Variability of antigenic epitopes of the fusion protein of Newcastle disease virus

Systems theory has been critiqued by a number of feminist writers who felt that it did not adequately address the issues of violence and male domination in families. This essay argues that systems theory describes the world from an "exogenic" perspective--the scientific world of nature, which is intrinsically amoral. In the exogenic world all causality is circular, as nature maintains a system that has survived for billions of years. Bateson found "mind" to be within the system of nature, implying that mind must also be amoral. However, most people view the world from an "endogenic" perspective, a personal construction of reality molded by the environment in which they live, and which inevitably incorporates morality. Humans believe that violence is wrong, not for intellectual reasons, but for moral reasons. Implications for therapy are presented. A postmodern or constructivist position is taken as a way to acknowledge the influence of relationships on problems and definitions of problems, while allowing for a moral or legal consensus to pervade the therapeutic enterprise.     

Role of cotranslational disulfide bond formation in the folding of the hemagglutinin-neuraminidase protein of Newcastle disease virus

The role of cotranslational disulfide bond formation in the folding pathway of the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus was explored. Electrophoresis of pulse-labeled HN protein in the presence or absence of reducing agent showed that, characteristic of many glycoproteins, the nascent HN protein contains intramolecular disulfide bonds. As reported by Braakman et al. (EMBO J. 11, 1717-1722, 1992), incubation of cells in dithiothreitol (DTT) blocked the formation of these bonds. Removal of DTT after a pulse-label allowed for the subsequent formation of intramolecular disulfide bonds and folding of the molecule as assayed by the appearance of conformationally sensitive antigenic sites and by the formation of disulfide-linked dimers. However, the t1/2 for the formation of a conformationally sensitive antigenic site after synthesis in the presence of DTT was over twice that of the control. Furthermore, the order of appearance of the antigenic sites was different from the control, suggesting that inhibition of cotranslational disulfide bond formation altered the folding pathway of the protein. Similar results were obtained in a cell-free system containing membranes. The HN protein forced to form intramolecular disulfide bonds posttranslationally had no detectable neuraminidase or cell attachment activity, suggesting that the protein had an abnormal conformation.     

A diagnostic immunoassay for Newcastle disease virus based on the nucleocapsid protein expressed by a recombinant baculovirus

A recombinant baculovirus containing a cDNA clone encoding the nucleocapsid (NP) protein of Newcastle disease virus (strain Ulster 2C) has been used to infect insect cells (Spodoptera frugiperda). High levels of overexpressed NP protein were observed, comprising up to 40% of total cellular protein, which were subsequently shown to be antigenic. Nucleoprotein derived from the crude soluble lysate of infected insect cells has been used in an indirect ELISA to detect the presence of anti-NDV antibodies in a cohort of chicken sera. Data produced from these tests indicated a good correlation between ELISA titre and haemagglutination inhibition test data. The test was not affected by interference from background cellular proteins nor by cross-reactivity with non-NDV poultry pathogens. Additionally, the test did not generate false-positive readings.     

Restoration of wild-type virus by double recombination of tombusvirus mutants with a host transgene

Nicotiana benthamiana plants transformed with the coat protein gene of tomato bushy stunt virus (TBSV) failed to elicit effective virus resistance when inoculated with wildtype virus. Subsequently, R1 and R2 progeny from 13 transgenic lines were inoculated with a TBSV mutant containing a defective coat protein gene. Mild symptoms typical of those elicited in nontransformed plants infected with the TBSV mutant initially appeared. However, within 2 to 4 weeks, up to 20% of the transgenic plants sporadically began to develop the lethal syndrome characteristic of wild-type virus infections. RNA hybridization and immunoblot analyses of these plants and nontransformed N. benthamiana inoculated with virus from the transgenic lines indicated that wild-type virus had been regenerated by a double recombination event between the defective virus and the coat protein transgene. Similar results were obtained with a TBSV deletion mutant containing a nucleotide sequence marker, and with a chimeric cucumber necrosis virus (CNV) containing the defective TBSV coat protein gene. In both cases, purified virions contained wild-type TBSV RNA or CNV chimeric RNA derived by recombination with the transgenic coat protein mRNA. These results thus demonstrate that recombinant tombus-viruses can arise frequently from viral genes expressed in transgenic plants.     

71-kilodalton heat shock cognate protein acts as a cellular receptor for syncytium formation induced by human T-cell lymphotropic virus type 1

We previously reported that the region corresponding to amino acids 197 to 216 of the gp46 surface glycoprotein (gp46-197) served as a binding domain for the interaction between gp46 and trypsin-sensitive membrane components of the target cell, leading to syncytium formation induced by human T-cell lymphotropic virus type 1 (HTLV-1)-bearing cells. Our new evidence shows that the 71-kDa heat shock cognate protein (HSC70) acts as a cellular receptor for syncytium formation. Using affinity chromatography with the peptide gp46-197, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we isolated three components (bands A, B, and C) from MOLT-4 cell lysate which exhibited specific interactions with gp46 and inhibitory activities for syncytium formation induced by HTLV-1-bearing cells. Band A and B components were identified as HSC70 and beta-actin, respectively, through amino acid sequencing by tandem mass spectrometry and immunostaining with specific monoclonal antibodies. Band C is likely to be a nonprotein component, because full activity for syncytium formation was seen after extensive trypsin digestion. Anti-HSC70 monoclonal antibody clearly blocked syncytium formation in a coculture of HTLV-1-bearing cells and indicator cells, whereas no inhibition was seen with anti-beta-actin monoclonal antibody. Furthermore, flow cytometric analysis indicated that anti-HSC70 antibody reacted with MOLT-4 cells. Thus, we propose that HSC70 expressed on the target cell surface acts as a cellular acceptor to gp46 exposed on the HTLV-1-infected cell for syncytium formation, thereby leading to cell-to-cell transmission of HTLV-1.     

The potential of various HIV-1 mutants to inhibit the replication of wild-type virus

Transluminal placement of a stent graft in patients with an abdominal aortic aneurysm is a new endovascular technique that offers a potentially less invasive and less risky alternative to open surgery. Complications after stent graft placement are not infrequent, but in most cases secondary endovascular intervention is successful. We describe a late major leak in the aneurysmal sac caused by a distal migration of the iliac limb of a bifurcated graft. This late complication was successfully treated by covered stent placement, excluding and thrombosing completely the reformed aneurysm.     

Preferential cytotoxic effect of Newcastle disease virus on lymphoma cells

Susceptibility of lymphoma cells (Daudi, HD-Mar) to Newcastle disease virus toxicity was found to be higher than that of lymphoblastoid cells (Milstein) and of resting peripheral blood lymphocyte (PBL). Phytohemagglutinin- and/or pokeweed-mitogen-activated PBL however, exhibited, elevated sensitivity, similar to that of lymphoma cells. The level of cytotoxicity was monitored by cell viability, inhibition of DNA synthesis and release of 51Cr. When Daudi cells were mixed with PBL they were significantly more sensitive to the killing effect of the virus (70% mortality compared to 30% 30 h after infection, P < 0.05). The degree of sensitivity to viral cytotoxicity was unrelated to the efficacy of adsorption, which was similar for all cell lines as shown by immunofluorescent staining and flow cytometry. Also an influenza strain A/PR/8/34 (H1N1) adsorbed but did not affect the viability of any of the cells tested. Our results demonstrate that New-castle disease virus caused preferential damage to lymphoma cells as compared to non-cancerous normal cells.     

Passive protection of gnotobiotic calves using monoclonal antibodies directed at different epitopes on the fusion protein of bovine respiratory syncytial virus

Two neutralizing, fusion-inhibiting bovine monoclonal antibodies (MAbs; B4 and B13) directed at different epitopes on the fusion protein of respiratory syncytial virus (RSV) protected the lungs of gnotobiotic calves from RSV infection. The MAbs were administered intratracheally 24 h before the calves were challenged with bovine RSV. A third, nonneutralizing, non-fusion-inhibiting but complement-fixing MAb, B1, provided no significant protection against infection, and the disease was not exacerbated. Pneumonic consolidation and mean virus titer in lung 7 days after challenge were significantly lower in calves given the fusion-inhibiting MAbs than in either control calves or those given MAb B1. The proliferative bronchiolitis with syncytial formation and widespread distribution of RSV antigen in the lower respiratory tract of the B1-treated and control calves were indistinguishable and typical of experimental bovine RSV infection. Syncytia were markedly absent, and little or no viral antigen was detected in either the B4- or B13-treated calves.     

Characterization of CD4-gp120 activation intermediates during human immunodeficiency virus type 1 syncytium formation

The mechanism by which cells expressing HIV envelope glycoproteins progress from binding CD4+ cells to syncytia formation is not entirely understood. The purpose of these investigations was to use physical and biochemical tools (temperature shifts, soluble CD4, protease inhibitors, and a battery of anti-CD4 monoclonal antibodies) to isolate discrete steps during syncytia formation. Previously (Fu et al., J Virol 1993;67:3818), we found that preincubation of cells stably expressing HIV-1 gp 160 (TF228.1.16) with CD4+ SupT1 cells at 16 degrees C, a temperature that is nonpermissive for syncytia formation, resulted in an increased rate of syncytia formation when the cocultures were shifted to the syncytia-permissive temperature of 37 degrees C. We have since found that syncytia formation is further enhanced by shifting the cocultures from 16 to 4 degrees C prior to incubation at 37 degrees C. Together, these data suggest that two discrete states, which we term the first and second activation intermediates (FAI and SAI), are involved in syncytia formation. We have found that acquisition of the FAI (by preincubation at 16 degree C) is sensitive to some serine protease inhibitors (PI), soluble CD4 (sCD4), shedding of gp120, and anti-CD4 monoclonal antibodies (MAb) directed toward the CDR-1/2 and CDR-3 regions of domain 1 on CD4. Expression of the FAI (formation of syncytia by shifting from 16 to 37 degrees C) remains sensitive to sCD4, shedding of gp120, and MAb directed toward CDR-1/2 but is less sensitive to MAb that bind CDR-3 and is insensitive to PI. Similarly, acquisition of the SAI (shifting cocultures from 16 to 4 degrees C), is sensitive to sCD4, shedding of gp120, and MAb directed toward CDR-1/2. In contrast, expression of the SAI (shifting cocultures from 16 to 4 to 37 degrees C) is sensitive only to MAb directed toward CDR-1/2 and cannot be blocked by sCD4, shedding of gp120, or PI. These data allow us to propose that syncytia formation, mediated by HIV-1 envelope glycoproteins, proceeds by a multistep cascade.     

Role of carbohydrate processing and calnexin binding in the folding and activity of the HN protein of Newcastle disease virus

In order to study central neuronal components involved in subcutaneous (s.c.) bee venom-induced persistent pain (a new tonic pain model), we use Fos immunostaining technique to study the spatial and temporal patterns of neuronal activity in the spinal cord of anesthetized rats. Following intraplantar bee venom injection, Fos-like immunoreactive (ir) neurons were only seen from L1 to S3 rostrocaudally with distinct distribution at L4-5 segments. At segments of L1-2 and S1-3, Fos-ir labelings were diffusely and symmetrically distributed on both sides of the gray matter; however, at L4-5 segments, Fos-ir neurons were densely localized in medial portion of laminae I-II, less densely in laminae V-VI and a few in laminae VII and X ipsilateral to the injection side. No Fos labeling was seen in ventral horn of the spinal cord at L4-5 segments. Fos protein began to express only within lamina I at 0.5 h, but increased over the whole dorsal horn at 1 h and reached peak labeling at 2 h after bee venom. Expression of c-Fos in laminae I-II decreased at 4 h, and completely disappeared at 24 h, however, labeling in laminae V-VI disappeared much slowly and existed even at 96 h after bee venom. Within laminae III-IV, Fos-ir neurons could not be seen at 0.5 h, but began to be seen at 1 h and appeared to exist even at 24 h after bee venom. Systemic morphine suppressed c-Fos expression dose-dependently in both superficial and deep layers of dorsal horn and the latter region was much more sensitive to morphine than the former one. The present results demonstrated that prolonged neuronal activities in superficial and deep layers of dorsal horn were essential to mediation of bee venom induced tonic pain and may have different roles in generation and/or modulation of spontaneous pain and hyperalgesia and allodynia.     

Pathogenesis of wild-type and leaderless foot-and-mouth disease virus in cattle

Four calves were experimentally infected via aerosol with foot-and-mouth disease virus. Two were infected with a wild-type virus derived from a full-length infectious clone (A12-IC), and two were infected with a clone-derived virus lacking the leader gene (A12-LLV2), with euthanasia and tissue collection at 24 and 72 h postexposure (hpe). Clinical disease was apparent only in the animal given A12-IC and euthanized at 72 hpe. In situ hybridization revealed that the animal infected with A12-IC and euthanized at 24 hpe had abundant viral nucleic acid in the lung, present in clusters of positive cells in the respiratory bronchiolar epithelium and associated subepithelial regions. At 72 hpe in the A12-IC-infected calf, viral nucleic acid in the lung was present in interstitial areas, and in addition, viral nucleic acid was detectable in epithelial tissues around histologically apparent vesicles. In animals infected with A12-LLV2, viral nucleic acid was detectable in the lung at both 24 and 72 hpe, but staining revealed a more localized distribution with less nucleic acid than was found in animals given A12-IC. Therefore, it appears that after aerosol exposure to A12-IC, early replication is in the region of the lung, with subsequent dissemination to distal sites. In comparison, the A12-LLV2 virus is much less widely disseminated in the lung at 24 hpe, with no lesions or virus detectable in secondary sites at 72 hpe. The greatly reduced pathogenicity of A12-LLV2 may make it an excellent candidate for a modified live viral vaccine.     

Observations on the transmissibility of lentogenic strains of Newcastle disease virus: significance of variables

Virus strain and age of chicken influenced the transmissibility of lentogenic strains of Newcastle disease virus (NDV). The ability of LaSota, B1, V4, CT, F, and Ulster strains to spread from cages of oronasally inoculated chickens to adjacent cages of susceptible chickens was assessed by virus isolation, serology, and immunity to challenge with virulent NDV. Although all inoculated chickens were immune to challenge, the immunity of contact chickens ranged from 100% for LaSota and CT strains to 0% for Ulster strain. The transmissibility of B1 and V4 strains for chickens 1, 4, 8, and 16 weeks old was assessed by within-cage contact infection, exposure to contaminated food and water containers, and exposure to air from infected chickens. Serology and immunity to challenge with virulent virus were used as criteria. Differences in transmissibility were observed for the strain of virus used, route of exposure, and age of chickens. Care must be used in interpreting the significance of strain differences until the effect of variables can be minimized by further improvements in design of the test procedure.     

Protective immune response of chickens against Newcastle disease, induced by the intranasal vaccination with inactivated virus

Intranasal vaccination of chickens with inactivated Newcastle disease virus (NDV) induced both local and systemic antibody responses, resulting in protection against intranasal challenge with a lethal dose of a virulent NDV strain. The immune response was enhanced by the use of cholera toxin B subunit (CTB) as an adjuvant and only small amounts of the challenge virus were recovered from the birds vaccinated together with CTB. On the other hand, subcutaneous vaccination with the same antigen induced only a serum antibody response in chickens, allowing the challenge virus to replicate in the sinus. The present results indicate that secretory antibodies induced on the respiratory mucosal surface by intranasal vaccination with inactivated NDV protected chickens from lethal infection by inhibiting virus replication at the portal of entry for the virus.     

Evidence of genetic heterogeneity of some lentogenic Newcastle disease virus strains

Two distinguishable subpopulations were recovered from chickens infected with lentogenic strains of Newcastle disease virus originally ioslated from chickens in Australia and Ireland. In both instances, a subpopulation that rapidly eluted from chicken erythrocytes was recovered predominantly from tracheal swabs, and a slow-eluting subpopulation from cloacal swabs. The two subpopulations differed in thermostability of the hemagglutinatin and mean survival time of infected chicken embryos, as well as in elution pattern. The heterogeneity characteristic of the Aust-V4 strain and NI-Ulster strain could not be demonstrated in B1, NJ-LaSota, and ENG-F, lentogenic strains commonly used in vaccines. The question is discussed of whether standard vaccine strains are also hetrogeneous if a technique for separating stock populations is found, and the significance of possible heterogeneity to the effectivenss of the vaccines.     

Membrane-induced step in the activation of Sendai virus fusion protein

Peptides derived from conserved heptad-repeat regions of several viruses have been shown recently to inhibit virus-cell fusion. To find out their possible role in the fusion process, two biologically active heptad-repeat segments of the fusion protein (F) of Sendai virus, SV-150 (residues 150-186), and SV-473 (residues 473-495) were synthesized, fluorescently labeled and spectroscopically characterized for their structure and organization in solution and within the membrane. SV-150 was found to be 50-fold less active than SV-473 in inhibiting Sendai virus-cell fusion. Circular dichroism (CD) spectroscopy revealed that in aqueous solution, the peptides are self-associated and adopt low alpha-helical structure. However, when the two peptides are mixed together, their alpha-helical content significantly increases. Fluorescence studies, CD, and polarized attenuated total reflection infrared (ATR-FTIR) spectroscopy showed that both peptides, alone or as a complex, bind strongly to negatively charged and zwitterionic phospholipid membranes, dissociate therein into alpha-helical monomers, but do not perturb the lipid packing of the membrane. The ability of the peptides to interact with each other in solution may be correlated with antiviral activity, whereas their ability to interact with the membrane, together with their location near the fusion peptide and the transmembrane domain, suggests a revision to the currently accepted model for viral-induced membrane fusion. In the revised model, in the sequence of events associated with viral entry, the two heptad-repeat sequences may assist in bringing the viral and cellular membranes closer, thus facilitating membrane fusion.     

Physicochemical dissociation of CD4-mediated syncytium formation and shedding of human immunodeficiency virus type 1 gp120

The mechanism of CD4-mediated fusion via activated human immunodeficiency virus type 1 (HIV-1) gp41 and the biological significance of soluble CD4 (sCD4)-induced shedding of gp120 are poorly understood. The purpose of these investigations was to determine whether shedding of gp120 led to fusion activation or inactivation. BJAB cells (TF228.1.16) stably expressing HIV-1 envelope glycoproteins (the gp120-gp41 complex) were used to examine the effects of pH and temperature on sCD4-induced shedding of gp120 and on cell-to-cell fusion (syncytium formation) with CD4+ SupT1 cells. sCD4-induced shedding of gp120 was maximal at pH 4.5 to 5.5 and did not occur at pH 8.5. At physiologic pH, sCD4-induced shedding of gp120 occurred at 22, 37, and 40 degrees C but neither at 16 nor 4 degrees C. In contrast, syncytia formed at pH 8.5 (maximally at pH 7.5) but not at pH 4.5 to 5.5. At pH 7.5, syncytia formed at 37 and 40 degrees C but not at 22, 16, or 4 degrees C. Preincubation of cocultures of TF228.1.16 and SupT1 cells at 4, 16, or 22 degrees C before the shift to 37 degrees C resulted in similar, increased, or decreased syncytium formation, respectively, compared with the control. Furthermore, an activated intermediate of CD4-gp120-gp41 ternary complex may form at 16 degrees C; this intermediate rapidly executes fusion upon a shift to 37 degrees C but readily decays upon a shift to the shedding-permissive but fusion-nonpermissive temperature of 22 degrees C. These physicochemical data indicate that shedding of HIV-1 gp120 is not an integral step in the fusion cascade and that CD4 may inactivate the fusion complex in a process analogous to sCD4-induced shedding of gp120.