Molecular findings and virological assessment of bladder papillomavirus infection in cattle.

Bovine and ovine papillomaviruses (BPVs - OaPVs) are infectious agents that have an important role in bladder carcinogenesis of cattle. In an attempt to better understand territorial prevalence of papillomavirus genotypes and gain insights into their molecular pathway(s), a virological assessment of papillomavirus infection was performed on 52 bladder tumors in cattle using droplet digital polymerase chain reaction (ddPCR), an improved version of conventional PCR. ddPCR detected and quantified BPV DNA and mRNAs in all tumor samples, showing that these viruses play a determinant role in bovine bladder carcinogenesis. OaPV DNA and mRNA were detected and quantified in 45 bladder tumors. BPV14, BPV13, BPV2, OaPV2, OaPV1, and OaPV3 were the genotypes most closely related to bladder tumors. ddPCR quantified BPV1 and OaPV4 DNA and their transcripts less frequently. Western blot analysis revealed a significant overexpression of the phosphorylated platelet derived growth factor ß receptor (PDGFßR) as well as the transcription factor E2F3, which modulate cell cycle progression in urothelial neoplasia. Furthermore, significant overexpression of calpain1, a Cys protease, was observed in bladder tumors related to BPVs alone and in BPV and OaPV coinfection. Calpain1 has been shown to play a role in producing free transcription factors of the E2F family, and molecular findings suggest that calpain family members work cooperatively to mutually regulate their protease activities in cattle bladder tumors. Altogether, these results showed territorial prevalence of BPV and OaPV genotypes and suggested that PDGFßR and the calpain system appeared to be molecular partners of both BPVs and OaPVs.

Molecular detection of transcriptionally active ovine papillomaviruses in commercial equine semen.

Virological evaluation was performed on equine semen to detect the presence of papillomaviruses (PVs) using droplet digital polymerase chain reaction (ddPCR) as the aim of this study was to investigate whether the sperm from asymptomatic stallions harbors ovine papillomaviruses (OaPVs). Twenty-seven semen samples were analyzed, 18 of which were commercially acquired. The remaining nine samples comprising semen and peripheral blood, were collected from nine stallions with no apparent signs of PV-related diseases during clinical examination at the Didactic Veterinary University Hospital (DVUH) of Naples. OaPV was detected in 26 semen samples. OaPV1 was the most prevalent virus infecting equine semen. OaPV1 infected 21 semen samples (~80.8%) and showed a high number of DNA and RNA copies per microliter. qPCR was used to detect OaPV1 DNA in the 18 semen samples. ddPCR was used to detect and quantify the expression of OaPV2, OaPV3, and OaPV4. qPCR failed to detect DNA for these genotypes. Additionally, ddPCR was used to detect the transcriptionally active OaPV1 in six blood and semen samples from the same stallion. ddPCR failed to detect any nucleic acids in OaPVs in peripheral blood samples from the three stallions. In one semen sample, ddPCR detected OaPV1 DNA but failed to detect any nucleic acid in the remaining two semen samples, and peripheral blood from the same animals of the remaining 18 semen samples was not available, OaPV1 and OaPV4 were responsible for nine and five single infections, respectively. No single infections with either OaPV3 or OaPV4 were seen.

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.

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.

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.

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.

Molecular Epidemiology of Ovine Papillomavirus Infections Among Sheep in Southern Italy.

Ovine papillomaviruses (OaPVs) were detected and quantified, for the first time, using droplet digital polymerase chain reaction (ddPCR) and real-time quantitative PCR (qPCR) via blood samples of 165 clinically healthy sheep. OaPV DNA was detected in 126 blood samples (~76.4%). DdPCR detected OaPV DNA in 124 samples; in only two additional samples positive for real-time qPCR, ddPCR failed to detect the presence of any OaPVs. In 70 of the positive samples (~55.6%), a single OaPV infection was observed, 12 of which were caused by OaPV1 (~17.1%) and 14 by OaPV2 (20%). OaPV3 was responsible for 19 single infections (~27.1%), and OaPV4 for 25 single infections (~35.7%). Multiple OaPV coinfections were observed in 56 (~44.4%) positive samples. OaPV coinfections caused by two genotypes were observed in 31 positive samples (~55.4%), with dual OaPV3/OaPV4 infection being the most prevalent as seen in 11 blood samples. In addition, five OaPV1/OaPV4, four OaPV1/OaPV2, four OaPV2/OaPV3, four OaPV1/OaPV3, and three OaPV2/OaPV4 dual coinfections were also detected. OaPV coinfections by triple and quadruple genotypes were detected in 24 (~42.8%) and only one (~1.8%) of coinfected blood samples, respectively. Multiple infections caused by OaPV1/OaPV3/OaPV4 genotypes were the most prevalent, as observed in 12 (50%) blood samples harboring triple OaPV infections. This study showed that ddPCR is the most sensitive and accurate assay for OaPV detection and quantification thus outperforming real-time qPCR in terms of sensitivity and specificity. Therefore, ddPCR may represent the molecular diagnostic tool of choice, ultimately providing useful insights into OaPV molecular epidemiology and field surveillance.

Bovine Delta Papillomavirus E5 Oncoprotein Interacts With TRIM25 and Hampers Antiviral Innate Immune Response Mediated by RIG-I-Like Receptors.

Persistent infection and tumourigenesis by papillomaviruses (PVs) require viral manipulation of various of cellular processes, including those involved in innate immune responses. Herein, we showed that bovine PV (BPV) E5 oncoprotein interacts with a tripartite motif-containing 25 (TRIM25) but not with Riplet in spontaneous BPV infection of urothelial cells of cattle. Statistically significant reduced protein levels of TRIM25, retinoic acid-inducible gene I (RIG-I), and melanoma differentiation-associated gene 5 (MDA5) were detected by Western blot analysis. Real-time quantitative PCR revealed marked transcriptional downregulation of RIG-I and MDA5 in E5-expressing cells compared with healthy urothelial cells. Mitochondrial antiviral signalling (MAVS) protein expression did not vary significantly between diseased and healthy cells. Co-immunoprecipitation studies showed that MAVS interacted with a protein network composed of Sec13, which is a positive regulator of MAVS-mediated RLR antiviral signalling, phosphorylated TANK binding kinase 1 (TBK1), and phosphorylated interferon regulatory factor 3 (IRF3). Immunoblotting revealed significantly low expression levels of Sec13 in BPV-infected cells. Low levels of Sec13 resulted in a weaker host antiviral immune response, as it attenuates MAVS-mediated IRF3 activation. Furthermore, western blot analysis revealed significantly reduced expression levels of pTBK1, which plays an essential role in the activation and phosphorylation of IRF3, a prerequisite for the latter to enter the nucleus to activate type 1 IFN genes. Our results suggested that the innate immune signalling pathway mediated by RIG-I-like receptors (RLRs) was impaired in cells infected with BPVs. Therefore, an effective immune response is not elicited against these viruses, which facilitates persistent viral infection.

Detection and quantification of bovine papillomavirus DNA by digital droplet PCR in sheep blood.

Highly pathogenic bovine papillomaviruses (BPVs) were detected and quantified for the first time using digital droplet polymerase chain reaction (ddPCR) by liquid biopsy in 103 clinically healthy sheep. Overall, ddPCR detected BPVs in 68 blood samples (66%). BPV infection by a single genotype was revealed in 61.8% of the blood samples, and BPV coinfection by double, triple or quadruple genotypes was observed in 38.2% of liquid biopsies. The BPV-2 genotype was most frequently seen in sheep, whereas BPV-1 was the least common. Furthermore, ddPCR was very useful for detection and quantification; the BPV-14 genotype was observed for the first time in ovine species, displaying the highest prevalence in some geographical areas (Apulia). In 42 of the positive samples (61.8%), a single BPV infection was observed, 26 of which were caused by BPV-2 (61.9%) and 7 by BPV-13 (16.7%). BPV-14 was responsible for 7 single infections (16.7%) and BPV-1 for 2 single infections (4.7%). Multiple BPV coinfections were observed in the remaining 26 positive samples (38.2%), with dual BPV-2/BPV-13 infection being the most prevalent (84.6%). BPV infection by triple and quadruple genotypes was also observed in 11.5% and 3.8% of cases, respectively. The present study showed that ddPCR, a biotechnological refinement of conventional PCR, is by far the most sensitive and accurate assay for BPV detection compared to conventional qPCR. Therefore, ddPCR displayed an essential diagnostic and epidemiological value very useful for the identification of otherwise undetectable BPV genotypes as well as their geographical distributions and suggesting that animal husbandry practices contribute to cross-species transmission of BPVs.

The diagnostic value of the droplet digital PCR for the detection of bovine deltapapillomavirus in goats by liquid biopsy.

In the present study, the highly pathogenic bovine deltapapillomavirus (δPV) was investigated by liquid biopsy in blood samples of 168 clinically normal goats using both droplet digital PCR (ddPCR) and quantitative real-time PCR (qPCR). Overall, ddPCR discovered BPV E5 DNA in ~ 61.3% of the blood samples examined, while real-time qPCR revealed the virus in ~ 10.7% of the same samples. Moreover, ddPCR showed BPV E5 DNA in ~ 78.8% of blood samples from goats that were in close contact with cattle and in 20% of blood samples from goats living in closed pens without any contact with cattle. In addition, ddPCR unmasked a single BPV genotype in ~ 59.2% and multiple genotypes in ~ 40.8% of goats harbouring BPV DNA, while real-time qPCR detected single genotypes in ~ 17% and multiple genotypes in ~ 1%. Of the BPV co-infections detected by ddPCR, 28 (~67%) involved two genotypes, eight (~19%) three genotypes and six (~14%) four genotypes. In contrast, real-time qPCR revealed BPV co-infection by two genotypes in only one sample and failed to detect co-infection by three or four genotypes. BPV2 and BPV13 were the most prevalent viruses responsible for single and multiple co-infections, respectively. The present study showed that ddPCR is promising method for circulating bovine papillomavirus DNA detection and quantification and suggested that animal husbandry practices contribute to cross-species transmission of BPVs.

Digital droplet PCR for the detection and quantification of circulating bovine Deltapapillomavirus.

In this study, the digital droplet polymerase chain reaction (ddPCR) was used to quantify circulating bovine papillomavirus (BPV; genus: Deltapapillomavirus) DNA levels in blood samples from 25 clinically normal cows and 15 cows with chronic enzootic haematuria due to papillomavirus-associated bladder tumours. ddPCR detected BPV DNA in 95% of all the samples (i.e. in 24 of the clinically normal cows and 14 of the diseased animals), whereas quantitative real-time PCR (qPCR) detected it in only 57.5% of the same blood samples, with percentage differences between ddPCR and qPCR being statistically significant (p-value ≤ .05), according to chi-squared test. Furthermore, ddPCR detected BPV infections by a single genotype and by multiple genotypes in 37% and 63% of the cows, whereas qPCR detected these in 16% and 16%. Of the two assays, ddPCR was the more sensitive and accurate clinical diagnostic tool, allowing the detection of otherwise undetectable BPV genotypes, and consequently, a higher number of BPV co-infections. qPCR failed to detect many BPV co-infections by multiple genotypes. Therefore, ddPCR may be an essential tool for improving diagnostic procedures, allowing the identification of the genotypic distribution of BPV and a better understanding about the territorial divergence, if any, of the BPV prevalence in different areas. No significant differences in the blood viral load estimations were observed between the two animal groups, suggesting that the bloodstream could be a site of primary infection. Finally, as BPV DNA was detected in cows affected by non-invasive urothelial tumours, including papilloma and papillary urothelial neoplasms of low malignant potential, the circulating BPVs appeared to be independent of the status of urothelial neoplasms. Therefore, unlike in humans, circulating BPVs cannot be an actual prognostic marker of urothelial tumours in cows.

Prohibitin 2 is Involved in Parkin-Mediated Mitophagy in Urothelial Cells of Cattle Infected with Bovine Papillomavirus.

Prohibitin 2 (PHB2), an inner mitochondrial membrane (IMM) protein, has recently been identified as a novel receptor involved in parkin-mediated mitophagy. In the field of veterinary medicine, the role of PHB2 in parkin-mediated mitophagy was described, for the first time, in urothelial cells of cattle, naturally infected with bovine papillomavirus (BPV). The BPV2 and BPV13 E5 oncoprotein, responsible for abortive infections in urothelial cells, was detected by RT-PCR. Severe ultrastructural abnormalities of the inner mitochondrial membrane were detected using transmission electron microscopy. PHB2 formed a functional complex with PHB1. PHB2 was significantly overexpressed in mitochondrial fractions from urothelial mucosa samples taken from cattle harbouring BPV infection. PHB2 overexpression could be attributed to mitochondrial dysfunction, as its expression levels in the cytosolic, microsomal, and nuclear fractions were seen to be unmodified. Immunoprecipitation studies revealed the interaction between PHB2 and phosphorylated forms of both PINK1 and parkin. Furthermore, PHB2 interacted with LC3-II, a marker of autophagosomal membranes and autophagy receptors, such as p62 and optineurin. PHB2 was shown to interact with transcription factor EB (TFEB), which is activated following parkin-mediated mitophagy, and embryonic stem cell-expressed Ras (ERAS), a constitutive protein coded by ERas. Western blot analysis revealed a significant overexpression of unphosphorylated TFEB in mitochondrial and nuclear fractions from urothelial mucosa samples from cattle suffering from BPV infection. Finally, PHB2 interacted with ERAS, believed to be involved in mitophagosome maturation. Taken together, the molecular and ultrastructural findings of this study suggested that BPV infection is responsible for parkin-dependent mitophagy, in the pathway of which PHB2 plays a crucial role.

Bovine papillomavirus E5 oncoprotein upregulates parkin-dependent mitophagy in urothelial cells of cattle with spontaneous papillomavirus infection: A mechanistic study.

This study aimed to provide mechanistic insights into mitophagy pathway associated with papillomavirus infection in urothelial cells of cattle. The elimination of mitochondria via autophagy, termed mitophagy, is an evolutionarily conserved mechanism for mitochondrial quality control and homeostasis. PINK1/parkin-mediated mitophagy, a ubiquitin-dependent selective autophagy of dysfunctional mitochondria, has been described here, for the first time, in urothelial cells from 25 bladder cancers in cattle infected by bovine papillomavirus (BPV). The expression of BPV-2 and BPV-13 E5 oncoprotein was detected by RT-PCR. Abnormal mitochondria delimited by expanding phagophores, were peculiar ultrastructural features of neoplastic urothelial cells. High levels of mitochondrial phosphorylated PINK1/parkin were observed in neoplastic urothelial cells infected by BPVs. Phosphoparkin interacted with mitofusin 2 (Mfn2) and ubiquitin (Ub), which confirmed that Mfn2 is a parkin receptor at the mitochondrial level, where parkin interacted also with Ub. Furthermore, parkin established a complex that was comprised of optineurin, p62, LC3, laforin, and embryonic stem cell-expressed Ras (ERAS), that interacted with BPV E5 oncoprotein, and Bag3, which, in turn, regulated the formation of a complex composed of Hpc70/Hsp70, CHIP, an HSC70-interacting E3 ubiquitin ligase. It is conceivable that ERAS is involved in mitophagosome maturation via phosphatidylinositol 3-kinase (PI3K) pathway. Bag3, in association with Hsc70/Hsp70, may contribute to the transport and degradation of CHIP-ubiquitinated cargo as this complex recognises ubiquitinated cargos and transports them to aggresomes to be degraded. Furthermore, Bag3 may be involved in mitophagosome formation as it interacted with synaptopodin 2, which is known to play a role in mitophagosome biogenesis.

Biological interactions of biocompatible and water-dispersed MoS2 nanosheets with bacteria and human cells.

Two dimensional materials beyond graphene such as MoS2 and WS2 are novel and interesting class of materials whose unique physico-chemical properties can be exploited in applications ranging from leading edge nanoelectronics to the frontiers between biomedicine and biotechnology. To unravel the potential of TMD crystals in biomedicine, control over their production through green and scalable routes in biocompatible solvents is critically important. Furthermore, considering multiple applications of eco-friendly 2D dispersions and their potential impact onto live matter, their toxicity and antimicrobial activity still remain an open issue. Herein, we focus on the current demands of 2D TMDs and produce high-quality, few-layered and defect-free MoS2 nanosheets, exfoliated and dispersed in pure water, stabilized up to three weeks. Hence, we studied the impact of this material on human cells by investigating its interactions with three cell lines: two tumoral, MCF7 (breast cancer) and U937 (leukemia), and one normal, HaCaT (epithelium). We observed novel and intriguing results, exhibiting evident cytotoxic effect induced in the tumor cell lines, absent in the normal cells in the tested conditions. The antibacterial action of MoS2 nanosheets is then investigated against a very dangerous gram negative bacterium, such as two types of Salmonellas: ATCC 14028 and wild-type Salmonella typhimurium. Additionally, concentration and layer-dependent modulation of cytotoxic effect is found both on human cells and Salmonellas.