- Volume 79, Issue 4, 1998

Mice deficient in the inducible nitric-oxide synthase (iNOS), constructed by gene-targeting, were significantly more susceptible to herpes simplex virus (HSV)-1 infection, displayed a delayed clearance of virus from the dorsal root ganglia (DRG) and exhibited an increase in the frequency of virus reactivation in DRG compared with similarly infected heterozygous mice. The infected iNOS-deficient mice developed enhanced Th1-type immune re- sponses and their spleen cells produced higher concentrations of IL-12 than similarly infected heterozygous mice. This finding suggests that iNOS plays an important role in resistance against HSV-1 infection. Furthermore, nitric oxide (NO) may block the development of Th1 cells via inhibition of IL-12 synthesis and thereby play a role in immune regulation.

Although intramuscular (i.m.) injection of DNA encoding glycoprotein D (gD) of bovine herpesvirus-1 (BHV-1) induces immune responses in cattle, this route of delivery is inefficient. Here we assessed three parameters that may enhance the efficacy of a gD DNA vaccine in cattle. First, the immune response generated by i.m. injected plasmid expressing a secreted form of gD (tgD) was determined and found to be very similar in magnitude to the response induced by gD-expressing plasmid. Secondly, gD- and tgD-expressing plasmids were administered by intradermal (i.d.) immunization, which resulted in a superior immune response to the secreted form, but no improvement in the response to the membrane-associated form. However, the form of gD used for immunization did not influence the immunoglobulin subtype, the ratio of antigen- specific IgG1 to IgG2 being approximately 4:1.

Finally, the effect of promoter strength was assessed by replacing the Rous sarcoma virus (RSV) promoter, which was used in the original experiments, with the human cytomegalovirus immediate early promoter and first intron A (HCMV/IA). Although upon transfection in vitro the HCMV/IA promoter appeared to be stronger than the RSV promoter, there was only a 2-fold higher antibody response in vivo upon i.d. injection of cattle. Protection against virus challenge was obtained in the calves immunized i.d. with tgD-encoding plasmid, as shown by a significant reduction in weight loss, virus excretion, temperature response and clinical disease. No significant protection was observed in the animals vaccinated i.d. with the gD-expressing plasmid, which correlates with the lower level of immunity pre-challenge.

Two mutant CVI988 Marek’s disease virus (MDV) strains were developed in which a part of ORF L1 was replaced by lacZ with the SV40 early promoter. These mutant strains, CVIL1LacZ-A and -B, were inoculated into chickens to test the hypothesis that ORF L1 is involved in the induction and/or maintenance of latency. Mutant virus could be reisolated from lymphocytes obtained from chickens during both the lytic and latent phase of infection, indi cating that ORF L1 is not essential for the induction and/or maintenance of latency or the reactivation from latency. β-Galactosidase-positive lymphocytes were detected during the latent infection demonstrating that the SV40 early promoter can be active in recombinant MDV strains during latent infection. Although the insertion of lacZ was stable in cell culture, recombination within lacZ and the BamHI-L fragment was observed during in vivo infection.

Glycoprotein M (gM) constitutes one of the rare examples of a nonessential glycoprotein conserved throughout all herpesvirus subfamilies. Whereas gM in wild-type pseudorabies virus (PrV) strains carries an M-glycan, gM of the attenuated strain Bartha is not glycosylated due to a point mutation in the N-glycosylation motif. Since PrV Bartha lacks glycoproteins E and I and carries a mutated gC, we analysed glycosylation of gM in isogenic PrV glycoprotein deletion mutants. Whereas gM was glycosylated normally in most mutants, two independent gC deletion mutants and a gI mutant expressed a nonglycosylated form of gM. DNA sequence analyses revealed the presence of point mutations in the N-glycosylation consensus motif. Surprisingly, mutations in strain Bartha, the two gC- deletion mutants and the gI mutant proved to be different, although all affected the N-glycosylation motif. Thus, our data show that different, apparently independent point mutations cause expression of nonglycosylated gM.

Glycoprotein H/glycoprotein L (gH/gL) complexes of herpesviruses are required for fusion of infecting virions with host cell membranes. In human cytomegalovirus (HCMV), neutralizing monoclonal antibodies (MAb) specific for gH inhibit the transfer of a fluorescent probe to the host cell from labelled virus particles. In similar fashion, in the present study, neutralizing gH-specific MAb inhibited HCMV- induced fusion-from-without in monolayers of both human embryonic fibroblasts and continuous astrocytoma cells (U373). No fusion was detected in cells co-infected with defective recombinant adenovirus vectors that elicited high-level expression of gH and gL, indicating that surface-expressed gH was not intrinsically fusogenic. However, when such cells were superinfected with HCMV that gave fusion- from-without, the resulting cell-to-cell fusion was considerably enhanced. Thus, under our experimental conditions, gH/gL on the cell surface functioned to increase membrane fusion once this was initiated by other components in the virus envelope.

During immunodeficiency after sublethal haemato- ablative treatment, cytomegalovirus (CMV) infection interferes with haematopoietic reconstitution and can cause lethal bone marrow (BM) aplasia. The in vivo model of murine CMV infection has identified the BM stroma as the principal target site of CMV in the haematopoietic cord. The infected cell type is the reticular stromal cell which forms the stromal network and produces essential haemopoietins, such as stem-cell factor (SCF). The expression of SCF was found to be reduced in the infected stroma, but the stromal network was not disrupted and the number of infected stromal cells was too low to explain the functional deficiency. These facts call for a negatively regulating cytokine that is induced by the infection and that potentiates the direct effect of infection by down-regulating haemopoietins in un infected bystander cells. Recent work has suggested that transforming growth factor (TGF)-β1 might be the cytokine involved in CMV-induced BM aplasia. We show here that murine CMV indirectly induces the accumulation of mature TGF-β1 in uninfected renal tubular epithelial cells and TGF-β1 transcription in BM stromal cells, whereas infected renal glomerular and interstitial cells, hepatocytes and BM stromal cells do not coexpress mature TGF-β1. Antiviral CD8 T-cell therapy prevented BM aplasia and also prevented the down-regulation of stromal SCF and interleukin-6 gene expression. Interestingly, however, the CD8 T cells did not preclude the up-regulation of mature TGF-β1, but proved to be inducers of TGF-β1 gene expression in BM stroma. These findings suggest that TGF-β1 is not the mediator of BM aplasia.

Vaccinia virus (VV) produces two antigenically distinct infectious virions, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Structurally, EEV consists of an IMV with an additional outer membrane containing proteins that are absent from IMV. EEV is important for virus dissemination both in vitro and in vivo. Studies of EEV entry have been hampered by having two infectious virions and by the rupture of the EEV outer membrane in the majority of EEV virions during their purification. To overcome these problems, we have developed a novel approach to study VV entry that is based on confocal microscopy and does not require EEV purification. This assay relies on immunofluorescent staining and detection of individual, intracellular, uncoated virus cores. By this method, we show that EEV entry, in contrast to IMV, is dependent on a low- pH pathway and that the IMV enwrapped inside the EEV exhibits a low-pH fusogenic activity. Together with neutralization data demonstrating that exposure to low pH disrupts the EEV outer membrane, this study strongly supports a model for EEV entry which consists of binding, endocytosis, low-pH- induced disruption of the EEV outer membrane and fusion of the exposed IMV with the endosomal membrane releasing the core into the cytosol. The roles of the EEV outer membrane in virus dissemination and virus entry are discussed in relation to this model.

It has been suggested that the beet yellows closterovirus (BYV)-encoded p65 protein, a homologue of HSP70 cell chaperones, plays a role as a virus movement protein (MP). To test this hypothesis, we used two types of complementation experiments with plant viruses containing the triple gene block (TGB) of MP genes. In one, the BYV p65 gene was cloned into a 35S promoter plasmid and introduced into Nicotiana benthamiana plants by microprojectile bombardment along with the 35S promoter- driven GUS gene-tagged cDNA of a transport- deficient potexvirus mutant. Transient expression of p65 complemented the mutant as visualized by the significant increase in the number of cells expressing the GUS reporter gene in the infection foci. In the other test, the p65 gene was inserted into the infectious cDNA of the hordeivirus RNAβ component to replace either the 58 kDa MP gene or the whole TGB. Inoculation of Chenopodium quinoa and Chenopodium amaranticolor plants with the T7 transcripts of the chimeric RNA/, together with the hordeivirus RNAα and RNAγ, caused symptomless infection in inoculated leaves detected by hybridization of the total leaf RNA with a specific cDNA probe. The ability of BYV p65 to substitute for the potexvirus or hordeivirus MPs provides direct evidence for its involvement in the cell-to-cell movement of closterovirus infection.

Transmission of zucchini yellow mosaic virus (ZYMV) by aphids was examined by introducing mutations within the highly conserved proline-threonine-lysine (PTK) motif of the helper component proteinase (HC-Pro) using a cDNA full-length clone. Replacement of proline by alanine (ATK) in the PTK motif abolished transmission almost completely both from plants and from membranes. Substitution of the basic lysine by glutamic acid (PTE) did not reduce the rate of transmission compared with the wild- type. Replacement of threonine by valine (PVK) or serine (PSK) resulted in a rate of transmission that was lower than that of the wild-type. The rate was lower for PSK than for PVK. Western blot comparison did not permit attribution of HC-Pro functionality in transmission to its level in the host. The HC-Pro of strains that effected transmission (with the wild- type PTK motif, and with the mutated PTE and PVK motifs) could also bind in vitro to virions of ZYMV. HC-Pro with a PSK motif, which was less effective in assisting transmission, could bind only weakly to virions, while HC-Pro of the almost non-transmissible strains (with PAK and ATK motifs) did not bind at all. Interestingly, positive binding was recorded for transmission-defective ZYMV-Ct, which has a PTK motif but has glutamic acid instead of lysine in the lysine-leucine-serine-cysteine (KLSC) motif. These findings support the ‘bridge hypothesis’, and confirm the binding of the HC-Pro to the virion. The possible role of the PTK and KLSC motifs in binding to the virus and to the mouthparts of the aphid is discussed.

The genome of satellite tobacco mosaic virus (STMV) adapted to tobacco mosaic virus (TMV), tomato mosaic virus or green tomato atypical mosaic virus consistently had two single base deletions at positions 1 and 61, corresponding to bases A and G, respectively, as compared to the type-strain genome which is naturally adapted to tobacco mild green mosaic virus (TMGMV). Transcript RNAs (STMVtmv) from clone pSTMVTMV which captured the deletions at positions 1 and 61 were infectious when co-inoculated to tobacco plants with either TMV or TMGMV at infection frequencies of > 90%. Two new STMV variants were created to investigate whether both deletions were essential for adaptation to TMV. These were STMVTMGMV ΔA1, which had the A at position 1 (A1) deleted, and STMVTMGMV ΔG61, which lacked G61. STMVTMGMV ΔA1 was infectious (75% frequency) in the presence of either TMV or TMGMV. Virion RNA of STMVTMGMV ΔA1 lost G61 after one infection cycle with TMV. This deletion did not occur in co-infections with TMGMV. STMVTMGMV ΔG61, like the clone STMVTMGMV, was infectious (100% frequency) with TMGMV but TMV did not support this clone. When Nicotiana benthamiana protoplasts were transfected with STMVTMGMV, STMVtmgmv ΔA1 or STMVTMV, STMV replicated when TMGMV was the helper virus. STMVTMVand STMVTMGMV ΔA1 replicated in the presence of helper TMV, but STMVTMGMV did not, the same result as in whole plants. The deletion of A1 is thus essential for initial STMV adaptation to TMV and the eventual deletion of G61 is a predicted additional change.

Complete DNA-A sequences of nine Pakistani geminivirus isolates from leaf curl-affected cotton (CLCuV-PK) or from okra, and the partial sequences of several additional isolates were determined. Sequences of isolates from cotton were of four types. Isolates from leaf curl-affected okra had virtually the same sequences as those from cotton. Isolates from yellow vein mosaic-affected okra were of two types (OYVMV types 201 and 301), both distinct from but closely related to the virus isolates from cotton. Of these six types, two types of CLCuV- PK are the most closely related but another (CLCuV- PK type 72b) is the most distinct. Of the encoded proteins, coat protein (CP) is the most strongly conserved (92–100%aminoacid sequence identity), and AC4 protein the most variable (41–87%).

The 5′ and 3′ halves of the intergenic region of some isolates had different affinities and occurred in seven combinations, suggesting that recombination had occurred and that the origin of replication was a favoured recombination site. Similarly, the first 1520 nt of CLCuV-PK type 804a DNA resembled those of OYVMV type 301 DNA but the remaining 1224 nt were very different. The AC1 (Rep) gene and 5′ part of the intergenic region of CLCuV-PK type 72b closely resembled those of OYVMV type 301, whereas the rest of the sequence did not. The cotton leaf curl epidemic in Pakistan is caused by several distinct variants, with recombination events involving OYVMV and other unspecified gemini- viruses having probably been involved in their evolution.

Tubular structures containing bacilliform virions were observed in cell-free extracts of Commelina diffusa infected with Commelina yellow mottle badnavirus (CoYMV). The exterior of the tubule reacted with antibodies to CoYMV movement protein, but not with antibodies to virus coat protein. Similar tubular structures containing bacilliform particles were also observed in ultrathin sections of CoYMV- infected C. diffusa. These tubular structures traversed the cell wall at points where this was thickened or protruded. No similar structures were observed in healthy C. diffusa. These observations support the hypothesis that the virion-containing tubular structures observed in cell-free extracts are the same as those observed in situ, that these structures are composed, at least in part, of virus movement protein, and that they play a role in the cell-to-cell trafficking of virions of CoYMV.

Autographa californica nucleopolyhedrovirus (Ac- MNPV) does not replicate in Bombyx mori cells (Bm5, BmN). We have shown previously that when a short DNA sequence within AcMNPV ORF95, which encodes the viral helicase P143, is replaced with the collinear region of B. mori nucleopolyhedrovirus (BmNPV), AcMNPV gains the ability to replicate in Bm5 cells. To determine the mutational events in the p143 gene required to allow AcMNPV replication in B. mori cells, AcMNPV recombinants produced in Sf9 cells were screened in vivo in B. mori larvae, which are more permissive to baculo- virus infection than B. mori cell lines. Eight combinations of mutations were tested and characterization of viral DNA extracted from dead larvae showed that amino acid changes at position 564 and 577 are required to kill B. mori larvae.

Transmissible spongiform encephalopathies are closely linked to the accumulation of a pathological isoform of a host-encoded prion protein (PrPC), designated PrPSc. In an attempt to generate mono- and polyclonal antibodies to ruminant PrP, 32 mice were vaccinated with peptide vaccines which were synthesized according to the amino acid sequence of ovine PrP. By this approach five PrP-reactive polyclonal antisera directed against four different domains of the protein were stimulated. Spleno- cytes of mice which had developed PrP-reactive antibodies were used for the generation of monoclonal antibodies (MAbs). Obtained PrP-specific MAbs were directed to three different domains of ruminant PrP which differed from the three previously described major MAb binding sites in rodent PrP. MAbs exhibited reactivity with non-denatured ruminant PrPC in ELISA and immunoprecipitation and with denatured ovine and bovine PrPSc in immunoblot. Cross-reactivity was observed with PrPC of nine other mammalian species and with pathological PrP preferably of ruminants and weakly with that of hamster and mouse. The generated MAbs will be useful tools for the development of diagnostic tests for BSE and scrapie as well as for pathogenesis studies of these diseases.