RNA-dependent RNA Polymerase Gene Research Articles

Molecular epidemiology and genetic diversity of norovirus among hospitalized children with acute gastroenteritis in Tianjin, China, 2018\u20132020

BackgroundNorovirus (NoV) is a major cause of viral acute gastroenteritis (AGE) in children worldwide. Epidemiological analysis with respect to the virus strains is limited in China. This study aimed to investigate the prevalence, patterns, and molecular characteristics of NoV infection among children with AGE in China.MethodsA total 4848 stool samples were collected from children who were admitted with AGE in Tianjin Children’s Hospital from August 2018 to July 2020. NoV was preliminarily detected using real-time reverse transcription polymerase chain reaction (RT-PCR). Partial sequences of the RNA-dependent RNA polymerase (RdRp) and capsid genes of positive samples were amplified by conventional RT-PCR and then sequenced. The NoV genotype was determined by online Norovirus Typing Tool Version 2.0, and phylogenetic analysis was conducted using MEGA 6.0.ResultsThe prevalence of NoV was 26.4% (1280/4848). NoV was detected in all age groups, with the 7–12 months group having the highest detection rate (655/2014, 32.5%). NoV was detected during most part of the year with higher frequency in winter than other seasons. Based on the genetic analysis of RdRp, GII. Pe was the most predominant genotype detected at 70.7% (381/539) followed by GII.P12 at 25.4% (137/539). GII.4 was the most predominant capsid genotype detected at 65.3% (338/518) followed by GII.3 at 26.8% (139/518). Based on the genetic analysis of RdRp and capsid sequences, the strains were clustered into 10 RdRp–capsid genotypes: GII.Pe-GII.4 Sydney 2012 (65.5%), GII.P12-GII.3 (27.2%), GII.P16-GII.2 (1.8%), GII.P12-GII.2 (0.2%), GII.P17-GII.17 (1.1%), GII.Pe-GII.3 (1.8%), GII.Pe-GII.2 (1.1%), GII.Pe-GII.1 (0.4%), GII.16-GII.4 Sydney 2012 (0.7%), and GII.P7-GII.6 (0.2%). The predominant NoV genotypes changed from GII.Pe-GII.4 Sydney 2012 and GII.P12-GII.3 between August 2018 and July 2019 to GII.Pe-GII.4 Sydney 2012 and GII.P16-GII.2 between August 2019 and July 2020. The patients with GII.Pe-GII.4 Sydney 2012 genotype were more likely to suffer from vomiting symptom than those with GII.P12-GII.3.ConclusionsNoV is an important pathogen responsible for viral AGE among children in China. GII.Pe-GII.4 Sydney 2012 and GII.P12-GII.3 were major recombinant genotypes. Knowledge of circulating genotypes and seasonal trends is of great importance for disease prevention and surveillance.

Discovery and Characterization of a Novel Bipartite Botrexvirus From the Phytopathogenic Fungus Botryosphaeria dothidea.

In this study, we describe a novel positive, single-stranded (+ss) RNA mycovirus, named Botryosphaeria dothidea botrexvirus 1 (BdBV1), from a phytopathogenic fungus Botryosphaeria dothidea showing abnormal morphology and attenuated virulence. BdBV1 is phylogenetically related to Botrytis virus X (BotVX) and is the second potential member of the proposed genus Botrexvirus in the family Alphaflexiviridae. However, it differs from the monopartite BotVX in that BdBV1 possesses a bipartite genome comprised of two ssRNA segments (RNA1 and RNA2 with lengths of 5,035 and 1,063 nt, respectively). BdBV1 RNA1 and RNA2 encode putative RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes, which share significant identity with corresponding genes in both fungal and plant viruses. Moreover, open reading frames (ORFs) 2–4 of BdBV1 RNA1 shared no detectable identity with any known viral proteins. Immunosorbent electron microscopy (ISEM) analysis using an antibody against the virus CP generated in vitro revealed that BdBV1 is encapsidated in filamentous particles. A comparison of the biological effects of BdBV1 infection on symptoms and growth in isogenic lines of virus-free and virus-infected B. dothidea revealed that BdBV1 is probably involved in reduced growth and virulence of the host fungus. This study describes and characterizes a novel bipartite botrexvirus, which is closely related to uni- and multi-partite fungal and plant viruses and contributes useful information to a better understanding of virus evolution.

A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a major threat to public health. Rapid molecular testing for convenient and timely diagnosis of SARS-CoV-2 infections represents a challenge that could help to control the current pandemic and prevent future outbreaks. We aimed to develop and validate a multiplex and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using lyophilized LAMP reagents for sensitive and rapid detection of SARS-CoV-2. LAMP primers were designed for a set of gene targets identified by a genome-wide comparison of viruses. Primer sets that showed optimal features were combined into a multiplex RT-LAMP assay. Analytical validation included assessment of the limit of detection (LoD), intra- and inter-assay precision, and cross-reaction with other respiratory pathogens. Clinical performance compared to that of real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) was assessed using 278 clinical RNA samples isolated from swabs collected from individuals tested for COVID-19. The RT-LAMP assay targeting the RNA-dependent RNA polymerase (RdRp), membrane (M), and ORF1ab genes achieved a comparable LoD (0.65 PFU/mL, CT=34.12) to RT-qPCR and was 10-fold more sensitive than RT-qPCR at detecting viral RNA in clinical samples. Cross-reactivity to other respiratory pathogens was not observed. The multiplex RT-LAMP assay demonstrated a strong robustness and acceptable intra- and inter-assay precision (mean coefficient of variation, 4.75% and 8.30%). Diagnostic sensitivity and specificity values were 100.0% (95% CI: 97.4–100.0%) and 98.6% (95% CI: 94.9–99.8%), respectively, showing high consistency (Cohen’s kappa, 0.986; 95% CI: 0.966–1.000; p<0.0001) compared to RT-qPCR. The novel one-step multiplex RT-LAMP assay is storable at room temperature and showed similar diagnostic accuracy to conventional RT-qPCR, while being faster (<45 min), simpler, and cheaper. The new assay could allow reliable and early diagnosis of SARS-CoV-2 infections in primary health care. It may aid large-scale testing in resource-limited settings, especially if it is integrated into a point-of-care diagnostic device.

Natural infection of covert mortality nodavirus affects Zebrafish (Danio rerio)

Covert mortality nodavirus (CMNV), a novel aquatic pathogen, causes viral covert mortality disease (VCMD) in shrimps and also known to infect farmed marine fish. To date, there has no report regarding the ability of this virus to infect freshwater fish. In this study, we screened and discovered CMNV-positive freshwater zebrafish individuals by reverse transcription-nested PCR (RT-nPCR). The sequence of CMNV amplicons from zebrafish was found to share 99% identity with RNA-dependent RNA polymerase (RdRp) gene of the original CMNV isolate. Histopathological examination of the CMNV-positive zebrafish samples revealed extensive vacuolation and karyopyknosis lesions in the retina of the eye and the midbrain mesencephalon. CMNV-like virus particles were visualized in these tissues under transmission electron microscope. Different degrees of pathological damages were also found in muscle, gills, thymus and ovarian tissues. Strong positive signals of CMNV probe were observed in these infected tissues by in situ hybridization. Overall, all results indicated that zebrafish, an acknowledged model organism, could be infected naturally by CMNV. Thus, it is needed to pay close attention to the possible interference of CMNV whether in assessment of toxic substances, or in studying the developmental characterization and the nerval function, when zebrafish was used as model animal.

Selective and electronic detection of COVID-19 (Coronavirus) using carbon nanotube field effect transistor-based biosensor: A proof-of-concept study

In this work, we propose and demonstrate a carbon nanotube (CNT) field-effect transistor (FET) based biosensor for selective detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). CNT FETs were fabricated on a flexible Kapton substrate and the sensor was fabricated by immobilizing the reverse sequence of the RNA-dependent RNA polymerase gene of SARS-CoV-2 onto the CNT channel. The biosensors were tested for the synthetic positive and control target sequences. The biosensor showed a selective sensing response to the positive target sequence with a limit of detection of 10 fM. The promising results from our study suggest that the CNT FET based biosensors can be used as a diagnostic tool for the detection of SARS-CoV-2.

Rational Engineering of the DNA Walker Amplification Strategy by Using a Au@Ti3C2@PEI-Ru(dcbpy)32+ Nanocomposite Biosensor for Detection of the SARS-CoV-2 RdRp Gene.

The detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for preventing and controlling infectious diseases and disease treatment. In this work, a Au@Ti3C2@PEI-Ru(dcbpy)32+ nanocomposite-based electrochemiluminescence (ECL) biosensor was rationally designed, which realized sensitive detection of the RNA-dependent RNA polymerase (RdRp) gene of SARS-CoV-2. In addition, a DNA walker was also used to excise the hairpin DNAs under the action of Nb.BbvCI endonuclease. Furthermore, model DNA–Ag nanoclusters (model DNA–AgNCs) were used to quench the initial ECL signal. As a result, the ECL biosensor was used to sensitively detect the SARS-CoV-2 RdRp gene with a detection range of 1 fM to 100 pM and a limit of detection of 0.21 fM. It was indicated that the ECL biosensor had a great application potential for clinical medical detection. Furthermore, the DNA walker amplification also played a reliable candidate strategy for other detection methods.

Copper-Coated Polypropylene Filter Face Mask with SARS-CoV-2 Antiviral Ability.

Face masks will be used to prevent pandemic recurrence and outbreaks of mutant SARS-CoV-2 strains until mass immunity is confirmed. The polypropylene (PP) filter is a representative disposable mask material that traps virus-containing bioaerosols, preventing secondary transmission. In this study, a copper thin film (20 nm) was deposited via vacuum coating on a spunbond PP filter surrounding a KF94 face mask to provide additional protection and lower the risk of secondary transmission. Film adhesion was improved using oxygen ion beam pretreatment, resulting in cuprous oxide formation on the PP fiber without structural deformation. The copper-coated mask exhibited filtration efficiencies of 95.1 ± 1.32% and 91.6 ± 0.83% for NaCl and paraffin oil particles, respectively. SARS-CoV-2 inactivation was evaluated by transferring virus-containing media onto the copper-coated PP filters and subsequently adding Vero cells. Infection was verified using real-time polymerase chain reaction and immunochemical staining. Vero cells added after contact with the copper-coated mask did not express the RNA-dependent RNA polymerase and envelope genes of SARS-CoV-2. The SARS-CoV-2 nucleocapsid immunofluorescence results indicated a reduction in the amount of virus of more than 75%. Therefore, copper-coated antiviral PP filters could be key materials in personal protective equipment, as well as in air-conditioning systems.

Core RNA Interference Genes Involved in miRNA and Ta-siRNA Biogenesis in Hops and Their Expression Analysis after Challenging with Verticillium nonalfalfae.

RNA interference is an evolutionary conserved mechanism by which organisms regulate the expression of genes in a sequence-specific manner to modulate defense responses against various abiotic or biotic stresses. Hops are grown for their use in brewing and, in recent years, for the pharmaceutical industry. Hop production is threatened by many phytopathogens, of which Verticillium, the causal agent of Verticillium wilt, is a major contributor to yield losses. In the present study, we performed identification, characterization, phylogenetic, and expression analyses of three Argonaute, two Dicer-like, and two RNA-dependent RNA polymerase genes in the susceptible hop cultivar Celeia and the resistant cultivar Wye Target after infection with Verticillium nonalfalfae. Phylogeny results showed clustering of hop RNAi proteins with their orthologues from the closely related species Cannabis sativa, Morus notabilis and Ziziphus jujuba which form a common cluster with species of the Rosaceae family. Expression analysis revealed downregulation of argonaute 2 in both cultivars on the third day post-inoculation, which may result in reduced AGO2-siRNA-mediated posttranscriptional gene silencing. Both cultivars may also repress ta-siRNA biogenesis at different dpi, as we observed downregulation of argonaute 7 in the susceptible cultivar on day 1 and downregulation of RDR6 in the resistant cultivar on day 3 after inoculation.

First Report on Natural Infection of Nodavirus in an Echinodermata, Sea Cucumber (Apostichopus japonicas).

Cross-species transmission of emerging viruses happens occasionally due to epidemiological, biological, and ecological factors, and it has caused more concern recently. Covert mortality nodavirus (CMNV) was revealed to be a unique shrimp virus that could cross species barrier to infect vertebrate fish. In the present study, CMNV reverse transcription-nested PCR (RT-nPCR)-positive samples were identified from farmed sea cucumber (Apostichopus japonicas) in the CMNV host range investigation. The amplicons of RT-nPCR from sea cucumber were sequenced, and its sequences showed 100% identity with the RNA-dependent RNA polymerase gene of the original CMNV isolate. Histopathological analysis revealed pathologic changes, including karyopyknosis and vacuolation of the epithelial cells, in the sea cucumber intestinal tissue. The extensive positive hybridization signals with CMNV probe were shown in the damaged epithelial cells in the in situ hybridization assay. Meanwhile, transmission electron microscopy analysis revealed CMNV-like virus particles in the intestine epithelium. All the results indicated that the sea cucumber, an Echinodermata, is a new host of CMNV. This study supplied further evidence of the wide host range of CMNV and also reminded us to pay close attention to its potential risk to threaten different aquaculture animal species.

Evaluation of the basic assay performance of the GeneSoc® rapid PCR testing system for detection of severe acute respiratory syndrome coronavirus 2

In the ongoing coronavirus disease 2019 (COVID-19) pandemic, PCR has been widely used for screening patients displaying relevant symptoms. The rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enables prompt diagnosis and the implementation of proper precautionary and isolation measures for the patient. In the present study, we aimed to evaluate the basic assay performance of an innovative PCR system, GeneSoC® (Kyorin Pharmaceutical Co. Ltd., Tokyo, Japan). A total of 1,445 clinical samples were submitted to the clinical laboratory, including confirmed or suspected cases of COVID-19, from February 13 to August 31. Specimen types included nasopharyngeal swabs. The sampling was performed several times for each patient every 2-7 days. Using this system, sequences specific for SARS-CoV-2 RNA could be detected in a sample within 10-15 min using the microfluidic thermal cycling technology. Analytical sensitivity studies showed that GeneSoC® could detect the target sequence of the viral envelope and RNA-dependent RNA-polymerase (RdRp) genes at 5 and 10 copies/μL, respectively. The precision of the GeneSoC® measurements using clinical isolates of the virus at a concentration of 103 copies/μL was favorable for both the genes; within-run repeatability and between-run reproducibility coefficient of variation values were less than 3% and 2%, respectively; and the reproducibility of inter-detection units was less than 5%. Method comparison by LightCycler® 480 showed the positive and negative agreement to be 100% [(174/174) and (1271/1271), respectively]. GeneSoC® proved to be a rapid and reliable detection system for the prompt diagnosis of symptomatic COVID-19 patients and could help reduce the spread of infections and facilitate more rapid treatment of infected patients.

Ultra-fast and recyclable DNA biosensor for point-of-care detection of SARS-CoV-2 (COVID-19)

Rapid diagnosis and case isolation are pivotal to controlling the current pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, a label-free DNA capacitive biosensor for the detection of SARS-CoV-2 that demonstrates real-time, low-cost, and high-throughput screening of nucleic acid samples is presented. Our novel biosensor composed of the interdigitated platinum/titanium electrodes on the glass substrate can detect the hybridization of analyte DNA with probe DNA. The hybridization signals of specific DNA sequences were verified through exhaustive physicochemical analytical techniques such as Fourier transform infrared (FT-IR) spectrometry, contact-angle analysis, and capacitance-frequency measurements. For a single-step hybridized reaction, the fabricated kit exhibited significant sensitivity (capacitance change, ΔC = ~2 nF) in detecting the conserved region of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) gene with high sensitivity of 0.843 nF/nM. In addition to capacitive measurements, this selective detection was confirmed by the fluorescence image and intensity from a SARS-CoV-2 gene labeled with a fluorescent dye. We also demonstrated that the kits are recyclable by surface ozone treatment using UV irradiation. Thus, these kits could potentially be applied to various types of label-free DNA, thereby acting as rapid, cost-effective biosensors for several diseases.

Development and validation of RT-PCR assays for testing for SARS-CoV-2.

The recent emergence and rapid global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demonstrates the urgent need for laboratory-developed assays for clinical diagnosis and public health interventions in the absence of commercial assays. We outline the progression of reverse-transcriptase polymerase chain reaction (RT-PCR) assays that were developed and validated at the Alberta Precision Laboratories, Public Health Laboratory, Alberta, Canada, to respond to this pandemic. Initially, testing was performed using SARS-CoV-2-specific and pan-coronavirus gel-based assays that were soon superseded by real-time RT-PCR assays targeting the envelope and RNA-dependent RNA polymerase genes to accommodate the high anticipated volumes of samples. Throughput was further enhanced by multiplexing the different targets together with the co-detection of an internal extraction control. These assays are comparable in sensitivity and specificity to the assays recommended by the World Health Organization and the US Centers for Disease Control and Prevention. The availability of real-time RT-PCR assays early in the pandemic was essential to provide valuable time to local health authorities to contain transmission and prepare for appropriate response strategies.

Development of a multiplex Loop-Mediated Isothermal Amplification (LAMP) assay for on-site diagnosis of SARS CoV-2.

A newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), has spread rapidly from its epicenter in China to more than 150 countries across six continents. In this study, we have designed three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer sets to detect the RNA-dependent RNA polymerase (RdRP), Envelope (E) and Nucleocapsid protein (N) genes of SARS CoV-2. For one tube reaction, the detection limits for five combination SARS CoV-2 LAMP primer sets (RdRP/E, RdRP/N, E/N, RdRP/E/N and RdRP/N/Internal control (actin beta)) were evaluated with a clinical nasopharyngeal swab sample. Among the five combination, the RdRP/E and RdRP/N/IC multiplex LAMP assays showed low detection limits. The sensitivity and specificity of the RT-LAMP assay were evaluated and compared to that of the widely used Allplex™ 2019-nCoV Assay (Seegene, Inc., Seoul, South Korea) and PowerChek™ 2019-nCoV Real-time PCR kit (Kogenebiotech, Seoul, South Korea) for 130 clinical samples from 91 SARS CoV-2 patients and 162 NP specimens from individuals with (72) and without (90) viral respiratory infections. The multiplex RdRP (FAM)/N (CY5)/IC (Hex) RT-LAMP assay showed comparable sensitivities (RdRP: 93.85%, N: 94.62% and RdRP/N: 96.92%) to that of the Allplex™ 2019-nCoV Assay (100%) and superior to those of PowerChek™ 2019-nCoV Real-time PCR kit (RdRP: 92.31%, E: 93.85% and RdRP/E: 95.38%).

A Hyper-Attenuated Variant of Rift Valley Fever Virus Generated by a Mutagenic Drug (Favipiravir) Unveils Potential Virulence Markers.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that causes Rift Valley fever (RVF), a zoonotic disease of wild and domestic ruminants, causing serious economic losses and a threat to human health that could be controlled by vaccination. Though RVF vaccines are available for livestock, no RVF vaccines have been licensed for veterinary use in non-endemic countries nor for human populations in RVF risk areas. In a recent work, we showed that favipiravir, a promising drug with antiviral activity against a number of RNA viruses, led to the extinction of RVFV from infected cell cultures. Nevertheless, certain drug concentrations allowed the recovery of a virus variant showing increased resistance to favipiravir. In this work, we characterized this novel resistant variant both at genomic and phenotypic level in vitro and in vivo. Interestingly, the resistant virus displayed reduced growth rates in C6/36 insect cells but not in mammalian cell lines, and was highly attenuated but still immunogenic in vivo. Some amino acid substitutions were identified in the viral RNA-dependent RNA-polymerase (RdRp) gene and in the virus encoded type I-interferon (IFN-I) antagonist NSs gene, in catalytic core motifs and nuclear localization associated positions, respectively. These data may help to characterize novel potential virulence markers, offering additional strategies for further safety improvements of RVF live attenuated vaccine candidates.

Development of a reverse transcription (RT) polymerase chain reaction (PCR) method for the detection of human norovirus in bivalve molluscs.

Noroviruses are significant seafood-borne pathogens, commonly associated with the consumption of filter feeding bivalve molluscs. Here, we report the development of a reverse transcription polymerase chain reaction (RT-PCR) method using primers based on the RNA-dependent RNA polymerase gene of norovirus genogroup II (NoV GII). Samples of bivalves were processed for the concentration of virus and extraction of RNA, followed by reverse transcription PCR. A total of 50 molluscan shellfish samples were analyzed, of which 16 samples yielded positive amplifications of norovirus nucleic acid. The PCR method described here, involving a single set of primers, is useful for rapid screening of shellfish for NoV GII.

Genetic diversity and expanded host range of astroviruses detected in small mammals in Singapore

Astroviruses are a genetically diverse group of viruses that infect a wide range of hosts, including small mammals. Small mammals were trapped at 19 sites across Singapore from November 2011 to May 2014. Pooled oropharyngeal and rectal swabs (n = 518) and large intestine tissue (n = 107) were screened using a PCR to detect the presence of Astrovirus RNA-dependent RNA polymerase (RdRp) gene. Astroviruses were detected in 93 of 625 (14.9%) of samples tested, with eight of 11 species of rats, shrews, and squirrels testing positive. This is the first detection of astroviruses in seven species (Callosciurus notatus, Mus castaneus, Rattus tanezumi, Rattus tiomanicus, Sundamys annandalei, Suncus murinus and Tupaia glis). Phylogenetic analysis of 10 RdRp gene sequences revealed that astroviruses from Singapore small mammals fall in three distinct clades, one that is specific to the common treeshrew (Tupaia glis), and two comprised of multiple species. One of these includes viruses from the cave nectar bat (Eonycteris spelaea), two rodent species, and a squirrel, suggesting that virus spillover from bats to small mammals may have occurred. Our results show an increased host range for astroviruses and highlight their potential for intra- and inter-species transmission.

Suitcase Lab for Rapid Detection of SARS-CoV-2 Based on Recombinase Polymerase Amplification Assay.

In March 2020, the SARS-CoV-2 virus outbreak was declared as a world pandemic by the World Health Organization (WHO). The only measures for controlling the outbreak are testing and isolation of infected cases. Molecular real-time polymerase chain reaction (PCR) assays are very sensitive but require highly equipped laboratories and well-trained personnel. In this study, a rapid point-of-need detection method was developed to detect the RNA-dependent RNA polymerase (RdRP), envelope protein (E), and nucleocapsid protein (N) genes of SARS-CoV-2 based on the reverse transcription recombinase polymerase amplification (RT-RPA) assay. RdRP, E, and N RT-RPA assays required approximately 15 min to amplify 2, 15, and 15 RNA molecules of molecular standard/reaction, respectively. RdRP and E RT-RPA assays detected SARS-CoV-1 and 2 genomic RNA, whereas the N RT-RPA assay identified only SARS-CoV-2 RNA. All established assays did not cross-react with nucleic acids of other respiratory pathogens. The RT-RPA assay’s clinical sensitivity and specificity in comparison to real-time RT-PCR (n = 36) were 94 and 100% for RdRP; 65 and 77% for E; and 83 and 94% for the N RT-RPA assay. The assays were deployed to the field, where the RdRP RT-RPA assays confirmed to produce the most accurate results in three different laboratories in Africa (n = 89). The RPA assays were run in a mobile suitcase laboratory to facilitate the deployment at point of need. The assays can contribute to speed up the control measures as well as assist in the detection of COVID-19 cases in low-resource settings.

Entropy-driven amplified electrochemiluminescence biosensor for RdRp gene of SARS-CoV-2 detection with self-assembled DNA tetrahedron scaffolds

Dependable, specific and rapid diagnostic methods for severe acute respiratory syndrome β-coronavirus (SARS-CoV-2) detection are needed to promote public health interventions for coronavirus disease 2019 (COVID-19). Herein, we have established an entropy-driven amplified electrochemiluminescence (ECL) strategy to detect the RNA-dependent RNA polymerase (RdRp) gene of SARS-CoV-2 known as RdRp-COVID which as the target for SARS-CoV-2 plays an essential role in the diagnosis of COVID-19. For the construction of the sensors, DNA tetrahedron (DT) is modified on the surface of the electrode to furnish robust and programmable scaffolds materials, upon which target DNA-participated entropy-driven amplified reaction is efficiently conducted to link the Ru (bpy)32+ modified S3 to the linear ssDNA at the vertex of the tetrahedron and eventually present an “ECL on” state. The rigid tetrahedral structure of the DT probe enhances the ECL intensity and avoids the cross-reactivity between single-stranded DNA, thus increasing the sensitivity of the assays. The enzyme-free entropy-driven reaction prevents the use of expensive enzyme reagents and facilitates the realization of large-scale screening of SARS-CoV-2 patients. Our DT-based ECL sensor has demonstrated significant specificity and high sensitivity for SARS-CoV-2 with a limit of detection (LOD) down to 2.67 fM. Additionally, our operational method has achieved the detection of RdRp-COVID in human serum samples, which supplies a reliable and feasible sensing platform for the clinical bioanalysis.

Late Male-Killing Viruses in Homona magnanima Identified as Osugoroshi Viruses, Novel Members of Partitiviridae.

Late male-killing, a male-specific death after hatching, is a unique phenotype found in Homona magnanima, oriental tea tortrix. The male-killing agent was suspected to be an RNA virus, but details were unknown. We herein successfully isolated and identified the putative male-killing virus as Osugoroshi viruses (OGVs). The three RNA-dependent RNA polymerase genes detected were phylogenetically related to Partitiviridae, a group of segmented double-stranded RNA viruses. Purified dsRNA from a late male-killing strain of H. magnanima revealed 24 segments, in addition to the RdRps, with consensus terminal sequences. These segments included the previously found male-killing agents MK1068 (herein OGV-related RNA16) and MK1241 (OGV-related RNA7) RNAs. Ultramicroscopic observation of purified virions, which induced late male-killing in the progeny of injected moths, showed sizes typical of Partitiviridae. Mathematical modeling showed the importance of late male-killing in facilitating horizontal transmission of OGVs in an H. magnanima population. This study is the first report on the isolation of partiti-like virus from insects, and one thought to be associated with late male-killing, although the viral genomic contents and combinations in each virus are still unknown.

Genomic characterization of a nebovirus strain with a novel RdRp genotype in yaks

Neboviruses (NeVs) are important causative agents of calf diarrhea that belong to the family Caliciviridae. In this study, we investigated the genomic characteristics of a NeV strain from yaks that has a novel RdRp genotype. The complete genome of this strain (YAK/NRG-A9/19/CH) is 7454 nt in length and shares 68.3%-79.7% nt sequence identity with those of other NeVs. The RNA-dependent RNA polymerase (RdRp) gene of this strain shares 66.5%-78.5% nt sequence identity (74.0%-89.3% aa sequence identity) with the eight available complete NeV RdRp sequences, and a phylogenetic analysis based on these sequences showed that the new strain formed an independent branch, indicating that the RdRp of strain YAK/NRG-A9/19/CH may represent a novel RdRp genotype of NeV. These results contribute to a further understanding of the molecular characteristics and genetic evolution of NeVs.