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Constitutively Expressed IFITM3 Protein in Human Endothelial Cells Poses an Early Infection Block to Human Influenza Viruses Xiangjie Sun, Hui Zeng, Amrita Kumar, Jessica A. Belser, Taronna R. Maines, Terrence M. Tumpey Immunology and Pathogenesis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA

ABSTRACT

A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans. IMPORTANCE

Avian influenza viruses, including H5N1 and H7N9, have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary endothelial damage are known to be present during severe human infections, the role of pulmonary endothelial cells in the pathogenesis of avian influenza virus infections is largely unknown. By comparing human seasonal influenza strains to avian influenza viruses, we provide greater insight into the interaction of influenza virus with human pulmonary endothelial cells. We show that human influenza virus infection is blocked during the early stages of virus entry, which is likely due to the relatively high expression of the host antiviral factors IFITMs (interferon-induced transmembrane proteins) located in membrane-bound compartments inside cells. Overall, this study provides a mechanism by which human endothelial cells limit replication of human influenza virus strains, whereas avian influenza viruses overcome these restriction factors in this cell type.

I

nfluenza A viruses are important respiratory pathogens in humans and are responsible for approximately 250,000 to 500,000 fatal cases of influenza during annual epidemics worldwide (1). Occasionally, influenza A viruses of novel strains or subtypes against which the general human population has no preexisting immunity emerge and cause severe pandemics, as was demonstrated in 1918, 1957, 1968, and, most recently, in 2009 (2). Meanwhile, certain influenza A viruses of avian origin are capable of crossing host species barriers, resulting in sporadic infection in humans. Among these viruses, highly pathogenic avian influenza (HPAI) H5N1 viruses cause the highest mortality rate in humans, approximately 60% based on WHO reports (3). While exhibiting reduced mortality in humans, low-pathogenicity avian influenza (LPAI) viruses of the H7N9 subtype have also been associated with severe disease, with over 700 reported cases since their initial detection in humans in 2013 (4, 5). Human influenza A viruses primarily target epithelial cells in the upper respiratory tract due to their abundant expression of ␣-2,6-linked sialic acids, the preferred receptors for human influenza viruses (1). However, pandemic influenza viruses (including

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the 1918 and 2009 H1N1 viruses) or recently isolated HPAI H5N1 viruses possess the ability to replicate in human lower respiratory tract tissues and induce exacerbated innate immune responses (6–9). This is demonstrated by early recruitment of inflammatory leukocytes to the lung and excessive cytokine production, ultimately leading to acute respiratory distress syndrome (ARDS) and high mortality rates (10, 11). While the molecular mechanisms of severe illness caused by influenza virus infection have not been completely uncovered, it is believed that aberrant proinflamma-

Received 29 June 2016 Accepted 27 September 2016 Accepted manuscript posted online 5 October 2016 Citation Sun X, Zeng H, Kumar A, Belser JA, Maines TR, Tumpey TM. 2016. Constitutively expressed IFITM3 protein in human endothelial cells poses an early infection block to human influenza viruses. J Virol 90:11157–11167. doi:10.1128/JVI.01254-16. Editor: B. Williams, Hudson Institute of Medical Research Address correspondence to Terrence M. Tumpey, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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tory cytokine production and the resulting damage to the epithelial-endothelial barrier of the pulmonary alveolus play an important role in the development of severe disease (12). Recently, it has been revealed that pulmonary endothelial cells are central orchestrators of cytokine production and leukocyte recruitment in mice inoculated with the 2009 pandemic H1N1 virus (13). The work suggests that despite not representing a primary site for influenza virus replication, pulmonary endothelial cells contribute to the severity of the infection (13). Moreover, in vitro studies have shown that influenza virus infection can upregulate the expression of several endothelial adhesion molecules (14, 15), which may facilitate extravasation of neutrophils and macrophages into the alveoli. The persistent influx of such inflammatory cells can lead to damage of the epithelial-endothelial barrier by releasing reactive oxygen species, cytokines, and neutrophil extracellular traps (16). Additionally, pulmonary endothelial cells are susceptible to HPAI H5N1 virus infection in vitro in an envelope-dependent manner and express high levels of proinflammatory cytokines upon infection, whereas most human influenza viruses display only limited infectivity under these conditions (17–19). However, the exact molecular mechanism governing how selected highly pathogenic H5N1 viruses, but not human influenza viruses, possess the ability to replicate and induce excessive cytokine production is still largely unknown. IFITMs (interferon-induced transmembrane proteins) were first identified as type I and type II interferon (IFN)-induced proteins in 1984 (20) and comprise a family of small, 10- to 15-kDa proteins. In humans, there are four functional IFITM genes: the IFITM1, IFITM2, IFITM3, and IFITM5 genes, with IFITM4 being a pseudogene (21). Although previous studies on IFITMs mainly focused on their roles in embryonic development, their functions as host antiviral factors were only recently discovered by RNA interference genomic screening for host factors involved in influenza virus infection (22). It was subsequently revealed that IFITMs can restrict the early stages of replication of a wide variety of viruses, including influenza virus (22), flavivirus (dengue and West Nile viruses) (23, 24), filovirus (Marburg virus and Ebola viruses) (23), and coronavirus (23). Among all the IFITM members present in humans, IFITM3 has shown to be the most potent antiviral factor in restricting influenza virus infection, as IFITM3 knockout mice displayed enhanced morbidity and mortality associated with seasonal or 2009 pandemic H1N1 influenza virus infection (25, 26). Furthermore, the single-nucleotide polymorphism (SNP) (12252-C) in the IFITM3 gene, which results in decreased IFITM3 protein expression, has been linked with a higher risk of hospitalization among individuals infected with seasonal or 2009 pandemic H1N1 virus, as well as the novel H7N9 virus (26, 27). In this study, we investigated the mechanism by which human endothelial cells limit replication of human influenza viruses and how avian influenza viruses overcome these restriction factors in this cell type. Our results show that human influenza virus infection is blocked during the early stages of virus entry, precisely, after hemifusion, likely due to the relatively high constitutive expression of the host antiviral factor IFITM3 located in endosomal and lysosomal compartments. Conversely, certain avian influenza viruses may circumvent this restriction by fusing at a higher pH in early endosomes or by other, unknown mechanisms. Overall, our study suggests human hosts are able to restrict influenza virus infection in pulmonary endothelial cells partially by constitutively

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expressing high levels of IFITM proteins before interferon induction. MATERIALS AND METHODS Cells and viruses. Primary human lung blood microvascular endothelial cells (HMVEC) (Lonza, Walkersville, MD), immortalized human lung microvascular endothelial cells (HULEC), and human umbilical vein endothelial cells (HUVEC) (obtained from the Scientific Resources Program, CDC, Atlanta, GA) were cultured in endothelial cell basal medium 2 (EBM-2) (Lonza) with supplements as previously described (17). Human brain vascular endothelial cells (HBVEC), kindly provided by Monique Stins, Johns Hopkins University, were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 10% Nu-Serum (Fisher Scientific), 2 mM L-glutamine, 1 mM sodium pyruvate, 1⫻ minimal essential medium (MEM) nonessential amino acids, MEM vitamins, and penicillin-streptomycin (100 U/ml). Human bronchial epithelium (Calu-3) cells were cultured in Eagle’s MEM supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, and nonessential amino acids. Human lung epithelial A549 cells and Madin-Darby canine kidney (MDCK) cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS. Primary human bronchial epithelial (NHBE) cells were grown in serum-free and hormone-supplemented bronchial epithelial growth medium according to the manufacturer’s instructions (Lonza). The A549 cells stably expressing IFITM3 or the vector pQCXIP (Clontech) were originally developed and kindly provided by Gregory B. Melikyan and Mariana Marin, Emory University, and were maintained with 1.5 mg/ml of puromycin. Influenza viruses A/Puerto Rico/8/1934 (PR8) (H1N1), A/Vietnam/ 1203/2004 (VN/04) (H5N1), A/Anhui/1/2013 (Anhui/1) (H7N9), A/ chicken/Vietnam/NCVD-1156/2011 (CK/11) (H9N2), A/Netherlands/ 219/2003 (NL/03) (H7N7), A/shoveler/Egypt/00215-NAMRU3/2007 (Shov/07) (H7N9), A/Brisbane/59/2007 (Bris/07) (seasonal H1N1), A/ Panama/2007/1999 (Pan/99) (seasonal H3N2), and 2009 pandemic A/Mexico/4482/2009 (Mex/09) (H1N1) were grown in the allantoic cavities of 10-day-old embryonated hen’s eggs for 24 to 48 h at 33°C to 37°C. A/Brisbane/59/2007 (Bris/07) (seasonal H1N1) and 2009 pandemic A/Mexico/4482/2009 (Mex/09) (H1N1) were propagated in MDCK cells as described previously (28). Allantoic fluid or cell culture supernatant was clarified by centrifugation, aliquoted, and stored at ⫺70°C; virus titers were determined by standard plaque assay using MDCK cells. All research with HPAI H5, H7, and H9 subtype viruses was conducted under biosafety level 3 (BSL3) containment, including enhancements required by the U.S. Department of Agriculture and the National Select Agent Program. The reassortant viruses bearing HA and NA genes from VN/VN/04 (H5N1) or Anhui/1 (H7N9) virus and internal genes from the PR8 donor virus (VN:PR8 and Anhui:PR8) (kindly provided by Li-Mei Chen, Influenza Division, CDC) were propagated in the allantoic cavities of 10-dayold embryonated hen’s eggs for 48 h at 35°C under BSL2⫹ laboratory conditions. Virus purification. PR8 viruses were propagated in the allantoic cavities of 10-day-old embryonated hen’s eggs and then concentrated and purified by equilibrium density centrifugation through a 30 to 60% linear sucrose gradient as previously described (29). The concentrations of purified virus were determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA) and then diluted to 2 mg/ml in phosphatebuffered saline (PBS) for virus labeling. Virus infection. HULEC or A549 cells were incubated with influenza virus at multiplicities of infection (MOI) ranging from 1 to 5 in viral infection medium (base culture medium supplemented with 0.3% bovine serum albumin [BSA]) for 1 h, followed by washing with viral infection medium, and cultured with fresh infection medium at 37°C in a 5% CO2 atmosphere for 8 h (time of peak expression) before being fixed with 4% paraformaldehyde for 20 min. Viral infectivity was quantified based on the percentage of nucleoprotein (NP)-positive cells by indirect immuno-

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fluorescence microscopy (counting at least 200 cells per infection) using a mouse monoclonal anti-influenza A virus NP clone A1 and A3 blend (Millipore, Billerica, MA). Acid-induced fusion assay at the cell plasma membrane (acid bypass assay). Influenza viruses at an MOI of 5 were bound to A549 cells and HULEC on ice for 1 h, washed twice with prechilled viral infection medium to remove any unbound virus particles, and then incubated with prewarmed fusion buffer (20 mM HEPES, 2 mM CaCl2, 150 mM NaCl, 20 mM citric acid monohydrate-sodium citrate tribasic dehydrate, pH 5.0) for 2 min at 37°C to induce virus fusion at the cell surface. Following acid treatment, the cells were washed twice with prechilled viral infection medium and then incubated for 8 h with viral infection medium containing 30 mM NH4Cl to prevent viral infection via endocytic pathways. Virus infection mediated by viral fusion at the cell surface was quantified based on the percentage of NP-positive cells, using immunofluorescence microscopy. Virus internalization assay. Concentrated and purified influenza PR8 viruses were labeled with biotin and used in a virus internalization assay as described previously (30). Briefly, 1 ml of purified virus (2 mg/ml) was incubated with 10 mM Sulfo-NHS-SS-biotin (Pierce) for 2 h at 4°C, followed by ultracentrifugation to remove any unincorporated biotin. The labeled viruses were resuspended in PBS at a concentration of 1 mg/ml and filtered through a 0.22-␮m filter before use. During the virus internalization assay, 10 ␮l of biotin-labeled virus was incubated with cells in viral infection medium for 1 h at 4°C, followed by washing with ice-cold viral infection medium to remove any unbound virus particles. To induce internalization, the virus-bound cells were shifted to 37°C for 1 h in viral infection medium. To distinguish cell surface-bound virus particles from internalized virions, cells were incubated with excess quantities of unconjugated streptavidin (2 mg/ml; Thermo Fisher Scientific Inc., Rockford, IL) on ice after internalization to mask biotins from surface-bound viruses, after which the cells were fixed and permeabilized with 0.5% Triton X-100 and visualized by immunofluorescence microscopy by staining with Alexa 488-conjugated streptavidin (Thermo Fisher Scientific Inc.). Virus fusion assay. Concentrated and purified PR8 virus at 100 ␮g/ml was labeled with R18 and SP-DiOC18 (Thermo Fisher Scientific Inc.) at final concentrations of 0.4 ␮M and 0.2 ␮M, as described previously (31, 32). Ten microliters of labeled PR8 viruses was bound to cells grown on 24-well plates on ice for 1 h, followed by washing three times with ice-cold viral infection medium, before the plates were transferred to a 37°C incubator with 0.5% CO2 for 1.5 h to induce viral internalization and fusion. The cells were fixed with 4% paraformaldehyde (PFA) and stained with DAPI (4=,6-diamidino-2-phenylindole) before being scanned with an LSM 710 Zeiss inverted confocal microscope at wavelengths of 510 to 525 nm (green) and 575 to 640 nm (red) simultaneously. Viral fusion in endosomes is indicated by a fluorescence color shift from the red to the green channel. Detection of influenza virus nucleocapsid uncoating. PR8 viruses at an MOI of 40 were bound to HULEC and A549 cells on ice for 1 h in viral infection medium, after which the virus-bound cells were incubated at 37°C for 3.5 h following washing with viral infection medium. The cells were fixed, permeabilized, and stained with the anti-M1 monoclonal antibody HB64 (ATCC, Manassas, VA) by indirect immunofluorescence microscopy. siRNA transfection. HULEC and A549 cells grown in 24-well plates were transfected with a mixture of IFITM3 small interfering RNA (siRNA) (catalog number 284737) or negative-control siRNA (catalog number AM4611) and Lipofectamine 2000 (Thermo Fisher Scientific Inc.) following the manufacturer’s recommended transfection procedures as described previously (33); the knockdown effect on the IFITM3 protein expression level was confirmed by Western blotting and by immunofluorescence microscopy with rabbit anti-human IFITM3 antibody (catalog number PA5-11274; Thermo Fisher Scientific Inc.) at 72 h posttransfection.

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Immunofluorescence microscopy and confocal imaging. Images were captured with a Zeiss Axioskop 2 fluorescence microscope with a 20⫻ or 40⫻ objective lens or an LSM 710 Zeiss inverted confocal microscope with a 40⫻, 63⫻, or 100⫻ oil objective lens and processed using Zen 2010 (Zeiss) and Adobe Photoshop (Adobe Inc.). The colocalization coefficient was analyzed with Zen 2010 and calculated based on the formula described by Manders et al. (34) from an average of at least 10 individual cells. For colocalization studies, the following reagents were used: rabbit anti-EEA1 antibody (Cell Signaling), CellLight Rab7-green fluorescent protein (GFP) (ThermoFisher Scientific), rabbit anti-human CD107a (LAMP-1) and anti-human CD63 antibodies (BD Biosciences), and mouse anti-EEA1 and LAMP-1 from Santa Cruz Biotechnology Inc. Western blotting. Human endothelial cells and epithelial cells from either cell lines or primary culture were grown to confluence in T-75 flasks. The cells were trypsinized and resuspended at 2 ⫻ 106/ml before being spun down and lysed in 2⫻ Laemmli sample buffer (Bio-Rad) containing 5% ␤-mercaptoethanol. The samples were boiled at 95°C for 5 min before being loaded into a 4 to 15% Mini-Protean Tris-glycine precast gel (Bio-Rad). Rabbit anti-IFITM3 (ThermoFisher Scientific) and anti-␤-actin (Sigma-Aldrich) antibodies were used to detect IFITM3 and actin expression in cell lysates, respectively. Influenza virus pH optimum of fusion. Analysis of the pH threshold of fusion was evaluated by virus-induced hemolysis assay as described by Shelton et al. (35). Briefly, viruses at 128 HA units (HAU) per 50 ␮l or PBS mock control was mixed with 50 ␮l of 1% (vol/vol) turkey red blood cells and incubated at 4°C in a 96-well plate for 1 h; then, the mixtures were pelleted and resuspended in 100 ␮l of fusion buffer (20 mM HEPES, 2 mM CaCl2, 150 mM NaCl, 20 mM citric acid monohydrate-sodium citrate tribasic dehydrate) with various pH values (from 4.8 to 7.4) at 37°C for 1 h to trigger hemolysis mediated by viral fusion activity. The released hemoglobin was measured as the optical density at 405 nm (OD405). The OD value from mock PBS control at each pH value was subtracted to calculate hemolysis. The maximal hemolysis at a given pH was normalized to 100%, and the hemolysis at other pH values was expressed as a percentage of the maximal hemolysis.

RESULTS

A group of selected avian influenza viruses exhibited extended tropism in human pulmonary endothelial cells. It has been documented previously that human influenza viruses of the H1N1 and H3N2 subtypes possess low infectivity in human pulmonary endothelial cells, whereas HPAI H5N1 viruses are able to infect and replicate efficiently in these cells (17–19). In order to further examine the tropism of influenza viruses in pulmonary endothelial cells, we infected HULEC with human or avian influenza viruses for 8 h; viral infectivity was measured by NP expression and examined by immunofluorescence microscopy. Different subtypes were used for HULEC infection, and parallel viral infections in human lung epithelial A549 cells, which are highly susceptible to human and avian influenza viruses, were included. The amount of virus corresponding to an MOI of 1 to 5, which produced 80 to 95% infectivity in A549 cells, was chosen to infect HULEC, and virus infectivity in both cell types was determined (Fig. 1A). To directly compare viral infectivity among different viruses, viral infection efficiency was expressed as the ratio of the percentage of NP-positive HULEC versus that of NP-positive permissive A549 cells. As shown in Fig. 1B, human influenza viruses of H1 and H3 subtypes exhibited very limited viral infection rates in HULEC with an infection ratio below 0.1 compared to viral infection rates in A549 cells. However, unlike human influenza viruses, the avian influenza viruses, VN/04 (HPAI H5N1), Anhui/1 (LPAI H7N9), and CK/11 (LPAI H9N2), could efficiently infect HULEC at an infection ratio of 0.65 or greater. Interestingly, the H7 viruses

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FIG 1 Influenza virus infectivity in HULEC. Influenza viruses at an MOI of 1 to 5, i.e., seasonal PR8 (H1N1), Bris/07 (H1N1), Pan/99 (H3N2) and 2009 pandemic Mex/09 (H1N1), VN/04 (H5N1), A/Anhui/1 (H7N9), CK/11 (H9N2), NL/03 (H7N7), Shov/07 (H7N9), and Anhui:PR8 and VN:PR8, were used to infect HULEC and A549 cells for 8 h, and viral infectivity was quantified based on the percentage of NP-positive cells by immunofluorescence microscopy (counting at least 200 cells per infection). (A) Viral infectivity in HULEC and A549 cells. (B) Ratios of viral infectivity in HULEC versus A549 cells. The error bars represent the standard deviations (SD) of the mean from three independent experiments. Statistical analysis was performed to compare the ratio of viral infectivity in HULEC versus A549 cells between human and avian influenza viruses by an unpaired t test with Welch’s correction.

NL/03 (HPAI H7N7) and Shov/07 (LPAI H7N9), the latter of which shares high sequence homology with the HA and NA genes from Anhui/1 virus, showed only intermediate infectivity in HULEC, as their infectivity in these cells was approximately 35 to 40% of that observed in A549 cells (Fig. 1). To better understand which viral genes are involved in the extended tropism of avian influenza viruses in HULEC, recombinant PR8 viruses harboring the HA and NA genes from Anhui/1 or VN/04 (lacking the polybasic cleavage site) were tested in the viral infection assay. Both recombinant influenza viruses showed enhanced viral infectivity in HULEC compared to PR8 virus, albeit at slightly lower infection rates than wild-type (wt) Anhui/1 or VN/04 virus (Fig. 1). This suggests that the HA and NA genes represent the main determinants of influenza virus tropism in HULEC. Taken together, our results show that, unlike human H1N1 and H3N2 viruses, avian influenza viruses of the H5, H7, and H9 subtypes have gained extended cellular tropism in human pulmonary endothelial cells in an apparently viral envelope protein-dependent manner. Human influenza viruses can infect endothelial cells by fusing at the cell surface. To elucidate the stage of human influenza virus infection that is blocked in endothelial cells, we first set out to determine whether virus infectivity could be rescued by forcing human influenza viruses to fuse at the endothelial cell surface. For this purpose, viruses were incubated with endothelial cells on ice for 1 h to allow virus binding, and then the cells were exposed to pH 5.0 buffer to force fusion between surface-bound influenza viruses and the host plasma membrane. In this assay approach, if viruses are able to bind and fuse at the cell surface, the viral nucleocapsid will be directly released into the cytosol instead of being transported through endocytic pathways, as occurs during normal viral entry. At 8 h after inducing fusion at the cell surface, viral infectivity in the presence of 30 mM NH4Cl, which was used to block viral entry via the endocytic pathway, was quantified based on NP staining. As a control, viral infection in permissive A549 cells was used to demonstrate that PR8 (H1N1) and Pan/99 (H3N2) viruses could achieve approximately 75% and 95% infectivity, respectively, by inducing fusion at the cell surface upon exposure to pH 5.0, but not pH 7.4, conditions (Fig. 2). Interestingly, although PR8 and Pan/99 viruses showed only very low infectivity in HULEC by the normal viral entry route, the infection

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rates of both viruses in HULEC reached up to 95% upon inducing fusion at the cell surface, comparable to their infectivity in A549 cells under the same conditions (Fig. 2). These findings indicate that human influenza viruses can bind to endothelial cells and fuse at the cell surface in low-pH environments at efficiencies comparable to those in permissive A549 cells, thus providing the means for the viral genome to enter the cytoplasm and initiate replication. Our results from this acid bypass assay confirmed that the infection block to human influenza virus in HULEC occurs downstream of viral binding but upstream of viral replication in the host nucleus. Human influenza viruses can become internalized and initiate hemifusion in endosomes but fail to uncoat during entry into endothelial cells. During influenza virus entry into host cells, the virus first binds to cellular receptors and then becomes internalized by endocytosis into endosomal compartments in which the viral membrane fuses with the endosomal membrane to release the viral contents into the cytosol and initiate replication. The fusion between the influenza virus membrane and host endosomal membrane starts with lipid mixing (hemifusion), which is then followed by fusion pore formation (36). After we determined that the block in human influenza virus infection in endothelial cells occurs downstream of viral binding but upstream of viral replication in the nucleus, we next performed stepwise entry assays to determine the exact step at which the infection is inhibited. First, we examined whether human influenza virus can bind and be internalized by endocytosis in HULEC. For this purpose, we used biotin-labeled PR8 virus in a virus binding and internalization assay as described previously (30). As shown in Fig. 3A (left column), PR8 virus was able to bind to both HULEC and A549 cells by staining with Alexa 488-streptavidin following virus incubation with cells at 4°C for 1 h. This further confirms that the virus has no defect in binding to HULEC compared to A549 cells. As expected, the signal from cell surface-bound virions could be blocked by preincubation with excessive amounts of unconjugated streptavidin (Fig. 3A, second column from left). Upon inducing internalization at 37°C for 1 h, internalized viruses stained with Alexa 488-streptavidin following unconjugated streptavidin preincubation were visible in both HULEC and A549 cells (Fig. 3A, right column), suggesting that PR8 virus can become internalized into HULEC with an efficiency similar to that in A549 cells.

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FIG 2 Human influenza virus infection in HULEC induced by fusing at the cell surface (acid bypass infection). Cell surface-bound PR8 H1N1 and Panama H3N2 viruses at an MOI of 5 were exposed to fusion buffer (pH 5.0 or 7.4) at 37°C for 2 min, after which the cells were cultured for 8 h in the presence of 30 mM NH4Cl to block virus fusion in the endocytic pathway. (Left) Immunofluorescence microscopy (20⫻ objective lens). Cells were stained with anti-NP antibody (green) and DAPI (blue) for nuclei. (Right) Viral infectivity expressed as the mean percentage of NP-positive cells from three independent experiments. The error bars represent SD. Two-way analysis of variance (ANOVA) was done to compare viral infectivity at pH 5.0 and at pH 7.4, ***, P ⬍ 0.001.

As PR8 virus demonstrated efficient binding and internalization into both HULEC and A549 cells, we next investigated if PR8 virus could fuse in the endosomes of endothelial cells. Following previously established dual-wavelength imaging methods to monitor influenza virus fusion (31), we labeled purified PR8 virus with dual lipophilic dyes, R18 (red) and SP-DiOC18 (green). In this assay, lipid mixing between the viral membrane and host endosomal membrane correlates with the fluorescence color shift from red to green due to the release of self-quenching DiOC18 and dissolution of fluorescent resonance energy transfer (FRET) from DiOC18 to R18 in the labeled viruses. As shown in Fig. 3B (top row), upon binding at 4°C, cell-bound viruses could be visualized only in the red channel (R18) in HULEC. Upon inducing internalization for 1.5 h, the green fluorescence signal was increased, indicating that PR8 virus can initiate hemifusion (lipid mixing) in HULEC (Fig. 3B, middle row). As a negative fusion control, virus internalization was induced in the presence of 30 mM NH4Cl to inhibit endosomal acidification; as a result, internalized viruses could no longer fuse in HULEC, as demonstrated by the lack of green signal in Fig. 3B (bottom row). PR8 virus fusion in A549 cells was included as a positive control, with results obtained in

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this cell line generally similar to those observed for HULEC (data not shown). Following confirmation of successful viral lipid mixing in endosomes of endothelial cells, we next monitored uncoating of the nucleocapsid by M1 protein staining with HB64 antibody in the presence of 1 mM cycloheximide to prevent synthesis of new viral proteins (37). The HB64 antibody recognizes the epitope in M1 protein, which becomes more accessible once the M1 protein is dispersed into the cytosol (38). In control A549 cells at 3.5 h postinternalization, PR8 M1 protein displayed bright dispersed cytoplasmic signals, indicating that viruses had undergone uncoating events, releasing M1 proteins into the cytosol (Fig. 3C). In contrast, in HULEC, the M1 protein exhibited a punctate staining pattern, suggesting the M1 protein was confined in vacuole-like compartments and failed to be released into the cytosol. In order to determine the location of M1 proteins in HULEC, we performed colocalization immunostaining with an early endosome marker (EEA1) and a late endosome/lysosome marker (LAMP-1). The overlap coefficient of M1 with EEA1 was 0.34, and it was 0.73 for M1 with LAMP-1 (Fig. 3C), demonstrating that PR8 virus M1 protein was mainly trapped in late endosome/lysosome compart-

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FIG 3 PR8 virus binding, internalization, fusion, and uncoating in HULEC and A549 cells. (A) Biotin-labeled PR8 virus binding and internalization. Cell surfacebound PR8 virus following binding at 4°C for 1 h was visualized by Alexa-488 streptavidin (strep) (left column); the signal from surface-bound PR8 viruses could be blocked by preincubation with unconjugated streptavidin (1 mg/ml) (second column from left). Upon inducing internalization at 37°C for 1 h, the cells were incubated with unconjugated streptavidin to block the signal from surfacebound viruses, and then the cells were fixed and permeabilized before being stained with Alexa-488 streptavidin for internalized PR8 virus visualization (right column). Both cell surface-bound and internalized viruses were shown by Alexa488 streptavidin staining without unconjugated preincubatioin (third column from left). The images were taken under a 63⫻ oil objective lens. (B) Virus-mediated lipid mixing in endosomes. R18 (red) and SP-DiOC18 (green) dual-colorlabeled PR8 viruses induced internalization at 37°C for 1.5 h; the lipid mixing (hemifusion) between the viral membrane and the host endosomal membrane was indicated by increased intensity of green fluorescence due to the release of self-quenching DiOC18 and dissolution of FRET. The images were taken under a 63⫻ oil objective lens. (C) (Top) PR8 virus uncoating. PR8 virus M1 proteins were visualized by immunofluorescence using anti-M1 antibody (HB64) at 3.5 h postinternalization and DAPI (blue) for nuclei (40⫻ objective lens). (Bottom) The PR8 virus M1 proteins (green) in HULEC were colocalized with the early endosome marker EEA1 (red) and the lysosomal marker LAMP-1 (red), and the overlap coefficients (ranging from 0 to 1, with 1 representing perfectly colocalized

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ments during entry into HULEC. Taken together, these results show that the blocking of human influenza virus infection in HULEC is at a posthemifusion step; as a result, virus can no longer uncoat nucleocapsid and release the viral genome into the cytosol. The virus replication restriction factor IFITM3 is constitutively expressed in human endothelial cells but at a significantly lower level in epithelial cells. The infection block in HULEC at a posthemifusion stage prompted us to investigate whether the IFITM3 protein, which has recently been suggested to block influenza virus infection at the same stage (39), plays a similar role in influenza virus infection in endothelial cells. IFITM proteins are generally expressed in cells at a basal level but can be significantly induced by type I and type II IFNs (20). We first examined by Western blotting whether human endothelial cells constitutively express IFITM3 without interferon induction. As shown in Fig. 4A, cell lysates from endothelial cells, including HULEC, HMVEC, and HUVEC, showed high levels of endogenous IFITM3 protein expression; HBVEC expressed a much lower level of IFITM3 protein. Compared to endothelial cells, human lung epithelial cells, including A549 and Calu-3 cells, failed to display IFITM3 protein expression under the same detection conditions, and NHBE cells had only marginal IFITM3 protein expression compared to that in HULEC. Using immunofluorescence microscopy, we next examined the locations of endogenous IFITM3 proteins in HULEC. In this cell type, IFITM3 showed a punctate staining pattern close to nuclear regions. In a colocalization study, we found that the overlap coefficient between IFITM3 and the early endosome marker EEA1 was 0.76, and it was 0.67 between IFITM3 and the late endosome/ lysosome marker LAMP-1, indicating endogenous IFITM3 is mainly located in endosomal compartments (Fig. 4B). Taken together, our results show that, unlike most respiratory epithelial cells, human pulmonary endothelial cells constitutively express high levels of IFITM3 in endosomes, which may potentially restrict influenza virus infection. IFITM3 knockdown by siRNA in HULEC partially enhances human influenza virus infection. To determine whether constitutively expressed IFITM3 proteins are involved in the restriction of influenza virus infection in HULEC, we used silencing RNA (siRNA) to knock down IFITM3 expression in HULEC prior to virus infection. As shown in Fig. 5A, 100 pmol of IFITM3 siRNA could significantly downregulate IFITM3 expression, as demonstrated by Western blotting (Fig. 5A). Following viral infection with PR8 virus, IFITM3 siRNA-transfected cells showed significantly enhanced viral infectivity (P ⬍ 0.001), with approximately 12-fold (21% versus 1.7%) and 8-fold (47% versus 5.8%) increases compared to cells transfected with negative-control siRNA at MOI of 0.25 and 1, respectively (Fig. 5B). However, knocking down IFITM3 expression in HULEC had a less significant effect on Anhui:PR8, as Anhui:PR8 virus infectivity increased from 28% to 35% at an MOI of 0.25 and increased from 46% to 56% at an MOI of 1 (Fig. 5B). The different restriction effects of endogenously expressed IFITM3 in HULEC on human and avian influenza viruses seem to be cell type specific. Thus, IFITM3 proteins

pixels) of the green and red signals were calculated with the program Zen from Zeiss and are marked in the right corners of the images. The images were taken with an LSM 710 Zeiss inverted confocal microscope under a 40⫻ objective lens with digital zoom 2. The scale bars represent 10 ␮m.

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FIG 4 Constitutive IFITM3 expression in human endothelial cells. (A) IFITM3 expression in endothelial cell lysates (HULEC, HMVEC, HUVEC, and HBVEC) and epithelial cell lysates (Calu-3, A549, and NHBE cells) was detected by anti-IFITM3 antibody in a Western blot assay; actin expression monitored by anti-␤-actin antibody blotting was used as an internal loading control. Actin and IFITM3 protein expression levels in the Western blot were quantified with a ChemiDoc MP system with Image Lab software. The relative IFITM3 protein expression was plotted as the mean ratio of the intensity of the IFITM3 signal to that of the actin internal control from three independent experiments. (B) Localization of IFITM3 in HULEC. The endogenous IFITM3 proteins (red) in HULEC were colocalized with the early endosome marker EEA1 (green) and the lysosomal marker LAMP-1 (green), and the overlap coefficients of the green and red signals were calculated with the program Zen from Zeiss and marked in the right corners of the images. DAPI (blue) was used to stain nuclei. 63⫻ oil objective lenses were used in microscopy.

overexpressed in A549 cells exhibited similar degrees of inhibition of all the influenza viruses we tested (Fig. 5C), which was consistent with the results of a previous study (22). Our results demonstrate that constitutively expressed IFITM3 proteins are able to selectively restrict human influenza virus infection in endothelial cells but have a less potent effect on certain avian influenza viruses.

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A higher virus fusion threshold is not associated with escape from IFITM3 restriction. Thus far, we have shown that human influenza virus entry into endothelial cells is blocked at a posthemifusion step, partially due to the constitutive expression of IFITM3. However, certain avian influenza viruses, including HPAI H5N1, LPAI H9N2, and novel LPAI H7N9 viruses, show significantly higher infection rates than H1N1 and H3N2 human

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FIG 6 pH fusion thresholds for influenza viruses. Influenza viruses at 128 HAU were incubated with 1% turkey erythrocytes at 4°C for 1 h before fusion was induced with 100 ␮l of fusion buffer at pHs ranging from 4.8 to 7.4. The hemolysis was measured as the OD405 from duplicate samples, and the maximal hemolysis was normalized to 100%. The hemolysis efficiencies at various pHs were expressed as percentages of the maximal hemolysis. The graph represents the average hemolysis efficiencies from three independent experiments. The pH thresholds for fusion (the pH range at which 50% of maximal hemolysis was achieved) are shown in the table. Viral infectivities in HULEC were classified as high, low, or intermediate based on the viral infection rates shown in Fig. 1.

FIG 5 Constitutively expressed IFITM3 proteins restrict PR8 virus infection in HULEC. (A) Different amounts (12.5 to 100 pmol) of IFITM3 siRNA and negative-control siRNA were transfected in HULEC, and the expression of IFITM3 was examined at 72 h posttransfection by Western blotting with antiIFITM3 antibody; anti-actin antibody was included as an internal loading control. (B) HULEC transfected with 100 pmol of IFITM3 siRNA for 72 h were infected with PR8 or Anhui:PR8 virus at MOI of 0.25 and 1 for 8 h before viral infectivity was quantified based on NP staining by immunofluorescence microscopy. The graph represents the mean infectivities of three independent experiments, with the error bars showing SD. Two-way ANOVA statistical analysis was performed to compare viral infectivities between IFITM3 siRNA and negative-control siRNA groups; ***, P ⬍ 0.001; *, P ⬍ 0.05. (C) A549 cells stably expressing IFITM3 or vector pQCXIP were infected with the indicated influenza viruses at an MOI of approximately 1, and viral infectivity at 8 h postinfection was quantified by NP-positive cells in immunofluorescence microscopy. Shown are the means of three independent experiments with SD. Two-way ANOVA statistical analysis was performed to compare viral infectivities in IFITM3 and vector-expressing A549 cells.

influenza viruses in endothelial cells. One hypothesis for how avian influenza viruses circumvent this infection block is that avian influenza viruses can fuse in early endosomes at relatively high pH values before traveling to late endosomes, where IFITM3 proteins have been suggested to be located and to restrict influ-

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enza virus entry. To test this possibility, we used a virus-mediated hemolysis assay to compare viral fusion pHs for selected human and avian influenza viruses. As shown in Fig. 6, human H1N1 influenza viruses, including PR8, Bris/09, and Mex/09, preferentially fused at a relatively low pH (⬍5.3). In contrast, the viruses that displayed the highest infectivity in HULEC (Anhui/13 [H7N9] and VN/04 [H5N1] viruses) had a fusion threshold of approximately pH 5.8 (Fig. 6). The H7 viruses NL/03 (H7N7) and Shov/07 (H7N9), which showed intermediate infection rates in HULEC, possessed slightly lower pH fusion thresholds (pH 5.5) than the Anhui/13 (H7N9) and VN/04 (H5N1) viruses. However, human Pan/99 (H3N2) virus shared a similar pH fusion profile with VN/04 virus despite the different infection rates of these two viruses in HULEC (Fig. 1 and 5). Similarly, CK/11 (H9N2) virus had a pH fusion range similar to that of human H1N1 viruses but was able to infect HULEC with much higher efficiency than H1N1 virus. Our results suggest that although having a relatively high fusion threshold might prompt some avian influenza viruses, like Anhui/1 and VN/04, to fuse early before reaching late endosomes to avoid IFITM3 restriction, other avian influenza viruses, such as CK/11 virus, may explore other, unknown routes to escape this restriction in endothelial cells.

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DISCUSSION

Human pulmonary cells constitute approximately 30% of the total cell population in the lung, providing the lining for the network of capillaries in the alveolus (40). Recently, pulmonary endothelium dysfunction caused by cytokines released from alveolar epithelial cells and leukocytes has been suggested to play an important role in virus-induced lung damage in severe cases of influenza virus infection (41). Additionally, given the close proximity of the pulmonary endothelium and alveolar epithelium, endothelial cells exposed to influenza virus particles released from infected neighboring epithelial cells may lead to cell death, increased endothelial permeability, and vascular destruction, further contributing to epithelial-endothelial barrier damage (41). However, previous studies in vitro have shown that pulmonary endothelial cells are not susceptible to infection with most human influenza viruses, as only HPAI H5N1 viruses were able to replicate efficiently and induce excessive production of proinflammatory cytokines (17, 19). Here, we extend previous work on influenza virus tropism in human pulmonary endothelial cells by identifying cellular and viral factors that contribute to viral infection in this cell type. We showed that human pulmonary endothelial HULEC are less permissive to human influenza virus infection than a subset of avian influenza viruses, including highly pathogenic H5N1 (VN/ 04), LPAI H7N9, and H9N2 viruses. Despite limited expression of ␣-2,6-linked sialic acid residues (17), the receptor for human influenza viruses, these viruses are still able to bind to endothelial cells with efficiency comparable to that of binding on epithelial cells. This was shown not only by the biotin-labeled-virus binding assay (Fig. 3A), but also by an acid bypass infection assay (Fig. 2). We further showed that human PR8 virus could be internalized and successfully initiate hemifusion in endothelial cells but could not uncoat and release the viral genome into the cytosol to initiate subsequent replication processes. Although human influenza virus infection is blocked at the early stages of viral entry in endothelial cells, this does not necessarily mean that the pulmonary endothelium cannot be activated through direct contact with human influenza viruses. It has been previously reported that a UVinactivated, replication-deficient human seasonal H3N2 virus was capable of causing a significant reduction in endothelial permeability by inducing degradation of the tight-junction protein Claudin 5. This was accomplished without causing cytotoxic effects on the endothelium, and binding of the virus alone was not sufficient to induce permeability changes (14). We postulate that host pattern recognition receptors (PRR) residing in host endocytic pathways, such as Toll-like receptor 7 (TLR7) or TLR8 in endosomes, might be involved in recognizing viral components and activating signal transductions in endothelial cells in a viral-replication-independent manner (42). Despite the fact that avian influenza viruses, such as HPAI H5N1 and LPAI H7N9, were able to replicate efficiently in endothelial cells and to induce strong cytokine production in vitro, further studies are needed to elucidate their exact role in viral pathogenesis in vivo, as postmortem analysis of patients who succumbed to H5N1 infection rarely showed endotheliotropism (6). In this study, we revealed that, unlike human bronchial epithelial Calu-3 and alveolar basal epithelium-derived A549 cells, human primary (HMVEC) or transformed (HULEC) pulmonary endothelial cells, as well as HUVEC, constitutively express high levels of IFITM3 without IFN induction, whereas NHBE cells

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showed only intermediate levels of IFITM3 expression. Although comprehensive immunohistochemical staining for IFITM3 expression in human lung tissues is still lacking, various levels of endogenous IFITM3 expression in mouse respiratory epithelial cells and pulmonary endothelial cells have been revealed (25). This suggests that endogenous expression of IFITM3 may be tissue or cell type specific; accordingly, higher endogenous expression of IFITM3 might provide a certain degree of advantage in protecting the host from influenza virus infection prior to interferon production. Although the antiviral activity of IFITM3 has been well established, the exact mechanism by which IFITM3 restricts viral infection is not yet fully understood. Early evidence suggests that IFITM3 restricts viral-membrane hemifusion, possibly by affecting the membrane’s molecular order and fluidity (43) or by modulating cholesterol homeostasis in the endosome by interacting with VAPA (vesicle-associated membrane proteinassociated protein A), resulting in inhibition of viral fusion (44). However, it was subsequently found that IFITM3 restricts viral infection by blocking the formation of fusion pores following virus-endosome hemifusion, possibly by modulating endosomalmembrane rigidity (39). Furthermore, IFITM3 subcellular localizations, which can be modulated by protein posttranslational modification, has proven to be important for its antiviral activities, and depending on the cell types and stimulation status, IFITM3 may localize differently in cells (45). In unstimulated WI-38 human primary fibroblasts, the majority of IFITM3 resides in the ER and becomes distributed in a vesicular pattern throughout the cell upon IFN exposure (22). When overexpressed in A549 cells, IFITM3 predominantly localizes in enlarged compartments shared with markers of endosomes and lysosomes (23). In our study, we showed that the constitutively expressed IFITM3 in HULEC mainly localized in endosomal and lysosomal compartments, as indicated by their colocalization with the early endosome and late endosome/lysosome markers EEA1 and LAMP-1, respectively, suggesting that endogenous IFITM3 proteins in HULEC already reside in the compartments involved in the influenza virus entry pathway and might be intrinsically primed for blocking influenza virus infection. However, we did not observe IFITM3-enriched large compartments, as previously revealed in overexpressed or IFN-induced A549 cells; whether this reflects a difference between cell types or between endogenously expressed versus overexpressed IFITM3 requires further investigation (39). In our study, we demonstrated that PR8 viral infection in HULEC was blocked after a hemifusion step. This suggests a potential role for endosome-residing constitutively expressed IFITM3 proteins in this block, based on our findings that knocking down IFITM3 expression with siRNA significantly improved PR8 virus infectivity in HULEC. However, the improved infectivity in HULEC upon IFITM3 siRNA treatment is still lower than that in A549 cells, suggesting a role for other restriction factors in endothelial cells. Moreover, a subset of avian influenza viruses from the H5, H7, and H9 subtypes seemed to be able to partially escape this IFITM3 restriction block in endothelial cells, as IFITM3 siRNA knockdown had only a moderate effect on improving the infectivity of a recombinant PR8 virus bearing the Anhui/1 H7N9 HA and NA. Future studies exploring whether interferon-induced IFITM3 in pulmonary endothelial cells, which may have different subcellular locations, has an effect on avian influenza virus infection similar to that of constitutively expressed IFITM3 are warranted. Most influenza viruses have a pH fusion range of 5.0 to 5.5,

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which matches the pH in the late endosome (46). However, certain influenza viruses, such as the novel H7N9 virus Anhui/1, have a higher pH threshold (pH 5.8) for fusion; such viruses may be able to fuse in early endosomes, which have pH values ranging from 5.4 to 6.2, depending on the cell type (47, 48). IFITM3 is mostly located in late endosomes or lysosomes when overexpressed or upon stimulation by IFN (23, 49) and has less inhibitory effects on viruses, such as vesicular stomatitis virus (VSV), that preferentially fuse at early endosomes or late endosome/multivesicular bodies (50); therefore, IFITM3 might be less potent in restricting influenza viruses, which can fuse in early endosomes. In our study, we did observe that VN/04 H5N1 and Anhui/13 H7N9 viruses, which showed efficient viral replication in HULEC, had a higher pH threshold (pH ⬎ 5.5) for fusion, indicating that the viruses could potentially escape IFITM3 restriction by fusing early, before reaching late endosomes. However, CK/11 H9N2 virus was able to achieve high infectivity in HULEC despite having a low pH fusion threshold, suggesting that certain influenza viruses may have developed different strategies to counteract IFITM3 restriction in endothelial cells. Altogether, we demonstrated that human influenza virus infection in human pulmonary endothelial cells is less efficient than that of selected strains of H5, H7, and H9 avian influenza viruses, partially due to the high constitutive expression of IFITM3 in late endosomes and lysosomes, which may block the procession from hemifusion to fusion pore formation during the viral entry process. In comparison, certain avian influenza viruses were able to escape the restriction in endothelial cells by fusing early in the endocytic pathway at a higher pH value or by other, unknown mechanisms. However, we cannot rule out the possibility that other, unidentified restriction factors may exist in human pulmonary endothelial cells that confer their resistance to influenza virus infection. An additional factor to be mentioned is the binding preference of the laboratory-adapted human influenza PR8 virus, with ␣-2,6- and ␣-2,3-linked sialic acid dual receptor binding specificity. This virus was able to bind endothelial cells (followed by internalization of the virus), but whether PR8 viruses are transported into the same endocytic compartments as avian influenza viruses, which preferentially bind to ␣-2,3-linked sialic acids, is unknown. Previously, it had been suggested that HPAI H5N1 influenza viruses with different receptor binding specificities might be sensed or recognized differently in human monocyte-derived macrophages and dendritic cells, subsequently leading to distinct signaling cascades (51). Whether influenza virus receptor specificity is involved in human pulmonary endothelial cell restriction to human influenza viruses requires further study.

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ACKNOWLEDGMENTS The findings and conclusions in this report are ours and do not necessarily represent the official position of the Centers for Disease Control and Prevention. X. Sun was supported by the Oak Ridge Institute for Science and Education.

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FUNDING INFORMATION This work, including the efforts of Xiangjie Sun, was funded by HHS | Centers for Disease Control and Prevention (CDC). 18.

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