Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506
Performing Department
Diagnostic Medicine & Pathobiology
Non Technical Summary
The current state of the viruses associated with bovine respiratory and enteric diseases is not complete or updated. Our preliminary research suggests a high prevalence of novel or uncommon viruses in porcine and bovine diagnostic samples that require further identification and characterization to determine their association to disease. We seek to characterize and describe viruses associated with bovine enteric and respiratory disease using genome sequencing, phylogenetic analyses, recombinant protein and antibody testing. The results of this research will be communicated to veterinarians clients as well as used to develop or update diagnostic assays.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Next-generation sequencing (NGS) is a cutting-edge sequencing technology which processes thousands to millions of DNA templates in parallel (massively parallel), decreasing cost per base and limiting hands-on time. Conventional 'Sanger' sequencing incorporates dideoxynucleotides, one nucleotide at a time, such that each incorporation stops the reaction cycle. Unlike the conventional 'Sanger' sequencing approach, NGS sequences via bridge amplification followed by florescent visualization using a camera. In addition, NGS applications can be used to sequence templates 'de novo' or without prior knowledge of the template sequence, therefore, allowing the sequencing of novel or uncommon viral pathogens within samples.During routine NGS diagnostic testing, we identified a high rate of co-infection (48.3%) of clinical swine enteric samples with 1 or more uncommon viruses including porcine kobuvirus, astrovirus, sapovirus, enterovirus and teschovirus. Furthermore, of the positive samples, four kobuvirus, five astrovirus, three enterovirus, three sapovirus and one teschovirus near-complete or complete genomes were extracted from the deep sequence data. More interestingly, we were able to identify an extremely divergent picobirnavirus in 29% of the swine enteric samples. Molecular characterization of the two-segmented genome indicated the RNA-dependent-RNA polymerase encoding segment is <50% similar at the nucleotide level to the closest picobirnavirus and the capsid encoding segment is <50% similar at the amino acid level to other picobirnaviruses. Although little is known regarding the pathogenicity of this virus, it is thought that picobirnavirus is a bacteriophage functioning in signaling and propagation of bacteria in the host animal.In parallel, we screened bovine respiratory diagnostic samples via RT-PCR for bovine kobuvirus which identified (>10%) positivity. While little is currently understood regarding the interactions and associations of the virome in livestock, we seek to;Identify common viruses in bovine enteric and respiratory samples submitted to the KSVDL using NGSCharacterize novel or uncommon viruses identified using both molecular and protein techniquesAssess the epidemiology associated with the dissemination and maintenance of these viral pathogens using the KSVDL's connection to clients
Project Methods
The Sequence-Independent-Single-Primer-Amplification (SISPA) metagenomic sequencing method we utilize in our lab was previously implemented to identify and characterize a variety of novel viruses including porcine circovirus 3, atypical porcine pestivirus, porcine posaviruses, porcine parainfluenza virus 1 and porcine parvovirus 7. Using this sequencing technique as well as the vast library of diagnostic samples, we plan to identify and describe the virome within 40 bovine enteric and 40 respiratory samples. Following the initial identification of the pathogen, we will use targeted primers to complete the genomes and screen within samples for prevalence studies. Molecular characterization will be performed following the genome completions using bioinformatics analyses of closest reference sequences available in Genbank via phylogenetic tree, amino acid changes and protein homology predictions (iTasser software). Virus isolation will be attempted on all positive samples using in-house diagnostic techniques.Antibody targets and antibody prevalence will be assessed via indirect elisa with recombinant proteins. Recombinant proteins will be synthesized using an in-house protein expression system which involves synthesizing the protein sequence into the pUC57 vector with a 6x His tag (Genscript), transfecting the vector into the appropriate expression E.coli (BL-21), amplifying and expressing the protein and purifying the protein (Ni-NTA his-tag purification). The purified protein will then be used to coat ELISA plates to assess antibody prevalence. The ELISA test will be evaluated based on Sample/positive values.