In order to improve the detection efficiency and accuracy of microfluidic chip, a magnetic beads preset technology were designed by using double permanent magnets as external magnetic field and the motion characteristics of preset magnetic beads were studied. The control principle of magnetic beads preset technology was introduced in detail, and the control structure was designed. The coupled field characteristics for magnetic beads in microchannels were analyzed, and the motion models of magnetic beads were established based on the magnetic beads preset technology, including capture motion and mixing motion. The relationship between the magnetic field force and the flow velocity for capturing magnetic bead, and the mixing time under the influence of flow field and magnetic field were derived. The magnetic beads preset technology effect was verified by experiments and numerical simulations were developed to analyze the influence of aspect ratio of permanent magnet on magnetic field. The study showed that the accuracy and efficiency of the magnetic bead control in the microchannel could be better realized by the magnetic beads preset technology. The derivation of the magnetic bead motion model can understand the motion characteristics of the magnetic bead more clearly, facilitate accurate control of the magnetic bead, and improve the success rate of the microfluidic detection. At present, magnetic bead is attracting more and more attention5, and is widely used in biomedical science11. For example, the magnetic beads with special surface treatment can be used to form a new complex by nonspecific or specific combination with the corresponding target materials, and can be separated under the control magnetic field. Then the separated magnetic beads are eluted by the eluent to complete DNA extraction13. The key of magnetic bead control is to set a reasonable magnetic field to achieve the effect that magnetic beads can be effectively captured and fully mixed with the reaction solution under the flow field influence.Magnetic bead is a kind of magnetic material with particle size between 1 and 100 nm, which not only has unique surface effect, volume effect, quantum size effect, functional groups like other general nano material, but also exhibits superparamagnetism, magnetic responsiveness, high coercivity14 integrated an iron–PDMS microstructure next to a microfluidic channel and placed between two external permanent magnets by injecting a mixture of iron powder and PDMS into a prefabricated channel. The magnetized iron–PDMS microstructure generates localized and strong forces on the magnetic particles to enhanced separation of the particles. Zhu et al.15 built a specific magnetic field in a desired chamber bonded with PDMS and PMMA sheets by embedding soft magnets of iron powders in the chip, and magnetized by an external magnetic field to form the nucleic acid capture platform based on magnetic beads. By improving the liquid flow rate and magnetic field distribution, the nucleic acid extraction efficiency was increased by about 80%, and the serpentine flow channel was used to increase the mixing efficiency of magnetic beads and reaction solution. Serra et al.16 realized the construction of high-throughput sequencing library based on microfluidic chip and the capture of nucleic acids of different sizes by integrating soft magnetic structure and pressure control valve. This method realized higher nucleic acid extraction efficiency (> 96.5%) than the traditional magnetic bead method, with the application advantages of high-throughput, automation and compatibility with downstream sequencing. Munaz et al.17 developed a simple microfluidic platform for separating diamagnetic particles with different sizes by using parallel ferromagnetic fluid, so that ferromagnetic fluid with predetermined concentration of magnetic nanoparticles can promote negative magnetophoresis. The result shows that the maximum separation efficiency of particles for 3.2 and 4.8 μm was 78% and 75% respectively. Zhang et al.19 both designed a special 3D structure in the microfluidic chip to improve the collision probability between magnetic beads and reaction liquid, so as to improve the mixing efficiency.Numerous of methods have been reported in the literature on controlling magnetic beads to improve magnetic beads capture efficiency and mixing efficiency. Zhou et al.Current research focuses on the design of control magnetic field, especially how to increase the magnetic field gradient to enhance the magnetic field force and improve the capture efficiency, and how to increase the length and complex of flow channel to improve the mixing efficiency. And the magnetic beads are mostly introduced from the outside of microfluidic chip, so the magnetic beads control system is usually complex, which is not conducive for the popularization of microfluidic detection technology. In addition, the motion characteristics of magnetic beads is the basis of the control magnetic field design, which is an important research direction of microfluidic detection.This paper presents a magnetic beads preset technology, and analyzes the feasibility of magnetic beads preset technology and the motion characteristics of preset magnetic beads, in order to improve the efficiency of microfluidic detection and simplify the microfluidic detection equipment.22. This paper focuses on the control motions about magnetic beads capture and magnetic beads mixing with the solution, which involves two problems: the capture efficiency and mixing efficiency, as shown in Fig. The functions of magnetic beads are different in microfluidic detection, but what they all have in common is to realize the accurate control of magnetic beads in micro scale and enhance mass transfer efficiency by use of magnetic field. Therefore, the dynamic characteristics and transmission pattern of magnetic beads in the micro channel under the effect of magnetic field are important research on the control of magnetic beads23 and fluid viscous resistance24. In our study, the magnetic beads were used to be endogenous of microfluidic chip, which can fully ensure magnetic beads capture efficiency.Magnetic beads are generally exogenous for microfluidic chips, thus it is necessary to capture the magnetic beads to the designated position in chip channel and wait for the subsequent reactions, as shown in Fig. The magnetic beads mixing motion refers to the relative motion between magnetic beads and reaction solution, as shown in in Fig. 26 exist: (1) the integrated coil will generate heat, and a new heat source will be introduced on the chip, which will be difficult to adapt to the temperature sensitive sample; (2) the magnetic induction generated by MEMS is not high, so the separation efficiency is low; (3) the processing of MEMS is complicated and difficult; (4) the chip used in MEMS is usually opaque, which will be unfavorable for optical detection. However, the external permanent magnet system is relatively simple, which will not affect microchannel design and will be easy to achieve magnetic field control. In our study, a kind of external permanent magnet system was designed and the motion characteristics of magnetic beads based on the system were analyzed.According to the differences of magnetic field sources, the research about magnetic bead control technology is mainly divided into two categories: micro-electro-mechanical system (MEMS) and external permanent magnet system. Generally speaking, although the MEMS technology has higher integration, the following problemsMagnetic beads preset technology, mainly including the method of magnetic beads preset and the control structure of preset magnetic beads, is to pre place the magnetic beads into the microfluidic chip, and in this way, magnetic beads can directly participate in the reaction when the reaction occurs.28. Microfluidics detection is a micro total analysis system completing a series of biochemical experiments on a microchip, such as biological samples preparation, DNA extraction, PCR reaction, on-chip detection and so on31. Compared with traditional detection methods, microfluidic detection is obviously advantageous. Microfluidic can handle a small quantity of samples to speed up the synthesis and analysis and reduce the consumption of reagents. The high surface area mass ratio and heat transfer coefficient of microfluidic can significantly shorten the time required for reaction and analysis to improve detection efficiency. The laminar flow characteristics in microfluidics can accurately control and optimize the fluid motion. Microfluidics devices which are compact, miniaturized, highly automated, and allow parallel analysis, can achieve high-throughput, large-scale and rapid detection.Microfluidics is a technology using microchannels with sizes of 10–100 μm to handle or manipulate microfluidics34. The structure of microfluidic chip designed with preset magnetic beads is shown in Fig. The platform of microfluidic detection is microfluidic chipPreset coating mixing. Put coating solution (such as PVA) and magnetic beads into vortex oscillator to fully mix to ensure mixing solution uniform density.Preset coating spraying. The quantitative mixing solution was evenly coated on the bottom of magnetic beads preset groove on the chip, and the thickness of the magnetic beads preset coating can be adjusted as needed, but it should not exceed 1/3 of the magnetic bead preset groove depth.Preset coating drying. Place the chip with preset coating into the blast drying oven at the temperature of 50–70 ℃ for 20–30 min. After the water in PVA solution was volatilized, the magnetic beads preset coating will be formed, which can wrap magnetic beads and effectively adhere to the bottom of magnetic bead preset groove.Microfluidic chip assembly. Make the film bonding on the chip to complete preset magnetic beads encapsulation, and paste the permanent magnet on the back of the chip to complete the assembly of magnetic beads preset microfluidic chip.The process of magnetic beads preset is shown in Fig. The functions of preset magnetic beads control structure mainly include the capture of preset magnetic beads during the dissolution of magnetic beads coating and the full mixing between preset magnetic beads and reaction solution.Figure Full mixing of magnet beads and adsorbent (sample solution) or eluent is the premise to ensure the effect of adsorption or elution. Magnetic beads preset technology adopted double-sided permanent magnet system, and the magnetic beads mixing motion was realized by the difference of magnetic field intensity caused by the interaction between preset permanent magnet (fixed weaker permanent magnet) and the movable permanent magnet (stronger permanent magnet). As shown in Fig. After the magnetic beads preset coating was dissolved, the magnetic beads were forced on the bottom of magnetic beads preset groove by preset permanent magnet 1, and the initial position of movable permanent magnet 4 was located at the bottom of base guide groove, so the influence of movable permanent magnet on the magnetic beads could be ignored. After the adsorbent or eluent was filled, the movable permanent magnet bracket 5 and the movable permanent magnet 4 were raised. Because the magnetic field strength of movable permanent magnet was much stronger than that of the preset permanent magnet, the adsorption force of movable permanent magnet for the magnetic beads gradually increased with the rise of movable permanent magnet, and the magnetic beads began to leave the bottom of the magnetic beads preset groove and moved towards the film direction, which will realize the downward mixing of the magnetic beads with the adsorbent or eluent. When the movable permanent magnet moved downward, the adsorption force for magnetic beads gradually weakened, and the adsorption force of preset permanent magnet for magnetic beads became the main adsorption force again. The magnetic beads moved to the bottom of magnetic beads preset groove, which realized the upward mixing between the magnetic beads and the adsorbent or eluent. The control of magnetic beads mixing motion could be adjusted by changing the motion speed for adjusting mixing speed. The motion track of magnetic beads can be controlled by changing the size of movable permanent magnet. The suspension distribution of magnetic beads can be realized by changing the relative position of movable permanent magnet and preset permanent magnet. Magnetic beads mixing motion control platform is shown in Fig. 35. When the capture amount is insufficient, the detection requirements are hard to satisfy. When the capture amount is too large, the detection cost will increase, which will affect the promotion of microfluidic detection technology. The magnetic beads preset technology can accurately quantify the magnetic beads in the chip to effectively ensure the optimal amount of magnetic beads using for microfluidic detection.The coupling effect between flow field and magnetic field should be considered for magnetic beads capture in microfluidic chip, and the motion of magnetic beads capture is complex, and difficult to accurately control the amount of magnetic beads captureMicrofluidic detection involves the supply of different reaction liquid, the design of different mechanical control devices , and design of different field control systems . However, the size of microfluidic chip is very small, which leads to the higher integration of microfluidic detection device, so the operation structure design for microfluidic chip is more complex due to the limitation of space. And if a certain operation structure for microfluidic chip can be reduced, it will greatly simplify the microfluidic devices.With magnetic beads preset technology, the magnetic beads are pre-placed in the chip, reducing the magnetic beads feedings mechanism and the complexity of microfluidic device.The scanning electron microscope (SEM) of magnetic beads preset coating is shown in Fig. Materials and equipment: Quartz glass square hole capillary with inner diameter of 1 × 1 mm; Nd-Fe-B strong permanent magnet with external dimension of 20 × 10 × 3 mm; Video microscope, ZX-H1400, Shenzhen Zhongwei Kechuang Technology Co., Ltd.Experimental methods: a wire with smooth surface, good centrality and diameter of 0.6 mm was placed in the quartz glass tube in advance. A certain concentration of PVA solution mixed evenly with magnetic beads was inhaled into a needle tube, then the PVA/magnetic beads mixture were injected into the quartz glass tube. After drying, the wire was carefully taken out from the quartz glass tube to form a preset magnetic beads quartz glass tube. The quartz glass tube was placed in the self-made bracket with one end connected with the plunger pump and the other end led into the beaker through the teflon tube. A permanent magnet was set under the quartz glass tube, and a video microscope was set in front of the quartz glass tube. After all the preparations were done, start the plunger pump to make the distilled water pass through the quartz glass tube at a certain flow rate and observe the dissolution effect.For the convenience of observation, quartz glass square hole capillary was used as the carrier for preset magnetic beads to observe the effect of dissolution and capture.The dissolving process of preset magnetic beads is shown in Fig. It should be verified whether there was change in the function of magnetic beads mixed with PVA solution or not. The magnetic beads were prefabricated with 5%, 10%, 15%, 20% PVA coating solution respectively, then dissolved and removed PVA to acquire preset magnetic beads. The preset magnetic beads adsorbed the same plasmid DNA containing HBV genome, then the extracted DNA was detected by PCR. After reaction, 5 μL reaction products were detected by the electrophoresis with 2% agarose gel to evaluate whether PVA affected the adsorption ability of magnetic beads. The electrophoretic detection of magnetic beads adsorption ability under different PVA concentration are shown in Fig. The dynamic characteristics of magnetic particles are mainly determined by two factors in microfluidic chips, the gradient magnetic field applied externally and the flow field in the microchannel. Therefore, the motion characteristics of magnetic beads in the microchannel mainly involve the theory of magnetostatics, hydrodynamics and Newton's second law.36; magnetic field is mild and non-destructive to most living cells. In magnetic beads preset technology, compound magnetic field effect of different permanent magnets on the magnetic beads is mainly considered.Magnetic field is the main way to control material transport in microfluidic chip. Compared with electric field, magnetic field has unique advantages: magnetic force is not affected by sample concentration, pH value and other parameters; No heat is generated by permanent magnet; Magnetic field can penetrate most of the materials such as glass and polymerWhen the magnetic beads enter the chip from outside, the capture efficiency of magnetic beads is closely related to the flow rate. When the flow rate is low, the capture efficiency is high, but the efficiency of biological analysis system is low. When the flow rate is high, the capture efficiency is low. Although the suitable flow rate and capture efficiency can be achieved with optimizing, using the magnetic beads preset technology can weaken the influence of flow rate, focus on the influence of static flow field on magnetic beads, and simplify the analysis model.38. According to Newton's second law:m is the mass of particle, τ is the position vector diameter of particle, and Fm, Fd, Fg, Ff are the magnetic force, viscous force, gravity and buoyancy. Since magnetic force and viscous force are much greater than gravity and buoyancy, the model is simplified as formula .The forces on magnetic bead are shown in Fig. Fm can be expressed as:μ0 is the vacuum permeability, M is the field dependent magnetization of magnetic bead, Vp is the volume of magnetic bead, and H is the space magnetic field strength. When the particle is saturated magnetized, its field dependent magnetization is the saturation magnetization, that is, M = Ms.Magnetic force H is the mode of magnetic field intensity, H = |h|, and f(H) satisfies the equation:R is the radius of the magnetic bead.In general, the field dependent magnetization follows the formula:B is the magnetic flux density.Because The magnetic field force on magnetic bead depends not only on the magnetic induction intensity, but also on the magnetic field gradient. Magnetic bead tends to move towards the maximum magnetic field intensity in the non-uniform magnetic field. The magnetic force in different directions are as follows:η is the dynamic viscosity of fluid, r is the radius of magnetic bead, vf is the velocity of fluid, vp is the velocity of magnetic bead, and fd is the hydrodynamic resistance coefficient.In microfluidic system with low Reynolds number, the viscous force acting on particles can be obtained by Stokes law:s is the distance between the magnetic bead and the wall of microchannel. When the magnetic bead is far away from the wall, fD = 1; When the magnetic bead is near the wall, fD is slightly greater than 1.When the wall effect is considered, the expression of hydrodynamic resistance coefficient is as follows:The time required for magnetic bead to reach a new equilibrium is extremely small. It can be considered that the motion of magnetic bead in the microchannel is always in a Quasi equilibrium state. The motion equation of the magnetic bead can be simplified as:By introducing Eqs.  and 9),,9), we gThe velocity components in each direction can be expressed as:When the magnetic beads with a certain velocity flows go through the microchannel, the magnetic beads will be shifted and separated from the fluid by magnetic force. Considering that the width to height ratio of the microchannel is large, the separation process can be simplified as a 2D model.Ff is the friction force between magnetic bead and the channel upper surface. When the magnetic bead is stable in the capture position, Fmx1 = 0 and vP = 0. The condition for the successful capture of magnetic beads under the velocity vf is as follows:The capture motion includes magnetic beads captured by permanent magnet 1 (preset permanent magnet) after magnetic beads released and fixed on the upper surface of the channel during solution washing. The force analysis of magnetic bead capture is shown in Fig. f is the friction coefficient between the magnetic bead and the flow channel. Introduce Eqs. 15\documeuce Eqs. , and getLet Therefore, only when the z-direction magnetic force produced by permanent magnet 1 is greater than [a] times of the liquid flow rate, the magnetic beads can be effectively captured.vfz is considered as 0.The mixing motion between magnetic beads and solution can be divided into upward mixing motion and downward mixing motion, which are driven by different permanent magnets. Because of using preset magnetic beads technology, Fmz1 > Fd, considering the quasi equilibrium state of magnetic bead motion:The upward mixing motion is achieved by permanent magnet 1. At this time, the permanent magnet 2 falls far enough, so the influence of its magnetic field on magnetic bead can be ignored. The force analysis of upward mixing motion of magnetic bead is shown in Fig. Q, and integrate both sides:t1 is the time required for upward mixing motion.The microchannel height is fmz2 > fmz1 + Fd, considering the quasi equilibrium state of magnetic bead motion:The downward mixing motion is achieved by permanent magnet 2. The force analysis of downward mixing motion of magnetic bead under the influence of the magnetic field of permanent magnet 1 is shown in Fig. The downward velocity of magnetic bead is:t2 is the time required for downward mixing motion.Integrate both sides:t1 and t2. After the magnetic beads are constant, the mixing efficiency depends on the following factors: (1) the smaller fluid dynamic viscosity, the higher mixing efficiency; (2) the greater magnetic field intensity of preset permanent magnet, the shorter upward mixing motion time, but the downward mixing motion needs to be considered comprehensively; (3) the greater magnetic field intensity difference, the shorter downward mixing motion time; (4) the smaller channel height, the higher mixing efficiency. After the type of magnetic beads, the size and quantity of flow channel and the type of different reaction solutions are determined, the specification of permanent magnet should be reasonably selected to ensure the magnetic beads motion effect, simplify the device structure and reduce the structure size.The mixing efficiency of magnetic beads is determined by Through the derivation of the upward mixing movement time and downward mixing movement time of preset magnetic beads, the time required for a single full mixing movement can be clearly obtained, and the total time required for the mixing movement can be obtained according to the number of up-and-down movements through related DNA extraction and elution effect experiment, which provides a basis for the determination of relevant parameters of the control magnetic field.w = 0.15 mm and height h = 0.15 mm. The material of microfluidic channel was set as air, which could truly simulate the impact of the environment. The boundary condition of the air was set as magnetic insulation, and the grid was divided according to the standard. The relevant parameters are: relative permeability of permanent magnet 1, vacuum permeability 4π × 10−7 H A−2, permanent magnet x axial remanence 0, permanent magnet y axial remanence ± 0.4 T.In the magnetic beads preset technology, the most important is how to select the appropriate permanent magnet to ensure the capture efficiency and mixing efficiency. We have developed a method to simulate the magnetic field by using COMSOL and verify the variation characteristics of magnetic field by changing the aspect ratio of permanent magnet in the microfluidic channel. As shown in Fig. The spatial distribution of magnetic field is shown in Fig. x = 1 mm, and the magnetic field intensity in y-direction reaches the maximum. Since the magnetic field force is directly proportional to the magnetic field gradient, it can be inferred that the magnetic force in x-direction will have the maximum near x = 1 mm. Therefore, the magnetic beads will tend to move in the central region of the permanent magnet.The magnetic field intensity component generated by permanent magnet in microchannel centerline is shown in Fig. x components of microchannel centerline with different aspect ratio are shown in Fig. w of the permanent magnet remains constant, the aspect ratio has no effect on the curve shape of the magnetic field intensity, which is a sinusoidal curve. The maximum value of x-direction magnetic field intensity and gradient magnetic field increase with the increase of magnet height h, but when the height of permanent magnet increases to a certain value, the increase of magnetic field intensity in the channel decreases gradually until it keeps constant. Therefore, the aspect ratio of permanent magnet can be increased appropriately to obtain greater magnetic field force and improve the magnetic beads capture and mixing efficiency.Magnetic field A magnetic beads preset technology was provided, and the feasibility of its application in microfluidic chip was verified. Based on the analysis of magnetic beads control motion, the motion model of preset magnetic beads was established, including capture motion and mixing motion. The mixing motion was decomposed into upward mixing motion and downward mixing motion, and the relationship between capture condition, mixing time and coupling field was derived, which can provide a calculation and design basis for simulation and control of preset magnetic beads. However, in the derivation of mixing time, the influence of movable permanent magnet was completely ignored in the upward mixing motion. And the time of downward mixing motion was calculated based on the movable permanent magnet moving to the closest position to the chip, but the equilibrium position should be located in a certain position during the ascending motion of movable permanent magnet. The actual mixing time should be shorter than the calculation time, that is, the velocity of movable permanent magnet need to be considered for a more accurate magnetic beads motion control time. In addition, our study ignored the influence of magnetic force in the direction of flow channel, which can be considered in the follow-up study to deeply analyze the distribution trajectory of magnetic beads along the flow channel.


Rodents (order Rodentia), followed by bats (order Chiroptera), comprise the largest percentage of living mammals on earth. Thus, it is not surprising that these two orders account for many of the reservoirs of the zoonotic RNA viruses discovered to date. The spillover of these viruses from wildlife to human do not typically result in pandemics but rather geographically confined outbreaks of human infection and disease. While limited geographically, these viruses cause thousands of cases of human disease each year. In this review, we focus on three questions regarding zoonotic viruses that originate in bats and rodents. First, what biological strategies have evolved that allow RNA viruses to reside in bats and rodents? Second, what are the environmental and ecological causes that drive viral spillover? Third, how does virus spillover occur from bats and rodents to humans? Hepacivirus genus have been isolated from reservoirs such as dogs, rodents, and horses [Globalization, environmental and anthropogenic changes provide ample opportunities for spillover and emergence of zoonotic diseases ,2,3. Thed horses . The cord horses ,15; but Hantaan orthohantavirus (HTNV) and Sin Nombre orthohantavirus (SNV)) and arenaviruses , Lassa mammarenavirus (LASV), and Argentinian mammarenavirus (formerly Junin virus (JUNV))) found circulating in the Americas or Africa (Nipah henipavirus (NiV) and Hendra henipavirus (HeV) are limited to the geographical range of the bat species  followed by bats (order Chiroptera). According to the Mammal Diversity Database maintained by the American Society of Mammologists, there are 2590 extant species within the Rodentia and 1430 extant species within the Chiroptera. Together, these represent 63% of the 6410 mammalian species known to exist today. Orders Rodentia and Chiroptera diverged approximately 96 million years ago (MYA) according to the most recent available published data (65 studies ca. December 2020) in TimeTree ,17. Bothaviridae . Chiroptaviridae ,23,24,25oviridae ,32,33. Ooviridae . For somonfirmed ,35,36,37In the following section, we discuss how biological factors and lifestyle traits contribute to the ability of bats and rodents to serve as a reservoir for an RNA virus. For bat and rodent reservoirs of RNA viruses, multiple characteristics may contribute to their ability to serve as a reservoir ,39,40,41Lyssavirus, has been reported [Artibeus jamaicensis, the Jamaican fruit-eating bat, with a high dose (not the low dose) of Tacaribe mammarenavirus (TCRV) [Calomys callosus (large vesper mouse) [C. callosus infected with MACV produce only 5% of the expected number of viable offspring, although having normal estrus cycles. MACV does not cause illness or death in either adult or juvenile mice, and as with other arenaviruses, it infects a variety of cell types. Although infectious virus has been recovered from most major organs, the kidney and spleen are the important target organs for arenavirus replication in rodents [Peromyscus maniculatus) in the USA [Myodes glareolus) populations harboring Puumala orthohantavirus (PUUV) surveyed in 55 areas over three years in Finland [Numerous studies have reported that with some exceptions , many RNA viruses may infect and persist in bats and rodents without causing disease ,44,45,46reported ,48,49. Hreported . In addis (TCRV) . TCRV has (TCRV) . In studr mouse) . In addir mouse)  found th rodents ,55. For  the USA  and in b Finland .Rousettus leschenaultii) were experimentally infected with a wild-caught Betacoronavirus isolated from the lesser short-faced fruit bat (Cynopterus brachyotis) in the Philippines, but these animals showed no clinical signs; viral RNA was only detected in the intestines and not in the liver, kidney, lung, spleen or brain. Similarly, Munster et al. [Rousettus aegyptiacus, were infected with Marburg marburgvirus (MARV), and no deaths, overt signs of morbidity, or gross lesions were identified, although, microscopic pathological changes were seen in the liver of infected bats [Pteropus poliocephalus) with NiV and reported infection, and pathological examination of tissues did not confirm the presence of the virus with associated pathology so that lesions could not be attributed to viral infection. Woon et al. [Pteropus alecto) with HeV and showed virus replication in the lungs and an inflammatory response, but no viral shedding nor pathology. Experimental infection of Egyptian rousette bats with the paramyxovirus, Sosuga pararubulavirus, showed infection and virus shedding, but no morbidity nor mortality. However, they report subclinical disease in a subset of tissues [Although it is clear that accepted, or presumed reservoir hosts, have an immunological response to virus infection, no apparent disease is detected in experimental or natural infections of certain hosts with viruses such as some coronaviruses, henipaviruses, some hantaviruses, paramyxoviruses, and filoviruses. In an interesting study by Watanabe et al.  laborator et al.  experimeted bats ,62,63,64ted bats , wild-cated bats  infectedn et al.  also inf tissues .Uniquely for bats, one proposed explanation for the absence of disease for some viral infections is the “flight as fever” hypothesis which posits that the high metabolic rate required for flight stimulates a fever response ,69. DailThe ability of bats and rodents to modulate the immune response to viral infection has been an active area of investigation and has been recently reviewed elsewhere ,45,74,75Most viruses are indirectly sensitive to the induction of IFNs following infection as they induce the upregulation of ISGs; the proteins transcribed from the ISGs actively engage in an antiviral response that directly or indirectly thwart virus replication. Thus, many RNA viruses have evolved strategies to suppress the IFN response and studies suggest similarity of the innate immune signaling pathways in bats and rodents ,79, althEptesicus fuscus, cells stimulated with poly(I:C); however, tumor necrosis factor-alpha, TNF-alpha, was much lower, and suppressed. In one study with Egyptian rousette bats experimentally infected with MARV, Guito et al. [Research on bat IFNs suggested diversity in the expression of IFN-alpha. In the black flying fox (reservoir of henipaviruses), and the lesser short-nosed fruit bat IFN-alpha is expressed constitutively . This iso et al.  observedMicrotus arvalis) and bank vole for hantaviruses will provide a valuable tool to exploring virus-reservoir host interaction [Two studies have examined henipaviruses in an immortalized cell line derived from the black flying fox ,85. The eraction . The Myxovirus resistance (Mx) proteins are ISGs that are well-studied in in-bred laboratory rodents and shown to directly inhibit many RNA viruses (reviewed in ). In wilZaire ebolavirus (ZEBOV), vesicular stomatitis virus and Rift Valley fever phlebovirus (RVFV) [Pipistrellus pipistrellus did not show in vitro antiviral activity against RVFV, suggesting functional differences may exist among bats. Direct examination of immune signaling in primary bat cells from a reservoir with the virus they harbor has not been reported for any of these viruses. The Mx1 gene was cloned from three families of bats: Pteropodidae, Phyllostomidae, and Vespertilionidae . Examinas (RVFV) . HoweverMyotis lucifugus) may potentially be more than 70,000 specificities [Bats potentially have more VDJ germline gene segments when compared to other mammals and with this lies the potential for bats to have a much larger naive immunoglobulin repertoire. For example, the naive B cell repertoire of the little brown bat  infection of animals without a functional T cell response  succumb rapidly to infection and diseases suggesting that cell-mediated immunity plays an important role in controlling infection [Rattus norvegicus) and SNV-infected deer mouse [For some hantaviral infections, the differences in immune responses between reservoir and nonreservoir host are evident in the cytotoxic T lymphocytes (CTL) response . nfection . Mouse mnfection  were usenfection . They shnfection . Viral rnfection ,100,101.nfection  proposednfection ,50. Treger mouse ,103. In er mouse . As with hantaviruses, the outcome of arenavirus infection is mediated by CTL responses ,105,106.For bats, less is known about the T cell responses following viral infection due to the lack of available immunological reagents; however, some groups have begun phenotypically and functionally characterizing bat lymphocyte subsets ,117. GomHomo sapiens. Some, such as the straw-colored fruit bat  are tree-roosters and long-distance migrators. However, the majority of massively colonial species are cave-roosters, where they congregate in maximally close proximity [, bent-winged bats, Miniopterus spp. (Miniopteridae), Egyptian slit-faced bats, , and several species of free-tailed bats ) [Unlike most rodents, most species of bats are social, roosting in groups which may range from a few individuals to colonies of many millions of individuals, including the largest single-species aggregations of mammals on earth except for roximity . Examplessidae)) ,123,124.Pteropus spp.  and Rhinolophus spp. (SARS-CoV related viruses) [Bat species that live in social groups, whether in small groups or in massive colonies with close contact, must play a key role in viral maintenance in the population. In large permanent colonies, viral transmission can potentially follow the birthing cycle of pups, in which an increase in population size with naive (newborn) members reduces herd immunity, allowing for viruses to be reestablished cyclically within a colony ,126. Recviruses) . These bCalisher et al.  list andMastomys natalensis, natal multimammate mice, the reservoir of LASV, are found in high abundances within human dwellings, which likely drives the higher rate of human LASV infections [for Akodon montensis [As with bats, the behavioral and demographic characteristics of rodents may contribute to their capacity to serve as reservoirs for harboring and maintaining viruses. In comparison, trade-offs between reproduction and lifespan constitute the life-history of rodents. Rodents generally exhibit an r-selected life history, characterized by early sexual maturity and large litter sizes , which lfections . Even onfections , and micfections . Moreoveontensis . In geneontensis  found evArenaviridae and Hantaviridae persist for the lifetime of the reservoir host, with the viruses present at relatively low levels in the host [Guanarito mammarenavirus, JUNV, LASV, LCMV, MACV, and Morogoro virus, studies have shown an age-dependency in the duration of infection among reservoir hosts [Some viruses within the the host ,142,143.ir hosts ,150,151.ir hosts ,150,151.ir hosts ,65.0, which represents the average number of secondary infections possible from a primary infected individual [0 falls below 1.0 and remains below 1.0 for several generations, then the virus will eventually be eliminated from the population. We have limited information on the R0 of RNA viruses in bat and rodent populations and how it is affected by physiological and environmental factors as well as age, the duration of infection, and animal behavior.Regardless of whether the virus persists in its reservoir host, the maintenance of virus in the population depends on the value of the basic reproduction number, Rdividual . When th−3 to 10−5 per round of RNA synthesis [−4 rate of misincorporation results in, on average, one error per round of replication. As discussed by Peck and Lauring [All the viruses discussed herein replicate and transcribe their genomes using an RNA dependent RNA polymerase (RdRp) encoded in their genomes. The RdRp and other viral proteins that support replication do not usually correct misincorporation of nucleosides—an exception being coronaviruses and other members of its family that can proofread mismatched base pairings during replication. Thus, replication and transcription have a high rate of error ranging from 10ynthesis ,154,155. Lauring , the mea Lauring . Genetic Lauring ,158,159. Lauring ,160,161.Paradoxurus hermaphroditus) or humans [Examples of genetic mutations in viruses harbored by wildlife reservoirs that emerge and play a role in promoting reservoir-to-human and sustained human-to-human transmission are few but often involve the binding and/or entry of the virus to host cells, enhanced replicative ability in the new hosts, and/or suppression of innate immunity. Mutations enhancing reservoir-to-human transmission were reported in the first SARS-CoV spillover, where mutations in the virus’s spike protein increased the protein’s binding affinity to angiotensin converting enzyme 2 (ACE2) receptor of either the intermediary host, Asian palm civets , human expansion into habitat , and land fragmentation  are some of the key drivers which seemingly alter wildlife populations and hence the balance of the virus in reservoir populations and drive spillover ,165,166.2) [The ecological drivers of outbreaks by emerging and re-emerging zoonotic viruses are not well understood due to the challenges in designing controlled field studies to understand how each of these specific factors drive pathogen presence and prevalence in wild animal populations, and during an outbreak, surveillance is usually not conducted with this in mind. For example, during the outbreak of hantavirus pulmonary syndrome (HPS) in the southwestern USA in 1993 researchers surveyed animals in the vicinity of the trailers and homes of infected persons. This resulted in the identification of the deer mouse, as the reservoir of the causative agent, SNV. In nature, the prevalence of hantaviruses in rodent populations can vary from undetectable to 40% in the primary rodent reservoir ,173,174.2) . This ra2) . Retrosp2) . Nearly 2) .It has been suggested that the most important drivers of spillover of bat-borne RNA viruses to humans is the anthropogenic encroachment into natural habitats of bats through urbanization and agriculture ,179,180.Clearly, reservoir population density and the prevalence of the virus in the population contribute to the probability of increased prevalence of the virus in its reservoir and the probability of spillover. In general; however, very little is known for many viruses in terms of how these viruses are maintained within their natural reservoirs, the normal prevalence of virus in wildlife, genetic variation within host and communities, and what drives increased spread in reservoir populations. In addition to natural history study of reservoir-virus populations in their native habitats, experimental field studies would help identify factors that change virus–host population dynamics.The majority of spillover events from a wild rodent  or bat  species In the following, we briefly review the above scenarios of spillover that resulted in human cases of disease and characteristics of these events. Rabies lyssavirus, genus Lyssavirus, was the first bat-borne virus discovered, which in retrospect is unsurprising because 50% of the currently known viruses carried by bats are rhabdoviruses [oviruses ,192. Theoviruses . Dr. Pawoviruses . In the oviruses . Accordioviruses . In addioviruses ,196,197.Human cases of infection have been associated with consumption of their reservoirs for both bats and rodents. In certain areas, eating rodent bushmeat is considered a delicacy; therefore, human transmission can also occur by exposure to their bodily fluids  durNyctereutes procyonoides) from live markets may have played a crucial role in outbreaks of human diseases such as those caused by SARS-CoV [Some outbreaks in humans involving paramyxoviruses, coronaviruses, and filoviruses have occurred through contact with an intermediate host that acts as an amplifier host ,202,203.SARS-CoV . Since cSARS-CoV . The batSARS-CoV ,202. In SARS-CoV ,207,208.Chlorocebus pygerythrus) that were imported from Uganda [MARV emerged in 1967 in Marburg and Frankfurt, Germany, as well as in Belgrade, Serbia, when 31 cases were reported of an unknown disease to which seven patients succumbed . Epidemim Uganda ,211. Them Uganda ,213,214.Zaire ebolavirus (ZEBOV) emerged in Zaire (now the Democratic Republic of Congo) and resulted in 318 cases with a mortality rate of 88% [In September 1976, e of 88% . The fire of 88% . Outbreae of 88% ,217. Bate of 88% ,37. While of 88% ,219,220.e of 88% .Henipavirus associated with outbreaks of human disease, HeV and NiV; these viruses are carried by multiple species of the bats commonly referred to as flying foxes (Pteropus spp.) [There are two species of the us spp.) ,224,225.us spp.) ,201. Sinus spp.) ,227,228.us spp.) ,230. The first outbreak of NiV occurred in Malaysia during 1998 in pigs and humans, with 265 human cases  and the culling of nearly one million pigs ,232. TheEpidemiological evidence suggests the indirect mode of transmission (aerosolized excreta) from rodents or bats to humans is similar for many zoonotic viruses. The literature supports indirect contact via inhalation of rodent excrement or direct contact with rodents such as rodent bites (discussed above) as the main routes of transmission to humans . These eIn South America, human cases of HPS, Argentine hemorrhagic fever (AHF), and Bolivian hemorrhagic fever (BHF) have been associated with agricultural activities. In the following we briefly discuss two notable outbreaks of arenaviruses associated with agricultural activity. Seven species of arenaviruses are known to cause hemorrhagic fever in humans in South America and Africa, presumably through the inhalation of aerosolized infectious excreta ,241,242.Calomys musculinus (dry lands vesper mouse) and C. laucha , are endemic to the Humid Pampas, a farming region in central east Argentina [C. musculinus is believed to be the primary reservoir of JUNV, although antibody to the virus is also detected in C. laucha [After its isolation in 1959, JUNV was the second arenavirus (following LCMV in 1933 in the USA) recognized to cause human illness, AHF ,244. Thergentina . C. musc. laucha . Rodent . laucha . These oC. callous [C. callosus in a span of 4 months, demonstrating that this rodent can acquire high population densities around human habitations. During 18 months of research in Beni, Bolivia cases of BHF were reported in every month of the year, with a distinct increase from January to May a period of intense agricultural activity (rainy season) [C. callosus invades homes during the rainy seasons. However, only a handful of cases have occurred since the 1960′s. In 2007–2008, an outbreak of five cases was reported among farmers in Bolivia [Around the same time as JUNV was recognized, human outbreaks of MACV associated with Bolivian hemorrhagic fever (BHF) were reported in Bolivia . The res callous . Outbrea callous . It was  callous . Rodent  season) . It was  season) . Since i Bolivia .After the first cases of MARV through direct contact with nonhuman primates, several other large outbreaks occurred throughout central and southern Africa; however, in contrast to the interaction with an intermediate nonhuman primate host discussed above, these cases were associated with patients visiting caves ,252,253.Sudan ebolavirus (SUDV) likely originated in a cotton factory in Nudar, Sudan in June 1976. In the five months that followed, 284 cases were reported with a mortality rate of 53%. It is suggested that exposure occurred via excreta from bats roosting in this factory [The first human cases of  factory .M. natalensis tends to aggregate in homes in search for food [Hylomyscus pamfi, in Nigeria and Guinea multimammate mouse, Mastomys erythroleucus, in both Nigeria and Guinea were recognized as additional hosts [Cases of Lassa fever were recognized in the 1950s; however, the virus causing the illness, LASV, was not identified until 1969 when three nurses died from it in a mission hospital in Nigeria ,256. Manfor food . These rfor food . More real hosts .In the first outbreak of SNV in the summer of 1993 through 1995 in the Four Corners states of the southwestern USA, 69% of the hantavirus pulmonary syndrome (HPS) cases had exposures closely associated with peridomestic activities in homes that showed signs of rodent infestation . A retroThe first recorded outbreak of hemorrhagic fever with renal syndrome (HFRS) associated with the hantavirus, HTNV occurred in soldiers during the Korean War in the early 1950′s and probably resulted from outdoor activities such as sleeping in tents and performing military exercises . SimilarLujo mammarenavirus involved a 36-year-old female travel agent who worked on a peri-urban farm near Lusaka, Zambia. Following illness, she was flown to Johannesburg, South Africa to be treated at a private hospital. There, four healthcare workers were exposed and became ill and three died [Andes orthohantavirus (ANDV) which have occurred in Argentina and Chile [Most human infections with hantaviruses and arenaviruses are associated with indirect exposure of rodent excreta. However, viruses in each genus have had suspected or documented cases of person-to-person transmission. In one report of illness from MACV, a 20-year-old female nursing student living in Cochabamba, Bolivia became ill following travel to her hometown in Fortaleza in Beni Department in December 1970 . Upon reree died . Among tnd Chile ,278,279.nd Chile . The outnd Chile . HoweverThe number of novel or reemerging RNA viruses which cause infections in humans increases each year. While most of these zoonotic infections do not lead to a pandemic, the global public health burden of all combined infections is high. The 2002–2003 SARS-CoV epidemic resulted in more than $40 billion in losses despite causing fewer than 1000 deaths . PredictIn addition to the natural reservoirs, intermediate spillover hosts  play an important role in the transmission network of viruses to humans. As such, intermediate hosts acting as a bridge between natural reservoirs and humans warrants greater attention for advancing our understanding of virus transmission and maintenance from these hosts. In addition to surveillance of bats, efforts need to focus on intermediate hosts as many bat viruses’ transmission to humans relies on these amplifier hosts. Metagenomic surveillance of potential intermediate hosts would provide valuable information on circulating and newly introduced viruses. Maintaining such samples in tissue biorepositories for wildlife specimens in museums is critical for retrospective analyses. In areas where reservoirs and intermediate hosts are known, control measures should be used to keep these reservoirs in check, such as preventing bats and rodents from contaminating food sources. Controlling the further spread of outbreaks is an important aspect of study to prevent isolated outbreaks from progressing into epidemics or pandemics. One essential feature to controlling outbreaks is understanding transmission routes to limit further spread, e.g., aerosol with SARS-CoV-2 and exposure to infected bodily fluids in the case of EBOV. Controlling virus transmission during outbreak scenarios depends on virus population dynamics within the reservoir and intermediate hosts. Understanding virus populations and their variants within reservoir and intermediate hosts are important as inherent mutations and mutations acquired through transmission may lead to an increase in viral fitness, allowing for spillover and increased transmission, as seen in the case of SARS-CoV and the Asian palm civet. Additionally, host genetics needs to be investigated as host genetic background can influence infection outcome. Some host population members can have a higher likelihood of transmitting a virus compared to others within the same population . Efforts focused on virus surveillance, virus–host interaction, and virus population changes can be complicated as reservoirs, intermediate hosts, and the viruses they harbor lack necessary reagents for their study, so development of these reagents are essential. In summary, we have addressed (1) why zoonotic viruses reside in bat and rodent reservoirs, (2) environmental and ecological causes driving spillover, and (3) shared and unique characteristics of previous epizootic events. Further investigation of these topics is a long-term process that will take vigorous effort, but continuous investigation will provide an effective means to understand the relationship between spillover and emergence of bat- and rodent-borne RNA viruses in their hosts.


The increasing risk from viral outbreaks such as the ongoing COVID-19 pandemic exacerbates the need for rapid, affordable and sensitive methods for virus detection, identification and quantification; however, existing methods for detecting virus particles in biological samples usually depend on multistep protocols that take considerable time to yield a result. Here, we introduce a rapid fluorescence in situ hybridization (FISH) protocol capable of detecting influenza virus, avian infectious bronchitis virus and SARS-CoV-2 specifically and quantitatively in approximately 20 min, in virus cultures, combined nasal and throat swabs with added virus and likely patient samples without previous purification. This fast and facile workflow can be adapted both as a lab technique and a future diagnostic tool in enveloped viruses with an accessible genome. As a respiratory infection caused by the coronavirus SARS-CoV-2, it first surfaced in China in late 2019 and has since resulted in over 4 million deaths worldwide. Contact tracing and rapid testing have been important tools to limit the spread of disease among the human population.With over 200 million confirmed cases so far, COVID-19 is the most devastating worldwide pandemic since the Spanish Flu of 19184. Due to its high sensitivity and specificity, reverse transcriptase-mediated polymerase chain reaction (RT-PCR) represents the gold standard in SARS-CoV-2 testing. However, an RT-PCR test takes several hours to complete, and can only be performed in a laboratory setting, as it requires viral lysis and RNA purification procedures and therefore sample collection, transportation and reception. This routine commonly delays test results by 24 h or more. As a promising alternative to RT-PCR, isothermal nucleic acid amplification methods such as loop-mediated isothermal amplification (LAMP) usually yield results within 1 h11. However, both RT-PCR and LAMP testing have been compromised by supply chain issues due to expensive production and storage conditions of the required reagents. Rapid antigen-detection tests based on immunoassays and lateral flow formats have been developed to detect SARS-CoV-2, but these can suffer from low sensitivities12. Thus, alternative testing methods that yield quick results with low material costs are still urgently needed.Currently, the most common diagnostic tests for a SARS-CoV-2 infection are based on nucleic acid amplification, antigen detection and serology tests16. FISH has been used to detect and identify pathogenic microbes, including bacteria18 and viral infections, e.g. HIV, Epstein-Barr virus and Dengue inside infected cells22.Fluorescence in situ hybridization (FISH) is a cytological technique that exploits the selective binding of fluorescently labeled oligo- and poly-nucleotides  to complementary sections of DNA and RNA inside fixed cells and tissues for the purpose of detection, quantification and spatial localization by a fluorescence microscope. In a medical context, FISH has predominantly been used to detect chromosomal aberrations and rearrangements in fixed cells, including translations, insertions and deletions24. By increasing the number of fluorophores that bind one RNA transcript, detection of single transcripts became possible, giving rise to single molecule FISH (smFISH)26. Typical smFISH applications use an array of fluorescently labelled DNA oligonucleotides (probes) that bind to several sites on the viral genome, thereby accumulating several fluorescent dye molecules on a viral RNA, making it visible as a bright, diffraction-limited spot, while unspecific binding to the surface will lead to a darker background of single probes. Further, by utilizing smFISH probes that carry spectrally distinct fluorophores, more than one RNA species can be imaged simultaneously27. In virology, single viral genomes or gene segments can be detected by smFISH leading to new insights into the molecular mechanism of infection, replication and assembly of influenza viruses30. Similar studies have been performed in other viruses, e.g., rotavirus, bunyavirus and human leukaemia virus33. Importantly, isolated virus particles can also be visualized by smFISH34.RNA FISH has been used extensively to visualize gene transcription in situ35. By increasing the concentrations of FISH probes and optimizing fixation conditions, RNA FISH hybridization times could be reduced to 5 min36. Moreover, choosing target sequences that do not form secondary structures can greatly increase hybridization rates37.Using smFISH, single viral RNA molecules can be detected without the need for enzymatic amplification. However, typical FISH assay times still take several hours to an entire day5 PFU/ mL and 102 PFU/ mL, respectively, in a 20-min assay. Further, we demonstrated that inactivated SARS-CoV-2 particles could be detected in the same way. Moreover, we showed that virus particles could be detected in nasopharyngeal swabs. By employing a dual-labeling strategy, we were able to suppress background caused by misidentified bright, fluorescent spots. Taken together, our data suggest that smFISH can be used as a powerful means to quickly and easily detect, identify and quantify a wide range of virus strains in both research and diagnostic applications.In this study, we introduce a rapid viral FISH protocol (rvFISH) for the detection of virus particles; the development of this improved assay depended on a systematic analysis of the efficiency of the hybridization reaction as we reduced the number of experimental steps and the hybridization time. We were able to detect influenza particles and infectious bronchitis virus (IBV), an avian coronavirus, down to a concentration of 1035 /mL would contain 20,000 PFU. Out of the total surface, only an area of ~ 3 × 105 µm2 was imaged, which would result in the detection of 200 PFU. However, the total number of virus particles is known to typically exceed the PFU by orders of magnitude, depending on the virus strain39.In our FISH setup, we applied a 20 µL of sample to a surface area of ~ 3 × 10To determine the labeling efficiency of this standard viral smFISH protocol, we calculated the number of probes bound to the neuraminidase (NA) gene of the influenza virus strain A/WSN/1933 (WSN). To achieve this, we immobilized the virus particles, stained them using a Quasar570-labelled FISH probe array comprising 48 individual 20-nucleotide (nt) probes Table , and use, the evaporation temperature was increased to 50 °C; at this temperature, a 20 µL sample took 10 min to evaporate, compared to 30 min in the standard protocol at room temperature. If the evaporation temperature was too high, e.g. 100 °C, the FISH signal was drastically reduced or absent. Evaporation was the most efficient method of immobilization, while interaction-based methods, e.g. PEG-Biotin, resulted in lower particle counts. Coating the glass surface with poly-l-lysine or chitosan did not increase the immobilization count, while occasionally resulting in a higher background from single probes. To reduce the hybridization time, we first considered that our results /mL) and IBV (106 PFU/mL). For both stocks, experiments were performed and tested using the complementary FISH probe array (positive sample) and an array that targeted one of the other two stocks (negative control). We obtained bright signals for both virus strains when the complementary FISH probe sets were used; in contrast, we obtained only low or no background in the presence of the non-complementary probes  infectious bronchitis virus (IBV), comprising 32 individual 20-nt DNA probes Table . The proWe also generated distinct intensity distributions for the detected fluorescent spots in both positive sample and negative control data sets Fig. B. While 5 PFU/ mL for WSN and 102 PFU/ mL for IBV 40 or lateral flow assays (LOD ~ 102 PFU/ mL for SARS-CoV-2)41.Next, we determined the detection limit of the rvFISH assay from the fluorescence intensity distributions. To quantify the number of particles, we corrected their intensities for day-to-day fluctuations in laser power (about ± 20%) by normalizing them to the approximate intensity value of a single fluorophore, given by the median intensity value of the negative sample with the lowest virus concentration. Spots within an intensity range of 15–40 single fluorophores were counted as particles. While particles were present in both positive and negative samples, the number of detected particles was significantly higher  in positive samples down to a concentration of 10IBV Fig. C,D. A dincy Fig. C. The dency Fig. B. Overal6 PFU/ mL) that was diluted to 104 PFU/mL. Similar to WSN and IBV, a high number of bright particles was only seen in the presence of the SARS-CoV-2 probes  and Quasar650 (red), respectively. The probes of both sets bind along the NA segment in an alternating manner Fig. A. Howeve42. To determine how the throat swab medium and any biological material from the patient affects the detection limit, we serially diluted the swab with 0.9% NaCl. To directly compare the particle counts, we added IBV to a final concentration of 104 PFU/mL in all samples, a concentration that represents a compromise between a high number of detected virus particles and low noise. All samples were clarified by centrifugation and the rvFISH protocol was performed on them as described above. Additionally to IBV probes  and SC2 (Ct = 27), showed elevated particle counts when compared to the negative samples 41. The discrepancy in LOD between the two virus strains can be explained by the observation that in general, not more than one in 10,000 cultured coronavirus particles is thought to be infective, while the particle-to-PFU ratio in influenza ranges from 10:1 to 100:139. Therefore, qRT-PCR with a detection limit of 102 RNA copies/mL can still be considered at least 100 times more sensitive than our assay in its current form. However, not all RNA molecules in a virus sample might be accessible to PCR amplification, a scenario that is less likely for the binding of multiple FISH probes to an RNA target. To further lower the LOD of the rvFISH assay, the number of false-positive particles from fluorescent noise needs to be reduced, while the sampled area needs to be increased.The LOD was defined as the lowest tested virus concentration with statistically significant difference  between a sample with a complementary probe set and a sample with a non-complementary control probe set. The LOD of our assay was 1046. Here, deep learning may be a future option to recognize and eliminate false-positive particles from our FISH analysis.Regarding the reduction of false positives in the negative control, we note that, in our FISH data, we usually discovered ~ 1 bright fluorescent spot per FOV that was identified as a virus particle by the detection algorithm, thereby limiting the sensitivity of our assay. We also demonstrated the success of a dual-color labelling approach, which used co-localization analysis to reduce the number of false-positive particles fivefold. The false positives can be reduced further by employing both a higher probe concentration and a larger probe array. The rationale in using more probes is that in a negative sample, false-positive particles become steadily less frequent at higher intensities; if virus particles have more probes bound to them, they will be brighter and therefore better distinguishable from most of the noise. Further, in a previous study, we used deep learning to detect virus particles in culture supernatants and clinical samples by staining their surface with fluorescent oligonucleotides, we used the difference in particle counts between positive and negative samples at an IBV concentration of 102 PFU/ mL to estimate that we could observe ~ 150 particles per dataset. This means that the theoretical detection limit in the absence of any noise would be around 100 PFU/ mL, as the sampled area will contain less than one particle on average for lower concentrations. However, we only sampled an area of 0.33 mm2, compared to the total area of 28 mm2. Thus, sampling the entire area would increase the theoretical sensitivity by two orders of magnitude, comparable to RT-PCR. To achieve sampling of the total area in a reasonable time frame, the sampling velocity would have to be increased by using an objective with lower magnification, e.g., by using an objective with a magnification of 20×, instead of 100×, we can sample a 25-fold larger area in the same amount of time. However, depending on the power of the microscope, the use of weaker objectives might require enhancing the signal from the virus particles (see previous paragraph) due to their lower numerical aperture and light collection efficiency.Regarding the increase of sampled area48. The microscopy skills required from the testing personnel or experimenter can be reduced by automated acquisition, auto-focusing, or having a microscope with a fixed axial position.A practical limitation for the wide-spread application of our approach is the fact that a fluorescence microscope is required. In this study, we used a commercial instrument capable of detecting fluorescence from single molecules, that we used to calibrate the detection limit for virus particles. However, as brighter particles (like fluorescent beads) can be used for this calibration, single molecule detection is not a requirement, and virus particles (which bind ~ 20 probes on average) should already be detectable by less sensitive microscopes. Improving particle brightness by expanding the probe arrays would bring down the technical requirements even more, to the point where mobile microscopy equipment might be usedA second limitation to virus detection by FISH is the accessibility of the viral genome to the hybridization probes that is expected to vary with genome architecture. In influenza, the viral RNA is coated by a nucleoprotein that associates with RNA, but does not appear to affect the hybridization efficiency noticeably, as our data shows Fig. . Even thApart from potentially detecting a wide range of virus strains, the rvFISH protocol will be a versatile tool to design experiments to investigate hybridization of oligonucleotides to virus particles in detail and thus, provide a foundation for the optimization of other hybridization-based techniques like PCR and LAMP.49. Briefly, WSN virus was grown in Madin-Darby bovine kidney (MDBK) cells. The cell culture supernatant  was collected and the viruses were titred by plaque assay, yielding a virus titre of 3.3 × 108 plaque forming units (PFU)/mL. IBV (Beau-R strain)50 was grown in chicken kidney (CK) cells and titred by plaque assay (1 × 106 PFU/mL). Viruses were inactivated by addition of 0.2% formaldehyde before use. SARS-CoV-2 was grown in Vero E6 cells. The cell culture supernatant  was collected and titred by plaque assay, yielding a virus titre of 1.05 × 106 PFU/mL. The virus was inactivated by addition of 4% formaldehyde before use.H1N1 A/WSN/1933 (WSN) influenza and coronavirus IBV have been described previouslyStellaris FISH probe arrays were ordered from Biosearch Technologies . The probes were designed using Stellaris’ online design tool, specifying the probe length as 20 nt and the maximum probe number per array as 48 (Tables S1–S3). The sequences of the NA segment in WSN and RdRP gene in CoV were selected as the target regions for the probes.2 stream. Subsequently, the dried coverslips were cleaned for 30 min in a plasma cleaner (Harrick) at high plasma strength for at least 30 min. Silicone gaskets  were placed on the plasma-treated coverslips.Coverslips (Menzel) were sonicated in 2% Hellmanex for 15 min, rinsed with MilliQ water 5 times, sonicated in water for another 5 min, rinsed with water 5 times and dried under an N35. A virus stock diluted in 0.5% NaCl was added to a plasma-treated coverslip and evaporated at room temperature and washed with phosphate buffered saline (PBS). The immobilized virus was fixed with 4% formaldehyde in PBS for 10 min and subsequently permeabilized with 0.5% Triton X-100 in PBS for 15 min. After washing with 2 × saline sodium citrate (SSC), the surface was blocked with hybridization buffer 1 ) for 30 min at 37 °C. Subsequently, the sample was incubated with 1 µM FISH probes in HB1 for 1–3 h at 37 °C. After hybridization, the sample was cleared with 2 × SSC, 10% formamide, 1% RNasin Plus and washed three times with 2 × SSC.This protocol is a modified version of a previously described protocolA virus stock diluted in 0.5% NaCl was added to a plasma-treated coverslip and evaporated at 50 °C for 10 min and washed with 2 × SSC. The immobilized virus was incubated with 1 µM FISH probes in HB2  for 10 min at 37 °C and washed once with 2 × SSC after hybridization.42. Experiments using throat swab material were performed in accordance with the Declaration of Helsinki and approved by the Medical Sciences Interdivisional Research Ethics Committee of the University of Oxford (R76273/RE001). Informed consent was obtained from the throat swab donor. The samples were clarified by centrifugation at 13,000×g for 5 min prior to use.A swab was taken from an apparently healthy individual according to instructions published by Public Health England51, followed by clarification at 13,000×g for 5–10 min and diluted 1:10 in viral resuspension buffer  prior to use.Ethical approval was obtained for the use of anonymized oropharyngeal specimens from patients for the diagnosis of SARS-CoV-2 . Specimens were maintained in Copan Universal Transport Medium (UTM) before being inactivated in a 1% final concentration of formaldehyde (Pierce) for 5 min at room temperature2 for the green (532 nm) laser when taking single-color images, and at 624 kW/cm2 for the green laser and 390 kW/cm2 for the red (647 nm) laser, when taking dual-color images. To automate the task and ensure no bias in the selection of FOVs, the whole sample was scanned using the multiple acquisition capability of the microscope; 81 FOVs were imaged in 3 min. For photobleaching experiments, the exposure time was decreased to 100 ms and the laser power (532 nm) was increased to 2.34 mW/cm2.All experiments were performed on a commercially available Nanoimager fluorescence microscope (Oxford Nanoimaging). The sample was imaged using total internal reflection fluorescence (TIRF) microscopy. The laser illumination was focused at an angle of 54° with respect to the default position. Images of a field of view (FOV) measuring 80 × 49 µm were taken with an exposure time of 500 ms, with a laser intensity kept constant at 780 kW/cm–7). The values of the fit were then normalized to the average intensity of a pixel and multiplied with the grey values of each raw image. Moreover, the average value of the fit was used as the fluorescent background of the images.All microscope data was processed using MatLab2019b. To correct the images for uneven illumination, an image was generated from the median pixel values across all FOVs and fitted with a cubic smoothing spline . The median value of all non-zero pixels in the filtered image was multiplied by three and used as a threshold to binarize the filtered image. The ‘bwpropfilt’ function was used to exclude objects smaller than 5 pixels (px). The intensity-weighted centroids of the remaining regions were calculated from the grey values of the original image using the ‘regionprops’ function (Steve Eddins). The intensity of the fluorescent spot was determined as the background-subtracted intensity of a bounding box with a diameter of 5 px around the centroid.Supplementary Information.


Background: Viruses cause various human diseases, some of which become pandemic outbreaks. This study synthesized evidence on antiviral medicinal plants in Africa which could potentially be further studied for viral infections including Coronavirus disease 2019 (COVID-19) treatment.Methods: PUBMED, CINAHIL, Scopus, Google Scholar, and Google databases were searched through keywords; antiviral, plant, herb, and Africa were combined using “AND” and “OR”. In-vitro studies, in-vivo studies, or clinical trials on botanical medicine used for the treatment of viruses in Africa were included.Results: Thirty-six studies were included in the evidence synthesis. Three hundred and twenty-eight plants were screened for antiviral activities of which 127 showed noteworthy activities against 25 viral species. These, were Poliovirus (42 plants), HSV (34 plants), Coxsackievirus (16 plants), Rhinovirus (14plants), Influenza (12 plants), Astrovirus (11 plants), SARS-CoV-2 (10 plants), HIV (10 plants), Echovirus (8 plants), Parvovirus (6 plants), Semiliki forest virus (5 plants), Measles virus (5 plants), Hepatitis virus (3 plants), Canine distemper virus (3 plants), Zika virus (2 plants), Vesicular stomatitis virus T2 (2 plants). Feline herpesvirus (FHV-1), Enterovirus, Dengue virus, Ebola virus, Chikungunya virus, Yellow fever virus, Respiratory syncytial virus, Rift Valley fever virus, Human cytomegalovirus each showed sensitivities to one plant.Conclusion: The current study provided a list of African medicinal plants which demonstrated antiviral activities and could potentially be candidates for COVID-19 treatment. However, all studies were preliminary and in vitro screening. Further in vivo studies are required for plant-based management of viral diseases. Viruses cause various human diseases of which several such as Ebola, HIV/AIDS, and Hepatitis B are hard to treat. Many pandemic outbreaks in world history were caused by a viral infection. The Spanish flu pandemic of 1918, the deadliest in history, infected an estimated 500 million people worldwide; which is about one-third of the planet’s population, and killed an estimated 20 million to 50 million people (1). In recent years, pandemics have arisen and have also been contained using various approaches. For example, Ebola virus outbreak between 2013 and 2016 with 11323 deaths , CoronavThe use of natural medicinal agents dates back to human prehistory where plants formed the basis of traditional medicine (TM) systems. Traditional medicine refers to health practices, approaches, knowledge, and beliefs incorporating plant, animal, and mineral-based medicines, spiritual therapies, manual techniques, and exercises which are applied singularly or in combination to treat or to diagnose and prevent illnesses or maintain well-being . TraditiSeveral herbal medicines have been used to treat viral infections traditionally for a long time. Some studies have reported the inhibitory effect of medicinal plant extracts against several viruses. Some of these studies were conducted on HIV, herpes simplex virus, hepatitis B virus, and poliovirus. For example, ethnobotanical studies in Africa described the treatment of viral hepatitis with traditional medicine in Africa . FurtherThis review was conducted using database searches and followed statements for Reporting Systematic Reviews and Meta-Analyses .Data were collected from MEDLINE/PUBMED, CINAHIL, Google Scholar, and Scopus databases. No language limitations were applied to reduce selection bias and Google was used to translate articles published in other languages than English. The search strategy used the following terms with appropriate Boolean operators; (“virus diseases” OR (“virus” AND “diseases”) OR “virus diseases” OR  OR “viral infection”) OR (“poliovirus” OR “poliovirus” OR HSV OR (“simplexvirus” OR “simplexvirus” OR (“herpes” AND “simplex” AND “virus”) OR “herpes simplex virus”) OR  OR “human influenza” OR “influenza”) OR (astro AND (“viruses” OR “viruses” OR “virus”)) OR (“parvovirus” OR “parvovirus”) OR (“rhinovirus” OR “rhinovirus”) OR  OR (“hiv"OR “hiv”) OR (“hiv”OR “hiv” OR (“human” AND “immunodeficiency” AND “virus”) OR “human immunodeficiency virus”) OR (semiliki AND (“forests”OR “forests” OR “forest”) AND (“viruses”OR “viruses” OR “virus”)) OR (“measles virus”OR (“measles” AND “virus”) OR “measles virus”) OR (“hepatitis viruses”OR (“hepatitis” AND “viruses”) OR “hepatitis viruses” OR (“hepatitis” AND “virus”) OR “hepatitis virus”) OR (“zika virus”OR (“zika” AND “virus”) OR “zika virus”) OR ((“vesicular stomatitis indiana virus”OR (“vesicular” AND “stomatitis” AND “indiana” AND “virus”) OR “vesicular stomatitis indiana virus” OR (“vesicular” AND “stomatitis” AND “virus”) OR “vesicular stomatitis virus”) AND T2) OR (“coronavirus disease 2019” OR “COVID-2019″) AND “herbal medicine” OR “traditional medicine” OR “oriental medicine” OR “Chinese medicine” OR “African medicine” OR “herbal formula” OR herb AND”) AND (“ AND (“africa"OR “africa”) AND “OR” AND  OR “african continental ancestry group” OR “african”) AND countries).http://etd.aau.edu.et, http://erepository.uonbi.ac.ke). EndNote reference manager was used to remove the duplications of references before screening. Either in vitro studies or in vivo studies or clinical trials of herbal medicine on African medicinal plants were included. Studies were eligible for inclusion if they were conducted to determine antiviral activities using available scientific methods and conducted on medicinal plants in Africa. Studies conducted on medicinal plants outside of Africa were excluded from the study. Review articles and ethnobotanical studies were also excluded. Eligibility assessment was conducted by TB and SD independently and disagreement between authors was resolved by discussion.We included original research articles and unpublished dissertations from their inception to 2020. The unpublished dissertations were obtained from university website , HSV (34 plants), Coxsackievirus (16 plants), Rhinovirus (14plants), Influenza (12 plants), Astrovirus (11 plants), SARS-CoV-2 (10 plants), HIV (10 plants), Echovirus (8 plants), Parvovirus , Measles virus (5 plants), Hepatitis virus (3 plants), Canine distemper virus (3 plants), Zika virus (2 plants), Vesicular stomatitis virus T2 (2 plants). Feline herpes virus (FHV-1), Enterovirus, Dengue virus, Ebola virus, Chikungunya virus, Yellow fever virus, Respiratory syncytial virus, Rift Valley fever virus, Human cytomegalovirus each showed sensitivities to one plant –4. Isola2 plants,oe ferox , a polysLinearis , Asiaticasiatica , Catechiutescens .This study summarized the antiviral activities of African medicinal plants. Forty two African medicinal plants showed noteworthy activities against poliovirus and twenty four against HSV.Recently, 10 African medicinal plants from Morocco showed noteworthy activities against SARS-CoV-2 (58). However, there is no currently available published study on Africa medicinal plants demonstrating clinical effectiveness. In contrast, China has developed several Chinese herbal medicines (CHM) and produced numerous clinical studies and publications. There is a daring absence of published studies on herbal medicine use in Africa in comparison to the actual magnitude of its practice. Many Africans are using one or another type of African traditional medicine either for prevention or treatment of COVID-19.Artemisia annua called COVID Organics which was even exported abroad , a highly defined hit-list of seven compounds. Furthermore, four nontoxic, druggable plant-derived alkaloids and terpenoids that bind to the receptor-binding site and catalytic dyad of SARS-CoV-2 3CLpro were identified. More than half of the selected top 20 alkaloids and terpenoids had a binding affinity for the 3CLpro of the SARS-coronaviruses that surpassed reference inhibitors. The 6-oxoisoiguesterin from Bisnorterpenes had the highest binding affinity to the 3CLpro of SARS-CoV-2 while 20-epi-isoiguesterinol from Bisnorterpenes, isoiguesterin from Bisnorterpenes, 20-epibryonolic acid from Cogniauxia podolaena was the top docked compounds to 3CLpro of SARS-CoV and MERS-CoV. The study revealed that natural agents from the alkaloids and terpenoids class of compounds are capable of inhibiting the 3CLpro with a high inhibitory pattern to both SARS-CoV-2 and SARS-CoV , a natural product from the plant, acts on chemokine receptors and inhibits the entry of HIV  were identified. The previous study also showed that multiple phorbol esters had anti-HIV-1 activities  and funcin vitro with a CC50 value of more than 100 μg/ml. These were aqueous extracts from Carissa edulis (Apocynaceae), Prunus africana Kalkman (Rosaceae) and the methanol extract from Acacia mellifera Benth (Fabaceae). Extracts of C. edulis exhibited the highest activity; an over 12.15% inhibition rate relative to the negative control. P. africana and A. mellifera extract demonstrated 5% inhibition and 2.15% inhibition respectively, relative to controls. Further confirmation of the activity of these plants using the quantitative real-time PCR technique showed the aqueous extract of C. edulis and the methanol extract of A. mellifera exhibited sustained activity over a range of plant extract concentrations from 31.25 μg/ml to 125 μg/ml. The evaluation of the EC50 the two plant extracts exhibiting notable anti–HBV activity using this technique yielded; C. edulis’ EC50 was 331.6 μg/ml while that of A. mellifera was 295.0 μg/ml  against influenza A virus exhibited antiviral effect. Most effective result were obtained from Rapanea melanophloeos methanol leaf extract (EC50 = 113.3 μg/ml) and Pittosporum viridiflorum methanol, 100 and 30% ethanol and acetone leaf extracts   . Ethiopi A virus  (Table 2Carissa edulis (Apocynaceae) has shown significant anti-HSV activity in vitro and in vivo (in vitro with minimal cell cytotoxicity (Lannea schweinfurthii, Combretum adenogonium, Ficus sycomorus, and Terminalia mollis showed strong antiviral activity against Herpes Simplex Virus type 1. Out of 42 Egyptian medicinal plants, Ephedra alata and Moringa peregrina are found to have antiviral activity against HSV. Also, the results revealed that Capparis sinaica, Tamarix nilotica, and Cyperus rotundus are found to have a virucidal effect against HSV(In sub-Saharan Africa, high prevalence rates between 60 and 80% in young adults have been recorded in population-based studies. It is usually managed by antiviral drugs such as a nucleoside analog acyclovir. However, resistance to ACV has been reported mainly among immunocompromised patients . Medicin in vivo . The exttoxicity . The extainst HSV.in vitro possible dose range, duration of action and in vivo pharmacodynamics properties cannot be established.The current study is only a preliminary study where some studies reported naively. As all studies In conclusion, African medicinal plants pose significant antiviral activities and could potentially be candidates for viral disease treatment and/or management. It is imperative therefore that research on currently available African medicinal plants be highly recommended. Outcomes from such studies would potentially lead to breakthrough discoveries for the management and/or treatment of COVID-19 and various other viral infections upon appropriate optimization.


Geminiviridae, genus Begomovirus) are DNA viruses transmitted in a circulative, persistent manner by the whitefly Bemisia tabaci (Gennadius). As revealed by their wide host range (more than 420 plant species), worldwide distribution, and effective vector transmission, begomoviruses are highly adaptive. Still, the genetic factors that facilitate their adaptation to a diverse array of hosts and vectors remain poorly understood. Mutations in the virus genome may confer a selective advantage for essential functions, such as transmission, replication, evading host responses, and movement within the host. Therefore, genetic variation is vital to virus evolution and, in response to selection pressure, is demonstrated as the emergence of new strains and species adapted to diverse hosts or with unique pathogenicity. The combination of variation and selection forms a genetic imprint on the genome. This review focuses on factors that contribute to the evolution of Begomovirus and their global spread, for which an unforeseen diversity and dispersal has been recognized and continues to expand.Begomoviruses (family  Cressdnaviricota) is the group of single-stranded DNA (ssDNA) viruses encoding a replication-associated protein (Rep) that appears to have originated from a common ancestor , an East African cassava mosaic virus Uganda (Uganda variant) to a much higher-population density on the restricted green areas of severely affected leaves and an increased migration rate of infective adults.The fecundity of different se hosts , and cul disease . The mai disease  shows to disease . The fecPapaya leaf curl virus, can cause cotton leaf curl disease in Pakistan but only when associated with a particular DNA-β molecule [One other fundamental area that needs clarification is the role and mode of interaction of the newly discovered circular ssDNA satellites with each other, their helper viruses, and their role in BGV epidemiology. These DNA satellites share no significant sequence homology with their helper BGV sequences and are of various types. The epidemiological role of DNA-β satellite molecules seems to be in extending the host range of BGVs. For example, at least five diverse BGV species, including molecule .Little is known about the selection pressures that seem to operate and drive BGV evolution towards increased virulence and an extended host range. However, the genomes of BGVs show extreme plasticity, leading to rapid evolution in response to changing cropping systems. Genetic factors determining virulence, host adaptation, and suppression of defense responses are under positive selection ,87. In tPrevious findings indicate that additional virus-induced driving forces for BGV epidemics might be the alteration of plant biochemistry so that infected plants emit volatiles as vector attractants, alter feeding behavior, enhance vector fertility , and allBGVs co-evolve with their hosts and vectors in diverse environments and face selection pressure for any host–vector combination to maintain genomic organization and protein functions that facilitate vector transmissibility, replication, and movement ,101. BasInvestigation on the molecular diversity of BGV populations prerequisites to focus the population rather than ‘molecular’ level , as simpIn summary, these few illustrations highlight the practicality and importance of beneficial mutations in the plant viral genome, providing acquaintance behind the resistant outbreak. Therefore, understanding the influence of the evolutionary direction of virus populations is vital for developing more durable strategies to control begomoviral diseases in crop fields. The abundance of these mutations is manifold, and it is secure to accept that many of the viral determinants in BGVs have not yet been identified. In conjunction with enhancements of practical genome engineering approaches, novel viral functions will be discovered. Genetic determinants facilitate virus variation, evolution, and adaptation to diverse hosts and environments. The potential of BGVs to evolve rapidly by acquiring genes from other BGVs or viruses of different genera enhances further complications. Consequently, a commencing point is mutations , recombination, and reassortment (in segmented viruses). While these mutations may occur randomly, selection separates beneficial from unfavorable and neutral mutations. Selection is enforced by the host, the environment, and their interaction. Mutations that provide a beneficial advantage are more likely to be fixed in the genome and accumulate to higher than random frequencies in areas of the genome that contribute to robustness by enhancing stability, transmission, replication competence, escape from immunity, suppression of immune responses, or a combination. Unquestionably, there will be no specific solution to monitoring these epidemics, and the human impact on BGV evolution can only be minimalized by constraining or altering some practices that have led to the advent of these viruses and their vector populations.Our review illustrates that we are only beginning to comprehend the tripartite interactions between BGVs, vectors, and host plants. The perpetual resurgence of new recombinant strains of TYLCV or any other BGV species might lead to resistance breaking, efficient vector transmission, and expansion of the host range, thus being a significant threat to crop production and disease management. In the future, it would be imperative to study a multidisciplinary approach, such as the combined study of host and geography, vector/human-mediated dispersal, and molecular interaction fundaments of begomovirus-satellite disease complexes, for the in-depth understanding of their expanding virosphere. It would help to determine the viral determinants of vital importance, broader infectivity, and a potential antibody target.


Plant viruses can be genetically modified to generate chimeric virus particles (CVPs) carrying heterologous peptides fused on the surface of coat protein (CP) subunits as vaccine candidates. However, some factors may be especially significant in determining the properties of chimeras. In this study, peptides from various sources and of various lengths were inserted into the Bamboo mosaic virus-based (BaMV) vector CP N-terminus to examine the chimeras infecting and accumulating in plants. Interestingly, it was found that the two different strains Foot-and-mouth disease virus (FMDV) VP1 antigens with flexible linker peptides (77 or 82 amino acids) were directly expressed on the BaMV CP, and the chimeric particles self-assembled and continued to express FMDV antigens. The chimeric CP, when directly fused with a large foreign protein (117 amino acids), can self-fold into incomplete virus particles or disks. The physicochemical properties of heterologus peptides N-terminus, complex strand structures of heterologus peptides C-terminus and different flexible linker peptides, can affect the chimera accumulation. Based on these findings, using plant virus-based chimeras to express foreign proteins can increase their length limitations, and engineered plant-made CVP-based vaccines have increasing potential for further development as novel vaccines. Molecular agricultural technology, utilizing plants as bioreactors to produce valuable proteins, has increased in recent years ,2,3. AmoHowever, CVP production in plants has several limitations . DirectlBamboo mosaic virus (BaMV) is a filamentous rod consisting of single-stranded positive-sense RNA. BaMV CP reveals flexible N- and C-terminal extensions, allowing deformation while maintaining structural integrity . The 35 The infectious recombinant constructs were constructed by inserting various polypeptide nucleotide sequences at the 5′-terminus of the truncated CP open reading frame of the vector pBS-d35CP  followinhttp://web.expasy.org/protparam/; accessed on 8 January 2012). These computed parameters include the theoretical pI, number of charged residues and grand average of hydropathicity . The computational predictions of disulfide bond connectivity were estimated using the DiANNA 1.1 web server [The physical and chemical properties of the peptides were calculated using the ProtParam program on the ExPASy server  plants with four or two fully expanded leaves by gently rubbing carborundum-dusted leaves with cotton swabs. The inoculated C. quinoa leaves were harvested at 10 days post infection (dpi). The inoculated N. benthamiana leaves and systemic-infected leaves were harvested at 14 and 21 dpi, respectively. Proteins extracted from the inoculated and systemic-infected leaves were separated using 12% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes  and reacted with antsera against BaMV CP [influenza A virus M2 (Abcam ab56086), FMDV VP1 [Infectious cDNA clones of recombinant chimeric viruses were inoculated onto the local lesion host reported . These p BaMV CP , influenFMDV VP1 , anti-FMThe accumulation levels of chimeric viruses were analyzed using an indirect enzyme-linked immunosorbent assay (ELISA). ELISA was performed as previously described, with minor modifications . Total iC. quinoa leaves at 10 dpi was collected and dropped onto glow-discharged carbon-coated copper grids. The grids were then negatively stained with 2% uranyl acetate and examined using transmission electron microscopy (Philips CM 100 Bio) at 80 kV. BaMV and BS-d35CP virions were examined using the same method as the control.Methods used for the examination of chimeric BVP1s (various FMDV VP1 epitopes expressed on BaMV) virions by SSEM were performed as previously reported . Copper C. quinoa and N. benthamiana. The infectious cDNA clones of pBS-d35CP, pBVP1 [C. quinoa and N. benthamiana, as previously reported [C. quinoa or N. benthamiana leaves at 10 dpi, the leaves were excised and mechanically transferred to healthy C. quinoa and N. benthamiana, respectively. The procedure mentioned above was repeated three times, and the progeny viruses BVP1-7A7, BVP1-7A9 and BVP1-7AG9 on C. quinoa and N. benthamiana leaves were assayed each time to examine the stability of the chimeric virus after successive passages in plants. Total protein contents were analyzed via Western blotting to confirm the stability of BVP1s.The stability of the BVP1s chimeric viruses was tested using P, pBVP1 , pBVP1-9reported . After st-test, where p < 0.01, was considered statistically significant.Differences between groups were evaluated using Student’s To determine the ability of individual peptides to influence infectivity and chimeric virus accumulation, peptides of different lengths and amino acid compositions were selected to directly express foreign peptides on BaMV CVP surfaces. Heterologous peptide nucleotide sequences were constructed into the 5′-terminus of the CP plasmid pBS-d35CP , as descC. quinoa. Chimeric BaMV infectious clones developed symptoms at 10 dpi. To further verify that CP generated from chimeric BaMV-inoculated plants contained various antigenic epitopes, Western blotting analysis was performed using specific antisera against BaMV CP, influenza A virus M2, FMDV VP1, or JEV EDIII, respectively. The inoculated leaves were then examined to evaluate the presence of these CVP accumulations through ELISA. The chimeras were divided into six groups: no-infection, 1–20%, 21–40%, 41–60%, 61–80%, and 81–100% infection with BS-d35CP, respectively  [The pI value of a protein molecule can be predicted by adding the number of positively charged residues and protonated amino termini and subtracting the number of negatively charged residues and deprotonated carboxyl termini. We estimated the pI value and charge of the heterologous peptides using the ProtParam program ry 2012) . The res12 . The Protein structure is determined by the amino acid sequence, as well as a multitude of interactions, localized within a secondary structural element, and points of contact in longer regions. Heterologous peptides fused to chimeric virus CPs that contain a high proportion of helix and strand structures, or peptide C-terminus-containing complex strand structures, have the potential to affect the infection and accumulation ability of the chimeras . Therefosindbis virus [betanodavirus [In addition, disulfide bonds form between the sulfur atoms of two cysteine side chains in a protein and may stabilize the folded state enthalpically through favorable local interactions. Intramolecular disulfide bonding is important for CP conformation, for example, potato virus X (PVX) , sindbisis virus , and betodavirus . In thisodavirus . Based oWe identified the important features of foreign proteins fused on BaMV CP, such as hydrophilicity, pI values, charges, fragment lengths and secondary structure characteristics, which have obvious effects on the accumulation of chimeras. Two analogous polypeptide fragments from FMDV O serotype porciniphilic strain (O/TAW/97) VP1 epitopes, with a charge:pI:hydropathicity value of +1:8.10:-0.811, and from PanAsia strain (O/TAW/99) VP1 epitopes, with a charge:pI:hydropathicity value of +2:9.10:-0.468, were used. Furthermore, we added a flexible (AAA or AAAGGGGS) spacer  between First, we compared the differences between pBVP1-7A9 and pBVP1-9A7, or pBVP1-7AG9 and pBVP1-9AG7 to determine the impact of N or C-terminus polypeptides on chimeric virus infection and accumulation. Second, the differences between pBVP1-7A9 and pBVP1-7AG9, or pBVP1-9A7 and pBVP1-9AG7 were compared to determine the impact of the flexible (AAA or AAAGGGGS) spacer or peptide length. BVP1-7A7 and BVP1-9A9 viruses were constructed as controls, and BVP1-9A7A7 was constructed from three fragments .N. benthamiana was inoculated with chimeric viruses. Total protein was extracted from the leaves inoculated with H2O (mock), pBS-d35CP, pBVP1, pBVP1-9, pBVP1-7A7, pBVP1-9A9, pBVP1-7A9, pBVP1-9A7, pBVP1-7AG9, pBVP1-9AG7, or pBVP1-9A7A7, and were subjected to SDS-PAGE , pBS-d35CP, pBVP1, pBVP1-9, pBVP1-7A7, pBVP1-9A9, pBVP1-7A9, pBVP1-9A7, pBVP1-7AG9, pBVP1-9AG7, or pBVP1-9A7A7 were analyzed using ELISA, and the results were categorized into six main groups as previously described  fused to the N-terminus of BaMV CP did not affect its ability to self-assemble into CVPs, and that BVP1-7A7, BVP1-9A9, BVP1-7A9, BVP1-9A7, BVP1-7AG9, and BVP1-9AG7 CVPs appeared to be typical flexuous rod-shaped, similar to the wild-type BaMV virions  was observed as incomplete virus particles or disks, as shown in C. quinoa or N. benthamiana leaves to generate P0 inoculants. The inoculants were then subjected to three sequential transmissions (P1–P3) in C. quinoa and N. benthamiana  ,39, PVX This study aimed to investigate the BaMV CVPs and BaMV CP N-terminus, as PVX was predicted to be exposed at the virion surface ,43 and mThe filamentous PVX virion contains approximately 1300 identical CP subunits , and mosA limitation of CVPs is virus assembly failure, when large amino acid length peptides are incorporated into the CP. Only peptides less than 25 amino acids were successfully introduced into the TMV CP . MoreovePrevious studies found that the flexibility of foreign proteins may also affect the recombinant virus. Linkers have been shown to have an increasingly important role in the construction of stable bioactive fusion proteins. Linkers can connect proteins together, improve the production of fusion proteins and increase expression yield . InteresMutual restrictions between virus and host interactions ,51,52 coFrom the results of the current study, we can conclude that the N-terminal of CP plays a pivotal role in the morphology, stability and accumulation of most flexible filamentous plant viruses. This study found that the N-terminal hydrophilic portion is an important factor in virion stability and causes changes in the chimeric virus accumulation level. Another important factor is the CP secondary structure, which changes to a disulfide bond. Other important factors include the length of the inserted foreign peptide, the pI value and charge of the inserted foreign peptide N-terminus, and the presence of foreign proteins containing the signal for host interactions. These factors have different effects on chimeric virus accumulation. Furthermore, adding suitable flexible spacers with foreign proteins can effectively improve chimeric virus accumulation. These findings indicate that peptide display technology, aimed at the mass production of pharmaceutically interesting heterologous sequences in plants, may also be a useful tool to unravel the complexity of plant-virus interactions during the infection cycle.To our knowledge, this is the first report to demonstrate CVPs with increased fragment length that directly express foreign proteins. The limited ability of plant virus-based vectors to express longer foreign peptides on CVPs has negatively impacted their utility for applications requiring longer peptides. This is also an important production limitation for the importance of various vaccines using plant CVPs. Therefore, this study provides new insights into effective and useful CVP-based vaccine candidate production in animals and humans.