Structures of the Hepaci-, Pegi-, and Pestiviruses envelope proteins suggest a novel membrane fusion mechanism.

Enveloped viruses encode specialised glycoproteins that mediate fusion of viral and host membranes. Discovery and understanding of the molecular mechanisms of fusion have been achieved through structural analyses of glycoproteins from many different viruses, and yet the fusion mechanisms of some viral genera remain unknown. We have employed systematic genome annotation and AlphaFold modelling to predict the structures of the E1E2 glycoproteins from 60 viral species in the Hepacivirus, Pegivirus, and Pestivirus genera. While the predicted structure of E2 varied widely, E1 exhibited a very consistent fold across genera, despite little or no similarity at the sequence level. Critically, the structure of E1 is unlike any other known viral glycoprotein. This suggests that the Hepaci-, Pegi-, and Pestiviruses may possess a common and novel membrane fusion mechanism. Comparison of E1E2 models from various species reveals recurrent features that are likely to be mechanistically important and sheds light on the evolution of membrane fusion in these viral genera. These findings provide new fundamental understanding of viral membrane fusion and are relevant to structure-guided vaccinology.

Development and application of an indirect ELISA for detection of antibodies against emerging atypical porcine pestivirus.

BACKGROUND: Atypical porcine pestivirus (APPV) is a newly discovered swine pestivirus, which can cause congenital tremor and high mortality in newborn piglets and subclinical infection in adult pigs, leading to significant impacts on the pig industry. Currently, there is no approved serological method to assess APPV infection status in pig farms. METHODS: In this study, the envelope glycoprotein E2 of APPV was highly expressed in suspension HEK293 cells, and further an indirect enzyme-linked immunosorbent assay based on the recombinant E2 protein (E2-iELISA) was developed and evaluated. RESULTS: The reaction parameters of the E2-iELISA were optimized, and the cutoff value was determined to be 0.2 by analyzing S/P values of 165 negative sera against APPV that were confirmed by virus neutralization test (VNT). Specificity test showed that the method had no cross-reaction with other common swine viruses. The E2-iELISA was evaluated using a panel of swine sera, and showed high sensitivity (113/120, 94.2%) and specificity (65/70, 92.9%), and the agreement rate with VNT was 93.7% (178/190). Subsequently, the E2-iELISA was utilized to investigate the seroprevalence of APPV in pig herds of China. When detecting 1368 pig serum samples collected from nine provinces in China, the overall seroprevalence of APPV was 73.9% (1011/1368). CONCLUSION: Our findings suggest that the E2-iELISA is specific and sensitive, and could be a valuable tool for serological surveillance of APPV infection in pigs.

Endogenous Viral Elements in Shrew Genomes Provide Insights into Pestivirus Ancient History.

As viral genomic imprints in host genomes, endogenous viral elements (EVEs) shed light on the deep evolutionary history of viruses, ancestral host ranges, and ancient viral-host interactions. In addition, they may provide crucial information for calibrating viral evolutionary timescales. In this study, we conducted a comprehensive in silico screening of a large data set of available mammalian genomes for EVEs deriving from members of the viral family Flaviviridae, an important group of viruses including well-known human pathogens, such as Zika, dengue, or hepatitis C viruses. We identified two novel pestivirus-like EVEs in the reference genome of the Indochinese shrew (Crocidura indochinensis). Homologs of these novel EVEs were subsequently detected in vivo by molecular detection and sequencing in 27 shrew species, including 26 species representing a wide distribution within the Crocidurinae subfamily and one in the Soricinae subfamily on different continents. Based on this wide distribution, we estimate that the integration event occurred before the last common ancestor of the subfamily, about 10.8 million years ago, attesting to an ancient origin of pestiviruses and Flaviviridae in general. Moreover, we provide the first description of Flaviviridae-derived EVEs in mammals even though the family encompasses numerous mammal-infecting members. This also suggests that shrews were past and perhaps also current natural reservoirs of pestiviruses. Taken together, our results expand the current known Pestivirus host range and provide novel insight into the ancient evolutionary history of pestiviruses and the Flaviviridae family in general.

DNAJC14-Independent Replication of the Atypical Porcine Pestivirus.

Atypical porcine pestiviruses (APPV; Pestivirus K) are a recently discovered, very divergent species of the genus Pestivirus within the family Flaviviridae. The presence of APPV in piglet-producing farms is associated with the occurrence of so-called "shaking piglets," suffering from mild to severe congenital tremor type A-II. Previous studies showed that the cellular protein DNAJC14 is an essential cofactor of the NS2 autoprotease of all classical pestiviruses. Consequently, genetically engineered DNAJC14 knockout cell lines were resistant to all tested noncytopathogenic (non-cp) pestiviruses. Surprisingly, we found that the non-cp APPV can replicate in these cells in the absence of DNAJC14, suggesting a divergent mechanism of polyprotein processing. A complete laboratory system for the study of APPV was established to learn more about the replication of this unusual virus. The inactivation of the APPV NS2 autoprotease using reverse genetics resulted in nonreplicative genomes. To further investigate whether a regulation of the NS2-3 cleavage is also existing in APPV, we constructed synthetic viral genomes with deletions and duplications leading to the NS2 independent release of mature NS3. As observed with other pestiviruses, the increase of mature NS3 resulted in elevated viral RNA replication levels and increased protein expression. Our data suggest that APPV exhibit a divergent mechanism for the regulation of the NS2 autoprotease activity most likely utilizing a different cellular protein for the adjustment of replication levels. IMPORTANCE DNAJC14 is an essential cofactor of the pestiviral NS2 autoprotease, limiting replication to tolerable levels as a prerequisite for the noncytopathogenic biotype of pestiviruses. Surprisingly, we found that the atypical porcine pestivirus (APPV) is able to replicate in the absence of DNAJC14. We further investigated the NS2-3 processing of APPV using a molecular clone, monoclonal antibodies, and DNAJC14 knockout cells. We identified two potential active site residues of the NS2 autoprotease and could demonstrate that the release of NS3 by the NS2 autoprotease is essential for APPV replication. Defective interfering genomes and viral genomes with duplicated NS3 sequences that produce mature NS3 independent of the NS2 autoprotease activity showed increased replication and antigen expression. It seems likely that an alternative cellular cofactor controls NS2-3 cleavage and thus replication of APPV. The replication-optimized synthetic APPV genomes might be suitable live vaccine candidates, whose establishment and testing warrant further research.

The NS4A Protein of Classical Swine Fever Virus Suppresses RNA Silencing in Mammalian Cells.

RNA interference (RNAi) is a significant posttranscriptional gene silencing mechanism and can function as an antiviral immunity in eukaryotes. However, numerous viruses can evade this antiviral RNAi by encoding viral suppressors of RNA silencing (VSRs). Classical swine fever virus (CSFV), belonging to the genus Pestivirus, is the cause of classical swine fever (CSF), which has an enormous impact on animal health and the pig industry. Notably, little is known about how Pestivirus blocks RNAi in their host. In this paper, we uncovered that CSFV NS4A protein can antagonize RNAi efficiently in mammalian cells by binding to double-stranded RNA and small interfering RNA. In addition, the VSR activity of CSFV NS4A was conserved among Pestivirus. Furthermore, the replication of VSR-deficient CSFV was attenuated but could be restored by the deficiency of RNAi in mammalian cells. In conclusion, our studies uncovered that CSFV NS4A is a novel VSR that suppresses RNAi in mammalian cells and shed new light on knowledge about CSFV and other Pestivirus. IMPORTANCE It is well known that RNAi is an important posttranscriptional gene silencing mechanism that is also involved in the antiviral response in mammalian cells. While numerous viruses have evolved to block this antiviral immunity by encoding VSRs. Our data demonstrated that the NS4A protein of CSFV exhibited a potent VSR activity through binding to dsRNA and siRNA in the context of CSFV infection in mammalian cells, which are a conservative feature among Pestivirus. In addition, the replication of VSR-deficient CSFV was attenuated but could be restored by the deficiency of RNAi, providing a theoretical basis for the development of other important attenuated Pestivirus vaccines.

Genome characteristics of atypical porcine pestivirus from abortion cases in Shandong Province, China.

BACKGROUND: Atypical porcine pestivirus (APPV) is a novel, highly variable porcine pestivirus. Previous reports have suggested that the virus is associated with congenital tremor (CT) type A-II in piglets, and little information is available about the correlation between the virus and sow abortion, or on coinfection with other viruses. In China, reported APPV strains were mainly isolated from South China and Central China, and data about the APPV genome from northern China are relatively scarce. METHODS: Eleven umbilical cords, one placenta, and one aborted piglet, were collected from aborted sows of the same farm in Shandong Province of northern China. Nucleic acids were extracted from the above samples, and subsequently pooled for viral metagenomics sequencing and bioinformatics analysis. The viral coexistence status and complete genome characteristics of APPV in Shandong Province were determined. RESULTS: In abortion cases, APPV was present with Getah virus, porcine picobirnavirus, porcine kobuvirus, porcine sapovirus, Po-Circo-like virus, porcine serum-associated circular virus, porcine bocavirus 1, porcine parvovirus 1, porcine parvovirus 3 and porcine circovirus 3, etc. The first complete genome sequence(11,556 nt) of APPV in Shandong Province of northern China, was obtained using viral metagenomics and designated APPV-SDHY-2022. Comparison with Chinese reference strains revealed that the polyprotein of APPV-SDHY-2022 shared 82.6-84.2%, 93.2-93.6%, and 80.7-85% nucleotide identity and 91.4-92.4%, 96.4-97.7%, and 90.6-92.2% amino acid identity with those of the Clade I, Clade II and Clade III strains, respectively. Phylogenetic analysis based on the complete polyprotein CDS and NS5A sequences concluded that APPV-SDHY-2022 belongs to Clade II. Analysis of the NS5A nucleotide sequences revealed homology of greater than 94.6% for the same isoform, 84.7-94.5% for different isoforms of the same clade and 76.8-81.1% for different clades. Therefore, Clade II was further divided into three subclades, and APPV-SDHY-2022 belonged to subclade 2.3. Members of Clade II have 20 unique amino acids in individual proteins, distinguishing them from Clade I and Clade III members. The E2 protein showed the greatest diversity of putative N-glycosylation sites with 9 patterns, and APPV-SDHY-2022 along with other Chinese APPV strains shared the conserved B-cell conformational epitope residues 39E, 70R, 173R, 190K and 191N of the E2 protein. CONCLUSIONS: We reported viral coexistence and the first complete genome sequence of APPV from abortion cases and from Shandong Province. The new APPV isolate belongs to an independent branch of Clade II. Our results increase the molecular and epidemiological understanding of APPV in China.

Isolation and artificial production of atypical porcine pestivirus, using the swine-kidney-derived cell line SK-L.

Congenital tremor (CT) in piglets was first reported in 1922, and although the causative pathogen was unknown for many years, atypical porcine pestivirus (APPV) was recently shown to be the cause. APPV is difficult to isolate, and there have been few reports of APPV isolated from field materials. Here, we successfully isolated infectious particles from a tonsillar emulsion from a CT-affected piglet using the established swine-kidney-derived cell line SK-L. In addition, we produced APPV artificially using these cells. Thus, SK-L cells are useful for both isolation and artificial production of APPV.

Screening for atypical porcine pestivirus in Swedish boar semen used for artificial insemination and a characterisation of the seminal RNA microbiome including the virome.

BACKGROUND: This study aimed to characterise the RNA microbiome, including the virome of extended semen from Swedish breeding boars, with particular focus on Atypical porcine pestivirus (APPV). This neurotropic virus, associated with congenital tremor type A-II in piglets, was recently demonstrated to induce the disease through insemination with semen from infected boars. RESULTS: From 124 Artificial Insemination (AI) doses from Swedish breeding boars, APPV was detected in one dose in addition to a sparse seminal RNA virome, characterised by retroviruses, phages, and some fecal-associated contaminants. The detected seminal microbiome was large and characterized by Gram-negative bacteria from the phylum Proteobacteria, mainly consisting of apathogenic or opportunistic bacteria. The proportion of bacteria with a pathogenic potential was low, and no antimicrobial resistance genes (ARGs) were detected in the datasets. CONCLUSION: Overall, the results indicate a good health status among Swedish breeding boars. The detection of APPV in semen raises the question of whether routine screening for APPV in breeding boars should be instigated.

Constant testing for Pestivirus in cell lines reveals different routes of contamination.

Pestivirus can contaminate cell cultures and sera and cause serious problems that evolve the integrity of studies, confidence in diagnostic results, and safety of human and animal vaccines. Contaminations by Pestivirus and other viruses may occur at any time and regular assays of monitoring in cell cultures and your supplies are necessary. This study aimed to analyze the phylogeny of Pestivirus detected from cell cultures, calf serum, and standard strains of three laboratories in Brazil that carry out frequent tests for the monitoring of cellular contaminations. These samples were submitted to phylogenetic analysis to understand the genetic relationship between contaminants occurring in these facilities. As result, the Pestivirus found in samples were Bovine viral diarrhea virus (BVDV-1 and BVDV-2), Hobi-like viruses (often named BVDV-3), and Classical swine fever virus (CSFV), and the phylogenetic analysis help us to infer at three possible routes of contamination in this work.

Development and Evaluation of Antigen-Specific Dual Matrix Pestivirus K ELISAs Using Longitudinal Known Infectious Status Samples.

Pestivirus K, commonly known as atypical porcine pestivirus (APPV), is the most common cause of congenital tremor (CT) in pigs. Currently, there is limited information on the infection dynamics of and immune response against APPV and no robust serologic assay to assess the effectiveness of preventative measures. To that end, known infection status samples were generated using experimental inoculation of cesarean-derived, colostrum-deprived pigs. Pigs (2 per pen) were inoculated with minimum essential medium (n = 6; negative control) or APPV (n = 16). Serum, pen-based oral fluid samples, and nasal swabs were collected through 70 days postinoculation (dpi). The immune response to recombinant APPV Erns, E2, or NS3 antigens was evaluated using both serum and oral fluids via indirect enzyme-linked immunosorbent assays (ELISAs). APPV was detected by real-time reverse transcription-PCR (RT-qPCR) in all oral fluid and serum samples from APPV-inoculated animals by 24 and 35 dpi, respectively. All samples remained genome positive until 70 dpi. Detection of nasal shedding was less consistent, with APPV being detected by RT-qPCR in all inoculated animals at 42, 49, and 56 dpi. Antibodies were first detected in oral fluids at 14 dpi, 10 days before serum detection, and concurrently with the first oral fluids RT-qPCR detection. Across sample types and time points, the Erns ELISA outperformed the other targets. In conclusion, both oral fluid and serum APPV Erns ELISAs can be used to economically evaluate the individual and herd status prior to and following intervention strategies.

Development of a quadruple PCR-based gene microarray for detection of vaccine and wild-type classical swine fever virus, African swine fever virus and atypical porcine pestivirus.

BACKGROUND: Classical swine fever (CSF), African swine fever (ASF), and atypical porcine pestivirus (APPV) are acute, virulent, and contagious viral diseases currently hampering the pig industry in China, which result in mummification or stillbirths in piglets and mortality in pigs. Diagnostic assays for the differentiation of infection and vaccination of CSFV, in addition to the detection of ASFV and APPV, are urgently required for better prevention, control, and elimination of these viral diseases in China. METHODS: A quadruple PCR-based gene microarray assay was developed in this study to simultaneously detect wild-type and vaccine CSFV strains, ASFV and APPV according to their conserved regions. Forty-two laboratory-confirmed samples, including positive samples of 10 other swine viral diseases, were tested using this assay to confirm its high specificity. RESULTS: This assay's limit of detections (LODs) for the wild-type and vaccine CSFV were 6.98 and 6.92 copies/µL. LODs for ASFV and APPV were 2.56 × 10 and 1.80 × 10 copies/µL, respectively. When compared with standard RT-PCR or qPCR for CSFV (GB/T 26875-2018), ASFV (MARR issue No.172), or APPV (CN108611442A) using 219 clinical samples, the coincidence was 100%. The results showed that this assay with high sensitivity could specifically distinguish ASFV, APPV, and CSFV, including CSFV infection and immunization. CONCLUSION: This assay provides a practical, simple, economic, and reliable test for the rapid detection and accurate diagnosis of the three viruses and may have good prospects for application in an epidemiological investigation, prevention, and control and elimination of these three diseases.

Genetic characterization of atypical porcine pestivirus from neonatal piglets with congenital tremor in Hubei province, China.

BACKGROUND: Atypical porcine pestivirus (APPV) is a single-stranded RNA virus with high genetic variation that causes congenital tremor (CT) in newborn piglets, belonging to the genus Pestivirus of the family Flaviviridae. Increasing cases of APPV infection in China in the past few years would pose severe challenges to the development of pig production. In view of the high genetic variability of APPV, the genetic characteristics of APPV in Hubei province was determined. METHODS: 52 tissue samples from 8 CT-affected newborn piglets were collected at two different periods in the same pig farm in Hubei province. Viral nucleic acid was extracted to detect pathogens that can cause CT in piglets or other common clinical pathogens by RT-PCR. Haematoxylin and eosin (HE) staining, immunohistochemical (IHC) analysis, and qRT-PCR were performed to observe histopathological changes and histological distribution, and detect the viral load of APPV in CT-affected piglets. The full-length genome of APPV was obtained and sequence analysis was conducted to determine the phylogenetic relationship. RESULTS: Histopathological observation and histological distribution analysis showed that the histological lesions and distribution of APPV were mainly in central nervous system (CNS) tissues and immune tissues. Viral load analysis revealed that the viral copy number was higher in the cerebellum, submaxillary lymph nodes, tonsil, and serum than in other tissues. Phylogenetic analysis showed that CH-HB2020 and CH-HB2021 belonged to Clade I.3, and is most closely related to APPV_CH-GX2016. Sequence alignment based on APPV encoding sequences (CDS) showed that the nucleotide identities of CH-HB2020 or CH-HB2021 with Clade I, Clade II, and Clade III strains were 83.5-98.6%, 83.1-83.5%, and 81.1-81.4%, respectively, while the amino acid identities were 91.9-99.2%, 91.2-95.3%, and 90.77-91.4%, respectively. No recombination event was observed in CH-HB2020 or CH-HB2021 strains. CONCLUSIONS: These findings enhance our understanding of the pathogenesis of APPV and may provide potential molecular evidence for its prevalence and transmission.

A new (old) bovine viral diarrhea virus 2 subtype: BVDV-2e.

Bovine pestiviruses are members of the species Pestivirus A (bovine viral diarrhea virus 1, BVDV-1), Pestivirus B (BVDV-2) or Pestivirus H (HoBiPeV). To date, BVDV-2 isolates/strains have been classified into three subtypes (a-c) by phylogenetic analysis, and an additional subtype (d) has been proposed based on 5' untranslated region (UTR) secondary structures. In a previous study, we identified some BVDV-2 sequences in the GenBank database that could not be classified as subtype a, b or c by phylogenetic analysis of their genomes, UTRs or individual genes. Here, we performed a detailed study of these sequences and assessed whether they might represent a distinct BVDV-2 subtype. Initially, we collected 85 BVDV-2 complete/near-complete genomes (CNCGs) from GenBank and performed a "proof of equivalence" between phylogenetic analyses based on CNCGs and open reading frames (ORFs), which showed that ORFs may be reliably used as a reference target for BVDV-2 phylogeny, allowing us to increase our dataset to 139 sequences. Among these, we found seven sequences that could not be classified as BVDV-2a-c. The same was observed in the phylogenetic analysis of CNCGs and viral genes. In addition, the seven non-BVDV-2a-c sequences formed a distinct cluster in all phylogenetic trees, which we propose to term BVDV-2e. BVDV-2e also showed 44 amino acid changes compared to BVDV-2a-c, 20 of which are in well-defined positions. Importantly, an additional phylogenetic analysis including BVDV-2d and a pairwise comparison of BVDV-2e and BVDV-2d sequences also supported the difference between these subtypes. Finally, we propose the recognition of BVDV-2e as a distinct BVDV-2 subtype and encourage its inclusion in future phylogenetic analyses to understand its distribution and evolution.

Development of a one-step multiplex qRT-PCR assay for the detection of African swine fever virus, classical swine fever virus and atypical porcine pestivirus.

BACKGROUND: African swine fever virus (ASFV), classical swine fever virus (CSFV) and atypical porcine pestivirus (APPV) have caused great economic losses to the swine industry in China. Since coinfections of ASFV, CSFV and APPV occur in certain pig herds, it is necessary to accurately and differentially detect these pathogens in field-collected samples. In this study, a one-step multiplex real-time quantitative reverse transcription-polymerase chain reaction (multiplex qRT-PCR) was developed for the simultaneous and differential detection of ASFV, CSFV and APPV. RESULTS: The one-step multiplex qRT-PCR presented here was able to simultaneously detect ASFV, CSFV and APPV but could not amplify other viruses, including porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), porcine parvovirus (PPV), porcine epidemic diarrhoea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PRoV), porcine deltacoronavirus (PDCoV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV-1), BVDV-2, etc. The limit of detection (LOD) of the assay was 2.52 × 101 copies/µL for ASFV, CSFV and APPV. A repeatability test using standard recombinant plasmids showed that the intra- and interassay coefficients of variation (CVs) were less than 2%. An assay of 509 clinical samples collected in Guangxi Province, southern China, from October 2018 to December 2020 showed that the positive rates of ASFV, CSFV and APPV were 45.58, 12.57 and 3.54%, respectively, while the coinfection rates of ASFV and CSFV, ASFV and APPV, CSFV and APPV were 4.91, 1.38, 0.98%, respectively. Phylogenetic analysis based on the nucleotide sequences of the partial ASFV p72 gene showed that all ASFV strains from Guangxi Province belonged to genotypes I and II. CONCLUSION: A one-step multiplex qRT-PCR with high specificity, sensitivity and repeatability was successfully developed for the simultaneous and differential detection of ASFV, CSFV and APPV.

Congenital tremor and splay leg in piglets - insights into the virome, local cytokine response, and histology.

BACKGROUND: Atypical porcine pestivirus (APPV) is a neurotropic virus associated with congenital tremor type A-II. A few experimental studies also indicate an association between APPV and splay leg. The overarching aim of the present study was to provide insights into the virome, local cytokine response, and histology of the CNS in piglets with signs of congenital tremor or splay leg. RESULTS: Characterization of the cytokine profile and virome of the brain in piglets with signs of congenital tremor revealed an APPV-associated upregulation of Stimulator of interferon genes (STING). The upregulation of STING was associated with an increased expression of the gene encoding IFN-α but no differential expression was recorded for the genes encoding CXCL8, IFN-ß, IFN-γ, IL-1ß, IL-6, or IL-10. No viral agents or cytokine upregulation could be detected in the spinal cord of piglets with signs of splay leg or in the brain of piglets without an APPV-infection. The histopathological examination showed no lesions in the CNS that could be attributed to the APPV-infection, as no difference between sick and healthy piglets could be seen. CONCLUSION: The results from this study provide evidence of an APPV-induced antiviral cytokine response but found no lesions related to the infection nor any support for a common causative agent.

First detection of emerging HoBi-like Pestivirus (BVD-3) among some persistently infected dairy cattle herds in Egypt.

Bovine viral diarrhoea virus (BVDV) is a serious veterinary health concern worldwide. We conducted this study to determine the prevalence of persistent infections (PI) and identify the current strain among some dairy cattle herds in Egypt. A total of 240 serum samples were collected from six Egyptian provinces. Between 2019 and 2020, samples were tested by Enzyme linked immunosorbent assay (ELISA) for detection of PI animals, and then molecular characterization was performed. Six calves were found PI with a prevalence of 2.5% (6/240). Using molecular characterization, HoBi-like Pestivirus (BVD-3) was successfully identified in Egypt for the first time. Based on the BVD-3 reference strains on Genbank, the detected strains had an identity ranging from 98.8 to 99.6%. Partial nucleotide sequence of the 5'UTR gene for six tested samples was submitted to Genbank with accessions: OM324396, OM324397, OM324398, OM324399, OM3243100, and OM3243101.

Classical swine fever (CSF): towards new challenges / La peste porcine classique (PPC) : vers de nouveaux défis

Classical swine fever (CSF) is a highly contagious swine-specific disease which may have a huge economic impact for porcine production. CSF is caused by a virus belonging to the Pestivirus genus, which has expanded for the past 5 years with the discovery of new species whose genetic proximity to the CSF virus could further complicate laboratory diagnosis. The various forms of the disease, and in particular the increased frequency of attenuated forms, linked to an evolution of CSF virus strains towards a reduction in their virulence, delay clinical diagnosis. Thus, a long period may elapse before an outbreak is detected, allowing the virus to circulate longer, with the risk of spreading to distant geographical areas. Efforts must be maintained in terms of surveillance and diagnostic tools development in order to detect CSF virus infection early and thus limit the spread of the disease and facilitate control measures.

La peste porcine classique (PPC) est une maladie très contagieuse, spécifique des suidés, qui continue à constituer une menace pour la production porcine malgré un statut indemne de la plupart des pays de l'Union européenne. La PPC est causée par un virus de la famille des Flaviviridae appartenant au genre Pestivirus, en extension depuis les cinq dernières années avec la découverte de nouvelles espèces, notamment chez le porc ou autres animaux de rente dont la proximité génétique avec le virus de la PPC pourrait davantage compliquer le diagnostic de laboratoire. La diversité des formes de la maladie, et notamment la fréquence accrue des formes atténuées et inapparentes liée à une évolution des souches du virus de la PPC vers une réduction de leur virulence, retarde le diagnostic clinique. Ainsi, une longue période peut s'écouler avant la détection d'un foyer, permettant au virus de la PPC de circuler plus longuement, avec le risque de diffuser vers des zones géographiques éloignées des premiers cas d'infection. Les efforts doivent être maintenus en termes de surveillance et de développement d'outils de diagnostic afin de détecter précocement une infection par le virus de la PPC et ainsi limiter la propagation de la maladie et faciliter les mesures de contrôle.

A novel NS3/4A protease dependent cleavage site within pestiviral NS2.

Pestiviruses like bovine viral diarrhoea virus (BVDV) and classical swine fever virus (CSFV) belong to the family Flaviviridae. A special feature of the Flaviviridae is the importance of nonstructural (NS) proteins for both genome replication and virion morphogenesis. The NS2-3-4A region and its regulated processing by the NS2 autoprotease and the NS3/4A protease plays a central role in the pestiviral life cycle. We report the identification and characterization of a novel internal cleavage in BVDV NS2, which is mediated by the NS3/4A protease. Further mapping using the NS2 of BVDV-1 strain NCP7 showed that cleavage occurs between L188 and G189. This cleavage site represents a novel sequence motif recognized by the NS3/4A protease and is conserved between the pestivirus species A, B and D. Inhibition of this internal NS2 cleavage by mutating the cleavage site did not cause obvious effects on RNA replication or virion morphogenesis in cultured cell lines. Accordingly, this novel internal NS2 cleavage adds an additional layer to the already complex polyprotein processing of Pestiviruses and might further extend the repertoires of the multifunctional NS2. However, unravelling of the functional relevance of this novel processing event in NS2, therefore, awaits future in vivo studies.

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.

Downstream Sequences Control the Processing of the Pestivirus Erns-E1 Precursor.

Like other enveloped viruses, pestiviruses employ cellular proteases for processing of their structural proteins. While typical signal peptidase cleavage motifs are present at the carboxy terminus of the signal sequence preceding Erns and the E1/E2 and E2/P7 sites, the Erns-E1 precursor is cleaved by signal peptidase at a highly unusual structure, in which the transmembrane sequence upstream of the cleavage site is replaced by an amphipathic helix. As shown before, the integrity of the amphipathic helix is crucial for efficient processing. The data presented here demonstrate that the E1 sequence downstream of this cleavage site is also important for the cleavage. Carboxy-terminal truncation of the E1 moiety as well as internal deletions in E1 reduced the cleavage efficiency to less than 30% of the wild-type (wt) level. Moreover, the C-terminal truncation by more than 30 amino acids resulted in strong secretion of the uncleaved fusion proteins. The reduced processing and increased secretion were even observed when 10 to 5 amino-terminal residues of E1 were left, whereas extensions by 1 or 3 E1 residues resulted in reduced processing but no significantly increased secretion. In contrast to the E1 sequences, a 10-amino-acid c-myc tag fused to the Erns C terminus had only marginal effect on secretion but was also not processed efficiently. Mutation of the von Heijne sequence upstream of E2 not only blocked the cleavage between E1 and E2 but also prevented the processing between Erns and E2. Thus, processing at the Erns-E1 site is a highly regulated process.IMPORTANCE Cellular signal peptidase (SPase) cleavage represents an important step in maturation of viral envelope proteins. Fine tuning of this system allows for establishment of concerted folding and processing processes in different enveloped viruses. We report here on SPase processing of the Erns-E1-E2 glycoprotein precursor of pestiviruses. Erns-E1 cleavage is delayed and only executed efficiently when the complete E1 sequence is present. C-terminal truncation of the Erns-E1 precursor impairs processing and leads to significant secretion of the protein. The latter is not detected when internal deletions preserving the E1 carboxy terminus are introduced, but also these constructs show impaired processing. Moreover, Erns-E1 is only processed after cleavage at the E1/E2 site. Thus, processing of the pestiviral glycoprotein precursor by SPase is done in an ordered way and depends on the integrity of the proteins for efficient cleavage. The functional importance of this processing scheme is discussed in the paper.