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Volume 77,
Issue 12,
1996
Volume 77, Issue 12, 1996
- Review Article
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Variation on a theme of Creutzfeldt-Jakob disease: implications of new cases with a young age at onset
More LessCreutzfeldt-Jakob disease (CJD) belongs to a group of human and animal diseases, distinguished from each other by differences in their clinical and neuropathological presentation or aetiology, but known collectively as the prion diseases. Prion diseases are unique amongst transmissible diseases in that the transmissible ‘agent’ does not appear to contain any nucleic acid and the only protein known to be associated with infectivity is host-coded. Prion disease is endemic in certain species, notably humans and sheep. It has also appeared in epidemic form in humans: for example, amongst tribal peoples of Papua New Guinea, where the disease is known as kuru, and in Western societies as a consequence of iatrogenic misadventure. Epidemic prion disease also occurs in animals: for example, in cattle [as the current epidemic of BSE (bovine spongiform encephalopathy) in Britain (Wells & Wilesmith, 1995)] and in farmed mink (Marsh, 1992).
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- SGM Special Lecture
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Biological consequences of human immunodeficiency virus type 1 envelope polymorphism: does variation matter?
More LessHuman immunodeficiency virus type 1 (HIV-1) establishes persistent infections in humans, in most cases leading to the development of AIDS. HIV-1 infects CD4+ lymphocytes, monocytes and dendritic cells in the peripheral blood and lymphoid organs, and microglia in the central nervous system (Gartner et al., 1986; Koenig et al., 1986; Pope et al., 1994). This virus tropism correlates with expression of the cell surface antigen CD4, which has been shown to be the principal receptor interacting with the virus surface glycoprotein, gp 120 (Dalgleish et al., 1984; Klatzmann et al., 1984). However, cell surface expression of CD4 alone is not sufficient to confer susceptibility to infection by HIV-1. Recently, several members of the chemokine receptor family of G-protein coupled seven transmembrane spanning proteins were identified as additional coreceptors (Alkhatib et al., 1996; Choe et al., 1996; Deng et al., 1996; Doranz et al., 1996; Dragic et al., 1996; Feng et al., 1996).
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- Articles
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- Animal
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- RNA viruses
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A novel neutralization epitope on the ‘thumb’ subdomain of human immunodeficiency virus type 1 reverse transcriptase revealed by a monoclonal antibody
We have prepared a MAb, 7C4, which inhibits the RNA-dependent DNA polymerase activity of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT); this MAb has allowed identification of a previously unknown neutralizing epitope of RT. Analysis of the epitope and of the mechanism of polymerase inhibition revealed that 7C4 acts by interfering with the interaction between RT and the template-primer. 7C4 recognizes a discontinuous epitope on the two α-helices, αH and αI, that make up the ‘thumb’ subdomain of RT. The existing crystallographic model of HIV-1 RT suggests that the ‘thumb’ subdomain, together with the ‘fingers’ and ‘palm’, form a nucleic-acid-binding cleft in the 66 kDa subunit of RT and that αH is in contact with the primer strand of the template-primer. The extent of inhibition of enzyme activity produced by 7C4 correlates with the reported primer-length-dependency of template-primer binding to RT. Inhibition by 7C4 was competitive with respect to the template-primer and mixed with respect to the substrate. Binding of 7C4 to RT was prevented by preincubation of the enzyme with high concentrations of template-primer but not with substrate. Thus, the 7C4 epitope apparently exists on part of the template-primer binding site of the αH and αI regions of the ‘thumb’ subdomain. This neutralization epitope is a logical target for the development of new types of HIV-1 RT inhibitors.
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Two neutralizing anti-V3 monoclonal antibodies act by affecting different functions of human immunodeficiency virus type 1
Monoclonal antibody (MAb) ICR41.1i (rat IgG2a) is specific for a conformation-dependent epitope of human immunodeficiency virus type 1 (HIV-1) V3, and MAb F58 (mouse IgG1) recognizes the peptide IXXGPGR, at the tip of the V3 loop. Both MAbs neutralized HIV-1 strain IIIB in C8166 and HeLa-T4(CD4) cells. Neutralization by either MAb did not inhibit attachment of virus to target cells as determined by FACS analysis, ELISA or immuno-fluorescence, and such attachment was absolutely dependent on the availability of CD4 molecules. F58 inhibited virus-induced cell-cell fusion, and reduced internalization of virions in direct proportion to neutralization. In contrast, ICR41.1i had no effect on HIV-1-mediated cell fusion or on internalization of virus. It was concluded that MAb F58 neutralized infectivity by inhibiting fusion of the virus with the cell and internalization of the viral core, and that ICR41. 1i neutralized by inhibiting a post-fusion-internalization event. The possible mechanism by which a neutralizing antibody binds to the V3 loop and affects the function(s) of structures inside the virion is discussed. Lastly, post-attachment neutralization (PAN) was investigated. F58 mediated PAN at 21 °C and 35 °C. However, ICR41. 1i gave PAN at 21 °C but not at 35 °C, suggesting that a temperature-dependent event affecting the V3 loop had abrogated neutralization. Overall, it appears that antibodies to different epitopes within the V3 loop neutralize by affecting very different functions of the virus.
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In vitro cytocidal effects of human immunodeficiency virus type 1 Nef on unprimed human CD4+ T cells without MHC restriction
More LessWe have previously shown that the C-terminal region of human immunodeficiency virus type 1 (HIV-1) Nef antigen present on the outer surface of virus-infected cells has an affinity for uninfected T cells and that the Nef protein is responsible for T cell death. To exclude completely the possibility of MHC restriction of this cytotoxic activity, the in vitro cytotoxic potential of HIV-1 Nef against various CD4+ T cell lines as well as naive T lymphocytes was investigated using a baculovirus expression system. Insect cells expressing myristoylated Nef on their cell surface were shown to kill a proportion of CD4+ T cells within 8 h. However, N-terminal truncated and unmyristoylated Nef proteins were not present on the outer surface of insect cell membranes and failed to show any killing activity. Monoclonal antibodies against the C-terminal region of Nef inhibited cytolysis. Thus, we conclude that specific Nef-mediated cytolysis is induced by contact with unprimed CD4+ T lymphocytes without MHC restriction.
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CD8+ cells from asymptomatic human immunodeficiency virus-infected individuals suppress superinfection of their peripheral blood mononuclear cells
More LessMost human immunodeficiency virus (HIV)-infected individuals show evidence of infection by only one strain of the virus despite possible frequent contact with multiple strains. The reason(s) for the emergence of a dominant strain of virus in HIV-infected people and the mechanism(s) which prevent other strains from establishing an infection is not known. In the present study, we demonstrate that peripheral blood mononuclear cells (PBMC) of asymptomatic HIV-infected individuals can resist productive infection by HIV-1 and HIV-2 strains. Although the PBMC of these individuals are resistant to superinfection, their CD4+ cells are susceptible to infection. Moreover, two weeks after infection of their PBMC in culture, the superinfecting virus can be recovered from isolated CD4+ cells. When CD8+ cells from asymptomatic individuals are added to the superinfected CD4+ cells, replication of the exogenously introduced virus is inhibited. In contrast, PBMC from individuals who have progressed to disease (Progressors) do not resist superinfection and their CD8+ cells do not show the antiviral activity which controls productive HIV infection. These findings suggest that CD8+ cells suppressing HIV replication in infected individuals may be critical in preventing the establishment of infection by other strains of HIV by blocking virus replication.
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Effect of cyclosporin A on the replication cycle of human immunodeficiency virus type 1 derived from H9 and Molt-4 producer cells
More LessThe effect of cyclosporin A (CsA) on the replication of human immunodeficiency virus type 1 (HIV-1) was studied. CsA treatment inhibited virus production in chronically infected H9 and Molt-4 cells. CsA treatment of HeLaCD4-LTR/β-gal cells or extracellular viruses also inhibited infection (IC50 1 µg/ml). The intracellular CsA-binding molecule cyclophilin A was detected in HIV-1 derived from chronically infected H9 cells, but it was present at a substantially lower level in HIV-1 derived from chronically infected Molt-4 cells. The low level of cyclophilin A in viral particles derived from Molt-4 cells correlated well with their substantially lower infectivity as assayed on HeLaCD4-LTR/β-gal cells. CsA treatment of infected cells showed a dose-dependent reduction of cyclophilin A incorporation into virions; the amount of cyclophilin A incorporation was found to be dependent on the producer cell type.
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Rapid development of vaccine protection in macaques by live-attenuated simian immunodeficiency virus
Convincing data on experimental vaccines against AIDS have been obtained in the simian immuno-deficiency virus (SIV) macaque model by preinfection with a virus attenuated by a nef deletion. To investigate the efficacy of a nef deletion mutant of SIVmac32H called pC8 as a live-attenuated vaccine after shorter preinfection periods and to learn more about the nature of the immune protection induced, eight rhesus monkeys were infected intravenously with the pC8 virus. All monkeys became persistently infected, exhibiting low cell-associated viral loads, but strong cellular and, in terms of binding antibodies, strong humoral antiviral responses. Two of eight pC8-infected monkeys developed an immuno-deficiency and were not challenged. Sequence analysis of their nef revealed complete replenishment of the deletion. The other six monkeys, two preinfected for 42 weeks and four for 22 weeks, were challenged with pathogenic spleen-derived SIV. Complete protection was achieved in four vaccinees. Virus was consistently detected in two vaccinees from the 22-week-group challenge, however, they remained clinically healthy over a prolonged period. Protection from challenge virus infection or a delayed disease development seemed to be associated with a sustained SIV-specific T helper cell response after challenge. Thus, a sterilizing immunity against superinfection with pathogenic SIV can be induced even after a relatively short waiting period of 22 weeks. Nevertheless, such a vaccine raises severe safety concerns because of its potential to revert to virulence.
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Regulation of human endogenous retrovirus-K Gag expression in teratocarcinoma cell lines and human tumours
More LessHuman endogenous retrovirus-K (HERV-K) Gag protein is produced by both Tera 1 and PA-1 (ovarian teratocarcinoma) cells, but only Tera 1 cells release the protein in the form of particles. It was unclear how Gag production was regulated in these cell types. Although both Tera 1 and PA-1 cells express Gag, demethylation upon treatment with 5-azacytidine (5-AZC) or exposure to the chromatin-modifying agent n-butyrate resulted in an increase in Gag protein levels only in Tera 1 cells. Consistent with this cell type-specific over-expression of Gag in response to demethylation, exposure to 5-AZC caused undermethylation of the gag gene and adjacent 5′LTR only in Tera 1 but not PA-1 or Raji cells. Similarly and importantly, under-methylation of gag sequences and expression of Gag were also correlated in primary human testicular tumours. These results therefore suggest that endogenous retroviral elements are subject to regulation through the methylation of CpG dinucleotides.
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Jaagsiekte retrovirus establishes a disseminated infection of the lymphoid tissues of sheep affected by pulmonary adenomatosis
More LessJaagsiekte retrovirus (JSRV) is an exogenous type D-related retrovirus specifically associated with a contagious lung cancer of sheep (sheep pulmonary adenomatosis; SPA). Recently, epithelial tumour cells in the lungs of SPA-affected sheep were identified as major sites of JSRV replication by immunological techniques and RT-PCR amplification of part of JSRV gag. JSRV was not detected outside the lungs and their draining lymph nodes. However, low levels of JSRV expression in non-respiratory tissues could have been masked by co-amplification of endogenous JSRV-related sequences, which were differentiated from JSRV by the lack of a Scal restriction site in the PCR product. To further investigate the pathogenesis of SPA, an exogenous virus-specific hemi-nested PCR was developed utilizing primers in the U3 region of JSRV LTR, where major differences between endogenous and exogenous sequences exist. This technique was shown to be ⩾ 105-fold more sensitive than the previous gag PCR/Scal digestion method. Using this new assay the tissue distribution of JSRV in sheep with natural and experimentally induced SPA was analysed. Proviral DNA and JSRV transcripts were found in all tumours and lung secretions of SPA-affected sheep (n = 22) and in several lymphoid tissues. The mediastinal lymph nodes draining the lungs were consistently demonstrated to be infected by JSRV (10/10). JSRV transcripts were also detected in spleen (7/9), thymus (2/4), bone marrow (4/8) and peripheral blood mononuclear cells (3/7). Proviral DNA was also detected in these tissues although in a much lower proportion of cases. JSRV was not detected in 27 samples from unaffected control animals (n = 15).
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Sequence analysis and transcriptional activity of the LTR of OLV-CU1, a North American ovine lentivirus
More LessAlthough ovine lentiviruses have been described in the United States since the early part of this century, North American strains of sheep lentiviruses remain relatively uncharacterized at the molecular level. The LTR of a North American ovine lentivirus, OLV-CU1, was found to be closely related at the molecular and functional levels to visna virus, the Icelandic ovine lentivirus. Sequence analysis of the LTR revealed high identity to other ovine and caprine lentiviruses in key regulatory elements of the upstream promoter region (-25 to -115). However, the R region of the LTR was much less homologous. Transcriptional control of OLV-CU1 in transient transcriptional assays required a conserved putative AP-4 region and possibly an AP-1 like element in the upstream promoter region for moderate to high levels of transcription, much like visna virus. In contrast to visna virus, the down-stream region beyond the transcriptional start site was required for virus-specific transactivation.
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Trojan Horse macrophages: studies with the murine lactate dehydrogenase-elevating virus and implications for sexually transmitted virus infection
More LessPrevious studies have suggested that monocytes or macrophages may mediate internal virus spread. For the present study, the tissue distribution and infectious potential of dye-labelled and/or lactate dehydrogenase-elevating virus (LDV)-infected murine macrophages were determined. Murine peritoneal macrophages were labelled with the fluorescent carbocyanine tracking dye Dil, injected into mice, and the tissue distribution of Dil-labelled cells was determined by fluorescence analysis of frozen sections. Mice receiving intravenous (i.v.) or intraperitoneal injections of Dil-labelled macrophages displayed rapid and broad tissue distribution of the labelled cells. Intravaginal injection of Dil-labelled macrophages resulted in penetration into the placentas, but not the fetuses, of pregnant mice. When macrophages were LDV-infected and Dil-labelled prior to i.v. injection into pregnant mice, they homed to various tissues including the placenta, but were not found in fetuses. Intravaginal injection of LDV-infected macrophages resulted in systemic LDV infection, even though the free-virus dose was less than the minimum infectious dose by this route. Neither polyclonal nor monoclonal IgG anti-LDV antibodies protected mice from vaginal infection with cell-associated virus, and LDV-immune complexes were themselves infectious by the vaginal route. These results show that exogenous macrophages are widely distributed following parenteral injection, penetrate locally to placentas after intravaginal injection, and are capable of acting vaginally as relatively efficient virus infection-delivery vehicles. Thus, ‘Trojan Horse’ macrophages are potentially infectious vehicles both for internal virus spread and for animal-to-animal transmission.
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Evolutionary analysis of variants of hepatitis C virus found in South-East Asia: comparison with classifications based upon sequence similarity
Variants of hepatitis C virus (HCV) have been classified by nucleotide sequence comparisons in different regions of the genome. Many investigators have defined the ranges of sequence similarity values or evolutionary distances corresponding to divisions of HCV into types, subtypes and isolates. Using these criteria, novel variants of HCV from Vietnam, Thailand and Indonesia have been classified as types 7, 8, 9, 10 and 11, many of which can be further subdivided into between two to four subtypes. In this study, this distance-based method of virus classification was compared with phylogenetic analysis and statistical measures to establish the confidence of the groupings. Using bootstrap resampling of phylogenetic trees in several subgenomic regions (core, E1, NS5) and with complete genomic sequences, we found that one set of novel HCV variants (‘types 7, 8, 9 and 11’) consistently grouped together into a single clade that also contained type 6a, while ‘type 10a’ grouped with type 3. In contrast, no robust higher-order groupings were observed between any of the other five previously described HCV genotypes (types 1–5). In each subgenomic region, the distribution of pairwise distances between members of the type 6 clade were consistently bi-modal and therefore provided no justification for classification of these variants into the three proposed categories (type, subtype, isolate). Based on these results, we propose that a more useful classification would regard all these variants as subtypes of type 6 or type 3, even though the level of sequence diversity within the clade was greater than observed for other genotypes. Classification by phylogenetic relatedness rules out simple sequence similarity measurements as a method for assigning HCV genotypes, but provides a more appropriate description of the evolutionary and epidemiological history of a virus.
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Low pH-induced pore formation by spike proteins of enveloped viruses
More LessExposure of Aedes albopictus cells infected with Semliki Forest virus (SFV; Togaviridae) to mildly acidic pH (5.6) results in a dramatic increase in the host cell membrane permeability due to pore formation by the virus spike proteins. Identical results were obtained when the cells were infected with two other viruses, Sindbis virus (SIN, Togaviridae) and vesicular stomatitis virus (VSV, Rhabdoviridae). This permeability change could also be observed on isolated virions of SFV, SIN and VSV by measuring the influx of propidium iodide, a nucleic acid-specific fluorescent marker, into the virions. This influx was dependent on the presence of the ectodomains of the viral spikes and could be hampered by zinc ions. Furthermore, haemagglutinin, a membrane protein of influenza A virus (Orthomyxoviridae), expressed in Aedes cells induced a change in membrane permeability identical to that induced by the spike proteins of SFV, SIN and VSV when exposed to low pH. Thus acid-induced membrane permeability changes produced by spike proteins of three different virus families could be demonstrated in infected cells as well as in virions. Therefore, the low pH-induced pore formation by viral spike proteins seems to be more than an event specific for togaviruses and might well be an inherent property of enveloped viruses that use the endocytotic pathway to infect a cell.
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Mapping the neutralizing epitopes on the glycoprotein of infectious haematopoietic necrosis virus, a fish rhabdovirus
More LessTwelve neutralizing monoclonal antibodies (MAbs) against the fish rhabdovirus, infectious haematopoietic necrosis virus (IHNV), were used to select 20 MAb escape mutants. The nucleotide sequence of the entire glycoprotein (G) gene was determined for six mutants representing differing cross-neutralization patterns and each had a single nucleotide change leading to a single amino acid substitution within one of three regions of the protein. These data were used to design nested PCR primers to amplify portions of the G gene of the 14 remaining mutants. When the PCR products from these mutants were sequenced, they also had single nucleotide substitutions coding for amino acid substitutions at the same, or nearby, locations. Of the 20 mutants for which all or part of the glycoprotein gene was sequenced, two MAbs selected mutants with substitutions at amino acids 230–231 (antigenic site I) and the remaining MAbs selected mutants with substitutions at amino acids 272–276 (antigenic site II). Two MAbs that selected mutants mapping to amino acids 272–276, selected other mutants that mapped to amino acids 78–81, raising the possibility that this portion of the N terminus of the protein was part of a discontinuous epitope defining antigenic site II. CLUSTAL alignment of the glycoproteins of rabies virus, vesicular stomatitis virus and IHNV revealed similarities in the location of the neutralizing epitopes and a high degree of conservation among cysteine residues, indicating that the glycoproteins of three different genera of animal rhabdoviruses may share a similar three-dimensional structure in spite of extensive sequence divergence.
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The genome sequence of the virulent Kabete ‘O’ strain of rinderpest virus: comparison with the derived vaccine
More LessWe have compared the complete genome sequences of the vaccine strain of rinderpest virus and the virulent strain from which it was derived. Only 87 bases differed between the two genomes (0.55%). Possibly significant differences in amino acid sequence were found in the N, P, F, H and L proteins. A number of differences were also found in the leader region (3′ end of the genome), whilst the trailer region appears to be more conserved. In addition, the length of the genome was found in both cases to be 15882, an exact multiple of six, fulfilling predictions made earlier based on work with Sendai and measles viruses.
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Nucleotide sequence of the gene encoding the viral polymerase of avian pneumovirus
More LessWe report here the nucleotide sequence of the L gene of avian pneumovirus (APV). This is the second pneumovirus L gene and the second avian paramyxovirus L gene, following that of Newcastle disease virus, to be sequenced. The APV L gene is 6099 nucleotides long and encodes a single large ORF of 2004 amino acids. This makes the APV L protein the smallest to be described for any non-segmented, negative-strand RNA virus. The protein contains six linear non-contiguous domains, a putative ATP-binding site and four polymerase motifs previously described for the L proteins of negative-strand RNA viruses. Phylogenetic analysis of domain III of 14 different L proteins suggests the pneumo-viruses to be as distant in evolutionary terms from the other members of the Paramyxoviridae as are the Filoviridae.
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Persistent infection of mammalian cells by Rift Valley fever virus
More LessInfection of mammalian cells with Rift Valley fever virus (RVFV) leads generally to the production of virus and cell death. In this paper we examined the fate of Vero cells infected with three strains of RVFV and observed that, while a large proportion of cells exhibited a clear cytopathic effect (CPE), a small but significant fraction did not undergo a lytic infection but was able to proliferate and establish a persistent infection. Several independent RVFV persistently infected cell lines have been established and passaged for more than 1 year after infection with a virulent strain (ZH548) and two attenuated strains (C13 and MP12). Although the viruses used for the primary infection were plaque-purified, we do not know whether defective-interfering particles were responsible for the establishment of the persistent infection. The persistently infected cells became resistant to superinfection with RVFV but not with other viruses and shed low amounts of infectious, lytic and non-lytic virus during a limited number of passages. In all the passages tested, the three genomic segments or related products were synthesized as well as the structural nucleoprotein N and glycoproteins G1 and G2. Abnormal defective RNAs were detected, migrating faster or slower than their respective counterparts. The faster-migrating RNAs were internally deleted, some of them possessing only the very terminal part of the 5′ genomic end.
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Isolation and characterization of Tula virus, a distinct serotype in the genus Hantavirus, family Bunyaviridae
A Vero E6 cell culture isolate of Tula virus (TUL), a hantavirus first detected in European common voles (Microtus arvalis and M. rossiaemeridionalis) by RT-PCR was obtained after initial passaging of TUL-infected vole lung samples in laboratory-colonized M. arvalis. TUL was defined as a classical serotype by a cross-focus-reduction neutralization test (FRNT) and was also shown to be distinct from other hantaviruses by haemagglutination inhibition assay. The sequences of S, M and partial L genome segments of the isolate were determined: the S segment was 99.9% identical to the original rodent-derived sequence. Serological evidence for a previous TUL infection was obtained from the serum of a blood donor living near a TUL focus in Moravia, Czech Republic, showing at least a 16-fold higher FRNT titre to TUL as compared to Puumala or other hantaviruses.
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