- Volume 76, Issue 9, 1995

Development in mammalian cells of a recombinant expression system that mimics the rotavirus capsid assembly process would be advantageous for studying the structural requirements for particle formation. To this end, we investigated the ability of a recombinant vaccinia virus system to produce double-layered virus-like particles. The genes coding for VP2 and VP6 proteins of the human rotavirus strain Wa were cloned and used to generate recombinant vaccinia viruses. Metabolic labelling of CV-1 cells infected with these recombinant viruses followed by immunoprecipitation with a polyclonal antiserum directed to Wa virus showed that VP2 and VP6 were efficiently expressed. The recombinant proteins were similar in size and immuno-reactivity to authentic rotavirus proteins. Biochemical and electron microscopy analyses demonstrated that simultaneous expression of VP2 and VP6 in mammalian cells resulted in the formation of intracellular spherical particles resembling double-layered rotavirus particles.

We have analysed the potential capacity of hybrid porcine parvovirus (PPV) capsids to present foreign epitopes to the immune system. Foreign sequences were introduced into the N and C termini of PPV VP2, which was previously shown to assemble spontaneously into parvovirus-like particles. The integrity of the C terminus was shown to be essential for preserving the structure of the capsid and therefore could not be used for epitope fusion. In contrast, insertion of sequences corresponding to T and B cell poliovirus epitopes in the N terminus did not alter the formation of particles. Moreover, the chimeric capsids containing the C3:T epitope were able to induce a T cell response in vivo. However, hybrid particles containing the C3:B epitope fused to the N terminus did not induce any peptide-specific antibody response, suggesting that the inserted B cell epitope was not exposed at the surface of the particles. These results show that the N terminus in PPV empty capsids is not an adequate site for insertion of B cell epitopes, but may be useful for T cell epitope presentation and suggest that the N terminus is located in an internal position.

All alpha herpesviruses of known DNA sequence have been found to encode a protein with similarities to immediate early protein Vmw110 (ICP0) of herpes simplex virus type 1 (HSV-1). The conserved portion of this family of proteins is a characteristic zinc binding module, known as a RING finger or C3HC4 domain. Examples of RING finger domains occur in many other proteins of diverse evolutionary origin and function. Recently, the solution structure of the equine herpesvirus 1 (EHV-1) RING finger protein, encoded by gene 63, has been solved. To investigate whether this structure could be considered to be a paradigm of herpesvirus RING domains, we have constructed a recombinant HSV-1 which expresses the EHV-1 gene 63 protein (EHVg63) in place of Vmw110. Comparison of the growth properties of the recombinant with those of wild-type and Vmw110-defective viruses indicates that EHVg63 is able to fulfil partially, but not completely, the roles of Vmw110 during virus growth in tissue culture.

We have determined if a defect at entry of the human pathogen herpes simplex virus type 1 (HSV-1) into cultured porcine cells extends to HSV-2 and if the poor susceptibility of porcine cells for these viruses is indicative of in vivo species tropism. HSV-1 replicates poorly in swine testis (ST) and other porcine cells which lack a functional non-heparan sulphate receptor(s) required for virus entry. By several criteria, ST cells resist infection by either HSV-1 or HSV-2. Infection can be restored if normal entry is bypassed by PEG-mediated virion-cell membrane fusion. Neither HSV serotype infects, replicates or produces clinical symptoms in infant pigs. No virus was isolated from any of multiple sites and seroconversion did not occur. The in vitro defect in porcine cells blocking HSV entry correlates with, and is likely to be at least partly responsible for, in vivo resistance of pigs to infection.

Recently, data demonstrating that CD4 is an essential component of the receptor for human herpesvirus 7 (HHV-7) as well as for human immunodeficiency virus have been accumulating. Since gangliosides and phorbol esters are known to induce selective down-modulation of cell surface CD4 expression, it might be expected that treatment with these agents would interfere with HHV-7 infection of CD4+ T cells. The present study, undertaken to verify this possibility, demonstrated that addition of monosialoganglioside-GM1 or 12-O-tetradecanoylphorbol 13-acetate effectively induced disappearance of CD4 from the cell surface and also reduced HHV-7 infectivity, as judged by the CPE on virus-infected cells and studies of indirect immunofluorescence, TCID50 and semi-quantitative PCR of the HHV-7 genome. Taken together with previous studies, the present data strongly suggest that the CD4 molecule is a critical component of the receptor for HHV-7.

To gain insight into the role of glycoprotein E of bovine herpesvirus 1 (BHV-1), we compared the distribution of wild-type (wt) BHV-1 with that of a gE deletion mutant (gE−) in calves after intranasal inoculation. The wt-infected calves had severe clinical signs, but the gE−-infected calves were virtually free of clinical signs. At 3, 4, 7, 8, 44, 45, 50 and 51 days post-infection (p.i.), one calf from each group was killed and tissues were collected for virus isolation and PCR analysis. At 3, 4, 7 and 8 days p.i., infectious virus could be isolated only from the nasopharyngeal mucosa, parotid gland and nearby lymphoid tissues for both the wt- and gE−-infected calves. At 3 and 4 days p.i., virus titres in these tissues were comparable in both the wt- and gE−-infected calves. However, the virus titres were significantly reduced at 7 and 8 days p.i. in the gE−-infected calves, but not in the wt-infected calves. Semi-quantitative PCR analysis revealed that for the entire infection period (3 to 51 days p.i.) significantly more BHV-1 DNA was detected in the trigeminal ganglia (TG) of the wt-infected calves than in those of the gE−-infected calves. We conclude that the gE− mutant infects the same limited number of tissues as wt BHV-1, but for a shorter period.

Vaccinia virus (VV) inhibits the presentation of certain epitopes from influenza virus nucleoprotein (NP), haemagglutinin (HA) and non-structural 1 (NS1) proteins to CD8+ cytotoxic T lymphocytes (CTL) by an unknown mechanism. We have investigated whether VV genes B13R and B22R, which encode proteins with amino acid similarity to serine protease inhibitors (serpins), are involved in this process. Recombinant VVs were constructed which express influenza virus proteins HA, NP or NS1 and which lack serpin gene B13R or both B13R and B22R. The lysis of cells infected with these viruses by influenza virus-specific CD8+ CTL was compared to the lysis of cells infected with viruses expressing both the influenza proteins and the serpin genes. Cytotoxicity assays showed that deletion of the VV serpin genes B13R and B22R and other genes between B13R and B24R did not increase the level of lysis, indicating that these genes are not involved in inhibition of antigen presentation of the epitopes tested.

In order to determine the cause of cellular death of lymphocytes in pigs with acute African swine fever and the relationships between African swine fever virus (ASFV) and interstitial cells, ten pigs were inoculated with a highly virulent strain of ASFV (Malawi’83) and samples taken for ultrastructural study of hepatic and renal interstitial tissues. We demonstrated death by apoptosis of lymphocytes and virus replication in fibroblasts, smooth muscle cells and endothelial cells in the interstitial tissues of pigs inoculated with ASFV. From day 5 onwards, apoptotic lymphocytes and intense virus replication in hepatic interstitial macrophages and fibroblasts were observed. By day 7, apoptotic lymphocytes and virus replication in macrophages, interstitial capillary endothelial cells and fibroblasts in the kidney were observed. Virus replication was also seen in smooth muscle cells of hepatic and renal arterioles and venules. Our results suggest that mononuclear phagocyte system (MPS) cell activation, and the resulting release of cytokines, could induce apoptosis of lymphocytes and virus replication in non-MPS cells.

The organization of the major (L1) and minor (L2) proteins in the human papillomavirus capsid is still largely unknown. In this study we analysed the disulphide bonding between L1 proteins and the association of L2 proteins with capsomers using virus-like particles obtained in insect cells by co-expression of the L1 and L2 genes of human papillomavirus type 33. About 50% of the L1 protein molecules in these particles (1.29 g/cm3) formed disulphide-bonded trimers. Reduction of the intermolecular disulphide bonds by dithiothreitol (DTT) treatment caused disassembly of virus-like particles into capsomers. This indicates that disulphide bonds between capsomers at the threefold symmetry positions of the capsid are essential for the assembly of the papillomavirus capsid. In contrast, the L2 protein was not engaged in intermolecular disulphide bonding. The L2 protein remained associated with capsomers on disassembly by treatment with DTT. When the disassembly was carried out in 0.65 m-NaCl, complete L2 protein molecules bound preferentially to capsomer oligomers, whereas truncated L2 protein molecules bound only to monomers. In 0.15 m-NaCl only complete L2 protein molecules remained bound to capsomers. This indicates that different regions of the L2 protein molecule are differentially involved in the association of the papillomavirus capsid.

A viroid present in very low titres was isolated from symptomless field broad bean plants. It was identified as a variant of citrus exocortis viroid in the T2, V and C domains. Infection of several hosts resulted in a change in the composition of the viroid population. Serial passage through tomato and back to the host of origin, broad bean, resulted in major changes in replication efficiency, host range and pathogenicity. The unique nucleotide sequence differences identified in the original broad bean variant were not conserved after passage through alternative hosts. The effects of these sequence variations on viroid secondary structure result in nonpathogenic viroid variants which can remain unnoticed in certain plant species but may act as reservoirs of viroid disease.

The systemic movement and replication of banana bunchy top virus (BBTV) DNA component one were investigated. Strand-specific RNA probes and PCR were used to indicate the presence of the virus in various parts of infected banana plants during infection on the basis of dsDNA replicative intermediates of BBTV. The strand-specific probes were not only able to detect the presence of the virus but also gave an indication of where the virus replicated. The results using both the virion sense and complementary to virion sense specific probes were essentially the same indicating that BBTV initially replicated for a short period at the site of inoculation, and subsequently moved down the pseudostem to the basal meristematic region and ultimately into the roots and newly formed leaves. The virus was detected in the leaves formed prior to inoculation after 21 days using PCR but was not detected by the RNA probes. This indicated that the virus had the ability to move into these leaves but may not have replicated or accumulated to significant levels. The appearance of multimeric forms of BBTV suggested that the virus may have replicated via a rolling circle mechanism. Additionally, BBTV DNA component one did not appear to replicate in its aphid vector, Pentalonia nigronervosa.

We have previously demonstrated that African cassava mosaic virus (ACMV) DNAs A and B efficiently complement the systemic spread of tomato golden mosaic virus (TGMV) DNA A when co-agroinoculated onto Nicotiana benthamiana. Here, we show that a mixture of an ACMV DNA A AC2 mutant and DNA B that is normally unable to systemically infect N. benthamiana can do so at low frequency when co-agroinoculated with TGMV DNA A. Analysis of viral DNA showed that the AC2 mutation was retained during infection. The mixture of genomic components was sap transmissible, indicating that systemic infectivity is not specifically attributable to the use of agroinoculation. In the presence of TGMV DNA A, ACMV coat protein as well as the DNA B gene products BV1 and BC1 were detected in systemically infected tissues. The results demonstrate that dysfunctional AC2 can be complemented in planta by its TGMV homologue AL2.

Two Spanish plum pox virus (PPV) isolates, 5.15 and 3.3, were used in transmission experiments involving the aphid vector Myzus persicae, with woody and herbaceous host plants. These isolates differ in the size of their coat protein (CP) and sequence analysis revealed that isolate 3.3 has a 15 amino acid deletion near the N terminus of the CP, affecting the same positions as in a previously reported non-aphid-transmissible PPV isolate from Germany. Aphid transmission experiments showed that isolate 5.15 was transmitted from infected plants whereas isolate 3.3 was not. In contrast, both isolates were readily aphid-transmitted when acquired through artificial membranes from purified virus preparations supplemented with purified helper component (HC) obtained from potato virus Y-infected plants. This indicates that non-transmissibility of isolate 3.3 may be due to a defect in the HC rather than in the CP.

Tomato ringspot nepovirus (TomRSV) produces a 45 kDa movement protein and a 58 kDa coat protein in infected plants. Accumulation of the movement protein in relation to that of the coat protein was studied in infected protoplasts using a monoclonal antibody against the movement protein and polyclonal antibodies against the coat protein. Unlike most other viral movement proteins, the TomRSV movement protein was present at late stages of infection. Pulse-chase labelling experiments revealed that the release of the movement protein from the precursor polyprotein was coordinated with that of the coat protein. However, the movement protein was less stable than the coat protein in the extractable fraction of the protoplasts. The expression pattern of the TomRSV movement protein is discussed in the light of the proposed mechanism of cell-to-cell movement of virus-like particles through tubular structures composed of the movement protein.

The complete nucleotide sequence of citrus tatter leaf capillovirus (CTLV lily strain) was determined. It is 6496 nucleotides long, excluding the 3′-terminal poly(A) tract, and contains two putative overlapping open reading frames (ORFs). ORF1 (positions 37-6354) encodes a potential polyprotein of molecular mass 242 kDa. ORF2 (positions 4788-5750) codes for a 36 kDa protein. The 242 kDa polypeptide contains several non-structural protein domains (i.e. methyltransferase, NTP-binding helicase, papain-like proteinase and polymerase) and, at its C terminus, the putative coat protein. The N-terminal region of the 36 kDa protein displays sequence similarity to the cell-to-cell movement proteins of the ‘30 K superfamily’. Such a genome structure is conserved between CTLV and apple stem grooving capillovirus. Capped transcripts from a plasmid containing the complete sequence of CTLV, with a T7 RNA promoter, successfully infected Chenopodium quinoa plants and caused symptoms characteristic of CTLV. Uncapped transcripts were non-infectious.

The smallest defective interfering RNA (DI-2) of cymbidium ringspot tombusvirus (CyRSV) was used to identify the cis-acting sequences necessary for its replication by making a series of deletions throughout the 404 nt long molecule and testing the biological activity of mutants. Deletion or substitution of the conserved sequence blocks (A, B and C) always yielded inactive molecules. The deletion of only a few nucleotides could be tolerated beyond the natural deletion sites in blocks A and B. However, either half of block C1 (34 nt) and the first 25 nt of C2 (102 nt) could be deleted without loss of infectivity. It was also demonstrated that either one of the two halves of block C1 was specifically required for replication. We suggest that the last 77 nt of the viral genome and either half of block C1 represent the complementary strand promoter sequence recognized by the viral replicase.