D9194.PDF

Rev. sci. tech. Off. int. Epiz., 1 9 9 8 , 1 7 (1), 231-248
Genetic control of host resistance to flavivirus
infection in animals
(1
G.R. Shellam (1) M.Y. S a n g s t e r -
2)
& N. U r o s e v i c
(1)
(1) Department of Microbiology, University of Western Australia, Nedlands, Perth, WA 6907, Australia
(2) Present address: Department of Immunology, St Jude Children's Research Hospital, 332 North Lauderdale,
Memphis, 38105-2724 Tennessee, United States of America
Summary
Flaviviruses a r e s m a l l , e n v e l o p e d R N A v i r u s e s w h i c h a r e g e n e r a l l y t r a n s m i t t e d by
a r t h r o p o d s t o a n i m a l s a n d m a n . A l t h o u g h f l a v i v i r u s e s c a u s e i m p o r t a n t d i s e a s e s in
d o m e s t i c a n i m a l s a n d m a n , flaviviral i n f e c t i o n of a n i m a l s w h i c h c o n s t i t u t e t h e
n o r m a l v e r t e b r a t e r e s e r v o i r m a y b e mild or s u b - c l i n i c a l , w h i c h s u g g e s t s t h a t
s o m e a d a p t a t i o n b e t w e e n virus a n d host m a y h a v e o c c u r r e d . W h i l e this
possibility is difficult t o s t u d y in w i l d a n i m a l s , e x t e n s i v e s t u d i e s using l a b o r a t o r y
m i c e h a v e d e m o n s t r a t e d t h e e x i s t e n c e of i n n a t e , f l a v i v i r u s - s p e c i f i c r e s i s t a n c e .
R e s i s t a n c e is h e r i t a b l e a n d is a t t r i b u t a b l e t o t h e g e n e Flv , w h i c h is l o c a t e d o n
c h r o m o s o m e 5 in this s p e c i e s . T h e m e c h a n i s m of r e s i s t a n c e is a t p r e s e n t
u n k n o w n , b u t a c t s e a r l y a n d limits t h e r e p l i c a t i o n of f l a v i v i r u s e s in cells. W h i l e
s o m e e v i d e n c e s u p p o r t s a role f o r Flv in e n h a n c i n g t h e p r o d u c t i o n of d e f e c t i v e
interfering v i r u s ; t h e r e b y restricting t h e p r o d u c t i o n of i n f e c t i o u s v i r u s , o t h e r
r e p o r t s s u g g e s t t h a t Flv i n t e r f e r e s w i t h e i t h e r v i r u s R N A r e p l i c a t i o n or R N A
p a c k a g i n g . R e c e n t r e s e a r c h s u g g e s t s t h a t c y t o p l a s m i c proteins bind t o t h e viral
r e p l i c a t i o n c o m p l e x and t h a t allelic f o r m s of t h e s e proteins in r e s i s t a n t m i c e m a y
r e s t r i c t t h e p r o d u c t i o n of i n f e c t i o u s p r o g e n y .
r
r
r
A p p a r e n t r e s i s t a n c e t o f l a v i v i r u s e s h a s b e e n d e s c r i b e d in o t h e r v e r t e b r a t e s ,
a l t h o u g h it r e m a i n s t o b e s e e n if this is a t t r i b u t a b l e t o a h o m o l o g u e of Flv .
N o n e t h e l e s s , k n o w l e d g e g a i n e d of t h e c h a r a c t e r i s t i c s a n d f u n c t i o n of Flv in m i c e
should b e a p p l i c a b l e t o o t h e r host s p e c i e s , a n d i m p r o v e m e n t of r e s i s t a n c e t o
flaviviral infection in d o m e s t i c a n i m a l s by s e l e c t i v e b r e e d i n g or g e n e t e c h n o l o g y
m a y ultimately be possible.
r
r
Keywords
A n i m a l diseases - Arboviruses - Flaviviruses - Genetics - Innate resistance.
Background
The flaviviruses (from the Latin flavus, 'yellow') are named
after the type species, yellow fever (YF) virus, which occupies
a pre-eminent position in the history of virology. The virus of
yellow fever was the first ultra-microscopic filterable agent
proved to be the cause of human disease ( 5 8 ) , and the
demonstration that yellow fever was transmitted by a
blood-sucking mosquito
made YF virus
the
first
arthropod-borne virus, or arbovirus, known to infect man. YF
was also the first arbovirus to be cultivated in the laboratory.
1950s, researchers recognised that arboviruses could be
separated serologically into two groups (group A and group B)
on the basis of their reactions in laboratory tests which
employed
haemagglutination
and
haemagglutination-
inhibition.
The family Togaviridae was created to incorporate the group A
and group B arboviruses which shared a similar mode of
•transmission and physicochemical properties, these being an
RNA genome, a lipid envelope and a nucleocapsid with cubic
symmetry. The group A viruses became the genus Alphavirus,
and the group B viruses became the Flavivirus
From the 1930s onwards, the number of arboviruses
identified steadily increased and studies on the antigenic
relationships between them were commenced. In the early
this
family.
However,
the
identification
genus within
of significant
differences between viruses in these two genera, particularly
the modes of replication, genome structure, gene order and
232
Rev. sci. tech. Off. int. Epiz, 17 (1)
virus morphogenesis, led to the creation of the new
Flaviviridae family to incorporate the Flavivirus genus ( 9 7 ) .
More recently, the Pestivirus genus, which contains bovine
diarrhoea virus, hog cholera virus and border disease of
sheep, and the 'hepatitis C-like viruses' genus (now the Hepaci
genus) which contains hepatitis C virus, have been added to
the Flaviviridae. Neither the pestiviruses nor hepatitis C virus
are arboviruses, and will not be discussed further in this
review.
For a comprehensive discussion of the history of the
Togaviridae and Flaviviridae, the reader is referred to the
review by Porterfield ( 5 7 ) . The current status of the
Flaviviridae is described in the report of the International
Committee on Taxonomy of Viruses (51).
The family Flaviviridae comprises 6 9 viruses, of which 67 are
arboviruses or are closely related to these viruses (Table I). Of
the arboviruses, 3 4 are transmitted by mosquitoes, 19 are
tick-borne, 12 are zoonotic agents transmitted between bats
or rodents without known arthropod vectors and 2 have
transmission cycles which have not been characterised.
detergents. Virions are stable at slightly alkaline pH (8.0) and
are inactivated above 40°C (62).
The viral genome comprises a single molecule of linear,
positive-sense single-stranded RNA (ssRNA) of approximately
10.7 kilobases (kb) (range 10.4-11.1 kb) which serves as the
viral messenger RNA (mRNA). The genome has a single, long,
open reading frame which encodes a polyprotein which is
proteolytically cleaved into ten viral proteins. The structural
proteins (C, M and E ) are encoded at the 5 ' end of the
genome, while the non-structural proteins (NS1, NS2A,
NS2B, NS3, NS4A, NS4B and NS5) are encoded at the 3 ' end.
Certain non-structural proteins may function as proteases,
helicases and polymerases in flavivirus replication.
Virus replication
Flaviviruses replicate in mammalian, avian and arthropod
cells and their replication strategy has been recently reviewed
by Rice (62). Their life-cycle is illustrated in Figure 1. Briefly,
virions bind to receptors on the cell surface through the viral E
Flaviviruses are an important cause of human and animal
disease. Thirteen of the 69 flaviviruses are a significant cause
of disease in man, and a larger number have been associated
with sporadic cases. Flavivirus diseases range from febrile
illnesses to life-threatening haemorrhagic fevers, encephalitis
and hepatitis. The most significant threats to human health
are yellow fever, dengue (DEN) and Japanese encephalitis (JE)
viruses, which are amongst the most important viral
pathogens of the developing world (48).
Flaviviruses are also pathogenic for domestic or wild animals
of economic importance, and establish infection in a range of
other vertebrate species in the wild. The infection of animals
by flaviviruses, and the genetic control of host resistance to
flaviviruses in animals, are the main topics of this review, and
will be discussed in detail.
Characteristics of members of
the Flavivirus genus
The flavivirus virion consists of a spherical nucleocapsid core
surrounded by a lipid envelope which has small surface
projections. Virions are spherical with a diameter of 4 0 to 5 0
nm. There are three structural proteins: the envelope (E)
protein, the transmembrane (M) protein and the capsid (C)
protein. The glycosylated E protein is located in the virion
envelope. Its crystal structure has been described recently.
The glycosylated M protein is also associated with the
envelope. Mature virions have a buoyant density in CsCl of
1.22 to 1.24 g/cm and are composed of 6% RNA, 6 6 %
protein, 9% carbohydrate and 17% lipid, which is derived
from the host cell. Because the envelope contains lipid,
flaviviruses can be rapidly inactivated by organic solvents and
3
Fig.
1
The life-cycle of flaviviruses
The steps of the virus life-cycle are as follows:
- entry into the cell and uncoating
- polyprotein synthesis on polyribosomes and processing on the membranes
of the endoplasmic reticulum
- virus RNA replication, including the replicative form (RF), replicative
intermediate (RI), replicative complex (RC) and final product virion RNA
(vRNA)
N denotes the cell nucleus. Host proteins are thought to bind to stem-loop
structures on the genomic viral RNA
233
Rev. sci. tech. Off. int. Epiz., 17 (1)
Table I
Vectors, hosts and diseases associated with the arthropod-borne flaviviruses
Modified from Monath and Heinz (48)
Virus (abbreviation)
(c)
Absettarov
Alfuy
Apoi
Aroa
Bagaza
Banzi (BAN)
Bouboui
Bussuquara
Cacipacore
Carey Island
Dakar bat
Dengue 1 (DEN)
Dengue 2
Dengue 3
Dengue 4
Edge Hill
Entebbe bat
Gadgets Gully
Hanzalova
Hypr
llheus (ILH)
Israel turkey meningo-encephalitis
Japanese encephalitis (JE)
Jugra
Jutiapa
Kadam
Karshi
Kedougou
Kokobera
Koutango
Kumlinge
Kunjin
Kyasanur Forest disease
Langat
Louping ill (LI)
(c)
(c)
(c)
Meaban
Modoc
Montana myotis leukoencephalitis
Murray Valley encephalitis (MVE)
Naranjal
Negishi
Ntaya
Omsk haemorrhagic fever
Phnom-Penh bat
Powassan (POW)
Rio Bravo
Rocio
Royal Farm
Russian spring summer encephalitis (RSSE)
Saboya
St Louis encephalitis (SLE)
Sal Vieja
San Perlita
Saumarez Reef
Sepik
Sokuluk
Spondweni
Stratford
Tembusu
Tyuleniy
Uganda S
Usutu
Wesselsbron
West Nile (WN)
Yaounde
Yellow fever (YF)
Zika
Vector
(a)
Tick
Mosquito
?Mosquito
Mosquito
Mosquito
Mosquito
Mosquito
Mosquito
Mosquito
Mosquito
Mosquito
Mosquito
Tick
Tick
Tick
Mosquito
Mosquito
Mosquito
Mosquito
Tick
Tick
Mosquito
* Mosquito
Mosquito (?tick)
Tick
Mosquito
Tick
Tick
Tick
Tick
Mosquito
Mosquito
Tick
Mosquito
Tick
Tick (mosquito)
Mosquito
Tick
Tick
?Phlebotomine
Mosquito (tick)
Tick
Mosquito
Principal
vertebrate host
Geographic
distribution
Rodent
Bird
Rodent
Europe
Australia-New Guinea
Asia
South America
Africa
Africa
Africa
South America
South America
Asia
Africa
World-wide
World-wide
World-wide
World-wide
Australia-New Guinea
Africa
Australia-New Guinea
Europe
Europe
South America
Middle East, Africa
Asia, Australia-New Guinea
Asia
Central America
Africa, Middle East
Asia
Africa
Australia-New Guinea
Africa
Europe
Australia-New Guinea
Asia
Asia
Europe
Rodent
Bird
Bat
Bat
Human, monkey
Human, monkey
Human, monkey
Human, monkey
?Marsupial
Bat
?Bird
Rodent
Rodent
Bird
Bird
Bird, pig
Bat
Rodent
?Rodent
?Rodent
Macropods
Rodent
Rodent
Bird
Rodent
?Rodent
Bird, rodent
Bird
Rodent
Bat
Bird
?Rodent
Rodent
Bat
Rodent
Bat
Bird
Bird
Rodent, bird
Rodent
Bird
Rodent
Rodent
Bird
Bat
Mosquito
Mosquito
Mosquito
Tick
Mosquito
Mosquito
Mosquito (tick)
Mosquito (tick)
Mosquito
Mosquito (tick)
Mosquito
?Marsupial
?Bird
Bird
Bird
Bird
?Rodent, sheep
Bird
Rodent, bird
Monkey
Monkey
a Parentheses indicate Isolation from alternate vector, but uncertain role In natural transmission cycle
b) Disease In economically Important animals
c) Viruses closely related or identical to Russian spring-summer encephalitis
d)laboratory infection only
e) Disease following experimental infection for cancer therapy
Europe
North America
North America
Australia-New Guinea
South America
Asia
Africa
Asia
Asia
North America, Asia
North America
South America
Asia
Asia, Europe
Africa
North America,
Central America,
South America
North America
North America
Australia-New Guinea
Australia-New Guinea
Asia
Africa
Australia-New Guinea
Asia
Asia, North America
Africa
Africa
Africa, Asia
Africa, Europe, Asia
Africa
Africa, South America
Africa, Asia
Human
disease
Animal disease
Animal disease
+
(d)
+
+
+
+
+
+
+
+
+
+
+
+
+
Turkey
Pig, horse
+
+
+
+
+
+
+
Horse
(b)
+
+
Sheep, pig, horse,
dog, goat, deer,
grouse
+
+
?Horse
+
+
Musk-rat
+
+
+
+
+
+
+
+
+
+
+
+
Sheep
Horse
(b)
234
protein. The availability of receptors for the E protein is
thought to determine tissue and cell tropisms and the host
range for flaviviruses. Viruses are internalised by the process
of receptor-mediated endocytosis into vesicles from which
nucleocapsids are thought to be released into the cytoplasm,
and are uncoated prior to translation of the genomic RNA,
which occurs in association with the rough endoplasmic
reticulum.
The primary translation product is the polyprotein, which is
cleaved at specific sites by host and viral proteases to produce
the structural and non-structural proteins. The pre-M protein
is the glycosylated precursor of the structural protein M and
cleavage of pre-M is delayed, occurring at approximately the
same time as virion release. The non-structural proteins
participate in the cleavage of the polyprotein (especially the
NS2B-NS3 complex, which exhibits serine protease activity)
and in RNA replication, since NS3 and NS5 are enzymatic
components of the viral RNA replicase.
After the incoming genomic mRNA has been translated, RNA
replication begins in the cytoplasm, principally in the
perinuclear region of the cell in association with membranes
of the endoplasmic reticulum. The 3 ' non-coding region of
the genome contains a predicted 3 ' terminal stem-and-loop
(SL) structure, which is common to most flaviviruses and may
serve as a promoter for the initiation of viral negative-strand
synthesis. Complementary negative strands are synthesised
first and serve as templates for the synthesis of genome-length
positive-stranded molecules. This process employs a
semi-conservative mechanism involving a double-stranded
replicative form (dsRF) consisting of positive- and
negative-stranded viral RNA, and a replicative intermediate
(RI) containing the dsRF with single-stranded viral RNA
attached to it. The dsRF is believed to serve as the template for
the synthesis of positive strand RNA, which replaces the
existing positive-strand RNA in the dsRF (18, 9 6 ) .
In the replication of a number of RNA viruses, host cell
proteins bind to the viral RNA replication complex. For
flaviviruses, the 3 ' SL structure of genomic viral RNA has been
shown to bind host cell proteins which are predicted to be
important for the replication of the viral RNA (4). This will be
discussed further in relation to the mechanism of genetically
controlled resistance to flaviviruses.
Virion morphogenesis occurs in association with intracellular
membranes, and the envelope appears to be acquired by
intracellular budding. Nascent virions are believed to be
transported from the endoplasmic reticulum to the cell
surface by vesicular transport through the secretory pathway
of the host cell (62). The release of virions occurs mainly by
exocytosis.
In vertebrate cells, the latent period (i.e., the time taken for an
infected cell to produce new virus) is 1 2 - 1 6 h, and virus
production continues over three to four days.
Rev. sci. tech. Off. int. Epiz., 17(1)
Effect of flavivirus infection on
host cells
Flavivirus infection commonly induces vacuolation and
proliferation of intracellular membranes in vertebrate cells
(50), and is cytocidal in many cell types, although persistent
infection may also occur. Host cell RNA and protein synthesis
is not markedly inhibited by flavivirus infection.
Cytocidal infections are uncommon in arthropod cells, and
persistent infections can be established in mosquito cells.
Mosquitoes exhibit a life-long chronic infection and produce
very high levels of virus in the salivary gland (62).
Interaction of flaviviruses with
vectors and hosts
The three components necessary for the transmission of
flaviviruses and other arboviruses are the vertebrate host, the
virus and the arthropod vector. The vectors and main
venebrate hosts for each of the flaviviruses are described in
Table I. A description of the role of particular arthropod
vectors in the transmission of the major flaviviruses is
provided in Monath and Heinz (48).
Thse vertebrate host plays a pivotal role in virus transmission.
By enabling the virus to replicate to sufficient levels for the
establishment of viraemia, the host serves as a virus reservoir
for blood-feeding vectors which, upon feeding, become
infected and biologically transmit the virus by biting other
susceptible vertebrates. A single viraemic host may serve as a
source of infection for a number of arthropods vectors, thus
amplifying the transmission of the virus.
Birds and mammals are the principal vertebrate hosts for
flaviviruses. Only occasionally, such as with yellow fever or
dengue viruses, are humans important in virus transmission,
and most human flavivirus infections are incidental to the
maintenance of these viruses in zoonotic cycles.
A detailed consideration of the role of the arthropod vector
and vertebrate host in the transmission of flaviviruses is
beyond the scope of this review, and the interested reader is
referred to a comprehensive treatment of this subject by
Monath ( 4 6 ) . However, several topics of relevance to the
infection of vertebrate hosts and the genetic control of host
resistance to flaviviruses are reviewed briefly below.
Selection of vertebrate hosts by arthropods
The vertebrate host must be readily available to vectors for
acquisition of a virus or for transmission. The characteristics
of vertebrates that attract arthropods include the colour,
intensity, movement and size of the host, and emanations
from the host such as carbon dioxide, humidity, temperature,
Rev. sci. tech. Off. int. Epiz., 17 (1)
amino acids, sex hormones, lactic acid and aggregation
pheromones released by feeding arthropods ( 7 3 ) . Arthropods
seem to use multiple cues to detect vertebrate hosts. However,
some of these host factors are likely to be genetically regulated
and may indirectly influence susceptibility to infection. An
interesting example of this is the malaria vector Anopheles
gambiae, which preferentially feeds on human subjects of
blood group O ( 9 8 ) .
The role of host viraemia in virus transmission
The amplification of flavivirus infections during horizontal
transmission is critical for the maintenance of these viruses in
nature; each infected host must serve as a source of infection
for multiple arthropods. For mosquitoes, whose feeding time
is very brief, the blood meal must contain sufficient virus to
establish midgut infection. Infected hosts involved in
transmission cycles usually exhibit viraemia titres of
> 5 log units/ml ( 4 8 ) . On the other hand, ticks feed over a
much longer time, and recent reports demonstrate that host
infection and viraemia may not be required for amplification
of tick-borne arbovirus infections. Tick-borne encephalitis
vims can be transmitted efficiently from infected to uninfected
ticks co-feeding on a vertebrate host in the absence of
detectable viraemia ( 3 8 , 5 2 ) .
10
The effect of host age and sex
The effect of age on the pathogenesis of flavivirus infections
has been studied extensively in the laboratory. New-born
mice are more susceptible to lethal encephalitis than adults,
and when inoculated peripherally remain susceptible to
encephalitis until approximately three weeks of age. While
this reflects in part the maturation of host protective responses
to limit the neuroinvasiveness of the virus, it may also be
associated with neuronal maturity, since immature neurons
are more susceptible to flavivirus infection than mature
neurons (53). In the field, younger animals might be expected
to develop higher viraemias and therefore to contribute more
to virus transmission by mosquitoes than older animals. This
principle has been demonstrated in birds for J E virus (72).
The effect of sex on host infection is less clear-cut, although
sexually mature adult female mice were more resistant to
St Louis encephalitis (SLE) virus-induced encephalitis than
males (1).
Genetic control of host resistance to infection
This topic is the major focus of this review, and is dealt with
below.
Diseases of animals caused by
flavivuses
Eight flaviviruses are pathogenic for domestic or wild animals
of economic importance (Table I). The review by Monath and
Heinz provides details of these diseases (48).
235
The most significant of these eight flaviviruses is J E virus,
which causes disease in horses and pigs. In horses, mortality is
low. In the more common, sub-clinical form of the disease,
fever, slow movement and jaundice occur after an incubation
period of eight to ten days. In the severe form of the disease,
horses develop fever, jaundice, petechial haemorrhaging and
paralysis. In pigs, infection may lead to encephalitis and
stillbirth or abortion and decreased fecundity as a result of
decreased spermatogenesis. Birds and pigs are the main
amplifying hosts and provide a source for infection of
mosquitoes. Cattle are often infected with JE virus, but exhibit
only low viraemia and do not perpetuate virus transmission.
Louping ill (LI) virus is a flavivirus of the tick-borne
encephalitis virus complex which is restricted to Great Britain
and Ireland and is transmitted by Ixodes ricinus ticks. This
vims commonly causes disease in sheep and red grouse,
which can be fatal. A clinical disease has also been described
in cows, pigs, goats, deer and dogs.
Wesselsbron vims induces disease in sheep in southern
Africa. Infection is associated with abortion and the death of
new-born lambs and pregnant ewes. Mortality may also be
high in infected goats. However, cattle do not develop
significant disease, despite evidence of widespread
seroconversion. The transmission cycle involves Aedes
mosquitoes. The species of vertebrate hosts important for
transmission is not known, although domestic livestock
develop high viraemias.
Israel turkey meningo-encephalitis virus was first isolated
from domestic turkeys in Israel. The vims produces a
progressive paralysis with meningo-encephalitis, which
results in 1 0 % - 1 2 % mortality. The vims is largely restricted to
the Middle East and the vector is thought to be a mosquito.
Omsk haemorrhagic fever vims is restricted to central Russia.
The vims is a member of the tick-borne encephalitis complex.
Although ticks have been implicated in transmission, direct
rodent-to-rodent transmission may also occur. The vims
causes disease in musk rats and widespread mortalities have
been reported. Hunters may become infected by direct
contact with tissues or secretions of infected musk rats.
West Nile (WN) vims infects horses, and encephalitis caused
by this vims has been reported in horses in France and Egypt.
In a study performed in southern Africa, 3 7 % of dogs
displayed seropositivity to the vims, but experimental studies
suggest that dogs develop only a mild illness and a low
viraemia, and are not important in vims transmission.
Similarly, cattle in Africa frequently show serological evidence
of infection but do not develop disease or viraemia. Birds are
susceptible to infection. The vims has been isolated from wild
birds in many areas, high rates of seropositivity have been
reported and birds are considered to be the major amplifying
host.
236
While Murray Valley encephalitis (MVE) virus and Kunjin
virus infect horses, and Kunjin virus has been associated with
encephalitis, disease is rare and the extent of morbidity in
horses and other animals such as dogs is not known.
Rev. sci. tech. Off. int. Epiz., 17(1)
resistance to bacteria and viruses was separately inherited
(92). Resistance to LI and SLE viruses appeared to be
conferred by an autosomal dominant gene. This was the first
demonstration that a host gene could control resistance to
disease induced by an animal virus.
Infection of other animals
A number of vertebrate hosts are involved in the maintenance
of flaviviruses in nature (Table 1). Amongst these, rodents,
birds, primates and bats are common although in some cases
the vector involved has not been identified. There appear to
have been relatively few studies of experimental infection of
the important host species with the exception of non-human
primates, which were studied in depth in the period
1930-1950, at which time the ecology of yellow fever virus
was being elucidated (27). More information is needed on the
ability of flaviviruses to establish infection and induce disease
in the relevant vertebrate host species to confirm their role in
the virus life-cycle. The reader is referred to several reviews of
flavivirus infection of a variety of non-human host species in
the wild (8, 1 2 , 8 0 , 8 3 ) .
While flavivirus infection in the vertebrate reservoir is usually
mild, disturbance to habitats can have disastrous
consequences. The significant mortality of monkeys infected
with Kyasanur Forest disease virus in the forests of Mysore,
southern India, has been attributed to the introduction of
cattle into this environment, which greatly increased the
number of hosts for the tick vector of this virus (8).
The preference for rodents as hosts by over 2 0 members of the
Flaviviridae
(Table I) is of interest, but whether the
phenomenon of genetic control of host resistance which has
been extensively studied in Mus domesticus is applicable to
other rodent species or to other vertebrates remains to be
determined.
Historical background to
genetically controlled resistance
to flavivirus infection in mice
When
compared
to
virus-susceptible
(VS) mice,
virus-resistant (VR) mice exhibited lower brain titres of SLE
virus following i.e. or intranasal (i.n.) inoculation, and brain
cell cultures derived from new-born resistant mice produced
lower yields of SLE virus than comparable cultures from
susceptible mice. The BSVR and BRVR lines were also found
to be resistant to Russian spring summer encephalitis (RSSE)
virus. However, resistance was restricted to certain viruses,
and both VR and VS mice were equally susceptible to
vesicular stomatitis virus, rabies virus and lymphocytic
choriomeningitis (LCM) virus.
In 1952, Sabin discovered that the Princeton Rockefeller
Institute (PRI) line of albino mice was uniformly resistant to
i.e. inoculation of the vaccine strain of YF virus (17D-YF)
virus, and that resistance was clearly inherited as an
autosomal dominant allele ( 6 3 ) . The replication of 17D-YF
virus was shown to be restricted in the brains of the resistant
mice when compared to susceptible Swiss mice. Similar
effects were seen for WN, J E , SLE, DEN and RSSE viruses. In
contrast, the multiplication of a large group of other viruses
was unaffected in the brains of PRI mice ( 6 3 , 6 4 , 65). This
indicated that the resistance of PRI mice was effective only
against a group of related viruses, now known as flaviviruses.
In 1 9 6 5 , to facilitate the characterisation of resistance,
Groschel and Koprowski introduced the resistance gene
carried by PRI mice into the flavivirus-susceptible inbred
strain C3H/He to create the congenie resistant strain C3H7RV
(25). Congenicity was indicated by the reciprocal exchange of
skin grafts. Although the inbred C3H/RV strain shares about
99.8% of the C3H/He genome (25), the two strains differ not
only at the flavivirus resistance locus, but also in
chromosomal segments representing many genes on either
side of this locus. At the resistance locus, the alleles
determining resistance and susceptibility have been
designated F¡v and Flv , respectively (24).
r
Studies of genetically controlled, innate resistance to
flavivirus-infection have a long history. In 1931, Sawyer and
Lloyd reported that different strains of mice varied in their
susceptibility to YF virus following intracerebral (i.e.)
challenge ( 7 1 ) . The Det strain was shown to possess
hereditary factors for resistance, although the mode of
inheritance could not be determined. In 1 9 3 3 , Webster
described the development of bacteria-resistant and
bacteria-susceptible lines from random-bred albino mice and,
by selective breeding using LI virus as a selective agent,
established bacteria-susceptible-virus-susceptible (BSVS),
bacteria-susceptible-virus-resistant (BSVR), bacteria-resistantvirus-susceptible (BRVS) and bacteria-resistant-virus-resistant
(BRVR) lines, which demonstrated for the first time that
s
Nature of resistance due to the
flvr/gene
Outcome of infection
On many occasions, flavivirus-resistant mice have been
shown to survive virus doses that would be lethal to
susceptible animals. Mice have been infected by the i.e., i.n.,
intraperitoneal (i.p.) and subcutaneous (s.c.) routes.
However, the resistance conferred by the F!v gene is not
absolute, and large doses of a virulent flavivirus have been
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237
Rev. sci. tech. Off. int. Epiz., 17 (1)
shown to produce a high level of mortality in
flavivirus-resistant mice ( 2 3 , 2 9 , 3 3 , 7 0 , 9 4 ) , although the
onset of disease symptoms and the time of death is delayed in
resistant mice when compared to susceptible mice ( 2 3 , 3 3 ) .
Growth of virus in vivo
The first evidence that flavivirus-resistant and -susceptible
mice differed in the ability to support flavivirus replication
was provided in 1 9 3 6 by Webster and Clow, who reported
reduced brain titres of SLE virus in resistant mice (94). This
observation has been confirmed by many subsequent studies
using doses of flaviviruses which have been either sublethal or
lethal for resistant mice. Hanson et al. compared W N virus
replication in the brains of adult C3H/RV and C3H/He mice
infected i.e. with a dose of virus which was sufficient to kill all
the mice (29). Virus titres rose rapidly to a high level in the
C3H/He mice. However, the appearance of infectious virus
was delayed in the C3H7RV mice and the maximum brain titre
of WN virus attained in these mice was about 3 l o g units
lower than in C3H/He mice. The replication of Banzi (BAN)
vims was also compared in the brains of adult C3H/RV and
C3H/He mice at an i.e. dose which was lethal for both strains
(3). From 32 h post infection, the mean titres of BAN virus in
the C3H/RV mice remained 1.0-1.5 l o g units lower than in
the C3H/He mice. However, statistical significance was
attained only sporadically. Compared to W N virus, the
replication of BAN virus appeared to be less restricted in
resistant mice. The viraemic state of flavivirus-resistant and
-susceptible mice at intervals after the i.p. inoculation of WN
vims was also compared ( 2 3 ) . Within 1 0 to 12 h after
infection, the blood titres of virus fell to undetectable levels in
both resistant and susceptible mice. However, termination of
the primary viraemia in susceptible mice was immediately
followed by a secondary viraemia of approximately 3 6 h
duration, and these mice succumbed to encephalitis within
six days of infection. In contrast, no secondary viraemia was
detected in resistant mice, which remained asymptomatic.
Johnson and Mims have postulated that viral invasion of the
central nervous system following peripheral infection is
dependent on viral replication at extraneural sites producing a
viraemia of sufficient magnitude and duration ( 3 6 , 3 7 ) . The
absence of a secondary viraemia in flavivirus-resistant mice
(23) suggested that extraneural cell populations in these mice
did not support W N virus replication.
10
10
Growth of virus in vitro
Resistance to flavivirus infection is expressed at the level of
individual cells. Brain cell cultures derived from new-born
resistant mice produced lower yields of W N virus than
comparable cultures from susceptible mice ( 2 3 ) , which
confirmed earlier work with SLE virus ( 9 5 ) . A diminished
ability to support W N virus replication was also displayed by
splenic macrophages ( 2 3 , 9 0 ) , peritoneal macrophages ( 2 3 ,
25, 29, 78) and mouse embryo fibroblasts (MEF) ( 1 9 , 2 9 )
from flavivirus-resistant mice.
Specificity of resistance
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There is considerable evidence that the effect of the Flv gene
is flavivirus-specific. Early evidence of virus specificity was
provided by studies of the VR mouse strains ( 9 3 ) . The
resistance of these mouse strains to LI, SLE and RSSE viruses
was not evident against vesicular stomatitis, rabies and LCM
viruses (17, 5 4 , 9 3 ) . However, the resistance gene carried by
the VR mouse strains may not be the same as the Flv gene.
The most comprehensive evidence that the Flv gene confers
resistance only to flaviviruses was compiled by Sabin in his
work with the PRI mouse strain (65), which was the source of
the Flv gene carried by C3H/RV mice (23).
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r
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Sabin examined a large number of viruses and found that the
replication-inhibiting factor in PRI mice only affected the
growth of a group of related viruses, now known as
flaviviruses (65). The development of the congenie C3H/RV
and C3H/He mouse strains ( 2 5 ) has enabled the effect of the
Flv gene to be studied against a controlled genetic
background. However, these mouse strains have been used
mainly to investigate the mechanism of action of the Flv gene,
and little additional specificity data has been produced. Since
the studies performed by Sabin ( 6 5 ) , in vivo and in vitro
studies have added only BAN, Ilheus (ILH) and the Australian
flaviviruses MVE, Kunjin, Alfuy and Kokobera viruses to the
list of flaviviruses known to b e affected by the Flv gene ( 2 9 ,
33, 67; M.Y. Sangster, unpublished observations). The action
of the Flv gene against many of the large number of
flaviviruses remains to be tested. The flaviviruses and
non-flaviviruses which have been tested for susceptibility to
the effect of the Flv gene are listed in Table II.
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r
r
r
r
r
Although the Flv gene has been shown to have some effect on
the growth of all flaviviruses examined so far, different
flaviviruses are not uniformly affected by the expression of the
resistance gene. For example, adult resistant mice survived at
least 1,000 times the i.e. dose of SLE virus fatal for susceptible
mice ( 9 4 ) . In contrast, by i.e. lethal d o s e ( L D ) titration,
adult C3H7RV mice were only about 16-fold more resistant
than C3H/He mice to BAN virus (33).
50
50
r
There is also some evidence that the effectiveness of the Flv
gene varies for different strains of a particular flavivirus.
Following the i.e. infection of flavivirus-resistant PRI mice, the
resultant mortality depended on the strain of WN, J E and SLE
virus used (63), and virus strain differences were also recently
observed with MVE virus in the ability of mice heterozygous
for Flv to survive lethal i.e. challenge ( 7 0 ) . Furthermore,
passaging the virus in the brains of flavivirus-susceptible mice
increased the virulence for PRI mice. Sabin suggested that
flavivirus variants may emerge which are relatively unaffected
by the inherited resistance mechanism in PRI mice. Evidence
that this does occur was provided by the isolation of a W N
virus mutant from a persistently infected culture of MEF
derived from C3H/RV mice (10). The mutant replicated more
efficiently than the parental virus in resistant cells, although it
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Rev. sci. tech. Off. int. Epiz, 17(1)
238
Table II
Viruses which have been tested for susceptibility to the effect of the
Flvr gene in mice
The Flv gene was found to restrict the growth of every flavivirus tested, but
no effect on any non-flavivirus was demonstrated
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Non-flaviviruses
Flaviviruses
Flaviviridae
St Louis encephalitis
Japanese encephalitis
West N i l e
Banzi
Dengue
Ilheus
Yellow fever
Louping i l l
Tick-bome encephalitis
Murray Valley encephalitis
Kunjin
Alfuy
Kokobera
Arenaviridae
Lymphocytic choriomeningitis
Bunyaviridae
Rift Valley fever
Sandfly fever
Kununurra
Picornaviridae
Poliovirus
Rhabdoviridae
Rabies
Togaviridae
Western equine encephalitis
Eastern equine encephalitis
Venezuelan equine encephalitis
Semliki Forest virus
Sindbis (b)
Chikungunya
Ross River
Herpesviridae
Herpes simplex
Murine cytomegalovirus
(a)
(a)
(a)
(3,b)
(a)
(a)
(c)
(a)
(g)
(b)
(a)
(a,d)
(e)
(a)
(a)
(f)
(g)
(g)
(g)
(a)
(a)
(a,c)
(b)
(g)
(a)
(g)
(a)
(b)
(c)
(d)
(e)
(f)
(g)
production of IFN ( 2 9 , 8 2 , 90). Results consistent with the in
vivo experiments have been obtained in in vitro experiments
in which W N virus-infected cultures of susceptible MEF
produced more IFN than resistant cultures (19). Hanson et al.
suggested that resistant cells were more sensitive than
susceptible cells to the action of IFN, the increased sensitivity
being flavivirus-specific ( 2 9 ) . In support of this contention,
the treatment of resistant and susceptible MEF and
macrophages with exogenous IFN before infection was
reported to cause a greater suppression of the replication of
the flaviviruses W N virus and ILH virus in the resistant cells
(29). In contrast, the IFN-mediated inhibition of alphaviruses
(Sindbis virus and chikungunya virus) and vesicular
stomatitis virus occurred to an equal extent in resistant and
susceptible cells. Darnell and Koprowski reported similar
observations (19).
A.B. Sabin (65)
B.Hanson et al. (29)
R.O. Jacoby and P.N.Bhatt|33)
D. Groschel and H. Koprowski (25)
L.T. Webster (93)
M.Y. Sangster and G.R. Shellam (67)
M.Y. Sangstet (unpublished observations)
was not totally insusceptible to the resistance mechanism, and
the virus did not produce disease in resistant mice.
Mechanism of resistance due to
the Flvr gene
Infection of cells
Compared to susceptible cells, resistant cells are unlikely to be
less readily infected by flaviviruses. A number of investigators
have found that comparable cultures of resistant and
susceptible cells produced similar yields of infectious virus
early in the course of infection ( 1 9 , 2 3 , 2 9 , 90), and a similar
percentage of MEF from resistant and susceptible mice
displayed WN virus-positive immunofluorescence during the
first 2 4 h after infection (19). This suggested that the uptake
of virus and the initial stages of replication proceeded equally
well in both resistant and susceptible cells.
Interferon
The role of interferon (IFN) in the mechanism of resistance to
flavivirus infection has received much attention. Vainio et al.
suggested that IFN played no role in inherited flavivirus
resistance ( 9 0 ) . Reduced flavivirus titres in the brains of
resistant mice were found not to reflect an earlier or enhanced
The involvement of IFN in the expression of flavivirus
resistance was further investigated using antibody to type 1
IFN. Immediately before flavivirus challenge, mice were
treated intravenously (i.v.) with sheep anti-mouse IFN (type 1)
immunoglobulin ( 1 4 ) . However, the resistance of C3H7RV
mice to i.e. 17D-YF virus infection or i.p. W N virus infection
was not abolished. Notably, the same anti-IFN antibody
abrogated the resistance of A2G mice (orthomyxovimsresistant) to i.e. challenge with neurotropic influenza vims,
i.n. challenge with pneumotropic influenza virus and i.p.
challenge with hepatotropic influenza virus ( 2 8 ) . In vitro,
continuous exposure to anti-IFN serum caused an increase in
virus production by both resistant and susceptible MEF
cultures ( 1 4 ) . However, the resistant cells continued to
produce less virus than the susceptible cells. Analogous
results were obtained when viral genomic RNA synthesis was
measured. The expression of inherited resistance to flavivirus
infection clearly was not abrogated in vivo or in vitro by
treatment with antibody to IFN.
Defective interfering virus
The production in culture of defective virus which is unable to
replicate because it lacks a complete genome is a feature of
many viruses, including flaviviruses. The defective viruses,
which are generated by growth at high multiplicity of
infection, interfere with the replication of standard virus and
are hence described as defective interfering (DI) virus. To
replicate, DI viruses require the presence of a virus with a
complete genome (helper virus) which provides the missing
genome functions. DI virus reduces the titre of standard
(helper) virus, thus limiting its own production. In this
manner, DI virus can slow the spread of infection.
The nature of the genetic lesions in all DI viruses studied to
date appear to be deletions, often with associated inversions
and other sub-genomic rearrangements ( 5 5 ) . However, the
mechanism by which genetic material is deleted to form DI
nucleic acid is not well understood. The very nature of DI
nucleic acids suggests that they are the product of erroneous
239
Hev. sci. tech. Off. int. Epiz.. 17 (1)
replication of standard viral nucleic acid. De novo generation
is therefore likely to occur randomly in all virus-infected cells.
The tendency of DI viruses to enrich themselves at the
expense of standard virus is likely to be due to the preferential
replication of the DI nucleic acid. It appears that the DI
nucleic acids of at least some RNA viruses have an enhanced
affinity for the viral polymerase. As a result, the replication of
the DI RNA is favoured over the replication of the standard
viral RNA. The interfering capacity of DI RNA may well be
reflected in rearranged 3'-terminal sequences (21, 3 2 ) .
Recently, defective viral RNAs which lack about 2 0 % of the
genome were found to replicate and interfere with the
replication of the standard virus in Vero cells persistently
infected with MVE virus (39). These RNAs, although carrying
large deletions in the structural region, supported polyprotein
synthesis and processing, thus partially contributing to the
life-cycle of the virus in the cell. Deletions affected genes for
the viral prM, E and NS1 proteins, which suggests that DI
vims interferes with viral RNA synthesis in addition to causing
major interference at the level of virion assembly and release
(39).
The first evidence that cells from resistant C3H7RV mice
produced more DI vims than cells from susceptible C3H/He
mice was provided by Darnell and Koprowski (19), in whose
experiments a portion of the culture fluids from resistant and
susceptible W N vims-infected MEF was transferred every
three days to fresh cultures of the same type. The titres
remained relatively high during passage in susceptible
cultures, but dropped rapidly during passage in resistant
cultures. Interferon did not appear to be responsible for the
decrease. Serially passaged culture fluid from resistant cells,
but not from susceptible cells, interfered with standard W N
vims replication in both resistant and susceptible cells.
Brinton analysed the intracellular viral RNA synthesised in
WN vims-infected resistant and susceptible MEF and also the
RNA in virions released from these cells ( 1 0 , 1 1 ) . At all times,
between 10 and 72 h after infection, less viral RNA was
detected in resistant cells. Furthermore, the level of viral
protein synthesis in W N vims-infected resistant cells was
below that measured in susceptible cells. Most of the RNA in
extracellular W N vims liberated from susceptible cells was
40S. In contrast, the predominant RNAs associated with
virions released by W N vims-infected resistant cells were
small-sized RNA species, which may have represented the
deleted viral genomes characteristic of many DI particles.
These and other studies ( 1 1 ) , which compared W N vims
growth in MEF from C3H/RV and C3H7He mice, clearly
demonstrated that the host cell exerted a major influence on
the composition of the progeny viral population. In contrast
to susceptible cells, flavivirus-infected resistant cells appeared
to produce vims populations which contained a high
proportion of DI viruses. Brinton and
Fernandez
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hypothesised that the product of the Flv gene was a host
factor involved in flavivirus replication ( 1 5 ) . An interaction
between this host factor and the viral polymerase or the viral
template could affect the nature of viral RNAs synthesised in
resistant cells and could result in the increased production of
DI viruses in cells from resistant mice.
While in vitro studies suggest that the resistance conferred by
the Flv gene is a consequence of the production of DI
particles by resistant cells, there are few reports of the in vivo
production of DI particles by resistant mice. When BAN vims
was administered i.p. to adult mice, interfering vims was
detected in the brains of resistant mice but not susceptible
mice ( 8 1 ) . This difference appeared to be associated with a
greater production of interfering vims in the spleens of the
resistant mice. However, following i.e. infection, interfering
vims could not be demonstrated in the brains of resistant or
susceptible mice, even though restricted vims growth was
evident in the resistant mice.
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Furthermore, when the replication of MVE vims in the brains
of resistant C3H/RV and susceptible C3H/HeJ mice was
compared, a selective accumulation of defective viral RNA
was not detected in the brains of resistant mice using
Northern blot hybridisation analysis (89). This suggests that
the production of DI vims is not necessarily a major effect of
Flv -mediated resistance, and that the results of earlier studies
may reflect characteristics of certain cell culture conditions or
the vims strain used. Interestingly, during MVE vims
replication in the brain, the viral replicative form (RF) and
replicative intermediate (RI) RNA forms accumulated to a
greater extent in resistant C3H/RV than in susceptible
C3H/HeJ mice (89), thus suggesting the direct involvement of
a putative cellular resistance factor in inhibition of either virus
RNA replication or packaging. An in vitro flavivirus resistance
model using primary cell cultures from the peritoneal cavity
of adult susceptible and resistant mice is being developed to
elucidate further the resistance mechanism (78).
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Effect of cellular factors on virus replication
Recent evidence from studies of RNA-protein interactions
involving flaviviral RNA and cellular proteins supports the
role of a cytoplasmic protein(s) in the expression of flavivirus
resistance (13).
The binding of cellular proteins to viral riboprobes carrying
specific SL structures of viral 5 ' and 3 ' non-translated regions
in a gel-retardation assay has been used to identify cellular
proteins with a putative role in flavivirus replication. A
number of cellular proteins, including translation elongation
factor-1 alpha ( E F - l a ) , have been shown to interact
specifically with viral SL structures, thereby suggesting the
involvement of cellular proteins in different steps of the vims
life-cycle in the cell (4, 5 ) . Similarly, studies on RNA-protein
interactions between viral riboprobes complementary to viral
genomic RNA and cell lysates from resistant and susceptible
mice showed differential affinity of certain cellular proteins
240
Rev. sci. tech. Off. int. Epiz., 17 (1)
from resistant and susceptible mice in binding to structural
elements on viral RNA ( 7 6 ) . At present there is no data to
indicate whether these proteins co-localise to the same cellular
compartments that are usually invaded by flaviviruses during
their life-cycle in the cell, or whether genetic loci encoding
these proteins are localised on mouse chromosome 5 in the
region carrying the Flv locus.
Ontogeny of resistance due to
the FlVr gene
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The expression of the Flv gene from early in foetal
development is suggested by the restricted growth of WN
virus in resistant MEF compared to susceptible MEF (19, 2 9 ) .
Sabin found that PRI mice up to the age of two days
succumbed to i.e. challenge with 17D-YF virus ( 6 3 , 6 4 ) .
Beyond five days of age, the mice behaved like adult animals
and exhibited no morbidity. Nevertheless, the peak viral titres
in the brains of the suckling mice that did not survive
infection did not exceed the litres reached in the brains of
adult resistant mice. Thus, although the ability of resistant
mice to survive flavivirus infection was age-dependent,
restricted virus replication was evident from birth.
The survival of resistant mice following infection with BAN
virus has also been shown to be dependent on the age of the
mouse. C3H/RV mice were found to exhibit substantial
resistance to i.p. infection only from four weeks of age, while
adult C3H/He remained as susceptible as weanling mice.
r
However, the operation of the Flv gene alone is unlikely to
account for the age-dependent resistance of C3H/RV mice to
peripheral BAN virus infection. Mice which are not genetically
resistant to flavivirus infection are known to develop
resistance to the peripheral inoculation of many neurotropic
flaviviruses between three and four weeks of age (42, 47, 7 7 ) .
The ability of adult C3H/RV mice to survive i.p. BAN virus
infection is also likely to be related to these changes.
Presumably, host factors developing after the third week of
life act to supplement the resistance conferred by the Flv
gene.
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-susceptible adult mice to mount antibody or T lymphocytemediated responses to flavivirus infection (23, 3 3 , 3 4 , 74).
r
Although the Flv gene-mediated resistance does not operate
through specific immune responses, it is clear that humoral
and cell-mediated immune responses supplement the
resistance conferred by the Flv gene and are important, if not
essential, for the clearance of vims and recovery from
infection.
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Distribution of genetic
resistance to flaviviruses in mice
Laboratory strains
Early studies identified resistance to flaviviruses as an
inherited trait in only four strains of mice, namely Det (40),
BSVR, BRVR (93) and PRI (63). Studies of the mechanism of
inheritance of resistance in the Det strain were inconclusive,
but flavivirus resistance in the other mouse strains was
attributed to a single autosomal gene with a dominant effect.
A
flavivirus-resistant
strain C3H/RV, congenic with
susceptible C3H7He mice, was produced using the PRI strain
as the source of the resistance gene, now designated Flv (24,
25). None of the commonly used inbred strains of laboratory
mice appear to carry the Flv gene ( 2 0 , 6 8 ) . These studies
determined the percentage mortality in a range of inbred
mouse strains following i.e. 17D-YF vims or MVE virus
infection. With the exception of C3H/RV mice, a high level of
mortality was produced in all of the strains tested, namely
Swiss, A, AKR, B10.D2, BALB/c, C3H/He, C57BI76,
C57BL/10, DBA/2, SJL, SWR, CBA, CE, I/M, LP.RIII, NZW
and RIII. Furthermore, the time to death was similar in all of
the susceptible strains.
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r
There is very little evidence that mouse strains which do not
carry the Flv gene differ in their susceptibility to flavivirus
infection. The demonstration of such differences would
suggest that additional genetic factors are involved in
flavivirus resistance. Pogodina and Savinov determined the
i.p. and s.c. L D endpoints for a number of tick-borne
encephalitis virus strains in 7-8 g mice of the inbred strains
C57BL, BALB, C3H and the rarely used strain C3HA, and
reported that C57BL and BALB mice were more susceptible to
flaviviruses than C3H and C3HA mice ( 5 6 ) . Nevertheless,
there are no reports that adult mice of these strains differ in
their susceptibility to flavivirus infection. Interestingly,
peritoneal macrophages obtained from CBAT6/T6 mice after
treatment with either thioglycollate or bacillus Bilié-CalmetteGuérin (BCG) supported the growth of WN vims significantly
better than comparable cells from C57BL/6 mice (16).
However, there is no evidence to indicate that this difference
reflects greater resistance of C57BL/6 mice to flavivirus
infection.
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5 0
Specific immune responses
r
Many studies conducted using mice which lacked the Flv
gene have shown that recovery from flavivirus infection is
dependent on a specific immune response involving antibody
and cell-mediated immune responses to viral antigens ( 3 1 ,
45). The resistance conferred by the Flv gene must be
independent of specific immune responses, since it operates
in vitro in MEF ( 1 9 ) and in new-born mice which are
immunologically incompetent ( 6 3 , 6 4 ) . This conclusion is
supported by studies which were unable to demonstrate
differences between the capacity of flavivirus-resistant and
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Rev. sci. teck Off. int. Epiz. 17(1)
Using JE viras, Miura et al. reported that adult mice of several
strains varied in resistance following i.p. inoculation, with
C3H being highly susceptible, C57BL/6 moderately resistant
and BALB/c very resistant to lethal infection (43). However,
the differences were not apparent following i.e. inoculation.
Evidence that resistance was attributable to peripheral
immune mechanisms which differed among these strains was
subsequently obtained (44), and the regulation of this form of
resistance was shown to be due to a single autosomal
dominant gene, though it is not known whether this
resistance is flavivirus-specific. The resistance appears to be
unrelated to resistance mediated by Flv , which is readily
demonstrable in the brains of adult mice inoculated i.e. with
flaviviruses.
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Wild mice
Flavivirus resistance has been demonstrated in populations of
wild mice. Darnell et al. found that 1 0 0 % of a small number of
wild M. musculus domesticus
from several locations in
California survived i.e. infection with a dose of 17D-YF virus
which was lethal for C3H7He mice, as did eight of ten
laboratory-bred descendants of wild mice trapped in
Maryland (20). Although the wild mice from California were
not tested for anti-flavivirus antibodies, neutralising
antibodies to W N virus were not detected in sera collected
from twenty Maryland mice immediately after their capture.
Breeding studies indicated the presence in the Maryland
population of an autosomal dominant allele conferring
resistance and a recessive susceptibility allele. It was suggested
that a flavivirus resistance gene may confer a selective
advantage under natural conditions ( 2 0 ) , and that Powassan
(POW) virus, a tick-borne flavivirus, may exert a selective
pressure on populations of wild mice in the United States of
America (9).
In studies of 149 wild M. m. domesticus trapped in various
locations in Australia, approximately 2 0 % survived an i.e.
challenge with a dose of MVE virus which was uniformly
lethal in susceptible C3H/HeJ mice ( 7 0 ) . To test for a genetic
basis for this resistance, breeding studies were initiated using
the male wild mice which had survived vims challenge and
susceptible C3H/HeJ mice. A single, autosomal dominant
Flv -like gene appeared to be primarily responsible, but there
was evidence for additional inherited resistance factors.
r
Flavivirus resistance has also been identified in other
taxonomic groups of the house mouse complex; in randombred mice recently derived from wild-trapped mice, resistance
to lethal i.e. challenge was apparent in M. m. musculus,
M. m. molossinus,
M. spretus, M. spicilegus,
M. caroli
and M. cookii, and a genetic basis for this resistance
was demonstrated in M. m. musculus, M. spretus and
M. spicilegus (70).
Using the highly inbred strains MOLD/Rk and CASA/Rk,
which were derived from M. m. molossinus and M. m.
castaneus, respectively, resistance to MVE and YF virases was
shown t o be due to single autosomal dominant genes. In
CASA/Rk, resistance was attributable to a gene which was
identical to Flv , while in the MOLD/Rk strain, resistance was
attributable to a new allele of this gene, designated Flv
(68).
r
mr
To assist in the characterisation of the alleles of Flv, new
congenie mouse strains have been created in which the
resistance alleles from wild M. m. domesticus and MOLD/Rk
mice have been introduced into the genetic background of
susceptible C3H/HeJ mice (88).
There is a small amount of evidence which suggests that wild
mice are natural hosts of pathogenic flaviviruses. Since
mosquitoes are unlikely to feed on mice to any significant
extent under natural conditions ( 8 4 ) , any flaviviruses which
infected wild mice could be predicted to be transmitted by
ticks. House mice and other rodents have been found to carry
ticks (22, 6 6 ) , and the isolation of a virus, possibly the
tick-borne flavivirus TBE (tick-borne encephalitis) virus from
M. hortulanus in Russia has been reported (66). Evidence that
M. m. domesticus in California are the hosts of a tick-borne
flavivirus was provided by Hardy et al., who classified 8 of 9 7
plasma samples collected from wild mice as containing
antibodies to P O W or a closely related virus (30).
It is possible that natural flavivirus infection was a powerful
selective force which affected ancestral populations of wild
mice prior to the radiation of species within the genus Mus
(7). However, although many rodent species serve as the
principal vertebrate host for flaviviruses (Table I), the mouse
may no longer be an important host, perhaps because of the
evolution of effective resistance genes in mouse populations
or as a result of changes in the behaviour and habitat of mice
which have limited their contact with virus vectors.
Nevertheless, the flavivirus-specific resistance genes which
evolved in ancestral mice may still be retained in modem
mouse populations, even though the removal of selective
pressures may have resulted in the appearance and retention
of alleles which do not contribute to resistance.
Mapping the Flv locus
Although natural resistance to flaviviruses in mice has been
known for many years to be under the control of a single
genetic locus (64), the Flv locus has only been recently located
on mouse chromosome 5 ( 7 5 ) and mapped with respect to
other loci (69). Using a three-point backcross linkage analysis,
the Flv locus was initially mapped between the anchor locus
responsible for retinal degeneration (rd) and the glucuro­
nidase structural gene (Gus-s), in a region encompassing 10 to
23 centiMorgans (cM) of genetic distance (69, 8 5 ) . In the
mapping studies, two sets of backcross mouse progeny were
used; one derived from (C3H/HeJxC3H/RV)Fl mice
backcrossed to susceptible C3H/HeJ, and the other from
(BALB/cxC3H7RV)Fl backcrossed to susceptible BALB/c
mice. These mouse strains have also been reported to carry
242
other genetic differences in addition to the one at the Flv
locus, which were selected for during backcrossing. Those
differences are at the Ric locus controlling resistance to
Rickettsia tsutsugamushi ( 3 5 ) and at the rd locus controlling
retinal degeneration ( 7 5 ) , both of which were previously
mapped to mouse chromosome 5 ( 2 6 , 4 1 ) . These loci were
initially used to determine a chromosome 5 location of Flv by
identifying their linkage to Flv ( 6 9 , 8 5 ) . Further mapping,
using additional polymorphic microsatellite markers, showed
that these loci were positioned 7.4 and 8.6 cM proximal to
Flv, respectively, on a low resolution genetic map
encompassing Flv (Fig. 2 ) ( 8 6 ) . In low resolution genetic
mapping, twelve microsatellite markers which were
polymorphic between either the congenic C3H/HeJ and
Rev. sci. tech. Off. int. Epiz, 17 (1)
C3H/RV mice or C3H/RV and BALB/c mice were typed in 325
backcross mice and positioned relative to Flv, which
narrowed the region around Flv from 2 3 to 1 cM (Fig. 2) (86).
A minimal interval around Flv has been further narrowed to
approximately 0.45 cM by using an additional number of
microsatellite markers in high resolution genetic mapping
involving 1,325 backcross mice (87).
These studies have allowed identification of new, closely
linked microsatellite markers which have been chosen to
screen a mouse genomic library in yeast artificial
chromosomes and to produce a physical map around Flv
(N. Urosevic and G.R. Shellam, unpublished data). The
genetic mapping of Flv and current studies on physical
mapping are part of a strategy to clone flavivirus resistance
allele(s) by positional cloning. Cloning the mouse Flv gene
will facilitate elucidation of the resistance mechanism and will
assist in characterising gene homologues in other animal
species. Such knowledge could provide insights into the effect
of flavivirus resistance on the interaction between vertebrate
hosts and flaviviruses in nature.
Resistance to flaviviruses in
other species
Since there have been no comparable studies in other species,
the wider applicability of resistance mediated by Flv is
unknown and will not be possible before the molecular
characterisation of Flv
and identification of possible
homologues in other species. However, observations made on
flavivirus infections in various species of birds, rodents,
non-human primates and in man suggest that innate
resistance may be a more general phenomenon.
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Fig. 2
Genetic map of the chromosomal region around Flv on mouse
chromosome 5
The distances between genetic markers are expressed in centiMorgans. The
centromere is represented by a solid circle. The anchor loci are underlined
Experimental challenge of four species of grouse and one
pheasant species with LI virus revealed that while red grouse,
willow grouse and ptarmigan developed high viraemias and
succumbed to the virus, capercaillie survived and developed
viraemias at levels which were not considered sufficient to
infect the tick vector Ixodes ricinis ( 6 1 ) . Similarly, pheasants
survived challenge with this virus ( 6 0 ) . These results were
interpreted in terms of the known exposure of these species to
LI virus in their natural habitats during their evolution. In
Scotland, the woodland and forest species (pheasant and
capercaillie) had become resistant due to long exposure to the
virus, while the tundra and moorland species (ptarmigan and
red grouse) remained susceptible because the virus either had
not been introduced or was introduced only recently,
respectively, into these habitats. Likewise, the susceptible
willow grouse inhabits northern latitudes in Europe beyond
the normal range of the tick vector. As resistant capercaillie
exhibited lower viraemias than the susceptible red and willow
grouse and ptarmigan by as early as the first day after
infection, the resistance was proposed to be innate rather than
immune-mediated ( 6 1 ) . This is also a feature of resistance
mediated by Flv .
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243
Rev. sci. tech. Off. int. Epiz., 17(1)
Since a small percentage of red grouse survive LI virus
infection in the field ( 5 9 ) , studies to determine whether this
apparent resistance has a genetic basis would be of interest.
The principle that long-standing host-parasite relationships
are accompanied by minimal disease (6) may explain the
observation that for many reservoir hosts, flavivirus infections
are mild or clinically inapparent ( 7 9 ) . In particular, the
clinically mild infection by YF virus of species of monkeys in
Africa (2, 8 3 ) , compared with the usually fatal infection of
South American species of monkeys, has been used as
evidence that YF virus emerged in Africa (27).
transmission to biting arthropods without experiencing severe
disease themselves.
One of the many factors which may influence this is the
genetic control o f host resistance, whereby resistance genes
may protect the host against the development of lethal disease
while permitting some virus replication. Although no
experimental model of genetically-controlled resistance exists
among the vertebrates which are the major hosts for
flaviviruses, extensive studies in mice have provided evidence
for flavivirus-specific resistance which is mediated by a single,
autosomal dominant gene, Flv . Building on a long history of
research in this model, more recent studies have shown that
Flv exerts its effect not through the immune system but at the
level of the cells which are the targets for these viruses, and
have identified steps in viral replication which are affected by
Flv .
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Apparent resistance to YF virus was reported in seven species
of African rodents which survived i.e. or s.c. challenge with
doses in the range 4 , 0 0 0 - 2 0 0 , 0 0 0 L D (83). However, any
possible genetic basis for this resistance has not been
explored.
5 0
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While the possibility that Flv enhances the production of
defective interfering virus in resistant cells and thus limits the
production of fully infectious virus remains to be established
in vivo, recent research has identified cellular proteins which
bind to the viral replication complex, and has suggested that
allelic forms of these proteins present in resistant mice may
restrict the production of infectious progeny.
Development of
flavivirus-resistant animals
The susceptibility to flavivirus-induced diseases of sheep
(LI and Wesselsbron viruses), pigs JE virus) and turkeys
(Israel turkey meningo-encephalitis virus) suggests that
attempts should be made to develop the resistance of these
economically important species. A range of breeds might
usefully be screened for disease resistance to improve
domestic stock through selective breeding, although this
approach may not be fruitful since, for example, all breeds of
sheep tested to date have proved to be susceptible to LI virus
(H. Reid, personal communication). Another approach would
be the production of Flv transgenic animals once Flv has
been cloned and sequenced. Either Flv or homologues in the
species of interest could be considered. Similarly, attempts
have been made to develop transgenic pigs to be genetically
resistant to influenza virus through the effect of the murine
Mxl gene (49). However, this has not yet been successful.
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Conclusion
Flaviviruses are
an
important
family
of mostly
anthropod-borne viruses which infect a wide range of
vertebrates, including animals and man. However, the
interactions between flaviviruses and their vertebrate hosts is
complex, and is influenced by a number of factors which
affect the virus, the vectors and the hosts, many of which are
not well understood. Important amongst these is how animals
which participate in the zoonotic cycle as reservoir hosts allow
sufficient viral replication in their tissues for the purposes of
However, future research in this field will depend on the
molecular identification of the resistance gene. Flv has now
been mapped to mouse chromosome 5, and ongoing physical
mapping combined with other approaches may lead to the
identification of a candidate gene.
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The successful conclusion of these studies will provide
opportunities for searching for Flv homologues in other
species, especially those important in the zoonotic cycle. By
applying this knowledge to domestic animals which suffer
flavivirus-induced
disease, both the identification and
selective breeding of flavivirus-resistant stock and the
development of transgenic animals bearing the Flv gene or its
homologue should be possible. Similarly, it may also be
possible to test domestic animals for Flv -like resistance to
pestiviruses, again with the opportunity to develop
virus-resistant stock or to identify a target for anti-viral drugs.
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Rev. sci. tech. Off. int. Epiz., 17(1)
244
Le contrôle génétique de la résistance des animaux à l'infection
par les flavivirus
G.R. Shellam, M.Y. Sangster & N. Urosevic
Résumé
Les flavivirus sont de petits virus à A R N e n v e l o p p é , g é n é r a l e m e n t t r a n s m i s par
d e s a r t h r o p o d e s à l'animal et à l ' h o m m e . A l o r s q u e les flavivirus s o n t à l'origine de
m a l a d i e s g r a v e s c h e z les a n i m a u x c o m m e c h e z l ' h o m m e , l'infection à flavivirus
d ' a n i m a u x qui c o n s t i t u e n t le r é s e r v o i r v e r t é b r é n o r m a l p e u t ê t r e fruste voire
a s y m p t o m a t i q u e , ce qui signifie p e u t - ê t r e l ' e x i s t e n c e d'une a d a p t a t i o n entre le
virus et l'hôte. U n e telle possibilité est difficile à é t u d i e r c h e z les animaux
s a u v a g e s , m a i s d e s é t u d e s a p p r o f o n d i e s p o r t a n t sur d e s souris de laboratoire ont
d é m o n t r é l'existence d'une r é s i s t a n c e n a t u r e l l e e t s p é c i f i q u e a u x flavivirus. Cette
r é s i s t a n c e h é r é d i t a i r e est a t t r i b u é e a u g è n e Flv , situé sur le c h r o m o s o m e 5 de
c e t t e e s p è c e . Le m é c a n i s m e de r é s i s t a n c e , pour l'instant i n c o n n u e t qui intervient
à un s t a d e p r é c o c e , limite la r é p l i c a t i o n d e s flavivirus d a n s les c e l l u l e s . Alors que
c e r t a i n e s é t u d e s m o n t r e n t que le Flv j o u e un rôle d a n s l'amélioration de la
production d'un virus d é f e c t u e u x i n t e r f é r a n t , t o u t en limitant la production du
virus i n f e c t i e u x , d ' a u t r e s i n d i q u e n t q u e le F l v i n t e r v i e n t soit d a n s la réplication du
g é n o m e viral soit au n i v e a u de l ' e n v e l o p p e de l ' A R N . D ' a p r è s d e s recherches
r é c e n t e s , les p r o t é i n e s c y t o p l a s m i q u e s s e r a i e n t liées a u c o m p l e x e de réplication
virale e t les f o r m e s alléliques de c e s p r o t é i n e s c h e z les souris résistantes
p o u r r a i e n t r e s t r e i n d r e la multiplication du virus.
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La r é s i s t a n c e a p p a r e n t e a u x flavivirus a é t é d é c r i t e c h e z d ' a u t r e s v e r t é b r é s , mais
on ignore e n c o r e si elle est a t t r i b u a b l e à un h o m o l o g u e du g è n e Flv . Néanmoins,
les informations o b t e n u e s sur les c a r a c t é r i s t i q u e s et le rôle du F l v c h e z la souris
d e v r a i e n t être a p p l i c a b l e s à d ' a u t r e s e s p è c e s h ô t e s , e t u n e a m é l i o r a t i o n de la
r é s i s t a n c e à l'infection à flavivirus c h e z les a n i m a u x d o m e s t i q u e s , q u e c e soit par
s é l e c t i o n g é n é t i q u e ou g é n i e g é n é t i q u e , p o u r r a i t f i n a l e m e n t ê t r e e n v i s a g é e .
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Mots-clés
Arbovirus - Flavivirus - Génétique - Maladies animales - Résistance innée.
Control de la resistencia a la infección por flavivirus en especies
huéspedes mediante ingeniería genética
G.R. Shellam, M.Y. Sangster & N. Urosevic
Resumen
Los flavivirus son virus A R N p e q u e ñ o s y c o n e n v o l t u r a , q u e g e n e r a l m e n t e se
t r a n s m i t e n a los a n i m a l e s y al h o m b r e a t r a v é s de a r t r ó p o d o s . A u n q u e estos virus
c a u s a n i m p o r t a n t e s e n f e r m e d a d e s e n los a n i m a l e s d o m é s t i c o s y el h o m b r e , la
i n f e c c i ó n de los a n i m a l e s que c o n s t i t u y e n el r e s e r v o r i o v e r t e b r a d o habitual del
virus p u e d e revestir un c a r á c t e r leve o s u b c l í n i c o , h e c h o q u e p a r e c e indicar
algún tipo de a d a p t a c i ó n e n t r e el virus y el h u é s p e d . A u n q u e e s difícil comprobar
tal a d a p t a c i ó n en a n i m a l e s s a l v a j e s , la e x h a u s t i v a e x p e r i m e n t a c i ó n c o n ratones
de laboratorio ha v e n i d o a d e m o s t r a r la e x i s t e n c i a d e una r e s i s t e n c i a a los
flavivirus innata y e s p e c í f i c a . Esta r e s i s t e n c i a es h e r e d i t a r i a y atribuible al gen
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Flv ,
situado en el c r o m o s o m a 5 del r a t ó n . A u n q u e por a h o r a se d e s c o n o c e el
m e c a n i s m o que la g o b i e r n a , se s a b e q u e la r e s i s t e n c i a i n t e r v i e n e e n un momento
p r e c o z de la i n f e c c i ó n , y q u e a c t ú a limitando la r e p l i c a c i ó n i n t r a c e l u l a r de los
245
Rev. sci. tech. Off. int. Epiz., 17 (1)
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flavivirus. P o r u n l a d o , ciertos r e s u l t a d o s a b o n a n la t e s i s d e q u e el g e n Flv
e s t i m u l a la p r o d u c c i ó n d e virus d e f i c i e n t e s , limitando c o n ello la p r o d u c c i ó n d e
virus i n f e c c i o s o s . Otros d a t o s , e n c a m b i o , s u g i e r e n q u e e l Flv interfiere b i e n c o n
la r e p l i c a c i ó n del A R N vírico o bien c o n el e n s a m b l a j e del A R N . I n v e s t i g a c i o n e s
r e c i e n t e s p a r e c e n i n d i c a r q u e c i e r t a s p r o t e í n a s c i t o p l a s m á t i c a s s e u n e n al
c o m p l e j o d e r e p l i c a c i ó n viral, y q u e , e n los r a t o n e s r e s i s t e n t e s , l a s f o r m a s
a l é l i c a s d e e s a s p r o t e í n a s p o d r í a n a c t u a r r e d u c i e n d o la p r o d u c c i ó n d e v i r i o n e s
infecciosos.
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A u n q u e t a m b i é n e n otros v e r t e b r a d o s s e h a d e s c r i t o lo q u e p a r e c e s e r u n a
r e s i s t e n c i a a los flavivirus, q u e d a p o r v e r si t a l p r o p i e d a d p u e d e atribuirse a un
g e n h o m ó l o g o al Flv . Con t o d o , d e b e r í a s e r posible a p l i c a r a otras e s p e c i e s
h u é s p e d e s los c o n o c i m i e n t o s a c t u a l e s s o b r e las c a r a c t e r í s t i c a s y la f u n c i ó n del
Flvr e n el r a t ó n . C a b e e s p e r a r q u e a la postre ello p e r m i t a m e j o r a r la r e s i s t e n c i a
de los a n i m a l e s d o m é s t i c o s a las i n f e c c i o n e s f l a v i v i r a l e s , y a s e a p o r s e l e c c i ó n
a n i m a l o m e d i a n t e la i n g e n i e r í a g e n é t i c a .
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Palabras clave
Arbovirus - Enfermedades animales - Flavivirus - Genética - Resistencia innata.
•
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