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Little information is available for antibody levels against SARS-CoV-2 variants of concern induced by Omicron breakthrough infection and a third booster with an inactivated vaccine (InV) or Ad5-nCoV in people with completion of two InV doses. Plasma was collected from InV pre-vaccinated Omicron-infected patients (OIPs), unvaccinated OIPs between 0 and 22 days, and healthy donors (HDs) 14 days or 6 months after the second doses of an InV and 14 days after a homogenous booster or heterologous booster of Ad5-nCoV. Anti-Wuhan-, Anti-Delta-, and Anti-Omicron-receptor binding domain (RBD)-IgG titers were detected using enzyme-linked immunosorbent assay. InV pre-vaccinated OIPs had higher anti-Wuhan-, anti-Delta-, and anti-Omicron-RBD-IgG titers compared to unvaccinated OIPs. Anti-Wuhan-RBD-IgG titers sharply increased in InV pre-vaccinated OIPs 0-5 days postinfection (DPI), while the geometric mean titers (GMTs) of anti-Delta- and anti-Omicron-RBD-IgG were 3.3-fold and 12.0-fold lower. Then, the GMT of anti-Delta- and anti-Omicron-RBD-IgG increased to 35 112 and 28 186 during 11-22 DPI, about 2.6-fold and 3.2-fold lower, respectively, than the anti-Wuhan-RBD-IgG titer. The anti-Wuhan-, anti-Delta-, and anti-Omicron-RBD-IgG titers declined over time in HDs after two doses of an InV, with 25.2-fold, 5.6-fold, and 4.5-fold declination, respectively, at 6 months relative to the titers at 14 days after the second vaccination. Anti-Wuhan-, anti-Delta-, and anti-Omicron-RBD-IgG titers elicited by a heterologous Ad5-nCoV booster were significantly higher than those elicited by an InV booster, comparable to those in InV pre-vaccinated OIPs. InV and Ad5-nCoV boosters could improve humoral immunity against Omicron variants. Of these, the Ad5-nCoV booster is a better alternative.

Homologous booster, heterologous booster, and Omicron variants breakthrough infection (OBI) could improve the humoral immunity against Omicron variants. Questions concerning about memory B cells (MBCs) and T cells immunity against Omicron variants, features of long-term immunity, after booster and OBI, needs to be explored. Here, comparative analysis demonstrate antibody and T cell immunity against ancestral strain, Delta and Omicron variants in Omicron breakthrough infected patients (OBIPs) are comparable to that in Ad5-nCoV boosted healthy volunteers (HVs), higher than that in inactivated vaccine (InV) boosted HVs. However, memory B cells (MBCs) immunity against Omicron variants was highest in OBIPs, followed by Ad5-nCoV boosted and InV boosted HVs. OBIPs and Ad5-nCoV boosted HVs have higher classical MBCs and activated MBCs, and lower naïve MBCs and atypical MBCs relative to both vaccine boosted HVs. Collectively, these data indicate Omicron breakthrough infection elicit higher MBCs and T cells against SARS-CoV-2 especially Omicron variants relative to homologous InV booster and heterologous Ad5-nCoV booster.

Background: The relationship between air pollutants and pulmonary tuberculosis (PTB) in HIV-infected patients, an immunocompromised population, is unclear.Methods: The data of PTB cases among HIV-infected patients admitted to Guangzhou Eighth People’s Hospital, the local largest HIV/AIDS treatment clinic, as well as the meteorological factors and air pollutants between 2014 and 2019, were collected. The associations between air pollutants and PTB were evaluated in generalized linear model (GLM) and distributed lag non-linear model (DLNM). The robustness of results was tested by sensitivity analysis.Results: A total of 494 subjects were enrolled. PTB cases were decreased over time and significantly correlated with monthly mean concentrations of sulfur dioxide (SO2, r = 0.5, P < 0.001) but not other pollutants. In multi-pollutant GLM, SO2 was a risk factor of monthly PTB cases (relative risk [RR] =1.160, 95% confidence interval [CI]: 1.105-1.218) and fine particulate (PM2.5­) was a protective factor (RR = 0.971, 95% CI: 0.950-0.993). In single-pollutant DLNM, a 10-unit increase in SO2 concentration was significantly related to PTB cases within lag 0-6 months with the peak RR at lag 0 month (RR = 1.201, 95% CI: 1.028-1.403) and linearly decreased over lag months, and the cumulative effect reached the peak at lag 13 month (RR = 3.672, 95% CI = 1.052-12.81). In multi-pollutant DLNM, SO2 remained a risk factor of PTB within lag 0-3 months. PM2.5 was negatively associated with PTB at lag 18 month (RR = 0.900, 95% CI: 0.810-0.999 ) in single-pollutant DLNM and not significant in multi-pollutant DLNM.Conclusions: SO2 is identified as a major risk factor of PTB in HIV-infected patients.

Hepatitis C virus (HCV) genotype 3 is widely distributed, and genotype 3-infected patients achieve a lower cure rate in direct-acting antiviral (DAA) therapy and are associated with a higher risk of hepatic steatosis than patients with other genotypes. Thus, the study of the virology and pathogenesis of genotype 3 HCV is increasingly relevant. Here, we developed a full-length infectious clone and a subgenomic replicon for the genotype 3a isolate, CH3a. From an infected serum, we constructed a full-length CH3a clone, however, it was nonviable in Huh7.5.1 cells. Next, we systematically adapted several intergenotypic recombinants containing Core-NS2 and 5′UTR-NS5A from CH3a, and other sequences from a replication-competent genotype 2 a clone JFH1. Adaptive mutations were identified, of which several combinations facilitated the replication of CH3a-JFH1 recombinants; however, they failed to adapt to the full-length CH3a and the recombinants containing CH3a NS5B. Thus, we attempted to separately adapt CH3a NS5B-3′UTR by constructing an intragenotypic recombinant using 5′UTR-NS5A from an infectious genotype 3a clone, DBN3acc, from which L3004P/M in NS5B and a deletion of 11 nucleotides (Δ11nt) downstream of the polyU/UC tract of the 3′UTR were identified and demonstrated to efficiently improve virus production. Finally, we combined functional 5′UTR-NS5A and NS5B-3′UTR sequences that carried the selected mutations to generate full-length CH3a with 26 or 27 substitutions (CH3acc), and both revealed efficient replication and virus spread in transfected and infected cells, releasing HCV of 104.2 f.f.u. ml−1. CH3acc was inhibited by DAAs targeting NS3/4A, NS5A and NS5B in a dose-dependent manner. The selected mutations permitted the development of subgenomic replicon CH3a-SGRep, by which L3004P, L3004M and Δ11nt were proven, together with a single-cycle virus production assay, to facilitate virus assembly, release, and RNA replication. CH3acc clones and CH3a-SGRep replicon provide new tools for the study of HCV genotype 3.

Background: Hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis C, end-stage liver diseases, and hepatocellular carcinoma. The development of infectious HCV cell culture systems primarily relied on the replication enhancement effect of adaptive mutations. Although the mode of action may vary, those adaptive mutations could direct the study of virus-host interactions required for efficient virus infection. We previously identified a substitution D559G in NS5B (RNA dependent RNA polymerase) critical for the replication of HCV genomes. In this study, we set out to study whether D559G-NS5B specifically interacted with some host factors crucial for HCV infection.Methods: Through mass spectrometry analysis of immunoprecipitation mixture of ectopically expressed wild-type and D559G-mutated NS5B, we identified candidate factors showing potential interactions with NS5B and D559G-NS5B. The requirement of selected host factor in HCV infection in vitro was demonstrated by gene knockout, overexpression, virus infection, and co-immunoprecipitation approaches.Results: From the results of immunoprecipitation and mass spectrometry analysis, we selected protein phosphatase 2 regulatory subunit B’delta (PPP2R5D) for further characterization. Co-immunoprecipitation confirmed that both wild-type and D559G NS5B proteins interacted with PPP2R5D, but the interaction between D559G-NS5B and PPP2R5D was more efficient. Silencing of PPP2R5D decreased HCV infection, and knockout of PPP2R5D nearly eliminated HCV infection in Huh7.5 cells. Transient and stably overexpression of PPP2R5D in PPP2R5D-knockout cells restored HCV infection to a level close to that seen for wild-type Huh7.5 cells. Conclusions: PPP2R5D is required for HCV infection in cultured hepatoma cells, and PPP2R5D may function through binding to HCV NS5B. The underlying mechanism of PPP2R5D in the complete HCV life cycle requires further investigation.

Background Hepatitis C virus (HCV) infection increased the risk of hepatocellular carcinoma. Identification of host factors required for HCV infection will help to unveil the HCV pathogenesis. Adaptive mutations that enable the replication of HCV infectious clones could provide hints that the mutation-carrying viral protein may specifically interact with some cellular factors essential for the HCV life cycle. Previously, we identified D559G mutation in HCV NS5B (RNA dependent RNA polymerase) important for replication of different genotype clones. Here, we searched for the factors that potentially interacted with NS5B and investigated its roles in HCV infection. Methods Wild-type-NS5B and D559G-NS5B of HCV genotype 2a clone, J6cc, were ectopically expressed in hepatoma Huh7.5 cells, and NS5B-binding proteins were pulled down and identified by mass spectrometry. The necessity and mode of action of the selected cellular protein for HCV infection were explored by experiments including gene knockout or knockdown, complementation, co-immunoprecipitation (Co-IP), colocalization, virus infection and replication, and enzymatic activity, etc. Results Mass spectrometry identified a number of cellular proteins, of which protein phosphatase 2 regulatory subunit B’delta (PPP2R5D, the PP2A regulatory B subunit) was one of D559G-NS5B-pulled down proteins and selected for further investigation. Co-IP confirmed that PPP2R5D specifically interacted with HCV NS5B but not HCV Core and NS3 proteins, and D559G slightly enhanced the interaction. NS5B also colocalized with PPP2R5D in the endoplasmic reticulum. Knockdown and knockout of PPP2R5D decreased and abrogated HCV infection in Huh7.5 cells, respectively, while transient and stable expression of PPP2R5D in PPP2R5D-knockout cells restored HCV infection to a level close to that in wild-type Huh7.5 cells. Replicon assay revealed that PPP2R5D promoted HCV replication, but the phosphatase activity and catalytic subunit of PP2A were not affected by NS5B. Conclusions PPP2R5D interactes with HCV NS5B and is required for HCV infection in cultured hepatoma cells through facilitating HCV replication.

Currently, no vaccine to prevent hepatitis C virus (HCV) infection is available. A major challenge in developing an HCV vaccine is the high diversity of HCV sequences. The purpose of immunization with viral glycoproteins is to induce a potent and long-lasting cellular and humoral immune response. However, this strategy only achieves limited protection, and antigen selection plays a crucial role in vaccine design. In this study, we investigated the humoral immune responses induced by intraperitoneal injection of keyhole limpet hemocyanin conjugated with 4 highly conserved peptides, including amino acids [aa]317-325 from E1 and aa418-429, aa502-518, and aa685-693 from E2, or 3 peptides from hypervariable region 1 (HVR1) of E2, including the N terminus of HVR1 (N-HVR1, aa384-396), C terminus of HVR1 (C-HVR1, aa397-410), and HVR1 in BALB/c mice. The neutralizing activity against HCV genotypes 1-6 was assessed using the cell culture HCV (HCVcc) system. The results showed that the 4 conserved peptides efficiently induced antibodies with potent neutralizing activity against 3 or 4 genotypes. Antibodies induced by aa685-693 conferred potent protection (>50%) against genotypes 2, 4, and 5. Peptide N-HVR1 elicited antibodies with the most potent neutralization activities against 3 HCV genotypes: TNcc(1a), S52(3a), and ED43(4a). These findings suggested that peptides within HCV glycoproteins could serve as potent immunogens for vaccine design and development.

Zika virus (ZIKV) is an Aedes mosquitoes-transmitted flavivirus, and its infection may cause severe neurological diseases. A genetically stable infectious clone is essential for ZIKV research, however the toxicity and instability of the viral cDNA in bacteria potentially due to its bacterial promoter activity are major challenges. Here, we constructed a full-length cDNA clone for isolate ZG01 by introducing non-coding changes T1865C/A1868G to reduce the bacterial promoter activity. Wild-type and recombinant ZG01 were highly attenuated in Vero cells, thus we serially passaged wild-type ZG01 through neonatal mice and Vero cells to generate high-titer virus, from which four mutations (4m, C2178T/G2913A/T4991C/T10561C) were identified. Addition of 4m greatly enhanced the infectivity, as ZG01_4m released ZIKV of 107.0-107.5 plaque-forming unit (PFU)/ml in infected Vero and A549 cells. ZG01_4m resembled the infectivity of high-titer ZG01 in vitro and in vivo. Notably, ZG01_4m plasmid was genetically stable after multiple rounds of transformation-purification in bacteria. Using ZG01_4m, we identified a potential RNA-RNA interaction between 5'UTR and 3'UTR and demonstrated that the nucleotides involved were essential for ZIKV production. The genetically stable ZG01 cDNA clone provides a reliable tool for the study of this important virus, and the strategy used here is feasible for the development of reverse genetics systems for other ZIKV isolates and related flaviviruses.

BackgroundClopidogrel is an inactive prodrug, it catalyzed into its active form by Cytochrome 450 and Paraoxonase-1(PON-1). polymorphisms of genes encoding these enzymes will affect the efficacy of Clopidogrel. The main objective of our study was to investigate the association of CYP2C19*2, CYP2C19*3 and PON-1Q192R polymorphisms with Clopidogrel resistance and major adverse cardiac events in Jin Hua district in the middle of Zhe Jiang Province in China.MethodsOne hundred sixty coronary heart disease patients with percutaneous coronary intervention, who were followed-up for 1 year, were enrolled in our study. These patients were co-administered aspirin 100 mg/d and clopidogrel 75 mg/d following a loading dose of 300 mg. The ADP-induced platelet aggregation rate was measured by Platelet aggregator. Genotypes of CYP2C19*2, CYP2C19*3, PON-1Q192R were determined using Sanger sequencing in all patients. Various clinical data were collected.ResultsThe frequencies of CYP2C19*2, CYP2C19*3 and PON-1Q192R homozygous mutant genotypes were significantly lower in non-responders than those in responders. After for all variables, CYP2C19*2, CYP2C19*3 and PON-1Q192R independently increased the risk of clopidogrel resistance with adjusted ORs 46.65(95% CI,1.77–25.04;p = 0.005); 22.74(95% CI, 3.11–166.27;p = 0.002); 5.69 (95% CI,1.06–30.47;p = 0.042). Over a follow-up of 12 months, the incidence of major adverse cardiac events (MACE) in CYP2C19*1/*2, *1/*3, *2/*2, *2/*3 was significantly higher than no mutant genotype (18/40vs.2/63,3/9vs.2/63, 11/6vs.2/63, 7/1vs2/63, respectively). There was no significant correlation between PON-1Q192R mutant allele and MACE.ConclusionOur study was first time to report on CYP2C19 and PON-1 polymorphisms in Jin Hua population in the middle of Zhe Jiang province in China. The carriage of CYP2C19*2 or *3 mutant allele significantly reduced the platelet response to clopidogrel and increase the MACE. The carriage of PON-1 mutant allele also significantly reduced the platelet response to clopidogrel, but would not increase the major adverse cardiac events after 1 year follow-up.Trial registrationChiCTR, ChiCTR1800018316. Registered 11 September 2018 – prospective registered,http://www.chictr.org.cn/edit.aspx?pid=30927&htm=4.

Background: MiR-15a-3p has been reported as a tumor suppressor in several kinds of cancer, including cervical cancer and gastric cancer. However, the precise molecular mechanisms underlying its role in prostate cancer (PCa) remain largely unknown. Methods: The expression of miR-15a-3p was determined in PCa tissues and cell lines using quantitative real time PCR. The biological function of miR-15a-3p in PCa cells was investigated using a MTT assay, Edu staining and transwell assay. Moreover, luciferase reporter assay, quantitative real time PCR and western blotting were used to identify and verify the direct downstream target of miR-15a-3p. Results: We found that the expression of miR-15a-3p was down-regulated in both PCa tissues and cell lines. The in vitro results showed that miR-15a-3p overexpression suppressed cell proliferation, invasion, and epithelial-mesenchymal transition (EMT) via down-regulating Wnt/β-catenin signaling in PCa cells. Moreover, SLC39A7 was a direct downstream target of miR-15a-3p. Furthermore, SLC39A7 overexpression attenuated the effects of miR-15a-3p on cell proliferation, invasion, Wnt/β-catenin pathway and EMT molecules. Conclusions: In summary, our study indicated that miR-15a-3p inhibited the proliferation, invasion, and EMT process of PCa cells via targeting SLC39A7 and suppressing Wnt/β-catenin signaling pathway, which may represent a new therapeutic objective for PCa treatment.

Cancer-associated genes serve a crucial role in carcinogenesis. The present study aimed to investigate the mRNA expression levels of microspherule protein 1 (MCRS1) and MCRS2 in colorectal cancer (CRC) and their association with clinical variables. The mRNA expression levels of MCRS1 and MCRS2 were assessed by semi-quantitative reverse transcription polymerase chain reaction in the tumor and corresponding non-tumor tissues of 54 newly-diagnosed CRC patients, as well as in the normal colonic mucosa tissue of 19 age/gender-matched healthy controls. Immunofluorescence was also employed to identify the expression of MCRS1 in CRC tissues, while the concentration of serum carcinoembryonic antigen (CEA) was determined by an enzyme-linked immunoassay. The results identified a negative correlation between MCRS1 and MCRS2 expression levels (r=−0.3018, P=0.0266). MCRS1 mRNA expression was significantly increased and MCRS2 mRNA expression was decreased in CRC tissues compared with the levels in the corresponding normal tissues (both P

Extra‑hepatic manifestations are frequently observed in hepatitis C virus (HCV)‑infected patients; however the underlying mechanisms remain largely unknown. In the present study, the human glioblastoma SF268 cell line (the precise origin of the cell type is not clear) was infected with HCV using HCV‑positive serum, and viral replication was assessed by immunofluorescence, reverse transcription‑polymerase chain reaction (PCR), quantitative PCR and western blotting following infection. HCV core protein and HCV RNA were detected in HCV‑positive serum‑infected SF268 cells at day 4 post‑infection, while no infection was observed in cells exposed to HCV‑negative serum. The mean HCV RNA levels at day 4 post‑infection were up to 5.00 IU/ml log10; however, HCV RNA and immunostaining for core protein were negative when cultured to day 6 or longer. The data suggest that human glioblastoma SF268 cells were transiently infected with HCV. AKT serine/threonine kinase phosphorylation was also detected in HCV‑infected SF268 cells at day 4 post‑infection. To the best of our knowledge, this is the first demonstration that a human glioblastoma cell line can be infected with serum‑derived HCV. The results provide evidence that HCV infection can occur in cells of the central nervous system. Neurological disorder‑associated phosphoinositide 3‑kinase‑AKT signaling pathway was activated in parallel with HCV infection, suggesting that SF268 may serve as an in vitro model for investigating HCV‑nervous system cell interactions.

Genotype 1b strain Con1 represents an important reference in the study of hepatitis C virus (HCV). Here, we aimed to develop an advanced infectious Con1 recombinant. We found that previously identified mutations A1226G/F1464L/A1672S/Q1773H permitted culture adaption of Con1 Core-NS5A (C-5A) recombinant containing 5'UTR and NS5B-3'UTR from JFH1 (genotype 2a), thus acquired additional mutations L725H/F886L/D2415G. C-5A containing all seven mutations (C-5A_7m) replicated efficiently in Huh7.5 and Huh7.5.1 cells and had an increased infectivity in SEC14L2-expressing Huh7.5.1 cells. Incorporation of Con1 NS5B was deleterious to C-5A_7m, however Con1 5'UTR was permissive but attenuated the virus. Nucleotides G1, A4, and G35 primarily accounted for the viral attenuation without affecting RNA translation. C-5A_7m was inhibited dose-dependently by simeprevir and daclatasvir, and substitutions at A4, A29, A34, and G35 conferred resistance to miR-122 antagonism. The novel Con1 5'UTR-NS5A recombinant, adaptive mutations, and critical nucleotides described here will facilitate future studies of HCV culture systems and virus-host interaction.

T helper 17 (Th17) and Th22 cells regulate the development of tumors. However, their roles in the development of colorectal cancer (CRC) are still unclear. A total of 49 patients with CRC and 18 healthy controls (HC) were evaluated for the percentages of circulating Th17 and Th22 cells by flow cytometry. The concentrations of serum interleukin-17A (IL-17A), IL-22 and carcinoembryonic antigen (CEA) were examined. The levels of IL-17A and IL-22 in tumors were determined by real-time PCR. We found that the percentages of Th17 and Th22 cells in the CRC patients were significantly lower than that in the HC and were associated negatively with the pathological stages of CRC. The levels of IL-17A and IL-22 mRNA transcripts were lower in the tumor tissues, particularly in the advanced CRC. After the tumor resection, the percentages of circulating Th17 and Th22 cells increased. These data suggest that decreased Th17 and Th22 responses may be associated with the development of CRC.