- Volume 65, Issue 12, 1984

We evaluated the influence of host genetic factors on the course of murine cytomegalovirus (MCMV) lung infection after intranasal inoculation and on the development of interstitial pneumonitis after virus and cyclophosphamide (CP) administration. Susceptibility to virus replication in the lungs of various inbred murine strains was inherited as an autosomal dominant trait, which was associated with the H-2 locus. Similarly, the development of MCMV interstitial pneumonitis was inherited as an autosomal dominant, polygenic trait. Susceptibility to lung infection was necessary, but not sufficient for MCMV interstitial pneumonitis. Non-H-2-associated factors were also needed and probably related to CP metabolism or the character of immune recovery after CP administration. Therefore, inheritable host factors influence both susceptibility to MCMV lung infection and the genesis of MCMV interstitial pneumonitis.

Arildone (WIN 38020), a broad spectrum antiviral, aryl-β-diketone (4-[6-(2-chloro-4-methoxy)phenoxyl]hexyl-3,5-heptanedione), blocks the replication of human cytomegalovirus at a stage prior to the synthesis of virus-specific DNA. Inhibitory action was demonstrated against a number of virus isolates from neonates and immune-compromised patients. Intranuclear sites of virus replication, highlighted by DNA-staining methods or immunofluorescence, were absent after Arildone treatment and corresponded with the lack of ultrastructural changes associated with productive infection. The abundance of early antigens in cells treated with Arildone was evidence for expression of the viral genome and this was confirmed by detection of immediate-early viral proteins in the presence of the drug. The results suggest that Arildone prevents the replication of human cytomegalovirus at a stage after virion uncoating but prior to viral DNA synthesis.

The varicella-zoster virus-infected cell proteins (VZV-ICPs) against which IgG, IgM and IgA antibodies were made in the course of primary varicella-zoster virus (VZV) infection were analysed by the immune transfer method. IgG antibodies were made against one or more of 18 VZV-ICPs by patients with varicella. IgM antibodies were produced which reacted with 21 VZV-ICPs. The spectrum of IgG antibody production during the first week after the onset of infection was limited to an average of three VZV-ICPs while IgM antibodies which reacted with an average of seven VZV-ICPs were detectable in the acute phase of varicella. Equivalent VZV IgG or IgM antibody titres by radioimmunoassay did not correlate with a similar pattern of antibody specificity for VZV-ICPs by immune transfer. A detectable immune response to all VZV-ICPs was not required for the recovery of individual patients from primary VZV infection.

Subcellular fractionation of rat and human cells transformed by the adenovirus type 12 (Ad-12) EcoRI-C DNA fragment showed that the 41000 mol. wt. (41K) E1a and 52K E1b proteins were present in the nucleus and cytoplasm at approximately equal concentrations. The 18K E1b protein was associated with the nuclear, mitochondrial, lysosomal and membrane fractions. The 41K E1a protein was also associated with various cytoskeletal structures (probably microtubules and 10 nm filaments) in Ad-12-transformed cells. The Ad-12 E1 41K and 52K proteins have been partially purified from transformed and infected cells. Using these preparations the 52K protein has been shown to exist under non-reducing conditions and probably in vivo as a 100K dimer stabilized by intermolecular disulphide bonds. The 41K protein bound strongly to histones H1 and H4 but much more weakly to H2A, H2B and H3. It did not interact with other comparable basic proteins or with the cytoskeletal components actin, tropomyosin and calmodulin. Although the 41K E1a protein bound to histones in vitro it is probable that such an interaction may not occur in vivo as very little of the adenovirus protein co-purified with chromatin from transformed cells. None of the Ad-12 E1 proteins showed any ATPase or protein kinase activity.

Using fragments of the cloned coxsackievirus B3 (CB3) genome as hybridization probes, regions in the CB3 genome with widely varying homology to heterologous CB serotype genomes have been demonstrated. A region composed of about 3.2 to 3.5 kbp of the CB3 genome in the 5′ half of the map is essentially unique to CB3 and exhibits little or no homology with heterologous CB serotype RNAs. The non-coding terminal 5′ end of the CB3 genome is well-conserved among the CB serotype RNAs tested. A sequence in the P3 region of the CB3 genome is also well conserved among heterologous CB serotypes.

Variable amounts of cDNA were synthesized in vitro from RNA extracted from several flaviviruses, including the four prototype dengue (DEN) virus serotypes. The synthesis was carried out using an oligo(dT) primer, suggesting the presence of a short poly(A) region at or near the 3′ end of some flavivirus genomes. The DEN-1 and DEN-2 prototype strains produced the largest amount of cDNA and were therefore used to investigate further the relatedness of flavivirus genomes by cDNA-RNA hybridization. The flaviviruses studied are related to each other to some extent since the hybrids formed exhibited about 30% S1 nuclease resistance, but a closer relationship was detected between dengue viruses of serotype 1 and 4 and between dengue virus serotype 2 and Edge Hill virus. A monoclonal antibody to the envelope protein (V3) of dengue viruses reacted with Edge Hill virus, confirming the genetic relationship between the viruses.

An outer capsid glycoprotein of human rotavirus (serotype 1) was localized and characterized by use of lectins, electron microscopy and a modified Western blotting technique. Lectins with specificity for fucose, galactose, glucose and mannose were mixed with purified single- or double-shelled human rotavirus. Aggregates observed by electron microscopy were obtained with double-shelled virus and concanavalin A, the only tested lectin with mannose specificity. By SDS-polyacrylamide gel electrophoretic analysis nine structural polypeptides could be identified. Five of these polypeptides were components of the inner capsid (VP1, VP2, VP3, VP4, VP6) and four components of the outer capsid (VP5, VP7, VP8, VP9). When using a modified Western blotting technique employed for glycoprotein detection, only VP7 was found to be glycosylated. This glycoprotein could be identified in rotavirus from human stools as well as from cell cultures. Heterogeneity in molecular weight of VP7 was observed in different isolates. An unexpected heterogeneity of VP7 was seen within a human rotavirus strain. An unplaqued stock of virus was found to exhibit two distinguishable glycoprotein bands (VP7, VP7a). After 10 passages in MA-104 cells the same strain was found to exhibit only one glycoprotein band.

A rat phaeochromocytoma cell line, termed PC12, was used to study scrapie replication. These cells, in response to the addition of nerve growth factor (NGF), exhibit a number of neuronal properties including morphological differentiation, electrophysiological responsiveness, and neurotransmitter synthesis. Cultures were exposed to scrapie brain homogenate (strain 139A), harvested every week for up to 6 weeks, and assayed for scrapie infectivity. Scrapie replication in vitro was monitored by injecting scrapie agent-exposed NGF-treated PC12 cells into mice and measuring time intervals from injection to onset of clinical symptoms. Mouse incubation periods vary inversely with the amount of scrapie infectivity present. Cells harvested at 7 and 14 days after exposure to scrapie agent showed a decrease in the level of infectivity followed by an increase at subsequent time points. The increase in scrapie infectivity from early to late time intervals after agent exposure clearly indicated replication in vitro. A fusion agent was not necessary to establish infection, and the addition of mouse peritoneal macrophages caused a reduction in the yield of infectivity per culture. Examination of cells by phase-contrast microscopy failed to reveal any cytopathology.

Ten isolates of infectious haematopoietic necrosis from salmonid fishes of different locations on the West Coast of North America from California to Alaska were compared by plaque size, single-step growth curves at 15 and 18 °C, rate of appearance of cytopathic effects in cell cultures, and growth over a range of temperatures. All isolates were distinguishable on the basis of each growth characteristic examined. The CO isolate from the Sacramento River drainage of California was the most singular of the 10 because of its diminutive plaque size and sensitivity to slightly elevated temperatures. The mean plaque diameter of the 10 isolates increased as the latitude of the geographic source of the isolate increased. Although the maximum titre obtained by all isolates was depressed at temperatures above approximately 18 °C, half of the isolates were not inhibited by temperatures as low as 0.5 °C.

Rauscher virus (RV) induces acute erythroleukaemia and a myeloproliferative disease in adult mice. It consists of a replication-competent murine leukaemia virus (R-MuLV) which acts as a helper virus and a defective transforming component which causes spleen focus formation, Rauscher spleen focus-forming virus (R-SFFV). The integrated proviral DNA of R-SFFV was cloned molecularly. The cloned R-SFFV was compared to that of other viral components which are associated with RV-induced disease and also to the cloned Friend SFFV (F-SFFV) and the myeloproliferative sarcoma virus (MPSV), both of which expand the erythroid (F-SFFV, MPSV) and myeloid (MPSV) compartment on infection of adult mice. The genome of R-SFFV differs, if analysed by restriction enzymes, from R-MuLV in the 3′ end of the genome between the env gene and the long terminal repeat. The difference is most likely an alteration in the 3′ part of the gp70-coding region of the env gene. Comparison with Rauscher mink cell focus-inducing virus (R-MCF) suggests that R-SFFV is derived from R-MCF by substitution of the 3′ half of the env gene with a sequence of unknown origin. The molecularly cloned R-SFFV pseudotyped with Friend MuLV induces an increase in late erythroid precursor cells which still require erythropoietin for maturation. Host range studies of the molecularly cloned R-SFFV prove that the Fv-2 r locus is required but not sufficient to restrict RV-induced haemopoiesis in adult mice, thus suggesting that R-SFFV has a different target cell range than F-SFFV and is similar to MPSV.

Spontaneous osteomas in strain 101 mice, a strain which has a high incidence of benign bone tumours, harbour numerous C-type virus-like particles with pleomorphic characteristics. A cell-free extract from osteomas from two mice induced bone tumours, together with osteopetrosis and lymphomas, in newborn mice of the low incidence NMRI strain after a latent period of 12 to 15 months. When C3H embryo fibroblasts were infected with the osteoma extract, the resulting cell line produced virus (OA MuLVC) with a high titre. OA MuLVC was cloned by serial endpoint dilution and NIH 3T3 cells were productively infected. The resulting virus was named OA MuLVN. OA MuLVC and OA MuLVN also induced bone tumours, osteopetrosis and lymphomas 12 to 15 months after injection into newborn NMRI mice. The isolated virus showed typical characteristics of the murine retrovirus group. Fv-1 host range restriction assays classified the viruses as N-ecotropic and XC-positive. Tryptic p30 peptide analysis and RNase T1 fingerprint analysis of OA MuLVC and OA MuLVN indicated that OA MuLVC contains an Akv-like virus as well as additional components, whereas OA MuLVN is closely related to Akv, but not identical to it. Serological analysis of the envelope proteins using monoclonal antibodies also showed the virus to be similar, but not identical, to Akv virus.

Sera of individuals infected with adult T-cell leukaemia virus (ATLV) react predominantly with the polypeptides gp68, p24 and p19. These polypeptides were isolated from ATLV-infected MT-2 cells and virus. The radioiodinated polypeptides were used to quantify respective antibodies in individual ATLV carrier sera. Heteroantisera prepared in rabbits against isolated polypeptides facilitated studies on the biosynthesis of the core and envelope polypeptides of ATLV. Pulse-chase experiments revealed a polypeptide of mol. wt. 48000 (48K) as the precursor to the core polypeptides p24 and p19. A 28K polypeptide related to p19 appeared to be an early side-product of the gag gene or a translate of a defective viral message. Antiserum to the putative env gene product gp68 recognized gp68, gp66 and small amounts of gp62. In tunicamycin-treated cells gp68, gp66 and gp62 were no longer synthesized, but a 54K polypeptide reacted with antiserum to gp68. Polypeptide p54 is structurally related to gp68 and therefore apparently represents the unglycosylated form of gp68. Moreover, the apparent mol. wt. of p54 and p48 agree with those predicted for respective env and gag precursors from the nucleotide sequence of an ATLV provirus.

We have compared the structural polypeptides of an adult T-cell leukaemia (ATL) virus (ATLV) isolate from a Japanese patient with ATL with those of a similar virus derived from a Japanese macaque monkey. Both are distinct but related entities. Their core polypeptides p19 could not be distinguished, but p24, another core polypeptide, and their envelope glycopolypeptides differ. The human virus directs the synthesis of a single intracellular glycopolypeptide, gp68, while the macaque virus specifies two such glycopolypeptides, gp57 and gp50. Furthermore, the glycopolypeptides of both viruses are serologically distinct. Thus, these viruses represent subtypes of the ATLV family and the macaque virus is apparently not involved in human ATL.

By electron microscopy, particles of defective interfering Semliki Forest virus (DI SFV) had a mean diameter of 46.8 nm compared with 55.9 nm for standard virus particles, a decrease of 16%. The difference was confirmed by measurements of the two-dimensional projected areas of DI and standard virus particles. We examined nine different DI virus preparations produced by four to 13 undiluted passages in BHK cells and all were found to contain a majority of the smaller type of particle. Calculation of the absolute number of small particles showed that there were 130 particles per interfering unit measured by the inhibition of virus RNA synthesis. However, a more sensitive assay based on interference with virus protein synthesis gave a particle:interference ratio of 6.5.

The distribution of a lipophilic cation, tetraphenylphosphonium (TPP+), has been used to monitor changes in mitochondrial and plasma membrane potentials within human embryo fibroblasts infected with human cytomegalovirus. An increase in TPP+ accumulation was observed throughout the whole viral replication cycle, beginning 6 h after infection. This effect requires an active viral DNA and seems to be dependent on the early transcription of the viral genome. In the early phase of viral replication, the increase in TPP+ accumulation is insensitive to mitochondrial inhibitors and sensitive to ouabain, and could be due to a hyperpolarization of the plasma membrane. Later during the infectious cycle, enhanced accumulation of the lipophilic cation is sensitive to mitochondrial inhibitors.

Specific polyclonal antisera to human interferon-α1 (HuIFN-α1), human interferon-α2 (HuIFN-α2) and human lymphoblastoid interferon (HuIFN-αLy Namalwa) have been raised in rabbits and sheep. The antisera raised against HuIFN-α1 and HuIFN-α2 strongly neutralized the antiviral activity of their homologous IFN-α subtypes, but were less active against the heterologous IFN-α subtypes and preparations containing mixtures of IFN-α subtypes, e.g. human leukocyte interferon (HuIFN-αLe). Antisera raised against HuIFN-αLy Namalwa strongly neutralized the antiviral activity of all IFN-α-containing preparations and showed weak cross-reactivity with human interferon-β (HuIFN-β). Neither anti-HuIFN-α1 nor anti-HuIFN-α2 could be demonstrated to neutralize the antiviral activity of HuIFN-β. A number of monoclonal antibodies to HuIFN-α2 have been prepared and these were found to neutralize HuIFN-α2 antiviral activity to varying degrees, but not to neutralize the heterologous subtype HuIFN-α1, preparations containing mixtures of IFN-α subtypes or HuIFN-β.

We have established four murine hybridoma cell lines which secrete specific antibody to avian infectious bronchitis virus (IBV) strain Massachusetts M41. Two monoclonal antibodies reacted with the spike protein and two reacted with the membrane protein. The specificity of the monoclonal antibodies for the external structural proteins was detected by immunoprecipitations using radiolabelled virus. The reactions of the monoclonal antibodies showed that one (S1) of the two glycopolypeptides associated with the spike protein has a strain-specific region involved in neutralization and haemagglutination, and the membrane protein has antigenic determinants which are present on the three strains of IBV tested (M41, Beaudette and D41).