A Validatable Droplet Digital Polymerase Chain Reaction Assay for the Detection of Adeno-Associated Viral Vectors in Bioshedding Studies of Tears.

The use of viral vectors to treat genetic diseases has increased substantially in recent years, with over 2,000 studies registered to date. Adeno-associated viral (AAV) vectors have found particular success in the treatment of eye related diseases, as exemplified by the approval of voretigene neparvovec-rzyl. To bring new therapies to market, regulatory agencies typically request qualified or validated bioshedding studies to evaluate release of the vector into the environment. However, no official guidelines for the development of molecular based assays to support such shedding studies have been released by the United States Food and Drug Administration, leaving developers to determine best practices for themselves. The purpose of this protocol is to present a validatable protocol for the detection of AAV vectors in human tears by droplet digital polymerase chain reaction (ddPCR) in support of clinical bioshedding studies. This manuscript discusses current industry approaches to molecular assay validation and demonstrates that the method exceeds the target assay acceptance criteria currently proposed in white papers. Finally, steps critical in the performance of any ddPCR assay, regardless of application, are discussed.

Disparate Entry of Adenoviruses Dictates Differential Innate Immune Responses on the Ocular Surface.

Human adenovirus infection of the ocular surface is associated with severe keratoconjunctivitis and the formation of subepithelial corneal infiltrates, which may persist and impair vision for months to years following infection. Long term pathology persists well beyond the resolution of viral replication, indicating that the prolonged immune response is not virus-mediated. However, it is not clear how these responses are sustained or even initiated following infection. This review discusses recent work from our laboratory and others which demonstrates different entry pathways specific to both adenovirus and cell type. These findings suggest that adenoviruses may stimulate specific pattern recognition receptors in an entry/trafficking-dependent manner, leading to distinct immune responses dependent on the virus/cell type combination. Additional work is needed to understand the specific connections between adenoviral entry and the stimulation of innate immune responses by the various cell types present on the ocular surface.

Transcriptome profiling of alphaherpesvirus-infected cells treated with the HIV-integrase inhibitor raltegravir reveals profound and specific alterations in host transcription.

Anti-microbial compounds typically exert their action by directly interfering with one or more stages of the pathogen's life cycle. However, some compounds also have secondary effects on the host that aid in pathogen clearance. Raltegravir is a human immunodeficiency virus (HIV)-integrase inhibitor that has been shown to alter the host immune response to HIV in addition to its direct antiviral effect. Interestingly, raltegravir can also directly inhibit the replication of various herpesviruses. However, the host-targeted effects of this drug in the context of a herpesvirus infection have not been explored. Here, we used felid alphaherpesvirus 1 (FHV-1), a close relative of human alphaherpesvirus 1 (HHV-1) that similarly causes ocular herpes, to characterize the host-targeted effects of raltegravir on corneal epithelial cells during an alphaherpesvirus infection. Using RNA deep sequencing, we found that raltegravir specifically boosts the expression of anti-angiogenic factors and promotes metabolic homeostasis in FHV-1-infected cells. In contrast, few changes in host gene transcription were found in uninfected cells. Importantly, we were able to demonstrate that these effects were specific to raltegravir and independent of the direct-acting antiviral effect of the drug, since treatment with the DNA polymerase inhibitor phosphonoacetic acid did not induce these host-targeted effects. Taken together, these results indicate that raltegravir has profound and specific effects on the host transcription profile of herpesvirus-infected cells that may contribute to the overall antiviral activity of the drug and could provide therapeutic benefits in vivo. Furthermore, this study provides a framework for future efforts evaluating the host-targeted effects of anti-microbial compounds.

First demonstration of equid gammaherpesviruses within the gastric mucosal epithelium of horses.

Horses commonly develop gastric mucosal ulcers, similar to humans, a condition known as equine gastric ulcer syndrome (EGUS) that can lead to poor performance and lost training time and care expenses. Unlike humans, however, an infectious bacterial cause of ulcers has not been conclusively identified. Herpesviruses, while well-established causative agents of diseases such as cold sores, genital lesions, and certain types of cancer, have also been implicated in the development of a subset of gastric ulcers in humans. The presence of equid herpesviruses in the gastrointestinal tract and their potential contribution to EGUS has not been evaluated. Here, we provide the first evidence of equid gammaherpesviruses 2 and 5 (EHV-2 and -5) within the epithelium of the gastric mucosa of horses. These viruses were initially detected by a nested PCR screen of gastric tissue samples obtained from client- and university-owned horses with and without ulcers; however, no association with EGUS was found in this limited sample set. We then validated a highly sensitive in situ hybridization (ISH) assay and used this assay to characterize the distribution of these viruses in necropsy gastric tissue samples from five racehorses. Analyses revealed frequent EHV-2 and EHV-5 co-infections within the gastric mucosal epithelium, regardless of the ulcer status. These results are the first to demonstrate the presence of equid gammaherpesviruses in the gastric mucosa of horses and warrants further investigation to determine the contribution of these viruses to the development of EGUS and/or other gastrointestinal diseases.

Equine Mesenchymal Stromal Cells from Different Sources Efficiently Differentiate into Hepatocyte-Like Cells.

Adult equine hepatocytes have proven challenging to culture long term in vitro as they rapidly lose their morphology and functionality, thus limiting studies on liver function and response to disease. In this study, we describe for the first time the differentiation of equine mesenchymal stromal cells (MSC) from a variety of sources into functional hepatocyte-like cells (HLC). First, we differentiated equine umbilical cord blood (UCB)-derived MSC into HLC and found that these cells exhibited a distinct polygonal morphology, stored glycogen as visualized by periodic acid Schiff's reagent staining, and were positive for albumin and other hepatocyte-specific genes. Second, we demonstrated that UCB-HLC could be revived following cryopreservation and retained their phenotype for at least 10 days. Third, we differentiated three sets of MSC from bone marrow (BM), adipose tissue (AT), and peripheral blood (PB), matched within the same horse. We achieved a 100% differentiation success rate with BM, 0% with AT, and 66% with PB. An additional set of nine PB-MSC samples resulted in an overall success rate of 42% (n = 12), and age or gender did not seem to have an effect on the success of hepatic differentiation from that source. In a final set of experiments, we evaluated the use of these HLC as tools in different fields of biomedical research like virology, to study viral growth, and toxicology, to study chemicals with hepatic toxicity. Equine HLC were found susceptible for infection with the equine herpesviruses type 1 (EHV-1), -2, and -5, and exhibited a more sensitive dose-dependent response to arsenic toxicity than the commonly used human hepatocellular cell line HepG2. Taken together, these data indicate that equine MSC can be efficiently differentiated into HLC and these equine HLC could be a useful tool for in vitro studies.