Ocular Viral Diseases of Animals

MICROBIOLOGY – VIRUSES & ANTIVIRAL THERAPY

David J. Maggs BVSc (hons), DACVO Professor, Comparative Ophthalmology University of California- Davis VM-SRS, 2112 Tupper Hall Davis, CA 95616 [email protected]

Acknowledgements: The following notes are developed from an original set written by and generously gifted to me by my friend and mentor Mark Nasisse, DVM, DACVO. I have modified and updated them biennially; however, the core content (as well as my fascination with FHV-1) is due entirely to Mark and I am deeply indebted to him. I am also thankful to Dr. Eric Ledbetter for his assistance with the section on canine herpesvirus. Throughout, any errors are mine and I would be grateful if you brought them to my attention.

Vi'rus n. slimy liquid or poison

INTRODUCTION From the perspective of complexity and variability of pathogenic mechanisms, few ocular diseases are more fascinating than those initiated by viral infections. Because most is currently known about ocular diseases due to feline herpesvirus type 1 (FHV-1) and because it utilizes so many of these pathogenic mechanisms, most attention will be paid to this agent and its disease manifestations. A brief discussion of some historic details, general principles of viral pathogenesis, and specific mechanisms of tissue injury known to induce ocular lesions in animals is provided initially.

A little History Dorland describes a virus as “a minute infectious agent not resolved in a light microscope, characterized by a lack of independent metabolism and the ability to replicate only within living host cells.” This apparently simple sentence belittles the centuries of scientific endeavor that led up to proving the existence of viruses and establishing those defining characteristics. A brief review of those critical discoveries, especially in context of the understanding (and undoubtedly some ongoing misunderstanding!) we have of viruses today, is fascinating…

“A minute infectious agent not resolved in a light microscope, characterized by a lack of independent metabolism and the ability to replicate only within living host cells” Dorland’s Medical Dictionary

The relatively small size of viral particles meant that they were not truly “seen” until 1939. It took William Henle - an avante garde scientist and, by the way, the person after whom the Loop of Henle is named - to propose in 1840 that infectious particles too small to see with the light microscope may not only exist but could answer some of the dilemmas with which he was presented. Of course, these were seen as heretical thoughts and not well received! However, not long after this, people with names now familiar to us, such as Pasteur, Koch, and Lister added irrefutable evidence to Henle’s theorem – but for much larger infectious particles - bacteria. Illnesses we now know as caused by viruses remained an enigma for at least another half a century.

Around the turn of the 20th century, using what became known as tobacco mosaic virus, a number of researchers proposed the existence of a “filterable infectious agent” that caused discoloration of the commercially important tobacco plant. The finding that this agent could reproduce only in the presence of living tissue as opposed to bacteria, which could replicate in cell free media was a subsequent and equally critical discovery. Still these proposed agents could not be observed and so were considered to be fluids. Hence the first name for viruses – “contagium vivium fluidum” and the current name “virus” - derived from the Latin word for “slimy liquid or poison”. In 1898, the first animal virus– foot and mouth disease (FMD) virus was discovered. The first human virus – yellow fever virus – was not identified until 1901. However, it was another 40 years before viruses were actually

“seen” for the first time - using electron microscopy (1939). And so some of the early mysteries surrounding the existence and character of viruses began to be unraveled…

It is tempting to believe that these were all experiments conducted in an earlier time when instrumentation and techniques were “primitive” and that these sorts of misunderstandings were to be expected. That couldn’t happen now, could it?

“PCR made it easier to see that certain people are infected with HIV, and some of those people came down with symptoms of AIDS. But that doesn't begin even to answer the question, “Does HIV cause AIDS?” The mystery of that damn virus has been generated by the $2 billion a year they spend on it. You take any other virus, and you spend $2 billion, and you can make up some great mysteries about it too.” Kary Mullis [1993 Nobel Laureate and inventor of the polymerase chain reaction (PCR)]

“It’s the virus – stupid!” David Ho (Scientific Director, Aaron Diamond AIDS Research Center, NY and Time Magazine’s 1996 Man of the Year)

Pathogenesis of viral infections Pathogenesis is the means or mechanism by which a virus causes disruption of function and sometimes death within a cell, (and sometimes a tissue or the whole organism) that it infects. It is very important that we realize this can often not cause signs of disease in animals or sometimes even symptoms in human beings. This will become critical when we discuss feline herpesvirus type 1 (FHV-1) pathogenesis in cats. In other words, there is an extremely delicate balance that exists between health and disease and between viral virulence and host defenses. Disease results when the virus “wins”. However, with many viruses, we must also be open to the fact that excessive host defense mechanisms may not always be an advantage and can cause immunopathology or proliferative disease. Think of feline infectious peritonitis or infectious canine hepatitis (more on them later).

Recall that, by definition, for a virus to replicate, it must enter a cell and co-opt some of the host cell’s synthetic machinery and energy sources. This makes specific drug targets and organism clearance more difficult At least a superficial understanding of virus-cell interactions is therefore critical. However, for clinicians, it can sometimes be difficult to understand various cellular and sub-cellular events in virus-cell interactions. One scheme that may assist the clinician involves drawing analogies between similar cellular and “whole host” events (Table 1). Each step in the complex series of events that describes viral entry into, replication within, and release from a cell (or from the host organism) can then be considered a barrier to disease production. When the virus successfully overcomes all barriers or if the host has deficiencies in some barriers, then infection (but not always disease) results. Likewise, each step at the host or cellular level can be seen as a potential target for control or therapeutic measures. Therefore a basic understanding of these steps will assist the clinician in suspecting and diagnosing viral etiologies in their patients, implementing therapeutic and disease control methods, and offering clients an accurate prognosis.

Table 1. Comparison of the stages in viral pathogenesis at a “whole animal” and a cellular (and subcellular) level

Whole Animal Cellular/Subcellular Entry into host Adsorption Primary replication Penetration Spread throughout host Uncoating Cell and tissue tropism Transcription Host immune responses Translation Secondary replication Replication Cell injury Assembly Persistence/Latency/Genomic integration Release

At the individual patient-specific and population-specific (think “feline shelter” or “herd health”) levels, there are a number of distinct but very closely related clinical considerations:

• What is/are the route/s of infection? • What are the preferred sites (cells, tissues, organs) of primary/secondary replication? • How does the virus spread within the host (cell to cell, axonal, hematogenous, etc.)? • What are the major means of immunity/defense (humoral, cell-mediated, mucosal, etc.)? • Are some forms of immune response harmful to the host (immunopathology, immunosuppression)? • Does the virus establish persistent or latent infections? For how long? • How does the virus cause cellular injury (dysfunction) or death? • How is the virus excreted or “shed”? • How stable is the virus in the environment? • Etc.

At a cellular level, infection occurs when the viral particle (virion) finds and attaches (adsorbs) to an appropriate cell membrane receptor, penetrates into that cell, exposes (uncoats) its nucleic acids for transcription and protein production, replicates itself, and then assembles new virus particles ready for release from that cell and potential infection of another cell either nearby or at a secondary location. An essential consideration for the clinician is what injurious events occur intracellularly during this reproductive process?

Stated in these ways, it becomes more clear that “successful” infection of the whole animal (rather than just a “few” cells) with disease production can be considered uncommon and, when it occurs, a reflection of how well evolved that virus is for infection of that host or how susceptible that host is to infection by that virus.

The critical steps for clinicians are cell entry, viral transcription, and virally mediated pathology because these are what define host susceptibility, viral virulence, nature of the disease produced, sites of action for potential treatments, and means of control/prevention. Therefore a little time will be spent on these.

Cell Entry Viral entry, beginning with virus-cell receptor binding and continuing with cell membrane penetration, is essential if the virus is to gain access to host cell synthetic machinery. Enveloped viruses uncoat (lose their envelope) during penetration due to fusion of the viral envelope and the cell membrane. It is assumed that viruses have evolved to use cell receptors that are present for other reasons and that this, in part, also determines species specificity and cell tropism. Like all interactions with cell membrane receptors, this is undoubtedly a complex series of physical, electrical, and chemical interactions, and may involve multiple receptors and/or permissive “cofactors”. The old “key and lock” analogy is undoubtedly useful but represents a gross over-simplification of this process. Various host defense mechanisms and potential therapeutic endeavors could target this aspect of host-virus interaction. For example, lactoferrin (present in tears) exerts an antiviral effect against FHV-1 and many other viruses, which is quite distinct from its iron-binding antibacterial effect. Rather, it appears to be due to inhibition of viral adsorption to and subsequent entry into the host cell.1,2

Viral transcription Transcription of viral DNA utilizes host enzymes (“machinery”) and nucleic acids (“building blocks”). This is why viruses are incapable of independent existence and are obligate intracellular pathogens. Some viruses simultaneously down-regulate host cell transcription to ensure better access to this “machinery” and the “building blocks”. Undoubtedly a multitude of complex and very strictly controlled steps are responsible for the success of this process. Although only a few of these are well defined or understood, some have been useful targets for pharmacological intervention. This is discussed more fully in the later section on antiviral therapies. This intimate and intricate relationship between viral and host cell functions during viral transcription results in antiviral drugs being generally more toxic than most antibacterial drugs because they tend to have “overlapping” negative effects on host machinery as well as the viral machinery they are designed to target.

Virally-mediated pathology Perhaps of most interest to the clinician are the mechanisms by which viruses cause cell injury or dysfunction (and ultimately illness within a patient). Figure 1 is intended to summarize the most frequently encountered of these

mechanisms. In summary, once a virus enters a cell, one of three initial outcomes is possible. The infection may result in cell lysis (and death). This occurs as a result of successful viral replication and coincides with the release of viral progeny from the host cell and therefore is also referred to as a “productive” infection. This is typical of a primary infection with one of the alphaherpesviruses such as FHV-1 or canine herpes virus (CHV) and it is the cell lysis that causes the corneal and conjunctival ulceration visible clinically. Alternatively, viral replication may occur at an extremely low rate, with no or undetectable release of mature virus particles. In this case cell lysis is unlikely however viral antigen production may continue and “drive” an immunological reaction that may be equally or more devastating than cytolysis. This is believed to be responsible for some of the more chronic outcomes of alphaherpesvirus infections such as stromal keratitis in cats (and human beings), and is discussed more fully in that section of these notes. The final alternative is that viral replication is stopped completely (i.e., a non-productive infection results). In a non-productive infection, 3 further outcomes are possible: (1) firstly, the cell may eliminate the virus and continue as before, (2) alternatively the cell may eliminate the virus but subsequently die, (3) the third outcome is of most interest pathologically and results when viral DNA is “sequestered” within the cell with either subsequent oncogenic transformation (e.g., FeLV), or reactivation (e.g., FHV-1).

With that brief introduction to how viruses infect (and sometimes injure) host cells (and sometimes tissues and organs), let’s discuss some specific examples of ophthalmic importance. Unashamedly, the majority of these notes are spent discussing FHV-1, the disease syndromes it causes, and antiviral therapies directed at it.

FELINE HERPESVIRUS TYPE 1 (FHV-1, Feline Rhinotracheitis Virus)

VIRAL FEATURES AND EPIDEMIOLOGY Feline herpesvirus-1 is a DNA virus taxonomically classified in the Herpesviridae family; subfamily Alphaherpesvirinae. The hallmark biological features of the alphaherpesviruses are:- o Relatively “tight” species specificity o A short reproductive cycle o Rapid spread in culture o Efficient destruction (lysis) of infected cells o Capacity to establish latency (especially in sensory ganglia)

While FHV-1 persists for life in latently infected cats, it is extremely labile in the environment. It survives approximately 12 hours in dry environments and approximately 18 hours in more moist conditions and is susceptible to most disinfectants and to desiccation. With respect to multidose vials in your clinic, it is rapidly killed by fluorescein stain and proparacaine; however FHV-1 could be recovered from eyewash for 5 days.3 Because of this short environmental survival, the major route of infection is via direct transfer of virus-containing macrodroplets between mucosal surfaces (oral, nasal, conjunctival). Of importance to those of us needing to isolate such patients within our hospitals, is the observation that macrodroplets can be sneezed up to 1.3 meters. However it is believed that cats do not form aerosols of any of the respiratory viruses during normal respiratory movements. Likewise, due to the environmental instability of the virus and its susceptibility to most disinfectants, the importance of fomites (most notably our own hands!) can be limited by practicing adequate hygiene between patients.

The structure of FHV-1 is similar to that of other alphaherpesviruses. It consists of 136 kilobase pairs with a long component (104 kb) and short component (30 kb).4-6 The nucleocapsid is 1200 Å in diameter and surrounded by a protein coat (tegument) that is enveloped by glycoproteins. The best studied of all FHV-1-encoded enzymes is thymidine kinase (TK). The FHV-1 TK gene is located approximately 40% along the long unique component of the FHV-1 genome.7 The location (but not the sequence) of the TK gene is reasonably conserved among alphaherpesviruses. Restriction endonuclease analysis reveals whole-genome cleavage patterns to be well conserved among FHV-1 strains, suggesting that there is little diversity in the FHV-1 genome on a worldwide basis.8,9 On the basis of restriction analysis, 3 major FHV-1 genotypes appear to exist (C7301, F2 and C7805).10,11 The C7301 appears to be the most common type; represented by 64 of 78 isolates in one study.10 Little variability is seen in pathogenesis of various viral strains and some of this variability may actually represent different host immune responses to viruses of similar pathogenicity.12

The natural host range of FHV-1 appears restricted to domestic and wild felids.9,13-17 This is likely a function of virus entry to the cell and mediated by surface receptor interactions.18,19 An estimated 75-97% of the world’s cat population is seropositive, and the virus is considered to be responsible for 45% of all upper respiratory infections.20- 22

Viral Replication Viral replication occurs primarily within epithelium of the upper respiratory tract and eye; principally the 23 conjunctiva, nasal turbinates, and nasopharynx; with more limited replication in corneal epithelium. Like species- specificity, tissue-specificity is likely mediated by virus-host cell surface receptor interactions.18 The initiation of clinical disease is independent of the presence of conjunctival or upper respiratory flora as shown by infection of germfree cats;24 and a study of anaerobic bacterial involvement in ulcerative keratitis failed to show coincident presence of FHV-1 DNA [as assessed by the polymerase chain reaction (PCR)] or cultivable virus [as assessed using virus isolation (VI)]).25 However, the role of secondary bacteria in the severity and progression of ulcerative corneal disease should not be under-estimated.

At the cellular level, adsorption occurs between viral surface glycoproteins and specific cell receptor/s. Evidence shows that cell surface receptors containing heparan sulfate are critical for adsorption of FHV-126 and other related alphaherpesviruses. Subsequent events include uncoating, penetration of the viral capsid into the infected cell, release of viral DNA from the capsid, viral gene expression, transcription, synthesis of viral proteins, and either establishment of latency or assembly of viral progeny, and egress from the infected cell. It is during the process of leaving the cell that the viral progeny obtain their envelope and induce cytolysis.

At least 17 FHV-1-specific peptides and 7 glycoproteins have been identified.18 Gene expression during FHV-1 replication occurs in 3 phases; immediate early (IE), early, and late. Products of IE genes are important for the regulation of viral reproduction and latency, and are capable of transactivating heterologous viral and cellular proteins. FHV-1 has 3 major immunogenic glycoproteins (gp) that are present predominantly in the viral envelope.27,28 Of these, gD (originally called gp60) is the viral hemagglutinin that elicits HI antibodies.29 In addition to being major targets for neutralizing antibodies, viral glycoproteins, particularly gC, are essential for attachment to cells and subsequent adsorption.30-32 Use of small interfering RNAs targeting gD reveal how critical it is for infectivity.33-35 The article by Maeda et al18 provides a more thorough review of properties and functions of FHV-1 glycoproteins.

Latency During acute replication within peripheral epithelial cells, virus particles ascend axons of sensory neurons to the associated ganglia where they establish lifelong latency in the majority (probably about 80%) of cats.36 In the case of ocular, nasal and facial epithelium, the regional sensory ganglion is the trigeminal ganglia.37-42 The definition of latency has evolved as new techniques have become available. Originally, viral latency was described as a period of total absence of clinically or histologically detectable inflammation. Virologically, latency describes a period during which virus cannot be cultured; i.e., a non-productive, non-lytic phase. Molecular biology techniques have permitted investigation of viral transcription, which is extremely limited during latency. The major gene expressed during this period is the “latency-associated transcript” (LAT). FHV-1 LAT has been characterized41 and identified within trigeminal ganglia but not cornea of cats without signs of ocular disease37 suggesting that although virus can be found in normal feline corneas, true corneal latency has not yet been proven. However, the relatively consistent detection of virulent and viable virus in the corneas of normal cats (especially within shelters)43 has important clinical implications for corneal transplantation and diagnostic testing in cats.

Periodic reactivation of latent FHV-1 occurs after stress; either physiological (e.g., re-housing, transport, parturition/lactation, etc.), or pharmacological (corticosteroid or epinephrine administration).36,44 Corticosteroid administration has been advocated as a method of detecting carriers in endemic populations.44 The reactivated virus is believed to descend the same sensory nerve axons to reach the peripheral epithelial tissues again; the so-called “round trip theory”. Clearly this provides a highly adapted and successful means of perpetuating an environmentally labile virus within a host population. This viral reactivation may be associated with varying degrees of recrudescent disease; alternatively viral shedding can occur in the absence of clinical signs. Note that the virus is reactivated while the disease is recrudescent. The molecular aspects of reactivation and recrudescence are reviewed by Maes.45

The so-called “round trip” axonal transport theory led to the assumption that viremia is unimportant in the pathogenesis of FHV-1 in cats. However, in other species infected with herpesviruses (and in cats infected with viruses other than FHV-1), viremia allows dissemination of the virus to all organs, and can cause abortion, encephalitis, disseminated intravascular coagulation, and generalized organ failure.46-49 In fact, FHV-1 DNA has been detected in the blood of cats exhibiting clinical signs of herpetic disease50-52 as well as apparently normal cats,52,53 and in distant sites such as bone or tendon in some cats that underwent mucosal infection.54 However, these have not been consistent findings.55 We have examined this in cats undergoing experimental primary infection or natural disease presumed to be due to herpetic reactivation.56 In that study,56 FHV-1 DNA was detected at least once in blood of all cats between 2 and 15 days after inoculation. However, FHV-1 DNA was never detected and live virus was not isolated (cultured) from blood mononuclear cell samples from any adult shelter cat undergoing presumed recrudescent disease. Therefore it appears that a brief period of viremia may be important in the pathogenesis of primary herpetic disease but maybe not during recrudescence.56

Persistent viral state An additional viral state distinct from latency (without disease), and primary or recrudescent cytolytic disease has been proposed for HSV-1 and may be relevant in FHV-1-related disease. This has been called a “persistent” state. The persistent states mimic some aspects of latency – especially the inability to culture viable (virulent) virus. However, persistent virus is distinct from latent virus because (a) it is associated with (and likely induces) a chronic inflammatory response by the host and (b) because genes in addition to LATs are expressed. It also differs from recrudescent disease because viral antigens cannot be detected and virus cannot be cultured.57 Persistent HSV-1 has been demonstrated in the cornea, conjunctiva, and eyelid skin of mice infected with HSV-1.57,58 The role that persistent virus may have in chronic immunopathological diseases associated with FHV-1 such as stromal keratitis, chronic conjunctivitis, herpetic dermatitis, and potentially anterior uveitis requires investigation. However, studies

utilizing PCR in cats known to be infected with FHV-1 but not showing clinical signs have identified viral DNA in trigeminal ganglia, autonomic ganglia, optic nerves, olfactory bulbs, vestibular ganglia, conjunctiva, and corneas of latently infected cats.37,38,40,43,59-62 Although this virus may have been latent, gene expression, antigen immunohistochemistry, and histologic responses were not always assessed. In one study, FHV-1 DNA was detected in almost 50% of corneas of cats without clinical evidence of ocular disease; however none contained detectable LATs suggesting that a state other than latency may explain viral presence in the cornea of these cats.37 The subsequent finding of virulent and viable virus in the corneas of normal cats (albeit within a shelter)43 reopens this issue and has important clinical implications for diagnostic testing and corneal transplantation in cats. Figure 2 illustrates and summarizes the viral states associated with latency, and primary, recrudescent, and persistent infections.

Incubation and shedding The incubation period for FHV-1 after initial exposure is 2-10 days but experimentally it has been shown that this and the severity of the disease induced are both dose-dependent. Viral shedding has been observed in the ocular, oropharyngeal and nasal secretions as early as 24 hours after inoculation and can persist continuously for 1-3 weeks. Subsequent, intermittent, shedding is characteristic of the lifelong carrier state.

Immune Responses to FHV-1 Serum neutralizing antibody responses occur to several major viral glycoproteins (60, 68, and 105 KD molecular weight).27,28 Neutralizing titers are usually of low magnitude (< 1:64) after primary infection and may rise with re- exposure or recrudescent disease. However there is a questionable relationship between titer magnitude and disease status, due perhaps in part to the inability of serological methods to discriminate between exposure to vaccine and wild-type virus.20 Similarly, correlation between circulating antibody titer and protection from disease is not clear.63 This may reflect the less major role that systemic humoral immunity is presumed to play relative to that of surface (mucosal) and cell-mediated arms of the immune response.64,65 Humoral immunity to FHV-1 has been shown to diminish the severity of infections, but appears incapable of preventing infection or the establishment of latency.66,67 Following vaccination, detectable titers appear to persist for at least 4 years and to be associated with a 52% reduction in clinical signs upon re-exposure.68 Seroconversion may occur faster in FHV-1-naïve cats receiving a subcutaneously-administered attenuated vaccine than in those receiving a modified-live FVRCP vaccine.69 Vaccines and vaccination are discussed more fully in the section on immunotherapy below.

COMPARATIVE SIGNIFICANCE Infection of human beings with herpes simplex virus type 1 (HSV-1) is almost identical in most respects to FHV-1 infection of cats:

• Primary infection manifests by 5 years of age, most commonly producing self-limiting upper respiratory signs. • Ninety percent of people are seropositive by age 15, and circulating antibodies are found in 95% of adults. • Recovery from primary infection is almost always spontaneous. • In the USA, 30 million people are affected, and 100 million episodes of labial, genital, and ocular infections occur annually. There are five hundred thousand cases of recurrent HSV keratitis annually, and HSV-1 is the number one infectious cause of corneal blindness in developed countries. (Chlamydia trachomatis is the leading infectious cause of corneal blindness in developing nations).

These similarities mean that much information can be gleaned from one virus and applied to the other. A recent review By Dr. Maes provides a valuable summary.45 In the case of therapies, the vast majority of measures used for FHV-1 have been “borrowed” from HSV-1. In some situations their efficacy has actually been tested in FHV-1 and sometimes found to be as expected from data generated with HSV-1.70-72 In other cases, these investigations have highlighted the toxicity73 or poor efficacy74 (or both) of certain compounds. This reminds us that knowledge gained

from the study and treatment of HSV-1 can improve our approach to cats with FHV-1 only if that information is carefully investigated, thoroughly tested, and judiciously applied.

Whenever we take a drug developed for the treatment of HSV-1 in humans and use it for treating a cat infected with FHV-1 we are making 2 giant leaps of faith unless placebo- controlled, masked, prospective trials of its safety (for cats) and efficacy (against FHV-1) have been performed.

PATHOGENIC MECHANISMS

Primary Infection Virus replicates in epithelium of nasal mucosa, conjunctiva, tonsil, and turbinates. Tissue damage is due to viral cytolysis. The ability of FHV-1 to induce rapid and severe lysis of susceptible cells presumably is responsible for symblepharon formation in young cats. FHV-1 replicates to a limited extent in corneal epithelium where it can produce dendritic lesions.75,76 The cause of the branching pattern is unknown, but is considered to be pathognomonic for herpetic infections of all species.75,77 Dendritic corneal lesions occur in a biphasic pattern on days 3 and 12 of primary infection, the latter peak likely reflecting virus released from replication within and rupture of conjunctival epithelium. Little, if any, viral replication occurs within the corneal stroma.76

Recrudescent disease Recrudescent disease occurs following periods of viral reactivation in only some latently infected animals. The severity of disease and the tissues involved in these recrudescent episodes range widely among individuals and even among disease episodes. Conjunctivitis is usually milder and less ulcerative than seen in the acute infection. However, substantial conjunctival thickening and hyperemia can occur secondary to inflammatory cell infiltration. Corneal infections may again involve epithelial tissues, in which case dendritic and later geographic corneal ulceration may be seen, as in primary infections. Stromal involvement is also reasonably common. Data produced for FHV-1 (like HSV-1) suggest that stromal damage is immunopathological (i.e., immune-mediated but not necessarily autoimmune) in origin. Histologic observations from cats with chronic, naturally-occurring stromal keratitis reveal fibrosis, collagen degeneration, and numerous lymphocytes, plasma cells, and macrophages.78 Experimentally, stromal keratitis does not occur during primary infection unless the normal immune response is suppressed by corticosteroids. In this scenario, the development of stromal keratitis is preceded by chronic epithelial ulceration and delayed viral clearance (with prolonged viral shedding), and acquisition of viral antigen by the corneal stroma. The subsequent stromal accumulation of polymorphonuclear cells, and T- and B-lymphocytes, correlates temporally with the return of normal immune function, eventually leading to stromal inflammation and fibrosis. The events surrounding experimental FHV-1 stromal keratitis are most compatible with a delayed type 78 hypersensitivity response (Th1 cell-mediated, macrophage effector cells). Despite the intensity of study of HSK and the consensus regarding its immunological mechanism, there is still a degree of controversy regarding the antigen responsible for initiating and maintaining the immunological response. The difficulty isolating virus from the cornea, the clinical response of patients to topical corticosteroid application and their lack of response to antiviral therapy, and the so-called immunologically privileged features unique to the cornea have caused some authors to suggest that auto-immunity is responsible.79,80 The suggested mechanisms are autoantigens (host antigens altered due to viral infection) or molecular mimicry (i.e., viral sensitization of the immune system to extremely similar but unaltered host antigens). By contrast, the detection of persistent virus in these tissues, and the demonstration of similar mechanisms of inflammation in non-corneal tissues such as eyelid skin and conjunctiva have provided evidence that undetectable viral antigens may be responsible.57,58

FHV-1-Associated Disease Syndromes

Keratoconjunctivitis Sicca (KCS). In one study, experimental FHV-1 infection was associated with decreased STT results in cats.76 Although the mechanism was not determined, 2 mechanisms were proposed: (i) secretory ductule destruction or obstruction as a result of conjunctivitis, or (ii) destruction of lacrimal acini as a result of viral lacrimal adenitis. More recently, there has been contradictory evidence that FHV-1 infection increases STT values

in association with reduced tear film quality; specifically, reduced tear film break-up time (TFBUT), mucin content of the tear film, and conjunctival goblet cell density (GCD).50,81 Following primary experimental infection in cats, TFBUT and GCD remained abnormal for at least 1 month following infection, despite apparent normalization of clinical and histological examination findings suggesting that FHV-1 infection induces persistent qualitative tear film abnormalities that are not detected without measurement of TFBUT or GCD.81 This suggests that mucinomimetic therapy should continue after apparent clinical recovery from FHV-1 infection and until TFBUT has returned to normal. Despite the remarkable antiviral efficacy of famciclovir, it does not reduce this marked goblet cell depletion suggesting that mucinomimetic therapy should be used in addition to an antiviral drug.82

Corneal Sequestration. Experimentally, chronic FHV-1 infection can result in corneal sequestration.76 However, the prevalence of detectable FHV-1 in samples collected from cats with sequestra has varied widely in the clinical setting,51,59,60,83-85 and the link between FHV-1 and sequestra is not proven to be causative. In the author’s opinion, sequestration is a non-specific response to stromal exposure or damage and FHV-1 is just one possible cause of this. This is borne out by identification of FHV-1 DNA less frequently in sequestra from Persian and Himalayan cats than those from domestic shorthaired cats that had better lid anatomy and function.60 This suggests that other non-viral causes of sequestration are more likely or prevalent in brachycephalic breeds.

Eosinophilic Keratitis. Clinical studies have suggested a link between FHV-1 infection and eosinophilic keratitis,86 and PCR testing of ocular surface scrapings from cats with cytology-confirmed eosinophilic keratitis has revealed FHV-1 DNA in 55-76%60,87 of cases. By contrast, PCR performed on tears collected using a STT strip failed to detect FHV-1 DNA in 10 cats with cytologically-proven eosinophilic keratitis.88 In one study,87 FHV-1 DNA was more likely to be detected if corneal ulceration preceded the diagnosis of the eosinophilic keratitis. As with corneal sequestra, the role of the virus in the initiation of this disease has not been determined.

Symblepharon. There is little question that symblepharon can be a sequela to severe primary FHV-1 infection. It is commonly seen in young animals, and presumably occurs as a result of widespread epithelial cytolysis with exposure of the conjunctival substantia propria and sometimes also the corneal stroma. It is the author’s belief that FHV-1 is the predominant cause of symblepharon formation in cats and that other infectious agents are unlikely to cause symblepharon formation.

Uveitis. HSV-1 is a well-documented cause of uveitis in humans.89 Given the shared biological behavior of these 2 alphaherpesviruses, we examined the role of FHV-1 in feline idiopathic uveitis.90 The PCR assay used demonstrated FHV-1 DNA in the aqueous humor of 12/86 cats, all but one of which had uveitis. The same study also used ELISA to examine FHV-1-specific antibody concentrations in aqueous humor and serum. While seropositivity did not vary among cats, intraocular antibody production, as determined by a Goldman-Witmer coefficient (C-value) > 1, was detected only in cats with uveitis. Additionally, a C-value > 8, which is frequently quoted as a more clinically useful indicator of intraocular antibody production, was found only in cats with idiopathic uveitis.90 A subsequent investigation also demonstrated FHV-1 DNA could be detected in the aqueous humor of cats and more often in the blood of cats with uveitis than those without uveitis.52 And an experimental inoculation model revealed that viral DNA could be found within, and live virus isolated from, the uveal tract (and retina) of all 4 cats at 6 and 10 but not 30 days after inoculation.62 Taken together, these data suggest that intraocular FHV-1 infection occurs and that, at least in some cats, stimulates a specific local intraocular antibody response. Because the trigeminal nerve supplies the uveal tract, it is possible that virus may reactivate spontaneously or via induction and arrive in the uvea (and aqueous humor) via the “round trip theory”, as for surface ocular disease. Viral pathogenic mechanisms similar to those reported in surface disease are therefore plausible explanations for the uveal pathology seen. That is, virally mediated cytolysis and immunopathological responses directed at auto or viral antigens are possible. However, proving a casual association remains difficult.

Dermatitis. Periodically, FHV-1 has been identified as a cause of dermatological lesions, particularly those involving facial skin of domestic91-94 and wild15,17 felidae. This is not surprising when one considers the marked epithelial tropism of this virus and the reliability with which HSV-1 causes dermal lesions.58 Unlike for ocular disease where a high rate of viral shedding in normal cats dramatically reduces the diagnostic utility of PCR, FHV-1 PCR appears to be extremely useful for herpetic dermatitis. In one study,94 FHV-1 DNA was detected in all 9 biopsy specimens from 5 cats with herpetic dermatitis but in only 1 of 17 biopsy specimens from the 14 cats with nonherpetic dermatitis, and in none of 21 biopsy specimens from 8 cats without dermatitis. When results of histologic examination were used as the gold standard in this study, sensitivity and specificity of the PCR assay were

100% and 95%, respectively. We concluded that FHV-1 DNA can be detected in the skin of cats with herpetic dermatitis, that the virus may play a causative role in the disease, and that this PCR assay may be useful in confirming a diagnosis of herpetic dermatitis.94 An Italian group using a different PCR assay to assess biopsies from cats with a variety of eosinophilic dermatopathies found FHV-1 DNA in 12/64 biopsies.95

Interactions with Other Agents

There appears to be a real but relatively minor relationship between chronic FHV-1 diseases and co-infection with FeLV and FIV.96 Cats with chronic FHV-1 infection are more likely to be infected with FeLV or FIV than normal cats. Experimentally, FIV-infected cats exposed to FHV-1 develop more severe disease, however, the length of the illness or level of FHV-1 shedding is not different.97 In vitro, FHV-1 has been shown to enhance activation of gene sequences of FIV.98,99 Co-infection rates of FHV-1 with other agents of upper respiratory disease such as FCV, Chlamydia felis, Bordetella bronchiseptica, and Mycoplasma spp. range widely,83,96,100-108 and the likely clinical significance of coinfections is not known. No potential interactions between FHV-1, Bartonella henselae, and Toxoplasma gondii in the induction of uveitis in cats have been detected.109

DIAGNOSIS OF FHV-1 OCULAR DISEASE

A major paradox exists with respect to the diagnosis of FHV-1.20 Cats experiencing primary FHV-1 infection shed virus in sufficient quantities that viral detection is relatively easy. However, clinical signs during this phase of infection tend to be characteristic and self-limiting, making definitive diagnosis less necessary. By contrast, during the more chronic FHV-1-associated syndromes, the diversity and ambiguity of clinical signs make viral identification more desirable, especially if specific antiviral therapy is being considered. However, the elusive nature of the virus in these chronic syndromes makes this difficult. Indeed, the diagnosis of FHV-1 in individual cats represents one of the greatest challenges in the management of chronic FHV-1-related diseases.

Although the extreme sensitivity (and specificity) of PCR has improved detection of virus, it has also confirmed that virus can be demonstrated in up to 49% of apparently normal cats.37 This must be considered when interpreting results of diagnostic assays in individual cats with disease. Additionally, we know that HSV-1 (and therefore possibly FHV-1) can be stimulated to reactivate by irritation of the peripheral sensory neurons.110-112 Therefore, it is possible that virus detected at a peripheral site in a diseased animal may be there as a result rather than a cause of the disease being investigated. Finally, PCR assay in common usage can differentiate vaccine from wild-type virus.113 Therefore, whenever virus is detected in a cat with disease, there are at least 4 possible explanations:

1. Its presence is coincidental (i.e., unrelated to the primary disease process) 2. Its presence is a consequence of the primary disease process 3. It is the cause of the primary disease process 4. It is vaccine virus

Whether virus is found or not (i.e. irrespective of the PCR test result), the clinician must still decide whether specific antiviral treatment is warranted. Currently, other than clinical acumen, there is no “test” that will answer that question!

Perhaps one of the best ways to diagnose FHV-1 is to maintain a strong clinical suspicion of its involvement in any cat with ocular surface disease and to be well aware of its classical clinical features, but to be questioning of its role in that disease process whenever it is detected using any currently available diagnostic assays.

The following discussion highlights suggestive and pathognomonic clinical signs and the role of laboratory assays for FHV-1 diagnosis. Currently-available tests rely on demonstration of an immunological response (usually in serum) to the organism, or detection of whole, cultivable virus by virus isolation (VI), its antigens by immunofluorescent antibody test (IFA), or its DNA by PCR.114

Clinical Signs. Primary ocular FHV-1 infection of the FHV-naïve host is characterized by conjunctival hyperemia (sometimes with conjunctival ulceration), serous ocular discharge that becomes purulent by day 5-7 of infection, mild chemosis, and moderate to marked blepharospasm.76 Primary infection is always associated with typical signs of upper respiratory infection. The uncomplicated clinical course usually is only 10-14 days; however tear film changes may persist longer than 1 month.81 The only pathognomonic clinical sign during primary infection is the presence of dendritic lesions,77 which are inconsistently observed, especially without rose bengal/lissamine green stain. Dendritic corneal ulcers, erosions, or scars are also considered a pathognomonic clinical feature in recrudescent FHV-1 infections.

The major causes of feline keratoconjunctivitis are infectious in nature and the major differential diagnoses are FHV-1 and Chlamydia felis (previously Chlamydophila felis and, before that, Chlamydia psittaci). Based upon Koch’s postulates115 and epidemiological studies in naturally infected cats,106 feline calicivirus (FCV) has traditionally been considered an unlikely and minor primary conjunctival pathogen106,115 and is not a recognized corneal pathogen.115 However, cats with marked upper respiratory tract disease due to FCV will sometimes have conjunctivitis as part of that syndrome, especially when they reside in shelters.103 As a result of the difficulty interpreting diagnostic tests for FHV-1, one of the most important steps in the diagnostic process and instead of (in my opinion) requesting laboratory confirmation is “old fashioned” clinical suspicion. Data presented in Table 2 are intended to assist with the distinction of primary infection with C. felis or FHV-1. Differentiation of chronic or recurrent syndromes utilizes the same criteria but is more difficult.

Table 2. Clinical signs that may assist with differential diagnosis of the cause of acute keratoconjunctivitis in cats.

Clinical Signs FHV-1 C. felis Conjunctival hyperaemia +++ ++ Chemosis + +++ Conjunctival ulceration +/- - Keratitis +/- - Dendrites Pathognomonic - ++ (primary) Respiratory signs/malaise +/- +/- (reactivation)

Cytology. In humans, the presence of multinucleate epithelial cells is considered compatible with HSV-1 infection. In cats this appears not to be the case. Although intranuclear inclusions are very common during primary FHV-1 infection, they are seen infrequently in clinical cases.83,96,105 The inflammatory cell infiltrate seen in acute FHV-1 infections consists predominantly of neutrophils and is of little diagnostic significance.96 Intranuclear inclusions are extremely helpful in histologic diagnosis of herpetic dermatitis.92-94

Immunofluorescence Antibody Testing. Immunofluorescent antibody (IFA or FA) testing was one of the original methods used to detect FHV-1 in cats.116 Samples for IFA testing can be gathered by conjunctival or corneal scraping or from biopsy sections. Smears should be acetone-fixed to preserve reactive epitopes. The IFA test utilizes an anti-FHV-1 antibody which is directly or indirectly labeled with a fluorescent marker and observed with a fluorescent microscope. Therefore, it is essential to collect samples for IFA testing before the instillation of fluorescein dye, as this will interfere with interpretation of the assay.117 The principal reported limitations of the IFA test for FHV-1 diagnosis include lack of sensitivity76 and, like all other viral detection methods, the number of normal cats which are positive by this assay. In one study, the IFA test was positive in 50% of cats in which acute FHV-1 infection was suspected and 36% in which chronic FHV- 1 infection was suspected; however 28% of clinically normal cats were also IFA positive.20 In addition, interpretation of IFA test results requires subjective judgment by the technician and discerning true versus non- specific fluorescence can affect test accuracy. Additionally, because a positive IFA result is dependent upon the identification of a fluorescing cell, sensitivity is greatly diminished unless an adequate number of cells is examined. Finally, a positive IFA test is also dependent upon there being recognizable viral epitopes. In chronic, naturally occurring infections, it is possible that viral epitopes are bound by secretory antibody, and unavailable for binding with the diagnostic antibody. Due to these limitations and the availability of alternate diagnostic methods, IFA

testing appears to be of limited clinical application in ophthalmic practice.

Virus isolation. Virus isolation (VI) has long been the gold standard for alphaherpesvirus diagnosis. Because the virus replicates so rapidly and produces characteristic cytopathic effects in cell culture, VI is relatively rapid and simple to perform. Although many cell lines may be used, the most common is Crandell-Rees feline kidney (CRFK) cells. Samples should be collected with Dacron swabs with plastic handles since cotton and alginate swabs, as well as wooden swab handles, can be inhibitory to some herpesviruses.118-120 Samples must also be collected before application of fluorescein or rose bengal (and likely lissamine green) stains as vital stains can inhibit viral replication.3 Ideally, samples should be adsorbed onto cells as soon as possible after collection to avoid viral death. If samples cannot be processed immediately, they should be maintained at refrigerator temperature (4oC) pending adsorption. The best approach is to moisten the swab with viral transport media, swab the conjunctival sac aggressively, and break the swab off into a tube containing 1-2 ml of the viral transport media. After vortexing, aliquots of the sample are allowed to adsorb onto confluent layers of CRFK cells, after which the media is replenished, the flask incubated, and observed daily for cytopathic effect. Freezing and re-thawing samples can result in loss of viral titer sufficient to preclude a positive test result. Despite its sensitivity, VI is not routinely advocated for clinical specimens due to the logistical difficulty of transporting and processing samples in a timely fashion. Additionally (and as for other viral detection tests), FHV-1 can be detected in approximately 11%20-24%43 of normal cats and only 0%43-18%20 of cats showing clinical evidence of FHV-1-related disease. Due to the number of normal cats that shed FHV-1, VI is not useful in the diagnosis of individual cats but remains a useful research and epidemiological tool.

Serology. Unfortunately, serologic titers have not proven useful in the diagnosis of FHV-1 infection.20 Although there are several reasons for this, 2 major reasons are the inability to separate vaccinal titers from response to wild-type virus and the fact that FHV-1 titers tend to be of low magnitude (especially when measured by serum neutralization), even after primary infection. Interest in vaccinology has stimulated new investigations of the value of titers for predicting resistance to infection.68,121

Serum Neutralization Depending upon which assay methods are used, serum neutralizing (SN) titers reach only 1:16 - 1:64, 30-60 days after exposure. Although SN titers predictably rise after experimental reactivation of latent virus, titers in cats with recrudescent infections rarely achieve great magnitude. Likewise, no correlation was demonstrated between FHV-1 (SN) seropositivity or titer magnitude and the likelihood of detecting virus or the presence of clinical signs.20 In a prior study,96 serum titer was found to be inversely proportional to the likelihood of getting a positive IFA or VI result perhaps confirming that serum antibodies can make virus difficult to detect using VI or IFA.

Immunoassays (ELISA’s) Immunoassays have been used for detecting HSV-1 antigen or antibody.122,123 A commercial ELISA for HSV-1 (Herpchek®) utilizes a polyclonal capture antibody and a biotinylated monoclonal anti-HSV detector antibody and has a sensitivity of 90%, and a specificity of 99% when compared with tissue culture methods.123 To date, a FHV-1 antigen-capture ELISA has not been developed. However, ELISA’s for the detection of antibodies to viral nucleocapsid124 and nuclear125 antigens have been described. These tests are more sensitive than SN and at least one was used to successfully detect FHV-1-specific antibody in aqueous humor and CSF, as well as serum.90,124 Seroprevalence, as assessed by ELISA in one study,20 was high (97%) and did not vary significantly between normal cats and cats suspected to be undergoing acute or chronic FHV-1 infections; nor did it correlate with the likelihood of detecting virus using IFA or VI. In that study, mean ELISA titer was significantly greater in clinically normal cats, while cats with chronic disease had the lowest mean titers, suggesting that IgG titers may be in some way protective. Importantly however, vaccination status was not considered in that study and serologic methods are unable to differentiate between titers arising from natural or vaccination exposure.

Polymerase Chain Reaction (PCR) The advent of PCR technology has significantly improved detection of ocular pathogens in veterinary and human medicine.126,127 The principle of the PCR assay is that small amounts of DNA (conceivably one molecule) can be exponentially amplified under appropriate conditions. The specificity of PCR can be increased through a process termed “nesting”, in which the product generated from the first series of cycles is cycled again in the presence of a second set of primers that identify a shorter, internal sequence of template DNA. Nesting also dramatically

increases PCR sensitivity, often to an extent where the potential for contamination (and consequently the false positive rate) is unacceptably high. This introduces the concept that assay sensitivity and diagnostic sensitivity may be inverse at higher levels of detection of FHV-1 because of the potential for contamination and because of the number of normal animals that shed virus in very low quantities. Based upon in vitro assessment, it appears that (unlike VI or IFA) prior application of a topical anesthetic agent or fluorescein stain does not inhibit PCR results for FHV-1; however these results need to be verified in vivo.128

A large number and variety of PCR assays for the detection of FHV-1 have been described and these have produced a very wide range of data regarding FHV-1 detection in normal and abnormal eyes (Table 3).

Table 3. Reported PCR detection rates for various tissue types, pathology, and sample types. (Results are reported on a per eye or per sample basis; not on a per cat basis)

Tissue and Pathology PCR Detection Rates Swab/brush Scraping Biopsy Normal cornea 6%,60 32%,43 46%,59 49%37 Normal conjunctiva 3%,102 8%,53 20%,100 31%55 12%59 Sequestrum 27%85 18%,59 44%,51 55%60 Feline eosinophilic 55%,87 76%60 keratitis/conjunctivitis Conjunctivitis 10%,105 14%,55 18%,50 54%59 33%100 Diseased but not specified 18%,129 25%,103 33%,130 47%,108 58%,106 89%,129 91%131

Although these studies were conducted on different populations with different clinical syndromes in different geographic areas, it is likely that some variation is also due to sampling and testing methodologies that might be controlled for by the clinician collecting and shipping the sample and the laboratory processing and reading out the sample. For example, variability among labs has been shown,132 whereas variation among sampling sites seems less important.106 Minimization of such variation will enable the clinician to better interpret the test result for their individual patient. Not unexpectedly, a major component of variability in PCR test result appears to be attributable to the PCR assay used, with calculated detection rates for the same samples tested with 6 different assays varying from 29-86%, and sample results being concordant in only 9 of 15 conjunctival biopsies.133 The type of sample collected, as well as the way in which the DNA is extracted and the PCR assay is performed also appears to be important.134,135 Although PCR may be used to look for FHV-1 DNA in any biological sample, the test is expected to be more likely to detect virus as more host cells are tested because FHV-1 is an obligate intracellular virus. Therefore, it is expected that biopsy samples are more likely to be positive than samples gathered by surface scraping, which in turn are more likely to be positive than swab samples; however this has not been tested yet. We did however compare cytobrushes and swabs and concluded that a swab was preferable to a cytobrush and that it should be submitted in a dry tube. Once it arrives at the lab, a defined volume (rather than a defined mass) of sample extract should be submitted to PCR.134 By contrast, sample handling and shipping protocols appear to exert less influence on FHV-1 PCR assay results and shipping at ambient temperature (not on ice) seemed appropriate.135

The main “take-home” message is that with shedding of FHV-1 at the peripheral surfaces of up to 50% of normal cats, individual testing seems of little benefit.37 Partly for this reason, quantitative PCR (qPCR) methods for FHV-1 detection have been assessed in experimentally infected animals.136 The authors’ hypothesis was that there is a critical concentration of viral DNA within host tissues that, below which, is likely to represent coincident or consequential virus, and above which is more likely to represent causative virus. The authors demonstrated good correlation between results of conventional methods of quantifying virus (viral titration) and the qPCR. Subsequently, SPF cats experimentally infected for the first time showed gradually reducing viral loads, as has been previously demonstrated using less sensitive techniques, such as VI. Relatively low-cellularity (STT and cytobrush) ocular samples from 20 cats which were demonstrating upper respiratory or ocular signs (keratitis and conjunctivitis) and in which natural FHV-1 infection was suspected then were assessed using the same technology. Reasonable correlation was again demonstrated between viral titer and DNA concentrations and, based on these

data, the authors characterized cats into three “stages” of disease. However, another group has since reported the clinical utility of a qPCR method in cats without history of conjunctivitis, cats with presumed herpetic conjunctivitis that had resolved at least 3 weeks prior to testing and those with active presumed herpetic conjunctivitis. They found no difference among the 3 groups in either the number of cats that tested positive or the amount of FHV-1 DNA detected.102 Most recently severity of histologically graded rhinitis was found to be significantly correlated with amount of FHV-1 DNA detected using qPCR;131 however whether these results are repeatable or applicable for ocular signs is not clear.

TREATMENT OF FHV-1 OCULAR DISEASE

Targets for Antiviral Pharmacology. Conceivably any step in the virus replicative process could be a target for antiviral intervention. These include adsorption, penetration, uncoating, early transcription, early translation, replication, late transcription, late translation, virus assembly, and release from the host cell. To date however, most of the effective antiviral therapies that have been developed target viral enzymes and proteins responsible for DNA synthesis. For a review of many available agents and their proposed mechanisms of action see this article by De Clercq,137 and for their specific applications in feline medicine, see the review by Thomasy & Maggs.138

Specific Antiviral Agents and FHV-1. FHV-1 is susceptible to inhibition by all commercially available antiviral ophthalmic medications studied thus far, however their safety in cats is not readily predicted from their behavior in other host species (principally humans), and their efficacy against FHV-1 is not predicted from their efficacy against other viruses (principally HSV-1 and - 2). In addition, there are no antiherpetic drugs currently approved in the USA for use against FHV-1 in cats. All those in common use are “borrowed” from the human pharmacy.

Whenever we take a drug developed for the treatment of HSV-1 in humans and use it to treat a cat infected with FHV-1 we are making 2 giant leaps of faith unless placebo-controlled, masked, prospective trials of its safety (for cats) and efficacy (against FHV-1) have been performed. For these reasons, careful in vitro investigation of efficacy against FHV-1, followed by safety and pharmacokinetic trials, subsequent placebo-controlled efficacy studies in experimental animals, and finally judicious clinical trials in client-owned animals should always precede widespread clinical use and anecdotal reporting.

Although opinions vary as to when and why antiviral therapy should be instituted in cats believed to be experiencing herpetic diseases, it appears reasonable that antiviral agents should be considered when signs are severe, persistent, or recurrent, particularly when there is corneal involvement, and especially ulceration. Because epithelial replication, latency and reactivation, and persistence are such interdependent and sequential phases of herpetic disease, interruption of any one of them is expected to limit the virus’ abilities to cause subsequent disease. Therefore, aggressive treatment of FHV-1-associated disease may limit disease progression and minimize frequency and severity of recurrences. This seems to be borne out by data that shows a significant direct (positive) correlation between clinical score and viral DNA copy number 30 days after experimental infection, which suggests that the quantity of viral DNA that becomes latently established in the ganglia is related to the severity of the acute clinical infection.62

Some important general concepts about antiviral agents assist with prescribing and expectations of this class of drugs: . Because viruses reside intracellularly and utilize host cellular machinery, antiviral agents tend to exhibit greater host toxicity than do antibacterial drugs. This sometimes limits topical application of these drugs but especially limits their systemic use. . All antiviral agents to date are virostatic; therefore they require replicating virus and must be dosed (orally or topically) relatively frequently. . No antiviral drug has proven antibacterial activity. . Antiviral drugs safe in humans are not necessarily safe in cats.

. Antiviral drugs effective against human herpesviruses are not necessarily effective against FHV-1. . Antiviral prodrugs metabolized to their active form by humans are not predictably metabolized by cats.

The effect of some antiviral drugs on FHV-1 replication in vitro has been studied and their potency relative to each other and relative to HSV-1 reported (Table 4). Antiviral efficacy is expressed as the IC50 (or the concentration at which viral replication is inhibited by 50% ). Note, therefore, that a lower IC50 equates to a more efficacious antiviral drug.

Table 4. Efficacy of various antiviral drugs against FHV-1 and HSV-1. Efficacy is reported as median (range) concentration (µM) at which in vitro viral replication is inhibited by 50% (IC50), therefore a lower IC50 equates to greater efficacy. Drugs are ranked (left to right) in order of decreasing efficacy against FHV-1. Note the different ranking for HSV-1.

IC50 (µM) A HPMP PMED BDV IDU GCV PCV TFU CDV VDB D ACV PFA A AP U V 150 14 18 19 19 187 5.6 8.9 (16- FHV-1 0.23 (1.2- 14 (5.1- (0.67- (7.9- 21 73 (140- (4.3-6.8) (5.2-13) 1110 130) 30.1) 1350) 168) 230) 00) HSV-1 22 2.1 0.39 1.5 6.9 0. 3 0.5 19 30 21 0.8 68 72,139, 70,139, 72,142,1 142,14 72,139,144 70,139, 142, 72,142, Ref 139,140 70,72,141 72,142,143 142,148 150,15 45,147,1 70,151 5,147,1 -147 149 154 149 1 52,153 49,150, 155

Abbreviations: ACV: Acyclovir; ADV: Adefovir; BDVU: Bromovinyldeoxyuridine; CDV: Cidofovir; GCV: Ganciclovir; HPMPA: (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine; IDU: Idoxuridine; PCV: Penciclovir; PFA: Foscarnet; PMEDAP: 9-(2-phosphonylmethoxyethyl)-2, 6-diaminopurine; TFU: Trifluridine; VDB: Vidarabine.

The following antiviral agents have been studied to varying degrees for their efficacy against FHV-1, their pharmacokinetics in cats, and/or their safety and efficacy in treating cats infected with FHV-1.

Idoxuridine (5-iodo-2'-doexyuridine; IDU) is a thymidine analogue originally developed for treatment of humans infected with herpes simplex virus (HSV) type 1. It differs from thymidine by having a single iodide substitution at the 5 position of the pyrimidine ring. Following intracellular phosphorylation, it competes with thymidine for incorporation into viral DNA rendering the resultant virus incapable of replication. IDU is a nonspecific inhibitor of DNA synthesis, affecting any process requiring thymidine. Host cells, therefore, are similarly affected, systemic therapy is not possible, and corneal toxicity can occur. It has been used as a topical (ophthalmic) 0.1% solution or 0.5% ointment. This drug is reasonably well tolerated by most cats and seems efficacious in many. It is no longer commercially available in the USA but can be obtained from a compounding pharmacist. No veterinary data regarding dose are available but, based on pharmacologic principles and data from humans, it likely should be applied to the affected eye at least 5-6 times daily. In a retrospective case series of cats with ocular disease attributed to FHV-1, 0.1% idoxuridine solution was used every 4-6 hours with improvement or resolution of clinical signs in 3 cats and no improvement or worsening in 4 cats.156

Vidarabine (Adenine arabinoside; 9-β-D-arabinofuranasyladenine; Ara-A®; Vira-A®) Vidarabine is an adenosine analogue that, following triphosphorylation, appears to affect viral DNA synthesis by interfering with DNA polymerase. However, like idoxuridine, vidarabine is non-selective in its effect and so is associated with notable host toxicity – especially if administered systemically. Because it affects a viral replication step different from that targeted by idoxuridine, vidarabine may be effective in patients whose disease seems resistant to idoxuridine. As a 3% ophthalmic ointment, vidarabine often appears to be better tolerated than many of the antiviral solutions. Where it is not available commercially, it can be obtained from a compounding pharmacist. Like idoxuridine, no veterinary data are available regarding dose but it should probably be applied to the affected eye at least 5-6 times daily. In a

retrospective case series of cats with ocular disease attributed to FHV-1, 3% vidarabine ointment was used every 4 to 6 hours with improvement noted in 1 cat and no improvement or worsening noted in 2 cats.156

Trifluridine (trifluorothymidine; 5-trifluoromethyl-2'-deoxyuridine; Viroptic®, generic) is a fluorinated nucleoside analogue of thymidine. Its specific mechanism of action against HSV-1 (for which it was developed) is not completely understood and has not been studied in FHV-1. However, following intracellular phosphorylation it reduces DNA synthesis via inhibition of thymidilate synthetase. It is too toxic to be administered systemically but topically-administered trifluridine is considered one of the most effective drugs for treating HSV-1 keratitis. This is in part due to its superior corneal epithelial penetration.157 In vitro, it is also one of the more potent antiviral drugs for FHV-1. It is commercially available in the USA as a 1% ophthalmic solution that based on human recommendations should probably be applied to the affected eye at least 5-6 times daily. Unfortunately, it is expensive, and often causes marked ocular irritation. In a retrospective case series of cats with ocular disease attributed to FHV-1, 1% trifluridine solution was used every 4-8 hours with improvement in 1 cat and no improvement or worsening in 2 cats.156

Acyclovir (9-(2-hydroxyethoxymethy)guanine; ACV; Zovirax®) is the prototype of a group of antiviral drugs known as acyclic nucleoside analogues. Members of this group of antiviral agents all require 3 phosphorylation steps for activation. The first of these steps must be catalyzed by a viral enzyme, thymidine kinase. This fact increases their safety and permits them to be systemically administered to humans. Unfortunately, the activity of the FHV-1 thymidine kinase enzyme on acyclovir is much less than that of the HSV-1-encoded enzyme, which likely explains the relative lack of efficacy of ACV against FHV-1.145,158 The second and third phosphorylation steps must be performed by host enzymes. To my knowledge the affinity of these enzymes for the acyclic nucleoside analogues has not been studied in cats. In addition to relatively low antiviral potency against FHV-1, acyclovir has poor bioavailability and is potentially toxic when systemically administered to cats. Oral administration of 50 mg/kg acyclovir to cats was associated with peak plasma levels of only 33 μM (approximately one third the IC50 for this virus).74 Common signs of toxicity are referable to bone marrow suppression. With the advent of the apparently safer and more effective famciclovir, administration of acyclovir seems more difficult to justify. Acyclovir is also available as an ophthalmic ointment in some countries which, while it may overcome systemic toxicity concerns, need not necessarily increase antiviral efficacy against FHV-1. Regardless, a 0.5% ointment used 5 times daily in naturally infected cats was associated with a median time to resolution of clinical signs of 10 days.150 Cats treated only 3 times daily took approximately twice as long to resolve and did so only once therapy was increased to 5 times daily. Taken together, these data suggest that frequent (at least 5 times daily) topical application of acyclovir may produce concentrations at the corneal surface that exceed the IC50 for this virus but are not associated with clinically appreciable toxicity. There are also in vitro data suggesting that interferon exerts a synergistic effect with acyclovir that could permit an approximately 8-fold reduction in acyclovir dose.159 In vivo investigation and validation of these data are needed.

Valacyclovir (L-valine, 2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy]ethyl ester, monochloride; Valtrex®) is another acyclic nucleoside analogue. As a prodrug of acyclovir, it is - in humans and cats - more efficiently absorbed from the gastrointestinal tract than acyclovir is and, following absorption, is converted to 160 acyclovir by a hepatic hydrolase. Plasma concentrations of acyclovir that surpass the IC50 for FHV-1 can be achieved after oral administration of this drug. However, in cats experimentally infected with FHV-1, valacyclovir induced fatal hepatic and renal necrosis, along with bone marrow suppression, and did not reduce viral shedding or clinical disease severity.73 Therefore, despite its superior pharmacokinetics, valacyclovir should never be administered to cats. This also reminds us how toxic acyclovir is in cats once a sufficiently high plasma concentration is achieved.

Ganciclovir (Cytovene®, Zirgan®) is another acyclic nucleoside analogue that, like acyclovir, requires triphosphorylation to achieve its active form with the first phosphorylation step mediated by viral thymidine kinase. It appears to be approximately 10-fold more effective against FHV-1 than is acyclovir.72 It is available for oral or intravenous administration in humans, where it is associated with more severe neurologic toxicity, neutropenia, and bacterial infections than is acyclovir.161,162 Like acyclovir, a prodrug of ganciclovir (valganciclovir) is available. To my knowledge, the safety and pharmacokinetics of ganciclovir or valganciclovir have not yet been studied in cats. An ophthalmic gel preparation (Zirgan®; 0.15%) is available for human use but is currently very expensive in the USA. However there are anecdotal reports of good efficacy and tolerability in cats with FHV-1 in Europe (where it is cheaper). Given the in vitro efficacy of ganciclovir, this product definitely warrants efficacy and safety trials in

cats. However, to the author’s knowledge, neither the safety nor pharmacokinetics of valganciclovir or ganciclovir in any form has been reported in cats.

Penciclovir and Famciclovir Penciclovir (9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine; BRL39123; Denavir®, Vectavir®) is a nucleoside deoxyguanosine analogue with a similar mechanism of action to acyclovir and with potent antiviral activity against a number of human herpesviruses. Like acyclovir, it requires viral and cellular phosphorylation but is highly effective against FHV-1 in vitro145-147 and in vivo.82 In a rabbit model of human HSV-1 keratitis, a 3% penciclovir ointment administered once, twice or four times daily decreased epithelial keratitis severity.163 Thus, a topical ophthalmic penciclovir ointment may be effective in cats with FHV-1 keratitis and/or conjunctivitis, but, to the authors’ knowledge, there are no commercial or compounded preparations available for ophthalmic use. Penciclovir is available as a 1% dermatologic cream for humans, but that should not be applied to the eye.

Famciclovir (2-(2-(2-amino-9H-Purin-9-yl)ethyl)-1,3-propanediol diacetate; Famvir®) is a highly bioavailable prodrug of penciclovir, which – once absorbed – is metabolized to penciclovir. In humans this metabolism is complex; requiring di-deacetylation to BRL42359, in the blood, liver, or small intestine, with subsequent oxidation to penciclovir by aldehyde oxidase in the liver.164-166 Neither famciclovir nor BRL42359 has any in vitro antiviral activity against FHV-1,146 therefore complete metabolism to penciclovir is required. However, hepatic aldehyde oxidase activity in cats is about 2% of that seen in humans and lower than in any other species reported to date.167 Famciclovir pharmacokinetics in the cat are extremely complex and nonlinear (i.e., doubling of famciclovir dose does not lead to doubling of plasma penciclovir concentration) presumably due to saturation of the hepatic oxidase.168,169 As a result very high plasma concentrations of BRL42359 accumulate in the cat.169 Fortunately, this compound demonstrates very little cytotoxicity in vitro.146 Table 5 summarizes the pharmacokinetic data available to date for penciclovir in tears and plasma of cats receiving one of numerous famciclovir dose regimens. Tissue concentration data are not yet available.

Table 5. Maximum (Cmax) and minimum plasma and tear penciclovir concentrations and time to plasma and tear Cmax in cats administered a variety of famciclovir doses at various dose frequencies.

Plasma [PCV] Plasma Tear [PCV] Tear Dose (ng/mL) PCV (ng/mL) FCV dose PCV frequency Tmax Css(min Cmax Css(min) Cmax Tmax (h) (h) ) 9-18 BID 330 64 5.3 ND ND ND mg/kg170 TID 680 180 3.8 ND ND ND

30 BID 2010 345 1.3 305 65 4.7 mg/kg169 TID 1755 570 1.7 395 160 2.0 40 BID 1945 445 2.3 375 55 3.3 mg/kg169 TID 2210 790 2.5 750 150 2.6 90 BID 2720 630 2.7 680 200 2.3 mg/kg169 TID 2015 905 2.7 555 275 3.0

Abbreviations: BID: twice daily; Cmax: maximum observed drug concentration; Css(min): minimum observed drug concentration during the dosing interval at steady state; FCV: famciclovir; ND: not done; PCV: penciclovir; TID; thrice daily; Tmax: time to C max.

In addition to these pharmacokinetic data, recommendation of an appropriate famciclovir dose requires: • Knowledge of whether penciclovir concentrations in plasma, tears, or the infected tissues themselves are most relevant • Selection of an appropriate target penciclovir concentration based on in vitro IC50s (which reportedly range from 304 to 3500 ng/mL.)72,139,146,147 • Knowledge of whether the targeted IC50 should be exceeded by the trough or the peak penciclovir concentrations, and for how long.

Together, these uncertainties have led to much controversy about the optimum famciclovir dose in cats, with reported doses ranging from 8 mg/kg once daily171 to 140 mg/kg thrice daily.172

The following data are provided to inform dose selection.

In the only masked, placebo-controlled efficacy trial to date, cats known to be infected with FHV-1 and given approximately ~90 mg famciclovir/kg thrice daily per os achieved an approximate peak plasma penciclovir concentration of 2100 ng/mL.82 Relative to control cats, treated cats had significantly reduced clinical signs, decreased serum globulin concentrations, reduced histologic evidence of conjunctivitis, decreased viral shedding, and reduced serum FHV-1 titers, as well as increased goblet cell density.82 A subsequent study showed that administration of a single dose of 40 mg/kg to uninfected healthy cats achieved nearly identical plasma penciclovir concentrations to those achieved with a single dose of 90 mg/kg.173 A third study174 revealed that cats receiving 40 mg/kg thrice daily had tear penciclovir concentrations likely to be effective against FHV-1 (using a target IC50 of 304 ng/mL)145 for at least 3 hours after each dose (i.e., for ≥ 9 hours/day). In the most comprehensive pharmacokinetic study to date, healthy cats were administered famciclovir at 30, 40 or 90 mg/kg twice or thrice daily, and plasma and tear famciclovir, BRL42359, and penciclovir concentrations were measured.169 This resulted in the recommendation that cats should receive 90 mg famciclovir/kg twice daily because this regimen achieved comparable plasma and tear penciclovir concentrations to those achieved with 90 mg/kg thrice daily, whereas the lower doses tested did not result in adequate tear penciclovir concentrations, even when administered thrice daily.

Perhaps most revealing so far, are data from a retrospective study comparing outcomes when famciclovir was administered thrice daily to cats with presumed herpetic disease at approximately 40 (n = 33 cats) or 90 mg/kg (n = 26 cats).172 Median duration of therapy required for clinical improvement was significantly longer in cats administered 40 versus 90 mg/kg. Furthermore, cats in the 90 mg/kg group showed significantly greater and faster improvement than did cats in the 40 mg/kg group. The reduction in treatment duration with the higher famciclovir dose was estimated to decrease overall client costs due to a reduction in total famciclovir administered (and potentially the number of recheck examinations required). These data, suggest that 90 mg/kg TID is clinically and cost effective. Meanwhile, pharmacokinetic data[50] 169 suggest that tear and plasma penciclovir concentrations are similar whether cats receive 90 mg famciclovir /kg 2 or 3 times daily. Taken together, data from these 2 studies169,172 suggest that 90 mg famciclovir/kg twice daily is likely to be effective in treating cats with herpetic disease. These data, combined with those from pharmacokinetic studies suggest that 90 mg/kg twice daily is likely to be effective in treating cats with FHV-1. Adverse events (most commonly gastrointestinal) potentially attributable to famciclovir were reported in 17% of cats receiving 40 or 90 mg famciclovir/kg thrice daily, but the prevalence was not different between the 2 dose groups.172 Assessing all in vivo tolerance data for famciclovir, this drug appears to be markedly safer than acyclovir and valacyclovir - the only other systemic antiviral drugs to be orally administered to cats.73,74,82,169,170,172-174 However, patients administered famciclovir should be closely monitored, and assessment of a complete blood count, serum biochemistry panel and urinalysis should be considered in cats with known intercurrent concurrent disease or cats expected to receive famciclovir for long periods. As in humans,175 reduction in dose frequency should be considered in cats with renal insufficiency.

Cidofovir is a cytosine analogue that requires 2 host-mediated phosphorylation steps but does not require virally- mediated phosphorylation. Its safety arises from its relatively high affinity for viral DNA polymerase compared with human DNA polymerase.176 It is available in injectable form in the United States and is administered intravenously or intravitreally to humans infected with herpesviruses; principally cytomegalovirus. This drug has also been topically applied as a 0.5% or 1.0% solution in experimental animal models of human herpetic keratoconjunctivitis and found to be equally effective when administered only twice daily as trifluridine was when administered 4-9 times daily.163,177 This is believed to be due to the long tissue half-lives of the metabolites of this drug.178 The efficacy of a 0.5% solution compounded in methylcellulose artificial tears and applied topically twice daily to cats experimentally infected with FHV-1 was associated with reduced viral shedding and clinical disease in a prospective, masked placebo-controlled study.179 There are occasional reports of its experimental topical use in humans being associated with stenosis of the nasolacrimal drainage system components,180,181 and it is not commercially available as an ophthalmic agent in humans. Therefore, although the in vitro and short-term in vivo efficacy of cidofovir against FHV-1 are proven, cats should be monitored for nasolacrimal cicatrization. Unlike most compounded agents, there are also some excellent data on the efficacy over time and using various storage conditions for cidofovir. Cidofovir 0.5% compounded in normal saline (not methylcellulose as was studied in vivo) retained efficacy under all combinations of storage conditions tested: in plastic or glass; refrigerated (4C), or frozen at -20C or -80C; and for 1, 2, 4, or 6 months.182 Importantly, safety was not studied.

Lambda-carrageenan is a red seaweed extract containing sulfated polysaccharides with demonstrated antiviral

activity against numerous enveloped viruses including HSV-1. Activity of λ-carrageenan against FHV-1 replication in vitro and in vaccinated cats exposed for the first time to wild type FHV-1 has been examined in a placebo- controlled masked study.183 λ-carrageenan demonstrated some antiviral activity in vitro when used prior to but not following viral adsorption. Safety/tolerance trials revealed no adverse effects in uninfected cats when 1 drop of a 250 µg/mL (or less concentrated) solution was applied topically 4 times per day. Efficacy was tested using 1 drop of a 250 µg/mL λ-carrageenan solution applied before and after infection (n = 6 cats) or after infection only (n = 6 cats). The drug did not reduce clinical signs, regardless of whether it was applied prior to and after infection or only after infection and a significant reduction in virus isolation was noted only on day 21 following inoculation. Other plant extracts with antiviral activity have also been studied in vitro.184

Leflunomide is an immunosuppressive agent that appears to have some antiviral effects against human herpesviruses including HSV-1.185 Its effects against FHV-1 in vitro have been reported.186 Simultaneous inoculation of CRFK cells with virus and treatment with leflunomide was associated with a significant and dose- dependent reduction in plaque number and - at higher concentrations - viral load. However (curiously) viral load was not reduced in a dose dependent manner at very low concentrations of this drug. At higher concentrations, some cytotoxicity was observed. Electron microscopy revealed an apparent failure in viral assembly, especially of the tegument and external membrane, which may indicate the mode of action of this drug.186

Lactoferrin is a mammalian iron-binding glycoprotein that has antibacterial, antifungal, antiprotozoal, and antiviral properties. It is produced by mucosal epithelial cells of many mammalian species and is present in secretions such as tears, saliva, seminal and vaginal fluids, and in low concentrations in plasma. Breast milk, especially colostrum, is a major source of lactoferrin. Lactoferrin has been demonstrated to exert a very potent antiviral effect against FHV-1 replication in vitro.2 Further analysis of the data in this study reveals that lactoferrin appears to inhibit FHV-1 adsorption to the cell surface and/or penetration of the virus into the cell. At a molecular level, this may occur due to interaction of lactoferrin with receptors on the cell surface or by direct neutralization or inhibition of the viral particle.

The interferons (IFNs) are a group of cytokines that have diverse immunological and antiviral functions. The IFNs are divided into 4 groups; α, β, γ, and ω interferons, and numerous subtypes. Viral infection stimulates cells to secrete IFN into the extracellular space where it binds to specific receptors on neighboring cells and, through mechanisms not fully understood, prevents or limits the spread of infection (i.e., it is not virucidal). Knowledge and consideration of this mode of action allows the clinician to set and maintain reasonable expectations of how effectively the IFNs may work when used therapeutically, and in which patients with which stage of disease they might be expected to be most effective.

Although IFNs may play important physiological roles in the control of viral infections, in vitro and clinical trials investigating potential therapeutic applications have produced conflicting and not solidly supportive results. In in vitro studies, 1 x 105 - 5 x 105 IU/ml of recombinant human IFNα or recombinant feline IFNω significantly reduced FHV-1 titer and/or cytopathic effect while not producing any detectable cytotoxic changes in the feline corneal cell line187 or CRFK cells188 on which the virus was grown. At higher concentrations, the effect of the recombinant feline IFNω was greater than that of IFNα.188 In a separate in vitro study, notable synergistic activity against FHV-1 was demonstrated when 10 - 62.5 µg/mL of acyclovir was combined with 10 or 100 IU/mL of human recombinant IFNα. The combined use of the 2 compounds did not cause increased cytotoxicity but permitted a nearly eightfold reduction in the dose of acyclovir required to achieve maximal inhibition of FHV-1. Significant synergistic interactions resulted when the IFNα was given before or after infection at the lower doses of acyclovir; however pretreatment was more effective.159

To my knowledge, there are relatively few peer-reviewed, placebo-controlled, prospective clinical trials of IFN administration to SPF cats experimentally infected with FHV-1(the gold standard for the other antiviral products described here). One study utilized 10,000 IU of recombinant feline IFNω administered topically (OU) q 12 hours and 2,000 IU administered PO q 24 hours.189 Based on data generated in studies utilizing other viruses and knowledge of the mode of action of IFNs, IFN administration in this feline study189 was initiated 2 days prior to viral inoculation but was not continued after inoculation. No beneficial effects were shown. The effects of very high-dose systemic administration of IFNα prior to experimental FHV-1 infection have also been studied; 108 IU/kg were administered subcutaneously BID on two consecutive days prior to inoculation.190 Although disease was not prevented, cumulative clinical scores were lower for cats treated with IFNα. An abstract has also been presented

detailing preliminary low-dose oral data.191 Cats received 1, 5 or 25 IU/cat IFNα PO q 24 hrs 24 and 48 hours after inoculation. Scores for disease severity were significantly lower in cats receiving 5 or 25 units than in control cats. A study of 16 cats naturally infected with FeLV, FIV, or both and living in a shelter assessed the effect of recombinant feline IFNω therapy in a non placebo-controlled trial. The treatment protocol involved 3 cycles of injections at Days 0, 14 and 60; each treatment cycle consisted of 5 subcutaneous injections of 1 MU/kg once daily for 5 days. Although cats tended to shed less FHV-1 by Day 60 than on admission, it is unclear if this was due to IFN administration. Finally, in a prospective, randomized, placebo-controlled, double-masked clinical trial,192 37 cats were randomly assigned to receive placebo or recombinant feline IFNω (2.5 MU/kg) subcutaneously, followed by 0.5 MU topically at 8-h intervals via the conjunctiva, intranasally, and orally for 21 days. All cats received additional treatment with antibiotics, expectorants, and inhalation of nebulized physiological saline with camomile. Clinical signs were evaluated over 42 days. All cats demonstrated improvement in clinical signs during the course of the study, with no significant difference in any of the assessed variables when comparing the two groups. Given the lack of rigorous supporting data to date, further research is necessary before use of this group of compounds can be recommended, especially in the more chronic or recrudescent clinical syndromes in those client-owned cats commonly seen by ophthalmologists.

Two additional areas of controversy exist regarding IFN administration. Firstly, the stability of INFα once thawed appears to be very limited and should be addressed with respect to dispensing practices for this drug. There has also been some concern raised regarding the efficacy of this drug when given orally. Since IFN is a simple peptide, it is presumably digested by gastric peptidases. However, the proposed route of absorption following oral administration is across the oropharyngeal mucosa, from where it is believed to be active in surrounding and distant lymphoid tissue. The dissemination of IFN activity is believed to occur via direct cell-to-cell transfer of the antiviral state. This provides a mechanism whereby low concentrations of IFN are able to produce potent antiviral activity even at sites distant from the route of entry, and are amplified via a cascade effect.193

Small (or short) interfering RNAs (siRNAs) are short (about 20 nucleotide), double-stranded sections of RNA designed to transfect a cell and interfere with (“knockdown”) expression of specific gene(s). To overcome the short- lived effect of transfection of native siRNAs, they can be incorporated into plasmids and thus extend their longevity, especially within rapidly dividing cells. Three studies have now been published investigating the effects of these agents on FHV-1.33-35 An initial in vitro study of siRNAs that targeted the glycoprotein and DNA polymerase genes of FHV-1 showed that the siRNAs directed against the FHV-1 glycoprotein D (gD) gene had some antiviral effect and warranted further study.33 A subsequent in vitro study assessed efficacy of some additional siRNAs alone or in combination and revealed that combined use of siRNAs targeting the FHV-1 DNA polymerase and gD genes was effective.34 However, intracellular delivery of these agents is essential but proving complex. While delivery agents that facilitate delivery of siRNAs into corneal cells in vitro have been developed and these were nontoxic in vitro and nonirritating when applied topically to normal cats’ eyes, thus far they have failed to deliver the siRNAs into corneal cells following topical application in vivo, perhaps due to rapid removal from the ocular surface by tears.35

Immunotherapy. A commercial hyperimmune serum containing antibodies against FCV, FHV-1, and feline panleukopenia virus is available in Europe for treatment of cats with upper respiratory disease and has been assessed in a randomized, placebo-controlled, double-masked clinical trial.194 Forty two cats received hyperimmune serum (n = 22) or saline (n = 20) for 3 consecutive days as well as symptomatic treatment. The serum was administered subcutaneously once daily and topically into eyes, nostrils, and mouth every 8 h. Clinical signs were recorded daily for 8 days and again on day 21. Clinical signs in both groups improved significantly over time (P < 0.001) with treated cats improving earlier (Day 3) than placebo-treated cats (Day 7). In a separate study,195 2 days post inoculation with FHV-1, 9 cats received placebo (n=3), or 10 (n=3) or 30 (n=3) mg/kg of chimeric antibodies developed against FHV-1 in hybridoma cells. Treated cats at both doses showed remarkable reduction in clinical signs. Pre-treatment of cats 15 days before inoculation had similar results.196

Probiotics. To date, I am aware of one prospective, placebo-controlled study of probiotic use for FHV-1 infection in cats.197 Cats were experimentally infected with FHV-1 for another study,179 and then administered as a dietary supplement an immune-enhancing probiotic (Enterococcus faecium strain SF68) or placebo. All cats in both groups showed such minimal evidence of disease that a statistically significant difference between groups could not be demonstrated.

Anti-inflammatory therapy. The use of anti-inflammatory drugs in the management of FHV-1 infections remains controversial.198 However, a return to basic virology and a review of the literature makes some comments possible. Recall that FHV-1 produces disease by (at least) two very different mechanisms that require markedly different (almost opposite) therapeutic approaches. Cytolytic infection represents active viral replication and is often ulcerative. Immunomodulation at this point is almost certainly contraindicated. By contrast, immunopathological (or immune-mediated) injury (such as feline eosinophilic keratitis) is mediated by host inflammatory responses “driven” by persistent viral and/or auto antigens.

The important therapeutic implication is that the immune-mediated disease may involve no or low-grade viral replication and so antiviral agents may not be effective as sole therapeutic agents. However, anti-inflammatory therapy may be indicated (presumably in addition to antiviral agents) for the treatment of immunopathological herpetic diseases.

Corticosteroids Systemic administration of corticosteroids is a well-established and reliable means of inducing viral reactivation from latency.44 This must be considered whenever these drugs are considered in the clinical management of other disease in FHV-1-infected cats. The ability of locally-administered corticosteroids to exacerbate self-limiting primary conjunctival infection and to sometimes induce chronic herpetic keratitis has also been well established.76 Complications seen in corticosteroid treated eyes included deeper and more persistent corneal ulcers, corneal edema, corneal vascularization, sequestrum or band keratopathy formation, and protracted viral shedding. Topical corticosteroids are therefore contraindicated in primary ocular FHV-1 infection. The specific adverse effects of corticosteroids in ocular viral infection are diverse and include inhibition of RNA and DNA, suppression of protein synthesis, decreased antigen presentation by T cells, decreased production of IL2, and impairment of macrophage and cellar immune functions. Therefore, from a basic virological standpoint, corticosteroid administration is difficult to support if active viral replication is occurring. However, because corneal opacification in chronic stromal keratitis occurs secondary to an exuberant immune response, corticosteroids may be indicated in such cases. It would seem prudent, however, to always treat concurrently with a topical antiviral.

NSAIDs and Cyclosporine The potential complications from using corticosteroids have prompted interest in the use of non-steroidal anti- inflammatory drugs (NSAIDs) for managing the inflammatory effects of ocular FHV-1 infection. Although flurbiprofen has been shown to exert some in vitro antiviral effect,199 HSV-1 clinical trials have demonstrated equivocal efficacy. In fact, in rabbits, flurbiprofen has been shown to exacerbate viral keratitis.200 Due to its relatively specific T-cell suppression, cyclosporine therapy has been investigated but has produced equivocal results. Although cyclosporine is capable of suppressing inflammatory events operative in viral stromal keratitis, this drug is also capable of impairing viral clearance from the eye, and beneficial immune responses. In vitro, cyclosporine exerts a dose dependent effect on HSV-1 replication.201 In some experimental model systems, however, cyclosporine therapy resulted in more severe and persistent keratitis.202 In clinical trials, cyclosporine and trifluridine were used in combination to treat HSK in humans with good results,203 and cyclosporine was used (often along with other therapies) for cats with eosinophilic keratitis, typically with good results.204 Other than that, use of NSAIDs and cyclosporine in chronic feline herpetic disease has been inadequately studied and I am unaware of any studies examining the effects of tacrolimus on ocular herpetic infections in any species. This suggests that use of these agents should, as a minimum, be restricted by the same principles that govern the use of corticosteroids in HSK.

Lysine Therapy. Lysine is perhaps the best studied and yet maybe one of the more controversial of all of the other compounds with proven or putative antiviral efficacy against FHV-1 in cats. As with the antiviral drugs, initial interest arose from in vitro data and clinical trials in humans. Lysine’s antiviral effect is believed to arise because arginine is an essential amino acid for FHV-1205 and HSV-1206,207 replication, and assumes that lysine antagonizes arginine availability to or utilization by these viruses during protein synthesis. This was hypothesized to affect protein synthesis of the virus more than the host because viral proteins had a higher arginine-to-lysine content than did human (and feline) proteins.208 And recent analysis suggests that the difference in feline versus FHV-1 protein amino acid content is minimal.209 This may explain why in vitro replication of HSV-1 206,207 and FHV-1205 is notably suppressed in the presence of markedly elevated lysine concentrations in combination with exceptionally low arginine concentrations, but that this is not borne out with more physiologic amino acid concentrations.210 Alternate mechanisms may

explain the suppression of viral replication attributable to lysine supplementation. Firstly, lysine and arginine competitively inhibit the transport of each other, suggesting that these amino acids share a common transport system.211 Secondly, lysine induces arginase, an enzyme that causes the catabolic degradation of arginine.212,213 This enzyme could be responsible for a further increase in the intracellular lysine-to-arginine ratio. In vivo data in cats are also contradictory. Stiles, et al began by demonstrating the safety and efficacy of oral administration of lysine to cats prior to primary exposure to FHV-1.214 Cats receiving 500 mg L-lysine every 12 hours per os (beginning 6 hours prior to experimental inoculation with FHV-1) exhibited less severe conjunctivitis than cats receiving placebo. However, viral shedding, as determined by VI, did not differ between groups. Subsequently, we demonstrated that oral lysine supplementation helps to prevent viral reactivation and/or shedding in latently infected cats.215 Despite significant elevations in plasma lysine concentration, no change in plasma arginine concentration or any ill effects attributable to lysine administration were observed in either study (other than perhaps some mild and reversible GI disturbance in Stiles’ study). I am aware of only one study in which bolus administration of lysine has been tested in naturally infected cats.216 This study examined the effects of lysine in 144 cats residing in a humane shelter. Cats received oral boluses of 250 mg (kittens) or 500 mg (adult cats) of lysine once daily for the duration of their stay at the shelter and outcomes were compared with those of an untreated control group. No significant treatment effect was detected on the incidence of infectious upper respiratory disease (IURD), the need for antimicrobial treatment for IURD, or the interval from admission to onset of IURD. However it was not determined if and to what extent these cats were shedding or infected with FHV-1 (or other pathogens).

Bolus administration of lysine is sometimes impractical, for several reasons. Cats frequently require very protracted therapy (life-long in some cases), and often live in multicat environments in which all cats should be treated. In some multicat situations, twice-daily oral administration techniques may stimulate further viral reactivation through stress or facilitate transfer of infectious organisms among cats by operators. Thus, we studied the safety and efficacy of incorporating lysine into cat food. Results of an initial safety trial were encouraging.217 Cats fed a diet supplemented with up to 8.6% (dry matter) lysine showed no signs of toxicity, had normal plasma arginine concentrations, and had normal food intake. Mean plasma lysine concentration of these cats was increased to levels similar to that achieved with bolus administration. In a subsequent study,218 25 cats with enzootic upper respiratory tract disease were fed a diet supplemented to 5.1% lysine while 25 cats remained on a basal ration (~1% lysine). The study was conducted for 52 days at the beginning of which cats were subjected to rehousing stress (which is known to cause viral reactivation). Ironically, food (and therefore lysine) intake decreased coincident with peak disease and viral presence. As a result, cats did not receive lysine at the very time they needed it most. Perhaps partly because of this, disease in cats fed the supplemented ration was more severe than that in cats fed the basal diet. In addition, viral shedding was more frequent in cats receiving the supplemented diet. To further assess these unexpected outcomes, we performed a similarly designed experiment219 in a local human shelter with a more consistent “background” level of stress and with greater numbers enrolled compared to the initial rehousing study.218 We enrolled 261 cats; each for 4 weeks. Despite plasma lysine concentration in treated cats being greater than that in control cats, again more treated cats than control cats developed moderate to severe disease and shed FHV-1 DNA at certain points throughout the study.219

In summary, there is considerable variability among these studies, especially with respect to methodology, study population, and dose and method of lysine administration. However, taken together, data from these studies suggest that lysine is safe when orally administered to cats and, provided that it is administered as a bolus, may reduce viral shedding in latently infected cats and clinical signs in cats undergoing primary exposure to the virus. However, the stress of bolus administration in shelter situations may well negate its effects and data do not support dietary supplementation. Unfortunately, no clinical trials have been conducted in the very group in which this drug is commonly used - client-owned cats with recurrent herpetic disease. Currently, I let clients help me decide whether they feel a trial of lysine in their cat was effective. I always administer L-lysine at 500 mg twice daily, and ensure that clients do not add it to food for animals to “graze” over an extended period. I also make owners aware that this is usually only an adjunctive or palliative therapy and that administration of antiviral drugs may also be necessary to gain better control of signs. Unlike the protocol for HSV-1-infected humans, owners of cats receiving lysine for FHV- 1 should not be advised to restrict their cat’s arginine intake. Feeding a single arginine-deficient meal to a cat can induce fatal encephalopathy.220 By adopting a common protocol of administering lysine to client-owned cats, it is hoped that well designed, double-masked studies in clinically relevant populations will be forthcoming. Such data would be especially relevant if generated in populations with variable vaccination history, intercurrent disease, physiologic stresses, and high turnover of cats of diverse genetic composition and with varied exposure to infectious diseases.

Vaccination. Vaccination plays a critical role in reduction of clinical signs attributable to FHV-1 in immunologically naïve animals. However, its role in treatment of FHV-1 infection remains controversial. A number of the basic virological issues already introduced, along with data from duration of immunity (DOI) studies bear particular relevance to discussions regarding the benefits and risks of FHV-1 vaccination. Because FHV-1 generally causes self-limiting disease with high morbidity but rare mortality, the benefits of vaccination must outweigh this relatively low risk.

The perfect vaccine for FHV-1 would prevent clinical evidence of disease and block establishment of latency in cats challenged with wild-type virus for a long period following vaccination. Additionally, it would do this without causing adverse side effects and without establishing latency and being shed itself. Currently, such a vaccine does not exist.

Vaccination Route, Immunology, and Safety Typical vaccination strategies for FHV-1 have included use of modified-live, inactivated, or genetically modified virus administered by injection (subcutaneously or intramuscularly) or topically (conjunctivally and/or intranasally). (By the way, some topical vaccines were sometimes mislabeled as “intraocular” vaccines!) Topically administered vaccines appear to have certain merit in the prevention of FHV-1 because the virus gains entry, replicates, causes pathology, and recurs at mucosal sites and because mucosal associated lymphoid tissue (MALT) of the conjunctiva and respiratory tract fulfills distinct and frequently advantageous immunological functions (Table 6).

Table 6. Comparison of the mucosal and systemic routes of FHV-1 vaccination.

Feature Vaccine Route Mucosal Systemic Onset of immunity Rapid Slower Post-vaccinal sarcoma Not recognized Possible Post vaccinal disease 2-4% Rare Potential for spread of vaccine virus Possible Unlikely Generation of mucosal immunity + ? Generation of systemic immunity + + Anamnestic response demonstrated ? +

Mucosal vaccination induces systemic immunity and mucosal immunity both at the site of inoculation and at distant mucosal sites. The onset of mucosal immunity is relatively rapid, with development of partial immunity within 2 days and complete immunity in approximately 4 days.221 In addition, it is suggested that maternal immunity is less likely to interfere with mucosal vaccination than systemic vaccination allowing relatively early vaccination of kittens believed to be at risk. When compared with injectable vaccines, mucosal vaccination is believed to be associated with a higher but infrequent (1.6-4.3%) rate of usually mild side effects. Additionally, mucosal administration of vaccines would not be expected to induce postvaccinal sarcoma formation, whereas the development of postvaccinal sarcomas that are spatially and temporally related to trivalent (panleucopenia/ calicivirus/ herpesvirus) vaccine administration has been reported.222 There may also be some additive effect from simultaneous mucosal and subcutaneous vaccine administration relative to subcutaneous administration alone.223 Finally, there is some early evidence that cats vaccinated with a modified live intranasal FHV-1/feline calicivirus vaccine developed nonspecific protection against subsequent challenge with the completely unrelated pathogen – Bordetella bronchiseptica.224

Vaccination and Latency Although, some vaccines have been suggested to decrease viral load within the trigeminal ganglia,66,225 a vaccine that prevents disease or establishment of latency has not been developed. In fact, there are some disturbing data that suggest some vaccines may themselves become latent within and reactivate from the ganglia.226 Cats in this study were vaccinated topically with a commercially-available, temperature-sensitive mutant virus and monitored for FHV-1 shedding using VI and PCR for 13 weeks. During this period, half were challenged with a field strain of FHV-1 at 28 days post vaccination, and viral reactivation was induced in all cats at 56 days post vaccination. All cats shed the vaccine virus from oropharyngeal or conjunctival tissues at multiple times following vaccination; most

for the duration of the experiment (13 weeks). Vaccination did not significantly reduce the amount of virus shed following challenge with wild type virus, and vaccine virus comprised 32% of all virus shed. Vaccination also did not significantly reduce the amount of virus shed following reactivation. At necropsy, vaccine virus was identified in the same tissues and at the same frequency as wild type virus. Persistent or latent virus was detected in neuronal (trigeminal ganglia, optic nerve, olfactory bulb) and extra-neuronal (cornea, nasal turbinates, and tonsil) sites. Taken together, these data suggest that this intranasal vaccination did reduce clinical evidence of disease but did not limit establishment of latency, reactivation from latency, or viral shedding following challenge. The finding that vaccine virus itself establishes latency and is shed from conjunctival fornices and the oropharynx suggests that it could be a source of antigen and thereby cause or exacerbate chronic herpetic disease, infect in-contact cats, or be detected by diagnostic assays and alter their interpretation.113 Another pilot study assessed FHV-1 shedding 7 days prior to and 28 days following intranasal vaccination and found it to be infrequent during both time periods.227

Vaccination and Disease Severity Given, the questionable ability of vaccination to act as an epidemiological control tool via inhibition of latency, the major role for FHV-1 vaccination appears to be diminishing disease severity. Most vaccine studies have tended to emphasize the dramatic reduction in severity of clinical disease when animals are challenged at varying periods after vaccination. A study of duration of immunity for FHV-1 vaccination administered subcutaneously to young kittens provides further interesting information.68 While antibody titers appeared to wane rapidly between 6 and 7.5 years post vaccination, cats showed a rapid and exaggerated anamnestic humoral response upon challenge - even in the absence of a detectable pre-challenge titer. Even 7.5 years after vaccination, clinical evidence of disease following challenge was reduced by approximately half and fatalities were completely prevented. Importantly though from an epidemiological standpoint, vaccination did not reduce the amount or duration of virus shed following challenge. I am unaware of any reports demonstrating that vaccination is beneficial in cats that are already infected with FHV-1; however this is an area of growing interest, especially in light of early evidence that intranasal vaccines may infer resistance of a nonspecific nature.224

FELINE INFECTIOUS PERITONITIS

Virus Features FIP is taxonomically positioned in the family Coronaviridae. This is a large enveloped RNA virus, and the largest of all RNA genomes. For a review see the paired articles by Pedersen.{Pedersen, 2014 #5763;Pedersen, 2014 #5764} No citation available yet). In that article, Pedersen succinctly summarizes the frustrations regarding this virus: “After a half century, FIP remains one of the last important infections of cats for which we have no single diagnostic test, no vaccine and no definitive explanations for how virus and host interact to cause disease. How can a ubiquitous and largely non-pathogenic enteric coronavirus transform into a highly lethal pathogen? What are the interactions between host and virus that determine both disease form (wet or dry) and outcome (death or resistance)? Why is it so difficult, and perhaps impossible, to develop a vaccine for FIP? What role do genetics play in disease susceptibility?” FIP has now also been diagnosed in ferrets.

The internal mutation theory, whereby a ubiquitous feline enteric coronavirus (FECV) mutates into a FIPV, is strongly supported by the literature, and at least three specific mutations have now been associated with the FECV- to-FIPV biotype conversion. The working hypothesis is that the FECV-to-FIPV transition involves positive selection for mutants that are increasingly fit for replication in macrophages and unfit for replication in enterocytes. The ultimate target cell is a distinct population of precursor monocytes/macrophages that have a specific affinity for the endothelium of venules in the serosa, omentum, pleura, meninges and uveal tract. Variability in genetic susceptibility has been identified in at least one breed of cats, but the genetics appear to be highly complex and cannot explain the entire disease incidence. Rather, it seems likely that FIP results from a confluence of numerous viral, host and environmental cofactors.

Pathogenic Mechanisms Infection with the FECV results in mild, self-limiting, and presumably often unnoticed gastrointestinal signs. In a small percentage of cats mutation to the FIPV occurs. Although FIP can affect cats of any age, it is diagnosed most commonly in cats less than 3 years of age and especially from 4-16 months of age.228 Diseases is seen most commonly in cats living within multicat settings. Mortality is extremely high although some cats can live with the disease for weeks to months. In cats with relatively mild presenting signs of the dry form of FIP, the 1-year survival

rate was 5%.{Pedersen, 2014 #5764} The basic pathology involves vasculitis and associated pyogranulomatous perivascular inflammation, both of which are thought to be due to an Arthus-type reaction (virus-antibody- complement).229,230 The immunopathogenesis of FIP is extremely complex and still not well understood. In his latest review, Pedersen provides an articulate summary: “It is widely assumed that immunity, when it occurs, is largely cell-mediated and that the production of antibodies is counterproductive. Antibodies enhance the uptake and replication of FIPVs in macrophages and also contribute to a type III hypersensitivity (antibody-mediated or Arthus- type) vasculitis.228 It is also assumed that much of the pathology occurring in FIP is associated with how macrophages respond to viral infection and how the immune system of the host responds to the infected cells. In this scenario, the effusive form of FIP results from a failure to mount T cell immunity in the face of a vigorous B cell response. At the opposite extreme, cats that resist disease presumably mount a vigorous cell-mediated immune response that is able to overcome any negative effects of antibodies. Cats with the dry form of FIP represent an intermediate state involving a cellular response that is partially effective in containing the virus to a relatively small number of macrophages in a few focal sites within specific target organs. The two forms of FIP are somewhat interchangeable; when it has been observed in experimental infection, the dry form always follows a brief bout of effusive disease. In the terminal stages of naturally occurring dry FIP, immunity can completely collapse and the disease reverts to a more effusive form. Although this scenario fits what is known about FIP, it must be emphasized that much of this scheme awaits confirmation and there are large gaps to be filled.” Ocular lesions occur more commonly with the dry form of FIP, result from the same mechanisms operative systemically, and are estimated to occur in 25% of infected cats. The principal target tissue is the uveal tract, particularly the choroid; the optic nerve may also be involved. Characteristic clinical signs are aqueous flare, keratic precipitates, iris swelling and color changes, hyphema, and retinal hemorrhages, perivascular exudates and detachment.231-233

Diagnosis of FIP Due to cross reactivity between the enteric coronavirus, serology is not useful as a sole test for diagnosing FIP infection. Rather, histopathological and immunohistochemical examination of affected tissues has long been considered the only reliable form of diagnosis. A large retrospective study appraised the commonly used diagnostic tests for FIP and calculated their positive (PPV) and negative (NPV) predictive values with respect to histopathologically confirmed cases.234 Individual serologic and hematological tests all had low diagnostic value. However, in cats with clinical signs suggestive of FIP, the presence of lymphopenia, hyperglobulinemia, and an antibody titer of 160 or greater had a PPV of 89%, whereas the absence of all 3 of these findings had a NPV of 99%. Nested RT-PCR directed toward a unique gene coding for peplomer protein E2 of the FIP virus produced sensitivity and specificity of 91.6% and 94% respectively.235 However, no diagnostic assay at this stage reliably differentiates FeCV from FIP virus. This, along with knowledge that FeCV (and FIP) can be detected in circulating blood cells, means that results of so-called “FIP” PCR conducted on aqueous humor samples collected from cats with uveitis must be interpreted with caution.

FELINE IMMUNODEFICIENCY VIRUS

Virus Features The taxonomic classification of FIV is the family - Retroviridae, Subfamily - Lentivirinae, Group - Lentiviruses. FIV consists of a 100-nm diameter, enveloped virion. The genome consists of single-stranded RNA.

Pathogenic Mechanisms FIV Retinopathy. FIV can cause geographic areas of thinning of the sensory retina, characterized ophthalmoscopically by hyper-reflectivity. Flame hemorrhages may also be present. Histologic findings are degeneration of all retinal layers with minimal inflammation. The lesion has been seen in naturally and experimentally infected cats, suggesting it is due to FIV and not opportunistic infections. Lesions occur during periods of higher viremia. These lesions have developed during the acute phase of infection (first 6 months) in experimentally infected cats, and during the AIDS-like stages in natural infection. The pathogenesis of this lesion is probably the same as the microvascular retinopathy that occurs in HIV infection, and the neurological degeneration that occurs in FIV infected cats and HIV infected humans. Two hypotheses have been proposed.

1. Neurotoxin; both FIV and HIV are capable of enhancing excitatory amino acid toxicity in cultured fetal neurons. This toxicity can be blocked using N-methyl-D-aspartate (NMDA) receptor antagonists. Glial cells must be present however for the toxicity to occur, suggesting the effect of the virus is indirect and mediated by

glial cells. These viruses do not appear to infect neurons, but can infect microglia, and possibly astrocytes.

2. The second hypothesis is that FIV/HIV results in increased vascular permeability and altered hemodynamics. The finding of cotton wool spots and retinal hemorrhages suggests vascular compromise does occur, but the pathogenesis of these lesions and their importance remains unclear.

FIV Anterior Uveitis. FIV is capable of causing anterior uveitis that can be transient or chronic and may persist despite anti-inflammatory therapy and lead to secondary glaucoma. Mononuclear inflammatory cell infiltrate adjacent to the base of the iris and anterior ciliary body are characteristic (but of course not pathognomonic) histologic features. The pathogenesis is unknown. Lappin et al suggests that opportunistic Toxoplasma infection plays a role.236 It is not known if this is a primary FIV-induced lesion or predominantly the result of opportunistic disease. FIV can be isolated from the aqueous humor of infected cats.

FIV-Associated Anterior Hyalitis (vitritis). An accumulation of WBC's in the anterior vitreous, especially around the lens zonules, may be seen in FIV infected cats, often producing a "snow bank" appearance.237 Although the lesion is not pathognomonic, most cats are FIV positive. The lesion is often an incidental finding and not associated with discomfort or other signs of anterior segment disease. Lens luxation and secondary glaucoma can result. Histologically these cells have been shown to be predominantly plasma cells. The pathogenesis of this lesion is not known but presumably arises from a pars planitis.

FIV-Associated Conjunctivitis. Mild, self-limiting conjunctivitis may be seen in acute FIV infection. FIV infection may also worsen the course of FHV-1 infection.97,238

FIV-Associated Anisocoria. Mild to moderate mydriasis may be seen in association with FIV infection, and both motor and sensory abnormalities have been described. The changes are usually permanent and slowly progressive. The pathogenesis is not known, but possibly related to CNS disease. Similar problems have been seen in HIV patients.

FIV-Associated Neoplasia. FIV-infected cats have a much higher rate of B-cell lymphoma than uninfected cats. These tumors tend to occur at atypical sites such as the eye or CNS, and can occur without lymph node or bone marrow involvement. Intestinal and renal lymphomas have developed in experimentally-infected SPF cats. The lymphomas examined to date using virus culture, PCR, and flow cytometry have been negative for virus, as is typical for HIV. The pathogenesis is unknown, but may be secondary to chronic type 1 immune activation occurring after HIV infection. A study that utilized nested PCR failed to demonstrate FIV DNA in any of 18 feline globes enucleated due to histologically confirmed uveal melanoma.239

FELINE LEUKEMIA VIRUS

Virus Features FeLV is in the family Retroviridae, subfamily Oncovirinae, Group Mammalian C-Type. It has an enveloped RNA genome.

Pathogenic Mechanisms Systemic. Viremia seeds virus to lymphoid and epithelial tissue, and bone marrow. Clinical signs are related to ability of the virus to induce hematological changes, immunosuppression, and tumor formation.

Ocular. The only ocular changes definitively linked to FeLV are retinal dysplasia, tumor formation, retinal hemorrhages, and bizarre pupillary changes.240-246

Retinal dysplasia: Exposure of fetal and neonatal kittens to FeLV can result in retinal dysplasia, which occurs secondary to diffuse retinal inflammation. Infection causes progressive disorganization and necrosis of retinal tissues, with subsequent reorganization into cell clumps and dysplastic rosettes. RPE proliferation and migration into the sensory retina were also described. The mature retina demonstrated full thickness folds and tubes associated with folding of the RPE. After experimental inoculation, retinal tumor formation is also possible.

Anemic retinopathy: Retinal hemorrhages have been seen in cats with FeLV and were originally attributed to

anemia. However a prospective study of the prevalence of retinal hemorrhage in dogs with anemia, thrombocytopenia, neither, or both reveals that anemia alone is not associated with retinal hemorrhage.247 Thus, further assessment of FeLV-infected cats with this finding is warranted.

Lymphosarcoma: Lymphosarcoma, especially of the anterior uvea, but also involving the conjunctival and retrobulbar tissues reflects the oncogenic effect of this virus. This is sometimes seen in the absence of identifiable tumor elsewhere in the body.

“Spastic Pupil” Syndrome: Although the data supporting this are limited, in all probability this represents FeLV infection of the autonomic nerves (parasympathetic portion of CN III). A wide variety of spontaneously changing pupillary abnormalities is possible.

Corneal Latency: In a study of normal corneas, FeLV DNA was demonstrated (using PCR) in 65% (11/17) FeLV- positive cats (as determined by p27 ELISA) and none of 17 FeLV-negative cats.248 Virus was localized to corneal epithelium using immunohistochemical staining techniques. This information must be considered when selecting suitable donor cats for corneal grafting procedures.

FELINE PANLEUKOPENIA

Virus Features This virus is in the family Parvoviridae, Genus - Parvovirus. Characteristics: Non-enveloped virions, icosohedral symmetry, 18-26 nm in diameter, virions have 32 capsomeres. Replication and assembly take place in the nucleus. Virions are very stable.

Pathogenic Mechanism. Dysplastic ocular development has been attributed to intrauterine infection of kittens with the panleukopenia virus.249 In one case report of a 6-week-old kitten, lesions were characterized by retinal thinning, loss of the normal architecture, and rosette formation.250 The virus was recovered from the cerebrum. Cerebellar hypoplasia was also present. Hydrancephaly has also been reported in association with perinatal panleukopenia infection.251 The optic nerves were small, with increased perineural connective tissue.

FELINE SARCOMA VIRUS (FeSV)

Virus Features Taxonomic classification: Family - Retroviridae, Subfamily - Oncovirinae, Group - Mammalian C-type. Horizontally transmitted oncogenic RNA virus. Replication-defective variant of FeLV.

Pathogenic Mechanisms Experimental Sarcoma Virus Uveitis. In experimental studies, FeSV induced anterior uveitis 37 days after subcutaneous injection.252 Iritis, posterior synechia, and cataracts were seen. The cellular infiltrate was lymphocytic and plasmacytic. Detection of large quantities of virus in the aqueous humor suggests that intraocular viral replication occurs, but inflammatory changes have been attributed to virus-antibody interactions, or a cell-mediated immune response.

Experimental Sarcoma Virus-Induced Uveal Melanoma. Intracameral injection of FeSV induces formation of uveal melanoma in cats.253,254 Tumors develop between 40 and 60 days after injection, and are composed of spindle and epithelioid cells. The tumors eventually enlarge to fill the anterior chamber. The posterior segment is involved only late in the course of the disease. More recently, nested PCR was used to examine globes enucleated due to feline uveal melanoma. DNA common to FeLV and FeSV was detected in only 3/36 globes.239

FeSV-Associated Fibrosarcomas. Fibrosarcomas of the eyelids in young cats have been associated with the oncogenic FeSV virus.243 However, in a study using PCR and immunohistochemistry, no association between FeLV or FeSV and ocular sarcoma could be demonstrated in biopsies from 6 cats.255

CANINE HERPESVIRUS

Virus Features Taxonomic classification: Family - Herpesviridae (DNA), subfamily Alphaherpesvirinae. Host Range: Canine only. Viral replication is similar to FHV-1.

Pathogenic Mechanism The virus shows marked host specificity for canidae.19 Traditionally, CHV-1 infection in dogs has been described as causing one of two relatively clearly defined syndromes believed to depend almost exclusively on the age of the affected dog – severe and often fatal systemic disease (sometimes with ocular signs) in young puppies, and mild conjunctivitis, vaginitis, or upper respiratory disease in adults.

Young, immunologically naïve puppies are believed to become infected in utero, or following perinatal contact with contaminated reproductive and respiratory secretions. Experimental infection of newborn puppies is associated with keratitis, uveitis, retinal necrosis and optic neuritis.256,257 The lesions are the result of virally-induced inflammation. Experimentally infected puppies develop panuveitis with the presence of intranuclear inclusion bodies within 4 days of infection. Necrosis, retardation of maturation, and reorganization into folds and tubes occur throughout the retina. Depigmentation and vacuolization of the RPE, with subsequent folding and hypertrophy are also seen. In some animals, ectopic retina was found within cystic spaces in the optic nerve. Death in puppies may occur from disseminated viral replication in parenchymal organs and the CNS. Like FHV-1 infection, lifelong neural latency is believed to occur in many affected animals with immunosuppression causing reactivation. By contrast, clinical signs in adults are typically very mild and include transient vaginitis, mild upper respiratory disease, or conjunctivitis.

However, reports have suggested that this clear age-related demarcation is not necessarily reliable with older dogs getting the fatal systemic forms of the disease and puppies and older getting mild ocular disease. For example, an adult dog receiving chemotherapy for lymphoma experienced the severe disseminated syndrome usually seen in puppies infected with CHV-1.258 Meanwhile, corneal disease has been described in 3 adult dogs with naturally- acquired CHV-1 infection.259,260 These dogs had classic dendritic ulcers, as well as nonspecific signs of ocular surface inflammation, which responded to cessation of topical immunomodulating drugs and initiation of topically administered antiviral agents. CHV-1 was isolated from ocular swabs. All dogs had altered local and/or systemic immunity (diabetes mellitus, KCS, topical administration of corticosteroids or cyclosporine, etc.). These cases suggest that these unusual presentations may, at least in part, be due to more regular local (ocular) and systemic immunosuppression of pet dogs as a result of or during treatment of other chronic diseases. However it is unlikely to be that simple. For example, the typical fatal form of hepatic necrosis described originally in only young puppies has been described in an apparently immunocompetent adult (9yo) dog.261 Likewise, a natural outbreak of CHV-1- induced ocular disease was described in a colony of 27 juvenile dogs but in the absence of the usual fatal systemic clinical syndromes and in which no source of immunosuppression was noted.262 Although, in affected dogs within the colony, marked bilateral conjunctivitis was a consistent sign (sometimes with petechiae), many dogs also developed ulcerative (26%) - often dendritic (19%) - or perilimbal stromal nonulcerative keratitis (19%). Antiviral treatment was not initiated. Another adult dog was presented with partial limbal stem cell deficiency and neurotrophic keratitis subsequent to a protracted episode of CHV-1 dendritic ulcerative keratitis (metaherpetic disease).263

Despite these striking clinical reports and the susceptibility of the canine cornea to CHV-1 infection in vitro,264 canine herpetic disease and shedding at the ocular surface still appear to occur infrequently when compared with the prevalence of FHV-1 on the feline cornea or conjunctiva.265 For example, samples collected from 50 dogs without surface ocular disease and 50 dogs with non-dendritic corneal ulcers all were negative when tested for the presence of CHV-1.259 In a separate virologic survey assessing the prevalence of CHV-1 (and 6 other viruses) in conjunctival samples from dogs with or without conjunctivitis, no virus (of any type) was ever detected in dogs without conjunctivitis.266 In dogs with conjunctivitis, CHV-1 (5 dogs) or CAV-2 (2 dogs) was detected by VI and/or PCR occasionally but significantly more commonly than in normal dogs. Further evidence of the degree of natural resistance to corneal, systemic and severe recurrent ocular disease in most dogs is provided by a study in which adult SPF beagles were infected with CHV-1 with or without prior disturbance of the corneal epithelial surface and/or subconjunctival corticosteroid administration.267 Dogs developed mild to moderate bilateral conjunctivitis, shed virus, and seroconverted but corneal disease was not noted, epithelial disturbance or corticosteroid administration did not worsen signs, and all dogs spontaneously recovered without evidence of spontaneous

reactivation or recrudescence for 224 days post inoculation.267 Likewise, a single fraction 50 Gy radiation dose administered to the ocular surface by strontium-90 beta radiotherapy did not result in detectable recurrent ocular CHV-1 infection in mature dogs with experimentally induced latent infection.268

By contrast, pharmacologically induced reactivation appears to be important in the pathogenesis of this virus, but highly dependent upon drug chosen and route administered. Dogs latently infected with CHV-1 via experimental inoculation 8 months previously and subsequently administered prednisolone systemically (3 mg prednisolone/kg/day x 7 days)269 experienced bilateral ocular disease (83%), viral shedding (50%), and 4-fold elevations in serum CHV-1 SN titers (100%). The same dogs where stimulated 12 months later (20 months after inoculation) by using topically applied 1% prednisolone acetate (1 drop OU QID x 28 days) and did not experience viral reactivation (detectable by quantitative PCR or SN) or recrudescent disease (detectable by slit lamp examination or ocular confocal microscopy).270 When the potent immunosuppressive agent cyclophosphamide was administered at 200 mg/m2 to mature dogs latently infected with CHV-1, evidence of myelosuppression was noted on Day 7, but no ocular signs suggestive of recurrent CHV-1 were noted clinically or with confocal microscopy, and ocular CHV-1 shedding was not detected by PCR.271 Finally, 8 mature Beagles with experimentally-induced latent CHV-1 infection proven to be reactivatable when prednisolone was systemically administered received 0.2% cyclosporine ointment in both eyes twice daily for 16 weeks. This failed to produce detectable virus at the ocular surface or signs of herpetic disease.265 (For a recent review of the ocular effects of this virus see the article by Ledbetter EC.)272

To date, very little work has been done to define preferred antiviral drugs for dogs with herpetic disease. In his original case series, Ledbetter recounts cases improving while on topically applied idoxuridine or trifluridine,259 and a separate case report describes improvement of a dog treated with cidofovir.260 In a subsequent controlled study,273 the in vitro efficacy of cidofovir against CHV-1 was established, and a 0.5% solution was applied topically BID for 14 days in dogs with experimentally-induced recurrent ocular CHV-1 infection. Although cidofovir displayed similar in vitro antiviral activity against CHV-1 and HSV-1, and treated dogs had significantly reduced durations of ocular viral shedding compared to the vehicle group, ocular disease scores were significantly higher in dogs receiving cidofovir compared to those in the vehicle group. Marked conjunctival pigmentation and blepharitis were also detected in the cidofovir group, but not the vehicle group. Conjunctival and corneal leukocyte infiltration scores determined by in vivo confocal microscopy were significantly higher in the cidofovir group. By contrast, a placebo- controlled experimental infection model revealed that trifluridine applied 4-6 times daily reduced clinical signs and viral shedding in latently infected dogs undergoing pharmacologic reactivation.274

CANINE DISTEMPER VIRUS (Carre's Disease, 1905)

Virus Features Taxonomic classification: Family - Paramyxoviridae (RNA), Genus - Morbillivirus. Structure: Negative strand RNA virus. Consists of 2 structural modules; an internal ribonucleoprotein core (nucleocapsid) containing single stranded viral RNA genome, and an outer spherical lipoprotein envelope.

Pathogenic Mechanism General. Direct cytolysis. Target tissues are epithelial and neural.

Systemic. Route of infection is via the respiratory tract. Virus then spreads from oropharynx to regional lymph nodes and tonsil. Viremia disseminates virus to lymph tissues throughout the body. Virus persists in epithelial tissues of dogs unable to mount a protective immune response.

Ocular. Tissues affected are conjunctival epithelium, lacrimal gland, retina, optic nerve and optic tracts. In the earliest report, retinitis, optic neuritis, and destruction of the optic tracts were described.275 Acute retinitis is characterized by congestion, edema of the nerve fiber layer and inner plexiform layer, and perivascular cuffing.276-278 Intranuclear inclusions may be seen in retinal glia. Sequelae are optic nerve degeneration, and focal to diffuse areas of retinal degeneration characterized ophthalmoscopically in the tapetal fundus by hyperreflectivity, and by RPE pigment loss in the non-tapetal fundus. Distemper inclusions have also been found in the ciliary body of a dog.279 In a subsequent retrospective study, 41% (9/22) of dogs with distemper were noted to have retinochoroiditis, and 54% were positive by IFA testing of conjunctival smears.280

CANINE ADENOVIRUS 1 (Infectious Hepatitis Virus, Rubarth's Disease, 1947)

Virus Features Taxonomic classification: Family - Adenoviridae (DNA). Adenoviridae are divided into two genera, Mastadenovirus and Aviadenovirus. The former includes canine adenovirus. Structure: Non-enveloped, regular icosahedrons, 65-80 nm in diameter. Capsid is composed of 250 subunits (capsomeres). Replicative Cycle: Attachment, penetration, uncoating, transcription, gene expression, protein synthesis, and assembly of virions.

Pathogenic Mechanisms Systemic. Natural infection occurs via the oropharynx, with subsequent spread to regional lymph nodes. Principal tissues involved are the liver, kidney, lymph nodes, and vascular endothelium.

Ocular. Viral invasion of the eye occurs in acute infection. Mild anterior uveitis results from virus replication in vascular endothelium. Corneal lesions developed between 7 and 21 days after experimental subcutaneous inoculation.281 Moderate to severe iridocyclitis was the consistent finding in all eyes that developed edema. The presence of a loose fibrin clot in the anterior chamber was a consistent finding in opaque eyes. Using IFA, viral antigen was identified in the vascular endothelium of the iris, trabecular endothelial cells, and macrophages. Virus was also found in corneal endothelium. Uveal infiltrate includes lymphocytes and plasma cells. Relatively low levels of virus were recovered from the eyes compared to other organs.

Ocular damage has been suggested to be predominantly mediated by a Type III immune response (Arthus reaction).282,283 Twenty-four dogs were inoculated with ICH; 9 dogs received virulent virus subcutaneously, and 15 dogs received attenuated virus intravenously. After 4-8 days, aqueous humor was aspirated and the anterior chambers injected with 0.2 ml of anti-ICH serum. Fourteen of 24 dogs developed eye disease, and in 2, corneal edema was severe. Purified viral antigen and anti-ICH serum was then injected into the anterior chamber of 8 dogs; intense iritis with severe edema occurred. The reaction was found to be more severe if complement was added to the immune complexes. In a subsequent study, viral replication was found to occur consistently in the corneal endothelium.284 Virus infection was found to occur not by contagious spread, but as the result of endothelial adsorption of virus that was released into the aqueous humor. It was concluded that parenteral inoculation with ICH does not in itself cause severe or permanent corneal endothelial cell dysfunction.

Prevalence. Ocular disease is estimated to occur in 20% of naturally occurring cases, and in 0.4% of dogs given modified live vaccine. Afghan hounds have been suggested to be predisposed to more severe ocular lesions after inoculation with attenuated CAV-1 vaccine than are beagles.285

EQUINE HERPESVIRUS

Virus Features Family: Herpesviridae.

To date, five genetically distinct equine herpesviruses (EHV) have been definitively identified: EHV-1 through EHV-5. As more accurate molecular techniques are used, it becomes obvious that previous reports often failed to differentiate between some of these viruses. Until relatively recently, EHV-1 and 4 were considered subtypes 1 and 2, respectively of EHV-1, while EHV-2 and 5 were not treated separately in the literature until around 1988, and some papers since then have continued to inadequately differentiate the two viruses.286,287 Interpretation of older data is therefore often difficult or impossible.

Although equine herpesviruses are frequently incriminated as ocular pathogens, little is known about their true role, and much evidence is based upon response to therapy. In fact, Koch’s postulates have rarely been demonstrated for the ocular signs currently blamed on the equine herpesviruses.288,289 A survey of ocular lesions and herpesvirus serology in a herd of 266 Lipizzaner horses in Austria sheds further light on the prevalence of these viruses but reinforces the difficulty of associating them with ocular syndromes.290 EHV-2 DNA was identified in 167 (62.8%) of the horses and EHV-5 in 136 (51.1%) horses; 105 (39.5%) were co-infected with EHV-2 and EHV-5. Horses carrying EHV-2 or EHV-5 DNA were significantly younger than the negative group. No significant association between any ocular disease pattern and presence of EHV-2 or EHV-5 was detected. A closely related study in the

same population of horses revealed no association of ocular lesions and herpesviral infection status when horses were examined serially every 6 months for 18 months.291 A smaller study in Northern California assessed 12 horses suspected of having herpetic ocular surface disease and 6 normal horses.292 Prevalence of EHV-1, -2, -4, and -5 DNA as assessed by qPCR did not differ significantly between control horses and those with idiopathic keratoconjunctivitis. Neither EHV-1 nor EHV-4 DNA was detected in any normal or affected horse; EHV-2 DNA was detected in 2/12 affected horses but in no normal horses. EHV-5 DNA was commonly found in ophthalmically normal horses and horses with idiopathic keratoconjunctivitis. Taken together these data suggest that (just as in the cat) diagnostic testing in individual cats is of extremely limited value.

Antiviral Efficacy Topically applied antiviral agents have been used to successfully treat herpetic keratitis in the horse, although no drugs are currently labeled for equine use. These drugs have undergone little testing with respect to their efficacy against the equine herpesviruses particularly EHV-2; however a recent review is comprehensive and very helpful for the alphaherpesviruses (EHV-1, -3, and -4) but has little information regarding EHV-2 and -5).293 When equine- specific data are not available, expected efficacy and application frequency are usually extrapolated from data generated in humans infected by HSV-1 and cats with FHV-1. Some general comments gained from those sources are likely to apply to horses and equine herpesviruses. All of the currently available antiviral agents are virostatic in action and therefore should be applied frequently. Although the treatment regimen recommended for humans (two- hourly application throughout the waking hours until the ulcer has healed) is usually not possible in horses, as frequent application as possible is recommended and therapy should be initiated early in the disease course. A minimum of four applications daily is probably indicated. The antiviral agents are variably toxic to corneal and conjunctival epithelium. In some cases this is noted as an apparent stinging reaction when the medications are applied. In more marked situations, delayed ulcer healing may occur.

There have been some studies assessing the in vitro antiviral efficacy against equine herpesviruses of some of the agents commonly used for human and feline herpesviruses. These are reviewed by Vissani.293 For EHV-1, these can be summarized (from most to least effective) as: ganciclovir cidofovir > acyclovir penciclovir > adefovir > foscarnet > brivudin.293,294 The majority of clinical reports for ophthalmic disease describe topical application of either idoxuridine or trifluridine for the treatment of herpetic ≅keratitis in horses. Some≅ reports suggest antiviral drugs developed for HSV-1 and used in the treatment of FHV-1 are effective.295-297 Acyclovir given orally to horses is 298,299 294 poorly bioavailable and reaches plasma concentrations that do not exceed the in vitro IC 50 for EHV-1. By contrast, oral administration of the acyclovir prodrug – valacyclovir – achieves concentrations within the sensitivity range of EHV- 1.299,300 In spite of these promising results, studies in horses experimentally infected with EHV-1 have not yielded consistently positive data.293,301 Plasma penciclovir concentrations achieved after horses received a single oral dose of 302 20 mg famciclovir/kg were sufficient to exceed the IC 50 for EHV-1; however, to my knowledge, none of these drugs have been tested in a prospective placebo-controlled therapeutic efficacy trial and tear pharmacokinetics have not been studied as they have in cats.82,174 The latter parameter seems particularly relevant in horses given that equine herpetic keratitis often lacks a robust vascular response. None of the antiviral agents has known antibacterial properties and therefore horses with ulcerative keratitis should probably be treated with a topical antibiotic agent in addition to any antiviral agent.

Clinical Syndromes Ocular signs associated with equine herpesviruses may be divided into three broad categories: • Primary ocular disease in the absence of systemic signs - for example, keratoconjunctivitis due to EHV-2 infection or chorioretinitis due to EHV-1. • Ocular signs secondary to neurological disease - for example, keratitis secondary to facial nerve paralysis or blindness secondary to cerebral vasculitis seen with EHV-1. • Ocular signs as a minor and self-limiting component of a more serious upper respiratory syndrome - such as mild and transient conjunctivitis seen in horses with severe rhinopneumonitis due to EHV-1 or -4 infection.

Keratoconjunctivitis Herpetic conjunctivitis is seen in association with respiratory disease syndromes following infection with EHV-1 or -4, and alone or with keratitis with EHV-2. Clinical signs are non-specific and all may occur as outbreaks. In the case, of EHV-1 or –4, bilateral conjunctivitis is usually a relatively minor feature of a significant respiratory infection. Younger animals are more severely affected.

In horses with keratitis/conjunctivitis or both (in the absence of respiratory signs), some data seems supportive of a role for EHV-2. For example, the prevalence of EHV-2 in horses with keratoconjunctivitis (12 of 27) was significantly higher than in clinically healthy horses (2 of 12).303 Response to treatment with trifluridine in 9 of 16 cases further supported an etiological role for EHV-2. Similarly, herpetic keratitis due to corneal replication of virus has been suggested to occur with EHV-2 only.296,297,304 This syndrome seems to be diagnosed more commonly in the United Kingdom than in other parts of the world, although the reason for this is unclear. Herpetic keratitis, and often keratoconjunctivitis, may occur in individual horses or as small outbreaks, especially in young horses, and is often unilateral. Concurrent respiratory signs are usually absent or very mild. Recurrences are likely. Superficial ulcerative and non-ulcerative keratitis is described and may represent different stages of the same disease process. Corneal opacities range widely in appearance from ring-shaped lesions, a more generalized stippling or “orange- peel” appearance, or reticulated or lace-like (dendritic) networks. In all cases, their multifocal nature is highly suggestive. Variable corneal stromal inflammatory cell infiltration, edema, and neovascularization have been described. Uveitis, if present, appears to represent “reflex uveitis” mediated by the axonal reflex and is usually manifest as a miotic pupil without flare.

Corticosteroids are contraindicated in the treatment of ulcerative disease in the horse and active herpetic disease in all species studied so far. Some reports exist of topical or systemic corticosteroids causing exacerbation or recurrence of herpetic keratitis in horses. For these reasons, it seems wise to avoid the use of topical or systemic corticosteroids in horses with active herpetic keratitis. The use of topical non-steroidal anti-inflammatory agents is controversial but should also probably be best avoided in herpetic keratitis.

Anterior uveitis frequently occurs with herpetic keratitis and is a significant component of the ocular pain seen in this disease. This usually responds very well to one or two applications of topical applications of atropine ointment. Atropine should be given to effect (i.e., until wide mydriasis is achieved) and then given as often as necessary to maintain pupil dilation. Frequently, only once or twice weekly application is required.

Ocular Signs Secondary to Neurological Disease Infection is generally sporadic but dramatic when it occurs. Ocular signs secondary to EHV-1 myeloencephalitis include nystagmus, blindness, strabismus, ptosis, exposure keratitis, optic neuritis, and retinal hemorrhage. These ocular signs represent manifestations of altered neurological function rather than virally induced ocular pathology. The ocular signs may result from CNS or peripheral cranial nerve injury, often secondary to vasculitis. The etiology of the exposure keratitis is presumably related to injury to the trigeminal (CN V) and/or facial (CN VII) nerve with resultant inability to sense corneal dryness, stimulate tear production by the lacrimal gland, and/or blink. Additionally, CN V is probably responsible for the supply and release of various trophic factors essential for normal corneal health. This means that the perceived need for, production of, and dispersion of tears and other corneal neurotrophic factors can all be affected leading to profound exposure keratitis.

Uveitis Anterior uveitis or diffuse chorioretinitis has been described in horses in which EHV-1 infection has been diagnosed or experimentally induced. The exact causal association however remains undefined. In one case,305 a foal experimentally infected with EHV-1 developed severe visual impairment and bilateral, chronic diffuse chorioretinitis approximately one month after experimental inoculation with EHV-1. Anterior uveitis was not seen and a causal association with EHV-1 was not demonstrated. A separate report306 describes an otherwise typical outbreak of neurological disease and neonatal mortality attributed to EHV-1 in which a number of affected foals had evidence of anterior uveitis in addition to more classic pneumonic and/or gastrointestinal signs. Clinical signs sometimes included blindness and hypopyon, both of which resolved. In these animals, uveitis may be a manifestation of a more generalized vasculitis or may relate to super-infection with another pathogen secondary to EHV-1-mediated immunosuppression. An experimental study307 provides more definitive evidence that EHV-1 is a cause of multifocal chorioretinal lesions (typically classic “bullet hole” lesions in the peripapillary fundus) in 50- 90% of experimentally infected horses. The lesions were evident between 3 weeks and 3 months after infection. The authors proposed that this was a vascular (ischemic) event. Chorioretinitis, optic neuritis, vitritis, retinal vasculitis, and encephalitis have also been described in camelids infected with EHV-1.308,309

OTHER VIRUSES CAPABLE OF OCULAR INVOLVEMENT IN THE HORSE

Equine viral arteritis - conjunctivitis, hypopyon, iridocyclitis, and keratitis. African horse sickness - conjunctival and eyelid edema. Equine adenovirus infection - conjunctivitis. Borna Disease - retinal infection, optic neuritis. Rabies - viral replication in retina and optic nerves. Equine viral encephalitis - blindness due to encephalitis. Equine infectious anemia - conjunctival hemorrhages.

BOVINE HERPESVIRUS I (Infectious Bovine Rhinotracheitis, IBR)

Virus Features Taxonomic classification: Family - Herpesviridae, Subfamily - Alphaherpesvirinae. Structure, replication, and tissue damage is typical of that of other alphaherpesviruses.

Pathogenic Mechanisms Systemic. Upper respiratory disease, tissue damage by direct cytolysis.

Ocular. Ocular disease may occur with or without respiratory signs. Conjunctivitis is the predominant feature, with formation of white conjunctival plaques composed of mononuclear cells (plasma cells and lymphocytes).310-312 Keratitis is also possible.

BOVINE VIRUS DIARRHEA VIRUS (BVD; Mucosal Disease)

Virus Features Taxonomic classification: Family - Togaviridae, Genus - Pestivirus (mucosal disease viruses). Virus structure: Lipid containing envelope with surface projections surrounding a spherical nucleocapsid. Virions are 40-70 nm in diameter. Replication takes place in the cytoplasm, and assembly is assumed to occur during budding through the plasma membrane.

Pathogenic Mechanism Ocular lesions occur as a result of exposure of the fetus to the virus between days 76 and 150 of gestation. Characteristic lesions are retinal dysplasia, cataracts, and microphthalmia.313,314 Reported histologic findings include swollen and multi-layered lens epithelium, microphthalmia, diffuse cataracts, retrolental fibrovascular membranes containing cartilage, and retinal dysplasia. Retinal dysplasia is described as severe, with absence of normal retinal architecture, diffusely fibrosed nerve fiber layer, and gliotic optic nerves. Mononuclear cell infiltration may also be seen.

MALIGNANT CATARRHAL FEVER (MCF)

Virus Features Taxonomic classification: Family - Herpesvirinae, Subfamily - Alcelaphinae.

Pathogenic Mechanisms General. Pansystemic vasculopathic disease of cloven-hoofed animals worldwide.315 Peracute form: fever, hemorrhagic gastroenteritis, and death in 1-3 days of onset of clinical signs. Alimentary form: fever, diarrhea, diffuse exanthema, lacrimation, lymphadenopathy. Head and eye form: typical form seen in Africa, mucopurulent nasal discharge, corneal opacity, edema of head and neck, and neurological manifestations. Mild form: nasal and lacrimal discharge, dermatitis, and lymphadenopathy (usually results in recovery).

Ocular. Lymphocytic vasculitis of retinal, scleral, uveal vessels. Anterior uveitis is associated with corneal edema and keratitis. Predominantly a lymphocytic infiltrate, contrasting the disease from BVD, TEME, IBR, and IBK.315 Immunological mechanism suspected; Type IV (DTH) mechanism proposed.

MISCELLANEOUS CAUSES OF VIRAL INDUCED OCULAR DISEASE IN CATTLE:

Rabies - non-suppurative retinitis.

BLUETONGUE

Virus Features Taxonomic classification: Family - Reoviridae, Genus - Orbivirus. Virus structure: Non-enveloped spherical virion, 70 nm in diameter, with 2 concentric capsids, the inner being icosohedral. RNA genome.

Pathogenic Mechanism Systemic. Virus replicates in hematopoietic cells and vascular endothelium.

Ocular. Similar to BVD (both calves and lambs).316-318 Vaccination of ewes during the first trimester of gestation (attenuated live vaccine) produces necrotizing retinitis and CNS damage. Target tissues are brain, spinal cord, and retina. The severity of the disease depends on when in gestation the ewes are vaccinated. In lambs infected at 50-58 days, early lesions affect predominantly the retinal vasculature (endothelial nuclei rounded and vesicular).

MAREK'S DISEASE

Virus Features Gamma herpesvirus of chickens.

Viral Pathogenesis Systemic. Virus is slowly cytopathic but has ability to transform T lymphocytes to cause tumor formation. Systemic disease includes neurolymphomatosis (range paralysis), acute Marek's disease, and ocular lymphomatosis.

Ocular. Severe panuveitis to ocular tumor formation.319

REFERENCES

1. Marchetti M, Longhi C, Conte MP, Pisani S, Valenti P, Seganti L. Lactoferrin inhibits herpes simplex virus type 1 adsorption to Vero cells. Antiviral Res. 1996;29(2-3):221-231. 2. Beaumont SL, Maggs DJ, Clarke HE. Effects of bovine lactoferrin on in vitro replication of feline herpesvirus. Vet Ophthalmol. 2003;6(3):245-250. 3. Storey ES, Gerding PA, Scherba G, Schaeffer DJ. Survival of equine herpesvirus-4, feline herpesvirus-1, and feline calicivirus in multidose ophthalmic solutions. Vet Ophthalmol. 2002;5(4):263-267. 4. Grail A, Harbour DA, Chia W. Restriction endonuclease mapping of the genome of feline herpesvirus type 1. Arch.Virol. 1991;116(1-4):209-220. 5. Rota PA, Maes RK, Ruyechan WT. Physical characterization of the genome of feline herpesvirus-1. Virology. 1986;154(1):168-179. 6. Tai SH, Niikura M, Cheng HH, Kruger JM, Wise AG, Maes RK. Complete genomic sequence and an infectious BAC clone of feline herpesvirus-1 (FHV-1). Virology. 2010;401(2):215-227. 7. Nunberg JH, Wright DK, Cole GE, et al. Identification of the thymidine kinase gene of feline herpesvirus: use of degenerate oligonucleotides in the polymerase chain reaction to isolate herpesvirus gene homologs. J Virol. 1989;63(8):3240-3249. 8. Horimoto T, Limcumpao JA, Xuan X, et al. Heterogeneity of feline herpesvirus type 1 strains. Arch.Virol. 1992;126(1-4):283-292. 9. Scherba G, Hajjar AM, Pernikoff DS, et al. Comparison of a cheetah herpesvirus isolate to feline herpesvirus type 1. Arch.Virol. 1988;100(1-2):89-97. 10. Maeda K, Kawaguchi Y, Ono M, Tajima T, Mikami T. Restriction endonuclease analysis of field isolates of feline herpesvirus type 1 and identification of heterogeneous regions. J Clin.Microbiol. 1995;33(1):217-221. 11. Maeda K, Kawaguchi Y, Ono M, Tajima T, Mikami T. Comparisons among feline herpesvirus type 1 isolates by immunoblot analysis. J Vet Med Sci. 1995;57(1):147-150. 12. Hamano M, Maeda K, Mizukoshi F, et al. Experimental infection of recent field isolates of feline herpesvirus type 1. J Vet Med.Sci. 2003;65(8):939-943. 13. Swanson WF, Maggs DJ, Clarke HE, et al. Assessment of viral presence in semen and reproductive function of frozen-thawed spermatozoa from Pallas' cats (Otocolobus manul) infected with feline herpesvirus. J Zoo.Wildl.Med. 2006;37(3):336-346. 14. Shibly S, Schmidt P, Robert N, Walzer C, Url A. Immunohistochemical screening for viral agents in cheetahs (Acinonyx jubatus) with myelopathy. Vet Rec. 2006;159(17):557-561. 15. Junge RE, Miller RE, Boever WJ, Scherba G, Sundberg J. Persistent cutaneous ulcers associated with feline herpesvirus type 1 infection in a cheetah. J.Am.Vet Med.Assoc. 1991;198(6):1057-1058. 16. van Vuuren M, Goosen T, Rogers P. Feline herpesvirus infection in a group of semi-captive cheetahs. J.S.Afr.Vet.Assoc. 1999;70(3):132-134. 17. Munson L, Wack R, Duncan M, et al. Chronic eosinophilic dermatitis associated with persistent feline herpes virus infection in cheetahs (Acinonyx jubatus). Vet Pathol. 2004;41(2):170-176. 18. Maeda K, Horimoto T, Mikami T. Properties and functions of feline herpesvirus type 1 glycoproteins. J Vet Med Sci. 1998;60(8):881-888. 19. Arii J, Kato K, Kawaguchi Y, Tohya Y, Akashi H. Analysis of herpesvirus host specificity determinants using herpesvirus genomes as bacterial artificial chromosomes. Microbiology and Immunology. 2009;53(8):433- 441. 20. Maggs DJ, Lappin MR, Reif JS, et al. Evaluation of serologic and viral detection methods for diagnosing feline herpesvirus-1 infection in cats with acute respiratory tract or chronic ocular disease. Journal of the American Veterinary Medical Association. 1999;214(4):502-507. 21. Ellis TM. Feline respiratory virus carriers in clinically healthy cats. Aust.Vet.J. 1981;57(3):115-118. 22. Studdert MJ, Martin MC. Virus diseases of the respiratory tract of cats. 1. Isolation of feline rhinotracheitis virus. Aust.Vet J. 1970;46(3):99-104. 23. Li Y, Van Cleemput J, Qiu Y, Reddy VR, Mateusen B, Nauwynck HJ. Ex vivo modeling of feline herpesvirus replication in ocular and respiratory mucosae, the primary targets of infection. Virus Res. 2015;210:227-231. 24. Hoover EA, Rohovsky MW, Griesemer RA. Experimental feline viral rhinotracheitis in the germfree cat. Am J Pathol. 1970;58(2):269-282. 25. Ledbetter EC, Scarlett JM. Isolation of obligate anaerobic bacteria from ulcerative keratitis in domestic animals. Vet Ophthalmol. 2008;11(2):114-122.

26. Maeda K, Yokoyama N, Fujita K, Maejima M, Mikami T. Heparin-binding activity of feline herpesvirus type 1 glycoproteins. Virus Res. 1997;52(2):169-176. 27. Maes RK, Fritsch SL, Herr LL, Rota PA. Immunogenic proteins of feline rhinotracheitis virus. J.Virol. 1984;51(1):259-262. 28. Burgener DC, Maes RK. Glycoprotein-specific immune responses in cats after exposure to feline herpesvirus-1. Am.J.Vet.Res. 1988;49(10):1673-1676. 29. Maeda K, Kawaguchi Y, Ono M, et al. A gD homologous gene of feline herpesvirus type 1 encodes a hemagglutinin (gp60). Virology. 1994;202(2):1034-1038. 30. Spatz SJ, Rota PA, Maes RK. Identification of the feline herpesvirus type 1 (FHV-1) genes encoding glycoproteins G, D, I and E: expression of FHV-1 glycoprotein D in vaccinia and raccoon poxviruses. J.Gen.Virol. 1994;75 ( Pt 6):1235-1244. 31. Maeda K, Horimoto T, Norimine J, et al. Identification and nucleotide sequence of a gene in feline herpesvirus type 1 homologous to the herpes simplex virus gene encoding the glycoprotein B. Arch.Virol. 1992;127(1-4):387-397. 32. Svennerholm B, Jeansson S, Vahlne A, Lycke E. Involvement of glycoprotein C (gC) in adsorption of herpes simplex virus type 1 (HSV-1) to the cell. Arch.Virol. 1991;120(3-4):273-279. 33. Wilkes RP, Kania SA. Use of interfering RNAs targeted against feline herpesvirus 1 glycoprotein D for inhibition of feline herpesvirus 1 infection of feline kidney cells. American journal of veterinary research. 2009;70(8):1018-1025. 34. Wilkes RP, Kania SA. Evaluation of the effects of small interfering RNAs on in vitro replication of feline herpesvirus-1. American journal of veterinary research. 2010;71(6):655-663. 35. Wilkes RP, Ward DA, Newkirk KM, Adams JK, Kania SA. Evaluation of delivery agents used for introduction of small interfering RNAs into feline corneal cells. American journal of veterinary research. 2013;74(2):243-247. 36. Gaskell RM, Povey RC. Experimental induction of feline viral rhinotracheitis virus re-excretion in FVR- recovered cats. Vet Rec. 1977;100(7):128-133. 37. Townsend WM, Stiles J, Guptill-Yoran L, Krohne SG. Development of a reverse transcriptase-polymerase chain reaction assay to detect feline herpesvirus-1 latency-associated transcripts in the trigeminal ganglia and corneas of cats that did not have clinical signs of ocular disease. American journal of veterinary research. 2004;65(3):314-319. 38. Weigler BJ, Babineau CA, Sherry B, Nasisse MP. High sensitivity polymerase chain reaction assay for active and latent feline herpesvirus-1 infections in domestic cats. Vet Rec. 1997;140(13):335-338. 39. Gaskell RM, Dennis PE, Goddard LE, Cocker FM, Wills JM. Isolation of felid herpesvirus I from the trigeminal ganglia of latently infected cats. J.Gen.Virol. 1985;66 ( Pt 2):391-394. 40. Reubel GH, Ramos RA, Hickman MA, Rimstad E, Hoffmann DE, Pedersen NC. Detection of active and latent feline herpesvirus 1 infections using the polymerase chain reaction. Arch.Virol. 1993;132(3-4):409-420. 41. Ohmura Y, Ono E, Matsuura T, Kida H, Shimizu Y. Detection of feline herpesvirus 1 transcripts in trigeminal ganglia of latently infected cats. Arch.Virol. 1993;129(1-4):341-347. 42. Nasisse MP, Davis BJ, Guy JS, Davidson MG, Sussman W. Isolation of feline herpesvirus 1 from the trigeminal ganglia of acutely and chronically infected cats. J Vet Intern Med. 1992;6(2):102-103. 43. Stiles J, Pogranichniy R. Detection of virulent feline herpesvirus-1 in the corneas of clinically normal cats. J Feline Med Surg. 2008;10(2):154-159. 44. Hickman MA, Reubel GH, Hoffman DE, Morris JG, Rogers QR, Pedersen NC. An epizootic of feline herpesvirus, type 1 in a large specific pathogen-free cat colony and attempts to eradicate the infection by identification and culling of carriers. Lab Anim. 1994;28(4):320-329. 45. Maes R. Felid herpesvirus type 1 infection in cats: a natural host model for alphaherpesvirus pathogenesis. ISRN Vet Sci. 2012;2012:495830. 46. Simons FA, Vennema H, Rofina JE, et al. A mRNA PCR for the diagnosis of feline infectious peritonitis. J Virol.Methods. 2005;124(1-2):111-116. 47. Nawynck HJ, Pensaert MB. Cell-free and cell-associated viremia in pigs after oronasal infection with Aujeszky's disease virus. Vet Microbiol. 1995;43(4):307-314. 48. Kimura H, Futamura M, Kito H, et al. Detection of viral DNA in neonatal herpes simplex virus infections: frequent and prolonged presence in serum and cerebrospinal fluid. J Infect.Dis. 1991;164(2):289-293. 49. van der Meulen KM, Favoreel HW, Pensaert MB, Nauwynck HJ. Immune escape of equine herpesvirus 1 and other herpesviruses of veterinary importance. Vet Immunol.Immunopathol. 2006;111(1-2):31-40. 50. Lim CC, Cullen CL. Schirmer tear test values and tear film break-up times in cats with conjunctivitis. Vet

Ophthalmol. 2005;8(5):305-310. 51. Cullen CL, Wadowska DW, Singh A, Melekhovets Y. Ultrastructural findings in feline corneal sequestra. Vet Ophthalmol. 2005;8(5):295-303. 52. Powell CC, McInnis CL, Fontenelle JP, Lappin MR. Bartonella species, feline herpesvirus-1, and Toxoplasma gondii PCR assay results from blood and aqueous humor samples from 104 cats with naturally occurring endogenous uveitis. J Feline Med Surg. 2010;12(12):923-928. 53. Cullen CL, Lim C, Sykes J. Tear film breakup times in young healthy cats before and after anesthesia. Vet Ophthalmol. 2005;8(3):159-165. 54. Swenson CL, Gardner K, Arnoczky SP. Infectious feline herpesvirus detected in distant bone and tendon following mucosal inoculation of specific pathogen-free cats. Veterinary microbiology. 2012;160(3-4):484-487. 55. Burgesser KM, Hotaling S, Schiebel A, Ashbaugh SE, Roberts SM, Collins JK. Comparison of PCR, virus isolation, and indirect fluorescent antibody staining in the detection of naturally occurring feline herpesvirus infections. J Vet Diagn.Invest. 1999;11(2):122-126. 56. Westermeyer HD, Thomasy SM, Kado-Fong H, Maggs DJ. Assessment of viremia associated with experimental primary feline herpesvirus infection or presumed herpetic recrudescence in cats. American journal of veterinary research. 2009;70(1):99-104. 57. Mitchell WJ, Gressens P, Martin JR, DeSanto R. Herpes simplex virus type 1 DNA persistence, progressive disease and transgenic immediate early gene promoter activity in chronic corneal infections in mice. J Gen.Virol. 1994;75 ( Pt 6):1201-1210. 58. Maggs DJ, Chang E, Nasisse MP, Mitchell WJ. Persistence of herpes simplex virus type 1 DNA in chronic conjunctival and eyelid lesions of mice. J Virol. 1998;72(11):9166-9172. 59. Stiles J, McDermott M, Bigsby D, et al. Use of nested polymerase chain reaction to identify feline herpesvirus in ocular tissue from clinically normal cats and cats with corneal sequestra or conjunctivitis. American journal of veterinary research. 1997;58(4):338-342. 60. Nasisse MP, Glover TL, Moore CP, Weigler BJ. Detection of feline herpesvirus 1 DNA in corneas of cats with eosinophilic keratitis or corneal sequestration. American journal of veterinary research. 1998;59(7):856- 858. 61. Parzefall B, Schmahl W, Fischer A, Blutke A, Truyen U, Matiasek K. Evidence of feline herpesvirus-1 DNA in the vestibular ganglion of domestic cats. Veterinary journal. 2010;184(3):371-372. 62. Townsend WM, Jacobi S, Tai SH, Kiupel M, Wise AG, Maes RK. Ocular and neural distribution of feline herpesvirus-1 during active and latent experimental infection in cats. BMC Vet Res. 2013;9:185. 63. Johnson RP, Povey RC. Vaccination against feline viral rhinotracheitis in kittens with maternally derived feline viral rhinotracheitis antibodies. J.Am.Vet Med.Assoc. 1985;186(2):149-152. 64. Tham KM, Studdert MJ. Clinical and immunological responses of cats to feline herpesvirus type 1 infection. Vet Rec. 1987;120(14):321-326. 65. Tham KM, Studdert MJ. Antibody and cell-mediated immune responses to feline herpesvirus 1 following inactivated vaccine and challenge. Zentralbl.Veterinarmed.[B]. 1987;34(8):585-597. 66. Orr CM, Gaskell CJ, Gaskell RM. Interaction of an intranasal combined feline viral rhinotracheitis, feline calicivirus vaccine and the FVR carrier state. Vet Rec. 1980;106(8):164-166. 67. Orr CM, Gaskell CJ, Gaskell RM. Interaction of a combined feline viral rhinotracheitis-feline calicivirus vaccine and the FVR carrier state. Vet.Rec. 1978;103(10):200-202. 68. Scott FW, Geissinger CM. Long-term immunity in cats vaccinated with an inactivated trivalent vaccine. Am.J.Vet.Res. 1999;60(5):652-658. 69. Lappin MR. Feline panleukopenia virus, feline herpesvirus-1 and feline calicivirus antibody responses in seronegative specific pathogen-free kittens after parenteral administration of an inactivated FVRCP vaccine or a modified live FVRCP vaccine. J Feline Med Surg. 2012;14(2):161-164. 70. Nasisse MP, Guy JS, Davidson MG, Sussman W, De Clercq E. In vitro susceptibility of feline herpesvirus-1 to vidarabine, idoxuridine, trifluridine, acyclovir, or bromovinyldeoxyuridine. American journal of veterinary research. 1989;50(1):158-160. 71. Maggs DJ, Collins BK, Thorne JG, Nasisse MP. Effects of L-lysine and L-arginine on in vitro replication of feline herpesvirus type-1. Am.J.Vet.Res. 2000;61(12):1474-1478. 72. Maggs DJ, Clarke HE. In vitro efficacy of ganciclovir, cidofovir, penciclovir, foscarnet, idoxuridine, and acyclovir against feline herpesvirus type-1. American journal of veterinary research. 2004;65(4):399-403. 73. Nasisse MP, Dorman DC, Jamison KC, Weigler BJ, Hawkins EC, Stevens JB. Effects of valacyclovir in cats infected with feline herpesvirus 1. American journal of veterinary research. 1997;58(10):1141-1144. 74. Owens JG, Nasisse MP, Tadepalli SM, Dorman DC. Pharmacokinetics of acyclovir in the cat. J Vet

Pharmacol Ther. 1996;19(6):488-490. 75. Nasisse MP. Feline herpesvirus ocular disease. Vet Clin.North Am.Small Anim Pract. 1990;20(3):667-680. 76. Nasisse MP, Guy JS, Davidson MG, Sussman WA, Fairley NM. Experimental ocular herpesvirus infection in the cat. Sites of virus replication, clinical features and effects of corticosteroid administration. Investigative Ophthalmology and Visual Science. 1989;30(8):1758-1768. 77. Roberts SR, Dawson CR, Coleman V, Togni B. Dendritic keratitis in a cat. J.Am.Vet Med.Assoc. 1972;161(3):285-289. 78. Nasisse MP, English RV, Tompkins MB, Guy JS, Sussman W. Immunologic, histologic, and virologic features of herpesvirus-induced stromal keratitis in cats. American journal of veterinary research. 1995;56(1):51-55. 79. Avery AC, Zhao ZS, Rodriguez A, et al. Resistance to herpes stromal keratitis conferred by an IgG2a-derived peptide. Nature. 1995;376(6539):431-434. 80. Deshpande SP, Zheng M, Lee S, Rouse BT. Mechanisms of pathogenesis in herpetic immunoinflammatory ocular lesions. Vet Microbiol. 2002;86(1-2):17-26. 81. Lim CC, Reilly CM, Thomasy SM, Kass PH, Maggs DJ. Effects of feline herpesvirus type 1 on tear film break-up time, Schirmer tear test results, and conjunctival goblet cell density in experimentally infected cats. American journal of veterinary research. 2009;70(3):394-403. 82. Thomasy SM, Lim CC, Reilly CM, Kass PH, Lappin MR, Maggs DJ. Evaluation of orally administered famciclovir in cats experimentally infected with feline herpesvirus type-1. American journal of veterinary research. 2011;72(1):85-95. 83. Volopich S, Benetka V, Schwendenwein I, Mostl K, Sommerfeld-Stur I, Nell B. Cytologic findings, and feline herpesvirus DNA and Chlamydophila felis antigen detection rates in normal cats and cats with conjunctival and corneal lesions. Vet Ophthalmol. 2005;8(1):25-32. 84. Andrew SE, Tou S, Brooks DE. Corneoconjunctival transposition for the treatment of feline corneal sequestra: a retrospective study of 17 cases (1990-1998). Vet Ophthalmol. 2001;4(2):107-111. 85. Grahn BH, Sisler S, Storey E. Qualitative tear film and conjunctival goblet cell assessment of cats with corneal sequestra. Vet Ophthalmol. 2005;8(3):167-170. 86. Morgan R, Abrams K, Kern T. Feline Eosinophilic Keratitis: A Retrospective Study of 54 Cases: (1989- 1994). Veterinary and Comparative Ophthalmology. 1996;6(2):131-134. 87. Dean E, Meunier V. Feline eosinophilic keratoconjunctivitis: a retrospective study of 45 cases (56 eyes). J Feline Med Surg. 2013;15(8):661-666. 88. Allgoewer I, Schaffer EH, Stockhaus C, Vogtlin A. Feline eosinophilic conjunctivitis. Vet Ophthalmol. 2001;4(1):69-74. 89. Liesegang TJ. A community study of ocular herpes simplex. Curr Eye Res 1991;10 Suppl:111-115. 90. Maggs DJ, Lappin MR, Nasisse MP. Detection of feline herpesvirus-specific antibodies and DNA in aqueous humor from cats with or without uveitis. American journal of veterinary research. 1999;60(8):932-936. 91. Suchy A, Bauder B, Gelbmann W, Lohr CV, Teifke JP, Weissenbock H. Diagnosis of feline herpesvirus infection by immunohistochemistry, polymerase chain reaction, and in situ hybridization. J Vet Diagn.Invest. 2000;12(2):186-191. 92. Hargis AM, Ginn PE. Feline herpesvirus 1-associated facial and nasal dermatitis and stomatitis in domestic cats. Vet Clin.North Am Small Anim Pract. 1999;29(6):1281-1290. 93. Hargis AM, Ginn PE, Mansell JE, Garber RL. Ulcerative facial and nasal dermatitis and stomatitis in cats associated with feline herpesvirus 1. Veterinary Dermatology. 1999;10:267-274. 94. Holland JL, Outerbridge CA, Affolter VK, Maggs DJ. Detection of feline herpesvirus 1 DNA in skin biopsy specimens from cats with or without dermatitis. Journal of the American Veterinary Medical Association. 2006;229(9):1442-1446. 95. Persico P, Roccabianca P, Corona A, Vercelli A, Cornegliani L. Detection of feline herpes virus 1 via polymerase chain reaction and immunohistochemistry in cats with ulcerative facial dermatitis, eosinophilic granuloma complex reaction patterns and mosquito bite hypersensitivity. Vet Dermatol. 2011;22(6):521-527. 96. Nasisse MP, Guy JS, Stevens JB, English RV, Davidson MG. Clinical and laboratory findings in chronic conjunctivitis in cats: 91 cases (1983-1991). Journal of the American Veterinary Medical Association. 1993;203(6):834-837. 97. Reubel GH, George JW, Barlough JE, Higgins J, Grant CK, Pedersen NC. Interaction of acute feline herpesvirus-1 and chronic feline immunodeficiency virus infections in experimentally infected specific pathogen free cats. Vet Immunol.Immunopathol. 1992;35(1-2):95-119. 98. Kawaguchi Y, Miyazawa T, Horimoto T, et al. Activation of feline immunodeficiency virus long terminal

repeat by feline herpesvirus type 1. Virology. 1991;184(1):449-454. 99. Kawaguchi Y, Norimine J, Miyazawa T, Kai C, Mikami T. Sequences within the feline immunodeficiency virus long terminal repeat that regulate gene expression and respond to activation by feline herpesvirus type 1. Virology. 1992;190(1):465-468. 100. Rampazzo A, Appino S, Pregel P, Tarducci A, Zini E, Biolatti B. Prevalence of Chlamydophila felis and feline herpesvirus 1 in cats with conjunctivitis in northern Italy. J Vet Intern.Med. 2003;17(6):799-807. 101. Cai Y, Fukushi H, Koyasu S, Kuroda E, Yamaguchi T, Hirai K. An etiological investigation of domestic cats with conjunctivitis and upper respiratory tract disease in Japan. J Vet Med Sci. 2002;64(3):215-219. 102. Low HC, Powell CC, Veir JK, Hawley JR, Lappin MR. Prevalence of feline herpesvirus 1, Chlamydophila felis, and Mycoplasma spp DNA in conjunctival cells collected from cats with and without conjunctivitis. American journal of veterinary research. 2007;68(6):643-648. 103. Gerriets W, Joy N, Huebner-Guthardt J, Eule JC. Feline calicivirus: a neglected cause of feline ocular surface infections? Veterinary ophthalmology. 2012;15(3):172-179. 104. Wieliczko AK, Ploneczka-Janeczko K. Feline herpesvirus 1 and Chlamydophila felis prevalence in cats with chronic conjunctivitis. Pol J Vet Sci. 2010;13(2):381-383. 105. Hillstrom A, Tvedten H, Kallberg M, Hanas S, Lindhe A, Holst BS. Evaluation of cytologic findings in feline conjunctivitis. Vet Clin Pathol. 2012. 106. Schulz C, Hartmann K, Mueller RS, Helps C, Schulz BS. Sampling sites for detection of feline herpesvirus-1, feline calicivirus and Chlamydia felis in cats with feline upper respiratory tract disease. J Feline Med Surg. 2015;17(12):1012-1019. 107. Litster A, Wu CC, Leutenegger CM. Detection of feline upper respiratory tract disease pathogens using a commercially available real-time PCR test. Veterinary journal. 2015;206(2):149-153. 108. von Bomhard W, Polkinghorne A, Lu ZH, et al. Detection of novel chlamydiae in cats with ocular disease. American journal of veterinary research. 2003;64(11):1421-1428. 109. Powell CC, Kordick DL, Lappin MR. Inoculation with Bartonella henselae followed by feline herpesvirus 1 fails to activate ocular toxoplasmosis in chronically infected cats. J Feline Med Surg. 2002;4(2):107-110. 110. Anderson WA, Margruder B, Kilbourne ED. Induced reactivation of herpes simplex virus in healed rabbit corneal lesions. Proc.Soc.Exp.Biol.Med. 1961;107:628-632. 111. Hill TJ, Blyth WA, Harbour DA. Trauma to the skin causes recurrence of herpes simplex in the mouse. J Gen.Virol. 1978;39(1):21-28. 112. Harbour DA, Hill TJ, Blyth WA. Recurrent herpes simplex in the mouse: inflammation in the skin and activation of virus in the ganglia following peripheral stimulation. J Gen.Virol. 1983;64 (Pt 7):1491-1498. 113. Maggs DJ, Clarke HE. Relative sensitivity of polymerase chain reaction assays used for detection of feline herpesvirus type 1 DNA in clinical samples and commercial vaccines. American journal of veterinary research. 2005;66(9):1550-1555. 114. Nasisse MP, Weigler BJ. The diagnosis of ocular feline herpesvirus infection. Vet.Comp.Ophthalmol. 1997;7(1):44-51. 115. Hoover EA, Kahn DE. Experimentally induced feline calicivirus infection: clinical signs and lesions. J.Am.Vet Med.Assoc. 1975;166(5):463-468. 116. Carlson JH, Scott FW. An immunofluorescence diagnostic test for feline viral rhinotracheitis. Am.J.Vet.Res. 1978;39(3):465-467. 117. da Silva Curiel JMA, Nasisse MP, Hook RR, Wilson HH, Collins BK, Mandell CP. Topical fluorescein dye: effects on immunofluorescent antibody test for feline herpesvirus keratoconjunctivitis. Prog.Vet.Comp.Ophthalmol. 1991;1(2):99-104. 118. Johnson C, Johnson F. Effect of Sampling Swab Materials on Detection of Herpes Simplex Virus. 1995. 119. Crane LR, Gutterman PA, Chapel T, Lerner AM. Incubation of swab materials with herpes simplex virus. J Infect.Dis. 1980;141(4):531. 120. Hanson BR, Schipper IA. Effects of swab materials on infectious bovine rhinotracheitis virus. American journal of veterinary research. 1976;37(6):707-708. 121. Lappin MR, Andrews J, Simpson D, Jensen WA. Use of serologic tests to predict resistance to feline herpesvirus 1, feline calicivirus, and feline parvovirus infection in cats. Journal of the American Veterinary Medical Association. 2002;220(1):38-42. 122. Dunkel EC, Pavan-Langston D, Fitzpatrick K, Cukor G. Rapid detection of herpes simplex virus (HSV) antigen in human ocular infections. Curr.Eye Res. 1988;7(7):661-666. 123. Wu TC, Zaza S, Callaway J. Evaluation of the Du Pont HERPCHEK herpes simplex virus antigen test with clinical specimens. J.Clin.Microbiol. 1989;27(8):1903-1905.

124. Dawson DA, Carman J, Collins J, Hill S, Lappin MR. Enzyme-linked immunosorbent assay for detection of feline herpesvirus 1 IgG in serum, aqueous humor, and cerebrospinal fluid. J.Vet.Diagn.Invest. 1998;10(4):315-319. 125. Satoh Y, Iizuka K, Fukuyama M, et al. An enzyme-linked immunosorbent assay using nuclear antigen for detection of feline herpesvirus 1 antibody. J Vet Diagn.Invest. 1999;11(4):334-340. 126. Adleberg JM, Wittwer C. Use of the polymerase chain reaction in the diagnosis of ocular disease. Curr.Opin.Ophthalmol. 1995;6(3):80-85. 127. Pfeffer M, Wiedmann M, Batt CA. Applications of DNA amplification techniques in veterinary diagnostics. Vet.Res.Commun. 1995;19(5):375-407. 128. Segarra S, Papasouliotis K, Helps C. The in vitro effects of proxymetacaine, fluorescein, and fusidic acid on real-time PCR assays used for the diagnosis of Feline herpesvirus 1 and Chlamydophila felis infections. Veterinary ophthalmology. 2011;14 Suppl 1:5-8. 129. Hara M, Fukuyama M, Suzuki Y, et al. Detection of feline herpesvirus 1 DNA by the nested polymerase chain reaction. Vet Microbiol. 1996;48(3-4):345-352. 130. Polak KC, Levy JK, Crawford PC, Leutenegger CM, Moriello KA. Infectious diseases in large-scale cat hoarding investigations. Veterinary journal. 2014;201(2):189-195. 131. Burns RE, Wagner DC, Leutenegger CM, Pesavento PA. Histologic and molecular correlation in shelter cats with acute upper respiratory infection. Journal of Clinical Microbiology. 2011;49(7):2454-2460. 132. Sandmeyer LS, Waldner CL, Bauer BS, Wen X, Bienzle D. Comparison of polymerase chain reaction tests for diagnosis of feline herpesvirus, Chlamydophila felis, and Mycoplasma spp. infection in cats with ocular disease in Canada. Canadian Veterinary Journal. 2010;51(6):629-633. 133. Maggs DJ, Clarke HE, Smith SJ. Comparison of 6 PCR protocols for detection of feline herpesvirus (FHV-1) DNA in clinical samples and commercial vaccines. 2004/10/20/. 134. Westermeyer HD, Kado-Fong H, Maggs DJ. Effects of sampling instrument and processing technique on DNA yield and detection rate for feline herpesvirus-1 via polymerase chain reaction assay. American journal of veterinary research. 2008;69(6):811-817. 135. Clarke HE, Kado-Fong H, Maggs DJ. Effects of temperature and time in transit on polymerase chain reaction detection of feline herpesvirus DNA. J Vet Diagn.Invest. 2006;18(4):388-391. 136. Vogtlin A, Fraefel C, Albini S, et al. Quantification of Feline Herpesvirus 1 DNA in Ocular Fluid Samples of Clinically Diseased Cats by Real-Time TaqMan PCR. J Clin.Microbiol. 2002;40(2):519-523. 137. De Clercq E. Antivirals: past, present and future. Biochem Pharmacol. 2013;85(6):727-744. 138. Thomasy SM, Maggs DJ. A review of antiviral drugs and other compounds with activity against feline herpesvirus type 1. Veterinary ophthalmology. 2016. 139. Williams DL, Fitzmaurice T, Lay L, et al. Efficacy of antiviral agents in feline herpetic keratitis: results of an in vitro study. Curr Eye Res. 2004;29(2-3):215-218. 140. Andrei G, Snoeck R, Goubau P, Desmyter J, De Clercq E. Comparative activity of various compounds against clinical strains of herpes simplex virus. Eur J Clin Microbiol Infect Dis. 1992;11(2):143-151. 141. Babiuk LA, Meldrum B, Gupta VS, Rouse BT. Comparison of the antiviral effects of 5-methoxymethyl- deoxyuridine with 5-iododeoxyuridine, cytosine arabinoside, and adenine arabinoside. Antimicrob.Agents Chemother. 1975;8(6):643-650. 142. van der Meulen K, Garre B, Croubels S, Nauwynck H. In vitro comparison of antiviral drugs against feline herpesvirus 1. BMC Vet Res. 2006;2:13. 143. Chen ZJ, Liu WG, Song JZ. [Experimental studies of 9-(1, 3-dihydroxy-2-propoxymethyl)-guanine(DHPG) against herpes simplex virus]. Yao Xue Xue Bao. 1989;24(5):331-334. 144. Lauter CB, Bailey EJ, Lerner AM. Microbiologic assays and neurological toxicity during use of adenine arabinoside in humans. The Journal of infectious diseases. 1976;134(1):75-79. 145. Hussein IT, Menashy RV, Field HJ. Penciclovir is a potent inhibitor of feline herpesvirus-1 with susceptibility determined at the level of virus-encoded thymidine kinase. Antiviral Res. 2008;78(3):268-274. 146. Groth AD, Contreras MT, Kado-Fong HK, Nguyen KQ, Thomasy SM, Maggs DJ. In vitro cytotoxicity and antiviral efficacy against feline herpesvirus type 1 of famciclovir and its metabolites. Veterinary ophthalmology. 2014;17(4):268-274. 147. Hussein IT, Field HJ. Development of a quantitative real-time TaqMan PCR assay for testing the susceptibility of feline herpesvirus-1 to antiviral compounds. Journal of Virological Methods. 2008;152(1- 2):85-90. 148. De Clercq E, Sakuma T, Baba M, et al. Antiviral activity of phosphonylmethoxyalkyl derivatives of purine and pyrimidines. Antiviral Res. 1987;8(5-6):261-272.

149. Gadler H, Larsson A, Solver E. Nucleic acid hybridization, a method to determine effects of antiviral compounds on herpes simplex virus type 1 DNA synthesis. Antiviral Res. 1984;4(1-2):63-70. 150. Williams DL, Robinson JC, Lay E, Field H. Efficacy of topical aciclovir for the treatment of feline herpetic keratitis: results of a prospective clinical trial and data from in vitro investigations. Veterinary Record. 2005;157(9):254-257. 151. Ayisi NK, Gupta SV, Babiuk LA. Combination chemotherapy: interaction of 5-methoxymethyldeoxyuridine with trifluorothymidine, phosphonoformate and acycloguanosine against herpes simplex viruses. Antiviral Res. 1985;5(1):13-27. 152. Sandmeyer LS, Keller CB, Bienzle D. Effects of cidofovir on cell death and replication of feline herpesvirus- 1 in cultured feline corneal epithelial cells. American journal of veterinary research. 2005;66(2):217-222. 153. Bronson JJ, Ghazzouli I, Hitchcock MJ, Webb RR, 2nd, Martin JC. Synthesis and antiviral activity of the nucleotide analogue (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine. J Med Chem. 1989;32(7):1457- 1463. 154. Bestman-Smith J, Boivin G. Herpes simplex virus isolates with reduced adefovir susceptibility selected in vivo by foscarnet therapy. J Med Virol. 2002;67(1):88-91. 155. Collins P. The spectrum of antiviral activities of acyclovir in vitro and in vivo. J Antimicrob.Chemother. 1983;12 Suppl B:19-27. 156. Stiles J. Treatment of cats with ocular disease attributable to herpesvirus infection: 17 cases (1983-1993). Journal of the American Veterinary Medical Association. 1995;207(5):599-603. 157. O'Brien WJ, Edelhauser HF. The corneal penetration of trifluorothymidine, adenine arabinoside, and idoxuridine: a comparative study. Invest Ophthalmol.Vis.Sci. 1977;16(12):1093-1103. 158. Hussein IT, Miguel RN, Tiley LS, Field HJ. Substrate specificity and molecular modelling of the feline herpesvirus-1 thymidine kinase. Archives of Virology. 2008;153(3):495-505. 159. Weiss RC. Synergistic antiviral activities of acyclovir and recombinant human leukocyte (alpha) interferon on feline herpesvirus replication. American journal of veterinary research. 1989;50(10):1672-1677. 160. de Miranda P, Burnette TC. Metabolic fate and pharmacokinetics of the acyclovir prodrug valaciclovir in cynomolgus monkeys. Drug Metab Dispos. 1994;22(1):55-59. 161. Ernst ME, Franey RJ. Acyclovir- and ganciclovir-induced neurotoxicity. Ann Pharmacother. 1998;32(1):111- 113. 162. Burns LJ, Miller W, Kandaswamy C, et al. Randomized clinical trial of ganciclovir vs acyclovir for prevention of cytomegalovirus antigenemia after allogeneic transplantation. Bone Marrow Transplant. 2002;30(12):945- 951. 163. Kaufman HE, Varnell ED, Thompson HW. Trifluridine, cidofovir, and penciclovir in the treatment of experimental herpetic keratitis. Arch.Ophthalmol. 1998;116(6):777-780. 164. Clarke SE, Harrell AW, Chenery RJ. Role of aldehyde oxidase in the in vitro conversion of famciclovir to penciclovir in human liver. Drug Metab Dispos. 1995;23(2):251-254. 165. Vere Hodge RA, Sutton D, Boyd MR, Harnden MR, Jarvest RL. Selection of an oral prodrug (BRL 42810; famciclovir) for the antiherpesvirus agent BRL 39123 [9-(4-hydroxy-3-hydroxymethylbut-l-yl)guanine; penciclovir]. Antimicrob.Agents Chemother. 1989;33(10):1765-1773. 166. Filer CW, Allen GD, Brown TA, et al. Metabolic and pharmacokinetic studies following oral administration of 14C-famciclovir to healthy subjects. Xenobiotica. 1994;24(4):357-368. 167. Dick RA, Kanne DB, Casida JE. Identification of aldehyde oxidase as the neonicotinoid nitroreductase. Chemical Research in Toxicology. 2005;18(2):317-323. 168. Sebbag L, Thomasy SM, Knych HK, Maggs DJ. Pharmacokinetics of famciclovir, penciclovir, and BRL 42359 following oral administration of famciclovir to cats Veterinary ophthalmology. 2013;16(6):E 45. 169. Sebbag L, Thomasy SM, Woodward AP, Knych HK, Maggs DJ. Pharmacokinetic modeling of penciclovir and BRL42359 in plasma and tears so as to optimize oral famciclovir dosing recommendation in cats. . American journal of veterinary research.In Press. 170. Thomasy SM, Maggs DJ, Moulin NK, Stanley SD. Pharmacokinetics and safety of penciclovir following oral administration of famciclovir to cats. American journal of veterinary research. 2007;68(11):1252-1258. 171. Malik R, Lessels NS, Webb S, et al. Treatment of feline herpesvirus-1 associated disease in cats with famciclovir and related drugs. J Feline Med Surg. 2009;11(1):40-48. 172. Thomasy SM, Shull O, Outerbridge CAL, et al. Oral administration of famciclovir for treatment of spontaneous ocular, respiratory or dermatologic disease attributed to feline herpesvirus type-1: A retrospective review in 59 client-owned cats. . Journal of the American Veterinary Medical Association.In Press.

173. Thomasy SM, Whittem T, Bales JL, Ferrone M, Stanley SD, Maggs DJ. Pharmacokinetics of penciclovir in healthy cats following oral administration of famciclovir or intravenous infusion of penciclovir. American journal of veterinary research. 2012;73(7):1092-1099. 174. Thomasy SM, Covert JC, Stanley SD, Maggs DJ. Pharmacokinetics of famciclovir and penciclovir in tears following oral administration of famciclovir to cats: a pilot study. Veterinary ophthalmology. 2012;15(5):299- 306. 175. Boike SC, Pue MA, Freed MI, et al. Pharmacokinetics of famciclovir in subjects with varying degrees of renal impairment. Clin Pharmacol Ther. 1994;55(4):418-426. 176. Neyts J, Snoeck R, Schols D, Balzarini J, De Clercq E. Selective inhibition of human cytomegalovirus DNA synthesis by (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine [(S)-HPMPC] and 9-(1,3-dihydroxy-2- propoxymethyl)guanine (DHPG). Virology. 1990;179(1):41-50. 177. Romanowski EG, Bartels SP, Gordon YJ. Comparative antiviral efficacies of cidofovir, trifluridine, and acyclovir in the HSV-1 rabbit keratitis model. Invest Ophthalmol.Vis.Sci. 1999;40(2):378-384. 178. Neyts J, Snoeck R, Balzarini J, De Clercq E. Particular characteristics of the anti-human cytomegalovirus activity of (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC) in vitro. Antiviral Res. 1991;16(1):41-52. 179. Fontenelle JP, Powell CC, Veir JK, Radecki SV, Lappin MR. Effect of topical ophthalmic application of cidofovir on experimentally induced primary ocular feline herpesvirus-1 infection in cats. American journal of veterinary research. 2008;69(2):289-293. 180. Sherman MD, Feldman KA, Farahmand SM, Margolis TP. Treatment of conjunctival squamous cell carcinoma with topical cidofovir. Am J Ophthalmol. 2002;134(3):432-433. 181. Gordon YJ, Romanowski EG, Araullo-Cruz T. Topical HPMPC inhibits adenovirus type 5 in the New Zealand rabbit ocular replication model. Invest Ophthalmol.Vis.Sci. 1994;35(12):4135-4143. 182. Stiles J, Gwin W, Pogranichniy R. Stability of 0.5% cidofovir stored under various conditions for up to 6 months. Veterinary ophthalmology. 2010;13(4):275-277. 183. Stiles J, Guptill-Yoran L, Moore GE, Pogranichniy RM. Effects of lambda-carrageenan on in vitro replication of feline herpesvirus and on experimentally induced herpetic conjunctivitis in cats. Invest Ophthalmol.Vis.Sci. 2008;49(4):1496-1501. 184. Bagla VP, McGaw LJ, Eloff JN. The antiviral activity of six South African plants traditionally used against infections in ethnoveterinary medicine. Veterinary microbiology. 2012;155(2-4):198-206. 185. Knight DA, Hejmanowski AQ, Dierksheide JE, Williams JW, Chong AS, Waldman WJ. Inhibition of herpes simplex virus type 1 by the experimental immunosuppressive agent leflunomide. Transplantation. 2001;71(1):170-174. 186. Williams CR, Sykes JE, Mehl M, et al. In vitro effects of the active metabolite of leflunomide, A77 1726, on feline herpesvirus-1. American journal of veterinary research. 2007;68(9):1010-1015. 187. Sandmeyer LS, Keller CB, Bienzle D. Effects of interferon-alpha on cytopathic changes and titers for feline herpesvirus-1 in primary cultures of feline corneal epithelial cells. American journal of veterinary research. 2005;66(2):210-216. 188. Siebeck N, Hurley DJ, Garcia M, et al. Effects of human recombinant alpha-2b interferon and feline recombinant omega interferon on in vitro replication of feline herpesvirus-1. American journal of veterinary research. 2006;67(8):1406-1411. 189. Haid C, Kaps S, Gonczi E, et al. Pretreatment with feline interferon omega and the course of subsequent infection with feline herpesvirus in cats. Vet Ophthalmol. 2007;10(5):278-284. 190. Cocker FM, Howard PE, Harbour DA. Effect of human alpha-hybrid interferon on the course of feline viral rhinotracheitis. Vet Rec. 1987;120(16):391-393. 191. Nasisse MP, Halenda RM, Luo H. Efficacy of low dose oral, natural human interferon alpha (nHuIFN ) in acute feline herpesvirus 1 (FHV-1) infection, a preliminary dose determination trial. 1996/10/21/; Wailea, HI. 192. Ballin AC, Schulz B, Helps C, Sauter-Louis C, Mueller RS, Hartmann K. Limited efficacy of topical recombinant feline interferon-omega for treatment of cats with acute upper respiratory viral disease. Veterinary journal. 2014;202(3):466-470. 193. Weigent DA, Langford MP, Stanton GJ, Blalock JE. Interferon-induced transfer of viral resistance by human B and T lymphocytes. Cell Immunol. 1984;87(2):678-683. 194. Friedl Y, Schulz B, Knebl A, Helps C, Truyen U, Hartmann K. Efficacy of passively transferred antibodies in cats with acute viral upper respiratory tract infection. Veterinary journal. 2014;201(3):316-321. 195. Umehashi M, Imamura T, Akiyama S, Kimachi K, Tokiyoshi S, Mikami T. Post-exposure Treatment of Cats

with Mouse-Cat Chimeric Antibodies against Feline Herpesvirus Type 1 and Feline Calicivirus. J Vet Med Sci. 2002;64(11):1017-1021. 196. Umehashi M, Imamura T, Akiyama S, et al. Pre-exposure treatment of cats with anti-FHV-1 and anti-FCV mouse-cat chimeric antibodies. J.Vet Med.Sci. 2003;65(5):563-566. 197. Lappin MR, Veir JK, Satyaraj E, Czarnecki-Maulden G. Pilot study to evaluate the effect of oral supplementation of Enterococcus faecium SF68 on cats with latent feline herpesvirus 1. J Feline Med Surg. 2009;11(8):650-654. 198. Nasisse MP. Issues in Ophthalmology: Anti-Inflammatory Therapy in Herpesvirus Keratoconjunctivitis. Progress in Veterinary and Comparative Ophthalmology. 1990;1(1):63-65. 199. Hendricks RL, Barfknecht CF, Schoenwald RD, Epstein RJ, Sugar J. The effect of flurbiprofen on herpes simplex virus type 1 stromal keratitis in mice. Invest Ophthalmol.Vis.Sci. 1990;31(8):1503-1511. 200. Trousdale MD, Dunkel EC, Nesburn AB. Effect of flurbiprofen on herpes simplex keratitis in rabbits. Invest Ophthalmol.Vis.Sci. 1980;19(3):267-270. 201. Vahlne A, Larsson PA, Horal P, et al. Inhibition of herpes simplex virus production in vitro by cyclosporin A. Arch.Virol. 1992;122(1-2):61-75. 202. Meyers-Elliott RH, Chitjian PA, Billups CB. Effects of cyclosporine A on clinical and immunological parameters in herpes simplex keratitis. Invest Ophthalmol.Vis.Sci. 1987;28(7):1170-1180. 203. Gunduz K, Ozdemir O. Topical cyclosporin as an adjunct to topical acyclovir treatment in herpetic stromal keratitis. Ophthalmic Res. 1997;29(6):405-408. 204. Spiess AK, Sapienza JS, Mayordomo A. Treatment of proliferative feline eosinophilic keratitis with topical 1.5% cyclosporine: 35 cases. Veterinary ophthalmology. 2009;12(2):132-137. 205. Maggs DJ, Collins BK, Thorne JG, Nasisse MP. Effects of L-lysine and L-arginine on in vitro replication of feline herpesvirus type-1. Am J Vet Res. 2000;61(12):1474-1478. 206. Tankersley RW, Jr. Amino Acid Requirements of Herpes Simplex Virus in Human Cells. Journal of bacteriology. 1964;87:609-613. 207. Griffith RS, DeLong DC, Nelson JD. Relation of arginine-lysine antagonism to herpes simplex growth in tissue culture. Chemotherapy. 1981;27(3):209-213. 208. Kaplan AS, Shimono H, Ben-Porat T. Synthesis of proteins in cells infected with herpesvirus. 3. Relative amino acid content of various proteins formed after infection. Virology. 1970;40(1):90-101. 209. Bol S, Bunnik EM. Lysine supplementation is not effective for the prevention or treatment of feline herpesvirus 1 infection in cats: a systematic review. BMC Vet Res. 2015;11(1):284. 210. Cave NJ, Dennis K, Gopakumar G, Dunowska M. Effects of physiologic concentrations of l-lysine on in vitro replication of feline herpesvirus 1. American journal of veterinary research. 2014;75(6):572-580. 211. Banos G, Daniel PM, Pratt OE. Saturation of a shared mechanism which transports L-arginine and L-lysine into the brain of the living rat. J Physiol (Lond). 1974;236(1):29-41. 212. Stutz MW, Savage JE, O'Dell BL. Relation of dietary cations to arginine-lysine antagonism and free amino acid patterns in chicks. J Nutr. 1971;101(3):377-384. 213. Hunter A, Downs CE. The inhibition of arginase by amino acids. J Biol.Chem. 1945;157 427-446. 214. Stiles J, Townsend WM, Rogers QR, Krohne SG. Effect of oral administration of L-lysine on conjunctivitis caused by feline herpesvirus in cats. American journal of veterinary research. 2002;63(1):99-103. 215. Maggs DJ, Nasisse MP, Kass PH. Efficacy of oral supplementation with L-lysine in cats latently infected with feline herpesvirus. American journal of veterinary research. 2003;64(1):37-42. 216. Rees TM, Lubinski JL. Oral supplementation with L-lysine did not prevent upper respiratory infection in a shelter population of cats. J Feline Med Surg. 2008;10(5):510-513. 217. Fascetti AJ, Maggs DJ, Kanchuk ML, Clarke HE, Rogers QR. Excess dietary lysine does not cause lysine- arginine antagonism in adult cats. J.Nutr. 2004;134(8 Suppl):2042S-2045S. 218. Maggs DJ, Sykes JE, Clarke HE, et al. Effects of dietary lysine supplementation in cats with enzootic upper respiratory disease. J Fel Med Surg. 2007;9(2):97-108. 219. Drazenovich TL, Fascetti AJ, Westermeyer HD, et al. Effects of dietary lysine supplementation on upper respiratory and ocular disease and detection of infectious organisms in cats within an animal shelter. American journal of veterinary research. 2009;70(11):1391-1400. 220. Morris JG, Rogers QR. Ammonia intoxication in the near-adult cat as a result of a dietary deficiency of arginine. Science. 1978;199(4327):431-432. 221. Cocker FM, Gaskell RM, Newby TJ, Gaskell CJ, Stokes CR, Bourne FJ. Efficacy of early (48 and 96 hour) protection against feline viral rhinotracheitis following intranasal vaccination with a live temperature sensitive mutant. Vet Rec. 1984;114(14):353-354.

222. Burton G, Mason KV. Do postvaccinal sarcomas occur in Australian cats? Aust.Vet J. 1997;75(2):102-106. 223. Reagan KL, Hawley JR, Lappin MR. Concurrent administration of an intranasal vaccine containing feline herpesvirus-1 (FHV-1) with a parenteral vaccine containing FHV-1 is superior to parenteral vaccination alone in an acute FHV-1 challenge model. Veterinary journal. 2014;201(2):202-206. 224. Bradley A, Kinyon J, Frana T, Bolte D, Hyatt DR, Lappin MR. Efficacy of intranasal administration of a modified live feline herpesvirus 1 and feline calicivirus vaccine against disease caused by Bordetella bronchiseptica after experimental challenge. J Vet Intern Med. 2012;26(5):1121-1125. 225. Sussman MD, Maes RK, Kruger JM. Vaccination of cats for feline rhinotracheitis results in a quantitative reduction of virulent feline herpesvirus-1 latency load after challenge. Virology. 1997;228(2):379-382. 226. Weigler BJ, Guy JS, Nasisse MP, Hancock SI, Sherry B. Effect of a live attenuated intranasal vaccine on latency and shedding of feline herpesvirus 1 in domestic cats. Arch.Virol. 1997;142(12):2389-2400. 227. Ruch-Gallie RA, Veir JK, Hawley JR, Lappin MR. Results of molecular diagnostic assays targeting feline herpesvirus-1 and feline calicivirus in adult cats administered modified live vaccines. J Feline Med Surg. 2011;13(8):541-545. 228. Pedersen NC. A review of feline infectious peritonitis virus infection: 1963-2008. J Feline Med Surg. 2009;11(4):225-258. 229. Wolfe LG, Griesemer RA. Feline infectious peritonitis. Pathol.Vet. 1966;3(3):255-270. 230. Hardy WD, Jr., Hurvitz AI. Feline infectious peritonitis: experimental studies. Journal of the American Veterinary Medical Association. 1971;158(6):Suppl. 231. Slauson DO, Finn JP. Meningoencephalitis and panophthalmitis in feline infectious peritonitis. Journal of the American Veterinary Medical Association. 1972;160(5):729-734. 232. Doherty MJ. Ocular manifestations of feline infectious peritonitis. Journal of the American Veterinary Medical Association. 1971;159(4):417-424. 233. Disque DF, Case MT, Youngren JA. Feline infectious peritonitis. Journal of the American Veterinary Medical Association. 1968;152(4):372-375. 234. Sparkes AH, Gruffydd-Jones TJ, Harbour DA. An appraisal of the value of laboratory tests in the diagnosis of feline infectious peritonitis. J Am Anim Hosp Assoc. 1994;30:345-350. 235. Gamble DA, Lobbiani A, Gramegna M, Moore LE, Colucci G. Development of a nested PCR assay for detection of feline infectious peritonitis virus in clinical specimens. J Clin.Microbiol. 1997;35(3):673-675. 236. Chavkin MJ, Lappin MR, Powell CC, Roberts SM. Seroepidemiologic and clinical observations of 93 cases of uveitis in cats. Progress Vet.& Comp.Ophth. 1992;2(1):29-36. 237. English RV, Davidson MG, Nasisse MP, Jamieson VE, Lappin MR. Intraocular disease associated with feline immunodeficiency virus infection in cats. Journal of the American Veterinary Medical Association. 1990;196(7):1116-1119. 238. Yamamoto JK, Hansen H, Ho EW, et al. Epidemiologic and clinical aspects of feline immunodeficiency virus infection in cats from the continental United States and Canada and possible mode of transmission. Journal of the American Veterinary Medical Association. 1989;194(2):213-220. 239. Stiles J, Bienzle D, Render JA, Buyukmihci NC, Johnson EC. Use of nested polymerase chain reaction (PCR) for detection of retroviruses from formalin-fixed, paraffin-embedded uveal melanomas in cats. Vet Ophthalmol. 1999;2(2):113-116. 240. Scagliotti RH. Current concepts in veterinary neuro-ophthalmology. Vet Clin.North Am Small Anim Pract. 1980;10(2):417-436. 241. Brightman AH, Macy DW. Pupillary abnormalities associated with the feline leukemia complex. Feline Practice. 1977;7:23-27. 242. Albert DM, Lahav M, Colby ED, Shadduck JA, Sang DN. Retinal neoplasia and dysplasia. I. Induction by feline leukemia virus. Invest Ophthalmol.Vis.Sci. 1977;16(4):325-337. 243. Hardy WD, Jr. Immunodiffusion studies of feline leukemia and sarcoma. Journal of the American Veterinary Medical Association. 1971;158(6):Suppl. 244. Fischer CA. Retinopathy in anemic cats. Journal of the American Veterinary Medical Association. 1970;156(10):1415-1427. 245. Meincke JE. Reticuloendothelial malignancies, with intraocular involvement in the cat. Journal of the American Veterinary Medical Association. 1966;148(2):157-161. 246. Carlton WW. Intraocular lymphosarcoma in two siamese cats. J Am Anim Hosp Assoc. 1976;12:83. 247. Shelah-Goraly M, Aroch I, Kass PH, Bruchim Y, Ofri R. A prospective study of the association of anemia and thrombocytopenia with ocular lesions in dogs. Veterinary journal. 2009;182(2):187-192. 248. Herring IP, Troy GC, Toth TE, Champagne ES, Pickett JP, Haines DM. Feline leukemia virus detection in

corneal tissues of cats by polymerase chain reaction and immunohistochemistry. Vet Ophthalmol. 2001;4(2):119-126. 249. MacMillan A. Retinal dysplasia and degeneration in the young cat: Feline panleukopenia virus as an etiological agent, University of California, Davis; 1974. 250. Percy DH, Scott FW, Albert DM. Retinal dysplasia due to feline panleukopenia virus infection. Journal of the American Veterinary Medical Association. 1975;167(10):935-937. 251. Greene CE, Gorgasz EJ, Martin CL. Hydranencephaly associated with feline panleukopenia. Journal of the American Veterinary Medical Association. 1982;180(7):767-768. 252. Lubin JR, Albert DM, Essex M, de Noronha F, Riis R. Experimental anterior uveitis after subcutaneous injection of feline sarcoma virus. Invest Ophthalmol.Vis.Sci. 1983;24(8):1055-1062. 253. Niederkorn JY, Shadduck JA, Albert D, Essex M. Serum antibodies against feline oncornavirus-associated cell membrane antigen in cats bearing virally induced uveal melanomas. Invest Ophthalmol.Vis.Sci. 1981;20(5):598-605. 254. Albert DM, Shadduck JA, Craft JL, Niederkorn JY. Feline uveal melanoma model induced with feline sarcoma virus. Invest Ophthalmol.Vis.Sci. 1981;20(5):606-624. 255. Cullen CL, Haines DM, Jackson ML, Peiffer RL, Grahn BH. The use of immunohistochemistry and the polymerase chain reaction for detection of feline leukemia virus and feline sarcoma virus in six cases of feline ocular sarcoma. Vet Ophthalmol. 1998;1(4):189-193. 256. Albert DM, Lahav M, Carmichael LE, Percy DH. Canine herpes-induced retinal dysplasia and associated ocular anomalies. Invest Ophthalmol. 1976;15(4):267-278. 257. Percy DH, Carmichael LE, Albert DM, King JM, Jonas AM. Lesions in Puppies Surviving Infection with Canine Herpesvirus. Veterinary Pathology. 1971;8:37-53. 258. Malone EK, Ledbetter EC, Rassnick KM, Kim SG, Russell D. Disseminated Canine Herpesvirus-1 Infection in an Immunocompromised Adult Dog. J Vet Intern Med. 2010. 259. Ledbetter EC, Riis RC, Kern TJ, Haley NJ, Schatzberg SJ. Corneal ulceration associated with naturally occurring canine herpesvirus-1 infection in two adult dogs. Journal of the American Veterinary Medical Association. 2006;229(3):376-384. 260. Gervais KJ, Pirie CG, Ledbetter EC, Pizzirani S. Acute primary canine herpesvirus-1 dendritic ulcerative keratitis in an adult dog. Veterinary ophthalmology. 2012;15(2):133-138. 261. Gadsden BJ, Maes RK, Wise AG, Kiupel M, Langohr IM. Fatal Canid herpesvirus 1 infection in an adult dog. J Vet Diagn Invest. 2012;24(3):604-607. 262. Ledbetter EC, Kim SG, Dubovi EJ. Outbreak of ocular disease associated with naturally-acquired canine herpesvirus-1 infection in a closed domestic dog colony. Veterinary ophthalmology. 2009;12(4):242-247. 263. Ledbetter EC, Marfurt CF, Dubielzig RR. Metaherpetic corneal disease in a dog associated with partial limbal stem cell deficiency and neurotrophic keratitis. Veterinary ophthalmology. 2013;16(4):282-288. 264. Harman RM, Bussche L, Ledbetter EC, Van de Walle GR. Establishment and characterization of an air- liquid canine corneal organ culture model to study acute herpes keratitis. J Virol. 2014;88(23):13669-13677. 265. Ledbetter EC, da Silva EC, Kim SG, Dubovi EJ, Schwark WS. Frequency of spontaneous canine herpesvirus-1 reactivation and ocular viral shedding in latently infected dogs and canine herpesvirus-1 reactivation and ocular viral shedding induced by topical administration of cyclosporine and systemic administration of corticosteroids. American journal of veterinary research. 2012;73(7):1079-1084. 266. Ledbetter EC, Hornbuckle WE, Dubovi EJ. Virologic survey of dogs with naturally acquired idiopathic conjunctivitis. Journal of the American Veterinary Medical Association. 2009;235(8):954-959. 267. Ledbetter EC, Dubovi EJ, Kim SG, Maggs DJ, Bicalho RC. Experimental primary ocular canine herpesvirus- 1 infection in adult dogs. American journal of veterinary research. 2009;70(4):513-521. 268. Nicklin AM, McEntee MC, Ledbetter EC. Effects of ocular surface strontium-90 beta radiotherapy in dogs latently infected with canine herpesvirus-1. Veterinary microbiology. 2014;174(3-4):433-437. 269. Ledbetter EC, Kim SG, Dubovi EJ, Bicalho RC. Experimental reactivation of latent canine herpesvirus-1 and induction of recurrent ocular disease in adult dogs. Veterinary microbiology. 2009;138(1-2):98-105. 270. Ledbetter EC, Kice NC, Matusow RB, Dubovi EJ, Kim SG. The effect of topical ocular corticosteroid administration in dogs with experimentally induced latent canine herpesvirus-1 infection. Exp Eye Res. 2010. 271. Mundy P, da Silva EC, Ledbetter EC. Effects of cyclophosphamide myelosuppression in adult dogs with latent canine herpesvirus-1 infection. Veterinary microbiology. 2012;159(1-2):230-235. 272. Ledbetter EC. Canine herpesvirus-1 ocular diseases of mature dogs. N Z Vet J. 2013;61(4):193-201. 273. Ledbetter EC, Spertus CB, Pennington MR, Van de Walle GR, Judd BE, Mohammed HO. In Vitro and In Vivo Evaluation of Cidofovir as a Topical Ophthalmic Antiviral for Ocular Canine Herpesvirus-1 Infections

in Dogs. J Ocul Pharmacol Ther. 2015;31(10):642-649. 274. Spertus CB, Mohammed HO, Ledbetter EC. Effects of topical ophthalmic trifluridine in dogs with experimental recurrent 1 ocular canine herpesvirus-1 infection. American journal of veterinary research. In Press. 275. Coats G. Retinal degeneration following distemper in a dog. Proc.Royal Soc.Med. 1913;7:14. 276. Fischer CA. Retinal and retinochoroidal lesions in early neuropathic canine distemper. Journal of the American Veterinary Medical Association. 1971;158(6):740-752. 277. Jubb KV, Saunders LZ, Coates HV. The intraocular lesions of canine distemper. J Comp Pathol. 1957;67:21-29. 278. Parry HB. Degeneration of the dog retina. IV Retinopathies associated with dog distemper-complex virus infections. Br.J.Ophthalmol. 1954;38:295. 279. Render JA, Carlton WW, Vestre WA. Canine distemper inclusions in the ciliary body. Journal of the American Veterinary Medical Association. 1982;181(2):164-165. 280. Thomas WB, Sorjonen DC, Steiss JE. A retrospective evaluation of 38 cases of canine distemper encephalomyelitis. J Am Anim Hosp Assoc. 1993;29:129-133. 281. Carmichael LE. The pathogenesis of ocular lesions in of infectious canine hepatitis. I. Pathology and virologic observations. Pathol.Vet. 1964;1:73-95. 282. Carmichael LE, Medic BL, Bistner SI, Aguirre GD. Viral-antibody complexes in canine adenovirus type 1 (CAV-1)ocular lesions: leukocyte chemotaxis and enzyme release. Cornell Vet. 1975;65(3):331-351. 283. Carmichael LE. The pathogenesis of ocular lesions of infectious canine hepatitis. II. Experimental ocular hypersensitivity produced by the virus. Pathol.Vet. 1965;2(4):344-359. 284. Aguirre G, Carmichael L, Bistner S. Corneal endothelium in viral induced anterior uveitis. Ultrastructural changes following canine adenovirus type 1 infection. Arch.Ophthalmol. 1975;93(3):219-224. 285. Curtis R, Barnett KC. Canine adenovirus-induced ocular lesions in the Afghan hound. Cornell Vet. 1981;71(1):85-95. 286. Agius CT, Nagesha HS, Studdert MJ. Equine herpesvirus 5: comparisons with EHV2 (equine cytomegalovirus), cloning, and mapping of a new equine herpesvirus with a novel genome structure. Virology. 1992;191(1):176-186. 287. VanDevanter DR, Warrener P, Bennett L, et al. Detection and analysis of diverse herpesviral species by consensus primer PCR. J Clin.Microbiol. 1996;34(7):1666-1671. 288. Gleeson LJ, Studdert MJ. Equine herpesviruses. Experimental infection of a foetus with type 2. Aust.Vet J. 1977;53(8):360-362. 289. Borchers K, Wolfinger U, Ludwig H, et al. Virological and molecular biological investigations into equine herpes virus type 2 (EHV-2) experimental infections. Virus Res. 1998;55(1):101-106. 290. Rushton J, Tichy A, Brem G, Druml T, Nell B. Ophthalmological findings in a closed herd of Lipizzaners. Equine Vet J. 2013;45(2):209-213. 291. Rushton JO, Kolodziejek J, Tichy A, Nowotny N, Nell B. Clinical course of ophthalmic findings and potential influence factors of herpesvirus infections: 18 month follow-up of a closed herd of lipizzaners. PLoS One. 2013;8(11):e79888. 292. Hollingsworth SR, Pusterla N, Kass PH, Good KL, Brault SA, Maggs DJ. Detection of equine herpesvirus in horses with idiopathic keratoconjunctivitis and comparison of three sampling techniques. Veterinary ophthalmology. 2015;18(5):416-421. 293. Vissani MA, Thiry E, Dal Pozzo F, Barrandeguy M. Antiviral agents against equid alphaherpesviruses: Current status and perspectives. Veterinary journal. 2016;207:38-44. 294. Garre B, van der MK, Nugent J, et al. In vitro susceptibility of six isolates of equine herpesvirus 1 to acyclovir, ganciclovir, cidofovir, adefovir, PMEDAP and foscarnet. Vet Microbiol. 2007;122(1-2):43-51. 295. Smith KO, Galloway KS, Hodges SL, et al. Sensitivity of equine herpesviruses 1 and 3 in vitro to a new nucleoside analogue, 9-[[2-hydroxy-1-(hydroxymethyl) ethoxy] methyl] guanine. American journal of veterinary research. 1983;44(6):1032-1035. 296. Miller TR, Gaskin JM, Whitley RD, Wittcoff ML. Herpetic keratitis in a horse. Eq.Vet.J. 1990;Supplement 10:15-17. 297. Matthews AG, Handscombe MC. Superficial keratitis in the horse: Treatment with the antiviral drug idoxuridine. Eq.Vet.J. 1983;Supplement 2:29-31. 298. Bentz BG, Maxwell LK, Erkert RS, et al. Pharmacokinetics of acyclovir after single intravenous and oral administration to adult horses. J Vet Intern.Med. 2006;20(3):589-594. 299. Garre B, Shebany K, Gryspeerdt A, et al. Pharmacokinetics of acyclovir after intravenous infusion of

acyclovir and after oral administration of acyclovir and its prodrug valacyclovir in healthy adult horses. Antimicrob.Agents Chemother. 2007;51(12):4308-4314. 300. Maxwell LK, Bentz BG, Bourne DW, Erkert RS. Pharmacokinetics of valacyclovir in the adult horse. J Vet Pharmacol.Ther. 2008;31(4):312-320. 301. Garre B, Gryspeerdt A, Croubels S, De Backer P, Nauwynck H. Evaluation of orally administered valacyclovir in experimentally EHV1-infected ponies. Veterinary microbiology. 2009;135(3-4):214-221. 302. Tsujimura K, Yamada M, Nagata S, et al. Pharmacokinetics of penciclovir after oral administration of its prodrug famciclovir to horses. J Vet Med Sci. 2010;72(3):357-361. 303. Kershaw O, von Oppen T, Glitz F, Deegen E, Ludwig H, Borchers K. Detection of equine herpesvirus type 2 (EHV-2) in horses with keratoconjunctivitis. Virus Res. 2001;80(1-2):93-99. 304. Thein P. The association of EHV-2 infection with keratitis and research on the occurrence of equine coital exanthema (EHV-3) of horses in Germany. In: Bryans, Gerber H, eds. Equine Infectious Diseases IV. Princeton, NJ: Vet Publ Inc; 1978:33-41. 305. Slater JD, Gibson JS, Barnett KC, Field HJ. Chorioretinopathy associated with neuropathology following infection with equine herpesvirus-1. Vet Rec. 1992;131(11):237-239. 306. McCartan CG, Russell MM, Wood JL, Mumford JA. Clinical, serological and virological characteristics of an outbreak of paresis and neonatal foal disease due to equine herpesvirus-1 on a stud farm. Vet Rec. 1995;136(1):7-12. 307. Hussey GS, Goehring LS, Lunn DP, et al. Experimental infection with equine herpesvirus type 1 (EHV-1) induces chorioretinal lesions. Vet Res. 2013;44:118. 308. Rebhun WC, Jenkins DH, Riis RC, Dill SG, Dubovi EJ, Torres A. An epizootic of blindness and encephalitis associated with a herpesvirus indistinguishable from equine herpesvirus I in a herd of alpacas and llamas. Journal of the American Veterinary Medical Association. 1988;192(7):953-956. 309. House JA, Gregg DA, Lubroth J, Dubovi EJ, Torres A. Experimental equine herpesvirus-1 infection in llamas (Lama glama). J Vet Diagn Invest. 1991;3(2):137-143. 310. Bistner SI, Rubin LF, Saunders LZ. The ocular lesions of bovine viral diarrhea-mucosal disease. Pathol.Vet. 1970;7(3):275-286. 311. Rebhun WC, Smith JS, Post JE, Holden HR. An outbreak of the conjunctival form of infectious bovine rhinotracheitis. Cornell Vet. 1978;68(3):297-301. 312. Timoney PJ, O'Connor PJ. An outbreak of the conjunctival form of infectious bovine rhinotracheitis virus infection. Vet Rec. 1971;89(13):170. 313. Wyand DS, Helmboldt CF, Nielsen SW. Malignant catarrhal fever in ehite-tailed deer. Journal of the American Veterinary Medical Association. 1971;159(5):605-610. 314. Kahrs RF, Scott FW, de Lahunte A. Congenital cerebella hypoplasia and ocular defects in calves following bovine viral diarrhea-mucosal disease infection in pregnant cattle. Journal of the American Veterinary Medical Association. 1970;156(10):1443-1450. 315. Whiteley HE, Young S, Liggitt HD, DeMartini JC. Ocular lesions of bovine malignant catarrhal fever. Vet Pathol. 1985;22(3):219-225. 316. Silverstein AM, Osburn BI, Prendergast RA. The pathogenesis of retinal dysplasia. Am J Ophthalmol. 1971;72(1):13-21. 317. Osburn BI, Johnson RT, Silverstein AM, Prendergast RA, Jochim MM, Levy SE. Experimental viral-induced congenital encephalopathies. II. The pathogenesis of bluetongue vaccine virus infection in fetal lambs. Lab Invest. 1971;25(3):206-210. 318. Osburn BI, Silverstein AM, Prendergast RA, Johnson RT, Parshall CJ, Jr. Experimental viral-induced congenital encephalopathies. I. Pathology of hydranencephaly and porencephaly caused by bluetongue vaccine virus. Lab Invest. 1971;25(3):197-205. 319. Smith TW, Albert DM, Robinson N, Calnek BW, Schwabe O. Ocular manifestations of Marek's disease. Invest Ophthalmol. 1974;13(8):586-592.