Molecular Epidemiology and Evolution of Influenza Viruses Circulating within European Swine between 2009 and 2013.

UNLABELLED: The emergence in humans of the A(H1N1)pdm09 influenza virus, a complex reassortant virus of swine origin, highlighted the importance of worldwide influenza virus surveillance in swine. To date, large-scale surveillance studies have been reported for southern China and North America, but such data have not yet been described for Europe. We report the first large-scale genomic characterization of 290 swine influenza viruses collected from 14 European countries between 2009 and 2013. A total of 23 distinct genotypes were identified, with the 7 most common comprising 82% of the incidence. Contrasting epidemiological dynamics were observed for two of these genotypes, H1huN2 and H3N2, with the former showing multiple long-lived geographically isolated lineages, while the latter had short-lived geographically diffuse lineages. At least 32 human-swine transmission events have resulted in A(H1N1)pdm09 becoming established at a mean frequency of 8% across European countries. Notably, swine in the United Kingdom have largely had a replacement of the endemic Eurasian avian virus-like ("avian-like") genotypes with A(H1N1)pdm09-derived genotypes. The high number of reassortant genotypes observed in European swine, combined with the identification of a genotype similar to the A(H3N2)v genotype in North America, underlines the importance of continued swine surveillance in Europe for the purposes of maintaining public health. This report further reveals that the emergences and drivers of virus evolution in swine differ at the global level. IMPORTANCE: The influenza A(H1N1)pdm09 virus contains a reassortant genome with segments derived from separate virus lineages that evolved in different regions of the world. In particular, its neuraminidase and matrix segments were derived from the Eurasian avian virus-like ("avian-like") lineage that emerged in European swine in the 1970s. However, while large-scale genomic characterization of swine has been reported for southern China and North America, no equivalent study has yet been reported for Europe. Surveillance of swine herds across Europe between 2009 and 2013 revealed that the A(H1N1)pdm09 virus is established in European swine, increasing the number of circulating lineages in the region and increasing the possibility of the emergence of a genotype with human pandemic potential. It also has implications for veterinary health, making prevention through vaccination more challenging. The identification of a genotype similar to the A(H3N2)v genotype, causing zoonoses at North American agricultural fairs, underlines the importance of continued genomic characterization in European swine.

Pathway analysis in blood cells of pigs infected with classical swine fever virus: comparison of pigs that develop a chronic form of infection or recover.

Infection of pigs with CSFV can lead to either acute disease, resulting in death or recovery, or chronic disease. The mechanisms by which CSFV manipulates the pig's first line of defence to establish a chronic infection are poorly understood. Therefore, pigs were infected with moderately virulent CSFV, and whole blood was collected on a regular basis during a period of 18 days. Using whole-genome microarrays, time-dependent changes in gene expression were recorded in blood cells of chronically diseased pigs and pigs that recovered. Bioinformatics analysis of regulated genes indicated that different immunological pathways were regulated in chronically diseased pigs compared to recovered pigs. In recovered pigs, antiviral defence mechanisms were rapidly activated, whereas in chronically diseased pigs, several genes with the potential to inhibit NF-κB- and IRF3/7-mediated transcription of type I interferons were up-regulated. Compared to recovered pigs, chronically diseased pigs failed to activate NK or cytotoxic T-cell pathways, and they showed decreased gene activity in antigen-presenting monocytes/macrophages. Remarkably, in chronically diseased pigs, genes related to the human autoimmune disease systemic lupus erythematosus (SLE) were up-regulated during the whole period of 18 days. CSFV pathology in kidney and skin resembles that of SLE. Furthermore, enzymes involved in the degradation of 1,25-dihydroxyvitamin D3 and of tryptophan to kynurenines were expressed at different levels in chronically diseased and recovered pigs. Both of these chemical processes may affect the functions of T helper/regulatory cells that are crucial for tempering the inflammatory response after a viral infection.

Seroprevalence of Schmallenberg virus antibodies among dairy cattle, the Netherlands, winter 2011-2012.

Infections with Schmallenberg virus (SBV) are associated with congenital malformations in ruminants. Because reporting of suspected cases only could underestimate the true rate of infection, we conducted a seroprevalence study in the Netherlands to detect past exposure to SBV among dairy cattle. A total of 1,123 serum samples collected from cattle during November 2011-January 2012 were tested for antibodies against SBV by using a virus neutralization test; seroprevalence was 72.5%. Seroprevalence was significantly higher in the central-eastern part of the Netherlands than in the northern and southern regions (p<0.001). In addition, high (70%-100%) within-herd seroprevalence was observed in 2 SBV-infected dairy herds and 2 SBV-infected sheep herds. No significant differences were found in age-specific prevalence of antibodies against SBV, which is an indication that SBV is newly arrived in the country.

Dynamics of virus excretion via different routes in pigs experimentally infected with classical swine fever virus strains of high, moderate or low virulence.

Classical swine fever virus (CSFV) is transmitted via secretions and excretions of infected pigs. The efficiency and speed of the transmission depends on a multitude of parameters, like quantities of virus excreted by infected pigs. This study provides quantitative data on excretion of CSFV over time from pigs infected with a highly, moderately or low virulent strain. For each strain, five individually housed pigs were infected. Virus excretion was quantified in oropharyngeal fluid, saliva, nasal fluid, lacrimal fluid, faeces, urine and skin scraping by virus titration and quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRRT-PCR). Infectious virus was excreted in all secretions and excretions of pigs infected with the highly and moderately virulent strain, while excretion from pigs infected with the low virulent strain was mostly restricted to the oronasal route. Pigs infected with the highly virulent strain excreted significantly more virus in all their secretions and excretions over the entire infectious period than pigs infected with the moderately or low virulent strains. An exception were the pigs that developed the chronic form of infection after inoculation with the moderately virulent strain. During the entire infectious period, they excreted the largest amounts of virus via most secretions and excretions, as they excreted virus continuously and for a long duration. This study highlights the crucial role chronically infected pigs may play in the transmission of CSFV. Furthermore, it demonstrates the importance of discriminating between strains and the clinical appearance of infection when using excretion data for modelling.

Quantification of classical swine fever virus in aerosols originating from pigs infected with strains of high, moderate or low virulence.

During epidemics of classical swine fever (CSF), the route of virus introduction into a farm is often unclear. One of the suggested routes is via the air. Under experimental conditions, airborne transmission over a short distance seems possible, but analysis of outbreak data is still inconclusive. For a better understanding of the role of airborne transmission, quantitative information is needed on concentrations of virus emitted by infected pigs. This was studied in four groups of 10 pigs in which three pigs were inoculated with either a low virulent strain (Zoelen), a low or high dose of a moderately virulent strain (Paderborn), or a highly virulent strain (Brescia). The other seven pigs in each group served as contact pigs. At several moments after infection, air samples were obtained using gelatine filters. Infectious virus and viral RNA were detected in the air of rooms housing the pigs infected with the moderately and highly virulent strains with titres of 10(1.2) to 10(3.0)TCID(50)/m(3) of infectious virus, and 10(1.6) to 10(3.8)TCID(50)equiv./m(3) of viral RNA. It was observed that the higher the dose or virulence of the virus strain used for inoculation of the pigs, the sooner virus could be detected in the air samples. This is the first study describing the quantification of (infectious) CSFV in air samples of rooms housing infected pigs, enabling to quantify the contribution of individual infected pigs to virus concentrations in aerosols. This can be used as input for quantitative models of airborne spread over large distances.

Survival of classical swine fever virus at various temperatures in faeces and urine derived from experimentally infected pigs.

Indirect transmission of classical swine fever virus (CSFV) can occur through contact with mechanical vectors, like clothing and footwear or transport vehicles, contaminated with the secretions or excretions of infected pigs. A prerequisite for indirect transmission is survival of the virus on the mechanical vector. Consequently, to obtain more insight into these transmission routes, it is important to know how long the virus remains viable outside the host. In this study we examined the survival of classical swine fever virus in faeces and urine derived from pigs intranasally inoculated with a highly or moderately virulent CSFV strain. Faeces and urine were collected between days 5 and 36 post-inoculation, and stored at 5, 12, 20, and 30 degrees C. Next, the virus titres were determined in the samples by virus titration, and a random selection of these samples was also analyzed by quantitative real-time reverse transcription polymerase chain reaction (qRRT-PCR) to determine the viral RNA decay. Survival curves were generated, and it was shown that the inactivation rate was inversely related to the storage temperature. Average half-life values were between 2 and 4 days at 5 degrees C, and between 1 and 3h at 30 degrees C. Significant differences were observed in survival between virus strains in faeces, however, not in urine. The reduction in viral RNA during the entire study period was limited. This study provided detailed information on survival of CSFV in excretions of infected pigs, which can be used to improve control measures or risk-analysis models.

Detection and quantification of classical swine fever virus in air samples originating from infected pigs and experimentally produced aerosols.

During epidemics of classical swine fever (CSF), neighbourhood infections occurred where none of the 'traditional' routes of transmission like direct animal contact, swill feeding, transport contact or transmission by people could be identified. A hypothesized route of virus introduction for these herds was airborne transmission. In order to better understand this possible transmission route, we developed a method to detect and quantify classical swine fever virus (CSFV) in air samples using gelatine filters. The air samples were collected from CSFV-infected pigs after experimental aerosolization of the virus. Furthermore, we studied the viability of the virus with time in aerosolized state. Three strains of CSFV were aerosolized in an empty isolator and air samples were taken at different time intervals. The virus remained infective in aerosolized state for at least 30 min with half-life time values ranging from 4.5 to 15 min. During animal experiments, concentrations of 10(0.3)-10(1.6)TCID(50)/m(3) CSFV were detected in air samples originating from the air of the pig cages and 10(0.4)-10(4.0)TCID(50)/m(3) from the expired air of infected animals. This is the first study describing the isolation and quantification of CSFV from air samples originating from infected pigs and their cages, supporting previous findings that airborne transmission of CSF is feasible.

Influence of Age and Dose of African Swine Fever Virus Infections on Clinical Outcome and Blood Parameters in Pigs.

African swine fever (ASF) is a fatal disease for domestic pigs, leading to serious economic losses in countries where ASF is endemic. Despite extensive research, efficient vaccines against ASF are lacking. Since peripheral blood cells are important mediators for vaccines, we study the impact of ASF on blood parameters in pigs with different ages and infected with different doses of ASF virus. Four different groups were studied: (1) 12 weeks of age/low virus dose; (2) 12 weeks of age/high virus dose; (3) 18 weeks of age/low virus dose; and (4) 18 weeks of age/high virus dose. By varying in age and/or ASFV inoculation dose, we monitor blood parameters during different degrees of disease. Thirty percent of the pigs survived the infection with a moderately virulent strain of African swine fever virus (ASFV). Animals that did survive infection were generally older, independent from the inoculation dose used. A firm reduction in many different cell types at 3-5 days postinfection (DPI) was accompanied by an increase in body temperature, followed by clinical signs and mortality from day 6 PI. While blood parameters generally normalized in survivors, γδ T cells and IL-10 levels could be related to mortality. These conclusions should be considered in new approaches for protection against ASF.

Pre-screening of crude peptides in a serological bead-based suspension array.

Most serological assays detect antibody responses in biological samples through affinity of serum antibodies for antigens provided in the assay. Certain antigens, however, may be difficult to produce and/or may contain unwanted epitopes. In these cases, a practical alternative may be the use of peptides as representatives for specific epitopes. Peptides can be obtained after purification in large quantities for a modest price, but screening of a large set of peptides during development may be relatively expensive. To cut costs of screening peptides for a new serological assay, the concept was investigated of using cheap non-purified (crude) peptides instead of purified peptides. Peptides were selected that represent three well-described linear epitopes of viral proteins: VP2 of canine parvovirus (CPV), gp41 of human immunodeficiency virus (HIV) and E2 of classical swine fever virus (CSFV). Crude and purified biotinylated peptides with either a short or long spacer between the biotin and the epitope were used to test their capability to bind antibodies in a bead-based suspension array. The results show that, in a bead-based suspension array, crude peptides can function as antigen for specific monoclonal antibodies, and that the acquired signals are less than with purified peptides. CSFV-derived crude peptides were also able to detect specific antibodies in swine serum, indicating the applicability of crude peptides for pre-screening large numbers of different peptides during the development of serological peptide-based assays.

Towards a peptide-based suspension array for the detection of pestivirus antibodies in swine.

Classical swine fever (CSF) is a highly contagious and lethal disease in swine. Serological tests for the diagnosis of CSF need not only to detect antibodies against CSFV, but also need to differentiate these from antibodies against other pestiviruses. To investigate the possibilities of specific peptide-based serology, various synthetic peptides that represent a well-described linear epitope of the CSFV E2 protein (TAVSPTTLR) were used to test the viability of a peptide-based suspension array for the detection of antibodies against pestiviruses in swine. The results show that N-terminally biotinylated peptides can bind to avidin conjugated beads, and function in detection of the corresponding monoclonal antibody WH303. There are indications that the length of the spacer between epitope and biotin affect the efficiency of the peptide-antibody interaction. A protocol was established that enables probing for antibodies in porcine sera, where neutravidin-blocking of serum and the use of empty control beads for normalization was crucial. With a set of porcine sera with antibodies against various pestiviruses, the proof of concept of a peptide-based suspension array for specific detection of antibodies against pestiviruses in porcine sera was demonstrated.

High turnover drives prolonged persistence of influenza in managed pig herds.

Pigs have long been hypothesized to play a central role in the emergence of novel human influenza A virus (IAV) strains, by serving as mixing vessels for mammalian and avian variants. However, the key issue of viral persistence in swine populations at different scales is ill understood. We address this gap using epidemiological models calibrated against seroprevalence data from Dutch finishing pigs to estimate the 'critical herd size' (CHS) for IAV persistence. We then examine the viral phylogenetic evidence for persistence by comparing human and swine IAV. Models suggest a CHS of approximately 3000 pigs above which influenza was likely to persist, i.e. orders of magnitude lower than persistence thresholds for IAV and other acute viruses in humans. At national and regional scales, we found much stronger empirical signatures of prolonged persistence of IAV in swine compared with human populations. These striking levels of persistence in small populations are driven by the high recruitment rate of susceptible piglets, and have significant implications for management of swine and for overall patterns of genetic diversity of IAV.

Quantification of different classical swine fever virus transmission routes within a single compartment.

During outbreaks of classical swine fever (CSF), CSF virus (CSFV) can be transmitted via different routes. Understanding these transmission routes is crucial in preventing the unlimited spread of the virus in a naïve population, and the subsequent eradication of the virus from that population. The objectives of the present study were to quantify virus transmission within a compartment, differentiating between transmission within a pen, transmission between pens via contact through (open) pen partitions, and transmission via the air. Furthermore, the possible contribution of each of these routes to infection of individual pigs was quantified. A CSFV outbreak was mimicked in a compartment housing 24 pigs in six different pens. Two pigs in one pen were inoculated with the moderately virulent Paderborn strain, and virus transmission to other pigs was followed in time. Virus transmission rates for transmission via the air (ß of 0.33 (0.14-0.64) per day) and transmission between adjacent pens (ß of 0.30 (0-0.88) per day) were comparable, but significantly lower than for virus transmission within a pen (ß of 6.1 (0.86-18) per day). The route via the air created new focal points of infection, from which virus transmission continued through other routes. This shows that, at least within a compartment, transmission via the air is expected to play a relevant role in the fast spread of the virus after an initial slow start. This will have consequences for efficacy of intervention measures, including vaccination during an outbreak.

No evidence of African swine fever virus replication in hard ticks.

African swine fever (ASF) is caused by African swine fever virus (ASFV), a tick-borne DNA virus. Soft ticks of the genus Ornithodoros are the only biological vectors of ASFV recognized so far. Although other hard ticks have been tested for vector competence, two commonly found tick species in Europe, Ixodes ricinus and Dermacentor reticulatus, have not been assessed for their vector competence for ASFV. In this study, we aimed to determine whether virus replication can occur in any of these two hard tick species (I. ricinus and/or D. reticulatus), in comparison with O. moubata (the confirmed vector), after feeding them blood containing different ASFV isolates using an improved in vitro system. DNA quantities of ASFV in these infected hard ticks were measured systematically, for 6 weeks in I. ricinus, and up to 8 weeks in D. reticulatus, and the results were compared to those obtained from O. moubata. There was evidence of virus replication in the O. moubata ticks. However, there was no evidence of virus replication in I. ricinus or D. reticulatus, even though viral DNA could be detected for up to 8 weeks after feeding in some cases. This study presents the first results on the possible vector competence of European hard (ixodid) ticks for ASFV, in a validated in vitro feeding setup. In conclusion, given the lack of evidence for virus replication under in vitro conditions, D. reticulatus and I. ricinus are unlikely to be relevant biological vectors of ASFV.

European surveillance network for influenza in pigs: surveillance programs, diagnostic tools and Swine influenza virus subtypes identified in 14 European countries from 2010 to 2013.

Swine influenza causes concern for global veterinary and public health officials. In continuing two previous networks that initiated the surveillance of swine influenza viruses (SIVs) circulating in European pigs between 2001 and 2008, a third European Surveillance Network for Influenza in Pigs (ESNIP3, 2010-2013) aimed to expand widely the knowledge of the epidemiology of European SIVs. ESNIP3 stimulated programs of harmonized SIV surveillance in European countries and supported the coordination of appropriate diagnostic tools and subtyping methods. Thus, an extensive virological monitoring, mainly conducted through passive surveillance programs, resulted in the examination of more than 9 000 herds in 17 countries. Influenza A viruses were detected in 31% of herds examined from which 1887 viruses were preliminary characterized. The dominating subtypes were the three European enzootic SIVs: avian-like swine H1N1 (53.6%), human-like reassortant swine H1N2 (13%) and human-like reassortant swine H3N2 (9.1%), as well as pandemic A/H1N1 2009 (H1N1pdm) virus (10.3%). Viruses from these four lineages co-circulated in several countries but with very different relative levels of incidence. For instance, the H3N2 subtype was not detected at all in some geographic areas whereas it was still prevalent in other parts of Europe. Interestingly, H3N2-free areas were those that exhibited highest frequencies of circulating H1N2 viruses. H1N1pdm viruses were isolated at an increasing incidence in some countries from 2010 to 2013, indicating that this subtype has become established in the European pig population. Finally, 13.9% of the viruses represented reassortants between these four lineages, especially between previous enzootic SIVs and H1N1pdm. These novel viruses were detected at the same time in several countries, with increasing prevalence. Some of them might become established in pig herds, causing implications for zoonotic infections.

Identification and characterization of mimotopes of classical swine fever virus E2 glycoprotein using specific anti-E2 monoclonal antibodies.

Classical swine fever virus (CSFV) shares high nucleic acid and amino acid sequence homology with the other members of the pestivirus genus, namely bovine viral diarrhea virus and border disease virus. All three viruses are able to infect swine and generate cross reactive antibodies, which is problematic during differential diagnosis for classical swine fever (CSF). Toward the development of a new generation of CSF specific diagnostic tools, monoclonal antibodies specific for CSFV were mapped using phage display technology. Six mimotopes were identified, some of which were found to be antigenic and/or specific for CSF when used as coating antigens in an ELISA for the detection of CSF antibodies in swine serum. Two mimotopes in particular termed V2-2 and V7-1 recognized numerous strains of CSF antisera and bound fewer BVD and BD antisera compared to a commercial CSF antibody ELISA. These two mimotopes may be useful to the pestivirus field in the development of a highly specific CSF antibody ELISA as well as in the development of other potential diagnostic technologies.

Classical swine fever and avian influenza epidemics: lessons learned.

Experience with recent large-scale epidemics of Classical Swine Fever and Avian Influenza--among others in the Netherlands--have teached us several lessons that should prepare us better for future outbreaks. Among others, improving early detection of outbreaks--by using syndrome surveillance systems--is a key factor, in which farmers and veterinary practitioners have an imminent role. A major step in this respect is facilitation of the use of exclusion diagnostics without closing down the farm in clinical situations with non-specific clinical signs observed in sick animals. The hesitance of farmers and veterinary practitioners to report a suspect clinical situation on a livestock farm and how to facilitate that process is another major issue. Furthermore, the importance of communication between the field and the laboratory with respect to post mortem examination will be highlighted, and the need for outbreak simulation exercises with neighbouring countries in order to be better prepared, will be discussed.