Case Report Pathogenesis of Fulminant Monkeypox with Bacterial Sepsis After Experimental Infection with West African Monkeypox Virus in a Cynomolgus Monkey

Int J Clin Exp Pathol 2014;7(7):4359-4370 www.ijcep.com /ISSN:1936-2625/IJCEP0000874

Case Report Pathogenesis of fulminant monkeypox with bacterial sepsis after experimental infection with West African monkeypox virus in a cynomolgus monkey

Noriyo Nagata1, Masayuki Saijo2, Michiyo Kataoka1, Yasushi Ami3, Yuriko Suzaki3, Yuko Sato1, Naoko Iwata-Yoshikawa1, Momoko Ogata2, Ichiro Kurane2, Shigeru Morikawa4, Tetsutaro Sata1, Hideki Hasegawa1

Departments of 1Pathology, 2Virology 1, 3Division of Experimental Animals Research, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan; 4Department of Veterinary Science, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan Received May 23, 2014; Accepted July 1, 2014; Epub June 15, 2014; Published July 1, 2014

Abstract: The pathogenesis of severe human monkeypox, which causes systemic and fulminant infections, is not clear. This study presents a case repot of fulminant monkeypox with bacterial sepsis after experimental infection with monkeypox virus in a cynomolgus monkey (Macaca fascicularis). In our previous study (Saijo et al., 2009, J Gen Virol), two cynomolgus monkeys became moribund after experimental infection with monkeypox virus Liberia strain, West African strain. One exhibited typical monkeypox-related papulovesicular lesions. The other monkey presented fulminant clinical symptoms with a characteristic flat red rash similar to that found in smallpox, which is associated with extremely high fatality rates. In this study, we found that the monkey with flat red rash had high levels of viremia and neutropenia, as well as high plasma levels of pro-inflammatory cytokines and chemokines compared with the other monkey. Monkeypox virus replicates in epithelial cells and macrophages in various organs. Sepsis due to Gram-positive cocci was confirmed histopathologically in the monkey with flat red rash. The lack of inflammatory response in the lesion suggested that the monkey with sepsis experienced strong immune suppression during the viral infection. The neutropenia and excessive inflammatory cytokine responses indicate that neutrophils play key roles in the pathogenesis of systemic and fulminant human monkeypox virus infections with sepsis.

Keywords: Animal model, cynomolgus monkey, cytokine, monkeypox, neutropenia

Introduction the USA outbreak, but some pediatric patients developed serious complications that could Human monkeypox, i.e., monkeypox virus infec- have resulted in death [8]. The disease course tion of humans, is the most important ortho- is often milder than that of smallpox. However, poxvirus infection at present now that small- the mortality rate with monkeypox infection has pox, which is caused by Variola virus, has been been reported as 1.5-17% in Africa [3]. eradicated. Monkeypox virus is a zoonotic agent in rodents, nonhuman primate species, The clinical features of classical smallpox with a and humans. Outbreaks of human monkeypox high mortality rate, i.e., Variola major, are clas- occur occasionally in remote villages in central sified as ordinary type, modified type, Variola and western Africa near tropical rainforests sine eruptione, hemorrhagic type, and flat type [1-6]. In 2003, an outbreak of human monkey- [9, 10]. The hemorrhagic type, with widespread pox with fever and skin eruptions occurred in hemorrhages in the skin and mucous mem- Midwestern USA. The patients were found to be branes, and the flat type, where the pustules infected with a West African strain of monkey- remain flat, were usually fatal. The pathophysi- pox virus caused by close contact with sick pet ological processes of these fulminant types of prairie dogs (Cynomys spp.), which may have smallpox are not well understood. Cynomolgus been in contact with wild rodents imported (Macaca fascicularis) and rhesus macaques from Ghana [7, 8]. There were no deaths during have been used as nonhuman primate models Fulminant monkeypox in macaque of smallpox and orthopoxvirus infections a Luminex 100TM (Luminex Co., Austin, TX) [11-17]. with a Human Cytokines 25-plex Antibody Bead Kit (BioSource Invitrogen, Camarillo, CA), The virulence and pathophysiology of monkey- according to the manufacturer’s instructions. pox two strains, Zr-599 and Liberia (Congo All of the infection experiments using cynomol- Basin and West African monkeypox virus gus monkeys were conducted under biosafety strains, respectively), were evaluated in cyno- level 3 conditions according to the guidelines molgus monkeys [18], which showed that for animal experiments at the National Institute Zr-599 was more virulent than Liberia. The clini- of Infectious Diseases. All of the procedures cally advanced stage was characterized by pox- used in this study were approved as biosafety viral papulovesicular rashes on days 7-9 post- level 3 and 2 by the Committees for Biosafety, inoculation (p.i.). Three Zr-599-inoculated mon- Animal Experiments, and Handling, and the keys and one Liberia-inoculated monkey were Ethical Regulations of the National Institute of moribund during days 13-18 p.i. The other mon- Infectious Diseases, Tokyo, Japan. keys recovered and survived for the observa- tional period of 22 days p.i. However, one Histopathology, immunohistochemistry, and Liberia-inoculated monkey did not exhibit typi- double-immunofluorescence staining cal rashes, although it was debilitated. A post- mortem pathological examination on day 10 Paraffin-embedded tissues, including skin, p.i. showed that the monkey had sepsis with lymph node, spleen, thymus, lung, heart, liver, Gram-positive cocci. The present study investi- kidney, intestine, testis, and brain, were used gated the clinical, immunological, and histo- for the histopathological examinations and for pathological features of the monkey with fulmi- the immunohistochemical detection of mon- nant monkeypox to elucidate the pathogenesis keypox virus antigen using anti-Vaccinia virus and risk factors related to this severe orthopox- rabbit antibody, which was prepared as virus infection with sepsis. described previously [19]. The antigen was retrieved by autoclaving for 15 min at 121°C in Materials and methods citric acid solution at pH 6.0. Formalin-fixed lung tissue was subjected to electron micro- Animal experiments scopic analysis using Epon 812-embedded This study used four adult cynomolgus mon- ultra-thin sections. To characterize the virus- keys (Tsukuba Primate Research Centre, infected cells, paraffin-embedded tissues were National Institute of Biomedical Innovation, subjected to a double-immunofluorescence Tsukuba, Japan), which were infected with mon- staining procedure using a rabbit antiserum keypox virus Liberia strain at a dose of 106 against Vaccinia virus, cytokeratin monoclonal plaque-forming units via subcutaneous injec- mouse antibody (clone MAB1611, Chemicon, tion, as reported previously [18]. After experi- CA), and a monocyte monoclonal mouse anti- mental infection, the animals were examined body (clone Mac387). The antigens were until 21 days p.i. to assess their clinical mani- retrieved by autoclaving in the retrieval solution festations and hematological parameters. at 121°C for 15 min, at pH 9.0 (Nichirei, Tokyo, During the clinical observation period, two mor- Japan). The sections were incubated with the ibund animals (#4567 and #4625) were sacri- first monoclonal antibody against cytokeratin ficed under anesthesia and autopsied to per- or monocytes, followed by incubation with anti- form pathological examinations on days 10 and serum against the Vaccinia virus antigen. The 18 p.i. The complete blood cell counts in the virus antigen-binding sites, and cytokeratin and peripheral blood collected in sodium heparin- monocytes were detected using goat anti-rab- treated tubes were measured using an autoan- bit Alexa Fluor 488 (Molecular Probes, Eugene, alyzer (Cell Tuck, Nihon Koden, Tokyo, Japan), OR) and goat anti-mouse Alexa Fluor 586 and the neutrophil, lymphocyte, monocyte, (Molecular Probes), respectively. The sections eosinophil, and basophil counts were deter- were incubated with the antibodies for 30 min mined by microscopic analysis. To evaluate the at 37°C, before mounting in SlowFade Gold immune responses of the monkeys infected antifade reagent with 4’,6-diamidino-2-phenyl- with monkeypox virus, the cytokine and chemo- indole (DAPI) (Molecular Probes) and the imag- kine levels in the plasma were measured using es were captured using a fluorescence micro-

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Figure 1. Clinical course, including the rash, viremia, and hemograms, of cynomolgus monkeys (#4639 and #4650, recovered monkeys; #4567 and #4625, moribund monkeys) after subcutaneous inoculation with monkeypox virus. A: Clinical images showing the rash on a surviving monkey (#4650, left panel) and a moribund monkey (#4567, right panel) on days 9 or 10 post-inoculation. B: The viral loads (number of copies/ml) in the whole blood of monkeys were measured using quantitative real-time PCR. Time course of the hematological changes in the white blood cell, neutrophil, lymphocyte, and monocyte counts (number of cells/µl) of the monkeys. scope (IX71; Olympus, Tokyo, Japan) equipped period. However, one monkey (#4567) had a with a Hamamatsu high-resolution digital B/W red rash without the typical papulovesicular CCD camera (ORCA2; Hamamatsu Photonics, rash evolution, which is generally observed in Hamamatsu, Japan). ordinary type monkeypox, and it was moribund on day 10 p.i. (Figure 1A, right panel). Analysis Results of the virus genome in the serum using quantitative real-time PCR, as reported previously Clinical features of fulminant monkeypox [19], showed that the peak viremia level of the recovered monkeys was <107 gene copies/ml All four monkeys exhibited clinical illness after on day 7 p.i., whereas that of the moribund subcutaneous inoculation with Liberia, includ- monkey (#4567) was >108 gene copies/ml on ing low activity, lethargy, and rash. Three mon- day 10 p.i. (Figure 1B). In addition, the peak keys had typical poxviral papulovesicular rash- viremia remained continuous in another mori- es on days 7-9 p.i. (Figure 1A, left panel). One bund monkey (#4625) after 7 days p.i. The total monkey (#4625) was moribund on day 18 p.i. blood cell count showed that moribund monkey but two monkeys (#4639 and #4650) survived #4567 had counts of <5,000 neutrophils/µl until the end of the 3-week clinical observation during the observation period (Figure 1B). By

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Figure 2. Changes in the chemokine (A), cytokine (B), and colony-stimulating factor (C) responses of cynomolgus monkeys after subcutaneous monkeypox inoculation.

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Figure 3. Histopathological features of the skin and lungs of the two moribund monkeys, #4625 (A-C) and #4567 (D-O), on days 18 and 10 post-inoculation (p.i.), respectively. (A-C) Pock lesion from the skin showing necrosis with severe inflammatory infiltration in the epidermis and dermis (A). Diffuse infiltration of mononuclear cells and neutrophils (B). Virus antigen-positive cells detected in the lesion (C). (D-F) Skin: spongiform degeneration (D, asterisks) and ballooning degeneration (E, higher magnification of D). The degenerate epithelial layer was positive for virus antigen (F, sequence section of E). (G-L) Lung: poxvirus infections in the bronchi and alveoli. Stratified and degenerate bronchiolar epithelial cells (G, H). Lack of inflammatory responses in the alveolar area(I). Virus antigen-positive cells detected in the bronchi and alveoli (J-L). (A, B, D, E, G-I) Hematoxylin-eosin staining. (C, F, J-L), Immunoperoxi- dase test using a polyclonal antibody against Vaccinia virus. (G, J) Original magnification ×10. (A, D, G, J, bar = 200 µm), ×100. (B, C, E, F, H, I, K, L, bar = 20 µm). (M-O) Electron microscopic analysis of alveolar macrophages in the lungs of moribund monkey #4567 on day 10 p.i. Virus particles are visible in the degenerate alveolar macrophages (M). (N), bar = 2 µm. Immature (M inset, bar = 200 nm) and mature (O, bar = 200 nm) virions in the cytoplasm of alveolar cells. contrast, the other three monkeys had counts Histopathological features of fulminant mon- >5,000 neutrophils/µl, which peaked at around keypox 15,000 neutrophils/µl at day 15 p.i. The neutrophil level decreased suddenly in #4652 Moribund monkey #4625 had a typical poxviral monkey on day 14 p.i. and the animal was mori- papulovesicular rash, where the histological bund. Interestingly, the lymphocyte counts findings indicated that the rash was swollen increased in all of the monkeys at day 10 p.i., with necrotic epithelial layers and severe including the moribund monkey. inflammatory reactions Figure ( 3A, 3B). Numerous neutrophils, mononuclear cells, and Plasma cytokine levels poxvirus antigen-positive cells were detected in Moribund monkey #4567 had high concentra- the lesion (Figure 3B, 3C). The histopathology tions of monocyte chemoattractant protein 1 detected swollen keratinocytes (ballooning (MCP-1/CCL2), IL (interleukin)-1-receptor ant- degeneration) in the skin of moribund monkey agonist (IL-Ra), and IL-6 in its plasma at the #4567 (Figure 3D, 3E). The inflammatory moribund endpoint on days 9 and 10 p.i. (Figure response was very slight or lacking in the skin 2A, 2B). Moribund monkey #4625 had extreme- lesions of moribund monkey #4567. The degen- ly high concentrations of MCP-1/CCL2 and erate epithelial cells were positive for the virus macrophage inflammatory protein-1 α (MIP-1α/ antigen according to the immunohistochemis- CCL3) in the plasma at the moribund endpoint try (Figure 3F). Stratified and degenerated on days 10, 14, and 18. The concentrations of bronchiolar epithelial cells with very slight monokine-induced interferon-γ (MIG/CXCL9), inflammatory infiltration were also present in IL-8, tumor necrosis factor-α (TNF-α), interferon the lungs of moribund monkey #4567 (Figure (IFN)-α, IFN-γ, and IL-13 in moribund monkey 3G, 3H). The pathological findings for the two #4567 were at least three times higher than monkeys (#4639 and #4650) were described those in the surviving monkeys at days 9 or 10 in a previous study [18]. The immunohistochem- p.i. (Figure 2A, 2B). The levels of IFN-α ical analysis showed that the degenerate epi- increased in moribund monkey #4625 after 12 thelial cells in the bronchi were positive for the days p.i. The levels of anti-inflammatory cyto- virus antigen (Figure 3J, 3K). There were no kine IL-10 were undetectable within 10 days p.i. obvious changes in the alveolar area, such as in all monkeys, but they increased after 14 inflammatory responses or detached cells days p.i. in the surviving monkeys (Figure 2B). (Figure 3I). However, there were large amounts The high levels of MIP-1α/CCL3 and IL-12 p40 in the plasma were the same as those found in of virus antigen-positive cells in the alveoli the surviving monkeys after the infection, according to the immunohistochemistry (Figure whereas the level of regulated on activation 3L). Surprisingly, virus antigen-positive cells normal T cell expressed and secreted (RANTES/ were found in all of the lung lobes in moribund CCL5) was lower in monkey moribund #4567 monkey #4567. Formalin-fixed lung tissue was (Figure 2A, 2B). These results show that the subjected to electron microscopic analysis levels of macrophage-, neutrophil-, and IFN- using Epon 812-embedded ultra-thin sections related pro-inflammatory chemokines and cyto- to confirm viral replication in the lungs. The kines were elevated significantly at the mori- sequential stages of virus particle develop- bund endpoint in monkey #4567. ment, including immature and mature virions,

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Figure 4. Histopathological features of the lymph node (A), liver (B), and testis (C) of moribund monkey #4625 on day 18 post-inoculation (p.i.), and those of the lymph node (D), spleen (E), bone marrow (F), epicardium (G), visceral pleura (H), and liver (I) of moribund monkey #4567 on day 10 p.i. Sinus enlargement and virus antigen-positive cells were present in the lymph node (A). Kuppfer cells were positive for virus antigen in the liver (B). Pock lesion in the testis (C). Severe inflammatory cell infiltration occurred in the surrounding focal necrosis (C, plus). Hemorrhage and necrosis were observed in the lymph node and spleen (D and E, asterisks). Lymphocytes were reduced in the follicular area (D and E). Myeloblasts and megakaryocytes were reduced and necrotic substances were observed in the bone marrow (F). Edema and slight inflammatory infiltration were observed in the epicardium (G) and visceral pleura (H). Virus antigen-positive cells in the lesion (D-H, insets). Swollen hepatocytes and amphophilic cytoplasmic inclusion bodies were detected with virus antigens in the liver (I, inset, arrows). (A–I) Hematoxylin-eosin staining. Insets, immunoperoxidase test using a polyclonal antibody against Vaccinia virus. Original magnification: ×10 (A, C, D, E; bar = 200 µm); ×100 (B, F, G, H, I, and all insets; bar = 20 µm). were observed in the cytoplasm of alveolar severe lymphocyte depletion in the lymphoid macrophages (Figure 3M-O). tissues, including the lymph nodes, spleen, thymus, and bone marrow, suggest that moribund The histopathological analysis detected swol- monkey #4567 experienced severe immune len monocytes in the sinus of the lymph node in suppression (Figure 4D-F). Hemorrhage and moribund monkey #4625, which were virus necrosis were also observed in the lymphoid antigen-positive cells (Figure 4A). The liver did tissues (Figure 4D-F). Edema and slight inflam- not exhibit any histopathological changes, matory infiltration with mononuclear cells were although the Kupffer cells were positive for observed in the epicardium and visceral pleura virus antigen whereas the hepatocytes were membrane of moribund monkey #4567 (Figure not (Figure 4B). The pock lesions in the testis 4G, 4H). Antigen-positive cells were detected in comprised necrosis and granulomatous inflam- the lymphoid tissues and serous membrane by matory reactions, including virus antigen-posi- immunohistochemistry. Amphophilic cytoplas- tive cells (Figure 4C). The lack of inflammatory mic inclusion bodies were found in the swollen responses in the skin and lung lesions, and the hepatocytes of the liver in moribund monkey

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Figure 5. Double-immunofluorescence staining to detect monkeypox-infected cells in moribund monkey #4567 on day 10 post-inoculation. The monkeypox virus antigen (red) colocalized with MAB1611-positive epithelial cells (green) in the skin, liver, and intestine. MAC387-positive monocytes (green) and monkeypox virus antigen (red) colocalized in the same cells.

#4567, which were positive for the viral antigen alveoli, were also positive for virus antigen (Figure 4I). Intralesional bacterial colonies were (Figure 5). The Mac387-positive macrophages detected in the lungs, bone marrow, and brain in the lung alveoli, lymph node, bone marrow (data not shown). These results suggest that (Figure 5), spleen, and liver (data not shown) moribund monkey #4567 suffered from a sys- were virus antigen-positive. temic monkeypox infection with Gram-positive coccal sepsis. Discussion

Double-immunofluorescence staining was con- Previous analyses of the histopathological ducted to detect monkeypox virus-susceptible appearance of vaccination-inoculation sites in cells in moribund monkey #4567 (Figure 5). biopsies have shown that papulovesicular der- The cytokeratin-positive epithelial cells in the matitis comprises vacuolated epithelium, skin (Figure 5), bronchi in the lung, intestine, edema of the papillae, hemorrhage, mononu- and liver (data not shown), but not those in the clear and polymorphonuclear cells, and peri-

4367 Int J Clin Exp Pathol 2014;7(7):4359-4370 Fulminant monkeypox in macaque vascular infiltration [10]. The histopathological system directly [23-26]. Monkeypox virus features of ordinary type monkeypox virus shares 14 immunomodulatory genes with infections comprise fibrinonecrotic inflamma- Variola virus, which affect IFNs, TNFs, interleu- tory reactions with macrophage and neutrophil kins, complement, and chemokines. In addi- infiltration in the viral replication sites, includ- tion, monkeypox virus encodes two additional ing the skin, respiratory tract, gastrointestinal proteins compared with Variola virus, which tar- tract, lymphoid organs, mucosal surfaces, and get the innate immunity pathway [25]. genital organs [11-13, 18]. In the present study, however, inflammatory infiltration was observed Increased numbers of leukocytes in the blood, rarely in the skin, lung, and liver, although abun- including lymphocytes, neutrophils, and mono- dant virus antigens were detected in these cytes, appear to be crucial for the immune sites in moribund monkey #4567. The absence response when surviving monkeypox virus of inflammatory infiltration in the skin may have infections. However, moribund monkey #4567 caused the general lack of papulovesicular der- had lower numbers of neutrophils compared matitis in moribund monkey #4567 and it was with the surviving monkeys. In addition, mon- also the cause of the widespread systemic viral key #4652 exhibited a sudden increase in its infection. In another study, 1/15 monkeys inoc- neutrophil levels on day 14 p.i. and it was mori- ulated with aerosolized Zaire strain monkeypox bund. The hematological and histopathological virus appeared to exhibit secondary bacterial analyses suggested that the moribund monkey septicemia, whereas the others died of bron- #4567 had myelodysplasia-related neutrope- chopneumonia [13]. The septic monkey had nia as an underlying condition. Previously, a preexisting subacute-to-chronic portal hepati- Vaccinia virus infection mouse model demon- + + + tis, bacterial septicaemia, and terminal differ- strated that CD11b Ly6C Ly6G cells, including ential interference contrast [13]. neutrophils and macrophages, produce type I IFNs and large quantities of reactive oxygen The presence of neutropenia in moribund mon- species [27]. Mice with depleted Ly6G+ cells key #4567 after infection might explain the and an absence of reactive oxygen species also development of the systemic and fulminant exhibited severe tissue damage after infection. monkeypox infection, including sepsis. Chem- Furthermore, a previous study showed that otherapy-induced neutropenia occurs in some monocytes and granulocytes were positive for cancer patients and is caused by bacterial poxvirus antigen after intravenous infection infections [20]. In septic shock syndrome, bac- with monkeypox virus in rhesus macaques, terial lipopolysaccharide induces high levels of according to flow cytometry analysis 28[ ]. the pro-inflammatory cytokine IL-6 and pro- In conclusion, the moribund monkey #4567 inflammatory chemokine IL-8 [21, 22]. Septic exhibited neutropenia and excessive inflamma- patients also have high levels of TNF-α, IL-1 tory responses after experimental infection receptor antagonists, and IL-2 receptors. with the Liberia strain of monkeypox and con- Moribund monkey #4567 had high levels of comitant bacterial sepsis. These reactions IL-6 and IL-8 in its serum. Thus, the high expres- might have promoted the development of sys- sion levels of IL-6 and IL-8 were probably inflam- temic and fulminant monkeypox. Thus, neutro- matory responses to bacterial shock syndrome phils play a key role in the resolution of system- rather than the monkeypox infection. In addi- ic monkeypox virus infection. This animal model tion, the increased levels of MIG/CXCL-9 and will facilitate a better understanding of the macrophage- and/or T cell-related cytokines pathogenesis of fulminant orthopoxvirus (TNF-α, IFN-γ, and IL-13) from day 7 p.i. infections. appeared to be related to the bacterial co- infection. Interestingly, both moribund mon- Acknowledgements keys (#4567 and #4625) had high levels of macrophage-related chemokines (MCP-1/ We thank our colleagues, especially Ms A CCL2 and MIP-1α/CCL-3) and IFN-α from days Harashima and Ms M Fujino. This work was 6 to 10 p.i., which appeared to be associated supported in part by research grants from the with the systemic monkeypox infection. Ministry of Health, Labor, and Welfare of Japan Orthopoxviruses encode multiple immunomod- (H22-Shinko-Ippan-009 and H26-Shinkogyosei- ulatory proteins that hijack the host immune Shitei-002), and from the Japanese Health

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