Immunosupression Features In Filovirus Infected Animals

E.Ryabchikova, L.Kolesnikova, Yu. Rassadkin

State Research Center of Virology and Biotechnology "Vector" Koltsovo Novosibirsk Region 633159 RUSSIA

Filoviral Marburg and Ebola infections are severe hemorrhagic fevers. Damage to blood clotting and vessel permeability are the most prominent clinical features in humans. In fatal Marburg and Ebola infections, humans and monkeys generally die with no evidence of an immune response; however, details of immune system damage are poorly investigated [Peters et al., 1996].

The goal of our study was to identify morphological features of the immune system impairment in monkeys infected with Marburg (Popp strain) and Ebola (Zaire strain) viruses. Green monkeys were parenterally infected with 100 LD50 of Marburg virus or Ebola virus. The samples of spleen, mesenteric, inguinal and peribronchial lymph nodes, lymphoid tissues of respiratory and gastrointestinal tracts were obtained daily, post infection (PI), and prepared for light and electron microscopy.

We found development of early pathological changes in lymphoid tissue in all sites of its anatomical locations in all the studied animals on days 2-3 PI: mitotic activities of lymphoid cells had ceased; swelling of macrophage and stromal cells had developed. Disintegration of dendritic (antigen-presenting) cells was found on days 3-4. These pathological changes increased during the course of both infections. At the terminal stages of Marburg and Ebola infections, we found severe lesions of monkey lymphoid tissue:

The histopathological pattern was complicated by additional damage to vascular endothelium, thrombosis and diapedesis of erythrocytes. The lymphocytes remained unchanged during the infection.

Marburg and Ebola viruses reproduced in macrophages and few fibroblasts in lymphoid tissue. Lymphoid cells are not able to reproduce filoviruses. The first Marburg virus infected macrophage cells were recognized in lymphatic tissue in splenic white pulp and mesenteric lymph nodes on day 5 PI. In the case of Ebola infection, first infected cells were identified in white pulp on day 3 PI and in lymph nodes on day 4 PI. The number of infected cells increased during the infection and numerous viral particles were situated in the intercellular space.

Reproduction of Marburg and Ebola viruses in macrophage cells in guinea pigs was shown in our previous studies [Ryabchikova E. et al., 1996; 1996a]. Macrophage cells play a significant role in regulation of various physiological and pathological reactions. Reproduction of filoviruses in these cells undoubtedly is important in the pathogenesis of Marburg and Ebola infections.

Other features of immune system damage are swelling and destruction of macrophages and stromal cells. Destruction of macrophages and stromal cells was observed from days 4-5 PI. No stromal and macrophage cells remained unchanged at the terminal stages of Marburg and Ebola infection in monkeys and guinea pigs [Ryabchikova E. et al., 1994; 1996a]. It is noteworthy that both infected and uninfected cells disintegrated. Macrophage and stromal cells disintegrated even at the location sites of lymphoid tissue that showed no obvious signs of viral infection. This was particularly true for lymphoid nodules and follicles of lung and intestine. The widespread disintegration of stromal and macrophage cells may be of importance in filoviral pathogenesis because both cell types are directly involved in regulation of immune response.

We observed total destruction of dendritic cells in Marburg and Ebola viruses infected monkeys from day 4 PI. The same observation was made during the studies of guinea pigs infected with Marburg and adapted Ebola viruses [Ryabchikova E. et al., 1996; 1996a]. Necrosis of dendritic cells, undoubtedly, aggravated the course of the disease because precisely they are responsible for primary presentation of virus antigens to immunocompetent cells.

A feature of lethal Marburg and Ebola infections is the absence of leukocyte reaction to infected cells. We did not observed migration of leukocytes to infected cells and their contact in the liver and other visceral organs. In the case of nonlethal Ebola infection in guinea pigs, clear leukocyte reaction to infected hepatocytes was found [Ryabchikova et al., 1996]. In guinea pigs aerogenically infected with Marburg virus, neutrophils and lymphocytes migrated to the liver infected cells [Ryabchikova E. et al., 1996 a]. However, the effector reaction did not develop and both leukocytes and infected cells remained morphologically unchanged.

Lymphopenia is a consistent feature of Ebola and Marburg infection in monkeys and guinea pigs [Luchko et al., 1995; Lub et al., 1995] and also in humans [Peters C.J. et al., 1996]. We examined bone marrow of Ebola virus infected monkeys and found severe damage to hemopoiesis, namely cell necrosis, cell depletion, diapedesis of erythrocytes, hemorrhages, a sharp decrease in the number of dividing and differentiating cells, and swelling of endotheliocytes. There was a breakdown of stromal cells and macrophages in the sections of bone marrow. The same changes were found in bone marrow of Marburg virus infected guinea pigs [Ryabchikova et al., 1996a].

Involution of lymphoid follicles developed in monkeys over the course of Marburg and Ebola infection. Rapid involution was manifested as a 69% reduction in the relative volume of the germinative centers of the follicles of the mesenteric lymph nodes during the first two days of Ebola infection. The reduction was mainly due to a decrease in the number of follicles and, to a lesser extent, in their size. The increasing lymphoid depletion became obvious from days 3-4 in both Marburg and Ebola infections. The lymphoid depletion was not caused by necrosis of lymphocytes. At the last days of infection, few visible follicles of lymph nodes and splenic white pulp had morphological appearances suggesting their waning function: small size, homogeneous germinal centers without blast cells. The B-zones were more affected than the T-zones in lymph nodes. However, the T-zones showed no signs of functional activity. Taken together, our studies revealed severe damage to lymphoid tissue in Marburg and Ebola viruses infected monkeys, which indicates that the function of immune system is greatly affected. The changes are multicomponent and each component itself can have a significant impact on the function of the immune system. In lethal filoviral infections the impairment is manifested in monkeys and guinea pigs at the morphological level as suppression of the cellular and humoral links of immunity: plasma cells are not formed and T-cells fail to react with infected targets. The following features of immune system damage are identical in lethal Ebola and Marburg infections in both guinea pigs and monkeys: lymphoid depletion, filovirus reproduction in macrophage cells, blocking of mitosis and plasma cells formation, and an absence of leukocyte attack to infected cells. All these features are present in the aerosol form of Marburg infection, except the last. This difference may be due to the additional time it takes the virus to enter the blood when infected aerogenically. Most interesting is that this is the first description of the pathological changes and impairment of the immune system in these viral infections. In the lethal infections, the inflammatory reaction does not develop, meaning that the immune defense is blocked. But in nonlethal infection, the leukocytes attack the infected cells and result in the formation of inflammatory foci.

References

Lub M. et al. - Probl. Virol. (Russian) - 1995. - N4, p. 158-161.
Luchko S. et al. - Bull. Exp. Biol. Med. (Russian) - 1995. - N 9, p. 302-304.
Peters C.J. et al. -In: Fields Virol., Third Ed. - 1996. - 1161-1176.
Ryabchikova E. et al. - Bull. Exp. Biol. Med. (Russian) - 1994. - N 4, p. 430 434.
Ryabchikova E. et al. - Arch.Virol. - 1996. - v.141, p. 909-921.
Ryabchikova E. et al. - Arch.Virol. - 1996a. - v.141, p. 2177-2190.

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