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Congenital tremors are neurological disorders of newborn piglets that may lead to serious health consequences, including increased mortality. Many reports have indicated that atypical porcine pestivirus (APPV), first identified in 2015, is a possible cause of this condition. In different countries, APPV was detected in farms affected by piglets’ congenital tremors. There is a lack of data regarding the presence of this virus in Poland. However, swine veterinarians report outbreaks of congenital tremor with unestablished aetiology nationwide. Therefore, this study aimed to verify the presence of APPV in piglets with congenital tremors in Poland. Samples of blood (2), faecal swabs (7), brain (7), cerebellum (5), spinal cord, (7) lymph nodes (7), tonsil (7), thymus (6), tongue (7), heart (7), lung (7) and liver (7) were collected from piglets. Form sows’ blood (5) and oral fluid (3, pooling samples) were collected. All samples have been derived from a commercial breeding farm reporting a case of congenital tremor and subjected to the qPCR via commercial kit (EXOone Atypical Porcine Pestivirus, Exopol, Spain). Six out of seven piglets tested positive for APPV, with the highest viral loads detected in the cerebellum and tonsils. All samples from sows were negative, emphasising the virus’s specificity to piglets. To the authors’ knowledge, this is the first report confirming APPV’s presence in Poland. Given the potentially significant impact of APPV infections on swine health and production economics, further research evaluating the epidemiology and pathogenicity of APPV, as well as identifying preventive measures, is sorely needed. [ABSTRACT FROM AUTHOR]

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

Abstract 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-β, IFN-γ, IL-1β, 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.

Abstract 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.

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-β, IFN-γ, IL-1β, 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Background Congenital tremor (CT) type A-II is a neurological disorder characterized by tremor of the head and body of newborn piglets. The suggested causative agent of the disease is the recently found atypical porcine pestivirus (APPV). The virus has been detected in piglets suffering from congenital tremor in central Europe, South and North America and in China but no studies has so far been performed in the Nordic countries. The overarching goal of this study was to investigate if APPV is present in the brain tissue of Swedish piglets suffering from congenital tremor. From June 2017 – June 2018, 15 piglets from four Swedish farms with ongoing outbreaks of congenital tremor and 13 piglets with splay leg originating from four different farms, were investigated for presence of APPV RNA in brain tissue. Matched healthy control piglets (n = 8) were also investigated. Two APPV-specific RT-qPCR methods targeting the NS3 and NS5B region, respectively, were used. A retrospective study was performed on material from Swedish piglets with congenital tremor sampled in 2004 (n = 11) and 2011/2012 (n = 3) using the described APPV-specific RT-qPCR methods. The total number of piglets with signs of CT in this study was 29. Results Atypical porcine pestivirus-RNA was detected in 93% (27/29) of the piglets suffering from congenital tremor. All piglets with congenital tremor from 2004 (n = 11) and 2012 (n = 3) were PCR-positive with respect to APPV, whereas, all of the healthy controls (n = 11) were negative. The piglets with congenital tremor sampled 2017–2018 had an odds ratio of 91.8 (95% CI 3.9128 to 2153.7842, z = 2.807, P = 0.0050) to test positive for APPV by qRT-PCR compared to the healthy piglets (Fishers exact test p

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. [ABSTRACT FROM AUTHOR]

Atypical porcine pestivirus (APPV) has recently been reported to be associated with congenital tremor in newborn piglets. Only limited information is available about the prevalence at herd level in endemically infected herds. Therefore, the aim of this study was to determine the within‐herd prevalence of APPV in a sub‐clinically infected sow herd in Switzerland and to analyse associations between the serological status as well as the age and sex of the pigs, litter number and days after the last insemination. In a census sampling, blood was collected from 125 sows, aged 180 days or older, and six boars. Sera were examined applying an indirect APPV‐specific ELISA to individual sera and an APPV RT‐PCR targeting the NS3 encoding regions of APPV to pools of five. The APPV antibody status was classified into low (S/P value ≤ 0.5), intermediate (S/P value = 0.5–1) and high reactivity (S/P value > 1.0). None of the pooled serum samples was positive for specific genome fragments of APPV. Of the 131 samples, 53.4% were highly reactive, 39.7% showed an intermediate reactivity, and 6.9% showed a low reactivity in the indirect ELISA, that is, were serologically negative. Significant associations between the S/P values and the age of the pigs (p <.001), the litter number (p <.001) and the numbers of days after the last insemination (p =.0188) were observed. The results indicate that this sow herd was previously infected with APPV, while viremia was not detected in any of the adult pigs. This might explain the absence of clinical signs in the suckling pigs. Potential reinfection and circulation of APPV in this sow herd might be due to semen from commercial boar studs or APPV‐positive animals in the absence of specific clinical signs. [ABSTRACT FROM AUTHOR]

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Background: Congenital tremor (CT) type A-II is a neurological disorder characterized by tremor of the head and body of newborn piglets. The suggested causative agent of the disease is the recently found atypical porcine pestivirus (APPV). The virus has been detected in piglets suffering from congenital tremor in central Europe, South and North America and in China but no studies has so far been performed in the Nordic countries. The overarching goal of this study was to investigate if APPV is present in the brain tissue of Swedish piglets suffering from congenital tremor. From June 2017 – June 2018, 15 piglets from four Swedish farms with ongoing outbreaks of congenital tremor and 13 piglets with splay leg originating from four different farms, were investigated for presence of APPV RNA in brain tissue. Matched healthy control piglets (n = 8) were also investigated. Two APPV-specific RT-qPCR methods targeting the NS3 and NS5B region, respectively, were used. A retrospective study was performed on material from Swedish piglets with congenital tremor sampled in 2004 (n = 11) and 2011/2012 (n = 3) using the described APPV-specific RT-qPCR methods. The total number of piglets with signs of CT in this study was 29. Results: Atypical porcine pestivirus-RNA was detected in 93% (27/29) of the piglets suffering from congenital tremor. All piglets with congenital tremor from 2004 (n = 11) and 2012 (n = 3) were PCR-positive with respect to APPV, whereas, all of the healthy controls (n = 11) were negative. The piglets with congenital tremor sampled 2017–2018 had an odds ratio of 91.8 (95% CI 3.9128 to 2153.7842, z = 2.807, P = 0.0050) to test positive for APPV by qRT-PCR compared to the healthy piglets (Fishers exact test p < 0.0001). These findings make it interesting to continue investigating APPV in the Swedish pig-population. Conclusion: This is the first description of atypical porcine pestivirus in piglets suffering from congenital tremor type A-II in Sweden and the Nordic countries. The virus has been present in the Swedish pig population since at least 2004. [ABSTRACT FROM AUTHOR]

Congenital tremor (CT) is a neurological disease that affects new-born piglets. It was described in 1922 and six different forms, designated type AI-V and type B, are described based on the causative agents, as well as specific histological findings in the central nervous system (CNS). The various forms present with identical clinical signs consisting of mild to severe tremor of the head and body, sometimes complicated with ataxia. By definition, all A-forms have hypomyelination of the CNS, whereas there are no histopathological lesions with the B-form. The cause of the A-II form was long unknown, however, at present several different viruses have been proposed as the causative agent: porcine circovirus-II (PCV-II), astrovirus, PCV-like virus P1, and atypical porcine pestivirus (APPV). Currently, APPV is the only virus that has been proven to fulfill Mokili's Metagenomic Koch's Postulates. Following infection of the pregnant sow, the virus passes the placental barrier and infects the fetus. Interestingly, no clinical signs of disease have been associated with APPV in adult pigs. Furthermore, other viruses cannot be ruled out as additional potential causes of CT. Given the increased interest and research in CT type A-II, the aim of this review is to summarize current knowledge. [ABSTRACT FROM AUTHOR]

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.

Congenital tremor (CT) type A-II in piglets is caused by an emerging atypical porcine pestivirus (APPV), which is prevalent in swine herds and a serious threat to the pig production industry. This study aimed to construct APPV E2 subunit vaccines fused with Fc fragments and evaluate their immunogenicity in piglets. Here, APPV E2Fc and E2ΔFc fusion proteins expressed in Drosophila Schneider 2 (S2) cells were demonstrated to form stable dimers in SDS-PAGE and western blotting assays. Functional analysis revealed that aE2Fc and aE2ΔFc fusion proteins could bind to FcγRI on antigen-presenting cells (APCs), with the affinity of aE2Fc to FcγRI being higher than that of aE2ΔFc. Moreover, subunit vaccines based on aE2, aE2Fc, and aE2ΔFc fusion proteins were prepared, and their immunogenicity was evaluated in piglets. The results showed that the Fc fusion proteins emulsified with the ISA 201VG adjuvant elicited stronger humoral and cellular immune responses than the IMS 1313VG adjuvant. These findings suggest that APPV E2 subunit vaccines fused with Fc fragments may be a promising vaccine candidate against APPV.

A novel pestivirus species was discovered in a piglet-producing farm in Austria during virologic examinations of congenital tremor cases. The emergence of this novel pestivirus species, provisionally termed Linda virus, in domestic pigs may have implications for classical swine fever virus surveillance and porcine health management.

Atypical porcine pestivirus (APPV) is an RNA virus newly discovered from swine in Asia, Europe, and the Americas. This novel virus has been confirmed as the cause of congenital tremor (CT) in piglets, which causes extensive economic losses to the swine industry. To investigate the genetic diversity and evolutionary relationship of APPV in China, 83 piglet samples with severe CT clinical signs were obtained from 12 commercial swine farms in 3 provinces of Southern China. RT-PCR revealed that the positive rates of APPV were as high as 100% (12/12) for the swine farms and 90.4% (75/83) for the samples. Subsequently, 21 positive samples and 3 positive samples were selected for partial E2 gene and complete polyprotein gene sequencing, respectively. Phylogenetic analysis showed that 62.5% of the sequences belonged to a novel APPV clade provisionally named genotype 3, which showed 81.0–82.1% sequence identity to genotypes 1 and 2. Amino acid sequence alignment showed that E2 protein of genotype 3 has three specific mutation sites, namely I19V, Y82F, and N107G. The results of the present study demonstrate that a novel APPV subgenotype, which is widely distributed in severe CT clinical samples in Southern China, was genetically diverse. We advocate for the inclusion of genotype 3 during revision of the APPV typing method.

Recently, piglets from a high-health status farm began exhibiting congenital tremors, high preweaning mortality and incidence of splayed legs. Postmortem histological examination identified a small number of scattered white matter vacuoles in the cerebellum and underlying brainstem of affected piglets. Presence of potential viral sources associated with this neurologic condition was initially infirmed using quantitative PCR for atypical porcine pestivirus (APPV), porcine teschovirus, and porcine sapelovirus. Using metagenomic analysis, APPV was identified as the main microbial species in serum obtained from piglets affected by congenital tremor. These piglets had higher preweaning mortality rates (46.4% vs. 15.3%) and incidence of splayed legs (33.0% vs. 0.8 %) compared to unaffected piglets. Piglets affected by congenital tremor had higher viral titer (P < 0.15) and larger birth weights (P < 0.05) compared to normal litter mates. Whole-genome sequencing and genome assembly of the novel APPV strain (MK728876) was carried out using Oxford Nanopore and related bioinformatics pipelines. Phylogenic analysis demonstrated that this strain along with other completely sequenced APPV strains were grouped into 2 clades, both including strains-inducing congenital tremor. Strains appear to cluster based on region but there were still significant differences within regions. Future research needs to address potential underdiagnosis due to genetic diversity but also to understand mode of transmission, variation in virulence, and the role of host genetics in APPV susceptibility. [ABSTRACT FROM AUTHOR]

Atypical porcine pestivirus (APPV) is an RNA virus newly discovered from swine in Asia, Europe, and the Americas. This novel virus has been confirmed as the cause of congenital tremor (CT) in piglets, which causes extensive economic losses to the swine industry. To investigate the genetic diversity and evolutionary relationship of APPV in China, 83 piglet samples with severe CT clinical signs were obtained from 12 commercial swine farms in 3 provinces of Southern China. RT-PCR revealed that the positive rates of APPV were as high as 100% (12/12) for the swine farms and 90.4% (75/83) for the samples. Subsequently, 21 positive samples and 3 positive samples were selected for partial E2 gene and complete polyprotein gene sequencing, respectively. Phylogenetic analysis showed that 62.5% of the sequences belonged to a novel APPV clade provisionally named genotype 3, which showed 81.0–82.1% sequence identity to genotypes 1 and 2. Amino acid sequence alignment showed that E2 protein of genotype 3 has three specific mutation sites, namely I19V, Y82F, and N107G. The results of the present study demonstrate that a novel APPV subgenotype, which is widely distributed in severe CT clinical samples in Southern China, was genetically diverse. We advocate for the inclusion of genotype 3 during revision of the APPV typing method. [ABSTRACT FROM AUTHOR]

Atypical porcine pestivirus (APPV) is a single-stranded RNA virus from the family Flaviviridae, which is linked to congenital tremor (CT) type A-II in newborn piglets. Here, we retrospectively investigated the molecular evolution of APPV on an affected herd between 2013 and 2019. Monitoring was done at regular intervals, and the same genotype of APPV was found during the entire study period, suggesting no introductions from outside the farm. The nucleotide substitutions over time did not show substantial amino acid variation in the structural glycoproteins. Furthermore, the evolution of the virus showed mainly purifying selection, and no positive selection. The limited pressure on the virus to change at immune-dominant regions suggested that the immune pressure at the farm might be low. In conclusion, farms can have circulation of APPV for years, and massive testing and removal of infected animals are not sufficient to clear the virus from affected farms.

The genus Pestivirus, which belongs to the family Flaviviridae, includes ssRNA+ viruses responsible for infectious diseases in swine, cattle, sheep, goats, and other domestic and wild animals. Recently, several putative pestiviruses species have been discovered and characterized in mammalian species (giraffe pestivirus, antelope pestivirus, HoBi virus, Bungowannah virus, and Linda virus); one of these is a genetically distinct pestivirus, named atypical porcine pestivirus (APPV), discovered using the next-generation sequencing technology. APPV has been detected in piglets with congenital tremor (CT) from four different continents, including North America, South America, Europe, and Asia. There is strong evidence that experimental inoculation and in field outbreaks involving APPV induce CT in piglets. Additionally, splay leg (SL) syndrome has been observed concurrently with CT, and it was induced by APPV in experimental studies and some field cases. Animals with a persistent and/or chronic infection condition can shed the virus over time. Viral-RNA is frequently detected in different tissues from CT-piglets; however, high loads of APPV are detected most consistently in central nervous tissue. Moreover, the APPV genome has been recently detected in semen and preputial swabs from boar studs, as well as in serum and tissue samples from wild boars and domestic adult pigs, all known to be clinically healthy. Phylogenetic analysis revealed that the APPV sequence (complete or partial polyprotein) exhibits high genetic diversity between viral strains detected in different countries and formed independent clusters according to geographic location. Additional studies are needed to evaluate the molecular detection and sero-prevalence of APPV around the world. Lastly, more research is needed to understand clinical presentations associated with APPV infection, as well as the economic losses related to the virus in pig production worldwide.

Abstract Atypical porcine pestivirus (APPV) have been discovered in swine herds from three provinces in China, suggesting a wide distribution in China. This study reports the occurrence of three novel APPV strains in China. They were detected from newborn piglets with clinical signs of congenital tremors (CT) in Guangdong Province, China. The complete genomic sequences of three novel APPV strains exhibited only 80.5%–84.1% nucleotide sequences homology with other APPV reference sequences available in GenBank. Phylogenetic analysis showed that these novel APPV strains formed independent branch from the American, German, Netherlandish, Australian and other Chinese strains. These results will help us better understand the epidemiology and genetic characteristics of APPV in China. Highlights • Three novel atypical porcine pestivirus (APPV) strains were detected in China. • The homology exhibited only 80.5%–84.1% with other APPV reference sequences. • These APPV strains formed independent branch from other APPV reference sequences. [ABSTRACT FROM AUTHOR]

As one of emerging porcine viruses, atypical porcine pestivirus (APPV) was found in three continents since it emerged in 2015. It is now thought as the causative agent for congenital tremor type A‐II in piglets. At the end of 2017, two APPV strains were identified from piglets with congenital tremor in Guangxi and Yunnan, China. The genome of APPV GX04/2017 strain was so far determined to be 11,534 nucleotides (nt) in length and contains a single open reading frame (ORF) encoding a polyprotein comprising 3,635 amino acids. Comparative analysis of ORF, Npro, E2, and NS3 gene sequences revealed that the APPV GX04/2017 strain shares nucleotide sequence identities of 82.8%–92.8% with other APPV strains, while YN01/2017 strain is 79.4%–97.4% homology to the others. Phylogenetic analysis showed that the APPV GX04/2017 and YN01/2017 are two novel APPV strains with the highest homology to each other, and relative high similarity to the APPV 000515 and JX‐JM01 strains in genome sequence. The current findings provide updated information about APPV epidemiology and divergence in China, which would certainly help to establish reliable diagnosis and surveillance programs for APPV. [ABSTRACT FROM AUTHOR]

Atypical porcine pestivirus (APPV) is a recently identified RNA virus within the Flaviviridae family, causing congenital tremor (CT) in the piglets of infected sows. We have investigated 25 cases of CT from 2005, 2007, 2010 and 2016–2018, originating from six different farms. RT‐PCR has been performed on these samples and all of the affected piglets were positive to APPV. Our phylogenetic analysis showed that Hungarian strains show a high degree of variability and are clustered into five distinct lineages. Four strains originating from one farm have shown exceptional similarity (99.9%) to an Austrian sequence, whereas another one from a different herd was grouped close to a Chinese strain (96.4% similarity). Our results suggest multiple events of introduction of the virus from various sources into Hungary. This is the first report of the presence and clinical relevance of APPV in the Hungarian pig population. [ABSTRACT FROM AUTHOR]

Recently, a novel atypical porcine pestivirus (APPV) in pig was reported. In this study, two APPV strains, APPV-China/GZ01/2016 (GZ01) and APPV-China/GD-SD/2016 (GD-SD), were identified in two newborn piglet herds with congenital tremor from China. The open reading frame of the two strains shared an 83.5% nucleotide identity. Phylogenetically, the APPV strains were placed into two groups: GZ01 belonged to group I and GD-SD belonged to group II. A high viral load was detected in the cerebellum (quantification cycles < 26). Further studies should be carried out to thoroughly elucidate the development of congenital tremors caused by APPV. [ABSTRACT FROM AUTHOR]

Summary: Atypical porcine pestivirus (APPV) has been detected in piglets with congenital tremor (CT) from three different continents including North America, Europe and Asia. Thirteen piglets from four farms in two different states in Brazil with CT were sampled. Viral RNA was detected by quantitative real‐time PCR in the cerebellum or cerebellum and spinal cord in the 100% of the piglets with CT, and APPV was not detected in any tissue sample from clinically non‐affected piglets with the exception of the cerebellum of one piglet from Farm A. Piglets with CT had an odds ratio of 99.0 (95% CI 3.4, 2823.8; p = .0072) compared to piglets without CT to test positive for APPV by qRT‐PCR. A subset of positive samples was selected for sequencing of the NS3 gene. Phylogenetic analysis revealed that Brazilian sequences of the NS3 formed an independent cluster and had the highest sequence identity with a sequence from the United States. This is the first identification of APPV infection in piglets with CT in South America. [ABSTRACT FROM AUTHOR]

Summary: Outbreaks of trembling and shaking were reported among pigs at two pig farms in Jiangsu Province, China. Serum and tissue samples tested positive for porcine circovirus‐like virus P1 and negative for classical swine fever virus, porcine circovirus type 2, astrovirus and porcine pestivirus using PCR/RT‐PCR and immunohistochemical techniques. High P1 viral genome loads were identified in sera, brain and lymph node tissue samples by qPCR. In addition, one of the most notable pathological changes was dissolution of the nucleus in Purkinje cells. The results of this study provide molecular evidence of an association between congenital tremor in pigs and P1 virus. [ABSTRACT FROM AUTHOR]

Pestiviruses are highly variable RNA viruses. A growing number of novel pestiviruses has been discovered in domestic and wild species in the last two decades. Recently, a novel atypical porcine pestivirus (APPV) linked with the development of congenital tremor (CT) in neonatal pigs was described in Europe and the Americas. Here, the first Asian APPV complete polyprotein coding sequence was assembled from serum samples from newborn piglets affected with CT in Southern China, and termed APPV_GD. 14 organ samples from affected piglets were analyzed by quantitative RT-PCR (qRT-PCR) to investigate the tissue tropism of APPV, and 135 serum samples from pigs from 10 farms were used for identifying APPV in adult pigs. The highest genome loads were found in submaxillary lymph nodes, and PCR-based detection showed that APPV genomes were present in seven samples from five farms. A phylogenetic tree was constructed based on the full-length genomes of the pestiviruses, and APPV_GD appeared on a new branch with another newly discovered APPV. Nucleotide identity analysis demonstrated that APPV_GD shared the highest nucleotide sequence identity with a German APPV. Bayesian inference was performed using 25 partial sequences of the APPV NS5B gene (528 bp) isolated from four countries in recent years. According to this analysis, the most recent common ancestor (tMRCA) of the current APPV strains might have emerged in Germany and then diversified and spread to Asia, the Americas, and other countries in Europe. However, the result of bayesian inference could change when more APPV strains are isolated in the future. The present study is the first to report APPV in China and infers the origin and dissemination of the current strains of the virus.

In 2015, a new pestivirus was described in pig sera in the United States. This new “atypical porcine pestivirus” (APPV) was later associated with congenital tremor (CT) in newborn piglets. The virus appears to be distributed worldwide, but the limited knowledge of virus diversity and the use of various diagnostic tests prevent direct comparisons. Therefore, we developed an APPV-specific real-time RT-PCR assay in the 5′UTR of the viral genome to investigate both retro- and prospectively the strains present in Switzerland and their prevalence in domestic pigs. Overall, 1080 sera obtained between 1986 and 2018 were analyzed, revealing a virus prevalence of approximately 13% in pigs for slaughter, whereas it was less than 1% in breeding pigs. In the prospective study, APPV was also detected in piglets displaying CT. None of the samples could detect the Linda virus, which is another new pestivirus recently reported in Austria. Sequencing and phylogenetic analysis revealed a broad diversity of APP viruses in Switzerland that are considerably distinct from sequences reported from other isolates in Europe and overseas. This study indicates that APPV has already been widely circulating in Switzerland for many years, mainly in young animals, with 1986 being the earliest report of APPV worldwide.

Summary: Recently, a putative new pestivirus species, provisionally named as Atypical Porcine Pestivirus (APPV), was associated with the congenital tremor in piglets in North America and consequently in Europe and Asia. The present research aimed to describe the detection and characterization of APPV employing NS5B gene partial sequencing, gross pathology and histologic examination of piglets displaying congenital tremor from two different farms of Southern Brazil. No gross lesions were observed, and the histological findings revealed moderate vacuolization of the white matter of the cerebellum. RT‐PCR followed by DNA sequencing and a phylogenetic analysis confirmed the presence of APPV in samples from the two farms, which the samples were distinct in nature. Phylogenetic reconstruction reinforced the high genetic variability within the APPVs previously reported. This is the first report of APPV in South America suggesting that this new group of viruses may be widespread in swine herds in other countries as it is in Brazil. [ABSTRACT FROM AUTHOR]

This study aimed to provide information regarding viral pathogenesis and molecular epidemiology linked with recently reported atypical porcine pestivirus (APPV) strains and to determine the circulation of APPV in Spain from 1997 to 2016. Two-day-old piglets with moderate-severe congenital tremor ( CT) from a Spanish farm were received for diagnostic purposes. Sera, nasal and rectal swabs and tissue samples were collected. qRT- PCR was performed in these samples, and a retrospective study to detect APPV RNA was carried out using a serum collection from 1997 to 2016. APPV genome was identified with high and moderate RNA loads in different tissues of the CT affected pigs. High APPV RNA load was detected in lymphoid organs, suggesting that these constitute a target for APPV replication. In 89 of the 642 retrospectively analysed samples (13.9%), APPV genome was detected. CT cases were related to the presence of APPV in viraemic piglets below 1 week of age, in which the viral RNA load was the highest. A considerable number of animals between 4 and 14 weeks of age and some 1-week-old piglets were viraemic in the absence of CT, which can act as carriers of the virus. The relative risk of APPV and CT was 8.5 ( CI 95% 5.8-12.5). Thus, our data show that APPV infection is epidemiologically related to CT. Phylogenetic analysis from 1615 NS2-3 nucleotides showed only one defined APPV clade, grouping the most phylogenetically related strains from Europe and China. Of this clade, there are other strains from Europe, USA and China. This data confirm the high APPV genetic diversity, not being able to cluster this virus according to the geographic area. Our result showed that APPV has been circulating in Spain at least since 1997, being the earliest date of detection of this virus worldwide and suggesting that APPV may be widespread. [ABSTRACT FROM AUTHOR]

Congenital tremor type A-II in piglets has been regarded as a transmissible disease since the 1970s, possibly caused by a very recently-described virus: atypical porcine pestivirus (APPV). Here, we describe several strains of APPV in piglets with clinical signs of congenital tremor (10 of 10 farms tested). Piglets on a farm with no history of congenital tremor were PCR-negative for the virus. To demonstrate a causal relationship between APPV and disease, three gilts were inoculated via intramuscular injection at day 32 of pregnancy. In two of the three litters, vertical transmission of the virus occurred. Clinical signs of congenital tremor were observed in APPV-infected newborns, yet also two asymptomatic carriers were among the offspring. Piglets of one litter were PCR-negative for the virus, and these piglets were all without congenital tremors. Long-term follow up of farm piglets born with congenital tremors showed that the initially high viremia in serum declines at five months of age, but shedding of the virus in feces continues, which explains why the virus remains present at affected farms and causes new outbreaks. We conclude that trans-placental transmission of APPV and subsequent infection of the fetuses is a very likely cause of congenital tremor type A-II in piglets. [ABSTRACT FROM AUTHOR]

The present study demonstrates the presence of porcine circovirus type 2 (PCV2) antigen in the brains of neonatal piglets with congenital tremor. Piglets with clinical signs of congenital tremor were collected from a commercial swine herd for post-mortem examination. Brain tissue was collected from the piglets and was fixed in 10 % neutral buffered formalin, embedded in paraffin, and 4-μm thick sections were obtained. The PCV2 antigens were identified in the paraffin-embedded brain tissue sections by immunohistochemistry and light microscopy. The piglet preweaning mortality rates from birth to 14 and 21 days of age were 24.1 and 27.6 %, respectively. No typical gross lesions of PCV2 infection were observed in any of the piglets; however, PCV2 DNA was detected in the heart of a piglet. Immunostaining of PCV2 antigen was detected in neurons throughout the brain. It can be concluded that PCV2 antigen is present in the brain tissue of neonatal piglets with congenital tremor. Additional studies are required to determine the association between PCV2 expression and brain function. [ABSTRACT FROM AUTHOR]

Background Congenital tremor is associated with demyelination of the brain and spinal cord and is clinically noted as outbreaks of trembling and shaking in newborn piglets during a limited time-period. Six forms of the disease have been described, where form AII may be caused by an, as yet, unidentified viral infection. This study aimed to investigate the presence of astrovirus and circovirus by sequencing and polymerase chain reaction (PCR) analysis and by relating the findings to the occurrence of disease and lesions in the brain, in 4-6 days-old piglets obtained from a clinical outbreak of congenital tremor. Results In piglets with congenital tremor, there were mild to moderate vacuolar changes of the white matter in the cerebrum, brain stem and cerebellum. In healthy piglets, less conspicuous vacuolar changes were detected. One healthy and one diseased piglet were positive for porcine circovirus type 2. The nested pan-PCR showed the presence of astrovirus in at least one brain region in all piglets and by sequencing, two different porcine astrovirus lineages were identified. Conclusions The results do not support previous studies identifying porcine circovirus type 2 as the cause of congenital tremor. The demonstration of astrovirus in the brain of piglets suffering from congenital tremor is interesting. However, astrovirus was demonstrated in both healthy and diseased individuals and therefore, further studies are warranted to determine the possible involvement of astrovirus in the pathogenesis of congenital tremor in pigs. [ABSTRACT FROM AUTHOR]

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.

Congenital tremor (CT) type A-II in piglets is caused by an emerging atypical porcine pestivirus (APPV), which is prevalent in swine herds and a serious threat to the pig production industry. This study aimed to construct APPV E2 subunit vaccines fused with Fc fragments and evaluate their immunogenicity in piglets. Here, APPV E2Fc and E2ΔFc fusion proteins expressed in Drosophila Schneider 2 (S2) cells were demonstrated to form stable dimers in SDS-PAGE and western blotting assays. Functional analysis revealed that aE2Fc and aE2ΔFc fusion proteins could bind to FcγRI on antigen-presenting cells (APCs), with the affinity of aE2Fc to FcγRI being higher than that of aE2ΔFc. Moreover, subunit vaccines based on aE2, aE2Fc, and aE2ΔFc fusion proteins were prepared, and their immunogenicity was evaluated in piglets. The results showed that the Fc fusion proteins emulsified with the ISA 201VG adjuvant elicited stronger humoral and cellular immune responses than the IMS 1313VG adjuvant. These findings suggest that APPV E2 subunit vaccines fused with Fc fragments may be a promising vaccine candidate against APPV. [ABSTRACT FROM AUTHOR]

A neonatal congenital tremor (CT) syndrome that sporadically occurs in Belgian piglets is described. The results of an epidemiologic follow-up survey on affected farms is presented. Experimental trials to provoke this syndrome by inoculation of a brain suspension, derived from CT-piglets, in pregnant sows by intramuscular and intrauterine way, partly succeeded. Histological and biochemical studies on brain tissue of these piglets indicated that this form of CT should be classified as an A2 form. Using serologic and virologic examinations we were however not able to identify the probably infectious CT-agent. Zusammenfassung Typ A2 Congenitaler Tremor beim Ferkel Es wird ein sporadisch in belgischen Schweinebeständen vorkommendes Syndrom mit congenitalem Tremor (CT) beschrieben. Die Ergebnisse einer entsprechenden epidemiologischen Untersuchung werden ebenfalls dargestellt. Versuche, das Syndrom durch intramuskuläre oder intrauterine Inokulation einer von CT-Ferkeln stammenden Hirnsuspension bei den Ferkeln trächtiger Muttersauen zu induzieren, verliefen zum Teil erfolgreich. Nach histologischen und biochemischen Untersuchungen am Hirn dieser Ferkel dürfte es sich bei der betreffenden Form des CT um die Typ-A2-Form handeln. Mittels serologischer und virologischer Untersuchungen konnte jedoch das wahrscheinlich infektiöse CT-Agens nicht identifiziert werden. [ABSTRACT FROM AUTHOR]

Atypical porcine pestivirus (APPV), an RNA virus member of the Flaviviridae family, has been associated with congenital tremor in newborn piglets. Previously reported quantitative polymerase chain reaction (qPCR)-based assays were unable to detect APPV in novel cases of congenital tremor originated from multiple farms from U.S. Midwest (MW). These assays targeted the viral polyprotein coding genes, which were shown to display substantial variation, with sequence identity ranging from 58.2% to 70.7% among 15 global APPV strains. In contrast, the 5'-untranslated region (5' UTR) was found to have a much higher degree of sequence conservation. In order to obtain the complete 5' UTR of the APPV strains originated from MW, the 5' end of the viral cDNA was obtained by using template switching approach followed by amplification and dideoxy sequencing. Eighty one percent of the 5' UTR was identical across 14 global and 5 MW strains with complete or relatively complete 5' UTR. Notably, some of the most highly conserved 5' UTR segments overlapped with potentially important regions of an internal ribosome entry site (IRES), suggesting their functional role in viral protein translation. A newly designed single qPCR assay, targeting 100% conserved 5' UTR regions across 19 strains, was able to detect APPV in samples of well documented cases of congenital tremor which originated from five MW farm sites (1-18 samples/site). As these fully conserved 5' UTR sequences may have functional importance, we expect that assays targeting this region would broadly detect APPV strains that are diverse in space and time., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

• Hypomyelination is a rare consequence of in utero BVDV infection in cattle. • A high prevalence congenital tremor and hypomyelination BVD outbreak is described. • The complete genome of a BVDV causing hypomyelination is reported for the first time. • Type and number of viral genomic mutations differ between brain and blood samples. • Genetic variability and host factors contributed to the unusual BVD outbreak. Hypomyelination is a rare consequence of in utero bovine viral diarrhoea virus (BVDV) infection. We describe a BVDV outbreak in a naïve Holstein dairy herd in northern Italy, with an unusually high prevalence of calves with neurological signs, generalised tremors and ataxia. Histological analysis showed that hypomyelination was the predominant lesion and that the most typical BVDV neuropathological findings (e.g. cerebellar hypoplasia) were absent. Virological and molecular analyses showed that non-cytopathic BVDV genotype 1b was associated with the calves' neurological signs and excluded other viruses responsible for congenital infection or neurological disorders. Whole-genome sequencing of BVDVs from the brain of a calf with neurological signs and the whole blood of a persistently infected herd-mate with no such sign showed >99.7 % sequence identity. Analysis of the quasispecies distribution revealed the greatest variation rates in regions coding for the structural proteins E1 and E2. Variation was slightly greater in the brain- than in the blood-derived sequence and occurred at different sites, suggesting the occurrence of distinct evolutionary processes in the two persistently infected calves. Molecular characterisation of BVDV genomes from five other calves with neurological signs from the same farm confirmed that the E1 and E2 regions were the most variable. Several factors, including genetic variability and host factors, appear to have contributed to the observed unique BVDV disease phenotype, characterised by hypomyelination and neurological signs. [ABSTRACT FROM AUTHOR]

Atypical porcine pestivirus (APPV) is a single-stranded RNA virus from the family Flaviviridae, which is linked to congenital tremor (CT) type A-II in newborn piglets. Here, we retrospectively investigated the molecular evolution of APPV on an affected herd between 2013 and 2019. Monitoring was done at regular intervals, and the same genotype of APPV was found during the entire study period, suggesting no introductions from outside the farm. The nucleotide substitutions over time did not show substantial amino acid variation in the structural glycoproteins. Furthermore, the evolution of the virus showed mainly purifying selection, and no positive selection. The limited pressure on the virus to change at immune-dominant regions suggested that the immune pressure at the farm might be low. In conclusion, farms can have circulation of APPV for years, and massive testing and removal of infected animals are not sufficient to clear the virus from affected farms. [ABSTRACT FROM AUTHOR]