Canad. M. A. J.

Sept. 1, 1958, vol. 79

KOCH'S POSTULATES AND THE
MODERN ERA IN VIRUS
RESEARCH
C. R. ROBINSON,* M.A., M.B.,
B.Chir. ( Cantab.), Toronto
WITH THE WIDESPREAD USE of new techniques, a
new era has emerged in the field of virus investigation,
and nearly one hundred antigenically distinct
viruses have been isolated and identified since
1947.
These "new" viruses include the poliomyelitis,
Coxsackie and ECHO viruses, the croup associated
(CA) virus of infants, myxoviruses such as the
Sendai virus, haemadsorption (HA), adenoviruses
and other respiratory viruses, and the virus of
cytomegalic inclusion disease. These viruses are
widely disseminated, there is a high incidence of
carriers, and it is difficult to prove that the viruses
isolated are not fortuitously present in the patient.
Whereas the earlier phase of virus research might
be described as coming under the heading of
"Illness in Search of Viruses", the present position
has aptly been described as "Viruses in Search of
Disease".4
It may be claimed that as viruses multiply only
in the cells of the body they do in fact cause an
infection even when no clinical illness is manifested.
This problem is illustrated best by the
enteric virus group, which includes as its members
the poliomyelitis, Coxsackie and ECHO agents
which are found in the intestinal tract of patients
as well as apparently healthy persons. The problem
also concerns the adenoviruses, isolated from tonsils
and adenoids, and the herpes simplex virus.
In the case of the enteric viruses, an isolation of
the virus from the stool assumes greater significance
if similar isolations can be made from the blood or
sites remote from the gastro-intestinal tract such
as the cerebrospinal fluid, brain or other tissues.
Since there are many antigenic types of each of
the individual members of the enteric group, the
presence of antibodies to one or more of the
serotypes, as a result of past infection, is common.
It is necessary therefore to demonstrate a significant
rise of serum antibodies to the infecting
strain before concluding that the presence of antibody
indicates current infection.
The relationship of a virus infection to a specified
clinical illness may be difficult to assess even when
a virus isolation has been obtained, and an immunological
response demonstrated. This problem
presents itself when two illnesses occur at a relatively
short interval, caused by two distinct viruses.
The viruses of the enteric group persist in the stool
for over a month after infection; it is easily seen
that the occurrence of an additional infection by a
closely related agent within that period of time
FitzGerald Memorial Fellow, School of Hygiene, University
of Toronto. Present address: Department of Pathology,
Ottawa General Hospital.
ROBINSON: KOCHS POSTULATES AND VIRUSES 387
would cause a diagnostic dilemma. In such
an instance, two viruses would be isolated, and a
specific immunological response to each would be
demonstrated. It would be extremely difficult to
say from such evidence which virus was causing
the more recent of the illnesses.
Still another problem encountered when assessing
the relation of a virus to a given illness is
caused by the frequent absence of a distinctive
clinical picture. For example, the enteric viruses
commonly cause a mild featureless illness, and
similar problems often arise with the arthropod-
borne (Arbor) viruses and many other viruses.
The ease with which certain of these newer
viruses can be isolated from human beings has
thus raised problems very similar to those which
confronted the bacteriologist of an earlier generation
seeking to prove the etiological role of a
micro-organism in a given disease. What then are
the criteria which must be fulfilled in order that
a particular virus may be accepted as the cause
of a particular clinical syndrome? In the field of
bacteriology Koch's postulates2 have been used as
guiding principles in the determination of the
pathogenic role of a micro-organism, since the
latter part of the 19th century, although Koch did
not enunciate them in the categorical form in
which they are usually quoted.5
They run in general terms as follows:
"1. The organism should be found in all cases
of the disease in question, and its distribution in
the body should be in accordance with the lesions
observed.
"2 The organism should be cultivated outside the
the body of the host in pure culture for several
generations.
"3. The organism so isolated should reproduce
the disease in other susceptible animals."
During the last few years of intensive work on
virus diseases, it has become apparent that Koch's
postulates are too rigid to be anplied without
qualification to viruses, for the following reasons:
1. Nowadays our conception of the distribution
of most viruses is that they are found in many cases
of minor infection which show little resemblance
to the fully developed disease, as well as in a
certain proportion of apparently healthy persons.
Viruses can seldom be recovered from every case
of the disease and furthermore are too small to be
seen in specific lesions, although intracellular inclusions
are produced by some.
2. It must further be appreciated that viruses, unlike
bacteria, do not grow on inanimate media, but
require for their multiplication, living cells; such
media are provided by an experimental animal, an
embryonated egg, or a tissue culture. In the earlier
days of virus research a "pure culture" could only
be described in terms of a specific infection produced
in an experimental animal. Such hosts are
however subject to virus diseases of their own, or
may carry 'latent" viruses. More recently, by the
use of the embryonated egg, it has been possible to
"388 ROBINSON: KOCH'S POSTULATES AND VIRUSES
identify specific lesions, such as characteristic
pocks on the chorio-allantoic membrane. Within
the last year or so, by the use of the Dulbecco
tissue culture technique, plaques can be recognized.
It is now possible to obtain a "pure culture" of
virus by subculture of either pocks or plaques.
.3. Finally, there is often difficulty in finding an
experimental animal which is susceptible to virus
infection.
In 1937, Rivers3 suggested a revision of
Koch's postulates more applicable to viruses. Although
he altered the letter of the law the spirit
remains unchanged, the basic tenet being that the
virus must be shown to occur in the sick individual,
inot as an incidental or accidental finding, but as
the cause of the disease under investigation. The
suggestions, since referred to as "Rivers' postulates",
may be stated as follows:
1. A specific virus must be found associated with
a disease with a degree of regularity; it is not
obligatory to demonstrate the presence of a virus
in every case of the disease produced by it. The
existence of virus carriers is recognized.
2. The virus must always be found at the proper
time in specific lesions, since they are intimately
connected with host cells. They may also be found
in the blood as a phenomenon of overflow from the
tissues. It is not essential that a virus be grown on
lifeless media or in modified tissue culture.
3. A transmissible infection must be produced
with a degree of regularity in a susceptible experimental
host by the inoculation of material free from
ordinary microbes or rickettsiwe and obtained from
the patient suffering from the nattural disease. If
characteristic features such as paralysis or inclusions
are exhibited by the natural disease they
are sought for in the experimental animal.
4. By the use of proper controls and by the
demonstration of an increase of specific antibodies
it must be shown that the viruLs was neither
fortuitously present in the patient nor accidentally
"picked up" in experimental animals.
Within the space of 20 years, however, and
mainly as the result of the demonstration of the
widespread prevalence of the newer viruses, which
often do not fulfil these postulates, an elaboration
of Rivers's postulates has become necessary, for the
following reason. Many of the illnesses which are
currently being intensively investigated, niotablv
the enteric virus infections, are mild, and infection
is usually manifested only by the excretion of virus
in stool and throat. The worker is thus perforce
obliged to work mainly with stool specimens as a
source for isolating the virus; and as has been
mentioned previously, the enteric viruses are widely
disseminated, of numerous serological types, and
nixtures
often occur.
These facts prompted Huebner, to define that
which he succinctly describes as a "Bill of Rights
for Prevalent Viruses", which might be said to
constitute a "guarantee against the imputation of
guilt by simple association just as, in the political
Canad. M. A. J.
Sept. 1, 1958, vol. 79
arena, the reaffirmation of such a principle periodically
seems to be necessary".
Huebner has emphasized the value of carefully
planned epidemiological studies. The salient
features of this epidemiological approach, which
is to a large extent indirect, must be to enhance
the etiological significance of the association of a
particular virus with a clinical illness by comparison
with observations made in a control population.
The value of such information as virus isolation
from the stool, which by itself is not of diagnostic
significance, will be greatly enhanced by proof of
the absence of illness and virus in control populations.
The controls used differ in their composition
according to the type of study which is performed.
The "cross-sectional" study is the form of investigation
most commonly practised, as in the investigation
of outbreaks of illness and epidemics. The control
is of a geographical type, normal individuals
being selected who have not had direct contact
with the group being investigated. The "longitudinal"
type of study involves the investigation
of a relatively static population over a long period
of time. The population being investigated acts as
a control for itself (temporal type of control), the
incidence of viruses and the type of illness
prevalent being compared from time to time, as for
example oIn a regular monthly basis. This method
of investigatioin is of advantage in demonstrating
the time relationship of the virus isolation to the
onset of clinical illness. The presence or absence
of similar syndromes unrelated to the particular
virus isolated can thus be clearly shown.
It is a sober thought for contemporary virologists
that the conditions needed to prove that a virus
causes a disease must now require the employment
of a wide range of laboratory tests, and in
addition specific epidemiological data.
As physicians, however, we are able to recognize
certain viirus syndromes with almost complete
certainty, and in such cases laboratory tests are
rarely required. Such examples are smallpox,
chickenpox, mumps, typical cases of infectious
hepatitis, herpes simplex and other virus illnesses
which give a characteristic clinical picture. On the
one hand, molluscum contagiosum has characteristic
clinical features and the diagnosis may be
confirmed with certainty by histological examination
of the lesion. As a further example, viruses
may be isolated from specific lesions of the brain
at autopsy, as in cases of encephalitis, or pathognomonic
inclusions may be present, as in the liver
in yellow fever. Certain viruses such as the rabies
virus will, when isolated, be accepted as the cause
of illness without reserve, for in man this virus is
always associated with disease. In these instances
the etiology of the disease is established without
recourse to satisfying any further conditions. On
the other hand, particularly when one deals with
the newer virises, the very mildness of the illness
may preclude the examination of any specimen
other than stool or throat swab, and as has been