hepatitis E virus
More LessUsing a faecal suspension with high load of Hepatitis E virus (HEV) (2.0×107 copies ml−1, genotype 3), we developed an efficient cell-culture system for HEV in a hepatocarcinoma cell line (PLC/PRF/5). HEV progeny released in the culture medium were passaged five times successively in PLC/PRF/5 cells. The initial day of appearance and load of HEV detectable in the culture supernatant after inoculation were dependent on the titre of seed virus in the inoculum. When 6.4×104 copies of HEV were inoculated on monolayers of PLC/PRF/5 cells in six-well microplates, HEV RNA was first detected in the culture medium on day 14 post-inoculation and increased to 9.1×105 copies ml−1 on day 60. When 8.6×105 copies of HEV were inoculated, HEV RNA was initially detected on day 12 and reached the highest titre of 8.6×107 copies ml−1 on day 60. HEV incubated at temperatures higher than 70 °C did not grow in PLC/PRF/5 cells, while HEV incubated at 56 °C for 30 min was infectious. Convalescent serum samples with IgM-class HEV antibodies obtained from patients infected with HEV of genotype 1, 3 or 4 neutralized the genotype 3 virus, indicating that HEV antibodies are broadly cross-reactive. Serum samples obtained from patients 8.7 or 24.0 years after the onset of HEV infection also prevented the propagation of HEV in PLC/PRF/5 cells, suggesting the presence of long-lasting HEV antibodies with neutralizing activity in individuals with past HEV infection.
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Detection and characterization of infectious Hepatitis E virus from commercial pig livers sold in local grocery stores in the USA
More LessHepatitis E virus (HEV) is a zoonotic pathogen of which pigs are reservoirs. To determine the presence of HEV RNA in commercial pig livers sold in local grocery stores in the USA, 127 packages of commercial pig liver were purchased and tested by a universal RT-PCR assay capable of detecting all four known HEV genotypes. Among the 127 livers tested, 14 were positive for HEV RNA. Sequence and phylogenetic analyses revealed that the 14 isolates all belonged to genotype 3. An animal study was subsequently conducted in pigs to determine whether the PCR-positive pig livers still contained infectious virus. The results showed that pigs inoculated with two of the three PCR-positive pig-liver homogenates became infected, as evidenced by the detection of faecal virus shedding, viraemia and seroconversion. The data demonstrated that commercial pig livers sold in grocery stores are contaminated by HEV and that the contaminating virus remains infectious, thus raising a public-health concern for food-borne HEV infection.
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Development and characterization of an infectious cDNA clone of the virulent Bucyrus strain of Equine arteritis virus
Strains of Equine arteritis virus (EAV) differ in the severity of the disease that they induce in horses. Infectious cDNA clones are potentially useful for identification of genetic determinants of EAV virulence; to date, two clones have been derived from a cell culture-adapted variant of the original (Bucyrus) isolate of EAV, and it has previously been shown that recombinant virus derived from one of these (rEAV030) is attenuated in horses. A complete cDNA copy of the genome of the virulent Bucyrus strain of EAV has now been assembled into a plasmid vector. In contrast to rEAV030, recombinant progeny virus derived from this clone caused severe disease in horses, characterized by pyrexia, oedema, leukopenia, high-titre viraemia and substantial nasal shedding of virus. The availability of infectious cDNA clones that produce recombinant viruses of different virulence to horses will facilitate characterization of the virulence determinants of EAV through reverse genetics.
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Recombinant swine beta interferon protects swine alveolar macrophages and MARC-145 cells from infection with Porcine reproductive and respiratory syndrome virus
More LessSwine beta interferon (swIFN-β) produced in HEK 293 cells infected with a recombinant, replication-defective human adenovirus 5 (Ad5) encoding the swIFN-β gene was tested for antiviral activity against Porcine reproductive and respiratory syndrome virus (PRRSV). MARC-145 cells were incubated overnight with dilutions of supernatant fluids from HEK 293 cells infected with Ad5-swIFN-β or with an Ad5 control virus (Ad5-Blue). Treated cells were infected with PRRSV; MARC-145 cells incubated with Ad5-Blue supernatants developed cytopathic effects (CPE), whereas those incubated with swIFN-β showed no CPE. To confirm the antiviral activity of swIFN-β, culture fluids from Ad5-swIFN-β-infected cells were affinity-purified on a Sepharose–anti-swIFN-β matrix, and the resulting fractions exhibited antiviral activity upon infection with PRRSV. The antiviral effects were specific, as they were blocked by mAbs against swIFN-β. Additional cultures of MARC-145 cells treated with swIFN-β-containing supernatants or affinity-purified swIFN-β were infected with PRRSV and tested by real-time RT-PCR for viral RNA in culture supernatants at various times post-inoculation. These experiments confirmed the protective effects of swIFN-β. swIFN-β was also tested for antiviral activity on porcine alveolar macrophages (PAMs) obtained by bronchoalveolar lavage from PRRSV-negative swine. PAMs were treated with dilutions of swIFN-β or Ad5-Blue culture fluids, infected with PRRSV and tested for viral RNA by real-time RT-PCR. The viral load data showed a dose-dependent protection in swIFN-β-treated PAMs, whereas no protection was evident from Ad5-Blue culture fluids. The data demonstrate that swIFN-β protects both MARC-145 cells and PAMs from PRRSV infection.
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Genomic analysis of diverse rubella virus genotypes
More LessBased on the sequence of the E1 glycoprotein gene, two clades and ten genotypes of Rubella virus have been distinguished; however, genomic sequences have been determined for viruses in only two of these genotypes. In this report, genomic sequences for viruses in an additional six genotypes were determined. The genome was found to be well conserved. The viruses in all eight of these genotypes had the same number of nucleotides in each of the two open reading frames (ORFs) and the untranslated regions (UTRs) at the 5′ and 3′ ends of the genome. Only the UTR between the ORFs (the junction region) exhibited differences in length. Of the nucleotides in the genome, 78 % were invariant. The greatest observed distance between viruses in different genotypes was 8.74 % and the maximum calculated genetic distance was 14.78 substitutions in 100 sites. This degree of variability was similar among regions of the genome with two exceptions, both within the P150 non-structural protein gene: the N-terminal region that encodes the methyl/guanylyltransferase domain was less variable, whereas the hypervariable domain in the middle of the gene was more divergent. Comparative phylogenetic analysis of different regions of the genome was done, using sequences from 43 viruses of the non-structural protease (near the 5′ end of the genome), the junction region (the middle) and the E1 gene (the 3′ end). Phylogenetic segregation of sequences from these three genomic regions was similar with the exception of genotype 1B viruses, among which a recombinational event near the junction region was identified.
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Effect of the phosphatidylinositol 3-kinase/Akt pathway on influenza A virus propagation
More LessThe phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway has attracted much recent interest due to its central role in modulating diverse downstream signalling pathways associated with cell survival, proliferation, differentiation, morphology and apoptosis. An increasing amount of information has demonstrated that many viruses activate the PI3K/Akt pathway to augment their efficient replication. In this study, the effect of the PI3K/Akt signalling pathway on influenza virus propagation was investigated. It was found that Akt phosphorylation was elevated in the late phase of influenza A/PR/8/34 infection in human lung carcinoma cells (A549). The PI3K-specific inhibitor LY294002 could suppress Akt phosphorylation, suggesting that influenza A virus-induced Akt phosphorylation is PI3K-dependent. UV-irradiated influenza virus failed to induce Akt phosphorylation, indicating that viral attachment and entry were not sufficient to trigger PI3K/Akt pathway activation. Blockage of PI3K/Akt activation by LY294002 and overexpression of the general receptor for phosphoinositides-1 PH domain (Grp1-PH) led to a reduction in virus yield. Moreover, in the presence of LY294002, viral RNA synthesis and viral protein expression were suppressed and, possibly as a consequence of low NP and M1 protein level, viral RNP nuclear export was also suppressed. These data suggest that the PI3K/Akt signalling pathway plays a role in influenza virus propagation.
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Antibodies specific to the HA2 glycopolypeptide of influenza A virus haemagglutinin with fusion-inhibition activity contribute to the protection of mice against lethal infection
More LessFour monoclonal antibodies (mAbs) recognizing distinct antigenic sites on the HA2 glycopolypeptide of influenza virus A/Dunedin/4/73 (H3N2) have been tested for in vivo protection. When applied intravenously before infection, three of them increased the survival of BALB/c mice infected with 1 LD50 homologous virus. The protection resulted simultaneously in 2 days earlier clearance of virus from the lungs. These three antibodies inhibited the fusion activity of virus in previous in vitro experiments. One of them, specific to N-terminal aa 1–38 of the HA2 glycopolypeptide, was also tested for protection against the heterologous virus A/Mississippi/1/85 (H3N2). Protection similar to that against the homologous virus was observed. The fourth mAb, without fusion-inhibition activity, did not protect mice. It is concluded that antibodies specific to the antigenically conserved HA2 glycopolypeptide that exhibit fusion-inhibition activity can contribute to the protection of infected mice and mediate more effective recovery from infection.
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Mapuera virus, a rubulavirus that inhibits interferon signalling in a wide variety of mammalian cells without degrading STATs
More LessMapuera virus (MPRV) is a paramyxovirus that was originally isolated from bats, but its host range remains unknown. It was classified as a member of the genus Rubulavirus on the basis of structural and genetic features. Like other rubulaviruses it encodes a V protein (MPRV/V) that functions as an interferon (IFN) antagonist. Here we show that MPRV/V differs from the IFN antagonists of other rubulaviruses in that it does not induce the proteasomal degradation of STAT proteins, key factors in the IFN signalling cascade. Rather, MPRV/V prevents the nuclear translocation of STATs in response to IFN stimulation and inhibits the formation of the transcription factor complex ISGF3. We also show that MPRV/V blocks IFN signalling in cells from diverse mammalian species and discuss the IFN response as a barrier to cross-species infections.
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Identification of novel canine rabies virus clades in the Middle East and North Africa
Four novel phylogenetic clades of canine rabies virus (RABV) variants have been identified in the Middle East and North Africa. The three novel Middle Eastern clades comprise RABV isolates from the borders between Israel and neighbouring countries. The North African clade (Africa 4) comprises four RABV isolates from Egypt and one from Israel. We characterized various RABV lineages antigenically by using a panel of monoclonal antibodies to the nucleoprotein (N) and phylogenetically by analysis of entire N gene sequences. The estimated mean substitution rate for the N gene alignment (2.7×10−4 substitutions per site per year) is comparable with previous estimates for RABV. The application of a molecular clock indicates the emergence of current canine RABV diversity to have occurred at about the same time (approx. 1870) in the Middle East and Europe, following divergence from established lineages in Africa and Asia.
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Thioaptamer decoy targeting of AP-1 proteins influences cytokine expression and the outcome of arenavirus infections
Viral haemorrhagic fever (VHF) is caused by a number of viruses, including arenaviruses. The pathogenesis is believed to involve dysregulation of cytokine production. The arenaviruses Lassa virus and Pichinde virus have a tropism for macrophages and other reticuloendothelial cells and both appear to suppress the normal macrophage response to virus infection. A decoy thioaptamer, XBY-S2, was developed and was found to bind to AP-1 transcription factor proteins. The P388D1 macrophage-like cell line contains members of the AP-1 family which may act as negative regulators of AP-1-controlled transcription. XBY-S2 was found to bind to Fra-2 and JunB, and enhance the induction of cytokines IL-6, IL-8 and TNF-α, while reducing the binding to AP-1 promoter elements. Administration of XBY-S2 to Pichinde virus-infected guinea pigs resulted in a significant reduction in Pichinde virus-induced mortality and enhanced the expression of cytokines from primary guinea pig macrophages, which may contribute to its ability to increase survival of Pichinde virus-infected guinea pigs. These data demonstrate a proof of concept that thioaptamers can be used to modulate the outcome of in vivo viral infections by arenaviruses by the manipulation of transcription factors involved in the regulation of the immune response.
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Phylogenetic analysis of Heliothis armigera cytoplasmic polyhedrosis virus type 14 and a series of dwarf segments found in the genome
More LessFull-length nucleotide sequences for the genome segments (S1–S6) of Heliothis armigera cytoplasmic polyhedrosis virus type 14 (HaCPV-14) have been characterized. Each segment consists of a single open reading frame with conserved motifs AGAA and AGCU at the 5′ and 3′ ends, respectively. Comparison of the proteins of HaCPV-14 with those of other members of the family Reoviridae suggests that S1 encodes an RNA-dependent RNA polymerase (RdRp), whilst S2 encodes a major capsid protein of the virus. Phylogenetic analysis of RdRps from 16 viruses in the family Reoviridae reveals that the genera Cypovirus and Oryzavirus may have originated from a common insect virus ancestor. A series of viable dwarf segments originating from S5 of HaCPV-14 has been identified. Analysis of the predicted secondary structures for these dwarf segments suggests that the signals essential for replication and packaging are located within the terminal sequences of these segments.
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Correlation between the induction of Th1 cytokines by an attenuated equine infectious anemia virus vaccine and protection against disease progression
More LessThe equine infectious anemia virus (EIAV) donkey-leukocyte attenuated vaccine (DLV) has been used to protect against equine infectious anaemia (EIA) disease for several decades in China. The attenuated mechanism and immunological protective mechanisms remain to be elucidated. To identify responses that correlate with the protection against disease, we immunized horses with DLV, followed by challenge with an EIAV wild-type strain LN. All vaccinated horses were asymptomatic and had a low level of virus replication (<10 copies ml−1). The expression level of cytokines including gamma interferon, interleukin 2 and 12 in DLV immunized horses was 5–100-fold higher than that in non-vaccinated controls (n=4, P<0.01). After challenge with virulent LN, horses vaccinated with DLV showed lower viral loads (<103 copies ml−1) with no temperature increase, except for one transient febrile episode in one animal. In contrast, horses in the non-vaccinated control group experienced much higher viral loads (>107 copies ml−1) and intermittent febrile episodes. Cytokine production in the DLV-vaccinated horses increased and attained a plateau level at approximately 50 days post-vaccination, and exceeded 107 copies per 107 peripheral blood mononuclear cells (PBMCs) 1–3 months post-challenge. However, non-vaccinated control horses died after several fever episodes (⩾39 °C), which coincided with higher viral load (106–107 copies ml−1) and lower cytokine production (<104 copies per 107 PBMCs). The results indicate that high levels of EIAV-specific cytokines induced by the attenuated EIAV vaccine may contribute to the protective immune response against EIA disease.
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- DNA viruses
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Identification of transcripts and protein products of the UL31, UL37, UL46, UL47, UL48, UL49 and US4 gene homologues of avian infectious laryngotracheitis virus
More LessIn the present study, the transcription and protein expression of seven genes of infectious laryngotracheitis virus (ILTV) were investigated: UL31 and UL37 possess homologues in all known avian and mammalian herpesviruses, whereas UL46–UL49 and US4 are only conserved in most alphaherpesviruses. A peculiarity of the ILTV genome is the translocation of UL47 from the unique long region to a position upstream of US4 within the unique short region. Northern blot analyses revealed that all of the analysed genes were transcribed most abundantly during the late (γ) phase of replication, but the only true late (γ2) gene was UL47. Using monospecific rabbit antisera, the protein products of all of the genes could be detected and localized in ILTV-infected cells. Considerable amounts of the UL31, UL47 and UL48 gene products were found in the cell nuclei, whereas the other proteins were restricted largely to the cytoplasm. Like the respective tegument proteins of other herpesviruses, the UL37 and UL46–UL49 gene products of ILTV were incorporated into virus particles, whereas the UL31 protein and the glycoprotein encoded by US4 (gG) were not detectable in purified virions. It was also demonstrated that the UL48 protein of ILTV is able to activate an alphaherpesvirus immediate-early gene promoter, which is also a typical feature of other UL48 homologues. Taken together, these results indicate that the functions of all of the investigated ILTV proteins are related to those of their homologues in other alphaherpesviruses.
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The UL47 gene of avian infectious laryngotracheitis virus is not essential for in vitro replication but is relevant for virulence in chickens
More LessThe genome of infectious laryngotracheitis virus (ILTV) exhibits several differences from those of other avian and mammalian alphaherpesviruses. One of them is the translocation of the conserved UL47 gene from the unique long (UL) to the unique short (US) genome region, where UL47 is inserted upstream of the US4 gene homologue. As in other alphaherpesviruses, UL47 encodes a major tegument protein of ILTV particles, whereas the US4 gene product is a non-structural glycoprotein, gG, which is secreted from infected cells. For functional characterization, an ILTV recombinant was isolated in which US4 together with the 3′-terminal part of UL47 was replaced by a reporter gene cassette encoding green fluorescent protein. From this virus, UL47 and US4 single-gene deletion mutants without foreign sequences were derived and virus revertants were also generated. In vitro studies revealed that both genes were non-essential for ILTV replication in cultured cells. Whereas US4-negative ILTV exhibited no detectable growth defects, maximum virus titres of the double deletion mutant and of UL47-negative ILTV were reduced about 10-fold compared with those of wild-type virus and rescued virus. Experimental infection of chickens demonstrated that UL47-negative ILTV was significantly attenuated in vivo and was shed in reduced amounts, whereas wild-type and rescued viruses caused severe disease and high mortality rates. As all immunized animals were protected against subsequent challenge infection with virulent ILTV, the UL47 deletion mutant might be suitable as a live-virus vaccine.
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Brn-3a suppresses pseudorabies virus-induced cell death in sensory neurons
More LessSensory neurons of the trigeminal ganglion (TG) are of crucial importance in the pathogenesis of many alphaherpesviruses, constituting major target cells for latency and reactivation events. We showed earlier that a subpopulation of porcine TG neurons, in contrast to other porcine cell types, is highly resistant to cell death induced by infection with the porcine alphaherpesvirus pseudorabies virus (PRV). Here, we report that expression of Brn-3a, a neuron-specific transcription factor implicated in cell survival of sensory neurons, correlates with the increased resistance of TG neurons towards PRV-induced cell death. In addition, overexpression of Brn-3a in the sensory neuronal cell line ND7 markedly increased resistance of these cells to PRV-induced cell death. Hence, Brn-3a may play a hitherto uncharacterized role in protection of sensory neurons from alphaherpesvirus-induced cell death, which may have implications for different aspects of the alphaherpesvirus life cycle, including latency/reactivation events.
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Evaluation of the vaccine potential of an equine herpesvirus type 1 vector expressing bovine viral diarrhea virus structural proteins
Bovine viral diarrhea virus (BVDV) is an economically important pathogen of cattle that is maintained in the population by persistently infected animals. Virus infection may result in reproductive failure, respiratory disease and diarrhoea in naïve, susceptible bovines. Here, the construction and characterization of a novel vectored vaccine, which is based on the incorporation of genes encoding BVDV structural proteins (C, Erns, E1, E2) into a bacterial artificial chromosome of the equine herpesvirus type 1 (EHV-1) vaccine strain RacH, are reported. The reconstituted vectored virus, rH_BVDV, expressed BVDV structural proteins efficiently and was indistinguishable from parental vector virus with respect to growth properties in cultured cells. Intramuscular immunization of seronegative cattle with rH_BVDV resulted in induction of BVDV-specific serum neutralizing and ELISA antibodies. Upon experimental challenge infection of immunized calves with the heterologous BVDV strain Ib SE5508, a strong anamnestic boost of the neutralizing-antibody response was observed in all vaccinated animals. Immunized animals presented with reduced viraemia levels and decreased nasal virus shedding, and maintained higher leukocyte counts than mock-vaccinated controls.
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Extensive sequence variation exists among isolates of murine cytomegalovirus within members of the m02 family of genes
More LessMurine cytomegalovirus (MCMV) is a widely used model for human cytomegalovirus (HCMV) and has facilitated many important discoveries about the biology of CMVs. Most of these studies are conducted using the laboratory MCMV strains Smith and K181. However, wild-derived isolates of MCMV, like HCMV clinical isolates, exhibit genetic variation from laboratory strains, particularly at the ends of their genomes in areas containing known or putative immune-evasion and tropism genes. This study analysed the nucleotide sequence of the m02–m05 region, within the m02 gene family, of a number of laboratory and wild-derived MCMV isolates, and found a large degree of variation in both the sequence and arrangement of genes. A new open reading frame (ORF), designated m03.5, was found to be present in a number of wild isolates of MCMV in place of m03. Two distinct isolates, W8 and W8211, were found to possess both m03 and m03.5. Both m03 and m03.5 had early transcription kinetics and the encoded proteins could be detected on the cell surface, consistent with a possible role in immune evasion through binding to host-cell proteins. These data show that gene duplication and sequence variation occur within different isolates of MCMV found in the wild. As this variation among strains may alter the function of genes, these findings should be considered when analysing gene function or host–virus interactions in laboratory models.
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Full-length EBNA1 mRNA-transduced dendritic cells stimulate cytotoxic T lymphocytes recognizing a novel HLA-Cw*0303- and -Cw*0304-restricted epitope on EBNA1-expressing cells
Epstein–Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is an attractive target for immunotherapy against EBV-associated malignancies because it is expressed in all EBV-positive cells. Although CD8+ cytotoxic T-lymphocyte (CTL) epitope presentation is largely prevented by its glycine–alanine-repeat domain (GAr), the use of mRNA-transduced dendritic cells (DCs) would offer the advantage of priming EBNA1-specific CTLs. After stimulation with GAr-containing EBNA1-transduced monocyte-derived DCs, two EBNA1-specific CTL clones, B5 and C6, were isolated successfully from a healthy donor. These CTLs recognize peptides in the context of HLA-B*3501 and HLA-Cw*0303, respectively. A novel epitope, FVYGGSKTSL, was then identified, presented by both HLA-Cw*0303 and -Cw*0304, which are expressed by >35 % of Japanese, >20 % of Northern Han Chinese and >25 % of Caucasians. The mixed lymphocyte–peptide culture method revealed that FVYGGSKTSL-specific CTL-precursor frequencies in HLA-Cw*0303- or -Cw*0304-positive donors were between 1×10−5 and 1×10−4 CD8+ T cells. Moreover, both CTL clones inhibited growth of HLA-matched EBV-transformed B lymphocytes in vitro, and B5 CTLs produced a gamma interferon response to EBNA1-expressing gastric carcinoma cells in the context of HLA-Cw*0303. These data demonstrate that EBNA1 mRNA-transduced DCs may be useful tools for inducing EBNA1-specific CTLs that might be of clinical interest for CTL therapy of EBV-associated malignancies.
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Bovine papular stomatitis virus encodes a functionally distinct VEGF that binds both VEGFR-1 and VEGFR-2
More LessBovine papular stomatitis virus (BPSV), a member of the genus Parapoxvirus, causes proliferative dermatitis in cattle and humans. Other species of the genus cause similar lesions, the nature of which has been attributed, at least in part, to a viral-encoded vascular endothelial growth factor (VEGF) that induces vascularization and dermal oedema through VEGF receptor-2 (VEGFR-2). The results of this study showed that BPSV strain V660 encodes a novel VEGF and that the predicted BPSV protein showed only 33–52 % amino acid identity to VEGFs encoded by the other species of the genus. BPSV VEGF showed higher identity to mammalian VEGF-A (51 %) than the other parapoxviral VEGFs (31–46 %). Assays of the purified BPSV VEGF (BPSVV660VEGF) demonstrated that it was also functionally more similar to VEGF-A, as it showed significant binding to VEGFR-1 and induced monocyte migration. Like VEGF-A and the other viral VEGFs, BPSVV660VEGF bound VEGFR-2 with high affinity. Sequence analysis and structural modelling of BPSVV660VEGF revealed specific residues, outside the known receptor-binding face, that are predicted either to influence VEGF structure or to mediate binding directly to the VEGFRs. These results indicate that BPSVV660VEGF is a biologically active member of the VEGF family and that, via its interaction with VEGFR-2, it is likely to contribute to the proliferative and highly vascularized nature of BPSV lesions. This is also the first example of a viral VEGF acting via VEGFR-1 and influencing haematopoietic cell function. These data suggest that BPSVV660VEGF is an evolutionary and functional intermediate between VEGF-A and the other parapoxviral VEGFs.
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Deletion of a major neutralizing epitope of human papillomavirus type 16 virus-like particles
More LessHuman papillomavirus type 16 (HPV-16) is a major cause of human cancer. Effective prophylactic vaccines are based on type-specific neutralizing antibodies. A major neutralizing epitope has been defined by the monoclonal antibody H16.V5. To investigate the importance of this epitope for overall immunogenicity of HPV-16, HPV-16 virus-like particles devoid of the H16.V5 epitope were engineered by site-directed mutagenesis of ten non-conserved, surface-exposed residues. Removal of the H16.V5-defined epitope had only a marginal effect on antigenic reactivity with antibodies in sera from infected subjects, but affected immunogenicity in experimental immunization of mice, with reduced induction of both antibody responses and CTL responses.
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Resolution of cervical dysplasia is associated with T-cell proliferative responses to human papillomavirus type 16 E2
The ‘high-risk’ human papillomaviruses (HPVs) cause persistent infections of the anogenital region that may resolve spontaneously following activation of a protective immune response. The aim of this study was to determine whether cell-mediated immunity (CMI) to the early protein E2 was associated with disease regression and to establish whether E2 CMI and antibodies to L1 virus-like particles (VLPs) were associated markers of immunity to HPV. Lymphoproliferative responses to histidine-tagged E2 and antibody responses to VLPs were measured in patients with persistent cervical dysplasia, those whose disease had recently resolved and normal controls. Resolvers had significantly higher E2-specific lymphoproliferative responses when compared with normal controls or persisters, whereas there was no significant difference between the persisters and the normal controls. The T cells stimulated by E2 secreted high levels of gamma interferon (IFN-γ), consistent with a type 1 helper (Th1) phenotype. VLP IgG responses were associated with current or previous HPV infection, but not with disease regression or a lymphoproliferative response to E2. Major histocompatibility complex class I-restricted T cells secreted IFN-γ following stimulation with E1, and E2 peptides were detected more frequently in the persister group. The data showed that lymphoproliferative responses to E2 with a cytokine profile indicative of Th1 are associated with disease resolution, supporting the development of a therapeutic vaccine that activates this type of response for the treatment of individuals with pre-existing disease.
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Seroprevalence of human papillomaviruses and Chlamydia trachomatis and cervical cancer risk: nested case–control study
A nested case–control study of invasive and in situ cervical cancer was performed within a community-based cohort of 13 595 Taiwanese women assembled in 1991, with a follow-up period of 9 years. Baseline serum or plasma samples were analysed for antibodies against human papillomavirus (HPV) types 6, 16 and 18 and Chlamydia trachomatis. In total, 114 cases (42 incident cases identified during follow-up and 72 prevalent cases identified at baseline) and 519 matched controls were included in the study. HPV-16 seropositivity was strongly associated with cervical cancer (OR=6.33; 95 % CI 3.45–11.62). Overall, C. trachomatis was not associated with cervical cancer, but was associated with cervical cancer in analyses restricted to incident cases of cancer (OR=2.94; 95 % CI 1.17–7.42) or to cases in which serum samples were analysed (OR=3.13; 95 % CI 1.16–8.47). An antagonistic interaction between HPV-6 and -16 was found in a multiplicative model. These results suggest that different HPV types might interfere in cervical carcinogenesis and that C. trachomatis is associated with cervical cancer in prospective studies, and support the notion that HPV-16 seropositivity is strongly associated with cervical cancer.
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Avian polyomavirus mutants with deletions in the VP4-encoding region show deficiencies in capsid assembly and virus release, and have reduced infectivity in chicken
More LessAvian polyomavirus (APV) is the causative agent of an acute fatal disease in psittacine and some non-psittacine birds. In contrast to mammalian polyomaviruses, the APV genome encodes the additional capsid protein VP4 and its variant VP4Δ, truncated by an internal deletion. Both proteins induce apoptosis. Mutation of their common initiation codon prevents virus replication. Here, the generation of replication competent deletion mutants expressing either VP4 or VP4Δ is reported. In contrast to infection with wild-type virus, chicken embryo cells showed no cytopathic changes after infection with the mutants, and induction of apoptosis as well as virus release from the infected cells were delayed. Electron microscopy revealed the presence of a high proportion of small particles and tubules in preparations of the VP4 deletion mutant, indicating a scaffolding function for VP4. Wild-type and mutant viruses elicited neutralizing antibodies against APV after intramuscular and intraperitoneal infection of chicken; however, VP4-specific antibodies were only detected after infection with wild-type virus. Using the oculonasal route of infection, seroconversion was only observed in chickens infected with the wild-type virus, indicating a strongly reduced infectivity of the mutants. Based on the biological properties of the deletion mutants, they could be considered as candidates for APV marker vaccines.
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- Plant
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Use of pentapeptide-insertion scanning mutagenesis for functional mapping of the plum pox virus helper component proteinase suppressor of gene silencing
More LessHelper component proteinase (HC-Pro) of Plum pox virus is a multifunctional potyvirus protein that has been examined intensively. In addition to its involvement in aphid transmission, genome amplification and long-distance movement, it is also one of the better-studied plant virus suppressors of RNA silencing. The first systematic analysis using pentapeptide-insertion scanning mutagenesis of the silencing suppression function of a potyvirus HC-Pro is presented here. Sixty-three in-frame insertion mutants, each containing five extra amino acids inserted randomly within the HC-Pro protein, were analysed for their ability to suppress transgene-induced RNA silencing using Agrobacterium infiltration in transgenic Nicotiana benthamiana plants expressing green fluorescent protein. A functional map was obtained, consisting of clearly defined regions with different classes of silencing-suppression activity (wild-type, restricted and disabled). This map confirmed that the N-terminal part of the protein, which is indispensable for aphid transmission, is dispensable for silencing suppression and supports the involvement of the central region in silencing suppression, in addition to its role in maintenance of genome amplification and synergism with other viruses. Moreover, evidence is provided that the C-terminal part of the protein, previously known to be necessary mainly for proteolytic activity, also participates in silencing suppression. Pentapeptide-insertion scanning mutagenesis has been shown to be a fast and powerful tool to functionally characterize plant virus proteins.
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Recombination and gene duplication in the evolutionary diversification of P1 proteins in the family Potyviridae
More LessGenome structure and sequence are notably conserved between members of the family Potyviridae. However, some genomic regions of these viruses, such as that encoding the P1 protein, show strikingly high variability. In this study, some partially conserved motifs were identified upstream of the quite well-conserved protease domain located near the P1 C terminus. The irregular distribution of these motifs suggests that the potyviral P1 proteins have undergone complex evolutionary diversification. Evidence was found of recombination events in the P1 N-terminal region, similar to those reported in potyviruses of the bean common mosaic virus subgroup, but also affecting other potyviruses. Moreover, intergeneric recombination events affecting potyviruses and ipomoviruses were also observed. Evidence that these recombination events could be linked to host adaptation is provided. Specific sequence features and differences in net charge help to classify the P1 proteins of members of the family Potyviridae into two groups: those from potyviruses and rymoviruses and those from tritimoviruses. The ipomovirus Cucumber vein yellowing virus has two P1 copies arranged in tandem, the most N-terminal one being of the potyvirus type and the other being of the tritimovirus type. These findings suggest that both recombination and gene duplication have contributed to P1 evolution and helped to facilitate successful adaptation of members of the family Potyviridae to a wide range of host species.
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Central domain of a potyvirus VPg is involved in the interaction with the host translation initiation factor eIF4E and the viral protein HcPro
More LessUsing recombinant proteins produced in bacteria or in infected plants, interactions between the VPg and HcPro of Lettuce mosaic potyvirus (LMV) and between LMV VPg and the lettuce translation initiation factor 4E, the cap-binding protein (eIF4E), were demonstrated in vitro. Interaction with eIF4E and HcPro both involved the same VPg central domain. The structure of this domain in the VPg context was predicted to include an amphiphilic α-helix, with the amino acids related to biological functions in various potyviruses exposed at the hydrophilic side.
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Synergistic pathogenicity of a phloem-limited begomovirus and tobamoviruses, despite negative interference
More LessIn contrast to previous observations on phloem-limited geminiviruses supported in movement andaccumulation by RNA viruses such as cucumo- and tobamoviruses, tissue infiltration by Abutilon mosaic virus (AbMV) was enhanced by neither Tobacco mosaic virus nor Tomato mosaic virus (ToMV) in two different hosts, Nicotiana benthamiana and tomato. Both tobamoviruses exerted a negative effect on the DNA virus, resulting in a decrease in AbMV accumulation and significantly reduced infectivity in N. benthamiana. Despite these unexpected molecular observations, a striking synergistic enhancement in pathogenicity occurred with respectto stunting and necrosis. In situ hybridization revealed that this was not due to any alteration of tissue infiltration by AbMV, which also remained limited to the phloem in the mixed infections. Transgenically expressed ToMV 30K movement protein was not able to induce phloemescape of AbMV in tomato plants and did not lead to any obvious change in begomovirus symptomatology.
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- Other Agents
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Prions spread via the autonomic nervous system from the gut to the central nervous system in cattle incubating bovine spongiform encephalopathy
To elucidate the still-unknown pathogenesis of bovine spongiform encephalopathy (BSE), an oral BSE challenge and sequential kill study was carried out on 56 calves. Relevant tissues belonging to the peripheral and central nervous system, as well as to the lymphoreticular tract, from necropsied animals were analysed by highly sensitive immunohistochemistry and immunoblotting techniques to reveal the presence of BSE-associated pathological prion protein (PrPSc) depositions. Our results demonstrate two routes involving the autonomic nervous system through which BSE prions spread by anterograde pathways from the gastrointestinal tract (GIT) to the central nervous system (CNS): (i) via the coeliac and mesenteric ganglion complex, splanchnic nerves and the lumbal/caudal thoracic spinal cord (representing the sympathetic GIT innervation); and (ii) via the Nervus vagus (parasympathetic GIT innervation). The dorsal root ganglia seem to be subsequently affected, so it is likely that BSE prion invasion of the non-autonomic peripheral nervous system (e.g. sciatic nerve) is a secondary retrograde event following prion replication in the CNS. Moreover, BSE-associated PrPSc was already detected in the brainstem of an animal 24 months post-infection, which is 8 months earlier than reported previously. These findings are important for the understanding of BSE pathogenesis and for the development of new diagnostic strategies for this infectious disease.
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Dynamics and genetics of PrPSc placental accumulation in sheep
Placentae from scrapie-affected ewes are an important source of contamination. This study confirmed that scrapie-incubating ewes bearing susceptible genotypes could produce both abnormal prion protein (PrPSc)-positive and -negative placentae, depending only on the PRP genotype of the fetus. The results also provided evidence indicating that scrapie-incubating ARR/VRQ ewes may be unable to accumulate prions in the placenta, whatever the genotype of their progeny. Multinucleated trophoblast cells appeared to play a key role in placental PrPSc accumulation. PrPSc accumulation began in syncytiotrophoblasts before disseminating to uninucleated trophoblasts. As these result from trophoblast/uterine epithelial cell fusion, syncytiotrophoblast cells expressed maternal and fetal PrPC, whilst uninucleated trophoblast cells only expressed fetal PrPC. In ARR/VRQ scrapie-infected ewes, expression of the ARR allele by syncytiotrophoblasts appeared to prevent initiation of PrPSc placental deposition. The absence of prions in affected ARR/VRQ sheep placentae reinforces strongly the interest in ARR selection for scrapie control.
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Comparison of CR36, a new heparan mimetic, and pentosan polysulfate in the treatment of prion diseases
Sulfated polyanions, including pentosan polysulfate (PPS) and heparan mimetics, number among the most effective drugs that have been used in experimental models of prion disease and are presumed to act in competition with endogenous heparan sulfate proteoglycans as co-receptors for prion protein (PrP) on the cell surface. PPS has been shown to prolong the survival of animals after intracerebral perfusion and is in limited use for the experimental treatment of human transmissible spongiform encephalopathies (TSEs). Here, PPS is compared with CR36, a new heparan mimetic. Ex vivo, CR36 was more efficient than PPS in reducing PrPres in scrapie-infected cell cultures and showed long-lasting activity. In vivo, CR36 showed none of the acute toxicity observed with PPS and reduced PrPres accumulation in spleens, but had only a marginal effect on the survival time of mice infected with bovine spongiform encephalopathy. In contrast, mice treated with PPS that survived the initial toxic mortality had no detectable PrPres in the spleens and lived 185 days longer than controls (+55 %). These results show, once again, that anti-TSE drugs cannot be encouraged for human therapeutic trials solely on the basis of in vitro or ex vivo observations, but must first be subjected to in vivo animal studies.
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Abnormal prion protein in the pituitary in sporadic and variant Creutzfeldt–Jakob disease
By using high-sensitivity Western blotting and immunohistochemistry, pituitary glands from patients with sporadic and variant Creutzfeldt–Jakob disease (sCJD and vCJD, respectively) were analysed for the presence of the protease-resistant form of the prion protein (PrPres). PrPres was detected in a greater proportion of vCJD pituitaries than sCJD pituitaries and was localized predominantly in the neurohypophysis. PrPres was also detected in a recurrent pituitary adenoma from an sCJD patient. Immunohistochemical analysis showed sparse positive labelling, predominantly in folliculostellate cells, in vCJD and sCJD adenohypophyses. The PrPres glycosylation pattern in the vCJD neurohypophyses showed a predominance of the unglycosylated band, which differed markedly from patterns found in all other vCJD tissues. The presence of PrPres in the pituitary of CJD patients at autopsy suggests that human growth hormone-related iatrogenic CJD may have indeed resulted from infectivity in collected pituitaries rather than necessarily from contamination of pituitary pools by adjacent brain tissue.
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- Phage
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Bacteriophage P4 sut1: a mutation suppressing transcription termination
In the Escherichia coli satellite phage P4, transcription starting from PLE is prevalently controlled via premature termination at several termination sites. We identified a spontaneous mutation, P4 sut1 (suppression of termination), in the natural stop codon of P4 orf151 that, by elongating translation, suppresses transcription termination at the downstream t151 site. Both the translational and the transcriptional profile of P4 sut1 differed from those of P4 wild-type. First of all, P4 sut1 did not express Orf151, but a higher molecular mass protein, compatible with the 303 codon open reading frame generated by the fusion of orf151, cnr and the intervening 138 nt. Moreover, after infection of E. coli, the mutant expressed a very low amount of the 1.3 and 1.7 kb transcripts originating at PLE and PLL promoters, respectively, and terminating at the intracistronic t151 site, whereas correspondingly higher amounts of the 4.1 and 4.5 kb RNAs arising from the same promoters and covering the entire operon were detected. Thus the sut1 mutation converts a natural stop codon into a sense codon, suppresses a natural intracistronic termination site and leads to overexpression of the downstream cnr and α genes. This correlates with the inability of P4 sut1 to propagate in the plasmid state. By cloning different P4 DNA fragments, we mapped the t151 transcription termination site within the 7633–7361 region between orf151 and gene cnr. A potential stem–loop structure, resembling the structure of a Rho-independent termination site, was predicted by mfold sequence analysis at 7414–7385.
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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 83 (2002)
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Volume 82 (2001)
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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 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)