- Volume 71, Issue 6, 1990

A panel of monoclonal antibodies which react with empty non-infectious type 3 poliovirus particles (C antigen) but not infectious virus (D antigen) were characterized for their reactivity with C antigen particles derived from neutralization-resistant virus strains which had single amino acid substitutions at each of the antigenic sites. Antibodies were identified which failed to bind to variant viruses with modifications at each of antigenic sites 2b, 3b and 4 indicating that the same amino acid sequences involved in the neutralization of infectious virus are also present on the surface of noninfectious particles but in different configurations.

Fourteen neutralizing monoclonal antibodies recognizing human rhinovirus (HRV) type 2 have been used to select a total of 51 virus escape mutants. Crossresistance analysis of the mutants, together with RNA sequencing and identification of amino acid substitutions, have revealed three neutralization sites on the virus surface. Two of these appear to correspond to the NIm-IA and NIm-II sites described for HRV-type 14, although there are also substantial differences. The third site has not been described previously.

Temperature-sensitive (ts) mutants of influenza virus strain A/Udorn/72 (H3N2 subtype) with lesions in RNA segment 8 exhibited intrasegmental complementation, and were divided in two complementation groups (HI and H2) on MDCK cells. The nucleotide sequence of segment 8 was determined for three of these mutants. The HI strains, ICR1629 and SPC45, have a single amino acid substitution in the coding region of the non-structural protein NS1, whereas the H2 strain, ICR516, has a substitution in the NS2- coding region. With both NS1 ts mutants, the synthesis of two late proteins, the matrix protein (Ml) and haemagglutinin (HA), was greatly reduced and NS1 synthesis also decreased at 40 °C (non-permissive temperature) compared to that at 34 °C (permissive temperature). The synthesis of each virus-specific RNA was analysed using a quantitative hybridization method. However, at 40 °C, the levels of individual mRNAs including those for the late proteins, were almost the same as those at 34 °C, and attained the wild-type levels later in the infection (5 h postinfection) when the synthesis of the late proteins and the NS1 protein was severely reduced. The observations suggest that the NS 1 protein, which is a nuclear protein, is involved in some post-transcriptional processes in the synthesis of the late proteins and the NS1 protein.

Plasmid vectors were constructed to facilitate the insertion and expression of a foreign gene in the haemagglutinin (HA) gene locus of vaccinia virus. Five unique cloning sites adjacent to the P7.5 promoter of vaccinia virus permit the rapid insertion of a foreign sequence coding for a protein into these plasmids. This vector system provides a simple procedure to select recombinant viruses because they can be readily identified on the basis of their HA-defective phenotype. Recombinant vaccinia viruses expressing influenza virus HA were constructed to characterize the possible use of this system. The recombinant viruses did express the influenza HA through the authentic pathway of biosynthesis. In addition to having immunological characteristics similar to the authentic influenza HA, the expressed HA was found to possess haemagglutinating, haemadsorption and acid-inducible fusion activities. These findings demonstrate the usefulness of this eukaryotic vector system.

The nucleotide sequences of the large open reading frame (ORF) from segment A of three European strains of infectious bursal disease virus (IBDV) have been determined using cDNA clones. This ORF of 3036 nucleotides encodes the virion proteins as a polyprotein in the following order: VP2, VP4, VP3. The nucleotide sequences determined have been compared to each other and to the published sequence of an Australian strain. The four strains are closely related, the greatest difference between two strains being 7.7% at the nucleotide level and 2.7% at the amino acid level. Comparisons show that there is a tight cluster of amino acid changes in the virion protein VP2. This variable region corresponded to the region where binding of a neutralizing monoclonal antibody has previously been mapped. A region in the centre of the segment, corresponding to the N terminal of VP4, was found to be completely conserved. Amino acid changes were spread fairly evenly through VP3 and there was no indication of a variable region as found in VP2.

Four major antigenic sites have been delineated on the spike protein (S) of the porcine enteric coronavirus transmissible gastroenteritis virus (TGEV) in previous topological studies using monoclonal antibodies (MAbs). Correlation of these sites with the physical structure of the protein was achieved by use of different approaches. Recombinant pEX plasmids directing the synthesis of various fused S polypeptides were constructed. A hybrid protein containing nine S-specific residues (363 to 371) was shown to express site C epitopes. The other sites were localized through study of the antigenic activity of fragments generated by controlled cleavage of the native protein with different endopeptidases. Two identified cleavage products of 26K and 13K, immunoreactive to site A-B- and site D-specific MAbs respectively, could be aligned on the S primary structure according to N-terminal sequence data. This led us to propose that the major neutralization domain A-B is contained in a region of approximately 200 residues with residue 506 as its N boundary. Similarly, site D epitopes should be located within a stretch of 130 residues, starting at 82 residues from the N terminus. Point mutations identified by direct RNA sequencing of neutralization-resistant mutants were consistent with the proposed location of these sites.

Seven neutralizing monoclonal antibodies (N-MAbs) were generated to an Australian isolate of bluetongue virus serotype 1 [BTV-1 (Aust)]. At least five of the N- MAbs were specific for epitopes on the outer coat protein VP2 and one was capable of binding SDS- treated protein in a Western blot. Six of the N-MAb panel bound and four of these neutralized BTV-1 (South Africa). None of the N-MAbs neutralized other Australian or South African BTV serotypes. However four of the N-MAb panel bound to a majority and two others bound with varying efficiency, to a more restricted but significant number of heterologous serotypes. Thus epitopes involved in the definition of one BTV serotype may be preserved on other serotypes but not be involved in their neutralization. To investigate the association between these epitopes and factors governing their expression, pools of neutralization- escape variants of BTV-1 (Aust), selected using each of six N-MAbs, were tested in virus neutralization and ELISA binding assays against the N-MAb panel. Each N-MAb displayed a unique reaction pattern with all 25 variants tested. All variants except one showed resistance to neutralization and/or reduced binding with at least three heterologous N-MAbs indicating the N- MAb-defined epitopes were mutually interactive. All 25 variants demonstrated increased resistance to neutralization by a bovine antiserum to BTV-1 (Aust). In total, the results from the variants revealed that the N-MAbs define seven distinct, interdependent neutralization epitopes which form at least part of a major neutralization domain on BTV. A majority of variants bound at least four and up to six N-MAbs, yet still resisted neutralization by them. This observation and the reaction of N-MAbs with BTV-1 (South Africa) and heterologous serotypes suggested that the conformation of the VP2 protein determines whether epitopes, conserved within the BTV serogroup, are involved in neutralization of individual serotypes of the virus.

The replication and polypeptide synthesis of Abadina virus, a member of the Palyam serogroup of orbi-viruses, was studied. The first virus-specified proteins could be demonstrated 2 to 4 h post-infection (p.i.) by immunoprecipitation. The rate of synthesis increased rapidly until 12 h p.i. after which it remained fairly constant until 18 h p.i. when it began to decline. Host cell protein synthesis shutoff was incomplete. Twelve virus-induced polypeptides were identified in infected cell lysates, ranging in Mr from 36K to 143K. Three small polypeptides (M r 15K to 20K) identified in homologous immunoprecipitation studies are also thought to be virus-specified. Nine structural proteins were identified, four being major components of the purified virion. Partial proteolysis was used to demonstrate homology between some proteins. Pulse-chase experiments provided no evidence for a precursor-product relationship between any of the Abadina virus proteins. A non-structural protein was found to be phosphorylated.

The gene encoding the hepatitis delta virus structural antigen (HDAg) was linked to a neomycin resistance gene in a retrovirus expression vector, and human HepG2 cells were transfected with the recombinant plasmid. A stable cell line was cloned that expressed HDAg in the nuclei of 100% of cells, in a pattern indicating a close relationship with cell nucleoli. Analysis of partially purified recombinant HDAg by HPLC showed an Mr in the range of 7 × 105 to 2 × 106, which appeared to contain conformation-dependent epitopes, whereas the density of the antigen was 1·19 g/ml by equilibrium centrifugation in caesium chloride, and in rate zonal centrifugation it sedimented with a value of SOS, close to that of particulate hepatitis B virus surface antigen. Immunoblotting demonstrated a single polypeptide with an Mr of 24K which corresponded to the smaller of the two HDAg-specific polypeptides present in infected sera. The recombinant HDAg polypeptide was shown to be a RNA-binding protein with specificity for both genomic and antigenomic species of hepatitis delta virus RNA.

Human papillomavirus (HPV) type 16 is implicated in the aetiology of anogenital dysplasia which may progress to malignancy. HPV-16 DNA is actively transcribed in cervical carcinomas, the most abundant transcripts being from the E6 and E7 early open reading frames. The E7 protein has been shown to have transforming activity in vitro. In this report we define four immunodominant B epitopes within the protein corresponding to the E7 gene, using a panel of murine monoclonal antibodies. Three epitopes are linear and lie within the N-terminal region of the molecule, and are unique to the HPV-16 E7 protein. One epitope is non-linear and presumed to be conformational. At least three of the four epitopes of the E7 protein are detectable by immunoprecipitation from an HPV-16- infected cervical carcinoma cell line. The demonstrated immunogenicity of the E7 protein allows us to deduce that this molecule may be a potential candidate for incorporation in a vaccine against cervical cancer.

Murine herpesvirus 68 (MHV-68) is a naturally occurring herpesvirus of small free-living rodents. In order to facilitate the molecular characterization of the virus genome, a library of cloned restriction fragments has been produced and restriction enzyme cleavage maps deduced for the enzymes BamHI EcoRI and HindIII. The MHV-68 genome comprises a region of unique DNA of approximately 118 kbp which is flanked by variable numbers of a 1·23 kb repeat unit. The organization of the MHV-68 genome is, therefore, most similar to that of the lymphotropic γ2 group of herpesviruses which include herpesvirus saimiri and herpesvirus ateles.

Short nucleotide sequence analysis of seven restriction fragments of murine herpesvirus 68 (MHV-68) DNA has been undertaken and used to determine the overall genome organization and relatedness of this virus to other well characterized representatives of the alpha-, beta- and gammaherpesvirus subgroups. Nine genes have been identified which encode amino acid sequences with greater similarity to proteins of the gammaherpesvirus Epstein-Barr virus (EBV) than to the homologous products of the alphaherpesviruses varicella-zoster virus and herpes simples virus type 1 or the betaherpesvirus human cytomegalovirus. In addition, the genome organization of MHV-68 is shown to have an overall collinearity with that of the gammaherpesviruses EBV and herpesvirus saimiri. In common with these viruses, dinucleotide frequency analysis of MHV-68 coding sequences reveals a marked reduction in CpG dinucleotide frequency thus implicating a dividing cell population as the site of latency in vivo.

The synthetic nonapeptide YAGAVVNDL [identical to the nine carboxy-terminal amino acids of the small subunit of herpes simplex virus (HSV)-encoded ribonucleotide reductase (RR)] was found to inhibit the RR activity induced by equine herpesvirus type 1 subtype 1 (EHV-1). Parallel experiments with HSV type 1 (HSV- l)-encoded RR established that the concentration of peptide required to inhibit 50% of the RR activity was 28 μm for both enzymes. The optimum pH for the EHV-1 enzyme was found to be between 8·0 and 8·1 which is the same as that of HSV-1 RR. By use of antisera made against peptides corresponding to different regions of the large subunit (RR1) of the HSV-1 enzyme and monoclonal antibodies directed against HSV-1 RR1 we have obtained evidence which suggests that the EHV-1 large subunit has an Mr of approximately 90000 and lacks the N-terminal domain which is so far unique to HSV.

Two sites within the short region origin of DNA replication (oris) in herpes simplex virus type 1 (HSV-1) which bind the product of the UL9 gene have previously been identified. One of these sites (site I) contains an 11 bp sequence which is also present in oris of varicella-zoster virus, and the other (site II) includes a related element differing in two positions. A third sequence (motif III), which lies close to binding site I, differs from the site I element at only a single position. We have deleted specifically each of these three 11 bp sequences from within functional copies of HSV-1 oris and have examined the effects on origin activity and binding of the UL9 protein. Gel retardation analyses confirmed the important roles of the regions deleted from sites I and II in interacting with the UL9 protein. In transient replication assays, copies of oris lacking the site I or II elements exhibited undetectable or residual (4 to 8 %) activity respectively. The UL9 protein did not bind to motif III even in the absence of site I sequences, although removal of the motif III sequence caused a small reduction in oris activity. A single base change which converted the sequence within binding site I to that of motif III was sufficient to abolish both the interaction of the UL9 gene product at this locus and the replicative ability of oris. Therefore, interaction of the UL9 protein with binding site I is essential for origin activity, but the presence of binding site II is also required for efficient replication.

Herpes simplex virus (HSV) particles contain a factor which can shut off host protein synthesis during the earliest stages of infection. The efficiency of shutoff varies between different strains of virus, type 2 strains being generally more active than type 1 strains. However, HSV-2 strain HG52 is deficient in host cell shutoff. We have investigated the basis of the different shutoff phenotypes of a strong shutoff strain (HSV-2 strain G), a weak shutoff virus (HSV-1 strain 17 syn+) and HG52 by comparative DNA sequence analysis of gene UL41, which encodes the virion-associated host shutoff factor. The results show that the UL41 genes of strains G and 17 are 86 % homologous and that the lack of shutoff by HG52 is likely to be explained by a frameshift mutation within its UL41 coding sequence.

Rat glioma C6 cells persistently infected with measles subacute sclerosing panencephalitis (SSPE) virus (C6/SSPE) express the viral membrane proteins haem- magglutinin (HA) and F on their cell surface as well as the intracellular proteins N, P and M. Previously we have shown that the addition of a polyclonal antibody against the HA antigen to the growth medium of C6/SSPE cells led to a gradual loss of all viral antigens. Here we show that the addition of a monoclonal antibody (MAb K83) leads only to a transient decrease in viral antigens during the first three passages. After the third passage viral antigens start to increase and after five passages they produce more antigens than at the premodulation level. At this point of the MAb treatment, MAb K83 no longer recognized the HA antigen on the surface of the cells and in virus particles produced by these cells in contrast with polyclonal antibodies or other MAbs against the HA antigen. The results suggest that specific variants of the SSPE virus with an altered HA antigen were selected by the MAb treatment.

A panel of 10 monoclonal antibodies produced after immunization with two porcine subgroup I rotavirus strains (OSU and A46), and directed against the major inner capsid protein (VP6), fell into six patterns of reactivity when tested against a collection of human and animal group A rotavirus strains. Monoclonal antibodies of pattern 1 recognized all rotavirus strains. Antibodies of patterns 2 and 3 recognized all subgroup II strains and some, but not all, subgroup I strains. Pattern 4 antibodies identified all subgroup I strains and two strains (H2, equine; CC117, porcine) not reactive with reference subgroup monoclonal antibodies (strains non-I non-II). Pattern 5 antibody exhibited the same reactivity as pattern 4 except for not recognizing the non-I non-II equine strain. Pattern 6 antibodies reacted exclusively with subgroup I and non-I non-II rotaviruses of porcine origin. By competitive binding assays, monoclonal antibodies of patterns 4, 5 and 6 appeared to recognize a single antigenic site, which included at least three overlapping epitopes. In immunoblots all monoclonal antibodies, except one, recognized only the trimeric, but not the monomeric form of VP6.

A subgenomic restriction fragment from cDNA prepared from Coxsackie B2 virus (CVB2) RNA was subeloned into a riboprobe vector allowing the production of enteroviral group-specific RNA probes complementary to either the positive (genomic) or negative (template) strand of enteroviral RNA. These ribo- probes were used to follow productive infection of cultured cells by CVB2; as expected, positive strand RNA was synthesized in approximately 100-fold excess over negative strand. RNA was extracted from muscle biopsy samples from patients with chronic fatigue syndrome and probed for the presence of enteroviral RNA. In cases where enteroviral RNA was detected the amounts of positive and negative strands of enteroviral RNA were approximately equal, in contrast to the situation in lytic infection of cultured cells. This suggests that entrovirus pesistence in muscle is due to a defect in control of viral RNA synthesis.