ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2015, Vol. 41, No. 1, pp. 46–56. © Pleiades Publishing, Ltd., 2015. Original Russian Text © I.V. Zhirnov, V.A. Ryabinin, A.N. Sinyakov, V.A. Ternovoy, A.N. Shikov, 2015, published in Bioorganicheskaya Khimiya, 2015, Vol. 41, No. 1, pp. 54–66.
Prototype of Oligonucleotide Microarray for Detection of Pathogens Relating to Arena and Filoviridae Families I. V. Zhirnova, V. A. Ryabinina, A. N. Sinyakova, 1, V. A. Ternovoyb, and A. N. Shikovb a
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, pr. Lavrent’eva 8, Novosibirsk, 630090 Russia b State Research Center of Virology and Biotechnology “Vector,” Koltsovo, Novosibirsk oblast, Russia Received, February 19, 2014; in final form, March 5, 2014
Abstract—A prototype of an oligonucleotide microarray for detection of the Lassa, Junin, Machupo, Gua narito viruses (Arenaviridae family), Ebola and Marburg viruses (Filoviridae family) has been presented. The design of primers and probes for hybridization was performed using an original approach based on the anal ysis of amino acid sequences of virus proteins (nucleocapsid protein for the Junin, Guanarito, and Machupo viruses and RNAdependent RNApolymerase for the Lassa, Ebola, and Marburg viruses) with the subse quent decoding of the identified unique peptides in the corresponding sets of oligonucleotides. Keywords: arenaviruses, filoviruses, microarray, minor groove DNAbinding ligand, diagnostics DOI: 10.1134/S1068162014050136 1
INTRODUCTION According to the classification of the World Health Organization, group I pathogens include infectious agents associated with severe and often fatal human diseases, for which in most cases there is no effective treatment and preventive measures [1]. In particular, this group contains viruses causing hemorrhagic fever, i.e., Lassa (LASV), Junin (JUNV), Machupo (MACV), and Guanarito (GTOV) and the Ebola (EBOV) and Marburg (MARV) viruses. The rapid laboratory diag nosis is very important for the organization of antiepi demic measures in cases when an assumption can occur about infections associated with these patho gens. Therefore, the development of diagnostic test systems for the rapid and reliable identification of the corresponding pathogens is the urgent task. Group I pathogens are often diagnosed serologi cally [2]. When diagnostics uses IgM, which rapidly emerges in the patients’ blood, testing is complicated because of crosshybridization between different types of arenaviruses. Although neutralizing antibodies are much more selective, they appear too late for early diagnosis. For example, neutralizing antibodies are not formed for several weeks in the blood of patients infected with the Lassa virus. At the same time, infec tion with group I pathogens often leads to death at 7– 10 days post infection. Test systems based on the polymerase chain reac tion (PCR) can be used long before neutralizing anti bodies appear in the patients’ blood. These tests were
1 Corresponding
author: phone: +7(383)3635173, +7(383)3635153; email:
[email protected].
elaborated in a number of works [3, 4]. However, this method also has significant drawbacks. Because of high variability of viruses, each virus strain requires the development of the individual diagnostic test contain ing certain amplification primers and a fluorescent probe to detect an amplicon. For example, the detec tion of the Lassa, Junin, Machupo, Guanarito, Ebola, and Marburg viruses was performed using 41 types of diagnostic kits [4]. It means that 41 laboratory analyses are necessary to reveal a particular virus type, which is very expensive and timeconsuming. In recent years, the design of analytical microarrays have become promising and quite intensively devel oped methods for diagnostics. In a short time, this direction has found application in various fields from fundamental research in molecular biology and molecular evolution to practical applications in medi cine, pharmacology, ecology, and forensics [5, 6]. The possibility of the parallel analysis of one sam ple on the set of discriminating probes on a microarray is particularly important for the diagnosis of infections associated with group I pathogens since this approach can significantly reduce the analysis time. To date, a method for detecting of the Lassa virus has been developed [7], which uses PCR followed by hybridization of formed amplicons on the microarray. The microarray contains 47 probes (24–34mer oligo nucleotides), which genotype all known strains of the Lassa virus. This method allows for the simultaneous detection of all strains of this virus that significantly saves the analysis time. The drawback of this method is that it provides the analysis of only the Lassa virus and does not detect another viruses of group I pathogens.
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The arena and filoviruses can be revealed by GreeneChipVr [8]. Microarrays of this type contain 60mer oligonucleotides as typing probes. Usually the typing was performed using three targets in the virus genome including the nucleotide sequences of the structural proteins. The sample preparation and its analysis are quite laborious and timeconsuming pro cesses; they include the isolation of RNA, reverse transcription with disseminated seed containing a primer for subsequent amplification of cDNA, and two PCR cycles, one of which is for the amplification of the formed cDNA and the other is for the introduc tion of a specific sequence for the complementary interaction with dendrimers containing the fluores cent labels. The analysis of the hybridization results is performed by a special program. In general, the GreeneChipVr diagnostics is quite expensive. More over, the use of 60mer oligomers as hybridization probes inevitably leads to a loss of specificity of the probes [9, 10]. To overcome the problems of the specificity in the detection of virus pathogens, Affimetrics, inc. (United States) along with Institut Pasteur (France) develop the universal resequencing microchip PathogenID v2.0 [11]. This microchip is used for the detection of a number of highly pathogenic viruses particularly belonging to group I pathogens. The reliability of the detection of the viruses with this microchip varies from very high (>97% for the Lassa virus Guinea) to low (28.7% for Lassa virus Ivory Coast). The authors note that some highly divergent viruses cannot be revealed with this microchip [11]. In general, in spite of a number of obvious advan tages of the GreeneChipVr and PathogenID micro chips, they are expensive to operate and do not always provide the necessary selectivity and reliability in the detection of the group I pathogens. The goal of this work is to develop the prototype of a highly specialized hybridization microarray capable of revealing only the group I pathogens. RESULTS AND DISCUSSION The efficiency of microarray diagnostics based on the hybridization of an analyzed DNA with discrimi nating oligonucleotide probes depends on the correct choice of the latter. The search of probes is solved rather simply for genomic sequences, which are very different in the primary structure, e.g., in the case of genusspecific probes, and in most cases does not rep resent any problem [12]. However this task is signifi cantly complicated when analyzing structurally related nucleotide sequences, which can occur in the case of identical genes within the family (Arena or Filoviridae). Moreover, there are additional complex ities associated with highly variable genomes within a single species of the above pathogens. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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The researchers of the laboratory of medicinal chemistry (ICBFM SB RAS, Russia) elaborated a new approach to the calculation of hybridization oligonu cleotide probes for highly variable viral genomes, which is based on the analysis of the sequences of viral proteins. This approach was successfully used for genotyping influenza A virus [13]. It was decided that the same method be used in this work to calculate hybridization probes in order to design the oligonucle otide microchip for revealing the target infection agents. In fact, this approach is based on finding the antigenic determinants of the analyzed viral proteins. The calculation of the probes includes the follow ing steps: • Collection of the data on the amino acid and nucleotide sequences for JUNV, GTOV, MACV, LASV, EBOV, and MARV. • Selection of the peptide fragments of the ana lyzed viral protein, which are common for all strains and isolates of the given pathogen. • Elimination from the resultant set of peptides of those amino acid sequences, which are present in other target viruses. • Calculation of oligonucleotide probes based on the structure of the chosen peptides. • Selection of the set of oligonucleotide probes optimal in size, which most likely allows for identify ing the given virus. • Elimination from the resultant set of oligonucle otide probes those sequences which can be probes for the other target viruses when changing one or two point substitutions. • Selection of oligonucleotide probes with optimal (in the certain range) melting temperature of the duplex formed by the probe and analyzed DNA. We used in this work the GenBank data (http://www.ncbi.nlm.nih.gov) and Protein Sequence Database for Pathogenic Arenaviruses (http:// epitope.liai.org:8080/projects/arena) [14] for nucleo capsid protein and RNAdependent RNA polymerase (for JUNV, MACV, GTOV, and LASV, respectively) and GenBank data on RNAdependent RNA poly merase for EBOV and MARV. The choice of the ana lyzed protein in all cases was determined by the factor of the maximal representation of the corresponding amino acid sequences in the databases. The chosen amino acid sequences for all strains and isolates for each of the target viruses were used to reveal the identical regions of seven amino acids (the longer peptides lead to the decrease in the number of probes common for the analyzed virus, and the shorter peptides lead to less specific probes [12]). It appeared to be impossible to find peptides common for all strains and isolates of each of the target viruses because of both the variability of amino acid sequences and the Vol. 41
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presence of partially sequenced sequences in data bases. Therefore, we select peptides presented in more than 90% of the analyzed amino acid sequences for each of the pathogens. The next step was the selection of the peptide sets for JUNV, GTOV, MACV, LASV, EBOV, and MARV. Peptides presented in the analyzed amino acid sequences of other target pathogens were eliminated from the corresponding set of each virus. The resultant six sets of 7mer peptides specific for JUNV, GTOV, MACV, LASV, EBOV, and MARV were converted to the set of 21mer oligonucleotides encoding these peptides. Using the scheme for the selection of probes [12], these oligonucleotides were used to obtain sets of oligonucleotides for discrimination of the target viruses. To reduce a the number of possible false positive results, the resultant set of oligonucleotide probes for each pathogen was examined for the presence of struc tures, which can be converted to probes for the other target viruses when changing one or two point substi tutions. These structures were eliminated from the set. The partial alignment of the melting temperature of duplexes formed by analyzed DNA and chosen oli gonucleotide probes was achieved by changing (short ening or lengthening) the length of the latter. In this work we chose the temperature interval from 60°C to 62°С. The modified in this way probes for each patho gen were examined for the presence of the sequences complementary to the other target viruses. According to the above principles, six sets of oligo nucleotide probes were selected to detect the JUNV, GTOV, MACV, LASV, EBOV, and MARV viruses (Table 1). The number of probes for revealing the par ticular target virus varied from 7 to 25. In total, 94 probes were chosen. The creation of the microarray diagnostic test sys tem to discriminate target viruses has a sense only if there is a possibility to perform the multiplex PCR at a certain stage of the analysis. A critical point in this sit
uation is the number of primers used. A large number of primers can increase the undesirable interaction between them, which reduces the efficiency of multi plex PCR. The universal primers for amplification of the target fragments of the genome of the arena and filoviruses contain a significant amount of degeneracy points, which leads to the significant heterogeneity of these primers. The number of individual oligonucle otides in the universal primers can exceed 1500 units. The total number of oligonucleotides involved in the amplification of the given DNA region can be suffi ciently decreased by reducing their length and, thereby, the number of degeneracy points. However, the use of this approach leads to the decrease in the melting temperature of heteroduplexes formed by a primer and a template, which negatively influences the PCR efficiency. One of the approaches to stabilize DNA duplexes is their complex formation with sequencespecific minor groove ligands [15]. For the first time, we syn thesized the oligonucleotide conjugates with ATspe cific minor groove ligands [16], which became wide spread in PCR. In order to increase the melting tem perature of DNA duplexes between a template and amplification primers, we decided to use double stranded oligopyrrole ligands with antiparallel orien tation of the oligocarboxamide strands [17] because these ligands ensured the maximal thermostability of duplexes. Due to a significant increase in the thermostability of DNA duplexes, this approach allowed one to signif icantly decrease the length of the oligonucleotide primers and, as a consequence, their total number. The synthesis of the conjugates was carried out using short oligodeoxyribonucleotides (15–22 mers) containing three (A or T) and one (G or C) nucleotide quartet at the 5’ end. The minor groove ligand Нγ PyPyPyγPyPyPyβDp [18] was attached to the 5' end of these oligonucleotides.
O N
O
HN
O
N
N H
H N
O N
N O
O
N H
H N N
O
NH2
N H
H N
H N
H N
NH+
N O
O
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Table 1. Sequences of oligonucleotide probes (5' → 3') for revealing JUNV, GTOV, MACV, LASV, EBOV, and MARV viruses No.
JUNV
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
TGTTGAGATGGCATTGTTCCAACCTGC CTGTTGAAATGGCATTGTTCCAACCTGCA ACTGACCCTGTTGAGATGGCATTGTTC ACCGATCCTGTTGAGATGGCATTGTTC CCACTGATCCTGTTGAGATGGCATTGTTC GCTACTGATCCTGTTGAGATGGCATTGTTC CGATGACATCAGGAAGTTGTTTGACCTCCAT ATCAGAGAGGCAGTGGGGAAACTC GTCATTAGAGAGGCAGTGGGGAAACTC CTGTCATTAGAGAAGCAGTGGGGAAACTC TTTGCTGCCTCCAGACATGGTTGT CATCAGAGAAGCAGTGGGGAAACTTGAC CTTGCTGCCTCCAGACATGGTTGT GATGACATCAGGAAGCTGTTTGACCTCC CGATGATATCAGGAAGCTGTTTGACCTCCAT GGAAGCTATTTGACCTCCATGGAAGAAGAGATCT CAGGAAACTATTTGACCTCCATGGAAGAAGAGATCT GACCCTGTTGAGATGGCGTTGTTC GTOV
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
TCAAATCTTTCTGAAATGCAAGAAGCGATCGTAAAGG TTGGCAGTTTTCCAACCTTCTTCAGGAAAC TTGGCTGTTTTCCAACCTTCCACAGGAAA GAGCTGGCAGTTTTCCAACCTTCTTCAG ATCAGACTATCAAATCTTTCTGAAATGCAAGAAGCGATC CATCAGATTGTCAAATCTTTCTGAAATGCAAGAAGCGATC TCTCTGAGATGCAAGAGGCAATTGTGAAAG GAGTTGGCAGTATTCCAACCTTCTTCAGG TTGGCAGTTTTCCAACCCTCTTCAGG CCTCAGGAAATTATGTGCACTGTTTCAGAAAACCTC GGAGTTAGCAGTTTTTCAACCTTCTTCAGGAAACT TCTGAGATGCAAGAAGCAATCATAAAGGAAGCAATG TTGGCAGTTTTCCAACCTTCCTCAGGAAA GAGTTGGCAGTGTTCCAACCTTCTTCAG TATGTGCACTGTTTCAGAAAACCTCATGATGAGAA ATCAGACTATCAAACCTTTCTGAAATGCAAGAGGCG TGAAATGCAAGAAGCGATCATAAAGGAAGCAATGAAGAA MACV
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
GCTGTAAAGAAGTTAGACCCCACAAATACACTGTG CCAGCCAATAAGCATTATATTCACTGTTTTAGAAAGCCACAT ATCAGCCAGTCAATAAGCATTATATTCACTGTTTTAGAAAGCC AAGATATGGTGATTACAACTCAAGGTTCTGACGACATAAG ACAAGACATGGTGATTACAACTCAAGGTTCTGAC CACAAGATATGGTGATTACAACTCAAGGTTCTGACG CTTACTGCCACAAGATATGGTGATTACAACTCAAGG GGTTTACTACCACAAGATATGGTGATTACAACTCAAGGTT GACATGGTGATCACAACTCAAGGTTCTGATGA CAAGATATGGTGATCACAACTCAAGGTTCTGATGAC GCCAGCCAACAAGCATTATATTCATTGTTTTAGAAAGCC GTTGTATCAACCAGCCAACAAACATTACATTCACTGT CTGTAAAGAAGTTAGATCCCACAAATACACTGTGGCT GCTGTAAAGAAGTTAGACCCCACTAATACACTGTG CTGTAAAGAAGTTGGATCCCACAAACACACTGT
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Table 1. (Contd.) No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
LASV TGCTATCCTTTCCATGAAATTGAATGTTTCATTGGCAC CACACGTCTCCTACAGTATGGATCACAGTAAGT AGTTATCAGGGAGTTGCCTGAACAACGA TTGGCACATGTCTCCTATAGCATGGATCATAGTAAGT GAATGTTTCTTTAGCTCATGTTTCCTACAGCATGGATC TACAGTTATCAGGGAGTTGCCTGAACAATGAGA GAGTTTGCAACTATCAGGTAGTTGTTTAAACAATGAGAGAGAG AATGAAGCTGAATGTCTCATCGGCACATGT CTTATCAATGAAGCTGAATGTCTCATCAGCACATGTTTC CATTGACTCTGCAATTATCTGGGAGTTGTTTAAATAGTGAGAAAG AGTTTGCAATTGTCAGGGAGTTGTTTGAACAAC GCGCTAAGTATGCAATTGTCAGGCAGTTGTTTAAAT CCTTGGCTCATGTGTCTTACAGTATGGATCATAGT TAAGCCTACAGTTATCAGGGAGTTGCCTGAAT GGTTGTCTTGTCAAAAGACTTGCAGGCA GCTATCTTGTCTATGAAACTCAATGTCTCTTTGGCACA CAGTTGTCAGGGAGTTGTCTCAATAATGAGAAGG ATGTTTCATTGGCACATGTCTCCTATAGTATGGATCATAGT CTCATTGGCACATGTGTCTTATAGCATGGATCATAG GAATGTCTCTTTAGCACATGTCTCTTATAGTATGGACCAC GAATGTCTCATCAGCACATGTTTCATATAGCATGGATCA GAATGTGTCTCTAGCGCATGTCTCTTATAGTATGGATC CTGAATGTTTCTTTAGCTCATGTTTCCTATAGCATGGATCATAG TGAATGTCTCATCGGCACATGTATCATATAGTATGGAC GAGGCATTGAGTTTGCAATTGTCAGGTAGTTG EBOV
1 2 3 4 5 6 7 8 9 10 11 12
ACACAAGTGATGATTTTGGTGAGCATGCC CACACAAGTGATGATTTTGGTGAGAATGCTACTGTTAGA GAATTCACAGCTCCCTTCATCAAATATTGCAACCA AGATATGAGTTTACAGCACCTTTTATAGAATATTGCAACCGTTG GCCACAGTTAGAGGGAGTAGCTTTGTAACTGATTTA CGTGAGCAAAAAGAAAGCTTATTGCATCAAGCAT ACTGTTAGAGGCAGCAGTTTTGTTACCGA CACCACACAAGTGACGATTTCGGTGA GCCACAGTTAGAGGGAGTAGTTTTGTAACTGATTTAG AAGCATCCTGGCACCATACAAGTGATGATTT CAAGCATCTTGGCATCATACGAGTGATGATTTC AGCGCGAGCAAAAAGAAAGCCTTTTG MARV
1 2 3 4 5 6 7
TTCTTGGCACCACAATTCAGCAAGCATAG TGGATGCATTTCTTAATACCACTATGCTATATGCATGTCAG GAATGCCATAGTAAGAGGTGCAAGTTTTGTTACTGATC GGGAACAGAAAGAAGCTTTATTACATCAGGCTTCTTG CTTGCCTTCCGATATGAATTTACACGGCATTTCATA CGCCTTCCGATATGAATTTACACGGCATTTCATA CGCTATAGTAAGGGGTGCAAGTTTTGTTACTGATC RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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Table 2. Sequences of oligonucleotide primers (5' → 3') used in the work No.
Primer sequence (5' → 3')*
1
ACAAGTGTCGATTTAAA
2
ACAAGTGTCGATTTAAC
3
AAGTGTCGATTTAACTG
4
ACAAGTGTTGATTTAAATG
5
ACAAGTATCGATTTAAATG
6
ACAAGTGTCGATTTATAT
7
ACAAGTGTTGATCTGA
8
ACAAGTATTGACCTGA
9
ACAAGCATTGATCTGA
10
ACTAGCATTGATCTGA
11
ACAAGTGTTGATCTAAC
12
ACAAATGTTGATCTAACA
13
AAGTGTTGATTTGACAA
14
ACAAGTGTCGATTTG
15
ACAAGTATTGATCTGAC
16
AGTTTAAGATCTCTTCTC
17
AGTTTAAGATCCCTTCT
18
TCAACCAGCTTAAGAT
19
TAACATCAACCAGTTTG
20
AGTTTAAGATCTCTTCTTC
21
AACAGTGATCACTGAA
22
TGAACAGTAATAACTGAAC
23
TGAACAGTGATAACTGA
24
TGAACAGTGATGACT
25
TTGAACAGTGACAAC
26
TGATGACTGAATTTTGT
27
AATCTGCTGATCATAGA
28
TTTGTTGATCATAGAGTC
29
TTTGCTGATCATAGAGT
30
AATGTTAAAGAATTTGTGTTT
31
TATGTTAAAGAATCTTTGTTTT
32
ATGTTGAAGAATCTCTG
33
AATGCTAAAAAACTTATGTTT
34
AATGTTAAAAAACTTGTGTT
35
AATGCTGAAGAATTTGT
36
AATGTTGAAGAACTTATGT
37
AATGTTGAAGAATTTGTG
38
AATGCTTAAAAATCTCTGT
39
ATGTTAAAAAACCTGTGT
40
TATGTTAAAAAACCTTTGC
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Primer type
Virus(es)
Direct
JUNV, GTOV, MACV
Reverse
JUNV
Reverse
GTOV
Reverse
MACV
Direct
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Table 2. (Contd.) No.
Primer sequence (5' → 3')*
Primer type
Virus(es)
41 42 43 44 45 46 47 48 49 50
TATGTCAGCTTGCAA AATCACGTCCTTTGA TGATATCTGCTTGCA TAGTCCCTCCCTTTA TATCAGCTTGCAAGT AATCTCTTCCTTTGATG AATCCCTTCCTTTTATATC AATCTCTTCCTTTAATGTC AATCCCTCCCTTTTATAT ATCAGCTTGTAGATCAT
Reverse
LASV
51
ATCAGCTTGTAAGTCA
Reverse
LASV
52 53 54 55 56 57 58
ATGATGGTTGTCACT TGATGGTAGTTACGG ATGATGGTTGTTACTG ATGATGGTTGTCACA TGATGGTAGTCACAG ATGATGGTTGTGACA ATGATGGTTGTAACAG
Direct
EBOV, MARV
59 60 61 62 63 64 65
TGTTATACATTGATTATCAC AATGCACTGATTGTC TGATACATTGATTATCCC TGTAATACATTGATTATCCC AGTTATACATTGATTGTCA TGTTATACATTGGTTATCA TGATGCATTGATTATCA
Reverse
EBOV, MARV
*Each primer contains ligand HgPyPyPygPyPyPybDp covalently attached to the 5' end.
The structures of the resultant conjugates are pre sented in Table 2. These conjugates were divided in two groups, i.e., corresponding to direct (33 struc tures) and reverse (32 structures) primers. The oligo nucleotide derivatives of each group were combined in equimolar ratio and used in PCR. The PCR reaction was carried out using simultaneously all primers for amplification of group I pathogens. The amplification with the simultaneous introduction of the fluorescent label (Cy5) was performed in the asymmetric regime to obtain predominantly single stranded products for the subsequent hybridization on the microarray. Configuration of the used microarray containing the probes for JUNV, GTOV, MACV, LASV, EBOV, and MARV is shown in Fig. 1. The oligonucleotide probes contained aminohexyl linker at the 3' end, which provided their attachment to the glass slide modified with phenyldiisothiocyan ate [19]. Marker oligonucleotide (dТ)8 bearing the Cy5 label at the 5' end and the aminohexyl linker at the 3' end was used as a print control.
The developed prototype of the microarray was tested using samples representing amplicons of the fragment of the gene encoding nucleocapsid protein of the JUNV (strain XJ#44), GTOV (strain VHF1750), and MACV (strain Carvallo) viruses and amlicons of the fragment of the gene encoding RNAdependent RNA polymerase of the LASV (strain CSF), MARV (strain Popp), and ZEBOV (Zaire ebolavirus, strain Mayinga) viruses. The evaluation of the results in this work was per formed using the mean fluorescence value of spots (Y) taken as the ratio of the sum of the fluorescence inten sities of spots corresponding to the probes for the given virus to the number of these spots [12]: ⎛ Y = ⎜ ⎝
N
∑ i
⎞ I i⎟ /N, ⎠
where Ii is the fluorescence intensity of the spot i for the given virus, N is the number of spots corresponding to the probes of the given virus.
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Figure 2 shows examples of the hybridization pat terns and corresponding histograms of the mean fluo rescence of the spots. The presented examples demon strate that the developed microarray allows for the unambiguous discrimination of the target viruses. In some cases the observed crosshybridization does not reduce the accuracy of the interpretation of the data because of a significant difference (in order of magni tude) between the mean fluorescence values of the spots. It was previously found that human DNA, mon key DNA, and cDNA of measles virus do not give unspecific reactions in PCR with amplification prim ers for filoviruses [19]. In addition, we examined the influence of sum mary human DNA on the specificity of the identifica tion of target viruses. For this purpose, summary human DNA (0.5–5.0 μg) or cDNA of measles virus (0.5–5.0 μg) were used in PCR in the presence of the primers for the target viruses. The reaction mixture containing the fluorescent label was hybridized with the typing probes on the microarray. The nonspecific hybridization was not observed in both cases. We evaluated the analytical sensitivity of microar ray identification of the Ebola virus, which was deter mined using 10fold dilutions of the positive control samples obtained from cultural viruscontaining liq uid with a known CTID virus titer. The sensitivity limit of the microarray evaluation was 2 × 104 gene equivalents in the analyzed sample. Thus, the results show the applicability of the pre viously developed method of the probe selection based on the analysis of the protein sequences [13] to design the prototype of the oligonucleotide microarray for the diagnostics of arena and filoviruses. EXPERIMENTAL We used plasmid DNA pCR2.1 (Invitrogen, United States) and pUC18 (Medigen, Russia); human DNA (Medigen, Russia); 2,2'dipyridyl disulfide (Aldrich, United States), triphenylphosphin (Fluka, United States), dUTPCy5 (Biosan, Russia), and Taq polymerase (Sibenzyme, Russia). TOP10 E. сoli cells (Invitrogen, United States) were used for molecular transformation. cDNA of the measles virus (MVs/ Blagoveshensk.RUS/18.10/2) isolated from the patient’s nasopharyngeal swab was used as a control. Oligonucleotide probes and primers were synthe sized on a ASM800 synthesizer (Biosset, Russia) by the standard method. Microarrays were prepared on phenylisothiocyanate glass slides [2] by the method of contact printing using a BioOdyssey Calligrapher MiniArrayer spotter (BioRad, United States). Each slide contained three similar microarray probes suit able for the simultaneous hybridization of three samples. The format of the subarray was 12 × 11 spots (Fig. 1) with the diameter of each spot being 360 μm. Positive control samples for the detection of the Marburg and Ebola viruses were obtained by the RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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JUNV
GTOV
MACV
LASV
EBOV MARV Spots corresponding to virusspecific probes Spots corresponding to marker oligonucleotide Probe is absent
Fig. 1. Microarray (12 × 11 spots) for revealing group I pathogens related to the Arena and Filoviridae family. Each row contains probes for revealing the corresponding virus (JUNV, GTOV, MACV, LASV, EBOV, or MARV).
method of the molecular transformation of competent bacterial E.coli cells by pCR2.1 plasmids containing DNA fragments corresponding to the region of the L gene of the Marburg and Ebola viruses [19] followed by the isolation and purification of plasmid DNA from the bacterial cells. The positive control samples for the detection of the arenaviruses were obtained on the basis of recombinant plasmid pUC bearing the virusspecific insertions. The cultivation of viruses and the preparation of cDNA were carried out as described in [19]. We used Vero cells from the cell culture collection in the State Research Center of Virology and Biotech nology Vector (SRC VB Vector, Russia) and Eagle’s MEM medium containing a double set of amino acids and vitamins (Eagle’s MEM × 2AAV) produced in SRC VB Vector. Virus was cultivated in the stationary regime on the Vero cell monolayer in culture flasks 1 L in volume. The multiplicity of infection was 0.01–0.1 pfu per cell. The virus was adsorbed on cells for 30 min at 37°С, followed by the addition of Eagle’s MEM × 2AAV medium (150 mL) containing 0.06% glutamine, 2% fetal bovine serum, and antibiotics (100 u/mL of benzylpenicillin and 100 μg/mL of streptomycin). Cells were incubated at 37°С until a cytopathic effect of the virus was tested (on the average for five days). The cultural viruscontaining liquid was used to iso late the virus. The preliminary inactivation of the samples and the isolation of RNA were carried out under the conditions regulated by methodical conditions MC 1.3. 256909 Vol. 41
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ZHIRNOV et al. (a) Y 45000 35000 25000 15000 5000 0 JUNV GTOV MACV LASV EBOV MARV (b) Y 45000 35000 25000 15000 5000 0 JUNV GTOV MACV LASV EBOV MARV (c) Y 40000 30000 20000 10000 0 JUNV GTOV MACV LASV EBOV MARV (d) Y 45000 35000 25000 15000 5000 0 JUNV GTOV MACV LASV EBOV MARV Fig. 2. Patterns of hybridization of amplicons of the fragment of the nucleocapsid gene of viruses JUNV (strain XJ#44) (a), GTOV (strain VHF1750) (b), and MACV (strain Carvallo) (c) and amplicons of the fragment of the RNAdependent RNA polymerase gene of viruses LASV (strain CSF) (d), ZEBOV (strain Mayinga) (e), and MARV (strain Popp) (f) on microarray (67°С, 1.5 h). Corresponding histograms of mean fluorescence values of spots (Y, in relative units). Measurement error was evaluated as the mean deviation calculated in accordance with [21]. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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Y 20000 15000 10000 5000 0 JUNV GTOV MACV LASV EBOV MARV
(f) Y 50000 40000 30000 20000 10000 0 JUNV GTOV MACV LASV EBOV MARV
Fig. 2. (Contd.)
(“Organization of laboratories, which use nucleic acid amplification techniques when working with material containing microorganisms of I–IV groups of pathoge nicity”). Viral RNA was isolated from the cultural virus containing liquid using the Reagent kit for DNA/RNA isolation from serum and plasma (Lytech Co., Russia) according to the manufacturer’s instruction. Reverse transcription was carried out using the RevertaL reagent kit (Central Research Institute of Epidemiology, Russia) according to the manufacturer’s instruction. Human DNA and cDNA of the measles virus (MVs/Blagoveshensk.RUS/18.10/2) were used as control samples containing no DNA of JUNV, GTOV, MACV, LASV, EBOV, and MARV. Amplification of the target gene fragments was per formed using the primers conjugated with the minor groove carboxypyrrole ligand [18] (see RESULTS AND DISCUSSION section). Synthesis of conjugates was carried out by the fol lowing method. Triphenylphosphin (15 mg), 2,2' dipyridyldisulfide (15 mg), and 4,4'N,Ndimethyl aminopyridine (7–8 mg) were added to a dried cetav lon salt of oligodeoxyribonucleotides (0.2 μmol) in 100 μL of DMSO. The mixture was stirred for 20 min, followed by the addition of carboxamidopyrrole ligand НγPyPyPyγPyPyPyβDp (2 mg) and diiso RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
propylethylamine (2–3 mL). The reaction mixture was stirred for 20 min and left for 20 h at room tempera ture. The conjugate was precipitated by 2% LiClO4 in acetone (1.2 mL) and centrifuged for 5 min at 1400 rpm. The supernatant was removed; the residue was washed with acetone (3 × 0.5 mL), dried in air, and dissolved in bidistilled water (1 mL). The oligonucleotide deriva tive was isolated by RT HPLC on a Agilent 1100 Series chromatograph (Aglient Technologies, United States) in a linear gradient of acetonitrile (from 0 to 70%) in aqueous solution of 20 mM triethylammonium ace tate for 40 min at a flow rate of 2 mL/min on a RPC18 column (4 × 250 mm, particles of 10 μm). Preferentially singlestranded amplicons were pre pared by PCR in the asymmetric regime [11]. The length of the formed amplicons was 370–480 bp. PCR was performed in the reaction mixture (50 μL) con taining 1× SE buffer (Sibenzyme, Russia) for Taq polymerase (60 mM TrisHCl, 25 mM KCl, 10 mM 2mercaptoethanol, 0.1% Triton X100, pH 8.5), 200 nM dATP, dCTP, dGTP, 70 nM dTTP, 50 nM dUTPCy5, 130 nmol of direct primers and 1300 nmol of reverse primers, 5 mM MgCl2, 4 μL of cDNA solu tion containing the virus (from 2 × 103 to 2 × 106 gene equivalents, and 1 U of Taq polymerase. PCR was car ried out in an iCycler thermocycler (BioRad, United States) using the following protocol: 95°С for 3 min; Vol. 41
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35 cycles by 95°С for 45 s; 65°С for 30 s; 72°С for 1 min. The reaction was monitored by the method of horizontal electrophoresis in 1.5% agarose gel in 1× TAE buffer (40 mM TrisHCl, 1 mM EDTA, pH 7.6) fol lowed by ethydium bromide staining of the products. The fluorescentlabeled amplicons were isolated from the excess of dUTPCy5 by gelfiltration on Qiagen columns (Qiagen, Germany), dried at 60°С in a DNA 120 SpeedVac vacuum concentrator (ThermoSavant, United States) and used for the following hybridization. The dried amplicon sample was dissolved in dis tilled water (100 μL), followed by the addition of 10 μL of 2× hybridization buffer (1× hybridization buffer contains 6× SSC (0.9 М NaCl, 0.09 М sodium citrate, 3 mM EDTA), 5× Denhardt’s solution, and 0.1% Tween20). Before loading on the slide, the reaction mixture was heated for 5 min at 97°С and cooled in ice. Hybridization was carried out in a hybridization chamber (ArrayIt, United States) at 67°С for 1.5 h. The slide was then washed successively in solutions of 6× SSC, 2× SSC + 0.1% SDS, 2× SSC, and 1× SSC and dried by centrifugation. Slide scanning was per formed on a ScanArray Express 2.0 slide scanner (PerkinElmer, United States) at the wavelength of the exciting laser (633 nm). The computer analysis of the scanning results was performed using the ScanArray Express v. 4 program (Perkin Elmer, United States). ACKNOWLEDGMENTS This work was supported by project no. 24.60 “Development of the Method of Nonfluorescent Microarray Diagnostics” of the Program of RAS “Fundamentals of Technology of Nanostructures and Nanomaterials”. REFERENCES 1. Prakticheskoe rukovodstvo po biologicheskoi bezopas nosti v laboratornykh usloviyakh (Practical Guidelines for Biological Safety under Laboratory Conditions), Geneva: Vsemirnaya organizatsiya zdravookhraneniya, 2004. 2. Ivanov, A.P., Bashkirtsev, V.N., and Tkachenko, E.A., Arch. Virol., 1981, vol. 67, pp. 71–74. 3. Gibb, T.R., Norwood, D.A.Jr., Woollen, N., and Hen chal, E.A., J. Clin. Microbiol., 2001, vol. 39, pp. 4125– 4130. 4. Trombley, A.R., Wachter, L., Garrison, J., Buckley Beason, V.A., Jahrling, J., Hensley, L.E., Schoepp, R.J., Norwood, D.A., Goba, A., Fair, J.N., and Kulesh, D.A., Am. J. Trop. Med. Hyg., 2010, vol. 82, pp. 954–960. 5. Microarrays in Clinical Diagnostics, Joos, T.O. and For tuna, P., Eds., Humana Press Inc., Methods Mol. Med., 2005, vol. 144.
6. Lagraulet, A., J. Lab. Automat., 2010, vol. 15, pp. 405– 413. 7. Gunther, S.J., Clin. Microbiol., 2012, vol. 50, pp. 2496– 2499. 8. Palacios, G., Quan, P.L., Jabado, O.J., Conlan, S., Hirschberg, D.L., Liu, Y., Zhai, J., Neil, Renwick N., Hui, J., Hegyi, H., Grolla, A., Strong, J.E., Towner, J.S., Geisbert, T.W., Jahrling, P.B., BuchenOsmond, C., Ellerbrok, H., SanchezSeco, M.P., Lussier, Y., For menty, P., Stuart, T., Nichol, S.T., Feldmann, H., Briese, T., and Lipkin, W.I., Emerging Infect. Dis., 2007, vol. 13, pp. 73–81. 9. Militon, C., Rimour, S., Missaoui, M., Biderre, C., Barra, V., Hill, D., Mone, A., Gagne, G., Meier, H., Peyretaillade, E., and Peyret, P., Bioinformatics, 2007, vol. 23, pp. 2550–2557. 10. Rimour, S., Hill, D., Militon, C., and Peyret, P., Bioin formatics, 2005, vol. 21, pp. 1094–10103. 11. Filippone, C., Marianneau, P., Murri, S., Mollard, N., AvsicZupanc, T., Chinikar, S., Despres, P., Caro, V., Gessain, A., Berthet, N., and Tordo, N., Clin. Micro biol. Infect., 2013, vol. 19, pp. E118–E128. 12. Ryabinin, V.A., Shundrin, L.A., Kostina, E.B., Laassri, M., Chizhikov, V., Shchelkunov, S.N., Chu makov, K., and Sinyakov, A.N., J. Med. Virol, 2006, vol. 78, pp. 1325–1340. 13. Ryabinin, V.A., Kostina, E.V., Maksakova, G.A., Ne verov, A.A., Chumakov, K.M., and Sinyakov, A.N., PloS ONE, 2011, vol. 6, no. 4. 14. Bui, H.H., Botten, J., Fusseder, N., Pasquetto, V., Mothe, B., Buchmeier, M.J., and Sette, A., Immunome Researc, vol. 3, no. h. 2007, pp. 1–8. 15. Gursky, G.V., Zasedatelev, A.S., Zhuze, A.L., Khor lin, A.A., Grokhovsky, S.L., Streltsov, S.A., Surovaya, A.N., Nikitin, S.M., Krylov, A.S., Retchin sky, V.O., Mikhailov, M.V., Beabealaschvilli, R.S., and Gottikh, B.P., Cold Spring Harbor Symp. Quant. Biol., 1983, vol. 47, pp. 367–378. 16. Sinyakov, A.N., Lokhov, S.G., Kutyavin, I.V., Gamper, H.B., and Meyer, R.B., J. Am. Chem. Soc., 1995, vol. 117, no. 17, pp. 4995–4996. 17. Mrksich, M., Wade, W.S., Dwyer, T.J., Geierstanger, B.H., Wemmer, D.E., and Dervan, P.B., Proc. Natl. Acad. Sci. USA, 1992, vol. 89, pp. 7586–7590. 18. Ryabinin, V.A. and Sinyakov, A.N., Russ. J. Bioorg. Chem., 1998, vol. 24, pp. 531–536. 19. Shikov, A.N., Ternovoi, V.A., Sementsova, A.O., Agafonov, A.P., and Sergeev, A.N., RF Patent No. 2458143. 20. Guo, Z., Guilfoyle, R.A., Thiel, A.J., Wang, R., and Smith, L.M., Nucleic Acids Res., 1994, vol. 22, pp. 5456–5465. 21. Gordon, A.J. and Ford, R.A., A Handbook of Practical Data, Techniques, and References, New York: Wiley, 1972.
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Translated by A. Levina
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