Enhancing the understanding of coinfection outcomes: Impact of natural atypical porcine pestivirus infection on porcine reproductive and respiratory syndrome in pigs.

Atypical porcine pestivirus (APPV) is a novel member of the Pestivirus genus detected in association with congenital tremor (CT) type A-II outbreaks and from apparently healthy pigs, both as singular infection and as part of multi-pathogen infections. 'Classical' pestiviruses are known to cause immunosuppression of their host, which can increase susceptibility to secondary infections, severely impacting health, welfare, and production. To investigate APPV's effect on the host's immune system and characterise disease outcomes, 12 piglets from a natural APPV CT type A-II outbreak were experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV), a significant porcine pathogen. Rectal temperatures indicating febrile responses, viremia and viral-specific humoral and cellular responses were assessed throughout the study. Pathological assessment of the lungs and APPV-PRRSV co-localisation within the lungs was performed at necropsy. Viral co-localisation and pathological assessment of the lungs (Immunohistochemistry, BaseScope in situ hybridisation) were performed post-mortem. APPV status did not impact virological or immunological differences in PRRSV-infected groups. However, significantly higher rectal temperatures were observed in the APPV+ve/PRRSV+ve group over four days, indicating APPV increased the febrile response. Significant differences in the lung consolidation of the apical and intermediate lobes were also present, suggesting that APPV co-infection may augment lung pathology.

Influence of bovine pestivirus heterogeneity on serological responses to 10 different commercial vaccine formulation.

Bovine Pestivirus typically involves one or more organ systems, with clinical manifestations ranging from mild to severe fatal systemic illness that lead to significant reproductive, productive, and economic losses. Vaccines face the challenge of addressing the significant variability of pestiviruses, which affects the interaction between viral antigens and the immune system's ability to provide protection. This study aimed to evaluate the serological responses against bovine viral diarrhea virus 1 (Pestivirus A) and Pestivirus B induced by 10 commercial vaccines, including one recombinant (vaccine E), two modified live (MLV multivalent, vaccine I, and MLV monovalent, vaccine J), and seven killed vaccines (KLV, vaccines A to H). Additionally, we evaluated the cross-reactivity between Pestivirus A and B from vaccines and HoBi-like pestivirus (Pestivirus H). In Phase 1, guinea pigs were used to screen for non-MLVs. They were divided into nine groups (n=6 each) and received two doses (⅕ of bovine dose) of eight different non-MLV on Days 0 and 21. Serum samples were collected on Days 0 and 30 for serological analyse. In Phase 2, Holstein × Gir heifers (n= 45) were divided into five groups, comprising 6-9 animals. They were vaccinated either once with MLVs or twice with the top non-MLVs screened in Phase 1. Serum samples were harvested on d0 (vaccination day) and d60 (60 days after the first dose) for MLV and non-MLV. Specific antibody titers were assessed virus neutralization (VN) and transformed in log2 for statistical analysis using PROC-MIXED. Significant effects were observed for vaccine groups, time points, and their interactions concerning neutralizing antibodies against Pestivirus A and B in both Guinea pigs and heifers. The Phase 1 study revealed serological responses against Pestivirus A exclusively in non-MLV D (85.33±13.49) and E (72.00±19.26). In the bovine study, the KLD vaccine D (72.00±15.10), recombinant vaccine E (90.66±25.85), and MLV I (170.66±28.22) resulted in an average of neutralizing antibodies against Pestivirus A that exceeded the protective threshold (≥ 60). However,individual analysis of heifers showed a higher frequency of animals presenting titers of Pestivirus A Ab surpassing 32 following vaccination with MLV I and J. None of the vaccine formulations in either study elicited a protective immune response against Pestivirus B or demonstrated cross-reactivity against Pestivirus H.

Isolation and Molecular Evidence of Tunisian Sheep-like Pestivirus (Pestivirus N) in Persistently Infected Sheep in Northern Italy, 2023.

Over the last few decades, several pestiviruses have been discovered in ruminants, pigs, and, more recently, in non-ungulate hosts. Consequently, the nomenclature and taxonomy of pestiviruses have been updated. The Tunisian sheep-like pestivirus (TSV, Pestivirus N) is an additional ovine pestivirus genetically closely related to classical swine fever virus (CSFV). In this study, during a survey of pestivirus infections in ovine farms in the Lombardy region of Northern Italy, we identified and isolated a pestivirus strain from a sheep that was found to belong to Pestivirus N species based on its genomic nucleotide identity. The sheep itself and its lamb were found to be persistently infected. We performed molecular characterization and phylogenetic analysis of three viral genomic regions (a fragment of 5'-UTR, partial Npro, and the whole E2 region). In conclusion, these results confirmed circulating TSV in Northern Italy after notification in Sicily, Italy, and France. Correlation with Italian, Tunisian, and French strains showed that detection might have resulted from the trading of live animals between countries, which supports the need for health control measures.

New Generation Self-Replicating RNA Vaccines Derived from Pestivirus Genome.

While mRNA vaccines have shown their worth, they have the same failing as inactivated vaccines, namely they have limited half-life, are non-replicating, and therefore limited to the size of the vaccine payload for the amount of material translated. New advances averting these problems are combining replicon RNA (RepRNA) technology with nanotechnology. RepRNA are large self-replicating RNA molecules (typically 12-15 kb) derived from viral genomes defective in at least one essential structural protein gene. They provide sustained antigen production, effectively increasing vaccine antigen payloads over time, without the risk of producing infectious progeny. The major limitations with RepRNA are RNase-sensitivity and inefficient uptake by dendritic cells (DCs), which need to be overcome for efficacious RNA-based vaccine design. We employed biodegradable delivery vehicles to protect the RepRNA and promote DC delivery. Condensing RepRNA with polyethylenimine (PEI) and encapsulating RepRNA into novel Coatsome-replicon vehicles are two approaches that have proven effective for delivery to DCs and induction of immune responses in vivo.

Porcine low-density lipoprotein receptor plays an important role in classical swine fever virus infection.

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.

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.

Establishment of a superinfection exclusion method for pestivirus titration using a recombinant reporter pestiviruses.

Pestiviruses are classified into two biotypes based on their cytopathogenicity. As the majority of pestivirus field isolates are noncytopathogenic, their titration requires alternative methods rather than direct observation of cytopathogenic effects, such as immunostaining using specific antibodies or interference with cytopathogenic strains. However, these methods require microscopic observation to assess virus growth, which is time- and labor-intensive, especially when handling several samples. In this study, we developed a novel luciferase-based pestivirus titration method using the superinfection exclusion phenomenon with recombinant reporter pestiviruses that possessed an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). In this method, swine kidney cells were inoculated with classical swine fever virus (CSFV) and superinfected with the reporter CSFV vGPE-/HiBiT 5 days postinoculation. Virus titer was determined based on virus growth measured in luminescence using the culture fluid 3 days after superinfection; the resultant virus titer was comparable to that obtained by immunoperoxidase staining. Furthermore, this method has proven to be applicable for the titration of border disease virus (BDV) by superinfection with both the homologous reporter BDV and heterologous reporter CSFV, suggesting that this novel virus titration method is a simple technique for automated virus detection based on the luciferase system.

Comparison of bovine viral diarrhea virus detection methods: Results of an international proficiency trial.

Bovine viral diarrhea virus (BVDV), one of the most important infectious cattle diseases globally, is being combated in multiple countries. The main source for virus transmission within herds and especially to unaffected cattle farms are life-long persistently infected (PI), immunotolerant animals. Therefore, the early identification of PI calves is a major pillar of disease control programs. In addition, rapid and reliable virus identification is necessary to confirm the causative agent in acute clinical cases. Here, we initiated an international interlaboratory proficiency trial in order to evaluate BVDV detection methods. Four ear notch samples and four sera were provided to the participating veterinary diagnostic laboratories (n = 40). Two of the ear notches and two sera contained BVDV and two ear notches and one serum were negative for pestiviruses. The remaining serum was positive for the ovine border disease virus (BDV). The sample panel was analyzed by an ERNS-based ELISA for antigen detection, diverse real-time RT-PCR (RT-qPCR) assays and/or virus isolation. Occasionally, additional typing of the virus strains was performed by sequencing or specific antibody staining of the obtained cell culture isolates. While the antigen ELISA allowed reliable BVDV diagnostics, infectious virus could be isolated only in just under half of the attempts (43.33%). RT-qPCR enabled the sensitive detection of pestiviruses, though an impact of the extraction method on the resulting quantification cycle values was observed. In general, subsequent typing of the detected virus strains is required to differentiate BVDV from BDV infections. In conclusion, for BVDV identification in clinical cases or in the context of disease control, RT-qPCR methods or ERNS antigen ELISAs should be preferentially used.

Structural comparison of typical and atypical E2 pestivirus glycoproteins.

Pestiviruses, within the family Flaviviridae, are economically important viruses of livestock. In recent years, new pestiviruses have been reported in domestic animals and non-cloven-hoofed animals. Among them, atypical porcine pestivirus (APPV) and Norway rat pestivirus (NRPV) have relatively little sequence conservation in their surface glycoprotein E2. Despite E2 being the main target for neutralizing antibodies and necessary for cell attachment and viral fusion, the mechanism of viral entry remains elusive. To gain further insights into the pestivirus E2 mechanism of action and to assess its diversity within the genus, we report X-ray structures of the pestivirus E2 proteins from APPV and NRPV. Despite the highly divergent structures, both are able to dimerize through their C-terminal domain and contain a solvent-exposed ß-hairpin reported to be involved in host receptor binding. Functional analysis of this ß-hairpin in the context of BVDV revealed its ability to rescue viral infectivity.

An investigation into the transmission and control of pestivirus in sheep in Australia.

Border disease virus (BDV) is a member of the pestivirus genus that primarily affects sheep, causing reproductive losses through abortion, still births and the birth of weak lambs. The key characteristic of this disease is the birth of persistently infected (PI) lambs which, after surviving transplacental infection, are born antibody negative, yet virus positive, and thus shed the virus for their entire life and are the primary source of spread within a flock. The cornerstones of BDV control are detection and elimination of PI animals, biosecurity measures to prevent re-infection, and surveillance programs. Recommendations for the control of BDV in sheep are centred around the approach to bovine viral diarrhoea virus (BVDV), the prominent cattle pestivirus species, due to a lack of specific research into BDV control and elimination. In this study, two aspects of a BDV control program were investigated: the effectiveness of the BVDV vaccine, Pestigard®, and the rate of seroconversion in a flock deliberately exposed to known PI lambs. The vaccine appeared to be safe, and the optimal dose was the full cattle dose (2 mL). While vaccination induced high virus neutralising titres to BVDV when administered as either a quarter, half or full dose registered for cattle, the BDV titres achieved were low and unlikely to prevent transplacental infection. In a second study, after exposure of between 2 and 15 days exposure to two PI lambs in confined conditions, only 3 of 66 previously naïve sheep demonstrated seroconversion. This demonstrated a very low rate of transmission and suggested that deliberate exposure to PI lambs at low-risk times for less than 15 days was not likely to be an effective means of achieving seroconversion throughout a flock and, therefore, not provide protection against BDV challenge during gestation.

Isolation and Molecular Characterization of Atypical Porcine Pestivirus Emerging in China.

Atypical porcine pestivirus (APPV) is a recently discovered and very divergent species of the genus Pestivirus within the family Flaviviridae, which causes congenital tremor (CT) in newborn piglets. In this study, an APPV epidemiological investigation was conducted by studying 975 swine samples (562 tissue and 413 serum samples) collected from different parts of China from 2017 to 2021. The results revealed that the overall positive rate of the APPV genome was 7.08% (69/975), among which 50.7% (35/69) of the samples tested positive for one or more other common swine viruses, especially porcine circovirus type 2 (PCV2) with a coinfection rate of 36.2% (25/69). Subsequently, a novel APPV strain, named China/HLJ491/2017, was isolated in porcine kidney (PK)-15 cells for the first time from a weaned piglet that was infected with both APPV and PCV2. The new APPV isolate was confirmed by RT-PCR, sequencing, immunofluorescence assay, and transmission electron microscopy. After clearing PCV2, a pure APPV strain was obtained and further stably propagated in PK-15 cells for more than 30 passages. Full genome sequencing and phylogenetic analysis showed that the China/HLJ491/2017 strain was classified as genotype 2, sharing 80.8 to 97.6% of its nucleotide identity with previously published APPV strains. In conclusion, this study enhanced our knowledge of this new pestivirus and the successful isolation of the APPV strain provides critical material for the investigation of the biological and pathogenic properties of this emerging virus, as well as the development of vaccines and diagnostic reagents.

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.

[Pestiviruses in sheep and goats in Austria: Options for integration into the bovine viral diarrhea (BVDV) monitoring program]. / Pestiviren bei Schafen und Ziegen in ­Österreich: Möglichkeiten zur ­Einbindung in die BVDV-Überwachung.

INTRODUCTION: After the successful eradication of the bovine viral diarrhea virus (BVDV) in cattle in Austria, the risk of infections with the border disease virus (BDV) remains. Both viruses belong to the pestivirus genus. BDV infections lead to false-positive results in BVDV surveillance. This can be attributed to the contact to small ruminant populations. In particular, keeping cattle together with sheep or goats on a farm or alpine pasture are significant risk factors. Between 2015 and 2022, BDV type 3 was detected in 15 cattles in Austria. These animals were almost exclusively persistently infected calves. However, a positive antibody result for pestiviruses can lead to an extremely time-consuming and costly, and not always successful search for the source of the infection if no active virus excretor is found. This study documents how small ruminants can be integrated into pestivirus monitoring with a manageable amount of work and costs. 23 406 sheep and goat samples from two brucellosis surveillance programs in small ruminants were analyzed retrospectively. Blood samples were examined using pestivirus real-time pool RT-PCR (qPCR). Direct virus detection of BDV-3 was achieved in 40 sheep from five different federal states. Over the entire investigation period a further 37 detections of BDV-3 were found in cattle, sheep and goats outside of this study throughout Austria. This study accounts for 52 % of all border disease detections from 2015 to 2022. By including small ruminants in pestivirus monitoring, the disruptive factor BDV and the risk of its introduction into cattle herds can be significantly minimized in the future.

INTRODUCTION: Après l'éradication réussie du virus de la diarrhée virale bovine (BVDV) chez les bovins en Autriche, le risque d'infections par le virus de la Border Disease (BDV) demeure. Ces deux virus appartiennent au genre des pestivirus. Les infections par le BDV entraînent des résultats faussement positifs dans la surveillance du BVDV. Ce phénomène peut être attribué aux contacts avec les populations de petits ruminants. En particulier, la détention de bovins avec des moutons ou des chèvres sur une exploitation ainsi que les pâturages alpins sont des facteurs de risque importants pour les infections. Entre 2015 et 2022, le BDV de type 3 a été détecté chez 15 bovins en Autriche. Ces animaux étaient presque exclusivement des veaux infectés de manière persistante. Cependant, un résultat positif aux anticorps contre les pestivirus peut conduire à une recherche extrêmement longue et coûteuse et pas toujours fructueuse de la source de l'infection si aucun excréteur de virus actif n'est trouvé. Cette étude montre comment les petits ruminants peuvent être intégrés dans la surveillance des pestivirus avec une quantité de travail et des coûts gérables. À cette fin, 23 460 échantillons d'ovins et de caprins provenant de deux programmes de surveillance de la brucellose chez les petits ruminants ont été utilisés de façon rétrospective. Les échantillons de sang ont été examinés à l'aide de la RT-PCR en temps réel des pestivirus (qPCR). La détection directe du virus BDV-3 a été réalisée chez 40 moutons provenant de cinq länder différents. Sur l'ensemble de la période d'investigation (2015 ­ 2022), 37 autres détections de BDV-3 ont été effectuées chez des bovins, des ovins et des caprins en dehors de cette étude, dans toute l'Autriche. Cette étude représente 52 % de toutes les détections de Border Disease entre 2015 et 2022. En incluant les petits ruminants dans la surveillance des pestivirus, le facteur de perturbation qu'est le BDV et le risque de son introduction dans les troupeaux de bovins peuvent être considérablement minimisés à l'avenir.

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.

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.

Attachment, Entry, and Intracellular Trafficking of Classical Swine Fever Virus.

Classical swine fever virus (CSFV), which is a positive-sense, single-stranded RNA virus with an envelope, is a member of the Pestivirus genus in the Flaviviridae family. CSFV causes a severe and highly contagious disease in pigs and is prevalent worldwide, threatening the pig farming industry. The detailed mechanisms of the CSFV life cycle have been reported, but are still limited. Some receptors and attachment factors of CSFV, including heparan sulfate (HS), laminin receptor (LamR), complement regulatory protein (CD46), MER tyrosine kinase (MERTK), disintegrin, and metalloproteinase domain-containing protein 17 (ADAM17), were identified. After attachment, CSFV internalizes via clathrin-mediated endocytosis (CME) and/or caveolae/raft-dependent endocytosis (CavME). After internalization, CSFV moves to early and late endosomes before uncoating. During this period, intracellular trafficking of CSFV relies on components of the endosomal sorting complex required for transport (ESCRT) and Rab proteins in the endosome dynamics, with a dependence on the cytoskeleton network. This review summarizes the data on the mechanisms of CSFV attachment, internalization pathways, and intracellular trafficking, and provides a general view of the early events in the CSFV life cycle.

Dynamics of Infection of Atypical Porcine Pestivirus in Commercial Pigs from Birth to Market: A Longitudinal Study.

Atypical porcine pestivirus (APPV) was found to be associated with pigs demonstrating congenital tremors (CT), and clinical signs in pigs have been reproduced after experimental challenge. Subsequently, APPV has been identified in both symptomatic and asymptomatic swine of all ages globally. The objective of this research was to perform a longitudinal study following two cohorts of pigs, those born in litters with pigs exhibiting CT and those born in litters without CT, to analyze the virus and antibody dynamics of APPV infection in serum from birth to market. There was a wide range in the percentage of affected pigs (8-75%) within CT-positive litters. After co-mingling with CT-positive litters at weaning, pigs from CT-negative litters developed viremia that was cleared after approximately 2 months, with the majority seroconverting by the end of the study. In contrast, a greater percentage of pigs exhibiting CT remained PCR positive throughout the growing phase, with less than one-third of these animals seroconverting. APPV RNA was present in multiple tissues from pigs in both groups at the time of marketing. This study improved our understanding of the infection dynamics of APPV in swine and the impact that the immune status and timing of infection have on the persistence of APPV in serum and tissues.

Pestivirus A Bovine viral diarrhea virus type 1 species genotypes circulating in China and Turkey.

Background: Pestivirus A Bovine viral diarrhea virus type 1 (BVDV-1) is a heterogeneous species within the genus, affecting cattle and other ruminants, with economic impact on livestock production. Aim: The study aimed to update the taxonomy of the Pestivirus A, BVDV-1 species and to verify the clustering of the strains reported as genotype 1v, originating from different countries. Methods: Recently deposited strains from China, Turkey, and Iran have been evaluated by the palindromic nucleotide substitutions (PNS) genotyping method. Results: Based on secondary structure analysis of the 5'-UTR sequences, strains reported as 1v from China were clustered as sub genotype 1.7.3 (1o). Genotype 1.19 (1w) was restricted to China and genotype 1.21 (1v) was present only in Turkey and Iran. Conclusion: The application of the PNS method clarified the taxonomical status of strains, revealing the homonymy of genetically different clusters. Furthermore, these observations indicated geographic segregation in the Pestivirus A species, and confirmed the occurrence of new atypical genetic variants, with potential implications on control and prophylaxis.

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.

Generation and Efficacy of Two Chimeric Viruses Derived from GPE- Vaccine Strain as Classical Swine Fever Vaccine Candidates.

A previous study proved that vGPE- mainly maintains the properties of classical swine fever (CSF) virus, which is comparable to the GPE- vaccine seed and is a potentially valuable backbone for developing a CSF marker vaccine. Chimeric viruses were constructed based on an infectious cDNA clone derived from the live attenuated GPE- vaccine strain as novel CSF vaccine candidates that potentially meet the concept of differentiating infected from vaccinated animals (DIVA) by substituting the glycoprotein Erns of the GPE- vaccine strain with the corresponding region of non-CSF pestiviruses, either pronghorn antelope pestivirus (PAPeV) or Phocoena pestivirus (PhoPeV). High viral growth and genetic stability after serial passages of the chimeric viruses, namely vGPE-/PAPeV Erns and vGPE-/PhoPeV Erns, were confirmed in vitro. In vivo investigation revealed that two chimeric viruses had comparable immunogenicity and safety profiles to the vGPE- vaccine strain. Vaccination at a dose of 104.0 TCID50 with either vGPE-/PAPeV Erns or vGPE-/PhoPeV Erns conferred complete protection for pigs against the CSF virus challenge in the early stage of immunization. In conclusion, the characteristics of vGPE-/PAPeV Erns and vGPE-/PhoPeV Erns affirmed their properties, as the vGPE- vaccine strain, positioning them as ideal candidates for future development of a CSF marker vaccine.