47, No. 2, pp.

139-274 1972

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MEMORANDA: VIRUS-ASSOCIATED -__ _______

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4iNDEX MARG2 1
INU MA 21

WORLD HEALTH ORGANIZATION

ORGANISATION MONDIALE DE LA SANTI
GENEVE
Memoranda
Virus-associated immunopathology:
animal models and implications for human disease*
1. Effects of viruses on the immune system, immune-complex diseases,
and antibody-mediated immunologic injury
The tissue damage caused by virus infection has been traditionally explained by the
ability of viruses to multiply in cells and thereby injure or destroy them. Recent evidence
suggests, however, that lesions may also be caused by the host's immune response to viral
antigens and that the immune system itself may be perturbed by some viruses. This memo-
randum reviews recent developments in viral immunopathology, with special reference
to animal model systems, and indicates the possible relevance of the new concepts and
techniques for certain diseases of man. Certain viruses, notably the leukaemia viruses
and some of those causing persistent infections, depress the host's ability to mount an anti-
body response to antigens, while other viruses may enhance the antibody response. Cell-
mediated immunity may also be depressed. Another immunopathological manifestation
of virus infection is immune-complex disease. When viruses or their antigens persist in the
circulation they combine with specific antibody, and the resulting complexes lodge in various
sites, especially the kidney. Further combination with complement leads to the release of
tissue-damaging substances. A third condition associated with virus infection is antibody-
mediated immunologic injury. Both oncogenic and non-oncogenic viruses frequently
induce new antigens on the surface of the cells they invade. When antibody attaches to
these antigens in the presence of complement, the cells are destroyed.

The lesions associated with virus infections have
traditionally been explained by the ability of viruses
to replicate in cells and hence cause cell injury and
even death. However, recent studies indicate that
virus-associated tissue damage may be due in part
to the immune response of the host to viral antigens.
The properties of viruses are seemingly ideal for
producing immunopathological damage. Viruses are
foreign antigens and, being self-replicating, can conti-
nue to produce antigen for long periods of time.
Certain viruses are also known to be able to induce
new antigens on the surface of cells they infect.
The host's immune system can respond to these
antigens.
In view of these properties, immunopathological
changes may be initiated by a number of different
mechanisms in the course of virus infection:
(1) Certain viruses can infect the cells of the
immune system and cause direct immunologic
* This memorandum was prepared by the signatories
listed on page 262.
2894 - 257
derangements. Many processes and parameters of
immune function may be thus affected, including
graft rejection, the induction of immunologic toler-
ance, antibody production, graft-versus-host reac-
tions, lymphocyte transformation, immunoglobulin
levels, phagocytosis, and delayed-type skin reactions.
(2) The host's immune response to viral antigens
can lead to the formation of virus-antibody com-
plexes capable of reacting with anti-immunoglobu-
lins, rheumatoid factor, and the components of
complement.
(3) New antigens produced by viruses on infected
cell surfaces can interact with specific antiviral anti-
body plus complement, thus causing cell destruction.
(4) Recent findings suggest that sensitized lympho-
cytes can also react with virus-induced cell surface
antigens and destroy the cell. Furthermore, cell-
mediated (or antibody-mediated) immune responses
to viruses may result in the release or activation of
biological mediators causing immunopathological
changes.
258 MEMORANDA
(5) An autoimmune response may be produced if
the virus (a) releases host-cell antigens, (b) alters
host-cell antigens and act as a " helper determinant ",
or (c) depresses the host genome, thus increasing the
production of embryonic or other antigens.
In addition, the genetic makeup of the host, while
not a mechanism of producing immunopathological
damage, can influence the nature and severity of
injury incurred during virus infection (Notkins et al.,
1970).
In some infections, such as lymphocytic chorio-
meningitis, the immune response of the host may
be the principal cause of the pathological mani-
festations while in other infections it may be of
less importance. In most if not all virus infections,
the host's immune response probably contributes
somewhat to the pathological picture. It should be
emphasized, however, that in the majority of cases
the overall effect of the immune response is more
likely to be beneficial than harmful.
Recent studies on virus-induced immunopatho-
logical reactions in domestic and experimental ani-
mals have led to the development of concepts and
technical methods that may be useful in investi-
gating certain viral diseases in man, including hepa-
titis. Progress in the field of viral immunopathology
has been rapid, and it was felt that a summary and
critical review of present knowledge would encourage
its wider application to clinical problems. Only
selected references have been included, since the
breadth of the subject made a complete review of
the literature impracticable. Suggestions for further
lines of investigation in viral immunopathology in
general and in viral hepatitis in particular will be
offered in Part 2 of this Memorandum, to be pub-
lished later.
EFFECTS OF VIRUSES ON THE IMMUNE SYSTEM
It has long been known that certain virus infec-
tions can alter the morphology of lymphoid organs.
Electron microscopy studies have demonstrated the
presence of virus particles in cells of the lympho-
reticular system, such as macrophages, lymphocytes,
neutrophils, thymocytes, Kupffer cells, and stem
cells. More recent investigations have shown that
certain viruses are able to replicate in macrophages
(e.g., arboviruses, murine hepatitis virus, lactate
dehydrogenase virus (LDV], and herpes simplex
virus (HSV]) while others can replicate in lymphocytes
(e.g., lymphocytic choriomeningitis virus [LCMV],
leukaemia viruses, and Epstein-Barr virus [EBVD.
Several viruses appear to replicate only in lympho-
cytes that have undergone blast transformation fol-
lowing exposure to specific antigen or phytohaemag-
glutinin. Not all infections of the immune system,
however, result in cell destruction; some lead to a
persistent infection. For example, infection with
EBV can result in the establishment of a continuous
lymphoid cell line in vitro, while infection with the
leukaemia viruses may be followed by malignant
transformation.
Recent studies indicate that certain virus infections
can affect the function of the immune system. These
investigations have utilized the immune response to a
variety of antigens unrelated to the infecting virus
in order to evaluate immunologic function. Mur-
ine leukaemia viruses have received the most atten-
tion. These viruses usually depress the immune
system, under certain circumstances to a significant
extent (Dent, 1972). For example, the number of
antibody-producing cells as determined by the
haemolytic plaque test (Jeme) may reportedly be
depressed by as much as 99 %. In general, infection
prior to the injection of antigen was found to result
in immunodepression, whereas infection after antigen
administration had considerably less effect. The
degree of immunodepression was dependent on the
dose of virus and on the nature and concentration
of the particular antigen. Moreover, some evidence
has been adduced that the leukaemia viruses (parti-
cularly Friend virus) can exert " selectively " depres-
sive effects, i.e. that they produce a greater depression
of the 7S than of the 19S immune response. Selective
effects also have been described in connexion with
other viruses. Infection with Aleutian disease virus
(ADV) can result in the appearance in the serum
of an excess of monoclonal immunoglobulin. It
also has been claimed that LDV and LCMV can
produce an acute and " selective " depression of
T cells, but these results need to be confirmed
and extended. Several non-oncogenic viruses (e.g.,
ADV, LCMV, and Junin virus) are also able to
depress the humoral immune response. In addition,
certain viruses, such as LDV, LCMV, and Venezue-
lan equine encephalitis virus (VEEV), can prevent
the development of experimentally-induced immuno-
logic tolerance.
Although most studies of viral effects have been
concerned with the humoral immune response, recent
investigations ofcell-mediated immunity and reticulo-
endothelial function demonstrate that these too can
be depressed. For example, allograft rejection is
profoundly depressed in animals infected with Gross
leukaemia virus and mildly depressed in animals in-
 VIRUS-ASSOCIATED IMMUNOPATHOLOGY. 1
fected with LDV. A number of viruses, including
the Rauscher and Friend viruses and those causing
measles and rubella, have been shown to inhibit
blast transformation of lymphocytes (Dent, 1972).
Not all viruses exert depressant effects on the
immune system. Several, such as LDV and VEEV,
can act as adjuvants and potentiate the immune
response to certain antigens.
A number of mechanisms have been postulated
to explain the immunodepressive effect of certain
virus infections (Allison, 1972; Notkins et al., 1970).
These include (1) virus-induced changes in the
uptake and processing of antigens, possibly by
alteration of cell surfaces; (2) depression of nucleic
acid and protein (antibody) synthesis; (3) destruction
of antibody-producing cells or their precursors;
(4) alteration of thymic function; (5) acceleration of
immunoglobulin catabolism; (6) antigenic compe-
tition; and (7) lymphocytolysis as a result of increased
adrenocortical secretion. Possible explanations of
the immunologic enhancement associated with
virus infections include (1) altered uptake and pro-
cessing of antigens; (2) increase in the number of
antibody-producing cells or their precursors; and
(3) enhanced metabolism of antibody-producing
cells.
The effects of virus infections on immune function
may have several important pathological repercus-
sions. Virus-induced immunodepression might allow
certain infections to persist, thereby adding to the
antigenic load and increasing the likelihood of
immunopathological consequences (e.g.. immune-
complex disease). Moreover, depression of the im-
mune response might trigger or enhance the growth
of certain tumours. Virus-induced potentiation of
immune response might also have immunopatho-
logical consequences, such as the development of
autoimmune disorders (WHO Scientific Group on
Factors Regulating the Immune Response, 1970).
Recommendations
(1) A systematic evaluation of the effects of viruses
on immune function should be undertaken. A num-
ber of viruses should be studied and a standard
set of immune function tests should be employed.
Among the factors that deserve special investigation
are antigen types (e.g., thymus-dependent versus
non-thymus-dependent), antigen dose, and the time
relationship between infection and antigen adminis-
stration.
(2) The effects of virus infection on different cell
types (e.g., macrophages, T and B lymphocytes)
259
should be studied in greater detail, with morphologic
changes perhaps serving as an indication of functional
alterations. Since differences in terminology often
make it difficult to assess reports of pathological
changes in lymphoid tissue, all modifications of the
lymphoid organs should be described according to
standardized criteria. Efforts at standardization are
currently being supported by the World Health
Organization.
(3) An attempt should be made to ascertain whether
viruses can in fact exert selective effects on immune
function, e.g., by depressing 7S versus 19S antibody,
or by affecting T cell function as opposed to B cell
function (Allison et al., 1971). The possibility should
also be looked into that the immune response to
the virus may itself be impaired if the infecting virus
damages more or less selectively the cells responding
to the viral antigens. If this proves to be the case,
virus-induced immunodepression might conceivably
be highly instrumental in prolonging certain virus
infections, such as murine leukaemia, hepatitis, sub-
acute sclerosing panencephalitis, or infections caused
by LDV, LCMV, or ADV.
IMMUNE-COMPLEX DISEASES
It is well known that the persistence of antigen-
antibody complexes in the circulating blood can
lead to serum sickness, as manifested by glomerulo-
nephritis, polyarteritis, urticaria, arthralgia, and
arthritis. Recently, it has been shown in animals that
viruses can persist in the bloodstream in the form
of virus-antibody complexes, and that the deposition
of these complexes in the kidney can produce an
immune-complex type of glomerulonephritis.
Infectious virus-antibody complexes have been
detected in the blood of animals with murine leukae-
mias and those infected with LDV, ADV, and
LCMV (Mellors et al., 1969; Oldstone & Dixon,
1969; Notkins et al., 1966; Porter et al., 1969;
Oldstone & Dixon, 1971b, respectively). Prelimi-
nary evidence suggests that infectious complexes
also exist in the bloodstream of horses infected with
equine infectious anaemia virus (EIAV) (McGuire
et al., 1971). Immunopathological studies have re-
vealed the presence of viral antigens, specific
antiviral antibody, and complement in the kidneys
of these animals (Oldstone & Dixon, 1971b).
Severe glomerulonephritis has been found in
LCMV carrier mice (Hotchin & Collins, 1964;
Oldstone & Dixon, 1969, 1971b). The severity of
the disease appears to be related to the strain of
9
260 MEMORANDA
the mouse, the amount of LCMV, and the amount
of antiviral antibody (Oldstone & Dixon, 1969).
Aleutian disease of mink also is characterized by
severe glomerulonephritis (Porter et al., 1969). All
mink appear to be susceptible to infection by ADV,
but those homozygous for the Aleutian gene develop
a more severe form of the disease, characterized by
heavy deposition of virus, antibody, and complement
in the glomeruli. However, relatively mild glomeru-
lar lesions are seen in mice infected with LDV.
In humans, circulating Australia antigen can exist
in the form of antigen-antibody complexes (Zucker-
man, 1971). One case of immune-complex nephritis
with deposition of Australia antigen, IgG, and
complement in the glomeruli has been reported,
and in 4 cases of hepatitis autopsy disclosed the
presence of Australia antigen, IgG, IgM, and comple-
ment in glomerular capillaries.
There is generally little evidence that vasculitis
can be caused by virus-antibody complexes, but
vascular lesions suggestive of polyarteritis nodosa
and containing immunoglobulins have been reported
late in the course of infection with ADV and EIAV.
Recently, polyarteritis nodosa has been described
in patients with circulating Australia antigen (Gocke
et al., 1971); in one such case, Australia antigen,
immunoglobulin, and complement were detected
in the arterial wall (Gocke et al., op. cit.). In 5
cases of fatal hepatitis, Australia antigen, immuno-
globulin, and complement were found in the intima
of arterioles exhibiting changes typical of periarte-
ritis. It has also been suggested that immune com-
plexes may be causally involved in the urticaria and
arthritis (Alpert et al., 1971) sometimes associated
with hepatitis.
Although deposition of circulating immune com-
plexes appears to be the most likely explanation of
these findings, the possibility has not been excluded
that viral antibody might attach to viral antigens
released locally from infected cells or to virus-induced
antigens on the surface of infected cells (see section
entitled " Antibody-mediated immunopathological
injury "). In several autopsy studies of patients
with various forms of hepatitis, intracellular and
extracellular deposits of Australia antigen, immuno-
globulins and complement were reportedly found
in liver parenchymal and Kupffer cells (Nowos-
lawski et al., 1972). In these cases, immunoglobulins
directed specifically against Australia antigen were
eluted from the liver with 2.5 M thiocyanate. At
present, however, there is very little information
available to pinpoint the factors responsible for
producing virus-induced immune-complex disease.
Whether the causal factor is the size of the complex,
the nature of the viral antigen, the amount or type
of antibody, the attachment of accessory factors
such as complement (Winchester et al., 1971) or
rheumatoid factor (Notkins, 1971; Winchester et al.,
1971; Ziegenfuss et al., 1971), or the rate at which
the antigen and antibody turn over (in the glomeru-
lar lesions) remains to be determined.
To date, virus-induced immune-complex disease
has been attributed to the deposition of virus-
antibody complexes during persistent virus infections
(Oldstone & Dixon, 1971b). Conceivably, immune-
complex disease also could occur from the repeated
deposition of such complexes during various acute
and recurrent virus infections. It should be empha-
sized that, in addition to virion-antibody complexes,
antibody bound to virus-induced membrane anti-
gens, soluble viral antigens, and viral nucleoproteins
might contribute to the pool of circulating immune
complexes.
The mechanism of tissue injury associated with
deposition of virus-antibody complexes is presu-
mably similar to that involved in the deposition of
nonviral antigen-antibody complexes. It is known
that activation of the complement sequence by
immune complexes can effect the release of sub-
stances that have the capacity to increase vascular
permeability, contract smooth muscle, and attract
polymorphonuclear and mononuclear leucocytes.
These factors would seem to play a role in the tissue
injury associated with immune-complex disease.
In addition, it has been postulated that immune
complexes might activate components of the clotting
system and thereby cause the deposition of fibrin.
Recommendations
(1) The presence of immune complexes in the
kidney, arterial walls, or other tissues should be
confirmed by demonstrating viral antigens, specific
antiviral antibody, and complement in the lesions
by immunofluorescence. If, however, the antigen
cannot be detected because antigenic sites have been
saturated by antiviral antibody, the antibody
should if possible be dissociated by standard tech-
niques (e.g., acid buffer, pH 2.0-3.0). The eluted
antigen or antibody may be characterized by im-
munodiffusion, complement fixation, virus neutrali-
zation, or other techniques.
(2) Attempts should be made to recover and iden-
tify infectious virus from the kidney, extrarenal
 VIRUS-ASSOCIATED IMMUNOPATHOLOGY. 1
tissue, and circulating blood by standard virus
isolation techniques. To determine whether the iso-
lated virus exists in the form of an infectious virus-
antibody complex, the anti-immunoglobulin neu-
tralization technique should be used.
(3) Efforts should also be made to detect non-
infectious virus-antibody complexes in the circu-
lation. Upon incubation with the Clq component
of complement or rheumatoid factor (Winchester
et al., 1971), these complexes may precipitate out
demonstrably. Conversely, incubation in an acid
buffer may dissociate the complexes and permit the
viral antigens and specific antiviral antibody to be
identified as described in (1) above.
(4) If virus cannot be recovered by any of the
above techniques, the animals should be immunized
with isolated complexes and their sera tested for
antibodies to a variety of viruses.
(5) When DNA-anti-DNA complexes are pre-
sent in the glomeruli, an endeavour should be made
to distinguish between viral nucleic acids and nucleic
acids of nonviral origin.
(6) Since glomerulonephritis of differing degrees of
severity can be produced in the same host by different
viruses (e.g., LCMV versus LDV), attention should
be focused on the factors involved in the initiation
and production of immune-complex disease. It would
be desirable to develop models to study the clearance
of virus-antibody complexes from the bloodstream
and the rate at which these complexes deposit and
turn over in the kidney.
(7) Animals with infections characterized by per-
sistent or recurrent viraemia (e.g., feline leukaemia,
African swine fever, hog cholera, and avian lympho-
matosis) should be examined for antiviral antibody
circulating virus-antibody complexes, and immune-
complex nephritis.
(8) A major effort should be made to elucidate the
role of immune complexes in the pathogenesis of
viral hepatitis in man.
ANTIBO)Y-MED)IATED IMMUNOLOGIC INJURY
In the last decade it has been shown that the
transformation of cells by oncogenic viruses results
in the appearance of new antigens on the cell sur-
face and that immune responses to these antigens
may be involved in tumour rejection. Non-onco-
genic viruses can also produce new antigens on the
surfaces of infected cells, but the biological signi-
261
ficance of these antigens has received relatively little
attention. Evidence is now beginning to emerge,
however, suggesting that the interaction of specific
antiviral antibody and complement with surface
antigens induced by non-oncogenic viruses can lead
to cell destruction and may contribute to the patho-
genesis of the lesions associated with certain virus
infections.
In vivo, the best experimental evidence that anti-
body can play such a pathogenetic role comes from
the demonstration that the passive administration
of specific antiviral antibody to animals infected with
LCMV (Oldstone & Dixon, 1970), ADV, or Japanese
B encephalitis virus produces or intensifies the charac-
teristic lesions associated with these infections. In
addition, it has been speculated that the interaction
of specific antiviral antibody and complement with
antigens induced by the respiratory syncytial,
measles, hepatitis, dengue, and equine infectious
arteritis viruses may be partly responsible for the
pathological picture seen in these infections. Another
suggestion has been that the passive attachment of
virus, antiviral antibody, and complement to the
surface of platelets or erythrocytes may result in
cell injury and might give rise to some of the haema-
tologic abnormalities associated with virus infections,
such as dengue shock syndrome (Russell, 1971)-
the most severe form of dengue haemorrhagic fever-
and equine infectious anaemia.
The strongest evidence that antiviral antibody and
complement can injure virus-infected cells has been
produced by in vitro experiments. It has been shown
that the infection of cells with viruses that do not
produce cytologic injury (rabies (Wiktor et al.,
1968), LCMV) (Oldstone & Dixon, 1971a), or with
viruses that ultimately do cause cell damage (HSV,
vacciniavirus, influenzavirus, Newcastle disease
virus [NDV] (Brier et al., 1970) is followed by the
appearance of new antigens on the surface of the
infected cells and that the interaction of specific
antiviral antibody and complement with these anti-
gens can produce immunologic injury. In the
absence of either specific antiviral antibody or com-
plement, such injury does not occur. The degree of
injury may depend on a number of factors (Brier et
al., 1970), including (1) the density of viral antigens
on the infected cell surfaces; (2) the inherent sus-
ceptibility of the cells to lysis by complement;
(3) the nature and concentration of the antiviral
antibody; (4) the ratio of complement-fixing to
non-complement-fixing antibody in the particular
serum; and (5) the presence of inhibitors, such as
262 MEMORANDA
anti-immunoglobulins or rheumatoid factor, that
might block complement-fixing sites on the antiviral
antibody. The appearance of viral antigens might
in turn be related to other factors, such as the phase
of the cell cycle or coinfection with a second virus.
If a particular virus produces few or no new anti-
genic sites on the surface of cells or if these antigenic
sites are far apart, complement-mediated cell de-
struction may not occur. If, however, the density
of virus-specific antigens on the cell surface should
rise during the course of an infection, this would
increase the likelihood of complement-mediated cell
destruction. Fluctuations in the density of viral
antigens on the infected cell surface might contribute
heavily to the pathogenesis of lesions associated with
" slow virus " infections (Porter, 1971).
Implications
The attachment of antiviral antibody in sublytic
concentrations to the surface of infected cells may
conceivably be instrumental in deciding the fate of
the cell. On the one hand, the attachment of anti-
viral antibody might accelerate phagocytosis of the
infected cell by activated macrophages. On the other
hand, antiviral antibody might prevent sensitized
lymphocytes from recognizing or reacting with the
viral antigens and thereby inhibit the cell-mediated
immune response. In virus infections, this might
prove to be the counterpart of "blocking" or
" enhancing " antibody.
Under certain circumstances the destruction of
virus-infected cells by antiviral antibody and comple-
ment may be more beneficial than harmful to the
host. Antibody-mediated cell destruction may be one
of the mechanisms by which the host combats those
viruses that tend to elude neutralization by spread-
ing directly from cell to cell. Moreover, the destruc-
tion of cells that are actively producing virus would
slow down viral replication and release or expose
the infectious virus within the cell to neutralizing
antibody. Thus, in virus infections, antibody-
mediated cell destruction may fulfil many of the
functions that have been postulated for cell-mediated
immunity and may serve as a complementary or
supplementary defence mechanism. In addition,
in vitro experiments suggest that the release of one
or more chemotactic-generating factors (Brier et al.,
1970) from infected cells and/or the interaction
between antiviral antibody and viral antigens can
activate the complement sequence and cause the
release of mediators able to attract polymorpho-
nuclear and mononuclear leucocytes.
Recommendations
(1) Although many investigators have speculated
that immunologic injury may contribute to the
pathological picture in certain virus infections, it
has been difficult to isolate and evaluate this pheno-
menon in vivo. The release of 51Cr from virus-
infected cells by antiviral antibody and complement
provides a simple, objective, and quantitative tech-
nique for studying immunologic injury in vitro.
With this technique it should be possible to (a)
investigate a variety of viruses; (b) evaluate virus-
induced immunologic injury in different types of
cell; (c) compare the roles of cytolytic and non-
cytolytic antibody in the serum of patients during
the course of various virus infections; (d) determine
whether biological mediators are released or acti-
vated as a result of the interaction of antiviral
antibody and complement with viral antigens; and
(e) investigate the relationship between antibody-
mediated and cell-mediated destruction of infected
cells.
(2) In vivo studies should be extended and experi-
mental models developed. Additional studies should
be conducted to evaluate the results of the passive
administration of cytolytic antiviral antibody to
infected animals with normal and depleted levels of
complement. Efforts should be made to demonstrate
the presence of antiviral antibody and complement
on the surface of injured cells at the site of the lesion.
Further thought should be given to the potential
beneficial or harmful effects of passive protection
with immunoglobulins containing cytolytic antiviral
antibody or with vaccines (e.g., rabies vaccine)
that might induce cytolytic antibody.
(3) Attempts should be made to compare the
in vitro and in vivo effects of antibody and comple-
ment on the lysis of virus-infected cells. Whether the
attachment of nonlytic antibody to infected cells
can inhibit the cell-mediated immune response should
be investigated.
* *
*
A. C. ALLISON, Clinical Research Centre, Northwick
Park Hospital, Harrow, Middlesex, England
W. I. B. BEVERIDGE, Veterinary Public Health Consultant,
World Health Organization, Geneva, Switzerland
W. C. COCKBURN, Chief Medical Officer, Virus Diseases,
World Health Organization, Geneva, Switzerland
JUNE EAST, Department of Environmental Carcino-
genesis, Imperial Cancer Research Fund, Mill Hill,
London, England
 VIRUS-ASSOCIATED IMMUNOPATHOLOGY. 1
H. C. GOODMAN, Chief Medical Officer, Immunology,
World Health Organization, Geneva, Switzerland
H. KOPROWSKI, The Wistar Institute of Anatomy and
Biology, Philadelphia, Pa., USA
P.-H. LAMBERT, World Health Organization Haematology
Research Unit, Cantonal Hospital, Geneva, Switzerland
J. J. vAN LOGHEM, Department of Immunopathology,
University of Amsterdam, The Netherlands
RtSUMA
ETATS IMMUNOPATHOLOGIQUES INDUITS PAR LES VIRUS: MODLLES ANIMAUX
ET RELATIONS AVEC LES MALADIES HUMAINES:
1. EFFETS DES VIRUS SUR LE SYSTEME IMMUNITAIRE, MALADIES DUES A DES IMMUNCO)APLEXES
ET LtSIONS IMMUNOLCGIQUES PROVCQUVES PAR L'INTERMtDIA]RE DES ANTICORPS
On sait depuis longtemps que les virus produisent
des lesions en endommageant et parfois en detruisant
les cellules a l'int6rieur desquelles ils se multiplient.
Plus recemment, on a decouvert que des alterations
tissulaires peuvent aussi r6sulter d'interactions entre le
virus et le systeme immunitaire de l'h6te. L'etude de
diverses maladies des animaux a permis de deceler un
certain nombre de mecanismes immunopathologiques
responsables des lesions provoquees par des infections
virales. Ces mecanismes - ainsi que les concepts et les
techniques issus de ces recherches - scnt d6crits dans la
lre et dans la 2e partie du present memorandum. Leurs
consequences eventuelles au regard des maladies
humaines sont examinees et plusieurs de leurs appli-
cations sont envisag6es.
Un premier type de lesion immunologique est diu
aux effets directs exerc6s par certains virus sur le systWme
immunitaire. Des virus, notamment les virus des leuc&
mies et des virus responsables d'i nfections de longue dur6e,
diminuent la capacite de production des anticorps chez
l'h6te; d'autres agissent en renforgant la reponse immuni-
taire a divers antigenes. Dans certains cas, il est manifeste
que les virus inhibent 6galement l'immunite a support
cellulaire. Cette action des virus sur la fonction immuni-
taire pourrait avoir de nombreuses et importantes cons&
quences du point de vue clinique; c'est ainsi que l'affai-
blissement de l'immunit6 serait susceptible de favoriser
le d6veloppement de tumeurs.
Les maladies dues a des immuncomplexes representent
une autre forme de l6sion immun opathologique provoqu6e
par des virus. Dans les infections virales persistantes,
les anticorps specifiques se combinent parfois aux virus
REFERENCES
Allison, A. C. (1971) Immunity against viruses. In:
The scientific basis of medicine, annual review for 1971,
London, Athlone Press (in press)
263
P. A. MIESCHER, Haematology Division, Cantonal Hospi-
tal, Geneva, Switzerland
C. A. MIMMs, Microbiology Department, The John Curtin
School of Medical Research, Australian National Uni-
versity, Canberra City, Australia
A. L. NOTKNS, Chief, Virology Section, National Institute
of Dental Research, Bethesda, Md., USA
G. TORRIGIANI, Medical Officer, Immunology, World
Health Organization, Geneva, Switzerland
ou aux antiggenes viraux pour former des immuncom-
plexes qui sont ensuite deposes dans divers endroits de
l'organisme, et en particulier dans le rein. Dans cette
dernie eventualite, on peut voir apparaitre ult6rieure-
ment une glomerule-nephrite caus&e par la combinaison
des immuncomplexes et du complement entrainant la
liberation de substances qui lesent les tissus. Les depots
d'immunocomplexes dans la paroi des petites arteres
peuvent provoquer des lesions vasculaires rappelant celles
de la periarterite noueuse.
Enfin, un troisieme type de lesion immunologique
est celui realise a l'intervention des anticorps. I1 s'agit
de la reaction produite lors de la fixation des anticorps
spcifiques sur les antigenes cellulaires de surface induits
par les virus. Le complement, normalement present, peut
alors leser les cellules et meme provoquer leur lyse.
On connalt un certain nombre de virus, en dehors des
virus oncogenes, qui produisent des antigenes a la surface
des cellules dans lesquelles ils ont penere. L'exp6ri-
mentation in vitro montre qu'en l'absence d'anticorps
sp&cifiques ou de complement il ne se produit aucune
l6sion immunologique. Dans certaines conditions, la
destruction des cellules infectees par le virus se revele,
en depit du dommage cause, favorable pour l'h6te
en ralentissant ou en arretant la multiplication du virus.
Dans d'autres cas, et notamment lorsqu'un grand nombre
de cellules d'un organe vital sont atteintes, la lesion peut
avoir des consequences graves et me me fatales.
D'autres formes d'interaction entre les virus et le
systeme immunitaire entrainant egalement des lesions
sont decrites dans la 2e partie du mimorandum.
Allison, A. C. et al. (1971) Cooperating and controlling
functions of thymus-derived lymphocytes in relation
to autoimmunity, Lancet, 2, 135
264 MEMORANTA
Alpert, E. et al. (1971) The pathogenesis of arthritis
associated with viral hepatitis, New Engl. J. Med.,
285, 185
Brier, A. M. (1972) Inhibition or enhancement of immu-
nological injury of virus infected cells. Proc. nat.
Acad. Sci. (Wash.) (in press)
Brier, A. M. et al. (1970) Inflammation and herpes
simplex virus: release of a chemotaxis-generating factor
from infected cells, Science, 170, 1104
Dent, P. (1972) Immunodepression by oncogenic viruses,
Progr. med. Virol., 14, 1
Gocke, D. J. et al. (1971) Vasculitis in association with
Australia antigen. J. exp. Med., 134, 330S
Hotchin, J. & Collins, D. N. (1964) Glomerulonephritis
and late onset disease of mice following neonatal virus
infection. Nature (Lond.), 203, 1357
McGuire, T. C. et al. (1971) Immunofluorescent locali-
zation of equine infectious anemia virus in tissue.
Amer. J. Path., 62, 283
Mellors, R. C. et al. (1969) Further implication of murine
leukaemia-like virus in the disorders of NZB mice,
J. exp. Med., 129, 1045
Notkins, A. L. et al. (1966) Infectious virus-antibody
complex in the blood of chronically infected mice,
J. exp. Med., 124, 81
Notkins, A. L. et al. (1970) Effect of virus infections
on the function of the immune system, Ann. Rev.
Microbiol., 24, 525
Notkins, A. L. (1971) Infectious virus-antibody com-
plexes: interaction with anti-immunoglobulins, comple-
ment, and rheumatoid factor, J. exp. Med., 134, 41S
Nowoslawski, A. et al. (1972) Australia antigen and
hepatitis: pathogenic considerations and practical
implications, Recent Adv. clin. Path. (in press)
Oldstone, M. B. A. & Dixon, F. J. (1969) Pathogenesis
of chronic disease associated with persistent lymphocy-
tic choriomeningitis viral infection. I. Relationship
of antibody production to diease in neonatally infected
mice. J. exp. Med., 129, 483
Oldstone, M. B. A. & Dixon, F. J. (1970) Pathogenesis
of chronic disease associated with persistent lympho-
cytic choriomeningitis viral infection. II. Relationship
of the anti-lymphocytic choriomeningitis immune
response to tissue injury in chronic lymphocytic
choriomeningitis disease, J. exp. Med., 131, 1
Oldstone, M. B. A. & Dixon, F. J. (1971a) The immune
response in lymphocytic choriomeningitis viral infection.
In: P. A. Miescher, ed., Immunophathology: VIth
International Symposium, 1970, Basel, Schwabe, p. 391
Oldstone, M. B. A. & Dixon, F. J. (1971b) Immune
complex disease in chronic viral infections. J. exp.
Med., 134, 32S
Porter, D. D. et al. (1969) The pathogenesis of Aleutian
disease of mink. I. In vivo viral replication and the
host antibody response to viral antigen. J. exp. Med.,
130, 575
Porter, D. D. (1971) A quantitative view of the slow
virus landscape, Progr. med. Virol., 13, 339
Russell, P. K. (1971) Immunopathologic mechanisms in
dengue shock syndrome. In: B. Amos, ed., Proceedings
of the First International Congress of Immunology,
Washington, New York, Academic Press, pp. 831-838
WHO Scientific Group on Factors Regulating the Im-
mune Response (1970) Wld Hlth Org. techn. Rep. Ser.,
No. 448
Wiktor, T. J. et al. (1968) Immune lysis of rabies virus-
infected cells, J. Immunol., 101, 1271
Winchester, R. J. et al. (1971) Occurrence of y-globulin
complexes in serum and joint fluid of rheumatoid
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factors as reagents for their demonstration, J. exp.
Med., 134, 286S
Ziegenfuss, J. F. Jr. et al. (1971) Rheumatoid factor and
Australia antigen. New Eng. J. Med., 284, 1104
Zuckermann, A. J. (1971) The immunopathology of viral
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Memoranda
Virus-associated immunopathology:
animal models and implications for human disease*
2. Cell-mediated immunity, autoimmune diseases, genetics, and implications for
clinical research
Part 2 of this memorandum describes further mechanisms whereby the interaction of
a virus with the host's immune system may lead to tissue damage. Cell-mediated immunity
plays a vital role in promoting recovery from virus infections, but under some circumstances
tissue damage may be caused by the reaction of immune cells with viral antigens. When
mice are infected with lymphocytic choriomeningitis virus neonatally or as adults while
receiving immunosuppressive drugs, widespread invasion of cells is seen but there is little
overt disease. If, however, normal adults are infected or if immune cells are transfused
into tolerant mice, cell injury and death follow. Viruses have long been suspected of contri-
buting to the pathogenesis of autoimmune diseases. Antibodies directed against normal cell
constituents have been reported in several virus infections. Viruses may conceivably un-
mask or release host antigens, alter host antigens and act as " helper determinants ",
or perhaps in other ways provoke immune responses against normal body constituents.
The immunopathological manifestations caused by viruses may also be influenced by the
host's genetic makeup. Certain observations indicate that, in addition to controlling
susceptibility to virus infection, genetic factors partly determine the effectiveness of the
immune response. The memorandum calls attention to the possible implications of these
concepts and findings for clinical research. Some of the diseases of animals and man that
serve as models for studies of virus-associated immunopathology are briefly described.

Part 1 of this memorandum on viral immuno-
pathology (Bull. Wld Hlth Org., 1972) dealt with
the direct effects of viruses on the immune system
and their role in immune-complex disease and anti-
body-mediated immunological injury. In Part 2
the relationship of viruses to cell-mediated immunity
and autoimmune diseases is examined, the genetic
aspects of the host's response to virus infection are
reviewed, and some implications of the new con-
cepts and techniques in viral immunopathology for
clinical research are explored. Annex 1 describes
a number of animal and human disease models
in use in this field.
CELL-MEDIATED IMMUNITY
There is increasing evidence that in some virus
infections the cell-mediated immune response plays
a central role in recovery from infection (Allison,
1972; Blanden, 1971; Glasgow, 1970; WHO Scien-
* This memorandum was prepared by the signatories
listed on page 270.
tific Group on Cell-Mediated Immune Responses,
1969), promoting the sterilization of tissues and the
resolution of lesions. Under some circumstances,
however, the reaction of immune cells with viral
antigens, either free or on the surface of infected
cells, may cause tissue damage. The laboratory
model of cell-mediated immunopathology that has
received the most study is lymphocytic choriomenin-
gitis virus (LCMV) infection of mice (Oldstone &
Dixon, 1970a).
LCMV-induced immunopathological damage
In adult mice infected with LCMV, viral replication
occurs in many tissues, including the viscera and
meninges. The infection is noncytopathic, and the
animals remain free of clinical signs until the onset
of an immune response 5-7 days after infection.
Associated with this response are inflammatory cell
infiltrations occurring at the visceral and meningeal
sites of viral replication and leading to pathological
changes, overt clinical signs, and death. Inhibition
of the immune response by neonatal thymectomy,

2895 265
266 MEMORANDA
irradiation, or the administration of antilymphocyte
serum (ALS) (Mims & Tosolini, 1969; Wiktor &
Koprowski, unpublished data) or cytotoxic drugs
can prevent the acute disease. Mice infected with
LCMV neonatally or congenitally also develop
widespread infection but their immune response
to the virus is poor. No clinical signs are seen in
these animals during the early phase of the infection 1
and late in life chronic glomerulonephritis develops
as a result of the deposition of virus-antibody
complexes in the kidney (see Bull. Wld Hlth Org.,
1972).
Attempts have been made to determine whether
it is the antibody-mediated or the cell-mediated
component of the immune response that causes the
pathological changes in mice infected as adults with
LCMV. In vitro experiments have shown that when
spleen cells from LCMV-immune mice are added to
LCMV-infected cells, the latter will be destroyed
(Lundstedt, 1969). Cell destruction is probably
mediated by a cytotoxic factor that is released
when spleen cells from LCMV-immune mice interact
with LCMV or LCMV-infected mouse fibroblasts
(Oldstone & Dixon, 1970b).
The design of the corresponding in vivo experi-
ments was as follows. Cyclophosphamide 2 was
given to adult mice infected with LCMV (Cole et
al., 1972), thus inhibiting the immune response,
and a persistent clinically inapparent infection was
established. When immune spleen cells were in-
jected into these mice, the animals died and acute
pathological changes comparable to these occurring
in the natural infection were seen. Since the trans-
fusion of immune serum produced less severe
pathological changes, it was concluded that the
injected cells had reacted with infected host cells to
create a cell-mediated immunopathological condi-
tion. In congenitally infected carrier mice, in con-
trast, the transfer of immune spleen cells failed to
1 In addition to LCMV, some other microorganisms are
known to produce less severe clinical signs in newborn
than in older animals, e.g., the agents of many protozoal
diseases and of heartwater. The latter is caused by a myco-
plasma-like organism that grows in vascular endothelium
and other sites. Infections of very young lambs or calves
cause only mild clinical signs, even when numerous organisms
are present for many months, whereas infections of older
animals are severe and often fatal. The transfer of lymphocytes
from old to young infected animals precipitates overt signs
of disease in the latter. Although there may be other ex-
planations-including maternal antibody-for the relative
insusceptibility of young animals to some infectious diseases,
the possible role of immunopathological reactions in adults
with the disease should always be borne in mind.
2 2-bis(2 - chloroethyl) amino]tetrahydro - 2H- 1,3,2-oxaza-
phosphorine 2-oxide.
yield any clinical or histologic evidence of tissue
damage. High titres of neutralizing and complement-
fixing antibody were produced in the recipient carrier
mice so that, under these circumstances too, the ani-
mals remained free of clinical signs. Other experi-
ments, however, have shown that LCMV antibody
plus complement is cytotoxic for LCMV-infected
cells in vitro (Oldstone & Dixon, 1971a), and
tissue lesions have been detected after the injec-
tion of large amounts of antibody into carrier
animals. The possible immunopathological action
of antibody was also suggested by the finding
that complement depletion by means of cobra
venom made LCMV considerably less lethal for
adult mice. However, mice deficient in the C5
component of complement showed normal suscepti-
bility to LCMV.
In summary, these results suggest that cell-
mediated immunity may be an important factor in
LCMV-induced immunopathological injury, al-
though they do not rule out the possibility that anti-
body may also play a role.
Evidence of cell-mediated immunity in other virus
infections
Certain experiments have indicated that cells per-
sistently infected with measles or mumps virus
(Speel et al., 1968) may be destroyed in vitro by
incubation with spleen cells obtained from specifi-
cally immunized animals, but these studies need to
be confirmed and expanded. It has also been sug-
gested that cell-mediated immune responses may be
responsible for the rashes associated with certain
infections (e.g., measles) and might account for the
skin lesions seen in rabbits infected with Shope
fibroma (Tompkins et al., 1970) and rabbit pox
viruses. Indirect evidence from patients with im-
munologic deficiencies also points to the importance
of cell-mediated immunity in certain virus infections,
but in others (e.g., enterovirus infections) cell-
mediated immunity may be relatively insignificant.
There is some evidence that sensitized lymphocytes
after reacting specifically with viral antigen may re-
lease biological mediators, such as migration inhi-
bition factor (MIF), lymphotoxin, interferon, and
factors chemotactic for polymorphonuclear and
mononuclear leucocytes. Some mediators also acti-
vate macrophages, which will then show an increased
capacity to take up and kill bacteria, protozoa, and
possibly viruses. In addition, activated macrophages
may interact with viral antigens on the surface of
infected cells and in this way, perhaps with the help
 VIRUS-ASSOCIATED IMMUNOPATHOLOGY. 2
of cell-bound antibody, may play a role in the host's
defence against virus infections (Tompkins et al.,
1970) while at 'the same time contributing to the
production of lesions (Allison, 1972).
Recommendations
(1) In vitro experiments to test for cell-mediated
cytotoxicity of the type described above should
be carried out in more virus infections. Caution
should be exercised in interpreting negative results,
however, because much may depend on the type of
infected target cell used.
(2) The distribution and density of viral antigens
on the surface of infected cells should be studied
thoroughly, since these are the targets of-cell-medi-
ated immunopathology. Immunofluorescence tech-
niques are relatively simple to use; alternatively,
the topographic localization of antigen can be ele-
gantly studied (Aoki et al., 1970) at the ultrastructural
level by the use of reconstituted antibody molecules
reacting with both viral antigen and marker particles.
(3) Since lysis of cells by sensitized lymphocytes
might be prevented by a " blocking antibody "
adsorbed on the surface of the target cells or by
antigen-antibody complexes adsorbed on the sensi-
tized lymphocytes or target cells (Hellstrom & Hell-
str6m, 1970), it would be desirable to investigate
this phenomenon in virus infections where cell-
mediated immunity may be involved.
(4) Sensitized lymphocytes may exert much of
their protective or other immunologic effects by
recruiting activated macrophages into infected areas.
Further studies are therefore needed of the role
played by macrophages not only in the expression and
pathological consequences of cell-mediated immunity
but also in the induction of the immune response to
virus infections.
(5) Attempts should be made to assess the impor-
tance of cell-mediated immune responses in the
pathogenesis of natural infections of animals. A
useful approach would be to treat animals with
cyclophosphamide, a drug that suppresses both
antibody- and cell-mediated immunity (Cole et al.,
1972), and then infect them with the virus to be stu-
died. If cyclophosphamide treatment were found to
decrease the pathogenicity of the infection without
affecting viral growth in the tissues, then the respec-
tive parts played by immune cells and antibody could
be investigated. Infected cyclophosphamide-treated
animals could be given either cells or serum from
267
normal or immune donors and the ensuing patho-
logical changes and disease could then be recorded.
(6) Cells found to be immunocompetent, as shown
by blast transformation after exposure to virus,
should be transferred to cyclophosphamide-treated
animals as in (5) with a view towards elucidating
their role.
(7) Since the mechanism of recovery from some
infections, notably human hepatitis, is still obscure,
an analysis of cell-mediated immunity against speci-
fic antigens should be made. One approach would
be to obtain lymphocytes (Rosenberg et al., un-
published data) from patients recovering from hepa-
titis, expose the cells in vitro to Australia antigen,
and determine whether they undergo blast trans-
formation.
AUTOIMMUNE DISEASES 1
Autoimmunity is the general term used to describe
an immune response, either antibody- or cell-
mediated, against normal body constituents. The
presence of autoantibodies to certain host antigens,
such as thyroglobulin, DNA, mitochondria, and
microsomes, is relatively common, especially in older
individuals and does not always lead to overt patho-
logical changes. However, certain autoantibodies,
particularly those directed against surface compo-
nents of such cells as erythrocytes, frequently pro-
duce severe autoimmune disease. Other autoimmune
diseases, such as Hashimoto's disease (a chronic
and progressive thyroiditis), pernicious anaemia, and
adrenalitis, are accompanied by infiltrations of
mononuclear cells, and cell-mediated responses are
believed to contribute to these pathological mani-
festations.
The role of viruses in the etiology or pathogenesis
of autoimmune disease has long been suspected
(Lindenmann & Klein, 1967). When considering
this problem, however, one must distinguish between
immune reactions directed against virus-specified
antigens and those directed against host antigens
(true autoantigens). Such distinctions may be diffi-
cult to make in practice, especially where viruses
are transmitted from mother to offspring and are
present throughout life. Furthermore, while a virus
can often be proved to be present, formal proof of
its absence is far more difficult to obtain.
1 Further information on some diseases of animals in
which autoimmune manifestations may be related directly
or indirectly to an underlying virus infection (in some cases
surmised but not proved) is given in Annex 1.
268 MEMORANDA
Theoretically, virus infections could precipitate
autoimmune reactions in a variety of ways: (1) The
virus itself might provide antigens (e.g., viral nucleo-
protein) cross-reacting with host antigens; (2) viruses
might unmask or release antigens from damaged
cells-autoantibodies directed against soluble nuclear
components have been found, for example, after
infectious mononucleosis; (3) viruses might alter
host-cell antigens and act as " helper determinants "
(Allison et al., 1971); (4) viruses might derepress host-
cell antigens (e.g., embryonic antigens); and (5)
viruses might affect the proliferation or responses of
immunocompetent cells or their precursors (see Bull.
Wld Hlth Org., 1972).
For many years it has been speculated that myxo-
viruses may cause autoimmune disease. These
viruses contain neuraminidase, an enzyme known
to produce antigenic modification in host cells.
Incubation of erythrocytes with certain myxoviruses
results in the unmasking of T-agglutinins, and im-
munization of animals with the altered red cells can
raise serum titres of antibody to these antigens. In
addition, elevated T-agglutinin levels have been ob-
served following natural myxovirus infection, but
no obvious autoimmune manifestations have been
reported. Thus, the relationship between myxovirus
infection and autoimmune disease remains unclear.
It has been shown that animals recovered from
tumours destroyed by lysis after infection with non-
oncogenic viruses (Koprowski et al., 1957), or im-
munized with homogenates (Lindenmann & Klein,
1967) are more resistant to subsequent challenge
with viable uninfected tumour cells than are animals
immunized with uninfected tumour homogenates.
Several investigators have postulated that the virus
modifies the surface antigens of the tumour by
introducing' a " helper determinant " (Hirsch et al.,
1968). These helper determinants would act in much
the same way as a hapten to increase the host's
ability to mount an effective immune response and
reject the tumour. Similarly, it has been suggested
that viruses acting as helper determinants might
initiate an autoimmune response to antigens of the
host's own cells. An alternative hypothesis is that
host antigens released from virus-infected cells or
host antigens incorporated into the envelope of the
maturing virion might reach immunologically com-
petent cells and stimulate the production of specific
antibody. These hypotheses have been proposed
to explain a number of diseases, including subacute
thyroiditis and postinfection encephalitis, but firm
experimental evidence is still lacking.
Antibodies to normal cell constituents have been
reported in a variety of other diseases known or
suspected to be caused by viruses. Antinuclear
antibodies (ANA) have been observed in Aleutian
disease of mink, equine infectious anaemia (Hen-
son et al., 1970; Squire, 1968), systemic lupus ery-
thematosus (SLE) in dogs and man (Lewis &
Schwartz, 1971; Blomjous & Feltkamp-Vroom,
1971), hepatitis in man (Zuckerman, 1971), as well as
in New Zealand Black (NZB) mice (Blomjous &
Feltkamp-Vroom, 1971). Antibodies to erythrocytes
have been reported in Aleutian disease, SLE, NZB
mice and EIA (Squire, 1968). Antibodies to mito-
chondria and smooth muscle have been found in
hepatitis, and antibody to thyroglobulin has been
detected in dogs with SLE. In some of these condi-
tions-e.g., SLE, a viral etiology has not been estab-
lished, while in others it is still far from clear whether
the autoimmune manifestations are a direct or an in-
direct consequence cf the virus infection. Even in the
widely studied NZB syndrome, many questions still
remain concerning the relationship between the C-
type particles observed in the tissue of these ani-
mals and the rise in antibody to erythrocytes and
nucleic acids (East, 1970).
Recently, several electron microscopy studies have
detected tubular structures resembling the nucleo-
capsids of myxoviruses or paramyxoviruses in the
glomeruli and synovia of patients with SLE (Melczer
et al., 1962; Gyorky et al., 1969; Sinkovics, 1971),
in the skin of patients with scleroderma and derma-
tomyositis (Norton et al., 1970). and in the thymus
of patients with myasthenia gravis and Hashimoto's
disease (Loghem, 1965). Although these diseases are
believed to have an autoimmune component, it is
not known how their pathogenesis may be related to
the virus-like particles.
Recommendations
(1) A clear distinction should be made between
immune responses to viral antigens (which should
not be considered under the topic of autoimmunity)
and immune responses to host or modified host
antigens.
(2) Assay methods should be developed and stan-
dardized for the detection and quantification of both
the humoral and the cellular immune response to host
antigens.
(3) The ability of a virus to cause autoimmune
disease should be determined by inoculating animals
with a cell-free preparation of purified virions.
 VIRUS-ASSOCIATED IMMUNOPATHOLOGY. 2
(4) In diseases of unknown etiology where auto-
immune manifestations have been demonstrated or
are strongly suspected, a search for a viral etiology
should be initiated. In humans, SLE, rheumatoid
arthritis and subacute thyroiditis appear to be good
ecandidates for study (see below). In animals, canine
(Lewis & Schwartz, 1971) and feline SLE (Slauson
et al., 1971) should receive further attention.
GENETICS
Genetic differences in the susceptibility of animals
to virus infections have been well established. Gene-
tic factors linked to the major histocompatibility loci
of the mouse may determine the susceptibility of the
animals to infection with, e.g., LCMV and some
of the oncogenic viruses (WHO Scientific Group on
the Genetics of the Immune Response, 1968). When
mice of the H-2k haplotype are exposed to oncogenic
viruses such as the Gross (WHO Scientific Group
on the Genetics of the Immune Response, 1968),
Friend, Rauscher, or Moloney virus, they develop
leukaemia, whereas mice of the H-2b haplotype show
much less sensitivity to these viruses. Susceptibility
to various types of RNA avian leucosis virus is also
genetically determined (WHO Scientific Group on
the Genetics of the Immune Response, 1968), and
a single dominant factor seems to account for the
resistance of one strain of mice to influenza virus
(WHO Scientific Group on the Genetics of the
Immune Response, 1968). A single gene also is res-
ponsible for the resistance of mice to group B arbo-
virus infections (Hanson & Koprowski, 1969).
That there are genetic differences in the immune
response of the host to a variety of nonviral antigens
has also been well documented. In some cases, the
magnitude of the immune responses to defined anti-
gens is under the control of allelic genes that are
closely linked to major histocompatibility genes.
It is possible that the nature and magnitude of the
immune response to viral antigens may likewise be
under genetic control and may affect immunopatho-
logical manifestations. Although genetic control
has not been documented with viral antigens, certain
animal experiments are consistent with this possi-
bility. The association of the IR-1 gene with the
H-2k haplotype may indicate that " genetic resistance "
to oncogenic viruses is expressed by the magnitude
of the immune response of the animal host (Fed.
Proc., 1972). Similarly, the genetic resistance of
C57BL mice to ectromelia virus is thought to be
reflected by their greater immunologic reactivity to
269
the virus (Schell, 1960). Conversely, the weaker
immune response of C57BL mice to LCMV is
believed to be responsible for the less severe LCMV-
induced immunopathological lesions in this strain
(Tosolini & Mims, 1971; Mims & Tosolini, 1969).
In man, there is some evidence that differences in
the immune response may be a factor in susceptibility
to hepatitis (Blumberg et al., 1969; Ceppellini et al.,
1970), Indian childhood cirrhosis (Nyak et al., 1972),
and subacute sclerosing panencephalitis (SSPE)
(Lischner et al., 1972), but it is not certain whether
the depressed immune response is genetically deter-
mined and precedes the infection or whether it is a
result of the infection.
Recommendations
(1) In view of the correlation in mice between
H-2 type and susceptibility to RNA virus and the
analogy between H-2 and human histocompatibility
antigens (HL-A), studies are now in progress to
search for an association between HL-A phenotype
and susceptibility to diseases with a suspected viral
etiology, such as Hodgkin's disease and acute leu-
kaemia. The preliminary studies on SLE should
be continued and extended to include rheumatoid
arthritis, myasthenia gravis, and eventually other
autoimmune diseases of possible viral etiology.
(2) Tests for cell-mediated immunity are not well
standardized, as shown by the conflicting reports not
only on SSPE (Lischner et al., 1972), but also
Hodgkin's disease and other conditions. It is re-
commended that tests be carried out under uniform
conditions, using internationally standardized rea-
gents (Bull. Wld Hlth Org., 1971). The World
Health Organization may be able to facilitate the
establishment and dissemination of standard tests
for cell-mediated immunity. Such tests should be
undertaken, for instance, in regions where the inci-
dence of Indian childhood cirrhosis is high (see
above).
(3) Studies to investigate possible linkage relation-
ships between hereditary diseases and genetic markers
present in blood cells and serum proteins should be
encouraged.
IMPLICATIONS FOR CLINICAL RESEARCH
Examples of each of the principal virus-associated
immunopathological mechanisms discussed in Parts 1
and 2 of this memorandum are known in experimen-
tal animals and provide an opportunity for detailed
270 MEMORANDA
analysis of the factors involved. While it would
not be possible to conduct similar research on human
subjects, the knowledge acquired in animal studies
may be applicable to human disease.
In certain virus infections of man, immunopatho-
logical complications are suspected of contributing
to the disease syndrome. These infections include
hepatitis, virus-related haemorrhagic fevers (dengue
almost certainly, and perhaps Argentinian haemor-
rhagic fever), respiratory syncytial virus infection,
infectious mononucleosis, and possibly SSPE. More-
over, immunopathological mechanisms may be in-
volved in causing the skin lesions-e.g., rashes-
associated with some of the common virus infections
of man, such as measles and vaccinia.
Conversely, there are some recognized immuno-
pathological diseases in which viruses are suspected
of playing a role. For instance, in immune-complex
glomerulonephritis of man the antigen may ulti-
mately prove to be viral in origin. Virus-like particles
have been found in patients with SLE (as well as in
a dog with SLE); these may be involved in the
pathogenesis of the disease syndrome. More exten-
sive investigations are needed to clarify the role of
viruses in the etiology of these diseases. Viruses have
also been suspected in the etiology of rheumatoid
arthritis, but thus far no proof of this has been
obtained. The chronic arthritis associated with
chlamydial disease in animals and with Reiter's
disease in man may have immunopathological
components but it is unlikely that these diseases are
useful models for rheumatoid arthritis. A possible
viral etiology involving immunopathological mecha-
nisms has also been suggested for endocrine diseases,
e.g., subacute thyroiditis, and haemolytic anaemia.
In conclusion, the theoretical concepts and techni-
cal methods summarized in this memorandum may
RtSUMP-
tTATS IMMUNOPATHOLOGIQUES INDUITS PAR LES VIRUS: MODALES' ANIMAUX ET RELATIONS
AVEC LES MALADIES.HUMAINES: 2. IMMUNITE A SUPPORT CELLULAIRE,
MALADIES AUTO-IMMUNES, GENETIQUE ET CONStQUENCES POUR LA RECHERCHE CLINIQUE
Dans cette deuxieme partie du memorandum, on decrit
d'autres mecanismes par lesquels l'interaction d'un virus
et du systeme immunitaire de 1'hote est susceptible de
provoquer des lesions tissulaires.
L'immunite a support cellulaire joue un role vital
dans la r6sistance aux infections virales et dans le pro-
cessus de guerison mais, dans certains cas, la reaction
entre les cellules immunes et les antigenes viraux, libres
be usefully applied to the study of suspected immuno-
pathological manifestations in human disease, in-
cluding autoimmune reactions. The experimental
finding that a disease can be prevented or attenuated
by the use of immunosuppressive drugs strongly
implies thatit has an immunopathological component.
However, caution should be exercised in attempting
to use immunosuppression in human patients, be-
cause of the risk of increasing the ability of the virus
to replicate and thereby disseminate the infection.
* *
A. C. ALLISON, Clinical Research Centre, Northwick
Park Hospital, Harrow, Middlesex, England
W. I. B. BEVERIDGE, Veterinary Public Health Consultant,
World Health Organization, Geneva, Switzerland
W. C. COCKBURN, Chief Medical Officer, Virus Diseases,
World Health Organization, Geneva, Switzerland
JUNE EAST, Department of Environmental Carcinogenesis,
Imperial Cancer Research Fund, Mill Hill, London
NW7 18D, England
H. C. GOODMAN, Chief Medical Officer, Immunology,
World Health Organization, Geneva, Switzerland
H. KOPROWSKI, The Wistar Institute of Anatomy and
Biology, Philadelphia, Pa., USA
P.-H. LAMBERT, World Health Organization Haematology
Research Unit, Cantonal Hospital, Geneva, Switzerland
J. J. VAN LOGHEM, Department of Immunopathology,
University of Amsterdam, Netherlands
P. A. MLESCHER, Haematology Division, Cantonal Hospi-
tal, Geneva, Switzerland
C. A. MIMMs, Microbiology Department, The John
Curtin School of Medical Research, Australian Natio-
nal University, Canberra City, Australia
A. L. NOTKINS, Chief, Virology Section, National Institute
of Dental Research, Bethesda, Md., USA
G. TORRIGIANI, Medical Officer, Immunology, World
Health Organization, Geneva, Switzerland
ou fixes a la surface des cellules infectees, peut leser
gravement les tissus et meme entrainer la mort. L'infec-
tion de souris (pendant la p6riode neonatale ou, a 1'etat
adulte, apr6s administration de medicaments immuno-
suppresseurs) par le virus de la choriomeningite lympho-
cytaire determine une invasion massive des cellules par
le virus, mais les animaux ne pr6sentent que tres peu
de sympt6mes cliniques de maladie pendant de longues
 VIRUS-ASSOCIATED IMMUNOPATHOLOGY. 2
p6riodes. Si on leur administre ensuite par transfusion
des cellules immunes, les l6sions tissulaires apparaissent
et la mort survient, la r6action etant similaire a celle
qui succede a l'infection de la souris adulte normale
par le meme virus.
On pressent depuis longtemps que les virus jouent un
r6le dans l'etiologie ou la pathog6nie de certaines
maladies auto-immunes, et les r6sultats de recherches
recentes plaident en faveur de cette hypothese. On a
signale l'apparition d'anticorps dirig6s contre les cons-
tituants normaux de la cellule au cours de plusieurs
infections virales. On admet, comme explications les plus
plausibles, que a) certains antigenes viraux seraient sem-
blables aux antigenes de l'h6te; b) les virus pourraient
r6veler la pr6sence d'antigenes cellulaires ou les liberer;
c) les virus pourraient modifier les antigenes cellulaires
de l'h6te et/ou se comporter comme des * determinants
accessoires *. La microscopie electronique a recemment
detecte des structures de type viral dans les tissus d'ani-
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Allison, A. C. et al. (1971) Cooperating and controlling
functions of thymus-derived lymphocytes in relation
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Aoki, T. et al. (1970) G (Gross) and H-2 cell surface
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Blomjous, F. J. E. M. & Feltkamp-Vroom, T. M. (1971)
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271
maux et de malades atteints de certaines affections a
composante immunologique, comme le lupus erythe-
mateux diss6mine et la maladie de Hashimoto.
Les differences g6netiques entre individus, au sein
d'une espece, peuvent influencer les manifestations immu-
nopathologiques dues aux virus. D'apres certaines obser-
vations, il apparait que les facteurs g6netiques controlent
non seulement la receptivite a l'infection virale - la
receptivite a l'infection par les virus de la leuc6mie, par
exemple, est liee it certains antiganes h6reditaires d'histo-
compatibilite - mais encore qu'ils conditionnent en
partie l'aptitude de l'hote a edifier une reponse immum-
taire a l'egard des virus et d'autres antigenes.
Le memorandum formule des recommandations rela-
tives aux investigations futures dans le domaine de
l'immunopathologie chez l'homme et chez des modeles
animaux et suggere differentes orientations pour la
recherche. Certaines affections humaines et animales A
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272 MEMORANDA
Melczer, M. et al. (1962) Data on the etiology of lupus
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Mims, C. A. & Tosolini, F. A. (1969) Pathogenesis of
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Norton, W. L. et al. (1970) Endothelial inclusions in
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Oldstone, M. B. A. & Dixon, F. J. (1970a) Pathogenesis
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ponse to tissue injury in chronic lymphocytic chorio-
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Annex 1
Murine autoimmune haemolytic anaemia
Haemolytic disease begins to develop in some
mice of the NZB strain at the age of 4-5 months;
direct Coombs reactions increase in incidence and
intensity until the majority of animals are positive at
age one year. These serologic reactions are accom-
panied by anaemia of insidious onset and variable
degree and by progressive reticulocytosis and spleno-
megaly. The circulating autoantibody reacts with
the erythrocytes of normal strains of mice in the
indirect Coombs test. Impairing the cell-mediated
immune responses by neonatal thymectomy neither
delays nor prevents Coombs conversion, which shows
that the reaction is antibody-mediated. Persistent
high levels of IgM are found.
C-type viruses, very similar to known viruses of
the murine leukaemia group, are regularly observed
in conventional and germ-free NZB embryos and
in animals of the NZB strain throughout life. Pre-
sumably, the virus is transmitted via the germ-cells
or placenta, or both, in the same way as the indi-
Sinkovics, J. G. (1971) Virus-like particles in systemic
lupus erythematosus, New Engl. J. Med., 284, 107
Slauson, D. 0. et al. (1971) Naturally occurring immune
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103, 131
Speel, L. F. et al. (1968) An immuno-cytopathogenic
interaction between sensitized leukocytes and epi-
thelial cells carrying a persistent noncytocidal myxo-
virus infection, J. Immunol., 101, 409
Squire, R. A. (1968) Equine infectious anaemia: a model
of immunoproliferative disease, Blood, 32, 157
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Zuckerman, A. J. (1971) The immunopathology of viral
hepatitis associated with Australia antigen. In: P. A.
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genous Gross virus of other spontaneously "highly
leukaemic strains ". The NZB virus possesses anti-
gens of the Gross type. The fact that germ-free
NZB mice also develop autoimmune haemolytic
anaemia suggests that the stimulus precipitating
autoimmunity is intrinsic and might be related to the
animals' own endogenous murine leukaemia virus.
There is still no convincing experimental evidence
that the virus in the form of cell-free filtrates can
reproduce the autoimmune disease in either syngeneic
or allogeneic recipients.
Male or female NZBs can, nevertheless, transmit
the disease to their hybrid offspring when mated
with normal partners, and since the transmission
patterns cannot be easily interpreted in simple genetic
terms a viral etiology must still be suspected. Coombs
reactions do not develop in other highly leukaemic
strains of mice. This implies that the NZBs react
differently to their Gross leukaemia virus, as indi-
cated by the observation that they produce cytotoxic
and possibly virus-neutralizing antibody. If their
thymocytes are sensitized to viral antigens, a helper
 VIRUS-ASSOCIATED IMMUNOPATHOLOGY. 2 273
effect may contribute to autoimmunity. However, have a good immune reactivity to DNA-like anti-
on the basis of present evidence, it is still a matter gens. In contrast, in NZB x NZW F1 hybrids infected
for conjecture whether the virus contributes to the with lactic dehydrogenase virus, depression of natural
development of autoimmunity in NZB mice. Gross virus infection leads to an inhibition of the
renal disease.
Murine SLE
Moderate to severe membranous glomerulo- Human SLE
nephritis with proteinuria develops in both germ- The distinctive features of human SLE are the
free and conventional NZB mice as they age, but anti-DNA antibodies and the immune complexes
only a few experience severe histologic damage and containing DNA present in the kidneys. Tubular
renal failure. However the lesions of F1 (NZBx structures resembling the nucleocapsids of myxo- or
New Zealand White [NZWD hybrids are more severe, paramyxoviruses have frequently been observed by
the majority of F1 females dying within a year. electron microscopy in the endothelial cells of renal
The hybrids spontaneously develop high titres of glomeruli and, less often, in the lymphocytes and
circulating anti-DNA, which together with DNA synovia of patients with SLE. Similar particles have
and complement accumulates in the mesangium and been found in skin biopsies of patients with sclero-
glomerular capillaries. Antibodies to RNA also derma and dermatomyositis, in the thymus of patients
circulate in young animals. Immunization of very with myasthenia gravis or Hashimoto's disease, and
young hybrids with single stranded DNA can hasten in the stomach of one person with pernicious anae-
the appearance of ANA and cause a fatal acceleration mia. The particles are situated in the cytoplasm of
of the glomerulonephritis. Synthetic double-stran- epithelial and endothelial cells and fibroblasts; they
ded RNA without adjuvant provokes the formation may be localized around the nucleus or lie free in
of anti-RNA and/or anti-DNA complexes and, with the dilated cisternae of the endoplasmic reticulum.
adjuvant, exacerbates the kidney disease and forms There is evidence that they contain RNA.
complexes in the kidneys.
Other strains spontaneously develop ANA, some
in very high incidence, and are able to produce speci- Canine SLE
fic ANA when suitably immunized. The titres of A canine counterpart of human SLE has been
ANA obtained vary with the strain, but high titres described, characterized by LE cells, circulating
are sometimes seen. The severe kidney disease of ANA, and fatal glomerulonephritis, associated with
the NBZ x NZW F1 hybrids is only one aspect of a severe Coombs-positive haemolytic anaemia often
their intrinsic hyperreactivity to some specific anti- accompanied by thrombocytopenic purpura. Poly-
genic stimuli, including DNA. A contribution arthritis, antibody to thyroglobulin, rheumatoid
by the endogenous Gross virus is suggested by the factor, and hyperglobulinaemia are also found. A
fact that glomerulonephritis still develops in germ- breeding colony of affected dogs has now been
free NZB mice, in which type-specific viral antigen established, and the offspring of affected parents, or
and anti-viral antibody are also present in the glo- an affected mother, exhibit multiple serologic abnor-
merular deposits. The most likely explanation is malities and thymic lesions but no clinical signs as
that the Gross virus triggers or reinforces the auto- yet of SLE. The data obtained to date do not point
immune reaction, resulting in glomerulonephritis. to a simple pattern of genetic inheritance, and the
However, the problem has yet to be solved, and the possibility of a vertically transmitted infectious agent
source of the DNA is still uncertain. In view of the has to be considered. One dog examined has had
presence of an RNA-dependent DNA polymerase virus-like particles in renal glomerular endothelium
in C-type particles, it is conceivable that the RNA similar to those reported in human SLE. An SLE-
in the virions may be transcribed into DNA pro- like condition has also been observed in cats.
viruses, thus providing material which either induces
or reacts with ANA. Equine infectious anaemia
There is increasing evidence that two factors are
involved in the pathogenesis of the lupus type of This is a disease produced by a virus transmitted
glomerulonephritis in mice. Mice carrying a large mechanically by insects and characterized by vascular
amount of Gross virus develop anti-DNA antibody lesions, anaemia, and glomerulonephritis. Spleno-
and a lupus-type glomerulonephritis, provided they megaly and lymphadenopathy are accompanied by
274 MEMORANDA
proliferation of atypical plasma cells; serum IgM
increases concomitantly with clinical episodes but
high titres may persist in chronic cases. The virus,
growing in cultured lymphocytes, is very similar
although not identical in ultrastructure to C-type
particles of leukaemia viruses. The infected horses
are C3-deficient, and complement coats the erythro-
cytes of some animals at various stages of the
disease. It is possible that virus antibody-complexes
on the surface of the erythrocytes account for the
presence of complement, in which case there is no
need to postulate the presence of true autoimmunity.
This condition would then be considered among the
immune-complex diseases.
Mycoplasma infection
Mycoplasma pneumoniae infection in man is regu-
larly followed by the transient appearance of cold
autoantibody with specificity against the I or i blood
group antigen. It has also been reported that anti-
bodies to some cell components of lung tissue are
present, and this finding would be worth confirming.
Antibodies with anti-I specificity have been repro-
duced in one experiment by inoculating M. pneumo-
niae into rabbits. Claims that rheumatoid arthritis is
related to infection by M. fermentans have not been
substantiated, and there is as yet no strong evidence
for a viral etiology of this disease.
Contents Table des matieres
Geochemical environments, trace elements, and cardiovascular diseases-
R. Masironi, A. T. Miesch, M. D. Cra4ford, & E. I. Hamnilton ... . 139
The role of permissible limits for hazardous airborne substances in the working
environment in the prevention of occupational disease-Theodore F. Hatch 151
Public health importance of rodents in South America-R. B. Mackenzie . . 161
A micrometabolic inhibition test for the estimation of poliovirus neutralizing
antibodies- Vassiliki G. Kyriazopoulou & Eleanor Bell . . . . . . . . . 171
Differences in the severity of physical signs in the right and left eyes of patients
with trachoma in Syria and Burma-P. G. Winkler, U Ko Lay, F. A. Assaad,
& T. K. Sundaresan ....................... . 177
Cutaneous responses to smallpox revaccination with calf lymph and the effect of
fluorocarbon purification of the vaccine-M. F. Polak, J. Huisman, J. M.
Bos, & A. C. Hekker . . . . . . . . . . . . . . . . . . . . . . . 185
An attempt to culture Mycobacterium leprae in cell-free, semisynthetic, soft
agar media-Toyoho Murohashi & Konosuke Yoshida . . . . . . . . . 195
A second international cooperative investigation into thioacetazone side effects:
2. Frequency and geographical distribution of side effects-A. B. Miller,
A. J. Nunn, D. K. Robinson, Wallace Fox, P. R. Somasundaram, & Ruth Tall 211
Report of the 1966-67 cholera vaccine trial in rural East Pakistan: 4. Five years
of observation with a practical assessment of the role of a cholera vaccine in
cholera control programmes-Wiley H. Mosley, K. M. A. Aziz, A. S. M.
Mizanur Rahman, A. K. M. Alauddin Chowdhury, Ansaruddin Ahmed, &
M. Fahimuddin ......................... . 229
Methods for dissecting dry insects and insects preserved in fixative solutions or
by refrigeration-Ernest U. Ungureanu . . . . . . . . . . . . . . . 239
Seasonal changes in the larvel populations of Aedes aegypti in two biotopes in
Dar es Salaam, Tanzania-Milan Trpis . . . . . . . . . . . . . . . 245

Memoranda
Virus-associated immunopathology: animal models and implications for human
disease: 1. Effects of viruses on the immune system, immune-complex
diseases, and antibody-mediated immunologic injury . . . . . . . . . . 257
Virus-associated immunopathology: animal models and implications for human
disease: 2. Cell-mediated immunity, autoimmune diseases, genetics, and
implications for clinical research . . . . . . . . . . . . . . . . . . 265