Comparison of circulation patterns of mumps virus in the Netherlands and Spain (2015-2020).

Background: Mumps is a viral infection mainly characterized by inflammation of the parotid glands. Despite of vaccination programs, infections among fully vaccinated populations were reported. The World Health Organization (WHO) recommends molecular surveillance of mumps based on sequencing of the small hydrophobic (SH) gene. The use of hypervariable non-coding regions (NCR) as additional molecular markers was proposed in multiple studies. Circulation of mumps virus (MuV) genotypes and variants in different European countries were described in the literature. From 2010 to 2020, mumps outbreaks caused by genotype G were described. However, this issue has not been analyzed from a wider geographical perspective. In the present study, sequence data from MuV detected in Spain and in The Netherlands during a period of 5 years (2015- March 2020) were analyzed to gain insights in the spatiotemporal spread of MuV at a larger geographical scale than in previous local studies. Methods: A total of 1,121 SH and 262 NCR between the Matrix and Fusion protein genes (MF-NCR) sequences from both countries were included in this study. Analysis of SH revealed 106 different haplotypes (set of identical sequences). Results: Of them, seven showing extensive circulation were considered variants. All seven were detected in both countries in coincident temporal periods. A single MF-NCR haplotype was detected in 156 sequences (59.3% of total), and was shared by five of the seven SH variants, as well as three minor MF-NCR haplotypes. All SH variants and MF-NCR haplotypes shared by both countries were detected first in Spain. Discussion: Our results suggest a transmission way from south to north Europe. The higher incidence rate of mumps in Spain in spite of similar immunization coverage in both countries, could be associated with higher risk of MuV exportation. In conclusion, the present study provided novel insights into the circulation of MuV variants and haplotypes beyond the borders of single countries. In fact, the use of MF-NCR molecular tool allowed to reveal MuV transmission flows between The Netherlands and Spain. Similar studies including other (European) countries are needed to provide a broader view of the data presented in this study.

Phylodynamics of Highly Pathogenic Avian Influenza A(H5N1) Virus Circulating in Indonesian Poultry.

After its first detection in 1996, the highly pathogenic avian influenza A(H5Nx) virus has spread extensively worldwide. HPAIv A(H5N1) was first detected in Indonesia in 2003 and has been endemic in poultry in this country ever since. However, Indonesia has limited information related to the phylodynamics of HPAIv A(H5N1) in poultry. The present study aimed to increase the understanding of the evolution and temporal dynamics of HPAIv H5N1 in Indonesian poultry between 2003 and 2016. To this end, HPAIv A(H5N1) hemagglutinin sequences of viruses collected from 2003 to 2016 were analyzed using Bayesian evolutionary analysis sampling trees. Results indicated that the common ancestor of Indonesian poultry HPAIv H5N1 arose approximately five years after the common ancestor worldwide of HPAI A(H5Nx). In addition, this study indicated that only two introductions of HPAIv A(H5N1) occurred, after which these viruses continued to evolve due to extensive spread among poultry. Furthermore, this study revealed the divergence of H5N1 clade 2.3.2.1c from H5N1 clade 2.3.2.1b. Both clades 2.3.2.1c and 2.3.2.1b share a common ancestor, clade 1, suggesting that clade 2.3.2.1 originated and diverged from China and other Asian countries. Since there was limited sequence and surveillance data for the HPAIv A(H5N1) from wild birds in Indonesia, the exact role of wild birds in the spread of HPAIv in Indonesia is currently unknown. The evolutionary dynamics of the Indonesian HPAIv A(H5N1) highlight the importance of continuing and improved genomic surveillance and adequate control measures in the different regions of both the poultry and wild birds. Spatial genomic surveillance is useful to take adequate control measures. Therefore, it will help to prevent the future evolution of HPAI A(H5N1) and pandemic threats.

Outbreaks of mumps genotype G viruses in the Netherlands between October 2019 and March 2020: clusters associated with multiple introductions.

BACKGROUND: From October 2019-March 2020, several clusters of mumps cases were identified in the Netherlands. Our objective was to describe cluster-associated mumps virus transmission using epidemiological and molecular information in order to help future mumps outbreak investigation and control efforts. METHODS: An epidemiological cluster includes ≥ 2 mumps cases with at least an epidemiological-link to a laboratory-confirmed mumps case. A molecular group includes ≥ 2 mumps cases with identical mumps virus sequences. Cases with symptom onset date between 1 October 2019 and 31 March 2020 reported through the National Notifiable Diseases Surveillance System were included. We described epidemiological and clinical characteristics of mumps cases. Sequence data was obtained from selected regions of mumps virus genomes (2270 nucleotides). Associations between epidemiological and molecular information were investigated. RESULTS: In total, 102 mumps cases were notified (90% laboratory-confirmed, 10% epidemiologically-linked). 71 out of 102 cases were identified as part of an epidemiological cluster and/or molecular group. Twenty-one (30%) of 71 cases were identified solely from epidemiological information, 25 (35%) solely from molecular surveillance, and 25 (35%) using both. Fourteen epidemiological clusters were identified containing a total of 46 (range: 2-12, median: 3) cases. Complete sequence data was obtained from 50 mumps genotype G viruses. Twelve molecular groups were identified containing 43 (range: 2-13) cases, dispersed geographically and timewise. Combined information grouped seven epidemiological clusters into two distinct molecular groups. The first lasting for 14 weeks, the other for 6. Additionally, one molecular group was detected, linked by geography and time but without an epidemiological-link. CONCLUSIONS: Combined epidemiological and molecular information indicated ongoing mumps virus transmission from multiple introductions for extended time periods. Sequence analysis provided valuable insights into epidemiological clustering. If combined information is available in a timely manner, this would improve outbreak detection, generate further insight into mumps transmission, and guide necessary control measures.

Genetic Analysis Reveals Differences in CD8+ T Cell Epitope Regions That May Impact Cross-Reactivity of Vaccine-Induced T Cells against Wild-Type Mumps Viruses.

Nowadays, mumps is re-emerging in highly vaccinated populations. Waning of vaccine-induced immunity plays a role, but antigenic differences between vaccine and mumps outbreak strains could also contribute to reduced vaccine effectiveness. CD8+ T cells play a critical role in immunity to viruses. However, limited data are available about sequence variability in CD8+ T cell epitope regions of mumps virus (MuV) proteins. Recently, the first set of naturally presented human leukocyte antigen Class I (HLA-I) epitopes of MuV was identified by us. In the present study, sequences of 40 CD8+ T cell epitope candidates, including previously and newly identified, obtained from Jeryl-Lynn mumps vaccine strains were compared with genomes from 462 circulating MuV strains. In 31 epitope candidates (78%) amino acid differences were detected, and in 17 (43%) of the epitope candidates the corresponding sequences in wild-type strains had reduced predicted HLA-I-binding compared to the vaccine strains. These findings suggest that vaccinated persons may have reduced T cell immunity to circulating mumps viruses due to antigenic differences.

An efficient molecular approach to distinguish chains of measles virus transmission in the elimination phase.

Measles viruses continue to spread globally, despite the availability of a safe and effective vaccine. Molecular surveillance of measles virus has become an essential tool to demonstrate whether cascades of infections in a certain region or country are the result of endemic spread or the repeatedly introduction of the virus in contained outbreaks. Currently, molecular surveillance of measles viruses worldwide is mainly based on 450 nucleotides of the C-terminal region of the nucleoprotein (N450). However, as a result of the disappearance of particular measles virus clades over the past decades, this gene segment does not provide sufficient resolution anymore to answer these questions. To increase the molecular resolution, sequence data were collected from three regions of the measles virus genome, the partial non-coding region between the M and F gene (M-F NCR4465-4754), partial H gene (H8022-8621) and the partial L gene (L10724-11438) for measles viruses detected in 2018 and 2019 in the Netherlands. Analysis of obtained sequence data indicated that sequencing of these three regions resulted in an increase in molecular resolution for measles virus genotype B3 and D8 viruses, two of the four global genotypes currently predominant in the European region. Furthermore, this improved resolution was sufficient to support an epidemiology characterized by repeat introduction of measles virus rather than endemic virus spread. In conclusion, sequencing of the M-F NCR4465-4754, H8022-8621 and L10724-11438 regions of the measles virus is an efficient and useful approach for molecular surveillance of measles viruses.

Co-detection of the measles vaccine and wild-type virus by real-time PCR: public health laboratory protocol.

In rare cases vaccination with the measles virus vaccine genotype A (MeVA) may cause a vaccine reaction with clinical signs similar to infection with wild-type measles virus (MeVwt). Rapid differentiation between MeVA and MeVwt infection is important for taking adequate public health measures. Recently, a few MeVA real-time reverse-transcription quantitative PCR methods (RT-qPCRs) were described that can distinguish between MeVA and MeVwt. However, detection of MeVA does in theory not exclude infection with MeVwt. In the present study, we established a protocol for determination of co-infections with MeVA and MeVwt. To this end, MeVA RT-qPCRs were used in combination with the routine measles virus (MeV) RT-qPCR, and the results suggested that the differences between the RT-qPCR Ct values (delta Ct, ∆Ct) could be used as criteria. Subsequently, we tested samples from vaccine-associated measles cases that were confirmed by genotyping. In addition, experimental mixtures of MeVA and MeVwt were tested in different concentrations. All tested MeVA clinical samples had ∆Ct ≤3.6. The results of experimental mixtures showed a mean ∆Ct ≤2.8 for genotype A alone and >3.2 when combined with either genotype B3 or D8. The results of a receiver operator characteristic analysis indicated that the optimum ∆Ct for use as a cut-off value was 3.5, while with ∆Ct values of 2.9 and 3.7 sensitivity and specificity were respectively 1.00. Thus, ∆Ct could be used to exclude the presence of MeVwt if MeVA is detected and ∆Ct is <2.9, while ∆Ct >3.7 were highly suggestive of co-infection and ≥2.9 ∆Ct <3.7 warranted additional confirmation, such as next-generation sequencing. This RT-qPCR-based protocol could be used for the exclusion of infection with MeVwt in cases with vaccine-associated measles reaction, crucial for the timely implementation of public health prevention and control measures.

Hemagglutinin Traits Determine Transmission of Avian A/H10N7 Influenza Virus between Mammals.

In 2014, an outbreak of avian A/H10N7 influenza virus occurred among seals along North-European coastal waters, significantly impacting seal populations. Here, we examine the cross-species transmission and mammalian adaptation of this influenza A virus, revealing changes in the hemagglutinin surface protein that increase stability and receptor binding. The seal A/H10N7 virus was aerosol or respiratory droplet transmissible between ferrets. Compared with avian H10 hemagglutinin, seal H10 hemagglutinin showed stronger binding to the human-type sialic acid receptor, with preferential binding to α2,6-linked sialic acids on long extended branches. In X-ray structures, changes in the 220-loop of the receptor-binding pocket caused similar interactions with human receptor as seen for pandemic strains. Two substitutions made seal H10 hemagglutinin more stable than avian H10 hemagglutinin and similar to human hemagglutinin. Consequently, identification of avian-origin influenza viruses across mammals appears critical to detect influenza A viruses posing a major threat to humans and other mammals.

Molecular epidemiology of mumps viruses in the Netherlands, 2017-2019.

Mumps cases continue to occur, also in countries with a relatively high vaccination rate. The last major outbreaks of mumps in the Netherlands were in 2009-2012 and thereafter, only small clusters and single cases were reported. Molecular epidemiology can provide insights in the circulation of mumps viruses. The aims of the present study were to analyze the molecular epidemiology of mumps viruses in the Netherlands in 2017-2019 and to compare the phylogenetic trees built from sequence data of near complete mumps virus genomes or from the SH gene and non-coding regions (SH+NCRs). To this end, Sanger sequence data from SH+NCRs were analyzed from 82 mumps genotype G viruses. In addition, near complete genomes were obtained from 10 mumps virus isolates using next-generation sequencing. Analysis of SH+NCRs sequences of mumps genotype G viruses revealed the presence of two major genetic lineages in the Netherlands, which was confirmed by analysis of near complete genomes. Comparison of phylogenetic trees built with SH+NCRs or near complete genomes indicated that the topology was similar, while somewhat longer branches were present in the phylogenetic tree with near complete genomes. These results confirm that analysis of SH + NCRs sequence data is a useful approach for molecular surveillance. Furthermore, data from recent mumps genotype G viruses might indicate (intermittent) circulation of mumps genotype G viruses in the Netherlands in 2017-2019.

[The role of children in the transmission of SARS-CoV-2]. / De rol van kinderen in de transmissie van SARS-CoV-2.

OBJECTIVE: To determine whether children play a role in the transmission of SARS-CoV-2 to other children and adults, and to gain insight into symptomatic and asymptomatic infections in children. DESIGN: Analysis of national COVID-19 notifications and prospective observational study in families with children. METHOD: Information about COVID-19 patients and their contacts was obtained from the registration systems used by the public health services. In an ongoing study, patients with COVID-19 were asked to participate if they have a family with children. On two occasions nose-throat swabs and blood were collected for PCR analysis and determination of antibodies against SARS-CoV-2. RESULTS: The notifications suggest that transmission finds place mainly between adults and to a lesser extent between parents and children. For the family study, data were available from 54 households with a total of 227 participants. In families of a confirmed COVID-19 patient, children between 1 and 11 years were less often positive in PCR and serology than older children and adults. CONCLUSION: The study gives no indications that children play an important role in the transmission of SARS-CoV-2. Children can indeed become infected, but transmission mainly takes place between adult peers and from adult family members to children. Transmission among children or from children to adults, as is known in influenza, appears to be less common. Ongoing studies should provide important information for further decision-making on control measures, such as closure of schools.

Oral fluid: Non-invasive alternative for parvovirus B19 diagnosis?

BACKGROUND: Infections with parvovirus B19 (B19V) have been associated with a wide range of disease manifestations of which erythema infectiosum (fifth disease) in children is most common. Clinical signs following infection of children with B19V can be similar to measles and rubella. Laboratory detection of B19V infections is based on detection of B19V-specific IgM antibodies by enzyme immunoassay (IgM-EIA) and/or B19V DNA by quantitative PCR (qPCR) on blood samples. The need for invasive sampling can be a barrier for public health diagnostics. OBJECTIVES: To evaluate the use of a dual target B19V-qPCR directed against the NS1 and VP2 of B19V on oral fluid samples as a non-invasive alternative for laboratory diagnosis of B19V infections in children below 12 years of age with exanthema. STUDY DESIGN: Oral fluid and serum samples were collected from 116 children with exanthema. All serum samples were tested by IgM-EIA/IgG-EIA, while all oral fluid and 56 serum samples were tested by B19V-qPCR. RESULTS: B19V-specific IgM antibodies were detected in 25 of 116 children in the study. B19V DNA was detected in oral fluid in 17 of the 25 children who were IgM positive, as well as two children who were IgM-equivocal or negative. The child with the equivocal IgM had a high quantity of B19V DNA in oral fluid (7 log IU/ml), compatible with an acute B19V infection. The IgM-negative child was IgG-positive and 4 log IU/ml B19V DNA was detected in the oral fluid sample, suggesting an acute infection and a falsely negative IgM. Sample size calculations indicated that oral fluid samples for qPCR should be collected from 2 to 3 children during outbreaks of exanthema to achieve similar sensitivity as IgM-EIA for one child (≥0.9) to confirm or exclude B19V. CONCLUSIONS: Results indicate that oral fluid samples are a suitable public health alternative for detection of B19V infections, potentially lowering the barriers for sampling.

Reassortments among Avian Influenza A(H5N1) Viruses Circulating in Indonesia, 2015-2016.

Highly pathogenic avian influenza (HPAI) A(H5N1) viruses have been circulating since 2003 in Indonesia, with major impacts on poultry health, severe economic losses, and 168 fatal laboratory-confirmed human cases. We performed phylogenetic analysis on 39 full-genome H5N1 virus samples collected during outbreaks among poultry in 2015-2016 in West Java and compared them with recently published sequences from Indonesia. Phylogenetic analysis revealed that the hemagglutinin gene of all samples belonged to 2 genetic groups in clade 2.3.2.1c. We also observed these groups for the neuraminidase, nucleoprotein, polymerase, and polymerase basic 1 genes. Matrix, nonstructural protein, and polymerase basic 2 genes of some HPAI were most closely related to clade 2.1.3 instead of clade 2.3.2.1c, and a polymerase basic 2 gene was most closely related to Eurasian low pathogenicity avian influenza. Our results detected a total of 13 reassortment types among HPAI in Indonesia, mostly in backyard chickens in Indramayu.

Optimizing molecular surveillance of mumps genotype G viruses.

Mumps viruses continue to cause sporadic cases and outbreaks in countries with a high vaccination coverage for mumps. Molecular surveillance of mumps viruses can be supportive to elucidate the origin and transmission routes of mumps virus in case of an outbreak. Currently, molecular surveillance is worldwide primarily focused on sequencing of the small hydrophobic (SH) gene. However, few studies have already shown that additional genes or regions contribute to the resolution of the sequence data in such a way that mumps cases that seem to be linked to the same source on basis of the SH sequence, appear to be linked to another source or chain of transmission. Notably, this sequence information was recently extracted from the hemagglutinin-neuraminidase (HN) and fusion (F) genes (total 3364 nucleotides), or from the sum of the three non-coding regions (NCRs; total 1954 nt) between the nucleocapsid protein, phosphoprotein, matrix protein and F protein, but also from the complete genome. Here, sequence data from NCRs were compared with that of the HN and F gene, using mumps genotype G viruses detected in the Netherlands between 2010 and 2018. Results of this study indicate that NCRs sequence data provided similar or slightly better sequence resolution compared to the HN and F genes for most viruses. For molecular surveillance of currently circulating mumps genotype G viruses is sequencing of SH in combination with NCRs currently a useful approach.

Comparison of Different In Situ Hybridization Techniques for the Detection of Various RNA and DNA Viruses.

In situ hybridization (ISH) is a technique to determine potential correlations between viruses and lesions. The aim of the study was to compare ISH techniques for the detection of various viruses in different tissues. Tested RNA viruses include atypical porcine pestivirus (APPV) in the cerebellum of pigs, equine and bovine hepacivirus (EqHV, BovHepV) in the liver of horses and cattle, respectively, and Schmallenberg virus (SBV) in the cerebrum of goats. Examined DNA viruses comprise canine bocavirus 2 (CBoV-2) in the intestine of dogs, porcine bocavirus (PBoV) in the spinal cord of pigs and porcine circovirus 2 (PCV-2) in cerebrum, lymph node, and lung of pigs. ISH with self-designed digoxigenin-labelled RNA probes revealed a positive signal for SBV, CBoV-2, and PCV-2, whereas it was lacking for APPV, BovHepV, EqHV, and PBoV. Commercially produced digoxigenin-labelled DNA probes detected CBoV-2 and PCV-2, but failed to detect PBoV. ISH with a commercially available fluorescent ISH (FISH)-RNA probe mix identified nucleic acids of all tested viruses. The detection rate and the cell-associated positive area using the FISH-RNA probe mix was highest compared to the results using other probes and protocols, representing a major benefit of this method. Nevertheless, there are differences in costs and procedure time.

Exploring contacts facilitating transmission of influenza A(H5N1) virus between poultry farms in West Java, Indonesia: A major role for backyard farms?

Highly pathogenic avian influenza virus (HPAIV) H5N1 has been reported in Asia, including Indonesia since 2003. Although several risk factors related to the HPAIV outbreaks in poultry in Indonesia have been identified, little is known of the contact structure of farms of different poultry production types (backyard chickens, broilers, layers, and ducks). This study aims to quantify the contact rates associated with the movement of people, and movements of live birds and products and equipment that affect the risk of HPAIV H5N1 transmission between poultry farms in Indonesia. On 124 poultry farms in 6 districts in West Java, logbooks were distributed to record the movements of farmers/staff and visitors and their poultry contacts. Most movements in backyard chicken, commercial native chicken, broiler and duck farms were visits to and from other poultry farms, whilst in layer farms visits to and from poultry companies, visits to egg collection houses and visit from other poultry farms were most frequent. Over 75% of persons visiting backyard chicken and duck farms had previously visited other poultry farms on the same day. Visitors of backyard chicken farms had the highest average contact rate, either direct contact with poultry on other farms before the visits (1.35 contact/day) or contact during their visits in the farms (10.03 contact/day). These results suggest that backyard chicken farms are most at risk for transmission of HPAIV compared to farms of the other poultry production types. Since visits of farm-to-farm were high, backyard farms could also a potential source for HPAIV transmission to commercial poultry farms.

Wild ducks excrete highly pathogenic avian influenza virus H5N8 (2014-2015) without clinical or pathological evidence of disease.

Highly pathogenic avian influenza (HPAI) is essentially a poultry disease. Wild birds have traditionally not been involved in its spread, but the epidemiology of HPAI has changed in recent years. After its emergence in southeastern Asia in 1996, H5 HPAI virus of the Goose/Guangdong lineage has evolved into several sub-lineages, some of which have spread over thousands of kilometers via long-distance migration of wild waterbirds. In order to determine whether the virus is adapting to wild waterbirds, we experimentally inoculated the HPAI H5N8 virus clade 2.3.4.4 group A from 2014 into four key waterbird species-Eurasian wigeon (Anas penelope), common teal (Anas crecca), mallard (Anas platyrhynchos), and common pochard (Aythya ferina)-and compared virus excretion and disease severity with historical data of the HPAI H5N1 virus infection from 2005 in the same four species. Our results showed that excretion was highest in Eurasian wigeons for the 2014 virus, whereas excretion was highest in common pochards and mallards for the 2005 virus. The 2014 virus infection was subclinical in all four waterbird species, while the 2005 virus caused clinical disease and pathological changes in over 50% of the common pochards. In chickens, the 2014 virus infection caused systemic disease and high mortality, similar to the 2005 virus. In conclusion, the evidence was strongest for Eurasian wigeons as long-distance vectors for HPAI H5N8 virus from 2014. The implications of the switch in species-specific virus excretion and decreased disease severity may be that the HPAI H5 virus more easily spreads in the wild-waterbird population.

Changing Role of Wild Birds in the Epidemiology of Avian Influenza A Viruses.

Waterbirds are the main reservoir for low pathogenic avian influenza A viruses (LPAIV), from which occasional spillover to poultry occurs. When circulating among poultry, LPAIV may become highly pathogenic avian influenza A viruses (HPAIV). In recent years, the epidemiology of HPAIV viruses has changed drastically. HPAIV H5N1 are currently endemic among poultry in a number of countries. In addition, global spread of HPAIV H5Nx viruses has resulted in major outbreaks among wild birds and poultry worldwide. Using data collected during these outbreaks, the role of migratory birds as a vector became increasingly clear. Here we provide an overview of current data about various aspects of the changing role of wild birds in the epidemiology of avian influenza A viruses.

Deaths among Wild Birds during Highly Pathogenic Avian Influenza A(H5N8) Virus Outbreak, the Netherlands.

During autumn-winter 2016-2017, highly pathogenic avian influenza A(H5N8) viruses caused mass die-offs among wild birds in the Netherlands. Among the ≈13,600 birds reported dead, most were tufted ducks (Aythya fuligula) and Eurasian wigeons (Anas penelope). Recurrence of avian influenza outbreaks might alter wild bird population dynamics.

Pathological findings in the red fox (Vulpes vulpes), stone marten (Martes foina) and raccoon dog (Nyctereutes procyonoides), with special emphasis on infectious and zoonotic agents in Northern Germany.

Anthropogenic landscape changes contributed to the reduction of availability of habitats to wild animals. Hence, the presence of wild terrestrial carnivores in urban and peri-urban sites has increased considerably over the years implying an increased risk of interspecies spillover of infectious diseases and the transmission of zoonoses. The present study provides a detailed characterisation of the health status of the red fox (Vulpes vulpes), stone marten (Martes foina) and raccoon dog (Nyctereutes procyonoides) in their natural rural and peri-urban habitats in Schleswig-Holstein, Germany between November 2013 and January 2016 with focus on zoonoses and infectious diseases that are potentially threatening to other wildlife or domestic animal species. 79 red foxes, 17 stone martens and 10 raccoon dogs were collected from traps or hunts. In order to detect morphological changes and potential infectious diseases, necropsy and pathohistological work-up was performed. Additionally, in selected animals immunohistochemistry (influenza A virus, parvovirus, feline leukemia virus, Borna disease virus, tick-borne encephalitis, canine adenovirus, Neospora caninum, Toxoplasma gondii and Listeria monocytogenes), next-generation sequencing, polymerase chain reaction (fox circovirus) and serum-neutralisation analysis (canine distemper virus) were performed. Furthermore, all animals were screened for fox rabies virus (immunofluorescence), canine distemper virus (immunohistochemistry) and Aujeszky's disease (virus cultivation). The most important findings included encephalitis (n = 16) and pneumonia (n = 20). None of the investigations revealed a specific cause for the observed morphological alterations except for one animal with an elevated serum titer of 1:160 for canine distemper. Animals displayed macroscopically and/or histopathologically detectable infections with parasites, including Taenia sp., Toxocara sp. and Alaria alata. In summary, wildlife predators carry zoonotic parasitic disease and suffer from inflammatory diseases of yet unknown etiology, possibly bearing infectious potential for other animal species and humans. This study highlights the value of monitoring terrestrial wildlife following the "One Health" notion, to estimate the incidence and the possible spread of zoonotic pathogens and to avoid animal to animal spillover as well as transmission to humans.

Norovirus Infection in Harbor Porpoises.

A norovirus was detected in harbor porpoises, a previously unknown host for norovirus. This norovirus had low similarity to any known norovirus. Viral RNA was detected primarily in intestinal tissue, and specific serum antibodies were detected in 8 (24%) of 34 harbor porpoises from the North Sea.

Influenza A (H10N7) Virus Causes Respiratory Tract Disease in Harbor Seals and Ferrets.

Avian influenza viruses sporadically cross the species barrier to mammals, including humans, in which they may cause epidemic disease. Recently such an epidemic occurred due to the emergence of avian influenza virus of the subtype H10N7 (Seal/H10N7) in harbor seals (Phoca vitulina). This epidemic caused high mortality in seals along the north-west coast of Europe and represented a potential risk for human health. To characterize the spectrum of lesions and to identify the target cells and viral distribution, findings in 16 harbor seals spontaneously infected with Seal/H10N7 are described. The seals had respiratory tract inflammation extending from the nasal cavity to bronchi associated with intralesional virus antigen in respiratory epithelial cells. Virus infection was restricted to the respiratory tract. The fatal outcome of the viral infection in seals was most likely caused by secondary bacterial infections. To investigate the pathogenic potential of H10N7 infection for humans, we inoculated the seal virus intratracheally into six ferrets and performed pathological and virological analyses at 3 and 7 days post inoculation. These experimentally inoculated ferrets displayed mild clinical signs, virus excretion from the pharynx and respiratory tract inflammation extending from bronchi to alveoli that was associated with virus antigen expression exclusively in the respiratory epithelium. Virus was isolated only from the respiratory tract. In conclusion, Seal/H10N7 infection in naturally infected harbor seals and experimentally infected ferrets shows that respiratory epithelial cells are the permissive cells for viral replication. Fatal outcome in seals was caused by secondary bacterial pneumonia similar to that in fatal human cases during influenza pandemics. Productive infection of ferrets indicates that seal/H10N7 may possess a zoonotic potential. This outbreak of LPAI from wild birds to seals demonstrates the risk of such occasions for mammals and thus humans.