Biomedical subjects

David H Kingsley

Publications and source records attributed to David H Kingsley.

12 recordsLinked to original sources

PubMed2006

An RNA extraction protocol for shellfish-borne viruses.

The GPTT virus RNA extraction method, originally developed for extraction of human norovirus and hepatitis A virus RNAs from contaminated shellfish, was evaluated for extraction of RNA from Aichi virus strain A846/88 (AiV), coxsackievirus strains A9 (CAV9) and B5 (CBV5), murine norovirus (strain MNV-1), and the norovirus surrogate, feline calicivirus (FCV) strain KCD, for the purpose of RT-PCR detection within seeded oyster (Crassostrea virginica) extracts. The RT-PCR equivalent sensitivities observed within seeded oysters as compared to virus stocks were 0.68, 6.8, 26, 5.6, and 14.5 RT-PCR(50) units when assaying 10% of total RNA extracted from seeded oyster extracts for CAV9, CBV5, AiV, FCV, and MNV-1, respectively. For oysters exposed to virus-contaminated seawater, the detection equivalent sensitivities observed were 680, 68, 2600, 560, and 14.5 RT-PCR(50) for CAV9, CBV5, AiV and FCV, and MNV-1, respectively. These results indicate that the GPTT method can be used as a general viral RNA extraction method for multiple picornaviruses and caliciviruses that could potentially contaminate shellfish.

Animals↗

PubMed2006

Inactivation of a norovirus by high-pressure processing.

Murine norovirus (strain MNV-1), a propagable norovirus, was evaluated for susceptibility to high-pressure processing. Experiments with virus stocks in Dulbecco's modified Eagle medium demonstrated that at room temperature (20 degrees C) the virus was inactivated over a pressure range of 350 to 450 MPa, with a 5-min, 450-MPa treatment being sufficient to inactivate 6.85 log(10) PFU of MNV-1. The inactivation of MNV-1 was enhanced when pressure was applied at an initial temperature of 5 degrees C; a 5-min pressure treatment of 350 MPa at 30 degrees C inactivated 1.15 log(10) PFU of virus, while the same treatment at 5 degrees C resulted in a reduction of 5.56 log(10) PFU. Evaluation of virus inactivation as a function of treatment times ranging from 0 to 150 s and 0 to 900 s at 5 degrees C and 20 degrees C, respectively, indicated that a decreasing rate of inactivation with time was consistent with Weibull or log-logistic inactivation kinetics. The inactivation of MNV-1 directly within oyster tissues was demonstrated; a 5-min, 400-MPa treatment at 5 degrees C was sufficient to inactivate 4.05 log(10) PFU. This work is the first demonstration that norovirus can be inactivated by high pressure and suggests good prospects for inactivation of nonpropagable human norovirus strains in foods.

Animals↗

PubMed2006

Inactivation of hepatitis A virus by high-pressure processing: the role of temperature and pressure oscillation.

Inactivation of hepatitis A virus (HAV) in Dulbecco's modified Eagle medium with 10% fetal bovine serum was studied at pressures of 300, 350, and 400 MPa and initial sample temperatures of -10, 0, 5, 10, 20, 30, 40, and 50 degrees C. Sample temperature during pressure application strongly influenced the efficiency of HAV inactivation. Elevated temperature (> 30 degrees C) enhanced pressure inactivation of HAV, while lower temperatures resulted in less inactivation. For example, 1-min treatments of 400 MPa at -10, 20, and 50 degrees C reduced titers of HAV by 1.0, 2.5, and 4.7 log PFU/ml, respectively. Pressure inactivation curves of HAV were obtained at 400 MPa and three temperatures (-10, 20, and 50 degrees C). With increasing treatment time, all three temperatures showed a rapid initial drop in virus titer with a diminishing inactivation rate (or tailing effect). Analysis of inactivation data indicated that the Weibull model more adequately fitted the inactivation curves than the linear model. Oscillatory high-pressure processing for 2, 4, 6, and 8 cycles at 400 MPa and temperatures of 20 and 50 degrees C did not considerably enhance pressure inactivation of HAV as compared with continuous high-pressure application. These results indicate that HAV exhibits, unlike other viruses examined to date, a reduced sensitivity to high pressure observed at cooler treatment temperatures. This work suggested that slightly elevated temperatures are advantageous for pressure inactivation of HAV within foods.

Animals↗

PubMed2005

High-pressure inactivation of hepatitis A virus within oysters.

Previous results demonstrated that hepatitis A virus (HAV) could be inactivated by high hydrostatic pressure (HHP) (D. H. Kingsley, D. Hoover, E. Papafragkou, and G. P. Richards, J. Food Prot. 65:1605-1609, 2002); however, direct evaluation of HAV inactivation within contaminated oysters was not performed. In this study, we report confirmation that HAV within contaminated shellfish is inactivated by HHP. Shellfish were initially contaminated with HAV by using a flowthrough system. PFU reductions of >1, >2, and >3 log(10) were observed for 1-min treatments at 350, 375, and 400 megapascals, respectively, within a temperature range of 8.7 to 10.3 degrees C. Bioconcentration of nearly 6 log(10) PFU of HAV per oyster was achieved under simulated natural conditions. These results suggest that HHP treatment of raw shellfish will be a viable strategy for the reduction of infectious HAV.

Animals↗

PubMed2005

Temperature and treatment time influence high hydrostatic pressure inactivation of feline calicivirus, a norovirus surrogate.

Interest in high hydrostatic pressure processing as a nonthermal pasteurization process for foods continues to increase. Feline calicivirus (FCV), a propagable virus that is genetically related to the nonpropagable human noroviruses, was used for detailed evaluation of the high pressure processing parameters necessary for virus inactivation. Pressure inactivation curves of FCV strain KCD in Dulbecco's modified Eagle medium with 10% fetal bovine serum were obtained at 200 and 250 MPa as a function of time at room temperature. Pressure inactivation curves at 200 and 250 MPa also were determined as a function of temperature ranging from --10 to 50 degrees C at treatment times of 4 and 2 min, respectively. Tailing was observed for inactivation as a function of treatment time, indicating that the linear model was not adequate for describing these curves. The two nonlinear models, the log logistic and Weibull functions, consistently produced better fit to inactivation curves than did the linear model. The mean square errors were 0.381 for the log logistic model, 0.425 for the Weibull model, and 1.546 for the linear model. For inactivation as a function of temperature, FCV was most resistant to pressure at 20 degrees C. Temperatures above and below 20 degrees C significantly increased pressure inactivation of FCV. A 4-min treatment of 200 MPa at --10 and 50 degrees C reduced the titer of FCV by 5.0 and 4.0 log units, respectively; whereas at 20 degrees C the same treatment only reduced the titer by 0.3 log units. These novel results point to the potential for using temperatures above and particularly below room temperature to lower the pressure needed to cause the desired level of virus inactivation.

Animals↗

PubMed2004

Inactivation of selected picornaviruses by high hydrostatic pressure.

The potential of high hydrostatic pressure processing (HPP) to inactivate Aichi virus (AiV), human parechovirus-1 (HPeV-1), and the coxsackievirus strains A9 and B5 was investigated. For coxsackievirus A9 (CAV9), a 5-min HPP treatment in minimum essential growth medium (MEM) supplemented with 10% fetal bovine sera (FBS) resulted in 3.4-, 6.5-, and 7.6-log(10) tissue culture infectious dose (50%) (TCID(50)) reductions at 400, 500, and 600megaPascals (MPa), respectively. For HPeV-1, a 5-min treatment in MEM with 10% FBS resulted in reductions of 1.3-, 4.3-, and 4.6-log(10) TCID(50) at 400, 500, and 600MPa, respectively; however, AiV and coxsackievirus B5 (CBV5) in MEM supplemented with 2 and 10% FBS, respectively, remained fully infectious after a 5-min treatment at 600MPa. These data establish that different picornaviruses have widely variable pressure inactivation thresholds in response to HPP.

Culture Media↗

PubMed2004

Membrane fusion of secretory vesicles of the sea urchin egg in the absence of NSF.

The role of cytosolic ATPases such as N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) in membrane fusion is controversial. We examined the physiology and biochemistry of ATP and NSF in the cortical system of the echinoderm egg to determine if NSF is an essential factor in membrane fusion during Ca(2+)-triggered exocytosis. Neither exocytosis in vitro, nor homotypic cortical vesicle (CV) fusion required soluble proteins or nucleotides, and both occurred in the presence of non-hydrolyzable analogs of ATP. While sensitive to thiol-specific reagents, CV exocytosis is not restored by the addition of cytosolic NSF, and fusion and NSF function are differentially sensitive to thiol-specific agents. To test participation of tightly bound, non-exchangeable NSF in CV-CV fusion, we cloned the sea urchin homolog and developed a species-specific antibody for western blots and physiological analysis. This antibody was without effect on CV exocytosis or homotypic fusion, despite being functionally inhibitory. NSF is detectable in intact cortices, cortices from which CVs had been removed and isolated CVs treated with ATP-gamma-S and egg cytosol to reveal NSF binding sites. In contrast, isolated CVs, though all capable of Ca(2+)-triggered homotypic fusion, contain less than one hexamer of NSF per CV. Thus NSF is not a required component of the CV fusion machinery.

Adenosine Triphosphate↗

PubMed2004

A SYBR green, real-time RT-PCR method to detect and quantitate Norwalk virus in stools.

A simple, single tube, hot start, real-time reverse transcription-PCR (rt RT-PCR) technique using SYBR green fluorescence was developed for the detection of genogroup I, cluster 1 Norwalk virus (NV) in stools. Sample dilution and heat release of viral RNA was effective as an alternative to more complex procedures to extract viruses from stool specimens. Real-time RT-PCR was applied to 68 stool isolates from patients participating in a NV volunteer study. First derivative melt curves were used to verify NV amplicon and to rule out the presence of primer dimer or spurious product. A dilution end-point standard curve was developed to semi-quantitate minimum virus levels and the results showed the number of RT-PCR amplifiable NV as high as 6.16 x 10(10)g(-1) of stool. The application of these methods was instrumental in identifying three asymptomatic patients who shed viruses in their stools, thus demonstrating a carrier state among seemingly healthy individuals. This study serves as a model for the development of rapid and specific detection, verification, and quantitation procedures for other Noroviruses in stools.

Benzothiazoles↗

PubMed2004

Pressure inactivation kinetics of phage lambda cI 857.

Inactivation curves of phage lambda cI 857 inactivated by high hydrostatic pressure were obtained at three pressure levels (300, 350, and 400 MPa) in buffered media and ultrahigh-temperature 2% reduced fat milk. Pressurization of phage lambda in buffered media at 300 MPa for 300 min, 350 MPa for 36 min, and 400 MPa for 8 min reduced the titer of phage lambda by 7.5, 6.7, and 7.7 log, respectively. Pressurization of phage lambda in milk at 300 MPa for 400 min, 350 MPa for 80 min, and 400 MPa for 20 min reduced the titer of phage lambda by 5.4, 6.4, and 7.1 log, respectively. Tailing was observed in all inactivation curves, indicating that the linear model was not adequate for describing these curves. Among the three nonlinear models studied, the Weibull and log-logistic models consistently produced best fits to all inactivation curves, and the modified Gompertz model the poorest. Because there were no significant differences in the values of shape factor (n) for suspension medium buffer, we reduced the number of parameters in the Weibull model from two to one by setting n at the mean value. The simplified Weibull model produced a fit comparable to the full model. Additionally, the simplified Weibull model allowed predictions to be made at pressures different from the experimental pressures. Menstruum was found to significantly affect the pressure resistance of phage lambda. Comparison of pressure inactivation of hepatitis A virus and phage lambda indicated that phage lambda is more sensitive to pressure than hepatitis A virus in Dulbecco's modified Eagle medium with 10% fetal bovine sera.

Animals↗

PubMed2003

Persistence of hepatitis A virus in oysters.

We investigated the ability of hepatitis A virus (HAV) to persist for up to 6 weeks in Eastern oysters (Crassostrea virginica). Viral RNA was detected by reverse transcription-polymerase chain reaction 6 weeks after 16 h of exposure to 90,000 PFU (180 PFU/ml of seawater) of HAV. Assaying for infectious virus in oysters that received a daily feeding of phytoplankton recovered 3,800, 650, and 500 PFU of HAV 1, 2, and 3 weeks after contamination with 90,000 PFU of HAV, respectively. However, no infectious HAV was isolated from oysters 4, 5, or 6 weeks after contamination. These results support the position that shellfish depuration is insufficient for the complete removal of infectious viruses. Extended relay times (in excess of 4 weeks) may be required to produce virologically safe shellfish.

Animals↗

PubMed2002

Detection of both hepatitis A virus and Norwalk-like virus in imported clams associated with food-borne illness.

Hepatitis A virus (HAV) and Norwalk-like virus (NLV) were detected by reverse transcription-PCR in clams imported into the United States from China. An epidemiological investigation showed that these clams were associated with five cases of Norwalk-like gastroenteritis in New York State in August 2000 (Food and Drug Administration Import Alert 16-50). They were labeled "cooked" but appeared raw. Viral RNA extraction was performed by using dissected digestive tissues rather than whole shellfish meats; this was followed by glycine buffer elution, polyethylene glycol precipitation, Tri-Reagent treatment, and purification of poly(A) RNA with magnetic beads coupled to poly(dT) oligonucleotides. We identified HAV and NLV as genotype I and genogroup II strains, respectively. Both viruses have high levels of homology to Asian strains. An analysis of fecal coliforms revealed a most-probable number of 93,000/100 g of clam meat, which is approximately 300-fold higher than the hygienic standard for shellfish meats.

Amino Acid Sequence↗

PubMed2002

Inactivation of hepatitis A virus and a calicivirus by high hydrostatic pressure.

Potential application of high hydrostatic pressure processing (HPP) as a method for virus inactivation was evaluated. A 7-log10 PFU/ml hepatitis A virus (HAV) stock, in tissue culture medium, was reduced to nondetectable levels after exposure to more than 450 MPa of pressure for 5 min. Titers of HAV were reduced in a time- and pressure-dependent manner between 300 and 450 MPa. In contrast, poliovirus titer was unaffected by a 5-min treatment at 600 MPa. Dilution of HAV in seawater increased the pressure resistance of HAV, suggesting a protective effect of salts on virus inactivation. RNase protection experiments indicated that viral capsids may remain intact during pressure treatment, suggesting that inactivation was due to subtle alterations of viral capsid proteins. A 7-log10 tissue culture infectious dose for 50% of the cultures per ml of feline calicivirus, a Norwalk virus surrogate, was completely inactivated after 5-min treatments with 275 MPa or more. These data show that HAV and a Norwalk virus surrogate can be inactivated by HPP and suggest that HPP may be capable of rendering potentially contaminated raw shellfish free of infectious viruses.

Caliciviridae↗