Test systems for the laboratory detection and differentiation of Powassan virus from tick-borne encephalitis virus (Flaviviridae: Orthoflavivirus) using RT-PCR and RT-qPCR

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Introduction

Orthoflavivirus powassanense (Powassan virus, POWV) and Orthoflavivirus encephalitidis (tick-borne encephalitis virus, TBEV) belong to the genus Orthoflavivirus of the family Flaviviridae. Orthoflavivirus virions are enveloped, 50 nm in diameter [1]. The orthoflavivirus genome is represented by (+)RNA approximately 11 kb in length [2]. It encodes one open reading frame (ORF) flanked by 5' and 3' untranslated regions (UTR). The ORF is processed into three structural proteins (C, prM and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) [3].

TBEV causes a disease that affects the human central nervous system. It is widespread in Europe and Northeast Asia. There are three main subtypes: "European" (Eur), "Siberian" (Sib), and "Far Eastern" (FE) [4], and several recently described subtypes: "Baikalian" (Bkl) 1 and 2, "Himalayan", as well as the "Obskaya" strain, which is genetically distant from the other subtypes [5]. The main vectors of TBEV are ticks of the genus Ixodes. The European subtype of TBEV is common in Western Europe, where it is transmitted by I. ricinus. The main vector of the Siberian, Far Eastern, and Baikalian subtypes in Siberia and the Russian Far East is the tick I. persulcatus [4, 5]. Ticks of the genera Haemaphysalis and Dermacentor may be additional vectors of TBEV [6, 7].

POWV causes a rare but severe neuroinvasive disease, with clinical characteristics similar to tick-borne encephalitis virus infection [8]. There are two POWV lineages [9]. Lineage I POWV is transmitted by ticks I. cookei and I. marxi in the United States and Canada [10]. Lineage II POWV, also called deer tick virus (DTV), was discovered in 1996. It is also distributed in the United States and Canada and is divided into Northeast and Midwest clades. The vector for this lineage is the tick I. scapularis [11]. In the Russian Far East, POWV was first isolated in 1972 from the tick H. longicornis (neumanni) [12] and then from the tick I. persulcatus [13]. Studies of POWV in the Russian Far East have shown that all POWV strains isolated between 1972 and 2006 are very similar to the Canadian LB strain. Only lineage I of the Powassan virus is currently detected in Russia [8]. The two virus lineages differ by ≈ 15% at the nucleotide level and by ≈ 5% at the amino acid level in the C/prM/E protein regions [14].

In the Russian Far East, there is an overlap of POWV and TBEV infection foci, and there is also a common vector for virus transmission – the tick I. persulcatus. The TBEV infection rates of I. persulcatus ticks collected between 1999 and 2014 in the Far East was 7,9 ± 0,7% [6]. The prevalence of POWV in ticks has not yet been determined; therefore, its contribution to seasonal morbidity is unknown. As early as 1977, antibodies to both POWV and TBEV were observed in rodents and birds in forested zones of the Russian Far East [15]. In another study [16], antibodies to both POWV and TBEV were detected in 4,3% of 117 people bitten by a tick. All of this demonstrates the need to monitor POWV in the Russian Far East and to develop a specific test system for distinguishing these viruses.

There are commercial test systems available for the detection of TBEV via the polymerase chain reaction (PCR), and several more laboratory test systems have been developed [17–19]. Various RT-PCR and RT-qPCR [20] systems for detecting POWV have also been developed [9]. Effectiveness of both TBEV and POWV systems in mixtures has not been established.

The aim of this work is to develop a test system for the differential detection of POWV and TBEV in a mixture, as well as to select oligonucleotide pairs that amplify large sections of POWV and TBEV structural regions from mixed samples.

Materials and methods

Viruses

All the viruses used in the work were taken from the institute's collection and used without additional passages. POWV strain Pow-24 (GenBank number: MG652438) was isolated from a pool of I. persulcatus ticks collected in Primorsky Krai in 1975. TBEV strain EK-328 (Sib) (DQ486861) was isolated from I. persulcatus ticks in Estonia in 1972. We used several members of the Orthoflavivirus genus (TBEV: strains SofjinKGG (FE) (GU121963), DV-936k (FE) (GU125722), Absettarov (Eur) (KU885457), 178/79 (Bkl-1) (EF469661) and 886/84 (Bkl-2) (EF469662); Loping ill virus (LIV): strain S1; West Nile virus (WNV): strain HP-90 lineage 1 (PX444461); Japanese encephalitis virus (JEV): strain JaGAr 01 (AF069076)) in the form of culture fluid from infected in pig embryo kidney (PEK) cells or brain suspensions of infected mice. Poliovirus (strain Sabin I) was used as an internal control [18] for extraction and reverse transcription (RT) in RT-qPCR.

Design of the differentiating primer systems for RT-qPCR and RT-PCR

The POWV RT-qPCR and RT-PCR systems were designed based on the alignment of selected POWV and TBEV strains (Table 1). Nucleotide sequences were aligned via the MEGA X program [21] using the Muscle algorithm, adhering to the standard parameters. Through this alignment, we manually selected the most divergent regions between the two viruses and the most conserved regions of the genomes from among strains of the same virus.

 

Table 1. Strains of POWV and TBEV used for alignment

Таблица 1. Штаммы ПОВВ и ВКЭ, используемые для выравнивания

POWV ПОВВ		TBEV ВКЭ
Strain name Название штамма	GenBank number Номер GenBank	Strain name Название штамма	GenBank number Номер GenBank
Pow-24	MG652438	EK-328c	MH094241
Ternay	HQ231415	Irkutsk-12	JN003209
Ulysses	HQ231414	Est54	GU183384
LB	L06436	Latvia-1-96	GU183382
64-7062 isolate POWANY64	HM440563	Lesopark 11	KJ701416
Spassk-9	EU770575
Partizansk/2006	EU543649
Nadezdinsk-1991	EU670438

 

Oligonucleotides

The sequences of the primers and probe for POWV detection via RT-qPCR are shown in Table 2. All oligonucleotides used in the work were synthesized by "Syntol" (Russia).

 

Table 2. Oligonucleotides for detection of Powassan virus via RT-qPCR

Таблица 2. Олигонуклеотиды для детекции вируса Повассан методом ОТ-ПЦР-РВ

Name Название	Sequence Последовательность	Type Тип	Position in the genome*, nt Позиция в геноме*, нт
PowTestF	5'-CCTTCACATGAGAGGGCGTC-3'	Forward Прямой	351–370
PowTestR2	5'-CGTGACGCAAGAGTAGGTGA-3'	Reverse Обратный	572–591
Pow_TestProbe	R6G-GCGGGCCAGTGGAAGGGACGC-BHQ-1	Probe Зонд	474–494
Pow_681r	5'-AGACCTTTTCCCCCTAGAT-3'	Primer for RT Праймер для ОТ	687–705

Note. * – genome positions are given for the strain Pow-24 (MG652438).

Примечание. * – позиции в геноме указаны по штамму Pow-24 (MG652438).

 

For the specific detection of TBEV strain EK-328 in the mixture, the RT-qPCR test system developed earlier [18] was used (Table 3).

 

Table 3. Oligonucleotides used for the amplification of TBEV and poliovirus in RT-qPCR

Таблица 3. Олигонуклеотиды, задействованные для амплификации ВКЭ и полиовируса в ОТ-ПЦР-РВ

Oligonucleotide Олигонуклеотид	Sequence Последовательность	Type Тип	Position in the genome, nt Позиция в геноме, нт
1. TBEV* 1. ВКЭ*
TBEL1	5'-TCTGAGGGAGACACACTTGG-3'	Forward Прямой	7672–7691
TBER1	5'-GTGCGCCTGTAAACAAAGAA-3'	Reverse Обратный	7735–7754
TBEP1	FAM-TCCTTGGTGCAGCTGTTCAGCC-BHQ-1	Probe Зонд	7709–7730
GTB1R	5'-CCATTCCGGCTCTGAACTTG-3'	Primer for RT Праймер для ОТ	8037–8056
2. Poliovirus** 2. Полиовирус**
PVL1	5'-GGCAGACGAGAAATACCCAT-3'	Forward Прямой	7121–7140
PVR1	5'-CGAACGTGATCCTGAGTGTT-3'	Reverse Обратный	7209–7228
PVP1	ROX-TTGATTCATGAATTTCCTTCATTGGCA-BHQ-2	Probe Зонд	7159–7185
PVR1	5'-CGAACGTGATCCTGAGTGTT-3'	Primer for RT Праймер для ОТ	7209–7228

Note. * – genome positions are listed by strain ЕК-328с (MH094241); ** – genome positions are listed according to [18].

Примечание. * – позиции в геноме указаны по штамму ЕК-328c (MH094241); ** – позиции в геноме указаны в соответствии с [18].

 

Table 4 shows the oligonucleotide sequences used to amplify large regions of the POWV (strain Pow-24) genome and TBEV (strain EK-328) genome via RT-PCR.

 

Table 4. Primer pairs designed to distinguish POWV and TBEV in a mixture using RT-PCR

Таблица 4. Пары праймеров, подобранные для дифференциации ПОВВ и ВКЭ в смеси методом ОТ-ПЦР

Group Группа	Oligonucleotide Олигонуклеотид	Type of primer Тип праймера	Position in the genome, nt Позиция в геноме, нт
Primers for Powassan virus* Праймеры для вируса Повассан*
I	Kgg 65 f 5'-AGATTTTCTTGCACGT-3'	Forward Прямой	1–16**
	Disc_Pow 919r 5'-GGCCCTAGACTCAACGC-3'	Reverse Обратный	901–917
II	Disc_Pow 852f 5'-AACAAGCTTCTAACTGCC-3'	Forward Прямой	817–834
	Disc_Pow 1889r 5'-GCTGTCCACAGGAACTCTCT-3'	Reverse Обратный	1871–1890
III	Disc_Pow 1782f 5'-GCCAGTGTGGAGGGCCAG-3'	Forward Прямой	1747–1764
	Disc_Pow 2669r 5'-CTTCTCCCTCACTCAGCA-3'	Reverse Обратный	2651–2668
	Pow3668 5'-CGCACGATCTCCTCCACTC-3'	Primer for RT Праймер для ОТ	3632–3650
Primers for tick-borne encephalitis virus*** Праймеры для вируса клещевого энцефалита***
I	Kgg 65 f 5'-AGATTTTCTTGCACGT-3'	Forward Прямой	1–16
	Disc_EK_930r 5'-GGAGCCAGGCAGAAGAG-3'	Reverse Обратный	946–962
II	Disc_EK 863f 5'-AACAAACTACTTGCCCTG-3'	Forward Прямой	862–879
	Disc_EK 1900r 5'-CTATCTGTTGGAGTCCGCC-3'	Reverse Обратный	1916–1934
III	Disc_EK 1793f 5'-GCGCACATTGATGGAACA-3'	Forward Прямой	1792–1809
	Disc_EK 2677r 5'-CGTCTCCCTCTGCCAAGG-3'	Reverse Обратный	2693–2710
	Kgg 30 5'-TGGTGCTCCTCACAGAAGC-3'	Primer for RT Праймер для ОТ	3349–3367

Note. * – genome positions are given for the strain Pow-24 (MG652438); ** – the position is indicated according to the L06436 genome; *** – genome positions are given for the strain ЕК-328c (MH094241).

Примечание. * – позиции в геноме указаны по штамму Pow-24 (MG652438); ** – позиция указана по геному L06436; *** – позиции в геноме указаны по штамму ЕК-328c (MH094241).

 

RNA isolation and reverse transcription

TRI Reagent LS (#T3934, Sigma-Aldrich, USA) was used to isolate RNA from samples according to the manufacturer's protocol. Briefly, 375 µl of TRI Reagent LS was added to 125 µl of the sample. Internal control (IC) (poliovirus, 8 × 10⁴ copies) and 1 μg of total RNA from PEK cell culture were added to normalize the amount of total RNA in each sample during RT-qPCR [22]. RT was performed using MMLV reverse transcriptase (#SK022L, "Evrogen", Russia) according to the manufacturer's protocol. The primers used in the RT reaction are presented in Table 2 and Table 3 for each virus. An RT reaction was carried out for TBEV (POWV) and IC in the same tube. A random hexamer primer (R6) was used in the RT reaction to test for the presence of orthoflaviviruses in the sample.

To determine optimal oligonucleotide amplification conditions for sequencing large regions of the POWV and TBEV genomes, the primers Pow3668 and Kgg 30 were used in an RT reaction to generate POWV complementary DNA (cDNA) and TBEV cDNA, respectively. The sequences of these primers are presented in Table 4.

Polymerase chain reaction

PCR was performed in DNAEngine (BioRad, USA) using DreamTaq DNA polymerase (#EP0701, Thermo Fisher Scientific, Lithuania) according to the manufacturer's protocol. Reaction cycle: 1) denaturation for 30 seconds at 95 °С; 2) annealing of primers for 30 seconds at a temperature selected separately for each pair of primers; and 3) elongation at 72 °С for an experimentally selected time for each pair of primers. This reaction cycle was repeated 40 times during the PCR. Before starting the PCR, pre-denaturation was performed at 95 °С for 10 minutes.

Orthoflavivirus amplification was carried out using panflaviviral primers cFD2 (5'-GTGTCCCAGCCGGCGGTGTCATCAGC-3') and MAMD (5'-AACATGATGGGRAARAGRGARAA-3') [23] to test for the presence of viruses in the sample. The reaction cycle is shown in Table 5.

 

Table 5. Amplification program for PCR with universal orthoflavivirus primers

Таблица 5. Цикл амплификации для ПЦР с универсальными праймерами для ортофлавивирусов

Amplification step Этап амплификации	Instruction Инструкция
1	Hold, 95 °C, 8 min
2	Hold, 95 °C, 20 seconds
3	Hold, 53 °C, 40 seconds
4	Hold, 72 °C, 1 min
5	Goto 2, 39 times
6	Hold, 72 °C, 7 min

 

Polymerase chain reaction with real-time detection

qPCR was performed on a C1000 Thermal Cycler using #KG2532 strips (Kirgen, China) and a CFX96 Real-Time System (BioRad, USA) for fluorescent signal detection. The reaction mixture used for the TBEV or poliovirus systems included 2,5 µl dNTP (2,5 mM), 2,5 µl 10x buffer, 2,5 µl MgCl₂ (25 mM), 2 µl each of forward and reverse primers (5 pmol/µl), 1 µl of the probe (5 pmol/µl) (Table 3), 10,25 µl ddH₂O, 0,25 µl SynTaq polymerase (5 units/µl), and 2 µl of the analyzed sample. For the POWV system, the composition described above was used, but the amount of MgCl₂ added was 1,875 µl, and the amount of ddH₂O added was 10,875 µl. The primers and probe for POWV are indicated in Table 2. Reagents for the qPCR (#R-412) were provided by "Syntol" (Russia). The reaction cycles for TBEV and poliovirus are shown in Table 6. Reaction cycle for POWV: 1) denaturation for 15 seconds at 95 °С; 2) annealing of primers and probe for 40 seconds at 63 °С with plate read. This reaction cycle was repeated 40 times during the qPCR. Before starting the qPCR, pre-denaturation was performed at 95 °С for 3 minutes. The obtained data were analyzed using the Bio-Rad CFX Manager 3.1 program.

 

Table 6. RT-qPCR amplification program for TBEV or poliovirus

Таблица 6. Цикл амплификации для ВКЭ или полиовируса в ОТ-ПЦР-РВ

Amplification step Этап амплификации	Instruction Инструкция
1	Hold, 95 °C, 5 min
2	Hold, 95 °C, 15 seconds
3	Hold, 60 °C, 45 seconds
4	Plate read
5	Goto 2, 39 times

 

Sample preparation for Sanger sequencing and bioinformatics analysis

First, analytical electrophoresis was conducted in 1,7% agarose gel on TAE buffer with the GeneRuler 100 bp Plus DNA Ladder marker (#SM0321, Thermo Fisher Scientific, Lithuania). Then, preparative electrophoresis was carried out in 1% agarose gel. Bands of the required lengths were cut for subsequent DNA extraction using the QIAquick Gel Extraction Kit (#28706, QIAGEN, Germany) according to the manufacturer's protocol.

The DNA was sequenced with the ABI PRISM BigDye Terminator v. 3.1 reagent kit (#4337454, Thermo Fisher Scientific, Lithuania), using the ABI PRISM 3500 (Applied Biosystems, USA) automated DNA sequencer. Chromatographic analysis was performed using the SeqMan v. 7.0.0 program from the DNAStar software package.

Preparation of standard samples of Powassan virus RNA

The primers BHT7PowF (5'-ATGACTGGATCCTAATACGACTCACTATAGGAGATTTTCTTGCACGTGT-3') and Pow_1116r (5'-AGGCAGTATTCTCTGGTTTC-3') were used to amplify Powassan virus cDNA in PCR. The PCR product was purified using the QIAquick Gel Extraction Kit (#28706, QIAGEN, Germany). Then, the PCR product was subjected to in vitro transcription using T7 RNA polymerase (#EP0111, Thermo Fisher Scientific, Lithuania) according to the manufacturer's protocol. The obtained RNA was purified in a 5–20% sucrose density gradient. The concentration of purified RNA was determined using a SmartSpec Plus spectrophotometer (BioRad, USA), and the fraction with the highest RNA concentration was selected. This fraction's RNA was isolated using TRI Reagent LS (#T3934, Sigma-Aldrich, USA), and final RNA concentration was measured. Then 10-fold dilutions were prepared and used as standards for RT-qPCR.

Results

Differentiating primer system for RT-qPCR

The PowTestF and PowTestR2 primers and the Pow_TestProbe probe (Table 2) for POWV were designed based on the alignment of the genome sequences of several strains of POWV and several strains of TBEV (Table 1, Fig. 1).

 

Fig. 1. Fragments of the alignment of genome sequences of several strains of Powassan and tick-borne encephalitis viruses with the primers (PowTestF – blue frame, PowTestR2 – red frame) and probe (Pow_TestProbe – green frame) marked on it.

Рис. 1. Фрагменты выравнивания геномных последовательностей нескольких штаммов вирусов Повассан и клещевого энцефалита с отмеченными праймерами (PowTestF – синяя рамка, PowTestR2 – красная рамка) и зондом (Pow_TestProbe – зеленая рамка).

 

Testing the specificity of primers detecting Powassan virus

Six strains of TBEV belonging to the European, Siberian, Far Eastern and two Baikalian subtypes, as well as other orthoflaviviruses (WNV, JEV and LIV) that occur in the same area as POWV, were used to test the specificity of the primers via the RT-qPCR method. The presence of viruses in the samples was confirmed (Fig. 2) using the universal orthoflavivirus primers cFD2 and MAMD [23]. The same viruses and negative controls were then tested using RT-qPCR, with the primer pair PowTestF/PowTestR2 and the probe Pow_TestProbe. This testing confirmed that developed RT-qPCR system specifically amplified POWV, with no detection of other viruses or negative controls (Fig. 3).

 

Fig. 2. Gel electrophoresis (with inversion) of PCR products of orthoflavivirus samples with panflavivirus primers. 1 – POWV (Pow-24), 2 – TBEV (ЕК-328), 3 – TBEV (DV-936k), 4 – TBEV (SofjinKGG), 5 – TBEV (Absettarov), 6 – TBEV (178/79), 7 – TBEV (886/84), 8 – WNV (Hp-90), 9 – JEV (JaGAr 01), 10 – LIV (S1), 11 – k⁻ (culture fluid), 12 – k^- (brain suspension). М – marker. Size of target products = 250 bp.

Рис. 2. Гель-электрофорез ПЦР-продуктов проб, содержащих ортофлавивирусы, с панфлавивирусными праймерами. 1 – ПОВВ (Pow-24), 2 – ВКЭ (ЕК-328), 3 – ВКЭ (DV-936k), 4 – ВКЭ (SofjinKGG), 5 – ВКЭ(Absettarov), 6 –ВКЭ (178/79), 7 –ВКЭ (886/84), 8 – WNV (Hp-90), 9 – JEV (JaGAr 01), 10 – LIV (S1), 11 – к⁻ (культуральная жидкость), 12 – к⁻ (мозговая суспензия). М – маркер. Размер целевых продуктов = 250 bp.

 

Fig. 3. RT-qPCR of orthoflaviviruses with primers PowTestF/PowTestR2 and probe Pow_TestProbe. Blue lines (A, B) – two replicates of the sample containing POWV; lines beyond the threshold (B) – strains of TBEV (ЕК-328; DV-936k; SofjinKGG; Absettarov; 178/79; 886/84), other orthoflaviviruses (WNV (Hp-90), JEV (JaGAr 01), LIV (S1)) and k⁻.

Рис. 3. ОТ-ПЦР-РВ ортофлавивирусов с праймерами PowTestF/PowTestR2 и зондом Pow_TestProbe. Синии линии (A, B) – две повторности проб, содержащих ПОВВ; кривые, находящиеся под пороговой линией (B) – штаммы ВКЭ (ЕК-328; DV-936k; SofjinKGG; Absettarov; 178/79; 886/84), другие ортофлавивирусы (WNV (Hp-90), JEV (JaGAr 01), LIV (S1)) и к⁻.

 

Obtaining standards for RT-qPCR

Standard samples with known amounts of POWV RNA were prepared as described above. They were subsequently utilized to create a calibration curve and used to determine the sensitivity of the method. The lowest amount of cDNA that was detected during amplification in RT-qPCR was taken as the system's limit of sensitivity, which was 10² copies/sample. The amplification efficiency was 99,2% (r² = 1) (Fig. 4).

 

Fig. 4. Calibration curve for the quantitation of Powassan virus cDNA.

Рис. 4. Калибровочная кривая для количественного определения кДНК вируса Повассан.

 

The developed system included internal control. We tested the POWV system in the RT reaction using two variants: for the first variant, the RT reaction for POWV and IC was carried out simultaneously in the same tube; the second reaction was conducted in two separate test tubes. We found no differences in sensitivity under these conditions.

Testing the specificity of amplification in the presence of another virus

We developed a system for the quantitative measurement of POWV and the quantitative ratio of POWV to TBEV in mixed samples. For this reason, we propose the use of the POWV system presented in this article and the TBEV system described earlier [18]. We studied the effect of the presence of TBEV on the detection of the POWV amount and vice versa.

To test the specificity of the primers for each of the systems, samples containing the following amounts of virus(es) were used: 1) 10⁷ copies/ml of the target virus; 2) 10⁷ copies/ml of the target virus + 10⁷ copies/ml of the heterologous virus; 3) 10⁵ copies/ml of the target virus; 4) 10⁵ copies/ml of the target virus + 10⁷ copies/ml of the heterologous virus; and 5) 10⁷ copies/ml of the heterologous virus. No amplification was observed in samples containing only a heterologous virus. Moreover, the number of copies of the target virus was the same regardless of whether a heterologous virus was present in the sample (Table 7).

 

Table 7. Results of the amplification of samples containing one virus or a mixture of viruses in different concentrations

Таблица 7. Результаты амплификации проб, содержащих один вирус или смесь вирусов в различных концентрациях

Sample Проба	log copies/mL* log копий/мл*
POWV detection system Система детекции ПОВВ
2 × 107 copies/mL POWV 2 × 107 копий/мл ПОВВ	7.08 ± 0.65
2 × 107 copies/mL POWV + 8 × 107 copies/mL TBEV 2 × 107 копий/мл ПОВВ + 8 × 107 копий/мл ВКЭ	6.72 ± 0.88
2 × 105 copies/mL POWV 2 × 105 копий/мл ПОВВ	5.08 ± 0.60
2 × 105 copies/mL POWV + 8 × 107 copies/mL TBEV 2 × 105 копий/мл ПОВВ + 8 × 107 копий/мл ВКЭ	4.82 ± 1.50
8 × 107 copies/mL TBEV 8 × 107 копий/мл ВКЭ	No signal detected Нет сигнала детекции
TBEV detection system Система детекции ВКЭ
8 × 107 copies/mL TBEV 8 × 107 копий/мл ВКЭ	7.56 ± 0.30
8 × 107 copies/mL TBEV + 2 × 107 copies/mL POWV 8 × 107 копий/мл ВКЭ + 2 × 107 копий/мл ПОВВ	7.47 ± 0.13
8 × 105 copies/mL TBEV 8 × 105 копий/мл ВКЭ	5.51 ± 0.14
8 × 105 copies/mL TBEV + 2 × 107 copies/mL POWV 8 × 105 копий/мл ВКЭ + 2 × 107 копий/мл ПОВВ	5.43 ± 0.09
2 × 107 copies/mL POWV 2 × 107 копий/мл ПОВВ	No signal detected Нет сигнала детекции

Note. * – three repetitions were performed. The values are given in the format AVERAGE ± CONFIDENCE.T.

Примечание. * – были выполнены три повторные амплификации. Значения приведены в формате «СРЗНАЧ ± ДОВЕРИТ.Т».

 

Thus, testing the primers for POWV and TBEV shows that the presence of another virus in the mixture does not distort the measurement result, which allows us to accurately determine the amount of one virus in the presence of another.

Selection of oligonucleotides for amplification of large regions of POWV and TBEV genomes via RT-PCR

In addition to virus quantification, the ability to sequence the structural region of a genome is useful for phylogenetic analysis of orthoflaviviruses. Primer sequences (Table 4) for the amplification of large regions of the POWV and TBEV genomes were selected manually according to an alignment of the genomes of several POWV and TBEV strains.

Selected oligonucleotides (schematically shown in Fig. 5) were designed to amplify three overlapping PCR products for use in the sequencing of the whole structural part of the genome.

 

Fig. 5. Schematic representation of the selected primer sets for POWV and TBEV according to the genomes.

Рис. 5. Схематичное изображение подобранных групп праймеров для ПОВВ и ВКЭ в соответствии с геномами.

 

The sample variants used for system optimization are presented in Table 8.

 

Table 8. The cDNA samples used in the PCR with the oligonucleotide pairs under study

Таблица 8. Образцы кДНК, используемые в ПЦР с исследуемыми парами олигонуклеотидов

№	Sample name Название пробы	Sample preparation Пробоподготовка
1	POWV cDNA кДНК ПОВВ	POWV RNA (Pow3668 in RT) РНК ПОВВ (Pow3668 в ОТ)
2	TBEV cDNA кДНК ВКЭ	TBEV RNA (Kgg 30 in RT) РНК ВКЭ (Kgg 30 в ОТ)
3	cDNA of virus mixture (POWV + TBEV) 1 кДНК смеси вирусов (ПОВВ + ВКЭ) 1	POWV RNA + TBEV RNA (Pow3668 in RT) РНК ПОВВ + РНК ВКЭ (Pow3668 в ОТ)
4	cDNA of virus mixture (POWV + TBEV) 2 кДНК смеси вирусов (ПОВВ + ВКЭ) 2	POWV RNA + TBEV RNA (Kgg 30 in RT) РНК ПОВВ + РНК ВКЭ (Kgg 30 в ОТ)
5	POWV cDNA + TBEV cDNA кДНК ПОВВ + кДНК ВКЭ	POWV RNA (Pow3668 in RT) + TBEV RNA (Kgg 30 in RT) 1 : 1 РНК ПОВВ (Pow3668 в ОТ) + РНК ВКЭ (Kgg 30 в ОТ) 1 : 1

 

Each pair of analyzed oligonucleotides (Table 4) was tested in PCR with the above-mentioned samples. Sanger sequencing was used to confirm the specificity of amplification. We selected the annealing temperature (Tm) and elongation time at which the oligonucleotides specific to POWV did not amplify the TBEV genome and vice versa (Fig. 6). The Tm for oligonucleotides of groups I and II varied from 47 to 53 °С; and for oligonucleotides of group III, this varied from 50 to 57 °С. The elongation time was initially 1'30". For group I oligonucleotides, the elongation time was optimized due to the presence of non-specific bands at the standard elongation time (Fig. 7). All bands of target length were verified by Sanger sequencing.

 

Fig. 6. Gel electrophoresis of PCR products of POWV cDNA, TBEV cDNA, and mixtures of these viruses with the analyzed primers of three groups. А – PCR products with POWV primers; В – PCR products with TBEV primers. Designations: 1 – POWV cDNA; 2 – TBEV cDNA; 3 – cDNA of virus mixture 1; 4 – cDNA of virus mixture 2; 5 – POWV cDNA + TBEV cDNA; М – marker; k⁻ – negative control. The red arrow indicates the products of the target length.

Рис. 6. Гель-электрофорез продуктов ПЦР кДНК ПОВВ, кДНК ВКЭ и смесей этих вирусов с анализируемыми праймерами трех групп. А – продукты ПЦР с праймерами на ПОВВ; В – продукты ПЦР с праймерами на ВКЭ. Обозначения: 1 – кДНК ПОВВ; 2 – кДНК ВКЭ; 3 – кДНК смеси вирусов 1; 4 – кДНК смеси вирусов 2; 5 – кДНК ПОВВ + кДНК ВКЭ; М – маркеры; к⁻ – отрицательный контроль. Красной стрелкой указаны продукты таргетной длины.

 

Fig. 7. Gel electrophoresis of PCR products of TBEV cDNA, POWV cDNA, and a mixture of these viruses with the analyzed primers of the first group. Wells 1–10 – PCR products with primers for TBEV; well 11 – marker GeneRuler DNA Ladder Mix (#SM0333, Thermo Fisher Scientific, Lithuania); wells 12–21 – PCR products with primers for POWV; wells 10 and 21 – negative controls. Samples 1, 2, and 3 contain TBEV cDNA with a Tm of 47, 49.6, and 53 ºС, respectively; samples 4, 5, and 6 contain POWV cDNA with the same Tm; samples 7, 8, and 9 contain a mixture of TBEV and POWV cDNAs with the same Tm. Samples 12, 13, and 14 contain POWV cDNA with a Tm of 47, 49.6, and 53 ºС, respectively; samples 15, 16, and 17 contain TBEV cDNA with the same Tm; samples 18, 19, and 20 contain a mixture of TBEV and POWV cDNAs with the same Tm. The red arrow indicates the target length products.

Рис. 7. Гель-электрофорез ПЦР-продуктов кДНК ВКЭ, кДНК ПОВВ и смеси этих вирусов с анализируемыми праймерами первой группы. Лунки 1–10 – ПЦР-продукты с праймерами на ВКЭ; лунка 11 – маркер GeneRuler DNA Ladder Mix (#SM0333, Thermo Fisher Scientific, Литва); лунки 12–21 – ПЦР-продукты с праймерами на ПОВВ. Лунки 10 и 21 – отрицательные контроли. Пробы 1, 2 и 3 содержат кДНК ВКЭ с Tm = 47; 49,6 и 53 ºС, соответственно; пробы 4, 5 и 6 содержат кДНК ПОВВ с теми же Tm; пробы 7, 8 и 9 содержат смесь кДНК ВКЭ и ПОВВ с теми же Tm. Пробы 12, 13 и 14 содержат кДНК ПОВВ с Tm = 47; 49,6 и 53 ºС, соответственно; пробы 15, 16 и 17 содержат кДНК ВКЭ с теми же Tm; пробы 18, 19 и 20 содержат смесь кДНК ВКЭ и ПОВВ с теми же Tm. Красной стрелкой указаны продукты таргетной длины.

 

As a result of our experiment, optimal amplification conditions were selected for each group of oligonucleotides, which are presented in Table 9.

 

Table 9. Amplification conditions with oligonucleotides for sequencing of large regions of the POWV and TBEV genomes

Таблица 9. Условия амплификации олигонуклеотидов для секвенирования больших участков геномов ПОВВ и ВКЭ

Primer group Группа праймеров	Belonging to the virus Принадлежность вирусу	Oligonucleotides Олигонуклеотиды	Tm, °С	Elongation time Время элонгации
I	POWV ПОВВ	Kgg65f–Disc_Pow 919r	50	30"
	TBEV ВКЭ	Kgg65f–Disc_EK 930r	53
II	POWV ПОВВ	Disc_Pow 852f–Disc_Pow 1889r	47	1'30"
	TBEV ВКЭ	Disc_EK 863f–Disc_EK 1900r
III	POWV ПОВВ	Disc_Pow 1782f–Disc_Pow 2669r	57	1'30"
	TBEV ВКЭ	Disc_EK 1793f–Disc_EK 2677r	52

 

Under optimal conditions, amplification with primers of groups II and III allowed us to obtain only the target virus. Amplification of the target virus using primers of group I is observed when the virus is present in the mixture. However, when target virus is not present, low-level amplification of a heterologous virus can be observed (Fig. 6, group I: A – well 2, B – well 1). It is likely due to the same primer at the 5' end (Kgg 65 f) being used for both systems, since the sequences of the POWV and TBEV genomes in this region are very similar; therefore, it is impossible to select another primer that would distinguish these viruses. It is important to note that the oligonucleotides used in the RT reaction for both POWV and TBEV are not discriminatory. When a heterologous primer is used for RT, accumulation of the target virus PCR product is detected (Fig. 6, well 4 for all A and well 3 for all B).

Discussion

Currently, many systems and commercial kits identify TBEV and POWV. However, in most cases, the primers in these systems are selected for conserved regions of orthoflaviviruses, which can lead to false-positive results. To our knowledge, there are no specialized systems for the differential detection of TBEV and POWV in a mixture.

The objective of this study was to develop a test system for monitoring and molecular epidemiology of POWV. Primers in existing test systems for detecting POWV often target the most conserved region of the orthoflavivirus genome – the non-structural protein NS5 [9]. However, for the stated purposes, this region of the genome is unsuitable, since primers from this region may amplify both POWV and TBEV. Based on in silico data from [9], test systems targeting the NS5 protein showed good results both in detecting all POWV lineages and in detecting each lineage separately. The same study [9] showed that test systems targeting structural proteins E and C are likely to be lineage-specific. Our RT-qPCR test system is focused on lineage I POWV, which is widespread in the Russian Far East, where mixed foci of POWV and TBEV are present [16]. The developed system shows specificity, with no false-positive signals in RT-qPCR with different subtypes of TBEV or other orthoflaviviruses (WNV, JEV, LIV). This enables rapid and accurate detection of POWV in the laboratory, which facilitates the processing of collected materials in areas where both TBEV and POWV are endemic.

The detection limit of viral cDNA was 100 copies in the qPCR reaction. When analyzing a mixture of POWV and TBEV, both the POWV and TBEV systems only showed amplification of the target virus, and the presence of another virus in the mixture did not affect the detection signal. The use of an internal control allows for accurate analysis of the quality of the isolated material and the efficiency of the reverse transcription reaction. We used poliovirus since it is routinely used in our laboratory as an internal control. If desired, cellular RNA can be used as an internal control, since in our experiment samples from uninfected culture fluid and uninfected brain suspension did not have amplification signals in RT-qPCR.

In this work, oligonucleotides were selected for the amplification of large regions of the POWV and TBEV genomes using RT-PCR in three fragments, extending from the 5' UTR to the NS1 protein. These amplification products, obtained using the indicated oligonucleotides, can be used for the phylogenetic analysis of orthoflaviviruses, as well as to confirm the presence of POWV or TBEV in samples from the sympatry zone by sequencing. Each primer group that was specifically selected and analyzed correctly identified the target virus in the mixture, even when group I primers were used for both viruses. In the presented work, strains of lineage II POWV (DTV) were not tested since all isolated strains of POWV in Russia are 99,8% similar to the Canadian strain LB, which belongs to lineage I POWV [8]. The design of the studied oligonucleotides was aimed specifically at lineage I POWV. The detection of lineage II POWV is possible, but this needs to be tested in practice.

Conclusion

A laboratory technique based on PCR has been developed that allows to detect the POWV in samples containing other orthoflaviviruses.

About the authors

Maria P. Fedina

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis)

Email: mariafedinamf@gmail.com
ORCID iD: 0009-0000-1530-2050

Junior Researcher in Laboratory of Biology of Arboviruses

Russian Federation, Moscow

Roman I. Ivannikov

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis)

Email: ivannikov_ra@mail.ru
ORCID iD: 0000-0002-1358-5824

Laboratory Research Assistant in Laboratory of Biology of Arboviruses

Russian Federation, Moscow

Galina G. Karganova

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis)

Email: karganova@bk.ru
ORCID iD: 0000-0002-8901-6206

Doctor of Biological Sciences, Professor, Leading Researcher and Head of Laboratory of Biology of Arboviruses

Russian Federation, Moscow

Alexander G. Litov

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis)

Author for correspondence.
Email: novosti-wxo@yandex.ru
ORCID iD: 0000-0002-6086-3655

PhD, Leading Researcher in Laboratory of Biology of Arboviruses

Russian Federation, Moscow

References

Supplementary files