Lassa Virus Targeting of Anterior Uvea and Endothelium of Cornea and Conjunctiva in Eye of Guinea Pig Model Joy M. Gary, Stephen R. Welch, Jana M. Ritter, JoAnn Coleman-McCray, Thanhthao Huynh, Markus H. Kainulainen, Brigid C. Bollweg, Vaunita Parihar, Stuart T. Nichol, Sherif R. Zaki, Christina F. Spiropoulou, Jessica R. Spengler Lassa virus (LASV), a hemorrhagic fever virus endemic to ocular structures targeted by LASV, and the clinical impli- West Africa, causes conjunctivitis in patients with acute dis- cations of ocular infection are unknown. ease. To examine ocular manifestations of LASV, we histo- In viral hemorrhagic fever disease, ocular manifesta- logically examined eyes from infected guinea pigs. In fatal tions are not limited to LF and are well described for infec- disease, LASV immunostaining was most prominent in the tion with Ebola virus (EBOV) (11,12), Marburg virus (13), anterior uvea, especially in the filtration angle, ciliary body, and Rift Valley fever virus (RVFV) (14–16). Recently, the and iris and in and around vessels in the bulbar conjunctiva and peripheral cornea, where it co-localized with an endo- implications of viral persistence in the eye and other immu- thelial marker (platelet endothelial cell adhesion molecule). noprivileged sites have been highlighted in Ebola virus dis- Antigen was primarily associated with infiltration of T-lym- ease (EVD) (12,17). The possibility of LASV persistence phocytes around vessels in the anterior uvea and with new in the eye is unknown, as is the extent of chronic pathologic vessel formation at the peripheral cornea. In animals that changes secondary to infection that could result in long- exhibited clinical signs but survived infection, eyes had little term functional abnormalities. to no inflammation and no LASV immunostaining 6 weeks Inbred Strain 13 guinea pigs almost uniformly die of after infection. Overall, in this model, LASV antigen was re- disease after LASV infection with the prototypic 1976 stricted to the anterior uvea and was associated with mild Josiah strain without requiring serial adaptation (18). chronic inflammation in animals with severe disease but In addition, we recently described nonlethal disease in was not detected in survivors. Strain 13 guinea pigs infected with a 2015 isolate from a person with LF imported to New Jersey, USA, from assa virus (LASV) is the etiologic agent of Lassa fe- Liberia (LASV 812673-LBR-USA-2015, or LASV- Lver (LF), a viral hemorrhagic fever endemic to West NJ2015 [19]). To investigate ocular manifestations Africa. Incidence of LF in areas to which it is endemic is of LASV infection in animals that died of or survived ≈100,000–300,000 cases annually, of which ≈5,000 are infection, we collected samples from animals infected fatal (1). After an incubation period of 7–21 days (2,3), with either LASV-Josiah or LASV-NJ2015. LASV loads disease onset is gradual and includes fever, weakness, my- and distribution, and associated ocular histopathology, ositis, and ulcerative pharyngitis that may progress to myo- were assessed in these animals. carditis, pneumonitis and pleuritis, and encephalopathy and hemorrhage (2). The most well-documented sequela of LF Material and Methods is hearing loss (4–8). Ocular involvement in acute LF includes conjunctivi- Ethics Statement tis and conjunctival edema (9). In addition, transient blind- All animal procedures were approved by the Centers ness has been described in humans recovering from LASV for Disease Control and Prevention (CDC; Atlanta, GA, infection (3,10). The extent of viral presence in the eye, the USA) Institutional Animal Care and Use Committee (IA- CUC; #2833SPEGUIC) and conducted in accordance Author affiliation: Centers for Disease Control and Prevention, with the Guide for the Care and Use of Laboratory An- Atlanta, Georgia, USA imals (20). CDC is fully accredited by the Association DOI: https://doi.org/10.3201/eid2505.181254 for Assessment and Accreditation of Laboratory Animal Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 5, May 2019 865 RESEARCH Care International. Procedures conducted with LASV or transcription PCR (qRT-PCR) targeting a strain-specific LASV-infected animals were performed in the CDC Bio- nucleoprotein gene sequence (primer and probe sequences safety Level 4 laboratory. available on request from the authors), and normalized to 18S RNA levels. We determined viral small (S) segment Virus copy numbers using standards prepared from in vitro–tran- Recombinant LASV-Josiah, based on the sequence of an scribed S segment RNA. isolate obtained in 1976 from the serum of a 40-year- old man hospitalized at Songo Hospital in Sebgwena, Histochemical Staining and Sierra Leone (21,22), was rescued in BSR-T7/5 cells Immunohistochemical Analysis and passaged twice in Vero-E6 cells (GenBank acces- Tissue specimens were fixed in 10% neutral buffered for- sion nos. HQ688673.1, HQ688675.1). Recombinant malin and subjected to gamma irradiation (2 × 106 rad). LASV 812673-LBR-USA-2015 (LASV-NJ2015), based Formalin-fixed tissues from all guinea pigs were routinely on the sequence of an isolate obtained in 2015 from a processed, embedded in paraffin, sectioned at 4 µm, and 55-year-old man who died of LF in New Jersey after stained with hematoxylin and eosin. A veterinary patholo- returning from Liberia, was rescued in BSR-T7/5 cells gist visually assessed inflammation within the eye as min- and passaged twice in Vero-E6 cells (19) (GenBank ac- imal (few scattered lymphocytes around vessels), mild cession nos. MG 812650, MG812651). We determined (small clusters of lymphocytes around vessels or within focus-forming units and 50% tissue culture infectious the filtration angle), or moderate (noticeably more intense dose (TCID50) titers in Vero-E6 cells by immunofluores- infiltrates of lymphocytes within the eye). A marked re- cence assays using an in-house anti-LASV monoclonal sponse, which we did not observe in these animals, would antibody mix targeting nucleoprotein and glycoprotein 2 have comprised tissue architecture disrupted by inflam- (SPR628), with TCID50 titers calculated using the meth- matory cells. od of Reed and Muench (23). We conducted immunohistochemical (IHC) assays using indirect immunoalkaline phosphatase detection on Guinea Pig Infections 4-µm sections. Colorimetric detection of attached anti- Sixteen strain 13/N guinea pigs (8 male, 8 female, 6 months bodies was performed using the Mach 4 AP polymer kit to >3 years of age) were obtained from our breeding colo- (Biocare Medical, https://biocare.net) at room temperature, ny at CDC. Age- and sex-matched 13/N guinea pigs were except for heat-induced epitope retrieval. Using either Re- inoculated subcutaneously with 104 focus-forming units veal or EDTA buffer, we conducted heat-induced epitope ≈ × 4 (equivalent to 2 10 TCID50) of LASV, either LASV- retrieval using the NxGen decloaker (Biocare Medical) at Josiah (10 animals) or LASV-NJ2015 (6 animals). Four 110°C for 15 min. All slides were blocked in Background animals infected with LASV-Josiah served as unvaccinated Punisher (Biocare Medical) for 10 min and incubated with controls in parallel studies (24). All animals were housed primary antibody for 30 min. Antibodies used were anti- individually and given daily fresh vegetable enrichment, CD3 (diluted 1:100 in EDTA buffer [#NCL-L-CD3–565; hay, commercial guinea pig chow, and water as desired. Leica Biosystems, https://www.leicabiosystems.com), an- Experienced CDC veterinarians or animal health techni- ti-CD79a (1:100, EDTA buffer [#NCL-L-CD79a-22; Leica cians assessed animal health. Animals were humanely eu- Biosystems]), and a mouse monoclonal antibody targeting thanized with isoflurane vapors and sodium pentobarbital LASV glycoprotein 2 at 1:1,000 (CDC). Mach 4 Probe was (SomnaSol Euthanasia-III solution; Henry Schein Animal applied for 10 min, followed by Mach 4 AP polymer for 15 Health, https://www.henryscheinvet.com) once clinical min (Biocare Medical). The antibody/polymer conjugate illness scores (including piloerection, ocular discharge, was visualized by applying Fast Red Chromogen dissolved weight loss >25%, changes in mentation, ataxia, dehydra- in Naphthol Phosphate substrate buffer to tissue sections tion, dyspnea, or hypothermia) indicated the animal was in for 20 min (Thermo Fisher Scientific). Appropriate nega- the terminal stages of disease, or at the completion of study tive control serum was run in parallel. Slides were counter- 41 days postinfection (dpi). stained with Mayer’s hematoxylin (Poly Scientific, https:// www.polyrnd.com) and blued in lithium carbonate (Poly Quantitative Reverse Transcription PCR Scientific). Positive controls included formalin-fixed, par- RNA was extracted from blood and homogenized tissue affin-embedded Vero-E6 cells infected with LASV, tissue samples using the MagMAX-96 Total RNA Isolation Kit from a human with LF, and guinea pig spleen and liver (for (Thermo Fisher Scientific, https://www.thermofisher.com) inflammatory cell and cell lineage markers). A veterinary on a 96-well ABI MagMAX extraction platform with a pathologist scored IHC staining on a scale of 0 (no IHC DNase-I treatment step, according to the manufacturer’s staining seen) to 4 (abundant, intense IHC staining within instructions. RNA was quantified by a quantitative reverse structures in the eye). 866 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 5, May 2019 Lassa Virus in Eye of Guinea Pig Model Table. Summary of ocular LASV staining and histopathologic