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Volume 72,
Issue 9,
1991
Volume 72, Issue 9, 1991
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The structural polypeptide VP3 of infectious bursal disease virus carries group- and serotype-specific epitopes
More LessTwo independent non-overlapping epitopes could be demonstrated on the structural protein VP3 of infectious bursal disease virus by non-neutralizing monoclonal antibodies produced against serotypes I and II. Both serotypes have one epitope in common, whereas the second epitope is distinct for serotype I and serotype II.
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Nucleotide sequence of the genes encoding the matrix protein of two wild-type measles virus strains
More LessThe nucleotide sequences of the matrix protein (M) genes of two wild-type measles virus (MV) isolates (JM and CM) have been determined and shown to differ in 56 positions; 31 of these differences are located in the non-coding region and 25 in the coding region of the gene. Most (80%) of the mutations in the coding region are changes to the third base of a codon. A maximum parsimony analysis of the available M gene nucleotide sequences allowed the construction of a tree with at least three lineages or subtypes. One wild-type strain (JM) was very similar to a subacute sclerosing panencephalitis virus strain (case B); the second wild-type strain, CM, showed nucleotide sequence similarity with MV from a case of measles inclusion body encephalitis. Both wild-type virus sequences are distinct from those so far determined for vaccine strains.
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Sequence characterization of the matrix protein genes of parainfluenza virus types 4A and 4B
The complete nucleotide sequences of the matrix protein (M) genes of parainfluenza virus types 4A and 4B (PIV-4A and -4B) were determined from cDNA of the mRNA, and found to be 1548 bases in length, exclusive of poly(A) sequences. The sequences contained a large open reading frame of 1146 nucleotides encoding 362 amino acids. A high degree of identity (96.1%) was observed between the amino acid sequences of PIV-4A and PIV-4B M. These M sequences were compared with those of 10 other paramyxoviruses and a phylogenetic tree was constructed.
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Molecular relationships between human parainfluenza virus type 2, and simian viruses 41 and 5: determination of nucleoprotein gene sequences of simian viruses 41 and 5
The nucleotide sequences of cDNAs of the simian virus 5 (SV5) nucleoprotein (NP) gene, and the 3′ end of the genome and NP gene of SV41 were determined. The open reading frames of the SV5 and SV41 NP genes encode polypeptides with M rs of 56582 and 60575, respectively, values which are consistent with those estimated by SDS-PAGE. The NP of human parainfluenza virus type 2 (hPIV-2) was more closely related to that of SV41 (amino acid sequence identity 70.5%) than that of SV5 (57.0%); the amino acid sequence identity between the NPs of SV41 and SV5 was 63.3%. The sequence of the 3′ end of the genome of SV41 showed a high level of similarity to that of hPIV-2, the terminal 18 nucleotides being identical. It is concluded from these findings that SV41 is related most closely to hPIV-2, even though SV5 had been thought to be an animal type of hPIV-2.
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The unique region of the human herpesvirus 6 genome is essentially collinear with the U l segment of human cytomegalovirus
More LessThe entire genome of human herpesvirus 6 (HHV-6) strain U1102 was cloned as overlapping fragments in cosmid and plasmid vectors. Cleavage maps were constructed for the restriction endonucleases BamHI, EcoRI, NotI and SmaI. The genome of HHV-6 U1102 is a linear dsDNA of 163 kbp, consisting of a long unique 142 kbp region flanked by direct terminal repeats of 10.5 kbp. Short stretches (290 to 470 nucleotides) of DNA, four from the terminal repeats and 55 from the U l region, were sequenced and compared by computer with the known herpesvirus amino acid sequences. Homologies were found for 10 open reading frames that are scattered over the U l region of HHV-6. Their relative positions and orientations indicate that the unique region of HHV-6 is essentially collinear with the U l region of human cytomegalovirus (HCMV), but is not collinear with the other human herpesviruses. It confirms and extends earlier observations that HHV-6 is more closely related to the β-herpesvirus HCMV, suggesting that HHV-6 may be considered as the prototype of a new β2-herpesvirus subgroup.
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Conserved domains of glycoprotein B (gB) of the monkey virus, simian agent 8, identified by comparison with herpesvirus gBs
More LessThe herpesvirus simian agent 8 (SA8) gene which corresponds to the herpes simplex virus (HSV) gene encoding glycoprotein B (gB) was localized, cloned and sequenced. Comparison of its deduced amino acid sequence with those of its counterparts in 12 other distinct herpesviruses was used to evaluate their homology and phylogenetic relationship. The results emphasized that SA8 gB is more closely related to those of HSV-1 and -2, and bovine herpesvirus 2 than to the homologous proteins of other herpesviruses. Furthermore, the alignment showed several regions of domains conserved in the closely related sequences, including four conserved in all the herpesvirus gB sequences examined. The conservation of 10 cysteine residues and most of the proline residues, as well as several potential N-glycosylation sites, suggested that the secondary and tertiary structures of these gBs were similar.
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The RL neurovirulence locus in herpes simplex virus type 2 strain HG52 plays no role in latency
More LessWe have demonstrated that the variant JH2604 of the herpes simplex virus type 2 (HSV-2) strain HG52 is completely avirulent in BALB/c mice following intracranial inoculation, with an LD50 of > 107 p.f.u./mouse compared to the wild-type LD50 of < 102 p.f.u./mouse. In JH2604, a 1.5 kbp deletion extends from the DR1/Ub junction of the ‘a’ sequence to 511 bp upstream of the 5′ end of IE1 in both long repeats. We have since constructed a second variant (2701) in which only 850 bp are removed from the RL. This deletion lies entirely within the sequences deleted in JH2604 and leaves intact most of a short 189 bp open reading frame (ORF) highly conserved between HSV-1 and HSV-2. Like JH2604, 2701 shows wild-type growth characteristics and is neither host range-nor temperature-restricted. This was most noteworthy in the case of mouse 3T6 cells. 2701 has an LD50 of 5×105 p.f.u./mouse on intracranial inoculation, a value intermediate between those of HG52 and JH2604. In assays for intracranial replication, JH2604 exhibits no detectable growth with a rapid decline in virus titre, 2701 shows limited growth over the first 24 to 36 h post-inoculation before the titre again declines and HG52 grows rapidly, reaching a high titre until the mice die. Taken together these results suggest that a region of the genome upstream of IE1 encodes a gene product essential for HSV replication in neurons of the central nervous system. It is highly likely that the conserved ORF is in an important region of a polypeptide essential for neurovirulence, although the upstream sequences present in 2701 but absent from JH2604 must also play a role. Although JH2604 and 2701 are avirulent, they both establish latent infection in the dorsal root ganglia of BALB/c mice and reactivate in vitro in a manner indistinguishable from HG52. This suggests a distinct separation of the factors involved in neurovirulence and the establishment of/reactivation from latency.
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Sequence analysis of the herpes simplex virus type 1 strain 17 variants 1704, 1705 and 1706 with respect to their origin and effect on the latency-associated transcript sequence
More LessThe precise endpoints of the deletions/insertions in three variants (1704, 1705 and 1706) of herpes simplex virus type 1 (HSV-1) strain 17 have been determined by dideoxynucleotide sequence analysis. The analysis was undertaken to discover whether the three variants had arisen from the same initial event and the extent of the deletions with respect to the latency-associated transcripts (LATs) and the proposed LAT promoter region. It is not possible from the deletion boundaries to determine unequivocally whether the three variants had arisen from the same recombination event although 1706 could be descended from 1705 by illegitimate recombination. The results demonstrate that spontaneous deletions can occur at random within R l , the extent of the deletions in U l is constrained by the essential nature of U l genes in vitro but is otherwise arbitrary and deletions in 1704 completely remove both copies of the LAT promoter region and in IR l extend into the 5′ end of the LAT sequence.
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Amplification of the Moloney murine leukaemia virus genome and its possible role in facilitation of chemical carcinogenesis in normal rat kidney cells
More LessIn a previous study we have shown that a single infectious particle of Moloney murine leukaemia virus per cell is sufficient to facilitate chemical carcinogenesis in normal rat kidney cells. When these cells are exposed to the carcinogen after a low number of passages post-infection (p.i.), cell transformation becomes apparent only after many subsequent passages. On the other hand, when exposure is done after a high number of passages p.i., cell transformation can be detected in the treated culture or at the next passage. It is thus evident that whereas the carcinogenic effect is rapid, the viral effect becomes apparent only after a long period of latency. Here we provide evidence that this viral effect requires multiple proviruses and that the long latent period reflects the time needed for a sufficient accumulation of proviruses in some of the cells. This accumulation may result from multiple rounds of superinfection by virions released into the culture medium, although we cannot exclude other mechanisms of provirus amplification. Our data also suggest that this amplification enhances virus production.
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A synthetic peptide elicits antibodies reactive with the external glycoprotein of human T cell lymphotropic virus type I
More LessA synthetic peptide derived from the external glycoprotein of human T cell lymphotropic virus type I (HTLV-I) (Env-5; amino acids 242 to 256) reacted with IgG antibodies in serum specimens from HTLV-I-infected individuals. C-terminal residues of Env-5 were crucial for the antibody reactivity. Polyclonal rabbit antibodies to Env-5 did not inhibit syncytium formation but such antibodies reacted specifically with gp68env and gp46env glycoproteins of HTLV-I in an immunoblot analysis. Immunoprecipitation of the surface-labelled MT-2 (HTLV-I-infected) cell line with anti-Env-5 precipitated the gp68env precursor protein. It was concluded that peptide Env-5 mimics a surface-exposed epitope on the HTLV-I external glycoprotein.
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The entire nucleotide sequence of foxtail mosaic virus RNA
More LessThe nucleotide sequence of the RNA genome of foxtail mosaic virus (FMV), a member of the potexvirus family, is 6151 nucleotides long, exclusive of a poly(A) tail. The RNA contains five principal open reading frames (ORFs), designated from the 5′ terminus as encoding proteins with M r values of 152.3K (ORF1), 26.4K (ORF2) which overlaps an 11.3K (ORF3) product, 5.8K (ORF4) which overlaps a 28.8K readthrough protein (ORF5A) which leads into the coat protein cistron of 23.7K (ORF5). The sizes and composition of the proteins encoded by the ORFs are generally similar to those found in other potexviruses; the least similar is the coat protein which nonetheless retains apparently critical consensus regions. The 5′ terminus of the previously reported 0.9 kb subgenomic (sg) RNA was determined by S1 nuclease mapping and shown to begin with the sequence GAAGA, 43 nucleotides upsteam from the first nucleotide of the coat protein initiation codon. The positions of the 5′ end of this sgRNA and of that deduced from the nucleotide sequence for a 1.9 kb sgRNA are entirely consistent with the previously published sizes of these sgRNAs.
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Nucleotide sequence of raspberry bushy dwarf virus RNA-2: a bicistronic component of a bipartite genome
More LessNorthern blot analysis with cDNA probes to RNA-3 (1 kb) of raspberry bushy dwarf virus (RBDV) revealed extensive sequence homology with RBDV RNA-2 (2.2 kb). Nucleotide sequencing showed that RNA-2 contains two large open reading frames (ORFs), of 1074 (5′ ORF) and 822 (3′ ORF) bases. The 3′ ORF is virtually identical in sequence to RNA-3, which encodes the M r 30509 (30K) coat protein. The 5′ ORF encodes an M r 38860 (39K) protein which slightly resembles the 32K protein encoded by RNA-3 of alfalfa mosaic virus (AIMV). RBDV RNA-2 resembles AIMV RNA-3 in being bicistronic and encoding a coat protein at the 3′ end. Comparing RNA-1 and RNA-2 of RBDV, only the 18 3′-terminal nucleotides are identical in sequence but the 3′-terminal 71 nucleotides of each RNA species have the potential to form similar stem-loop structures.
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Evolutionary relationships in the cucumoviruses: nucleotide sequence of tomato aspermy virus RNA 1
More LessRNA 1 of the V strain of tomato aspermy virus (TAV) consists of 3410 nucleotides and contains one open reading frame (ORF) of 2982 nucleotides, resembling RNA 1 of cucumber mosaic virus (CMV) strains Q and Fny (68% and 66% identical, respectively) and of brome mosaic virus (BMV) (41% identical). In comparisons between amino acid sequences, three conserved regions (N-terminal, C-terminal and central) between TAV and each CMV were found. The N- and C-terminal regions were also conserved with BMV, and contained, respectively, consensus motifs for methyltransferases and for nucleic acid helicases. The 5′ and 3′ non-coding sequences were highly similar to those of TAV RNA 2. When the sequences for the genomic RNAs of the V and C strains of TAV, and of their encoded products, are compared with those reported for CMV strains representing either subgroup I (Fny-CMV) or subgroup II (Q-CMV) of CMV, it was found that the different virus-encoded proteins are conserved differently between these three viruses. Also, the divergence between TAV and both CMV subgroups has proceeded at different rates for the different ORFs. On the whole, the divergence between TAV and CMV is of the same order as that found between CMV subgroups I and II, which suggests that TAV, Q-CMV and Fny-CMV could be considered as representing three equivalent subgroups of a taxonomic entity.
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The phylogeny of RNA-dependent RNA polymerases of positive-strand RNA viruses
More LessRepresentative amino acid sequences of the RNA-dependent RNA polymerases of all groups of positive-strand RNA viruses were aligned hierarchically, starting with the most closely related ones. This resulted in delineation of three large supergroups. Within each of the supergroups, the sequences of segments of approximately 300 amino acid residues originating from the central and/or C-terminal portions of the polymerases could be aligned with statistically significant scores. Specific consensus patterns of conserved amino acid residues were derived for each of the supergroups. The composition of the polymerase supergroups was as follows. I. Picorna-, noda-, como-, nepo-, poty-, bymo-, sobemoviruses, and a subset of luteoviruses (beet western yellows virus and potato leafroll virus). II. Carmo-, tombus-, dianthoviruses, another subset of luteoviruses (barley yellow dwarf virus), pestiviruses, hepatitis C virus (HCV), flaviviruses and, unexpectedly, single-stranded RNA bacteriophages. III. Tobamo-, tobra-, hordei-, tricornaviruses, beet yellows virus, alpha-, rubi-, furoviruses, hepatitis E virus (HEV), potex-, carla-, tymoviruses, and apple chlorotic leaf spot virus. An unusual organization was shown for corona- and torovirus polymerases whose N-terminal regions were found to be related to the respective domains of supergroup I, and the C-terminal regions to those of the supergroup III polymerases. The alignments of the three polymerase supergroups were superimposed to produce a comprehensive final alignment encompassing eight distinct conserved motifs. Phylogenetic analysis using three independent methods of tree construction confirmed the separation of the positive-strand RNA viral polymerases into three supergroups and revealed some unexpected clusters within the supergroups. These included the grouping of HCV and the pestiviruses with carmoviruses and related plant viruses in supergroup II, and the grouping of HEV and rubiviruses with furoviruses in supergroup III.
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Tomato spotted wilt virus L RNA encodes a putative RNA polymerase
The complete nucleotide sequence of the large (L) genome segment of tomato spotted wilt virus (TSWV) has been determined. The RNA is 8897 nucleotides long and contains complementary 3′ and 5′ ends, comprising 62 nucleotides at the 5′ end and 66 nucleotides at the 3′ end. The RNA is of negative polarity, with one large open reading frame (ORF) located on the viral complementary strand. This ORF corresponds to a primary translation product of 2875 amino acids in length, with a predicted M r of 331500. Comparison with the polymerase proteins of other negative-strand viruses indicates that this protein most likely represents the viral polymerase. The genetic organization of TSWV L RNA is similar to that of the L RNA segments of Bunyamwera and Hantaan viruses, animal-infecting representatives of the Bunyaviridae.
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Heterologous encapsidation in mixed infections among four isolates of barley yellow dwarf virus
F. Wen and R. M. ListerWe used immunohybridization and ELISA to investigate heterologous encapsidation (transcapsidation and phenotypic mixing) between paired isolates of barley yellow dwarf virus (BYDV) in doubly infected oat plants, Avena sativa L. cv. Clintland 64. Virions in samples extracted from plants doubly infected with two viruses were trapped with an antibody specific to one virus, and the nucleic acids of the trapped virions were identified with a cDNA probe specific to the other. Heterologous encapsidation was found in mixed infections between isolates NY-RPV and NY-MAV-PS1, NY-RPV and P-PAV, NY-RMV and NY-MAV-PS1, P-PAV and NY-MAV-PS1, and NY-RPV and NY-RMV. Heterologous encapsidation between NY-RPV and P-PAV, and between NY-RPV and NY-MAV-PS1, occurred in one direction, while the heterologous encapsidation between P-PAV and NY-MAV-PS1 occurred in both directions. Further analysis by heterologous ELISA and immunohybridization assays with immunoprecipitated samples demonstrated that trans-capsidation was the predominant type of heterologous encapsidation in mixed infections of NY-RPV and P-PAV, NY-RPV and NY-MAV-PS1, and NY-RMV and NY-MAV-PS1; phenotypic mixing was the predominant type of heterologous encapsidation in mixed infections of P-PAV and NY-MAV-PS1. Phenotypic mixing was also detected in mixed infections of NY-RPV and NY-RMV. These results suggest that among BYDV isolates transcapsidation is more common between distantly related isolates than between more closely related isolates, and phenotypic mixing is more common between more closely related isolates than distantly related isolates.
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Influence of the C terminus of the small protein subunit of bean pod mottle virus on the antigenicity of the virus determined using monoclonal antibodies and anti-peptide antiserum
More LessMiddle component particles of bean pod mottle virus (BPMV) containing small protein subunits with a cleaved C terminus were used to produce monoclonal antibodies (MAbs). All MAbs were specific for cryptotopes, i.e. epitopes present only on dissociated BPMV protein. The MAbs reacted more strongly with virus protein preparations containing the cleaved form of the small subunit than with preparations containing only the uncleaved form. It seems that the presence of additional residues at the C terminus of the intact small subunit interferes with antibody binding. Antibodies raised against synthetic peptides corresponding to the C terminus of the uncleaved small subunit reacted with both intact virions and dissociated subunits. This C-terminal region seems to play a dominant role in the antigenicity of the virus.
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Molecular analysis of rice dwarf phytoreovirus segment S11 corresponding to wound tumour phytoreovirus segment S12
More LessThe complete nucleotide sequence of rice dwarf phytoreovirus (RDV) genome segment S11 was determined. S11 is 1067 nucleotides long. There is an inverted repeat of 10 bp adjacent to the conserved 5′-terminal hexanucleotide (5′ GGUAAA 3′) and 3′-terminal tetranucleotide (5′ UAGU 3′) sequences. A single large open reading frame found in the plus strand of S11 begins with the first AUG codon (bases 6 to 8) and extends for 567 bases. Evolutionary relatedness between RDV S11 and wound tumour phytoreovirus S12 based on amino acid sequence similarity (25.8%) was found. In addition to the first AUG triplet, RDV S11 possesses a second in-phase AUG triplet (positions 30 to 32) nearby, which conforms to the Kozak consensus sequence. Two forms of the protein were identified by using an in vitro transcription and translation system in which a tailored full-length cDNA was the initial template. The abolition of the first AUG codon by site-directed mutagenesis resulted in disappearance of the larger translation product. These results strongly suggest that the two products are translated from the first and second AUG codons. Whether the two proteins are expressed in vivo is at present unclear.
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Outer capsid protein heterogeneity of rice dwarf phytoreovirus
More LessThe 46K outer capsid protein encoded by RNA segment S8 and the 42K polypeptide, previously thought to be the segment S9-encoded structural protein, were isolated from a rice dwarf phytoreovirus purified preparation, and then analysed by peptide mapping and electroblot-ELISA. Staphylococcus aureus V8 protease peptide mapping patterns of the 42K and 46K proteins were similar. Two monoclonal antibodies (MAbs), obtained after immunization with virus particles dissociated by 0.1% SDS, were each specific for both the 42K and 46K proteins. Furthermore, the MAbs bound common peptide fragments which were generated by digestion of the 42K and 46K proteins with V8 protease or proteinase K. These results strongly suggest that the 42K protein is not a gene product of S9 but a product overlapping with the 46K outer capsid protein. Whether the two proteins are functionally distinct remains to be determined.
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Effect of recombinant beet necrotic yellow vein virus with different RNA compositions on mechanically inoculated sugarbeets
More LessBeet necrotic yellow vein virus (BNYVV) inocula with different RNA compositions were prepared from infectious transcripts of RNAs 3 and 4 and the Rg 1 isolate, which has a genome consisting only of RNAs 1 and 2. The recombinant viruses were inoculated on 6- to 8-day-old sugarbeet seedlings by ‘vortexing’. Inocula containing RNAs 1 and 2 or 1, 2 and 4 produced some growth reduction, but the most dramatic effects, with yield reductions of about 95% in a highly susceptible variety, were seen when RNA 3 was also present in the inoculum. Under these conditions the side roots were brown and brittle and often deteriorated, but ‘root beardedness’ was not observed. This might be due to the fact that our experiments were done in the absence of Polymyxa betae. Alternatively, the heavy inoculation at a very young age may either have weakened the plants to such an extent that extensive root proliferation was impaired or it may have led to rapid deterioration of the proliferating rootlets, which would therefore be lost prior to or during removal of the tap roots from the soil. In the presence of RNA 3 the virus concentrations in tap roots were markedly increased suggesting that this RNA facilitates the multiplication and/or spread of the virus in root tissues.
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