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.

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.

Genome-wide association analysis for porcine reproductive and respiratory syndrome virus susceptibility traits in two genetic populations of pigs1.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen that continues to threaten swine industry sustainability. The complexity and high genetic diversity of PRRSV has prevented vaccines from conferring adequate protection against disease outbreaks. Genome-wide association analyses of PRRSV experimentally infected pigs representing two genetic lines (n = 174 to 176) revealed two major genomic regions accounting for ~1.2% of the genetic variation in PRRSV-specific antibody level in serum or lung. The major region for serum antibody was mapped to SSC7 near the SLAII complex, which has also been implicated in susceptibility to other swine viral pathogens. Haplotype substitution analysis uncovered potential DQB1 haplotypes associated with divergent effects. A novel major region for lung antibody was mapped to the proximal end of SSC17 with the top SNP overlapping two genes, PRAG1 and LONRF1. Sequencing LONRF1 uncovered polymorphisms within the coding region that may play a role in regulating PRRSV-specific antibody production in lung tissue following PRRSV infection. These data implicate novel host genomic regions (SSC17) that influence PRRSV-specific immune response as well as a common region (SSC7) potentially involved in susceptibility to multiple viral pathogens.

A 16.7 kb deletion in Sipa1l3 is associated with juvenile cataract in mice.

Congenital or juvenile cataract is a disease condition in which opacification of the lenses is present at birth or manifests early in life. It has been attributed to different monogenic factors with a high degree of heterogeneity and is often studied using mouse models. A spontaneous mutation was identified in a mouse line selected for heat loss that influenced lens formation and resulted in juvenile cataracts in mice homozygous for the recessive allele. Genetic dissection of this selection line by combining high-density genotypes and homozygosity mapping uncovered a 906 kb fragment on MMU7 encompassing 21 SNPs split into two groups of consecutive, homozygous segments specific to the cataract phenotype. Haplotype analysis revealed a 197.5 kb segment unique to cataract-affected mice that included a single known transcript consisting of the first 14 exons of Sipa1l3. In this region, we discovered a deletion of 1114 bp at the mRNA level, spanning four coding exons, predicted to produce a truncated Sipa1l3 protein lacking a portion of a Rap-GAP domain and two other potentially vital domains. At the genome level, the deletion consisted of 16,733 bp. Genotyping across different samples confirmed that only affected mice were homozygous for the deletion and normal mice were either heterozygous or homozygous for the wild-type allele. Further studies will be required to determine the impact of the truncated Sipa1l3 domains on eye development.