Rapid detection of Akabane virus by a novel reverse transcription loop-mediated isothermal amplification assay (RT-LAMP)

Background Akabane disease, caused by Akabane virus, is an insect-transmitted disease of ruminants that is primarily characterized by fetal damage. Methods and results In this study, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of Akabane virus was successfully developed. The primers were designed to target the highly conserved fragment of nucleoprotein from the Akabane virus. The results indicate that the assay is highly specific and sensitive with a detection limit of 5.0 TCID50 /mL within a 60-min incubation time. A total of 126 abortive samples collected from Xinjiang province were detected by the established RT-LAMP. The results of RT-LAMP assay showed 96.8% agreement with the semi-nested RT-PCR. Conclusion This study is to first to develop a rapid, sensitive, and accurate method for the detection of Akabane virus, which may be used to screen clinical samples in developing countries or regions.


Background
Akabane virus, the causative agent of enzootic bovine arthrogryposis and hydranencephaly, is an arbovirus in the genus Orthobunyavirus and the Simbu sero-group of the family Bunyaviridae. This particular virus is transmitted among animals by hematophagous arthropod vectors such as mosquitoes and culicoides biting midges [1]. The infection of Akabane virus in pregnant cows can lead to a series of endemic and abnormal deliveries such as abortion, premature birth, stillbirth, and birth of abnormal calves [1][2][3][4][5]. Between 1972 to 1975, more than 40,000 cattle were infected in Japan [6], which resulted in a huge economic loss to the cattle industry. To date, Akabane disease is widely prevalent in Japan, Korea, Israel, Central Africa, and Australia [1,4,5,7,8].
Although laboratory diagnostic techniques for Akabane disease such as viral neutralization test (VNT), enzymelinked immunosorbent assays (ELISAs) [9][10][11][12], immunoperoxidase staining [13,14], dot immunobinding assay [15], nested reverse transcription polymerase chain reaction (RT-PCR) [16], multiplex RT-PCR [17] and real-time RT-PCR [18] have been established, most of the current serological tests have cross-reactions with other related viruses, particularly those in the simbu serogroup [9,15,19]. PCR-based molecular techniques need specialized equipment and may not be readily available in diagnostic laboratories of underdeveloped and developed countries [16]. Viral isolation from aborted fetus is rarely successful because of an elicited immune response that inhibits virus replication. Therefore, it is necessary to develop new assays for the detection of Akabane virus in the clinic.
In this study, we demonstrated that loop-mediated isothermal amplification (LAMP) provides a new method for molecular diagnosis [20]. LAMP has been widely used to detect different pathogens because of its rapid detection [21,22]. The aim of this study is to develop a rapid and accurate molecular tool for detecting Akabane virus in clinical specimens.

Results and discussion
The results demonstrated that only Akabane virus was detectable by RT-LAMP assay ( Figure 1). In contrast, this assay did not detect the other tested viral strains. Following AccIII digestion, the amplification products appeared as 2 fragments with sizes of 33 and 180 bp ( Figure 2). The results also showed that the RT-LAMP assay is highly sensitive as it can detect a template at 10 -5 dilution with a detection limit of 5.0 TCID 50 /mL ( Figure 3). RT-LAMP exhibited high concordance with the semi-nested RT-PCR for Akabane virus detection in clinical samples (Table 1). Fifty-one out of 126 samples were positive for the Akabane virus using both RT-LAMP and semi-nested RT-PCR assays. One negative sample from semi-nested RT-PCR was positive by RT-LAMP assay, and 3 negative samples from RT-LAMP assay were positive by semi-nested RT-PCR. An additional seventy-one samples also were negative from both assays. Accordingly, the cohen's kappa coefficient for these two assays was 96.62%.
In this study, we report a novel and rapid molecular detection technique named RT-LAMP assay to detect Akabane virus from clinical samples with high specificity (Figure 2). This assay can distinguish Akabane virus from other four viruses known to cause abortion in cattle (bovine herpesvirus 1, bovine parvovirus, bluetongue virus, and bovine viral diarrhea virus). In addition, the developed RT-LAMP is simple and rapid compared to other molecular detection assays (single RT-PCR, nested RT-PCR, or real-time RT-PCR). Therefore, RT-LAMP has great potential for use as a field test to detect Akabane viral infection.
A critical factor when performing the RT-LAMP assay is to select a conserved nucleic acid fragment for the design of specific primers. In the present study, 25 sequences of the nucleoprotein gene of Akabane virus reported in GenBank were analyzed, and a highly conserved fragment was chosen as the target region ( Figure 4). Two pairs of specific primers (two inner primers and two outer primers) were designed using the Primer Explorer Version 4 software. On these bases, these primers were further analyzed by Blast N software. The rigorous selection and analysis of high conservation fragment are conducive to the specificity of established RT-LAMP.
The detection of positive amplification of RT-LAMP can be accomplished by agarose gel electrophoresis and real-time monitoring using a turbidimeter. In addition, amplification of the target gene can also be visualized by a fluorescent intercalating dye. In this study, SYBR Green I was used for detection of the amplified products by observing color change. The orange color of the dye will fluoresce green under natural light with positive amplification. For the cases with no amplification, the orange color of the dye is retained.
We show that the RT-LAMP assay using 126 clinical samples demonstrated that the RT-LAMP assay is high sensitive and accurate, similar to the semi-nested RT-PCR assay. The detection method also demonstrated that the Akabane virus infection is common in calves for various geographical areas of Xinjiang province. Thus, preventive measures are recommended for the cattle industry.
In conclusion, a novel RT-LAMP assay was successfully developed for the rapid detection of Akabane virus genomic RNA in clinical samples from abortive cases. The assay is highly sensitive and specific. This rapid, easy-to-operate, and sensitive assay can be used for the diagnosis and epidemiological surveillance of Akabane virus infection in resource-limited countries or regions.

Design of specific RT-LAMP primers
Nucleoprotein (N) gene sequences of 25 different strains/isolates of Akabane virus were obtained from GenBank, and the homology was analyzed using the molecular software DNAMAN. The conserved fragment with high homology was chosen to be the target region ( Figure 4) which was used to design the Akabane virus RT-LAMP primers by the Primer Explorer V4 software (http://primerexplorer.jp/e/) (Table 2), as previously described by Notomi et al. [20].

RNA extraction
Total RNA was extracted from Akabane virus-infected BHK-21 cells or clinical samples with SV-total RNA Isolation System (Promega, Mannheim, Germany), according to the manufacturer's protocol. The RNA was eluted in 20 μL of RNase-free water containing 0.04%

RT-LAMP
The 2 μL of template RNA, and 6.5 μL of distilled water. The reaction mixture was incubated at 63°C for 60 min in a heating block, followed by heating at 80°C for 2 min to terminate the reaction. After the reaction, a 10 μL aliquot of the RT-LAMP product was subjected to electrophoresis in 2.0% agarose gel and visualized by staining with 0.5 μg/mL of ethidium bromide under ultraviolet light. For visual fluorescence detection, 1 μL of a 10-fold dilution of SYBR Green I (1000×) was added into the reaction mixture, and the positive amplification was determined according to the color change (orange and green in a negative and positive reaction, respectively).

Analytical sensitivity and specificity of RT-LAMP
To evaluate the specificity of the RT-LAMP reaction, four viruses known to cause abortion in cattle, bovine herpesvirus 1 (strain 758), bovine parvovirus (strain BF15), bluetongue virus (strain CSIRO 154) and bovine viral diarrhea virus (strain NADL) were used. To determine the sensitivity of RT-LAMP assay, the culture supernatant containing 5×10 5 TCID 50 /mL of Akabane virus was diluted 10-fold and amplified by RT-LAMP assay. For further confirmation, the amplified products were confirmed by DNA sequencing and digested with restriction enzyme AccIII (Promega) and their sizes of digested products were analyzed by electrophoresis in 3% agarose gels. Visualization was performed by straining with ethidium bromide.

Semi-nested RT-PCR
The semi-nested RT-PCR was performed using the specific primers N1 (outer sense primer), N2 (antisense primer) and N3 (inner antisense primer) (   10 min. 2 μL of the first amplification product was used as DNA template for each of the 25 μL secondary amplifications. The conditions and concentrations of the secondary amplification were identical to those of the primary, except for the primers (N1 and N3) and extension at 72°C for 30 s. The PCR products of the secondary amplification round were analyzed by 3% gel electrophoresis and stained with ethidium bromide, and confirmed by DNA sequencing.

Evaluation of the RT-LAMP Using Clinical Samples
The clinical samples were subjected to total RNA extraction as described above. The RT-LAMP assay was used to detect the presence of Akabane virus. In addition, semi-nested RT-PCR was employed to examine the presence of Akabane virus and for testing the consistency between the two assays (RT-LAMP and semi-nested RT-PCR). The Cohen's kappa coefficient between RT-LAMP and semi-nested RT-PCR was calculated according to the following formula.

Ethical approval
The experiments were carried out in accordance with the guidelines issued by the Ethical Committee of Shihezi University.