- Volume 71, Issue 11, 1990
Volume 71, Issue 11, 1990
- Review Article
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Structure, function and evolution of picornaviruses
More LessConclusionThis review illustrates how molecular and structural analyses have contributed to a greater understanding of the rich biological diversity seen in the picornaviruses. In many cases the second phase of studies, based on increasingly powerful genetic engineering, biochemical and immunological techniques is now well under way and beginning to reveal the depth of the vast store of information which still lies encoded cryptically in the sequences and structures which have been determined. The advances made already suggest that the next few years will be even more fruitful than those since 1981 when the first picornavirus sequence was published.
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- Animal
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Type 3 poliovirus/Finland/1984 is genetically related to common Mediterranean strains
More LessStrains of poliovirus type 3 isolated in Finland in 1984 and 1985 (P3/Fin/84) are known to differ considerably from the type 3 vaccine strains in both nucleotide sequence and antigenic properties. In the search for the origin of the outbreak we first tested 80 type 3 strains that had been isolated elsewhere in the world during the years 1953 to 1986. An oligonucleotide probe complementary to a highly variable 17 nucleotide interval in the 5′ non-coding region of the genomic RNA of P3/Fin/84 reacted with five strains. Also it was revealed that two of the latter five strains were related to the P3/Fin/84 strains in two separate genomic regions compared after partial RNA sequencing. One of them was isolated in Switzerland in 1980 and the other in Turkey in 1981. The Swiss strain was from a patient who had recently returned from a journey to various Mediterranean countries. Consequently, 16 other strains isolated in the late 1970s and early 1980s in Europe or in the Mediterranean countries were studied in detail by partial genomic sequencing and with neutralizing monoclonal antibodies. Two separate regions of the genome were compared by sequencing and corresponding dendrograms were constructed. The Switzerland and Turkey strains were found to be the strains most closely related to the viruses of the 1984 Finland epidemic. These results indicate that type 3 poliovirus strains related to P3/Fin/84 had been circulating in Mediterranean countries since the late 1970s.
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Antigenic structure of chimeras of type 1 and type 3 poliovirus involving antigenic site 1
Chimeric polioviruses have been prepared in which part of the antigenic site 1-encoding sequence of the Sabin strain of type 1 poliovirus has been replaced by sequences based on those found in the homologous region of the Sabin type 3 strain. The chimeras were analysed for their reaction with polyclonal and monoclonal antibodies raised against type 1 and type 3 viruses, and with polyclonal antipeptide sera, as well as for their immunogenicity in animals. The effectiveness with which the type 3 site was presented antigenically varied in ways which were partially predictable, based on the behaviour of type 3 mutants with monoclonal antibodies. However, other factors were implicated which may include conformational effects and other components of the site in addition to those altered in the chimeras. The ability of the chimeras to induce antibodies reacting with type 3 polioviruses paralleled their antigenic reactivity, and evidence is presented for the induction of strain-specific antibodies.
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Cleavage specificity of the poliovirus 3C protease is not restricted to Gln-Gly at the 3C/3D junction
More LessThe 3C protease of poliovirus is distinguished from that of all other picornaviruses in that it only cleaves at Gln-Gly amino acid pairs within the viral polyprotein. To determine whether this strict cleavage specificity is an intrinsic property of the poliovirus 3C protease, amino acid substitutions were introduced at one of the Gln-Gly cleavage sites. Oligonucleotide-directed site- specific mutagenesis of an infectious poliovirus type 1 (Mahoney strain) cDNA was used to change the Gln-Gly site at the 3C/3D junction of the polyprotein into Gln-Val, Gin-Ala, Gln-Ser or Gin-Pro. The effects of these substitutions were studied in vivo after transfection of primate cells by the mutated cDNAs. The Gln-Gly to Gin-Pro substitution was lethal for virus growth, and the corresponding altered 3CD polypeptide expressed in insect cells using a recombinant baculovirus vector did not appear to undergo autocleavage. The Gln-Gly to Gln-Val change was also lethal, although production of virus was occasionally observed as a result of reverse mutations. Mutants with Gin-Ala and Gln-Ser sequences were viable, indicating that these dipeptides can be cleaved by the poliovirus protease in vivo. However, processing at the 3C/3D junction occurred relatively inefficiently in the case of the Gln-Ser virus. Furthermore, the Gln-Gly to Gin-Ala substitution seemed to result in an additional cleavage event within the N-terminal part of polypeptide 3D.
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Borna disease virus-induced meningoencephalomyelitis caused by a virus-specific CD4+ T cell-mediated immune reaction
More LessAfter intracerebral inoculation of Borna disease virus (BDV), Lewis rats develop a persistent infection of the central nervous system which is pathohistologically represented by perivascular encephalitic lesions predo-minantly in the grey matter. In previous studies it has been shown that a cell-mediated immune response causes Borna disease (BD). In order to define further the immune cell responsible for this immunopathologi-cal disease, a BDV-specific T cell line, NM1, was established and cultured in vitro. Phenotypically this T cell line was characterized by cytofluorometry as CD4-positive (CD4+). Proliferation assays with syngeneic and allogeneic antigen-presenting cells, and blocking experiments with monoclonal antibodies, revealed major histocompatibility complex class II antigens to be restriction elements. After passive transfer of this virus-specific CD4+ T cell into immunosuppressed BDV-infected recipients, full-blown disease could be induced. Immunohistological examination of the cells involved in perivascular inflammatory infiltrates in BDV-infected rats and in recipients of the NM1 T cell line revealed a dominance of macrophages and CD4+ T cells. The presence of these cells in encephalitic lesions strongly suggests a delayed type of hypersensitivity reaction as the pathogenetic mechanism of BD.
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Multiplication of virulent and demyelinating Semliki Forest virus in the mouse central nervous system: consequences in BALB/c and SJL mice
More LessThe sites of multiplication in the mouse central nervous system (CNS) of the virulent L10 strain of Semliki Forest virus (SFV) and the L10-SFV-derived demyelinating M9 mutant were determined using both BALB/c and SJL mouse strains. In situ hybridization (ISH), using a cRNA probe to an SFV non-structural sequence, and immunogold-silver staining (IGSS), using polyclonal anti-SFV rabbit IgG, were the techniques utilized. For L10-SFV, viral RNA and antigen were detected in neurons and glial cells of both mouse strains. For BALB/c mice infected with M9-SFV, both neuronal and glial cell infection was less extensive than that obtained with L10. ISH or IGSS were generally not sensitive enough to detect viral RNA and antigen, respectively, in M9-SFV-infected SJL mice. M9-SFV multiplied to a similar titre in primary cultures of glial cells derived from either BALB/c or SJL mice. Following infection with M9-SFV, small plaques of demyelination in the CNS and occasional small aggregates of mononuclear leukocytes in the leptomen- inges persisted for up to 12 months in SJL mice but not BALB/c mice. This was not associated with detectable persistence of infectious virus, viral antigen or viral RNA in the CNS.
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Effects of different biological response modifiers on interferon expression in bacterial lipopolysaccharide (LPS)-responsive and LPS-hyporesponsive mouse peritoneal macrophages
More LessWe have previously shown that the antiviral state of explanted mouse peritoneal macrophages (PM) decays during in vitro culture and that this decay is much more rapid in Lps d PM than it is in Lps n PM. Moreover, Lps n PM can transfer the antiviral state to other cells, whereas Lps d PM cannot. In vitro treatment of Lps n PM with different agents [i.e., bacterial lipopolysaccharide (LPS), interferon (IFN)-γ, tumour necrosis factor (TNF)-α, macrophage colony-stimulating factor (M-CSF) and antibody to Mac-1 antigen] induced an antiviral state to vesicular stomatitis virus (VSV) which was inhibited by antibodies to IFN-β. Treatment of Lps n PM with LPS or IFN-γ resulted in greater accumulation of IFN-β mRNA, whereas no change in the barely detectable levels of IFN-α mRNA was observed. Marked accumulation of IFN-β mRNA was also observed in PM after TNF-α treatment. M-CSF and IFN-γ (but not LPS) also induced an IFN-mediated antiviral state in Lps d PM. Low levels of spontaneous transcription of IFN-β mRNA were detected in nuclei from Lps d PM. Treatment of Lps d PM with IFN-γ for 3 h resulted in the accumulation of IFN-β mRNA without any concomitant increase in the transcription of the IFN-β gene, as determined by run-on transcription assays with isolated nuclei. The addition of as little as 1 international unit/ml of IFN-γ to PM resulted in a 100-fold inhibition of VSV yield. As antibodies to IFN-α/β inhibited only a portion of the IFN-γ-induced antiviral state, such an antiviral state might reflect the synergism between IFN-γ and endogenous IFN-β. In fact, the addition of low doses of both IFN-γ and IFN-β to either Lps n or Lps d PM resulted in synergistic antiviral effects. In vivo treatment of Lps d mice with granulocyte-macrophage (GM)-CSF, M-CSF, IFN-γ or Newcastle disease virus rendered peritoneal cells capable of transferring an antiviral state. These results indicate that (i) various stimuli can induce IFN-β production by PM, (ii) Lps d PM spontaneously transcribe low levels of IFN-β mRNA, even though they cannot transfer an antiviral state, (iii) different stimuli, but not LPS, induce a normal IFN response in Lps d PM, (iv) IFN-γ increases the accumulation of IFN-β mRNA in Lps d PM by post-transcriptional mechanisms and (v) IFN-γ may act synergistically with endogenous IFN-β in inducing a potent antiviral state to VSV in PM.
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Monoclonal antibodies to rabbit haemorrhagic disease virus and their use in the diagnosis of infection
More LessHybridomas producing monoclonal antibodies (MAbs) to rabbit haemorrhagic disease virus (RHDV) were prepared. Using Western blot (WB) analysis, the MAbs obtained were divided into two groups, one reacting with the major structural proteins of M r 61K and 38K, and the other giving negative reactions. Both groups of MAbs, however, reacted specifically with RHDV in ELISA and by immunoperoxidase (IP) and immunofluorescence (IF) tests with infected cells. As demonstrated by WB using RHDV-specific MAbs and a MAb to feline calicivirus (FCV) strain F9, the major structural (capsid) proteins of RHDV and FCV have very similar sizes (M r 61K and 38K compared to 62K to 64K and 40K respectively). No cross-reactions of MAbs with proteins of the other virus were observed in WB analysis, ELISA, IP tests or IF. The high specificity and sensitivity of RHDV-specific MAbs make them suitable for the routine IP and IF diagnosis of RHDV in liver cells of rabbits dying after natural or experimental infections.
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Porcine respiratory coronavirus differs from transmissible gastroenteritis virus by a few genomic deletions
More LessThe genome organization of porcine respiratory coronavirus (PRCV), a newly recognized agent which has a close antigenic relationship to the enteropathogenic transmissible gastroenteritis virus (TGEV), was studied. Genomic RNA from cell-cultured PRCV (French isolate RM4) was used to produce cDNA clones covering the genomic 3′ end to the start of the spike (S) glycoprotein gene (7519 nucleotides). Six open reading frames (ORFs) were identified that allowed the translation of three coronavirus structural proteins and three putative non-structural (NS) polypeptides, homologous to TGEV ORFs designated NS3-1, NS4 and NS7. Pairwise alignment of PRCV nucleotide and amino acid sequences with sequence data available for three TGEV strains revealed a 96% overall homology. However, the genome of PRCV exhibited two important distinctive features. The first was that the S gene lacked 672 nucleotides in the 5′ region and encoded a truncated form of the S polypeptide, and secondly, the first NS ORF downstream of the S gene was predicted to be non-functional as a consequence of a double deletion. The significance of genomic deletions with respect to tissue tropism and evolution of coronaviruses is discussed.
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Location of epitopes on the major core protein p24 of human immunodeficiency virus
More LessAntibody-binding sites were mapped on all overlapping nonapeptides of the major core protein p24 of human immunodeficiency virus type 1 (HIV-1) using murine monoclonal antibodies (MAbs) and sheep and rabbit polyclonal antibodies raised against HIV-1/H9 (strain IIIB) viral lysate and antibodies obtained from humans infected with HIV-1. The binding sites were mapped to various distinct regions of this protein. After superimposition of the antibody-binding sites on a proposed model of p24 of HIV-1, these sites appeared to be located on the surface of the protein on loops, turns and coils of p24 but, unexpectedly, not on the major part of the predicted ‘puff. Little reaction was found with the inaccessible anti-parallel β-barrel. These results are the first experimental evidence for the validity of the structure proposed for p24 of HIV-1.
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Identification of operationally overlapping and independent cross-reactive neutralization regions on human rotavirus VP4
More LessCross-reactive neutralization epitopes on VP4 of human rotavirus (HRV) were analysed by the use of VP4-specific neutralizing monoclonal antibodies (N-MAbs) and MAb-resistant mutants. Seven anti-VP4 N-MAbs obtained in this study by using HRV serotypes 1 and 3 as immunizing antigens showed a variety of cross-reactivity patterns to 20 HRV strains with different serotype specificity in neutralization tests and a broader cross-reactivity to them was found for four N-MAbs in an enzyme-linked immunosorbent assay. On the basis of the reactivity patterns against rotaviruses in neutralization tests, these seven N-MAbs were classified into four groups. Cross-neutralization tests using a total of 12 pairs of MAbs and resistant mutants, including five pairs which had been prepared previously, showed that VP4 of HRV (strain KU) contained two independent antigenic regions. One, region C1, was recognized by a single MAb (YO-2C2) and the other was made up of two antigenic regions (C2 and C3) which overlapped operationally. Identification of amino acid substitution sites on VP4 of representative mutants of HRV strain KU indicated that amino acid positions 385 or 392 and 428 or 433 were critical for the C2 and C3 regions, respectively. These results suggested that regions C2 and C3 exist as conformational antigenic sites.
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Antigenic and biochemical characterization of bovine rotavirus V1005, a new member of rotavirus serotype 10
Bovine rotavirus (BRV) VI005 is serologically distinct from rotavirus serotypes 1, 2, 3, 4, 5, 6, 8 and 9. BRV VI005 showed cross-reactions with BRV B223, the American prototype of serotype 10 rotavirus, and with BRV E4049, a British serotype 10 isolate. BRV V1005 was, however, not neutralized by four monoclonal antibodies directed against VP7 of BRV B223. Twoway cross-reactions were observed between BRV VI005 and a reassortant rotavirus containing the VP4 from BRV UK. In addition the major tryptic cleavage product of VP4, VP5*, from BRV V1005 is indistinguishable by peptide mapping and its isoelectric point from the homologous protein of BRV UK, but is clearly different from VP5* of BRV NCDV. The peptide map of VP7 from BRV VI005 differed from that obtained for VP7 of BRV UK.
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Characteristically distinct isolates of the nuclear polyhedrosis virus from Spodoptera litura
More LessMore than 100 isolates were plaque-purified to examine the genetic variations in four wild stocks of Spodoptera litura. Nuclear polyhedrosis virus (NPV) collected in Japan. These isolates were characterized by their in vitro host range in three established insect cell lines, growth characteristics, polyhedral protein, DNA restriction endonuclease pattern and DNA hybridization. The isolates were separated into four distinct groups: (I) isolates corresponding to Autographa californica NPV, (II and IV) two different groups of isolates of S. littoralis NPV which had been previously characterized and (III) isolates with no correspondence to any reported virus group. Of the S. litura NPV wild stocks, two were mixtures of more than two different groups of NPVs. We have discussed the advantage of having a mixture of different NPV groups in the same wild virus stocks.
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Expression of avian leukaemia virus env-gp85 in Spodoptera frugiperda cells by use of a baculovirus expression vector
We studied the genetic expression of gp85 of avian leukaemia virus (ALV) subgroup A in a baculovirus/insect cell system. 5′-terminal sequences of the gag gene were added to precede the ALV gp85 sequence and a stop codon was introduced at the boundary of gp85 and gp37. The resulting construct was then cloned into the baculovirus transfer vector pAcYM1, which contains the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Cells of the insect Spodoptera frugiperda (Sf9) were cotransfected with the resulting recombinant transfer vector pAc85 and infectious AcNPV/E2 DNA. After cotransfection, recombinant baculovirus that lacked the polyhedrin gene and expressed gp85 was selected from the supernatant and used to infect Sf9 cells. The expression of the gp85 gene peaked 3 days after infection, but expression products were not released into the culture medium even though the signal peptide had been cleaved. Owing to incomplete N-glycosylation in the insect cells the largest gp85 product had an M r of only 65000. In immunofluorescence tests and immunoblots the recombinant gp85 products reacted with polyclonal and monoclonal antibodies directed against ALV gp85 of subgroup A. Chickens inoculated with crude lysates of Sf9 cells infected with gp85-expressing recombinant baculovirus developed antibodies directed against ALV gp85. These antibodies were not capable of neutralizing ALV.
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Purification and characterization of the major group-specific core antigen VP7 of bluetongue virus synthesized by a recombinant baculovirus
More LessThe major core protein, VP7, of bluetongue virus serotype 10 (BTV-10) has been purified from insect cells infected with a genetically manipulated recombinant baculovirus. The high level expression of VP7 (in excess of 100 mg per litre of culture) and its presence in the soluble fraction of infected cells following lysis by detergent has allowed the purification of the protein virtually to homogeneity (95%) by a simple two-step procedure of ammonium sulphate fractionation and ion-exchange chromatography. The purified antigen is highly immunogenic and has been shown in an ELISA to be reactive with antisera of 24 BTV serotypes (1 to 24) as well as with an antiserum raised to African horsesickness virus type 4 (AHSV-4), a representative of another serogroup of orbiviruses. In confirmation of these data a monospecific antiserum raised with the expressed product has been shown by Western blot analyses to react with other BTV serotypes as well as with two serotypes of epizootic haemorrhagic disease virus (EHDV-1 and EHDV-2), a closely related orbivirus. The data indicated that VP7 is a highly conserved protein amongst BTV serotypes and at least partly conserved amongst three serogroups of orbiviruses.
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Purification and characterization of the infectious hypodermal and haematopoietic necrosis virus of penaeid shrimps
More LessInfectious hypodermal and haematopoietic necrosis (IHHN) is one of the most important viral diseases of cultured penaeid shrimps and is potentially a limiting factor in the development of farming projects for some species of these shrimps. Although the IHHN agent was recognized early as being viral in origin, attempts to characterize it were inconclusive because of difficulties in obtaining sufficient amounts of purified virions to permit its characterization. Recent improvements of purification procedures have allowed the physic chemical characterization of this virus. Purified IHHNV is a non-enveloped icosahedral particle averaging 22 nm in diameter, exhibiting a mean buoyant density of 1·40 g/ml in CsCl. The genome is a single molecule of ssDNA with an estimated size of 4·1 kb by molecule length measurement in transmission electron microscopy. As determined by SDS-PAGE, the particle contains four polypeptides with M rs of 74K, 47K, 39K and 37·5K, respectively. From its characteristics, this virus could be a member of the Parvoviridae family.
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The production of human parvovirus capsid proteins in Escherichia coli and their potential as diagnostic antigens
More LessWe have expressed a number of polypeptides derived from the capsid proteins of the human parvovirus B19 in Escherichia coli. These include native VP1 (84K) and VP2 (58K) proteins and also fusions to β- galactosidase containing differing amounts of the amino terminus of the VP 1/2 polypeptide. Although each of these was expressed at high levels and the majority were produced as full-length proteins, only one was soluble. This soluble polypeptide, pi32, is a β- galactosidase fusion protein that includes 145 amino acids from B19 which are entirely derived from the region unique to VP1. Despite containing such a small portion of VP1, which itself constitutes only 4% of total capsid protein, p132 reacted with all our known anti-B19 IgM-positive human serum samples. We conclude that this region contains epitopes which must be prominently exposed on the intact virus. We have demonstrated the use of this recombinant antigen in a simple diagnostic assay for B19-specific antibodies which can be used for initial screening of human serum samples. In a survey of 103 serum specimens, our ELISA positively identified all samples (19/19) which were positive by IgM antibody capture radioimmunoassay. The recombinant p132 antigen is efficiently produced and readily purified from E. coli, and its use as a diagnostic antigen should increase the availability of routine clinical testing for human parvovirus infection.
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Structure and composition of a family of human cytomegalovirus glycoprotein complexes designated gC-I (gB)
More LessMurine rhonoclonal antibodies (MAbs) were made to the 52000 (gp52) and the 93000 to 130000 Mr (gp93–130) glycoproteins from a human cytomegalovirus (HCMV) glycoprotein complex designated gC-I or the gB homologue. MAbs recognizing either gp52 or gp93–130 could immunoprecipitate unreduced gC-I complexes from non-ionic detergent extracts of HCMV. Western blotting was performed with immunoaffmity- purified gC-I complexes which were reduced prior to analysis. MAbs made against gp52 recognized gp52 and a 158000 Mr glycoprotein (gpl58). MAbs which recognized gp93–130 in a Western blot also reacted with gpl58, which is a gC-I precursor glycoprotein. The origin of gp93–130 was demonstrated by the reactivity of our gp93–130 MAbs with a recombinant protein containing the N-terminal portion of the gB gene. These data are consistent with the hypothesis that gp52 and gp93–130 are generated from the same high Mr precursor by proteolysis. MAbs recognizing either gp52 or gp93–130 neutralized Towne strain HCMV, but MAbs recognizing gp52 required complement to neutralize whereas MAbs recognizing gp93–130 did not. It was also determined that gp93–130 and gpl58 have detectable amounts of 0-linked glycans but gp52 does not, showing a difference in the glycosyla- tion of these glycoproteins. Analysis of gC-I disulphide bonds showed that two types were present, one which was very susceptible to reduction and a second which was less susceptible. These complexes could consist of very susceptible inter-complex disulphide bonds and less susceptible intra-complex disulphide bonds.
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A herpes simplex virus type 1 recombinant with both copies of the Vmw175 coding sequences replaced by the homologous varicella-zoster virus open reading frame
More LessVaricella-zoster virus (VZV) gene 62 encodes a protein with a predicted Mr of 140000 (VZV 140K) that shares considerable amino acid homology with the immediate early (IE) regulatory protein Vmwl75 of herpes simplex virus type 1 (HSV-1) and is believed to be its functional equivalent. We have tested this hypothesis by insertion of VZV gene 62 (expressed from the HSV- 1 IE3 promoter) into both IE3 gene loci in the short region repeats of the HSV-1 genome. The parent virus used for this manipulation was D30EBA, which is a variant of HSV-1 from which the majority of the Vmwl75 coding sequences have been deleted. Like other HSV-1 viruses lacking Vmwl75 function, D30EBA is able to grow only in cell lines which express Vmwl75 constitutively. The resulting recombinant virus, HSV-140, is able to propagate (but unable to form obvious plaques) on normal cell lines. The properties of HSV-140 were studied by monitoring the time course of polypeptide expression and DNA replication during normal infection. We found that at high multiplicity HSV-140 synthesized apparently normal amounts of many viral polypeptides but that the expression of certain late genes was reduced; this slight defect may be related to less efficient DNA replication by HSV-140. At low multiplicity HSV-140 expressed viral proteins inefficiently. Surprisingly, VZV 140K was produced in large amounts at later times of a normal infection, indicating that the polypeptide fails to autoregulate the IE3 promoter. The results strongly suggest that VZV 140K is able to perform most of the functions of Vmwl75 during growth of HSV-1, but that differences in detail lead to less efficient virus growth.
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Definition of murine T helper cell determinants in the major capsid protein of human papillomavirus type 16
More LessThree murine major histocompatibility complex (MHC) class II-restricted T cell determinants were identified in the major capsid protein L1 of human papillomavirus (HPV) type 16. Peptides derived from HPV-16 L1, which contain putative T cell epitopes located by a predictive algorithm, were synthesized and tested for lymphoproliferative activity by direct immunization, followed by in vitro assay of responses to peptides or recombinant HPV-16 L1. The MHC restriction of the stimulatory peptides was determined using blocking monoclonal antibodies against class II molecules. The responses, which were specific for the priming peptides alone, cross-reacted with recombinant L1 but not with analogous peptides derived from other HPV types.
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Multiple interactions between cellular factors and the non-coding region of human papillomavirus type 16
More LessThe interaction between cellular factors and the noncoding region (NCR) of human papillomavirus type 16 (HPV-16) has been studied using different approaches: DNase I hypersensitive sites (HSS) analysis of HPV-16 chromatin in SiHa cells, footprinting (FP) of the NCR in vitro with nuclear extracts from SiHa cells and band shift analysis of synthetic oligonucleotides corresponding to the HSS and footprint sites with nuclear extracts from a variety of cells. These analyses have shown that cellular DNA-binding factors (CDBFs) bind to at least 13 sites, FP-A to M, over approximately 500 bp in the 3′ half of the NCR. FP-E to H overlap with the keratinocyte-dependent enhancer and most sites contain the consensus sequence A TGCCAAA T, which is similar to both the cytokeratin promoter element AARCCAAA and the ubiquitous CCAAT motif. FP-E appears to be the target for C/EBP and AP1 binding, but FP-F, G and H appear to bind novel specific cellular factors. The CDBF binding to FP-G has been tentatively identified as the same factor binding to the cytokeratin promoter element.
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Identification of seroreactive regions of the human papillomavirus type 16 proteins E4, E6, E7 and L1
Small fragments of the DNA of human papillomavirus type 16 (HPV-16) were randomly cloned into the bacteriophage fd which expresses the resulting peptides as part of its capsid. Antisera raised against different HPV-16 fusion proteins were used for screening of the phage clones and the reacting peptides were determined by sequencing the inserted HPV-16 DNA fragments of the positive recombinants. Seroreactive regions of the proteins derived from the E4, E6, E7 (two regions) and LI (three regions) open reading frames could be found by this approach. Of these seven regions, four were defined by at least two overlapping inserts, thus limiting the domains to between 10 and 15 amino acids. In the case of the E4 open reading frame, the same region identified by immunoscreening was also found when synthetic overlapping octapeptides were tested by ELISA with the anti-E4 antiserum. Using an approach to predict ‘receptor-like’ regions within the respective proteins, five of the seven regions were also identified. From the data on these regions, synthetic peptides were produced and used for the detection of antibodies against HPV-16 proteins in human sera by ELISA.
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Synthetic peptides derived from E7 region of human papillomavirus type 16 used as antigens in ELISA
More LessNine overlapping peptides (20 amino acid) covering the entire sequence of early antigen E7 of human papillomavirus type 16 (HPV-16) were synthesized and tested as antigens with human sera in ELISA. Five of these peptides (no. 1 to 5 counting from the N terminus of the E7 protein) reacted with a pool of sera from HPV-16-infected individuals (as determined by molecular hybridization with their biopsy specimens); one (no. 5) was also reactive with pools of HPV-18- and HPV-6- or 11-infected individuals. Sera from 24 patients with cervical intraepithelial neoplasia (CIN) and from 29 invasive cervical carcinoma (INCA) patients were tested for the presence of antibodies reactive with peptides, no. 1 to 4 covering amino acids 1 to 50 and with peptide no. 5 covering amino acids 41 to 60. Only one of the sera from CIN patients was reactive with peptides no. 1 (amino acids 1 to 20) and no. 4 (amino acids 31 to 50). However, the majority of these sera reacted with peptide no. 5. The occurrence of this antibody was only slightly less frequent in sera from healthy subjects compared to CIN patients. On the other hand, sera from the INCA patients were reactive with the peptides no. 1 to 3 more frequently than the sera from matched control subjects. Positive reactions of sera from INCA patients were most frequently seen with no. 2; 24% of these sera but only 7% of the controls were reactive with no. 2 peptide. The present data suggest that no. 1 to 3 are HPV-16-specific, whereas no. 5 is broadly cross-reactive.
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Expression of the full-length products of the human papillomavirus type 6b (HPV-6b) and HPV-11 L2 open reading frames by recombinant baculovirus, and antigenic comparisons with HPV-11 whole virus particles
More LessThe L2 open reading frames (ORFs) of human papillomavirus (HPV) types 6b and 11 were expressed as full-length non-fusion proteins in Spodoptera frugi- perda (Sf-9) cells using recombinant baculovirus. Both proteins were detected on Western blots as immuno- reactive bands which migrated with apparent Mr s of 76K and 78K, respectively, and contained both crossreactive and type-specific epitopes, as determined by polyclonal antisera directed against defined subregions of the HPV-6b and HPV-11 L2 ORFs. In addition, the minor capsid protein of HPV-11 particles co-migrates with the HPV-11 L2 ORF product and is immunore- active with HPV-11 L2-specific antisera. These observations indicate that the anomalous electrophoretic mobilities of papillomavirus L2 ORF proteins can be explained without invoking post-transcriptional processing events and that the minor capsid protein of HPV-11 is antigenically and biophysically related to the HPV-11 L2 ORF product.
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Characterization of BK virus variants rescued from human tumours and tumour cell lines
Episomal BK virus (BKV) DNA was detected in primary human brain tumours, in Kaposi’s sarcoma and in cell lines from brain tumours, Ewing sarcoma and osteogenic sarcoma. Infectious BKV was rescued from several tumours and tumour cell lines by transfection of total cellular DNA into human embryonic fibroblasts. Restriction endonuclease and nucleotide sequence analysis showed that all the rescued viruses are similar to BKV-IR, a BK variant previously isolated from a human tumour of pancreatic islets, indicating that a specific BKV strain may be associated with certain types of human tumours. All the variants contain a putative transposable element in the regulatory region of the viral genome. This region has mutagenic properties and enhancing activity in transformation, suggesting a possible role of these variants in tumour induction or progression.
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Immunoelectron microscopy on the topographical distribution of the poliovirus receptor
More LessThe topographical distribution of the poliovirus receptor on the cell surface was demonstrated by immunoelectron microscopy using monoclonal antibodies and immunogold markers. The receptor appeared in small clusters, which were randomly distributed over the cell surface and along cellular processes. The distribution pattern of the clusters corresponded to that of adsorbed and immunogold- labelled poliovirus particles and suggests a multivalent organization of poliovirus binding sites. Freezefracturing and ultrathin sectioning did not reveal any specific ultrastructures within the plasma membrane at labelled receptor areas. Incubation of native cells with anti-receptor antibodies did not remove the receptor molecule from the cell surface nor did it induce ultrastructural alterations within the plasma membrane. The antibody-receptor complexes exhibited lateral mobility within the plasma membrane and were able to aggregate into large immune complexes after incubation with a second ligand.
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A synthetic peptide derived from the amino acid sequence of canine parvovirus structural proteins which defines a B cell epitope and elicits antiviral antibody in BALB c mice
More LessSynthetic peptides, recombinant fusion proteins and mouse monoclonal antibodies were used to delineate a B cell epitope of the VP′2 structural protein of canine parvovirus (CPV). Although this epitope is not preferentially recognized in the normal antibody response to CPV, virus-specific antibodies could be induced in BALB/c mice with a synthetic peptide representing the epitope. The potential of this non-dominant B cell epitope to induce antiviral immunity in the presence of maternal CPV-specific antibodies, is discussed.
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Nucleotide sequence of feline panleukopenia virus: comparison with canine parvovirus identifies host-specific differences
More LessThe nucleotide sequence of feline panleukopenia virus (FPV) strain 193 was determined and compared with the sequence of canine parvovirus (CPV) strain N and partial sequences of FPV strain Carl and CPV strain b. Base differences were identified at 115 positions in these 5·1 kb genomes and predicted amino acid differences occurred at 40 positions. The two overlapping capsid protein genes contained almost twice as many base differences as the single non-structural protein gene (49 compared to 26) and about the same ratio was calculated for predicted amino acid differences (27 compared to 13). The 27 variant amino acids in the capsid proteins were clustered at three sites in the primary sequence, whereas 10 of the 13 variant amino acids in the non-structural protein occurred in the 130 C-terminal amino acids. The two FPV strains differed consistently from the two CPV strains at 31 bases: 12 base changes in the capsid protein genes resulted in six amino acid changes, six base changes in the nonstructural protein gene resulted in three amino acid changes, and 13 base changes occurred in the noncoding sequence.
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Physicochemical analysis of the hepatitis B virus core antigen produced by a baculovirus expression vector
More LessThe hepatitis B virus particle consists of an envelope carrying the surface antigen of the virus and an internal capsid consisting of the core antigen (HBcAg). The internal capsid contains the circular, partially dsDNA genome and the viral polymerase. Empty core particles have been produced in Spodoptera frugiperda cells using a recombinant baculovirus vector, YMIKTc, that expresses a 21· 4K derivative of the HBcAg gene. The particles have been purified to homogeneity by caesium chloride density gradient centrifugation followed by glycerol gradient centrifugation. Physicochemical analysis of the core particles showed that they exhibited a sedimentation coefficient (s0 20,w) of 82·5S and a diffusion coefficient (D) of 1 ·28 × 10−7 cm2/s. The M r obtained by substitution of these values in the Svedberg equation was 5·8 × 106, using a partial specific volume of 0·73 ml/g for the viral protein as estimated from the amino acid composition. The M r determined from sedimentation equilibrium analyses was 6·3 × 106. Spectrophotometric and metabolic labelling analyses failed to detect nucleic acids in the core preparations. The data are at variance with the prediction that cores exhibit a T=3 symmetry and contain some 180 subunits. The results suggest that the baculovirus- expressed cores may contain up to 300 subunits of HBcAg protein.
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Biological characterization of recombinant vaccinia viruses in mice infected by the respiratory route
More LessA murine model based on infection by the respiratory route has been used to study the pathogenesis of recombinant vaccinia viruses. The neurovirulent Western Reserve (WR) strain and the Wyeth smallpox vaccine strain were used as vectors. Recombinant viruses were constructed by insertion of the Epstein- Barr virus membrane glycoprotein 340 gene into the thymidine kinase (TK) gene of each vaccinia virus. Intranasal inoculation of DBA/2 mice with 106 pockforming units (pk.f.u.) of the WR strain was lethal but mice survived similar infection with the WR recombinant virus. Each virus was recovered from lung, blood and brain but, unlike wild-type virus, the recombinant virus was subsequently cleared. No deaths occurred after similar infection with the Wyeth strain or the Wyeth recombinant virus. There was limited growth of the Wyeth strain in the respiratory tract, low levels of virus in the blood and only sporadic recovery in brain extracts. The Wyeth recombinant virus was cleared rapidly with little viraemia or detectable infection of the central nervous system. No phenotypic character determined in vitro could be related consistently to the virulence of wild-type and recombinant viruses. Although the lethal character of the WR strain was affected by its TK+ phenotype, mice survived infection by intranasal inoculation with 106 pk.f.u. of WR TK+ recombinant viruses which either expressed the human interleukin 2 gene or had a deficient vaccinia virus growth factor gene.
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Infectious cucumber mosaic virus RNA transcribed in vitro from clones obtained from cDNA amplified using the polymerase chain reaction
More LessFull-length cDNA to RNA 1, RNA 2 and RNA 3 of cucumber mosaic virus strain Q (CMV-Q) was amplified using the polymerase chain reaction (PCR). The first-strand primer contained a BamHI site and sequences complementary to the 3′ terminus of the RNA, The second-strand primers contained a BamHI site, a T7 promoter and sequences corresponding to the 5′ terminus of each RNA. After cleavage with BamHI, the PCR products were cloned into the BamHI site of the vector pEMBL9(+). Five clones of each RNA were selected and RNA transcripts were synthesized in vitro from each clone using T7 RNA polymerase. The constructs were designed to allow transcription to initiate precisely at the 5′ terminus of each RNA. All the transcripts were found to be infectious when inoculated onto Nicotiana tabacum cv. Samsun plants in sets of three, corresponding to RNA 1, RNA 2 and RNA 3. Of the transcript sets, four induced symptoms indistinguishable from symptoms induced by CMV-Q RNAs. However a fifth transcript set induced much more severe symptoms. Plasmids were also constructed to allow synthesis of transcripts with one or two additional G residues at the 5′ terminus of each RNA. Although the yields of such transcripts synthesized in vitro with T7 RNA polymerase were higher, their infectivity was lower than that of those with no additional residues at their 5′ termini.
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Expression of plant virus genes in animal cells: high-level synthesis of cowpea mosaic virus B-RNA-encoded proteins with baculovirus expression vectors
More LessThe baculovirus expression system has been used to produce non-structural proteins encoded by bottom- component RNA (B-RNA) of cowpea mosaic virus (CPMV). For this, cDNAs containing the 60K, 87K, 110K and 170K protein coding sequences were each provided with an ATG start codon and the cDNA containing the 60K coding sequence with a TAA stop codon immediately downstream of the coding sequence. Recombinant baculoviruses were retrieved which harboured the modified B-cDNA sequences under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (/lcNPV). Upon infection of Spodoptera frugiperda cells with these recombinant baculoviruses, proteins were produced which were indistinguishable from the viral proteins found in CPMV-infected plants as judged by their migration in polyacrylamide gels and their reactivity with CPMV-specific antisera. Specific processing of CPMV polyproteins in cells infected with the 110K- and 170K-encoding baculovirus recombinants proved that the CPMV-encoded 24K protease activity contained in these polyproteins is active in these cells. Approximately 10% of the 110K protein was processed into 87K and 24K proteins and the 170K protein almost completely into the 110K, 87K, 84K, 60K and 24K polypeptides. In S. frugiperda cells infected by recombinant AcNPVs harbouring the 87K or 110K coding sequences, the CPMV-specific proteins amounted to 10 to 20% of the total cellular protein content, whereas in cells infected by recombinants encoding the 60K and 170K polypeptides the amounts of CPMV-specific proteins synthesized were much lower. Northern blot analysis indicated that the low-level synthesis of the 60K and 170K polypeptides was not due to inferior transcription of the cloned genes but was probably the result of inefficient translation of the RNAs derived from these constructs. It is concluded that plant virus genes can be efficiently expressed in an animal cell expression system to yield proteins that are structurally and, in at least one case (24K protein), functionally identical to the authentic plant virus proteins.
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Detection and typing of maize streak virus and other distantly related geminiviruses of grasses by polymerase chain reaction amplification of a conserved viral sequence
More LessThe application of the polymerase chain reaction DNA amplification technique to the detection and typing of isolates of maize streak virus (MSV) and other related geminiviruses of grasses is described. The oligonucleotide primers used for amplification were 17-mers which contained a number of degeneracies. An approximately 250 base pair fragment was amplified from all geminivirus-infected grass and cereal samples tested. The amplification reaction was specific, working down to a concentration of 50 fg/ml of MSV-specific plasmid-cloned DNA and with a 10−9 dilution of MSV- infected maize DNA extract. DNA could also be amplified from distantly related geminiviruses, including two different sugarcane viruses, digitaria streak virus and another as yet uncharacterized virus of a Panicum sp. Amplified DNA from a Mauritian sugarcane isolate (SSV-M) was cloned and sequenced. Sequence comparison and phylogenetic analysis showed that this sequence differed sufficiently from the analogous region of other geminiviruses for SSV-M to be considered a distinct virus. The use of the polymerase chain reaction for the amplification of gemini- and other virus genomes or genomic fragments for typing, mapping, phylogenetic analysis and taxonomy is discussed.
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The molecular cloning and nucleotide sequencing of the 3′-terminal region of Ornithogalum mosaic virus
More LessDNA complementary to the 3′-terminal 3684 nucleotides of the Ornithogalum mosaic potyvirus (OMV) genome was cloned and sequenced. The sequence consisted of a single large open reading frame which probably starts upstream of the cloned region. By comparison to other sequenced potyviruses, it was estimated that the clone contained the 3′ non-coding (3′-NC) region, the coat protein (CP) gene and the large nuclear inclusion protein (NIb) gene, as well as approximately 85% of the small nuclear inclusion protein (NIa) gene. The 3′-NC region of 274 nucleotides showed 38% to 45% similarity to the corresponding regions of other potyviruses. The putative CP gene could encode a 253 amino acid coat protein with a calculated M r of 28807. Analysis of the amino acid sequences of OMV and other potyvirus proteins showed similarities of 66% to 77% for CP, 72% to 73% for NIb and 63% to 71% for NIa proteins. These data, as well as phylogenetic analysis of the CP sequences, suggested that OMV is a typical but taxonomically distinct potyvirus.
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Sequence of the 3′-terminal region of turnip mosaic virus RNA and the capsid protein gene
A sequence of 1801 nucleotides originating from the 3′ end region of turnip mosaic virus (TuMV) RNA was cloned using the polymerase chain reaction and found to contain one long open reading frame (ORF). The amino acid sequence of three different regions of the isolated TuMV capsid protein (including the NH2 terminus) was determined and these partial sequences were found in the translation product predicted to be encoded by the large ORF. The data suggested that the TuMV capsid protein was a product arising from the maturation of a larger polyprotein, as observed for other potyviruses. Furthermore, the putative cleavage site corresponded to a glutamine-alanine dipeptide, a site commonly used in plant virus polyprotein processing. The capsid protein cistron was composed of 864 nucleotides and corresponded to a region encoding 288 amino acids with a calculated M r of 33186; the adjacent 3′ non-coding region was 667 nucleotides long. The deduced amino acid sequence of the TuMV capsid protein is closely related to other potyvirus capsid proteins, with most of the variation being found within the NH2-terminal region.
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