Hamster Lung Research Articles

Pathomorphological changes in the lung tissues of Syrian hamsters during experimental infection with SARS‐COV‐2

The new coronavirus infection caused by SARS‐CoV‐2 is a dangerous respiratory disease. The aim of the investigation was to study the pathomorphological changes in lungs of Syrian hamsters infected with SARS‐CoV‐2 as is necessary for adequate therapy and prevention of the disease.Syrian hamsters of the SPF category were infected intranasally with a dose of 5×105TCID50 SARS‐CoV‐2/human/RUS/Nsk‐FRCFTM‐1/2020). Animals were euthanised on days 3, 6, 10, 14 and 21 after infection and the lungs were collected. To determine virus replication real‐time RT‐PCR was used. The lungs was used for histological examination. Statistics was performed using Statistics for Windows 10 software.Specific pathological lesions characterising the inflammatory process were detected in all groups of animals. The severity of pathological changes and the volume of lung tissue lesions varied depending on the stage of the disease. The most severe lesions were observed on days 6–10. By day 21, the process of organ structure restoration was noticed.The study showed that pathological changes in the Syrian hamster’s lungs caused by experimental infection with the SARS‐CoV‐2 were multifaceted and complex. In mild cases, congestion and infiltration are observed, and in severe cases, atelectasis and pulmonary edema were life‐threatening. The most severe lesions in lungs were noted on days 6–10. By day 21, the process of restoration was observed.

Evaluating cytotoxicity and genotoxicity of oil extracted from visceral fat of Caiman yacare (Daudin, 1802) in Chinese hamster lung fibroblast in vitro

In previous studies, the oil extracted from the visceral fat of Caiman yacare (Daudin, 1802) demonstrated a wound-healing effect on the skin of Wistar rats. To enhance knowledge our about the mechanism underlying this effect, we analysed the oil’s toxicological potential in vitro. Cytotoxicity, genotoxicity, pro-oxidant, and antioxidant activities were evaluated in a V79-4 cell line. The oil was obtained using the Soxhlet method, and the proportions of the fatty acid profile was previously identified 43.74 % saturated and 34.65 % unsaturated fatty acids. Protocol 487 of the Organisation for Economic Co-operation and Development (OECD) was employed for cell line selection and concentrations. Cytotoxicity was determined using the MTT assay and clonogenic survival. Pro-oxidant and antioxidant activities were analysed using flow cytometry. Genotoxicity was evaluated using comet and micronucleus assays. The oil did not demonstrate cytotoxicity up to a concentration of 500 µg/mL. At concentrations of 250 and 500 µg/mL, the oil exerted a protective effect against oxidative stress and showed genotoxic effects only at the highest concentration (2000 µg/mL). Like other oils of interest for human health, the oil extracted from the visceral fat of C. yacare demonstrated low toxicological potential in vitro.

Ultrapotent class I neutralizing antibodies post Omicron breakthrough infection overcome broad SARS-CoV-2 escape variants

The spread of emerging SARS-CoV-2 immune escape sublineages, especially JN.1 and KP.2, has resulted in new waves of COVID-19 globally. The evolving memory B cell responses elicited by the parental Omicron variants to subvariants with substantial antigenic drift remain incompletely investigated. Using the single B cell antibody cloning technology, we isolated single memory B cells, delineated the B cell receptor repertoire and conducted the pseudovirus-based assay for recovered neutralizing antibodies (NAb) screening. We analyzed the cryo-EM structures of top broadly NAbs (bnAbs) and evaluated their invivo efficacy (golden Syrian hamster model). By investigating the evolution of human B cell immunity, we discovered a new panel of bnAbs arising from vaccinees after Omicron BA.2/BA.5 breakthrough infections. Two lead bnAbs neutralized major Omicron subvariants including JN.1 and KP.2 with IC50 values less than 10ng/mL, representing ultrapotent receptor binding domain (RBD)-specific class I bnAbs. They belonged to the IGHV3-53/3-66 clonotypes instead of evolving from the pre-existing vaccine-induced IGHV1-58/IGKV3-20 bnAb ZCB11. Despite sequence diversity, they targeted previously unrecognized, highly conserved conformational epitopes in the receptor binding motif (RBM) for ultrapotent ACE2 blockade. The lead bnAb ZCP3B4 not only protected the lungs of hamsters intranasally challenged with BA.5.2, BQ.1.1 and XBB.1.5 but also prevented their contact transmission. Our findings demonstrated that class I bnAbs have evolved an ultrapotent mode of action protecting against highly transmissible and broad Omicron escape variants, and their epitopes are potential targets for novel bnAbs and vaccine development. A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Broad protection and respiratory immunity of dual mRNA vaccination against SARS-CoV-2 variants

While first-generation, spike (S)-based COVID-19 vaccines were effective against early SARS-CoV-2 strains, the rapid evolution of novel Omicron subvariants have substantially reduced vaccine efficacy. As such, broadly protective vaccines against SARS-CoV-2 are needed to prevent future viral emergence. In addition, it remains less clear whether peripheral immunization, especially with mRNA vaccines, elicits effective respiratory immunity. Our group has developed a nucleoside-modified mRNA vaccine expressing the nucleocapsid (N) protein of the ancestral SARS-CoV-2 virus and has tested its use in combination with the S-based mRNA vaccine (mRNA-S). In this study, we examined efficacy of mRNA-N alone or in combination with mRNA-S (mRNA-S+N) against more immune evasive Omicron variants in hamsters. Our data show that mRNA-N alone induces a modest but significant protection against BA.5 and that dual mRNA-S+N vaccination confers complete protection against both BA.5 and BQ.1, preventing detection of virus in the hamster lungs. Analysis of respiratory immune response in mice shows that intramuscular mRNA-S+N immunization effectively induces respiratory S- and N-specific T cell responses in the lungs and in bronchoalveolar lavage (BAL), as well as antigen-specific binding IgG in BAL. Together, our data further support mRNA-S+N as a potential pan-COVID-19 vaccine for broad protection against current and emerging SARS-CoV-2 variants.

Evidence for Safety of The Dietary Ingredient Agmatine Sulfate as Assessed by Mutagenicity and Genotoxicity Studies

Agmatine, 1-Amino-4-guanidinobutane, is a ubiquitous naturally occurring molecule present in low amounts in a wide variety of foodstuff. Clinical trials have demonstrated the safety of oral agmatine sulfate and have led to its development as an effective dietary ingredient for promoting resilient nerve functions. Although clearly required, the mutagenic and genotoxic effects of agmatine have not been previously reported. The present study, therefore, undertook to assess the safety profile of agmatine using currently accepted in vitro and in vivo mutagenicity and genotoxicity tests. The test item was G-Agmatine®, a proprietary brand of agmatine sulfate. Using the bacterial reverse mutation assay (Ames test), the study found that G-Agmatine® has no mutagenic effects. It had no clastogenic effects as observed by the in vitro chromosomal aberration test using Chinese Hamster lung cells. And it lacked genotoxic effects as evidenced by the lack of increased frequency of micronucleated polychromatic immature erythrocytes following oral administration in the mouse micronucleus test. Taken together with previously published data, results of the present study further support the safety of agmatine sulfate as a dietary ingredient.

Efficacy of molnupiravir and interferon for the treatment of SARS-CoV-2 in golden Syrian hamster.

The mortality and hospitalization rate by COVID-19 dropped significantly currently, but its seasonal outbreaks make antiviral treatment still vital. The mortality and hospitazation rate by COVID-19 dropped significantly currently, but its seasonal ourbreaks make antiviral treatment still vital. In our study, syrian golden hamsters were treated with molnupiravir and interferons (IFNs) after SARS-CoV-2 infection. Their weight changes, pathological changes, virus replication and inflammation levels were evaluated. In the IFNs single treatment, only IFN-α group reduced viral load (p < 0.05) and virus titer in hamster lungs. The TNF-α expression decreased significantly in both IFNs treatment at 2dpi. Histological and immunofluorescence results showed lung damage in the IFNs groups were milder at 4dpi. In the molnupiravir/IFN-α combination treatment, weight loss and virus replication in lung were significantly decreased in the mono-molnupiravir group and combination group (p < 0.05), the expression of IL-6, TNF-α, IL-1β and MIP-1α also decreased significantly (p < 0.05), but the combination treatment was not more effective than the mono-molnupiravir treatment. Histological and immunofluorescence results showed the lung damage and inflammation in mono-molnupiravir and combination groups were milder. In summary, IFNs treatment had anti-inflammatory effect against SARS-CoV-2, only IFN-α showed a weak antiviral effect. Molnupiravir/IFN-α combination treatment was effective against SARS-CoV-2 but was not superior to mono-molnupiravir treatment. IFN-α could be considered for immunocompromised patients to stimulate and activate early immune responses.

The small molecule inhibitor of SARS-CoV-2 3CLpro EDP-235 prevents viral replication and transmission in vivo

The COVID-19 pandemic has led to the deaths of millions of people and severe global economic impacts. Small molecule therapeutics have played an important role in the fight against SARS-CoV-2, the virus responsible for COVID-19, but their efficacy has been limited in scope and availability, with many people unable to access their benefits, and better options are needed. EDP-235 is specifically designed to inhibit the SARS-CoV-2 3CLpro, with potent nanomolar activity against all SARS-CoV-2 variants to date, as well as clinically relevant human and zoonotic coronaviruses. EDP-235 maintains potency against variants bearing mutations associated with nirmatrelvir resistance. Additionally, EDP-235 demonstrates a ≥ 500-fold selectivity index against multiple host proteases. In a male Syrian hamster model of COVID-19, EDP-235 suppresses SARS-CoV-2 replication and viral-induced hamster lung pathology. In a female ferret model, EDP-235 inhibits production of SARS-CoV-2 infectious virus and RNA at multiple anatomical sites. Furthermore, SARS-CoV-2 contact transmission does not occur when naïve ferrets are co-housed with infected, EDP-235-treated ferrets. Collectively, these results demonstrate that EDP-235 is a broad-spectrum coronavirus inhibitor with efficacy in animal models of primary infection and transmission.

Evaluating Molecular Mechanism of Viral Inhibition of Aerosolized Smart Nano-Enabled Antiviral Therapeutic (SNAT) on SARS-CoV-2-Infected Hamsters.

Smart Nano-enabled Antiviral Therapeutic (SNAT) is a promising nanodrug that previously demonstrated efficacy in preclinical studies to alleviate SARS-CoV-2 pathology in hamsters. SNAT comprises taxoid (Tx)-decorated amino (NH2)-functionalized near-atomic size positively charged silver nanoparticles (Tx-[NH2-AgNPs]). Herein, we aimed to elucidate the molecular mechanism of the viral inhibition and safety of aerosolized SNAT treatment in SARS-CoV-2-infected golden Syrian hamsters. High-resolution transmission electron microscopy (HR-TEM) coupled with energy dispersive spectroscopy (EDS) and ELISAs showed SNAT binds directly to the SARS-CoV-2 virus by interacting with intact spike (S) protein, specifically to S2 subunit. SNAT (≥1 µg/mL) treatment significantly lowered SARS-CoV-2 infections of Calu-3 cells. Extraction-free whole transcriptome assay was used to detect changes in circulatory micronome in hamsters treated intranasally with SNAT (two doses of 10 µg/mL of 2 mL each administered 24 h apart). Uninfected hamsters treated with SNAT had altered circulatory concentrations of 18 microRNAs (8 miRNAs upregulated, 10 downregulated) on day 3 post-treatment compared to uninfected controls. SNAT-induced downregulation of miR-141-3p and miR-200b-3p may reduce viral replication and inflammation by targeting Ythdf2 and Slit2, respectively. Further, SNAT treatment significantly lowered IL-6 expression in infected hamster lungs compared to untreated infected hamsters. Taken together, we demonstrate that SNAT binds directly to SARS-CoV-2 via the S protein to prevent viral entry and propose a model by which SNAT alters the cellular miRNA-directed milieu to promote antiviral cellular processes and neutralize infection. Our results provide insights into the use of low-dose intranasally delivered SNAT in treating SARS-CoV-2 infections in a hamster model.

An evaluation of the genotoxicity and 90-day repeated dose oral toxicity in rats of Porphyridium purpureum.

Interest in microalgae products for use in food is increasing, as demands for sustainable and cost-effective food choices grow due to the escalating global population and increase in climate-related struggles with agriculture. Toxicological assessments of some species of microalgae have been conducted, but there were little data available for the oral consumption of the red microalgae Porphyridium purpureum and no data on genotoxicity. This article articulates a genotoxicity assessment and a 90-day repeated dose oral toxicity study in rats performed according to OECD guidelines. Under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used in the bacterial reverse mutation test. Similarly, the test item did not induce structural chromosomal aberrations in V79 hamster lung cells. The test item also did not cause chromosomal damage in bone marrow of mice in the mammalian micronucleus test. The no observed adverse effect level (NOAEL) of the 90-day repeated dose oral toxicity study in rats was determined to be the highest dose tested, 3000 mg/kg bw/day. These data add to the body of evidence regarding the safety of P.purpureum for human consumption.

Metabolomic characterization of monoclonal antibody-producing Chinese hamster lung (CHL)-YN cells in glucose-controlled serum-free fed-batch operation.

The fast-growing Chinese hamster lung (CHL)-YN cell line was recently developed for monoclonal antibody production. In this study, we applied a serum-free fed-batch cultivation process to immunoglobulin (Ig)G1-producing CHL-YN cells, which were then used to design a dynamic glucose supply system to stabilize the extracellular glucose concentration based on glucose consumption. Glucose consumption of the cultures rapidly oscillated following three phases of glutamine metabolism: consumption, production, and re-consumption. Use of the dynamic glucose supply prolonged the viability of the CHL-YN-IgG1 cell cultures and increased IgG1 production. Liquid chromatography with tandem mass spectrometry-based target metabolomics analysis of the extracellular metabolites during the first glutamine shift was conducted to search for depleted compounds. The results suggest that the levels of four amino acids, namely arginine, aspartate, methionine, and serine, were sharply decreased in CHL-YN cells during glutamine production. Supporting evidence from metabolic and gene expression analyses also suggest that CHL-YN cells acquired ornithine- and cystathionine-production abilities that differed from those in Chinese hamster ovary-K1 cells, potentially leading to proline and cysteine biosynthesis.

Barcoded SARS-CoV-2 viruses define the impact of time and route of transmission on the transmission bottleneck in a Syrian hamster model.

The transmission bottleneck, defined as the number of viruses that transmit from one host to infect another, is an important determinant of the rate of virus evolution and the level of immunity required to protect against virus transmission. Despite its importance, SARS-CoV-2's transmission bottleneck remains poorly characterized, in part due to a lack of quantitative measurement tools. To address this, we adapted a SARS-CoV-2 reverse genetics system to generate a pool of >200 isogenic SARS-CoV-2 viruses harboring specific 6-nucleotide barcodes inserted in ORF10, a non-translated ORF. We directly inoculated donor Syrian hamsters intranasally with this barcoded virus pool and exposed a paired naïve contact hamster to each donor. Following exposure, the nasal turbinates, trachea, and lungs were collected, viral titers were measured, and the number of barcodes in each tissue were enumerated to quantify the transmission bottleneck. The duration and route (airborne, direct contact, and fomite) of exposure were varied to assess their impact on the transmission bottleneck. In airborne-exposed hamsters, the transmission bottleneck increased with longer exposure durations. We found that direct contact exposure produced the largest transmission bottleneck (average 27 BCs), followed by airborne exposure (average 16 BCs) then fomite exposure (average 8 BCs). Interestingly, we detected unique BCs in both the upper and lower respiratory tract of contact animals from all routes of exposure, suggesting that SARS-CoV-2 can directly infect hamster lungs. Altogether, these findings highlight the utility of barcoded viruses as tools to rigorously study virus transmission. In the future, barcoded SARS-CoV-2 will strengthen studies of immune factors that influence virus transmission.

Genotoxicity and cytotoxicity evaluation of a heat-not-burn product

‘Heat-not-burn’ products (HnBP) contain lower levels of harmful substances than traditional cigarettes, but the use of these products warrants further toxicological evaluation. We have compared the cytotoxicity and genotoxicity of a heat-not burn product with conventional cigarettes, in vivo and in vitro. Male Sprague Dawley rats were exposed to mainstream smoke from conventional cigarettes or a HnBP, for 4 or 28 days, followed by isolation of bone marrow polychromatic erythrocytes (PCE) and histological examination of the testes. Chinese hamster lung fibroblast cells were exposed in vitro to total particulate matter from cigarette smoke obtained through Cambridge filters. The cytotoxicity and genotoxicity of total particulate matter were assessed by the neutral red uptake assay, chromosome aberration assay, in vitro micronucleus test, comet assay, and Ames assay. In the short-term exposure rat models, only the conventional-cigarettes group showed a significant increase in the ratio of micronuclei to total PCE. There was no significant difference in rat testis histology in the long-term exposure models. In vitro, in the neutral red uptake assay, the HnBP product showed lower cytotoxicity than conventional cigarettes. Conventional cigarettes showed greater genotoxicity in the chromosome aberration assay, high-dose Ames tests with exogenous metabolic activation, and micronucleus tests. In summary, our results suggest that HnBP have lower cytotoxicity and genotoxicity than conventional cigarettes.

A novel glycoglycerolipid from Holotrichia diomphalia Bates: Structure characteristics and protective effect against DNA damage

A lipidated polysaccharide, HDPS-2II, was isolated from the dried larva of Holotrichia diomphalia, which is used in traditional Chinese medicine. The molecular weight of HDPS-2II was 5.9 kDa, which contained a polysaccharide backbone of →4)-β-Manp-(1 → 4,6)-β-Manp-(1 → [6)-α-Glcp-(1]n → 6)-α-Glcp→ with the side chain α-Glcp-(6 → 1)-α-Glcp-(6 → linked to the C-4 of β-1,4,6-Manp and four types of lipid chains including 4-(4-methyl-2-(methylamino)pentanamido)pentanoic acid, 5-(3-(tert-butyl)phenoxy)hexan-2-ol, N-(3-methyl-5-oxopentan-2-yl)palmitamide, and N-(5-amino-3-methyl-5-oxopentan-2-yl)stearamide. The lipid chains were linked to C-1 of terminal α-1,6-Glcp in carbohydrate chain through diacyl-glycerol. HDPS-2II exhibited DNA protective effects and antioxidative activity on H2O2- or adriamycin (ADM)-induced Chinese hamster lung cells. Furthermore, HDPS-2II significantly ameliorated chromosome aberrations and the accumulation of reactive oxygen species (ROS), reduced γ-H2AX signaling and the expressions of NADPH oxidase (NOX)2, NOX4, P22phox, and P47phox in ADM-induced cardiomyocytes. Mechanistically, HDPS-2II suppressed ADM-induced up-regulation of NOX2 and NOX4 in cardiomyocytes, but not in NOX2 or NOX4 knocked-down cardiomyocytes, indicating that HDPS-2II could relieve intracellular DNA damage by regulating NOX2/NOX4 signaling. These findings demonstrate that HDPS-2II is a new potential DNA protective agent.

3D imaging of SARS-CoV-2 infected hamster lungs by X-ray phase contrast tomography enables drug testing

X-ray Phase Contrast Tomography (XPCT) based on wavefield propagation has been established as a high resolution three-dimensional (3D) imaging modality, suitable to reconstruct the intricate structure of soft tissues, and the corresponding pathological alterations. However, for biomedical research, more is needed than 3D visualisation and rendering of the cytoarchitecture in a few selected cases. First, the throughput needs to be increased to cover a statistically relevant number of samples. Second, the cytoarchitecture has to be quantified in terms of morphometric parameters, independent of visual impression. Third, dimensionality reduction and classification are required for identification of effects and interpretation of results. To address these challenges, we here design and implement a novel integrated and high throughput XPCT imaging and analysis workflow for 3D histology, pathohistology and drug testing. Our approach uses semi-automated data acquisition, reconstruction and statistical quantification. We demonstrate its capability for the example of lung pathohistology in Covid-19. Using a small animal model, different Covid-19 drug candidates are administered after infection and tested in view of restoration of the physiological cytoarchitecture, specifically the alveolar morphology. To this end, we then use morphometric parameter determination followed by a dimensionality reduction and classification based on optimal transport. This approach allows efficient discrimination between physiological and pathological lung structure, thereby providing quantitative insights into the pathological progression and partial recovery due to drug treatment. Finally, we stress that the XPCT image chain implemented here only used synchrotron radiation for validation, while the data used for analysis was recorded with laboratory μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mu$$\\end{document}CT radiation, more easily accessible for pre-clinical research.

Invivo evaluation of Andrographis paniculata and Boesenbergia rotunda extract activity against SARS-CoV-2 Delta variant in Golden Syrian hamsters: Potential herbal alternative for COVID-19 treatment

The ongoing COVID-19 pandemic has triggered extensive research, mainly focused on identifying effective therapeutic agents, specifically those targeting highly pathogenic SARS-CoV-2 variants. This study aimed to investigate the in vivo antiviral efficacy and anti-inflammatory activity of herbal extracts derived from Andrographis paniculata and Boesenbergia rotunda, using a Golden Syrian hamster model infected with Delta, a representative variant associated with severe COVID-19. Hamsters were intranasally inoculated with the SARS-CoV-2 Delta variant and orally administered either vehicle control, B. rotunda, or A.paniculata extract at a dosage of 1000 mg/kg/day. Euthanasia was conducted on days 1, 3, and 7 post-inoculation, with 4 animals per group. The results demonstrated that oral administration of A. paniculata extract significantly alleviated both lethality and infection severity compared with the vehicle control and B. rotunda extract. However, neither extract exhibited direct antiviral activity in terms of reducing viral load in the lungs. Nonetheless, A. paniculata extract treatment significantly reduced IL-6 protein levels in the lung tissue (7278 ± 868.4 pg/g tissue) compared to the control (12,495 ± 1118 pg/g tissue), indicating there was a decrease in local inflammation. This finding is evidenced by the ability of A. paniculata extract to reduce histological lesions in the lungs of infected hamsters. Furthermore, both extracts significantly decreased IL-6 and IP-10 mRNA expression in peripheral blood mononuclear cells of infected hamsters compared to the control group, suggesting systemic anti-inflammatory effects occurred. In conclusion, A. paniculata extract's potential therapeutic application for SARS-CoV-2 arises from its observed capacity to lessen inflammatory cytokine concentrations and mitigate lung pathology.

Protective effect of luteolin against oxidative stress‑mediated cell injury via enhancing antioxidant systems.

Physiological stress such as excessive reactive oxygen species (ROS) production may contribute normal fibroblasts activation into cancer‑associated fibroblasts, which serve a crucial role in certain types of cancer such as pancreatic, breast, liver and lung cancer. The present study aimed to examine the cytoprotective effects of luteolin (3',4',5,7‑tetrahydroxyflavone) against hydrogen peroxide (H2O2)‑generated oxidative stress in lung fibroblasts. To examine the effects of luteolin against H2O2‑induced damages, cell viability, sub‑G1 cell population, nuclear staining with Hoechst 33342, lipid peroxidation and comet assays were performed. To evaluate the effects of luteolin on the protein expression level of apoptosis, western blot assay was performed. To assess the antioxidant effects of luteolin, detection of ROS using H2DCFDA staining, O2‑ and ·OH using electron spin resonance spectrometer and antioxidant enzyme activity was performed. In a cell‑free chemical system, luteolin scavenges superoxide anion and hydroxyl radical generated by xanthine/xanthine oxidase and the Fenton reaction (FeSO4/H2O2). Furthermore, Chinese hamster lung fibroblasts (V79‑4) treated with H2O2 showed a significant increase in cellular ROS. Intracellular ROS levels and damage to cellular components such as lipids and DNA in H2O2‑treated cells were significantly decreased by luteolin pretreatment. Luteolin increased cell viability, which was impaired following H2O2 treatment and prevented H2O2‑mediated apoptosis. Luteolin suppressed active caspase‑9 and caspase‑3 levels while increasing Bcl‑2 expression and decreasing Bax protein levels. Additionally, luteolin restored levels of glutathione that was reduced in response to H2O2. Moreover, luteolin enhanced the activity and protein expressions of superoxide dismutase, catalase, glutathione peroxidase, and heme oxygenase‑1. Overall, these results indicated that luteolin inhibits H2O2‑mediated cellular damage by upregulating antioxidant enzymes.

Further preclinical characterization of molnupiravir against SARS-CoV-2: Antiviral activity determinants and viral genome alteration patterns

The SARS-CoV-2 pandemic has highlighted the need for broad-spectrum antiviral drugs to respond promptly to viral emergence. We conducted a preclinical study of molnupiravir (MOV) against SARS-CoV-2 to fully characterise its antiviral properties and mode of action. The antiviral activity of different concentrations of MOV was evaluated ex vivo on human airway epithelium (HAE) and in vivo in a hamster model at three escalating doses (150, 300 and 400 mg/kg/day) according to three different regimens (preventive, pre-emptive and curative). We assessed viral loads and infectious titres at the apical pole of HAE and in hamster lungs, and MOV trough concentration in plasma and lungs. To explore the mode of action of the MOV, the entire genomes of the collected viruses were deep-sequenced. MOV effectively reduced viral titres in HAE and in the lungs of treated animals. Early treatment after infection was a key factor in efficacy, probably associated with high lung concentrations of MOV, suggesting good accumulation in the lung. MOV induced genomic alteration in viral genomes with an increase in the number of minority variants, and predominant G to A transitions. The observed reduction in viral replication and its mechanism of action leading to lethal mutagenesis, supported by clinical trials showing antiviral action in humans, provide a convincing basis for further research as an additional means in the fight against COVID-19 and other RNA viruses.

Genes involved in the limited spread of SARS-CoV-2 in the lower respiratory airways of hamsters may be associated with adaptive evolution.

Novel respiratory viruses can cause a pandemic and then evolve to coexist with humans. The Omicron strain of severe acute respiratory syndrome coronavirus 2 has spread worldwide since its emergence in late 2021, and its sub-lineages are now established in human society. Compared to previous strains, Omicron is markedly less invasive in the lungs and causes less severe disease. One reason for this is that humans are acquiring immunity through previous infection and vaccination, but the nature of the virus itself is also changing. Using our newly established low-volume inoculation system, which reflects natural human infection, we show that the Omicron strain spreads less efficiently into the lungs of hamsters compared with an earlier Wuhan strain. Furthermore, by characterizing chimeric viruses with the Omicron gene in the Wuhan strain genetic background and vice versa, we found that viral genes downstream of ORF3a, but not the S gene, were responsible for the limited spread of the Omicron strain in the lower airways of the virus-infected hamsters. Moreover, molecular evolutionary analysis of SARS-CoV-2 revealed a positive selection of genes downstream of ORF3a (M and E genes). Our findings provide insight into the adaptive evolution of the virus in humans during the pandemic convergence phase.IMPORTANCEThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has spread worldwide since its emergence in late 2021, and its sub-lineages are established in human society. Compared to previous strains, the Omicron strain is less invasive in the lower respiratory tract, including the lungs, and causes less severe disease; however, the mechanistic basis for its restricted replication in the lower airways is poorly understood. In this study, using a newly established low-volume inoculation system that reflects natural human infection, we demonstrated that the Omicron strain spreads less efficiently into the lungs of hamsters compared with an earlier Wuhan strain and found that viral genes downstream of ORF3a are responsible for replication restriction in the lower respiratory tract of Omicron-infected hamsters. Furthermore, we detected a positive selection of genes downstream of ORF3a (especially the M and E genes) in SARS-CoV-2, suggesting that these genes may undergo adaptive changes in humans.

Comparative spatial proteomics between SARS-CoV-2 infected lungs of Roborovski dwarf hamsters and Syrian hamsters

Comparative spatial proteomics between SARS-CoV-2 infected lungs of Roborovski dwarf hamsters and Syrian hamsters

Toxicity Assessment of a No-Pain Pharmacopuncture Extract Using a Standard Battery of In Vitro Chromosome Aberration Tests.

Genotoxicity is evaluated through a chromosomal aberration test using cultured mammalian cells to determine the toxicity of no-pain pharmacopuncture (NPP), which has recently been used to treat musculoskeletal pain disorders in Korean medical clinical practice. An initial test was performed to determine the dosage range of the NPP, followed by the main test. In this study, NPP doses of 10.0, 5.0, and 2.5%, and negative and positive controls were tested. An in vitro chromosome aberration test was performed using Chinese hamster lung cells under short-term treatment with or without metabolic activation and under continuous treatment without metabolic activation. Compared with the saline negative control group, NPP did not significantly increase the frequency of chromosomal abnormalities in Chinese hamster lung cells, regardless of the presence or absence of metabolic activation. Additionally, the number of cells with structural chromosomal abnormalities was significantly higher in the positive control group than that in the negative control group that received saline. Based on the above results, the chromosomal abnormality-producing effect of NPP was determined to be negative under these test conditions.