(-)-Epigallocatechin-3-gallate inhibits the replication cycle of hepatitis C virus.

(-)-Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea. In this study, we found that hepatitis C virus (HCV) infection was significantly suppressed by EGCG in an HCV cell culture (HCVcc) system using a JFH1-GFP chimeric virus, with a 50 % effective concentration (EC(50)) of 17.9 µM. The inhibitory activity of EGCG was confirmed by monitoring HCV RNA and protein expression levels in Huh7.5.1 cells infected with the JFH1 virus. Moreover, we demonstrated that the inhibitory mechanisms of EGCG were attributable to the suppression of both the HCV entry and RNA replication steps, although EGCG had little effect on translation directed by the viral internal ribosome entry site (IRES). Furthermore, HCV could be rapidly eliminated from cell cultures after two and five passages in the presence of 50 and 25 µM EGCG, respectively. These results indicate that EGCG is a potential candidate as a preventive and antiviral drug for HCV infection.

Washed microbiota transplantation reduces serum uric acid levels in patients with hyperuricaemia.

BACKGROUND: Previous studies have found that hyperuricaemia (HUA) is closely related to intestinal flora imbalance. AIM: The current study investigated the effects and safety of washed microbiota transplantation (WMT) on serum uric acid (SUA) levels in different populations. METHODS: A total of 144 patients who received WMT from July 2016 to April 2020 in the First Affiliated Hospital of Guangdong Pharmaceutical University and had SUA data before treatment were selected. Changes in SUA levels before and after treatment were retrospectively reviewed based on short-term and mid-term effects of WMT regimens. SUA levels measured in the last test within 3 mo after the first WMT represented the short-term effect, and SUA levels measured in the last test within 3-6 mo after the first WMT represented the mid-term effect. The patients were divided into an HUA group (SUA > 416 µM) and a normal uric acid (NUA) group (SUA ≥ 202 µM to ≤ 416 µM) based on pretreatment SUA levels. RESULTS: Average short-term SUA levels in the HUA group decreased after WMT (481.00 ± 99.85 vs 546.81 ± 109.64 µM, n = 32, P < 0.05) in 25/32 patients and returned to normal in 10/32 patients. The short-term level of SUA reduction after treatment moderately correlated with SUA levels before treatment (r = 0.549, R² = 0.300, P < 0.05). Average SUA levels decreased after the first and second courses of WMT (469.74 ± 97.68 vs 540.00 ± 107.16 µM, n = 35, and 465.57 ± 88.88 vs 513.19 ± 78.14 µM, n = 21, P < 0.05). Short-term and mid-term SUA levels after WMT and SUA levels after the first, second and third courses of WMT were similar to the levels before WMT in the NUA group (P > 0.05). Only 1/144 patients developed mild diarrhea after WMT. CONCLUSION: WMT reduces short-term SUA levels in patients with HUA with mild side effects but has no obvious effect on SUA levels in patients with NUA.

Combinational benefit of antihistamines and remdesivir for reducing SARS-CoV-2 replication and alleviating inflammation-induced lung injury in mice.

COVID-19 is an immune-mediated inflammatory disease caused by SARS-CoV-2 infection, the combination of anti-inflammatory and antiviral therapy is predicted to provide clinical benefits. We recently demonstrated that mast cells (MCs) are an essential mediator of SARS-CoV-2-initiated hyperinflammation. We also showed that spike protein-induced MC degranulation initiates alveolar epithelial inflammation for barrier disruption and suggested an off-label use of antihistamines as MC stabilizers to block degranulation and consequently suppress inflammation and prevent lung injury. In this study, we emphasized the essential role of MCs in SARS-CoV-2-induced lung lesions in vivo, and demonstrated the benefits of co-administration of antihistamines and antiviral drug remdesivir in SARS-CoV-2-infected mice. Specifically, SARS-CoV-2 spike protein-induced MC degranulation resulted in alveolar-capillary injury, while pretreatment of pulmonary microvascular endothelial cells with antihistamines prevented adhesion junction disruption; predictably, the combination of antiviral drug remdesivir with the antihistamine loratadine, a histamine receptor 1 (HR1) antagonist, dampened viral replication and inflammation, thereby greatly reducing lung injury. Our findings emphasize the crucial role of MCs in SARS-CoV-2-induced inflammation and lung injury and provide a feasible combination antiviral and anti-inflammatory therapy for COVID-19 treatment.

SARS-CoV-2-triggered mast cell rapid degranulation induces alveolar epithelial inflammation and lung injury.

SARS-CoV-2 infection-induced hyper-inflammation links to the acute lung injury and COVID-19 severity. Identifying the primary mediators that initiate the uncontrolled hypercytokinemia is essential for treatments. Mast cells (MCs) are strategically located at the mucosa and beneficially or detrimentally regulate immune inflammations. In this study, we showed that SARS-CoV-2-triggered MC degranulation initiated alveolar epithelial inflammation and lung injury. SARS-CoV-2 challenge induced MC degranulation in ACE-2 humanized mice and rhesus macaques, and a rapid MC degranulation could be recapitulated with Spike-RBD binding to ACE2 in cells; MC degranulation altered various signaling pathways in alveolar epithelial cells, particularly, the induction of pro-inflammatory factors and consequential disruption of tight junctions. Importantly, the administration of clinical MC stabilizers for blocking degranulation dampened SARS-CoV-2-induced production of pro-inflammatory factors and prevented lung injury. These findings uncover a novel mechanism for SARS-CoV-2 initiating lung inflammation, and suggest an off-label use of MC stabilizer as immunomodulators for COVID-19 treatments.

Histone deacetylase 3 promotes innate antiviral immunity through deacetylation of TBK1.

TANK-binding kinase 1 (TBK1), a core kinase of antiviral pathways, activates the production of interferons (IFNs). It has been reported that deacetylation activates TBK1; however, the precise mechanism still remains to be uncovered. We show here that during the early stage of viral infection, the acetylation of TBK1 was increased, and the acetylation of TBK1 at Lys241 enhanced the recruitment of IRF3 to TBK1. HDAC3 directly deacetylated TBK1 at Lys241 and Lys692, which resulted in the activation of TBK1. Deacetylation at Lys241 and Lys692 was critical for the kinase activity and dimerization of TBK1 respectively. Using knockout cell lines and transgenic mice, we confirmed that a HDAC3 null mutant exhibited enhanced susceptibility to viral challenge via impaired production of type I IFNs. Furthermore, activated TBK1 phosphorylated HDAC3, which promoted the deacetylation activity of HDAC3 and formed a feedback loop. In this study, we illustrated the roles the acetylated and deacetylated forms of TBK1 play in antiviral innate responses and clarified the post-translational modulations involved in the interaction between TBK1 and HDAC3.

[Research progress on human sarcocystosis].

Human sarcocystosis (both the intestinal and muscular forms) may be emerging as a significant, foodborne zoonotic infection in southeast Asia and southwest of China. This review summarizes recent findings in classification of Sarcocystis spp, epidemiologic features, clinical symptoms, diagnosis, treatment and prevention in human infections.

Zoonotic origins of human coronavirus 2019 (HCoV-19 / SARS-CoV-2): why is this work important?

The ongoing pandemic of coronavirus disease 2019 (COVID-19), caused by infection with human coronavirus 2019 (HCoV-19 / SARS-CoV-2 / 2019-nCoV), is a global threat to the human population. Here, we briefly summarize the available data for the zoonotic origins of HCoV-19, with reference to the other two epidemics of highly virulent coronaviruses, SARS-CoV and MERS-CoV, which cause severe pneumonia in humans. We propose to intensify future efforts for tracing the origins of HCoV-19, which is a very important scientific question for the control and prevention of the pandemic.

NKG2A is a NK cell exhaustion checkpoint for HCV persistence.

Exhaustion of cytotoxic effector natural killer (NK) and CD8+ T cells have important functions in the establishment of persistent viral infections, but how exhaustion is induced during chronic hepatitis C virus (HCV) infection remains poorly defined. Here we show, using the humanized C/OTg mice permissive for persistent HCV infection, that NK and CD8+ T cells become sequentially exhausted shortly after their transient hepatic infiltration and activation in acute HCV infection. HCV infection upregulates Qa-1 expression in hepatocytes, which ligates NKG2A to induce NK cell exhaustion. Antibodies targeting NKG2A or Qa-1 prevents NK exhaustion and promotes NK-dependent HCV clearance. Moreover, reactivated NK cells provide sufficient IFN-γ that helps rejuvenate polyclonal HCV CD8+ T cell response and clearance of HCV. Our data thus show that NKG2A serves as a critical checkpoint for HCV-induced NK exhaustion, and that NKG2A blockade sequentially boosts interdependent NK and CD8+ T cell functions to prevent persistent HCV infection.

Hepatitis B virus infection: Defective surface antigen expression and pathogenesis.

Hepatitis B virus (HBV) infection is a global public health concern. HBV causes chronic infection in patients and can lead to liver cirrhosis, hepatocellular carcinoma, and other severe liver diseases. Thus, understanding HBV-related pathogenesis is of particular importance for prevention and clinical intervention. HBV surface antigens are indispensable for HBV virion formation and are useful viral markers for diagnosis and clinical assessment. During chronic HBV infection, HBV genomes may acquire and accumulate mutations and deletions, leading to the expression of defective HBV surface antigens. These defective HBV surface antigens have been found to play important roles in the progression of HBV-associated liver diseases. In this review, we focus our discussion on the nature of defective HBV surface antigen mutations and their contribution to the pathogenesis of fulminant hepatitis B. The relationship between defective surface antigens and occult HBV infection are also discussed.

Free radical scavenging and antioxidant enzymes activation of polysaccharide extract from Nostoc sphaeroides.

Nostoc sphaeroides Kuetzing has been used as a traditional medicine in China to treat a variety of ailments. This research identified the antioxidant activities of polysaccharide extract from Nostoc sphaeroides. The extract, which contains 46.2% carbohydrates, exhibited an effective scavenging capability on superoxide radical, hydroxyl radicals in non site-specific as well as site-specific assays, and also performed lipid peroxidation inhibition in a dose-dependent manner. Polysaccharide extract had no 1,1-diphenyl-2-picrylhydrazyl radical scavenging potential at all test concentrations. Activities of superoxide dismutase, catalase, and glutathione peroxidase in human embryo kidney 293 cells were increased effectively when Nostoc sphaeroides extract was applied. These results suggested that the use of N. sphaeroides in treating ailments may be based on the antioxidant capacities of polysaccharide composition.

[Effects of hFRNK on E-cadherin/beta-catenin in colon cancer cells in vitro].

OBJECTIVE: To explore the effects of human FRNK gene on E-cadherin/beta-catenin complex in colon cancer cell line Colo320WT cells stimulated with extrinsic gastrinl7. METHODS: AdEasy system was used to construct pAdhFRNK expressing human FRNK gene by recombination in E. coli. BJ5283. pCR3.1/GR plasmid expressing gastrin receptor CCK-2 was transfected into colon cancer cell line Colo320 cells by Lipofectamine 2000 and expressing stably CCK-2R clones were screened by G418 (500 pg/ml). The expression levels of gastrin receptor in Colo320 cells and the transfected Colo320WT cells were assayed by RT-PCR. Colo320WT cells were treated by 10(-8) mol/L gastrinl7 for 12 h; and after Colo320WT cells were infected by pAdhFRNK (MOI: 100) for 2 d the cells were treated by gastrin17 for 12 h again. The expression levels of E-cadherin and beta-catenin in TX-100 soluble fraction and TX-100 insoluble fraction of Colo320WT cells were assayed by co-immunoprecipation and Western blot. E-cadherin and beta-catenin's distribution in Colo320WT cells were detected by immunocytochemistry. RESULTS: When 10(-8) mol/L gastrin17 stimulated Colo320WT cells for 12 h, the expression levels of E-cadherin and beta-catenin in TX-100-soluble fraction decreased apparently, while the expression levels of E-cadherin and beta-catenin in TX-100-insoluble fraction increased markedly. When pAdhFRNK infected Colo320WT cells for 2 d and 10(-8) mol/L gastrin17 treated the cells for 12 h, the expression levels of E-cadherin and beta-catenin in TX-100-soluble fraction increased apparently again, and the expression levels of E-cadherin and beta-catenin in TX-100-insolutble fraction decreased markedly. Immunocytochemistry showed that the distribution of E-cadherin and beta-catenin was translocated from plasma membrane into cytoplasm and nucleus in the cells stimulated with gastrinl7, and after the cells were infected with pAdhFRNK and stimulated by gastrinl7 again. beta-catenin was mainly observed in cytoplasm and little nuclear immunoreactivity. CONCLUSION: An adenovirus vector pAdhFRNK can inhibit abnormal distribution of E-cadherin and beta-catenin in the gastrin17-stimulated cells. The mechanism is probably that hFRNK can disphosphorylate phosphorylated FAK and block FAK pathway.

Construction of recombinant baculovirus Ac-CMV-hSox9 for gene therapy of intervertebral disc degeneration.

OBJECTIVE: To construct the recombinant baculovirus Ac-cytomegalovirus (CMV)-hSox9 for gene therapy of intervertebral disc degeneration. METHODS: Bac-to-Bac system was used for the construction of baculovirus Ac-CMV-hSox9. The cDNA of hSox9 was first cloned into a plasmid vector under the control of CMV promotor to generate the donor plasmid pFastBacDuljgreen fluorescene protein (GFP)-CMV (pFGC)-hSox9. The resultant plasmid was transformed into DH10Bac cells and then the transformation mixture was spread on Luria-Bertani (LB) agarose culture medium containing isopropyl-beta-D-thiogalactoside (IPTG), X-gal, gentamicin, kanamycin and tetracycline. The white colonies were selected and cultured for amplification, and the hSox9Bacmid DNA was extracted. After verification, recombinant baculovirus Ac-CMV-hSox9 was obtained through transfecting Sf 21 cells. The expression of hSox9 gene in the intervertebral disc cells in rabbits was determined by Western blotting and immunohistochemical staining. RESULTS: Polymerase chain reaction (PCR) confirmed the presence of hSox9 gene in the recombinant baculovirus and the Sf 21 cells transfected by the baculovirus showed the expression of fluorescence protein. Western blotting and immunohistochemical staining analysis indicated that exogenous hSox9 gene was expressed in the disc cells. CONCLUSIONS: The successful construction of the recombinant baculovirus Ac-CMV-hSox9 and the confirmation of the target gene expression provides a novel expression vector system for basic research and clinical treatment of intervertebral degenerative disc disease.

[A comparative study on isoenzymes of Sarcocystis spp. from cattle and water buffaloes].

OBJECTIVE: To analyze the zymogram of peroxidase (PER) and phosphoglucose isomerase (PGI) of three species of Sarcocystis. METHODS: The collected parasites were homogenized and fragmented by ultrasonication. After centrifugation, the supernatants were analyzed by isoelectric focusing electrophoresis. RESULTS: The isolates of S. cruzi from infected water buffalo and cattle all showed identical enzyme profiles, 7 bands of PER at pH 4.44-6.98 and 6 bands of PGI at pH 4.66-6.53; and same with the isolates of S. hirsuta. 5 bands of PER at pH 4.97-7.15 and 4 bands of PGI at pH 4.70-6.51. The zymograms among S. cruzi, S. hirsuta and S. fusiformis were different considerably. CONCLUSION: The data support the hypothesis that both water buffalo and cattle are the natural intermediate hosts of S. cruzi and S. hirsuta at the gene level. S. cruzi, S. hirsuta and S. fusiformis are different species.

Genetic variation of hepatitis B virus and its significance for pathogenesis.

Hepatitis B virus (HBV) has a worldwide distribution and is endemic in many populations. Due to its unique life cycle which requires an error-prone reverse transcriptase for replication, it constantly evolves, resulting in tremendous genetic variation in the form of genotypes, sub-genotypes, and mutations. In recent years, there has been considerable research on the relationship between HBV genetic variation and HBV-related pathogenesis, which has profound implications in the natural history of HBV infection, viral detection, immune prevention, drug treatment and prognosis. In this review, we attempted to provide a brief account of the influence of HBV genotype on the pathogenesis of HBV infection and summarize our current knowledge on the effects of HBV mutations in different regions on HBV-associated pathogenesis, with an emphasis on mutations in the preS/S proteins in immune evasion, occult HBV infection and hepatocellular carcinoma (HCC), mutations in polymerase in relation to drug resistance, mutations in HBV core and e antigen in immune evasion, chronicalization of infection and hepatitis B-related acute-on-chronic liver failure, and finally mutations in HBV x proteins in HCC.

A taxonomic re-appraisal of Sarcocystis nesbitti (Protozoa: Sarcocystidae) from the monkey Macaca fascicularis in Yunnan, PR China.

The first detection of Sarcocystis nesbitti Mandour, 1969 in the Chinese mainland is reported and the morphology of the sarcocyst is described in detail. The parasite was detected in the monkey, Macaca fascicularis, maintained on a monkey farm in Yunnan Province; the infection may have occurred via faecal contamination from local rats, mice and/or birds. S. nesbitti was characterized as follows: a macroscopic sarcocyst, length of the cyst up to 2 mm; cyst wall smooth, thin and no perpendicular protrusion is seen under the light microscope; border of cyst wall wavy, primary cyst wall thin (38-65 nm) and invaginated; ground substance about 0.5-0.76 microm thick with electron-dense granules and concentric spherical bodies. The cyst wall is described as type 1 by electron microscopy. It is suspected that S. nesbitti may utilize Macaca mulatta, M. fascicularis, Cercocebus atys, and Papio papionis, as well as human as intermediate hosts. The taxonomy of S. nesbitti is re-appraised in the light of a consideration of possible experimental artefacts and examination of the past literature. Evidence is presented that S. nesbitti may be one of the species infecting humans in South Asia and that the monkey may be a potential reservoir host.

A PCR-based RFLP analysis of Sarcocystis cruzi (Protozoa: Sarcocystidae) in Yunnan Province, PR China, reveals the water buffalo (Bubalus bubalis) as a natural intermediate host.

A polymerase chain reaction-based restriction fragment length polymorphism (RFLP) approach is used to examine Sarcocystis cruzi-like taxa from the atypical intermediate host, water buffalo, in Yunnan, People's Republic of China. The loci examined lie within the 18S rRNA gene. A total of 15 water buffalo isolates are compared with those of 10 S. cruzi from cattle. RFLP patterns for the S. cruzi isolates from cattle and the S. cruzi-like taxon from water buffalo are found to be identical with all the 12 restriction enzymes used. Interpopulation variation between samples from Kunming and Gengma (Yunnan) is found to be undetectable at these loci for both S. cruzi and the S. cruzi-like taxon. But RFLPs are found between the S. cruzi taxa and S. suihominis from pigs at the same study sites. These findings support the hypothesis that S. cruzi is able to use the water buffalo as an intermediate host and is not restricted to cattle as was previously supposed.

Control of hepatitis B virus replication by interferons and Toll-like receptor signaling pathways.

Hepatitis B virus (HBV) infection is one of the major causes of liver diseases, affecting more than 350 million people worldwide. The interferon (IFN)-mediated innate immune responses could restrict HBV replication at the different steps of viral life cycle. Indeed, IFN-α has been successfully used for treatment of patients with chronic hepatitis B. However, the role of the innate immune response in HBV replication and the mechanism of the anti-HBV effect of IFN-α are not completely explored. In this review, we summarized the currently available knowledge about the IFN-mediated anti-HBV effect in the HBV life cycle and the possible effectors downstream the IFN signaling pathway. The antiviral effect of Toll-like receptors (TLRs) in HBV replication is briefly discussed. The strategies exploited by HBV to evade the IFN- and TLR-mediated antiviral actions are summarized.

[Localization of AcMNPV NLA genes in Sf9 cells].

Nuclear actin which plays a key role in many nucleic processes has become a research hotspot. Baculovirus is the only reported pathogen using nuclear actin to replicate and proliferate. However, little is known about the mechanism of monomeric G-actin accumulation within nuclei of baculovirus-infected cells. It has been reported that AcMNPV ie-1, pe38, ac4, he65, ac102, and ac152 could be required for mediating nuclear localization of G-actin from transiently transfected results in TN-368 cells. In this paper, we found that IE1, AC152, PE38, AC102 localized in the whole cell and PE38, AC102 localized in the nuclear mainly, while both AC4 and HE65 localized in cytoplasm and could be mediated into the nucleus by AC102 and IE1 respectively for the first time. And ie-1 or pe38, ac4, he65 could mediate nuclear G-actin to accumulate partly, while these four genes were sufficient for recruiting G-actin accumulation within the nucleus when driven by promoter OpIE2. Determining the functions of each of these AcMNPV NLA gene products will advance our understanding of baculovirus biology and function of nuclear actin.

[Cloning and functional research of Arp2/3-P40/ARPC1 subunit of Sf9 cells].

The baculovirus-induced actin polymerization is mainly associated with the virus nucleocapsid protein P78/83, which is homologous with WASP proteins that can activate Arp2/3 complex and induce the actin polymerization. In order to explore the role of Arp2/3 complex in the baculovirus replication, the P40 subunit of Arp2/3 complex from Sf9 (Spodoptera frugiperda 9) cell line was cloned and characterized. Immunofluorescent microscopy assay indicated that P40 was recruited to the inner-side of nuclear membrane during virus infection, which was in accordance with nuclear F-actin distribution in virus-infected cells as documented in our previous research, suggesting P40 could be used to track Arp2/3 complex subcellular distribution changes during virus infection. In addition, co-immunoprecipitation assay demonstrated that P40 interacted with P78/83 only in virus-infected cells, suggesting that actin polymerization induced by P78/83-Arp2/3 complex during baculovirus infection was regulated by some unidentified virus factors.

The protamine-like DNA-binding protein P6.9 epigenetically up-regulates Autographa californica multiple nucleopolyhedrovirus gene transcription in the late infection phase.

Protamines are a group of highly basic proteins first discovered in spermatozoon that allow for denser packaging of DNA than histones and will result in down-regulation of gene transcription[1]. It is well recognized that the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) encodes P6.9, a protamine-like protein that forms the viral subnucleosome through binding to the viral genome[29]. Previous research demonstrates that P6.9 is essential for viral nucleocapsid assembly, while it has no influence on viral genome replication[31]. In the present study, the role of P6.9 in viral gene transcription regulation is characterized. In contrast to protamines or other protamine-like proteins that usually down-regulate gene transcription, P6.9 appears to up-regulate viral gene transcription at 12-24 hours post infection (hpi), whereas it is non-essential for the basal level of viral gene transcription. Fluorescence microscopy reveals the P6.9's co-localization with DNA is temporally and spatially synchronized with P6.9's impact on viral gene transcription, indicating the P6.9-DNA association contributes to transcription regulation. Chromatin fractionation assay further reveals an unexpected co-existence of P6.9 and host RNA polymerase II in the same transcriptionally active chromatin fraction at 24 hpi, which may probably contribute to viral gene transcription up-regulation in the late infection phase.