key: cord-0690023-hlxx780q
authors: Komurasaki, Yusuke; Nagineni, Chandrasekharam N.; Wang, Yun; Hooks, John J.
title: Virus RNA Persists within the Retina in Coronavirus-Induced Retinopathy
date: 1996-08-15
journal: Virology
DOI: 10.1006/viro.1996.0442
sha: 08d735bb53c3af46320aad799286c81a44606d86
doc_id: 690023
cord_uid: hlxx780q

Abstract The murine coronavirus, mouse hepatitis virus (MHV), JHM strain, induces a biphasic retinal disease in adult BALB/c mice. In the early phase, Day 1 to Day 7, a retinal vasculitis is noted which is associated with the presence of viral proteins and infectious virus. In the late phase, Day 10 to Day 140, a retinal degeneration is associated with the absence of viral proteins, infectious virus, and inflammatory cells. The purpose of this study was to determine if viral RNA persists within the retina during the retinal degenerative phase of the disease. BALB/c mice were inoculated by the intravitreal route with 104.0TCID50/5 μl of virus. The presence of viral RNA was detected byin situhybridization with a viral cDNA probe and viral proteins were identified by immunocytochemical staining. During the acute phase of the infection, viral RNA was found in the retina, RPE, ciliary body epithelium, and the iris epithelium. During the late phase of the infection, viral RNA was almost exclusively found within the retina and RPE and not in the anterior segment of the eye. Within the retina, viral RNA was detected in the ganglion cell layer, the inner retina, the outer retina, and the RPE cell. Immunocytochemical staining identified viral protein within the retina only from Day 1 to Day 8. This ocular disease was also associated with a persistent systemic infection. Both viral RNA and viral proteins were identified within the liver during the first 8 days. However, only viral RNA was detected in the liver from Day 8 to Day 60. These studies demonstrate that MHV established an acute infection (Day 1–8) where infectious virus and viral proteins were identified. This was followed by a persistent infection within the retina and liver where only viral RNA were detected byin situhybridization.

Retinal degenerative disorders consist of a diverse degeneration in the absence of vasculitis or inflammation. This degenerative process was associated with a group of diseases frequently associated with a genetic predisposition; however, many cases are of unknown reduction of the photoreceptor layer, loss of interphotoreceptor retinoid binding protein, abnormalities in the reticause. Viruses may trigger some of these pathologic pronal pigment epithelium (RPE), and retinal detachments. cesses. In fact, viral infections often precede acute

The development of the degenerative phase of the dismultifocal placoid pigment epitheliopathy and acute macease was controlled by a genetic predisposition of the ular neuroretinopathy, and therefore are under suspicion host and was associated with the development of antias causative agents (1-2). In addition, herpes simplex retinal and anti-RPE cell autoantibodies (11). virus and varicella zoster virus have been implicated in One of the intriguing aspects of this disease process acute retinal necrosis (3, 4) and cytomegalovirus freis the nature of viral clearance. The acute phase of the quently induces a retinitis in immunosuppressed individdisease was associated with the presence of viral prouals (5). Retinal degenerative changes were also seen teins and the detection of infectious virus within the retin Creutzfeldt-Jacob disease and in subacute sclerosing ina (8, 9) . However, after Day 8, infectious virus and viral panencephalitis (6, 7). In order to evaluate the varied proteins were not detected. At this time, anti-virus neuways by which viruses may trigger retinal degenerative tralizing antibody were readily detected within the sera processes, we have developed a model of virus-induced of infected animals. In the absence of infectious virus, retinopathy.

retinal degenerative changes became apparent at Day The murine coronavirus, mouse hepatitis virus (MHV), 10 and continued for months. The purpose of this study induces an acute and chronic ocular disease in BALB/c was to determine if the virus persisted within the retina mice (8) (9) (10) . In the early, acute phase, 1 to 7 days after and other tissues during the course of this disease proinoculation, a mild retinal vasculitis was observed. The cess. In situ hybridization was selected as a way to demsecond stage was seen by Day 10 and progresses for onstrate virus persistence and to identify the cellular loseveral months. This stage was characterized by a retinal cation of the virus. The JHM strain of mouse hepatitis virus (MHV-JHM) was obtained from American Type Culture Collection in mouse L2 cells. Viral infectivity titrations were performed on L2 cells propagated in 96-well microtiter plates. The infectivity of stock virus was 10 5.6 TCID 50 /0.1 ml. Clone 2-2, which contained cDNA representing MHV-A59 genes 4-6 cloned into PstI site of pGEM-1 (Promega, Madison, WI), was provided by Dr. Susan R. Weiss (University of Pennsylvania, Philadelphia, PA) (12) . The insert was excised from the vector and purified by gel electrophoresis. cDNA labeling with digoxigenin-11-dUTP was carried out by the random primed method using a commercially available kit (Boehringer Mannheim, Indianapolis, IN).

Intravitreal inoculation of 12-week-old male BALB/c mice was performed as previously described (8, 9) . Mice ization, the slides were deparafinized, rehydrated, digested with proteinase K, and air-dried. Twenty-five microliters of prehybridization solution (50% deionized peroxidase procedure, as described previously (8, 9) . Rabbit polyclonal antibody to MHV (F88, provided by Dr. formamide, 11 Denhardt's solution, 41 SSC (11 SSC is 150 mM NaCl plus 15 mM sodium citrate), 10% dextran Katherine Holmes, Uniformed Services Univ Health Sci, Bethesda, MD) or normal rabbit sera were used. sulfate, and 100 mg/ml denatured salmon sperm DNA) was added to each slide, followed by incubation for 1 hr In order to evaluate the detection of viral RNA by in situ hybridization, we first studied JHM virus replication at room temperature. Five microliters of the labeled probe was added to the slides (final concentration of labeled in L2 cells. The virus cDNA probe hybridized to virus RNA in the infected cultures. In contrast, no reactivity cDNA was 100 ng/ml). Coverslips were placed on slides and the probe DNA was denatured on a hot plate at 90Њ was noted in the untreated L2 cells. Moreover, RNase treatment of infected cultures inhibited reactivity and in-for 10 min. The slides were cooled on ice and hybridization was carried out for 16 hr at 37Њ in a humidified box.

cubation of the infected cultures with plasmid cDNA resulted in no reactivity (data not shown). Analysis of in situ After removal of coverslips the slides were washed in 21 SSC twice for 10 min at room temperature, 11 SSC hybridization for viral RNA in JHM virus infected BALB/c mouse eyes is shown in Fig. 1 . When uninfected BALB/ for 30 min at room temperature with mild agitation, and 0.11 SSC twice for 10 min at 42Њ. Negative controls, c mouse eye sections were incubated with the viral cDNA probe, no reactivity was observed (Fig. 1A) . In contrast, RNase A (Boehringer Mannheim) treated tissue sections, digoxigenin-labeled linearized pBR328 (Boehringer when JHM virus-infected BALB/c mouse eye sections were incubated with the viral cDNA probe, a positive Mannheim) as nonhomologous probe, and in situ hybridization without probe were processed similarly.

signal was noted within the retina (Fig. 1B ). If the virusinfected eye sections were pretreated with RNAase, the The slides were washed twice for 5 min in buffer 1 (100 mM Tris-HCl, pH 7.5, 150 mM NaCl), then they were reactivity with the viral probe was inhibited. Likewise, when JHM virus-infected BALB/c mouse eyes were incu-immersed in buffer 2 (0.5% blocking reagent solution, Boehringer Mannheim) for 60 min. After brief dipping bated with plasmid cDNA, no reactivity was noted. This assay system was then used to evaluate mouse in buffer 2, they were incubated with anti-digoxigenin antibody (1 in 500 dilution with buffer 2) conjugated to eyes harvested from 1 to 60 days after JHM virus inoculation. By in situ hybridization, the viral RNA was detected alkaline phosphatase for 1 hr. This was followed by two 15-min washes in buffer 1 and immersion in buffer 3 (100 within the retina from Day 1 to Day 60. The mean number of positive signals of reactivity with the viral cDNA probe mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl) for 3 min. Visualization was performed with nitroblue tetrazo-are summarized in Fig. 2 . Multiple areas of viral RNA were detected within the eye throughout the 60-day pe-lium salt and 5-bromo-4-chloro-3-indolyl phosphate solution in a dark sealed box for up to 16 hr and the reaction riod, while the maximal number of positive signals were noted between Day 5 and Day 10. We next evaluated the was stopped by immersion in buffer 4 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) before mounting in Aqua-Mount cellular distribution of viral RNA within the eye. As is seen in Table 1 , the majority of viral RNA was detected (Lerner Laboratories, Pittsburgh, PA). Paraffin sections were also assayed for viral proteins by an immuno-in the retina. Low levels of viral RNA were detected in The data presented indicates that the viral RNA reviral cDNA probe ( Table 2 ). The retina was classified into mains within the retina throughout the 60-day period of four areas: ganglion cell layer, inner retina, outer retina, analysis. Since our earlier studies showed that infectious and RPE. The inner retina contains the inner plexiform virus and viral antigen was not detected within the retina layer, the inner nuclear layer, and Muller cells. The outer after Day 7, we wanted to compare the two assay sysretina consists of the outer plexiform layer, outer nuclear tems on the same samples (8, 9) . Both viral RNA and layer, photoreceptor inner and outer segments, and viral protein were detected in 100% of the eyes removed Muller cells. The Muller cell which is known to allow during Day 1 to Day 6 postinoculation. Between Day 8 JHM virus replication actually transverses the retina and and Day 60, viral RNA was detected in 100% of the retiis therefore a part of the inner and outer retina area.

nas. However, viral antigen was detected in only one of four eyes removed 8 to 10 days postinoculation and was seen in the retina, viral protein could be detected within the liver at Day 1 through Day 8 postinoculation. How-tected in meningial cells up to 77 days after inoculation. Thus the persistent infection was associated with the ever, after Day 8, viral protein was not detected by immunocytochemical staining even though viral RNA was de-CNS and not the liver in this model system (26) . In addition to these studies which identify persistent infection tected by in situ hybridization (data not shown).

In this study we demonstrated that JHM virus estab-in mice, MHV persistent infections have also been detected in the rat and monkey CNS (27, 28) . lished a persistent infection within the retina of BALB/ c mice. Throughout the 60-day period, viral RNA was

The data presented here identified that virus RNA was detected within the retina for 8 weeks after infectious detected by in situ hybridization within the retina. Moreover, evaluation of the liver revealed a similar virus and viral proteins were detected. The loss of infectious virus and viral proteins was correlated with the pattern of viral persistence, indicating that the virusinduced retinopathy was associated with a systemic activation of several components of the immune system. Anti-virus neutralizing antibodies and anti-retinal autoan-persistent virus infection. The technique of in situ hybridization allowed us to identify some of the cell types tibodies were observed in the sera 6 to 7 days after infection (8, 11, 29) . This may be a critical period for the within the retina which contained the viral RNA. During the acute phase of the infection, viral RNA was found virus to avoid the immune surveillance (30, 31) . In this model system of murine coronavirus-induced retinopa-in the retina, RPE, ciliary body epithelium, and the iris epithelium. During the late phase of the infection, viral thy, the continuous presence of viral RNA and the possible production of low levels of viral proteins may contrib-RNA was almost exclusively found within the retina and RPE and not in the anterior segment of the eye.

ute to the multifaceted pathogenic processes within the retina. Within the retina, viral RNA was detected in the ganglion cell layer, the inner retina, and the outer retina. The Muller cell is considered the most prominent glial 

system (CNS) and liver (18). For example MHV infection 62

mice resulted in an acute necrotizing encephalitis fol

cover infectious virus or detect viral antigens (21). Perl-Raton, FL. man and coworkers also identified MHV, JHM strain, viral 11. Hooks

These investigators suggested that persis

Lab. Invest. tence may be maintained due to viral spread from cell

to cell thereby escaping immune elimination (24, 25)

the liver during the first 7 days after inoculation. However

after Day 21 viral RNA and proteins were not detected 16. Hooks

In ''Concepts in Viral Patho