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Am. J. Trop. Med. Hyg., 76(4), 2007, pp. 785–790 Copyright © 2007 by The American Society of Tropical Medicine and Hygiene

DHORI (: ) INFECTION IN MICE: A MODEL OF THE PATHOGENESIS OF SEVERE ORTHOMYXOVIRUS INFECTION

ROSA I. MATEO, SHU-YUAN XIAO, HAO LEI, AMELIA P. A. TRAVASSOS DA ROSA, AND ROBERT B. TESH* Departments of Pathology and of Internal Medicine and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas

Abstract. After intranasal, subcutaneous, or intraperitoneal infection with (DHOV), adult mice devel- oped a fulminant and uniformly fatal illness with many of the clinical and pathologic findings seen in mice infected with H5N1 highly pathogenic avian A virus. Histopathologic findings in of DHOV-infected mice consisted of hemorrhage, inflammation, and thickening of the interstitium and the alveolar septa and alveolar edema. Extra- pulmonary findings included hepatocellular necrosis and steatosis, widespread severe fibrinoid necrosis in lymphoid organs, marked loss and karyorrhexis, and neuronal degeneration in . Similar systemic histopathologic findings have been reported in the few fatal H5N1 cases examined at autopsy. Because of the relationship of DHOV to the influenza , its 2 status, and its similar pathology in mice, the DHOV- model may offer a low-cost, relatively safe, and realistic animal model for studies on the pathogenesis and management of H5N1 virus infection.

INTRODUCTION (Indianapolis, IN). The animals were cared for in accordance with guidelines of the Committee on Care and Use of Labo- Since the appearance of human disease caused by avian ratory Animals (Institute of Laboratory Animal Resources, 1 influenza A H5N1 viruses in Hong Kong in 1997, there has National Resource Council) under an animal use protocol been renewed interest in the pathogenesis of highly patho- approved by the University of Texas Medical Branch. 2–13 genic (HPAI) viruses. One of the major Virus assay. samples from the infected mice were deterrents to research with H5N1 and other HPAI viruses is titrated by plaque assay in monolayer cultures of Vero cells, that the biosafety, security, and agricultural regulations gov- as described previously.19 Serial 10-fold dilutions of the blood erning their use effectively restrict experimental animal work from 10−1 to 10−6 were prepared in phosphate-buffered saline, to a very few high security laboratory facilities. Safer and pH 7.4, containing 10% fetal bovine serum (PBS). Duplicate more accessible animal models are needed. wells of 24-well microplate cultures of Vero cells were inocu- The family Orthomyxoviridae currently consists of five gen- lated with 100 ␮L of each dilution. After virus absorption for era: Influenzavirus A, Influenzavirus B, Influenzavirus C, 1 hour and addition of an agar overlay, the cultures were 14 15,16 Thogotovirus, and Isavirus. Felipe and others previ- incubated at 37°C; a second overlay containing 1% neutral ously reported that two , Dhori and Thogoto, red was added 72 hours later, and plaques were counted 6 were lethal to mice inoculated intraperitoneally and that they days after inoculation. Virus titers were calculated as the produced pathologic lesions in the animals similar to those number of plaque forming units (PFU) per milliliter of blood. described with mouse-adapted strains of influenza virus. To Experimental design. A total of 30 mice was used; the ani- further study this observation, experiments in which ICR mals were divided into three groups of 10 animals each. The mice were inoculated intraperitoneally, subcutaneously, or in- three groups were inoculated with a DHOV stock containing tranasally with Dhori virus were carried out, and the clinical 107.9 PFU/mL, administered by the intraperitoneal (IP), sub- outcome, level of viremia, and histopathology after infection cutaneous (SC), or intranasal (IN) routes. Mice infected by by the three routes were determined. This report describes the IP and SC routes received 100 ␮L of a 1:2 dilution of the our findings that indicate that Dhori virus, a biosafety level 2 virus stock (∼106.6 PFU). Animals infected by the IN route 17 (BSL-2) non-select agent, could serve as a safe alternative were first anesthetized with Halothane (Halocarbon Labora- model in mice for studies on the pathogenesis and therapy of tories, River Edge, NJ), and two drops (∼100 ␮L) of the stock HPAI virus infection. virus solution were instilled into the nares. After infection, each mouse was examined daily for signs of illness, and 100 ␮L of blood was taken from the retro-orbital MATERIALS AND METHODS sinus for virus assay. Whole blood was diluted 1:10 in PBS in Virus. The prototype strain (IG 611313) of Dhori virus marked vials and frozen at −80°C for subsequent titration. If (DHOV) was used in this study; it was originally isolated from an animal died or appeared moribund, a necropsy was per- dromedarii collected from in formed, and samples of the heart, , , spleen, kidney, State, , in 1961.18 The virus used to infect the mice had adrenal glands, thymus, and cervical lymph nodes, as well as been passaged four times previously by intracerebral inocu- the brain, were collected and fixed in 10% buffered formalin lation of suckling mice. for 48 hours, before being transferred to 70% ethanol for Animal. The mice used in this study were 8- to 11-week-old storage until processing. females (ICR strain), obtained from Harlan Sprague-Dawley Histopathologic and immunohistochemical methods. After fixation, the tissues were processed for routine paraffin em- bedding and sectioning. Histologic sections of 4–5 ␮m thick- * Address correspondence to Robert B. Tesh, Department of Pathol- ness were made and stained with hematoxylin and eosin 20 ogy, University of Texas Medical Branch, 301 University Blvd., (H&E) and evaluated microscopically. Selected tissue sec- Galveston, TX 77555-0609. E-mail [email protected] tions were also studied immunohistochemically, using a 785 786 MATEO AND OTHERS

DHOV hyperimmune mouse ascitic fluid (1:100 dilution) as the primary antibody. A mouse-on-mouse ISO-IHC labeling kit (InnoGenex, San Ramos, CA) was used, according to the manufacturer’s instructions and a published protocol.20 The primary antibody was incubated with the sections at room temperature for 2 hours. Tissue sections from uninfected mice were used as negative controls. To further validate the speci- ficity of the immunohistochemical staining, the primary anti- body was replaced by an irrelevant mouse antibody (anti- ), which yielded no staining.

RESULTS

Clinical manifestations and mortality. Within 24 hours after infection, the 10 mice in the IP group showed signs of illness (ruffled fur, emaciation, lethargy, and labored breathing). All FIGURE 2. Mean daily levels of viremia (log10 PFU/mL) and SD in of these animals were dead or moribund by the fourth day. mice after infection with DHOV by the intraperitoneal, subcutane- ous, and intranasal routes. Mice in the SC and IN groups appeared well during the first 2 days after infection (p.i.); by the third day, they had devel- oped signs of illness similar to the IP group, and all were dead by Day 6 p.i. Figure 1 shows survival of mice in the three some (Figure 4A). Many of the lungs showed thickening of groups. The onset of illness and time of death were earlier in the interstitium and the alveolar septa, with increased cellu- the IP group, but the clinical manifestations and ultimate out- larity accompanied by scattered degenerating cells with frag- come (death) were similar in the three groups. mented nuclei (apoptosis; Figure 4B). These pulmonary changes may have been responsible for clinical signs of respi- Viremia. The mean daily virus titers (log10 PFU/mL) ob- tained on blood samples from mice in the three groups are ratory distress in the mice. shown in Figure 2. Except for a transient low-level viremia in There was widespread, severe fibrinoid necrosis involving one mouse in the IN group on the first day, the animals did the lymphoid organs, namely, the thymus (Figure 5A), spleen not develop a detectable viremia until the second or third day (Figure 5B and C), and in a few cases when available, thoracic p.i. In general, the highest levels of viremia were in the IP and abdominal lymph nodes. It was characterized by marked group; however, these animals were all dead or moribund by karyorrhexis of the (with cellular and nuclear Day 4 p.i. Mice in the SC and IN groups had a slightly longer condensation and fragmentation), resulting in a diffuse loss of viremia until their death on the sixth day p.i. lymphocytes, which was replaced by pink, amorphous fibrin- Histopathologic and immunohistochemical studies. The oid material. Sometimes scattered tingible body macrophages of some animals in the IN group showed scattered mild in a “blue” background were observed in the spleen (Figure neuronal degeneration, focally involving the cortex, hippo- 5C), representing increased phagocytosis of apoptotic lym- campus, cerebellum (Purkinje cells), and brainstem. This was phocytes. In addition, areas containing activated lymphocytes rather inconsistent. The central white matter near the hippo- were noted, characterized by marked enlargement of cells campus, subcortical region of the cerebellum, and brainstem and nuclei, with prominent nucleoli. Some of the cells at the contained prominent vacuolation (Figure 3), caused by con- center of the lymphoid follicles and periarteriolar lymphatic densation and shrinkage of neurons. sheath manifested necrosis, with preservation of the overall The lungs of the IN-infected animals consistently showed follicular architecture (follicular fibrinoid necrosis; Figure multifocal hemorrhagic pneumonitis, with alveolar edema in 5B). The liver initially showed mild foamy change, which quickly progressed to multifocal necrapoptosis of hepatocytes with no inflammation (Figure 5D) and to severe coagulative zonal necrosis (Figure 5E). The adrenal glands of the IN-infected animals showed only minimal changes, from a few scattered degenerating cells in the cortical layer to small necrotic foci (no more than three necrotic cells), to large areas of cortical necrosis (Figure 5F). No significant abnormalities were observed in the heart, pan- creas, or kidney. The histologic findings in the SC and IP groups were similar to those of the IN-infected mice. There was increased mono- nuclear infiltration, particularly in the brainstem. In the liver, multifocal hepatic necrosis was consistently seen; early micro- vesicular steatosis was seen in about one half of the animals. As noted above, there were no abnormalities seen in the heart, pancreas (only examined from a few animals), or kid-

FIGURE 1. Survival curves in mice infected with DHOV by the ney. Focal cortical cell necrosis was seen in adrenals in about .group). one half of the SC- and IP-infected animals/10 ס intraperitoneal, subcutaneous, and intranasal routes (N DHORI VIRUS PATHOGENESIS IN MICE 787

FIGURE 3. Histopathology of brain of a mouse infected with DHOV. The section is of brainstem and shows neuronal degenera- tion, increased mononuclear inflammatory cells, and vacuolation of the neuropil.

Immunohistochemically, specific Dhori viral was detected in many organs, with almost all of the antigen in the nuclei of the cells (Figure 6). Although a few scattered neu- rons were found to be positive (Figure 6A), positive staining was more readily seen in the lungs, thymus, spleen, liver, adrenals (Figure 6B–F), and lymph nodes (data not shown). Of these latter organs, staining in the liver was the most in- tense (Figure 6E).

DISCUSSION

DHOV has a wide distribution in the Old World; it has FIGURE 4. Histopathology in lungs of DHOV-infected mice. A, been isolated in southern and eastern Europe, Central Asia, Pulmonary edema. B, Necrosis, inflammatory cell (predominantly and Africa from ticks, mainly of the Hy- macrophage and lymphocyte) infiltration in the interstitium and focal alomma.18,21,22 Antibodies against DHOV have been de- hemorrhages. tected in large domestic animals (camels, , and ), as well as in living in endemic regions.18,23 Five hu- man cases of accidental aerosol infection with DHOV were mus, thoracic and abdominal lymph nodes, liver, spleen, and reported among a group of laboratory workers in the former to a lesser degree, in adrenals of the DHOV-infected mice. USSR.24 These individuals developed an acute febrile illness One of the most striking findings was the lymphotropism of characterized by , retro-orbital pain, , gen- the virus; DHOV-infected mice had extensive lymphocytic eral , and a prolonged period of convalescence; two loss. The process manifested initially as numerous lympho- of these people also developed mild symptoms of cranial cytes with nuclear fragmentation and tingible body macro- nerve involvement with radicular syndrome.24 phages (Figure 5C), and later as large areas of effacement of The genus Thogotovirus currently is made up of four vi- lymphoid areas with fibrinoid changes in the spleen and thy- ruses: Thogoto, Dhori, Batken, and Araguari.22,25 The first mus (Figure 5A and B). It seems that apoptosis was involved three viruses are thought to be -transmitted22; less is in the earlier phase of the lymphocytic loss; as the infection known about the natural of Araguari virus.25 progresses, more cells and multiple factors become involved, The thogotoviruses contain six or seven segments of linear, incorporating the fibrinoid necrosis as part of the - negative sense ssRNA,14 and they share many morphologic, esis. Furthermore, the virus seemed to either stimulate or genetic, and biochemical similarities with the influenza vi- activate lymphocytes, resulting in the appearance of large ruses, including the ability to block the expression of mam- numbers of immunoblast-like cells in the spleen and abdomi- malian genes.4,25–28 Results of this study indicate nal lymph nodes (Figure 5C). In this regard, it resembled the that the pathology of DHOV infection in mice is also similar pathology of Epstein-Barr virus infection.35 Other au- to that produced by influenza viruses in mice.7,29–34 thors9,11–13 have reported similar findings in fatal human Results of our study confirm the earlier findings of Felipe cases of H5N1 virus infection. Focal necrosis of lymph nodes and others,15 namely that DHOV infection in mice produces and atrophy of the splenic white pulp have been observed in a systemic disease with involvement of multiple organs. His- these patients, along with marked hemophagocytosis. topathologic changes were observed in the lungs, brain, thy- Experimental studies with influenza viruses in animal mod- 788 MATEO AND OTHERS

FIGURE 5. Histopathology of selected other organs in DHOV-infected mice. A, Thymus showing severe fibrinoid necrosis, with near total depletion of lymphocytes. B, Spleen exhibiting fibrinoid necrosis of most of the central portion of a periarteriolar lymphoid sheath. C, Spleen exhibiting enlargement of lymphocytes, along with individual lymphocytic apoptosis. D, Liver exhibiting apoptosis of scattered hepatocytes (arrowheads), which progresses to large areas of zonal necrosis (E). F, Focal necrosis in the cortical layer of an adrenal gland. els have also reported multiple organ involvement.2,6,7,29,33,34 that had been selected for hepatotropism, Haller29 described Intranasal inoculation of some avian and the progressive histologic changes occurring in of the viruses resulted in systemic disease involving the lungs, brain, mice after IP inoculation of the virus. Infected mice devel- liver, and kidney.6,29,33,34 Two recent studies in inocu- oped scattered hepatic lesions ranging from focal to wide- lated with H5N1 influenza virus found a similar infectivity spread liver cell necrosis, with almost no inflammatory cell pattern.2,10 In addition to the expected pulmonary pathology infiltration. The hepatocellular necrosis was characterized by (acute , bronchopneumonia and interstitial pneu- acidophilic changes, nuclear pyknotic and karyorrhectic monia with suppurative exudates, epithelial necrosis, and in- changes, and sometimes Councilman bodies.29 Similar cyto- tra-alveolar edema), the infected ferrets also showed neuro- logic changes were observed in our DHOV-infected mice. nal degeneration with inflammatory infiltration of the brain Autopsies on fatal human cases of H5N1 virus infection have and hepatocellular necrosis.2 Using an avian influenza virus also revealed extensive liver involvement with central lobular DHORI VIRUS PATHOGENESIS IN MICE 789

FIGURE 6. Immunohistochemical detection for Dhori viral in tissues of infected mice. A, Brain, a few scattered small neurons in the granule layer in the cerebellar cortex are positive. B, Lung, many interstitial cells are stained. C, Thymus, residual antigen positive cells in the largely necrotic area. D, Spleen, a few splenic macrophages and lymphocytes are positive. E, Liver, many antigen-positive hepatocytes. F, Adrenal, a focus of positive antigen stain in cortical cells. Note that the positive antigen straining (red color) is mostly intranuclear. necrosis, fatty changes, extra-medullary hematopoiesis, and known about the pathogenesis of the H5N1 virus or the activated Kupffer cells with hemophagocytic activity.9,11–13 mechanism of the severe human disease it causes. Although > Severe human cases of H5N1 virus infection also have bio- 120 human deaths caused by the H5N1 virus have been re- chemical evidence of liver dysfunction.1,36 ported, the pathogenesis of the disease in people is still poorly Based on the above comparisons, we believe that the understood, in part because it has been difficult to get tissues DHOV mouse model has many similarities in terms of tissue from fatal cases. In many of the Asian countries where most distribution and pathology to H5N1 influenza virus infection of the human deaths have occurred, cultural issues prevent in the mouse and models. It also has many similarities autopsies of victims. In addition, the biosafety, security, and to the pathology observed in the few human cases that have agricultural restrictions regulating use of the H5N1 virus in been examined histopathologically. Despite worldwide con- experimental animal studies have effectively limited such cern about a possible H5N1 , little is yet work to a few high security containment laboratories. Be- 790 MATEO AND OTHERS cause of the taxonomic relationship of DHOV to the influ- the International Committee on the of Viruses. San enza viruses, its BSL-2 status, and its similar pathology in Diego, CA: Academic Press; 681–693. mice, the DHOV mouse model may offer a lower-cost, safer, 15. Felipe AR, Peleteiro MC, Rebedo de Andrade H, 1990. Dhori virus induced lesions in mice. Acta Virol 34: 578–581. and realistic animal model for studies on the pathogenesis and 16. Felipe AR, Peleteiro MC, Monath TP, Calisher CH, 1988. Patho- management of H5N1 influenza virus infection. logical findings in mice infected with Thogoto virus, a tick- borne orthomyxovirus. Acta Virol 30: 337–340. Received October 17, 2006. Accepted for publication December 22, 17. CDC/NIH, 2006. 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