Search Results (13)

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 (N^pro). 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 N^pro 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. Full article

Bovine viral diarrhea virus (BVDV), a pestivirus in the family Flaviviridae, is a major livestock pathogen. Horizontal transmission leads to acute transient infections via the oronasal route, whereas vertical transmission might lead to the birth of immunotolerant, persistently infected animals. In both cases, BVDV exerts an immunosuppressive effect, predisposing infected animals to secondary infections. E^rns, an immunomodulatory viral protein, is present on the envelope of the virus and is released as a soluble protein. In this form, it is taken up by cells and, with its RNase activity, degrades single- and double-stranded (ds) RNA, thus preventing activation of the host’s interferon system. Here, we show that E^rns of the pestiviruses BVDV and Bungowannah virus effectively inhibit dsRNA-induced IFN synthesis in well-differentiated airway epithelial cells cultured at the air–liquid interface. This activity was observed independently of the side of entry, apical or basolateral, of the pseudostratified, polarized cell layer. Virus infection was successful from both surfaces but was inefficient, requiring several days of incubation. Virus release was almost exclusively restricted to the apical side. This confirms that primary, well-differentiated respiratory epithelial cells cultured at the air–liquid interface are an appropriate model to study viral infection and innate immunotolerance in the bovine respiratory tract. Furthermore, evidence is presented that E^rns might contribute to the immunosuppressive effect observed after BVDV infections, especially in persistently infected animals. Full article

Some viruses can suppress superinfections of their host cells by related or different virus species. The phenomenon of superinfection exclusion can be caused by inhibiting virus attachment, receptor binding and entry, by replication interference, or competition for host cell resources. Blocking attachment and entry not only prevents unproductive double infections but also stops newly produced virions from re-entering the cell post-exocytosis. In this study, we investigated the exclusion of superinfections between the different pestivirus species. Bovine and porcine cells pre-infected with non-cytopathogenic pestivirus strains were evaluated for susceptibility to subsequent superinfection using comparative titrations. Our findings revealed significant variation in exclusion potency depending on the pre- and superinfecting virus species, as well as the host cell species. Despite this variability, all tested classical pestivirus species reduced host cell susceptibility to subsequent infections, indicating a conserved entry mechanism. Unexpectedly, pre-infection with atypical porcine pestivirus (APPV) increased host cell susceptibility to classical pestiviruses. Further analysis showed that APPV can infect SK-6 cells independently of ADAM17, a critical attachment factor for the classical pestiviruses. These results indicate that APPV uses different binding and entry mechanisms than the other pestiviruses. The observed increase in the susceptibility of cells post-APPV infection warrants further investigation and could have practical implications, such as aiding challenging pestivirus isolation from diagnostic samples. Full article

Linda virus (LindaV) was first identified in a pig farm in Styria, Austria in 2015 and associated with congenital tremor (CT) type A-II in newborn piglets. Since then, only one more LindaV affected farm was retrospectively discovered 10 km away from the initially affected farm. Here, we report the recent outbreak of a novel LindaV strain in a farrow-to-finish farm in the federal state Carinthia, Austria. No connection between this farm and the previously affected farms could be discovered. The outbreak was characterized by severe CT cases in several litters and high preweaning mortality. A herd visit two months after the onset of clinical symptoms followed by a diagnostic workup revealed the presence of several viremic six-week-old nursery pigs. These animals shed large amounts of virus via feces and saliva, implying an important epidemiological role for within- and between-herd virus transmission. The novel LindaV strain was isolated and genetically characterized. The findings underline a low prevalence of LindaV in the Austrian pig population and highlight the threat when introduced into a pig herd. Furthermore, the results urge the need to better understand the routes of persistence and transmission of this enigmatic pestivirus in the pig population. Full article

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. Full article

Marker or DIVA (differentiation of infected from vaccinated animals) vaccines are beneficial tools for the eradication of animal diseases in regions with a high prevalence of the designated disease. Bovine viral diarrhea virus (BVDV)-1 (syn. Pestivirus A) is a flavivirus that infects predominantly cattle resulting in major economic losses. An increasing number of countries have implemented BVDV eradication programs that focus on the detection and removal of persistently infected cattle. No efficient marker or DIVA vaccine is yet commercially available to drive the eradication success, to prevent fetal infection and to allow serological monitoring of the BVDV status in vaccinated farms. Bungowannah virus (BuPV, species Pestivirus F), a related member of the genus Pestivirus with a restricted prevalence to a single pig farm complex in Australia, was chosen as the genetic backbone for a marker vaccine candidate. The glycoproteins E1 and E2 of BuPV were substituted by the heterologous E1 and E2, which are major immunogens, of the BVDV-1 strain CP7. In addition, the candidate vaccine was further attenuated by the introduction of a deletion within the N^pro protein coding sequence, a major type I interferon inhibitor. Immunization of cattle with the chimeric vaccine virus BuPV_ΔN^pro_E1E2 CP7 (modified live or inactivated) followed by a subsequent experimental challenge infection confirmed the safety of the prototype strain and provided a high level of clinical protection against BVDV-1. The serological discrimination of vaccinated cattle could be enabled by the combined detection of BVDV-1 E2- in the absence of both BVDV NS3- and BVDV E^rns-specific antibodies. The study demonstrates for the first time the generation and application of an efficient BVDV-1 modified double marker vaccine candidate that is based on the genetic background of BuPV accompanied by commercially available serological marker ELISA systems. Full article

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. Full article

Bovine viral diarrhea virus (BVDV), a pestivirus which exists in the two distinct species BVDV-1 (syn. Pestivirus A) and BVDV-2 (syn. Pestivirus B), is the causative agent of one of the most widespread and economically important virus infections in cattle. For economic as well as for animal health reasons, an increasing number of national BVDV control programs were recently implemented. The main focus lies on the detection and removal of persistently infected cattle. The application of efficient marker or DIVA (differentiation of infected from vaccinated animals) vaccines would be beneficial for the eradication success in regions with a high BVDV prevalence to prevent fetal infection and it would allow serological monitoring of the BVDV status also in vaccinated farms. Therefore, a marker vaccine based on the cytopathic (cp) BVDV-1b strain CP7 was constructed as a synthetic backbone (BVDV-1b_synCP7). For serological discrimination of vaccinated from infected animals, the viral protein E^rns was substituted by the heterologous E^rns of Bungowannah virus (BuPV, species Pestivirus F). In addition, the vaccines were attenuated by a deletion within the type I interferon inhibitor N^pro protein encoding sequence. The BVDV-2 vaccine candidate is based on the genetic sequence of the glycoproteins E1 and E2 of BVDV-2 strain CS8644 (CS), which were introduced into the backbone of BVDV-1b_synCP7_ΔN^pro_E^rns Bungo in substitution of the homologous glycoproteins. Vaccine virus recovery resulted in infectious cytopathic virus chimera that grew to titers of up to 10⁶ TCID₅₀/mL. Both synthetic chimera BVDV-1b_synCP7_ΔN^pro_E^rns Bungo and BVDV-1b_synCP7_ΔN^pro_E^rns Bungo_E1E2 BVDV-2 CS were avirulent in cattle, provided a high level of protection in immunization and challenge experiments against both BVDV species and allowed differentiation of infected from vaccinated cattle. Our study presents the first report on an efficient BVDV-1 and -2 modified live marker vaccine candidate and the accompanying commercially available serological marker ELISA system. Full article

Bungowannah virus is a novel pestivirus identified from a disease outbreak in a piggery in Australia in June 2003. The aim of this study was to determine whether infection of pregnant pigs with Bungowannah virus induces the clinical signs and gross pathology observed during the initial outbreak and how this correlates with the time of infection. Twenty-four pregnant pigs were infected at one of four stages of gestation (approximately 35, 55, 75 or 90 days). The number of progeny born alive, stillborn or mummified, and signs of disease were recorded. Some surviving piglets were euthanased at weaning and others at ages up to 11 months. All piglets were subjected to a detailed necropsy. The greatest effects were observed following infection at 35 or 90 days of gestation. Infection at 35 days resulted in a significant reduction in the number of pigs born alive and an increased number of mummified foetuses (18%) and preweaning mortalities (70%). Preweaning losses were higher following infection at 90 days of gestation (29%) and were associated with sudden death and cardiorespiratory signs. Stunting occurred in chronically and persistently infected animals. This study reproduced the clinical signs and gross pathology of the porcine myocarditis syndrome and characterised the association between the time of infection and the clinical outcome. Full article

Reverse genetics systems are powerful tools for functional studies of viral genes or for vaccine development. Here, we established DNA-launched reverse genetics for the pestivirus Bungowannah virus (BuPV), where cDNA flanked by a hammerhead ribozyme sequence at the 5′ end and the hepatitis delta ribozyme at the 3′ end was placed under the control of the CMV RNA polymerase II promoter. Infectious recombinant BuPV could be rescued from pBuPV-DNA-transfected SK-6 cells and it had very similar growth characteristics to BuPV generated by conventional RNA-based reverse genetics and wild type BuPV. Subsequently, DNA-based E^RNS deleted BuPV split genomes (pBuPV∆E^RNS/E^RNS)—co-expressing the E^RNS protein from a separate synthetic CAG promoter—were constructed and characterized in vitro. Overall, DNA-launched BuPV genomes enable a rapid and cost-effective generation of recombinant BuPV and virus mutants, however, the protein expression efficiency of the DNA-launched systems after transfection is very low and needs further optimization in the future to allow the use e.g., as vaccine platform. Full article

Bungowannah virus is a novel porcine pestivirus identified in a disease outbreak in Australia in 2003. The aim of this study was to determine the outcome of infection of the pregnant pig with this virus. Twenty-four pregnant pigs were infected at days 35, 55, 75 or 90 of gestation. Blood, tonsillar and rectal swabs were collected from each pig at birth and then weekly until euthanasia or death. Tissues were sampled at necropsy. Viral load was measured by real-time reverse-transcription polymerase chain reaction (qRT-PCR) and antibody levels in serum by peroxidase-linked immunoassay. Bungowannah virus was detected in the serum and excretions of all infected pigs at birth regardless of the stage of gestation at which infection occurred. Persistent infections occurred following infection prior to the development of foetal immunocompetence. Unexpectedly some animals infected at day 55 of gestation later cleared the virus and seroconverted. Viraemia and viral shedding resolved quickest following infection in late gestation. Full article

Bungowannah virus is a pestivirus known to cause reproductive losses in pigs. The virus has not been found in other species, nor is it known if it has the capacity to cause disease in other animals. Eight sheep, eight calves and seven pregnant cows were experimentally infected with Bungowannah virus. It was found that sheep and calves could be infected. Furthermore, it was shown that the virus is able to cross the bovine placenta and cause infection of the foetus. These findings demonstrate the potential for species other than pigs to become infected with Bungowannah virus and the need to prevent them from becoming infected. Full article

Several novel porcine pestiviruses that are linked to disease outbreaks in commercial pig farms were discovered during recent years. Bungowannah pestivirus (BuPV; new species Pestivirus F) causes sudden death in young pigs, but has only ever been isolated in the Australian region Bungowannah. Atypical porcine pestivirus (APPV; new species Pestivirus K) on the other hand has been found in multiple countries worldwide and is potentially linked to congenital tremor, a disease that causes considerable production problems in pig farms. To assess the seroprevalences of both viruses in German commercial farms during the years 2009/10 and 2018, two approaches were selected. Antibodies against Pestivirus F were detected by a traditional in-house indirect immunofluorescence test against the culture-grown virus isolate, while for the detection of Pestivirus K-specific antibodies, a newly developed test system utilizing a chimeric construct of bovine viral diarrhea virus 1 (BVDV-1; species Pestivirus A) containing the E1 and E2 encoding sequences of APPV was established. A total of 1115 samples originating from 122 farms located in seven German federal states were investigated. Antibodies against Bungowannah virus could not be detected, confirming the absence of this virus in other regions than the initially affected Australian pig farm complex. In contrast, antibodies against APPV were highly prevalent throughout Germany at both investigated time points. The seroprevalence at the state level fluctuated to some degree, but the overall percentage remained stable, as is to be expected for an endemic pestivirus lacking any form of control measures. Full article