Introduction
One of the most common perceptions of a virus is to look at it as an
enemy, a pathogen, a "disease-causing germ". In veterinary virology,
this usually translates into "XY virus causes a devastating (severe,
economically important) disease in this or that animal species and may
(even) be transmitted to man". Indeed, many viruses have been recognized
as the causative agents of diseases, such as Rinderpest, Foot-and-Mouth
Disease, Malignant Catarrhal Fever, African and Classical Swine Fever,
Pseudorabies, Transmisssible Gastroenteritis, Canine Distemper, Feline
Parvovirosis, Rabies, and Influenza - to name but a few. On the other
hand, viruses with little or no known pathogenicity did not stir much
interest, were neglected at best, if not ridiculed in the scientific
community. Examples include bovine herpesvirus type 4, canine parvovirus
1, porcine circovirus, toroviruses, and lentiviruses in general, at
least before the advent of the AIDS epidemic.
Indeed, research was focused mainly on the disease-causing viruses.
Adaptation of viruses to grow in cell culture and the increasing knowledge
of molecular biology have contributed most to the expanding field of
virology in the second half of the 20th century.
As illustrated in Fig. 1, the number of appearances of the keyword "virus"
in Medline from 1960 to 1999 reveals two stepwise increases, the first
one in the mid-sixties reflecting the progress made in molecular biology
or, more specifically, in genetic engineering. The second step in the
mid-seventies coincides with the Asilomar conference, where scientists
declared their willingness to carry the responsibility for their work,
particularly in the field of genetic engineering. With the advent of
the AIDS epidemic, an exponential increase of virus-related literature
can be observed, which levelled into a plateau by the mid-nineties.
In 1999 the keyword "virus" was found 11.686 times, not even reaching
the number of quotes in 1994 (11.788 times). Is this statistic heralding
the doom of virology?
Emerging viruses and virus-like agents
The emergence of new viruses is not uncommon, as Table 1 shows. New
viruses can be assigned to two categories at least: (i) newly evolved
ones, like the influenza virus H5N1 Hong Kong [22]
or the porcine respiratory coronavirus (PRCV) [14,
19], and (ii) newly discovered ones,
like the Australian bat lyssavirus [10],
the porcine circoviruses type 1 [28,
29] and type 2 [2],
or Nipahvirus [5]. It may remain
obscure into which category a particular virus fits, and one may safely
assume that there are many viruses out there, which have not (yet) been
recognized as disease agents.
So why should we perpetuate the dogma that viruses are always disease
agents? On the other hand, should we really consider some viruses harmless,
only because we have not (yet) seen their association with disease?
Co-evolution of viruses and host defence mechanisms
In our daily research, we use viruses to study the host's defence system
and employ immunological tools to examine viruses. Despite of this mutual
usefulness, the interaction between viruses and their hosts is most
often pictured as a battle, a war-like situation. Therefore, only a
total victory, the complete destruction of the enemy, i.e. total clearance
of the virus from the organism, the population, the world, is considered
as a basis for lasting peace. In veterinary virology, this view is reflected
in political strategies to protect farm animals from highly contagious
disease agents, such as those recorded in List A of the Office International
des Épizooties (O.I.E.). Once the label "DANGEROUS!" has been
attached to a particular virus, it can be removed only with great difficulty,
mostly only after a change in the political climate. This fact leads
to strange situations, as e.g. in the pestivirus field, where classical
swine fever virus (CSFV) is in List A of the O.I.E. As a consequence,
CSFV-infected herds must be destroyed and control measures must be taken
to prove that the virus has been eliminated from the farm, the region,
the country in question. In contrast, the closely related bovine virus
diarrhoea virus (BVDV) and border disease virus - also pestiviruses
- are considered as innocuous and consequently neglected by the veterinary
authorities. This example emphasizes the most important argument, the
seeming truism that neither the virus nor its host are static entities.
Who can tell whether the vacuum created by the extinction of CSFV will
not be filled soon by another virus?
The first encounter between a new or exotic virus and a particular host
may result in a fatal outcome for both. This situation is illustrated
by reports of encounters between seemingly new viruses and unprepared
hosts, recently [5,18]
and in the past (e.g. [23, 24]).
The host's inadequate defence and/or the virus' poor adaptation may
result in death of the infected organism, and the virus would soon become
extinct if it destroyed the susceptible population too rapidly. Hence,
to survive in nature, any virus needs the host's functional defence
as much as the host itself - both the host and the virus are on a path
of co-evolution (Fig. 2) (e.g. [12],
and references therein). Again: both are essential, the defence mechanisms
for the survival of host and virus, and the virus for the constant education
and evolution of the defence mechanisms.
A dramatic example of this co-evolution, the HIV/AIDS epidemic, is
occurring before our eyes. In Botswana, a shocking 35.8% of adults are
infected with HIV http://www.unaids.org/fact_sheets/files/Africa_Eng.html
, which means that Africa will probably be repopulated by progeny of
HIV-resistant survivors of the infection. Genetic determinants for long-term
survival of AIDS have indeed been described [4,31],
as have viral determinants, which influence the same [1].
In my opinion, the eradication of a given virus will create an empty
ecological niche, soon to be taken by another agent, probably by one
less adapted to the host than the original virus. Thus, if extermination
of viruses is not the ultimate goal of veterinary virology, what is
its goal? Obviously, the direction of co-evolution is influenced by
external factors; understanding these factors is essential for steering
the co-evolution of host and virus a course that is beneficial for humans
and animals.
Development of vaccines
Modified live (mlv) and inactivated viral vaccines have a long history.
In recent years, however, it has become customary to start developing
and producing vaccines as soon as a new virus has been identified and
adapted to growth in cell culture. In my view, this has resulted in
a number of useless, if not dangerous vaccine preparations. One remarkable
example is the field application of a modified live African Swine Fever
(ASF) virus vaccine in Spain and Portugal, which almost led to the perpetuation
of ASF on the Iberian peninsula ([12],
and references therein).
Another example is the case of a canine coronavirus (CCV) vaccine. Although
no disease could be associated with CCV infection [26],
much effort was invested into developing a vaccine. To demonstrate its
efficacy, immunized and control dogs were treated with dexamethasone
after challenge, which resulted in diarrhoea in most (80%) control animals,
as well as in some (15%) vaccinees [9].
Using this approach it was shown that dogs could be protected from a
disease that does not even exist in nature.
The influenza viruses - much underestimated in veterinary virology -
may serve as a third example. Between 1985 and 1991, several subtype
H1N1 influenza A viruses had been isolated from patients in Mongolia.
Characterization by sequence analysis [3]
showed that these isolates were most closely related to strains isolated
from diseased camels in the same region at the same time. The camel
viruses were apparently derived from a UV-inactivated reassortant vaccine
(PR8/USSR/77) that had been prepared for humans (in Leningrad, 1978)
and used in the Mongolian population. The evidence was convincing: a
reassortant between two human influenza virus strains had caused severe
epizootics in camels, a species previously unknown to be a natural host
for influenza A viruses.
Which lessons should we take home from these incidents? I suggest the
following: (i) there is a need for better vaccines (Tab. 2) (ii) vaccine
development should take the natural co-evolution of viruses and their
hosts into account (iii) results obtained in model studies should be
interpreted with caution (iv) it is very clear that mistakes have occurred
in the past and more are bound to happen in the future. However, these
mistakes should not discourage us. Vaccine development should continue
with prudence and forethought, keeping the Japanese proverb in mind:
"Vision without action is a daydream. Action without vision is a nightmare".
Indeed, progress has recently been achieved in developing novel vaccination
strategies, epitomized by DNA or RNA vaccines (reviewed in [15])
and by the BAC-VAC principle [25].
BAC-VAC refers to an artificial bacterial chromosome harbouring the
entire genome of herpes simplex virus type 1 (fHSVDpac),
with minor deletions. The most important deletion concerns the signals
for packaging of the viral DNA into virions. This bacterially cloned
viral DNA is infectious and leads to replication, protein synthesis
and virus particle formation upon gene gun application. However, the
progeny is not infectious, because the viral DNA cannot be packaged.
This single-cycle replication was found to induce cytotoxic T-cells
(CTL), antibodies, and protection in a manner almost indistinguishable
from immune reactions after mlv immunization - and it proved superior
to inactivated or conventional DNA vaccines. Specifically, 1.5 mg
of fHSVDpac coated to gold particles yielded
the same amount of CTLs as 109 TCID50 of DISC
HSV-1 [17], but five times higher
antibody titers and antibodies of all isotypes; gB-specific CTLs could
even be measured without previous restimulation in vitro. BAC-VAC immunized
mice were protected against an intracerebral (i.c.) challenge infection
with 200 LD50 of HSV-1, which leads to disease and death
in control animals. Protection was cell-mediated, since serum transfer
had no effect.
Apart from its potential to induce an immunity similar to that after infection,
BAC-VAC is intrinsically safe: if ever the packaging signals would be
restored by recombination during co-infection with a wild type virus,
the immune system would profit from a lead over the multiplication of
the recombinant, and development of disease or even spread of the vaccine
virus would be quite unlikely.
Gene therapy
Casually, viruses have been paraphrased as "jumping genes" or "bad news
in an envelope". Both references acknowledge their potential for gene
therapeutic applications. From an opposite vantage point, a host species
may profit from a virus as a population control agent, as a biological
weapon that destroys competitors for limited natural resources by means
of what we may flippantly call "contagious gene therapy"; these viruses
may occasionally wreak havoc when a species is targeted, which did not
undergo co-evolutionary adaptation (Table 3).
However, a virus infection may also be looked at more favourably, as
a special form of "gene therapy". The host acquires the genome of an
infecting virus, and in the course of the ongoing virus-host interactions,
a plethora of host genes is regulated. This occurs in a variety of manners,
depending on the virus and the infected organism, and includes the multifaceted
cascade of immune responses (see e.g. [11,30].
Ultimately, the infected organism will not only survive but also have
acquired a selective advantage through an improved immune defence. In
a long-term perspective, such virus-host interactions lead into the
mentioned co-evolutionary pathway, with advantages on both sides. Thus
viruses may also be viewed as movable genes or extrinsic genotypes of
a host. At the same time, the virus profits from this mutuality by constantly
probing for the opening of new ecological niches. In this case, the
virus cannibalises genomic information from its host and modifies it
in a manner that suits its replication and survival in nature.
From this perspective, the virus could be looked at as a companion,
a helping hand, and it would seem plausible to ask one's friend for
even more help. Recently, Luo and Salzman (2000) have compared the efficiency
with the toxicity of DNA delivery systems [16].
They found that most often efficient delivery was obtained only at the
expense of high toxicity (Fig. 3). The lowest relative toxicity associated
with the highest efficiency was accredited to microinjection. Needless
to say that viruses easily surpass the efficiency of manual microinjection.
In Table 4 I have listed some advantages and disadvantages of viral vectors. In most cases advantages in one aspect are counterbalanced by disadvantages in other aspects.
Collaborative efforts between clinicians, gene therapy specialists
and virologists have resulted in helpervirus-free vectors, which combine
the advantages of different systems [6,20].
For example the large transgene capacity of up to 160 kbp and ability
to target neuronal cells of herpes simplex virus amplicons was successfully
combined with elements of the adeno-associated virus (AAV), which stands
for sustained gene expression and the possibility to integrate the transgene
at a specific location of the human chromosome 19 [7].
Moreover, a herpes simplex virus-Epstein-Barr virus hybrid amplicon
was used to convert cells to producers of retrovirus vectors, which
were enveloped for either ectotropic or amphotropic cell targeting in
gene therapeutic applications [21].
These vectors are not only tools for the treatment of humans suffering
from certain genetic diseases. They may be important for the analysis
and modification of virus-host interactions in general. Thus herpetic
stromal keratitis (HSK) is considered as virus-induced but sustained
by the immune system [27]. The ongoing
ocular inflammation can be suppressed by the topical administration
of IL-10 DNA [8]. This report indicated
that ongoing immunopathological events can be influenced by local gene
therapy. There is an enormous potential for helpervirus-free amplicon
vectors to modulating immunopathological diseases. By using them for
gene delivery, most caveats associated with conventional viral vectors,
as listed in Table 4, would appear manageable.
This may seem a truism, but I still want to emphasise: an enormous amount
of virological knowledge will have to be collected before such visionary
concepts will find their way into medical or veterinary applications.
Conclusions
Viruses are not necessarily disease agents; on the other hand, viruses
currently not associated with disease may not be that harmless. Viruses
and their hosts co-evolve, which has lead to more successful viruses
in the face of improved host defence mechanisms. New and better vaccines
are needed but should not be developed without taking the co-evolution
of viruses and their hosts into account. The use of viruses as vectors
for gene therapy is not without risks - after all, they are disease
agents. However, if treated with respect and studied with care and foresight,
they carry the promise of future mediators to improved human and animal
health and welfare. Cell cultures and animal models had their glorious
dominance in the past and may still be valuable in the times to come.
However, veterinary virology will continue to play an important role,
because animal viruses can be studied in their natural hosts, and creative
ideas may spark from this fortunate situation. To speak with James Richard
Broughton: "The only limits are, as always, those of vision". The world-wide
community of veterinary virologists must combine its knowledge with
that of specialists in immunology, molecular biology, gene therapy,
and especially with clinicians. Only then will it be possible to achieve
things previously thought to be inconceivable. To conclude with another
quote, by Robert Kennedy: I wish that you may dream of things that never
were, and ask why not?
Acknowledgments
Special thanks to all those who took their time to talk to me about
their views on virology, particularly to Ernst Peterhans, Bernard Roizman,
Cornel Fraefel, David Paton, Esteban Domingo, Etienne Thiry, Hans Koblet,
Luis Enjuanes, Marian C. Horzinek, Mark Suter, Peter Nettleton, Ricco
Wittek, Robert Webster, Robert Wyler, and to the many participants in
discussions after my presentation of these thoughts at the International
Congress of the European Society for Veterinary Virology (ESVV) in Brescia,
Italy (2000).
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