Effects of Lianhuaqingwen Capsules in adults with mild-to-moderate coronavirus disease 2019: an international, multicenter, double-blind, randomized controlled trial.

BACKGROUND: In a randomized trial, Lianhuaqingwen (LHQW) capsule was effective for accelerating symptom recovery among patients with coronavirus disease 2019 (COVID-19). However, the lack of blinding and limited sample sizes decreased the level of clinical evidence. OBJECTIVES: To evaluate the efficacy and safety of LHQW capsule in adults with mild-to-moderate COVID-19. METHODS: We conducted a double-blind randomized controlled trial in adults with mild-to-moderate COVID-19 (17 sites from China, Thailand, Philippine and Vietnam). Patients received standard-of-care alone or plus LHQW capsules (4 capsules, thrice daily) for 14 days. The primary endpoint was the median time to sustained clinical improvement or resolution of nine major symptoms. RESULTS: The full-analysis set consisted of 410 patients in LHQW capsules and 405 in placebo group. LHQW significantly shortened the primary endpoint in the full-analysis set (4.0 vs. 6.7 days, hazards ratio: 1.63, 95% confidence interval: 1.39-1.90). LHQW capsules shortened the median time to sustained clinical improvement or resolution of stuffy or runny nose (2.8 vs. 3.7 days), sore throat (2.0 vs. 2.6 days), cough (3.2 vs. 4.9 days), feeling hot or feverish (1.0 vs. 1.3 days), low energy or tiredness (1.3 vs. 1.9 days), and myalgia (1.5 vs. 2.0 days). The duration to sustained clinical improvement or resolution of shortness of breath, headache, and chills or shivering did not differ significantly between the two groups. Safety was comparable between the two groups. No serious adverse events were reported. INTERPRETATION: LHQW capsules promote recovery of mild-to-moderate COVID-19 via accelerating symptom resolution and were well tolerated. Trial registration ChiCTR2200056727 .

Kinetics of antigen-specific IgM/IgG/IgA antibody responses during Zika virus natural infection in two patients.

Understanding of kinetics of antibody responses is crucial for developing rapid serological tests and studying the mechanisms of Zika virus (ZIKV) infection. Most of the serological diagnostic assays previously published are based on either IgM or IgG titer, little is known on the level of IgA antibody in saliva and urine. In this study, we investigated the kinetics of IgM/IgG/IgA antibody responses in serum, saliva, and urine obtained from two ZIKV infected individuals from as early as the second day of onset of symptoms to as long as 2 years postinfection. Other than detecting robust early IgM response, long lasting IgG response, we discovered strong early IgA response specific for ZIKV in saliva in both patients. This unique observation provides a novel strategy and scientific basis for the development of noninvasive rapid tests for ZIKV infection.

The clinical significance of myeloid-derived suppressor cells in dengue fever patients.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) play immunosuppressive roles in cancers and some infectious diseases; however, their role in dengue fever (DF) remains unknown. This study evaluated the clinical significance of MDSCs in DF patients. METHODS: This study comprised 178 non-severe DF patients, 20 non-dengue fever (NDF) controls, and 30 healthy donors. The DF patients were divided into the following five groups based on the fever duration from its onset to the day of sample collection: fever duration of 1-2, 3-4, 5-6, 7-8, and > 9 days. Among these DF patients, 14 were monitored for eight days, and their peripheral blood samples were collected every two days. The mononuclear cells were isolated and analyzed using flow cytometry. The correlation between the MDSCs and clinical and immunological indicators of the DF patients was evaluated using Spearman analysis. RESULTS: The count of the peripheral blood MDSCs, especially monocytic MDSCs, of the 178 DF patients were dramatically higher than those of the NDF and healthy controls, and remarkably decreased with the fever duration. Moreover, the MDSC count correlated with some indicators, including the dengue viral load (rho = 0.367, p < .001), body temperature (rho = 0.263, p = .005), prothrombin time (rho = 0.475, p < .001), CD4+ T cell number (rho = - 0.317, p < .001), CD8+ T cell number (rho = - 0.361, p < .001), "programmed cell death protein 1" (PD-1) (rho = - 0.347, p < .001), "T cell immunoglobulin domain and mucin domain-3" (Tim3) (rho = - 0.258, p = .001), interferon-α (IFN-α) (rho = 0.43, p < .001), and "regulated upon activation normal T-cell expressed and secreted" (RANTES) (rho = 0.278, p = .019). Furthermore, the level of arginase-1, but not nitric oxide, was higher in the DF patients than in the healthy controls and was closely related to the number of MDSCs (rho = 0.265, p = .024). CONCLUSIONS: Our study reveals a significant correlation between MDSCs and DF clinical indicators, posing MDSCs as potential target cells for DF treatment.

The Protective Effects of the A/ZJU01/ PR8/2013 Split H7N9 Avian Influenza Vaccine Against Highly Pathogenic H7N9 in BALB/c Mice.

BACKGROUND/AIMS: Since the first case of novel H7N9 infection was reported, China has experienced five epidemics of H7N9. During the fifth wave, a highly pathogenic H7N9 strain emerged. In order to assess whether the H7N9 vaccine based on A/Zhejiang/DTID-ZJU01/2013(H7N9) was effective in protecting against highly pathogenic H7N9, we conducted this study. METHODS: Groups of mice were immunized twice by intraperitoneal injection with 500 µl of either split vaccine alone or MF59-adjuvanted vaccine. Serum was collected 2 weeks after the second vaccine booster. The hemagglutinin inhibition test was conducted on vaccine seed and highly pathogenic H7N9 to evaluate the neutralization of highly pathogenic H7N9. We also immunized mice and challenged them with highly pathogenic H7N9. Mice were observed for illness, weight loss, and death at 1 week and 2 weeks post-infection. Then, the mice were sacrificed and lungs were removed. Antibody responses were assessed and pathological changes in the lung tissue were evaluated. RESULTS: The ability of serum to neutralize highly pathogenic H7N9 was reduced. In mice, highly pathogenic H7N9 was more virulent than A/Zhejiang/DTID-ZJU01/2013(H7N9). After challenge with highly pathogenic H7N9, all mice died while mice challenged with A/Zhejiang/DTID-ZJU01/2013(H7N9) all recovered. The A/ZJU01/PR8/2013 split H7N9 avian influenza vaccine was able to protect against infection with highly pathogenic H7N9 in mice, with or without MF59. Moreover, H7N9 vaccine adjuvanted with MF59 produced high antibody levels, which lead to better protection. CONCLUSIONS: The A/ZJU01/PR8/2013 split H7N9 avian influenza vaccine based on A/Zhejiang/DTID-ZJU01/2013(H7N9) is effective in protecting against highly pathogenic H7N9. H7N9 vaccine adjuvanted with MF59 offers better protection against infection with highly pathogenic H7N9. In order to make the H7N9 vaccine applicable to humans, further clinical trials are required to evaluate MF59 adjuvanted vaccine. Meanwhile, the vaccine strain should be updated based on the highly pathogenic H7N9 gene sequence.

Complete genome sequence of dengue virus serotype 2 Cosmopolitan genotype strain in Guangdong, China.

Here we report the complete genome sequence of a dengue virus serotype 2 (DENV-2) strain, GZ40, isolated in Guangdong, China, in 2010. A phylogenetic analysis classified GZ40 into the Cosmopolitan genotype, while previous Chinese DENV-2 isolates belong to the Asian I genotype. The reemergence of the Cosmopolitan genotype of DENV-2 in China deserves further investigation.

Complete genome sequence of a dengue virus serotype 4 strain isolated in Guangdong, China.

Here we report the first complete genome sequence of a dengue virus serotype 4 genotype II strain, GZ30, isolated in Guangzhou, Guangdong Province, China, in 2010. The sequence information provided herein will help us to understand the molecular epidemiology of dengue virus and predict the risk of severe diseases in mainland China.

[Differential response to pegylated interferon plus ribavirin combination therapy in chronic hepatitis C and HIV/HCV co-infected patients].

OBJECTIVE: To investigate the potential differences in response to pegylated interferon (Peg-IFN) plus ribavirin (RBV) combination therapy in patients with hepatitis C virus (HCV) mono-infection and human immunodeficiency virus (HIV)/HCV co-infection. METHODS: Seventy HIV/HCV patients and sixty HCV patients, were administered a 48-week course of Peg-IFN + RBV. The HCV load was tested by the COBAS automatic viral load analysis system (lower limit of quantification = 15 IU/ml) at treatment weeks 0 (baseline), 4, 12, 24, and 48 and at week 24 after drug withdrawal. The patients were also genotyped by sequencing for the host-encoded interleukin (IL)-28B single nucleotide polymorphisms (SNPs) related to HCV Peg-IFN + RBV therapy outcome: rs8099917, rs12979860 and rs12980275. In addition, the HCV-encoded NS5B gene region was genotyped by nested-PCR and sequencing followed by BLAST searching of the Los Alamos National Laboratory HCV database. The significance of between-group differences in response to therapy and roles of SNPs were evaluated by statistical analyses. RESULTS: The ratio of sustained virological response (SVR) was significantly lower in the HIV/HCV co-infected patients than the HCV mono-infected patients (32.9% vs. 71.7%; P less than 0.001). While the HIV/HCV co-infected patients did not show a significant difference in SVR ratio achieved between individuals infected with the HCV-1 genotype and the non-HCV-1 genotype (30.8% vs. 33.3%; P = 1.000), the HCV mono-infected patients did (86.1% vs. non 50.0%, P = 0.002). Moreover, the SVR ratio was higher in the HCV-1 genotype HCV mono-infected patients than in the HIV/HCV-1 genotype co-infected patents (30.8% vs. 86.1%; P less than 0.001). The different IL-28B genotypes were not significantly correlated to the PEG-IFN+RBV therapy response of either HCV mono-infected patients or HIV/HCV co-infected patients (P more than 0.05). CONCLUSION: HCV mono-infected patients respond better to Peg-IFN + RBV therapy than HIV/HCV co-infected patients. The HCV-1 genotype may promote this therapy response in HCV mono-infected patients, but the IL-28B genotypes appear to play no significant role.

[Study on the copy numbers and mRNA expression levels of the programmed death-1 gene in chronic hepatitis B patients].

OBJECTIVE: To study the copy numbers and mRNA expression levels of the Programmed Death-1 gene in chronic hepatitis B patients and to analyze the differences of the copy numbers and mRNA expression levels of the gene in patients with different clinical outcomes. METHODS: Real time PCR was adopted to detect the PD-1 gene copy numbers and their mRNA expressions in peripheral blood mononuclear cells (PBMCs) from 27 samples from healthy donors in Control group, 31 samples from chronic asymptomatic HBV carriers (ASC, n=31), 19 samples from chronic severe hepatitis B patients (CSH, n=19) and 29 samples from Primary hepatitis B Virus-related hepatocarcinoma (PHC, n=29). The differences and relationship of copy numbers and their mRNA expression levels among those groups were compared and analyzed by adopting Chi-square test and Rank sum test. RESULTS: PD-1 gene copy number deviated from 0 copy to 3 copies among all the 106 samples. In control group, ASC group, CSH group and PHC group, the percentages of cases of haploid (single) were 37.0%, 35.5%, 26.3% and 6.9%, respectively, the percentages of cases of diploid (double) were 55.5%, 58.0%, 63.2% and 82.8%, respectively, and the percentages of cases of triploid (triple) were 3.7%, 6.5%, 10.5% and 10.3%, respectively. The percentage of cases of polyploid (diploid and triploid) in control group, ASC group, CSH group and PHC group were 59.3%, 64.5%, 73.7% and 93.1%, respectively. The different distribution of PD-1 gene copy number of polyploid was significant in total samples (x2=9.583, P<0.05). Compared with Control Group and ASC group, the percentage of cases of polyploid in PHC group was lower with the x2 equals to 8.985 and 7.215 respectively and both with P less than 0.05. The difference between the two groups was statistically significant. The mean PD-1 gene copy numbers for these four groups were 1.59+/-0.63, 1.70+/-0.52, 1.84+/-0.60 and 2.00+/-0.37 while the median were 0.002 54, 0.002 72, 0.002 55 and 0.001 33 respectively. Except the control group, there was a uptrend in the other three groups while PD-1 gene mRNA expression presented a downtrend. The mean of PD-1 gene copy numbers of 2 and their mRNA expression levels were 19.59, 32.57 and 33.22 for PHC, CSH and ASC groups among which PHC group had the lowest value, there was significant differences found in the comparison with F=5.395 and P<0.05. CONCLUSION: PD-1 gene copy numbers and their mRNA expression levels were different in chronic HBV infected patients with different transformation. It is valuable to follow up the patients with more than 1 copy number of PD-1 gene in long term.

[Analysis of hepatitis C virus (HCV) subtypes in HIV/HCV co-infected and HCV mono-infected individuals in Guangdong province].

OBJECTIVE: To explore the transmission routes, genotypes/subtypes distribution and genetic character of HCV in HIV/HCV co-infected and HCV mono-infected individuals in Guangdong Province. METHODS: Reverse transcription (RT) nested PCR was performed to amplify the HCV NS5B gene region from 95 HIV/HCV co-infected and 99 HCV mono-infected individuals lived in Guangdong province. The PCR products were then sequenced for HCV subtyping. Genetic analysis was done by MEGA4 software. RESULTS: (1) HIV/HCV co-infected individuals infected HCV mostly through injection drug use (IDU, 78.9%), the HCV subtypes were identified as 6a (53.7%), 3a (17.9%), 1b (15.8%), 3b (11.6%) and 1a (1.0%) respectively, the genetic distance within subtype 1b was longer than those within other subtypes, the predominant HCV subtype in HIV/HCV co-infected individuals infected through IDU was 6a (60.0%). (2) HCV mono-infected individuals infected HCV mostly through blood or blood products transfusions (80.8%), the HCV subtypes were identified as 1b (67.7%), 6a (17.2%), 3a (6.1%), 2a (5.0%), 3b (2.0%), 4a (1.0%) and 5a (1.0%) respectively, the genetic distance within subtype 1b was also longer than those within other subtypes, the predominant HCV subtype in HCV mono-infected individuals infected through blood or blood products transfusions was 1b (76.2%). CONCLUSION: The diversity of HCV subtypes in HIV/HCV co-infected and HCV mono-infected individuals in Guangdong Province was high, both the major transmission route and HCV subtype between HIV/HCV co-infected individuals and HCV mono-infected individuals were different.

Kinetic Characteristics of Neutralizing Antibody Responses Vary among Patients with COVID-19.

OBJECTIVE: The coronavirus disease 2019 (COVID-19) pandemic continues to present a major challenge to public health. Vaccine development requires an understanding of the kinetics of neutralizing antibody (NAb) responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: In total, 605 serum samples from 125 COVID-19 patients (from January 1 to March 14, 2020) varying in age, sex, severity of symptoms, and presence of underlying diseases were collected, and antibody titers were measured using a micro-neutralization assay with wild-type SARS-CoV-2. RESULTS: NAbs were detectable approximately 10 days post-onset (dpo) of symptoms and peaked at approximately 20 dpo. The NAb levels were slightly higher in young males and severe cases, while no significant difference was observed for the other classifications. In follow-up cases, the NAb titer had increased or stabilized in 18 cases, whereas it had decreased in 26 cases, and in one case NAbs were undetectable at the end of our observation. Although a decreasing trend in NAb titer was observed in many cases, the NAb level was generally still protective. CONCLUSION: We demonstrated that NAb levels vary among all categories of COVID-19 patients. Long-term studies are needed to determine the longevity and protective efficiency of NAbs induced by SARS-CoV-2.

[Subcellular mimical localization of the ATP synthase B subunit from Clonorchis sinensis under different conditions of cell cycling].

OBJECTIVE: To investigate the subcellular localization of ATP synthase b subunit from Clonorchis sinensis under different conditions of Hela cell cycling, and the effect of this protein on the expression of its encoding-gene and homologous host genes. METHODS: pEGFP-N1-CsATP-synt_B and the vector pEGFP-N1 were transfected into Hela cells, respectively. Transfected cells were synchronized in G0/G1 by serum starvation, G1/S, S cells by double thymidine block, and G2/M cells by thymidine-Nocodozale block. After synchronization, the subcellular localization of the expressed fusion protein was observed with a laser confocal microscope. The expression level of this fusion protein in cells was detected by flow cytometry (FCM). The expression of CsATP-synt_B and HomoATP-synt_B in different cell cycle phases accessed by RT-PCR. RESULTS: FCM results indicated that in the G0/G1 phase the expression of pEGFP-N1 vector was decreased significantly, while pEGFP-N1-CsATP-synt_B expression showed an upward trend. In the other phases of cell cycle, the protein expression was similar in the above two kinds of plasmids. The intact CsATP-synt_B was expressed in mitochondria in the G0/G1, S, and G2/M phases and nucleus during G1/S phase. After the fusion proteins entered the nucleus, the mRNA expression of CsATP-synt_B and HomoATP-synt_B increased significantly. CONCLUSION: CsATP-synt_B can be expressed in the nucleus during G1/S phase, and regulated by the cell cycle and energy requirements.

Pre-S Deletions are Predominant Quasispecies in HIV/HBV Infection: Quasispecies Perspective.

BACKGROUND: Combined HIV infection can accelerate HBV-induced liver disease. It is known that HBV Pre-S deletion is closely related to HBV-associated terminal liver disease in HBV mono-infection. Currently, data on HBV Pre-S quasispecies feature deletion in HIV/HBV co-infected patients are lacking. METHODS: The characteristics and blood samples of patients with chronic HBV infection were collected and classified into an HIV/HBV co-infection group and an HBV mono-infection group according to HIV antibody results before treatment. HBV DNA in serum was extracted. The HBV Pre-S region was amplified by nested-PCR and was further T-A cloned. Using the standard sequence of the matched genotype HBV as a reference, BioEdit 7.0 software was employed for sequence alignment. RESULTS: HBV Pre-S regions were successfully amplified from 147 patients, including 71 cases in the HIV/HBV co-infected group and 76 cases in the HBV mono-infected group. The proportion of the HIV/HBV co-infected group with Pre-S quasispecies deletion was lower than that of the HBV mono-infected group. By analyzing the frequency of Pre-S quasispecies in the two groups, the frequency of Pre-S quasispecies in HIV/HBV co-infected patients with Pre-S quasispecies was higher than HBV mono-infected patients. The frequency of Pre-S quasispecies deletion of the S protein promoter region in the HIV/HBV co-infected group was significantly higher than that in the HBV mono-infected group. CONCLUSION: High-frequency Pre-S quasispecies deletions are predominant in HIV/HBV co-infected patients; however, low-frequency Pre-S deletions are predominant in HBV mono-infected patients, providing a reference for the pathogenesis of the accelerated progression of liver disease in HIV/HBV co-infection.

Assessing the association between platelets and immune recovery in HIV/HBV co-infected patients: A long-term cohort in Asia.

BACKGROUND: Previous studies have reported that platelet count is associated with the progression of liver disease caused by hepatitis B virus (HBV), but there have been no reports on whether platelet count is associated with immune recovery in HIV/HBV co-infected patients. METHODS: A retrospective analysis was conducted on 167 HIV-infected patients whose continuously highly active antiretroviral therapy (HAART) strategy was lamivudine +tenofovir+ efavirenz, of which 75 were HIV/HBV co-infected patients and 92 were HIV mono-infected patients. The biochemical examination results and demographic characteristics of all patients before HAART were collected, and routine blood test results (including platelet count) and immune cell count (including CD4 cells count) after all time points of HAART were obtained. All patients were observed until 72 months. CD4 cells count of 350 or 500 cells/µl 72 months after HAART served as the boundary for judging the immune reconstruction effect. RESULTS: The basic characteristics of HIV/HBV co-infected patients and HIV mono-infected patients were matched. All patients had a good viral response (HIV RNA <20 copies/ml, HBV DNA < 100 copies/mL) and immune response during HAART. The platelets with poor immune recovery in HIV/HBV co-infected patients were also maintained at an apparent lower level than that in patients with good immune recovery. However, this phenomenon was not found in HIV mono-infected patients. The platelet level at many time points after HAART therapy in HIV/HBV co-infected patients can predict the effect of immune recovery at 72 months after HAART. CONCLUSION: The platelet counts of HIV/HBV co-infected patients were correlated with CD4 counts during the follow-up of HAART. These results suggest that the mechanisms associated with thrombocytopenia may be involved in the regulation of immune recovery after treatment in HIV/HBV co-infected patients.

Novel pathogenic characteristics of highly pathogenic avian influenza virus H7N9: viraemia and extrapulmonary infection.

The H7N9 virus mutated in 2017, resulting in new cases of highly pathogenic avian influenza (HPAI) H7N9 virus infection. H7N9 was found in a viraemic patient in Guangdong province, China. The present study aimed to clarify the pathogenic characteristics of HPAI H7N9. Virus was isolated from the plasma and sputum of the patient with HPAI H7N9. Liquid phase chip technology was used to detect the plasma cytokines from the infected patient and healthy controls. Mice were infected with strains A/Guangdong/GZ8H002/2017(H7N9) and A/Zhejiang/DTID-ZJU01/2013(H7N9) to observe the virus's pathogenic characteristics. Serum and brain tissue were collected at 2, 4, and 6 days after infection. The viruses in serum and brain tissue were detected and isolated. The two strains were infected into A549 cells, exosomes were extracted, and virus genes in the exosomes were assessed. Live virus was isolated from the patient's plasma. An acute cytokine storm was detected during the whole course of the disease. In animal experiments, A/Guangdong/GZ8H002/2017(H7N9) was more pathogenic than A/Zhejiang /DTID-ZJU01/2013(H7N9) and resulted in the death of mice. Live virus was isolated from infected mouse serum. Virus infection was also detected in the brain of mice. Under viral stress, A549 cells secreted exosomes containing the entire viral genome. The viraemic patient was confirmed to have an HPAI H7N9 infection. A/Guangdong/GZ8H002/2017(H7N9) showed significantly enhanced toxicity. Patient deaths might result from cytokine storms and brain infections. Extrapulmonary tissue infection might occur via the exosome pathway. The determined pathogenic characteristics of HPAI H7N9 will contribute to its future treatment.

Antiretroviral Long-Term Efficacy and Resistance of Lopinavir/Ritonavir Plus Lamivudine in HIV-1-Infected Treatment-Naïve Patients (ALTERLL): 144-Week Results of a Randomized, Open-Label, Non-Inferiority Study From Guangdong, China.

Dual therapy with lopinavir/ritonavir (LPV/r) plus lamivudine (3TC) has been demonstrated to be non-inferior to the triple drug regimen including LPV/r plus two nucleoside reverse transcriptase inhibitors (NRTIs) in 48-week studies. However, little is known about the long-term efficacy and drug resistance of this simplified strategy. A randomized, controlled, open-label, non-inferiority trial (ALTERLL) was conducted to assess the efficacy, drug resistance, and safety of dual therapy with LPV/r plus 3TC (DT group), compared with the first-line triple-therapy regimen containing tenofovir (TDF), 3TC plus efavirenz (EFV) (TT group) in antiretroviral therapy (ART)-naïve HIV-1-infected adults in Guangdong, China. The primary endpoint was the proportion of patients with plasma HIV-1 RNA < 50 copies/ml at week 144. Between March 1 and December 31, 2015, a total of 196 patients (from 274 patients screened) were included and randomly assigned to either the DT group (n = 99) or the TT group (n = 97). In the primary intention-to-treat (ITT) analysis at week 144, 95 patients (96%) in the DT group and 93 patients (95.9%) in the TT group achieved virological inhibition with plasma HIV-1 RNA <50 copies/ml (difference: 0.1%; 95% CI, -4.6-4.7%), meeting the criteria for non-inferiority. The DT group did not show significant differences in the mean increase in CD4+ cell count (247.0 vs. 204.5 cells/mm3; p = 0.074) or the CD4/CD8 ratio (0.47 vs. 0.49; p = 0.947) from baseline, or the inflammatory biomarker levels through week 144 compared with the TT group. For the subgroup analysis, baseline high viremia (HIV-1 RNA > 100,000 copies/ml) and genotype BC did not affect the primary endpoint or the mean increase in CD4+ cell count or CD4/CD8 ratio from baseline at week 144. However, in patients with genotype AE, the DT group showed a higher mean increase in CD4+ cell count from baseline through 144 weeks than the TT group (308.7 vs. 209.4 cells/mm3; p = 0.038). No secondary HIV resistance was observed in either group. Moreover, no severe adverse event (SAE) or death was observed in any group. Nonetheless, more patients in the TT group (6.1%) discontinued the assigned regimen than those in the DT group (1%) due to adverse events. Dual therapy with LPV/r plus 3TC manifests long-term non-inferior therapeutic efficacy, low drug resistance, good safety, and tolerability compared with the first-line triple-therapy regimen in Guangdong, China, indicating dual therapy is a viable alternative in resource-limited areas. Clinical Trial Registration: [http://www.chictr.org.cn], identifier [ChiCTR1900024611].

[Tissular and subcellular localization of the ATP synthase B subunit in Clonorchis sinensis].

OBJECTIVE: To illustrate the distribution of ATP synthase b subunit in the tissue of Clonorchis sinensis adult and its subcellular mimical localization in HeLa cells. METHODS: With the antiserum against recombinant CsATP-synt_B protein raised from SD rats as primary antibody, paraffin sections of the adult of C. sinensis were processed by the method of fluorescent immunohistochemistry to observe the distribution of CsATP-synt_B protein in adult worm. According to the prediction by bioinformatics of the definite mitochondrial targeting sequence (MTS) and probable Bipartite nuclear localization signals (NLS_BP)in CsATP-syntB sequence, recombinant pEGFP-N1 plasmids containing the intact and three defective CsATP-synt_B sequence with single defect of MTS or NLS_BP or double defect respectively were constructed. The recombinant plasmids and the control plasmid-pEGFP-N1, pEYFP-Mito and H2B-CFP, were transfected into the HeLa cells by Lipofectamine 2000 reagent and the subcellular location of the GFP fusion protein was observed with confocal microscopy. RESULTS: The CsATP-synt_B protein appeared to distribute all over the adult worm, especially abundant on the acetabulum, ovary, vitellarium and tegument. The intact CsATP-synt_B was definitely expressed in mitochondria and/or nucleus of infected HeLa cells, whereas the MTS-deleted mutant only in cytoplasma and nucleus, the NLS_BP-deleted mutant in mitochondria and cytoplasm, and the double defect mutant only in cytoplasm. CONCLUSION: The distribution of CsATP-synt_B in adult is accord with that of mitochondria, and mainly exits in the organs and the tissues of active energy metabolism. This study first predicted and confirmed that CsATP-synt_B can be expressed in the nucleus.

[Cloning and expression of the F0-ATP synthase B chain of Clonorchis sinensis and immunogenicity identification of the recombinant protein].

OBJECTIVE: To clone and express the Clonorchis sinensis F0-ATP synthase b chain (CsF0-ATP-synt_B) gene and analyze immunogenicity of the recombinant protein. METHODS: The coding region F0-ATP synthase b chain gene with the mitochondrial targeting sequence (MTS) removed was amplified with PCR using the cloned plasmid as template, and the product was cloned into the prokaryotic expression vector pET-28a(+), transformed into E. coli BL21 (DE3) and induced with IPTG. The expressed product was purified by Ni-IDA affinity chromatography,and analyzed by SDS-PAGE for its expression and identified by Western blotting for its immunogenicity. RESULTS: The coding sequence of the F0-ATP synthase b-chain like gene removed off the MTS contains 813 base pairs encoding 271 amino acids with a theoretical molecular weight of 31,171.9. PCR, double enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pET-28a (+)-CsF0-ATP-synt_B was constructed successfully, and the resolvable expression was obtained in E.coli BL21. Highly purified recombinant protein was prepared through affinity chromatography. The recombinant protein could be recognized by the immune serum of the SD rat immunized with the recombinant protein. CONCLUSION: The CsF0-ATP-synt_B like gene has been efficiently expressed in prokaryotic expression system with immunogenicity.

[Gene copy number, mRNA transcription and protein expression of PD-1 gene in primary hepatocarcinoma patients].

OBJECTIVE: To study the gene copy number, mRNA transcription and protien expression of programmed cell death 1 (PD-1) gene in primary hepatocellular carcinoma (PHC) patients and normal control individuals (NC) who are anti-HBs positive, and to investigate the variations in PD-1 gene copy numbers and its relationship with PHC. METHODS: Real-time PCR was adopted to detect the PD-1 gene copy numbers and their mRNA expressions in peripheral blood mononuclear cells (PBMCs) from 24 samples of PHC patients and 26 of NC. Protein expression level of PD-1 on CD8+ T was analyzed by flow cytometry. RESULTS: In terms of number of PD-1 gene copy numbers, the percentage of cases of haploid (single) was 34.62% and 4.17% in PHC group and control group respectively while the percentage of cases of diploid (double) was 61.54% and 95.83% respectively. The difference between the two was statistically significant (chi2 = 7.639, P = 0.006). The rate of cases with double PD-1 gene copy numbers was found to be higher in patients with PHC than in control group. It was also found that the average expression of PD-1 mRNA was 2.35E-03 in control group and 1.23E-03 in PHC group. The expression level was significant lower in PHC group than that in control group when compared by using Mann-whitey technic (U = 153, P = 0.009). Furthermore, the frequency of PD-1 protein expression on CD8+ T cells was 3.72 +/- 0.32 in control group and 16.13 +/- 1.68 in PHC group. The level of PD-1 mRNA expression was higher in PHC and significant differences was shown between two groups (t = -7.073, P = 0.000). CONCLUSIONS: Our study suggests that the variation in PD-1 gene copy number may trigger primary hepatocellular carcinoma to HBV carriers. The relationship between the variation of PD-1 gene copy numbers and its association with primary hepatocellular carcinoma is worth further focus.

Genetic diversity analysis of Penicillium marneffei isolated from AIDS patients in Guangdong, China using randomly amplified polymorphic DNA.

BACKGROUND: Penicillium marneffei (P. marneffei) is an emerging pathogenic fungus that can cause invasive mycosis in patients with AIDS. The epidemiological features of P. marneffei infection in AIDS patients in Guangdong province remain unclear so far. This study aimed to investigate the genetic diversity within a population of 163 P. marneffei isolates obtained from AIDS patients and search for the dominant clinical strains in Guangdong province. METHODS: One hundred and sixty-three P. marneffei isolates obtained from AIDS patients in Guangdong province during January 2004 and December 2009 were studied by randomly amplified polymorphic DNA (RAPD) using two random primers (H2 and H22). The degree of similarity between samples was calculated through similarity coefficients from RAPD fragment data and the dendrogram was assessed using the unweighted pair group method with arithmetic mean (UPGMA). RESULTS: Two primers showed a high degree of discrimination and good stability. Primer H2 yielded eight different patterns (H2-1 to H2-8) among 163 isolates with the discriminatory power being 0.413. Primer H22 identified seven types (H22-1 to H22-7) among 163 isolates with the discriminatory power being 0.467. Genetic similarity coefficients based on RAPD data among 163 P. marneffei isolates ranged from 0.681 to 0.957, 61.96% of which were no less than 0.83. The discriminatory power of the two primers was 0.524. One hundred and sixty-three P. marneffei isolates were clustered into nine distinct groups (groups I to IX) at the similarity coefficient value of 0.83 and group I was the most common, including 101 strains (61.96%). CONCLUSION: The RAPD analyses could provide important information as to the degree of genetic diversity and the relationship among clinical P. marneffei isolates, revealing genetic polymorphism and dominant strains.