Is There a Risk for Introducing Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Through the Legal Importation of Pork?

Since the appearance of porcine reproductive and respiratory syndrome virus (PRRSV) in the late 1980s, the virus has become endemic throughout the world, with only the countries of Sweden, Switzerland, Finland, Norway, Australia, and New Zealand historically free of PRRS virus. Biosecurity is maintained largely through restrictions on the importation of pigs and semen. The risk for a PRRSV outbreak via the legal importation of fresh/chilled/frozen pork from PRRSV-positive countries remains controversial. However, examination of the historical record shows that countries retained a PRRSV-negative status during the importation of more than 500,000 tons of fresh/chilled/frozen pork from PRRSV-positive trading partners. This review describes some of the unique properties of PRRSV, including the poor stability of the virus in the environment, the low probability for airborne transmission, and the inability to sustain infections in feral swine, which make PRRSV a poor candidate for disease introduction through the legal importation of pork.     

Survival of Porcine Reproductive and Respiratory Syndrome Virus in Pork Products

There is a risk of virus transmission through contaminated pork, and many viruses are considered potential hazards for both humans and livestock. The risk of transmission may be elevated with importation/exportation of meat between countries globally. Survival of porcine reproductive and respiratory syndrome virus (PRRSV) in different pork products has not been studied. The present study evaluated PRRSV survival in four different products: fresh sausage, ham, bacon, and acidified sausage prepared with experimentally contaminated pork. These products were prepared according to standard methods used by the manufacturers of pork products, and then stored at room temperature, 4 °C and ?20 °C. PRRSV was detected only in fresh sausage for up to 15 days at 4 °C and for 30 days at ?20 °C. No PRRSV was detected at any temperature in any of the other three products. These preliminary data provide valuable information for the pork processing industry, as well as in planning for import/export of these products among different countries.     

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.     

Isolation and Genomic Sequence of Hepatitis A Virus from Mixed Frozen Berries in Italy

Hepatitis A virus (HAV) was detected in two samples of mixed frozen berries linked to Italian hepatitis A outbreak in April and September 2013. Both viruses were fully sequenced by next-generation sequencing and the genomes clustered with HAV complete genomes of sub-genotype IA with nucleotide identities of 95–97 %.     

Characteristics of Filoviridae: Marburg and Ebola Viruses

Filoviruses are enveloped, nonsegmented negative-stranded RNA viruses. The two species, Marburg and Ebola virus, are serologically, biochemically, and genetically distinct. Marburg virus was first isolated during an outbreak in Europe in 1967, and Ebola virus emerged in 1976 as the causative agent of two simultaneous outbreaks in southern Sudan and northern Zaire. Although the main route of infection is known to be person-to-person transmission by intimate contact, the natural reservoir for filoviruses still remains a mystery.     

Detection and Typing of Norovirus from Frozen Strawberries Involved in a Large-Scale Gastroenteritis Outbreak in Germany

During September/October 2012, a norovirus gastroenteritis outbreak affecting about 11,000 people occurred in Germany. Epidemiological studies suggested that frozen strawberries represented the vehicle of infection. We describe here the analysis of frozen strawberries for the presence of norovirus. Samples were taken by applying a stratified subsampling scheme. Two different methods for virus extraction from strawberries were compared. First, viruses were eluted from strawberries under alkaline conditions and concentrated using a polyethylene glycol precipitation. Second, ultrafiltration was applied for concentration of viruses rinsed off of the berries. In both cases, RNA was extracted and analyzed by real-time RT-PCR. Application of the ultrafiltration method generally resulted in a lower detection rate. Noroviruses were detected in 7/11 samples derived from the lot of strawberries implicated in the outbreak using the precipitation method. Typing of norovirus revealed three different genotypes including a combination of norovirus genotype II.16 (viral polymerase) and II.13 (viral capsid). This genotype combination was also found in some of the patients that were involved in the outbreak, but that had not been reported in Germany so far. In conclusion, heterogeneously distributed noroviruses in frozen strawberries can be detected by applying an optimized combination of sampling procedures, virus extraction methods, and real-time RT-PCR protocols. The detection of several different genotypes in the strawberries may suggest contamination from sewage rather than from a single infected food handler.     

Design and Application of Nucleic Acid Standards for Quantitative Detection of Enteric Viruses by Real-Time PCR

Synthetic multiple-target RNA and DNA oligonucleotides were constructed for use as quantification standards for nucleic acid amplification assays for human norovirus genogroup I and II, hepatitis E virus, murine norovirus, human adenovirus, porcine adenovirus and bovine polyomavirus. This approach overcomes the problems related to the difficulty of obtaining practical quantities of viral RNA and DNA from these viruses. The quantification capacity of assays using the standards was excellent in each case (R ² > 0.998 and PCR efficiency > 0.89). The copy numbers of the standards were equivalent to the genome equivalents of representative viruses (murine norovirus and human adenovirus), ensuring an accurate determination of virus presence. The availability of these standards should facilitate the implementation of nucleic acid amplification-based methods for quantitative virus detection.     

Effect of the Shellfish Proteinase K Digestion Method on Norovirus Capsid Integrity

Norovirus outbreaks are associated with the consumption of contaminated shellfish, and so efficient methods to recover and detect infectious norovirus in shellfish are important. The Proteinase K digestion method used to recover norovirus from shellfish, as described in the ISO 15216, would be a good candidate but its impact on the virus capsid integrity and thus infectivity was never examined. The aim of this study was to assess the impact of the Proteinase K digestion method, and of the heat treatment component of the method alone, on norovirus (genogroups I and II) and MS2 bacteriophage capsid integrity. A slightly modified version of the ISO method was used. RT-qPCR was used for virus detection following digestion of accessible viral RNA using RNases. MS2 phage infectivity was measured using a plaque assay. The effect of shellfish digestive glands (DG) on recovery was evaluated. In the presence of shellfish DG, a reduction in MS2 phage infectivity of about 1 log₁₀ was observed after the Proteinase K digestion method and after heat treatment component alone. For norovirus GII and MS2 phage, there was no significant loss of genome following the Proteinase K digestion method but there was a significant 0.24 log₁₀ loss of norovirus GI. In the absence of shellfish DG, the reduction in MS2 phage infectivity was about 2 log₁₀, with the addition of RNases resulting in a significant loss of genome for all tested viruses following complete Proteinase K digestion method and the heat treatment alone. While some protective effect from the shellfish DG on viruses was observed, the impact on capsid integrity and infectivity suggests that this method, while suitable for norovirus genome detection, may not completely preserve virus infectivity.     

One-year Surveillance of Human Enteric Viruses in Raw and Treated Wastewaters,Downstream River Waters,and Drinking Waters

Human enteric viruses are a major cause of waterborne diseases, and can be transmitted by contaminated water of all kinds, including drinking and recreational water. The objectives of the present study were to assess the occurrence of enteric viruses (enterovirus, norovirus, adenovirus, hepatitis A and E virus) in raw and treated wastewaters, in rivers receiving wastewater discharges, and in drinking waters. Wastewater treatment plants’ (WWTP) pathogen removal efficiencies by adenovirus quantitative real-time PCR and the presence of infectious enterovirus, by cell culture assays, in treated wastewaters and in surface waters were also evaluated. A total of 90 water samples were collected: raw and treated wastewaters (treated effluents and ultrafiltered water reused for industrial purposes), water from two rivers receiving treated discharges, and drinking water. Nested PCR assays were used for the identification of viral DNA/RNA, followed by direct amplicon sequencing. All raw sewage samples (21/21), 61.9 % of treated wastewater samples (13/21), and 25 % of ultrafiltered water samples (3/12) were contaminated with at least one viral family. Multiple virus families and genera were frequently detected. Mean positive PCRs per sample decreased significantly from raw to treated sewage and to ultrafiltered waters. Moreover, quantitative adenovirus data showed a reduction in excess of 99 % in viral genome copies following wastewater treatment. In surface waters, 78.6 % (22/28) of samples tested positive for one or more viruses by molecular methods, but enterovirus-specific infectivity assays did not reveal infectious particles in these samples. All drinking water samples tested negative for all viruses, demonstrating the effectiveness of treatment in removing viral pathogens from drinking water. Integrated strategies to manage water from all sources are crucial to ensure water quality.     

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.     

Rethinking the Significance of Reovirus in Water and Wastewater

The genus Orthoreovirus contains nonenveloped viruses with double-stranded gene segments encased in a double-layered icosahedral capsid shell. These features constitute major determinants of virion stability in the environment and virion resistance against physical and chemical agents. Reovirus (ReoV) is the general term most commonly used for all virus strains that infect humans and nonhuman animals. Several studies have demonstrated the frequent occurrence of ReoV in wastewaters and natural waters, including surface and ground waters from different geographical areas. Most of these studies have reported higher concentrations of ReoV than any other enteric virus analyzed. They are more commonly isolated in chlorine-disinfected wastewaters than other enteric viruses, and appear to survive longer in water. The ability of ReoV to form large aggregates, even with different types of enteric viruses (e.g., poliovirus) and their ability to undergo mechanisms of gene segment reassortment among different serotypes may also explain their greater stability. Different approaches have been applied for concentration of ReoV from water; however, the recovery efficiency of the filtration methods has not been fully evaluated. Recently, molecular methods for identification of ReoV strains and quantification of virus genome have been developed. Studies have shown that the overall detection sensitivity of ReoV RNA is enhanced through initial replication of infectious virions in cell culture. More studies are needed to specifically address unresolved issues about the fate and distribution of ReoV in the environment since this virus is not commonly included in virological investigations.     

The Effect of Heat on the Physicochemical Properties of Bacteriophage MS2

The differences in physicochemical characteristics between infectious and non-infectious viral particles are poorly known. Even for heat, which is known as one of the most efficient treatments to inactivate enteric viruses, the global inactivation mechanisms have not been described yet. Such knowledge would help distinguish between both types of particles and therefore clarify the interpretation of the presence of viral genomes in food after heat treatment. In this study, we examined in particular the differences in electrostatic charge and hydrophobicity between the two particle types. MS2 phage, a common surrogate for enteric viruses, was used as a model virus. The heat-induced inactivation process of the infectious phages caused hydrophobic domains to be transiently exposed and their charge to become less negative. The particles also became progressively permeable to small molecules such as SYPRO Orange dye. The presence of non-infectious phage particles in which the genome was not accessible to RNases has been clearly demonstrated. These observations were done for MS2 phages exposed to a temperature of 60 °C. When exposed to a temperature higher than their critical temperature (72 °C), the particles were disrupted and the genome became available for RNases. At lower temperatures, 60 °C in this study, the transient expression of hydrophobic domains of remaining infectious phages appeared as an interesting parameter for improving their specific detection.     

大连湾和大窑湾表层沉积物病毒分布特点

为了揭示近岸沉积物环境中的病毒-细菌关系,以及探讨病毒对污染环境的响应,本研究利用SYBR Green I染色计数法,分别对2012年5月(春季)、8月(夏季)和11月(秋季)大连湾和大窑湾表层沉积物中的病毒和细菌丰度进行了检测。结果表明:大连湾表层沉积物病毒丰度的季节变化表现为夏季春季秋季,最高值1.59108VLP/g(湿重)出现在夏季;最小值1.53106VLP/g出现在春季。平面分布春季为湾中 湾顶 湾口,夏季和秋季分布特点为湾中 湾口 湾顶。病毒和细菌有显著相关性(R2=0.80,n=20)。而大窑湾表层沉积物病毒丰度的季节变化则变现为夏季秋季春季,最高值7.08107VLP/g出现在夏季,最小值1.16106VLP/g出现在春季。大窑湾区域分为湾内和湾外,3个季节的平面分布均表现为湾外湾内。病毒和细菌的关联性较大连湾差(R2=0.50,n=12)。大连湾及大窑湾表层沉积物病毒与细菌之比分别为4.12和4.09。两个海湾沉积物病毒分布有所差异,可能与沉积物环境有关系,有待进一步研究。     

污水新型冠状病毒监测研究进展及启示

新冠肺炎疫情暴发以来,全球多个国家和地区频繁在污水中检测出新型冠状病毒核酸. 开展基于污水流行病学的污水新型冠状病毒监测,可作为人群新冠肺炎监测的重要补充,获得的核酸浓度数据和序列突变分析结果,可为新冠肺炎疫情早期预警、早期发现无症状感染者、评估感染规模、监测人群流行趋势、开展病毒溯源调查、制定防控政策等提供科学依据. 鉴于此,本文系统介绍了国内外污水中新型冠状病毒的来源及影响病毒存活的主要因素,归纳了常用的污水新型冠状病毒富集浓缩和核酸检测方法,概括了全球已开展的监测项目、进展及现存科学问题,指出了现有研究的局限性,包括缺乏污水中新型冠状病毒的分布特征及感染性研究、预测预警模型开发与应用不足等. 为我国下一阶段新冠肺炎疫情或者未来可能出现的突发重大疫情的精准防控提供借鉴与参考,提升我国应对传染病及非传染病的预测预警、流行规模评估及精准施策的能力.      

历史传染病疫情的环境与气候特征初探及对新冠肺炎疫情的思考

新型冠状病毒肺炎（COVID-19,简称“新冠肺炎”）疫情的暴发与流行对人类社会安全造成严重危害,同时也考验着世界各国公共卫生系统应对大型突发性传染病的防控能力.对历史传染病疫情暴发和流行的环境与气候特征进行总结,对于新冠肺炎疫情的科学研究及防控具有重要的参考价值.结果表明:①历史上人际传播的冠状病毒科、正粘病毒科传染病多暴发于北半球亚热带季风气候地区及冬春季节,而黄病毒科传染病多暴发在热带地区及高温多雨的夏秋季节.②全球变暖和极端天气催生影响传染病的暴发及传播.③人类对生态系统平衡的影响,迫使病毒宿主栖息地迁移和不同病毒宿主聚集,增加病毒变异概率和传染病暴发风险.在此基础上,提出了对新冠肺炎疫情的思考,认为适宜的气候因素可能利于疫情的暴发与流行,而热带国家疫情的暴发则说明需要重新审视气候、环境条件及生态因素对新型冠状病毒的影响.研究结果将为新冠肺炎疫情的防控和未来传染病疫情的预测及阻断提供参考和借鉴.      

Comprehensive Study on Enteric Viruses and Indicators in Surface Water in Kyoto,Japan, During 2014–2015 Season

Certain enteric viruses that are present in the water environment are potential risk factors of waterborne infections. To better understand the impact of viruses in water, both enteric viruses and their potential indicators should be comparatively investigated. In this study, occurrences of GI- and GII-noroviruses (NoVs), sapovirus (SaV), rotavirus (RoV), Aichi virus 1 (AiV-1), enterovirus (EV), and pepper mild mottle virus (PMMoV) were quantitatively determined in surface water samples in Japan. Additionally, the genotype distribution of GI- and GII-NoVs was determined using a next-generation amplicon sequencing. PMMoV was the most abundant virus regardless of season and location, indicating its usefulness as an indicator for the viral contamination of water. Other potential indicators, AiV and EV, were less abundant than GII-NoV. Viruses other than PMMoV showed seasonality, i.e., EV and other viruses (NoVs, SaV, RoV, and AiV-1) became prevalent during summer and winter, respectively. SaV showed a relatively high abundance at a location that was affected by untreated wastewater. Regarding NoV genotypes, GI.1, GI.2, GI.4, GI.5, GI.6, GII.3, GII.4, GII.6, and GII.17 were found from the surface water samples. GII.4 and GII.17 seemed to have contributed to the high abundance of GII-NoV in the samples. Interestingly, GII.17 strains became prevalent in the water samples before becoming prevalent among gastroenteritis patients in Japan. These findings provide further insights into the properties of viruses as contaminants in the water environment.     

Assessment of Water Quality in a Border Region Between the Atlantic Forest and an Urbanised Area in Rio de Janeiro,Brazil

The preservation of water resources is one of the goals of the designation of parks that act as natural reservoirs. In order to assess the impact of the presence of humans in an environmental preservation area bordering urban areas, the presence of four pathogenic enteric viruses [group A rotavirus (RV-A), norovirus (NoV), human adenoviruses (HAdV), and hepatitis A virus (HAV)], as well as the physico-chemical parameters, and Escherichia coli levels were assessed in riverine water samples. From June 2008 to May 2009, monthly monitoring was performed along the Engenho Novo River. RV-A, NoV, and HAdV were observed in 29 % (31/108) of the water samples, with concentrations of up to 10³ genome copies/liter. The natural occurrence of infectious HAdV was demonstrated by Integrated Cell Culture-PCR (ICC-PCR). This study confirms the suitability of using the detection of fecal-oral transmitted viruses as a marker of human fecal contamination in water matrices and indicates the spread of pathogenic viruses occurring in an alleged area of environmental protection.