- Volume 87, Issue 12, 2006
Volume 87, Issue 12, 2006
- Animal
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- RNA viruses
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De novo infection and propagation of wild-type Hepatitis C virus in human T lymphocytes in vitro
More LessWhile exploring previous findings that ex vivo treatment of lymphoid cells from Hepatitis C virus (HCV)-infected individuals with T cell-stimulating mitogens augments detection of the residing virus, an in vitro HCV replication system was established, in which mitogen-induced T cell-enriched cultures served as HCV targets and the derived T cells multiplied virus during repeated serial passage. HCV replication was ascertained by detecting HCV RNA positive and negative strands, HCV NS5a and E2 proteins, release of HCV virions and nucleocapsids (confirmed by immunoelectron microscopy) and de novo infection of mitogen-induced T cells prepared from healthy donors. Further, affinity-purified normal human T lymphocytes were also susceptible to HCV infection in vitro and HCV replication was detected in pure T cells isolated from a patient with chronic hepatitis C. These results document that T cells can support propagation of HCV both in vivo and in vitro. The infection system established offers a valuable tool for in vitro studies on the entire cycle of HCV replication, virus cytopathogenicity and evaluation of antiviral agents against wild-type HCV in the natural host-cell milieu.
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A hepatitis C virus (HCV) NS3/4A protease-dependent strategy for the identification and purification of HCV-infected cells
As a tool for the identification and/or purification of hepatitis C virus (HCV)-infected cells, a chimeric form of the Gal4VP16 transcription factor was engineered to be activated only in the presence of the HCV NS3/4A protease and to induce different reporter genes [choramphenical acetyltransferase (CAT), green fluorescent protein (GFP) and the cell-surface marker H-2Kk] through the (Gal4)5-E1b promoter. For this, the NS5A/5B trans-cleavage motif of HCV of genotype 1a was inserted between Gal4VP16 and the N terminus of the endoplasmic reticulum (ER)-resident protein PERK, and it was demonstrated that it could be cleaved specifically by NS3/4A. Accordingly, transient transfection in tetracycline-inducible UHCV-11 cells expressing the HCV polyprotein of genotype 1a revealed the migration of the Gal4VP16 moiety of the chimera from the ER to the nucleus upon HCV expression. Activation of the chimera provoked specific gene induction, as shown by CAT assay, first in UHCV-11 cells and then in Huh-7 cells expressing an HCV replicon of genotype 1b (Huh-7 Rep). In addition, the GFP reporter gene allowed rapid fluorescence monitoring of HCV expression in the Huh-7 Rep cells. Finally, the chimera was introduced into Huh-7.5 cells infected with cell culture-generated HCV JFH1 (genotype 2a), allowing the purification of the HCV-infected cells by immunomagnetic cell sorting using H-2Kk as gene reporter. In conclusion, the Gal4VP16 chimera activation system can be used for the rapid identification and purification of HCV-infected cells.
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CD8+ T cell-mediated immune responses in West Nile virus (Sarafend strain) encephalitis are independent of gamma interferon
More LessThe flavivirus West Nile virus (WNV) can cause fatal encephalitis in humans and mice. It has recently been demonstrated, in an experimental model using WNV strain Sarafend and C57BL/6 mice, that both virus- and immune-mediated pathology is involved in WNV encephalitis, with CD8+ T cells being the dominant subpopulation of lymphocyte infiltrates in the brain. Here, the role of activated WNV-immune CD8+ T cells in mouse WNV encephalitis was investigated further. Passive transfer of WNV-immune CD8+ T cells reduced mortality significantly and prolonged survival times of mice infected with WNV. Early infiltration of WNV-immune CD8+ T cells into infected brains is shown, suggesting a beneficial contribution of these lymphocytes to recovery from encephalitis. This antiviral function was not markedly mediated by gamma interferon (IFN-γ), as a deficiency in IFN-γ did not affect mortality to two strains of WNV (Sarafend and Kunjin) or brain virus titres significantly. The cytolytic potential, as well as precursor frequency, of WNV-immune CD8+ T cells were not altered by the absence of IFN-γ. This was reflected in transfer experiments of WNV-immune CD8+ T cells from IFN-γ −/− mice into WNV-infected wild-type mice, which showed that IFN-γ-deficient T cells were as effective as those from WNV-immune wild-type mice in ameliorating disease outcome. It is speculated here that one of the pleiotropic functions of IFN-γ is mimicked by WNV-Sarafend-mediated upregulation of cell-surface expression of major histocompatibility complex antigens, which may explain the lack of phenotype of IFN-γ −/− mice in response to WNV.
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Avian virulence and thermostable replication of the North American strain of West Nile virus
The NY99 genotype of West Nile virus (WNV) introduced into North America has demonstrated high virulence for American crows (AMCRs), whilst a closely related WNV strain (KEN-3829) from Kenya exhibits substantially reduced virulence in AMCRs [ Brault, A. C., Langevin, S. A., Bowen, R. A., Panella, N. A., Biggerstaff, B. J., Miller, B. R. & Nicholas, K. (2004) . Emerg Infect Dis 10, 2161–2168]. Viruses rescued from infectious cDNA clones of both the NY99 and KEN-3829 strains demonstrated virulence comparable to that of their parental strains in AMCRs. To begin to define parameters that might explain the different virulence phenotypes between these two viruses, temperature-sensitivity assays were performed for both viruses at the high temperatures experienced in viraemic AMCRs. Growth curves of the two WNV strains were performed in African green monkey kidney (Vero; 37–42 °C) and duck embryonic fibroblast (DEF; 37–45 °C) cells cultured at temperatures that were tolerated by the cell line. Unlike the NY99 virus, marked decreases in KEN-3829 viral titres were detected between 36 and 120 h post-infection (p.i.) at temperatures above 43 °C. Replication of KEN-3829 viral RNA was reduced 6500-fold at 72 h p.i. in DEF cells incubated at 44 °C relative to levels of intracellular virus-specific RNA measured at 37 °C. In contrast, replication of virus derived from the NY99 infectious cDNA at 44 °C demonstrated only a 17-fold reduction in RNA level. These results indicated that the ability of WNV NY99 to replicate at the high temperatures measured in infected AMCRs could be an important factor leading to the increased avian virulence and emergence of this strain of WNV.
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MCP-1, a highly expressed chemokine in dengue haemorrhagic fever/dengue shock syndrome patients, may cause permeability change, possibly through reduced tight junctions of vascular endothelium cells
Vascular leakage, one hallmark of dengue haemorrhagic fever (DHF) and dengue shock syndrome, has been linked to the mediators secreted from cells in the circulatory system. In this study, extremely high expression levels of monocyte chemoattractant protein-1 (MCP-1) were found in the plasma of DHF patients compared with low MCP-1 expression levels in the plasma of enterovirus 71-infected patients. It was also found that MCP-1 expression was induced in dengue virus 2 (DV2)-infected monocytes and lymphocytes, but not in liver or endothelial cells. Exposing monolayers of human umbilical vein endothelial cells (HUVECs) to recombinant human MCP-1 (rhMCP-1) or to the culture supernatant of DV2-infected human monocytes increased the vascular permeability of the cells. MCP-1-neutralizing monoclonal antibody only partially prevented monolayer permeability change. Consistently, the distribution of the tight junction protein ZO-1 on the cellular membranes of HUVECs was disrupted by rhMCP-1 or by the conditioned medium of DV2-infected monocytes. In summary, it was found that the increased permeability and disrupted tight junctions of human vascular endothelium cells were effected through a mechanism partially dependent on MCP-1, which was secreted by DV2-infected monocytes and lymphocytes.
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Mutations in the palm region of a plus-strand RNA virus polymerase result in attenuated phenotype
More LessThe three-dimensional structure of RNA-dependent RNA polymerases (RdRps) is highly conserved among RNA viruses. In a previous study, a unique set of mutant strains of Bovine viral diarrhea virus was obtained, encompassing either a genomic deletion of six codons or duplications of between 1 and 45 codons; these mutations affect different parts of the palm region, the most conserved part of RdRps containing the catalytic centre. In the present study, a detailed characterization of the RdRp mutant viruses was performed, demonstrating different degrees of a small-plaque phenotype in cell culture, correlating with significantly reduced viral RNA synthesis and delayed virus replication. Taken together, the results of this study demonstrate a surprising flexibility within the palm region of a plus-strand RNA virus RdRp, resulting in viral attenuation in vitro. This interesting insight into an essential viral protein may have implications for the development of vaccines and attenuated viral vectors.
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Phosphorylation of human respiratory syncytial virus P protein at threonine 108 controls its interaction with the M2-1 protein in the viral RNA polymerase complex
More LessThe human respiratory syncytial virus (HRSV) P protein is phosphorylated, with different turnover rates, at several serine (S) and threonine (T) residues. The role of phosphothreonines in viral RNA synthesis was studied by using P protein substitution variants and the HRSV-based minigenome pM/SH. By using liquid chromatography coupled to ion-trap mass spectrometry, it was found that P protein T108 was phosphorylated by addition of a high-turnover phosphate group. This phosphorylation occurs in P protein expressed transiently and during HRSV infection. The results suggest that phosphorylation at P protein T108 affects M2-1 transcriptional activities, because this modification prevents interaction between the P and M2-1 proteins. Therefore, P protein phosphorylation–dephosphorylation at T108 could distinguish the role of the P protein in viral transcription and replication.
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Exchange of P/V genes between two non-cytopathic simian virus 5 variants results in a recombinant virus that kills cells through death pathways that are sensitive to caspase inhibitors
The paramyxovirus Simian virus 5 (SV5) is largely non-cytopathic in human epithelial and fibroblast cells. WF-PIV has been described previously as a naturally occurring SV5 variant that encodes P and V proteins differing from the wild-type (WT) SV5 proteins in eight and five amino acid positions, respectively. In this study, it is shown that WF-PIV is like WT SV5 by being largely non-cytopathic in A549 lung epithelial cells. However, substitution of the WF-PIV P/V gene into the background of WT SV5 resulted in a hybrid virus (P/V-WF) that induced apoptotic cell death not seen with either of the parental viruses. The kinetics of HeLa cell killing and induction of apoptosis by the P/V-WF chimera differed from those of the previously described P/V-CPI− chimera by being slower and less extensive. HeLa cell killing by the P/V-WF chimera was effectively reduced by inhibitors of caspase-9, but not of caspase-8. These results demonstrate that an exchange of P/V genes from two non-cytopathic SV5 variants can produce apoptosis-inducing chimeras, and that the role of the SV5 P/V gene products in limiting apoptosis can be dependent on expression in the context of a native viral genome.
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A single amino acid substitution in the V protein of Nipah virus alters its ability to block interferon signalling in cells from different species
More LessThe V protein of the paramyxovirus Nipah virus (NiV) has been shown to antagonize the interferon (IFN) response in human cells via sequestration of STAT1 and STAT2. This study describes a mutant of the NiV V protein, referred to as V(AAHL), that is unable to antagonize IFN signalling and demonstrates that a single amino acid substitution is responsible for its inactivity. The molecular basis for this was identified as a failure to interact with STAT1 and STAT2. It was also shown that NiV V, but not V(AAHL), was functional as an IFN antagonist in human, monkey, rabbit, dog, horse, pig and bat cells, which suggests that the ability of NiV to block IFN signalling is not a major constraint that prevents this virus from crossing species barriers.
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Recent H5N1 avian Influenza A virus increases rapidly in virulence to mice after a single passage in mice
To evaluate the potential pathogenicity to mammals of the recent H5N1 avian Influenza A virus, viruses recovered from dead mice infected with A/chicken/Yamaguchi/7/2004 isolated in Japan were examined. All recovered viruses from the brains of dead mice infected with this strain (without any prior adaptation to mice) had substituted the amino acid at position 627 of the PB2 protein from glutamic acid to lysine. Their mouse lethality had increased by approximately 5×104 times over that of the original virus. Histopathological analysis reinforced the finding that these variants caused more rapid and severe damage to mice than the original virus. This revealed that it might be useful to characterize the recovered virus to assess its potential pathogenicity to mammals.
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Cultured skin fibroblast cells derived from bluetongue virus-inoculated sheep and field-infected cattle are not a source of late and protracted recoverable virus
A recent hypothesis to explain the recurrence of bluetongue disease after winter seasonal absences of the vector has suggested a role for persistent infection of sheep. This report presents combined independent work from two laboratories investigating the possible recovery of Bluetongue virus (BTV) over a protracted period after infection of both sheep and cattle. Prior to infection with either cell-culture-adapted or non-culture-adapted BTV, sheep were subjected to a preliminary exposure to Culicoides sp. insects, which reportedly facilitates recovery of virus from infected sheep several months post-infection (p.i.). A series of skin biopsies at different intervals p.i. was used to establish skin fibroblast (SF) cultures from which attempts were made to detect virus by isolation and by molecular and immunological methods. Also examined was the effect on virus recovery of additional exposure to Culicoides sp. prior to skin biopsy during the post-inoculation period. A herd of cattle sentinels for surveillance of natural BTV infection in northern Australia was monitored prospectively for seroconversion. Evidence of infection initiated attempted virus recovery by establishing SF cultures. It was found that in both cattle and sheep there was not a protracted period over which BTV could be recovered from SF cultures. The data do not support a general hypothesis that BTV persists in either sheep or cattle.
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In vitro and in vivo identification of structural and sequence elements in the 5′ untranslated region of Ectropis obliqua picorna-like virus required for internal initiation
More LessEctropis obliqua picorna-like virus (EoPV) is a newly described insect virus that is classified as a putative member of the genus Iflavirus. The virus possesses a large, positive-sense RNA genome encoding a single polyprotein that shares physicochemical properties with those of members of the family Picornaviridae. The 5′ untranslated region (5′ UTR) plays an important role in picornavirus translation initiation, as it contains an internal ribosome entry site (IRES) that mediates cap-independent translation. To investigate translation in EoPV, an extensive range of mutations were engineered within the 5′ UTR and the effects of these changes were examined in vitro and in vivo by using a bicistronic construct. Results showed that deletions within the first 63 nt had little impact on IRES activity, whilst core IRES function was contained within stem–loops C and D, as their removal abrogated IRES activity significantly. In contrast to these findings, removal of stem–loop G containing two cryptic AUGs caused a remarkable increase in IRES activity, which was further investigated by site-directed mutagenesis at these two positions. It was also confirmed that initiation of protein synthesis occurs at AUG6 (position 391–394) and not at the AUG immediately downstream of the polypyrimidine tract. Mutation of the polypyrimidine tract (CCTTTC) had a slight effect on EoPV IRES activity. Furthermore, mutations of the RAAA motif led to a decrease in IRES activity of approximately 40 % in vitro, but these results were not supported by in vivo experiments. In conclusion, this study reveals that the EoPV IRES element is unique, although it has features in common with the type II IRESs.
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Characterization of the 5′ internal ribosome entry site of Plautia stali intestine virus
More LessThe RNA genome of Plautia stali intestine virus (PSIV; Cripavirus, Dicistroviridae) contains two open reading frames, the first of which is preceded by a 570 nt untranslated region (5′ UTR). The 5′ UTR was confirmed to be an internal ribosome entry site (IRES) using an insect cell lysate translation system: translation of a second cistron increased 14-fold in the presence of the 5′ UTR and a cap analogue did not inhibit translation of the second cistron. Deletion analysis showed that 349 bases corresponding to nt 225–573 in the PSIV genome were necessary for internal initiation. The PSIV 5′ IRES did not function in rabbit reticulocyte lysate or wheatgerm translation systems; however, the intergenic IRES for capsid translation of PSIV was functional in both systems, indicating that the 5′ IRES and the intergenic IRES have distinct requirements for their activities. Chemical and enzymic analyses of the 5′ IRES of PSIV indicate that its structure is distinct from that of Rhopalosiphum padi virus. Because 5′ IRES elements in some dicistroviruses have been reported to be active in plant and mammalian cell-free translation systems, there appears to be variation among dicistroviruses in the mechanism of translation initiation mediated by 5′ IRES elements.
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- DNA viruses
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Early herpes simplex virus type 1 infection is dependent on regulated Rac1/Cdc42 signalling in epithelial MDCKII cells
The aim of this study was to understand how molecular determinants of epithelial cells influence initial infection by herpes simplex virus type 1 (HSV-1). Upon infection of the epithelial MDCKII cell line, enhanced association of virus particles with cells forming actin protrusions was observed, suggesting a putative role of actin dynamics in HSV-1 infection. Thus, the impact of the small Rho-like GTPases Rac1, Cdc42 and RhoA acting as key regulators of actin dynamics was addressed. Endogenous Rac1 and Cdc42 were temporarily activated at 15 and 30 min after HSV-1 infection. When constitutively active Cdc42 or Rac1 mutants were expressed transiently, a significant decrease in infectivity was observed, whereas expression of RhoA mutants had no influence. Furthermore, dominant-negative Cdc42 led to decreased infectivity, whereas dominant-negative Rac1 had no effect. So far, the study of potential effectors indicated that Rac1/Cdc42 mutants inhibited infectivity independently of p21-activated kinase (Pak1). The inhibitory effect of Rac1/Cdc42 mutant expression on HSV-1 infection was characterized further and it was found that binding, internalization and transport of HSV-1 were not affected by expression of Rac1/Cdc42 mutants. Thus, these results provide the first evidence for a role of Rac1/Cdc42 signalling during early HSV-1 infection and suggest a mechanism relying on virus-induced regulation of Rac1/Cdc42 activities.
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Competition and complementation between thymidine kinase-negative and wild-type herpes simplex virus during co-infection of mouse trigeminal ganglia
More LessLaboratory strains of herpes simplex virus lacking thymidine kinase (TK) cannot replicate acutely to detectable levels in mouse trigeminal ganglia and do not reactivate from latency. However, many pathogenic clinical isolates that are resistant to the antiviral drug acyclovir are heterogeneous populations of TK-negative (TK−) and TK-positive (TK+) viruses. To recapitulate this in vivo, mice were infected with mixtures of wild-type virus and a recombinant TK− mutant in various ratios. Following co-infection, the replication, number of latent viral genomes and reactivation efficiency of TK+ virus in trigeminal ganglia were reduced in a manner related to the amount of TK− virus in the inoculum. TK+ virus did not always complement the acute replication or increase the number of latent viral genomes of TK− mutant in mouse ganglia. Even so, TK+ virus could still confer the pathogenic phenotype to a TK− mutant, somehow providing sufficient TK activity in trans to permit a TK− mutant to reactivate from latently infected ganglia.
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Pseudorabies virus particles lacking tegument proteins pUL11 or pUL16 incorporate less full-length pUL36 than wild-type virus, but specifically accumulate a pUL36 N-terminal fragment
More LessProteins of the virion tegument of alphaherpesviruses are involved in protein–protein interactions, which play important roles in virus morphogenesis. Seven single-gene deletion mutants of Pseudorabies virus were analysed for alterations in the overall composition of the virion beyond the loss of the targeted protein. The UL36 protein (pUL36) was present in equal amounts in wild-type virions and mutants lacking pUL21, pUL49, pUL51, pUS3 or pUS8. Virions lacking pUL11 or pUL16 incorporated less full-length pUL36 than wild-type particles, but contained increased amounts of an N-terminal fragment of pUL36 that is present only in traces in wild-type virus and the other mutants.
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Genome sequences of two frog herpesviruses
More LessThe sequences of two frog herpesviruses, Ranid herpesvirus 1 and Ranid herpesvirus 2, were determined. They are respectively 220 859 and 231 801 bp in size and contain 132 and 147 predicted genes. The genomes are related most closely in the central regions, where 40 genes are conserved convincingly. Nineteen of these genes are also conserved in a fish herpesvirus, Ictalurid herpesvirus 1. The terminal regions of the genomes are largely not conserved and contain many of the 15 families of related genes present in each genome. The frog herpesviruses are unique among sequenced herpesviruses in that the three exons of the gene encoding the putative ATPase subunit of terminase are not specified by the same DNA strand and in that they encode a putative DNA (cytosine-5-)-methyltransferase and have extensively methylated genomes.
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Murine gammaherpesvirus-68 glycoprotein B presents a difficult neutralization target to monoclonal antibodies derived from infected mice
More LessPersistent viruses disseminate from immune hosts. They must therefore resist neutralization by antibody. Murine gammaherpesvirus-68 (MHV-68) represents an accessible model with which to address how resistance to neutralization is achieved and how overcoming it might improve infection control. The MHV-68 glycoprotein B (gB), like that of other herpesviruses, is a virion protein that is essential for infectivity. As such, it presents a potential neutralization target. In order to test whether virus-induced antibodies reduce virion infectivity by binding to gB, monoclonal antibodies (mAbs) were derived from MHV-68-infected mice. gB-specific mAbs were common, but only an IgM specific for the gB N terminus reduced virion infectivity significantly. It inhibited MHV-68 entry into BHK-21 cells at a post-binding step that was linked closely to membrane fusion. Reducing the mAb to IgM monomers compromised neutralization severely, suggesting that a pentameric structure was crucial to its function. Antibody treatment never blocked BHK-21 cell infection completely and blocked the infection of NMuMG epithelial cells hardly at all. Virions saturated with antibody also remained infectious to mice. Thus, the MHV-68 gB presents at best a very difficult target for antibody-mediated neutralization.
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High levels of retroperitoneal fibromatosis (RF)-associated herpesvirus in RF lesions in macaques are associated with ORF73 LANA expression in spindleoid tumour cells
Two distinct lineages of rhadinoviruses related to Kaposi's sarcoma (KS)-associated herpesvirus (KSHV; Human herpesvirus 8), the causative agent of KS, have been identified. In macaques, the RV1 lineage is represented by retroperitoneal fibromatosis (RF) herpesvirus (RFHV), the homologue of KSHV, whilst the RV2 lineage is represented by rhesus rhadinovirus (RRV), a more distantly related virus. Real-time quantitative PCR was used to estimate the loads of RV1 and RV2 rhadinoviruses in simian acquired immunodeficiency syndrome-associated RF (SAIDS-RF), a neoplasm of macaques with similarities to AIDS-associated KS. Both RV1 and RV2 rhadinoviruses were detected in macaques with RF. The RV1 loads were 220- to 4300-fold higher in RF tumours than in spleen, showing a strong tumour association (mean loads of 1 800 000 vs 2900 copies per 106 cells in tumours and spleen, respectively). In contrast, RV2 loads in the RF tumours were 100-fold lower than RV1 loads and showed similar levels in tumours and spleen (mean loads of 16 000 vs 24 000 copies per 106 cells, respectively). Immunostaining with antibodies reactive against RFHV ORF73 latency-associated nuclear antigen (LANA) showed intense nuclear staining of the spindleoid RF tumour cells. Correlation of viral load and the number of LANA-positive cells indicated that RF tumour cells contained multiple copies of the RFHV genome per cell. This pattern of infectivity is similar to that seen in KS tumours latently infected with KSHV. Our study demonstrates similarities in the biology of KSHV and RFHV and supports a role for RFHV in the aetiology of SAIDS-RF.
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E1A promoter of bovine adenovirus type 3
More LessConserved motifs of eukaryotic gene promoters, such as TATA box and CAAT box sequences, of E1A of human adenoviruses (e.g human adenovirus 5) lie between the left inverted terminal repeat (ITR) and the ATG of E1A. However, analysis of the left end of the bovine adenovirus 3 (BAdV-3) genome revealed that the conserved sequences of the E1A promoter are present only in the ITR. As such, the promoter activity of ITR was tested in the context of a BAdV-3 vector or a plasmid-based system. Different regions of the left end of the BAdV-3 genome initiated transcription of the red fluorescent protein gene in a plasmid-based system. Moreover, BAdV-3 mutants in which the open reading frame of E1A was placed immediately downstream of the ITR produced E1A transcript and could be propagated in non-E1A-complementing Madin–Darby bovine kidney cells. These results suggest that the left ITR contains the sole BAdV-3 E1A promoter.
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Retention of 1.2 kbp of ‘novel’ genomic sequence in two European field isolates and some vaccine strains of Fowlpox virus extends open reading frame fpv241
More LessThe emergence of variant fowlpox viruses (FWPVs) and increasing field use of recombinants against avian influenza H5N1 emphasize the need to monitor vaccines and to distinguish them from field strains. Five commercial vaccines, two laboratory viruses and two European field isolates were characterized by PCR and sequencing at 18 loci differing between attenuated FP9 and its pathogenic progenitor. PCR failed to discriminate between the viruses and sequence determination revealed no significant differences at any locus, except for a polymorphic locus encompassed by deletion 24 (9.3 kbp) in FP9. Surprisingly, ‘novel’ previously unreported sequence (spanning 1.2 kbp) was found in both European field isolates and three of the vaccines. It was absent from the other two vaccines, removed by a 1.2 kbp deletion identical to that surprisingly also observed in the completely sequenced genome of FPV USDA. This locus (H9) adds a potentially useful tool for discriminating between FWPV field isolates and vaccines.
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Detection of the prototype of a potential novel genus in the family Papillomaviridae in association with canine epidermodysplasia verruciformis
More LessEpidermodysplasia verruciformis (EV) is a rare human genetic predisposition to develop flat warts, some of which subsequently undergo cancer transformation. Some human papillomaviruses (HPVs), i.e. HPV 5 and 8, have been associated with cancer development as a sequela of EV. As similar diseases have been observed in dogs, it was hypothesized that unknown canine papillomaviruses (CPVs) may exist and that they may be present in cases of canine EV. Consequently, DNA was extracted from a malignant lesion of a dog with EV and circular DNA was amplified by multiple-primed rolling-circle amplification (RCA). Indeed, sequence determination and analysis of the RCA-amplified and cloned DNA from a malignant canine EV lesion resulted in the detection and primary description of a third CPV (CPV3). Typical papillomavirus genes were identified, with deduced amino acid similarities ranging from 20 to 57 % for E1, E2, E6, E7, L1 and L2, respectively. According to the sequence of the L1 gene, which is used for papillomavirus classification, the new isolate meets the majority of criteria needed to declare detection of a novel genus among the papillomaviruses. Thus, CPV3 may represent the prototype of this novel genus. As the novel virus was found in a dog in association with lesions reminiscent of human EV, it should be interesting to test in the future whether this condition can be reproduced in experimental animals. If such were the case, a new model for EV could be established.
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Isolation and characterization of the first American bottlenose dolphin papillomavirus: Tursiops truncatus papillomavirus type 2
A novel papillomavirus (PV) was isolated from a genital condyloma of a free-ranging bottlenose dolphin inhabiting the coastal waters of Charleston Harbor, SC, USA: Tursiops truncatus papillomavirus type 2 (TtPV2). This novel virus represents the first isolated North American cetacean PV and the first American bottlenose dolphin PV. After the viral genome was cloned, sequenced and characterized genetically, phylogenetic analyses revealed that TtPV2 is most similar to the only published cetacean PV isolated and characterized thus far, Phocoena spinipinnis PV type 1 (PsPV1). A striking feature of the genome of TtPV2, as well as that of PsPV1, is the lack of an E7 open reading frame, which typically encodes one of the oncogenic proteins believed to be responsible for malignant transformation in the high-risk mucosotropic human papillomaviruses (HPVs). TtPV2 E6 contains a PDZ-binding motif that has been shown to be involved in transformation in the case of high-risk genital HPVs.
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Short regions of sequence identity between the genomes of human and rodent parvoviruses and their respective hosts occur within host genes for the cytoskeleton, cell adhesion and Wnt signalling
More LessOur understanding of the mechanism(s) of pathogenesis and persistence of vertebrate parvoviruses remains incomplete. With the recent availability of the complete genome sequences of human, rat and mouse, and the ability to search these sequences and to locate matches to exact genomic regions, further insight into the interaction of parvoviruses with their human and rodent hosts is possible. To determine the extent and nature of sequence identity between candidate parvoviruses and their respective hosts, blast searches of the genome sequences of adeno-associated virus, parvovirus B19, mouse parvovirus, the prototype strain and immunosuppressant variant of minute virus of mouse, Kilham rat virus and rat parvovirus were performed against the genome(s) of their respective hosts (human, rat and mouse) using the resources of the NCBI and the Celera Discovery System. Regions of identity and similarity were mapped to their precise location in their particular host genome. For each virus, between one and 12 identical regions were found. Each identical region was 17–26 nt and was generally found at multiple sites within the particular host genome. These identical regions were predominantly located in non-coding regions of particular host genes and in intergenic regions. The ontology of host genes in which identical regions were found for each of the nine virus–host interactions highlighted several pathways/processes, including the cytoskeleton, cell adhesion and Wnt signalling. Within each virus species, these homologous regions were highly conserved (100 % identity in 16 out of 23 alignments where more than one sequence was available). All of these aspects suggest a particular advantage to the viruses of the presence of these sequences.
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- Plant
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Tomato yellow spot virus, a tomato-infecting begomovirus from Brazil with a closer relationship to viruses from Sida sp., forms pseudorecombinants with begomoviruses from tomato but not from Sida
More LessGeminiviruses are characterized by a circular, single-stranded DNA genome and twinned icosahedral particles. Begomoviruses (whitefly-transmitted geminiviruses) are a major constraint to crop production worldwide. In Brazil, tomato-infecting begomoviruses emerged as serious pathogens over the last 10 years, due to the introduction of a new biotype of the insect vector. Tomato yellow spot virus (ToYSV) is a newly described begomovirus originally isolated from tomato, but phylogenetically closer to viruses from Sida sp. A study was performed to determine the viability of pseudorecombinants formed between the DNA components of ToYSV and other weed- and tomato-infecting begomoviruses from Brazil. Despite its closer relationship to weed-infecting viruses, ToYSV was only capable of forming viable pseudorecombinants with tomato viruses. An infectious pseudorecombinant formed between ToYSV DNA-A and tomato crinkle leaf yellows virus (TCrLYV) DNA-B induced severe symptoms in Nicotiana benthamiana. This was attributed, at least in part, to the fact that the origins of replication of both components had identical Rep-binding sequences. However, this was not the case for another infectious pseudorecombinant formed between tomato golden mosaic virus (TGMV) DNA-A and ToYSV DNA-B, which have different Rep-binding sequences. These results reinforce the notion that pseudorecombinant formation cannot be explained solely on the basis of phylogenetic relationships and conserved iteron sequences, and suggest that the TGMV Rep protein may be more versatile in terms of recognizing heterologous DNA components than that of ToYSV.
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Multiple virus resistance at a high frequency using a single transgene construct
RNA silencing is a natural antiviral defence in plants, which can be exploited in transgenic plants for preprogramming virus recognition and ensuring enhanced resistance. By arranging viral transgenes as inverted repeats it is thus possible to obtain strong repression of incoming viruses. Due to the high sequence specificity of RNA silencing, this technology has hitherto been limited to the targeting of single viruses. Here it is shown that efficient simultaneous targeting of four different tospoviruses can be achieved by using a single small transgene based on the production of minimal sized chimaeric cassettes. Due to simultaneous RNA silencing, as demonstrated by specific siRNA accumulation, the transgenic expression of these cassettes rendered up to 82 % of the transformed plant lines heritably resistant against all four viruses. Thus RNA silencing can be further improved for high frequency multiple virus resistance by combining small RNA fragments from a series of target viruses.
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- Fungal
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Synergism between a mycoreovirus and a hypovirus mediated by the papain-like protease p29 of the prototypic hypovirus CHV1-EP713
More LessInfection of the chestnut blight fungus, Cryphonectria parasitica, by the prototypic hypovirus Cryphonectria hypovirus 1-EP713 (CHV1-EP713) or by the type member, Mycoreovirus 1-Cp9B21 (MyRV1-Cp9B21), of a novel genus (Mycoreovirus) of the family Reoviridae results in hypovirulence, but with a different spectrum of phenotypic changes. The former virus depresses pigmentation and conidiation dramatically, whilst the latter virus has little effect on these processes. This study showed that double infection by the two viruses resulted in a phenotype similar to that of CHV1-EP713 singly infected colonies, but with further decreased levels of host conidiation and vegetative growth and increased levels of MyRV1-Cp9B21 genomic dsRNA accumulation (twofold) and vertical transmission (sixfold). In contrast, CHV1-EP713 RNA accumulation was not altered by MyRV1-Cp9B21 infection. It was also found that the papain-like cysteine protease p29, encoded by CHV1-EP713 ORF A, contributes to the phenotypic alterations and transactivation of MyRV1-Cp9B21 replication and transmission. Chromosomally expressed p29 was able to increase MyRV1-Cp9B21 vertical transmission by more than twofold and genomic RNA accumulation by 80 %. Transactivation was abolished by Cys→Gly mutations at p29 residues 70 and 72 located within the previously identified symptom-determinant domain required for suppression of host pigmentation and sporulation and p29-mediated in trans enhancement of homologous Δp29 mutant virus RNA replication. Transactivation was not altered by Ser substitutions at the p29 protease catalytic residue Cys162. These results indicated a link between p29-mediated enhancement of heterologous virus accumulation and transmission and p29-mediated host symptom expression. The role of p29 as a suppressor of RNA silencing is discussed.
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- Other Agents
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Immunological characterization of abnormal prion protein from atypical scrapie cases in sheep using a panel of monoclonal antibodies
More LessAfter the implementation of an active surveillance programme for scrapie in sheep in the EU, the number of diagnosed classical scrapie cases rose sharply and a novel kind of so-called atypical scrapie case was discovered. These atypical scrapie cases display unusual features concerning the distribution of the abnormal prion protein (PrPSc) in the brain, a distinct electrophoretic profile of PrPSc and an inconsistent reaction pattern in the currently used rapid tests. In this report, PrPSc of two German atypical sheep scrapie cases was characterized by epitope mapping using a panel of 18 monoclonal antibodies that were directed against epitopes located throughout the prion protein. This analysis suggests that PrPSc derived from atypical scrapie cases and treated with proteinase K is largely composed of an 11 kDa fragment (previously referred to as the 12 kDa band) and of polymeric fragments thereof. The 11 kDa band corresponds to a prion protein fragment spanning approximately aa 90–153 and may therefore represent a novel PrPSc type.
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Prion protein in the cerebrospinal fluid of healthy and naturally scrapie-affected sheep
The aim of this study was to characterize the cerebrospinal fluid (CSF) prion protein (PrP) of healthy and naturally scrapie-affected sheep. The soluble form of CSF PrPC immunoblotted with an anti-octarepeat and an anti-C terminus mAb showed two isoforms of approximately 33 and 26 kDa, corresponding to the biglycosylated and unglycosylated isoforms of brain PrPC. Neither the mean concentration nor the electrophoretic profile of CSF PrP differed between healthy and scrapie-affected sheep, whereas a slightly increased resistance of CSF PrP to mild proteolysis by proteinase K was evident in the CSF of scrapie-affected sheep. No difference in susceptibility to proteolysis was observed between the two ARR and VRQ genetic variants of the purified prokaryote recombinant PrP. It was concluded that the physicochemical properties of PrPC in the CSF could be altered during scrapie and that these changes might reflect the physiopathological process of prion disease.
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A case–control study of scrapie Nor98 in Norwegian sheep flocks
More LessScrapie is a fatal, neurological disease of sheep and goats and belongs to the transmissible spongiform encephalopathies. In 1998, a new type of scrapie, designated scrapie Nor98, was detected in Norway. Scrapie Nor98 differs from classical scrapie in the distribution of pathological changes and of the scrapie prion protein, the Western blot profile of the prion protein, and with isolated cases usually being observed in the case flocks. In 2004, a case–control study was conducted on scrapie Nor98 with 28 cases and 102 randomly selected controls. The questionnaire included questions on demographic data, animal contact between sheep flocks, indirect contact with equipment, use of concentrate feed and supplemental feeds, and use of medicines and vaccines. The data were analysed by using logistic regression with the sheep flock as the statistical unit. In the final model, the detection of scrapie Nor98 was related to the practice of not removing all afterbirths, the use of vitamin and mineral feed supplements, the absence of concentrate feed of swine or poultry on the farm and the presence of dogs on the farm. The results show that the epidemiology of scrapie Nor98 differs from that of classical scrapie in that no risk factors that indicate transmission of scrapie Nor98 between flocks by movement or direct contact between animals were found. Furthermore, the association between scrapie Nor98 and mineral intake shown herein should be explored further. Although the possibility that scrapie Nor98 has a low transmissibility between animals under natural conditions cannot be ruled out, the results would also be in accordance with a spontaneous aetiology.
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Infectious agent of sheep scrapie may persist in the environment for at least 16 years
More LessIn 1978, a rigorous programme was implemented to stop the spread of, and subsequently eradicate, sheep scrapie in Iceland. Affected flocks were culled, premises were disinfected and, after 2–3 years, restocked with lambs from scrapie-free areas. Between 1978 and 2004, scrapie recurred on 33 farms. Nine of these recurrences occurred 14–21 years after culling, apparently as the result of environmental contamination, but outside entry could not always be absolutely excluded. Of special interest was one farm with a small, completely self-contained flock where scrapie recurred 18 years after culling, 2 years after some lambs had been housed in an old sheep-house that had never been disinfected. Epidemiological investigation established with near certitude that the disease had not been introduced from the outside and it is concluded that the agent may have persisted in the old sheep-house for at least 16 years.
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Ovine prion protein variant A136R154L168Q171 increases resistance to experimental challenge with bovine spongiform encephalopathy agent
More LessSusceptibility and incubation periods of transmissible spongiform encephalopathies, such as scrapie in sheep, are modulated by the PrP gene. The standard model of association between ovine PrP genetics and classical scrapie susceptibility is based on PrP genotypes with respect to codons 136, 154 and 171, e.g. alanine–arginine–glutamine (ARQ). It is demonstrated here that a proline to leucine substitution in codon 168 of the ovine PrP protein gene is associated with increased resistance to experimental bovine spongiform encephalopathy (BSE) inoculation. The ARL168Q PrP allele was found in heterozygous ARP168Q/ARL168Q sheep that have so far survived intravenous BSE challenge three times longer than BSE-challenged homozygous ARP168Q/ARP168Q sheep, which develop disease in around 700 days. In contrast, the L141F polymorphism does not appear to be associated with susceptibility to intravenous BSE challenge.
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A novel, resistance-linked ovine PrP variant and its equivalent mouse variant modulate the in vitro cell-free conversion of rPrP to PrPres
More LessPrion diseases are associated with the conversion of the normal cellular prion protein, PrPc, to the abnormal, disease-associated form, PrPSc. This conversion can be mimicked in vitro by using a cell-free conversion assay. It has recently been shown that this assay can be modified to use bacterial recombinant PrP as substrate and mimic the in vivo transmission characteristics of rodent scrapie. Here, it is demonstrated that the assay replicates the ovine polymorphism barriers of scrapie transmission. In addition, the recently identified ovine PrP variant ARL168Q, which is associated with resistance of sheep to experimental BSE, modulates the cell-free conversion of ovine recombinant PrP to PrPres by three different types of PrPSc, reducing conversion efficiencies to levels similar to those of the ovine resistance-associated ARR variant. Also, the equivalent variant in mice (L164) is resistant to conversion by 87V scrapie. Together, these results suggest a significant role for this position and/or amino acid in conversion.
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Synergistic and strain-specific effects of bovine spongiform encephalopathy and scrapie prions in the cell-free conversion of recombinant prion protein
This study describes the conversion of murine PrPC by PrPSc from three different mouse scrapie strains (ME7, 87V and 22A) and from a mouse-passaged bovine spongiform encephalopathy (BSE) strain (BSE/Bl6). This was demonstrated by a modified, non-radioactive, cell-free conversion assay using bacterial prion protein, which was converted into a proteinase K (PK)-resistant fragment designated PrPres. Using this assay, newly formed PrPres could be detected by an antibody that discriminated de novo PrPres and the original PrPSc seed. The results suggested that PrPres formation occurs in three phases: the first 48 h when PrPres formation is delayed, followed by a period of substantially accelerated PrPres formation and a plateau phase when a maximum concentration of PrPres is reached after 72 h. The conversion of prokaryotically expressed PrPC by ME7 and BSE prions led to unglycosylated, PK-digested, abnormal PrPres fragments, which differed in molecular mass by 1 kDa. Therefore, prion strain phenotypes were retained in the cell-free conversion, even when recombinant PrPC was used as the substrate. Moreover, co-incubation of ME7 and BSE prions resulted in equal amounts of both ME7- and BSE-derived PrPres fragments (as distinguished by their different molecular sizes) and also in a significantly increased total amount of de novo-generated PrPres. This was found to be more than twice the amount of either strain when incubated separately. This result indicates a synergistic effect of both strains during cell-free conversion. It is not yet known whether such a cooperative action between BSE and scrapie prions also occurs in vivo.
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Transmission and characterization of bovine spongiform encephalopathy sources in two ovine transgenic mouse lines (TgOvPrP4 and TgOvPrP59)
More LessTransgenic mice expressing the prion protein (PrP) of species affected by transmissible spongiform encephalopathies (TSEs) have recently been produced to facilitate experimental transmission of these diseases by comparison with wild-type mice. However, whilst wild-type mice have largely been described for the discrimination of different TSE strains, including differentiation of agents involved in bovine spongiform encephalopathy (BSE) and scrapie, this has been only poorly described in transgenic mice. Here, two ovine transgenic mouse lines (TgOvPrP4 and TgOvPrP59), expressing the ovine PrP (A136 R154 Q171) under control of the neuron-specific enolase promoter, were studied; they were challenged with brainstem or spinal cord from experimentally BSE-infected sheep (AA136 RR154 QQ171 and AA136 RR154 RR171 genotypes) or brainstem from cattle BSE and natural sheep scrapie. The disease was transmitted successfully from all of these sources, with a mean of approximately 300 days survival following challenge with material from two ARQ-homozygous BSE-infected sheep in TgOvPrP4 mice, whereas the survival period in mice challenged with material from the ARR-homozygous BSE-infected sheep was 423 days on average. It was shown that, in the two ovine transgenic mouse lines, the Western blot characteristics of protease-resistant PrP (PrPres) were similar, whatever the BSE source, with a low apparent molecular mass of the unglycosylated glycoform, a poor labelling by P4 monoclonal antibody and high proportions of the diglycosylated form. With all BSE sources, but not with scrapie, florid plaques were observed in the brains of mice from both transgenic lines. These data reinforce the potential of this recently developed experimental model for the discrimination of BSE from scrapie agents.
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Passage of chronic wasting disease prion into transgenic mice expressing Rocky Mountain elk (Cervus elaphus nelsoni) PrPC
Chronic wasting disease (CWD) of elk (Cervus elaphus nelsoni) and mule deer (Odocoileus hemionus) is one of three naturally occurring forms of prion disease, the others being Creutzfeldt–Jakob disease in humans and scrapie in sheep. In the last few decades, CWD has spread among captive and free-ranging cervids in 13 US states, two Canadian provinces and recently in Korea. The origin of the CWD agent(s) in cervids is not known. This study describes the development of a transgenic mouse line (TgElk) homozygous for a transgene array encoding the elk prion protein (PrPC) and its use in propagating and simulating CWD in mice. Intracerebral injection of one mule deer and three elk CWD isolates into TgElk mice led to disease with incubation periods of 127 and 95 days, respectively. Upon secondary passage, the incubation time was reduced to 108 and 90 days, respectively. Upon passage into TgElk mice, CWD prions (PrPSc) maintained the characteristic Western blot profiles seen in CWD-affected mule deer and elk and produced histopathological modifications consistent with those observed in the natural disease. The short incubation time observed on passage from cervid to mouse with both mule deer and elk CWD brain homogenates and the demonstrated capacity of the animals to propagate (mouse to mouse) CWD agents make the TgElk line a valuable model to study CWD agents in cervid populations. In addition, these results with this new transgenic line suggest the intriguing hypothesis that there could be more than one strain of CWD agent in cervids.
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- Jgv Direct
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Proteolytic maturation of replicase polyprotein pp1a by the nsp4 main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp7
More LessThe positive-stranded RNA genome of the arterivirus Equine arteritis virus (order Nidovirales) encodes the partially overlapping replicase polyproteins pp1a (1727 aa) and pp1ab (3175 aa). Previously, three viral proteinases were reported to cleave these large polyproteins into 12 non-structural proteins (nsps). The chymotrypsin-like viral main proteinase residing in nsp4 is responsible for eight of these cleavages. Processing of the C-terminal half of pp1a (the nsp3–8 region) was postulated to occur following either of two alternative proteolytic pathways (the ‘major’ and ‘minor’ pathways). Here, the importance of these two pathways was investigated by using a reverse-genetics system and inactivating each of the cleavage sites by site-directed mutagenesis. For all of these pp1a cleavage sites, mutations that prevented cleavage by the nsp4 proteinase were found to block or severely inhibit EAV RNA synthesis. Furthermore, our studies identified a novel nsp4 cleavage site (Glu-1575/Ala-1576) that is located within nsp7 and is conserved in arteriviruses. The N-terminal nsp7 fragment (nsp7α) derived from this cleavage was detected in lysates of both EAV-infected cells and cells transiently expressing pp1a. Mutagenesis of the novel cleavage site in the context of an EAV full-length cDNA clone proved to be lethal, underlining the fact that the highly regulated, nsp4-mediated processing of the C-terminal half of pp1a is a crucial event in the arterivirus life cycle.
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Volumes and issues
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Volume 105 (2024)
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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)