Due to the overlapping epidemiology and clinical manifestations of flaviviruses, differential diagnosis of these viral diseases is complicated, and the results are unreliable. There is perpetual demand for a simplified, sensitive, rapid and inexpensive assay with less cross-reactivity. The ability to sort distinct virus particles from a mixture of biological samples is crucial for improving the sensitivity of diagnoses. Therefore, we developed a sorting system for the subsequent differential diagnosis of dengue and tick-borne encephalitis in the early stage. We employed aptamer-modified polystyrene (PS) microspheres with different diameters to specifically capture dengue virus (DENV) and tick-borne encephalitis virus (TBEV), and utilized a traveling surface acoustic wave (TSAW) device to accomplish microsphere sorting according to particle size. The captured viruses were then characterized by laser scanning confocal microscopy (LSCM), field emission scanning electron microscopy (FE-SEM) and reverse transcription-polymerase chain reaction (RT‒PCR). The characterization results indicated that the acoustic sorting process was effective and damage-free for subsequent analysis. Furthermore, the strategy can be utilized for sample pretreatment in the differential diagnosis of viral diseases. Flaviviruses, the leading cause of arthropod-borne diseases in humans, are single-stranded RNA, enveloped viruses belonging to the Flaviviridae family. Flaviviruses include 53 recognized virus species, of which 40 species are known to cause disease in humans. The most well-known members include dengue virus (DENV), Zika virus (ZIKV), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), tick-borne encephalitis virus (TBEV) and Langat virus (LGTV)2. Among these flaviviruses, DENV is one of the most important mosquito-borne viruses, and TBEV is among the most common tick-transmitted pathogens. There are many flu-like initial symptoms, including fever, fatigue, general malaise, headache and muscular pain, at the early stage of flavivirus infection3. Therefore, differential diagnosis of dengue and tick-borne encephalitis (TBE) in the early stage based on clinical symptoms alone is unreliable4. Inaccurate diagnosis may cause unnecessary hospital admission and in turn increase the burden on health care systems. Conventional laboratory diagnostic assays have long been used as tools for detecting DENV in clinical practice. Nonetheless, these detection methods, e.g., virus isolation–based molecular assay methods, are time-consuming, difficult, and require skilled technicians and central laboratory facilities. The major problem with serological methods for DENV detection is their strong predisposition to cross-react with antibodies, which are produced in response to other simultaneous and previous flavivirus infections 5. Therefore, there is a perpetual demand for a simplified, sensitive, rapid and inexpensive assay with less cross-reactivity. To meet the demand, the ability to sort distinct virus particles from a mixture of biological samples is crucial for improving the sensitivity of the test.Viral infections are a grave threat to public health as well as the global economy due to their high pathogenicity for humans and animals. Viral infections mostly occur through contaminated water, food, bodily fluids, and even air, spread rapidly and result in the death of humans and animals worldwide6. Similar to the conformational recognition between antibodies and antigens, aptamers bind to their cognate targets with high specificity and affinity through various types of interactions, such as van der Waals forces, hydrogen bonds, electrostatic interactions and bonds between complementary bases9. Due to their striking advantages over traditional antibodies, such as ease of selection, low cost of synthesis, high stability-specificity-sensitivity, and potential for modifications and tagging, aptamers are considered potential alternatives to monoclonal antibodies in many fields, including diagnostics, therapeutics, cell imaging, drug delivery, selective chromatography, biosensing, and biomarker discovery10. The characteristic properties of the aptamers previously mentioned make them valuable tools for research in viral diagnosis and therapy against viral infection as well. Many previous studies have used aptamers to capture and detect viruses such as human papillomavirus (HPV)11, hepatitis B virus (HBV)12, hepatitis C virus (HCV)14, norovirus (NoV)17, ZIKV19, and DENV21. Fletcher et al.20 developed an aptamer-based biosensor using DNA aptamers for the DENV genome, generating fluorescence to confirm the presence of dengue sequences. Basso et al.21 reported an aptasensor composed of γ-Fe2O3(SAMN) nanoparticles and modified with gold nanoparticles (AuNPs) conjugated to aptamers that can specifically bind to four dengue serotypes.Due to the nanometer size of virus particles, it is difficult to sort distinct virus particles from biological samples directly. To sort the specific virus, aptamer-modified solid substrates  were developed to capture different viruses. Aptamers are RNA or ssDNA molecules of approximately 20 to 80 bases with a specific conformational affinity toward target molecules, including proteins, small molecules, metal ions, bacterial cells, viruses, cancer cells, and even tissues23, electrical24, magnetic25, optical26, and mechanical systems27. Among the various techniques, acoustofluidics, the integration of acoustics and microfluidics, can separate particles depending on their different physical properties, such as density, size, and compressibility. Acoustofluidic separation offers a highly controllable, label-free, contactless, damage-free and biocompatible approach that relies on the differential effect of acoustic streaming and radiation forces acting on particles suspended in a liquid30. It has increasingly been applied in the separation of different biological targets from complex fluids, such as proteins32, circulating tumor cells (CTCs)33, viruses34 and small extracellular vehicles (EVs)41. Sung et al.31 simultaneously separated proteins of three kinds  via TSAW-driven ARF in the proposed acoustofluidic device, which can be used as a promising tool for various protein-based clinical diagnostic assays. Wu et al.33 introduced a high-throughput, acoustic-based CTC-separation device that used tilted-angle standing surface acoustic waves for ex vivo CTC growth studies and CTC phenotypic studies. They further achieved the successful isolation and phenotypic characterization of CTCs from clinical patient samples with metastatic prostate cancer. Later, the same group reported a standing surface acoustic wave (SSAW) device to separate EVs and lipoproteins based on their acoustic properties, further expanding the capability of acoustofluidic separation technology for separating submicron-scale objects35. The study of Ku et al.37 proposed a noncontact platform that makes use of ultrasonic wave scattering between micrometer-sized seeding particles and nanoparticles of interest in a resonant cavity for EV enrichment. The platform named acoustic trapping can be used to isolate EVs, including microvesicles and exosome-size vesicles, from cell culture conditioned media, human urine, and plasma samples. Most studies mentioned above have focused on separating one special biological target from complex fluids, but few studies have been concerned with sorting multiple different targets from samples.When sorting micro- or nanoparticles, microfluidic devices exhibit several advantages, including the capability of being integrated into various drive systems, including acousticIn this study, we developed an efficient multiple virus sorting method based on aptamer-modified microspheres in a traveling surface acoustic wave (TSAW) device. Here, we employed aptamer-modified polystyrene (PS) microspheres with different diameters to specifically capture DENV and TBEV and utilized a TSAW device to accomplish microsphere sorting according to particle size. The sorted viruses were characterized by laser scanning confocal microscopy (LSCM), field emission scanning electron microscopy (FE-SEM) and reverse transcription-polymerase chain reaction (RT‒PCR) to confirm that the strategy was suitable for subsequent tests.Carboxy polystyrene (PS) microspheres were purchased from Unibead Scientific Co. Ltd. . N-hydroxysulfosuccinimide (Sulfo-NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Thermo. Phosphate buffer solution (PBS) (pH 7.4), trihydroxymethyl aminomethane-hydrochloric acid (Tris-HCl) and 2-(N-morpholino) ethanesulfonic acid hydrate (MES) buffer  were purchased from Leagene Biotechnology . Dengue virus (DENV), tick-borne encephalitis virus-like particles (TBEV), Japanese encephalitis virus (JEV), and coxsackie virus B3 (CVB3) were obtained from the Fourth Military Medical University. Bovine serum albumin (BSA) and fetal bovine serum (FBS) were purchased from Sangon Biotechnology .42. The width and height of the polydimethylsiloxane (PDMS) microchannel are 300 μm and 80 μm, respectively. To fabricate the acoustofluidic device, the photoresist  was patterned on the transparent LiNbO3 substrate by standard lithography, then metal layers  were deposited on the substrate by electron beam evaporation . Finally, lift-off technology was used to obtain the target IDTs. The PDMS microchannel was fabricated by standard soft lithography technology. The microchannel mold was fabricated on the silicon wafer using SU8 photoresist. PDMS was mixed with the curing agent at a ratio of 10:1 and poured into the microchannel mold. After curing for 4 h at 85 °C, the PDMS microchannel was peeled off from the mold and cut into an appropriate size. The PDMS microchannel was bonded with a thin PDMS film (50 μm thick) after oxygen plasma treatment. Finally, to fabricate the TSAW device, the PDMS microchannel was aligned on the IDTs with oil as a waveguide layer. This type of prebonded microchannel permits easier replacement of the microchannel and the IDTs and greatly reduces the fabrication cost of the acoustofluidics device. The structure and image of the TSAW device are shown in Fig. The main structure parameters of IDTs in the TSAW device design we need to consider are the aperture, the width and the pitch of each electrode finger, and the number of finger pairs (N). Normally, the size of the width and pitch of each linear electrode finger is designed with the same value in microfluidic devices. The focused interdigital transducers (FIDTs) with arc electrodes in our microfluidic device contain 15 pairs of focused electrodes, and the acoustic aperture is 1 mm. The designed width and pitch of each electrode is 20 μm, corresponding to a TSAW wavelength of 80 μm. The degree of the arc of the FIDTs is set to 60°, and the induced SAW is considered to have smaller energy attenuation, higher focused energy and longer propagating distance43.Aptamers used for virus capture and primers applied in the RT‒PCR procedure were synthesized by Sangon Biotechnology . The sequences are listed in Table First, a 50 μL solution of carboxy-coated PS microspheres  was washed three times with PBS buffer to remove the storage solvent completely and resuspended in 500 μL of MES buffer . Then, 10 mg of EDC and 10 mg of Sulfo-NHS were added to the above MES buffer and reacted for 15 min at room temperature. Then, the PS microspheres were washed twice quickly with PBS buffer and resuspended in 500 μL of MES buffer . Here, the washing procedure can remove the excess reagent and is useful for avoiding the influence of hydrolysis or excessive EDC reaction on aptamer modification. A 10 μL solution of DENV capture aptamer (5 µM) was added into the above solution and reacted at room temperature for 2 h with shaking at 180 r/min. Finally, the aptamer-modified PS microspheres (PS15-A) were blocked with 300 μl of 3% BSA for 30 min, suspended in 500 μL of PBS (pH 7.4), and finally stored at 4 °C for further use. The blocking procedure is beneficial to reduce nonspecific adsorption and binding on the microsphere surface. In accordance with the aforementioned method, the TBEV capture aptamer was conjugated on the 2 μm PS microspheres to prepare the complexes (PS2-A). The conjugation between the aptamer and the PS microspheres was verified by Fourier transform infrared spectroscopy (FTIR) on a Nicolet iS10 .The schematic workflow of the acoustofluidic sorting of DENV and TBEV particles is shown in Fig. 28. Here, we used a typical TSAW on 128° rotated Y-cut lithium niobate substrates generated by a single IDT oriented at a certain angle with respect to the X-direction of the crystal. The TSAW propagated toward the PDMS microchannel and radiated into the fluid at a Rayleigh angle θR, and θR was approximately 22°, as calculated by the following Eq.  and acoustic wave frequency (f). Here, we employed 2 μm and 15 μm PS microspheres to sort different viruses, and the values of κ were 0.21 to 2 μm and 1.57 to 15 μm, respectively, under the 50 MHz TSAW. This means that only particles with a size of 15 μm could be affected by the radiation force and induce a differential streamline for particles under TSAW. The PS microsphere-Aptamer-DENV (PS15-A-DENV) and PS microsphere-Aptamer-TBEV (PS2-A-TBEV) complexes were sorted from the mixed virus solution in the PDMS microchannel by TSAW. Briefly, the mixed sample solution was injected into the center inlet of the PDMS microchannel. Two PBS sheath flows were injected into the adjacent inlets, and the sample solution was forced into a narrow and straight line. The flow rate in the microchannel was controlled within 2000-2800 μl per hour by regulating the inlet pressure. To achieve high separation efficiency, the limit was calculated as 2800 μl per hour in the situation, in which the maximum tolerable power input was set on the IDTs. When the IDTs were turned on with a frequency of 46 MHz and an input power of 8 W, the PS15-A-DENV complexes were driven into the PBS stream and collected in outlet 1. The rest of the sample solution containing PS2-A-TBEV was collected in outlet 2.The collected PS15-A-DENV complexes in outlet 1 were labeled with the DENV marker aptamer conjugated with fluorophore 6-FAM, while the collected PS2-A-TBEV composites in outlet 2 were labeled with the TBEV mark aptamer conjugated with fluorophore Texas Red. Then, the labeled complexes were observed by laser scanning confocal microscopy (LSCM) and FS5 spectrofluorometry (Edinburgh Instruments). At the same time, samples containing Japanese encephalitis virus (JEV) and Coxsachie virus B3 (CVB3) were used as parallel controls to ensure the specific recognition ability of the DENV capture aptamer and the TBEV capture aptamer.The surface morphologies of the PS15-A-DENV complexes and PS2-A-TBEV complexes were obtained by field emission scanning electron microscopy . The PS particles were dispersed on the cooper mesh and observed by FE-SEM. Reverse transcription-polymerase chain reaction (RT‒PCR) was employed to detect DENV NS5 and YFV NS5 genetic expression to further confirm the successful capture of DENV and TBEV. Briefly, the viruses captured by the PS-A were lysed to extract the RNAs, and then RNAs were converted into their complementary DNA (cDNA) sequences by reverse transcriptase, followed by amplification of the newly synthesized cDNA by standard polymerase chain reaction (PCR) procedures. The PCR product was detected by real-time fluorescent quantitative PCR on a CFX 96 Touch , and the results were analyzed by Bio-Rad CFX Manager3.43. All the selected aptamers have classic hairpin structures that help the aptamers recognize and capture targets. The DENV capture aptamer and mark aptamer were both specific for the four dengue serotypes. Their efficacy and specificity were verified in the work of Chen et al.45 The TBEV capture aptamer and mark aptamer can bind TBEV effectively and specifically46.The secondary structure of the aptamers was analyzed by the Mfold web platform and is shown in Fig. 47. As shown in Fig. −1 and 1068 cm−1 of the benzene ring in the polystyrene and the characteristic peaks of 2849 cm−1 and 2923 cm−1 of the -CH2- group in the PS microspheres. Compared to the control PS microspheres, the microsphere samples coupled with the aptamer produced an additional 1420 cm−1 characteristic peak of amide bond -C–N- stretching vibration, 1600 cm−1 characteristic peak of -NH- flexural vibration, and 1715 cm−1 characteristic peak of C and G bases before and after virus capture. The amide -C–N- bond was formed between the aptamer 5′ end conjugated -NH2 group and the -COOH group modified on the PS microspheres. A slight difference in the FTIR spectrograms was obtained after virus capture. This may be because the main components of virus, RNA, and proteins share most groups with the BSA and aptamer on the modified PS microspheres.The conjugation between the aptamer and the PS microspheres was verified by Fourier transform infrared spectroscopy (FTIR)The PS microsphere-Aptamer-DENV (PS15-A-DENV) and PS microsphere-Aptamer-TBEV (PS2-A-TBEV) complexes were sorted from the mixed virus solution in the PDMS microchannel by a TSAW device. As shown in Fig. The PS microspheres collected from outlet 1 and outlet 2 were labeled with 6-FAM coupled aptamer (DENV mark aptamer) and Texas Red coupled aptamer (TBEV mark aptamer), respectively. Subsequently, the labeled samples were observed by laser scanning confocal microscopy (LSCM) excited at 488 nm and 561 nm. The obtained confocal microscopy photographs are shown in Fig. However, LSCM is regularly used for qualitative detection. Due to the smaller scale and lower distinguishability of 2 μm PS microspheres, red fluorescence was more difficult to detect by LSCM than green fluorescence. To achieve quantitative detection of fluorescence, we employed an FS5 spectrofluorometer to obtain the fluorescence intensity of the samples in FBS and PBS to simulate the physiological environment. In this work, we adopted fluorescence intensity for semiquantitative detection of different viruses. As shown in Fig. 48, while the TBEV particles were approximately 50 nm in diameter with a smooth surface49.The SEM photographs of PS15-A-DENV and PS2-A-TBEV complexes were obtained by FE-SEM to visually confirm the capture of virus and are shown in Fig. Relative DENV NS5 gene expression was detected by RT‒PCR with actin as the housekeeping gene. As shown in Fig. In conclusion, we developed an efficient sorting method based on specific capture of virus by aptamer-modified microspheres and acoustofluidic separation, which is effective and damage-free for subsequent analysis. By this method, contactless and biocompatible separation of two different viruses was accomplished in this work. By modifying the PS microspheres with aptamers, the specification of the captured viruses is labeled by the size of the particles, which corresponds to different radiation forces in the same acoustic field. The acoustofluidic device using TSAW was introduced to complete the sorting of PS microspheres with different diameters. A 15 μm PS microsphere purity of 98.46 ± 2.56% and a 2 μm PS microsphere purity of 99.67 ± 0.57% were obtained after sorting samples containing 23.53 ± 8.77% 15 μm PS microspheres. The combination of aptamer-modified PS microspheres and TSAW devices can prevent matrix interference, reduce detection cost, simplify operation and make it easier to sort viruses. The sorted PS microspheres were detected by LSCM, SEM and RT‒PCR. The observed fluorescence signal and relative genetic expression were positively correlated with the virus concentration, which demonstrates little effect of TSAW sorting on subsequent genetic testing and fluorescence detection. It follows that the sorted virus particles can be employed for subsequent biological detection, such as cell culture, ELISA, Western blotting and PCR tests, which have been used for a long time as tools for the diagnosis of viral diseases in clinical practice. Successful sorting of distinct virus particles from a mixture of biological samples is favorable for improving the detection sensitivity. This work shows a promising method for sample pretreatment in the differential diagnosis of viral diseases.50. The sorting method based on specific capture of virus by aptamer-modified microspheres and acoustofluidic separation here can be expanded to multiple virus sorting, and the number of sorted virus species depends on the size differentiation of microspheres by surface acoustic waves. Furthermore, the sorted PS microspheres can be labeled with fluorescent-coupled aptamers for rapid detection of different viruses. Further studies will be carried out to develop a simplified, sensitive, rapid, inexpensive, and less cross-reactive multiplexed detection method for the differential diagnosis of viral diseases.In addition, multiplexed tests covering multiple infectious agents are especially needed in the case of diseases with indistinct clinical symptoms or where coinfections are possible, such as in fever with acute onsetsumpplmentary


Tomato brown rugose fruit virus (ToBRFV) is a contact-transmitted tobamovirus affecting many tomato growing regions of the world. This study investigated the effects of different glasshouse surfaces on the survival of the virus; the efficacy of different disinfectants; and heat treatment against ToBRFV . A bioassay followed by ELISA was used to check virus viability. ToBRFV survived for at least 7 days on all surfaces tested and on some for at least 6 months. The virus survived for over two hours on hands and gloves. Hand washing was shown to be unreliable for the removal of the virus. Glutaraldehyde and quaternary ammonium compound disinfectants were effective at one hour on all surfaces. Some other disinfectants were effective at one hour of contact time, on all surfaces except concrete. Sodium hypochlorite was partially effective against ToBRFV, even on concrete. A 5 min soak of plastic trays in water at 90 °C was effective at denaturing ToBRFV; however, 5 min at 70 °C was not. Heating infected sap showed the thermal inactivation point to be 90 °C, confirming the hot water treatment results and showing that deactivation was due to the heat treatment and not a washing effect of the water. Tobamovirus, family Virgaviridae. As with other members of the genus, the virus is expected to have virions that are thermally stable and survive for long periods of time in plant sap [Tm-22 resistance gene that confers varietal resistance to other tobamoviruses, such as tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV) [Capsicum annuum) [Tomato brown rugose fruit virus (ToBRFV) is an emerging viral pathogen that has been reported infecting tomatoes throughout Europe, North America, China and the Middle East ,2. Tomatlant sap . The virs (ToMV) ,5. The v annuum) .Given the threat that the virus presents to tomato production worldwide, the virus has become the subject of regulatory controls ,7. The kTobamovirus) and found that some of these were effective against liquid inoculum preparations over short exposure times; however, this work did not study the potential impact of surface substrates on the efficacy of disinfectant regimes. It has previously been demonstrated that the substrate surface can influence the survival of contact-transmissible viral pathogens. For example, potato spindle tuber viroid  has been shown to remain infectious for a greater length of time on leather or string than on surfaces such as plastic or metal [There has been less focus on the practical aspects of outbreak management, such as understanding how resilient ToBRFV is with regard to natural degradation, and also the efficacy of disinfectants at reducing the risk of carry-over infection through prophylactic use or re-infection after an outbreak. Samarah et al.  reportedor metal . There aor metal . Whilst or metal ,20 focusTobamovirus, it would be expected that ToBRFV would be robust, with long retention of infectivity  and resistance to disinfection by comparison to other viral pathogens outside of this genus. It is essential to identify effective routine disinfection regimes for prophylactic hygiene treatments. Therefore, this study reports a series of experiments that aimed to determine the survival of ToBRFV and the efficacy of disinfection approaches on a range of surfaces commonly found in a glasshouse environment, including human skin, steel, aluminium, hard plastic, polythene, glass and concrete, with disinfection approaches including thermal deactivation and a range of chemical active ingredients. As a member of the genus The aim of the work described here was to ascertain the viable survival time and determine disinfection efficacy, including thermal inactivation, for ToBRFV. This included a range of experiments to determine survival on a range of surfaces, including hands, gloves, concrete, glass, steel, aluminium, hard plastic and polythene. The efficacy of disinfectants was determined on each of these surfaces. Thermal inactivation was determined both by soaking contaminated plastic surfaces in hot water and confirmed by heating sap from infected plants. The infectivity of sap residues was measured by a bioassay and confirmed via ELISA.2 on a range of different surfaces , by using a cotton bud to produce a thin visible layer of sap. In additional experiments , the surfaces were also contaminated by lightly rubbing with infected leaves from the same source plants. To account for potential differences in hand surfaces and washing techniques, two people of different ages, one male and one female, were selected to carry out the experiments. ToBRFV  was inoculated onto tomato plants, cv. Moneymaker. Leaf material was collected two to three weeks after inoculation and confirmed positive by real time RT-PCR using the specific ToBRFV assay of Menzel and Winter , amendedFor both the survival and disinfection experiments, virus viability was demonstrated by taking swabs at various time points and testing by bioassay and ELISA. Once the inoculum was dry, swabs were taken after initial contamination to show that the initial inoculum was viable. In the case of survival studies, further swabs were taken at specified time points. In the case of hand washing, disinfection and heat treatment studies, swabs were taken post-treatment as specified below. At the end of setting up each experiment, a swab was taken from the buffer and inoculated onto test plants, as a control, to show that the buffer had not become contaminated.Nicotiana tabacum plants cv. White Burley (approximately 5 weeks from sowing). The inoculated leaves were marked by a hole from a pipette tip prior to inoculation. Gloves were worn and changed between each replicate. The N. tabacum plants were then covered with a bread bag , placed on individual plastic saucers to avoid cross-contamination and placed in a glasshouse at 20 to 25 °C for 2 to 3 weeks. N. tabacum was used as a test plant as it is susceptible to ToBRFV and rapidly shows symptoms on the inoculated leaves.Cotton buds, soaked in phosphate buffer pH7 containing celite , a mild abrasive powder, were used to take swabs from different surfaces. Swabs were taken by rubbing the surface with the cotton bud and then these were gently rubbed onto the leaves of N. tabacum leaf was used as a negative control. Samples with absorbance values greater than three times the negative control’s absorbance value were classified as positive. ELISA was performed to confirm that any lesions seen were due to the virus and not from mechanical damage or phytotoxic effects of the disinfectants. Following visual assessment, the inoculated leaves were removed and tested by ELISA for ToBRFV using antisera from DSMZ  according to the manufacturers’ instructions. An uninfected  For each variable, e.g., surface and time, three swabs were taken, and each inoculated onto an individual test plant to give three repetitions for each sample point. All experiments were performed in duplicate at different time points to determine whether results could be consistently generated.Survival was investigated by exposing skin and gloved hands (nitrile-type disposable gloves) to infected sap, and secondly by rubbing the hands with infected leaves. The bare hand and gloved hand were swabbed at 15 min intervals up to 1 h and then 30 min intervals up to 2 h. A range of glasshouse surfaces  were surface-contaminated with ToBRFV-infected leaf sap. A picking crate from a tomato grower was used as the hard plastic surface. The surfaces were kept at ambient temperature throughout the trial (approximately 20 °C) and swabs were taken at different time periods (ranging from 2 h to 6 months) and inoculated onto test plants, to ascertain the length of time for which the virus survived on different surfaces. ®, Mölnlycke, Milton Keynes, UK) and water and medicated hand wash followed by an alcohol gel .Hands were rubbed for approximately 5 s with leaves from ToBRFV-infected tomato plants. Hands were then washed for 30 s or 1 min using the following washes: water only, water and household soap, water and medicated hand wash   and left for 1 min, before more swabs were taken and inoculated onto test plants. To investigate whether any inactivation of the virus, after hot water treatment, was due to the heat treatment or also a washing effect of the water, ground ToBRFV-infected tomato sap  was placed in 1.5 mL micro-centrifuge tubes  suspended in a water bath, for 5 min, at various temperatures ranging from 70 °C to 95 °C; 70 °C is the temperature used for pasteurisation and 90 °C is the thermal deactivation temperature for cucumber green mottle mosaic virus (CGMMV) . After iAs with the survival on glasshouse surfaces experiment, the six surfaces were contaminated with ToBRFV-infected leaf sap. Once the sap on the surfaces was dry, swabs were taken from the surfaces and inoculated onto test plants, to show that the virus was infectious. The surfaces were then sprayed with a disinfectant using a domestic spray bottle , at the recommended rate, and left for various contact times before swabs were taken and inoculated onto test plants. The disinfectants included in the study were chosen because they are widely available to growers and cover several different groups of active compounds. Menno Florades  was chosen as it is approved for use as a plant protection product in the European Union . Huwa-SaInitially, a 1 min contact time was investigated for several disinfectants , sodium hypochlorite and Virkon S)); however, as this short contact time was ineffective, a 60 min contact time was used for all the disinfectants. Minimum effective contact times were then investigated for the disinfectants that showed the best results at 60 min.N. tabacum test plants, to check for viability.Swabs were taken from different surfaces contaminated with ToBRFV-infected sap before and after being sprayed with Virkon and Unifect G  , equipment and surfaces encountered in routine work  and also human skin . The posToBRFV remained viable on all glasshouse surfaces after seven days, infecting all test plants in both experimental repetitions. From 28 days post-contamination, in some cases, there was a difference in the number of positive plants between the two repetitions of the experiments. The virus was still active on most surfaces after 90 days, except for aluminium. Results for concrete were inconsistent: it was found to be negative for ToBRFV after 28 days but positive again after 90 days. ToBRFV was still infectious in sap swabs from glass, hard plastic and polythene even after 180 days, although not all plants were infected in both trial repetitions, indicating some drop in infectivity. Overall, the results show that there is a good chance that ToBRFV will survive on surfaces for (at least) 28 days, and on some surfaces for at least 6 months. ®, York, UK) and hand wash followed by antibacterial gel all appeared to have some limited effect on reducing the levels of infectious ToBRFV on the hands. The results from investigations on the efficacy of hand washing at removing ToBRFV from hands are presented . Washing® plus an antibacterial hand gel appeared successful at removing ToBRFV. Water plus Hibiscrub® was only partially successful at 1 min. Washing contaminated hands for 1 min in water alone, water plus household soap and water with HibiscrubThe results presented in All the disinfectants tested  at a 1 min exposure time failed to give control of ToBRFV . SubsequUnifect G and Virocid   were the most effective on all surfaces at deactivating the virus. These glutaraldehyde/quaternary ammonium products and sodium hypochlorite were the only products to be effective on concrete consistently. Even the increased concentration of Huwa-San , which was effective on all other surfaces, was not effective on concrete. Differing results were seen for Virkon S (a.i. potassium peroxymonosulfate) on concrete, with the first experiment giving 100% effectiveness and the repeat experiment showing two out of three plants infected post-treatment. Virkon S was effective on all other surfaces with a 1 h contact time.Menno Florades (a.i. Benzoic acid) was mostly effective on all surfaces except concrete. Sodium hypochlorite is partially effective at denaturing ToBRFV on polythene, glass and stainless steel and is completely effective against ToBRFV on other surfaces. Huwa-San (3% a.i.), Jet 5 (a.i. peroxyacetic acid) and trisodium orthophosphate (TSOP) were not very effective against ToBRFV with a 1 h treatment. Hard plastic and polythene were potentially the surfaces that the disinfectants deactivated ToBRFV on most successfully. Stainless steel, aluminium and glass were varied in their results, with all disinfectants showing a general reduction in infectivity. Concrete was the surface where the disinfectants had the least effect. Overall results show that Unifect G and Virocid, disinfectants containing glutaraldehyde and quaternary ammonium compounds as the active ingredients, reduced the infectivity of ToBRFV the most after 60 min, with Virkon S (potassium peroxymonosulfate), Menno Florades (a.i. benzoic acid) and a high concentration of Huwa-San (12.5% a.i.) (a.i. hydrogen peroxide) also resulting in a large reduction in infectivity. However, on all surfaces, there was only a small reduction in ToBRFV infectivity after treatment with Huwa-San at 3%.Treatment with Unifect G, for one hour, was shown to be 100% effective against ToBRFV on all surfaces , and furAll indicator plants inoculated from surfaces after treatment with Unifect G, after 10 min contact time, were negative by ELISA for ToBRFV. However, all swabs taken directly from the surfaces were positive by real-time RT-PCR . Whilst There have been a limited number of studies published on the efficacy of disinfectant treatments. Two of these studies have focused on seed treatments to reduce the risk of entry to a production system ,16. A thToBRFV will remain infectious on both skin and disposable (nitrile-type) gloves for at least two hours. Tomato plants are repeatedly handled throughout their production cycle for side-shoot removal, stringing, fruit picking and other activities. The length of survival of the virus underlines the potential for the virus to rapidly spread through a crop via normal production practices. Additionally, the length of time in which the virus can contaminate hands highlights the need for effective biosecurity measures on entry to the production facility. Hand washing results were very variable, even when repeating the same washing conditions, suggesting that it may be the physical act of washing the hands that removes the majority of the virus and not the chemicals used. However, this may also be due to different levels of the virus being deposited on the hands from rubbing infected leaves, or different hand washing techniques used by individuals. As hand washing is unreliable, to achieve the thorough elimination of the virus, a wash of more than 30 s is required, which would be difficult to apply and enforce in practice. In practice, the worker’s hands would be highly contaminated and a 30 s or 1 min hand wash would be unlikely to eliminate the ‘green sap’ from hands. The wearing of gloves and changing them as often as needed would eliminate the need to monitor hand washing times. These hand washing results were consistent with the findings of Chanda et al. , who fouThe survival results presented here demonstrate that the virus remains infectious on some glasshouse surfaces as a potential inoculum for at least 6 months. These data also indicate that the survival rate of the virus is dependent on the surface on which it is present. This difference in survival between different surfaces is similar to the results found by Mackie et al.  looking Effective disinfection regimes are essential for post-outbreak clean-up as well as for prophylactic routine disinfection. The efficacy of several different disinfectants was evaluated on a range of surfaces representative of those commonly found in a glasshouse or nursery. The products included in the study were chosen because they are widely available to growers and cover several different groups of active compounds . These dSodium hypochlorite is partially or completely effective at denaturing ToBRFV depending on the surface. This active ingredient also reduces seed-borne virus transmission: Davino et al.  found 2.https://nrm.co.nz/wp-content/uploads/2017/09/Virkon-S-UsageGuide.pdf, accessed on 13th September 2023). The effectiveness of 1% Virocid at the deactivation of ToBRFV matches the findings of Chanda et al. [Huwa-San, at the concentration recommended for surface disinfection, was ineffective against ToBRFV at the recommended contact time. At the concentration recommended for use in large nebulising systems (cold foggers), it was effective against ToBRFV at 1 h, except on concrete. Davino et al.  found thIn this study, trisodium orthophosphate was not totally effective against ToBRFV on any of the surfaces. Chanda et al.  found siSoaking contaminated tray sections in hot water at 90 °C for 5 min deactivated the virus, but 70 °C was not effective, which is consistent with findings on other tobamoviruses; for example, CGMMV in sap is inactivated by 10 min at 90 °C . Hot watThe results from testing swabs by real-time RT- PCR show that there are issues when using this method for the detection of ToBRFV post-disinfection. Viral RNA that does not appear to be biologically active was found to still be detectable, by real-time RT-PCR. Swab testing can provide extra assurance to growers that the virus is absent and as a management tool, giving early warning of an outbreak and determining how far the virus has spread within a glasshouse. However, due to ToBRFV detection after disinfection, despite inactivation, it is not recommended that swab testing by PCR is carried out after crop clean-up or to declare eradication. If swab testing is carried out in the event of an outbreak after clean-up, it would be recommended to also have swabs tested by inoculation onto test plants to give more confidence in the clean-up procedure.It must be noted that ground infected sap was added to each surface, and this may be an artificially high amount of virus. Moreover, the surfaces were not washed before disinfection, which would physically remove some of the virus. The disinfectants tested were all used at the recommended rates, except Huwa-San 12.5% a.i.), but were not all used at the recommended contact times, as the aim was to find a contact time that was useful in as many situations as possible. An issue that arose briefly during this experiment was the inconsistency of the positive controls showing virus symptoms on indicator plants. During the disinfection of contaminated surfaces experiment, the positive controls, which were indicator plants inoculated with swabs taken from the different ToBRFV-contaminated surfaces, before the surfaces were sprayed with disinfectant, did not show clear virus symptoms. As the virus had previously been shown to survive on all surfaces for at least 7 days and up to 6 months, it was very unusual that the controls were not positive after less than an hour on each surface. The most likely explanation for this is the light levels in the glasshouse where the test plants were kept after inoculation. These test plants were kept in the glasshouse with LED lights in December, when the general light levels were very low. The International Seed Federation protocol on the detection of ToBRFV in seed [.5% a.i.,It would be of interest to look at other time points for the disinfection work and to determine other methods to establish the difference between detection and viability to avoid having to perform extensive bioassays.ToBRFV is very stable, but there are possible ways to denature the virus to prevent it from spreading and infecting plants. Several products had success across multiple surfaces, the most difficult surface being concrete, where greater care to disinfect should be taken. Gloves are recommended alongside hand washing, to control both the spread of viruses such as ToBRFV and other contact transmitted pathogens. Hot water treatment seems sufficient for plastic trays. While denaturing of the virus is possible, it is likely that ToBRFV detection will persist, as swabs of disinfected surfaces can still give positive results when tested by real-time RT-PCR. Therefore, real-time RT-PCR is not suitable to indicate the eradication of ToBRFV following an outbreak, due to persisting molecular detection. These studies here take no account of the “food-safe” aspects of the disinfectants and growers should consider potential residues when formulating disinfection strategies. The extended contact times required for the efficacy of some of the disinfectants investigated in this study will provide practical challenges to growers in implementing effective disinfection regimes. However, the focus of cleaning and disinfection protocols should be the eradication of this virus, as well as other contact-transmitted pathogens, such as PSTVd. At present, there are no studies on overarching disinfection protocols, and it would be useful for future work to consider combination treatments for the range of potential pathogen threats in the tomato glasshouse.


Bifidobacterium animalis subsp. lactis (BB-12) in comparison to the conventional plain alginate beads. Within the scope of this study, the encapsulation of BB-12 through emulsification followed by external gelation was performed. For this purpose, a variety of alginate-based blends, composed of conventional and novel materials, were used. The results demonstrated that alginate beads incorporating 1% carrageenan or 2% nanocrystalline cellulose provided great protection to the viability of the probiotic bacteria during refrigerated storage , as well as in vitro simulation of the gastrointestinal tract . The incorporation of glycerol into the formulation of the beads improved the protective efficiency of the beads to the BB-12 cells during frozen storage, increasing significantly their viability compared to the plain alginate beads. Beads made of milk, alginate 1%, glucose 5%, and inulin 2% provided the best results in all cases. The microstructure of beads was assessed through SEM analysis and showed absence of free bacteria on the surface of the produced beads. Consequently, the encapsulation of BB-12 through emulsification in a complex encapsulating system was proved successful and effective.Aim of this work was the development of integrated and complex encapsulating systems that will provide more efficient protection to the probiotic strain  Additionally, they have been widely incorporated into various fermented foods and dairy products . The con-glycose  or even -glycose . In contBifidobacterium animalis subsp. lactis (BB-12), was selected to be examined, as it involves mild conditions and presents low cost and high cellular retention [Rodrigues et al.  encapsulactis BB-2, was seThe emulsification technique includes the dispersion of probiotic cells in a water-based polymer suspension (discontinuous phase), which is then added in an appropriate amount of oil (continuous phase) in order to form a water-in-oil emulsion; it is substantially based on the association and interactions between the discontinuous and continuous phase. The subsequent addition of a calcium chloride solution leads to the insolubilization of the water-soluble polymer and the formation of gels beads within the oil phase, thus encapsulating the probiotic bacteria. The beads produced by emulsification may be of a wide range of shapes and sizes, whereas their diameter may be sufficiently small, even below 300 μm . This te2+ chelating agents and monovalent ions or harsh conditions [Biocompatible and non-toxic materials are investigated for the incorporation of encapsulated products in food matrices . In partnditions . In ordenditions  or corn nditions . Howevernditions  or freeznditions , 19, sinnditions . Intensenditions –23.l-cysteine-HCl) is examined. The occurring blends are evaluated and compared regarding their effectiveness, in terms of protecting BB-12 cells during refrigerated or frozen storage as well as their transition through a simulated gastrointestinal system.In this work, the elaboration of integrated and complex encapsulating systems consisting of sodium alginate, other hydrocolloid materials , milk and/or milk proteins, glucose, and prebiotics (inulin) is investigated. Additionally, the incorporation of cryoprotectants (glycerol) or oxygen scavengers . Sodium alginate, calcium chloride, xanthan gum, κ-carrageenan, pectin, inulin, glucose, and Tween 80 were provided by Sigma-Aldrich Chemie GmbH ; glycerol was purchased from Lach-Ner ; and nanocrystalline cellulose (CNC) from CelluForce . Olive pomace oil was kindly provided by MINERVA S.A.  and sweet whey with the following specifications: humidity 1%, fat content 1%, protein content 10.2%, lactose (hydrated) 75%, ash content 7.3%, was kindly provided by ION S.A. . Whole-fat milk was obtained from the local market. The materials for the microbiological analyses, such as MRS agar, Ringer’s solution, citric acid, and disodium phosphate, were acquired from Merck , whereas l-cysteine-HCl, neomycine sulfate, nalidixic acid, lithium chloride, and paromomycine sulfate by Thermo Fischer Scientific . Pepsin, pancreatin, and bile extract were obtained from Acros Organics .The probiotic culture of 2 solution was also sterilized at 121 °C for 15 min and cooled at ambient temperature. The glassware required for the encapsulation procedure was also sterilized and cooled under the same conditions. The probiotic strain BB-12 was incorporated at a concentration of 5% w/v into the encapsulating blend in order to form the aqueous phase. The oil phase was prepared by mixing Tween 80 (1.5% w/v) with olive pomace oil. Subsequently, the aqueous phase was dispersed into the oil phase at a ratio of 1:3. For the formation of the emulsion, the mixture was homogenized by a high-speed homogenizer  at 1200 rpm for 5 min. Subsequently, a 0.5 M CaCl2 solution was slowly titrated to the emulsion under magnetic stirring, in order to cross-link the water-soluble polymers and form particles within the oil phase. The formed beads were allowed to harden/cross-link for 30 min under magnetic stirring at room temperature and were then harvested by centrifugation , washed with sterilized distilled water, and stored in sterile conical tubes at 4 °C. The above-described procedure was performed under aseptic conditions.The solutions of the various encapsulating blends were prepared according to the formulations presented in Table l-cysteine hydrochloride and 0.5% v/v NNLP . After 72 h of anaerobic incubation at 37 °C, cell counts were determined and expressed as log cfu g−1.In order to evaluate the survival of BB-12 during the encapsulation process, cell counts were determined after the emulsification. Cell counts were obtained by determining the number of cfu in 1 g of beads. For this purpose, 1 g of the produced beads was suspended in 9 mL of citrate–phosphate buffer (pH 7.0) and disaggregated in a stomacher until cells were completely released. Samples were 10 times serially diluted in Ringer’s solution and plated between 2 layers of modified MRS agar with 0.3% v/v The encapsulation yield (EY) is a measurement combining the entrapment efficacy and the survival of viable cells during encapsulation, and was calculated as follows :o is the theoretical number of cells estimated according to the number of probiotic cells added prior to the encapsulation.The beads containing the encapsulated BB-12 cells were stored at 4 °C and –18 °C for a 30-day period. Their survival was evaluated at 10-days intervals through microbiological analysis, as described in “−1 in HCl 0.1 N, pH 2.0) and incubated at 37 °C for 90 min. Subsequently, 2.5 mL of simulated intestinal fluid (SIF)  was added to the incubated mixture that was adjusted to pH 5.0 and incubated at 37 °C for 30 min. Finally, the pH was adjusted to 6.5 and the incubation was continued for another 90 min. Bacterial enumeration was executed as described in “This analysis was conducted according to our previous study  based onA scanning electron microscope  at an accelerating voltage of 25 kV was used to characterize the shape and the external surface of the beads produced according to the various formulations. The beads were freeze-dried, fixed in stubs with double-sided copper tape, and coated with a thin gold layer (180 s at a current of 40 mA) using a Baltzer evaporator  before being observed in the microscope.All experimental results were submitted to analysis of variance (ANOVA) using the Statistica Software version 12 . When significant differences were observed, the Duncan’s test was applied in order to compare means at a 5% significance level. The experiments were performed in triplicate, the measurements were replicated 3 times, and their mean values are presented.A variety of encapsulating blends were used for the encapsulation of the probiotic strain BB-12 through emulsification, as described in “p < 0.05) higher EY values, up to 98.9%. The utilization of milk for the development of milk-alginate beads (AM) provided the most satisfactory EY values, reaching up to 99.2%. The effectiveness of the combination of these materials is attributed to the development of a denser and more stable grid that is able to retain and protect a greater number of probiotic cells. The above results come in agreement with other researchers that attempted the reinforcement of the alginate system with other materials, such as starch. Martin et al. [Lactobacillus (L.) casei and Bifidobacterium (B.) bifidum in alginate-gelatinized starch beads, with or without chitosan coating, achieving very high EY values of 96.4–98.1%. Furthermore, in the current study, the additional incorporation of inulin or l-cysteine-HCl did not significantly enhance the encapsulation efficiency.On the other hand, the incorporation of xanthan gum (AX), κ-carrageenan (AC), and nanocrystalline cellulose (ACNC) in the alginate beads, as well as whey in combination with pectin (AWP) provided significantly . Values with different superscripts are significantly different. Small lettered superscripts are used to differentiate values between rows (different encapsulating agents), while capital lettered superscripts to differentiate values between columns (different additives).Values shown are means ± standard deviations , exceeding 42.9% and 50.3%, respectively, by the end of the 30-day storage  the BB-12 cells’ survival in all cases, leading to 0.9–22.5% higher survival rates compared to samples containing alginate only. Our results are in agreement with other researchers that observed improved viability during storage by encapsulating various probiotic strains in particles containing inulin [The BB-12 cells encapsulated in alginate alone or in alginate with inulin suffered significant reductions in their viability  the survival of the probiotic cells during frozen storage compared to those encapsulated in plain alginate beads . In all cases, significant viability loss of 3.1–6.2 log cfu g−1 occurred during the first days of storage due to the sudden exposure of the BB-12 cells to the injurious low temperature. The formation of ice crystals that provokes damage to the membrane structure of the probiotic cells and, thus, changing their physiological state can lead to cells’ death [−1 for up to 20 days of storage. As in the case of refrigerated storage, whey-pectin, carrageenan, CNC, and milk provided the highest protection of BB-12 viability during frozen storage as well. In the case of frozen storage, in particular, greater survival rates (57.1%) were achieved when the cryoprotectant glycerol (AGl) was included into the encapsulating mixture  as well as during the entire storage period. The results presented in Fig. s’ death . In the p < 0.05) during frozen storage by increasing the survival by 0.6–24.4%. Raddatz et al. [l-cysteine-HCl demonstrated slightly increased stability during storage at –18 °C. Optimal results were achieved through the combination of milk, alginate, inulin and l-cysteine-HCl (AML-cys), as even after 30 days of storage the bacterial load was maintained at 6.1 log cfu g−1. Similar results are reported by Sousa et al. [l-cysteine-HCl was incorporated.The addition of inulin in the alginate beads also improved the viability of BB-12 . The alginate, alginate-xanthan gum, and alginate-glycerol beads with  or without incorporated inulin  presented the lowest bacterial loads, with survival rates of 22.3–29.3% at the end of the in vitro GI simulation. The addition of xanthan gum or glycerol in the mixture did not significantly affect the viability of BB-12 during the in vitro simulation. Similar results regarding the protective effect of glycerol in probiotic bacteria under GI conditions were also found by Sultana et al. [L. casei 01, through emulsification, in alginate-starch blends and observed that the higher the percentage of incorporated starch into the alginate beads, the greater its survival under simulated GI conditions. The protective effect of the alginate-starch blend was also observed by Sabikhi et al. [Lactobacillus acidophilus at different concentrations of bile salts . Gerez et al. [Lactobacillus rhamnosus CRL 1505 in pectin or whey protein-pectin beads through emulsification and coated the occurring particles with whey protein for enhanced protection, achieving significantly higher survival rates of the encapsulated cells than the free cells when exposed to simulated GI conditions. Moreover, Zou et al. [−1 in the survival of B. bifidum F-35 by further addition of pectin in the alginate beads. However, no significant improvement was observed when starch was added in the alginate mixture. Furthermore, the incorporation of inulin into the encapsulating blends significantly enhanced (p < 0.05) the viability and increased the survival rates by up to 7.0%. Our results are in agreement with other researchers that reported the beneficial effect of inulin under GI conditions [l-cysteine-HCl in the encapsulating blends provided only a slight increase of the survival rates (0.4–0.7%) when exposed to simulated GI conditions.For the assessment of the coating materials’ efficacy, the encapsulated cells were further exposed to in vitro simulated gastrointestinal conditions as described in “a et al. . On the i et al.  who examz et al.  encapsulu et al.  reportednditions , 30, 31.The scanning electron microscopy (SEM) analysis micrographs for the beads produced with different encapsulating blends are presented in Fig. However, for the needs of the SEM analysis, the beads were first subjected to freeze drying. This resulted to samples with irregular shape and size. Thus, the initially soft and smooth surface of beads turned into a rough one with irregular concavities and wrinkles, due to the removal of water from the hydrogel. This sponge-like external structure occurs due to the fast sublimation of the frozen water from the beads, leading to pores formed in the place of the ice crystals . AccordiIn order to provide a more detailed approach of samples’ microstructure, a randomly chosen sample is presented in Fig. The encapsulation of BB-12 cells through emulsification, in most cases, improved the survival of the BB-12 cells both during storage or transit through the GI tract. Alginate on its own was not efficient in maintaining probiotics’ viability. On the other hand, its combination with certain conventional (carrageenan) or novel (CNC) materials enhanced the protective properties of the occurring beads. The best results were provided when water was replaced by milk during the encapsulation process (AM). Interestingly, these materials, due to the dense structure of the beads produced, were effective not only in protecting BB-12 at low storage temperatures (4 °C and −18 °C) but also during the in vitro simulation of the GI tract. Consequently, the emulsification with the use of encapsulating blends proposed in this study may significantly maintain the viability of probiotic bacteria during both storage and simulated GI conditions in a simple and cost-effective manner. The proposed encapsulation systems can be studied for the enrichment of food products, as they are promising protective matrices for BB-12 cells. Thus, they will be probably able to provide viability enhancement of BB-12 cells during food manufacturing and storage, as well as during simulated GI conditions.


Brucella abortus is a bacterial pathogen causing bovine brucellosis worldwide. This facultative extracellular–intracellular pathogen can be transmitted to humans, leading to a zoonotic disease. The disease remains a public health concern, particularly in regions where livestock farming is present. The two-component regulatory system BvrR/BvrS was described by isolating the attenuated transposition mutants bvrR::Tn5 and bvrS::Tn5, whose characterization led to the understanding of the role of the system in bacterial survival. However, a phenotypic comparison with deletion mutants has not been performed because their construction has been unsuccessful in brucellae and difficult in phylogenetically related Rhizobiales with BvrR/BvrS orthologs. Here, we used an unmarked gene excision strategy to generate a B. abortus mutant strain lacking both genes, called B. abortus ∆bvrRS. The deletion was verified through PCR, Southern blot, Western blot, Sanger sequencing, and whole-genome sequencing, confirming a clean mutation without further alterations at the genome level. B. abortus ∆bvrRS shared attenuated phenotypic traits with both transposition mutants, confirming the role of BvrR/BvrS in pathogenesis and membrane integrity. This B. abortus ∆bvrRS with a non-antimicrobial marker is an excellent tool for continuing studies on the role of BvrR/BvrS in the B. abortus lifestyle. Brucella organisms are Gram-negative facultative extracellular–intracellular pathogens that cause brucellosis, a neglected zoonotic disease with a worldwide distribution. Brucella abortus infects cattle and exhibits a strong tropism for the reproductive system, causing abortion, infertility, decreased milk production, reproductive failure, epididymitis, and hence, economic losses. Humans are accidental hosts, initially developing an acute febrile disease that could become a chronic infection with osteoarticular, gastrointestinal, hepatobiliary, pulmonary, genitourinary, cardiovascular, and neurological complications [ications .Brucellae lack classical virulence factors, and their pathogenicity depends on their ability to invade, survive, and replicate inside host professional and non-professional phagocytes. During their intracellular lifecycle, brucellae avoid the endocytic pathway and redirect their trafficking to a replicative compartment derived from the endoplasmic reticulum ,3. ThereB. abortus kanamycin-resistant transposition mutants, one in bvrR and the other in bvrS. These mutants were selected based on an association of brucellae virulence with peculiar membrane properties rarely present simultaneously in other Gram-negative pathogens [in vivo [In brucellae, the TCS BvrR/BvrS comprises the sensor membrane protein BvrS and its cognate cytoplasmic response regulator BvrR. This TCS was described in 1998 through the isolation and phenotypic characterization of two athogens , like a athogens . Accordiathogens ,6, and Bathogens , an oute[in vivo , pathway[in vivo , and vir[in vivo ,6.Rhizobiales having orthologs of BvrR/BvrS, like the Sinorhizobium meliloti TCS ExoS/ChvI [Agrobacterium tumefacient TCS ChvG/ChvI [Bartonella henselae TCS BatR/BatS [B. abortus double-null mutant strain in bvrR and bvrS called B. abortus ∆bvrRS. The phenotype of B. abortus ∆bvrRS was attenuated in the cell culture model like the one described for both transposition mutants, validating the role of BvrR/BvrS in virulence.The use of transposition mutants encoding antimicrobial resistance, although not ideal, has being an alternative to the construction of mutants in this TCS, which has proven difficult to generate in brucellae ,12 and ixoS/ChvI ,15,16,17hvG/ChvI ,19,20, aatR/BatS . TherefoatR/BatS . Here, wB. abortus were carried out according to the “Reglamento de Bioseguridad de la CCSS 39975–0”, 2012, after the “Decreto Ejecutivo #30965-S”, 2002, and research protocol SIA 0652–19 approved by the National University, Costa Rica. The strains and plasmids used in this study are listed in 2, 10 mM MgSO4, and 20 mM glucose), Columbia Agar supplemented with 5% (v/v) of sheep blood (CBA), and Luria–Bertani (LB) medium. When required, the following supplements were added to the different culture media: 10% (w/v) sucrose (Suc), 30 µg/mL kanamycin (Km), 100 µg/mL spectinomycin (Spc), and 30 µg/mL chloramphenicol (Cm). All bacterial cultures were incubated at 37 °C, under constant shaking at 200 rpm when necessary. The cultures were inoculated with 7 × 109 CFU in a final volume of 20 mL TSB to obtain growth curves. The optical densities were measured at 420 nm every 2 to 4 h.Bacterial strains, growth conditions, and plasmids. All procedures involving live B. abortus ΔbvrRS. The genes bvrR (BAW_12006) and bvrS (BAW_12007) of the parental strain B. abortus 2308W were mutated using a previously described [bvrR upstream (Up) fragment from approximately 1 kb to the second codon of the bvrR coding sequence (coordinates 2010312 to 2011285) was amplified with the primers bvrRS-Up-For and bvrRS-Up-Rev (bvrS downstream (Dn) fragment containing the last two codons of the bvrS coding region to approximately 1 kb downstream (coordinates 2013910 to 2014911) was amplified with the primers bvrRS-Dn-For and bvrRS-Dn-Rev , and the Dn fragment was cut with PstI . Both fragments were treated with polynucleotide kinase in a ligase buffer and included in a single ligation mix with BamHI/PstI-digested pNPTS138 (sacB for sucrose (Suc) counterselection. The resulting plasmid was called pΔbvrRS (bvrR and bvrS (coordinates 2011286 to 2013909). Subsequently, 1–3 μL of pΔbvrRS in distilled water was electroporated into 40 μL of B. abortus 2308W competent cells prepared as described in [TM cell electroporator using the following parameters: 2.5 kV at 400 ohms and 50 mF. After electroporation, 1 mL of SOC medium was added, and the cells were incubated overnight. Then, 100–200 μL volumes were plated on CBA + Km to select the clones harboring pΔbvrRS. The plates were incubated for 6–10 days. Single colonies were selected, transferred into TSB, and incubated overnight. Volumes of 50, 100, and 200 μL were plated on TSA + 10% Suc to counterselect clones integrating the Up–Dn construct into the chromosome of the parental strain by allelic exchange. The plates were incubated for 3–6 days. Single colonies were picked, and exact replicas were cultured on TSA + 10% Suc and TSA+ Km, to select SucR and KmS clones that were expected to have lost both bvrRS genes and the suicidal plasmid. The plates were incubated for 2–3 days. The SucR and KmS clones were screened by colony PCR with the primers bvrRS-Con-For and bvrRS-Con-Rev  and are not expected to amplify any PCR product in the mutant strain. Mutant clones selected based on the presence of the 709 bp Con-amplicon and the absence of the 63 bp bv1-amplicon, were subjected to biochemical identification tests and further confirmation by DNA Sanger sequencing, Southern blot, Western blot, and whole-genome sequencing, as described below.The construction of escribed ,25, non-S-Up-Rev . A bvrS S-Dn-Rev . The Up pNPTS138 , a suici pΔbvrRS  and lackribed in ,25. Elec-Con-Rev  in a rea primers . The “bvB. abortus 2308W. The performed tests included urease (1 h and 24 h), oxidase, H2S production, nitrate reduction, and sensitivity to thionine (20 µg/mL) (24 h and 72 h) and basic fuchsin (20 µg/mL) (24 h and 72 h). The presence of a smooth LPS was verified through the acriflavine agglutination test as described in [Biochemical identification. Basic biochemical tests were performed as described in  using thribed in .® Genomic DNA Purification Kit . Purified DNA was double digested with 25 U of BshTI (AgeI) and 12.5 U of MlsI (MscI) restriction enzymes , respectively, cutting the B. abortus 2308W genomic DNA at positions 2011220 (−60 from bvrR start codon) and 2014898 (+983 from bvrS stop codon) and, respectively, generating 3679 bp and 1055 bp fragments in the parental strain and in the B. abortus ∆bvrRS mutant strains. The digested DNA of each strain was separated through electrophoresis in a 0.6% agarose gel for 2 h at 100 V, and the gel was blotted onto a nylon membrane . The probe was generated using the primers bvrRS-South . Probe labeling, hybridization, and detection were performed using the DIG DNA Labeling and Detection kit .Southern Blot. The mutation was confirmed by a Southern blot as described in , with a RS-South , amplifyWestern Blot. The BvrS, BvrR, and Omp25 expressions were assessed as described in , with mobvrR and bvrS. The regions recombined during the deletion of the bvrR and bvrS genes were also sequenced. The upstream recombination region was amplified with pckA primers .Sanger sequencing. The Con-amplicon of the mutant strain was Sanger sequenced to confirm the absence of  primers , obtaini primers , obtainiB. abortus 2308W and B. abortus ∆bvrRS was obtained using the Wizard® Genomic DNA Purification Kit  from liquid cultures grown to the stationary phase. Genomic libraries were prepared with the rapid barcoding sequencing SQK-RBK004 kit  for sequencing on a minION platform using the MinKNOW software version 22.08.4, according to the manufacturers’ recommendations. Base calling and the conversion of the raw data to the FASTQ format were performed with Guppy v.3.6.0., while the reads’ quality was verified with nanoplot 1.40.2 [B. abortus 2308W and B. abortus ∆bvrRS, respectively. The resulting complete-genome assemblies at the chromosome level were compared using BLAST [B. abortus ∆bvrRS and B. abortus 2308W. Genome content coverage was also verified by read mapping against the assembled genome using the long-read mapping pipeline Vulcan 1.0.3 [Whole-genome sequencing. For whole-genome sequencing, genomic DNA from t 1.40.2 . Low-quat 1.40.2 . Adaptert 1.40.2  and Chect 1.40.2 . The prong BLAST  with blaan 1.0.3 . Genomican 1.0.3 .B. abortus strains in the exponential growth phase as described in [B. abortus CFUs was determined at 0, 24, and 48 h after infection.Gentamicin-protection assay and intracellular replication quantification. Murine RAW 264.7 macrophages (ATCC TIB-71) or HeLa epithelial cells (ATCC clone CCl-2) were cultivated and infected with ribed in ,27. The Membrane integrity tests. The sensitivity to the bactericidal action of non-immune serum was tested as described in , with anStatistical analyses. The Kruskal–Wallis test for multiple comparisons was used as described in .bvrR and bvrS using an unmarked gene excision approach. The colony PCR screening of the SucR and KmS clones revealed four potential mutant clones lacking bvrR and bvrS, numbered 3, 4, 41, and 42  and thionine (20 µg/mL) at 72 h; and negative reactions to nitrate reduction and resistance to fuchsin (20 µg/mL) and thionine (20 µg/mL) at 24 h.In this study, we constructed a null mutant with a deletion in , and 42 . Clone 4B. abortus ∆bvrRS excluded crystal violet, did not agglutinate with acriflavine, and agglutinated with antibodies against smooth LPS, indicating the conservation of the S-LPS phenotype as the parental strain. In TSB, B. abortus ∆bvrRS consistently exhibited a prolonged lag phase in all independent replicas, retarding entry into the log phase and catching up with B. abortus 2308 W in the stationary phase  without further alterations in the genome (BLAST comparison between the strain 2308W and the mutant strain confirmed the deletion of the e genome .B. abortus ∆bvrRS, we assessed its ability to replicate intracellularly in HeLa cells and RAW macrophages. As shown in B. abortus 2308W was able to replicate intracellularly in both types of cells, but B. abortus ∆bvrRS and the transposition mutants, B. abortus bvrR::Tn5 and B. abortus bvrS::Tn5, did not, confirming attenuation in the cell model.To characterize the virulence phenotype of B. abortus ∆bvrRS, we first performed a susceptibility to polymyxin B test. As shown in B. abortus ∆bvrRS and both transposition mutants, B. abortus bvrR::Tn5 and B. abortus bvrS::Tn5, were lower by at least two dilutions of difference than those of the parental strain, suggesting altered membrane integrity. All the MICs increased by at least two dilutions of difference compared to that at pH 7.0, indicating a higher resistance under acidic conditions.To assess the membrane integrity of B. abortus ∆bvrRS was susceptible to the action of a non-immune serum, unlike the parental strain (B. abortus ∆bvrRS displayed a lack of expression of this outer membrane protein, unlike B. abortus 2308W (Then, we confirmed that l strain a, and weus 2308W b.bvrRS genes as essential [Rhizobiales was consistently reported as difficult [B. abortus mutant strain with a deletion in both bvrRS genes. Different experimental approaches confirmed the in-frame double deletion, and the whole-genome sequencing results demonstrated a clean null mutation without further alterations in the genome.The role of the TCS BvrR/BvrS in the virulence of brucellae has been studied in transposition mutants with a kanamycin-resistance marker in the inserted transposon . Construssential . Later issential , which iifficult ,19,20,21ifficult ,25 to coB. abortus ∆bvrRS revealed a smooth LPS, unaltered biochemical characteristics, retarded lag growth phase defects, inability to replicate intracellularly in cell models, susceptibility to polymyxin B and to human non-immune serum, and lack of expression of Omp25. These results are consistent with previous studies about the role of the TCS BvrR/BvrS that were performed using the transposition mutants B. abortus bvrR::Tn5 and B. abortus bvrS::Tn5 [The phenotypic characterization of vrS::Tn5 ,7,37.B. abortus null mutant in the bvrR/bvrS genes was successfully obtained through allelic replacement. This novel mutant does not harbor any resistant marker gene and will be useful for further studies on the TCS BvrR/BvrS.In conclusion, a


Plant polyphenols with a catechol structure can form covalent adducts with meat proteins, which affects the quality and processing of meat products. However, there is a lack of fast and effective methods of characterizing these adducts and understanding their mechanisms. This study aimed to investigate the covalent interaction between myofibrillar protein (MP) and caffeic acid (CA), a plant polyphenol with a catechol structure, using molecular probe technology. The CA-MP adducts were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and detected via Western blot and LC-MS/MS analyses. The Western blot analysis revealed that various specific adducts were successfully enriched and identified as bands around 220 kDa, 45 kDa, and two distinct bands between 95 and 130 kDa. Combined with the LC-MS/MS analysis, a total of 51 peptides were identified to be CA-adducted, corresponding to 31 proteins. More than 80% of the adducted peptides carried one adducted site, and the rest carried two adducted sites. The adducted sites were located on cysteine (C/Cys), histidine (H/His), arginine (R/Arg), lysine (K/Lys), proline (P/Pro), and N-terminal (N-Term) residues. Results showed that the covalent interaction of CA and MP was highly selective for the R side chain of amino acids. Moreover, the adducts were more likely to form via C-N bonding than C-S bonding. This study provides new insights into the covalent interaction of plant polyphenols and meat proteins, which has important implications for the rational use of plant polyphenols in the meat processing industry. Plant polyphenols are natural antioxidants that can protect meat products from oxidative deterioration by scavenging free radicals and inhibiting peroxidation ,2. HowevPrevious studies have shown that CA adduction can affect the structure and gel properties of myofibrillar protein (MP) ,4. HowevMolecular probe technology is a powerful method for identifying the modification of proteins by natural active compounds  in complex systems. This method involves labeling the active compound with a molecular probe that can bind strongly to the target proteins and carry a detectable tag, such as fluorophores, isotopes, biotin, or bio-orthogonal groups ,11. The In this study, we synthesized a biotinylated derivative of caffeic acid (BioC) that can act as a molecular probe to identify protein targets responsible for caffeic acid’s covalent modification. Based on the molecular probe technology, we aimed to identify protein–phenol adducts in meat proteins. A method to enrich, isolate, and identify adducts was firstly established and its feasibility was validated; it was then applied to identify and preliminarily quantify specific phenol–protein adducts in the MP system. Combined with LC-MS/MS, the adducted peptides and amino acid sites were also comprehensively identified and quantified. Finally, the mechanism of the adduction reaction between CA and MP was explored based on the adducted sites and their relative abundance. This study provides new insights into the covalent interactions of plant polyphenols and meat proteins and their implications for meat quality and processing.Caffeic acid and streptavidin magnetic beads were both obtained from Cell Signaling Technology . Bovine serum albumin, biotin, trypsin, and polyvinylidene fluoride membranes were purchased from Sigma-Aldrich . An anti-biotin antibody (abs153474) was obtained from Absin Bioscience Inc. . Goat anti-rabbit IgG was purchased from Beijing Cowin Biotech Co., Ltd. . Skimmed milk powder was obtained from Sinopharm Chemical Reagent Co., Ltd. . Other reagents were purchased from Genscript Biotech Co., Ltd.  unless otherwise specified.v/v) and recrystallized with methylene chloride/petroleum ether. Next, intermediate 1 was dissolved in dichloromethane, the treatment of which with methanolic hydrochloric acid solution for 15 h in an ice bath and subsequent concentration generated the desired intermediate 2. Finally, the amidation of intermediate 2 with D-(+)-biotin in a similar way as above afforded the BioC  as a control. All the samples were incubated at 4 °C for 12 h. Then, the BSA-BioC adducts in the system were isolated and characterized using the method described in Section 2.3Bovine serum albumin (BSA), as a model protein, has been widely studied in various fields due to the diversity of its physiological functions . ConsequSamples were loaded onto SDS-PAGE equipment according to the method of Liu  with a 4A Western blot analysis was used to detect bands of the BioC-adducted proteins according to Pi . Brieflym/z; the precursor intensity threshold was set at 5.0e4, and a maximum injection time of 20 ms was used. For each full MS scan, the top 10 most abundant precursor ions were selected for MS2 with an isolation window of 1.6 m/z and a higher-energy collision dissociation with a normalized collision energy of 30. MS2 spectra were collected at a resolution of 15,000. The target value was 5.0e3 with a maximum fill time of 20 ms and a dynamic exclusion time of 30 s.Samples were prepared using a filter-aided sample preparation (FASP) workflow, as described by Wiśniewski et al. . The pep21H28N4O5S)  and the possible degradation product of the BioC (C9H8O4)  were specified as variable modifications. A q-value (FDR) cutoff for the precursor and protein level was applied at 1%. The processed data output was then exported to Excel for further usage.Raw data were processed and analyzed using Proteome Discoverer (PD) 2.4 SP1  with default settings. PD was set up to search the database from NCBI. Human keratin and trypsin sequences were used as the contaminated database for proteomic searching. Trypsin was used as the digestion enzyme. Carbamidomethyl (C) was specified as the fixed modification. Oxidation (M), acetyl (Protein N-term), the BioC ; We developed a novel method for the enrichment and separation of phenol–protein adducts by using a BioC rather than CA in the reaction system to label proteins that can adduct with CA. The BioC is a biotinylated derivative of CA which can be recognized and captured by streptavidin magnetic beads with high specificity and affinity. However, to avoid the possible adsorption of other molecules on the surface of the streptavidin magnetic beads, non-specific washing steps are often required . TherefoAs shown in As shown in p < 0.05). Specifically, the Actin-BioC and band-2-BioC adducts were firstly observed at a low BioC concentration (50 µM). MHC-BioC and band-1-BioC were observed at a BioC concentration of 250 µM. With an increasing BioC concentration, the intensity of all of adducts was significantly increased (p < 0.05). The results illustrate that the formation of MP-BioC adducts has concentration-dependent characteristics.In p < 0.05) when the CA concentration increased. When the CA concentration was 16 times higher than the BioC concentration (4 × 103 µmol/g MP), the binding of the BioC to MP was almost completely blocked.The characteristic protein bands identified in the concentration-dependent experiments decreased gradually when we added different concentrations of highly active ligands. This was an effective way to confirm specific protein bands. In our study, we further tested the covalent interaction between molecule probes and highly active ligands that were added simultaneously to establish a competition control which could prevent the molecule probes from binding to target proteins via competition from highly active ligands. The addition of different concentrations of highly active ligands may lead to a gradual fading of the characteristic protein bands identified in concentration-dependent experiments, which was an effective method of determining specific protein bands. In this study, to further validate the covalent interaction between the BioC and MP, we allowed the BioC or CA to compete for binding to MP with or without an excess of the competitive inhibitor CA, and the BioC concentration was kept at 250 µM. The protein bands at 220 kDa and 45 kDa, as well as two strong protein bands between 95 and 130 kDa, depended on the concentration and could be stopped by the competitive inhibitor CA. This indicated that all these protein bands were specific MP-BioC adducts. More importantly, the results showed that the BioC and MP interacted in the same way as the CA and MP. Since the BioC was modified from CA and had the same biological activity as CA , it was To further explore the formation mechanism of MP-BioC adducts, the BioC-adducted proteins in the MP sample (1250 µM) were identified using an Orbitrap Exploris 480 mass spectrometer. After filtering data with a score > 10, a total of 31 BioC-adducted proteins were found, including myosin, actin, troponin, and tropomyosin . Six BioIn the molecular mass range of 95–130 kDa, the BioC modification of alpha-actinin-3 isoform X2 (103.2 kDa), glycogen phosphorylase muscle form (97.2 kDa), and muscle glycogen phosphorylase partial  occurred at the N-Term site , and that of FH1/FH2 domain-containing protein 1 isoform X1 (129.2 kDa) was located at two P sites (SPFPPPPPPAAP*LP*PSAPDGLALPTK). In addition, the alpha2 chain of type I collagen (129.1 kDa) had two adducted sites P and R (GEVGPAGPNGFAGPAGAAGQP*GAKGER*GTK). Furthermore, lower-molecular-mass adducts were identified, such as C-U-editing enzyme APOBEC-2 (25.8 kDa), GTP-binding protein SAR1b (22.4 kDa), and troponin I (21.3 kDa). BioC modifications of them occurred at the C or N-Term sites, and the adducted peptides involved were TFLC*YVIEAQSK, ELNARPLEVFMC*SVLK, and MSADAMLK, respectively.Myosin, an important component of MP, has a molecular structure rich in charged amino acids and cysteines , which aAltogether, 51 BioC-adducted peptides were identified among 31 MP-BioC-adducted proteins . BioC adp < 0.05).Distribution of adducted sites: In unoxidized phenolic compounds, hydroxyl groups are excellent hydrogen donors, which do not directly form irreversible covalent bonds with amino acids but allow for the formation of hydrogen bonds between phenols and carboxyl groups in proteins. Under alkaline conditions, hydroxyl groups are oxidized and converted into the active derivative quinone. The latter then undergoes irreversible covalent addition reactions with the sulfhydryl or amino groups of susceptible amino acid residues in proteins ,36. SomeThe sequence motif of adducted sites: The full-length sequence of the different adducted peptides was searched in the NCBI according to the gene number before selecting the adducted site and 10 amino acids on each side of it to form a polypeptide with 21 amino acids. The sequence logo (seqlogo) was drawn using Weblogo (Version 2.8.2) to further explore the sequence motif of the adducted sites. The height (%) of each letter in the seqlogo corresponds to the frequency of the occurrence of amino acid residues at that location, and that letters in each position are arranged in order of conservatism from the largest to smallest, which makes it easy to identify conserved sequences from the top letters. As shown in 2 group  rather than the -SH group (C adducted site) of MP, resulting in various phenol–protein adducts. Our method provides a powerful tool for the isolation and identification of phenol–protein adducts in meat protein systems as well as the comprehensive analysis of the types and numbers of adducted sites, which reveal the mechanism of the covalent binding of phenols to proteins.In this study, we developed and validated a novel method based on molecular probe technology to accurately identify phenol–protein adducts in meat protein systems, using BSA as a model protein. Our method involves the use of a BioC, a biotinylated derivative of CA which can covalently bind to MP in the same manner as CA. It was demonstrated that the BioC, a biotinylated derivative of CA, can interact with MP in the same way as CA, forming covalent bonds with specific amino acid residues. By using an LC-MS/MS analysis, 31 CA-adducted proteins, 51 CA-adducted peptides, and 6 kinds of CA-adducted sites were identified. Furthermore, it was revealed that CA prefers to react with the -NHAlthough phenolic compounds have many beneficial effects on the quality, safety, and functionality of meat and meat products, there are some potential drawbacks and risks that need to be considered. One of the major challenges of using phenolic compounds in the meat industry is the oxidation of phenolic compounds with the formation of quinones, which could affect the quality, safety, and functionality of meat and meat products. The oxidation of phenolic compounds and the formation of quinones are influenced by many factors, such as the type, concentration, source of phenolic compounds, pH, temperature, oxygen, metal ions, enzymes, and other food components of the meat system, and the processing and storage conditions of the meat products. In this study, covalent interactions between plant polyphenols and meat proteins were investigated. However, only low-molecular-weight polyphenols were used, which may differ from high-molecular-weight polyphenols, such as proanthocyanidins and more complex tannins, in their interaction patterns and biological effects. High-molecular-weight polyphenols might form more stable covalent complexes with proteins. A future investigation is therefore required to take into account the molecular characteristics of polyphenols and the environmental factors that may influence the interactions between polyphenols and proteins and their effects on meat quality.


The recently detected virus in eastern China in 2018 led to some health concerns, especially with the global trend of spreading viruses. As a new RNA-detected genus of the henipavirus family was found in Eastern China, the number of patients affected has reached 35 through zoonotic spread, with symptoms ranging from simple fever to fatal affection of vital organs such as the brain, liver, and kidneys. Researchers have found that shrew animals might be a potential reservoir for the Langya virus; however, data is still limited regarding human-to-human transmission. Current efforts by the Chinese Health Ministry and the Taiwan Centers for Disease Control and Prevention to deduct the spread of the virus and track its origin by trying to sequence the disease genome are evident. With all this in mind, the recommendation to face this new novel virus revolves around protecting the most vulnerable population at risk of being infected, such as farmers, and preventing the spread of the virus. Efforts must be directed toward screening animals for henipavirus and diving more deeply into the etiology of how this virus has spread to humans to help understand the spread of zoonotic viruses in the future. Henipavirus genus. According to the Centers for Disease Control and Prevention, five other viruses were previously identified in this genusNew England Journal of Medicine, this newly identified pathogen is mostly related to the Mojiang virusInfectious disease specialists have long warned that environmental damage and climate change will increase the risk of ‘zoonotic spillovers’, or the spread of infections from animals to humans. In the midst of current efforts worldwide to tackle the ongoing monkeypox virus outbreak and coronavirus disease 2019 (COVID-19) pandemic, a new animal virus known as the Langya henipavirus (LayV) has been discovered in humans in Eastern ChinaIt is worth mentioning that scientists have limited information regarding the mode of transmission of LayVIn December 2018, as part of a program to monitor humans who experienced fevers after encountering animals, researchers used a sample from a throat swab to discover the novel virus ‘via metagenomic analysis and subsequent virus isolation’.Pteropus spp.). Their transfer to mammals eventually led to their ability to infect human beingsTo determine which animal species was the source of the LayV, the researchers tested several animals for the presence of the virusNew England Journal of Medicine, published a paper detailing the new virus’s detailsThe current Langya outbreak comes against a background of debates around the origin of the severe acute respiratory syndrome coronavirus 2 virus. One of the several points raised in the scientific and public communities is the slow rate at which the Chinese government alerted the world about the emergence of an unusual infectionSince farmers made up the majority of the 35 cases recorded, comprehensive sanitation measures should be adopted in the field of animal husbandry. Disease surveillance revealed no shared exposure sources or close contact between infected individuals, indicating that human infection may have happened sporadically, according to the research teamsSince the novel LayV was detected in late 2018, dozens of patients have been infected with little information on the origin of reported cases and the viral exposure through interhuman transmission. This has left the Chinese health care system with concerns concerning tracking the virus and sequencing its genome. Unfortunately, this new virus lacks a targeted treatment and a protective vaccine, despite the fact that doctors are trying different antivirals to treat the symptoms. Prevention seems to be the key to avoiding this viral conflict. Moreover, experts have mentioned that the likelihood of this disease transforming into a pandemic is still little to none. However, with the observed COVID-19 pandemic, more efforts must be initiated to target the origin and curb the spread of the infection. We highly recommend implementing a targeted, robust system to face this virus and putting some effort into activating efficient health policies to deduct the viral load from known zoonotic reservoirs. Appropriate investment in vaccine-development research might save countries from potential outbreaks in vulnerable sites such as farms and sites where human–animal interaction occurs.Not applicable.Not applicableWe have not received any financial support for this manuscript.O.U.: conceptualization, project administration, writing – review and designing, reviewed and edited the first draft, and reviewed and edited the final draft. J.W.: reviewed and edited the second draft. S.S.: reviewed and edited the third draft. All authors contributed in data collection and assembly, manuscript writing, final approval of manuscript. Figure 1 was drawn and analyzed by authors S.A. and O.U. Figure 2 was drawn and analyzed by authors A.R. and O.U.The authors declare no conflicts of interest.Name of the registry: not applicable.Unique identifying number or registration ID: not applicable.Hyperlink to your specific registration (must be publicly accessible and will be checked): not applicable.Olivier Uwishema: principal investigator.Not applicable.Not commissioned, externally peer reviewed.