W.H.M. van der Poel

'Schmallenberg virus' - a novel orthobunyavirus emerging in Europe

In 2011, a novel orthobunyavirus of the Simbu serogroup, the Schmallenberg virus (SBV), was discovered using a metagenomic approach. SBV caused a large epidemic in Europe in ruminants. As with related viruses such as Akabane virus, it appears to be transmitted by biting midges. Transplacental infection often results in the birth of malformed calves, lambs and goat kids. In more than 5000 farms in Germany, The Netherlands, Belgium, France, UK, Italy, Spain, Luxembourg, Denmark and Switzerland acute infections of adult ruminants or malformed SBV-positive offspring were detected, and high seroprevalences were seen in adult ruminants in the core regions in The Netherlands, Germany and Belgium. The discovery of SBV, the spread of the epidemic, the role of vectors, the impact on livestock, public health issues, SBV diagnosis and measures taken are described in this review. Lessons to be learned from the Schmallenberg virus epidemic and the consequences for future outbreaks are discussed.

Hepatitis E Virus RNA in Commercial Porcine Livers in The Netherlands

Human hepatitis E virus (HEV) infections by genotype 3 strains in industrialized countries are hypothesized to be caused by pigs. To examine this hypothesis, the potential health risks of transmission routes should be examined. Possible foodborne transmission was studied by quantifying the presence and infectivity of HEV in commercial porcine livers in The Netherlands. A comparison of four tissue disruption and seven RNA extraction methods revealed that mechanical disruption followed by silica-based RNA extraction gave the highest RNA yields and was therefore employed on commercial porcine livers. Four (6.5%) of 62 porcine livers were HEV RNA positive by reverse transcriptase PCR and Southern blot hybridization. Each positive liver was estimated to contain approximately 65 PCR-detectable units per g. Sequences were obtained for three of four positive livers and classified as HEV genotype 3. Ninety-three percent similarity to Dutch human HEV sequences and 97% similarity to Dutch swine HEV sequences were observed. To determine whether positive livers contained infectious HEV particles, extracts from livers with known HEV RNA sequences were inoculated intravenously in pigs. Two control pigs were included: one was inoculated with a high dose known to result in infection (10(4) PCR-detectable units of HEV RNA), and the other was inoculated with a lower concentration of virus that equaled the concentration of PCR-detectable units in commercial livers ( approximately 20 PCR-detectable units). Infection was observed in the high-dose control, but not in other pigs, suggesting a dose-dependent response in pigs. Hence, the implications of HEV RNA in commercial porcine livers in The Netherlands are unknown. However, HEV RNA is present in commercial porcine livers, and sufficient heating of porcine livers before consumption as precautionary measure is recommended.

Bayesian estimation of hepatitis E virus seroprevalence for populations with different exposure levels to swine in The Netherlands.

Hepatitis E virus (HEV) is ubiquitous in pigs worldwide and may be zoonotic. Previous HEV seroprevalence estimates for groups of people working with swine were higher than for control groups. However, discordance among results of anti-HEV assays means that true seroprevalence estimates, i.e. seroprevalence due to previous exposure to HEV, depends on choice of seroassay. We tested blood samples from three subpopulations (49 swine veterinarians, 153 non-swine veterinarians and 644 randomly selected individuals from the general population) with one IgM and two IgG ELISAs, and subsets with IgG and/or IgM Western blots. A Bayesian stochastical model was used to combine results of all assays. The model accounted for imperfection of each assay by estimating sensitivity and specificity, and accounted for dependence between serological assays. As expected, discordance among assay results occurred. Applying the model yielded seroprevalence estimates of approximately 11% for swine veterinarians,approximately 6% for non-swine veterinarians and approximately 2% for the general population. By combining the results of five serological assays in a Bayesian stochastical model we confirmed that exposure to swine or their environment was associated with elevated HEV seroprevalence.

A one-year intensified study of outbreaks of gastroenteritis in The Netherlands

In 2002, in The Netherlands a national study of gastroenteritis outbreaks was performed. Epidemiological information was collected by the Public Health Services (PHS) and the Food Inspection Services (FIS) using standardized questionnaires. Stool samples were collected for diagnostic testing. For foodborne outbreaks, food samples were taken. In total, 281 gastroenteritis outbreaks were included, mainly from nursing homes and homes for the elderly (57%), restaurants (11%), hospitals (9%) and day-care centres (7%). Direct person-to-person spread was the predominant transmission route in all settings (overall 78%), except for restaurant outbreaks where food was suspected in almost 90% (overall in 21% of outbreaks). The most common pathogen was norovirus (54%), followed by Salmonella spp. (4%), rotavirus group A (2%), Campylobacter spp. (1%) and only incidentally others. In conclusion, most outbreaks were reported from health and residential institutions, with norovirus as the dominant agent. Control should aim at reducing person-to-person spread. In foodborne outbreaks norovirus was common, due to contamination of food by food handlers. Salmonella, as the second foodborne pathogen, was mainly associated with raw shell eggs. These results stress the continuous need for food safety education, complementary to governmental regulation.

Schmallenberg Virus in Culicoides Biting Midges in the Netherlands in 2012

A total of 130 pools of Culicoides biting midges collected between May and September 2012 in the Netherlands were assayed for Schmallenberg virus (SBV). The Culicoides midges were caught in the same area as where in 2011 a high proportion of Culicoides pools tested positive for SBV, in majority with a high viral load (Ct values between 20 and 30). Two of a total of 42 pools comprising 50 midges/pool of the Obsoletus complex from the 2012 collection tested weak positive (Ct values: 34.96 and 37.66), indicating a relatively low viral load. On an individual midge level, the proportion of SBV-infected Culicoides of the Obsoletus complex caught in the same area and in a comparable period of the year was significantly lower in 2012 (0.1% = 1 per 1050 tested) compared with 2011 (0.56% = 13 per 2300 tested).

Outbreak of Porcine Epidemic Diarrhea Virus in Portugal, 2015

Summary An outbreak of porcine epidemic diarrhea virus (PEDV) in the South of Portugal in January 2015 and the spread of PEDV northwards in the territory are described. Comparative analysis of the amplified sequences showed a very high (99.0%) identity with the PEDV variant most recently reported in the United States and also show complete (100%) identity to the strains recently reported in Germany, supporting the hypothesis that a unique strain is currently circulating in Europe. The origin of this PEDV variant still needs to be elucidated and further studies in the remaining European countries may contribute to the knowledge.

Age-dependent differences in the pathogenesis of bovine respiratory syncytial virus infections related to the development of natural immunocompetence

The severity of respiratory syncytial virus (RSV) infections appears to differ with age in both humans and bovines. A primary RSV infection in naïve infants and in young calves runs a more severe course when they are 1-6 months old than in their first month of life. The relative lack of clinical signs in the first month of age may be due to high levels of maternally derived neutralizing antibodies or low exposure to infectious virus. This study examined whether age-dependent differences in the pathogenesis of bovine RSV (bRSV) between neonatal and young calves may be due to differences in age-dependent immunocompetence. To study the effect of age and immune parameters on bRSV disease in neonatal and young calves, neonatal (1-day-old) calves without maternally derived antibodies were infected experimentally with bRSV and the severity of disease and immune responses were evaluated in comparison with disease in similar 6-week-old infected calves. Neonatal calves had more extensive virus replication and lung consolidation, but lower pro-inflammatory [in particular tumour necrosis factor alpha (TNF-α)] responses, specific humoral immune responses, lung neutrophilic infiltration and clinical signs of disease than 6-week-old calves. The lack of correlation between virus replication and clinical signs suggests an important role of pro-inflammatory cytokines, in particular TNF-α, in the disease. The capacity to produce pro-inflammatory TNF-α appeared to increase with age, and may explain the age-dependent differences in RSV pathogenesis.

Replication of hepatitis E virus in three-dimensional cell culture.

Hepatitis E is an acute, viral hepatitis epidemic in developing regions, but which is detected with increasing frequency in sporadic form in developed regions. Pigs and possibly some other mammals are considered reservoirs of zoonotic infection with hepatitis E virus (HEV). However, whilst the relative significance of potential transmission routes from pigs to people is still unclear, the consumption of raw or undercooked pig meat has been implicated as a source of HEV infection. The lack of information about HEV zoonotic transmission is due in part to the difficulties of in vitro propagation of HEV. The Rotating Wall Vessel (RVW) has been described as a useful tool for the culture of cell lines in a 3-dimensional (3D) configuration. The aim of this work was to develop a 3D cell culture system for HEV to facilitate studies into the viability of virions contaminating pig tissues. This study, demonstrated that HEV can replicate efficiently in the RWV in human hepatoblastoma PLC/PRF/5 cells for up to 5 months not only by real time RT-PCR but also by detection of complete virions via electron microscopy. Furthermore, the replication of HEV progeny was observed by detecting HEV RNA by RT-PCR. The progeny were able to infect fresh 3D cultures, showing that this method is able to produce infectious hepatitis E virions.

Molecular Epidemiology of Bat Lyssaviruses in Europe

Bat rabies cases in Europe are principally attributed to two lyssaviruses, namely European bat lyssavirus type 1 (EBLV‐1) and European bat lyssavirus type 2 (EBLV‐2). Between 1977 and 2011, 961 cases of bat rabies were reported to Rabies Bulletin Europe, with the vast majority (>97%) being attributed to EBLV‐1. There have been 25 suspected cases of EBLV‐2, of which 22 have been confirmed. In addition, two single isolations of unique lyssaviruses from European insectivorous bats were reported in south‐west Russia in 2002 (West Caucasian bat virus) and in Germany in 2010 (Bokeloh bat lyssavirus). In this review, we present phylogenetic analyses of the EBLV‐1 and EBLV‐2 using partial nucleoprotein (N) gene sequences. In particular, we have analysed all EBLV‐2 cases for which viral sequences (N gene, 400 nucleotides) are available (n = 21). Oropharyngeal swabs collected from two healthy Myotis daubentonii during active surveillance programmes in Scotland and Switzerland also yielded viral RNA (EBLV‐2). Despite the relatively low number of EBLV‐2 cases, a surprisingly large amount of anomalous data has been published in the scientific literature and Genbank, which we have collated and clarified. For both viruses, geographical relationships are clearly defined on the phylogenetic analysis. Whilst there is no clear chronological clustering for either virus, there is some evidence for host specific relationships, particularly for EBLV‐1 where more host variation has been observed. Further genomic regions must be studied, in particular for EBLV‐1 isolates from Spain and the EBLV‐2 isolates to provide support for the existence of sublineages.

Detection and characterization of hepatitis e virus in domestic pigs of different ages in Portugal.

This study represents the primary hepatitis E virus (HEV) surveillance in domestic pigs in Portugal, five pig farms were investigated in 5 different Portuguese regions, ten faecal samples were collected at four different stages of the production. All faecal samples were tested for hepatitis E virus by real‐time RT‐PCR. At least one sample from each farms of all age groups tested positive for HEV. The prevalence in the pig herds varied from 10% to 30% and the mean prevalence was 32% in weaners, 20% in growers, 32% in fatteners and 4% in adult dry sows. Phylogenetic analysis of the detected HEV sequences indicated that the circulating virus strains belong under the genotype 3.