HCV-RNA positivity in peripheral blood mononuclear cells of patients with chronic HCV-infection: does it really mean viral replication?

AIM: To analyze the association of HCV-RNA with peripheral blood mononuclear cells (PBMC) and to answer the question whether HCV-RNA positivity in PBMC is due to viral replication. METHODS: HCV-RNA was monitored in serum and PBMC preparations from 15 patients with chronic HCV infection before, during and after an IFN-alpha therapy using a nested RT/PCR technique. In a second approach, PBMC from healthy donors were incubated in HCV positive plasma. RESULTS: In the IFN-alpha responding patients,HCV-RNA disappeared first from total RNA preparations of PBMC and then from serum. In contrast, in relapsing patients, HCV-RNA reappeared first in serum and then in PBMC. A quantitative analysis of the HCV-RNA concentration in serum was performed before and after transition from detectable to non detectable HCV-RNA in PBMC-RNA and vice versa. When HCV-RNA was detectable in PBMC preparations, the HCV concentration in serum was significantly higher than the serum HCV-RNA concentration when HCV-RNA in PBMC was not detectable. Furthermore, at no time during the observation period was HCV specific RNA observed in PBMC, if HCV-RNA in serum was under the detection limit. Incubation of PBMC from healthy donors with several dilutions of HCV positive plasma for two hours showed a concentration dependent PCR positivity for HCV-RNA in reisolated PBMC. CONCLUSION: The detectability of HCV-RNA in total RNA from PBMC seems to depend on the HCV concentration in serum. Contamination or passive adsorption by circulating virus could be the reason for detection of HCV-RNA in PBMC preparations of chronically infected patients.

RNA interference and antiviral therapy

RNA interference (RNAi) is an evolutionally conserved gene silencing mechanism present in a variety of eukaryotic species. RNAi uses short double-stranded RNA (dsRNA) to trigger degradation or translation repression of homologous RNA targets in a sequence-specific manner. This system can be induced effectively in vitro and in vivo by direct application of small interfering RNAs (siRNAs), or by expression of short hairpin RNA (shRNA) with non-viral and viral vectors. To date, RNAi has been extensively used as a novel and effective tool for functional genomic studies, and has displayed great potential in treating human diseases, including human genetic and acquired disorders such as cancer and viral infections. In the present review, we focus on the recent development in the use of RNAi in the prevention and treatment of viral infections. The mechanisms, strategies, hurdles and prospects of employing RNAi in the pharmaceutical industry are also discussed.

The Biochemical Impact by Covalent Shielding of the Anionic Oxygen of the Phosphate Group in DNA and RNA as Methylated Phosphotriester Linkage on the Inhibition of DNA Duplication and on HIV-1 RNA Viral Infectivity Has Been Seriously Overlooked

With the help of model experiments, we are able to offer a detailed proposal for the inhibition of DNA duplication and no inhibition of RNA viral infectivity. As a backbone, we introduced methyl phosphotriester (MPTE). Duplex formation according to the traditional Watson and Crick base-pairing: [(MPTE)n−1 DNA] * DNA and [(MPTE)n−1 DNA] * RNA, where n = number of DNA and RNA bases. However, in the latter case, inhibition is obtained by reduction of the number of MPTE linkages, as is confirmed with model experiments and under biological conditions with micro (mi)RNA substrates. The latter results have recently been published. One or more single MPTEs are disseminated over different places of DNA without neighbour MPTEs (Prof. Wen-Yih Chen and his group, Taiwan).

HIV-1 RNA Viral Load, CD4 Count and Some Haematological Parameters of People Living with HIV in the Enugu Metropolis

Background: It is widely known that the human immune-deficiency virus (HIV) induces biochemical and physiological changes in affected persons. Consequently, the overall aim of this study was to evaluate the HIV-1 RNA viral load, CD4 count, and certain haematological parameters among HIV treatment-na?ve subjects in the Enugu metropolis of Nigeria. Materials and Methods: A total of 252 HIV-infected, ART-native subjects (≥18) attending the University of Nigeria Teaching Hospital (UNTH) in Ituku-Ozalla, Enugu were recruited for this study and were made up of 157 (62.3%) females and 95 (37.7%) males. A total of 250 HIV-negative subjects were used as control subjects (100 males and 150 females). Blood samples were collected from all the participants and their HIV-1 status was confirmed by an immunoblot confirmatory test. Their haematological parameters and CD4 count were evaluated, while the HIV-1 viral load was only assessed on confirmed HIV-positive subjects. Results: There was female predominance (62.3%) among these HIV-positive subjects. The mean age of HIV-positive subjects was 39.16 ± 10.08 years while the mean age of the control subjects was 34.8 ± 8.6 years. The age group of 31 - 40 years (102/252 (40.5%)) constituted most of the test subjects. The total white blood cells (TWBC) (6.05 ± 5.46), lymphocyte counts (36 ± 14), haemoglobin concentrations (Hb) (9.85 ± 7.36) and the CD4 counts (242 ± 228) of the HIV-infected subjects showed a significant difference when compared with their control counterpart values of TWBC (4.5 ± 0.568), lymphocytes (39.67 ± 8.2), Hb (13.48 ± 1.5), and CD4 counts (807 ± 249) (p 0.05). Anaemia, lymphocytopenia, and thrombocytopenia were the haematological abnormalities seen in the HIV-positive subjects. HIV viral load correlated with haemoglobin concentration, CD4 count, lymphocyte count, and neutrophil count (p Conclusion: Prognostic factors, such as haemoglobin concentrations, CD4 counts, lymphocyte counts, and neutrophil counts can be used to monitor patients’ viral loads since they correlate with the latter;furthermore, age is a factor that should be considered in the management of HIV-positive patients.

Down-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference

Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation.Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or aftermalignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper wedemonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression inairway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targetinghuman IL-8 in cultured airway epithelial cells (IB3-1, Cftr-/-; C38, Cftr-corrected) stimulated with TNF-α, IL-1β orheat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reducedby shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-κB, or on the protein levels ofIκB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for thetreatment of inflammatory diseases.

Structure-function relationship in viral RNA genomes: The case of hepatitis C virus

The acquisition of a storage information system beyond the nucleotide sequence has been a crucial issue for the propagation and dispersion of RNA viruses. This system is composed by highly conserved, complex structural units in the genomic RNA, termed functional RNA domains. These elements interact with other regions of the viral genome and/or proteins to direct viral translation, replication and encapsidation. The genomic RNA of the hepatitis C virus(HCV) is a good model for investigating about conserved structural units. It contains functional domains, defined by highly conserved structural RNA motifs, mostly located in the 5'-untranslatable regions(5'UTRs) and 3'UTR, but also occupying long stretches of the coding sequence. Viral translation initiation is mediated by an internal ribosome entry site located at the 5' terminus of the viral genome and regulated by distal functional RNA domains placed at the 3' end. Subsequent RNA replication strongly depends on the 3'UTR folding and is also influenced by the 5' end of the HCV RNA. Further increase in the genome copy number unleashes the formation of homodimers by direct interaction of two genomic RNA molecules, which are finally packed and released to the extracellular medium. All these processes, as well as transitions between them, are controlled by structural RNA elements that establish a complex, direct and long-distance RNARNA interaction network. This review summarizes current knowledge about functional RNA domains within the HCV RNA genome and provides an overview of the control exerted by direct, long-range RNA-RNA contacts for the execution of the viral cycle.

Carrimycin inhibits coronavirus replication by decreasing the efficiency of programmed–1 ribosomal frameshifting through directly binding to the RNA pseudoknot of viral frameshift-stimulatory element

The pandemic of SARS-CoV-2 worldwide with successive emerging variants urgently calls for small-molecule oral drugs with broad-spectrum antiviral activity.Here,we show that carrimycin,a new macrolide antibiotic in the clinic and an antiviral candidate for SARS-CoV-2 in phase III trials,decreases the efficiency of programmed–1 ribosomal frameshifting of coronaviruses and thus impedes viral replication in a broad-spectrum fashion.Carrimycin binds directly to the coronaviral frameshift-stimulatory element(FSE)RNA pseudoknot,interrupting the viral protein translation switch from ORF1a to ORF1b and thereby reducing the level of the core components of the viral replication and transcription complexes.Combined carrimycin with known viral replicase inhibitors yielded a synergistic inhibitory effect on coronaviruses.Because the FSE mechanism is essential in all coronaviruses,carrimycin could be a new broad-spectrum antiviral drug for human coronaviruses by directly targeting the conserved coronaviral FSE RNA.This finding may open a new direction in antiviral drug discovery for coronavirus variants.

PPM1D Silencing by Lentiviral-mediated RNA Interference Inhibits Proliferation and Invasion of Human Glioma Cells

To construct a lentiviral shRNA vector targeting human protein phosphatase 1D magnesium-dependent（PPM1D） gene and detect its effectiveness of gene silencing in human gliomas,specific siRNA targets with short hairpin frame were designed and synthesized.DNA oligo was cloned into the pFU-GW-iRNA lentiviral expression vector,and then PCR and sequencing analyses were conducted to verify the constructs.After the verified plasmids were transfected into 293T cells,the lentivirus was produced and the titer of virus was determined.Real-time quantitative PCR and Western blot were performed to detect the PPM1D expression level in the infected glioma cells.PCR and Western blot analyses revealed the optimal interfering target,and the virus with a titer of 6×10^8 TU/mL was successfully packaged.The PPM1D expression in human glioma cells was knocked down at both mRNA and protein levels by virus infection.The expression of PPM1D mRNA and protein was decreased by 76.3% and 87.0% respectively as compared with control group.The multiple functions of human glioma cells after PPM1D RNA interference were detected by flow cytometry and cell counting kit-8（CCK-8）.Efficient down-regulation of PPM1D resulted in significantly increased cell apoptosis and reduced cell proliferation and invasion potential in U87-MG cells.We have successfully constructed the lentiviral shRNA expression vector capable of stable PPM1D gene silencing at both mRNA and protein levels in glioma cells.And our data gave evidence that the reduced cell growth observed after PPM1D silencing in glioma cells was at least partly due to increased apoptotic cell death.

Reverse genetics: Unlocking the secrets of negative sense RNA viral pathogens

Negative-sense RNA viruses comprise several zoonotic pathogens that mutate rapidly and frequently emerge in people including Influenza, Ebola, Rabies, Hendra and Nipah viruses. Acute respiratory distress syndrome, encephalitis and vasculitis are common disease outcomes in people as a result of pathogenic viral infection, and are also associated with high case fatality rates. Viral spread from exposure sites to systemic tissues and organs is mediated by virulence factors, including viral attachment glycoproteins and accessory proteins, and their contribution to infection and disease have been delineated by reverse genetics; a molecular approach that enables researchers to experimentally produce recombinant and reassortant viruses from cloned cD NA. Through reverse genetics we have developed a deeper understanding of virulence factors key to disease causation thereby enabling development of targeted antiviral therapies and well-defined live attenuated vaccines. Despite the value of reverse genetics for virulence factor discovery, classical reverse genetic approaches may not provide sufficient resolution for characterization of heterogeneous viral populations, because current techniques recover clonal virus, representing a consensus sequence. In this review the contribution of reverse genetics to virulence factor characterization is outlined, while the limitation of the technique is discussed withreference to new technologies that may be utilized to improve reverse genetic approaches.

Construction of a lentiviral vector for RNA interference of human VIM gene and its silencing effect in pancreatic cancer cells

Objective： To construct a lentiviral expression vector for RNA interference （RNAi） of human VIM gene; and assess its gene silencing effect in pancreatic cancer cell line Panc-1. Methods： Three pairs of human VIM gene short hairpin RNA（shRNA） sequences were designed using a software available on-line and one pair came from document. After synthesis and annealing, four double-stranded oligonucleotides （dsOligo） were cloned into the pGCL-GFP/U6 plasmid, which were subsequently confirmed by polymerase chain reaction （PCR） and DNA sequencing analysis. Real-time PCR and Westemblotting were used to screen the effective pGCL-GFP-shRNA plasmid in 293T cells, then the most effective one was packed into the recombinant lentivirus Lv-VIM-shRNA with lentiviral packing materials pHelper 1.0 and pHelper 2.0 in 293T cells. The titer of lentivirus was determined by hole-by-dilution titer assay. The silencing effect of Lv-VIM-shRNA in Panc-1 calls were validated by real-time PCR and Western-blotting. Results： An effective Lv-VIM-shRNA was successfully constructed. The titer of lentivirus was determined on 2× 10^9TU/mL. The expressions of VIM mRNA and vimentin were down-regluated in the Panc-1 cells infected with Lv-VIM-shRNA. Conclusion： An effective Lv-VIM-shRNA could inhibit the expression of VIM gene in Panc-1 cells in vitro, which provides a tool for investigating the role of VIM gene in the signaling pathway involved in tumorigenesis and progression of pancreatic cancer and searching new therapeutic targets.

LXRα gene downregulation by lentiviral-based RNA interference enhances liver function after fatty liver transplantation in rats

Steatotic liver grafts, although accepted, increase the risk of poor posttransplantation liver function. However, the growing demand for adequate donor organs has led to the increased use of so-called marginal grafts. Liver X receptor alpha （LXRα） is important in fatty acid metabolism and inter- related with the specific ischemia-reperfusion injury in fatty liver transplantation. This study aimed to investigate whether LXRa RNA interference （RNAi） could improve the organ func- tion of liver transplant recipients. METHODS： Fifty Sprague-Dawley rats were fed with a high-fat diet and 56% alcohol. The livers of these animals had greater than 60% macrovesicular steatosis and were used as liver do- nors. The experimental donors were treated with 7×10^7 TU LXRα-RNAi-LV of a mixture injection and control donors with negative control-LV vector injection into the portal vein 72 hours before the operation. The effects of LXRa-RNAi-LV were assessed by serum aminotransferases, histology, immunostain- ing, and protein levels. The transcription of LXRα mRNA was assessed by reverse transcription-polymerase chain reaction. RESULTS： Compared with controls, LXRa RNAi inhibited the expression of LXRα at the mRNA （0.53±0.03 vs 0.94±0.02, P〈0.05） and protein levels （0.51±0.08 vs 1.09±0.12, P〈0.05）. LXRa RNAi also decreased the expressions of sterol regula- tory element-binding protein lc （SREBP-Ic） and CD36. LXRa RNAi consequently reduced fatty acid accumulation in hepa- tocytes. Compared with control animals, LXRα RNAi-treated group had lower serum alanine aminotransferase, aspartate aminotransferase, interleukin-1β, and tumor necrosis factor- alpha levels and milder pathologic damages. TUNEL analysisrevealed a significant reduction of apoptosis in the livers of rats treated with LXRa-RNAi-LV, and overall survival as determined by the Kaplan-Meier method was improved among rats treated with LXRα-RNAi-LV （P〈0.05）. CONCLUSION： LXRa-RNAi-LV treatment significantly down- regulated LXRa expression and improve steatotic liver graft function and recipient survival after a fatty liver transplanta- tion in rats.

多发性骨髓瘤患者血清lncRNA PITPNA-AS1、lncRNA NORAD水平及其与患者预后的关系

目的:分析多发性骨髓瘤(MM)患者血清长链非编码RNA(lncRNA)PITPNA反义RNA1(PITPNA-AS1)、lncRNA NORAD水平及其与患者预后的关系。方法:分别选择本院收治的96例MM患者和96例来本院健康查体的志愿者作为试验组和对照组,时间在2018年1月至2020年12月期间;采用实时荧光定量PCR(RT-qPCR)法检测两组血清中lncRNA PITPNA-AS1、lncRNA NORAD的相对表达量;采用Pearson相关分析分析血清lncRNA PITPNA-AS1与lncRNA NORAD水平的相关性;采用Kaplan-Meier生存分析分析血清lncRNA PITPNA-AS1、lncRNA NORAD水平与MM预后的关系;采用多元Cox回归分析MM患者预后的影响因素。结果:试验组血清lncRNA PITPNA-AS1、lncRNA NORAD水平显著高于对照组(P<0.05)。Ⅲ期MM患者血清lncRNA PITPNA-AS1、lncRNA NORAD水平显著高于Ⅰ期和Ⅱ期,Ⅱ期显著高于Ⅰ期(P<0.05)。MM患者血清lncRNA PITPNA-AS1与lncRNA NORAD水平呈正相关(r=0.636,P<0.05)。lncRNA PITPNA-AS1和lncRNA NORAD高表达患者3年总生存率均低于低表达患者(χ~2值分别为8.065、11.937,P值分别为0.005、0.001)。ISS分期较高、lncRNA PITPNA-AS1高水平、lncRNA NORAD高水平是MM患者预后的危险因素(P<0.05)。结论:MM患者血清lncRNA PITPNA-AS1、lncRNA NORAD水平异常升高,且与患者预后关系密切。

Effects of lentiviral RNA interference-mediated downregulation of integrin-linked kinase on biological behaviors of human lens epithelial cells

AIM：To investigate the effects of lentivirus（LV）mediated integrin-linked kinase（ILK）RNA interference（RNAi）on biological behaviors of human lens epithelial cells（LECs）.·METHODS：Human cataract LECs and immortalized human LEC line,human lens epithelial（HLE）B-3 cells were transfected by lentiviral vector expressing ILKspecific short hairpin RNA（sh RNA）and then stimulated by transforming growth factor-β（TGF-β）,the silencing of ILK gene and protein was identified by reverse transcription-polymerase chain reaction（RT-PCR）and Western blot methods;biological behaviors including cell cycle and apoptosis,cell morphology,α-smooth muscle actin（SMA）stress fiber formation and cell migration were examined.·RESULTS：Remarkable decreases of ILK protein expression were detected in LECs carrying lentiviral ILK-sh RNA vector;flow cytometry revealed arresting of cell cycle progression through the G1/S transition and higher apoptosis rate in ILK-RNAi-LV transfected cells.Lessα-SMA stress fiber formation and migration was observed in ILK-RNAi-LV transfected LECs.·CONCLUSION：The present study demonstrated that ILK was an important regulator for LECs proliferation and migration.LV mediated ILK RNAi is an effective way todecrease ILK-regulated cell growth by arresting cell cycle progression and increasing cell apoptosis,as well as,to prevent cell migration by inhibiting TGF-βinducedα-SMA stress fiber formation.Thus,LV mediated ILK RNAi might be useful to prevent posterior capsular opacification.

LncRNA NORAD通过miR-513b-5p/GREM1轴调节颅内动脉瘤血管平滑肌细胞增殖、迁移、侵袭和凋亡的作用机制

目的:探讨长链非编码RNA DNA损伤诱导的非编码RNA(LncRNA NORAD)通过miR-513b-5p/GREM1轴调节颅内动脉瘤血管平滑肌细胞(VSMC)增殖、迁移、侵袭和凋亡的机制。方法:采用实时荧光定量聚合酶链式反应(PCR)法检测人颅内动脉瘤组织和正常组织中LncRNA NORAD、miR-513b-5p及GREM1表达。体外分离培养人VSMC,随机分为对照组、LncRNA NORAD siRNA组、miR-513b-5p mimics组、共转染(LncRNA NORAD siRNA+miR-513b-5p inhibitor)组、共转染阴性对照(LncRNA NORAD siRNA阴性对照+miR-513b-5p inhibitor阴性对照)组,分组转染后,采用实时荧光定量PCR法检测各组细胞LncRNA NORAD、miR-513b-5p及GREM1 mRNA表达;采用细胞计数试剂盒(CCK-8)和免疫荧光染色检测各组细胞增殖情况;采用Hoechst 33342染色和免疫荧光染色检测各组细胞凋亡情况;采用细胞划痕实验和Transwell实验检测各组细胞迁移、侵袭情况;采用免疫印记实验检测各组细胞上皮间充质转化(EMT)标志蛋白神经钙黏素(N-cadherin)、E-钙黏素(E-cadherin)、波形蛋白(Vimentin)表达;采用双荧光素酶报告实验分析VSMC中LncRNA NORAD对miR-513b-5p、miR-513b-5p对GREM1的靶向调控。结果:与正常组织比较,颅内动脉瘤组织LncRNA NORAD、GREM1 mRNA表达明显升高(P<0.05),miR-513b-5p表达明显降低(P<0.05)。与对照组比较,LncRNA NORAD siRNA组、miR-513b-5p mimics组细胞GREM1 mRNA表达、增殖率、Ki67阳性率、迁移率、侵袭数及N-cadherin、Vimentin蛋白表达降低(P<0.05),miR-513b-5p表达、凋亡率及Bax/Bcl-2、E-cadherin蛋白表达升高(P<0.05);共转染阴性对照组各指标差异无统计学意义(P>0.05)。与LncRNA NORAD siRNA组比较,共转染组细胞GREM1 mRNA表达、增殖率、Ki67阳性率、迁移率、侵袭数及N-cadherin、Vimentin蛋白表达升高(P<0.05),miR-513b-5p表达、凋亡率及Bax/Bcl-2、E-cadherin蛋白表达降低(P<0.05)。结论:敲低LncRNA NORAD可通过上调miR-513b-5p表达而降低GREM1表达,从而抑制VSMC增殖与侵袭迁移,并促使其凋亡。

子宫内膜癌组织中lncRNA HOXA-AS2、FOXD2-AS1、CRNDE的表达与患者临床病理特征及预后的关系

目的探讨子宫内膜癌组织中长链非编码RNA HOXA-AS2(lncRNA HOXA-AS2)、长链非编码RNA FOXD2-AS1(lncRNA FOXD2-AS1)、长链非编码RNA CRNDE(lncRNA CRNDE)的表达与患者临床病理特征及预后的关系。方法收集2017年10月至2020年2月于该院住院手术的119例子宫内膜癌患者术中切除的子宫内膜癌的癌组织及癌旁组织标本。回顾性分析组织中HOXA-AS2、FOXD2-AS1、CRNDE相对表达水平,以及三者表达与患者临床病理特征、3年生存率的关系。结果子宫内膜癌患者癌组织HOXA-AS2、FOXD2-AS1、CRNDE相对表达水平均高于癌旁组织,差异有统计学意义(P<0.05)。子宫内膜癌患者癌组织HOXA-AS2、FOXD2-AS1、CRNDE相对表达水平两两之间均呈正相关(r_(HOXA-AS2 vs.FOXD2-AS1)=0.384,P=0.001;r_(HOXA-AS2 vs.CRNDE)=0.576,P<0.001;r_(FOXD2-AS1 vs.CRNDE)=0.326,P=0.003)。HOXA-AS2、FOXD2-AS1、CRNDE高表达组中国际妇产科学联合会分期为Ⅲ+Ⅳ期、发生淋巴结转移、浸润深度为深层、分化程度为低分化的患者所占比例高于低表达组,差异有统计学意义(P<0.05)。子宫内膜癌患者癌组织HOXA-AS2低表达组3年生存率(52/60,86.67%)高于子宫内膜癌患者癌组织HOXA-AS2高表达组(40/59,67.79%),差异有统计学意义(χ^(2)=6.039,P<0.05);子宫内膜癌患者癌组织FOXD2-AS1低表达组3年生存率(53/59,89.83%)高于子宫内膜癌患者癌组织FOXD2-AS1高表达组(39/60,65.00%),差异有统计学意义(χ^(2)=10.456,P<0.05);子宫内膜癌患者癌组织CRNDE低表达组3年生存率(51/60,85.00%)高于子宫内膜癌患者癌组织CRNDE高表达组(41/59,69.49%),差异有统计学意义(χ^(2)=4.079,P<0.05)。HOXA-AS2、FOXD2-AS1、CRNDE是子宫内膜癌患者死亡的危险因素(P<0.05)。结论子宫内膜癌的癌组织HOXA-AS2、FOXD2-AS1、CRNDE的表达与患者临床病理特征及预后密切相关。

Comparable outcomes but higher risks of prolonged viral RNA shedding duration and secondary infection in cancer survivors with COVID-19: A multi-center, matched retrospective cohort study

Results Sixty-one cancer survivors and 183 matched non-cancer patients were screened from 2,828 COVID-19 infected patients admitted to 4 hospitals in Wuhan,China.The median ages of the cancer survivor cohort and non-cancer patient cohort were 64.0(55.0–73.0)and 64.0(54.0–73.5),respectively(P=0.909).Cancer survivors reported a higher incidence of symptom onset than non-cancer patients.Fever(80.3%vs.65.0%;P=0.026)was the most prevalent symptom,followed by cough(65.6%vs.37.7%;P<0.001),myalgia,and fatigue(45.9%vs.13.6%;P<0.001).The risks of the development of severe events(adjusted hazard ratio[AHR]=1.25;95%confidence interval[CI]:0.76–2.06;P=0.378)and mortality(relative risk[RR]=0.90,95%CI:0.79–1.04;P=0.416)in the cancer survivor cohort were comparable to those of the matched non-cancer patient cohort.However,the cancer survivor cohort showed a higher incidence of secondary infection(52.5%vs.30.1%;RR=1.47,95%CI:1.11–1.95;P=0.002)and a prolonged viral RNA shedding duration(32 days[IQR 26.0–46.0]vs.24.0 days[IQR 18.0–33.0];AHR=0.54;95%CI:0.38–0.80;P<0.05).Conclusion Compared to non-cancer patients,cancer survivors with COVID-19 exhibited a higher incidence of secondary infection,a prolonged period of viral shedding,but comparable risks of the development of severe events and mortality.It is helpful for clinicians to take tailored measures to treat cancer survivors with COVID-19.

Small RNA deep sequencing reveals the presence of multiple viral infections in cucurbit crops in Guangdong,China

Viral diseases are among the most critical damaging factors that impose a global threat to the cucurbit industry.China is the world’s leading country for the production and consumption of cucurbits.Guangdong,a province in southern China dominated by the tropical and subtropical climate,favors the survival of different plant viruses and their vectors.Five main cucurbit crops showing various disease symptoms were surveyed and collected to identify viruses infecting cucurbits in Guangdong during 2018–2020.In the field,the incidence ranged from 5-30%,or even 60-100% in the case of severely infected cucurbits.A total of 357 symptomatic samples were collected and subsequently screened for cucurbit viruses by small RNA deep sequencing and assembly(sRSA).Seventeen virus species belonging to 10 genera were identified in the five main cucurbit crops.The most common viruses were papaya ringspot virus(PRSV;Potyvirus),zucchini tigre mosaic virus(ZTMV;Potyvirus),zucchini yellow mosaic virus(ZYMV;Potyvirus),and watermelon silver mottle virus(WSMoV;Orthotospovirus),with infection rates of 24.4,19.0,17.1,and 14.3%,respectively.Notably,the most prevalent viruses were melon yellow spot orthotospovirus(MYSV)in cucumber,PRSV in squash,cucumber green mottle mosaic virus(CGMMV;Tobamovirus)in bottle gourd,WSMoV in white gourd,and ZYMV in luffa.Mixed infections were prevalent,and the types of mixed infections varied substantially in different cucurbit crops.Moreover,the full-length nucleotide sequences of watermelon green mottle mosaic virus(WGMMV),CGMMV,and watermelon virus A(WVA;Wamavirus)identified in bottle gourd were cloned and analyzed.This study is the first reporting WGMMV infecting bottle gourd in China mainland.In summary,the results demonstrate that in Guangdong,the most prevalent viruses belong to potyviruses,orthotospoviruses,and tobamoviruses groups.The findings will facilitate agricultural researchers and farmers to plan and implement effective disease control strategies aiming at timely detection and management of cucurbit-infecting viral pathogens.

Adenoviral vector mediated-expression of caspase-3 siRNA on apoptosis induced by lipopolysaccharide

Objective： To construct the recombinant adenovirus expressing small RNA of rats caspase-3 and observe the down-regulation effect of caspase-3 in neurons induced by lipopolysaccharide（LPS） in vitro. Methods： pShuttleHl-siCas3 containing Oligo DNA of the targeting sequences and pEGFPC1-Cas3 containing caspase-3 and EGFP sequences were constructed respectively, pShuttleH 1-siCas3 and pEGFPC 1-Cas3 were co-transfected to the 293 cells by liposomes to determine interfering efficacy by flow cytometry, pShuttleHl-siCas3 was linearized and transformed into E. coli B J5183 cells containing backbone plasmid pAdEasy-1. The recombinant plasmid was transfected into 293 cells to package the adenovirus Ad-siCas3. The titers of adenovirus were determined by the specific 50% tissue culture infection dosage method. After virus infected the cultured hippocampus neurons, LPS-induced apoptosis and caspase-3 mRNA expression were observed. Results： It was identified that the sequence of target gene was correctly inserted into the genome of virus. The expression of green fluorescence protein was reduced by pShuttleHl-siCas3 in 293 cells. The titer of recombinant adenovirus was 1.06×10^10pfu/ml. After virus infection, caspase-3 mRNA was greatly reduced and neurons apoptosis was suppressed. Conclusion： The recombinant adenovirus expressing rats caspase-3 siRNA were successfully constructed, which may probably be further used in pain therapy by its anti-apoptosis effect.

Construction and Characterization of Lentiviral shRNA Expression Vector Targeting Rat CD40 Gene in Dendritic Cells

To construct a lentiviral shRNA vector targeting rat CD40 gene and detect its effectiveness of gene silencing in dendritic cells（DCs）, specific siRNA targets with short hairpin frame were designed and synthesized according to the mRNA sequence of rat CD40 gene. DNA oligo was cloned into lentiviral expression vector, and then PCR and sequencing analyses were conducted to verify the constructs. The verified plasmids were transfected into 293T cells that over-express recombinant CD40 in order to select the most effective siRNA targets, shRNA lentiviruses from the selected constructs were propagated and harvested with a virus packaging system, and the virus titers were determined. Western blot and Real-time PCR were performed to determine CD40 expression level in the virusinfected dendritic cells. PCR and sequencing analyses reveal that shRNA plasmids of four targets were successfully constructed. The optimal interfering target was selected, and the virus with a titer of 5 × 10^7 TU/mL was successfully packaged. CD40 expression in rat DCs was knockdown at both mRNA and protein levels by virus infection. In comparison to that of control groups, CD40 mRNA expression and protein expression were decreased by 60.9% and 61.2%, respectively. We have successfully constructed recombinant lentiviral shRNA expression vector targeting rat CD40 gene that can effectively down-regulate CD40 gene expression at mRNA and protein levels in rat DC.

Long noncoding RNA negative regulator of antiviral response contributes to pancreatic ductal adenocarcinoma progression via targeting miR-299-3p

BACKGROUND Pancreatic ductal cancer(PDAC)has high malignancy and poor prognosis.Long noncoding RNAs(lncRNAs)are associated with high levels of malignancy,including PDAC.However,the biological and clinical significance of negative regulator of antiviral response(NRAV)in PDAC is unclear.AIM To study the regulatory role of lncRNA NRAV in PDAC.METHODS GEPIA analyzed lncRNA NRAV and miRNA(miR-299-3p)expression levels in PDAC tissues and measured them in PDAC cells by quantitative measurements in real time.The specific role of NRAV and miR-299-3p in cell proliferation and transfer potential was evaluated by cell formation analysis,Cell Counting Kit-8 and Transwell analysis.The relationship between NRAV and miR-299-3p was studied by predictive bioinformatics,RNA immunoassay,and fluorescence enzyme analysis.In vivo experiments included transplantation of simulated tumor cells under naked mice.RESULTS The expression level of lncRNA NRAV was higher in both tumor tissues and cell lines of PDAC and was negatively associated with the clinical survival of PDAC patients.Functionally,overexpression of NRAV promoted cell proliferation and metastasis of PDAC cells,while knockdown of NRAV reversed these effects.Finally,NRAV was performed as a molecular sponge of miR-299-3p.Moreover,overexpression of miR-299-3p could reverse the promoting effects of NRAV on cell proliferation and metastasis of PDAC cells.CONCLUSION NRAV facilitates progression of PDAC as a molecular sponge of miR-299-3p and may be a potential molecular marker for diagnosis and treatment of PDAC.