- Volume 72, Issue 8, 1991

Analysis of the restriction site polymorphism (RSP) of human parvovirus B19 using 12 restriction endonucleases (REs) recognizing four or five bp sequences (4- or 5-bp REs) revealed a significant difference between strains previously classified as being of the same genome type, and a relationship between two strains of different genome types, thereby indicating a global spread of B19 virus strains. These findings demonstrated the advantage of this set of 4- and 5-bp REs for the calculation of the degree of genetic diversity and clearly it is necessary to amend the taxonomy of B19 virus strains using these REs. We examined the nucleotide (nt) sequence between nt 3141 and 3411, at the N terminus of the VP2 protein coding region, in 12 B19 virus strains. The pattern of distribution of nucleotide differences between the strains confirmed the classification by RSP analysis. Between nt 3293 and nt 3364, a region in which an antigenic epitope may be encoded, there was no evidence of a nucleotide change causing an amino acid change. Thus, the amino acid sequence in this potential epitope is probably conserved.

The replicative form (RF) DNA of chicken anaemia agent (CAA) was isolated and cloned into bacterial plasmids. After religation of the cloned CAA DNA and transfection into MDCC-MSB1 cells, the DNA could induce c.p.e. characteristic of that caused by CAA, and an antigen was produced which gave positive immunofluorescence when detected with an anti-CAA serum. Sanger sequencing of the 2298 bp genome revealed several open reading frames (ORFs); the major ORF encoded a polypeptide of 51.8K. In SDS-PAGE of CAA viral particles a 50K protein has been reported as the only detectable viral protein. The genomic region downstream of the major ORF had several predicted GC-rich inverted repeats, a poly(A) signal and four copies of an 18 bp repeat element. Database searches did not reveal any sequence with homology to the viral genomic DNA, nor to the amino acid sequence of any of the ORFs, apart from the N-terminal 40 amino acids of the major ORF which showed a limited similarity to the structure of protamines.

The haemagglutinins (HAs) of five H3 influenza A viruses isolated from domestic ducks and one from a goose in southern China were analysed antigenically and genetically. The patterns of reactivity of two of the duck viruses and the goose virus with a panel of monoclonal antibodies to 10 different epitopes on the H3 HA were similar to those of influenza viruses isolated from wild ducks and pigs, as well as those of the earliest human H3 viruses. The other three isolates from domestic ducks were different from each other and from these viruses antigenically. Sequence analysis revealed that the HA genes of the two duck viruses and the goose virus were closely related to those of isolates from wild ducks and pigs; the identities between the deduced amino acid sequence of the HA of one of the isolates from domestic ducks and those of isolates from a wild duck and a pig were 98.7% and 99.5%, respectively. The antigenic and genetic similarity between these H3 HAs suggests that in southern China, the hypothetical influenza epicentre, domestic ducks may have played a role in the introduction of avian influenza viruses to pigs from feral ducks. The findings also support the hypothesis that the pig was a ‘mixing vessel’, producing a new human pandemic strain, A/Hong Kong/68 (H3N2), by genetic reassortment.

The virulent avian influenza virus A/Ty/Ont/7732/66 (H5N9) (Ty/Ont) causes severe destruction of the lymphoid cells in infected birds. Previous studies have suggested that viral infection of macrophages may be involved. However, Ty/Ont failed to replicate productively in primary cultures of chicken macrophages. Therefore, in an effort to develop an in vitro system for our studies, we examined the susceptibility of an avian macrophage cell line, HD11, to Ty/Ont. We found that Ty/Ont replicated in the HD11 cells to high titres, as measured by haemagglutination (HA) assays and infectivity yields. To determine whether this property was unique to Ty/Ont, we also examined the replication of influenza viruses representative of all 13 HA subtypes and an attenuated variant of Ty/Ont. All of the tested viruses replicated in HD11 cells; the avirulent strains required the presence of trypsin in the culture medium whereas virulent viruses and the attenuated variant of Ty/Ont did not. These results suggest that the HD11 cells can support the replication of a wide variety of influenza viruses and that this continuous avian cell line may prove useful for in vitro studies on these viruses.

The expression of biotype-specific epitopes in cells infected with cytopathic (cp) and non-cytopathic (ncp) bovine viral diarrhoea virus (BVDV) was analysed by immunofluorescence. Four monoclonal antibodies (MAbs) directed against different epitopes on the viral glycoprotein gp48 were used. With cells infected with cpBVDV strain NADL, the four MAbs yielded a strong and granular cytoplasmic fluorescence. The same pattern was observed when cells were infected with ncpBVDV 7443 with two of the MAbs (BVD/C12, BVD/C42). In contrast, reactivity with the other two MAbs (BVD/C38, BVD/C46) was restricted to a narrow perinuclear zone. These biotype-specific differences were not observed either with a gp53-specific MAb, or with an MAb specific for the nonstructural protein p125/p80. Double immunofluorescence staining of living cells with a polyclonal BVDV-specific serum and with the MAbs revealed that expression of viral proteins on the surface of cells infected with cp- or ncpBVDV, respectively, was not detectable.

To estimate the particle size of hepatitis C virus (HCV), a major causative agent of post-transfusion non-A, non-B hepatitis, we filtered plasma or serum samples through microporous cellulose fibres with different pore sizes. The amount of HCV particles in samples before and after filtration was determined by a quantitative reverse transcriptase polymerase chain reaction (PCR) method. Since there is no quantitative biological assay for HCV, except for that in chimpanzees, the HCV titre obtained from the PCR method was used in an equation constructed previously for application to filtration experiments with a flavivirus which is distantly related to HCV. The particle was estimated to be between 30 and 38 nm in diameter, although the possibility remained that larger HCV particles or HCV aggregates with a diameter of more than 39 nm might exist. Double-step filtration through microporous cellulose fibres with a pore size of 35 nm reduced the HCV content to below levels detectable by our PCR method, indicating that it is possible to eliminate HCV particles by simple filtration techniques.

We have previously generated neutralization-resistant variants of Tacaribe virus in the presence of a monoclonal antibody (MAb) specific for the envelope glycoprotein. The envelope glycoprotein precursor (GPC) genes of two variant viruses were sequenced following polymerase chain reaction amplification of a specific region of the Tacaribe virus S RNA, and compared with the GPC gene of the parental virus. Multiple nucleotide changes in the 3′ half of the GPC gene were identified in the variants, suggesting that this part of the gene codes for the envelope glycoprotein of Tacaribe virus recognized by the MAb. Both variants showed unique amino acid substitutions up to 166 residues apart, suggesting that the most likely basis for neutralization resistance was a change in an epitope in which the critical residues are juxtaposed by conformation rather than by proximity in coding.

A bovine enterovirus, MZ-468, showed cytopathic effects on cell line F-647a, which was established by coculture of human T cell lymphotropic virus type 1-transformed MT-2 cells and X-irradiated rabbit lymphocytes. Microcalorimetric assay showed that residual, viable, MZ-468-infected F-647a cells produced less heat than non-infected cells. The therapeutic effects of MZ-468 infection were examined in rabbits in which adult T cell-like leukaemia (ATL) had been induced by inoculation of F-647a cells (1 × 108 cells). Six newborn rabbits were separated into three groups: group A was inoculated with F-647a cells only; group B was treated with MZ-468 at the time of inoculation with cells; group C was treated with the same amount of virus 24 h after the inoculation with cells and then once every 4 days. Both of the animals in group A and one in group C died 10 and 11 days, and 22 days, respectively, after the inoculation with cells. Both rabbits in group B and one in group C survived for more than 4 months. The rabbits that died were examined pathologically; leukaemic infiltrations were found in the lungs of the group B rabbits, and in the lungs, spleens and livers of both group A rabbits. Two identical experiments produced almost the same findings. These results suggest that bovine enterovirus might be used clinically to prolong the life-span of ATL patients.

Naturally occurring defective interfering RNAs (DI-RNAs) greatly reduce the accumulation of their helper virus in vivo, but are rarely associated with plant positive-strand RNA viruses. Deletion mutants pRNA-2 M/S and pRNA-2 E/S, derived from brome mosaic virus (BMV) genomic RNA-2, replicated in a manner dependent on BMV RNA-1 and -2, and effectively interfered with their accumulation in barley protoplasts. Based on their mode of replication, these mutant RNAs have been termed parasitic RNAs (pRNAs). When present with RNA-1 and -2 at low inoculum amounts, pRNA-2 M/S and pRNA-2 E/S reduced the level of replication of RNA-2, the parental RNA, by 37% and 64%, respectively. Greater amounts of pRNA in the inoculum completely eliminated the replication of both RNA-1 and -2. Mutations that prevented translation of truncated proteins from the pRNAs did not affect interference, but those that reduced pRNA replication decreased their ability to interfere with genomic RNA replication. At a molar pRNA: genomic RNA inoculum ratio of 1.5:1, pRNA-2 E/S reduced the accumulation of all helper virus RNAs by > 60%. This occurred in the presence of wild-type RNA-3 or ΔSGP RNA-3, a deletion mutant of RNA-3 that lacks the subgenomic promoter necessary for coat protein expression, demonstrating that the interference mediated by the pRNAs was not effected by encapsidation. These data indicate that the expression of pRNAs that function as artificial DI-RNAs in transgenic plants may be an approach for inducing resistance to virus infection which is applicable to a wide range of plant viruses.

A spontaneous red clover necrotic mosaic virus mutant, TpM-341, was isolated by multiple passage of Czechoslovakian isolate TpM-34 in beans, followed by three cycles of single lesion isolation in cowpea and in Chenopodium quinoa. The symptoms induced in cowpea by TpM-34 and TpM-341 differed. TpM-34 gave rise to chlorotic lesions which expanded with time, often becoming confluent with adjacent lesions, and developed necrotic margins; the plants became systemically infected. TpM-341 induced necrotic lesions which, once developed, did not expand further; plants did not become systemically infected. Analysis of pseudorecombinants formed between the RNAs of TpM-34 and TpM-341 showed that RNA 2 deter-mined the difference in symptoms. Comparison of the nucleotide sequence of the open reading frames (ORFs) encoding the P2 movement proteins of the two isolates revealed only one difference, a deletion of an A residue in a sequence of four A residues (nucleotides 790 to 793). Construction of full-length TpM-34 and TpM-341 RNA 2 cDNA clones, from which infectious RNA 2 could be transcribed in vitro, and in vitro mutagenesis of a cDNA clone of TpM-341, confirmed that the difference in the symptoms induced by TpM-341 was caused by the loss of this A residue. This single base deletion was predicted to cause a translational frame-shift in the P2 ORF causing the loss of 88 amino acids at the C terminus which were replaced by a sequence of 34 different amino acids, producing a truncated P2 protein. In vitro translation of RNA 2 transcribed from cDNA clones showed that RNA with four or three A residues starting at nucleotide 790 produced proteins of M r 36K and 30K respectively, in agreement with the predictions based on the nucleotide sequence.

The 3′ end of the RNA-2 of arabis mosaic virus (ArMV) was cloned and sequenced. The N-terminal amino acid sequence of the virion coat protein was determined by Edman degradation and the corresponding coding region identified. This gene was modified at the 5′ and 3′ ends by use of mismatched primers in the polymerase chain reaction (PCR), in order to facilitate the cloning of the gene, and to provide it with a methionine initiation codon. The modified cloned gene was expressed in transgenic plants, recombinant baculovirus-infected insect cells and bacteria. Both the insect cells and the plants expressing the modified coat protein gene contained empty virus-like particles (VLPs) similar to the empty virus shells found in plants infected with ArMV. These VLPs were not detected in the Escherichia coli expressing the coat protein. Analysis of the primary amino acid sequence in the ArMV coat protein revealed extensive regions of identity with that of grapevine fanleaf virus. Patterns in these identities may reflect a three-domain organization of the proteins.

An antiserum, elicited by a synthetic peptide coupled to bovine serum albumin, reacted specifically with the non-structural 16K protein of tobacco rattle virus. The protein was detected in extracts of systemically infected Nicotiana clevelandii leaves, but only in those made with the aid of SDS, urea and 2-mercaptoethanol. Immunogold labelling of ultrathin sections showed that the protein was mainly associated with nuclei, but was also present in the cytoplasm. These observations suggest that the 16K protein binds to macromolecular components of infected cells, especially in nuclei, but do not clarify its function.

An examination of the genomic strategy of pea enation mosaic virus (PEMV) RNA 1 has verified strong organizational and sequence relationships between PEMV and the beet western yellows-potato leafroll luteovirus subgroup. Sequence analysis of RNA 1 demonstrated five predominant open reading frames (ORFs). The extreme 5′ ORF encodes a 34K product of unknown function. The second ORF encodes an 84K product which overlaps 90% of ORF 1 (in a unique reading frame) and is expressed by internal initiation beginning at the second start codon from the 5′ terminus. This protein contains a protease-like motif characteristic of serine- and cysteine-based proteases, suggesting involvement in post-translational processing of viral translation products. The third ORF is characterized by a number of RNA polymerase motifs and a helicase-like motif typical of RNA-dependent RNA polymerases. It overlaps (out of frame) the ORF 2 product and is proposed to be expressed by a frameshift fusion of the ORF 2 and ORF 3 products. The fourth ORF encodes the viral coat protein, and is immediately followed in frame by a 33K ORF thought to represent the aphid transmission subunit of the PEMV virion. Northern blot analysis of polysome-associated RNA suggests that both products are expressed from an 1800 nucleotide subgenomic mRNA, with the 33K product expressed as a read-through fusion with the coat protein monomer.

The potato leafroll luteovirus protein of M r 17K (pr17), which is encoded by an open reading frame on the 3′ half of the viral genome, was expressed by using bacterial expression vector systems. Fusion proteins were obtained for the full-length viral protein as well as its N-terminal acidic (GST/pr17N) and C-proximal (GST/pr17C) basic domains and used in nucleic acid-binding studies. Filter-bound as well as soluble pr17 bound to single-stranded RNA or DNA. The binding domain was shown to reside in the basic C-proximal part of the polypeptide, whereas the N-terminal acidic domain did not show any affinity for nucleic acid. These biochemical properties of pr17 together with its structural features suggest a regulatory role for this protein during virus replication.

The mode of expression of the overlapping genes of the triple block positioned internally in potato virus X (PVX) RNA was examined. The results of In vitro translation of synthetic RNA transcripts and natural PVX-specific methylmercuric hydroxide-denatured dsRNAs suggest that the 25K protein is expressed as a single translation product of the 2.1 kb subgenomic (sg) RNA and that both the 12K and 8K proteins are expressed from the same 1.4 kb sgRNA.