- Volume 56, Issue 1, 1981

Sigma virus isolates have been extracted from samples of Drosophila melanogaster collected in the wild and some of the virus characteristics measured. Most of the virus isolates shared rather similar features after injection and had ‘medium’ values for incubation time, transovarial transmission pattern, and sensitivity to genes for resistance to the virus. One single serotype was found, similar to that of the laboratory strains already tested.

The transient, sublethal infection produced by intracerebral inoculation of the Flury high egg passage (HEP) strain of rabies virus into adult mice was converted into a lethal one (approx. 80 to 100% mortality) by administering 150 mg/kg cyclophosphamide (CY) 2 days after infection. Immunosuppressed, infected animals showed no immunological response to rabies and died 15 to 20 days after infection. However, mortality was reduced to 12% when suppressed mice were adoptively immunized, 4 days after infection, with an intravenous injection of 60 × 106 spleen cells from rabies-immune syngeneic donors. The lymphocytes obtained early after donor immunization (4 to 11 days) reduced mortality, whereas those obtained late (16 to 32 days after immunization) were not effective. The ability of donor cells to protect animals corresponded very closely with donor cytotoxic T lymphocyte (CTL) activity. Within 4 days after immune cell transfer, serum neutralizing antibody and CTL levels in recipients were comparable to those found in virus-infected control animals.

Immune donor cells were fractionated into thymus-derived (T-enriched) and bone marrow-derived (B-enriched) subsets. The T and B subsets reduced mortality to 32% and 34% respectively. CTL and serum neutralizing antibody responses could be detected in these animals, although they appeared later than in mice treated with unfractionated immune spleen cells. The present study demonstrates that both B and T lymphocytes are required for optimum clearance of rabies from the central nervous system (CNS) and suggests a functional role for rabies-specific CTL in vivo.

SV40 infection of Muntiacus muntjak cells (ATCC, CCL: 157) resulted in abortive transformation with formation of T antigen and induction of cellular DNA replication in the absence of virus production. These cells were resistant to stable transformation by SV40 regardless of the route of infection, including microinjection of virus into cell nuclei. The present studies show that T antigen-containing cells persist and that the number of T antigen-positive cells remains constant in infected cultures, which is reflected in the nearly constant fraction of T antigen-positive cells in stationary cultures and a decrease of the fraction of T antigen-positive cells in proliferating cultures. These data suggest that following cell division only one daughter cell on the average can maintain a detectable level of T antigen. The cell cycle kinetics of proliferating muntjac cultures were not altered by the abortively transforming infection, and infectious progeny was made following cell fusion with permissive CV-1 cells. It is suggested that the few copies of viral DNA that do persist are present in a ‘plasmid-like’ state as non-integrated DNA. In this way SV40 can appear to be lost from the majority of the cells in a culture but actually be conserved in a small population of cells for a long time. In such a state a papovavirus might go undetected in vitro as well as in vivo by even the most sensitive immunological and molecular techniques.

Persistently infected cultures have been established from Vero cells surviving primary infection with Tacaribe virus (Vero-T). The growth rate and morphological characteristics of the persistently infected cells were indistinguishable from normal Vero cells. Virus release declined during the first 6 passages, a cyclical pattern was observed between passages 6 and 16, and subsequently no virus infectivity could be detected. Co-cultivation with normal RK-13 or Vero cells enhanced virus yield from virus-producing cultures of Vero-T cells (passage 15), but the addition of susceptible cells had no effect on non-producer Vero-T cultures (passage 19). Only a small proportion (<1%) of the persistently infected cells tested during the first 16 passages produced infectious virus. The virus released during the early stages of persistence was temperature-sensitive if grown at 40 °C, more thermolabile at 50 °C than parental virus, and unable to initiate a persistent infection in Vero cells. Vero-T cells consistently showed refractoriness to homotypic Tacaribe virus superinfection and a selective graded resistance to other arenavirus replication. The possible use of viral susceptibility of persistently infected cultures as marker of antigenic relationship among Tacaribe complex viruses is considered.

A four-layer solid phase enzyme-immunoassay (EIA) and radioimmunoassay (RIA) techniques were applied for the type-specific detection of parainfluenza type 1, 2 and 3 virus antigens in sonicated nasopharyngeal specimens of patients with acute respiratory disease. Guinea-pig antiviral immunoglobulins on solid phase were used as the capture antibodies, rabbit antiviral immunoglobulins as the secondary antibodies, and horseradish peroxidase-labelled swine anti-rabbit immunoglobulins (EIA), or 125I-labelled sheep anti-rabbit IgG (RIA) as the indicator antibodies. A total of 174 nasopharyngeal specimens collected by mucus extractor were tested, and the results were compared with those obtained by a routinely used immunofluorescence (IF) technique. The same number of positive specimens were achieved by the EIA and the RIA and 3/4, 4/4 and 19/20 immunofluorescence (IF)-positive nasopharyngeal specimens were positive by the parainfluenza type 1, 2 and 3 immunoassays respectively. In addition, four parainfluenza type 1 and three parainfluenza type 3 virus-positive specimens were found by the immunoassays out of 146 parainfluenza IF-negative specimens. The type-specificities of the parainfluenza immunoassays were confirmed by showing that no cross-reactions occurred when purified immunizing antigens and the EIA-and RIA-positive clinical specimens were cross-tested. The results indicate that parainfluenza type-specific antigens can be detected directly in nasopharyngeal specimens by the immunoassays and the preliminary findings with a small number of positive specimens suggest that these assays have a diagnostic potential which is similar or slightly better than the IF techniques.

Restriction endonucleases EcoRI, BamHI, HindIII, BglI, BglII, HpaI and PstI recognized 48 cleavage sites in EDS adenovirus strain B8/78 DNA, whereas CELO virus DNA was cut into 61 fragments by the same enzymes. No similarity could be detected in the restriction patterns obtained from the two avian adenoviruses. The calculated mol. wt. of B8/78 DNA was 22.6 × 106 [about 34.2 kilobases (kb)] and that of CELO virus DNA was 28.9 × 106 (about 43.7 kb). The fragments generated from B8/78 DNA by EcoRI and BamHI were physically mapped.

We investigated the ultrastructural development and maturation of cytomegalovirus (CMV) nuclear inclusions (NIs) in human embryo thyroid cells at 1 to 144 h post-infection. At 5 h, most cells had rounded from an initial fibroblastic appearance and contained early NIs. At 24 h, early NIs were larger and better defined. At 48 h, although early NIs were still present, most cells had larger and presumably more mature NIs. These latter NIs consisted of several subunits, each made up of a fibrillar network enclosing an electron-lucent area which contained coarse and delicate granules. Also, at 48 h, virus particles were first seen in the nucleoplasm. At 72 h, in cells with more developed NIs, virus particles were closely associated with the fibrillar network. Between 96 and 144 h, the NIs reached maximum size and were made up of numerous subunits. The results indicate that two types of NIs coexist during CMV infection. The appearance of the early and late NIs coincides with the reported peaks of CMV DNA synthesis and thus may explain the biphasic pattern of DNA synthesis in CMV infection. Morphogenetic features of the NIs conform with the hypothesis that synthesis of CMV DNA may occur in the centre of each NI subunit and that the fibrillar network represents condensing capsid proteins.

The experiments show that 30 virus-induced or virus-specified proteins were synthesized in Raji cells after superinfection with Epstein—Barr virus (EBV) derived from P3HR1 cells. Using a combination of pulse labelling, application of cycloheximide blocks at different times post-infection, treatment with amino acid analogues and inhibition of DNA synthesis it was shown that three groups of proteins appear in Raji cells after superinfection; the synthesis of the proteins in any one group appears to be coordinately regulated. Amongst the six virus-induced proteins which were synthesized immediately after release from an early cycloheximide block one would expect to find those proteins essential for the transition from EBNA to EA synthesis. Using human sera with differing specificities for the various antigen groups 11 proteins were identified as being specifically precipitated by sera having high titres against the EBV-induced early antigen complex.

We have characterized Herpesvirus tamarinus (HT), a virus of New World primates related to herpes simplex virus. HT virion DNA cross-hybridized appreciably with herpes simplex virus type 1 (HSV-1) DNA. The HT-HSV-1 cross-hybridizing DNA sequences were distributed over the length of the genome; some DNA sequences, however, were apparently more homologous than others. HT virion DNA was estimated from the sum of restriction endonuclease fragments to have a mol. wt. of 98 × 106. Only 1 molar fragments were found with some restriction endonucleases and 0.5 and 1 molar fragments were found with other restriction endonucleases but no evidence was found for 0.25 molar fragments. Independent isolates of HT from different species of South American primates gave nearly identical restriction endonuclease cleavage patterns of virion DNA. Although cross-reacting antigenic proteins of HT and HSV-1 were not detected by neutralization or immunofluorescence tests, cross-reacting antigenic proteins were detected by SDS-polyacrylamide gel electrophoresis of immunoprecipitates of 35S-labelled infected cell extracts.

At least 12 virus-induced DNA-binding proteins with mol. wt. ranging from 14 × 103 to 119 × 103 have been isolated from frog virus (FV 3)-infected fathead minnow cells by DNA affinity chromatography. Two enzymic activities, DNA-dependent DNA polymerase and endodeoxyribonuclease, were present in the DNA-binding proteins; these enzymic activities were similar to those induced by FV 3 in infected cells. A single species of DNA-binding protein with a mol. wt. of 36000 had very high affinity for single-stranded DNA, but a low one for double-stranded DNA. Proteins with such characteristic affinity for single-stranded DNA destabilize the DNA helix and are essential for viral DNA replication. Thus, the 36000 mol. wt. DNA-binding protein is a candidate for such a role in FV 3 DNA replication.

The predominant RNAs in purified VSV particles are 42S and 4S in size. The 4S RNA is host transfer RNA that did not incorporate detectable radiolabel during VSV infection and was detected by in vitro labelling. Surprisingly, when BHK cells were prelabelled for 30 to 54 h before infection, the incorporation of [3H]uridine and [32P]orthophosphate into virus tRNAs remained very low and virus tRNAs were found to have a 5-to 15-fold lower specific activity than the total host tRNA, the value depending, in part, upon the period of prelabelling. Two-dimensional gel electrophoresis and partial sequence analysis indicated that the virus tRNAs include most species of host tRNA and no singly predominant species was apparent. Transfer RNAs are packaged by several enveloped viruses, but we have not found 4S RNA in reovirus, which lacks an envelope. We suggest that VSV contains a membrane-associated population of tRNA which has a slower rate of turnover than the total population of cellular tRNA.

The survival of interferon (IFN)-treated cells after encephalomyocarditis (EMC) virus infection depends on both the concentration of interferon and the multiplicity of infection (m.o.i.) used. The cell survived EMC infection if a high IFN/m.o.i. ratio was used in the experiment, whereas cell death took place at low IFN/m.o.i. ratios, even if IFN is also present during infection. Analysis by polyacrylamide gel electrophoresis of the proteins synthesized in IFN-treated cells subsequently infected with EMC indicated that no virus proteins were detected at either low or high multiplicities of infection. However, at low m.o.i. the cell survived and continued synthesizing cellular proteins exclusively, whereas at high m.o.i. a drastic shut-off of host protein synthesis took place. Virus which had been inactivated by u.v. irradiation was unable to cause the shut-off of host protein synthesis, either in control or in IFN-treated cells. This result suggests that some virus gene expression occurs in cells treated with IFN, although no virus protein synthesis was detected. The synthesis of virus RNA was also strongly inhibited after treatment of cells with IFN.

The integrity of the cell membrane in control and in IFN-treated cells was studied by analysing the 86Rb+ ion leakage, the thymidine pool, the choline uptake and the entry of the translation inhibitor hygromycin B, to which cells are impermeable, at different times after EMC infection. The results obtained indicate that the early membrane leakiness observed after virus infection is not prevented by IFN treatment. However, the development of late leakiness to 86Rb+ ions, thymidine and hygromycin B was not observed in IFN-treated cells.

Interferon production in Namalwa cells, a human lymphoblastoid line, was enhanced by lowering the incubation temperature after induction. The optimum conditions for this effect were established. At the lower temperature interferon synthesis proceeded at a lower rate but continued for longer. Interferon mRNA was shown to be associated with the polysomes for longer after induction. Addition of drugs that inhibit transcription did not prevent the increased production of interferon. Thus, the increased production of interferon is due to the prolonged translation of interferon mRNA.

The capacity of an egg-grown Sendai virus preparation to induce interferon in the human lymphoblastoid cell Namalwa is dependent on its passage history. Virus which has been serially passaged at high dilution is a poor inducer, whereas virus serially passaged undiluted is a good inducer. Such a good inducer preparation has a low infectivity to haemagglutination ratio as the result of a high content of defective-interfering (DI) particles. Using DI particles purified on glycerol gradients, it is shown that for the induction of maximum interferon titres both infectious and DI particles are required. DI particles alone induce little or no interferon. Addition of DI particles to fully infectious Sendai virus preparations increased the interferon yield obtained from Namalwa cells some 60- to 100-fold.

The 79000 mol. wt. measles virion membrane glycoprotein G has been isolated from purified measles virus. Ultracentrifugation of 2% Triton X-100-treated measles virus produced a soluble supernatant fraction containing both G and F, the other external viral membrane protein. Lentil lectin-Sepharose and Sephacryl S-300 column chromatography of this fraction gave a pure preparation of G protein. Sucrose density-gradient centrifugation and SDS-polyacrylamide gel electrophoresis revealed that G was isolated from the virion membrane in the form of a disulphide-linked dimer. Antiserum prepared against purified G reacted only with the G polypeptide of measles virus in a slab gel antibody overlay technique. The antiserum also exhibited haemagglutination inhibition, virus neutralization and haemolysis inhibition activities.

A measles virus (Halle SSPE isolate) induced ring plaque phenomenon (concentric rings of living and dead cells) has been shown to be associated with the temperature-dependent production of interfering particles. The interfering particles have been purified by potassium tartrate linear gradient centrifugation and have buoyant densities of 1.15 g/ml (M particle) and 1.06 g/ml (L particle) respectively. Both interfering particle populations have been shown to decrease the yield of wild-type measles virus from infected cells by 50% when co-infection experiments were performed. Neither the M or L particle population interfered with the growth of VSV in the same host-cell system.

Growth of influenza B virus was found to be greatly restricted in Vero cells compared with that in MDCK cells. The analysis of protein synthesis in infected cells showed that the synthesis of M protein is selectively inhibited in abortive infection. Cell fractionation experiments demonstrated that the viral glycoproteins, HA and NA, migrate from rough membranes via smooth membranes to plasma membranes in abortive as well as in productive cells. These results suggest that intracellular migration of influenza B virus glycoproteins occurs independently of the synthesis of M protein although M protein synthesis appears to be required for the formation of virus particles.

An immunoperoxidase-based quantitative assay has been developed for infectious units of Autographa californica nuclear polyhedrosis virus infecting TN-368 cells. Major benefits of this assay over existing quantitative assays for baculoviruses are that it is not dependent upon polyhedra production, it is clear-cut and easy to score, and results are obtained within 24 h.

Foot-and-mouth disease virus particles were observed by electron microscopy in the cytoplasm of alveolar secretory cells of the bovine mammary gland after contact exposure of uninfected cows to pigs with foot-and-mouth disease. Virus, contained in membrane-limited vesicles, was released from the basal and peranuclear portions of the cell into the intracellular and extracellular spaces by an exocytotic mechanism similar to that of the release of the milk-fat globule. Virus was released into the lumen from the apical portion of the cell both by membrane-limited vesicles and by the merocrinal exocytosis of casein-associated virus. The lytic release of virus was observed in 20% of the preparations observed.