Comparative analysis of SARS-CoV-2 variants Alpha (B.1.1.7), Gamma (P.1), Zeta (P.2) and Delta (B.1.617.2) in Vero-E6 cells: ultrastructural characterization of cytopathology and replication kinetics

Abstract This study compares the effects of virus-cell interactions among SARS-CoV-2 variants of concern (VOCs) isolated in Brazil in 2021, hypothesizing a correlation between cellular alterations and mortality and between viral load and transmissibility. For this purpose, reference isolates of Alpha, Gamma, Zeta, and Delta variants were inoculated into monolayers of Vero-E6 cells. Viral RNA was quantified in cell supernatants by RT‒PCR, and infected cells were analyzed by Transmission Electron Microscopy (TEM) for qualitative and quantitative evaluation of cellular changes 24, 48, and 72 hours postinfection (hpi). Ultrastructural analyses showed that all variants of SARS-CoV-2 altered the structure and function of mitochondria, nucleus, and rough endoplasmic reticulum of cells. Monolayers infected with the Delta variant showed the highest number of modified cells and the greatest statistically significant differences compared to those of other variants. Viral particles were observed in the cytosol and the cell membrane in 100 % of the cells at 48 hpi. Alpha showed the highest mean particle diameter (79 nm), and Gamma and Delta were the smallest (75 nm). Alpha and Gamma had the highest particle frequency per field at 48 hpi, while the same was observed for Zeta and Delta at 72 hpi and 24 hpi, respectively. The cycle threshold of viral RNA varied among the target protein, VOC, and time of infection. The findings presented here demonstrate that all four VOCs evaluated caused ultrastructural changes in Vero-E6 cells, which were more prominent when infection occured with the Delta variant.

Synthesis and biological activities of chlorin e6-based conjugate of fluorouracil as dual-mode antitumor photosensitizer / 药学实践杂志

Objective To design and synthesize the conjugate (compound 1) of chlorin e6 (compound 3) with fluorouracil (5-Fu) as novel pH-responsive dual-mode antitumor photosensitizer by acyl hydrazone bond coupling, based on literature reports that combination of 5-Fu and photosensitizer possess synergistic anti-tumor effect, and investigate its photodynamic antitumor activity and mechanism. Methods Lead compound 3 was obtained by alkali degradation with 25% KOH-CH3OH on pheophorbide a (compound 4) which was prepared through acid hydrolysis of chlorophyll a in crude chlorophyll extracts from silkworm excrement. Reflux reaction of 5-Fu with P2S5 in pyridine formed crude 4-thio-5-fluorouracil which was followed to react with hydrazine hydrate (N2H4·H2O) in CH3OH to give 5-fluorouracil-4-hydrazone (compound 2). Then, treatment of compound 3 i.e. acid alkali degradation product of chlorophyll a in silkworm excrement with EDC·HCl generated its 171- and 152 cyclic anhydride which was followed to directly react with intermediate compound 2 to successfully get title compound 1. In addition, its pH-responsive 5-Fu release and photodynamic antitumor activity and their mechanisms in vitro were investigated. Results Compound 1 could responsively release 5-Fu at pH 5.0, with a cumulative release rate of 60.3% within 24 h. It exhibited much higher phototoxicity against melanoma B16-F10 and liver cancer HepG2 cells than talaporfin and its precursor compound 3, with IC50 value being 0.73 μmol/L for B16-F10 cells and 0.90 μmol/L for HepG2 cells, respectively. Upon light irradiation, it also could significantly induce cell apoptosis and intracellular ROS level and block cell cycle in S phase. Its structure was confirmed by UV, 1H-NMR, ESI-MS and elemental analysis data. Conclusion The conjugate compound 1 of compound 3 and 5-Fu has the advantages of strong PDT anticancer activity, high therapeutic index (i.e. dark toxicity/phototoxicity ratio) and responsively release 5-Fu at pH 5.0 etc. which shows “unimolecular” dual antitumor effects of PDT and chemotherapy and is worthy of further research and development.

Effect of silencing E6-associated protein on level of p53 protein in human papilloma virus negative cervical cancer cells / 中国生物制品学杂志

@#Objective To investigate the effect of silencing E6-associated protein(E6AP)on the level of p53 protein in human papilloma virus(HPV)negative cervical cancer cells(C33A cells).Methods The siRNA sequence silencing E6AP(siE6AP)and silencing control disordered siRNA sequence(siControl)were transfected into C33A cells with the mediation of LipofectamineTM2000 transfection reagent respectively.The silencing effect of siRNA on E6AP and the expression of p53and cleaved-caspase-3 proteins were detected by Western blot.Results The levels of E6AP protein in C33A cells of siE6AP group were significantly lower(t =-4.597,P<0.05),while the levels of p53 and cleaved-caspase-3 proteins were significantly higher than those of siControl group(t = 4.533 and 7.099 respectively,each P<0.05).Conclusion Silencing of E6AP significantly increased the expression of p53 protein in C33A cells,indicating that silencing of E6AP may restore the activity and function of p53 protein in C33A cells.

Mechanism of nitidine chloride inducing apoptosis of cervical cancer cells by inhibiting p53 ubiquitination degradation / 中国药理学通报

Aim To investigate the effect of nitidine chloride (NC) on the apoptosis of cervical cancer cells and its mechanism. Methods Cervical cancer cell lines HeLa and SiHa were selected as subjects. The cytotoxicity of NC and its inhibitory effect on cell growth were detected by CCK-8 assay and clone formation assay. The effect of NC on the apoptosis of cervical cancer cells was detected by TUNEL assay, and the expression of apoptosis-related proteins was detected by Western blot. The effects of NC on the interaction between p53 and E6AP protein, the level of p53 ubiquitination modification and the stability of p53 protein in cervical cancer cells were analyzed by immunoprecipi-tation assay, ubiquitination assay and Western blot assay. Results NC could significantly inhibit the proliferation and induce apoptosis of cervical cancer cells. NC could inhibit the interaction between tumor suppressor protein p53 and E6AP in cervical cancer cells, reduce the level of p53 ubiquitination modification, delay the degradation of p53 and increase the expression level of p53 protein. Conclusions NC can inhibit the ubiquitination and degradation of p53, improve the expression level of p53 protein, restore its tumor suppressor function, and thus play an anti -cervical cancer role.

Brain endothelial cell-derived extracellular vesicles with a mitochondria-targeting photosensitizer effectively treat glioblastoma by hijacking the blood‒brain barrier

Glioblastoma (GBM) is the most aggressive malignant brain tumor and has a high mortality rate. Photodynamic therapy (PDT) has emerged as a promising approach for the treatment of malignant brain tumors. However, the use of PDT for the treatment of GBM has been limited by its low blood‒brain barrier (BBB) permeability and lack of cancer-targeting ability. Herein, brain endothelial cell-derived extracellular vesicles (bEVs) were used as a biocompatible nanoplatform to transport photosensitizers into brain tumors across the BBB. To enhance PDT efficacy, the photosensitizer chlorin e6 (Ce6) was linked to mitochondria-targeting triphenylphosphonium (TPP) and entrapped into bEVs. TPP-conjugated Ce6 (TPP-Ce6) selectively accumulated in the mitochondria, which rendered brain tumor cells more susceptible to reactive oxygen species-induced apoptosis under light irradiation. Moreover, the encapsulation of TPP-Ce6 into bEVs markedly improved the aqueous stability and cellular internalization of TPP-Ce6, leading to significantly enhanced PDT efficacy in U87MG GBM cells. An in vivo biodistribution study using orthotopic GBM-xenografted mice showed that bEVs containing TPP-Ce6 [bEV(TPP-Ce6)] substantially accumulated in brain tumors after BBB penetration via transferrin receptor-mediated transcytosis. As such, bEV(TPP-Ce6)-mediated PDT considerably inhibited the growth of GBM without causing adverse systemic toxicity, suggesting that mitochondria are an effective target for photodynamic GBM therapy.

Assessment of the diagnostic value and prognosis of different detection markers in endocervical adenocarcinoma / 中华肿瘤杂志

Objective: To study the diagnostic value of different detection markers in histological categories of endocervical adenocarcinoma (ECA), and their assessment of patient prognosis. Methods: A retrospective study of 54 patients with ECA in the Cancer Hospital, Chinese Academy of Medical Sciences from 2005-2010 were performed. The cases of ECA were classified into two categories, namely human papillomavirus-associated adenocarcinoma (HPVA) and non-human papillomavirus-associated adenocarcinoma (NHPVA), based on the 2018 international endocervical adenocarcinoma criteria and classification (IECC). To detect HR-HPV DNA and HR-HPV E6/E7 mRNA in all patients, we used whole tissue section PCR (WTS-PCR) and HPV E6/E7 mRNA in situ hybridization (ISH) techniques, respectively. Additionally, we performed Laser microdissection PCR (LCM-PCR) on 15 randomly selected HR-HPV DNA-positive cases to confirm the accuracy of the above two assays in identifying ECA lesions. Receiver operating characteristic (ROC) curves were used to analyze the efficacy of markers to identify HPVA and NHPVA. Univariate and multifactorial Cox proportional risk model regression analyses were performed for factors influencing ECA patients' prognoses. Results: Of the 54 patients with ECA, 30 were HPVA and 24 were NHPVA. A total of 96.7% (29/30) of HPVA patients were positive for HR-HPV DNA and 63.3% (19/30) for HR-HPV E6/E7 mRNA, and 33.3% (8/24) of NHPVA patients were positive for HR-HPV DNA and HR-HPV E6/E7 mRNA was not detected (0/24), and the differences were statistically significant (P<0.001). LCM-PCR showed that five patients were positive for HR-HPV DNA in the area of glandular epithelial lesions and others were negative, which was in good agreement with the E6/E7 mRNA ISH assay (Kappa=0.842, P=0.001). Analysis of the ROC results showed that the AUC of HR-HPV DNA, HR-HPV E6/E7 mRNA, and p16 to identify HPVA and NHPVA were 0.817, 0.817, and 0.692, respectively, with sensitivities of 96.7%, 63.3%, and 80.0% and specificities of 66.7%, 100.0%, and 58.3%, respectively. HR-HPV DNA identified HPVA and NHPVA with higher AUC than p16 (P=0.044). The difference in survival rates between HR-HPV DNA (WTS-PCR assay) positive and negative patients was not statistically significant (P=0.156), while the difference in survival rates between HR-HPV E6/E7 mRNA positive and negative patients, and p16 positive and negative patients were statistically significant (both P<0.05). Multifactorial Cox regression analysis showed that International Federation of Obstetrics and Gynecology (FIGO) staging (HR=19.875, 95% CI: 1.526-258.833) and parametrial involvement (HR=14.032, 95% CI: 1.281-153.761) were independent factors influencing the prognosis of patients with ECA. Conclusions: HR-HPV E6/E7 mRNA is more reflective of HPV infection in ECA tissue. The efficacy of HR-HPV E6/E7 mRNA and HR-HPV DNA (WTS-PCR assay) in identifying HPVA and NHPVA is similar, with higher sensitivity of HR-HPV DNA and higher specificity of HR-HPV E6/E7 mRNA. HR-HPV DNA is more effective than p16 in identifying HPVA and NHPVA. HPV E6/E7 mRNA and p16 positive ECA patients have better survival rates than negative.

Optimización de una técnica de PCR convencional para la amplificación de la región LCR y el gen E6 del virus del papiloma humano tipo 16

El cáncer de cuello uterino es el cuarto cáncer más frecuente en mujeres en el mundo y a nivel mundial, el VPH 16 se encuentra presente en aproximadamente el 60% de los casos. A la fecha, las variantes de VPH 16 se clasifican en 4 linajes y 16 sublinajes asociándose algunas variantes con severidad de lesión. La secuenciación de la región LCR y del gen E6 es utilizada para la clasificación de variantes. Por ello, el objetivo fue optimizar 2 PCRs convencionales para detectar la región LCR y una PCR para el gen E6. Para ello, se utilizaron muestras positivas para VPH 16, cebadores específicos para la región LCR y el gen E6. Se probaron las reacciones a diferentes temperaturas de anillamientos. La concentración de MgCl2, dNTP y cebadores fueron determinadas siguiendo las recomendaciones del fabricante de la enzima ADN polimerasa utilizada. Para la amplificación de la región LCR y el gen E6 del VPH 16, se observaron mejores resultados a una temperatura de anillamiento de 57°C y 50°C, respectivamente. La concentración de MgCl2 utilizada en ambas reacciones fue de 1,5mM, la de dNTP 0,2mM y la de cebadores 0,2uM. La optimización de la reacción de PCR permitirá obtener amplicones aptos para secuenciación, a fin de determinar las variantes génicas y posteriormente evaluar funcionalidad y actividad transcripcional que puedan estar relacionadas con la patogénesis cervical.

Cervical cancer is the fourth most common cancer in women in the world and worldwide HPV 16 is present in approximately 60% of cases. To date, HPV 16 variants are classified into 4 lineages and 16 sublineages, with some variants being associated with lesion severity. Sequencing of the LCR region and the E6 gene is used for variant classification. Therefore, the objective was to optimize two conventional PCRs to detect the LCR region and one PCR for the E6 gene. For this purpose, HPV 16 positive samples, specific primers for the LCR region and the E6 gene were used. The reactions were tested at different alignment temperatures. The concentration of MgCl2, dNTP, and primers was determined following the recommendations of the manufacturer of the DNA polymerase enzyme used. For amplification of the LCR region and the HPV 16 E6 gene, the best results were observed at an annealing temperature of 57°C and 50°C, respectively. The concentration of MgCl2 used in both reactions was 1.5mM, dNTP 0.2mM and primers 0.2uM. The present optimization will be used to sequence the amplified products to determine the variants and subsequently evaluate the functionality and transcriptional activity in order to relate it to cervical pathogenesis.

Mechanisms underlying synergistic induction and promotion of cutaneous squamous cell carcinoma in nude mice by ultraviolet light and human papillomavirus 16 E6 / 中华皮肤科杂志

Objective:To establish a xenograft model of cutaneous squamous cell carcinoma (CSCC) in nude mice, and to explore mechanisms underlying synergistic induction and promotion of CSCC in nude mice by ultraviolet radiation and human papillomavirus (HPV) infection.Methods:The human CSCC A431 cells were divided into 3 groups, namely HPV16 E6 overexpression group (LV-OE-HPV16 E6 group) transfected with adenovirus-mediated HPV16 E6 gene, empty vector group transfected with empty adenovirus vectors, and blank control group remaining untransfected. Using serum-free Dulbecco′s modified Eagle′s medium (DMEM) , A431 cells in the empty vector group and LV-OE-HPV16 E6 group were prepared into single-cell suspensions, which were subcutaneously inoculated into the left buttocks of SKH-1 nude mice separately, namely empty vector group ( n = 16) and LV-OE-HPV16 E6 group ( n = 16) . Tumor growth was observed and recorded for the mice every 3 days. When the tumor size reached 150 mm 3, the modeling was considered successful. After successful modeling, 8 mice in each group were irradiated with ultraviolet light at a dose of 1 440 mJ·cm -2·d -1 for 12 minutes each time, the other 8 mice in each group received no ultraviolet radiation, that is to say, all the 32 mice were divided into 4 groups: empty vector group, empty vector + UV group, LV-OE-HPV16 E6 group, and LV-OE-HPV16 E6 + UV group. After 4-week radiation, these nude mice were sacrificed, tumor weight and volume were measured, a tumor growth curve was drawn, immunohistochemistry study, Western blot analysis and real-time fluorescence-based quantitative PCR (qRT-PCR) were conducted to determine the protein and mRNA expression of Wnt1 and β-catenin in CSCC tissues collected from nude mice, respectively. For normally distributed measurement data, analysis of variance was used for intergroup comparisons, and least significant difference- t test for multiple comparisons; for non-normally distributed measurement data, rank sum test was used for intergroup comparisons. Results:Compared with the empty vector group (2.20 ± 0.24 g) , the tumor weight significantly increased in the empty vector + UV group (2.90 ± 0.36 g, t = 4.39, P < 0.001) , LV-OE-HPV16 E6 group (3.19 ± 0.32 g, t = 6.77, P < 0.001) , and LV-OE-HPV16 E6 + UV group (4.41 ± 0.18 g, t = 20.11, P < 0.001) ; the tumor volume was also significantly higher in the empty vector + UV group (1 033.12 ± 400.15 mm 3, t = 1.90, P < 0.001) , LV-OE-HPV16 E6 group (1 119.21 ± 447.57 mm 3, t = 2.21, P < 0.001) , and LV-OE-HPV16 E6 + UV group (1 464.29 ± 409.98 mm 3, t = 4.22, P < 0.001) than in the empty vector group (688.94 ± 319.31 mm 3) . Immunohistochemical study showed no significant difference in the protein expression of Wnt1 and β-catenin among the 4 groups ( F = 0.76, 0.71, respectively, both P > 0.05) ; Western blot analysis showed significant differences in the protein expression levels of Wnt1 and β-catenin among the 4 groups ( F = 16.74, 49.90, respectively, both P < 0.05) , which were significantly higher in the LV-OE-HPV16 E6 + UV group than in the empty vector group, empty vector + UV group and LV-OE-HPV16 E6 group (all P < 0.05) . qRT-PCR showed a significant difference in the mRNA expression of Wnt1 and β-catenin among the 4 groups ( F = 7.77, 8.38, respectively, both P<0.05) , and the LV-OE-HPV16 E6 + UV group showed significantly increased Wnt1 mRNA expression levels compared with the empty vector group, empty vector + UV group and LV-OE-HPV16 E6 group (all P < 0.05) . Conclusion:Ultraviolet radiation and HPV infection showed synergistic effect on the induction and promotion of CSCC.

Clinical analysis on the application value of human papillomavirus E6/E7 mRNA testing for screening under 30 years old / 中国医师进修杂志

Objective:To evaluate the clinical value of human papillomavirus (HPV) E6/E7 mRNA testing in cervical cancer screening under 30 years old.Methods:The clinical data of 330 young women (less than 30 years old) with minor abnormalities of thinprep cytologic test (TCT) screening for in Dalian Women′s and Children′s Medical Center (Group) Chunliu Region from January 2020 to June 2021 were retrospectively analyzed. Among them, 165 patients underwent HPV DNA typing test (DNA group), and 165 patients underwent HPV E6/E7 mRNA typing test (mRNA group). The positive rate of cervical intraepithelial neoplasia (CIN) Ⅱ and above (CIN Ⅱ +) was compared between two groups, and the positive rates of CIN Ⅱ + in different high risk HPV types. Results:The positive rate of high risk HPV types in mRNA group was significantly lower than that in DNA group: 32.7% (54/165) vs. 47.9% (79/165), and there was statistical difference ( χ2 = 7.87, P<0.05). There was no statistical difference in the incidence of CIN Ⅱ + of patients with positive of high risk HPV types between DNA group and mRNA: 45.6% (36/79) and 59.3% (32/54), P>0.05. There was no statistical difference in the incidence of CIN Ⅱ + of patients with HPV 16, 18 or 45 positive between DNA group and mRNA group: 38.5% (10/26) and 6/10, P>0.05. The incidence of CIN Ⅱ + of patients without HPV 16, 18 or 45 positive in mRNA group was significantly higher than that in DNA group: 60.9% (28/46) vs. 41.3% (26/63), and there was statistical difference ( P<0.05). Conclusions:Without increasing the rate of missed diagnosis, HPV E6/E7 mRNA testing plays an important shunt role in women under 30 years old, and the predicted value of CIN Ⅱ + is higher for patients who are not infected with HPV16/18/45 with HPV E6/E7 mRNA testing.

Isolation and identification of a severe acute respiratory syndrome coronavirus 2 strain in feces from COVID-19 patients in Shanghai / 上海预防医学

ObjectiveTo isolate and study the biological characteristics of severe acute respiratory syndrome coronavirus 2 （SARS-CoV-2） from feces of coronavirus disease 2019 （COVID-19） patients. MethodsVero E6 cells were used for virus isolation and the isolated strains were tested by nucleic acid test， immunofluorescence test， virulence test and whole genome sequencing. 50% tissue culture infective dose （TCID50） was calculated after the cell cultures of each generation were collected ResultsEight fecal specimens were inoculated with Vero E6 cells after treatment and cultured for 48 h. One specimen showed obvious cytopathic effect on Vero E6 cells. One SARS-CoV-2 out of 8 fecal samples from COVID-19 patients were isolated， and separation rate was 12.5%. The TCID50 of P1， P2 and P3 were 104.0/0.2 mL， 104.5/0.2 mL and 104.75/0.2 mL， respectively. Only one of the 8 stool samples had SARS-CoV-2 virus replication and amplification， and the Ct value of the nucleic acid detection was about 10. The sequence of the isolation was more than 99.99% homologous with that of Wuhan-Hu-1（GenBank MN908947）. ConclusionThe SARS-CoV-2 strain is isolated from the fecal samples of COVID-19 cases and is confirmed by genomic sequencing and immunofluorescence test， which indicates the presence of live virus in feces of COVID-19 cases.

Establishment and identification of human immortalized keratinocytes stably expressing human papillomavirus type 16 E6/E7 gene / 中华皮肤科杂志

Objective:To construct human immortalized keratinocytes stably expressing human papillomavirus type 16 (HPV16) E6/E7 gene, and provide a cell model for studying mechanisms underlying HPV16 E6/E7-induced cell immortalization and malignant transformation.Methods:Primary human foreskin keratinocytes (HFKs) were isolated by sequential two-step enzymatic digestion. Cultured HFKs were stably transfected with a HPV16 E6/E7 gene-overexpressing lentiviral vector LV5-HPV16 E6/E7, and consecutively cultured for more than 30 passages. Then, immortalized keratinocytes were screened out and divided into 3 groups: (1) blank control group: second-passage primary HFKs; (2) experimental group: HFKs transfected with LV5-HPV16 E6/E7 at different passages, and the second-passage primary HFKs transfected with LV5-HPV16 E6/E7 were referred to as A0 cells, thereafter, the transfected HFKs were named according to their passage number, such as A1, A2, ... A30; (3) positive control group: the HPV16-positive cervical cancer cell line SiHa. Real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis were performed to determine the mRNA expression of HPV16 E6/E7 and protein expression of HPV16 E6/E7 and CK14, respectively, in the blank control group, experimental group and positive control group. Cell counting kit-8 (CCK8) assay and Transwell insert invasion assay were conducted to assess the cellular proliferative and invasive activity. In vivo tumor formation experiment in nude mice was conducted to investigate the tumorigenicity of A30 cells in the experimental group and SiHa cells in the positive control group. Results:Primary HFKs were successfully isolated. After the primary HFKs were transfected with the recombinant plasmid LV5-HPV16 E6/E7, the blank control group showed no fluorescence in the cells, but showed senescent phenotypes after serial passages, while in the experimental group, the volume and morphology of A30 cells were similar to those of the primary HFKs with the proportion of fluorescence-positive cells being 100%. Compared with the blank control group, the experimental group showed significantly increased mRNA expression levels of HPV16 E6 and E7 in A1, A10, A20 and A30 cells (HPV16 E6: t = 7.12, 8.07, 6.53, 5.66, P < 0.001, < 0.001, = 0.001, = 0.005, respectively; HPV16 E7: t = 3.20, 4.29, 3.75, 4.22; P = 0.024, 0.008, 0.013, 0.014, respectively) . The protein expression of HPV16 E6/E7 was absent in the blank control group, but was observed in A30 and SiHa cells. CCK8 assay showed that the proliferative activity of A10, A20 and A30 cells was significantly higher than that of the blank control group ( t = 6.49, 7.55, 9.43, P = 0.003, 0.002, 0.001, respectively) , while there was no significant difference in the proliferative activity between A1 cells and the blank control group ( t = 2.40, P = 0.074) . Transwell insert invasion assay showed that A30 cells could not cross the basement membrane, but SiHa cells could pass through the basement membrane and were stained blue. Two months after the inoculation with A30 cells in the nude mice, no visible tumors were found, which was confirmed by a histological study. Subcutaneous tumors were formed in the nude mice after the inoculation with SiHa cells. Conclusion:Human immortalized keratinocytes were successfully established by lentivirus-mediated transfection with HPV16 E6/E7 gene, and can serve as an ideal cell model for HPV-related research.

Establishment and evaluation of a fluorescent antibody assay against varicella-zoster virus membrane antigen based on Vero E6 cells / 中国输血杂志

【Objective】 To establish and evaluate a fluorescent antibody to membrane antigen (FAMA) method for detecting antibodies against varicella-zoster virus (VZV) based on Vero E6 cells. 【Methods】 Based on the adapted VZV-Oka-E6 strain that VZV-Oka live attenuated varicella vaccine strain grew in Vero E6 cells, Vero E6 cells were infected with VZV-Oka-E6 of three different doses (104.65, 104.95 and 105.25 TCID50), and the cytopathic effect was observed under a microscope to determine the optimal infection dose of VZV-Oka-E6 strain. Then the detectable sensitivity of the infected cell antigen slides prepared after fixing the infected cells with different fixatives was compared to determine the optimal fixative. As a result, the FAMA method based on Vero E6 cells for the determination of neutralizing anti-VZV has been developed. The established FAMA assay was used to detect the international standard for varicella zoster immunoglobulin with different titers to determine the sensitivity of the assay. The specificity of the assay was evaluated by detecting specific antibodies against human herpes simplex virus (HSV) type 1 and type 2. The neutralizing anti-VZV antibodies of the international standard for varicella zoster immunoglobulin were detected using VZV-infected cell antigen slides prepared in the same batch and four different batches, respectively, to determine the intra-assay repeatability and inter-assay repeatability. The international standard for varicella zoster immunoglobulin with three known titers was detected to evaluate the relative accuracy of this assay. The anti-VZV titers in 20 apheresis plasma samples were determined with the newly established FAMA test and ELISA test, respectively, and the detection results of the two methods were compared using Spearman’s correlation test. 【Results】 The optimal infection dose of the VZV-Oka-E6 strain in FAMA assay was determined to be 105.25 TCID50, and acetone precooled at -20℃ was used as the fixative. The FAMA test has a high sensitivity with a detecting limit of 31.25 mIU/mL for neutralizing anti-VZV titers. The negative result was observed when detecting herpes simplex virus (HSV) type 1 and 2 specific antibodies. The international standard for varicella zoster immunoglobulin was detected by VZV infected cell antigen slides prepared in the same batch and 4 different batches, with the coefficient of variation being 29.95% and 26.71%, respectively. The detection value of the international standard for varicella zoster immunoglobulin with three different titer levels was consistent with their theoretical value. The correlation coefficient of the detection results of 20 apheresis plasma samples by the FAMA test and ELISA test was 0.268. 【Conclusion】 The VZV FAMA assay has good sensitivity, specificity, repeatability, and relative accuracy in detecting neutralizing anti-VZV titers. It can be applied for detecting neutralizing anti-VZV titers in apheresis plasma samples as well as the varicella-zoster immunoglobulin (VZIG) preparations.

Morphology and morphogenesis of SARS-CoV-2 in Vero-E6 cells

BACKGROUND The coronaviruses (CoVs) called the attention of the world for causing outbreaks of severe acute respiratory syndrome (SARS-CoV), in Asia in 2002-03, and respiratory disease in the Middle East (MERS-CoV), in 2012. In December 2019, yet again a new coronavirus (SARS-CoV-2) first identified in Wuhan, China, was associated with a severe respiratory infection, known today as COVID-19. This new virus quickly spread throughout China and 30 additional countries. As result, the World Health Organization (WHO) elevated the status of the COVID-19 outbreak from emergency of international concern to pandemic on March 11, 2020. The impact of COVID-19 on public health and economy fueled a worldwide race to approve therapeutic and prophylactic agents, but so far, there are no specific antiviral drugs or vaccines available. In current scenario, the development of in vitro systems for viral mass production and for testing antiviral and vaccine candidates proves to be an urgent matter. OBJECTIVE The objective of this paper is study the biology of SARS-CoV-2 in Vero-E6 cells at the ultrastructural level. METHODS In this study, we documented, by transmission electron microscopy and real-time reverse transcription polymerase chain reaction (RT-PCR), the infection of Vero-E6 cells with SARS-CoV-2 samples isolated from Brazilian patients. FINDINGS The infected cells presented cytopathic effects and SARS-CoV-2 particles were observed attached to the cell surface and inside cytoplasmic vesicles. The entry of the virus into cells occurred through the endocytic pathway or by fusion of the viral envelope with the cell membrane. Assembled nucleocapsids were verified inside rough endoplasmic reticulum cisterns (RER). Viral maturation seemed to occur by budding of viral particles from the RER into smooth membrane vesicles. MAIN CONCLUSIONS Therefore, the susceptibility of Vero-E6 cells to SARS-CoV-2 infection and the viral pathway inside the cells were demonstrated by ultrastructural analysis.

Diagnostic value of serum miRNA-9-5p, miRNA-21-5p and miRNA-3923 in cervical cancer / 肿瘤研究与临床

Objective:To investigate the diagnostic value of serum miRNA-9-5p (miR-9-5p), miRNA-21-5p (miR-21-5p) and miRNA-3923 (miR-3923) in patients with cervical cancer.Methods:The data of 100 cervical cancer patients in Shanxi Provincial Cancer Hospital from July 2016 to June 2018 (the experimental group) and 100 healthy subjects (the healthy control group) during the same period were collected. The real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) combined with probe hybridization was used to detect the expression levels of human papillomavirus (HPV) E6/E7 mRNA in paraffin-embedded tissues of patients with cervical cancer and in cervical exfoliated cells of the healthy control people. Ct value ≤ 40 cycles was considered as HPV E6/E7 positive. Serum samples from 3 patients with cervical cancer and 3 healthy people were taken out; microRNA (miRNA) Array was used to detect the expression level of 384 miRNA; the differential expression of miRNA was screened out and cluster analysis was performed, and then the screened miRNAs was verified by using qRT-PCR. Finally, the receiver operation characteristics (ROC) curves of screened miRNAs alone and HPV E6/E7 alone or the combination of three miRNAs and HPV E6/E7 in the diagnosis of cervical cancer were drawn to make comparison of diagnostic efficacy.Results:Among the paraffin-embedded tissues from 100 patients with cervical cancer, there were 65 cases (65%) HPV E6/E7 positive; in the healthy control group, HPV E6/E7 in cervical exfoliated cells of 100 people was negative. A total of 248 differentially expressed miRNAs were detected from the serum samples in 3 patients with cervical cancer and 3 healthy people. The cluster analysis finally identified 16 abnormally regulated miRNAs. qRT-PCR verification confirmed that differences in the expression levels of miR-9-5p, miR-21-5p and miR-3923 in the healthy control group and cervical cancer group were statistically significant (all P < 0.01), and then the three were selected to make diagnosis of cervical cancer. The expression levels of miR-9-5p, miR-21-5p and miR-3923 in HPV E6/E7 positive cervical cancer group were higher than those in the healthy control group (all P < 0.05), expression levels of miR-21-5p and miR-3923 in HPV E6/E7 negative cervical cancer group were increased ( P = 0.008, P = 0.038); expression levels of the three miRNAs in HPV E6/E7 positive group were higher than those in HPV E6/E7 negative group (all P < 0.05). ROC curve analysis showed that the area under the curve (AUC) of miR-3923 was the biggest (0.843), the specificity was the highest (82%, the cut-off value was 2.88) and the sensitivity of miR-21-5p was the highest (85%, the cut-off value was 4.08) when miR-9-5p, miR-21-5p and miR-3923 were respectively applied to diagnose the cervical cancer; AUC (0.924), the sensitivity and the specificity (85%, 94%; the cut-off value was 4.04) of the combination of the three indicators were higher than those of the single indicator in the diagnosis of cervical cancer. AUC was 0.766 when HPV E6/E7 was kused alone to diagnose. The diagnostic efficacy of HPV E6/E7 combined with miR-9-5p, miR-21-5p, miR-3923 respectively was further improved, the corresponding AUC was 0.914, 0.848, 0.932, respectively; the diagnostic efficacy of the combination of the four indicators was the highest (AUC was 0.942). Conclusion:miR-9-5p, miR-21-5p and miR-3923 may be helpful in the diagnosis of cervical cancer.

Human papillomavirus type 18 E5 oncoprotein cooperates with E6 and E7 in promoting cell viability and invasion and in modulating the cellular redox state

BACKGROUND High-risk human papillomaviruses (HR-HPVs) are the etiological agents of cervical cancer. Among them, types 16 and 18 are the most prevalent worldwide. The HPV genome encodes three oncoproteins (E5, E6, and E7) that possess a high transformation potential in culture cells when transduced simultaneously. In the present study, we analysed how these oncoproteins cooperate to boost key cancer cell features such as uncontrolled cell proliferation, invasion potential, and cellular redox state imbalance. Oxidative stress is known to contribute to the carcinogenic process, as reactive oxygen species (ROS) constitute a potentially harmful by-product of many cellular reactions, and an efficient clearance mechanism is therefore required. Cells infected with HR-HPVs can adapt to oxidative stress conditions by upregulating the formation of endogenous antioxidants such as catalase, glutathione (GSH), and peroxiredoxin (PRX). OBJECTIVES The primary aim of this work was to study how these oncoproteins cooperate to promote the development of certain cancer cell features such as uncontrolled cell proliferation, invasion potential, and oxidative stress that are known to aid in the carcinogenic process. METHODS To perform this study, we generated three different HaCaT cell lines using retroviral transduction that stably expressed combinations of HPV-18 oncogenes that included HaCaT E5-18, HaCaT E6/E7-18, and HaCaT E5/E6/E7-18. FINDINGS Our results revealed a statistically significant increment in cell viability as measured by MTT assay, cell proliferation, and invasion assays in the cell line containing the three viral oncogenes. Additionally, we observed that cells expressing HPV-18 E5/E6/E7 exhibited a decrease in catalase activity and a significant augmentation of GSH and PRX1 levels relative to those of E5, E6/E7, and HaCaT cells. MAIN CONCLUSIONS This study demonstrates for the first time that HPV-18 E5, E6, and E7 oncoproteins can cooperate to enhance malignant transformation.

Clinical significance of human papillomavirus 16/18 E6 protein detection in shunting and prognosis of patients with atypical squamous cells of undetermined significance and low-grade squamous intraepithelial lesions / 肿瘤研究与临床

Objective To evaluate the value of human papillomavirus (HPV) 16/18 E6 protein detection in shunting and prognosis in patients with atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions (LSIL). Methods A total of 98 patients with ASCUS or LSIL from the Affiliated Cancer Hospital of Shanxi Medical University between May 2014 and May 2015 were selected as the subjects. All of them received the thin-cytologic test (TCT), HPV DNA, HPV16/18 E6 protein tests and colposcopy examination. After 3-year follow-up of patients with cervical intraepithelial neoplasia (CIN) grade Ⅰor bellow lesions diagnosed by biopsy and 30 negative controls, the above tests were performed again. The efficacies of all the tests were analyzed. The value of CIN grade Ⅱ or above was predicted. Results The sensitivity, specificity, positive predictive value and negative predictive value in predicting CIN grade Ⅱor above lesions of HPV16/18 E6 protein , HPV DNA and HPV16/18 DNA was 30.8％, 95.3％, 50.0％, 90.0％, respectively; 84.6％, 37.6％, 17.2％, 94.1％, respectively and 61.5％, 67.1％, 22.2％, 91.9％, respectively in shunting study. The relative risk (RR) of CIN grade Ⅱor above lesions in patients with positive HPV16/18 E6 protein, persistent positive HPV16/18 DNA and positive HPV16/18 DNA was 13.429, 10.231 and 8.343, respectively in the follow-up study. Odds ratio (OR) of HPV16/18 E6 positive protein presenting persistent positive HPV16/18 DNA was 34.833 (95％ CI 5.020-241.711). Conclusions In patients with ASCUS and LSIL, the specificity and positive predictive value of HPV16/18 E6 protein in predicting CIN grade Ⅱ or above lesions are higher than those of HPV DNA and HPV16/18 DNA. Moreover, these patients with HPV16/18 E6 protein positive have a higher risk of developing CIN grade Ⅱ or above lesions and persistent positive HPV16/18 DNA.

Application of AHPV and Its Genotyping in Low-grade Squamous Intraepithelial Lesions / 实用妇产科杂志

Objective:To investigate the application of Aptima high-risk human papillomavirus (HR-HPV) E6/E7 mRNA (AHPV) and its genotyping (GT) in the risk assessment of low-grade squamous intraepithelial lesions (LSIL).Methods:The AHPV and its genotype (AHPV-GT) incervical exfoliated cells were detectedin 529 women with LSIL.The DNA-Based Hybrid Capture 2 HPV Test (HC2-HPV), colposcopy and cervical biopsy were performedsimultaneously.Results:① In 529 patients with LSIL, the positive rate of HC2-HPV in the group of ＜30 years old was significantly higher than that in the group of ≥30 years old (92.2％ vs 83.6％, P=0.026).There was no significant difference in AHPV positive rate among different age groups (82.5％ vs 77.7％, P=0.284).No significant difference of the genotyping (AHPV-GT) was detected between the two groups, either.In the 529 cases, 83 cases of HSIL+were confirmed by histology.81 cases (97.6％) were AHPV positive in the patients with HSIL+;② Compared with other 11 positive types of HR-HPV, the incidence of HSIL+in GT+ women increased significantly (P＜0.05).In the group ≥30 years old, the OR value of HSIL+exposure risk of AHPV16 positive women was the highest (141.00), which was significantly higher than that of 18/45+, GT +, AHPV + (P=0.005, 0.000, 0.000).However, in the group of ＜30 years old, the OR value of HSIL+exposure risk of AHPV16 positive women was 8.50, which showed no significant difference from that of 18/45+ and AHPV-(P=1.000, 0.070).③ In group over 30 years old, the specificity of detecting HSIL+by AHPV was higher than that by HC2-HPV (P＜0.05).There was no significant difference in detection specificity between AHPV and HC2-HPV in women under 30 years old.Conclusions:AHPV and its GT detection are reliable methods for colposcopic screening and risk stratification in women aged over 30 years old with LSIL, more attention should be focused on AHPV16 positive.Better biological markers should be explored for younger women.

Application and clinical significance of human papilloma virus E6/E7 mRNA detection in cervical atypical glandular cells / 中国医师进修杂志

Objective To investigate the application and clinical significance of human papilloma virus (HPV) E6/E7 mRNA detection in cervical atypical glandular cells (AGC). Methods Four hundred and forty-five cervical AGC patients diagnosed by thin-layer liquid-based cytology in the Maternity Affiliated Hospital of Dalian Medical University from January 2014 to March 2018 were collected. Histological follow-up data and HPV E6/E7 mRNA detection results were analyzed, and histological differences in HPV E6/E7 mRNA positive and negative patients were compared. Results The histological result of 445 patients with cervical AGC showed that negative was in 306 cases (68.76%), and clinical significant lesion was in 129 cases (28.99%). In 445 patients with cervical AGC, HPV E6/E7 mRNA result was positive in 121 cases (27.19%), among whom the positive rate of HPV 16 and 18/45 type was 54.55% (66/121); HPV E6/E7 mRNA result was negative in 324 cases (72.81% ), including 13 non-cervical lesions. The negative rate of histological results in HPV E6/E7 mRNA negative patients was significantly higher than that in HPV E6/E7 mRNA positive patients: 91.05% (295/324) vs. 9.09% (11/121), and there was statistical difference (P＜0.01); the rates of adenocarcinoma in situ (AIS), high-grade squamous intraepithelial lesion (HSIL) and cervical adenocarcinoma of histological result in HPV E6/E7 mRNA positive patients were significantly higher than those in HPV E6/E7 mRNA negative patients: 40.50% (49/121) vs. 1.23% (4/324), 44.63% (54/121) vs. 1.23% (4/324), 3.31% (4/121) vs. 0.31% (1/324), and there were statistical differences (P＜0.01). The sensitivity of HPV E6/E7 mRNA in detecting clinical significant lesion of cervical AGC patients was 82.95% (107/129), the specificity was 95.57% (302/316), positive predictive value was 88.43% (107/121), and negative predictive value was 93.21% (302/324). Conclusions The histological result of cervical AGC shows that the amount of negative patients is significantly higher than clinical significant lesion. For cervical AGC patients with HPV E6/E7 mRNA negative results, conservative follow-up can be adopted after excluding extracervical lesions and fully assessing the risk of cervical lesions. However, the cervical AGC patients with HPV E6/E7 mRNA positive results need further examination to detect lesion and choose treatment earlier.

Chemical constituents from the aerial parts of saururus chinensis() / 中国药学杂志

OBJECTIVE: To study the chemical constituents from the aerial parts of Saururus chinensis. METHODS: The compounds were extracted by supercritical CO2 extraction and isolated by various chromatographic methods, such as silica gel, MCI and pre-HPLC. The structures were elucidated by physico-chemical constants and spectroscopic methods. RESULTS: Fourteen compounds were isolated and identified as N-p-trans-coumaroyltyramine (1), linarin (2), blumenol A (3), mannitol (4), 5,7-dyhydroxyl-3,4′-bimethoxyflavonoids (5), 6,7-dimethoxy-4-hydroxy-1-naphthalene formic acid (6)3-hydroxy-4-methoxy benzoic acid (7), (2R,3R)-2,3-dihydro-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-3-methylbenzofuran-5-aldehyde (8), (6S,7E)-6-hydroxy-4,7-megastigmadien-3,9-dione (9), veratric acid (10), p-hydroxybenzaldehyde (11), syringaldehyde (12), 3,4,5-trimethoxybenzoic acid (13) and physcion (14). CONCLUSION: Compounds 1-13 are isolated from this plant for the first time.

Human papillomavirus and genome instability: from productive infection to cancer

Infection with high oncogenic risk human papillomavirus types is the etiological factor of cervical cancer and a major cause of other epithelial malignancies, including vulvar, vaginal, anal, penile and head and neck carcinomas. These agents affect epithelial homeostasis through the expression of specific proteins that deregulate important cellular signaling pathways to achieve efficient viral replication. Among the major targets of viral proteins are components of the DNA damage detection and repair machinery. The activation of many of these cellular factors is critical to process viral genome replication intermediates and, consequently, to sustain faithful viral progeny production. In addition to the important role of cellular DNA repair machinery in the infective human papillomavirus cycle, alterations in the expression and activity of many of its components are observed in human papillomavirus-related tumors. Several studies from different laboratories have reported the impact of the expression of human papillomavirus oncogenes, mainly E6 and E7, on proteins in almost all the main cellular DNA repair mechanisms. This has direct consequences on cellular transformation since it causes the accumulation of point mutations, insertions and deletions of short nucleotide stretches, as well as numerical and structural chromosomal alterations characteristic of tumor cells. On the other hand, it is clear that human papillomavirus-transformed cells depend on the preservation of a basal cellular DNA repair activity level to maintain tumor cell viability. In this review, we summarize the data concerning the effect of human papillomavirus infection on DNA repair mechanisms. In addition, we discuss the potential of exploiting human papillomavirus-transformed cell dependency on DNA repair pathways as effective antitumoral therapies.