Photochemical inactivation of Pseudomonas aeruginosa

Adaptability to a broad range of environments together with relatively high resistance to antibiotics and to disinfectants makes Pseudomonas aeruginosa a concern in hospitals and in public health. We investigated whether UVA-mediated photochemical inactivation of P. aeruginosa could be accomplished with high efficiency while at the same time preserving the sensitivity of subsequent diagnostic tests. We characterized dose responses and bactericidal kinetic rates of 5-iodonaphthyl 1-azide (INA) and of amotosalen (AMO) as these substances exposed to UVA are known to inactivate germs with minimal impact to blood products or to viral antigens. Neither UVA without photochemicals nor INA or AMO in the dark inactivated bacteria. We found that AMO was ca 1000-fold more effective in inactivating P. aeruginosa cells than INA under similar conditions. Photoinactivation with either INA or AMO at conditions that abolished bacterial infectivity did not impair polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) testing. For comparison, similar titers of Bacillus atrophaeus spores (a surrogate for B. anthracis) remained unaffected at conditions that reduced the survival of P. aeruginosa below detection levels. The results presented in this study should assist in improved methods to inactivate P. aeruginosa in environmental, clinical and forensic samples without impairing subsequent nucleic acid- or immune-based analysis.     

PHOTOINACTIVATION (365 NM) OF VACCINIA AND HERPES SIMPLEX VIRUSES INDUCED BY A NEW BUILT-IN DNA PHOTOSENSITIZER: 4-THIOTHYMIDINE

The thymidine analogue 4-thiothymidine (s⁴T) strongly absorbs light at wavelengths in the UVA range (Λ_max 335 nm) and we have examined the photoinactivation of vaccinia and herpes simplex viruses grown in the presence of this nucleoside. The cells used in this study (Vero, mouse 1D-TK⁺) were able to grow at the same rate when cultured in the presence of 2 mM s⁴T or 2 mM thymidine, albeit at a slower rate than control cells. Consistent with this finding, viruses grown in the presence of1–4 mM s⁴T were obtained in reduced yield but retained full infectivity. Both viruses were specifically inactivated by irradiation with 365 nm light and their photosensitivity, as measured by the initial slope of the inactivation curve, increased in parallel with the concentration of s⁴T added to the culture medium. More than 90% of vaccinia virus grown in the presence of 4 mM s⁴T was inactivated. Organomercurial agarose chromatography of sheared DNA isolated from vaccinia virus grown in the presence of 2 mM s⁴T showed that approximately 2.5% of DNA fragments were specifically retained, as compared to 0.2% for control DNA. This value corresponds to at least one s4T residue incorporated per 30 000 nucleotides of vaccinia virus DNA. In fact, it is likely that this ratio is actually approximately 10 times higher because of the incomplete retention of control thiolated oligodeoxynucleotides. The incorporation of s⁴T into vaccinia virus DNA was required for photoinactivation as (1) the expression of a viral or cellular thymidine kinase was required to confer photosensitivity, and (2) virus plaque reduction assays revealed that maximal photosensitivity coincided with the first rounds of viral DNA replication. The photo-inactivated virus was unable to induce detectable synthesis of several early proteins after infection of cells. These data show that s⁴T is incorporated into the DNA of vaccinia virus grown in the presence of the analogue and then behaves as a built-in UVA light photosensitizer.     

Characterization of the Effects of Aryl-azido Compounds and UVA Irradiation on the Viral Proteins and Infectivity of Human Immunodeficiency Virus Type 1

Hydrophobic UV-activatable compounds have been shown to partition into the hydrophobic region of biological membranes to selectively label transmembrane proteins, and to inactivate enveloped viruses. Here, we analyze various UV-activatable azido- and iodo-based hydrophobic compounds for their ability to inactivate a model-enveloped virus, human immunodeficiency virus (HIV-1 MN). Treatment of HIV-1 with 1,5-diazidonapthalene (DAN), 1-iodo, 5-azidonaphthalene (INA), 1-azidonaphthalene (AzNAP) or 4,4′-diazidobiphenyl (DABIPH) followed by UVA irradiation for 2 min resulted in complete viral inactivation, whereas treatment using analogous non–azido-containing controls had no effect. Incorporation of an azido moiety within these hydrophobic compounds to promote photoinduced covalent reactions with proteins was found to be the primary mechanism of viral inactivation for this class of compounds. Prolonged UVA irradiation of the virus in the presence of these azido compounds resulted in further modifications of viral proteins, due to the generation of reactive oxygen species, leading to aggregation as visualized via Western blot analysis, providing additional viral modifications that may inhibit viral infectivity. Furthermore, inactivation using these compounds resulted in the preservation of surface antigenic structures (recognized by neutralizing antibodies b12, 2g12 and 4e10), which is favorable for the creation of vaccines from these inactivated virus preparations.     

DIFFERENTIAL EFFECTS OF PHOTOACTIVE FURANYL COMPOUNDS ON VIRUS FUNCTIONS

Abstract— Five photoactive furanyl compounds were investigated for their activities against viruses. The two furanocoumarins used were 8-methoxypsoralen (8-MOP) and angelicin; two furanochromones, visnagin and khellin, and the furanoquinoline, dictamnine, were also used. The DNA-containing herpes virus murine cytomegalovirus (MCMV) and the RNA-containing togavirus, Sindbis virus, were the target viruses. All five compounds inactivated both viruses in the presence of UVA, although Sindbis virus was much less sensitive. The relative order of antiviral potency was 8-MOP > dictamnine > visnagin > angelicin > khellin. Dictamnine however was slightly more effective than 8-MOP against Sindbis virus. None of the treatments affected the structural integrity of MCMV, nor did they interfere with the normal transit of the virus into host cells or the localisation of the viral genome in the cell nucleus. Some early viral gene functions were expressed but the viruses did not replicate.     

Inactivation of Vaccinia Virus by Natural Sunlight and by Artificial UVB Radiation

This study determined the sensitivity of vaccinia virus, an orthopox virus commonly used as a surrogate for variola virus (etiological agent of smallpox), exposed to UVB radiation emitted by a solar simulator, or to direct natural sunlight. The data obtained indicate that: (1) the virucidal effect of natural sunlight can be mimicked adequately by an artificial light source with similar spectral characteristics in the UVB, (2) viral sensitivity to UVB or to solar radiation can be correlated with experimental data previously obtained with UVC, (3) the correlation factor between virus inactivation by solar radiation (measured at 300 ± 5 nm) and by UVC (254 nm) is between 33 and 60, and (4) the sensitivity of viruses either dry on glass surfaces or in liquid suspension is similar when in the presence of similar amounts of cellular debris and growth media. The findings reported in this study should assist in estimating the threat posed by the persistence of virus during epidemics or after an accidental or intentional release.     

ANTIVIRAL ACTIVITY OF THE PHOTOACTIVE THIOPHENE α-TERTHIENYL

The naturally occurring thiophene, α-terthienyl, was investigated for phototoxicity against several viruses and a line of mouse cells. The compound was extremely phototoxic to the two-membrane-containing animal viruses, murine cytomegalovirus (MCMV) and Sindbis virus (SV). Antiviral activity was detected at 10⁵μg/m in the presence of UVA. However, no effect was seen in the absence of UV-A, even at 0.1 μg/m of αT. Mouse cells were much more resistant to αT, as was the bacterial virus T4, which does not contain a membrane. Murine CMV, which had been inactivated by αT and UVA, penetrated mouse cells efficiently; but the viral DNA could not replicate, and late viral proteins were not made. Thus viral gene expression was inhibited in the photoinactivated virus. In order to account for all these data we suggest that αT may interact with viral proteins in addition to membrane lipids.     

Whole‐Genome Sequencing of a Single Viral Species from a Highly Heterogeneous Sample

Metagenomic studies suggest that only a small fraction of the viruses that exist in nature have been identified and studied. Characterization of unknown viral genomes is hindered by the many genomes populating any virus sample. A new method is reported that integrates drop‐based microfluidics and computational analysis to enable the purification of any single viral species from a complex mixed virus sample and the retrieval of complete genome sequences. By using this platform, the genome sequence of a 5243 bp dsDNA virus that was spiked into wastewater was retrieved with greater than 96 % sequence coverage and more than 99.8 % sequence identity. This method holds great potential for virus discovery since it allows enrichment and sequencing of previously undescribed viruses as well as known viruses.     

ANTIVIRAL ACTIVITY OF GILVOCARCIN V PLUS UVA RADIATION

Abstract Gilvocarcin V (GV), a coumarin, is a nucleic acid photosensitizer that is phototoxic to bacteria and mammalian cells at picomolar levels in the presence of near-UV radiation (UVA). We evaluated the effectiveness of GV plus UVA for inactivation of several viruses, including herpes simplex virus, type 1 (HSV) and the bacterial viruses φX174, T7, PRD1 and φ6. Some inactivation of the bacterial viruses was observed with UVA radiation alone (4–50% survival at 26 kJ/m²). Additional photosensitized inactivation was observed only with T7 and φ6 at 2.0 μ M GV. On the other hand, HSV was photoinactivated with concentrations of GV three orders of magnitude lower (1.0 n M ). Similar to the case with UV (254 nm) inactivation, the GV-UVA survival curve for HSV indicated multicomponent inactivation kinetics, which could not be explained by photobleaching of GV. The wide range of photosensitivities of these viruses to GV cannot be adequately explained by models based only on viral nucleic acid content or presence of lipid envelopes.     

Phototoxicity against tumor cells and Sindbis virus by an octahedral rhodium bisbipyridyl complex and evidence for the genome as a target in viral photoinactivation

An octahedral rhodium complex (cis-dichloro(dipyrido[3,2a-2',3'c]phenazine)(1,10-phenanthroline)rhodium(III) chloride; DPPZPHEN) has been prepared that can penetrate tumor cell membranes and the Sindbis viral capsid. The compound is phototoxic to these entities when irradiated with UVA light. Model studies with calf thymus and supercoiled plasmid DNA indicate that the complex can both bind with, and nick, nucleic acid. Analysis of Sindbis virus, following irradiation with the metal complex, confirmed that the viral genome was rendered noninfectious by this treatment.     

Selection of recombinant vaccinia viruses (Tian Tan strain) expressing hepatitis B virus surface antigen by using beta-galactosidase as a marker

We constructed a plasmid that contains a small piece of DNA with two vaccinia promoters running in opposite directions--a promoter from a late gene encoding an 11 K polypeptide (P11) and a promoter from an early gene encoding 25K (P25). These promoters were isolated from the Tian Tan strain of vaccinia virus and were flanked by the thymidine kinase (TK) sequence of the same virus. Genes encoding the hepatitis B virus surface antigen (HBsAg) and the Escherichia coli beta-galactosidase (LacZ) were inserted downstream of the 11 K and 25 K promoters respectively so that coexpression plasmids were constructed. Recombinant vaccinia viruses were selected directly by picking blue plaques formed under overlaying agarose medium containing X-gal. HBsAg was expressed to high level by these recombinant viruses. These recombinant viruses showed reduced virulence on rabbit skin and induced anti-HBs after intradermal inoculation of rabbits.     

Virus Particles Monitored by Fluorescence Spectroscopy: A Potential Detection Assay for Macromolecular Assembly¶

Native fluorescence spectroscopy was used for in situ investigations of two lipid‐containing bacteriophages from the cystovirus family as well as their Pseudomonad host cells. Both the viruses φ6 and φ12 and their bacterial host proteins contain the amino acid tryptophan (trp), which is the predominant fluorophore in UV. Within proteins, trp's structural environment differs, and the differences are reflected in their spectroscopic signatures. It was observed that the peak of the trp emission from both viruses was at 330 nm, a significantly shorter wavelength than trp in either the Pseudomonad host cells or the amino acid's chemical form. This allowed us to monitor the viral attachment process and subsequent lytic release of progeny virus particles by measurement of the trp emission spectra during the infection process. This work demonstrates that fluorescence may offer a novel tool to detect viruses and monitor viral infection of cells and may be part of a biodefense application.     

THE UNUSUAL UVA-DEPENDENT ANTIVIRAL PROPERTIES OF THE FUROISOCOUMARIN, CORIANDRIN

The novel furoisocoumarin, coriandrin, which was found recently to possess an interesting combination of photobiological properties, was investigated for antiviral activity in the presence and absence of UVA (long-wavelength ultraviolet radiation). In contrast to results obtained with other antiviral furocoumarins, such as 8-MOP (8-methoxypsoralen), coriandrin was much more phototoxic to the RNA-virus Sindbis virus than to the DNA-virus murine cytomegalovirus, although both viruses were substantially more sensitive to this compound than they were to 8-MOP. Human immunodeficiency virus, HIV-1, was also susceptible to coriandrin + UVA. Another unexpected finding was that viruses without membranes were completely resistant to coriandrin. This suggests that a membrane component was a target for the compound. The antiviral activity of coriandrin was profoundly inhibited by serum components in the reaction mixtures, which suggests that the compound may have a strong affinity for certain protein or lipid materials, although maximal interference was only obtained when all components of the reaction mixture, virus, coriandrin and serum, were irradiated simultaneously. Thus it appears that coriandrin has unusual antiviral properties that would not be predicted from its chemical similarity to furocoumarins.     

Antiviral properties of photosensitizers

Abstract— We have studied the antiviral properties of three different groups of photo-sensitizers, viz. (i) various furyl compounds; (ii) β-carboline alkaloids; (iii) thiophenes and their acetylene derivatives. In general the antiviral potency of the furyl compounds correlated with their ability to produce DNA photoadducts. Among the naturally occurring β-carboline alkaloids, harmine was considerably more potent (in the presence of long wavelength UV radiation, UVA) than several other harmane-related compounds. Slight alterations in chemical structure had profound effects on their antiviral activities. Harmine was shown to inactivate the DNA-virus murine cytomegalovirus (MCMV) by inhibiting viral gene expression, although other targets may also exist. Several eudistomins, carboline derivatives isolated from a tunicate, were also photoactive against viruses. Various plant thiophenes and polyacetylenes were studied in detail. These compounds also required UVA for antiviral activity, and some of them were extremely potent against viruses with membranes, e.g. α-terthienyl, which showed significant activity at only 10^-5μg/ml. When MCMV had been treated with α-terthienyl plus UVA, the virus retained its integrity and penetrated cells normally; but the virus did not replicate. More than 30 additional thiophenes have recently been evaluated, including many synthetic ones, and some of these are even more potent than a-terthienyl. We believe that certain thiophenes possess potential therapeutic value and should be tested against model virus infections in animals.     

The use of FTIR microscopy for evaluation of herpes viruses infection development kinetics

The kinetics of Herpes simplex infection development was studied using an FTIR microscopy (FTIR-M) method. The family of herpes viruses includes several members like H. simplex types I and II (HSV I, II), Varicella zoster (VZV) viruses which are involved in various human and animal infections of different parts of the body. In our previous study, we found significant spectral differences between normal uninfected cells in cultures and cells infected with herpes viruses at early stages of the infection. In the present study, cells in cultures were infected with either HSV-I or VZV and at various times post-infection they were examined either by optical microscopy or by advanced FTIR-M. Spectroscopic measurements show a consistent decrease in the intensity of the carbohydrate peak in correlation with the viral infection development, observed by optical microscopy. This decrease in cellular carbohydrate level was used as indicator for herpes viruses infection kinetics. This parameter could be used as a basis for applying a spectroscopic method for the evaluation of herpes virus infection development. Our results show also that the development kinetics of viral infection has an exponential character for these viruses.     

Removal of poliovirus type 1 from a protein mixture using an immunoaffinity chromatography column.

An immunoaffinity matrix was prepared using human polyclonal antibody (Intragam) attached to Sepharose 4B activated with CNBr. The immunoaffinity matrix was then assessed with regard to its capacity to remove viruses. The challenge virus, poliovirus type 1 was loaded in high titre in either PBS or a preparation derived from human plasma known as supernatant II + III. This fraction is depleted of IgG and is used to prepare human albumin. It was shown that an average greater than 5 logs of spiked virus were removed in one passage through the column. This type of approach may prove useful as a viral removal method in biopharmaceutical manufacturing.     

Activation, exposure and penetration of virally encoded, membrane-active polypeptides during non-enveloped virus entry

Host cell entry by influenza and other enveloped viruses is well characterized, however, the manner in which non-enveloped viruses deliver their genome across host cell membranes in the absence of membrane fusion remains unresolved. The discovery of short, membrane altering, amphipathic or hydrophobic sequences in several non-enveloped virus capsid proteins such as the gamma (gamma) peptide of nodaviruses and tetraviruses, VP4 and the N-terminal region of VP1 of picornaviruses, micro1N of reoviruses, and protein VI of adenoviruses suggests that these small peptides facilitate breaching of the host membrane and the delivery of the viral genome into the host cell. In spite of conspicuous differences in entry among non-enveloped virions, the short stretches of membrane active regions are associated with similar, entry-related events including: I) proteolytic cleavage of a precursor capsid protein resulting in increased dynamic character and/or accessibility of these peptides; II) structural changes in the virus capsid triggered by receptor binding and/or low pH in entry compartments, resulting in peptide exposure; III) externalized peptides interact with host membranes and disrupt them, facilitating delivery of the viral genome inside the host cell. Here we discuss the membrane alteration activity in non-enveloped viruses with reference to the gamma peptide of nodaviruses. Virtually all of the characteristics of gamma are shared by analogous peptides in other non-enveloped viruses, making it a simple prototype for comparative purposes.     

单病毒示踪

病毒是对人类健康威胁最大的病原之一,由其引发的病毒性疾病对人民健康、国家安全和社会经济构成重大威胁.病毒感染机制研究对病毒性疾病的防控及治疗具有重大意义.病毒侵染宿主细胞的动态过程涉及病毒组分与多种细胞组分或细胞器间复杂的相互作用,但是传统手段无法对该动态过程进行实时跟踪研究.单病毒示踪技术作为一种可以实时原位示踪单颗...     

ANALYSIS OF VIRAL DNA, PROTEIN AND ENVELOPE DAMAGE AFTER METHYLENE BLUE, PHTHALOCYANINE DERIVATIVE OR MEROCYANINE 540 PHOTOSENSITIZATION

Abstract— Although numerous photosensitizers have been used experimentally to decontaminate viruses in cellular blood components, little is known about their mechanisms of photoinactivation. Using M13 bacteriophage and vesicular stomatitis virus (VSV) as model viruses, we have investigated alteration of the viral genome, protein and envelope after phototreatment. Methylene blue (MB) and aluminum phthalocyanine tetrasulfonate (AlPcS₄) phototreatment inactivated bacteriophage M13 and decreased the fraction of single-stranded circular genomic DNA (sc-DNA) by converting it to linear form. This conversion was enhanced by treating the extracted DNA with piperidine at 55°C. Piperidine-labile breaks were well correlated to phage survival (5.1% sc-DNA at 1.7% phage survival for MB) under conditions where only minor differences were seen in the relative abundance of M13 coat protein on sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE). Neither aluminum phthalocyanine (AlPc) nor merocyanine 540 (MC540) inactivated M13 nor were there significant changes observed in DNA and coat protein. Methylene blue, AlPcS₄ and AlPc inactivated VSV and inhibited fusion of the virus envelope to Vero cells at pH 5.7 (i.e. with plasma membrane). However, the degree of this inhibition was small compared to the extent of virus inactivation (43% inhibition vs 4.7 log10 or 99.998% inactivation, for MB). In contrast, an antibody to VSV G-spike protein inhibited fusion at pH 5.7 by 52% with a concomitant decline in VSV infectivity of 0.15 log₁₀ (30%). Few changes were observed in the relative abundance of G protein for MB and AlPcS₄ phototreated samples and no additional protein bands were observed on SDS-PAGE. Phototreatment did not appreciably change the relative fusion ability at pH 7.2 (via the endocytotic pathway). These results collectively suggest that nucleic acid may be an important target for photoinactivation of these model viruses by MB and AlPcS₄.     

PSORALEN-MEDIATED VIRUS PHOTOINACTIVATION IN PLATELET CONCENTRATES: ENHANCED SPECIFICITY OF VIRUS KILL IN THE ABSENCE OF SHORTER UVA WAVELENGTHS

Abstract— Treatments with psoralens and long-wavelength ultraviolet radiation (UVA, 320–400 nm; PUVA) have shown efficacy for virus sterilization of platelet concentrates (PC). Our laboratory has employed the psoralen derivative 4'-aminomethyl-4, 5', 8-trimethylpsoralen (AMT), and we have found that platelet integrity is best preserved when rutin, a flavonoid that quenches multiple reactive oxygen species, is present during AMT/UVA treatment of PC. In this report, we examine the effects of different UVA spectra under our standard PC treatment conditions (i.e. 50 μg/mL AMT, 0.35 mM rutin and 38 J/cm² UVA). Added vesicular stomatitis virus (VSV; ≥5.5 log₁₀) was completely inactivated with the simultaneous maintenance of the platelet aggregation response (>90% of control) when a UVA light source with transmission mainly between 360 and 370 nm (narrow UVA1) was used. In contrast, with a broad-band UVA (320–400 nm; broad UVA) light source, the aggregation response was greatly compromised (<50% of control) with only a minor increase in the rate of VSV kill. With this lamp, platelet function could be improved to about 75% of the control by adding a long-pass filter, which reduced the transmission of shorter (≤345 nm) UVA wavelengths (340–400 nm; UVA1). At equivalent levels of virus kill, aggregation function was always best preserved when narrow UVA1 was used for PUVA treatment. Even in the absence of AMT, and with or without rutin present, narrow UVA 1 irradiation was better tolerated by platelets than was broad UVA. Our results suggest that for PUVA treatment of PC, the UVA dose range in which complete virus kill is obtained with the simultaneous maintenance of in vitro platelet function is smallest with broad UVA irradiation and largest with narrow UVA1. Thus, the virus specificity of PC treatment with AMT, UVA and quenchers can be further enhanced by the exclusion of the shorter UVA wavelength range.     

ANTIVIRAL EFFECT OF HARMINE, A PHOTOACTIVE β-CARBOLINE ALKALOID

Abstract— Harmine, a β-carboline alkaloid, was found to be an efficient photoactive agent against the DNA-containing murine cytomegalovirus (MCMV) and the RNA containing Sindbis virus, a togavirus, both of which contain membranes. In contrast the host cells themselves were relatively resistant. The MCMV was investigated in more detail. Virus which had been inactivated by harmine plus UVA retained its structural integrity and was fully capable of penetrating host cells, following which the viral genome entered the cell nucleus. The viral genes which normally code for the switch-off in thymidine kinase activity arid for the normal cytopathic effects were not expressed; however the viral geneis responsible for the cytotoxic effect due to high multiplicities of infection was still expressed, indicating that there may be a differential block of some viral genes