Browsing by Author "Stanton, James B."

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    Determining the role of natural SARS-CoV-2 infection in the death of domestic pets: 10 cases (2020-2021)
    Carpenter, Ann; Ghai, Ria R.; Gary, Joy; Ritter, Jana M.; Carvallo, Francisco R.; Diel, Diego G.; Martins, Mathias; Murphy, Julia; Schroeder, Betsy A.; Brightbill, Kevin; Tewari, Deepanker; Boger, Lore; Gabel, Julie; Cobb, Robert; Hennebelle, Janemarie; Stanton, James B.; McCullough, Kathryn; Mosley, Yung-Yi C.; Naikare, Hemant K.; Radcliffe, Rachel; Parr, Boyd; Balsamo, Gary; Robbins, Brent; Smith, David; Slavinski, Sally; Williams, Carl; Meckes, Doug; Jones, Dee; Frazier, Tony; Steury, Kelley; Rooney, Jane; Torchetti, Mia; Wendling, Natalie; Currie, Dustin; Behravesh, Casey Barton; Wallace, Ryan M. (2021-11-01)
    OBJECTIVE To establish a pathoepidemiological model to evaluate the role of SARS-CoV-2 infection in the first 10 companion animals that died while infected with SARS-CoV-2 in the US. ANIMALS 10 cats and dogs that tested positive for SARS-CoV-2 and died or were euthanized in the US between March 2020 and January 2021. PROCEDURES A standardized algorithm was developed to direct case investigations, determine the necessity of certain diagnostic procedures, and evaluate the role, if any, that SARS-CoV-2 infection played in the animals' course of disease and death. Using clinical and diagnostic information collected by state animal health officials, state public health veterinarians, and other state and local partners, this algorithm was applied to each animal case. RESULTS SARS-CoV-2 was an incidental finding in 8 animals, was suspected to have contributed to the severity of clinical signs leading to euthanasia in 1 dog, and was the primary reason for death for 1 cat. CONCLUSIONS AND CLINICAL RELEVANCE This report provides the global community with a standardized process for directing case investigations, determining the necessity of certain diagnostic procedures, and determining the clinical significance of SARS-CoV-2 infections in animals with fatal outcomes and provides evidence that SARS-CoV-2 can, in rare circumstances, cause or contribute to death in pets.
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    Rapid virulence prediction and identification of Newcastle disease virus genotypes using third-generation sequencing
    Butt, Salman L.; Taylor, Tonya L.; Volkening, Jeremy D.; Dimitrov, Kiril M.; Williams-Coplin, Dawn; Lahmers, Kevin K.; Miller, Patti J.; Rana, Asif M.; Suarez, David L.; Afonso, Claudio L.; Stanton, James B. (2018-11-22)
    Background Newcastle disease (ND) outbreaks are global challenges to the poultry industry. Effective management requires rapid identification and virulence prediction of the circulating Newcastle disease viruses (NDV), the causative agent of ND. However, these diagnostics are hindered by the genetic diversity and rapid evolution of NDVs. Methods An amplicon sequencing (AmpSeq) workflow for virulence and genotype prediction of NDV samples using a third-generation, real-time DNA sequencing platform is described here. 1D MinION sequencing of barcoded NDV amplicons was performed using 33 egg-grown isolates, (15 NDV genotypes), and 15 clinical swab samples collected from field outbreaks. Assembly-based data analysis was performed in a customized, Galaxy-based AmpSeq workflow. MinION-based results were compared to previously published sequences and to sequences obtained using a previously published Illumina MiSeq workflow. Results For all egg-grown isolates, NDV was detected and virulence and genotype were accurately predicted. For clinical samples, NDV was detected in ten of eleven NDV samples. Six of the clinical samples contained two mixed genotypes as determined by MiSeq, of which the MinION method detected both genotypes in four samples. Additionally, testing a dilution series of one NDV isolate resulted in NDV detection in a dilution as low as 101 50% egg infectious dose per milliliter. This was accomplished in as little as 7 min of sequencing time, with a 98.37% sequence identity compared to the expected consensus obtained by MiSeq. Conclusions The depth of sequencing, fast sequencing capabilities, accuracy of the consensus sequences, and the low cost of multiplexing allowed for effective virulence prediction and genotype identification of NDVs currently circulating worldwide. The sensitivity of this protocol was preliminary tested using only one genotype. After more extensive evaluation of the sensitivity and specificity, this protocol will likely be applicable to the detection and characterization of NDV.
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    Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples
    Butt, Salman L.; Erwood, Eric C.; Zhang, Jian; Sellers, Holly S.; Young, Kelsey T.; Lahmers, Kevin K.; Stanton, James B. (2020-03)
    Infectious bronchitis (IB) causes significant economic losses in the global poultry industry. Control of IB is hindered by the genetic diversity of the causative agent, infectious bronchitis virus (IBV), which has led to the emergence of several serotypes that lack complete serologic cross-protection. Although serotyping requires immunologic characterization, genotyping is an efficient means to identify IBVs detected in samples. Sanger sequencing of the S1 subunit of the spike gene is currently used to genotype IBV; however, the universal S1 PCR was created to work from cultured IBV, and it is inefficient at detecting multiple viruses in a single sample. We describe herein a MinION-based, amplicon-based sequencing (AmpSeq) method that genetically categorized IBV from clinical samples, including samples with multiple IBVs. Total RNA was extracted from 15 tracheal scrapings and choanal cleft swab samples, randomly reverse transcribed, and PCR amplified using modified S1-targeted primers. Amplicons were barcoded to allow for pooling of samples, processed per manufacturer's instructions into a 1D MinION sequencing library, and then sequenced on the MinION. The AmpSeq method detected IBV in 13 of 14 IBV-positive samples. AmpSeq accurately detected and genotyped both IBV lineages in 3 of 5 samples containing 2 IBV lineages. Additionally, 1 sample contained 3 IBV lineages, and AmpSeq accurately detected 2 of the 3 lineages. Strain identification, including detection of different IBVs from the same lineage, was also possible with this AmpSeq method. Our results demonstrate the feasibility of using MinION-based AmpSeq for rapid and accurate identification and lineage typing of IBV from oral swab samples.