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.

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.

Atypical Porcine Pestivirus Circulation and Molecular Evolution within an Affected Swine Herd.

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.

Characterization of the Humoral Immune Response Induced after Infection with Atypical Porcine Pestivirus (APPV).

Atypical porcine pestivirus (APPV) is a widely distributed pathogen causing congenital tremor (CT) in piglets. So far, no data are available regarding the humoral immune response against APPV. In this study, piglets and their sows from an affected herd were tested longitudinally for viral genome and antibodies. APPV genome was detected in the majority of the piglets (14/15) from CT affected litters. Transient infection of gilts was observed. Kinetics of Erns- and E2-specific antibodies and their neutralizing capacity were determined by recently (Erns) and newly (E2) developed antibody ELISAs and virus neutralization assays. Putative maternally derived antibodies (MDA) were detected in most piglets, but displayed only low to moderate neutralizing capacity (ND50 ≤ 112). Horizontal APPV transmission occurred when uninfected and infected piglets were mingled on the flat deck. Horizontally infected piglets were clinically inapparent and showed only transient viremia with subsequently consistently high E2 antibody levels. For piglets from CT affected litters, significantly lower neutralizing antibody titers were observed. Results indicate that E2 represents the main target of neutralizing antibodies. Characterization of the humoral immune response against APPV will help to provide valuable serological diagnosis, to understand the epidemiology of this novel pathogen, and to implement tailored prevention strategies.

Detection of atypical porcine pestivirus genome in newborn piglets affected by congenital tremor and high preweaning mortality1.

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.

12 novel atypical porcine pestivirus genomes from neonatal piglets with congenital tremors: A newly emerging branch and high prevalence in China.

Atypical porcine pestivirus (APPV), a newly discovered member of the genus Pestivirus, is considered to be associated with congenital tremors (CT) in piglets. From June 2016 to January 2018, 440 serum and tissue samples from CT-affected piglets in Anhui, Guangdong and Guangxi provinces were collected to detect APPV. The results showed a high level of 63.4% preference for APPV in 27 swine farms and complicated co-infection cases between APPV and other 12 swine viruses. Meanwhile, 12 novel APPV genomes were screened and identified. Results showed that complete genomes, Npro and NS5A genes of these novel 12 APPV sequences revealed 80.5%-99.8%, 78%-100% and 76.9%-99.8% nucleotide identities, respectively. Phylogenetic analyses based on sequences of full-length genomes, Npro and NS5A genes of APPV indicated three well-defined clades including a newly emerging branch in China. This study provides novel epidemiological information of APPV in China.

Molecular characterization of two novel atypical porcine pestivirus (APPV) strains from piglets with congenital tremor in China.

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.

Identification and characterization of atypical porcine pestivirus genomes in newborn piglets with congenital tremor in China.

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.

Congenital infection with atypical porcine pestivirus (APPV) is associated with disease and viral persistence.

In 2013, several Austrian piglet-producing farms recorded outbreaks of action-related repetitive myoclonia in newborn piglets ("shaking piglets"). Malnutrition was seen in numerous piglets as a complication of this tremor syndrome. Overall piglet mortality was increased and the number of weaned piglets per sow decreased by more than 10% due to this outbreak. Histological examination of the CNS of affected piglets revealed moderate hypomyelination of the white substance in cerebellum and spinal cord. We detected a recently discovered pestivirus, termed atypical porcine pestivirus (APPV) in all these cases by RT-PCR. A genomic sequence and seven partial sequences were determined and revealed a 90% identity to the US APPV sequences and 92% identity to German sequences. In confirmation with previous reports, APPV genomes were identified in different body fluids and tissues including the CNS of diseased piglets. APPV could be isolated from a "shaking piglet", which was incapable of consuming colostrum, and passaged on different porcine cells at very low titers. To assess the antibody response a blocking ELISA was developed targeting NS3. APPV specific antibodies were identified in sows and in PCR positive piglets affected by congenital tremor (CT). APPV genomes were detected continuously in piglets that gradually recovered from CT, while the antibody titers decreased over a 12-week interval, pointing towards maternally transmitted antibodies. High viral loads were detectable by qRT-PCR in saliva and semen of infected young adults indicating a persistent infection.

Atypical Porcine Pestivirus: A Possible Cause of Congenital Tremor Type A-II in Newborn Piglets.

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.

Concurrent peste des petits ruminants virus and pestivirus infection in stillborn twin lambs.

Concurrent infection with peste des petits ruminants virus (PPRV) and pestivirus was diagnosed in stillborn twin lambs. With the flock history, the findings of epidermal syncytial cells and necrotizing bronchitis/bronchiolitis prompted testing for PPRV infection, and PPRV antigen was detected by immunohistochemistry (IHC) in the skin, lungs, kidneys, rumen, and thymus. Macroscopic anomalies that were typical of border disease included scoliosis, brachygnathism, prognathism, arthrogryposis, hydranencephaly, cerebellar hypoplasia, and hairy fleece; pestiviral antigen was detected by IHC in the brain, liver, lungs, and kidneys. Tissues from both lambs were positive by reverse transcriptase-polymerase chain reaction (RT-PCR) for PPRV and pestivirus. To the authors' knowledge, PPR has not been reported previously as a congenital infection or in combination with pestiviral infection.

Congenital infection of pigs with ruminant-type pestiviruses.

Congenital infections of pigs were induced with two ruminant-type pestiviruses isolated from pigs. One of the viruses was bovine viral diarrhoea virus-like and the other border disease virus-like. Both produced symptoms similar to those observed with low virulence strains of classical swine fever virus. A striking effect of persistent virus infection in post-natal life was stunting in viraemic animals. It was also shown that a congenitally infected pig shed virus for 2.5 years and in sufficient quantity to infect other pigs, even by indirect contact. Unlike ruminants, congenitally infected pigs sometimes had persistent viraemia but eventually eliminated the virus. Clearance of virus from the blood was related to the appearance of neutralizing antibodies. However, clearance from the tissues sometimes took as much as 5 months longer than from the blood.