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This is to certify that the
thesis entitled

MOLECULAR PATHOLOGY OF FHV—1 INFECTION WITHIN
FELINE CORNEA, CONJUNCTIVA, LACRIMAL GLAND.
NICTITANS GLAND. TRIGEMINAL GANGLION AND CILIARY

Master of
Science

 

GANGLION

presented by

SUSAN JACOBI

has been accepted towards fulfillment
of the requirements for the

degree in Department of Small Animal
Clinical Sciences

 

 

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5/08 K'lProlecc8PresICIRC/DateDue,indd

 

MOLECULAR PATHOLOGY OF FHV-l INFECTION WITHIN FELINE
CORNEA, CONJUNCTIVA, LACRIMAL GLAND, NICTITANS GLAND,
TRIGEMINAL GANGLION AND CILIARY GANGLION
By

Susan Jacobi

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE

Department of Small Animal Clinical Sciences

2008

ABSTRACT
MOLECULAR PATHOLOGY OF FHV-l INFECTION WITHIN FELINE

CORNEA, CONJUNCTIV A, LACRIMAL GLAND, NICTITAN S GLAND,
TRIGEMINAL GANGLION AND CILIARY GANGLION

By

Susan Jacobi

Feline herpesvirus 1 (FHV-l) is one of the major causes of feline upper
respiratory tract and ocular disease. Despite intensive research in this area, the
pathogenesis of FHV-l infection within ocular and neural tissues remains poorly
understood.

In this study we utilized virus isolation, a quantitative real-time polymerase
chain reaction, histopathologic examination, and in situ hybridization to quantify the
amount F HV-l present and evaluate the associated morphological changes within the
corneas, conjunctiva, lacrimal glands, nictitans glands, trigeminal ganglia, and ciliary
ganglia of experimentally inoculated specific pathogen free cats during both the acute
and latent phases of infection. We determined F HV -1 is able to infect not only
conjunctiva] epithelium, corneal epithelium, and trigeminal ganglion, but also the
ciliary ganglion. The ciliary ganglion may serve as an additional site of latency for
FHV-l and could explain the preponderance of ocular lesions noted during
recrudescent infections. However additional studies are required to explore this theory.
The theory that lacrimal adenitis contributes to the development of KCS was not
supported as there was no evidence of FHV-l infection or secondary inflammation

within the glandular tissues at any time.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................................... V
LIST OF FIGURES ................................................................................................. VI
1. CHAPTER - FELINE HERPESVIRUS 1 LITERATURE REVIEW .............. 1
1.1 Feline herpesvirus 1 — Classification ..................................................... 1
1.2 Feline herpesvirus 1 — Characteristics .................................................... 1
1.3 Feline herpesvirus 1 — Replication ......................................................... 2
1.4 Feline herpesvirus 1 — Latency .............................................................. 4
1.5 Feline herpesvirus 1 disease - Epidemiology and pathogenesis .............. 6
1.6 Feline herpesvirus 1 disease — Other species .......................................... 9
1.7 Feline herpesvirus 1 disease - Systemic manifestations ........................ 13
1.8 Feline herpesvirus 1 disease - Ocular manifestations ........................... 14
1.8.1 Conjunctivitis .......................................................................... 14
1.8.2 Keratitis ................................................................................... 15
1.8.3 Stromal keratitis ....................................................................... 16
1.8.4 Keratoconjunctivitis sicca ........................................................ 18
1.8.5 Ophthalmia neonatorum and symblepharon ............................. 20
1.8.6 Corneal sequestrum .................................................................. 20
1.8.7 Eosinophilic keratitis ............................................................... 21
1.8.8 Anterior uveitis ........................................................................ 22

1.9 Feline herpesvirus 1 disease — Diagnosis ............................................. 23
1.9.1 Virus isolation ......................................................................... 23
1.9.2 Serology .................................................................................. 24
1.9.3 Fluorescent antibody test .......................................................... 25
1.9.4 PCR assays .............................................................................. 26
1.9.5 In situ Hybridization ................................................................ 28
1.9.6 FHV-l detection in different tissues ......................................... 28

1.10 Feline herpesvirus 1 disease - Treatment and prevention ..................... 31
1.11 Antiviral therapy ................................................................................. 33
1.11.1 Nucleoside analogues ............................................................... 33
1.11.2 Acyclovir ................................................................................. 34
1.11.3 Valacyclovir ............................................................................ 35
1.11.4 Ganciclovir .............................................................................. 35
1.11.5 Penciclovir ............................................................................... 36
1.11.6 Famciclovir .............................................................................. 37
1.11.7 Idoxuridine .............................................................................. 37
1.11.8 Trifluridine .............................................................................. 38
1.11.9 Vidarabine ............................................................................... 38
1.11.10 Cidofovir ................................................................................. 39
1.11.11 Bromovinyldeoxyuridine ......................................................... 39
1.11.12 Ribavirin .................................................................................. 40
1.12 Efficacy of nucleoside analogues against FHV-l ................................. 40
1.13 Non-nucleoside antivirals .................................................................... 42
1.13.1 Foscamet ................................................................................. 42
1.13.2 L-Lysine .................................................................................. 42

iii

1.13.3 Interferons ............................................................................... 45

1.13.4 Bovine lactoferrin .................................................................... 46
1.13.5 Vaccination .............................................................................. 47
1.14 Conclusions ......................................................................................... 50
2. CHAPTER — ANATOMY AND HISTOLOGY OF TISSUES
RELEVANT TO THIS STUDY ................................................................... 52
2.1 Cornea ................................................................................................ 52
2.2 Conjunctiva ......................................................................................... 53
2.3 Lacrimal gland .................................................................................... 53
2.4 Nictitans gland .................................................................................... 54
2.5 Trigeminal ganglion ............................................................................ 55
2.6 Ciliary ganglion .................................................................................. 56
3. CHAPTER - HY POTHESES AND SPECIFIC AIMS .................................. 57
3.1 Hypotheses .......................................................................................... 57
3.2 Specific aims ....................................................................................... 57
4. CHAPTER — RESEARCH PROTOCOL, RESULTS, AND
DISCUSSION .............................................................................................. 59
4.1 Introduction ......................................................................................... 59
4.2 Materials and Methods ........................................................................ 61
4.2.1 Animals ................................................................................... 61
4.2.2 Inoculation and clinical scoring ................................................ 61
4.2.3 Samples ................................................................................... 62
4.2.4 Virus isolation ......................................................................... 63
4.2.5 Quantitative real time PCR ...................................................... 63
4.3 Results ................................................................................................ 71
4.3.1 Clinical features ....................................................................... 71
4.3.2 Virus isolation ......................................................................... 72
4.3.3 Quantitative real time PCR ...................................................... 73
4.3.4 Histopathology ......................................................................... 74
4.3.5 In situ hybridization ................................................................. 78
4.4 Discussion ........................................................................................... 80
APPENDICES ........................................................................................................ 87
5. APPENDIX A ............................................................................................. 88
6. APPENDIX B .............................................................................................. 89
7. APPENDIX C .............................................................................................. 94
8 APPENDIX D ............................................................................................. 95
9. BIBLIOGRAPHY ...................................................................................... 101

iv

Table 4.1.

Table 4.2.

Table 4.3.

Table 4.4.

Table 5.1.

Table 7.1.

Table 7.2.

Table 7.3.

Table 8.1.

Table 8.2.

Table 8.3.

Table 8.4.

Table 8.5.

Table 8.6.

LIST OF TABLES

Clinical scores ......................................................................................... 72
Summarized results of virus isolation (positive/total) .............................. 73
Summarized results of quantitative real time PCR (positive/total) ........... 74
Summarized results of in situ hybridization ............................................. 78
USDA Scoring Method for FHV-l .......................................................... 88
Virus isolation day 6 post inoculation ...................................................... 94
Virus isolation day 10 post inoculation .................................................... 94
Virus isolation day 30 post inoculation .................................................... 94
PCR results of cornea (Copy no. / 100 ng DNA) ..................................... 95
PCR results of conjunctiva (Copy no. / 100 ng DNA) ............................. 96
PCR results of lacrimal gland (Copy no. / 100 ng DNA) ......................... 97
PCR results of nictitans gland (Copy no. / 100 ng DNA) ......................... 98
PCR results of trigeminal ganglia (Copy no. / 100 ng DNA) ................... 99
PCR results of ciliary ganglia (Copy no. / 20 ng DNA) ......................... 100

LIST OF FIGURES

Images in this thesis are presented in color.

Figure 4.1. Standard melt curve chart. ..................................................................... 65
Figure 4.2. Standard melt peak chart ........................................................................ 65
Figure 4.3. Standard amplification chart, 10 fold dilutions of 106 genomic

copies of FHV-l, triplicate. .......................................................................... 66
Figure 4.4. PCR standard curve. .............................................................................. 66
Figure 4.5. FHV-l pellet pre-treated with different protocols ................................... 68
Figure 4.6. FHV-l pellet pre-treated via micowaving for 10 minutes using an

antibody dilution of 1:250. ........................................................................... 69
Figure 4.7. Conjunctiva] sample from cat # 12. (HE) ............................................... 76
Figure 4.8. Corneal sample from cat # 9. (HE) ......................................................... 76
Figure 4.9. Lacrimal gland sample from cat # 2. (HE) ............................................. 77
Figure 4.10. Trigeminal ganglion sample from cat # 1. (HE) ................................... 77
Figure 4.11. In situ hybridization of the conjunctiva] sample from cat # 12 .............. 79
Figure 4.12. In situ hybridization of the corneal sample from cat # 4. ...................... 79
Figure 6.1. Results of the clinical examination: sneezing. ........................................ 89
Figure 6.2. Results of the clinical examination: elevated body temperature. ............. 89
Figure 6.3. Results of the clinical examination: dehydration. ................................... 90
Figure 6.4. Results of the clinical examination: anorexia. ........................................ 90
Figure 6.5. Results of the clinical examination: ptyalism. ........................................ 91
Figure 6.6. Results of the clinical examination: oral ulceration. ............................... 91
Figure 6.7. Results of the clinical examination: nasal discharge. .............................. 92
Figure 6.8. Results of the clinical examination: blepharospasm ................................ 92
Figure 6.9. Results of the clinical examination: ocular discharge. ............................ 93
Figure 6.10. Results of the clinical examination: conjunctivitis ................................ 93

1. CHAPTER - FELINE HERPESVIRUS 1 LITERATURE REVIEW

1.1 Feline herpesvirus 1 — Classification

The family of the herpesviridae is comprised of different subfamilies:
alphaherpesvirinae, betaherpesvirinae, gammaherpesvirinae, and an unidentified
subfamily.l Herpesvirz'dae are enveloped viruses that have an icosahedral capsid
containing a linear double stranded DNA.1 Feline herpesvirus 1 (FHV-l) has been
classified as a member of the varicellovirus genus of the subfamily
alphaherpesvirz’nae.2'5 Prototypical members of this subfamily are human
herpesvirus-1 (herpes simplex virus, HSV-l, genus simplexvirus), and human
varicella-zoster virus (VZV, genus varicellovirus).1 Alphaherpesvirinae relevant to
the veterinary field include equine herpesvirus 1 and 4 (EHV-l and 4), bovine
herpesvirus-1 (BHV-l), canine herpesvirus-1 (CHV-l), pseudorabies virus (PRV,
PHV-l), and gallid herpesvirus-2 (GHV-Z, Marek’s disease virus).6’7 Feline
herpesvirus-1 is closely related genetically and antigenetically to CHV-l and phocid
(seal) herpesvirus-1 (PhV-l), and cross-protection between feline and PhV-l has been

reportedg'l6

1.2 Feline herpesvirus 1 - Characteristics

The size of feline herpesvirus particles is 120-180 nm in diameter.17 Like most
alphaherpesviruses, FHV-l is composed of four major structural elements including
an electron-opaque core, an icosahedral capsid, a tegument, and an envelope."7 A
single molecule of linear double stranded DNA is wrapped around the fibrous spool-
1ike core, which is anchored inside the capsid.1 The capsid is a structural characteristic
of all herpesviruses, 100 nm in diameter, and consists of 150 hexameric and 12

- r,4,7,1 . -
pentamenc capsomers. 7 The nucleocapsrd is surrounded by an electron dense

1,4,7

amorphous material, called the tegument. The outer layer of the virion is formed

by a fragile lipoprotein envelope, which carries viral glycoproteins.l

The physical structure of the FHV -1 genome is similar to the genomes of other
alphahennesvirinae.18 Rota et a1., first characterized the genome of FHV-l strain C-27,
the strain utilized in the present study, and determined that FHV-l DNA was
approximately 134 kb and was composed of a long (L) and a short (S) segment. The
long segment (U1) was 104 kb in size and was composed of unique DNA.18 The
adjacent S segment was approximately 30 kb in size and contained a central portion of
unique DNA (Us) which was approximately 8kb in size.18 The Us region was
bounded by inverted repeat sequences which were 11 kb in size.18 Later Grail et a1.,
determined the size of the genome of F HV-l strain B-927 to be 126 kb, composed of

a long unique region of 99 kb and a short region of 27 kb containing a unique

sequence of 8-9 kb flanked by inverted repeats of 7-8.5 kb.19

1.3 Feline herpesvirus 1 — Replication

The replication cycle of HSV-1 has been studied extensively. Most other
alpha-herpesvirina follow a similar pattern. The different phases of replication are
initiation, early gene expression, DNA replication, late gene expression, assembly,
and release. 1’7’20

Epithelial cells of the nasal mucosa, conjunctiva, tonsils, and nasal turbinates
are the target for primary infection by F HV -1 .21 During the initiation phase, multiple
proteins participate in the attachment of the virus to the host cell.1 Three viral
glycoproteins, gB, gC, and gD, are essential for HSV-l infection.l An FHV-l gB
homolog has been identified and sequenced.22 Via gB and gC the virion binds to

heparin sulfate or chondroitin sulfate proteoglycans of the extracellular matrix of the

host cell.l To facilitate cellular entry, gD binds to a cellular co-receptor."20 This

2

contact initiates fusion of viral and plasma membranes.1 Viral nucleocapsids then
attach to microtubules and are transported to the nucleus.23 The viral nucleocapsid
docks at the nuclear pore, releasing viral DNA into the nucleus. L7

Gene expression of the FHV-l genome is believed to be similar to that of
HSV-1 in human cells.“25 VP16, a tegument protein, interacts with host transcription
proteins to stimulate transcription of immediate-early genes by host RNA polymerase
II."26 Immediate-early mRNAs are spliced and transported to the cytoplasm where
they are translated. The immediate-early proteins (or-proteins) are transported to the
nucleus where they activate transcription of early genes and regulate transcription of
immediate-early genes.l Early gene transcripts are transported to the cytoplasm. The
early proteins (B-proteins) fimction primarily in DNA replication and production of
substrates for DNA synthesis. Some fl-proteins are transported to the nucleus, while
others function in the cytoplasm.1

DNA replication and recombination produces long, concatemeric DNA, the
template for late gene expression. Late mRNAs are transported to the cytoplasm
where they are translated.1 Late proteins (“y-proteins) function primarily as viral
structural proteins or participate in virus assembly and particle egress. The precursor
glycoproteins are also transported to the Golgi apparatus for further modification and
processing. Mature glycoproteins are transported to the plasma membrane of the
infected cell. 'y-proteins are transported to the nucleus for assembly of the
nucleocapsid and DNA packaging. Newly replicated DNA is packaged into
nucleocapsids. '

DNA containing nucleocapsids, together with some tegument proteins, bud
from the inner nuclear membrane into the perinuclear lumen, acquiring an envelope.1

These immature enveloped virions fuse with the outer nuclear membrane, releasing

the nucleocapsid into the cytoplasm.1 The structure is then transported to a late trans-
Golgi compartment or endosome that contains mature viral membrane proteins.1 Here
the particles acquire an envelope containing mature viral envelope proteins and the
complete tegument layer.1 The enveloped virus particle then buds into a vesicle that is
transported to the plasma membrane for release by exocytosis.1 Enveloped viruses
tend to spread from cell to cell, which accounts for the formation of plaques and

. . . . . . 27-29
syncytra seen during vrrus rsolatron 1n cell culture.

1.4 Feline herpesvirus 1 -— Latency

Feline herpesvirus-1, similar to other herpesviruses can establish latency, a
phase where replication is limited. The virus may become latent for the lifetime of the
host. Three general properties characterize latent infections: expression of productive
cycle viral genes is absent or inefficient; immune detection of the cell harbouring the
latent genome is reduced or eliminated; and the viral genome persists intact so that at
some later time a productive acute infection can be initiated.1 In the latent state, the
genome can be maintained as a non-replicating chromosome in a non-dividing cell
such as a neuron (e.g., HSV-l, VZV); become an autonomous, self replicating
chromosome in a dividing cell (e.g., Epstein-Barr virus), or be integrated into a host
chromosome (e.g., adeno-associated virus).1 During latency, a balance among the
regulators of viral and cellular gene expression must be maintained. Therefore only a
restricted set of viral gene products is produced.l

For (Jr-herpesviruses it was generally believed that sensory neurons within
ganglia were the primary site of latency.30 However, more recent studies have shown
herpes simplex latency within the autonomic pathways of mice.31 The trigeminal
ganglion is considered a primary site of latency following ocular, oral, or intranasal

infection.32’33 Once latency is established, viral DNA persists in neurons for the life of

4

an infected host.30 It is unclear if latency induces neurologic injury.3'0 However, it has
been shown that during reactivation from a latent infection, inflammation and
destruction of neurons and surrounding tissue can occur.“35

In neurons latently infected with HSV-l the only abundantly transcribed gene
is the latency-associated transcript (LAT).36’37 Therefore, LATs provide molecular
markers for latently infected cells.3°’38 The length of the primary LAT transcripts is
8.3 to 8.5 “3.39,“, LAT overlaps the viral genes for ICPO and ICP34.5 in an anti-sense
direction.”37 It has been suggested that LAT is a regulatory RNA that represses
expression of ICPO.37 Although the LATs are first detectable at the peak of an acute
infection, they are present at much higher levels during latency with high levels in

37““ Several studies suggested that LATs play a role in

trigeminal ganglia.
establishment of latency. It has been demonstrated that HSV-l LATs enhances
establishment of latency in mice.42’43 Furthermore, it has been found, that LATs
repress productive viral gene expression in trigeminal ganglia of mice during acute
infection.“45 Although LATs are not mandatory for the virus to establish latency,
viral mutants that are unable to express LATs are reactivation deficient“6 Therefore
LATs may play a role in reactivation from latency. Different studies using various
LAT mutants have shown that LATs increase the induced and spontaneous
reactivation phenotypes in the rabbit ocular model“’48 and the induced reactivation
phenotype in mice.42’49'52

LATs also have anti-apoptotic features and maintain latency by promoting the
survival of infected neurons.53’54 Gupta et al.55 showed that microRNA encoded by the
HSV-l LAT gene confers resistance to apoptosis. The anti-apoptotic activity of LATs

is important during the latency-reactivation cycle. Because infected neurons would

normally be eliminated by cytotoxic T cells, which kill their target cells in part by

apoptosis, high levels of LAT within latently infected neurons may prevent their
elimination.56 A functional relationship between the ability of LATs to promote cell
survival and their ability to enhance spontaneous reactivation has been
demonstrated.57

Primary and recrudescent herpetic disease is more prevalent among immune-
compromised individuals.58 The fact that viral reactivation is associated with immune
suppression and the persistence of T cells and cytokines in latently infected human
trigeminal ganglia suggest that T cells play a role in control of the virus at the site of
latency.5860 Theil et al.59 found T cells positive for CD3 and CD8 markers as well as
CD68 positive macrophages in 30 of 42 trigeminal ganglia from 21 healthy humans.
The presence of these cells correlated constantly with the occurrence of HSV-1 LATs.
Uninfected trigeminal ganglia showed no immune cell infiltration. Quantitative real
time PCR revealed an elevated cytokine/chemokine expression (IFN-y, TNF-or,
RANTES, and IP-lO) in the latently infected trigeminal ganglia demonstrating that
latent herpesviral infection in humans is accompanied by a chronic inflammatory
process, but without neuronal destruction. It seems likely that the immune response

maintains viral latency and inhibits viral reactivation.

1.5 Feline herpesvirus 1 disease - Epidemiology and pathogenesis

Feline herpesvirus-1 is also known as feline rhinotracheitis virus. Feline viral
rhinotracheitis was first recognized in 1957 in 5 to lO-week-old farm kittens that were
being raised in a laboratoryzw Since then it has been recognized as a disease with
world-wide distribution within the cat population. Epidemiologic studies indicate that
as many as 50-75% of cats have serologic evidence of exposure to the virus.(’2’63 Other

studies estimate that over 90% of cats are sero-positive for the virus;64 at least 80% of

infected individuals remain latently infected for life; and approximately 45% of
latently infected cats shed virus throughout life.65

Reactivation of virus may occur spontaneously but is most likely after stress.66
Re-excretion of F HV-l could be stimulated in more than 50% of cats recovered from
F HV-l infection through systemic administration of corticosteroids or a change in
housing.67 Detectable viral shedding does not immediately follow the stress.66 A lag
phase of 4-11 days (mean 7.2 days) precedes the shedding of infectious virus.65 The

period of shedding can range in duration from 1-13 days (mean 6.5 days).65'67

“”68 although in some cases carriers show

Shedding carriers are mainly asymptomatic,
recrudescence of mild clinical signs while they are shedding.69 During episodes of
viral shedding infectious virus is present within oro-nasal and conjunctival secretions
and serves as source of infection to other cats.”67

The enveloped virion is relatively fragile and very susceptible to heat, drying,
or disinfectants.7°’7| At room temperature FHV-l can only persist in the enviromnent
for 18-24 hours in relatively dry conditions.7°‘72’73 Transmission of FHV-] between
cats occurs via direct contact, fomites, or macrodroplets which can be sneezed about
1.3 m.74 Management of infected cats within a hospital situation requires appropriate
separation of the cats and routine hygiene practices for personnel and facilities.75 The
virus survives less than one hour in Ophthalmic solutions used for routine diagnostics
testing, such as fluorescein stain and topical ocular anesthetics. 76 However, the virus
may persist for up to 5 days in eye wash solutions.76

Young cats are most susceptible to primary infection, especially around 8-12
weeks of age when maternal antibodies wane.70 Cats with underlying disease (feline

leukaemia virus (FeLV), feline immunodeficiency virus (F IV)) are also very

susceptible to FHV-l infection.77 Due to limited ability of FHV-l to replicate at

temperatures of 37 °C or higher, the primary sites of replication are the cooler
epithelial surfaces.70 The natural routes of infection are via nasal, oral, and
conjunctival mucous membranes.“’70 In the acute phase of infection virus replication
occurs predominantly in the mucous membranes of the nasal septum, turbinates,
nasopharynx, and tonsils; other tissues including conjunctivae, mandibular lymph
nodes and the upper trachea are also often involved.66’78’79 The infection is most
commonly restricted to superficial epithelial tissue, which may include the corneal
epithelium-’3'80 Since viral replication is usually restricted to areas of lower body
temperature, viremia is rare. It has only been occasionally reported and the virus
appears to be associated with peripheral blood leucocytes.79’81 Generalized disease is
predominantly seen in debilitated animals or neonatal kittens.70‘82’83

As the virus replicates its cytopathic effect creates areas of multifocal
epithelial necrosis leading to inflammation with neutrophilic infiltration and fibrin

- 27,6l,6 ,7 .72,79
exudation. 6 0

Secondary bacterial infection often worsens the condition.70
Incubation periods can range from 2-10 days.27 The clinical signs usually resolve
within 10-14 days.27 Detection of infectious virus is possible as early as 24 h post
infection via oropharyngeal and nasal swabs. The virus generally persists for 1-3
weeks, although detection of viral DNA by PCR might be possible for longer.”85

In severe cases osteolytic changes within the turbinate can be caused by the
virus.66 Following intravenous inoculation of young kittens a predilection of the virus
for regions of skeletal growth, including the turbinates has been shown.86
Experimentally it also has been shown that intravaginal instillation of the virus in

pregnant queens can lead to vaginitis and congenitally infected kittens.87 Intravenous

inoculation of pregnant queens led to transplacental infection and abortion.88

1.6 Feline herpesvirus 1 disease — Other species

Feline herpesvirus 1 has been described to possess a very narrow host range.89
However, FHV-l can exist within other non-domestic felids or even non-felidae as
well.

In 1971 Thomson et al.90 reported the isolation of a herpesvirus from three
Cheetahs (Acinonyx jubatus) living at the Lion Park at Warragamba, New South
Wales. Two of the cats developed conjunctivitis and rhinitis.90 The third cat showed
no clinical symptoms and it was suggested that this animal may have been a carrier.90
Virus was isolated from the eyes, nose, and mouth, but not from the rectum. The
isolates were identified a herpesvirus by their production of cytopathic effects,
including giant cells and Cowdry type A inclusion bodies, and their appearance on
electron microscopy.90

In 1984 a disease outbreak occurred at the St. Louis Zoological Park, affecting
two of three queen-reared Cheetah cub siblings.91 The cubs had been vaccinated with
a killed virus product at three-week intervals from ages 3.5 to 18 weeks.91 By the age
of three months they developed chronic, multifocal, ulcerative cutaneous lesions,
which were refractory to treatment.” Excisional biopsies suggested a diagnosis of
eosinophilic granuloma, but intranuclear inclusions characteristic of herpesvirus were
also found.g1 Herpesvirus was also found in these samples by transmission electron
microscopy. Virus isolation revealed a virus that induced cytopathology similar to that
of alphaherpesviruses. The presence of a herpesvirus was suggested, which was
named cheetah herpesvirus (ChHV).91 In order to compare this virus with FHV-l,
further tests (dot blot hybridization, restriction endonuclease digested DNA
electrophoretic profiles, antigenic comparison by serum neutralization and western

immunoblotting analysis, SDS polyacrylamide gel electrophoresis comparison of viral

9

polypeptides) revealed that the ChHV isolate was closely related to FHV -1.91

However, at least two detectable differences in the Kpn I digestion patterns were
considered to be significant because of the remarkable stability of FHV-l and lack of
restriction fragment length polymorphism.92 Whether or not ChHV is truly different
from FHV-l remains to be investigated.

In 1977 Boever et a].93 reported viral rhinotracheitis in 4 of 5 clouded leopards
(Felis nebulosa) housed at the St. Louis Zoo. The leopards had clinical signs which
consisted of serous nasal and ocular discharge and sneezing. Two of the cats also had
ulcerative glossitis. One pregnant animal developed vaginitis and aborted. Nasal and
rectal swabs were obtained for virus isolation from three severely affected animals
that died due to progression of their illness. A cytopathogenic agent, which was
identified as feline rhinotracheitis virus, was isolated from the nasal swab of one
animal.93

Truyen et a]. in 199094 reported an acute outbreak of encephalomyelitis in 10
lions from a safari park in Germany. Three lions were euthanized and different
diagnostic tests (histopathology, virus isolation, serum neutralization) were performed.
Apart from mild swelling and reddening of the tonsils, alterations were mainly found
in the brain. The pathological diagnosis was non-suppurative polioencephalomyelitis
with demyelinization of the spinal cord. In the nuclei of some glial cells small
intranuclear, acidophilic, round and ovoid structures suggestive of intranuclear
inclusion bodies were found. Virus isolated from the tonsils of one animal
demonstrated cytopathic effects. The cytopathic agent was serologically characterized
as FHV -1. However, FHV-l was not isolated from any brain tissues. Ultimately it was

concluded that FHV-l was unlikely to be the cause of the encephalitis outbreak.94

10

Surveys of antibody titers in free—ranging lions in Africa revealed a
seroprevalence for FHV—l of 67% and 91% in Etosha National Park and Kruger
National Park, respectively.”96 Antibodies to FHV -1 were found in 309/310 samples
in lions living within Serengeti National Park and Ngorongoro Crater.97

In 1991, Eberle et a].98 evaluated sera from 19 free-ranging bobcats (Lynx
rufus) to determine the seroprevalence of FHV-l via an enzyme linked
immunosorbent assay (ELISA). All samples were negative suggesting that FHV-l
was uncommon or not present at all in this species. Four free-ranging bobcats were
captured, and ELISA for anti- FHV -1 antibodies and attempts at virus isolation from
oropharyngeal swabs were negative. The cats were inoculated by instilling 200 u] of
the viral suspension in each nostril. None of the animals developed clinical signs of
disease. However, virus was isolated from the oropharynx and nostrils on days 3-4, 7-
8, and 14-15 post-inoculation. Although the bobcats did produce anti-FHV-l IgG,
titers were generally lower than those seen in domestic cats.98 The experiment
demonstrated that bobcats are susceptible to FHV-l infection, but clinical signs of
disease do not accompany the infections. Paul-Murphy et al."9 demonstrated a
seroprevalence of 19% for FHV-l in 58 mountain lions (F elis concolor) in California.

In 1999, Pratelli et a].100 reported an outbreak of respiratory tract disease
characterized by sneezing, nasal and ocular mucopurulent discharge, anorexia and
prostration in three tigers and one leopard from the Fasano Zoo in southern Italy.
Nasal swabs were obtained from all four animals for virus isolation. Characteristic
cytopathic effects and intranuclear inclusions were observed. Virus neutralization
tests using a goat antiserum, monospecific to FHV-l, identified the isolates as F HV -1
strains. Restriction patterns were compared with those of a vaccine] and a field strain

of FHV-l. Restriction patterns obtained with PstI and BamHI revealed that the

11

genomes of the four isolates, the field strain, and the vaccination strain were identical.
It could not be concluded if the disease was caused by a FHV-l strain reactivated in a
wild felid or by a strain transmitted by domestic cats.100

Nakamura et al.101 investigated the seroprevalence of F HV-l in domestic cats
(F elis catus) and leopard cats (F elis bengalensis) in Vietnam. All cats were
considered to be unvaccinated. In the Hanoi area only 1.4% (1/69) of domestic cats
were positive for FHV-l antibodies, compared to 44% (22/50) in the Ho Chi Minh
City area. Of the tested leopard cats, 11.1% (1/9) were positive in the Hanoi area,
whereas 50% (2/4) were positive in the Ho Chi Minh City area. The authors suggested
that the higher cat population density in Ho Chi Minh City area explained in part the
higher prevalence of HIV-1.

Everrnann et al.102 isolated a herpesvirus from dogs with diarrhea. Virus
neutralization tests revealed a relation of the isolated viruses to F HV-l. Rota et 31.103
compared five herpesvirus isolates from these dogs to the DNA and polypeptide
patterns of FHV-l. The genomes of these five canine isolates showed restriction
enzyme digestion patterns that were nearly identical to those of FHV -1, but
completely different from CHV. The disease potential of these canine F HV-l-like
isolates remained unclear in this study.

1.104 isolated FHV-l from the thymus and spleen of a 17 week old

Kramer eta
cross breed dog with a history of distemper-like illness, diabetes mellitus, and
pancreatic atrophy. Following culture of the virus in Crandell feline kidney cells,

three healthy puppies were inoculated intravenously and intranasally with the viral

suspension. No clinical signs were produced.

12

In a study investigating spotted hyenas, only one of 240 animals had serum
antibodies against FHV-l.105 This suggests that hyenas unlikely play a role as a

reservoir for FHV-l in the Serengeti ecosystem.

1.7 Feline herpesvirus 1 disease - Systemic manifestations

Feline herpesvirus-1 infection causes upper respiratory tract symptoms, ocular
manifestations and reproductive failure.106 The primary, acute disease affects kittens
more commonly and more severely. Following an incubation period of 24-48 hours, a
sudden onset of sneezing, followed by profuse serous nasal and ocular discharge,
hypersalivation, fever, anorexia and lethargy may be observed.”1’27’70’79’106 Due to
secondary bacteria] invasion, the discharge may progress from serous to
mucopurulent.106 In severe cases, the virus may invade the lungs leading to viral
pneumonia and possibly secondary bacteria] pneumonia.70’82’83’107 Bordetella
bronchiseptica has been identified as a frequent secondary invader.108 Primary FHV—l
infection is self limiting in most cases, and symptoms regress within 10-14 days.70
Chronic sinusitis may follow severe primary infection and may result in necrosis and
distortion of the turbinates.“66

In rare cases of FHV-l infection, oral ulceration has been observed. However,
oral ulceration is more commonly associated with feline calicivirus (FCV)
infection.69’106 As a rare sequela to FHV-l infection, neurological signs have been
described.109 Abortion has been associated with FHV-l infection. However,
experimental studies have suggested that abortion is most likely due to the severe
systemic illness, rather than a direct effect of the virus itself.88 Hickman et a].110
investigated a natural outbreak of F HV-l infection in a specific pathogen free cat

colony. No cases of abortion were seen, even in severely affected pregnant queens. 1 '0

l3

Cutaneous ulceration has been described with FHV—l infection in domestic
cats and also in Cheetahsm'l '6 Histopathologic examination of the lesions reveals

”5’1'6 Typically a marked eosinophilic

necrotizing and ulcerative changes.
inflammatory response is noted within these lesions. However, the infiltrate may also
be neutrophilic in some cases.1 1 H '6 Holland et al.117 showed that FHV -1 DNA can be
detected in the skin of cats with herpetic dermatitis and suggested that the virus may

play a causative role in the disease. Furthermore, a PCR assay for FHV -1 was shown

to be useful in the diagnostic process with high sensitivity and specificity.”7
1.8 Feline herpesvirus 1 disease - Ocular manifestations

1.8.1 Conjunctivitis

Feline herpesvirus-1 infection is the most common cause of conjunctivitis in
cats and is often accompanied by respiratory disease.‘18 Bilateral conjunctivitis
characterized by blepharospasm, chemosis, serous ocular discharge, and hyperemia is
the most common ocular manifestation during acute FHV-l infection.4’2l”0’71"19‘122
Diffuse necrosis of the conjunctiva] epithelium and intranuclear inclusions within
conjunctiva] epithelial cells are present by day four post-infection.4 Around day 8
post-infection, cytopathic effects of the virus cause the conjunctiva] epithelium to
slough.4 The serous ocular discharge can progress over several days to mucopurulent
discharge as polymorphonuclear cells as well as low numbers of macrophages
infiltrate the conjunctiva] stroma.4’120 Bacteria] infections may worsen the
mucopurulent discharge, and pseudodiphtheritic membrane formation may occur.1 '8

Most cats recover within 10-20 days with no other ocular sequelaef“120 However,

more severe infection or immunosuppression may result in chronic or recurrent

14

conjunctivitis.123 F HV-l infection in adult cats typically causes conjunctiva]
hyperemia, intermittent blepharospasm, and mild serous ocular discharge. '24
Conjunctiva] cytologic findings depend on the duration of the disease process
and the presence or absence of other infectious agents. 1 18 The predominant cell type is
the lymphocyte, although macrophages are also a common finding. l 18"” With chronic
or secondary infection, more neutrophils and giant epithelial cells may be seen.1 18“”
Epithelial intranuclear inclusion bodies may occur, but are rarely observed in clinical

C3868.121

1.8.2 Keratitis

Epithelial keratitis commonly occurs during primary infection in young cats,
which resolves spontaneously in most cases.125 Feline herpesvirus-l induced corneal
ulceration in adult cats is likely due to reactivation of the virus.l ”"20 This condition is
usually accompanied by varying degree of conjunctivitis.70 Systemic
immunosuppression, environmental stress, and systemic administration of
corticosteroids have been associated with reactivation of latent virus.12°‘125 In the adult
cat, herpetic keratitis can be unilateral or bilateral and is usually not associated with
upper respiratory disease.”8’125 Corneal edema may precede the formation of comea]

ulcers. 1 18

During the acute stage of ocular infection, the corneal lesions tend to be
microdendritic.”9 Early corneal ulcers may also be dendritic. However, they often
progress quickly to larger geographic ulcers.4’70 Dendritic shaped corneal ulcers are
the classical pattern of corneal ulcerations induced by FHV-1.4’7°’”9’126"27 They result
from the direct cytopathic effect of the virus”:5 .127 Sometimes, ulcers may also occur
as numerous punctate lesions, that may coalesce to form a larger superficial ulcer.“8

Occasionally herpetic ulcerations progress, likely due to secondary bacterial infection,

to involve deeper stromal layers or cause corneal perforation.7O Chronic ulcers may

15

incite a corneal vascular response, although the intensity varies greatly.70 FHV-l
induced corneal ulcerations may resolve spontaneously, whereas others may develop

. . 70
rnto indolent ulcers.

1.8.3 Stromal keratitis

Herpetic stromal keratitis caused by HSV-l is the leading cause of infectious
corneal blindness in the industrialized world. Changes associated with stromal
keratitis are thought to be due to the inflammatory response to chronic or repeated
recrudescent infection. Several observations support the conclusion that the
pathogenesis of herpetic stromal keratitis is immune mediated, including the
observation that this manifestation of ocular herpetic disease is uncommon in humans
who are immunodeficient.128 Furthermore, large numbers of inflammatory cells
infiltrate the cornea, including polymorphonuclear neutrophils, macrophages,
Langerhans cells, natural killer cells, plasma cells and T cellsm"32 The importance
of the immune response in stromal keratitis is also supported by observations that in T
cell-deficient mice, ocular infection with HSV-l was not associated with the
development of marked stromal disease. '33

The exact immunological mechanism of the development of stromal keratitis
is not known. Originally the chronic changes believed to result from immune complex
deposition because immunoglobulins and herpesvirus particles were found in the
stromal” However, there is evidence, that a chronic low-level viral infection persists
within stromal keratocytes providing a continuous stimulus to antigen-specific T
lymphocytes that kill the keratocytes by a cytotoxic mechanism.I35 Furthermore, CD4
T cells reactive against HSV-l antigens have been isolated from chromic stromal

keratitis lesionsm’137 The pathogenesis of corneal scarring and vascularization in

stromal keratitis is still uncertainm’139 HSV-l infection can induce production of

16

different angiogenic factors such as vascular endothelial growth factor (VEGF),
matrix metalloproteinases (MMP) 2 and 9, platelet-derived grth factor (PDGF), B-
fibrosing growth factor (bFGF), macrophage inflammatory protein 2 (MIP-2) and
monocyte chemotactic protein 1 (MCP—l).'4°’142 These factors are mainly produced by
polymorphonuclear neutrophils. However, VEGF is also expressed in corneal
epithelial and stromal cells. It appears to be produced by cells close to those infected
by the virus.140 It is possible that interleukin 1 (IL-1) and interleukin 6 (IL-6),
produced initially by infected cells, induced the production of VEGF.

Stromal keratitis is a rare manifestation of corneal FHV -1 infection.“’80
However, due to its potential to cause vision threatening corneal scarring, it is
considered the most significant manifestation of feline ocular herpetic disease.4 It is
usually associated with chronic epithelial ulceration and characterized by stromal
edema, deep vascularization and the presence of inflammatory cells.4 In
experimentally induced ocular feline herpesvirus-1 infection, the virus was observed
to preferentially infect conjunctiva] epithelium. However, FHV-l was able to replicate
in corneal epithelium, and only minimal viral antigen was detected in the corneal

stroma . 80

When cats were treated with subconjunctival administration of
betamethasone followed by topical application of FHV-l to the scarified cornea, a
stromal keratitis developed, characterized by geographic epithelial ulceration,
interstitial edema, deep vascularization, and inflammatory cell infiltratesfl’80

To elaborate the mechanism through which stromal disease occurs, Nasisse et
a].143 studied the histologic, immunologic, and virologic features of FHV-l induced
keratitis in 18 experimentally infected cats. Stromal disease was induced by local

immunosuppression via application of three consecutive daily subconjunctival

injections of 4 mg betamethasone prior to inoculation with FHV-l .143 Prior to the

17

development of stromal keratitis, prolonged absence of the corneal epithelium,
decreased numbers of circulating lymphocytes, decreased mitogen responses, and
acquisition of viral antigen by the corneal stroma were noted.'43 The first cells to
arrive in infected corneas were neutrophils. The subsequent arrival of B and T
lymphocytes was supportive of a specific antiviral immune response.143 Fibrosis and
scarring developed secondary to the stromal inflammation. The authors concluded,
that it is probable that the corticosteroid-mediated suppression of the normal immune
function allowed uncontrolled local virus replication, which combined with delayed
corneal epithelial healing led to stromal accumulation of virus.143 The development of

stromal keratitis was likely a result of the immune response to viral antigen.‘43

1.8.4 Keratoconjunctivitis sicca

Decreased tear production in cats has long been suspected to be a potential
sequelae to FHV-l infection.”9 Clinical signs can include conjunctiva] hyperemia, a
dry appearance of the cornea, cornea] epithelial hyperplasia and corneal ulceration.120
The cause remains speculative, but it has been assumed that excretory duct occlusion
or lacrimal adenitis is responsible.4’70’80 In most cats, keratoconjunctivitis sicca is
transient, with tear production increasing with resolution of active FHV-l related
disease.70 However, a decrease in tear production associated with FHV-l infection
can also be permanent.“’70 Experimentally, a temporary keratoconjunctivitis sicca
developed in 5 of 10 cats treated pre-inoculation with three consecutive daily
subconjunctival injections of betamethasone.80 Aqueous tear production returned to
normal in all cats by day 60 post-infection.

Using specific fluorescent antibody test and virus isolation, Bistner, et a].
failed to demonstrate the presence of FHV-l within the lacrimal gland.121 However,

using a PCR assay, Reubel et a], detected FHV-l DNA in two of five lacrimal glands

18

of latently infected cats, as defined by negative virus isolation from swabs of oral and
ocular secretions. 144 Fluorescein labeled antibody has been used to examine human
lacrimal glands during both acute and asymptomatic stages of HSV-1 corneal
infection. Viral antigen was detected in 2/3 patients with acute disease and in 4/7
asymptomatic patients with a history of previous herpetic infection.145 A case of
dacryoadenitis was described in an immunodeficient patient.‘46 A biopsy sample from
the right lacrimal gland was positive on immunohistochemisty for HSV-l. However,
virus isolation was negative for HSV-l, VZV, cytomegalovirus, and enterovirus, but
positive for adenovirus. The patient was treated with systemic antiviral therapy
(acyclovir). The patient recovered with the exception of mild bilateral dry eye
symptoms. Duration of the dry eye could not be evaluated because the patient was lost
to follow-up.

Lim and Cullen147 evaluated Schirmer tear test values and tear film break up
times in cats with conjunctivitis. Lower mean tear film break up times, but not lower
Schirmer tear test values were found in cats that had FHV—l DNA detected by PCR in
their blood compared to cats that did not. The authors concluded that tear film
instability may play a role in the pathogenesis of feline conjunctivitis, although it is
not clear if it is a predisposing factor or a result of it.

In a recent study the effect of primary FHV-l infection on tear film break up
time and Schirmer tear test values, as well as the relationship between these
parameters and conjunctival goblet cell density, histological inflammation, and
clinical disease severity was evaluated.148 Tear film break up time and goblet cell
density decreased dramatically during the first week following viral inoculation,
continued to decline until 15 and 21 days post-inoculation and remained abnormal for

the duration of the study (29 days)!“ The total disease score and clinical and

19

histological evidence of conjunctivitis increased in all 6 cats during the first week, but
began to normalize thereafter. Schirmer tear test values remained elevated for 29 days

following inoculation, likely due to the continued tear-film instability. ”8

1.8.5 Ophthalmia neonatorum and symblepharon

Ophthalmia neonatorum as a presentation of early FHV-l infections was

Lm Two to four week old kittens with ocular lesions

initially described by Bistner et a
had generalized disease as well characterized by rhinitis, tracheitis,
bronchopneumonia, and hepatic necrosism Infection with FHV-l before the eyelids
are open can result in mucopurulent conjunctivitis which causes distension of the
eyelids. ‘20

Symblepharon formation, the adhesion of conjunctiva to itself or to the cornea,
may be observed as a sequela to FHV-l infection.“’120 The profound epithelial
necrosis caused by FHV-l precedes this condition, leading to adhesions and scar

- 4.19.120
formation. 1

1.8.6 Corneal sequestrum

Feline corneal sequestrum (FCS) is a disease unique to the catm’lso

characterized by degeneration of collagen and accumulation of brown
pigment.“8"51’152 The pigment appears to be water soluble and not melanin or
hemosiderin.”(’)’151’153 However, Featherstone et al.”4 found particles within corneal
sequestra which were consistent with the appearance of melanin particles. The clinical
appearance is characteristic. Lesions are variable in pigmentation, round to oval
shaped and usually located centrally or paracentrally.'49’155 Corneal vascularization,
118,119,156

perilesiona] stromal edema, and inflammation are common in chronic disease.

Clinical signs include blepharospasm, epiphora, enophthalrnos, elevation of the

20

nictitans, chemosis and hyperemia of the conjunctiva as well as photophobia and

149,150,157,158 The

decreased activity. condition can occur unilaterally or

149,150,158 118,119,150,158,159 118.158

bilaterally. Predispositions for Persian, Himalayan,

160 158 118,150,158

Colorpoint, American Shorthair and Siamese cats are reported.

Histopathologically the sequestrum is characterized by stromal degeneration, cell loss
and dessication, often surrounded by a zone of inflammatory cells.”8’151'153’156’161

The etiology of FCS is unknown. Feline herpesvirus-1 DNA has been detected
in 18-55. 1% of keratectomy specimens from cats with corneal sequestrum using

polymerase chain reaction (PCR).'62"63 Using immunofluorescent antibody tests

(IF A), FHV-l was detected in 22.9% of corneal sequestra examined.‘58

1.8.7 Eosinophilic keratitis

Infection with F HV-l has also been implicated in the development of
eosinophilic keratitis. "’4 Clinically, the patient presents with a pink to white, irregular,
vascularized lesion in the peripheral limba] area that may invade the superficial
cornea, conjunctiva, and nictitating membrane and be present unilaterally or
bilaterally.“3’”9’I65 Cytologic examination usually demonstrates the presence of
eosinophils, mast cells, lymphocytes, or plasma cellsm’166 Histopathological findings
are chronic granulomatous inflammation characterized by infiltration of plasma cells,
lymphocytes, numerous eosinophilic granulocytes and occasionally mast cells. 1 19’1“

Using IFA, corneal scraping specimens from cats affected with eosinophilic
keratitis were positive for FHV-l in 9 of 27 cases (33.3%).164 Nasisse at al.163
detected F HV-l DNA using PCR assay in 76.3% of corneal scrapings from cats with

eosinophilic keratitis, but only in 5.9% of those from clinically normal cats. The

higher detection rate in the second study is explained by the higher sensitivity of PCR

21

compared to IF A. The role of FHV-l in the development of eosinophilic keratitis has

to be further elucidated. '20

1.8.8 Anterior uveitis

Herpes infections that involve the anterior uvea have been described in
humans and are generally not associated with simultaneous or past keratitis.167
Utilizing PCR and hybridization techniques, Bustos et al. detected HSV-l DNA in
20/30 (66.6%) human ciliary ganglia from patients with no known active HSV-l

‘68 This suggests that the ciliary ganglion may be an

infection at the time of death.
additional site of HSV-1 latency in humans.168 Latent HSV-l was also found in ciliary
ganglia from experimentally infected mice.31 This suggests that anterior uveitis may
result from virus reactivation within the ciliary ganglia rather than within the

1.169 concluded from another study, that

trigeminal ganglion. However, Labetoulle eta
infection of the iris and the ciliary body results from transfer of virus through the
sympathetic system via the superior cervical ganglion. If additional sites of latency
within the autonomic system exist in cats, a possible role of FHV-l in feline anterior
uveitis could be explained.

Maggs et a].170 utilized a PCR assay to detect FHV-l DNA in the aqueous
humor of 11 of 73 cats (14%) with idiopathic anterior uveitis or uveitis due to
T oxoplasma gondii. Intraocular production of FHV-l antibodies was noted in 22/44
cats with idiopathic uveitis and 11/29 cats with toxoplasma uveitis, whereas
intraocular production of FHV-l antibodies was never detected in cats without
uveitis.170 In a study by Powell et a].,171 four specific pathogen free kittens were
inoculated with FHV-l by use of an atomizer administering V2 of the volume into the

oropharynx and ‘A of the volume into each nostril. Two of the cats developed mild

anterior uveitis 11 and 25 days post-infection respectively that resolved within one

22

week of appearance. No posterior segment lesions were detected.l7l These findings

suggests that F HV-l may play a role in feline anterior uveitis.

1.9 Feline herpesvirus 1 disease - Diagnosis

The characteristic clinical signs and the self-limiting nature of most acute
phase FHV-l infections often make obtaining a definitive diagnosis less critical.75’120
Application of Rose Bengal stain, a vital dye that stains dead and degenerating cells,
to the ocular surface can be helpful to identify early dendritic lesions prior to the
disruption of the corneal epitheliumno’lm‘I72 Application of sodium fluorescein to the
ocular surface will identify corneal epithelial disruption.120 Conjunctiva] cytology
may be performed in acute, primary F HV-l infections particularly for the presence of
intranuclear inclusion bodies.”8 May-Gruenwald-Giemsa stain has been
recommended for identifying intranuclear inclusions.121 Conjunctiva] cytology is
rarely diagnostic because the predominant cell type observed is the neutrophil and
intranuclear inclusions are infrequent.126

The diversity of clinical signs in chronic FHV-l disease make a definitive

diagnosis with viral identification more desirable/5’120

Tests that are currently
available either rely on detection of the organism or demonstration of an immunologic
response to the organism. The methodologies available include virus isolation (VI),
detection of virus neutralizing serum antibodies (SN), enzyme-linked immunosorbent

assay (ELISA), immunofluorescent antibody testing (IFA), or polymerase chain

reaction assays (PCR).75

1.9.1 Virus isolation

In 1958, Crandell and Maurer21 isolated a feline virus associated with

intranuclear inclusion bodies, which was designated as the C-27 virus. In 1959, the

23

cytopathogenic features of feline rhinotracheitis virus in tissue cultures of feline renal
cells were described as: development of intranuclear inclusion bodies, formation of
giant cells, and a slight reduction in the metabolic rate of infected cells.28 Virus
isolation (VI) has been the gold standard for the diagnosis of alphaherpesviruses.75
Due to F HV-l ’5 rapid replication and production of characteristic cytopathic effects
in cell culture, V1 is relatively simple to perform provided viable virus was collected
and transported to the laboratory.75 Dacron or cotton swabs are recommended to
collect the samples as alginate swabs can be inhibitory to some herpesviruses.173 After

collection of the sample, it should be refrigerated and shipped promptly.75

1.9.2 Serology

Regardless of methodology, serologic titers are not considered useful in the
diagnosis of FHV-l infection because of the high rate of vaccination against F HV-l
with resultant systemic antibody titers.70’75’123 Therefore titers are unlikely beneficial,
because serology does not differentiate between immune response to vaccination or
natural infection.75 In one study, no correlation between F HV -1 seropositivity or titer
magnitude and the likelihood of detecting virus or presence of clinical signs could be
demonstrated.64

The serum neutralization (SN) test is commonly used to detect F HV-l
infection in catsm’m"76 However, FHV-l is difficult to detect with this method,
because of the poor irnmunogenicity of the virus.177 The presence of antibody in
serum suggests prior infection but does not necessarily correlate with clinical
disease.178 On the other hand FHV -1 has been documented in ocular tissues of many
cats negative for antibody by serum neutralization test. '79

Enzyme-linked immunosorbent assays (ELISA) for detection of feline

herpesvirus-1 antibody have been developedm’178 These tests are more sensitive than

24

serum neutralization.178 However in one study, seroprevalence, as assessed by ELISA
did not vary significantly between normal cats and cats suspected to be undergoing
acute or chronic FHV-l infections, nor did it correlate with the likelihood of viral

detection utilizing IFA or VI.64

1.9.3 Fluorescent antibody test

Fluorescent antibody tests can be performed as a direct test (polyclonal
fluorescein conjugated antibody) or an indirect test (polyclonal unlabelled antibody,
which then reacts with a conjugated antibody). The antibody then detects FHV-l
epitopes on the cell surface.75 However, the indirect test is more sensitive than the

180

direct test. Samples are usually obtained by conjunctiva] or corneal scraping or by

biopsy.75"2° It is necessary to collect the sample prior to topical application of
fluorescein dye, as this will interfere with the interpretation of the test result.181
Carlson and Scott found the test specific and sensitive for diagnosing cats showing
clinical sings of FHV—l infection.182 In contrast, other studies found different
limitations of the fluorescent antibody test, such as lack of significant differences in
detection rates between acutely or chronically affected and normal animalsf’4 and the
subjective nature of the laboratory interpretation.“126 Particularly in chronic
infections, it is possible that viral epitopes may be bound to host antibody, which
results in a false negative test.126 Burgesser et a].183 determined the sensitivity and
specificity for indirect immunofluorescence to be 29% and 96%, respectively,

compared to PCR. These limitations and the availability of other diagnostic methods,

make fluorescent antibody tests appear to be of limited clinical application.75

25

1.9.4 PCR assays

Us“ and

The polymerase chain reaction was introduced in 1987 by Mullis et a
dramatically changed the field of molecular biology as well as disease diagnosis in
man and animals. To obtain a sample for FHV-l detection, host cells need to be
sampled, since the virus is an obligate intracellular agent.75 Biopsy samples are
recommended.75 However, in other studies, FHV-l DNA was obtained by the use of
Schirmer tear test strips or microbrush swabs.34’185 Laboratories often suggest the
samples are placed in a small volume of a physiological fluid such as phosphate
buffered saline, frozen, and shipped overnight on ice for analysis.” However, Maggs
et a].186 assessed the effect of shipping temperature on results of one PCR assay for
detection of FHV-l DNA. PCR results did not differ for identical samples sent
overnight on ice or mailed over several days at ambient daytime maximum
temperatures between 17 an 47 °C.'86

Multiple PCR assays, some of which are commercially available, have been
described for use in the detection of FHV-l DNA.85‘144’183"87"89 PCR analysis for
F HV-l is commonly based on DNA-amplification of the thymidine kinase gene.126’l44
PCR and nested PCR are highly sensitive for the detection of active or latent FHV-l

- - 2 .144, , . . .
infections.I 6 162 183 These PCR techmques have shown to be more sensrtrve than

the use of immunofluorescent antibody testing or virus isolation, but detection limits

,l44,l , 7- 9
vary greatly.“ 8318 I8

Nested PCR utilizes a second primer pair internal to the initial primer. Stiles et
a].188 demonstrated a nested PCR assay to be more sensitive for identification of F HV-
] in cats with conjunctivitis than virus isolation or fluorescent antibody tests. In
another study comparing virus isolation, single PCR, and nested PCR for the detection

of FHV-l in ocular and nasal secretions, nested PCR was shown to be 4.8 times more

26

'89 When 22 samples that were negative for virus isolation

sensitive than single PCR.
were assayed, 19/22 (86.4%) were positive by nested PCR and 2/22 (9%) were
positive by single PCR.189

Real time PCR not only allows detection of, but also the quantification of a
given DNA sequence.190 Accumulation of PCR product is measured through a
labelled fluorogenic probe.190 Advantages of real time PCR over conventional PCR
include simultaneous quantification of input target DNA within the PCR process, no
necessity for post-amplification steps, and therefore reduced chance for cross
contamination.190 The procedure is fast and highly efficient.190 A real time PCR assay
was developed for the quantification of FHV-l DNA in ocular fluid samples of
clinically diseased cats.84 A good correlation between viral titer and quantitative PCR
was observed during early infection.84 In the second stage of disease, the viral titers
decreased, while the PCR signal remained high, and in the final stage of the disease,
virus isolation was negative and the PCR signal decreased consistently.84 The authors
concluded that comparing the results of virus isolation and quantitative PCR in
consecutive samples would allow one to track the course of the infection.84 Low et
al.‘91 assessed the prevalence of FHV-l DNA in cats with and without conjunctivitis
using conventional PCR and quantitative PCR. The concordance between the two
assays for detection of FHV-l DNA was 92.5%. '91 No significant difference for FHV-
l prevalence was found between cats with conjunctivitis and cats without
conjunctivitis. '91

Another study compared six published PCR assays for detection of FHV-l
DNA in 15 clinical samples and five vaccines licensed for use in preventing FHV-l
associated disease.192 Detection rates for the clinical samples varied between 29% and

86%.192 All six PCR assays detected FHV-l DNA in all five vaccines tested.192

27

Therefore, a positive PCR results cannot differentiate a vaccinal strain from natural
strains and does not differentiate sub-clinically infected cats from those with clinical

disease as a result of FHV-l infection.“192

1.9.5 In situ Hybridization

In order to obtain information regarding latent F HV-l infection, Ohmura et
al.193 utilized in situ hybridization to detect FHV-l LATs in the trigeminal ganglia of

1.194 performed in situ hybridization with a

experimentally infected cats. Suchy et a
digoxigenin-labeled double-stranded DNA probe for FHV-l on different tissues from
a severely affected cat. Hybridization signal was found only in nuclei of intact
epithelial cells in the basal layers of the epidermis, hair follicles of the eyelids, nose,
superior lip and paw, as well as in conj unctival epithelium of palpebrae and nictitating
membrane and in a few superficial cells of both corneas.I94 Kim and Yi'95 optimized
an in situ hybridization protocol for the detection of F HV -1 DNA by using microwave
irradiation as pre-treatment. Although this technique may be of limited value in
diagnosis of clinical cases, it offers great potential in studying FHV-l infection of
different tissues as in situ hybridization allows for specific localization of the virus
and evaluation of tissue morphology simultaneously. Sensitivity of in situ
hybridization largely depends on many variables in the utilized protocol including
fixation of the tissue, exposure of target nucleic acid, selection of probe, type of probe
labelling, denaturation of doublestranded DNA, hybridization conditions, and

detection conditions. 196

1.9.6 FHV-l detection in different tissues

Despite the high incidence of F HV-l infection within the feline population and

many investigations into its pathology, much remains unknown. For example, FHV-l

28

latency is stated to occur only in the trigeminal ganglia.2 During the latent period,
clinical signs are absent and the virus cannot be isolated using standard cell culture
techniquesfm‘197 However, several studies have detected FHV-l DNA by PCR in
13%188 to 31%183 of conjunctival and 5.9%,163 46%, '62 and 49%198 of comeal samples

from clinically normal cats. Despite the PCR positive results of conjunctival samples,

183 1188

FHV-l was detected either infrequently or not at al by virus isolation (VI) or
fluorescent antibody (FA) staining. In these samples, F HV -1 was not quantified, nor
was the active or latent viral status or specific viral location within each tissue
identified.

It is known that FHV-l demonstrates a tropism for conjunctival epithelium
and also replicates within corneal epithelium.”80 In one study, immunohistochemical
staining of corneal and conjunctival epithelial cells revealed the diffuse presence of
viral antigen during the acute stage of infection.80 In cats with experimentally induced
stromal keratitis, viral antigen persisted within the corneal stroma between collagen
lamellae.I43 However these cats were immunosuppressed by three consecutive daily
subconjunctival injections of betamethasone prior to inoculation with the virus. In
clinically normal cats with PCR detectable virus, whether the virus persists in
epithelial cells, becomes localized in the underlying stroma, or has any effect on the
surrounding tissues is not known. Viral location is critical, as acquisition of viral
antigen by the corneal stroma is the factor currently thought to precede development

of stromal keratitis.143

The ability of the virus to infect other ocular tissues has not
been widely explored.
Studies to identify sites of F HV-l latency have concentrated particularly on

the trigeminal ganglion as this is an important site of viral latency for many other

herpesviruses.l97’199 Gaskell et al.‘99 isolated FHV-l from the trigeminal ganglia of

29

3/ 17 cats latently infected with field virus using a tissue fragment culture technique.
F HV-l could not be detected in four cats previously infected with a vaccine strain of
FHV-l or in four cats which were administered the vaccine and 48 hours later were
inoculated with field virus. 199 The authors suggest that the vaccine may prevent latent
infection.

Nasisse et a1.200 isolated FHV-l in trigeminal ganglia from 5/26 latently
infected cats using a similar tissue culture method. FHV-l was also isolated from the
trigeminal ganglia from 4/4 cats with active infection.200

Reubel et al.144 developed and utilized a PCR assay to detect F HV-l in various
tissues in latently infected cats. Latency was defined by concurrent negative virus
isolation for those tissues. FHV-l was detected by PCR in optic nerves (6/6),
trigeminal ganglia (8/9), olfactory bulbs (7/9), nasal turbinates (7/9), corneas (6/6),
lacrimal glands (2/5), salivary glands (1/9), oral fauces (3/7), tonsils (1/9), cerebrum
(1/9), conjunctiva (1/9), and mesenteric lymph node (1/9). No virus was detected by
PCR in cerebral cortex, thymus, trachea, lung, liver, spleen, kidney, or peripheral
blood monocular cells. However, 5/9 cats received intravenous methylprednisolone
acetate 10 days prior to necropsy. In all five cats virus could be isolated from oral
swabs obtained at the time of necropsy. In these cases, it is likely that a recrudescent
infection was present at the time of euthanasia. In 4/4 cats viral DNA was detected
utilizing PCR from oral swabs. Vaginal swabs were negative in all 5 cats.

In a similar study conducted by Weigler et al.85 various tissues from 10 cats
latently infected with FHV-l were evaluated for the presence of FHV-l by PCR.

Latency was confirmed by concurrent negative virus isolation. All 20 trigeminal

ganglia, 15/20 optic nerves, and 6/20 corneas were positive for F HV -1 DNA on PCR.

30

FHV -1 was detected in 4/10 optic chiasma and 2/9 olfactory bulbs. Virus was not
detected within the uvea.

Detection of latency associated transcripts (LATs) is probably the most
accurate way to truly define the latent stage of infection.”197 With the detection of
LATs it is also possible to distinguish true latency from low-level persistent
infection.“ Using in situ hybridization and northern blot analysis, Ohmura et a1.193
detected transcripts of opposite strand to the immediate-early mRNA in the trigeminal
ganglia of 3/5 experimentally infected cats.

In another study,198 FHV-l DNA was detected by PCR in 45/92 (48.9%)
corneas and in 38/92 (41.3%) trigeminal ganglia of cats without clinical signs of
FHV -1 infection. The investigators also developed a reverse transcriptase PCR (RT—
PCR) to detect FHV -1 LATs. None of the 39 tested corneas and four of the 16 tested
trigeminal ganglia had positive results for F HV-l LATs.198

These studies to identify FHV -1 sites of latency suggest that FHV-l follows a

pattern similar to HSV-l during latency. However, studies to evaluate whether F HV-l

infects and establishes latency in autonomic ganglia have not yet been undertaken.

1.10 Feline herpesvirus 1 disease - Treatment and prevention

Upper respiratory tract infection during the acute stage of the disease is in
most cases self-limiting. Systemic broad-spectrum antibiotics are indicated to prevent
or treat secondary bacterial infections.66’70’71 Supportive care is also very important.120
It is necessary to keep oro-nasal pathways clear. Frequent removal of nasal and ocular
discharge is recommended.7l"06’202 Intravenous or subcutaneous fluid administration

66,71,202

can become necessary to achieve rehydration. Nutritional support is indicated

in cases of severe anorexia.“202

31

Corneal ulceration should be treated with topical broad spectrum antibiotics to
prevent secondary bacterial infections, and topical antiviral therapy should be
considered.6‘r”70’l '8’203 In kittens with primary FHV-l infection with corneal ulceration
or early symblepharon formation, topical antiviral therapy can be beneficial.(’6’70’203 In
these cases frequent mechanical break-down of the symblepharon is also
recommendedm’203 In older cats with recurring bouts of FHV—l conjunctivitis,
therapy depends on the severity, duration, and frequency of the outbreaks.7°’203 Mild
transient conjunctivitis does not require specific treatment, as it is usually self-
limitingm‘120 If the conjunctivitis does not resolve or a corneal ulcer is present, topical
antiviral therapy, as discussed below, is indicatedm’l 18’125 Mechanical debridement of

the corneal ulcer can be helpfulm’203

to enhance the healing by removal of any loose
epithelium, as well as removal of viral particles. Specific antiviral and adjunctive
therapy, such as oral supplementation with the amino acid L-lysine, or application of
interferons are described below in detail.

Topical application of corticosteroids is the recommended initial treatment in
cases of eosinophilic keratitis;1 '8'125 however, in cats where an association with F HV-
1 is suspected, other treatment options should be considered insteadm’204 Oral
administration of megestrol acetate, 5 mg/day during the first week and 2.5-5
mg/week for maintenance dosage as treatment of eosinophilic keratitis has been

recommended. 1 '9’204

Due to the potential of serious systemic side effects, such as
temperament changes, polyphagia, diabetes mellitus, iatrogenic Addison’s syndrome,
endometritis, cystic endometrial hyperplasia, mammary hypertrophy and neoplasia,
megestrol acetate is not recommended as the initial choice of therapy.”"”205 Topical

204,206

application of cyclosporine can be used alternatively. In a recent study by Spiess

et a1.,206 33 cats were treated with topical cyclosporine 1.5% two or three times daily.

32

83% of these cats showed marked improvement and 6% showed mild improvement of
the disease. While, three animals showed no response, the likely cause was thought to
be poor owner compliance. Two animals developed blepharitis, which resolved after

treatment.

1.1 1 Antiviral therapy

A multitude of antiviral drugs are licensed for treatment of viral diseases in
humans. As a result of the many similarities between HSV-l and FHV-l, several of
the drugs are employed in the treatment of herpetic disease in cats. However,
differences in the biologic behaviour of these viruses support the need for continued
research into and development of antiviral drugs specifically for use against F HV-l in
cats.

Frequent administration of the antiviral compounds is crucial in order for

therapy to be effective.207

This is often a problem for cat owners, as many of the
available topical antivirals are topically irritating.120 The presently available antiviral
drugs are also virustatic. Therefore the drugs are effective only during the replicative
phase of infection and latent FHV-l remains unaffected."207’208 Precise targeting of
viral specific replicative processes is difficult as the virus utilizes host cell machinery

207 For many antiviral agents, the mechanism of action does not

for replication.
adequately differentiate infected from non-infected cells, which can lead to host

toxicity.207

1.11.1 Nucleoside analogues

The structure of these medications is similar to the nucleosides required for
viral and host DNA and RNA synthesis. However, due to slight modifications the

nucleoside analogues are able to disrupt the viral replication cycle.207 Following an

33

intracellular phosphorylation, the nucleoside analogues are incorporated into newly
synthesized mammalian and viral DNA strands.1 DNA synthesis is then disrupted by
mechanisms that are similar for all nucleoside analogues and are described in further

detail below.

1.11.2 Acyclovir

Acyclovir (9-(2-hydroxyethoxymethyl)guanine (acycloguanosine)) is similar
to guanosine, but it contains an acyclic sugar group.l Acyclovir is a specific, non-
toxic drug, that is highly efficacious against herpes simplex virus and, to some extent,
varicella zoster virus.l Activation of this pro-drug requires three kinases to be present
in the cell to convert acyclovir to a triphosphate derivate, the actual antiviral drug.1
The first step, phosphorylation of acyclovir into the monophosphate, is catalyzed by a
herpesvirus-specific thymidine kinase.209'2” Cellular kinases perform the two
remaining phosphorylation steps to produce acyclovir triphosphate."207 Acyclovir
triphosphate has a greater affinity for the viral DNA polymerase than the naturally
occurring deoxyguanine triphosphate, and is therefore preferentially incorporated into
growing DNA via a competitive mechanism."207

The primary activities of acyclovir are related to its ability to decrease viral
replication by chain termination of replication DNA by inactivating viral DNA
polymerase.207 As acyclovir lacks the 3'- hydroxyl group present in the cyclic sugar
ring of naturally occurring nucleotides, the growing DNA chain terminates, and
further DNA replication is blocked."207 When acyclovir triphosphate is bound to the
DNA template in the presence of the next nucleotide to be attached, the viral DNA
polymerase is inactivated by the formation of an irreversible complexm’213 Further
DNA replication is shut down by these mechanisms; therefore, no further production

of infectious virions is possible.207

34

Acyclovir is highly specific for cells infected with herpes simplex virus or
varicella zoster virus.1 The specificity depends on the virus-encoded kinase.l
Differences or mutations in the viral thymidine kinase prevent the initial step of
monophosphorylation leading to antiviral resistance.207 Since the development of
acyclovir in 1974, many variations of chain-terminating nucleoside analogues have
been developed in order to increase efficacy and bioavailability.1 Information

regarding efficacy of acyclovir against FHV—l is described below.

1.11.3 Valacyclovir

Valacyclovir (L-valine, 2-[(2-amino-l ,6-dihydro-6-oxo-9H-purin-9-
yl)methoxy]ethyl ester, monohydrochloride) is the hydrochloride salt of the L-valyl

1,207,214

ester derivate of acyclovir. Following oral administration, valacyclovir is

nearly completely converted by a liver hydrolase to acyclovir and L-valine during

25'2” It is therefore considered to be a pro-pro-drug.l

intestinal or hepatic metabolism.
The mean absolute oral bioavailability of acyclovir after administration of
valacyclovir is four-fold higher (54.4 i 9.1%)214’2'8 than the limited oral

bioavailability of acyclovir (15 to 21%).219

The bioavailability of valacyclovir in cats
is 2.3-fold greater than that of acyclovir;220 however, severe systemic side effects such
as bone marrow suppression, hepatic necrosis, and renal tubule epithelial necrosis

have been documented.221 Due to its extremely toxic properties, valacyclovir is

considered unsuitable for use in cats.221

1.11.4 Ganciclovir

Ganciclovir (9-(1,3-dihydroxy-2-propoxymethyl)guanine) is an acyclic
guanosine analogue, which is structurally closely related to acyclovir.2°7'222 Following

phosphorylation to its triphosphorylated form, ganciclovir inactivates viral DNA

35

polymerase by competing with deoxyguanosine triphosphate for incorporation into
the replicating DNA.207 Its incorporation into viral DNA-strands also results in

223 The toxicity of ganciclovir is greater than that of

termination of elongation.
acyclovir in vivo and in vitro. It can cause severe neutropenia in humans.207 Similar to
acyclovir, the oral bioavailaility is limited. Therefore a valine ester of ganciclovir
(valganciclovir) has been synthesized.222 Like valacyclovir, valganciclovir will be

converted to release the parent compound, ganciclovir.222

1.11.5 Penciclovir

Penciclovir (9-(4-hydroxy-3 -hydroxymethyl-but-1-yl)guanine) was first

2.224 The activity of penciclovir against several herpesviruses was

synthesized in 197
first evaluated in 1987.225 It is an acyclic guanosine nucleoside analogue with a
structure similar to acyclovir.207’222’225 Like acyclovir and ganciclovir, penciclovir
must be initially phosphorylated by a viral thymidine kinase, followed by two more
phosphorylation steps to reach its active form, penciclovir-triphosphate.207’222 This
active form has been shown to inhibit herpesvirus DNA polymerase. However the
possibility exists for internal incorporation of penciclovir residues into viral DNA and

further chain extension.226

The first phosphorylation step of penciclovir to
penciclovir-monophosphate is more efficient than the phosphorylation of acyclovir.
However, the penciclovir-triphosphate formed in infected cells is a less effective
inhibitor of herpes simplex virus DNA polymerase than acyclovir-triphosphate.227
Eamshaw et al.227 showed that for HSV-l DNA polymerase, the K for penciclovir
triphosphate is about lOO-fold greater than that for acyclovir triphosphate. Therefore,
a greater drug concentration is needed to inhibit viral DNA polymerase and hence

inhibit the virus. Although penciclovir showed a similar spectrum of antiviral activity

and selectivity when compared to acyclovir, the oral bioavailability was even lower

36

than that of acyclovir.222 Attempts at improving bioavailability led to the development

. . 2
of famcrclovrr.22

1.11.6 Famciclovir

F amciclovir (Diacetyl-6-deoxy-9-(4-hydroxy-3 -hydroxymethyl-but—1 -
yl)guanine) is the diacetyl derivate of penciclovir.228 Following oral administration of

famciclovir, it is efficiently converted to penciclovir.229

The absolute bioavailability
of penciclovir from famciclovir following a single oral dose is 77%. The final
products are penciclovir and the harmless by-product, acetic acid.229 Administered
orally, famciclovir is highly effective against HSV-l in a variety of different animal
models.230 In a quantitative study on the prevention of HSV-1 latency in mice,
significantly less latent virus was detected in the ganglia of mice treated with

231

famciclovir as compared to those treated with valacyclovir. Oral administration of

famciclovir appears to be well tolerated in cats.232

1.11.7 Idoxuridine

Idoxuridine (IDU, 5-iodo-2'-deoxyuridine) was the first available antiviral
agent, initially synthesized in 1959.233 It is still used in veterinary medicine. In human
medicine it has largely been replaced by newer agents, such as acyclovir.207
Idoxuridine is a thymidine analogue, which does not require a virally encoded
thymidine kinase for phosphorylation."211 Since it can be phosphorylated by both

1’2” Idoxuridine has a

cellular and viral kinases, it has less specificity than acyclovir.
direct inhibitory effect on virus-specific DNA polymerase causing suppression of
viral DNA synthesis.211 Idoxuridine also causes disruptive replicative events as

incorporation of the thymidine analogue into newly synthesized viral DNA produces

abnormal viral proteins?” When administered systemically, it combines with cellular

37

DNA and can cause severe systemic side effects, including neoplastic changes,

2” Ophthalmic preparations are often compounded as

genetic mutations, or infertility.
0.5% ointment or 0.1% solution,234 which are well tolerated and reasonably effective

in the treatment of FHV-l disease in cats.207

1.11.8 Trifluridine

Heidelberger et al. first synthesized 5-trifluoromethyl-Z-deocyuridine (TFT) in
1964.235 Like idoxuridine, trifluridine is structurally similar to deoxythymidine, a
pyrimidine nucleoside used in DNA synthesis.207 It acts by incorporation into viral
DNA, followed by inhibition of transcription of late virus-specific DNA?” As with
idoxuridine, trifluridine is also independent of viral thymidine kinase for
phosphorylation because it can be phosphorylated by cellular or viral kinases to its
active form.233 Trifluridine is commercially available as a 1% ophthalmic solution.234

Clinically in cats, it is less well tolerated than idoxuridine or Vidarabinem'234

1.11.9 Vidarabine

Vidarabine (9-Beta-d-rabinofuranosyladenine (Ara-A, adenine arabinoside))
was first synthesized in 1960 as a potential anti-neoplastic agent.2'l In 1964, its
antiviral properties were reported.236 Vidarabine is a nucleoside analogue of
adenosine, which does not require viral thymidine kinase for phosphorylation and

237 It selectively inhibits the replication of DNA viruses and some RNA

activation.
viruseszm’z” Following the three successive phosphorylation steps performed by host
cellular enzymes, it is a competitive inhibitor of cellular and viral DNA polymerases,

but has more potency against viral enzymes.21 "237 Systemic use is limited by its toxic

neurologic and gastrointestinal side effects.”1 Vidarabine is available as a 3%

38

ophthalmic ointment with a recommended application frequency of every three

234

hours. Clinically, it is reasonably tolerated by cats.207

1.1 1.10 Cidofovir

Cidofovir ((S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine) was first
described in 1987 as a broad-spectrum anti-DNA viral agent active against
herpesviruses (HSV-1, HSV-2, VZV, CMV), adeno- and poxviruses.238 Cidofovir
does not require viral thymidine kinase for phosphorylation as cellular host enzymes
perform the two phosphorylation steps required to reach its active form.207
Nephrotoxicity of cidovovir when given orally necessitates limitation of the dosage in
humans.222 l-[((S)-2-Hydroxy-2-Oxo-l ,4,2-Dioxaphosphorinan-5-yl)Methyl]cytosine
is the chemically stable pro-drug of cidofovir, which has reduced nephrotoxicity as
compared to cidofovir in rats239 and in humans.240 Within the cells, the pro-drug is
converted to cidofovir by an intracellular cyclic CMP phosphodiesterase.24' To
increase the limited oral bioavailability, alkoxyalkyl esters have been prepared.242

Cidofovir was able to reduce clinical disease and viral shedding in cattle with
bovine herpesvirus type 1 infection.243 Topical administration of cidofovir was highly

effective in rabbit models in the treatment of experimental herpetic keratitis?“247

1.1 1 .1 1 Bromovinyldeoxyuridine

Bromovinyldeoxyuridine, BVDU [(E)-5-(2-bromovinyl-2'-deoxyuridine)] is a

pyrimidine analogue of the nucleoside thymidine.207

For phosphorylation it requires
viral-encoded thymidine kinase like acyclovir.207 F HV -1 thymidine kinase does not
effectively phosphorylate this agent, making BVDU relatively ineffective against

FHV—l .207 Because of the low potency, BVDU is not used in veterinary medicine.207

39

1.11.12 Ribavirin

1-Beta-d-ribofuranosyl-l,2,4-triazole—3ocarboxamide, (ribavirin or virazole) is
a triazole purine analogue, which inhibits a broad spectrum of RNA and DNA
viruses.“ Following phosphorylation to its 5'-monophosphate form, it directly
inhibits the synthesis of guanine nucleosides. Ribavirin triphosphate also blocks ATP
and GTP binding to RNA polymerase.248 Systemic administration may lead to a
number of side effects, such as extravascular hemolysis, bone marrow suppression,
gastrointestinal toxicity, and central nervous signsm’248 These side effect make it

unsuitable for clinical use.

1.12 Efficacy of nucleoside analogues against FHV-l

1.249 evaluated the in vitro susceptibility of feline herpesvirus-1 to

Nasisse et a
various antiviral drugs and found the relative potency of these compounds was
trifluridine >> idoxuridine > Vidarabine > bromovinyldeoxyuridine >> acyclovir.
Maggs and Clarke250 conducted a study in order to establish the in vitro efficacy
against FHV-l of ganciclovir, cidofovir, penciclovir and foscamet. Furthermore the
results were compared to the in vitro efficacy of acyclovir and idoxuridine. The
relative potency of these compounds was idoxuridine = ganciclovir >> cidofovir >
penciclovir >> acyclovir >> foscamet. The authors concluded that clinical trials with
ganciclovir, cidofovir and penciclovir are warranted.

Van der Meulen et al.25 I compared the in vitro efficacy of different antiviral
drugs against FHV-l. Ganciclovir, 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine
(PMEDAP) and cidofovir were most potent; adefovir and foscamet were

interrnediately potent; and acyclovir was least potent. However, all antiviral drugs

were able to significantly reduce plaque size in Crandell-Rees feline kidney (CRFK)

4O

cells when compared with the untreated control. None of the compounds were toxic
for the CRFK cells at antiviral concentrations.

A study to assess the effect of cidofovir on viability of feline corneal epithelial
cells, replication of FHV—l, and virus-induced cytopathic changes revealed that
cidofovir in vitro was highly efficacious against FHV-l infection of a primary culture
of feline corneal epithelial cells, but had cytostatic effects on the cultured cells.252 A
recent study by Fontenelle et al.253 found that a twice daily application of 0.5%
cidofovir solution in both eyes significantly decreased the amount of viral shedding,
as determined by quantitative real time PCR, and the severity of clinical disease in
cats with experimentally induced ocular FHV -1 infection.

Owens et al.220 studied the pharmacokinetics of acyclovir and valacyclovir in
cats. Nephrotoxicity and bone marrow suppression were observed with repeated
dosing of acyclovir and valacyclovir in cats with experimentally induced FHV-l
infection. F uthermore, neither compound appeared efficacious against F HV-l
infection. In another study conducted to determine whether orally administered
valacyclovir could be used safely and effectively to treat cats with primary F HV-l
infection, severe systemic side effects such as coagulative necrosis of the renal tubular
epithelium, centrilobular atrophy and hepatic necrosis, and severe bone marrow

(1.221

depression were note The experiment had to be terminated after 12 days for

humane reasons. Therefore treatment of cats with systemic acyclovir or valacyclovir

is not recommended. However, Williams et al.254

evaluated the efficacy of topical
ophthalmic acyclovir applied five times daily as a treatment for FHV-l keratitis in
cats and concluded that topical acyclovir can have a beneficial effect against FHV-l

conjunctivitis and keratitis.

41

A study evaluating the pharmacokinetics and safety of penciclovir following
oral administration of famciclovir in cats revealed that famciclovir dosages of 15
mg/kg orally administered every eight hours are unlikely to result in plasma
penciclovir concentrations with activity against FHV-l.232 No adverse side effects
with the oral administration of famciclovir were observed. However, anecdotal reports
suggestive favourable responses after administration of 6-10 mg/kg orally of

famciclovir every 12 to 24 hours to clinically affected animals.232

1.13 Non-nucleoside antivirals

1.13.1 Foscarnet

Phosphonoformic acid (PF A) is a pyrophosphate analogue inhibiting in a non-
competitive way the pyrophosphate binding site of DNA polymerase?” Foscamet
does not require phosphorylation. Its efficacy is therefore not dependent on viral
thymidine kinase.1 It has been shown to have activity against herpesviruses, human
immunodeficiency virus, and other RNA and DNA viruses.255 Foscamet is
administered intravenously, and due to its severe renal toxicity it is recommended
only for life threatening infections for which other antiviral drugs are no longer

250

effective.1 Maggs and Clarke investigated the antiviral activity of foscamet against

FHV-l in vitro and reported it to be relatively ineffective.

1.13.2 L-Lysine

Tankersley256 in 1964 studied the amino acid requirements of herpes simplex
virus in human cells. Results showed that cells infected with herpes simplex virus
deprived of arginine support neither cytopathic effects nor virus replication. However,

when arginine was replaced, a prompt and extensive infection followed. Lysine was

42

not required but rather exhibited a partially inhibitory effect upon virus
multiplication.256
A later study confirmed that the yield of several DNA viruses in cell culture is

257 Arginine deprivation

largely dependent on the presence of arginine in the medium.
in the media of HSV cell cultures causes a selective decrease in the synthesis of viral
macromolecules. It seems that deprivation of arginine may be more detrimental to
virus multiplication than restriction of other amino acids because several specific viral
proteins are large in size and rich in arginine.257

A study evaluating the relationship of arginine-lysine antagonism to herpes
simplex grth in tissue culture concluded that arginine deficiency suppressed HSV
replication while lysine antagonized the viral growth-promoting action of arginine.258
Walsh et al.259 investigated lysine’s therapeutic response when administered to
patients with herpes simplex and found lysine to be an effective form of treatment for
herpetic infection. L-lysine was shown to reduce the occurrence of, severity of and
healing time for recurrent HSV infectionm’m

Multiple studies have investigated the mechanism of the arginine-lysine
antagonism. Kadirvel and Kratzer262 concluded that the lysine-induced arginine
deficiency is a metabolic effect rather than an inhibition of gastrointestinal tract
absorption of arginine. Another study supports arginine and lysine competition for a

(1.263 Therefore an increase of L—lysine plasma

common transport system in the bloo
concentration saturates the transport mechanism and thereby reduces the availability
of arginine.

Arginine is an essential amino acid for cats and cannot be restricted in feline

diets due to their extreme sensitivity to an arginine deficiency. Morris and Rogers264

fed cats that were fasted overnight a single meal of a complete amino acid diet

43

without arginine. Signs of severe hyperammonemia developed and one cat died 4.5

l.264 The authors suggest that the rapidity of the onset

hours after ingestion of the mea
of symptoms arises from a combination of l.) a rapid depletion of free arginine in the
liver and therefore of omithine in the postabsorbtive state, and 2.) by inhibition of
arginine release from protein catabolism after feeding as a result of the anabolic
response of incoming amino acids.

Maggs et al.265 examined the effect of various concentrations of L-lysine and
L-arginine on in vitro replication of feline herpesvirus-1. Results showed that arginine
had a substantial grth promoting effect on F HV -1 and supplementation of the viral
culture medium with lysine attenuated this effect. Data indicated that high
concentrations of L-lysine reduce in vitro replication of FHV-l in media containing
low concentrations of arginine.265

Cats that received oral administration of L-lysine 500 mg every 12 hours
beginning 6 hours prior to inoculation of FHV-l developed less severe conjunctivitis
after inoculation than cats receiving placebo.266 Plasma L-lysine and arginine levels
were measured prior to the study and on days 3, 14, and 22, which was 1 day after
cessation of oral administration of L-lysine. Mean plasma L-lysine concentrations
were significantly higher than in untreated cats. Arginine plasma concentrations did
not differ significantly. The authors did note that if food was withheld prior to oral
administration of 500 mg or 1000 mg L-lysine, 50% of the cats vomited within 1
hour.266 Therefore L-lysine should be given with a small amount of food.

Another study examined the effect of orally administered L-lysine 400 mg
once daily on clinical signs of FHV—l and ocular shedding of virus in latently infected

267

cats The authors found decreased viral shedding but no significant change in

clinical signs during reactivation. Plasma L-lysine and arginine concentrations were

44

assessed on day 30 at 3 and 24 hours following L-lysine administration. Three hours
following administration, the mean plasma lysine concentration was significantly
increased compared to the control group. Mean plasma arginine concentration did not
vary significantly between treated and untreated cats. No significant difference in
mean plasma lysine or arginine concentrations were detected between the treatment
and the control group 24 hours after administration of L-lysine. These data suggest

that twice-daily administration of L-lysine would be more beneficial.

1.13.3 Interferons

Interferons (IFNs) are glycoproteins produced by cells in response to viral
infection.268 They are members of the cytokine family and are important mediators of
the initial, innate and adaptive immune system.1 In addition to their antiviral
properties, interferons possess anti-proliferative and immune modulating properties.l
Although the protein was described in 1954,269 it was named interferon in 1957 due to
its viral interference.270 Human interferons are reported to have antiviral efficacy

271-273

against herpes simplex virus as sole agents or administered in combination with

”“76 Topical application early in the course of HSV-1 infection

nucleoside analogues.
reduces the severity and duration of oral or genital herpes277 and shortens the period
of viral shedding.278 Using topical human leucocyte interferon 3x1045 units/ml twice
daily in humans with HSV, dendritic ulcers healed more rapidly and viral shedding
was significantly inhibited.279 A study comparing the in vitro effect of an undefined
feline interferon and two recombinant human alpha-interferons on feline herpesvirus-
1, feline calicivirus, and vesicular stomatitis virus found that treatment with these

interferons reduced the viral yield for each of these three viruses as compared with

that of the control cultures.280

45

Sandmeyer et al.281 investigated the effect of interferon-alpha on viability of
feline corneal epithelial cells, replication of feline herpesvirus-1, and virus-induced
cytopathic changes in primary cultures of feline corneal epithelial cells. Interferon-
alpha did not have cytotoxic effects and at a concentration of 105 fU/ml was
moderately effective against FHV-l infection in primary cell cultures of feline corneal
epithelial cells.28' In another study, the effect of recombinant feline interferon omega
(rFeIFN-w) administered topically and orally prior to subsequent F HV-l infection in

d.282 No beneficial effects of pre-treatment with rFeIFN-w in the

cats was investigate
course of primary FHV-l infection in cats was observed.282 A cat with a longstanding
feline herpesvirus-induced facial dermatitis was successfully treated with rFeIFN-
(0.283 The cat received 1.5 million units (MU)/kg on day 0. Half of the dose was
injected perilesionally and intradennally, the other half subcutaneously on the lateral
thorax. Great improvement was noted after only 2 days. The cat received consecutive
injections on days 2, 19, 21, and 23. Four months later at the last recheck examination,
the lesion had significantly regressed.

Considering the different results of the various studies or case reports, further
investigations and controlled clinical trials would be helpful to assess the in viva
therapeutic effect of interferons against FHV-l infection. Route of application and

time point of application in relation to viral infection as well as type of interferon may

play an important role for its efficacy.

1.13.4 Bovine lactoferrin

Lactoferrin is an iron binding glycoprotein that has been shown to possess

1 284-287 1 288-290 1 29] 1292-295
9 9 9

antibacteria antifunga antiprotozoa and antivira properties.

Mucosal cells of many mammalian species produce lactoferrin. Fujihara et al.295

reported a significant decrease in HSV-l titers following topical administration of 1%

46

lactoferrin using a mouse corneal infection model. Another study also found bovine
lactoferrin to have potent antiviral activity against HSV-l infection by competing
with the virus for cellular binding sites.296

Bovine lactoferrin was also effective against FHV -1 in cell culture.297 A
further study found bovine lactoferrin to have an inhibitory effect on the in vitro
replication of F HV-l prior to and during, but not following viral adsorption.298 When
CRFK cells were exposed to lactoferrin prior to or during viral adsorption, FHV -1
replication was inhibited by 87-96%. No cytotoxic effect of lactoferrin was observed.
The authors concluded that lactoferrin may inhibit F HV-l adsorption to the cell

surface and/or the penetration of the virus into the cell. Controlled clinical studies to

evaluate the in vivo therapeutic effect against F HV-l infections are warranted.

1.13.5 Vaccination

Vaccines against FHV-l have been available for a number of years. There are
several types of F HV-l vaccines commercially available, which are invariably given
in association with feline calicivirus (FCV) vaccinesf’6 Modified live virus (MLV),
and adjuvanted inactivated virus vaccines are available for parental administration. In
some countries, intranasal MLV vaccines are available.(’6 Currently available vaccines
induce reasonable protection against disease in previously unexposed cats.“"97
However, although severe disease is uncommon in a vaccinated cat, milder forms of
FHV-l disease can occur, especially given a high challenge dosem

None of the currently available vaccines appear to completely protect against
F HV -1 infection or the development of the carrier state. However, both viral shedding
and latency load of wild type virus after viral challenge may be reduced in vaccinated

cats compared to unvaccinated controls.'97‘299'3°‘

47

Cocker et al.302 concluded from their study that intranasal vaccination with a
cold adapted strain of FHV-l two days prior to challenge gave partial protection, and
vaccination four days before challenge gave complete protection against feline viral
rhinotracheitis. Weigler et 31.301 evaluated the biology of intranasal vaccination with a
temperature sensitive FHV-l mutant. Twelve cats were vaccinated with a temperature
sensitive mutant strain of F HV -1 intranasally. Four weeks post vaccination, 6 cats
were challenged with a virulent FHV-l field strain. Eight weeks post vaccination, all
12 cats were subjected to a stress protocol (intravenous injection of 30 mg
cyclophosphamide followed by 10 mg dexamethasone 24 hours later) in order to
reactivate latent infections. Cats were euthanized 13 weeks post vaccination. Utilizing
PCR and VI it was shown that all 12 cats shed virus throughout the 4 weeks following
vaccination. Although no clinical signs were observed in the 6 cats that were
challenged, FHV-l was recovered 4 weeks post challenge. Following euthanasia
F HV-l during latency was detected in trigeminal ganglia, optic nerves, corneas, optic
chiasma, olfactory bulbs, palatine tonsils and nasal turbinates. The frequency
distribution of FHV-l detected by PCR was identical for vaccine-only and vaccine-
challenge groups. Therefore, latent infection with vaccine virus can be established
following intranasal vaccination. The authors were not able to differentiate vaccine
strain from challenge strain of F HV-l by the methods utilized in this study.

Sussman et a1.300 performed quantitative PCR to detect latent wild-type F HV-l
in the trigeminal ganglia, olfactory bulbs, and brain stems of cats either unvaccinated
or subcutaneously vaccinated with FHV-lCV, F HVfi-galglgEA, or FHV-lSA prior to
challenge with a virulent FHV-l field strain. Latent FHV-l could be detected at
significantly higher concentrations in trigeminal ganglia from cats that were either not

vaccinated or vaccinated with FHVB-galglgEA than in ganglia from cats that were

48

vaccinated with either FHV-lSA or FHV-ICV. In the olfactory bulbs or brain stems
only extremely low levels of FHV-l DNA could be detected. Another study found
that administration of a single dose of a commercially available FHV-l intranasal
vaccine within several days prior to exposure lessened clinical signs of disease and
F HV-l shedding compared to unvaccinated cats.299

Vaccines against FHV-l are generally safe, and although occasional mild
transient clinical signs can follow their use, it is unclear if this is due to the FHV-l or

303 Kruger et al.304 reported that cats vaccinated subcutaneously

the F CV component.
or intranasally with high doses of a recombinant FHV-l strain responded with only
mild clinical signs and developed strong immunity against subsequent virulent virus
challenge. Care should be taken to avoid inadvertent oronasal administration of
attenuated FHV-l vaccines labelled for intramuscular or subcutaneous administration
as disease may be induced.7"304'306 This occurs because vaccines labelled for
intramuscular or subcutaneous administration are truly virulent strains. When
administered intramuscularly or subcutaneously, virulence is completely masked.
When this commercially available vaccine is administered by the oronasal route to
cats disease is induced as seen with natural infection.304

A number of genes have been targeted for the deve10pment of recombinant
FHV-l vaccines. These targeted genes mainly encode enzymes involved in DNA
synthesis, such as thymidine kinase and ribonucleotide reductase genes, and a number
of glycoprotein genes.197 Yokoyama et 31.307 developed a recombinant FHV-l
attenuated live vaccine, which was deleted in a defined region within the thymidine
kinase gene. This attenuated live vaccine caused only mild symptoms and provided

siginificant protection against challenge with the parent F HV-l strain. The deletion

also served as a genetic marker for PCR assays and allowed for easy detection of and

49

differentiation of the vaccinal strain. The level of protection was later improved
through the addition of a late promoter gC in the construct.308

Glycoproteins that are essential can be developed for use as subunit vaccines;
whereas genes of non-essential glycoproteins can be targeted for the construction of

‘97 Recombinant poxviruses and baculoviruses that express the F HV-

deletion mutants.
l glycoprotein D have been constructed to characterize its properties and potential as

an immunogen.309’3 '0 Also, multiple FHV-l deletion/insertional mutants have been

300,304,31 l 312

developed including gE-gl deletion mutants, a gI insertional mutant, and an
insertional mutant of ORF2, downstream of glycoprotein C313 These mutants are
generally less virulent and are reasonably protective against disease, especially when
administered via the oro-nasal route.66 However, since the protection offered by these

new mutants is not superior to conventionally attenuated vaccines, none has been

marketed so far.66

1.14 Conclusions

Herpesviruses are of major medical importance across all species. Despite
extensive research in the past 150 years, many details of the biological nature of the
different herpesviruses still have to be elucidated. FHV -1 remains a widespread
pathogen within the feline population worldwide. Expansion of our current
knowledge and understanding will potentially offer new perspectives for prevention
and therapy of the multiple manifestations of feline herpetic disease.

Expanded knowledge of the herpesvirinae also becomes critical because of
their potential new applications. A large area of current research focuses on the
utilization of HSV-1 as a viral vector for gene therapy and its potential as an oncolytic
virus. If cats and humans will be purposely inoculated with the herpesvirinae as

therapeutic agents, we must ensure that all ramifications of that infection are known.

50

Through genetic engineering and recombinant DNA techniques, the
herpesvirinae are being utilized in the creation of novel vaccines which function as
delivery agents. The delivery vaccine consists of an attenuated live vector in which
foreign genetic information from one or more other pathogens has been incorporated
in order to induce protective immunity against those pathogens and potentially against
the field type variants of the vector.314 Recombinant F HV-l has been evaluated as a

. . . . . . . . -1
vector for rmmunrzatron agarnst feline rmmunodeficrency vrrus,315 3 8

308,319,320 321

calicivirus, and toxoplasmosis.

Viral oncolysis, the destruction of neoplastic cells by replicating viruses is a
promising strategy for cancer treatment. While numerous viruses may be used for this
type of therapy, HSV-l is an attractive vector due to its high infectivity, ease of
genetic engineering, and a large transgene capacity.322 Positron emission tomography
(PET) has become a useful tool for tracking sites of viral replication.323 HSV
thymidine kinase serves as the PET reporter gene. The PET scan provides a novel,
non-invasive, easily repeatable instrument to ascertain the location and magnitude of

viral replication and tumor lysis.322

51

2. CHAPTER — ANATOMY AND HISTOLOGY OF TISSUES RELEVANT

TO THIS STUDY

2.1 Cornea

The cornea is the anterior continuation of the sclera, the fibrous tunic of the
globe. The transition zone between the cornea and the sclera is called the limbus. The
careful arrangement of the fibrous structure and the absence of blood vessels and
pigment allow transmission of light. The cat cornea is slightly conical and has a rather
steep curvature with a radius of 8-9 mm.324 The shape of the cornea is almost circular
with the horizontal diameter (16 mm) slightly greater than the vertical diameter (15.5

).324 This shape allows 40-42 diopters of light refraction.325 In the cat the central

mm
thickness of the cornea varies only slightly from the periphery. The average overall
thickness is 0.62 mm — 0.68 mm.324‘325

The most anterior of the corneal layers, the corneal epithelium, is a
continuation of the conjunctiva, and is composed of nonkeratinized, stratified
squamous epithelium.324‘326 The epithelium has one basal layer of columnar cells, two

324 The most

or three layers of polyhedral cells, and 7-10 layers of squamous cells.
superficial layer of squamous cells is in direct contact with the precomeal tear film
and has a microplicated surface.327

The corneal stroma (substantia propria) comprises 90% of the thickness of
the cornea — approximately 0.55 mm in the catm’325 Parallel bundles of collagen
fibrils form lamellae, with each lamella running the entire diameter of the cornea.325
The collagen fibrils within a lamella are parallel, but between lamellae, they vary in
direction.325 The fixed fibrocytes of the cornea, the keratocytes, lie flattened between

325

the lamellae. These cells and their extensions form and maintain the stromal

52

325

lamellae. The precise organization of the corneal stroma is the most important

factor in maintaining corneal clarity.3’25‘327
Descemet’s membrane, a homogeneous, acellular membrane, is the
exaggerated basement membrane of the corneal endothelium.325 It is produced
throughout life, becoming thicker as the animal ages.325 In cats, the thickness of
Descemet’s membrane ranges from 8-12 “111.324
The innermost layer of the cornea, the endothelium, is a single layer of
flattened hexagonal cells.325’327 Its thickness in cats is 3-4 pm.”4
The avascular cornea is nourished by the capillary loops at the limbus, the pre-
corneal tear film, and the aqueous humor.327 Branches of the ciliary nerves that arise

from the ophthalmic nerve, a branch of the trigeminal nerve innervate the cornea.327

2.2 Conjunctiva

The anterior corneal epithelium is continuous with the bulbar conjunctiva at
the corneoscleral junction.327 The epithelium of the lower nasal and middle fomix and
the lower tarsal portion of the palpebral conjunctiva contain the highest density of
goblet cells.325 The bulbar conjunctiva essentially lacks goblet cells.325 Two layers are
distinguished within the substantia propria of the conjunctiva, a superficial adenoid
layer, which contains lymphatic follicles and glands, and a deep, fibrous layer. The

325

fibrous layer contains the nerves and vessels. A superficial and a deep plexus of

lymphatics are present in the conjunctiva.325 Drainage extends to the parotid lymph
nodes and the submaxillary lymph nodes.325
2.3 Lacrimal gland

The lacrimal gland lies dorsolaterally enclosed within the periorbita.324‘325 The

gland is 1 mm thick with an equal width and a length of 14 mm.324 The thickness is

53

324

maximal centrally and tapers towards the periphery. Its surface is slightly lobulated,

324

and its anterior edge lies very close to the limbus. The lacrimal gland is

324

tubuloalveolar and of serous quality. It is composed of columnar cells and the

secreting acini are arranged in lobules of different sizes, supported by a relatively

324

sparse stroma of connective tissue. The lumen of the interlobular ducts is

324

approximately 30 pm in diameter. The lateral artery, a branch of the internal

24
d.3

maxillary artery serves the lacrimal glan Two lacrimal veins enter into the

external rete, which is drained by the ophthalmic vein from which it drains into the
external jugular vein.324 The lacrimal nerve, a branch of the ophthalmic division of the

trigeminal nerve, receives sympathetic fibers via the internal carotic plexus and

parasympathetic fibers via the pterygopalatine ganglion.328

2.4 Nictitans gland

The gland of the nictitans surrounds the caudal end of the shaft of the
cartilagenous plate of the third eyelid.325 It is thin, and has a flat roughly triangular
shape with its apex pointing caudally towards the apex of the orbit.324 Most of the

gland lies on the palpebral side of the cartilage, where it is 1 to 1.5 mm thick, whereas

324

on the bulbar side the gland is very thin. It is a serous gland in the cat.325 Although

324

the acini are positive for PAS suggesting a seromucoid type. The nictitans gland is

24
(1.3

much more richly endowed with septa than the lacrimal glan These septa carry

324

many prominent interlobular ducts and blood vessels. The ducts within the gland

324 Many fat cells are found within the septa.324 The

have diameters up to 60 um.
nictitans gland receives its parasympathetic innervation via the infratrochlear nerve, a
branch of the nasociliary nerve which is a branch of the ophthalmic division of the

trigeminal nerve.328

54

2.5 Trigeminal ganglion

329

The trigeminal nerve is the 5th cranial nerve. It has both motor and sensory

329

. . . . . 2 , 29
components. The Radzx sensorza rs much larger than the Radzx motorra.3 8 3 The

trigeminal nerve enters the trigeminal canal on the rostromedial aspect of the petrosal

9
bone}2

Within this canal is the large trigeminal ganglion. It contains sensory cell
bodies of the afferent, sensory fibersm’329 The three branches of the trigeminal nerve
emerge from the trigeminal ganglion: the ophthalmic nerve, the maxillary nerve, and
the mandibular nervem’329 The Radix motoria, which also contains afferent,
proprioceptive fibers, crosses the trigeminal ganglion medially and unites rostrally
with one sensory branch forming the mandibular nerve, which is a mixed nervem’329

The ophthalmic nerve consists of sensory axons from the eyelids, the globe
and the nose.329 The nerve passes through the orbital fissure of the skull. Within the
periorbita, it divides into three branches: the frontal nerve, lacrimal nerve, and
nasociliary nerve.329 These branches innervate the skin of the upper eyelid, lacrimal
gland, cornea, conjunctiva, nasal cavity, and the skin of the nostril.329

The maxillary nerve is sensory to the lower eyelids, nasal mucosa, upper teeth,
upper lip, and nose, and its distal branches contain postganglionic parasympathetic
axons (via fibers from the pterygopalatine ganglion) that innervate the lacrimal, nasal,
and palatine glandsm’329 The nerve leaves the cranial cavity through the round
foramen, alar canal, and rostral alar foramen and courses rostrally to the maxillary

329

foramen. In the pterygopalatine fossa the maxillary nerve gives rise to the

zygomatic nerve, pterygopalatine nerve, and infraorbital nerve.329

The mandibular nerve is both motor and sensory. It exits the cranial cavity

328

through the oval foramen. The nerve divides into multiple branches: masseteric

nerve, lateral and medial pterygoid nerves, tensor tympani nerve, tensor veli palatine

55

nerve, auriculotemporal nerve, the inferior alveolar nerve, the mylohyoid nerve, and
the lingual nervem’329 The mandibular nerve is sensory to the buccal cavity, tongue,
teeth of the lower jaw, the lower lip, in part the skin of the head, and mucosa of the

intraosseous part of the external ear canal.329

2.6 Ciliary ganglion

The oculomotor nerve is the 3rd cranial nerve. It consists of somatic efferent
neurons that innervate several of the striated extraocular muscles (dorsal rectus
muscle, medial rectus muscle, ventral rectus muscle, ventral oblique muscle, and parts
of the retractor bulbi muscle) and visceral efferent neurons (parasympathetic) that
innervate ocular smooth muscle.‘7’28’329 The nerve exits the cranial cavity through the

. , 9
orbrtal fissue.328 32

Upon entering the orbit, the nerve divides into a smaller dorsal
branch and a ventral branchm’329 The ventral branch continues rostrally passing
lateral and slightly ventral to the optic nerve, where it terminates in a number of
branches to the medial rectus, ventral rectus, and ventral oblique musclesm’329 At its
termination is the ciliary ganglion where the preganglionic parasympathetic fibers

329

synapse with postganglionic neurons. The two short ciliary nerves contain the

postganglionic axons, innervating the ciliary muscle and the iris sphincter muscle of

the eye.328-330

56

3. CHAPTER - HYPOTHESES AND SPECIFIC AIMS

This investigation was designed to follow infection with FHV-l through the
acute phase and into latency. The viral quantity, location, replicative status, and local
tissue effect of the virus were assessed through virus isolation, quantitative PCR, in
situ hybridization, and routine histopathologic examination of corneas, conjunctiva,
lacrimal glands, nictitans glands, trigeminal ganglia, and ciliary ganglia at various

time points during the infectious process.

3.1 Hypotheses

1. F HV-l DNA is present during both the acute and latent stages within the
corneas, conjunctiva, lacrimal glands, nictitans glands, trigeminal ganglia, and
cilary ganglia of experimentally infected cats.

2. FHV-l infects lacrimal tissue and leads to inflammation within the lacrimal

gland and nictitans gland.

3.2 Specific aims

l. Localization of F HV-l within the cornea, conjunctiva, lacrimal gland, and
nictitans gland by in situ hybridization.

2. Quantification of F HV -1 within cornea, conjunctiva, lacrimal gland, nictitans
gland, trigeminal ganglion, and cilary ganglion during the acute and latent
stages of infection by use of a real-time PCR assay.

3. Use of virus isolation to determine whether the F HV-l is active or latent when

detected in the tissues using the real-time PCR assay.

57

4. Assessment of local tissue effect of the virus within cornea, conjunctiva,
lacrimal gland, nictitans gland, trigeminal ganglion, and cilary ganglion during

the acute and latent stages of infection.

58

4. CHAPTER — RESEARCH PROTOCOL, RESULTS, AND DISCUSSION

4.1 Introduction

Feline herpes virus 1 (FHV-l) infection is widespread within the cat
population.4 Herpesviral ocular disease can be quite severe. Typical acute
manifestations include ocular discharge, blepharospasm, conjunctivitis, keratitis, and

4,79

corneal ulceration. Chronic ocular manifestations and sequelae may include

persistent conjunctivitis, symblepharon, stromal keratitis and possibly corneal
sequestration and eosinophilic keratitis.77'80’163

Decreased tear production in cats has long been suspected to be a potential
sequela to FHV-l infection.33 I The cause remains speculative, but it has been assumed
that either excretory duct occlusion or lacrimal adenitis is responsiblefl’80 To the
authors knowledge, histopathologic examination of the lacrimal tissues has not been
performed to support either of these theories. Experimentally, a temporary
keratoconjunctivitis sicca developed in 5 of 10 cats treated pre-inoculation with three
consecutive daily subconjunctival injections of betamethasone.80 However, aqueous
tear production returned to normal in all cats by day 60 post-infection.80 Bistner, et
al.m failed to demonstrate the presence of F HV-l within the lacrimal gland utilizing
specific fluorescent antibody staining or virus isolation. Interestingly, fluorescein-
labelled antibody has been used to examine human lacrimal glands and revealed
HSV-1 viral antigen in 2/3 patients with acute herpetic keratitis and in 4/7
asymptomatic patients with a history of previous herpetic infection.145

The potential for recrudescent infection is high because the virus remains
latent in approximately 80% of infected cats and 50% of FHV-l recovered cats will

shed virus spontaneously or in response to stress.65 During the latent period clinical

signs are absent and the virus cannot be isolated using standard cell culture

59

techniques.38 FHV-l DNA has been detected utilizing PCR assays in the nasal
turbinates, oral fauces, tonsils, salivary glands, mesenteric lymph nodes, conjunctiva,
corneas, lacrimal glands, optic nerves, trigeminal ganglia, olfactory bulbs, and cerebra
of latently infected cats.85’I44 Latency was confirmed by concurrent negative virus
isolation.38

Furthermore, using in situ hybridization and northern blot analysis of
trigeminal ganglia, Ohmura et al. detected transcripts of the strand opposite to the

immediate-early mRNA in cats latently infected with FHV-l.193

These transcripts
likely represent latency associated transcripts (LATs) which are the only transcripts
present during latency and may therefore serve as markers of latency.”38 Townsend
et al.198 also detected LATs utilizing a reverse transcriptase PCR assay in 4/16
trigeminal ganglia of clinically normal cats. However, to the authors knowledge
histologic examination of the latently infected neural tissues to determine the effect of
the F HV -1 infection has not been performed.

In humans with a history of previous herpes simplex-l (HSV-l) infection, the
ciliary ganglion has been identified as a site of latency. Bustos et al.168 identified and
detected HSV-l utilizing PCR in 20/30 ciliary ganglia from human cadavers with no
history of active herpetic lesions at the time of death. However, the ciliary ganglion
has not been evaluated for presence of FHV-l during either active or latent infections
in cats.

The objectives of this study are to utilize virus isolation, a quantitative real-
time polymerase chain reaction assay, histopathologic examination, and in situ

hybridization to localize and quantify the amount of F HV-l present and evaluate the

associated morphological changes within the corneas, conjunctiva, lacrimal glands,

60

nictitans glands, trigeminal ganglia, and ciliary ganglia of experimentally inoculated

specific pathogen free cats during both the acute and latent phases of infection.
4.2 Materials and Methods

4.2.1 Animals

Fifteen 6-month-old female SPF cats were obtained from a commercial vendor.
Cats were housed in individual cages with treatment and control groups in separate
rooms (Biocontainment Level-2) with controlled temperature, humidity, and lighting.
All personnel donned sterile disposable Tyvek coveralls, gloves, head covers, and
shoes covers upon entering a room. Cats were fed a combination of dry and moist
diets. All cats were acclimated for 7 days before viral inoculation.

A11 cats were determined to be serologically negative for FHV -1 virus
neutralizing antibody prior to inoculation. Blood samples were collected on days 7, 14,
and 21 post inoculation from the group 3 cats for evaluation of FHV-l virus

neutralizing antibodies.

4.2.2 Inoculation and clinical scoring

Cats were anesthetized via mask administration of halothane and oxygen.
Twelve cats were infected by instilling 1.05 ml (1x105 TCIDso/ml) of the C-27 strain
of FHV-l into the lefl and right conjunctival sacs (350 pl each), external nares (150 pl
each), and oropharynx (50 ul) of each cat. The other 3 cats served as controls. The
C27 strain of FHV -1 was obtained from a commercial source (ATCC, Manassas, VA)
and was certified to be a purified suspension.

Clinical signs including fever, sneezing, ocular or nasal discharge, anorexia,

dyspnea, and dehydration were scored daily for each cat as described (U .S.

61

Department of Agriculture, Animal and Plant Health Inspection Service, National
Animal Veterinary Services Laboratory, 1985) by 3 investigators (Jacobi, Wasserrnan,
Townsend) (Appendix A). Previously published work304 had established that all
animals should be exhibiting ocular signs on day 6, all animals should be at the peak
of ocular and respiratory signs on day 10, and latency should be established with
resolution of clinical signs by day 30. Therefore, on days 6 (group 1), 10 (group 2)
and 30 (group 3), four inoculated and one control cats were humanely euthanized and
samples were immediately collected. All experiments were performed in full
compliance with the ARVO Statement for the Use of Animals in Ophthalmic and
Vision Research and with approval from the Michigan State University Animal Care

and Use Committee.

4.2.3 Samples

Aseptic, bilateral sample collection (corneas, conjunctiva, lacrimal glands,
nictitans glands, trigeminal ganglia, ciliary ganglia) occurred immediately following
euthanasia. Particular care was taken to avoid cross contamination between tissue
samples by alcohol dipping and flaming of the instruments for each use.

Tissue samples or entire ganglia from the right side were placed in Eppendorf
tubes containing one sterile homogenizing bead with 800 u] of Bovamick’s buffer and
homogenized for further virus isolation and PCR analysis. Tissue samples from the
left cornea, conjunctiva, lacrimal gland, and nictitans gland were divided. One half
was placed immediately in 10% formalin; the other V2 was frozen at -80 °C. Due to
their limited size, the entire left trigeminal ganglion and ciliary ganglion were placed

immediately in 10% formalin.

62

4.2.4 Virus isolation

Following homogenization in the Qiagen Mixer mill (QIAGEN Inc., USA)
and collection of a 200 pl aliquot for DNA extraction, the remaining suspension was
transferred to a 15 ml conical tube. The volume was brought to 3 ml with Eagle
minimal essential medium (EMEM) supplemented with 10% fetal bovine serum
(FBS). The homogenate was then filtered (0.45 pm). Filtered tissue extracts (500 pl)
were inoculated onto monolayers of Crandell feline kidney (CRFK) cells grown in 24-
well microtiter plates. All cultures were performed in duplicate.

The cultures were observed daily for five days with an inverted microscope
(Nikon Eclipse TSlOO) for the cytopathic effects (CPE) characteristic of FHV-l. A
sample was considered positive for FHV -1 only if it produced CPE within both
culture wells. If only one culture was positive, another culture was performed for the
sample, again in duplicate. The result was then considered positive if CPE was

produced in one or both of the repeat cultures.

4.2.5 Quantitative real time PCR

4.2.5.1 DNA extraction

A mixture of 100 pl tissue homogenate and 100 pl phosphate buffered saline
(PBS) was utilized for DNA extraction. Extraction controls, performed for each
extraction, consisted of 200 pl PBS. Purification of DNA was performed using a
commercially available kit (DNeasy Tissue Kit, QIAGEN, Valencia, CA) in
accordance with the manufacturer’s protocol. The samples were eluted with 100 pl
Buffer AB (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0). DNA was quantified using a

spectrophotometer (NanoDrop 1000, Therrno scientific, Wilmington, DE).

63

4.2.5.2 PCR amplification of F HV-l

A real-time SYBR Green-based PCR specific for FHV -1 has been previously
described.332 The primers for this assay target a 478-bp fragment of the FHV-l gE
gene. The sequence of the forward primer is 5’-GGT CAT GTG TAA TGT TGA CG-
3’, and the reverse primer is 5’- GTC TTT GGT TCT GAT GAG AG-3’.3“ The
sensitivity of this assay is 10 viral genome copies. The diagnostic utility of the assay
has been confirmed by its ability to amplify the targeted DNA fragment of 48 known
clinical isolates of FHV-l. The 50 ul real time PCR reaction mixture consisted of 25
pl 2x SYBR Green mix (Taq DNA polymerase, dNTPs, SYBR Green I, 5.0 mM
MgC12; QIAGEN QuantiTect SYBR Green PCR Kit, Valencia, CA), 1 u] forward
primer (25 pmol/ul), 1 ul reverse primer (25 pmol/ul), 100 ng of template DNA and
ddeO. PCR amplification was performed on an iCyclerTM iQ® System (Bio-Rad
Laboratories, Hercules, CA) with the following cycling conditions: pre-denaturation
at 95 °C for 15 min, followed by 40 cycles of 94 °C for 30 sec, 50 °C for 30 sec and
72 °C for l min. Amplification was continuously monitored in real time by
quantifying the amount of fluorescence emitted at 530 nm at each annealing step.
Following the final elongation step at 72 °C for 60 seconds, a post-PCR melt curve
analysis was performed, amplicon melt temperatures were determined by raising the
temperature in 0.5 °C increments from 55 °C to 95 °C and monitoring changes in
emitted fluorescence (Figures 4.1 and 4.2). All reactions were conducted in duplicate.
DNA extraction controls and reagent controls were included in each run. A standard
curve of threshold cycle (Ct) in relation to starting viral copy number was constructed
(Figures 4.3 and 4.4). The standard curve for F HV-l was generated by a series of 10-
fold dilutions (10l - 106) of FHV-l DNA. Samples for the standard curve were

assayed in triplicate for each run and served also as the positive control.

64

Melt Curve Chart: Data 2006-05-15 2150.0pd

RFU

 

55 60 65 70 80 85 90 95

Temperature, Celsius
Figure 4.1. Standard melt curve chart.

Melt Peak Chart: Data 2006-05-15 2150.0pd

-d( RFU )ldT

 

55 6O 65 7O 75 80 85 90 95

Temperature, Celsius
Figure 4.2. Standard melt peak chart.

65

Melt Peak Chart: Data 2006-05—1 5 2150.0pd

2200
2000
1 800
1 600
1400
1 200
1 000
800
600
400
200
O

 

 

PCR Base Line Subtracted Curve Fit RFU

0 5 10 15 20 25 30 35 40 45
Temperature, Celsius

Figure 4.3. Standard amplification chart, 10 fold dilutions of 106 genomic copies of
FHV-l, triplicate.

0 Standard
40
35 -

 

 

i-
if

:7

 

1111

25 *
20

15......4......
O 2 4 6 8

Log Starting Quantity, copy number

 

 

IL

Threshold Cycle

1

 

 

 

L111
4
q

 

+ SYBR IE=85.6% R"2=0.987 slope=-3.725 y-int=39...

PCR Standard Curve: Data 2006-05-15 2150.0pd
Figure 4.4. PCR standard curve.

66

4.2.5.3 Statistical analysis

Data were analysed using the Wilcoxon Rank -Sum test for non Gaussian data
distribution. Following Bonferroni’s adjustment for multiple comparisons, the level of
significance was p < 0.017. For all tissues mean copy numbers of the 3 time points

(day 6, day 10, and day 30 post inoculation) were compared.

4.2.5.4 Histopathology

Following fixation in 10% formalin, tissues were paraffin-embedded,
sectioned at 5 pm, deparaffinized, and stained with hematoxylin and eosin (HE). All

samples were examined with a light microscope.

4.2.5.5 In situ hybridization

In situ hybridization was performed to specifically localize FHV-l within the
cornea, conjunctiva, lacrimal gland, and nictitans gland. The in situ protocol was first
optimized by evaluating various pretreatment methods: 1.) baking for 30 min at 60 °C
prior to deparaffinization of the section followed by enzymatic digestion (100 ul/ml
proteinase K in TBS buffer (50 mM Tris HCl, pH 7.4, 10 mM EDTA, 10 mM NaCl))
for 15 minutes at 37 °C; 2.) enzymatic digestion following deparaffinization without
previous baking; 3.) microwaving: slides covered with 10 mM Tris HCl, pH 8.1 + 1
mM EDTA buffer were microwaved at full power for 5 or 10 minutes. Also antibody
dilutions of 1:250 and 1:500 were evaluated (Figure 4.5). All optimization was
performed on F HV-l pellets. Superior results were obtained by pre-treating via
microwaving for 10 minutes and using an antibody dilution of 1:250 (Figure 4.6).

This pretreatment protocol was used for all tissue sections.

67

‘ ~0-

niés ‘ Is an...)

 

.‘ gm '
Figure 4. 5. FHV- 1 pellet pre-treated with different protocols.
A. Enzymatic digestion with proteinase K using an antibody dilution of 1:500.
B. Enzymatic digestion with proteinase K using an antibody dilution of 1:250.
C. Baking for 30 min at 60 °C prior to deparaffinization of the section followed by
enzymatic digestion with proteinase K using an antibody dilution of 1:250.
D. Micowaving for 10 minutes using an antibody dilution of 1:500.

68

    

  

  

‘ — ‘ , A - F . n - r
r .- _ v - ._ ‘ .
. o , 1,. I,
.'f _ t ‘ . : . at, ' “F
r , , > - ‘r-O . 4

pre-treated via micowaving for 10 minutes using an

[.8 1.3.- ‘.
Figure 4.6. FHV-l pellet
antibody dilution of 1:250.

Comea, conjunctiva, lacrimal gland, and nictitans gland were examined in
duplicate. The FHV-l DNA probe used targets a portion of the gE gene. The 30-mer
synthetic oligonucleotide probe with the sequence 5' CGA TGA CAG AAG GTG
CCG CAC TTT TAG TCG ATG GG 3' was synthesized and labeled with
digoxigenin-l l-dUTP by a commercial supplier (IDTDNA, Coralville, IA). Positive
(FHV-l pellet) and negative controls (canine nictitating membrane, F HV-l pellet with
no probe applied) were included with each batch of slides.

Paraffin sections were prepared on silane-coated slides. Sections were then
dewaxed in xylene (2 x 10 min), taken through a series of graded ethanols (l x 5 min

in 100%, 95%, 75%, and 50%) and washed in nanopure water (2 x 5 min).195 Sections

 

were then placed in Tris/41., ' J' ' ‘ ‘ " acid (EDTA) buffer (10 mM Tris

HCl, pH 8.1 + 1 mM EDTA) and microwaved at full power continuously for 10

69

minutes. Sections were allowed to cool for 15 minutes and nanopure water was
gradually added over 5 minutes to reduce the temperature. ‘95 Tissues were then fixed
in 4% paraformaldehyde in phosphate buffered saline (PBS) for 10 minutes.333 Slides
were allowed to air dry and the tissue sections were layered with 75 pl of
prehybridization solution containing 4X saline sodium citrate (SCC); 50 mM
NaHzPO4/Na2HPO4, pH 7.0; 1 mM EDTA; 5X Denhardt’s solution (1% Ficoll, 1%
polyvinylpyrrolidone and 1% bovine serum albumin); 10 pl sonicated salmon sperm
DNA(500 pg/ml); formamide to 25%; and nanopure water. The solution was held in
place by the use of a liquid blocker (PAP PEN, Invitrogen, Carlsbad, CA).
Prehybridization was allowed for 60 minutes at room temperature. Then hybridization
solution consisting of the prehybridization solution and 200 ng/ml DIG labeled probe
was layered over each section. Slides were heated for 6 minutes at 95 °C and
immediately quenched on ice for 1 minute. Hybridization followed at 25 °C overnight
in a humidified chamber. Sections were then washed twice in 2X SCC for 5 minutes
and 0.1X SCC for 10 minutes at room temperature. Slides were rinsed briefly in
buffer 1 (100 mM Tris HCL, 150 mM NaCl, pH 7.5). The tissues were then covered
with buffer 2 (100 mM Tris HCL, 150 mM NaCl, pH 7.5, and 0.5% blocking reagent)
and cover-slipped with parafilm. Slides were allowed to sit for 15 minutes at room
temperature. Following removal of the cover slips, slides were rinsed briefly in buffer
1. Then, 30 pl of 1:250 anti-DIG AP, Fab fragment antibody conjugate was layered
onto the tissues, which were cover-slipped with parafilm, and placed in a humidified
chamber at 25 °C for 2 hours. Slides were then washed twice for 10 minutes in buffer
1 and allowed to equilibrate for 5 minutes in buffer 3 (0.1 M Tris HCL, pH 9.5, 0.1 M
NaCl) at room temperature. Then 20 pl of 1:50 NBT/BCIP solution was layered onto

each tissue, cover-slipped with glass cover slips, and covered with aluminum foil to

 

prevent illumination. Color was allowed to develop overnight at 25 °C. Following
removal of the cover-slips, the reaction was stopped by rinsing the slides briefly in 10
mM Tris HCl, pH 8.1 + 1 mM EDTA. Slides were then washed in nanopure water.
Neutral red counter-stain was layered over each tissue for 1 minute and then rinsed
with nanopure water. Slides were then cover-slipped (Irnmu-mount, Shandon,

Pittsburgh, PA) and evaluated under a light microscope.

4.3 Results

4.3.1 Clinical features

All inoculated cats euthanized on day 30 post inoculation developed antibodies
against F HV-l with titers ranging from 1:32 to 1:128. The control cat did not develop
antibodies against FHV-l. None of the control cats showed any signs of disease at any
time. Sneezing developed in 11/12 inoculated cats with a peak between day 3 and 9
post-inoculation. None of the cats developed a cough, rales, open mouth breathing, or
corneal or cutaneous ulceration. Blepharospasm of variable severity and duration was
noted in 10/ 12 inoculated cats. All 12/ 12 inoculated cats developed serous or in some
cases mucopurulent ocular discharge of variable duration and severity between days
3-14 post-inoculation. Conjunctivitis was found in all 12/ 12 inoculated cats of
variable duration and severity starting on day 3 post-inoculation. Other signs noted in
variable severity and duration were nasal discharge in 10/12 inoculated cats,
salivation in 3/ 12 inoculated cats and oral ulceration in 9/ 12 inoculated cats. Two of
the 12 inoculated cats developed mild signs of dehydration, 6/ 12 cats developed a
transient, mild anorexia, and 5/ 12 cats had a transient pyrexia (>102.5 °F) between

days 3 and 7 post-inoculation. None of the cats required or received any treatment

71

during the study. See clinical scores in Table 4.1. Detailed data are shown in

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Appendix B.

Day of euthanasia post inoculation Cat number Clinical score

Day 6 1 8
2 7
12 4
4 5
5 0

Day 10 6 24
7 19
8 16
9 31
10 0

Day 30 11 20
3 44
13 27
14 20
15 0

 

 

 

Table 4.1. Clinical scores.

4.3.2 Virus isolation

None of the tissues were positive on virus isolation for the control cats. Virus

isolation from the four inoculated cats euthanized on day 6 and 10 post-inoculation

72

 

demonstrated 2/4 positive trigeminal ganglia, 2/4 positive ciliary ganglia, and 4/4
positive lacrimal glands, nictitans glands, corneas, and conjunctiva respectively. Virus
isolation from the four inoculated cats euthanized on day 30 post-inoculation
demonstrated only 1 positive sample; a corneal specimen. All other tissues from the
group 3 cats were negative for virus isolation of FHV-l. Results are summarized in

the Table 4.2. Data are shown in detail in Appendix C.

Cornea 4/4 4/4
'unctiva 4/4 4/4

Tri ' 2/4 2/4
C' ' ' 2/4 2/4

Table 4.2. Summarized results of virus isolation (positive/total)

 

4.3.3 Quantitative real time PCR

FHV-l DNA could not be detected in any of the tissue samples from the
control cats. In all infected animals FHV -1 DNA could be detected in cornea,
conjunctiva, trigeminal ganglia and ciliary ganglia on days 6 and 10 post inoculation.
All inoculated cats had positive trigeminal ganglia on day 30. In addition, one cat was
positive for FHV-l DNA in the corneal, conjunctival, and ciliary ganglion samples on
day 30. The three remaining inoculated cats were negative for those tissue samples on
day 30. (Table 4.3)

Copy numbers per 100 ng DNA were similar for the corneal samples in all cats
on day 6 and day 10. In conjunctival samples, copy numbers tended to be lower on
day 10 than on day 6 post-inoculation. For the trigeminal ganglion and the ciliary
ganglion, copy numbers tended to be increased on day 10 compared to day 6.
However, statistical analysis using the Wilcoxon Rank-Sum test with a level of

significance of p < 0.017 following Bonferroni’s adjustment for multiple comparisons,

73

showed that none of the comparisons between the 3 different time points were
significant.

PCR results for the cornea, conjunctiva, lacrimal gland and the nictitans gland
tissues for cats # 4, 7, and 12 revealed positive extraction controls. PCR for these
samples was not repeated for the glandular tissues due to the fact that upon histologic
examination, conjunctival tissue was found to be adherent to the glandular material
and could have resulted in false positive results because of possible presence of F HV-
1 in the conjunctiva rather than in the gland itself. PCR was repeated for the corneal
and conjunctival tissues of cats # 4, 7, and 12 in duplicate. Results are summarized in

the Table 4.3. Data are shown in detail in Appendix D.

10 30
Cornea 4/4 4/4 1/4
tiva 4/4 4/4 2/4

Tri ' 4/4 4/4 4/4
C' ' ° 4/4 4/4 1/4

Table 4.3. Summarized results of quantitative real time PCR (positive/total)

 

4.3.4 Histopathology

None of the control cats showed any histomorphologic abnormalities in any of
the tissues examined. Conj unctival changes characterized by epithelial cell attenuation
or loss and lymphoplasmacytic inflammation were seen in all 12 inoculated cats
(Figure 4.7), although the changes were milder in cats euthanized on day 30 post-
inoculation. In these cats areas of conjunctival re-epithelialization were present. All
corneal sections were normal except for those from one cat (cat # 9) euthanized on
day 10 post-inoculation. Multiple, solitary, degenerate epithelial cells and a mild
lymphoplasmacytic infiltrate characterized the changes within the corneal section
(Figure 4.8).

74

The glandular tissues within all sections of lacrimal and nictitans glands
appeared normal (Figure 4.9). However, histopathologic examination revealed that
multiple specimens of lacrimal and nictitans gland contained the adjacent conjunctiva.
The conjunctiva in these samples exhibited changes consistent with those noted in all
other conjunctival samples.

All 4 trigeminal ganglion samples obtained on day 6, 3 samples obtained on
day 10, and 1 sample obtained on day 30 post-inoculation revealed histopathologic
changes including ganglion cell necrosis, drop out or loss of ganglion cells, increased
numbers of glial cells, and lymphoplasmacytic inflammation (Figure 4.10). All ciliary
ganglia evaluated appeared normal. Due to the small size of the ganglia from one cat
euthanized on day 10, no appropriate section of the trigeminal ganglion could be
obtained. From 1 cat (day 10) and 2 cats (day 30 post inoculation) no appropriate

section of the ciliary ganglion could be obtained.

75

 

r

v-2-
Figure 4.8. Corneal sample from cat # 9. (HE)

 

Figure 4.7. Conjunctiva] sample from cat # l2. (HE)

76

:. .- r- . . W '3‘ '
I! ”HWLMO??'%§L£" 1 ~ 14%
.. .7?va ‘ - P'

-'.' ,.

in _.
cat # 2. HE

 

. " pr . . ‘V r , f ' 3 j: " '
.. a; _‘-. {5.9 g ‘ 3.: “14.2%: .. ‘
Figure 4.10. Trigeminal ganglion sample from cat # 1. (HE)

4.3.5 In situ hybridization

Results of in situ hybridization are summarized in Table 4.4. A blue to brown
reaction product confined to the nuclei of intact epithelial cells characterized a
positive hybridization signal. None of the control cats showed positive results in any
of the tissue samples. In conjunctival samples the positive cells were found adjacent
to areas of inflammation (Figure 4.11). No inflammatory changes were noted adjacent
to the positive superficial corneal epithelial cells (Figure 4.12) Hybridization signals
were not detected within the lacrimal or nictitans glands. However, positive
hybridization signals were detected within the overlying conjunctiva. Corneal samples
of cat # 2 were not available for evaluation via in situ hybridization. The tissue quality
of all 4 samples from the group 3 (day 30) cats was very poor. One cat appeared to
have a positive hybridization signal in the cornea, but this result may be spurious due

to the poor tissue morphology.

 

ISH

 

Cat N o. Cornea Con'unctiva Lacrimal Nictitans
J Gland Gland

 

1 + + - -

 

2 Not avail.

 

Day6 12 -

 

+++
I
I

4 +

 

5 (control) -

 

6 -

 

7 - - - -

 

Day 10 8 - - - -

 

9 - - - -

 

 

 

 

10 (control) - - - -

 

 

11
3
Day 30 13 Poor tissue morphology
14

15 (control)

 

 

 

 

 

 

 

Table 4.4. Summarized results of in situ hybridization

78

 

.933

A
a

My

it“

 

" . 50 pm ‘5'

,-

Figure 4.11. In situ hybridization of the conj unctival sample from cat # 12.

 

50 pm

Figure 4.12. In situ hybridization of the corneal sample from cat # 4.

79

4.4 Discussion

Clinical signs of natural as well as experimentally induced FHV -1 infection
can be quite severe.70’79 In the present study infected cats generally exhibited mild
signs of upper respiratory and ocular disease. A possible explanation for the mild
clinical signs compared to natural infection or experimental infection with other viral
strains could be viral attenuation over time leading to a reduced pathogenicity of the
C27 strain of FHV-l.

Similar to observations by Bistner et al.12 I no clinical signs of keratitis were
observed in any of the cats at any point in time during this primary infection.
However, fluorescein staining and Rose Bengal staining of the cornea were not
performed daily. Therefore very subtle lesions may not have been detected.

Oral ulcers are reported to occur rarely in association with feline herpes virus
1 infection and are more commonly associated with feline calicivirus infectionm’106
Although the areas of oral ulceration noted were generally small and transient, 75% of
inoculated cats were affected. It is possible those small ulcers are overlooked in
clinical cases or that field strains do not induce development of oral ulceration. The
oropharyngeal administration of 50 pl of viral suspension may also represent a greater
viral inoculum than generally reaches those tissues during natural infection.

At 30 days post-inoculation, latency should be established with resolution of
clinical signs.304 Latency has been previously defined as the absence of detectable
FHV-l as assessed by virus isolation, but detectable viral DNA using PCR
assays.85 ’144 In this study, virus isolation was performed immediately following tissue
harvest. Infectious virus was isolated from all ocular tissues and 50% of the ganglia
on day 6 and day 10 post-inoculation. This is consistent with virus shedding as

expected with active disease. On day 30 all tissue samples from 3 of the inoculated

80

cats were negative on V1. This is consistent with establishment of latency, which has
previously been shown to occur by this time post inoculation.304 However, F HV-l
could be isolated from the corneal sample of one cat. From the same cat FHV-l DNA
could also be detected using quantitative real-time PCR in the cornea, conjunctiva,
and both the ciliary and trigeminal ganglia. Although the cat did not show any clinical
signs at the time of euthanasia, these results suggest the cat was either suffering a
recrudescent infection or failed to clear the primary infection. A similar scenario has
been noted in studies by Stiles et a1.162 and Townsend et al.198 in which F HV -1 DNA
was detected in the corneas of clinically normal cats. In these studies virus isolation
was not performed. However, in the study by Townsend et al.198 LATs could not be
detected in the cornea, suggesting the infection was not latent.

Histopathological examination revealed typical changes for the conjunctival
tissues in all inoculated cats. However, glandular tissues from all lacrimal and
nictitans glands were normal. These results do not support the suggestion by Nasisse4
and Nasisse et al.80 that lacrimal adenitis is responsible for FHV-l related
keratoconjunctivitis sicca (KCS). As inflammatory changes were noted in the
overlying conjunctiva, perhaps occlusion of the excretory ductules is the cause of
FHV-l related KCS. Clinically none of the cats demonstrated clinical signs consistent
with KCS. However, tear production was not evaluated in this study. Inoculation with
a more virulent strain of F HV-l might cause KCS to develop and better elucidate the
cause.

Conjunctival tissue exhibiting changes consistent with conjunctivitis was
noted in sections of the lacrimal and nictitans glands. Therefore, the results of the
virus isolation for these tissues are suspect, since FHV-l could be isolated from

conjunctiva during the acute phase. Unfortunately lacrimal tissue could not be

81

harvested without contamination from the overlying conjunctiva. For this reason,
quantitative real-time PCR was not repeated for the glandular tissue samples with
positive extraction controls.

All trigeminal ganglia examined histopathologically during the acute
infectious state showed signs of lymphoplasmacytic inflammation. However, only 1
trigeminal ganglion obtained from a cat on day 30 showed histomorphological
changes; a drop out of ganglion cells, but no concurrent inflammation. These results
suggest that the trigeminal ganglia developed transient inflammation during the acute
infection. The absence of lymphocytes during the latent phase is very interesting.
Current studies of HSV-1 in man suggest lymphocytes play a key role in preventing
reactivation of the virus within neuronal tissues“0

In contrast to the inflammation seen within the acute phase trigeminal ganglia,
no histomorphologic changes could be detected in any of the ciliary ganglia. This
occurred despite a 50% isolation rate on V1 and a 100% detection rate on quantitative
real time PCR during the acute phase of FHV-l infection. It is possible that localized
inflammatory changes do not occur or they were missed on histopathologic
examination due to the limited number of sections obtained from the ganglia.

The F HV-l DNA copy numbers in corneal samples as determined by
quantitative real-time PCR were similar in samples obtained on day 6 to samples
collected on day 10 post-inoculation. Except for one cat’s corneal sample in which
recrudescent or persistent infection is suspected, all other cats euthanized on day 30
had negative tissue samples. DNA copy numbers in conjunctival tissues tended to be
lower on day 10 than on day 6. Therefore it is possible that the infection clears from
the conjunctiva before it clears from the cornea. For the trigeminal ganglia a transient

increase on day 10 was noted before the viral copy number decreased by day 30. A

82

similar phenomenon of detectable viral DNA increase on day 10 was noted in the
ciliary ganglia. Therefore we would speculate that these ganglia reach their peak viral
load later than the surface ocular tissues. In contrast to findings in a previous study300
F HV-l DNA copy number within the trigeminal ganglia during latency was only
slightly lower than during active infection. In that study quantification was carried out
using an automated electrochemiluminescence detector. The higher sensitivity of the
quantitative real time PCR assay utilized in the present study could explain the
difference in viral copy numbers between the two studies. However, our results are
similar to those seen in murine models of HSV-1.3%336 On day 30 a low copy number
of FHV-l DNA was detected only in the ciliary ganglion from the cat with a
suspected recrudescent or persistent infection. All other ciliary ganglia were negative.
From these data we cannot conclude that the ciliary ganglion is a site of latency for
F HV-l in cats. Further investigation will be necessary.

All cats euthanized on day 6 post-inoculation were positive on ISH within
corneal and/or conjunctival tissues. In contrast none of the cats euthanized on day 10
post-inoculation had a clearly positive result in any of the tissues. The positive control
samples processed at the same time demonstrated strong positive hybridization signals.
Furthermore, these tissues were processed with other tissues in which positive
hybridization signals were detected. These results are somewhat surprising as virus
isolation for conjunctival and corneal samples was positive for all inoculated cats
euthanized on days 6 and 10. Furthermore, viral load as determined by quantitative
real time PCR was similar for ISH-positive and -negative cats euthanized on day 6
and 10.

The strongest positive hybridization signal was detected within conjunctival

epithelial cells from cat #12 euthanized on day 6 post-inoculation. This cat also had

83

the highest viral load within corneal and conjunctival tissue samples based upon the
RT-PCR results. However, no positive signal was found within this cat’s cornea.
FHV -1 positive cells were generally sparse in positive tissues. Therefore it would be
possible to have examined sections that did not contain cells harboring sufficient virus
to produce a detectable positive hybridization signal. Also, cats with severe
conjunctivitis had necrotized or very attenuated epithelium. This leaves less intact
epithelial cells in which to detect the virus. The overall sensitivity of the ISH to detect
FHV-l utilizing this protocol was poor when compared to the sensitivity of the RT-
PCR assay. A possible explanation could be the localized distribution of FHV-l
infected cells within the tissues.

No virus could be detected with this method within glandular tissue samples
examined. This is consistent with the normal tissue morphology shown in standard
histopathologic examination. However, F HV-l in low quantities could be present.
Furthermore the clinical signs seen in this study were generally mild. Therefore it is
possible that F HV-l infects lacrimal and nictitans glands in more severe infections.

All tissues of cats euthanized on day 30 showed poor cellular morphology in
the sections examined by ISH. The morphology of the same tissues was excellent for
standard histology. This is difficult to explain, since these tissues were processed for
the ISH in different runs together with other tissues that demonstrated good
morphology. It could be possible that fixation of the tissues post euthanasia was either
insufficient or excessive and the intense treatment during ISH led to the
morphological changes noted. Repeating the ISH for these tissues with varying
conditions could help to explain the changes found.

In future studies, additional tissues such as uvea, retina, optic nerve, olfactory

bulb, brainstem, cerebellum, cerebrum (visual cortex), optic chiasm, cranial cervical

84

ganglion, otic ganglion, pterygopalatine ganglion, mandibular salivary gland,
submandibular lymph node, parotid salivary gland, parotid lymph node, nasal
turbinates, tonsil, lung, trachea, oral fauces, and retropharyngeal lymph node could be
examined to identify other targets of FHV-l during both the acute and latent stage of
infection. Herpesvirinae have been detected within some of these tissues during
studies of HSV-1 infection in murine models31 and pseudorabies virus infection in
pigletsm‘338 The presence of virus within these tissues could expand our
understanding of the pathology induced by FHV-l infection and the patterns of
recrudescence noted clinically.

Modulation of apoptosis may be a key factor allowing herpesvirinae to
establish latency.339'340 However, the ability of FHV-l infection to influence cellular
apoptosis has not been investigated. If FHV-l infection can block apoptosis of
infected cells, this is a mechanism by which the host defenses are evaded. Apoptosis
could be assayed by measuring caspase-3 activity. Caspase activation is thought to be
the most specific indicator of apoptosis and caspase 3, the key converging enzyme is
ubiquitously distributed making it an excellent choice for a marker of apoptosis.34'

More sensitive methods to detect FHV-l could be utilized including in situ
PCR or laser capture of individual cells for PCR analysis. Furthermore, a different
FHV-l strain exhibiting higher virulence than C27 could be used for inoculation. This
could also facilitate the detection rate and help to identify further targets of FHV-l.

In conclusion, this study confirmed the ability of F HV-l to acutely infect not
only conjunctival epithelium, corneal epithelium, and trigeminal ganglion, but also
the ciliary ganglion. The ciliary ganglion may serve as an additional site of latency for
FHV-l and could explain the preponderance of ocular lesions noted during

recrudescent infections. However additional studies to prove this hypothesis are

85

required. The copy number within latent trigeminal ganglia was much higher than
those previously reported due to the increased sensitivity of the assay utilized. The
copy numbers reported are similar to those found for HSV-1 in murine models of
infection. However, despite an initial lymphoplasmacytic inflammatory response and
persistence of the virus, the ganglia appeared normal by day 30 post-inoculation. This
lack of inflammation is in direct contrast with the current theory that lymphocytes are
a key factor in regulation of the latent state. The theory that lacrimal adenitis
contributes to the development of KCS was not supported as there was no evidence of

FHV-l infection or secondary inflammation within the glandular tissues at any time.

86

APPENDICES

87

5. APPENDIX A

 

USDA Scoring Method for FHV-la

 

 

 

 

 

 

 

 

 

 

 

 

Clinical signs 1 Days I Score
Fever
103.0 to 103.9 °F 1 each day
104.0 to 104.9 °F 2 each day
2105 °F 3 each day
Conjunctivitis
Serous discharge only 1 to 3 l
a 2
Mucopurulent discharge 1 to 2 2
3 to 5 4
26 6
Rhinitis
Serous discharge only 1 to 3 l
a 2
Mucopurulent discharge 1 to 2 2
3 to 5 4
26 6
Sneezing 1 each day
Dyspnea
Audible rales 2 each day
Coughing 2 each day
Open mouth breathing 3 each day
Depression
Anorexia 1 each day
Dehydration l to 2 3
2 4
Hypothermia <99 °F 2 each day
Oral ulcers (linguinal or oral mucosa)
1 ulcer <4 mm 1 to 5 2
6 to 9 3
210 4
Multiple ulcers <4 mm 1 to 4 3
5 to 8 5
2 7
Ulcer or ulcers >4 mm 1 to 4 5
5 to 8 7
3 9
Salivating 1 each day
Extenal ulcers (lip or nares)
Nonbleeding ulcer 4
Bleeding ulcer 6
Death 15

 

a US. Department of Agriculture, Animal and Plant Health Inspection Service,

National Animal Veterinary Services Laboratory (1985).
Table 5.1. USDA Scoring Method for FHV-l

88

 

6. APPENDIX B

sneezing

ca'123456789

15
14
13
3
11
10

(O

.s
.rrorurburmuoo

 

Figure 6.1. Results of the clinical examination: sneezing.

elevated body temperature

C31

15
14
13
3
11
10

123456789

    

(D

a
ANNAO‘IO’VQ

Figure 6.2. Results of the clinical examination: elevated body temperature.

89

dehydration

can123456789

    

15
14
13
3
11
1o

(.0

.a
ANN-“mafia

Figure 6.3. Results of the clinical examination: dehydration.

anorexia
cat

15
14
13
3
11
10
9

123456789

.3
ANNAU’IO’NQ

 

Figure 6.4. Results of the clinical examination: anorexia.

90

ptyalism

cat123456789

15
14
13
3
11
10

ID

.5
ANNAUIQVQ

 

Figure 6.5. Results of the clinical examination: ptyalism.

oral ulceration
cat

15
14
13
3
11
10

123456789

(D

A
ANNAU‘IU’VQ

 

Figure 6.6. Results of the clinical examination: oral ulceration.

91

nasal discharge
cat

15
14
13
3
11
10
9

123456789

—I
gumbmmwm

 

Figure 6.7. Results of the clinical examination: nasal discharge.

blepharospasm
W 1 2 3 4 5 6 7 8 9
15
14
13
3
11
1o

(.0

_I
ANN‘U‘O’NO’

 

Figure 6.8. Results of the clinical examination: blepharospasm.

92

ocular discharge

cat1234567 9
15
14
13
3
11
1o
9

  
  

d
ANN‘U’IU’NO

Figure 6.9. Results of the clinical examination: ocular discharge.

conjunctivitis

cat123456789
15
14
13
3
11
1o
9

.n
ANNAUIOINQ

Figure 6.10. Results of the clinical examination: conjunctivitis.

93

7. APPENDIX C

DAY 6

 

Sample ControlS Cat4 Cat 1 Cat2 Cat 12

 

— + +

Trigeminal Ganglia
Ciliary Ganglia —
Lacrimal Gland —

Cornea —

++++

Conjunctiva —

Nictitating Gland — +

+++++
++++

++++

 

Table 7.1. Virus isolation day 6 post inoculation

DAY 10

 

Sample Control 10 Cat6 Cat7 Cat8 Cat9

 

Trigeminal Ganglia — + + _ _
Ciliary Ganglia —
Lacrimal Gland —

Cornea —

++++

Conjunctiva —

Nictitating Gland —

+ + + + +
++++
++++

+

 

Table 7.2. Virus isolation day 10 post inoculation

DAY 30

 

Sample Control 15 Cat 3 Cat 11 Cat 13 Cat 14

 

Trigeminal Ganglia — — _ _ _
Ciliary Ganglia — — _ _ _
Lacrimal Gland — — _ ._ _
Cornea - _ + _ _
Conjunctiva — _ _ _ _

Nictitating Gland — — _ .. _

 

Table 7.3. Virus isolation day 30 post inoculation

94

8. APPENDIX D

 

 

 

 

 

Cat # Repeat#1 Repeat#2 AveragL Std Dev
ML

1 172,000 224,000 198,000 36,770
2 1,370,000 1,510,000 1,440,000 98,995
4 56,500 86,900 71,700 21,496
12 269,000 384,000 326,500 81,317
5 O O 0 0
DAY 10 P1.

6 226,000 298,000 262,000 50,912
7 73,400 74,500 73,950 778

8 177,000 223,000 200,000 32,527
9 189,000 247,000 218,000 41,012
10 O 0 0 0
DAY 30 Pl.

3 O 0 0 0

1 1 22,600 28,000 25,300 3,818
13 0 O 0 0

14 0 0 0 0

15 O O O 0

 

 

 

Table 8.]. PCR results of cornea (Copy no. / 100 ng DNA)

95

 

 

 

 

 

Cat # Repeat#1 Repeat#2 Average Std Dev
DAY 6 P1.

1 125,000 174,000 149,500 34,648
2 610,000 648,000 629,000 26,870
4 434,000 317,000 375,500 82,731
12 2,510,000 2,410,000 2,460,000 70,71 1
5 0 O O 0
DAY 10 Pl.

6 4,200 5,510 4,855 926

7 619,000 493,000 556,000 89,095
8 16,500 19,600 18,050 2,192
9 27,500 29,500 28,500 1,414
10 O O O 0
DAY 30 P1.

3 0 O 0 O

11 105 122 114 12

13 O O O

14 5 14 9 6

15 0 O 0 O

 

 

Table 8.2. PCR results of conjunctiva (Copy no. / 100 ng DNA)

96

 

 

 

 

 

 

 

 

 

 

 

Table 8.3. PCR results of lacrimal gland (Copy no. / 100 ng DNA)
Marked fields: contamination of sample, assay not repeated.

97

 

 

 

 

 

 

 

 

 

 

Table 8.4. PCR results of nictitans gland (Copy no. / 100 ng DNA)
Marked fields: contamination of sample, assay not repeated.

98

 

 

 

 

Cat # Repeat#1 Remain Average Std Dev
DAY 6 P1.

1 16,500 16,200 16,350 212

2 12,200 13,000 12,600 566

4 14,100 16,700 15,400 1,838
12 10,800 10,600 10,700 141

5 0 0 O 0
DAY 10 P1.

6 17,500 31,800 24,650 10,1 12
7 57,000 37,700 47,350 13,647
8 13,400 13,200 13,300 141

9 50,400 52,400 51,400 1,414
10 0 0 0 0
DAY 30 P1.

3 15,900 34,200 25,050 12,940
11 3,110 3,130 3,120 14

13 5,370 5,790 5,580 297

14 13,500 16,100 14,800 1,838
15 0 0 0 O

 

 

 

Table 8.5. PCR results of trigeminal ganglia (Copy no. / 100 ng DNA)

99

 

 

 

 

 

 

Cat # Repeat#l Repeat#2 Average Std Dev

DAY 6 P1.

1 239 192 216 33
2.440 1.630 2.035 573

4 5.660 3.230 4.445 1.718

12 149 65 107 59

5 0 0 0 0

DAY 10 P1.

6 5.130 4.560 4.845 403

7 26.000 47.600 36.800 15.274

8 685 512 599 122

9 410 364 387 33

10 O O 0 0

DAY 30 P1.

3 0 0 0 0

11 100 115 108 11

13 O 0

l4 0 O

15 0 0 0 0

 

 

Table 8.6. PCR results of ciliary ganglia (Copy no. / 20 ng DNA)

100

 

10.

11.

12.

13.

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