Autonomously Replicating RNAs of Bungowannah Pestivirus: ERNS Is Not Essential for the Generation of Infectious Particles.

Autonomously replicating subgenomic Bungowannah virus (BuPV) RNAs (BuPV replicons) with deletions of the genome regions encoding the structural proteins C, ERNS, E1, and E2 were constructed on the basis of an infectious cDNA clone of BuPV. Nanoluciferase (Nluc) insertion was used to compare the replication efficiencies of all constructs after electroporation of in vitro-transcribed RNA from the different clones. Deletion of C, E1, E2, or the complete structural protein genome region (C-ERNS-E1-E2) prevented the production of infectious progeny virus, whereas deletion of ERNS still allowed the generation of infectious particles. However, those ΔERNS viral particles were defective in virus assembly and/or egress and could not be further propagated for more than three additional passages in porcine SK-6 cells. These "defective-in-third-cycle" BuPV ΔERNS mutants were subsequently used to express the classical swine fever virus envelope protein E2, the N-terminal domain of the Schmallenberg virus Gc protein, and the receptor binding domain of the Middle East respiratory syndrome coronavirus spike protein. The constructs could be efficiently complemented and further passaged in SK-6 cells constitutively expressing the BuPV ERNS protein. Importantly, BuPVs are able to infect a wide variety of target cell lines, allowing expression in a very wide host spectrum. Therefore, we suggest that packaged BuPV ΔERNS replicon particles have potential as broad-spectrum viral vectors.IMPORTANCE The proteins NPRO and ERNS are unique for the genus Pestivirus, but only NPRO has been demonstrated to be nonessential for in vitro growth. While this was also speculated for ERNS, it has always been previously shown that pestivirus replicons with deletions of the structural proteins ERNS, E1, or E2 did not produce any infectious progeny virus in susceptible host cells. Here, we demonstrated for the first time that BuPV ERNS is dispensable for the generation of infectious virus particles but still important for efficient passaging. The ERNS-defective BuPV particles showed clearly limited growth in cell culture but were capable of several rounds of infection, expression of foreign genes, and highly efficient trans-complementation to rescue virus replicon particles (VRPs). The noncytopathic characteristics and the absence of preexisting immunity to BuPV in human populations and livestock also provide a significant benefit for a possible use, e.g., as a vector vaccine platform.

Immunoglobulin G of systemic sclerosis patients programs a pro-inflammatory and profibrotic phenotype in monocyte-like THP-1 cells.

OBJECTIVES: Functional IgG autoantibodies against diverse G protein-coupled receptors, i.e. antibodies with agonistic or antagonistic activity at these receptors, are abundant in human serum. Their levels are altered in patients with SSc, and autoantibodies against angiotensin II receptor 1 (ATR1) and endothelin receptor A (ETA) have been suggested to drive SSc by inducing the chemokines CXCL8 and CCL18 in the blood. The objective of our study is to profile the effect of IgG in SSc (SSc-IgG) on the production of soluble mediators in monocytic cells. METHODS: Monocyte-like THP-1 cells were stimulated with SSc-IgG and their secretome was analysed. Furthermore, the significance of major pro-inflammatory pathways for the induction of CXCL8 and CCL18 in response to SSc-IgG was assessed by a pharmacological approach. RESULTS: Stimulation with SSc-IgG significantly alters the secretome of THP-1 cells towards a general pro-inflammatory and profibrotic phenotype, which includes an increase of CCL18 and CXCL8. The consequent expression profiles vary depending on the individual donor of the SSc-IgG. CCL18 and CXCL8 expression is thus regulated differentially, with AP-1 driving the induction of both CCL18 and CXCL8 and the TAK/IKK-ß/NF-κB pathway and ERK1/2 driving that of CXCL8. CONCLUSIONS: Our results suggest that SSc-IgG contributes to the generation of the pro-inflammatory/profibrotic tissue milieu characteristic of SSc by its induction of a respective phenotype in monocytes. Furthermore, our results highlight AP-1 as a critical regulator of gene transcription of CCL18 in monocytic cells and as a promising pharmacological therapeutic target for the treatment of SSc.

Bungowannah virus in the affected pig population: a retrospective genetic analysis.

Bungowannah virus, which belongs to the genus Pestivirus within the family Flaviviridae, has been associated with myocarditis and a high incidence of stillbirths in pigs. In 2003, the virus was initially detected in a large pig farming complex on two separate sites in New South Wales, Australia. Until now, it has not been detected at other locations. Despite a program of depopulation and disinfection, the virus could be only eradicated from one of the affected farm complexes, the Bungowannah unit, but became endemic on the second complex, the Corowa unit. In the present study, the genetic variability of virus isolates collected between 2003 and 2014 in the endemically infected population has been retrospectively investigated. Phylogenetic analysis carried out based on sequences of the E2 and NS5B coding regions and the full-length open-reading frame revealed that the isolates from the different farm sites are closely related, but that samples collected between 2010 and 2014 at the Corowa farm site clustered in a different branch of the phylogenetic tree. Since 2010, a high-genetic stability of this RNA virus within the Corowa farm complex, probably due to an effective adaptation of the virus to the affected pig population, could be observed.

Single-Round Infectious Particle Production by DNA-Launched Infectious Clones of Bungowannah Pestivirus.

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 ERNS deleted BuPV split genomes (pBuPV∆ERNS/ERNS)-co-expressing the ERNS 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.

Seroprevalences of Newly Discovered Porcine Pestiviruses in German Pig Farms.

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.