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Influenza viruses cause considerable morbidity and mortality, whether in the context of annual epidemics, sporadic pandemics, or outbreaks of avian influenza virus. For hepatitis C virus (HCV), an estimated 170 million people are chronically infected worldwide. These individuals are at high risk of developing progressive liver injury or hepatocellular carcinoma. Since the efficacy of currently approved antiviral drugs is threatened by emerging viral resistance and the cost remains high, new antiviral drugs are still required. By utilizing a structure-based approach, novel substituted indole-flutimide heterocyclic derivatives (1,2-annulated indolediketopiperazines) were rationally designed, synthesized and evaluated as influenza PA endonuclease inhibitors. The compounds were also tested for their antiviral effect against HCV. All N-hydroxyimides were potent PA endonuclease inhibitors while displaying low cytotoxicity. Compound 6 proved to be the most active analogue, while the most favorable indole substitution was fluorine at position 8 (compound 18). The chloro-derivative 24 showed additional potent anti-HCV activity and exhibited remarkable selectivity (>19). In accordance with the SAR data, removal of the hydroxyl group from the imidic nitrogen (compound 26) caused a complete loss of activity against influenza PA endonuclease as well as HCV. © The Royal Society of Chemistry 2016.

Low oxygen tension exerts a significant effect on the replication of several DNA and RNA viruses in cultured cells. In vitro propagation of hepatitis C virus (HCV) has thus far been studied under atmospheric oxygen levels despite the fact that the liver tissue microenvironment is hypoxic. In this study, we investigated the efficiency of HCV production in actively dividing or differentiating human hepatoma cells cultured under low or atmospheric oxygen tensions. By using both HCV replicons and infection-based assays, low oxygen was found to enhance HCV RNA replication whereas virus entry and RNA translation were not affected. Hypoxia signaling pathway-focused DNA microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) analyses revealed an upregulation of genes related to hypoxic stress, glycolytic metabolism, cell growth, and proliferation when cells were kept under low (3% [vol/vol]) oxygen tension, likely reflecting cell adaptation to anaerobic conditions. Interestingly, hypoxia-mediated enhancement of HCV replication correlated directly with the increase in anaerobic glycolysis and creatine kinase B (CKB) activity that leads to elevated ATP production. Surprisingly, activation of hypoxia-inducible factor alpha (HIF-alpha) was not involved in the elevation of HCV replication. Instead, a number of oncogenes known to be associated with glycolysis were upregulated and evidence that these oncogenes contribute to hypoxia-mediated enhancement of HCV replication was obtained. Finally, in liver biopsy specimens of HCV-infected patients, the levels of hypoxia and anaerobic metabolism markers correlated with HCV RNA levels. These results provide new insights into the impact of oxygen tension on the intricate HCV-host cell interaction.

Hepatitis C virus (HCV) infection is a major cause for chronic liver disease and hepatocellular carcinoma. The HCV-ARF/core+1 protein is an alternative product of HCV core-encoding sequence of unknown biological function. Highly purified HCV core and ARF/core+1 recombinant proteins from HCV genotype 1a and HCV-ARF/core+1 recombinant protein from HCV genotype 3a were expressed in Escherichia coli. Using an enzyme-linked immunosorbent assay, we assessed the prevalence of anti-ARF/core+1 antibodies in 90 chronic hepatitis C patients infected with HCV genotypes 1a/1b or 3a, treated with pegylated interferon (Peg-IFN-a-2a) plus ribavirin. Samples derived from 92 healthy blood donors were used as negative controls. All HCV-RNA-positive serum samples reacted with core 1a antigen, while 15 (37.5%) of 40 and 14 (28%) of 50 patients infected with HCV-1a/1b and HCV-3a, respectively, were found to have anti-ARF/core+1 antibodies into their serum before treatment initiation. These antibodies were persistently present during treatment follow-up and linked to elevated levels of HCV-RNA at baseline. © 2011 Blackwell Publishing Ltd.

The core region of the hepatitis C virus (HCV) genome possesses an overlapping ORF that has been shown to encode a protein, known as the alternate reading frame protein (ARFP), F or core+1. The biological role of this protein remains elusive, as it appears to be non-essential for virus replication. However, a number of independent studies have shown that the ARFP/F/core+1 protein elicits humoral and cellular immune responses in HCV-infected individuals and interacts with important cellular proteins. To assess the significance of the core+1 humoral response in HCV-infected patients, we examined the prevalence of anti-core+1 antibodies in sera from patients with hepatocellular carcinoma (HCC) in comparison with chronically HCV-infected individuals without HCC. We produced two HCV core+1 histidine-tagged recombinant proteins for genotypes la (aa 11-160) and 1b (aa 11-144), as well as a non-tagged highly purified recombinant core+1/S protein (aa 85-144) of HCV-1b. Using an in-house ELISA, we tested the prevalence of core+1 antibodies in 45 patients with HCC in comparison with 47 chronically HCV-infected patients without HCC and 77 negative-control sera. More than 50% of the serum samples from HCC patients reacted with all core+1 antigens, whereas <26% of the sera from the non-HCC HCV-infected individuals tested positive. No core+1-specific reactivity was detected in any of the control samples. In conclusion, the high occurrence of anti-core+1 antibodies in the serum of HCC patients suggests a role for the ARFP/F/core+1 protein in the pathogenesis of HCC.

The aim of this study was to evaluate the possible association of human papillomaviruses (HPV) with the development of squamous cell lung carcinomas (SqCLCs). Tissue material from 52 cases of SqCLCs were studied, and the data were evaluated according to the degree of differentiation, HPV presence and type. Analysis was performed by polymerase chain reaction (PCR) method using consensus primers, and the results were confirmed by subsequent Southern blot hybridization. Overall, the results showed 69% positivity (n=32). Forty-one cases were examined for the presence of specific HPV types (6/11 and 16/18) by hybridization of the PCR products with P-32-labelled probes. HPV 6/11 types were detected in 6 of the 29 positive cases (20.6%). HPV 16/18 types were the most prevalent types, and were detected in 11/29 cases (37.9%: 4/10 of well-differentiated cases, 6/25 of moderately and 1/6 of poorly differentiated carcinomas). Our results confirm the possibility that HPV might play a role in the development of SqCLCs and suggest a possible relation of high-risk HPV16/18 types to tumour differentiation.

Background and Objectives: More than 30 different human papillomavirus (HPV) types infect the anogenital mucosa and are responsible for a variety of benign, premalignant, and malignant lesions including cervical cancer, The goal of this study was to determine the distribution of individual HPV types in various grades of cervical precancerous lesions cinema in patients from Greece, Study Design: Specimens were analyzed for HPV-DNA sequences by polymerase chain reaction and Southern blot hybridization, Polymerase chain reaction analysis was performed with consensus and type-specific primers. Restriction length fragment polymorphism analysis and/or hybridization of the general primer polymerase chain reaction product were used for HPV typing. Results: In cervical carcinomas HPV-16 was found in 56%, HPV-18 in 23%,and HPV-31 in 6% of the HPV-positive patients, In precancerous lesions HPV-16 was found in 13% of low-grade squamous intraepithelial lesions (LG-SIL) as compared with 41% of high-grade squamous intraepithelial lesions (LG-SIL) patients, HPV-18 was found at similar frequency both in LG-SIL (13%) and HG-SIL (14%), HPV-31 and HPV-33 were detected at moderate levels both in LG-SIL (11%) and in HG-SIL (14%). In addition, HPV-53 and HPV-66 were detected at low frequency in LG-SIL (2%), whereas HPV-51 was found only in HG-SIL (4%), Finally, HPV-6 was associated with 13% of LG-SIL. Conclusions: Overall, the prevalence rate of the genital HPV types was in the range previously described for many western countries but the HPV-18 positivity was higher than that reported for most European countries.

Fifty biopsies from high-grade squamous intraepithelial lesions (HG-SIL) and 14 cervical carcinoma biopsies from Greek women were screened for the presence of human papillomavirus (HPV) DNA sequences by Southern blot hybridization and by the polymerase chain reaction (PCR) for the presence of different HPV types. In high-grade SIL, HPV DNA sequences were detected in 44 of 50 biopsies with the following distribution: 36% HPV 16, 12% HPV 18, 6% HPV 31, 6% HPV 33, 4% HPV 51, and 24% unclassified HPV types. In cervical carcinoma biopsies, 13 of 14 specimens were positive for HPV DNA sequences. Six biopsies were positive for HPV 16, three were positive for HPV 18, and four contained unclassified HPV types. Overall, of the total 64 biopsies, 57 (89%) were positive for HPV DNA sequences. Of these, Southern blot hybridization alone detected HPV DNA sequences in 39 cases, whereas by PCR 18 additional specimens were found to be positive for HPV. Among the HPV 16-positive biopsies, two samples produced a Pstl banding pattern very similar but not identical to that of HPV 16 prototype and were referred to as HPV 16 isolates. One HPV 16 isolate appears to carry a mutation within the carboxy-terminal half of the L2 gene that results in the loss of a Pstl site. The other HPV 16 isolate had a similar Pstl banding pattern to that previously reported as HPV 16 'variant' found in Cape Town [Williamson et al., 1989, Journal of Medical Virology 28:146-149, 1994, Journal of Medical Virology 43:231-237.] and in Italy [Li Vigni et al., 1994, 2nd international Congress of Papillomavirus in Human Pathology (Abstracts), p 100.].