Virus inactivation under the photodynamic effect of phthalocyanine zinc(II) complexes.

Various metal phthalocyanines have been studied for their capacity for photodynamic effects on viruses. Two newly synthesized water-soluble phthalocyanine Zn(II) complexes with different charges, cationic methylpyridyloxy-substituted Zn(II)- phthalocyanine (ZnPcMe) and anionic sulfophenoxy-substituted Zn(II)-phthalocyanine (ZnPcS), were used for photoinactivation of two DNA-containing enveloped viruses (herpes simplex virus type 1 and vaccinia virus), two RNA-containing enveloped viruses (bovine viral diarrhea virus and Newcastle disease virus) and two nude viruses (the enterovirus Coxsackie B1, a RNA-containing virus, and human adenovirus 5, a DNA virus). These two differently charged phthalocyanine complexes showed an identical marked virucidal effect against herpes simplex virus type 1, which was one and the same at an irradiation lasting 5 or 20 min (Δlog=3.0 and 4.0, respectively). Towards vaccinia virus this effect was lower, Δlog=1.8 under the effect of ZnPcMe and 2.0 for ZnPcS. Bovine viral diarrhea virus manifested a moderate sensitivity to ZnPcMe (Δlog=1.8) and a pronounced one to ZnPcS at 5- and 20-min irradiation (Δlog=5.8 and 5.3, respectively). The complexes were unable to inactivate Newcastle disease virus, Coxsackievirus B1 and human adenovirus type 5.

Inactivation of the infectivity of two highly pathogenic avian influenza viruses and a virulent Newcastle disease virus by ultraviolet radiation.

Exposure of a virulent isolate of Newcastle disease virus (NDV) and two highly pathogenic avian influenza (HPAI) viruses, one of H7N1 subtype and the other H5N1 subtype, to a continuous ultraviolet B flux of approximately 90µW/cm(2), which models solar ultraviolet radiation, resulted in an exponential decline in infectivity with time. The time taken for a reduction in titre of 1 log10 median tissue culture infectious dose for each virus was: NDV, 69 min; H7N1 HPAI virus, 158 min; and H5N1 HPAI, virus 167 min.

A preliminary study on viral decontamination of chicken serum using gamma-irradiation.

A preliminary experiment was carried out to determine whether a decontamination procedure using gamma irradiation, similar to that adopted in the European guideline for bovine serum contaminated by pestivirus, could be applied to chicken serum. Chicken sera spiked with known amounts of enveloped and non-enveloped chicken viruses were gamma irradiated. The remaining live viruses were then measured by titration and the virus reduction capacity of the irradiation process was established for both enveloped and non-enveloped virus models. In parallel with the irradiation procedure, a classical in vivo extraneous agent test was also evaluated in order to see if it has the capacity to detect low enough levels of live viruses to be used for testing irradiated serum. The results suggest that the principles of the bovine serum decontamination procedure may be applied to chicken serum. Further studies are required to determine if this process would provide an acceptable solution for the viral 'decontamination' of chicken serum.

Combined action of virus injection and local tumor irradiation on tumor growth in mice.

The dynamics of SCCVII transplantable tumor growth in C3H/H mice was determined after local tumor irradiation and/or virus (NDV LaSota) i.p. injection. The virus applied alone significantly suppressed tumor growth, particularly until the 19th day after tumor transplantation. Local irradiation with 30 Gy resulted in tumor disappearance followed with its regrowth about 15 days later. However, if the virus was injected after the irradiation, there was no tumor growth until the end of the 31 day observation period. It should be noted that virus application prior to local irradiation did not have any additional influence on tumor growth. Thus, the pronounced efficacy of virus applied after tumor irradiation deserves attention. It is possible that the virus injected after irradiation induced a chain of cytokine production joining the action of tumor destruction induced by irradiation. This should be further studied in clarifying the approaches to combined tumor therapy with possible cell-free vaccine production.

Stimulation of human natural interferon-alpha response via paramyxovirus hemagglutinin lectin-cell interaction.

A lectin-carbohydrate recognition event without enzymatic function is proposed as molecular basis for an important innate immune response to enveloped viruses. It involves the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus (NDV) and sialic acid expressing cellular receptors on human natural interferon (IFN) alpha producing cells. This conclusion is based on two types of experimental evidence: (a) strong UV irradiation of NDV, which destroyed the cell binding and hemadsorption (HAd) but not the neuraminidase (NA) activity of HN, also destroyed its IFN-alpha inducing activity; (b) DNA transfectants expressing HN mutant molecules with greatly reduced NA but not HAd activity induced IFN-alpha while transfectants expressing HN mutant molecules with greatly reduced NA and HAd activity were incapabable of inducing IFN-alpha in human peripheral blood mononuclear cells. The results clarify molecular mechanisms involved in pattern recognition during innate immune responses.

Cytopathic effect inhibition assay for canine interferon activity.

Conditions for cytopathic effect (CPE) inhibition assay of canine interferon (IFN) activity in Madin-Darby canine kidney (MDCK) cells and in canine tumor cell line A72 was investigated using the New Jersey strain of vesicular stomatitis virus (VSV). The culture supernatant from canine splenocytes stimulated with ultraviolet-irradiated Newcastle disease viruses was used as reference IFN. MDCK cells were resistant for growth of VSV when the cells were confluent. Full CPE was observed only in a sparsely growing culture. Canine IFN activity could be assayed on less than 10(4) MDCK cells/well of a 96-well microplate, and more than 10(5) TCID50/ml of VSV was required. In A72 cells, VSV growth was not as dependent on cell density as in MDCK cells, requiring 10(3) TCID50/ml of VSV. MDCK-VSV system showed a higher IFN sensitivity than A72-VSV, whereas reproducibility was higher for the latter than the former. Based on these findings, A72-VSV system for canine IFN assay is recommended for practical use due to its easy handling characteristics.

Regulatory mechanisms of MuRantes and CRG-2 chemokine gene induction in central nervous system glial cells by virus.

In this report we characterize the induction mechanisms of two chemokine genes, MuRantes and crg-2, the murine homologs of human RANTES and IP-10, respectively, in primary rat astrocytes and microglia by the neurotropic paramyxovirus, Newcastle Disease Virus (NDV). The time course for NDV induction of both MuRantes and crg-2 genes in astrocytes and microglia was similar, with peak mRNA expression at 10-12 h. Unlike crg-2, MuRantes mRNA was not induced by IFN-gamma. MuRantes and crg-2 are transcriptionally induced by noninfectious, UV-irradiated NDV in astrocytes and microglia, unlike TNFalpha gene transcription, which is induced only by live NDV. These data indicate that signals generated through virus-receptor interaction on the target cell surface suffice to initiate MuRantes and crg-2 gene transcription in the absence of viral replication. In astrocytes, MuRantes mRNA accumulation in response to NDV was completely blocked by tyrosine kinase inhibitors, and partially by PKC and PKA inhibitors, whereas crg-2 mRNA accumulation was significantly inhibited by PKC inhibitors, but minimally or not affected by inhibitors of tyrosine kinase or PKA activity. These kinase inhibitors also affected MuRantes and crg-2 gene transcription in similar patterns to those observed for mRNA levels, but did not reduce the mRNA stability. Therefore, the signals required for mRNA accumulation appear to operate at the level of transcription. Efficient transcription of MuRantes and crg-2 genes may require different sets of transcriptional proteins and enhancers that are regulated by different signaling pathways activated by NDV.

Effects of ultraviolet radiation on structural components of enveloped RNA viruses

The interaction of ultraviolet radiation and virus particles of Western Equine Encephalomyelitis Virus (WEE) and Newcastle Disease Virus (NDV) which have respectively RNA of positive (RNA+) and negative (RNA-) polarity as genomes, was studied using purified particles. The purified virus preparations were irradiated at a range from 1,000 to 6,000 joules per m2 with posterior analysis of their propagation in primary cell cultures of chicken embryos. It could be observed that a radiation dose of to 4,500 joules per m2 could induce 10(9) TCID50 per ml as minimal loss of titer for WEE virus and NDV. The hemagglutination assay was used as a toll to evaluate the alterations caused by UV radiation on the molecular arrangement of virus proteins. Alterations of the virus hemagglutinating activity were only observe when radiation levels higher than 6,000 joules per m2 were used. The results from hemolysis assays showed the importance of the loss of the envelope integrity and the damages to nucleoprotein structures during the inactivation process, when we used radiation doses higher than 6,000 joules per m2. This model of study can increase our comprehension of the radiation effects on the cell physiology and biological components of the cell membranes.

The effect of a mesogenic and a lentogenic Newcastle disease virus strain on Burkitt lymphoma Daudi cells.

The destructive effect of Newcastle disease virus (NDV) strains on Burkitt lymphoma Daudi cells was investigated. Interaction of an active and UV-inactivated mesogenic strain (Roakin), as well as an active attenuated lentogenic strain (B1), grown in the allantoic sac of embryonated eggs, at high multiplicity, caused inhibition in cellular DNA synthesis and arrest in cell multiplication, eventually killing of the cells. The lentogenic strain cultivated in chicken fibroblasts exhibited only a moderate activity. The mechanism of the cytolytic effect is presumably linked to the increase in cell membrane permeability indicated by the elevation in 51Cr release. Thus it appears that the massive adsorption and/or penetration of viral particles, active or UV-inactivated (or possibly a toxic component that resides in the virion), damages the plasma membrane and may be responsible for the killing of the cells.

Complete regression of human neuroblastoma xenografts in athymic mice after local Newcastle disease virus therapy.

BACKGROUND: Neuroblastoma is the most common pediatric extra-cranial solid cancer. Using conventional therapies, children older than 1 year of age with advanced neuroblastoma have a poor prognosis. The development of new approaches for treating such children with neuroblastoma continues to be one of the most important goals today in pediatric oncology. Despite numerous anecdotal reports of human tumor regression during viral infections, the use of viruses to directly lyse neuroblastoma cells has never been reported as a potential therapy. Newcastle disease virus (NDV) has been shown to replicate in and kill cultured human and rat neuroblastoma cells but not normal human fibroblasts. PURPOSE: Our purpose was to determine if this selective killing of human neuroblastoma (IMR-32) cells is maintained during the in vivo treatment of established tumors. METHODS: Two experiments were performed using NDV strain 73-T. Athymic mice with subcutaneous IMR-32 human neuroblastoma xenografts (6-12 mm) were treated intralesionally with live NDV, UV-inactivated NDV, or phosphate-buffered saline (PBS). To study virus replication in situ, mice were given intratumoral or intramuscular injections of NDV. These mice were then killed at various times, and the amount of infectious virus present in tumor or muscle was determined. RESULTS: After one injection of live NDV, 17 of 18 tumors regressed completely, whereas rapid tumor growth occurred in all 18 mice treated with PBS and in all nine mice treated with UV-inactivated NDV (P < .0001). The one tumor that showed only a partial response to a single injection regressed completely after a second NDV treatment. Six months following virus-induced regression, only one tumor had recurred. No significant acute or chronic side effects of live NDV were noted in athymic mice given doses up to 500 times that used in this study. Virus levels increased more than 80-fold between 5 and 24 hours in virus-injected tumors (P < .04), while no infectious virus was produced in NDV-injected muscle tissue. CONCLUSIONS: NDV 73-T appears to replicate selectively in human IMR-32 neuroblastoma xenografts, leading directly to a potent antitumor effect as demonstrated by long-lasting, complete tumor regression occurring after a single local injection of virus. IMPLICATION: These experiments may provide an important step in the development of new therapeutic approaches to challenging cancers such as neuroblastoma.

Double-stranded ribonuclease coinduced with interferon.

Double-stranded (ds) RNA and many viruses are inducers of interferon (IFN), the latter presumably because they contain, or can form, dsRNA. Concomitant with the induction of IFN in chicken embryo cells was the induction of a novel double-stranded ribonuclease (dsRNase), which was released into the medium and continued to accumulate long after IFN production ceased. Only avian cells (chicken, quail, turkey, or duck) expressed high levels of this dsRNase; mammalian, turtle, or fish cells did not. Production of the nuclease was inducer dose-dependent. Optimum pH and cation requirements distinguished it from other dsRNase activities. Degradation of dsRNA was endonucleolytic. Activity resided in a molecule of an Mr of approximately 34,500. Low levels of a single-stranded (ss) RNase activity were inseparable from the dsRNase. The role for a dsRNA-inducible dsRNase released from cells is unknown.

Purification and characterization of chick interferon induced by viruses.

Chick interferon (IFN), produced in primary chick embryo (CE) cells stimulated by u.v.-irradiated Newcastle disease virus, was partially purified by two-step chromatography using both controlled pore glass and Blue Sepharose. The specific activity of the IFN increased about 500-fold by this method and the final recovery from starting material was more than 95%. The partially purified IFN was analysed by SDS-PAGE, and two peaks of IFN activity were observed. The molecular weight represented by the sharp peak was estimated to be 18 000 (18K) and a broad peak was found at 20K to 30K. Glycosidase treatment before SDS-PAGE resulted in disappearance of the broad peak and increased the activity of the 18K peak. Anti-CE IFN rabbit serum and a monoclonal antibody against the CE IFN neutralized the antiviral activity of all IFN samples prepared under various conditions.

Interferon induction by viruses. X. A model for interferon induction by Newcastle disease virus.

Unirradiated Newcastle disease virus (NDV, strain AV) induced high levels of interferon (IFN) in primary chick embryo cells if the cells were 'aged' in vitro for 6 to 7 days. Dose (multiplicity)-response (IFN yield) curves, carried out in the presence of anti-NDV serum to prevent cycling infection, revealed that stocks of NDV-AV contain about sevenfold more IFN-inducing particles (IFP) than infectious particles (PFP). These non-infectious IFP were responsible for nearly all IFN induction in 'aged' cells, since PFP were determined to be incapable of inducing IFN. In contrast, with mouse L(Y) cells as hosts, about one-third the number of particles as there are PFP appeared to score as IFP. Heat and u.v. radiation (254 nm) inactivated NDV IFP and PFP activity at the same rate whether tested in chick or mouse cells, implying that virion-associated transcription is required to induce IFN. A model is proposed to account for the generation of IFN-inducing particles from infectious NDV following u.v. irradiation, and their subsequent inactivation at high doses of radiation. The model defines a series of u.v. targets in the NDV genome that regulate the expression of IFN-inducing particle activity in 'unaged' chick embryo cells.

Temperature-sensitive mutants of newcastle disease virus affecting interferon induction.

Temperature-sensitive (ts) mutants of Newcastle disease virus (NDV) were isolated and studied for interferon (IFN) induction in primary chick embryo (CE) cells. At the non-permissive temperature (41 degrees C), there was no viral RNA synthesis or IFN induction by u.v.-treated virions except for ts-3 (RNA+), which did synthesize RNA at 41 degrees C, and whose u.v.-treated virions did induce IFN at this temperature. Another mutant (ts-4) induced IFN without irradiation, at the permissive temperature (37 degrees C). The minimum u.v. target size for IFN inducibility was unaffected by the mutation and corresponded to about 5% of the genome required for the expression of infectivity. These results support the hypothesis that the appearance of NDV RNA immediately after infection (primary transcription) plays a key role in IFN induction.

UV irradiation analysis of complementation between, and replication of, RNA-negative temperature-sensitive mutants of Newcastle disease virus.

Random UV irradiation-induced lesions destroy the infectivity of Newcastle disease virus (NDV) by blocking downstream transcription from the single viral promoter. The nucleocapsid-associated polypeptides most likely to be involved in RNA synthesis are located at the extreme ends of the genome: NP and P are promoter proximal genes, and L is the most distal gene. We attempted to order the two temperature-sensitive (ts) RNA-negative (RNA-) mutant groups of NDV by determining the UV target sizes for the complementing abilities of mutants A1 and E1. After UV irradiation, E1 was unable to complement A1, a result compatible with the A mutation lying in the L gene. In contrast, after UV irradiation, A1 was able to complement E1 for both virus production and viral protein synthesis, with a target size most consistent with the E mutation lying in the P gene. UV-irradiated virus was unable to replicate as indicated by its absence in the yields of multiply infected cells, either as infectious virus or as particles with complementing activity. After irradiation, ts mutant B1 delta P, with a non-ts mutation affecting the electrophoretic mobility of the P protein, complemented E1 in a manner similar to A1, but it did not amplify the expression of delta P in infected cells. This too is consistent with irradiated virus being unable to replicate despite the presence of the components needed for replication of E1. At high UV doses, A1 was able to complement E1 in a different, UV-resistant manner, probably by direct donation of input polypeptides. Multiplicity reactivation has previously been observed at high-multiplicity infection by UV-irradiation paramyxoviruses. In this case, virions which are noninfectious because they lack a protein component may be activated by a protein from irradiation virions.

The in vitro effects of Newcastle disease virus on the metabolic and antibacterial functions of human neutrophils.

Live Newcastle disease virus (NDV) was used to investigate the in vitro effects of a viral infection on phagocytosis, chemiluminescence generation, superoxide production, oxygen consumption, NADPH-oxidase activity, and intracellular killing of bacteria by Ficoll-Hypaque separated human neutrophils. Phagocytosis of oil red O particles by NDV-treated PMN was inhibited by 50%. Chemiluminescence by PMN was inhibited 79% after zymosan stimulation and 86% after tetradeconyl phorbol acetate stimulation. Superoxide generation was inhibited by 68%. Oxygen consumption was inhibited in the presence of NDV by 37% after stimulation with phorbol myristate acetate, while membrane-associated NADPH-enzyme activity was decreased by 19%. The percent of surviving intracellular S. aureus was significantly elevated in NDV-treated PMN after 60 and 120 min of incubation. Purified bacterial neuraminidase markedly suppressed chemiluminescence, while neuraminic acid blocked the effects of the virus. These observations suggest that infections with myxoviruses may suppress a number of vital neutrophil functions. It appears that the effects may be partly mediated by the interaction of viral neuraminidase with the external neutrophil membrane.

[The influence of artificial infrared radiation and microwaves upon mucopolysaccharides, protamine sulphate, and mucin in virus-infected organ cultures (author's transl)]. / Einfluss von künstlich erzeugten Infrarotstrahlen und Mikrowellen auf Mucopolysaccharide, Protaminsulfat und Mucin in virus-infizierten Organkulturen.

Infrared radiation and 1-2 micron microwaves influenced the infectivity of Newcastle Disease Virus (NDV) upon chick embryo tracheal tissue in such a way that the expected destruction of ciliated epithelium turned out to be incomplete or did not take place at all, respectively. Due to the fact that direct damaging of viruses following such radiation was not shown and that it was our intention to arrange the experimental set-up as if natural rhinolaryngeal conditions had induced the infection of the mucous membrane of the respiratory tract a study was performed in order to establish the radiation influence on mediator substances involved in the virus infection of mucous membranes. The mucopolysaccharides, chondroitine sulphate and hyaluronic acid, as well as mucin served as models; in addition protamine sulphate was used for reasons of comparison. The influence of the above substances upon NDV-infection in organ cultures and the effect of electromagnetic waves upon such influence were studied. By choosing a virus concentration of 10(-8)/ml on chick embryo tracheal tissue it was established that the application of infrared radiation (Osram Siccatherm Infrared Radiator,- 1-2 micron) and cm-waves (Klystron Raytheon Comp. Mass. USA, - 1.35 cm) for a length of 10 min. inhibited Newcastle Disease Virus (NDV) infectivity. The suspension fluid was treated with infrared and the tissue with cm-waves. Previous experiments revealed that direct radiation influence upon viruses cannot be taken for granted which is why the agents, chondroitine sulphate (polyanion) 5-10 microgram/ml, and hyaluronic acid (polyanion) 10-50 microgram/ml, were used in order to study such influence upon NDV-infected tracheal mucous membrane. In addition, protamine sulphate (polycation), 5-10 microgram/ml, and mucin were used. All the above mentioned substances influenced viral infectivity in organ cultures-expressed in terms of quotients: quotient 1 means no influence on the motility of the ciliated mucous membrane cells and quotient 0.01 means the complete destruction of the cells. Chondroitine sulphate inhibited strong but promoted weak virus infectivity, hyaluronic acid inhibited strong virus infectivity, protamine sulphate inhibited strong but promoted weak virus infectivity and mucin promoted weak virus infectivity in accordance with the used virus concentrations (Table 2). Under the influence of infrared radiation the mediator substances exercised a different influence upon viral infectivity: 25 times out of 32 experiments chondroitine sulphate was inhibited and hyaluronic acid increased in 8 out of 12 experiments (Table 2). The influence of protamine sulphate on viral infectivity was found to be increased 39 times and inhibited 14 times out of 54 experiments, mucin was inhibited 6 times. Furthermore, it is to be added that additional radiation with cm-waves increased the influence of infrared radiation (Table 1)...

Interferon production by individual L cells.

Using a protected centre technique in which agarose prevents the diffusion of interferon from individual producing cells, we have shown that essentially every cell in a monolayer of mouse L cells can be induced to produce interferon by infection with Newcastle disease virus (NDV). The amount of interferon produced by individual cells appeared to be highly variable, even when cloned cells and viruses were used. U.v.-irradiated virus lost its capacity to induce interferon in L cells and to infect chick embryo fibroblasts at the same rate. A small proportion of cells (1 X 10-6 to 10 X 10-6) appeared to produce interferon constitutively. This fraction was increased threefold by u.v. irradiation of the cells, and up to 10-fold by exposing cells to the mutagen ethyl methane sulphonate.

Interferon induction with Newcastle disease virus in FS-4 cells: effect of priming with interferon and of virus inactivating treatments.

Inoculation of human FS-4 cells with Newcastle disease virus (NDV) resulted in the induction of two distinct interferon responses, one that peaked at about 5 hr (early response) and one that reached a maximum between 10 to 24 hr after inoculation (second response). The early interferon response was enhanced by previous treatment of the cells with interferon (priming), whereas the second response decreased after interferon treatment in a dose-dependent manner. The early response diminished with decreasing multiplicities of infection, the magnitude of the second response in unprimed cells was relatively independent of the dose of NDV employed. The early interferon response was sensitive to inhibition by actinomycin D for only 1 hr after inoculation. In marked contrast, the second response remained sensitive to inhibition by actinomycin D until 12 hr after inoculation. The ability of NDV to induce the second response was greatly diminished by irradiation of the virus with ultraviolet light or by its treatment with hydroxylamine, whereas the ability to stimulate the early response was relatively resistant to these virus-inactivating treatments. Treatment of NDV with hydroxylamine abolished the virus to induce the second response at the same rate as it destroyed infectivity. The results suggest the existence of at least two distinct mechanisms of interferon induction by NDV; the early response is triggered either by a virion component or by a product of primary transcription, whereas induction of the second response requires the expression of some functions of the virus not needed for triggering the early response.