Rapid and reversible reduction of junctional permeability in cells infected with a temperature-sensitive mutant of avian sarcoma virus

The transformed or normal phenotype of cultured normal rat kidney cells infected with a temperature-sensitive mutant of avian sarcoma virus is conditional on the temperature at which the cells are grown. Using dye injection techniques, we show that junction-mediated dye transfer is also temperature-sensitive. The extent and rate of transfer between infected cells grown at the transformation-permissive temperature (35 degrees C) is significantly reduced when compared to infected cells grown at the nonpermissive temperature (40.5 degrees C) or uninfected cells grown at either temperature. Infected cells subjected to reciprocal temperature shifts express rapid and reversible alterations of dye transfer capacities, with responses evident by 15 min and completed by 60 min for temperature shifts in either direction. These results suggest that altered junctional capacities may be fundamental to the expression of the ASV-induced, transformed phenotype.     

Biochemical transformation of mouse cells by herpes simplex virus type 2: enhancement by means of low-level photodynamic treatment.

The biochemical transformation of thymidine kinase-deficient cells by UV-inactivated herpes simplex virus is enhanced by low-level photodynamic treatment of the infected cells. At the concentration of proflavine used, the virus was not inactivated and both virus and cellular DNA syntheses were only marginally inhibited. The observed enhancement of the transfer of a virus gene to the cell genome suggests a possible cocarcinogenic role for photodynamically active dyes at very low concentrations.     

Induction of human cell DNA synthesis by herpes simplex virus type 2.

The effect of herpes simplex virus type 2 (HSV-2) infection on the synthesis of DNA in human embryonic fibroblast cells was determined at temperatures permissive (37 C) and nonpermissive (42 C) for virus multiplication. During incubation of HSV-2 infected cultures at 42 C for 2 to 4 days or after shift-down from 42 to 37 C, incorporation of (3H)TdR into total DNA was increased 2-to 30-fold as compared with mock-infected cultures. Analysis of the (3H)DNA suggested that host cell DNA synthesis was induced by HSV-2 infection. Induction of host cell DNA synthesis by HSV-2 also occurred in cells arrested in DNA replication by low serum concentration. The three strains of HSV-2 tested were capable of stimulating cellular DNA synthesis. Virus inactivated by UV irradiation, heat, or neutral red dye and light did not induce cellular DNA synthesis, suggesting that an active viral genome is necessary for induction.     

FLOW CYTOMETRIC ANALYSES OF VIRAL INFECTION IN TWO MARINE PHYTOPLANKTON SPECIES, MICROMONAS PUSILLA (PRASINOPHYCEAE) AND PHAEOCYSTIS POUCHETII (PRYMNESIOPHYCEAE)

Cell characteristics of two axenic marine phytoplankton species, Micromonas pusilla (Butscher) Manton et Parke and Phaeocystis pouchetii (Hariot) Lagerheim, were followed during viral infection using flow cytometry. Distinct differences between noninfected and infected cultures were detected in the forward scatter intensities for both algal species. Changes in side scatter signals on viral infection were found only for P. pouchetii. Chlorophyll red fluorescence intensity per cell decreased gradually over time in the infected cultures. DNA analyses were performed using the nucleic acid–specific fluorescent dye SYBR Green I. Shortly after infection the fraction of algal cells with more than one genome equivalent increased for both species because of the replication of viral DNA in the infected cells. Over time, a population of algal cells with low red autofluorescence and low DNA fluorescence developed, likely representing algal cells just prior to viral lysis. The present study provides insight into basic virus–algal host cell interactions. It shows that flow cytometry can be a useful tool to discriminate between virus infected and noninfected phytoplankton cells.     

Plasmodium-infected blood cells analyzed and sorted by flow fluorimetry with the deoxyribonucleic acid binding dye 33258 Hoechst.

Red cells from Plasmodium berghei infected mouse blood can be sorted on the basis of their DNA content with the bisbenzimidazole dye 33258 Hoechst. The optimal conditions for dye uptake have been established and with these conditions uninfected cells are nonfluorescent and can be completely separated from infected cells which exhibit fluorescence in almost direct proportion to the number of parasite nuclei (i.e. DNA) they contain. The number of fluorescent cells detected and their fluorescence intensity is shown to be dependent on the dye concentration and the incubation medium being used. At least a proportion of the infected cells sorted from each fluorescence peak in the cell distribution retain their infectivity in vivo with some, but not all, conditions of labeling. This technique is being used to separate minor cell populations from infected blood for biochemical and immunochemical analyses and to screen human samples for malaria infected cells.     

Effect of low-NaCl medium on the envelope glycoproteins of Sindbis virus

Lowering the NaCl concentration of the medium inhibits the release of Sindbis virus from infected chicks cells at a stage after the nucleocapsids have bound to the membranes of the infected cells. The failure of trypsin treatment to release the inhibited virus and the ratio of the proteins in the inhibited cells make it seem likely that the inhibited virus is all intracellular. Experiments using antisera specific for E1 and E2, the envelope glycoproteins of Sindbis, suggest that the inhibitory effect of low-salt medium is mediated through an effect on E2. Lactoperoxidase radioiodination experiments indicate that, even when cleaved from PE2, E2 is not exposed on the surface of low-NaCl-treated chick cells.     

Superinfection exclusion in cells infected with hepatitis C virus

Superinfection exclusion is the ability of an established virus infection to interfere with infection by a second virus. In this study, we found that Huh-7.5 cells acutely infected with hepatitis C virus (HCV) genotype 2a (chimeric strain J6/JFH) and cells harboring HCV genotype 1a, 1b, or 2a full-length or subgenomic replicons were resistant to infection with cell culture-produced HCV (HCVcc). Replicon-containing cells became permissive for HCVcc infection after treatment with an HCV-specific protease inhibitor. With the exception of cells harboring a J6/JFH-FLneo replicon, infected or replicon-containing cells were permissive for HCV pseudoparticle (HCVpp) entry, demonstrating a postentry superinfection block downstream of primary translation. The surprising resistance of J6/JFH-FLneo replicon-containing cells to HCVpp infection suggested a defect in virus entry. This block was due to reduced expression of the HCV coreceptor CD81. Further analyses indicated that J6/JFH may be toxic for cells expressing high levels of CD81, thus selecting for a CD81(low) population. CD81 down regulation was not observed in acutely infected cells, suggesting that this may not be a general mechanism of HCV superinfection exclusion. Thus, HCV establishes superinfection exclusion at a postentry step, and this effect is reversible by treatment of infected cells with antiviral compounds.     

Induction of cell surface expression of HLA antigens by human IFN-gamma encoded by recombinant vaccinia virus

A recombinant vaccinia virus (VV) encoding human IFN-gamma (VV-huIFN-gamma) was constructed and its effects on MHC Ag expression in human and murine cells in vitro analyzed by flow cytometry. At high multiplicities of infection (5 pfu/cell) the IFN-gamma expressed by vaccinia was not able to overcome the profound decrease of MHC concentration, normally associated with VV infection, in any of the cells tested. However, at successively decreasing multiplicities of infection, a gradual increase in MHC class I concentration above control levels was observed in human 143B cells but not in murine L929 cells, thus indicating that the species specificity of IFN-gamma is preserved in VV-huIFN-gamma-infected cells. We infer from these data that the IFN-gamma secreted by infected 143B cells is able to exert an MHC upregulating effect on uninfected cells in the vicinity. Antiviral activity of the IFN-gamma expressed by the virus was also assessed. Pretreatment for 24 to 48 h of human 143B cells with IFN-gamma containing supernatants had a significant antiviral effect comparable to rhuIFN-gamma. However, when added 1 h after virus infection, antiviral activity was much less evident. Also, the IFN-gamma secreted by infected 143B cells in monolayers infected at low multiplicity did not efficiently inhibit spread of infection to other cells in the vicinity.     

Vectorial release of poliovirus from polarized human intestinal epithelial cells.

Polarized epithelial cells represent the primary barrier to virus infection of the host, which must also be traversed prior to virus dissemination from the infected organism. Although there is considerable information available concerning the release of enveloped viruses from such cells, relatively little is known about the processes involved in the dissemination of nonenveloped viruses. We have used two polarized epithelial cell lines, Vero C1008 (African green monkey kidney epithelial cells) and Caco-2 (human intestinal epithelial cells), infected with poliovirus and investigated the process of virus release. Release of poliovirus was observed to occur almost exclusively from the apical cell surface in Caco-2 cells, whereas infected Vero C1008 cells exhibited nondirectional release. Structures consistent with the vectorial transport of virus contained within vesicles or viral aggregates were observed by electron microscopy. Treatment with monensin or ammonium chloride partially inhibited virus release from Caco-2 cells. No significant cell lysis was observed at the times postinfection when extracellular virus was initially detected, and transepithelial resistance and vital dye uptake measurements showed only a moderate decrease. Brefeldin A was found to significantly and specifically inhibit poliovirus biosynthetic processes by an as yet uncharacterized mechanism. The vectorial release of poliovirus from the apical (or luminal) surface of human intestinal epithelial cells has significant implications for viral pathogenesis in the human gut.     

Selective inhibitory effects of hybrid liposomes on the growth of HIV type 1-infected cells in vitro

A good correlation between fifty-percent inhibitory concentration (IC(50)) of hybrid liposomes (HL) composed of dimyristoylphosphatidylcholine and polyoxyethylene(n) dodecyl ether on the growth of MOLT-4/IIIB cells (MOLT-4 cells chronically infected with human immunodeficiency virus (HIV)) and the membrane fluidity of HL was obtained. Furthermore, the huge enhancement of virus production was observed in the latently HIV-infected (J(22)-HL-60) cells after the treatment with HL.     

Novel antiviral activity found in the media of Sindbis virus-persistently infected mosquito (Aedes albopictus) cell cultures.

Aedes albopictus (mosquito) cells persistently infected with Sindbis virus for a period of 6 months release into the medium a low-molecular-weight material capable of specifically reducing the yields of Sindbis virus during the "acute phase" of infection in mosquito cells. The antiviral activity was produced in detectable levels at 3 days after infection, and its concentration in the extracellular medium increased thereafter. The antiviral activity was inactivated by treatment with the enzyme protease K and heat. It was not activated by treatment with antibody prepared against extracts of Sindbis virus-infected BHK-21 cells. The antiviral activity differs from interferon produced by vertebrate cells in that it is virus specific as well as cell specific.     

Pseudorabies Virus Infection Alters Neuronal Activity and Connectivity In Vitro

Alpha-herpesviruses, including human herpes simplex virus 1 & 2, varicella zoster virus and the swine pseudorabies virus (PRV), infect the peripheral nervous system of their hosts. Symptoms of infection often include itching, numbness, or pain indicative of altered neurological function. To determine if there is an in vitro electrophysiological correlate to these characteristic in vivo symptoms, we infected cultured rat sympathetic neurons with well-characterized strains of PRV known to produce virulent or attenuated symptoms in animals. Whole-cell patch clamp recordings were made at various times after infection. By 8 hours of infection with virulent PRV, action potential (AP) firing rates increased substantially and were accompanied by hyperpolarized resting membrane potentials and spikelet-like events. Coincident with the increase in AP firing rate, adjacent neurons exhibited coupled firing events, first with AP-spikelets and later with near identical resting membrane potentials and AP firing. Small fusion pores between adjacent cell bodies formed early after infection as demonstrated by transfer of the low molecular weight dye, Lucifer Yellow. Later, larger pores formed as demonstrated by transfer of high molecular weight Texas red-dextran conjugates between infected cells. Further evidence for viral-induced fusion pores was obtained by infecting neurons with a viral mutant defective for glycoprotein B, a component of the viral membrane fusion complex. These infected neurons were essentially identical to mock infected neurons: no increased AP firing, no spikelet-like events, and no electrical or dye transfer. Infection with PRV Bartha, an attenuated circuit-tracing strain delayed, but did not eliminate the increased neuronal activity and coupling events. We suggest that formation of fusion pores between infected neurons results in electrical coupling and elevated firing rates, and that these processes may contribute to the altered neural function seen in PRV-infected animals.     

Anti-Influenza A Virus Effect of Hypericum perforatum L. Extract

To study the antiviral effect of Hypericum perforatum L. extract (HPE) on influenza A virus (IAV) (H1N1) in vitro and in vivo. Cytopathic effect (CPE) and neutral red (NR) dye uptake were used to examine the antiviral effect of HPE on Madin Darby Canine Kidney (MDCK) cells which were infected with IAV in vitro. HPE was effective against influenza A virus (IAV) in vitro, with a 50% effective concentration (EC50) of 40 μg/mL. The mean 50% cytotoxic concentration (CC50) in the MDCK used in these experiments was 1.5 mg/mL. Ribavirin was run in parallel with EC50 values of 5.0 μg/mL; the mean CC50 for ribavirin was 520 μg/mL. Oral gavage administrations of HPE or ribavirin to mice infected with the IAV were highly effective in preventing death, slowing the decline of arterial oxygen saturation, inhibiting lung consolidation and reducing lung virus titers. The minimum effective dose of HPE in these studies was 31.25 mg/kg/day, which was administered twice daily for 5 d beginning 4 h prior to virus exposure. Below a dosage of 2000 mg/kg/day, almost all treated mice survived, which suggests that HPE is of low toxicity. Ribavirin's minimum effective dose was 40 mg/kg/day with the LD50 determined to be 200 mg/kg/day. Delay of the initiation of either HPE or ribavirin therapy, using approximately 1/3 LD50 dose each time, could still be protective as late as 48 h after exposure to the IAV. While both agents appeared to have similar efficacy against IAV infections, HPE was considered to be less toxic and may warrant further evaluation as a possible therapy for influenza.     

Polarization of allogeneic T-cell responses by influenza virus-infected dendritic cells

The developing immune response in the lymph nodes of mice infected with influenza virus has both Th1- and Th2-type characteristics. Modulation of the interactions between antigen-presenting cells and T cells is one mechanism that may alter the quality of the immune response. We have previously shown that the ability of dendritic cells (DC) to stimulate the proliferation of alloreactive T cells is changed by influenza virus due to viral neuraminidase (NA) activity. Here we show that DC infected with influenza virus A/PR/8/34 (PR8) stimulate T cells to produce different types of cytokines in a dose-dependent manner. Optimal amounts of the Th1-type cytokines interleukin-2 (IL-2) and gamma interferon (IFN-gamma) were produced from T cells stimulated by DC infected with low doses of PR8, while the Th2-type cytokines IL-4 and IL-10 were produced only in response to DC infected with high doses of PR8. IL-2 and IFN-gamma levels corresponded with T-cell proliferation and were dependent on the activity of viral NA on the DC surface. In contrast, IL-4 secretion required the treatment of T cells with NA. Since viral particles were released only from DC that are infected with high doses of PR8, our results suggest that viral NA on newly formed virus particles desialylates T-cell surface molecules to facilitate a Th2-type response. These results suggest that the activity of NA may contribute to the mixed Th-type response observed during influenza virus infection.     

Duck Hepatitis B Virus cccDNA Amplification Efficiency in Natural Infection Is Regulated by Virus Secretion Efficiency

Previous mutation based studies showed that ablating synthesis of viral envelope proteins led to elevated hepadnaviral covalently closed circular DNA (cccDNA) amplification, but it remains unknown how cccDNA amplification is regulated in natural hepadnaviral infection because of a lack of research system. In this study we report a simple procedure to prepare two identical duck hepatitis B virus inocula, but they possess 10-100-fold difference in cccDNA amplification in infected cell culture. We demonstrate that the infected cells with higher cccDNA amplification significantly reduce the virus secretion efficiency that results in higher accumulation of relaxed circular DNA (rcDNA) and DHBsAg in the cells. The infected cells with lower cccDNA amplification significantly increase the virus secretion efficiency that leads to lower intracellular rcDNA and DHBsAg accumulation. In contrast with the findings generated in the mutation based experimental system, the regulation of cccDNA amplification in natural hepadnaviral infection bypasses direct regulation of the cellular envelope proteins concentration, instead it modulates virus secretion efficiency that ultimately impacts the intracellular rcDNA concentration, an important factor determining the destination of the synthesized rcDNA in infected cells.     

Inactivation of viruses with photoactive compounds.

The transmission of human immunodeficiency virus (HIV-1) and other enveloped virus by blood transfusion is a major concern. Photosensitive dyes such as hematoporphyrin derivative (HPD), dihematoporphyrin ether (DHE), benzoporphyrin derivatives (BPD), extended ring porphyrins, sapphyrins and texaphyrins, and various cyanines were used with viral cultures to test the feasibility of using those light-excitable dyes to kill virus. A photodynamic flow cell was used to irradiate viral suspensions or viral infected cells in culture media or in whole blood. Herpes virus (HSV-1) was used to screen compounds. Effective compounds were subsequently tested for their ability to kill HIV-1, CMV, and SIV in culture medium and in blood and proved to effectively kill free virus and infected cells at significant viremias. Irradiation was achieved with a filtered xenon light source and/or tunable dye laser. Concentrations of dyes at 10 times viral kill dose were irradiated in blood which was tested for damage to erythrocytes (RBC), platelets, and blood proteins. No damage to RBC, complement factors, and immunoglobulins was evident immediately after photodynamic treatment. Platelet condition is minimally modified with time. Photodynamic treatment of blood appears to be a feasible means of eradicating virus and some protozoans from blood.     

Study of the effect of parotitis vaccine on DNA repair in human lymphocytes

The effect of parotitis vaccine virus (strain L-3) on the DNA repair synthesis induced by 4-nitroquinoline-1-oxide has been studied. The efficiency of the repair synthesis depends on individual properties of the human body, viral multiplicity and concentration of the mutagen. A two-fold increase in DNA repair synthesis was obtained after infection of cells with low viral multiplicity (0.001 HADU50 per cell) and using 2.5 x 10(-7) M concentration of the mutagen A ten-fold increase in mutagen concentration affecting the infected cells was accompanied by the inhibition of DNA repair synthesis. Lymphocytes from children studied 7 days after vaccination by the attenuated virus did not reveal any changes in DNA repair synthesis as compared with the cells from nonvaccinated children.     

Improved method for detection of cellular transcripts by in situ hybridization. Detection of virus-specific RNA in rous sarcoma virus-infected cells by in situ hybridization to cDNA

Optimal conditions for the detection of complex RNA sequences in individual cells by in situ hybridization have been determined by using in vitro cultured quail embryonic cells infected with Rous sarcoma virus and a single-stranded 3H-cDNA probe of high specific activity complementary to the RSV genome. It is shown that fixation of target tissue can be suitably achieved by using glutaraldehyde at low concentration, and subjecting cytological preparations to heat post-fixation treatment. Conditions for removing unhybridized radioactive probe molecules by means of S1 nuclease are reported.     

The glycoprotein products of varicella-zoster virus gene 14 and their defective accumulation in a vaccine strain (Oka).

Many characteristics of the putative protein encoded by varicella-zoster virus (VZV) open reading fram (ORF) 14 indicate that it is a glycoprotein, which has been designated gpV. To identify the protein products of the gene, the coding sequences were placed under the control of the vaccinia virus p7.5 promoter and recombinant vaccinia viruses were constructed. Heterogeneous polypeptides with molecular weights of 95,000 to 105,000 (95K to 105K polypeptides) were expressed in cells infected by a vaccinia virus recombinant (vKIP5) containing ORF 14 from VZV Scott but were not expressed by control vaccinia viruses. These polypeptides were recognized by antibodies present in human sera that contained high levels of anti-VZV antibodies. Conversely, antisera raised in rabbits inoculated with vKIP5 reacted specifically with heterogeneous 95K to 105K polypeptides present in VZV Scott-infected but not uninfected cells; these polypeptides show a patchy plasma membrane fluorescence pattern in VZV Scott-infected cells. These same antisera neutralized VZV strain Scott infectivity in the absence of complement. Endoglycosidase F treatment of isolated gpV polypeptides and tunicamycin treatment of cells infected with the vKIP5 recombinant indicated that the polypeptides were glycosylated. Three sets of data imply that the VZV strain Oka, which has been used to produce a live attenuated virus vaccine, accumulates low levels of gpV polypeptides relative to wild-type strains: (i) blocking of antibodies in human sera with excess VZV Oka-infected cell antigen yielded residual antibodies which were reactive with the 95K to 105K gpV polypeptides expressed in cells infected by VZV strain Scott and by the vKIP5 vaccinia virus recombinant, but not with Oka-infected cell polypeptides; (ii) antisera raised to vKIP5 detected very low levels of reactive polypeptides made in VZV Oka-infected cells and neutralized VZV Oka virus much less efficiently than VZV Scott; and (iii) comparisons of the reactivity of sera from live attenuated virus vaccine vaccinees with sera derived from patients recovering from wild-type infections indicated greatly reduced levels of gpV-specific antibodies in some vaccinees.     

Large-scale propagation of a replication-defective adenovirus vector in stirred-tank bioreactor PER.C6 cell culture under sparging conditions

Large-scale propagation of replication-defective adenovirus vectors has not been well studied to date. One of the challenges for efficient propagation at large scale is to overcome the sensitivity of virus infected cells to gas sparging required for oxygenation and CO(2) removal. In our initial experiments, it was observed that productivity of an adenovirus vector was significantly reduced under sparging conditions as compared to nonsparged, i.e., surface-aerated controls in serum-free cultures. Investigations led to the identification of a buffer containing surfactant (Polysorbate-80, PS-80) that was included in the virus seed stock formulation and introduced through virus infection into the culture at a very low concentration as the cause of the reduced virus productivity. This finding was not obvious and trivial, as neither uninfected sparged nor infected nonsparged PER.C6 trade mark cells in serum-free cultures were affected by the buffer at such a low PS-80 concentration of 0.00025% (v/v), which is a common component of serum-free cell culture media. These results strongly suggest that virus-infected cells behave very differently from uninfected cells under sparging conditions. To mitigate the deleterious effects of sparging, the virus seed stock was prepared in the absence of the buffer containing PS-80. At the same time, the concentration of Pluronic-F68 (PF-68) in the serum-free medium was increased to 1 g/L, at which cell growth and metabolism were unaffected, even though this measure alone did not result in virus productivity improvement. Only by implementing the two measures together was virus productivity loss completely eliminated under sparging conditions. After demonstration of the process robustness in 2-L bioreactors, this adenovirus propagation process was successfully scaled up to 250 L in a 300-L bioreactor under the worst-case sparging conditions projected for 10,000-L scale.