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 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.

[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.

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.

Whole-Genome-Sequence-Based Evolutionary Analyses of HoBi-like Pestiviruses Reveal Insights into Their Origin and Evolutionary History.

HoBi-like pestivirus (HoBiPeV), classified under Pestivirus H species, is an emerging cattle pathogen of high economic impact. However, the origin and evolution of HoBiPeV are not very clear due to a lack of full genomic sequences from diverse clades. This study aimed to determine full-genome sequences of HoBiPeV strains of three novel clades (c, d and e) and perform full-genome-based genetic and evolutionary analyses. Bayesian phylogenetic analyses herein confirmed the existence and independent evolution of four main HoBiPeV clades (a, c, d and e) globally, with genetic divergence ranging from 13.0% to 18.2%. Our Bayesian molecular clock estimates revealed that HoBiPeV most likely originated in India, with a dated tMRCA of 1938 (1762-2000), evidencing a more recent origin of HoBiPeV. The evolution rate of HoBiPeV was estimated to be 2.133 × 10-3 subs/site/year at full-genome level but varied widely among individual genes. Selection pressure analyses identified most of the positively selected sites in E2. Additionally, 21.8% of the ORF codon sites were found under strong episodic diversifying selection, providing first evidence of negative selection in HoBiPeV evolution. No recombination event was evident for HoBiPeV-c, d and e strains. These findings provide new insights into HoBiPeV origin and evolutionary history for better understanding the epidemiology and host-pathogen interactions and stimulate vaccine research.

Improvement of bovine pestiviral diagnosis by the development of a cost-effective method for detecting viral RNA in fresh specimens and samples spotted in filter papers.

Bovine pestiviruses are the causative agents of bovine viral diarrhea, a disease that causes severe economic losses in cattle. The aim of this study was to improve their diagnosis by developing a RT-qPCR to detect bovine pestiviruses A, B and H; and to set up a protocol for collecting, shipping and preserving bovine pestiviral RNA on filter papers. The developed RT-qPCR showed high sensitivity in detecting these viruses in different matrices: viral stocks, semen and serum samples. With regard to the possibility of using the technique to test serum pools, it was possible to identify a positive serum sample within a pool containing 30 sera. In addition to evaluating the qPCR from fresh samples, the use of filter papers to sow bovine samples was analyzed. The sampling method on two different filter papers using bovine blood drops was a useful alternative for diagnostic purposes and allowed to preserve pestiviral RNA for up to 12 months under refrigeration.

Learning and strategic imitation in modelling farmers' dynamic decisions on bovine viral diarrhoea vaccination.

Considering human decision-making is essential for understanding the mechanisms underlying the propagation of real-life diseases. We present an extension of a model for pathogen spread that considers farmers' dynamic decision-making regarding the adoption of a control measure in their own herd. Farmers can take into account the decisions and observed costs of their trade partners or of their geographic neighbours. The model and construction of such costs are adapted to the case of bovine viral diarrhoea, for which an individual-based stochastic model is considered. Simulation results suggest that obtaining information from geographic neighbours might lead to a better control of bovine viral diarrhoea than considering information from trade partners. In particular, using information from all geographic neighbours at each decision time seems to be more beneficial than considering only the information from one geographic neighbour or trade partner at each time. This study highlights the central role that social dynamics among farmers can take in the spread and control of bovine viral diarrhoea, providing insights into how public policy efforts could be targeted in order to increase voluntary vaccination uptake against this disease in endemic areas.

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.

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.

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.

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.

HoBi-like pestivirus in 2 cases of fatal respiratory disease of feedlot cattle in Argentina.

HoBi-like pestivirus (HoBiPeV) is an emerging virus that has been detected in cattle and other ruminants. We diagnosed 2 cases of fatal bovine respiratory disease complex (BRDC) associated with infection with HoBiPeV in a feedlot in Argentina. The main findings in 2 steers autopsied were interstitial bronchopneumonia (case 1) and fibrinous bronchopneumonia (case 2). HoBiPeV was detected by RT-PCR in lungs of both animals and by immunohistochemistry in case 2. Phylogenetic analysis showed that both strains clustered within the "Brazilian-Italian" clade. In case 2, Mannheimia haemolytica was isolated from the lung. There is scant information about the contribution of HoBiPeV to the pathogenesis of BRDC. To our knowledge, HoBiPeV has not been reported previously in association with M. haemolytica pneumonia. Our findings further support the involvement of HoBiPeV in cases of BRDC and contribute to understanding the synergy of this etiologic agent in the pathogenesis of BRD, which is critical for the development of appropriate preventive strategies.

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.

Development and use of a droplet digital PCR (ddPCR) assay to achieve sensitive and fast atypical porcine pestivirus detection.

Atypical porcine pestivirus (APPV) is a recently discovered RNA virus, which mainly caused congenital tremor in piglets. Droplet digital PCR (ddPCR) is an absolute quantitative method that does not rely on the standard curve but has high sensitivity and accuracy. The present study aimed to develop a ddPCR detection assay for APPV. Furthermore, we evaluated the limit of detection, sensitivity, specificity and reproducibility of the ddPCR and real-time quantitative PCR (qPCR) and tested 135 clinical samples to calculate the detection rate of the two methods. The results showed that both methods had a strong linear relationship and quantitative correlation. The ddPCR assay had a minimum detection limit of 0.15 copies/µL for APPV, with a sensitivity 100 times that of qPCR. We tested clinical samples and found that the APPV ddPCR had a 27.4% positive detection rate, noticeably higher than that of the qPCR (14.8%). Additionally, the APPV ddPCR method had excellent repeatability and specificity. In brief, our study provided a novel, feasible and sensitive diagnostic technique to identify and monitor APPV.

New Emergence of the Novel Pestivirus Linda Virus in a Pig Farm in Carinthia, Austria.

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.

First molecular evidence of border disease virus in wild boars in Turkey.

Molecular studies on viral diseases in wildlife are limited in Turkey. Pestiviruses infect domestic animals such as pig, cattle, sheep, goats and many other wild ungulates. Cross-species transmission of pestiviruses between wildlife and domestic livestock is a subject of recent concern where wild ungulates are in close contact with domestic ruminants. The International Committee on Virus Taxonomy (ICTV) has named the genus Pestivirus, which belongs to the Flaviviridae family, using the format Pestivirus A, Pestivirus B, Pestivirus C, and so on. Pestivirus A-D replaces Bovine viral diarrhea virus-1 (BVDV-1), Bovine viral diarrhea virus-2 (BVDV-2), Classical swine fever virus (CSFV) and Border disease virus (BDV) respectively. During the 2013-2014 hunting season, a total of 40 samples were collected from wild boars (Sus scrofa ferus) in the area of Western Mediterranean Turkey. In the samples, nucleic acids were investigated for pestivirus, Aujeszky's disease virus, Borna disease virus, coronavirus, mastadenovirus and rotavirus. RT-PCR was performed using primary sets to detect specific partial gene region specific to each virus. Sequence analysis was performed on a positive sample. Phylogenetic analysis revealed that the positive sample, TR/Burdur/13/Boar3, belonged to BDV genotype 1 (Pestivirus D). The first molecular findings of BDV in wild boars in Turkey are reported in this study. This study highlights the importance of further research into diseases that might be transmitted from wild boars to ruminants in Turkey.

Detection and localization of atypical porcine pestivirus in the testicles of naturally infected, congenital tremor affected piglets.

Atypical porcine pestivirus (APPV) belongs to the genus Pestivirus within the family Flaviviridae. Recently, APPV has been identified as the causative agent of congenital tremor (CT) type AII. The disease is a neurological disorder that affects newborn piglets and is characterized by generalized trembling of the animals and often splay legs. CT is well known worldwide, and the virus seems to be highly prevalent in major swine producing areas. However, little is known about the epidemiology of the infection, transmission and spread of the virus between herds. Here, we show the high prevalence of APPV in processing fluid samples collected from Hungarian pig herds which led us to investigate the cellular targets of the virus in the testicles of newborn piglets affected by CT. By the development of an RNA in situ hybridization assay and the use of immunohistochemistry on consecutive slides, we identified the target cells of APPV in the testicle: interstitial Leydig cells, peritubular myoid cells and smooth muscle cells of medium-sized arteries. Previous studies have shown that APPV can be found in the semen of sexually mature boars suggesting the role of infected boars and their semen in the transmission of the virus similar to many other members of the Flaviviridae family. As in our case, the virus has not been identified in cells beyond the Sertoli cell barrier, further studies on infected adult boars' testicles and other reproductive glands are needed to analyze the possible changes in the cell tropism of APPV that might contribute to its prolonged extraction by the semen beyond the period of viraemia.

Atypical porcine pestivirus-A widespread virus in the Swedish wild boar population.

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

Detection and genetic analysis of a novel atypical porcine pestivirus from piglets with congenital tremor in Japan.

Atypical porcine pestivirus (APPV), which has been confirmed to be associated with congenital tremor (CT) in pigs, is a newly discovered porcine virus that has been found in the Americas, Europe and Asia; however, no report of APPV in Japan has been published. We identified an APPV in the central nervous system of Japanese piglets with CT and firstly determined and analysed the complete genome sequence. Phylogenetic analysis using the complete genome nucleotide sequence of the Japanese APPV, named Anna/2020, and those of APPVs from the NCBI database showed that APPVs were divided into three genotypes (genotypes 1 to 3), and that Anna/2020 clustered with the genotype 3 APPV strains, but distantly branched from these strains. Pairwise complete coding region nucleotide sequence comparisons revealed that there was 94.0%- 99.7% sequence identity among the genotype 3 strains, while Anna/2020 showed 87.0%-89.3% identity to those genotype 3 strains, suggesting that Anna/2020 represents a novel APPV lineage within genotype 3. Retrospective examinations using RT-PCR revealed one genotype 1 and two novel genotype 3 APPVs from pigs without CT, and that novel genotype 3 APPVs have been prevalent in Japan since at least 2007.

Molecular characterization of recent HoBi-like pestivirus isolates from cattle showing mucosal disease-like signs in India reveals emergence of a novel genetic lineage.

Natural infections with HoBi-like pestivirus (HoBiPeV) have been detected in cattle in South America, Europe and Asia with a range of clinical manifestations including fatal mucosal disease (MD). In India, although HoBiPeV in cattle has been reported, there is no information on currently circulating HoBiPeV strains and associated severe clinical disease. Between September 2018 and December 2019, suspected cases of bovine viral diarrhoea with severe disease in cattle were noticed in farmers' small holdings in Tamil Nadu State. To determine the extent of pestivirus infection, blood, serum, nasal or oral swab samples of 46 cattle from 18 villages were tested. Based on the real-time RT-PCR, antigen ELISA and nucleotide sequencing results, pestivirus was detected in nine cattle from eight villages in two districts and all pestiviruses were identified as HoBiPeV. All nine HoBiPeV-infected cattle displayed clinical signs resembling MD and HoBiPeV isolates (n = 9) obtained were characterized at genetic and antigenic level. Phylogenetic analyses based on 5'-untranslated regions (5'-UTR), Npro and combined 5'-UTR-Npro gene sequences revealed that eight HoBiPeV isolates clustered into a clade, distinct from all reported HoBiPeV clades (a-d), whereas one belonged to HoBiPeV-d clade, thus providing evidence of emergence of a novel HoBiPeV clade (e). This was also supported by HoBiPeV-e clade-specific amino acid substitutions in Npro and the antigenic reactivity patterns. The study demonstrates the existence and independent evolution of five HoBiPeV clades (four main clades) globally and surprisingly three exclusive to India. Also we confirm first HoBiPeV occurrence in southern India with predominant prevalence of HoBiPeV-e strains. Besides demonstrating increased HoBiPeV genetic diversity, here we show association of HoBiPeV with severe clinical disease involving fatalities highlighting impact of HoBiPeV on cattle health. The emergence of a novel HoBiPeV lineage provides new insights on global HoBiPeV epidemiology and genetic diversity reiterating the need for continuous monitoring of HoBiPeV in India.