Gamma Irradiation as an Effective Method for Inactivation of Emerging Viral Pathogens.

Gamma irradiation using a cobalt-60 source is a commonly used method for the inactivation of infectious specimens to be handled safely in subsequent laboratory procedures. Here, we determined irradiation doses to safely inactivate liquid proteinaceous specimens harboring different emerging/reemerging viral pathogens known to cause neglected tropical and other diseases of regional or global public health importance. By using a representative arenavirus, bunyavirus, coronavirus, filovirus, flavivirus, orthomyxovirus, and paramyxovirus, we found that these enveloped viruses differed in their susceptibility to irradiation treatment with adsorbed doses for inactivation of a target dose of 1 × 106 50% tissue culture infectious dose (TCID50)/mL ranging from 1 to 5 MRads. This finding seemed generally inversely correlated with genome size. Our data may help to guide other facilities in testing and verifying safe inactivation procedures.

Low Temperature and Low UV Indexes Correlated with Peaks of Influenza Virus Activity in Northern Europe during 2010⁻2018.

With the increasing pace of global warming, it is important to understand the role of meteorological factors in influenza virus (IV) epidemics. In this study, we investigated the impact of temperature, UV index, humidity, wind speed, atmospheric pressure, and precipitation on IV activity in Norway, Sweden, Finland, Estonia, Latvia and Lithuania during 2010⁻2018. Both correlation and machine learning analyses revealed that low temperature and UV indexes were the most predictive meteorological factors for IV epidemics in Northern Europe. Our in vitro experiments confirmed that low temperature and UV radiation preserved IV infectivity. Associations between these meteorological factors and IV activity could improve surveillance and promote development of accurate predictive models for future influenza outbreaks in the region.

Influenza virus subpopulations: interferon induction-suppressing particles require expression of NS1 and act globally in cells; UV irradiation of interferon-inducing particles blocks global shut-off and enhances interferon production.

Influenza virus populations contain several subpopulations of noninfectious biologically active particles that are measured by the unique phenotypes they express. Two of these subpopulations were studied: (1) interferon (IFN)-inducing particles (IFP) and (2) IFN induction-suppressing particles (ISP). ISP are dominant in cells coinfected with one or more IFP; they completely suppress IFN production in cells otherwise programmed to induce it. Influenza virus ISP were shown to act in host cells in a nonspecific and global manner, suppressing IFN induction independent of the family of viruses serving as IFN inducers. ISP must be present within the first 3 h of coinfection with IFP to be maximally effective; by 7 hpi IFN induction/production is refractory to the action of superinfecting ISP. UV target and thermal inactivation analyses revealed that ISP activity was dependent solely on the expression of the NS gene. Low doses of UV radiation enhanced by ∼10-fold the already high IFN-inducing capacity of a virus that expressed truncated NS1. There was no change in the number of IFP, implying that the production of IFN/cell had increased. We postulated that preventing degradation of cellular RNA pol II by viral polymerase prolonged the transcription of cellular mRNA, including IFN mRNA, to enhance the IFN-inducing capacity of the cell without any increase in the number of IFP. These studies point to the dueling roles of IFP and ISP in modulating IFN induction/production, the former activity being critical to the efficacy of live attenuated influenza vaccines.

Orthogonal inactivation of influenza and the creation of detergent resistant viral aggregates: towards a novel vaccine strategy.

BACKGROUND: It has been previously shown that enveloped viruses can be inactivated using aryl azides, such as 1-iodo-5-azidonaphthalene (INA), plus UVA irradiation with preservation of surface epitopes in the inactivated virus preparations. Prolonged UVA irradiation in the presence of INA results in ROS-species formation, which in turn results in detergent resistant viral protein fractions. RESULTS: Herein, we characterize the applicability of this technique to inactivate influenza. It is shown that influenza virus + INA (100 micromolar) + UVA irradiation for 30 minutes results in a significant (p < 0.05) increase in pelletablehemagglutinin after Triton X-100 treatment followed by ultracentrifugation. Additionally, characterization of the virus suspension by immunogold labeling in cryo-EM, and viral pellet characterization via immunoprecipitation with a neutralizing antibody, shows preservation of neutralization epitopes after this treatment. CONCLUSION: These orthogonally inactivated viral preparations with detergent resistant fractions are being explored as a novel route for safe, effective inactivated vaccines generated from a variety of enveloped viruses.

Cytokine induction in human cord blood lymphocytes after pulsing with UV-inactivated influenza viruses.

Mitogenic activity of UV-inactivated influenza viruses in cord blood lymphocytes (CBL), as measured by cytokine release, was investigated. Using prototype viruses of subtype H3N2 (A/Aichi/68), H2N2 (A/Japan/57), and H1N1 (A/Puerto Rico/34) for influenza A virus, and B/Lee/40 for influenza B virus, the results indicated that both Th1 and Th2 cytokines were induced. Stimulation indices were significantly higher for IFNgamma, IL-4 and IL-10 by influenza A viruses than by influenza B virus. Stimulation indices for IL-2 and IL-6 were lower, as these two cytokines were spontaneously released by cord blood lymphocytes in culture. Alignment of the amino acid sequences of the HA for the viruses used in this study indicated that influenza B virus lacked sequence homology to the antigenic sites identified for influenza A virus. Therefore, the antigenic sites may play a role in the mitogenic property, and cord blood lymphocytes could provide a system to compare this property for clinical isolates of influenza virus.

Inactivation of infectious salmon anaemia virus, viral haemorrhagic septicaemia virus and infectious pancreatic necrosis virus in water using UVC irradiation.

The UVC irradiation doses necessary for a 99.9% (3-log) inactivation of 3 different fish pathogenic viruses diluted in freshwater/seawater and wastewater from a fish processing plant were determined. The results showed that both infectious salmon anaemia virus (ISAV) and viral haemorrhagic septicaemia virus (VHSV) were very sensitive to UVC irradiation, showing a 3-log reduction of infectivity in freshwater of 33 +/- 3.5 and 7.9 +/- 1.5 J m(-2), respectively, while that of infectious pancreatic necrosis virus (IPNV) was substantially higher, 1188 +/- 57 J m(-2). Using ISAV as a model, a comparison of the effect of UVC irradiation on virus isolation versus reverse transcription polymerase chain reaction (RT-PCR) showed that considerably higher UVC doses, depending on the length of the amplified product, were necessary to abolish RT-PCR detection of viral RNA.

Erythrocytes enhance the immunogenicity of oral vaccination with gamma irradiated influenza virus: increasing the dose of irradiation results in a significant diminution of lung IgA response.

Previous studies have demonstrated that the ability of gamma-irradiated whole influenza virus to prime for specific anti-influenza antibody responses was dramatically enhanced when delivered in a complex with chicken red blood cell ghosts (cRBC). The purpose of this study was to investigate the effect of increasing the dose of gamma irradiation used to inactivate A/Queensland/6/72 virus on the ability of the virus-cRBC complex to prime for specific influenza responses. Spleen cell proliferation studies confirmed the enhancing effect of the cRBC carrier for oral vaccination with irradiated virus. Cells from mice vaccinated with 30 kGy-irradiated virus only did not respond to influenza stimulation in vitro, whereas cells from mice vaccinated with irradiated virus+cRBC showed significant increases in proliferation to antigen exposure. No significant antibody response or challenge virus clearance was observed in mice orally vaccinated with irradiated (13.1 or 30 kGy) virus alone, even when the dose was increased significantly. Oral vaccination with live virus (+/-cRBC) primed for significant influenza specific IgA responses in the lungs, in addition to IgG responses in the lungs and sera. The dose of irradiation used to inactivate the virus was found to be critical to the profile of antibody response when the virus was delivered in a complex with cRBC. Oral vaccination of Swiss mice with 13.1 or 30 kGy virus (+cRBC) primed for significant serum and lung IgG responses. Lung IgA responses for 13.1 kGy+cRBC vaccinated mice were detected, but 30 kGy+cRBC vaccinated Swiss and CBA/H mice had no significant lung IgA response. The abrogation of IgA response, however, did not lessen the clearance of live challenge virus in outbred mice, suggesting a primary role for IgG and/or CTL response in the control of influenza virus infection post oral vaccination. To ensure direct comparison of virus alone and virus+cRBC treatments, the concentration of virus complexed to the cRBC was determined.

Characterization of influenza virus-induced death of J774.1 macrophages.

The mechanism and role of influenza virus (IV)-induced pathogenesis of macrophages during respiratory infection are ill defined. Reported here are findings on IV-induced cytopathic effects (CPEs) for an in vitro experimental system using the murine macrophage cell line J774.1. CPE was elicited by 0.2 or greater multiplicity of infection (m.o.i.). CPEs showed a lag of 6-8 h postinfection and occurred most rapidly between 6 and 12 h. J774.1 cells did not support productive IV replication, but immunofluorescence demonstrated that IV protein synthesis occurred. Light microscopy and DNA staining showed that after death cells had very condensed cytoplasm and nuclei. Cell remnants were surrounded by intact plasma membrane (PM) as demonstrated by exclusion of a membrane-impermeant dye. Time-lapse video microscopy recordings between 6 and 10 h postinfection showed sequential structural changes, including previously undescribed events. Notable changes were a rapid cytokinesis (zeiosis; "cell boiling"), followed by nuclear shrinkage, and an unusual transient blebbing of the PM. DNA fragmentation occurred after 12 h, producing a wide size range. UV-inactivated virus failed to induce CPEs, and CPE was blocked by amantadine. N-Acetylcysteine and pyrrolidine dithiocarbamate, but not other inhibitors of reactive oxygen intermediates, reduced or blocked the CPE. Most changes observed are those attributed to apoptotic processes rather than necrotic cell death. The kinetics and inhibitor effects suggest that IV infection and replication must be initiated to activate CPEs.

Radiation inactivation analysis of fusion and hemolysis by vesicular stomatitis virus.

Radiation inactivation analysis was used to determine the size of the functional unit responsible for fusion of vesicular stomatitis virus (VSV) with cardiolipin or phosphatidylcholine-phosphatidylethanolamine (1:1) liposomes, and for VSV-induced hemolysis. When radiation-insensitive background values were subtracted, the calculated functional units for all three activities were similar, ranging from 866 to 957 kDa, equivalent to about 15 G protein molecules. This is in striking contrast to results of similar studies with influenza and Sendai viruses, in which the functional unit corresponded in size to a single fusion protein monomer, and suggests that VSV fusion may occur by a different mechanism.

Influenza virus-specific cytotoxic T-cell recognition: stimulation of nucleoprotein-specific clones with intact antigen.

The present study was undertaken to investigate the role of 'antigen processing' in influenza virus-specific cytotoxic T (Tc)-cell recognition. H-2 Db-restricted Tc-cell clones specific for the 1934 influenza nucleoprotein (NP) were tested in an in vitro proliferation assay for the recognition of intact virus, purified protein or peptide antigen. Inactivated virus required further 'processing', which was inhibited by either NH4Cl treatment or paraformaldehyde fixation of antigen-presenting cells (APC). Purified NP, by contrast, was readily presented by both normal peritoneal exudate cells and H-2 Db gene 'transfected' L cells. The response was not inhibited by either NH4Cl or prior paraformaldehyde treatment of APC. Peptone-induced peritoneal exudate cells presented ineffectively unless treated with NH4Cl or prefixed with paraformaldehyde. Comparison of the responses to either purified protein or a synthetic peptide implies that the epitope recognized by the three NP specific clones is not 'cryptic' and, therefore, that the purified protein, in this case, does not require 'processing'.

Cell-mediated lysis of heat-inactivated influenza virus-coated murine targets.

The involvement of inoculated virus antigens in the induction of target susceptibility to cytotoxic T lymphocyte (CTL)-mediated lysis was investigated using heat-inactivated influenza virus, PR8 strain, and various inhibitors in comparison to the cases for live or ultraviolet (u.v.)-irradiated influenza and Sendai viruses. Induction of target susceptibility with heated PR8 was not inhibited by cycloheximide and actinomycin D as in the case of u.v.-irradiated Sendai virus, whereas live virus and u.v.-irradiated PR8 were inhibited under conditions which suppress protein synthesis. Induction of target susceptibility with the live and inactivated PR8 tested was suppressed in the presence of chloroquine, contrary to the case of Sendai virus, and was dependent on the cleavage type of influenza virus haemagglutinin. These findings suggest that the viral target antigens recognized by CTL in heated PR8-coated targets came from inoculated virus proteins, whereas those in PR8-infected or u.v.-irradiated PR8-coated targets involved newly synthesized viral proteins. The former viral target antigens seem to be transferred or processed from the endosome, depending on low pH fusion in the endosomes into which they were engulfed. In this point, the induction of viral target antigens with heated PR8 was different from that induced by u.v.-inactivated Sendai virus. Targets made with heated PR8 were recognized by cross-reactive CTL over the HA subtype.

Principles of selective inactivation of viral genome. I. UV-induced inactivation of influenza virus.

Photosensitivity of infectious, haemagglutinating and neuraminidase activities of influenza virus has been determined experimentally. The photosensitivity of infectious activity of type B (strain Hongkong/8/73) influenza virus ws found to be higher than that of type A strains NIB-4 (N3N2-3), Leningrad/399/76 (H3N2-3) and NIB-6 (H1N1). The data obtained may be used for the determination of conditions of UV-irradiation for preparation of UV-inactivated antiviral vaccines.