Preparation of MS2-based nanoparticles as control and standard materials for the molecular detection of dengue virus serotypes
Introduction
Dengue is the most rapidly spreading mosquito-borne virus in the world. The latest world-wide survey indicated 390 million cases of dengue infections per year (Bhatt et al., 2013). Apart from indigenous cases in tropical and sub-tropical regions, recently imported cases from epidemic areas could also potentially cause a dengue pandemic. In 2014, part of the dengue epidemic of Guangdong in China was caused by cases imported from Malaysia and Singapore, that then spread to 20 other cities (Huang et al., 2016a), even as far as Japan (Quam et al., 2016). Imported cases were also reported in America (Moncayo et al., 2015), Italy (Quam et al., 2015), and Pakistan (Wesolowski et al., 2015). In consideration of the threat to human health and the substantial economic cost caused by dengue virus (DENV) (Guzman et al., 2010, Martina et al., 2009, Shepard et al., 2013), effective measures should be taken to control its transmission. These measures should include epidemiological surveillance by governments (De Simone et al., 2004, Guo et al., 2016, WHO, 2009), and efficient and accurate detection of DENV in laboratories (Guo et al., 2016, WHO, 2009).
Various DENV serotypes are transmitted to humans through the bites of infected Aedes mosquitoes, principally Ae. Aegypti (WHO, 2009). Their genomes code for a core protein, precursor membrane protein, envelope protein, and nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) (Guzman et al., 2010). Various real-time polymerase chain reaction (RT-PCR) assays have been developed for DENV detection and serotype identification (Chien et al., 2006, De Simone et al., 2004, Hue et al., 2011, Johnson et al., 2005, Muñoz-Jordán et al., 2009). However, when detecting the DENV, several factors, such as nucleic acid extraction techniques, detection methods, instruments, commercial assays used, and disparate individual operators can cause variations in a laboratory’s performance. This makes results obtained from different laboratories non-comparable. Furthermore, many RT-PCR assays have not been validated for accuracy and quality; approximately 10% of published methods show optimal performance, while more than 80% lack sensitivity and/or specificity information (Domingo et al., 2010). Notably, the quantitative (q)RT-PCR assay is highly sensitive to amplicon contamination, and false-positive results may occur. Meanwhile, RNA degradation in the detection process of DENV can result in false-negative results and affect the laboratory performance.
Although there are various problems associated with detecting DENV, no effective measures have been taken to monitor the performance of laboratories and detection assays for this virus in China because of the difficulty in obtaining sufficient clinical samples to prepare control materials. Although quantification of the virus in clinical samples is helpful for improving the ability of laboratories to detect the virus, there are few studies on quantification of the DENV in the world. Considering the above conditions, in this study we constructed DENV-1 to 4 nanoparticle expression systems to produce corresponding MS2-based virus-like particles. We then assessed their suitability as standards for the quantitative detection of DENV-1 to 4, as well as to serve as control materials for assessing the performance of laboratories using qRT-PCR assays across China.
Section snippets
Synthesis of DENV-1 to 4 segments
Based on previous studies (Callahan et al., 2001, Chien et al., 2006, Hu et al., 2010, Hue et al., 2011, Ito et al., 2004, Johnson et al., 2005, Kim et al., 2015, Lau et al., 2015, Leparc-Goffart et al., 2009, Parida et al., 2005, Santiago et al., 2013, Teoh et al., 2015, Waggoner et al., 2013), four genome segments from standard strains of DENV-1 to 4 were obtained from GenBank (accession nos., EU848545.1, KM204118, AY662691.1, and AY947539.1, respectively) and synthesized by Sangon Biotech
Characteristics of DENV-1 to 4 nanoparticles
One percent agarose gel electrophoresis revealed four bands of digested DENV-1 to 4 nanoparticles about 1 kb in size. Undigested nanoparticles were less than 250 bp because the free nucleic acids adsorbed on the surface (Fig. 2a). Polyacrylamide gel electrophoresis revealed an intense protein band at 14 kDa in each sample, exactly the molecular weight of the MS2-based nanoparticle (Fig. 2b). Furthermore, nanoparticles approximately 30 nm in diameter were clearly observed by transmission electron
Discussion
Currently, laboratory detection of DENV mainly targets the infected virus itself or viral markers present in patient serum such as virus-encoded antigens, viral nucleic acids, or virus-induced antibodies (WHO, 2009). Among these, a nucleic acid detection assay, developed 20 years ago (Lanciotti et al., 1992), is a more sensitive and specific tool, and plays an important role in detecting dengue infection. When detecting DENV using qRT-PCR, there are several steps that might affect the
Competing interests
The authors have declared that no competing interests exist.
Funding
This work was supported by the Special Fund for Health-Scientific Research in the Public Interest from the National Population and Family Planning Commission of the People’s Republic of China [No. 201402018].
Acknowledgments
We thank all the laboratories that participated in this program. We also thank all the commercial kit manufacturers for providing the dengue detection reagents in the study.
References (48)
- et al.
Dengue virus surveillance: the co-circulation of DENV-1, DENV-2 and DENV-3 in the State of Rio de Janeiro, Brazil
Trans. R. Soc. Trop. Med. Hyg.
(2004) - et al.
Use of armored RNA as a standard to construct a calibration curve for real-time RT-PCR
J. Virol. Methods
(2005) - et al.
Governmental supervision and rapid detection on dengue vectors: an important role for dengue control in China
Acta Trop.
(2016) - et al.
Validation of an internally controlled one-step real-time multiplex RT-PCR assay for the detection and quantitation of dengue virus RNA in plasma
J. Virol. Methods
(2011) - et al.
Clinical diagnosis of early dengue infection by novel one-step multiplex real-time RT-PCR targeting NS1 gene
J. Clin. Virol.
(2015) - et al.
Quantification of human astroviruses in sewage using real-time RT-PCR
Res. Microbiol.
(2004) - et al.
Development and validation of real-time one-step reverse transcription-PCR for the detection and typing of dengue viruses
J. Clin. Virol.
(2009) - et al.
Assessment of different commercial RNA-extraction and RT-PCR kits for detection of hepatitis A virus in mussel tissues
J. Virol. Methods
(2004) - et al.
Molecular mechanism of RNA phage morphogenesis
Int. J. Biochem.
(1994) - et al.
The three-dimensional structures of two complexes between recombinant MS2 capsids and RNA operator fragmentsreveal sequence-specific protein-RNA interactions
J. Mol. Biol.
(1997)
The global distribution and burden of dengue
Nature
Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays
J. Mol. Endocrinol.
Development and evaluation of serotype- and group-specific fluorogenic reverse transcriptase PCR (TaqMan) assays for dengue virus
J. Clin. Microbiol.
Development of real-time reverse transcriptase PCR assays to detect and serotype dengue viruses
J. Clin. Microbiol.
Quantification and duration of viraemia during hepatitis A infection as determined by real-time RT-PCR
J. Viral Hepat.
2nd International external quality control assessment for the molecular diagnosis of dengue infections
PLoS Negl. Trop. Dis.
Dengue: a continuing global threat
Nat. Rev. Microbiol.
The crystal structure of a high affinity RNA stem-loop complexed with the bacteriophage MS2 capsid: further challenges in the modeling of ligand-RNA interactions
RNA
Anchored pan dengue RT-PCR and fast sanger sequencing for detection of dengue RNA in human serum
J. Med. Virol.
Epidemiology and characteristics of the dengue outbreak in Guangdong, Southern China, in 2014
Eur. J. Clin. Microbiol. Infect. Dis.
Detection of infectious dengue virus by selective real-time quantitative polymerase chain reaction
Virol. Sin.
Development and evaluation of fluorogenic TaqMan reverse transcriptase PCR assays for detection of dengue virus types 1 to 4
J. Clin. Microbiol.
Serotype-specific detection of dengue viruses in a fourplex real-time reverse transcriptase PCR assay
J. Clin. Microbiol.
Dissecting the key recognition features of the MS2 bacteriophage translational repression complex
Nucleic Acids Res.
Cited by (5)
External quality assessment of molecular testing of 9 viral encephalitis-related viruses in China
2021, Virus ResearchCitation Excerpt :Compared with current quality control materials, such as DNA plasmids and patient-derived RNA samples, the MS2-based nanoparticles we have established not only solve the problems associated with DNA plasmids, but also do not reflect the RNA extraction process and the lack of limited RNA samples. MS2-based nanoparticles have been widely used in the EQA programs for various viruses, such as influenza virus, dengue fever virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Fu et al., 2017; Wang et al., 2021; Zhang et al., 2018). Most of the participants in this EQA were commercial kit manufacturers and international travel health care centers.
One-plasmid double-expression system for preparation of MS2 virus-like particles packaging SARS-CoV-2 RNA
2023, Frontiers in Cellular and Infection MicrobiologyRecombinant Retroviral Particles: Technology of Poduction and Application as Positive Controls for PCR Diagnostics of Dangerous Viral Infections
2020, Problemy Osobo Opasnykh InfektsiiApplications of digital PCR in clinical microbiology
2018, Advanced Techniques in Diagnostic Microbiology