Standard Plaque Assay Research Articles

Counting cytopathogenic virus adsorbed to cellulose nitrate membrane filters as a simple method for counting viruses in raw sewage and sewage effluents

Using the VIRMETADEN (acronym derived from virus adsorption enumeration) method to count cytopathogenic viruses adsorbed to cellulose nitrate membrane filters from prefiltered and decontaminated sewage samples was shown to be feasible. The numbers of naturally occurring enteroviruses recovered by the VIRMETADEN method were significantly higher than those obtained by standard plaque assay. For prefiltration of sewage samples, low protein binding polyvinylidene fluoride (PVDF) membranes of 0.65 and 5 μm pore size proved to be at least as good as prefilters used previously, both in the volume that could be filtered and in the low virus retention. Regarding decontamination, 0.22 μm pore size PVDF membranes were as good as chloroform treatment. Alternatively, decontamination with 0.05% phenol followed by filtration through 0.65 μm PVDF membrane filters proved to be an excellent method for clarifying and decontaminating the sample prior to enumerating viruses using the VIRMETADEN method. It allows good recovery and greater decontaminated sample volumes. The application of VIRMETADEN to decontaminated raw sewage and secondary effluent gave recoveries of approximately 100% of vaccinal polioviruses seeded in sewage. The advantage of this approach is that the same method is applicable to sewage affluents and effluents with a simple and fast method, which implies lower material and labour costs.

Duck hepatitis B photoinactivation bydimethylmethylene blue in RBC suspensions.

Dimethylmethylene blue (DMMB) has been used to photoinactivate a number of model viruses, including VSV, in RBC suspensions under conditions that preserve in vitro RBC properties during storage. The relative sensitivity of duck HBV (DHBV) and VSV to photoinactivation by DMMB was investigated by performing an indirect immunofluorescence assay (IFA) using primary duck hepatocyte (PDH) cultures or a standard plaque assay for the respective viruses. DMMB was added to 45-percent Hct, WBC-reduced, oxygenated AS-3 RBCs at 10-, 1-, and 0.1-microM concentrations. Samples (1-mm thick) were illuminated with 5.4-mW per cm(2) of red light for 2 or 9 seconds. Unilluminated samples without DMMB or with 10 microM DMMB served as control. DHBV and VSV were rapidly photoinactivated by DMMB in a concentration and light-dose-dependent fashion. Neither virus was substantially inactivated by incubation with DMMB in the dark. For a given light exposure, DHBV required a concentration of DMMB one-one hundredth that of VSV to achieve approximately the same level of inactivation. DHBV appears to be considerably more sensitive than VSV to DMMB photoinactivation. Photoinactivation in 45-percent Hct RBCs can be achieved in seconds by using micromolar quantities of dye.

Intracellular Chelation of Iron by Bipyridyl Inhibits DNA Virus Replication: RIBONUCLEOTIDE REDUCTASE MATURATION AS A PROBE OF INTRACELLULAR IRON POOLS

The efficient replication of large DNA viruses requires dNTPs supplied by a viral ribonucleotide reductase. Viral ribonucleotide reductase is an early gene product of both vaccinia and herpes simplex virus. For productive infection, the apoprotein must scavenge iron from the endogenous, labile iron pool(s). The membrane-permeant, intracellular Fe(2+) chelator, 2,2'-bipyridine (bipyridyl, BIP), is known to sequester iron from this pool. We show here that BIP strongly inhibits the replication of both vaccinia and herpes simplex virus, type 1. In a standard plaque assay, 50 microm BIP caused a 50% reduction in plaque-forming units with either virus. Strong inhibition was observed only when BIP was added within 3 h post-infection. This time dependence was observed also in regards to inhibition of viral late protein and DNA synthesis by BIP. BIP did not inhibit the activity of vaccinia ribonucleotide reductase (RR), its synthesis, nor its stability indicating that BIP blocked the activation of the apoprotein. In parallel with its inhibition of vaccinia RR activation, BIP treatment increased the RNA binding activity of the endogenous iron-response protein, IRP1, by 1.9-fold. The data indicate that the diiron prosthetic group in vaccinia RR is assembled from iron taken from the BIP-accessible, labile iron pool that is sampled also by ferritin and the iron-regulated protein found in the cytosol of mammalian cells.

Determination of bovine adenovirus-3 titer based on immunohistochemical detection of DNA binding protein in infected cells

DNA sequence coding for a portion of DNA binding protein (amino acids 3–58) of bovine adenovirus type-3 (BAV-3) was cloned and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase. The fusion protein was affinity purified and used to immunize rabbits. Immunoprecipitation and Western blot analysis showed that the antiserum could specifically recognize a protein of 48 kDa in BAV-3-infected cells, which was produced both in early and late phases of BAV-3 life cycle. Based on the ability of antiserum to recognize DNA binding protein, a novel assay for BAV-3 quantitation was established. The assay is less time consuming and can be performed on a wide variety of bovine cells. In addition, virus titers determined by this assay are comparable to the standard plaque assay.

Incidence and Severity of Herpetic Stromal Keratitis: Impaired by the Depletion of Lymph Node Macrophages

HSV-1 induced stromal keratitis (HSK) is an immune-mediated disease. The role of macrophages in this process is still unclear. In this study we investigated the influence of specific macrophage depletion from the spleen and the submandibular lymph nodes by dichloromethylene diphosphonate liposomes (Cl2MDP-LIP) on the course of HSV-1 keratitis. BALB/c mice were infected corneally with 105PFU of HSV-1 (KOS). Groups of mice received Cl2MDP-LIP 7 and 2 days prior to infection. Cl2MDP-LIP were given by various routes: intravenously (i.v.) for macrophage depletion in the spleen; subcutaneously for macrophage depletion in the submandibular regional lymph node (s.c.); or both i.v. and s.c. The development of HSV-1 keratitis was evaluated clinically and histologically. A standard plaque assay from the infected eyes was used to measure virus clearance. Seventy-nine percent of the HSV-1-infected control mice (n=14) developed severe stromal keratitis by day 14 p.i. The development of stromal keratitis was inhibited by Cl2MDP-LIP given s.c. (64%;n=14;P<0.05), i.v. and s.c. (50%;n=14;P<0.05), but not by i.v. treatment alone (77%;n=13). After s.c., i.v. and s.c. Cl2MDP-LIP injection, histologically the corneal stroma had a decrease in inflammatory cell infiltration by day 14 p.i. compared to the control group, and the DTH response was reduced. The healing of epithelial HSV-1 keratitis and the virus clearance were not affected significantly. These results indicate an important function of macrophages in the course of HSV-1 keratitis. Virus replication in the eye does not appear to be affected by monocytes/macrophages of lymph nodes and spleen. In contrast, the immunopathological process of stromal HSV-keratitis that results in corneal destruction is profoundly accelerated by macrophages in the lymph nodes.

Characterization of DNA Binding Protein of Porcine Adenovirus Type 3

To identify and characterize the protein encoded by the E2A region of porcine adenovirus (PAV)-3, DNA sequence coding for a portion (amino acids 102–457) of the DNA binding protein (DBP) open reaching frame was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase protein of Schistosoma japonica. The affinity-purified fusion protein was used to immunize rabbits. Immunoprecipitation/Western blot analysis demonstrated that the antisera specifically recognized a protein of 50 kD in PAV-3-infected cells. Immunoperoxidase staining detected the DBP protein predominantly in the nucleus of the cells. Western blot analysis demonstrated that DBP was detected as early as 6 h after infection and remained detectable throughout the infection. Based on these results, a novel assay for quantitation of PAV-3 was established. The assay is less time consuming and can be performed in different porcine cells. In addition, virus titers determined by this assay are comparable to the standard plaque assay.

Zinc salts inactivate clinical isolates of herpes simplex virus in vitro.

Using a standard plaque assay and clinical isolates of herpes simplex virus (HSV), we have tested the ability of zinc salts to inactivate HSV. Virus was treated by incubation at 37 degrees C with zinc salts in morpholinepropanesulfonic acid-buffered culture medium and was then diluted and plated onto CV-1 cells for detection and quantitation of remaining infectious virus. Of 10 randomly chosen clinical isolates (five HSV type 1 [HSV-1] isolates and five HSV-2 isolates), seven were inactivated >98% by treatment in vitro with 50 mM zinc gluconate for 2 h and nine were inactivated >97% by treatment with zinc lactate. The effect was concentration dependent. With an HSV-1 isolate, 50 mM zinc gluconate or zinc lactate caused 100% inactivation, 15 mM caused 98 to 99% inactivation, and 5 mM caused 63 to 86% inactivation. With an HSV-2 isolate, 50 and 15 mM zinc gluconate caused 30% inactivation and 5 and 1 mM caused less than 9% inactivation, whereas 50 and 15 mM zinc lactate caused greater than 92% inactivation and 5 and 1 mM caused 37 and 26% inactivation, respectively. The ability of the zinc salts to inactivate HSV was not related to pH in the pH range of 6.1 to 7.6 since inactivation by zinc gluconate or zinc lactate in that pH range was 99.7 to 100% with a 2-h treatment with 50 mM zinc salt. Short (5-min) treatments of selected isolates with zinc gluconate, zinc lactate, zinc acetate, or zinc sulfate yielded inactivation rates of 0 to 55%.

Development of a highly sensitive quantitative competitive PCR assay for the detection of murine cytomegalovirus DNA

Viral persistence and molecular latency are characteristic of infection by the human cytomegalovirus (HCMV). Using the murine cytomegalovirus (MCMV) as a model for human infection, a quantitative-competitive polymerase chain reaction (QC-PCR) assay was developed to detect and quantify MCMV-DNA in the salivary glands of infected mice. The QC-PCR detected high numbers of MCMV DNA copies in the absence of infectious virus. By comparing the DNA content and the results obtained from a standard semiquantitative plaque assay, it is concluded that 1 plaque-forming unit (pfu) is the equivalent of approximately 1500 viral genomes. By day 42-post infection (pi) 4×10 3 copies of DNA/1 mg tissue were sufficient to reactivate infectious virions after cyclophosphamide immunosupression. By day 90 pi, however, when the DNA load was decreased to <1.2×10 2, reactivation was not observed. These results indicate that viral reactivation will occur when the number of infectious DNA copies is equivalent about 2–3 pfu. This quantitative test may therefore help to detect CMV and the risk of reactivation in immunosupressed patients.

Nasal quantity of respiratory syncytical virus correlates with disease severity in hospitalized infants.

To evaluate the relationship between nasal quantity of respiratory syncytial virus (RSV) and disease severity in hospitalized infants without underlying cardiopulmonary disease or immunodeficiency. Nasal aspirates were obtained from hospitalized infants <24 months of age with recently identified RSV infection and evaluated for RSV quantity by a standard plaque assay on HEp-2 cell monolayers. Subjects were classified as having "severe" disease if they required mechanical ventilation at the time of sample collection and as having "nonsevere" disease if they did not. Linear modeling was used to determine the relationship between nasal RSV quantity and various independent variables, including disease severity. Nasal aspirates from 39 patients were evaluated. Age, gender and mean duration of time from symptom onset to sample acquisition (5 days) were similar between the severe (n = 15) and nonsevere (n = 24) groups. Significantly more infants were born at <35 weeks gestation in the severe disease group (7 of 15 vs. 3 of 24, P = 0.017), and infants born at <35 weeks gestation were significantly more likely to be of non-Caucasian ethnicity than were infants born at > or =35 weeks gestation (8 of 10 vs. 12 of 29, P = 0.035). The linear model found that higher nasal RSV quantities were associated with severe disease [mean +/- SEM, 5.06 +/- 0.34 log plaque-forming units (pfu)/ml vs. 3.91 +/- 0.35 log pfu/ml, P = 0.022], gestational age > or =35 weeks (5.44 +/- 0.27 log pfu/ml vs. 3.52 +/- 0.45 log pfu/ml, P = 0.002) and non-Caucasian ethnicity (5.16 +/- 0.30 log pfu/ml vs. 3.80 +/- 0.37 log pfu/ml, P = 0.006). Nasal RSV quantity correlates with disease severity in hospitalized infants with recently identified RSV infection.

Macrophage-depletion influences the course of murine HSV-1 keratitis

Purpose. Herpes simplex virus type 1 infection of the cornea induces an immune-mediated disease termed “herpes stromal keratitis” (HSK) that is a major cause of blindness. In this study we investigated the influence of macrophage depletion by Cl 2 MDP encapsulated in liposomes (Cl 2 MDP-LIP) on the course of HSV-1 keratitis. Methods. The corneas of BALB/c mice were infected with 10 5 PFU of HSV-1 (KOS strain). Mice groups received sub-conjunctival PBS or Cl 2 MDP-LIP injections 7 and 2 days prior to infection. The eyes were studied clinically, histologically and immunohistochemically with F4/80 antibody at various time points after treatment. Clearance of the virus from the HSV-infected eyes was measured with a standard plaque assay. Results. After subconjunctival Cl 2 MDP-LIP treatment, the HSV-1-induced epithelial keratitis was more severe (P < 0.05). The virus titers were significantly higher after macrophage depletion (day 7, P < 0.005). Stromal keratitis developed in 78.6% of HSV-1 infected PBS treated control mice (n = 14) by day 14 after infection. By subconjunctival Cl 2 MDP-LIP treatment (n = 14) the incidence of stromal keratitis was reduced to 42.9%, and the keratitis was less severe (P < 0.05). Conclusions. The data demonstrate an influence of macrophages on the course of HSV-1 keratitis in mice. Macrophage depletion influence the viral replication in the cornea and the immune-mediated process of HSK.

A simple methodological approach for counting and identifying culturable viruses adsorbed to cellulose nitrate membrane filters.

We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they covered the entire surface of each filter and penetrated through the pores. When such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method and a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters. Polioviruses in aqueous suspensions were then concentrated by adsorption to cellulose membrane filters and were subsequently counted without elution, a step which is necessary when the commonly used methods are employed. The pore size of the membrane filter, the sample contents, and the sample volume were optimized for tap water, seawater, and a 0.25 M glycine buffer solution. The numbers of viruses recovered under the optimized conditions were more than 50% greater than the numbers counted by the standard plaque assay. When ceftazidime was added to the assay medium in addition to the antibiotics which are typically used, the method could be used to study natural samples with low and intermediate levels of microbial pollution without decontamination of the samples. This methodological approach also allowed plaque hybridization either directly on cellulose nitrate membranes or on Hybond N+ membranes after the preparations were transferred.

Clinical virology of Ebola hemorrhagic fever (EHF): virus, virus antigen, and IgG and IgM antibody findings among EHF patients in Kikwit, Democratic Republic of the Congo, 1995.

Ebola hemorrhagic fever (EHF) patients treated at Kikwit General Hospital during the 1995 outbreak were tested for viral antigen, IgG and IgM antibody, and infectious virus. Viral antigen could be detected in virtually all patients during the acute phase of illness, while antibody was not always detectable before death. Virus was also isolated from patients during the course of their febrile illness, but attempts to quantify virus in Vero E6 cells by standard plaque assay were often unsuccessful. IgG and IgM antibody appeared at approximately the same time after disease onset (8-10 days), but IgM persisted for a much shorter period among the surviving convalescent patients. IgG antibody was detectable in surviving patients through about 2 years after onset, the latest time that samples were obtained. Detection of Ebola virus antigens or virus isolation appears to be the most reliable means of diagnosis for patients with suspected acute EHF, since patients with this often-fatal disease (80% mortality) may not develop detectable antibodies before death.

Toxicology and humoral immunity assessment of decamethylcyclopentasiloxane (D5) following a 1-month whole body inhalation exposure in Fischer 344 rats.

D5 is a low-molecular-weight cyclic siloxane used for industrial and consumer product applications. The objective of the present study was to assess potential toxic and immunomodulatory consequences of inhalation exposure to D5. Male and female Fischer 344 rats (25/group) were exposed by whole body inhalation to 0, 10, 25, 75, or 160 ppm D5 6 h/day, 7 days/week for 28 days. Clinical signs, body weights, and food consumption were recorded. On the day following the final exposure, 10 rats/group/sex were euthanized and a complete necropsy performed. Following a 14-day nonexposure recovery period, the remaining 5 rats/sex/group were necropsied. Body and organ weights were obtained and a complete set of tissues was taken for histopathology. Samples were also collected for serum chemistry, hematology, and urinalysis. Immunotoxicology-designated rats (10/sex/group) were immunized with sheep erythrocytes (sRBC) 4 days prior to euthanasia and cyclophosphamide (CYP) was administered i.p. to positive controls on days 24 through 28. The anti-sRBC antibody-forming cell (AFC) response was evaluated in a standard plaque assay. Blood was also collected for examination in the anti-sRBC enzyme-linked immunosorbant assay (ELISA). D5 exposure did not modulate humoral immunity, while the internal control, CYP, produced the expected suppression of the AFC response. D5 exposure caused no adverse effects on body weight, food consumption, or urinalysis parameters. Serum alkaline phosphatase (SAP) was significantly decreased in females at terminal (12%, 160 ppm) and recovery sacrifice. A significant increase in the liver-to-body weight ratio was observed in female animals at the end of exposures (13%, 160 ppm), but was not noted in recovery animals from the same exposure group. In males, significant increases in liver-to-body weight (5%) and thymus-to-body weight (14%) ratios were also noted at the high dose at terminal sacrifice and were not present at recovery. At recovery only, a significant increase in spleen-to-body weight ratios (14 and 17%; 25 and 160 ppm, respectively) was noted. At the end of exposure, histopathological analysis indicated an increased incidence and severity of nasal (Level 1) goblet cell proliferation. Focal macrophage accumulation in the lung was also observed to be increased in incidence in both sexes at 160 ppm. At the end of the recovery period, the effects in both of these organs appeared to be reversible. In summary, D5 inhalation exposure did not alter humoral immunity and caused only minor, transient changes in hematological, serum chemistry, and organ weight values. Histopathological changes were confined to the respiratory tract and appeared to be reversible. The no observed effect level for systemic toxicity, based primarily on the liver weight changes, was 75 ppm.

Formation of submicron colloidal particles from marine bacteria by viral infection

Formation of submicron colloidal particles from marine bacteria by viral infection

Herpes simplex type 1: lacZ recombinant viruses. I. Characterization and application to defining the mechanisms of action of known antiherpes agents

Recombinant viruses with the lacZ gene placed under the control of the HSV-1 ICP4, TK and gD regulatory regions were constructed by recombination into the TK locus of HSV-1. Difficulty in isolating ICP4 and gD recombinant viruses with high level, regulated expression of β-galactosidase was overcome by the use of HSV-1 translational initiation sequences of these genes in place of vector-derived sequences. β-Galactosidase expression displayed the kinetics particular to each viral class. The maximal expression of β-galactosidase from the recombinant viruses within a 22-h period (m.o.i. 5) (relative to the ICP4 virus) was gD(3) > gC(2) > ICP4(1) > TK(0.5). The ICP4 virus produces easily quantifiable levels of β-galactosidase activity for multiplicities of infection from 5 × 10 −4 through 5 over 48 h postinfection. At multiplicities of infection between 2 and 5, ICP4-driven activity was measurable within 2 h postinfection from a monolayer of 3 × 10 4 Vero cells in microtiter wells. Mechanisms of inhibition of several antivirals were probed by using the regulated expression of β-galactosidase from the ICP4 virus as a marker for viral growth. An experimental antiviral (E3925, IC 50 1 μg/ml) and a neutralizing gD MAb (DUP55306, IC 50 0.6 μg/ml) acted prior to immediate early synthesis, consistent with inhibition of viral entry or uncoating. IFN-γ inhibited expression of immediate-early synthesis, while having no effect on viral entry. IC 50 values for E3925 obtained using either the ICP4 or gD viruses at m.o.i. 0.005, were in good agreement with those obtained by standard plaque assays, but were determined in only 1 day, using a microtiter plate format. Thus, these reporter viruses are useful tools for defining the mechanisms of action of antiherpes agents, while quantitatively reproducing the results for IC 50 determinations from standard plaque assays within 24 h in a microtiter plate format.

A SIMPLE AND RAPID METHOD FOR THE DETECTION AND ENUMERATION OF PHAGES OF BACTEROIDES FRAGILIS HSP40 IN FOODS AND SEWAGE1

ABSTRACTThe phage‐host model of Bacteroides fragilis HSP40 has been documented as a potential index of human sewage and enteroviruses in sewage samples. However, this organism is an obligate anaerobe, which significantly limits the convenience of this model. The objectives of this study were to develop a simple method for the detection and enumeration of phages of B. fragilis HSP40 without using cumbersome anaerobic methods, to compare the novel method to the standard plaque assay (PA), and to enumerate phages naturally occurring in foods and sewage using both methods. Oxyrase enzymeTM (OE) was used at 0.5 U/ml in the growth medium of B. fragilis HSP40 before aerobic incubation. A double tube for phage (DTP) was developed and compared to the standard PA throughout the study. An enrichment procedure for samples was tested using 0.5 U/ml OE. Results indicated that the DTP could detect plaque forming units (PFU) of phages of B. fragilis HSP40 within 12–18h, with a sensitivity of 1 PFU/ml. The need for anaerobic incubation was totally eliminated by using OE and applying the DTP. Phage counts using DTP and PA were highly correlated (R2= 0.99–1.00) in spiked peptone water and ground beef. Enrichment of samples with OE enhanced phage recovery from spiked ground beef. With the DTP and PA, phages of B. fragilis HSP40 were detected from sewage, oysters, mushrooms and chicken. No such phage was detected from fresh ground beef before and after enrichment with either method. The DTP method was simpler than the PA, and it can be recommended for rapid screening of foods for fecal contamination and potential presence of viruses.

Mannan‐Binding Protein and Bovine Conglutinin Mediate Enhancement of Herpes Simplex Virus Type 2 Infection in Mice

A broad range of plant lectins have recently been shown to inhibit the infectivity of herpes simplex virus type 1 (HSV-1) in vitro. We decided to investigate the role of mammalian lectins in infection with herpes simplex virus. Two lectins, conglutinin and mannan-binding protein (also called mannose-binding protein, MBP), belonging to the collectin family of lectins, were examined. Four week-old BALB/c mice were injected subcutaneously with 100 micrograms bovine conglutinin or 50 micrograms human MBP 1 day before intravenous infection with 5 x 10(4) PFU of herpes simplex virus type 2 (HSV-2). A three-fold increase in virus titre of the liver was observed on day 3 of the infection in the mice pretreated with conglutinin or MBP, whereas no effect was seen on days 1 and 5. In a standard plaque assay using Vero cells we were not able to demonstrate reproducibly either infection inhibition or infection enhancement, when virus was pre-incubated with differing concentrations of the collectins. The concentrations used were similar to those used by us in vivo, and by others in in vitro experiments showing inhibition of the infectivity of HSV-1 with plant lectins. In an ELISA with HSV-2 antigens captured on anti-HSV-2 antibodies, calcium-dependent and carbohydrate inhibitable binding of the collectins was observed. Our results indicate that the effect of endogenous mammalian collectins in vivo may not be neutralization as suggested by the data using plant lectins. Instead, the previously described opsonizing activity of the mammalian collectins may provide the virions with an alternative port of entry into cells leading to infection enhancement.

Synthesis and anti-herpes activity of some a-ring functionalized dehydroabietane derivatives

Dehydroabietic acid 5 was converted to the exocyclic olefin 7 by applying the Barton decarboxylation protocol. The latter served as key intermediate for the stereoselective synthesis of a variety of ring-A functionalized derivatives. Compounds 13 and 16 turned out to be active against the herpes virus (HSV-2) in the standard plaque assay and seem to interfere with an early event in viral replication.

Permeability of latex and thermoplastic elastomer gloves to the bacteriophage φX174

Background: Most studies challenging the integrity of the glove barrier have compared the permeability of vinyl and latex gloves. However, no studies of a new nonlatex, nonvinyl thermoplastic elastomer have been reported. This pilot study therefore compared the protective barriers provided by latex and thermoplastic elastomer surgical gloves against penetration of the bacteriophage φX174. Methods: Twenty thermoplastic elastomer gloves and 25 commercially available latex gloves (20. brand 1, 5 brand 2) were filled with a sterile serum surrogate and exposed to the φX174 virus in a flask, with shaking for 180 minutes at 37° C. Aliquots of 5 ml were withdrawn at baseline, 30, 60, 120, and 180 minutes and assayed by a standard plaque assay. The remaining contents of the gloves were then tested by an extremely sensitive qualitative assay (plaque assay without dilution of the sample). Results: With the standard plaque assay, virus was detected in 30% of the brand 1 latex gloves, in 80% of the brand 2 latex gloves, but in none of the thermoplastic elastomer gloves. The qualitative assay, which can detect even a single virus in the entire glove contents, had positive results for 30% of the thermoplastic elastomer gloves, 70% of the brand 1 gloves, and 100% of the brand 2 gloves. Conclusions: Despite the small sample, the results of this stringent assay suggest that the mechanical barrier offered by thermoplastic elastomer gloves is equal to or better than that provided by the latex gloves tested. Clinical studies are needed to evaluate thermoplastic elastomer gloves, which may withstand mechanical stress better than latex or vinyl. Thermoplastic elastomer gloves may therefore be a desirable alternative for health care workers in high-risk settings or for individuals with latex allergies.

The retrograde tracer Fluoro-Gold interferes with the infectivity of herpes simplex virus

Fluoro-Gold has been used previously to identify those trigeminal ganglion cells that innervate the central cornea. To examine the effects of Fluoro-Gold treatment on infection and spread of HSV in vivo, we measured the number of plaque forming units recovered from trigeminal ganglia 3 or 5 days after corneal scratch and inoculation with Fluoro-Gold and HSV. Treatment with Fluoro-Gold reduced the amount of virus recovered after retrograde transport 63% at 3 days and 28% at 5 days after inoculation. When we examined trigeminal ganglion sections from animals treated with HSV and Fluoro-Gold, we found the number of neurons double labeled with antibodies that recognize HSV and Fluoro-Gold was only 13% of all Fluoro-Gold labeled neurons. This was significantly fewer cells that we had anticipated, on the basis of double labeling experiments with wheat germ agglutinin combined with Fluoro-Gold. The effects of varying doses of the retrograde tracer, Fluoro-Gold on Herpes simplex virus (type 1) (HSV) infectivity were also assayed in vitro using a standard viral plaque assay. At 1 × 10 −3 mg/ml Fluoro-Gold there was no effect on the number of plaque forming units. At 5 × 10 −1 mg/ml the number of plaques was reduced about 67%. We conclude that Fluoro-Gold interferes with productive HSV infection in vivo and in vitro after retrograde transport of HSV by neurons.