SARS-CoV-2, like other coronaviruses, builds a membrane-bound replication organelle to enable RNA replication 1 . The SARS-CoV-2 replication organelle is composed of double-membrane vesicles (DMVs) that are tethered to the endoplasmic reticulum (ER) by thin membrane connectors 2 , but the viral proteins and the host factors involved remain unknown. Here we identify the viral non-structural proteins (NSPs) that generate the SARS-CoV-2 replication organelle. NSP3 and NSP4 generate the DMVs, whereas NSP6, through oligomerization and an amphipathic helix, zippers ER membranes and establishes the connectors. The NSP6(ΔSGF) mutant, which arose independently in the Alpha, Beta, Gamma, Eta, Iota and Lambda variants of SARS-CoV-2, behaves as a gain-of-function mutant with a higher ER-zippering activity. We identified three main roles for NSP6: first, to act as a filter in communication between the replication organelle and the ER, by allowing lipid flow but restricting the access of ER luminal proteins to the DMVs; second, to position and organize DMV clusters; and third, to mediate contact with lipid droplets (LDs) through the LD-tethering complex DFCP1-RAB18. NSP6 thus acts as an organizer of DMV clusters and can provide a selective means of refurbishing them with LD-derived lipids. Notably, both properly formed NSP6 connectors and LDs are required for the replication of SARS-CoV-2. Our findings provide insight into the biological activity of NSP6 of SARS-CoV-2 and of other coronaviruses, and have the potential to fuel the search for broad antiviral agents. SARS-CoV-2 extensively rearranges host cellular membranes into replication organelles that provide a microenvironment conducive to RNA synthesis and protection from host sensor and defence systems 1,2 . The 16 viral NSPs that are released from polyproteins pp1a and pp1ab by 2 viral proteases include 13 cytosolic proteins, which are involved in RNA replication, and 3 transmembrane proteins, NSP3, NSP4 and NSP6. Studies on other coronaviruses suggest that NSP3 and NSP4, with a hitherto undefined contribution from NSP6, are responsible for generating the replication organelles 3-6 . Despite considerable advances in our understanding of the ultrastructure of the SARS-CoV-2 replication organelle 2,7,8 , mechanistic insights into its biogenesis have so far been limited. In particular, there is at present-to our knowledge-no information on the role of NSP6 in this process. Of note, six SARS-CoV-2 'variants of concern' (VOCs) (Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Eta (B.1.525), Iota (B.1.526) 9 and Lambda (C.37) 10 ) share a three-amino-acid deletion in NSP6 (NSP6(ΔSGF)), in addition to the more noted mutations in the spike protein; this finding adds further impetus to the need to examine the role of NSP6 in the biogenesis of replication organelles and in the replication of SARS-CoV-2. NSP6 induces ER zipperingWe tagged SARS-CoV-2 NSP6 at either the N or the C terminus. C-terminally tagged NSP6 showed a diffuse distribution in the ER (Fig. 1a and Extended Data Fig. ...

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An inverted pyramidal metasurface was designed, fabricated, and studied at the nanoscale level for the development of a label-free pathogen detection on a chip platform that merges nanotechnology and surface-enhanced Raman scattering (SERS). Based on the integration and synergy of these ingredients, a virus immunoassay was proposed as a relevant proof of concept for very sensitive detection of hepatitis A virus, for the first time to our best knowledge, in a very small volume (2 μL), without complex signal amplification, allowing to detect a minimal virus concentration of 13 pg/mL. The proposed work aims to develop a high-flux and high-accuracy surface-enhanced Raman spectroscopy (SERS) nanobiosensor for the detection of pathogens to provide an effective method for early and easy water monitoring, which can be fast and convenient.

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SummaryHepatitis E virus (HEV) is a zoonotic pathogen with a worldwide distribution, and infects several mammalian species, including pigs and wild boars, which are recognized as its natural reservoirs. The virus causes a usually self-limiting liver disease with a mortality rate generally below 1%, although mortality rates of 15%-25% have been recorded in pregnant woman. Chronic infections can also occur. The prevalence of HEV has been extensively studied in wild boars and pigs in northern Italy, where intensive pig herds are predominantly located. In contrast, few data have been collected in south-central Italy, where small pig herds are surrounded by large regional parks populated with heterogeneous wild fauna. In this study, 291 liver samples from wild boars caught in south-central Italy were analysed with the molecular detection of viral RNA. Our results confirm the circulation of HEV in these animals, with a mean prevalence of 13.7% (40 of 291). A nucleotide sequence analysis showed that the HEV strains were highly conserved within the same geographic areas. The wild boar HEV strains belonged to the HEV-3c subtype, which is frequently described in wild boars, and to an uncommon undefined subtype (HEV-3j-like).The viral prevalence detected is concerning because it could represent a potential risk to hunters, meat workers and consumers of wild boar liver and derivative products. The hypothesized inter-species transmission of HEV to pigs and the possibility that the virus maintains its virulence in the environment and the meat chain also present potential risks to human health, and warrant further investigations in the near future.