Heartland virus infection in hamsters deficient in type I interferon signaling: Protracted disease course ameliorated by favipiravir

Virology 511 (2017) 175–183

Contents lists available at ScienceDirect

Virology journal homepage: www.elsevier.com/locate/yviro

Heartland virus infection in hamsters deficient in type I interferon signaling: Protracted disease course ameliorated by favipiravir

MARK

Jonna B. Westovera, Johanna D. Rigasa,b, Arnaud J. Van Wetterea,b, Rong Lia, Brady T. Hickersona, Kie-Hoon Junga, Jinxin Miaoa,c, Erin S. Reynoldsd, Bettina L. Conradb, ⁎ Skot Nielsona, Yousuke Furutae, Saravanan Thangamanid, Zhongde Wanga, Brian B. Gowena, a

Utah State University, 5600 Old Main Hill, Logan, UT 84322, USA Utah Veterinary Diagnostic Laboratory, 950 E. 1400 N., Logan, UT 84341, USA c Zhengzhou University, 100 Kexue Ave., Zhengzhou Shi, Henan Sheng, People's Republic of China d University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555, USA e Research Laboratories, Toyama Chemical Co., Ltd., Toyama 930-8508, Japan b

A R T I C L E I N F O

A BS T RAC T

Keywords: Heartland virus Phlebovirus Severe fever with thrombocytopenia syndrome virus Favipiravir Animal model Type I interferon

Heartland virus (HRTV) is an emerging tick-borne virus (Bunyaviridae, Phlebovirus) that has caused sporadic cases of human disease in several central and mid-eastern states of America. Animal models of HRTV disease are needed to gain insights into viral pathogenesis and advancing antiviral drug development. Presence of clinical disease following HRTV challenge in hamsters deficient in STAT2 function underscores the important role played by type I interferon-induced antiviral responses. However, the recovery of most of the infected animals suggests that other mechanisms to control infection and limit disease offer substantial protection. The most prominent disease sign with HRTV infection in STAT2 knockout hamsters was dramatic weight loss with clinical laboratory and histopathology demonstrating acute inflammation in the spleen, lymph node, liver and lung. Finally, we show that HRTV disease in hamsters can be prevented by the use of favipiravir, a promising broad-spectrum antiviral in clinical development for the treatment of influenza.

1. Introduction Heartland virus (HRTV) is a member of the family Bunyaviridae (genus Phlebovirus) and an emerging tick-borne agent recently discovered in the United States (US). It is most closely related genetically to severe fever with thrombocytopenia syndrome virus (SFTSV) now endemic in regions of China, South Korea and Japan (Liu et al., 2014; Matsuno et al., 2013; McMullan et al., 2012). HRTV was first isolated in Missouri in 2009 from two hospitalized farmers that presented with fever, fatigue, diarrhea, leukopenia, and thrombocytopenia (McMullan et al., 2012). With just over 30 reported cases and despite serological evidence of widespread prevalence of HRTV across midwestern and eastern US (CDC, 2017; Bosco-Lauth et al., 2015; Riemersma and Komar, 2015), the virus appears to be less virulent than SFTSV causing mostly subclinical or mild illness. Death associated with HRTV disease appears to be linked to advancing age and underlying conditions that likely exacerbate the infection and disease process (Oklahoma State Department of Health, 2014; Fill et al., 2016; Muehlenbachs et al., 2014).

⁎

Corresponding author. E-mail address: [email protected] (B.B. Gowen).

http://dx.doi.org/10.1016/j.virol.2017.08.004 Received 2 May 2017; Received in revised form 1 August 2017; Accepted 2 August 2017 0042-6822/ © 2017 Elsevier Inc. All rights reserved.

HRTV has a single-stranded RNA genome consisting of three segments. The large and the medium segments are negative sense and encode the RNA-dependent-RNA polymerase and the glycoproteins Gn and Gc, respectively. The small segment encodes the nucleocapsid and nonstructural (NSs) proteins in an ambisense configuration (McMullan et al., 2012). The NSs proteins of related phleboviruses are known to be important virulence factors involved in the subversion of the host innate immune response to viral infection (Wuerth and Weber, 2016). The NSs protein from closely related SFTSV has been shown to sequester STAT proteins involved in interferon (IFN) signaling pathways (Ning et al., 2015). Consistent with these findings, we recently demonstrated that hamsters with an inactivating deletion in STAT2 are highly susceptible to SFTSV challenge (Gowen et al., 2016). Moreover, AG129 mice lacking functional IFN-α/β and γ receptors were recently found to be highly susceptible to HRTV infection (Bosco-Lauth et al., 2016). Herein we describe studies investigating the pathogenesis and natural history of HRTV infection in STAT2 knockout (KO) hamsters and the use of favipiravir, a broad-spectrum antiviral currently being clinically eval-

Virology 511 (2017) 175–183

J.B. Westover et al.

2.5. Viral titers

uated in Japan as a treatment for SFTS, as a potential therapeutic option for HRTV disease (Furuta et al., 2013; UMIN, 2016).

Virus titers were assayed using a previously described infectious cell culture assay (Gowen et al., 2007). Briefly, tissue samples were homogenized in MEM and the clarified homogenates and serum were serially titrated on Vero E6 cells. Viral cytopathic effect (CPE) was determined 11 days after plating and the 50% endpoints were calculated as described (Reed and Muench, 1938). The lower limit of detection (LLD) for serum samples was 1.67 log10 CCID50/ml. The LLD for tissues ranged from 2.5 to 3.5 log10 CCID50/g.

2. Materials and methods 2.1. Animals The development of the STAT2 KO golden Syrian hamsters has been previously described (Fan et al., 2014). Male and female STAT2 KO hamsters (4–5 weeks of age) were used for all experiments and obtained from the breeding colony at Utah State University (USU). The animals were fed autoclaved Harlan Lab Block and sterilized tap water ad libitum and acclimated for approximately one week in the biosafety level 2 containment facility prior to virus challenge. All animal procedures complied with USDA guidelines and were conducted at the AAALAC-accredited Laboratory Animal Research Center at USU.

2.6. Serum biochemistry A comprehensive 18-parameter metabolic blood biochemistry analysis was performed on all serum samples to evaluate liver and kidney function, as well as electrolyte and fluid balance. All tests were performed per the manufacturer's recommendations using a DRICHEM 4000 (Heska).

2.2. Virus

2.7. Hematology

HRTV, strain MO-4, was obtained from Robert Tesh (World Reference Center for Emerging Viruses and Arboviruses, The University of Texas Medical Branch). The virus stock (2.5 × 106 50% cell culture infectious doses (CCID50)/ml; 1 passage in Vero E6 cells) was derived from a clarified cell culture lysate preparation and diluted in minimum essential medium (MEM) for inoculations.

Anticoagulated whole-blood samples were analyzed using an automated complete blood count (CBC) instrument (Advia 120 Hematology Analyzer from Siemens Healthcare Diagnostics Inc.). To confirm the CBC analysis, blood smears were also prepared for manual total WBC estimates and differential cell counts performed by a board-certified veterinary clinical pathologist. The smears were also evaluated for morphological features such as toxic changes and platelet clumping. Toxic changes (basophilia, vacuolation, Dohle bodies, and granulation) are diagnostic features that can be observed in neutrophils to gain further insights into the inflammatory response. They occur while neutrophils are formed in the bone marrow under a significant cytokine influence (Stockham and Scott, 2008), and were graded on a scale of 0– 3 with 0 being no changes and 3 being marked toxic changes.

2.3. Susceptibility of STAT2 KO Hamsters to HRTV Hamsters (n = 22) were grouped by weight and sex to minimize differences across experimental groups. In the initial susceptibility study, 4 animals in each group were inoculated by subcutaneous (s.c.) injection of 0.2 ml containing 50, 500, 5000 or 50,000 CCID50 of HRTV and 2 hamsters were sham-infected with MEM. The hamsters were observed for 21 days for morbidity and mortality. For all studies, animal weight data were reported as the mean weights of the hamsters relative to their starting weights prior to infection. A second study was conducted in which both heterozygous and homozygous STAT2-deficient hamsters were challenged s.c with the three highest doses of HRTV used in the initial study. The hamsters (n = 14) were grouped so that each challenge dose group had one heterozygous animal and three homozygous for the STAT2 deficiency, while minimizing weight and sex differences between groups. Two heterozygous hamsters were used for sham-infections. All hamsters were observed for 17 days for morbidity and mortality.

2.8. Histopathology and IHC Tissue samples of spleen, liver, kidney, heart, lung, brain, intestine, and submandibular lymph nodes were preserved in 10% neutral buffered formalin for histopathologic examination by a board-certified veterinary pathologist. In tissues where lesions were present, severity was scored on a scale of 0–4 with 0 indicating no lesions and 4 indicating severe lesions. Spleen, liver, kidney, heart, lymph node and intestine from the moribund HRTV-infected hamster and a shaminfected control were also processed for IHC following standard protocols (Hermance et al., 2016; Santos et al., 2016). The presence of HRTV antigen was detected in the fixed tissues using a mouse monoclonal antibody against the nucleoprotein (Calvert and Brault, 2015).

2.4. Pathogenesis and natural history of HRTV infection in STAT2 KO hamsters

2.9. In vitro antiviral activity of favipiravir Homozygous STAT2 KO hamsters were challenged s.c. with 5 × 104 CCID50 HRTV and randomly assigned to groups (n = 3/group) designated for sacrifice on specified days post-infection (p.i.). Groups were sacrificed on days 2, 5, 7, 9, 11 and 13 p.i. and a single shaminfected control per day on days 2, 7 and 13 p.i. Prior to sacrifice, whole blood was collected from anesthetized animals by retro-orbital bleed into K3 EDTA-coated tubes for hematologic analysis. Following euthanasia, hamsters were exsanguinated by cardiac puncture to obtain serum for biochemistry and viral titer analyses. Urine was collected to determine urine specific gravity. Anticoagulated blood and bone marrow smears were prepared for manual white blood cell (WBC) counts and cytology. Tissue samples of liver, spleen, kidney, heart, lung, brain, small intestine, and lymph nodes were collected for infectious virus titer determination, histopathology and immunohistochemistry (IHC).

Virus yield reduction (VYR) assays were used to evaluate the efficacy of favipiravir (Toyama Chemical Co., Ltd.) and ribavirin (ICN Pharmaceuticals), against HRTV infection in Vero E6 cells. Briefly, subconfluent cells in 96-well microplates were infected at a multiplicity of infection of 0.01 and treated with half-log dilutions of favipiravir or ribavirin in culture medium supplemented with 2% fetal bovine serum. Plates were incubated at 37 °C and 5% CO2 for 7 days and frozen at − 80 °C. After thawing, cell lysates were titrated by endpoint dilution (Reed and Muench, 1938). The 50% cell cytotoxic dose (CC50) was determined by neutral red dye uptake in uninfected, drug-treated cells. The 90% effective concentration (EC90) was determined by regression analysis and represents the concentration of drug that reduced the virus yield by one log10 unit. The selectivity index (SI) was calculated using the formula: SI = CC50/EC90. 176

Virology 511 (2017) 175–183

J.B. Westover et al.

2.10. In vivo antiviral efficacy study STAT2 KO hamsters (n = 35) were sorted by sex and weight to minimize differences between treatment groups. Hamsters (n = 8/ treatment group, n = 3 for sham-infected group) were challenged s.c. with 5 × 104 CCID50 of HRTV and treated orally, twice daily with favipiravir (300 or 150 mg/kg/day), ribavirin (75 mg/kg/day) or the 0.4% carboxymethyl cellulose vehicle placebo. The 10-day treatment regimens were initiated one day post-HRTV challenge. Predetermined subsets of animals (n = 3/treatment group) were sacrificed on day 9 of infection for analysis of serum, liver, spleen, and kidney viral titers. The remaining hamsters continued to receive treatments and were observed for 21 days for morbidity and mortality. Sham-infected animals were included as controls for weight gain (n = 1) and background for day 9 virus titer assays (n = 2). 2.11. Statistical analysis The Mantel-Cox log-rank test was used for analysis of Kaplan-Meier survival curves. One-way ANOVA with Dunnett's posttest was used to correct for multiple comparisons of blood chemistry, hematology and viral titer data compared to the sham-infected animals; two-way ANOVA was used to compare the daily weights. All statistical evaluations were done using Prism 7 (GraphPad Software). 3. Results 3.1. Susceptibility of STAT2 KO hamsters to HRTV Wild-type golden Syrian hamsters are refractory to disease and develop no detectable viremia following HRTV challenge (Bosco-Lauth et al., 2016). To investigate whether STAT2 deficiency would render hamsters susceptible to HRTV, STAT2 KO hamsters (n = 4/group) were inoculated with serial dilutions of virus (50,000, 5000, 500 or 50 CCID50) or sham-infected and observed for signs of illness. With the exception of a single animal that succumbed on day 18 p.i. at the 5000 CCID50 challenge dose, there was no other mortality observed (Fig. 1A). All hamsters challenged with 500 CCID50 of HRTV or more started losing weight by day 4 p.i. with the animals infected with 50,000 CCID50 having the most rapid weight loss (Fig. 1B). The affected hamsters had varying degrees of reduced activity and ruffled fur coinciding with the observed weight loss. Most of the surviving animals started gaining weight between days 10–13 p.i. To further investigate the role of STAT2 in the control of HRTV infection, STAT2 KO hamsters (n = 9) and heterozygous littermates (n = 3) with a single functional STAT2 allele were challenged in parallel. All homozygous STAT2 KO hamsters started to lose weight by approximately 5–6 days p.i. (Fig. 2). In contrast, the heterozygous animals showed no signs of illness and continued to gain weight similarly to the sham-infected STAT2 KO hamsters, indicating that a single functional copy of the STAT2 gene is sufficient to effectively control HRTV infection and prevent disease.

Fig. 1. Titration of HRTV in STAT2 KO hamsters. A) Percent survival of animals infected i.p. with the indicated dose of HRTV (n = 6 for the 50 CCID50 challenge group, n = 4 for all other virus challenge groups). B) Weight data are represented as the group mean and standard deviation of the percent change in weight of surviving animals relative to their starting weights on the day of challenge. Sham-infected controls (n = 2) are shown for comparison.

Serum and tissues were analyzed for the presence of infectious virus during the course of the HRTV infection. All 3 animals on day 5 had a low-level viremia (102.7, 103.5, and 104.5 log10 CCID50/ml), but virus was undetectable in the tissues early on (Fig. 4). We detected low viral titers in some tissues (spleen, liver, heart, lung, and lymph node) in a couple of the hamsters from the day 7 group, and one of those animals had barely detectable viremia (102 log10 CCID50/ml). The moribund animal from the day 13 group that was euthanized on day 9 was an outlier that presented with viral titers in all tissues except the liver, brain (not shown) and serum (Fig. 4). HRTV antigen was present in spleen (Fig. 5D), kidney, heart, lung, small intestine and lymph node by IHC (not shown), and inflammatory lesions were observed mainly in the spleen (Fig. 5B) and lymph node (not shown) of the day 9 moribund animal. Infectious virus was undetectable in all other day 9, 11 and 13 hamsters. Several serum biochemistry tests summarized in Supplemental Table 1 suggested the presence of liver disease in HRTV-infected hamsters. ALP was significantly elevated in the animals sacrificed on days 7 and 9 p.i. compared to the sham-infected controls. Also consistent with liver insufficiency, products synthesized in the liver including GLU and ALB were dramatically decreased on days 7 and 9 p.i. Similarly, TP and CA were also reduced in the day 7 and 9 animals, consistent with the majority of TP being comprised of ALB and that at least 50% of CA is bound to ALB (Killingsworth, 1979). CHOL, another product synthesized by the liver, was also decreased on days 7 and 9, but not significantly. Other serum biochemistry indicators of liver pathology were not significantly affected.

3.2. Natural history and pathogenesis of HRTV infection in STAT2 KO hamsters To gain insights into the pathogenesis of HRTV infection, a natural history study was designed to investigate several virologic, clinical, and laboratory parameters during acute infection in STAT2 KO hamsters by sacrificing 3 hamsters each day on days 2, 5, 7, 9, 11 and 13 p.i. A challenge dose of 5 × 104 CCID50 was selected based on the titration experiment. As expected, dramatic weight loss due to HRTV infection was observed compared to the sham-infected hamsters (Fig. 3). In the day 13 group, 1 of the 3 animals was moribund on day 9 p.i. and another succumbed prior to the time of sample collection. Thus, the moribund animal was humanely euthanized and included in the day 9 sacrifice cohort. 177

Virology 511 (2017) 175–183

J.B. Westover et al.

Fig. 2. Longitudinal analysis of weight change in homozygous and heterozygous STAT2 KO hamsters challenged with HRTV. Individual tracings of the weights of animals challenged s.c. with A) 50,000 CCID50, B) 5000 CCID50, or C) 500 CCID50 of HRTV are shown with two sham-infected control STAT2 KO hamsters for comparison. Weight data are represented as the percent change in weight relative to starting weights on the day of challenge.

decreased MG and PHOS on days 7 and 9 (also day 11 for MG) p.i., which could be the result of a loss of appetite as the animals became sick. The hematologic analysis is summarized in Supplemental Table 2. Erythrocyte parameters including RBC, MCHC, and RDW did not significantly differ between HRTV-infected and sham-infected control animals. HGB and HCT showed a significant decrease beginning on day 9. Typically, RBC and HCT should have the same degree of change as both parameters indicate red cell mass. A discrepancy between HCT and RBC might suggest an artifactual alteration in the red cell size as this parameter is a calculated value using the MCV. It was speculated that the decreased MCV was an artifact secondary to the low blood volume when mixed with the anticoagulant solution prior to hematologic evaluation. This process can result in shrinkage of erythrocytes. In an attempt to address this, the packed cell volume (PCV) should be used for future experiments as this parameter is physically measured by centrifugation of the sample, and it is a more accurate assessment of the RBC mass when compared to the HCT. Consistent with systemic inflammation, leukocytosis was evident throughout most of the HRTV infection with markedly increased WBC counts present 7–9 days p.i. (Supplemental Table 2). An increase in mature and immature (band) neutrophil numbers and a decrease in lymphocytes was evident during the course of the infection. This resulted in an inversion of neutrophils to lymphocytes ratio, further proof of a profound inflammatory response in the hamsters, with day 7 p.i. being the most pronounced. No thrombocytopenia was observed; however, a significant increase in platelets (thrombocytosis) was found on day 11 p.i. and is in line with the strong inflammatory response. Analysis of blood smears showed toxic changes in neutrophils which are also indicative of a vigorous systemic inflammation (Supplemental

Fig. 3. Temporal analysis of weight change during the natural history study of the HRTV infection in STAT2 KO hamsters. Animals were weighed prior to s.c. challenge with 5 × 104 CCID50 of HRTV and randomly assigned to groups for sacrifice on days 2, 5, 7, 9, 11, or 13. Sham-infected controls were sacrificed on days 2, 7, and 13 p.i. The data are represented as the group mean and standard deviation of the percent change in weight of surviving animals relative to their starting weights on the day of challenge.

There was no evidence of renal disease in HRTV-infected animals as CRE, BUN and urine specific gravity (data not shown) did not significantly deviate from the sham-infected control animals (Supplemental Table 1). HRTV-infected animals did have significantly 178

Virology 511 (2017) 175–183

J.B. Westover et al.

Fig. 4. Infectious viral titers during the course of the HRTV infection in STAT2 KO hamsters. Prior to s.c. infection with 5 × 104 CCID50 of HRTV, three animals per day were designated for sacrifice on days 2, 5, 7, 9, 11, or 13 p.i. One animal in the day 13 group succumbed from the infection prior to time of sacrifice and another was found moribund on day 9. This is reflected in the graph with a single data point for day 13 and the additional moribund animal (green up-side-down triangle) shown on day 9. Serum and tissue samples were analyzed for viral titers in hamsters sacrificed on the indicated day of infection. Results are shown as the group mean and unique symbols in each sacrifice group represent values for the same animal across all tissues. No virus was detected in any of the brain samples (not shown).

179

Virology 511 (2017) 175–183

J.B. Westover et al.

Fig. 5. Histology and IHC detection of viral antigen in the moribund HRTV-infected hamster. Hematoxylin and eosin stained spleen sections from A) sham-infected control hamster and B) HRTV-infected, moribund hamster (day 9 p.i.) with neutrophilic inflammation and necrosis of the splenic white pulp. Scale bar = 20 µm, 600× magnification. Immunohistochemical HRTV antigen detection in spleen sections from the respective C) a sham-infected control hamster and D) the HRTV-infected, moribund hamster with splenocytes exhibiting immunoreactivity. Scale bars = 20 µm, 400× magnification. Hematoxylin counterstain.

ribavirin, against HRTV was first evaluated in cell culture by VYR (Fig. 7A). The EC90 for favipiravir was 2.8 μg/ml ± 0.3 (17.5 μM) and 18.7 μg/ml ± 1.9 (76.6 μM) for ribavirin. The CC50 for both compounds was > 1000 μg/ml (Fig. 7B). Thus, favipiravir had an SI value of > 405 while ribavirin's SI was > 60. Having determined that favipiravir and ribavirin are highly active against HRTV in vitro, an experiment was designed to evaluate their efficacy in the STAT2 KO hamster HRTV infection model. Several doses of favipiravir and a single dose of ribavirin were tested with 8 hamsters per treatment group. Treatment with either dose of favipiravir completely prevented weight loss in the HRTV-infected animals (Fig. 8A). In contrast, hamsters receiving ribavirin or the placebo began to lose weight on day 5 p.i. until day 12 when they started recovering from the infection. A slight dosedependent effect was seen as the 150 mg/kg/day favipiravir group mean weight increase was less than that of the animals receiving the higher dose. Fig. 8B reflects the percent weight change recorded on day 11 for each animal relative to their respective day 4 weights. Animals that received 300 mg/kg/day favipiravir gained, on average, 15% in weight from day 4 to day 11 p.i. The hamsters in the 150 mg/ kg/day treatment group gained approximately 10% during the same time period. This equates to a highly significant 20–25% difference compared to the 10% weight loss measured in the ribavirin- and placebo-treated animals over the same time period. The effect of the antiviral drug treatments on reducing day 9 viral titers was evaluated in pre-determined subsets (n = 3) of STAT2 KO hamsters infected and treated in parallel to those observed for weight loss. Animals treated with either dose of favipiravir had no detectable virus in serum, liver, spleen, or kidney tissues (data not shown). Only one ribavirin-treated hamster had 1-2 log10 CCID50 virus above the assay detection limit in its serum, liver, and kidney tissue, and a single placebo-treated animal had approximately 2.5 log10 CCID50 virus above the detection limit in its serum, spleen, and kidney tissue.

Table 2). The toxic changes were most dramatic through day 9, with all animals in the day 11 and 13 sacrifice groups having no observable changes. Histopathologic evaluation of all collected tissues demonstrated inflammatory lesions affecting primarily the spleen, lymph node and, to a lesser intensity, lung, liver, and heart (summarized in Fig. 6). The liver lesions consisted of mild to moderate, multifocal, periportal and random, acute, neutrophilic hepatitis. Splenic lesions consisted of moderate to severe, multifocal, acute neutrophilic splenitis affecting both the white and red pulps. The lungs presented with neutrophilic interstitial pneumonia, but no or minimal accompanying parenchymal injury. Heart lesions consisted of multifocal neutrophilic epicarditis and/or myocarditis. We were limited in the successful collection of submandibular lymph nodes to animals in the later stages of disease, likely due to enlargement and easier visualization of the nodes at this time. The lymph node lesions consisted of severe neutrophilic inflammation. No lesions were found in the kidney, intestinal tract, brain, or skeletal muscle. Histopathologic lesions corroborated well with the CBC and serum biochemistry results indicating liver injury and an acute systemic inflammatory response. Remarkably, despite significant inflammatory lesions, little to no infectious virus was present in these tissues with the exception of the moribund animal euthanized on day 9 (Fig. 4). Bone marrow smears were examined for each animal. There was no evidence of necrosis indicative of viral CPE on bone marrow cells. All bone marrows had normal cellularity; however, an increased percent of immature granulocytes with an increased myeloid to erythroid ratio was evidence that the bone marrow was responding to the increased demand for inflammatory cells in response to infection in other organs. 3.3. Antiviral activity of favipiravir and ribavirin against HRTV The activity of the broadly active nucleoside analogs, favipiravir and 180

Virology 511 (2017) 175–183

J.B. Westover et al.

Fig. 6. Histopathology findings summary from STAT2 KO hamsters challenged with HRTV represented as a heat map. Lesions were scored as 0 = no lesions, 1 = minimal, 2 = mild, 3 = moderate, and 4 = severe. No lesions were found in the kidney, intestinal tract, or brain. Lymph nodes for several animals were not analyzed for histopathology due to limited sample availability and are represented by blank spaces in the table. The moribund animal that was included in the day 9 analyses is marked with an asterisk (*).

4. Discussion Based on the close phylogenetic relationship between HRTV and SFTSV (Matsuno et al., 2013; McMullan et al., 2012), we hypothesized that hamsters deficient in type I IFN signaling would be highly susceptible to HRTV infection similarly to that which was reported for SFTSV (Gowen et al., 2016). Our findings, however, indicate that HRTV causes only moderate disease in STAT2 KO hamsters, which appears to be largely driven by the severe inflammatory response to the infection, and characterized by notable weight loss, reduced activity and limited ruffling of fur. The disease course was protracted with the majority of animals surviving the infection. The generally less severe HRTV infection in the immune deficient STAT2 KO hamsters is consistent with the idea that the virus, despite evidence of its presence across many midwestern and eastern states (Bosco-Lauth et al., 2015; Riemersma and Komar, 2015), seldom causes severe disease that would require hospitalization in healthy individuals. HRTV causes a systemic infection in STAT2 KO hamsters, but viremia was only detected at low levels on the second day p.i. Moreover, evidence of viral replication in tissues was only observed in a few animals suggesting that it may have been limited by the immune response to the site of inoculation and the draining lymph node. Human cases of HRTV disease generally present with marked leukopenia and thrombocytopenia (Pastula et al., 2014), but these clinical features were not evident in the hamster model. To the contrary, WBC counts were elevated throughout the study, and although there was a trend towards reduced platelet levels on day 7 p.i., significantly higher levels were found on day 11 when animals generally were beginning to regain lost weight. Collectively, the clinical chemistries, hematology and histopathology findings demonstrated a systemic inflammatory disease that resulted in a 10- to 14-day illness from which most animals recovered. The prominent neutrophilia

Fig. 7. In vitro inhibition of HRTV by favipiravir and ribavirin. A) Virus titers were determined by endpoint titration of cell lysates from Vero E6 cell cultures infected with HRTV (MOI of 0.01) and treated with increasing concentrations of favipiravir and ribavirin. Linear regression analysis conducted on yields of infectious HRTV titers in cell lysates collected at 7 days post-infection revealed a clear dose response with an EC90 of 2.8 μg/ml for favipiravir and 18.7 μg/ml for ribavirin. B) Cytotoxicity data in uninfected cells treated in parallel are presented as the percent cell viability after a 7-day incubation compared to untreated controls. Data are representative of multiple experiments. Means and standard deviations from experimental replicates (n = 3) are shown.

181

Virology 511 (2017) 175–183

J.B. Westover et al.

strains underscores the importance of type I and type II IFN signaling in preventing severe infection and mortality. AG129 mice, which are devoid of IFN-α/β and γ receptors, were the only animals found to be highly susceptible to disease by the same strain (MO-4) of the virus used in our studies, with 80% lethality observed at a challenge dose of 10 PFU (Bosco-Lauth et al., 2016). Hematologic analysis was not performed in the AG129 mouse model and histopathologic lesions were limited to increased numbers of mononuclear cells in the spleen. Considering our findings in STAT2 KO hamsters, IFN-γ responses may contribute substantially to the control of HRTV infection. Indeed, we have found the virus to be lethal in AG129 mice, but cause little to no disease in IFN-α/β receptor KO (IFNAR-/-) mice (Supplemental data Fig. 1). This suggests that known antiviral functions of IFN-γ likely play an important role in combatting HRTV infection (Schroder et al., 2004). Further characterization of the immune response in rodent models and humans is needed to gain insights into the observed differences. Finally, there are currently no antivirals available for the treatment of HRTV infection. Our data showed promising in vitro and in vivo efficacy results in our evaluation of favipiravir, a broad-spectrum antiviral with demonstrated activity against the closely related SFTSV infection in mice and hamsters (Gowen et al., 2016; Tani et al., 2016). Further, the compound has also been shown to be highly active against another pathogenic phlebovirus, Rift Valley fever virus (Caroline et al., 2014; Scharton et al., 2014). Favipiravir is currently approved as an anti-influenza drug in Japan and is being evaluated as a therapy for SFTSV (UMIN, 2016). Phase 3 clinical trials for influenza have been completed in the US and approval could make it available for off-label use for treating severe cases of HRTV infection. Funding This work was supported by funding from the National Institutes of Health (HHSN272201000039I) and the Next-Generation Bio-Green 21 Program (PJ01107703 to ZW). ST is supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health Grant 1R21AI113128-01A1. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.virol.2017.08.004. References

Fig. 8. Percent weight change in STAT2 KO hamsters challenged s.c. with 5 × 104 CCID50 HRTV during the course of the efficacy experiment. Hamsters in each treatment group (n = 5) received the indicated doses of favipiravir, ribavirin or placebo (p.o., twice daily for 10 days) beginning 1 day p.i. A) The data are represented as the group mean and standard deviation of the percent change in weight of surviving animals relative to their starting weights on the day prior to challenge. A sham-infected animal is included for comparison. By repeated measures two-way ANOVA, the hamster weights were significantly higher (P < 0.05) in the favipiravir groups compared to placebo- (days 6–21) and ribavirin-treated animals (days 7–18). B) Percent weight change on day 11 relative to respective day 4 weights. ***P < 0.001 compared to ribavirin or placebo-treated animals. Tx; treatment.

Bosco-Lauth, A.M., Panella, N.A., Root, J.J., Gidlewski, T., Lash, R.R., Harmon, J.R., Burkhalter, K.L., Godsey, M.S., Savage, H.M., Nicholson, W.L., Komar, N., Brault, A.C., 2015. Serological investigation of heartland virus (Bunyaviridae: phlebovirus) exposure in wild and domestic animals adjacent to human case sites in Missouri 2012–2013. Am. J. Trop. Med. Hyg. 92, 1163–1167. Bosco-Lauth, A.M., Calvert, A.E., Root, J.J., Gidlewski, T., Bird, B.H., Bowen, R.A., Muehlenbachs, A., Zaki, S.R., Brault, A.C., 2016. Vertebrate host susceptibility to heartland virus. Emerg. Infect. Dis. 22, 2070–2077. Calvert, A.E., Brault, A.C., 2015. Development and characterization of monoclonal antibodies directed against the nucleoprotein of heartland virus. Am. J. Trop. Med. Hyg. 93, 1338–1340. Caroline, A.L., Powell, D.S., Bethel, L.M., Oury, T.D., Reed, D.S., Hartman, A.L., 2014. Broad spectrum antiviral activity of favipiravir (T-705): protection from highly lethal inhalational Rift Valley Fever. PLoS Negl. Trop. Dis. 8, e2790. CDC, 2017. Heartland Virus. Fan, Z., Li, W., Lee, S.R., Meng, Q., Shi, B., Bunch, T.D., White, K.L., Kong, I.K., Wang, Z., 2014. Efficient gene targeting in golden Syrian hamsters by the CRISPR/Cas9 system. PLoS One 9, e109755. Fill, M.A., Compton, M.L., McDonald, E.C., Moncayo, A.C., Dunn, J.R., Schaffner, W., Bhatnagar, J., Zaki, S.R., Jones, T.F., Shieh, W.J., 2016. Novel clinical and pathologic findings in a heartland virus-associated death. Clin. Infect. Dis.. Furuta, Y., Gowen, B.B., Takahashi, K., Shiraki, K., Smee, D.F., Barnard, D.L., 2013. Favipiravir (T-705), a novel viral RNA polymerase inhibitor. Antivir. Res. 100, 446–454. Galani, I.E., Andreakos, E., 2015. Neutrophils in viral infections: current concepts and caveats. J. Leukoc. Biol. 98, 557–564. Gowen, B.B., Wong, M.H., Jung, K.H., Sanders, A.B., Mendenhall, M., Bailey, K.W.,

observed in STAT2 KO hamsters is not a significant finding in human cases of HRTV infection. The presence of neutrophilic inflammation in several hamster organs correlates well with the neutrophilia and was also observed in a related phlebovirus infection model (Gowen et al., 2016). Although the role of neutrophils in the control of viral infections is still unclear (Galani and Andreakos, 2015), it is possible that the neutrophilic inflammatory response induced by HRTV infection may substantially contribute to virus clearance, as supported by the inconsistent and relatively low levels of infectious virus present in hamster tissues. Results from a recently published study investigating HRTV infection in a number of different vertebrate species and several mouse 182

Virology 511 (2017) 175–183

J.B. Westover et al.

inclusion bodies. J. Virol. 89, 4227–4236. Oklahoma State Department of Health, 2014. Oklahoma State Health Department Confirms First Case and Death of Heartland Virus. Pastula, D.M., Turabelidze, G., Yates, K.F., Jones, T.F., Lambert, A.J., Panella, A.J., Kosoy, O.I., Velez, J.O., Fisher, M., Staples, E., Centers for Disease, C., Prevention, 2014. Notes from the field: Heartland virus disease – United States, 2012–2013. MMWR Morb. Mortal. Wkly. Rep. 63, 270–271. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493–497. Riemersma, K.K., Komar, N., 2015. Heartland virus neutralizing antibodies in vertebrate wildlife, United States, 2009–2014. Emerg. Infect. Dis. 21, 1830–1833. Santos, R.I., Hermance, M.E., Gelman, B.B., Thangamani, S., 2016. Spinal cord ventral horns and lymphoid organ involvement in powassan virus infection in a mouse model. Viruses, 8. Scharton, D., Bailey, K.W., Vest, Z., Westover, J.B., Kumaki, Y., Van Wettere, A., Furuta, Y., Gowen, B.B., 2014. Favipiravir (T-705) protects against peracute Rift Valley fever virus infection and reduces delayed-onset neurologic disease observed with ribavirin treatment. Antivir. Res. 104, 84–92. Schroder, K., Hertzog, P.J., Ravasi, T., Hume, D.A., 2004. Interferon-gamma: an overview of signals, mechanisms and functions. J. Leukoc. Biol. 75, 163–189. Stockham, S.L., Scott, M.A., 2008. Fundamentals of Veterinary Clinical Pathology 2nd ed.. Blackwell Pub, Ames, Iowa. Tani, H., Fukuma, A., Fukushi, S., Taniguchi, S., Yoshikawa, T., Iwata-Yoshikawa, N., Sato, Y., Suzuki, T., Nagata, N., Hasegawa, H., Kawai, Y., Uda, A., Morikawa, S., Shimojima, M., Watanabe, H., Saijo, M., 2016. Efficacy of T-705 (Favipiravir) in the treatment of infections with lethal severe fever with thrombocytopenia syndrome virus. mSphere, 1. UMIN, 2016. Clinical Study of Favipiravir for Patients with Severe Fever with Thrombocytopenia Syndrome. Wuerth, J.D., Weber, F., 2016. Phleboviruses and the Type I interferon response. Viruses, 8.

Furuta, Y., Sidwell, R.W., 2007. In vitro and in vivo activities of T-705 against arenavirus and bunyavirus infections. Antimicrob. Agents Chemother. 51, 3168–3176. Gowen, B.B., Westover, J.B., Miao, J., Van Wettere, A.J., Rigas, J.D., Hickerson, B.T., Jung, K.H., Li, R., Conrad, B.L., Nielson, S., Furuta, Y., Wang, Z., 2016. Modeling severe fever with thrombocytopenia syndrome virus infection in golden Syrian hamsters: importance of STAT2 in preventing disease and effective treatment with favipiravir. J. Virol.. Hermance, M.E., Santos, R.I., Kelly, B.C., Valbuena, G., Thangamani, S., 2016. Immune cell targets of infection at the tick-skin interface during powassan virus transmission. PLoS One 11, e0155889. Killingsworth, L.M., 1979. Plasma protein patterns in health and disease. CRC Crit. Rev. Clin. Lab. Sci. 11, 1–30. Liu, Q., He, B., Huang, S.Y., Wei, F., Zhu, X.Q., 2014. Severe fever with thrombocytopenia syndrome, an emerging tick-borne zoonosis. Lancet Infect. Dis. 14, 763–772. Matsuno, K., Weisend, C., Travassos da Rosa, A.P., Anzick, S.L., Dahlstrom, E., Porcella, S.F., Dorward, D.W., Yu, X.J., Tesh, R.B., Ebihara, H., 2013. Characterization of the Bhanja serogroup viruses (Bunyaviridae): a novel species of the genus Phlebovirus and its relationship with other emerging tick-borne phleboviruses. J. Virol. 87, 3719–3728. McMullan, L.K., Folk, S.M., Kelly, A.J., MacNeil, A., Goldsmith, C.S., Metcalfe, M.G., Batten, B.C., Albarino, C.G., Zaki, S.R., Rollin, P.E., Nicholson, W.L., Nichol, S.T., 2012. A new phlebovirus associated with severe febrile illness in Missouri. N. Engl. J. Med. 367, 834–841. Muehlenbachs, A., Fata, C.R., Lambert, A.J., Paddock, C.D., Velez, J.O., Blau, D.M., Staples, J.E., Karlekar, M.B., Bhatnagar, J., Nasci, R.S., Zaki, S.R., 2014. Heartland virus-associated death in tennessee. Clin. Infect. Dis. 59, 845–850. Ning, Y.J., Feng, K., Min, Y.Q., Cao, W.C., Wang, M., Deng, F., Hu, Z., Wang, H., 2015. Disruption of type I interferon signaling by the nonstructural protein of severe fever with thrombocytopenia syndrome virus via the hijacking of STAT2 and STAT1 into

183