- Volume 71, Issue 9, 1990
Volume 71, Issue 9, 1990
- Animal
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Prediction and identification of a T cell epitope in the fusion protein of measles virus immunodominant in mice and humans
More LessAmino acid residues 288 to 302 of the fusion protein of measles virus were predicted by a variety of methods to represent a putative T cell epitope. This sequence was synthesized and the peptide was injected into mice of six inbred strains to test this possibility. Lymphocytes from peptide-immunized mice from all six H-2 disparate strains were able to mount a proliferative response following in vitro culture with the peptide. In addition, lymphocytes from three strains also proliferated in the presence of live measles virus. The peptide also behaved as a B cell epitope in that immunization with free peptide in adjuvant resulted in anti-peptide antibody production in all mouse strains. However, these antibodies did not react with the virus in either a solid-phase immunoassay or a virus neutralization assay. Peripheral blood lymphocytes from 10 laboratory personnel with a prior history of exposure to measles virus were tested in a proliferation assay with the peptide and with the virus. Lymphocytes from all 10 individuals proliferated in response to culture with the virus and those from eight responded to the peptide. These results give further support to the concept of permissive interaction of antigenic peptides with a wide range of class II major histocompatibility complex molecules both in mice and man and indicate the possibility of designing peptides that could be used as components of a synthetic vaccine for use in man.
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The antigenic structure of dengue type 1 virus envelope and NS1 proteins expressed in Escherichia coli
More LessThe antigenic structures of the envelope protein, E, and the non-structural protein, NS1, of dengue type 1 virus (DENI) have been studied in the form of recombinant fusion proteins expressed in Escherichia coli. Deletion analysis was used to identify two distinct antigenic domains in E that reacted with subsets of antiviral monoclonal antibodies (MAbs). Domain I of E extends from amino acid residues (aa) 76 to 93 of E; domain II extends from aa 293 to 402 and contains an essential disulphide bridge. MAbs also reacted with several determinants clustered near the N terminus of the NS 1 protein (aa 57 to 126). Recombinant fusion proteins containing E. coli trpE sequences and most of the sequences for either E or NS1 were immunogenic in mice. The antibodies elicited by the E fusion protein reacted with a portion of the protein containing domain II, whereas antibodies elicited by the NS1 fusion protein did not react with the antigenic determinants defined by our MAbs.
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Partial nucleotide sequence of South American yellow fever virus strain 1899/81: structural proteins and NS1
More LessWe have partially cloned and sequenced the genome of a Peruvian yellow fever virus isolate (1899/81) and compared the nucleotide and deduced amino acid sequences of this strain with the previously published sequence of the West African yellow fever virus strain Asibi. In the 3594 base region sequenced, which contains the structural genes (C, M, E), all but the 72 3′-terminal nucleotides of the NS1 gene and 108 nucleotides of the 5′ non-coding region, 515 nucleotide substitutions were detected. Nucleotide divergence was lowest in the 5′ non-coding region, 2·8%, compared with an average rate of 14·7% in the coding regions. Over 91 % of the 512 nucleotide changes in the coding region were silent; 44 amino acid substitutions resulted. The capsid protein was the least conserved, whereas the M protein was the most highly conserved (6·7% and 1·3% divergence, respectively). The envelope protein had 18 amino acid changes (3·7% divergence), one of which created an additional site for potential glycosylation of the 1899/81 virus. NS1 protein divergence (3·9%) was similar to that seen in the E protein. Of the 44 amino acid substitutions found, 34 (77%) were conservative. The highest number of nonconservative differences occurred in the envelope glycoprotein. These changes may significantly affect the antigenic and biological functions of the viruses.
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Persistent infection of a glioma cell line generates a Theiler’s virus variant which fails to induce demyelinating disease in SJL/J mice
More LessTheiler’s murine encephalomyelitis virus (TMEV) induces demyelinating disease which is associated with persistent virus infection of the central nervous system. To study the interaction between TMEV and host cells, we infected the G26-20 glioma cell line in vitro, and this resulted in a lytic infection in which most, but not all, cells were killed. Surviving cells divided and formed a viable monolayer in which a small proportion of cells displayed viral cytopathic effects. Levels of virus produced by these cultures over a 6 month period fluctuated between 6 and 8 log10 p.f.u./ml as measured by viral plaque assay. Similarly, the percentage of cells producing both viral antigen and viral RNA, as measured by a simultaneous immunoperoxidase/in situ hybridization technique, varied between 5 and 30%. Although persistently infected cultures were susceptible to challenge by both vesicular stomatitis virus and herpes simplex virus, they were resistant to infection by homologous viruses. Interferon activity was not identified. TMEV isolated from passage 12 produced smaller plaques than wild-type Daniels strain virus (wt-DAV) on L-2 cell monolayers. In contrast to demyelination induced in SJL/J mice after intracerebral inoculation with wt-DAV, mice infected with the small plaque variant virus failed to develop viral persistence or chronic demyelination. However, following immunosuppression by total body irradiation, SJL/J mice infected with the small plaque variant developed viral persistence but no demyelination. Characterization of the biochemical and molecular determinants of the variant will lead to a better understanding of determinants important in viral persistence.
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Echoviruses include genetically distinct serotypes
More LessWe have studied the genetic relationships of echoviruses using nucleotide sequencing and hybridization analysis. The nucleotide sequence of the echovirus 11 (EV11) P2 and P3 regions, which encode the nonstructural proteins, was shown to resemble closely those of coxsackie B viruses (CBV) and coxsackievirus A9 (CAV9). EV11, CBV and CAV9 have a similar organization in the 3′ non-coding region when compared to polioviruses and CAV21. In contrast, the 3′ end of EV22 shares only minimal sequence homology with other sequenced enteroviruses, and the 3′ non-coding region has a unique secondary structure. Thirty-three echovirus reference strains were tested by nucleic acid hybridization using cDNA probes from the genomes of EV6, 11, 18 and 22. It was shown that a great majority of the strains belongs to the same subgroup as serotypes 6, 11 and 18, whereas EV22 and EV23 are genetically not closely related to this major subgroup.
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Detection and differentiation of picornaviruses in clinical samples following genomic amplification
A polymerase chain reaction (PCR) assay was used to detect and differentiate picornaviruses (PVs), using primers homologous to the 5′ non-coding and VP2 regions of the PV genome. The PCR resulted in a 530 bp PCR product for human rhinoviruses (HRVs) and a 650 bp product for polioviruses, coxsackieviruses (CV) or echoviruses. The PCR assay could detect as little as 1 p.f.u. of virus in either cerebrospinal fluid (CSF) or stool, using ethidium bromide-stained gels. Standard strains of poliovirus, CV, echovirus and HRV were detected, with the exception of echovirus type 22. In contrast, heterologous viruses, such as herpes simplex virus, human cytomegalovirus, adenovirus, influenza virus and rotavirus, as well as human and monkey cell DNA, were not amplified. In nasal swabs taken from patients with respiratory infections, the PCR detected 27 of 28 HRV isolation-positive specimens. All specimens from which viruses other than HRVs were isolated were negative by PCR. The PCR definitively identified poliovirus and CVs from the CSF or stool of patients with aseptic meningitis, as well as CV in the pericardial fluid of a patient who had suffered a myocardial infarction. Specimens taken from patients with similar pathologies, and from which heterologous viruses were isolated, were uniformly negative by PCR.
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Inhibitory effect of protein kinase C inhibitor on the replication of influenza type A virus
More LessThe growth of influenza virus A/PR/8/34 in MDCK cells was inhibited by l-(5-isoquinolinesulphonyl)- 2-methylpiperazine dihydrochloride (H7) which is a potent inhibitor of protein kinase C, but not by an effective inhibitor of cyclic nucleotide-dependent protein kinases. Analysing the inhibitory effect of H7 during the replication cycle of influenza virus, we found that the primary transcripts were sufficiently synthesized in infected cells exposed to H7. The primary transcripts synthesized in the presence and absence of H7 were active in directing the synthesis of viral polypeptides both in a cell-free system and in the system containing H7. In the system where infected cells were exposed to H7, the viral positive-sense RNAs were also significantly amplified 6 h after infection. However, the synthesis of viral proteins other than nucleoprotein from viral primary or amplified (secondary) mRNAs was extremely restricted. The synthesis of host cellular proteins in mock-infected cells was significantly retained in the presence of H7. These results suggest that the selective inhibition of influenza virus translation following the transcription of viral mRNA was induced by H7 in infected cells.
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Influenza B virus mRNA synthesis in vivo: efficient transcription of vRNAs 1, 2 and 3
More LessInfluenza B virus genomic RNA (vRNA) segments encoding polymerase proteins were shown to be efficiently transcribed in vivo, unlike those of influenza A virus. The results are discussed in connection with encoding polymerase proteins were shown to be the primary structure of the 3′ends of vRNA segments.
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Induction of cervical neoplasia in the mouse by an extract of cells infected by varicella-zoster virus
More LessSince several human herpesviruses, including varicella-zoster virus (VZV), have been demonstrated to transform mammalian cells in vitro, VZV was tested in a mouse model of virus-induced cervical neoplasia to determine whether it is oncogenic in vivo. Herpes simplex viruses types 1 and 2 and cytomegalovirus have been previously shown to induce cervical neoplasia in this mouse model. VZV was propagated in WI-38 cell cultures and inactivated by ultraviolet irradiation. Control material was prepared in an identical manner from uninfected cell cultures. Cotton tampons, saturated with inactivated virus or control material, were inserted into the vaginas of C57BL mice three times a week for 60 weeks. Cervical dysplasia was detected in 40 % and invasive carcinoma in 34 % of virus-exposed mice by histological examination. No lesions were detected in control animals. These observations indicate that VZV, or some product of virus-infected cells, is oncogenic in vivo for the mouse cervix.
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Identification of antigenic sites on pseudorabies virus glycoprotein gp50 implicated in virus penetration of the host cell
M. Eloit, H. Bouzghaia and B. TomaFive monoclonal antibodies specific for glycoprotein gp50 of pseudorabies virus were used to make a topographical map of gp50 and to determine the biological function of the different antigenic domains. Three antigenic domains were identified by competition binding assays and additivity assays (IA, IB, II). Domain IA corresponds to a continuous epitope, whereas domains IB and II consist of one or several discontinuous epitopes, identified by their resistance to heat or reducing treatments. Domains IA and IB correspond to sites highly involved in virus neutralization. Neutralization experiments by monoclonal antibodies recognizing domains IA and IB and performed before or after adsorption of virions to cells showed that these domains have a role in penetration of virus into the cell.
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Increased antibody responses to human papillomavirus type 16 L1 protein expressed by recombinant vaccinia virus lacking serine protease inhibitor genes
The L1 gene of human papillomavirus type 16 (HPV- 16) driven by the vaccinia virus major late 4b gene promoter has been inserted into three different sites of the vaccinia virus genome. Insertion into the thymidine kinase (TK) gene was achieved by selection of TK− mutants in BUdR on TK− cells. Insertion into two vaccinia virus serine protease inhibitor (serpin) genes was achieved by co-insertion of the Escherichia coli xanthine guanine phosphoribosyltransferase gene linked to the vaccinia virus 7·5K promoter and selection of mycophenolic acid-resistant recombinant viruses. Each recombinant virus expressed a 57K L1 protein at similar levels and with similar kinetics. However, immunization of mice with these recombinant viruses induced different levels of antibody to the LI protein. Viruses lacking serpin genes B13R and B24R induced significantly higher antibody levels than did viruses lacking the TK gene. The presence of functional B13R and B24R gene products is therefore somehow immunosuppressive at least for antibody responses to the LI protein of HPV-16.
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The sequence of the nucleocapsid protein (N) gene of Piry virus: possible domains in the N protein of vesiculoviruses
More LessA number of independent cDNA clones of the Piry virus N gene message were identified and sequenced. From the resulting sequences and previously published data, we derived the sequence of the mRNA for this protein. Sequence similarities of the translated region of Piry virus with that of other viruses suggest that Piry virus is as distantly related to Chandipura virus as it is to the vesicular stomatitis viruses of Indiana and New Jersey serotypes. Based on the relative conservation of the amino acid sequence of the nucleocapsid protein of these vesiculoviruses, the N protein can be subdivided into at least three regions, possibly indicative of functional domains.
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Categorizing some early and late transcripts directed by the Autographa californica nuclear polyhedrosis virus
More LessUsing an SI mapping assay on RNA from Spodoptera frugiperda cells infected by the Autographa californicanuclear polyhedrosis virus in the presence and absence of cycloheximide and aphidicolin, we can distinguish three classes of transcripts. First, there are those whose synthesis is blocked by the DNA synthesis inhibitor aphidicolin and which are therefore late transcripts. These include the late transcript of the 39K gene and a late leftward transcript across the XhoI site in the HindIII-F region. Second, there are those whose synthesis is not blocked by aphidicolin, but whose accumulation is inhibited by the protein synthesis inhibitor cycloheximide and which are therefore presumably delayed early genes. These include the p26 transcript(s), the early 39K transcript and the a2 transcript in the HindIII-K/Q region. Third, there are those whose accumulation is not affected or is enhanced by cycloheximide. These are not necessarily immediate early transcripts, but their response to cycloheximide is clearly different from that of those in the second class. They include the α1, and α3 transcripts in the HindIII-K/Q region and the early leftward transcript across the XhoI site in the HindIII-F region.
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Nucleotide sequence and genomic organization of melon necrotic spot virus
More LessCloned cDNA copies of the genomic RNA of melon necrotic spot virus (MNSV) have been sequenced and the sizes and locations of predicted viral proteins have been deduced. The genome consists of at least 4262 nucleotides and the positive strand contains three to five open reading frames (ORFs) which may be expressed. The 5′ proximal ORF encodes a 29K protein (p29) and terminates with an amber codon which may be read through to produce an 89K protein (p89). Two small centrally located ORFs each encode a 7K protein (p7A and p7B). As p7A is in frame with p7B, readthrough of the amber codon terminating p7A may occur, producing a 14K protein (pi4). The 3′ proximal ORF encodes the 42K coat protein. The genomic organization of MNSV, its probable translation strategy and the amino acid sequences of its putative proteins closely resemble those of known carmoviruses, suggesting that MNSV should be classified as a member of the carmovirus group. Unusual properties of the putative MNSV replicase (p89) suggest that MNSV should be classified in a new virus supergroup with several other viruses sharing these properties.
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Nucleotide sequence and structural analysis of two satellite RNAs associated with chicory yellow mottle virus
More LessThe two satellite RNAs associated with CYMV infections were sequenced. The larger (sCYMV-L1) has only linear molecules 1145 nucleotides long, a poly(A) tail, a long open reading frame (ORF) coding for a protein of M r 39636 resembling in composition those of other large nepovirus satellite RNAs, a 5′ leader sequence of 16 nucleotides and a 3′ non-coding region of 40 nucleotides. In vitro translation of sCYMV-L1 yielded a protein product with a size that corresponded to that predicted from the sequence. The smaller satellite (sCYMV-S1) is 457 nucleotides long, has no ORF of significant length and no in vitro messenger activity. Both linear and circular forms of this satellite RNA were detected in infected tissues. Comparison of the sCYMV-S1 primary structure with the sequences of other small nepoviral satellites reveals large regions of homology. Analysis of the secondary structures derived from the sequences of the plus and minus strands suggests possible consensus sequences for their self-cleavage.
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Cucumber mosaic virus satellite RNA (Y strain): analysis of sequences which affect yellow mosaic symptoms on tobacco
More LessPlants infected with cucumber mosaic virus (CMV) (KIN strain) produce a mild mosaic disease on tobacco whereas infections of CMV with satellite RNA (strain Y) cause a severe yellow mosaic. Analysis of recombinant and mutant forms of satellite RNA identified a site (nucleotides 185/186) in the Y satellite RNA that affects the ability to induce the yellow mosaic in combination with CMV but not with tomato aspermy virus. The location of this site with respect to other mutations in the satellite RNA indicated that polypeptides, which may be encoded by the satellite RNA, have no role in induction of yellow mosaic symptoms. The symptom induction is therefore an effect of the satellite RNA on the host plant with the intervention of the helper virus. In the course of the mutation analysis of satellite RNA we detected several secondary mutations which arose in planta. Two of these were deletions of more than 80 nucleotides. Other forms of mutant satellite RNA were non-functional even though the modifications involved nucleotides completely within the large secondary deletions. These data imply complex intramolecular interactions in the satellite RNA.
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Complete nucleotide sequence of clover yellow mosaic virus RNA
The entire genomic RNA of clover yellow mosaic virus was sequenced from cDNA clones and run-off cDNA transcripts. The genomic RNA is 7015 nucleotides in length [excluding a 3′ poly(A) tail], with six open reading frames (ORFs) greater than 150 nucleotides in length. The first five ORFs encode proteins of M r 19IK, 26K, 12K, 6-5K and 28K, respectively. The sixth ORF lies completely within ORF1 and codes for a protein of M r 14K. The capsid protein coding region (M r 23K) is found within ORF5 which encodes the M r 28K protein. Proteins encoded by ORFs 1 to 3 and ORF5 show strong homology with proteins of other potexviruses, especially papaya mosaic virus.
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Molecular genetic analyses of the soybean mosaic virus NIa proteinase
More LessRecombinant DNA molecules containing cDNA to a soybean mosaic virus (SMV) RNA genome were constructed and partial nucleotide sequences determined for two cDNA inserts, pSMV-34 and pSMV-35. Comparison of the predicted amino acid sequence encoded by the pSMV-34 cDNA insert to other potyvirus protein sequences revealed extensive homology with the region of the genome encoding the NIa proteinase, with conservation of the amino acids proposed to form the catalytic triad of the active site. Cell-free transcription and translation of the cloned cDNA sequence containing the NIa open reading frame and flanking sequences revealed that NIa proteinase sequences, which were expressed as part of a high Mr precursor, were able to undergo proteolytic processing. Alteration of the codon for one of the putative active site residues by site-directed mutagenesis eliminated processing and resulted in the accumulation of a high Mr precursor. Based on predicted amino acid sequences at five putative cleavage sites within the SMV polyprotein, a consensus SMV NIa proteinase cleavage sequence of Glu/Asn- Xaa-Val-Xaa-Xaa-Gln ↓ Gly/Ser was proposed. The SMV NIa proteinase and its putative cleavage sites maintained motifs found in other potyviruses.
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Dependence of groundnut rosette virus on its satellite RNA as well as on groundnut rosette assistor luteovirus for transmission by Aphis craccivora
More LessTransmission of groundnut rosette virus (GRV) by Aphis craccivora is known to depend on the additional presence in the source plants of a luteovirus, groundnut rosette assistor virus (GRAV). Naturally occurring isolates of GRV contain a satellite RNA which is the main cause of rosette symptoms in groundnut, different variants of the satellite being responsible for the green and chlorotic forms of rosette. In extensive glasshouse tests, GRAV-dependent transmission of GRV by A. craccivora occurred only from groundnut plants infected with satellite-containing isolates of GRV. This was true whether the GRV isolates were from groundnut plants from Nigeria or Malawi with either the green or chlorotic forms of rosette and whether they contained homologous or heterologous satellites. Aphid transmission of GRV therefore depends not only on the presence of GRAV but also on that of the GRV satellite RNA. This probably explains why satellite-free isolates of GRV have not been found in nature. This is the first report of satellite RNA mediating aphid transmission of a plant virus.
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Transgenic Nicotiana debneyii expressing viral coat protein are resistant to potato virus S infection
More LessThe coat protein gene from potato virus S (PVS) was introduced into Nicotiana debneyii by leaf disc transformation using Agrobacterium tumefaciens. Transgenic plants expressing the viral coat protein were highly resistant to subsequent infection by the ME strain of PVS as indicated by an absence of symptom development and a lack of accumulation of virus in both the inoculated and upper leaves. As in reported experiments with plants expressing potato virus X coat protein, plants expressing PVS coat protein were also protected from inoculation with PVS RNA. These results provide further evidence that coat protein- mediated protection for these two groups of viruses, which share similar genome organizations, may involve inhibition of some early event in infection other than or in addition to virus uncoating.
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