紫外线和次氯酸钠对Escherichia coli和Poliovirus的消毒作用

本研究选用大肠杆菌(Escherichia coli)和脊髓灰质炎病毒(poliovirus)分别作为典型的细菌和病毒,利用培养和定量PCR的检测技术,对比研究紫外线消毒和次氯酸钠消毒对细菌和病毒的作用特点.结果表明:脊髓灰质炎病毒比大肠杆菌更难被灭活,达到1-log所需的氯剂量分别为19.2 mg·L~(-1)·min和10.14 mg·L~(-1)·min;所需的紫外线剂量分别为6.37 m J·cm~(-2)和1.81 m J·cm~(-2).定量PCR方法检测大肠杆菌和脊髓灰质炎病毒达到1-log的核酸损伤所需的紫外线剂量和氯剂量要比培养法高出1~2数量级,紫外线消毒对脊髓灰质炎病毒的RNA损伤量明显大于对大肠杆菌的DNA损伤,病毒的单链RNA对紫外线的敏感性更强,该结果与培养法正好相反.达到1-log核酸损伤脊髓灰质炎病毒所需的紫外线剂量为135 m J·cm~(-2),大肠杆菌所需的剂量为270.3 m J·cm~(-2),核酸损伤需要更多的消毒剂量,可能由于消毒过程微生物进入活性但处于非可培养状态(VBNC),以及灭活对微生物其他分子的损伤和微生物死后核酸的持续性.     

Survival of Coronaviruses in Water and Wastewater

The advent of severe acute respiratory syndrome and its potential environmental transmission indicates the need for more information on the survival of coronavirus in water and wastewater. The survival of representative coronaviruses, feline infectious peritonitis virus, and human coronavirus 229E was determined in filtered and unfiltered tap water (4 and 23°C) and wastewater (23°C). This was compared to poliovirus 1 under the same test conditions. Inactivation of coronaviruses in the test water was highly dependent on temperature, level of organic matter, and presence of antagonistic bacteria. The time required for the virus titer to decrease 99.9% (T_99.9) shows that in tap water, coronaviruses are inactivated faster in water at 23°C (10 days) than in water at 4°C (>100 days). Coronaviruses die off rapidly in wastewater, with T_99.9 values of between 2 and 4 days. Poliovirus survived longer than coronaviruses in all test waters, except the 4°C tap water.     

Inactivation of SARS-CoV-2 by Ozonated Glycerol

We evaluated the SARS-CoV-2-inactivation activity of ozonated glycerol (OG). When a viral solution with 1% fetal bovine serum (FBS) was mixed with test solutions at a ratio of 1:19 and incubated for 20 s, OG with ozone concentrations of over 1000 ppm inactivated ≥ 94.38% of the virus. Extension of the reaction time to 1 h led to the inactivation of ≥ 99.82% of the virus (the viral titer was below the detection limit). Extension to 24 h resulted in concentrations over 200 ppm OG inactivating ≥ 99.87% of the virus (the viral titers were below the detection limit). Next, viral solutions with 1, 20, and 40% FBS were mixed with test solutions at a ratio of 1:19 and incubated for 5 min. Whereas the virucidal activity of 500 ppm OG was very limited in the presence of 1% FBS (79.47% inactivation), it increased in the presence of 20 and 40% FBS (95.13 and 97.95% inactivation, respectively; the viral titers were not below the detection limit). Meanwhile, over 1000 ppm OG inactivated ≥ 99.44% of the virus regardless of the FBS concentration (the viral titers were below the detection limit). Extension of the reaction time to 1 h led to 500 ppm OG inactivating ≥ 99.91 and ≥ 99.95% of the virus with 20 and 40% FBS, respectively (the viral titers were below the detection limit). These results suggested that OG might be useful as a virucidal agent against SARS-CoV-2.

     

纳米TiO2-光催化灭活水中噬菌体MS2

提出了一种简易的水中病毒灭活方法,以噬菌体MS2为模式病毒,探讨了紫外光照射时间、TiO2浓度、紫外光强度对自来水中MS2灭活效率的影响;评价了太阳光代替紫外光用于光催化灭活病毒的可行性.研究结果表明,聚乙烯(PE)材质的样品袋能更好地透过紫外光;紫外光照射6h,50mg/L的TiO2可以灭活自来水中7.95Log的MS2;紫外光强度在45μW/m2以下时,灭活率随强度的提高而增加;在溪水中,50mg/L的TiO2仅有4.87Log的灭活效率. 在太阳光下,50mg/L的TiO2在自来水和溪水中对MS2的灭活效率差异不明显,都达到了6.1Log以上,表明太阳光-TiO2灭活水中的病毒可行且高效.     

Virus Genome Quantification Does not Predict Norovirus Infectivity After Application of Food Inactivation Processing Technologies

When determining the effect of food processing on the infectivity of any contaminating virus, it is necessary to distinguish unambiguously between infectious and non-infectious viruses present. However, this can be difficult in the particular case of noroviruses (NoVs) because no reliable cell culture model is available. The aim of this study was to assess the use of molecular methods—RT real-time PCR (RT-qPCR) and enzymatic treatment (ET) coupled to RT-qPCR—to quantify the infectivity of NoV after application of various inactivating food-processing technologies. RT-qPCR and ET-RT-qPCR gave significantly different (P < 0.01) results concerning the reduction in viral genome counts by all inactivation procedures and conditions used, except for HHP treatment at 600 MPa for 5 min. These findings indicate that the ET prior to RT-qPCR has an effect on the estimation of the reduction of virus genome counts, and may eliminate genomes of affected virus particles. However, no correlation was found between the results obtained by ET-RT-qPCR and those obtained by cell culture. Therefore, the effect is presumably only partial, and not adequate to allow accurate estimation of virus inactivation. Consequently, our results indicate that the quantification of virus genomes by PCR, regardless of prior ET, is not adequate for establishing virus inactivation and/or infectivity. In addition, our results also illustrate that the general effect of virus inactivation is not directly correlated to effects on the integrity of virus genome and protein capsid. Presumably, inactivation by food processing is the consequence of effects on proteins involved in adhesion and invasion stages.     

Thermal Inactivation of Human Norovirus Surrogates

We investigated the thermal inactivation profiles of murine norovirus (MNV), Hepatitis A virus (HAV), and feline calicivirus (FCV), which are surrogates for the study of human noroviruses. Thermal inactivation of MNV and FCV were evaluated at 37, 50, and 60°C and HAV at 37, 50, 60, and 70°C. All viral surrogates were relatively stable at 37°C. MNV and FCV decimal reduction times (D-values) at 50°C were statistically significantly different (P < 0.05) with MNV being more stable. Both surrogates had comparable, low D-values at 60°C. HAV had significantly higher (P < 0.05) D-values than both MNV and FCV at 50 and 60°C. Overall, the infectivity assay results indicate that HAV is resistant to thermal inactivation while MNV is moderately resistant and FCV is least resistant.     

Heat Inactivation of Hepatitis A Virus in Shellfish Using Steam

Shellfish are an important cause of foodborne viral illness. Consumer-friendly cooking recommendations for shellfish could improve food safety and decrease the risk for infection from contaminated products. Thermal inactivation parameters were established for hepatitis A virus (HAV) in mussels and validated with cooking experiments. Steaming for only 2–5 min was not sufficient to inactivate HAV in mussels in all layers of a steamer. Steaming mussels for 6 min was sufficient to inactivate HAV in all layers. These cooking guidelines produce shellfish with a reduced risk for foodborne virus transmission.     

Drinking water biotic safety of particles and bacteria attached to fines in activated carbon process

In this paper, the drinking water biotic safety of particles and bacteria attached to fines in activated carbon process was investigated by actual treatment process and advanced treatment pilot trial with granular activated carbon. In the experiment, the particles were detected by IBR particle calculating instrument, the activated carbon fines were counted on the basis of the most probable number (MPN) with a microscope, the total number of bacteria was analyzed between the conventional agar culture medium and the one with R2A, and the bacteria attached to activated carbon fines was resolved by the homogenization technique. The experimental results showed that the average total number of particles was 205 CNT/mL in the activated carbon effluent during a filter cycle, of which the number of particles with sizes > 2 μm was 77 CNT/mL more than the present particle control criterion of the American drinking water product standard (50 CNT/mL). The backwash of low density and long duration lowered particle number in the effluent. The MPN of activated carbon fines in the effluent was between 400 and 600 CNT/L, which accounted for less than 5‰ of the total particles from activated carbon filtration for a poor relative level (R ² = 0.34). The microorganisms in activated carbon effluent consisted mostly of heterotrophic bacillus and the total bacteria number was five times as high as that of the inflow, i.e. the effluent from sand filter. The actual bacteria number may be truly indicated by the detection technique with R2A culture medium compared with the traditional agar cultivation. The inactivation efficiency of bacteria attached to activated carbon fines was less than 40% under 1.1 mg/L of chlorine contacting for 40 min. Results showed that the particles and bacteria attached to activated carbon fines may influence drinking water biotic safety, and that the effective control measures need to be further investigated.     

免疫磁珠分离与实时定量PCR技术联合检测水中轮状病毒的研究

建立了一种免疫磁珠分离技术联合实时定量PCR快速定量检测水中轮状病毒的方法.通过制备能够分离水中轮状病毒的特异免疫磁珠,优化分离条件,建立了免疫磁珠分离前处理方法,并与逆转录、实时定量PCR结合,成功用于水中轮状病毒的检测.研究发现,在1 mL水样中加入10 μL轮状病毒免疫磁珠、0.25 μL Tween 20、孵育2 h可达到较好的分离效果;免疫磁珠可用于3%牛肉浸膏等常用病毒洗脱液中的轮状病毒的分离,表明该分离技术能与已有的病毒浓集方法良好地整合.免疫磁珠分离技术与实时定量PCR联合用于检测水中轮状病毒,全过程需时约5 h,检测限为1×10⁴　copies/mL(相当于3～4　PFU/mＬ),检测结果与细胞病变试验检测结果有良好的线性相关关系（R²=0.981 6）,表明其能较好地表征水样的病毒感染风险.对接种已知量的轮状病毒的污水处理厂二级出水、再生水、地表水、自来水等水样的实验表明,该法可用于各种水样中轮状病毒的检测.     

Survival and Inactivation by Advanced Oxidative Process of Foodborne Viruses in Model Low-Moisture Foods

Enteric viruses, such as human norovirus (NoV) and hepatitis A virus (HAV), are the major causes of foodborne illnesses worldwide. These viruses have low infectious dose, and may remain infectious for weeks in the environment and food. Limited information is available regarding viral survival and transmission in low-moisture foods (LMF). LMFs are generally considered as ready-to-eat products, which undergo no or minimal pathogen reduction steps. However, numerous foodborne viral outbreaks associated with LMFs have been reported in recent years. The objective of this study was to examine the survival of foodborne viruses in LMFs during 4-week storage at ambient temperature and to evaluate the efficacy of advanced oxidative process (AOP) treatment in the inactivation of these viruses. For this purpose, select LMFs such as pistachios, chocolate, and cereal were inoculated with HAV and the norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV), then viral survival on these food matrices was measured over a four-week incubation at ambient temperature, by both plaque assay and droplet-digital RT-PCR (ddRT-PCR) using the modified ISO-15216 method as well as the magnetic bead assay for viral recovery. We observed an approximately 0.5 log reduction in viral genome copies, and 1 log reduction in viral infectivity for all three tested viruses following storage of select inoculated LMFs for 4 weeks. Therefore, the present study shows that the examined foodborne viruses can persist for a long time in LMFs. Next, we examined the inactivation efficacy of AOP treatment, which combines UV-C, ozone, and hydrogen peroxide vapor, and observed that while approximately 100% (4 log) inactivation can be achieved for FCV, and MNV in chocolate, the inactivation efficiency diminishes to approximately 90% (1 log) in pistachios and 70% (< 1 log) in cereal. AOP treatment could therefore be a good candidate for risk reduction of foodborne viruses from certain LMFs depending on the food matrix and surface of treatment.

     

Low Pathogenic Avian Influenza Viruses (H3N8, H5N6): In Vitro Influence of <Emphasis Type="SmallCaps">d</Emphasis>,<Emphasis Type="SmallCaps">l</Emphasis>-Lactic Acid and Sodium Chloride on Infectivity and Virus Persistence in Short Fermented Raw Poultry Sausage

Since highly pathogenic avian influenza virus H5N1 emerged in 1997, avian influenza is considered one of the most important infectious diseases globally. In respect of virus transmission to humans, the consumption of raw poultry products remains of serious concern. In this study, data about survival time and inactivation kinetics of two low pathogenic avian influenza virus (AIV) strains (H3N8, H5N6) in short fermented raw sausage were obtained. In addition, the impact of the preserving factors d,l-lactic acid and sodium chloride on virus infectivity was evaluated through in vitro studies. Virus infectivity was confirmed in embryonated chicken eggs. Inactivation of H3N8 was seen in d,l-lactic acid solutions (0.15 and 0.20%, pH 4.40–4.70 and pH 3.80–3.91) at both temperatures (20 vs. 4°C) during 3 days of exposure. However, infectious virus particles could still be detected after exposure to 0.1% d,l-lactic acid (pH 5.80–5.99). In all NaCl solutions (2, 6 and 12% w/v), infectivity of the H3N8 strain decreased steadily but reduction of the virus titre increased significantly with higher temperature. In raw sausages, decline in virus titre was observed for both strains during ripening and storage. Thereby, decline of virus infectivity was dependent on time and temperature with a more marked effect at higher temperatures (22 vs. 7°C). At refrigeration (7°C), both viruses maintained infectivity over 14 days. Results indicate that appropriate processing of short fermented raw poultry sausage is likely to reduce risk of virus exposure due to adequate inactivation of AIV during ripening and storage.     

Early Days of Food and Environmental Virology

In July 1962, the author joined the Food Research Institute (FRI), then at the University of Chicago, to become its food virologist. There was a limited record of waterborne viral disease outbreaks at the time; recorded data on foodborne outbreaks were fewer still. Laboratory environmental (water and wastewater) virology was in its infancy, and food virology was in gestation. Detection of viruses was most often attempted by inoculation of primary primate cell cultures, with observation for plaque formation or cytopathic effects. Focus was initially on enteroviruses and reoviruses. Environmental and food samples had to be liquefied if not already in liquid form; clarified to remove solids, bacteria, and fungi; and concentrated to a volume that could be tested in cell culture. Cytotoxicity was also a concern. Studies at the FRI and some other laboratories addressed all of these challenges. The FRI group was the World Health Organization’s Collaborating Center for Food Virology for many years. Other topics studied were virus inactivation as functions of temperature, time, matrix, disinfectants, and microbial action; peroral and ex-vivo infectivity; and the suitability of various virus surrogates for environmental monitoring and inactivation experiments. Detection of noroviruses and hepatitis A virus required molecular methods, most often RT-PCR. When it was found that inactivated virus often gave the same RT-PCR signal as that of infectious virus, sample treatments were sought, which would prevent false-positive test results. Many laboratories around the world have taken up food and environmental virology since 1962, with the result that a dedicated journal has been launched.     

Naturally Occurring Flavonoids Against Human Norovirus Surrogates

Naturally occurring plant-derived flavonoids are reported to have antibacterial, antiviral, and pharmacological activities. The objectives of this study were to determine the antiviral effects of four flavonoids (myricetin, l-epicatechin, tangeretin, and naringenin) on the infectivity of food borne norovirus surrogates after 2 h at 37 °C. The lab-culturable surrogates, feline calicivirus (FCV-F9) at titers of ~7 log₁₀ PFU/ml (high titer) or ~5 log₁₀ PFU/ml (low titer) and murine norovirus (MNV-1) at ~5 log₁₀ PFU/ml, were mixed with equal volumes of myricetin, l-epicatechin, tangeretin, or naringenin at concentrations of 0.5 or 1 mM, and incubated for 2 h at 37 °C. Treatments of viruses were neutralized in cell culture medium containing 10 % heat-inactivated fetal bovine serum, serially diluted, and plaque assayed. Each treatment was replicated thrice and assayed in duplicate. FCV-F9 (low titer) was not found to be reduced by tangeretin or naringenin, but was reduced to undetectable levels by myricetin at both concentrations. Low titer FCV-F9 was also decreased by 1.40 log₁₀ PFU/ml with l-epicatechin at 0.5 mM. FCV-F9 at high titers was decreased by 3.17 and 0.72 log₁₀ PFU/ml with myricetin and l-epicatechin at 0.5 mM, and 1.73 log₁₀ PFU/ml with myricetin at 0.25 mM, respectively. However, MNV-1 showed no significant inactivation by the four tested treatments. The antiviral effects of the tested flavonoids are dependent on the virus type, titer, and dose. Further research will focus on understanding the antiviral mechanism of myricetin and l-epicatechin.     

Solar Disinfection of Water for Inactivation of Enteric Viruses and its Enhancement by Riboflavin

Solar disinfection (SODIS) has been described as a cheap and effective method of treating contaminated water to inactivate pathogenic microorganisms. In this study, SODIS was assessed for its efficacy in inactivating three enteric viruses (coxsackievirus B3, coxsackievirus B5 and poliovirus), either on its own or in the presence of riboflavin as a disinfection enhancer. On its own, SODIS produced a reduction of virus infectivity of 4–6 log₁₀ in 6 h. In the presence of riboflavin, inactivation was more rapid in all viruses studied, and with coxsackievirus B5 and poliovirus an extra 1–2 log₁₀ increase in reduction of infectivity was observed after 6 h exposure. This study provides a practical example of low technology methods which could be utilised to provide safe drinking water in various circumstances.     

生物硝化池污水中硝化细菌的快速定量研究

实验采用聚合酶链式反应（PCR）技术与最大几率数法（MPN）相结合的MPN－PCR法对生物硝化池污水中的硝化细菌进行快速定量。所用的一对PCR引物是在对硝化细菌的16SrRNA基因进行系统比较的基础上设计合成的，可以扩增出大小为388bp的DNA片段。以从生物硝化池污水中抽提的含硝化细菌DNA的混合DNA为模板，进行PCR扩增并确定合适的扩增条件。运用MPN－PCR法进行定量检测的整个过程可在几小时之内完成。     

Temperature Dependent Depuration of Norovirus GII and Tulane Virus from Oysters (Crassostrea gigas)

Raw oysters are considered a culinary delicacy but are frequently the culprit in food-borne norovirus (NoV) infections. As commercial depuration procedures are currently unable to efficiently eliminate NoV from oysters, an optimisation of the process should be considered. This study addresses the ability of elevated water temperatures to enhance the elimination of NoV and Tulane virus (TuV) from Pacific oysters (Crassostrea gigas). Both viruses were experimentally bioaccumulated in oysters, which were thereafter depurated at 12 °C and 17 °C for 4 weeks. Infectious TuV and viral RNA were monitored weekly for 28 days by TCID₅₀ and (PMAxx-) RT-qPCR, respectively. TuV RNA was more persistent than NoV and decreased by?<?0.5 log₁₀ after 14 days, while NoV reductions were already?>?1.0 log₁₀ at this time. For RT-qPCR there was no detectable benefit of elevated water temperatures or PMAxx for either virus (p?>?0.05). TuV TCID₅₀ decreased steadily, and reductions were significantly different between the two temperatures (p?<?0.001). This was most evident on days 14 and 21 when reductions at 17 °C were 1.3–1.7 log₁₀ higher than at 12 °C. After 3 weeks, reductions?>?3.0 log₁₀ were observed at 17 °C, while at 12 °C reductions did not exceed 1.9 log₁₀. The length of depuration also had an influence on virus numbers. TuV reductions increased from?<?1.0 log₁₀ after seven days to?>?4.0 log₁₀ after 4 weeks. This implies that an extension of the depuration period to more than seven days, possibly in combination with elevated water temperatures, may be beneficial for the inactivation and removal of viral pathogens.

     

杂食性鱼类排泄物中藻类光能活性研究

借助叶绿素荧光技术,通过对杂食性鱼类鲫鱼(Crucian Carp)和金鲫鱼(Gold Crucian Carp)摄食微囊藻(Microcysis aeruginosa)后排泄物的培养,从鱼类排泄物藻类叶绿素荧光活性的角度探讨了杂食性鱼类在控藻上的应用.结果表明,鱼类摄食对微囊藻生长及叶绿素荧光参数均有显著影响(P<0.05).鲫鱼组的叶绿素荧光参数ΦPSII从第3d开始,Fv/Fo、Fv/Fm、ETR和qP从第5d开始随培养时间的延长而增加,而NPQ始终呈下降趋势.金鲫鱼组的叶绿素荧光参数(Fv/Fo、Fv/Fm、ΦPSII、ETR、qP)在培养期间一直呈降低趋势.鲫鱼组的Chl a浓度与细胞密度在培养期间先下降再上升,最后恢复至对照组水平,且Chl a浓度同部分叶绿素荧光参数(Fv/Fo、Fv/Fm、ΦPSII)呈现极显著正相关(P<0.01);金鲫鱼组的Chl a浓度与细胞密度于实验期间均下降,实验期间一直低于对照组,且两参数均与叶绿素荧光参数(Fv/Fo、Fv/Fm、ΦPSII、ETR、qP)呈极显著正相关(P<0.01).可见,微囊藻经鲫鱼摄食后,其叶绿素光合及生长活性经过短暂的下降后会逐渐恢复,而金鲫鱼可有效降低微囊藻活性,但金鲫鱼作为一种观赏鱼类,不适宜在大面积水体放养.