- Volume 71, Issue 1, 1990

Eight monoclonal antibodies (MAbs) prepared against the flaviviruses Saint Louis encephalitis, dengue 2 and dengue 3 viruses all recognized epitopes on the envelope protein of the prototype flavivirus, yellow fever (YF) virus. Three of these MAbs with flavivirus group-common specificity and two MAbs with a flavivirus-subgroup specificity were found to distinguish wild-type YF viruses from YF 17D-204 vaccine virus, but not from the closely related 17DD vaccine virus, nor from the French neurotropic vaccine virus. This pattern of reactivity was seen only with viruses grown in Aedes albopictus C6/36 cells and not with viruses grown in vertebrate cells (SW13 and Vero cells), where all five MAbs recognized epitopes on both wild-type and 17D-204 viruses. Examination of adult A. aegypti mosquitoes infected with the same YF viruses as above gave a different pattern of results to those in C6/36 cells. Thus, epitope expression differs between mammalian and arthropod cells and between arthropod cells in vitro and in vivo. Neutralization tests showed that all five MAbs would neutralize wild-type Asibi virus grown in SW13 cells, but not Asibi virus grown in C6/36 cells, nor 17D-204 vaccine virus grown in either cell type. Therefore, it is concluded that when YF virus is grown in mosquito cells, wild-type virus is antigenically and biologically distinct from the 17D- 204 vaccine virus.

The coding region of the gene for the nucleocapsid protein of Lassa virus has been inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) using the transfer vector pAcYMl, so that expression of the foreign DNA is under the control of the promoter of the AcNPV polyhedrin gene. Infection of cultured Spodoptera frugiperda cells with recombinant virus resulted in the synthesis of high levels of a protein that was indistinguishable from the authentic Lassa virus protein by SDS gel electrophoresis and immunoblotting with a variety of specific immune sera and monoclonal antibodies (MAbs). The kinetics of appearance of the protein were comparable to those of polyhedrin production in wild-type AcNPV-infected cells. The recombinant material was antigenic when used in ELISA for Lassa virus-specific antibodies, reacting well with MAbs specific for the nucleocapsid protein and with sera from experimentally infected guinea-pigs. The recombinant ELISA was able to clearly distinguish sera from human cases of Lassa fever against a panel of known negative sera of African origin. Recombinant-infected insect cells were an effective substitute for mammalian cells infected with Lassa virus itself in the immunofluorescence assay for Lassa virus-specific antibodies. This system offers attractive alternatives to the use of Lassa virus-infected materials as reagents in diagnostic procedures.

Six temperature-sensitive mutants of vesicular stomatitis virus (VSV) were isolated from the central nervous system (CNS) of athymic nude mice. The nude mice had been reconstituted with syngeneic T lymphocytes and then infected with a temperature-sensitive mutant of VSV, tsG31-KS5 VSV, for 20 days. In BHK-21 cells incubated at 38 °C, the normal body temperature of mice, all six CNS virus clones had diminished RNA synthesis, when compared to RNA production in BHK-21 cells incubated at 31 °C. In contrast, the original tsG31-KS5 VSV mutant synthesized more RNA at 38 °C than it did at 31 °C. In vitro transcription assays were exploited to discern which viral protein(s) was functionally accountable for the abated synthesis of RNA of the CNS VSV isolates. The ribonucleoprotein complexes from the CNS VSV isolates were disrupted and template (N protein and RNA) and enzyme (L and NS proteins) fractions were purified. In vitro transcription assays were performed with template fractions of the brain isolates, added to enzyme fractions either wild-type wt VSV or tsG31-KS5 VSV, or with template fractions of wt VSV or tsG31-KS5 VSV mixed with enzyme fractions of the CNS isolates. The template fraction was responsible for the decrease in RNA synthesis in all six of the brain-isolated clones. When the template fractions of wt or tsG31-KS5 VSV were mixed with enzyme fractions of all the CNS-derived VSV, except BP5A VSV, leader sequence RNA and large Mr transcripts were transcribed. One clone, BP5A VSV, did not synthesize RNA when mixed with either template or enzyme of wt VSV, and probably had more than one functional mutation that influenced viral RNA synthesis.

The primary multiplication site of VVTGgRAB, a recombinant vaccinia virus (VV) expressing the rabies virus G glycoprotein, was studied in comparison with that of the parental VV Copenhagen strain, after oral administration to foxes. Foxes were fed with 108 TCID50 of either VVTGgRAB or VV and were sacrificed 12, 24, 48 or 96 h after inoculation. Both viruses were detected by viral isolation in the tonsils during the first 48 h after inoculation at titres between 102 and 104·3 TCID50/ml. Indirect immunofluorescence confirmed the presence of the virus in tonsils of some of the foxes. The polymerase chain reaction allowed the detection of VVTGgRAB in the tonsils of both of two foxes tested after 24 h, three of three foxes after 48 h, in the buccal mucosa of one of two foxes tested after 24 h and two of three foxes after 48 h and in the soft palate of one of two foxes tested after 24 h and one of three foxes after 48 h. VV was detected in the tonsils of one fox tested after 48 h, in the buccal mucosa of another fox tested after 24 h, and in the first fox after 48 h by the same reaction. Foxes were inoculated with virus isolated from fox tonsils 24 h after oral administration (with or without cell culture amplification) to perform back passages. No virus could be isolated in either case after this passage. The innocuity of VVTGgRAB was also demonstrated when foxes were inoculated with passaged virus.

In order to begin to elucidate the genomic basis of the attenuation of mouse-adapted, poliovirus type 2 strain W-2 (PV2/W-2), we have cloned and sequenced the virus and compared it with the virulent, mouse-adapted PV2/Lansing strain. In addition, we have performed computer-generated comparisons of PV2/W-2 to the non-mouse-adapted, attenuated PV2/Sabin strain to determine whether mutational patterns occur that result in attenuation. The PV2/W-2 genome is 7434 nucleotides in length, which is three bases shorter than PV2/Lansing. The 5′ non-coding region of PV2/W-2 is 747 nucleotides in length (compared to 744 in PV2/Lansing) and shares 98·8% identity with PV2/Lansing and 82·3% identity with PV2/Sabin. Overall, the PV2/W-2 polyprotein (2205 amino acids) is two amino acids shorter than that of either PV2/Lansing or PV2/Sabin (2207 amino acids). These contiguous deletions fall within the P3-D region (polymerase). Within these 2205 amino acid residues only 26 differences were observed between PV2/W-2 and PV2/Lansing (98-8% identity), whereas 92 occurred between PV2/W-2 and PV2/Sabin (95·8% identity). The 3′ non-coding region of PV2/W-2 is 72 nucleotides in length and shares 100% identity with PV2/Lansing and 98-6% identity with PV2/Sabin. Amino acid changes in the capsid protein region occurred in neutralization sites 1 and 3, areas previously shown to be important for pathogenicity. The cleavage site between non-structural proteins P2-C/P3- A consisted of a glutamine-serine pair, in contrast to other sequenced polioviruses which have a glutamine- glycine dipeptide.

The fusion glycoprotein of the Edinburgh strain of respiratory syncytial (RS) virus was cloned from infected cell mRNA. A full-length clone was subjected to sequence analysis, and compared with other strains of RS virus. The inferred primary amino acid sequence was used to generate a nested set of overlapping peptides spanning the mature protein. Peptides were synthesized on polyethylene pins and examined for their reactivity towards high titre human antisera.

Decameric peptides spanning the highly conserved region between amino acids Lys and Ala (positions 470 to 490), reacted strongly with the sera and a detailed study with hexameric peptides located the epitope to FPSDEF, at positions 483 to 488. Replacement synthesis analysis revealed that Pro at position 484 and Glu at 487 were critical components of the binding site and could not be substituted.

The antiviral effects of 6-diazo-5-oxo-l-norleucine (L-DON) on the replication of human parainfluenza virus type 2 (HPIV-2), mumps and vesicular stomatitis viruses were studied. L-DON suppressed growth of these viruses and, in particular, HPIV-2 in four cell types. L-DON was not toxic to the cells at the active dose and did not significantly inhibit cellular macromolecular synthesis. The L-DON-sensitive step of HPIV-2 replication was considered to be relatively early. The NP, P and M proteins were, although at a low level, clearly detectable in HPIV-2-infected Vero cells treated with L-DON, whereas the HN and F proteins were scarcely detected by either immunostaining or immunoprecipitation, indicating that L-DON mainly decreased the amounts of viral glycoproteins. Furthermore, Northern blot hybridization showed that secondary transcription of virus RNA was also inhibited.

The mechanism of neutralization of a type A influenza virus by polyclonal IgM was similar for both tracheal epithelial and BHK cells. Maximum neutralization was only 90% and most (70%) of the virus failed to attach to inoculated cells. The remainder attached to N-acetylneuraminic acid receptors but was not internalized. IgM aggregated virus, but only at an IgM:virus ratio below the level required for neutralization. Failure to detect any loss of infectivity associated with aggregation suggested that aggregates were unstable. Monoclonal polymeric IgA neutralized virus more efficiently on BHK cells (99·9%) than the equivalent amounts of IgM (90%). Otherwise the mechanisms of IgA and IgM neutralization were similar, except that IgA-induced aggregation was coincident with loss of infectivity and may thus have contributed to it. However, IgA-neutralized virus attached to tracheal epithelial cells more efficiently than infectious virus, initially using a neuraminidase-sensitive receptor, but then becoming neuraminidase-resistant. Whether the latter IgA-virus complexes were internalized or attached to a neuraminidase-resistant receptor is not known. This use of differentiated murine cells with murine IgA gave neutralization data that differed qualitatively from those obtained with the same antibody and undifferentiated hamster cells.

Synthetic peptides that mimic antigenic determinants of viral proteins were used in vaccine studies of feline leukaemia virus (FeLV) infection. Immunoreactive epitopes on FeLV gp70 and pl5E were predicted according to the criteria of their terminal position, hydrophilicity and the probability of them constituting helical structures. Nineteen peptides, consisting of seven to 19 amino acid residues, were synthesized, of which two peptides were derived from the FeLV subtype A, 16 from subtype B and one from subtype C. Rabbits were immunized with individual peptides coupled to keyhole limpet haemocyanin and the specificity and biological activities of these hyperimmune sera were determined by ELISA, Western blotting, virus neutralization and cytotoxicity assays. All sera reacted specifically with the immunizing peptide. Twelve of the 19 peptides induced antibodies against purified gp85 and antibodies to 11 peptides reacted with the whole virus. One peptide representing the carboxy terminus of the transmembrane protein pl5E, and two peptides derived from the external glycoprotein gp70 elicited neutralizing antibodies, whereas antisera against four peptides enhanced virus infection in vitro. None of the peptide antisera mediated complement lysis of FeLV-infected cells.

Envelope glycoproteins, gpl20 and gp41, of the human immunodeficiency virus type 1 (HIV-1) elicit immune responses, including virus-neutralizing antibodies, which are expected to play a role in the defence against HIV-1 infection. Subregions of the gpl20/gp41 sequence have immunosuppressive effects or may be implicated in autoimmune responses. Some of the immunodominant epitopes of gpl20/gp41 do not contribute to protective immunity and act as immunological decoys. These circumstances emphasize the need to select from gpl20/gp41 regions inducing protective responses. Towards this goal, 30 peptides covering approximately 87 % of the HIV-1 strain BH10 gpl20/gp41 sequence were synthesized. Antibodies in rabbit and human anti-HIV-1 sera recognized 28 and nine of the peptides, respectively, indicating that most of the gpl20/gp41 sequence is immunogenic and secondly, that the antibody response to HIV-1 is restricted in infected humans. Most of the peptides, without conjugation to carriers, elicited high levels of anti-peptide (endpoints 1: > 104) and anti-gpl20/gp41 (endpoints 1: ≥ 103) antibodies. The highest levels of virus-neutralizing antibodies were elicited by peptide 306 to 338 from a hypervariable loop of gpl20. Additional peptides from the full-length hypervariable loop (303 to 338) of HIV-1 BH10 and from 20 additional HIV-1 isolates were recognized differentially by human anti-HIV, suggesting that success of passive immunization may depend on a match between administered antibodies and the challenging HIV-1 strain, and also that active immunization with selected peptides from a hypervariable region of distinct HIV-1 isolates should be explored further as a method for prophylaxis against infection.

Biological interactions between human cytomegalovirus (HCMV) and the human immunodeficiency virus type 1 (HIV-1) were analysed in transfection and infection experiments, carried out in a human osteogenic sarcoma cell line (HOS) and in the same cell line chronically infected with HCMV (E155). When HOS and El55 cells were transfected with recombinant plasmids containing the HIV long terminal repeat (LTR) linked to the bacterial chloramphenicol acetyl- transferase (CAT) gene, LTR-directed CAT expression was 20 times higher in El55 cells than in HOS cells. HOS cells co-infected with HCMV and HIV-1 showed enhanced production of the HIV-1 p24 antigen. In reciprocal experiments, an increase in HCMV immediate early gene expression was observed when HCMV- infected HOS cells and El55 cells were either transfected with a recombinant plasmid containing the HIV transactivator gene (pTAT), or when infected with HIV-1. DNA hybridization analysis of E155 and HCMV-infected HOS cells revealed higher levels of HCMV DNA in cells transfected with pTAT than in cells transfected with other non-specific recombinant plasmids. El55 cells transfected with pTAT also produced higher titres of infectious HCMV than control cultures of El55 cells transfected with other recombinant plasmids, including pMTAT carrying a mutant tat gene. The functional reciprocity in vitro between HCMV and HIV is discussed with respect to its possible implications for the clinical development of AIDS.

The major immediate early enhancer of human cytomegalovirus (HCMV) is known to exert a strong constitutive transcription stimulation in a broad spectrum of cells. This basal activity can be augmented considerably by elevated levels of intracellular cAMP in a cell type-specific manner. Cyclic AMP induction was observed in several lymphoid cell lines and in HeLa cells. One of the functionally important enhancer sequence modules, the 19 bp repeat element, mediates this effect as a cAMP-responsive element (CRE). It acts more efficiently than the corresponding sequence from the human chorionic gonadotropin gene. It is suggested that protein kinase C is involved in the pathway which leads to the activation of CRE- containing genes in lymphoid cells. Gel retardation assays indicated that similar, but not identical complexes are formed between nuclear protein extracts and the CREs of HCMV and the gonadotropin gene.

We prepared a murine monoclonal antibody reactive to a human cytomegalovirus (HCMV)-induced nuclear protein with an Mr of 68000. Expression of the 68K protein was compared with the major immediate early (IE) 72K protein in various cell types after infection with HCMV or microinjection of plasmid DNA containing the major IE gene. The 68K nuclear protein was detected 2 to 3 h after appearance of the 72K protein in human embryonal lung (HEL) cells infected with HCMV. The 68K protein was distributed throughout the cytoplasm in the late phase of infection, while the 72K protein remained chiefly in the nucleus. The 68K protein was barely detected in the cells under IE conditions by immunoprecipitation, but, together with the 72K protein, it was expressed after microinjection of cloned DNA, containing only the major IE region (region 1), into the nuclei of HEL cells. The 72K protein was expressed in nuclei 2 h after microinjection, whereas the 68K protein was detected 4 to 5 h after the injection. The 68K protein was expressed after microinjection in non-permissive rodent fibroblasts or non-permissive transformed human cells in which these proteins were not expressed after viral infection. Immunoprecipitations after chase-labelling from IE conditions or after partial digestions suggested that the 68K protein is neither a degradation nor a modification product of the major IE 72K protein.

We prepared a murine monoclonal antibody reactive to a human cytomegalovirus (HCMV)-induced nuclear protein with an Mr of 68000. Expression of the 68K protein was compared with the major immediate early (IE) 72K protein in various cell types after infection with HCMV or microinjection of plasmid DNA containing the major IE gene. The 68K nuclear protein was detected 2 to 3 h after appearance of the 72K protein in human embryonal lung (HEL) cells infected with HCMV. The 68K protein was distributed throughout the cytoplasm in the late phase of infection, while the 72K protein remained chiefly in the nucleus. The 68K protein was barely detected in the cells under IE conditions by immunoprecipitation, but, together with the 72K protein, it was expressed after microinjection of cloned DNA, containing only the major IE region (region 1), into the nuclei of HEL cells. The 72K protein was expressed in nuclei 2 h after microinjection, whereas the 68K protein was detected 4 to 5 h after the injection. The 68K protein was expressed after microinjection in non-permissive rodent fibroblasts or non-permissive transformed human cells in which these proteins were not expressed after viral infection. Immunoprecipitations after chase-labelling from IE conditions or after partial digestions suggested that the 68K protein is neither a degradation nor a modification product of the major IE 72K protein.

We prepared a murine monoclonal antibody reactive to a human cytomegalovirus (HCMV)-induced nuclear protein with an Mr of 68000. Expression of the 68K protein was compared with the major immediate early (IE) 72K protein in various cell types after infection with HCMV or microinjection of plasmid DNA containing the major IE gene. The 68K nuclear protein was detected 2 to 3 h after appearance of the 72K protein in human embryonal lung (HEL) cells infected with HCMV. The 68K protein was distributed throughout the cytoplasm in the late phase of infection, while the 72K protein remained chiefly in the nucleus. The 68K protein was barely detected in the cells under IE conditions by immunoprecipitation, but, together with the 72K protein, it was expressed after microinjection of cloned DNA, containing only the major IE region (region 1), into the nuclei of HEL cells. The 72K protein was expressed in nuclei 2 h after microinjection, whereas the 68K protein was detected 4 to 5 h after the injection. The 68K protein was expressed after microinjection in non-permissive rodent fibroblasts or non-permissive transformed human cells in which these proteins were not expressed after viral infection. Immunoprecipitations after chase-labelling from IE conditions or after partial digestions suggested that the 68K protein is neither a degradation nor a modification product of the major IE 72K protein.

We prepared a murine monoclonal antibody reactive to a human cytomegalovirus (HCMV)-induced nuclear protein with an Mr of 68000. Expression of the 68K protein was compared with the major immediate early (IE) 72K protein in various cell types after infection with HCMV or microinjection of plasmid DNA containing the major IE gene. The 68K nuclear protein was detected 2 to 3 h after appearance of the 72K protein in human embryonal lung (HEL) cells infected with HCMV. The 68K protein was distributed throughout the cytoplasm in the late phase of infection, while the 72K protein remained chiefly in the nucleus. The 68K protein was barely detected in the cells under IE conditions by immunoprecipitation, but, together with the 72K protein, it was expressed after microinjection of cloned DNA, containing only the major IE region (region 1), into the nuclei of HEL cells. The 72K protein was expressed in nuclei 2 h after microinjection, whereas the 68K protein was detected 4 to 5 h after the injection. The 68K protein was expressed after microinjection in non-permissive rodent fibroblasts or non-permissive transformed human cells in which these proteins were not expressed after viral infection. Immunoprecipitations after chase-labelling from IE conditions or after partial digestions suggested that the 68K protein is neither a degradation nor a modification product of the major IE 72K protein.

During herpes simplex virus type 1 (HSV-1) latent infection of the mouse trigeminal ganglion there is limited viral gene expression. The latency-associated transcripts (LAT) map approximately to the PstI-MluI fragment within the BamHI B and BamHI E fragments (long repeat regions) of the viral genome. Additional weak hybridization signals have been detected by in situ hybridization that correspond to transcription from HSV-1 DNA fragments adjacent to the PstI-MluI fragment. We mapped the region encoding this additional transcription. This minor latency-associated RNA (m-LAT) was shown to map to a group of contiguous fragments (approximately 8-3 kb of DNA), which are adjacent to the 3′ end of LAT and to a (2-0 kb) fragment adjacent to the 5′ end of the LAT. Using single-stranded probes in in situ hybridization experiments, we showed that the KpnI-BamHI and BamHI-SacI regions of m-LAT are transcribed in a rightward direction within the long internal repeat region. This low abundance RNA may be related to the previously described LAT.

The restriction map of the bovine herpesvirus 4 (BHV- 4) genome (V. Test strain) was established for the restriction enzymes EcoBI, BamHI and HindIII by analysis of clones from a lambda library (Sau3AI partial digestion) and from a plasmid library (EcoRI fragments). One genome unit was defined as the length of the unique central part, flanked at both ends by one of the terminal tandem repeats called polyrepetitive DNA (prDNA) and was estimated to be 113 ± 2 kbp. A restriction map of the prDNA of the V. Test strain showed internal 200 bp tandem repeats of different sequences. This region in the prDNA was highly polymorphic between BHV-4 strains, even in a viral DNA preparation from a plaque-purified strain. The right junction between the repeated and the unique sequence of the genome occurred at an almost constant site, but the left junction contained a modified prDNA and was variable between BHV-4 strains. The unique central part of the genome was very similar in the four strains under consideration, with a few variations due to the presence or absence of a restriction site and four length variations were observed, located at positions 0·006 to 0·034 (left end), 0·211 to 0·225, 0·864 to 0·881 and 0·962 to 0·984 (right end). The total length variation of 1 genome unit does not exceed 1 kbp.

Simian virus 40 (SV40)-based shuttle vectors, containing the SV40 late genes, can be packaged as infectious pseudovirions. In terms of their function as bacterial plasmids, modifications in the overall size of these plasmids can be tolerated within a very wide range, which has allowed us to determine the requirements for SV40 encapsidation, free of the more stringent limitations of SV40 virus. Monkey COS7 cells were transfected with over- and undersized SV40-based shuttle virus plasmids and their progeny have been analysed to follow the stability and evolution of these genomes. Two of the three plasmids analysed undergo recombination, generating molecules with sizes of between 4·0 and to 4·8 kb which were selected after multiple lytic cycles. This size range may correspond to the DNA lengths preferentially packaged in SV40 capsids. The structure of the rearranged plasmids indicates that there is a strong selective pressure for genomes that retain the functions necessary for replication and virus production. Depending on the parent DNA, two main classes of rearrangements were generated: duplications in tandem with the SV40 origin of replication and deletions. Both classes are probably a result of selective size and replicative advantages, which are then biologically amplified during plasmid transmission as virus particles.

The highly restricted host range of JC virus (JCV) has made it difficult to study the biology of this common human papovavirus. To increase our understanding of the tissue specificity of this virus, we have examined the expression of the T antigen (T-Ag) in primary and established cell lines from various tissues of transgenic mice containing the JCV early region. In contrast to earlier results from a simian virus 40-containing transgenic mouse, there was no T-Ag expression in mesenchymal fibroblasts derived from two lines of JCV-transgenic mice. Instead, we isolated T-Ag- positive (T-Ag+) cells that had characteristics consistent with a neural crest origin. Furthermore, primary brain cultures contained many T-Ag+ astrocytes, but no expression was detected in macrophages, epithelial cells, neuronal cells nor, surprisingly, in oligodendrocytes. Continued passage of these cultures resulted in vigorously growing glial fibrillary acidic proteinpositive, T-Ag+ astrocytes. Thus, the strict tissue specificity of JCV expression was maintained, despite the fact that the viral genome pre-existed in every tissue of these transgenic mice and these constraints on expression were preserved even when cells were explanted in vitro.

Human papillomavirus (HPV) types 6 and 11 are usually found in benign genital lesions and laryngeal papillomas. However, the occasional occurrence of their DNAs in carcinomas of the genital tract and larynx suggests that they have some tumorigenic activity. In this paper, we have examined the cotransforming and transactivation activities of the E7 genes from these virus types and show that they cooperate with ras to transform primary cells, but at a greatly reduced level compared to HPV-16 E7.Although the efficiencies of transformation in vitro by HPV-6 and HPV-11 are low, it is striking that the cells that are transformed are highly tumorigenic in vivo in immunocompetent animals. Transactivation studies using the adenovirus E2 promoter demonstrated that both HPV-11 E7 and HPV-16 E7 could stimulate transcription to a similar degree. These results separate the transactivation and co-transforming activities of HPV E7 genes.