No abstract

Qβ phages infect Escherichia coli in the human gut by recognizing F-pili as receptors. Infection therefore occurs under reducing conditions induced by physiological agents (e.g. glutathione) or the intestinal bacterial flora. After excretion in the environment, phage particles are exposed to oxidizing conditions and sometimes disinfection. If inactivation does not occur, the phage may infect new hosts in the human gut through the oral route. During such a life cycle, we demonstrated that, outside the human gut, cysteines of the major protein capsid of Qβ phage form disulfide bonds. Disinfection with NaClO does not allow overoxidation to occur. Such oxidation induces inactivation rather by irreversible damage to the minor proteins. In the presence of glutathione, most disulfide bonds are reduced, which slightly increases the capacity of the phage to infect E. coli in vitro Such reduction is reversible and barely alters infectivity of the phage. Reduction of all disulfide bonds by dithiothreitol leads to complete capsid destabilization. These data provide new insights into how the phages are impacted by oxidizing-reducing conditions outside their host cell and raises the possibility of the intervention of the redox during life cycle of the phage.

Pathogenic enteric viruses and bacteriophages such as Qβ and MS2 are transmitted through the fecal-oral route. However, oxidants such as peroxynitrite (ONOOH) and hypochlorous acid (HClO) can prevent new infection by inactivating infectious viruses. Their virucidal effect is well recognized, and yet predicting the effects of oxidants on viruses is currently impossible because the detailed mechanisms of viral inactivation remain unclear. Our data show that ONOOH and HClO cross-linked the capsid proteins and RNA genomes of Qβ and MS2 phages. Consistently, the capsids appeared intact by transmission electron microscopy (TEM) even when 99% of the phages were inactivated by oxidation. Moreover, a precise molecular study of the capsid proteins shows that ONOOH and HClO preferentially targeted capsid protein regions containing the oxidantsensitive amino acid C, Y, or W. Interestingly, the interaction of these amino acids was a crucial parameter defining whether they would be modified by the addition of O, Cl, or NO 2 or whether it induced the loss of the protein region detected by mass spectrometry, together suggesting potential sites for cross-link formation. Together, these data show that HClO and ONOOH consistently target oxidant-sensitive amino acids regardless of the structural organization of Qβ and MS2, even though the phenotypes change as a function of the interaction with adjacent proteins/RNA. These data also indicate a potential novel mechanism of viral inactivation in which cross-linking may impair infectivity.

Human noroviruses (HuNoVs) are the leading cause of viral foodborne outbreaks worldwide. To date, no available methods can be routinely used to detect infectious HuNoVs in foodstuffs. HuNoVs recognize Histo-Blood Group Antigens (HBGAs) through the binding pocket (BP) of capsid protein VP1, which promotes infection in the host cell. In this context, the suitability of human HBGA-binding assays to evaluate the BP integrity of HuNoVs was studied on GII.4 virus-like particles (VLPs) and GII.4 HuNoVs during natural ageing at 20 °C and heat treatments. Our results demonstrate that this approach may reduce the over-estimation of potential infectious HuNoVs resulting from solely using the genome detection, even though some limitations have been identified. The specificity of HBGA-binding to the BP is clearly dependent on the HGBA type (as previously evidenced) and the ionic strength of the media without disturbing such interactions. This study also provides new arguments regarding the ability of VLPs to mimic HuNoV behavior during inactivation treatments. The BP stability of VLPs was at least 4.3 fold lower than that of HuNoVs at 20 °C, whereas capsids of both particles were disrupted at 72 °C. Thus, VLPs are relevant surrogates of HuNoVs for inactivation treatments inducing significant changes in the capsid structure.