- Volume 79, Issue 12, 1998

Tupaia herpesviruses (THVs) have been isolated from malignant lymphomas and from degenerating lung or spleen cell cultures of tree shrews (Tupaia spp.), but because of a lack of genetic information the final classification of THVs is still open. In the present work the viral DNA polymerase (DPOL) gene was mapped within the genome of the different THV strains using PCR and degenerate oligonucleotide primers. Nucleotide sequences of the DPOL genes of THV strains 1 to 5 were determined and used for comparative analyses. The transcriptional activity of the THV-2 DPOL gene was confirmed by RT-PCR. It was found that the different THV strains are very closely related to each other. When compared to other herpesviruses the highest amino acid sequence identities detected were with DPOLs of the murine and human cytomegaloviruses. These results justify the conclusion that THVs are members of the subfamily Betaherpes- virinae.

Human herpesvirus-8 (HHV-8) DNA sequences have been identified in all forms of Kaposi’s sarcoma (KS), a cancer found primarily in adult AIDS patients. We have identified HHV-8 strains in a rare human immunodeficiency virus (HlV)-negative form of KS, which is endemic in children in parts of sub-Saharan Africa. This was shown in Zambia, where we also had identified HHV-8 sequences in blood from HIVnegative febrile children without KS. In order to investigate the relationship of these Zambian strains to each other and to those from other forms of KS, we compared them to strains we have characterized from European AIDS KS (Denmark) and all published sequences from all forms of KS. Four distinct genomic regions were examined by PCR and sequencing: ORF26, ORF75, gH and K1. The results showed a distinct grouping of strains from both sets of Zambian children in all genomic regions studied, but which was most pronounced in the K1 glycoprotein gene. This gene was highly variable, encoding up to 25% amino acid sequence variation. In contrast, the Zambian groups were closely related to each other, with only 2% variation. Similar results were found in comparisons to the K1 sequences from HIV-positive febrile infants or KS children. The data raise the possibility that in areas where rare childhood endemic KS occurs, geographical variation in HHV-8 may relate to differences in virulence or transmission.

Apoptosis induced by H-1 parvovirus infection was investigated in C6 rat glioblastoma cells and in newborn rats. Apoptotic changes, such as chromatin condensation, the appearance of apoptotic nuclear bodies and oligonucleosomal DNA ladders, were observed in infected C6 cells 2 days after infection. Inhibitor assay results suggest that a caspase-3-dependent apoptosis activation pathway is induced by H-1 virus infection in C6 cells. Observations made in vivo revealed that the number of apoptotic cells increased in the infected cerebellum, coinciding with known virus infection sites.

Chicken anaemia virus (CAV) expresses three proteins, VP1, VP2 and VP3, but its capsid contains only the VP1 protein. In this paper, we report that for production of the neutralizing epitope, cosynthesis of (recombinant) VP1 and VP2 has to take place. We show via immunofluorescence that recom- binant-baculovirus-infected Sf9 cells synthesizing VP1 (or VP2) alone react very poorly with CAV- specific neutralizing antibodies. In contrast, Sf9 cells co-infected with VP1- and VP2-recombinant baculo-viruses, or infected with a single recombinant baculovirus co-expressing both VP1 and VP2, react strongly with the neutralizing antibodies. Furthermore, immunoprecipitation assays show that VP1 and VP2 interact directly with each other, which indicates that the non-structural protein VP2 might act as a scaffold protein in virion assembly. Recombinant baculovirus expressing VP1 and VP2 is, therefore, a potential production system for a subunit vaccine against CAV infection.

The p73 proteins α and β were identified based on their similarity to the tumour suppressor gene product p53. p53 and the p73 proteins activate transcription from p53-responsive promoters. The large T antigen of simian virus 40 (SV40) forms a specific complex with p53 and inhibits p53-medi- ated transcription. Here we show that the large T antigens from SV40 and JC virus strongly reduce the transcriptional activity of p53 but do not detectably affect the ability of the p73 proteins to transactivate. p53 but not the p73 proteins associate with SV40 T antigen in vitro. Finally, p53 colocalizes with a cytoplasmic mutant of SV40 T antigen, whereas both variants of p73 fail to colocalize with cytoplasmic T antigen. These results indicate that T antigen selectively binds and inactivates p53 but does not detectably affect the p73 proteins.

Recently a new human virus, TT virus (TTV) was identified in the serum of a patient with posttransfusion hepatitis of unknown aetiology. Comparative sequence analysis of a 222 nt fragment of ORF1 of TTV was performed to assess the genomic variability of this virus. Phylogenetic analysis of the nucleotide sequences of 76 TTV isolates collected in 17 countries segregated them into two major groups: TTV 1 and TTV 2. The TTV 1 group comprised two distinct subgroups, which corresponded to previously described TTV subtypes 1a and 1b. The TTV 2 group was separated into four main branches, two of which included sequences previously provisionally attributed as TTV types 2 and 3. Bootstrap resampling, however, did not support the reliability of this grouping, suggesting that the isolates in the TTV 2 group should be considered as subtypes of a single type rather than different TTV types.

Three maize streak virus (MSV) isolates were derived from an MSV population used to assess the response to infection of maize cultivars. Isolate SP1 was obtained from this population through short acquisition and inoculation periods (1 and 5 min, respectively), using a single Cicadulina mbila vector. Isolate SP2 was derived from SP1 after transmission to a wild perennial host (Coix lacryma-jobi), on which it was maintained for about 4 years without insect transmission. Isolate N2A, the most pathogenic isolate, was obtained from the initial population after serial passages on almost completely resistant inbred maize lines. The complexity of each isolate was analysed by RFLP analysis and sequencing based on 120 SP1 clones, 36 SP2 clones and 40 N2A clones. All three isolates were composed of different but related clones, consistent with a quasispecies structure. The mutations were distributed throughout the genome. Mutation frequencies, based on all available sequences, were 3·8 × 10 4 for SP1, 10· 5 × 10 4 for SP2 and 6· 9 × 10 4 for N2A. As expected from the bottleneck selection step, the intra-isolate variability of SP1 was relatively low. Comparison between SP1 and SP2 showed that SP1 heterogeneity increased during maintenance on the wild host. Furthermore, the consensus sequences of SP1 and SP2 differed by two non-synonymous substitutions in the complementary sense gene repA. N2A had a relatively low degree of heterogeneity, but was composed of several sub-populations. The results reflect the influence of the mode of selection of MSV isolates on their quasispecies organization, i.e. distribution of variants, and master sequence.

Four further circular ssDNA components (C7–C10), about 1 kb in size and structurally similar to the previously described components (C1–C6) found associated with a Syrian (Sy) isolate of faba bean necrotic yellows virus (FBNYV), have been identified. Similar to C1 and C2, two of the new components (C7 and C9) encode putative replication- associated (Rep) proteins of 33 ·2 and 32·7 kDa, respectively, the former of which is 90% identical to the C10 Rep protein of milk vetch dwarf virus (MDV). C8 encodes a putative protein (17 4 kDa) whose function is unknown, but which is highly conserved between FBNYV and the other nanoviruses MDV, subterranean clover stunt virus and banana bunchy top virus. The putative protein (19·7 kDa) encoded by C10 contains an LXCXE motif, which is also present in the homologues of its relatives, suggesting that they may all interact with plant retinoblastoma-like proteins. Sequence information for seven components of an Egyptian (Eg) FBNYV isolate indicated that six of them share > 96% identity with FBNYV-Sy. However, the C1 Rep protein of FBNYV-Eg was only 63·5% identical to that of FBNYV-Sy, but was 88·3 % identical to the MDV-C2 Rep protein. We conclude that the FBNYV genome consists of seven to ten components, six of which encode non-Rep proteins. All ten components of the FBNYV genome, except the Rep components C2 and C9, had closely related counterparts in the MDV genome. In spite of this similarity, FBNYV and MDV appear to be distinct virus species.

Milk vetch dwarf virus (MDV) is a member of the proposed genus Nanovirus, and its genome is composed of multiple, circular ssDNA components of about 1 kb. We have cloned and sequenced ten ssDNA components and designated them MDV-C1 to C10. Each DNA component contains one potential major open reading frame, and contains a putative stem-loop structure in the non-coding region. Notably, four components (C1, C2, C3 and C10) encode distinct replication-associated (Rep) proteins of 33 kDa, which show only limited (42–57%) amino acid identity. The six other components encode proteins with calculated molecular masses ranging from 12·7 to 19·7 kDa. Comparison of the sequences with those of other nanoviruses reveals that MDV is closely related to faba bean necrotic yellows virus (FBNYV) and subterranean clover stunt virus (SCSV). Six putative MDV genome products, including one Rep and five non-Rep proteins, show high (70·4–90·9%) amino acid identity to the corresponding six FBNYV proteins, whereas two other Rep proteins encoded by MDV-C2 and C3 are 82·3% and 73·0% identical to those encoded by SCSV-C2 and C6, respectively. These results indicate that MDV, FBNYV and SCSV have diverged from a common origin, which had multiple Rep components. In addition, the putative proteins encoded by MDV-C4 and its homologues contain a consensus retinoblastoma-binding motif, suggesting that they may be involved in controlling the host cell cycle.

Mutations of K → E in the highly conserved ‘KITC’ motif of the potyvirus helper component (HC) protein result in loss of HC function in aphid transmission, presumably because of inability to interact with virions, stylets or both. In this study we show that HC of potato virus C (PVC), a naturally occurring variant of potato virus Y (PVY) that has the K → E mutation, lacks the ability to be retained in stylets, whereas PVY HC is retained. The K → E mutation in either PVC or a site-directed mutant of tobacco etch virus (TEV) did not hinder binding to capsid protein, nor did deletion of the N-terminal 107 aa of TEV HC. An additional mutation, F → L at aa 10 of TEV HC, which renders HC non-functional but does not affect binding to capsid protein, is reported. Collectively, the results suggest that the N-terminal domain is required for interaction of HC with stylets rather than for binding to virions.

The potato A potyvirus (PVA)-encoded proteins P1, HC-Pro, P3, CI, VPg, NIaPro, NIb and coat protein (CP) were expressed as 6 × His-tagged recombinant proteins in Escherichia coli and purified to homogeneity. RNA binding was tested using purified proteins in Northwestern and liquid assays. PVA proteins except P3 bound to positive- and negativesense transcripts prepared from the nontranslated 5′ - and 3′ -regions of PVA genomic RNA and to full- length transcripts of PVA RNA. RNA binding by these proteins showed no sequence specificity since they also bound to various non-PVA control RNAs. Binding properties of P1, HC-Pro, CI and NIaPro are consistent with previous studies carried out on a few other potyviruses, but the binding of VPg, NIb and CP to RNA reveal novel interactions between RNA and potyvirus proteins. Furthermore, the RNA- binding properties of all major proteins of a potyvirus have not been reported previously.

Transgenic pea lines carrying the replicase (NIb) gene of pea seed-borne mosaic potyvirus (PSbMV) were generated and used in experiments to determine the effectiveness of induced resistance upon heterologous isolates. Three pea lines showed inducible resistance in which an initial infection by the homologous isolate (PSbMV-DPD1) was followed by a highly resistant state. Resistance was observed in plants in either the homozygous or hemizygous condition and resulted in no overall yield loss despite the initial infection. Resistance was associated with a loss of both viral and transgene RNA, which is indicative of a mechanism based upon post-transcriptional gene silencing. There was no correlation between the steady-state levels of transgene RNA and ability of the plants to show resistance. To test the specificity of the resistance, plants were also inoculated with the most distantly related sequenced PSbMV isolate, NY. PSbMV-NY varied between experiments in its ability to induce resistance, suggesting that the sequence identity in the NIb gene is borderline for the specificity required for triggering gene silencing. Upon challenge inoculation of virus-free recovered leaves, the specificity of the induced resistance varied between the two isolates and indicated that the virus and transgene additively determined the resistant state. These results suggest that the sequence requirements for triggering gene silencing may differ from those involved in the degradation process.

In contrast to most alfalfa mosaic virus (AMV) strains (YSMV, S, M and 425), AMV strains VRU and 15/64 can form abnormally long virus particles, an ability which has been linked to the coat protein (CP). In order to study this phenomenon, the CP-encoding RNAs 3 of AMV strains VRU and 15/64 were cloned and fully sequenced. Comparative sequence analyses of AMV RNA 3 sequences derived from different strains revealed two non-conservative amino acid substitutions, Ser66 and Leu175, which occur exclusively in the closely related VRU- and 15/64-CPs. When these amino acid alterations were introduced into the CP of AMV strain 425 unusually long virus particles were assembled. This confirms that amino acids Ser66 and Leu175 of the CPs of AMV strains VRU and 15/64 are involved in the formation of tubular virus particles.

Antigenic sites in the cucumber mosaic virus (CMV) coat protein (CP) have been identified using a polyclonal antiserum prepared against glutaral- dehyde-fixed virions. Antibodies were used to screen a random peptide library of heptamers displayed on the surface of a bacteriophage. Eight of 36 (22%) sequenced phage clones had inserts resembling a putative virion surface domain of the CMV CP. This region has the sequence LETDEL, corresponding to amino acids 194–199 in the Fny- CMV CP. The binding of phage clones to Fny-CMV antiserum was inhibited by a synthetic peptide representing this region. Six of 36 (17%) phage clones contained sequences corresponding to a C- terminal sequence in the Fny-CMV CP, which is thought to be internal in assembled virions. This sequence, EHQRIPTSGV, represents amino acids 206–215 and all but the P residue were observed in at least one clone. Four of 36 (11%) sequenced phage clones carried sequences that matched a portion of the sequence RLLLPDSV, corresponding to amino acids 89–96 in the Fny-CMV CP. This region was also identified as the antigenic site recognized by a monoclonal antibody (MAb23C10E4). Eleven percent of the phage (4 of 36) contained sequences matching at least three amino acids of the N-terminal region in the CMV CP. The positions of the antigenic sites seen in this study are consistent with a predicted structure for the CMV CP.

This is the first report of sequence from Fiji disease fijivirus (FDV), the type member of the genus Fijivirus of the family Reovi ridae. FDV genome segment (S9) comprised 1843 nt and contained two non-overlapping ORFs, separated by a 57 nt intergenic region. S9 ORF 1 comprised 1008 ntand encoded a 335-amino-acid polypeptide (predicted molecular mass 38·6 kDa), while ORF 2 comprised 627 nt and encoded a 208-amino-acid polypeptide (predicted molecular mass 23·8 kDa). The 5′ and 3′ non-coding regions were 49 and 102 nt, respectively. The S9 terminal sequences were 5′ AAGU-UUUU------UGUC 3′, and located immediately adjacent to these sequences were 12 bp imperfect inverted repeats. The entire S9 ORF 1 and the hydrophilic regions of S9 ORF 2 were each expressed as a fusion protein with the maltose-binding protein in Escherichia coli. Antibodies produced against the ORF 1 fusion protein reacted strongly with a protein of approximately 39 kDa present in both crude extracts of FDV-infected sugarcane and partially purified FDV preparations. In contrast, antibodies raised against the modified ORF 2 fusion protein did not react with any proteins in the same samples. Further, polyclonal antibodies produced against partially purified FDV reacted with the ORF 1, but not the modified ORF 2, fusion protein. These results indicate that FDV S9 ORF 1 encodes a major structural protein, while ORF 2 probably encodes a non-structural protein.

Nucleotide sequences were determined for two hop stunt viroid-related Group II citrus viroids characterized as either a cachexia disease non-pathogenic variant (CVd-IIa) or a pathogenic variant (CVd-IIb). Sequence identity between the two variants of 95·6% indicated a conserved genome with the principal region of nucleotide difference clustered in the variable (V) domain. Full-length viroid RT-PCR cDNA products were cloned into plasmid SP72. Viroid cDNA clones as well as derived RNA transcripts were transmissible to citron (Citrus medica L.) and Luffa aegyptiaca Mill. To determine the locus of cachexia pathogenicity as well as symptom expression in Luffa, chimeric viroid cDNA clones were constructed from segments of either the left terminal, pathogenic and conserved (T1-P-C) domains or the conserved, variable and right terminal (C-V- T2) domains of CVd-IIa or CVd-IIb in reciprocal exchanges. Symptoms induced by the various chimeric constructs on the two bioassay hosts reflected the differential response observed with CVd-IIa and -IIb. Constructs with the C-V-T2 domains region from clone-IIa induced severe symptoms on Luffa typical of CVd-IIa, but were non-symptomatic on mandarin as a bioassay host for the cachexia disease. Constructs with the same region (C-V-T2) from the clone-IIb genome induced only mild symptoms on Luffa, but produced a severe reaction on mandarin, as observed for CVd-IIb. Specific site- directed mutations were introduced into the V domain of the CVd-IIa clone to construct viroid cDNA clones with either partial or complete conversions to the CVd-IIb sequence. With the introduction of six site-specific changes into the V domain of the clone- IIa genome, cachexia pathogenicity was acquired as well as a moderation of severe symptoms on Luffa.

The prion protein (PrP) gene modulates the incidence and incubation periods of transmissible spongiform encephalopathies of sheep, goats, mice and man. Here, a new caprine PrP allele encoding the shortest naturally occurring PrP protein so far described is reported. This variant contains only three instead of the usual five copies of a short peptide repeat [Pro-Gln/His-Gly-Gly-Gly-(Gly)-Trp- Gly-Gln] characteristic of PrP, with an additional Trp to Gly substitution in codon 102. Fifteen out of 111 genotyped goats carried the novel PrP allele and 14 survived without signs of disease for at least 4 years. One goat heterozygous for the polymorphism was challenged experimentally with SSBP/1-scrapie and succumbed after an unusually long incubation period.