Recent research at the University of Nebraska-Lincoln integrated next-generation sequencing and metagenomic analyses to uncover the source of recent cases of congenital tremor in newborn piglets observed in research and commercial farms across Nebraska. The research was undertaken after the initial diagnostic testing of a batch of affected piglets, based on quantitative Polymerase Chain Reaction (qPCR), was negative for a number of viruses known to induce neurological conditions such as atypical porcine pestivirus (APPV), porcine teschovirus and porcine sapelovirus. However, the histopathologic examination uncovered higher incidences of scattered white matter vacuoles in the cerebellum and underlying brainstem, explaining some of the observed symptoms.
In order to identify the potential source of the observed symptoms, we employed a novel DNA sequencing approach, Oxford Nanopore Technology, used successfully in the human field to efficiently identify novel strains of viruses across the world. The instrument is portable, fits in the palm of a hand, and is highly suitable for field applications. Briefly, viral nucleic acids were isolated from serum obtained from affected piglets followed by massive sequencing of microbial nucleic acids. The obtained sequences were subjected to metagenomic analysis in which sequences from affected piglets were compared to microbial reference genome sequences available in databases. A new APPV strain was identified as the main viral species in the serum obtained from piglets affected by congenital tremor.
Sequence data suggested that initial APPV diagnostic test was negative due to lack of specificity of the qPCR assay. Using the Nanopore sequencing data, a new specific qPCR assay was designed which provided the sensitivity required for a diagnostic test of the new APPV strain. The new assay detected APPV in newborn piglets exhibiting congenital tremor as well as in their unaffected littermates but not in piglets from unaffected litters or in dams. At 30 days of age, the majority of the piglets (69.6 percent) still had detectable levels of APPV. The signs of tremor were absent from most pigs at 50 days of age (91.6 percent) with a suggestive decrease in viremia also being observed.
After initial cases of congenital tremor, a controlled exposure data set was generated using maternal crossbred gilts (n=91) subjected to fetal fluids of sows which had previously produced infected litters. In the exposed sows, 45 percent of the litters and 30.8 percent of all piglets were affected by tremor. Piglets affected by congenital tremor had higher preweaning mortality rates (46.4 percent versus 15.3 percent) compared to unaffected piglets, in both affected and unaffected litters. Mortality of the unaffected littermates from affected litters was higher (24.2 percent versus 12.7 percent) compared to piglets from unaffected litters. More than half (53.7 percent) of the mortality in the affected piglets was observed in the first day of life.
A significant relationship was observed between the occurrence of congenital tremor and birth weight across and within litters. Piglets from unaffected litters had larger average birth weight (1.30 ± 0.02 kg) compared to piglets exhibiting tremor (1.22 ± 0.02 kg) and their unaffected littermates (1.09 ± 0.03 kg). As expected, piglets affected by congenital tremor had higher viral titer compared to normal littermates.
An important relationship was also uncovered between the frequency of congenital tremor and splayed legs. Presence of splayed legs was observed in the majority of the litters affected by congenital tremor (73.2 percent), while the incidence was limited (6 percent) in the unaffected litters. The average frequency of splayed legs in the litters affected by congenital tremor was 22.4 percent, while in the unaffected litters the average frequency was marginal (0.5 percent). In general, the presence of splayed legs in piglets affected by tremor was larger (33 percent) compared to unaffected piglets (0.8 percent).
Generating whole-genome sequencing data and complete viral genome assembly directly from clinical samples is challenging due to relatively low viral titer. To completely sequence the genome of the new APPV strain our group designed an approach that provided efficient enrichment and genome coverage of the low-abundance viral genetic material isolated from clinical samples and rapid-turn around in detecting a novel strain. To assess sequence diversity and the evolutionary relationship among APPV genomes, the viral RNA and predicted polyprotein sequences of the novel strain was compared with the genomes of 30 other APPV strains isolated across the globe. This analysis indicated a high overall nucleotide diversity scattered across the viral APPV genome; this diversity is the reason why initially piglets affected by congenital tremor tested negative for APPV. As the number of farm sources and samples obtained across the state increases, the new qPCR assay proved also ineffective due to the highly diverse APPV genomes. As a result, sequencing data of additional strains obtained recently led to the design and evaluation of a Universal qPCR assay that provides reliable APPV testing despite the genetic diversity of the virus.
Future research needs to address the lack of understanding of the mode of transmission, gestational time of infection that leads to clinical symptoms and the role of host genetics in APPV susceptibility. A paper describing these results in detail was recently published in the Journal of Animal Science (https://academic.oup.com/jas/article/97/10/4093/5545417/).
Researchers: Kylee M. Sutton, University of Nebraska-Lincoln Animal Science Department; Kevin K. Lahmers, Virginia-Maryland College of Veterinary Medicine Department of Biomedical Sciences and Pathology and Virginia Tech Animal Laboratory Services; Seth P. Harris, University of Nebraska-Lincoln Veterinary Diagnostic Center, School of Veterinary Medicine and Biomedical Sciences; Hiruni R. Wijesena, University of Nebraska-Lincoln Animal Science Department; Benny E. Mote, University of Nebraska-Lincoln Animal Science Department; Stephen D. Kachman, University of Nebraska-Lincoln Department of Statistics; Tudor Borza, Dalhousie University Department of Plant, Food and Environmental Sciences; and Daniel C. Ciobanu, University of Nebraska-Lincoln Animal Science Department and School of Biological Sciences. For more information, contact Ciobanu at email@example.com.