[Using real-time PCR to evaluate the effect of viral inactivation by Methylene Blue with visible light].

To investigate the feasibility of using Real-Time PCR to evaluate the effectiveness of Sindbis virus inactivation by Methylene Blue with visible light. Sindbis virus was treated by Methylene Blue with different intensity of visible light and the transcribed cDNA was quantified by Real-Time PCR. Residual infectivity of treated virus was tested by cell infection method as parallel control at the same time. The residual infectivity of virus decreased from 6.50 lgTCID50/mL to under the limit of detection as light intensity increased. Meanwhile, the quantity of virus cDNA decreased significantly (P < 0.05), which correlated to the decline of virus infectivity (R2 > 0.98). Methylene Blue with visible light could cause lesion to nucleic acid of Sindbis virus, the extent of which was light intensity-dependent and correlated to the decrease of virus infectivity. The results demonstrated that Real-Time PCR can be a useful tool for evaluating effect of virus inactivation after Methylene Blue treatment with light.

Photoinactivation of sindbis virus infectivity without inhibition of membrane fusion.

Photoinactivation of enveloped viruses is commonly associated with damage to fusion proteins and inhibition of membrane fusion capacity. Here we show that photobleaching of Sindbis virus labeled with the membrane localized dye, R18 (octadecyl rhodamine B) causes a dramatic loss of infectivity without observable changes in low-pH triggered membrane fusion to liposomes. Sindbis labeled with DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) also maintains low-pH triggered membrane fusion capacity, but in contrast to R18, extensive photobleaching of DiI-labeled virus has little effect on infectivity. Electrophoretic gel analysis suggests no cross-linking of viral fusion proteins following photobleaching of dye-labeled Sindbis. These observations have implications for live-cell, single particle tracking studies of dye-labeled Sindbis virus. Our observations suggest that R18 and DiI have different propensities for spontaneous flip-flop in lipid bilayers.

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.

Virus inactivation and protein recovery in a novel ultraviolet-C reactor.

BACKGROUND AND OBJECTIVES: Ultraviolet-C (UVC) irradiation is a viral-inactivation method that was dismissed by many plasma fractionators as a result of the potential for protein damage and the difficulty in delivering uniform doses. A reactor with novel spiral flow hydraulic mixing was recently designed for uniform and controlled UVC treatment. The objective of this study was to investigate virus inactivation and protein recovery after treatment through the new reactor. MATERIALS AND METHODS: Virus- and mock-spiked Alpha1-proteinase inhibitor (Alpha1-PI) solutions were treated with UVC. The virus samples were assayed for residual infectivity and amplified by the polymerase chain reaction (PCR). The mock-spiked samples were assayed for protein integrity. RESULTS: Greater than 4 log10 of all test viruses were inactivated, regardless of the type of nucleic acid or presence of an envelope. Unlike previous studies, viruses with the smallest genomes were found to be those most sensitive to UVC irradiation, and detection of PCR amplicons > or = 2.0 kb was correlated to viral infectivity. Doses that achieved significant virus inactivation yielded recovery of > 90% protein activity, even in the absence of quenchers. CONCLUSIONS: The results demonstrate the effectiveness of UVC treatment, in the novel reactor, to inactivate viruses without causing significant protein damage, and confirm the utility of large PCR amplicons as markers for infectious virus.

Induction of apoptosis by Sindbis virus occurs at cell entry and does not require virus replication.

Sindbis virus (SV) is an alphavirus that causes encephalitis in mice and can lead to the apoptotic death of infected cells. To determine the step in virus replication during which apoptosis is triggered, we used UV-inactivated SV, chemicals that block virus fusion or protein synthesis, and cells that do and do not express heparan sulfate, the initial binding molecule for SV infection of many cells. In initial experiments, UV-inactivated neuroadapted SV (NSV) induced apoptosis in Chinese hamster ovary (CHO) cells lacking heparan sulfate in the presence of cycloheximide. When fusion of prebound UV-inactivated NSV was rapidly induced at the plasma membrane by exposure to acidic pH, apoptosis was induced in CHO cells with or without heparan sulfate in the presence or absence of cycloheximide in a virus dose-dependent manner. In N18 neuroblastoma cells, the relative virulence of the virus strain was an important determinant of apoptosis induced by UV-inactivated SV. Treatment of N18 cells with monensin to prevent endosomal acidification an hour before, but not 2 h after, exposure to live NSV blocked the induction of cell death, as did treatment with NH(4)Cl or bafilomycin A1. These studies indicate that SV can induce apoptosis at the time of fusion with the cell membrane and that virus replication is not required.

Equine arteritis virus subgenomic RNA transcription: UV inactivation and translation inhibition studies.

The expression of the genetic information of equine arteritis virus (EAV), an arterivirus, involves the synthesis of six subgenomic (sg) mRNAs. These are 5' and 3' coterminal since they are composed of a leader and a body sequence, which are identical to the 5' and 3' ends of the genome, respectively. Previously, it has been suggested that cis-splicing of a genome-length precursor RNA is involved in their synthesis. This was reevaluated in a comparative analysis of the sg RNA synthesis of EAV, the coronavirus mouse hepatitis virus (MHV), and the alphavirus Sindbis virus. UV transcription mapping showed that the majority of the EAV sg RNAs made at later stages of infection is not derived from a genome-length precursor. However, complete independence of sg RNA synthesis from that of genomic RNA was never observed during the course of infection. The possibility that this resulted from UV irradiation-induced effects on the synthesis of the viral replicase was investigated by inhibiting translation using cycloheximide. For EAV, ongoing protein synthesis was found to be more important for the synthesis of sg RNA than for that of genomic RNA. In general, MHV transcription was extremely sensitive to translation inhibition, whereas EAV genomic RNA synthesis became independent of de novo protein synthesis late in infection.

Inactivation of free and cell-associated human immunodeficiency virus in platelet suspensions by aminomethyltrimethylpsoralen and ultraviolet light.

BACKGROUND: It has previously been reported that 40 micrograms per mL of aminomethyltrimethylpsoralen (AMT) plus 2.4 to 7.2 J per cm2 of ultraviolet A (UVA) light inactivated 4 to 6 log10 of several model viruses in platelet suspensions. This inactivation was achieved while satisfactory levels of platelet count, pH, morphology, aggregation, and hemostatic effectiveness were maintained. STUDY DESIGN AND METHODS: The efficacy of this procedure for inactivating free and intracellular human immunodeficiency virus (HIV), including integrated proviral sequences, was studied. RESULTS: The kinetics of inactivation for free HIV (4-5 log10 kill with 1.2-4.8 J/cm2) were similar to those obtained for the previously studied viruses. For studies on cell-associated virus, H9 cells productively infected with HIV were added to platelet suspensions and treated with the above regimen of AMT and UVA. The phototreated cells were then cocultivated with uninfected H9 cells for 4 weeks and supernatants were assayed by enzyme-linked immunosorbent assay for HIV p24. No evidence of HIV replication was detectable for cells receiving as little as 2.4 J per cm2 of UVA irradiation in the presence of AMT. Further, it has been demonstrated that stably integrated sequences from the HIV proviral env gene can no longer be amplified by polymerase chain reaction after 1.2 J per cm2 of UVA (with 40 micrograms/mL AMT) exposure. CONCLUSION: These data suggest that AMT and UVA is an effective antiviral treatment for free and cell-associated HIV in platelet suspensions.

Structure-activity studies of photoactivated antiviral and cytotoxic tricyclic thiophenes.

The photoactivated antiviral and cytotoxic activities of the naturally occurring thiophene, alpha-terthienyl (1), and 15 synthetic analogues were evaluated against murine cytomegalovirus and Sindbis virus, and murine mastocytoma cells. After irradiation with near UV light, alpha-terthienyl and most of its analogues had significant toxicity, with minimum inhibitory concentrations in the range of 0.02-40 microM. In the absence of near UV irradiation, only one analogue had antiviral activity and five were cytotoxic. The most active analogues were those containing carboxylic acid, hydroxyl, or cyano substituents. Quantitative structure-activity relationship analysis of thiophene phototoxicity suggested that the rate of singlet oxygen production is the primary determinant of antiviral and cytotoxic activities. For phototoxicity against murine cytomegalovirus, a significant role for hydrophobicity was also demonstrated. Tricyclic thiophenes show significant potential for photochemotherapy of viral infections and cancer, and further evaluation in animal models is recommended.

Screening of medicinal plants from Yunnan Province in southwest China for antiviral activity.

In an ethnopharmacological screening of medicinal plants used in Yunnan province of China, ethanol extracts from 31 plant species were assayed for inhibition of murine cytomegalovirus and Sindbis virus infections. Parallel assays were carried out with and without exposure to UVA radiation to test for photo-mediation of activity. Antiviral activity was observed with 16 of the plant extracts. Eight plant extracts have been selected for further studies, with the objective of characterizing the antiviral constituents.

Interferon induction by viruses. IV. Sindbis virus: early passage defective-interfering particles induce interferon.

We have shown that a single defective-interfering (DI) particle of early (5th) passage Sindbis virus induces maximal amounts of interferon in an 'aged' primary chick embryo cell. The capacity of such DI particles to induce interferon is inactivated by small amounts of u.v. radiation (i/e dose = 232 ergs/mm2). The I/e dose for inactivation of the interferon-inducing capacity of infectious virus particles is 399 ergs/mm2 and for infectivity is 101 ergs/mm2. Pre-treatment with interferon blocks formation of interferon in response to either DI or infectious virus particles. Our results suggest that Sindbis virus genes must be expressed to form the interferon inducer, which is presumably a molecule of double-stranded (ds)RNA. We postulate that for interferon induction, the genomic RNA which codes for genes G and A must be translated into products whose concerted action produces a dsRNA molecule upon synthesis of a segment of RNA complementary to the genome. The RNA from early passage DI particles is sufficiently large (25S, 1.6 x 10(6) mol. wt.) to accommodate these genes, whereas the RNA from the late passage DI particles (20S, 1.0 x 10(6) mol. wt.) is not. Late (15th) passage DI particles do not induce interferon formation.

Interferon induction by viruses. II. Sindbis virus: interferon induction requires one-quarter of the genome--genes G and A.

We have measured the amounts of interferon formed by chick cells 'aged' in vitro in response to different amounts of infectious wild-type Sindbis virus. Our results suggest that one plaque-forming unit is enough to induce maximum interferon formation. With higher m.o.i. the yield of interferon is less. To inactivate the interferon-inducing activity of Sindbis virus, four times more u.v.-radiation was needed than to inactivate the infectivity of the virus. This suggests that only 25% of the virus genome need be intact in order to induce interferon. Temperature-sensitive Sindbis virus mutants from the three RNA+ complementation groups, C. D and E, gave rise to interferon in chick cells incubated at a non-permissive temperature, Similarly, mutants from two of the RNA- groups, B and F, gave rise to interferon, but not mutants from groups G and A. We conclude that no pre-formed inducer of interferon is present in Sindbis virus. It appears, however, that genes G and A represent a special one-quarter of the genome which must be functional in order to synthesize an interferon-inducing moiety. We suggest that this moiety is a double-stranded RNA molecule formed after synthesis of a segment of RNA complementary to the genome.

Synthesis of alphavirus-specified RNA.

UV irradiation of chicken fibroblasts infected with Semliki Forest or Sindbis virus has been used to investigate the mechanism of synthesis of 42S and 26S RNA, the major plus-strand virus-specified RNAs formed during the multiplication of standard virus particles. From an analysis of the kinetics of UV inactivation of the synthesis of these two RNAs, we conclude (i) that 26S RNA is formed by internal transcriptive initiation from a point about two-thirds of the way from the 3' end of the 42S negative-strand template; (ii) that there exists a population of plus-strand synthesizing complexes whose members are each capable of synthesizing both 42S and 26S RNA; and (iii) that, on a time-averaged basis, each complex in wild-type virus-infected cells contains one virus polymerase mediating 42S RNA synthesis and three mediating 26S RNA synthesis. The RNA phenotypes of 15 RNA(-)ts mutants of Sindbis virus have been examined after temperature shift to the restrictive temperature. Under these conditions, cells infected with three mutants, N2, N7, and E268, synthesized four to six times as much 42S RNA (relative to 26S RNA) as wild-type virus-infected cells. These studies were extended by examining, in detail, the RNA and polypeptide phenotypes of mutants N2 and E268. These experiments showed that, in N2- and E268-infected cells, one of the virus-specified nonstructural (NS) polypeptides (NS p89; H. Brzeski and S. I. T. Kennedy, J. Virol. 22:420-429, 1977) is thermolabile after shift up to restrictive temperature. This finding, together with the observation that, after shift, the 26S/42S RNA ratio in N2-infected cells changes markedly in favor of 42S RNA synthesis, leads us to conclude that, of the three NS polypeptides, NS p89 modulates 26S RNA synthesis.

Genetic exclusion and stable complementation of Sindbis virus.

In an effort to enhance genetic interactions by eliminating spatial or physical barriers between variants of Sindbis virus MgCl2 was used to aggregate infecting viral particles. Mixing viral samples in a 1:1 ratio with 0.5 M MgCl2 produced maximal reduction in plaque forming units (PFU) with minimal cell damage due to MgCl2. Aggregate size was determined to be about 7 PFU. Samples taken at 3,5 and 10 hours after infection with mixed aggregates composed of large and small plaque forming virus indicated that only one type of genome was represented among the progeny particles. In addition, aggregation enhanced complementation and the progeny were stable after several cycles of sonication and passage.

Comparison of the sensitivity to ultraviolet irradiation of reovirus 3 and some viruses of the Kemerovo group.

The kinetics of UV inactivation of the tick-borne Kemerovo (strain R-10) and Lipovnik (strain Lip-91) viruses which have been preliminarily classified as possible members of the Reovirus group was examined. Reovirus 3 and Sindbis virus served as reference double-stranded RNA and single-stranded RNA viruses, respectively. The parameters of UV inresembled those of Reovirus 3. This is consistent with their tentative classification as reovirus-like viruses.

Homologous interference induced by Sindbis virus.

Homologous interference during Sindbis virus infection has been investigated. Prior infection of either chicken embryo fibroblast or BHK(21) cell cultures results in reduced yields of progeny virions of the superinfecting genotype. This reduction in yield results from a reduction in the number of cells in the cultures capable of producing the superinfecting genotype. The development of interference parallels the attachment kinetics of Sindbis virus. Interference requires an active viral genome since the activity is sensitive to inactivation by ultraviolet light, and an RNA(-) mutant, ts-24, fails to induce interference under nonpermissive conditions. However, ts-6, an RNA(-) mutant belonging to a different complementation group, and the RNA(+) mutants, ts-2 and ts-20, interfere at both permissive and nonpermissive temperatures.

Effects of actinomycin D and ultraviolet and ionizing radiation on Pichinde virus.

Actinomycin D (0.05 mug/ml) suppresses the synthesis of ribosomal RNA of baby hamster kidney (BHK21) cells. The production of infectious Pichinde virus was enhanced in the presence of actinomycin D, although the production of virus particles was not substantially different from cultures inoculated in the absence of the drug. By prelabeling BHK21 cells with (3)H-uridine and then allowing the virus to replicate in the presence of actinomycin D, it was possible to show that ribosomal RNA synthesized prior to infection was incorporated into the virion. A single-hit kinetics of inactivation of Pichinde virus was observed with ultraviolet light, suggesting that the virus contains only a single copy of genome per virion. Comparison of the inactivation kinetics by gamma irradiation of Pichinde virus with Sindbis and rubella virus indicated that the radiosensitive genome of Pichinde virus was about 6 x 10(6) to 8 x 10(6) daltons. This value is greater than the 3.2 x 10(6) daltons which was estimated by biochemical analysis. One possible explanation considered is that the ribosomal RNA of host cell origin is functional and accounts for the differences in genome size estimated by the two methods.