The severe combined immunodeficiency (scid) mouse. A laboratory model for the analysis of Lyme arthritis and carditis.

We report that the spirochete B. burgdorferi induces progressive polyarthritis and carditis in mice with severe combined immunodeficiency syndrome (scid) but not in normal C.B-17 mice. The onset and severity of the disease were dependent on (a) the viability; (b) the infectivity; and (c) the dose of inoculated B. burgdorferi organisms. Infective spirochetes were isolated from both blood and joints of inoculated scid mice. These findings suggest that B. burgdorferi-induced chronic arthritis and carditis in mice develops independently of lymphocyte function and makes the scid mouse an attractive laboratory model to study the role of the immune system in experimental Lyme Borreliosis.

[Robert Koch's unpublished and unfinished experiments on sheep-pox (author's transl)]. / Nicht veröffentlichte unvollendete Versuche Robert Kocks über Schafpocken

The archives of the Robert Koch Institute include a casket with preparations and handwritten notes by Robert Koch (Fig. 1). He made these preparations during his time as a rural doctor between October of 1878 and September of 1880. They refer to an outbreak of sheep-pox at Rackwitz, a place near his practice at Wollstein (Fig. 2). This work has not been published; we know of it from one of Robert Koch's private letters (11). To reconstruct his working scheme and reasoning, we consulted particularly his reports on rinderpest experiments which he began in 1896 (6). The preparations from this casket which had been stained with Bismarck brown (according to Weigert) date back to a period when Robert Koch developed the foundations of bacteriology and they are evidence of his preparedness to accept new operational procedures (1, 2, 3). Thus, we have to assume that these preparations were to serve as evidence of a bacteriological etiology of sheep-pox. A wrong conclusion as to associations between the superinfection present and etiology of the disease (7) was ruled out by maintaining his own postulate. Simultaneously with this preparation work, Robert Koch performed animal experiments (11). His experience from these studies was utilized later on in his rinderpest experiments (6). On account of his confrontation with viral disease - which had its starting point in his unpublished work on sheep-pox - Robert Koch stated his postulate to be valid in the same manner as if bacteriological etiology had been demonstrated (4,6). The importance of these preparations is also seen in the interpretation of viral tissue damage, i.e. increase of macrophages and plasma cells with subsequent necrosis (9) characteristic of vira infection (Figs. 3, 4).

[Sulphated glycosaminoglycans as virus inhibitors. 3rd communication: therapy of viral diseases by means of glycoasaminoglycanpolysulphates. Establishment of fundamentals in experiments with laboratory animals (author's transl)]. / Sulfatierte Glycosaminoglycane als Virusinhibitoren. 3. mitteilung: zur ermittlung der Grundlagen einer Therapie von Viruskrankheiten mit Glycosaminoglycanpolysulfaten im Tierversuch

Following the in vitro and in vivo demonstration of their inhibitory effect upon 17 D yellow fever virus (Comm. I and II) it has been tried to demonstrate the therapeutic effect of three GAGPS (L1, L5, L8)1 in experimental animals. It had been found that L1 possessed the strongest inhibitory action and L5 the lowest toxicity. L8 served as control substance with different chemical structure. Mice that had been intracerebrally infected with 50 to 100 LD50 yellow fever virus were subsequently treated with L1, L5 and L8 by i.v., i.p., i.m., and oral routes. At first it was found by cytophotometric measurements that the i.c. applicated substances accumulated in the nerve cells of the hippocampus major, the cerebellum (Purkinje cells) and the cortex; the uptake was nearly doubled if a mixture with virus was used (Table 1). Following preliminary experiments to determine the adequate quantity of virus, five experiments were performed in the order mentioned. In the first series were treated groups of 30 animals after intracerebral infection with 100 mug/0.02 ml L1 by the i.m. and i.p. routes respectively, beginning from the first day p.i. for a period of seven days (Table 2). A certain difference of the rate of deaths and surfivals was seen between the treated and untreated groups. Among the treated mice delayed death was a prominent occurrence (Fig. 1). A second experiment involving a double dose of L1a (200 mug/0.02 ml) from another batch of GAGPS showed no better effect (Table 3). An explanation was given by the fact that L1a demonstrated a moderate toxicity with high doses about 5000 mug/ml in the i.c. control (Table 4 and 5). A graphic representation of both experiments can be found in Figs. 2a and 2b. The relative low virus input in the third series as shown in the virus control impedes additionly clearcut results. In the fourth experiment the infected mice were treated with GAGPS doses between 250 and 2500 mug/ml; L1 was administered by the oral, L5 and L8 by the intraveneous route. The death rate of the animals treated with low doses of L1 (250-1000 mug/ml) is diminished clearly and there was a significant difference between treated and untreated mice when L5 and L8 were applied (Table 6). Fig. 3 shows the graphic representation of experiment four. The good results of treatment were confirmed by histopathological findings (Table 7). There was a clear difference in the kind and quantal distribution of cerebral lesions in treated and untreated mice. In the last series L1 was administered by the i.v., L5 and L8 by the oral route (Table 8). Although the virus dose given in this series was rather low a protective effect was seen with low doses of L1 (312 mug/ml) and L5 )500 and 1000 mug/ml). Also these results were confirmed by histopathological examination. In summary, the GAGPS L1, L5 and L8 were found to have a clear therapeutic effect upon the experimental encephalitis of mice caused by infection with 17 D yellow fever virus, in the case of experiment four with statistical significance.

[A contribution to infection with yellow fever virus 17D in chick embryos (author's transl)]. / Ein Beitrag zur Gelbfiebervirus-17D-Infektion in Hühnerembryonen

During the production of yellow fever virus 17 D vaccine from chick embryos, few embryos die in the time between injection of the eggs and harvest of the embryos. In order to answer the question whether the death of the embryos is due to the infection or to the injury caused by the injection, it is necessary to examine the embryos macro- and microscopically for pathological changes. 8 and 9 days old chick embryos were inoculated into the amniotic cavity with different concentrations of yellow fever virus 17 D (table 1). The embryos were removed from the eggs between the 2nd and 7th day after inoculation. Heart, lung, kidney, brain, liver, and spleen were removed. Macroscopically observable pathological symptoms of the embryos and their organs were recorded. Sections of the organs were histologically investigated. Some embryos died soon after the infection, others on the 5th and 6th day of incubation (table 2). Their death was dependent on the dosage of the inoculum. Macroscopically, some embryos showed oedemas, petechiae on head and trunk, haemorrhages of liver and kidney, enlargement of liver and spleen, and a yellow discolouration of the liver (table 3). Microscopically, liver and brain showed the greatest pathological changes; heart and kidney were also affected, whereas lungs and spleen seemed to be unaffected. The following pathological changes were observed: slight fatty degeneration, oedemas, vascular inflammation, perivascular infiltrates, diffuse infiltrations, infiltrations in form of small nodules and necroses (table 3 and figures 1-12). the severity of the symptoms was evaluated using arbitrary units. These units are summarized on table 3 according to time of occurrence p.i., to virus dilution, to organ, and to type of symptom. An index of pathological changes was allived by dividing the sum of units by the number of organs. The maximum value of the index was demonstrable in liver and brain (table 4). Figure 13 adn table 5 show the development of the lesions during the infection. The maximum value of the index was reached on the 5th day p.i. The most significant pathological changes, as necrosis and perivascular infiltrate, were mainly observed from the 5th day p.i. on (table 6). The perivascular infiltrates were found in heart and brain, the necroses in brain and liver (table 7).

[Sulphated glycosaminoglycans as virus inhibitors. 2nd communication: Inhibitory effect of glycosaminoglycanpolysulphates on yellow fever virus 17 D in animal experiments (author's transl)]. / Sulfatierte Glycosaminoglycane als Virusinhibitoren. 2. Mitteilung: Hemmwirkung von Glycosaminoglycanpolysulfaten auf Gelbfiebervirus 17 D im Tierversuch

Glycosaminoglycanpolysulfates (GAGPS) have virus inhibiting properties demonstrable by means of tissue culture in the plaque method. In brain preparations of children who had died of a hyperpyretic toxicosis, cell necroses were found corresponding to the picture of tissue culture plaques. The question arose from these observations whether this inhibiting effect of GAGPS can perhaps also be demonstrated in vivo. In animal experiments, cell necroses corresponding to those of the infant brain could be observed during the course of a 17 D yellow fever encephalitis in mice. The Luitpoldt-Werk Munich placed to our disposal 13 different GAGPS for tests. Each of these substances was tested in 210 mice (fig. 1). Virus dilutions (LD 50/ml) were mixed to the same volume with the indicated concentrations of substance directly before vaccination. The differing LD 50 doses is due to the fact that each ampoule contains a different content of virus.) The toxicity of all substances is practically zero (table 2a, 2b). The effect of the inhibiting substances was evaluated at first by means of a deviation of the rate between alive and dead animals (table 1). The statistical significance of the effect of some substances was that high so that an inhibition of the virus replication has obviously to be considered. The significance for L1 and L4 - they are chemically very similar - is higher than 0.001 (table 4). The virus inhibiting effect of the substances was controlled by histopathology. 31 brains of mice were dissected and histologically evaluated; the lesions of the brains were examined and recorded (table 3). The effect of the substances was measured by absence or diminution of the lesions. The most effective substance was L1 as far as its concentration was higher than the critical limit of 625 gamma/ml.

Quantitative determination of the disintegration of nerve cells in the cortex caused by viral encephalitis (17 D-yellow fever) (Preliminary communication).

The counting of nerve cells give an answer to the question whether there is a significant loss of cells in the cortex in the course of viral encephalitis. For normal animals, average cell numbers of 275.42 were counted; for infected animals divided into two groups depending on the severity of illness, 144.95 and 192.87 cells, respectively (p = 0.0001).

Relation between decreased mental efficiency in mice and the presence of cerebral lesions after experimental encephalitis caused by yellow fever virus.

The behavior of 1,072 mice that had recovered from encephalitic infection with intracerebrally injected yellow fever virus 17D and of 216 normal mice was tested in a maze and on a horizontal rod rotating around its axle. Infected animals needed more time (average, 8.90 min) to find their food in a maze than did normal animals (average, 4.37 min). Infected mice were able to stay on the rotating rod for a shorter time (average, 6.4 seconds) than were normal animals (average, 9.0 seconds). The correlation between the concentration of virus injected and the performance of the mice was confirmed by the extent of lesions found by histologic study: animals that had anatomic lesions after surviving encephalitic infection showed abnormal behavior.

[Quantitative determination of the disintegration of nerve cells in the cortex caused by viral encephalitis (17 D-yellow fever), as a basis for the evaluation of the pathological processes in the central nerve system (author's transl)]. / Quantitative Bestimmung der Zerstörung von Nervenzellen im Cortex nach Virusencephalitis (17 D-Gelbfieber), eine Grundlage zur Beurteilung pathologischer Prozesse im ZNS.

A loss of nerve cells in the cortex after encephalitis was reported already in the classical work by Nissl, Spielmeyer, and Spatz. A loss of nerve cells will become only noticeable if it amounts to at least 50%. But as such clear pictures are rarely found, estimations were always considered as doubtful and incorrect. Not only the number of cells is important in consideration of the morphological change in the cortex but also the size of cells. The development of a new apparatus made is possible to consider two structural parameters: the surface and the perimeter of cells. In 497 histological serial preparations obtained from 43 mouse brains we determined the number, the surface, and the perimeter of nerve cells. 39 animals were infected intracerebraly with yellow fever 17 D; 4 normal animals served as controls. Among the infected animals, 8 were treated with a mucopolysaccharide. The cells were counted within a determined area (standard unity); this area was taken from the angle between the curbura exterior and the sulcus anterior-posterior of the brain. There was a significant difference between the number of nerve cells in normal (278) and in infected (202) animals. The animals treated with mucopolysaccharide showed a normal quantity of nerve cells but surface and perimeter corresponded to the data of the infected ones. The surface of normal animals was at 23.39, that of infected at 14.29. There was also a significant difference with regard to the cell perimeter: normal 14.97, infected 12.02. This means a shrinking of cells. The cell shrinkage revealed that the nerve cells were affected. The measurement of these three parameters presents new and exact statistical findings which enable a reconsideration of neurovirulence.

The portals of entry of herpes simplex virus infections.

In the present paper different possible portals of entry into the body for Herpes simplex Virus are examined. The possibility to infect the cornea with HsV without a preceding scarification was established. Scanning microscopy clearly showed the lesions of the infected cornea and a parallelity between the concentration of the inoculum and the spread of lesions. The i.c., i.m. and i.p. portals of entry are compared as to their capacity to produce encephalitis. The titration of virus may also be realized in the animal experiment. The intragastric portal of entry could be clearly demonstrated. So it is possible to explain herpes encephalitis in human newborns from mothers infected with Herpes virus.

[Sulphated glycosaminoglycans as virus inhibitors. 4th communication: clinical aspects of zoster with the proposal of a new treatment (author's transl)]. / Sulfatierte Glycosaminoglycane als Virusinhibitoren. 4. Mitteilung: Zur Klinik der Zoster-Erkankung mit einem neuen Therapeivorschlag

On the base of plaque inhibition tests in at least three virus-host-systems and successful treatment of mice with experimentally induced yellow fever encephalitis a well known sulphated glycosaminoglycan (GAGPS) "L5" was used as therapeutic agent in cases of zoster infection in man. The material was applied as 1% solution in water continuously on lint and rewettet for several days until nearly complete healing was achieved. The clinical course of 18 patients of former years (12 women, 6 men) was compared with 26 cases (19 women, 7 men) treated additionally with the new substance. All of them were carefully observed in the Rudolf-Virchow-Hospital in Berlin. Besides of registrating data as age and sex of the patients and the seasonal distribution of cases the course of illness was analyzed. The mean duration before admission to the hopsital was 4,9 days in the control group and 7,2 days in the L5-patients. Signs of organ diseases possibly acting as trigger mechanism for the remanifestation of the zoster were nearly twice in number in L5 cases than in the controls. Despite of this unfavorable stiutation the main parameters showed a clear tendency to return earlier to normal values in the GAGPS group. The fever was shortened. The blood lymphocytes normalized better with mean absolute values of 2400 in the L5-group and 3029 per m(3) in the control cases. The fall of the cell number in the liquor cerebrospinalis was more rapidly in the GAGPS treatment. The mean stay in the hospital was 33,2 days in L5 cases and 44,6 days in control patients respectively in the main group of age between 60 and 80 years. One death in the GAGPS group corresponded to four in the control group. In the local skin area the edema disappeared rapidly with beginning of treatment. Confluent vesicles flattened within 24 to 48 hours and no further efflorescences were seen. Pain diminished in few days. The good results justify continued trials.

[Abnormality in the behaviour of mice after an experimental encephalitis by yellow fever virus 17 D. Experiment model(author's transl)]. / Feststellung einer Verhaltensabnormität bei Mäusen nach experimenteller Enzephalitis durch Gelbfiebervirus 17 D. Modellversuch

Viral encephalitides in infants are very often followed by serious mental manifestations. To be able experimentally to investigate this pathological phenomenon, we established the following model. Mice having recovered from an intracerebral infection with yellow fever virus 17 D (routine test for the potency of yellow fever vaccine) and giving the impression of normal and healthy animals were examined for their psychical behaviour. As unit of measurement we took the time for running through a labyrith (fig. 1). At the entrance of the labyrith in a box there was placed the population of animals of one cage (6 animals in maximum) and at the opposite side there was deposited the food. The animals going to be examined had been without food for 24 hours. The time was taken having passed from the moment of placing the animals up to the moment of their nibbing at the food. We examined 11 groups of mice having past routine tests for the potency of the yellow fever vaccine. One of them had been tested twice at an interval of 54 days (table 1). The 791 mice which had passed the encephalitic infection showed an average running time of 8.90 min. The 164 controls, however, had an average running time of 4.37 min (table 2). This difference is significant (p=greater than 1/1000). The average running time is proportional to the injected virus quantity (table 3). The significance of each single virus dilution to the group of normal animals is always greater than 1/1000. The significance is more than 1/100 for each single virus dilution between 10(-1) and 10(-3) compared with the virus dilution of 10(-5). Immediately after the clinical period (21 days), no difference could be observed between the single virus dilutions. After 76 days only, the test showed the above mentioned differences (table 4). Therefore, the mice having found their food after a long running time had more serious postencephalitic lesions than those having had a shorter running time. To prove this statement, we examined histologically brains of 14 mice which had found their food after 20 min on an average and those of 16 mice having had an average running time of 6.58 min. The histological evaluation was performed in regard to eight pathological characteristica. The results were recorded on tables for each slide with a histological preparation. For this we used squared paper (fig. 2, 3). For each brain of mice, the so-called "index of lesions" was calculated by the proportion of labelled and all squares. .....

[Herpes Simplex Keratitis under the Scanning lectron Microscope(author's transl)]. / Keratitis dendritica unter dem Raster-Elektronenmikroskop

In 12 rabbits a dendritic keratitis could be provoked by applying a suspension of herpes viruses (Herpes virus hominis -- HVH type 1) onto their previously both uviolised and non-uviolised corneae. 4 days later ophthalmectomy was performed and the corneae were examined by the scanning electron microscope. Macroscopically, both in the uviolised eyes and the control eyes punctiform lesions of the corneae were observed. After short ray-treatment (3 min) no differences were present between the uviolised and non-uviolised eyes. At longer time of exposure (7--15 min), however, a more distinct alteration occurred in the uviolised eye. The scanning-electron-microscopical picture showed the dendritic lesions to be composed of numerous circular foci of 100 to 200 mu. There were no differences in the lesions of the uviolised and non-uviolised corneae. The fact that after ultraviolet ray-treatment a great part of the epithelial cells is sloughed off, but, nevertheless, the same dendritic lesions develop, contradicts the assumption of a morphological substrate of the epithelial cells being the inducing factor.

The influence of nicotinamide, tryptophan, and methionine upon galactosamine-induced effects in the liver.

After administration of D-galactosamine-HCl alterations in liver cells - histologically resembling hepatitis - occur. During this process several biochemical changes are demonstrable. The formation of these alterations may be prevented by combined administration of nicotinamide + L-methionine or DL-tryptophan + L-methionine. This had been confirmed by histology as well as by determination of GOT and GPT activity in the serum.

A mouse model for Borrelia burgdorferi infection: pathogenesis, immune response and protection.

Viable Borrelia burgdorferi (B. burgdorferi) organisms induce a chronic infection associated with arthritis, carditis and hepatitis in severe combined immunodeficiency (scid) mice but not in most of the adult mice from the various immunocompetent inbred strains tested. Furthermore, we have found that experimental inoculation of normal mice with B. burgdorferi organisms leads to the generation of antibodies and T cells specific for various spirochetal antigens including the outer surface proteins A and B (OspA, OspB) as well as flagellin. The assumption of a protective role of the immune response during B. burgdorferi infection in mice is supported by our recent findings that passively transferred B. burgdorferi-specific immune mouse sera as well as monoclonal antibodies to OspA are able to prevent the development of the disease in scid mice. We show now that purified OspA protein both in its native and recombinant form is immunogenic and that the antibodies generated are able to confer protection to scid mice against B. burgdorferi infection.

Demonstration of antigen-specific T cells and histopathological alterations in mice experimentally inoculated with Borrelia burgdorferi.

Antigen-specific T-cell responses and histopathological changes were studied in mice experimentally inoculated with Borrelia burgdorferi B31. Inbred mice with different H-2 haplotypes and/or different genetic backgrounds were inoculated with B. burgdorferi organisms and tested for antigen-specific T-cell responses in vivo (delayed-type hypersensitivity [DTH]) and in vitro (T-cell proliferation). Comparable DTH responses were found after inoculation with either inactivated (in the presence of adjuvants) or viable microorganisms in all mouse strains, except BALB/c, irrespective of the H-2 haplotype (b, d, k, or s) tested and the sex of the animals. Moreover, in mice presensitized to B. burgdorferi, DTH responses could be induced only with antigen preparations derived from the corresponding strain but not with those obtained from either related spirochetes such as Treponema phagedenis and Leptospira interrogans or unrelated bacteria such as Mycobacterium tuberculosis. T cells isolated from lymph nodes or spleens of mice previously sensitized to B. burgdorferi but not those from naïve mice could be induced for antigen-specific proliferation in vitro, as revealed by [3H]thymidine incorporation. Histopathological examination of mice inoculated with viable B. burgdorferi organisms revealed significant perivascular infiltrates consisting mainly of mononuclear and a few polymorphonuclear leukocytes in different organs (brain, heart, lungs, liver, and kidneys) and the appearance of giant multinucleated cells within the spleen similar to those found in human skin specimens of patients suffering from cutaneous manifestations of Lyme disease. Our findings suggest that mice are a suitable animal model with which to study the immune response to B. burgdorferi and the pathogenesis of Lyme disease.

Myositis in mice inoculated with Borrelia burgdorferi.

The authors describe the appearance of myositis in immunocompetent and immunodeficient mice after subcutaneous inoculation with Borrelia burgdorferi by histology and immunohistology. Experimental infection of mice 1) causes inflammation of striated but not smooth muscles, 2) affects the entire musculoskeletal system, and 3) is characterized by perivascular and interfacicular infiltration of mononuclear leukocytes in the striated muscle leading to necrosis as well as disruption of muscle fibers. The lesions found in striated muscle specimens were most pronounced in immunodeficient (SCID), less severe in T-cell-deficient nu/nu (BALB/c, C57BL/6) and marginal to moderate or almost not present in immunocompetent AKR/N and C.B-17 mice, respectively.

Prednisolone reduces experimental arthritis, and inflammatory tissue destruction in SCID mice infected with Borrelia burgdorferi.

Glucocorticosteroids (GC) are widely used as anti-inflammatory agents. The effects of Prednisolone on the development of Borrelia (B.) burgdorferi-induced clinical arthritis and organ inflammation was studied in severe combined immunodeficiency (SCID) mice. The drug was administered orally at a dose of 3, 10 and 30 mg/kg, starting shortly before experimental infection of the mice. A dose dependent inhibition of arthritic joint swelling was observed. Full protection was obtained with 30 mg/kg until 21 days after infection, subsequently, mild joint swelling developed but progression and severity of the disease was considerably less than in the other treated as well as in the untreated mice. Inhibition of clinical arthritis coincided with reduction of inflammatory cell infiltration in the joints, liver and muscle. Prednisolone was ineffective when application was initiated after arthritis was fully developed, i.e., 22 days after infection. Since the activated endothelium plays a critical role in development of inflammatory lesions, the expression of the cellular adhesion molecules (CAMs) E-selectin, P-selectin, ICAM-1 and VCAM-1 was determined in vitro using the bEnd3 endothelial cell line. Stimulation with a sonicated B. burgdorferi preparation in the presence of the water-soluble compound Prednisolone-21-hemisuccinate considerably reduced expression of ICAM-1, and marginally also of E-selectin, whereas the level of P-selectin and VCAM-1 remained unaltered. Thus, downregulation of ICAM-1 might be a critical factor in Prednisolone-mediated inhibition of B. burgdorferi-induced inflammation; the flare up of the disease after the initial protection indicates that additional therapy, e.g. with antibiotics, is necessary.

Studies on early events of Borrelia burgdorferi-induced cytokine production in immunodeficient SCID mice by using a tissue chamber model for acute inflammation.

Lyme arthritis, one of the common features of Borrelia burgdorferi infection in the human, is associated with the production of various monocyte derived cytokines. To investigate the expression and regulation of cytokines during the acute phase of spirochete induced inflammation, a perforated Teflon chamber was implanted under the dorsal skin of severe combined immunodeficiency (SCID) and immunocompetent co-isogenic C.B-17 mice. The histology of the surrounding chamber tissue exhibited sterile inflammation with several features reminiscent of an inflamed synovium, i.e. infiltration of polymorphonuclear and mononuclear cells, fibroblast-like cells and neovascularization. The experimental inoculation of Borrelia burgdorferi into the chamber resulted in the production of TNF-alpha, IL-1 and IL-6 into the chamber exudate, in both the immunodeficient, disease susceptible SCID and the immunocompetent, disease resistant C.B-17 mice. Peak levels of TNF-alpha were reached at 2 hours and of IL-1 and IL-6 at 6 hours after infection; by 24 hours, cytokine levels were only marginal (IL-1, IL-6) or non-detectable (TNF-alpha). Experimental infection by s.c. injection distant from the tissue chamber led to colonization of the spirochetes into the chamber, suggesting a tropism of the bacteria for this tissue. Thus, this model provides a system for studying acute events of Borellia burgdorferi induced cytokine regulation in a complex cellular, synovium-like environment that the bacterium encounters in vivo.

Perforin is essential for control of ectromelia virus but not related poxviruses in mice.

Lack of perforin renders the relatively resistant mouse strain C57BL/6 highly susceptible to the natural mouse pathogen ectromelia virus, a cytopathic orthopoxvirus. This is indicated by increased mortality, elevated virus titers and pathology in liver and spleen, and increased levels of liver enzymes in blood. Cowpox virus on the other hand is more virulent in the presence of perforin than in its absence. An additional lack of granzyme A which together with perforin is a constituent of cytoplasmic granules from cytotoxic T cells increases the virulence of cowpox virus.