Varroa destructor mite and the viruses that this ectoparasite transmits are generally considered as the bees’ most important biological threats. Almost all efforts to remedy this dual infection have so far focused on the control of the Varroa mite alone and not on the viruses it transmits. In the present study, the sanitary control of breeding queens was conducted on eggs taken from drone brood for 4 consecutive years (2015–2018). The screening was performed on the sideline of an ongoing breeding program, which allowed us to estimate the heritabilities of the virus status of the eggs. We used the term ‘suppressed in ovo virus infection’ (SOV) for this novel trait and found moderate heritabilities for the presence of several viruses simultaneously and for the presence of single viral species. Colonies that expressed the SOV trait seemed to be more resilient to virus infections as a whole with fewer and less severe Deformed wing virus infections in most developmental stages, especially in the male caste. The implementation of this novel trait into breeding programs is recommended.Honey bees are under pressure due to abnormal high colony death rates, especially during the winter. The infestation by the  However, honey bees are under pressure due to abnormal high colony death rates, especially during the winter2. There are several possible explanations for the increased mortality, but the infestation by the Varroa destructor mite and the viruses that this ectoparasite transmits are generally considered the most important biotic threats4. The mite was found to be an efficient vector of viruses even to the extent that initially the clinical symptoms caused by the Deformed wing virus (DWV) were wrongly attributed to the mite5. The Varroa mite and DWV interact in many different ways, resulting in an increase in DWV virulence7 and colony mortality12. These effects are partially due to the mutualistic symbiotic relationship between both, in which the mite provides transmission of the virus when it feeds on the bee, whereas the virus undermines the immunity of the honey bee by interfering with NF-κB signaling, possibly facilitating the mite’s trophic activity13. Almost all efforts to remedy this dual infection have so far focused on the control of the Varroa mite alone. Indeed, several mite-control strategies have been set up in beekeeping practice that rely on acaricide medication or other treatments, biotechnical apicultural methods and selection for Varroa resistance14. The control of the viruses transmitted by the mite is therefore achieved almost exclusively indirectly, by limiting the severity of the mite infestation. One exception to this is the treatment with double-stranded RNA to provoke a targeted antiviral immune response based on RNA interference, but this is rarely used in beekeeping practice16.The economic value of the honey bee can be almost entirely attributed to its pollination services for agricultural cropsVarroa mite is described as horizontal transmission, as it is from one individual to another from the same generation by either direct or indirect contact. Virus transmission through bodily contact, trophallaxis, feeding and common flower visits are also categorized under horizontal transmission. On the other hand, vertical transmission refers to the passing of an infectious agent from parent to offspring via eggs and semen. It has been demonstrated that DWV vertical transmission occurs predominantly by virus particles adhering to the surface of the egg (transovum) rather than intracellularly17. Queens can become infected by trophallaxis and during natural mating18 or instrumental insemination19, and both the drone’s endophallus20 and semen21 can be the source of virus infection. In contrast to worker brood and drone brood, queen brood cells have an extraordinary low Varroa infestation level22, so it is unlikely that the queen becomes virus infected by vectoring. Virus infection of the queens can contribute to queen failure18, which seems to be an important cause of colony collapse24. The queen’s ovaries can become heavily infected25 leading to typical pathological reactions with discoloration26.The transmission of viruses between bees by the vectoring 27. Here we present the continuation of this study in which the sanitary control of the queens was conducted on eggs taken from drone brood for 4 consecutive years (2015–2018), before finally estimating the heritabilities of the virus status of the eggs. The term ‘suppressed in ovo virus infection’ (SOV) was given to this novel trait. We hypothesized that queens that were capable of clearing a visceral virus infection and consequently deposited virus-free eggs, would pass this predisposition of protective anti-viral immune responses on to their descendants. Drone brood eggs were our target samples as they are unfertilized and thus carry only the alleles of the breeding queen.Because virus infection of a queen can influence her performances and represents an important source of infection for her progeny by vertical transmission, we started in 2012 a sanitary control of the breeding queens from Flemish beekeepers in the North of Belgium following a non-destructive approach. Freshly laid eggs taken from worker brood were examined for the commonly occurring bee viruses and led to the observation that vertical transmission occurs rather frequentlyFifty four Flemish queen breeders volunteered drone eggs to determine the virus status of the queens (from 2015 to 2018). The dataset contained 625 samples of 560 Carnica queens, 25 Buckfast queens and 23 queens of unknown subspecies  for DWV (except in 2016) and SBV and the occurrence of these viral species fell to its lowest level in 2018 with only 6.1 and 4.5% of the samples being positive, respectively  with some 10,000 colonies annually. Table The heritability for SOV/TVS was estimated to be 0.25. The estimated heritabilities for three individual viruses were of the same order of size. The estimate for SOV/BQCV was larger but due to the limited size of the dataset the standard errors of all estimates were high. Nevertheless it may be concluded that the different forms of SOV were moderately heritable.p-values) for the fixed effect for queen’s virus loads were presented in Table The statistical model used to estimate the heritability also included the virus score of the dam. This is a fixed effect and represents the non-genetic  effect of the virus score of the dam on that of the daughter, as expressed in their respective drone eggs. The significance levels  = 12.4, p < 0.01] and for the larva  and pupa developmental stage . Across all castes, 53% of the samples taken from SOV colonies were DWV positive, compared to 64% in the control group . The outcome was more pronounced with the threshold value set at 109 viral genomic copies per sample, representing samples with a severe DWV load. We found that such harmful DWV infections hardly occur in the colonies selected for the SOV trait (1%), whereas in the control colonies they were found in 14% of the samples . The number of severe infections is significantly lower across all samples of workers and drones, and for the pupae and adults of the two castes [all worker samples: Chi-squared  = 15.3, p < 0.01; worker pupae: Chi-squared  = 5.3, p < 0.05; adult workers: Chi-squared  = 14.1, p < 0.01; all drone samples: Chi-squared  = 12.4, p < 0.01; drone pupae: Chi-squared  = 4.2, p < 0.05; adult drones: Chi-squared  = 8.5, p < 0.01; Fig. N = 286) = 4.648, p < 0.05; see also Fig. p = 0.285). Together with the equal distribution of the SOV group and the control group between the apiaries we could assure that the trait rather than the environment or the circumstances at the apiary was here the determining factor.We subsequently determined the impact of the SOV trait on the DWV and SBV infection levels of four developmental stages of drones and workers of 4 SOV colonies (SOV group) in comparison to 4 control colonies (control group). The colonies were kept at 4 apiaries, with 1 SOV colony and 1 control colony each. Due to the extremely low SBV infection levels, these results were not informative  association.The present study suggests for the first time that the honey bee’s potential to control virus infections is heritable. There are some observations of isolated We hypothesized that the virus status of the egg is a reflection of the health status—and by extension of the immune potential—of the queen, though we never verified this. As this study was interwoven in a running breeding program, in which breeders tested the offspring of their most precious queens, this was impossible to do. However, we did verify what the SOV trait means in terms of virus infections in the different developmental stages and castes of the colony. And the outcome was very promising: it seems that the honey bee egg holds the key of resilience to virus infection. The SOV trait provides no sterile immunity against viruses as they are still present, but some yet unknown mechanism seems to avoid that later exposures to—for instance—DWV will end with harmful infection levels.34. Bees like other insects have developed an antiviral immunity that relies on RNA interference (RNAi) among other pathways35. In an earlier study we found that the expression level of key components of the RNAi machinery are downregulated in heavily virus infected colonies36. We then suggested that viral RNAi suppressors mute the anti-viral immunity, a process that previously was described for the bees’ Israeli acute paralysis virus37. The differences in expression profiles could, however, also be explained as a functional RNAi response that keeps viruses under control in the subgroup with the lowest virus load. No such responses have been studied in honey bee queens yet, but it seems plausible that RNAi plays a role in the expression of the SOV trait. In Caenorhabditis elegans it has been demonstrated that the antiviral RNAi response not only inhibits vertical transmission, but also promotes transgenerational inheritance of antiviral immunity. Such heritable antiviral immunity seems to be mediated by small RNAs38. If similar mechanisms would occur in honey bees, the predisposition of an effective antiviral immunity of the queens could be inherited to the worker bees and protect the colony from virus related pathologies, as suggested by our in depth study of the virus load of four SOV colonies and their controls. Given the fact that viruses play a crucial role in the collapse of honey bee colonies, the implications could be tremendous. Moreover, if worker bees are capable of clearing a virus infection the overall virus contamination level of the colony will drop gradually, a process from which even bees of neighboring colonies that lack the protective genetic profile will benefit. Our data are suggesting this is happening as also in the DV + Q subgroup both the DWV and SBV infections dropped considerably over the last 2 years.The immunity of the queen against viruses certainly has also a social and behavioral component, but once a queen has acquired a virus infection, the propagation of the virus and the disease progression happens as in worker beesin ovo virus infection’ and demonstrated that the trait is heritable through the genotype of the queen, and not that of the worker, and can be expressed against several viruses simultaneously or against each of the viruses individually. The estimated heritability seems to be moderate with a value of about 0.25. The trait has a beneficial effect on the virus load of the colony as a whole with fewer and less severe DWV infections and its implementation into breeding programs is recommended.In conclusion, we discovered a new trait that renders honey bee eggs free of virus infection. We used the term ‘suppressed Flemish queen breeders provided between 1 and 45 samples each (see Supplementary Table Varroa control. In each apiary one SOV colony and one control colony was selected based on the sanitary report of the queen in the previous year (2018) as determined by RT-PCR (see below) on a pooled egg sample. Of each colony 10 freshly laid eggs (pooled sample), 10 stretched larvae, 10 red-eyed pupae and 10 adults were collected for each caste (drones and workers). Where possible guard worker bees were selected in order to minimize the risk of sampling bees drifted from other colonies. Three drone larva samples could not be collected as one queen showed an irregular drone laying pattern and eventually ceased laying drone brood around the beginning of the sampling period. Placement of a drone brood frame in the hive to evoke the onset drone brood laying yielded insufficient success. Samples were kept in a cold chain as described here above.At the end of the study  and with the queens’ sanitary reports in hands, we selected 4 apiaries for an in depth study of the viral infection levels across different developmental stages and castes of the honey bee. The apiaries were managed by experienced beekeepers following standard beekeeping practices, including The eggs were homogenized in the presence of zirconium beads in 0.5 ml QIAzol lysis reagent (Qiagen). For larvae, pupae and adults 1 ml QIAzol lysis reagent was used. RNA was extracted using the RNeasy Lipid tissue mini kit (Qiagen) according to the manufactures instructions and finally eluted in 30 µl elution buffer. The concentration of the total RNA was measured with a Nanodrop (Isogen). Using random hexamer primers, 200 ng RNA was retro-transcribed with the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific).2; 0.2 mM dNTPs each; 1.2 U HotStarTaq Plus DNA polymerase (Qiagen) and 2 μl cDNA product. PCR assays were performed using the following cycling conditions: 95 °C—5 min; 94 °C—30 s, 55 °C—30 s, 72 °C—1 min, 35 cycles; final elongation 72 °C—10 min, hold 4 °C. PCR amplicons were separated by electrophoresis using 1.5% agarose gels stained with ethidium bromide and visualized under UV light.The eggs were examined by uniplex RT-PCR assays for the presence of viruses of the ABPV complex, BQCV, DWV and SBV. We used honey bee β-actin as a control gene. All PCR reaction mixtures contained: 2 μM of each primer (see Supplementary Table −1). Each plate included a no target control (NTC). Viral loads of SBV and DWV were quantified using absolute quantification methods based on a standard curve obtained through a eightfold 5 × serial dilution of viral plasmid loads that ranged between 104–1010 copies/µl. All data were analyzed using CFX Manager 3.1 software (Bio-Rad). Baseline correction and threshold setting were performed using the automatic calculation offered by the same software. Maximum accepted quantification cycle (Ct) difference between replicates was set to one Ct. The successful amplification of the reference gene β-actin was used to confirm the integrity of samples throughout the entire procedure.The comparison of the virus load across developmental stages was done by a uniplex qRT-PCR or real-time PCR using Platinum SYBR Green qPCR SuperMix-UDG (Thermo Scientific). Each reaction consisted of 0.4 µM of each primer  or used to produce drones for instrumental insemination. The pedigrees were traced back to 2005 using historical information in the data base yik stands for the observation ;kth test location in which an apiary in another year is considered to be a different test location. There were 101 test locations with 1 to 18 records each;The linear animal model describing the observations was as in Eq. .1\documThe presence of both Although in the model all effects were estimated simultaneously, one might say that all queens with observations contributed to estimate the effect of test location, that cases where queen and dam have an observation contributed to the estimation of environmental similarity and that the genetic relationships between queens allowed to estimate 40. We analysed the models assuming a binomial distribution of the phenotypes (Table The statistical analyses were carried out using ASRemles Table  with an 40.Heritabilities for the different traits were estimated according to Eq. .2\docume40 with a model that only included the overall mean, the effect of test location and the random residual effect.The dataset was too small to estimate genetic correlations. Conceptually for each observation the deviation was computed from the estimate of the fixed effect of test location and pairwise Pearson correlations were computed between those deviations. The correlations and their standard errors were estimated using ASReml9 DWV genomic copies per bee was used as a threshold to distinguish ‘severely infected bees’. This threshold is reported in multiple studies as the transition between covert to overt infection43. For analyses of the differences in the number of DWV infections and severe DWV infections the Chi-squared test or the Fisher’s exact test was used depending on the sample size. Differences in infection loads between groups were analyzed by one-way ANOVA. A possible disproportional effect of individual hives on the number of infections was analyzed using the Mann–Whitney test.To improve data compliance with statistical tests and data visualization, viral loads of all samples were log10 transformed. Data analysis and visualization were performed using ‘R’ version 3.6.1. The level of 10Supplementary Information.


Science shows that D614G increases SARS-CoV-2 spread in cultured human nasal epithelium and enhances transmission in a hamster model.1It has been unclear why SARS-CoV-2 with a D614G mutation in the spike protein became dominant early in the COVID-19 pandemic. A recent study by Hou et al. published in The COVID-19 pandemic has claimed more than two million lives and has wreaked havoc on the global economy. The causative agent, SARS-CoV-2, is believed to have been transmitted from an animal reservoir, most likely bats, to humans and such a zoonotic spillover is not unprecedented. In 2002, SARS-CoV crossed the species barrier and caused the SARS epidemic that was associated with over 8000 cases. SARS-CoV and SARS-CoV-2 share several biological commonalities, particularly with regard to viral entry into target cells. The spike (S) proteins of both viruses use the cellular protein ACE2 as a receptor and the cellular protease TMPRSS2 for S protein priming. However, there are also major differences. The most prominent one is the efficient replication of SARS-CoV-2 in the upper respiratory tract and its high human-to-human transmissibility that was not observed for SARS-CoV.2 and D614G is present in variant VOC 202012/01 , which has become highly prevalent in Southeast England. D614G was associated with increased viral loads in the upper respiratory tract2 and augmented transmissibility of D614G-bearing viruses has been suspected. However, increased transmissibility remained to be demonstrated and the underlying mechanism remained to be explored. Moreover, it has been suggested that D614G might be associated with increased S protein stability and particle infectivity but might have little impact on antibody-mediated neutralization.4 However, confirmation with authentic SARS-CoV-2 was largely lacking.The S protein is a major determinant of coronavirus tropism and likely also intraspecies transmissibility. Interestingly, SARS-CoV-2 with mutation D614G in the S protein became dominant early in the pandemic1 The group employed reverse genetics to generate SARS-CoV-2 harboring mutation D614G. The G614 virus entered cell lines with higher efficiency than the D614 virus but this did not translate into increased multicycle replication for at present unclear reasons.1 Different findings were obtained with primary respiratory epithelium ex vivo: G614 virus replicated to higher titers than D614 virus in human nasal, but not small or large airway epithelium and outcompeted D614 virus (Fig. 1 In mouse and hamster models for SARS-CoV-2 infection of humans, no appreciable differences in D614 and G614 virus replication and pathogenesis were observed although hamsters infected with G614 virus lost slightly more weight than D614 virus-infected animals.1 In contrast, the G614 virus was transmitted faster between hamsters than the D614 virus (Fig. 1A seminal study by Hou et al. provided important insights into the role of D614G in SARS-CoV-2 infectivity, spread, pathogenesis, and transmission.4 These findings have been confirmed with authentic SARS-CoV-2 in this study1 and might be explained by G614 S trimers being more frequently in a conformation suitable for ACE2 binding as compared to D614 trimers.3 The increased viral replication in human nasal epithelium ex vivo is in keeping with the results of a recent study5 and with the increased viral load in the upper respiratory tract of human patients infected with the G614 virus.2 The increased viral replication of the G614 virus in the upper respiratory tract may augment human-to-human transmissibility and account for the global dominance of viruses bearing D614G but the underlying mechanism remains elusive. One can speculate that G614 might augment the usage of attachment-promoting factors or TMPRSS2-related S protein-activating proteases mainly expressed in the upper respiratory tract. In addition, D614G may be associated with increased particle stability.5Studies with pseudotyped particles had initially suggested that D614G increases cell entry.1 The replication of the G614 virus in the nasal epithelium of hamsters used for transmission experiments was not increased relative to the D614 virus (Fig. 5 Similarly, the G614 virus outcompeted the D614 virus in the nasal epithelium of infected animals.5 Hou et al. moved these analyses a critical step forward by demonstrating that the G614 virus is transmitted faster than the D614 virus.1 Thus, contact animals exposed to the G614 virus showed viral replication in the nasal epithelium earlier than animals exposed to the D614 virus (Fig. The work by Hou et al. demonstrates for the first time that although the D614G exchange has little if any impact on pathogenesis it promotes transmission.1 Thus, no appreciable difference in the neutralization of D614 and G614 virus by sera from COVID-19 patients or monoclonal antibodies was observed.1 A separate study reached similar conclusions,3 while another one suggested that G614 viruses might be moderately more susceptible to antibody-mediated neutralization.5 While these minor discrepancies might be due to differences in the experimental systems used, both studies clearly indicate that G614 will not compromise SARS-CoV-2 control by the humoral immune response.Concerns have been raised that the global dominance of the G614 virus may compromise the efficacy of vaccines, which have been generated using the D614 virus. Hou et al. demonstrate that this concern is unfounded.More information is needed to fully understand how D614G promotes transmission. Thus, the molecular mechanism underlying increased replication of G614 viruses in nasal epithelial cells remains largely open and we offered potential explanations above. Further, the interplay of D614G with other mutations in the S protein remains to be explored. Finally, it is important to better link cell culture and clinical data to understand how and why D614G-bearing viruses drive the pandemic.In sum, Hou et al. as well as previous studies show that SARS-CoV-2 can acquire mutations that might increase viral spread in the human population and it will be imperative to monitor the circulating viruses for such mutations.


The response of the opti-MMA system was characterized in several optical configurations to develop a theoretical description of its sensitivity and dynamic range. The speed, low-cost, and sensitivity of this system promises to allow MMAs to be readily applied in in vitro diagnostics and biosensing.Micromagnetic arrays (MMAs) have proven to be powerful tools for controlling the transport and separation of bioanalytes, i.e., they allow bioanalyte-superparamagnetic (SPM) bead complexes of specific size and magnetization to be moved in a synchronized manner that is precisely controlled with the orientation of an external magnetic field. This article presents a laser-photodetector system for the simple detection of individual SPM beads moving on a specific region of an MMA. This system detects the SPM beads through the change in intensity of reflective light as they move from the highly reflective micromagnetics to the supporting substrate. We demonstrate that this opti-MMA system allowed the size, number, and magnetic and optical properties of the SPM beads to be rapidly determined for regions > 49 µm The increased reaction rates and surface area of microscopic bead suspensions are particularly useful for lab on a chip (LOC) applications resulting in improve sampling speed and sensitivity4. SPM bead manipulation in microscale devices can easily be performed with localized magnetic fields generated by permanent magnets or electromagnets6. More generally, SPM beads have been used for a range of essential operations in LOC devices, e.g., transport, separation, sorting, mixing and detection. Demonstrations using SPM beads in LOC devices include DNA extraction7, bead sorting8, cell separation10, rare cell isolation12, fluidic control15, and biomarker detection18. The simplicity of these magnetically driven operations is, however, counterbalanced by the tendency of the beads to form aggregates inside a microfluidic device and the risk of mechanical disruption of rare and fragile cells20.Superparamagnetic (SPM) microscopic beads are a powerful tool for in vitro diagnostics and lab-scale biotechnology separation including the isolation of nucleic acids, proteins, virus, and cells4 and 105 T/m, in a localized volume. Micromagnetic arrays (MMAs) composed of these permanent magnets have been used to capture and control the transport of SPM beads with micron precision, i.e., the combination of the MMA and an external rotating magnetic field generates a travelling magnetic field wave (TMFV) that translates across the MMA at the speed of rotation of the applied field25. The SPM beads are trapped in the local magnetic field maxima and at low rotational frequencies are transported at the same velocity as the TMFV. In nonlinear magnetophoresis (NLM), the beads travel synchronously with the external field (“phase-locked regime”) until a critical frequency, ωc, is reached, which is approximately 20 Hz for 2.8 mm diameter SPM beads with a saturation magnetization of 11.2 ± 1 Am2kg−1 when high quality micromagnets are used. When the rotational frequency is higher than ωc the beads are no longer synchronized with the rotating field, and they enter a “phase-slipping” regime in which they exhibit a non-linear response to the TMFV and their velocity starts decreasing. Further increasing the rotational frequencies results in bead immobilization on the MMA at a frequency ωi, which for high quality micromagnets is approximately 39 Hz23. The critical frequency is the frequency at which the population of SPM beads is observed to de-synchronize with the external field, and has a direct dependence on the size and the magnetization of the beads. Thus, above ωc it is possible to observe different bead behavior due to non-uniformities in size and magnetization. The population of beads in the “phase-slipping” regime increases as the rotation frequency increases until at ωi all beads are in this regime21. The local magnetic field of the micromagnets also dominates the bead-bead interactions for moderate bead magnetizations preventing the formation of aggregates and chains23. This has allowed MMAs to be used for a variety of precise single-particle operations, e.g., the transport of magnetic beads has been controlled with nm-precision, rapid-multiplexed separation has been achieved in mL-scale volumes, pumping-mixing has been demonstrated in microchannels without external fluidic systems, and automated detection of magnetic beads and biological targets has been performed24.Microfabricated permanent magnets patterned inside LOC devices provide a means to create strong magnetic field gradients, between 1027, Hall effect30, tunneling magnetoresistance31, giant magnetoresistance35, and biochemiresistor sensors36. These technologies are very sensitive but limited in scope by cost, complexity, need for calibration, and limited sensitivity. Recently, the detection of magnetic beads and bioanalyte attachments has been successfully implemented with optical methods39. For example, beads and cells patterned on a specifically functionalized surfaces were detected by comparing the transmitted and diffracted light intensity38. This approach, however, requires a high degree of control of the chemistry of the surface, which ultimately limits its application for biosensing. We have found that micromagnets are highly reflective, acting as micro-mirrors, forming the basis for the detection of optically active SPM beads40. In this article we demonstrate that SPM beads modulate the reflected intensity of light so that it is possible to extract information about the number, size, velocity and the presence of a biological target. We describe an opti-MMA detection system based on the controlled motion of the SPM beads on the micro-mirrors that is capable of reaching single bead sensitivity through the selection of specific lasers and fields of view (FOV). The sensitivity of this detection system was determined for different NLM operating conditions, i.e., in the phase-locked and phase-slipping mode, and forming the basis for a theoretical model for the response of this detection system.On-chip SPM bead detection for biosensing has been demonstrated using optical microscopyxz-plane, laser source, dichroic mirrors, collimating lens, objective lens, and a photodetector for real-time detection of the reflected light intensity. As described previously, the rotating magnetic field orthogonal to the axis of flow on the F-NLM chip was generated with three electromagnets configured in two axes, as shown in Fig. 22. The solenoids were composed of 570 coils surrounding a cylindrical iron core (ASTM A536 ductile iron) that had a diameter of 60 mm and length of 150 mm. Two synchronized sinusoidal signals with a 90° phase difference were generated with a two-channel function generator . This signal was amplified to the desired current using two programmable amplifiers  and supplied to the electromagnets assembled in the x and z axes. This generated an elliptical, rotating magnetic field with amplitude of 48 and 29 G in the x direction and z directions, respectively. The uniformity of the magnetic field generated by the electromagnets on the micromagnet array was > 96% in the axial direction and > 83% in the radial direction.Figure a, and 8 µm in center-to-center distance, d) on a silicon substrate22. A positive photoresist based lift-off process was used to fabricate the cobalt micromagnets with a high degree of dimensional control. The micromagnet array was composed of 5.0 ± 0.1 μm diameter circular magnets arranged in a square lattice with an 8.0 ± 0.1 μm center-to-center distance. The micromagnets were created by thermally depositing 5 nm chromium, 70 nm cobalt, and 5 nm chromium on the patterned photoresist. This thickness of cobalt was chosen to produce the highest values of critical and immobilization frequencies. After lift-off the resulting micromagnets were inspected with scanning electron microscopy and atomic force microscopy to determine their z and xy-dimensions with a 1 and 10 nm resolution, respectively. The micromagnet arrays were subsequently coated with a 600 nm thick spin-on-glass (SOG) layer  to provide chemical protection.The MMA consists of a periodic array of cobalt micromagnets  and 1,963 µm2 (FOV2) illumination area whereas the 20× objective produced a 38,013 µm2 (FOV3) and 3.02 mm2 (FOV4) illumination area. The light reflected from the MMA was collected onto a high bandwidth silicon photodetector with built-in amplifier . The output signal from the photodetector was recorded and digitally filtered by using a third order Butterworth filter on the data acquisition card .The motion of SPM beads on the MMA was recorded with a high-speed camera , as shown in Fig. 41. The magnetic properties of the M270 beads have been measured using SQUID magnetometry and the saturation magnetization was 11.2 ± 1 Am2kg−1 and volumetric magnetic susceptibility at fields of less than 1,000 Oe was 0.1723. The beads were used as provided at a defined concentration between 103 to 106 bead per mL of phosphate buffered saline with 0.1% of Tween 20 . A thin layer of casein was adsorbed on the MMA surface to minimize attractive forces with the SPM beads. The motion of these beads on the MMA was consistent with previous studies from this laboratory23.The commercially available SPM magnetic beads used in this study were 2.8 µm in diameter and coated with a monolayer of protein . These beads have been used by this group and others in the study of micromagnetic behavior due to their uniform size, i.e., the coefficient of variation of the radius as < 3%, and defined magnetic properties22. In this study, we often only used the two x-electromagnets, i.e., EMx1 and EMx2, producing a magnetic field oscillating in the x-direction. In this configuration the beads did not travel across the MMA but rather oscillated from the center of a micromagnet to the center of the region between that micromagnets, as shown in Fig. P1) and position 2 (P2) on the MMA, in the “phase-locked” regime. In the “phase-slipping” regime the SPM beads became immobilized at the edge of the micromagnets, as shown in Fig. ΔA, was found to decrease as the frequency of the external magnetic field approached ωc until they were immobilized at the edge of the micromagnet when ω = ωi22. Using this approach, it was possible to use a fixed number of beads simplifying analysis.Figure 4 to 107 SPM beads/ml was placed on the MMA and covered by a glass microscope coverslip. The programmable external magnetic field was activated, and the bead motion was simultaneously monitored with the photodetector and high-speed camera.The detection process was performed as follows. A 10 µL volume of 10ω, was varied from 0 to 100 Hz. Five distinct regimes of behavior were identified that are characteristic of the ω and the number of the SPM beads. In regions (i) and (vi) an external magnetic field was not applied to the MMA and the photodetector signal was characterized by a background signal that was set by the number of beads in the optical sensing area and optical properties of the system. In region (ii) a low-frequency of rotation of the magnetic field, i.e., ω < 19 Hz, was applied to the MMA producing a periodic photodetector signal. In this range of ω the beads moved between the micro-mirror and silicon substrate in a phase-locked mode that was synchronized with ω, as illustrated in Fig. Vmin ~ 785 mV) corresponded to all the beads positioned on-top of the micromagnets, P1 in Fig. Vmax ~ 815 mV) corresponded to all the beads positioned on the silicon substrate, i.e., between adjacent micro-mirrors, P2 as shown Fig. Figure iii) the beads motion started to decouple from the external magnetic field at frequency equal to and higher than the critical frequency, ωc. At this frequency, optical microscopy revealed that the beads started to become immobilize on the edge of micro-mirror and oscillated with an amplitude, ΔA, as shown in Fig. iii), which was a result of the fact that ΔA decreased as ω increased22. As the ω increased further it reached the immobilization frequency, ωi, at which all the beads were in a phase-slipping mode and thus immobilized of the MMA. The critical and immobilization frequency of the 2.8 µm SPM beads were determined to be 19 ± 2 Hz and 40 ± 2 Hz, respectively. Region (iv) corresponded to the signal as the frequency was increased from 20 to 39 Hz. In this region, ωc ≤ ω < ωi and the amplitude of the signal continued decreasing as the oscillation frequency increased. The decreased ΔA resulted from an increasing portion of the beads entering a phase-slipping regime. In region (v), ω increased from 40 to 100 Hz where ω ≥ ωi. In this region, all the beads were in the phase-slipping regime having a minimum oscillation motion at the edge of micro-magnets. This produced an average signal of ~ 800 mV approximately halfway between Vmin and Vmax with a peak-to-peak frequency ~ 5 mV. This resulted from the fact that SPM beads were trapped in a position between the silicon and micro-mirror, as shown in Fig. In region . The relationship between nominalized voltage and the quantity of SPM was similar for all four FOVs, i.e., Vmin/Vmax decreased in a linear fashion with the number of SPM beads in the FOV. The sensitivity, S, of the optical detection systems was defined as–5 per bead, respectively. These results suggest that the sensitivity of the detection system was higher for optical systems with a smaller field of view. For instance, a single 2.8 µm bead could be easily detected in FOV1 while the limit of detection was two beads in FOV2, four beads in FOV3, and thirty-two beads in FOV4. Although the sensitivity of detection in larger fields of view was low, they had a wider area of detection and thus were capable of higher throughput, e.g., the numbers of detectable SPM beads in FOV4 varied from 32 to more than 800.The magnitude of the photodetector signal was studied for four optical configurations, i.e., FOV1, 2, 3 and 4, as a function of the number of the beads in the sensing area. Figure ω for specific densities of SPM beads in the four different FOVs. For each optical system the Vmin/Vmax was constant at low frequencies for a specific quantity of beads until the critical frequency was reached. This is consistent with the model of SPM beads moving in the phase-locked mode at low frequencies that was described above. The ωc was found to be 19 ± 2 Hz for all bead and optical configurations except for 3 beads in FOV1. This exception can be attributed to the propensity of the beads to form clusters on a single micromagnet under these conditions. The Vmin/Vmax increased for ω higher than critical frequency until it reaches a constant value that was determined by the total number of beads in the FOV. It was also observed that the more beads that were in the FOV the larger the Vmin/Vmax change in the frequency range ωc< ω < ωi. Taken together these results demonstrated that the magnitude of the photodetector signal at a specific frequency could be used to determine the number of beads in the FOV for a specific optical detection configuration.Figure Vmin and Vmax are determined as the beads move in a synchronous manner under the external magnetic in either the phase-locked, ω < ωc , or phase-slipping, ω ≥ ωi, regime. In the frequency regime between these two limits, i.e., ωc ≤ ω < ωi, the motion of the beads was influenced by variations in their physical properties, i.e., size and magnetization, and the optical signal was a function of ω.A model has been developed to describe the signal of the opti-MMA detection system for a defined number of SPM beads as a function of the mode of transport. This model is based on describing the change in the photodetector signal as the SPM beads moved from the reflective micromagnets to the supporting silicon substrate, and a Vo = Rs G Ω Po, where Rs is the responsively of photodetector, G is the transimpedance gain, Ω is a scale factor and Po is the input light power to photodetector. The photodetector power was proportional to the reflected light intensity, Po = IoRo, where Io is the laser intensity at the surface of the MMA and Ro is the effective reflectance of the MMA. When SPM beads are on MMA their strong absorption and scatter behavior must be considered. If the reflectance of the MMA were uniform the opti-MMA signal without bead motion would beRp is the effective reflectance of the N beads of area Ap. The optical adsorption of the SPM beads will be determined by their physical properties, and we anticipate it will be proportional to their loading with iron oxide nanoparticles and follow a second order scale law with radius. The optical scattering of the SPM beads lies in the Mie scattering regime and thus also follow a second order scale law with radius,The photodetector signal was determined by the optical properties of the system. In the case where there are no beads on the surface of the MMA the photodetector signal was ζ(t), which was defined as the overlap of the beads with the micro-mirrors at different time points. In the case in which all beads moved in a synchronized phase-locked manner ζ(t) = cos(ωt/2). Thus, the value of ζ(t) is equal to 1 and 0 at P1 (θxz = 0˚) and P2 (θxz = 180˚), respectively, as shown in Fig. N beads on the MMA may expressed asRSi-Rp. The minimum and maximum output voltages can be expressed in their simplest forms to beIn the phase-locked transport mode, the motion of the beads was described by a frequency dependent overlap parameter, Vmin/Vmax that were carried-out based Eq. (4). The empirical and materials parameters used for these calculations are listed in Table R2 values are presented in the Fig. Vmin/Vmax values that were consistently larger than the experimental results for FOVs 2–4. Figure Vmin/Vmax values.Figure ω > ωc, the motion of beads was not fully synchronized with external field. In this regime the frequency, the photodetector signal was determined by ΔA and the population of beads in the phase-slipping regime. Based on the analysis of the speed of a SPM bead in the NLM system we defined.C was a constant obtained empirically21. This behavior can be defined using a second overlap parameterµ is the mean and σ is the standard deviation obtained experimentally. Figure At higher external field frequencies, ω > ωc can be expressed in its simplest form in terms of N(ω), ζ(t) and Thus, the photodetector signal for Vmin/Vmax were carried-out as a function of ω based Eq. , which is a function of the external magnet field. The primary sources of error in this assumption are the interactions of the beads with the MAA surface and each other that lead to transport behavior that is not consistent with Eq. , materials properties , and transport of the SPM beads in the rotating external magnetic field.Vo, Vmin and Vmax, were proportional to the response of photodetector, the transimpedance gain, and the input light power to photodetector, which is dependent on both Po and FOV. A small FOV produced higher bead sensitivity, S, while a larger FOV had a lower sensitivity due to the larger reflective area of the MMA relative to the size of a SPM bead. This meant that the signal-to-noise ratio (ΔV/ΔVnoise) for the detector increased from 37, 10, 0.57 and 0 dB for FOV 1, 2, 3 and 4, respectively, where the noise amplitude was determined experimentally to be 1.5 mV. The sensitivity of the optical detection system made it possible to detect a single bead for FOV1/2, two beads for FOV3, and twenty-eight beads for FOV4.The optical detection system signals, i.e., Ro) is the sum of the relative reflectance of the micromagnets (V/Vmax) should increase as S increased from 0.0007 to 0.0027 per bead (data not presented). This leads us to conclude that the sensitivity of the optical detection system can be increased sufficiently to detect individual 2.8 µm SPM beads in a field of view of > 40,000 µm2 by modification of the MMA optical properties. It is also theoretically possible to increase the sensitivity of the optical detection system by using smaller micromagnets.The sensitivity of the optical detector was also closely related to physical properties of the MMA, SPM beads and the sample cell. The reflectance of the MMA . These results confirm that the optical signal decreases for smaller beads but suggest it scales like the second power of radius, although the optical adsorption of the smaller beads is quite a bit higher due it the higher loading of SPM nanoparticles. Obviously, there are other optical effects of relevance to the effective reflectance of SPM beads on the micromagnetic mirrors that are not accounted for in this simple model.The sensitivity of the detection scheme was also closely related to the size and effective reflectance of SPM bead used in the detection scheme. The optical signal should be proportional to the area of the bead, as shown in Eq. . The effωc < ω < ωi, to determine the physical properties of the SPM bead-analyte complex, including size and magnetization. Such a measurement could be completed in a matter of seconds, as shown in Fig. The highest sensitivity of the opti-MMA detection system was achieved when the SPM beads were transported in the phase-locked regime. A decrease in the sensitivity of the detection system will result from variations in the size, transport behavior, or aggregation state of the SPM beads. The physical properties of SPM beads can, however, be determined if the opti-MMA signal can be measured across a range of frequencies. For example, the optical signal could be measured at low frequencies to determine the number of beads in a FOV and then the frequency could then be increased, i.e., 2 and in principle can be used to determine its size, magnetic properties and interaction with an analyte. We have also demonstrated that an optimized MMA design can be implemented to increase sensitivity to a single 2.8 µm bead over a 40,000 µm2 area or an 850 nm bead in a 49 µm2 area. In principle, such a system will allow as many as 10,0000 2.8 µm bead to be monitored per second using a FOV4 detection area and there is also the possibility of use multiple colors for multiplexing. These performance characteristics are similar to flow cytometry with the noted difference that the SPM bead transport is precisely controlled by magnetophoresis. These results and a recent bioanalytical demonstration40 lead us to conclude that optical-magnetophoresis provides a promising approach to rapid, cost-effectively, and sensitive detection of biological analytes with minimum consumption of reagents.In summary, the magnitude of an opti-MMA photodetector signal has been used to identify a single 2.8 µm diameter SPM bead in a field of view of 2,000 µmSupplementary Information.


Cryptococcus neoformans and Cryptococcus gattii are the two most common species that cause human cryptococcosis. These two species of Cryptococcus have differences in pathogenicity, diagnosis, and treatment. Cryptococcal infections are usually difficult to identify because of their slow growth in vitro. In addition, the long detection cycle of Cryptococcus in clinical specimens makes the diagnosis of Cryptococcal infections difficult. Here, we used positively charged silver nanoparticles (AgNPs+) as a substrate to distinguish between C. neoformans and C. gattii in clinical specimens directly via surface-enhanced Raman scattering (SERS) and spectral analysis. The AgNPs+ self-assembled on the surface of the fungal cell wall via electrostatic aggregation, leading to enhanced SERS signals that were better than the standard substrate negatively charged silver nanoparticles (AgNPs). The SERS spectra could also be used as a sample database in the multivariate analysis via orthogonal partial least-squares discriminant analysis. This novel SERS detection method can clearly distinguish between the two Cryptococcus species using principal component analysis. The accuracy of the training data and test data was 100% after a tenfold crossover validation.There are approximately 1 million cryptococcal infections per year among HIV+ individuals, resulting in nearly 625,000 deaths.  On average, there are 1 million cryptococcal infections per year among individuals living with HIV/AIDS, resulting in nearly 625,000 deaths . Therefore, Cryptococcus infection has attracted increasing attention. Cryptococcus neoformans and Cryptococcus gattii are the two most common species causing human cryptococcosis2; Cryptococcus species need to be identified to guide and predict treatment3. Gattii and Eeckels reported the first case of C. gattii infection in 1970. Since then, there have only been a few reports of its occurrence. C. gattii was initially called C. neoformans var. gattii because its characteristics were very similar to those of C. neoformans. In 2002, Kwon-Chung et al.4 found that there were some differences in phenotype, biology, and genetic taxonomy between C. neoformans and C. neoformans var. gattii ; thus, C. gattii was reclassified as a single species.A report by the United Nation's HIV/AIDS (Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome) program and the World Health Organization (C. neoformans and C. gattii5. Polymerase chain reaction (PCR), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS), gene chips, and second-generation sequencing technology have all been used for clinical identification of Cryptococcus and can distinguish between C. neoformans and C. gattii; however, the equipment costs are very high10. The surface-enhanced Raman scattering (SERS) effect refers to the Raman scattering signal of adsorbed molecules due to the enhancement of an electromagnetic field on or near the sample surface, or in the excitation region of specific metal conductors or sol that have been specially prepared11. SERS offers several advantages for fungal identification that can make up for the deficiencies of the above detection methods. For example, SERS spectra offer spectral fingerprints unique to the constituent molecules. SERS offers qualitative and quantitative detection of the analyte because it has high specificity12. The SERS spectral bands are narrow with high resolution of spectral peaks, thus minimizing fluorescence background interference13. The characteristic spectra facilitate simultaneous detection of multiple components. Surface-enhanced Raman spectroscopy offers biological testing and is relatively inexpensive and portable14.Traditional detection methods for cryptococcus, such as ink staining and antigen kit testing, are relatively fast but cannot distinguish between C. neoformans and C. gattii detection. Although SERS can identify pathogens, further development and improvement is needed before clinical application. This is particularly true for cryptococcus—a special fungal pathogen that contains a capsule16.It is surprising that there are no reports using SERS for 18. The most common approach uses 1% sodium citrate mixed with 1.69 mM AgNO3 with heating at 100 °C19. The enhancement and reproducibility with anionic analytes are low due to electrostatic repulsion from the sodium citrate on the AgNPs21. We first measured the zeta potential of C. neoformans and C. gattii and found that both had negative charges on their surfaces. We previously prepared positively charged silver nanoparticles (AgNPs+) with a NaBH4 reducing agent; the AgNPs+ could adsorb onto Cryptococcus via electrostatic interactions but the stability of this substrate was relatively poor22. To improve the stability of the substrate, AgNPs+ were synthesized with cetyl trimethylammonium bromide (CTAB) as a cationic surfactant23. Herein, we report a rapid detection method for Cryptococcus by SERS using positively charged silver nanoparticles for the first time. After obtaining Cryptococcus spectral data, we used orthogonal partial least-squares discriminant analysis (OPLS-DA), an orthogonal, partial further-squares discriminant analysis algorithm, to analyze the spectral data.Silver nanoparticles (AgNPs) are widely used as standard SERS substratesC. neoformans and C. gattii are negatively charged. Figure + and that the AgNPs+ are spherical. Figure + zeta potential is positive. The AgNPs particle size is 78.8 nm and the AgNPs+ particle size is 105.7 nm, as shown in Fig. + are shown in Fig. + were adsorbed onto the surface of C. neoformans and C. gattii.Figure Cryptococcus neoformans was used to verify the Raman enhancement effect of the AgNPs+. The same volume of AgNPs and AgNPs+ were mixed with the same volume of fungal liquid at a 1:1 ratio, and their Raman signal intensities were compared under the same excitation power and integration time. Figure +. Therefore, the AgNPs+ was used as the enhancement substrate to distinguish the two species of Cryptococcus.C. neoformans and C. gattii. The red line is the mean value of the SERS spectrum of C. neoformans, the black line is from the SERS spectrum of C. gattii, and the blue line is the difference between them. As shown in Fig. C. neoformans and C. gattii were similar, and the Raman shifts at 655, 790, 906, 1,008, 1,204 and 1,332 cm−1 were significantly enhanced. The physiological structure and biochemical properties of C. neoformans and C. gattii are very similar. This explains why C. gattii remained a variant of C. neoformans until 20024. However, their peaks have differences at 655 cm−1, 790 cm−1, 1,008 cm−1, and 1,332 cm−1. The peak bands at 655 cm−1 are assigned to Guanine and tyrosine, the peak located at 790 cm−1 is assigned to Cytosine and uracil, the peak located at 1,008 cm−1 is assigned to Phenylalanine, galactomannan and C–C aromatic ring stretching, and the peak located at 1,332 cm−1 is assigned to Adenine, guanine and CH deformation.The Raman spectra were averaged from the Raman spectra of each Cryptococcus species; the x coordinate is the Raman shift, and the y coordinate is the Raman intensity. Figure C. neoformans and C. gattii, as the fingerprints are different between the two species of cryptococcus. OPLS-DA based on the SIMCA14.1 software was performed on the Raman spectrum data from all measurements. Figure 24. The data points are well separated on both sides of the central axis. There is no intersection and they are concentrated on both sides. The OPLS-DA model 1 (Table 2X (cum) = 0.986, R2Y (cum) = 0.999, and Q2 (cum) = 0.998, indicating that the quality parameters of the model were good. Here, R2X represents the interpretation rate of the model in the X-axis direction, i.e., 98.6%. Term R2Y describes the model in the Y-axis direction, i.e., 99.9%, and Q2 is the prediction rate of the model, i.e., 99.8%25.OPLS-DA was used to further distinguish C. neoformans and C. gattii were divided into 10 parts. Nine parts were successively used as training data, and one part served as the test data. The average accuracy of the training and test data were obtained after 10 operations. Tables 27.The classification ability of the model was evaluated using a tenfold crossover validation with SIMCA14.1. All the SERS spectrum data of C. neoformans infection accounts for the majority of cryptococcosis, whereas C. gattii infection is relatively rare28. C. neoformans causes infections mainly in the central nervous system; pulmonary infection accounts for only 35% of all infected patients. In an in vivo microscopy experiment conducted by Ngamskulrungroj et al.31, there was no significant difference in the clinical symptoms between patients with these two cryptococcal infections, and a differential diagnosis based on clinical symptoms was difficult34. However, C. gattii requires longer periods of antifungal therapy, which motivates our efforts for discriminating between the two species.Cryptococcosis is a global, invasive fungal infectious disease caused by Cryptococcus. Treatment of this disease is still extremely challenging. 5. Although the ink detection method of cerebrospinal fluid (CSF) is simple and fast, distinguishing between C. neoformans and C. gattii is difficult2. Cryptococcal antigen kits can use latex agglutination and enzyme-linked immunosorbent assays to study the cryptococcal polysaccharide capsular antigen (CrAg) composition in blood. This method offers rapid diagnosis within 2–3 h, and it is more sensitive than direct staining microscopy. However, most of the antiserum used is based on the C. neoformans antigen, and it is possible to miss the detection of C. gattii35. Antigen clearance kinetics are slower than treatment response, and continuous monitoring has limited value. Regarding molecular biology methods, PCR gene sequences using specific gene segments can be used for identification and typing. These are sensitive and specific and can be used to genotype C. neoformans and C. gattii36. However, PCR is time-consuming and prone to contamination37. MALDI-TOF MS can also classify cryptococci and can reliably display the genetic and evolutionary relationships between species and within species. MALDI-TOF MS is more accurate and faster than gene sequencing38. However, it is expensive and only research hospitals can support a mass spectrometer. Gene chips are also expensive and difficult to use in routine clinical care, and second-generation sequencing requires bioinformatics professionals.Traditional methods include ink staining and antigen kit testingC. neoformans and C. gattii10, SERS exhibits high specificity with low costs and fast analysis times. SERS can quickly identify and distinguish C. neoformans and C. gattii. Here, AgNPs+ were formed from AgNO3 with CTAB and NaBH4. We found that the AgNO3 concentration could be used to tune the size of the AgNPs+35. This method is simple and easy to repeat. Its repeatability compensates for the difficult nanomaterial synthesis. The preparation is convenient and can be easily promoted and applied in a clinical setting. The SEM image in Fig. +. The AgNPs+ were spherical, colloidally stable, and approximately 110 nm in diameter. The AgNPs+ served as the SERS substrate. We measured the spectra of high-quality C. neoformans and C. gattii, and Fig. C. neoformans and C. gattii.All current cryptococcus detection methods have limitations, and this study used surface enhanced Raman spectroscopy to detect cryptococcus. Versus other detection methods for C. neoformans and C. gattii. All the data points were distributed on both sides of the central axis with no intersection, indicating that OPLS-DA could distinguish the SERS spectra of C. neoformans and C. gattii well. In addition, as shown in Table The spectra for the two cryptococcus show few differences because they are closely related. Therefore, it is necessary to use an OPLS-DA analysis tool. Figure We have initiated the application of SERS for cryptococcus detection. SERS can be used for rapid and nondestructive detection of Cryptococcus with relatively low equipment cost. Due to the special properties of the capsule, cryptococcus is difficult to detect relative to other fungi such as candida and mold. Therefore, many detection methods that can be applied to detect common fungi cannot be used to detect cryptococcus. This highlights the universal applicability of Raman spectroscopy to detect pathogenic fungi. The OPLS-DA data shows that Raman can clearly distinguish between two cryptococcus species with very similar properties, which is an excellent result.C. neoformans and C. gattii. The positively charged nanoparticles are shown to increase the intensity of the Raman spectra acquired from Cryptococcus. A classification model to discriminate the SERS spectra of C. neoformans and C. gattii was then built using OPLS-DA. This model is capable of explaining over 99% of the variance in the data and achieves a 99.8% predictive rate of discrimination. These results indicate that a SERS-based diagnostic tool may be a powerful technique for diagnosing cryptococcosis.We report a novel detection method to identify and discriminate This study has been approved by the ethics review committee of Shanghai hospital, China. Comply with the World Medical Association Declaration of Helsinki, the relevant Chinese laws and regulations, and other ethical principles to protect the rights and interests of the subjects, as well as the regulations of the ethics committee of Shanghai (accession number: 2013SMMU-LL013). Signed informed consent were acquired from all patients with the admission number: 277311, 296013, 454964 and 484728.3 (99%) was purchased from Beijing Modern Oriental Trading Co., Ltd., NaBH4 (99%), CTAB was from Sigma-Aldrich Trading Co., Ltd. , and ammonium hydroxide (NH4OH) was from Xilong Chemical Group Co., Ltd. (China). All commercial chemical reagents were used directly without further purification. The monocrystalline Si used to detect the Raman signal was supplied by Zhejiang Lijing Silicon Materials Co., Ltd. (China). In addition, the Sabouraud Dextrose agar (SDA) medium used to culture Cryptococcus was purchased from Shanghai Comaga Microbial Technology Co., Ltd. (China). The Milli-Q water was obtained from Millipore Systems Corp. (USA).AgNOThe ultraviolet–visible-light (UV–Vis) spectrophotometer was a Shimadzu 2600 . Morphology studies used a Hitachi h-7650  scanning electron microscope (SEM), a Zeiss thermal field emission scanning electron microscope-Merlin , and a JEOL 2010 high-resolution transmission electron microscope . The zeta potentials were measured with a Zetasizer Nano ZSP analyzer . SERS measurements used the i-raman Plus bws465-785 h Raman spectrometer . Other instruments included a MiniSpin centrifuge  and a zncl-bs230 *230 heated electromagnetic stirrer .+ colloids were prepared according to previous methods by van Lierop et al.17 and Chen et al.18. The AgNPs+ diameters could be increased to more than 100 nm18. Solution A was prepared first, as follows: 3.26 mM AgNO3, 0.4 M NH4OH, and 0.5 mM CTAB (cetyl trimethyl ammonium bromide) were added to 20 mL of ultrapure water. Solution B was then prepared as follows: 8 mM NaBH4 and 0.5 mM CTAB were added to 20 mL of ultrapure water. Solutions A and B were placed in an ice bath for 15 min to slow the reaction. Solution A was then poured into a 50-mL conical flask that was vigorously stirred on a heated electromagnetic stirrer at 700 rpm. Solution B was then poured into solution A dropwise and stirred on a heated electromagnetic mixer for 4.5 h; this process was repeated in an ice bath. The conical flask was sealed during the stirring process to prevent the silver from being oxidized. The stirring was stopped after 4.5 h, and the mixture was heated at 100℃ for 12 min to remove the remaining NH3 and NaBH4. The heat was then turned off, and the stirring was continued until the mixture cooled to room temperature. Ultrapure water was then added to a total volume of 40 mL; 1.5 mL of the solution was placed in a 1.5 mL polystyrene tube and centrifuged at 7,500 rpm for 7 min. After centrifugation, 1.4 mL of the supernatant was discarded, and the remaining solution was mixed. The resulting AgNPs+ were stored in a refrigerator at 4 °C.AgNPsC. neoformans and C. gattii were collected as cerebrospinal fluid (CSF) at Shanghai Changzheng Hospital, China. The specimens were from various media including blood plate medium, chocolate agar plate medium, sabouraud dextrose agar (SDA) medium, or isolated from a single colony. Selected fungal colonies were cultured for 48 h in ambient air at 25 °C in SDA medium. The fungal suspension was identified using India ink staining, a lateral flow assay (LFA) for cryptococcal antigen (CrAg) detection  and using an ABI_3730xl sequenator  to specifically sequence the intergenic spacer 1 (IGS1) region of the nuclear ribosomal rRNA gene of the fungal culture.Clinical isolates of Cryptococcus gattii is very clinically rare. Two strains of C. neoformans and two strains of C. gattii were selected and grown in SDA medium for 48 h at 25 °C. Approximately 50 μL of Cryptococcus colonies were mixed with 100 μL of ultrapure water and oscillated for 2 min to ensure complete mixing. The number of C. neoformans and C. gattii in the solution was calculated by a fungal counter as 1.03 × 109 and 1.05 × 109 cells mL–1, respectively, followed by mixing of 10 μL of fungus liquid with 10 μL of the pre-prepared AgNPs+ as shown in Fig. C. neoformans and C. gattii. The Cryptococcus mixtures were placed on a silicon chip and studied with a microscope; 20 different fields-of-view were studied. The total acquisition time of the SERS spectrum was 45 min, including the time of centrifugation of the substrate, sample preparation, and spectrum testing.The Raman spectrometer used in this experiment has a maximum laser power of 275 mW at 785 nm with a light spot diameter of 105 μm. The excitation power was 20% of the maximum power, and the integration time was 15 s. We used the white light function to find the darkest area on the wafer, and 20 replicate SERS spectra were recorded. The processed spectra were used to compare the average Raman data from 2X and R2Y represent the interpretation rate of the established model to the X and Y matrix, respectively; term Q2 indicates the prediction ability of the model24. The fitting accuracy of the model improves as R2 and Q2 approach 1. Lower values of R2 and Q2 lead to worse model fitting accuracy. A fitting accuracy of R2 and Q2 above 0.5 (50%) is good, and higher than 0.4 is acceptable.The OPLS-DA spectral data were collected with SIMCA 14.1  software. OPLS-DA is a supervised discriminant analysis method that predicts differences in fungi species by establishing the relationship model between the expression level of metabolites and the fungi species. The OPLS-DA model is established for comparisons between two groups, and the parameter evaluation of the model is presented in the form of a table in which RC. neoformans and C. gattii were divided into 10 parts; nine parts were successively used as training data and one part served as the test data. After 10 operations, the average accuracy of the training and test data was obtained. The mean value of the accuracy (or error rate) of the results of the tenfold crossover validation was used to estimate the accuracy of the algorithm.The accuracy of the OPLS-DA model for classification of unknown samples was predicted via a tenfold crossover validation. The classification ability of the model was also evaluated by a tenfold crossover examination. The SERS data of all


Author summary: In global terms, LSD has been termed a “neglected disease” due to its historic natural occurrence of being restricted to Africa and, occasionally, Israel. However, after its slow spread throughout the Middle East, the disease is now experiencing a resurgence of research interest following a recent and rapid spread into more northern latitudes. Given the dearth of solid findings on potential transmission mechanisms, no efficient or reliable control program currently exists, which does not involve the use of live attenuated vaccines or stamping out policies – both of which are controversial for implementation in non-endemic regions or countries. The vector-borne mode is the only working concept currently available, but with scarce evidence to support the aggressive spread northwards – except for human-assisted spread, including legal or illegal animal transportation. The emergence of outbreaks is not consistently linked to weather conditions, with the potential for new outbreaks to occur and spread rapidly. Here, for the first time, we provide evidence for indirect contact-mode transmission for a naturally-occurring recombinant LSDV isolated from the field. In an insect-proof facility, we obtained solid evidence that the novel LSDV strain can pass to in-contact animals. Given the recombinant nature of the virus utilised, its genetic background relating to the observed transmission pattern within the study needs to be delineated.The transmission of “lumpy skin disease virus” (LSDV) has prompted intensive research efforts due to the rapid spread and high impact of the disease in recent years, especially in Eastern Europe and Balkan countries. In this study, we experimentally evaluate the vaccine-derived virulent recombinant LSDV strain (Saratov/2017) and provide solid evidence on the capacity of the virus for transmission in a vector-proof environment. In the 60-day long experiment, we used inoculated bulls (IN group) and two groups of in-contact animals (C1 and C2), with the former (C1) being in contact with the inoculated animals at the onset of the trial and the latter (C2) being introduced at day 33 of the experiment. The infection in both groups of contact animals was confirmed clinically, serologically and virologically, and viremia was demonstrated in blood, nasal and ocular excretions, using molecular tools. Further studies into LSDV biology are a priority to gain insights into whether the hypothesized indirect contact mode evidenced in this study is a  Capripoxvirus within the family Poxviridae . It contains a double-stranded, covalently-linked linear DNA genome enveloped by a lipid bilayer. Mature capripox virions have an oval profile and large lateral bodies, and they are 320 ×260 nm on average in size1. The viral genome is approximately 151 kb in size, encoding 156 putative genes2. LSD virus (LSDV) genes share a high degree of co-linearity and amino acid identity (average of 65%) with genes of other known mammalian poxviruses, particularly suipoxvirus, yatapoxvirus, and leporipoxvirus. Within the genus Capripoxvirus, which also includes sheep pox and goat pox viruses, genetic identities are at least 96%3. Generally, based on full-genome sequences deposited in GenBank, LSDV exists as two genetic lineages: field isolates and live attenuated vaccine strains, although evidence for a naturally occurring recombinant between a vaccine strain and field strain has also been reported4.Lumpy skin disease (LSD) virus belongs to the genus 5, with one documented isolation from sheep7, although a limited number of other ruminant species, such as antelope, also appear to be susceptible. In recent decades, the disease has been documented in both Southern and Northern hemispheres due to a rapid expansion of its range, although historically, it was restricted to the African continent10. The unprecedented incursions into new territories and resurgence of LSD creates an objective need for joint efforts to investigate the obscure nature of LSDV transmission11.Displaying a strict and narrow host range, LSDV mainly infects cattle and water buffaloes12, whereas the predominant transmission pathway for sheep pox and goat pox viruses is via aerosols. However, importantly, other poxviruses are most commonly spread by direct contact rather than by transmission by arthropod vectors such as by contaminated insects during biting of their hosts. Moreover, poxvirus infection can also occur through ingestion, parenteral inoculation, or droplet or aerosol exposure to mucous membranes or broken skin. Some poxviruses can be transmitted by fomites (inanimate objects). Transmission pathways may vary among poxviruses even within a genus13. For orthopoxviruses that infect humans, infection occurs via exposure to aerosols or droplets (variola virus) or through close personal contact. Poxviruses from the Parapoxvirus genus (e.g. orf virus or milker’s nodule virus) can pass from one animal to another through direct or indirect contact. Unfortunately, there are no available studies addressing the issue whether closely related capripoxviruses actually employ different routes of spread.Initial evidence and studies suggested that direct or indirect contact (without vectors) are ineffective routes for LSDV transmission15. Unfortunately, transmission studies conducted to date using arthropod species which may be involved in transmission are mostly species which are restricted to the Southern hemisphere , which complicates analysis of transmission potential of local species in climatically new geographic areas in the Northern hemisphere. Moreover, LSD outbreaks are not only reported within the warmer summer period, optimal for arthropod blood meal search activity, but also outside of it.This observation thus points to a possible non-vector-borne route for spread of the virus . Studies using a small cohort of animals were conducted in the past to show that direct contact transmission between infected and naïve animals is possible, but at an extremely low efficiency rate16. New work to replicate the findings has not yet been encouraged. Nevertheless, field evidence suggests that successful transmission can be achieved when naïve animals are allowed to share a drinking trough with severely infected animals18.Transmission of LSDV is surmised to occur through mechanical vector-borne spread via insect or tick bites as most outbreaks occur during the warmer  summer months when potential vector species numbers are high21. Attenuated vaccine strains have also been claimed to be devoid of transmission capacity, but recent field evidence argues to the contrary23.This supports the common hypothesis that direct contact does not appear to be an effective route for LSDV transmission. In addition, recent experiments with various field strains did not result in successful contact transmissionIn this environment of uncertainty for LSD spread, elucidation of the exact transmission mode/s could contribute significantly towards improving control and eradication programs.4, in an experimental setting and for the first time conclusively demonstrate non-vector-borne transmission of the virus.In this paper, we report on work performed to evaluate transmission of the naturally occurring vaccine-derived virulent recombinant strain of LSDV Saratov/201722. The strain was obtained from the FGBI ARRIAH depository and refreshed using two rounds of passaging in goat testis cells. To prepare the final inoculum virus, the refreshed virus was subjected to polymerase chain reaction (PCR) amplification of different loci of vaccine and field strain genomes to confirm the identity of this virus strain4.The vaccine-derived virulent recombinant LSDV strain, LSDV Saratov/2017, was isolated by FGBI ARRIAH researchers from a cow presenting with severe clinical signs of LSDThe animal experiment, as well as the euthanasia procedure, were approved by the Ethics Committee of the Federal Center for Animal Health, Russia (Permit Number: №2/1-21082018) and conducted in strict accordance with Directive 2010/63/EU on the protection of animals used for scientific purposes.ad libitum. The experiment was carried out in an insect-proof facility. To detect any possible dipteran presence, indoor blood-feeding insect UV light traps and sticky traps were mounted at regular intervals on the walls of the facility. The animals were also examined for the presence of ticks.The initial experimental group consisted of 10 bulls of the Russian Black Pied breed aged 6-8 months. The animals were consecutively numbered from 1 to 10 in a random fashion and maintained in Animal Biosafety Level 3 housing with a 12-hourly light-dark cycle, relative humidity of 30% to 70%, temperature of 23 to 26 °C and all animals were monitored twice daily by the veterinary staff. Water and feed were provided 50/ml of the recombinant virus, LSDV Saratov/2017, intravenously  and the remaining five animals with odd numbers were mock inoculated  with phosphate-buffered saline (PBS)  each received 2 ml of 5 log TCIDThe animals were monitored daily for the presence of fever until day 52 and clinical signs of LSD until the end of the experiment. To evaluate the time course of virus shedding and viremia, nasal and ocular discharges, skin scabs, blood and semen were collected and tested for virus presence using PCR until day 50.To assess the clinical picture and collect samples, all experimental animals were first stunned using a penetrative captive bolt to induce unconsciousness, followed by the administration of “Adilinum super”  at day 61 p.i. at a recommended dose of 5 mg/kg according to the drug use instruction approved by the Russian Federal Service for Veterinary and Phytosanitary Surveillance in 2008. At the recommended dose, the Adilinum mechanism of action provides painless and rapid euthanasia: cerebral death commences first followed by circulatory collapse.Only skin lesions were used for virus isolation and culture. They were ground finely and added to sterile saline at a mass/volume (m/v) ratio of 1:10, followed by repeated freeze–thaw cycles at −80 °C and room temperature (3 times) to disrupt cell membranes. The processed samples were clarified at 1,500 rpm for 15 minutes (min). The supernatant was removed, mixed with antibiotics  at room temperature for 90 min and then inoculated onto ovine testis or goat gonad cells (70–80% confluency for both), as follows: after removal of the growth medium from flasks containing the cell cultures and washing twice with Hanks’ medium, a 0.1 mL volume of inoculum was added. The flasks were incubated at 37 °C for 90 min to ensure virus adhesion. Once the incubation period was completed, 10 mL of maintenance medium supplemented with 1.0 mL fetal bovine serum was added. The flasks were again incubated at 37 °C. The inoculated cell cultures were observed daily for cytopathic effect (CPE). The cells were then harvested when 80% CPE was observed and lysed to release the virus, using repeated freeze- thaw cycles (3×). The presence of LSDV was confirmed using real-time PCR.24, with a few modifications. The test was performed on ovine testis cells using 2 replicates. The volume of inocumlum virus was 100 µl into each well. The neutralization index was considered negative if less than or equal to1:8.Virus neutralization in flat-bottomed microplates (96 wells) was conducted using the protocol described previouslyA double-antigen ELISA for the detection of antibodies against capripoxviruses, including lumpy skin disease virus (LSDV), sheeppox virus (SPPV) and goatpox virus (GTPV) in serum or plasma from cattle, sheep, goats or other susceptible species , was used to confirm exposure of the bulls to LSDV infection. Serum samples were collected from infected (IN) and in-contact animals (C1 and C2)  showing clinical signs at 0, 42 and 60 days p.i. The ELISA was performed according to the manufacturer’s recommendations. Reactions were measured as optical density (OD) readings at 450 nm using a Tecan Sunrise absorbance microplate reader (Switzerland). The antibody responses were represented as sample-to-positive (S/P) ratios (percentages), calculated as follows: S/P% ratio = (sample OD– negative control OD)/(positive control OD– negative control OD) × 100%. S/P ratios ≥ 30% were considered as being positive.The samples were aseptically handled and processed as 10% homogenates in PBS. A 200 μL aliquot was used for total nucleic acid extraction using the QIAamp DNA Mini Kit , following the manufacturer’s recommendations.et al.25. The fluorogenic probe was labeled at the 5′ end with the FAM reporter dye and with BHQ as a quencher at the 3′ end. Selected primers and probes were synthesized by Syntol . PCR was performed using a Rotor-Gene Q  instrument and the following thermal-cycling profile: 95 °C for 10 min, followed by 45 cycles at 95 °C for 15 seconds (s) and 60 °C for 60 s. The final reaction volume was 25 μL containing 10 pmol of each primer, as well as 5 pmol of the probe, 25 mM MgCl2, 5 μL 5× PCR Buffer , 1 μL of 10 pmol dNTPs , and deionized water to make up the final volume. Samples were tested and results interpreted according to the protocol, as previously described25.Sample extracts were analyzed using real-time PCR for the presence of LSDV DNA, as previously described by Sprygin The baseline average temperature for the animals before the trial was 38.4 °C. All animals infected with LSDV at the outset of the trial (group IN) had fevers up to 40.7 °C (range 39.0 °C to 40.7 °C) on p.i. day 3, indicating virus replication and circulation. By day 8 p.i., the body temperatures of all the inoculated bulls rose above 39.6 °C and their temperatures remained high until p.i. day 20, and thereafter moderate declines were measured to within the range of 38.1 to 39.6 °C in most of the infected animals up to around day 40 p.i. – thereafter a second bout of fever (bi-phasic) was measured in most of the group (except for NI-8) with temperatures rising to a high of 41.5 °C (IN-2) on day 41 p.i., tailoring off to close to normal levels from day 48 p.i. Fig. .Figure 1On the other hand, the control (C1) in-contact animals that did not receive virus at initial inoculation, maintained normal body temperatures of around 38.6 °C until day 19 p.i., and thereafter their temperatures also showed increases up to 41.1 °C , which decreased slightly on day 46 p.i., but then rising again (bi-phasic fever) in most animals (except C1-1) up until the end of the trial, i.e., day 61.In C2 animals, which were introduced from day 33 p.i., their body temperatures increased from as early as five days post-introduction , also showing a bi-phasic pattern of fever, with the second phase starting around day 49 p.i. (16 days post-introduction), with a high of 40.9 °C being recorded for animal C2-3.There was a significant level of edema in the inoculated bulls (IN group) in their subscapular axillary and popliteal lymph nodes, except in bull IN-6 – this animal exhibited edema in its dewlap area and joints, accompanied by lesions on its muzzle, and general weakness Figs.  and 3. IViremia, as assessed using qPCR on blood samples, was evident as early as p.i. day 7 in the infected animals IN-6), peaking by p.i. days 11-13 (only detectable on day 13 in IN-8), followed by a slow decline after p.i. day 20, until p.i. day 38 in all animals in this group   animals (except in C1-1), which were not inoculated, viremia was measurable using qPCR only after p.i. day 27, to a varying extent, and reached a plateau between p.i. day 38 and 41 (4 out of 5 bulls), which corresponds to days 25–28 following the peak viremia in infected (IN) bulls. Viremia in IN and C1 animals became undetectable after p.i. day 43 .For the C2 group, except for a low-level detectable viremia five days post-introduction in C2-3, no viremia was detectable in blood for the remaining animals, or at any other time point during the trial for this group, even though a low-level bi-phasic fever was measured. However, low levels of viral DNA was detectable from nasal and/or ocular secretions from all animals from this group at some point during their inclusion in the trial .Virus shedding in inoculated bulls was initially observed as early as p.i. day 11 in nasal discharges, and this lasted intermittently, at least until p.i. day 38. Ocular shedding was also observed in nos. 2, 4, 6 and 10, starting from day 13 p.i. , Fig. 7.The dynamics of virus shedding in the two groups in contact with each other at the outset of the trial (IN and C1) showed no statistically significant differences (p > 0.05). The nasal shedding was more durable in IN bulls versus C1 animals with no statistical significance in virus loads. The ocular shedding was comparable in time between the two groups and did not differ statistically Figs.  and 8.In the inoculated bulls, by p.i. day 11, there was a significant appearance of lesions (0.5 to 1 cm diameter) on the shoulders of three of them . In addition, in these bulls in this group, except IN-2, the scrotum and ventral aspects of the abdomen showed typical foci of erosions. Bull IN-2 showed erosions in only the groin and scapula areas, with the temperature remaining steady at an average of 40 °C. By day 15, there was fulminating disease presentation in infected bull nos. 4, 6 and 10 with symptoms of multiple lumps, coalescing together over the entire body (up to 3 cm in diameter), erosions in the foci up to 2 cm in diameter, and lesions in the scapula and sides as large as 1.5 cm in diameter. Erosions were also observed on their muzzles and associated epithelia. Bull no. 8 displayed similar symptoms, but their appearance was delayed. By p.i. day 21, the size of the lesions had enlarged to 2.5 cm in diameter in inoculated bull nos. 4, 6 and 10, and their nasal epithelia were hyperaemic. Also, there were isolated foci of necrotic lesions in the scrota of these animals.For the C1 bulls, by day 28 p.i., three of them exhibited small erosions in their nasal epithelia and in their muzzles Fig. , during Blood, nasal and ocular discharges were positive for LSDV DNA from these animals, as assessed using qPCR. Even though bull C1-1 was asymptomatic, it was positive for the presence of virus using qPCR analysis. The presence of virus in ocular shedding became detectable only from p.i. day 28 , while virus shedding in nasal excretions was evident mostly after 35 days p.i. S–8. OculaFrom day 38 of the experiment, i.e., five days after introducing the C2 animals, viral DNA was detectable from them in blood (viremia), ocular and nasal discharges –8. SheddInoculated (IN) animal nos. 4, 6 and 10 had persistent clinical symptoms, with virus shedding in secretions, till p.i. day 37. But, the inoculated bulls nos. 2 and 8 were asymptomatic, without fever, despite delivering positive PCR results for the presence of virus. All the infected (inoculated) bulls stopped shedding the virus thereafter, with erosions displaying complete scarring and their edemas being cleared, but with their lymph nodes remaining enlarged (as detectable to the touch) up to p.i. day 45.On the other hand, most of the C1 bulls  displayed clinical signs until day 54. Their skin lesions became necrotic and erupted, with persistent fever between 39 and 40 °C. At p.i. day 50, these bulls developed additional skin lesions, hyperemic nasal epithelia, mild edema in their jaws and dewlaps, and enlarged lymph nodes in their necks. They also had enlarged lymph nodes that were painful to the touch. There was still detectable virus shedding, at this time.ELISA testing was conducted to assess the levels of seropositivity of the animals to the virus, and it was confirmed that at day 0 before the inoculation, all animals were seronegative Table . By p.i.26. However, novel features arising in new chimeric progeny as a result of these events may include drastic phenotypic changes such as a shift in host range, pathogenicity and in transmission pathways27. The first field evidence of capripoxvirus recombination between a virulent and vaccine strain was published recently, with the new virus clustering outside both distinct groups of vaccine and field strains4. Despite the genome delineation, the degree to which the genome rearrangements contributed to new phenotypic characteristics remains to be elucidated. In this study, we follow up on the novel recombinant strain, LSDV Saratov/2017, in susceptible hosts (bulls) and demonstrate clearly for the first time non-vector in-contact transmission of this isolate with a focus on the time course of viremia and virus shedding in different groups of experimental animals.Recombination is key towards molecular evolution of viruses. Many viruses appear to have diverged from a common ancestor through genetic exchanges and reassortments to expand their diversity, likely including capripoxviruses20) – in this case, efficient non-vector-assisted transmission. Previous experiments with field strains never showed convincing proof that LSDV can transmit to susceptible hosts through air-borne or other non-vector contact modes21. By contrast, the findings of the current study showed that the recombinant virus was able to transmit horizontally without arthropod assistance, as neither flying insects nor ticks were detected throughout the trial period in the indoor containment facility. The first group of in-contact animals (C1) possibly became infected via indirect contact through sharing food and water troughs, while actual physical contact with adjacent animals to their left and right was also possible and may have resulted in direct contact transmission via the mucosa resulting from virus shedding. Drinking troughs have already been blamed for indirect transmission of LSDV in an insect-free setting17, however the complexity of transmission is far from being fully comprehended18.The experiment was designed to gain an insight into whether the novel vaccine-derived virulent recombinant LSDV strain, with some genes restored to the yet unknown wild-type parental strain, such as the KSGP-like LSDV isolate, could exert new features surmised to be absent in the normal field parental strains  previously showed that DNA in blood was detectable between 6 and 15 days p.i., only. In the same study, oral and nasal shedding was reported between 12 and 18 days p.i., whereas in our study this window was from 11 to 38 days p.i., which is much longer.Interestingly, the viremia in the IN bulls was observed between 7 and 22 days p.i., up to 38 days p.i., whereas Babiuk 21. However, the presumed indirect contact  route as evidenced by C1 bulls in this study deserves further attention in experimental settings to delineate the complex nature of LSDV transmission. The first evidence of viremia in C1 bulls was observed at day 29 p.i. , nasal and ocular shedding did not differ significantly between IN and C1 animals (p > 0.05), but the duration of viremia and nasal shedding was twice as long in IN animals versus C1 animals, except for ocular shedding where the duration was comparable S. It is l29 p.i. S, which i29 p.i. S.Concerning the C2 group, these animals also became infected via an in-contact mode as evidenced by clinical signs, PCR and virus isolation. Although this group had a significantly lower virus load (viremia) versus the IN and C1 groups (p < 0.05), the nasal and oral discharge loads in this group did not differ significantly from either of the other groups (p > 0.05) Figs. –8. As thOf note is that the ELISA and VNT results indicate that the C2 group did not seroconvert, even after 27 days, possibly due to the shorter time available for antibodies to be elicited in this group compared to the other groups Table , whereasIn addition, the localization and dissemination of skin lesions merits further discussion. From our experience, inoculated animals usually develop generalized disease with lesions breaking out evenly over the entire body, including scrotum, muzzle and epithelium, and within the same basic time period. This observation was also true for the IN bulls in this study. The C1 animals, in contrast, displayed a dramatically different pattern, where skin lesions first appeared and were initially restricted to the neck and head regions only, with no lesions in the scrotum or remainder of the body. However, eventually new lesions did develop, spreading throughout the remainder of the animals, down to the tail Fig. . This is4. Since this virus clearly showed contact transmission, it follows that all LSDVs should be capable of spreading by the same mode to varying degrees. Along with the anticipated vector-borne mode of transmission, with no single vector species yet clearly identified as being responsible for transmission, the direct or indirect contact modes have far reaching implications regarding control and eradication. The previously pursued intravenous pathway resulting from vector blood-feeding, has failed to explain outbreaks occurring when cold or dry weather conditions do not permit potential vector flight activity1. The likely reality is that a number of modes probably come into play, with the dominant one leading to an outbreak dependent on the specific circumstances and environmental and biological factors present immediately prior to the onset of the outbreak. This aspect warrants urgent attention and research efforts in the context of the current low-level research base on LSD, especially in non-endemic countries, to gain better insights to enable more adequate and targeted control practices to be developed and implemented.Another important aspect of the current findings begs the question as to which genomic loci of the recombinant virus likely contributed to enhanced severity and improved capacity for contact transmission. Although identification of the particular genetic targets determining the virus phenotype was outside the scope of the current study, a number of the vaccine-derived genes within the recombinant virus, which reverted to the wild-type genotype following recombination, warrant further studyde novo-created feature absent from both parental stains of the novel LSDV or whether it maintained a low profile, but was activated by genetic alterations following recombination.Assimilating the evidence gained from this study, a contact mode of LSDV transmission has been clearly demonstrated for a novel recombinant LSDV recently recovered in the field. Coupled to other alternative modes like vector-assisted spread, these findings may contribute to future risk analysis and complement approaches to combating the threat this disease poses. The findings presented herein will lay a justifying basis for revisiting the control strategies currently in place, considering the danger from the emergence of hybrids, should the use of live vaccines be resorted to, especially in non-endemic countries. Further studies into LSDV biology are warranted to delineate the reasonable question if the contact mode demonstrated here is a Overall, the current approach to combat LSD and the virus, generally looks at insect vector control, besides vaccination and restriction of cattle movement, while food, water or litter are often overlooked as being ineffective sources of infection. In this scenario, control strategies need to be revisited for development of a more complex approach involving new parameters to consider in risk analyses. Moreover, contact transmission mitigates the factor of seasonality, which is linked to insect activity and widens the possibilities for spread regardless of the presence of biting insects.Supplementary Information.