- Volume 66, Issue 8, 1985

Haemagglutinin prepared from influenza virus A/Memphis/1/71 by bromelain digestion was centrifuged through continuous sucrose gradients buffered at pH 7.4 or pH 4.9. From these gradients were isolated two forms of the protein which displayed different equilibrium sedimentation properties. One species behaved as a molecule with a mol. wt. of 190000, the other with a mol. wt. of 70000. These results are consistent with the separation of trimeric and monomeric haemagglutinin. A comparison of their antigenic properties, using monoclonal antibodies raised against intact virus, showed that major antigenic differences occur between the two forms of haemagglutinin. None of the monoclonal antibodies reacted with haemagglutinin denatured by reduction and alkylation.

Direct biochemical evidence for the existence of mutations in five of the RNA segments of the A/Leningrad/134/57 cold-adapted 47th passage mutant as compared with its wild-type progenitor has been obtained using two techniques. T1 oligonucleotide mapping of total viral RNA as well as of individual RNA segments revealed changes in RNAs 4, 5 and 6. Analysis of S1 nuclease-treated RNA–RNA hybrids on polyacrylamide gels revealed changes in at least one of the polymerase genes as well as in RNAs 4, 5, 6 and 7. These findings provide a direct demonstration for the existence of multiple mutations in the cold-adapted mutant vaccine strain.

An indirect enzyme-linked immunosorbent assay (ELISA) which uses monoclonal antibody as solid-phase immunosorbent was developed to measure specific polypeptides of respiratory syncytial virus (RSV). The assay was used to examine 43 nasopharyngeal (NP) aspirates from RSV-positive infants that had been examined previously for RSV by culture, direct immunofluorescence, and polyclonal antibody ELISA. Frozen NP aspirates were serially diluted and examined for the 66K mol. wt. fusion glycoprotein (F), the 84K large surface glycoprotein (G) and the 41K nucleoprotein (N) by monoclonal capture ELISA. F protein was detected in all 43 specimens, G protein was detectable in 20 (47%) and N protein in 22 (51%) of 43 NP aspirates. In specimens with detectable G and N proteins, F was detected by endpoint titration at approximately tenfold greater dilutions than either G or N. In 19 sequential NP aspirates from five patients with RSV infection, F was present in higher titre throughout infection. In 20 cases, matching cell culture isolates were examined by immunofluorescence with strain-specific monoclonal antibodies. Three of 20 isolates showed strain-specific differences by their lack of reaction with anti-G monoclonal antibody. Titration of the 20 cell culture isolates by monoclonal antibody capture ELISA showed the relative amount of F and N proteins to be equal in all cases, whereas levels of G protein tended to be slightly lower. Reconstruction experiments with NP aspirates demonstrated that degradation of F and N proteins did not occur in NP aspirates, but that G protein antigenicity appeared to be affected by nasal secretions. When compared with cell culture-grown material, nasal secretions contained abundant F protein but a surprisingly low concentration of N protein.

Scrapie-associated fibrils (SAF) were isolated and purified from animals infected with three different scrapie agents: ME7 and 139A in mice, and 263K in hamsters. Mouse ME7 and 139A SAF differed from hamster 263K SAF in morphology, sedimentation rate and protein composition. SAF from the three scrapie agents were distinguishable from each other by their sensitivity to proteinase K digestion. SAF copurified with infectivity in both the hamster and mouse systems. SAF appear to be a unique class of structures which are related but specific for each individual scrapie agent. These properties may correlate with the biological and pathological differences seen among these agents.

Five lines of chickens maintained as specific pathogen-free flocks in Australia were characterized in relation to endogenous antigens and endogenous avian leukosis virus expression. Embryos of line N were predominantly of C/E phenotype, uniformly positive for group-specific antigen and chick helper factor (gs+chf+) and 38% expressed endogenous virus at a very low titre. Embryos of line M4 were uniformly of C/ABE phenotype and were either gs+chf+ or gs−chf+. Line W19 embryos segregated for susceptibility to viruses of subgroup A, B and D and were either of C/E or C/ABE phenotype. The majority of W19 embryos were gs+chf+ with a small proportion being gs+chf−. Line I13 embryos were either of C/0 or C/ABE phenotype, uniformly gs−chf− and 44% of embryos expressed endogenous virus at a low titre. Line S segregated for susceptibility to subgroup E virus and embryos were either of C/E or C/0 phenotype, while the majority of embryos from line S were gs−chf− with some embryos being gs+chf+ or gs−chf+. The degree of interference of gs+chf+ and gs−chf+ phenotypes with subgroup E virus infection was identical with the interference patterns of classical gs+chf+ and gs−chf+ phenotypes. The resistance to infection with avian sarcoma viruses of subgroups E in lines N and M4, and to a degree in line W19, was highly associated with the presence of chf. Resistance to subgroup E virus was independent of chf in lines S and I13, probably being under the control of an independent locus. Cellular restriction of endogenous virus replication existed in all subgroup E virus-susceptible cells of line I13 in contrast to cells of line S which supported replication of endogenous virus. The phenotype of chicken cells for the expression of endogenous gs antigen and chf could accurately be predicted from the test performed on whole blood cells.

A total of seven different isolates of Hantaviruses from five mammalian species and six geographical areas were examined by a double sandwich ELISA with biotin-avidin amplification system. Three serotypes were recognized by comparing antigen titres to homologous and heterologous antibody and blocking antibody titres to homologous and heterologous antigens. Serotype 1 included four strains: Hantaan virus isolated from Apodemus agrarius in South Korea, Hupei-I isolated from acute-phase serum from a patient with epidemic haemorrhagic fever (EHF) in China, SR-11 isolated from a laboratory rat associated with an EHF outbreak in Sapporo, Japan, and Tchoupitoulas isolated from a wharf rat (Rattus norvegicus) trapped in New Orleans, La., U.S.A. Serotype 2 included two strains of nephropathia epidemica virus, Hällnäs-I and Puumala, isolated from Clethrionomys glareolus trapped in nephropathia epidemica foci in Sweden and Finland, respectively. Serotype 3 included one strain, Prospect Hill-I, isolated from Microtus pennsylvanicus, trapped in Frederick, Md., U.S.A.

In this paper, we show that the pattern of expression of the human hepatitis B surface antigen (HBs Ag) gene, transfected along with a dominant selectable marker into mammalian cells, is complex. In human hepatoma (HepG2) cells, late transient expression occurs and permanent expression takes place at high frequencies in the selected clones. In HeLa and human xeroderma pigmentosum (GM4312A)-derived cells, the late transient expression is barely seen or absent and permanent expression is only seen in a few selected clones. In monkey kidney Vero cells, late transient expression has been described and we show in this report that only 5% of the selected clones are capable of expressing HBs Ag in a permanent manner. In most of the Vero clones, the absence of HBs Ag expression is mainly due to HBs Ag gene rearrangements. We have selected and amplified more than 500 transfected Vero clones and have characterized in detail one clone (GAR1412) which is a permanent high-level HBs Ag expressor.

Tacaribe virus stocks were prepared which induced definite lytic responses in Vero cells infected at multiplicities giving synchronous infection. Under these conditions, the first signs of cytopathic effect (c.p.e.) appeared at about 30 h post-infection and cell lysis occurred after 40 h. Before the onset of cytopathic changes, the virus induced inhibition of host cell protein, DNA and RNA (primarily rRNA) synthesis. These were designated c.p.e. (+) virus stocks. The effect of virus on host cell macromolecular synthesis and development of c.p.e. were not related to the virus isolate, but to the conditions under which the virus was produced. Thus, from a single virus clone, working stocks were derived which could or could not induce inhibition of host cell functions and c.p.e. development. The virus stocks that did not induce inhibition are defined as c.p.e. (−). Analysis of [3H]leucine-labelled proteins from Vero cells infected with either the c.p.e. (+) or the c.p.e. (-) virus stocks revealed synthesis of two virus-specific polypeptides migrating with mobilities corresponding to mol. wt. 68000 and 79000. Thse are presumed to correspond, respectively, to the nucleoprotein and to the minor polypeptide p79. In cells infected with the c.p.e. (+) virus stock, the virus-specific polypeptides were synthesized at times when there was a drastic inhibition of host cell protein synthesis. The yield of infectious progeny during the first 24 h of infection is similar in Vero cells infected with either the c.p.e. (+) or the c.p.e. (−) virus stocks. The proportion of defective interfering particles was much higher in the c.p.e. (−) than in the c.p.e. (+) virus stocks. The results presented here are the first demonstration that an arenavirus affects the biosynthetic machinery of the host cell.

Mice previously latently infected with the F strain of herpes simplex virus type 1 (HSV-1) can be successfully colonized with a second virus strain if HSV-2 is introduced at the same peripheral site as HSV-1. On the other hand, HSV-1 strains seemed able mutually to exclude establishment of latency with each other. Mice (3 months or 3 years after nasal infection) latently infected with HSV-1 were thus superinfected with HSV-2. The mice were sacrified 2 days post-infection when HSV-2 replication in the ganglia was found to have commenced. Ganglia were homogenized immediately and virus was plaqued on permissive cells. HSV-1 plaques were regularly obtained among HSV-2 plaques as assessed by staining with an enzyme-linked immunosorbent using a type-specific monoclonal antibody recognizing glycoprotein C of HSV-1. DNA from this virus had identical restriction endonuclease patterns (EcoRI, BamHI and HindIII) to the F strain used to infect the animals latently. HSV-1 was not retrieved from ganglia of controls superinfected with a neuroadapted vaccinia virus or were mock-superinfected. The results suggest that it is possible to superinfect a latently infected ganglionic neuronal cell with a heterotypic HSV strain and that the subsequently introduced HSV-2 can act in trans to induce reactivation of latent HSV-1.

The related triterpenoid compounds carbenoxolone sodium (CBX) and cicloxolone sodium (CCX) have been investigated in clinical trials for treatment of herpes simplex virus (HSV) infections. When the drugs were tested in vitro, two dose-related effects on BHK cells became apparent: the rate of cell growth was reduced and the drugs exhibited cytotoxicity at high concentrations. Flow 2002 cells, in contrast, were apparently unaffected by all drug concentrations tested. The effect of up to 3 days incubation with 100 µm-CCX on BHK cells was reversible. The presence of 500 µm-CBX or 300 µm-CCX during the HSV replication cycle reduced the infectious virus yield to less than 0.01%: CCX is the more potent anti-herpes agent. The contribution made by cytotoxicity to the overall antiviral effect (measured by 24 h yield) was negligible in Flow 2002 cells, and was relatively unimportant in BHK cells. The amount of HSV-1 or HSV-2 adsorbing to pretreated BHK cells was reduced by 20% and 40% respectively at the highest drug concentrations. Neither 500 µm-CBX nor 300 µm-CCX treatment for 24 h completely inhibited HSV-1 replication, but HSV-2 replication was abolished. The drugs appear to be continuously active throughout the infectious cycle. Infectious HSV particles appeared to become inactivated during or soon after egress from the cell. The two triterpenoid drugs lowered the number of virus particles made, and to a much greater extent reduced the infectious virus yield; thus, the progeny virus quality is greatly diminished. HSV-2 infections were more readily inhibited by either CCX or CBX than were HSV-1 infections.

Varicella-zoster virus (VZV)-infected cell surface proteins were investigated using extrinsic radiolabelling of the cell surface, immunoprecipitation of detergent-solubilized extract of the same cell surface and fractionation of the immunoprecipitates using SDS-PAGE. Glycosylated proteins were identified by their affinity for Ricinus communis lectin. Six glycoproteins with apparent mol. wt. of 170K, 105K, 93K, 81K, 53K and 45K were identified. The 170K glycoprotein was shown to be disulphidelinked since under reducing conditions for SDS-PAGE it was cleaved to a protein of 63K mol. wt. The IgG responses to these glycoproteins during various clinical circumstances are described. In acute sera from all chickenpox patients and in the majority of acute shingles sera, antibodies reactive with glycoproteins could not be detected. In chickenpox convalescence, antibodies reactive with glycoproteins of mol. wt. 170K, 105K, 53K and 45K were identified, whilst during zoster convalescence antibodies to all six were prominent. Antibodies to the disulphide-linked glycoprotein persisted for many years following both the primary disease and its reactivation. Disseminated zoster was associated with significantly low levels of antibodies to these surface glycoproteins.

Varicella-zoster virus glycoprotein gp1/gp3 was purified by affinity chromatography using anti-gp1/gp3 monoclonal antibody 19.1 linked to CNBr-activated Sepharose CL-4B. Rabbits immunized with purified glycoprotein gp1/gp3 developed monospecific neutralizing antibody.

Three BALB/c monoclonal antibodies capable of neutralizing herpes simplex virus type 1 (HSV-1) were used to prepare rabbit anti-idiotypic antisera. Affinity-purified antibodies from four of these rabbits were used to immunize mice by repeated subcutaneous injection over a period of 6 to 7 weeks: the mice were then challenged with HSV-1 subcutaneously in the ear pinna. Measurement of ear swelling showed that prior administration of anti-idiotypic serum could generate dose-dependent delayed-type hypersensitivity responses.

Somatic cell hybrids between rat XC(HPRT−) cells, non-permissive for herpes simplex virus type 1 (HSV-1) infection, and permissive mouse L(TK−) cells were constructed and karyotyped. Infection of these hybrid cells by HSV-1 strains F and MP revealed that they were susceptible to the virus. The amounts of virus produced by the hybrid cells, as well as the cytopathic effect observed, was very similar to that of the parental L(TK−) cells. Our results suggest that failure of HSV-1 to replicate in XC cells is more likely to be due to the absence of cellular elements required for efficient virus multiplication rather than to the presence of blocking or inhibiting factors.

The DNA sequence homology between bovine herpesvirus type 1 (BHV-1) and pseudorabies virus (PRV) was examined. Reciprocal cross-hybridization of viral DNA labelled by nick translation to Southern blots of restriction endonuclease-digested DNA detected homologous sequences dispersed throughout the genomes of the two viruses. The DNA-DNA hybrids formed were stable under high-stringency wash conditions. Sequences of a 32P-labelled PRV DNA fragment probe were found to hybridize only to a specific region of the BHV-1 genome, suggesting that the detected sequence homology was not due to fortuitous hybridization of G + C-rich sequences. As measured by liquid reassociation kinetics the homology between these two viruses was approximately 8%.

HindIII fragments D to P of DNA from a Chinese vaccine strain (Tian Tan) of vaccinia virus have been molecularly cloned into the plasmid pAT153 at the unique HindIII site. The Chinese strain DNA differs from a non-vaccine American strain (WR) in having an additional HindIII fragment (P). Twelve HindIII D clones and 12 HindIII F clones of the Chinese strain were analysed by digestion by EcoRI, BamHI, PstI and XhoI. Two forms of D (designated a and b) and of F (a and b) were demonstrated. In each the differences were detected as the presence of an additional EcoRI site, in Da and Fa. The HindIII Fa and Fb fragments of the Chinese strain were shown to differ significantly from the WR strain in their restriction site maps.

A 2.3 kb cDNA was cloned from human T-cell leukaemia virus [HTLV(MT-2)] virion RNA using a vector system, as plasmid pHTLV 707. The restriction endonuclease map of pHTLV 707 revealed that the insert contained the 5′ half of the env gene and a portion of the pX region of HTLV, corresponding to the subgenomic RNA derived from 32S defective HTLV. Nucleotide sequence analysis of pHTLV 707 indicated that the clone contained an open reading frame for a 60K mol. wt. protein including the upstream and entire pX IV region. A rabbit antibody raised against a synthetic decapeptide deduced from the nucleotide sequence at the carboxyl terminus of the pX IV region immunoprecipitated gp68, and also 80K and 40K proteins.