The emergence of highly contagious influenza A virus strains, such as the new H1N1 swine influenza, represents a serious threat to global human health. Efforts to control emerging influenza strains focus on surveillance and early diagnosis, as well as development of effective vaccines and novel antiviral drugs. Herein we document the anti-influenza activity of the anti-infective drug nitazoxanide and its active circulating-metabolite tizoxanide and describe a class of second generation thiazolides effective against influenza A virus. Thiazolides inhibit the replication of H1N1 and different other strains of influenza A virus by a novel mechanism: they act at post-translational level by selectively blocking the maturation of the viral hemagglutinin at a stage preceding resistance to endoglycosidase H digestion, thus impairing hemagglutinin intracellular trafficking and insertion into the host plasma membrane, a key step for correct assembly and exit of the virus from the host cell. Targeting the maturation of the viral glycoprotein offers the opportunity to disrupt the production of infectious viral particles attacking the pathogen at a level different from the currently available anti-influenza drugs. The results indicate that thiazolides may represent a new class of antiviral drugs effective against influenza A infection.

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dRotaviruses, nonenveloped viruses presenting a distinctive triple-layered particle architecture enclosing a segmented doublestranded RNA genome, exhibit a unique morphogenetic pathway requiring the formation of cytoplasmic inclusion bodies called viroplasms in a process involving the nonstructural viral proteins NSP5 and NSP2. In these structures the concerted packaging and replication of the 11 positive-polarity single-stranded RNAs take place to generate the viral double-stranded RNA (dsRNA) genomic segments. Rotavirus infection is a leading cause of gastroenteritis-associated severe morbidity and mortality in young children, but no effective antiviral therapy exists. Herein we investigate the antirotaviral activity of the thiazolide anti-infective nitazoxanide and reveal a novel mechanism by which thiazolides act against rotaviruses. Nitazoxanide and its active circulating metabolite, tizoxanide, inhibit simian A/SA11-G3P[2] and human Wa-G1P[8] rotavirus replication in different types of cells with 50% effective concentrations (EC 50 s) ranging from 0.3 to 2 g/ml and 50% cytotoxic concentrations (CC 50 s) higher than 50 g/ml. Thiazolides do not affect virus infectivity, binding, or entry into target cells and do not cause a general inhibition of viral protein expression, whereas they reduce the size and alter the architecture of viroplasms, decreasing rotavirus dsRNA formation. As revealed by protein/protein interaction analysis, confocal immunofluorescence microscopy, and viroplasm-like structure formation analysis, thiazolides act by hindering the interaction between the nonstructural proteins NSP5 and NSP2. Altogether the results indicate that thiazolides inhibit rotavirus replication by interfering with viral morphogenesis and may represent a novel class of antiviral drugs effective against rotavirus gastroenteritis. R otaviruses are complex nonenveloped viruses belonging to the Reoviridae family. The rotavirion has a distinctive triple-layered particle (TLP) architecture that surrounds a genome composed of 11 segments of double-stranded RNA (dsRNA) encoding six structural viral proteins (VPs) and six nonstructural proteins (NSPs) (1, 2). The capsid structure comprises an inner-core shell of VP2 dimers and an intermediate shell formed by trimers of the major structural protein VP6, which interacts with both the VP2 core protein and the outer shell constituted by the VP4 protein (the rotavirus spikes, which express P-serotype epitopes), and VP7 glycoprotein trimers, which express G-serotype epitopes (2). The P and G serotypes represent independently segregating neutralization epitopes imparting immunity to infection. VP7, which is the second most abundant protein in the virion, is cotranslationally glycosylated as it is inserted into the endoplasmic reticulum (ER) membrane via a cleavable signal sequence found at the N terminus of the protein (1, 2). Rotaviruses exhibit a unique morphogenetic pathway. Double-layered particles (DLPs) are assembled in the cytoplasm at special areas termed viroplasms and the...

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