- Volume 59, Issue 1, 1982

Tris(hydroxymethyl)aminomethane (tris) inactivates Lactobacillus lactis bacteriophage LL-H in vitro. The inactivation is caused by DNA ejection. This effect occurs in tris-HCl buffer only. Both pH and temperature affect the sensitivity of the phage to the tris treatment. The divalent cation Mg2+ prevented the inactivating effect of tris at concentrations about 103-fold lower than for the monovalent cation K+.

The envelope protein E (formerly designated V3) of the flavivirus Kunjin was not labelled with radioactive galactose, mannose or glucosamine during virus growth in Vero cells. On electrophoresis through polyacrylamide gels containing SDS, the envelope (E) protein migrated more rapidly than related intracellular virus-specified glycoproteins. Furthermore, E had a density in CsCl solution consistent with that of a protein lacking carbohydrate, and did not bind to concanavalin A-agarose. In contrast, the envelope glycoprotein of Murray Valley encephalitis virus (MVEV) did bind to concanavalin A under similar conditions and was readily labelled with radioactive mannose. These results suggested that the E protein of Kunjin virus was not glycosylated, a feature not shared with MVEV and West Nile virus (WNV), whose properties were consistent with the presence of oligosaccharides attached to the envelope proteins. When Kunjin virions were labelled with radioactive glucosamine, the label was contained in GP19 (formerly NV2). The glycopeptides derived by Pronase digestion of GP19 from Kunjin virions were larger than those derived from GP19 obtained from infected cells.

The metabolic fate of human leukocyte interferon (HuIFN-α) was studied after intravenous injection into rats and cynomolgus monkeys. At various intervals the animals were sacrificed and the HuIFN-α content determined in serum and various tissues. HuIFN-α quickly disappeared from the circulation and was found mainly in the kidneys, in which levels were at least 7- to 10-fold higher than in the liver, spleen, lungs, heart, brain and muscles. No interferon was detected in urine. Subcellular fractionation of kidney revealed that the mitochondrial-lysosomal fraction (15000 g) had a high HuIFN-α content. It was also found that HuIFN-α was rapidly inactivated by two types of proteinases found in the lysosomal fractions of rat, monkey and human kidneys, with an optimal pH of 3 to 4. The inactivation was partially inhibited by either pepstatin or leupeptin. Inactivation was totally prevented by a mixture of both inhibitors. Since it is known that interferon is scantily excreted in urine, our findings suggest that the kidney serves as a main site for its degradation.

The presence of an RNA-dependent RNA polymerase was demonstrated in purified infectious pancreatic necrosis virus (IPNV). The enzyme was active in vitro without any pretreatment of the virus. Optimum activity was shown at 30 °C, pH 8 and in the presence of 6 mm-magnesium ions. Approx. 50% of the polymerase product remained associated with the dsRNA template of the virions. The remainder was found as extravirion ssRNA broken down to 5S to 7S fragments by virus-associated RNase(s). Although the addition of bentonite considerably reduced the amount of RNA synthesized, it protected the ssRNA product from degradation. This, in turn, permitted the synthesis of small amounts of ssRNA, which when analysed by sucrose gradient centrifugation or polyacrylamide gel electrophoresis behaved identically to the 24S single-stranded virus mRNA produced in infected cells. The virion polymerase was not stimulated by S-adenosyl-l-methionine or the addition of cellular or capped reovirus ssRNA. Several other modifications of the assay system were tried in an attempt to increase 24S RNA synthesis, but with little success. When [3H]uridine-labelled virus was used in the polymerase reaction, some labelled 24S ssRNA was obtained, indicating that in vitro transcription may proceed by a semi-conservative (displacement) mechanism.

The Hallé subacute sclerosing panencephalitis (SSPE) measles virus isolate and its plaque-purified progeny were investigated to determine whether any unusual properties could be associated with its ability to cause persistent infection. Three types of plaque-purified progeny were isolated. One population appeared to be similar in biological and biochemical properties to laboratory-adapted measles virus and had the ability to induce syncytia (syn+). A second population (syn−) plaqued more efficiently at 39 °C than at 33 °C, did not cause normal cell fusion at either temperature, and produced particles that interfered with the replication of other measles virus isolates in vivo and in vitro. This syn− virus was further plaquepurified to eliminate the interfering particles, producing the syn− P2 virus. This virus also plaqued more efficiently at 39 °C than at 33 °C, but caused cell fusion only at 39 °C. Both syn− viruses and the parental virus were significantly less virulent in vivo than the syn+ virus and caused a more prolonged infection. Biochemical analysis showed that the syn− P2 population produced particles that banded at two different densities in potassium tartrate gradients; both densities were less than those of the standard laboratory measles virus and the syn+ virus. Although the syn− P2 virus did not cause cell fusion at 33 °C, [35S]methionine labelling demonstrated that the haemolysin/cell fusion protein was present in syn− P2 virions. The production of interfering particles, the inability to cause cell fusion at 33 °C, and the cold-sensitive nature of the syn− population appear to play a role in the ability of the Hallé SSPE virus to establish persistent infection.

Simian sarcoma-associated virus (SSaV) was repeatedly passaged on three human cell lines. The proteins of the progeny virus were analysed for the presence of variant polypeptides. Occasionally, a few variant polypeptides were observed. One-dimensional peptide maps of the major virus protein p30 revealed no modifications after 25 cycles of infection on the three cell lines studied. The peptide map of Pr65gag of virus grown through 25 passages on a human chondrosarcoma cell line was slightly different from that of the virus stock before passaging. The relative amount of the virus protein p30 as compared to p18 and p16 (possibly the SSaV equivalents of p15E and p12E) was variable depending on the host cell. Virus grown on Daudi cells was relatively deficient in p18 and p16. These virus particles were morphologically altered and had a low infectivity.

We have examined the expression of type C RNA viruses in different Friend erythroleukaemic cell types, distinguished on the basis of increasingly malignant characteristics, which arise during the ageing of mice inoculated with the polycythaemic Friend virus complex. Most early appearing Friend cells (type I) expressed ecotropic virus, but cells of later malignant types showed decreased and variable expression. In general, the more malignant cells released less ecotropic virus. Xenotropic virus was detected in low numbers from type I, II, and IV cells. Two viruses were cloned from type II tumour cells: a xenotropic virus (II clone 1) and an N-tropic ecotropic virus (II clone 2). No pathogenic activity was found when II clone 1 was inoculated into newborn and adult DBA/2J and NIH/Swiss mice observed for up to 20 months, whereas II clone 2 caused a rapid anaemic erythroleukaemia in both N- and B-type newborn mouse strains. It caused a similar form of leukaemia in susceptible N-type adult mice, but at a lower frequency and with a longer latency (usually >5 months). This finding demonstrated a lack of NB restriction in newborn mice. The virus was much less active in DBA/2J mice from which it had been originally cloned; it also appeared to cause lymphoma or to shorten the latency of spontaneous lymphoma in DBA/2J mice.

Bovine leukaemia virus (BLV) was found to agglutinate mouse erythrocytes. Under optimal conditions, including the use of neuraminidase-treated erythrocytes, 200 µg/ml of BLV purified from the supernatant fluid of BLV-infected bat cells had haemagglutinating titres of about 512 units. BLV haemagglutination was drastically affected by pH and temperature; maximum agglutination occurred at pH 6 and 4 °C. That the BLV haemagglutinin is a glycoprotein was suggested by the fact that trypsin, potassium periodate or neuraminidase, but not lipid solvents or phospholipase C, significantly reduced the haemagglutinating (HA) activity of purified BLV. Furthermore, purified BLV glycoprotein of mol. wt. 51000 (gp51) had HA activity. The receptors for BLV on mouse erythrocytes were inactivated by proteolytic enzymes but not by sodium deoxycholate or potassium periodate. Neuraminidase treatment of erythrocytes increased their agglutinability fourfold. Haemagglutination is a relatively sensitive test for detecting BLV glycoprotein because 0.4 µg/ml of glycoprotein can be detected by this method. The pH and temperature sensitivity of the BLV HA reaction and specificity for mouse erythrocytes distinguish BLV from that of equine infectious anaemia virus and murine leukaemia virus, the other C type retroviruses known to have HA activity.

Mice inoculated intracerebrally (i.c.) with a mutant strain of HSV were found to develop cataracts 1 to 2 months after inoculation. Cataract formation was subsequently shown to follow an acute retinitis which commenced within 1 week of inoculation. The mutant had been selected for high resistance to the nucleoside analogue acyclovir and has been shown previously to be defective in the induction of thymidine kinase and also to express an altered DNA polymerase. The LD50 for mice inoculated i.c. was > 105 p.f.u. compared with approx. 7 p.f.u. for the parental strain. Studies of virus replication following i.c. inoculation with a sublethal dose of the mutant revealed that only small amounts of infectious virus were produced in the brain, but during a period from 6 to 12 days after inoculation vigorous replication occurred in retinal tissue, producing very high titres of virus.

The WC11 isolate of malignant catarrhal fever virus (MCFV) was purified by a method employing phase separation of the virus in an aqueous polymer system. Virus was grown in primary bovine thyroid cells. The virus released into the extracellular medium was purified. Initial concentration and partial purification of MCFV occurred after separation of the virus into the dextran phase following the addition of 10% (w/v) polyethylene glycol and 8% (w/v) dextran T10 to the extracellular virus. Further purification was achieved by centrifugation into a 20% (w/v) Ficoll cushion followed by centrifugation to equilibrium by flotation in a 15 to 36% (w/w) CsCl or 30 to 60% (w/w) sucrose gradient. Virus recovery was monitored using a plaque assay on bovine thyroid cells and ranged from 3 to 23% of the input extracellular virus.

Human cytomegalovirus (HCMV) DNA was digested with restriction endonucleases and the fragments characterized with respect to molecular weight and relative mole proportions. The terminal fragments were identified by digesting HCMV DNA with exonucleases before restriction endonuclease treatment and subsequent gel analysis. The HindIII fragments of HCMV DNA were cloned in Escherichia coli and recombinant plasmids were characterized by digestion with restriction endonucleases and by molecular hybridization with HindIII, BglII and XbaI fragments of the virus genome. Data from these experiments were used to construct physical maps of HCMV DNA for the HindIII, BglII and XbaI restriction endonucleases. The terminal regions of the genome and the region containing fragment HindIII M were shown to be heterogeneous.

After infection with Sendai virus or Newcastle disease virus (NDV) strain F, human osteosarcoma MG63 cells produced large amounts of interferon-β. Both interferon production and overall protein synthesis were strongly inhibited by hypertonic salt. Interferon mRNA synthesis, however, was little affected by hypertonic salt up to twice normal salt concentrations, although cellular RNA synthesis was inhibited under these conditions. The results are compared to those obtained with polyriboinosinic acid:polyribocytidylic acid copolymer [poly(rI).poly(rC)] inductions of MG63 cells.

Influenza B virus exhibited haemolytic activity at low pH, but the pH profile for its activity varied from strain to strain. The results of selective heat-inactivation of the enzyme activity of neuraminidase (NA) or elimination from virions of the enzyme-active portion of the NA molecule by trypsin digestion, suggest that proteolytically cleaved haemagglutinin, but not enzyme activity of NA, is essential for haemolysis by influenza B virus.

The antigenic determinants of the haemagglutinin and haemolysin antigens of measles virus were located at the surface of HEp2 cells infected with measles virus and on measles virions released from these cells, using immunoelectron microscopy. Antisera specific for haemagglutinin or haemolysin antigen and peroxidase-conjugated antiglobulin were used. Treatment of the infected cells with trypsin removed the virus spikes and prevented binding by the anti-haemagglutinin serum, while the reaction with anti-haemolysin serum was unaltered. This suggests that the antigenic determinants for measles haemagglutinin reside on the spike, while the antigenic determinants for haemolysin reside on, or are close to, the virus membrane.

The effect of monensin, a monovalent ionophore, on La Crosse virus particle formation and polypeptide synthesis was examined. Monensin inhibited the release of virus particles (both infectious and non-infectious) from infected BHK-21 cells. Monensin had no detectable effect on the synthesis of polypeptides G1, G2 and N.

An assay for rabies glycoprotein antigen based on single-radial-immunodiffusion (SRD) is described. Rabies glycoprotein antigen at concentrations of 0.7 µg/ml or greater (approx. 1 international unit, IU) produced well-defined SRD reaction zones in immunoplates containing antibody to purified glycoprotein. Plots of zone area against relative antigen concentration were linear. The method was found to be of suitable sensitivity for in vitro potency assays of inactivated cell culture rabies vaccines. Qualitative differences were detected between rabies vaccines prepared by two different methods when these were analysed in sucrose gradients for glycoprotein antigen associated with intact virions or in ‘soluble’ form associated with subviral structures. In vaccines prepared by zonal ultracentrifugation the glycoprotein was totally associated with intact virus, whilst in those prepared by ultrafiltration comparable quantities of subviral antigen were also detected. The SRD test appears to have considerable potential for assays of the antigenic content of rabies vaccines and has the advantage of reducing reliance on conventional in vivo tests for immunogenicity which employ infectious virus.

Simian foamy virus (SFV) pseudotypes of vesicular stomatitis virus have been successfully produced and their host range characterized. The availability of these pseudotypes has permitted the development of a rapid, quantitative assay to measure neutralizing antibody titres to SFV that has proved useful in a sero-epidemiological study.