- Volume 77, Issue 8, 1996
Volume 77, Issue 8, 1996
- Review Article
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Molecular biology of the feline immunodeficiency virus auxiliary genes
More LessFeline immunodeficiency virus (FIV) causes a slowly progressive multiorgan disease in infected cats and shows considerable pathogenic similarities to the human immuno-deficiency virus type 1 (HIV-1), the causative agent of AIDS (Miyazawa & Mikami, 1993; Pedersen et al., 1987; Yamamoto et al., 1988). It is therefore considered a useful small-animal model for HIV-1-induced AIDS studies. FIV belongs to the family Retroviridae, a group of small, enveloped positive-strand RNA viruses. These viruses have an enzyme, reverse transcriptase, which enables them to replicate their RNA genome through a DNA intermediate (Coffin, 1992). FIV is a member of the genus Lentivirus and is closely related in biological characteristics and genome organization to other mammalian lentiviruses, including HIV, simian immunodeficiency virus (SIV), bovine immunodeficiency-like virus (BIV), equine infectious anaemia virus (EIAV), visna virus and caprine arthritis-encephalitis virus (CAEV) (Narayan & Clements, 1990).
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- Animal
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- RNA viruses
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Feline immunodeficiency virus can infect a human cell line (MOLT-4) but establishes a state of latency in the cells
Infectivity of feline immunodeficiency virus (FIV) in feline and human lymphoblastoid cell lines was examined using homogeneous populations of FIV derived from infectious molecular clones of strains TM2 and Petaluma, and two recombinant chimeric clones carrying gag, pol, vif and ORF-A from the heterologous virus. FIV from the clones with the env region of the Petaluma strain was shown to infect and establish provirus in a human lymphoid cell line (MOLT-4), although the FIV-infected cells did not produce any infectious viruses. By treatment of the infected MOLT-4 cells with a phorbol ester, infectious virus was rescued. To examine which stage of the life-cycle of FIV is blocked in these cells, we analysed transcription of FIV-14 in the cells by RT-PCR. FIV-specific RNA expression could not be detected. These results strongly suggest that latency of the virus in MOLT-4 cells is due to a failure in transcription.
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The structure and phylogeny of a new family of human endogenous retroviruses
More LessA novel endogenous retrovirus (ERV) designated XA34 was isolated from a human glioma cDNA library using low stringency hybridization with an ERV-9 env probe. Southern blot hybridizations with human genomic DNA revealed the presence of approximately 16 genomic copies closely related to XA34. Sequencing of a 2303 bp cDNA clone of XA34 showed that it belongs to a new ERV family. The XA34 ERV has recombined with an ERV-9-like retrovirus resulting in a truncated ERV-9-like env region that ends with an Alul-like 3′ LTR. By using PCR, we isolated ≈ 940 bp pol fragments from three additional members of this family, XA35, XA36 and XA37. A fifth member, XA38, was isolated and sequenced as a 4729 bp genomic clone. The genomic XA38 clone spans from pol towards the 3′ flanking region. The XA38 virus contains a more cryptic env region. The XA38 env is truncated in the transmembrane region and the virus then ends with three Alu repeats. Southern blot studies with human, chimpanzee, orangutan and squirrel monkey DNA show the presence of the XA34 family in all these species. That both the New and Old World monkeys have this ERV family means that the integration and/or amplification in the primate germ-line of XA34 probably took place about 40–45 million years ago. The phylogeny and the closest relatives to ERV XA34 are discussed.
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The human immunodeficiency virus type 1 regulatory protein Tat inhibits interferon-induced iNos activity in a murine macrophage cell line
More LessHuman immunodeficiency virus type 1 (HIV-1) infection is frequently associated with concurrent infection by opportunistic pathogens, against which production of nitric oxide by host macrophages provides a first line of defence. We have investigated whether regulatory HIV-1 proteins, such as Tat, can modulate the activity of the inducible nitric oxide synthase (iNos) gene when expressed in stable transfectant lines of RAW264.7 cells. A bioassay for Tat, based on transactivation of an HIV-1 LTR-CAT reporter gene, allowed selection of Tat-expressing cells. Parental and Tat-expressing macrophages accumulated identical levels of nitrite following lipopolysaccharide (LPS) stimulation. Interferon γ (IFN-γ) stimulation however, resulted in reduced levels of nitrite accumulation as a direct consequence of Tat expression. Conditioned media from Tat-expressing cells reduced the level of nitrite accumulation in parental cells following IFN-γ stimulation but not stimulation with LPS. These results implicate HIV-1 Tat as a modulator of the IFN-γ-specific signal transduction pathways leading to iNos expression.
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Genomic and biological alteration of a human immunodeficiency virus type 1 (HIV-1)-simian immunodeficiency virus strain mac chimera, with HIV-1 Env, recovered from a long-term carrier monkey
A macaque monkey infected with NM-3, a human immunodeficiency virus type 1 (HIV-1)-simian immunodeficiency virus strain mac (SIVmac) chimeric virus with env, rev, tat and vpu derived from HIV-1 and LTR, gag, pol, vif and vpx derived from SIVmac, became a long-term carrier (more than 2.8 years). This monkey produced neutralizing antibodies to the original NM-3 as well as to the parental HIV-1. The virus recovered at 116 weeks replicated more rapidly and productively in macaque peripheral blood mononuclear cells than the original virus. The recovered virus was not neutralized either by antibodies raised early in the monkey or by a neutralizing monoclonal antibody that recognizes the V3 loop of HIV-1 Env, whereas both the early antibodies and the monoclonal antibody neutralized the original NM-3. Analysis of the virus genomic population revealed a few common mutations in the V3 region that caused amino acid changes. These data are consistent with the hypothesis that the virus escaped from the early antibodies and that the observed mutations contributed to this, as with HIV-1-infected humans. The observed mutations could equally well be the result of adaptation to simian cells. These results suggest that the HIV-1-SIVmac chimeric virus will be useful for investigating genetic variation of HIV-1 env and alteration of biological properties in vivo in relation to the host immune response.
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Fine-specificity of cytotoxic T lymphocytes which recognize conserved epitopes of the Gag protein of human immunodeficiency virus type 1
Human immunodeficiency virus type 1 (HIV-1) Gag-specific cytotoxic T lymphocyte (CTL) responses were studied in seven seropositive long-term asymptomatic individuals (CDC A1) with stable CD4 counts for more than 8 years. Using a set of partially overlapping peptides covering the whole Gag, five 15–20-mer peptides were found to contain CTL epitopes. Further characterization of these epitopes revealed a new HLA-A25-restricted CTL epitope in p24, p24203–212 ETINEEAAEW. This region of Gag is highly conserved in clades B and D of HIV-1. Naturally occurring amino acid sequences, containing p24203D (consensus HIV-1 clades A, C, F, G and H) or p24204I (HIV-2ROD) were not recognized by CTL recognizing the index peptide. No virus variants with mutations in this sequence were found in peripheral blood mononuclear cells from the HIV-1-infected individual concerned during the 8 year observation period, indicating that the virus had not escaped from the observed CTL response.
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Human antibodies that neutralize primary human immunodeficiency virus type 1 in vitro do not provide protection in an in vivo model
More LessRecently, conflicting data have been published about the ability of antibodies which efficiently neutralize T cell-adapted human immunodeficiency virus type 1 (HIV-1) strains to neutralize primary HIV-1 strains in vitro and in vivo. Here we present data indicating that such antibodies fail to neutralize primary HIV-1 strains in vivo. To this end, a newly developed chimeric human-to-mouse model was used, in which several aspects of primary HIV-1 infection are mimicked. Poly- and monoclonal anti-bodies protected the grafted human cells, in a dose-dependent way, from infection with T cell-adapted HIV-1 in this system. A human monoclonal antibody specific for the CD4 binding domain that efficiently neutralizes HIV-1 IIIB in vitro did not protect the human graft from HIV-1 IIIB infection. None of the antibodies provided protection in the in vivo model against infection with primary HIV-1 strains, although they were able to neutralize these same strains in vitro.
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Cell death induced by cytopathic bovine viral diarrhoea virus is mediated by apoptosis
More LessCells infected with two closely related isolates of bovine viral diarrhoea virus (BVDV), one cytopathic (CP) and one non-cytopathic (NCP), were examined for signs of apoptosis. The results from labelling DNA using terminal transferase and biotinylated dUTP and by observing oligonucleosomal-sized DNA fragmentation indicated that the CP strain of BVDV induced apoptosis in cell culture but the NCP strain did not. Induction of apoptosis correlated with infected cells undergoing apoptosis rather than interactions between infected and uninfected cells and the induction of apoptosis by CP BVDV was a dominant trait in cells co-infected with both types of virus.
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Difference in virus-binding activity of two distinct receptor proteins for mouse hepatitis virus
More LessThe receptor proteins, MHVR1 (Bgp C or splice variant of mmCGM1 containing two ectodomains) and MHVR2 (mmCGM2) have been reported to be functional receptors for MHV, although there was a significant difference in their virus-binding activity as determined by virus overlay protein blot assay (VOPBA). To compare the receptor function of these proteins, their virus-binding capacities were tested by using soluble forms of the proteins which lacked the transmembrane and intracytoplasmic domains. To estimate the amounts of these proteins expressed, an epitope of influenza HA protein, for which specific monoclonal antibody was available, was used as a tag. Recombinant soluble MHVR1 and MHVR2, expressed in RK 13 cells using recombinant vaccinia virus were secreted into the culture fluids of infected cells expressing these proteins. The inhibitory effect on virus infectivity of MHVR1 was shown to be about 500-fold higher than that of MHVR2. A similar disparity was observed in virus binding by VOPBA. These two proteins worked as functional receptors when they were expressed on resistant BHK-21 cells. However, the efficiency of MHV infection in BHK-21 cells expressing MHVR1 was about 30-fold higher, as compared with those expressing MHVR2. These data show that the receptor function of MHVR1 was significantly higher than that of MHVR2 and suggests that the difference in susceptibility between SJL and BALB/c mice might be due to the specific receptor protein expressed in those animals.
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European brown hare syndrome virus: molecular cloning and sequencing of the genome
More LessThe genome of the European brown hare syndrome virus (EBHSV), a calicivirus related to rabbit haemorrhagic disease virus (RHDV), was fully sequenced. It was 7442 bases long and contained two ORFs. In RHDV, the 5′ large ORF (ORF1) is predicted to encode a polyprotein precursor to the non-structural and capsid proteins. The small ORF (ORF2) encodes a predicted protein of 12 kDa. Alignment of sequences of EBHSV and RHDV showed 71% nucleotide identity; the changes were uniformly scattered over the whole genome. Minor differences could be detected when comparing two EBHSV sequences, indicating that EBHSV could vary to the same extent as RHDV. Four cleavage sites previously identified on the RHDV polyprotein were conserved in EBHSV. These sequencing data clearly show that EBHSV and RHDV share a similar genomic organization and confirm that EBHSV and RHDV are two distinct members within the family Caliciviridae.
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Genetic and phylogenetic clustering of enteroviruses
Genetic and phylogenetic analysis of enteroviruses showed that in the 5′NCR enteroviruses formed three clusters: polioviruses (PVs), coxsackievirus A type 21 (CAV21), CAV24 and enterovirus type 70 (ENV70) formed one cluster; coxsackievirus B isolates (CBVs), CAV9, CAV16, ENV71, echovirus type 11 (EV11), EV12 and all partially sequenced echoviruses and swine vesicular disease virus (SVDV) belonged to another cluster and bovine enteroviruses (BEVs) formed the third cluster. In the capsid coding region five clusters were seen: PVs, CAV21 and CAV24 formed one cluster (PV-like); ENV70 formed a cluster of its own; all CBVs, CAV9, EV11, EV12 and SVDV formed the third cluster (CBV-like); CAV16, CAV2 and ENV71 belonged to the fourth cluster (CAV16-like) and BEVs formed their own cluster (BEV-like). In the 3′NCR the same clusters were seen as in the coding region suggesting a close association of the 3′NCR with viral proteins while the cellular environment may be more important in the evolution of the 5′NCR. Secondary structures were predicted in the 3′NCR, which showed two different patterns among the five clusters. A potential pseudoknot region common in all five clusters was identified. Although the BEV-like viruses formed a separate cluster in all genomic regions, in the coding region they seem to be phylogenetically related to the CAV16-like viruses.
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Equine rhinovirus serotypes 1 and 2: relationship to each other and to aphthoviruses and cardioviruses
G. Wutz, H. Auer, N. Nowotny, B. Grosse, T. Skern and E. KuechlerEquine rhinoviruses (ERVs) are picornaviruses which cause a mild respiratory infection in horses. The illness resembles the common cold brought about by rhinoviruses in humans; however, the presence of a viraemia during ERV-1 infection, the occurrence of persistent infections and the physical properties are all more reminiscent of foot-and-mouth disease virus (FMDV). cDNA cloning and sequencing of the genomes of ERV-1 and ERV-2 between the poly(C) and poly(A) tracts showed that the serotypes are heterogeneous. Nevertheless, the genomic architecture of both serotypes is most similar to that of FMDV. Indeed, a comparison of the derived protein sequences of ERV-1 shows that their identity is greatest to FMDV. In contrast, most ERV-2 proteins are more related to encephalomyocarditis virus (EMCV) proteins than they are to FMDV or ERV-1. These results place ERV-1 alongside FMDV in the aphthovirus genus of the picornavirus family and indicate that this virus may serve as a model system for examining the biology of FMDV.
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Monoclonal antibodies raised against infectious haematopoietic necrosis virus (IHNV) G protein and a cellular 90 kDa protein neutralize IHNV infection in vitro
Immune sera were obtained from four rainbow trout that had survived natural infection by infectious haematopoietic necrosis virus (IHNV), and five monoclonal antibodies (MAbs) were prepared against a Korean isolate of IHNV, IHNV-PRT. These immune sera and MAbs were characterized in terms of IHNV-neutralizing properties and reactivity in Western blots with the viral proteins of IHNV-PRT. All five MAbs and four immune sera neutralized IHNV-PRT to various extents. Antibodies in these immune sera recognized two structural proteins of IHNV, G and M1, and one protein with a molecular mass of 90 kDa. Of the five MAbs, three (AB9, AF6 and AG6) recognized the IHNV G protein, and the other two (AB7 and BC2) recognized the 90 kDa protein. The 90 kDa protein was found to be a cellular protein constitutively expressed at low levels in fish cells and expression of this protein was augmented by infection with IHNV and heat shock. MAbs specific to four stress proteins, hsp60, hsp70, hsp90 and grp94, failed to bind to this 90 kDa protein. MAbs AB9 and AB7 reacted fairly broadly with six different IHNV strains. Together, these results indicate that (1) two IHNV proteins, G and M1, and a 90 kDa cellular protein are immunogenic, (2) G and the 90 kDa proteins contain neutralizing epitopes, and (3) the epitopes recognized by MAbs AB9 and AB7 are conserved among the six different IHNV strains.
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Semi-permissive replication and functional aspects of the immune response in a cotton rat model of human parainfluenza virus type 3 infection
A cotton rat (Sigmodon fulviventer) model of human parainfluenza virus type 3 (HPIV-3) infection was used to study patterns of HPIV-3 replication in naive and immune hosts. Growth curves revealed that nasal and pulmonary tissues of naive animals were semi-permissive for virus replication, with amounts of progeny virus proportional to inoculating doses. In naive animals there was a total eclipse in nasal tissues beginning 4 h after inoculation. By contrast, there was only partial eclipse of virus in pulmonary tissues, most pronounced at 1 h after inoculation. Immune animals demonstrated a delayed eclipse in pulmonary tissues upon rechallenge. Infection with very low doses of HPIV-3 induced complete protection against high-dose challenge in the absence of systemic neutralizing antibody, suggesting a significant role for other systemic or local immune effectors.
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Mutational analysis of the influenza virus A/Victoria/3/75 PA protein: studies of interaction with PB1 protein and identification of a dominant negative mutant
More LessThe RNA polymerase activity and PB1 binding of influenza virus PA mutants were studied using an in vivo-reconstituted polymerase assay and a two hybrid system. Deletions covering the whole PA protein abolished polymerase activity, but the deletion of the 154 N-terminal amino acids allowed PB1 binding, indicating that the PA protein N terminus is not absolutely required for this interaction. Further internal or C-terminal deletions abolished PB1 interaction, suggesting that most of the protein is involved in this association. As a novel finding we showed that a single amino acid insertion mutant, PAI672, was responsible for a temperature-sensitive phenotype. Mutant PAS509, which had a serine insertion at position 509, bound to PB1 like wild-type PA but did not show any polymerase activity. Over-expression of PAS509 interfered with the polymerase activity of wild-type PA, identifying PAS509 as a dominant negative mutant.
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Large outbreak of swine influenza in southern Japan caused by reassortant (H1N2) influenza viruses: its epizootic background and characterization of the causative viruses
In the winter of 1989 and the spring of 1990, there were large outbreaks of respiratory disease in two swine herds in Nagasaki Prefecture, southern Japan. Serological surveillance indicated that the majority of swine possessed antibodies to swine influenza virus H1 haemagglutinin and neuraminidase of early H3N2 influenza virus strains. Eight viruses were isolated from swine that showed typical clinical symptoms of influenza. The haemagglutinin and neuraminidase of these isolates were closely related to those of swine H1N1 and early human H3N2 viruses, respectively. At least two types of haemagglutinin antigens, distinguished by two monoclonal antibodies, were involved in the outbreaks. Evolutionary analyses indicated that the haemagglutinin gene of the H1N2 reassortants was closely related to those of a recent swine lineage (A/sw/HK/1/74 and A/sw/Ehime/1/80 viruses). However, the neuraminidase genes of the H1N2 reassortants were similar to those of swine N2 viruses which in turn are related to early human H3N2 viruses. A comparison of partial nucleotide sequences revealed that the six other genes of A/sw/Nagasaki/1/89 were derived from those of swine H1N1 virus.
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The S RNA genomic sequences of Inkoo, San Angelo, Serra do Navio, South River and Tahyna bunyaviruses
More LessThe complete nucleotide sequences of the small (S) genomic RNA segments of five California (CAL) serogroup bunyaviruses (two Inkoo virus strains, San Angelo virus, Serra do Navio virus, South River virus and Tahyna virus) were determined. In agreement with previously published data concerning CAL serogroup viruses, the nucleocapsid (N) and non-structural (NS s ) proteins were encoded in over-lapping open reading frames (ORFs). All N protein ORFs were 708 nucleotides in length and encoded a 235 amino-acid gene product. The NS s ORFs were either 279 or 294 nucleotides in length, which would encode 92 or 97 amino-acid proteins, respectively. Comparative analysis of the nucleotide sequences and amino acids corresponding to the nucleocapsid protein resulted in a predicted relationship among these viruses that generally agreed with those determined by serology. The only exception was Inkoo virus, where comparisons based on the S RNA sequence and partial M RNA sequence suggest that this virus is more similar to Jamestown Canyon virus of the Melao complex than it is to viruses such as Tahyna and La Crosse viruses of the California encephalitis complex.
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Inkoo and Tahyna, the European California serogroup bunyaviruses: sequence and phylogeny of the S RNA segment
Inkoo (INK) and Tahyna (TAH) viruses, European representatives of the California serogroup (CAL), genus Bunyavirus, family Bunyaviridae, are transmitted by mosquitoes and frequently infect man. The S segments of INK and TAH prototype strains were amplified, cloned and sequenced. INK S consists of 986 and TAH S of 977 nucleotides (nt) coding for a nucleocapsid protein of 235 amino acids (aa) and, in an overlapping reading frame, for a nonstructural protein of 92 or 97 aa, respectively. By S segment sequences and phylogenetic analysis INK was seen to be most closely related to Jamestown Canyon virus, isolated in the USA (92.4% nt and 96.6% aa identity), which is currently classified in a different subcomplex within the CAL viruses. TAH was genetically closest to Lumbo virus, isolated in Mozambique (89.0% nt and 94.1% aa identity). The data suggest that genetic variation within the CAL viruses is less related to geographical distance than to similarity in ecological cycles.
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Large RNA segment of Dugbe nairovirus encodes the putative RNA polymerase
More LessThe nucleotide sequence of the large (L) RNA segment of Dugbe (DUG) virus (Nairovirus, Bunyaviridae) was determined, completing the first entire genome sequence of a nairovirus. The L segment comprised 12255 nucleotides, making a total genome size of 18855 nucleotides, and the ends showed identity with the ends of the medium (M) and small (S) genomic segments. A single open reading frame (ORF) was present in the viral complementary strand, sufficient to encode a protein of 459 kDa. The predicted protein sequence showed the core polymerase motifs characteristic of the RNA-dependent RNA polymerases of segmented negative-stranded viruses. Comparison of the conserved motifs with the corresponding region of other segmented negative-strand viruses showed a closer relationship between nairoviruses and phleboviruses than with other Bunyaviridae or with other virus families. However, the core polymerase was the only function that could be assigned to a region of the DUG L gene.
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- DNA viruses
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Sequence variability among different parvovirus B19 isolates
Parvovirus B19 is the causative agent of a variety of clinical manifestations, ranging from asymptomatic to severe infection. The basis for this complex pattern of B19-associated diseases is as yet poorly understood. In general there are two different possibilities: firstly, the infected individuals may have a genetic or acquired predisposition, which renders them susceptible for a certain course of infection; secondly, differences in the B19 genome may result in different outcomes of infection. In order to investigate this second possibility we have partially sequenced the genomes of 20 different B19 isolates derived from serum samples from patients with various B19-associated diseases. Four distinct regions, which cover nearly half of the genome and include parts of the coding regions of all three major B19 proteins - NS1, VP1 and VP2, were selected for sequencing. Comparisons between the different extracted virus isolates at the DNA and protein levels revealed that isolates from patients with persistent parvovirus B19 infection show a tendency towards higher genome variability with respect to isolates derived from persons with acute infection.
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Species specificity for transduction of cultured cells by a recombinant Lulll rodent parvovirus genome encapsidated by canine parvovirus or feline panleukopenia virus
More LessWe previously reported that a recombinant genome derived from the autonomous rodent parvovirus LuIII could be pseudotyped with capsids of the closely related viruses, H1 and minute virus of mice. To determine whether this was also possible with less related viruses, LuIII recombinant genomes containing a luciferase reporter were cotransfected into permissive cells together with plasmids expressing the capsid proteins of either feline panleukopenia virus (FPV) or its host range variant, canine parvovirus (CPV). We observed efficient packaging of the recombinant DNA into transducing virions that displayed the cell tropism of the virus that supplied the capsid. Thus, the FPV- and CPV-pseudotyped virions were able to transduce a feline cell line but they showed no transducing activity for the human NB324K line, which is permissive for Lulll. The transducing activity of the pseudotyped viruses was not inhibited by neuraminidase treatment of the permissive recipient cells, in contrast to that of virions packaged using LuIII capsid proteins. Furthermore, canine A72 cells (permissive for CPV but not FPV) were efficiently transduced by CPV-packaged but not by FPV-packaged LuIII recombinant genomes. Pseudotyped recombinants will be useful for elucidating parvovirus host range determinants since they enable the packaged DNA and each of the capsid proteins to be supplied independently. They should also facilitate control over the targeting of parvovirus vectors for gene transfer.
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Laboratory production of infectious stocks of rabbit oral papillomavirus
Several small, raised lesions from the underside of the tongue of domestic rabbits were isolated, and an extract prepared and tested for the presence of rabbit oral papillomavirus (ROPV). Two weeks after inoculation of this extract into the underside of rabbit tongues, multiple small discrete, grey-white nodules were observed that reached a maximum size of 2 mm in diameter by 5 weeks. These lesions showed typical ROPV pathology, and nuclei stained positive for papillomavirus (PV) group-specific antigen (GSA) by immunocytochemistry. Tissue fragments from rabbit tongues were incubated with a suspension of ROPV and placed subrenally into athymic mice. After 60 days, cysts were removed, sections cut for histology, and a virus stock prepared. GSA staining and in situ hybridization demonstrated that the xenografts were morphologically transformed with areas showing strong nuclear staining for viral capsid antigen and ROPV DNA. Extracts prepared from the pooled xenografts contained infectious ROPV as demonstrated by inoculation into the undersurface of tongues of non-immune New Zealand White rabbits. The results demonstrated that stocks of infectious ROPV can be prepared in the athymic mouse xenograft system for use in studies on the experimental transmission of a mucosal-targeting animal papillomavirus.
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Antibodies to human papillomavirus type 11 virus-like particles in sera of patients with genital warts and in control groups
More LessWe analysed by ELISA a total of 478 human sera for the presence of antibodies to HPV-11 virus-like particles. The sera were obtained from patients with current genital warts (group CO), from males attending the hospital for fertility disorders (group MA), from blood donors (group BD) and from patients hospitalized for reasons unrelated to HPV infections (group HO). Antibody prevalence was higher in male patients of group CO (23.0%) as compared to males of groups MA (3.2%; P < 0.0001), HO (5.3%; P = 0.01) and BD (16.7%; NS). In addition, there was a significant difference in antibody titre between the males of group CO compared to group MA. Within the whole sample the absorbance of sera from females was higher than in specimens from males (P < 0.0001). A small subset of the sera was also tested by radioimmunoprecipitation assay (RIPA). There was good agreement between the data obtained by ELISA and RIPA.
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Characterization of the helicase and ATPase activity of human papillomavirus type 6b E1 protein
Human papillomavirus type 6b (HPV-6b) is one of the most common causes of human genital warts, an important sexually transmitted disease. Discovery of antiviral therapies for this condition has been hampered by the inability to propagate the virus using standard tissue culture techniques and through difficulties in expressing sufficient recombinant viral proteins in vitro. Replication of papillomavirus DNA requires to viral proteins, E1 and E2. In an effort to establish assays to discover compounds active against this virus, we have co-expressed HPV-6b E1 and E2 proteins in insect cells. We demonstrate that the two proteins form a heteromeric complex which can be purified by sequence-specific DNA affinity chromatography. We also demonstrate that the complex has both E1-associated ATPase and ATP-dependent DNA helicase activity and report further characterization of these functions.
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Common epitope on protein VI of enteric adenoviruses from subgenera A and F
More LessWestern blot analysis with monoclonal antibodies, produced in response to immunization with gradient-purified adenovirus 41 (Ad41) virions, identified two epitopes of interest on protein VI of enteric adenoviruses. One epitope is unique to subgenus F adenoviruses (Ad40 and Ad41); the other epitope is common to subgenus A (Ad12, 18 and 31) and subgenus F(Ad40, 41) adenoviruses but is not shared by representative serotypes of subgenera B (Ad3 and 7), C (Ad1, 2 and 5), D (Ad8) or E (Ad4). Alignment of the deduced amino acid sequence of the genes encoding the protein VI precursor (pre-VI) of Ad40 and Ad41 (subgenus F), Ad12 and Ad31 (subgenus A), Ad2 and Ad5 (subgenus C) shows that the N-terminal one-third and C-terminal 23 amino acids of pre-VI are highly conserved. Within the central domain, pre-VI of subgenus F serotypes is more closely related to that of subgenus A serotypes than to pre-VI of the non-enteric subgenus C adenoviruses (Ad2 and Ad5). By expressing random oligonucleotide fragments of the Ad41 protein VI gene as part of a T7 gene 10 fusion protein, the two epitopes of interest were mapped to within the same 14 amino acid region in the central domain of protein VI. Given the association of subgenera A and F adenoviruses with paediatric gastroenteritis, the epitope shared by these serotypes may be functionally significant with respect to gut tropism. In addition, this epitope is potentially valuable as a target for the detection of enteric adenoviruses in clinical specimens.
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Activation of the protease from human adenovirus type 2 is accompanied by a conformational change that is dependent on cysteine-104
More LessAdenovirus codes for a protease the activity of which can be regulated in vitro by an 11 residue peptide (GVQSLKRRRCF) derived from another viral protein, pVI. Three cysteine residues, one in the activating peptide and two in the protease (C104 and C122), play a central role in both activation and catalysis. Expression of protease mutants in insect cells has shown that C104 is not essential for proteolytic activity. GVQSLKRRRCF also caused a concentration-dependent increase in tryptophan fluorescence of protease expressed in Escherichia coli that paralleled the increase in proteolytic activity, indicating that activation was accompanied by a conformational change. Tryptophan fluorescence of C104S was not increased by the addition of GVQSLKRRRCF, nor was the fluorescence of wild-type protease increased by the addition of the peptide analogues where cysteine is replaced by aspartic acid or serine, suggesting that C104 is involved in activation and C122 in catalysis.
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Mutations in the envelope gene of hepatitis B virus variants co-occurring with antibody to surface antigen in sera from patients with chronic hepatitis B
More LessThree clones of hepatitis B virus (HBV) DNA were propagated from sera of each of five patients with chronic hepatitis B who possessed hepatitis B surface antigen (HBsAg) and antibody to HBsAg in their serum. The clones were sequenced within the envelope gene (the preS1, preS2 regions and the S gene). Clones from four patients had various missense mutations involving codons 124–147 of the S-gene which encode amino acids in the loop structures that form the conformational, common antigenic determinant of HBsAg. Clones from three patients had Asn-130 (Gly in the wild-type), which generated a potential N-glycosylation site, Asn-Thr-Ser, spanning amino acids 130–132 of the S-gene product. In addition, clones from one patient had Arg-145 (Gly in the wild-type), which has been reported in escape mutants of HBV. One of the three clones from another patient had Ser-126 in place of Ile or Thr in wild-type HBV, but the remaining two had no mutations known to affect expression of the common determinant of HBsAg. The remaining patient possessed HBsAg of subtype adr and anti-HBs specific for the w determinant. Clones from this patient did not reveal any mutations which are known to affect the common antigenic determinant of HBsAg.
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Severe outcome of hepatitis B virus (HBV) infection and lack of HBV e antigen-defective virus emergence in patients homozygous for HLA class I alleles
In the Mediterranean region almost all patients with hepatitis B virus (HBV)-related cirrhosis are anti-HBV e antigen (anti-HBeAg)-positive and carriers of HBeAg-negative virus mutants. The six members of a family who acquired HBV infection were recently studied: two siblings developed cirrhosis with persistence of HBeAg positivity, whereas their parents and two more siblings cleared the virus. The two cirrhotic patients showed homozygosity for HLA class I by phenotype, which is a rare occurrence in the general population, while the other family members were heterozygous for HLA class I. The sequencing analyses of the entire viral DNAs isolated from both cirrhotic patients showed that the two viral genomes were almost identical and no mutation preventing HBeAg synthesis or viral gene expression was present. These findings might suggest that homozygosity for HLA class I molecules might be responsible for an insufficient response to the virus, favouring chronic outcome of the infection and the long-lasting persistence of HBV populations that produce HBeAg.
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Protease-activated lymphoid cell and hepatocyte recognition site in the preS1 domain of the large woodchuck hepatitis virus envelope protein
More LessA site capable of strictly host- and cell type-specific recognition was identified in the preS1 domain of woodchuck hepatitis virus (WHV) through the use of antipeptide antisera generated against the extreme N-terminal fragment of the large virus envelope protein. The crucial determinant of this binding site was mapped to amino acids 10–13. Although a synthetic analogue of the site was highly immunogenic, natural WHV envelope did not display the site activity unless it was modified by proteolysis or acidic pH treatment, indicating an internal location of the determinant in viral envelope. Synthetic peptides encompassing the sequence of this site bound woodchuck lymphoid cells and hepatocytes in a species-restricted manner which followed characteristics of a specific ligand-receptor interaction, although their ability to interact with lymphoid cells was considerably greater than that for hepatocytes. In WHV-infected animals, a netural antibody to the identified cryptic cell-binding site arose independently of that directed against epitopes of unmodified virus envelope and its appearance constituted the earliest immunovirological indicator of virus invasion. Our results demonstrated that the preS1 domain of the large WHV envelope protein is endowed with the species- and cell type-specific recognition site which is protected against antibody surveillance by the natural tertiary structure of the protein and we suggest that proteolytic cleavage is required to induce the binding activity.
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Immediate early protein IE63 of herpes simplex virus type 1 binds RNA directly
More LessThe herpes simplex virus type 1 (HSV-1) immediate early protein IE63 acts post-transcriptionally to affect RNA 3′-processing and splicing. Functional domains such as the RGG box and zinc-finger motifs potentially provide the protein with RNA binding capacity. Here, IE63 protein expressed in E. coli, purified by affinity chromatography and used in RNA binding assays, demonstrated similar binding to RNA substrates containing poly(A) sites from different temporal classes of HSV-1 genes, RNA containing splice site recognition sequences and RNA containing no recognized processing motifs. Competition binding assays showed that IE63 binding could be competed out, suggesting that IE63 binds RNA weakly. HSV-1 infection results in an increase or stabilization in vitro of protein binding to poly(A) site-containing RNAs; IE63 is required for this effect. RNA binding assays combining purified IE63 with protein from mock-infected and HSV-1 infected nuclear extracts demonstrated no effect on protein-RNA binding patterns.
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Role of cis-acting sequences of the ICPO promoter of herpes simplex virus type 1 in viral pathogenesis, latency and reactivation
More LessA mutant herpes simplex virus type 1, termed ΔTfi, with a 350bp deletion of the Sp1, NF-κB, TAATGARATs, C/EBP and F2 DNA-binding elements from -420 to -70 relative to the transcriptional start site of ICPO (Vmw 110), was generated and characterized. The efficiency of plating of ΔTfi was reduced on Vero cells and it expressed correctly initiated ICPO RNA in the presence of cycloheximide, although RNA levels were 2.5-fold lower than with wild-type (KOS) and marker-rescued (ΔTfiR) viruses. This was consistent with a demonstrated reduction in ICPO protein expression for ΔTfi at early times post-infection and a 3-fold reduction in ICPO-dependent transactivation of the ICP6 promoter. KOS, ΔTfi and ΔTfiR replication in murine corneas and trigeminal ganglia were comparable when measured on a complementing cell line, but ΔTfi titres appeared 15- to 50-fold lower when measured on Vero cells. ΔTfi was correspondingly less virulent than wild-type or marker-rescued viruses in both immunocompetent and SCID mice. ΔTfi, however, established and reactivated from latency with efficiencies comparable to wild-type and marker-rescued viruses. These results demonstrate that although this deletion of the ICPO promoter results in lower levels of ICPO in vitro and decreased virulence in vivo, the establishment of and reactivation from latency are unaffected. This indicates that elements which regulate ICPO expression and virulence during acute infection may be distinct from those involved in reactivation.
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Characterization of the genes, including that encoding the viral proteinase, contained in BamHI restriction fragment 9 of the pseudorabies virus genome
More LessWe describe the nucleotide sequence, transcription pattern and open reading frames (ORFs) located on BamHI restriction fragment 9 (0.406–0.435 map units) in the unique long segment of the pseudorabies virus (PRV) genome. The fragment contains three nested genes with a common 3′ end. The 5′ ends of the corresponding 0.9, 1.7 and 3.3 kb mRNAs have been mapped. Fragment BamHI-9 contains three complete ORFs, ORF1, ORF2 and ORF2.5. ORF1, which is within the 3.3 kb transcript, encodes a protein with an apparent molecular mass of 60 kDa which is homologous to the product of the herpes simplex virus type 1 UL25 gene. The 1.7 kb mRNA contains ORF2, whose product is homologous to the herpesvirus proteinases, while the 0.9 kb transcript contains ORF2.5, which probably encodes the assembly protein precursor. ORF2 was identified as the PRV proteinase gene following expression in E. coli, using the product of ORF2.5 as the substrate protein.
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Identification of a 56 kDa putative bovine herpesvirus 1 cellular receptor by anti-idiotype antibodies
More LessPolyclonal anti-idiotype antibodies (anti-ids) to anti-bovine herpesvirus 1 (BHV-1) MAbs blocked virus infection in cell cultures, indicating that they contain internal images of a viral attachment protein(s). In the present study anti-id (anti-83) of BHV-1 gB was used as a probe to detect the cellular receptor. Anti-id specifically identified a 56 kDa protein in radioimmunoprecipitation analysis (RIPA) of Madin-Darby bovine kidney (MDBK) cell membranes suggesting the involvement of this cell surface component in BHV-1 binding. Anti-83 pretreated with MAb 83 failed to identify the 56 kDa cellular component proving its specificity for the reacting epitope of MAb 83. The recognition of 56 kDa protein by anti-id was inhibited by prior incubation of radiolabelled membrane proteins with BHV-1 suggesting that the ligands competed for the same binding sites on the cells. 35S-Radiolabelled BHV-1 virions also bound a 56 kDa protein from purified MDBK cell membrane proteins in a virus overlay protein blot assay. RIPA using anti-id as probe detected the 56 kDa protein in permissive MDBK cells but not in non-permissive bat lung cells. The protein nature of the 56 kDa component was confirmed by protease treatment of membranes which resulted in abolition of the 56 kDa signal in RIPA. In addition, purified 56 kDa protein inhibited biotinylated BHV-1 attachment in flow cytometry. These findings indicate that the 56 kDa protein identified by anti-id is a putative receptor for BHV-1.
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Induction of apoptosis in epithelial cells by Epstein—Barr virus latent membrane protein 1
More LessEpstein—Barr virus (EBV) induces human B cell transformation and is closely associated with naso-pharyngeal carcinoma. The expression of an EBV latent membrane protein, LMP-1, protects B cells from apoptosis by up-regulating the expression of a cellular oncogene, bcl-2. LMP-1 also transforms rodent fibroblasts and affects the differentiation, morphology and growth of human and rodent epithelial cells. In this report, we describe a novel finding that high level expression of the LMP-1 gene in a human epithelial cell line (RHEK-1) induces apoptosis, characterized by chromosomal DNA fragmentation in the transfected cells. In particular, such an effect was more apparent under serum starvation. We also found that in the transfected RHEK-1 cells, LMP-1 expression neither affected bcl-2 expression nor led the cells to grow in semisolid soft agar medium. These results indicate that LMP-1 may participate in the development of EBV-associated epithelial malignancy via a mechanism different from that seen in B cell or fibroblast transformation.
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Functional analysis of C-terminal deletion mutants of Epstein—Barr virus thymidine kinase
Thymidine kinase (TK) activity was detected following expression of the TK gene of Epstein—Barr virus (EBV) using the pET expression plasmid and E. coli BL21(DE3)pLysS. To study the amino acid residues required at the C terminus of the EBV TK protein for enzymatic activity, a series of C-terminal deletion mutants was generated by direct truncation, linker insertion or PCR mutagenesis to create stop codons at particular sites. Deletion of nine residues from the C terminus caused a 35% reduction in TK activity, while a ten-residue deletion completely abolished the activity. A single point mutation at residue Cys570, corresponding to Cys336 of herpes simplex virus TK, did not alter the TK activity. Single amino acid changes within the last seven to ten residues also did not affect activity. The results indicate that maintenance of the conformation of the C terminus is important for enzyme activity.
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Cloning, restriction endonuclease mapping and partial sequence analysis of the genome of human herpesvirus 7 strain JI
More LessHuman herpesvirus 7 (HHV-7) is a recently isolated herpesvirus that has been shown to be related to human cytomegalovirus and human herpesvirus 6 and to be a member of the betaherpesvirus subgroup. Here we report the cloning, restriction endonuclease mapping and partial sequence analysis of HHV-7 strain JI DNA. Virus particles were obtained from the supernatant of infected SupT1 cells, the DNA isolated by proteinase K treatment-phenol extraction, and full-length viral DNA was purified and isolated on a pulsed-field gel. Aliquots of this highly purified material were treated in the following ways: (i) sonicated and end-repaired to create short randomly sheared fragments for cloning into M13mp18-Smal vector DNA; (ii) cut with EcoRI for cloning into EcoRI-cut λZAPII or λDASHII vectors; (iii) cut with BamHI for cloning into BamHI-cut λZAP-Express or λDASHII vectors. Partial nucleotide sequencing of the M13 clones followed by detection of open reading frames and their translation allowed the identification of homologues through FASTA searches of the database. Relevant M13 clones were used as probes to isolate corresponding λ phage clones, which could tentatively be mapped to the genome on the basis of presumed genetic collinearity between HHV-7 and HHV-6. Genomic ‘walking’ between EcoRI and BamHI λ genomic libraries enabled overlapping neighbouring clones to be identified and mapped. Each of these clones was analysed to map BamHI, EcoRI, Sall, Smal and Xhol restriction endonuclease sites to provide complete endonuclease maps for the entire genome.
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- Insect
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Characterization of the Lymantria dispar nucleopolyhedrovirus 25K FP gene
More LessThe Lymantria dispar nucleopolyhedrovirus (LdMNPV) gene encoding the 25K FP protein has been cloned and sequenced. The 25K FP gene codes for a 217 amino acid protein with a predicted molecular mass of 24870 Da. Expression of the 25K FP protein in a rabbit reticulocyte system generated a 27 kDa protein, in close agreement with the molecular mass predicted from the nucleotide sequence. The gene is located between 40.3 and 40.8 map units on the viral genome. It is respect to the circular map at late times during the infection cycle from a consensus baculovirus late promoter. The LdMNPV and Autographa californica nucleopolyhedrovirus (AcMNPV) 25K FP proteins exhibit 52% amino acid identity with several regions showing greater than 75% identity. Homologues to the AcMNPV orf59 and orf60 were also identified upstream (with respect to the genome) of the 25K FP gene in LdMNPV and exhibit 52% and 45% amino acid identity, respectively.
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- Plant
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Tomato golden mosaic virus open reading frame Al4 is genetically distinct from its C4 analogue in monopartite geminiviruses
More LessTomato golden mosaic virus (TGMV) is a bipartite geminivirus with six well-characterized genes. An additional open reading frame (ORF), AL4, lies within the essential AL1 gene. Recent studies of monopartite, dicot-infecting geminiviruses have revealed that mutations in their analogous C4 ORFs have host-specific effects on infectivity, symptomatology, virus movement and/or viral DNA accumulation. We have investigated whether TGMV has a similar host-specific requirement for AL4. The phenotypes of three TGMV al4 mutants were determined in a range of hosts, which included species that revealed c4 mutant phenotypes for monopartite geminiviruses. Each TGMV al4 mutant was indistinguishable from wild-type TGMV in all hosts tested. Additional analyses of double mutants revealed no evidence for functional redundancy between AL4 and the AL3, or AR1 genes. In contrast to the putative C4 proteins of monopartite geminiviruses, TGMV AL4, if it is expressed, is either non-functional, or functionally redundant with an essential TGMV gene product.
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Frequent occurrence of recombinant potyvirus isolates
More LessWe have performed a systematic search for recombination in the region encoding coat protein and the 3′ non-translated region in natural isolates of potyviruses, the largest group of plant RNA viruses. The presence of recombination, and the localization of the cross-over points, were confirmed statistically, by three different methods. Recombination was detected or suspected in 18 out of 109 potyvirus isolates tested, belonging to four out of eight virus species, and was most prevalent in potato virus Y, clear in bean common mosaic virus, and possible in bean yellow mosaic and zucchini yellow mosaic viruses. Recombination was not detected in the four other potyvirus species tested, including plum pox virus, despite the availability of numerous sequences for this last species. Though it was not specifically researched, no evidence for inter-specific recombination was found. For several reasons, including the fact that only a minor portion of the genome was analysed, the above figures certainly represent an underestimate of the extent of recombination among isolates of potyviruses, which might thus be a common phenomenon.
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The 5′-terminal region of a tombusvirus genome determines the origin of multivesicular bodies
More LessMultivesicular bodies (MVB) are membranous cytoplasmic inclusions that are invariably associated with tombusvirus infections regardless of the virus species, the host, or the tissue examined. MVB are virus-induced structures since they are absent from tissues of healthy plants and are always present both in infected plants and protoplasts. MVB derive from peroxisomes in cells infected by a number of tombusviruses including cymbidium ringspot virus (CymRSV) and from mitochondria in cells infected by another tombusvirus, carnation Italian ringspot virus (CIRV). By using common restriction sites in full-length infectious clones, hybrid clones of these two viruses were constructed. In addition, a mutant of CIRV was prepared in which the protein encoded by the first open reading frame was shortened by 22 amino acids. All mutant transcripts were viable and infected Nicotiana benthamiana plants. Infected leaf tissue samples were collected, processed for thin sectioning, and observed in the electron microscope. The origin of MVB was shown to be under the control of the 5′ region of the viral genome. A sequence as short as about 600 nucleotides in ORF 1 contained the determinants for formation of MVB from peroxisomes or mitochondria.
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Immunological characterization of rice tungro spherical virus coat proteins and differentiation of isolates from the Philippines and India
More LessRice tungro spherical virus (RTSV) has an RNA genome of more than 12 kb with various features which classify it as a plant picornavirus. The capsid comprises three coat protein (CP) species, CP1, CP2 and CP3, with predicted molecular masses of 22.5, 22.0 and 33 kDa, respectively, which are cleaved from a polyprotein. In order to obtain information on the properties of these proteins, each was expressed in E. coli, purified as a fusion to the maltose-binding protein and used for raising a polyclonal antiserum. CP1, CP2 and CP3 with the expected molecular masses were detected specifically in virus preparations. CP3 is probably the major antigenic determinant on the surface of RTSV particles, as was shown by ELISA, Western blotting and immunogold electron microscopy using antisera obtained against whole virus particles and to each CP separately. In some cases, especially in crude extracts, CP3 antiserum detected several other proteins (40–42 kDa), which could be products of CP3 post-translational modification. No serological differences were detected between the three CPs from isolates from the Philippines, Thailand, Malaysia and India. The CP3-related 40–42 kDa proteins of the Indian RTSV isolate have a slightly higher electrophoretic mobility (42–44 kDa) and a different response to cellulolytic enzyme preparations, which allows them to be differentiated from south-east Asian isolates.
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- Other Agents
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Sequential appearance and accumulation of pathognomonic markers in the central nervous system of hamsters orally infected with scrapie
More LessBoth infectivity and TSE-specific amyloid protein (also referred to as protease resistant- or prion protein, PrP) are pathognomonic markers for transmissible spongiform encephalopathies (TSE). This paper presents a new densitometric method for the quantification of TSE-specific amyloid protein and its application to studying the pathogenesis of scrapie in Syrian hamsters after infection with scrapie strain 263K. A first study established a close correlation between infectivity and TSE-specific amyloid protein with a doubling time of 2–2.6 days in the brain and cervical spinal cord for both markers. The ratio of infectivity and TSE-specific unit during the course of infection. A subsequent study addressed the temporal-spatial spread of infection in the central nervous system by tracing the accumulation of the pathological protein. The pathogenetic process was first detected in the spinal cord between vertebrae T4 and T9, and then showed an anterograde and retrograde spread with a rate of 0.8–1⊙0 mm/day. There were also some indications for a possible alternative route of spread of infection from the periphery to the brain, other than via the spinal cord. Involvement of the spleen did not appear essential for the early pathogenesis in hamsters orally infected with the 263K strain of scrapie.
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Replication of scrapie in spleens of SCID mice follows reconstitution with wild-type mouse bone marrow
More LessSCID mice are resistant to intraperitoneal infection with 103 and 104 intracerebral ID50 units of ME7 scrapie agent whereas they develop disease after intracerebral challenge. However, higher doses introduced, by intraperitoneal or subcutaneous routes, produce disease. Immunocompetent mice of the same strain (CB20) developed scrapie following either intracerebral or intraperitoneal infection. Bioassay of spleens from SCID mice infected with 10−1 dilutions of ME7 scrapie by intraperitoneal, intracerebral or abdominal subcutaneous injection showed traces or low levels of infectivity in spleen. However, subcutaneous injection beneath the skin of the neck failed to infect the spleen. CB20 bone marrow reconstitution of SCID mice resulted in the regeneration of a normal lymphoid architecture in the spleen. Spleens from these reconstituted mice, infected intracerebrally with a 10−1 dilution of ME7 contained high levels of infectivity. These results suggest that the ability to replicate scrapie agent in spleen or lymphoid tissue depends on the restoration of normal lymphoid structure and in particular the presence of differentiated follicular dendritic cells. The possibility that SCID mice can select minor strains of scrapie which are normally unrecognized in cloned ME7 is discussed.
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Protease-resistant PrP deposition in brain and non-central nervous system tissues of a murine model of bovine spongiform encephalopathy
More LessInfectivity within the central nervous system has been demonstrated by the transmission of bovine spongiform encephalopathy (BSE) from affected cattle to inbred laboratory mice. Sedimentable, protease-resistant PrP (PrPSc) has also been extracted from BSE-affected cattle brain. Both infectivity and PrPSc have been reported in the lymphoreticular tissues of sheep and mice clinically and preclinically affected with scrapie. Neither infectivity nor PrPSc has yet been detected in non-neural tissues of naturally occurring, clinical cases of BSE in cattle. We have used a murine model of BSE (301V isolate in VM/Dk mice) to investigate when and where PrPSc accumulates. PrPSc was detected both in brain and in extraneural sites prior to the onset of clinical symptoms. This murine BSE model differs, however, in four important aspects from our previously published findings for murine scrapie models: (a) PrPSc was found relatively late into the incubation period; (b) after intracerebral inoculation, PrPSc was found in brain before it was found in other tissues; (c) no PrPSc was found in most of the spleens from clinically affected animals after intracerebral inoculation; and (d) even after intra-peritoneal infection, PrPSc was detected in brain first.
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- Corrigendum
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