Resistance of livestock to viruses: mechanisms and strategies for genetic engineering

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Resistance of livestock to viruses: mechanisms and

strategies for genetic engineering

Js Gavora

To cite this version:

Review

Resistance of livestock

to

viruses:

mechanisms and

_strategies

for

_{genetic engineering}

JS Gavora

Centre

_for

Food and Animal

_{Research, Agriculture}

and

_{Agri-Food Canada, Ottawa,}

ON KIA OC6 Canada

(Received

26 March

1996; accepted

13

_August

₁₉₉₆₎

Summary - This communication aims to inform readers from research and

_industry

about the

_{possibilities}

of

_{developing genetic engineering strategies}

for

_improvement

of resistance to viruses in livestock. It

_briefly

reviews coevolution of hosts and

parasites, principal

elements of virus-host interactions,

existing

resistance

_mechanisms,

and conventional

methods for

_improvement

of disease resistance. Research results from

_{genetic engineering}

of

new resistance mechanisms in both

_plants

and

_animals,

as well as

_{investigation}

of

_possible

risks and

_{’biological}

cost’ of such mechanisms are summarized as a

_background

for the

discussion of

_{prerequisites}

and

_strategies

for future

_{genetic engineering}

of resistance to

viruses in livestock. It is concluded

_that,

while conventional

_breeding

methods will remain the

_{principal approach}

to the

_improvement

of disease resistance, in some instances the

introduction of new,

genetically engineered

resistance mechanisms may be

justified.

livestock

_/

virus

_/

resistance mechanism

_/

genetic engineering

Résumé - Résistance des animaux de ferme aux virus: mécanismes et _stratégies de

génie génétique. Cette mise au _point vise à

_informer

les chercheurs et les

_{professionnels}

des

possibilités

qu’offre

le

génie génétique

pour améliorer la résistance aux virus des

animaux de

_ferme.

Le _{rapport passe en} revue la coévolution

_{hôté-parasité,}

les _principau!

aspects des interactions

_virus-hôte,

les mécanismes de résistance existants et les méthodes

classiques

d’amélioration de la résistance avx maladies. Les résultats des recherches sur

la mise en ceuvré _par

_{génie génétique}

de nouveaux mécanismes de résistance tant animale que

végétale

sont

résumés,

ainsi que l’étude des

risques possibles

et du « coût

biologique»

»

de ces mécanismes. Ces considérations constituent la toile de

fond

de la discussion sur

les conditions

_requises

et les

_stratégies

_pour, à

l’avenir,

améliorer par

génie génétique

la résistance aux virus chez les animaux de

ferme.

La conclusion tirée est que, à côté des méthodes

classiques

de sélection qui resteront la

principale

voie

_{d’amélioration,}

dans

certains cas il peut être

_justifié

d’introduire de nouveaux mécanismes de résistance par

génie génétique.

INTRODUCTION

Maximum survival of

_livestock,

with

_good

health and well

_being

are conditions for efficient animal

_{production. Many}

of the current livestock disease

_problems

that

prevent

the realization of this

optimal production goal

are caused

_by

viruses,

described

_by

Peter Medawar as

_&dquo;pieces

of bad news

_wrapped

in

_protein

coat&dquo;. This

review deals with

possible

new,

genetic engineering strategies

for the

_improvement

of resistance to viruses in livestock. Since work on

_{genetic engineering}

of disease

resistance is more advanced in

_plants

than in

_livestock,

information on research in

plants

is also reviewed.

The use of livestock for

food,

fibre and draft over hundreds of _years has led to

a

_significant

influence

by

humans on the evolution of domesticated animal

_species.

Some of the

_changes

induced

_by

artificial selection

_parallel

in their

_significance

speciation.

A modern

_meat-type

chicken can be viewed as a

_species

different from a

modern

_egg-type

chicken. Similar differences exist between breeds of

_dairy

and beef cattle. This

_{’genetic engineering’}

of livestock was achieved

_through

the

_long-term

use

of conventional

_{genetic improvement}

methods. It can be

argued

that gene transfer

represents

just

another

phase

in the

_development

of

_genetic

_engineering

of livestock

and that it would be foolish not to take

_advantage

of the new

technologies.

Thus

introduction of new mechanisms of disease resistance in livestock

_by

_gene transfer

may be viewed as a

_logical

continuation of the creative influence of humans on the

evolution of farm animals and birds that could benefit mankind

_by

_improvements

in food

_safety

and

_production

_efficiency.

Increased disease resistance will also

_improve

the welfare of livestock. The latter _{consequence may} make this

_type

of

_genetic

engineering

more

_acceptable

to the

_general

_public

than other

_types

of gene transfer. If there is one attribute that is common to

_viruses,

it is the lack of

_uniformity

in all

_aspects

of their existence.

_{Nevertheless,}

this review

_attempts

to find

_general

elements and common

_patterns

in the

subject

discussed. As

background

for the

discussion of the

_subject,

the article deals

_briefly

with coevolution of hosts and

parasites

and

_principal

elements of virus-host

interactions,

and reviews

_past

im-provement

of disease resistance in

_plants

and livestock

_by

conventional

_breeding

and

_{genetic engineering,}

as well as the

_potential

_{’biological}

cost’ of

_genetic

manip-ulation. It includes

_{prerequisites}

for and

_principles

of the

design

of new resistance

mechanisms,

and proposes

possible strategies

for the introduction of disease

resis-tance mechanisms

_by

_gene transfer.

The main

_goal

of this review is to inform readers from both research and

_industry

about this area of

_long-term

interest to animal

_agriculture

and outline the

potential

use of the

_concept

of new resistance mechanisms for the benefit of mankind and

improvement

of animal welfare.

COEVOLUTION OF HOSTS AND VIRUSES

Basic

_{understanding}

of the

_parallel

evolution of viruses and their hosts

provides

a

useful

_starting

_point

for the consideration of

_strategies

for

_genetic

_engineering

of

new mechanisms of resistance.

Therefore,

principal

elements of the coevolution of

Viruses are

obligatory,

intracellular

_parasistes

with limited _genome sizes that

code for functions the virus cannot

_adopt

from host cells

_(Strauss

et

_al,

_1991).

Viruses have their own

_{evolutionary histories, independent}

of those of their hosts.

It is not clear whether viruses had a

single

or

multiple

_origin.

The

_origin

of a virus

is defined as that time when its

_replication

and evolution became

_independent

of the macromolecules from which it was derived

_(Strauss

et

_al,

_1991).

Viruses may have arisen

(1)

by

selection from an

organelle;

(2)

from cellular DNA or

RNA

_components

that donate macromolecules which

_gain

the

_ability

to

_replicate

and evolve

_{independently;}

or

(3)

from

self-replicating

molecules.

_Polymers

of

ribonucleotides can contain both the information

_required

and the functional

capacity

to form a

self-replicating

_system

(Watson

et

_al,

_1987).

The main mechanisms of viral evolution are

_mutation,

_{recombination,}

and

gene

duplication.

Viruses have a _very short

_generation

interval and

high

mu-tation rate. For

_example,

the mutation rate of a chicken retrovirus is

10-

5

nucleotide/replication cycles - approximately eight

orders

_higher

than that of the host cell genome

(Dougherty

and

Temin,

1988).

Nevertheless,

the virus

_always

re-tains its

_origin

of

_replication.

Recombination has also a

_large

role in viral evolution

because it allowed viruses to

_{’try out}

new _{gene combinations’.} An

example

of an

unusual

_acquisition

of genes

by

a virus are three tRNA _{genes in}

bacteriophage

T4 -a

_type

of _gene

_only

observed in

_eukaryotes

_(Gott

et

_al,

_{1986). Although}

it is

_possible

that _{the genes evolved within}

T4,

the

phage

may also have

acquired

the genes from

an

_eukaryotic

host

(Michel

and

Dujon, 1986). Similarly

some retroviruses such as

Rous sarcoma virus

_acquired

_oncogenes for _{their genome.}

In

_general,

DNA viruses are more stable than RNA viruses and do not cause

rapidly moving pandemics

as is the rule for RNA

_viruses;

in

_contrast,

DNA viruses tend to establish

_persistent

or latent infections which _may lead to

_malignant

transformations

_(Strauss

et

_al,

_1991).

_Exceptions

to the

_general

rule include the

herpesvirus

of Marek’s

disease,

a DNA virus that can cause

_rapidly

_moving

disease outbreaks in

_chickens,

and the avian leukosis

_viruses,

RNA viruses that exhibit a

period

of

_latency

and seldom cause

_high

_mortality.

A disease of the host is not an

evolutionary goal

of the

_parasite.

Compatibility

is

_preferable

to

_{incompatibility.}

Subclinical infections are _common;

_they

are the rule - diseases the

_exception.

There is no selective

advantage

to the virus in

_making

the host

_ill,

unless the disease aids in the transmission of the virus to new

_hosts,

such as in the case of diarrhea. In some

_instances,

disease may also result from

an overzealous immune

_system.

Hence the

_interplay

between microbes and hosts

should not

_necessarily

be seen as an

ongoing

battle but as a coevolution of

_species

(Pincus

et

_al,

_1992).

PRINCIPAL ELEMENTS OF VIRUS-HOST INTERACTIONS

General considerations

Susceptibility (in

the narrow

_sense)

is the

_capacity

of cells to become infected. For a

virus to survive and

_reproduce,

essential viral genes have to ensure:

(1)

_replication

enzymes, to

actually

replicating

the viral genome,

although

even the most

self-dependent

viruses use some host cell function in the _process;

₍₂₎

_packaging

of the

genome into virus

particle -

viral

_proteins

do the

_{packaging, although}

host

_proteins

may

complex

with viral ones in the process; and

(3)

alteration of the structure

or function of the infected cell - the effects _{may range} from cell destruction to

subtle,

but

_{significant changes}

in function and

_antigenic

_specificity

of infected cells. In

_general,

once it

_enters,

no virus leaves a cell

unchanged.

During

their

_replication,

viruses

_exploit

host cell molecules at the expense of the cells. There are three

_types

of viral infection

(Knipe, 1991). (1)

In

_{nonproductive}

cases the infection is blocked because the cell lacks a

_component

essential for viral

replication.

The viral _genome may be lost or remain

_integrated

in the host _genome.

The cell may or may not survive or, if

growth

properties

of the cells are altered

_by

the

_{virus, oncogenic}

transformation may take

place.

(2)

Productive infection is when the cell

_produces

the virus

_but,

as a _consequence, dies and

_lyses.

(3)

Productive

infection is when the cell survives and continues to

_produce

the virus.

The levels of

_injury

to the cells

_resulting

from viral infection range from no

visible effects to cell death and include inclusion

_body

or

_syncytium

formation

and cell

lysis.

In most instances cell

_injury

is a _consequence of _{processes necessary}

for virus

_replication

but at least in one known

_instance,

the

_penton

_protein

of the

adenovirus,

which has no known _purpose in the viral

_cycle,

causes

_cytopathic

effects

in

_monolayer

cells

_(Valentine

and

_Pereira,

_1965).

Genetic

_{engineering strategies}

that

_{prevent entry}

of viruses into host cells would

be effective

_against

all three

_types

of viral infection. Other

_strategies

discussed

below can deal with various

_stages

of viral life

cycles

and would

accordingly

affect

the outcome of viral infection.

To

_provide

a basis for the examination of the

_{opportunities}

to devise and

genetically

engineer

new resistance

mechanisms,

the viral life

cycle

that consists

of three fundamental

_steps,

_{attachment, penetration,}

and

_replication

_(Roizman,

1991)

will be examined _{in sequence.}

Attachment

of virus to the

host

cell

Attachment of the virus to the host cell

_is,

in most

_instances,

_through

a

specific

binding

of a virion

_protein,

the

_{antireceptor,}

to a constituent of the cell

_surface,

the

receptor.

Complex

viruses,

such as

_vaccinia,

_may have more than one

_species

of

_antireceptor

or

_{antireceptors}

_may have several

_domains,

each

_reacting

with a

different

_receptor.

Mutations of

_receptors

_may cause a loss of the

capacity

of a

receptor

and

_antireceptor

to interact and thus lead to resistance to viral infection. It seems

_likely

that mutations in

_{antireceptors}

_preventing

viral attachment will be

automatically

eliminated from viral

evolution,

unless

they

are able to interact with

a substitute host.

The number of

_receptors

for which information is

_accumulating

is

_rapidly

increasing. Examples

in table I show that

_receptors

are

mostly glycoproteins.

Not all

cells in a

_{susceptible organism}

_express viral

_receptors,

a

_phenomenon

that _may limit

susceptibility.

Even

_though

our

_{understanding}

of

receptors

is still at an

_early

_stage,

it is obvious that viral

_receptors

are molecules that have a normal

_{physiological}

While there is a

_great

deal of

variability

in the

types

of molecule in viral

_receptors,

some cell surface molecules are used

_{by multiple,}

often unrelated viruses

(table I).

When viewed across host

_species,

for

_{example, histocompatibility}

molecules are

receptors

for both Semliki-Forest

_togavirus

and human

_coronavirus;

sialic acid residues serve as

_receptors

for both the influenza

_myxovirus

and

_reoviruses,

_although

there are rotaviruses that do not

_require

their presence

(Mendez

et

al,

1993)

and low

_{density lipoproteins}

_(LDL)

are

_receptors

for both the human minor cold

_picorna

virus and avian leukosis viruses.

Viruses

_compete

with molecules that

_require

_receptors

for a

_{physiological}

func-tion of the host. For

_example,

LDL and the human minor rhinovirus

_compete

for

LDL

_receptors

_(table

_I),

and cells with

_{down-regulated}

LDL

_receptor

_expression

yield

much less virus than

_up-regulated

cells

_(Hofer

et

_al,

_1994).

Viruses tend to use

abundant molecules as

_receptors,

so that reduction in

_availability

of the molecules

for the

_{physiological}

function is not

_lethal,

or molecules whose function can be

substituted

_by

other molecules. There are alternative viral

_strategies

to deal with the

_receptor

_problem.

The

_part

of the

_{sodium-independent}

_transporter

of cationic amino

_acids,

used as the

_receptor

for

_ecotropic

bovine leukemia virus

_(table

_I),

is

different from the

_part

of the

_protein

_directly

involved in the amino-acid

transport

function. Thus the

_{physiological}

function of the

_receptor

can

_{continue, despite}

bind-ing

of virus to the

_receptor

_(Wang

et

_al,

_1994).

Another

_example

_confirming

this

possibility

is the

sodium-dependent

transporter

of

_inorganic

phosphate

that serves as the

_receptor

for the

gibbon

_ape leukemia virus

(table I).

Productive infection of

cells

_expressing

this

_receptor

results in

_{complete blockage}

of the

_uptake

of

_inorganic

phosphate

mediated

_by

the

_receptor.

_{Nevertheless,}

the infection is not

_cytotoxic.

Hence,

there is

_likely

more than one

_phosphate

_transport

mechanism in these cells

(Olah

et

_al,

_1994).

This

aspect

of viral

_strategies

may open up

possibilities

to block the

_receptor

_sites,

thus

_preventing

_entry

of a virus without serious

impairment

of

physiological

function of the

_receptor.

The

_receptor

for

_herpes

_simplex

virus

_exemplifies

a situation of

special

interest

from the

_point

of view of future

_engineering

of disease resistance. The viral

receptor

heparan

sulfate is

_present

on cell surfaces but

_body

fluids also contain

_heparin

and

heparin-binding

proteins,

either of which can

_prevent

_binding

of

_{herpes simplex}

virus to cells

_(Spear

et

_al,

_1992).

Hence

_spread

of the virus is

_likely

influenced

by

both immune _response and the

_probability

that the virus will be

_entrapped

and inhibited from

_binding

to cells

_by

extracellular forms of the

_receptor

_(heparin

or

_heparan

sulfate).

_Similarly,

soluble molecules of the CD4

_receptor

for human

immunodeficiency

virus,

as well as

_fragments

of the critical CD4 domains can inhibit

infection

_(Smith

et

_al,

_1987).

It has been

_suggested

that a secreted

_receptor

for avian leukosis virus

_{might similarly}

be able to neutralize the virus

_(Bates

et

_al,

_1993).

Penetration of a virus into the cell

Penetration of a virus into the cells is

_usually

an

_{energy-dependent}

_process that occurs almost

instantly

after attachment. As summarized

_by

Roizman

_(1991),

penetration

can occur as

(1)

translocation of the entire virus

_particle

across the

in-side

_{cytoplasmatic}

vacuoles;

or

(3)

fusion of the cell membrane with the virion

envelope.

Non-enveloped

viruses

_penetrate

host cells

_by

the first two processes.

Uncoating

of the virus

_particle

takes

_place

after

_penetration.

For some

_viruses,

such

as

_{orthomyxoviruses}

and

_{picorna viruses,}

divestiture of the

_{protective envelope}

or

capsid

takes

_place

_upon their

_entry

into cells. For

_others,

such as

_{herpes viruses,}

the

_capsid

is

_{transported along}

the

_{cytoplasmic cytoskeleton}

into nuclear pores. With

_reoviruses,

_only

a

_portion

of the

_capsid

is removed and the viral _genome expresses all its functions even

_though

it is never

fully

released from the

capsid.

While several

_{genetic engineering strategies}

to

_prevent

attachment of viruses to

host cells can be devised and are

_{proposed below, strategies}

to

_prevent

_penetration

of viruses attached to cells are much less obvious.

Virus

_{multiplication}

Viruses use _many

_strategies

for

_replication

_leading

to

_{(1) encoding}

and

_organization

of viral genomes,

(2)

expression

of viral genes,

(3)

replication

of viral genes, and

(4)

assembly

and maturation of _{viral progeny. The}

_key

event in these _processes is

the

_synthesis

of viral

_proteins.

_Regardless

of its

_{size, organization,}

or

_composition,

a virus must

_present

to the cell’s

_{protein synthesizing}

mechanisms an mRNA that

the cell

_recognizes

and translates.

The interaction between the viral cell attachment

_protein

and host-cell

recep-tors is the

_principal

determinant of

_tropism,

but there are other factors involved.

For retroviruses and

_{papovaviruses,}

_cis-acting

elements of the viral _{genome, gene}

enhancers,

which are

_usually

50-100

_bp

in size and often

repeated

in

_tandem,

stimu-late

_{transcription}

_(Serfling

et

_al,

_1985).

_They

_may serve as an

_entry

_point

for RNA

polymerase

II. Enhancers _may be both

_{cell-type-specific}

and

cell-differentiation-specific,

in that

_they

function

_mainly

in certain cell

_types

_(Tyler

and

_Fields,

_1991).

For avian

_{retroviruses,}

enhancer

_regions

within the

_long

terminal

_repeat

_(LTR)

are an element of the viral genome that determines cell

tropism

of disease

expression

(Brown

et

_al,

_1988).

The cell

_imposes

three constraints on the virus at the

_point

of virus

multipli-cation.

₍₁₎

The cell may lack enzymes to

_synthesize

mRNA off the viral RNA

genome, or _may lack _enzymes to transcribe viral DNA.

₍₂₎

_Eukaryotic

host cell

protein-synthesis

machinery

translates

_only

_{monocistronic messages} and does not

recognize

internal initiation sites within mRNA. As a _consequence the virus must

synthesize

either a

_separate

mRNA for each _gene or an

mRNA

_encompassing

a

’polyprotein’

to be later cleaved.

₍₃₎

The

_expression

of viral

_proteins

is _in compe-tition with cellular _genes. Viruses evolved

strategies

that either confer

competitive

advantage

to viral mRNA or abolish translation of cellular mRNAs.

The host _range of a virus defines both the kinds of tissue or cells and animal

species

in which a virus can enter and

_multiply

_{(Roizman, 1991).}

_Receptors

_may be

_species

_specific.

For

_example,

the

_poliovirus

_receptor

is

_only

found on

_primate

mammalian cells

_(McLaren

et

_al,

_1959).

A

_{tissue-specific}

_receptor

is

_exemplified

by

the CD4

_receptor

for the HIV

_virus,

which is

_present

_only

on

_{T-lymphocytes}

(table I).

Species-specifity

of

_receptors

is one of the

_components

of non-host

Other virus—cell interactions

Infection with some viruses leads to inhibition of

_{transcription}

of cellular

protein-coding

genes

by

host

polymerase

II,

possibly through

competition

for

transcription

between cellular and viral genes.

Herpes

simplex

virions contain a

_{transcriptional}

activator

_complex

_(Post

et

_al,

_1981),

while adenovirus

_provides

a

_trans-acting

EIA _gene

_{product responsible}

for increased

_polymerase

activity

after adenovirus

infection

_{(Nevins, 1986).}

Viruses can also induce or _express new

DNA-binding

proteins.

Thus a retrovirus encodes a

_homolog

to cellular

_{transcription}

factor AP-1

(Bohmann

et

_al,

_1987).

Splicing

of viral mRNA _{precursors is}

_{accomplished by}

cellular _enzymes. Influenza and retroviruses can

_regulate

the extent of the

_splicing,

adenovirus inhibits matu-ration of cellular

_mRNA,

and influenza virus

transcription

complexes

intervene in the host mRNA maturation

_{(Knipe, 1991).}

Many

viral mRNAs are

_capped,

in that

_they

contain a

_single

_major

initiation site

near their 5’

end,

and their translation is similar to that of host mRNA.

However,

inhibition of host mRNA translation

_provides

the virus with increased

_availability

of ribosomal units. Thus

herpes

simplex

and

poxvirus

degrade

cellular mRNA to decrease its translation

_(Inglis,

_1982;

Fenwick and

_McMenamin,

_1984).

Other mechanisms include

_competition

for the host translational

apparatus

by

production

of

_large

amounts of viral

_mRNA,

or viral mRNA with

higher

affinity

to ribosomes than cellular mRNA

_{(Knipe, 1991)}

and

_changes

in

the

_specificity

of host translational

apparatus;

for

example,

extracts from

poliovirus-infected

cells translate

_poliovirus

but not host mRNA

_(Rose

et

_al,

_1978).

Both RNA and DNA viruses cause inhibition of host-cell DNA

synthesis (Knipe,

1991).

Eukaryotic

cell

_proteins

contain

_signals

that

_target

them to a

_specific

cell

compartment

or

organelle.

Viral

_proteins

_may also contain similar

_signals

for their

localization within the cell. Viral

_proteins

make use of cellular

_{chaperone proteins}

to

secure their _proper

_{folding. Similarly,}

_many

_{post-translational}

modifications of viral

proteins

are

performed

by

cellular _enzymes. For

example,

tissue-specific

_proteases

cleave

_specific

_proteins

on the virion surface thus

_facilitating

virion

_infectivity

(Scheid

and

_Choppin,

_1988).

Maintenance of viral DNA in the host cell and release of progeny virus There are two

_types

of mechanism for

_maintaining

viral DNA in the host cell:

₍₁₎

virus DNA is

_integrated

into the cellular _genome, eg, in

retroviruses;

or

(2)

viral

DNA is maintained as extrachromosomal circular molecule in the infected

cell,

eg,

Epstein-Barr virus,

or bovine

_papilloma

virus. Viruses that

_persist

in the

_body

may cause

_damage,

and

_prevention

of

_persistence

_may be the next best defence if

prevention

of virus

entry

is

_impossible.

Persistence is

_usually

in differentiated cells that remain

_{morphologically unchanged}

but _may lose their differentiated or

’luxury’ function,

as well as their homeostasis. Persistent viruses can

_negatively

influence host cells in two _ways:

₍₁₎

_{virus presence} and

replication

causes

damage

resulting

in a selective

_{disadvantage;}

and

₍₂₎

in such a _way that the virus will

_gain

an

_{evolutionary advantage}

for which there will be selection _pressure to maintain.

Alternatively,

some viruses

_undergo

a

_latency

_stage

in their life

cycle

that seems to

Enveloped

viruses move from infected cells either

by

_budding

through

the

plasma

membrane or

_by

secretion vesicles

containing

virus

_particles

within the

plasma

membrane

_{(Knipe, 1991).}

_{Non-enveloped}

viruses are

mostly

released

by lysis

of the

cells but

_they

can also leave without cell

lysis

as in Simian virus 40

_(Norkin

and

Ouelette, 1976).

Spread

of virus

_through

the host

_body

To facilitate their survival and

spread

throughout

the

_body,

some viruses have

evolved

_strategies

to modulate the immune response of their host to their

favor,

a

_phenomenon

_recently

reviewed

_{by Fujinami}

_(1994).

Virus infection can lead to

development

of immune responses

against

the host’s own tissues and viruses can

also code for

_proteins,

_homologous

to cellular

_proteins,

that

_modify

the host’s immune _response. For

_{example, Epstein-Barr}

virus

_produces

a BCRF1

_protein

similar to the interleukin IL-10

_protein

_{(a cytokine-inhibiting factor)}

that inhibits the

_production

of IL-2 and

_IL-3,

tumor necrosis

_factor,

_gamma

_interferon,

and

macrophage-granulocyte colony-stimulating

factor. The

_herpes

simplex

virus-1

(HSV-1)

but not HSV-2 can interfere with the

_complement

_system

_by

producing

a

_protein

that acts as a

_receptor

for the

_component

of the

_complement

cascade.

Virus infections can also interfere

_directly

with the

_major

histocompatibility

_system

(MHC).

Cytomegalovirus

encodes an MHC class I

_{heavy-chain homolog}

that limits

expression

of the cellular class I molecules on cell surfaces and this _may reduce

killing

of infected cells

_by

host defences.

EXISTING RESISTANCE MECHANISMS Non-host resistance

Most animal and

_plant

_species

are resistant to the

_great

_majority

of viruses. Non-host resistance is the

_rule,

_{susceptibility}

the

_{exception. However,}

the nature of

non-host resistance is not

_sufficiently

understood to

_{fully explore}

the

_{incompatibility}

between viruses and non-hosts

_(Wilson,

_1993).

Nevertheless,

it is certain that we, as well as all

_animals,

are

_{&dquo;continuously}

bathed in a sea of

_microbes,

_yet

harmed

_by

a

relatively

few&dquo;

(Oldstone, 1993).

To

_coexist,

viruses and their hosts

have

established,

to a

_greater

or lesser

_degree,

an

equilibrium.

In

_general,

normal

coevolution of

_parasites

and their hosts is from

_{disoperation, through exploitation,}

to toleration and from facultative to

_{obligatory mutualism,}

but

_genetic

_changes

_may also

_bring

reversals to this _process

_{(Dobzhansky, 1959).}

None of the

_strategies

for the creation of _new,

_{genetically engineered}

viral resistance mechanisms

_proposed

in this article are derived from non-host resistance.

_{Nevertheless,}

a brief discussion of the

subject

is included to stimulate further

_exploration

of this

_{widespread phenomenon}

as the

possible

basis for

_protection

of livestock

_against

viruses.

Some

_knowledge

of non-host resistance mechanisms is

_emerging

from

experi-mentation with

_plant

viruses that infect

_permissible

but

_normally

resistant cells

_by

bypassing

the resistance barrier

_(Dawson

and

_{Hilf, 1992).}

Viral host _{range is}

deter-mined

_by

interactions between

_existing

viral _gene

_products

and

_{corresponding}

host

not determined

_by

a

particular

_gene

product

that enables the virus to overcome host

defences but

_by

a ’fit’ between viral _gene

_product

and _{certain gene}

products

of the

host. There are two

_general

_{prerequisites}

for successful infection:

₍₁₎

Presence of all

conditions necessary for viral infection. Absence of the conditions results in

_’passive

resistance mechanisms’ in

_plants,

that tend to be recessive or

_incompletely

dom-inant.

₍₂₎

Absence of successful host defences.

_Adaptation

mechanisms of viruses that enable them to infect

_potential

hosts

_protected

_by

non-host mechanisms _may

include an

ability

to overcome a host block

_by

a mutation or recombination with another

_virus,

or

_acquisition

by

the virus of

capabilities formerly

_provided

by

the

hosts that are not available in resistant

_plants.

A virus can

_capture

such

_genetic

information from the host. Non-immune mechanisms

There are _many mechanisms of resistance to viral diseases. For our _purposes,

emphasis

will be

_placed

on non-immune mechanisms. Of

_particular

interest in this review are those mechanisms that

_prevent

the

_entry

of viruses into host cells. Viral

receptors

can be variable so that some alleles of the

_receptor

_may make the

potential

host resistant to viral infection.

_However,

it is

_{only rarely}

that resistance to infection is observed in otherwise

_susceptible

host

_species.

This indicates that

_during

virus-host

_coevolution,

viruses tend to utilize

_{evolutionarily}

stable molecules as

_receptors.

Resistance to infection

_by

_parvovirus

B 19 in some humans is due to lack of a

specific

virus

_receptor.

_People

who do not have the

_erythrocyte

P

_{antigen parvovirus}

receptor

(Brown

et

_al,

₁₉₉₃₎

are

_naturally

resistant to the virus

_(Brown

et

_al,

1994).

Another

_example

is resistance to coronaviruses in mice. A monomeric

_protein

has been identified as a

_receptor

for mouse

hepatitis

virus on intestinal and liver

cells. The _presence of this

_receptor

_{appears to} be the

_principal

determinant of

susceptibility

to infection

_(Boyle

et

al,

1987).

Similar variation in viral

_receptors

is

observed in

_genetic

resistance to avian leukosis virus

_(ALV)

infection in chickens

(Payne,

1985).

The ALV

_receptors,

which

_belong

to the

_family

of

_receptors

for LDL

_(Bates

et

al,

1993),

include recessive alleles that do not allow viral

entry

into

potential

host cells and render some chickens resistant to the virus. The

_receptor

for

_subgroup

A ALV was shown to map to

TVA

*

S

known as the dominant _gene for

susceptibility

to

_subgroup

A virus

_(Bates

et

_al,

_1994).

Susceptibility

of cells to infection needs to be

_{distinguished}

from

_{permissiveness,}

which can be defined as the

_ability

of a cell to

_support

viral

_replication.

For

_example,

chick cells are not

_susceptible

to

_poliovirus

but are

_permissive

to its

_replication

following

their transfection with

_poliovirus

RNA

_{(Roizman, 1991).}

Such cells are

potential

hosts for a

_virus,

_providing

a mutation

_provides

means for the virus to enter the cells.

In

_laboratory

_mice,

alleles at the Fv-4 locus determine

_{susceptibility}

to infection with

_ecotropic

murine leukemia viruses and the resistance is dominant in hetero-zygous mice

(Ikeda

and

Odaka, 1983).

A viral

protein gp70

normally

interacts with the viral

receptors

on cells.

However,

in resistant

_mice,

the

specific

_receptor

on cell

envelope

also controls resistance to infection

_{by subgroup}

E virus

_(Robinson

et

_al,

1981).

Resistance of mice to certain strains of influenza virus is a dominant trait

associated with the allele Mx on chromosome 16

(Staehli

et

_al,

_1986).

The resistance is mediated

_by

action of

_alpha-

and beta-interferons that induce Mx

protein

expression

which inhibits

_synthesis

of viral mRNA

_(Krug

et

_al,

_1985).

A recent review of

_{natural, ’preimmune’}

resistance loci in mice

_(Malo

and

Skamene,

1994)

includes genes

controlling

resistance to influenza

virus,

cytomegalo-virus,

ecromelia,

Friend leukemia

_virus,

mink cell

_{focus-forming}

_virus,

_Moloney

leukemia,

radiation

leukemia,

and Rous sarcoma virus. The resistance _genes

repre-sent a

_variety

of mechanisms that do not involve viral

receptors.

For

_example,

the Cmvl _gene, associated with resistance to

_{cytomegalovirus,}

_appears to control host responses mediated

by

natural killer and

inflammatory

response cells.

Similarly,

the resistance loci in Friend leukemia control the

_{susceptibility}

of

target

cells to viral

replication.

Immune mechanisms

It is not the purpose of this review to

_provide

a detailed account of immune mechanisms that

_protect

against

virus infection. The brief text below will

_give

only

a

general

outline of immune _responses and

examples

of how the

_system

_may

be influenced

_by

viruses.

Acquired

immune responses involve

_phagocytic,

humoral and cell-mediated

systems.

Only

the cell-mediated immune response that is

especially

effective

against

cells

_{containing actively replicating}

virus

_and,

as a

_rule,

is the most

_important

defence

_against

viral infections will be discussed

_briefly.

The cellular immune

system

becomes sensitized to viral infection

_only

after viral

_proteins

are

_degraded

to short linear

_{peptide epitopes}

that become

_complexed

with class I or II

_major

histocompatibility complex

proteins.

The

_{resulting complexes}

are

transported

to

cell

_surface,

where

_they

are

_presented

as ’non-self’ entities to

_{T-lymphocytes.}

If the viral

_antigen

has not

_previously

encountered the T-cell

_repertoire

of the

_host,

the initial

_{antigen-specific}

activation event

_requires

appearance of

MHC-peptide

complexes

on

_{antigen-presenting}

cells. But if activated

_{T-cells, previously}

sensitized

to the viral

_epitopes

are

available,

then a broader class of

_{antigen-presenting}

cells can be

_targeted

for clearance

_{by cytotoxic}

T cells. In both

_events,

the

_ability

to

discriminate self molecules from the viral

_{epitopes depends}

on the

_presentation

of the non-self

_peptide

to T-cells in

_{specific peptide-binding}

grooves of the MHC molecules on

_{antigen-presenting}

cells.

McFadden and Kane

₍₁₉₉₄₎

summarized how DNA viruses

_perturb

the MHC and alter immune

_recognition.

A number of gene

products

of DNA viruses have been identified as

directly affecting

MHC

_expression

or

antigen

_{presentation,}

whereas

RNA viruses interact with MHC

_by

indirect mechanisms. Most DNA viruses are

able to modulate cellular

_immunity.

It seems that _many viral _gene

_products

remain

to be identified _among the open

reading

frames of as

yet

unknown function that

exists in these viruses. Besides a trivial

_strategy

of

_hiding

DNA molecules in

cells,

such as neurons that lack MHC surface

molecules,

viruses can

_modify

MHC

There is now evidence that viruses can combat antiviral effector T cells

_directly

by blocking

their antiviral

activity

(Bertoletti

et

_al,

_1994).

In humans infected with HIV-1 and

hepatitis

B

_viruses,

_naturally

_occurring

variants of

_epitopes

recognized by cytotoxic

T

_lymphocytes

_{may act} as

_antagonists

in vivo because

the

_{corresponding peptides}

prevent

a

_cytotoxic

T cell response.

Although exactly

how the

_antagonists

function is not

_known,

it is evident that the presence of these

antagonists

prevents

the T cell from

_performing

its function.

Endogenous

viruses

_represent

a

_separate

phenomenon

with

regards

to the immune

_system.

As a

_rule,

the host is

_{completely immunologically}

tolerant to

endogenous

viruses.

However,

antibodies

against

endogenous

retroviruses were

found in mice

_(Miyazawa

et

_al,

_1987).

How the immune

_system

makes antibodies

against

endogenous

retroviral gene

products

is unknown but this

ability

may relate to the

_expression

_{of such genes after the establishment} of

_{immunological}

tolerance

to

_endogenous

retroviral

_{antigens expressed}

earlier in life

_(Miyazawa

and

_Fujisawa,

1994).

A similar

_delay

in

_expression

of the

_endogenous

viral gene ev-6 has been described in chickens

_{(Crittenden, 1991)}

and may serve as a model for construction

of similar

_{’self-vaccinating’}

_transgenes

in the future.

Pathogen-mediated

resistance

Given the

_potential

benefits that can be derived from the use

_by

the host of

parts

of a

_pathogen’s

_genome to induce

_resistance,

the

_paucity

of

_{pathogen-mediated}

resistance mechanisms in nature is

_surprising.

The situation

_begs

the

_question

whether evolution exhausted all such

_{possibilities}

in the

_development

of host

defences.

_Why

did certain mechanisms

_develop

and others not? A reason for the

absence or rare occurrence of

_{pathogen-mediated}

defence mechanisms may be that

they

encompass some

_disadvantage

for the host.

One

_example

in which a viral genome has become an

_integral

_part

of the host are

_endogenous

_proviruses

found in germ cells of all vertebrates. For

_example,

in the

_laboratory

mouse

_{endogenous proviruses}

occupy more than

0.5%

of the

cellular DNA

_(Pincus

et

_al,

_1992).

_{In the genomes} of

_chickens,

there are several

families of retrovirus-related

_permanent

insertions. In the most

_thoroughly

studied

family

of

_endogenous

viral _genes, there are more than 20

_endogenous

_proviruses

in various

parts

of the _genome

_{(Crittenden, 1991).}

The _presence of some of these

proviruses

may interfere in the

spread

of the

generally non-pathogenic endogenous

virus

_{produced by}

other such

_{proviruses. However,}

the

_endogenous

_proviruses

do not

protect

the host

_against

infection with similar but more

harmful,

_pathogenic

exogenous viruses. On the

contrary,

the

_{antigenic similarity}

between the

_products

of the

_endogenous

_proviruses

and the exogenous viral

antigens

reduces the

_ability

of birds with certain

_types

of these

_proviruses

to mount an immune _response

_against

the exogenous virus

(Crittenden

et

_{al, 1984;}

Gavora et

_al,

_1995b).

A

_possible

reason

why

other

_endogenous

_proviral

sequences did not evolve as resistance mechanisms

CONVENTIONAL METHODS FOR IMPROVEMENT OF

RESISTANCE AND POSSIBLE ADVANTAGES OF GENETICALLY

ENGINEERED RESISTANCE MECHANISMS

Genetic variation is a

_{prime prerequisite}

for

_genetic

_change

_by

selection. As a

general

rule, genetic

variation exists in the

ability

of livestock to tolerate infectious

diseases. And it was this variation that allowed

_populations

of domestic animals

and birds to survive under continuous exposure to

_{rapidly evolving}

disease

_agents.

Before

domestication,

disease resistance of

_today’s

livestock

_species

was influenced

by

natural selection and the current status of variable resistance to

_multiple

disease

agents

can be considered to be the result of a _response to the selection pressure of

multiple pathogens.

As a _consequence of

_{domestication,}

a

_significant

new element that entered this

evolutionary

system

was artificial selection for characters that benefit humans as users of livestock.

Simultaneously, housing

conditions evolved towards increased

concentration of animals and birds and thus

_{provided opportunities}

for

_spread

of

pathogens. Improved

disease

prevention

and control measures now

provide

some

compensation

for the

_larger

_population

sizes used in current

_production

_systems.

Selection for disease resistance

plays

a

_relatively

minor but

_increasingly

impor-tant role in livestock

_improvement.

The choice of selection criteria and the

_emphasis

they

receive in the context of total selection _pressure available to a

_practical

breeder are decided

by

market demands and economic considerations. Disease resistance traits receive attention from the breeders

_mainly

when a

_specific

disease is a

_major

cause of economic loss.

Although

in most instances

_{existing genetic}

variation

_provides

an

adequate

basis

for resistance

_selection,

selection _may not

_always

be

_practised.

Such selection is

expensive

because the

_expression

of resistance traits

_requires

_exposure of selection candidates or their relatives to the disease

_agent.

This is

_why

industries

prefer

to look for indirect selection

_techniques

that do not

_require

_{pathogen challenge.}

Recent

developments

in gene

mapping

provide good

prospects

for progress in this direction. Indirect selection for resistance to the

_herpesvirus

of Marek’s disease in

chickens,

by

increasing

the

_frequency

of the ’resistant’

_major

_{histocompatibility haplotypes,}

is one

_example

of such a

_technique.

It has been

_{practised by}

most of the world’s

poultry

breeding

companies

over the

_past

two decades

_{(Gavora, 1990).}

Conventional

_procedures

for direct and indirect selection for disease resistance will in the foreseeable future be the main route for

_{genetic improvement}

of disease resistance. One

_disadvantage

of their

_application

is the

_{general absence,}

with rare

exceptions

mentioned

above,

of

_genetic

variation in resistance to infection. Thus

genetic improvements

in disease resistance

_by

conventional means lead

_mostly

to better resistance of livestock to disease

_{development -}

a situation where the

organism

becomes infected but tolerates the

_pathogen

and reduces its ill effects.

Hence

_development

of new

_genetic

mechanisms that

prevent entry

of a

_pathogen

into the

host,

or otherwise

substantially

_improve

the

_position

of the host in

the

_{pathogen-host}

interaction is

_justified.

While conventional selection leads to

quantitative improvement

of

resistance,

the new mechanisms would

represent

a

qualitative

change

that,

at least in some

_instances,

will

_justify

the

_large

effort and

to be stable and remains effective

_despite

evolution of the

_pathogen

and functions without harmful effects on the animal’s

_production

_capacity.

_Improvement

in the

welfare of the modified livestock will be an

_automatic,

additional benefit.

In _crops

Despite large

differences between animals and

_plants,

sufficient similarities exist in their resistance mechanisms to

_justify

examination of the situation in

_plants

with

_regards

to

_{genetic engineering}

of viral resistance. For

example,

normal virus

replication

requires

a subtle balance of virus and host coded

_proteins,

_present

in

critical relative concentrations at

_specific

times and locations.

Therefore,

Wilson

(1993)

suggests

that any

unregulated

superimposition

of

_protein

or nucleic acid

species

interacting

with the virus can result in

_plants

in an

_apparently

virus-resistant

_phenotype.

The results from

_{experimentation}

with animal cells into which

a viral _gene was inserted indicate that a similar situation _may also exist in animals

(Gavora

et

_al,

_1994).

The idea that viral

_components

contained in

_plants

_might

interfere with virus

infection was first

_proposed

well before _gene transfer

_techniques

became available

(Hamilton, 1980)

and the

_concept

of

_{pathogen-derived}

resistance was first

_put

forward in a formal statement

_by

Sanford and Johnston

_(1985).

There are several

approaches

to the introduction of disease resistance

_by

gene transfer in

plants

(Fitchen

and

_Beachy,

_1993).

_They

include transfers of

_segments

of viral _genome

encoding

capsid

or coat

_proteins,

viral _sequences

_encoding

_proteins

that _may be subunits of viral

_replicase,

_sequences

_incapable

of

_encoding

_proteins,

entire genomes of

_defective,

_interfering

_viruses,

and

_complete

_genomes of mild virus strains. The

transgenes

may act on initiation of

infection, replication

of

virus, spread

of infection

throughout

the

_plant,

and

_symptom

_development.

The level of

_protection

derived from the

_transgene

_{ranges from low} to

_high

and its breadth of host _range from broad to narrow. The available data are not sufficient to

_firmly

establish the molecular mechanisms of the

_protection.

In

_{general, although}

a viral _{sequence may} confer

resistance in one virus-host

system,

an

analogous

_sequence from a different virus

in another virus-host

_system

may not be effective.

Protection conferred

_by

_sequences

_encoding

viral coat

_proteins

The

_{conceptual simplicity}

of the

_approach

and

_availability

of virus coat gene sequences facilitated broad

implementation

of this

strategy.

Fichten and

_Beachy

(1993)

list 19

_{published examples}

of this

_approach.

It is

_unlikely

that a

single

mechanism accounts for the observed resistance of the

_transgenic

_plants

but

regardless

of the mode of the

_transgene

_action,

resistance results from a block in an

_early

event in the infection process

(Fichten

and

Beachy,

1993).

In resistance

to some viruses other than tobacco

_mosaic,

it seems that accumulation of the coat

protein

transgene

RNA,

rather than the virus coat

protein

itself is

_responsible

for resistance. Resistance has been observed even in

_plants

that transcribed a

translation-incompetent

coat

_protein

mRNA

_(Kawchuk

et

_{al, 1991;}

De Haan et

_al,

1992).

It seems that even in the absence of

understanding

of its

_mechanism,

the