- Volume 81, Issue 1, 2000

The open reading frame B438L, located within the EcoRI B fragment of the African swine fever virus genome, is predicted to encode a protein of 438 amino acids with a molecular mass of 49·3 kDa. It presents a cell attachment RGD (Arg–Gly–Asp) motif but no other significant similarity to protein sequences in databases. Northern blot and primer extension analysis showed that B438L is transcribed only at late times during virus infection. The B438L gene product has been expressed in Escherichia coli, purified and used as an antigen for antibody production. The rabbit antiserum specific for pB438L recognized a protein of about 49 kDa in virus-infected cell extracts. This protein was synthesized late in infection by all the virus strains tested, was located in cytoplasmic virus factories and appeared as a structural component of purified virus particles.

The hepatitis B virus (HBV) genotype was determined in a total of 121 plasma samples collected in France and the US from patients chronically infected with HBV. HBV genotype A was predominant in this collection, appearing in 66 samples (54%), while genotypes B, C, D, E and F occurred in 4 (3%), 14 (12%), 23 (19%), 1 (1%) and 0 (0%) of samples, respectively. However, the genotype of a total of 13 (11%) samples (2 from France, 11 from the US) could not be determined with the methodology used. Sequence analysis, and subsequent phylogenetic analysis of the complete genome and the individual open reading frames, showed that the virus isolate from these samples was 3248 bp long and, phylogenetically, did not cluster with any of the known genotypes. This strain was provisionally called HBV genotype G. Virus isolates that were obtained from geographically separated regions like France and the US were closely related to each other. All virus strains analysed contained some characteristic differences when compared to genotype A: a translational stop codon at aa 2 and 28 of the preCore region; a 36 nt (12 aa) insert in the amino-terminal part of the Core antigen (HBcAg); a 2 aa deletion in the carboxy-terminal part of HBcAg; and a 1 aa deletion in the preS1 open reading frame. The deduced amino acid sequence of HBsAg suggests that this newly discovered genotype G strain belongs to serological group adw2.

Mutations in the hepatitis B virus (HBV) genome have so far been investigated in cross-sectional or short-term longitudinal studies. Information about long-term changes is lacking due to the difficulty of sampling over long observation periods. In this study, a retrospective approach was used that allowed the analysis of changes in the viral genome from transmission to late stages of infection without the requirement for sampling early during this period. The entire viral genome was sequenced from serum samples of three mothers and their 10 adult children, who presumably had been infected vertically. The emergence of mutations between birth and sampling (mean 26·5 years) was assessed by comparing the individual sequences with the sequence of the strain assumed to have been transmitted. The mean differences from this sequence were 0·02 and 0·28% in seven asymptomatic and one symptomatic hepatitis B e antigen (HBeAg)-positive carriers, respectively, and 0·62 % in five HBeAg-negative carriers. Mutations occurred throughout the genome and 88% of the mutations caused amino acid substitutions spread over all genes. In HBeAg-negative carriers, the number of nucleotide and amino acid changes was independent of the severity of liver disease and, except the 1762AGG1764→TGA changes, no specific mutation was associated with liver disease. In conclusion, by using a novel method it was found that the entire HBV genome is extremely stable over long periods of time during the HBeAg-positive phase if the immune response (inflammation) is weak, whereas an average of 20 mutations emerged after development of hepatitis and/or loss of HBeAg without association with clinical outcome.

Sequences were recently obtained from four double-stranded (ds) RNAs from different plant species. These dsRNAs are not associated with particles and as they appeared not to be horizontally transmitted, they were thought to be a kind of RNA plasmid. Here we report that the RNA-dependent RNA polymerase (RdRp) and helicase domains encoded by these dsRNAs are related to those of viruses of the alpha-like virus supergroup. Recent work on the RdRp sequences of alpha-like viruses raised doubts about their relatedness, but our analyses confirm that almost all the viruses previously assigned to the supergroup are related. Alpha-like viruses have single-stranded (ss) RNA genomes and produce particles, and they are much more diverse than the dsRNAs. This difference in diversity suggests the ssRNA alpha-like virus form is older, and we speculate that the transformation to a dsRNA form began when an ancestral ssRNA virus lost its virion protein gene. The phylogeny of the dsRNAs indicates this transformation was not recent and features of the dsRNA genome structure and translation strategy suggest it is now irreversible. Our analyses also show some dsRNAs from distantly related plants are closely related, indicating they have not strictly co-speciated with their hosts. In view of the affinities of the dsRNAs, we believe they should be classified as viruses and we suggest they be recognized as members of a new virus genus (Endornavirus) and family (Endoviridae).

We showed previously that transgenic plants with the green fluorescent protein (GFP) gene fused to segments of the nucleocapsid (N) gene of tomato spotted wilt virus (TSWV) displayed post-transcriptional gene silencing of the GFP and N gene segments and resistance to TSWV. These results suggested that a chimeric transgene composed of viral gene segments might confer multiple virus resistance in transgenic plants. To test this hypothesis and to determine the minimum length of the N gene that could trans-inactivate the challenging TSWV, transgenic plants were developed that contained GFP fused with N gene segments of 24–453 bp. Progeny from these plants were challenged with: (i) a chimeric tobacco mosaic virus containing the GFP gene, (ii) a chimeric tobacco mosaic virus with GFP plus the N gene of TSWV and (iii) TSWV. A number of transgenic plants expressing the transgene with GFP fused to N gene segments from 110 to 453 bp in size were resistant to these viruses. Resistant plants exhibited post-transcriptional gene silencing. In contrast, all transgenic lines with transgenes consisting of GFP fused to N gene segments of 24 or 59 bp were susceptible to TSWV, even though the transgene was post-transcriptionally silenced. Thus, virus resistance and post-transcriptional gene silencing were uncoupled when the N gene segment was 59 bp or less. These results provide evidence that multiple virus resistance is possible through the simple strategy of linking viral gene segments to a silencer DNA such as GFP.

To evaluate the genetic diversity and understand the evolution of Yam mosaic virus (YMV), a highly destructive pathogen of yam (Dioscorea sp.), sequencing was carried out of the C-terminal part of the replicase (NIb), the coat protein (CP) and the 3′-untranslated region (3′-UTR) of 27 YMV isolates collected from the three main cultivated species (Dioscorea alata, the complex Dioscorea cayenensis–Dioscorea rotundata and Dioscorea trifida). YMV showed the most variable CP relative to eight other potyviruses. This high variability was structured into nine distant molecular groups, as revealed by phylogenetic analyses and validated by assessment of the molecular evolutionary noise. No correlation was observed between the CP and 3′-UTR diversities and phylogenies. The most diversified and divergent groups included isolates from Africa. The remaining groups clustered in a single clade and a geographical distinction between isolates from the Caribbean, South America and Africa was observed. The role of the host in the selection of particular isolates was illustrated by the case of a divergent cultivar from Burkina Faso. Phylogenetic topological incongruence and complementary statistical tests highlighted the fact that recombination events, with single and multiple crossover sites, largely contributed to the evolution of YMV. We hypothesise an African origin of YMV from the yam complex D. cayenensis–D. rotundata, followed by independent transfers to D. alata and D. trifida during virus evolution.

Relationships among the amino acid sequences of viral movement proteins related to the 30 kDa (‘30K’) movement protein of tobacco mosaic virus – the 30K superfamily – were explored. Sequences were grouped into 18 families. A comparison of secondary structure predictions for each family revealed a common predicted core structure flanked by variable N- and C-terminal domains. The core consisted of a series of β-elements flanked by an α-helix on each end. Consensus sequences for each of the families were generated and aligned with one another. From this alignment an overall secondary structure prediction was generated and a consensus sequence that can recognize each family in database searches was obtained. The analysis led to criteria that were used to evaluate other virus-encoded proteins for possible membership of the 30K superfamily. A rhabdoviral and a tenuiviral protein were identified as 30K superfamily members, as were plant-encoded phloem proteins. Parsimony analysis grouped tubule-forming movement proteins separate from others. Establishment of the alignment of residues of diverse families facilitates comparison of mutagenesis experiments done on different movement proteins and should serve as a guide for further such experiments.

cDNA copies of the coat protein (CP) gene of Indian peanut clump virus (IPCV)-H were introduced into cells of Nicotiana benthamiana or Escherichia coli by transformation with vectors based on pROKII or pET respectively. In both plant and bacterial cells, IPCV CP was expressed and assembled to form virus-like particles (VLP). In plant extracts, the smallest preponderant particle length was about 50 nm. Other abundant lengths were about 85 and about 120 nm. The commonest VLP length in bacterial extracts was about 30 nm. Many of the longer VLP appeared to comprise aggregates of shorter particles. The lengths of the supposed ‘monomer’ VLP corresponded approximately to those expected for encapsidated CP gene transcript RNA. Immunocapture RT–PCR, using primers designed to amplify the CP gene, confirmed that the VLP contained RNA encoding IPCV-H CP. The results show that encapsidation does not require the presence of the 5′-terminal untranslated sequence of the virus RNA and suggest that if there is an ‘origin of assembly’ motif or sequence, it lies within the CP gene. When transgenic plants expressing IPCV-H CP were inoculated with IPCV-L, a strain that is serologically distinct from IPCV-H, the virus particles that accumulated contained both types of CP.

The complete nucleotide sequences of apple mosaic virus RNA 1 and 2 have been characterized. Apple mosaic virus RNA 1 is 3476 nucleotides in length and encodes a single large open reading frame (ORF), whereas apple mosaic virus RNA 2 is 2979 nucleotides in length and also encodes a single ORF. The amino acid sequences encoded by RNA 1 and 2 show similarity to all of the other ilarviruses for which sequence data are available, but both are more closely related to alfalfa mosaic virus (AMV) than to other ilarviruses. Points of similarity include the absence of ORF 2b, present on the RNA 2 of all previously characterized ilarviruses. The close relationship to AMV also occurs in the movement protein, encoded by RNA 3, but not with the coat protein. These data suggest that the present taxonomy should be revised, and that AMV should be considered an aphid-transmissible ilarvirus.

Carnation Italian ringspot tombusvirus encodes a protein, referred to as 36K, that possesses a mitochondrial targeting signal and two transmembrane segments which are thought to anchor this protein to the outer membrane of the mitochondrial envelope of infected plant cells. To determine the topology of the virus protein inserted in the cell membrane, as well as the sequence requirements for targeting and insertion, an in vivo system was set up in which this could be analysed in the absence of productive virus infection. The 36K protein was expressed in the yeast Saccharomyces cerevisiae in native form or fused to the green fluorescent protein. Using a fluorescence microscope, large green-fluorescing cytoplasmic aggregates were visible which stained red when cells were treated with the vital stain MitoTracker, which is specific for mitochondria. These aggregates were shown by electron microscopy to be composed of either mitochondria or membranes. The latter type was particularly abundant for the construct in which the green fluorescent protein was fused at the N terminus of the 36K protein. Immunoelectron microscopy demonstrated that the viral protein is present in the anomalous aggregates and Western blot analysis of protein extracts showed 36K to be resistant to alkaline, urea or salt extraction, a property of integral membrane proteins.

Stem cuttings were collected in Cameroon from cassava plants displaying cassava mosaic disease (CMD) symptoms. The nature of the viruses present was determined by using the PCR with primers specific for the coat protein (CP) genes of African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV). All samples were infected by ACMV and eight of the 50 samples were infected by both ACMV and an EACMV-like virus. The complete nucleotide sequences of DNA-A and -B of representative ACMV and EACMV-like viruses were determined. The DNA-A component of the EACMV-like virus contained evidence of recombination in the AC2–AC3 region and DNA-B also contained evidence of recombination in BC1. However, both components retained gene arrangements typical of bipartite begomoviruses. When Nicotiana benthamiana plants were doubly inoculated with these Cameroon isolates of ACMV and EACMV (ACMV/CM, EACMV/CM) by using sap from cassava plants or infectious clones, the symptoms were more severe than for plants inoculated with either virus alone. Southern blot analysis of viral DNAs from infected plants showed that there were significantly higher levels of accumulation of both ACMV/CM components and, to a lesser extent, of EACMV/CM components in mixed-infected plants than in singly infected plants. These results strongly suggest the occurrence of a synergistic interaction between the two viruses.

Green fluorescent protein (GFP)-tagging was used to determine the intracellular localization pattern of the proteins encoded by banana bunchy top virus (BBTV) DNA-3, -4 and -6. The protein encoded by BBTV DNA-4, which possesses a hydrophobic N terminus, was found to localize exclusively to the cell periphery while the proteins encoded by BBTV DNA-3 and -6 were found in both the nucleus and the cytoplasm. Co-expression of the DNA-4 protein and the proteins encoded by BBTV DNA-3 and -6 revealed that the DNA-4 protein was able to re-locate the DNA-6 protein, but not the DNA-3 protein, to the cell periphery. The 29 amino acid N-terminal hydrophobic region of the DNA-4 gene product appeared to be essential for specific localization of this protein since deletion of this region abolished its ability to localize to the cell periphery. These results indicate that BBTV may utilize a system analogous to that of the begomoviruses with the BBTV DNA-6 protein acting as a nuclear shuttle protein (NSP) while the DNA-4 protein transports the NSP–DNA complexes to the cell periphery for intercellular transport. The protein encoded by BBTV DNA-5 was found to contain an LXCXE motif and yeast two-hybrid analysis revealed that the DNA-5 protein has retinoblastoma (Rb)-binding activity. This activity was dependent on an intact LXCXE motif since specific mutations to either the C or E residue completely abolished Rb-binding activity. These results indicate that the gene product of BBTV DNA-5 is an Rb-binding-like protein and may play an important role in host-cell cycle manipulation.