- Volume 63, Issue 2, 1982

Two plaque-size variants of the neurotropic JHM strain of mouse hepatitis virus have been isolated from the virus stock after eight serial passages in suckling mouse brain. One variant, JHM-DL, produces large plaques, while the other, JHM-DS, produces small plaques in tissue culture. DS replicates more slowly, has a lower virus yield in vitro, and is less virulent for mice than DL. They also differ in their pathogenicity for mice: JHM-DL infection results in acute encephalomyelitis while JHM-DS infection results in demyelination. Oligonucleotide fingerprint analysis of the RNA genomes of these two variants revealed that they had almost identical genetic sequences. Each variant, however, had a unique oligonucleotide spot not found in the other. The unique spot of the large plaque variant, JHM-DL, was localized at approximately 3 to 5 kb from the 3′ end, while the JHM-DS unique spot was mapped at 14 to 15 kb from the 3′ end of the genome. We have further shown that these oligonucleotide changes are not correlated with the plaque morphology. These two viruses may be useful for studying the molecular basis of virus-induced demyelination.

Deletions in the cloned thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1), strain 17 syn +, were produced by two methods. Removal of a 506 base pair fragment from between the unique SstI and BglII restriction endonuclease sites of pTK1 (HSV-1 BamHI p cloned in pAT153) and subsequent transformation of Escherichia coli resulted in the isolation of 50 deleted plasmids. Sequential digestion of pTK1 with BglII and nuclease BAL 31 followed by ligation and recleavage with BglII resulted in the isolation of 31 deleted plasmids. Three clones, pTK2, pTK3 and pTK4, obtained following BglII and SstI treatment of pTK1 were recombined with wild-type (wt) HSV-1 (17) syn + DNA in baby hamster kidney (BHK) cells to produce TK− deletion mutants HSV-1 (17) TK 1301, HSV-1 (17) TK 1302 and HSV-1 (17) TK 1303 respectively. 5-Bromo-2′-deoxyuridine, 5-bromo-2′-deoxycytidine and 9-(2-hydroxyethoxymethyl)guanine were used to reduce the background of TK+ virus in heterogeneous recombinant stocks analysed for the presence of TK− recombinants. All recombinant clones isolated produced a small syncytial plaque morphology in BHK cells. The mutants HSV-1 (17) TK 1301 and HSV-1 (17) TK 1302 were TK−, failed to produce polypeptides of molecular weights 43000 and 19000 found in wt-infected cells and demonstrated one-step growth curves different from wt virus and the TK− mutant HSV-1 (17) dPyk−7. Superinfection studies with HSV-1 (17) TK 1301, HSV-1 (17) TK 1302, HSV-1 (MDK) and HSV-1 (17) dPyk−7 indicated that all TK− mutants except dPyK−7 produce a trans-acting gene product which can switch on the transforming HSV-1 TK gene.

The production and properties of three monoclonal antibodies, designated LP1, LP4 and AP2, directed against non-glycosylated polypeptides of herpes simplex virus type 2 are described. LP1 is specific for polypeptide VP16 and cross-reacts with HSV-1; LP4 reacts with the major DNA-binding protein and is type-specific. AP2 is directed against the major capsid antigen of HSV-1 and HSV-2.

The infectivity of herpes simplex virus, type 1, strain ANG progeny DNA from standard virus infections and of progeny DNA from infections involving defective-interfering virus particles (DI DNA) was compared in transfection assays. No difference in infectivity of virus DNA isolated either from infected cells or from progeny virus was found for a given type of infection. However, the values for the two types of infection differed markedly, with DI progeny DNA being less infectious by more than 2 log10. The low infectivity was mainly due to the presence of interfering DNA molecules in DI progeny DNA, regardless of whether intracellular DNA or DNA extracted from mature virions was analysed. The interfering capacity of DI progeny DNA did not depend on the integrity of the genomes. The physical proximity provided by simultaneous precipitation of infectious and of interfering DNA is an important factor influencing the degree to which DI DNA interferes. Interference by DI DNA in the transfection assay can be partly reversed by the addition of XbaI fragments of standard DNA; in control experiments this fragmented DNA was shown to lead to a reduction rather than to an enhancement of the infectivity of standard virus DNA.

The herpes simplex virus type 1(HSV-1) strain ANG, unlike the majority of HSV-1 isolates, does not cause lethal encephalitis in various inbred mouse strains, when applied in doses up to 2 × 107 plaque-forming units intraperitoneally, intravenously, intravaginally, orally, or via the foot pads. We studied and compared the progress of infection by this non-lethal strain and by a lethal HSV-1 strain, and the components of the host defence mechanism involved. If injected intracerebrally, HSV-1 ANG replicated efficiently in mouse brain cells and led to encephalitis. Upon systemic or peripheral infection, it replicated in several mouse organs with a virulence similar to lethal HSV-1 isolates. It was clear that transport of HSV-1 ANG to the central nervous system (CNS) or replication in CNS tissue is efficiently restricted after peripheral infection. Conceivably, infection with lethal HSV-1 strains proceeds in two distinct steps, virus replication at the site of infection and in the spleen and, secondly, transport to CNS tissue and propagation in CNS cells. This second step is apparently blocked in infections of DBA/2 mice by HSV-1 ANG and can thus be studied separately. The blocking mechanism was not a function of interferon induction or sensitivity, nor was it due to an enhanced NK cell activation. Experiments with silica-treated mice, and with homozygous nude mice, which lacked T-lymphocytes, suggested that the observed restriction in virus transfer is independent of T-cell and macrophage functions. Yet, newborn mice were fully susceptible to intraperitoneal infection with HSV-1 ANG, suggesting that age-dependent defence mechanisms, the nature of which needs to be further examined, are of relevance in the restriction of peripheral infection by HSV-1 ANG in adult mice.

Production of circulating interferon (IFN) was measured in inbred mouse strains following intravenous injection of herpes simplex virus type 1 (HSV-1). IFN titres reached maximal levels 2 to 3 h after injection of virus and a 10-fold difference was found between C57BL/6 mice and BALB/c, as high and low producers respectively. Mendelian analysis revealed that HSV-induced IFN production is governed by several loci, one of which is X-linked. The strain distribution pattern obtained from results in recombinant inbred lines and the results obtained in the congenic B6-C-H-28c-If-11 strain furthermore indicated an absence of close linkage to If-1. It is concluded that the levels of HSV-induced early IFN production are influenced by several autosomal loci and one X-linked locus.

Splenic leukocytes derived from N- and P-line chickens exposed to turkey herpesvirus (HVT-4), and from UCD-003 chickens exposed to the non-oncogenic SB-1 clone of Marek's disease virus were fractionated into subpopulations at various days post-exposure. The level of infection in these fractions was determined by enumeration of plaque-forming units after co-cultivation of leukocytes with virus-permissive chicken embryo fibroblasts. At 5 days post-exposure most HVT-4-infected lymphocytes were found to be of intermediate to low buoyant density (1.040 to 1.065 g/ml) and not to have detectable Fc receptors. They possessed surface Ia but were not IgM-bearing. At 15 days post-exposure HVT-4-infected lymphocytes were found to be of intermediate to high buoyant density (1.070 to 1.080 g/ml) and, at this time, virus isolation rates (p.f.u./106 cells) from fractions enriched in cells bearing Fc receptors were approximately equal to those leukocyte suspensions depleted of Fc receptor-bearing cells. Furthermore, the percentage reduction in infectivity caused by depletion of Ia-bearing cells at 15 days post-exposure was less than that at 5 days post-exposure. At both 5 and 15 days post-exposure carbonyl iron treatment failed to remove HVT-4-infected lymphocytes. These characteristics were similar whether HVT-4-infected lymphocytes were derived from N- or P-line chickens. SB-1-infected lymphocytes derived from UCD-003 chickens at 6, 7 and 14 days post-exposure were detected with equal frequency in fractions enriched or depleted of cells possessing Fc receptors. SB-1-infected lymphocytes for the most part lacked surface Ia and IgM, and were not depleted by carbonyl iron treatment. From these data it was concluded that most HVT-4- and SB-1-infected lymphocytes detectable by virus isolation were neither B cells nor macrophages.

Tritium-labelled, purified herpes simplex virus (HSV) enveloped particles produced in the presence of tunicamycin (TM) or 2-deoxy-d-glucose (DG) adsorbed to GMK cells equally as well as standard virus. However, in the presence of TM or DG, there was a reduced transport of virus DNA to cell nuclei and an increased sensitivity of attached particles to proteinase K. The reduced infectivity of HSV produced in the presence of glycosylation inhibitors is therefore probably due to an impairment in the fusion of virus envelope with plasma membranes.

Because of the association of measles virus with persistent infections such as subacute sclerosing panencephalitis, and its possible involvement in multiple sclerosis, we have investigated the capacity of this virus to establish chronic infections in human peripheral blood lymphocytes (PBLs). We have demonstrated that a latent, persistent infection of human PBLs with measles virus results in low levels of infectious virus production in which large amounts of virus-induced interferon could be detected. Further, treatment of these silently infected cells with an anti-human leukocyte interferon serum results in a productive measles virus infection. The mechanism by which the anti-interferon serum shifts the virus-cell interaction from persistence to productive infection is discussed.

We have examined the relationship between induction of interferon (IFN) and prostaglandin E (PGE) production by poly(rI).poly(rC) in cultured human foreskin fibroblasts (FS11). Hydrocortisone and dexamethasone (2.5 × 10−7 m), which are known inhibitors of PGE synthesis, significantly decreased the induction of both IFN and PGE in IFN-pretreated (primed) cells. Desoxycorticosterone, progesterone and estradiol were devoid of this activity. Hydrocortisone also blocked the induction of IFN by double-stranded RNA (dsRNA), cycloheximide and actinomycin D in FS11 cells. Arachidonic acid overcame the inhibitory effect of hydrocortisone on PGE production, but failed to restore IFN production in the presence of the steroid. Moreover, the prostaglandin synthetase inhibitors, indomethacin, aspirin and flufenamic acid, did not change IFN production by dsRNA in primed FS11 cells, although prostaglandin synthesis was abolished. Although the induction of IFN and PGE by poly(rI).poly(rC) might be consequences of the same initial event in the cell, the accumulation of PGE does not seem to have a regulatory effect on the synthesis of IFN in this system.

We have tested the effect of the culture medium of chicken embryo fibroblasts (CEF) transformed by Rous sarcoma virus (RSV) on plasminogen activator (PA) activity of normal and RSV-infected cells. The results obtained showed that fibrin digestion by cultures of normal uninfected and RSV-infected cells directly attached to a fibrin substrate was increased when the cells were exposed to the supernatant of an 18 h-culture of CEF transformed by RSV. Addition of cycloheximide to the supernatant completely abolished this effect. Similarly, exposure of both normal and RSV-infected cells to the culture medium of RSV-transformed CEF resulted in a marked increase of the soluble PA activity present in the supernatant. This effect, however, was not abolished by inhibition of protein synthesis. For both the cell-bound and soluble PA activities of normal and RSV-infected cells the stimulatory effect was transient, being maximal between 8 and 12 h of exposure to the transformed cell-conditioned medium and disappearing after 24 h. A possible correlation with transformation-enhancing factor(s), previously reported to be present in the culture medium of transformed cells, is discussed.

Inoculation of inactivated 146S foot-and-mouth disease virus particles into guineapigs elicited the formation of neutralizing antibody and the serum had a 10-fold higher titre in radioimmunoassay (RIA) with 146S particles than with the 12S virus subunit. In contrast, a single inoculation of the 12S subunit or the isolated polypeptide VP1 elicited the formation of antibody having a much lower titre in RIA with the 146S particle than with the 12S subunit and low or undetectable neutralizing activity. However, sera from guinea-pigs given two or more inoculations of the 12S subunit or VP1 had neutralizing activity. The level in the anti-VP1 serum was lower than that in the anti-12S serum and both were much lower than that in animals receiving two inoculations of the 146S particle. The neutralizing activity elicited by the three antigens was absorbed by the homologous antigen. In contrast, neither the 12S subunit nor VP1 absorbed the anti-146S neutralizing antibody and VP1 did not absorb the anti-12S subunit neutralizing antibody. However, the 12S subunit partly absorbed the neutralizing activity elicited by VP1. The results are compatible with a model in which the 146S particle elicits a spectrum of neutralizing antibodies which are completely absorbed by the homologous particle but only partially by the 12S subunit or VP1. The results are discussed in relation to the structural features required for the production of neutralizing antibody.

DNA from African swine fever (ASF) virus was isolated and was characterized by two restriction enzymes, SmaI and EcoRI. Although both enzymes can distinguish Vero cell-adapted ASF isolates by characteristic restriction endonuclease cleavage patterns, all ASF isolates examined exhibited a high degree of similarity, as measured by co-migration of most of the DNA fragments. The molecular weight of ASF DNA, based on size estimates of DNA fragments from cleavage patterns, ranged from 93 × 106 to 100 × 106. Virus genome heterogeneity was observed in uncloned, cell culture-adapted ASF isolates as well as in a plaque-purified virus after serial passage in Vero cells. In contrast to the rather minor differences in restriction pattern among the Vero cell-adapted isolates, a major alteration in restriction endonuclease cleavage sites was observed during adaptation of the wild-type virus to cell culture.

Human embryonic fibroblasts (HEF) have been transformed by BK virus (BKV) DNA and by u.v.-inactivated or live BKV alone or in association with methyl-cholanthrene (MTC). The transformed cells produced BKV large T and small t antigens as well as the cellular 53 kdal protein, detected by immunofluorescence and immunoprecipitation. After an initial phase of lysis and virus shedding, virus or its coat protein antigen could not be detected in transformed cells. All human transformed cell lines could be superinfected by BKV or BKV DNA, but their susceptibility to superinfection was 20- to 500-fold lower than normal HEF. BKV could be rescued by fusion of transformed cells with normal HEF or Vero cells and by transfection of normal HEF with total DNA and DNA extracted from the Hirt supernatant of transformed cells. Blot hybridization analysis of DNA from transformed cells showed a considerable amount of free BKV DNA in monomeric and polymeric forms. Integrated BKV DNA was absent in most cell lines but present in only small amounts in BKV-transformed cells treated with MTC. Analysis of free BKV DNA with various restriction endonucleases and by blot hybridization showed that monomeric forms were complete BKV genomes, whereas polymers contained both complete and defective or rearranged BKV DNA. Transformation of HEF was also obtained with a 3.7 kilobase (kb) fragment of the BKV genome, produced by sequential digestion of BKV with the restriction endonucleases HhaI and EcoRI. This fragment extends clockwise on the virus genome from 0 to 72.2 map units and contains the entire early region. Blot hybridization analysis of cells transformed by the HhaI/EcoRI 3.7 kb fragment showed two separate integrations of BKV sequences without free virus DNA.

Restriction mapping of polyoma virus DNA in mammary tumours of athymic mice gave patterns that varied with the tumour examined. These reflected differences in both the organization and the state of integration of virus genomes in the host chromosomes. All tumours contained tandemly integrated full-length and defective virus genomes. Some tumours also contained unintegrated virus DNA molecules, some full-length and others defective. The deletions were localized in the virus genomic sequences coding for the distal part of the large T antigen. After the first transplantation, the organization of polyoma virus genomes in tumours remained essentially unchanged through four successive transplantations. The tumour cells that initially contained free virus DNA molecules continued to possess such molecules during serial transplantations. The virus DNA molecules in transplanted tumours lacking unintegrated virus genomes were more methylated than in tumours containing unintegrated virus genomes.

Inkoo virus (a bunyavirus) was grown in BHK-21 cells and labelled with [35S]methionine or [3H]mannose. [35S]Methionine labelled the two envelope glycoproteins G1 (M r = 125000) and G2 (M r = 35000), as well as the nucleocapsid protein N (M r = 25000). Only G1 and G2 were labelled with the sugar precursor. The [3H]mannose-labelled virus was solubilized with detergent and digested with Pronase. The structure of the labelled glycopeptides originating from the mixture of G1 and G2 was studied by degrading the glycans stepwise with specific exo- and endoglycosidases, and by analysing the products by both gel and paper chromatography, as well as lectin-affinity chromatography. Three classes of N-glycosidic glycans were found: complex glycans with the monosaccharide sequence (NeuNAcαGalβGlcNacβ)≥2 (Man)3 (GlcNAc)2 (occurrence of fucose was not studied), high mannose-type chains with the average structure (Man)4-6 (GlcNAc)2, and endoglycosidase H-resistant small glycans which were partly susceptible to mannosidase. These latter types of oligosaccharide chains are a novel finding among virus glycoproteins. The relative ratio of the three types of oligosaccharide chains was roughly 4.6:1:1 respectively. The G1 glycoprotein carried most of the sugar chains, since it contained 85% of the [3H]mannose label. The results are discussed in relation to the site of virus maturation at smooth-surfaced vesicles in the Golgi region.

Prostaglandins of the A series potently inhibited the production of vaccinia virus in mouse L fibroblasts. With the highest non-toxic dose of PGA1, 4 µg/ml, the replication of the virus was inhibited by 95.3%. The antiviral activity was dose-dependent and specific for the A series. At the dose used, PGA1 was not toxic to uninfected cells and did not alter cell metabolism as measured by DNA, RNA and protein synthesis. PGA1 did not influence the adsorption of the virus by the host cells and the antiviral activity was not dependent on the presence of PGA1 during the early stages of infection. PGA treatment delayed and partially inhibited virus DNA synthesis and, while it did not produce any change in the pattern of protein synthesis in uninfected cells, it altered both the rate and the pattern of virus protein synthesis. We conclude that PGA1 selectively inhibits one or more steps involved in the replication of vaccinia virus in mouse L fibroblasts.

The biosynthesis of IgM by the Epstein—Barr virus-negative RAMOS lymphoblastoid cell line infected with an influenza A virus, fowl plague virus Dobson strain (FPV-B), was investigated. The results show that FPV infection of RAMOS cells slightly inhibited overall cellular protein synthesis only at 24 h after infection, despite the synthesis of FPV-specific proteins. However, even at this time, the synthesis and secretion of IgM were not affected by virus infection. Secreted IgM contained a reduced amount of sialic acid. The quantity of the asialylated IgM increased proportionally to the amount of enzymically active neuraminidase, suggesting that the asialylation of IgM is due to the action of virus neuraminidase. No such asialylated IgM was observed in RAMOS cells infected with measles virus, which does not possess neuraminidase. These results, together with a previous observation of ours that asialylated immunoglobulins acquire an altered antigenicity, suggest that the modulation of enzyme activities in B lymphocytes in response to an exogenous aggression may lead to disturbances in the structure and in the antigenic properties of immunoglobulins.

Virus structural antigens were localized within a line of monkey kidney (MA104) cells infected with the simian rotavirus SA11 using electron microscopic immunoperoxidase techniques. When hyperimmune guinea-pig anti-SA11 serum was used, virus particles, membranes of virus-associated endoplasmic reticulum, and viroplasmic inclusions were most heavily labelled. A general cytoplasmic reaction (ribosomes, intracytoplasmic membranes, etc.) with anti-SA11 serum was also observed, but nuclei were unstained. In addition, several other virus-induced structures were found to contain rotavirus proteins, including convoluted smooth membrane within the endoplasmic reticulum, aberrant virus-like particles, and 15 to 20 nm diam. cytoplasmic tubules. Monospecific antiserum to VP7 (outer capsid glycoprotein, mol. wt. 38000) reacted strongly with virus particles and the virus-associated endoplasmic reticulum, but reacted poorly with viroplasmic inclusions. The nucleus and general cytoplasm were unstained with anti-VP7. In contrast, monospecific antisera to VP2 and VP6 (inner capsid proteins, mol. wt. 94000 and 41000 respectively) reacted very strongly with viroplasmic inclusions. Virus particles, endoplasmic reticulum and cytoplasmic ribosomes were also labelled with these sera. These results indicate that rotavirus inner capsid proteins are synthesized throughout the cytoplasm and become concentrated in viroplasmic inclusions, while the outer capsid glycoprotein is synthesized primarily on ribosomes of the rough endoplasmic reticulum. Thus, the outer capsid layer appears to be acquired during virus budding into cisternae of the endoplasmic reticulum.