DHORI VIRUS (ORTHOMYXOVIRIDAE: THOGOTOVIRUS) 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 Dhori virus (DHOV), adult mice devel-oped a fulminant and uniformly fatal illness with many of the clinical and pathologic findings seen in mice infected withH5N1 highly pathogenic avian influenza A virus. Histopathologic findings in lungs of DHOV-infected mice consisted ofhemorrhage, 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 lymphoidorgans, marked lymphocyte loss and karyorrhexis, and neuronal degeneration in brain. Similar systemic histopathologicfindings have been reported in the few fatal human H5N1 cases examined at autopsy. Because of the relationship ofDHOV to the influenza viruses, its biosafety level 2 status, and its similar pathology in mice, the DHOV-mouse modelmay offer a low-cost, relatively safe, and realistic animal model for studies on the pathogenesis and management ofH5N1 virus infection.

INTRODUCTION

Since the appearance of human disease caused by avianinfluenza A H5N1 viruses in Hong Kong in 1997,1 there hasbeen renewed interest in the pathogenesis of highly patho-genic avian influenza (HPAI) viruses.2–13 One of the majordeterrents to research with H5N1 and other HPAI viruses isthat the biosafety, security, and agricultural regulations gov-erning their use effectively restrict experimental animal workto a very few high security laboratory facilities. Safer andmore accessible animal models are needed.

The family Orthomyxoviridae currently consists of five gen-era: Influenzavirus A, Influenzavirus B, Influenzavirus C,Thogotovirus, and Isavirus.14 Felipe and others15,16 previ-ously reported that two thogotoviruses, Dhori and Thogoto,were lethal to mice inoculated intraperitoneally and that theyproduced pathologic lesions in the animals similar to thosedescribed with mouse-adapted strains of influenza virus. Tofurther study this observation, experiments in which ICRmice were inoculated intraperitoneally, subcutaneously, or in-tranasally with Dhori virus were carried out, and the clinicaloutcome, level of viremia, and histopathology after infectionby the three routes were determined. This report describesour findings that indicate that Dhori virus, a biosafety level 2(BSL-2) non-select agent,17 could serve as a safe alternativemodel in mice for studies on the pathogenesis and therapy ofHPAI virus infection.

MATERIALS AND METHODS

Virus. The prototype strain (IG 611313) of Dhori virus(DHOV) was used in this study; it was originally isolated fromHyalomma dromedarii ticks collected from camels in GujaratState, India, in 1961.18 The virus used to infect the mice hadbeen passaged four times previously by intracerebral inocu-lation of suckling mice.

Animal. The mice used in this study were 8- to 11-week-oldfemales (ICR strain), obtained from Harlan Sprague-Dawley

(Indianapolis, IN). The animals were cared for in accordancewith guidelines of the Committee on Care and Use of Labo-ratory Animals (Institute of Laboratory Animal Resources,National Resource Council) under an animal use protocolapproved by the University of Texas Medical Branch.

Virus assay. Blood samples from the infected mice weretitrated by plaque assay in monolayer cultures of Vero cells,as described previously.19 Serial 10-fold dilutions of the bloodfrom 10−1 to 10−6 were prepared in phosphate-buffered saline,pH 7.4, containing 10% fetal bovine serum (PBS). Duplicatewells of 24-well microplate cultures of Vero cells were inocu-lated with 100 �L of each dilution. After virus absorption for1 hour and addition of an agar overlay, the cultures wereincubated at 37°C; a second overlay containing 1% neutralred was added 72 hours later, and plaques were counted 6days after inoculation. Virus titers were calculated as thenumber of plaque forming units (PFU) per milliliter of blood.

Experimental design. A total of 30 mice was used; the ani-mals were divided into three groups of 10 animals each. Thethree groups were inoculated with a DHOV stock containing107.9 PFU/mL, administered by the intraperitoneal (IP), sub-cutaneous (SC), or intranasal (IN) routes. Mice infected bythe IP and SC routes received 100 �L of a 1:2 dilution of thevirus stock (∼106.6 PFU). Animals infected by the IN routewere first anesthetized with Halothane (Halocarbon Labora-tories, River Edge, NJ), and two drops (∼100 �L) of the stockvirus solution were instilled into the nares.

After infection, each mouse was examined daily for signs ofillness, and 100 �L of blood was taken from the retro-orbitalsinus for virus assay. Whole blood was diluted 1:10 in PBS inmarked vials and frozen at −80°C for subsequent titration. Ifan animal died or appeared moribund, a necropsy was per-formed, and samples of the heart, lung, liver, spleen, kidney,adrenal glands, thymus, and cervical lymph nodes, as well asthe brain, were collected and fixed in 10% buffered formalinfor 48 hours, before being transferred to 70% ethanol forstorage until processing.

Histopathologic and immunohistochemical methods. Afterfixation, the tissues were processed for routine paraffin em-bedding and sectioning. Histologic sections of 4–5 �m thick-ness were made and stained with hematoxylin and eosin(H&E) and evaluated microscopically.20 Selected tissue sec-tions were also studied immunohistochemically, using a

* Address correspondence to Robert B. Tesh, Department of Pathol-ogy, University of Texas Medical Branch, 301 University Blvd.,Galveston, TX 77555-0609. E-mail rtesh@utmb.edu

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

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DHOV hyperimmune mouse ascitic fluid (1:100 dilution) asthe primary antibody. A mouse-on-mouse ISO-IHC labelingkit (InnoGenex, San Ramos, CA) was used, according to themanufacturer’s instructions and a published protocol.20 Theprimary antibody was incubated with the sections at roomtemperature for 2 hours. Tissue sections from uninfected micewere 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-West Nile virus), which yielded no staining.

RESULTS

Clinical manifestations and mortality. Within 24 hours afterinfection, the 10 mice in the IP group showed signs of illness(ruffled fur, emaciation, lethargy, and labored breathing). Allof these animals were dead or moribund by the fourth day.Mice in the SC and IN groups appeared well during the first2 days after infection (p.i.); by the third day, they had devel-oped signs of illness similar to the IP group, and all were deadby Day 6 p.i. Figure 1 shows survival of mice in the threegroups. The onset of illness and time of death were earlier inthe IP group, but the clinical manifestations and ultimate out-come (death) were similar in the three groups.

Viremia. The mean daily virus titers (log10 PFU/mL) ob-tained on blood samples from mice in the three groups areshown in Figure 2. Except for a transient low-level viremia inone mouse in the IN group on the first day, the animals didnot develop a detectable viremia until the second or third dayp.i. In general, the highest levels of viremia were in the IPgroup; however, these animals were all dead or moribund byDay 4 p.i. Mice in the SC and IN groups had a slightly longerviremia until their death on the sixth day p.i.

Histopathologic and immunohistochemical studies. Thebrains of some animals in the IN group showed scattered mildneuronal degeneration, focally involving the cortex, hippo-campus, cerebellum (Purkinje cells), and brainstem. This wasrather inconsistent. The central white matter near the hippo-campus, subcortical region of the cerebellum, and brainstemcontained prominent vacuolation (Figure 3), caused by con-densation and shrinkage of neurons.

The lungs of the IN-infected animals consistently showedmultifocal hemorrhagic pneumonitis, with alveolar edema in

some (Figure 4A). Many of the lungs showed thickening ofthe interstitium and the alveolar septa, with increased cellu-larity accompanied by scattered degenerating cells with frag-mented nuclei (apoptosis; Figure 4B). These pulmonarychanges may have been responsible for clinical signs of respi-ratory distress in the mice.

There was widespread, severe fibrinoid necrosis involvingthe lymphoid organs, namely, the thymus (Figure 5A), spleen(Figure 5B and C), and in a few cases when available, thoracicand abdominal lymph nodes. It was characterized by markedkaryorrhexis of the lymphocytes (with cellular and nuclearcondensation and fragmentation), resulting in a diffuse loss oflymphocytes, which was replaced by pink, amorphous fibrin-oid material. Sometimes scattered tingible body macrophagesin a “blue” background were observed in the spleen (Figure5C), representing increased phagocytosis of apoptotic lym-phocytes. In addition, areas containing activated lymphocyteswere noted, characterized by marked enlargement of cellsand nuclei, with prominent nucleoli. Some of the cells at thecenter of the lymphoid follicles and periarteriolar lymphaticsheath manifested necrosis, with preservation of the overallfollicular architecture (follicular fibrinoid necrosis; Figure5B).

The liver initially showed mild foamy change, whichquickly progressed to multifocal necrapoptosis of hepatocyteswith no inflammation (Figure 5D) and to severe coagulativezonal necrosis (Figure 5E).

The adrenal glands of the IN-infected animals showed onlyminimal changes, from a few scattered degenerating cells inthe cortical layer to small necrotic foci (no more than threenecrotic 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 similarto 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 theheart, pancreas (only examined from a few animals), or kid-ney. Focal cortical cell necrosis was seen in adrenals in aboutone half of the SC- and IP-infected animals.

FIGURE 1. Survival curves in mice infected with DHOV by theintraperitoneal, subcutaneous, and intranasal routes (N � 10/group).

FIGURE 2. Mean daily levels of viremia (log10 PFU/mL) and SD inmice after infection with DHOV by the intraperitoneal, subcutane-ous, and intranasal routes.

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Immunohistochemically, specific Dhori viral antigen wasdetected in many organs, with almost all of the antigen in thenuclei of the cells (Figure 6). Although a few scattered neu-rons were found to be positive (Figure 6A), positive stainingwas 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 hasbeen isolated in southern and eastern Europe, Central Asia,and Africa from ticks, mainly species of the genus Hy-alomma.18,21,22 Antibodies against DHOV have been de-tected in large domestic animals (camels, horses, and goats),as well as in humans living in endemic regions.18,23 Five hu-man cases of accidental aerosol infection with DHOV werereported among a group of laboratory workers in the formerUSSR.24 These individuals developed an acute febrile illnesscharacterized by headache, retro-orbital pain, myalgia, gen-eral weakness, and a prolonged period of convalescence; twoof these people also developed mild symptoms of cranialnerve involvement with radicular syndrome.24

The genus Thogotovirus currently is made up of four vi-ruses: Thogoto, Dhori, Batken, and Araguari.22,25 The firstthree viruses are thought to be tick-transmitted22; less isknown about the natural transmission of Araguari virus.25

The thogotoviruses contain six or seven segments of linear,negative sense ssRNA,14 and they share many morphologic,genetic, and biochemical similarities with the influenza vi-ruses, including the ability to block the expression of mam-malian interferon genes.4,25–28 Results of this study indicatethat the pathology of DHOV infection in mice is also similarto that produced by influenza viruses in mice.7,29–34

Results of our study confirm the earlier findings of Felipeand others,15 namely that DHOV infection in mice producesa systemic disease with involvement of multiple organs. His-topathologic changes were observed in the lungs, brain, thy-

mus, thoracic and abdominal lymph nodes, liver, spleen, andto a lesser degree, in adrenals of the DHOV-infected mice.One of the most striking findings was the lymphotropism ofthe virus; DHOV-infected mice had extensive lymphocyticloss. The process manifested initially as numerous lympho-cytes with nuclear fragmentation and tingible body macro-phages (Figure 5C), and later as large areas of effacement oflymphoid areas with fibrinoid changes in the spleen and thy-mus (Figure 5A and B). It seems that apoptosis was involvedin the earlier phase of the lymphocytic loss; as the infectionprogresses, more cells and multiple factors become involved,incorporating the fibrinoid necrosis as part of the pathogen-esis. Furthermore, the virus seemed to either stimulate oractivate lymphocytes, resulting in the appearance of largenumbers of immunoblast-like cells in the spleen and abdomi-nal lymph nodes (Figure 5C). In this regard, it resembled thepathology of Epstein-Barr virus infection.35 Other au-thors9,11–13 have reported similar findings in fatal humancases of H5N1 virus infection. Focal necrosis of lymph nodesand atrophy of the splenic white pulp have been observed inthese patients, along with marked hemophagocytosis.

Experimental studies with influenza viruses in animal mod-

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

FIGURE 4. Histopathology in lungs of DHOV-infected mice. A,Pulmonary edema. B, Necrosis, inflammatory cell (predominantlymacrophage and lymphocyte) infiltration in the interstitium and focalhemorrhages.

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els have also reported multiple organ involvement.2,6,7,29,33,34

Intranasal inoculation of some avian and equine influenzaviruses resulted in systemic disease involving the lungs, brain,liver, and kidney.6,29,33,34 Two recent studies in ferrets inocu-lated with H5N1 influenza virus found a similar infectivitypattern.2,10 In addition to the expected pulmonary pathology(acute bronchiolitis, bronchopneumonia and interstitial pneu-monia with suppurative exudates, epithelial necrosis, and in-tra-alveolar edema), the infected ferrets also showed neuro-nal degeneration with inflammatory infiltration of the brainand hepatocellular necrosis.2 Using an avian influenza virus

that had been selected for hepatotropism, Haller29 describedthe progressive histologic changes occurring in livers of themice after IP inoculation of the virus. Infected mice devel-oped scattered hepatic lesions ranging from focal to wide-spread liver cell necrosis, with almost no inflammatory cellinfiltration. The hepatocellular necrosis was characterized byacidophilic changes, nuclear pyknotic and karyorrhecticchanges, and sometimes Councilman bodies.29 Similar cyto-logic changes were observed in our DHOV-infected mice.Autopsies on fatal human cases of H5N1 virus infection havealso revealed extensive liver involvement with central lobular

FIGURE 5. Histopathology of selected other organs in DHOV-infected mice. A, Thymus showing severe fibrinoid necrosis, with near totaldepletion of lymphocytes. B, Spleen exhibiting fibrinoid necrosis of most of the central portion of a periarteriolar lymphoid sheath. C, Spleenexhibiting 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.

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necrosis, fatty changes, extra-medullary hematopoiesis, andactivated Kupffer cells with hemophagocytic activity.9,11–13

Severe human cases of H5N1 virus infection also have bio-chemical evidence of liver dysfunction.1,36

Based on the above comparisons, we believe that theDHOV mouse model has many similarities in terms of tissuedistribution and pathology to H5N1 influenza virus infectionin the mouse and ferret models. It also has many similaritiesto the pathology observed in the few human cases that havebeen examined histopathologically. Despite worldwide con-cern about a possible H5N1 influenza pandemic, little is yet

known about the pathogenesis of the H5N1 virus or themechanism of the severe human disease it causes. Although >120 human deaths caused by the H5N1 virus have been re-ported, the pathogenesis of the disease in people is still poorlyunderstood, in part because it has been difficult to get tissuesfrom fatal cases. In many of the Asian countries where mostof the human deaths have occurred, cultural issues preventautopsies of victims. In addition, the biosafety, security, andagricultural restrictions regulating use of the H5N1 virus inexperimental animal studies have effectively limited suchwork to a few high security containment laboratories. Be-

FIGURE 6. Immunohistochemical detection for Dhori viral antigens in tissues of infected mice. A, Brain, a few scattered small neurons in thegranule layer in the cerebellar cortex are positive. B, Lung, many interstitial cells are stained. C, Thymus, residual antigen positive cells in thelargely 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.

DHORI VIRUS PATHOGENESIS IN MICE 789

cause of the taxonomic relationship of DHOV to the influ-enza viruses, its BSL-2 status, and its similar pathology inmice, the DHOV mouse model may offer a lower-cost, safer,and realistic animal model for studies on the pathogenesis andmanagement of H5N1 influenza virus infection.

Received October 17, 2006. Accepted for publication December 22,2006.

Acknowledgments: The authors thank Hilda Guzman and PatrickNewman for fine technical assistance and Dora Salinas for help inpreparing the manuscript.Authors’ addresses: Rosa I. Mateo, Shu-Yuan Xiao, Hao Lei, AmeliaP.A. Travassos da Rosa, and Robert B. Tesh, Department of Pathol-ogy, University of Texas Medical Branch, 301 University Blvd.,Galveston, TX 77555-0609. E-mail rtesh@utmb.edu.

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