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

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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

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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 r

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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 r

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). r

r

r

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. r

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r

r

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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

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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 r

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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 r

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

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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

r

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. r

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). r

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

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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 r

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. r

-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. r

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. r

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. r

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 r

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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. r

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

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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

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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. r

r

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 . r

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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 . r

r

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. r

r

r

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. r

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. r

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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. r

r

r

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 . r

r

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 r

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

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r

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. r

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 . r

Palabras clave Arbovirus - Enfermedades animales - Flavivirus - Genética - Resistencia innata. •

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