- Volume 69, Issue 8, 1988

The nucleotide sequence of the P (phosphoprotein) gene of two strains of mumps virus has been determined from overlapping cDNA clones. The P gene contained a single open reading frame coding for a protein of 391 amino acids with a calculated M r of 41587, in good agreement with the value (40K to 45K) estimated from electrophoretic mobility on SDS-polyacrylamide gels. No open reading frame analogous to the C gene of other paramyxoviruses existed in the mumps virus P gene region. Comparison of the amino acid sequence of the mumps virus P protein with that of Newcastle disease virus showed a limited sequence homology.

Marked differences in the apparent M rs of the HN, NP and F proteins of simian virus 5 (SV5) and parainfluenza virus type 2 (PF-2) were revealed by SDS-PAGE. To examine the antigenic relationships between SV5, PF-2 and other paramyxoviruses, monoclonal antibodies (MAbs) specific to PF-2 were isolated. These antibodies had specificities for the HN, NP and P proteins and together with 54 MAbs to SV5 were tested for their ability to react with SV5, PF-2, PF-3, mumps and measles virus proteins. Most of these MAbs (55 out of 60) reacted with homologous virus only. However, five reacted with both SV5 and PF-2. These antibodies had reactivities to the NP, M and P proteins. Furthermore, one of the antibodies with reactivity to the P protein also reacted with mumps virus. Although none of the 21 MAbs with specificities for the HN protein of either SV5 or PF-2 cross-reacted with heterologous virus, some antigenic similarities between the HN protein of SV5 and PF-2 could be detected. This was demonstrated by raising a series of polyclonal antisera to purified preparations of SV5 or PF-2 HN proteins in BALB/c mice, and testing for their ability to neutralize both SV5 and PF-2 and also to immunoprecipitate the HN proteins of these viruses. Surprisingly, while low levels of cross-neutralizing antibody could be detected in some sera (e.g. neutralization of SV5 1:1600 and of PF-2 1:80), other sera with similar neutralization titres against homologous virus failed to neutralize heterologous virus. Furthermore, only a minority of the anti-HN antisera showed any immune-precipitating activity against the heterologous HN protein.

Measles virus envelope haemagglutinin (H) was purified rapidly with Triton X-100-solubilized virions by a two-step anion-exchange chromatography using fast protein liquid chromatography. The purity of the glycoprotein in its dimeric form was demonstrated by SDS-PAGE followed by silver staining or autoradiography. The purified H glycoprotein was further freed from contaminating detergent by dialysis of octylglucoside detergent. This purification procedure, together with subsequent lyophilization and storage at -70°C of the H glycoprotein which was incorporated into phospholipid vesicles allowed the full preservation of its haemagglutinating activity, its reactivity with a monoclonal anti-H antibody that recognized a conformational epitope and its capacity to elicit anti-H antibodies with haemagglutination-inhibiting and neutralizing activities.

The potential of immune-stimulating complexes (iscoms), a novel form of antigenic presentation, for the induction of protective immunity against morbillivirus infection was shown by immunizing dogs with canine distemper virus (CDV) iscoms, which contained the fusion (F) protein and a minor amount of the haemagglutinin of the virus. The immunized dogs developed CDV-neutralizing antibodies but, in contrast to non-immunized dogs, did not develop viraemia or clinical signs of infection upon intranasal challenge with the virulent Snyder Hill strain of CDV. Immunization of dogs with measles virus (MV) iscoms, prepared either from affinity-purified MV F protein or from purified whole virus, resulted in partial protection against challenge with CDV. The data presented clearly show that the iscom form of antigenic presentation may be considered a serious candidate for subunit vaccines against morbillivirus infection.

Fluctuation of oligo-2′,5′-adenylate synthetase (2-5AS) activity was examined in K562 cells infected with vaccine strains of measles virus (strains AIK-C and CAM-70) and mumps virus (strains Torii and Miyahara). Persistent infection was easily established in the mumps virus-infected cells without significant cytolysis or cell killing. In contrast, most of the cells infected with measles virus were killed by extensive cytolysis within 3 to 4 days. The small number of cells that did survive became persistently infected. That these persistently infected cells carried a virus antigen was confirmed by fluorescein isothiocyanate-labelled anti-measles virus rabbit antiserum and anti-mumps virus rabbit antiserum. The cells produced infectious progeny virus as well as interferon (IFN). Little induction of 2-5AS activity by IFN was demonstrated during the early stages of infection by these viruses. Similar results were observed in some of the persistently infected cells but not, however, K-CMP cells (K562 cells persistently infected with CAM-70) or K-MMP cells (K562 cells persistently infected with Miyahara). Failure to induce 2-5AS activity was unchanged in cells cultured for more than 6 months. The decrease of 2-5AS activity observed in K-MTP cells (K562 cells persistently infected with Torii) was the result of suppression of transcription of 2-5AS mRNA. On the other hand, a normal level of mRNA was found in K-AKP cells (K562 cells persistently infected with AIK-C). Therefore, it is suggested that the decrease of 2-5AS activity in K-AKP cells may be due to a failure to translate 2-5AS mRNA.

Experimental induction of malignant lymphomas can be achieved in the cottontop tamarin by inoculation with Epstein-Barr (EB) virus. This system provides an animal model for assessing the efficacy of vaccine protection against the virus which is intended to reduce the incidence of human tumours associated with EB virus infection, namely endemic Burkitt’s lymphoma and undifferentiated nasopharyngeal carcinoma. Cottontop tamarins have been vaccinated with the major envelope glycoprotein of EB virus, gp340, incorporated into immune-stimulating complexes (iscoms) and were thereby protected against a 100% lymphomagenic dose of virus. The gp340 iscoms are highly immunogenic, requiring only a few micrograms of immunogen to induce protective immunity and thus would be a strong candidate for further development as an EB virus vaccine for use in man.

Monoclonal antibodies directed against VP3, the envelope glycoprotein of lactate dehydrogenase-elevating virus (LDV), were found to neutralize a large proportion of the virus population. This effect of monoclonal anti-VP3 antibodies was significantly increased by a murine monoclonal rheumatoid factor, indicating that the same antiviral antibodies can either neutralize or sensitize different fractions of the virus. This observation could be explained by heterogeneity in LDV particles, resulting in diverse responses to antibodies and therefore to the persistence of the virus in vivo.

Non-neutralizing, serotype-specific anti-NS1 monoclonal antibodies partially protected passively immunized mice from lethal dengue 2 virus intracerebral challenge. There was no apparent correlation between complement-fixing activity and protective capacity among individual anti-NS1 monoclonal antibodies. Immunization with specific combinations of non-protective or partially protective antibodies resulted in prolonged survival or reduced mortality. Solid protection, equal to that achieved after immunization with neutralizing polyclonal antibody, was achieved only with an antibody pair which individually fixed complement to high titre with homologous virus. Some groups of mice had increased morbidity after immunization with combinations of protective monoclonal antibodies that bind to overlapping epitopes. These results may affect the design of recombinant dengue vaccines which may require the inclusion of serotype-specific antigenic domains.

Monoclonal antibodies (MAbs) were raised against gag proteins of human immunodeficiency virus type 1 (HIV-1), strain HTLV-IIIB. One of 29 antibodies was specific for p17 of HIV-1. Twenty of 28 MAbs reactive with the major core protein p24 of HIV-1 showed cross-reactivity with HIV-2, and five of these also detected the corresponding antigens of simian immunodeficiency virus (SIVmac). The MAbs were reactive in several tests, i.e. ELISA, immunostaining of Western blots, immunofluorescence, alkaline phosphatase-anti-alkaline phosphatase immunocytochemistry and immunoelectron microscopy. The submembrane protein p17 was clearly localized within the virion.

The binding site of a monoclonal antibody to the haemagglutinin-neuraminidase (HN) polypeptide of Newcastle disease virus (NDV) has been located. Complementary DNA or synthetic oligonucleotides corresponding to portions of the HN gene were cloned into the Escherichia coli vector pUC19 and fragments of the HN protein were thereby fused to the α-peptide of β-galactosidase. Western blot analysis of E. coli lysates containing expressed fragments of the HN cDNA or synthetic oligonucleotides identified an antibody-binding peptide (Asp-Glu-Gln-Asp-Tyr-Gln-Ile-Arg; amino acid residues 346 to 353). Nucleotide sequence analysis of an antibody-resistant mutant of NDV revealed a Glu (wild-type) to Lys (mutant) substitution within the above sequence. The methods described could be useful for the location of continuous epitopes of other polypeptides.

The major natural reservoir of Junin virus, the aetiological agent of Argentine haemorrhagic fever, is the cricetid Calomys musculinus. Neonatal animals experimentally infected with Junin virus (XJCl3 strain) developed typical disease and approximately 80% of them died. Most survivors become persistently infected. Antigenically variant viruses were isolated from the blood and brain of infected cricetids during the acute and chronic stages of the disease. These variants could be distinguished from the parental strain by kinetic neutralization assays using polyclonal antibodies. Some biological properties were shared with the parental virus strain including its virulence for newborn C. musculinus. These variant viruses may play a major role in chronic disease since we have shown that a viral isolate from an infected brain was poorly neutralized by serum obtained from the same animal.

Approaches to cell culture propagation of hepatitis A virus (HAV) have used either acute infection by passage of infected cell lysates or supernatants into uninfected cells or the passage of persistently infected cells. The findings presented here demonstrate that the growth and recovery of purified virus from foetal rhesus monkey kidney (FRhK4) cells persistently infected with HAV isolate HAS-15 decreased over a 2 to 3 month period. In contrast, high multiplicity acute infection of FRhK4 cells with purified HAS-15 HAV resulted in degeneration of the cell monolayer 2 to 3 weeks later. Large scale propagation of acutely infected cells followed by traditional picornavirus purification procedures reproducibly yielded milligram amounts of purified virus.

Double-stranded cDNA of potato virus X (PVX) genomic RNA has been cloned and sequenced. The sequence [6435 nucleotides excluding the poly(A) tract] revealed five open reading frames (ORFs) which were numbered one to five starting at the 5′ terminus of the RNA. They encoded proteins of M r 165588 (166K), 24622 (25K), 12324 (12K), 7595 (8K) and 25080 (coat protein), respectively. ORFs 1 and 2 were inphase coding regions. The ORF 1 product contained domains of homology with the tobacco mosaic virus 126K and 183K products. The ORF 2 and 3 products showed homologies with the barley stripe mosaic virus 58K and 14K proteins, the beet necrotic yellow vein virus 42K and 13K products and the white clover mosaic virus 26K and 13K products, respectively. The significance of these homologies with respect to putative functions of the PVX-encoded proteins are discussed.

A panel of mouse monoclonal antibodies (MAbs) against potato virus X (PVX) was obtained and three of these which had high affinity to the antigen were characterized in detail. These three antibodies defined two epitopes on PVX and recognized native virus, viral coat protein and denatured viral coat protein in various immunological assays. Two of the MAbs and rabbit anti-PVX polyclonal antibodies bound to the 68 amino acid N-terminal peptide of the PVX coat protein. This implies that the N terminus of the PVX coat protein is exposed at the virus surface and forms a highly immunogenic antigenic determinant. In double antibody sandwich (DAS) ELISA, MAbs and their horseradish peroxidase conjugates reacted with PVX at 10 to 20 ng/ml. Monoclonal antibodies to PVX reacted with virus in potato leaves and tubers and detected the virus in DAS ELISA in various combinations, including in combination with polyclonal antibodies.

Using immunogold cytochemistry, plasmodesmata have been identified as subcellular locations of the protein product (P1) of cauliflower mosaic virus gene 1 in infected turnip tissue. Thin sections, from tissue adjacent to chlorotic local lesions on systemically infected fully expanded leaves, were probed with antiserum to a protein product derived from a lacZ-gene 1 fusion, and with control sera. Modified plasmodesmata between infected mesophyll cells, and plasmodesmata in the end walls of phloem parenchyma cells were specifically labelled with anti-P1 serum. In the former case, the position of the label suggested that P1 was extracellular and had formed a structural component of the modified plasmodesmata. Cell walls at the corners of cells in both healthy and infected tissue were also labelled. Anti-P1 serum also reacted with nuclei and the leaf cuticle, in both healthy and infected tissue. These observations provide strong circumstantial evidence that P1 is involved in the cell-to-cell movement of cauliflower mosaic virus.

An assay system was developed to enable cauliflower mosaic virus (CaMV) isolate Cabb B-JI to be acquired by Myzus persicae feeding through membranes on crude extracts and subcellular fractions of infected turnip plants. Optimum transmission of the virus was from solutions containing 200 mm-Tris-HCl pH 7.6, 100 mm-EGTA and 50 mm-MgCl2. The rates of transmission by single aphids from such extracts were similar to or higher than those of single aphids from injected plants to healthy plants. Similar transmission efficiencies following feeds lasting 1 min, 15 min or 3 h show that the aphid-virus association is not diminished by long acquisition feeds. Subcellular fractionation showed that transmission was related to the presence of virus particles and aphid transmission factor (ATF) but not to the inclusion body protein. It is suggested that the transmitted agents were virus particles linked to ATF. Although figwort mosaic virus (FMV) was not transmitted by aphids from extracts of infected plants it became aphid-transmissible when the extracts were mixed with similar extracts of plants infected with CaMV-Cabb B-JI, perhaps because CaMV ATF assists in the transmission of FMV.

Particles of tobacco ringspot nepovirus from purified preparations were trapped on grids by immunosorbent electron microscopy and then either negatively stained, or freeze-dried and shadowed with uranium, for structural studies. Particle dimensions differed considerably with different stains and methods of preparation, but no obvious substructure was apparent. In contrast, particles which were freeze-dried and then shadowed exhibited either fivefold or threefold symmetry and had a structure resembling that of models made up of 60 units in clusters of five arranged in a T = 1 lattice. Both chemical and morphological evidence are compatible with this structure.