Detection of Langat virus by TaqMan real-time one-step qRT-PCR method

Langat virus (LGTV), one of the members of the tick-borne encephalitis virus (TBEV) complex, was firstly isolated from Ixodes granulatus ticks in Malaysia. However, the prevalence of LGTV in ticks in the region remains unknown. Surveillance for LGTV is therefore important and thus a tool for specific detection of LGTV is needed. In the present study, we developed a real-time quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) for rapid detection of LGTV. Our findings showed that the developed qRT-PCR could detect LGTV at a titre as low as 0.1 FFU/ml. The detection limit of the qRT-PCR assay at 95% probability was 0.28 FFU/ml as determined by probit analysis (p ≤ 0.05). Besides, the designed primers and probe did not amplify ORF of the E genes for some closely related and more pathogenic viruses including TBEV, Louping ill virus, Omsk hemorrhagic fever virus (OHFV), Alkhurma virus (ALKV), Kyasanur Forest Disease virus (KFDV) and Powassan virus (POWV) which showed the acceptable specificity of the developed assay. The sensitivity of the developed method also has been confirmed by determining the LGTV in infected tick cell line as well as LGTV- spiked tick tissues.

there is more than 80% of homology between the genome of TBEV and LGTV. The high percentage of antigenic similarity between TBEV and LGTV was the main reason that some investigators have studied LGTV as a potential candidate for vaccine development against TBEV. However, the current LGTV prevalence in ticks and humans in Malaysia is unknown. This suggests the importance of surveillance for the virus in the region and thus the need of a tool for specific detection of LGTV.
In the present study, we have designed and developed a TaqMan real-time quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) method for rapid detection of LGTV.

Results
Virus propagation and titration in Vero cells. The LGTV infectious titre was obtained by calculating the numbers of visible foci from focus forming assay (FFA) 3 days post infection (poi) in Vero cells. In this study, the LGTV infectious titre was 1.45 × 10 6 FFU/ml. The titre of this virus was used in determining the highest amount of LGTV RNA in the qRT-PCR which was 1.0 × 10 6 FFU/ml.

Generation of the clones for E gene of closely related viruses to
LGTV. The synthesized sequences for the E gene of Louping ill virus, OHFV, ALKV, KFDV, and POWV as the closely related viruses to LGTV were cloned in pET-51b(+ ) plasmid. The data from sequencing of the constructed clones have shown that all E genes were cloned successfully.
Generation of TBEV RNA. For further assessment of specificity for the developed qRT-PCR, against the TBEV as the closest virus with LGTV, a TBEV RNA was used and generated via in vitro transcription of pTNd/5′ containing E gene of TBEV. The in vitro transcription yielded TBEV RNA at a concentration of 187 ng/μ l. Amplification of a 532 bp DNA fragment by the TBEV RT-PCR assay indicated the presence of TBEV RNA (Fig. 1). The in vitro-transcribed TBEV RNA was used in the qRT-PCR for specificity evaluation.
Specificity of the LGTV qRT-PCR assay. Initially, the similarity between the designed primers/probe and the ORF for E gene of LGTV and other related viruses have been calculated theoretically. Multiple sequence alignment of the viral E gene sequences has been done using Clustal X 2.0 software and as it is shown in Table 1 the most significant similarity has been found between the designed primers/probe and E gene ORF for LGTV with 100% similarity. Whereas the similarity between the sequences of designed primers/probe with TBEV and other related viruses were not too significant (Table 1). Therefore, the chance of detection of each related viruses with synthesized primers and probe was not considerable theoretically. However, the obtained data in this part of the study have been confirmed using synthesized E genes of all related viruses.
The constructed clones for all synthesized E genes of related viruses in pET-51b(+ )vector have been evaluated by our developed LGTV qRT-PCR assay and as it is shown in Fig. 4b, there was no amplification for all those viruses with Eff % = 118.189 and R 2 = 0.998.
Nevertheless, the specificity of LGTV qRT-PCR also determined against in vitro-transcribed TBEV RNA. The Eff% of LGTV qRT-PCR against TBEV was 117.976 and R 2 was 0.999. There was no amplification for in vitro-transcribed TBEV RNA (Fig. 4c).

Sensitivity of the LGTV qRT-PCR assay.
To determine the sensitivity of the LGTV qRT-PCR assay, LGTV spiked crushed homogenized tick tissues and also LGTV infected IDE8 cells have been studied in two independent experiments. A homogenate of non-infected crushed tick tissue has been spiked with LGTV together with necessary controls including another crushed tick tissue sample, which has been spiked by LGTV and TBEV RNA as well as non-infected crushed tick tissue sample. As it is shown in Fig. 4d, our developed qRT-PCR method can detect LGTV but not TBEV RNA in both spiked samples. The Eff% of qRT-PCR was 108.91 and R 2 was 0.998.
As a further investigation to prove the sensitivity of our developed qRT-PCR, extracted RNA of LGTV from the LGTV-infected IDE8 cell line has been evaluated in 2 and 4 days poi. The amplification of the LGTV RNA in LGTV-infected IDE8 cell line has been shown successfully with increasing RNA level through the day 2 to 4 poi that were equivalent to the 406,268 FFU/ml of LGTV in day 2 post infection and 776,412 FFU/ml in day 4 post infection. The Eff% of LGTV qRT-PCR was 135.054 and the R 2 was 0.995. There was no amplification detected in the non-infected IDE8 cells as negative control for this assay (Fig. 4e).

Discussion
In the present study, a novel real-time qRT-PCR assay was developed for detection of LGTV TP21 strain. We demonstrated that the qRT-PCR assay could detect the LGTV at a titer as low as 0.1 FFU/ ml. Probit analysis determined that the 95% detection limit of the qRT-PCR assay was 0.28 FFU/ml. This showed that the developed qRT-PCR assay was sensitive. Besides, the sensitivity of the qRT-PCR was determined against viral spiked crushed homogenised tick tissues and viral infected IDE8 tick cell line with LGTV and LGTV/TBEV together. The purpose of spiking both LGTV and TBEV together  was to determine whether the designed and developed qRT-PCR in this study could differentiate the LGTV from TBEV as the closest virus in terms of genome homology in infected tick tissues and cell line or not. However, our data have proven that the developed qRT-PCR only can detect and amplify the LGTV RNA in both LGTV spiked samples specifically. The negative controls including non-spiked tick tissues and TBEV-RNA spiked tick tissues showed no amplification. This showed the sensitiveness of our developed qRT-PCR. Our data from LGTV-infected IDE8 cell line has shown that the LGTV RNA level has increased through the incubation time poi. This finding showed the capability of our developed method for quantification of LGTV in samples and also it has been proven that IDE8 cell line could be a proper tick cell line for LGTV replication and propagation. Interestingly, there was no amplification for non-LGTV infected IDE8 cells/crushed tick tissues as another evidence to prove the specificity of the developed qRT-PCR in this study.
In order to assess the specificity of the developed LGTV qRT-PCR assay in this study, the E gene for five closely related viruses including Louping ill virus, OHFV, ALKV, KFDV, and POWV were synthesized and cloned in pET-51b(+ ) vector followed by evaluation by developed LGTV qRT-PCR. We have shown that our developed qRT-PCR did not amplify the E gene of six closely related viruses despite of the genomic homology between all those viruses and LGTV that could be a proof for the specificity of the developed qRT-PCR. However, as one of the limitations of our study we did not have access to all those viruses in our lab.
Furthermore in vitro-transcribed TBEV RNA was used to evaluate specificity of LGTV qRT-PCR. From Fig. 4c there was no amplification of the in vitro-transcribed TBEV RNA. This showed that the developed qRT-PCR can detect LGTV RNA only. The percentage of identity of LGTV primers and probe against closely related viruses in Table 1 showed the specificity of the developed qRT-PCR.
Campbell and colleagues previously developed a RT-PCR assay for detection of LGTV using a primer set targeting the viral C-prM region. They tested the RT-PCR on LGTV-infected Vero cell tissue culture supernatant fluid. The RT-PCR primers however showed cross-reaction with TBEV 9 .In the present study, we developed a real-time qRT-PCR assay for specific detection of LGTV and unlike to previous study, the qRT-PCR primers and TaqMan probe designed in our study targeted regions of the viral E gene that are conserved among LGTV but vary from those of closely related TBEV. In addition, the qRT-PCR assay can be completed in a shorter time (approximately 40 min) than the RT-PCR as post-amplification analysis by gel electrophoresis is not required.
As one of the limitations for our study we did not use viable intact TBEV and other related viruses due to unavailability of those viruses in our laboratory and also our collaborator's laboratory. However, all mentioned viruses are categorized under the Biosafety Level 3 (BSL3) pathogens in Malaysia 13 as they are not prevalent in the region. Nevertheless, we have tried to evaluate the specificity of our developed method against synthesized E genes of all related viruses as an alternative solution following theoretical study by alignment of primers/probe with relative viral ORFs. Therefore, we suggest the further evaluation of our developed method using all related infectious viruses for the future studies. Nevertheless, the developed qRT-PCR assay in this study would provide a good start for surveillance and exploration of new LGTV samples in the region.
In conclusion, the qRT-PCR assay developed in our study is sensitive and specific for the rapid detection of LGTV. While there is limited information describing LGTV infection in humans and ticks, the qRT-PCR assay could eventually help to improve virological surveillance in the regions where LGTV is endemic. LGTV  Virus titration. Viral FFA using Vero cells determined virus infectivity titre of the LGTV. Cells were seeded at 6 × 10 5 cells/well in 24-well plates and cultured overnight as above. Growth medium was removed and 200 μ l of 10-fold serially diluted LGTV(1:10 to 1:10 9 ) in EMEM with 2% FBS was added to the wells. After 1 h of adsorption at 37 °C in the presence of 5% CO 2 , the medium was replaced with 500 μ l of overlay comprising EMEM containing 2% FBS and 1.5% carboxymethylcellulose (Sigma-Aldrich, USA). The cells were incubated for 3 days as above. After 3 days of incubation, foci of infected cells were detected by a peroxidase-based viral focus-staining assay. In brief, the overlay was discarded and cells were washed gently three times with phosphate buffered saline (PBS). The cells were fixed with 4% paraformaldehyde for 30 min at room temperature followed by three washes with PBS. 300 μ l of 1% IgepalCA-630 detergent (Sigma-Aldrich, St. Louis, MO, USA) was added to permeabilize the cells for 15 min at room temperature. Subsequently, the cells were washed three times with PBS and blocked with 3% skimmed milk (Sigma, St. Louis, MO, USA) in PBS for 2 h at room temperature. After another three washes with PBS, the cells were incubated with anti-flavivirus monoclonal antibody D14G2 (Merck Millipore) diluted 1:500 in 1% skimmed milk in PBS at 37 °C for 1 h. Cells were then washed three times with PBS and incubated with goat-anti-mouse IgG conjugated with horse-radish peroxidase (Merck Millipore) at a dilution of 1:250 in 1% skimmed milk in PBS for 1 h and 10 min at 37 °C. After washing three times with PBS, Metal-Enhanced DAB Substrate (Thermo Scientific Pierce, Rockford, IL, USA) was added to each well for 10 min to stain the virus foci. Foci were counted under a SMZ 1000 stereomicroscope (Nikon, Tokyo, Japan) and the virus titre was expressed as focus-forming units/ml (FFU/ml).
LGTV RNA was extracted from virus stock using the QIAmp Viral RNA Mini Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions and extracted RNA stored at − 80 °C until further use.

Synthesizing of E genes for LGTV-related viruses. The envelope gene of five closely related viruses:
Louping ill virus, OHFV, ALKV, KFDV, and POWV were used to determine the specificity of the LGTV qRT-PCR assay. The sequences for all viral E genes used in this study were retrieved from GenBank ( Table 2). The E genes of the five viruses were synthesized and cloned in pET-51b(+ ) vector by Genscript company (Genscript, Piscataway, NJ) followed by sequencing to show the accuracy of the gene synthesis.
Generation of TBEV RNA. The plasmid (pTNd/5′ ) containing cDNA derived from TBEV strain Neudoerfl was kindly provided by Professor Franz Heinz of the Department of Virology, Medical University of Vienna, Austria and used in this study to assess the specificity of the qRT-PCR assay for LGTV detection. The pTNd/5′ was transformed into NovaBlue competent cells (Novagen Inc. USA) for propagation. After overnight bacterial culture, the bacterial cells were lysed and the pTNd/5′ was extracted using a Qiagen Plasmid Extraction kit (Qiagen, Hilden, Germany).
The pTNd/5′ was linearized by digestion with NheI-HF (New England Biolabs, USA) at 37 °C for 1 h and 65 °C for 20 min. The digested pTNd/5′ was analyzed by agarose gel electrophoresis. The linearized pTNd/5′ was extracted and purified from the agarose gel using a QiaQuick Gel Extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Subsequently, the purified pTNd/5′ was transcribed by T7 RNA polymerase using a MEGAscript ® T7 Kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions. Briefly, 1 μ g of purified pTNd/5′ was added to a 20 μ l transcription reaction containing 2 μ l of each of ribonucleotide solutions (ATP, CTP, GTP, and UTP), 2 μ l of 10 × reaction buffer and 2 μ l of T7 RNA Polymerase enzyme mix. The reaction was incubated at 37 °C for 4 h. After in vitro transcription, the transcription product was treated with 1.0 μ l DNase and the concentration of RNA was measured using a NanoPhotometer (Implen).

Preparation of LGTV spiked tick lysate. Ticks were collected from Kampung Tumboh Hangat and
Kampung Sungai Perah, Perak. The ticks were stored in 70% ethanol for 3 min, 5% bleached for 3 min and washed twice with double distilled water for 3 min followed by drying on filter paper for 3 min. In next step, the sample was frozen in liquid nitrogen and powdered with a steel mortar and pestle followed by homogenizing the lysate in PBS. Following a brief spin, the homogenized tick tissue sample was spiked with 10 3 FFU/ml of LGTV and one sample spiked with LGTV and TBEV. The negative controls consisted of non-spiked tick tissues and also one sample with TBEV-spiked tick tissue. The RNA was extracted and stored at − 80 °C until further use.

Preparation of LGTV infected IDE8 cells. The sensitivity of LGTV qRT-PCR was evaluated via
LGTV RNA extracted from LGTV infected-IDE8 cells as well. IDE8 cells were seeded at 1 × 10 6 cells/ tube in flat-sided tube and cultured overnight as above. Growth medium was removed and 200 μ l of LGTV in L15 medium with 2% FBS was added into the tube. Cells in negative control tube were maintained with medium with no LGTV. After 1 h of adsorption at 32 °C, the cells were washed gently three times with serum free L15 medium and replaced with 3 ml of L15 medium with 2% FBS. The cells were incubated for 2 days and 4 days at temperature as above. After 2 days and 4 days of incubation, RNA of LGTV was extracted from IDE8 cells using the RNeasy ® Plus Mini Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. All RNA extraction was performed manually. The RNA was eluted in 40 μ l of RNase-free water and stored at − 80 °C until further use.
Design of LGTV-specific qRT-PCR primers and probe. The LGTV-specific primers and TaqMan probe used for the qRT-PCR assay were designed based on conserved regions of the E gene of LGTV. Sequences of the E genes from the closely related viruses TBEV, Louping ill virus, OHFV, ALKV, KFDV, and POWV 14 ( Table 2) were used during the design process to avoid cross-reactivity of the LGTV primers and probe. All the viral E gene sequences used in this study were retrieved from GenBank. Multiple sequence alignment of the viral E gene sequences was performed using Clustal X 2.0 15 .
Primers and probe analysis relative to LGTV E gene ORF. The E gene sequence of LGTV and closely related viruses were retrieved from GenBank (Table 2). Forward primer, reverse primer and TaqMan probe of LGTV qRT-PCR was analysed relative to LGTV E gene ORF at position of forward primer: 1607-1625; reverse primer: 1652-1671 and probe: 1628-1646. Percentage of identity was calculated to determine the specificity of the LGTV qRT-PCR primers and probe against closely related viruses.
Development of qRT-PCR assay. The qRT-PCR was performed in a total reaction volume of 12.0 μ l consisting of1 × TaqMan ® Fast Virus 1-step Master Mix (Applied Biosystems, USA), 1 × CustomTaqMan ® Gene Expression Assays (Applied Biosystems, USA), and 2 μ l of RNA template. The qRT-PCR standard curve, ranging from 1 to 10 6 FFU/ml, was generated from a 10-fold serial dilution of LGTV RNA extracted from the virus stock with known viral titer. All amplifications were performed in triplicate using the StepOnePlus ™ Real-Time PCR System (Applied Biosystems, USA) with the following amplification cycles: reverse transcription at 50 °C for 5 min, initial denaturation at 95 °C for 20 s, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. Raw data was analyzed with StepOne Software v2.2.1 to determine the amount of viral RNA based on the threshold cycles (Ct). The efficiency of the qRT-PCR was measured from the slope of the standard curve.
Detection limit of developed qRT-PCR assay. The detection limit of the qRT-PCR assay was assessed by using a panel of serially diluted viral RNA samples in nuclease-free water extracted from culture supernatant with known viral titers of 100, 50, 10, 5, 1, 0.5 and 0.1 FFU/ml (quantitated by viral titration assay). The qRT-PCR detection limit test was repeated nine times. A probit analysis was performed using IBM SPSS Statistics, version 21 (IBM Corporation, New York, United States) to calculate the detection limit of the qRT-PCR assay at 95% probability.