Expression of HCV genotype-4 core antigen in prokaryotic E. coli system for diagnosis of HCV infection in Egypt.

BACKGROUND: Egypt has a high prevalence of hepatitis C virus (HCV) infection with 92.5% of genotype-4. AIM: This study aimed to clone and express the core gene of HCV genotype-4 for using it to develop a highly sensitive, specific, and cost-effective diagnostic assay for detecting HCV infection. METHODS: Using synthetic HCV genotype-4 core gene, pET15b as E. coli expression vector, and 1 mM lactose as inducer, the HCV core protein (MW 17 kDa) was expressed in the form of inclusion bodies (IBs) that was purified and solubilized using 8 M guanidinium HCl. The recombinant core protein was in vitro refolded by a rapid dilution method for further purification using weak cation exchange liquid chromatography. The immunogenicity of the purified protein was tested by ELISA using 129 serum samples. RESULTS: The recombinant core protein was successfully expressed and purified. The results also showed that the in-house anti-HCV core assay is accurate, specific (~96.6%), and highly sensitive (~100%) in accordance with the commercial ELISA kit. CONCLUSION: The sensitivity, specificity, and reproducibility of the developed assay were high and promising to be used as a screening assay for detecting HCV infection.

A simple method to purify recombinant HCV core protein expressed in Pichia pastoris for obtaining virus-like particles and producing monoclonal antibodies.

In this study, we describe an optimized method of obtaining virus-like particles (VLPs) of the recombinant hepatitis C virus (HCV) core protein (HCcAg) expressed in yeast cells (Pichia pastoris), which can be used for the construction of diagnostic test systems and vaccine engineering. The described simplified procedure was developed to enable in vitro self-assembly of HCcAg molecules into VLPs during protein purification. In brief, the HCcAg protein was precipitated from yeast cell lysates with ammonium sulfate and renatured by gel filtration on Sephadex G-25 under reducing conditions. VLPs were self-assembled after the removal of the reducing agent by gel filtration on Sephadex G-25. Protein purity and specificity were evaluated by SDS-PAGE and immunoblotting analysis. The molecular mass of VLPs and their relative quantity were measured by HPLC, followed by confirmation of VLPs production and estimation of their shape and size by transmission electron microscopy. As a result, we obtained recombinant HCcAg preparation (with ~90% purity) in the form of VLPs and monomers, which has been used to produce hybridomas secreting monoclonal antibodies (mAbs) against HCcAg.

The value of p16 and HPV DNA in non-tonsillar, non-base of tongue oropharyngeal cancer.

BACKGROUND: Oropharyngeal squamous cell carcinoma (OPSCC) is dominated by tonsillar and tongue base carcinomas (TSCC/BOTSCC), but there are carcinomas at other sites, such as uvula/soft palate/pharyngeal wall here defined as other OPSCC. Human papillomavirus (HPV) positive TSCC/BOTSCC have favorable outcome, and the TNM-classification separates OPSCC into HPV mediated (p16INK4a overexpressing, p16+) and HPV unrelated OPSCC (p16INK4a non-overexpressing, p16-) cancer, but the prognostic role of p16+ in other OPSCC is unclear. AIMS/OBJECTIVES: This study therefore aimed to further investigate the prognostic role of p16+, presence of HPV DNA, or both combined in other OPSCC. MATERIAL AND METHODS: 195 other OPSCC, from patients diagnosed 2000-2018 were tested for p16, and/or presence of HPV DNA and the data correlated to outcome. RESULTS: Neither overall survival, nor disease free survival correlated to presence of p16+ or HPV DNA in other OPSCC. p16+ and HPV DNA presence were correlated (p < .0001), but the sensitivity of p16 as a surrogate marker for presence of HPV DNA was low (49%). CONCLUSIONS AND SIGNIFICANCE: The data suggest that p16+ (and p16+/HPV DNA) positive other OPSCC should be analyzed cautiously and possibly separately from the HPV mediated OPSCC staging group.

Canola oilseed- and Escherichia coli- derived hepatitis C virus (HCV) core proteins adjuvanted with oil bodies, induced robust Th1-oriented immune responses in immunized mice.

Induction of broad Th1 cellular immune responses and cytokines is crucial characteristics for vaccines against intracellular infections such as hepatitis C virus (HCV). Plants (especially oilseed tissues) and plant-immunomodulators (like oil bodies) offer cost-effective and scalable possibilities for the production of immunologically relevant and safe vaccine antigens and adjuvants, respectively. Herein, we provide data of the murine immunization by transgenic canola oilseed-derived HCV core protein (HCVcp) soluble extract (TSE) and Escherichia coli- derived rHCVcp in combination with Canola oil bodies (oil) compared to that of the Freund's (FA) adjuvant. Mice immunized by TSE+ oil developed both strong humeral (IgG) and Th1-biased cellular responses, manifested by high levels of IFN-γ and lower IgG1/IgG2a ratio and IL-4 secretion. Results of the intracellular cytokine staining indicated that TSE+ oil immunization in mice triggered both CD4+ and CD8+ T cells to release IFN-γ, while CD4+ cells were mostly triggered when FA was used. Analyses by qRT-PCR indicated that a combination of rHCVcp/TSE with oil body induced high levels of IL-10 cytokines compared to that of the FA adjuvant. These characteristics are important properties for the design of an HCV vaccine candidate and indicate the potential of Canola-derived antigen and oil bodies in addressing these concerns.

A Novel Cis-Acting RNA Structural Element Embedded in the Core Coding Region of the Hepatitis C Virus Genome Directs Internal Translation Initiation of the Overlapping Core+1 ORF.

Hepatitis C virus (HCV) genome translation is initiated via an internal ribosome entry site (IRES) embedded in the 5'-untranslated region (5'UTR). We have earlier shown that the conserved RNA stem-loops (SL) SL47 and SL87 of the HCV core-encoding region are important for viral genome translation in cell culture and in vivo. Moreover, we have reported that an open reading frame overlapping the core gene in the +1 frame (core+1 ORF) encodes alternative translation products, including a protein initiated at the internal AUG codons 85/87 of this frame (nt 597-599 and 603-605), downstream of SL87, which is designated core+1/Short (core+1/S). Here, we provide evidence for SL47 and SL87 possessing a novel cis-acting element that directs the internal translation initiation of core+1/S. Firstly, using a bicistronic dual luciferase reporter system and RNA-transfection experiments, we found that nucleotides 344-596 of the HCV genotype-1a and -2a genomes support translation initiation at the core+1 frame AUG codons 85/87, when present in the sense but not the opposite orientation. Secondly, site-directed mutagenesis combined with an analysis of ribosome-HCV RNA association elucidated that SL47 and SL87 are essential for this alternative translation mechanism. Finally, experiments using cells transfected with JFH1 replicons or infected with virus-like particles showed that core+1/S expression is independent from the 5'UTR IRES and does not utilize the polyprotein initiation codon, but it requires intact SL47 and SL87 structures. Thus, SL47 and SL87, apart from their role in viral polyprotein translation, are necessary elements for mediating the internal translation initiation of the alternative core+1/S ORF.

Genetic variations on 31 and 450 residues of influenza A nucleoprotein affect viral replication and translation.

BACKGROUND: Influenza A viruses cause epidemics/severe pandemics that pose a great global health threat. Among eight viral RNA segments, the multiple functions of nucleoprotein (NP) play important roles in viral replication and transcription. METHODS: To understand how NP contributes to the virus evolution, we analyzed the NP gene of H3N2 viruses in Taiwan and 14,220 NP sequences collected from Influenza Research Database. The identified genetic variations were further analyzed by mini-genome assay, virus growth assay, viral RNA and protein expression as well as ferret model to analyze their impacts on viral replication properties. RESULTS: The NP genetic analysis by Taiwan and global sequences showed similar evolution pattern that the NP backbones changed through time accompanied with specific residue substitutions from 1999 to 2018. Other than the conserved residues, fifteen sporadic substitutions were observed in which the 31R, 377G and 450S showed higher frequency. We found 31R and 450S decreased polymerase activity while the dominant residues (31 K and 450G) had higher activity. The 31 K and 450G showed better viral translation and replication in vitro and in vivo. CONCLUSIONS: These findings indicated variations identified in evolution have roles in modulating viral replication in vitro and in vivo. This study demonstrates that the interaction between variations of NP during virus evolution deserves future attention.

The pattern of antiviral protein expression induced by interferon λ1 in peripheral blood mononuclear cells of patients with chronic hepatitis C virus infection.

Interferon lambda was discovered in recent years to be an antiviral agent, and research on different aspects of this antiviral factor in viral infection and investigations of its effectiveness are also progressing. The immunological effects of interferon lambda on different cell populations is not precisely known, which may be due to its use of a heterodimeric receptor consisting of IL-10R2 and IFN-λR1, which are not broadly expressed in all types of cells. In the present study, signaling by interferon lambda and its effect on the expression of hepatitis C virus (HCV) proteins were measured, and the expression pattern of some antiviral proteins and IL-10 levels were investigated in peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from 50 patients with chronic genotype 1a HCV infection and 10 healthy individuals as controls. The PBMCs were treated with various doses of interferon lambda at different times of cultivation. Real-time PCR was used for relative quantification of Mxa, PKR, OAS, ISG15 and HCV core mRNAs. Expression of the NS5A protein was measured by flow cytometry, and IL-10 production was assessed by ELISA. A significant increase in the expression of mRNA encoding antiviral proteins and a decrease in the expression of mRNAs encoding the HCV core protein were observed when cells were treated with interferon lambda in an intermittent manner. The expression of HCV NS5A protein and interleukin 10 levels were also lower than in the control group. It was shown that the maximum antiviral effect of interferon lambda in PBMCs is dependent on the dose and treatment time.

Core gene insertion in hepatitis B virus genotype G functions at both the encoded amino acid sequence and RNA structure levels to stimulate core protein expression.

Hepatitis B virus genotype G possesses a 36-nucleotide (nt) insertion at the 5' end of core gene, adding 12 residues to core protein. The insertion markedly increased core protein level irrespective of viral genotype, with the effect reproducible using CMV-core gene construct. Here we used such expression constructs and transient transfection experiments in Huh7 cells to identify the structural bases. The insertion is predicted to create a stem-loop structure 14nt downstream of core gene AUG. A + 1 or + 2 frameshift into the 36nt mitigated enhancement of core protein level. Point mutations to disrupt or restore the stem-loop had opposite effects on core protein expression. Shifting the translation initiation site downstream or further upstream of the stem-loop rendered it inhibitory or no longer stimulatory of core protein expression. Therefore, both the reading frame and a properly positioned stem-loop structure contribute to marked increase in core protein expression by the 36-nt insertion.

Differential regulation of hepatitis B virus core protein expression and genome replication by a small upstream open reading frame and naturally occurring mutations in the precore region.

Hepatitis B virus (HBV) transcribes two subsets of 3.5-kb RNAs: precore RNA for hepatitis B e antigen (HBeAg) expression, and pregenomic RNA for core and P protein translation as well as genome replication. HBeAg expression could be prevented by mutations in the precore region, while an upstream open reading frame (uORF) has been proposed as a negative regulator of core protein translation. We employed replication competent HBV DNA constructs and transient transfection experiments in Huh7 cells to verify the uORF effect and to explore the alternative function of precore RNA. Optimized Kozak sequence for the uORF or extra ATG codons as present in some HBV genotypes reduced core protein expression. G1896A nonsense mutation promoted more efficient core protein expression than mutated precore ATG, while a +1 frameshift mutation was ineffective. In conclusion, various HBeAg-negative precore mutations and mutations affecting uORF differentially regulate core protein expression and genome replication.

Age-dependent PPARα activation induces hepatic sulfatide accumulation in transgenic mice carrying the hepatitis C virus core gene.

Sulfatides, a type of glycosphingolipid, are associated with carcinogenesis. Peroxisome proliferator-activated receptor α (PPARα) is involved in the regulation of sulfatide metabolism as well as in cancer development. We previously reported that transgenic (Tg) mice expressing hepatitis C virus core protein (HCVcp) exhibited age-dependent PPARα activation and carcinogenesis in liver. However, the metabolism of sulfatides in hepatocellular carcinoma is unknown. To examine the relationship between sulfatide metabolism, carcinogenesis, HCVcp, and PPARα, age-dependent changes of these factors were examined in HCVcpTg, PPARα inhibitor-treated HCVcpTg, and non-Tg mice. The sulfatide content in liver, the hepatic expression of two key enzymes catalyzing the initial and last reactions in sulfatide synthesis, the hepatic expression of known sulfatide-transferring protein, oxidative stress, and hepatic PPARα expression and its activation were age-dependently increased in HCVcpTg mice. The increased synthesis and accumulation of sulfatides and PPARα activation were significantly enhanced in liver cancer lesions. These changes were attenuated by PPARα inhibitor treatment and not observed in non-Tg mice. These results suggest that HCVcp-induced age-dependent PPARα activation increases synthesis of sulfatides and the resulting sulfatide accumulation affects HCV-related liver cancer. The monitoring of hepatic sulfatide content and the modulation of sulfatide generation by intervention using a PPARα inhibitor might be useful for the prediction and prevention of HCV-related hepatocarcinogenesis, respectively.

Development and validation of a staging system for HPV-related oropharyngeal cancer by the International Collaboration on Oropharyngeal cancer Network for Staging (ICON-S): a multicentre cohort study.

BACKGROUND: Human papillomavirus-related (HPV+) oropharyngeal cancer is a rapidly emerging disease with generally good prognosis. Many prognostic algorithms for oropharyngeal cancer incorporate HPV status as a stratification factor, rather than recognising the uniqueness of HPV+ disease. The International Collaboration on Oropharyngeal cancer Network for Staging (ICON-S) aimed to develop a TNM classification specific to HPV+ oropharyngeal cancer. METHODS: The ICON-S study included patients with non-metastatic oropharyngeal cancer from seven cancer centres located across Europe and North America; one centre comprised the training cohort and six formed the validation cohorts. We ascertained patients' HPV status with p16 staining or in-situ hybridisation. We compared overall survival at 5 years between training and validation cohorts according to 7th edition TNM classifications and HPV status. We used recursive partitioning analysis (RPA) and adjusted hazard ratio (AHR) modelling methods to derive new staging classifications for HPV+ oropharyngeal cancer. Recent hypotheses concerning the effect of lower neck lymph nodes and number of lymph nodes were also investigated in an exploratory training cohort to assess relevance within the ICON-S classification. FINDINGS: Of 1907 patients with HPV+ oropharyngeal cancer, 661 (35%) were recruited at the training centre and 1246 (65%) were enrolled at the validation centres. 5-year overall survival was similar for 7th edition TNM stage I, II, III, and IVA (respectively; 88% [95% CI 74-100]; 82% [71-95]; 84% [79-89]; and 81% [79-83]; global p=0·25) but was lower for stage IVB (60% [53-68]; p<0·0001). 5-year overall survival did not differ among N0 (80% [95% CI 73-87]), N1-N2a (87% [83-90]), and N2b (83% [80-86]) subsets, but was significantly lower for those with N3 disease (59% [51-69]; p<0·0001). Stage classifications derived by RPA and AHR models were ranked according to survival performance, and AHR-New was ranked first, followed by AHR-Orig, RPA, and 7th edition TNM. AHR-New was selected as the proposed ICON-S stage classification. Because 5-year overall survival was similar for patients classed as T4a and T4b, T4 is no longer subdivided in the re-termed ICON-S T categories. Since 5-year overall survival was similar among N1, N2a, and N2b, we re-termed the 7th edition N categories as follows: ICON-S N0, no lymph nodes; ICON-S N1, ipsilateral lymph nodes; ICON-S N2, bilateral or contralateral lymph nodes; and ICON-S N3, lymph nodes larger than 6 cm. This resembles the N classification of nasopharyngeal carcinoma but without a lower neck lymph node variable. The proposed ICON-S classification is stage I (T1-T2N0-N1), stage II (T1-T2N2 or T3N0-N2), and stage III (T4 or N3). Metastatic disease (M1) is classified as ICON-S stage IV. In an exploratory training cohort (n=702), lower lymph node neck involvement had a significant effect on survival in ICON-S stage III but had no effect in ICON-S stage I and II and was not significant as an independent factor. Overall survival was similar for patients with fewer than five lymph nodes and those with five or more lymph nodes, within all ICON-S stages. INTERPRETATION: Our proposed ICON-S staging system for HPV+ oropharyngeal cancer is suitable for the 8th edition of the Union for International Cancer Control/American Joint Committee on Cancer TNM classification. Future work is needed to ascertain whether T and N categories should be further refined and whether non-anatomical factors might augment the full classification. FUNDING: None.

Regulation of HepG2 cell apoptosis by hepatitis C virus (HCV) core protein via the sirt1-p53-bax pathway.

Hepatitis C virus (HCV) core protein stimulates many signaling pathways related to apoptosis inhibition resulting in hepatocellular carcinoma (HCC). It has been reported that sirt1 is involved in regulating apoptosis; therefore, we investigated the influence of HCV core protein on sirt1 expression and apoptosis in human HepG2 cells. Our study showed that HCV core protein inhibited apoptosis of HepG2 cells as well as caspase-3 expression and activity (P < 0.05). At the same time, sirt1 expression was increased at both the mRNA (P < 0.05) and protein (P < 0.05) levels. Furthermore, apoptosis inhibition was reversed when sirt1 was knocked down (P < 0.05). Our study provides further evidence that the sirt1-p53-Bax signaling pathway plays an important role in regulating the suppression of cell apoptosis induced by HCV core protein.

Expression of Hepatitis C Virus Core and E2 antigenic recombinant proteins and their use for development of diagnostic assays.

BACKGROUND: Early diagnosis of HCV infection is based on detection of antibodies against HCV proteins using recombinant viral antigens. The present study was designed to select, clone and express the antigenic regions of Core and E2 genes from local HCV-3a genotype and to utilize the antigenic recombinant proteins (Core & E2) to develop highly sensitive, specific and economical diagnostic assays for detection of HCV infection. METHODS: The antigenic sites were determined within Core and E2 genes and were then cloned in pET-28a expression vector. The right orientation of the desired inserted fragments of Core and E2 were confirmed via sequencing prior to expression and were then transformed in BL21 (DE3) pLysS strains of E. coli and induced with 0.5mM Isopropyl-b-D-thiogalactopyranoside (IPTG) for the production of antigenic recombinant proteins. The produced truncated antigens were then purified by Nickel affinity chromatography and were confirmed by western blotting, immunoblotting and enzyme-linked immunosorbent assay (ELISA). RESULTS: The expressed Core and E2 recombinant antigens were used to develop immunoblotting assay for the detection of anti-HCV antibodies in sera. With immunoblotting, a total of 93-HCV infected sera and 35-HCV negative individuals were tested for the presence of anti-HCV antibodies to the Core and E2 antigens. Recombinant antigen showed 100% reactivity against HCV infected sera, with no cross reactivity against HCV-negative sera. The immunoblot assay mixture of recombinant antigens (Core+E2) showed a strong reaction intensity in the test area (TA) as compared to the individual truncated Core and E2 recombinant antigens. In the in-house ELISA assay, mixed Core and E2 recombinant antigens showed 100% reactivity against a standardized panel of 150-HCV-positive sera and non reactivity against a standardized panel of 150 HCV-negative sera while also being non reactive to sera positive for other viral infections. The antigenic recombinant antigens also were tested for the 30 sera of known genotypes. The antigens did not detect antibodies to genotype-3a, but detected antibodies to all genotypes and did not discriminate them genotype wise. A panel of 175 of HCV-suspected serum samples was subjected to comparative analysis with our in-house ELISA assay and with commercial HCV screening assays. After subjecting the results to the formulas for determining the quality parameters, immunoblot assay had 100% sensitivity and specificity, while the ELISA assay had 100% sensitivity and 98.8% specificity as compared to commercially available assays. CONCLUSION: This study indicates that a mixture of Core and E2 antigens are potentially valuable antigens and there is the possibility of developing serological assays for monitoring HCV infection.

Expression of the novel hepatitis C virus core+1/ARF protein in the context of JFH1-based replicons.

Hepatitis C virus contains a second open reading frame within the core gene, designated core+1/ARF. Here we demonstrate for the first time expression of core+1/ARF protein in the context of a bicistronic JFH1-based replicon and report the production of two isoforms, core+1/L (long) and core+1/S (short), with different kinetics.

Regulation of core expression during the hepatitis C virus life cycle.

Core plays a critical role during hepatitis C virus (HCV) assembly, not only as a structural component of the virion, but also as a regulator of the formation of assembly sites. In this study, we observed that core is expressed later than other HCV proteins in a single viral cycle assay, resulting in a relative increase of core expression during a late step of the viral life cycle. This delayed core expression results from an increase of core half-life, indicating that core is initially degraded and is stabilized at a late step of the HCV life cycle. Stabilization-mediated delayed kinetics of core expression were also observed using heterologous expression systems. Core stabilization did not depend on its interaction with non-structural proteins or lipid droplets but was correlated with its expression levels and its oligomerization status. Therefore in the course of a HCV infection, core stabilization is likely to occur when the prior amplification of the viral genome during an initial replication step allows core to be synthesized at higher levels as a stable protein, during the assembly step of the viral life cycle.

Effects of human Parvovirus B19 and Bocavirus VP1 unique region on tight junction of human airway epithelial A549 cells.

As is widely recognized, human parvovirus B19 (B19) and human bocavirus (HBoV) are important human pathogens. Obviously, both VP1 unique region (VP1u) of B19 and HBoV exhibit the secreted phospholipase A2 (sPLA2)-like enzymatic activity and are recognized to participate in the pathogenesis of lower respiratory tract illnesses. However, exactly how, both VP1u from B19 and HBoV affect tight junction has seldom been addressed. Therefore, this study investigates how B19-VP1u and HBoV-VP1u may affect the tight junction of the airway epithelial A549 cells by examining phospholipase A2 activity and transepithelial electrical resistance (TEER) as well as performing immunoblotting analyses. Experimental results indicate that TEER is more significantly decreased in A549 cells by treatment with TNF-α (10 ng), two dosages of B19-VP1u and BoV-VP1u (400 ng and 4000 ng) or bee venom PLA2 (10 ng) than that of the control. Accordingly, more significantly increased claudin-1 and decreased occludin are detected in A549 cells by treatment with TNF-α or both dosages of HBoV-VP1u than that of the control. Additionally, more significantly decreased Na+/K+ ATPase is observed in A549 cells by treatment with TNF-α, high dosage of B19-VP1u or both dosages of BoV-VP1u than that of the control. Above findings suggest that HBoV-VP1u rather than B19 VP1u likely plays more important roles in the disruption of tight junction in the airway tract. Meanwhile, this discrepancy appears not to be associated with the secreted phospholipase A2 (sPLA2)-like enzymatic activity.

[Construction of recombinant adenoviral vector expressing genes of the conservative influenza proteins M2 and nucleoprotein].

Influenza is a highly contagious and one of the most massive infection diseases. General epidemiological significance has a strain, which belongs to subtype A. A high degree of genetic variety leads to the permanent changes in the antigenic structure of the influenza virus. Therefore, the current influenza vaccines require periodic updating of the composition of strains. Presently, it is important to develop a universal vaccine that can protect against different strains of influenza A virus at the same time and is based on the conserved antigens of the influenza virus. The recombinant adenovirus vectors expressing genes of conserved viral antigenes may be a promising candidate vaccine against influenza A. Using the method of the homologous recombination, we developed in this study recombinant adenovirus of fifth serotype that expresses genes of the ion channel M2 and nucleoprotein NP of the influenza virus A. Genes of the consensus protein M2 and NP of human influenza A virus were included into the structure of the viral genome. The expression of the antigens M2 and NP using recombinant adenovirus vector was detected by a Western blot assay. The immunogenicity of the developed recombinant adenovirus vector was demonstrated by the intranasal immunization of laboratory mice.

[The biological function of auto-induced expression of the hepatitis C virus soluble core protein].

OBJECTIVE: To investigate the biological role of auto-induced expression of hepatitis C virus (HCV) core protein (protein C) using a recombinant protein in an in vitro cell-based system. METHODS: The PCR-amplified full-length HCV protein C gene (573 bp) was inserted into the pET28a prokaryotic expression vector. The recombinant plasmid was transformed into BL21(DE3)pLysS E. coli to achieve high-concentration expression of the recombinant C protein by auto-induction. The recombinant protein C was purified by Ni-NTA affinity chromatography, and tested in a protein binding assay for its ability to bind the HCV NS3 protein. RESULTS: The transformed E. coli produced a large amount of recombinant protein C, as detected in the sonicated supernatant of the bacteria culture. The antigenic reactivity of the recombinant protein C was confirmed by western blotting. However, the recombinant protein C could not be purified by Ni-NTA affinity chromatography, but co-precipitated with the HCV NS3 protein. CONCLUSION: Soluble recombinant protein C was successfully expressed by auto-induction, and shown to interact with the HCV NS3 protein, which provides a novel insight into the putative biological activity of this factor in HCV-related molecular processes. Future studies of this recombinant HCV protein C's crystal structure and antigenicity may provide further clues to its biological function(s) and potential for clinical applications.

Dose- and time-dependent apoptosis induced by avian H9N2 influenza virus in human cells.

To understand human response to avian H9N2 influenza, we investigated the effects of the viral infection on A549, HepG2, and HeLa cells at low and high MOIs. To identify virus-host interplay, expression of Mx and NP genes was measured in the cells supernatants. Cell viability and apoptosis were evaluated by MTT assay, DNA fragmentation, and florescent staining. The virus titration and NP gene transcript levels indicate lower susceptibility of HeLa cell to H9N2 replication than other cells. Although H9N2 did produce a faster CPE in HepG2, high dose of the virus induced apoptosis within early stage of A549 infection. The DNA laddering was enhanced in the cell correlated with increase in virus transcripts. The undetectable to different regulation levels of Mx gene were observed in response to H9N2 infection suggesting that an insufficient antiviral defense in the noncompetent-IFN HepG2 cell promotes efficient viral replication. These results showed that the permissivity of HepG2 for H9N2 is comparable with A549; however, liver cells are not target tissue respond to the infection. These data revealed that the H9N2 virus induced apoptosis signaling via mitochondrial pathway in human alveolar epithelial cells, indicating that the induction may be associated with a dose-dependent manner.

Th1 and Th2 cytokine profiles induced by hepatitis C virus F protein in peripheral blood mononuclear cells from chronic hepatitis C patients.

Th1 and Th2 cytokine response has been confirmed to be correlated with the pathogenesis of HCV infection. The aim of the study is to investigate the Th1 and Th2 cytokine profiles induced by HCV alternate reading frame protein (F protein) in chronic hepatitis C patients. We assessed the immune responses specific to HCV F protein in 55 chronic HCV patients. IFN-γ, IL-2, IL-4 and IL-5 secretion by peripheral blood mononuclear cells (PBMC) post F protein stimulation were compared among HCV patients and healthy donors. Finally, the associations between HCV F protein and HLA class II alleles were explored. We found that the seroprevalence of anti-F antibodies in HCV-related hepatocellular carcinoma (HCC) patients was significantly higher than that of patients without HCC, but such a significant difference in humoral immune responses to F protein was not observed in HCV 1b-infected- and non-HCV 1b-infected-patients. Additionally, the PBMC proliferation of HCC patients was significantly lower than that of patients without HCC. Furthermore, F protein stimulation of PBMCs from F-seropositive patients resulted in Th2 biased cytokine responses (significantly decreased IFN-γ and/or IL-2 and significantly increased IL-4 and/or IL-5 levels) that reportedly may contribute to HCC progression and pathogenesis. However, no significant difference in the association between HCV F protein and HLA-DRB1*0201, 0301, 0405, 1001 and HLA-DQB1*0201, 0401, 0502, 0602 was observed in this study. These findings suggest that F protein may contribute to the HCV-associated bias in Th1/Th2 responses of chronic hepatitis C patients including the progress of HCC pathogenesis.