Thermal Inactivation of Hepatitis E Virus in Pork Products Estimated with a Semiquantitative Infectivity Assay.

Hepatitis E virus genotype 3 (HEV-3) is a food-borne pathogen causative of hepatitis E infections in humans. In Europe, HEV-3 is mainly transmitted through the consumption of raw or undercooked pork. In order to determine the effectiveness of control measures that can be taken in the industry or by the consumer, it is pivotal to determine the infectivity of HEV present in pork products after thermal food-processing steps. First, we implemented a method for the detection of infectious HEV-3c and HEV-3e in a cell culture medium and in extracts from inoculated pork products. Next, we investigated the effect of the thermal inactivation of HEV by mimicking food-processing steps specific for dried sausage and liver homogenate matrices. After four weeks, HEV-inoculated dried sausage subjected to 21 °C or lower temperatures was still infectious. For the liver homogenate, the highest HEV-3c/e inactivation of the conditions tested was observed at 71 °C for five min or longer. Finally, our method was able to successfully detect and estimate viral loads of infectious HEV in naturally infected pig livers. Our data provide a basis for the future use of the quantitative microbial risk assessment of infectious HEV in pork products that are subjected to thermal food processing steps.

Epidemiology of Pathogens Listed as Potential Bioterrorism Agents, the Netherlands, 2009‒2019.

We provide incidences (cases/10 million persons) in the Netherlands during 2009-2019 for pathogens listed as potential bioterrorism agents. We included pathogens from the highest categories of the European Medicines Agency or the US Centers for Disease Control and Prevention. Notifiable diseases and recently published data were used to calculate the average annual incidence. Coxiella burnetii had the highest incidence because of a Q fever epidemic during 2007-2010. Incidence then decreased to 10.8 cases/. Pathogens with an incidence >1 were Brucella spp. (2.5 cases), Francisella tularensis (1.3 cases), and Burkholderia pseudomallei (1.1 cases). Pathogens with an incidence <1 were hemorrhagic fever viruses (0.3 cases), Clostridium botulinum (0.2 cases), and Bacillus anthracis (0.1 cases). Variola major and Yersinia pestis were absent. The generally low incidences make it unlikely that ill-meaning persons can isolate these pathogens from natural sources in the Netherlands. However, the pathogens are stored in laboratories, underscoring the need for biosecurity measures.

Biosafety and biosecurity challenges during the COVID-19 pandemic and beyond.

As the world continues to battle the SARS-CoV-2 pandemic, it is a stark reminder of the devastation biological threats can cause. In an unprecedented way the global community saw a massive surge in the demand for diagnostic capacities, which had a substantial impact on biosafety and biosecurity. Laboratories had to cope with a surge in laboratory testing capacity, while resources and training possibilities were limited. In addition, the pandemic highlighted the impact biological threats can have, thereby giving rise to new dialogue about biosecurity and new biological threats. This paper aims to highlight some of the most pressing issues regarding biosafety and biosecurity observed during the COVID-19 pandemic with special focus on low and lower middle-income countries. The authors provide lessons learned, tools and recommendations to improve future biosafety and biosecurity and increase preparedness for the next global health crisis.

Complementarity of International Instruments in the Field of Biosecurity.

The COVID-19 pandemic has demonstrated the devastating impact of infectious disease outbreaks and the threat of emerging and re-emerging dangerous pathogens, independent of their origin. Natural, accidental, and deliberate disease outbreaks all need systems in place for an effective public health response. The best known international instrument in the field of public health is the WHO International Health Regulations (2005). Although the International Health Regulations are mainly focused on natural disease outbreaks, the actions to take to comply with them also contribute to biosecurity and non-proliferation. This paper examines in case of full implementation of the International Health Regulations, what other actions states should take to comply with international biosecurity instruments, including the Biological and Toxin Weapons Convention and United Nations Security Council Resolution 1540, to effectively prevent and defend against intentional biological threats. An overview of international instruments from different disciplines regarding biosecurity is presented. Furthermore, this paper clarifies the similarities between the international biosecurity instruments and addresses the additional requirements that instruments stipulate. From a detailed comparison between the instruments it can be concluded that, to adhere to all legally-binding international biosecurity instruments, specific non-proliferation and export control measures are necessary in addition to full implementation of the International Health Regulations. Additionally, an overview of non-legally binding instruments in the field of biosecurity is presented and practical implementation examples are highlighted. Compliance with legally binding instruments can be improved by precise guidance provided by non-legally binding instruments that are clear and attuned to the situation on the ground. To improve understanding of the existing international instruments, this paper aims to provide an overview of the international legal biosecurity framework to biosecurity experts, policymakers, civil servants, and practitioners. It offers possible practical applications for the politico-legal context and accommodates the enhancement of full employment of biosecurity resources for an improved multidisciplinary capacity to prevent, detect, and respond to infectious disease outbreaks.

Dual-Use Quickscan: A Web-Based Tool to Assess the Dual-Use Potential of Life Science Research.

Research on pathogenic organisms is crucial for medical, biological and agricultural developments. However, biological agents as well as associated knowledge and techniques, can also be misused, for example for the development of biological weapons. Potential malicious use of well-intended research, referred to as "dual-use research", poses a threat to public health and the environment. There are various international resources providing frameworks to assess dual-use potential of the research concerned. However, concrete instructions for researchers on how to perform a dual-use risk assessment is largely lacking. The international need for practical dual-use monitoring and risk assessment instructions, in addition to the need to raise awareness among scientists about potential dual-use aspects of their research has been identified over the last years by the Netherlands Biosecurity Office, through consulting national and international biorisk stakeholders. We identified that Biorisk Management Advisors and researchers need a practical tool to facilitate a dual-use assessment on their specific research. Therefore, the Netherlands Biosecurity Office developed a web-based Dual-Use Quickscan (www.dualusequickscan.com), that can be used periodically by researchers working with microorganisms to assess potential dual-use risks of their research by answering a set of fifteen yes/no questions. The questions for the tool were extracted from existing international open resources, and categorized into three themes: characteristics of the biological agent, knowledge and technology about the biological agent, and consequences of misuse. The results of the Quickscan provide the researcher with an indication of the dual-use potential of the research and can be used as a basis for further discussions with a Biorisk Management Advisor. The Dual-Use Quickscan can be embedded in a broader system of biosafety and biosecurity that includes dual-use monitoring and awareness within organizations. Increased international attention to examine pathogens with pandemic potential has been enhanced by the current COVID-19 pandemic, hence monitoring of dual-use potential urgently needs to be encouraged.

Systematic approach towards establishing a National Inventory of Dangerous Pathogens.

International regulations stipulate that countries need to organize their biosafety and biosecurity systems to minimize the risk of accidental (biosafety) or malicious intentional (biosecurity) release of dangerous pathogens. International Health Regulations (IHR) benchmarks from the WHO state that even for a level of limited capacity countries need to 'Identify and document human and animal health facilities that store/maintain dangerous pathogens and toxins in the relevant sectors and health professionals responsible for them'. This study provides a stepwise, systematic approach and best practices for countries to initiate a national inventory of dangerous pathogens. With a national inventory of dangerous pathogens a country can identify and document information in a dedicated electronic database on institutes that store or maintain dangerous pathogens. The systematic approach for the implementation of a national inventory of dangerous pathogens consists of four stages; identification, preparation, implementation, and maintenance and evaluation. In the identification phase, commitment of the relevant national ministries is to be established, and a responsible government entity needs to be identified. In the preparatory phase, a list of pathogens to be incorporated in the inventory, as well as a list of institutes to include, is to be agreed upon. In the implementation phase, the institutes are contacted, and the collected data is stored safely and securely in a electronical database. Finally, in the maintenance and evaluation phase meaningful insights are derived and reported to the relevant government authorities. Also, preparations for updates and modifications are undertaken, such as modifications of pathogen lists or institute lists. The approach and database, which is available from the authors, have been tested for the implementation of a national inventory of dangerous pathogens in multiple East-African countries. A national inventory of dangerous pathogens helps countries in strengthening national biosafety and biosecurity as well as in their compliance to IHR.

Surface chemistry-dependent antiviral activity of silver nanoparticles.

The toxicity towards viruses of silver nanoparticles (AgNPs) has been reported to be dependent on several factors such as particle concentration, size, and shape. Although these factors may indeed contribute to the toxicity of AgNPs, the results presented in this work demonstrate that surface chemistry and especially surface charge is a crucial factor governing their antiviral activity. Here, this work investigated the influence of capping agents representing various surface charges ranging from negative to positive. These AgNPs were capped with citrate, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP) mercaptoacetic acid (MAA) and (branched polyethyleneimine (BPEI). We show that AgNPs exhibited surface charge-dependent toxicity towards MS2 bacteriophages. Among the capping agents under investigation, BPEI capped AgNPs (Ag/BPEI) exhibited the highest reduction of MS2 resulting in ≥6 log10-units reductions, followed by 4-5 log10-units reductions with PVP and PEG capping's and 3-4 log10-units with MAA and citrate cappings. Bare nanoparticles reported a mere 1-2 log10-units reduction. Electrostatic interaction between the positively charged BPEI-coating and the negatively charged virus surface played a significant role in bringing the MS2 closer to toxic silver ions (Ag+). Further results obtained from TEM showed that Ag/BPEI nanoparticles could directly damage the structure of the MS2 bacteriophages. AgNPs and cationic capping agents' observed synergy can lead to much lower and much more efficient dosing of AgNPs for antiviral applications.

The Vulnerability Scan, a Web Tool to Increase Institutional Biosecurity Resilience.

The importance of vigilance within organizations working with high-risk biological material receives increasing attention. However, an in-depth and comprehensive tool, dedicated to increase awareness of potential risks and to assess an organization's current biosecurity vulnerabilities, has not been available yet. We developed the "Biosecurity Vulnerability Scan," a web tool that identifies biosecurity gaps in an organization based on eight biosecurity pillars of good practice. Although the tool aims primarily to assist biosafety and biosecurity officers, it can also be useful to researchers working with dangerous pathogens, their principal investigators, management, or those responsible for security issues in the life sciences. Results are only stored locally and are provided in an "overview report," which includes information on relevant risks and control measures. This can support well-substantiated decision-making on strengthening biosecurity measures within a specific organization. With this article, we aim to support institutes to increase their overall security resilience and to improve institutional biosecurity in particular by providing practical recommendations. The Biosecurity Vulnerability Scan is available at www.biosecurityvulnerabilityscan.nl.

Norovirus Outbreak Associated with Swimming in a Recreational Lake Not Influenced by External Human Fecal Sources in The Netherlands, August 2012.

Swimming in fecally contaminated recreational water may lead to gastrointestinal illness. A recreational water-associated outbreak of norovirus (NoV) infections affecting at least 100 people in The Netherlands occurred in August 2012. Questionnaire responses from patients indicated swimming in recreational lake Zeumeren as the most likely cause of illness. Most patients visited the lake during the weekend of 18⁻19 August, during which the weather was exceptionally warm (maximum temperatures 32⁻33 °C), and visitor numbers elevated. Patients, mostly children, became ill with gastroenteritis 1⁻6 days (median 2 days) after exposure. Four stool samples from patients were NoV GI positive. Subsurface sandy soil from one of the beaches where most patients swam was NoV GI positive; the water sample was negative. The epidemiological curve and the timeline of investigation based on reported symptoms demonstrate the difficulty in discovering the source in recreational water outbreaks. A NoV outbreak in a recreational lake that is not subjected to external fecal contamination sources shows the need for active communication about human shedding of viruses during and after diarrheal episodes and the advice to refrain from swimming, even a few weeks after the symptoms have resolved.

Quantitative farm-to-fork risk assessment model for norovirus and hepatitis A virus in European leafy green vegetable and berry fruit supply chains.

Fresh produce that is contaminated with viruses may lead to infection and viral gastroenteritis or hepatitis when consumed raw. It is thus important to reduce virus numbers on these foods. Prevention of virus contamination in fresh produce production and processing may be more effective than treatment, as sufficient virus removal or inactivation by post-harvest treatment requires high doses that may adversely affect food quality. To date knowledge of the contribution of various potential contamination routes is lacking. A risk assessment model was developed for human norovirus, hepatitis A virus and human adenovirus in raspberry and salad vegetable supply chains to quantify contributions of potential contamination sources to the contamination of produce at retail. These models were used to estimate public health risks. Model parameterization was based on monitoring data from European supply chains and literature data. No human pathogenic viruses were found in the soft fruit supply chains; human adenovirus (hAdV) was detected, which was additionally monitored as an indicator of fecal pollution to assess the contribution of potential contamination points. Estimated risks per serving of lettuce based on the models were 3×10(-4) (6×10(-6)-5×10(-3)) for NoV infection and 3×10(-8) (7×10(-10)-3×10(-6)) for hepatitis A jaundice. The contribution to virus contamination of hand-contact was larger as compared with the contribution of irrigation, the conveyor belt or the water used for produce rinsing. In conclusion, viral contamination in the lettuce and soft fruit supply chains occurred and estimated health risks were generally low. Nevertheless, the 97.5% upper limit for the estimated NoV contamination of lettuce suggested that infection risks up to 50% per serving might occur. Our study suggests that attention to full compliance for hand hygiene will improve fresh produce safety related to virus risks most as compared to the other examined sources, given the monitoring results. This effect will be further aided by compliance with other hygiene and water quality regulations in production and processing facilities.

Virus transfer proportions between gloved fingertips, soft berries, and lettuce, and associated health risks.

Multiple outbreaks of human norovirus (hNoV) have been associated with fresh produce, such as soft berries and lettuce. Even though food handlers are considered an important source for the introduction of hNoV into food chains, their contribution to public health risks associated with hNoV remains unknown. To assess to which extent food handlers contribute to the introduction and spread of hNoV in fresh produce chains quantitative virus transfer data are needed. We estimated transfer proportions of hNoV GI.4, GII.4, murine norovirus (MNV-1), a culturable surrogate of hNoV, and human adenovirus (hAdV-2), a human pathogen proposed as an indicator for human faecal pollution, between gloved fingertips and raspberries, strawberries, and lettuce, by quantitative RT-PCR and cell culture if applicable. Virus transfer proportions were corrected for virus-matrix specific recoveries, and variability and uncertainty of the parameters were estimated. Virus transfer from gloves to soft berries was generally lower as compared to lettuce, with mean transfer proportions ranging between 0.1 to 2.3% and 9 to 10% for infectious MNV-1 and hAdV-2, respectively. Transfer from produce to glove was mostly greater than transfer from glove to produce, adding to the likelihood of virus transfer due to cross contamination from contaminated produce via food handlers. HNoV GI.4 and hNoV GII.4 showed no significant difference between their mean transfer proportions. Using the estimated transfer proportions, we studied the impact of low and high transfer proportions on the public health risk, based on a scenario in which a food handler picked raspberries with contaminated fingertips. Given the made assumptions, we could show that for a pathogen as infectious as hNoV, low transfer proportions may pose a greater public health risk than high transfer proportions, due to a greater viral spread. We demonstrated the potential of food handlers in spreading hNoV in food chains, showing that prevention of virus contamination on food handlers' hands is crucial for food safety. Nevertheless, complete prevention of virus contamination on fresh produce cannot be achieved in reality, and reliable and effective intervention measures are consequently required. We estimated that, especially for low transfer proportions, a robust one log10-unit reduction of infectious hNoV on contaminated produce, and on food handlers' hands, could lower the public health risk substantially. Using the obtained data in quantitative risk assessment will aid in elucidating the contribution of food handlers in hNoV transmission.

Aichi virus in sewage and surface water, the Netherlands.

Detection of Aichi virus in humans was initially reported in Japan in 1989. To establish a timeline for the prevalence of Aichi virus infection among humans in the Netherlands, we conducted molecular analysis of archival water samples from 1987-2000 and 2009-2012. Aichi virus RNA was detected in 100% (8/8) of sewage samples and 100% (7/7) of surface water samples collected during 1987-2000 and 100% (8/8) of sewage samples and 71% (5/7) of surface water samples collected during 2009-2012. Several genotype A and B Aichi virus lineages were observed over the 25-year period studied, but the time course of viral genetic diversity showed recent expansion of the genotype B population over genotype A. Our results show that Aichi virus has been circulating among the human population in the Netherlands since before its initial detection in humans was reported and that genotype B now predominates in this country.

Persistence of human norovirus in reconstituted pesticides--pesticide application as a possible source of viruses in fresh produce chains.

The consumption of fresh produce is frequently associated with outbreaks of human norovirus (hNoV) disease. To prevent the contamination of fresh produce with hNoV, knowledge of the possible introduction sources of the viruses, such as water, is needed to be able to implement appropriate and efficient preventive measures. Contaminated water used to reconstitute pesticides could be a relevant source of infectious hNoV, determined by the initial level of virus contamination and the persistence of these viruses in reconstituted pesticides. We studied the persistence of hNoV GI.4, hNoV GII.4 and murine norovirus (MNV-1), the only culturable norovirus, in eight different pesticides after 0 and 2h. Virus concentrations were determined by reverse transcriptase PCR, and infectivity of MNV-1 was determined by endpoint dilutions followed by maximum likelihood estimations. MNV-1 was found to remain infectious in seven of the eight tested pesticides at the highest concentration applied in practice. In the presence of the insecticide Vertimec, MNV-1 infectivity decreased rapidly with a 1.9 log(10)-unit reduction at timepoint T(0). Also, the concentration of NoV GI.4 RNA decreased considerably with a 1.7 log(10)-unit reduction; whereas the detected PCR fragment of hNoV GII.4 remained stable. Assuming a similar persistence of infectious MNV-1 and hNoV we can conclude that water containing hNoV used to dilute pesticides may be an important source of infectious hNoV in fresh produce chains. The application of pesticides may therefore not only be a chemical hazard, but also a microbiological hazard for public health. The inclusion of antiviral substances in reconstituted pesticides may be appropriate to reduce the virological health risk posed by the application of pesticides.

Persistence of human norovirus GII.4 and GI.4, murine norovirus, and human adenovirus on soft berries as compared with PBS at commonly applied storage conditions.

Human noroviruses (hNoV) have been detected on soft fruits. Especially raspberries have been found to be associated with outbreaks of gastroenteritis suggesting persistence of hNoV on these fruits. Therefore, the persistence of hNoV GII.4 and GI.4, murine norovirus (MNV-1, a culturable surrogate for hNoV), and human adenovirus (hAdV, an indicator for human fecal contamination), on raspberries, strawberries and in phosphate buffered saline (PBS) at 4°C, 10°C and 21°C, mimicking commonly applied storage conditions was studied by molecular and cell culture techniques. Monophasic, biphasic and Weibull models were fitted to virus counts with maximum likelihood estimation. The tested viruses were persistent (≤0.5 log(10)-unit reduction in viral titer) under all studied conditions in PBS, at 4°C and 10°C on raspberries, and at 4°C on strawberries. The difference in viral persistence on raspberries and strawberries was most pronounced at 21°C. Here, infectious MNV-1 and hAdV particles decayed rapidly on strawberries with TFL-values (time for the first log(10)-unit reduction) of only 1day (95% CI of 0.6-1 and 0.8-1days, respectively). On raspberries, however, the TFL-value of infectious MNV-1 was found to likely exceed the shelf life of the berries with 3days (95% CI of 2.8-3.1days); hAdV remained infectious with only 0.3 log(10)-unit reduction (95% CI of 0.2-0.4) in viral titer. For hNoV GI, a TFL-value of 2days (95% CI 1-4days) was determined based on the targeted genome fragment, whereas the TFL-value of hNoV GII exceeded the shelf life of strawberries at 21°C. The greater viral persistence on raspberries as compared to strawberries, especially at 21°C, may at least in part explain why raspberries are more frequently associated with hNoV outbreaks than strawberries. Moreover, our results show that due to the high persistence of the virus already low contamination levels of the highly infectious hNoV may be associated with an infection risk of humans after consumption of raspberries. The estimated decay parameters and uncertainties of this study serve as important input requirements in the quantitative assessment of public health risks from the consumption of soft fruits.

QMRAspot: a tool for Quantitative Microbial Risk Assessment from surface water to potable water.

In the Netherlands, a health based target for microbially safe drinking water is set at less than one infection per 10,000 persons per year. For the assessment of the microbial safety of drinking water, Dutch drinking water suppliers must conduct a Quantitative Microbial Risk Assessment (QMRA) at least every three years for the so-called index pathogens enterovirus, Campylobacter, Cryptosporidium and Giardia. In order to collect raw data in the proper format and to automate the process of QMRA, an interactive user-friendly computational tool, QMRAspot, was developed to analyze and conduct QMRA for drinking water produced from surface water. This paper gives a description of the raw data requirements for QMRA as well as a functional description of the tool. No extensive prior knowledge about QMRA modeling is required by the user, because QMRAspot provides guidance to the user on the quantity, type and format of raw data and performs a complete analysis of the raw data to yield a risk outcome for drinking water consumption that can be compared with other production locations, a legislative standard or an acceptable health based target. The uniform approach promotes proper collection and usage of raw data and, warrants quality of the risk assessment as well as enhances efficiency, i.e., less time is required. QMRAspot may facilitate QMRA for drinking water suppliers worldwide. The tool aids policy makers and other involved parties in formulating mitigation strategies, and prioritization and evaluation of effective preventive measures as integral part of water safety plans.

Pathogenic Vibrio species in dutch shellfish destined for direct human consumption.

Vibrio parahaemolyticus is a common cause of shellfish-related gastroenteritis all over the world. V. parahaemolyticus and Vibrio alginolyticus have previously been detected in water samples from the Oosterschelde, a large inlet on the North Sea, which is used for both recreational purposes and shellfish production. In 2006, oysters (Crassostrea gigas) from a noncommercial oyster bed in the Oosterschelde and oysters bought in Dutch fish shops were tested for the presence of pathogenic Vibrio species; in 2007 and 2008, oysters (C. gigas) and mussels (Mytilus edulis) from Oosterschelde production areas were examined. Total Vibrio numbers were related to water temperatures to study joint patterns. Vibrio was found in oysters and mussels from the production areas, and levels ranged from 6 to 622 most probable number (MPN) per g in oysters and 6 to 62 MPN/g in mussels. Vibrio levels in oysters from fish shops were 231 to >333 MPN/g, whereas levels in noncommercial oysters ranged from 231 to >2,398 MPN/g. About 80% of the isolated strains were V. alginolyticus, and approximately 10% were identified as V. parahaemolyticus. Vibrio counts in shellfish samples increased with increasing water temperature and declined when water temperatures dropped; Vibrio was not detected when water temperatures declined to <13.5 degrees C. Based on the obtained results and the known high V. parahaemolyticus dose (>10(4) cells per serving of oysters) required for infection, it is concluded that the risk of gastrointestinal infections with V. parahaemolyticus through consumption of shellfish from the Oosterschelde production sites is presumably low.

Sources of hepatitis E virus genotype 3 in The Netherlands.

Non-travel-related hepatitis E virus (HEV) genotype 3 infections in persons in the Netherlands may have a zoonotic, foodborne, or water-borne origin. Possible reservoirs for HEV transmission by water, food, and animals were studied. HEV genotype 3/open reading frame 2 sequences were detected in 53% of pig farms, 4% of wild boar feces, and 17% of surface water samples. HEV sequences grouped within 4 genotype 3 clusters, of which 1 is so far unique to the Netherlands. The 2 largest clusters contained 35% and 43% of the animal and environmental sequences and 75% and 6%, respectively, of human HEV sequences obtained from a study on Dutch hepatitis E patients. This finding suggests that infection risk may be also dependent on transmission routes other than the ones currently studied. Besides the route of exposure, virus characteristics may be an important determinant for HEV disease in humans.

The course of hepatitis E virus infection in pigs after contact-infection and intravenous inoculation.

BACKGROUND: Worldwide, hepatitis E virus (HEV) genotype 3 is observed in pigs and transmission to humans is implied. To be able to estimate public health risks from e.g. contact with pigs or consumption of pork products, the transmission routes and dynamics of infection should be identified. Hence, the course of HEV-infection in naturally infected pigs should be studied. RESULTS: To resemble natural transmission, 24 HEV-susceptible pigs were infected either by one-to-one exposure to intravenously inoculated pigs (C1-pigs; n = 10), by one-to-one exposure to contact-infected pigs (C2-pigs: n = 7; C3-pigs: n = 5) or due to an unknown non-intravenous infection route (one C2-pig and one C3-pig). The course of HEV-infection for contact-infected pigs was characterized by: faecal HEV RNA excretion that started at day 7 (95% confidence interval: 5-10) postexposure and lasted 23 (19-28) days; viremia that started after 13 (8-17) days of faecal HEV RNA excretion and lasted 11 (8-13) days; antibody development that was detected after 13 (10-16) days of faecal HEV RNA excretion. The time until onset of faecal HEV RNA excretion and onset of viremia was significantly shorter for iv-pigs compared to contact-infected pigs, whereas the duration of faecal HEV RNA excretion was significantly longer. At 28 days postinfection HEV RNA was detected less frequently in organs of contact-infected pigs compared to iv-pigs. For contact-infected pigs, HEV RNA was detected in 20 of 39 muscle samples that were proxies for pork at retail and in 4 of 7 urine samples. CONCLUSION: The course of infection differed between infection routes, suggesting that contact-infection could be a better model for natural transmission than iv inoculation. Urine and meat were identified as possible HEV-sources for pig-to-pig and pig-to-human HEV transmission.

Estimation of hepatitis E virus transmission among pigs due to contact-exposure.

Locally acquired hepatitis E in humans from industrialized countries has been repeatedly suggested to originate from pigs. Pigs may serve as a reservoir of hepatitis E virus (HEV) for humans when a typical infected pig causes on average more than one newly infected pig, a property that is expressed by the basic reproduction ratio R(0). In this study, R(0) for HEV transmission among pigs was estimated from chains of one-to-one transmission experiments in two blocks of five chains each. Per chain, susceptible first-generation contact pigs were contact-exposed to intravenously inoculated pigs, subsequently susceptible second-generation contact pigs were contact-exposed to infected first-generation contact pigs, and lastly, susceptible third-generation contact pigs were contact-exposed to infected second-generation contact pigs. Thus, in the second and third link of the chain, HEV-transmission due to contact with a contact-infected pig was observed. Transmission of HEV was monitored by reverse transcriptase polymerase chain reaction (RT-PCR) on individual faecal samples taken every two/three days. For susceptible pigs, the average period between exposure to an infectious pig and HEV excretion was six days (standard deviation: 4). The length of HEV-excretion (i.e. infectious period) was estimated at 49 days (95% confidence interval (CI): 17-141) for block 1 and 13 days (95% CI: 11-17) for block 2. The R0 for contact-exposure was estimated to be 8.8 (95% CI: 4-19), showing the potential of HEV to cause epidemics in populations of pigs.