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Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

Little is known about coinfection among several hepatitis B virus (HBV) genotypes, although previous reports of recombination and genotype shifts indicate that this should occur. In the present study, we designed a method to identify mixtures of genotype A and another genotype, regardless of whether one of them predominates. Using this method, signs of genotypic coinfection were found in 20 (67%) of 30 hepatitis B e antigen-positive patients treated with interferon (IFN). In 8 of these patients, coinfection or genotype shifts were detectable by direct sequencing or standard preS genotyping. In most of these cases, genotype changes were detected after a >2-log decrease or increase of the HBV DNA level. The presence of genotype mixtures did not significantly influence IFN response. Because quasi-species selection may occur at or shortly after transmission, patients might acquire HBV infection from subjects who appear to be infected with a different genotype. This should be considered when tracing the source of HBV infection.

Therapy for chronic hepatitis B with interferon-alpha (IFN) may result in viral clearance and hepatitis B e seroconversion in 30-40% of patients. It is still unclear whether viral genetic variability influences response rates. However, certain core promoter mutations were recently associated with a better response to IFN. In the present study, the entire X region, including the core promoter, of hepatitis B virus (HBV) from 26 HBeAg-positive patients treated with IFN for 12 weeks, was sequenced. Serum samples pre-treatment, at end-of-treatment, and at follow-up of 18 sustained and 8 nonsustained responders were analyzed. Most patients were of European origin and had moderate aminotransferase elevation (mean 2.4 x upper limit of normal) and genotype A infection. Before treatment, 16 patients had an X region identical to a consensus sequence of the corresponding genotype; in the remaining 10 patients, a median of 1.5 mutations were found. After treatment, 1-4 new mutations (mean 1.8) had emerged in 5 patients. There was no association between specific mutations, or the number of mutations, and response to IFN. The low frequency of mutations indicates that analysis of this region is of limited clinical value and that emerging mutations in this region are not major determinants of response to treatment with IFN-alpha., (Copyright 2002 Wiley-Liss, Inc.)

Abstract: Hepatitis B virus infected patients on long-term lamivudine treatment are exposed to a 15–32% risk compounded annually of developing resistance mutations. Such resistance results in a progression of the liver damage caused by chronic hepatitis B, and may also impair the effect of other antivirals through cross-resistance. At present lamivudine is used frequently as monotherapy because of its relatively low price and negligible side effects. Thus, simple methods for identifying resistance mutations are required. A method based on real-time polymerase chain reaction with TaqMan chemistry is described. The method combines both primer specificity, in order to target wild type and mutant viral strains at codon 180, and a mixture of three minor groove binding probes distinguishing the YMDD wild type and the YVDD and YIDD variants at codon 204. The accuracy of the method was verified by concordance with results of direct sequencing and restriction fragment length polymorphism when examining 27 samples from five patients, in whom lamivudine resistance was known to have developed. This method is rapid, cost effective, and should prove useful for monitoring patients treated with lamivudine. [Copyright &y& Elsevier]

Describes two case studies in which hepatitis B antibodies-positive patients experienced a sudden increase of hepatitis B virus replication during highly active antiretroviral therapy. Medical history of the patients; Clinical and virological course of patients; Treatment of hepatitis B reactivation.

Except for few highly pathogenic viruses, no antiviral drug has been approved for treatment of viral infections in humans. Plant extracts, selected based on their ethno-medical use, represent an important source of compounds for the development of novel candidate antiviral drugs. This especially concerns plants with ethnomedical records on their use in treatment of viral infections. To identify and document medicinal plants used by traditional health practitioners (THPs) for treatment of respiratory infections and muco-cutaneous lesions in order to study their antiviral activity including identification of active components and elucidation of mode of antiviral activity. The ethno-medical survey was performed in the Kagera region of Tanzania. The THPs were asked for plants used for treatment of signs and symptoms of respiratory infections and watery muco-cutaneous blisters in oral and genital regions. The plants identified were successively extracted with n -hexane, ethyl acetate and water, and the extracts assayed for anti-respiratory syncytial virus (RSV), anti-herpes simplex virus 2 (HSV-2), and anti-human parainfluenza virus 2 (HPIV-2) activity in cultured cells. Antiviral components were separated by ethanol precipitation and CL-6B chromatography, and the mode of antiviral activity elucidated by the time-of-addition assay and selection for the virus variants resistant to antiviral plant extract. THPs identified fifteen plants used for treatment of respiratory infections and muco-cutaneous blisters. The water extract, but not n- hexane or ethyl acetate extracts, of six of these plants including Erythrina abyssinica stem bark, inhibited infectivity of two glycosaminoglycan-binding viruses i.e., RSV and HSV-2 but not the sialic acid binding HPIV-2. An activity-guided separation revealed that antiviral component(s) of water extract of E. abyssinica could be precipitated with ethanol. This sample potently and selectively inhibited RSV and HSV-2 infectivity in cultured cells with IC50 values of 2.1 μg/ml (selectivity index >476) and 0.14 μg/ml (selectivity index >7143) respectively. The sample exhibited inhibitory effect on the virus attachment to and entry into the cells by directly targeting the viral particles. Indeed, 10 consecutive virus passages in HEp-2 cells in the presence of this extract selected for a resistant RSV variant lacking the attachment, viral membrane-associated, G protein due to a stop codon at amino acid residue 33 (Leu33stop). Fractionation of the E. abyssinica extract on a CL-6B column revealed that anti-RSV and HSV-2 activity correlated with carbohydrate content. The most pronounced antiviral activity was associated with a carbohydrate containing ingredient of molecular mass of <5 kDa, which may polymerize to antiviral composites of up to 410 kDa. Altogether, the water extract of six medicinal plants showed anti-RSV and anti-HSV-2 activities. Extended studies of the stem bark of E. abyssinica identified antiviral components that potently and selectively inhibited infectivity of free RSV and HSV-2 particles, a feature of importance in topical treatment of these infections. This observation confirms ethno-medical information concerning the use of E. abyssinica extract for treatment of respiratory infections and herpetic lesions. [Display omitted] • Ethnomedical survey in Kagera region, Tanzania, identified 15 plants used for respiratory ailments and herpetic lesions. • Water extract of 6 plants exhibited potent anti-RSV and anti-HSV-2 activity while showing little or no cytotoxicity. • Carbohydrate-containing antiviral components affected free viral particles and selected for RSV deficient in G protein. [ABSTRACT FROM AUTHOR]