Pathobiology and Treatment of Viral Keratitis

Abstract

Keratitis is one of the most prevalent ocular diseases manifested by partial or total loss of vision. Amongst infectious (viz., microbes including bacteria, fungi, amebae, and viruses) and non-infectious (viz., eye trauma, chemical exposure, and ultraviolet exposure, contact lens) risk factors, viral keratitis has been demonstrated as one of the leading causes of corneal opacity. While many viruses have been shown to cause keratitis (such as rhabdoviruses, coxsackieviruses, etc.,), herpesviruses are the predominant etiologic agent of viral keratitis. This chapter will summarize current knowledge on the prevalence, diagnosis, and pathobiology of viral keratitis. Virus-mediated immunomodulation of host innate and adaptive immune components is critical for viral persistence, and dysfunctional immune responses may cause destruction of ocular tissues leading to keratitis. Immunosuppressed or immunocompromised individuals may display recurring disease with pronounced severity. Early diagnosis of viral keratitis is beneficial for disease management and response to treatment. Finally, we have discussed current and emerging therapies to treat viral keratitis.

1. Introduction

Keratitis is an inflammation of the cornea, the transparent layer of tissue that covers the pupil and iris (Liesegang. 2001a). In United States alone, annual expenditure on keratitis treatment is estimated at $175 million, of which $58 million and $12 million are endured by Medicare and Medicaid patients, respectively (Collier et al. 2014). Microbial or ulcerative keratitis is characterized by a defect in the corneal epithelium alongside suppurative stromal infiltration (Ni et al. 2015). Microbial keratitis is estimated to impact roughly 30,000 people in the US annually (Jeng et al. 2010). Both infectious and non-infectious causes have been associated with the etiology of microbial keratitis (Srinivasan et al. 2008). Corneal infection with viruses, bacteria, fungi, or parasites can result in infectious keratitis, a subset of microbial keratitis (Agrawal et al. 1994, Bharathi et al. 2002, Keay et al. 2006, Ray et al. 2015). The risk factors for non-infectious, microbial keratitis are varied and include ocular trauma, chemical exposure, contact lens usage, chronic inflammatory eye disease, and diabetes mellitus (Bialasiewicz et al. 2006, Collier et al. 2014, Parmar et al. 2006, Srinivasan et al. 2008). A 2014 Chinese cross-sectional study estimated the prevalence of infectious keratitis to be 0.192% which was broken down into 0.11%, 0.075%, and 0.007% for viral, bacterial, and fungal origins respectively (Song et al. 2014). Another 2014 study reported the prevalence of infectious keratitis to be 0.148% with 0.065%, 0.068%, and 0.015% due to viral, bacterial, and fungal causes (Cao et al. 2014). Representative images of different varieties of infectious keratitis are provided in Figure 1.

Figure 1.

Representative keratitis pictures of human eyes. (a) Fluorescein stained images of herpes simplex corneal keratitis (Azher et al. 2017). (b) A close-up whole eye image of human eye showing herpetic disciform keratitis (Vislisel. 2015). (c) Varicella zoster virus disciform keratitis (Vislisel. 2014). (d) Slit-lamp imaging of adenoviral keratitis (Wikipedia. 2020). (e) Image of a human eye with fungal keratitis caused due to Aspergillus (Vislisel. 2016). (f) Slit-lamp photography of Nocardia keratitis caused by Nocardia farcinia (Scruggs. 2017).

Viral keratitis (VK) is one of the most prevalent forms of infectious keratitis (Cao et al. 2014, Song et al. 2014). Of the different viruses which have been reported to cause keratitis, the alpha-herpesvirus herpes simplex virus (HSV) is the dominant cause (Carmichael. 2012, Farooq et al. 2010, Farooq and Shukla. 2012, Hjalgrim et al. 2007, Wharton. 1996). Other common agents of viral keratitis include the beta-herpesvirus cytomegalovirus (CMV), the alpha-herpesvirus varicella-zoster virus (VZV), and the gamma-herpesvirus Epstein-Barr virus (EBV). The alpha-subfamily has the greatest host range and establishes latency in sensory neurons, beta-subfamily has an intermediate host range and establishes latency primarily in lymphoid tissues, and the gamma-subfamily has a narrow host range and establish latency in only lymphoid tissues (Whitley, Richard J. 1996). In this chapter, we present evidence demonstrating the etiological role of these herpesviruses in the pathogenesis of viral keratitis. We describe the crosstalk of host-viral interactions during disease, including the host immune response to the virus, viral means of immune evasion, viral reactivation, and resolution. Finally, we provide a comprehensive update on diagnostics and therapeutic strategies to treat viral keratitis.

2. HSV

2.1. Incidence and Prevalence

There are two types of herpes simplex viruses: type 1 and type 2. HSV-1 is more commonly associated with ocular infection, while HSV-2 primarily infects the genitalia (Whitley, R. J. et al. 1998). HSV-1 is a ubiquitous herpesvirus that infects roughly two-thirds of the world’s population (Liesegang. 2001b). Approximately 65% of the US population is found seropositive for HSV-1 (Xu et al. 2006). Direct contact with body secretions, viz. saliva, tears, etc., of infected individuals is considered as key to the human-to-human spread of HSV-1. Recently, National Health and Nutrition Examination Survey data (2015–16) revealed a linear decrease in seroprevalence of HSV-1 from 1999–2000 to 2015–2016 for all races: from 52.4% to 36.9% for non-Hispanic white, 68.4% to 58.8% for non-Hispanic black, and 82.0% to 71.7% for Mexican-Americans (Koganti et al. 2019a, McQuillan Geraldine, Kruszon-Moran Deanna, Flagg Elaine, Paulose-Ram, Ryne. 2019, Xu et al. 2006). HSV-1 infection in children and adults is associated with entirely different disease manifestations. Ulcerative, painful stomatitis, overt fever, anorexia and local edema of oral mucosa are found in children, while pharyngitis and tonsillitis are common initial infection symptoms in adults. Interestingly, HSV-1 can attach to sensory nerves in the throat, move to the trigeminal ganglia (TG), and persist as lifelong latent form in TG (Koujah, L. et al. 2019). Reactivation of HSV-1 in adults is also associated with recurring herpes labialis - more commonly referred to as cold sores (Wald and Corey. 2007). However, in extremely rare cases (0.0001–0.0005% of the population), HSV-1 can cause encephalitis which is fatal in more than 70% of cases if left untreated (Wald and Corey. 2007). Herpes Simplex Encephalitis (HSE) initiates with normal cold-like symptoms followed by headache, confusion, nausea, fever, seizures, and drowsiness as a result of neurological deterioration (McQuillan et al. 2018, Wald and Corey. 2007).

Over 500,000 people suffer with HSV-1 ocular disease in US, which may result in conjunctivitis, iridocyclitis, acute retinal necrosis and keratitis in severe cases (Farooq and Shukla. 2012, Liesegang et al. 1989). Given a primary HSV-1 infection, ocular disease is expected in approximately 8% of the cases (Farooq and Shukla. 2012, Holland et al. 1992). A significant percentage (~15%) of patients with a primary HSV-1 ocular infection also develop severe complications such as blepharoconjunctivitis and dendritic ulcers (Darougar et al. 1985). The prevalence of virus in the western countries, disease prevalence is reported as 150 cases per 100,000 people, and the incidence varies between 10–30 cases per 100,000 people annually (Labetoulle et al. 2005, Liesegang et al. 1989, Young et al. 2010). The risk of developing HSK is calculated as 1% worldwide, and the global annual incidence of herpetic keratitis is reported to be approximately 1.5 million cases (Farooq and Shukla. 2012, Reynaud et al. 2017). Interestingly, HSK is a persistent disease with relatively high recurrence rates: 9.6% after 1 year, 22.9% after 2 years, 40.0% after 5 years, and 67% after 10 years (Farooq and Shukla. 2012, Labetoulle et al. 2005, Liesegang. 2001b, Shuster et al. 1981, Stanzel et al. 2014). Approximately 40% of patients with HSK will experience 2–5 relapses over their lifetimes (Wishart et al. 1987). Additional information regarding the epidemiology of HSK is provided in Table 1.

Table 1.

Statistics on the epidemiology of HSK.

Statistics	Prevalence and percentages	References
Prevalence of HSK in US	150 in 100,000, and 20K new cases annually	(Liesegang et al. 1989)
Annual incidence in western countries	10–30 per 100,000	(Labetoulle et al. 2005, Liesegang et al. 1989, Young et al. 2010)
% of people with antibodies to HSV-1 by middle age	More than 90%	(Liesegang. 2001b)
% of primary HSV-1 infections that are asymptomatic and not recognized	More than 90%	(Liesegang. 2001b)
% Recurrence at 1 yr, 2 yrs, 5 yrs, and 10 yrs	9.6%, 22.9%, 40.0%, 67.0%	(Farooq and Shukla. 2012, Labetoulle et al. 2005, Liesegang. 2001b, Shuster et al. 1981, Stanzel et al. 2014)
Global lifetime risk of developing HSK	1%	(Reynaud et al. 2017)
Patients who experience 2–5 relapses of HSK over their lifetime vs 6–15 relapses	40% vs 11%	(Wishart et al. 1987)
% of patients with HSK who develop severe complications	15%	(Darougar et al. 1985)
Incidence of ACV-resistant strains among immunocompetent patients	0–0.6%	(Christophers et al. 1998, Nugier et al. 1992)
Incidence of ACV-resistant strains among immunocompromised patients	3–6% with 14% for bone marrow transplant recipients	(Christophers et al. 1998, Nugier et al. 1992)

2.2. Pathobiology

The transmission of HSV-1 occurs through intimate contact between a seronegative and an actively shedding, seropositive individual via bodily fluids and tissue transplants (Whitley, R. J. et al. 1998). After attaching to host surfaces, the virus travels to the trigeminal ganglia- a latent site of infection, (Whitley, R. J. et al. 1998). Though the exact etiology of HSV-1 reactivation is not known, it is well-agreed that some host stressor induces the reactivation of HSV-1 in the TG, such as psychological stress, fatigue, immunosuppression, and UV exposure (Jones. 2003, Perng and Jones. 2010, Toma et al. 2008). The reactivated virus travels again through the trigeminal ganglia and ophthalmic nerves to the eye in a retrograde fashion, and the resultant chronic inflammation facilitates corneal damage (Koganti et al. 2019b). HSV-1 either sheds in a symptomatic or asymptomatic mode. During symptomatic shedding, the virus replicates in a controlled fashion and facilitates the recruitment of multiple cells, thereby resulting in the formation of large syncytial structures (Koujah, L. et al. 2019). Subsequently, the replicated viral genomes are assembled within virions. Towards the end of the infectious cycle, the cell bursts in a necroptotic fashion and disseminates the virus to the surrounding tissue (Koujah, L. et al. 2019).

Amongst the viruses contributing viral keratitis, the immune reactions are only well-studied in case of HSV-1-induced keratitis (Figure 2). During HSV-1 infection, the virus replicates in corneal epithelial cells and forms large syncytial structures (Pertel and Spear. 1996). These cells eventually burst in a necroptotic fashion and release HSV-1 virions in the surrounding tissues, which results in the formation of large keratitis lesions in different layers of the cornea: epithelium, stroma, and endothelium (Toriyama et al. 2014). Interestingly, cornea epithelial cells have shown to demonstrate distinct expressional dynamics of toll like receptors (TLRs) during HSV-1 infection (Jin et al. 2007). TLR-4, 7, 8, and 9 are significantly up-regulated in active keratitis (Jin et al. 2007). Knocking out TLR-2 has proven to be an effective strategy in inhibiting keratitis lesions in mice models (Sarangi et al. 2007). Conversely, TLR-4 knock out mice demonstrated more rapid development of severe keratitis lesions (Sarangi et al. 2007). In addition, TLR-9 antagonists play important roles in preventing exacerbation of disease during the spread of HSV-1 DNA. Although TLR-9 ligation with HSV-1 DNA initiates the strong immune responses for the clearance of HSV-1, it also promotes inflammatory processes which lead to cornea destruction (Krug et al. 2004, Sarangi et al. 2007, Takeda et al. 2011). Therefore, blockade of TLR-2 and TLR-9 and activation of TLR-4 by molecular mimics may be a novel, efficacious strategy to avoid innate immune mediated damage to cornea.

Figure 2.

HSV-1 life cycle and associated immune reactions during ocular infection. During the primary infection in the corneal epithelium, antigen-presenting cells such as dendritic cells activate the humoral and cellular immune pathways. Naïve T cells and NK cells become activated and target infected host cells for lysis. Likewise, B cells become activated and differentiate into memory B cells and plasma cells. Plasma B cells secrete antiviral antibodies to combat infection. Memory B and T cells remain at the site of latency to monitor reactivation and stimulate a quicker immune response once reactivation is triggered Immune infiltration in the corneal epithelium during recurrent infection creates inflammation, and chronic corneal inflammation can stimulate the development of keratitis.

Besides corneal epithelial cells, HSV-1 is trophic for DCs located in the corneal basement membrane (Albers et al. 1989). To decrease the HSV-1 load during primary ocular HSV-1 infection, Mott et al. immunized BALB/c or C57BL/6 mice with Fms-like tyrosine kinase 3 ligand (Flt3L) - a potent stimulatory factor for DCs (CD11⁺ CD8α⁺) in vivo (Mott et al. 2008). However, the enhanced number of DCs (more specifically CD11c⁺ CD8α⁺ cells) were associated with increased amount of HSV-1 latency (Mott et al. 2008). Moreover, adoptive transfer of CD11c⁺ CD8α⁻ DCs were associated with reduced HSV-1 latency. Bryant-Hudson and Carr have shown that the inhibition of programmed death 1 ligand (PD-L1) interactions with its receptor PD-1 stymied the activation of DCs and elevated viral titers (Bryant-Hudson and Carr. 2012). Furthermore, inactivation of DCs during HSV-1 infection was also found to be associated with the downregulation of various imperative molecules for DCs, such as CD80, CD86, CD54, CCR7, CXCR4, and MHC1 (Novak and Peng. 2005).

Akin to other viruses, HSV-1 crosstalk with immune cells decides the fate of prolonged infection dynamics or resolution. Innate immune system plays central role in limiting HSV-1 replication, which controls virus replication until the activation of antiviral adaptive immune response for the clearance of the HSV-1 (Cheng et al. 2000, Weber et al. 2006). HSV-1 replication in epithelial cells induce type 1 interferons that eventually leads to the activation of innate immune system (Egan et al. 2013). The activation of macrophages and neutrophils is one of the major responses to HSV-1 replication (Kodukula et al. 1999). Activation of macrophages in this pursuit secretes interferons and cytokines (Kather et al. 2010, Kodukula et al. 1999), whilst natural killer (NK) cell activation in this clinical setting induces the apoptosis of infected epithelial cells via interferon-γ and granzymes A and B (Ghiasi et al. 2000, Grubor-Bauk et al. 2008, Kassim et al. 2009).

Liu et al. highlighted the infiltration of various immune population at various time points of HSV-1 infection. They have shown that Mφ and γδ TCR+ T lymphocytes infiltrate the TG at 3–5 days of infection and TCR-αβ⁺ T (CD4⁺ and CD8⁺ T cells) infiltrate at 7–12 days of infection (Liu, T. et al. 1996). In cell depletion studies, people have demonstrated the significance of these cells types in the protection against HSV-1 infection. γδ TCR+ T cells reduce the severity of HSV-1-induced epithelial lesions and prevent the development of viral encephalitis during infection (Sciammas et al. 1997), while CD8+ T cells inhibit reactivation without destroying latently-infected neurons (Liu, Ting et al. 2000). γδ TCR+ T cells also produce IFN-γ which triggers the production of the pro-inflammatory cytokine TNF-α and nitric oxide (NO) (Jones-Carson et al. 1995). Furthermore, B cells are involved in the antigen presentation and secrete cytokines, during HSV-1 infection (Deshpande et al. 2000). The protective immunity conferred by these immune cell populations, either present in cornea or infiltrated after HSV-1 infection, helps in clearing the virus. However, these inflammatory reactions are responsible for corneal damage due to inflammatory microenvironment by secretion of diverse cytokines including IFN-α, IFN-β, and IFN-γ, IL-1α, IL-1β, TGF-β, TNF-α, IL-6, IL-8, IL-12, and IL-17 (Cathcart et al. 2011, Kaye and Choudhary. 2006, Remeijer et al. 2004).

HSV-1 ensures robust suppression of the immune response towards the earlier stages of infection and latency (Burgess and Mohr. 2018, Matundan and Ghiasi. 2019, Mogensen et al. 2004). Viral antigen presentation is reported to be inhibited by ICP47 and virion host shutoff protein (vhs). ICP47 competes with transporter associated with antigen processing TAP –which is involved in the translocation of antigen peptides into ER for the loading on newly synthesized MHC-I molecules (161–163). On the other hand, vhs is an mRNA-specific RNase that triggers rapid shutoff of host protein synthesis to inhibit new production of MHC-I for antigen presentation (164). As a result, inflammation of the tissue due to the infiltration of CD4+ and CD8+ T-cells is uncommon during the early stages of active viral replication. However, a robust immune response is seen towards the later stages of HSV-1 infection that leads to tissue inflammation, resulting in sequelae such as blepharitis, epithelial and stromal keratitis, and keratoconjunctivitis in the infected eye (Lobo et al. 2019).

2.3. Characteristics of Herpes Simplex Keratitis (HSK)

Depending on the corneal layer affected or neuronal cell infection, four subtypes of herpes simplex keratitis (HSK) are classified as epithelial, stromal, endothelial, or neurotrophic keratitis (Holland and Schwartz. 1999). Interstitial keratitis is a specific form of stromal keratitis where inflammation is localized to the stroma with minimal effects on the epithelium or endothelium. The diagnosis of HSK relies upon its clinical presentation during a slit-lamp examination (Azher et al. 2017, Darougar et al. 1985).

Epithelial keratitis comprises approximately 60% of ocular HSV cases (Lobo et al. 2019). Epithelial keratitis is reported to begin with granular spots, progressing to punctate lesions, and eventually causes branching dendritic lesions loaded with active viruses (Azher et al. 2017). Dendritic lesions are branching with terminal bulbs, which contain live virus (Lobo et al. 2019). The swollen borders of these dendritic lesions are the signature of intraepithelial cell infiltration (Shoji et al. 2016). These lesions can coalesce to form a geographic ulcer with discrete flat edges that can change conformation as infection spreads (Liesegang et al. 1989, Lobo et al. 2019). As a result, corneal sensation is diminished in the affected eye (Lobo et al. 2019). The appearance of these dendritic cells lesions in this clinical setting (epithelial HSK) make them suitable to be diagnosed by the slit-lamp examination (Darougar et al. 1985). However, immunofluorescence assays or PCR can be useful tools to confirm that HSV-1 is the cause of the symptoms observed (El-Aal et al. 2006). Epithelial lesions either may heal naturally or the treatment with nucleoside analogs (viz., acyclovir) can interfere with viral replication to prevent corneal damage by severe epithelial keratitis (Wilhelmus, Kirk R. 2015).

The stromal subtype of HSK accounts for ≥ 20–48% of ocular HSV cases (Remeijer et al. 2004) and shows the highest impact on vision loss via the scarring and neovascularization of the cornea compared to its other three subtypes (Lobo et al. 2019). Stromal HSK can be further sub-divided into disciform, necrotizing, and immune stromal keratitis (Shoji et al. 2016). Telltale signs of disciform keratitis are circular, focal stromal edema along with keratic precipitates and Descemet membrane folds (Lobo et al. 2019, Shoji et al. 2016). In necrotizing stromal keratitis, the stroma experiences dense, white infiltrates and edema due to the virus (Heiligenhaus et al. 2003). Furthermore, it can develop an abscess-like opacity and the eponymous necrosis of the stromal tissue (Toriyama et al. 2014). Epithelial ulcers may be present in this subtype as well as increased risk of stromal melting (Lobo et al. 2019). Immune stromal keratitis is characterized by inflammation of the stroma without damage to the epithelium. Immune rings, indicative of antigen-antibody immune complex deposits, are another manifestation of immune stromal keratitis (Lobo et al. 2019). Therein, immune stromal keratitis is the most common of the three stromal variants (Lobo et al. 2019).

Endotheliitis is the third form of HSK. Like disciform keratitis, endotheliitis presents with inflammation of the endothelium. Stromal opacity might also develop in this subtype of HSK, but it is mainly caused by endothelial dysfunction rather than stromal inflammation. Keratic precipitates, corneal edema, and anterior chamber inflammation are three characteristics of endotheliitis that can greatly aid diagnosis. Depending on the arrangement of keratic precipitates, endotheliitis can be further classified into one of four subtypes: linear, sectoral, disciform, and diffuse (Alfawaz. 2013). While HSK endotheliitis can present quite similarly to anterior uveitis, it can be distinguished by the presence of iritis and stromal edema (Carrillo-Arroyo et al. 2012).

Neurotrophic keratitis is the fourth subtype of HSK, wherein HSV damages the nerves which innervate the cornea, resulting in reduced sensitivity. Herpes infections make up 27% of all neurotrophic keratitis cases (Bonini et al. 2000). The clinical presentation of HSK can be divided into three stages. Although patients do not usually report pain, they can experience dry eyes, due to a loss of reflex tears, and photophobia in the first stage (Sacchetti and Lambiase. 2014). Superficial punctate keratopathy and corneal edema can also occur. In the second stage, patients may present with epithelial defects in the shape of ovals or circles along with stromal edema (Versura et al. 2018). In the third and most severe stage, corneal ulcers, scarring, or perforations can lead to a loss of vision and the release of aqueous humor (Sacchetti and Lambiase. 2014, Versura et al. 2018).

3. CMV

3.1. Incidence and Prevalence

CMV is a member of the beta-herpesvirus family and one of the leading causes of congenital infections worldwide (Manicklal et al. 2013).The transmission rate from a mother with a primary CMV infection to her child is between 30–40% (Carlson et al. 2010). Altogether, 1% of newborn infants in the United States test positive for CMV infections (Bonalumi et al. 2011, Carlson et al. 2010). The incidences of CMV positive live births in other developed countries were reported in the range of 0.3–2.4% (Alford et al. 1990, Peckham. 1991). CMV is also reported as the most common virus during the intrauterine infection. 5% to 15% of CMV positive live infants were found to be associated with sensorineural hearing loss, delay of psychomotor development, and visual impairment after the birth (Bonalumi et al. 2011) and approximately 20–30% of them die due to severe complications viz., disseminated intravascular coagulation, hepatic dysfunction, and subsequent bacterial infections (Pass et al. 2006, Raynor. 1993). Additionally, 40% live infants infected with CMV infections develop a panel of auditory, ocular, and CNS diseases including microcephaly, encephalitis, mental retardation, optic atrophy, and deafness among others (Ahlfors et al. 1986, Arribas et al. 1996, Carmichael. 2012, Furutate et al. 2011). Fortunately, the rate of transmission due to non-primary infections drops to 1–2.2% (Fowler et al. 1992, Kenneson and Cannon. 2007). These infections can rarely become severe but can result in pneumonia, mononucleosis, and meningitis (Beam et al. 2018, Dumoulin and Eyer. 2018, Rafailidis et al. 2007).

CMV is an emerging etiological agent of corneal endotheliitis. CMV-induced ocular pathologies range from corneal endotheliitis, episodic anterior uveitis, sector iris atrophy with iritis, chronic anterior uveitis, and ultimately to retinitis (Inoue, T. et al. 1998, Koizumi et al. 2006, Wilhelmus, K. R. et al. 1996). Interestingly, a study by Kandori et al. revealed that CMV has propensity towards corneal endotheliitis rather than epithelial or stromal keratitis. It was reported that 24.1% of cases with corneal edema demonstrating idiopathic corneal endotheliitis were positive for CMV DNA (copy number ranged from 6.3×10⁴ to 3.6×10⁶ per mL) (Kandori et al. 2010a). From a large cohort of Japanese patients, the development of CMV endotheliitis appears to be more frequent in middle-aged men (80.2% of the patients were male with mean age was 66.9 ± 10.9 years) (Koizumi et al. 2015). PCR of the aqueous humor of these patents was negative for HSV and VZV DNA (Koizumi et al. 2015).

3.2. Pathobiology

While not as frequent as HSK, CMV-induced keratitis has garnered significant attention in recent times due to dramatic surge in the number of diagnosed keratitis patients (Carmichael. 2012). CMV establishes latency in the blood of infected individuals and can be transmitted through various bodily fluids, blood transfusion, sexual intercourse, saliva and breast-feeding (Alfawaz. 2013, Hayes et al. 1972, Lang and Kummer. 1975, Peckham. 1991). Its flexible means of transmission results in up to 100% seroprevalence in certain parts of the globe (Cannon et al. 2011). Interestingly, viral tropism for CD34⁺ myeloid progenitor cells (predominant for CMV latency) and other cells residing in the pulmonary, corneal, retinal, and salivary tissues makes it one of the most ubiquitous and adaptable pathogens (Reeves and Sinclair. 2008). Thus, in immunocompromised and immunosuppressed hosts, opportunistic CMV infection is reported to reveal a wide gamut of symptoms: from asymptomatic viremia to CMV syndrome and tissue-invasive disease, primarily due to activation of type 1 immunity (Reeves and Sinclair. 2008).

During CMV-induced keratitis, the virus strategically replicates and assembles in a pseudo G1 phase before egressing from the host cells to infect the neighboring cells (Angelova et al. 2012). This replicative phenomenon of CMV has been shown to be involved during epithelial keratitis, stromal keratitis and endotheliitis with an opaque, branching, non-ulcerative epitheliopathy (Alfawaz. 2013). Furthermore, CMV show tissue tropism towards endothelial cells, smooth muscle cells, and myeloid cells (Sinzger et al. 2008). Interestingly, CMV has been demonstrated to infect the human placenta as the presence of CMV DNA has been detected in placental tissue at different times in gestation times- derived from uncomplicated pregnancies (Fisher et al. 2000, Trincado et al. 2005). The activation of transmitted CMV copies via maternal-fetal interface depends on the secretion of IL-10 from fetal cells invading the uterine wall and involved in the formation of placenta (Fisher et al. 2000, Roth et al. 1996). Latently infected maternal macrophages and/or dendritic cells (DCs) with CMV migrate through maternal-fetal interface and local immune suppressive environment maintained by IL-10 might drive its reactivation and subsequent transmission to the fetus. Interestingly, CMV exploits maternal derived low avidity anti–CMV antibodies, for the transmission of CMV virions. Binding of these antibodies with neonatal fragment crystallizable receptor (FcR), and the resultant transcytosis of these complexes across the syncytio-trophoblast has been proven as an imperative mechanism for CMV transmission to the fetus (Maidji et al. 2007). On the contrary, CMV infection downregulates HLA-C (Huard and Früh. 2000), which in-turn negatively regulates natural killer (NK) cell effector function. This allows NK cell-mediated killing of virally infected cells. Therefore, it appears that IL-10 enriched environment likely play the central role for CMV spread. This mechanism needs to be evaluated during course of keratitis. The involvement of TLR2, TLR9 and DC has already been revealed for CMV infection (Puttur et al. 2016, Szomolanyi-Tsuda et al. 2006), but the underlying mechanisms demands further investigation with respect to viral keratitis.

3.3. Characteristics of CMV Keratitis

Of studies examining CMV keratitis in immunocompetent patients, only CMV endotheliitis has been reported in the scientific literature (Oka et al. 2015). Clinical presentation of CMV endotheliitis is like HSK endotheliitis as characterized by coin-shaped keratic precipitates, corneal edema, and inflammation of the anterior chamber (Alfawaz. 2013, Büyüktepe and Yalçındağ. 2020). The outermost layers of the cornea are typically unaffected (Koizumi et al. 2008). Patients may also experience high intraocular pressure, endothelial cell loss, and anterior uveitis (Chee et al. 2008). CMV endotheliitis can have four sub-classifications: linear, sectoral, disciform, and diffuse. Both linear and sectorial classes have exhibited the symptoms of localized edema (Choi et al. 2013). The keratic precipitates are arranged in a linear sequence in the former case and spread throughout the swollen cornea in the later subtype (Alfawaz. 2013, Koizumi et al. 2008). The disciform variant has circular edema in the central or paracentral cornea accompanied with many keratic precipitates inside (Alfawaz. 2013). In the diffuse endotheliitis, edema and small KPs are present throughout the entire cornea (Alfawaz. 2013). KPs have been shown to be pigmented in certain cases (Tan and Tan. 2019, Wang, S. et al. 2010). A case report has described an “owl’s eye” morphology characteristic of CMV endotheliitis whereby confocal microscopy reveals a large, highly reflective region in the nucleus encircled in a ring of low reflection (Shiraishi et al. 2007).

CMV epithelial keratitis has been reported in an immunocompromised AIDs patient. Despite oral and topical antiviral treatments, opaque, branching, epitheliopathy without ulcers were observed (Wilhelmus, K. R. et al. 1996). The patient later developed stromal keratouveitis (Wilhelmus, K. R. et al. 1996). CMV stromal keratitis has been independently observed in an AIDS patient and was alleviated with systemic ganciclovir treatment (Inoue, T. et al. 1998). CMV keratitis has also occurred post keratoplasty of even immunocompetent patients (Büyüktepe and Yalçındağ. 2020, Kandori et al. 2010b, Tan and Tan. 2019, Wang, S. et al. 2010, Wehrly et al. 1995). CMV endotheliitis presents similarly to a graft rejection or failure post keratoplasty (Fernández López and Chan. 2017). In one case, the virus superficially infected keratocytes, and the impacted corneal button was removed to cure the infection (Wehrly et al. 1995). Long-term topical ganciclovir was used successfully in a case study to prevent recurring graft failure post Descemet membrane endothelial keratoplasty (Basilious and Chew. 2019). CMV endotheliitis can occur post laser in-situ keratomileusis (LASIK) surgery, and LASIK can reactivate keratitis episodes (Tan et al. 2016). In many of these cases, early identification and treatment of CMV keratitis prevented permanent damage to the cornea.

4. VZV

4.1. Incidence and Prevalence

The alpha-herpesvirus VZV used to be an ubiquitous infection in childhood with approximately 4 million cases each year, causing about 100 deaths annually (Meyer et al. 2000, Wharton. 1996). As with HSV, there was a considerable decline in VZV cases precipitously from the year 1995 to 2000 (Seward et al. 2002). Due to a high (~90%) VZV vaccination among children during that 5-year span, the incidence of VZV have drastically reduced in recent times (Seward et al. 2002). Consistent with this hypothesis, VZV rates in 2010 were 97% lower than in 1995. The mean incidence of VZV was estimated as 4–4.5 per 1,000 person-years (Yawn and Gilden. 2013). The prevalence of VZV is increasing over time as one Minnesotan study found the mean incidence rose from 3.2 per 1,000 person-years in 1996–1997 to 4.1 in 2000–2001 (Yawn and Gilden. 2013). Post childhood, VZV infections develop symptoms primarily in adults over 50 years old (Yawn and Gilden. 2013). Less than 10% of all infections occur in the immunocompromised, and roughly 3% require hospitalization (Yawn and Gilden. 2013).

Despite reducing the incidence of VZV infections, the live attenuated vaccine still allows the virus to establish latency and diseases later in life upon reactivation such as the highly contagious chickenpox and shingles (Amlie-Lefond and Gilden. 2016, Yawn and Gilden. 2013). Severe infections can result in strokes in both children and adults (Amlie-Lefond and Gilden. 2016). In rare cases, ocular VZV infections manifest as keratitis (Khodabande. 2009). However, most symptoms are resolved with antiviral and corticosteroid therapy (Khodabande. 2009).

4.2. Pathobiology

VZV causes a sight-threatening condition known as Herpes zoster ophthalmicus (HZO) - an infection of trigeminal nerve that supplies sensation (touch and pain) to the eye surface, eyelids, forehead, and nose (Miyakoshi et al. 2012). Approximately, 50–72% patients of VZV infected individuals demonstrate the signs of ocular involvement (Pavan-Langston. 1995). VZV is transmitted between individuals primarily through respiratory aerosol or droplet inhalation (Gershon and Gershon. 2013). It can also spread via contact with infectious lesions such as those which arise during chickenpox. Primary infection commonly occurs in the upper respiratory epithelium and can produce symptoms after incubating for a period between 10–21 days (Zerboni et al. 2014). Many VZV proteins including IE62, IE63, ORF47, and ORF66 inhibit interferon responses in host cells, suppressing the immune system to promote infection (Zerboni et al. 2014). Like HSV, VZV establishes latency in the TG and travels towards the corneal tissues for replication (Laing et al. 2018). VZV also possesses tropism for T cells, especially in the tonsils, along with dendritic cells (Zerboni et al. 2014). Reactivation of VZV can cause shingles, and reactivation of the virus within the ophthalmic nerve results in HZO. HZO contributes to approximately 25% of herpes zoster infections (Shaikh and Ta. 2002).

4.3. Characteristics of VZV Keratitis

While most HZO patients present with a periorbital vesicular rash, a fraction of them develop keratitis (Shaikh and Ta. 2002). VZV infection in corneal epithelium is clinically manifested by the appearance of pseudo-dendrites or punctate keratitis, which are smaller and lack a terminal bulb-like appearance as in case of HSV-1 infection. Corneal lesions have been described as gray, elevated, and pleomorphic (Chern et al. 1998, Hu et al. 2010). Patients with VZV keratitis report symptoms including pain, tearing, and a foreign-body sensation in the afflicted eye (Miyakoshi et al. 2012). Mild inflammation is observed without the increase in intraocular pressure (Miyakoshi et al. 2012). Corneal ulcers may be found with underlying dense anterior corneal stromal infiltration, leading to opacity in the infected eye (Miyakoshi et al. 2012). VZV keratitis has been reported to have disciform distributions when present in the stroma (Silverstein et al. 1997). Furthermore, the appearance of nummular (coin-shaped) keratitis in the superficial stroma and sclerokeratitis have been reported in patients possessing VZV DNA (Denier et al. 2020). A recent case series of seven children with mean age of age years old afflicted with VZV keratitis reported four cases with nummular keratitis and three cases with deep stromal diffuse keratitis (Denier et al. 2020). Furthermore, six of the seven children experienced a relapse by a median follow-up time of 31 months (Denier et al. 2020).

5. EBV

5.1. Incidence and Prevalence

EBV is a member of the gamma-herpesvirus family that, like the other herpesviruses described above, establish a lifelong infection. Children become universally seropositive at the age of 3–4 years in developing countries and at adolescence in developed countries (de-Thé et al. 1975, Haahr et al. 2004, Hjalgrim et al. 2007). The worldwide prevalence for EBV has been reported at ≥90% (Pembrey et al. 2013, Smatti et al. 2018). Interestingly, both age and educational level were found to be associated with increased seropositivity in one 2015 study from Taiwan (Chen, C. et al. 2015). Other studies have found women, the sexually active, those living in tropical countries, and smokers as being at risk for EBV infections (Balfour et al. 2013, Higgins et al. 2007, Levine et al. 2012). As of 2019, there is no vaccine available for the prevention of EBV (Cohen. 2018). EBV commonly causes upper respiratory infections in children and mononucleosis in young adults (Nowalk and Green. 2016). EBV is one of the rare viruses which is associated with certain lymphomas and carcinomas (Nowalk and Green. 2016). Furthermore, patients undergoing organ transplants can become afflicted with post-transplant lymphoproliferative disorder, which can result in symptoms ranging from mononucleosis to lymphoma (Nowalk and Green. 2016). Less commonly, EBV has been implicated in cases of retinitis and keratitis (Lin et al. 2015, Pflugfelder et al. 1990, Sato et al. 2018).

5.2. Pathobiology

EBV is primarily transmitted via contact with salivary excretions (Nowalk and Green. 2016). EBV is the primary cause of infectious mononucleosis (IM) and persists asymptomatically for lifetime in the most adult population (Smatti et al. 2018). It has been considered as the imperative risk factor for Hodgkin’s lymphoma and certain carcinomas (Jarrett. 2003, Mohl et al. 2016, Sathiyamoorthy et al. 2016). EBV can infect the B-cells, T-cells, epithelial cells, and myocytes, and is reported to maintain latency primarily in B-cells (Mohl et al. 2016, Sathiyamoorthy et al. 2016). Unlike HSV and CMV, EBV can transform B-cell populations which can lead to Burkitt, Hodgkin’s, and other types of lymphomas (Küppers. 2003). studies. EBV also spreads via intimate contact from asymptomatic seropositive individuals who actively shed virus. Though EBV does not cause symptomatic pathologies in most individuals, it has been shown to be associated with aggressive lymphoproliferative disorders in transplant/immunocompromised patients (Niller et al. 2008). One of the most potent stimuli for reactivation post latency is B-cell receptor (BCR) activation (Lv et al. 2017). Downstream signaling leads to ubiquitination status of many proteins, including those that impact RNA processing (Lv et al. 2017). Proteins involved in RNA processing significantly regulate the EBV life cycle, and the changes induced upon BCR activation contribute to viral reactivation (Lv et al. 2017). Additionally, psychological stress and subsequent weakening of host immunity can also stimulate EBV reactivation from latency (Kerr. 2019). The flavonoid luteolin was demonstrated to inhibit EBV reactivation by reducing the activity of immediate-early gene promoters (Wu et al. 2016).

Though relatively few studies examine EBV infections in the cornea, there exist some which shed light on the signaling pathways involved in its pathogenesis. The process by which EBV infections induce an epithelial-mesenchymal transition in the corneal epithelium was found to involve TGF-β1-induced Syk and Src kinases, downstream signaling of which activates the PI3K/AKT and ERK pathways (Park, G. B. et al. 2014). Another study in human corneal epithelial cells has discovered that EBV-encoded RNA 1 and 2 stimulates TLR3 and RIG-I expression which proceeds to activate TRIF/TRAF or RIP-1 signaling (Park, G. B. et al. 2015). These pathways result in the release of pro-inflammatory cytokines which may promote corneal damage and neovascularization during EBV keratitis (Park, G. B. et al. 2015).

5.3. Characteristics of EBV Keratitis

EBV keratitis is reported in children as young as 9 years old to young adults (Palay et al. 1993, Wong et al. 1987). Interestingly, episodes are independent of other common EBV systemic disease such as mononucleosis (Palay et al. 1993, Sajjadi and Parvin. 1994, Tanner. 1954). EBV keratitis can manifest as dendritic lesions of the epithelium or deeper infiltrates in the stroma (Matoba, A. Y. and Jones. 1987, Sajjadi and Parvin. 1994, Tanner. 1954). Symptoms associated with stromal keratitis includes the following: blurred vision, photophobia, irritation of the cornea, and multifocal or ring-shaped granular anterior stromal opacities that did not breach other regions of the stroma (Matoba, Alice Y. et al. 1986, Sajjadi and Parvin. 1994). In more severe cases, EBV keratitis can cause acute corneal necrosis and neovascularization of the cornea (Park, G. B. et al. 2014). Red coloring of the eye is typically not observed (Sajjadi and Parvin. 1994). Some patients have reported soft, pleomorphic infiltrates that afflicted all regions of the peripheral cornea with little to no neovascularization (Matoba, Alice Y. et al. 1986). Recently, patients with infectious mononucleosis have exhibited nummular keratitis or chronic relapsing interstitial keratitis with neovascularization (Iovieno et al. 2020, Pinnolis et al. 1980).

6. Other Viruses

Other than the four herpesviruses discussed before, there are a few additional viruses that have been reported to cause keratitis: HHV-7, Coxsackievirus, Rhabdovirus, and Adenovirus (Inoue, Tomoyuki et al. 2010, Jhanji, Vishal, MD, FRCOphth et al. 2015, Lim et al. 2014, Madhavan et al. 2002). However, these viruses are less frequently detected in the inflamed cornea compared to HSV and CMV or even VZV and EBV. Figure 3 shows different viruses detected in corneal tissues and their tropism to epithelial, stromal, and endothelial layers. Other viruses that have been implicated in causing interstitial keratitis are human T-lymphotropic virus-1 (HTLV-1), mumps, Rubeola (measles), vaccinia, and variola viruses (Altmann et al. 2010, Bhaskaram et al. 1986, Merle et al. 2002, Semba. 2003, Shibata et al. 2016). These may be useful as differential diagnosis in rare cases of infectious keratitis when the more common viruses are absent. The degree to which each case of infectious keratitis displays precipitates, opacity, and lesions varies greatly along with their corneal distribution.

Figure 3.

Model of HSV-1 infection and reactivation from latency to cause keratitis. Upon the initial infection of the corneal epithelium, the virus travels to the sensory ganglia to establish latency. Upon reactivation, it travels in a retrograde manner back to the eye. The resulting inflammation and immune infiltration in the cornea can lead to keratitis. Other viruses and the corneal layers at which they have been shown to cause keratitis are shown at the bottom of the figure (Gueudry et al. 2013, Holland and Schwartz. 1999, Inoue, Tomoyuki et al. 2010, Lim et al. 2014, Madhavan et al. 2002, Matoba, Alice Y. et al. 1986, Oka et al. 2015, Vislisel. 2014).

7. Clinical Diagnosis

Diagnosis of keratitis is profoundly dependent on a thorough slit lamp examination and subsequent laboratory testing. Corneal samples can be obtained from patients using a swab, spatula, scalpel, or a spud (Leck. 2009) and this specimen is then Gram stained and cultured. Gram stains detect the causative bacteria (60–75% accuracy) and fungi (35–90% accuracy) (Gopinathan et al. 2009). In contrast to bacterial or fungal keratitis, the diagnosis of viral keratitis is primarily made based on the clinical examination. Characteristic dendritic lesions are observed in many cases, a classic symptom of herpes simplex keratitis (Azher et al. 2017, Darougar et al. 1985). KPs with coin shapes are an analogous reference for CMV endotheliitis or VZV/EBV stromal keratitis in rarer cases (Alfawaz. 2013). Furthermore, keratitis could be secondary to another manifestation of viral disease such as periorbital vesicular rashes during VZV infection or mononucleosis during EBV infection. Other tests in the physical examination such as the Snellen Acuity Test or the penlight exam can give the clinician additional information with which to make diagnoses. When keratitis is suspected, a fluorescein stain can be used to assess damage to the epithelium (Wolffsohn et al. 2017). The dye will stain the corneal stroma, revealing lesions or gaps in the epithelial layer (Wolffsohn et al. 2017).

While the clinical presentation is sufficient for differential diagnosis, the viral genome should be isolated from the anterior chamber fluid and subsequently tested using PCR (Kandori et al. 2010a). Given the lack of pathogens in normal aqueous humor, a positive test is often sufficient to clinch the diagnosis so long as the test is performed during the onset of infection or reactivation (Alfawaz. 2013, Kandori et al. 2010b). The sensitivity of PCR declines later during infection (Alfawaz. 2013, Jd et al. 2006). For this reason, a negative test does not necessarily mean the virus is absent (Jd et al. 2006). PCR is highly specific to each virus based on the primers used. For example, in recurring infections, positive PCR results for EBV DNA and negative results for other viruses such as HSV, CMV, and VZV is considered sufficient evidence to confirm the diagnosis (Reinhard. 2009).

Aqueous humor can also be analyzed for the presence of antibodies using an enzyme linked immunosorbent assay (ELISA), and the Goldmann-Witmer coefficient (GWC) can be calculated (Alfawaz. 2013, Wang, Z. J. et al. 2016). A GWC > 3 is considered positive for antibody production (Ys et al. 2010). Antibody testing via Goldmann-Witmer coefficient (GWC) assays is another useful method to confirm the diagnosis as it can be performed at any time during the infection (Westeneng et al. 2007). For some common viruses, even healthy eyes will be positive for IgG antibodies. For example, EBV IgG antibodies are common in most healthy eyes due to its high prevalence in the population. Therefore, positive tests for EBV IgM antibodies are used to confirm a diagnosis of primary EBV keratitis (Reinhard. 2009).

PCR and GWC assays complement each other as diagnostic tools. One study found that, in cases of infectious keratitis, only 43% of infected specimens were positive with both PCR and GWC (Jd et al. 2006). 48% were positive only with GWC, and 9% were positive only with PCR (Jd et al. 2006). Another study examined the sensitivity of immunoblotting, GWC, and PCR for the diagnosis of ocular toxoplasmosis. Each technique used in isolation had a sensitivity of 81%, but the combination of any two increased the sensitivity to roughly 90% which rose again to 97% when all three were used (Fekkar et al. 2008). PCR of corneal scrapings can also be used to confirm the initial diagnosis (Austin et al. 2017, El-Aal et al. 2006, Inoue, Tomoyuki et al. 2010, Kandori et al. 2010a), while immunofluorescence tests are rarely used as their sensitivity is much lower than the other methods. Figure 4 shows a flowchart describing the differential diagnosis of viral keratitis.

Figure 4:

Flow chart showing a generic process to differentially diagnose viral keratitis from other types of ocular inflammatory diseases.

8. Treatment of Viral Keratitis

8.1. The Herpetic Eye Disease Studies

To evaluate the ability of corticosteroids to treat HSK, a randomized, double-masked, clinical trial known as the Herpetic Eye Disease Study (HEDS) was conducted. In 1994, the results were published and demonstrated that the adjuvant corticosteroid therapy in addition to trifluridine reduced the likelihood of developing stromal keratouveitis by 68% (Wilhelmus, Kirk R. et al. 1994). The HEDS found that corticosteroids significantly reduced the duration of stromal keratitis symptoms from 72 days to 26 days on average when compared to a placebo (Austin et al. 2017). No detrimental effect on visual outcome occurred with the treatment group at 6 months post randomization (Wilhelmus, Kirk R. et al. 1994). Shortly after, the HEDS-II studies were conducted to examine the role of acyclovir in treating herpetic eye disease. The HEDS-Epithelial Keratitis Trial found oral acyclovir, taken 400 mg at 5 times per day for three weeks, offered no additional benefits on top of topical trifluridine therapy in preventing the development of HSK (Barron et al. 1994). The HEDS-Acyclovir Prevention Trial found that a year of daily acyclovir administration reduced the relapse rates for ocular and orofacial HSV diseases (Wilhemus et al. 1998). These landmark studies made prophylactic use of acyclovir and adjuvant corticosteroids to prevent episodes of HSK staples of modern treatment.

8.2. Nucleoside analogs and glucocorticoids

Treatments for viral keratitis can be generally divided into two subcategories: antiviral nucleoside analogs and glucocorticoids (Table 2). One of the most common therapies is acyclovir (ACV), a nucleoside analog which acts as a competitive inhibitor of the viral DNA polymerase (Koganti et al. 2019a). Its affinity for viral DNA polymerase is roughly 200 times greater than its affinity for the host polymerase which contributes to its high specificity. ACV is generally well tolerated and an effective treatment for viral keratitis (Whitley, R. J. and Gnann Jr. 1992). It can be administered orally as topical formulations have a lower bioavailability on the cornea (Wilhelmus, Kirk R. 2015). ACV can treat active infections or used as prophylactic to prevent recurring episodes of reactivation. There are multiple analogs of ACV on the market: cidofovir, brincidofovir, ganciclovir, and valacyclovir, and trifluridine (Table 2). Each possesses a unique set of benefits and disadvantages compared to traditional ACV therapy. Acyclovir or an analog can be taken orally and daily to prevent recurrences of keratitis (Wilhemus et al. 1998). Prophylactic acyclovir therapy was reported to reduce recurrences of ocular disease by 32% as compared to 19% for patients taking placebos (Wilhemus et al. 1998). These findings were replicated in additional studies (Miserocchi et al. 2007, Uchoa et al. 2003).

Table 2:

Standard and non-standard medications for the treatment of viral keratitis. Standard medications are the first line of defense and given during the initial treatment of viral keratitis. Non-standard medications are given when the standard medications show little efficacy in controlling the disease.

Name:	Type of treatment modality:	Dosing Frequency:	Mechanism of action:	Benefits:	Disadvantages:
Cidofovir	Standard	5 mg/kg/week intravenously for two weeks. Then every alternate week.	Antiviral	Active against multiple viruses including adenoviruses and herpesviruses. More effective than gangiclovir	Nephrotoxicity when taken orally
Brincidofovir	Standard	5 mg/kg/week intravenously for two weeks. Then every alternate week.	Antiviral	Lipid ester form of cidofovir and can be taken orally	Diarrhea observed in pediatric patients
Ganciclovir	Standard	5 mg/kg every 12 hours	Antiviral	3% gel can be applied topically to avoid systemic usage	-
Acyclovir	Standard	800 mg five times daily for seven days	Antiviral	Taken orally to prevent active infection	Topical gels are not as effective
Valacyclovir	Standard	1000 mg three times daily for seven days	Antiviral	Reduced dosage when compared to acyclovir	-
Trifluridine	Standard	1% drop every 2 hours	Antiviral	Works more efficiently than acyclovir when used topically	Causes ocular irritation and inflammation during prolonged use
Prednisolone	Standard	0.1 % ophthalmic drops four times daily	Glucocorticoid	Reduces inflammation and pain	Causes blurred vision during administration. Should be administered alongside antiviral treatment
Dexamethasone	Standard	0.1 % ophthalmic drops four times daily	Glucocorticoid	Reduces inflammation and pain	Causes blurred vision during administration. Should be administered alongside antiviral treatment
Loteprednol	Standard	0.5 % ophthalmic drops four times daily	Glucocorticoid	Reduces inflammation and pain	Causes blurred vision during administration. Should be administered alongside antiviral treatment
Cyclosporine	Non-standard	0.05 % ophthalmic drops two times daily	Anti-inflammatory	Reduces inflammation and pain
Fluorometholone	Non-standard	0.1 % ophthalmic drops four times daily	Glucocorticoid	Effective against keratoconjuctivitis and episcleritis	Causes blurred vision during administration.
Rimexolone	Non-standard	1 % solution drops two times daily	Glucocorticoid	Can also be used to treat anterior uveitis, conjunctivitis in addition to keratitis	Causes blurred vision and foreign body sensation of eye

However, the nucleoside analogs possess weaknesses that limit their effectiveness. They are susceptible to drug resistance, especially in immunocompromised patients (Gateley et al. 1990, Jiang et al. 2016). Conversely, the prevalence of acyclovir-resistance strains of HSV in immunocompetent patients is approximately 0.1–3% (Piret and Boivin. 2011, Pottage and Kessler. 1995). A downside of long-term prophylactic usage of ACV is the increased chance of developing resistant, recurrent HSK (van Velzen et al. 2013). ACV-resistant strains of VZV in immunocompetent patients are even rarer as only one case has been reported (Gueudry et al. 2013). Resistance can be diagnosed when the patient’s condition remains constant or deteriorates despite a course of ACV (7–10 days for HSV and 10–14 days for VZV) and a viral infection is confirmed (Ogura et al. 2017, Toriyama et al. 2014). The best confirmation of ACV-resistance is a plaque reduction assay with an EC₅₀ value > 9 μM (Piret and Boivin. 2011). Recent advances have shown that qPCR can be a faster, easier alternative to the plaque reduction assay (Virók et al. 2017). When clinical treatment failure with nucleoside analogs is confirmed, alternatives to acyclovir should be prescribed. Foscarnet has been shown to be an effective treatment for acute retinal necrosis caused by an ocular infection with an ACV-resistant strain of HSV (Ogura et al. 2017). Additionally, topical trifluorothymidine was reported to improve a patient’s necrotizing keratitis due to ACV-resistant (Toriyama et al. 2014). Other alternatives to ACV include topical or intravenous administrations of cidofoir or imiquimoid (Martinez et al. 2006, Ronkainen et al. 2018). While topical administration of these therapies reduces the likelihood of resistance emerging, it remains a significant problem. Furthermore, acyclovir and trifluridine can cause nephrotoxicity and ocular disorders with extended usage (Carmine et al. 1982, Raymond Fleischer and Michael Johnson. 2010, Yildiz et al. 2013). Pregnant women are also discouraged from taking acyclovir due to the chance of chromosome mutations occurring.

Glucocorticoids are also prescribed for the treatment of keratitis. Examples include prednisolone, dexamethasone and loteprednol. They reduce inflammation and pain at the site of infection, but do not target the viral infection directly (Austin et al. 2017). For this reason, these drugs are often provided as adjuvant therapies. Glucocorticoids may cause blurry vision during administration and occasionally foreign body sensation in the eye. Given the drawbacks to both traditional antivirals and corticosteroids, there is a need for effective, novel therapies to viral keratitis.

8.3. Surgical Interventions

Surgical interventions are commonly used in patients to address corneal melting, a complication of necrotizing HSK or neurotrophic herpes keratitis (Tuli et al. 2018). Corneal gluing using a cyanoacrylate-based adhesive can be used to seal small, < 3 mm wide perforations in the cornea (Rana and Savant. 2013). Amniotic membrane transplants (AMTs) can both help neurotrophic ulcers heal and provide growth factors to promote migration of epithelial cells (Chen, H. J. et al. 2000). Furthermore, AMTs combat the thinning of the cornea and show promise in increasing corneal thickness for a year post operation (Nubile et al. 2011). Corneal transplantation or keratoplasty makes up yet another surgical intervention to improve visual acuity in keratitis patients (Serna-Ojeda et al. 2017). Up to 20% of penetrating keratoplasty operations are performed to manage cases of herpes keratitis, while roughly 5% of operations are due to corneal scarring (Wang, J. et al. 2011). Keratoplasty provides tectonic support to the cornea, but significant rates of rejections make it difficult for operations to provide long-term relief (Lyall et al. 2012). Corneal vascularization that often occurs during HSK raises the chances of rejection and even recurrent infections in the transplanted cornea (Kuffova et al. 2016). Oral acyclovir is often given after keratoplasty to reduce the rates of recurrent infection (Kanclerz and Alio. 2020). 6 months of ACV therapy post-penetrating keratoplasties is well supported in the literature although its usage for lamellar keratoplasties is less clear (Kanclerz and Alio. 2020).

Anterior lamellar transplants have better outcomes than penetrating keratoplasty operations, but they are inferior when patients have deep scarring of the cornea or endothelial involvement (Tuli et al. 2018). For a more comprehensive guide to other surgical interventions in herpetic keratitis patients, please refer to this excellent 2018 review (Tuli et al. 2018).

9. Experimental Therapies

A variety of experimental therapies that exist for HSV keratitis, each of which focuses on a different stage of the viral life cycle (Koganti et al. 2019a). For viral entry, there are three overarching groups of treatments: aptamers, cationic peptides, and humanized antibodies. An aptamer is a short oligonucleotide sequence that can assume a variety of structures and functions (Zhu et al. 2015). RNA aptamers targeting the viral glycoprotein D (gD) have been shown to reduce entry in vitro as measured by a plaque assay (Gopinath et al. 2012). DNA aptamers are cheaper to produce, more stable, and have shown success in inhibiting HSV-1 in ex vivo and in vivo corneal models (Yadavalli et al. 2017). However, the efficacy of aptamers in keratitis models specifically has not been demonstrated (Yadavalli et al. 2017). Similarly, humanized monoclonal antibodies have shown promising in vitro and in vivo results, but studies have not tested their efficacy in keratitis models yet (Bauer et al. 2017, Krawczyk et al. 2013).

Cationic peptides were reported to show efficacy in treating ocular HSV-1 infections. HSV-1 must bind to the anionic heparan sulfate (HS) prior to entering host cells. Positively charged peptides can bind to cell surface HS and thus interfere with viral attachment. The G1 and G2 classes of cationic peptides have repeatedly demonstrated promising outcomes using in vitro, ex vivo, and in vivo corneal models (Ali et al. 2012, Bultmann et al. 2007, Jaishankar et al. 2016, Park, P. J. et al. 2013, Tiwari et al. 2011). G1 peptides have alternating positive charges, while G2 peptides have repetitive stretches of cationic amino acids. Certain cationic peptides, such as retrocyclin-2 and TAT-Cd, have reported to be beneficial in reducing the severity of symptoms in a keratitis murine model when administered prophylactically or a few hours post-infection (Brandt et al. 2007, Jose et al. 2013). However, their efficacy deteriorates when applied 24 hours post-infection or longer (Brandt et al. 2007, Jose et al. 2013). Thus, they may play a limited role in treating patients that already have developed HSK.

While traditional therapies consist of nucleoside analogs, CRISPR/Cas9 technology may fulfill a critical niche in current treatment: the removal of latent viral genomes from the host, therefore preventing recurring keratitis episodes (Koujah, Lulia et al. 2019). Targeting viral genetic elements critical for productive and latent herpesvirus infection by CRISPR/Cas9 guided mutation has been successfully employed (200, 201, 202). In 2014, Wang and Quake succeeded in harnessing CRISPR/Cas9 to remove latent EBV genomes from patient-derived cells (Jianbin Wang and Stephen R. Quake. 2014). A unique feature of most of the HHV is their ability to encode microRNAs (miRNA), which facilitate viral persistence by immune evasion and perturbation of host antiviral defense (203, 204, 205, 206). Studies from our lab and others have shown marked accumulation of viral miRNAs in various diseases including cancers, oral infections, transplant rejections, etc. (207, 208, 209, 210, 211, 212, 213). Harnessing CRISPR/Cas9 to mutate miRNAs can impair viral life cycle switching, modulation of host cellular responses and immune evasion. In vitro studies using a latently infected gastric carcinoma cell line NSU-719 have highlighted promising potential of CRISPR/Cas9-mediated editing of EBV genome by targeting miRNAs BART5, BART6, and BART15 (200). Further development of this technology could lead to similar results in murine models and eventually humans. Given that more than two-thirds of patients afflicted with HSK will experience at least one reactivation episode 10 years post the primary infection, safe and reproducible therapy that reduces the likelihood of recurrence is of great value.

Concerning inhibiting viral translation, the PDK1 inhibitor BX795 may be utilized (Clark et al. 2009). BX795 inhibits the phosphorylation of Akt at Ser473, and the downstream effects result in the inhibition of viral protein synthesis (Jaishankar et al. 2018, Yadavalli et al. 2019). The efficacy of B795 has been demonstrated in vitro, ex vivo, and in vivo models of the cornea (Jaishankar et al. 2018, Yadavalli et al. 2019). This drug is effective when administered either therapeutically or prophylactically. These antiviral effects are coupled with a lack of toxicity at an effective dosage (Yadavalli et al. 2019). Given these attractive characteristics, BX795 may develop into an alternative to nucleoside analog therapy, extremely useful in patients with resistance to acyclovir or its derivatives.

In a recent study, an activated carbon-based drug delivery system termed DECON (Drug Encapsulated CarbON) was used to deliver acyclovir in a sustained manner. This delivery system was able to replace currently prescribed three-times per day dosage requirement to once every alternate day. DECON was also shown to trap free viruses and in the process release acyclovir from its nanopores. A recent review article thoroughly discusses the emerging therapies for ocular viral infection (Koganti et al. 2019a).

10. Conclusions

Keratitis is a highly prevalent ocular disorder worldwide, and viruses are the most common etiological agents of infectious keratitis. HSV, CMV, VZV, and EBV are frequently detected in patients with infectious keratitis. However, the bulk of the literature on viral keratitis concerns HSV and CMV keratitis. Keratitis resulting from infection with these viruses can have varied clinical manifestations unique to each virus. Therefore, rapid and accurate diagnosis is essential in disease management and prevent permanent damage to the cornea. Development of multiplex PCR or antigen-based technologies will facilitate differential diagnosis of VK and identify which virus(es) mediate underlying pathogenesis. Immune cells are critical in containing virus and immunocompetent individuals successfully thwart virus by eliciting potent antiviral responses. However, recurring diseases with adverse outcomes is often reported in immunocompromised/immunosuppressed individuals. Nonetheless, knowledge gaps remain in our understanding of immunomodulation by viruses causing keratitis and future studies focusing on delineating immune mediators in VK will reveal new insights. While nucleoside analogs like acyclovir are effective treatments, drawbacks of these drugs are reported, especially in immunocompromised patients. Identification of host pathways and immune cells targeted by viruses will enable development of novel therapeutic treatment modalities for patients suffering from viral keratitis.

Highlights.

1. VK is the most prevalent form of infectious keratitis with over 1 million annual cases of herpetic keratitis alone.

2. VK can cause four different types of keratitis: epithelial, stromal, endothelial, and neurotrophic keratopathy.

3. Necroptosis of the corneal tissue and immune cell infiltration contribute to the viral keratitis.

4. Nucleoside analogs and glucocorticoids are primary treatment options for patients with VK.