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.

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.

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.

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.

An emerging novel virus: Atypical porcine pestivirus (APPV).

Emerging porcine pestivirus diseases frequently challenge prevention and control strategies in the swine industry. Over the past decade, a few novel pestiviruses have been identified in pigs. This article focuses on the recently emerging atypical porcine pestivirus (APPV) that potentially threatens global swine herd health security. The virus was first identified in 2016, in the United States and thereafter, accumulated evidence shows that it is currently distributed in three continents. The clinical presentation of APPV-infected pigs is characterized by congenital tremor (CT) type A-II in piglets, while adult pigs may become persistent carriers and shedders. Here, a literature review is conducted to summarize the published findings in the virus genomic biology, transmission, epidemiology, pathogenesis, and diagnosis, which would shed light on acceleration of development of anti-APPV strategies.

Detection of atypical porcine pestivirus in Swedish piglets with congenital tremor type A-II.

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.

Prevalence and genome characteristics of atypical porcine pestivirus in southwest China.

Atypical porcine pestivirus (APPV) causes congenital tremor (CT) in piglets and has a wide geographical distribution. In this study, we evaluated APPV prevalence using 165 piglet sera from southwest China. Viral RNA was detectable by qRT-PCR in 43.6 % (17/39, 95 % CI 27.8-60.4 %) of piglets with CT, while viral RNA was not detected in the sera of any healthy piglets. The seven complete APPV genomes were obtained from distinct farms and were 11 269-11 459 nucleotides in length. The genomes of the seven strains shared 82.8-98 % identity with the APPV reference strains. Phylogenetic analysis of the complete genomes as well as E2 and Nrpo sequences revealed that the seven APPVs clustered into two groups: four strains belonged to genogroups A and D and three strains belonged to a novel APPV genotype, tentatively called genogroup E. This study provides important insights into the epidemiological features and genetic diversity of APPV.

Novel Pestivirus Species in Pigs, Austria, 2015.

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.

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.

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.

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.

Genetic diversity and detection of atypical porcine pestivirus infections.

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.

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.

Fc-Mediated E2-Dimer Subunit Vaccines of Atypical Porcine Pestivirus Induce Efficient Humoral and Cellular Immune Responses in Piglets.

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.

Bovine viral diarrhoea virus 1b infection associated with congenital tremor and hypomyelination in Holstein calves.

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.

Detection of Atypical Porcine Pestivirus in Piglets from Danish Sow Herds.

Atypical porcine pestivirus (APPV) was first discovered in North America in 2015 and was later shown to be associated with congenital tremor (CT) in piglets. CT is an occasional challenge in some Danish sow herds. Therefore, we initiated an observational case control study to clarify a possible relationship between CT and APPV in Danish pig production. Blood samples were collected from piglets affected by CT (n = 55) in ten different sow herds and from healthy piglets in five sow herds without a history of CT piglets (n = 25), as well as one sow herd with a sporadic occurrence of CT (n = 5). APPV was detected by RT-qPCR in all samples from piglets affected by CT and in three out of five samples from piglets in the herd with a sporadic occurrence of CT. In the herds without a history of CT, only one out of 25 piglets were positive for APPV. In addition, farmers or veterinarians in CT-affected herds were asked about their experience of the issue. CT is most often seen in gilt litters, and a substantial increase in pre-weaning mortality is only observed in severe cases. According to our investigations, APPV is a common finding in piglets suffering from CT in Denmark.

Investigation of congenital tremor associated with Classical swine fever virus genotype 2.2 in an organized pig farm in north-eastern India.

The present study describes an outbreak of Classical swine fever (CSF) in an organized pig farm followed by an episode of CSF virus (CSFV) associated congenital tremors in piglets. The outbreak was recorded in a newly procured herd of Hampshire pigs housed adjacent to the existing pigs of the farm. The recorded CSF outbreak caused a mortality of 100% in the newly procured and 54.28% in the existing herd. As the disease subsides, the clinically recovered boars were served naturally with Tamworth gilts. Though, the sows farrowed on usual gestation period, litters born to each sow showed congenital tremors and eventually died within 24 h of birth. Necropsy analysis of affected piglets was indicative of CSFV infection and was further confirmed using RT-PCR signifying a transplacental infection. The CSFV strains from the initial outbreak and post outbreak episode of congenital tremors were successfully isolated in PK-15 cells and detected in indirect FAT and RT-PCR. Phylogenetic analysis based on E2 gene and 5'NTR of CSFV grouped the isolates within the genotype 2.2 and revealed close resemblance with previously reported Indian isolates of CSFV genotype 2.2 origin. To the best of our knowledge, this is the first report of CSFV induced congenital form reported from India under natural conditions.

A review of congenital tremor type A-II in piglets.

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.

Genetic characterization and recombination analysis of atypical porcine pestivirus.

Atypical porcine pestivirus (APPV) is recognised as the etiology of congenital tremor (CT) Type A-II and poses a challenge to pig production. Here, we described a CT case in piglets caused by APPV infection in central China in 2017. Interestingly, different from a previous report, more CT litters were observed in the second and third parity sows compared to the first and fourth parity. Evolutionary analysis and recombination evaluation were conducted for the isolate and 61 APPV genomes were available in GenBank. Phylogenetic analysis revealed a high level of genetic variation of APPV and the coexistence of three clades (Clades I-III) in China. The isolate was clustered into Clade I, which seemed to be prevalent worldwide and displayed higher genetic variability (Subgroups 1-4) compared with Clade II and Clade III, both of which were only reported in China. Notably, three putative recombinants were identified and characterized in APPV. The recombination events occurred in inter-clades (Clade II and III) or intra-clades (Clade I). To the best of our knowledge, this study presents the first evidence of homologous recombination within Pestivirus K. These results provide new clinical presentations of APPV infection and may be helpful in better understanding the large amount of genetic variations in this genus.

Pestivirus K (Atypical Porcine Pestivirus): Update on the Virus, Viral Infection, and the Association with Congenital Tremor in Newborn Piglets.

The atypical porcine pestivirus (APPV) belongs to the species Pestivirus K of the genus Pestivirus and the family Flaviviridae, and it has been associated with congenital tremor (CT) type A-II in newborn piglets. Although APPV was discovered in 2015, evidence shows that APPV has circulated in pig herds for many years, at least since 1986. Due to the frequently reported outbreaks of CT on different continents, the importance of this virus for global pig production is notable. Since 2015, several studies have been conducted to clarify the association between APPV and CT. However, some findings regarding APPV infection and the measures taken to control and prevent the spread of this virus need to be contextualized to understand the infection better. This review attempts to highlight advances in the understanding of APPV associated with type A-II CT, such as etiology, epidemiology, diagnosis, and control and prevention measures, and also describes the pathophysiology of the infection and its consequences for pig production. Further research still needs to be conducted to elucidate the host's immune response to APPV infection, the control and prevention of this infection, and the possible development of vaccines.