Reverse Transcription-polymerase Chain Reaction Amplification Research Articles

First Report of Cotton Leafroll Dwarf Virus infection of Malvaviscus arboreus in China.

Cotton leafroll dwarf virus (CLRDV, genus Polerovirus, family Solemoviridae) has been reported to infect cotton in Brazil, Argentina, India, Thailand and Timor-Leste (Agrofoglio YC et al. 2017; Corrêa RL et al. 2005; Mukherjee et al. 2012; Ray et al. 2016; Sharman et al. 2015), and in the United States (Ali and Mokhtari et al. 2020; Avelar et al. 2019). It has also been recently reported to infect Cicer arietinum (chickpea) in Uzbekistan and Hibiscus syriacus in Korea (Igori et al. 2022; Kumari et al. 2020). In China, the natural infection of plants by CLRDV has not been reported previously. In August 2017, leaf samples were collected from a wild plant of Malvaviscus arboreus (Malvaceae) exhibiting symptoms including leaf yellowing and distorting in Tengchong County of Yunnan Province. Leaves were used for total RNA extraction using TRIzol Reagent (Invitrogen, USA). Small RNA library construction and deep sequencing was performed on illumina HiSeqTM 2000 platform by Novogene Bioinformatic Technology Co., Ltd (Beijing, China). A total of 11, 525, 708 raw reads were obtained and further computationally analyzed by Perl scripts. The adaptors were removed and the obtained 7, 520, 902 clean reads with size of 18- to 26-nucleotide (nt) were aligned with the GenBank virus RefSeq database using Bowtie software. These reads were mainly mapped to the genomes of hibiscus bacilliform virus (genusBadnavirus,familyCaulimoviridae), hibiscus chlorotic ringspot virus (genus Betacarmovirus, family Procedovirinae), hibiscus latent Singapore virus (genus Tobamovirus, family Virgaviridae) and CLRDV isolate ARG (accession no. GU167940). The average coverage depth of clean reads mapped to CLRDV genome was 97.76%. Contigs greater than 50 nt were used to search for similar sequences by BLASTx, and 107 contigs were annotated as homologous to CLRDV isolates. To confirm CLRDV infection, reverse transcription polymerase chain reaction (RT-PCR) was performed using the specific primer pair CLRDV-F (5'-TCCACAGGAAGTATCACGTTCG-3') and CLRDV-R (5'-CCTTGTGTGGTTTGATTCGTGA-3') designed based on two of these contigs well-aligned to the genome of CLRDV isolate ARG. An amplicon of 1095-bp size was amplified, and was sequenced by Sanger sequencing (TsingKe Biological Technology, Chengdu, China), and BLASTn search results showed a maximum nucleotide identity of 95.45% with CLRDV isolate CN-S5, an isolate obtained from soybean aphid host in China (accession no. KX588248). To obtain more information on this CLRDV isolate, four primer pairs were designed and used for RT-PCR amplification (TableS1). The amplicons with size of about 860-, 1400-, 3200- and 1100-bp, were obtained separately and assembled into a complete genome sequence up to 5, 865-nt in length (isolate YN, deposited under GenBank accession no. MN057665). BLASTn showed the highest nucleotide similarity of 94.61% with CLRDV isolate CN-S5. From 2018 to 2022, additional M. arboreus samples with leaf yellowing or curling symptoms (9 from Shapingba District in Chongqing City, 5 from Nanchong City in Sichuan Province, 9 from Kunming City and 12 from Tengchong County in Yunnan Province) were collected and tested for CLRDV by RT-PCR using primer pairs CLRDV-F/CLRDV-R. The nucleotide sequences of the CLRDV P0 gene in two samples from Tengchong County were obtained by Sanger sequencing and deposited under GenBank (CLRDV isolate TCSL1 P0 gene, accession no. OQ749809; CLRDV isolate TCSW2 P0 gene, accession no. OQ749809). To our knowledge, this is the first report of CLRDV naturally infecting Malvaviscus arboreus in China, thus extending the information on its geographical distribution and host range. Malvaviscus arboreus is a widely cultivated ornamental plant in Yunnan Province, China. The natural occurrence of CLRDV not only affects the ornamental value of Malvaviscus arboreus, but also poses a potential threat to cotton production in China. This study will assist further surveillance of CLRDV infection and future development of effective protection strategies against CLRDV in China.

Evaluation of reverse transcription-polymerase chain reaction and simultaneous amplification and testing for quantitative detection of serum hepatitis B virus RNA

BackgroundChronic hepatitis B virus (HBV) infection is one of the common infectious diseases in the world. HBV covalently closed circular DNA (cccDNA) is the initial template of HBV replication, which can exist in human hepatocytes for a long time and is difficult to be completely removed. It has been shown that HBV RNA can directly respond to the levels and transcriptional activity of cccDNA in hepatocytes and can be used as a surrogate marker of cccDNA transcriptional activity. At present, the detection techniques used for quantitative HBV RNA mainly include reverse transcription quantitative polymerase chain reaction (RT-qPCR) and simultaneous amplification and testing (SAT). MethodsIn this study, we verified the performance of the SAT method for detecting HBV RNA and the clinical effectiveness of SAT and RT-qPCR, and compared the correlation and consistency of the two detection methods for HBV RNA detection. ResultsThe results showed that the limit of detection for HBV RNA by SAT method was 50 copies/mL, with a linear range of 1 × 102-1 × 108 copies/mL. There was no difference in HBV RNA levels detected by the two methods. The correlation and consistency of the results were good, with the coefficient of determination of 0.7787 in HBeAg positive group and 0.8235 in HBeAg negative group. ConclusionsTherefore, this study confirmed that the SAT method and RT-qPCR for detecting HBV RNA have good agreement, which are both reliable methods to detect HBV RNA and can replace each other.

#6499 The CIRCRNA–MIRNA–MRNA REGULATORY NETWORK AND THE POTENTIAL ASSOCIATIONS WITH THE PATHOGENESIS OF SYSTEMIC LUPUS ERYTHEMATOSUS

Abstract Background and Aims This study aimed to explore the possible role of plasma and PBMCs circular RNA (circRNA) in systemic lupus erythematosus (SLE). Method Total RNA was extracted from blood plasma samples obtained from 10 patients with SLE and 10 healthy controls and subjected to microarray analysis to define the profile of circRNA expression. The quantitative reverse transcription-polymerase chain reaction amplification (qRT-PCR) was conducted. The overlapped circRNA between PBMCs and plasma was performed, the interactions with microRNAs were predicted, the miRNA target mRNA was predicted, and the GEO database was used. The Gene ontology and pathway analysis was performed. Results 131 upregulated and 314 significantly downregulated circRNAs were identified in the plasma of patients with SLE by the Fold change criteria (≥2.0) and P < 0.05. The qRT-PCR results showed that the expression of has-circRNA-102531, has-circRNA-103984, and has-circRNA-104262 was increased in plasma of SLE, and the expression of has-circRNA-102972, has-circRNA-102006, has-circRNA-104313 was decreased in plasma of SLE. 28 upregulated circRNAs and 119 downregulated circRNAs were overlapped from PBMCs and plasma, and ubiquitination was enriched. Furthermore, the circRNA-miRNA-mRNA network was constructed in SLE after analyzing dataset GSE61635 from GEO. The circRNA-miRNA-mRNA network comprises 54 circRNAs, 41 miRNAs, and 580 mRNAs. In addition, the TNF signaling pathway and the MAPK pathway were enriched from the mRNA of the miRNA target. Conclusion We described differentially expressed circRNAs in plasma and PBMCs, and the circRNA-miRNA-mRNA network was constructed. The network's circRNAs could be a potential diagnostic biomarker and potentially play an important role in the pathogenesis and development of SLE.

Molecular Characterization of Three Apple Geminivirus Isolates in Crabapples Detected in Inner Mongolia, China.

Apple geminivirus 1 (AGV) in the genus Maldovirus of the family Geminiviridae was first identified infecting apple trees in the year 2015 in China. In this work, we characterized three isolates of the AGV in the Chinese pearleaf crabapple (Malus asiatica) in Inner Mongolia Autonomous Region. The viruses were detected by Illumina sequencing and its existence was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) amplification of an AGV fragment. Between the three AGV isolates and the initially characterized AGV isolate PL2015, the nucleotide sequence identities of the complete genome ranged from 91.2 to 91.7%, of the coat protein gene (V1) ranged from 95.4% to 97.3%, and of the replicase gene (C1) ranged from 87.3% to 88.0%. Phylogenetic analysis indicated that the three isolates formed a monophyletic group together with the AGV, separated from the current genera in the family Geminiviridae. This is the first description of the AGV infecting crabapples.

Cloning and Characterization of Aquaporins 11 and Its Expression Analysis under Molt Cycle in <i>Eriocheir sinensis</i>

Aquaporins play important roles in water molecule transport, ion transport, and osmotic pressure regulation. In this study, the full-length cDNA of aquaporin from Chinese mitten crab ( Eirocheir sinensis ) was cloned for first time using reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends analyses. The full-length of AQP11 cDNA is 1746bp, with a 463bp 5’-untranslated region (UTR), a 476bp 3’-UTR, and an open reading frame (ORF) of 807 bp which encodes a 269 amino acid polypeptide. The molecular mass of the predicted protein is 29.46 kDa with an estimated PI of 5.38. Bioinformatics software analysis revealed that AQP11 gene contains 4 transmembrane domains, 2 NPV structural units. Homologous analysis showed that AQP11 of Eirocheir sinensis  has the highest homology to AQP11 of Litopenaeus vanname i .  Real-time quantitative RT-PCR showed that AQP11 gene could be detected in all tested tissues of Eirocheir sinensis , with the highest expression level in intestine, followed by the brain, muscle and thoracic ganglion, and the lowest expression level in the hepatopancreas, gills and blood. In the intestinal, the expression of AQP11 was significantly lower at the intermolt stage (stages C) and premolt stage (stage D), significantly enhanced and reached the maximal level at the ecdysis stage (stage E). On the other hand, the expression of AQP11 gene in muscle showed low expression in the intermolt stage (stage C), and it was increased gradually at the premolt stage (stage D), significantly enhanced and reached the maximal level at the ecdysis stage (E stage), and then reaching the postmolt stage(AB stage) decline. In summary, our results indicate that AQP11 may play an important role in molting of Enirocheir sinensis.

Isolation, Identification, and Genetic Phylogenetic Analysis of Two Different Genotypes of Bovine Parainfluenza 3 Virus in China.

Bovine parainfluenza virus 3 (BPIV3) is one of several viruses that contribute to bovine respiratory disease complex (BRDC). During this study, isolation of BPIV3 was attempted from 20 PCR-positive swabs by Madin-Darby Bovine Kidney (MDBK) cells. Nine samples showed obvious cytopathic lesions identified as BPIV3 by reverse-transcription polymerase chain reaction amplification and sequencing. The genomes of isolates XJ21032-1 and XJ20055-3 were sequenced using Illumina sequencing technology and determined to have lengths of 15,512 bp and 15,479 bp, respectively. Phylogenetic analysis revealed that isolate XJ21032-1 was genotype B, and isolate XJ20055-3 was genotype C. In addition, the two isolates had multiple amino acid changes in nucleocapsid protein, fusion protein, and hemagglutinin/neuraminidase, major antigenic proteins. This allows the further recognition of the presence of BPIV3 type B in Chinese cattle herds. We hope this will help trace the origin of BPIV3, improve the understanding of differences between genotypes, and provide data support for vaccine development.

First Report of Tobacco Mild Green Mosaic Virus Infecting Rehmannia glutinosa in China.

Rehmannia glutinosa (family Scrophulariaceae) is an important traditional medicinal plant, whose root is used to treat anemia, hemoptysis, and gynecological diseases in China (Matsumoto et al. 1989). This plant is native to China and cultivated in China, Korea, Japan, and northern Vietnam (Kwak et al. 2020). Viral diseases caused remarkable loss in the yield and quality of R. glutinosa (Ling et al. 2009). To date, ten viruses have been identified globally to infect R. glutinosa and seven of these viruses reported in China (Liu et al. 2018; Zhang et al. 2021). Most plants of R. glutinosa are infected with one or more of these viruses (Kwak et al. 2018; Zhang et al. 2004). In July 2020, a survey of the viral disease infecting R. glutinosa was conducted in commercial plantations of Wenxian, Wuzhi, Mengzhou, and Yuzhou counties in Henan Province, China. The disease symptoms included mosaic, chlorosis, leaf distortion, and the percentage of symptomatic plants was over 70% in the surveyed fields (n=9). Sixty leaf samples of symptomatic R. glutinosa plants were collected from nine cultivation fields in Wenxian, Wuzhi, Mengzhou, and Yuzhou counties (five to seven plants for each field). Total RNA was extracted from one pooled sample containing a portion of all above-mentioned leaf samples using RNAprep Pure Plant Plus Kit (TIANGEN Biotech, Beijing, China) and analyzed by high-throughput sequencing (HTS) to identify viral pathogens. A transcriptome library was generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA), and sequenced on an Illumina NovaSeq6000 sequencing system at Berry Genomics Corporation (Beijing, China). A total of 27,664,949 high-quality clean reads were obtained after trimming and used for contig assembly. The assembled contigs (n=109,180) were searched using Basic Local Alignment Search Tool (BLAST) at GenBank. BLASTn analysis showed that the R. glutinosa plants were infected with known viruses, including broad bean wilt virus, rehmannia mosaic virus, youcai mosaic virus, and cucurbit chlorotic yellows virus. In addition, one contig (6,418 nt in length) had a nucleotide sequence identity of 99.64% with the TN29 isolate of tobacco mild green mosaic virus (TMGMV, GenBank accession no. MF139550). To confirm the presence of this virus, sixty above-mentioned samples were screened by reverse transcription-polymerase chain reaction (RT-PCR) using the specific primer pairs (Supplementary Table1) TMGMG-CPF/TMGMG-CPR targeting a 545-nt fragment within the CP gene. Amplicons with expected sizes were detected from 47 of 60 samples but not from the negative control (virus-free healthy plant through the tip meristem culture). Seventeen amplicons (11#, 13#, 14#, 21#, 22#, 23#, 25#, 26#, 27#, 31#, 32#, 33#, 37#, 52#, 57#, 59#, and 60#) of TMGMV-CP were selected, and purified. The PCR products were cloned into the pMD19-T vector (TAKARA Biotech, Dalian, China) and sequenced. The sequences were deposited into the GenBank (accession nos. MZ395944 to MZ395960). The near-full-length genomic sequence of TMGMV-Rg14 isolate was obtained from one positive sample (sample no. 14) by RT-PCR amplification of two overlapping fragments using the following primer pairs: TMGMV-40F/TMGMV-3570R and TMGMV-3220F/TMGMV-6400R. The near-full-length genomic sequence of the TMGMV-Rg14 isolate was 6 304 nucleotides (nt) in length and deposited into GenBank (accession no. MZ395975). BLASTn analysis demonstrated that the TMGMV-Rg14 isolate shared a sequence identity ranging from 96.89% (AB078435) to 99.60% (MF139550) with the other TMGMV isolates. Furthermore, the virus-free healthy R. glutinosa plants were inoculated with sap from the positive sample (14#) to confirm the infection of TMGMV. Mosaic symptoms were induced on the systemically infected leaves of the inoculated plants 14 days post inoculation. The systemically infected leaves of inoculated plants were assayed by RT-PCR using the primer pairs TMGMV-CPF/CPR. Amplicons of expected size were detected from the inoculated plants but not from non-inoculated plants. To our knowledge, this is the first report of TMGMV infection on R. glutinosa. Further studies are necessary to select a suitable indicator plant for this TMGMV, its host range, and the symptoms it induces in single infection. Since R. glutinosa is cultivated by vegetative propagation, production of virus-free healthy plants is necessary. This study will help to generate virus-free healthy plants and prevent viral disease on R. glutinosa. Further study is needed to determine its pathological implications and economic impact on R. glutinosa in China.

Current State of Molecular and Serological Methods for Detection of Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family, is the etiological agent of an acute and devastating enteric disease that causes moderate-to-high mortality in suckling piglets. The accurate and early detection of PEDV infection is essential for the prevention and control of the spread of the disease. Many molecular assays have been developed for the detection of PEDV, including reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR (qRT-PCR) and loop-mediated isothermal amplification assays. Additionally, several serological methods have been developed and are widely used for the detection of antibodies against PEDV. Some of them, such as the immunochromatography assay, can generate results very quickly and in field conditions. Molecular assays detect viral RNA in clinical samples rapidly, and with high sensitivity and specificity. Serological assays can determine prior immune exposure to PEDV, can be used to monitor the efficacy of vaccination strategies and may help to predict the duration of immunity in piglets. However, they are less sensitive than nucleic acid-based detection methods. Sanger and next-generation sequencing (NGS) allow the analysis of PEDV cDNA or RNA sequences, and thus, provide highly specific results. Furthermore, NGS based on nonspecific DNA cleavage in clustered regularly interspaced short palindromic repeats (CRISPR)–Cas systems promise major advances in the diagnosis of PEDV infection. The objective of this paper was to summarize the current serological and molecular PEDV assays, highlight their diagnostic performance and emphasize the advantages and drawbacks of the application of individual tests.

First Report of Bean common mosaic virus Infecting Heavenly Bamboo (Nandina domestica) in China.

Heavenly bamboo (Nandina domestica) is an evergreen ornamental plant with worldwide distribution. In May 2018, seven out of twenty N. domestica plants showing virus-like symptoms, such as yellow mosaic and curling, were observed in Lin'an, Zhejiang province. To determine the causal agent, a small RNA library was constructed using the Small RNA v1.5 Sample Prep Kit (Illumina, San Diego, USA) with total RNA extracted from leaves of a symptomatic plant. The library was sequenced by the Solexa platform at BGI Genomics (Shenzhen, China). A total number of 21,071,675 high-quality reads of 17-28 nucleotides (nt) in length remained after trimming adapter sequences and quality control. Reads were assembled using Velvet 0.7.31 and Oases 0.2.07 with the k-mer value of 17 (Schulz et al. 2012). BlastN and BlastX search against the GenBank viral nonredundant sequence databases revealed fifty-six contigs homologous to bean common mosaic virus (BCMV; genus Potyvirus; family Potyviridae). No contig homologous to the genomic sequence of other plant-infecting viruses was identified. These contigs were further assembled into a 9,315-nt fragment by SeqMan Pro 7.1.0 in Lasergene package (DNASTAR, Madison, WI), which covered 92.68% of the genome of BCMV strain CT (BCMV-CT; GenBank accession no. KM076650). The genome of this BCMV isolate (BCMV-NTZ1) was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using primers designed based on assembled contigs with the Phusion® High-Fidelity DNA Polymerase (New England Biolabs, Beijing, China) and the FirstChoice® RLM-RACE Kit (Invitrogen, Carlsbad, USA), respectively. Amplicons were cloned and Sanger sequenced with three independent clones per amplicon. The genome is 10,052 nt in length excluding the poly-A tail (Genbank accession no. MZ670770) and shared the highest nt sequence identities with BCMV-CT (88.46%). The putative polyprotein shared 93.36% amino acid (aa) sequence identity with that of BCMV-CT. BCMV-NTZ1 also clustered with BCMV-CT in phylogenetic trees based on BCMV full genomes and aa sequences of coat protein. Five-leaf-stage seedlings of Nicotiana tabacum, N. benthamiana, Glycine max(Linn.) Merr., and Capsicum frutescens were mechanically inoculated with sap of BCMV-infected N. domestica leaves at fifteen plants per species. Seedlings of G. max developed virus-like (mosaic and leaf deformity) symptoms (7/15) at 15 days post-inoculation, while other plants remained symptomless throughout the experiment. Subsequent RT-PCR on all the plants using primers 27F1/14Rter and sequencing confirmed the presence and absence of BCMV-NTZ1 in all symptomatic G. max seedlings and other asymptomatic indicator plants, respectively. Subsequent RT-PCR survey further confirmed the association of BCMV with symptomatic heavenly bamboo samples but not asymptomatic plants (7/20). To the best of our knowledge, this is the first report of BCMV naturally infecting heavenly bamboo in China. N. domestica is susceptible to many viruses, e.g., cucumber mosaic virus, plantago asiatica mosaic virus, nandina stem pitting virus, apple stem grooving virus, and alternanthera mosaic virus (Barnett et al. 1973; Ahmed et al. 1983; Hughes et al. 2002, 2005; Tang et al. 2010; Wei et al. 2015). Our results indicate that N. domestica can also serve as an overwinter reservoir for BCMV and special attention should be paid to the damage it may cause.

West Nile Fever Virus Infection in Horses in São Paulo State, Brazil

Background: The West Nile virus (WNV) antibodies were reported in Brazil in the serum samples taken from horses and birds in the Midwest region and Paraíba state in 2008 and 2013, respectively. In 2014, the first human case was confirmed in a rural worker in the state of Piauí and, in 2018, the virus was isolated from the central nervous system of a horse with nervous symptoms in the state of Espírito Santo. The virus is a member of the Flaviviridae family of the genus Flavivirus (neurotropic), infecting several mammalian species, with humans and horses being the most susceptible. Approximately 35% of horses develop clinical signs, thus they are considered the best sentinels for this disease. The aim of this case report is to describe the first confirmed cases of West Nile Fever (WNF) in two horses in the state of São Paulo.Cases: Two horses with neurological symptoms were treated at the Veterinary Hospital of Cruzeiro do Sul University (São Paulo, SP), in 2019. Both horses came from neighboring regions that have a large Atlantic Forest preservation area and are also routes for migratory birds, known to be competent hosts for transmitting the West Nile Fever virus, such as the swallow, the falcon and the hawk. The first one had symptoms, such as weakness and sporadic seizures; however, after recovering, it was hospitalized a few days later due to kidney failure and laminitis. The second one showed incoordination, pelvic limb weakness, and was walking in circles, evolving to seizures. Both animals were euthanized, and their central nervous system samples and total blood samples were tested for rabies, herpes virus, and WNV; the first 2 tests showed negative results. Ribonucleic acids (RNA) were extracted from erythrocytes using the polymerase chain reaction (PCR) technique in-house. The WNV-specific reverse transcription-polymerase chain reaction amplification products were obtained using the nested PCR-multiplex PCR combination.Discussion: Since the 1940s, several WNF outbreaks have been reported around the world (Africa, Europe, Asia and Middle East). In the 2000s, the USA had the most amount of WNF cases reported; cases started being reported in Central and South America in the following years. The virus was identified for the first time in Brazil in 2014. Since then, our country is a route for migratory birds, with many states still having forests, several arboviruses are found such as WNF, which could become a public health problem. Both horses in the present study showed neurological signs and the horse that recovered had renal failure. Such signs are inconclusive, however, similar to those that occur in humans infected by the virus in its neurotropic form. The emergence of new diseases is an important aspect of public health. The literature is vast regarding the description of the pathogenesis, clinical signs, diagnosis, viral persistence and sequelae of WNF in humans, however, it is scarce regarding the viral persistence and sequelae of the disease in horses. Future studies are needed to understand the post-infection period in horses, as they are the most sensitive animals along with humans to this virus. Here, we report the first confirmed cases of WNF in the city of São Paulo to bring awareness about considering this disease while diagnosing horses with nervous system disorders.Keywords: encephalitis, horses, flavivirus, mosquito.Título: Infecção pelo vírus da Febre do Nilo Ocidental em equinos no Estado de São PauloDescritores: encefalite, equinos, flavivírus, mosquito.

A rapid water bath PCR combined with lateral flow assay for the simultaneous detection of SARS-CoV-2 and influenza B virus.

The outbreak of the coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant global health and economic threats to the human society. Thus, a rapid and accurate detection method for early testing and diagnosis should be established. In this study, a rapid water bath polymerase chain reaction (PCR) combined with lateral flow assay was developed to detect SARS-CoV-2 and influenza B virus simultaneously. A homemade automated transfer device equipped with reaction tube shuttled rapidly between two water baths at 98 °C and 53 °C to realize rapid PCR. After amplification, two-ended labeled PCR products were detected using the lateral flow strip with two test lines and streptavidin-conjugated quantum dot nanobeads. The fluorescence value was read using a handheld instrument. The established assay could complete reverse-transcription PCR amplification and lateral flow detection in 45 minutes. The detection limits were 8.44 copies per μL and 14.23 copies per μL for SARS-CoV-2 and influenza B virus, respectively. The coefficients of variation of the test strip were 10.10% for the SARS-CoV-2 and 4.94% for the influenza B virus, demonstrating the excellent repeatability of the experiment. These results indicated that the rapid PCR combined with lateral flow assay could detect SARS-CoV-2 and influenza B virus simultaneously at a short assay time and low cost, thereby showing the remarkable potential for the rapid and multiplex detection of respiratory viruses in resource-limited settings.

Bioinformatics analysis of tumor-educated platelet microRNAs in patients with hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies that seriously threaten global health. The primary reason for its grim prognosis is the lack of sensitive tools for early diagnosis. The purpose of the present study was to apply bioinformatics analysis to explore tumor-educated platelet (TEP) microRNA (miRNA) expression and its potential diagnostic utility in HCC.Methods: Twenty-five HCC patients and 25 healthy controls were included. RNA sequencing was utilized to screen miRNA alterations in platelets derived from HCC patients (n=5) and controls (n=5). Gene set enrichment analysis was performed to analyze the targeted mRNAs of differentially expressed miRNAs by using the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, aiming at main functions and pathways, respectively. We then verified the selected platelet miRNAs in another cohort by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) amplification.Results: A total of 250 differentially expressed miRNAs were identified, among which 111 were down-regulated and 139 were up-regulated. The functional enrichment analysis of differentially expressed miRNAs suggested that their target genes were involved primarily in pathways related to HCC. Expression levels of miR-495-3p and miR-1293 were further validated by qRT-PCR, which yielded results consistent with the sequencing analysis. The area under the receiver operating characteristic (ROC) curve of miR-495-3p and miR-1293 as diagnostic tests for HCC were 0.76 and 0.78, respectively.Conclusion: TEP miRNAs such as miR-495-3p and miR-1293 were differentially expressed in HCC patients, and may be involved in the pathophysiology of HCC.

Sequencing, Expression, and Functional Analyses of Four Genes Related to Fatty Acid Biosynthesis During the Diapause Process in the Female Ladybird, Coccinella septempunctata L.

The ladybird Coccinella septempunctata L., a predatory insect, serves as an excellent biological control agent against common agricultural pests. It undergoes a diapause phenomenon, during which a large amount of fat accumulates in the abdomen. A comprehensive analysis of this lipid accumulation can reveal the molecular mechanisms underlying diapause regulation, which can be exploited to improve the shipping and transport of the insect for agricultural applications. In this study, we compared the transcriptome of C. septempunctata during non-diapause, diapause, and post-diapause and screened four key genes related to lipid metabolism. The cDNA of these four relevant enzymes, acetyl-CoA carboxylase (ACC), long-chain fatty acid-CoA ligase (ACSL), elongase of very-long-chain fatty acids (ELO), and very-long-chain 3-oxoacyl-CoA reductase (KAR), were cloned using reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. Their expression profiles were analyzed during the preparation and maintenance phases of diapause and the post-diapause phase. The functions of these four key enzymes in diapause were further verified using RNA interference. All four genes were most closely related to the homeotic gene from Tribolium castaneum. The expression profiles of these four genes were significantly affected under diapause-inducing conditions; their expression level was the highest in the diapause preparation phase, and it gradually decreased with the diapause induction time. RNA interference showed that the target genes play important roles in fat storage during early diapause, and the decrease in their expression leads to a decrease in lipid content in C. septempunctata. These results indicate an important role of ACC, ACSL, ELO, and KAR in lipid accumulation. Our findings could help elucidate the production and accumulation of lipids by insects during the preparation for diapause and improve biological control.

The identification of circular RNAs from peripheral blood mononuclear cells in systemic lupus erythematosus

BackgroundThe diagnosis of systemic lupus erythematosus (SLE) is complicated. This study explores the expression of circular RNAs (circRNAs), which are closed non-coding RNAs in which the 5′ and 3′ ends are covalently linked and which work by sponging microRNAs. CircRNAs were extracted from peripheral blood mononuclear cells (PBMCs) of SLE patients to identify novel circRNA species that might be used for SLE diagnosis.MethodsMicroarray was applied to screening circRNAs changes in PBMCs obtained from SLE patients (n = 10) and healthy participants (n = 10), paired for age and sex. We then verified the selected circRNAs in PBMCs using quantitative reverse transcription-polymerase chain reaction amplification (qRT-PCR) in another cohort, including ten paired SLE patients and healthy participants. The correlation between the differential circRNAs and clinical pathology of SLE were analyzed.Results182 up-regulated and 563 significantly down-regulated circRNAs in PBMCs of patients with SLE were identified. Besides, the qRT-PCR results were consistent with the microarray results. The correlation analysis revealed that has_circRNA_100236, has_circRNA_102489, and has_circRNA_101413 were correlated with positive anti-dsDNA, thrombocytopenia, and positive IgG, respectively. Lastly, their miRNAs targets and the binding sites were predicted.ConclusionWe identified some dysregulated circRNAs in PBMCs from SLE patients, and these circRNAs may be developed as the novel biomarkers for the diagnosis of SLE.

Current Challenges for the Effective Management of the COVID-19 Pandemic.

Within a short period, the coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2) spread all over the globe and became the first pandemic of the present century. Early diagnostic tools and effective drugs are urgently needed to effectively manage the COVID-19 pandemic. Based on current literature, we provide recent updates on SARS-CoV-2 biology, available diagnostic methods, and therapeutic options for the management of COVID-19 pandemic. A literature survey was done using Google and PubMed and Web of Science to summarize the current updates on this topic. Current coronavirus diagnostic tests are reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR (qRT-PCR), and reverse transcription loop-mediated isothermal amplification (RT-LAMP) which detects the presence of specific genome sequence of virus. Existing antiviral drugs or new therapeutic options such as neutralizing antibody or plasma therapy are mostly used to restrict the virus growth with a limited success. As there is no specific treatment or vaccine available to limit the infection of SARS-CoV-2, we need to rely on the existing way to limit the disease. The first priority to fight COVID-19 is development of early diagnostic tools so that infected persons can be identified and further viral transmission can be blocked. Evaluation of existing drugs or identification of new therapeutic entities becomes the major challenge to deal with the present pandemic.

Memory B cells producing somatically mutated antiphospholipid antibodies are present in healthy individuals

AbstractAntiphospholipid antibodies (aPLs) are associated with thrombosis and recurrent abortions during autoimmune pathologies, but they are also produced in healthy individuals and during infectious diseases. To analyze the possible links between physiologic and pathologic aPLs, it is of importance to characterize normal aPL production. We took advantage of the known tropism of Epstein-Barr virus (EBV) for B cells in general, and memory B cells in particular, during primary infectious mononucleosis (IMN) in 3 patients to get access to anticardiolipin (aCL)-producing B cells. Flow cytometry analysis of these cells showed that, depending on the patient, 10% to 60% of immunoglobulin M (IgM) aCL-producing B cells express the CD27 marker of memory B cells. Single cell sorting of aCL B cells, followed by single cell reverse transcription-polymerase chain reaction (RT-PCR) amplification of their immunoglobulin variable region genes, showed that some of these cells produce mutated forms of aCL antibodies, confirming their memory B-cell origin. Considering that, during primary IMN, EBV infects and expands already pre-existing memory B cells, we conclude that healthy individuals have a discrete pool of aCL memory cells able to produce mutated forms of antibodies. The implications of this new information are discussed in light of different hypotheses regarding the origin of aCL. (Blood. 2003;102:2459-2465)

Amino acid substitutions in VP2, VP1, and 2C attenuate a Coxsackievirus A16 in mice

Coxsackievirus A16 (CVA16) is one of the major etiological agents of hand, foot and mouth disease (HFMD), a common acute infectious disease affecting infants and young children. Severe symptoms of the central nervous system may develop and even lead to death. Here, a plaque-purified CVA16 strain, L731-P1 (P1), was serially passaged in Vero cells for six times and passage 6 (P6) stock became highly attenuated in newborn mice. Genomic sequencing of the P1 and P6 revealed seven nucleotide substitutions at positions 1434 (C to U), 2744 (A to G), 2747 (A to G), 3161 (G to A), 3182 (A to G), 4968 (C to U), and 6064 (C to U). Six of these substitutions resulted in amino acid changes at VP2-T161 M, VP1–N102D, VP1-T103A, VP1-E241K, VP1-T248A, and 2C–S297F, respectively. P1-based infectious cDNA was generated to further investigate these virulent determinants. Independent reverse transcription-polymerase chain reaction (RT-PCR) amplifications for mutant constructions and plaque-purification of the P6 for isolation of variants were performed to determine dominant mutations and strains more related to attenuation. The virulent P1, attenuated P6, as well as a plaque purified strain (PP) and other four recombinant mutants, were inoculated into one-day-old BALB/c mice and the 50% lethal dose of each strain was determined. Comparison of virulence among these strains indicated that amino acid changes of VP1–N102D, VP1-E241K and 2C–S297F might be associated more closely with a high level attenuation of CVA16-L731-P6 than other mutations. Identification of novel residues associated with virulence may contribute to understanding of molecular basis of virulence of CVA16 and other enteroviruses.

Genome-Wide Identification of WRKY Genes in Artemisia annua: Characterization of a Putative Ortholog of AtWRKY40.

Artemisia annua L. is well-known as the plant source of artemisinin, a sesquiterpene lactone with effective antimalarial activity. Here, a putative ortholog of the Arabidopsis thaliana WRKY40 transcription factor (TF) was isolated via reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends in A. annua and named AaWRKY40. A putative nuclear localization domain was identified in silico and experimentally confirmed by using protoplasts of A. annua transiently transformed with AaWRKY40-GFP. A genome-wide analysis identified 122 WRKY genes in A. annua, and a manually curated database was obtained. The deduced proteins were categorized into the major WRKY groups, with group IIa containing eight WRKY members including AaWRKY40. Protein motifs, gene structure, and promoter regions of group IIa WRKY TFs of A. annua were characterized. The promoter region of AaWRKY group IIa genes contained several abiotic stress cis-acting regulatory elements, among which a highly conserved W-box motif was identified. Expression analysis of AaWRKY40 compared to AaWRKY1 in A. annua cell cultures treated with methyl jasmonate known to enhance artemisinin production, suggested a possible involvement of AaWRKY40 in terpenoid metabolism. Further investigation is necessary to study the role of AaWRKY40 and possible interactions with other TFs in A. annua.

Characterization and complete genome sequences of two novel variants of the family Closteroviridae from Chinese kiwifruit.

Two distinct closterovirus-like genome sequences (termed AdV-1 v1 and v2) were identified in Actinidia chinensis var. deliciosa ‘Miliang-1’ that had no disease symptoms using high-throughput sequencing. Using overlapping reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends, the genomic sequences of AdV-1 v1 and v2 were confirmed as 17,646 and 18,578 nucleotides in length, respectively. The two complete genomes contained 9 and 15 open reading frames, respectively, coding for proteins having domains typical of Closteroviridae, such as RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homolog (HSP70h) and coat protein (CP). Sequence analysis showed that the amino acid sequences of RdRp, HSP70h, and CP of the two variants exhibited high similarity (> 80%), while their genomic organization was somewhat different. This suggested that the two viral genomes identified here are variants of the family Closteroviridae in a single kiwifruit host. Furthermore, phylogenetic relationship analysis revealed that the two variants had a closer relationship with the unclassified virus Persimmon virus B (PeVB) and Actinidia virus 1 (AcV-1) than with other members of the family Closteroviridae, as did their genomic organization. It is speculated that the two variants, together with PeVB and AcV-1 belong to a new subfamily of Closteroviridae.

Molecular cloning, tissue distribution and function analysis of duck TLR7

Toll-like receptors (TLRs) play an important role in detecting pathogen-associated molecular patterns (PAMPs). Among the TLRs, TLR7 is involved in the recognition of antiviral compounds and single-stranded RNA. This study was designed to explore the structure and function of TLR7 in duck (Anas platyrhynchos), a natural host for avian influenza virus. Firstly, the full-length cDNA of Shaoxing egg-laying duck TLR7 (duTLR7) was obtained using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). It consisted of 38 base pairs (bp) 5′-untranslated region (UTR), 187 bp 3′-UTR, and 3270 bp open reading frame that encodes a single protein of 1089 amino acid residues. DuTLR7 shares high identity with TLR7 genes from other vertebrates. In healthy ducks, duTLR7 transcripts were broadly expressed in different tissues, with higher expression levels in the liver, kidney, and thymus. The highest relative transcript level of duTLR7 could be induced with R848 stimulation. In addition, overexpression of duTLR7 by stimulating with poly(I:C) significantly promoted IFN-β, NF-κB, IRF7, TRIF, Mx, STAT1 and STAT2 expressions. Taken together, these results suggest that TLR7 may play an important role in the innate immune response of ducks.