Wim H. M. van der Poel

Virus hazards from food, water and other contaminated environments

Abstract Numerous viruses of human or animal origin can spread in the environment and infect people via water and food, mostly through ingestion and occasionally through skin contact. These viruses are released into the environment by various routes including water run‐offs and aerosols. Furthermore, zoonotic viruses may infect humans exposed to contaminated surface waters. Foodstuffs of animal origin can be contaminated, and their consumption may cause human infection if the viruses are not inactivated during food processing. Molecular epidemiology and surveillance of environmental samples are necessary to elucidate the public health hazards associated with exposure to environmental viruses. Whereas monitoring of viral nucleic acids by PCR methods is relatively straightforward and well documented, detection of infectious virus particles is technically more demanding and not always possible (e.g. human norovirus or hepatitis E virus). The human pathogenic viruses that are most relevant in this context are nonenveloped and belong to the families of the C aliciviridae, A denoviridae, H epeviridae, P icornaviridae and R eoviridae. Sampling methods and strategies, first‐choice detection methods and evaluation criteria are reviewed.

Cluster of Cases of Acute Hepatitis Associated with Hepatitis E Virus Infection Acquired in The Netherlands

Increasing evidence suggests that hepatitis E virus (HEV) infection may occur in developed countries and that swine may act as a reservoir. We report a cluster of 2 confirmed cases and 1 presumptive case of hepatitis associated with HEV. The typed strain from 1 case was related to HEV strains found in North America and Europe, and it was also related to a cluster of swine HEV strains found in The Netherlands. Our findings indicate that locally acquired HEV infections in industrialized countries may be overlooked. Routine testing for HEV infection in patients with acute hepatitis in The Netherlands should be considered before a diagnosis of autoimmune hepatitis is reached and steroid therapy is initiated.

Seroprevalence of Schmallenberg Virus Antibodies among Dairy Cattle, the Netherlands, Winter 2011–2012

Seroprevalence was highest in the eastern part of the country, bordering Germany, where the virus was first identified.

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

BackgroundWorldwide, 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.ResultsTo 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.ConclusionThe 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.

First isolation of hepatitis E virus genotype 4 in Europe through swine surveillance in the Netherlands and Belgium.

Hepatitis E virus (HEV) genotypes 3 and 4 are a cause of human hepatitis and swine are considered the main reservoir. To study the HEV prevalence and characterize circulating HEV strains, fecal samples from swine in the Netherlands and Belgium were tested by RT-PCR. HEV prevalence in swine was 7–15%. The Dutch strains were characterized as genotype 3, subgroups 3a, 3c and 3f, closely related to sequences found in humans and swine earlier. The HEV strains found in Belgium belonged to genotypes 3f and 4b. The HEV genotype 4 strain was the first ever reported in swine in Europe and an experimental infection in pigs was performed to isolate the virus. The genotype 4 strain readily infected piglets and caused fever and virus shedding. Since HEV4 infections have been reported to run a more severe clinical course in humans this observation may have public health implications.

Detection of Noroviruses in Foods: A Study on Virus Extraction Procedures in Foods Implicated in Outbreaks of Human Gastroenteritis

Disease outbreaks in which foods are epidemiologically implicated as the common source are frequently reported. Noroviruses and enteric hepatitis A viruses are among the most prevalent causative agents of foodborne diseases. However, the detection of these viruses in foods other than shellfish is often time-consuming and unsuccessful. In this study, three virus concentration methods were compared: polyethylene glycol (PEG) plus NaCl, ultracentrifugation, and ultrafiltration. Two RNA extraction methods, TRIzol and RNeasy Mini Kit (Qiagen), were compared for detection of viruses in whipped cream and lettuce (as representatives of the dairy and vegetable-fruit food groups, respectively). A seeding experiment with canine calicivirus was conducted to determine the efficiency of each virus extraction procedure. The PEG-NaCl-TRIzol method was most efficient for the detection of viruses in whipped cream and the ultracentrifugation-RNeasy-Mini Kit procedure was best for detection on lettuce. Based on the seeding experiments, food items implicated in norovirus-associated gastroenteritis outbreaks were subjected to the optimal procedure for a specific composition and matrix. No noroviruses were detected in the implicated food items, possibly because the concentration of virus on the food item was too low or because of the presence of inhibitory factors. For each food group, a specific procedure is optimal. Inhibitory factors should be controlled in these procedures because they influence virus detection in food.

Emerging group-A rotavirus and a nosocomial outbreak of diarrhoea

A P[6]G9 group-A rotavirus caused a protracted hospital outbreak of neonatal diarrhoea in The Netherlands. The outbreak lasted 5 months with 52 cases and an average attack rate of 40%, 46 cases were in an incubator section for neonates under 1 month of age. Rotavirus P161G9 was detected by RT-PCR in stool samples from the 31 cases tested. Emergence of this genotype in Europe may have implications for neonates lacking protective maternal antibodies and for the development of rotavirus vaccines.

Hepatitis E Virus in Pork Liver Sausage, France

We investigated viability of hepatitis E virus (HEV) identified in contaminated pork liver sausages obtained from France. HEV replication was demonstrated in 1 of 4 samples by using a 3-dimensional cell culture system. The risk for human infection with HEV by consumption of these sausages should be considered to be high.

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.

Development of a virus neutralisation test to detect antibodies against Schmallenberg virus and serological results in suspect and infected herds.

BackgroundAt the end of 2011, a new orthobunyavirus, tentatively named Schmallenberg virus (SBV), was discovered in Germany. This virus has since been associated with clinical signs of decreased milk production, watery diarrhoea and fever in dairy cows, and subsequently also with congenital malformations in calves, lambs and goat kids. In affected countries, initial surveillance for the infection was based on examination of malformed progeny. These suspicions were followed up by real-time reverse transcription polymerase chain reaction (RT-PCR) on brain tissue. For epidemiological purposes, a serological assay was, however, needed.ResultsA virus neutralisation test (VNT) was developed and optimized, and subsequently evaluated. This VNT has a specificity of >99% and the sensitivity is likely also very close to 100%. The assay is highly repeatable and reproducible. The final assay was used to test for antibodies in cows, ewes and does from herds known to be infected or suspected to be so. Targets for sampling in these herds were the mothers of malformed offspring. In herds with an RT-PCR confirmed SBV infection, more than 94% (190 out of 201) of the ewes and 99% (145 out of 146) of the cows were seropositive. In herds with suspicion of SBV infection based on birth of malformed offspring only (no or negative RT-PCR), more than 90% (231 out of 255) of the ewes and 95% (795 out of 834) of the cows were seropositive. In goats, on the other hand, only a low number of seropositives was found: overall 36.4%, being 16 out of 44 goats tested.ConclusionsGiven the characteristics of this VNT, it can be used at a relative high throughput for testing of animals for export, surveillance, screening and research purposes, but can also be used as a confirmation test for commercially available enzyme-linked immunosorbent assays (ELISA’s) and for (relative) quantification of antibodies.Suspicions of SBV infections that were confirmed by RT-PCR were almost always confirmed by serology in cows. Due to individual registration and identification of cows and calves, affected offspring could almost always be traced back to the mother. Ewes on the other hand were not always the mothers of affected lambs, but were in many cases herd mates with unaffected lambs. This indicated a high within-herd seroprevalence of antibodies against SBV.