- Volume 77, Issue 7, 1996
Volume 77, Issue 7, 1996
- Review Article
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Potyviral HC-Pro: a multifunctional protein
More LessIntroduction. The genus Potyvirus, family Potyviridae, is the largest genus of plant viruses with 180 members or possible members (Brunt, 1992). Potyviruses are flexuous filamentous particles which contain a single-stranded RNA genome of positive polarity possessing a covalently linked 5′-terminal viral protein (VPg) and a 3′-terminal poly(A) tail (reviewed in Riechmann et al., 1992). They are transmitted from plant to plant by aphids in a non-persistent manner, and this process is dependent on the presence of two virus-encoded proteins (reviewed in Pirone, 1991). One of these, the helper component-proteinase (HC-Pro) has attracted renewed attention during the last few years due to its multifunctionality and to it being implicated in different steps of the potyvirus life cycle. The properties, as well as the established and postulated functions of this protein, are reviewed.
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- DNA viruses
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Intracellular distribution of the ORF4 gene product of varicella-zoster virus is influenced by the IE62 protein
Varicella-zoster virus (VZV) open reading frame 4-encoded protein (IE4) possesses transactivating properties for VZV genes as well as for genes of heterologous viruses. The major regulatory immediate-early protein of VZV (IE62) is a transactivator of VZV gene expression. In transfection assays, IE4 has been shown to enhance activation induced by IE62. To investigate the functional interactions underlying this observation, indirect immuno-fluorescence studies were undertaken to determine whether IE62 could influence IE4 intracellular localization in transfected cells. In single transfections, IE4 was predominantly found in cytoplasm. In cotransfection with IE62, the IE4 localization pattern was altered, with nuclear staining predominating over cytoplasmic staining. This effect was specific to the IE62 protein since the gene products of ORF63 and ORF61, which are also regulatory proteins, did not influence IE4 distribution. The use of IE62 mutants indicated that IE62 influence is independent of its transactivation function and that the integrity of regions 3 and 4 is required. IE62 remained nuclear whether IE4 was present or not. These observations underline differences in the regulation of gene expression between VZV proteins and their herpes simplex virus type 1 homologues. In infected cells, IE4 was only sometimes found to colocalize with IE62 in nuclei. This observation suggests that when all VZV proteins are present, complex interactions probably occur which could diminish the influence of IE62.
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Glycoprotein H (gII/gp108) and glycoprotein L form a functional complex which plays a role in penetration, but not in attachment, of bovine herpesvirus 1
The glycoproteins of bovine herpesvirus 1 (BHV-1) play important roles in the interactions between virions and target cells. A 108 kDa glycoprotein, designated gII or gp108, has been identified by two different panels of monoclonal antibodies. The gII- and gp108-specific monoclonal antibodies were shown to react with the same protein, which was identified by N-terminal sequencing as the homologue of herpes simplex virus type 1 (HSV-1) gH. When BHV-1 gH was purified by immunoadsorbent chromatography, gL was co-purified. The gH-gL complex induced the production of antibodies that neutralized virus infectivity and inhibited virus penetration. Affinity-purified gH-gL did prevent penetration, but not attachment of BHV-1, which suggests that the gH-gL complex is essential for penetration of BHV-1 into susceptible cells.
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Effect on substrate binding of an alteration at the conserved aspartic acid-162 in herpes simplex virus type 1 thymidine kinase
More LessDespite the extensive use of antiviral drugs for the treatment of herpesvirus infections and as prodrugs for ablative gene therapy of cancer, little structural information about the drug activating enzyme, herpes simplex virus type 1 thymidine kinase (TK), was available until recently. In the absence of the three-dimensional structure we sought to elucidate the function of the key aspartic acid residue (D162) present within a highly conserved tri-peptide motif that is thought to function in nucleoside binding. In this study we generated a mutant, D162Q, by site-directed mutagenesis, purified both the wild-type and mutant TKs to near homogeneity by single-step affinity chromatography and determined the kinetic parameters for thymidine, ATP, dTMP and dTTP interactions. A 12-fold increase in K m for thymidine by D162Q TK (K m = 6.67 µm) relative to wild-type enzyme (K m = 0.56 µm) was observed and the absence of any alteration in K m for ATP suggests that D162 participates in nucleoside binding. Furthermore, the K i for dTMP is significantly higher for D162Q TK than for HSV-1 TK which is indicative of a shared or overlapping binding site with thymidine. This assessment is further supported by the different inhibition patterns of D162Q and wild-type TKs observed using [α-32P]5-N3dUMP photoaffinity labelling in the presence of thymidine, ganciclovir or dTMP. Interestingly, the K i for dTTP was 30-fold lower for D162Q TK (K i = 2.2 µm) than for the wild-type enzyme (K i = 65.8 µm) which provides further evidence of the importance of D162 in TK function.
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Repression by RAZ of Epstein—Barr virus bZIP transcription factor EB1 is dimerization independent
More LessThe hallmark of Epstein—Barr virus (EBV) infection is the establishment of a viral genome transcription pattern called latency. The EBV BZLF1 gene product EB1 (also known as ZEBRA or Zta) is a transcription factor which is essential for the switch from latency to the lytic cycle. It has been proposed that latency is maintained (i) by the inhibition of EB1 translation via antisense hybridization of EBNA1 and EB1 hnRNAs, or (ii) by the inactivation of the EB1 activating function via the direct interaction of EB1 with ReIA, the retinoic acid receptor and p53, or via the titration of EB1 in RAZ:EB1 inactive heterodimers that are unable to bind to DNA. RAZ, a fusion protein which contains the EB1 C-terminal dimerization and DNA-binding domains fused to the N-terminal 86 amino acids of the EBV BRLF1 gene product R, has been described as a trans-dominant negative regulator of EB1-activated transcription. We demonstrate here that although RAZ efficiently represses EB1-mediated transcriptional activation, the amount of RAZ protein expressed is incompatible with repression through the titration of EB1 in inactive EB1:RAZ heterodimers. Furthermore, we also demonstrate that RAZ efficiently represses transcription activated by an EB1 mutant carrying the GCN4 homodimerization domain (EB1gcn4), despite the inability of RAZ and EB1gcn4 to form stable heterodimers.
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Glycoprotein H of human cytomegalovirus is a major antigen for the neutralizing humoral immune response
More LessA recombinant baculovirus expressing glycoprotein H (gpUL75) of human cytomegalovirus was used to examine the humoral immune response in naturally infected individuals. Recombinant baculovirus infected insect cells produced two forms of gH with molecular masses of 78–82 kDa and 94 kDa. The 94 kDa polypeptide was modified by high mannose oligosaccharide side-chains as shown by reduction in molecular mass after treatment with endoglycosidases H and F. The 78–82 kDa protein represented the non-glycosylated precursor which was resistant to the enzymes. In contrast to gH expressed in mammalian cells, the recombinant baculovirus expressed gH was transported to the cell surface. Glycoprotein H produced in insect cells was reactive with human convalescent sera and all tested neutralizing monoclonal antibodies recognizing either linear or conformational epitopes. Antibodies reacting with insect cell derived gH were detected in 96% of HCMV seropositive human sera. Using insect cells infected with the gH expressing recombinant baculovirus as immunoabsorbent, between 0% and 58% of the total virus neutralizing activity was removed from sera of individuals with a past HCMV infection. gH must therefore be considered a major antigen for the induction of neutralizing antibodies during natural infection.
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Processing of human cytomegalovirus glycoprotein B in recombinant adenovirus-infected cells
More LessIntracellular processing of human cytomegalovirus (HCMV) glycoprotein B (gB; gpUL55) expressed by a recombinant adenovirus (Ad-gB) was studied in human A549 cells as processing events could affect immunogenicity when such viruses are used as live-recombinant vaccines. Cleavage of [35S]methionine-labelled gp130 into gp93 and gp55 reached a maximum after a 3 h chase. Cleavage was completely inhibited by brefeldin A, suggesting that processing normally occurs as a late Golgi or post-Golgi event. Uncleaved gp130 remained completely sensitive to endo-β-N-acetylglucosaminidase H (Endo-H) in untreated cells following long chase periods, indicating high-mannose oligosaccharides at all of the 18 N-linked glycosylation sites (Asn-X-Ser/Thr) and retention in the endoplasmic reticulum. Endo-H analysis of gp55 from swainsonine-treated and untreated cells was consistent with glycosylation at all three potential sites, with two oligosaccharides remaining sensitive to Endo-H and one being processed to Endo-H resistance. The heavily glycosylated N-terminal gp93 subunit was not detected by [35S]methionine-labelling but was easily detected along with gp55 after labelling with [3H]mannose. No cleavage of gp130 was observed in analogous pulse-chase radiolabelling of Ad-gB-infected human fibroblasts, even though these cells are permissive for HCMV replication and can process the native gB molecule. Processing of gB in recombinant adenovirus-infected A549 cells was generally similar to that previously reported for native gB in HCMV-infected fibroblasts.
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Cloning and sequence analysis of the genes encoding DNA polymerase, glycoprotein B, ICP 18.5 and major DNA-binding protein of rat cytomegalovirus
More LessIn all herpesviruses a block of genes is present which is composed of the genes encoding DNA polymerase, glycoprotein B (gB), ICP18.5 and major DNA-binding protein (MDBP). Here we report the cloning and sequencing of this gene block from rat cytomegalovirus (RCMV). The gene block spans 13.3 kbp and contains the four genes in the order pol, gB, ICP18.5 and MDBP. A similar order of genes has previously been reported for human and murine cytomegaloviruses. The pol, gB, ICP18.5 and MDBP genes contain open reading frames which have the capacity to encode proteins of 1120, 914, 893 and 1281 amino acids, respectively. Comparison of the predicted amino acid sequences of the four RCMV proteins with the corresponding proteins of other herpesviruses revealed a close relationship between RCMV and other cytomegaloviruses, which corroborates the usefulness of the RCMV-rat model for studying cytomegalovirus biology.
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Location, DNA sequence and transcriptional analysis of the DNA polymerase gene of orf virus
More LessDegenerate oligonucleotides representing conserved regions of various DNA polymerases hybridized to a region located 26 kb from the left end of the orf virus (OV) strain NZ-2 genome. DNA sequence analysis of this region revealed a 3024 bp open reading frame able to encode a protein with 56% amino acid identity to the DNA polymerase of vaccinia virus (VAC) and with significant homology to other DNA polymerases. Early transcripts derived from the open reading frame were detected in RNA purified from OV-infected cells, and 5′ ends were mapped to a region 8–19 nt downstream from an A/T-rich sequence that resembles VAC early promoters. Unlike the VAC gene, the OV DNA polymerase makes almost exclusive use of G/C coding options. Attempts to substitute the activity of the OV DNA polymerase for its VAC counterpart were unsuccessful. This may indicate that the OV DNA polymerase is incompatible with VAC accessory proteins.
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Construction of recombinant myxoma viruses expressing foreign genes from different intergenic sites without associated attenuation
More LessTwo myxoma virus transient dominant selection vectors were constructed and used to generate recombinant viruses expressing single and double foreign gene insertions from intergenic sites. The intergenic insertion sites were located between the myxoma virus genes MJ2 (thymidine kinase) and MJ2a, and MA24 (β-subunit RNA polymerase) and MA27 (fusion protein) located approximately 60 and 113 kb from the left-end of the viral genome, respectively. Recombinant myxoma viruses expressing the IacZ gene from either intergenic insertion site retained wild-type virulence. However, expression of the gus gene reduced the virulence of the recombinant viruses in vivo. Northern blot analysis indicated that the major late mRNAs encoding the viral RNA polymerase subunit and fusion protein are both of discrete size. Insertion of a foreign gene under the control of a synthetic late promoter between the MA24 and MA27 genes results in a specific-sized major late transcript for the inserted foreign gene. The MA27 gene transcripts directed by these recombinant viruses are heterogeneous in size, implying the typical pattern of poxvirus late transcription by random 3′-termination prior to polyadenylation. The transcription studies suggest signals located downstream of the insertion site direct 3′-processing of late transcripts irrespective of the gene immediately upstream.
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Vaccination of cattle with bovine papillomavirus type 4 L2 elicits the production of virus-neutralizing antibodies
More LessProphylactic vaccination of cattle with the N terminus (L2a, aa 11–200) of the minor capsid protein L2 completely prevented bovine papillomavirus type 4 (BPV-4) infection of the alimentary canal. To investigate the mechanisms underlying protection from viral infection, sera from vaccinated animals were analysed in neutralization assays both in the nude mouse xenograft system and in cattle. BPV-4 retained its infectivity when incubated with pre-immune cattle sera, whereas, when incubated with immune sera from animals vaccinated with either whole L2 or its N terminus L2a, its infectivity was greatly reduced, indicating that the immune sera had neutralizing activity against the virus. This activity could be abrogated by absorbing the immune sera with L2 or L2a, thus indicating that virus neutralization was due to the presence in the immune sera of anti-L2 antibodies.
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Proliferative T cell responses to the human papillomavirus type 16 E7 protein in women with cervical dysplasia and cervical carcinoma and in healthy individuals
The levels of proliferative T cell responses to peptides representing the human papillomavirus type 16 (HPV-16) E7 protein have been measured using short-term T cell lines derived from peripheral blood of healthy women and those with cervical dysplasias and carcinoma of the cervix. In healthy individuals 47% (7/15) responded predominantly to the N- and C-terminal regions of the protein and 6/7 responders were to a single peptide between amino acids 80–94. In comparison 29% (9/31) of women with cervical dysplasia responded to HPV-16 E7, with a significantly reduced response to both the N- and C-terminal regions (P = 0.03 and 0.038, respectively). A higher proportion of responders was found in patients with high grade lesions (56%, 5/9) versus those with atypical or low grade histology (20%, 4/20) and the response to a single peptide between amino acids 75–94 was also increased in this patient group (P = 0.044). This may be a reflection of higher levels of current or previous exposure to HPV-16 in patients with high grade lesions. Correlation of T cell responses with HPV DNA type (detected by PCR of cervical biopsy tissue) showed that 3/9 (33%) HPV-16 DNA-positive individuals responded. This suggests that E7 may not be the dominant target of the immune response or that the response to E7 is down-regulated in these patients. In addition 4/18 (22%) HPV-16 DNA-negative individuals responded, suggesting that their T cells may have been primed by previous exposure to HPV-16 or that a cross-reactive response was detected. Proliferative T cell responses to both HPV-16 E7 and L1 were reduced in women with cervical carcinoma in comparison to those with cervical dysplasia and healthy controls. The observed down-regulation of responses to HPV-16 E7 in women with cervical dysplasia and cervical carcinoma may reflect an altered functional balance between subsets of T helper cells in HPV-16 infections.
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- Animal
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- RNA viruses
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Unimpaired function of a naturally occurring C terminally truncated vif gene product of human immunodeficiency virus type 1
More LessIn ∼ 10% of natural human immunodeficiency virus 1 (HIV-1) vif gene populations, sequences of shortened vif open reading frames with premature stop codons have been found. Here we report the functional analysis of two patient-derived vif genes. Vif45-2 encodes a C terminally truncated Vif protein of only 173 instead of 192 amino acids and additionally contains several rare amino acid substitutions which are in part shared by vifA65-5. HIV-1 pNL4-3-derived recombinant A45-2 and A65-5 virions were fully infectious in H9 cells and human PBMC, both known to be non-permissive for vif-defective HIV-1. Furthermore, A45-2 virions produced in primary human monocyte-derived macro-phages were infectious for MT-4 cells. This study unequivocally demonstrates that the C-terminal region (19 amino acids) of the Vif protein is dispensable for Vif function in the in vitro cell culture systems employed. Additionally, we investigated whether the Vif protein might be phosphorylated in vivo and obtained no evidence for this.
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Varying temperature-dependence of post-attachment neutralization of human immunodeficiency virus type 1 by monoclonal antibodies to gp120: identification of a very early fusion-independent event as a neutralization target
More LessNeutralization of human immunodeficiency virus type 1 by adding antibody after the virus has attached to the host cell (post-attachment neutralization: PAN) was investigated using three rat monoclonal antibodies (MAbs) directed against the outer domain of the membrane protein, gp120. Two of the MAbs are specific for the CD4-binding site region and one for the V3 loop. MAb ICR39.13g (CD4-binding site region-specific; IgG2b) effected PAN efficiently at temperatures from 4 to 35 °C. MAb ICR41.1i (V3 loop-specific; IgG2c) effected PAN only at temperatures of 24 °C and below. This suggests that its V3 epitope is masked by a change in gp120 which occurs at temperatures ⩾ 26 °C, or that the virion function which is inhibited by ICR41.1i and is responsible for neutralization has already operated at ⩾ 26 °C. Resistance to neutralization by ICR41.1i occurred within 20 min of shifting the temperature up to 35 °C. Finally, MAb ICR39.3b (CD4-binding site region-specific; IgG2b) did not give PAN at any temperature, indicating that neutralization can only occur if this MAb binds virus before it attaches to the cell. Thus, these studies identify at least one novel fusion-independent event, the neutralization target of a V3 MAb, which occurs very early in the initial stages of virus-cell interaction.
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Antigenicity of truncated forms of the human immunodeficiency virus type 1 envelope glycoprotein
More LessChinese hamster ovary (CHO) cell lines secreting a series of truncated forms of human immunodeficiency virus type 1 (HIV-1) IIIB (clone BH10) gp120 were assembled. Using purified glyco-proteins, we demonstrated the functional and structural integrity of these truncates by their reactivity with both sCD4 and anti-gp120 monoclonal antibodies (MAbs). Deletion of the V1, V2 and V3 regions had minimal effects on CD4 binding, but deletion of the NH2 terminus affected the folding of the truncated molecule. Deletion of either V1/V2 or V1/V2/V3 regions led to enhanced recognition by some, but not all, MAbs mapping to the CD4 binding site. In contrast, deletion of the V1/V2 regions had no effect on the ability of V3-specific MAbs to bind to the truncate. These results support the use of truncated forms of gp120 as components of potential HIV vaccines.
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Replication kinetics and cell tropism of an immunosuppressive feline leukaemia virus
More LessTo elucidate in vivo cell tropism and infection kinetics of an immunodeficiency-inducing isolate of feline leukaemia virus (FeLV-FAIDS), we quantified the two major genotypes comprising FeLV-FAIDS [the replication-competent common form (clone 61E) and the replication-defective variant (clone 61C)] in lymphocyte and leukocyte populations from infected cats. Micromagnetic separation of cell subsets, virus genome-specific PCR and flow cytometry were used to demonstrate the following sequence of events in infected animals: (i) very early replication of both 61E and 61C in CD4 T cells (provirus burden 0.2 to 1 copy/cell at 2–4 weeks post-infection); (ii) lower magnitude replication of both viruses in CD8 T cells and B cells during this initial phase of infection; (iii) plateauing of CD4 cell virus burden accompanied by escalation in CD8 and B cell provirus burdens after 4 weeks; (iv) extensive infection of haemopoietic and circulating myeloid cells. FeLV-FAIDS 61E and 61C replication kinetics and lymphocyte tropisms were similar in blood and lymph nodes, where provirus burdens ranged from 0.15 to 1.0 copy/cell. Moreover, virus infection was productive; 8–48% of blood lymphocytes, 35–81% of node lymphocytes and 53–98% of bone marrow cells expressed FeLV capsid antigen (p27 Gag). These findings suggest that the immunosuppressive potency of FeLV-FAIDS reflects the unique cytopathicity rather than unique cytotropism of its 61C (versus 61E) component.
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Characterization of the gene encoding core protein VP6 of two African horsesickness virus serotypes
More LessThe genes encoding the inner core protein VP6 of African horsesickness virus (AHSV) serotypes 3 and 6 have been cloned and sequenced. The genes are 1169 nucleotides in length and both encode a largely hydrophilic protein of 369 amino acids. The VP6 amino acid sequence is highly conserved between the two serotypes with an overall similarity of 95%. Comparison of the AHSV VP6 amino acid sequences with those of bluetongue virus serotype 10 VP6 revealed that it is 41 amino acids longer with an overall amino acid identity of 29%. The similarity is mainly confined to a short but highly conserved 13 amino acid region at the N terminus, a short seven amino acid region at the C terminus and a 22 amino acid region close to the C terminus. Within this last region is a smaller 11 amino acid region from 318 to 328 with a 91% similarity to the Rep helicase of Escherichia coli.
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Interference of reovirus strains occurs between the stages of uncoating and dsRNA accumulation
More LessInterference of wild-type reovirus growth by some temperature-sensitive (ts) mutant viruses under non-permissive conditions or by other wild-type isolates has been demonstrated; however, the stage of the virus replication cycle at which interference occurs has not been defined. Examination of the time-course of the yields of T1 Lang (T1L) dsRNA in the progeny of mixed infections of T1L with T3 Dearing (T3D) or with a panel of T3D ts mutants at a non-permissive temperature revealed that interference takes place by 8–10 h post-infection and occurs prior to or at the same time as accumulation of reovirus dsRNA. Taken together with our previous results, these data indicate that interference occurs during a window between virus uncoating and synthesis of dsRNA in the reovirus replication cycle, probably at the stage of assembly of primary reovirus particles.
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Pathogenesis of an attenuated and a virulent strain of group A human rotavirus in neonatal gnotobiotic pigs
More LessGnotobiotic (Gn) pigs were orally inoculated with Wa strain (G1P1A[P8]) human rotavirus (Wa HRV) serially passaged in Gn pigs (virulent) or cell culture (attenuated) to determine the median virus infectious dose (ID50) and to assess the site of infection and type and progression of morphological lesions and clinical responses induced by these two strains in Gn pigs. The ID50 of virulent Wa HRV was ⩽ 1 f.f.u. whereas the infectivity of attenuated Wa HRV had to be determined by seroconversion and was ∼ 1.3 × 106 f.f.u. Diarrhoea developed at 13 h post-inoculation (p.i.) in pigs inoculated with ∼ 105 f.f.u. of virulent Wa HRV and correlated with the presence of viral antigen within villous epithelial cells; villous atrophy developed later at 24 h p.i. and correlated with peak faecal viral titres; recovery from disease correlated with the return of morphologically normal villi. Virus, diarrhoea and villous atrophy were not detected in pigs inoculated with ∼ 2 × 108 f.f.u. attenuated Wa HRV although HRV-specific serum antibodies were present by 7 days p.i. These findings demonstrate that virulent Wa HRV infection in Gn pigs occurs primarily within intestinal villous epithelial cells with villous atrophy developing as a sequela to infection. However, factors other than villous atrophy appear to contribute to the early stages of HRV-associated disease expression in Gn pigs. The ability of the attenuated virus to elicit virus-neutralizing serum antibodies without disease or pathology indicates promise in the use of such strains for oral immunization.
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The region between the M and S genes of porcine haemagglutinating encephalomyelitis virus is highly similar to human coronavirus OC43
More LessThe nucleotide sequences of the regions between the membrane and spike protein genes of three strains of porcine haemagglutinating encephalomyelitis virus (HEV) were determined. A total of 739 (HEV strain 67N) and 751 (strains NT9 and VW572) nucleotides were sequenced. Two ORFs, potentially encoding proteins of 12.8 and 9.6 kDa, were identified. Pairwise comparisons with the corresponding ORFs in bovine coronavirus (BCV) and human coronavirus (HCV) OC43 revealed sequence similarities of greater than 88.5% at the nucleotide and 85.3% at the amino acid level for the 12.8 kDa ORF product. For the 9.6 kDa ORF product similarities were greater than 96.9% and 95.2%, respectively. An additional 12 nucleotide deletion upstream of the 12.8 kDa ORF start codon was found in HEV 67N compared to NT9 and VW572. These results reveal a genomic organization of HEV in the region analysed that is homologous to HCV OC43 but different from BCV.
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Molecular differences between wild-type Japanese encephalitis virus strains of high and low mouse neuroinvasiveness
More LessJapanese encephalitis (JE) virus strain P3 was highly neurovirulent and neuroinvasive in weanling mice whereas two other JE virus strains, SA14/USA and S892, were only neurovirulent. Infectivity titrations of brains and sera showed that P3 virus multiplied faster and reached a higher infectivity titre than S892 virus following inoculation of viruses by the intraperitoneal (i.p.) route. The p.f.u./LD50 was 101.7 and 106.2 for P3 and S892 viruses respectively, following i.p. inoculation, while JE virus strain SA14/USA did not kill mice when inoculated by this route (i.e. ⩾ 106.3 p.f.u./LD50). Nevertheless, the genomic similarity between P3 virus and strains SA14/USA and S892 was more than 97.8% at the nucleotide level and 99% at the amino acid level. Compared with S892 and SA14/USA viruses, P3 virus had 33 and 21 amino acid differences, respectively. The structural protein genes of P3 virus were more divergent than non-structural protein genes. Nine unique amino acids were found in the envelope protein gene. None of these amino acid differences were shared with other wild-type JE virus strains. Although we cannot identify the precise molecular determinants of virulence of JE virus, there were no unique amino acids in M, NS1, NS2A, NS3, NS4A and NS4B proteins of P3 virus compared with other wild-type viruses. Therefore, it appears that these proteins make no significant contribution to the high neuroinvasiveness of P3 virus. The structural proteins, and non-structural proteins NS2B and NS5 may be involved in increasing neurovirulence and neuroinvasiveness of P3 virus. P3 virus differed by several nucleotides in the 3′ non-coding region while no nucleotide difference was found in the 5′ non-coding region.
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Persistent infection of African buffalo (Syncerus caffer) with SAT-type foot-and-mouth disease viruses: rate of fixation of mutations, antigenic change and interspecies transmission
Transmission of a plaque-purified SAT-2 foot-and-mouth disease virus (FMDV) occurred erratically from artificially infected African buffaloes in captivity to susceptible buffaloes and cattle in the same enclosure; in some instances transmission occurred only after contact between persistently infected carriers and susceptible animals lasting a number of months. Because the rate at which FMDV mutations accumulated in persistently infected buffaloes was approximately linear (1.64% nucleotide substitutions per year over the region of the 1D gene sequenced), both buffaloes and cattle that became infected some months after the start of the experiment were infected with viruses that differed from the original clone. The nucleotide differences were reflected in significant antigenic change. A SAT-1 FMDV from a separate experiment inadvertently infected some of the buffalo in the SAT-2 experiment. The SAT-1 FMDV also accumulated mutations at a constant rate in individual buffaloes (1.54% nucleotide changes per year) but the resultant antigenic variation was less than for SAT-2. It is concluded that persistently infected buffaloes in the wild constantly generate variants of SAT-1 and SAT-2 which explains the wide range of genomic and antigenic variants which occur in SAT-1 and SAT-2 viruses in southern Africa.
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Enterovirus infection of the central nervous system of humans: lack of association with chronic neurological disease
We have searched, using a sensitive nested-PCR, for enterovirus RNA in cerebrospinal fluid and post mortem central nervous system (CNS) tissue from patients with previous poliomyelitis with or without late functional deterioration, patients with motor neuron disease (MND), and control patients with other neurological disease or without neurological disease. Enterovirus RNA was detected in patients with previous poliomyelitis and MND, but also in control patients with and without neurological disease. Our results do not provide any evidence that such enterovirus infection is related to late functional deterioration in patients with previous poliomyelitis, which could be attributed to other medical conditions in most instances, and do not support the hypothesis that MND is associated with enterovirus infection of the CNS. Nucleotide sequence analysis of enterovirus RNA sequences detected indicated that enteroviruses detected were of the non-polio type.
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Interactions between the ectodomains of haemagglutinin and CD46 as a primary step in measles virus entry
More LessRecombinant soluble forms of the ectodomains of measles virus haemagglutinin (sH) and of its receptor CD46 (sCD46) were obtained as a purified disulphide-bonded sH homodimer with an apparent molecular mass of 160 kDa and a purified sCD46 monomer with an apparent molecular mass of 60 kDa, without detectable contamination with moesin. Purified sH bound to purified and immobilized sCD46 and this binding was specifically inhibited by sCD46 in solution. sCD46 bound to wild-type H expressed on the cell surface and inhibited measles virus binding to CD46-expressing cells. Binding of sCD46 to cell surface H was increased about twofold when measles virus fusion protein was coexpressed with H. sH bound to wild-type cell surface CD46 and inhibited measles virus binding onto CD46-expressing cells. sCD46 also inhibited virus infection. Thus, the direct interaction between the ectodomains of H and CD46 is likely to be the primary event in measles virus infection.
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The immunological activity of a deletion mutant of influenza virus haemagglutinin lacking the globular region
More LessA deletion mutant of influenza virus haemagglutinin (HA; headless HA) lacking the globular region was expressed in CV-1 cells and detected with a monoclonal antibody, C179, which recognizes a conformational epitope in the middle of the stem region of HA and neutralizes all H1 and H2 subtypes. The cDNA coding for the headless HA was constructed from influenza virus A/Okuda/57 (H2N2), which was also used to select C179. The conformational epitope recognized by C179 was highly stable even after removal of the globular region. The survival rate of mice immunized with the headless HA and challenged with lethal influenza virus A/FM/1/47 (H1N1) was significantly higher than that of the control mice. The headless HA has the potential to induce cross-protection against influenza virus infection.
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Frequent occurrence of genetic reassortment between influenza C virus strains in nature
Previous studies of the haemagglutinin-esterase (HE) genes of various influenza C isolates suggested the existence of three distinct virus lineages (C/Yamagata/26/81-, C/Aichi/1/81- and C/Mississippi/80-related lineages) in Japan in the 1980s. Here we analysed the genetic properties of three strains (C/Yamagata/5/92, C/Miyagi/3/93 and C/Miyagi/4/93) isolated in Yamagata and Sendai Cities, Japan, in 1992/1993. Comparison of total or partial nucleotide sequences of the seven RNA segments of C/Yamagata/5/92 with those of 11 previous isolates suggested that the 1992 strain is a reassortant which inherited HE, P3, NP and M genes from a C/Mississippi/80-like virus and PB2, PB1 and NS genes from a C/pig/Beijing/115/81-like virus. Furthermore, it became evident that at least two (C/England/83 and C/Yamagata/9/88) of the 11 reference strains are also reassortants.
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Heterogeneity in the haemagglutinin gene and emergence of the highly pathogenic phenotype among recent H5N2 avian influenza viruses from Mexico
More LessMolecular changes in the haemagglutinin (HA)-coding regions and proteolytic cleavage sites from multiple H5N2 subtype viruses isolated during a recent outbreak of avian influenza (AI) in central Mexico have been characterized. Eighteen isolates, collected during a 15 month period (October 1993 to January 1995) from six central states, were sequenced. None of the 18 predicted HA1 amino acid sequences were identical and changes were not restricted to a specific region of the sequence. Phylogenetic analyses of the HA1 sequences demonstrated two virus lineages, designated Puebla and Jalisco, with sequence variation as high as 10.5% for amino acid and 6.2% for nucleotide sequences. During the latter months of the surveillance period, highly pathogenic (HP) strains of Al emerged causing lethal disease in commercial poultry flocks. In each of the HP strains isolated, the HA protein was cleaved in chicken embryo fibroblast cells in the absence of trypsin, and two alterations not found in earlier non-HP isolates were detected. In the HA protein, HP strains all had a glutamic acid → lysine substitution at amino acid position 324 and an insertion of arginine and lysine as new residues 325 and 326. The insertion appears to be due to a duplication of the nucleotide sequence AAAGAA at nucleotide positions 965–970 of the HA1-coding region. Computer-assisted secondary structure analyses place the target for the insertion in a predicted RNA stem-loop structure. A mechanism is suggested by which the polymerase duplicates the sequence.
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- Insect
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Expression of polydnavirus genes under polydnavirus promoter regulation in insect larvae infected with baculovirus recombinants
More LessWe have evaluated the use of baculoviruses to deliver Campoletis sonorensis polydnavirus (CsPDV) genomic DNA into lepidopteran larvae to facilitate the identification of functional CsPDV genes. Genomic fragments consisting of regulatory (promoter) and coding sequences for two CsPDV genes (VHv1.1 and WHv1.6) were used to generate CsPDV-baculovirus recombinants and evaluate the expression of genes under the regulation of the CsPDV promoters. Northern blot and primer extension studies established that CsPDV genes were expressed under the control of their own promoters in these CsPDV-baculovirus recombinants. Transcripts were detected as early as 4 h post-infection indicating that temporal activity of CsPDV promoters was retained. The VHv1.1 gene product as expressed from CsPDV-baculovirus recombinants was identical in size and in functional properties to that produced in CsPDV-infected insects. CsPDV-baculovirus recombinants may be useful for the screening and characterization of polydnavirus genes with functional activities that can only be evaluated in insect larvae.
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- Plant
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Switches in the mode of transmission select for or against a poorly aphid-transmissible strain of potato virus Y with reduced helper component and virus accumulation
More LessA poorly aphid-transmissible potato virus Y (PVY-PAT) variant emerged after several cycles of mechanical transmission of an initially aphid-transmissible (AT) isolate. Sequence analysis of the N-terminal region of the helper component-proteinase (HC-Pro) gene revealed a Lys to Glu change at a position previously found to abolish the HC-Pro aphid transmission activity in several potyviruses. Two cycles of aphid transmission allowed the virus population to evolve towards an AT form (PVY-ATnew) where a Glu to Lys change was observed. PVY-PAT produced lower amounts of coat protein and the accumulation of its HC-Pro in infected plants decreased from 7 to 28 days post-inoculation, as compared to PVY-ATnew. RT-PCR and restriction analysis showed that the two virus populations co-existed in the PVY-AT isolate and that the AT form was counter-selected during mechanical transmission. These observations suggest that the Lys to Glu substitution leads to decreased stability of HC-Pro resulting in poor transmissions by aphids, and further strengthen the idea that HC-Pro is involved in the accumulation of potyvirus in infected plants.
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Assembly of virus-like particles in insect cells infected with a baculovirus containing a modified coat protein gene of potato leafroll luteovirus
More LessDNA encoding the coat protein (P3) of a Scottish isolate of potato leafroll virus (PLRV) was inserted into the genome of Autographa californica nucleo-polyhedrovirus (AcNPV) such that the coat protein was expressed either in an unmodified form or with the addition of the amino acid sequence MHHHHHHGDDDDKDAMG at the N terminus (P3-6H). Insect cells infected with these recombinant baculoviruses accumulated substantial amounts of P3 and P3-6H. P3 could not be recovered from cell extracts unless it was denatured in SDS but a proportion of the P3-6H was recoverable in a soluble form in non-denaturing conditions. Immunogold labelling of sections of infected cells showed that P3 accumulated in nuclei in large amorphous bodies. In contrast, although much of the P3-6H also accumulated in nuclei, it formed virus-like particles (VLP) which were often grouped in close-packed, almost crystalline arrays. When electron microscope grids coated with antibodies to PLRV were floated on cell extracts containing P3-6H, VLP were trapped which were indistinguishable from PLRV particles trapped from extracts of PLRV-infected plants. The VLP co-sedimented in sucrose gradients with PLRV particles which suggests that the VLP contained RNA. VLP collected from sucrose density gradient fractions contained protein which reached with nickel chelated to nitrilotriacetic acid, a histidine-specific reagent. Cells infected with either recombinant baculovirus also synthesized a protein, with an M r of about 17000, which was shown to be the translation product of the P4 gene which is in the + 1 reading frame within the coat protein gene. This protein was also found in the nuclear fraction of infected cells but was more readily soluble than was P3.
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High resolution analysis of the readthrough domain of beet necrotic yellow vein virus readthrough protein: a KTER motif is important for efficient transmission of the virus by Polymyxa betae
More LessThe 5′-terminal cistron of beet necrotic yellow vein furovirus RNA 2 encodes the 21 kDa major viral coat protein and terminates with an amber stop codon which can undergo suppression to give rise to a 75 kDa readthrough (RT) protein referred to as P75. P75 is a minor component of virions and the 54 kDa RT domain following the coat protein sequence is important both for virus assembly and transmission by the fungal vector Polymyxa betae. To better define the regions of the RT domain involved in these two steps, RNA 2 transcripts encoding different in-frame RT domain deletion mutants were tested for their ability to form virions when inoculated to plants with the other viral RNAs and to be fungus-transmitted. All deletions in the N-terminal half of the RT domain interfered with virus assembly and partially or completely inhibited fungus transmission. A 411 nucleotide deletion within the C-terminal half of the RT domain did not inhibit assembly but blocked fungus transmission of the virus. Alanine scanning mutagenesis within the aforesaid 411 nucleotide subdomain identified a peptide motif (KTER) which is important for the fungus transmission process.
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Horseradish curly top virus is a distinct subgroup II geminivirus species with rep and C4 genes derived from a subgroup III ancestor
More LessThe complete nucleotide sequence (3080 nt) of an infectious DNA clone derived from the geminivirus horseradish curly top virus (HrCTV) has been determined. The relationship of HrCTV to other gemini-viruses was examined using dot matrix plots of nucleotide sequence similarities, and by phylogeny of predicted amino acid sequences of individual ORFs based upon parsimony or neighbour-joining methods. These analyses indicate that the V1 and V2 virion sense ORFs of HrCTV are most closely related to, yet distinct from, the corresponding ORFs of the subgroup II geminivirus beet curly top virus (BCTV). HrCTV also encodes a third virion sense ORF (V3) which is similar (72–74% amino acid identity) to the BCTV V3 ORF; however, the HrCTV V3 ORF has diverged in sequence to a greater extent relative to that observed among isolates of BCTV (98–100% amino acid identity). The HrCTV genome encodes only three complementary sense ORFs (C1, C2 and C4) and lacks a C3 ORF which is conserved among all other subgroup II and III geminiviruses characterized to date. Although the neighbour-joining analysis indicated that the HrCTV C2 ORF was distantly related to the C2 ORF of BCTV, the predicted amino acid sequence deduced from the HrCTV C2 ORF lacks the characteristic zinc-finger domain present in the transcriptional activating protein (TrAP) encoded by the subgroup III ORF AC2, which is also retained within the TrAP-related product of the BCTV C2 ORF. Surprisingly, the rep and C4 proteins encoded by HrCTV share a closer phylogenetic relationship to the corresponding proteins of the subgroup III geminivirus squash leaf curl virus (SLCV) than to BCTV. These results suggest that the HrCTV genome may have arisen by a recombination event between a BCTV-like subgroup II virus ancestor and an SLCV-like subgroup III virus ancestor. Possible mechanisms that may explain recombination events among geminiviruses are discussed.
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- Other Agents
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Scrapie infection can be established readily through skin scarification in immunocompetent but not immunodeficient mice
More LessScarification of the skin is a possible route of entry for scrapie infectivity in sheep, and for Creutz-feldt-Jakob disease agent in humans within the context of occupational exposure to infected brain in the autopsy room or laboratory. The effectiveness of skin scarification routes as portals of entry for infectivity had not previously been tested experimentally but this study has shown that these are efficient routes for establishing infection in mice using the 139A and ME7 strains of scrapie agent. Scarification had much the same efficiency as inoculation by the intraperitoneal, intravenous or perivenous routes but was not effective in immunocompromised (SCID) mice. It was concluded that replication of infectivity within the lymphoreticular system, which is precluded in SCID mice, is a necessary prerequisite for the development of infection in the central nervous system following inoculation via scarification.
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Strain specific and common pathogenic events in murine models of scrapie and bovine spongiform encephalopathy
The development of transmissible spongiform encephalopathies in experimental models depends on two major factors: the intracerebral accumulation of an abnormal, protease-resistant isoform of PrP (PrPres), which is a host protein mainly expressed in neurons; and the existence of different strains of agent. In order to make a distinction between pathogenic mechanisms depending upon the accumulation of host-derived PrPres and the strain-specific effects, we quantified and compared the sequence of molecular [PrPres and glial fibrillary acidic protein (GFAP) accumulation] and pathological events in the brains of syngeneic mice throughout the course of infection with two different strains of agent. The bovine spongiform encephalopathy (BSE) agent exhibits properties different from any known scrapie source and has been studied in comparison with a classical scrapie strain. Convergent kinetic data in both models confirmed the cause-effect relationship between PrPres and pathological changes and showed that PrPres accumulation is directly responsible for astrocyte activation in vivo. Moreover, we observed a threshold level of PrPres for this effect on astroglial cells. However, despite similar infectivity titres, the BSE model produced less PrPres than scrapie, and the relative importance of gliosis was higher. The comparison of the molecular and pathological features after intracerebral or intraperitoneal inoculation also revealed differences between the models. Therefore, the mechanisms leading to the targeting and the fine regulation of the molecular events seem to be independent of the host PrP and to depend upon the agent. The possible involvement of a regulatory molecule accounting for these specificities has to be considered.
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Volumes and issues
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Volume 105 (2024)
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Volume 1 (1967)