Biology subjects

Milewska, A.

Publications and source records attributed to Milewska, A..

7 recordsLinked to original sources

bioRxiv2026

A 10-year longitudinal study reveals bat-driven SARSr-CoV evolution

Bats are recognized reservoirs for several zoonoses and have an exceptional tolerance to viruses, likely linked with strong evolutionary selection in immune genes. Notably, in light of the emergence history of severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2 is hypothesized to have originated from Rhinolophus bats as well. In-depth studies are still needed, however, to unravel how SARS-related coronaviruses (SARSr-CoVs) persist and thrive in nature through evolution, virus-host interactions, and cross-species transmission. Here we present a pioneering 10-year longitudinal study of BBCoV-EPI1, a SARSr-CoV in Rhinolophus bats that revealed the complexity of virus-host interactions and the tempo at which new viral lineages emerge. Our results demonstrated the impacts of host life cycles (hibernation, female gathering, weaning juveniles, and mating periods) on strong seasonal oscillations in viral prevalence. These pronounced fluctuations drove viral diversification through bottlenecks, founder effects and Darwinian selection, counterbalanced by high mutation rates, fast gradualism, and recombination. We also found that host-cell enzymes are major contributors to BBCoV-EPI1 evolution in bats. The antiviral effect of APOBEC3 detected here in bats is weaker than it is in humans. Conversely, the ADAR enzyme had an unexpectedly strong and beneficial influence on viral evolution, even though the high mutation rate of RNA viruses is usually attributed to viral polymerases' low replication fidelity. We discovered that key BBCoV-EPI1 innovations in Rhinolophus bats were mostly located in proteins involved in cell-cycle hijack and in blocking bat cells' antiviral response, rather than in the Spike (S). We also showed that the BBCoV-EPI1 S protein efficiently uses angiotensin-converting enzyme 2 (ACE2) receptor to enter Rhinolophus bat cells, but not to enter human or sympatric species cells, due to amino-acid differences at the S-ACE2 interface. Last, this work identifies 101 critical amino-acid changes frequently associated with viral fitness shift, and paves the way for future experimental studies focusing on the capacity of SARSr-CoVs to adapt to hosts and cross the species barrier.

microbiology↗

bioRxiv2020

SARS-CoV-2 inhibition in human airway epithelial cells using a mucoadhesive, amphiphilic chitosan that may serve as an anti-viral nasal spray

There are currently no cures for coronavirus infections, making the prevention of infections the only course open at the present time. The COVID-19 pandemic has been difficult to prevent, as the infection is spread by respiratory droplets and thus effective, scalable and safe preventive interventions are urgently needed. We hypothesise that preventing viral entry into mammalian nasal epithelial cells may be one way to limit the spread of COVID-19. Here we show that N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ), a positively charged polymer that has been through an extensive Good Laboratory Practice toxicology screen, is able to reduce the infectivity of SARS-COV-2 in A549ACE2+ and Vero E6 cells with a log removal value of -3 to -4 at a concentration of 10 - 100 g/ mL (p < 0.05 compared to untreated controls) and to limit infectivity in human airway epithelial cells at a concentration of 500 g/ mL (p < 0.05 compared to untreated controls). GCPQ is currently being developed as a pharmaceutical excipient in nasal and ocular formulations. GCPQs electrostatic binding to the virus, preventing viral entry into the host cells, is the most likely mechanism of viral inhibition. Radiolabelled GCPQ studies in mice show that at a dose of 10 mg/ kg, GCPQ has a long residence time in mouse nares, with 13.1% of the injected dose identified from SPECT/CT in the nares, 24 hours after nasal dosing. With a no observed adverse effect level of 18 mg/ kg in rats, following a 28-day repeat dose study, clinical testing of this polymer, as a COVID-19 prophylactic is warranted.

pharmacology and toxicology↗

bioRxiv2020

Zika virus NS3 protease and its cellular substrates

AO_SCPLOWBSTRACTC_SCPLOWZika virus is a flavivirus discovered in 1947, but the association between Zika virus infection and brain disorders was not demonstrated until 2015 in Brazil. Infection mostly poses a threat to women during pregnancy, since it may cause microcephaly and other neurological dysfunctions in the developing fetus. However, infection is also associated with Guillain-Barre syndrome. The nonstructural NS3 protein is essential for virus replication because it helps to remodel the cellular microenvironment. Several reports show that this protease can process cellular substrates and thereby modify cellular pathways that are important for the virus. Herein, we explored some of the targets of NS3, but we could not confirm the biological relevance of its protease activity. Thus, although mass spectrometry is highly sensitive and useful in many instances, being also able to show directions, where cell/virus interaction occurs, we believe that biological validation of the observed results is essential.

microbiology↗

bioRxiv2020

The SARS-CoV-2 ORF10 is not essential in vitro or in vivo in humans.

SARS-CoV-2 genome annotation revealed the presence of 10 open reading frames (ORFs), of which the last one (ORF10) is positioned downstream the N gene. It is a hypothetical gene, which was speculated to encode a 38 aa protein. This hypothetical protein does not share sequence similarity with any other known protein and cannot be associated with a function. While the role of this ORF10 was proposed, there is a growing evidence showing that the ORF10 is not a coding region. Here, we identified SARS-CoV-2 variants in which the ORF10 gene was prematurely terminated. The disease was not attenuated, and the transmissibility between humans was not hampered. Also in vitro, the strains replicated similarly, as the related viruses with the intact ORF10. Altogether, based on clinical observation and laboratory analyses, it appears that the ORF10 protein is not essential in humans. This observation further proves that the ORF10 should not be treated as the protein-coding gene, and the genome annotations should be amended.

microbiology↗

bioRxiv2020

HTCC as a highly effective polymeric inhibitor of SARS-CoV-2 and MERS-CoV

The beginning of 2020 brought us information about the novel coronavirus emerging in China. Rapid research resulted in the characterization of the pathogen, which appeared to be a member of the SARS-like cluster, commonly seen in bats. Despite the global and local efforts, the virus escaped the healthcare measures and rapidly spread in China and later globally, officially causing a pandemic and global crisis in March 2020. At present, different scenarios are being written to contain the virus, but the development of novel anticoronavirals for all highly pathogenic coronaviruses remains the major challenge. Here, we describe the antiviral activity of previously developed by us HTCC compound (N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride), which may be used as potential inhibitor of currently circulating highly pathogenic coronaviruses - SARS-CoV-2 and MERS-CoV.

microbiology↗

bioRxiv2020

Replication of SARS-CoV-2 in human respiratory epithelium

SARS-CoV-2 emerged by the end of 2019 to rapidly spread in 2020. At present, it is of utmost importance to understand the virus biology and to rapidly assess the potential of existing drugs and develop new active compounds. While some animal models for such studies are under development, most of the research is carried out in the Vero E6 cells. Here, we propose fully differentiated human airway epithelium cultures as a model for studies on the SARS-CoV-2. Further, we also provide basic characteristics of the system.

microbiology↗

bioRxiv2020

Kallikrein 13: a new player in coronaviral infections.

Human coronavirus HKU1 (HCoV-HKU1) is associated with respiratory disease and is prevalent worldwide, but in vitro model for virus replication is lacking. Interaction between the coronaviral spike (S) protein and its receptor is the major determinant of virus tissue and host specificity, but virus entry is a complex process requiring a concerted action of multiple cellular elements. Here, we show that KLK13 is required for the infection of the human respiratory epithelium and is sufficient to mediate the entry of HCoV-HKU1 to non-permissive RD cells. We also demonstrated HCoV-HKU1 S protein cleavage by KLK13 in the S1/S2 region, proving that KLK13 is the priming enzyme for this virus. Summarizing, we show for the first time that protease distribution and specificity predetermines the tissue and cell specificity of the virus and may also regulate interspecies transmission. It is also of importance that presented data may be relevant for the emerging coronaviruses, including SARS-CoV-2 and may help to understand the differences in their zoonotic potential.

microbiology↗