The CSFV DNAChip: A novel diagnostic assay for classical swine fever virus

Journal of Virological Methods 204 (2014) 44–48

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The CSFV DNAChip: A novel diagnostic assay for classical swine fever virus Yong Kwan Kim a , Seong-In Lim a , Yoon-Young Cho a , Jae-Young Song a , JoonBae Kim b , Dong-Jun An a,∗ a b

Viral Disease Division, Animal and Plant Quarantine Agency, Anyang, Gyeonggi-do, 430-757, Republic of Korea Median Diagnostics Inc, Chuncheon, Gangwon-do, 200-883, Republic of Korea

a b s t r a c t Article history: Received 1 December 2013 Received in revised form 25 February 2014 Accepted 21 March 2014 Available online 31 March 2014 Keywords: Classical swine fever DNAChip Genotyping

A novel assay, the CSFV DNAChip, was developed to clearly and rapidly discriminate three genotypes of classical swine fever virus (CSFV). Total RNA was extracted from clinical samples and then subjected to a one-step reverse-transcription polymerase chain reaction (RT-PCR) using Cy3-labeled primers from the 5 non-coding region (NCR) of CSFV. Amplicons were hybridized to the CSFV DNAChip and fluorescence scanning was performed for detection of CSFV. A cut-off fluorescence intensity value of 5000 was determined by two-graph receiver operating curve (TG-ROC) analysis. The limit of detection values for the developed DNA chip assay were 0.313 ng/␮L for amplicon concentration and 1TCID50 /100 ␮L for virus titer. Using the developed DNA chip, 157 field samples (91 CSFV-positive and 66 CSFV-negative) were investigated. The genotypes determined by the CSFV DNAChip agreed completely with those determined by nucleotide sequence analysis of the viral genome. The developed CSFV DNAChip will be helpful in implementing a CSFV eradication strategy, as it provides a rapid and accurate diagnostic assay that can discriminate easily among CSFV genotypes. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Classical swine fever virus (CSFV) is a highly contagious and fatal disease of swine (Sus scrofa) that affects domestic pigs and wild boars (Vandeputte and Chappuis, 1999; Edwards et al., 2000). By the end of the 20th century, successful CSFV eradication had been achieved in many regions, including North America, Australasia, and parts of Northern Europe. However, CSFV remains widespread in many parts of the world, including South America, Eastern Europe, and Southeast Asia (Vargas Terán et al., 2004). CSFV is a member of the genus Pestivirus, which belongs to the Flaviviridae family. This genome, a single-stranded and positivepolarity RNA, is made up of a sequence of approximately 12,300 nucleotides. CSFV can be divided further into three different genotypes, which in turn are comprised of three or four subgroups (Greiser-Wilke et al., 1998). The World Organization for Animal Health (OIE) recommends the following methods for identification of CSFV in field samples.

∗ Corresponding author at: Animal and Plant Quarantine Agency, Anyang, Gyeonggi-do, 430-757, Republic of Korea. Tel.: +82 31 467 1782; fax: +82 31 467 1800. E-mail address: [email protected] (D.-J. An). http://dx.doi.org/10.1016/j.jviromet.2014.03.020 0166-0934/© 2014 Elsevier B.V. All rights reserved.

The first is a set of standard laboratory methods for diagnosis of viral diseases, based mainly on viral isolation in cell culture. The fluorescent antibody test (FAT) can also be used to detect CSFV antigen in cryostat sections of tissues. Immunoperoxidase staining using monoclonal antibodies developed for the differentiation of Pestivirus has also been used to detect CSFV in cryostat sections. Recent advances in molecular biology, including the polymerase chain reaction (PCR), multiplex PCR, and real-time PCR, have provided alternative methods for simultaneous and differential diagnosis of CSFV (Agüero et al., 2004; Hoffmann et al., 2005; Risatti et al., 2005; Giammarioli et al., 2008). Recently, a DNA chip assay was developed for the detection of viral pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV), foot and mouth disease virus (FMDV), and influenza virus (Liu et al., 2006; Cao et al., 2013; Nguyen et al., 2012). DNA chip assays enable the acquisition of a large amount of information within a short time. In addition, the DNA chip assay is relatively affordable and convenient in terms of the labor required. However, a common drawback of this method is low sensitivity and specificity due to the random arrangement of high surface-density immobilized probes (Nimse et al., 2013). To overcome these problems, a novel 9G DNAChip was developed. The distinctive feature of the 9G DNAChip is the use of nine guanine bases to generate lateral spacing between immobilized

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45

Table 1 Sequences of the probes for genotyping using the CSFV 9G DNAChip. Probe

Sequence 5 → 3 

Positiona

Specific target genotype

P1 P2

5 -GGGGGGGGGAAACAGGACTTAGACCACCCAGGG 5 -GGGGGGGGGAAACAGCACCCTATCAGGTCGTAC

150–170 304–324

G1, G2, and G3 G1, G2, and G3

P3

5 -GGGGGGGGGTTTACATAGCATCTCGAGGTGGG

210–229

G1 (virulent strain) and G2 (CW2002N, KH2002N1)



P4

5 -GGGGGGGGGTTTACATAGCATATCGAGGTGGG

210–229

G1 (vaccine strain)

P5

5 -GGGGGGGGGAAAATACTAGCCTGCTAGTGGGCC

329–349

G2 (Alfort)



P6

5 -GGGGGGGGGAAAATACTAGCCTGTTAGTGGGCC

329–349

G2 (GXWZ2N, CW2002N, KH2002N1)

P7 P8

5 -GGGGGGGGGAAACCCATCACGCAGTGTGATTTCAC 5 -GGGGGGGGGAAAATCTCGAGATGAGCTGCTGC

280–302 203–222

G3 G3

HC

5 -GGGGGGGGGAAACCTACTRTGGGCATGGCTAG

94–113

Hybridization control

a

Genome position relative to the ALD strain of CSFV (Accession No.: EU789580); G1: genotype 1; G2: genotype 2; G3: genotype 3; GGGGGGGGG: 9G for immobilization of the probes on the AMCA slide; AAA or TTT; vertical spacer group; HC: complementary sequence of Cy3-labeled forward primer.

probes, which provides a high degree of accessibility, leading to 100% target-specific hybridization with 100% specificity and 100% sensitivity (An et al., 2012). Thus, the 9G DNAChip has emerged as a powerful diagnostic tool in molecular biology. The present study utilized previous 9G DNAChip technology to generate the CSFV DNAChip and evaluated the sensitivity and specificity of this new chip for the rapid detection and genotyping of CSFV strains.

2. Materials and methods 2.1. Field samples A total of 157 blood samples were obtained from several provinces in South Korea from 1997 to 2005. Total RNA was extracted using the Qiagen RNeasy Mini Kit (Hilden, Germany) according to the manufacturer’s instructions. Of the 157 samples, 91 were confirmed to be positive and 66 were confirmed to be negative by RT-PCR. Sequencing analysis of the 5 non-coding region (NCR) was performed for classification of CSFV genotypes according to a previous study (Paton et al., 2000). Of the 91 CSFV-positive samples, 33, 53, and 5 samples were from the live attenuated vaccine strain (LOM), genotype 2, and genotype 3, respectively.

2.2. Probes Genotype-specific probes for CSFV were designed using sequences from the GenBank nucleotide database at the National Center for Biotechnology Information (NCBI). Eighteen genomic sequences of CSFV, including eight strains of genotype 1 (ALD, Brescia, Buri47, GPE, LOM-Japan, Shimen, LOM, and LOME− ), four strains of genotype 2 (Alfort, GXWZ2N, CW2002N, and KH2002N1) and six strains of genotype 3 (88039, 96939, 97009, 97347, JJ9811, and YI9908), were aligned using the CLUSTAL X alignment program. Appropriate sequences were selected and synthesized from BIONEER (Daejon, South Korea). Probe sequences corresponding to the three genotypes of CSFV are depicted in Table 1.

2.4. Design and preparation of the CSFV DNAChip The CSFV DNAchip developed in this study was exclusively made by Biometrix Technology Inc. (Chuncheon, South Korea). The CSFV DNAChip were prepared according to a previous report on a different 9G DNAChip (Song et al., 2011). The immobilization solutions (15% glycerol, 50 mM butyl amine, 600 mM NH4 Cl, pH 7.4), containing two common positive probe groups (P1 and P2) and six individual typing probe groups (P3, P4, P5, P6, P7, and P8) corresponding to the CSFV genotypes (Table 1), along with the hybridization control (HC) probes, were spotted on an aminocalix[4]arene (AMCA) slide using a Quarray2 microarrayer (Genetix Quarray, UK). The spots were arranged to make 5-by5 pixels. The probe concentration in each spot was 7 pmol/cm−2 . Immobilization probe density of 7 pmol/cm−2 was achieved using 1 nL of oligonucleotide probes (33 pmol/nL). The microarray slide was kept in an incubator (25 ◦ C, 50% relative humidity) for 4 h to immobilize the oligonucleotides. The slide was then suspended in blocking buffer (0.5% milk casein in 4× SSC, pH 7.4) at 25 ◦ C for 30 min to remove the excess oligonucleotides and deactivate the non-spotted area. The slide glass was rinsed with washing solutions A (0.01% Triton X-100 in 4× SSC, pH 7.4) and B (4× SSC, pH 7.4) for 5 min each, and dried using a commercial centrifuge (at 100 × g). The CSFV DNAChip were stored in Secure-SealTM hybridization chambers to prevent dehydration. 2.5. Cy3-labeled PCR The Cy3-labeled 5 NCR forward primer (5 -CTAGCCA TGCCCWYAGTAGG-3 ) and 5 NCR reverse primer (5 -CAGCTTCARYGTTGATTGT-3 ) were used for amplification, and were designed to be used with the CSFV DNAChip. The PCR mixture, with a total volume of 25 ␮L, consisted of 5 ␮L of extracted RNA, 0.4 ␮M of each primer, 10 mM dNTP mix, and Qiagen one-step RT-PCR enzyme mix (Hilden, Germany) in an amplification buffer. Amplification was performed as follows: 30 min at 45 ◦ C for reverse transcription; 15 min at 94 ◦ C, followed by 35 cycles of 1 min at 94 ◦ C, 10 s at 55 ◦ C, and 30 s at 72 ◦ C; with a final elongation step of 5 min at 72 ◦ C. Ten microliters of Cy3-labeled PCR product were used for further CSFV detection and genotyping using the CSFV DNAChip.

2.3. Preparation of reference strains 2.6. Measurement of amplicon concentrations for detection limit Three genotypes of CSFV, including two strains of genotype 1 (ALD and LOM), the CW2002N strain of genotype 2 (isolated from Cheorwon province in South Korea in 2002), and the YI9908 strain of genotype 3 (isolated from Yongin province in South Korea in 1998), were prepared and used to investigate the cross-reactivity among probes corresponding to these three CSFV genotypes.

Different concentration of amplicons differed in their ability to absorb light. To identify the detection limit of amplicons, the concentration of each amplicon was calculated according to the following formula;Absorbance (OD260 nm ) = e × C,where e is the extinction coefficient and C is the concentration of the amplicons.

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2.7. Hybridization procedure Briefly, each hybridization chamber of the CSFV DNAChip was covered with a mixture of 40 ␮L of the hybridization buffer solution (20% formamide, 0.01% Triton X-100, 4× SSC, pH 7.4) and 10 ␮L of Cy3-labeled PCR product. Afterwards, the mixture was incubated at 48 ◦ C for 60 min. The CSFV DNAChip was then rinsed with washing solutions A and B at 40 ◦ C for 1 min each to remove the excess target DNA and dried in a commercial centrifuge (100 × g). The fluorescent signal was measured using a ScanArray LITE scanner (GSI Lumonics, MA, USA) and quantified using Quant Array software (Packard Bioscience, CT, USA). 2.8. Measurement of sensitivity and specificity Sensitivity (true positive rate) and specificity (true negative rate) are methods to measure the proportion of true positive and negative results. Sensitivity was determined by the following formula: number of true positives/(number of true positives + number of false negatives). The specificity was determined by the following formula: number of true negatives/(number of true negatives + number of false positives).

3. Results 3.1. Cut-off value for the CSFV DNAChip The previously confirmed 91 CSFV-positive and 66 CSFVnegative samples were used as controls to set the cut-off value. The range of fluorescence intensity (FI) values for the positive controls exceeded 6000. According to two-graph receiver operating curve (TG-ROC) analysis, 84.6% (77/91) of the positive samples showed fluorescence intensities of more than 30,000, and 15.4% (14/91) of the positive samples showed fluorescence intensities that ranged between 6000 and 29,000. All of the negative samples (66/66) exhibited fluorescent intensities below 4000 (Table 2). Sensitivity and specificity were calculated according to variation in FI. Through TG-ROC analysis, the cut-off value was set at 5000. This value yielded sensitivity and specificity values of 100% (91/91) and 100% (66/66), respectively (Table 2).

P1

P1

C

B

B

C

B

B

P2

P2

B

B

B

B

B

B

P3

P3

C

P5

P5

C

P7

P7

P4

P4

B

B

B

B

B

B

B

B

C

P6

P6

C

P8

P8

Fig. 1. Probe-spotting scheme on the CSFV DNAChip. P1 and P2 probe: G1, G2, and G3; P3 probe: G1 (only virulent strain) and G2 (CW2002N, KH2002N1); P4 probe: G1 (LOM vaccine strain only); P5 probe: G2 (Alfort); P6 probe: G2 (GXWZ2N, CW2002N, and KH2002N1); P7 and P8 probes: G3; C: hybridization control; B: blank.

3.2. Cross-reactivity test Reference strains, including genotype 1 (ALD and LOM), genotype 2 (CW2002N), and genotype 3 (YI9908), were applied to the CSFV DNAChip to observe the cross-reactivity of probes corresponding to the three genotypes of CSFV. The spotting scheme on the CSFV DNAChip is depicted in Fig. 1. The live attenuated vaccine strain (LOM) and the genotype 1 (ALD), genotype 2 (CW2002N), and genotype 3 (YI9908) strains were reverse-transcribed and amplified by RT-PCR. Each amplicon was quantified and concentrations were adjusted so that equal concentrations of each amplicon were applied to the CSFV DNAChip. As depicted in Fig. 2, the amplicons were able to efficiently hybridize with type-specific oligonucleotide probes corresponding to their genotype, without cross-reactions.

3.3. Limit of detection by CSFV DNAChip The limit of detection (LOD) is the lowest quantity of CSFV that can be distinguished from the absence of CSFV. The detection limits for amplicon concentration and virus titers on the CSFV DNAChip were as low as 0.313 ng/␮L and 1TCID50 /100 ␮L, respectively (Supplemental Figs. 1 and 2).

Table 2 ROC curve for fluorescent intensity on the CSFV 9G DNAChip. FIa for P1 probe

Sample No. (157)

0–1000 1000–2000 2000–3000 3000–4000 4000–5000 5000–6000 6000–7000 7000–8000 9000–10,000 11,000–12,000 12,000–13,000 13,000–14,000 14,000–15,000 16,000–17,000 20,000–21,000 21,000–22,000 23,000–24,000 25,000–26,000 26,000–27,000 28,000–29,000 >30,000

45 12 7 2 0 0 1 0 1 0 0 0 1 1 3 1 2 1 1 2 77

a

FI: Fluorescent intensity.

RT-PCR Positive (91)

Negative (66)

– – – – – – 1 – 1 – – – 1 1 3 1 2 1 1 2 77

45 12 7 2 – – – – – – – – – – – – – – – – –

Sensitivity (%)

Specificity (%)

100.0 100.0 100.0 100.0 100.0 100.0 98.9 98.9 97.8 97.8 97.8 97.8 96.7 95.6 92.3 91.2 89.0 87.9 86.8 84.6 84.6

68.2 86.4 97.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0

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Fig. 2. Cross-reactivity test for the CSFV DNAChip. The respective fluorescence images resulted from hybridization of the immobilized probes with Cy3-labeled PCR products corresponding to each genotype of CSFV.

Table 3 Validation of CSFV genotyping discrimination. Isolation year

Sample No.

CSFV 9G DNAChip G1

1997 1998 2002 2003 2004 2005 Total

4 1 40 8 8 30 91

G2

4 1

3 30 33a

40 8 5 53

Sequencing after PCR

G3

5

G3 (n = 4) G3 (n = 1) G2 (n = 40) G2 (n = 8) G1 (n = 3a ) and G2 (n = 5) G1 (n = 30a ) 91

a

All strains of G1 were identified as the live attenuated vaccine strain (LOM) by both nucleotide sequence analysis and the CSFV 9G DNAChip assay.

3.4. Genotyping results obtained from the CSFV DNAChip As depicted in Table 3, five samples that belonged to genotype 3 were isolated in 1997 and 1998 from several provinces, including Gyeonggi, Jenbuk, and Chungnam. Since 2002, there has been a genotype shift from CSFV genotype 3 to genotype 2. In addition, three and thirty samples belonging to genotype 1 were newly isolated from the island of Jeju in 2004 and 2005, respectively. All strains of genotype 1 were identified by CSFV DNAChip assay as belonging to the LOM vaccine strain. Genotyping results from the CSFV DNAChip agreed completely with the results obtained by nucleotide sequence analysis (Table 3). 4. Discussion Previous study (Song et al., 2012a,b) found that the FI on DNAchip was maximal when probes were appended with nine consecutive guanine subunits (9G). Based on these findings, nine consecutive guanine bases were used to generate the CSFV DNAChip. The success of DNAChip depends not only on the density of the attached probes but also on the accessibility of the probes. In general, DNAChip surfaces are obtained by DNA immobilization techniques, such as covalent or non-covalent immobilization (Joos et al., 1997; Kumar et al., 2004; Nonglaton et al., 2004). Earlier, non-9G spotting methods could increase the concentration of the immobilized probes, leading to low hybridization yield and nonspecific interactions. However, appending the probes with nine consecutive guanines appears to enhance the accessibility of the probes (Song et al., 2011, 2012a,b; An et al., 2012). RT-PCR is an extremely powerful technique used to amplify any specific piece of RNA of interest. The main advantage of RT-PCR is that it is very sensitive. However, the sensitivity of RT-PCR is also its major disadvantage since very small amounts of contaminating RNA can also be amplified. Therefore, a common drawback of RT-PCR is the possibility of amplification of non-target RNA. Although the sensitivity of the CSFV DNAChip is similar to that of RT-PCR, it shows a relatively low non-specific interaction. In

addition, the genotypes determined by the CSFV DNAChip perfectly matched those determined by nucleotide sequence analysis. Furthermore, the CSFV DNAChip method is less labor-intensive and time-consuming than RT-PCR. The detection limit of the CSFV DNAChip is substantially impacted by signals from non-specific interactions. If false signals due to non-specific interactions are minimized, any signals higher than those of the negative control (NC) can be used for the effective detection of CSFV. Therefore, it is important to be sure that there are no non-specific interactions. The average fluorescence intensities on the non-spotted and spotted areas of the NC were approximately 700–800 and 1000–1100, respectively. The similarity in these two fluorescence intensities (non-spotted and spotted areas) clearly indicate that there are no non-specific interactions. Therefore, this novel DNAChip assay can be used for the effective detection of CSFV. In signal detection theory, a receiver operating characteristic (ROC) is a graphical plot that illustrates the performance of an assay as the discrimination threshold is varied. The area under the ROC curve (AUC) quantifies the overall ability of the test to discriminate between samples with and without the target pathogens. The maximum value for the AUC is 1.0, indicating a perfect test, while a minimum value has an AUC of 0.5, indicating a useless test (Brown and Davis, 2006). There are several scales for AUC value interpretation, but, in general, diagnostic assays with ROC curves of AUC ≤ 0.75 are not considered appropriate for use as diagnostic tests (Fan et al., 2006). For the CSFV DNAChip assay, the AUC was calculated as 1000, demonstrating its power as a tool for detection of CSFV. In the present study, P3 probes were originally generated to detect genotype 1 of CSFV. However, the target sequences of the P3 probes were also present in the sequences of CW2002N and KH2002N1, which belong to genotype 2. As shown in Fig. 2, CW2002N generates FI not only in the P6 but also in the P3 spotting area on the CSFV DNAChip. The Alfort strain, however, only generated FI in the P5 spotting area, as shown in Fig. 2. Therefore, if the FI was generated in the P5 or P6 spotting area, it could be considered to represent genotype 2, regardless of FI in the P3 spotting area. Recent CSFV outbreaks in China have mainly been associated with virulent isolates of genotypes 1.1, 2.1, 2.2, and 2.3 (Sun et al., 2013; Tu et al., 2001). Three reports have documented the occurrence of 1.1 and 2.2 genotypes in India (Desai et al., 2010; Patil et al., 2010; Nandi et al., 2011). In Vietnam, between 2002 and 2003, two subgroups of CSFV were detected; the majority were from subgroup 2.1 and the remainder were from subgroup 2.2 (Kamakawa et al., 2006). A phylogenetic analysis of CSFV in South Korea found that all field virus strains isolated before 2000 were from genotype 3. However, there has been a genotype shift from CSFV genotype 3 to genotype 2 since 2002 (Cha et al., 2007; Song et al., 2012a,b). In the present study, genotyping results from the CSFV DNAChip assay also identified that the field-collected virulent strain responsible for outbreaks in South Korea since 2002 belongs to genotype 2.

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5. Conclusions In conclusion, the CSFV DNAChip described here provides a sensitive and specific tool for clear discrimination between three CSFV genotypes, within 7 h. The CSFV DNAChip provides a useful method for overcoming several disadvantages and limitations of other molecular diagnostic techniques. As a rapid and accurate diagnosis assay, the CSFV DNAChip could aid in the implementation of the CSFV eradication strategy, as well as providing an easy method for discriminating among CSFV genotypes. Acknowledgements We deeply appreciate the technical assistance provided by Biometrix Technology Inc. This study was supported by a grant from the Animal and Plant Quarantine Agency (QIA), Ministry of Agriculture, Food and Rural Affairs, Republic of Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jviromet. 2014.03.020. References Agüero, M., Fernández, J., Romero, L.J., Zamora, M.J., Sánchez, C., Belák, S., Arias, M., Sánchez-Vizcaíno, J.M., 2004. A highly sensitive and specific gel-based multiplex RT-PCR assay for the simultaneous and differential diagnosis of African swine fever and Classical swine fever in clinical samples. Vet. Res. 35, 551–563. An, H., Song, K., Kim, J., Kim, J., Nguyen, V., Ta, V., Sayyed, D.R., Kim, T., 2012. HPV 9G DNA Chip: 100% clinical sensitivity and specificity. J. Clin. Microbiol. 50, 562–568. Brown, C.D., Davis, H.T., 2006. Receiver operating characteristic curves and related decision measures: a tutorial. Chemometr. Intell. Lab. Syst. 80, 24–38. Cao, Q., Fan, A., Klapperich, C., 2013. Microfluidic chip fabrication and method to detect influenza. J. Vis. Exp. 26, 73. Cha, S.H., Choi, E.J., Park, J.H., Yoon, S.R., Kwon, J.H., Yoon, K.J., Song, J.Y., 2007. Phylogenetic characterization of classical swine fever viruses isolated in Korea between 1988 and 2003. Virus Res. 126, 256–261. Desai, G.S., Sharma, A.R., Kataria, S.N., Barman, N.A., Tiwari, K., 2010. 5 -UTR-based phylogenetic analysis of Classical swine fever virus isolates from India. Acta Virol. 54, 79–82. Edwards, S., Fukusho, A., Lefevre, P., Lipowski, A., Pejsak, Z., Roehe, P., Westergaard, J., 2000. Classical swine fever: the global situation. Vet. Microbiol. 73, 103–119. Fan, J., Upadhye, S., Worster, A., 2006. Understanding receiver operating characteristic (ROC) curves. CJEM 8, 19–20. Giammarioli, M., Pellegrini, C., Casciari, C., De, Mia.G.M., 2008. Development of a novel hot-start multiplex PCR for simultaneous detection of classical swine fever virus, African swine fever virus, porcine circovirus type 2, porcine reproductive

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