- Volume 57, Issue 1, 1981

International Course of Veterinary Clinical Immunology

Ecole Nationale Veterinaire D’Alfort (22–27 March 1982)

To provide a week intensive course in veterinary clinical immunology laboratory techniques

Closing date for registration: 30 January 1982

Information: Dept. of Microbiology and Immunology, Ecole Nationale Veterinaire D’Alfort, 7, Avenue du Général de Gaulle, 94704 Maisons Alfort Cedex Tel: (1).375.92.11. poste 245

Proteus mirabilis phage 5006Mpa is a converting variant for ampicillin resistance of phage 5006M. We show here that the ampicillin resistance marker of transposon Tn1 is located on a 9.8% insertion with respect to the wild-type phage genome. This renders the 5006Mpa genome 5% oversized, albeit without loss of wild-type genetic material from the phage population.

Ribonucleotide reductase is an essential enzyme in mammalian DNA replication. In quiescent BHK-21/C13 cells exhibiting a low level of ribonucleotide reductase activity, infection with herpes simplex virus (HSV) resulted in the early induction of an altered ribonucleotide reductase. The extent of the induction was dependent upon the m.o.i. and could be diminished or prevented by u.v. treatment of the viral stock, or by inhibitors of mRNA synthesis or protein synthesis. The induction followed the same course of synthesis as viral thymidine kinase and DNA polymerase, and could thus be classified with them as a β polypeptide. These results suggested that the new activity was produced as a consequence of the virus genome expression. Comparisons of the properties of ribonucleotide reductase extracted from exponentially growing BHK-21/C13 cells showed that the HSV-induced enzyme differed from the cellular isozyme by its insensitivity to inhibition by dTTP, dATP or araATP and its resistance to high salt concentrations. On the other hand, the virus-induced enzyme and the cellular isozyme exhibited a similar sensitivity to hydroxyurea. Therefore, the reported inhibition of HSV DNA replication by hydroxyurea could be the result of inhibition of both HSV-induced and cellular reductase activities.

2-Deoxy-d-glucose (dGlc) was found to selectively inhibit virus DNA synthesis in human embryonic lung cells infected with human cytomegalovirus (HCMV). The effective concentration of dGlc was approx. 10-fold higher in culture medium containing glucose instead of sodium pyruvate. This inhibitory action of dGlc was fully reversible following replacement of the inhibitor medium by fresh medium after a 48 h treatment of infected cells. Virus DNA synthesis could be selectively inhibited by addition of dGlc even after initiation of HCMV DNA replication. In contrast, virus DNA synthesis in herpes simplex virus-infected cells was insensitive to dGlc. The drug was found to deplete HCMV-infected cells of uridine triphosphate and caused a progressive reduction of uridine incorporation into RNA. To substantiate a possible interference by dGlc with the expression and/or function of virus-induced, chromatin-associated factors essential for virus DNA replication, DNA synthesis of chromatin preparations from dGlc-treated, HCMV-infected cells was analysed. In contrast to preparations of untreated or phosphonoacetic acid (PAA)-treated, HCMV-infected cells, those of dGlc-treated cells lacked significant in vitro DNA-synthesizing activity; virus DNA was not synthesized by these preparations. Tunicamycin in the presence of low concentrations of dimethyl sulphoxide was also found to be effective in abolishing HCMV-induced DNA replication. It is thus suggested that dGlc interferes with the function of an ‘early’ chromatin-associated glycoprotein essential for virus DNA synthesis.

The oligosaccharide moieties of vesicular stomatitis virus glycoprotein from virus grown in four different cell lines have been characterized by sequential enzymic degradation followed by ion-exchange chromatography and analytical gel filtration. Whilst the same two peptide sites are glycosylated in all cell lines, the extent of sialylation of the oligosaccharides is, however, a function of the cell line in which the virus is produced. Using specific purified glycosidases for sequential degradation of glycopeptides obtained after Pronase digestion, the oligosaccharide structures from the different host cell lines appear similar. However, differential sensitivity of the glycopeptides to treatment with a partially purified mixture of endo- and exoglycosidases indicates that the oligosaccharide structures are not identical.

The interaction between mouse hepatitis virus 3 (MHV3) and cells was studied in order to investigate whether or not early events occurring after infection could be involved in the difference in virus replication seen between mouse strains with different genetic sensitivities to MHV3 infection. Kinetic data showed that MHV3 uptake by both macrophages and L cells was time- and temperature-dependent. In addition, treatment of cells with cytochalasin B or prostaglandin E1, prior to virus infection, resulted in a strong inhibition of sheep red blood cell phagocytosis without any effect on MHV3 uptake. Similar uptake of radiolabelled MHV3 was shown by whole spleen cells, purified T lymphocytes and thymocytes. Furthermore, no difference in 3H-labelled MHV3 uptake was seen between macrophages originating from resistant A/J mice, semi-susceptible (C57B1/6 × A/J)F1 and susceptible animals. These results indicate, therefore, that genetically related in vivo sensitivity toward MHV3 infection is not related to differential uptake of virus by cells.

An ecotropic type C retrovirus (D1-MuLV) isolated from SJL/J mice was injected into neonatally thymectomized SJL/J mice. There was no acceleration of development of reticulum cell neoplasm (RCN) in these mice as compared with the control, uninoculated but similarly thymectomized group. The incidence of RCN at 10 and 11 months after injection was 14.6% and 12.5% respectively. Two female mice inoculated with D1-MuLV developed mammary adenocarcinoma. There was persistence of high titres of the ecotropic virus associated with RCN and mammary tumour of SJL/J mice. Xenotropic virus (X-MuLV) was detected in spleens of normal SJL/J mice at ages 6 and 12 months (60% and 80% respectively) but not at other ages. The X-MuLV isolated from SJL/J mouse embryo cell cultures treated with 5-iodo-2′-deoxyuridine (SJL-MEF-X-MuLV) and that isolated from a spontaneous RCN (SJL-RCN-X-MuLV) were compared with NZB-X-MuLV (NZB mouse origin) and AT124-X-MuLV (NIH Swiss mouse origin) in regard to their host range, ion and primer-template preference by reverse transcriptase, virus interference and neutralization characteristics. Cross-neutralization and gp70 competitive radio-immunoassays showed that D1-MuLV is more closely related to AKR-MuLV than to Rauscher-MuLV and appears to share some virus envelope antigens with SJL-X-MuLVs. The type-specific gag gene product (p12) from Balb:virus-2 and NZB-X-MuLV was used in competitive radioimmunoassay, and SJL-RCN-X-MuLV was found to be more closely related to Balb:virus-2 (MuLV-Xα) than to NZB-X-MuLV (MuLV-Xβ).

The major antigens of a mouse mammary tumour virus (MMTV) isolated from the milk of exogenously infected MB+ Swiss mice were compared with the viral components of other MMTV strains by using the methodology developed by Teramoto et al., (1977a). The anti-gp47 (Swiss) serum differentiated between type- and group-specific antigenic determinants on the major glycoprotein; two distinct type-reactivities were demonstrated, one of them being shared by the MMTVs (Swiss) and (RIII), and the other by the MMTVs (C3H) and (GR). The MMTVs (Swiss) and (RIII) also reacted identically in a ‘type-specific’ assay using an anti-p28 (Swiss) serum, but a distinct reactivity was observed with the MMTV (C3H). In the isologous test where an anti-(total RIII) serum was used to bind intact, externally labelled RIII virions, the Swiss virus was seen to possess a type-specific determinant on its surface which distinguished itself from both MMTVs (RIII) and (C3H). The Swiss/fC57BL mice (which are devoid of the milk virus and for this reason are referred to as MB−), expressed only the internal virus antigens in their mammary glands. Under the ‘type-specific’ assay conditions, the p28 antigen present in the mammary gland extracts of the MB+ mice was indistinguishable from the p28 antigen purified from the Swiss virion, but was clearly distinct from the p28 reactivity present in the mammary gland extracts of the MB− mice. The p28 (MB−) antigen may thus represent the expression of an endogenous virus sequence different from the milk virus genome.

We have compared endogenous proviruses in DNA of chickens of 11 breeds by means of Southern’s technique. Many of the endogenous virus loci found were missing from the genomes of the extensively studied white leghorn chickens, although some of the proviruses and especially ev-1 appeared to be widely spread among different chickens. Most of the proviruses were similar to Rous-associated virus (RAV-O) as judged by the EcoRI digestion patterns. Several genetically different proviruses were found in the DNA of 28 brown leghorns. They contain neither ev-1 provirus in their genome, nor any other one common for all individuals. All four Italian partridge-coloured chickens examined appeared to be free from ALV-related sequences in their DNA showing the possibility of normal life without known endogenous proviruses. Possible causes of inter-breed differences of chicken endogenous proviruses are discussed.

Hepatitis B virus-related DNA was detected in the chromosomal DNA of three out of seven hepatocellular carcinomas and two out of five cirrhosis samples examined, by means of the blot-hybridization technique, described by Southern (1975). The integration patterns were not identical but some similarities raise the question of whether there are some preferred sites of viral integration.

Duplex RNA molecules made by hybridization of virion and mRNA of vesicular stomatitis virus (VSV) were digested with ribonuclease and separated into five size classes, each containing the gene and the mRNA for one of the VSV proteins. Denaturation of the duplexes yielded full size mRNA lacking poly(A) tails. Utilizing duplex formation between the RNAs from VSV temperature-sensitive (ts) mutants and their revertants and subsequent RNase digestion under varying salt conditions, specific cleavages within a certain duplex were seen for representative mutants from complementation groups III, IV and V. Specific cleavages were not seen for a group II mutant. From these results gene assignments cannot be made for group II; equivocal assignments are made for group III and clear assignments made for group IV and V. The assignment for the group V mutants, however, does not conform to expectations. Nevertheless, from these studies and other published ones, there is the suggestion that interactions may exist between the gene products of complementation groups II and V during VSV transcription and morphogenesis. These results also support the lack of transcriptional splicing for VSV mRNAs.

An assay for antiviral antibody based on antibody-dependent plaque enhancement (ADPE) in the macrophage cell line P388D1 is described which is as sensitive as, or more sensitive than a radioimmune assay. The method is applicable to a range of Togaviridae and Bunyaviridae although not to Picornaviridae and Herpesviridae. The variables affecting the assay are investigated.

Viruses isolated from ticks (Ixodes uriae) and a kittiwake (Rissa tridactyla) from a seabird colony at St. Abb’s Head, Scotland, were shown by complement fixation tests (CFT) to be antigenically related to the Uukuniemi and Kemerovo serogroups. Electron microscopic examination of cell cultures infected with the Kemerovo group viruses revealed particles characteristic of orbiviruses, 72 ± 3 nm in diam., with an inner core 37 ± 3 nm in diam., in association with intracytoplasmic, densely staining granular areas, and with fibrillar and tubular structures. Cell cultures infected with the Uukuniemi group viruses revealed characteristic bunyavirus particles, 94 ± 7 nm in diam., with a closely adherent envelope. Both orbi- and bunyaviruses were isolated from two tick pools and the kittiwake. A third tick pool contained an orbivirus which cross-reacted with the other isolates in CFT and fluorescent antibody tests, but was distinguished from them by neutralization tests.

Mouse thymic virus (TA) is a naturally occurring herpesvirus of laboratory and wild mice, which produces massive thymic necrosis when inoculated into newborn mice. Our histopathological study showed necrosis not only of the thymus but also of the spleen and lymph nodes which was noticeable by day 7 and complete by day 14. Both spleen and lymph nodes regenerated to an almost normal histological pattern by day 70. The results show that TA infects multiple lymphoid tissues causing massive necrosis in all, and is not limited to a single site, the thymus. TA infection was found to be a persistent herpesvirus infection in both the lymph nodes and spleen. During the period of acute infection, as necrosis increased, the response of cell suspensions of lymph nodes to the T cell mitogens concanavalin A and phytohaemagglutinin was virtually non-existent. Activity returned to normal as the histological repair progressed.

Human cytomegalovirus (HCMV), purified exclusively from the extracellular media, contained a DNA polymerase activity in addition to a protein kinase activity. The DNA polymerase expressed its maximum activity in the presence of 5 to 10 mm-MgCl2. The enzyme was able to use effectively activated calf thymus DNA, poly(dA).oligo(dT)12–18 and poly(dC).oligo(dG)12–18 as the template primers. The DNA polymerizing activity was eluted with 0.18 to 0.2 m-KCl from a phosphocellulose column. It was relatively resistant to phosphonoacetic acid inhibition even at a high concentration of 100 µg/ml with activated calf thymus DNA as the template primer, but the DNA polymerase activity was totally suppressed at this concentration when poly(dA).oligo(dT)12–18 was used as the template primer. The enzyme activity was inhibited by ammonium sulphate at 0.01 to 0.3 m with either activated calf thymus DNA or poly(dA).oligo(dT)12–18 as the template primer. The protein kinase has maximum activity in the presence of 10 to 20 mm-MgCl2, and preferred virion proteins as phospho-acceptor to protamine sulphate. Histone, caesin and bovine serum albumin (BSA) were found to be poor substrates. The phosphorylated protein pattern of the in vivo [32P]orthophosphate-labelled virions was not identical to that of the in vitro phosphorylated Nonidet P40-dissociated virions, although seven phosphorylated polypeptides did co-migrate in SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Procedures known to solubilize virions showed that the DNA polymerase and protein kinase were internal components of the virion.

A rabies virus persistent infection in BHK21 S13 cells was established and maintained in culture for more than 4 years. Initially, the cultures produced a large plaque virus similar to that produced by the original virus, but between the 10th and 20th passage, this was replaced by a small plaque variant. By the 200th passage, infectious virus could no longer be detected in the medium. After further cell passages (⩾ 300) no infectious particles could be detected in the medium. At various passage levels, the persistently infected cells were labelled with [35S]methionine and the virus antigens immunoprecipitated and analysed by polyacrylamide gel electrophoresis. No changes in the virus polypeptides were observed in the establishment of the persistent state. However, after the 20th passage (predominance of small plaque variant) there was an increase in the size of the glycoprotein. This was followed (164th passage) by a change in the M1 polypeptide which was subsequently further modified in the defective state (⩾ 300 passages). Virus isolated from the 400th passage by treatment of the cells with DEAE-dextran, was also modified in the glycoprotein and M1 polypeptides and contained less L polypeptide than the original virus. This virus grew more slowly, to a lower titre and was no longer pathogenic in suckling mice.

Monoclonal antibodies raised against the Hallé strain of measles virus were tested for their strain specificity using other measles virus isolates (Edmonston, Leningrad, Lec and two fresh isolates JT and Fasquelle). Monoclonal antibodies to L, HA and NP polypeptides, with one exception, reacted with all the measles virus strains tested; antibody from hybrid line 25 failed to react with the NP polypeptide of JT virus by immunofluorescence or radioimmunoprecipitation. Three other monoclonal antibodies, two reacting with NP and one with HA, although giving a positive immunofluorescence reaction, did not immunoprecipitate labelled antigen from JT virus-infected cells. The immunological relationship of canine distemper and measles viruses was also investigated with monoclonal antibodies. Two of the three anti-NP and three of the four anti-HA monoclonal antibodies reacted with canine distemper virus in an immunofluorescence reaction but only one (anti-NP) reacted in a radioimmunoprecipitation test.

Two-dimensional analysis of polypeptides from A/FPV/Rostock/34 (FP/R)-infected chick embryo fibroblast cells using non-equilibrium pH gradient gel electrophoresis followed by polyacrylamide gel electrophoresis, showed that nucleoprotein (NP) was the only detectable virus phosphorprotein and was present in both the nucleus and cytoplasm. The kinetics of accumulation of phosphorylated NP in the nucleus and cytoplasm were similar, suggesting that the presence or absence of phosphate groups did not control the entry of NP into the nucleus. In the course of this study, two-dimensional analysis of [35S]methionine-labelled FP/R-infected cells revealed some major differences from previously published work which are discussed.

The synthesis of virus-specific polypeptides and messenger RNA in cell cultures persistently infected with an isolate of measles virus from a patient with subacute sclerosing panencephalitis (SSPE) has been compared to that found in a lytic infection with the homologous virus. The persistent infection described here was chosen as its biological characteristics reflect those of virus-infected brain cells from SSPE patients. The synthesis of H, N and possibly F protein was seen in both lytic and persistent infections, but the synthesis of M protein was only detected in the lytic infection. However, messenger RNA isolated from either the lytic or persistent infection directed the synthesis in a cell-free translation system of all structural polypeptides, including M, and also three non-structural polypeptides, with mol. wt. of 34000, 30000 and 18000. Messenger RNAs coding for the virus-specific polypeptides were also shown to be polyadenylated. In addition, those polypeptides made in vitro which were antigenically related to the haemagglutinin, demonstrated structural changes after passage through a persistent infection.