Ixodes ricinus ticks with tick-borne encephalitis (TBE) and Kemerovo (KEM) virus, both known to be transmitted predominantly by ixodid ticks. Both methods permitted TBEV acquisition by ticks and we further confirmed virus trans-stadial transmission and onward transmission to a vertebrate host. However, only artificial feeding system allowed to demonstrate both acquisition by ticks and trans-stadial transmission for KEMV. Yet we did not observe transmission of KEMV to mice (IFNAR−/− or BALB/c). Artificial infection methods of ticks are important tools to study tick-virus interactions. When optimally used under laboratory settings, they provide important insights into tick-borne virus transmission cycles.Up to 170 tick-borne viruses (TBVs) have been identified to date. However, there is a paucity of information regarding TBVs and their interaction with respective vectors, limiting the development of new effective and urgently needed control methods. To overcome this gap of knowledge, it is essential to reproduce transmission cycles under controlled laboratory conditions. In this study we assessed an artificial feeding system (AFS) and an immersion technique (IT) to infect  In human and veterinary medicine, most tick-borne pathogens are transmitted by various hard ticks belonging to genera Ixodes, Haemaphysalis, Dermacentor, Hyalomma and Rhipicephalus and by certain soft ticks belonging to genera Argas and Ornithodoros. Among tick-borne pathogens, 170 tick-borne viruses (TBVs) were identified and belong to nine virus families and twelve virus genera. Viruses transmitted via tick bites can cause various symptoms in humans and animals, ranging from mild febrile illness to neurological disorders or even haemorrhagic fevers5.Ticks represent significant risks for human and animal health. Because they are obligate hematophagous ectoparasites and feed on diverse vertebrate hosts, they are considered as one of the most important vectors of zoonotic pathogens. Ticks can transmit a wide variety of bacteria, parasites and viruses6. The difficulties relate to the obligate hematophagous nature of hard ticks. Therefore, they need an animal host to feed on and reproduce, requiring lengthy feeding times and ingesting larger blood meals than any other known hematophagous arthropod. Tick-borne viruses received much less attention than viruses transmitted by mosquitoes, especially in terms of interaction with their arthropod vectors. Competent ticks acquire a given arbovirus during their feeding on infected vertebrate hosts or during co-feeding with infected ticks. The virus is then transmitted during the next blood meal after an extrinsic incubation period over which the virus will replicate and disseminate throughout the tick body. Hard ticks take only one blood meal per stage , meaning that the trans-stadial transmission of the virus is necessary for transmission to a new vertebrate host upon the following blood meal. Indeed, TBVs must survive changes occurring during moulting and maintain their infectivity into the following developmental stage7. Because transmission of viruses occurs when saliva is injected during feeding upon the host, the virus must reach and infect the tick salivary glands8. The complexity of the whole process starting with virus acquisition and progressing to infection of a new host, involves delicate molecular interactions between viruses and their vectors. Investigating these interactions facilitates the identification of elements and pathways used/abused by TBVs, which could be targeted to attempt blocking virus replication and control TBV-related diseases9. Therefore, reproducing transmission cycles of tick-borne viruses under controlled laboratory conditions remains an important prerequisite to study their interactions with respective vectors.The oversite and existing gaps in our knowledge of ticks and TBV are partly due to the difficulty of setting effective experimental models to assess vector-competence or study virus-tick interactions in general. In fact, establishing and maintaining laboratory tick colonies and performing experimental infections of ticks remain a challenge6. Several methodologies are in use in laboratories such as feeding on a live animal, capillary feeding, inoculation or immersion. Many of these techniques were used to infect ticks with bacteria and/or parasites. For viruses the most widely used methods are intrathoracic inoculation (bypasses the gut barrier), anal pore microinjection (does not bypass gut barrier) or feeding on a vireamic host13. The main aim of our study is to adapt/develop efficient tick infection methodologies, including an artificial feeding system (AFS)14 or an immersion technique (IT)15. Developing infection methods which allow selective infection of ticks with TBVs facilitates the study of pathogens of interest in the arthropod vector. The AFS relies on ticks feeding through an animal skin and has been validated as a model to infect ticks with Babesia divergens14, Babesia venatorum16, Bartonella henselae19 and Bartonella birtlesii20. The IT was validated to infect Ixodes scapularis with Borrelia burgdorferi15 and Langat virus (LGTV)21. Ticks were directly immersed into a suspension of the pathogen and further fed on a vertebrate host immediately thereafter. Tick infection models with tick-borne encephalitis virus  have been previously established and validated under laboratory settings. The main techniques include parenteral inoculation and co-feeding on mice24.In order to study the complex of ticks and the pathogens they transmit, laboratory tick infection methodologies have been developedIxodes ricinus is the primary vector of a wide range of pathogens and the most abundant and widely distributed tick species in Europe25. In our study, we used viruses belonging to genera Flavivirus or Orbivirus to infect I. ricinus. TBEV is known to be transmitted by I. ricinus and responsible for severe neurological illness of humans in Europe and Asia26. It was used as a positive control to assess the efficacy of both AFS and IT as infection methods. Kemerovo virus  is suspected to be the causative agent of encephalitis cases in humans in central Europe and Russia28. AFS and IT were used to assess vector competence of I. ricinus for KEMV. The virus has been isolated/detected in I. persulcatus and I. ricinus29. Up until this study, assessing vector competence of I. ricinus for KEMV has never been attempted. Assessing the efficacy of both infection techniques was based on the three criteria of vector competence: (i) virus acquisition by ticks, (ii) trans-stadial transmission, and (iii) transmission of the viruses to a vertebrate host.p value = 0.18) (Table p < 0.001) (Table p = 0.88). However, the success of trans-stadial transmission of TBEV from engorged larvae to moulted nymphs was higher using the AFS than the IT (p < 0.001) . Out of the 14 mice, twelve (86%) died or showed typical symptoms of TBEV and were hence euthanised at the humane endpoint of the disease. The mean survival time was 11.6 days (± 1.75) after infestation. The remaining two mice, which had survived, presented symptoms of TBEV but recovered ultimately.p < 0.001) (Table p < 0.001)  were collected. Engorged nymphs, mouse organs and blood samples were tested by real-time RT-PCR for KEMV RNA. All samples were tested negative ruling out the possibility of a KEMV transmission to the mice by the feeding ticks.Five to seven months post-moulting, nymphs were fed on 4 BALB/c and 4 IFNARp = 0.26). Trans-stadial transmission tended to be much better with TBEV than with KEMV (p = 0.06). The presence of KEMV or TBEV genomes in the ticks correlates with the uptake of viral particles by the larvae during artificial feeding or immersion. Acquisition using the IT was more efficient with TBEV than with KEMV (p < 0.001). Trans-stadial transmission of KEMV was not confirmed using this technique. The distribution of Ct values of engorged larvae and moulted nymphs is shown in Fig. p < 0.001). Levels of KEMV RNA appear to be similar in engorged larvae and nymphs after moulting (p > 0.05).We used TBEV experiments as positive controls for the feasibility of artificial infections with KEMV. As shown in Tables  p < 0.00. Trans-s p < 0.00. Trans-s14 and an IT21. I. ricinus is known to be a competent vector for TBEV32. This virus as a positive control to study the potential of the two infection methods for testing tick vector competence. We also assessed vector competence of I. ricinus for KEMV, a tick-borne zoonotic orbivirus. We have shown that TBEV can successfully infect I. ricinus using either methods and was efficiently passed from the infected ticks to a susceptible mouse model, as confirmed by the development of specific clinical signs and the detection of TBEV RNA in the mouse blood and organs. Our results also shown that the AFS is a more efficient method to infect ticks with TBEV than the IT. In addition, ticks were efficiently infected with KEMV using the AFS, while the IT failed to achieve infection. However, no KEMV transmission from infected ticks to mice as a model vertebrate host was observed.This study investigated two laboratory tick infection methods for tick-borne viruses, namely an AFS11. Though a versatile technique, intrathoracic injection bypasses the gut barrier and hence is far from representing a natural route of infection12. Oral feeding remains the gold standard to assess key elements of virus-ticks interaction, especially considering that the gut barrier is an important aspect of vector competence. For instance, Dhori and Dugbe viruses are two tick-borne viruses transmitted by Hyalomma dromedarii/H. marginatum or Amblyomma variegatum. Both viruses replicate efficiently in R. appendiculatus haemocoel and are transmitted trans-stadialy and to a vertebrate host when the virus is injected into the tick. However, this tick species is not a competent vector of these two viruses and no replication, trans-stadial transmission nor transmission to a vertebrate host were observed when ticks were orally infected using virus spiked blood33. However, for the immersion method, it is not clear whether the virus is taken up by oral absorption or diffuses into the tick body through various pores. This method has also the disadvantage of requiring live animals to feed ticks which have been immersed into the virus suspension. Nevertheless it is a low cost method which is easier to set up and is less time consuming than the AFS. For larvae, the IT lasts 4 days, which spans the immersion of the ticks and the feeding on mice. Yet, the major limitation of IT is the generation of cohorts of infected ticks with an equal pathogen burden. In this study, the number of infected ticks with either virus using the IT was lower than using AFS. For TBEV infection, we obtained 224 engorged ticks after immersion, with an estimated infection rate at 72%, whereas using AFS we obtained 2009 engorged ticks with a comparable infection rate. Comparatively, the AFS lasts a minimum of 7 days with daily blood changes. It is also noted that our results with TBEV or KEMV suggest that the efficacy of the immersion method could rely on the nature of the virus/virus family. Unlike TBEV, we did not observe trans-stadial transmission of KEMV from larvae to nymphs using IT, when such a transmission was demonstrated with AFS.Both infection methods used in this study, have their advantages and limitations. These techniques allow experimental tick infection without the requirement for viraemic animals They are also safe methods which avoid the use of glass needles required to inject a virus suspension into ticks6. AFS is a better method in terms of infection with TBEV and KEMV and it generates a larger cohort of infected ticks. In addition, it is an infection technique that is closer to the natural mode of tick infection (feeding on an infected viraemic vertebrate). However, it appears that the success of tick feeding is higher with live animals than upon using AFS. In addition, and despite good engorgement rates for control ticks in the KEMV experiment (56%), the observed engorgement rates with blood spiked with KEMV (29%) or with TBEV (22 to 23% ) were lower than those described in the scientific literature (ranging from 43 to 84.5%).19 Indeed, one of the limiting factor with this method is the unpredictability of feeding rates due to tick behaviour, as ticks may resist to feed, but this can also be a problem with feeding on live animals6.Using the IT, the number of obtained engorged ticks is limited for further analysis due to a limited number of ticks per animal and limited number of animals used for ethical reasons. By contrast, the AFS allows feeding of a higher number of ticksI. scapularis ticks with similar infection rates in engorged larvae of 96% and 87% in nymphs after moulting21. However, it was not the case with KEMV where the infection rates of larvae and nymphs after moulting were 26.25% and 0%, respectively. One possible explanation of the observed differences in infection efficiency can be the virus titre in the suspension used for immersion of larvae. In our study, the TBEV titre (2.5 × 108 PFU/mL) was 100-fold higher than KEMV titre (4.4 × 106 PFU/mL). In the LGTV study, the virus titre was 107 PFU/mL21. The nature of the virus may also provide additional explanations to the observed differences. TBEV and LGTV are both enveloped flaviviruses with a single-stranded RNA genome, while KEMV is an orbivirus having a dsRNA genome and is non-enveloped. Thus, the two groups of viruses differ substantially in terms of structure of the virus particles (enveloped versus non-enveloped) and particular mechanisms involved in cell entry (endocytosis versus fusion) and tissue dissemination39. Furthermore, during the feeding on a vertebrate host, ticks inject saliva that modifies the local environment at the bite location. It has been demonstrated that saliva plays a role in the transmission of tick-borne pathogens to a vertebrate host and also in the pathogens acquisition40. Certain molecules present in vector saliva substantially influence infectivity and survival of TBVs in the tick8, and it has been shown that salivary proteases enhance infectivity of insect-borne orbiviruses38. If we assume that KEMV was taken up through body pores other than the mouth parts, its infectivity may have been less efficient to the larval tissues. Consequently, as the IT is further from a natural mode of feeding than AFS, it might influence infection efficacy of specific viruses and AFS remains a better tool to study the virus-tick interactions particularly to evaluate vector competence.The IT was efficient with TBEV, as observed with significant infection rates in nymphs which moulted from immersed larvae (48%). This technique was successful with LGTV in I. ricinus in the field29. KEMV was isolated from these ticks in 1964 in Czechoslovakia41 and Vologda region in Russia in 197529. However, up until this study, vector competence studies of I. ricinus for KEMV has been never attempted. Our results cast reasonable doubt about I. ricinus being a natural vector for KEMV. We have shown that I. ricinus larvae can be infected with KEMV during a blood meal and remain infected after moulting (two weeks after) when infected using our AFS. Although a relatively high infection rate was observed in engorged larvae (83.3%), a low efficacy of trans-stadial transmission resulted in only 40% of infected nymphs two weeks after moulting was reported. The first trials of KEMV transmission were performed five months post-moulting and we did not observe transmission to mice. The virus seemed to have disappear from the nymphs which were found negative for KEMV RNA 5 months post-moulting. These results suggest that the ticks cleared the virus during this period. Arthropods have antiviral innate immune responses. The most robust antiviral response is RNA interference (RNAi) which targets viral RNA degradation, thus keeping virus replication in check42. This pathway is well known in mosquitoes but not clearly understood in ticks. A previously published study showed the involvement of Dcr and Ago proteins in the antiviral RNAi response after TBEV or LGTV infection of IDE8 tick cells (a cell line derived from I. scapularis)43. It is possible that I. ricinus antiviral immunity cleared the virus or even KEMV failed to disseminate from the site of entry into the tick to other tick organs, a process that is crucial to infect salivary glands and onward transmission to vertebrate hosts. Our studies showed that I. ricinus larvae were infected with the virus, which survived moulting into nymphs, but real-time RT-PCR detecting KEMV RNA was performed with RNA extract from whole ticks, and localisation of the virus within the nymphs has not be identified so far.Like TBEV, KEMV was associated with 44 and the loss of any potentially virus particles present on the surface of their bodies. However, it should be noted that the non-biological transmission by immersed larvae via a possible contamination of the mouthparts suspected for TBEV, was not observed with KEMV.The detection of KEMV RNA in larvae extract after immersion may have resulted from particles which remained on the body surface or from particles which have been taken up by the tick though body pores without further replication. Larvae moulting into nymphs is accompanied by a loss of the cuticle (apolysis)50. The I. ricinus ticks we used in our studies were from Slovakia. The prevalent serotypes of KEMV in Slovakia are Lipovnik (LIPV) and Tribeč (TRBV) virus as previously reported41, and both viruses has been isolated from I. ricinus. The KEMV strain used in this study was isolated in Russia in 1962 where several KEMV isolates were obtained from I. persulcatus27, the principal Ixodid tick in that geographical region51. Thus the geographical origin of the tick population could be a contributing factor to the observed results. We have currently ongoing studies with laboratory reared I. persulcatus larvae from Russia, which will provide clear cut information about the role of this tick species in maintaining KEMV in the field and evaluation of our experimental infection methods. Studies with I. ricinus from Russia would also provide additional valuable information as to whether geographical variants of this tick may influence competence. Alternatively, an assessment of vector competence of I. ricinus from Slovakia towards TRBV or LIPV could also shed light on potential genetic traits that are virus and tick population related.Moreover, interaction between arboviruses and their vector is a complex process. Vector competence is genetically determined and could rely on a particular virus strain and on a particular arthropod geographical strain. This has been shown previously for other arboviruses such as TBEV, bluetongue, Rift Valley fever virus or chikungunya virusI. ricinus and showed vector competence of I. ricinus for TBEV in the laboratory. Our preliminary results suggest that I. ricinus appears to be poorly competent for KEMV but further studies are required to confirm such a result. Both techniques presented in this study have their advantages and limitations but as the IT is further from a natural mode of feeding than AFS, it might influence infection efficacy of specific viruses and AFS remains a better tool to study the virus-tick interactions, tick co-infections  and more particularly to evaluate vector competence. These studies are essential to understand mechanisms of evolution, emergence/re-emergence of tick-borne pathogens, and to identify new methods to control tick-borne diseases for which an urgent public health response is needed.We have assessed two experimental techniques to infect ticks with tick-borne viruses. We validated two tick infection methods, an AFS and an IT, with TBEV using I. ricinus larvae . The virus was originally isolated from the brain of a patient who died of acute encephalitis in 196227. This isolate was further amplified once in suckling mouse brain and passaged twice in Vero cells in 1969. Before use, the initial stock of the virus was propagated three times in BSR cells.A KEMV virus stock was prepared in BSR cells (a clone of BHK-215 cells/well) in 24-well plates with Dulbecco's Modified Eagle Medium (DMEM) supplemented with penicillin (50 IU/mL), streptomycin (50 µg/mL) and 10% foetal bovin serum (FBS) at 37 °C under 5% CO2. Ten-fold serial dilutions of TBEV were prepared in serum-free DMEM. Two hundred microliters of each dilution were added per well for 1.5 h at 37 °C. Cells were covered with a carboxy-methyl cellulose (CMC) overlay in DMEM. After five days of incubation at 37 °C, cells were rinsed three times with phosphate-buffered saline (PBS) and fixed/stained with a 10% formaldehyde solution containing 0.1% crystal violet. TBEV titre was determined as 2.5 × 108 PFU/mL.TBEV titre was determined using plaque assay using either porcine PS or monkey kidney Vero E6 cells. On the day before the assay, cells were plated , to free virus particles from cell debris as described previously56. The mixture was shaken manually and centrifuged at 2000 g for 5 min. The aqueous phase, containing free virus particles, was carefully recovered. Briefly, BSR cells were plated (1 × 105 cells/well) in 24-well plates a day before running the assay in DMEM supplemented with penicillin (50 IU/mL) and streptomycin (50 µg/mL), 10% of FBS at 37 °C, 5% CO2. Ten-fold serial dilutions of the virus were prepared in serum-free DMEM and 250 µL of each dilution was added per well for 2 h at 37 °C. Cells were then covered with 1 mL of molten 1% low melting point agarose in DMEM. After incubation at 37 °C for 5 days, 2 mL of 10% formaldehyde was added to each well. Cells were stained with 0.1% naphthalene-black solution after removal of the agarose plugs, and plaques counted. The virus titre was determined as 4.4 × 106 PFU/mL.KEMV titre was also determined by plaque assay as described previouslyI. ricinus larvae derived from the 1st generation of ticks collected in the Murbach forest from the Alsace region in France, or from a laboratory colony of ticks initially collected in the Czech Republic. For KEMV infection studies, we used larvae from the 4th generation of a laboratory colony provided by the Institute of Zoology, Slovak Academy of Sciences, Bratislava, Slovakia. Ticks were maintained at 22 °C with 95% relative humidity and a 12 h light/dark cycle in the animal facilities as described in14. In each case, after egg-laying, females were tested for KEMV and TBEV RNA before larvae were used in the experiments.For TBEV infection studies, we used 57. TBEV-infected ticks from the artificial feeding experiments, were fed on adult (4–5 weeks old) type I interferon receptor knock-out mice ) kindly provided by Prof. Michel Aguet 59. These mice are also susceptible for TBEV infections and develop severe clinical signs ending by death around day 6 post-infection60.TBEV-infected ticks from immersion experiments were fed on three weeks old specific pathogen-free (SPF) female mice of the ICR (CD1) strain  known for their susceptibility to TBEV infections−/− female mice (10 weeks old) were used for feeding KEMV-infected ticks. BALB/c mice were used for feeding after immersing ticks in the viral suspension and for assessing the transmission of the virus to the vertebrate host. IFNAR−/− mice were used for assessing the virus transmission from the infected ticks to the host only. The use of these strains is warranted by previous findings. BALB/c mice are commonly used for infection with orbiviruses61, and IFNAR−/− usually develop severe clinical signs of infection with KEMV, including neurological signs, haemorrhage, and prostration . In addition, IFNAR−/− mice are well known to be susceptible for orbivirus infections62.BALB/c female mice (7 weeks old)  and IFNAR. The present research was conducted in compliance with all relevant European Union guidelines for work with animals and with the Czech or French national law guidelines on the use of experimental animals and protection of animals against cruelty. Experimental protocols were approved by the Committee on the Ethics of Animal Experimentation at the Institute of Parasitology, the Departmental Expert Committee for Approval of Projects of Experiments on Animals at the Czech Academy of Sciences (Permit No. 29/2016) and by ANSES-ENVA-UPEC Ethics Committee for Animal Experimentation .The mice were individually held in plastic cages with wood-chip bedding, fed ad libitum and kept in standardised conditions  at the SPF animal facilities of the Institute of Parasitology and ANSES14 was used to infect I. ricinus with TBEV and KEMV. The system, material and the setting are shown in Fig. 14. Commercial defibrinated sheep blood was purchased from Eurobio scientific (France). Before use, the blood was supplemented with 17 U/mL of heparin . Gentamicin  and Amphotericin B  at a final concentration of 10 mg/mL and 250 µg/mL, respectively. For infection studies, the blood was directly spiked with 105 PFU/mL for TBEV65 or 104 PFU/mL for KEMV55. For each experiment, control and infected settings were used in parallel. Ticks were placed in the tick chamber and enclosed with a mosquito mesh allowing them to breathe. To attract ticks, the setting was maintained at 37 °C by a circulating warmed water circuit  were used for virus-spiked or clean bloods. Approximately 3,000 larvae were placed in each chamber. Ticks for KEMV experimentation were a gift from colleagues in Slovakia. We thus used a single setting for KEMV-spiked blood and a single control setting each containing approximately 2,000 larvae, availability of ticks being here a limiting factor.The frequency of blood changes in the AFS was decided based on an initial assessment of virus infectivity in sheep blood spiked with cell-culture derived TBEV or KEMV.5 PFU/ml using a fresh TBEV suspension. Blood samples were recovered from the setting at 0, 4, 8, 10, 24 and 30 h and titrated by plaque assay as described above. TBEV was titrated at different time’s intervals after blood spiking. After 10 h post-infection, the titre decreased by about 1.5 log.TBEV infectivity in sheep blood was assessed over a time course of 30 h. The blood was spiked with 104 PFU/ml of fresh KEMV suspension and blood samples were recovered at 0, 4, 8, 10 and 24 h post-spiking and assessed by real-time RT-PCR. We observed a slight drop in KEMV genome copy numbers after 12 h  was placed at − 80 °C immediately after collection to further detect viral RNA.Ixodes scapularis with Borrelia burgdorferi15 and Langat virus21 and adapted here for TBEV and KEMV.The IT was initially developed to infect 6 PFU/mL for KEMV and 2.5 × 108 PFU/mL for TBEV) of 5 mL for 45 min at 37 °C. The fluid was pumped in and out using the plunger every 10 min to ensure immersion of all ticks. Ticks were then rinsed three times with PBS and centrifuged at 200 g for 30 s to remove the supernatant between each washing  strain were used for feeding TBEV-immersed ticks and eight female BALB/c mice were used for feeding KEMV-immersed ticks.To immerse ticks easily, the tip of a 1 mL syringe was cut and covered with a mosquito mesh attached with a rubber band. Between 100 and 200 larvae were placed in this syringe, inserted into another syringe of 10 mL, the tip of which was also cut , and finally inserted into a 50 mL centrifuge tube Fig. . Ticks wTicks were allowed to feed for four days. The first engorged larvae were collected on day three post-infestation and the latest on day four post-infestation. Symptoms of infection and/or mortality of mice were monitored throughout the experiments which lasted for 5 days post-infestation for KEMV and 11 days post-infestation for TBEV. Blood samples were collected only from mice used in the KEMV experiment. Engorged larvae were placed in flasks and maintained at a relative humidity of 95% at 22 °C. Between 9–12 engorged larvae per mouse were randomly chosen and stored at -80 °C to check for the presence of viral RNA.−/−  or CD1 (larvae immersed in TBEV suspension) mice. Nymphs which moulted from larvae infected with KEMV were fed on IFNAR−/− mice or BALB/c mice. Symptoms of the infection and/or mortality were recorded for each mouse throughout the experiments. At the end of the experiments , mouse blood and organs  were sampled . Presence of virus in mouse blood and tissue samples was assessed by real-time RT-PCR for TBEV and KEMV as described below. All procedures are summarised in Table To check if nymphs which were infected at the preceding larval stage can transmit the virus to a vertebrate host, ticks were placed on mice within a capsule system as described above. Nymphs which moulted from larvae infected with TBEV were fed on IFNARVirus acquisition by larvae, trans-stadial transmission from larvae to nymph, and transmission from nymphs to mice were assessed by detecting virus genome in both ticks and mouse organs and blood, using real time RT-PCR.For RNA extraction, ticks were homogenised individually , in 350 µL of lysis buffer and 3.5 µL of β-mercaptoethanol with six stainless-steel beads (2.8 mm diameter) using Precellys®24 Dual homogeniser  at 5500 × rpm for 20 s or a single sterile stainless-steel bead (5 mm diameter) in TissueLyzer II  at 30 Hz for 3 min. RNA was then extracted from individual ticks, pooled ticks or 40 µL of whole mouse blood using NucleoSpin® RNA extract II  as described by the manufacturer. All mouse organs were individually homogenised  in 1 mL of DMEM culture medium supplemented with 10% FBS with six stainless-steel beads using the Precellys®24 Dual homogeniser at 5500 rpm for 20 s. RNA was extracted from 500 µL of homogenate using NucleoSpin® RNA extract II kit . All RNA samples were eluted in 30 µL of RNase-free water and stored at − 80 °C until further used.67. Thus, before real-time RT-PCR, the dsRNA is heat denatured at 99 °C for 5 min using an appropriate heating system, such as a thermal cycler. Renaturation of dsRNA must be prevented by immediately quenching the tubes on wet ice. Reverse transcription was performed using the qSript cDNA Supermix kit  as previously described68. The reaction was carried out in a final volume of 5 µL containing 1 µL of qScript cDNA Supermix 5X, 3 µL of RNase-free water and 1 µL of RNA. The thermal cycling programme consisted of a single cycle at 25 °C for 5 min, followed by one cycle at 42 °C for 30 min and one final cycle at 85 °C for 5 min. DNA preamplification was performed in presence of primers KEMV_F -GTCAGACGGATTTTCGACCTC- and KEMV_R -GCGAGCCAGATCCCGATGT, targeting genome segment 2 (encoding the VP2 protein). The preamplification was carried out in a final volume of 5 µL, containing 1 µL of 5X Perfecta Preamp Supermix , 1.25 µL of primers solution , 1.5 µL of distilled water and 1.25 µL of KEMV cDNA. The PCR cycling programme consisted of one cycle at 95 °C for 2 min, 14 successive cycles of denaturation at 95 °C for 15 s and amplification at 60 °C for 4 min. The pre-amplified DNA were 1:2 diluted and stored at − 20 °C until further used. The real-time PCR was performed using the LightCycler® 480 Probes Master kit according to the manufacturer's instructions . The following primers and probe were used: KEMV_F , KEMV_R  and KEMV_P ACGGGCCAACACTCGTTCATCACAG 68. All samples were tested in duplicates. The PCR cycling programme consisted of one cycle at 95 °C for 30 s, 45 successive cycles at 95 °C for 10 s, 60 °C for 30 s and 72 °C for 1 s. The programme ended by a cooling cycle at 40 °C for 30 s.Tick or organ lysates were tested individually by real-time RT-PCR. Because the genome of KEMV consists of segmented double-stranded RNA, heat-denaturation is a prerequisite for a successful reverse transcription69. The PCR cycling programme consisted of one cycle of reverse transcription at 63 °C for 3 min, followed by one cycle at 95 °C for 30 s, 45 successive cycles at 95 °C for 10 s, 60 °C for 30 s and 72 °C for 1 s. The programme ended by a cooling cycle at 40 °C for 30 s.TBEV RNA was detected using either the genesig Tick-borne encephalitis virus Polyprotein Advanced Kit  together with OasigTM lyophilised OneStep qRT-PCR Mastermix  or LightCycler® 480 RNA Master Hydrolysis Probes kit following manufacturer's instructions . The following primers and probes, targeting the 3’ non coding region (3’NCR), were used: TBEV_F GGGCGGTTCTTGTTCTCC , TBEV_R ACACATCACCTCCTTGTCAGACT  and TBEV_P TGAGCCACCATCACCCAGACACA The authors confirm the study was carried out in compliance with the ARRIVE guidelines.Supplementary Figure S1.


Multiple antiviral immunities were developed to defend against viral infection in hosts. RNA interference (RNAi)-based antiviral innate immunity is evolutionarily conserved in eukaryotes and plays a vital role against all types of viruses. During the arms race between the host and virus, many viruses evolve viral suppressors of RNA silencing (VSRs) to inhibit antiviral innate immunity. Here, we reviewed the mechanism at different stages in RNAi-based antiviral innate immunity in plants and the counteractions of various VSRs, mainly upon infection of RNA viruses in model plant Arabidopsis. Some critical challenges in the field were also proposed, and we think that further elucidating conserved antiviral innate immunity may convey a broad spectrum of antiviral strategies to prevent viral diseases in the future. Caenothabditis elegan [The phenomenon of RNA silencing was first observed in plants in 1990 when the introduction of chalcone synthase transgene into petunia led to the suppression of endogenous homological genes . In 1998s elegan  and terms elegan ,4. Numers elegan ,6. RNAi s elegan . AdditioThere are two major classes of endogenous small RNA in plants: microRNAs (miRNAs) and siRNAs . miRNAs Upon pathogen infection, different sizes of pathogen RNA-derived siRNAs are also produced to induce RNAi-based antimicrobial immunity to confer host resistance ,11. RNAiHere, we will review the perception of viral RNA and the initiation of RNAi-based antiviral defense, production, and amplification of viral siRNA (vsiRNA) and the functions of antiviral RNAi effector Argonautes, with an emphasis on the recent progress in the field, challenging the existing questions on model plant Arabidopsis. Some VSRs of plant viruses and their functions were also summarized for a better understanding of the arms race between plant hosts and viruses. We apologize that some research progresses in the field may not be included.Viruses are almost the smallest organisms on Earth, with a classical structure in which genetic material, RNA or DNA, is packaged in coat protein. Viruses need to be paralyzed in the host and propagated using the materials and energy of the host. Unlike other microbial pathogens, pattern-recognition receptors (PRRs) are not found to perceive viruses on the cell membrane of the host. After virus enters the host cell, double-strand viral RNAs will be produced during viral replication through viral replicases, host Dicer proteins will perceive and dice the double-strand viral RNAs to produce 21–24 nt lengths of vsiRNA. Thus, Dicer protein may be regarded as a viral PRR used to initiate the RNAi-based antiviral pathway.Dicer belongs to the RNaseIII-like family and has highly conserved endonuclease in eukaryotes . In ArabDCL1 is mainly involved in the biogenesis pathway of 21 nt micro-RNAs (miRNA), which play essential roles in all aspects of plant development and plant responses to environmental stimuli . DCL1, Dtomato mosaic virus (ToMV) infection by producing 22 nt vsiRNA in tomatoes [DCL2 is responsible for processing exogenous double-stranded RNA (dsRNA) molecules or natural antisense siRNAs into 22-nt siRNAs in Arabidopsis ,36. Howetomatoes .DCL3 generates 24nt siRNAs to regulate RNA-dependent DNA methylation (RdDM) in transcriptional gene silencing (TGS) in Arabidopsis . Recentldcl4 mutant showed a phenotype of elongated, downward curled rosette leaves [DCL4 cleaves long endogenous dsRNA to produce 21 nt siRNAs, such as trans-acting siRNAs (ta-siRNAs), which are crucial for plant development ,49,50,51e leaves  and augme leaves ,54. In Rpotato spindle tuber viroid [Although each DCL is responsible for the production of distinct small RNA, they may function redundantly or hierarchically in RNAi-based antiviral innate immunity. For example, DCL4 was regarded as an endogenous suppressor to repress DCL2-mediated production of 22 nt siRNAs ,55; hower viroid . In addir viroid .α-β-β-β-α folds and two α-helices to interact with dsRNA [turnip yellow mosaic virus (TYMV) [drb3 mutant was hyper-susceptible to cabbage leaf curl virus (CaLCuV) and beet curly top virus (BCTV) infection, and the viral genome methylation was substantially reduced in drb3 [tobacco rattle virus (TRV), tomato bushy stunt virus (TBSV), potato virus X (PVX), and grapevine fanleaf virus (GFLV) [DsRNA-binding (DRB) proteins are also required for the proper perception and dicing of viral RNAs by DCLs . The Arath dsRNA ,63. DRBsth dsRNA . For exath dsRNA ,65,66. Ds (TYMV) . The drb in drb3 . DRB2 was (GFLV) .Caenorhabditis elegan. They exponentially generate viral dsRNA, which serves as DCLs substrates for vsiRNA biogenesis, probably using truncated viral RNAs as templates [After the perception and dicing of viral dsRNA through DCL, primary vsiRNA will be produced. However, adequate secondary vsiRNA must be produced through amplification for efficient antiviral defense. Host RNA-dependent RNA Polymerase (RdRP) proteins are core factors for secondary vsiRNA amplification in plants and emplates ,69,70.There are six RdRP proteins in Arabidopsis (RDR1 to RDR6). RDR1, RDR2, and RDR6 all share the C-terminal canonical catalytic DLDGD motif of eukaryotic RDRs and have orthologs in many plant species, while RDR3, RDR4, and RDR5 share an atypical DFDGD amino acid motif in the catalytic domain . RDR1, RRDR1 can be induced by the infection of the virus , viroid rdr6 mutant and sgs3 mutant display the same defects in antiviral defense and development [rice stripe virus (RSV) [RDR6 is constitutively-expressed in various tissues in Arabidopsis. RDR6 not only promotes RNAi-based antiviral innate immunity by mediating vsiRNA biogenesis, especially after the infection of RNA viruses, it also controls plant development by mediating the biogenesis of endogenous siRNA, such as tasiRNAs ,76. RDR6elopment ,81,82,83elopment  and contelopment . Besidesus (RSV) .RDR2 mainly associates with Pol IV to form a complex for transcribing short dsRNA precursors, which are cleaved by DCL3 to produce 24 nt siRNAs for directing DNA methylation ,87,88,89ala1/ala2 or avi2 mutant, production of secondary vsiRNAs was dramatically reduced. ALA1/ALA2 contain the typical P4-type ATPase structure (rdr6 3 (ENOR3) since it was also identified through a genetic screen from rdr6 background using another CMV mutant in which the 2b gene was deleted [Several novel factors involved in the amplification of secondary vsiRNA were also recently discovered. Antiviral RNAi-defective 1(AVI1)/aminophospholipid transporting ATPase 2 (ALA2), ALA1, and AVI2 were identified through a robust forward genetic screen using a cucumber mosaic virus (CMV) mutant in which start codons of VSR-2b were mutated ,93,94,95tructure  and may  deleted . AVI2, a deleted .RDRs and new factors such as ALA1/2 and AVI2 are widely conserved in plants and worms to ensure sufficient biogenesis of vsiRNA for efficient RNAi-based antiviral innate immunity. However, RDRs are absent in Drosophila, mice, and humans, in which a different mechanism was recently discovered for vsiRNA amplification through extrachromosomal circular DNA . WhethervsiRNA must be loaded to AGO effectors to form RISC, then targets complementary viral genomes to PTGS or TGS in RNAi-based antiviral innate immunity. Effector AGOs are evolutionarily conserved and widespread in eukaryotes, though absent in prokaryotes . They weago1 knockout mutants are lethal. Therefore, the function of AGO1 in RNAi-based antiviral innate immunity was only examined using hypomorphic AGO1 mutants, such as ago1-27, which still displayed severe developmental defects [ago2 mutant does not show defects in growth and development; AGO2 may solely regulate plant defense in Arabidopsis. It was reported that AGO2 prefers to bind vsiRNAs with 5′ terminal A and AGO1 prefers to U [ago1 mutant with intact catalytical activity was susceptible to viral infection [Pseudomonas syringae [MEMB12 to modulate exocytosis of antimicrobial PR proteins and increase antiviral activity [Ten AGOs are encoded in Arabidopsis ,112,113. defects . Unlike ers to U . AGO2 isers to U ,127,128.ers to U ,127, whinfection . AGO2 issyringae , and AGOactivity . Therefoago4 mutants was susceptible to the infection of BCTV due to the diminished hypermethylation on BCTV genome [ago4 mutants are susceptible to several RNA viruses, such as turnip crinkle virus (TCV), bamboo mosaic virus (BaMV), and plantago asiatica mosaic virus (PlAMV) [AGO4, AGO6, and AGO9 are the major effectors functioning in the RdDM pathway in Arabidopsis. AGO4, AGO6, and AGO9 were shown to bind 24 nt heterochromatic small interfering RNAs (het-siRNAs) and contribute to the RdDM pathway ,131. It V genome . Surpris (PlAMV) ,134,135 potato virus X (PVX) systemic infection, while AGO5 only plays a secondary role when AGO2 is overcome in the initially infected leaves [turnip mosaic virus (TuMV) [As to other AGO effectors, AGO5 together with AGO2 participate in reducing d leaves . AGO7 (ad leaves . AGO7 cad leaves . AGO10 cs (TuMV) , besidess (TuMV) .rice stripe tenuivirus (RSV), RSV coat protein (CP) triggers JA accumulation and upregulates JA-responsive transcription factor JAMYB to directly bind to the AGO18 promoter to activate AGO18 transcription [Interestingly, more than 10 AGO orthologs were found in some important crops such as rice and tomato, with 19 orthologs in rice and 15 in tomato. They can evolve to have differential functions in antiviral defense and development. For example, when infected with cription . AGO18 wcription . On the cription . Our unpIn the defense and counter-defense arm race between host plants and viruses, viruses evolve VSR proteins to inhibit RNAi-based antiviral innate immunity. VSRs target different steps of the RNAi-based antiviral pathway to counteract the conserved antiviral immunity  142,143143.tomato yellow leaf curl China virus (TYLCCNV), and P6 of rice yellow stunt virus (RYSV) interfere with the RDR1/6-dependent biogenesis of secondary vsiRNA [tomato yellow leaf curl geminivirus (TYLCV), P2 of RSV and P4 of rice stripe mosaic virus (RMSV) interacts with SGS3 to inhibit the biogenesis of secondary vsiRNA [A very common counteraction of VSRs is to impede vsiRNA amplification. For example, 2b of CMV, βC1 of y vsiRNA ,180,185.y vsiRNA ,182,189.y vsiRNA .cauliflower mosaic virus (CaMV) interacts with DRB4 to block dsRNA binding [tomato spotted wilt virus (TSWV) and Hc-Pro of potato virus Y (PVY), also bind to long viral dsRNA, which could block the sensing or processing of the viral RNAs by DCLs. Some other VSRs could directly target vsiRNA to inhibit RNAi-based antiviral innate immunity. For example, P19, the well-known VSR of tombusviruses, binds and sequesters vsiRNA, while RNase III of sweet potato chlorotic stunt crinivirus (SPCSV) binds and mediate vsiRNA degradation, and HC-Pro of zucchini yellow mosaic virus (ZYMV) decreases vsiRNA stability by disturbing vsiRNA methylation by HEN1. Disturbing the antiviral function of effector AGOs is another strategy used by some VSRs. For example, P0 of potato leafroll virus (PLRV) can mediate AGO1 degradation, and 2b of CMV can interfere with AGO1 and AGO4 and disturb their functions.Some VSRs were found to obstruct the perception or dicing of viral dsRNA. For example, CP of TCV could inhibit the dicing activity of DCL4 , and P6  binding . Some VStomato leaf curl Guangdong virus (ToLCGdV) can also interact with BAM1 to suppress PTGS and reverse methylation-mediated TGS [barley stripe mosaic virus (BSMV), targeted AUTOPHAGY PROTEIN7 (ATG7) to disrupt the ATG7–ATG8 interaction and promote viral infection [Surprisingly, unlike the above VSRs that counteract RNAi-based antiviral innate immunity, other mechanisms were found to antagonize antiviral responses by some VSRs. Recently, a study showed that VSR p19 can interact with the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1) and BAM2 to inhibit the cell-to-cell movement of RNA silencing ,194. VSRated TGS . In addiated TGS ,197. It ated TGS . Howevernfection .Now we know that almost all plant viruses, especially pathogenic plant viruses, possess one or more VSR. The existence of VSRs contributes to the successful aggression of viruses and viral epidemics; they also seriously hinder our appreciation of the indispensable antiviral innate immunity in plants and other eukaryotes.RNAi-based antiviral innate immunity is recognized as a fundamental antiviral innate immunity in plants and animals; some key components functioning in the antiviral pathway were found and are well characterized. However, our understanding of the exact mechanism of the antiviral pathway is far from complete since only a few novel components in the pathway were found, and some critical questions or challenges in the field are waiting to be tacked. For example, (1) whether a specific cellular structure exists for vsiRNA biogenesis and amplification, and (2) whether/how vsiRNAs move over long-distances to confer systematic resistance. (3) Additionally, the impact of function differentiation on the translation inhibition or slicing of different AGOs and their functional localization in cells are largely unknown. (4) The true function and mechanism of RNAi-based antiviral innate immunity in important agricultural crops are elusive and (5) the application of RNAi-based antiviral innate immunity in agricultural practice needs to be further explored. Therefore, further studies are required to dissect the vital antiviral innate immunity, and progress in the field could eventually lead to finding novel strategies or methods to prevent specific and broad-spectrum viral diseases in plants and even humans.


Virus screening and viral genome reconstruction are urgent and crucial for the rapid identification of viral pathogens, i.e., tracing the source and understanding the pathogenesis when a viral outbreak occurs. Next-generation sequencing (NGS) provides an efficient and unbiased way to identify viral pathogens in host-associated and environmental samples without prior knowledge. Despite the availability of software, data analysis still requires human operations. A mature pipeline is urgently needed when thousands of viral pathogen and viral genome reconstruction samples need to be rapidly identified.In this paper, we present a rapid and accurate workflow to screen metagenomics sequencing data for viral pathogens and other compositions, as well as enable a reference-based assembler to reconstruct viral genomes. Moreover, we tested our workflow on several metagenomics datasets, including a SARS-CoV-2 patient sample with NGS data, pangolins tissues with NGS data, Middle East Respiratory Syndrome (MERS)-infected cells with NGS data, etc. Our workflow demonstrated high accuracy and efficiency when identifying target viruses from large scale NGS metagenomics data. Our workflow was flexible when working with a broad range of NGS datasets from small (kb) to large (100 Gb). This took from a few minutes to a few hours to complete each task. At the same time, our workflow automatically generates reports that incorporate visualized feedback .Overall, our system enabled the rapid screening and identification of viral pathogens from metagenomics data, providing an important piece to support viral pathogen research during a pandemic. The visualized report contains information from raw sequence quality to a reconstructed viral sequence, which allows non-professional people to screen their samples for viruses by themselves (Additional file The online version contains supplementary material available at 10.1186/s12920-021-01138-z. In the last 20 years, many disease outbreaks have been attributed to viruses, from SARS, Ebola , 2, MERSNext-generation sequencing (NGS) techniques have recently switched from proof-of-concept studies to a routinely used tool in the clinical microbiology laboratory. It is now easy and efficient to generate metagenomics data for direct analysis of genetic materials in order to identify viruses and their abundances in various environments . HoweverBioinformatics tools used for virus identification either use amino acid profile (gene-based) approaches or nucleotide profile (reference-based) approaches. Gene-based identification methods are fast but not suitable for viruses with unknown marker genes . ReferenSince the SARS-CoV-2 outbreak, scientists from different fields have contributed various features to better understanding the novel coronavirus. At the initial stage, most researchers have focused on the viral genomes  using HISAT2. The host genome could be downloaded from the NCBI. We then used SAMtools  to filter unmapped reads in order to remove source sequences. For single-end reads, only unmapped  strands were retained. For paired-end reads, unmapped strands , unmapped mate strands , and unmapped double-strands  were retained. Unmapped reads were extracted and converted to FASTQ files using bam2fastq before virus identification.We employed the FastViromeExplorer  to screeftp://ftp.ncbi.nlm.nih.gov/refseq/release/viral/) and accession2taxid file (ftp://ftp.ncbi.nlm.nih.gov/pub/taxonomy/accession2taxid/nucl_gb.accession2taxid.gz). Further, they could generate the correct format of the requested viral reference files by using the necessary information from downloaded references.The trimmed FASTQ files were imported into FastViromeExplorer and short k-mers were mapped with the Kallisto index, i.e., “ncbi-virus-kallisto-index-k31.idx”. Matched virus information was taken from the taxonomy list “ncbi-viruses-list.txt”. These two viral reference files could be directly downloaded from the FastViromeExplorer website. However, since viral genomes increased rapidly every year, we provided the users a shell script (build_index.sh) used to update these viral reference files with the latest NCBI viral data as needed. Our shell script (build_index.sh) could build these viral reference files by downloading NCBI reference viral genomes  were extracted, sorted, and de-duplicated for calling single nucleotide variants (SNVs). We did not directly use the alignment output of Kallisto because FastViromeExplorer is a pseudo-alignment and hits depend on a short k-mer (31 bp as default). Although a short k-mer could be conducive to screening out virus’ calculation speed, it is not suitable for genome assembly. Actually, abnormal mutations  popped up when FastViromeExplorer alignments were used.SNV calling using BCFtoolsSNV calling was performed using the multi-pileup file (mpileup) module in BCFtools. De-duplicated reads for each potential virus were indexed using the SAMtools index. Genotype likelihoods were estimated using the mpileup module in BCFtools. SNVs were called via BCFtools.Consensus generation with BCFtoolsWe defined a three-step reference-based genome assembly for each individual potential virus identified using FastViromeExplorer:Consensus was generated using BCFtools consensus, where gaps and positions with low coverage were masked with ‘N’. Variants were output in the form of IUPAC ambiguity codes.Quality control of the NGS data: consisted of a table summarizing the quality of raw data collected from the outputs of FASTQC and FQCHECK, and figures to visualize the quality results were generated via FASTQC.Identities and compositions of the reads were visualized via a pie-chart using R to clarify the proportions of the low-quality reads, reads from hosts, and reads from viral genomes.Coverage of each viral genome were displayed as follows: (1) a bar-plot showing the counts and percentage of the top five viruses identified; (2) a table summarizing the coverage and species of each virus; and (3) a curve plot depicting the distribution of the counts across the individual viral genome if genome coverage was above 50%.A statistical summary of the assembled viral genomes showed the length and GC content of each virus and consensus genome of each viral genome.Outputs produced in the previous three steps were finally collected and evaluated to generate a PDF report. The report consisted of four parts:Plots were produced using R and tables were made by collecting summary information generated in previous steps (using shell and Python scripts). Plots and tables were embedded in a brew model, which was then converted from latex to PDF using texi2dvi. We recommend using R 3.5 and higher in the workflow. A full report demo can be found in the supplementary material.We tested VIW on several datasets downloaded from NGDC  and the EBI-ENA database  . ParvoviSamples 2 SRR10903401) and 3 (SRR10903402) were two COVID-19 cases from Wuhan  . As such0903401 aSamples 4–10 were downloaded from the NGDC database  . Samples 11–15 represented SRR1192017, SRR1191695, SRR1191876, SRR1191783, and SRR1192321. Samples were prepared by infecting human lung-derived MRC5 cells with MERS-CoV for 48 h, and profiled by high throughput sequencing (NCBI GSE56192 summary). In these cases, human sequences accounted for around 32.68–40.14% of total reads, and viral genome proportions were similar to the one in SARS-CoV-2 patients’ samples. Two MERS-CoV sequences were found in all five cases with coverage over 98% and average reads depth above 10,000, as shown in Tables The numbers of SNVs in these five samples were similar, since human cells were infected by the same laboratory MERS-CoV strain in vitro. As shown in Table Since these samples were over 8 Gb, it took much longer to run. The time used by the FastViromeExplorer assembly method was much shorter than the FastViromeExplorer + BWA method, and was much shorter than the BWA assembly method in all five cases.To test whether our workflow could be applied to different host species, we ran our workflow on pangolin samples because reports showed that SARS-CoV-2 might derive from pangolin . Thus, wAs shown in Table We also ran our workflow on datasets with different trimming conditions to test whether the trimming strategy would affect virus detection. We tested our datasets with q = 20, length = 20; q = 30, length = 30; and q = 40, length = 50. The meaning of q = 20 was that, for any base, if its Phred score was less than 20 , it would be trimmed off, and the meaning of length = 20 was that, after the base trimming, the whole read would be trimmed off if the total length of a read was less than 20 bp. The results showed that most viruses can be detected with q = 20 and length = 20. The disappearance of viruses in the other two conditions could be due to the cutoff of those virus sequences in the samples. Especially when we used q = 40 and length = 50 to trim the sample reads, all sequences in samples 1–10 were trimmed off, as shown in Additional file We tested our workflow, VIW, with samples of SARS-CoV-2 from human and pangolin to show its capability of handling sequencing data for multi-species. We test the workflow with samples of SARS-CoV-2 and MERS-CoV from human cell samples to show its capability to identify different viruses in samples. This workflow can be used widely in clinical studies. For example, it can be used for the SARS-CoV-2 epidemic as additional evidence for diagnosis. The workflow is very flexible and can be used on various NGS datasets for various purposes. Although there are other applications for virus detection purposes—e.g., toolset FastV, released by Zhang’s team —our workBecause we used reliable software to filter host sequences, removed adapters and low quality reads in the preprocessing steps, and identified viruses during the virus identification step, our biggest concern for the presented workflow is the accuracy of the virus assembly step. As for virus sequence alignment, although FastViromeExplorer did a good job identifying viruses, it cannot be used as a direct alignment method, since it might bring unstable assembly results, i.e., extremely large or small number of SNVs. BWA is a reliable software for alignment, because it considers a full length alignment of the reads to the reference genome. However, it takes a long time when the input is large. We therefore combined FastViromeExplorer  and BWA to fulfill the two tasks, i.e., virus detection and genome assembly, wherein it worked as a virus detector and a filter in order to reduce the amount of sequences need to be aligned by the BWA. On one hand, our workflow retained its alignment accuracy by using BWA. On the other hand, our tests demonstrated that the running speed of our default method was faster than using the BWA assembly directly. Therefore, even though we provide other alignment methods on GitHub, we highly recommend our default method, as it is suitable for most NGS data. In addition, the human-friendly report generated in our workflow systematically presents results in each step, and makes it more convenient for researchers to understand their NGS data. In summary, the advantages of our workflow are that it generates results quickly and accurately, and the results are easy to understand.Although our workflow is sensitive enough to detect viruses with only one read present in the sample, which happened in the Sendai virus for sample lung11, our workflow lacked a method to decide whether a virus actually existed in the sample. The workflow kept the Sendai virus in its report, as identified virus in sample lung11. Therefore, our workflow detected 7 pangolin lung samples with the Sendai virus. However, the fundamental study suggests that only 6 samples contained this virus. Since we only used online datasets for our tests, we could not verify this. Thence, further tests should be done to clarify the minimum coverage needed to determine the existence of a virus. Another problem of this workflow is that since it assembled viral sequences using known viral genome for reference, it could not assemble unknown viruses. For instance, pangolin samples lung7 and lung8 both reported the contamination of SARS-CoV-2, but with only 7.00% and 11.53% total coverage. It is not clear whether this virus found in the human being was SARS-CoV-2. If it is an unknown coronavirus, our workflow can only define it with its closest relative, but not a novel virus. In another words, when the coverage of a virus produced in our workflow is low, further tests and experiments are requested to identify the virus. This could be done with further experimental verification, or by testing more samples with our workflow.In conclusion, our workflow, VIW, can be used to identify viruses contained in a host genome. By running this workflow, users can obtain the following: a table that lists all potential viruses in their samples; a file with SNV information for each identified virus; a FA file with assembled viral genome; and a report visualizing the quality and composition of raw data, and information on the coverage and taxonomy of identified viruses. The workflow can be used as the first step to study viruses, where virus sequences are extracted and assembled from NGS raw data. In addition, the workflow can be used for clinical diagnoses.Additional file 1: An example of the final report generated by our pipeline.Additional file 2: Sample Sources.Additional file 3: Viruses identification among different reads quality cut off.


Arachnida, subclass Acari) are vectors of transmitting a broad range of pathogenic microorganisms, protozoa, and viruses affecting humans and animals. Liaoning Province is rich in forests where different animals and, abundant Haemaphysalis longicornis ticks exist.Ticks  reads showed significant sequence identity to known viruses. Sequence and phylogenetic analysis revealed that viral sequences showing a close relationship with Dabieshan tick virus, Aleutian mink disease virus, adeno-associated virus, Gokushovirus, avian gyrovirus 2 were present in the virome of these ticks. However, the significance of these viruses to human and animal health requires further investigation. Notably, an hepe-like virus, named tick-borne hepe-like virus sequence, was obtained and was highly prevalent in these ticks with a rate of 50%. Nevertheless, one constraint of our study was the limited geographical distribution of the sampled ticks.Our study offers an overview of the virome in ticks from a forest region of Liaoning Province and provides further awareness of the viral diversity of ticks.The online version contains supplementary material available at 10.1186/s12985-021-01632-x. Arachnida, subclass Acari) transmit a broad range of pathogenic microorganisms, protozoa, and viruses and are the second most common vectors of diseases affecting livestock, humans, and companion animals [Ticks  is the predominant species in Liaoning Province [Liaoning Province, situated in the northeastern region of China, is owned by a temperate monsoon climate region with plenty of rainfall and sunshine. Furthermore, it has 4.641 million hectares of forests and is rich in animal resources, with 827 species of animals, including amphibians, mammals, reptiles, and birds. Researchers found that tick richness correlated with forest size, even among locally common birds . Ticks aProvince , but theSince the necessity and feasibility, this study aims to study the diversity and evolutionary origin of viruses in ticks from a forest of Liaoning Province by a viral metagenomic approach.According to climatic conditions of summer-autumn favor tick proliferation, we collected 300 live adult ticks from June to August during 2015 in a big forest park of Dalian city in southern Liaoning .The collected ticks were divided into six groups Pool 01-06) based on their sampling month   to remove eukaryotic and bacterial cell-sized particles. The filtrate was treated for 60 min at 37 °C with a DNases mixture , benzonase (Novagen), and RNase (Fermentas) to digest unprotected nucleic acid , 19. Nuc5. Candidate viral hits were then contrasted to an in-house non-redundant (NVNR) protein database to remove false-positive viral hits. The NVNR database was compiled using non-viral protein sequences extracted from an NCBI nr fasta file . Contigs without significant BLASTx similarity to the viral proteome database were searched against viral protein families in the vFam database using HMMER3 to detect remote viral protein similarities.Paired-end reads of 250 bp generated by HiSeq were debarcoded using vendor software from Illumina. Clonal reads were removed, and low-sequencing-quality tails were trimmed using Phred. Adaptors were removed using the default parameters of VecScreen. The cleThe assembled contigs and unassembled reads in known taxonomy assignments obtained from the previous step were performed de novo assembly and reference mapping in Geneious version 2019.2.3  to acquiTo safeguard consistency in the library, the volume of each library for next generation sequencing (NGS) is based on the relative brightness intensity of electrophoretic bands. After the construction of the sequencing libraries, the quality of the libraries was confirmed by agarose gel electrophoresis (1% agarose). Eventually, library quantitation, including testing the concentration and fragment insertion size were performed before NGS sequencing.Standard precautions were used for all steps to prevent cross-sample contamination and nucleic acid degradation. Aerosol filter pipet tips were used to reduce the possibility of sample cross-contamination. All the materials  directly contacted with nucleic acid samples were RNase and DNase free. Nucleic acid samples were dissolved in DEPC treated water and RNase inhibitors were added.The sequence alignment files to build phylogenetic trees in Figs. H. longicornis. Ticks were pooled into six libraries based on the sampling period. All tick pools were sequenced by two lanes of Illumina HiSeq, resulting in 1,218,388 raw reads with an average of 203,064 raw reads per pool  domains  across the RDRP domain of TKHEV with corresponding members of Hepeviridae through BLASTx search. Unfortunately, no read is assigned to the capsid protein region of TKHEV in all pools. The RdRp is the only conserved-sequence domain across all RNA virus sequences and was used for phylogenetic inference [Although there was imparity in virus sequence composition among libraries, reads of the single-stranded negative-sense viruses were the most abundant in the tick libraries, comprising 69% of all viral reads. After De Nove Assembly and Mapping to reference sequences, we recovered a 5.3 kb segment encoding a putative > 1,700-amino acid protein, provisionally named tick-borne hepe-like virus (TKHEV) strain lntk2unc1   clustered into two major branches infecting vertebrates and invertebrates, based on three conserved domains, respectively. TKHEV was located in the branch of HEVs infecting invertebrates. TKHEV formed a distinct clade with Barns Ness breadcrumb sponge hepe-like viruses 1 and 2 (MF189998 and MF189999)  clades . Among tin China , 32. Theis ticks . Based ois ticks . DTV-ln  of DTVs , 34 , and the other two sequences belong to the subfamily Densovirinae infecting invertebrates [Dependoparvovirus were identified. Due to high identity with adeno associated virus, we tentatively named it tick adeno associated virus 1 (TKAAV1) strain lntk1parc2 (MW334986), tick adeno associated virus 2 (TKAAV2) strain lntk6parc2 (MW334987), and tick adeno associated virus 3 (TKAAV3) strain lntk6parc1 (MW334988)  strain lntk6denc1(MW353163) and tick densovirus 2 (TKDSV2) strain lntk6denc2 (MW353164). TKDSV1 formed an individual cluster before a group of viruses belonging to Iteradensovirus  strain lntk45anec1 (MW334984)  strain lntk5micc2 (MW334989), tick gokushovirus 2 (TKGV2) strain lntk5micc1(MW334990), tick microvirus 1 (TKMV1) strain lntk4micc2 (MW334991), tick microvirus 2 (TKMV2) strain lntk1micc1 (MW334992) and tick microvirus 3 (TKMV3) strain lntk2micc2 (MW334993) , with 99.4% amino acid sequence identity  reads belong to 18 viral families. Notably, we observed a phenomenon regarding the reads number from Pool02 is exceptionally high  and limited amino acid identity across the RDRP domain of this virus with a representative of Hepeviridae. The tick-borne tetravirus-like virus tentatively divided into the Alphatetraviridae, due to dissimilarity in sequence and host association, it likely represents a new family of arthropod-associated viruses [Hepeviridae are classified into two genera Orthohepevirus infecting mammals and avians, and Piscihepevirus, infecting invertebrates [Hepeviridae because it has a similar conserved genome structure highly divergent from the known members in the family  and Lihan tick virus formed a separate branch that was previous to a branch of DTV-ln. One of these (QKR71511) can cause severe symptoms, suggesting DTV-ln likely can infecting people and other animals.The next section of our study was concerned with the transmission of DTV. A strain of DTV (KM817666) was first discovered in  in 2014 . Researc viruses . EpidemiThe researchers found avian gyrovirus 2 (AGV2) in the serum of diseased chicken for the first time , but to The single most noteworthy observation to emerge from our study was that the sequences matched to mammalian viruses were found in the six libraries, such as TKAMPV, and TKAAV1-3 Additional file 2. Presence and abundances of viral families from different pools. (.csv)


Sexagenarians were discharged to home at a higher rate , experienced a longer operating room (OR) time , and had a differing overall hospital length of stay  compared to septuagenarians and octogenarians and older, respectively. Sexagenarians had 7% and 23% less expensive personnel costs from post-anesthesia care unit (PACU) to discharge (p < 0.0001), and 1% and 24% more expensive implant costs (p = 0.077) compared to septuagenarians and octogenarians and older, respectively. Sexagenarians had a lower total in-hospital cost for rTJAs by 0.9% compared to septuagenarians but 12% more expensive total in-hospital costs compared to octogenarians and older (p = 0.185). Multivariate linear regression showed that the implant cost , OR time , personnel cost from PACU through to discharge , and rTHAs  to be the strongest associations with overall costs. Focusing on the implant costs and OR times to reduce costs for all age groups for rTJAs is important to provide cost-effective VBHC.The demand for revision total joint arthroplasties (rTJAs) is expected to increase as the age of the population continues to rise. Accurate cost data regarding hospital expenses for differing age groups are needed to deliver optimal care within value-based healthcare (VBHC) models. The aim of this study was to compare the total in-hospital costs by decadal groups following rTJA and to determine the primary drivers of the costs for these procedures. Time-driven activity-based costing (TDABC) was used to capture granular hospital costs. A total of 551 rTJAs were included in the study, with 294 sexagenarians, 198 septuagenarians, and 59 octogenarians and older. Sexagenarians had a lower ASA classification (2.3 vs. 2.4 and 2.7;  Approximately 7 million Americans are living with a total hip or knee replacement . As patirTHAs and rTKAs are more costly and more demanding of provider and institutional resources than primary arthroplasty procedures ,8,9. RevThe primary aim of our study was to compare the cost differences for an rTJA episode of care among age groups by decades. We also sought to determine which factors contribute to hospital costs among these groups. We hypothesized that hospital cost differences would exist between decadal age groups for rTJAs.After internal review board (IRB) approval was obtained (#1629639-1), financial data were retrospectively collected between January 2018 through to May 2020 at our single-specialty orthopedic hospital for patients aged 60–69 years (sexagenarians), 70–79 years (septuagenarians), and 80 years and older (octogenarians and nonagenarians) who underwent either an rTHA or an rTKA. We collected data on patient demographics, including age, American Society of Anesthesiologists (ASA) classification, procedure type, and insurance, as well as discharge disposition, operating room (OR) time, length of stay (LOS), personnel cost of the post-anesthesia care unit (PACU) through to discharge, and implant and total in-hospital costs to compare the differences between decadal age groups. Financial data are presented as indexed values to protect institutional proprietary information.Time-driven activity-based costing (TDABC) was used with the help of a third-party, commercial cost-analysis database, Avant-garde Health , to determine the episode-of-care costs. TDABC is a modern cost-accounting methodology that is designed to improve patient value  and allop-value correction was applied and the significance threshold was set to p = 0.003 for our multiple procedure group comparisons.To compare the characteristics and outcomes between sexagenarians, septuagenarians, and octogenarians and older undergoing rTHAs and rTKAs, chi-square and analysis of variance (ANOVA) tests were used where appropriate for categorical and continuous data, respectively. Continuous quantitative variables are expressed as an average with a standard deviation after a normal distribution was confirmed via ANOVA residual plot analysis. Multivariate linear regression was used to estimate the relationship between the total in-hospital cost and other significant independent variables, such as OR time, implant cost, and length of stay. To protect confidential internal data, standardized beta coefficients and 95% confidence intervals were used to compare the strength of the effect of each individual independent variable to total in-hospital costs. A standardized regression coefficient is calculated by dividing a parameter estimate by the ratio of the sample standard deviation of the dependent variable to the sample standard deviation of the regressor. All statistical analyses were performed using SAS v9.4 . Significance was set at 0.05. A post-hoc Bonferroni n = 55) and nonagenarian (n = 4) patients (p = 0.0014) and were more often privately insured  compared to septuagenarians and octogenarians and older, respectively. Sexagenarians underwent a higher percentage of rTKA procedures, but this difference was not significant (p = 0.226).A total of 551 rTJAs were included in the study . Of thespatients . On averp < 0.0001) and experienced a longer OR time  compared to septuagenarians and octogenarians and older, respectively. Sexagenarians had a longer LOS compared to septuagenarians (2.8 days vs. 2.7 days) but a shorter LOS compared to octogenarians and older . Sexagenarians also had higher personnel costs from the PACU through to discharge by 1% compared to septuagenarians, but personnel costs were 22% less expensive than octogenarians and older (p = 0.0064). The decadal age groups did not differ in implant cost or total in-hospital costs for rTHAs .A total of 275 (49.9%) of the procedures were rTHAs . Among tp < 0.0001) and experienced longer LOS  compared to septuagenarians and octogenarians and older, respectively. The OR times between the decadal age groups were similar (p = 0.23).A total of 276 (50.1%) of the procedures were rTKAs . Among tp = 0.0001). The decadal age groups did not differ in implant cost or total in-hospital costs for rTKAs ; however, the octogenarian and older age group had 26% lower implant costs than sexagenarians, which was likely due to the eight lower-cost liner exchange procedures in this group.Sexagenarians had lower personnel costs from the PACU through to discharge by 16% compared to septuagenarians and 28% compared to octogenarians and older (p < 0.0001), experienced a longer OR time , shorter length of stay , lower personnel costs from the PACU through to discharge (p < 0.0001), and had 1% and 24% lower implant costs (p = 0.077) compared to septuagenarians and octogenarians and older, respectively (p = 0.185).Overall, sexagenarians were more often discharged to home  on the total in-hospital cost. The OR time was positively associated with the second-strongest effect  and the personnel cost PACU through to discharge was also positively associated with the third-strongest effect  on the total in-hospital cost. The rTHA procedure type was negatively associated with the total in-hospital cost . Decadal age group was not a significant factor after holding all other variables constant (p = 0.19 for septuagenarians and p = 0.45 for octogenarians and older).The results of the multivariate linear regression analysis of the total in-hospital costs are reported in By 2050, 10% of the United States’ elderly population 65 years and older will be persons 90 years or older . RevisioA predominant concern when considering revision arthroplasty in elderly patients is patient safety and the risk of perioperative complications based on medical comorbidities. On average, octogenarians and nonagenarians had a higher ASA classification than sexagenarians and septuagenarians. ASA ratings correlate with patient medical comorbidities and can be helpful in predicting perioperative risks and outcomes. Previous studies have looked at outcomes for nonagenarians and octogenarians in revision procedures ,15,16,17For rTHAs, nonagenarians and octogenarians require transfusions 33.13% and 24% of the time, respectively . HoweverFor rTKA, octogenarians have higher rates of blood transfusion and slightly longer length of stay compared to younger populations but there were no differences in mortality or readmission . For octIn primary THAs and TKAs, patients with private payers were shown to have fewer complications and lower inpatient mortality rates compared to patients with government payers ,19. For Previous studies examining the costs of an rTJA in octogenarians and nonagenarians are limited. For rTKAs, octogenarians and nonagenarians have been shown to have total hospital charges of 86,203 USD and 98,828 USD, respectively . For rTHFor both rTHAs and rTKAs, octogenarians and nonagenarians had a significantly longer length of stay, increased personnel costs from PACU through to discharge, and were more often discharged to skilled nursing facilities (SNFs) compared to the younger decade groups. Of note, the conservative Bonferroni-adjusted significance level of 0.003 to reduce the likelihood of a type I error was not reached for rTHA’s LOS and personnel costs. Despite this, the longer length of stay and increased personnel costs were likely related to more time-intensive nursing in PACU and on the floor due to comorbidities, and likely contributed to spending more time with physical therapists due to frailty. These physical therapy evaluations likely influenced their discharge disposition to locations other than home. All of these factors contributed to a higher total in-hospital cost. In order to measure the relative effect of these variables, we performed a multivariate linear regression model with standardized estimates. In our study, implant costs were shown to be the largest driver of in-hospital total costs. This is consistent with the literature showing that the TJA procedure implant price is the major driver of hospital costs ,25,26.Aside from the implant costs, the OR time, personnel cost from the PACU through to discharge, and procedure type were the most significant factors impacting the total in-hospital costs for revision procedures. Octogenarians and nonagenarians were found to have a decreased OR time compared to sexagenarians and septuagenarians for rTJAs overall. This was most likely a reflection of this patient group undergoing less complicated revisions, such as liner exchanges as opposed to full component exchange revisions. The decreased OR time had a significant effect on overall costs for these older patients, potentially offsetting the increased personnel costs needed for care. The impact of octogenarian and nonagenarian versus sexagenarian status was not associated with total costs after holding all other variables constant. This was consistent with our ANOVA results showing no difference in overall costs when comparing groups. The rTHA procedure type was associated with a significant decrease in overall costs, indicating that an rTHA is a less costly procedure than an rTKA among these groups.A strength of the current study includes a relatively large sample size of 353 rTJA procedures at a single orthopedic institution over a two-year period. Further, our methodology of TDABC has been shown to be a more accurate cost account for TJAs than traditional methods ,22. The In conclusion, overall hospital costs for revision TJAs were not significantly more expensive for octogenarians and nonagenarians compared to sexagenarians and septuagenarians. As life expectancy increases, an increase in older patients needing revisions of primary TJA procedures is expected. Higher implant costs were seen to be a primary driver of total hospital costs for revision TJAs, regardless of age group. As the country focuses on value, which is defined as the outcomes per dollar spent, identifying episode-of-care costs in older patients undergoing revision TJAs is critical in delivering value-based health care.


Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), and/or Sugarcane streak mosaic virus (SCSMV). The compound infection of mosaic has become increasingly serious in the last few years. The disease directly affects the photosynthesis and growth of sugarcane, leading to a significant decrease in cane yield and sucrose content, and thus serious economic losses. This review covers four aspects of sugarcane mosaic disease management: first, the current situation of sugarcane mosaic disease and its epidemic characteristics; second, the pathogenicity and genetic diversity of the three viruses; third, the identification methods of mosaic and its pathogen species; and fourth, the prevention and control measures for sugarcane mosaic disease and potential future research focus. The review is expected to provide scientific literature and guidance for the effective prevention and control of mosaic through resistance breeding in sugarcane.Mosaic is one of the most important sugarcane diseases, caused by single or compound infection of  Saccharum spp. hybrids), the most important sugar and energy crop originating in the tropics, is a perennial, high biomass herb ratoon C4 crop. It is widely cultivated in more than 100 countries or regions in the tropics and subtropics, with a total area of about 27 million hectares. The world annual output is about 1.95 billion tons of fresh cane, which provides nearly 80% of sugar, 60% of bioethanol, and a total economic value of 75 billion U.S. dollars . Furthermore, the pressed cane juice can be used to produce diesel, jet fuel, and other high-value products , and more than 100 species in 40 genera of the Gramineae family [Stenotaphrum secundatum  Kuntze] [Sorghum almum Parodi.) [Cucurbita moschata (Duch. ex Lam.) Duch.]  , ColumbuParodi.) , pumpkin) Duch.] , red-veironeura) , and candica L.) . SrMV casis cv.)  and cause family ,53,54.Gramineae hosts. In nature, transmission of SCMV and SrMV is primarily by several aphid vectors including Dactynotus ambrosiae P1 gene [P1 gene ,81,124.Mosaic disease can be identified by visual inspection of sugarcane leaves for symptoms when evaluating germplasm resistance ,125,126.Traditionally, the type of virus, host range and transmission mode are determined mainly based on the symptoms of the host plant after inoculation with the virus ,127. BefThe most prominent pathological feature of plant tissue cells infected by a virus of the Potyviridae family is the production of columnar inclusions. The feature has been used to distinguish among virus species. Edwardson  divided Miscanthus by Grisham et al. [Serological detection is a simple, rapid, and low-cost method for plant viruses, including agar gel immunodiffusion (AGID), electroblot immunoassay (EBIA), double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), direct antigen coating ELISA (DAC-ELISA), indirect ELISA, and dot enzyme-linked immunosorbent assay (Dot-ELISA)  ,130,131.m et al.  using inm et al.  used DACm et al.  establisSugarcane yellow leaf virus (SCYLV). Smith and Velde [PCR-based molecular techniques have been developed and widely used in the detection of the mosaic virus, such as direct-binding polymerase chain reaction (DB-PCR), reverse transcription PCR (RT-PCR), immunocapture RT-PCR (IC-RT-PCR), duplex immunocapture RT-PCR (D-IC-RT-PCR), one-step multiplex RT-PCR, real-time quantitative RT-PCR (qRT-PCR) and loop-mediated isothermal amplification (LAMP) . These mnd Velde  were ablnd Velde  developend Velde  reportednd Velde  demonstrnd Velde  developend Velde . The priS. officinarum, 10–15% from S. spontaneum, and 5–10% from recombinant chromosomes [Saccharum and its related genera have formed extremely rich and valuable germplasm resources [Saccharum hybrid clones with high resistance to mosaic disease have been identified by natural infection or artificial inoculation, such as SP70-1143, IACSP95-2078 [Selection and rational distribution of disease-resistant varieties are the most economic and effective prevention and control measures against these viruses. The genetic base of modern sugarcane cultivars is narrow, with about 80% from omosomes . During esources ,143, whiesources . In receP95-2078 , SWSwm1 P95-2078 , YG34, YP95-2078 , ROC16 [P95-2078 , GT03-23P95-2078 . The curn = 12 x = 100−130; genome size = ~10 Gb) is a highly complex autopolyploid and aneuploid crop, and a complete reference genome of modern sugarcane cultivated species is still lacking up to now [n = 2 x = 20; genome size = ~2300 Mb) [CP gene-mediated transfer since the first report of the introduction of the Tobacco mosaic virus (TMV) CP gene into tobacco in 1986 [SCMV-CP gene into sugarcane meristem and obtained chimeric transformed plants. Subsequently, Joyce et al. [Molecular marker-assisted breeding and genetic engineering improvements have helped promote the development of resistant varieties. Molecular marker-assisted breeding is an effective method to accelerate the breeding process of multi-resistant varieties. However, the technology is limited by the lack of markers closely linked to disease resistance. Sugarcane  ,163,164.2300 Mb) . The mos in 1986 . Smith e in 1986  used a ge et al. , Ingelbre et al. , and Sooe et al.  also suce et al. ,174,175.e et al. ,177,178.The application of virus-free plantlets through tissue culture can not only eliminate or slow down the incidence of mosaic disease, but also increase the sucrose content by more than 0.5% and the cane yield by 20–40% . In one To determine the technical specifications for the production and testing of sugarcane virus-free plantlets, leaves were collected from the virus-free plantlets produced by the heat treatment and axillary shoot tip culture technology in 2015, followed by tracking and PCR testing the leaves of 120 putative virus-free plantlets collected from different companies at different ecological demonstration sites in China . The PCRGood field management is another effective way to enhance plant resistance and reduce the spread of viruses. Specific measures include: (1) avoiding planting virus host crops around or in the sugarcane fields ; (2) the1 populations can be used for correlation analysis between genotypes and phenotypic traits. There are also successful cases on genetic engineering via gene gun bombardment or gene editing to improve disease resistance in sugarcane. Successfully edited alleles can be up to 49 copies simultaneously targeting the chlorophyll content gene, which draws a new blueprint for transforming the disease resistance pathway in sugarcane. We hope that the present review can provide scientific references and thoughts for the effective prevention and control of viruses in sugarcane.The cultivation and planting of resistant varieties are the most economic and effective methods to control sugarcane infecting viruses, and growers are most likely to adopt this technology. However, due to the diversity of pathogens, the highly complex genome of sugarcane, the wide segregation of traits among hybrid progenies, and the extremely low probability of excellent gene aggregation, sugarcane cross-breeding may have to rely on a huge population, for example, 1 to 1.2 million of plantlets to be planted annually in China. Therefore, it is very difficult to select good varieties that have both commercial value and mosaic resistance. In addition, field evaluation tests of virus-free plantlets from integrated detoxification technology in China have shown that the “virus-free” effect is not ideal, even if the plantlets were produced by a more effective method of combining heat treatment and axillary bud or shoot tip culture. In view of all these facts, the development of molecular markers associated with disease resistance genes should be the most effective way to breed sugarcane varieties resistant to different viruses. Sugarcane propagates asexually and F


A full factorial design with four factors  and polyamide 4,10 (PA410) in the polymer matrix, content percent of biocarbon (BioC), the temperature at which it was pyrolyzed and the presence of a chain extender (CE)), each factor with two levels (high and low), was carried out to optimize the mechanical properties of the resulting composites. After applying a linear model, changes in tensile strength, elongation at break and impact energy were not statistically significant within the considered material space, while the ones in the flexural modulus, the tensile modulus, density and heat deflection temperature (HDT) were. The two most influential factors were the content of BioC and its pyrolysis temperature, followed by the content of PPA. The affinity of PPA with a high-temperature biocarbon and the affinity of PA410 with a lower-temperature biocarbon, appear to explain the mechanical properties of the resulting composites. The study also revealed that the addition of CE hindered the mechanical properties. By maximizing the flexural modulus, tensile modulus and HDT, while minimizing the density, the optimal composite predicted is an 80 [PPA:PA410 (25:75)] wt% polymer composite, with 20 wt% of a BioC, pyrolyzed at a calculated 823 °C. To reduce the impacts that global climate change will have on human life, global warming must not exceed 1.5 °C. This means that many industries will have to adapt and change in order to reduce their carbon dioxide emissions . Among t2 emissions have decreased from 500 g/mile [311 g/km] of gasoline in the 1980s, to less than 400 g/mile [249 g/km] in the 2010s  and a high and low temperature at which the biocarbon was produced. The full factorial analysis was chosen to learn of any interaction that could affect any of the measured responses, as fractional designs can confound important interactions. A scheme of the 22 is less than 60% for tensile strength, elongation at break and the flexural modulus. This means that they were not considered relevant for further analysis, as the mean of all the values was a better predictor than the model itself.Before any analysis of the model results can be performed, the residuals of the different fitted values must be evaluated, in order to know if they follow a normal distribution. If no patterns in the residuals can be discerned, the linear model can be considered valid. In the case of the tensile and flexural modulus, as seen in However, in the case of HDT and density, as seen in λ) is chosen, selecting the most appropriate transformation for the data, based on a maximum value of lambda. This is shown in Equation (1):ith response variable and how it should be transformed, according to the specific value of λ. Although this method can be applied by hand, modern computers can calculate and estimate the maximum value of There are many techniques that can convert data from a non-linear model to a linear one. That is, these techniques will improve the fit of the model and ensure normal distribution of the residuals, with respect to the newly transformed and fitted model. In the paper by Box and Cox , a methoFrom the results in For the flexural modulus, as seen in The question arises as to why the CE did not improve these two mechanical properties, compared to preliminary testing and blends. Russo and Acierno found that the addition of a cross-linking agent actually worsened the creep properties of a PPA-based, 50 wt% glass fiber-filled composite , which tIn the case of HDT, Finally, for density, the statistically significant factors are the content of PPA, the content of BioC and the temperature of pyrolysis of BioC. The two-way interaction that is statistically significant is the content of BioC and the presence of CE; as is the three-way interaction between the content of PPA, the content of BioC and the presence of CE; as well as the three-way interaction between the content of PPA, the temperature of pyrolysis of BioC and the presence of the CE, as seen in One of the main interests of this paper is optimizing the mechanical properties of the composites, after establishing statistical methods that would allow us to predict the behaviour. The statistical analysis software can construct a model that allows the user to ask for the optimum composite under certain conditions. We limited the material space within the level of the factors already tested, so that there would be no extrapolation of data. We also set the program to maximize the flexural and tensile modulus, as well as the HDT, and to minimize the density. Additionally, the program asks for “Weight” and “Importance”. To avoid bias or skewing the prediction, we left all the values set at “1” for all factors. If a desired application or process required a specific value in a particular characteristic, the optimized composite would be different. The result of the optimization is shown in The result shows that the optimum composite within the material space and the conditions imparted upon the program is an 80 (PPA:PA410 (25:75)) wt% composite, with 20 wt% of a BioC pyrolyzed at 823 °C, and no CE, though this composite would have to be produced and tested in order to validate the model. As seen during the analysis of the statistical models, in many cases, the amount of PPA had a negative or slightly positive effect on the properties, so it is logical that a composite with a low amount of this polymer would be selected. Higher content of BioC resulted in high mechanical properties, so despite the increase in density, it is also logical that 20 wt% would be chosen by the algorithm. The temperature of pyrolysis of BioC also makes sense, as we saw that even at a low percentage of PPA, the BioC content enhances all mechanical properties. Finally, the presence of CE hindered the mechanical properties in all categories, so it is logical that it is absent from this optimization.PPA, grade Zytel  and PA 4,10  were dried for 4 h and 24 h, respectively, at 80 °C, in order to ensure the moisture content did not exceed the level recommended by the manufacturers . The biocarbon was produced from pine wood saw dust , by drying the material for at least 48 h at 80 °C, then pyrolyzed using a GLO 0/11-1G batch type industrial front-loading retort furnace  at 500 and 900 °C, respectively, using a heating rate of 5 °C/min, and a dwelling time of 1 h for both temperatures. After the removal of the pyrolyzed material from the furnace, the material was reduced in size using a commercial blender, and was then ball-milled for 2 h, using zirconium oxide vessels, each one utilizing 100 ceramic zirconium oxide balls and placing 52 g of the biocarbon in each one. The biocarbon was then ball-milled at 200 rpm, reversing the direction of the milling every half hour, for a total of 2 h. This material is what is denominated as BioC. Afterwards, BioC was dried at 80 °C for the 900 °C BioC or at 60 °C for the 500 °C BioC, for at least 24 h, before the processed blends were extruded.All the blends were weighted and pre-measured, to be hand mixed, according to the composition stipulated in The resulting samples, after being conditioned for at least 40 h, were tested for their flexural and tensile properties utilizing a universal testing machine 3382 , under the ASTM protocols D638-14 and D790-15, respectively. The tensile crosshead speed was 5 mm/min for all composites, while the flexural speed was 14 mm/min. The impact energy tests were run under ASTM D256, using notched samples, with an Izod pendulum, a model Zwick/Roell Impact tester . In all these studies, a total of 5 samples were utilized. Density was measured using an electronic Densimeter MDS-3000 , taking the average of two samples.For HDT, the machine DMA Q500  was used, with each sample being tested with a force calculated to exert 0.455 MPa of stress unto the sample, using the average of two samples, heating the samples at a rate of 2 °C/min, until a 0.22% strain was measured by the machine. The HDT measurement was taken at 0.2% strain.As these composites are novel, all processing conditions and parameters were kept the same, to measure the influence that each of the two-level factors had on the mechanical and thermal properties of the composites, without any interference from the processing conditions. This also helps avoid doubling the required number of composites, to keep a full factorial design, if a single of the processing conditions was varied.p ≤ 0.05 was used, that is, the alternative hypothesis cannot be rejected if this value is equal or less than 0.05. Each measurement of a composite was treated as a block, to increase the statistical power of the analysis. A linear model with all their factors and interactions was used initially for all the measured mechanical properties. Interaction plots were also calculated to see the effect of each factor on each property, as well as residual plots to check the normal distribution of the residuals versus the fitted values. The response variables analyzed were the tensile strength and modulus, the elongation at break, the flexural modulus, the impact energy, the HDT and the density of the composites.The program Minitab 17 was employed, utilizing ANOVA and interaction plots. ANOVA allows us to test multiple hypotheses for each property, in which the null hypothesis  is tested against the alternative hypothesis . A typical significance value of A central composite design could have been used to further estimate the optimum conditions if a nonlinear relationship is detected during the full factorial analysis. However, the required axial points could point to regions that would be outside the material space, such as using 0% content of BioC with two different temperatures. Box–Behnken designs can also be used for nonlinear relationships, which again, were not detected in the data considered for this paper. They also require three levels per factor, which would significantly increase the number of runs, leading to a fractional factorial design, with the disadvantage of confusing higher-order interactions.Of all the mechanical properties evaluated for the material space composed by wt% of PPA and BioC, the temperature at which BioC was pyrolyzed and the presence or absence of 0.5 phr of CE, only the tensile and flexural modulus as well as the HDT and density were affected in a statistically significant manner by the chosen factors. The linear model was considered adequate for all the response variables, despite the non-normal distribution of the residuals in the HDT and density models. Applying a Box–Cox transformation to HDT yielded an untransformed model. In the case of density, though it suggested transforming the linear model by substituting the response variable to the fifth power of the same variable, there was not enough of an increase in the explanation power for which to prefer it over the linear model.The only main factor that was considered statistically significant in all the mechanical characteristics was the content of BioC, as each response variable had its own set of significant factors and significant interactions. The main interaction that was statistically significant in all the composite characteristics was the two-way interaction between the content of PPA and the content of BioC.3 and an HDT of 179 °C, at 0.455 MPa at a 0.2% strain.An optimized composite was predicted by giving equal weight and importance to all factors, with the composition of 80 (PPA:PA410 (25:75)) wt% composite, with 20 wt% of a BioC pyrolyzed at 823 °C, and no CE, with a predicted flexural modulus of 3.25 GPa, a tensile modulus of 4.01 GPa, a density of 1.11 g/cmThe material space considered in this paper shows great potential for increasing the sustainability of pieces that require the use of a high temperature polymer like PPA, without compromising its mechanical properties, or increasing the density in a significant manner.