Application of Novel Sialoglyco-Particulates ... - ACS Publications

Article Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Application of Novel Sialoglyco Particulates Enhances the Detection Sensitivity of the Equine Influenza Virus by Real-Time Reverse Transcriptase Polymerase Chain Reaction Makoto Ogata,*,† Takashi Yamanaka,‡ Ami Koizumi,† Mao Sakamoto,† Rena Aita,† Hiroyuki Endo,† Takehiro Yachi,† Noriko Yamauchi,§ Tadamune Otsubo,∥ Kiyoshi Ikeda,∥ Tatsuya Kato,⊥ Enoch Y. Park,⊥ Hiroyuki Kono,¶ Manabu Nemoto,‡ and Kazuya I. P. J. Hidari*,△

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Department of Applied Chemistry and Biochemistry, National Institute of Technology, Fukushima College, 30 Nagao, Iwaki, Fukushima 970-8034, Japan ‡ Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke, Tochigi 329-0412, Japan § Department of Materials Science and Engineering, Graduate School of Science and Engineering, Ibaraki University, 4-12-1 Naka-narusawa-cho, Hitachi, Ibaraki 316-8511, Japan ∥ Department of Organic Chemistry, School of Pharmaceutical Sciences, Hiroshima International University, Kure-shi, Hiroshima, Japan ⊥ Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan ¶ Division of Applied Chemistry and Biochemistry, National Institute of Technology, Tomakomai College, Nishikioka 443, Tomakomai, Hokkaido 059-1275, Japan △ Department of Food and Nutrition, Junior College Division, University of Aizu, 1-1 Aza-Kadota, Yahata, Ikki-machi, Aizuwakamatsu, Fukushima 965-8570, Japan S Supporting Information *

ABSTRACT: Sialoglyco particulates carrying an N-glycolylneuraminyl-α-(2 → 3)-N-acetyllactosamine (Neu5Gcα2,3LacNAc) residue that displays a high level of affinity for the equine influenza virus (EIV) were generated using sialoglycopolypeptide and hexyl-containing hybrid silica particulates. The particulates were spherical with a diameter of approximately 950 nm and found to have good dispersibility in aqueous solution. Interaction between the sialoglyco particulates and the EIV was investigated by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) of the EIV genome captured on the particulates. The number of EIV-specific genes detected by rRT-PCR on a nasal swab obtained from infected horses clearly increased when the sample was treated with sialoglyco particulates. Our results show these novel sialoglyco particulates can be used as a highly sensitive tool for detecting low levels of EIV that were previously undetectable in the early or late stage of infection. KEYWORDS: carbohydrate, equine influenza virus, multivalency, particulate, sensitive detection

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swab.3,7 However, due to the rapid transmission of EIV from horse to horse, these tests are ineffective in preventing an outbreak of equine influenza. Thus, a quick and robust test to detect EIV from horse specimens, such as nasal swabs, is required to avoid an epidemic. Detection of equine influenza is currently carried out using rapid diagnostic kits that utilize an immunochromatography approach, but these tests have a low level of sensitivity.8 An rRT-PCR method with enhanced detection sensitivity has also been used for EIV diagnosis.9,10

INTRODUCTION

Equine influenza virus (EIV), classified as a strain of influenza A virus, is the causative agent of equine influenza, an acute respiratory disease of horses.1−3 Transmission of the virus occurs by inhalation of aerosols or direct contact with fomites, such as inanimate objects or people moving between infected and uninfected horses. The precise mode of infection is caused by the binding of EIV-hemagglutinins (EIV-HA) to sialoglyco receptors expressed in horses.3−6 Equine influenza, which is highly contagious, shows clinical symptoms that are similar to other respiratory diseases.1 An infection caused by equine influenza is confirmed by measuring the induction of serum antibody specific for EIV and isolating the virus from a nasal © XXXX American Chemical Society

Received: December 16, 2018 Accepted: January 27, 2019 Published: January 28, 2019 A

DOI: 10.1021/acsabm.8b00813 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

Article

ACS Applied Bio Materials Table 1. Characterization of Artificial Sialo-glycopolypeptides DS (%)a compounds

Neu5Gcα2,3LacNAc-

decyl-

kDab

poly[Neu5Gcα2,3LacNAc-β-O(CH2)5NH-/γ-PGA] (1) poly[Neu5Gcα2,3LacNAc-β-O(CH2)5NH-/CH3(CH2)9NH-/γ-PGA] (2)

21 21

0 44

2000 2340

Degree of substitution (DS) of Neu5Gcα2,3LacNAc derivatives with/without decyl groups based on the DP of glutamic acid residues of γ-PGA (set as 100%). Calculated from 1H NMR data at 45 °C. bCalculated kDa. a

methyl protons of 1-aminodecane. Poly[Neu5Gcα2,3LacNAc-β-O(CH2)5NH-[21%]/CH3(CH2)9NH-[44%]/γ-PGA] 2 was prepared in the following manner. Poly[Neu5Gcα2,3LacNAc-β-O(CH2)5NH-[21%]/γPGA] (1, 9.2 mg, average MW: 2 000 000) was first dissolved in 1.66 mL of 0.1 M carbonate/bicarbonate buffer (pH 10.0). Next, BOP (142 mg, 0.32 mmol) and HOBt (11.9 mg, 0.09 mmol) in dimethyl sulfoxide (DMSO, 3.3 mL) were added, and the resulting mixture was stirred at 25 °C for 15 min. 1-Aminodecane (11.7 μL, 0.06 mmol) was then added, and the mixture was incubated for a further 24 h at the same temperature with stirring. The reaction solution was loaded onto a disposable PD-10 Desalting Column (GE Healthcare, UK) that was pre-equilibrated with 10 mM phosphate-buffered saline (PBS) at pH 7.4. The target fraction was subsequently dialyzed for 3 days and then lyophilized after concentration to yield compound 2 (7.6 mg) (refer to Table 1, Figures S3 and S4). 1H NMR (D2O, 500.13 MHz at 318 K): δ 4.55−4.13 (1H, α-methine, γ-PGA), 4.19 (s, 2H, HOCH2CONH-″), 4.08−3.57 (21H, from sugar, H-α), 3.35−3.05 (H-ε, H-j), 2.85 (dd, 1H, J3″ax,3″eq 12.5, J3″eq,4″ 4.5 Hz, H-3″eq), 2.45 (2H, γ-methylene, γPGA), 2.38−1.98 (2H, β-methylene, γ-PGA), 2.09 (s, 3H, CH3CONH−), 1.88 (t, 1H, J3″ax,3″eq 12.5, J3″ax,4″ 12.5 Hz, H-3″ax), 1.55−1.00 (22H, H-β, H-γ, H-δ, H-b, H-c, H-d, H-e, H-f, H-g, H-h, Hi), 0.90 (3H, H-a). 13C NMR (D2O, 125.13 MHz at 318 K): δ 177−176 (carbonyl carbon of amide groups), 175.7 (HOOC″-), 105.5 (C-1′), 103.8 (C-1), 102.7 (C-2″), 81.6 (C-4), 78.3 (C-5′), 77.9 (C-3′), 77.5 (C-5), 75.4 (C-6″), 75.2 (C-3), 74.5 (C-8″), 72.9 (C-α), 72.2 (C-2′), 71.0 and 70.8 (C-4″, C-7″), 70.2 (C-4′), 65.4 (C-9″), 63.7 (C-6′, HOCH2CONH’’-), 63.0 (C-6), 57.9 (C-2), 56.5 and 53.6 (α-methine, γ-PGA), 54.3 (C-5″), 42.5 (C-3″), 42.1 (C-ε, C-j), 34.5 (C-c), 34.1 (γmethylene, γ-PGA), 32.3 and 32.0 (C-d, C-e, C-f, C-g, C-i), 31.0 (C-β, C-δ), 30.0 (β-methylene, γ-PGA), 29.7 (C-h), 25.2 (C-γ, C-b, CH3CONH−), 16.4 (C-a). Poly[Lac-β-NHCO(CH2)5NH-[21%]/ CH3(CH2)9NH-[32%]/γ-PGA] was prepared in a similar manner (Table S1 and Figure S5). Preparation of Sialoglyco Particulates with Poly[Neu5Gcα2,3LacNAc-β-O(CH 2 ) 5 NH-/CH 3 (CH 2 ) 9 NH-/γ-PGA]. Poly[Neu5Gcα2,3LacNAc-β-O(CH2)5NH-[21%]/ CH3(CH2)9NH-[44%]/γ-PGA] 2 immobilized particulate (Neu5Gcα2,3LacNAc particulate) was prepared as follows. To a solution of 0.13 μM poly[Neu5Gcα2,3LacNAc-β-O(CH 2 ) 5 NH- [ 2 1 % ] / CH3(CH2)9NH-[44%]/γ-PGA] in 30% DMSO solution of 99.9% pure DMSO with 70% distilled water (48.8 mL) was added a silica particulate (1.2 g), whose surface was made more hydrophobic by introduction of hexyl groups. The mixture was shaken at room temperature for 24 h. The particulate was extensively washed (5×) in distilled water and then blocked with 48.8 mL of 2% bovine serum albumin (BSA) solution at room temperature for 2 h. After washing as before the resulting sialoglyco particulate 3 (0.6 g) was suspended in distilled water and stored at 4 °C. Likewise, the multivalent Lac-carrying particulate was prepared from poly[Lac-β-NHCO(CH2)5NH-[21%]/ CH3(CH2)9NH-[32%]/γ-PGA] using the same protocol. Multivalent Neu5Gcα2,3LacNAc-Carrying Particulate Characterization. The particulate size in dispersion was measured using dynamic light scattering (DLS, Zetasizer Nano ZSP, Malvern, UK) in distilled water at 25 °C. Samples were prepared by dispersing the sialoglyco particulate 3 (1 mg) in water (2 mL) by ultrasonication for 1 min. Scanning electron microscopy (SEM, S-3400N, Hitachi High Technologies Co., Tokyo, Japan) was used for the observation of the glyco-particulate samples. Lectin Binding Assay. The multivalent Neu5Gcα2,3LacNAccarrying particulate (3, 1 mg/10 μL in distilled water) was added to 6.3

Nonetheless, detecting the low titer of virus during the early stage of EIV infection is still a major issue. Yamanaka et al. reported interspecies transmission of the H3N8 influenza A virus between horses and dogs.11 The study established that the binding of canine H3N8 influenza A virus to N-glycolylneuraminyl-α-(2 → 3)-galactose (Neu5Gcα2,3Gal) is important for the proliferation and pathogenicity of this virus in horses. Based on these findings, we recently developed a novel multivalent N-glycolylneuraminic acid (Neu5Gc) probe with good binding affinity against Neu5Gc-favoring influenza viral hemagglutinin (HA), such as EIV.12 This multivalent Neu5Gc probe comprises sialoglycopolypeptides containing multivalent Neu5Gcα2,3Galβ1,4GlcNAc (Neu5Gcα2,3LacNAc) moieties with a γ-polyglutamic acid (γ-PGA) backbone. In this study, we synthesized sialoglyco particulates utilizing sialoglycopolypeptide-containing multivalent Neu5Gcα2,3LacNAc moieties, which can act as a selective viral adsorbent for EIV. Efficient adsorption and concentration of EIV was achieved by preparing sialoglyco particulates using sialoglycopolypeptide with high affinity for the virus HA. We concluded that combining synthetic sialoglyco particulates and conventional virus detection techniques, such as rRT-PCR, can improve detection sensitivity even for difficult to detect viruses such as EIV.

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MATERIALS AND METHODS

Materials. γ-PGA-Na {average molecular weight (MW) of 990 000; degree of polymerization (DP) of 6557} from Bacillus subtilis was supplied by Meiji Food Materia Co., Ltd. (Tokyo, Japan). Hexylcontaining hybrid silica particulates,13 poly[Neu5Gcα2,3LacNAc-βO(CH2)5NH-/γ-PGA] (1, Neu5Gcα2,3LacNAc: 21%, kDa: 2000, Figure S1)12,14 and poly[Lac-β-NHCO(CH2)5NH-/γ-PGA] (Lac: 21%, kDa: 1550, Figure S2),15 were prepared by previously reported methods. Maackia amurensis agglutinin (MAA), fluorescein isothiocyanate conjugated MAA (FITC-MAA), Sambucus sieboldiana agglutinin (SSA), and Erythrina crista-galli agglutinin (ECA) were purchased from Cosmo Bio Co. Ltd. (Tokyo, Japan) or EY Laboratories Inc. (San Mateo, CA). Other chemicals were obtained from commercial sources. Analytical Methods. Water-suppressed 1H and 13C NMR spectra were recorded on a 2-channel 500 MHz Bruker AVIII spectrometer (Bruker BioSpin, GmbH, Bremen, Germany) operating at 500.13 and 125.13 MHz for 1H and 13C experiments, respectively. The spectrometer was equipped with a 5 mm z-gradient dual-resonance BBFO probe maintained at 318 K. The water-suppressed 1H NMR spectra were recorded by setting the 1H flip angle, repetition time, and number of scans to 30°, 4 s, and 256, respectively. 13C NMR spectra were obtained using a 13C flip angle, repetition time, and scan number of 30°, 1.5 s, and 184 320, respectively. Synthesis of Poly[Neu5Gcα2,3LacNAc-β-O(CH 2 ) 5 NH-/ CH3(CH2)9NH-/γ-PGA]. The primary amino group of 1-aminodecane was used to covalently attach the remaining α-carboxy groups of glutamic acid residues of compound 1 via conventional benzotriazol-1yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP)/ 1-hydroxybenzotriazole hydrate (HOBt) chemistry. The resulting ratio of substitutions of 1-aminodecane with glutamic acid residues of 1 was expressed as a percentage based on the relative intensities of the proton NMR signal areas of H-3″ methylene protons of Neu5Gc and terminal B


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Figure 1. Preparation of poly[Neu5Gcα2,3LacNAc-β-O(CH2)5NH-/CH3(CH2)9NH-/γ-PGA]-immobilized particulate. ̈ one-year-old horses by Briefly, EIV was used to infect four naive 7.9 inhalation (10 50% egg infectious dose/horse) on Day 0. Clinical samples were subsequently collected on a daily basis from 0 to 9 days. Virus Adsorption Using Neu5Gcα2,3LacNAc Particulate, Viral RNA Extraction, and rRT-PCR. Neu5Gcα2,3LacNAc particulate (3, 1 mg/10 μL in distilled water) was added to clinical samples (1400 μL) in a microtube and mixed by inversion. After incubation for 30 min at 4 °C, each suspension was centrifuged at 5500g for 5 min and the supernatant removed. The resulting precipitate was then suspended in PBS (140 μL). The copy numbers of EIV were determined by viral RNA extraction from 140 μL of either nontreated or treated suspensions using a QIAamp Viral RNA Mini Kit (QIAGEN, Tokyo, Japan) in accordance with the manufacturer’s protocol. Extracted viral RNA was eluted in a final volume of 60 μL. A 2 μL aliquot of the eluate was used as template for rRT-PCR with primers designed against the EIV-HA gene as described in previous reports.8,17 A cutoff value was set at ≤10 copies/reaction/2 μL of template.

μM MAA, SSA, and ECA (10 μL in 10 mM PBS), respectively, in a microtube. After incubation for 30 min at 4 °C, each suspension was centrifuged at 8000g for 2 min. The supernatants were analyzed by measuring the absorbance at 280 nm using a Thermo Scientific NanoDrop 2000 spectrophotometer (Wilmington, DE, USA). The amount of soluble lectin was determined by reference to a standard curve. Furthermore, interaction of 3 and FITC-labeled lectin (FITCMAA) was also analyzed. The multivalent Neu5Gcα2,3LacNAccarrying particulate (3, 1 mg/10 μL in distilled water) was added to 4 μM FITC-MAA (10 μL in 10 mM PBS) in a microtube. After incubation for 30 min at 4 °C, the suspension was centrifuged at 8000g for 2 min. The precipitated particles were washed (5×) in 10 mM PBS. Finally, the sample (0.1 mg/150 μL in 10 mM PBS) was observed using a FluoView confocal laser microscope (Fluoview FV10i, Olympus Co., Tokyo, Japan). The fluorochromes were analyzed in the green channel. Viruses. The EIV strain (A/equine/Malaysia/M201/2015) utilized in this study was propagated as described previously.8,16 Clinical Samples Generated from Experimentally Infected Horses. Virus challenge against horses, collection of clinical samples, and virus isolation were performed as described in previous reports.8,16 C


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Figure 2. Synthesis of poly[Neu5Gcα2,3LacNAc-β-O(CH2)5NH-/CH3(CH2)9NH-/γ-PGA].

Figure 3. 1H NMR spectrum and structure of compound 2. Solvent, D2O; temperature, 45 °C; sample concentration, 5 mg/160 μL; 500.13 MHz.

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RESULTS AND DISCUSSION Synthesis and Properties of Sialoglyco Particulates. Several synthetic glycoparticles carrying multivalent sialosides that target viral HAs have been reported in the literature as influenza virus inhibitors/detection probes using either gold,18−20 silver,21 polyglycerol,22,23 latex,24 or cyclic dextrins25 as the backbone. However, to date, these inhibitors or detection probes have been limited to glycoparticles obtained by immobilizing N-acetylneuraminic acid (Neu5Ac), a common sialic acid found on the surface of human cells. Furthermore, most of the glycoparticles were obtained by immobilizing sugar chains (monomer) in a multivalent manner to the particle carrier. By contrast, here we adopted a strategy of molecular design that involved constructing a spherical multivalent Neu5Gcα2,3LacNAc particulate by immobilizing sialoglycopolypeptides to a silica particulate whose surface was made

hydrophobic by addition of hexyl groups (Figure 1). Specifically, the sialoglycopolypeptide was immobilized to modified hydrophobic silica particulates by hydrophobic interaction. This was achieved by introduction of an alkyl chain (1-aminodecane) onto sialoglycopolypeptides (Figure 2). The amino function of 1-aminodecane was reacted with the remaining α-carboxy groups in the main chain of the sialoglycopolypeptide {poly[Neu5Gcα2,3LacNAc-β-O(CH2)5NH-/γ-PGA], 1} in the presence of the condensation reagents BOP and HOBt. In this study, sialoglycopolypeptide 1 with a molecular weight of 2 000 000 and degree of substitution (DS) of 21% Neu5Gcα2,3LacNAc glycoside was prepared and used as starting material for the glycan moiety of sialoglyco particulates. The reaction mixture was loaded onto a Sephadex G-25 M PD-10 column to separate target poly[Neu5Gcα2,3LacNAc-β-O(CH2)5NH-/CH3(CH2)9NH-/γ-PGA] 2 from low molecular D


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ACS Applied Bio Materials weight compounds. 1H and 13C NMR analyses were used to determine the structure of 2. In the 1H NMR spectrum of 2, a characteristic signal at δ = 0.90 ppm was assigned to the terminal methyl proton of the introduced decyl group (Figures 3 and S3). The extent of derivatization was assessed by integrating the resulting 1H NMR signals. By utilizing this approach, we calculated the degree of decylation of 2 to be 44% (Table 1). In the 13C NMR spectrum of 2, 13C NMR signals due to the decyl group appeared in addition to the signals arising from 1 (Figure S4). Specifically, analogous to the 1H NMR spectrum, a characteristic carbon signal from the terminal methyl group of the decyl moiety was observed at δ = 16.4 ppm. These data indicate that the condensation reaction between the remaining carboxy groups of 1 and amino group of 1-aminodecane had progressed as anticipated. Immobilization of the sialo sugar chain on the surface of the particulate was then carried out using compound 2. Hexyl-containing hybrid silica particulates, recently reported by Yamauchi et al., were used for the backbone of the target sialoglyco particulate.13 Specifically, the hexylcontaining hybrid silica particulates and compound 2 were reacted in an aqueous solution of DMSO at room temperature with shaking for 24 h. Thereafter, the target sialoglyco particulate 3 was prepared by performing particle washing and 2% BSA blocking (Figure 1). The hexyl-containing hybrid silica particulate used as backbone could not be dispersed in water (Figure 4A(a)). By

morphology of 3. The average diameter of 3 was determined to be 938 ± 38 nm, which is coincident with the DLS measurements (diameter 951 ± 30 nm) (Figure 4C). Taken together, these results demonstrate that the sialoglyco particulates do not undergo aggregation in aqueous solution. To evaluate the presence or absence of sialoglycan on the surface of synthetic particulate 3, we examined the interaction between 3 and a plant lectin MAA. MAA, from the seeds of the leguminous plant M. amurensis, specifically binds to α2,3sialylgalactose and is a useful tool for sialylated glycoconjugate studies.26−28 In addition, the interaction using SSA,29 which recognizes α2,6-sialylgalactose,30 and ECA,31 which recognizes galactose,32 as a comparison target was also evaluated. Initially, the interaction time between the lectin and synthetic particulate 3 was determined, which was maximal after ∼30 min. Subsequently, the interaction between 3 and each lectin (MAA, SSA, and ECA) after 30 min was evaluated (Figure 5A). From all the tested lectins, the multivalent Neu5Gcα2,3-

Figure 4. (A) Photograph of (a) precipitated hexyl particulate and (b) dispersed Neu5Gcα2,3LacNAc particulate 3 in water. (B) DLS of 3 was used to ascertain particulate sizes. (C) SEM image of 3. Scale bar: 1 μm.

contrast, the resulting sialoglyco particulate 3 showed a high degree of dispersibility (Figure 4A(b)). This observation suggests that the sialo sugar chains were immobilized on the surface of the hexyl-containing hybrid silica particulates. The dispersibility and particle shape of the prepared sialoglyco particulate 3 were determined by DLS in distilled water at 25 °C. The particulate preparation showed a low polydispersity index and a homogeneous distribution. The dispersion profile of the sialoglyco particulate gave a single narrow peak. The average diameter of the particles was 951 ± 30 nm in distilled water (Figure 4B). The diameter of 3 was almost identical to the hydrophobic hybrid silica particulate. Moreover, SEM analysis was used to provide direct evidence for the diameter and

Figure 5. (A) Effect of interaction time on Neu5Gcα2,3LacNAc particulate 3−MAA interaction. Conditions used for the interaction are described in the Materials and Methods. (B) Amount of each lectin adsorbed to 1 mg of 3. (C) Confocal microscopy assay using FITCMAA lectin with Neu5Gcα2,3LacNAc particulate 3. (a) Fluorescent micrographs of Neu5Gcα2,3LacNAc particulate 3 after interaction with FITC-MAA, (b) merged, and (c) bright image. Scale bars: 5 μm. E



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CONCLUSION We have developed a novel sialoglyco particulate 3 that acts as a potent and specific adsorbent for EIV. Synthesis of this particulate 3 was achieved by immobilization of glycopolypeptide 2 containing multivalent Neu5Gcα2,3LacNAc glycoside onto hydrophobic silica particulates via hydrophobic interactions. Furthermore, high sensitivity detection of EIV was realized by combining EIV specific adsorption to compound 3 with rRT-PCR. This is the first report describing the synthesis of sialoglyco particulates capable of selectively adsorbing EIV. We believe these particulates will be a valuable screening tool in equine quarantine systems.

LacNAc-carrying particulate 3 was found to react specifically with the MAA lectin (Figure 5B). On average, MAA lectin adsorbs 4.4 μg per 1 mg of the synthetic particulate 3. Furthermore, the presence of α2,3-sialylgalactose residues on the surface of compound 3 was also verified by confocal laser microscope observations after interaction with FITC-MAA (Figure 5C). These results indicate that compound 2 is readily immobilized on the surface of hexyl-containing hybrid silica particulates by hydrophobic interaction in an aqueous DMSO solution to form sialoglyco particulate 3. Sensitive and Direct Detection of Equine Influenza Virus Using a Combination of Sialoglyco Particulates and rRT-PCR. To assess the detection sensitivity of EIV in horse clinical samples we used rRT-PCR to compare changes in copy number of EIV before and after treatment with sialoglyco particulate 3 generated in this study. The difference in copy numbers log transformed on each day was analyzed by two-way ANOVA and Sidak multiple comparison tests as post hoc tests. P values less than 0.05 were considered significant in this study. The daily changes of the geometric means of copy numbers in the clinical samples before and after treatment are shown in Figure 6. In all cases, the copy number of EIV in clinical samples

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.8b00813.

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Table S1 and Figures S1−S6 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel.: +81 24 646 0823. Fax: +81 24 646 0825. E-mail: ogata@ fukushima-nct.ac.jp (M.O.) *Tel./Fax: +81 242 37 2427. E-mail: [email protected] (K.I.P.J.H.). ORCID

Makoto Ogata: 0000-0002-8319-1265 Enoch Y. Park: 0000-0002-7840-1424 Author Contributions ●

M.O. and K.I.P.J.H. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan [grant number 17K07781] (M.O.).

Figure 6. Daily changes of geometric mean copy numbers for clinical samples either untreated or treated with the particulate (n = 4). ●, Samples treated with Neu5Gcα2,3LacNAc particulate; ○, samples not treated with particulate. The lowest RNA copy number of rRT-PCR was 5 × 103 copies/mL (10 copies/test). Asterisk (*) indicates statistical significance (P < 0.001, two-way ANOVA and Sidak multiple comparison tests). Error bars: standard errors.

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REFERENCES

(1) Cullinane, A.; Newton, J. R. Equine Influenza − A Global Perspective. Vet. Microbiol. 2013, 167, 205−214. (2) Webster, R. G.; Bean, W. J.; Gorman, O. T.; Chambers, T. M.; Kawaoka, Y. Evolution and Ecology of Influenza A Viruses. Microbiol. Rev. 1992, 56, 152−179. (3) Wilson, W. D. Equine Influenza. Vet. Clin. North Am. Equine Pract. 1993, 9, 257−282. (4) Rogers, G. N.; Pritchett, T. J.; Lane, J. L.; Paulson, J. C. Differential Sensitivity of Human, Avian, and Equine Influenza A Viruses to a Glycoprotein Inhibitor of Infection: Selection of Receptor Specific Variants. Virology 1983, 131, 394−408. (5) Ito, T.; Kawaoka, Y. Host-Range Barrier of Influenza A Viruses. Vet. Microbiol. 2000, 74, 71−75. (6) Suzuki, Y.; Ito, T.; Suzuki, T.; Holland, R. E.; Chambers, T. M.; Kiso, M.; Ishida, H.; Kawaoka, Y. Sialic Acid Species as a Determinant of the Host Range of Influenza A Viruses. J. Virol. 2000, 74, 11825− 11831. (7) van Maanen, C.; Cullinane, A. Equine Influenza Virus Infections: An Update. Vet. Q. 2002, 24, 79−94. (8) Yamanaka, T.; Nemoto, M.; Bannai, H.; Tsujimura, K.; Kondo, T.; Matsumura, T.; Gildea, S.; Cullinane, A. Evaluation of Twenty-Two Rapid Antigen Detection Tests in the Diagnosis of Equine Influenza

taken after postinfection day 1 and treated with the particulate was significantly greater than before treatment (P < 0.001). Therefore, the treatment with particulate before testing for EIV by rRT-PCR enhances the sensitivity of the assay. As anticipated, EIV was not adsorbed to asialo particulates (Lac particulates). Interestingly, in one infected horse, it was shown that EIV can be clearly detected after sialoglyco-particulate processing for clinical samples taken on postinfection days 8 and 9, which would otherwise have been below the detection limit of rRT-PCR (Figure S6). At an early stage of infection (i.e., postinfection day 1 during the incubation period) no clinical symptoms are evident. Thus, increasing the diagnostic sensitivity of the assay by treating clinical samples with the particulate before testing for equine influenza will help prevent the spread of EIV at venues hosting events such as horseracing, horse auctions, and equestrian sports. F


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