Standard Plaque Assay Research Articles

Enzyme-Linked Immunosorbent Assay-Format Tissue Culture Infectious Dose-50 Test for Titrating Dengue Virus

A dengue nonstructural protein 1 (NS1) antigen capture enzyme-linked immunosorbent assay (ELISA)-based tissue culture infectious dose-50 (TCID50) test (TCID50-ELISA) was developed as an alternative to the standard plaque assay for titrating dengue virus. Virus titers obtained by TCID50-ELISA were comparable to those obtained by the plaque assay and by the traditional TCID50-cytopathic effect (CPE) test (TCID50-CPE), with a better reproducibility and a lower coefficient of variation. Quantitative comparison of TCID50-ELISA and TCID50-CPE resulted in a correlation coefficient of 0.976. Moreover, this new method showed a wider application to C6/36, Vero E6, BHK-21, and Vero cells compared with other titration methods. In summary, the novel TCID50-ELISA method described here provides a more reliable and more accurate alternative compared to the plaque assay and TCID50-CPE for titration of dengue virus.

Time-Dependent Effects of Pomegranate Juice and Pomegranate Polyphenols on Foodborne Viral Reduction

Pomegranate juice (PJ) and pomegranate polyphenolic extracts (PP) have antiviral effects against HIV-1, influenza, herpes, and poxviruses, and we recently demonstrated their effect against human noroviral surrogates. In the present study, the time-dependent effects of two commercial brands of PJ and PP at two concentrations (2 and 4 mg/mL) on the infectivity of foodborne viral surrogates (feline calicivirus FCV-F9, murine norovirus MNV-1, and MS2 bacteriophage) at room temperature for up to 1 h were evaluated. Each virus at ∼5 log(10) plaque-forming units (PFU)/mL was mixed with equal volumes of PJ, or PP at 4 or 8 mg/mL, and incubated for 0, 10, 20, 30, 45, and 60 min at room temperature. Viral titers after each treatment were determined by standardized plaque assays and compared with untreated controls. Virus titer reduction by PJ and PP was found to be a rather rapid process, with ≥50% of titer reduction occurring within the first 20 min of treatment for all three tested viruses. Within the first 20 min, titer reductions of 3.12, 0.79, and 0.23 log(10) PFU/mL for FCV-F9, MNV-1, and MS2, respectively, were obtained using PJ. FCV-F9, MNV-1, and MS2 titers were reduced by 4.02, 0.68, and 0.18 log(10) PFU/mL with 2 mg/mL PP and 5.09, 1.14, and 0.19 log(10) PFU/mL with 4 mg/mL PP, respectively, after 20 min. The mechanism of viral reduction by PJ and PP needs to be elucidated and clinical trials should be undertaken before recommending for therapeutic or preventive purposes.

Grape Seed Extract for Control of Human Enteric Viruses

Grape seed extract (GSE) is reported to have many pharmacological benefits, including antioxidant, anti-inflammatory, anticarcinogenic, and antimicrobial properties. However, the effect of this inexpensive rich source of natural phenolic compounds on human enteric viruses has not been well documented. In the present study, the effect of commercial GSE, Gravinol-S, on the infectivity of human enteric virus surrogates (feline calicivirus, FCV-F9; murine norovirus, MNV-1; and bacteriophage MS2) and hepatitis A virus (HAV; strain HM175) was evaluated. GSE at concentrations of 0.5, 1, and 2 mg/ml was individually mixed with equal volumes of each virus at titers of ∼7 log(10) PFU/ml or ∼5 log(10) PFU/ml and incubated for 2 h at room temperature or 37°C. The infectivity of the recovered viruses after triplicate treatments was evaluated by standardized plaque assays. At high titers (∼7 log(10) PFU/ml), FCV-F9 was significantly reduced by 3.64, 4.10, and 4.61 log(10) PFU/ml; MNV-1 by 0.82, 1.35, and 1.73 log(10) PFU/ml; MS2 by 1.13, 1.43, and 1.60 log(10) PFU/ml; and HAV by 1.81, 2.66, and 3.20 log(10) PFU/ml after treatment at 37°C with 0.25, 0.50, and 1 mg/ml GSE, respectively (P < 0.05) in a dose-dependent manner. GSE treatment of low titers (∼5 log(10) PFU/ml) at 37°C also showed viral reductions. Room-temperature treatments with GSE caused significant reduction of the four viruses, with higher reduction for low-titer FCV-F9, MNV-1, and HAV compared to high titers. Our results indicate that GSE shows promise for application in the food industry as an inexpensive novel natural alternative to reduce viral contamination and enhance food safety.

Detection of Staphylococcus aureus Using 15N-Labeled Bacteriophage Amplification Coupled with Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry

A novel approach to rapid bacterial detection using an isotopically labeled (15)N bacteriophage and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is introduced. Current phage amplification detection (PAD) via mass spectrometric analysis is limited because host bacteria must be inoculated with low phage titers in such a way that initial infecting phage concentrations must be below the detection limit of the instrument, thus lengthening incubation times. Additionally, PAD techniques cannot distinguish inoculate input phage from output phage which can increase the possibility of false positive results. Here, we report a rapid and accurate PAD approach for identification of Staphylococcus aureus via detection of bacteriophage capsid proteins. This approach uses both a wild-type (14)N and a (15)N-isotopically labeled S. aureus-specific bacteriophage. High (15)N phage titers, above our instrument's detection limits, were used to inoculate S. aureus. MALDI-TOF MS detection of the (14)N progeny capsid proteins in the phage-amplified culture indicated the presence of the host bacteria. Successful phage amplification was observed after 90 min of incubation. The amplification was observed by both MALDI-TOF MS analysis and by standard plaque assay measurements. This method overcomes current limitations by improving analysis times while increasing selectivity when compared to previously reported PAD methodologies.

MiR-101 regulates HSV-1 replication by targeting ATP5B

MicroRNAs (miRNAs) are short non-coding RNAs that negatively modulate gene expression at the post-transcriptional level and are known to be involved in the cross-talk between the host and virus. Using a standard plaque assay and real-time PCR method, we found that ectopic expression of miR-101 could significantly suppress herpes simplex virus-1 (HSV-1) replication, and that blocking endogenous miR-101 could increase viral progeny without affecting cell viability. Bioinformatics analysis indicates the 3′ untranslated region (UTR) of mitochondrial ATP synthase subunit beta (ATP5B) has a putative binding site for miR-101. MiR-101 can directly bind to ATP5B 3′UTR and negatively regulate ATP5B expression. Using a RNA interference technique, knockdown of ATP5B significantly inhibited HSV-1 replication, indicating that ATP5B functions as a pro-viral factor. The ectopic expression of ATP5B lacking the 3′UTR could override the suppressive effect of miR-101 on HSV-1 replication. A concordant inverse correlation between miR-101 and ATP5B was observed in HSV-1-infected HeLa cells. Up-regulation of miR-101 expression may play a role in repressing productive HSV-1 replication by targeting ATP5B. Exploring the role of host-encoded miRNA in the regulation of viral infection would enable us to better understand the intricate networks of host–pathogen interactions.

Inactivation of Human Enteric Virus Surrogates by High-Intensity Ultrasound

Foodborne viruses, especially human noroviruses, are recognized as leading causes of nonbacterial gastroenteritis worldwide. Development of effective inactivation methods is of great importance to control their spread. In this study, the effect of high-intensity ultrasound (HIUS) on the infectivity of three foodborne virus surrogates was investigated. The three surrogates, murine norovirus (MNV-1), feline calicivirus (FCV-F9), and MS2 bacteriophage, were diluted in phosphate-buffered saline (PBS) or orange juice to a titer of approximately 6 log(10) PFU/mL or approximately 4 log(10) PFU/mL. The ultrasound treatment was performed in duplicate by immersing the HIUS probe in virus-containing solution that was cooled in ice-water and sonicated at 20 kHz for 2, 5, 10, 15, 20, and 30 min with 30 sec on and 30 sec off. The infectivity of the recovered viruses after each ultrasound treatment was evaluated in duplicate using standardized plaque assays and compared to untreated controls. The results show that HIUS effectiveness depended on the virus type, the initial titer of the viruses, and the virus suspension solution. At titers of approximately 4 log(10) PFU/mL in PBS, feline calicivirus (FCV)-F9, MS2, and murine norovirus (MNV)-1 required 5-, 10-, and 30-min treatment, respectively, for complete inactivation. At initial titers of approximately 4 log(10) PFU/mL in orange juice, FCV-F9 required a 15-min treatment for complete inactivation and only a 1.55 log(10) PFU/mL reduction was achieved for MNV-1 in orange juice after 30-min treatment. Thus, inactivation by HIUS in orange juice was much lower than in PBS. Experiments using titers of approximately 6 log(10) PFU/mL showed decreased effects compared to those using titers of approximately 4 log(10) PFU/mL. These results indicate that HIUS alone is not sufficient to inactivate virus in food. Hurdle technologies that combine HIUS with antimicrobials, heat, or pressure should be explored for viral inactivation.

In Vitro Effects of Pomegranate Juice and Pomegranate Polyphenols on Foodborne Viral Surrogates

Pomegranate juice (PJ) has gained popularity because of its associated antioxidant, antimicrobial, anticancer, and anti-inflammatory properties. However, its effects against epidemiologically significant foodborne viruses have not been investigated. In the absence of culturable human noroviruses, feline calicivirus (FCV-F9), murine norovirus (MNV-1), and MS2 (ssRNA) bacteriophage were used as foodborne viral surrogates. The aim of this research was to study the effects of PJ and pomegranate polyphenols (PP) on foodborne viral infectivity. Viruses at high (∼ 7 log(10) PFU/mL) or low (∼ 5 log(10) PFU/mL) titers were mixed with equal volumes of PJ, 8, 16, and 32 mg/mL of PP, or water (control) and incubated for 1 h at room temperature. Viral infectivity after treatments was evaluated using standardized plaque assays. PJ decreased the titer of FCV-F9, MNV-1, and MS2 by 2.56, 1.32, and 0.32 log(10) PFU/mL, respectively, for low titers and 1.20, 0.06, and 0.63 log(10) PFU/mL, respectively, for high titers. Interestingly, FCV-F9 was undetectable after exposure to the three tested PP solutions using both low and high titers. MNV-1 at low initial titers was reduced by 1.30, 2.11, and 3.61 log(10) PFU/mL and at high initial titers by 1.56, 1.48, and 1.54 log(10) PFU/mL with 4, 8, and 16 mg/mL of PP treatment, respectively. MS2 at low initial titers was reduced by 0.41, 0.45, and 0.93 log(10) PFU/mL and at high initial titers by 0.32, 0.41, and 0.72 log(10) PFU/mL after 4, 8, and 16 mg/mL of PP treatment, respectively. PJ and PP resulted in titer reductions of foodborne virus surrogates after 1 h exposure, showing promise for use in hurdle technologies and/or for therapeutic or preventive use. To suggest the use of PJ and PP as natural remedies for foodborne viral illness prevention, their mechanism of action against viral infectivity needs to be further investigated.

Antiviral effects of cranberry juice and cranberry proanthocyanidins on foodborne viral surrogates – A time dependence study in vitro

Cranberry juice (CJ) and cranberry proanthocyanidins (PAC) are widely known for their antibacterial, antiviral, and pharmacological activities. The effect of CJ and cranberry PAC on the infectivity of foodborne viral surrogates, murine norovirus (MNV-1), feline calicivirus (FCV-F9), MS2 (ssRNA) bacteriophage, and ϕX-174 (ssDNA) bacteriophage after 0 min to 1 h at room temperature was evaluated. Viruses at titers of ∼5 log 10PFU/ml were mixed with equal volumes of CJ at pH 2.6, CJ at pH 7.0, 0.30 mg/ml CJ PAC, 0.60 mg/ml PAC, or water and incubated for 0, 10, 20, 30, 40, 50 min, and 1 h at room temperature. Infectivity was determined using standard plaque assays. The viral reduction rates of the four tested viruses were found to vary considerably. Among the tested viruses, FCV-F9 titers were decreased the most by ∼5 log 10PFU/ml within 30 min. MS2 titers were decreased the least by only ∼1 log 10PFU/ml after 1 h with CJ at pH 2.6 and 0.30 mg/ml PAC, and ∼0.5 log 10PFU/ml with CJ at pH 7.0 and 0.15 mg/ml PAC. MNV-1 and ϕ-X174 showed comparable titer reductions which was between that of FCV-F9 and MS2. In most cases, viral reduction within the first 10 min of treatment accounted for ≥50% of the total reduction. Transmission electron microscopy on FCV-F9 treated with CJ and PAC revealed structural changes. This study shows potential of using natural bioactive compounds for controlling foodborne viral diseases. Further studies are necessary to elucidate the mechanism of action of CJ components and to understand the differences in viral titer reduction profiles.

A Third-Generation Herpesvirus Is Effective Against Gastroesophageal Cancer

Gastroesophageal cancer remains a leading cause of cancer deaths and is uniformly fatal in patients presenting with metastases and recurrence. This study sets out to determine the effect of a third-generation, replication-competent, oncolytic herpes simplex type 1 virus containing transgenes encoding for a fusogenic membrane glycoprotein and Fcy::Fur, against gastroesophageal cancer. The cytotoxic effect of the virus was tested on human gastroesophageal cancer cell lines OCUM-2MD3, MKN-45, AGS, MKN-1, MKN-74, and BE-3 at sequential multiplicities of infection (MOI). Cytotoxicity was measured using a lactate dehydrogenase assay. Viral replication was tested by serially diluting supernatants from viral infections and titering on VERO cells via standard plaque assay. Correlations of cytotoxicity and viral replication were also investigated. All cell lines were susceptible to viral infection and demonstrated a dose-dependent effect, with greater and faster cytotoxicity at higher MOIs. Viral replication was supported in the cell lines tested, with peak titers by d 5, some supporting as high as >40,000× amplification. Cell lines with longer doubling times (>30 h) also achieved higher viral titers at a MOI of 0.1. Cell lines with shorter doubling times achieved 50% cell kill in fewer days, with an average of2.3 d for cell lines with doubling times under 30 h compared with 4.4 d for cell lines with doubling times over 30 h. These results suggest that this third-generation oncolytic herpesvirus can effectively infectand lyse gastroesophageal cancer cells and may provide a novel therapy against gastroesophageal cancer.

The effect of cranberry juice and cranberry proanthocyanidins on the infectivity of human enteric viral surrogates

The effect of cranberry juice (CJ) and cranberry proanthocyanidins (PAC) on the infectivity of human enteric virus surrogates, murine norovirus (MNV-1), feline calicivirus (FCV-F9), MS2(ssRNA) bacteriophage, and phiX-174(ssDNA) bacteriophage was studied. Viruses at high (∼7 log 10 PFU/ml) or low (∼5 log 10 PFU/ml) titers were mixed with equal volumes of CJ, 0.30, 0.60, and 1.20 mg/ml final PAC concentration, or water and incubated for 1 h at room temperature. Viral infectivity after treatments was evaluated using standardized plaque assays. At low viral titers, FCV-F9 was undetectable after exposure to CJ or the three tested PAC solutions. MNV-1 was reduced by 2.06 log 10 PFU/ml with CJ, and 2.63, 2.75, and 2.95 log 10 PFU/ml with 0.15, 0.30, and 0.60 mg/ml PAC, respectively. MS2 titers were reduced by 1.14 log 10 PFU/ml with CJ, and 0.55, 0.80, and 0.96 log 10 PFU/ml with 0.15, 0.30, and 0.60 mg/ml PAC, respectively. ϕ-X174 titers were reduced by 1.79 log 10 PFU/ml with CJ, and 1.95, 3.67, and 4.98 log 10 PFU/ml with PAC at 0.15, 0.30, and 0.60 mg/ml, respectively. Experiments using high titers showed similar trends but with decreased effects. CJ and PAC show promise as natural anti-virals that could potentially be exploited for foodborne viral illness treatment and prevention.

Effect of chitosan on the infectivity of murine norovirus, feline calicivirus, and bacteriophage MS2.

Chitosan is known to inhibit microorganisms of concern to plants, animals, and humans. However, the effect of chitosan on human enteric viruses of public health concern has not been extensively investigated. The purpose of this study was to determine the effect of chitosan on three human enteric viral surrogates: murine norovirus 1 (MNV-1), feline calicivirus F-9 (FCV-F9), and (ssRNA) bacteriophage MS2 (MS2). Chitosan oligosaccharide lactate (molecular weight of 5,000) and water-soluble chitosan (molecular weight of 53,000) at concentrations of 1.4, 0.7, and 0.35% were incubated at 37 degrees C for 3 h with equal volumes of each virus at high (approximately 7 log PFU/ml) and low (approximately 5 log PFU/ml) titers. Chitosan effects on each treated virus were evaluated with standardized plaque assays in comparison to untreated virus controls. The water-soluble chitosan at 0.7% decreased the FCV-F9 titer by approximately 2.83 log PFU/ml, with decreasing effects at lower concentrations, and also decreased MS2 at high titers by approximately 1.18 to 1.41 log PFU/ml, regardless of the concentration used. Chitosan treatments at the concentrations studied had no effect on MNV-1 at high titers. Chitosan oligosaccharide showed similar trends against the viruses, but to a lesser extent compared with that of water-soluble chitosan. When lower virus titers (approximately 5 log PFU/ml) were used, plaque reduction was observed for FCV-F9 and MS2, but not MNV-1. The use of higher-molecular-weight chitosan and at higher concentrations with longer incubation may be necessary to inactivate MNV-1. These results in the plaque reduction of human enteric virus surrogates by chitosan treatment show promise for its potential application in the food environment.

Evaluation of hydrogen peroxide gaseous disinfection systems to decontaminate viruses

This study assessed the efficacy of two commonly used gaseous disinfection systems against high concentrations of a resistant viral surrogate in the presence and absence of soiling. MS2 bacteriophage suspensions were dried on to stainless steel carriers and exposed to hydrogen peroxide vapour (HPV) and vapour hydrogen peroxide (VHP) gaseous disinfection systems. The bacteriophages were also suspended and dried in 10% and 50% of horse blood to simulate the virus being present in a spill of blood/bodily fluids in a hospital ward environment. Carriers were removed from the gaseous disinfectant at regular intervals into phosphate-buffered saline, vortexed and assayed using a standard plaque assay. The effectiveness of both the HPV and VHP systems varied with the concentration of the bacteriophage with HPV resulting in a 6log(10) reduction in 10 min at the lowest viral concentration [10(7) plaque-forming units (pfu)/carrier] and requiring 45 min at the highest concentration (10(9) pfu/carrier). For the VHP system a 30 min exposure period was required to achieve a 6log(10) reduction at the lowest concentration and 60-90 min for the highest concentration. The addition of blood to the suspension greatly reduced the effectiveness of both disinfectants. This study demonstrates that the effectiveness of gaseous disinfectants against bacteriophage is a function of the viral concentration as well as the degree of soiling. It highlights the importance of effective cleaning prior to gaseous disinfection especially where high concentration agents are suspended in body fluids to ensure effective decontamination in hospitals.

Development of an antibody-dependent enhancement assay for dengue virus using stable BHK-21 cell lines expressing FcγRIIA

Dengue virus (DENV) causes a wide range of symptoms, from mild febrile illness, dengue fever (DF), to severe life threatening illness, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Subneutralizing concentrations of antibody to DENV enhance DENV infection in FcγR positive cells. This phenomenon is known as antibody-dependent enhancement (ADE). ADE is considered to be a risk factor for DHF and DSS. To develop an ADE assay for DENV, two stable BHK-21 cell lines were established that express FcγRIIA (BHK-FcγRIIA). The BHK-FcγRIIA cell lines were used in an ADE assay with monoclonal antibody (4G2) to DENV, and DENV antibody-positive human sera. Virus growth was quantified directly in BHK-FcγRIIA cells with a standard plaque assay procedure. ADE was detected with monoclonal antibody (4G2) to DENV. ADE was also detected with DENV antibody-positive human sera, but not with DENV antibody-negative human sera. The new ADE assay using BHK-FcγRIIA cells is simple and practical, and is useful for defining the role of ADE in the pathogenesis of DENV infection.

Long-Range Reverse Transcription as a Useful Tool to Assess the Genomic Integrity of Norovirus

The effects of heat (72°C) and UV-light (254 nm) on the infectivity of murine norovirus-1 (MNV-1) and human norovirus (NoV) are reported. The infectivity of MNV-1 was measured using standard cell-culture plaque assays and was compared with a specific duplex RT-qPCR for MNV-1, which targets two fragments of the MNV genome, one 142 bp (short-range, sRT-qPCR) and a second 4.6 kb (long-range, loRT-qPCR) upstream from the reverse transcription (RT) priming site (poly-A tail). After the heat treatment of MNV neither the sRT- nor loRT-qPCR correlated with decreased infectivity as assessed by the plaque assay. However, after UV-exposure, the lo-qPCR showed decreased qPCR amplification, which became more pronounced with increased UV-exposure time, and reflected decreased infectivity as shown in the plaque assay. The sRT-qPCR amplification was not affected by UV-exposure. Similar results were obtained when challenging non-culturable human GI and GII NoV with UV-light. Using previously published RT-qPCR assays for GI and GII NoV, no decrease in RT-qPCR amplification was seen when using the respective reverse primers of the RT-qPCR assay in the RT reaction, whereas a significant decrease in qPCR amplification occurred when the RT reaction was primed at the poly-A tail about 2.3 kb from the qPCR amplification site. The results indicate that, in contrast to RT-qPCR with a long-range RT, RT-qPCR with a short-range RT does not reflect genomic integrity and therefore viral infectivity. Furthermore, human NoV appears to be highly sensitive to UV-irradiation which could lead to more efficient decontamination procedures.

A bioluminescence-based assay for enumeration of lytic bacteriophage

A bioluminescence-based assay for enumeration of lytic bacteriophage was developed. The assay consists of a bioluminescent Escherichia coli as the host bacterium, the lytic bacteriophage T4 and an automated luminometer measuring luminescence over time. The assay is based on the decrease in luminescence as the bioluminescent host cells are lysed by T4. The T4 concentration, bioluminescent E. coli concentration, phage suspension medium, and temperature (25 °C and 37 °C) were varied. There was a strong negative correlation between bioluminescence intensities and T4 phage concentrations at both room temperature ( R 2 = 0.993) and 37 °C ( R 2 = 0.970). Phage was detected more rapidly at 37 °C than at 25 °C. The detection limit was also lower when the assay was performed at 37 °C with a minimum detection level of 2.4 log CFU/ml compared to 3.4 log CFU/ml for 25 °C. The assay was used to determine thermal inactivation using T4 phages heated at 70 °C for 0 to 30 min, and phage concentrations were determined using the bioluminescence assay and a standard plaque assay. There was no significant difference between the two enumeration methods ( P > 0.01). This study suggests the bioluminescence-based assay can be used as an alternative for quantitatively monitoring phage infectivity, instead of conventional standard plaque assays.

Accurate single-day titration of adenovirus vectors based on equivalence of protein VII nuclear dots and infectious particles

Protein VII is an abundant component of adenovirus particles and is tightly associated with the viral DNA. It enters the nucleus along with the infecting viral genome and remains bound throughout early phase. Protein VII can be visualized by immunofluorescent staining as discrete dots in the infected cell nucleus. Comparison between protein VII staining and expression of the 72 kDa DNA-binding protein revealed a one-to-one correspondence between protein VII dots and infectious viral genomes. A similar relationship was observed for a helper-dependent adenovirus vector expressing green fluorescent protein. This relationship allowed accurate titration of adenovirus preparations, including wild-type and helper-dependent vectors, using a 1-day immunofluorescence method. The method can be applied to any adenovirus vector and gives results equivalent to the standard plaque assay.

In vivo biodistribution of a highly attenuated recombinant vesicular stomatitis virus expressing HIV-1 Gag following intramuscular, intranasal, or intravenous inoculation

Recombinant vesicular stomatitis viruses (rVSVs) are being developed as potential HIV-1 vaccine candidates. To characterize the in vivo replication and dissemination of rVSV vectors in mice, high doses of a highly attenuated vector expressing HIV-1 Gag, rVSV IN-N4CT9-Gag1, and a prototypic reference virus, rVSV IN-HIVGag5, were delivered intramuscularly (IM), intranasally (IN), or intravenously (IV). We used quantitative, real-time RT-PCR (Q-PCR) and standard plaque assays to measure the temporal dissemination of these viruses to various tissues. Following IM inoculation, both viruses were detected primarily at the injection site as well as in draining lymph nodes; neither virus induced significant weight loss, pathologic signs, or evidence of neuroinvasion. In contrast, following IN inoculation, the prototypic virus was detected in all tissues tested and caused significant weight loss leading to death. IN administration of rVSV IN-N4CT9-Gag1 resulted in detection in numerous tissues (brain, lung, nasal turbinates, and lymph nodes) albeit in significantly reduced levels, which caused little or no weight loss nor any mortality. Following IV inoculation, both prototypic and attenuated viruses were detected by Q-PCR in all tissues tested. In contrast to the prototype, rVSV IN-N4CT9-Gag1 viral loads were significantly lower in all organs tested, and no infectious virus was detected in the brain following IV inoculation, despite the presence of viral RNA. These studies demonstrated significant differences in the biodistribution patterns of and the associated pathogenicity engendered by the prototypic and attenuated vectors in a highly susceptible host.

Inhibition of Herpes Simplex Virus Type 1 Infection by Some Selected Antitumor Titanium(III)-Based Coordination Compounds

ABSTRACTUsing a standard plaque assay and clinical strains of Herpes Simplex virus type 1 (HSV 1), we have tested the ability of two titanium (Ti) - content complexes—Titanocene Dichloride (Cp2TiCl2) and Titanocene Y. Virus was treated by incubation at 37°C with both complexes in cell culture media and was then diluted and plated onto human embryonic lung fibroblast cells MRC-5 for detection and quantitation of remaining infectious virus. Of one randomly chosen clinical HSV 1 (TM strain), sensitive to acyclovir (ACV), was inactivated > 98% by treatment in vitro with 0.01 μM Titanocene Y for 2h. The effect was concentration dependent. With an HSV 1 strain, 0.01 μM Titanocene Y or Cp2TiCl2 caused 99% inactivation, 0.001μM caused 98 to 99% inactivation, and 0.0001μM caused 63 to 86% inactivation. Short (5 min) treatments of selected isolate with Titanocene Y or Cp2TiCl2 yielded inactivation rates of 0 to 55%. We assumed also that Titanocene Y or Cp2TiCl2 could be able to affect negatively not only virus DNA synthesis but also another virus target. The data obtained show that: (i) the 300bp relatively stable region during a short period of viral replication corresponding to ReIV is amplified in non-treated viral control, but not in mock cells and in negative control and (ii) the region was not amplified in Titanocene Y treated samples. The data show that the effect of the suppressive therapy of virus replication by Titanocene Y could be due to the suppression of immediate early Us1 and Us12 genes encoding essential for virus replication products α22—the protein affecting virus ability to replicate, and α47—the protein inhibiting MHC I antigen presentation.

Sex Differences in Clearance of HSV‐1 after Acute Ethanol Exposure

Sex hormones are known to regulate immunity, and a dichotomy exists in the immune response between the sexes. Ethanol is also recognized to alter the immune response and may affect clearance of infectious agents differently in men and women. To examine sex differences in ethanol's effect on virus clearance, we used an intransasal model of Herpes Simplex Virus Type I (HSV‐1) infection. Male and female C57BL/6 mice were treated with an acute dose of ethanol (2.9g/kg) designed to give blood alcohol levels of 300mg/dL at 30 minutes at which time they were inoculated with HSV‐1 (5x104pfu). The lungs were harvested and virus replication was assessed using standard plaque assays. Virus titers were significantly higher on day 2 and 6 post‐inoculation in the lungs of female mice given ethanol compared to female mice given saline (p &lt; 0.05) and male mice given saline or ethanol (p &lt; 0.05). Additionally, Th1 cytokines including IFN‐γ, TNF‐α, and IL‐12 were all found to be 2‐fold lower in the lungs of female mice given ethanol compared to their saline controls. No differences were observed in cytokine production between male mice given ethanol or saline. These data suggest that ethanol may inhibit IFN‐γ production in female mice leading to decreased virus clearance. Supported by NIH R01AA012034, T32AA013527, an Illinois Excellence in Academic Medicine Grant and the Dr. Ralph and Marian C. Falk Medical Research Trust.

Rab8, a Vesicular Traffic Regulator, Is Involved in Dengue Virus Infection in HepG2 Cells

Objective: The pathogenesis of dengue virus (DV) has not been completely clarified. Rab8 regulates vesicular traffic from Golgi to plasma membrane where DV is matured and then delivered by exocytosis. In this study, involvement of Rab8 in DV serotype 2 (DV2) infection was investigated in HpeG2 cells. Methods: Distributions of Rab8 and DV2, and the number of infection cells were observed by immunostaining. HepG2^Rab8AM and HepG2^Rab8DN cells were constructed to stably express a constitutively active mutant of Rab8 and a dominant negative mutant, respectively, which were assessed by flow cytometry. Production of infectious virions and the amounts of DV2 entry were detected by standard plaque assay. Viral RNA replication was detected by real-time RT-PCR. Results: Rab8 showed high co-localization with DV2 in HpeG2 cells and the amount of DV antigen-positive cells decreased in HepG2^Rab8AM and HepG2^Rab8DN cells. Also, progeny virus released from those cells was drastically reduced. Infectious virions produced in cells were also significantly reduced, while the viral RNA replication was down-regulated by a different level. Furthermore, viral entry into those cells was reduced by about 80%. Conclusions: Our data suggest that the function of Rab8 is important for DV2 infection, and Rab8 may be involved in DV2 infection.