Contribution to pathogenesis of accessory proteins of deadly human coronaviruses.

Coronaviruses (CoVs) are enveloped and positive-stranded RNA viruses with a large genome (∼ 30kb). CoVs include essential genes, such as the replicase and four genes coding for structural proteins (S, M, N and E), and genes encoding accessory proteins, which are variable in number, sequence and function among different CoVs. Accessory proteins are non-essential for virus replication, but are frequently involved in virus-host interactions associated with virulence. The scientific literature on CoV accessory proteins includes information analyzing the effect of deleting or mutating accessory genes in the context of viral infection, which requires the engineering of CoV genomes using reverse genetics systems. However, a considerable number of publications analyze gene function by overexpressing the protein in the absence of other viral proteins. This ectopic expression provides relevant information, although does not acknowledge the complex interplay of proteins during virus infection. A critical review of the literature may be helpful to interpret apparent discrepancies in the conclusions obtained by different experimental approaches. This review summarizes the current knowledge on human CoV accessory proteins, with an emphasis on their contribution to virus-host interactions and pathogenesis. This knowledge may help the search for antiviral drugs and vaccine development, still needed for some highly pathogenic human CoVs.

Viricidal Activity of Thermoplastic Polyurethane Materials with Silver Nanoparticles.

The use of diverse Ag-based nanoparticulated forms has shown promising results in controlling viral propagation. In this study, a commercial nanomaterial consisting of ceramic-coated silver nanoparticles (AgNPs) was incorporated into thermoplastic polyurethane (TPU) plates using an industrial protocol, and the surface composition, ion-release dynamics and viricidal properties were studied. The surface characterization by FESEM-EDX revealed that the molar composition of the ceramic material was 5.5 P:3.3 Mg:Al and facilitated the identification of the embedded AgNPs (54.4 ± 24.9 nm). As determined by ICPMS, the release rates from the AgNP-TPU into aqueous solvents were 4 ppm/h for Ag and Al, and 28.4 ppm/h for Mg ions. Regarding the biological assays, the AgNP-TPU material did not induce significant cytotoxicity in the cell lines employed. Its viricidal activity was characterized, based on ISO 21702:2019, using the Spring viraemia of carp virus (SVCV), and then tested against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The results demonstrated that AgNP-TPU materials exhibited significant (75%) and direct antiviral activity against SVCV virions in a time- and temperature-dependent manner. Similar inhibition levels were found against SARS-CoV-2. These findings show the potential of AgNP-TPU-based materials as a supporting strategy to control viral spread.

Pulsed-Xenon Ultraviolet Light Highly Inactivates Human Coronaviruses on Solid Surfaces, Particularly SARS-CoV-2.

In the context of ongoing and future pandemics, non-pharmaceutical interventions are critical in reducing viral infections and the emergence of new antigenic variants while the population reaches immunity to limit viral transmission. This study provides information on efficient and fast methods of disinfecting surfaces contaminated with different human coronaviruses (CoVs) in healthcare settings. The ability to disinfect three different human coronaviruses (HCoV-229E, MERS-CoV, and SARS-CoV-2) on dried surfaces with light was determined for a fully characterized pulsed-xenon ultraviolet (PX-UV) source. Thereafter, the effectiveness of this treatment to inactivate SARS-CoV-2 was compared to that of conventional low-pressure mercury UVC lamps by using equivalent irradiances of UVC wavelengths. Under the experimental conditions of this research, PX-UV light completely inactivated the CoVs tested on solid surfaces since the infectivity of the three CoVs was reduced up to 4 orders of magnitude by PX-UV irradiation, with a cumulated dose of as much as 21.162 mJ/cm2 when considering all UV wavelengths (5.402 mJ/cm2 of just UVC light). Furthermore, continuous irradiation with UVC light was less efficient in inactivating SARS-CoV-2 than treatment with PX-UV light. Therefore, PX-UV light postulates as a promising decontamination measure to tackle the propagation of future outbreaks of CoVs.

MERS-CoV ORF4b is a virulence factor involved in the inflammatory pathology induced in the lungs of mice.

No vaccines or specific antiviral drugs are authorized against Middle East respiratory syndrome coronavirus (MERS-CoV) despite its high mortality rate and prevalence in dromedary camels. Since 2012, MERS-CoV has been causing sporadic zoonotic infections in humans, which poses a risk of genetic evolution to become a pandemic virus. MERS-CoV genome encodes five accessory proteins, 3, 4a, 4b, 5 and 8b for which limited information is available in the context of infection. This work describes 4b as a virulence factor in vivo, since the deletion mutant of a mouse-adapted MERS-CoV-Δ4b (MERS-CoV-MA-Δ4b) was completely attenuated in a humanized DPP4 knock-in mouse model, resulting in no mortality. Attenuation in the absence of 4b was associated with a significant reduction in lung pathology and chemokine expression levels at 4 and 6 days post-infection, suggesting that 4b contributed to the induction of lung inflammatory pathology. The accumulation of 4b in the nucleus in vivo was not relevant to virulence, since deletion of its nuclear localization signal led to 100% mortality. Interestingly, the presence of 4b protein was found to regulate autophagy in the lungs of mice, leading to upregulation of BECN1, ATG3 and LC3A mRNA. Further analysis in MRC-5 cell line showed that, in the context of infection, MERS-CoV-MA 4b inhibited autophagy, as confirmed by the increase of p62 and the decrease of ULK1 protein levels, either by direct or indirect mechanisms. Together, these results correlated autophagy activation in the absence of 4b with downregulation of a pathogenic inflammatory response, thus contributing to attenuation of MERS-CoV-MA-Δ4b.

Nature of viruses and pandemics: Coronaviruses.

Coronaviruses (CoVs) have the largest genome among RNA viruses and store large amounts of information without genome integration as they replicate in the cell cytoplasm. The replication of the virus is a continuous process, whereas the transcription of the subgenomic mRNAs is a discontinuous one, involving a template switch, which resembles a high frequency recombination mechanism that may favor virus genome variability. The origin of the three deadly human CoVs SARS-CoV, MERS-CoV and SARS-CoV-2 are zoonotic events. SARS-CoV-2 has incorporated in its spike protein a furine proteolytic site that facilitates the activation of the virus in any tissue, making this CoV strain highly polytropic and pathogenic. Using MERS-CoV as a model, a propagation-deficient RNA replicon was generated by removing E protein gene (essential for viral morphogenesis and involved in virulence), and accessory genes 3, 4a, 4b and 5 (responsible for antagonism of the innate immune response) to attenuate the virus: MERS-CoV-Δ[3,4a,4b,5,E]. This RNA replicon is strongly attenuated and elicits sterilizing protection after a single immunization in transgenic mice with the receptor for MERS-CoV, making it a promising vaccine candidate for this virus and an interesting platform for vector-based vaccine development. A strategy could be developed for the design of RNA replicon vaccines for other human pathogenic coronaviruses.

Cross-neutralization activity against SARS-CoV-2 is present in currently available intravenous immunoglobulins.

Background: Cross-reactivity against human coronaviruses with Flebogamma® DIF and Gamunex®-C, two available intravenous immunoglobulins (IVIG), has been reported. In this study, these IVIG were tested for neutralization activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV and Middle East respiratory syndrome CoV (MERS-CoV). Materials & methods: Neutralization capacity of lots of IVIG manufactured prior to COVID-19 pandemic was assessed against these viruses in cell culture. Infectivity neutralization was quantified by percent reduction in plaque-forming units and/or cytopathic/cytotoxic methods. Results: All IVIG preparations showed neutralization of SARS-CoV-2 isolates. All IVIG lots produced neutralization of SARS-CoV. No IVIG preparation showed significant neutralizing activity against MERS-CoV. Conclusion: The tested IVIG contain antibodies with significant in vitro cross-neutralization capacity against SARS-CoV-2 and SARS-CoV, but not MERS-CoV. These preparations are currently under evaluation as potential therapies for COVID-19.

Canonical and Noncanonical Autophagy as Potential Targets for COVID-19.

The SARS-CoV-2 pandemic necessitates a review of the molecular mechanisms underlying cellular infection by coronaviruses, in order to identify potential therapeutic targets against the associated new disease (COVID-19). Previous studies on its counterparts prove a complex and concomitant interaction between coronaviruses and autophagy. The precise manipulation of this pathway allows these viruses to exploit the autophagy molecular machinery while avoiding its protective apoptotic drift and cellular innate immune responses. In turn, the maneuverability margins of such hijacking appear to be so narrow that the modulation of the autophagy, regardless of whether using inducers or inhibitors (many of which are FDA-approved for the treatment of other diseases), is usually detrimental to viral replication, including SARS-CoV-2. Recent discoveries indicate that these interactions stretch into the still poorly explored noncanonical autophagy pathway, which might play a substantial role in coronavirus replication. Still, some potential therapeutic targets within this pathway, such as RAB9 and its interacting proteins, look promising considering current knowledge. Thus, the combinatory treatment of COVID-19 with drugs affecting both canonical and noncanonical autophagy pathways may be a turning point in the fight against this and other viral infections, which may also imply beneficial prospects of long-term protection.