- Volume 78, Issue 9, 1997

Vaccination with DNA constructs encoding viral antigens has been shown to induce antiviral immunity in various model hosts. However, relevant natural virus-host systems have so far been analysed to only a very limited extent. To test the efficacy of DNA vaccination in an economically important large animal, pigs were immunized against Aujeszky’s disease, a serious virus infection caused by the alphaherpesvirus pseudorabies virus (PrV), which is characterized by severe central nervous and respiratory symptoms. After vaccination with plasmid vectors containing genes for immunogenic envelope glycoproteins C or D (gC or gD) of PrV under control of the major immediate early promotor of human cytomegalovirus, animals developed serum antibodies which recognized the respective antigen in immunoblot and exhibited neutralizing activity. Animals vaccinated with the gC expression plasmid were fully protected against a lethal challenge with PrV strain 75V19,and showed partial protection against the highly virulent NIA-3 strain. In contrast, protection was not observed after vaccination with the gD plasmid. Three intramuscular or intradermal immunizations with as little as 1 μg of gC plasmid DNA resulted in seroconversion and partial protection against lethal NIA- 3 infection. Specific antibodies were detected until at least 9 months after vaccination. In addition, a cellular immune response specific for gC could be demonstrated in proliferation assays of peripheral mononuclear lymphocytes. Our results thus demonstrate the potency of DNA vaccination for protection of large animals against a lethal virus infection.

Glycoprotein M (gM) is one of the very few nonessential glycoproteins conserved throughout the herpesvirus family. Despite this conservation little is known about its function in virus replication. To test for the importance of gM in vivo in a natural virus-host system, 6-week-old piglets were intranasally infected with a gM mutant of the alphaherpesvirus pseudorabies virus (PrV). Following infection virus excretion from the nasal mucosa was decreased ca. 100-fold compared to wild-type or revertant virus. Clinical signs were limited to transiently elevated temperature. In contrast, animals infected by wild-type or revertant virus exhibited high fever, severe respiratory symptoms and affliction of the central nervous system. Prior infection with gM PrV conferred protection against challenge infection and animals mounted an antibody response against gM after wild-type virus infection. Thus, gM is important for efficient virus replication in vivo and deletion of gM may contribute to development of live attenuated, genetically marked vaccines.

Varicella-zoster virus (VZV) genes 33 and 33·5 are predicted to encode the VZV proteinase and its substrate (theassembly protein) respectively. These genes were expressed in insect cells using recombinant baculovirus and it was confirmed that gene 33 encodes a proteinase capable of autoproteolytic processing at two positions. When VZV gene 33·5 was co-expressed with the VZV proteinase, processing of the VZV33·5 gene product was observed. A polyclonal antiserum to the VZV assembly protein domain highlighted a set of proteins in VZV infected HEL cells identical to those identified in insect cells expressing VZV genes 33 and 33·5. To facilitate further characterization of the VZV proteinase the enzyme was purified by affinity chromatography from an E. coli expression system and in vitro activity was observed.

Acute virus infections can induce immune deficiencies, as shown by immunosuppression to a variety of antigens and mitogens. Previously we observed that live bovine herpesvirus-1 (BHV-1) induced considerable lymphocyte death in culture, suggesting that the virus infected one or more cell populations. Our goal was to identify the cells infected by BHV-1 and the mechanism resulting in cell death. ConA activated cells were cultured with BHV-1 and stained with monoclonal antibodies specific for virus envelope glycoproteins (gB, gC and gD) and lymphocyte surface proteins (CD2, CD4 and CD8) and a molecule associated with γ/δ cells. Two- colour immunofluorescence revealed that virus glycoproteins were preferentially expressed on T lymphocytes of the CD4Mphenotype. Live virus was required for virus glycoprotein expression, and by 48 h considerable loss of CD4 expression was observed. To confirm virus replication, RNA was isolated from cells, reverse transcribed and amplified using primers to a 342 bp region of immediate- early and early genes (IER2.9/ER2.6) or a 392 bp region of an early gene (gD). Immediate-early/early gene products were detected in CD4 T lymphocytes but not in infectious virions. Lymphocyte apoptosis was observed by 7 h post-infection with increasing levels of cell death at 24–48 h after infection. These findings suggest that the loss of proliferating CD4 T cells during infection or vaccination with modified live vaccines provides the opportunity for secondary infections that commonly occur following BHV-1 infection.

A series of mutants of equine herpesvirus-1 (EHV-1) which contain deletions in non-essential genes was previously characterized in a murine intranasal infection model. One mutant, ED71 which was shown to be attenuated in the model, was further characterized by inoculation into pregnant mice. Despite the attenuation previously reported, intranasal inoculation of pregnant mice resulted in premature parturition and the birth of dead or dying foetuses. Furthermore, mice inoculated before pregnancy with the same mutant, and subsequently challenged 14 days after conception with wild-type virus, were not protected from abortion.

We have identified the human herpesvirus-6 variant B (HHV-6B)-specific neutralizing epitope on glycoprotein H (gH) which is recognized by monoclonal antibody (MAb) OHV3, with complement-independent neutralizing activity. HHV-6 gHs from HHV-6A (strain U1102) and HHV-6B (strain HST) were expressed in a T7-vaccinia virus transient expression system. OHV3 reacted with HST gH, but not with U1102 gH, in an immunoprecipitation assay and an indirect immunofluorescence assay. In addition, OHV3 reacted with chimeric gHs, formed between U1102 gH and HST gH, containing amino acids 272 to 422 of HST gH. Sequence comparison between U1102 and HST showed seven amino acid differences in this region. Site-specific mutations were introduced into these positions and then reactivity against OHV3 was investigated. The arginine at position 389 of HST gH was shown to be a determinant of the HHV-6B-specific reactivity of OHV3.

Epstein-Barr virus (EBV) glycoprotein gp110 has substantial structural and sequence homology with herpes simplex virus (HSV) gB and gBs of other alpha- and betaherpesviruses but unlike HSV gB localizes differently in infected cells and is absent from virions. To facilitate the analysis of EBV gp110, antisera were raised to fragments of gp110 expressed in a bacterial system. They recognized a protein of the predicted size in recombinant bacterial lysates, in lymphoblastoid cells and in recombinant vaccinia virus-gp110 infected cells. gp110 from all sources possessed a high-mannose type of N-glycosylation implying that gp110 has not passed through the Golgi. Immunofluorescence and immuno-electron microscopy confirmed this conclusion and demonstrated that, in contrast to HSV gB, the majority of immunoreactive gp110 was present at the nuclear membrane or endoplasmic reticulum (ER) but not at the cell membrane. Unexpectedly, a truncated version of gp110 lacking the hydrophobic C-terminal region, despite forming dimers analogous to HSV dimers, was transported in a similar manner to full-length gp110. Two chimeric proteins constructed by replacing the N- and C-terminal domains of gp110 with corresponding regions of gp340/220 were also transported to the nuclear membrane/ER. These data suggest that unlike HSV gB both the N- and C-terminal portions of EBV gp110 contain independent signals sufficient to direct the molecule to the ER/nuclear membrane. Specific transport of gammaherpesvirus gB homologues to the nuclear membrane, from where herpesviruses bud, suggests that they may be involved in the egress of virus from the nucleus.

Mature lymphomas produced in Rhode Island Red (RIR) chickens infected with the RB1B strain of Marek’s disease virus (MDV) were examined for the presence of viral DNA and RNA and expression of viral antigens. In situ hybridization showed that all tumours examined contained viral DNA in areas of lymphoid infiltration. In 3/5 tumours, there was a correlation between the number and distribution of cells expressing the Marek’s disease EcoRI-Q gene (meq) and those that carried the lymphoid cell marker AV37. Expression of the MDV-specific phos- phoprotein pp38 was infrequent in lymphomas but abundant in a splenic tumour which also expressed the viral glycoprotein gB. Northern blot analysis of lymphocyte fractions purified by immunoaffinity showed that CD4 andAV37 fractions from lymphomas expressed meq and the small RNA antisense to ICP4 (SAR). The results are consistent with the notion that transformed cells are CD4Mcells, carrying the AV37 marker and expressing meq and SAR.

Sequence variations in the E6/E7 (nt 34–880) and the L1 (nt 6584–7035) ORFs, and in the long control region (LCR) (nt 7289–93) of human papillomavirus type 16 (HPV-16) were analysed in five penile carcinoma biopsies obtained from Ugandan patients. Uganda is a country with a high incidence of genital cancers. All five isolates were classified as members of African-1 lineage (Af1) by phylogenetic analysis based on LCR sequences. The E6 gene phylogenetic analysis, however, showed that four isolates fell into a new subclass designated Af1-u. This subclass, characterized by three point mutations located at the 5′ end of the E6 gene with resulting changes in amino acids at positions 10 and 14, is distinguishable from the Af1 class by the absence of synonymous mutations at nt 286 and 289. The nonsynonymous substitution at nt 335 was present in three out of five samples. The E6 Af1 mutation pattern was present in only a single Ugandan HPV-16 isolate. Nucleotide sequence analysis of the E7 and L1 regions did not allow any Af1 subclass identification. The physical state of the viral DNA in these samples was characterized by PCR and Southern blot analysis. Oligonucleotides which enable amplification of the full length E2 region (nt 2734–3872) failed to amplify the target sequence in four out of five samples, suggesting disruption of the E2 ORF and integration of the HPV genome into the human DNA. Southern blot analysis confirmed the virus integration status. Our results contribute to the characterization of the HPV-16 ‘African lineages’ with the identification of the Af1- u subclass; furthermore, this is also the first report showing that in male genital cancers HPV-16 is integrated into the human genome with disruption of the E2 ORF.

A type-specific and neutralizing mouse MAb (V5) against human papillomavirus (HPV) type 16 capsids was found to block the serological reactivity of human sera with the corresponding capsids. Out of 352 human serum samples tested for the presence of IgG against HPV-16, more than 75% of reactive sera were completely blocked by the V5 antibody. Type-specific MAbs against HPV-6, −18 and −33 were also found to block serological reactivity with capsids of the corresponding HPV types for the majority of reactive human sera. The results suggest that most antibodies in human sera that are reactive with intact HPV capsids recognize the same or closely related major antigenic determinant(s).

Identification of in vitro immunogenicT-cell epitopes is important for the design of immunotherapeutics targeted to specific antigenic sites. To identify candidate cytotoxic T-lymphocyte (CTL) epitopes in the protease of human immunodeficiency virus type 1 (HIV-1) strain MN, we synthesized 9-mer and 10- mer peptides containing the HLA-A*0201 binding motif. Binding affinity of the peptides was measured by HLA-A*0201 up-regulation on T2 cells. Peptides with high binding-affinity were tested for their ability to stimulate primary CTLs from healthy HIVnegative blood donors. Peptide-specific CTLs were obtained from five out of six donors by stimulation with a 9-mer (LVGPTPVNI) or a 10-mer (VLVGPTPVNI) peptide derived from a highly conserved amino acid stretch in the C-terminal region of the protease. Addition of peptide-specific CTLs to acutely HIV-infected lymphocytes resulted in inhibition of p24 gag production. In conclusion, a highly conserved HIV protease peptide regularly elicits peptide-specific CTLs. Targeting immune responses against defined epitopes in non-variable regions may be a feasible way to minimize the risk of virus escape from immune surveillance.

The evolution of genomic RNA of human immunodeficiency virus type 1 (HIV-1), subtype A, was studied in three Rwandan mother-child pairs over a period of 12–30 months. In two pairs a homogeneous subtype A V3 sequence population was observed at seroconversion and the virus populations in the children resembled those in the mothers. One of these mother-child pairs was infected with an A/C recombinant virus (Ap17/Cp24). In the third pair, a heterogeneous V3 sequence population was observed in the maternal seroconversion sample but the V3 sequence population in the child’s sample was homogeneous. In each individual the intra- and intersample variation (between the seroconversion and follow-up samples) increased over time in both the V3 region and p179ag . Independent evolution for 1–2 years did not abolish the epidemiological relationship between virus populations in mother and child.

Tat strongly stimulates transcription of the human immunodeficiency type 1 (HIV-1) provirus by interacting with various cellular transcription factors, including TFIID. The results presented in this report indicate that the effect exerted by Tat also involves an interaction with TFIIB. A direct protein-protein interaction between Tat and TFIIB was observed in vitro. Detailed analysis of this interaction showed that the cysteine-rich and core domains of Tat bind to the N-terminal moiety of the general transcription factor. The role of the interaction between Tat and TFIIB in the activation of the entire HIV-1 promoter was analysed. Transfection experiments performed using a reporter construct containing the HIV-1 long terminal repeat fused to a reporter gene showed that overexpression of TFIIB progressively suppressed Tat-induced transcription. This effect was weakened by an increase in the intracellular con centration of Tat. A similar consequence of TFIIB overexpression was observed in a HeLa cell line stably transformed with a construct corresponding to the lacZ gene under the control of the HIV-1 promoter. Mutants of TFIIB which differed in their ability to interact with Tat and to function in basal transcription were analysed. The ability of TFIIB mutants defective for basal transcription to inhibit Tat-induced activity of the HIV-1 promoter depended on their capacity to interact with Tat. Mutants of TFIIB functional for basal transcription, but defective for the interaction with Tat, exhibited a dominant negative effect. From these data we propose a model in which interaction between Tat and both general transcription factors TBP and TFIIB maintains the transcriptional initiation complex in an active configuration.

The genetic diversity of 32 Italian isolates of feline immunodeficiency virus (FIV) was studied. Isolates were obtained from domestic cats living in different areas. Sequence data were obtained from a 308 bp fragment of the p25 region of the gag gene. Phylogenetic relationships among these sequences and previously published sequences were determined. All the Italian isolates could be assigned to subtype B; however, four isolates formed two separate clusters and may represent genetic out- liers. The reliability of classification results was confirmed by repeating the phylogenetic analysis with DNA sequences from the entire gag genes of two isolates and from the surface glycoprotein domain of the env gene of four isolates. It is concluded that the short segment of gag used permits reliable genotyping of FIV isolates. The study also shows that subtype B is largely prevalent in Italy.

The human T cell leukaemia virus type I (HTLV-I) Tax protein is an activator of viral and cellular gene expression. Tax does not bind DNA directly, but does interact with cellular DNA binding proteins. These interactions bring Tax to a specific group of promoters and may help to determine the specificity of Tax transactivation. Previous studies have demonstrated that the activity of Tax, when tethered to a given promoter, is enhanced by the presence of adjacent transcription factor binding sites. To examine the specificity of this augmentation, a series of transcription factor binding sites was tested for the ability to enhance the activity of a Gal-Tax fusion protein. The greatest increase in Gal-Tax activity was observed when an octamer binding site was placed adjacent to the Gal4 binding sites. However, the octamer binding site failed to independently function as a Tax responsive element in the absence of an adjacent Tax-tethering element. Oct-2 was not required for augmentation of Gal-Tax activity as this enhancement was observed in BHK- 21 cells, which lack Oct-2. The ability of octamer binding sites to augment transcription was specific for Gal-Tax, as compared to other transactivators. Taken together, these results demonstrate that the degree of Tax transactivation can be influenced by the elemental composition of the target promoter.

Two major genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) have been described, which correspond to the European and North American isolates. PRRSV nucleocapsid (N) protein has been identified as the most immunodominant viral protein. The N genes from two PRRSV isolates, Olot/91 (European) and Québec 807/94 (North American), were cloned and expressed in: (i) baculovirus under the control of the polyhedrin promoter and (ii) Escherichia coli using the pET3x system. The N protein from both isolates was expressed much more efficiently in E. coli as a fusion protein than in baculovirus. The antigenicity of the protein was similar in both systems and it was recognized by a collection of 48 PRRSV-positive pig sera. The antigenic structure of the PRRSV N protein was investigated using seven monoclonal antibodies (MAbs) and overlapping fragments of the protein expressed in E. coli. Four MAbs recognized two discontinuous epitopes that were present in the partially folded protein, or at least a large fragment comprising the first 78 residues. The other three MAbs revealed the presence of a common antigenic site localized in the central region of the protein (amino acids 50–66). This region is well conserved among different isolates of European and North American origin and is the most hydrophilic region of the protein. However, this epitope, although recognized by the MAbs and many pig sera, is not useful for diagnostic purposes. Moreover, none of the N protein fragments were able to mimic the antigenicity of the entire protein.

Nucleotide sequences of the envelope protein genes of 19 geographically and temporally distinct dengue (DEN)-4 viruses were determined. Nucleic acid sequence comparison revealed that the identity among the DEN-4 viruses was greater than 92%. Similarity among deduced amino acids was between 96 and 100%; in most cases identical amino acid substitutions occurred among viruses from similar geographical regions. Alignment of nucleic acid sequences followed by parsimony analysis generated phylogenetic trees, which indicated that geographically independent evolution of DEN-4 viruses had occurred. DEN-4 viruses were separated into two genetically distinct subtypes (genotypes). Genotype-1 contains viruses from the Philippines, Thailand and Sri Lanka; genotype-2 consists of viruses from Indonesia, Tahiti, the Caribbean Islands (Puerto Rico, Dominica) and Central and South America.

The complete nucleotide sequences of the genomes of dengue-1 virus virulent 45AZ5 PDK-0 and attenuated vaccine candidate strain 45AZ5 PDK-27 have been determined and compared with the dengue-1 virus Western Pacific (West Pac) 74 parent strain from which 45AZ5 PDK-0 was derived. Twenty-five (0·23%) nucleotide and 10 (0·29%) amino acid substitutions occurred between parent strain dengue-1 virus West Pac 74 and virulent strain 45AZ5 PDK-0, which was derived from the parent by serial passage in diploid foetal rhesus lung (FRhL-2) and mutagenized with 5-azacytidine.These substitutions were preserved in the 45AZ5 PDK-27 vaccine. 45AZ5 PDK-0 and PDK-27 strains, which differ by 27 passages in primary dog kidney (PDK) cells, show 25 (0·23%) nucleotide and 11 (0·32%) amino acid divergences. These comparative studies suggest that the changes which occurred between the West Pac 74 and 45AZ5 PDK-0 strains may alter the biological properties of the virus but may not be important for attenuation. Important nucleotide base changes responsible for attenuation accumulated between 45AZ5 PDK-0 and 27.

We compared the sequence of an envelope protein gene fragment from 21 temporally distinct West Nile (WN) virus strains, isolated in nine African countries and in France. Alignment of nucleotide sequences defined two groups of viruses which diverged by up to 29%. The first group of subtypes is composed of nine WN strains from France and Africa. The Austral-Asian Kunjin virus was classified as a WN subtype in this first group. The second group includes 12 WN strains from Africa and Madagascar. Four strains harboured a 12 nucleotide in-frame deletion. The loss of the corresponding four amino acids resulted in the loss of the potential glycosylation site present in several WN strains. The distribution of virus subtypes into two lineages did not correlate with host preference or geographical origin. The isolation of closely related subtypes in distant countries is consistent with WN viruses being disseminated by migrating birds.