- Volume 66, Issue 11, 1985

When microinjected as cloned DNA, the nucleoprotein (NP) of influenza virus A/NT/60/68 (H3N2) accumulated in the nuclei of Xenopus laevis oocytes, and cultured cells of rodent and primate origin. This accumulation appeared to be specific and a property of the NP itself (or conceivably NP in association with unknown cellular constitutents) since no other influenza virus components were present in DNA-injected cells. In the oocyte nucleus, clonally derived NP achieved an eightfold concentration over that in the cytoplasm. Such NP was full-length as judged by its mobility during PAGE and had the native conformation of H3N2 virus NP according to its reaction with a panel of monoclonal antibodies. NP appeared to be in the soluble fraction of the nucleus as it did not sediment under conditions which removed particulate matter from nuclear extracts. Microinjection of extracts of chick embryo fibroblast cells infected with A/FPV/Rostock/34 (H7N1) showed that exogenous NP had an affinity for the nucleus similar to that synthesized intracellularly from cloned NP DNA. This conclusion was supported by an experiment in which cloned NP from the oocyte nucleus re-entered the nucleus after injection into the cytoplasm of fresh oocytes. Injection of mRNA, extracted from chick embryo fibroblast cells infected with A/FPV/Rostock/34, into oocytes directed the synthesis of the viral proteins M (M r 28 000), and NS1 (M r 27 000) as well as NP (M r 56 000). While NP from this source concentrated in the nucleus as before, M merely associated with the nucleus without exceeding the cytoplasmic level. Even more remarkable was NS1; although in injected cells this protein is concentrated in nucleoli, in microinjected oocytes its nuclear concentration was threefold less than that in the cytoplasm, despite the very large number (> 1500) of nucleoli present in Xenopus oocytes. It seems likely that the karyophilic nature of M and NS1, unlike that of NP, is a property not of the proteins themselves, but of a complex which they form with some other product of the infected cell. These findings were repeated when extracts from infected chick embryo cells containing NP, M and NS1 proteins radiolabelled in vivo, were injected into the cytoplasm of oocytes.

In a search for myotropic viruses with a potential to initiate muscle autoimmunity, we found that two strains of influenza A virus, A/England/863/78 (H3N2) and the reassortant virus X-47 (H3N2), could infect human syncytial myotubes lytically. The X-47 strain could, in addition, infect unicellular precursor myoblasts. Intracellular viral protein synthesis was demonstrated by pulse-labelling studies in both cell types with both virus strains. By immunofluorescence and immunoelectron microscopy, viral antigens were demonstrated in infected muscle cells specifically identified by double staining with monoclonal antibodies to either of two independent muscle-specific antigens. However, using ‘co-capping’ techniques in conjunction with electron microscopy, there was no evidence of association between viral haemagglutinin and the acetylcholine receptor (one major target of autoimmunity to muscle cells) on the infected cell membrane.

A sub-immunizing dose of a synthetic peptide corresponding to the amino acids 141 to 160 region of protein VP1 from foot-and-mouth disease virus (FMDV), serotype O1, coupled to keyhole limpet haemocyanin (141-160KLH) has been shown to prime the immune system of guinea-pigs for an FMDV serotype-specific neutralizing antibody response to a second sub-immunizing dose of the same peptide. Optimal priming required an interval of 42 days between the priming dose and the booster dose. No priming was observed in the absence of adjuvant. The secondary response was not restricted by the carrier since animals primed with 141-160KLH could be boosted with uncoupled 141–160 or 141–160 coupled to tetanus toxoid. It has also been shown that uncoupled peptide 141–160 will prime for a neutralizing antibody response when it is incorporated into a relatively non-immunogenic carrier such as small unilamellar liposomes. These results indicate that the 141–160 peptide of FMDV, as well as containing an important neutralizing antibody site, can initiate its own T-helper cell response.

Antiviral effects of prostaglandins of the A series (PGAs) on Sendai, vaccinia and vesicular stomatitis viruses have previously been reported and a relationship between the antiviral actions of PGAs and interferons has been suggested. We have investigated the antiviral activity of PGAs on encephalomyocarditis (EMC) virus. Using single-cycle assays of virus replication our results indicate that PGAs only inhibit when present in the culture medium after the cells are infected, and that they are most effective during incubation periods including from 3 to 5 h post-infection. Furthermore, viral RNA synthesis is blocked in infected cells treated with PGA and, as a result, viral antigens are greatly reduced in the cytoplasm of the cells 5 h post-infection. Since the antiviral effect of PGAs is unperturbed by actinomycin D, when cellular RNA synthesis is greatly reduced, it appears unlikely that induction of new cellular proteins is the reason for the antiviral activity of PGAs. In separate experiments we were unable to demonstrate directly the induction of interferon, or of the two dsRNA-dependent enzymes, 2′,5′-oligoadenylate synthetase and protein kinase, which are greatly increased in interferon-treated cells. Thus, we conclude that the antiviral activity of PGAs is unrelated to the antiviral action of interferons and involves a unique mechanism independent of cellular protein synthesis.

The multiplication of the M9, A7 and L10 strains of Semliki Forest virus (SFV), both in weanling mice and primary mouse brain cell cultures, was compared. Following both intraperitoneal (i.p.) and intracerebral (i.c.) injection, the virulent L10 strain multiplied to higher titre in the mouse central nervous system (CNS) than did the less virulent M9 and A7 strains, whereas M9 multiplied to higher titre than A7. By the i.c. route, all three virus strains multiplied to higher titre than following i.p. injection. Multiplication of A7 and M9 in oligodendrocytes, but not neurons, was detected following i.c. injection. All three virus strains showed a tropism for cultured mouse glial cells rather than neurons. The L10 strain multiplied better in neurons than did A7 or M9. It is concluded that the mechanism of acute demyelination induced by the M9 and A7 strains is similar. Based on this and previous studies, it is proposed that infection of glial cells triggers immune-mediated demyelination. The virulence of the L10 strain is due to its ability to exceed a lethal threshold of damage to neurons before immune intervention can occur.

Exposure of rotavirus SA-11 to polyclonal neutralizing antibody from hyperimmunized guinea-pigs permitted selection of variants which were poorly neutralized by antisera against the parental virus. In one-way cross-neutralization experiments, at least 22 of 24 plaque-purified variants could be classified as belonging to a serotype different from that of the parent. Most antisera generated against the variants, however, readily neutralized the parental virus. This indicates that immunodominant neutralization epitopes in the parent differed from those in the variants. Changes in immunodominant epitopes caused the serotypic relationships between the variants and other strains of rotavirus to differ from those of the parental SA-11. The serotypic relatedness of human strain P (human serotype 3) was reduced while, in contrast to results found with the parental SA-11, several of the antisera against the variants recognized the bovine rotavirus NCDV as the same serotype. Causes for these changes are discussed.

Treatment of calves with bovine recombinant α1 interferon prior to challenge with bovine herpesvirus type 1 increased the animals’ ability to withstand a subsequent Pasteurella haemolytica challenge. The reduction in viral-bacterial synergy observed following interferon treatment did not appear to be due to a direct effect of the interferon on virus replication in the upper respiratory tract. Thus, even though interferon-treated animals shed slightly less virus from their nasal passages than did untreated animals, this reduction was not statistically significant. Furthermore, there was no difference in the level of intranasal interferon secreted by control or interferon-treated animals. These results suggest that interferon treatment does not affect the production of endogenous interferon. In contrast, a significant difference was observed between the number of days that control animals were sick, the levels of serum fibrinogen and the functional activity of polymorphonuclear neutrophilic granulocytes obtained from infected calves. These results suggest that bovine recombinant α1 interferon may have a greater immunomodulatory effect than a direct antiviral effect in this model. This is further supported by the observation that bovine herpesvirus type 1 is relatively resistant to the direct antiviral effect of bovine recombinant α1 interferon in vitro.

Human cell lines that contain and express the gene encoding the adenovirus type 5 DNA-binding protein (Ad5 DBP) are very useful for the isolation of adenovirus mutants with an altered DBP. In order to obtain these cells, human 143 tk− cells were transfected, using the calcium phosphate technique, with plasmids containing the Ad5 DBP gene and the herpes simplex virus thymidine kinase (HSV tk) gene as a selectable marker. Characterization of several tk+ transformants revealed that these cells did contain the HSV tk gene, but in none of these cells could Ad5 DBP DNA sequences be detected. However, when 143 tk− cells were co-transfected with a plasmid containing the Ad5 DBP gene and another plasmid carrying early region E1, integration of the Ad5 DBP gene in chromosomal DNA could be detected. Integration of Ad5 DNA sequences was also observed when transfection was performed with plasmids containing the Ad5 DBP gene and the long terminal repeat of Moloney murine leukaemia virus. By employing a radioimmunoassay it could be shown that DBP-related proteins were synthesized in two of the cell lines containing the Ad5 DBP gene. Since both cell lines support the growth of the temperature-sensitive viral DBP mutant, H5ts125, at the non-permissive temperature, the DBP-related proteins expressed in these cells must be functional.

The 78A1 isolate of Moloney murine sarcoma virus (78A1 Mo-MuSV) was cloned from a genomic library obtained from virus producer rat cells, in the lambda vector L47. Among the recombinants hybridizing with a probe specific for the v-mos sequences, we recovered a recombinant which contained leukaemia virus (MuLV) sequences and was able to transform both mouse and rat cells in transfection experiments. The cloned provirus could be rescued by both Mo-MuLV ecotropic and amphotropic viruses in mouse cells, but only with the amphotropic helper virus in rat cells. Comparative restriction mapping indicates that the 78A1 provirus is 200 bp longer than the HT1 provirus. The difference lies in the gag-pol junction region of Mo-MuSV. Other minor differences were found in the gag region, whereas the restriction patterns of the 3′ parts of the proviruses were identical.

A new isolate of Moloney murine sarcoma virus (Mo-MuSV), designated 78A1, has been molecularly cloned. The cloned genome, found to be larger than that of other known isolates of the same virus is close in size to that of the myeloproliferative sarcoma virus (MPSV), also a derivative of the original Mo-MuSV/Moloney murine leukaemia virus (Mo-MuLV) complex. Until now, MPSV was the only Mo-MuSV isolate known to be capable of inducing a myeloproliferative disease associated with a tumoural syndrome when injected intravenously into sensitive mice. We compared the biological activity of our cloned virus isolate (78A1) and that of another cloned Mo-MuSV virus (HT1) whose genome is slightly smaller than that of 78A1. The helper virus (Mo-MuLV) associated with the Mo-MuSV isolates was also injected alone as control. After injection into sensitive mice only the isolate 78A1, as well as MPSV caused a tumoural syndrome invading spleen, liver and other haematopoietic organs, and the appearance of granulo-macrophage precursors not requiring exogenous stimulating factors for their proliferation and differentiation. The 78A1 virus has a longer latency period (3 months) than MPSV (several days) and does not induce a typical myeloproliferative disease.

Cydia pomonella granulosis viruses (CpGV) from seven different sources in Europe, America and New Zealand were compared by restriction enzyme analysis. Most samples were indistinguishable from the Mexican isolate (CpGV-M). Isolates from Russia (CpGV-R) and England (CpGV-E) showed small genotypic differences. CpGV-E was shown to be a mixture of two variants, E1 and E2. CpGV-E1 was indistinguishable from CpGV-M. A physical map of CpGV-M was constructed for the enzymes EcoRI, BamHI, HindIII, SmaI and ApaI. A comparison of fragment profiles allowed construction of maps for CpGV-R and CpGV-E2. Relative to CpGV-M, CpGV-R had a single deletion of 2.4 kbp and CpGV-E2 was modified in one area resulting in an additional EcoRI site, a shift in a BamHI site and in total about 1 kbp more DNA. The map was orientated by locating the granulin gene using the cloned granulin gene from Trichoplusia ni GV as a probe. There was no significant difference between the infectivities of the Mexican, Russian and English isolates for neonate larvae.

A nuclear polyhedrosis virus (NPV) strain from Spodoptera exempta (SeMNPV-25 baculovirus) is a restriction endonuclease DNA map variant similar to Autographa californica NPV (AcMNPV baculovirus). Fourteen restriction endonuclease variants were identified and isolated from a SeMNPV baculovirus stock with 12 of the variants found at low frequency (< 3%). The DNA from each variant was compared to the prototype SeMNPV-25 for insertions, deletions and new restriction sites. Regions of variation were defined on the prototype SeMNPV-25 genome, and the nature of the variation within these regions was determined. These data are discussed and compared with the existing data on other variants of AcMNPV. A comparison of the physical maps revealed that all the SeMNPV variants were different from those reported for AcMNPV. Although the SeMNPV variants were distinctive, they were clearly genomic AcMNPV variants. The regions of the baculovirus genomic variation were identified, and three separate mechanisms are suggested for their generation. Five regions (hr1 to hr5) were associated with intragenic homologous viral sequences, five regions (vI to vIII) may be associated with the insertion of DNA sequences of cellular origin, and two regions (pI and pII) were associated with mutations resulting in the addition or loss of a PstI site. Physical maps were generated for SeMNPV variant regions vI, hr2, vII and vIII.

In this study we examined the role of secondary modifications in the expression of polyalbumin receptors by hepatitis B surface antigen (HBsAg) produced in vitro. Several clones isolated from 3T3 mouse fibroblasts after transfection with the hepatitis B virus genome produced HBsAg with marked variation in the expression of polyalbumin receptors. Treatment of the cells with glycosylation inhibitors (tunicamycin, glucosamine) resulted in loss of the 27 000 mol. wt. HBsAg glycopolypeptide, concomitantly with a marked increase in polyalbumin receptors. These data suggest that glycosylation regulates the activity of polyalbumin receptors associated with native HBsAg.

Antisera raised in rabbits and also for the first time in mice against scrapie-associated fibril (SAF) protein from hamster brain have been quantified by a modified ELISA technique (NC-ELISA) and used for a detailed analysis of SAF proteins obtained from hamster, mouse, and from patients who died of Creutzfeldt-Jakob disease. The antisera predominantly detected five bands in a Western blot analysis with apparent molecular weights of about 26 000 (26K), 24K, 20K, 18K and 16K. By gel electrophoresis these antigens seem to be identical in mouse, hamster and man. The amount of material in the various bands, however, varies according to host or agent. In control materials from healthy brain SAF protein was found to be absent even when this material was used in a 50-fold excess compared to diseased brain.

A protein with an apparent molecular weight of 14 500 (14.5K) was extractable from homogenates of Borna disease virus-infected brains and tissue cultures using high concentrations of detergent and salt and by differential centrifugation procedures. The protein, present in an aggregated form, was remarkably resistant to proteinase K. Specific antibodies prepared in the homologous system (rat) recognized the 14.5K protein from various sources (infected brain of rat, mouse or chicken, and tissue cultures), but did not neutralize infectivity nor stain Borna disease virus-specific antigens from in vitro or in vivo preparations. Post-infection immune sera from different animal species did not detect the protein. This 14.5K protein was infection-specific but not disease-specific, and is inferred to be part of an internal virion component.

An acid-labile interferon-α (IFN-α) exists in IFN produced in human peripheral leukocytes with Sendai virus. This activity was found in fractions of molecular weights about 100 000 (100K), 58K and 44K. The IFN activity in the 100K fraction was neutralized with anti-HuIFN-α serum alone, but activity in the 58K and 44K fractions was neutralized only by a mixture of anti-HuIFN-α and anti-HuIFN-β sera.

At physiological ionic strength, monoclonal antibody 35-1f4 has previously been shown to neutralize poliovirus type 1 by antibody-mediated polymerization. At low ionic strength, this antibody neutralized the virus by a hit-and-run mechanism: the virions were converted to non-infectious, empty capsids devoid of antibodies. These empty capsids resembled those formed by thermal denaturation of native polio virions in their sedimentation coefficient (80S), antigenicity (H) and isoelectric pH (6.3).