- Volume 73, Issue 3, 1992
Volume 73, Issue 3, 1992
- Animal
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Sulphate polyanions prolong the incubation period of scrapie-infected hamsters
The effect of the organic sulphated polyanions, pentosan sulphate (SP54), dextran sulphate 500 (DS500) and suramin, have been tested on golden Syrian hamsters infected with the 263K strain of scrapie by the intraperitoneal (i.p.) or the intracerebral route. SP54 had the greatest effect in prolonging the incubation period of the disease when administered within 2 h of the i.p. inoculum. The same amount of SP54 given 24 h after scrapie inoculation had a potent effect in some animals and no effect in others. This result suggests that SP54 inhibits the uptake of the scrapie agent into the nerve endings and/or carrier cells at the site of the inoculum, i.e. the peritoneum, and that this event occurs in about 24 h. DS500 had a similar although less potent effect (22.4 days delay during the incubation period) than SP54 (54.4 days) when administered within 2 h of scrapie injection by the i.p. route, and suramin had only a minimal effect (10 days). This study suggests that treatment of scrapie and related spongiform encephalopathies of animals and man is possible only before the agent has reached the clinical target areas of the brain.
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Demonstration of a hepatitis C virus-specific antigen predicted from the putative core gene in the circulation of infected hosts
An ELISA was used to detect a protein derived from the core gene of the hepatitis C virus (HCV) in human plasma. The solid phase antibody in the assay was a murine monoclonal antibody against a synthetic peptide deduced from the putative core gene of HCV (residues 39 to 74). An enzyme-labelled affinity-purified human antibody directed at another region within the HCV core (residues 5 to 23) was the second antibody tracer. The ELISA had a sensitivity capable of detecting a few ng/ml of the HCV core polypeptide expressed in Escherichia coli. Core antigen activity in plasma of infected hosts was detected after treatment of HCV RNA-rich fractions from buoyant density centrifugation with the detergent Tween 80. There was a direct correlation between core antigen ELISA values of a plasma fraction and intensities of polymerase chain reaction signals for HCV RNA. These observations are consistent with the proposal that the N-terminal sequence of the predicted polyprotein of HCV is a nucleocapsid protein, and that improved core antigen assays may correlate with viraemia.
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Typing hepatitis C virus by polymerase chain reaction with type-specific primers: application to clinical surveys and tracing infectious sources
Based on variation in nucleotide sequence within restricted regions in the putative C (core) gene of hepatitis C virus (HCV), four groups of HCV have been postulated in a panel of 44 HCV isolates. They were provisionally designated types I, II, III and IV. A method for typing HCV was developed, depending on the amplification of a C gene sequence by polymerase chain reaction using a universal primer (sense) and a mixture of four type-specific primers (antisense). HCV types were determined by the size of the products specific to each of them. Type II was found in HCV samples from 131 (82%) of 159 blood donors, more often than in those from 48 (60%) of 80 patients with non-A, non-B (NANB) liver disease in Japan (P < 0.01). In 11 haemophiliacs who had received imported coagulation factor concentrates, type I was found in five, as against type II in four. Double infection with two different HCV types was found in two patients with chronic NANB liver disease (types I and II; II and III) and two haemophiliacs (types I and II; I and III). HCV types were identical in mother and baby in each of two examples of perinatal transmission, and were also identical in donor and recipient in a case of accidental needle exposure.
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Extraordinarily low density of hepatitis C virus estimated by sucrose density gradient centrifugation and the polymerase chain reaction
More LessThe genomic RNA of hepatitis C virus (HCV) in the plasma of volunteer blood donors was detected by using the polymerase chain reaction in a fraction of density 1.08 g/ml from sucrose density gradient equilibrium centrifugation. When the fraction was treated with the detergent NP40 and recentrifuged in sucrose, the HCV RNA banded at 1.25 g/ml. Assuming that NP40 removed a lipid-rich surface coat from HCV, the 1.08 g/ml and 1.25 g/ml HCV RNA may correspond to intact HCV virions and nucleocapsids, respectively. The extraordinarily low density of the virion is unusual in comparison to the density of classified viruses.
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Baculovirus-expressed glycoprotein H of herpes simplex virus type 1 (HSV-1) induces neutralizing antibody and delayed type hypersensitivity responses, but does not protect immunized mice against lethal HSV-1 challenge
More LessWe have shown previously that herpes simplex virus type 1 (HSV-1) glycoprotein H (gH) expressed by a baculovirus recombinant is transported to the cell surface in the absence of other HSV-1 gene products, and that the expressed gH has an apparent M r similar to that of authentic HSV-1 gH. We report here that antibodies raised in mice to this baculovirus-expressed gH neutralize the infectivity of HSV-1 in vitro; this neutralizing activity was not complement-dependent. Mice vaccinated with gH also developed delayed type hypersensitivity (DTH) to HSV-1. This is the first report of expressed HSV-1 gH inducing neutralizing antibody or DTH responses in vaccinated animals. In contrast to the gH expressed in mammalian systems, the ability of this baculovirus-expressed gH to induce a neutralizing antibody response may be due to the inability of the mammalian expression system to transport gH to the cell surface. Despite inducing anti-HSV-1 neutralizing antibody and DTH responses, vaccination of mice with gH did not protect the mice against lethal intraperitoneal challenge with HSV-1.
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The herpes simplex virus type 1 tegument protein VP22 is encoded by gene UL49
More LessVP22 is a major tegument protein of herpes simplex virus type 1 and is highly phosphorylated in the infected cell. Indirect evidence exists to suggest that it is encoded by gene UL49, present in the BamHI F fragment of the genome. Using the polymerase chain reaction we have cloned the UL49 open reading frame into a mammalian expression vector under the control of the human cytomegalovirus immediate early gene promoter. After transfection into COS-7 cells expression of the gene product was detected by means of Western blotting and immunofluorescence. The results clearly indicate that the protein encoded by UL49 is VP22, and that in transfected cells it appears to have characteristics similar to those of the protein synthesized in infected cells.
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Comparison between in vitro neutralization titres and in vivo protection against homologous and heterologous challenge induced by vaccines prepared from two serologically distinct variants of foot-and-mouth disease virus, serotype A22
More LessGuinea-pigs were challenged with homologous or heterologous strains of foot-and-mouth disease virus (FMDV) following vaccination with baby hamster kidney (BHK) monolayer cell-adapted or BHK suspension cell-adapted strains of FMDV serotype A22 Iraq 24/64. The protection afforded by these vaccines was analysed as a function of antigen dose and the in vitro serum virus neutralization titres achieved. The results show that the level of neutralizing antibody induced that afforded 50% protection was similar for both vaccines in homologous or heterologous challenge situations. However, although the dose of antigen required to achieve this titre against homologous virus was similar for the two vaccines, approximately 20-fold more of the suspension cell-adapted virus was required to elicit a protective titre against heterologous challenge compared to the dose of monolayer cell-adapted virus required. A synthetic peptide representing the amino acid sequence 135 to 167 of VP1, which is identical in the A22 Iraq 24/64 variant viruses, was shown to induce protection against both homologous and heterologous virus challenge.
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Evolution of influenza B/Victoria/2/87-like viruses: occurrence of a genetically conserved virus under conditions of low epidemic activity
More LessNucleotide sequence analysis of the gene region coding for the HA1 domain of the influenza B virus haemagglutinin was performed on seven field strains isolated during the 1989 to 1990 season and two field strains isolated in 1985 and 1988 in Finland. All isolates were antigenically and genetically related to B/Victoria/2/87 virus and distinct from B/Yamagata/16/88 virus. The three strains isolated at the beginning of the 1989 1990 season in Turku were almost identical to an American variant (B/Texas/37/88-B/Ohio/10/88) of the previous season, whereas the four strains isolated later in the 1989 to 1990 season in Helsinki formed a new group of heterogeneous viruses. The phylogenetic tree compiled suggests that the two branches had evolved from a common origin, probably in 1987.
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Sequence analysis of M2 mRNA of bovine respiratory syncytial virus obtained from an F-M2 dicistronic mRNA suggests structural homology with that of human respiratory syncytial virus
More LessThe nucleotide sequences of the F and M2 mRNAs of strain A51908 of bovine respiratory syncytial virus (BRSV) were determined by sequencing cDNA of an intracellular dicistronic mRNA. Comparison of the F mRNA sequence with those of other BRSV strains showed that there was extensive sequence identity at both the nucleotide (95% identity) and amino acid (94% identity) levels. Alignment of the nucleotide and encoded amino acid sequences of M2 mRNA of BRSV with those of human respiratory syncytial virus (HRSV) M2 mRNA showed 69% identity at the nucleotide level and 80% identity at the amino acid level. The general features of BRSV F and M2 proteins are similar to those described previously for the HRSV proteins. The M2 mRNA of BRSV also contained a second internal, overlapping open reading frame (ORF) similar to one reported for HRSV. The predicted products of the second ORFs of BRSV and HRSV shared 43% amino acid identity. As described for HRSV, the 3′-terminal end of the M2 mRNA overlaps with the 5′ end of the L gene by 68 nucleotides. The identity between the N-terminal regions of the L proteins of BRSV and HRSV is 75%. In addition, the intergenic sequence of the F-M2 gene junction of BRSV was determined.
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Down-regulation of vesicular stomatitis virus transcription by the matrix protein of influenza virus
More LessThe matrix (M1) protein isolated from influenza A/WSN/33 virus, when reconstituted with ribonucleo-protein (RNP) cores of vesicular stomatitis virus (VSV), resulted in inhibition of VSV transcription in vitro. The presence of endogenous wild-type (wt) or mutant (tsO23) VSV matrix (M) protein on RNP cores did not prevent down-regulation of VSV transcription by reconstituted influenza virus M1 protein. In fact, endogenous VSV wt M protein augmented transcription inhibition by M1 protein reconstituted with RNP/M protein cores, whereas mutant tsO23 M protein endogenous to RNP cores had no effect on down-regulation of VSV transcription by M1 protein. These data suggest that VSV M protein and influenza virus M1 protein recognize two different sites on RNP cores responsible for down-regulation of VSV transcription. Monoclonal antibodies (MAbs) directed to epitope 2 of M1 protein had been previously shown to reverse transcription inhibition by M1 protein on influenza virus RNP cores, but the same epitope 2-specific MAb had little effect on transcription inhibition by M1 protein reconstituted with VSV RNP cores. VSV M protein bears a striking resemblance biologically and genetically to the M1 protein, including, as shown here, their capacity to bind viral RNA. However, the VSV wt M protein exhibited no capacity to down-regulate transcription by influenza virus RNP cores. The significance of these studies is the identification on VSV RNP templates of at least two separate sites for recognition of protein factors that repress VSV transcription.
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- Plant
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Viral RNA synthesis in tomato spotted wilt virus-infected Nicotiana rustica plants
More LessThe synthesis of viral RNA species in tomato spotted wilt virus-infected Nicotiana rustica plants was followed in terms of time and relative abundance. Systemic symptoms were visible after 4 days post-inoculation (p.i.), but viral (v) and viral-complementary (vc) strands of all three genomic RNA segments [large (L) RNA, medium (M) RNA and small (S) RNA] were detected from 2 days p.i. In addition, two subgenomic mRNAs, derived from S RNA, were detected. For the L RNA segment no subgenomic mRNAs were detected, suggesting that this segment is expressed via the synthesis of a genome-sized vc mRNA. A possible M-specific subgenomic mRNA was detected, showing a similar time course of appearance as the subgenomic mRNAs derived from the S RNA segment. Analysis of cytoplasmic RNA fractions revealed that both v and vc strands of all three genomic segments associate with the nucleocapsid protein into nucleocapsid structures, the vcRNA species being present in lower amounts. Intact, enveloped virus particles contained only the v strand of the L RNA segment and, surprisingly, both v and vc strands of the M and S RNA segment, though in different ratios.
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Epitope mapping on fragments of beet necrotic yellow vein virus coat protein
The location of five SDS-stable epitopes on the coat protein (CP) of beet necrotic yellow vein virus was determined by reacting Escherichia coli-expressed free CP, as well as fusion proteins (FP) containing fragments of the CP, with polyclonal and monoclonal antibodies on Western blots. Epitope 1, which has previously been found to be exposed on only one extremity of the virus particle, was located in the region between amino acids (aa) 1 and 7, i.e. on the N terminus of the CP. It was blocked when the N terminus of the CP was linked to a portion of the β-galactosidase sequence in an FP. Epitope 3, which has previously been found to be exposed on the opposite extremity of the particle, was located in the region between aa 37 and 59. Epitope 4, which is exposed along the entire length of the particle, occurs on the C terminus of CP (aa 183 to 188). Two previously unknown epitopes were identified in the regions between aa 115 and 125 and 125 and 140, respectively. The former was located on the same extremity of the particle as epitope 3, the latter became accessible only after denaturation of the particle. Nothing is known about the probably non-adjacent aa sequences that participate in the formation of the two SDS-labile epitopes (epitopes 2 and 5) which are found on one extremity and along the entire length of the particle, respectively.
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Nucleotide sequence analyses of peanut stunt cucumovirus RNAs 1 and 2
More LessThe nucleotide sequences of the RNAs 1 and 2 of the peanut stunt virus strain J (PSV-J) were determined and compared with those of the cucumber mosaic virus strain Y (CMV-Y, subgroup I), strain Q (CMV-Q, subgroup II) and the tomato aspermy virus strain V (TAV-V) at both the nucleotide and protein levels. RNA 1 of PSV-J consists of 3355 nucleotides (nt) and has one large open reading frame (ORF) which can encode the putative 1a protein of M r 112025. PSV-J RNA 1 and the la protein are 65 to 73% identical to those of CMV-Y and -Q, and 65 to 69% to those of TAV-V. RNA 2 of PSV-J contains 2946 nt and also has one large ORF which can encode the putative 2a protein of M r 93575. For RNA 2 and the 2a protein, identities between PSV-J and two strains of CMV are calculated to be 53 to 61%. When compared with TAV-V, the same degree of similarity as seen with CMVs is observed. The 1a protein has the consensus sequences found in some helicases and methyltransferases and the 2a protein includes a sequence which exists in several RNA-dependent RNA polymerases.
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Infectious in vivo transcripts of a plum pox potyvirus full-length cDNA clone containing the cauliflower mosaic virus 35S RNA promoter
More LessA full-length cDNA clone of an aphid non-transmissible isolate of plum pox potyvirus (PPV) was rendered biologically active when placed under the control of the cauliflower mosaic virus 35S RNA promoter and the nopaline synthase polyadenylation signal. The cDNA was constructed so that the exact 5′ end of the PPV RNA was present at the transcription initiation site. Inoculation of plasmid DNA onto Nicotiana benthamiana led to systemic infection, whereas local lesions were produced in Chenopodium amaranticolor and C. quinoa, typical of an infection with PPV. Examination of infected plants revealed PPV-specific virus particles as well as viral RNA, the coat protein and the non-structural large nuclear inclusion protein (NIb).
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- Fungal
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A North American hypovirulent isolate of the chestnut blight fungus with European isolate-related dsRNA
More LessWe have synthesized and mapped a cDNA library representing the one major dsRNA element associated with hypovirulence in strain NB58 of the chestnut blight fungus, Cryphonectria (=Endothia) parasitica, which was isolated from recovering chestnut trees in New Jersey, U.S.A. The linear dsRNA has a size of approximately 12.5 kbp and is polyadenylated at the 3′ terminus of one strand. Molecular hybridization experiments indicate that there is sequence similarity between the NB58 dsRNA and dsRNAs from European isolates of C. parasitica, but not among dsRNAs of NB58 and those associated with other North American isolates. Hybridization experiments with mapped cDNA clones representing different regions of the 12.5 kbp dsRNA indicate that the termini and the 3′-proximal two-thirds (relative to the plus strand) are more conserved among NB58 and the European isolates than the rest of the 5′-proximal one-third. Nucleotide sequence analysis of the termini of NB58 dsRNA suggests common organizational features between it and the dsRNA from French-derived strain EP713.
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