- Volume 89, Issue 4, 2008
Volume 89, Issue 4, 2008
- Animal
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- DNA viruses
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Effect of phosphorylation on the transactivation activity of Epstein–Barr virus BMRF1, a major target of the viral BGLF4 kinase
More LessModification of human herpesvirus DNA polymerase processivity factors (PFs) by phosphorylation occurs frequently during viral lytic replication. However, functional regulation of the herpesvirus PFs through phosphorylation is not well understood. In addition to processivity, the PF BMRF1 of Epstein–Barr virus can function as a transactivator to activate the BHLF1 promoter within the lytic origin of replication (oriLyt), which is assumed to facilitate DNA replication through remodelling viral chromatin structure. BMRF1 is known to be phosphorylated by the viral BGLF4 kinase, but its impact on BMRF1 function is unclear. Seven candidate BGLF4 target sites were predicted within a proline-rich region between the DNA-processivity and nuclear-localization domains of BMRF1. We show that four of these residues, Ser-337, Thr-344, Ser-349 and Thr-355, are responsible for the BGLF4-induced hyperphosphorylation of BMRF1. In functional analyses, a phosphorylation-mimicking mutant of BMRF1 shows similar nuclear localization, as well as DNA-binding ability, to the wild type; however, it displays stronger synergistic activation of the BHLF1 promoter with Zta. Notably, BGLF4 downregulates BMRF1 transactivation and enhances the transactivation activity of Zta and the synergistic activation of BMRF1 and Zta on the BHLF1 promoter. Our findings suggest that BGLF4 may modulate the activation of the oriLyt BHLF1 promoter coordinately through multiple mechanisms to facilitate optimal oriLyt-dependent viral DNA replication.
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Identification of envelope protein pORF81 of koi herpesvirus
Koi herpesvirus (KHV), an emerging pathogen causing mass mortality in koi and common carp, possesses the largest known herpesvirus genome of 295 kbp predicted to encode 156 different proteins. However, none of them has been identified or functionally characterized up to now. In this study, a rabbit antiserum was prepared against a bacterial fusion protein that permitted detection of the predicted type III membrane protein encoded by ORF81 of KHV. In Western blot analyses, the abundant ORF81 gene product of KHV exhibited an apparent mass of 26 kDa and appeared to be non-glycosylated. It could be localized in the cytoplasm of infected cells and in virion envelopes by indirect immunofluorescence and immunoelectron microscopy, respectively. The antiserum was also suitable for the detection of pORF81 in sections of gills, kidneys, hepatopancreas and skin of KHV-infected carp by immunohistochemistry.
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Effect of basal core promoter and pre-core mutations on hepatitis B virus replication
More LessThere are two hypotheses explaining a fulminant outcome after hepatitis B virus (HBV) infection, both of which may be applicable at the same time: (i) basal core promoter (BCP) mutations increase viral replication, allowing rapid spread of the virus through the liver, and (ii) pre-core (pre-C) mutations abrogating hepatitis B e antigen (HBeAg) synthesis remove its tolerogenic effect, leading to a vigorous immune response. This study investigated the effect of these mutations on virus replication efficiency and HBeAg production. Substitutions A1762T/G1764A and T1753C, C1766T and T1768A in the BCP region, and G1896A and G1899A in the pre-C region, were examined either alone or in combination, using a common genetic background. Huh7 cells were transfected with these constructs and real-time PCR was used to quantify released virion-associated and intracellular HBV DNA, pregenomic RNA and pre-C mRNA. In addition, culture supernatants were tested for hepatitis B surface antigen (HBsAg) and HBeAg. The double BCP mutation (A1762T/G1764A) and the pre-C mutations (G1896A, G1899A), either alone or in combination, had no appreciable effect on the replication capacity of the virus. In contrast, clones with mutations at positions 1766/1768, 1762/1764/1766 and 1753/1762/1764 exhibited increased-replication phenotypes. HBeAg was undetectable in all cultures transfected with constructs bearing the G1896A stop-codon mutation, as expected. In contrast, constructs with additional mutations in the BCP region had appreciably lower levels of HBeAg expression than the wild type. Thus, core promoter mutations other than those at 1762/1764 appear to upregulate viral DNA replication and, at the same time, greatly reduce HBeAg production.
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Association of serum and mucosal neutralizing antibodies to human papillomavirus type 16 (HPV-16) with HPV-16 infection and cervical disease
We investigated neutralizing antibodies to human papillomavirus type 16 (HPV-16) in serum and cervical washes from 84 women with normal cytology or cervical disease. Serum neutralizing antibodies were detected in 78 % of women infected at the cervix with HPV-16, compared with 35 % (P=0.002) of women infected with HPV-16-related types (α9 HPV types), 14 % (P<0.0001) of women infected with HPV-16 non-related types and none of HPV-uninfected women. A significant correlation between HPV-16 infection and serum HPV-16-neutralizing antibodies was observed (r s=0.97; P=0.032). Cervical neutralizing antibodies were detected in 38 % of women with HPV-16 infection and in 17 % of women infected with the HPV-16-related type HPV-31. Cervical neutralizing antibodies correlated with HPV-16 infection (r s=0.95; P=0.08), but not with cervical disease. Serum and cervical HPV-16 antibody responses were not affected significantly by human immunodeficiency virus type 1 infection. In conclusion, serum and cervical HPV-16-neutralizing antibodies were found to correlate with HPV-16 infection, but not with cervical disease.
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In vivo characterization of two granuloviruses in larvae of Mythimna separata (Lepidoptera: Noctuidae)
More LessThe pathogenicity of two granuloviruses (GVs), Xestia c-nigrum GV (XecnGV) and Pseudaletia unipuncta GV (PsunGV), was examined in Mythimna separata. Partial sequencing of the genome of PsunGV indicated that it is related closely to XecnGV, but considered to be a different species. PsunGV and XecnGV showed similar pathogenicity in terms of dose–mortality response and pattern of host mass changes following infection. Both GVs killed infected larvae in 2–3 weeks. Temporal changes in the concentrations of GV-specific DNA in the larval haemolymph were measured by using a real-time quantitative PCR. Viral DNA concentration increased quickly and reached a plateau at 60–72 h post-inoculation. Rates of budded virus (BV) production of each GV were estimated on the basis of viral DNA concentrations by a modified Gompertz model. The slopes of the estimated BV growth curves of both XecnGV and PsunGV in M. separata larvae were equivalent to that of Mamestra brassicae nucleopolyhedrovirus (NPV) in its original host, reported in our previous study. This suggested that BV production is not a major factor in the slower killing speed of GVs in comparison to NPVs. The GV-infected larvae survived for an additional 10 days or more after reaching a maximum level of BV concentration, and kept growing without pupation. These findings also suggested that the GVs have a unique mechanism to regulate the growth of host larvae.
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Open reading frame Bm21 of Bombyx mori nucleopolyhedrovirus is not essential for virus replication in vitro, but its deletion extends the median survival time of infected larvae
More LessIn this report, the open reading frame 21 (Bm21) of Bombyx mori nucleopolyhedrovirus (BmNPV), one of the unique genes of group I NPVs, was characterized. Bm21 is predicted to encode a protein of 55.8 kDa and was found to contain imperfectly conserved leucine-rich repeats. 3′ Rapid amplification of cDNA ends (3′RACE) showed that the transcript of Bm21 was first detected from 6 h post-infection and that it also encompassed the complete Bm20. 5′RACE revealed three transcription initiation sites, one of which mapped to the baculovirus early transcription motifs CGTGC and CAGT. Transient-expression and superinfection assays indicated that BM21 localized in the nucleus of infected BmN cells. To study the function of BM21, a Bm21-null virus was constructed using bacmid technology. Viral one-step growth curve analyses showed that the Bm21-null virus had similar budded virus production kinetics to those of the parental virus. Bioassay analyses showed that the median lethal concentration (LC50) of the Bm21-null virus was similar to that of the control virus; however, the median survival time (ST50) of the knockout virus was significantly longer than the control virus. These results indicate that BM21 is not essential for virus replication in vitro, but that deletion of the gene delays the killing of the infected larvae.
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A viral histone H4 encoded by Cotesia plutellae bracovirus inhibits haemocyte-spreading behaviour of the diamondback moth, Plutella xylostella
Wael Gad and Yonggyun KimHistone H4 is highly conserved and forms a central-core nucleosome with H3 in eukaryotic chromatin. Its covalent modification at the protruding N-terminal region from the nucleosomal core can change the chromatin conformation in order to regulate gene expression. A viral H4 was found in the genome of Cotesia plutellae bracovirus (CpBV). The obligate host of the virus is an endoparasitoid wasp, C. plutellae, which parasitizes the diamondback moth, Plutella xylostella, and interrupts host development and immune reactions. CpBV has been regarded as a major source for interrupting the physiological processes during parasitization. CpBV H4 shows high sequence identity with the amino acid sequence of P. xylostella H4 except for an extended N-terminal region (38 aa). This extended N-terminal CpBV H4 contains nine lysine residues. CpBV H4 was expressed in P. xylostella parasitized by C. plutellae. Western blot analysis using a wide-spectrum H4 antibody showed two H4s in parasitized P. xylostella. In parasitized haemocytes, CpBV H4 was detected predominantly in the nucleus and was highly acetylated. The effect of CpBV H4 on haemocytes was analysed by transient expression using a eukaryotic expression vector, which was injected into non-parasitized P. xylostella. Expression of CpBV H4 was confirmed in the transfected P. xylostella by RT-PCR and immunofluorescence assays. Haemocytes of the transfected larvae lost their spreading ability on an extracellular matrix. Inhibition of the cellular immune response by transient expression was reversed by RNA interference using dsRNA of CpBV H4. These results suggest that CpBV H4 plays a critical role in suppressing host immune responses during parasitization.
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- Plant
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Association of the transcriptional response of soybean plants with soybean mosaic virus systemic infection
More LessCompatible virus infection induces and suppresses host gene expression at the global level. These gene-expression changes are the molecular basis of symptom development and general stress and defence-like responses of the host. To assess transcriptional changes in soybean plants infected with soybean mosaic virus (SMV), the first soybean trifoliate leaf, immediately above the SMV-inoculated unifoliate leaf, was sampled at 7, 14 and 21 days post-inoculation (p.i.) and subjected to microarray analysis. The identified changes in gene expression in soybean leaves with SMV infection at different time points were associated with the observed symptom development. By using stringent selection criteria (≥2- or ≤−2-fold change and a Q value of ≤0.05), 273 (1.5 %) and 173 (0.9 %) transcripts were identified to be up- and downregulated, respectively, from 18 613 soybean cDNAs on the array. The expression levels of many transcripts encoding proteins for hormone metabolism, cell-wall biogenesis, chloroplast functions and photosynthesis were repressed at 14 days p.i. and were associated with the highest levels of viral RNA in the host cells. A number of transcripts corresponding to genes involved in defence were either downregulated or not affected at the early stages of infection, but upregulated at the late stages, indicating that the plant immune response is not activated until the late time points of infection. Such a delayed defence response may be critical for SMV to establish its systemic infection.
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Rapid evolutionary dynamics of zucchini yellow mosaic virus
More LessZucchini yellow mosaic virus (ZYMV) is an economically important virus of cucurbit crops. However, little is known about the rate at which this virus has evolved within members of the family Cucurbitaceae, or the timescale of its epidemiological history. Herein, we present the first analysis of the evolutionary dynamics of ZYMV. Using a Bayesian coalescent approach we show that the coat protein of ZYMV has evolved at a mean rate of 5.0×10−4 nucleotide substitutions per site, per year. Notably, this rate is equivalent to those observed in animal RNA viruses. Using the same approach we show that the lineages of ZYMV sampled here have an ancestry that dates back no more than 800 years, suggesting that human activities have played a central role in the dispersal of ZYMV. Finally, an analysis of phylogeographical structure provides strong evidence for the in situ evolution of ZYMV within individual countries.
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- Other Agents
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A species barrier limits transmission of chronic wasting disease to mink (Mustela vison)
Transmissible mink encephalopathy (TME) occurs as sporadic outbreaks associated with ingestion of feed presumably contaminated with some type of prion disease. Mink lack a species barrier to primary oral challenge with bovine spongiform encephalopathy, whereas they have a barrier to such challenge with scrapie. We investigated whether mink have a species barrier to chronic wasting disease (CWD) by performing primary intracerebral (IC) and primary oral challenge with CWD-positive elk brain. Primary IC challenge resulted in clinical disease in two of eight mink at 31–33 months incubation. Affected mink had spongiform vacuolation and astrocytosis within the central nervous system and immunoreactivity to disease-associated prion protein (PrPd) in brain, retina and lymph node. CWD IC recipients had significantly lower brain vacuolation and PrPd deposition scores, significantly lower cerebrocortical astrocyte counts and significantly higher hippocampal astrocyte counts than TME IC recipients. Primary oral challenge with CWD-positive elk brain (n=22) or with CWD-negative elk brain given IC (n=7) or orally (n=23) did not result in clinical or microscopic abnormalities during 42 months observation. Novel prion gene polymorphisms were identified at codon 27 (arginine/tryptophan) and codon 232 (arginine/lysine). This study shows that, whilst CWD can cause disease when given IC to mink, the lesions are not characteristic of TME, transmission is inefficient compared with TME and oral challenge does not result in disease. The demonstration of a species barrier in cervid-to-mustelid prion transmission indicates that mink are unlikely to be involved in natural CWD transmission.
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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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