- Volume 79, Issue 4, 1998
Volume 79, Issue 4, 1998
- Articles
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Human recombinant Puumala virus antibodies: cross-reaction with other hantaviruses and use in diagnostics.
A panel of seven human monoclonal Fabs against Puumala virus (PUU) nucleocapsid protein (N) was obtained by panning an antibody phage-display library prepared from the spleen of a PUU-immune individual. Three antibodies reacted in immuno-blotting and cross-reacted strongly with Tula and Sin Nombre virus recombinant N proteins. These antibodies mapped to the amino terminus of the N protein. One PUU glycoprotein 2 (G2)-specific Fab obtained against a novel epitope (G2c) cross-reacted with Khabarovsk virus but not with the other hantavirus serotypes. An N protein-specific Fab was successfully used as capture antibody to detect PUU-specific serum IgG and IgM antibodies in an enzyme immunoassay. The result demonstrates the usefulness of recombinant human Fabs as potential diagnostic tools.
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Pseudotype formation with La Crosse virus glycoproteins.
More LessPseudotype formation is a powerful tool for analysing mechanisms of virus neutralization and entry, since it allows for analysis of glycoprotein properties without the necessity for preparing recombinant genomes. Using recombinant vaccinia viruses, we prepared pseudotypes of La Crosse virus with recombinant glycoproteins cloned from the monoclonal antibody (MAb)-resistant variant V31. The resulting pseudotypes became partially resistant to MAb 807–31. Furthermore, when the V31 glycoproteins were incorporated into a second MAb-resistant variant (V33), the pseudotyped virus became sensitive to neutralization by the MAb (807–33) originally used in its selection. These results suggest a simple technique for the incorporation of glycoprotein mutations into bunya-viruses, allowing analysis of mechanisms of neutralization and other virus entry functions.
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Nucleotides in the panhandle structure of the influenza B virus virion RNA are involved in the specificity between influenza A and B viruses.
More LessInfluenza A and B viruses share common sequences and potentially similar panhandle structures in the terminal noncoding regions of virion RNA (vRNA). Interesting differences exist, however, in the number of conserved nucleotides at the 5′ and 3′ ends of the vRNAs, in base pairs constituting the panhandle duplex, and the length of uridine stretch (U stretch) juxtaposed to the RNA duplex. To analyse the contribution of these signals to the specificity between the two viruses, a transient ribonucleo-protein transfection method was used for the expression of the chloramphenicol acetyltransferase (CAT) reporter gene flanked by the noncoding nucleotides derived from influenza B vRNA. While the base pairing in the RNA duplex was primarily important for template activity, mismatch mutations G11 × G12′ and C12 × A13′ in the terminal RNA duplex region were utilized by influenza B virus, whereas these mutations were detrimental for influenza A virus. Different activity profiles were observed in the length preference of the RNA duplexes: maximum template activity was observed with 11 base pairs for influenza B virus, and 8 base pairs for influenza A virus. When the mutants with various lengths of U stretch were tested, highest CAT activities were observed with 5 to 7 uridine residues in influenza A virus, whereas in influenza B virus the activity was drastically decreased with 7 uridine residues. We suggest that the specific interaction of influenza virus RNA polymerase with these noncoding cis-acting signals in transcription of the RNA genome, along with unique coding strategies adopted by influenza B virus, has contributed to the divergence of these two closely related viruses.
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Sendai virus-like particles devoid of haemagglutinin-neuraminidase protein infect cells via the human asialoglycoprotein receptor.
More LessVirus-like particles with genetically defined envelope proteins were generated from cDNA in order to examine the requirement of Sendai virus haemagglutinin-neuraminidase (HN) protein for particle formation, and the role of fusion protein (F) in receptor binding and membrane fusion. Characterization of particles devoid of HN protein showed that particle formation was unimpaired by the absence of HN protein, indicating that HN protein is dispensable for virus assembly and budding. Infection studies further demonstrated that virus adsorption and penetration can be mediated solely by the F protein when the human asialoglycoprotein receptor is present at the surface of host cells.
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Immunological basis for protection in a murine model of tick-borne encephalitis by a recombinant adenovirus carrying the gene encoding the NS1 non-structural protein.
The humoral immune response to flaviviruses is mainly directed to the major envelope protein, E, and a glycosylated non-structural protein, NS1. Cell-mediated immune responses, however, appear to be directed mainly against non-structural proteins. Experiments described here show that a defective recombinant adenovirus (Rad51) containing the gene encoding the NS1 protein of tick-borne encephalitis virus can induce a strong protective immune response against several pathogenic tick-borne flaviviruses in an experimental animal model, and can enhance the efficacy of conventional vaccine preparations. A protective immune response against a lethal virus challenge can also be induced by the passive transfer of antibodies, B cells or T cells from animals vaccinated with Rad51. Raised levels of non-neutralizing antibodies and cytokines associated with a T helper cell-type 1 immune response are also observed. These data demonstrate the importance of non-structural viral proteins in the protective immune response against flaviviruses and support the use of non-structural viral proteins as vaccine components.
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Definition of the region on NS3 which contains multiple epitopes recognized by dengue virus serotype-cross-reactive and flavivirus-cross-reactive, HLA-DPw2-restricted CD4+ T cell clones.
More LessThe epitopes recognized by sixCD4 CD8 cytotoxic T lymphocyte (CTL) clones established from a dengue-3 virus-immune donor were defined. (i) Three CTL clones, JK10, JK34 and JK39, were crossreactive for dengue virus types 1–4. (ii) One clone, JK28, was cross-reactive for dengue virus types 1–4 and West Nile virus. (iii) Two clones, JK26 and JK49, were cross-reactive for dengue virus types 1–4, West Nile virus and yellow fever virus. The clones, except for JK49, recognized the same epitope on NS3 in an HLA-DPw2-restricted fashion. The smallest synthetic peptide recognized by the five CTL clones was a 10 aa peptide which comprises aa 255–264 on dengue virus NS3. JK49 recognized the overlapping epitope which comprises aa 257–266 in an HLA-DPw2-restricted fashion. Analysis of T cell receptor (TCR) usage by these T cell clones revealed that (i) JK10 and JK34 use Vα11,and JK34 and JK28 use Vβ23, and (ii) the amino acid sequences of the V(D)J junctional region of the TCR were different among these five CTL clones. There were, however, single amino acid conservations among TCRs of some of these T cell clones. These results indicate that the region on NS3 which comprises aa 255–266 contains multiple epitopes recognized by dengue serotype-cross-reactive and flavivirus-cross-reactive CD4 CTL in an HLA-DPw2-restricted fashion and that a single epitope can be recognized by T cells which have heterogeneous virus specificities.
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Low level or absent in vivo replication of hepatitis C virus and hepatitis G virus/GB virus C in peripheral blood mononuclear cells.
More LessTo investigate which subsets of peripheral blood mononuclear cells (PBMCs) are susceptible to infection with hepatitis C virus (HCV) and hepatitis G virus (HGV) or GB virus C (GBV-C), a PCR-based assay using tagged primers in the core region (HCV) and NS3 region (HGV/GBV-C) for the specific detection of negative strand (replicating) viral RNA sequences was developed. In liver biopsy samples both positive and negative strands of HCV RNA were detected, at levels ranging from 3 to 11 × 106 RNA copies per 106 cells and 3·7–4·2 × 103 copies per 106 cells respectively, while lower frequencies of positive strands of GBV-C/HGV RNA were detected (from 13 biopsies, the highest frequency was 7·3 × 103 per 106 cells). In no samples were negative RNA strands detected. To investigate extra-hepatic replication of HCV and GBV-C/HGV, CD4 , CD8 and B lymphocytes, monocytes and putative dendritic cell populations were separated from PBMCs from ten study subjects. Detection of positive strand HCV RNA was largely confined to B lymphocytes (at levels of up to 5 × 103 copies per 106 cells), while detection of negative strands was confined to a single subset (dendritic cells) of one of the study individuals. Similarly, GBV-C/HGV was detected at low levels in only twelve of twenty PBMC samples, while negative strands were uniformly absent. The low levels of HCV and GBV-C/HGV RNA in PBMCs suggest that these cells are at most a minor reservoir for virus replication. The absence of detectable replication of GBV-C/HGV suggests that the actual site of GBV-C/HGV replication remains to be discovered.
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A common RNA motif in the 3′ end of the genomes of astroviruses, avian infectious bronchitis virus and an equine rhinovirus.
More LessIn the 3′ non-coding region of the genomes of infectious bronchitis virus, an avian coronavirus and the picornavirus equine rhinovirus serotype 2, there is a motif with remarkable similarity, both in sequence and folding, to the second RNA stem-loop from the 3′ end of the genomes of human astro-viruses. This motif was also found in astroviruses of sheep, pig and turkey, suggesting that it is a common feature of all astroviruses. The conserved nature of the motif indicates that there has been strong selection for its preservation. There is significant homology between the regions flanking this motif in infectious bronchitis virus and a continuous RNA sequence at the same distance from the 3′ poly(A) tail in some related mammalian coronaviruses. These observations suggest that the presence of the motif in these three viral families is the result of at least two separate RNA recombination events.
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Induction of protective immunity in chickens vaccinated with infectious bronchitis virus S1 glycoprotein expressed by a recombinant baculovirus.
A recombinant baculovirus containing the S1 glycoprotein gene of the virulent nephropathogenic KM91 strain of infectious bronchitis virus (IBV) was constructed in order to investigate protective immunity in vaccinated chickens. Results from the protection test were evaluated by re-isolation of virus from the kidneys and tracheas of vaccinated chickens after challenge with strain KM91. After three immunizations, the recombinant S1 (rS1) glycoprotein induced 50% protection of the kidney, whilst inactivated KM91 induced 88% and 50% protection of the kidney and trachea, respectively. In chickens primed with the attenuated H120 vaccine strain, which is heterologous to KM91, the rS1 glycoprotein induced 83% protection of the kidney after two immunizations. Haemagglutination-inhi-bition titres were also increased in chickens immunized with the rS1 glycoprotein after three immunizations, and significantly higher titres were detected after challenge. These data indicate that the expressed rS1 glycoprotein alone can induce a protective immune response as well as an antibody response.
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Identification of mutations in the rotavirus protein VP4 that alter sialic-acid-dependent infection.
To explore further the role of VP4 as the rotavirus cell attachment protein, VP7 monoreassortants derived from the sialic-acid-dependent simian strain RRV and from the sialic-acid-independent human strains D, DS-1 and ST-3 were tested for susceptibility of infectivity of neuraminidase-treated MA-104 cells. Infectivity of RRV × D VP7 and RRV × ST-3 VP7 monoreassortants decreased when sialic acid was removed from the cell surface. However, of three separate RRV × DS-1 VP7 monoreassortants tested, only one was sialic-acid-dependent. Sequence analysis showed that both sialic-acid-independent strains contained a single amino acid change, Lys to Arg, at position 187. In addition, sialic-acid-independent infectivity was seen in one of 14 RRV VP4 neutralization escape mutants tested, and this strain was found to have a Gly to Glu change at amino acid position 150. These results indicate that positions 150 and 187 of VP4 play an important role in early rotavirus-cell interactions.
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Antimicrobial peptides melittin and cecropin inhibit replication of human immunodeficiency virus 1 by suppressing viral gene expression
Antimicrobial peptides are effectors of innate immunity, providing their hosts with rapid non-specific defence against parasitic invaders. In this report, the effects are assessed of two well-characterized antimicrobial amphipathic peptides (melittin and cecropin) on human immunodeficiency virus 1 (HIV-1) replication and gene expression in acutely infected cells at subtoxic concentrations. Production of infectious, cell-free virus was inhibited in a dose-dependent manner, with ID50 values in the range 0·9–1·5 μM for melittin and 2–3 μM for cecropin. Analysis of the effect of melittin on cell-associated virus production revealed decreased levels of Gag antigen and HIV-1 mRNAs. Transient transfection assays with HIV long terminal repeat (LTR)-driven reporter gene plasmids indicated that melittin has a direct suppressive effect on activity of the HIV LTR. HIV LTR activity was also reduced in human cells stably transfected with retroviral expression plasmids for the melittin or cecropin gene. It is concluded that antimicrobial peptides such as melittin and cecropin are capable of inhibiting cell-associated production of HIV-1 by suppressing HIV-1 gene expression.
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Replication of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus strain mac (SIVmac) and chimeric HIV-1/SIVmac viruses having env genes derived from macrophage-tropic viruses: an indication of different mechanisms of macrophage-tropism in human and monkey cells.
To investigate the transferability of macrophage (Mϕ)-tropism among primate lentiviruses, we constructed recombinants of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus strain mac (SIVmac) and chimeric HIV-1/SIVmac (SHIV) having env region Mϕ-tropic determinants. A recombinant HIV-1 having env partially derived from a Mϕ-tropic HIV-1 strain (JR-FL) replicated in human macrophages but not in monkey macrophages. Conversely, a recombinant SIVmac having env from a Mϕ-tropic strain (SIVmac316) replicated in monkey macrophages but not in human macrophages. A new SHIV (designated NM-3rN/JRFL) carrying the LTR and gag, pol, vif, vpx and nef of SIVmac and vpr, tat, rev, vpu and env of HIV-1 with env partially replaced by that of JR-FL was replication-competent in human macrophages but not in monkey macrophages. These results suggest that the Mϕ-tropic determinant is specific to each host species and that the mechanism of Mϕ-tropism is different between HIV and SIV.
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Endotoxin treatment of equine infectious anaemia virus-infected horse macrophage cultures decreases production of infectious virus.
More LessLentiviruses replicate in cells of the immune system, and activation of immune cells has been shown to modulate virus replication. To determine the effects of macrophage activation on replication of equine infectious anaemia virus (EIAV), primary horse macrophage cultures (HMCs) were established from 20 different horses, infected with an avirulent strain of EIAV, and stimulated with 5 μg/ml of bacterial endotoxin. Supernatants collected from HMCs were assayed for the presence of tumour necrosis factor (TNF-α) and for production of infectious virus. Results indicated that EIAV replication in vitro varied significantly (P ⩽ 0·0001) from horse to horse, regardless of the treatment of HMCs. Also, EIAV replication was significantly (P ⩽ 0·0001) decreased in HMCs stimulated with bacterial endotoxin as compared to untreated HMCs. No significant correlation was found between virus replication and production of TNF-α following treatment of virus-infected cells with bacterial endotoxin. However, when HMCs were treated with endotoxin prior to virus infection, inhibition of EIAV replication was proportional to increasing levels of endotoxin. PCR and RT-PCR were used to amplify EIAV proviral DNA and mRNA sequences, respectively, at various time-points following infection. The results indicated that the early events of EIAV replication, up to and including transcription of multiple-spliced mRNAs, were not inhibited by treatment of EIAV-infected macrophages with bacterial endotoxin. This suggests that endotoxin treatment inhibits a posttranscriptional step in the virus replication cycle.
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Antigenic variants of J subgroup avian leukosis virus: sequence analysis reveals multiple changes in the env gene.
K Venugopal, L M Smith, K Howes and L N PayneHPRS-103, the prototype of avian leukosis virus (ALV) subgroup J, was isolated in 1989 from meat-type chickens from commercial flocks where it induces myelocytic myeloid leukosis (ML). The HPRS-103 env gene differs considerably from other ALV subgroups but shows high identity (75–97%) to env-like sequences of the different members of the EAV family of endogenous avian retroviruses. Recently, we have isolated several viruses related to HPRS-103 from cases of ML. Although these isolates showed properties of ALV subgroup J, the majority of them resisted neutralization by HPRS-103-specific serum, suggesting antigenic variation. The nucleotide sequence of the env gene of the variant viruses showed several substitutions resulting in amino acid changes especially clustered in the variable regions hr1, hr2 and vr3. Analysis of the data suggests that selection pressure, probably from the immune response, is driving the antigenic variation among the isolates. Phylogenetic analysis of the sequences showed the evolutionary relationships of the isolates with HPRS-103 and the EAV family of endogenous avian retroviruses. The epidemiological significance of the antigenic variation and the emergence of variant viruses are discussed.
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Properties of tumour suppressor p53 in murine hepatocyte lines transformed by hepatitis B virus X protein.
Persistent infection by hepatitis B virus (HBV) correlates with the prevalence of hepatocellular carcinoma. It has recently been demonstrated that the complete viral genome very efficiently transforms the immortalized murine hepatocyte line FMH202 in vitro. Here it is shown that the viral transactivating protein X (HBx) is sufficient to transform FMH202 cells, albeit with lower efficiency. Clonal cell lines expressing HBx mRNA in moderate or high amounts grew in soft agar and formed tumours in nude mice. Growth efficiency in soft agar of HBx transformed cell lines was much lower than that of cell lines transformed with the complete genome, and latency of tumour induction in nude mice was significantly longer after inoculation of HBx than of HBV transformed FMH202 cell lines. A marker of complete transformation, p53, was found to be phosphorylated more strongly in HBx transfected cell lines than in controls, and a cellular kinase was found to be associated with p53 complexes from HBx transformed cell lines. p53 was of wild-type conformation and was located in the nucleus of transformed cells.
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Detection of new human papillomavirus sequences in skin lesions of a renal transplant recipient and characterization of one complete genome related to epidermodysplasia verruciformis-associated types.
More LessHuman papillomavirus (HPV) DNA, originally isolated from patients suffering from the skin disease epidermodysplasia verruciformis (EV), and a growing number of related sequences have recently been detected in a high percentage of benign and malignant skin lesions of both immunosuppressed and immunocompetent people. HPV L1 DNA fragments (374– 389 bp long) from a solar keratosis and a squamous cell carcinoma (SCC) of a renal transplant recipient were amplified, cloned and sequenced. In 54 clones, six different HPV sequences were identified. One of these six corresponded to the known type HPV-8 and two (RTRX3 and RTRX7) have been described previously in cutaneous lesions of immunosuppressed patients. The remaining three sequences were different from all known HPV types: an HPV-9-related sequence (77·4% identity), an RTRX2-related sequence (82·6% identity), and an HPV-22-related sequence (83·7% identity). These three sequences, representing putatively new HPV types, were named RTRX8, RTRX9 and RTRX10, respectively. RTRX7 was found in the majority of clones from both lesions. The complete genome of RTRX7 (7731 bp) was cloned as six overlapping subgenomic fragments, generated by nested PCR with DNA extracts from the SCC. RTRX7 showed a genome organization typical of HPVs associated with EV. The L1 DNA sequence differed by 15% from the corresponding region of its closest known relative, HPV- 12; thus, RTRX7 can be regarded as a new HPV type. RTRX7 DNA could not be detected by Southern blot hybridization with the homologous probe, indicating that the DNA concentration was below one copy per 10 cells in the investigated SCC.
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Human polyomavirus JC control region variants in persistently infected CNS and kidney tissue.
More LessThe question of a possible role for JC virus (JCV) genomic rearrangements in the pathogenesis of progressive multifocal leukoencephalopathy (PML) was addressed by analysis of the genomic complexity and the transcriptional control region (TCR) of the JCV DNA population in persistently infected CNS and kidney tissue. After cloning of full-length viral DNA, no extensive changes were detected in the coding regions of the JCV genome by restriction analysis suggesting an intact JCV DNA population. For further analysis of the distribution of JCV subtypes, the non-coding region was amplified by PCR. Molecular analysis revealed homogeneous JCV TCR populations in almost 50% of the individuals. Heterogeneity was found in two CNS samples with three and five different JCV subtypes, respectively, and in four kidney specimens with two TCR subtypes. Altogether, seven TCR subtypes were identified. One in each group represented single promoter element TCRs without duplication of sequences. The TCR of the major variant JCV-W1 was comparable in sequence and structure to that of the PML prototype JCV Mad-1 DNA. The identification of dominant PML-derived JCV TCR subtypes in most persistently infected individuals suggests that rearrangements of the JCV TCR can be associated with the persistent state of infection. However, it appears unlikely that PML-associated JCV subtypes are generated anew in each individual host in the course of persistence. The findings rather suggest that a limited number of stable JCV subtypes circulate in different geographical regions of the world.
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JC virus Type 1 has multiple subtypes: three new complete genomes.
More LessThe complete genomes of three new Type 1 strains of JC virus (JCV) from urine have been analysed. These were subtype 1A, subtype 1B and Type 4 as assigned from a short typing fragment in the VP1 gene. They differ from Mad1 (subtype 1A) by less than 10% of the DNA sequence. Based on its complete genome, the JCV Type 4 strain falls into a Type 1 subgroup. Type 4, with several Type 3-like sites in the short typing fragment, is a possible recombinant strain. The consensus of Type 1 DNA sequences is distinguished within the coding region from both Type 2 (strain GS/B) and five Type 3 (African and African American) strains at 64 sites. Most mutations are silent, but at 21 positions amino acid changes occur. Our findings define the subtypes of JCV Type 1 and support the validity of genotyping within the short VP1 fragment.
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Glycoprotein C-deficient mutants of two strains of herpes simplex virus type 1 exhibit unaltered adsorption characteristics on polarized or non-polarized cells.
More LessMutants of herpes simplex virus type 1 (HSV-1) strain SC16, lacking each of the dispensable glycoproteins C, G, E, I or J, were examined for their ability to infect the apical or basolateral surfaces of polarized human epithelial cells. None of the mutants was significantly different from the wild- type parent when assayed on either surface. Since a previous report had demonstrated that glycoprotein C (gC) was necessary for the infection of apical surfaces of polarized epithelium, a second gC-negative mutant was constructed on a background of HSV-1 strain HFEM. This mutant dis- played no phenotype when assayed on the apical surface. Furthermore, neither gC-negative mutant differed from its wild-type parent in its adsorption kinetics or specific infectivity on non-polarized Vero cells, a result which is inconsistent with the view that interactions between gC and cell surface proteoglycans constitute the initial adsorption process. Our findings thus conflict with previous reports and suggest that proposed functions of HSV-1 gC in the infection of polarized and non-polarized cells may be strain-dependent.
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Characterization of the protein encoded by gene UL49A of herpes simplex virus type 1.
More LessHerpes simplex virus type 1 (HSV-1) gene UL49A potentially encodes a primary translation product of 91 residues with a signal sequence at the N terminus and a membrane anchor domain near the C terminus. Mutants were generated in this gene and utilized to characterize the encoded protein on SDS-PAGE as a 6·7 kDa species which fractionated with infected cell membranes, was a relatively abundant virion component, and was not detectably O-glycosylated. The protein was identified by microsequencing as a 68 residue polypeptide formed by removal of 23 residues from the N terminus of the primary translation product. Cleavage of the signal sequence was also demonstrated by in vitro transcription and translation in the presence of microsomal membranes. The UL49A protein was efficiently solubilized along with envelope proteins by treatment of virions with a non-ionic detergent but only in the presence of a reducing agent, suggesting that it may be an envelope protein that is disulphide-linked to the tegument. It is apparent from mutational analysis that the 10 amino acid residues at the C terminus are not essential for synthesis of the protein, signal sequence cleavage, targeting to membranes and virions, linkage to the tegument and growth of virus in cell culture.
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Mice lacking inducible nitric-oxide synthase are more susceptible to herpes simplex virus infection despite enhanced Th1 cell responses.
Mice deficient in the inducible nitric-oxide synthase (iNOS), constructed by gene-targeting, were significantly more susceptible to herpes simplex virus (HSV)-1 infection, displayed a delayed clearance of virus from the dorsal root ganglia (DRG) and exhibited an increase in the frequency of virus reactivation in DRG compared with similarly infected heterozygous mice. The infected iNOS-deficient mice developed enhanced Th1-type immune re- sponses and their spleen cells produced higher concentrations of IL-12 than similarly infected heterozygous mice. This finding suggests that iNOS plays an important role in resistance against HSV-1 infection. Furthermore, nitric oxide (NO) may block the development of Th1 cells via inhibition of IL-12 synthesis and thereby play a role in immune regulation.
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Intradermal immunization with a bovine herpesvirus-1 DNA vaccine induces protective immunity in cattle.
Although intramuscular (i.m.) injection of DNA encoding glycoprotein D (gD) of bovine herpesvirus-1 (BHV-1) induces immune responses in cattle, this route of delivery is inefficient. Here we assessed three parameters that may enhance the efficacy of a gD DNA vaccine in cattle. First, the immune response generated by i.m. injected plasmid expressing a secreted form of gD (tgD) was determined and found to be very similar in magnitude to the response induced by gD-expressing plasmid. Secondly, gD- and tgD-expressing plasmids were administered by intradermal (i.d.) immunization, which resulted in a superior immune response to the secreted form, but no improvement in the response to the membrane-associated form. However, the form of gD used for immunization did not influence the immunoglobulin subtype, the ratio of antigen- specific IgG1 to IgG2 being approximately 4:1.
Finally, the effect of promoter strength was assessed by replacing the Rous sarcoma virus (RSV) promoter, which was used in the original experiments, with the human cytomegalovirus immediate early promoter and first intron A (HCMV/IA). Although upon transfection in vitro the HCMV/IA promoter appeared to be stronger than the RSV promoter, there was only a 2-fold higher antibody response in vivo upon i.d. injection of cattle. Protection against virus challenge was obtained in the calves immunized i.d. with tgD-encoding plasmid, as shown by a significant reduction in weight loss, virus excretion, temperature response and clinical disease. No significant protection was observed in the animals vaccinated i.d. with the gD-expressing plasmid, which correlates with the lower level of immunity pre-challenge.
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Open reading frame L1 of Marek’s disease herpesvirus is not essential for in vitro and in vivo virus replication and establishment of latency.
More LessTwo mutant CVI988 Marek’s disease virus (MDV) strains were developed in which a part of ORF L1 was replaced by lacZ with the SV40 early promoter. These mutant strains, CVIL1LacZ-A and -B, were inoculated into chickens to test the hypothesis that ORF L1 is involved in the induction and/or maintenance of latency. Mutant virus could be reisolated from lymphocytes obtained from chickens during both the lytic and latent phase of infection, indi cating that ORF L1 is not essential for the induction and/or maintenance of latency or the reactivation from latency. β-Galactosidase-positive lymphocytes were detected during the latent infection demonstrating that the SV40 early promoter can be active in recombinant MDV strains during latent infection. Although the insertion of lacZ was stable in cell culture, recombination within lacZ and the BamHI-L fragment was observed during in vivo infection.
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Different point mutations within the conserved N-glycosylation motif of pseudorabies virus glycoprotein M result in expression of a nonglycosylated form of the protein.
More LessGlycoprotein M (gM) constitutes one of the rare examples of a nonessential glycoprotein conserved throughout all herpesvirus subfamilies. Whereas gM in wild-type pseudorabies virus (PrV) strains carries an M-glycan, gM of the attenuated strain Bartha is not glycosylated due to a point mutation in the N-glycosylation motif. Since PrV Bartha lacks glycoproteins E and I and carries a mutated gC, we analysed glycosylation of gM in isogenic PrV glycoprotein deletion mutants. Whereas gM was glycosylated normally in most mutants, two independent gC deletion mutants and a gI mutant expressed a nonglycosylated form of gM. DNA sequence analyses revealed the presence of point mutations in the N-glycosylation consensus motif. Surprisingly, mutations in strain Bartha, the two gC- deletion mutants and the gI mutant proved to be different, although all affected the N-glycosylation motif. Thus, our data show that different, apparently independent point mutations cause expression of nonglycosylated gM.
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Human cytomegalovirus glycoprotein H/glycoprotein L complex modulates fusion-from-without
More LessGlycoprotein H/glycoprotein L (gH/gL) complexes of herpesviruses are required for fusion of infecting virions with host cell membranes. In human cytomegalovirus (HCMV), neutralizing monoclonal antibodies (MAb) specific for gH inhibit the transfer of a fluorescent probe to the host cell from labelled virus particles. In similar fashion, in the present study, neutralizing gH-specific MAb inhibited HCMV- induced fusion-from-without in monolayers of both human embryonic fibroblasts and continuous astrocytoma cells (U373). No fusion was detected in cells co-infected with defective recombinant adenovirus vectors that elicited high-level expression of gH and gL, indicating that surface-expressed gH was not intrinsically fusogenic. However, when such cells were superinfected with HCMV that gave fusion- from-without, the resulting cell-to-cell fusion was considerably enhanced. Thus, under our experimental conditions, gH/gL on the cell surface functioned to increase membrane fusion once this was initiated by other components in the virus envelope.
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Evidence against a key role for transforming growth factor-β1 in cytomegalovirus-induced bone marrow aplasia.
More LessDuring immunodeficiency after sublethal haemato- ablative treatment, cytomegalovirus (CMV) infection interferes with haematopoietic reconstitution and can cause lethal bone marrow (BM) aplasia. The in vivo model of murine CMV infection has identified the BM stroma as the principal target site of CMV in the haematopoietic cord. The infected cell type is the reticular stromal cell which forms the stromal network and produces essential haemopoietins, such as stem-cell factor (SCF). The expression of SCF was found to be reduced in the infected stroma, but the stromal network was not disrupted and the number of infected stromal cells was too low to explain the functional deficiency. These facts call for a negatively regulating cytokine that is induced by the infection and that potentiates the direct effect of infection by down-regulating haemopoietins in un infected bystander cells. Recent work has suggested that transforming growth factor (TGF)-β1 might be the cytokine involved in CMV-induced BM aplasia. We show here that murine CMV indirectly induces the accumulation of mature TGF-β1 in uninfected renal tubular epithelial cells and TGF-β1 transcription in BM stromal cells, whereas infected renal glomerular and interstitial cells, hepatocytes and BM stromal cells do not coexpress mature TGF-β1. Antiviral CD8 T-cell therapy prevented BM aplasia and also prevented the down-regulation of stromal SCF and interleukin-6 gene expression. Interestingly, however, the CD8 T cells did not preclude the up-regulation of mature TGF-β1, but proved to be inducers of TGF-β1 gene expression in BM stroma. These findings suggest that TGF-β1 is not the mediator of BM aplasia.
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Intracellular and extracellular vaccinia virions enter cells by different mechanisms.
More LessVaccinia virus (VV) produces two antigenically distinct infectious virions, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Structurally, EEV consists of an IMV with an additional outer membrane containing proteins that are absent from IMV. EEV is important for virus dissemination both in vitro and in vivo. Studies of EEV entry have been hampered by having two infectious virions and by the rupture of the EEV outer membrane in the majority of EEV virions during their purification. To overcome these problems, we have developed a novel approach to study VV entry that is based on confocal microscopy and does not require EEV purification. This assay relies on immunofluorescent staining and detection of individual, intracellular, uncoated virus cores. By this method, we show that EEV entry, in contrast to IMV, is dependent on a low- pH pathway and that the IMV enwrapped inside the EEV exhibits a low-pH fusogenic activity. Together with neutralization data demonstrating that exposure to low pH disrupts the EEV outer membrane, this study strongly supports a model for EEV entry which consists of binding, endocytosis, low-pH- induced disruption of the EEV outer membrane and fusion of the exposed IMV with the endosomal membrane releasing the core into the cytosol. The roles of the EEV outer membrane in virus dissemination and virus entry are discussed in relation to this model.
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Beet yellows closterovirus HSP70-like protein mediates the cell-to-cell movement of a potexvirus transport-deficient mutant and a hordeivirus-based chimeric virus.
It has been suggested that the beet yellows closterovirus (BYV)-encoded p65 protein, a homologue of HSP70 cell chaperones, plays a role as a virus movement protein (MP). To test this hypothesis, we used two types of complementation experiments with plant viruses containing the triple gene block (TGB) of MP genes. In one, the BYV p65 gene was cloned into a 35S promoter plasmid and introduced into Nicotiana benthamiana plants by microprojectile bombardment along with the 35S promoter- driven GUS gene-tagged cDNA of a transport- deficient potexvirus mutant. Transient expression of p65 complemented the mutant as visualized by the significant increase in the number of cells expressing the GUS reporter gene in the infection foci. In the other test, the p65 gene was inserted into the infectious cDNA of the hordeivirus RNAβ component to replace either the 58 kDa MP gene or the whole TGB. Inoculation of Chenopodium quinoa and Chenopodium amaranticolor plants with the T7 transcripts of the chimeric RNA/, together with the hordeivirus RNAα and RNAγ, caused symptomless infection in inoculated leaves detected by hybridization of the total leaf RNA with a specific cDNA probe. The ability of BYV p65 to substitute for the potexvirus or hordeivirus MPs provides direct evidence for its involvement in the cell-to-cell movement of closterovirus infection.
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Mutations in the HC-Pro gene of zucchini yellow mosaic potyvirus: effects on aphid transmission and binding to purified virions.
More LessTransmission of zucchini yellow mosaic virus (ZYMV) by aphids was examined by introducing mutations within the highly conserved proline-threonine-lysine (PTK) motif of the helper component proteinase (HC-Pro) using a cDNA full-length clone. Replacement of proline by alanine (ATK) in the PTK motif abolished transmission almost completely both from plants and from membranes. Substitution of the basic lysine by glutamic acid (PTE) did not reduce the rate of transmission compared with the wild- type. Replacement of threonine by valine (PVK) or serine (PSK) resulted in a rate of transmission that was lower than that of the wild-type. The rate was lower for PSK than for PVK. Western blot comparison did not permit attribution of HC-Pro functionality in transmission to its level in the host. The HC-Pro of strains that effected transmission (with the wild- type PTK motif, and with the mutated PTE and PVK motifs) could also bind in vitro to virions of ZYMV. HC-Pro with a PSK motif, which was less effective in assisting transmission, could bind only weakly to virions, while HC-Pro of the almost non-transmissible strains (with PAK and ATK motifs) did not bind at all. Interestingly, positive binding was recorded for transmission-defective ZYMV-Ct, which has a PTK motif but has glutamic acid instead of lysine in the lysine-leucine-serine-cysteine (KLSC) motif. These findings support the ‘bridge hypothesis’, and confirm the binding of the HC-Pro to the virion. The possible role of the PTK and KLSC motifs in binding to the virus and to the mouthparts of the aphid is discussed.
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Specific sequence changes in the 5′-terminal region of the genome of satellite tobacco mosaic virus are required for adaptation to tobacco mosaic virus.
More LessThe genome of satellite tobacco mosaic virus (STMV) adapted to tobacco mosaic virus (TMV), tomato mosaic virus or green tomato atypical mosaic virus consistently had two single base deletions at positions 1 and 61, corresponding to bases A and G, respectively, as compared to the type-strain genome which is naturally adapted to tobacco mild green mosaic virus (TMGMV). Transcript RNAs (STMVtmv) from clone pSTMVTMV which captured the deletions at positions 1 and 61 were infectious when co-inoculated to tobacco plants with either TMV or TMGMV at infection frequencies of > 90%. Two new STMV variants were created to investigate whether both deletions were essential for adaptation to TMV. These were STMVTMGMV ΔA1, which had the A at position 1 (A1) deleted, and STMVTMGMV ΔG61, which lacked G61. STMVTMGMV ΔA1 was infectious (75% frequency) in the presence of either TMV or TMGMV. Virion RNA of STMVTMGMV ΔA1 lost G61 after one infection cycle with TMV. This deletion did not occur in co-infections with TMGMV. STMVTMGMV ΔG61, like the clone STMVTMGMV, was infectious (100% frequency) with TMGMV but TMV did not support this clone. When Nicotiana benthamiana protoplasts were transfected with STMVTMGMV, STMVtmgmv ΔA1 or STMVTMV, STMV replicated when TMGMV was the helper virus. STMVTMVand STMVTMGMV ΔA1 replicated in the presence of helper TMV, but STMVTMGMV did not, the same result as in whole plants. The deletion of A1 is thus essential for initial STMV adaptation to TMV and the eventual deletion of G61 is a predicted additional change.
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Four DNA-A variants among Pakistani isolates of cotton leaf curl virus and their affinities to DNA-A of geminivirus isolates from okra.
More LessComplete DNA-A sequences of nine Pakistani geminivirus isolates from leaf curl-affected cotton (CLCuV-PK) or from okra, and the partial sequences of several additional isolates were determined. Sequences of isolates from cotton were of four types. Isolates from leaf curl-affected okra had virtually the same sequences as those from cotton. Isolates from yellow vein mosaic-affected okra were of two types (OYVMV types 201 and 301), both distinct from but closely related to the virus isolates from cotton. Of these six types, two types of CLCuV- PK are the most closely related but another (CLCuV- PK type 72b) is the most distinct. Of the encoded proteins, coat protein (CP) is the most strongly conserved (92–100%aminoacid sequence identity), and AC4 protein the most variable (41–87%).
The 5′ and 3′ halves of the intergenic region of some isolates had different affinities and occurred in seven combinations, suggesting that recombination had occurred and that the origin of replication was a favoured recombination site. Similarly, the first 1520 nt of CLCuV-PK type 804a DNA resembled those of OYVMV type 301 DNA but the remaining 1224 nt were very different. The AC1 (Rep) gene and 5′ part of the intergenic region of CLCuV-PK type 72b closely resembled those of OYVMV type 301, whereas the rest of the sequence did not. The cotton leaf curl epidemic in Pakistan is caused by several distinct variants, with recombination events involving OYVMV and other unspecified gemini- viruses having probably been involved in their evolution.
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Tubules containing virions are present in plant tissues infected with Commelina yellow mottle badnavirus.
More LessTubular structures containing bacilliform virions were observed in cell-free extracts of Commelina diffusa infected with Commelina yellow mottle badnavirus (CoYMV). The exterior of the tubule reacted with antibodies to CoYMV movement protein, but not with antibodies to virus coat protein. Similar tubular structures containing bacilliform particles were also observed in ultrathin sections of CoYMV- infected C. diffusa. These tubular structures traversed the cell wall at points where this was thickened or protruded. No similar structures were observed in healthy C. diffusa. These observations support the hypothesis that the virion-containing tubular structures observed in cell-free extracts are the same as those observed in situ, that these structures are composed, at least in part, of virus movement protein, and that they play a role in the cell-to-cell trafficking of virions of CoYMV.
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Two key mutations in the host-range specificity domain of the p143 gene of Autographa californica nucleopolyhedrovirus are required to kill Bombyx mori larvae.
More LessAutographa californica nucleopolyhedrovirus (Ac- MNPV) does not replicate in Bombyx mori cells (Bm5, BmN). We have shown previously that when a short DNA sequence within AcMNPV ORF95, which encodes the viral helicase P143, is replaced with the collinear region of B. mori nucleopolyhedrovirus (BmNPV), AcMNPV gains the ability to replicate in Bm5 cells. To determine the mutational events in the p143 gene required to allow AcMNPV replication in B. mori cells, AcMNPV recombinants produced in Sf9 cells were screened in vivo in B. mori larvae, which are more permissive to baculo- virus infection than B. mori cell lines. Eight combinations of mutations were tested and characterization of viral DNA extracted from dead larvae showed that amino acid changes at position 564 and 577 are required to kill B. mori larvae.
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Synthetic peptide vaccines yield monoclonal antibodies to cellular and pathological prion proteins of ruminants.
More LessTransmissible spongiform encephalopathies are closely linked to the accumulation of a pathological isoform of a host-encoded prion protein (PrPC), designated PrPSc. In an attempt to generate mono- and polyclonal antibodies to ruminant PrP, 32 mice were vaccinated with peptide vaccines which were synthesized according to the amino acid sequence of ovine PrP. By this approach five PrP-reactive polyclonal antisera directed against four different domains of the protein were stimulated. Spleno- cytes of mice which had developed PrP-reactive antibodies were used for the generation of monoclonal antibodies (MAbs). Obtained PrP-specific MAbs were directed to three different domains of ruminant PrP which differed from the three previously described major MAb binding sites in rodent PrP. MAbs exhibited reactivity with non-denatured ruminant PrPC in ELISA and immunoprecipitation and with denatured ovine and bovine PrPSc in immunoblot. Cross-reactivity was observed with PrPC of nine other mammalian species and with pathological PrP preferably of ruminants and weakly with that of hamster and mouse. The generated MAbs will be useful tools for the development of diagnostic tests for BSE and scrapie as well as for pathogenesis studies of these diseases.
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