AN ABSTRACT OF THE THESIS OF
IRWANDI YUSUF
in
for the degree of
VETERINARY MEDICINE
MASTER OF SCIENCE
presented on
March 15, 1993
Title: ANTIGENIC COMPARISON OF BOVINE, OVINE, EQUINE, AND
LLAMA ADENOVIRUS ES
Abstract approved:
Redacted for Privacy
Donald E. Mattson, D.V.M., Ph.D.
Fifteen adenoviruses from cattle, sheep, horses, and llamas were studied by
virus neutralization to determine their degree of antigenic similarity. Prototype vi-
ruses included bovine adenoviruses species 1-8, ovine adenoviruses species 5 and
6, and equine adenovirus species 1. Unclassified viruses that were compared to
the prototype viruses were isolated from different locations within Oregon and
were represented by bovine isolate 32CN, ovine isolates 47F and 475N, and llama
isolate 7649. Reciprocal virus neutralization tests were performed and the degree
of antigenic similarity, i.e., species differentiation was determined by criteria established by the International Committee for the Nomenclature of Viruses.
The study showed that many of the adenoviruses, both prototype and unclas-
sified, shared minor antigenic components with each other. Prototype viruses pos-
sessed major antigenic differences and, as previously demonstrated by other
investigators, should be classified as separate virus species. Bovine adenovirus
isolate 32CN was shown to be of the same species as ovine adenovirus isolate
475N, but neither isolate was similar to any of the prototype virus species studied.
Ovine adenovirus isolate 47F was shown to be of the same species as ovine adeno-
virus species 5 strain RTS 42. Llama adenovirus isolate 7649, while sharing minor
antigens with different viruses from cattle and sheep, was shown to be a dist'-^*
species. This represents the first species of adenovirus from llama.
ANTIGENIC COMPARISON OF BOVINE, OVINE,
EQUINE, AND LLAMA ADENOVIRUSES
by
Irwandi Yusuf
A THESIS
submitted to
Oregon State University
in partial fulfillment of the
requirements for the
degree of
Master of Science
Completed March 15, 1993
Commencement
June 1993
APPROVED:
Redacted for Privacy
Associate Professor of College of Veterinary Medicine in
Charge of Major
Redacted for Privacy
of College of Veteriry Medicine
Redacted for Privacy
Dean of Gfalduate Sch
Date thesis is presented:
Typed by Irwandi Yusuf for:
March 15, 1993
Irwandi Yusuf
Acknowledgments
With deepest and sincere gratitude, I would like to thankDr. Loren D. Roller,
dean of College of Veterinary Medicine Oregon State University for the
arrangements and assistance as well as personal encouragement he provided to
enable me to pursue MS' degree in Veterinary Science.
I wish to thankmy major professor, Dr. Donald E. Mattson, for his guidance,
encouragement, critical review and helpful- suggestions during my course of study,
research and thesis preparation. Se has a great share in enabling me to
understand advanced Immunology much deeper.
I am deeply grateful to 79cky 9. Baker of OS'U Veterinary Diagnostic
Laboratory for his continuous assistance during the research and thesis
completion. I received a great deal of laboratory skiffs from this comrade.
I also would like to thankAffison Bates of College of Veterinary Medicine
Library for her ad libitum assistance in retrieving articles for this thesis.
With deepest gratitude I thankmy mother and father, my wife and son for their
prayers, support, patience, and constant encouragement.
This study program was sponsored by Syiah Ruda 'University Foundation,
Indonesia and OS'U College of Veterinary Medicine, USA.
Ticapan Terimakosik
Terimakasih yang takterhingga dan penghargcran yang taus saya sampaikan
kepada Dr. Loren D. Koller, dekan College of Veterinary Medicine Oregon State
'University atas segala bantuan morif dan materi sehingga saya berhasil
menyelesaikan studi saya.
Demikian juga, kepada major professor saya, Dr. Donald E. Mattson, saya
sampaikan rasa terimakasih dan penghawaan yang setinggi-tingginya atas
bimbingan dan saran-saran beliau sewaktu saya menyelesaikan studi. Andif
beliau sangat besar bagi saya dalam memahami Immunology secara febih
mendafam.
Sayajuga merasa sangat berhutang du& kepada 79cky 9. Baker atas latihan,
petunjukdan bantuannya selama saya melakukan riset pada OS'U Veterinary
Diagnostic Laboratory. Berkat bantuan kamerad inifah saya dapat menguasai
beberapa tehnikceff culture dan diagnosa kevirusan di faboratorium.
I(epada Affison Bates, ahli kepustakaan pada OS'U College of Veterinary
Medicine, saya ucapkan terimakasih banyakatas akses ad libitum yang criberikan
kepada saya edam mengumpulkan bahan-bahan untuk thesis ini.
Terimakasih dan penghargaan yang ikhlas saya alamatkan kepada kedua orang
tua saya serta isteri dan anaksaya atas doa yang mereka panjatkan serta atas
ketabahan, dorongan Ian keikhlasan mereka.
Program studi ini disponsori ofeh Nyasan Universitas Syiah Ruda' dan College
of Veterinary Medicine Oregon State 'University, USA."
TABLE OF CONTENTS
CHAPTER
I.
INTRODUCTION
II. LITERATURE REVIEW
PAGE
1
3
Adenoviral Classification
3
Bovine Adenoviruses
6
Ovine Adenoviruses
8
Equine Adenovirus
15
Llama Adenovirus
15
III. MATERIALS AND METHODS
16
Media
16
Cell Cultures
16
Virus Source
17
Antiserum Production
18
Virus Neutralization Test
19
IV. RESULT
20
Bovine Adenoviruses
21
Bovine Adenovirus Isolate 32CN and Ovine Adenoviruses
22
Equine Adenovirus (EAV) Species 1
22
Llama Adenovirus Isolate 7649
23
V. DISCUSSION
24
VI. BIBLIOGRAPHY
27
LIST OF TABLES
TABLE
1.
List of cell cultures used for replicating bovine, ovine, llama, and
equine adenoviruses
2. Reciprocal virus neutralization of the bovine, ovine, llama, and
equine adenoviruses
PAGE
17
20
ANTIGENIC COMPARISON OF BOVINE, OVINE,
EQUINE, AND LLAMA ADENOVIRUSES
I. INTRODUCTION
The study of adenoviruses has drawn much attention since the first human
adenovirus was isolated in 1953.1" In human, these agents produce a wide variety
of disease syndromes associated with infection of the upper and lower respiratory
tract,
52 gastro-intestinal tract,"' "' and ocular tissue.' Adenoviral infections in
many animal species, as well as in humans, have generally been associated with a
variety of disease symptoms including: pharyngitis, bronchitis, bronchiolitis, and
conjunctivitis, keratoconjunctivitis, pneumonia, and pneumoenteritis.
Naturally occurring bovine adenovirus (BAV) infection usually causes a
mild upper respiratory disease or conjunctivitis. Bovine adenovirus species 3 can
cause a mild pneumoenteritis in calves.' Bovine adenovirus species 2 has been
shown to cause in utero infection in sheep.' Bovine adenoviruses have also been
isolated from calves with hemorrhagic gastroenteritis.' Canine adenoviruses
cause hepatitis and laryngotracheitis.7 Equine adenovirus (EAV) has been asso-
ciated with pneumonia in foals' and with fever in racehorses.'" Ovine adenoviruses (OAV) and caprine adenoviruses cause a wide variety of signs associated
with the respiratory and alimentary tract infection.' Finally, llama, as a newly
studied animal species appears to have its own adenovirus (LAV) that causes respiratory and alimentary tracts disorders. To date, only one llama adenovirus has
been discovered and the virus is untyped.76
The purpose of the current study was to characterize the antigenic profile of
four adenoviruses by the virus neutralization test. All four viruses were recovered
2
in Oregon: one adenovirus was isolated from the nasal cavity of a calf, two ovine
adenoviruses were isolated from lambs and one adenovirus was isolated from a
young llama (cria). Another purpose of this investigation was to confirm the antigenic profiles of available adenoviruses. The outcome of this study is expected to
facilitate the diagnosis of animal adenoviruses, as well as to provide direction for
selection of a vaccine candidate strain should it be considered necessary and feasi-
ble. Due to some limitations, this study did not involve swine adenoviruses, caprine adenoviruses and ovine adenoviruses species 1-4.
3
II. LITERATURE REVIEW
Adenoviral Classification
Rowe and coworkers first isolated a cytopathogenic agent from human ade-
noids and proposed the term Adenoid Degeneration agent in 1953.' Hilleman
and Werner isolated these viruses from patients with acute respiratory illness and
used the term Respiratory Illness agent.' Huebner and colleagues in 1954 proposed the term Adeno Pharyngeal Conjunctival agents as the group name."
In
1956 Enders proposed adenoviruses as the group name for these viruses." Lastly,
the International Committee on the Taxonomy of Viruses, a virology section of In-
ternational Association of Microbiological Societies, established the formerly genus adenovirus into the family Adenoviridae. This family is represented by two
genera which consists of Mastadenovirus (adenovirus of mammals) and Aviadenovirus (adenovirus of avians).38
The characteristics of adenovirus have been defined by Ginsberg,' Huebner
et al.," and Willner.1" Adenoviruses from all species are nonenveloped, icosahe-
dral, 700-800 A in diameter, of a density of 1.34 g/ml in Cs Cl, and of a particle
weight of about 175 x 106 daltons. All adenovirus contain linear double-stranded
deoxyribonucleic acid (DNA) of 29 x 106 daltons molecular weight.' The icosa-
hedral protein shell (capsid) has a 5:3:2 cubic symmetry pattern and consists of
240 hexons (each bound to six neighbors) and, at the apices, 12 pentons (each
bound to 5 neighbors) which carry fibers; a fiber on each penton.46 The hexons are
polygonal prisms with a central hole.' The pentons, which are more complex,
consist of similar polygonal base with a fiber attached. The fibers vary in length
(100 - 310 A) and are 20 A in diameter. The fibers have a terminal knob measuring 40 A in diameter."
4
Adenoviruses are resistant to the action of lipid solvents, such as ether,"
chloroform," fluorocarbons,' and deoxycholate53 indicating that the viruses lack
essential lipid. Adenoviruses are acid stable" and, in fact, are more stable at acid
than alkaline pH.' They are also relatively stable in homogenates of infected
cells."
Adenoviruses replicate in the epithelium or epithelial-like cells of cell
cultures.44Adsorption of these viruses to susceptible host cells is relatively slow.
With species 2 virus of human origin, approximately 70% of the viruses are adsorbed in six hours at 36 C. It is assumed that 12 hours are needed for maximum
adsorption."
Nucleic acid replication occurs in the nucleus. Protein is synthesized in cy-
toplasm, it then migrates to the nucleus where the virus is assembled." The assembly of viral subunits is very inefficient with only 10-15% of the viral DNA and
protein incorporated into viral particles. The remaining nucleic acid and protein
become parts of the intranuclear inclusions.' Both types A and B intranuclear in-
clusions are formed by various species of mammalian and avian adenoviruses.'''
The cytopathic effect of adenoviruses in susceptible cells in vitro is characterized
by rounding and clumping of the cells52'1" and detachment from the surface.85, 98, 116
These viruses also invoke an enhanced utilization of glucose in the cell cultures
leading to an increase in organic acids. Therefore, adenovirus-infected cell cultures become more acid than noninfected
55
Adenoviruses contain several antigens. By using a variety of serological
techniques, group, species, and subgroup-specific determinants can be observed
from the hexon polypeptide. Both group and subgroup-specific determinants can
be detected from the penton base protein, while subgroup and species specific de-
terminants can be found from the fiber.'" Formerly, the hexons, pentons, and
the fibers were called antigens A, B, and C, respectively.' The hexon antigen is
5
usually distinguished by complement fixation"' 101 or gel diffusion test,' and the
presence of group-specific antigen is a required criterion for inclusion of viruses
into the adenovirus group." Exceptions to this rule have been shown for avian
adenovirus by Ginsberg,' and subgroup 2 bovine adenovirus by Archetti and
Harsfall Jr.' The pentons particles have been confirmed to be identical with celldetaching factor,' early cytopathic factor,' and toxin-like materia1.36' 63 Human
adenoviruses species 1, 3, 7, 8, 12, 14, 16, 18, 21, 24, 31,113 simian adenoviruses
species SV7, SV20, SV33, SV34, SV37, SV38,56 avian adenovirus (CEL0)," in-
fectious canine hepatitis virus,' and bovine adenovirus species 3 strain WBR 123
are oncogenic in newborn experimentally-infected hamsters. Human adenovirus
species 12 is also oncogenic for suckling mice, mastomys,94 and rats. 54
There are now 41 distinct antigenic species of human adenoviruses.'" Infection has been associated with a wide variety of disease syndromes, including
conjunctivitis, pharyngitis, keratitis, bronchitis, bronchiolitis with pneumonia, cystitis and enteritis."' 86' 92 Infection is usually mild but is much more severe in chil-
dren and military recruits." Likewise, different adenovirus species vary in their
virulence with some strains more consistently inducing severe disease.''''
Adenoviruses are ubiquitous"' and the number of species isolated and char-
acterized is increasing with the passage of time. Animal for which adenoviruses
are described includes cattle, horses, sheep, goat, pigs, birds, and mice. In recent years researchers have also isolated adenovirus from buffaloes,' reptiles," and
llamas.' For the time being there are 41 human adenovirus species, 9 bovine spe-
cies, 6 ovine species, 2 caprine species, 4 porcine, 1 equine, 1 murine, 14 avian
species, and many other isolated but they have not yet been characterized.
Adenovirus infections are primarily host specific; however, cross species infections are not uncommon among closely related, e.g., sheep and cattle."
6
Bovine Adenoviruses
Bovine adenoviruses (BAV) were first isolated in the United States. Klein
et al. recovered two isolates (strain 10 and 19) from the feces of apparently
healthy cattle. These isolates have been designated as species 1 and species 2,
respectively. 61-62
Ide and his coworkers, isolated BAV species 1 from a calf with pneumonia
and enteritis in Canada. Calves inoculated intratracheally or intravenously with
this strain showed no clinical response, although an immune response did
develop.' BAV species 1 was also isolated from a calf with pneumoenteritis in
Norway.104
Darbyshire and coworkers isolated BAV species 3 (strain WBR 1) from the
conjunctiva of an apparently healthy cow in England.' When it was inoculated in
newborn calves, the virus was shown to invoke a mild clinical response character-
ized by pyrexia, respiratory distress, nasal and conjunctival discharges. Although
diarrhea was not a prominent sign of the disease, the virus was isolated from both
the respiratory and alimentary tracts.'
In calves inoculated with BAV species 1 (strain 10) and species 2 (strain
19), a mild respiratory disease was produced.
Gross and microscopic tissue
changes were generally similar, but less severe than those observed with BAV spe-
cies 3 (strain WBR 1) infection.' This finding suggested that species 3 is the most
virulent of the first three species of bovine adenoviruses.
Researchers in Hungary isolated BAV species 4 (strain THT/62) and BAV
species 5 (strain BA/65) from the nasal cavity, various organs, and intestinal contents of calves with pneumoenteritis.' 12 Btirki and coworkers also revealed that
BAV species 4 and 5 were associated with enzootic pneumoenteritis in Austria.'
In Japan, Tanaka and other investigators isolated a BAV (strain Nagano) from the
7
blood of a 25-month-old bull with pyrexia, anorexia, and soft feces!" Further studies showed that strain Nagano was serologically related to BAV species 4 (strain
THT/62).77
There were further reports on the discovery of adenoviruses. Rondhuis reported the isolation of BAV species 6 (strain 671130) which was recovered as a
latent virus from bovine testicular cell cultures.' Wilcox recovered two adenoviruses (strain KC1 and KC2) from cattle with conjunctivitis and keratoconjunctivitis in Australia.120
Inaba and coworkers isolated strain Fukuroi from a
three-year-old cow with pyrexia, anorexia, diarrhea, and respiratory disease in
Japan." Strain Fukuroi was later designated as species 7.78 Bartha et al. reported
the isolation of BAV species 8 (strain MISK/67) from calves with pneumoenteritis
in Hungary. This was shown to be partially related antigenically to BAV species
4, 5, and 6.13 Guenov and coworkers isolated BAV species 9 (strain Sofia 4/67)
from the kidney of an apparently healthy calf in Bulgaria." Mattson reported the
isolation of strain 5C from Oregon calves with pneumoenteritis. This strain was
serologically related to BAV species 3 (strain WBR 1).74 Alieva et al. recovered
an untyped adenoviruses from calves with pneumonia in Russia.' Lupini et al.
isolated an untyped adenoviruses from calves with respiratory symptoms: cough-
ing, dyspnea, and fever in Italy.'
On the basis of certain physical, chemical, and biological properties, Bartha
suggested dividing bovine adenoviruses into two subgroups. The strains in subgroup 1 (species 1, 2, 3) possess the soluble antigen common with human adenovi-
ruses, are inactivated at 56 °C for 30 minutes, replicate well in bovine kidney cell
cultures, form a single intranuclear inclusion which is irregular in shape, and are
readily isolated in the first passage of cell culture. The strains designated as mem-
bers of subgroup 2 (species 4, 5, 6, 7, strain BIL) have only traces of the soluble
antigen common with human adenoviruses, are partially inactivated at 56 °C for 30
8
minutes, replicate in bovine testicular cell cultures, form multiple intranuclear in-
clusions which are regular in shape, and are isolated only after a series of blind
passages in cell culture.' It was later shown, however, that heat inactivation dif-
ferences between the two subgroups appear to be variable and it was suggested
that this criterion be deleted for differentiation."' 74 In additional to what has been
proposed by Bartha, later, BAV species 9 and BAV species 8 were added into sub-
group 1 and subgroup 2, respectively.'
Ovine Adenoviruses
Currently, six antigenic species of ovine adenoviruses and two species of ca-
prine adenoviruses have been described.' These numbers will unquestionably increase as research continues.
Ovine adenovirus (OAV) have been isolated from numerous countries, the
percentage of adult animals possessing antibodies to these agents, indicating pre-
vious infection, varies from 60 to near 100 percent. Ovine adenoviruses are frequently isolated from apparently clinically normal lambs as well as from lambs
with a history of enteric and respiratory disease. Likewise, these viruses have
been shown to be etiologic agents of lamb pneumonia and diarrhea. Lambs exper-
imentally infected with OAV show signs of pyrexia, anorexia, hyperpnea, dyspnea, conjunctivitis, cough and diarrhea. Like BAV, some species of OAV have
been shown to produce a viremia in the dam resulting in fetal disease.'
It is now apparent that interspecies transmission of BAV, OAV and CAV
can occur. Sheep have been shown on several occasions to be naturally infected
with BAV.86 It also can be said that young animals appear to express signs of dis-
ease more consistently when infected with adenoviruses than do older animals.
Likewise, sheep experimentally infected with OAV followed by bacterial
9
pathogens develop more severe signs of disease and pathologic changes than when
infected with either microorganism individually.'
Darbyshire and Pereira suggested that sheep were naturally infected with
adenoviruses.' But apart from a preliminary report by Mc Ferran et al. in 1969,"
there was no previous report of isolation of sheep adenoviruses.
Successful attempts to isolate adenovirus from sheep were made as early as
1969 by Mc Ferran and coworkers in Ireland. On the basis of morphology and oth-
er physical and serological properties, and the fact that the viruses were isolated
from the feces of diseased sheep, the authors suggested their possible role in pro-
ducing pneumoenteritis in sheep.' Using lamb kidney cultures, Mc Ferran et al.
isolated three serologically distinct OAV from feces of sheep. Two of these three
isolated strains showed cross reactivity to adenovirus group antigens. Their work
suggested that adenoviruses can be recovered from both healthy and diseased animals. Detection of OAV from the feces suggested their involvement in enteric
infections."
Adenovirus group-specific antigens were detected in sheep and goats by
Darbyshire in the United Kingdom. Serum from different animals including sheep
and goats was tested in parallel with a human adenovirus species 5 antigen using
the Agar Gel Immuno Diffusion (AGID) test. Results showed that 1/103 sheep
and 33/50 goats had the precipitating antibodies against adenovirus group
antigen.'
Sharp et al. described a new adenovirus in sheep. They showed that this
new strain, 7769, was quite distinct antigenically from the three serotypes of OAV
previously isolated.
They were able to demonstrate that strain 7769 was not
neutralized by antiserums to BAV as were the three serospecies isolated earlier.
They inoculated the virus in pathogen-free lambs, and it replicated and stimulated
an immunological response without producing any clinical signs. This suggested
10
that strain 7769 was non-pathogenic or causes disease in conjunction with other
agents or factors."'
Bela and Palfi reported adenovirus infection in lambs in fattening facilities
and described the epizootiology of the respiratory syndrome and suggested the dis-
ease contributing factors. Looking for the viral involvement, the workers were
able to isolate an adenovirus, strain Het/3, from the nasal discharge of the affected
lambs.' In effort to confirm that the isolated Het/3 adenovirus strain was the etiological agent of respiratory disease in sheep, Belak et al. later conducted an exper-
imental study, which covered several aspects of the disease. They concluded that
the strain Het/3 is related to BAV species 2.'4
Thurley and colleagues described the causes of mild pneumonia of lambs in
New Zealand. According to their findings, beside bacterial, serum antibody titers
to two adenoviruses were found. Two serological distinct viruses (later confirmed
as OAV) were isolated from the feces and nasal secretions of sheep."2
In a seroprevalence and microbiological study of pneumonia of lambs in
New Zealand, beside describing bacterial isolation and lesions, Pfeffer and
coworkers reported the isolation of adenoviruses which were neutralized by antiserum to a local untyped strain of OAV (WV 757/75).93
New Zealand investigators, Davies and Humphreys, described the character-
ization of two strains of adenovirus which were recovered from sheep, in New
Zealand over a period of 10 months. Samples were collected from nasal secretions, feces and lungs of dead animal and propagated in lambs testicular (LT) cultures. Two distinctly different adenoviruses species were isolated, neither of them
agglutinated mouse, rat, guinea pig
,
sheep, cattle, and human "0" erythrocytes
but one of the isolates agglutinated chicken erythrocytes. Both strains were serologically distinct on the basis of cross neutralization test. However, their relation-
ship to the five established serospecies of ovine adenovirus was not determined."
11
The same authors experimentally infected 3 to 4 months old colostrum-deprived
lambs with an untyped OAV isolate strain (757/75). The virus which was administered intranasally and intratracheally, induced moderate clinical signs of respira-
tory tract disease. Pathological changes were limited to the respiratory tract. The
lambs experienced a viremia as the virus was detected in the blood up to 14 days
after infection?' The WV 757/75, together with PI-3, has also been found to infect
pregnant ewes and both the agent and antibody against it were passed to the fetuses. The newborns were shown to eventually suffer from the infection later in
their life when the maternal antibody abated.'
A group of Australian researchers described the presence of adenovirus in
sheep liver which died due to cycad poisoning.' Cross neutralization tests were
conducted with this isolate, designated as PI1537/82, and with the eight species of
BAV, six species of OAV and four species of porcine adenovirus. This PI1537/82
was only neutralized by antiserum to a New Zealand adenovirus species (WV 757)
and BAV 7 and was not neutralized by other adenovirus antiserums tested. Also,
the antiserum against PI1537/82 virus neutralized the WV 757 and BAV 7. Ade-
novirus group-specific antigen for PI1537/82 isolate was demonstrated by cross
immunofluorescence between PI1537/82 and OAV 4 and also confirmed in a reciprocal fashion, i.e., OAV 4 infected cells were stained by PI1537/82 antiserum.'
In an extensive study, Davies and colleagues observed the relationship and
course of disease when lambs were infected with ovine adenovirus followed by
Pasteurella haemolytica. Clinically, the group which received the virus followed
by the bacteria developed more severe signs of respiratory tract disease while the
groups that received the virus or bacteria alone had minor signs of disease. Simi-
larly, the number of bacteria from the group inoculated with Pasteurella haemolytica alone was low as compared to the group which received both bacteria and
virus. This showed that bacteria alone was eliminated efficiently from lungs by
12
body defense mechanisms. This study suggested that the OAV strain WV 419/75,
must be considered as another potential respiratory pathogen capable of initiating a
bacterial bronchopneumonia.'
Sharp et al. experimented with ovine adenovirus species 4 strain 7769 and
found it to be a primarily enteric virus. However, by mean of aerosol transmission, the virus was shown to replicate first in the respiratory tract followed by the
alimentary tract.'"
Palya and coworkers inoculated ovine adenovirus strain GY/14 and strain
Het/3 in colostrum-deprived 3 weeks-old-lambs. They found no animal died because of infection although the clinical signs of respiratory and alimentary tracts
involvement were evident. They concluded that GY/14 and Het/3 strains of ovine
adenovirus were similar in being able to induce the disease. However, GY/14
strain caused more severe clinical signs of disease."
NM et al. studied OAV strain GY/14 and noted that acute phase of infection was manifested by signs of respiratory tract disease with associated pathologic
changes. In the chronic phase of disease, signs were limited to reduced growth
rate, varying degrees of anorexia and intermittent pyrexia. Pathologic changes
were observed in the lungs and kidneys. Virus isolation was difficult in the chron-
ic phase of the disease as the organ involved e.g. lung explants had to be cultured
with ovine fetal cells. Lambs produced a good antibody response to the virus with
the titre varying from 1:32 to 1:128 on day 17 post inoculation. The authors con-
cluded that some lambs shed virus for prolonged periods of time for factors not
completely understood."
In 1983, two serospecies of adenoviruses were isolated from lambs assembled at a ram testing station in Central America.66 One isolate, RTS 151, was
pathogenic for colostrum-deprived lambs producing clinical signs of respiratory
tract disease and severe pathologic changes." In cross neutralization tests, one of
13
these viruses, RTS 42, was typed as OAV species 5;1." while RTS 151 was unre-
lated to OAV species 1 through 5 or to the BAV species 1-8. However, RTS 151
did possess the adenovirus group-specific antigen as demonstrated by agar gel pre-
cipitation test and further agar gel immunodiffusion test showed that a common
antigen was shared between BAV 3, RTS 42 and RTS 151 isolate.' This virus
was later typed as OAV species 6 strain RTS 151.1
Lehmkuhl et al. conducted a two year long seroepidemiological study on
lambs for the presence of common respiratory viruses of sheep. They reported that
infections due to OAV were substantial. The mean prevalence for both years of
blood collection shows; 95% for OAV 5, 87.2% for PI-3 virus, 84.5% for RSV,
41.7% for OAV 6, 8.7% for BVD virus, 5.4% for BHV-1 and 3.3% for ovine pro-
gressive pneumonia (OPP) virus. Ovine adenovirus species 6 had the highest infection rate on the basis of mean of two year serum analysis i.e., 42.8% (207/484)
as determined by an increase of equal to or greater than 4-fold serum antibody titre
as compared from 1st to 2nd sample. The infection rates for other viruses were;
31.1% for OAV 5 (RTS 151), 15.3% P1-3 virus, 5.6% for RSV, 0.6% for BVD virus and 0.4% for BHV-1.68
In an advanced study, Lehinkuhl and coworkers infected a group of one
week-colostrum-deprived lambs with the strain RTS 42 (OAV 5) and necropsied
them on different days after inoculation until 21 days PI. Virus was present in na-
sal secretions and lung tissues in all lambs killed between days 1-6 PI. However,
they were not able to recover the virus from any other organ including intestine.
Virus neutralization antibodies first appeared on day 6 PI and were high in the se-
rum of lambs killed on day 12 PI but the titers decreased in serums collected on
day 21 PI. Because the virus was not isolated from any other organ except the
lungs, the authors suggested that RTS 42 is a respiratory virus. The presence of
virus in feces can be explained by observation that RTS 42 is acid resistant and
14
may have replicated in the respiratory tract and passed through the digestive
tract.'
Dubey and Sharma reported the presence of OAV 1 in India. The virus was
isolated from the sheep with pneumoenteritis. Nasal and fecal samples were inoculated in embryonic lamb kidney (LK) cells in order to isolate the virus. Indian
sheep (4.05%), goats (7.25%) and cattle (9.15%) showed serum antibody titers
against the adenovirus.' Different cell cultures of sheep and goat origin supported
the growth of the adenovirus but there were some differences in showing CPE and
virus yield. Lung cell cultures showed a later CPE than kidney and testicular cultures, whereas the kidney cell cultures yielded higher titers than did testicular and
lung cell cultures.'
Bel& and Rusvai studied the occurrence of natural in utero adenoviral in-
fection in sheep. They isolated three adenoviral strains from the kidney of 174
sheep fetuses and showed their relationship to BAV 2. Neutralizing antibodies
against BAV 2 were present in 20% (5/25) blood samples of sheep fetuses. This
study proved that transplacental transfer of adenovirus infection is possible
naturally.'
Goyal and colleagues conducted a study for seroprevalence of antibodies to
seven viruses in Minnesota (USA) in healthy ewes. The researchers discovered
that 7.6% of the ewes tested vectored BAV 3."
LeaMaster and coworkers performed an epizootologic study of respiratory
tract diseases and isolated OAV 5 and 6 in recently weaned lambs.
Both sero-
types were found jointly infecting the lambs, which showed signs of respiratory
tract disease. The morbidity rate was 13% whereas the total mortality was 4.1%;
57.6% of which was due to pneumonia. Adenovirus was present in 36% of lung
specimens with pneumonia."
15
Equine Adenovirus
Few articles concerning equine adenoviruses (EAV) have been published.
However, EAV infection is not a major problem in horses. These viruses are generally pathogens of low virulence in normal healthy horses. When clinical disease
occurs, it is usually mild and limited to the upper respiratory system. Only in
immuno-compromised individuals do adenoviruses regularly cause severe clinical
disease. So far, the majority of isolations have proven those viruses to be of a
single serospecies designated equine adenovirus species 1. This adenovirus is re-
portedly present worldwide and is apparently species specific.'
A team of Japanese researchers recovered EAV from racehorses in Japan.
These horses had been showing chronic pyrexia for 6 years. However, the team
was unable to conclude that the clinical sign was due to EAV since there were also
some other viruses found in the same horses."'
Llama Adenovirus
Not much is known about llama adenoviruses, for llama is a newly studied
animal species. The work toward the investigation of viruses in llama has been
started at Oregon State University. Mattson and coworkers' have recently identi-
fied an adenovirus in llama with diarrhea and designated it as LAV 7649. This
adenovirus, however, has not been yet typed. According to Mattson, there are
several other isolates that are now being characterized; one of those was isolated in
Michigan. 76
Mattson et al., 1992: Unpublished Data.
16
III. MATERIALS AND METHODS
Media
Medium used for growth and maintenance of cell cultures and for dilution of
virus and serum was Minimal Essential Medium (MEM) with Ear les salts.' In
addition, MEM used in cell cultures contained 100 units per ml penicillin and 100
tig per ml streptomycin sulfate. Growth medium for cell cultures contained 10
percent fetal bovine serum, while maintenance medium contained 5 percent fetal
bovine serum. Serum used for cell cultures was shown to be free of antibodies to
the viruses being tested. A Tris-EDTA (TE) solution was used to resuspend purified virus and was composed of Tris (0.01M), EDTA (0.001M), and NaC1 (0.1 M)
at pH 7.5.
Cell Cultures
Primary bovine kidney (BK) and bovine spleen (BS) cell cultures were
propagated from a 6-month-old bovine fetus. Llama kidney (LLK) cell cultures
were propagated from a 4-month-old llama. Primary cell cultures were prepared
by the trypsin digestion method. Bovine turbinate (BT) cell and ovine fetal corneal cells (OFC) were received from the National Animal Disease Service Labora-
tory.'
Stock cultures of primary cells and cell lines were transferred with a
Hepes-EDTA-trypsin solution pH 7.5 (0.35 gram percent Hepes, 0.072 gram percent glucose, 0.022 gram percent KC1, 0.759 gram percent NaC1, 0.14 gram per-
cent sodium phosphate, 0.02 gram percent EDTA, and 100 mg percent trypsin).
Transferred cells were used to produce either additional stock cultures or cultures
in microtiter plates.
b
Gibco BRL, Life Technologies Inc., Grand Island, N.Y. Catalogue No. 410-1600 EL.
National Veterinary Service Laboratories. PO Box 844, Ames, Iowa 50010.
17
Virus Source
Bovine adenovirus (BAV) species 1-2 and 4-8 were purchased.d Ovine ade-
novirus species 5, strain RTS 42 (OAV 5) and species 6, strain RTS 151 (OAV 6)
were received from Dr. Howard Lehmkuhl.e Ovine adenovirus strain OAV 47F
and OAV 475N were isolated in Oregon from lambs."' Equine adenovirus species
1 (EAV 1) was received from the National Animal Disease Laboratory. Bovine
adenovirus species 3 strain 5C was isolated in Oregon from the conjunctiva of a
calf with conjunctivitis and dian-hea.'
Bovine adenovirus strain 32CN (BAV
32CN) was isolated from a 4-month-old calf with conjunctivitis! Llama adenovirus strain 7649 (LAV 7649) was isolated from the feces of a 2-week-old cria with
diarrhea.g
Stock virus was allowed to replicate in the following cell cultures
(Table 1):
Table 1. List of cell cultures used for replicating bovine, ovine, llama, and
equine adenoviruses.
Type of cell cultures
Adenovirus species
Bovine kidney (BK) cell culture
BAV 1 and BAV 2
Bovine turbinate (BT) cell culture
BAV 3 and BAV 32CN
Bovine spleen (BS) cell culture
BAV 4, BAV 5, BAV 6, BAV 7, BAV 8,
Ovine fetal corneal (OFC) cell culture
OAV 5, OAV 6, OAV 47F, and OAV
Llama kidney (LLK) cell culture
LAV 7649
Virus (10 ml suspension) was inoculated upon cell culture monolayers in
490 cm2 roller bottles. Virus was adsorbed for 4 hours at 37 C after which the
maintenance medium was added. Cultures were incubated at 37 C for 24 hours
d
American Type Culture Collection, 12301 Park lawn Dr. Rockville, MD 20850.
Howard Lehmkuhl, DVM, PhD., National Animal Disease Laboratory, Ames Iowa 50010
f
Donald Mattson, DVM, PhD., Personal Communication. College of Veterinary Medicine
Oregon State University, Corvallis, OR 97331.
Idem footnote f.
18
after cytopathic effect (CPE) involved nearly 100 percent of cells (usually 7 to 12
days). Cells were scraped from the flask, subjected to ultrasonic disruption for 45
seconds and clarified by centrifugation at 3000 times gravity for 20 minutes. The
supernatant fluid was placed in vials and frozen at minus 30 C until used. Virus
was titered in 10-fold dilution steps in 96- well flat bottom microtiter plates.h Titer
of the virus was calculated by the Reed-Muench formula.'
Antiserum Production
Virus replication for production of antiserum in rabbits was similar to that
which has been described. When culture flasks were ready to harvest, the cells
were scraped from the flask. The infected cells were centrifuged 3000 times grav-
ity for 20 minutes. The cell pellet was washed once in TE buffer and recentrifu-
gated. The pellet was resuspended in one tenth of the original volume in TE
buffer. The cells were subjected to ultrasonic disruption for 1 minute at 4 C. An
equal volume of trichlorotrifluoroethane (TCTFE) was added to the preparation
and the product was mixed in a blender for 2 minutes. The mixture was centrifuged 2000 times gravity for 15 minutes, and the supernatant fluid removed. Ten
milliliters of TE solution was added to the interphase and the product was blended
and centrifuged again, the two supernatant fluid layers were combined. Twenty
five milliliter of virus suspension was layered on a discontinous cesium chloride
gradient bilayers (top layer, 6 ml with a density of 1.2 g per ml and bottom layer, 8
ml with a density of 1.4 g per ml). The preparation was centrifuged 83,000 times
gravity for 90 minutes at 4 C in a SW 28 rotor. The opaque bands at the 1.4 g per
ml interface from all tubes were removed and pooled. The bands were mixed in
1.33 g per ml cesium chloride in TE solution to a total volume of 38 ml. The
preparation was centrifuged 83,000 times gravity for 36 hours at 4 C. The opaque
band was removed by side puncture, confirmed to be 1.3-1.34 g per ml density,
h
Catalogue No. 25861, Corning Glass Works. Corning, New York 14831.
19
and dialyzed against physiological buffered saline (pH 7.5) for 24 hours. The final
purification product was examined by negative staining in an electron microscope
to assure the presence of virus. The preparation was divided into 4 aliquots. Aliquot one was mixed with an equal amount of Freund's Complete Adjuvant and in-
jected subcutaneously (4 sites) and intramuscularly (1 site) in a New Zealand
white rabbit. Each different adenovirus prototype or untyped virus was injected
into different rabbits. Aliquots 2 - 4 were frozen until they were used at which
time they were thawed and mixed with an equal volume of Freund's Incomplete
Adjuvant. One aliquot each was injected subcutaneously (4 sites) on days 7, 14,
and 21. The rabbit was exsanguinated by cardiac puncture on day 35. Serum was
separated and heat-inactivated at 56 C for 30 minutes after which aliquots (3 ml
each) were stored at minus 30 C.
Virus Neutralization Test
Antigenic distinctiveness among the different adenoviruses was determined
by the virus neutralization (VN) test. Antiserums to the viruses were thawed and
diluted in 2-fold steps in 50 ill MEM in flat bottom microliter plates.' Each dilu-
tion series was performed in triplicate beginning with a 1:8 dilution of serum.
One hundred medium tissue culture infectious doses of virus in 50 ttl of MEM was
added to each well containing the diluted serum. Initial serum dilution was now
1:16. Plates were incubated at 37 C for 60 minutes after which 50 µl of MEM
containing 5 x 105 cells was added to each well. Finally, one drop of sterile mineral oil was added to each well to prevent drying. The plates were placed in a CO2
incubator (2.5% CO2 concentration) and incubated at 37 C. Cultures were examined for CPE after 7 to 8 days. Serum end point were determined by the Reed-
Muench formula.'
20
IV. RESULT
Virus neutralization tests indicated some cross-neutralizations occurred in
both intra-species and inter-species of animals. A complete summary of crossneutralizations is presented (Table 2).
Table 2. Reciprocal virus neutralization of the bovine, ovine, llama, and equine
adenoviruses.
Virus
BAV 1
BAV 2
Antiserum against
BAV BAV BAV BAV BAV BAV BAV BAV 32CN OAV OAV OAV OAV LAV EAV I
47F
6,183 600
32
353
27
118,696
BAV 3
7649
3,049
1,557
69
3,098
BAV 4
508
BAV 5
58
2,400
BAV 6
75
32
6,080
BAV 7
6,770
BAV 8
22
75
2,874
32CN
787
OAV 6
7,069
42,109
19,100
44,700
38
22
EAV 1
6,770
17,716
1,951
76
19,100
508
193,065
OAV 5
OAV
47F
OAV
475N
LAV
7649
475N
1,304
32
87
24
625
6,432
87
22
7,461
48
172,674
65,015
The numbers indicate reciprocal of the serum dilution. Titer that is equal or
higher than 1:22 is regarded positive. Highlighted cells are homologous tests.
Blank cells are negative, i.e., titer < 1:22.
21
Bovine Adenoviruses
BAV 1, which belongs to the subgroup 1, was neutralized not only by its
homologous antiserum (1:6,183) and BAV 2 (1:600), but also by OAV 6 (1:1,304)
and by the antiserum to the bovine adenovirus isolate 32CN (1:353). However,
the neutralizations of BAV 1 and BAV 2 viruses by both of the OAV 6 and 32CN
antiserums were one way. On the other hand, antiserum to BAV 1 one-sidedly
neutralized the OAV 47F virus, while its neutralization to BAV 2 virus (1:32) was
considerably weaker than the reciprocal (1:600).
BAV 2, a member of subgroup 1, was neutralized by its homologous antise-
rum (1:118,696) and by BAV 1 antiserum (1:32). This virus was also strongly
neutralized by OAV 5 antiserum (1:3,049) and OAV 47F antiserum (1:1,557).
The pattern of the neutralization by these antiserums was two-sided. The BAV 2
was also neutralized by 32CN and llama adenovirus (LAV) antiserums, 1:27 and
1:69, respectively. Nevertheless, these neutralizations were weak and only one
way.
Another member of BAV subgroup 1, BAV 3, was found not to cross-react
with the other viruses. This virus was only neutralized by its own antiserum
(1:3,096).
Bovine adenovirus species 4 and BAV 8, members of subgroup 2, had a
1:508 and 1:2874 end point by their own homologous antiserums, respectively.
These viruses were also found to have a two-way cross-neutralization to each oth-
er. The BAV 4 was neutralized by BAV 8 antiserum (1:58). Likewise, the antise-
rum to BAV 4 neutralized the BAV 8 (1:22). The BAV 8 also two-sidedly
interacted with BAV 5 and this antigenic interaction was rather weak. The antigenic relationship was 1:75 for both ways.
22
The BAV 5 was neutralized by its own homologous antiserum with a titer of
1:2400.
Besides being neutralized by BAV 8 antiserum, it was also partially
neutralized by OAV 475N. However, this neutralization was one-sided and the
titration end point for this reaction was 1:32.
Results of this study showed that BAV 6 and BAV 7 were merely solitaires.
They did not cross-react with any of the other viruses. The homologous antiserum
titrations for these viruses were 1:6080 and 1:6770, respectively.
Bovine Adenovirus Isolate 32CN and Ovine Adenoviruses
The 32CN virus was found to be neutralized by many antiserums. However,
this virus appeared to have a very close antigenic relationship to the OAV 475N.
Antiserum to the OAV 475N neutralized the 32CN at 1:19100 and, reversely, the
antiserum to the 32CN neutralized the OAV 475N at 1:44700. There was also a
moderately close two way-cross-neutralization between the 32CN and the OAV 6
at 1:508 and reversely at 1:951 end points. Furthermore, antiserum to OAV 475N
neutralized OAV 6 at 1:625 in a one-way fashion. The 32CN virus had a two-way
cross-reaction with LAV 7649, while the OAV 475N virus was one-sidedly
neutralized by LAV 7649 antiserums. But the magnitude of those cross-reactions
with LAV 7649 were considerably weak.
Equine Adenovirus (EAV) Species 1
This current study was only able to show a very weak antigenic reactivity of
the EAV to the 32CN. It was partially neutralized by the 32CN (1:22) and this
partial neutralization was one way. The homologous serum neutralization titer for
the EAV was 1:65015.
23
Llama Adenovirus Isolate 7649
It is evident from the data that llama adenovirus isolate 7649 is not closely
related antigenically with any of the other viruses. Even though it did possess
common antigenic determinants with some other adenoviruses, i.e., with 32CN
(1:38 vs. 1:87) and OAV 5 (1:32 vs 1:24), the degree of the relatednesses were
only minor. The antiserum to LAV isolate 7649 did neutralize the BAV 2, OAV
47F, and the OAV 475N at low level and in one-way fashion. The homologous
titer of the LAV isolate 7649 was 1:172,674.
24
V. DISCUSSION
Antigenic distinctiveness of adenovirus species is determined by the virus
neutralization test. A test virus is determined to be of the same species as a proto-
type virus if less than or equal to 16 antibody units of reference antiserum to the
prototype virus neutralizes a test virus. Using standard virus neutralization proce-
dures, one antibody unit is defined as the most dilute concentration of antiserum
that neutralizes 100 TCID50 of homologous virus. Virus neutralization also must
be demonstrated in a reciprocal fashion with 16 antibody units or less of heterologous antiserum being capable of neutralizing the prototype virus."'
Using the above parameters, this investigation has shown that ovine adeno-
virus species 5, strain RTS-42 is of the same antigenic species as Oregon isolate
OAV 47F, a 1:1 and 1:1 test and reciprocal antibody ratio (TRAR) was shown.
Likewise, Oregon isolate OAV 475N is of the same antigenic species as Oregon
isolate 32CN with a 1:1 and 1:4.3 TRAR. Since all ovine adenoviruses were not
available for comparison, it is not known if the 32CN - OAV 475N virus isolates
are of a previously unrecognized species or if they can be classified as one of the
recognized OAV species 1-4.
Early in the period when adenoviruses were first characterized, it was noted
that these viruses were relatively species specific both to the natural host and to
cell culture derived from the natural host.'' 99 While this is a vague indefinable
categorization and not suitable for precise taxonomic purposes, it is an important
biological characteristic which appears to hold true for all group members even
some 30 years after these viruses were characterized. These viruses have been
shown repeatedly to replicate and produce disease only in the natural host or
closely related genera. Likewise, adenovinises generally have the ability to undergo continuous propagation in cell cultures derived only from the natural host or its
25
close relatives. With large inoculum doses, the virus may induce a cytopathic ef-
fect in cell culture derived from an alien host, but such infection is generally
abortive."
The current research has reconfirmed the host specificity nature of adenoviruses by showing that adenoviruses from diverse species do not share common ma-
jor antigens. While this observation has been noted with most species previously,
this is the first study where the antigenic nature and cell culture specificity of a lla-
ma adenovirus has been studied. This study represents the first time an adenovirus
from llamas has been characterized antigenically. While additional adenovirus
from this species of animal may be recovered in the future, it has been shown that
LAV isolate 7649 was distinct antigenically from other viruses characterized in
this investigation.
Adenoviruses from such diverse species as equine, llama and members of
the Bovidae family (bovine and ovine) were shown not to share major antigenic
components. However, due to the complex antigenic nature of the adenoviral cap-
sid (where several antigens may be involved in the virus neutralization reaction),
some minor antigens were detected between adenoviruses from different species of
animals.
In the current study, two species of animals (bovine and ovine) are closely
related anatomically and physiologically and both are classified in the Bovidae
family of animals." This research has shown that bovines and ovines appear to be
able to be infected with isolate 32CN (a bovine isolate). Further, previous studies
conducted by others have shown that sheep can be infected with BAV species 2
and species 7.3'14
The antigenic composition of human adenoviruses has been studied more
extensively than any other species. Several human adenoviruses share common
antigenic determinants eventhough they are classified as different species. It is not
26
uncommon to have several members of the 41 species to be related antigenically in
a variable fashion. Some species are so closely related that infection is difficult to
diagnose by serologic testing, i.e., when a person becomes infected with one virus,
an antibody response ensues against the homologous and heterologous species
(antigenically related species) of virus (heterotypic antibody response). 118 This ob-
servation has epidemiological significance in human disease. In addition, knowledge of this immunologic response pattern also may have implications in possible
candidate species for vaccine production. The number of strains of virus for incorporation in a vaccine is obviously limited. Accordingly, it would be prudent to
use a species of virus with a broad heterotypic antigenic profile for the species of
animal in question, e.g., for cattle BAV species 8 and for sheep strain 32CN/OAV
475N.
27
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