- Volume 77, Issue 9, 1996

The gene encoding the muscovy duck parvovirus (DPV)-strain 89384 capsid proteins VP2 and VP3 was cloned in a baculovirus expression system and expressed in insect cells. The recombinant proteins were found to react with specific anti-DPV serum by Western blotting and to be located in the nucleus of insect cells (Sf9) as shown by immunofluorescence. Empty virus-like particles (VLPs) identical in size and appearance to DPV virions were observed by electron microscopy. The antigenicity and immunogenicity of the recombinant proteins were evaluated by ELISA and seroneutralization. Immunization of 3-week-old muscovy ducklings induced anti-DPV antibodies; neutralizing antibody titres were consistent with those observed in ducklings inoculated with a commercial inactivated vaccine. The way to develop these promising results is discussed.

Using the whole body section hybridization technique, we monitored the organ- and age-specific pattern of replication of hamster polyomavirus (HaPV) DNA in a colony of Syrian hamsters, which are susceptible to lymphoma induction. Three phases of viral infection and replication could be distinguished: first, a phase of acute infection characterized by high levels of replication of HaPV DNA in the haemopoietic organs and the liver. This culminated 5 to 7 days post-infection (p.i.); second, at 10 days p.i., a phase of viral clearance became evident; and finally, a third phase reflected both the restriction of HaPV replication in adult hamsters and the accumulation of HaPV DNA at sites of tumour development. A remarkable conformity was observed between the tissue specificity of viral replication and the induced tumour profile: high levels of replication of HaPV DNA were restricted to cells of the haemopoietic system and lymphoid tumours were induced. As shown by in situ hybridization, the viral infection in non-haemopoietic organs was due to the dissemination of HaPV-infected blood cells.

Simian virus 40 (SV40) binding to growth-arrested cells activated an intracellular signalling pathway that induced the up-regulation of the primary response genes c-myc, c-jun and c-sis within 30 min and of JE within 90 min. The up-regulation of the primary response genes occurred in the presence of cycloheximide and when UV-inactivated SV40 was adsorbed to cells. SV40 binding did not activate Raf or mitogen-activated protein kinase (MAP/ERK1), or mobilize intracellular Ca2+. The SV40-induced up-regulation of c-myc and c-jun was blocked by the tyrosine kinase inhibitor, genistein, and by the protein kinase C (PKC) inhibitor, calphostin C, but not by expression of the MAP kinase-specific phosphatase, MKP-1. These results suggest that the SV40-induced signalling pathway includes the activities of a tyrosine kinase and a Ca2+-independent isoform of PKC, but not of Raf or MAP kinase. Finally, SV40 infectious entry into cells was specifically and reversibly blocked by genistein.

T cell proliferative responses against human papillomavirus type 16 (HPV-16) E7 protein were studied in relation to HPV status over time in 51 women originally diagnosed with abnormal cervical cytology and participating in a follow-up study. HPV-16-positive patients were grouped as having either a persistent, a cleared or a fluctuating HPV-16 infection as determined by PCR in consecutive cervical smears up until the moment of testing. Positive proliferative responses against HPV-16 E7 were found in 15/26 patients with a persistent, cleared or fluctuating HPV-16 infection (57.7%). In contrast, 0/15 patients who had been typed HPV-negative during follow-up showed positive responses (P = 0.0005). Further analysis showed positive responses to be more frequent in patients with persistent HPV-16 infections and stable or progressing cervical lesions (8/9 patients reactive, 88.9%) as compared to patients with cleared or fluctuating HPV-16 infections and stable or regressing cervical lesions (7/17, 41.1%, P = 0.04). The relatively strong T cell proliferative responses against HPV-16 E7 observed in patients with a persistent HPV-16 infection and progressive cervical lesions indicate that the effectivity of such responses cannot be predicted and apparently depends on additional factors.

The regulation of human papillomavirus (HPV) late gene expression is difficult to analyse because the late proteins L1 and L2 are only produced in the upper layers of terminally differentiated keratinocytes. However, for the minor capsid protein L2 of HPV types 1, 6, 11 and 16, rare mRNAs or cDNAs starting 3′ of the E5 open reading frame (ORF) were previously described. In order to analyse whether the DNA region preceding the late ORFs (late upstream region, LUR) of HPV-16 and HPV-18 has promoter activity, transient transfection assays employing luciferase reporter constructs were performed. The results show that the LUR of HPV-16 and HPV-18 exhibits an orientation-dependent promoter activity in different cells. By analysing 3′-deletion mutants of the HPV-16 LUR, we identified 78 bp within the sequence between the E5 and L2 ORFs to be critical for the promoter activity. Furthermore, the analysis of a 5′-deletion mutant revealed a negative cis-regulatory element located within the E2 ORF. The HPV-16 early poly(A) signal is located downstream of the critical promoter region. Inactivation of this element by site-directed mutagenesis strongly enhanced luciferase activity. However, mutation of two potential TATA-binding protein (TBP) sites located within the critical promoter region did not abolish the activity. Altogether, these data indicate the possibility of a TATA-less promoter in the HPV-16 and HPV-18 LURs. Together with the early poly(A) signal, this potential promoter might be involved in the differentiation-dependent regulation of late gene expression.

The cysteine protease encoded by adenovirus type 2 contains eight cysteines, some of which are involved in catalysis and enzyme activation. Here we investigated the effects of oxidation, mercaptoethanol, dithiothreitol, diamide and protein disulphide isomerase on wild-type and mutant enzymes. Three isoforms of the enzyme were detected in infected cells and a fourth in preparations of purified recombinant enzyme. The latter isoform was absent in preparations of enzyme mutated at any of the three conserved cysteines, C-104, C-122 and C-126. Enzyme activity could be stimulated by agents other than the authentic activating peptide (pVIc), such as cysteamine, though less efficiently. Diamide at low concentrations stimulated the activity of the ts1 enzyme, but inhibited both ts1 and wild-type enzyme at higher concentrations. Protein disulphide isomerase failed to restore enzyme activity to the oxidized isoform. The present studies incombination with previous results using mutants appeared to rule out amino acids C-67, C-122, C-126 and C-127, leaving the two remaining semi-conserved C-17 and C-40 and the conserved C-104 as potential candidates for binding peptide pVIc.

Induction of programmed cell death has been described during infection with many different viruses. We have investigated the influence of African swine fever virus (ASFV) on apoptosis of different cell populations during in vitro and in vivo infection. We observed apoptosis in ASFV-infected monocyte/macrophage and peripheral blood mononuclear cell cultures. Apoptosis was demonstrated in these cells by DNA fragmentation, DNA staining and DNA-associated histone fraction detection assays. Flow cytometry analysis of infected cultures also showed morphological and functional alterations, including changes in the cell cycle and percentage of cell fractions stained with propidium iodide. After in vivo infection with three different virulent strains of ASFV, apoptosis of infected cells from the mononuclear phagocytic system and closely related elements from different tissues was observed. Additionally, infected pigs showed an intense degree of apoptosis of lymphocytes, which are not infected by the virus. In lymph nodes and other lymphoid organs, broad bands of apoptotic cells presented typical nuclear changes under light microscopy. The occurrence of DNA fragmentation was confirmed in these tissues using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling. These findings, together with the pathological observations in infected pigs of a depletion in cell populations in lymphoid organs, suggest that virus interference with programmed cell death plays a central role in pathogenesis of this disease, being responsible for lymphoid organ impairment in acute ASFV infection.

We have cloned and sequenced the Kpnl L and M restriction fragments of infectious laryngotracheitis virus (ILTV, gallid herpesvirus 1) DNA, which are localized adjacently at the right end of the unique long region of the genome. Within the 6930 bp DNA sequence six complete open reading frames (ORFs) were identified. The predicted amino acid sequences of four of them exhibit significant homologies to the UL5 (helicase), UL4, UL3 and UL2 (uracil-DNA glycosylase) genes, which are conserved in similar arrangement in all alpha-herpesvirus genomes characterized up to now. A short ORF of 72 codons between UL3 and UL4 of ILTV is positionally homologous to the UL3.5 gene present in the genomes of different members of the Varicellovirus genus of alphaherpesviruses, but not in herpes simplex virus. The predicted ILTV protein encoded upstream of UL2 possesses limited sequence homology to the UL1 gene product of herpesviruses, the structural glycoprotein L. The presence of a N-terminal signal sequence, a conserved N-glycosylation site and two conserved cysteine residues indicates a similar function of the putative ILTV protein. Upstream of the UL1 gene of ILTV the C-terminal part of an additional ORF designated as ULO was identified, which exhibits no significant homology to known herpesvirus genes. RNA analyses indicate the expression of all detected ILTV genes including ULO.

The open reading frames encoding bovine interleukin 2 (boIL-2) and bovine interleukin 4 (boIL-4) were integrated into the unique short segment of the genome of bovine herpesvirus 1 (BHV-1) and expressed under control of the murine cytomegalovirus (MCMV) immediate-early 1 (ie1) enhancer-promoter element or the MCMV early 1 (e1) promoter. Madin-Darby bovine kidney cells infected with the recombinant viruses secreted boIL-2 or boIL-4 into the culture medium. Secretion was inhibited by the presence of brefeldin A during the infection, indicating that export from the cells was dependent on a functional Golgi apparatus. Treatment of the secreted interleukins with N-glycosidase F reduced the apparent molecular mass of recombinant BHV-1-expressed boIL-2 from 22 kDa to 16 kDa and that of boIL-4 from 20 kDa to 13 kDa, which demonstrated that both cytokines contain N-linked oligosaccharides. Digestion with neuraminidase and O-glycosidase had no detectable effect on the apparent molecular masses, suggesting that BHV-1-expressed boIL-2 and boIL-4 are not, or only slightly, O-glycosylated. In vitro experiments demonstrated the biological activity of recombinant BHV-1-expressed boIL-2 and boIL-4 by their ability to maintain the proliferation of bovine 4325 T cells and activated bovine B cells, respectively. In conclusion, we show that boIL-2 and boIL-4 are secreted from recombinant BHV-1-infected cells as biologically active glycoproteins.

We have developed a panel of 14 monoclonal antibodies (MAbs) to POL, the catalytic subunit of herpes simplex virus type 1 (HSV-1) DNA polymerase encoded by gene UL30, and one MAb to the UL52 protein, another of the seven proteins essential for replication of HSV DNA. The approximate locations of the epitopes of the polymerase-specific MAbs were identified using truncated polymerase molecules, and the antibodies were characterized in a number of immunological assays allowing eight different specificities to be recognized. These MAbs, together with a polyclonal antibody raised in rabbits against a third DNA replication protein, ICP8, were used to localize the respective proteins by immunofluorescence in cells infected with wild-type HSV-1 or the DNA replication-defective mutants ambUL8 or 2-2. In BHK cells infected with ambUL8, a mutant with an amber termination codon within the coding region of gene UL8, the UL52 protein did not enter the nucleus, although ICP8 and POL entered the nucleus in a normal fashion. The failure of the UL52 protein to be correctly transported to the nucleus was also observed in both HFL and Vero cells infected with ambUL8. In contrast, UL52 protein was transported to the nucleus in BHK cells infected with wild-type HSV-1 or with 2-2, a mutant lacking a functional UL9 protein.

Herpes simplex virus type 1 (HSV-1) capsid assembly takes place in the nucleus of infected cells. However, when each of the outer capsid shell proteins, VP5, VP23 and VP26, is expressed in the absence of any other HSV-1 proteins, it does not localize to the nucleus but is distributed throughout the cell. We have previously shown that the HSV-1 capsid scaffolding protein, preVP22a, can relocate VP5 into the nucleus but does not influence the distribution of VP23. We now demonstrate that the outer capsid shell protein, VP19C, is able to relocate both VP5 and VP23 separately into the nucleus. However, nuclear localization of VP26 is only observed when VP5 is present together with either VP19C or preVP22a. Thus, pair-wise interactions involving all of the abundant capsid proteins have now been identified. Electron microscope examination of insect cells coinfected with recombinant baculoviruses expressing VP19C and VP5 reveals the presence of 70 nm diameter ‘capsid-like’ structures, suggesting that these two proteins can form the basic capsid shell.

tsBN2, a temperature-sensitive (ts) growth mutant of the hamster cell line BHK-21, has a point mutation in the RCC1 (regulator of chromosome condensation) gene, and prematurely enters mitosis at 39.5°C, a nonpermissive temperature. In this mutant at 39.5°C infectious progeny of herpes simplex virus type 1 (HSV-1) was not produced and replication of HSV-1 DNA was inhibited. HSV-1 DNA from virus particles is normally circularized upon infection, and circularized HSV-1 DNA molecules can serve as template for DNA replication. In tsBN2 at 39.5°C, HSV-1 DNA appeared to remain linear after infection, suggesting the obstruction of HSV-1 DNA circularization, which could account for failure of HSV-1 DNA replication. In transient replication assays performed in tsBN2 at 39.5°C, through superinfection with HSV-1 helper virus, there was no evidence of replication of circular DNA of the hybrid plasmid containing the HSV-1 replication origin. Production of mRNAs of HSV-1 early genes required for HSV-1 DNA replication was decreased in tsBN2 at 39.5°C. Therefore, RCC1 was assumed to be involved in the formation of an HSV-1 DNA configuration suitable for replication (that is circularization) and the supply of proteins required for replication of the circularized HSV-1 DNAs.

A quantitative competitive PCR assay for human herpesvirus 6 (HHV-6) was developed. Firstly, viral burden was determined in the blood of 25 healthy persons. Using 1 µg of DNA, the prevalence of HHV-6 was 36% (9/25). Eight persons had viral loads of ⩽ 32 HHV-6 genomes/µg DNA. The viral burden in the ninth individual was 1.2 × 106 HHV-6 genome copies/µg DNA, which remained constant over a period of 10 months. This demonstrates the persistence of a high HHV-6 load in the absence of apparent disease. Secondly, HHV-6 burden was determined in 100 post-mortem tissues from seven AIDS patients and three controls. For all tissues combined, there was a statistically significant higher median viral load in AIDS patients (56 copies/µg DNA, range 0–43 321) compared to controls (10 copies/µg DNA, range 0–423) (P = 0.04). The precision and reproducibility of this assay will allow hypotheses concerning the pathogenic potential of HHV-6 to be tested quantitatively.

Glycoprotein H (gH) of pseudorabies virus (PrV) is a structural component of the virion and forms a complex with another glycoprotein, gL. For a detailed analysis of the function of PrV gH, we isolated a gH-deficient mutant on trans-complementing gH-expressing cells after insertion of a β-galactosidase expression cassette into a partially deleted gH gene. The absence of gH did not affect primary or secondary attachment of PrV but the mutant was not infectious. The defect in infectivity could partially be overcome by experimentally induced membrane fusion using PEG, which suggests that gH was necessary for fusion between virion and cellular membranes. After intranasal inoculation into mice, the LD50 of complemented gH− PrV was more than four orders of magnitude higher than that of wild-type PrV. Infection of the respiratory epithelium was much less efficient with complemented gH− PrV as compared with rescued PrV, reflecting the lack of direct cell-to-cell spread. Complemented gH− PrV was able to penetrate into a few trigeminal and sympathetic first order neurons accessible from the nasal cavity, whereas transneuronal transfer in the second order neurons was not observed. In summary, gH is essential for entry and cell-to-cell spread in cell culture, and for propagation in the nervous system of mice. This substantiates the hypothesis that transneuronal spread in vivo and direct cell-to-cell spread in cell culture are governed by similar mechanisms.

Human cytomegalovirus (HCMV) genes expressed in the early phase of infection mediate the destabilization of nascent major histocompatibility complex (MHC) class I molecules in infected cells and thus prevent antigen presentation to CD8+ T lymphocytes. We report that HCMV genes interfere with the MHC class I pathway of antigen presentation by at least two mechanisms. Firstly, permissive infection of fibroblasts is characterized by a continuous decline in the capacity to translocate peptides from the cytosol into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). Inactivation of peptide transport is operative despite augmented TAP expression during HCMV infection. Secondly, TAP molecules fail to associate with MHC class I heavy chains indicating that HCMV early gene expression also interferes with MHC class I maturation. A temperature-sensitive mutant of HCMV, ts9, which lacks 15 kb of DNA encoding the genes US1–US15 of HCMV, had lost the capacity to interfere with MHC class I assembly and to inhibit the peptide translocation function of TAP. One of the genes deleted in ts9, US11, which was reported to downregulate the expression of MHC class I molecules, does not affect peptide transport by TAP. Therefore, we conclude that HCMV encodes at least two early gene functions that interfere with the MHC class I antigen presentation pathway.

A novel fusion assay was established to determine fusion activity with cocultivated human foreskin fibroblasts of stable transfectants derived from human astrocytoma cells (U373) expressing authentic or mutagenized human cytomegalovirus glycoprotein B (HCMV gB; gpUL55). Compared to transfectants expressing authentic HCMV gB, those expressing gB forms with a deletion of hydrophobic domain 1 (hd1; aa 714–747) or with deletions of specific segments in the cytoplasmic tail (aa 811–825 and 871–906) exhibited significantly reduced heterologous fusogenicity. HCMV gB-specific monoclonal antibodies (MAbs) as well as MAb against cellular annexin II prevented fusion of the transfectant expressing authentic gB. Comparable surface exposure of HCMV gB or its derivatives was demonstrated in all transfectants by FACS analysis. Our observations are compatible with the notion that indigenous fusion activity of HCMV gB depends on the extracellular hd1 domain and on the conformation of the cytoplasmic tail.

The nucleotide sequence of a 1200 bp DNA fragment of Spodoptera exigua nucleopolyhedrovirus (SeMNPV) was determined. This sequence contained a cluster of two open reading frames (ORFs), one coding for a viral ubiquitin (v-ubi) and another with homology to orf2 of Autographa californica (Ac) MNPV and Orgyia pseudotsugata (Op) MNPV. The v-ubi ORF is 240 nucleotides (nt) long, potentially encoding a protein of 80 amino acids with a predicted molecular mass of 9.4 kDa. The amino acid sequence of the v-ubi gene in SeMNPV has 75% and 81.6% identity with the v-ubi gene of AcMNPV and OpMNPV and approximately 84% with cellular ubiquitins. Northern blot analysis revealed three major small transcripts late in infection, of about 690, 550 and 400 nt long. Primer extension analysis showed that transcription started from within two consensus late promoter elements (TAAG), located at positions -6 and -30. The start site at position -4/-5 precedes the shortest leader eported to date for a baculovirus gene. The other ORF, xb187, was identified in the opposite orientation immediately upstream of the v-ubi gene. This ORF potentially encodes a 22 kDa protein with unknown function and about 60% amino acid similarity to the products of the orf2 genes of AcMNPV and OpMNPV. The SeMNPV xb187 ORF is transcribed late in infection via two transcripts, 1.2 kb and 770 nt long. The v-ubi-xb187 gene cluster is located at map unit (m.u.) 89 on the genome of SeMNPV. This is different from the position of an identical cluster in the AcMNPV and OpMNPV genomes, located at relative m.u. 20.

In the present study, a lepidopteran cell line (Ld-652Y, from the gypsy moth, Lymantria dispar) exposed to Hyposoter fugitivus polydnavirus (HfPV) was found to display a variety of cytopathic effects. These included a transient inhibition of cell proliferation, rounding up, aggregation and apoptosis. In addition, unusual paracrystalline structures appeared within the lumen of the rough endoplasmic reticulum; similar structures were observed in the spherulocytes of parasitized Malacosoma disstria. Following Coomassie Blue staining, two new cell-associated polypeptides were detected; one of these, an 8 kDa polypeptide, could also be observed following exposure of Ld-652Y cells to media taken from infected cultures or to cell-free haemolymph from parasitized M. disstria. After a period of 2–4 weeks, the L. dispar cell cultures were observed to largely recover from the effects of exposure to virus, and resumed proliferation; ‘transformed’ cell populations tended to form aggregates, and adhered less tightly to the substrate. Viral DNA was stably maintained in all recovered cell lines, possibly in chromosomally integrated form.