- Volume 71, Issue 9, 1990

Long-term persistence of the avian leukosis virus (ALV), the transformation-defective mutant of Prague strain Rous sarcoma virus subgroup C (td PR-C) was established in heterologous duck hosts after infection in mid-embryogenesis. Transient viraemia was observed for about 4 weeks after hatching and was lost in most of the infected ducks by about 6 months. Loss of viraemia was accompanied by the increasing synthesis of virus-neutralizing antibodies. In spite of strong virus-neutralizing antibodies, virus was detected by the cocultivation assay in duck tissues throughout the observation period up to 5 years. In the viraemic phase of infection, we found integrated proviruses in various tissues, preferentially in stomach muscle tissue and in the thymus. The long-term persistence of virus was frequently accompanied by liver necrosis and neoplastic diseases. Injection of td PR-C virus into early embryos resulted in more pronounced infection accompanied by an increased copy number of viral DNA per cell, high mortality and remarkable atrophy of thymus tissue in infected ducklings.

Simian immunodeficiency virus from macaques (SIVmac) is closely related in its structure and biological activity to human immunodeficiency virus, and is the best animal model for the acquired immunodeficiency syndrome. We investigated the kinetics of membrane fusion between SIVmac and phospholipid vesicles and the effects of various parameters on this process. Purified SIVmac was labelled with octadecyl rhodamine B chloride, and fusion was continuously monitored as the dilution of the probe in target membranes. These studies show that SIVmac fusion is strongly dependent upon the liposome composition. Fusion with pure cardiolipin (CL) liposomes is significantly faster than with CL/dioleoylphosphatidylcholine (DOPC) (3:7), phosphatidylserine (PS) or disialoganglioside (GDla)/ DOPC (1·5:8·5) vesicles. SIVmac does not fuse appreciably with pure DOPC liposomes. Reduction of pH from 7·5 to 4·5 greatly enhances the rate of SIVmac fusion with CL, CL/DOPC and PS membranes, but does not affect fusion with DOPC or GDla/DOPC membranes. Calcium stimulates viral fusion with CL liposomes, but not with CL/DOPC or DOPC liposomes. SIVmac fuses with human erythrocyte ghost membranes only slowly at reduced pH. Our results indicate that SIVmac can fuse with membranes lacking the known viral receptor, CD4. Although the mechanism of SIVmac fusion with model and biological membranes remains to be determined, the fusion activity of SIVmac shares similarities with other lipid- enveloped viruses such as Sendai and influenza viruses.

Sulphoevernan is a sulphated α-1 →3,1 → 4 polyglucan (M r 20000) with a helical structure. This compound effectively inhibits both human immunodeficiency virus type 1 (HIV-1) and type 2 infection of cells in vitro at concentrations around 0·5 µg/ml. Moreover, the compound completely inhibits HIV-1-induced syncytium formation at a concentration of 1 µg/ml. Competition experiments with 35S-labelled sulphoevernan revealed that the mannose-specific lectin from Narcissus pseudonarcissus prevented binding of sulphoevernan to HIV-1, whereas the antibody OKT4A did not reduce the amount of sulphoevernan bound to MT-2 cells. These data indicate that the non-cytotoxic polymer sulphoevernan binds to the virus rather than to the host cell. In vivo studies, using Rauscher leukaemia virus in NMRI mice, revealed that, at a daily dose of 20 mg/kg, the animals were protected against virus-induced increases in spleen weight. From these in vitro and in vivo data we conclude that sulphoevernan has potential in the treatment of acquired immunodeficiency syndrome.

Antisense RNA, which has a sequence complementary to mRNA, may provide the basis for antiviral therapies of high selectivity. We have explored the inhibitory effect of six antisense RNAs upon the replication of human immunodeficiency virus (HIV) in cell culture. We chose regions of the HIV genome to test whether sequences required for splicing or for translation initiation were more susceptible to antisense RNA interference. Our results suggest that inhibitory antisense RNAs contain sequences complementary to the AUG initiation codon of the tat gene and have a comparatively low tendency to form intramolecular base pairs which would interfere with intermolecular duplex formation. Inhibition can be substantial (over 70%) but is transient. Transience does not result from mutation of the input virus. Inhibition was not a consequence of the induction of interferon by antisense RNA-mRNA duplex formation. Our results suggest that at least part of the inhibitory effect is at the posttranscriptional level.

Six major epitopes have been recognized within the transmembrane gp41 molecule of human immunodeficiency virus type 1 (HIV-1). The immunodominant epitope is also recognized by antibodies in sera from laboratory personnel and is similar to a linear sequence of amino acids in the genome protein of two rhinovirus serotypes. The hypothesis is presented that immunodominance is produced by multiple priming of the host, following repeated infections with viruses unrelated to HIV-1, which share similar epitopes.

Aspartic proteinases from human immunodeficiency virus type 1 (HIV-1) and avian myeloblastosis virus (AMV) were found to interfere with microtubule assembly. Preincubation of the proteinases with purified brain microtubule proteins (tubulin and microtubule-associated proteins) at low ionic strength (pH 6·8), completely inhibited microtubule assembly. Analysis of microtubule proteins after incubation with proteinase showed no effect on tubulin but extensive cleavage of the microtubule-associated proteins 1 and 2 was observed. The digestion by the two proteinases differed. In the presence of HIV-1 proteinase, a fragment with an M r of approximately 300000 appeared, as well as at least three other new fragments, with M r values of 188000, 124000 and 73000. In the presence of AMV proteinase, the microtubule-associated proteins were extensively digested to many small fragments. The extending microtubule-associated proteins normally seen by electron microscopy on the microtubule surface disappeared after treatment with AMV proteinase. Our results show that retroviral proteinases are not restricted to cleavage of viral polyproteins in vitro. It is suggested that proteolysis of microtubular proteins by viral proteinases is an important step in viral pathogenicity and that it may be part of a mechanism causing degenerative effects in infected cells.

Recently, hepatitis B virus (HBV) replication in the absence of HBe antigenaemia has been attributed to HBV variants with a TAG stop codon in the distal pre-C region associated with one or two point mutations. We describe here a rapid detection method for the diagnosis of such HBeAg-negative HBV variants using selective oligonucleotide hybridization. The entire pre-C region was amplified by the polymerase chain reaction and hybridized under stringent conditions with non-mutated (M0), one (M1) and two (M2) point-mutated oligonucleotide probes. Of the 15 HBeAg-positive (group I) and 20 HBeAg-negative (group II) serum samples studied, 14 samples in group I and one sample in group II hybridized with M0 only and 18 samples in group II hybridized with M1 or M2, or both.The remaining two samples (from groups I and II, respectively) failed to hybridize with any of the three probes. DNA sequencing confirmed mixed distal pre-C sequences in samples hybridizing with more than one probe and also revealed novel mutations in the distal pre-C region of the two samples which failed to hybridize with any of the probes. The latter sample had a +2 frameshift and hence represented a new type of HBeAg-negative HBV variant. This method may therefore prove useful in the diagnosis of infections by HBeAg-negative HBV variants resulting from common mutations in the pre-C region, as well as for the identification of less common variants with novel mutations in the same region.

Blood-borne type non-A, non-B (NANB) hepatitis- associated microtubular aggregates protein was isolated and partially sequenced. The microtubular aggregates were isolated from the hepatocytes of NANB- infected chimpanzees and were found to have a buoyant density in sucrose solution of 1·21 to 1·23 g/ml. A single protein, recognized by our anti-microtubular aggregates monoclonal antibodies, was found to have an Mr of 44000 (p44). This p44 protein was not found in uninfected chimpanzees. We determined a partial amino acid sequence for p44, and showed that it has no homology to any known proteins.

A 1·7 kb cDNA encoding a novel antigen (p44; apparent Mr 44K) associated with non-A, non-B (NANB) hepatitis, was isolated from the hepatic cDNA library of a chimpanzee infected with NANB hepatitis. The library was screened with a monoclonal antibody against this antigen. The cDNA cloned contained an open reading frame encoding a 444 amino acid protein with an Mr calculated to be 50468. The cDNA hybridized to a 1·9 kb mRNA obtained from chimpanzee hepatocytes infected with either the NANB or hepatitis delta viruses. It hybridized weakly to mRNA from hepatitis B virus-infected hepatocytes, and not at all to mRNA from normal chimpanzee hepatocytes. Southern blot analysis revealed that p44 is a host protein in chimpanzees, and that an identical gene exists in the human genome.

Infection of tissue culture cells with vaccinia virus results in the specific secretion of several polypeptides into the medium. Previous studies identified a protein of approximate Mr 35000 (35K) which was secreted in large amounts at both early and late times after infection with the Evans strain. We now show that a related protein is secreted by the Lister strain but not by WR, Wyeth nor Tian Tan. The gene encoding the Lister strain 35K protein was mapped within the inverted terminal repeats of the genome. The DNA sequence of this region showed that the ends of this gene are very similar to previously published sequences flanking a gene of WR which encodes a protein of approximate Mr 7500 (7·5K). Our results suggest that the 7·5K polypeptide of WR may have arisen as a result of a deletion event and is a truncated form of the 35K Lister protein. Site-directed mutagenesis demonstrated that the 35K secreted protein encoded by Lister is not essential for growth in tissue culture.

The major capsid protein (MCP) of human cytomegalovirus (HCMV) was expressed in three portions as β-galactosidase fusion proteins, covering about 75 % of the open reading frame (ORF). Fusion protein SH 1 contained nucleotides 101 to 1243 of the ORF, fusion protein FS 1 contained nucleotides 1944 to 3089 and fusion protein SS 1 covered nucleotides 2624 to 3793. The recombinant proteins were tested for their immunoreactivity with human sera. Fusion protein FS 1 was found to represent the immunodominant region. The recombinant proteins were used to generate polyvalent rabbit antisera to investigate cross-reactivities with the major capsid protein (VP5) of herpes simplex virus type 1 (HSV-1). A monospecific antiserum raised against the fusion protein close to the N terminus of the MCP, as well as a monoclonal antibody and a monospecific rabbit antiserum directed against the viral MCP, cross-reacted with the VP5 as shown by immunoblotting and immunofluorescence. In order to detect common epitopes of the major capsid proteins of HCMV and HSV-1, the recombinant proteins were conjugated to CNBr-activated Sepharose and taken for purification of MCP antibodies from HCMV and HSV-1 seropositive individuals. Using this affinity chromatography method, cross-reactivity could be observed with HCMV- and HSV-positive human antisera in immunoblot experiments.

Proteins of purified virions of equine herpesvirus 4 (EHV-4; equine rhinopneumonitis), EHV-1 (equine abortion virus) and asinine herpesvirus 3 (AHV-3) were compared by metabolic labelling with [35S]methionine or [14C]glucosamine during growth of low passage virus in natural host cells (horse or donkey) and high passage virus in an appropriate cell line and analysis by SDS-PAGE. Approximately 25 different proteins (Mr 300K to 21·5K) were clearly resolved for each virus. The three viruses had similar profiles although significant differences were found. The proteins of the cell line-grown viruses were similar to their precursor viruses grown in natural host cells although some small differences, probably related to differences in glycosy- lation by the various cell types, were noted. Six or seven high abundance glycoproteins were identified for EHV- 4, EHV-1 and AHV-3. The profile of seven glycoproteins of AHV-3 was more similar to EHV-1 than to EHV-4. Antigenic relationships of the proteins of the three viruses were examined using radioimmunopreci- pitation (RIP) and Western blot analyses and a series of polyclonal sera raised in colostrum-deprived, specific pathogen-free (SPF) foals which were immunized with inactivated EHV-4 (foal 3) or EHV-1 (foal 1), challenged and cross-challenged; a polyclonal donkey serum to AHV-3 was also used. The ontogeny of the antibody response in the SPF foals was studied and the major immunogenic proteins, as determined by RIP, were correlated with previously determined serum neutralizing antibody titres. Antibodies were first detected 14 days after primary immunization and were directed to EHV-4 proteins of M, 113K, 75K and 56K or EHV-1 proteins of 110K, 78K, 60K and 58K. Antibodies to these same three (EHV-4) or four (EHV- 1) proteins, together with antibodies to the major capsid protein and proteins of 67K (EHV-4) and 87K (EHV-1) were detected in response to primary infection (control foal 2) and these sera had high neutralizing antibody titres. The antigens of the three viruses were extensively cross-reactive with immunodominant proteins in the Mr ranges 150K to 11 OK and 62K to 56K. However, cross-absorption of EHV-4 and EHV-1 SPF foal antisera indicated the presence of significant amounts of type-specific antibody.

Bovine herpesvirus 1 (BHV-1) has a linear DNA genome of about 135 kb which appears as two isomers, resulting from its short unique segment being present in the two possible orientations with respect to the large unique segment. BHV-1 also circularizes its DNA to form replicative molecules. Definition of the target sequences at the genomic termini involved in the recombination events during genomic replication and isomerization, as well as virus maturation, led to the discovery that 10% of the genome molecules have additional DNA sequences attached to the right-hand terminus, as shown by electron microscopy. Three such tails have been cloned molecularly; they differ in length and nucleotide sequence, and hybridization experiments demonstrate the cellular origin of two of the three tails. The evidence presented here is consistent with a proportion of the BHV-1 genomes recombining their DNA with cellular DNA during lytic infection.

The role of carbohydrate in the antigenic and immunogenic structure of bovine herpesvirus type 1 (BHV-1) glycoproteins gI and gIV was investigated. Deglycosy- lated proteins induced a significantly lower antibody response in rabbits than native glycoproteins suggesting that the immunogenicity of several epitopes on gI and gIV is carbohydrate-dependent. Loss of carbohydrate from gl also resulted in a significantly decreased ability to induce a serum neutralizing antibody response to BHV-1, due to modifications in three distinct carbohydrate-containing continuous epitopes. Similarly, in vitro lysis of BHV-1-infected cells was significantly reduced when antibodies raised against degly- cosylated gl were employed; this was attributed to changes in two of the three carbohydrate-dependent neutralizing epitopes on gI. The oligosaccharides may be directly involved as actual components of these continuous epitopes, rather than in stabilization of the conformation of the protein. In contrast, carbohydrate removal from gIV did not have a significant effect on the capacity to stimulate a neutralizing antibody response. Accordingly, none of the neutralizing epitopes on gIV appeared to be carbohydrate-dependent. Similarly, lysis of virus-infected cells was not significantly reduced when antibodies specific for deglycosy- lated rather than native gIV were used. In contrast to the humoral response, the delayed-type hypersensitivity response was stronger in rabbits immunized with deglycosylated proteins than in those inoculated with native glycoproteins gI or gIV. Consequently, the carbohydrates on gI and gIV may play a dual role in the host’s immune recognition and response by contributing to certain epitopes, but masking others. The implications for the development of a subunit vaccine against BHV-1 are discussed.

Monoclonal antibodies (MAbs) were produced against the G, M2 and N proteins of bovine ephemeral fever virus (BEFV) and 29 were selected for further study. Thirteen neutralizing MAbs were assigned to one conformation-independent and at least two conformation-dependent antigenic sites on the G protein by a competitive binding ELISA. The panel of MAbs were tested by neutralization and immunofluorescence with three strains of BEFV and three BEFV-related viruses. The results indicated that BEFV strains from different sources were not identical and that the M2 protein was the least variable of the proteins investigated. Passive protection studies in mice showed that the correlation between neutralizing titre and resistance to challenge was 0· 85 (P < 0· 001).

A DNA clone of RNA segment 8 (S8) of bluetongue virus type 10 (BTV-10), an orbivirus member of the Reoviridae family has been expressed to high levels (20 mg/1 × l09 cells) using an Autographa califomica nuclear polyhedrosis virus expression vector (pA- cYMl). The expressed protein is similar to the authentic BTV phosphoprotein NS2, in its size, antigenicity, and also the manner of phosphorylation (e.g. same peptides and residues). Both mammalian and insect cell-derived NS2 proteins are phosphory- lated at serine residues only. Using affinity column chromatography and a gel retardation assay, the expressed protein has been shown to possess ssRNA- binding ability, a property which is shown to be independent of the phosphorylation state of the protein. In immunoelectron micrographic studies, gold-labelled anti-expressed NS2 antibodies have been used to localize the NS2 protein within the viral inclusion bodies (VIBs) in BTV-infected mammalian cells. Large inclusion bodies, morphologically similar to VIBs, have been identified in the recombinant virus- infected Spodoptera frugiperda cells. These structures have been shown to react with gold-labelled anti-BTV- 10 antisera, demonstrating the first direct evidence of the origin of inclusion bodies in orbivirus infection.

The immunological relationships between distemper viruses, isolated from a seal and mink in Denmark and from a dog in Greenland, were investigated with 39 previously developed monoclonal antibodies (MAbs) directed against four major structural proteins of canine distemper virus (CDV). They were also investigated with 16 newly developed MAbs directed against the fusion (F) and large glycoprotein (named H in analogy with measles virus) of phocid distemper virus (PDV) isolated from a harbour seal (Phoca vitulina). These MAbs were reacted with the three different isolated viruses and with the LEC strain of measles virus, in ELISA and immunofluorescence tests. In addition, immunoprecipitation tests were carried out with some of the cross-reacting antibodies. All 55 MAbs reacted identically with distemper virus isolated from seals or mink. When the MAbs produced against CDV were tested, 37 of 39 antibodies reacted with a virus isolated from a sled dog diseased in an outbreak of distemper in Greenland prior to the epizootic among seals in the North Sea. Of the 39 antibodies, 25 reacted with PDV and distemper virus isolated from mink. Of these antibodies, only three of the nine antibodies directed against the H protein of CDV cross-reacted with PDV and distemper virus from mink. Eleven MAbs, reacting with six epitopes of the H protein of PDV, were produced. All 11 antibodies reacted with distemper virus from mink, two of the antibodies reacted with CDV and none reacted with measles virus. All five antibodies reacting with three different epitopes of the F protein of PDV reacted with distemper virus from mink and CDV. Of these five antibodies three, directed against two epitopes, reacted with measles virus. Of the two envelope proteins, the H protein shows pronounced immunological differences between PDV and CDV. In contrast, immunologically the F protein appears to be well conserved among morbilliviruses. It is concluded that the virus causing the epizootic in seals in the North Sea in 1988 may have infected mink on land, or, alternatively, the virus in the sea may have originated from virus-infected mink.

Antigenic relationships of simian virus 41 (SV41) to other paramyxoviruses were examined by immunopre-cipitation of isotope-labelled SV41-infected cell lysates with specific antisera. SV41 is closely related to the group comprising human parainfluenza virus 2 (HPIV-2), simian virus 5 (SV5), parainfluenza virus 4 and mumps virus. Slight cross-neutralization was detected between SV41, HPIV-2 and SV5. Anti-SV41 activities were detected in 21 of 1116 human serum specimens, indicating that a proportion of the human population is infected with SV41. The haemagglutinin-neuramini-dase of SV41 was preferentially immunoprecipitated by anti-SV41 positive sera.