Reciprocal competition binding assays have previously demonstrated that 20 of 24 human rhinovirus serotypes tested compete for a single cellular receptor. These studies suggested that the vast majority of rhinovirus serotypes utilize a single cellular receptor. With HeLa cells as an immunogen, a mouse monoclonal antibody was isolated which had the precise specificity predicted by the competition binding study. The receptor antibody was shown to protect HeLa cells from infection by 78 of 88 human rhinovirus serotypes assayed. In addition, the receptor antibody protects HeLa cells from infection by three coxsackievirus A serotypes. The receptor antibody was unable to protect cells from infection by a wide range of other RNA and DNA viruses. Using the receptor antibody and human rhinovirus type 15, we determined that the cellular receptor utilized by the vast number of human rhinovirus serotypes is present only on cells of human origin, with the exception of chimpanzee-derived cells. The receptor antibody has a strong affinity for the cellular receptor as evidenced by its rapid binding kinetics and ability to displace previously bound human rhinovirus virions from receptors. No viral variants were identified which could bypass the receptor blockage.
BALB/c mice were immunized with purified preparations of hepatitis A virus (HAV) isolated after 21 days of growth in LLC-MK2 cells. The HAV antigen was isolated from CsCI gradients and consisted primarily of the following three proteins as analyzed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining: VP-1 at 33,000 daltons, VP-2 at 29,000 to 30,000 daltons, and VP-3 at 27,000 daltons. The spleen cells isolated from two BALB/c mice, immunized with two inoculations of HAV, were fused with SP 2/0 myeloma cells and grown in hypoxanthine-aminopterin-thymidine medium. Of 270 hybridomas initially screened, 72 were positive for binding HAV by a noncompetitive radioimmunoassay. All 72 were tested for the ability to neutralize the infectivity of HAV in an in vitro cell culture assay that was adapted for microtiter plates and that used detergent-treated virus for improved neutralization sensitivity and newborn cynomolgus monkey kidney cells for rapid growth. Eighteen hybridomas were positive for neutralization; 16 remained stable. Of the 16, 9 were able to compete with labeled polyclonal serum for binding to HAV. The nine competing hybridomas could be separated into two groups which appear to be directed towards two different sites on HAV and could complement each other in the competitive radioimmunoassay against polyclonal sera. Of the original 16 neutralizing hybridomas, 4 were subcloned through two cycles of limit dilutions. All four monoclonal antibodies retained their original neutralizing and competitive properties; three were immunoglobulin G2a, and one was immunoglobulin Gl. All four monoclonal antibodies readily precipitate whole '25I-labeled HAV but are not able to recognize the disrupted proteins of the virus (as tested by immune precipitations of heat-and detergent-disrupted virions or Western blot analyses). However, the heterobifunctional cross-linking reagent toluene-2,4-diisocyanate was used to cross-link purified Fab fragments of two different monoclonal antibodies (2D2 and 6A5) to HAV before disruption. This reagent demonstrated a specific reaction of the monoclonal antibodies to the VP-1 of HAV, suggesting this major surface protein contains at least one of the major neutralization sites for HAV.
Chlorine dioxide (ClO(2)) is an antimicrobial agent available for commercial produce washing. This study examined the efficacy of ClO(2) at 5 parts per million (ppm) during spray washing of tomatoes (5.0 ml/s per fruit) for preventing Salmonella enterica transfer from inoculated roller brushes to fruit and wash runoff. Furthermore, the sanitizing effects of ClO(2) during spray washing at low and high flow rates (5.0 and 9.3 ml/s per fruit, respectively) on tomato surfaces (nonstem scar areas) with either newly introduced (wet) or overnight air-dried Salmonella inocula were investigated. Salmonella transfer from contaminated brushes to fruit surfaces was reduced 2.1 +/- 0.6 or 4.7 +/- 0.2 log cycles after spray washing with water for 40 s or with the ClO(2) solution for 10 s, respectively. Cross-contamination of Salmonella from brushes to wash runoff during fruit washing for 60 s decreased 5.9 +/- 0.3 log cycles when ClO(2) was used. Fruit washing using contaminated brushes and low flow-rate washing with either water or ClO(2) solution for 10 s reduced newly introduced Salmonella on fruit surfaces by 1.7 +/- 0.6 or 5.1 +/- 0.3 log cycles, respectively. For fruit surfaces with air-dried inocula, washing with water and using uncontaminated brushes for 10 to 40 s reduced Salmonella by 3.2 +/- 0.3 to 3.4 +/- 0.4 log cycles; and the reduction was significantly improved by using ClO(2), high flow rate, or a longer washing time. Washing with ClO(2) at tested flow rates for 10 to 60 s resulted in a 4.4 +/- 0.6 to 5.2 +/- 0.1 log reduction of air-dried Salmonella on fruit surfaces.
The potential of Salmonella population to rebound on non-washed and washed roma tomatoes and jalapeño peppers in humid storage at 4°C, 10°C, 15°C, 21°C, or 35°C for ≤12 days was investigated. The initial inoculation levels of Salmonella on peppers and tomatoes were 5.6 and 5.2 log CFU/cm(2), respectively. Air-drying of fruit surfaces resulted in contamination levels of 3.9 and 3.7 log CFU/cm(2) on inoculated peppers and tomatoes, respectively. At 21°C and 35°C, the levels of air-dried Salmonella inoculums on produce surfaces increased ≥2 log cycles, with the most rapid growth in the first 3 days. Mechanical washing on rollers (rinsing; R-treatment) or revolving brushes (rinsing and brushing; RB-treatment) with water decreased Salmonella counts by ≥2.5 log CFU/cm(2) on both peppers and tomatoes. After R- or RB-treatment, peppers stored at 21°C and 35°C permitted residual Salmonella (≤1.4 log CFU/cm(2)) to grow to 2.6-3.9 log CFU/cm(2). During storage, residual Salmonella (≤1.0 log CFU/cm(2)) on washed tomatoes increased to 3.1 log CFU/cm(2) at 35°C following R-treatment and 3.8 log CFU/cm(2) at 21°C following RB-treatment. Cold storage at 4°C and 10°C effectively prevented the proliferation of Salmonella on both washed and non-washed produce. The current study on jalapeño peppers and roma tomatoes demonstrated that Salmonella population can rebound on produce in humid storage before or after washing. The finding highlights the benefit of uninterrupted cold storage for safer produce operations.
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