Co-administration of chicken IL-2 alleviates clinical signs and replication of the ILTV chicken embryo origin vaccine by pre-activating natural killer cells and cytotoxic T lymphocytes.

IMPORTANCE: Chickens immunized with the infectious laryngotracheitis chicken embryo origin (CEO) vaccine (Medivac, PT Medion Farma Jaya) experience adverse reactions, hindering its safety and effective use in poultry flocks. To improve the effect of the vaccine, we sought to find a strategy to alleviate the respiratory reactions associated with the vaccine. Here, we confirmed that co-administering the CEO vaccine with chIL-2 by oral delivery led to significant alleviation of the vaccine reactions in chickens after immunization. Furthermore, we found that the co-administration of chIL-2 with the CEO vaccine reduced the clinical signs of the CEO vaccine while enhancing natural killer cells and cytotoxic T lymphocyte response to decrease viral loads in their tissues, particularly in the trachea and conjunctiva. Importantly, we demonstrated that the chIL-2 treatment can ameliorate the replication of the CEO vaccine without compromising its effectiveness. This study provides new insights into further applications of chIL-2 and a promising strategy for alleviating the adverse reaction of vaccines.

Characterization of Ebola Virus Mucosal Challenge Routes in Cynomolgus Macaques.

Zaïre ebolavirus (EBOV) causes Ebola virus disease (EVD), a devastating viral hemorrhagic fever in humans. Nonhuman primate (NHP) models of EVD traditionally use intramuscular infection with higher case fatality rates and reduced mean time-to-death compared to contact transmission typical of human cases of EVD. A cynomolgus macaque model of oral and conjunctival EBOV was used to further characterize the more clinically relevant contact transmission of EVD. NHPs challenged via the oral route had an overall 50% survival rate. NHPs challenged with a target dose of 1 × 102 PFU or 1 × 104 PFU of EBOV via the conjunctival route had 40% and 100% mortality, respectively. Classic signs of lethal EVD-like disease were observed in all NHPs that succumbed to EBOV infection including viremia, hematological abnormalities, clinical chemistries indicative of hepatic and renal disease, and histopathological findings. Evidence of EBOV viral persistence in the eye was observed in NHPs challenged via the conjunctival route. IMPORTANCE This study is the first to examine the Kikwit strain of EBOV, the most commonly used strain, in the gold-standard macaque model of infection. Additionally, this is the first description of the detection of virus in the vitreous fluid, an immune privileged site that has been proposed as a viral reservoir, following conjunctival challenge. The oral and conjunctival macaque challenge model of EVD described here more faithfully recapitulates the prodrome that has been reported for human EVD. This work paves the way for more advanced studies to model contact transmission of EVD, including early events in mucosal infection and immunity, as well as the establishment of persistent viral infection and the emergence from these reservoirs.

Vascular Endothelial Growth Factor Expression Patterns in non- Human Papillomavirus - Related Pterygia: An Experimental Study on Cell Spot Arrays Digital Analysis.

PURPOSE: The role of angiogenic factors -such as vascular endothelial growth factor (VEGF) - in the development and progression of pterygia lesions remains under investigation. In the current study, we analyzed VEGF protein expression in a series of pterygia and normal conjunctiva epithelia. METHODS: Using a liquid-based cytology assay, thirty (n = 30) cell specimens were obtained by applying a smooth scraping on conjunctiva epithelia and fixed accordingly. None of them had a history of Human Papillomavirus (HPV) infection. Similarly, the same process was applied also in normal conjunctiva epithelia (n = 10; control group). We constructed five (n = 5) slides each containing eight (n = 8) cell spots. An immunocytochemistry (ICC) assay was implemented. Digital image analysis was also performed for evaluating objectively the corresponding immunostaining intensity levels. RESULTS: All the examined pterygia cell samples over-expressed the marker. High staining intensity levels were detected in 15/30 (50%), whereas the rest 15/30 (50%) demonstrated moderate expression. Overall VEGF expression was statistically significantly higher in pterygia compared to normal conjunctiva epithelia (p=.0001). Concerning the other parameters, VEGF protein expression did not associate with the gender of the patients (p = 0.518), the presence of a recurrent lesion (p = 0.311), the anatomical location (p = 0.191) or with their morphology (p = 0.316). Interestingly, the recurrent lesions demonstrated the highest levels of VEGF expression. CONCLUSIONS: VEGF overexpression is a frequent event in pterygia playing a potentially central molecular role in the progression of the lesion. Cell spot array analysis -based on liquid cytology- seems to be an innovative, easy-to-use technique for analyzing a broad variety of molecules in multiple specimens on the same slide by applying different ICC assays.

Clinical Utility of COVID-19 Real Time-Polymerase Chain Reaction Testing of Ocular Tissues of Non-COVID-19 Cornea Donors Deemed Suitable for Corneal Retrieval and Transplantation.

PURPOSE: The purpose of this study was to evaluate the prevalence of SARS-CoV-2 in human postmortem ocular tissues of asymptomatic donors and its implications on our eye banking protocols. METHODS: The expression of SARS-CoV-2 RNA was assessed by reverse transcription-polymerase chain reaction in corneal rims and conjunctival tissues from 100 donors who were found suitable for transplantation as per the donor screening guidelines of the Global Alliance of Eye Bank Associations. The donor's clinical history and cause of death were assessed for secondary analysis. RESULTS: Of 200 ocular tissues (100 corneal and 100 conjunctival) from the same 1 eye of 100 surgical-intended donors, between September 2020 and April 2021, the overall positivity rate for SARS-CoV-2 was ∼1% (2/200). Both the ocular samples that tested positive were conjunctival biopsies (2/100, 2%), whereas corneal samples were negative (0/100, 0%) in both donors. The causes of donor death were trauma in 51 donors, suicide in 33, cardiac arrest in 7, electric shock in 5, metabolic cause in 2, malignancy in 1, and snake bite in 1. None of the donors had a medical history suggestive of COVID infection or possible contact. None of the recipients from the donors were reported to have any systemic adverse event after keratoplasty until the follow-up of 6 weeks. CONCLUSIONS: The overall prevalence of SARS-CoV-2 was 1% (2% for conjunctival and 0% for corneal samples, P value = 0.5) in the donors who were found suitable for cornea recovery and transplantation. The findings of exceptionally low positive rates in our samples validate the criticality of history-based donor screening and do not support the necessity of postmortem PCR testing as a criterion for procurement and subsequent use for corneal transplantation.

Characterization of SARS-CoV-2 Entry Factors' Expression in Corneal and Limbal Tissues of Adult Human Donors Aged from 58 to 85.

Purpose: Recent studies have shown the presence of SARS-CoV-2 entry factors on the ocular surface, identifying the eye as an additional entry route for the virus. Moreover, the coexpression of angiotensin-converting enzyme 2 (ACE2) with other SARS-CoV-2 entry factors [transmembrane protease serine 2 (TMPRSS2), transmembrane protease serine 4 (TMPRSS4), and dipeptidyl peptidase-4 (DPP4)] facilitates the virus infection. Methods: Here, we performed a study over 10 adult corneal and limbal tissues from human donors, both male and female between 58 and 85 years of age. Some of the main virus entry factors were analyzed and their expression was quantified and correlated with the age and sex of the donors through western blot. The receptors' localization was investigated through immunofluorescence. Results: Immunofluorescence confirmed the localization of ACE2 and TMPRSS2 on the ocular surface and showed, for the first time, the localization of TMPRSS4 and DPP4 in limbal and corneal epithelial superficial cells. The quantitative analysis showed that the expression of SARS-CoV-2 entry factors on corneal and limbal cells is likely to be modulated in an age-dependent manner, in agreement with the increased susceptibility to COVID-19 in the elderly. Moreover, we found a relationship between the expression of TMPRSS proteases with the activation state of limbal cells in 80-year-old donors. Conclusion: This study provides information on the expression of SARS-CoV-2 entry factors on the ocular surface of 10 adult human donors and is a first observation of a possible age-dependent modulation on corneal and limbal tissues. Our data pave the way to further investigate the susceptibility to the infection through the ocular surface in the elderly.

Viral S protein histochemistry reveals few potential SARS-CoV-2 entry sites in human ocular tissues.

Despite the reported low expression of the primary SARS-CoV-2 receptor ACE2 in distinct ocular tissues, some clinical evidence suggests that SARS-CoV-2 can infect the eye. In this study, we explored potential entry sites for SARS-CoV-2 by viral S protein histochemistry on various ocular tissues and compared the staining patterns with RNA and protein expression of TMPRSS2 and ACE2. Potential viral entry sites were investigated by histochemistry using tagged recombinant viral S protein on 52 ocular tissue samples including specimens of the cornea, conjunctiva, lid margin, lacrimal gland tissue, retina, choroid, and RPE. In addition, ACE2 and TMPRSS2 immunohistochemistry were performed on the same ocular tissue, each with distinct antibodies binding to different epitopes. Lung tissue samples were used as positive controls. Finally, bulk RNA sequencing (RNA-Seq) was used to determine the expression of ACE2 and its auxiliary factors in the tissues mentioned above. S protein histochemistry revealed a positive staining in lung tissue but absent staining in the cornea, the conjunctiva, eye lid samples, the lacrimal glands, the retina and the optic nerve which was supported by hardly any immunoreactivity for ACE2 and TMPRSS2 and scarce ACE2 and TMPRSS2 RNA expression. Negligible staining with antibodies targeting ACE2 or TMPRSS2 was seen in the main and accessory lacrimal glands. In contrast, ocular staining (S protein, ACE2, TMPRSS2) was distinctly present in pigmented cells of the RPE and choroid, as well as in the ciliary body and the iris stroma. S protein histochemistry revealed hardly any SARS-CoV-2 entry sites in all ocular tissues examined. Similarly, no significant ACE2 or TMPRSS2 expression was found in extra- and intraocular tissue. While this study suggest a rather low risk of ocular infection with SARS-CoV-2, it should be noted, that potential viral entry sites may increase in response to inflammation or in certain disease states.

Human Papillomavirus Related Neoplasia of the Ocular Adnexa.

Human papillomaviruses (HPV) are a large group of DNA viruses that infect the basal cells of the stratified epithelium at different anatomic locations. In the ocular adnexal region, the mucosa of the conjunctiva and the lacrimal drainage system, as well as the eyelid skin, are potential locations for HPV-related neoplasia. The role of HPV in squamous cell neoplasia of the ocular adnexa has been debated for several decades. Due to the rarity of all these tumors, large studies are not available in the scientific literature, thereby hampering the precision of the HPV prevalence estimates and the ability to conclude. Nevertheless, increasing evidence supports that defined subsets of conjunctival papillomas, intraepithelial neoplasia, and carcinomas develop in an HPV-dependent pathway. The role of HPV in squamous cell tumors arising in the lacrimal drainage system and the eyelid is still uncertain. Further, the potential of HPV status as a diagnostic, prognostic, or predictive biomarker in these diseases is a topic for future research.

COVID-19 Ocular Manifestations in the Early Phase of Disease.

Purpose: To evaluate and establishe the number of patients with ocular manifestations in the early phase of systemic COVID-19 infection.Methods: A cross-sectional study was performed in a COVID-19 referral center regarding ocular findings in patients with COVID-19 in the first few days of the disease. The patients were submitted to a clinical examination, an ophthalmic exam and a RT-PCR for SARS-COV-2.Results: Out of 1740 patients, we reported 108 patients with ocular manifestations. Forty-nine with markedly conjunctivitis had conjunctival swab positive for SARS-COV-2, four of them developed keratitis. There were mostly no evidence of retinopathy nor decrease in visual acuity. They had no marked clinical symptoms, which can contribute and demonstrate that the virus may cause ocular disease as an only finding or in the very early stage of the infection.Conclusion: Patients were in the first days of COVID-19 infection, presented ocular manifestations suggested to be related to the virus and need to be aware of the pathways of transmissions.

Increased Retinal Vessel Tortuosity Associated With Crimean-Congo Hemorrhagic Fever in Children.

BACKGROUND: To evaluate the ocular symptoms and findings of children diagnosed with Crimean-Congo hemorrhagic fever (CCHF). METHODS: In this prospective study, children diagnosed with CCHF who underwent a complete ophthalmologic examination during the hospitalization period were included. RESULTS: Twenty-four children with a mean age of 12.4 ± 3.6 years were included study. The most common ocular finding was conjunctival hyperemia and was observed in 50% of patients. Nine (37.4%) children had abnormalities in fundus examination. Two (8.3%) of them had dilated retinal veins, and 7 (29.1%) had tortuous retinal vessels. No significant difference was found between mild to moderate and severe disease groups in terms of ocular symptoms and ophthalmologic examination findings (P > 0.05, for all). CONCLUSIONS: The increased retinal vessel tortuosity was detected as a fundus examination finding in children with CCHF. Both ophthalmologists and pediatricians should be aware of the various ocular manifestations of CCHF for rapid diagnosis and management.

Elevated levels of Merkel cell polyoma virus in the anophthalmic conjunctiva.

The human ocular surface hosts a paucibacterial resident microbiome and virome. The factors contributing to homeostasis of this mucosal community are presently unknown. To determine the impact of ocular enucleation and prosthesis placement on the ocular surface microbiome, we sampled conjunctival swabs from 20 anophthalmic and 20 fellow-eye intact conjunctiva. DNA was extracted and subjected to quantitative 16S rDNA PCR, biome representational karyotyping (BRiSK), and quantitative PCR (qPCR) confirmation of specific organisms. 16S ribosomal qPCR revealed equivalent bacterial loads between conditions. Biome representational in silico karyotyping (BRiSK) demonstrated comparable bacterial fauna between anophthalmic and intact conjunctiva. Both torque teno virus and Merkel cell polyoma virus (MCPyV) were detected frequently in healthy and anophthalmic conjunctiva. By qPCR, MCPyV was detected in 19/20 anophthalmic samples compared with 5/20 fellow eyes. MCPyV copy number averaged 891 copies/ng in anophthalmic conjunctiva compared with 193 copies/ng in fellow eyes (p < 0.001). These results suggest that enucleation and prosthesis placement affect the ocular surface flora, particularly for the resident virome. As MCPyV has been shown to be the etiologic cause of Merkel cell carcinoma, understanding the mechanisms by which the ocular surface regulates this virus may have clinical importance.

An Eye on COVID-19: A Meta-analysis of Positive Conjunctival Reverse Transcriptase-Polymerase Chain Reaction and SARS-CoV-2 Conjunctivitis Prevalence.

SIGNIFICANCE: This analysis and review demonstrate that, although emerging data indicate that the prevalence of severe acute respiratory coronavirus 2 (SARS-CoV-2) on the ocular surface and coronavirus disease 2019 (COVID-19) conjunctivitis is rare, the ocular surface remains of interest as a potential inoculation and transmission site for SARS-CoV-2. Continued safety precautions should be taken as more data become available.COVID-19, caused by SARS-CoV-2, is a novel, global pandemic that has infected millions and, up to this point, caused more than two million fatalities worldwide. The ocular surface has become of interest as a possible vector for transmission by acting as a direct inoculation site, being a conduit for the virus into the respiratory system or as a method of transmission from potentially infected conjunctiva or tears. The components necessary for SARS-CoV-2 to theoretically infect ocular tissues are present: binding receptors (angiotensin-converting enzyme 2 and cluster of differentiation 147) and mechanisms for cell entry (transmembrane protease serine 2 and cathepsin L). This meta-analysis of COVID-19 prevalence data indicates that SARS-CoV-2 RNA has been infrequently found in conjunctival samples when tested with reverse transcriptase-polymerase chain reaction. This review estimates the prevalence of SARS-CoV-2 on the ocular surface and prevalence of conjunctivitis in patients with laboratory-confirmed COVID-19. There is much to be learned regarding ocular tropism of SARS-CoV-2.

The Presence of SARS-CoV-2 in Conjunctival Secretions of COVID-19 Patients.

Purpose: To evaluate the presence of SARS-CoV-2 in conjunctival secretions of COVID-19 patients.Material and Methods: In this retrospective study, the records were examined of patients who were treated in the hospital with the diagnosis of COVID-19 between March-May 2020 and were referred to the eye clinic due to ocular symptoms. Conjunctival swabs from both confirmed and suspected COVID-19 cases during hospitalization were analyzed.Results: A total of 35 patients (22 suspected, 13 laboratory-confirmed COVID-19) were referred to the eye clinic. Conjunctival swab samples from 3 patients yielded positive PCR results. These three patients were being treated in the intensive care unit, and all were suspected COVID-19 patients.Conclusion: SARS-CoV-2 may be detected in patients with suspected COVID-19. Even with conjunctivitis findings, SARS-CoV-2 may not be detected in most conjunctiva swab samples of COVID-19 patients.

An Update on COVID-19 Related Ophthalmic Manifestations.

Purpose: To summarize ophthalmic manifestations of coronavirus disease (COVID-19) reported in the literature thus far.Methods: The PubMed database was systematically searched through October 24, 2020, to identify relevant articles using the following search terms: ("COVID-19" OR "SARS-CoV-2") AND ("eye" OR "ophthalmology" OR "retina" OR "retinal findings" OR "cornea" OR "conjunctiva"). Only articles published in English were included in this review.Results: The reported prevalence of ophthalmic manifestations is generally low, but correlates positively with the severity of the disease. Most commonly reported ocular manifestations are conjunctivitis, conjunctival hyperemia and chemosis. Retinal findings include microhemorrhages and flame-shaped hemorrhages, cotton wool spots, dilated veins, and tortuous vessels.Conclusion: Considering the COVID-19 cases have reached pandemic dimensions and are surging, yet again, it is of utmost importance to determine its ophthalmic manifestations and prevent their vision threatening complications. Further studies are warranted to establish whether the retinal findings appear due to the COVID-19 or are an incidental finding in patients with a preexisting diabetic or hypertensive retinopathy.

The eye: “An organ that must not be forgotten in coronavirus disease 2019 (COVID-2019) pandemic”

The coronavirus family is a group of zoonotic viruses with some recognized reservoirs particularly some bats. A novel coronavirus emerged in the province of Wuhan (China) in December of 2019.The number of infected patient with serious respiratory infection quickly spread around the world to become a global pandemic. The clinical presentation and viral pathogenesis of the coronavirus disease named COVID-19 indicated that the virus is transmitted from person to person through infected droplets entering the respiratory mucosa. Close contact with infected individuals particularly in crowded environments has characterized the rapid spread of the infection.Clinical manifestations of the viral infection have mentioned the presence of some ocular findings such as conjunctival congestion, conjunctivitis and even corneal injury associated with the classical COVID-19 infection. Some animal models of different coronaviruses eye infections have described the viral pathogenesis through tear and conjunctival sampling. On the other hand, we are recommended protective measure to prevent contagion and limit the spread of the virus in health care professionals and contact lenses wearers. (AU)

Comparison of the simultaneous conjunctiva and oropharynx-nasopharynx swab results in patients applying to the SARS-CoV-2 outpatient clinic for the first time.

The aim is to comparatively evaluate the results of simultaneous conjunctiva and oropharynx-nasopharynx (ONP) swabs in patients who had presented to the outpatient department with a suspicion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). An ONP sample was obtained following bilateral conjunctiva swabs in 85 subjects with a contact history or symptoms but unknown SARS-CoV-2 status and with no ocular symptoms or findings. The results were evaluated according to the patient's symptoms and how the swab was taken. The conjunctiva swab was positive in 29 (34.1%) cases and the ONP swab in 20 (23.5%) cases. Both methods produced positive results in 11 (14.1%) cases. The mean cycle threshold (Ct ) value was 30.15 ± 3.41 in symptomatic cases and 33.62 ± 1.76 in asymptomatic cases (p = .008). The mean Ct value was 24.37 ± 3.48 when only the ONP swab was positive and 31.22 ± 1.99 when only the conjunctiva swab was positive. In cases that were positive by both methods, the mean Ct value was 25.21 ± 4.94 for the ONP swab and 30.29 ± 5.05 for the conjunctiva swab. We found higher SARS-CoV-2 detection rates with the conjunctiva swab than the ONP swab in cases with unknown SARS-CoV-2 status in the early period. In addition, the conjunctival viral load seemed to be higher in symptomatic cases than in asymptomatic cases. We, therefore, believe a conjunctiva swab could be an alternative method to detect SARS-CoV-2 at the time of the first presentation to the outpatient department.

In the eye of the storm: SARS-CoV-2 infection and replication at the ocular surface?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) first emerged in December 2019 and spread quickly causing the coronavirus disease 2019 (COVID-19) pandemic. Recent single cell RNA-Seq analyses have shown the presence of SARS-CoV-2 entry factors in the human corneal, limbal, and conjunctival superficial epithelium, leading to suggestions that the human ocular surface may serve as an additional entry gateway and infection hub for SARS-CoV-2. In this article, we review the ocular clinical presentations of COVID-19 and the features of the ocular surface that may underline the overall low ocular SARS-CoV-2 infection. We critically evaluate the studies performed in nonhuman primates, ex vivo organ culture ocular models, stem cell derived eye organoids and the differences in infection efficiency observed in different parts of human ocular surface epithelium. Finally, we highlight the additional work that needs to be carried out to understand the immune response of the ocular surface to SARS-CoV-2 infection, which can be translated into prophylactic treatments that may be applied to other organ systems.

Evaluation of Nasopharyngeal and Conjunctival Swab Samples of Hospitalised Patients with Confirmed COVID-19.

PURPOSE: To evaluate the results of conjunctival and nasopharyngeal swab tests in patients with confirmed COVID-19. METHODS: This prospective study included 45 patients who were hospitalized for confirmed COVID-19. Nasopharyngeal swab samples were obtained from the patients before hospitalization. Only one eye of each patient was randomly selected for-conjunctival sampling. All participants underwent a complete slit-lamp examination. Conjunctival and nasopharyngeal swab samples were analyzed by reversetranscriptase-polymerase-chain reaction (RT-PCR). RESULTS: Twenty seven (60%) of the patients were male and 18 (40%) were female. Conjunctival swab was positive in only one (2.22%) patient. None of the COVID-19 patients showed ocular changes and symptoms. There were no abnormalities of the ocular surface, anterior chamber or posterior segment at slit-lamp examination. CONCLUSIONS: The RT-PCR was not high positive in the conjunctiva as in nasopharyngeal swabs. Ocular changes were not common in COVID-19 patients.