Flavonoids derived from medicinal plants as a COVID-19 treatment.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19 disease. Through its viral spike (S) protein, the virus enters and infects epithelial cells by utilizing angiotensin-converting enzyme 2 as a host cell's receptor protein. The COVID-19 pandemic had a profound impact on global public health and economies. Although various effective vaccinations and medications are now available to prevent and treat COVID-19, natural compounds derived from medicinal plants, particularly flavonoids, demonstrated therapeutic potential to treat COVID-19 disease. Flavonoids exhibit dual antiviral mechanisms: direct interference with viral invasion and inhibition of replication. Specifically, they target key viral molecules, particularly viral proteases, involved in infection. These compounds showcase significant immunomodulatory and anti-inflammatory properties, effectively inhibiting various inflammatory cytokines. Additionally, emerging evidence supports the potential of flavonoids to mitigate the progression of COVID-19 in individuals with obesity by positively influencing lipid metabolism. This review aims to elucidate the molecular structure of SARS-CoV-2 and the underlying mechanism of action of flavonoids on the virus. This study evaluates the potential anti-SARS-CoV-2 properties exhibited by flavonoid compounds, with a specific interest in their structure and mechanisms of action, as therapeutic applications for the prevention and treatment of COVID-19. Nevertheless, a significant portion of existing knowledge is based on theoretical frameworks and findings derived from in vitro investigations. Further research is required to better assess the effectiveness of flavonoids in combating SARS-CoV-2, with a particular emphasis on in vivo and clinical investigations.

Physical and chemical effects of conventional microplastic glitter versus alternative glitter particles on a freshwater plant (Lemnaceae: Lemna minor).

Glitters are primary microplastics which are directly littered into the environment, yet the ecological effects have seldom been tested. When microplastics enter the environment, their physical presence and chemical leachate may alter the physiology of primary producers. Glitter can be composed of plastic or natural and/or biodegradable materials, often with additives. Three experiments were run for 14 days to separate chemical and physical effects of different types of glitter: polyethylene terephthalate (PET), biodegradable modified regenerated cellulose (MRC), synthetic mica, and a natural particle control (kaolinite) on several physical characteristics of Lemna minor (common duckweed). L. minor was exposed to either fresh (chemical and physical effects), leachate from glitter (chemical) or aged glitter (physical). Overall, there was little effect of PET, synthetic mica, kaolinite or of any aged glitter. High concentrations of fresh MRC glitters, however, decreased root length, biomass and chlorophyll content of L. minor. Some of these effects were also present when exposed to leachate from MRC glitters, but were less pronounced. Elemental analysis revealed the presence of metals in MRC glitters which may explain these responses. Short-term ecotoxicity of biodegradable glitters can arise due to their physical and chemical properties, but may lessen over time as their surface coating degrades.

Microfiber Release to Water, Via Laundering, and to Air, via Everyday Use: A Comparison between Polyester Clothing with Differing Textile Parameters.

Textiles are one of the major sources of microplastic pollution to aquatic environments and have also been reported in dry and wet atmospheric deposition. There is still a lack of information on the direct release of microfibers from garments to the air and on the influence of textile characteristics including structure, type of yarn, and twist. The present study examines microfiber emissions directly to the air and to water as a consequence of laundering. Polyester garments with different textile characteristics were examined including various material compositions, fabric structure, yarn twist, fiber type, and hairiness. Scaling up our data indicates release of microfibers per person per year to the air is of a similar order of magnitude to that released to wastewater by laundering. The lowest releases to both air and water were recorded for a garment with a very compact woven structure and highly twisted yarns made of continuous filaments, compared with those with a looser structure (knitted, short staple fibers, lower twist). Our results demonstrate for the first time that direct release of microfibers from garments to air as a consequence of wear is of equal importance to releases to water. Currently there is considerable interest in interventions focused on capture from wastewater. However, our results suggest more effective interventions are likely to result from changes in textile design that could reduce emissions to both air and water.

Microautophagy upregulation in cutaneous lymph nodes of dogs naturally infected by Leishmania infantum.

This is the first study showing an in vivo microautophagy upregulation by Leishmania infantum in dogs. Both Leishmania amastigotes and promastigotes were detected in the cytoplasm of many professional and nonprofessional phagocytic cells of popliteal lymph node of three dogs suffering from chronic cutaneous leishmaniasis. Ultrastructurally, parasites appeared to be wrapped by lysosomes and/or multivesicular bodies. Neither phagophores nor double-membraned vacuoles consistent with autophagosomes were observed. Transcription factor EB (TFEB), a key factor involved in lysosome biogenesis, showed a statistically significant increase in the total component when examined by western blot in samples from leishmaniotic dogs compared with samples from healthy dogs. Instead, phosphorylated TFEB showed unmodified expression levels both in leishmaniotic and healthy dogs. Furthermore, Hsc70 and endosomal sorting complex required for transport (ESCRT)-I, which are known to play a role in microautophagy, showed no variation in expression levels both in diseased and healthy animals. Vps4A/B, an evolutionary conserved ATPase responsible for ESCRT-I complex disassembly and MVB maturation, was statistically significantly overexpressed in lymph nodal samples from leishmaniotic dogs. Bag3 was downregulated in diseased dogs whereas CHIP, p62, and LC3-II did not show any variation in expression levels. The altered expression profile of Bag3 suggested an altered interaction of Bag3 with Hsc70 and CHIP, which usually form a molecular complex involved in autophagosome-lysosome pathways. Ultrastructural and molecular findings suggested that the microautophagy pathway is upregulated in lymph nodes of dogs suffering from a chronic natural infection by Leishmania infantum.

Heavy metals concentrations in some commercially key species from Sicilian coasts (Mediterranean Sea): Potential human health risk estimation.

Trace elements were measured in the edible tissues of target marine species (Sardina pilchardus, Mullus barbatus, Mullus surmuletus, Merluccius merluccius and Parapenaeus longirostris) collected in some of the main ports of the Sicilian coast (southern Italy) in order to evaluate the potential human health risk associated to their consumption. Cadmium, Pb and Hg were below the tolerable limits reported by the European Union in foodstuffs (Commission Regulation (EC) 1881/2006) except for Hg in P. longirostris from one sampling site. The health risk for consumers was assessed both for adults and children calculating the estimated weekly intake (EWI), the target hazard quotient (THQ) and the lifetime cancer risk (CR) for each element. The EWI did not exceed the provisional tolerable weekly intake (PTWI) recommended by the European Food Safety Authority and THQs were < 1 in most cases. Further, the probabilistic distributions of health hazard were evaluated through Monte-Carlo simulation which confirmed a non-carcinogenic risk. With some exceptions related to As exposure, the results obtained definitively confirm the goodness of the fishery commercial resources in one of the most important seafood market of the central Mediterranean Sea and represent a positive data for the assessment of the good environmental status for descriptor 9 of the Marine Strategy Framework Directive in this area of the basin.

Co-inertia multivariate approach for the evaluation of anthropogenic impact on two commercial fish along Tyrrhenian coasts.

Aliphatic hydrocarbon levels were determined by the GC/MS technique in fish livers of Engraulis encrasicolus (Ee) and Trachurus trachurus (Tt), collected from a particular area of the Mediterranean Sea, called GSA 10, which is located exactly in Tyrrhenian Sea between Campania coast and North Sicily coast. The aim was to evaluate their potential use as specific bioindicators towards this class of contaminants. Both Tt and Ee are considered to be pollution monitoring bioindicators, due to their dominance in marine communities and economic fishing interest. Ee showed a higher tendency to bioaccumulate TAHs, due to the lower quantity of fatty acids in liver tissues with respect to Tt. The area under study has been characterised a) chemically with the acquisition of temperature, oxygen and salinity profiles along the water column, and b) ecologically with the determination of amino acid contents in fish eyes, in order to gain information on the adaptation to environmental changes. Moreover, specific activities of two hydrolytic enzymes, such as alkaline phosphatase and peroxidase in fish epidermal mucus, together with lactate in blood plasma and cortisol levels, have been investigated for the first time, in order to obtain insights into the effects of hydrocarbons on animal welfare. A multiple co-inertia analysis was also applied to chemical and environmental parameters, in order to explore any possible correlation between different variables. The multivariate approach showed a clear spatial distribution between environmental and chemical variables in Ee, whilst there was an absence of a spatial trend in Tt. Moreover, the chemometric analysis showed a very high correlation between amino acid profiles and environmental variables for both species, confirming the possibility of being used as ecological welfare indices for short-term environmental variations.

Design and characterization of a chitosan physical gel promoting wound healing in mice.

In this study, a sterile and biocompatible chitosan (CHI) gel for wound healing applications was formulated. CHI powder was treated in autoclave (ttCHI) to prepare sterile formulations. The heat treatment modified the CHI molecular weight, as evidenced by GPC analysis, and its physical-chemical features. Differential scanning calorimetry studies indicated that the macromolecules, before and after thermal treatment, differ in the strength of water-polymer interaction leading to different viscoelastic and flow properties. Thermally treated CHI exhibited the following effects: (i) increased the proliferation and migration of human foreskin foetal fibroblasts at 24 h; (ii) accelerated wound healing (measured as area of lesion) at 3 and 10 days in an in vivo model of pressure ulcers. These effects were linked to the increase of the hydroxyproline and haemoglobin content as well as Wnt protein expression. Moreover, we found a reduction of myeloperoxidase activity and TNF-α mRNA expression. These observations suggest the potential of this novel CHI gel in wound healing and other therapeutic applications.

Synthesis and pharmacological evaluation of modified adenosines joined to mono-functional platinum moieties.

The synthesis of four novel platinum complexes, bearing N6-(6-amino-hexyl)adenosine or a 1,6-di(adenosin-N6-yl)-hexane respectively, as ligands of mono-functional cisplatin or monochloro(ethylendiamine)platinum(II), is reported. The chemistry exploits the high affinity of the charged platinum centres towards the N7 position of the adenosine base system and a primary amine of an alkyl chain installed on the C6 position of the purine. The cytotoxic behaviour of the synthesized complexes has been studied in A549 adenocarcinomic human alveolar basal epithelial and MCF7 human breast adenocarcinomic cancer cell lines, in order to investigate their effects on cell viability and proliferation.

Vertical Intrauterine Bovine and Ovine Papillomavirus Coinfection in Pregnant Cows.

There is very little information available about transplacental infections by the papillomavirus in ruminants. However, recent evidence has emerged of the first report of vertical infections of bovine papillomavirus (BPV) in fetuses from naturally infected, pregnant cows. This study reports the coinfection of BPV and ovine papillomavirus (OaPV) in bovine fetuses from infected pregnant cows suffering from bladder tumors caused by simultaneous, persistent viral infections. Some molecular mechanisms involving the binary complex composed of Eras and platelet-derived growth factor ß receptor (PDGFßR), by which BPVs and OaPVs contribute to reproductive disorders, have been investigated. A droplet digital polymerase chain reaction (ddPCR) was used to detect and quantify the nucleic acids of the BPVs of the Deltapapillomavirus genus (BPV1, BPV2, BPV13, and BPV14) and OaPVs belonging to the Deltapapillomavirus (OaPV1, OaPV2, and OaPV4) and Dyokappapapillomavirus (OaPV3) genera in the placenta and fetal organs (heart, lung, liver, and kidneys) of four bovine fetuses from four pregnant cows with neoplasia of the urinary bladder. A papillomaviral evaluation was also performed on the bladder tumors and peripheral blood of these pregnant cows. In all fetal and maternal samples, the genotype distribution of BPVs and OaPVs were evaluated using both their DNA and RNA. A BPV and OaPV coinfection was seen in bladder tumors, whereas only BPV infection was found in peripheral blood. The genotype distribution of both the BPVs and OaPVs detected in placentas and fetal organs indicated a stronger concordance with the viral genotypes detected in bladder tumors rather than in peripheral blood. This suggests that the viruses found in placentas and fetuses may have originated from infected bladders. Our study highlights the likelihood of vertical infections with BPVs and OaPVs and emphasizes the importance of gaining further insights into the mechanisms and consequences of this exposure. This study warrants further research as adverse pregnancy outcomes are a major source of economic losses in cattle breeding.

Effect of the age of garments used under real-life conditions on microfibre release from polyester and cotton clothing.

The release of microfibres from fabrics during laundering represents an important source of plastic and natural microfibres to aquatic environments. Garment age - how long the garment has been used - could be a key factor influencing the rate of release, yet most studies of microfibre shedding have only assessed newly manufactured products. To this end, we quantified microfibre release during laundering in domestic washing machines from polyester (PES) and cotton garments (n = 38) used in real-life conditions for periods between 1 and 31 years with different use intensities. In addition, to better understand the factors involved in microfibre releases, fibre composition (different PES percentages) and type of garments (T-shirts, polo shirts, uniforms, sports shirts, and sweatshirts) were examined. All garments released microfibres during washing, while the older garments presented higher releases for clothing with a PES/cotton blend. In general, older garments (15-31 years) released nearly twice as many fibres when washed than newer garments (1-10 years). The mass of microfibres released was consistently greater in garments with a higher proportion of cotton than PES (up to 1.774 mg g-1 in 2% PES and 0.366 mg g-1 in 100% PES fabrics), suggesting that cotton might be released more readily such that the relative proportion of PES in the garments could increase over time. Additionally, SEM images showed fibre damage, with fibres from the older garments exhibiting more peeling and splitting. While it is important to note that the overall environmental footprint is undoubtedly reduced by keeping garments in use for longer periods of time, older garments were shown to release more microfibres.

Anodonta cygnea, a freshwater swan mussel, exposed to diazinon: toxicity thresholds in behaviour and physiology.

Swan mussels (Anodonta cygnea) have been suggested as suitable bioindicators for the presence of pollutants in the environment. Application of the physiological and behavioral markers in these sessile species can be beneficial for environmental monitoring. The present study aimed to investigate the relationship between the behavioral disorders of movement and siphoning associated with the inhibition of tissue Acetylcholinesterase (AChE). For experiments, overally 120 bivalves of Anodonta cygnea (mean total length 80.33 ± 6.7 mm) were transported from the agricultural drains and canals in Sari county (Mazandaran Province, Iran) to our laboratory. First, the LC50-96 h of diazinon was estimated according to the Organization for Economic Co-operation and Development (OECD 1992) guideline with static water conditions. The sub-lethal toxicity pesticide experiments were conducted on the basis of the lowest observed effect concentration (LOEC) and the maximum acceptable toxicant concentration (MATC). The LC50-96 h, LOEC, and MATC values of diazinon were 85.2, 42.1, and 8.5 mg L- 1, respectively. Based on the observations of mussels' movement, the burrowing and displacement decreased with the concentration of toxicant in water. Moreover, the presence of diazinon in water and its exposure to experimental animals significantly reduces their siphoning rate. The RDA showed that the AChE activity had a higher correlation with the siphoning behavior than the movement behavior. The comparison of enzyme activity at different exposure and recovery times showed that there was a significant difference among the groups affected by the consumed pesticide (p = 0.001, between contrasts). The most remarkable morphometric characteristic was the siphon opening that was inversely correlated with the enzymatic activity. Studies in bioethics might benefit from paying attention to these traits that are directly related to the level of toxicity and behavioral adaptations required for animal survival.

Exploring the Impact of Contaminants of Emerging Concern on Fish and Invertebrates Physiology in the Mediterranean Sea.

In this historical context, the Mediterranean Sea faces an increasing threat from emerging pollutants such as pharmaceuticals, personal care products, heavy metals, pesticides and microplastics, which pose a serious risk to the environment and human health. In this regard, aquatic invertebrates and fish are particularly vulnerable to the toxic effects of these pollutants, and several species have been identified as bio-indicators for their detection. Among these, bivalve molluscs and elasmobranchs are now widely used as bio-indicators to accurately assess the effects of contaminants. The study focuses on the catshark Scyliorhinus canicular and on the Mediterranean mussel Mytilus galloprovincialis. The first one is a useful indicator of localised contamination levels due to its exposure to pollutants that accumulate on the seabed. Moreover, it has a high trophic position and plays an important role in the Mediterranean Sea ecosystem. The bivalve mollusc Mytilus galloprovincialis, on the other hand, being a filter-feeding organism, can acquire and bioaccumulate foreign particles present in its environment. Additionally, because it is also a species of commercial interest, it has a direct impact on human health. In conclusion, the increasing presence of emerging pollutants in the Mediterranean Sea is a serious issue that requires immediate attention. Bivalve molluscs and elasmobranchs are two examples of bio-indicators that must be used to precisely determine the effects of these pollutants on the marine ecosystem and human health.

Detection and Quantification of Nocardia crassostreae, an Emerging Pathogen, in Mytilus galloprovincialis in the Mediterranean Sea Using Droplet Digital PCR.

Nocardia crassostreae is a novel pathogen responsible for infections in oysters (Crassostrea gigas) and mussels (Mytilus galloprovincialis). N. crassostreae is also responsible for nocardiosis both in immunocompetent and immunocompromised patients. We investigated N. crassostreae DNA in mussels grown in marine sites of the Mediterranean Sea in the Campania Region. We examined 185 mussel pooled samples by droplet digital PCR (ddPCR) and real-time quantitative PCR (qPCR), each pool composed of 10 mussels and 149 individual mussels. ddPCR detected N. crassostreae DNA in 48 mussel pooled samples and in 23 individual mussel samples. qPCR detected N. crassostreae DNA in six pooled samples and six individual mussel samples. The two molecular assays for the detection of N. crassostreae DNA showed significant differences both in the pooled and in individual samples. Our study demonstrated that ddPCR outperformed real-time qPCR for N. crassostreae DNA detection, thus confirming that ddPCR technology can identify the pathogens in many infectious diseases with high sensitivity and specificity. Furthermore, in individual mussels showing histological lesions due to N. crassostreae, the lowest copy number/microliter detected by ddPCR of this pathogen was 0.3, which suggests that this dose could be enough to cause infections of N. crassostreae in mussels.

Prevalence and genotype distribution of caprine papillomavirus in peripheral blood of healthy goats in farms from three European countries.

Caprine papillomaviruses (ChPVs, Capra hircus papillomaviruses) were detected and quantified for the first time using droplet digital polymerase chain reaction (ddPCR) in blood samples of 374 clinically healthy goats from farms located in Italy, Romania, and Serbia. Overall, ddPCR revealed ChPV DNA in 78 of the 374 examined samples, indicating that ~21% of the goats harbored circulating papillomavirus DNA. In particular, in Italian goat farms, ChPV genotypes were detected and quantified in 58 of 157 blood samples (~37%), 11 of 117 samples from Serbian farms (~9.4%), and 9 of 100 from Romanian blood samples (9%). Blood samples from Italian goat farms showed a high prevalence of ChPV1, which was detected in 45 samples (28.6%). The ChPV2 genotype was detected in 13 samples (~8.3%). Therefore, significant differences in prevalence and genotype distributions were observed. On Serbian and Romanian farms, no significant differences were observed in the genotype prevalence of ChPVs. Molecular findings are consistent with ChPV prevalence, characterized by a territorial distribution similar to that of papillomaviruses in other mammalian species. Furthermore, this study showed that ddPCR is a very sensitive and accurate assay for ChPV detection and quantification. The ddPCR may be the molecular diagnostic tool of choice, ultimately providing useful insights into the molecular epidemiology and field surveillance of ChPV.

Stress related blood values in Scyliorhinus canicula as live-indicators of physiological status after bottom trawling capture activity.

The quantification of capture-related physiological stress is an important factor when assessing the potential for post-release survival in sharks that are incidentally captured. In the absence of these biological data and when the post-release fate is unknown, effective management plans cannot be formulated and may lead to highly susceptible shark populations being overfished. Here, we measured the levels of lactate, glucose, alanine amino transferase (ALT), aspartate amino transferase (AST), Ca2+, Na+ K+,Cl - Mg 2+ and Pi in the plasma of mature and immature lesser spotted dogfish (Scyliorhinus canicula, herein dogfish) which were incidentally captured at two depths (shallow: 50-200 m, and deep: 201-500 m) by bottom trawl off the coast of southern Sicily. These values were used as biomarkers and physiological indicators of the secondary stress response associated with capture. This study found that dogfish captured in deeper waters (below 200 m) had elevated levels of glucose, Na+, Ca2+ and K+ compared to those inhabiting depths less than <200 m. We hypothesize that the elevated levels of physiological stress in dogfish captured at greater depths may be related to the prolonged duration of the interactions with the fishing gear in the area off southern Sicily. Our findings provide new data on the capture-related stress in dogfish and increase the understanding of the potential for post-release survival in sharks captured at two depths by bottom trawl, information that is important for improving the general management plans for the fishery. However, our PC Analysis results revealed that Maturity have a positive contribution from the sample weight, sample length, ALT, AST and a negative contribution from Pi.

Possible etiological association of ovine papillomaviruses with bladder tumors in cattle.

INTRODUCTION: Bladder tumors of cattle are very uncommon accounting from 0.1% to 0.01% of all bovine malignancies. Bladder tumors are common in cattle grazing on bracken fern-infested pasturelands. Bovine papillomaviruses have a crucial role in tumors of bovine urinary bladder. AIM OF THE STUDY: To investigate the potential association of ovine papillomavirus (OaPV) infection with bladder carcinogenesis of cattle. METHODS: Droplet digital PCR was used to detect and quantify the nucleic acids of OaPVs in bladder tumors of cattle that were collected at public and private slaughterhouses. RESULTS: OaPV DNA and RNA were detected and quantified in 10 bladder tumors of cattle that were tested negative for bovine papillomaviruses. The most prevalent genotypes were OaPV1 and OaPV2. OaPV4 was rarely observed. Furthermore, we detected a significant overexpression and hyperphosphorylation of pRb and a significant overexpression and activation of the calpain-1 as well as a significant overexpression of E2F3 and of phosphorylated (activated) PDGFßR in neoplastic bladders in comparison with healthy bladders, which suggests that E2F3 and PDGFßR may play an important role in OaPV-mediated molecular pathways that lead to bladder carcinogenesis. CONCLUSION: In all tumors, OaPV RNA could explain the causality of the disease of the urinary bladder. Therefore, persistent infections by OaPVs could be involved in bladder carcinogenesis. Our data showed that there is a possible etiologic association of OaPVs with bladder tumors of cattle.

Protein network study of human AF4 reveals its central role in RNA Pol II-mediated transcription and in phosphorylation-dependent regulatory mechanisms.

AF4 belongs to a family of proteins implicated in childhood lymphoblastic leukaemia, FRAXE (Fragile X E site) mental retardation and ataxia. AF4 is a transcriptional activator that is involved in transcriptional elongation. Although AF4 has been implicated in MLL (mixed-lineage leukaemia)-related leukaemogenesis, AF4-dependent physiological mechanisms have not been clearly defined. Proteins that interact with AF4 may also play important roles in mediating oncogenesis, and are potential targets for novel therapies. Using a functional proteomic approach involving tandem MS and bioinformatics, we identified 51 AF4-interacting proteins of various Gene Ontology categories. Approximately 60% participate in transcription regulatory mechanisms, including the Mediator complex in eukaryotic cells. In the present paper we report one of the first extensive proteomic studies aimed at elucidating AF4 protein cross-talk. Moreover, we found that the AF4 residues Thr(220) and Ser(212) are phosphorylated, which suggests that AF4 function depends on phosphorylation mechanisms. We also mapped the AF4-interaction site with CDK9 (cyclin-dependent kinase 9), which is a direct interactor crucial for the function and regulation of the protein. The findings of the present study significantly expand the number of putative members of the multiprotein complex formed by AF4, which is instrumental in promoting the transcription/elongation of specific genes in human cells.

Evaluation of toxicity of Personal Care Products (PCPs) in freshwaters: Zebrafish as a model.

Personal care products (PCPs) are part of the large and growing family of emerging contaminants (ECs). Many daily products such as sunscreens, toothpaste, make-up products, perfume, and others, fall under this definition, and their use is increasing exponentially. Furthermore, the degradation of some components of these products is limited. Indeed, they are able to easily reach and accumulate in aquatic systems, representing a new class of contaminants. Moreover, due to their chemical properties, they can interfere at different biological levels, and for this reason, they need to be thoroughly investigated. We have reviewed the literature on PCPs, with a special focus on the adverse effects on the freshwater zebrafish (Danio rerio). The aim of this work is to provide a careful assessment of the toxicity of these compounds, in order to raise awareness for more conscious and responsible use.

Evidence of a novel cross-species transmission by ovine papillomaviruses.

Ovine papillomavirus (OaPV) comprises four genotypes; OaPV1, OaPV2 and OaPV4 are fibropapillomaviruses within the genus Deltapapillomavirus, whereas OaPV3 is an epitheliotropic virus that belongs to the genus Dyokappapapillomavirus. To date, all of them have been known to infect sheep only. OaPV1, OaPV2 and OaPV4 have been associated with ovine cutaneous and mucosal fibropapillomas, whereas OaPV3 is a key factor in the squamous cell carcinoma pathway of the sheep skin. Whole blood samples obtained from 128 cattle at public slaughterhouses were investigated using droplet digital polymerase chain reaction (ddPCR). ddPCR is a new-generation PCR technique that enables an accurate and absolute quantification of target molecules with high sensitivity and specificity. All OaPVs were detected by identification and quantification of nucleic acids using specific fluorescent probes. Of 128 blood samples, 100 (∼78%) showed OaPV infections. Further, 42, 35 and 23 blood samples showed single, double and triple OaPV infections, respectively. OaPV1 was responsible for 22 single infections, OaPV2 caused 16 single infections and OaPV3 and OaPV4 caused two single infections each. OaPV1 and OaPV2 were the most frequent ovine viruses in dual and triple infections. In many blood samples, both ovine deltapapillomavirus and dyokappapapillomavirus were found to be transcriptionally active, as shown by the detection and quantification of E5 oncogene transcripts for OaPV1, L1 transcripts for OaPV2, E6 and E7 transcripts for OaPV3 and E6 for OaPV4. OaPVs were found in the blood samples from cattle that shared grasslands rich in bracken ferns known to contain immunosuppressant substances. Furthermore, OaPVs were also found in cattle from intensive livestock farming without any contact with sheep. Because OaPV DNA was detected in both grass hay and corn silage, it is conceivable that these feed may be the viral sources.

Bovine delta papillomavirus E5 oncoprotein negatively regulates the cGAS-STING signaling pathway in cattle in a spontaneous model of viral disease.

Persistent infection and tumorigenesis by papillomaviruses (PVs) require viral manipulation of various cellular processes, including those involved in innate immune responses. The cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) pathway has emerged as an essential innate immune sensing system, that recognizes DNA and trigger potent antiviral effector responses. In this study, we found that bovine PV (BPV) E5 protein, the major oncoprotein of bovine delta PVs, interacts with STING but not with cGAS in a spontaneous BPV infection of neoplastic urothelial cells of cattle. Real-time RT-PCR revealed a significant reduction in both cGAS and STING transcripts in E5-expressing cells. Furthermore, western blot (WB) analysis failed to detect any variation in the expression of interferon-inducible protein 16 (IFI16), an upstream effector of the STING pathway. A ternary complex composed of E5/STING/IFI16 was also observed. Co-immunoprecipitation studies showed that STING interacts with a protein network composed of total and phosphorylated TANK-binding kinase 1 (TBK1), total and phosphorylated interferon regulatory factor 3 (IRF3), IRF7, IKKα, IKKß, IKKϵ, ELKS, MEKK3, and TAK1. RT-qPCR revealed a significant reduction in TBK1 mRNA levels in BPV-infected cells. WB analysis revealed significantly reduced expression levels of pTBK1, which is essential for the activation and phosphorylation of IRF3, a prerequisite for the latter to enter the nucleus to activate type 1 IFN genes. WB also revealed significantly down-expression of IKKα, IKKß, IKKϵ, and overexpression of IRF7, ELKS, MEKK3, and TAK1in BPV-positive urothelial cells compared with that in uninfected healthy cells. Phosphorylated p65 (p-p65) was significantly reduced in both the nuclear and cytosolic compartments of BPV-infected cells compared with that in uninfected urothelial cells. Our results suggest that the innate immune signaling pathway mediated by cGAS-STING is impaired in cells infected with BPV. Therefore, effective immune responses are not elicited against these viruses, which facilitates persistent viral infection and subsequent tumorigenesis.