Jerry R. Malayer

The present study was carried out to analyze the complete genome sequences of infectious bursal disease virus (IBDV) isolates obtained from field outbreaks in the southern regions of India. Bursal tissue samples were collected and screened by RT-PCR, targeting the VP2 gene. Positive samples were subjected to serological identification via AGID. Following this, eight samples (BGE14, BGE15, MDI14, THI14, EDE14, RPM14, VCN14, and NKL14) were subjected to virus isolation in 9 to 11-day-old embryonated chicken eggs, and their complete genomes were sequenced. Analysis of the VP2 hypervariable region (HVR) revealed that all eight isolates had five unique and highly conserved amino acids (A222, I242, Q249, I256, and S299). However, all the isolates reveal a substitution of Isoleucine by Valine at residue 294 (I294V). Furthermore, analysis of segment B from all Indian IBDV sequences revealed that the triplet amino acid pattern was NEG (residues 145–147) and the amino acid at position 242 was consistently D across all isolates. These findings suggest that segment B of the isolates in this study resembled that of vaccine strains and non-vvIBDV strains. Additionally, the presence of the signature D242 in all Indian isolates, characteristic of non-vvIBDV strains, implies a potential attenuation. Moreover, in the phylogenetic analysis of VP2-HVR, all isolates clustered with very virulent reference strains, while segment B clustered with classical attenuated strains. Notably, the phylogenetic analysis of VP2-HVR and VP1 of these viruses demonstrated genetic variances, suggesting evolutionary changes in segment B across all eight Indian isolates, likely indicative of natural genome reassortment resulting in these specific outbreaks in the flocks.

Introduction. With expanding demand for diagnostics, newer methodologies are needed for faster, user-friendly and multiplexed pathogen detection. Metagenome-based diagnostics offer potential solutions to address these needs as sequencing technologies have become affordable. However, the diagnostic utility of sequencing technologies is currently limited since analysis of the large amounts of data generated, are either computationally expensive or carry lower sensitivity and specificity for pathogen detection. Hypothesis/Gap Statement. There is a need for novel, user friendly, and computationally inexpensive platforms for metagenome sequence analysis for diagnostic applications. Methods. In this study, we report the use of MiFi^® (Microbe Finder), a computationally inexpensive algorithm with a user-friendly online interface, for accurate, rapid and multiplexed pathogen detection from metagenome sequence data. Detection is accomplished based on identification of signature genomic sequence segments of the target pathogen in metagenome sequence data. In this study we used bovine respiratory disease (BRD) complex as a model. Results and Conclusions. Using MiFi^®, multiple target bacteria and a DNA virus were successfully detected in a multiplex format from metagenome sequences acquired from bovine lung tissue. Overall, 51 clinical samples were assessed and MiFi^® showed 100 % analytical specificity and varying levels of analytical sensitivity (62.5 %–100 %) when compared with other traditional pathogen detection techniques, such as PCR. Consistent detection of bacteria was possible from lung samples artificially spiked with 10⁹–10⁴ c.f.u. of Mannheimia haemolytica .

Surveillance is one of the core activities of national organizations responsible for human, animal, or plant health, with the goal of demonstrating the absence of infection or infestation, determining the presence or distribution of infection or infestation, and/or detecting as early as possible exotic or emerging pests and pathogens that may be harmful to agriculture and the environment. Surveillance is a tool to establish absence of the pest or pathogen, monitor trends, facilitate the mitigation and control of infection or infestation, provide data for use in risk analysis, substantiate the rationale for sanitary measures, and provide assurances to trading partners, producers, and the public. The type of surveillance applied depends on the objectives of the surveillance, the available data sources, resources, and the outputs needed to support decision-making.

FENDRR (Fetal-lethal non-coding developmental regulatory RNA, LncRNA FOXF1-AS1) is a recently identified tumor suppressor long non-coding (LncRNA) RNA, and its expression has been linked with epigenetic modulation of the target genes involved in tumor immunity. In this study, we aimed to understand the role of FENDRR in predicting immune-responsiveness and the inflammatory tumor environment. Briefly, FENDRR expression and its relationship to immune activation signals were assessed in murine cell lines. Data suggested that tumor cells (e.g., C26 colon, 4T1 breast) that typically upregulate immune activation genes and the MHC class I molecule exhibited high FENDRR expression levels. Conversely, tumor cells with a generalized downregulation of immune-related gene expression (e.g., B16F10 melanoma) demonstrated low to undetectable FENDRR levels. Mechanistically, the modulation of FENDRR expression enhanced the inflammatory and WNT signaling pathways in tumors. Our early data suggest that FENDRR can play an important role in the development of immune-relevant phenotypes in tumors, and thereby improve cancer immunotherapy.

Genomic sequencing has played a major role in understanding the pathogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With the current pandemic, it is essential that SARS-CoV-2 viruses are sequenced regularly to determine mutations and genomic modifications in different geographical locations. In this study we sequenced SARS-CoV-2 from 5 clinical samples obtained in Oklahoma, USA during different time points of pandemic presence in the state. One sample from the initial days of the pandemic in the state and 4 during the peak in Oklahoma were sequenced. Previously reported mutations including D614G in S gene, P4715L in ORF1ab, S194L, R203K and G204R in N gene were identified in the genomes sequenced in this study. Possible novel mutations were also detected such as G1167V in S gene, A6269S and P3371S in ORF1ab, T28I in ORF7b, G96R in ORF8. Phylogenetic analysis of the genomes showed similarity to viruses from across the globe. These novel mutations and phylogenetic analysis emphasize the contagious nature of the virus.

Francisella tularensis(Ft) is a highly virulent, intracellular Gram-negative bacterial pathogen. Acute Ft infection by aerosol route causes pneumonic tularemia, characterized by nodular hemorrhagic lesions, neutrophil-predominant influx, necrotic debris, fibrin deposition, and severe alveolitis. Ft suppresses activity of neutrophils by impairing their respiratory burst and phagocytic activity. However, the fate of the massive numbers of neutrophils recruited to the infection site is unclear. Here, we show that Ft infection resulted in prominent induction of neutrophil extracellular traps (NETs) within damaged lungs of mice infected with the live attenuated vaccine strain of Ft(Ft-LVS), as well as in the lungs of domestic cats and rabbits naturally infected with Ft. Further, Ft-LVS infection increased lung myeloperoxidase (MPO) activity, which mediates histone protein degradation during NETosis and anchors chromatin scaffolds in NETs. In addition, Ft infection also induced expression of peptidylarginine deiminase 4, an enzyme that causes citrullination of histones during formation of NETs. The released NETs were found largely attached to the alveolar epithelium, and disrupted the thin alveolar epithelial barrier. Furthermore, Ft infection induced a concentration-dependent release of NETs from neutrophils in vitro. Pharmacological blocking of MPO reduced Ft-induced NETs release, whereas addition of H₂O₂ (a substrate of MPO) significantly augmented NETs release, thus indicating a critical role of MPO in Ft-induced NETs. Although immunofluorescence and electron microscopy revealed that NETs could efficiently trap Ft bacteria, NETs failed to exert bactericidal effects. Taken together, these findings suggest that NETs exacerbate tissue damage in pulmonary Ft infection, and that targeting NETosis may offer novel therapeutic interventions in alleviating Ft-induced tissue damage.

Musculoskeletal infections caused by bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa in children and adults can lead to adverse outcomes including a need for extensive surgical debridement and limb amputation. To enable targeted antimicrobial release in infected tissues, the objective of this study was to design and investigate novel elastin-like polypeptide (ELP)-based thermally sensitive liposomes in vitro. ELP biopolymers can change their phase behaviour at higher temperatures. We hypothesised that ELP-TSL will improve therapeutic efficacy by releasing antimicrobial payloads locally at higher temperatures (≥39 °C). ELP-TSL library were formulated by varying cholesterol and phospholipid composition by the thin film and extrusion method. A broad-spectrum antimicrobial (Ciprofloxacin or Cipro) was encapsulated inside the liposomes by the ammonium sulphate gradient method. Cipro release from ELP-TSLs was assessed in physiological buffers containing ∼25% serum by fluorescence spectroscopy, and efficacy against Staphylococcus aureus and Pseudomonas aeruginosa was assessed by disc diffusion and planktonic assay. Active loading of Cipro achieved an encapsulation efficiency of 40–70% in the ELP-TSL depending upon composition. ELP-TSL Cipro release was near complete at ≥39 °C; however, the release rates could be delayed by cholesterol. Triggered release of Cipro from ELP-TSL at ∼42 °C induced significant killing of S. aureus and P. aeruginosa compared to 37 °C. Our in vitro data suggest that ELP-TSL may potentially improve bacterial wound therapy in patients.