Identification of Newly Emerging Influenza Viruses by Surface

Nov 3, 2015 - (1) In addition to circulating seasonal influenza viruses, fatal new variants regularly emerge as threats. The recent 2009 flu pandemic ...
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Identification of Newly Emerging Influenza Viruses by SurfaceEnhanced Raman Spectroscopy Jae-young Lim,†,# Jung-soo Nam,‡,# Se-eun Yang,#,† Hyunku Shin,† Yoon-ha Jang,‡ Gyu-Un Bae,§ Taewook Kang,∥ Kwang-il Lim,*,‡ and Yeonho Choi*,†,⊥ †

Department of Bio-convergence Engineering, Korea University, Seoul, 136-713, Korea Department of Medical & Pharmaceutical Sciences, Sookmyung Women’s University, Seoul, 140-742, Korea § Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women’s University, Seoul, 140-742, Korea ∥ Department of Chemical & Biomolecular Engineering, Sogang University, Seoul, 121-742, Korea ⊥ School of Biomedical Engineering, Korea University, Seoul, 136-713, Korea ‡

S Supporting Information *

ABSTRACT: In this work, we demonstrate in situ virus identification based on surface-enhanced Raman scattering (SERS). We hypothesized that newly emerging influenza viruses possess surface proteins and lipids that can generate distinctive Raman signals. To test this hypothesis, SERS signals were measured from the surface of a noninfluenza virus, two different influenza viruses, and a genetically shuffled influenza virus. To ensure the safety for experimenters we constructed nonreplicating pseudotyped viruses that display main influenza virus surface components. Pseudotype with influenza virus components produced enhanced Raman peaks, on gold nanoparticles, that are easily distinguishable from those of pseudotype with a noninfluenza virus component, vesicular stomatitis virus G protein (VSVG). Furthermore, virus with the surface components of a newly emerging influenza strain, A/California/04/2009 (H1N1), generated Raman peaks different from those of viruses with components of the conventional laboratory-adapted influenza strain, A/WSN/ 33 (H1N1). Interestingly, the virus simultaneously displaying surface components of both influenza strains, a model mutant with genome reassortment, also produced a Raman signal pattern that is clearly distinguishable from those of each strain. This work highlights that SERS can provide a powerful label-free strategy to quickly identify newly emerging and potentially fatal influenza viruses.

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troscopy (SERS).6 Raman scattering can allow us to obtain fingerprint information on a molecule of interest.7−9 Raman signals can be markedly enhanced around metal nanostructures; thus, nanomaterial-based SERS has received much attention as the basis for molecular sensing and imaging. However, because there are only extremely small “hot-spot” in which Raman signals can be greatly enhanced, SERS has generally been used to detect molecules of less than a few nanometers in length.10,11 Influenza viruses are approximately 100 nm in diameter and thereby larger than the molecules that are conventionally analyzable by SERS. However, because viruses possess unique surface protein and lipid profiles on their outer layer, we hypothesized that viruses can generate detectable characteristic Raman signals when their individual surface molecules are adequately in contact with metal nanoparticles.12,13 Consequently, we believed that real-time influenza virus detection can be achieved by measuring the Raman signals from the surface molecules of influenza viruses because Raman scattering is a real-time and label-free detection method. Surface protein and lipid profiles are distinctive characteristics of each virus.

nfluenza viruses are enveloped viruses with seven- or eightsegmented genomes and are common human pathogens. These viruses infect 5% to 15% of the global human population annually, and a seasonal epidemic occurs almost every year, mainly in the winter. Influenza virus infection often leads to severe illness, causing 250 000 to 500 000 deaths per year globally.1 In addition to circulating seasonal influenza viruses, fatal new variants regularly emerge as threats. The recent 2009 flu pandemic caused by a new variant had persisted for longer than a year, eventually resulting in approximately 300 000 deaths.2 Rapid diagnosis of infection by such a new influenza strain is critical for controlling viral spread in its early stage because patients can be treated before the onset of severe illness or effectively quarantined until the relevant vaccines or drugs are available. In general, enzyme-linked immunosorbent assay (ELISA)3 and real-time reverse transcription polymerase chain reaction (RT PCR)4,5 analysis have been used to diagnose influenza virus infection. However, the requirement for predefined labels or probes such as target virus-specific antibodies and DNA oligos limits use of the conventional methods for the rapid identification of newly emerging influenza viruses. In this study, we developed an efficient, label-free influenza virus detection method using surface-enhanced Raman spec© XXXX American Chemical Society

Received: February 3, 2015 Accepted: November 3, 2015

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Figure 1. Schematic diagram of the rapid identification of influenza viruses via SERS. Three different groups of viruses, noninfluenza (displaying VSVG), influenza (displaying CAL HA and NA or WSN HA and NA), and shuffled influenza (displaying WSN HA and CAL NA) viruses, were analyzed by SERS. As an excitation light source, a 785 nm CW laser was used.

Figure 2. Effect of aggregating agents on SERS spectra: (a) scanning electron microscopy (SEM) images of GNP aggregates induced by CuSO4, MgCl2, and CaCl2. The scale bars correspond to 1 μm (main) and 500 nm (inset). (b) SERS spectra of R6G molecules on CuSO4-, MgCl2-, and CaCl2-induced GNP aggregates. (c) SERS intensity at 1511 cm−1. Each error bar corresponds to the standard deviation of 10 independent measurements.

differences in Raman signals with those of its parent influenza viruses (Figure 1). There are many conventional ways to make SERS substrates. For example, focused ion-beam method or photolithography can precisely make a nanoplasmonic structure such as fanshaped and nanoholes.16−18 However, these methods require complex steps and considerable time. Because we focused on the virus detection via SERS rather than making an optimal substrate for viruses, salt induced aggregation of 80 nm gold nanoparticles (GNPs) is considered as a cost-effective and simple fabrication method for our SERS substrates. As induce

As proof of our concept, we performed SERS measurements on viruses in three steps. First, we compared the Raman intensities of an influenza virus with those of a noninfluenza virus. As these two types of viruses have markedly different surface protein and lipid profiles, clearly distinguishable Raman signals should be obtained.14,15 In the next step, we determined the differences in Raman spectra between two different influenza viruses. Finally, as a model of a new variant, we constructed a genetically shuffled influenza virus from two different influenza viruses and confirmed the similarities and B

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Figure 3. Construction of viruses and analyses of viral morphologies and infectivities. (a) The process to construct pseudotyped viruses displaying surface proteins of recently emerging influenza virus (A/California/04/2009(H1N1)). This procedure involved the introduction of plasmids encoding viral components into packaging cells. (b) Transmission electron microscopy (TEM) images of constructed virus particles. The scale bars represent 100 nm. (c) Virally infected cells expressed eGFP, verifying the functionality of the constructed viruses. The figures represent the cases of viruses with VSVG (I), CAL HA+NA (II), WSN HA+NA (III), and WSN HA+CAL NA (IV), respectively.

coverage, would allow us to obtain the highest intensity SERS spectra. In our experiment, the CuSO4-induced aggregates actually produced clearer SERS spectra than other aggregates (Figure 2b). We also measured the SERS intensities for R6G on substrates that were prepared with different concentrations of GNP colloidal solution but a fixed concentration of CuSO4. As the concentration of GNP was increased, the shape of the GNP aggregates changed from 1D fractal structures to 3D multilayered structures. As the surface plasmon resonance frequency is closely related to the type, size, and shape of the substrate, the morphological change of substrate, which depends on GNP concentration, also caused the changes in its enhancement effects on SERS intensities. Our results showed that the highest SERS signals were obtained for the substrate fabricated using a 1.45 mM GNP colloidal solution (Figure S1c). A recently emerging influenza virus mutant from the 2009 flu pandemic, A/California/04/2009 (H1N1) virus (referred to as the CAL strain below), was chosen for our experiments.24 Because the viral infection could lead to severe illness in the

more efficiently monolayer can induce monolayer aggregation than monovalent ones and as halide anions repel analytes by strongly binding to the surface of GNPs, several representative salts, including CuSO4, MgCl2, and CaCl2, were applied for GNP spreading at the same ionic concentration, and then SERS spectra were measured from 1 μM rhodamine 6G (R6G)19−23 on chemically differently prepared GNP aggregates. When measured at 1511 cm−1, a CuSO4-induced substrate produced signals with intensities of 1.5- and 2.4-fold greater than those of MgCl2 and CaCl2-induced substrates, respectively. As shown in Figure 2a, salt-induced aggregates exhibited 25.7%, 10.5%, and 6.1% two-dimensional (2D) surface coverage in each. In general, with a large surface coverage, aggregated GNPs form one-dimensional (1D) and branched nanostructures rather than three-dimensional (3D) bulky structures. As a larger surface area is more likely to provide more “hotspots” and as the surface plasmon resonance of branched-type nanostructures is better suited to an excitation wavelength (785 nm) than are bulky submicrometer to micrometer structures, we supposed that CuSO4-induced aggregates, which have the largest surface C

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Analytical Chemistry experimenters, we needed to construct virus particles that lacked replication ability but still displayed the influenza virus surface proteins. We supposed that the surface proteins can actually interact with GNP substrates, emitting detectable enhanced Raman spectra. To achieve this goal, we took advantage of pseudotyping technology, which is especially useful for generating chimeric enveloped viruses. In this approach, the internal core containing the genome of a type A virus is decorated with the lipid membrane and envelope proteins of a type B virus (referred to as “virus A pseudotyped with the envelope proteins of virus B”).25 In our work, we hybridized the HIV-1 interior with the main envelope proteins of the CAL strain, hemagglutinin (HA), and neuraminidase (NA) (denoted here as VCAL HA+NA). The relevant methods (Figure 3a) are described in detail in the Materials and Methods section. For enhanced safety, the HIV-1 genome lacking all of the HIV-1 genes was utilized for packaging virus particles. For comparison in SERS analyses, we also constructed viruses displaying HA and NA of A/WSN/33 (H1N1), a widely used laboratory-adapted strain (referred to as the WSN strain below), (denoted as VWSN HA+NA), and envelope glycoproteins from a noninfluenza virus, vesicular stomatitis virus (VSV) (denoted as VVSVG).26 We first examined the morphologies of the packaged virus particles (VVSVG, VCAL HA+NA, VWSN HA+NA, and VWSN HA+CAL NA) by transmission electron microscopy (TEM). The virus particles were approximately 100 nm in diameter and possessed roughly spherical shapes as expected (Figure 3b). The TEM images indicate that physical virus particles can form from a combination of the HIV-1 internal core and influenza virus outer proteins. All four types of viruses were fully functional, demonstrating their ability to infect 293T cells with the expression of enhanced green fluorescent protein (eGFP) (Figure 3c). Those viruses were not replicative and were thereby unable to produce progeny particles but rather were designed to be able to infect cells only one time. Prior to the main SERS measurements for the target viruses we briefly optimized the size of GNP substrates. Four different GNPs of 10, 30, 50, and 80 nm in diameter, respectively, were tested as SERS substrates for the main target, VCAL HA+NA. As the GNP size was increased, stronger and sharper SERS peaks were, overall, obtained from the virus (Figure 4a). With GNP substrates smaller than 50 nm we could not obtain a significant level of Raman spectra. In contrast, GNP of 80 nm produced highly enhanced Raman spectra for the virus (Figure 4a). For a more quantitative comparison among the four substrates, SERS intensities at the two characteristic Raman shifts for the virus, 923 and 1356 cm−1, were measured. GNP of 80 nm could produce viral spectra, 9.6-fold greater than GNP of 30 nm, at both Raman shifts (Figure 4b). The same GNP size (80 nm) was also optimal for obtaining the SERS spectra for VWSN HA+NA, (Figure S2). To determine enhancement factor (EF) of our SERS substrate, we compared Raman intensities with SERS spectra of influenza viruses (VCAL HA+NA). The experiment results showed that EF was 2919 at 1573 cm−1 (Figure S5). GNP-based SERS of the viruses generated unique Raman shift patterns at different wavelengths depending on the surface proteins that were displayed on the virus particles (Figure 5a).27−29 Compared with the SERS spectra of the noninfluenza viruses displaying VSVG, virus particles displaying CAL HA and NA produced distinct peaks at 923 and 1356 cm−1. Not surprisingly, the two types of virus particles also shared the

Figure 4. Optimization of GNP size for better enhancing viral SERS signal. (a) The quality of SERS spectra highly depended on the GNP size. GNP substrates were applied at the same concentration independent of their size and aggregated at 10 mM CuSO4. (b) SERS intensities measured at 923 cm−1 and 1356 cm−1. Each error bar corresponds to the half standard deviation of 10 independent measurements. Raman signals from VCAL HA+NA were measured.

identical Raman shifts at 1231 and 1564 cm−1 (Figure 5a). These shared peaks would result from the common amino acids, lipid components, and submolecular domains in the two virus types. Our results indicate that newly emerging influenza viruses can be clearly distinguished from noninfluenza viruses by SERS peak positions and intensity distributions as viral fingerprints. When the viruses are packaged in the absence of envelope protein expression, budding of virus particles from the packaging cell surface should be inefficient. As expected, the supernatant from packaging cells in the absence of envelope protein expression did not contain infectious virus particles (Table S1) and did not generate a significant level of SERS spectra either (Figure 5a). In contrast, the supernatant of packaging cells in the presence of envelope protein expression contained infectious particles (Table S1) and produced clear SERS spectra patterns (Figure 5). Remarkably, the supernatant with VCAL HA+NA had a lower titer of infectious particles than other viruses (Table S1). Since an equivalent level of genomic VCAL HA+NA particles were detected in the supernatant in comparison with other viral cases, this low infectious particle yield is likely linked to the low infectivity of VCAL HA+NA for the chosen host cells, human embryonic kidney (HEK) 293T cells. A more challenging diagnostic problem is to distinguish between different influenza virus strains (e.g., distinguishing a newly emerging influenza mutant virus from a seasonal flu virus). For such a task, conventional diagnosis methods based D

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two viruses had peaks at the same wavelengths but showed different relative intensity distributions among the peaks. Interestingly, the extent of the difference in the Raman spectra between the viruses was not proportional to that in the amino acid sequences between them. For example, the virus particles displaying VSVG (VVSVG), which only possesses 10.5% (refer to the sequence alignment results in Figure 6a) and 12.6% homology with the CAL HA and NA, respectively, showed two Raman peaks in common with VCAL HA+NA, and only one unique peak (Figure 5a). In contrast, VWSN HA+NA, which displays surface proteins having 81.7% and 79.4% homology with the corresponding proteins of the CAL strain (Figures 6b and 5c), showed two common peaks with those of VCAL HA+NA and two unique peaks (Figure 5b). Moreover, the three mentioned viruses possess two common Raman peaks (Figure 5a,b and Table S2). These results highlight that Raman spectra likely capture key characteristics but only partial physical and chemical profiles of viral surface molecules, rather than the entire set of profiles.30 New influenza viruses have continuously emerged and are often severely pathogenic to humans. One of the main mechanisms involved in the development of such mutants is the reassortment of virus genome segments. When different virus strains coincidentally infect the same host cells, progeny virus particles can pick up mixed genome segments from multiple parental strains. The recent 2009 flu pandemic involved a variant that was generated from genome reassortment of different influenza viruses from swine, birds, and humans.31 We tested whether SERS can be used to identify such newly occurring mutants. We constructed virus particles that displayed both WSN HA and CAL NA as a model of genome-reassorted influenza viruses (VWSN HA+CAL NA) by introducing the genes encoding the two proteins into packaging cells. The overall SERS peak pattern from the new virus was different from that of either parental virus (VCAL HA+NA or VWSN HA+NA) (Figure 5 and Table S2).32,33 The SERS spectra for the model virus are the result of a mixing of SERS peaks of the two parental viruses (Table S2). Consequently, multiple Raman peak positions of the mutant matched those of VCAL HA+NA and VWSN HA+NA at 740, 923, 1107, and 1356 cm−1.

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CONCLUSION We demonstrated influenza virus detection by SERS, with the aim of a rapid and precise diagnosis of viral infection. As every enveloped virus has a unique set of surface proteins and lipids, viruses can generate their own unique enhanced Raman spectra by interacting with metal nanostructures. To determine whether SERS can be applied to detect influenza viruses, four different viruses (one noninfluenza virus (VVSVG), two influenza viruses (VCAL HA+NA, VWSN HA+NA), and one shuffled influenza virus (VWSN HA+CAL NA) were produced and tested. The SERS spectra from VVSVG showed one unique peak and two peaks matched with those from VCAL HA+NA. In the case of VWSN HA+NA, two peaks that were common to all of the viruses, and two unique peaks were obtained. Although further experiments with additional viruses are necessary, our results suggest that SERS can be used to distinguish not only noninfluenza viruses from influenza viruses but also one influenza virus from another. Moreover, a shuffled virus as a model for a new influenza virus (produced by virus genome segment reassortment) demonstrated a different SERS spectral pattern in terms of peak positions and intensities. On the basis of our results, we believe that our idea can be realized for rapid

Figure 5. SERS spectra of viruses. SERS spectra of (a) VCAL HA+NA (a newly emerged influenza virus) and VVSVG (noninfluenza virus), (b) VCAL HA+NA (a newly emerging influenza virus strain) and VWSN HA+NA (a lab adapted influenza virus strain), and (c) VWSN HA+CAL NA (a model genome-reassorted influenza virus). Black diamonds mean the SERS peaks of PBS at 638, 846, 994, and 1587 cm−1. Magenta circles represent the common peaks of the viruses at 1231 and 1587 cm−1.

on ELISA and RT PCR not only are laborious and timeconsuming but also require predefined, strain-specific probes. As such probes are not typically available at the early stage of the spreading of a new viral infection, the conventional methods have significant limitations with respect to identifying new influenza viruses. As an alternative, we applied our SERS system to effectively distinguish between viruses displaying surface proteins from one of two different influenza virus strains, VCAL HA+NA and VWSN HA+NA. The two viruses generated clearly distinct Raman peaks (Figure 5b). Whereas VCAL HA+NA demonstrated distinct peaks at 923 and 1356 cm−1, VWSN HA+NA exhibited distinct peaks at 740 and 1107 cm−1. In addition, the E

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Figure 6. Sequence alignment of viral surface proteins. Sequence alignment of (a) CAL HA and VSVG, (b) CAL HA and WSN HA, and (c) CAL NA and WSN NA. The perfectly matched amino acids between two aligned proteins of interest are colored in yellow. The amino acids that are not matched but chemically similar are colored in green. F

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a 50× objective lens (NA = 0.70), which was also used to collect the Raman signals (exposure time = 10 s).35,36 The average number of virus particle of VVSVG, VWSN HA+NA, VCAL HA+NA, and VWSN HA+CAL NA were estimated to be 11.12, 16.71, 17.01, and 12.35 in a focused laser spot, respectively (Table S1). Obtaining Raman Signals. All data processing was performed using MATLAB (MathWorks, Inc., Natick, MA) and OriginPro 8 (OriginLab Corporation). Prior to analysis, the spectra were smoothed using the Savitsky−Golay method with a second-order polynomial and window size of 5. More details about postprocessing method of SERS signals are described in Figure S4.

identification of pandemic influenza viruses. Furthermore, we expect that our method can be used to detect other new viruses, such as the MERS viruses.

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MATERIALS AND METHODS Preparation of SERS Substrates. Cover glasses were cleaned with piranha solution (3:1 of H2SO4 and H2O2), rinsed with deionized water (DIW), and dried with N2 gas. For SERS substrates, 80 nm spherical gold nanoparticles (GNPs, BBInternational) were mixed with 10 mM CuSO4 in a 50:1 v/v ratio on the cover glass. After complete drying, virus samples (50 μL) were added. Cell Culture. HEK 293T cells were grown in Iscove’s modified Dulbecco’s medium (Gibco) supplemented with 10% fetal bovine serum (FBS) at 37 °C and 5% CO2. Virus Packaging and Purification. To package pseudotyped virus particles, plasmids encoding the HIV-1 genome and internal components, such as Gag and Pol, and plasmids encoding viral envelope proteins were introduced into 293T cells via a calcium-phosphate-based transfection method. 293T cells were prepared in 10 cm dishes 1 day before transfection. During transfection, HIV-1 plasmids (10 μg of pFUGW, 5 μg of pMDLg/pRRE, and 1.5 μg of pRSV-Rev) were added to 293T cells. For packaging virus particles displaying VSVG, 3.5 μg of pcDNA3 IVS CMV-VSVG was also added. For packaging virus particles displaying the CAL strain (A/California/04/ 2009 H1N1) or the WSN strain (A/WSN/33 H1N1) HA and NA, 3.5 μg of pcDNA3 IVS CMV-CALHA (or -WSNHA), and 3.5 μg of pcDNA3 IVS CMV-CALNA (or -WSNNA) were also added. To package recombinant virus particles with the WSN strain HA and the CAL strain NA, 3.5 μg of pcDNA3 IVS CMV-WSNHA and 3.5 μg of pcDNA3 IVS CMV-CALNA were added to 293T cells. The virus particles were harvested from the supernatant of the packaging cells twice, 1 day and 2 days post-transfection, and concentrated in a 20% sucrose cushion by ultracentrifugation at 25 000 rpm and at 4 °C for 1.5 h. Titering of Virus Particles. Genomic titers of virus solutions were measured by real-time qPCR34 using CFX ConnectTM Real-Time System (Bio-Rad) and SYBR Green1 (Enzynomics). Each virus sample was analyzed with primers, 5′-AGCTTGCCTTGAGTGCTTCA-3′ and 5′-TGACTAAAAGGGTCTGAGGGA-3′. Infectious titers of packaged viruses were measured by counting the 293T cells that had been virally transduced to express eGFP via flow cytometry on FacsCanto II (BD Science) 5 days postinfection. Virus Infection. To effectively assess whether the packaged viruses were functional, we introduced the gene encoding eGFP into the viral genome and monitored intracellular eGFP expression postinfection. We seeded 10 000 293T cells 1 day before infection, innoculated virus solution into the medium above the cells during infection, and then imaged the cells 4 days postinfection. Unlike the viruses displaying VSVG, virus particles displaying influenza surface proteins require to be activated for infection by protease treatment as live influenza viruses do. The corresponding viruses were activated by incubation with TPCK-treated trypsin at 0.1 μg/μL and at 37 °C for 1.5 h during infection. SERS Measurements. After excitation by a 10 mW, 785 nm CW laser, SERS signals were collected with an inverted microscope (Axiovert Carl Zeiss) and sent to a spectrometer (SP2300, PI Acton). Our system was connected to a CCD detector cooled to −70 °C. The laser light was coupled through

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b02661. Additional spectra and experimental data (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions #

J.-y.L., J.-s.N., and S.-e.Y. equally contributed to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant Number HI14C2537). In addition, this work was supported by the Nano·Material Technology Development Program (Grant 2012M3A7B4034986) funded by the National Research Foundation and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (Grant No. 2011-0030700). We thank Hye-soo Han, Joohee Hong, and Yonghee Shin at Sogang University for their help in obtaining TEM images of pseudotyped virus particles. We also thank Dr. Kawaoka at the University of Wisconsin-Madison and Dr. Garcia-Sastre at Mount Sinai Hospital for providing us with genes that encode the influenza virus proteins.

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