A tiny viral protein, SARS-CoV-2-ORF7b: Functional molecular mechanisms. (preprint)

This study presents the interaction with human host metabolism of SARS-CoV-2 ORF7b protein (43 aa), using a Protein-Protein-Interaction Networks analysis. After pruning, we selected from BioGRID the 51 most significant proteins among 2,753 proven interactions and 1,708 interactors, specific to ORF7b. We used these proteins as functional seeds and we got a significant network of 551 nodes via STRING. We performed topological analysis and calculated topological distributions by Cytoscape. Seven high ranked proteins as hub and seven as bottleneck following a hub-and-spoke network-architectural-model were found. Through this interaction model, we identified significant GO-processes (5,057 terms in 15 categories) induced in human metabolism by ORF7b. High statistical significance processes of dysregulated molecular cell mechanisms by the action of ORF7b were discovered. We detected disease-related human proteins and their involvement in metabolic roles, how they relate in a distorted way to signaling and/or functional systems, in particular intra- and inter-cellular signaling systems and the molecular mechanisms that supervise to programmed cell death, with mechanisms similar to that of cancer metastasis diffusion. A cluster analysis showed 10 compact and significant functional clusters, where two of them overlap in a Giant-Connected-Components core of 206 total nodes. These two clusters contain most of the high-rank nodes. ORF7b mainly acts through these two clusters, inducing most of the metabolic dysregulation. We conducted a co-regulation, transcriptional analysis by hub and bottleneck proteins. This analysis allowed us to define the transcription factors and miRNAs that control the high-ranking proteins and the dysregulated processes, within the limits of the poor knowledge that these sectors still impose.

Targeting ATP2B1 impairs PI3K/Akt/Fox-O3 signaling and reduces SARS-COV-2 replication in vivo. (preprint)

ATP2B1 is a known regulator of calcium (Ca2+) cellular export and homeostasis. Diminished levels of extra- or intra-cellular Ca2+ content have been suggested to block SARS-CoV-2 replication. Here, we demonstrate that a newly nontoxic caloxin-derivative compound (PI-7) inhibits ATP2B1, reduces the extra- and intra-cellular Ca2+ levels and impairs SARS-CoV-2 replication and propagation (VOCs: Delta and Omicron 2), as also measured by inhibition of syncytia in vitro. Furthermore, a FOXO3 transcriptional site of regulation of expression at the 5 end of the ATP2B1 locus, together with a rare homozygous intronic variant in the ATP2B1 locus (rs11337717; chr12:89643729, T>C), are shown to be associated with severity of COVID19 (symptomatic versus asymptomatic patients). Here, we identify the mechanism of action during SARS-CoV-2 infection, which involves the PI3K/Akt signaling pathway, inactivation of FOXO3 (i.e., phosphorylation), and inhibition of transcriptional control of both membrane and reticulum Ca2+ pumps (ATP2B1 and ATP2A1 [i.e., SERCA1], respectively). The pharmacological action of compound PI-7 on sustaining both ATP2B1 and ATP2A1 expression reduces the intracellular cytoplasmic Ca2+ pool and thus negatively influences SARS-CoV-2 replication and propagation. As compound PI-7 shows a lack of toxicity, its prophylactic use as a therapy against the COVID19 pandemic is here proposed.

FIRST DESCRIPTION OF SEROLOGICAL EVIDENCE FOR SARS-CoV-2 IN CATTLE (preprint)

Following the coronavirus disease 2019 (COVID-19) epidemic peak in Ariano Irpino, Campania region (Italy), we tested cattle for the presence of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) on a cattle farm at which, prior to the investigation, 13 of the 20 farmworkers showed COVID-19-like symptoms, and one of them died. Twenty-four cows were sampled to detect SARS-CoV-2. All nasal and rectal swabs and milk samples were negative for SARS-CoV-2 RNA. Of the 24 collected serum samples, 11 were positive for SARS-CoV-2 nucleocapsid protein, 14 were positive for SARS-CoV-2 spike protein, and 13 were positive for SARS-COV-2-neutralising antibodies; all samples were negative for Bovine Coronavirus (BCoV), another betacoronavirus. To our knowledge, this is the first report of natural serological evidence of SARS-CoV-2 infection in cattle. We hypothesise that this may be a case of reverse zoonosis. However, the role of cattle in SARS-CoV-2 infection and transmission seems to be negligible.

SARS-CoV-2 Quantitative Real Time PCR and Viral Loads Analysis Among Asymptomatic and Symptomatic Patients: an Observational Study on an Outbreak in Two Nursing Facilities in Campania Region (Southern Italy) (preprint)

Background: In December 2019 an outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 was first observed in Wuhan, China. The virus has spread rapidly throughout the world creating a pandemic scenario. Several risk factors have been identified, such as age, gender, concomitant diseases as well as viral load. One of the key questions since the beginning of the pandemic, is the role of asymptomatic people in spreading SARS-CoV-2. An observational study in Southern Italy was conducted in order to elucidate the possible role of asymptomatic individuals related to their viral loads in the transmission of the virus within two nursing facilities. Methods: oro-nasopharyngeal swabs from 179 nursing health care workers and patients were collected. SARS-CoV-2 RT-qPCR was performed and viral loads were calculated by using standard curve. For positive results, a statistical correlation between viral loads, the presence/absence of symptoms, age and gender variables was investigated. Results: SARS-CoV-2 was confirmed in the 50.8% (n=91) of the cases. Median age of positive individuals resulted higher than negative ones. A statistically significant difference (p <.001) was observed for age and gender variables and over 65 years individuals showed higher susceptibility to SARS-CoV-2 infection than younger ones (OR=3.93) as well as female (OR=2.86). Among 91 tested positive, the 70.3% was symptomatic (n=64) whilst the 29.7% was asymptomatic (n=27). Median viral loads of asymptomatic individuals were found statistically significant higher than symptomatic ones (p=.001), while no influence was observed in age and gender variables.Conclusions: A range from 9.2% to 69% of confirmed SARS-CoV-2 cases remains asymptomatic, moreover, sporadic transmissions from asymptomatic people are reported, that makes their involvement an important issue to take into account in the spreading control of the virus. An asymptomatic clinical course was observed in the 29.7% of positive individuals, moreover, median viral loads resulted to be statistically significant when compared to symptomatic ones. Surely, such a relevant frequency should not be ignored in relation to the spread of the disease in an environment which has not only important intrinsic (age, gender, concomitant diseases) but also extrinsic factors such as high population density and close contacts. 

Long-chain polyphosphates impair SARS-CoV-2 infection and replication: a route for therapy in man (preprint)

Anti-viral activities of long-chain inorganic polyphosphates (PolyPs) against severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection were investigated. In molecular docking analyses, PolyPs interacted with several conserved angiotensin-converting enzyme (ACE)2 and RNA-dependent RNA polymerase (RdRp) amino acids. We thus tested PolyPs for functional interactions in vitro in SARS-CoV-2-infected Vero E6, Caco2 and human primary nasal epithelial cells. Immunofluorescence, qPCR, direct RNA sequencing, FISH and Immunoblotting were used to determine virus loads and transcription levels of genomic(g)RNAs and sub-genomic(sg)RNAs. We show that PolyP120 binds to ACE2 and enhances its proteasomal degradation. PolyP120 shows steric hindrance of the genomic Sars-CoV-2-RNA/RdRP complex, to impair synthesis of positive-sense gRNAs, viral subgenomic transcripts and structural proteins needed for viral replication. Thus, PolyP120 impairs infection and replication of Korean and European (containing non-synonymous variants) SARS-CoV-2 strains. As PolyPs have no toxic activities, we envision their use as a nebulised formula for oropharyngeal delivery to prevent infections of SARS-CoV-2 and during early phases of antiviral therapy.

Fatal Interstitial Pneumonia Caused by Bovine Coronavirus in Cows from Southern Italy (preprint)

An outbreak of winter disease, complicated by severe respiratory syndrome, occurred in January 2020 in a high production dairy cow herd located in a hilly area of the Calabria region. Of the 52 animals belonging to the farm, 5 (9.6%) died with severe respiratory distress, death occurring 3-4 days after the appearance of the respiratory signs (caught and gasping breath). Microbiological analysis revealed absence of pathogenic bacteria whilst Real-time PCR identified the presence of RNA from Bovine Coronavirus (BCoV) in several organs: lungs, small intestine (jejunum), mediastinal lymph nodes, liver and placenta. Since being the only pathogen identified, BCoV was hypothesized to be the cause of the lethal pulmonary infection. Like the other CoVs, BCoV is able to cause different syndromes. Its role in calfhood diarrhoea and in mild respiratory disease is well known: we report instead the involvement of this virus in a severe and fatal respiratory disorder, with symptoms and disease evolution resembling that of Severe Acute Respiratory Syndromes (SARS).