Biology subjects

Stanton, J. B.

Publications and source records attributed to Stanton, J. B..

6 recordsLinked to original sources

bioRxiv2024

Pandemic Risk Assessment for a Swine Influenza A Virus in Comparative Human Substrates

Swine influenza A viruses pose a public health concern as novel and circulating strains occasionally spill over into human hosts, with the potential to cause disease. Crucial to preempting these events is the use of a threat assessment framework for human populations. However, established guidelines do not specify what animal models or in vitro substrates should be used. We completed an assessment of a contemporary swine influenza isolate, A/swine/GA/A27480/2019, using animal models and human cell substrates. Infection studies in vivo revealed high replicative ability and a pathogenic phenotype in the swine host with replication corresponding to a complementary study performed in swine primary respiratory epithelial cells. However, replication was limited in human primary cell substrates. This contrasted with our findings in the Calu-3 cell line, which demonstrated a replication profile on par with the 2009 pandemic H1N1 virus. These data suggest selection of models is important for meaningful risk assessment. Article Summary LineA novel swine influenza A virus was rigorously assessed for pandemic potential in animal models and human surrogate culture models, illustrating a wide range in potential public health risk dependent on the model utilized.

microbiology↗

bioRxiv2023

Piscichuviral encephalitis in marine and freshwater chelonians: first evidence of jingchuviral disease

Chuviruses (family Chuviridae), which are in the recently discovered order Jingchuvirales, were first identified in arthropods in 2015 and have been detected through metagenomics in numerous invertebrates, but only a few vertebrates. With only few metagenomically based detections in vertebrates, their replication competency in vertebrates remained questioned, let alone their pathological significance. This study identified three novel chuviruses as the etiology of lymphocytic meningoencephalomyelitis in three wild aquatic turtles: an alligator snapping turtle (Macrochelys sp.), a Kemps ridley turtle (Lepidochelys kempii), and a loggerhead turtle (Caretta caretta). The application of random, deep sequencing successfully assembled the complete snapping turtle chuvirus-1 [STCV-1], Kemps ridley turtle chuvirus-1 [KTCV-1] genome, and loggerhead turtle chuvirus-1 [LTCV-1]) genome. The STCV-1 and KTCV-1 sequences were used to create custom RNAscope probes for in situ hybridization, which confirmed STCV-1, KTCV-1, and LTCV-1 (cross reactivity to the KTCV-1 probe) RNA within the inflamed region of the brain and spinal cord. STCV-1 and KTCV-1 were isolated on several turtle-origin cell lines. Phylogenetic analysis illustrated that all three viruses clustered with other vertebrate chuviruses, all within the genus Piscichuvirus. With more than 91% pairwise amino acid identity of the polymerase proteins, STCV-1, KTCV-1, and LTCV-1 belong to the same novel species, putatively named Piscichuvirus testudinae. This study demonstrates the first in situ evidence of chuviral pathogenicity in animals and only the second instance of jingchuviral isolation. The association of these chuviruses in three different chelonians with neurologic disease suggests the possibility that chuviruses are a significant, previously unrecognized cause of lymphocytic meningoencephalomyelitis in freshwater and marine turtles. Additional studies of these viruses are needed to elucidate their role in chelonians and the possibility of related viruses in other related hosts. ImportanceChuviruses have been identified in multiple animal species, including humans. However, most were identified metagenomically, and detection was not strongly associated with disease. This study provides the first evidence of chuviral disease in animals in diseased tissue: mononuclear meningoencephalomyelitis in three chelonians from three different genera, two distinct families. These pathogenic turtle chuviruses belong to the genus Piscichuvirus containing other non-mammalian vertebrate chuviruses and were classified together within a novel chuviral species. This study supports the need for further investigations into chuviruses to understand their biology, pathogenic potential, and their association with central nervous system inflammation in chelonians, other reptiles, and other vertebrates.

pathology↗

bioRxiv2021

Putative novel avian paramyxoviruses identified from wild bird surveillance (United States, 2016?2018) and retrospective analysis of APMV-2s and APMV-6s in GenBank

Avian paramyxoviruses (APMVs) (subfamily Avulavirinae) have been isolated from over 200 species of wild and domestic birds from around the world. The International Committee on Taxonomy of Viruses (ICTV) currently defines 22 different APMV species, with Avian orthoavulavirus 1 (whose viruses are designated as APMV-1) being the most frequently studied due to its economic burden to the poultry industry. Less is known about other APMV species, including limited knowledge on the genetic diversity in wild birds and there is a paucity of public whole genome sequences for APMV-2 to -22. The goal of this study was to use MinION sequencing to genetically characterize APMVs isolated from wild bird swab samples collected during 2016-2018 in the United States. Multiplexed MinION libraries were prepared using a random strand-switching approach using 37 egg-cultured, influenza-negative, hemagglutination-positive samples. Thirty-five APMV isolates that had complete polymerase coding sequences were speciated using ICTVs current Paramyxoviridae phylogenetic methodology. Viruses from APMV-1, -4, -6, -8 were classified, one putative novel species (Avian orthoavulavirus 23) was identified from viruses isolated in this study, two putative new APMV species (Avian metaavulavirus 24 and 27) were identified from viruses isolated in this study and from retrospective GenBank sequences, and two putative new APMV species (Avian metaavulavirus 25 and 26) were identified solely from retrospective GenBank sequences. Furthermore, co-infections of APMVs were identified in a subset of the samples. The potential limitations of the branch length being the only speciation criterion and the potential benefit of a group pairwise distance analysis are discussed. ImportanceMost species of APMVs are understudied and/or underreported and many species were incidentally identified from asymptomatic wild birds; however, the disease significance of APMVs in wild birds is not fully determined. The rapid rise in high-throughput sequencing coupled with avian influenza surveillance programs have identified 12 different APMV species in the last decade and have challenged the resolution of classical serological methods to identify new viral species. Currently, ICTVs only criterion for Paramyxoviridae species classification is the requirement of a branch length >0.03 using a phylogenetic tree constructed from polymerase (L) amino acid sequences. The results from this study identify one new APMV species, propose four additional new APMV species, and highlight that the criterion may have insufficient resolution for APMV species demarcation and that refinement or expansion of this criterion may need to be established for Paramyxoviridae speciation.

microbiology↗

bioRxiv2019

Randomly primed, strand-switching MinION-based sequencing for the detection and characterization of cultured RNA viruses

RNA viruses rapidly mutate, which can result in increased virulence, increased escape from vaccine protection, and false negative detection results. Targeted detection methods have a limited ability to detect unknown viruses and often provide insufficient data to detect coinfections or identify antigenic variants. Random, deep sequencing is a method that can more fully detect and characterize RNA viruses and is often coupled with molecular techniques or culture methods for viral enrichment. Viral culture coupled with third-generation sequencing were tested for the ability to detect and characterize RNA viruses. Cultures of bovine viral diarrhea virus, canine distemper virus, epizootic hemorrhagic disease virus, infectious bronchitis virus, two influenza A viruses, and porcine respiratory and reproductive syndrome virus were sequenced on the MinION platform using a random, reverse primer in a strand-switching reaction, coupled with PCR-based barcoding. Reads were taxonomically classified and used for reference-based sequence building using a stock personal computer. This method accurately detected and identified complete coding sequence genomes with a minimum of 20x coverage depth for all seven viruses, including a sample containing two viruses. Each lineage-typing region had at least 26x coverage depth for all viruses. Furthermore, analyzing the canine distemper virus sample through a pipeline devoid of canine distemper virus reference sequences modeled the ability of this protocol to detect unknown viruses. These results show the ability of this technique to detect and characterize dsRNA, negative- and positive-sense ssRNA, nonsegmented, and segmented RNA viruses.

pathology↗

bioRxiv2019

Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples

Infectious bronchitis (IB) causes significant economic losses in the global poultry industry. Control of infectious bronchitis 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. While serotyping by definition 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 mixed isolates. This paper describes a MinION-based AmpSeq method that genetically typed IBV from clinical samples, including samples with multiple isolates. Total RNA was extracted from fifteen 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 manufacturers instructions into a 1D MinION sequencing library, and 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 three of five samples containing two IBV lineages. Additionally, one sample contained three IBV lineages, and AmpSeq accurately detected two of the three. Strain identification, including detection of different strains from the same lineage, was also possible with this AmpSeq method. The results demonstrate the feasibility of using MinION-based AmpSeq for rapid and accurate identification and lineage typing of IBV from oral swab samples.

microbiology↗

bioRxiv2018

Rapid and sensitive virulence prediction and identification of Newcastle disease virus genotypes using third-generation sequencing

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. A highly sensitive amplicon sequencing (AmpSeq) workflow for virulence and genotype prediction of NDV samples using a third-generation, real-time DNA sequencing platform is described using both egg-propagated virus and clinical samples. 1D MinION sequencing of barcoded NDV amplicons was performed on 33 egg-grown isolates, (23 unique lineages, including 15 different NDV genotypes), and from 15 clinical swab samples from field outbreaks. Assembly-based data analysis was performed in a customized, Galaxy-based AmpSeq workflow. For all egg-grown samples, NDV was detected and virulence and genotype were predicted. For clinical samples, NDV was detected in ten of eleven NDV samples. Six of the clinical samples contained two mixed genotypes, of which the MinION method detected both genotypes in four of those samples. Additionally, testing a dilution series of one NDV sample resulted in detection of NDV with a 50% egg infectious dose (EID50) as low as 101 EID50/ml. This was accomplished in as little as 7 minutes of sequencing time, with a 98.37% sequence identity compared to the expected consensus. The high sensitivity, fast sequencing capabilities, accuracy of the consensus sequences, and the low cost of multiplexing allowed for identification of NDV of different genotypes circulating worldwide. This general method will likely be applicable to other infections agents.

molecular biology↗