The 3 nontranslated region of the genomes of Sindbis virus (SIN) and other alphaviruses carries several repeat sequence elements (RSEs) as well as a 19-nucleotide (nt) conserved sequence element (3CSE). The 3CSE and the adjoining poly(A) tail of the SIN genome are thought to act as viral promoters for negative-sense RNA synthesis and genome replication. Eight different SIN isolates that carry altered 3CSEs were studied in detail to evaluate the role of the 3CSE in genome replication. The salient findings of this study as it applies to SIN infection of BHK cells are as follows: i) the classical 19-nt 3CSE of the SIN genome is not essential for genome replication, long-term stability, or packaging; ii) compensatory amino acid or nucleotide changes within the SIN genomes are not required to counteract base changes in the 3 terminal motifs of the SIN genome; iii) the 5 1-kb regions of all SIN genomes, regardless of the differences in 3 terminal motifs, do not undergo any base changes even after 18 passages; iv) although extensive addition of AU-rich motifs occurs in the SIN genomes carrying defective 3CSE, these are not essential for genome viability or function; and v) the newly added AU-rich motifs are composed predominantly of RSEs. These findings are consistent with the idea that the 3 terminal AU-rich motifs of the SIN genomes do not bind directly to the viral polymerase and that cellular proteins with broad AU-rich binding specificity may mediate this interaction. In addition to the classical 3CSE, other RNA motifs located elsewhere in the SIN genome must play a major role in template selection by the SIN RNA polymerase.Viruses carrying RNA genomes cause devastating human illnesses, such as AIDS, rabies, poliomyelitis, hepatitis, and hemorrhagic fevers. There is little doubt that these viruses evolve rapidly in response to environmental and genetic pressures (25, 57). For example, hundreds of different genotypes of recombinant human immunodeficiency virus (HIV) genomes contribute to the growing AIDS epidemic (7). Viruses such as influenza undergo continuous genetic changes and escape host immune mechanisms (63). The polymerases and cellular factors that act upon RNA genomes are central to the survival and evolution of RNA viruses. Despite our expanding knowledge of the biology of RNA viruses, understanding the anatomy and biochemistry of the enzymes and factors that copy and modify the RNA genomes continues to be challenging. RNA genomes in general are believed to carry cis-acting RNA motifs that regulate RNA synthesis and genome amplification. Many RNA genomes, including those of all retroviruses, carry these regulatory RNA motifs at their genome termini (12, 57). However, results obtained from some animal and plant RNA viral systems suggest the occurrence of internal cis-acting motifs (2,16,19,38,40,41,50). Since the cis-acting RNA motifs presumably interact with viral and cellular factors, the evolution of these RNA motifs is thought to be constrained. In fact, the sequence and/or structure of these RNA mot...

A six-strain mixture of E. coli O157:H7 was exposed to 0 to 9 min of six treatments: (i) hydrostatic pressure (400 MPa) at 4°C;(ii) hydrostatic pressure and thymol at 4°C;(iii) thymol at 4°C;(iv) heat at 40°C;(v) hydrostatic pressure at 40°C; and (vi) hydrostatic pressure and thymol at 40°C. Pressure intensity level of 400 MPa and thymol concentration of 0.15% (w/v) were used for the experiments of inoculated pathogen (4.0 to 5.0 log CFU/mL) in a homogenate (10% nonsterilized beef in 90% sterilized distilled water). Temperature was precisely monitored by stainless steel water jacket surrounding pressure chamber (16 mL volume), mechanically linked to a refrigerated circulating water bath. Analyses of variance were conducted followed by Tukey-and Dunnett's-adjusted mean separations. Pathogen counts before treatment were 4.08 ± 0.7 log CFU/mL and were reduced (P < 0.05) to 0.67 ± 0.2 log CFU/mL after 6 min of pressure treatment. Thymol and mild heat (40°C) further augmented decontamination efficacy of pressure treatments where in their presence, the mesophilic background microbiota counts of pressure-treated samples after 3, 6, and 9 min were reduced (P < 0.05) by 2.1, 2.5, and 3.1 log CFU/mL, respectively. Results of the current study indicate that thymol and mild heat could enhance decontamination efficacy of elevated hydrostatic pressure for pasteurization of food commodities. This could be of great significance for industry practitioners to assure microbiological safety of a product and cost optimization by benefiting from synergism of antimicrobials, mild heat, and elevated hydrostatic pressure.

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Current study investigated effects of elevated hydrostatic pressure exposure in the presence of mild heat and natural antimicrobials against Staphylococcus aureus. Hydrostatic pressure of 350 to 550 MPa with nisin (5000 IU/mL), carvacrol, or caprylic acid (0.5% v/v) were applied for the reduction in four-strain mixture of S. aureus in HEPES buffer at 4 and 40 °C for up to 7 min. Results were statistically analyzed by ANOVA and D-values were additionally calculated using best-fitted linear model. Prior to exposure to treatments at 4 °C, counts of the pathogen were 7.95 ± 0.4 log CFU/mL and were reduced (p < 0.05) to 6.44 ± 0.3 log CFU/mL after 7 min of treatment at 450 MPa. D-value associated with this treatment was 5.34 min (R2 = 0.72). At 40 °C, counts were 8.21 ± 0.7 and 5.77 ± 0.3 log CFU/mL before and after the 7-min treatments, respectively. D-value associated with 40 °C treatment was 3.30 min (R2 = 0.62). Application of the antimicrobials provided additional pathogen reduction augmentation for treatments < 5 min. The results of the current study could be incorporated for meeting regulatory requirements such as Food Code, HACCP, and Preventive Control for Human Food of Food Safety Modernization Act for assuring microbiological safety of products against this prevalent pathogen of public health concern.

The inactivation of bacterial endospores continues to be the main curtailment for further adoption of high-pressure processing in intrastate, interstate, and global food commerce. The current study investigated the effects of elevated hydrostatic pressure for the inactivation of endospore suspension of three indicator spore-forming bacteria of concern to the food industry. Additionally, the effects of four bacteriocin/bactericidal compounds were studied for augmenting the decontamination efficacy of the treatment. Elevated hydrostatic pressure at 650 MPa and at 50 °C was applied for 0 min (untreated control) and for 3, 7, and 11 min with and without 50K IU of nisin, 224 mg/L lysozyme, 1% lactic acid, and 1% CitricidalTM. The results were statistically analyzed using Tukey- and Dunnett’s-adjusted ANOVA. Under the condition of our experiments, we observed that a well-designed pressure treatment synergized with mild heat and bacteriocin/bactericidal compounds could reduce up to >4 logs CFU/mL (i.e., >99.99%) of bacterial endospores. Additions of nisin and lysozyme were able, to a great extent, to augment (p < 0.05) the decontamination efficacy of pressure-based treatments against Bacillus amyloliquefaciens and Bacillus atrophaeus, while exhibiting no added benefit (p ≥ 0.05) for reducing endospores of Geobacillus stearothermophilus. The addition of lactic acid, however, was efficacious for augmenting the pressure-based reduction of bacterial endospores of the three microorganisms.