Jolanda D.F. de Groot-Mijnes

Natural History of a Recurrent Feline Coronavirus Infection and the Role of Cellular Immunity in Survival and Disease

The genus Coronavirus (family Coronaviridae, order Nidovirales) comprises a group of enveloped positive-strand RNA viruses of mammals and birds. With a genome of 27 to 31 kb, encoding an ∼750-kDa pp1ab replicase polyprotein, four structural proteins (S, M, N, and E) and up to five accessory nonstructural proteins, coronaviruses (CoVs) are the largest RNA viruses known to date (5, 11). In humans, they are mostly associated with mild enteric or respiratory infections, such as the common cold, and hence were long considered of modest clinical importance. However, the sudden emergence of severe acute respiratory syndrome (SARS) has sparked wide interest in CoV biology and pathogenesis (12, 22, 23, 34, 36). The more recent discovery of yet another human CoV, HCoV-NL63 (14, 45), also implicated in severe respiratory disease, further emphasizes the pathogenic potential of CoVs and stresses the need for the development of new prophylactic and therapeutic strategies. Among the most conspicuous clinicopathological findings reported for SARS are the protracted multiphasic course of the infection with recurrence of fever and disease after initial apparent improvement and a consistent CD4+ and CD8+ T-cell lymphopenia (4, 8, 23, 36, 44, 56). In this respect, there are striking similarities with a lethal CoV infection occurring in cats. Feline infectious peritonitis (FIP) is a progressive debilitating condition caused by FIP viruses (FIPVs) (for a review, see reference 9), pathogenic virulence mutants spontaneously arising from apathogenic feline enteric CoV field strains (18, 35, 49). Typical for the disease are the widespread pyogranulomatous lesions, which occur in various tissues and organs, including lung, liver, spleen, omentum, and brain (32, 50, 54). The infection of macrophages and monocytes is thought to be key to the pathogenic mechanism (40, 52). There is ample evidence for a crucial involvement of the immune system. A profound T-cell depletion from the periphery and the lymphatic tissues, observed in cats with end-stage FIP (16, 21), and the common occurrence of hypergammaglobulinemia (29, 50) are indicative of a severe virus-induced immune dysregulation (20). The humoral response against FIPV does not seem to be protective and can in fact lead to “early death syndrome,” a more fulminating and drastically shortened course of the disease (31, 52). Antibodies directed against the spike protein S, when present at subneutralizing titers, apparently opsonize the virus and enhance the infection of target cells via Fc receptor-mediated attachment (7, 19, 47). It is commonly believed that the control of infection and FIPV clearance are primarily achieved through cell-mediated immunity (CMI) (17, 35, 51). Here, we present a comprehensive study of the natural history and immunobiology of FIP, based upon longitudinal infection experiments performed with the highly virulent FIPV strain 79-1146. We show that FIPV causes a multiphasic recurrent infection with waves of enhanced FIPV replication coinciding with fever, weight loss, and a dramatic decline in peripheral CD4+ and CD8+ T-cell counts. Consistent with the notion that CMI is protective, we detected FIPV-specific Th1 T-cell responses in surviving animals with the spike protein S as the main CD8+ T-cell antigen. A model is discussed in which cellular immunity is counteracted by virus-induced T-cell depletion and in which the efficacy of the initial T-cell responses critically determines disease progression and the ultimate outcome of the infection.

Comparison of Rubella Virus- and Herpes Virus-Associated Anterior Uveitis Clinical Manifestations and Visual Prognosis

PURPOSE To compare the clinical characteristics and visual prognosis of patients with anterior uveitis (AU) and intraocular fluid analysis positive for rubella virus (RV), herpes simplex virus (HSV), or varicella zoster virus (VZV). DESIGN Retrospective, observational study. PARTICIPANTS The study included 106 patients with AU and positive polymerase chain reaction (PCR) results, Goldmann-Witmer coefficients (GWCs), or both, for RV (n = 57), HSV (n = 39), or VZV (n = 10). METHODS Clinical records of the included patients were analyzed retrospectively; demographic constitution, ophthalmologic characteristics, and visual prognosis were compared. MAIN OUTCOME MEASURES Age, gender, and diverse clinical and laboratory characteristics, including course and laterality of AU; prevalence of positive results for PCR, GWC, or both; conjunctival redness; corneal edema; history of keratitis; presence of keratic precipitates; synechiae; heterochromia; and grade of inflammation. In addition, complications and visual acuity at 1 and 3 years of follow-up were recorded. RESULTS All 3 types of viral AU were characterized by unilateral involvement (80%-97%). Rubella virus AU was characterized by younger age at onset and chronic course and typically was associated with cataract at presentation. Heterochromia was present in 23% of RV AU patients. Anterior uveitis associated with HSV or VZV occurred characteristically in older patients and frequently followed an acute course. Clinical features associated with herpetic AU included conjunctival redness, corneal edema, history of keratitis, and development of posterior synechiae. Herpes simplex virus AU often had severe anterior chamber inflammation, whereas the presence of vitritis was more common in RV AU and VZV AU. The prevalence of documented intraocular pressure (IOP) of more than 30 mmHg (25%-50%; P = 0.06) and development of glaucoma (18%-30%; P = 0.686) were similar in all 3 groups. Focal chorioretinal scars were seen in 22% of RV AU eyes, in 0% of HSV AU eyes, and in 11% of VZV AU eyes (P = 0.003). Visual prognosis was favorable for all 3 groups. CONCLUSIONS These observations identify clinical differences between RV AU, HSV AU, and VZV AU and may be of particular value to ophthalmologists who are unable to carry out intraocular fluid analysis to discriminate between these types of viral AU. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Intraocular Interleukin-17 and Proinflammatory Cytokines in HLA-A29–Associated Birdshot Chorioretinopathy

PURPOSE To determine the levels of 23 immune mediators in paired aqueous humor (AqH) and serum samples from patients with birdshot chorioretinopathy (BSCR). DESIGN Single-centre case-control study. METHODS A multiplex immunoassay was used to determine the levels of 23 immune mediators (T-cell, proinflammatory, and vascular-active mediators) in paired AqH and serum of 16 BSCR patients. The AqH of 11 age-related cataract controls served as controls. RESULTS AqH levels of the T-cell mediators interleukin (IL)-2 (P=.044) and IL-17 (P=.039) and proinflammatory mediators IL-1β (P=.032), IL-6 (P=.034), and tumor necrosis factor α (P=.041) were elevated compared with that of age-related cataract controls. The elevated intraocular levels of IL-1β, IL-17, and tumor necrosis factor α in BSCR samples were higher than their concurrent serum levels. A significant positive correlation of intraocular mediators was noted between IL-17 and both IL-2 (r=0.744; P<.0001) and IL-23 (r=0.921; P<.0001) and between IL-2 and IL-23 (r=0.776; P<.0001). AqH levels of vascular-active mediators were not distinct between the groups. CONCLUSIONS BSCR patients have elevated intraocular levels of proinflammatory and T cell-associated cytokines. Our results suggest the novel pathogenic concept that BSCR is an autoimmune inflammatory disease restricted to the eye and associated with elevated IL-17.

Identification of New Pathogens in the Intraocular Fluid of Patients With Uveitis

Purpose To determine infectious causes in patients with uveitis of unknown origin by intraocular fluids analysis. Design Case-control study. Methods Ocular fluids from 139 patients suspected of infectious uveitis, but negative for herpes simplex virus, varicella-zoster virus, cytomegalovirus, and Toxoplasma gondii by polymerase chain reaction and/or antibody analysis in intraocular fluids, were assessed for the presence of 18 viruses and 3 bacteria by real-time polymerase chain reaction (PCR). The ocular fluids from 48 patients with uveitis of known etiology or with cataract were included as controls. Results Positive PCR results were found for Epstein-Barr virus, for rubella virus, and for human herpesvirus 6 each in 1 patient and for human parechovirus in 4 patients. Of the human parechovirus–positive patients, 1 was immunocompromised and had panuveitis. The other 3 patients were immunocompetent and had anterior uveitis, all with corneal involvement. Conclusions Human parechovirus might be associated with infectious (kerato)uveitis.

Diagnostic Approach to Ocular Toxoplasmosis

Toxoplasmic retinochoroiditis is deemed a local event, which may fail to evoke a detectable systemic immune response. A correct diagnosis of the disease is a necessary basis for estimating its clinical burden. This is not so difficult in a typical clinical picture. In atypical cases, further diagnostic efforts are to be installed. Although the aqueous humor may be analyzed for specific antibodies or the presence of parasitic DNA, the DNA burden therein is low, and in rare instances a confirmation would necessitate vitreous sampling. A laboratory confirmation of the diagnosis is frustrated by individual differences in the time elapsing between clinical symptoms and activation of specific antibody production, which may result in false negatives. In congenital ocular toxoplasmosis, a delay in the onset of specific local antibody production could reflect immune tolerance. Herein, the authors attempt to provide a simple and practicable algorithm for a clinically tailored diagnostic approach in atypical instances.

Intraocular biomarker identification in uveitis associated with juvenile idiopathic arthritis.

PURPOSE To investigate the presence of biomarkers in aqueous humor (AH) from patients with uveitis associated with juvenile idiopathic arthritis (JIA). METHODS AH (N = 73) AND SERUM (N = 105) SAMPLES FROM 116 CHILDREN WERE ANALYZED USING SURFACE ENHANCED LASER DESORPTION/IONIZATION TIME OF FLIGHT MASS SPECTROMETRY (SELDI-TOF MS). THE SAMPLES WERE DIVIDED INTO THE FOLLOWING GROUPS JIA, silent chronic anterior uveitis (AU), other uveitis entities, and noninflammatory controls. Statistical biomarker identification was performed using the SELDI-ToF Biomarker Analysis Cluster Wizard followed by multivariate statistical analysis. Biochemical identification of biomarkers was performed by polyacrylamide gel protein separation, followed by liquid chromatography tandem mass spectrometry. ELISA was performed in a number of AH samples representing all four study groups. RESULTS In the JIA group, one AH protein peak at mass/charge (m/z) 13,762 had qualitative and quantitative differences in expression compared with the other uveitis entities and the controls, but not to the group of silent chronic AU. Its quantitative expression in AH of patients with JIA and other silent chronic AU was positively associated with uveitis activity. The protein at m/z 13,762 in AH was identified as transthyretin (TTR). The TTR concentration in AH differed significantly between the study groups (P = 0.006) with considerably higher TTR concentrations in JIA and silent chronic AU samples positive for m/z 13,762 than those of the other uveitis and control groups. CONCLUSIONS TTR is a potential intraocular biomarker of JIA- associated uveitis. Its role in the pathogenesis of silent chronic AU with and without arthritis needs further investigation.

Diagnostic pars plana vitrectomy and aqueous analyses in patients with uveitis of unknown cause

Purpose: To compare the yield of diagnostic pars plana vitrectomy (PPV) with the yield of aqueous analyses in patients with uveitis of unknown cause. Methods: Seventy-five consecutive patients (84 eyes) with uveitis involving posterior eye segment who undergo a diagnostic PPV from 2005 through 2009 were retrospectively reviewed. Vitreous specimens were simultaneously analyzed by microbiological culture, flow cytometry, and cytology as well as by polymerase chain reaction and for intraocular antibody production by Goldmann–Witmer coefficient. In 53 eyes, both aqueous and vitreous samples were assessed. The primary outcome measure was the comparison between vitreous and aqueous analyses. Results: Vitreous analysis was positive in 18 of 84 eyes (21%). Positive results indicated infectious uveitis in 12 of 18 cases (67%) and lymphoma in 6 of 18 (33%) cases. Of the 53 eyes with both aqueous and vitreous samples available, aqueous analysis revealed the diagnosis in 6 of 53 eyes and vitreous in 9 of 53 eyes. Unilateral uveitis (P = 0.022), panuveitis and uveitis posterior (P ⩽ 0.001), preoperative immunosuppressive therapy (P = 0.004), and increasing age (P = 0.018) were associated with an increased diagnostic yield of PPV. Overall, 1 year after PPV, median visual acuity improved from 20/200 to 20/80 (Snellen, P ⩽ 0.001). Of 18 patients who were on immunosuppressive treatment before PPV, 8 (44%) were able to stop immunosuppressive therapy during 1-year follow-up. The complications of PPV consisted predominantly of cataract development (33/65, 51%). Conclusion: Diagnostic PPV with the analysis of vitreous fluid by multiple laboratories for infectious and malignant disorders was useful in diagnosing uveitis of unknown cause. Previous aqueous analysis was especially valuable for the diagnosis of intraocular infections and may therefore decrease the number of patients who would otherwise undergo an invasive diagnostic PPV. Furthermore, PPV was associated with improved visual acuity and decreased use of immunosuppressive therapy.

Intraocular and plasma HIV-1 RNA loads and HIV uveitis.

Objective:The objective of this study was to analyze human immunodeficiency virus (HIV) dynamics across the blood–retinal barrier and to determine whether the high levels of HIV in the eye are associated with any ocular disorders in HIV-infected patients. Design:This study included a prospective case series of 40 HIV-positive patients with uveitis. Intervention:Clinical and laboratory examinations included plasma and intraocular HIV-1 RNA loads as well as the clinical manifestations of uveitis. Results:Intraocular HIV-1 RNA was detected in 32% (13/40) of HIV-positive patients with uveitis. Intraocular HIV-1 RNA loads were associated with high HIV-1 RNA plasma loads (P < 0.001) and not being on HAART therapy (P = 0.005). In addition, detectable intraocular HIV-1 RNA levels were higher in patients with the absence of retinal lesions (P = 0.008). In three patients, the HIV load in the eye largely exceeded that of plasma. These three patients had all bilateral anterior uveitis and/or vitritis without retinal lesions and exhibited no evidence of other intraocular infectious agents causing uveitis than HIV itself. Conclusion:The eye can form a sanctuary where HIV might replicate and cause an inflammatory reaction.

The diagnostic value of intraocular fluid analysis by polymerase chain reaction in Thai patients with uveitis

Uveitis is a major cause of severe visual impairment throughout the world and can be initiated by various infectious and non-infectious causes. Early recognition of specific infections is important as the treatment with antimicrobial agents might stop the progression or even cure the eye disease. To determine the infectious causes of uveitis in Thailand, intraocular fluid samples of 100 HIV-negative patients and 47 HIV-positive patients with uveitis were examined using real-time PCR analysis for herpes simplex virus, varicella zoster virus, cytomegalovirus and Toxoplasma gondii. Positive PCR results were found in 33/100 (33%) HIV-negative patients and in 33/47 (70%) HIV-positive patients with uveitis. In Thailand, cytomegalovirus was identified as the most frequent cause of infectious uveitis in both HIV-negative and HIV-positive patients (49 and 91%, respectively). PCR analysis of intraocular samples in uveitis was a valuable diagnostic assay. The pattern of uveitis observed in the Far East differs from that found in the West.

Human Immunodeficiency Virus-induced Uveitis : Intraocular and Plasma Human Immunodeficiency Virus-1 RNA Loads

OBJECTIVE To report on a human immunodeficiency virus (HIV)-infected patient with uveitis and an intraocular HIV1 RNA load largely exceeding that of plasma and no evidence of other intraocular infectious agents causing uveitis than HIV itself. DESIGN Interventional case report. PARTICIPANT A 37-year-old male HIV-infected patient with uveitis and no retinal manifestations. METHODS Clinical and laboratory examinations including extensive intraocular fluid analyses for various pathogens and HIV-1 RNA loads in the aqueous and plasma. MAIN OUTCOME MEASURES Results of aqueous analysis and ophthalmologic features. Correlations between the results of aqueous testing and clinical characteristics. RESULTS A 37-year-old patient presented with progressive uveitis. He was positive for HIV-1, and his HIV-1 RNA plasma load was 44,600 copies/mL. His intraocular HIV-1 RNA load was >1,900,000 copies/mL, which largely exceeded his concurrent plasma load. No evidence of infectious agents other than HIV itself was found, and the uveitis reacted promptly to solely the antiretroviral treatment. CONCLUSIONS Our findings suggest that HIV can locally replicate within the eye and cause an intraocular inflammatory reaction.