Climate change associated transformations of Arctic marine ecosystems are having detrimental impacts on Arctic endemic marine mammals. However, warming conditions are providing new habitats for temperate marine mammals, some of which are expanding into Arctic regions, posing a threat of novel pathogen introduction and disease transmission. We analyzed swab samples for Influenza A, morbilli- (Phocine distemper virus, PDV), and herpesvirus nucleic acid and performed microbiological screenings and serological analyses for antibodies for the same viral pathogens, the bacterial agents Brucella spp. and Leptospira spp. and the protozoan parasite Toxoplasma (T.) gondii from ringed (Pusa hispida), bearded (Erignathus barbatus), and the newly established harbor seals (Phoca vitulina) in Kongsfjorden, Svalbard. Antibodies against Phocine Herpesvirus-1 or antigenetically related phocine herpesviruses (PhHV) were detected in all species. Antibodies against Influenza A were only found in one ringed seal and antibodies against PDV were found in two harbor seals. Twelve harbor seals and two ringed seals tested positive for Herpesvirus DNA by PCR. Brucella spp. and Leptospira spp. antibodies were detected in multiple harbor seals and a single bearded seal. Three harbor, two ringed, and one bearded seal had antibodies against T. gondii. Our results indicate an overlap and possible transmission of pathogens between all investigated seal species. The introduction of new pathogens to the Arctic through temperate species' range expansions could have fatal consequences for immunologically naive Arctic seal populations and should be further investigated.

In 2019, a marine pestivirus, Phocoena pestivirus (PhoPeV), family Pestiviridae, was described for the first time. To date, the impact of PhoPeV infections on harbor porpoises is still unknown. Therefore, a screening study was carried out on harbor porpoises found dead on the German North Sea and Baltic Sea coasts during 2021-2025. Analysis of organ samples collected from 84 harbor porpoises by reverse transcription (RT)-PCR showed the presence of PhoPeV RNA in samples from five animals (5.9%). Infectious virus was isolated from two of these samples. Phylogenetic analysis showed a close relationship to previously reported PhoPeV isolates. Moreover, a virus neutralization assay has been established using organ liquid and organ homogenate matrices instead of the standard serum sample matrix. Samples from seven out of 72 animals tested positive for neutralizing antibodies against PhoPeV including four samples with an antibody titer ≥ 320 ND50. Cross-neutralization studies including classical swine fever virus (CSFV), border disease virus (BDV), as well as Bungowannah pestivirus (BuPV), and Linda virus (LindaV), both viruses being most closely related to PhoPeV, showed no significant serological relatedness to PhoPeV. In conclusion, both PhoPeV RNA and neutralizing antibodies against PhoPeV have been detected in animals found dead in the North Sea and Baltic Sea over the last 5 years, indicating a continuous circulation of PhoPeV in the harbor porpoise population for at least several years.

The first marine pestivirus, Phocoena pestivirus (PhoPeV), isolated from harbor porpoise, has been recently described. To further characterize this unique pestivirus, its host cell tropism and growth kinetics were determined in different cell lines. In addition, the interaction of PhoPeV with innate immunity in porcine epithelial cells and the role of selected cellular factors involved in the viral entry and RNA replication of PhoPeV were investigated in comparison to closely and distantly related pestiviruses. While Bungowannah pestivirus (BuPV), a unique porcine pestivirus closely related to PhoPeV, exhibits a broad cell tropism, PhoPeV only infects cells from pigs, cattle, sheep, and cats, as has been described for classical swine fever virus (CSFV). Viral titers correlate with the amount of intracellular PhoPeV-specific RNA detected in the tested cell lines. PhoPeV replicates most efficiently in the porcine kidney cell line SK6. Pestiviruses generally counteract the cellular innate immune response by degradation of interferon regulatory factor 3 (IRF3) mediated by the viral N-terminal protease (Npro). No degradation of IRF3 and an increased expression of the type 1 interferon-stimulated antiviral protein Mx1 was observed in porcine cells infected with PhoPeV whose genome lacks the Npro encoding region. Infection of a CD46-deficient porcine cell line suggested that CD46, which is implicated in the viral entry of several pestiviruses, is not a major factor for the viral entry of PhoPeV. Moreover, the results of this study confirmed that the cellular factor DNAJC14 plays a crucial role in viral RNA replication of non-cytopathic pestiviruses, including PhoPeV.

As a complementary tool for control of classical swine fever (CSF) in wild boar vaccine baits containing the attenuated and highly efficacious C-strain have been distributed in several countries. Several issues have been identified with the present available bait system, like relatively low uptake by piglets, melting point and mechanical stability. Hence, an alternative bait system has been developed including a more thermo-stable bait matrix and vaccine container. In the present study, the attractiveness and the capability of inducing an adequate immune response of this new bait system (IP) was compared with the present available product (CP) in two groups of 25 pigs each. The pigs of each group were offered the respective bait individually, and the animals were bled at 14 days prior to vaccination and 28 - and 42-days post vaccination (dpv). Blood samples were examined for antibodies in ELISA and virus neutralizing test (VNT). Two and one animal in the CP and IP group refused to consume the bait, respectively. The IP was significantly more rapidly consumed than the CP (p = 0.021). All animals that consumed a bait in both groups tested seropositive in VNT (≥10 ND50) at 28 dpv and reached antibody titers above the threshold for protective immunity (32 ND50) at 42 dpv. Hence, it can be concluded that the new bait system can induce an adequate immune response in pigs after the consumption of a single bait.

We present the first documented case of highly pathogenic avian influenza virus (HPAIV) subtype H5N5 in an Atlantic walrus (Odobenus rosmarus rosmarus). The animal was found dead in Svalbard, Norway, in 2023. Sequence analysis revealed the highest genetic similarity with virus isolates from different avian hosts.

The envelope glycoprotein E2 of pestiviruses plays a crucial role in viral entry and elicits a virus-neutralizing humoral immune response. Consequently, the epitopes recognized by monoclonal antibodies (mAbs) on E2 are a significant focus in pestivirus research and diagnostics. In this study, we characterized a panel of murine mAbs against the E2 protein of classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV), two major pathogens for swine and cattle, respectively. The majority of mAbs neutralized the virus in vitro and recognized conformational epitopes, which were also detected by sera from infected animals. Notably, binding to these epitopes was retained after low-pH treatment, although conformational epitopes were disrupted upon disulfide bond reduction. The epitopes of the anti-CSFV mAbs were located in various domains of E2, including the interdomain linker sequences. Conversely, all but one of the anti-BVDV mAb epitopes were located in domain A. Moreover, the reactivity of one mAb suggests a conformational interdependence among the linker sequences of pestivirus E2. The panel of mAbs characterized in this study holds potential to support basic research on the mechanism of early pestivirus invasion and to assist in the design of E2-based diagnostic tools and vaccines. IMPORTANCE:Classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV), which belong to the family Flaviviridae, cause economically significant diseases in pigs and cattle. The pestivirus glycoprotein E2 is located on the viral surface and is targeted by antibodies that neutralize virus infection. Due to its variability, E2 is a useful antigen for the development of diagnostic tests to differentiate between infections caused by different pestiviruses. In the present study, two panels of monoclonal antibodies (mAbs) specifically reactive with either CSFV or BVDV E2 were characterized. Interestingly, the majority of mAbs neutralized the respective virus in vitro. Epitope mapping revealed that the mAbs recognized low-pH-resistant epitopes of conformational nature located in different domains of CSFV E2 (anti-CSFV mAbs) or in domain A of BVDV E2 (anti-BVDV mAbs). The recombinant proteins along with the characterized mAbs have the potential to develop improved pestivirus-specific diagnostic tests and vaccines.

Several cellular factors have been reported to be required for replication of classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae. However, many steps of its replication cycle are still poorly understood. The low-density lipoprotein receptor (LDLR) is involved in cell entry and post-entry processes of different viruses including other members of the Flaviviridae. In this study, the relevance of LDLR in replication of CSFV and another porcine pestivirus, Bungowannah pestivirus (BuPV), was investigated by antibody-mediated blocking of LDLR and genetically engineered porcine cell lines providing altered LDLR expression levels. An LDLR-specific antibody largely blocked infection with CSFV, but had only a minor impact on BuPV. Infections of the genetically modified cells confirmed an LDLR-dependent replication of CSFV. Compared to wild type cells, lower and higher expression of LDLR resulted in a 3.5-fold decrease or increase in viral titers already 20 h post infection. Viral titers were 25-fold increased in LDLR-overexpressing cells compared to cells with reduced LDLR expression at 72 h post infection. The varying LDLR expression levels had no clear effect on permissivity to BuPV. A decoy receptor assay using recombinant soluble LDLR provided no evidence that LDLR may function as a receptor for CSFV or BuPV. Differences in their dependency on LDLR suggest that CSFV and BuPV likely use different mechanisms to interact with their host cells. Moreover, this study reveals similarities in the replication cycles of CSFV and other members of the family Flaviviridae that are dependent on LDLR.

Classical swine fever virus (CSFV) shares high antigenic homology with other members of the genus Pestivirus. Because several pestivirus species can also infect swine, eliciting cross-reactive antibodies, it is important to define CSFV-specific epitopes for the differential diagnosis of classical swine fever (CSF) by serology. For this purpose, epitope mapping of seven monoclonal antibodies (mAbs), recognizing sites on the D/A domain of glycoprotein E2, was performed using recombinant expressed antigenic domains and mutants of E2, as well as an overlapping peptide library. Three CSFV-specific epitopes, i.e., 780-IEEMGDDFGFGLCPF-794, 810-NGSAFYLVCPIGWTG-824, and 846-REKPF-850, were identified within the D/A domain of E2. Site-directed mutagenesis further confirmed that residues 783-MGD-785, 789-FGLCPF-794, 813-AFYLVCPIGWTG-824, and 846REK-848 were critical residues in these regions. In addition, a F789S difference within the epitope 780IEEMGDDFGFGLCPF-794 was responsible for the absence of binding of two mAbs to the E2 protein of the live attenuated CSFV vaccine strain Riems. Structural modeling revealed that, the three epitopes are located near each other, suggesting that they may form a more complex conformational epitope on the D/A domain in vivo. Six of the mAbs neutralized viruses of diverse genotypes, indicating that the target epitopes are involved in virus interaction with cells. The binding of CSFV to cells was significantly reduced after pre-incubation with either truncated E2 proteins comprising the D/A domain or with the CSFV-specific mAbs targeting the domain D/A. These epitopes identified on the D/A domain are important targets for virus neutralization that might be involved in the early steps of CSFV infection. These findings reveal potential candidates for improving the differential diagnosis of pestiviruses by serology.

The Svalbard white whale (Delphinapterus leucas) population is one of the smallest in the world, making it particularly vulnerable to challenges such as climate change and pathogens. In this study, serum samples from live captured (2001–2016) white whales from this region were investigated for influenza A virus (IAV) antibodies (Abs) (n = 27) and RNA (n = 25); morbillivirus (MV) Abs (n = 3) and RNA (n = 25); Brucella spp. Abs; and Toxoplasma gondii Abs (n = 27). IAV Abs were found in a single adult male that was captured in Van Mijenfjorden in 2001, although no IAV RNA was detected. Brucella spp. Abs were found in 59% of the sample group (16/27). All MV and T. gondii results were negative. The results show that Svalbard white whales have been exposed to IAV and Brucella spp., although evidence of disease is lacking. However, dramatic changes in climate and marine ecosystems are taking place in the Arctic, so surveillance of health parameters, including pathogens, is critical for tracking changes in the status of this vulnerable population.

Einleitung Massensterben von Wild- und Nutzgeflügel infolge von Infektionen mit aviären Influenza-A-Viren (AIV) unterstreichen die Relevanz weiterer Untersuchungen bezüglich Verbreitung und Wirtsspezifität potenziell zoonotischer Erreger. In der vorliegenden Studie wurde eine bisher selten beschriebene Infektion des zentralen Nervensystems (ZNS) mit hochpathogenem AIV (HPAIV) des Subtyps H5N8 in Seehunden nachgewiesen.

The genus Pestivirus within the family Flaviviridae comprises highly relevant animal pathogens such as bovine viral diarrhoea virus 1 and 2 (BVDV-1 and -2) classified into the two species Pestivirus A and Pestivirus B, respectively. First described in 2004, HoBi-like pestiviruses (HoBiPeV) represent emerging bovine pathogens that belong to a separate species (Pestivirus H), but share many similarities with BVDV-1 and -2. Additionally, two giraffe pestivirus (GPeV) strains both originating from Kenya represent another distinct species (Pestivirus G), whose members replicate very efficiently in bovine cells. In this study, we investigated the role of bovine complement regulatory protein 46 (CD46(bov)), the receptor of BVDV-1 and -2, in the entry of HoBiPeV and GPeV. For this purpose, bovine CD46-knockout and CD46-rescue cell lines were generated by CRISPR/Cas9 technology and subsequent trans-complementation, respectively. Our results provide strong evidence that the impact of CD46(bov) differs between viruses belonging to Pestivirus H and viruses representing Pestivirus G: CD46(bov) revealed to be a major cellular entry factor for HoBiPeV strain HaVi-20. In contrast, GPeV strain PG-2 presented as largely independent of CD46(bov), suggesting a different entry mechanism involving other molecular determinants which remain to be identified. In addition, we demonstrated that, similar to BVDV-1 and -2, virus isolates of both Pestivirus H and Pestivirus G are able to adapt to cell culture conditions by using heparan sulfate to enter the host cell. In conclusion, our findings show that different bovine pestiviruses use diverse mechanisms of host cell entry.

In brain tissue of three harbor seals of the German North Sea coast, high virus loads of highly pathogenic avian influenza virus (HPAIV) H5N8 were detected. Identification of different virus variants indicates high exposure to HPAIV circulating in wild birds, but there is no evidence for H5 specific antibodies in healthy seals. Replication of avian viruses in seals may allow HPAIV to acquire mutations needed to adapt to mammalian hosts as shown by PB2 627K variants detected in these cases.

Pestiviruses are widespread pathogens causing severe acute and chronic diseases among terrestrial mammals. Recently, Phocoena pestivirus (PhoPeV) was described in harbour porpoises (Phocoena phocoena) of the North Sea, expanding the host range to marine mammals. While the role of the virus is unknown, intrauterine infections with the most closely related pestiviruses— Bungowannah pestivirus (BuPV) and Linda virus (LindaV)—can cause increased rates of abortions and deaths in young piglets. Such diseases could severely impact already vulnerable harbour porpoise populations. Here, we investigated the presence of PhoPeV in 77 harbour porpoises, 277 harbour seals (Phoca vitulina), grey seals (Halichoerus grypus) and ringed seals (Pusa hispida) collected in the Baltic Sea region between 2002 and 2019. The full genome sequence of a pestivirus was obtained from a juvenile female porpoise collected along the coast of Zealand in Denmark in 2011. The comparative Bayesian phylogenetic analyses revealed a close relationship between the new PhoPeV sequence and previously published North Sea sequences with a recent divergence from genotype 1 sequences between 2005 and 2009. Our findings provide further insight into the circulation of PhoPeV and expand the distribution from the North Sea to the Baltic Sea region with possible implications for the vulnerable Belt Sea and endangered Baltic Proper harbour porpoise populations.

Classical swine fever virus (CSFV) shares high structural and antigenic homology with bovine viral diarrhea virus (BVDV) and border disease virus (BDV). Because all three viruses can infect swine and elicit cross-reactive antibodies, it is necessary to differentiate among them with regard to serological diagnosis of classical swine fever. To understand the mechanism of cross-reactivity, it is important to define common or specific epitopes of these viruses. For this purpose, epitope mapping of six monoclonal antibodies (mAbs) was performed using recombinant expressed antigenic domains of CSFV and BDV E2 proteins. One CSFV-specific conformational epitope and one CSFV and BDV common epitope within domain B/C of E2 were identified. Site-directed mutagenesis confirmed that residues G725 and V738/I738 of the CSFV-specific epitope and P709/L709 and E713 of the second epitope are important for mAbs binding. Infection of CSFV in porcine cells was significantly reduced after pre-incubation of the cells with the domain B/C of E2 or after pre-incubation of CSFV with the mAbs detecting domain B/C. 3D structural modeling suggested that both epitopes are exposed on the surface of E2. Based on this, the identified epitopes represent a potential target for virus neutralization and might be involved in the early steps of CSFV infection.

Apart from the established pestivirus species Pestivirus A to Pestivirus K novel species emerged. Pigs represent not only hosts for porcine pestiviruses, but are also susceptible to bovine viral diarrhea virus, border disease virus (BDV) and other ruminant pestiviruses. The present study focused on the characterization of the ovine Tunisian sheep-like virus (TSV) as well as Bungowannah virus (BuPV) and BDV strain Frijters, which were isolated from pigs. For this purpose, we performed genetic characterization based on complete coding sequences, studies on virus replication in cell culture and in domestic pigs, and cross-neutralization assays using experimentally derived sera. TSV forms a distinct phylogenetic group more closely related to Pestivirus C (classical swine fever virus, CSFV) than to Pestivirus D (BDV). In contrast to BDV and BuPV, TSV replicates by far more efficiently on ovine than on porcine cells. Nevertheless, pigs were susceptible to TSV. As a consequence of close antigenic relatedness of TSV to CSFV, cross-reactivity was detected in CSFV-specific antibody assays. In conclusion, TSV is genetically closely related to CSFV and can replicate in domestic pigs. Due to close antigenic relatedness, field infections of pigs with TSV and other ruminant pestiviruses can interfere with serological diagnosis of classical swine fever.

Pestiviruses are plus-stranded RNA viruses belonging to the family Flaviviridae. They comprise several important pathogens like classical swine fever virus and bovine viral diarrhea virus that induce economically important animal diseases. In 2017, the last update of pestivirus taxonomy resulted in demarcation of 11 species designated Pestivirus A through Pestivirus K. Since then, multiple new pestiviruses have been reported including pathogens associated with disease in pigs or small ruminants. In addition, pestivirus sequences have been found during metagenomics analysis of different non-ungulate hosts (bats, rodents, whale, and pangolin), but the consequences of this pestivirus diversity for animal health still need to be established. To provide a systematic classification of the newly discovered viruses, we analyzed the genetic relationship based on complete coding sequences (cds) and deduced polyprotein sequences and calculated pairwise distances that allow species demarcation. In addition, phylogenetic analysis was performed based on a highly conserved region within the non-structural protein NS5B. Taking into account the genetic relationships observed together with available information about antigenic properties, host origin, and characteristics of disease, we propose to expand the number of pestivirus species to 19 by adding eight additional species designated Pestivirus L through Pestivirus S.

Pestiviruses such as bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) belong to the family Flaviviridae and represent pathogens of outstanding veterinary relevance. Pestiviruses enter cells via receptor-mediated endocytosis. For entry in bovine cells, complement regulatory protein CD46bov serves as a cellular receptor for BVDV. In this study, the role of porcine CD46pig in cellular entry was investigated for the recently discovered atypical porcine pestivirus (APPV), CSFV, and Bungowannah virus (BuPV) in order to elucidate the observed differences in host cell tropism. A cell culture-adapted APPV variant, which shows enhanced viral replication in vitro, was generated and demonstrated a strict tropism of APPV for porcine cells. One of the porcine cell lines displayed areas of CD46pig-expressing cells and areas of nonexpressing cells, and one single cell line revealed not to express any CD46pig The CD46pig-deficient porcine lymphoma cell line, known to facilitate CSFV replication, was the only porcine cell line nonpermissive to APPV, indicating a significant difference in the entry mechanism of APPV and CSFV. Infection experiments with a set of genetically engineered CD46pig knockout cells confirmed that CD46pig is a major receptor of APPV as CD46bov is for BVDV. In contrast, it is apparently not an essential determinant in host cell entry of other porcine pestiviruses such as CSFV and BuPV. Existence of a CD46pig-independent entry mechanism illustrates that the pestiviral entry process is more diverse than previously recognized.IMPORTANCE Pestiviruses comprise animal pathogens such as classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV) that cause notifiable diseases with great economic impact. Several additional pestivirus species affecting animal health were recently identified, including atypical porcine pestivirus (APPV). APPV is associated with health problems in piglets and is highly abundant in pig populations worldwide. Complement control protein CD46 serves as a receptor for diverse bacterial and viral pathogens, including particular adenoviruses, herpesviruses, measles virus (MeV), and BVDV. Porcine CD46 (CD46pig) was suggested to be a major receptor for CSFV. Here, we identified remarkable differences in relevance of CD46pig during entry of porcine pestiviruses. Resembling BVDV, efficient APPV infection in cell culture depends on CD46pig, while other porcine pestiviruses can efficiently enter and infect cells in the absence of CD46pig Thus, the study provides insights into the entry process of these pathogens and may help to understand differences in their biology.

The recently identified causative agent of congenital tremor in domestic piglets, atypical porcine pestivirus (APPV), was detected in serum from Swedish wild boar. A previous study from Sweden described APPV in domestic piglets suffering from congenital tremor, but the APPV situation in the wild boar population was unknown. In this study, 595 serum samples from wild boar originating from 13 counties in the south and central parts of Sweden, collected between 2000 and 2018, were analysed for the presence of the APPV-genome and for antibodies against the APPV-glycoprotein Erns . The results revealed that APPV is highly abundant in the Swedish wild boar population; 12% (73/595) were APPV-genome positive in serum and 72% (433/595) of the tested wild boars displayed APPV-specific antibodies. The present study also shows that APPV has been present in the Swedish wild boar population since at least the year 2000. The viral sequences obtained from the wild boars were highly similar to those obtained from Swedish domestic pigs positive for APPV and suffering from congenital tremor, suggesting a viral exchange between wild boars and domestic pigs. The high proportion of viraemic and seropositive wild boar is indicative of wild boar being an important reservoir for APPV.

Classical swine fever (CSF) is among the most relevant viral epizootic diseases of swine. Due to its severe economic impact, CSF is notifiable to the world organisation for animal health. Strict control policies, including systematic stamping out of infected herds with and without vaccination, have permitted regional virus eradication. Nevertheless, CSF virus (CSFV) persists in certain areas of the world and has re-emerged regularly. This review summarizes the basic established knowledge in the field and provides a comprehensive and updated overview of the recent advances in fundamental CSFV research, diagnostics and vaccine development. It covers the latest discoveries on the genetic diversity of pestiviruses, with implications for taxonomy, the progress in understanding disease pathogenesis, immunity against acute and persistent infections, and the recent findings in virus-host interactions and virulence determinants. We also review the progress and pitfalls in the improvement of diagnostic tools and the challenges in the development of modern and efficacious marker vaccines compatible with serological tests for disease surveillance. Finally, we highlight the gaps that require research efforts in the future.