- Volume 72, Issue 1, 1991

Scrapie can be transmitted by novel infectious pathogens termed prions. No evidence for a scrapie-specific nucleic acid has been detected to date. To investigate amounts, types and sizes of nucleic acid molecules associated with prions in purified preparations, aliquots were deproteinized, and the nucleic acids analysed by PAGE and silver staining. Digestion with nucleases and exposure to Zn2+ prior to analysis substantially diminished the content of nucleic acids, but did not alter the prion titre indicating that those nucleic acids which were removed are not essential for infectivity. Since a single species of scrapie-specific nucleic acid could not be identified, we explored the unprecedented possibility of scrapie-specific nucleic acids of variable length which are biologically active. If such molecules of variable length exist then they might be hidden within the background smear on silver-stained gels after PAGE. A new procedure designated return refocusing gel electrophoresis (RRGE) was developed to identify heterogeneous nucleic acids in purified prion fractions. The content of variable length nucleic acids was reduced by a factor of 10 by exhaustive Bal 31 exonuclease digestion after dispersion of purified prions into detergent-lipid-protein complexes. For example, a typical sample after Bal 31 digestion contained approximately 4 ng of nucleic acid of variable length and 108.7 ID50 units of scrapie prion infectivity. Consideration of different models for a hypothetical scrapie-specific nucleic acid suggests that such a molecule would have to be: (i) quite small (< 100 nucleotides), (ii) possess a particle-to-infectivity ratio near unity or (iii) heterogeneous in size. Although our results do not eliminate the possibility that prions possess a scrapie-specific nucleic acid of variable length, they narrow considerably the spectrum of features specifying such a candidate molecule.

Murine monoclonal antibodies (MAbs) gp41-1 (IgG2a) and gp41-2 (IgG1), directed against the envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1), were produced and characterized. These MAbs recognized both gp160 and gp41 and reacted with divergent HIV-1 isolates. Surface binding assays using viable HIV-infected cells indicated that these MAbs were directed against surface-exposed epitopes. Both MAbs caused a reduction in reverse transcriptase activity. Syngeneic monoclonal anti-idiotypic antibodies (anti-ids) against gp41-1 were also generated. Six anti-ids (agp41-11 to agp41-16) were selected by ELISA using F(ab′)2 fragments of gp41-1; no reaction was observed when fragments from an irrelevant IgG2a MAb were used. Anti-ids were recognized by both gp41-1 and gp41-2 biotinylated MAbs. Competitive ELISA studies suggested that anti-ids were directed against at least three distinct idiotopes on gp41-1. All anti-ids reacted with idiotopes associated with both heavy and light chains and not with separated chains. The binding of MAbs gp41-1 and gp41-2 to HIV-infected cells was inhibited by each anti-id, except for the binding of gp41-2 which was not affected by the presence of agp41-12. Immunization of rabbits with agp41-11 and agp41-13 resulted in an antibody response against recombinant gp160. These studies indicated that these two anti-ids contain a surrogate image of the antigen recognized by gp41-1.

The C-terminal region of human immunodeficiency virus (HIV) reverse transcriptase (RT) contains the domain responsible for RNase H activity. To determine the importance of this RNase H domain, specific changes in the C-terminal region of a recombinant RT expressed in Escherichia coli were introduced by amino acid substitutions and specific deletions. The enzyme activities of purified wild-type and mutant RT/RNase H proteins, standardized for protein content, were compared by filter assays and thermal inactivation kinetics. A point mutation of His 539 → Asn produced an enzyme with a marked thermolabile RNase H function (nine-fold increase in inactivation), whereas RT function was only marginally more labile than that of the wild-type (two-fold). A second mutation, His 539 → Asp, impaired both enzyme activities to a similar degree (four- to five-fold). A C-terminal deletion of 19 amino acids (aa) (aa 540 to 558) and a C-terminal truncation of 21 aa (aa 540 to 560) reduced RT as well as RNase H activity. A 130 aa deletion enzyme exhibited no RNase H activity and insufficient RT activity to allow inactivation studies. Two mutants, the 19 aa deletion and His → Asn, were introduced into proviral HIV-1 DNA clones to determine whether changes in enzyme activity, particularly RNase H activity, affected virus infectivity. Both mutants were non-infectious, indicating that the C-terminal 19 to 21 amino acids and His 539 of the RT/RNase H protein are essential for HIV replication. These results are consistent with the assumption that RNase H is essential for the infectivity of HIV-1.

The proteins of bovine ephemeral fever virus (BEFV) were examined in purified virions and in infected BHK-21 cells. Five structural proteins were named L (180K), G (81K), N (52K), M1 (43K) and M2 (29K). The 81 K G protein incorporated [3H]glucosamine, was removed from virions by treatment with Triton X-100 and bound monoclonal antibodies which were both neutralizing and protective. Treatment of virions with Triton X-100 and 0.2 to 1.0 m-NaCl progressively released L, M1 and M2. The N protein remained associated with nucleocapsids in up to 2.5 m-NaCl. The glycoprotein (G), nucleoprotein (N) and matrix protein (M2) were phosphorylated. In BEFV-infected BHK-21 cells, five virus-induced proteins were detected from 12 h post-infection. The L, N, M1 and M2 proteins corresponded to those detected in virions whereas the G protein existed in two forms. In tunicamycin-treated cells these occurred as 67K and 71K non-glycosylated precursors. In the absence of tunicamycin, 77K and 79K glycosylated forms were further modified to produce the 81K virion G protein and a 90K cell-associated form. Five viral proteins were also detected in cells infected with the closely related Berrimah virus; the Berrimah virus G protein was also present in two forms.

The sequence of the fusion (F) glycoprotein of turkey rhinotracheitis virus (TRTV) has been deduced from cDNA clones derived from oligo(dT)-selected infected cell RNA. The protein consists of 538 amino acids, the F2 and F1 subunits containing 102 (including the F2-F1 connecting peptide RRRR) and 436 residues, respectively. Each subunit has one potential N-linked glycosylation site. The protein has 38 to 39% amino acid identity with the F protein of respiratory syncytial virus (Pneumovirus genus) but only about half that with members of the other two genera (Paramyxovirus and Morbillivirus) in the Paramyxoviridae family. This is the first sequence evidence to support the view that TRTV is a pneumovirus, the first avian member of the genus to be described.

The sequences of a region of the nucleocapsid protein gene, between nucleotides 1231 and 1686, encoding the C-terminal 151 amino acid residues of the nucleocapsid protein have been determined for 16 strains of measles virus. Analysis of this region showed that it is highly divergent (up to 7.2% divergence in the nucleotide sequence and 10.6% divergence in the amino acid sequence between most distant strains) and that several lineages of measles virus can be found to co-circulate at a given time. Some of the lineages show geographical restriction. The results for measles virus are similar to those reported for other human paramyxoviruses such as mumps virus, parainfluenza type 3 virus and the avian Newcastle disease virus.

A library of cDNA clones from mRNA of human parainfluenza type 2 virus (PIV2) was constructed and the nucleotide sequence of the fusion (F) glycoprotein gene determined. The F gene boundaries were obtained by primer extension sequencing on F mRNA and on viral genomic RNA. The mRNA coding for the F glycoprotein is composed of 1918 nucleotides. It contains a single large open reading frame that encodes a protein of 551 amino acids with an M r of 59586. The predicted PIV2 F protein contains the cleavage-activation site (amino acids 101 to 106) including two Arg and two Lys residues, where the protein is cleaved by host protease into F1 and F2 subunits by analogy with other paramyxoviruses. Three hydrophobic domains are recognized, the signal peptide (amino acids 1 to 21), that is cleaved off in the mature protein, the fusion peptide (amino acids 107 to 132) at the cleavage-generated N terminus of subunit F1 and the membrane anchorage region (amino acids 486 to 513) near the C terminus of the protein. The predicted F protein has six potential glycosylation sites and 10 of the 12 Cys residues present have conserved positions as compared with those of other paramyxovirus F proteins. Primer extension sequencing on viral RNA gave the 3′ end sequence as UAAAUUCU6 followed by the 5′ end of the haemagglutinin-neuraminidase (HN) gene thus establishing the order of genes coding for the viral glycoproteins of PIV2 as 5′-F-HN-3′.

We have studied changes of epitopes on the haemagglutinin molecule (HA) of H1N1 influenza viruses isolated between 1977 and 1986. For this purpose monoclonal antibodies (MAbs) were raised against the HA of the influenza A/England/333/80 and A/Yamagata/120/86 strain viruses. In order to define the amino acid residues responsible for the change of epitopes, we prepared several HA cDNAs modified by site-directed mutagenesis and cloned them into a simian virus 40 expression vector (SVHA). The substitution of glycine with serine at position 125c (suffix indicates presence in H1 but not in H3 subtype HAs) on the HA of the influenza A/USSR/90/77 strain virus resulted in the loss of epitope 110 (epitopes were named after MAbs) and created new epitopes 139 and 15, which were observed on the HA of A/England/333/80 and a few isolates from 1983. These new epitopes disappeared from the HA in some of the isolates in 1983 and most of the isolates in 1984 and 1986. The disappearance of epitopes 139 and 15 seems to be associated with the loss of epitope W18, which was identified on the HA of A/USSR/90/77. We suggested previously that amino acid residue 189 was involved in epitope W18. We therefore expressed an HA protein with two amino acid substitutions at positions 189 and 125c and found that the conversion of glutamine to lysine at position 189 in SVHA-67 prevented the expression of epitopes 139 and 15.

Monoclonal antibodies against the nucleoprotein (NP) and matrix (M) protein of influenza C virus were prepared and characterized. At least two non-overlapping or partially overlapping antigenic sites were delineated on each of the proteins by competitive binding assays. Western blot analysis showed that two antigenic sites on the M protein were highly resistant to conformational changes whereas two sites on NP were sensitive. No antigenic variation was seen in either the NP or the M protein when the reactivity of monoclonal antibodies with 23 different influenza C strains isolated over a 41 year period was studied by radioimmunoprecipitation and enzyme-linked immunosorbent assay. Immunofluorescence analysis with the monoclonal antibodies revealed that both the NP and M proteins migrated to the cell nucleus during the replication cycle of influenza C virus.

Release of α-amino[14C]isobutyric acid from ferret Mpf cells was promoted by staphylococcal α toxin, diphtheria toxin and streptolysin S. This release was enhanced to a significant extent if the cells had been previously infected with influenza virus strain A/Puerto Rico/8/34 (PR8, H1N1), although infection with virus alone did not increase the release of radiolabel as compared with that from untreated cells; inactivated virus had a similar enhancing action. The mechanism of enhancement is unclear but it occurs between 0.5 and 2 h post-inoculation and viral membrane/endosome membrane fusion is essential. Endotoxin had no effect on membrane permeability, either alone or with PR8. The relevance of these in vitro observations to the previously observed enhancement of toxin lethality by influenza virus in vivo is discussed.

Neutralizing monoclonal antibodies (MAbs) directed to the VP7 protein and neutralization-resistant mutants were used to analyse the antigenic structure of VP7 of human rotavirus serotype 1. Cross-neutralization tests using the MAbs and the resistant mutants indicated the existence of two functionally independent neutralization epitope regions (S1 and S2) on VP7. Region S1 corresponds to a single epitope domain of VP7 which has been detected previously. Two MAbs prepared in this study recognized the S1 region, and the resistant mutants they selected had amino acid substitutions at positions 94 or 213. On the other hand, region S2 is considered to be a novel epitope. Single or double amino acid substitutions were detected in the variable regions (amino acid positions 145, 217 and 221) and in the constant regions (positions 104, 201 and 291) of the VP7 protein of mutants selected by MAbs directed to the S2 region. It was suggested that the variable region E (amino acids 208 to 221) includes two independent neutralization sites, and that amino acid substitutions in the constant region of VP7 also affect serotype-specific neutralization epitopes. Neutralization epitopes on VP7 are considered to be highly dependent on the conformation of VP7.

A recombinant vaccinia virus vector was constructed which expressed the major surface glycoprotein G of human respiratory syncytial virus (RSV) and the thymidine kinase (tk) gene of vaccinia virus. The virulence of this tk+ recombinant virus was compared with that of a tk− recombinant and the wild-type (wt) virus after intranasal inoculation of mice. Respiratory infection with wt virus resulted in a lethal infection with widespread dissemination of virus. In contrast, infection with the tk− recombinant was not lethal and the virus had a reduced ability to disseminate to extrapulmonary tissue compared with wt virus. Insertion of the tk gene restored the virulence of the recombinant virus to the level of that of the wt virus. Despite a dramatic reduction in virulence of the tk− recombinant, virus could occasionally be recovered from the brains of mice. The expression of the attachment glycoprotein of RSV appeared to enhance the ability of the tk− recombinant virus to replicate in the lungs when compared with recombinants expressing fusion or nucleoprotein genes. The results confirm that inactivation of the tk gene results in a dramatic reduction of virulence for mice but suggest that there is still a potential danger of infection of the brain following intranasal administration of virus.

Herpes simplex virus type 1 (HSV-1) subgenomic sequences from 0.743 to 0.782 map units have been molecularly cloned as plasmid AT1 and shown to inhibit stable DNA-mediated gene transformation of Ltk− cells with the HSV-1 thymidine kinase (tk) gene. Here it is shown that AT1 also inhibits transient gene expression. Expression from the chloramphenicol acetyltransferase (CAT) gene under the control of either the HSV-1 tk gene or the Rous sarcoma virus (RSV) promoter was inhibited when cotransfected into Ltk− and CV-1 cells with equimolar amounts of AT1. AT1 was subcloned as three overlapping plasmids called AT1a, α27 and AT1b. The α27 plasmid encodes the HSV-1 immediate early gene, α27; AT1a possesses sequences that specify an open reading frame in HSV-1 strain KOS used in these studies, although the significance of this open reading frame is unknown; AT1b possesses the sequences for UL55 and UL56, also genes for which no function has been reported. No single subclone or pair of subclones demonstrated significant inhibition of transient gene expression. Cotransfection of all three subclones did result in inhibition of RSV-CAT gene expression, suggesting that information from each subclone is necessary. One of the three subclones, α27, contains the HSV-1 immediate early gene, α27, so the possibility that other immediate early genes could substitute for α27 was tested. Inhibition of RSV-CAT gene expression was also achieved by cotransfection of AT1a and AT1b with either an α0- or α4-containing plasmid, suggesting that the role of the α27-containing plasmid can be replaced by other alpha genes with trans-regulating capability. Finally, AT1a and AT1b linker insertion mutants have been constructed and used to study the role these plasmids play in mediating inhibition. These results suggest that AT1 contains HSV-1 functions in addition to that of α27 that interfere with gene expression.

C57BL/6 mice, which differ genetically from other strains by their resistance to herpes simplex virus type 1 (HSV-1) infection, were inoculated intraperitoneally with different doses of tumour necrosis factor α (TNF-α). Mice pretreated with 100 ng, or even 10 ng, of TNF-α showed prolonged survival compared to control mice that were infected with 107 p.f.u. of HSV-1. Significant protection was observed in mice injected 4 or 8 h prior to or after HSV-1 inoculation, respectively. Protection was also observed when mice which differed at their H-2 locus were treated with TNF-α after infection with HSV-1. Interferon could not be detected in the sera of mice at different time points after infection with HSV-1 or injection of TNF-α and there was no enhanced interferon titre in mice treated with both TNF-α and HSV-1, suggesting some interferon-independent protection. However, mice treated with TNF-α showed a marked activation of natural killer (NK) cells compared to untreated control mice or mice that were treated with HSV-1 alone. To test whether enhanced NK cell activity is responsible for TNF-α-induced protection, mice were injected with the NK cell-specific antibody anti-asialo Gm-1. In this experimental protocol the survival rate was almost unaffected, indicating that the observed protection was not due to activation of NK cells and that TNF-α is involved in the regulation of antiviral mechanisms other than the activation of interferons. Although additional production of interferon induced by TNF-α cannot be exclusded, an antiviral effect of TNF-α on the course of HSV-1 infection may be postulated from our data

The ability of eight neutralizing monoclonal antibodies (MAbs) specific for structural proteins of murine cytomegalovirus (MCMV) to protect mice passively against MCMV infection was examined to determine firstly whether a correlation existed between the neutralization titres of the MAbs in vitro and the protection afforded by the MAbs in vivo and, secondly, the contribution of the host towards neutralization by the MAbs in vivo. The reduction in MCMV titre in the livers of BALB/c and C57BL/10 mice by the MAbs closely correlated with their neutralization titres in vitro. However, in the spleens of BALB/c mice, in which MCMV replicates to high titre, almost all of the MAbs tested were ineffective in reducing MCMV replication. Indeed, a significant increase in splenic MCMV replication was observed in mice treated 24 h prior to MCMV replication with either neutralizing MAbs or polyclonal Ig. Each of six MAbs prophylactically protected between 66 and 100% of mice from an intraperitoneal challenge with 4 LD50 MCMV regardless of their neutralization titre in vitro. The persistence of MCMV replication in the salivary gland was not prevented by either polyclonal Ig or MAbs. Despite the absolute requirement for complement for the neutralization of MCMV in vitro, both polyclonal Ig and MAb 4F9 protected A/J mice, which are deficient in the fifth component of complement, as efficiently as they did complement competent BALB/c mice. These results demonstrate that MAbs specific for single MCMV polypeptides are protective in vivo. In addition, the extent to which the MAbs protected against MCMV could not be predicted from their immunoreactive or neutralizing titres in vitro or by their effect on splenic MCMV replication in vivo. Furthermore, these studies suggest that the mechanism(s) of neutralization of MCMV in vitro are different to those which act in vivo.

More than 50 fragments resulting from complete digestion of the DNA of human herpesvirus 6 (HHV-6, strain U1102) with BamHI, EcoRI, HindIII, KpnI, NruI, SalI or SmaI have been isolated as clones in M13, plasmid, cosmid and lambda vectors. Using these clones, maps have been constructed for the fragments produced by nine restriction enzymes from unit-length virus genomes and from their concatemeric precursors. The unit-length genome is a linear, double-stranded molecule of 161.5 kbp composed of a central segment of a largely unique sequence of 141 kbp (U) with a sequence of 10 kbp duplicated in the same orientation at both ‘left’ and ‘right’ genomic termini (i.e. ‘left’ and ‘right’ copies of the direct repeat; DRL and DRR). Adopting as standard an orientation in which the major capsid protein gene is ‘left’ of the gene for alkaline exonuclease, then the ‘right’ genome termini and DRL. U junctions occur close to or within repetitive (GGGTTA)n sequences. Repetitions of short sequence motifs are present in at least two other regions of the genome. One of these regions consists of a simple repeat (T C/G) of approximately 1.5 kbp in length and is unstable as clones in bacterial vectors. The second region is stably maintained in such vectors and consists of a tandem array of at least 25 copies of a 110 bp sequence containing a single KpnI site. Comparisons of fragments arising from unit-length DNA with those from virus DNA from the nuclei of infected cells have shown that the concatemeric junctions in intracellular DNA contain head-to-tail dimers of the terminal duplications (i.e. … U1.DRR1.DRL2.U2 …). The gross structure established here for the genome from the U1102 isolate of HHV-6 resembles closely that suggested by Pellett and his colleagues for the Z29 isolate and differs from that of the five previously characterized human herpesviruses. This structure of HHV-6 DNA bears a superficial resemblance to that proposed for DNA from channel catfish virus and equine cytomegalovirus.

Infectivity of human herpesvirus 6 (HHV-6) was physicochemically characterized. Virus particles were excreted from infected cord blood mononuclear cells into the culture supernatant at a concentration of 108 to 109 virus particles/ml. The virus particle/infectivity ratio of HHV-6 in the culture supernatant was approximately 103:1 and that in the sonicated infected cells was 104:1. Infectivity was stable below 37 °C and at neutral pH (7.0 to 8.5). These fundamental data may be useful for serological and biological research on HHV-6.

The DNAs of human papillomavirus (HPV) types 4, 16 and 18 have been detected in biopsies of normal and malignant human oral mucosa by Southern blot hybridization and the polymerase chain reaction (PCR). By the former technique, HPV-4, HPV-16 and HPV-18 DNAs were detected in three separate carcinomas but were found in adjacent dysplastic and normal tissue by the PCR only. The PCR technique also allowed detection of HPV-16 and HPV-18 DNA in additional carcinomas and normal samples. The oral HPV-4 DNA was molecularly cloned and extensive restriction analysis and nucleotide sequencing showed identity with the prototype HPV-4 DNA. The HPV-18 DNA detected by Southern blot hybridization showed an altered restriction pattern in the E1 region of the viral genome; however direct nucleotide sequencing of PCR products from the E6 open reading frame showed no sequence alterations in either normal or malignant samples.

The sequence of the gene encoding the major neutralization antigen (VP7) of the type member (bovine virus strain B223) of the proposed serotype 10 of rotavirus has been determined. This was done using a rapid new strategy involving direct sequencing by primer extension of cDNA generated from the relevant virus gene (gene 8) using a combined reverse transcription/polymerase chain reaction. The sequence obtained is 1062 bp in length and contains a single long open reading frame capable of encoding a protein of 326 amino acids. Comparison of the sequence to that of the corresponding gene of the other major virus serotype found in cattle, serotype 6, showed that it was approximately 25% divergent at the nucleotide level and 18% divergent at the amino acid level.

Biochemical characteristics of five rotavirus-like viruses isolated from striped bass (Morone saxatilis), turbot (Scophthalmus maximus), smelt (Osmerus mordax) and Atlantic salmon (Salmo salar) in North America and Europe were compared. The genome of each isolate was composed of 11 segments of dsRNA and each isolate had a unique electropherotype in polyacrylamide gels. Agarose gel electrophoresis showed similar RNA profiles for all four isolates from North America, whereas the RNA profile of the isolate from Europe was different. Analysis of virion proteins revealed that each virus had five structural proteins ranging in M r from 130000 to 34000. Each isolate had a unique polypeptide profile but their overall polypeptide patterns were similar. Reciprocal RNA-RNA blot hybridization demonstrated that all these rotavirus-like viruses cross-hybridized with each other except for the isolate from Europe which did not hybridize with the RNA from any of the other isolates. No genetic relationship was found between these rotavirus-like viruses of fish and a true group A rotavirus (SA11).