David Yang

Zhongshan Entry-Exit Inspection and Quarantine Bureau, 中山, Guangdong, China

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Publications (5)14.58 Total impact

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    ABSTRACT: Human noroviruses (HuNoVs) are the major cause of epidemic non-bacterial gastroenteritis. Although quantitative real-time RT-PCR (qRT-PCR) is widely-used for detecting HuNoVs, it only detects the presence of viral RNA and does not indicate viral infectivity. Human blood group antigens (HBGAs) have been identified as receptors/co-receptors for both HuNoVs and Tulane Virus (TV), and are crucial for viral infection. We propose that viral infectivity can be evaluated with a molecular assay based on receptor-captured viruses. In this study, we employed TV as a HuNoV surrogate to validate the HBGA-based capture-qRT-PCR method against the TCID50 method. We employed type-B HBGA on an immuno-well module to concentrate TV, followed by amplification of the captured viral genome by in situ qRT-PCR. We first demonstrate that this In Situ Capture qRT-PCR (ISC-qRT-PCR) method could effectively concentrate and detect TV. We then treated TV under either partial or full-inactivation conditions, and measured the remaining infectivity by ISC-qRT-PCR and tissue-culture-based amplification method (TCID50). We found that the ISC-qRT-PCR method could be used to evaluate virus inactivation deriving from damage to the capsid, and study interactions between the capsid and viral receptor. Heat, chlorine, and ethanol treatment primarily affect the capsid structure, which in turns affects the ability of the capsid to bind to viral receptors. Inactivation of the virus by these methods could be reflected by ISC-qRT-PCR method and confirmed by TCID50 assay. However, the loss of the infectivity caused by damage to the viral genome (such as from UV irradiation) could not be effectively reflected by this method. Despite this limitation, the ISC-qRT-PCR provides an alternative approach to determine inactivation of Tulane virus. A particular advantage of the ISC-qRT-PCR method is that it is also a faster and easier method to effectively recover and detect the viruses, as there is no need to extract viral RNA, nor need to transfer the captured virus from magnetic beads to PCR tubes for further amplification. Therefore, ISC-qRT-PCR can be easily adapted for use in automated systems for multiple samples.
    Applied and Environmental Microbiology 01/2014; · 3.95 Impact Factor
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    ABSTRACT: Human noroviruses (HuNoVs) are the major cause of nonbacterial gastroenteritis epidemics. The culturable feline calicivirus and murine norovirus have been used extensively as surrogates to study HuNoV biology, as HuNoV does not grow in vitro. Additional efforts to identify new surrogates are needed, because neither of these common surrogates are truly intestinal pathogens. The newly described Tulane virus (TV) is a typical calicivirus, it is isolated from macaque stools, is cultivable in vitro, and recognizes human histo-blood group antigens. Therefore, TV is a promising surrogate for HuNoVs. In this study, we evaluated the resistance or stability of TV under various physical and environmental conditions by measuring a 50% reduction of tissue culture infective dose (TCID50) by using a TV cell culture system. Due to the nature of this virus, it is hard to produce a high-titer stock through tissue culture. In our study, the maximal reduction in virus titers was 5D (D = 1 log) in heat-denaturation and EtOH experiments, and 4D in UV, chlorine, and pH-stability experiments. Therefore in this study, we defined the inactivation of TV as reaching a TCID50/ml of 0 (a 4- to 5-D reduction in TCID50, depending on the detection limit). TV was inactivated after incubation at 63°C for 5 min, incubation at 56°C for 30 min (5D), exposure to 60 mJ/cm(2) of UVC radiation (4D), or incubation at 300 ppm of free chlorine for 10 min (4D). TV was shown to be stable from pH 3.0 to 8.0, though an obvious reduction in virus titer was observed at pH 2.5 and 9.0, and was inactivated at pH 10.0 (4D). TV was resistant to a low concentration of EtOH (40% or lower) but was fully inactivated (5D) by 50 to 70% EtOH after a short exposure (20 s). In contrast, quantitative real-time PCR was unable to detect, or poorly detected, virus titer reductions between treated and untreated samples described in this study.
    Journal of food protection 04/2013; 76(4):712-8. · 1.83 Impact Factor
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    ABSTRACT: Water is an important route for human norovirus (HuNoV) transmission. Using magnetic beads conjugated with blood group-like antigens (HuNoV receptors), we developed a simple and rapid receptor-binding capture and magnetic sequestration (RBCMS) method and compared it to the existing negatively charged membrane absorption/elution (NCMAE) method for concentrating HuNoV from sewage effluent. RBCMS required 6-fold-less sample volume than the NCMAE method and also resulted in a significantly higher yield of HuNoV. The NCMAE and RBCMS concentrations of genogroup I (GI) HuNoV measured by quantitative reverse transcription-PCR (qRT-PCR) resulted in average threshold cycle (C(T)) values of 34.68 (8.68 copies, 252-fold concentration) versus 34.07 (13.05 copies, 477-fold concentration), respectively; the NCMAE and RBCMS concentrations of genogroup II (GII) HuNoV were measured as average C(T) values of 33.32 (24.7 copies, 239-fold concentration) versus 32.38 (46.9 copies, 333-fold concentration), respectively. The specificity of qRT-PCR was confirmed by traditional RT-PCR and an RNase I protection assay. The qRT-PCR signal from RBCMS-concentrated HuNoV treated with RNase I indicated that it was from encapsidated RNA and, probably, viable virus. In contrast, the qRT-PCR signal from NCMAE-concentrated HuNoV was not protected from RNase I and, likely, degradation. Both GI and GII HuNoV were detected from sewage effluent samples collected between April and July with average concentrations of 7.8 × 10(3) genomic copies per liter (gc/liter) and 4.3 × 10(4) gc/liter, respectively. No GI and <2% GII HuNoV were detected in sewage samples stored at room temperature for 4 weeks. We conclude that RBCMS requires less sample volume, has better recovery and sensitivity, and is faster than NCMAE for detection of HuNoV in sewage.
    Applied and Environmental Microbiology 11/2011; 78(2):429-36. · 3.95 Impact Factor
  • Peng Tian, David Yang, Robert Mandrell
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    ABSTRACT: Food contamination by human norovirus (hNoV) is a major cause of gastrointestinal disease. We evaluated the effectiveness of removing inoculated hNoV from the surfaces of raspberries and romaine lettuce by a simple wash in tap water and in different forms of electrolyzed water (EW), including acidic EW (AEW), neutral EW (NEW), and basic EW (BEW). A simple rinsing or soaking in water was able to remove >95% of hNoV from surface-inoculated raspberries. In contrast, only 75% of hNoV was removed from surface-inoculated romaine lettuce by rinsing in tap water. An AEW wash enhanced the binding of hNoV to raspberries and lettuce. Only 7.5% (±10%) and 4% (±3.1%) of hNoV were removed by AEW wash from surface-inoculated raspberries and lettuce, respectively. When raspberries and lettuce were prewashed with NEW or BEW prior to surface inoculation, an AEW wash likewise resulted in significantly less removal of hNoV compared with untreated samples. A prewash with AEW significantly decreased the removal of hNoV from raspberries and lettuce when they were washed with NEW, from 90.6 to 51% and from 76 to 51.3% , respectively. There are minimal or no improvements gained by use of any of the EWs instead of a regular tap water wash in removal of hNoV from produce. However, use of AEW shows a significant decrease in the removal of hNoV from contaminated produce compared with other water rinses. The ability to remove hNoV from different types of produce varies, possibly due to differences among types of ligand-like molecules that bind hNoV. The distribution of hNoV on raspberries and lettuce was studied using recombinant Norwalk-like particles (rNVLP). By immunofluorescence microscopy, we were able to observe binding of rNVLP only to vein areas of romaine lettuce, suggesting that the virus was binding to specific molecules in these areas. Random binding of rNVLP occurred only with raspberries prewashed with AEW or washed with AEW.
    Journal of food protection 08/2011; 74(8):1364-9. · 1.83 Impact Factor
  • Peng Tian, David Yang, Robert Mandrell
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    ABSTRACT: Human norovirus (NoV) outbreaks are major food safety concerns. The virus has to be concentrated from food samples in order to be detected. PEG precipitation is the most common method to recover the virus. Recently, histo-blood group antigens (HBGA) have been recognized as receptors for human NoV, and have been utilized as an alternative method to concentrate human NoV for samples up to 40 mL in volume. However, to wash off the virus from contaminated fresh food samples, at least 250 mL of wash volume is required. Recirculating affinity magnetic separation system (RCAMS) has been tried by others to concentrate human NoV from large-volume samples and failed to yield consistent results with the standard procedure of 30 min of recirculation at the default flow rate. Our work here demonstrates that proper recirculation time and flow rate are key factors for success in using the RCAMS. The bead recovery rate was increased from 28% to 47%, 67% and 90% when recirculation times were extended from 30 min to 60 min, 120 min and 180 min, respectively. The kinetics study suggests that at least 120 min recirculation is required to obtain a good recovery of NoV. In addition, different binding and elution conditions were compared for releasing NoV from inoculated lettuce. Phosphate-buffered saline (PBS) and water results in similar efficacy for virus release, but the released virus does not bind to RCAMS effectively unless pH was adjusted to acidic. Either citrate-buffered saline (CBS) wash, or water wash followed by CBS adjustment, resulted in an enhanced recovery of virus. We also demonstrated that the standard curve generated from viral RNA extracted from serially-diluted virus samples is more accurate for quantitative analysis than standard curves generated from serially-diluted plasmid DNA or transcribed-RNA templates, both of which tend to overestimate the concentration power. The efficacy of recovery of NoV from produce using RCAMS was directly compared with that of the PEG method in NoV inoculated lettuce. 40, 4, 0.4, and 0.04 RTU can be detected by both methods. At 0.004 RTU, NoV was detectable in all three samples concentrated by the RCAMS method, while none could be detected by the PEG precipitation method. RCAMS is a simple and rapid method that is more sensitive than conventional methods for recovery of NoV from food samples with a large sample size. In addition, the RTU value detected through RCAMS-processed samples is more biologically relevant.
    International journal of food microbiology 06/2011; 147(3):223-7. · 3.01 Impact Factor

Publication Stats

23 Citations
14.58 Total Impact Points

Institutions

  • 2011
    • Zhongshan Entry-Exit Inspection and Quarantine Bureau
      中山, Guangdong, China
    • United States Department of Agriculture
      • Agricultural Research Service (ARS)
      Washington, D. C., DC, United States