Neuropathology of H5N1 virus infection in ferrets

Veterinary Microbiology 156 (2012) 294–304

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Neuropathology of H5N1 virus infection in ferrets Peng Bi-Hung, Yun Nadezhda, Chumakova Olga, Zacks Michele, Campbell Gerald, Smith Jeanon, Smith Jennifer, Linde Seth, Linde Jenna, Paessler Slobodan* Galveston National Laboratory, Sealy Vaccine Center and Institute for Human Infections and Immunity, Department of Pathology, University of Texas Medical Branch (UTMB), Galveston, TX 77555-0609, United States

A R T I C L E I N F O

A B S T R A C T

Article history: Received 27 January 2011 Received in revised form 5 July 2011 Accepted 25 November 2011

Highly pathogenic H5N1 virus remains a potential threat to humans. Over 289 fatalities have been reported in WHO confirmed human cases since 2003, and lack of effective vaccines and early treatments contribute to increasing numbers of cases and fatalities. H5N1 encephalitis is a recognized cause of death in Vietnamese cases, and brain pathology is described in other human cases and naturally infected animals. However, neither pathogenesis of H5N1 viral infection in human brain nor post-infection effects in survivors have been fully investigated. We report the brain pathology in a ferret model for active infection and 18-day survival stages. This model closely resembles the infection pattern and progression in human cases of influenza A, and our report is the first description of brain pathology for longer term (18-day) survival in ferrets. We analyzed viral replication, type and severity of meningoencephalitis, infected cell types, and cellular responses to infection. We found viral replication to very high titers in ferret brain, closely correlating with severity of meningoencephalitis. Viral antigens were detected predominantly in neurons, correlating with inflammatory lesions, and less frequently in astrocytes and ependymal cells during active infection. Mononuclear cell infiltrates were observed in early stages predominantly in cerebral cortex, brainstem, and leptomeninges, and less commonly in cerebellum and other areas. Astrogliosis was mild at day 4 post-infection, but robust by day 18. Early and continuous treatment with an antiviral agent (peramivir) inhibited virus production to non-detectable levels, reduced severity of brain injury, and promoted higher survival rates. ß 2011 Elsevier B.V. All rights reserved.

Keywords: Influenza Virus Encephalitis Gliosis

1. Introduction Avian influenza H5N1 virus was first isolated in 1996 from a farmed goose in Guangdong Province of China. In 1997, the first outbreak was reported in poultry in farms and live animal markets, claiming 6 lives out of 18 human cases in Hong Kong (WHO, 2010a). This is the first known instance of human infection with this virus. After the first H5N1 storm, it was quiet for few years probably due to

* Corresponding author at: Department of Pathology, Galveston National Laboratory, UTMB, 301 University Boulevard, Galveston, TX 77555-1019, United States. Tel.: +1 409 747 0764; fax: +1 409 747 0762. E-mail address: [email protected] (S. Paessler). 0378-1135/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2011.11.025

the massive slaughtering of farm poultry. However, the H5N1 viruses were still circulating in geese and exchanging genetic material with other avian influenza viruses in wild birds. The new variants of H5N1 viruses resurfaced in the Hong Kong poultry markets in 2001 (Lipatov et al., 2003). In early 2003, again in Hong Kong, there were 2 human cases of H5N1 infection resulting in death after a recent travel to China (Peiris et al., 2004). In December 2003, an outbreak of A/H5N1 occurred among poultry in South Korea and afterwards in Vietnam, Japan, Thailand, Laos, Cambodia, China, Indonesia, and Malaysia in 2004 (WHO, 2010a). From 2003 to early March 2010, there were 289 deaths in the group of 488 WHO confirmed human cases of H5N1 worldwide (WHO, 2010b).

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Comparing the clinical features of the 1997 Hong Kong and the 2004 Southeast Asia outbreaks, virtually all patients presented with fever, cough, shortness of breath, and lower respiratory-tract symptoms. Pulmonary infiltrates were present in 61% of 18 patients in the 1997 Hong Kong outbreak and 100% of the 41 hospitalized patients from the 2004 outbreak. Progression to respiratory failure occurred in 70–100% of the subset of cases from 2004, but in only 44% of the 1997 cases. The overall mortality was 64%, with outbreak mortality ranging from 33% in the 1997 Hong Kong outbreak to 100% (four patients) in Cambodia in 2004 (Thomas and Noppenberger, 2007). The later outbreak included less typical presentations of the disease where gastrointestinal and encephalitic symptoms predominated. The first case reported in 2004 was a 39-yearold woman in Thailand who presented to the hospital with a 1-week history of fever, diarrhea, nausea, and vomiting, but without respiratory symptoms, and died the day after admission. Positive H5N1 infection was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) performed on nasopharnygeal aspirates. This was the first avian influenza case in a patient with predominantly gastrointestinal symptoms (Apisarnthanarak et al., 2004). Other instances include two pediatric cases in Vietnam, a 9-year-old girl and her 4-year-old brother, who presented with fever and watery diarrhea (greater than 10 episodes per day) and progressed to coma and death. The H5N1 virus was detected in the cerebrospinal fluid and acute H5N1 encephalitis was diagnosed in both cases (de Jong et al., 2005a). These cases were the first demonstration of direct central nervous system (CNS) involvement in human H5N1 infection. After the 2004 outbreak, H5N1 encephalitis has been documented in other naturally infected species besides humans. These species include native chickens, Japanese quail and ducks in Thailand (Antarasena et al., 2006), crows (Tanimura et al., 2006) and whooper swans (Ogawa et al., 2009) in Japan, tufted ducks in Sweden (Brojer et al., 2009), and swans in Germany (Teifke et al., 2007), tigers and two leopards at a zoo in Thailand (Keawcharoen et al., 2004), stone marten (Klopfleisch et al., 2007). Considering the finding of H5N1 encephalitis in natural infections in multiple species after the 2004 outbreak and the prevalence of rapid reassortment among influenza A subtypes, a serious concern exists that more neurovirulent strains of the H5N1 virus will appear in future outbreaks. Therefore, effective treatments for H5N1 infections are urgently needed because of the inefficient penetration of current agents through the blood–brain barrier (BBB). In addition, there is no current vaccine specifically directed against H5N1, although cross protection using vaccines for seasonal influenza is under investigation. The two neuraminidase inhibitors recommended for treatment of H5N1 infections are oseltamivir and zanamivir (WHO, 2006). However, these antiviral drugs were initially developed to treat uncomplicated seasonal influenza, and their efficacy in H5N1 infections is limited. Oseltamivir resistance has been found in patients after only a few days of treatment, and patients have died in spite of treatment (de Jong et al., 2005b). Aerosolized zanamivir needs a special device for administration, and is much less useful

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for severely ill patients who are unable to inhale it, or for those with pulmonary infections that are inaccessible to topical therapy (Medeiros et al., 2007). Peramivir, an injectable form of neuraminidase inhibitor, has exhibited inhibitory effects against influenza viruses which show resistant to zanamivir and oseltamivir (Mishin et al., 2005; Clinical_Studies_Info, 2006). A better understanding of the pathogenesis of H5N1 encephalitis is an essential requirement for the development of drugs and vaccines. Since autopsies were not performed on the two human encephalitis cases from Vietnam, knowledge of the pathogenesis of H5N1 encephalitis still relies on animal studies. Mice and ferrets have been reported to have high fatality rates and various degrees of brain damage after infection with H5N1 viruses isolated from poultry or humans (Govorkova et al., 2005; Zitzow et al., 2002; Maines et al., 2005; Rowe et al., 2003; Iwasaki et al., 2004). However, there is still not enough information to elucidate the neuropathogenesis of viral entry and spread, disease development, and response of the nervous system to infection. We report here a study using the intranasal infection model in the ferret, which produces a pulmonary syndrome similar to that of influenza A infection in the human (van Riel et al., 2006), to assess H5N1 encephalitis and how it is modified by treatment with a neuraminidase inhibitor. The infectious agent in this study is the highly pathogenic Vietnam strain of H5N1 (VN/04/1203) virus. The treatment used is peramivir, an injectable neuraminidase inhibitor that has exhibited inhibitory effects against influenza viruses that are resistant to zanamivir and oseltamivir (Mishin et al., 2005; Clinical_Studies_Info, 2006). Our endpoints for this study include viral replication, brain pathology, and cytokine production during the active infection, and pathologic changes including reactive gliosis in the later survival stages when virus is no longer detectable. 2. Materials and methods 2.1. Animals and infection Ferrets (Mustela putorius furo), were purchased from Marshall Farms (North Rose, New York). Animals were housed in a pathogen-free environment for a minimum of 2–7 days prior to treatment with antiviral drugs or infection with influenza A H5N1. Implantation of transponders for telemetric temperature recording was carried out in an Animal Biosafety Level (ABSL) 2 facility, and infection and drug treatment was performed at ABSL-4. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Texas Medical Branch (UTMB) and carried out according to National Institute of Health (NIH) guidelines. Prior to infection, blood was drawn for routine hematological and serological testing to rule out potential prior infections with the circulating strains of influenza. Influenza A/Vietnam/1203/04 was provided by the Influenza Laboratory at the U.S. Centers for Disease Control and Prevention in Atlanta, Georgia. All work with this virus isolate was approved by appropriate institutional offices and federal agencies (CDC/USDA), and was performed in

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the Robert E. Shope Laboratory at BSL-4 at the University of Texas Medical Branch (Galveston, TX). Virus stock for inoculation of animals was obtained by cultivation in embryonated chicken eggs (Charles River Laboratories, Wilmington, MA) at 37 8C for 20–36 h. Aliquots of harvested virus were stored at 80 8C until animal infection. Peramivir (BCX-1812, provided by BioCryst Pharmaceuticals, Inc. at Birmingham, AL) was diluted in sterile saline (0.9% sodium chloride), and this diluent stock was used as the ‘‘vehicle’’ control. Forty 6- to 8-week-old ferrets were infected intranasally with 1.7  104 TCID50 (median tissue culture infectious dose) of influenza A H5N1 (A/Vietnam/1203/04). Half of the infected ferrets were then treated with multiple doses of peramivir (30 mg/kg), or vehicle as control, at 1 h post infection (+1 h), and then daily for 4 days. Following infection and drug treatment, animals were monitored daily for up to 18 days post infection (dpi) for death and disease development. Five animals per group were randomly preselected to be euthanized at 4 and 6 dpi. These time points have been previously shown to be representative of the ‘‘active infection stage’’ (Yun et al., 2008). The remainder either were euthanized due to severe neurologic impairment or survived for the full 18 day period. Brain and lungs were removed from each euthanized ferret. The brain was divided in the midline sagittal plane, and one half brain and fresh lung, liver and spleen tissue were frozen for viral titer determination and RNA purification. Viral titers, survival statistics, and clinical progression for these animals have been previously reported (Yun et al., 2008). Numbers of ferrets with detectable virus and brain lesions are summarized in Table 1. In addition to the five ferrets which were euthanized according to the experimental schedule at 4 and 6 dpi, four ferrets from the vehicle group, labeled as the ‘‘paralysis’’ subgroup, were euthanized at 5 or 6 dpi (3 and 1 ferrets respectively) due to the development of severe neurologic impairment, manifested by paralysis. The remaining half brain was fixed in 10% buffered formalin for histology and immunofluorescence microscopy. 2.2. Histological processing and sectioning Formalin fixed half brains were blocked in the sagittal plan to achieve consistent paramedian sections. The tissue was processed for paraffin embedding using standard

histologic processing methods, and embedded in paraffin (Parapro XLT, melting point 55 8C, StatLab Medical Products, McKinney, TX). Sections were made at 5 mm thickness and stained with hematoxylin and eosin (H&E) by standard methods. 2.3. Histology and pathologic grading H&E slides of brain sections were examined by two experienced neuropathologists (GAC and BHP), who provided consensus descriptive histopathology and semiquantitative grading of the major pathologic changes. The pathologic changes graded included (1) meningitis in the form of leptomeningeal infiltrates of mononuclear cells (lymphocytes and monocytes), (2) parenchymal mononuclear cell infiltrates, including perivascular infiltrates and glial-microglial nodules, and (3) necrosis. For all parameters, the changes were graded for severity using a semi-quantitative score on a scale ranging from 0 to 3, defined as follows: 0 = no significant change, normal; 1 = mild or minimal changes; 2 = moderate or intermediate changes; and 3 = most severe changes. Assessment of severity took into consideration both the extent of changes as well as local intensity. 2.4. Immunofluorescence microscopy Brain sections (5 mm) were immunostained to detect viral antigens using antiserum to Influenza A/Vietnam/ 1203/04 developed in mice in our laboratory, and to identify reactive astrocytosis (proliferation or hyperplasia) and astrogliosis (hypertrophy) using antibody to glial fibrillary acidic protein (GFAP). This astrocyte-specific intermediate filament marker is useful no only to identify increased numbers of astrocytes, but also to demonstrate hypertrophic morphology and increased cytoplasmic signal due to increased production of glial filaments. After deparaffinization, tissue sections were incubated with 5% normal goat serum and 0.1% Tween 20 in 1 PBS (10 mM phosphate, 137 mM NaCl and 2.7 mM KCl, pH7.4) for 15 min to block non-specific binding, without antigen retrieval. Both primary antibodies, mouse H5N1 antiserum (1:300) and rabbit anti-GFAP (1:250, Sigma, St. Louis, MO), were added to the solution and incubated for 1 h at room temperature. Adjacent sections were incubated with irrelevant antibody (against vaccine strain Venezuelan equine encephalitis virus) as a negative control. Tissue sections were rinsed three times with 1 PBS in the presence of 0.1% Tween 20 before the secondary antibodies, goat anti-mouse Alexa-488

Table 1 Virus detection and histologic lesions. Treatment

Vehicle

Days post infection (# of ferrets) # of ferrets with detectable virus in lungs # of ferrets with detectable virus in brain # of ferrets with histologic brain lesions Mean histologic grade Brain regions involved

4 (5) 0 2 3 0.40 cb, bs, lm

Peramivir 6 (5) 3 1 2 0.37 cb, bs, cbl, lm

18 (3) 0 0 1 0.26 cb, bs, cbl, lm

P* (4) 4 4 4 1.75 cb, bs, cbl, lm

4 (5) 0 0 0 0.00 None

6 (5) 0 0 1 0.06 cb, bs

18 (7) 0 0 3 0.22 cb, bs, cbl, lm

P*: ferrets with severe illness and paralysis that were euthanized at 5 and 6 dpi. Brain regions: cb—cerebrum, cbl—cerebellum, bs—brainstem, lm— leptomeninges; see text for additional description.

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and goat anti-rabbit Alexa-595 (both 1:250, excitation wavelengths 488 and 595 nm respectively, Invitrogen), were added. After 1 h incubation, sections were rinsed three times and then counter-stained with DAPI (excitation wavelength at 350 nm, Sigma, St. Louis, MO). Positive fluorescent signal was visualized and photographs were taken using a fluorescence microscope (Olympus XL71) equipped with a digital camera (DP70). 2.5. Measurement of cytokine and chemokine production in ferret brains Total RNA was isolated from the frozen half brains of preselected ferrets. The thawed brain tissue was homogenized and treated with Trizol reagent (Invitrogen), followed by chloroform extraction and isopropanol precipitation. Reverse transcription was carried out with SuperScript III reverse transcriptase (Invitrogen). Briefly, a mixture of 2 mg of total RNA from each brain sample, 0.5 mM dNTPs (New England Biolabs), 5 ng random hexamer primers (Invitrogen), and nuclease free water were heated at 65 8C for 5 min, and followed by incubation on ice for 2 min. Subsequently, the total reaction volume was brought up to 40 ml with enzyme buffer, 5 mM dithiothreitol (DTT), RNase inhibitor (New England Biolabs), and 10 U reverse transcriptase. The reactions were incubated at room temperature for 10 min and reverse transcription was performed at 50 8C for 40 min. After the reactions were inactivated at 70 8C for 15 min, 1 ml of each resulting cDNA was amplified using real-time polymerase chain reaction (PCR) for quantitation. Each reaction (25 ml in total) included cDNA, the iTaq SYBR Green Supermix with ROX (Bio-Rad), nuclease-free water, and 0.4 mM of cytokine- or glyceraldehyde-3-phosphate (GAPDH)-specific primers. Primers were designed based on published sequences (Nakata et al., 2009). Real-time PCR was performed using the StepOne Real-Time PCR System (Applied Biosystems). Cycling conditions included an initial 2 min denaturation at 95 8C, followed by 40 cycles of denaturation (95 8C) and extension (60 8C). A disassociation analysis was performed between 60 8C and 95 8C with an increment of 0.2 8C per second to determine the melting temperature of the PCR product. Cycle thresholds were normalized to GAPDH mRNA levels to control for variations in the amounts of input RNA between samples. Transcripts were quantified in the experimental samples using a comparative approach between the experimental and control groups. 3. Results 3.1. H5N1 virus directly infects and replicates in the central nervous system Direct infection of H5N1 virus was demonstrated using immunofluorescence microscopy. The major cell type infected by H5N1 virus in the brain is the neuron. Infected ependymal cells and astrocytes are also observed, but with very low frequency and only during the first week (4– 6 dpi) of infection. In this active infection stage, neurons were positively labeled with fluorescence-conjugated

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antiserum, correlated with inflammatory lesions throughout the cerebrum, brainstem and cerebellum. At day 4, positively labeled neurons are also observed even though inflammatory lesions are not yet as prominent as at day 6. Fig. 1 illustrates labeled neurons in the cerebral cortex (upper left panel) and cerebellum (Purkinje neurons, upper right panel). Labeled ependymal cells, detected in only a few of the infected ferrets, are shown in the lower left panel. To identify infected astrocytes, we double-labeled brain sections with antiserum to viral antigens and antibody to the specific astrocyte marker GFAP. An infected astrocyte displaying positive labeling with viral antigens (green) in the nucleus and GFAP (red) in the cytoplasm is shown in the lower right panel of Fig. 1. There were no detectable levels (<1  104 TCID50) of virus found in either brains or lungs of the peramivirtreated group in the early stages, or in either group at the 18-day survivor stage. In the vehicle group, virus was detected as early as 4 dpi in the brains of 2 ferrets, but not in lungs until 6 dpi (3 ferrets). However, viral titers in the paralysis group were significantly higher than those in the 4 and 6 dpi groups in both brain and lung. The highest titer reached 1010 TCID50 (Yun et al., 2008). The severity of histologic brain lesions correlates directly with the viral titers, also peaking in the ‘‘paralysis group’’ (Table 1). 3.2. Direct infection of H5N1 in the CNS causes severe meningitis and encephalitis The predominant lesions observed in CNS are cellular infiltrates in leptomeninges and perivascular spaces (‘‘cuffing’’). In more severe lesions, the infiltrates exhibit angiocentric invasion in parenchyma and focal necrosis may be present. Infiltrates are mainly mononuclear, composed morphologically of lymphocytes and monocytes. Plasma cells do not represent a significant component. Where necrosis is present, neutrophils and nuclear debris are also observed. In these lesions, degenerating neurons and neuronophagia can be found, but nuclear or cytoplasmic inclusions are not observed. The most consistently involved regions are cerebral gray matter and brainstem, closely followed by leptomeninges. In the more severe cases, cerebellum is also involved (Table 1). Cortical lesions range from isolated infiltrates (perivascular or leptomeningeal) to more extensive involvement of meninges, cortex, and, in most severe cases, subcortical white matter. Lesions of subcortical or deep white matter or cerebral deep nuclei (hippocampus, corpus striatum, and thalamus) are rare and, when present, are restricted to isolated perivascular infiltrates. Dentate nucleus of cerebellum is involved in some of the more severe cases. To evaluate pathologic changes due to the CNS infection, we performed a semiquantitative grading analysis for encephalitis, meningitis and necrosis on H&E stained brain sections. Table 1 shows that pathologic changes can be seen as early as 4 dpi, and in every ferret of the paralysis group. Peramivir treatment reduced the occurrence of brain injury at the early stage from 9 of 14 in the vehicle group to 1 of 10 in the peramivir group. However, there is no significant difference between the two groups in number of animals involved at the 18-day

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Fig. 1. Cell types targeted by H5N1 virus in the brain. Cell types infected by H5N1 virus are identified by immunofluorescence microscopy using antiserum to viral antigens (green). The major target for the virus is neurons (cerebral cortical and cerebellar Purkinje neurons shown). Ependymal cells (seen lining a ventricle) and astrocytes can also be infected. Note that an infected astrocyte (arrow) is identified by double-labeling with viral antigens (present in nucleus) and glial fibrillary acidic protein (GFAP, astrocyte marker, red). Cell nuclei are stained with DAPI (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

survival stage. Examples of typical lesions, mononuclear cell meningitis and perivascular infiltrates in cerebral cortex (grades 3, 2 and 1), compared to the normal cerebral cortex (grade 0) are shown in Fig. 2A. Fig. 2B shows an example of a severe necrotizing lesion in the brainstem (grade 3). The mean grade for each group is listed in Table 1 and graphed in Fig. 2C. Overall, the vehicle group showed much more severe lesions compared to the peramivir treated group at the early active infection stage. Similar to frequency of involvement, there is no difference in severity between the two groups at the 18-day survival stage. At the earliest time point (4 dpi), three ferrets exhibit lesions but only two of them have detectable levels of virus. One animal with relatively low brain viral titer (1.3  104 TCID50), had lesions only in brain stem, while a second animal with higher viral titer (1.3  107 TCID50) had more widely distributed lesions. By 5 and 6 dpi, lesions were all widely distributed throughout the brain. 3.3. Astrocyte responses and cytokine production in the active infection stage

GFAP immunofluorescence studies revealed slightly increased fluorescence labeling in the brain in our vehicle group at 4 dpi (upper left panel, Fig. 3). At 6 dpi, not only was the number of positively stained cells increased, but also the level of staining in each cell increased tremendously (lower left panel). On the contrary, no astrocytosis or gliosis was observed in the peramivir treated group, which showed normal astrocyte morphology consisting of a small cap of GFAP immunoreactive intermediate filaments adjacent to the nucleus (right panels). In addition to astrocyte activation, synthesis and secretion of cytokines within the CNS may be important for the interaction between infected and non-infected cells, and also between the CNS and the immune system. Our analysis showed that mRNA levels of IFN-g, TNF-a, IL6, IL-8 and IL-10 in the vehicle group were significantly higher than those of the non-infected group. Furthermore, levels of IFN-g and IL-8 mRNA in the vehicle group were significantly higher than those of the peramivir treated group (Fig. 4). 3.4. Histologic changes at the 18-day survival stage

To assess the response of the CNS to H5N1 viral infection during the active infection stage, we analyzed the activation of astrocytes using GFAP immunofluorescence, and the syntheses of several pro-inflammatory cytokines and chemokines by quantitative RT-PCR.

To evaluate the changes in the CNS at the 18-day survival stage we examined H&E brain sections in addition to immunofluorescence microscopy for gliosis. As shown in Table 1, there were 1 (of 3) ferret in the vehicle and 3 (of

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Fig. 2. Semiquantitative grading of brain lesions in ferrets infected with H5N1 virus. (A) Examples of histologic grading of meningoencephalitis. Brain sections were stained with hematoxylin and eosin (H&E) to evaluate the inflammation resulting from infection. Histological lesions include mononuclear cell meningitis and perivascular infiltrates in cerebral cortex, cerebellum and brain stem. Examples of each grade (3, 2 and 1) and normal cerebral cortex (grade 0) are shown. (B) An example of a severe necrotizing lesion in the brain stem (grade 3). (C) Results of semiquantitative grading show higher average grades in the vehicle versus the peramivir-treated group in the active infection stages, but not in the 18-day survival stage.

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Fig. 3. Activation of astrocytes. Activation of astrocytes in response to H5N1 infection is assessed by immunofluorescence microscopy using antibody to the astrocyte-specific marker, GFAP (red). In the peramivir-treated group (right panels), no increased staining of GFAP is seen at either 4 or 6 dpi. However, the number of positive stained cells (at 6 dpi) and the levels of staining (at 4 and 6 dpi) are increased in the vehicle group (left panels). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

7) ferrets in the peramivir treated groups with histologic lesions at the 18-day survival stage. All inflammatory lesions in these ferrets were mild (grade 1), without necrosis, and there were no significant differences between the two groups. Mononuclear cell infiltrates at this stage were more diffuse and predominantly located in widely distributed areas of subcortical white matter (Fig. 5, right panel) compared to the distinctly focal and more severe lesions (involving meninges, gray and white matter) of the earlier stages in which viral antigen is detectable. In addition, subcortical white matter diffusely exhibited reactive microglia in the form of ‘‘rod’’ cells that were not specifically associated with inflammatory lesions. A few glial-microglial nodules were also observed in these brains, although they do not represent a significant component of the lesions. The few such lesions observed were located mainly in cerebellar dentate nucleus. One characteristic of the histologic changes of astrogliosis is that they remain evident over a long period of time, even after inflammatory reactions in the CNS have subsided. This characteristic provides a good marker for

prior reactive processes in the brain. We examined brain sections from all surviving ferrets at 18 dpi by GFAP immunofluorescence microscopy. Our analysis showed that the majority of these ferrets exhibited gliosis in various degrees of severity, but with no significant differences between the two groups. Examples of gliosis are shown in Fig. 6. Astrocytes in the white matter of cerebral hemisphere (left panels) showed increased GFAP immunoreactivity and number of immunoreactive processes. Bergmann astrocytes in the cerebellum (right panels) also show increased GFAP immunoreactivity and proliferation, in some cases in concert with Purkinje cell loss. 4. Discussion This study demonstrates that H5N1 virus can infect and replicate efficiently to very high titers in the CNS in the ferret model, and provides histological evidence that this infection causes meningoencephalitis resulting in death in severe cases. Peramivir treatment improved survival rates

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Fig. 4. Cytokine and chemokine production in the active infection stage. To determine the expression of proinflammatory cytokine and chemokine gene levels following H5N1 virus infection in ferrets, brains from animals infected with 1.7  104 TCID50 of influenza A/Vietnam/1203/04 (H5N1) virus were collected at 5–6 dpi. The brain homogenates were assayed for IL-6, IL-8, IFN-g, TNF-a and IL-10 gene expression. Controls were selected from animals that were not infected by virus. Our analysis strategy was to focus on up- or down-regulated gene expression during infection. Signals were normalized to the housekeeping gene GAPDH. This chart shows relative quantity (RQ) after normalization of mRNA levels. The mRNA levels of all cytokines and chemokines analyzed here were significantly elevated in the vehicle group compared to the control group, and peramivir treatment reduced these levels of gene expression. In addition, the level of gene expression correlates well with viral titer and histological lesions in each individual animal.

from 30% in the control group to 70% in the treated group (as reported previously; Yun et al., 2008), and reduced the severity of meningoencephalitis, levels of astrocyte activation, and cytokine production. It is well known that seasonal influenza viruses cause upper respiratory syndromes, but H5N1 viruses mainly target the lower respiratory tract in humans (Shinya et al., 2006). In fatal cases, the major cause of death has been

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Fig. 5. Diffuse mononuclear cell infiltrates (right panel) in cerebellar white matter of a peramivir-treated ferret compared with an uninvolved region (left panel) at 18 dpi. This lesion is an indication of a focus of ongoing inflammatory reaction in the late stage of infection in some animals even in the absence of detectable virus (H&E).

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severe pneumonia with few exceptions. As a cause of death in H5N1 infections, encephalitis is atypical, having been reported as the major lethal disease only in two siblings in Vietnam (de Jong et al., 2005a). No postmortem information is available from these two cases. Although we cannot directly compare our experimental results with these two cases, there are at least four published fatal human cases in which autopsies were performed and significant brain involvement was reported (To et al., 2001; Uiprasertkul et al., 2005; Gu et al., 2007; Zhang et al., 2009). The reported cause of death in these cases was respiratory syndrome with multiple organ failure. These four cases demonstrate various degrees and stages of brain pathology. The first case is a 13-year-old girl who suffered from severe pneumonia and survived for one month. Multiple microscopic lesions, consisting of demyelinated areas containing ‘‘reactive histiocytes’’, were described in the white matter of the cerebrum, but viral genomes were not detected (To et al., 2001). The character of the lesions in this case indicates a post-inflammatory stage after active infection has subsided that is similar to the reactive changes in the white matter of the longer surviving ferrets in our study. In those 18-day survivors, mild diffuse mononuclear infiltrates were found only in the white matter of brains from a few ferrets in both peramivir- and vehicle-treated groups, none of which had detectable levels of virus. These lesions may represent on-going inflammation due to persistent but undetectable low levels of infection even after peramivir treatment. On the other hand, continuation of the secondary repair response and probable continued death of injured neurons, even after resolution of the infection, may be the predominant processes. In the second human case, who survived 17 days after onset of the disease, small foci of necrosis were reported in the brain, also with no virus detected (Uiprasertkul et al., 2005). This finding differs from our and other groups’ experimental studies in which necrosis correlates with early lesions and high viral titers in brain. These animal models do differ from natural infections by the high doses of virus delivered intranasally, likely resulting in much higher levels of virus directly entering the brain. The last two human cases (survival 27 and 11 days respectively), provide some clues to the distribution of the H5N1 virus in CNS cells. Positive detection of viral antigens and genomes were scattered throughout the brain, but pathologic changes were not reported (Gu et al., 2007; Zhang et al., 2009). The H5N1 positive cells in the brain were mainly neurons, which is consistent with our findings. Infected astrocytes were mentioned and identified only by nuclear morphology in one animal study (Jang et al., 2009). We went further to conclusively identify the astrocyte as another target for H5N1 infection by using double immunofluorescence labeling of viral antigens and GFAP to definitively identify infected cells. In addition, we also demonstrated that ependymal cells can be infected by H5N1 virus. Ependymal cell infection may represent one factor responsible for the detection of virus in the cerebrospinal fluid in the acute encephalitis cases from Vietnam, although leakage of virus through the ependymal lining is also not excluded. Cerebrospinal fluid viral content may thus be an effective means of monitoring

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Fig. 6. Astrogliosis in the brain of surviving ferrets. In ferrets surviving 18 days post infection, immunofluorescence for GFAP demonstrates gliosis, a reaction to the resolving inflammatory lesion. Our results show that gliosis is found in survivors from both peramivir-treated (lower panels) and untreated animals (upper panels).

the severity of H5N1 meningoencephalitis. These reports of human cases did not, however, address the presence of gliosis. In our experimental model, we found gliosis to be a consistent and robust indicator of previous injury due to infection and inflammation. Immunofluorescence for GFAP revealed gliosis in the white matter in all long-surviving ferrets that developed any symptoms of infection (2 in the vehicle-treated group and 5 in the peramivir-treated group). The presence of reactive gliosis further supports the late stage post-inflammatory scenario, and should be evaluated in future autopsy studies of human cases. Despite the rarity of direct H5N1 infection in the human central nervous system, it is very common in naturally infected mammals and birds. The brain pathology described in experimental animals, naturally infected animals, and human cases is similar, and generally consistent with other types of viral encephalitis, consisting of infiltrates of mononuclear cells, both diffuse and perivascular, glial-microglial nodules, and necrosis in the severe cases. Several hypotheses exist regarding how the virus enters the central nervous system. Currently, most investigators believe that H5N1 viruses enter the CNS through cranial nerves, mainly into the brain stem, and then spread to the rest of the brain. These conclusions are derived from mouse models with intranasal inoculation (Iwasaki et al., 2004; Jang et al., 2009; Park et al., 2002). Other hypotheses emphasize the importance of the blood circulation and the olfactory pathway. With tight blood– brain barrier (BBB) protection, viruses can only enter the CNS through blood circulation when the integrity of the

BBB is compromised since no evidence exists that brain endothelium is a target for infection. Since animals used in all studies were in good health with intact BBB, and viral antigens have not been detected in perivascular locations during the early infection stage, the hypothesis of bloodborne transmission is less attractive. Although we have not specifically investigated the route of viral entry in ferrets, one of our ferrets with a low viral titer at the earliest time point had lesions exclusively in the brain stem, supporting a potential route of entry similar to the mouse model. However, our results do not rule out the possibility that virus can enter the CNS from nasal epithelium through the olfactory system or from blood after BBB integrity is compromised by proinflammatory cytokines in the later stages. The considerable variation in degree and patterns of brain injury reported in H5N1 virus infection in humans, naturally infected mammals and waterbirds, and experimental animal models may be related to the route of viral entry and the formulation of the viral inoculum. In most of the experimental models, animals are challenged intranasally with a relatively high dose of virus in a small volume of solution, which facilitates the attachment of viruses to the upper respiratory epithelium and may explain earlier and higher titers of virus in the brain than in the lungs. In human cases, on the other hand, a relatively lower dose of virus is suspected to enter the respiratory tract initially in the form of aerosol droplets that can be directly passed into the lungs where the cells have a relatively high affinity for the virus (van Riel et al., 2006). Because the immunologically

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privileged environment of the brain is maintained by limiting traffic of systemic immune cells through the BBB, systemic immune responses to brain infections are delayed (Becher et al., 2000). Also, a lower level of initial brain infection may not provoke robust inflammation. These factors help explain why brain lesions (meningitis, encephalitis and necrosis) in experimental animals are earlier and more severe than those in human cases. Differences in outcomes of brain infection between natural and experimental infection may also be influenced by the route of entry. In the examples of infections in wild species described above, moderate to severe encephalitis was reported in most of the subjects regardless of the presence of lung involvement, a finding that is also different from human cases. Most of these species (mammals and waterbirds) are thought to acquire H5N1 viruses mainly through ingesting contaminated food or water, and necropsy results reveal lesions in both the CNS and the gastrointestinal tract independent of pulmonary involvement. With this oral route of infection, viruses likely contact only a portion of the upper respiratory tract, delaying spread to the lungs, while spread to other systems through the gastrointestinal tract is more rapid. Brojer’s group provided supportive evidence for the spread of the H5N1 virus from the GI system to the CNS through the peripheral nervous system by analyzing the distribution of virus in infected tufted ducks in the fields (Brojer et al., 2009). Lesions and viral antigens were found in the brain, pancreas and the upper respiratory tract, and viral antigens were detected in peripheral nerve ganglia adjacent to the adrenal gland. These findings open the possibility of spread of infection to the CNS from the GI tract as well as from the upper respiratory tract through the peripheral nervous system, and emphasize the importance of examining peripheral and cranial nerve ganglia in experimental animals and autopsies of human cases. Our work corroborates the findings cited above that H5N1 is neurotropic, infecting predominantly neurons in CNS, but shows some degree of pantropism in infecting neuroglial cells (astrocytes and ependymal cells) at lower levels. We showed that the severity of meningoencephalitis correlated well with viral titers, and that gliosis was well developed by post infection day 18. Early and continuous treatment with an antiviral agent (peramivir) inhibited virus production to non-detectable levels, reduced the severity of brain injury, and promoted a higher survival rate, but our treatment regimen did not reduce the level of reactive gliosis at the late stage. Gliosis is one of the factors influencing late evolution of brain injury, and treatments that control its progression may lead to better survivor outcomes in terms of neurologic sequelae. Future investigations for which this model might provide valuable information impacting patient outcomes include better definition of antiviral treatment regimens with respect to late sequelae, and better understanding of the entry of virus and early events establishing infection in the brain. References Antarasena, C., Sirimujalin, R., Prommuang, P., Blacksell, S.D., Promkuntod, N., Prommuang, P., 2006. Tissue tropism of a Thailand strain of

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