Qiana Latrese Matthews

The Getah virus (GETV) is a mosquito-borne RNA virus in the Togaviridae family and the Alphavirus genus, associated with severe disease outbreaks in livestock across Asia-Pacific regions. Since its first isolation in Malaysia in 1955, GETV has been reported in Eurasia and the South Pacific, yet documented cases in the United States remain exceptionally rare, leaving major gaps in regional vector competence and surveillance data. To evaluate the potential of local mosquito populations as GETV carriers, field collections were conducted in Montgomery and Pike Road neighborhoods of Alabama from 2023 to 2024, capturing multiple mosquito species for molecular screening. Initial real-time qPCR assays on 200 pooled and individual samples suggested that approximate 30% of specimens demonstrated possible GETV carriage potential. To validate viral presence, two distinct primer sets were designed to amplify viral genome fragments. Mosquito RNA was reverse-transcribed into cDNA, followed by conventional PCR. The first PCR, targeting ∼280 bp, produced single or multiple amplicon bands in 32% of samples via gel electrophoresis. These positive cDNAs were re-amplified with a second primer pair targeting a ∼430 bp fragment from a separate genomic region, yielding confirmatory bands in 30% of specimens. Amplified products were purified and Sanger sequenced, revealing approximate 95% nucleotide similarity to the wild-type GETV reference genome. This study delivers the first field-based molecular evidence supporting GETV detection in Alabama mosquitoes, signaling either localized emergence or potential introduction of the virus. These findings underscore the need for expanded vector competence profiling, arboviral surveillance, and livestock disease preparedness in the southeastern United States, strengthening both public health readiness and state-level vector monitoring strategies.

Extracellular vesicles (EVs), nanosized particles approximately 20-1000 nm in size, are rapidly garnering attention for their therapeutic potential. Specifically, human breast milk-derived extracellular vesicles (HBMDEVs) have been shown to confer protection in experimental models of necrotizing enterocolitis, an intestinal disease that primarily affects premature infants, resulting in mortality as high as 50%. However, traditional extracellular vesicle isolation techniques, such as differential ultracentrifugation, size exclusion chromatography, etc., are either time-consuming or require specialized instrumentation, neither of which is practical in the clinical setting. Hence, there is a need for a simple isolation method that will allow for rapid reactive administration of HBMDEVs to patients. Proposed here is a combination of centrifugation of human breast milk to remove fat and cellular debris, coupled with rapid ultrafiltration of the remaining skim milk that produces high yield and functional extracellular vesicles. Using this technique may allow for the use of HBMDEVs as a practical therapeutic option in the clinical setting.

Extracellular vesicles (EVs), nanosized particles approximately 20-1000 nm in size, are rapidly garnering attention for their therapeutic potential. Specifically, human breast milk-derived extracellular vesicles (HBMDEVs) have been shown to confer protection in experimental models of necrotizing enterocolitis, an intestinal disease that primarily affects premature infants, resulting in mortality as high as 50%. However, traditional extracellular vesicle isolation techniques, such as differential ultracentrifugation, size exclusion chromatography, etc., are either time-consuming or require specialized instrumentation, neither of which is practical in the clinical setting. Hence, there is a need for a simple isolation method that will allow for rapid reactive administration of HBMDEVs to patients. Proposed here is a combination of centrifugation of human breast milk to remove fat and cellular debris, coupled with rapid ultrafiltration of the remaining skim milk that produces high yield and functional extracellular vesicles. Using this technique may allow for the use of HBMDEVs as a practical therapeutic option in the clinical setting.

Introduction: Coronavirus (CoV) is an extremely contagious, enveloped positive-single-stranded RNA virus, which has become a global pandemic that causes several illnesses in humans and animals. Hence, it is necessary to investigate viral-induced reactions across diverse hosts. Herein, we propose utilizing naturally secreted extracellular vesicles (EVs), mainly focusing on exosomes to examine virus–host responses following CoV infection. Exosomes are small membrane-bound vesicles originating from the endosomal pathway, which play a pivotal role in intracellular communication and physiological and pathological processes. We suggested that CoV could impact EV formation, content, and diverse immune responses in vitro. Methods: In this study, we infected A-72, which is a canine fibroblast cell line, with a feline coronavirus (FCoV) and canine coronavirus (CCoV) independently in an exosome-free media at 0.001 multiplicity of infection (MOI), with incubation periods of 48 and 72 h. The cell viability was significantly downregulated with increased incubation time following FCoV and CCoV infection, which was identified by performing the 3-(4,5-dimethylthiazo-1-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After the infection, EVs were isolated through ultracentrifugation, and the subsequent analysis involved quantifying and characterizing the purified EVs using various techniques. Results: NanoSight particle tracking analysis (NTA) verified that EV dimensions fell between 100 and 200 nm at both incubation periods. At both periods, total protein and RNA levels were significantly upregulated in A-72-derived EVs following FCoV and CCoV infections. However, total DNA levels were gradually upregulated with increased incubation time. Dot blot analysis indicated that the expression levels of ACE2, IL-1β, Flotillin-1, CD63, caspase-8, and Hsp90 were modified in A-72-derived EVs following both CoV infections. Conclusions: Our results indicated that FCoV and CCoV infections could modulate the EV production and content, which could play a role in the development of viral diseases. Investigating diverse animal CoV will provide in-depth insight into host exosome biology during CoV infection. Hence, our findings contribute to the comprehension and characterization of EVs in virus–host interactions during CoV infection.

Extracellular vesicles (EVs) have arisen as potential therapeutic tools in managing infectious diseases because EVs can regulate cell-to-cell signaling, function as drug transport mechanisms, and influence immune reactions. They are obtained from a myriad of sources, such as plants, humans, and animal cells. EVs like exosomes and ectosomes can be utilized in their native form as therapeutics or engineered to encompass antimicrobials, vaccines, and oligonucleotides of interest with a targeted delivery strategy. An in-depth understanding of host-pathogen dynamics provides a solid foundation for exploiting its full potential in therapeutics against infectious diseases. This review mainly offers an extensive summary of EVs, comprising their various origins, formations, and pathogen relationships. It further provides insights into the various techniques utilized in isolating and engineering these vesicles to target infectious diseases and how challenges involving large-scale production and cargo loading efficiency should be addressed for clinical application. Finally, preclinical and clinical implementations of EVs derived from animals, plants, and microorganisms are elucidated, stressing their promise for designing innovative antimicrobial approaches.

Introduction: Coronavirus (CoV) has become a public health crisis that causes numerous illnesses in humans and certain animals. Studies have identified the small, lipid-bound structures called extracellular vesicles (EVs) as the mechanism through which viruses can enter host cells, spread, and evade the host’s immune defenses. EVs are able to package and carry numerous viral compounds, including proteins, genetic substances, lipids, and receptor proteins. We proposed that the coronavirus could alter EV production and content, as well as influence EV biogenesis and composition in host cells.Methods: In the current research, Crandell-Rees feline kidney (CRFK) cells were infected with feline coronavirus (FCoV) in an exosome-free media at a multiplicity of infection (MOI) of 2,500 infectious units (IFU) at 48 h and 72 h time points. Cell viability was analyzed and found to be significantly decreased by 9% (48 h) and 15% (72 h) due to FCoV infection. EVs were isolated by ultracentrifugation, and the surface morphology of isolated EVs was analyzed via Scanning Electron Microscope (SEM).Results: NanoSight particle tracking analysis (NTA) confirmed that the mean particle sizes of control EVs were 131.9 nm and 126.6 nm, while FCoV infected-derived EVs were 143.4 nm and 120.9 nm at 48 and 72 h, respectively. Total DNA, RNA, and protein levels were determined in isolated EVs at both incubation time points; however, total protein was significantly increased at 48 h. Expression of specific protein markers such as TMPRSS2, ACE2, Alix, TSG101, CDs (29, 47, 63), TLRs (3, 6, 7), TNF-α, and others were altered in infection-derived EVs when compared to control-derived EVs after FCoV infection.Discussion: Our findings suggested that FCoV infection could alter the EV production and composition in host cells, which affects the infection progression and disease evolution. One purpose of studying EVs in various animal coronaviruses that are in close contact with humans is to provide significant information about disease development, transmission, and adaptation. Hence, this study suggests that EVs could provide diagnostic and therapeutic applications in animal CoVs, and such understanding could provide information to prevent future coronavirus outbreaks.

Introduction: Drug repurposing is fast growing and becoming an attractive approach for identifying novel targets, such as exosomes for cancer and antiviral therapy. Exosomes are a specialized class of extracellular vesicles that serve as functional mediators in intercellular communication and signaling that are important in normal physiological functions. A continuously growing body of evidence has established a correlation between the abnormal release of exosomes with various viral disease pathologies including cancer. Cells that are virus-infected release exosomes known to influence the process via the loading and transfer of viral components, such as miRNA, small (s) RNA, DNA, and proteins. Inhibition of exosome release may abate the spread and severity of viral infection, thus making exosomes an attractive target for antiviral therapies. We previously demonstrated the pharmacological inhibition of exosomes.Methods: Herein, we used a cell-based assay to determine the effect of Human adenovirus type 3 (HAdV3) on the exosome inhibition process by azole and Heparin derivatives. HAdV3-infected cells were treated with two concentrations of each inhibitor at different time points.Results: HAdV3 activities led to increased total sRNA, DNA, and exosome particle concentrations via particle tracking in the presence of Climbazole and Heparin relative to uninfected exosomes. In addition, there was an increased expression of classical markers such as ALG-2 interacting protein X (ALIX), and tetraspanin (CD63), (p < 0.05) and upregulated transcription factor interferon regulatory factor (IRF) 8 in the presence of HAdV3 after 24 hours (h) of treatment. Whereas higher concentrations of Climbazole and Heparin sodium salt were found to inhibit total exosome protein (p < 0.001) and exo-RNA (p < 0.01) content even in the presence of HAdV3 relative to infected exosomes only. Activities of HAdV3 in the presence of selected inhibitors resulted in the positive regulation of exosome related DNA damage/repair signaling proteins. Blocking exosome secretion partially obstructed viral entry. Immunological studies revealed that HAdV3 fiber protein levels in A549 cells were reduced at all concentrations of Climbazole and Heparin and both multiplicities of infections (p < 0.001).Discussion: Our findings suggest that while HAdV may bolster inhibited exosome content and release when modulating certain activities of the endosomal pathway mediators, HAdV entry might be constrained by the activities of these pharmacological agents.

The scientific interest in cannabis plants’ beneficial properties has recently sparked certain interest in the possible functional characterization of plant-derived extracellular vesicles (PDEVs). Establishing the most appropriate and efficient isolation procedure for PDEVs remains a challenge due to vast differences in the physio-structural characteristics of different plants within the same genera and species. In this study, we employed a crude but standard isolation procedure for the extraction of apoplastic wash fluid (AWF) which is known to contain the PDEVs. This method includes a detailed stepwise process of PDEV extraction from five (5) cultivars of cannabis plants, namely: Citrus (C), Henola (HA), Bialobrezenski (BZ), Southern-Sunset (SS), and Cat-Daddy (CAD). Approximately, 150 leaves were collected from each plant strain. In order to collect PDEV pellets, apoplastic wash fluid (AWF) was extracted from plants via negative pressure permeabilization and infiltration followed by high-speed differential ultracentrifugation. Particle tracking analysis of PDEVs revealed particle size distribution in the range of 20 to 200 nm from all plant strains, while PDEV total protein concentration from HA was higher than that of SS. Although HA-PDEVs’ total protein was higher than SS-PDEVs, SS-PDEVs’ RNA yield was higher than that of HA-PDEVs. Our result suggests that the cannabis plant strains contain EVs, and PDEV concentration from the cannabis plant could be age or strain dependent. Overall, the results provide a guide for the selection and optimization of PDEV isolation methods for future studies.

The continuous emergence of novel viruses and their diseases are a threat to global public health as there have been three outbreaks of coronaviruses that are highly pathogenic to humans in the span of the last two decades, severe acute respiratory syndrome (SARS)-CoV in 2002, Middle East respiratory syndrome (MERS)-CoV in 2012, and novel SARS-CoV-2 which emerged in 2019. The unprecedented spread of SARS-CoV-2 worldwide has given rise to multiple SARS-CoV-2 variants that have either altered transmissibility, infectivity, or immune escaping ability, causing diseases in a broad range of animals including human and non-human hosts such as companion, farm, zoo, or wild animals. In this review, we have discussed the recent SARS-CoV-2 outbreak, potential animal reservoirs, and natural infections in companion and farm animals, with a particular focus on SARS-CoV-2 variants. The expeditious development of COVID-19 vaccines and the advancements in antiviral therapeutics have contained the COVID-19 pandemic to some extent; however, extensive research and surveillance concerning viral epidemiology, animal transmission, variants, or seroprevalence in diverse hosts are essential for the future eradication of COVID-19.