Detection of New Bunyavirus RNA by Reverse Transcription–Loop-Mediated Isothermal Amplification

Abstract

dSevere fever with thrombocytopenia syndrome (SFTS) is a newly emerging and epidemic infectious disease in central and northeast China. It is caused by New Bunyavirus and carries an average 12% case fatality rate. Early and rapid detection is critical for prevention and control of New Bunyavirus infection, since no vaccine or antiviral drugs are currently available, and prevention requires careful attention to control of the suspected tick vector. In this study, a simple and sensitive reverse transcription-loopmediated isothermal amplification (RT-LAMP) assay was developed for rapid detection of New Bunyavirus. The detection limit of the RT-LAMP assay was approximately 10 3 50% tissue culture infective doses/ml of New Bunyavirus in culture supernatants, and no cross-reactive amplification of other viruses known to cause similar clinical manifestations was observed. The assay was further evaluated using 138 specimens from clinically suspected SFTS and 40 laboratory-proven hantavirus infection with fever and renal syndrome patients, and the assay exhibited 97% agreement compared to real-time RT-PCR and conventional RT-PCR. Using real-time RT-PCR as the diagnostic gold standard, RT-LAMP was 99% sensitive and 100% specific. The RT-LAMP assay could become a useful alternative in clinical diagnosis of SFTS caused by New Bunyavirus, especially in resource-limited hospitals or rural clinics of China. Since 2007, many patients in China have presented with an illness with clinical characteristics that include acute onset of fever with leukopenia, thrombocytopenia, elevated serum hepatic enzyme levels, gastrointestinal manifestations, neurological symptoms, and bleeding tendencies. The disease was initially suspected to be human granulocytic anaplasmosis (HGA) (1), but in 2009, difficulty in proving that etiology prompted syndromic characterization of the patients as having "severe fever with thrombocytopenia syndrome" (SFTS), while other potential etiologies were investigated (2). A novel bunyavirus was isolated from patient acute-phase serum samples and is now known as New Bunyavirus (NBV), severe fever with thrombocytopenia syndrome bunyavirus (SFTSV), or Huaiyangshan virus (HYSV) (1-3).As the clinical manifestations of SFTS are nonspecific, there is a need to develop assays that can differentiate this from various other infectious diseases, in particular, hemorrhagic fever with renal syndrome (HFRS) and HGA. Reliable rapid and accurate diagnostic tests are urgently required to identify and diagnose clinically suspected SFTS, to assess treatment efficacy, and for disease surveillance (4).Recently, a novel DNA amplification method called loop-mediated isothermal amplification (LAMP) has been applied for diagnosis of infectious diseases (5). The addition of reverse transcriptase makes it possible to amplify RNA sequences by LAMP (reverse transcription-loop-mediated isothermal amplification, or RT-LAMP). RT-LAMP has already been applied to the detection of several RNA viruses, such as enterovirus 71 (EV71), swineorigin inf...

Severe fever with thrombocytopenia syndrome: a newly discovered emerging infectious disease

“…A loop-mediated isothermal amplification assay targeting the S segment of SFTSV has been developed; with this assay, the entire procedure was completed within 30 min, and the sensitivity of the assay was 94.4% [54,55]. An RT-cross-priming amplification with a vertical flow visualization strip assay based on the M segment of SFTSV, with a sensitivity and specificity of 94.1% and 100.0%, respectively, has been developed [56]. A multiplex real-time RT-PCR assay has been developed for simultaneous detection of SFTSV, Hantaan virus, Seoul virus and Dengue virus for patients 2-12 days after the onset of symptoms; the sensitivity was 100% [57].…”

Colorimetric biosensors for point-of-care virus detections

“…(1) For human: severe acute respiratory syndrome coronavirus (SARS-CoV) [58,59], dengue viruses [60,61], hepatitis A viruses [12], hepatitis B viruses [7,8], hepatitis C viruses [62], human severe fever of bunya viruses with thrombocytopenia syndrome (SFTS) [63][64][65], influenza A H1N1 viruses infecting human [34], Zika [48], Enterovirus 71 for human hand, foot, and mouth disease (HFMD) [66], human immun-odeficiency virus 1 (HIV-1) [30], Zaire ebolaviruses [67], rotaviruses in fecal samples for acute viral gastroenteritis [68]. (2) For arthropod: Chikungunya viruses [11] and arthropodborne West Nile viruses [69].…”

Application of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for the detection of SFTSV-specific human IgG and IgM antibodies by indirect ELISA

“…Virus isolation from the blood of viremic patients is the direct evidence of SFTSV infection, however, it is time-consuming and needs high security biocontainment facility [1,2]. Detection of SFTSV genome could be achieved by different nucleic acid detection techniques such as reverse transcription-PCR (RT-PCR) [1,2], real-time RT-PCR [14,17], reverse transcriptionloop-mediated isothermal amplification assay (RT-LAMP) [18][19][20], reverse transcription-cross-priming amplification coupled (RT-CPA) with vertical flow (VF) visualization [21]. Although these techniques have high sensitivity and specificity in early diagnosis, the duration of viraemia in SFTSV infection is very short, generally 1-6 days after the disease onset [22].…”