Poxviridae family and mainly infects hatchling and juvenile Nile crocodiles. Most poxviruses encode inhibitors of the host antiviral protein kinase R (PKR), which is activated by viral double-stranded (ds) RNA formed during virus replication, resulting in the phosphorylation of eIF2α and the subsequent shutdown of general mRNA translation. Because CRV lacks orthologs of known poxviral PKR inhibitors, we experimentally characterized one candidate (CRV157), which contains a predicted dsRNA-binding domain. Bioinformatic analyses indicated that CRV157 evolved independently from other poxvirus PKR inhibitors. CRV157 bound to dsRNA, co-localized with PKR in the cytosol, and inhibited PKR from various species. To analyze whether CRV157 could inhibit PKR in the context of a poxvirus infection, we constructed recombinant vaccinia virus strains that contain either CRV157, or a mutant CRV157 deficient in dsRNA binding in a strain that lacks PKR inhibitors. The presence of wild-type CRV157 rescued vaccinia virus replication, while the CRV157 mutant did not. The ability of CRV157 to inhibit PKR correlated with virus replication and eIF2α phosphorylation. The independent evolution of CRV157 demonstrates that poxvirus PKR inhibitors evolved from a diverse set of ancestral genes in an example of convergent evolution.Crocodilepox virus (CRV) belongs to the  Poxviridae family are large double-stranded DNA viruses, which exclusively replicate in the host cytoplasm. The genomes of poxviruses range between 134 kb and 360 kb, and encode approximately 200 genes . R. Rkd celytoplasm B.The best test to assess whether CRV157 is a biologically meaningful PKR inhibitor is to insert it into a VACV strain that lacks its PKR inhibitors and determine whether it can rescue virus replication. We therefore inserted either CRV157, CRV157-K48A/K49A, or VACV E3L (as a positive control) into the replication-incompetent VACV strain VC-R4, which lacks both E3L and K3L. All of the transgenes were integrated into VC-R4 by a method that allows the seamless integration of transgenes into the E3L locus, and is driven by the same endogenous E3L promoter . To compSince eIF2α phosphorylation more directly demonstrates the effects of PKR inhibitors, we also monitored eIF2α phosphorylation levels in virus-infected RK13 cells by Western blot analysis B. To anaCrocodylidpoxvirus genus [Poxviruses infecting crocodilians pose threats for both farm-raised and wild populations ,14,16. Tus genus ,11. In-dus genus . To the Streptomyces sp. RNase III indicates that it might have been horizontally transferred from an unsampled bacterium. However, the currently available sequences do not allow us to infer the origin of CRV157 with high confidence. While most poxvirus proteins show higher sequence identities with eukaryotic proteins in bioinformatic analyses, some poxvirus proteins appear to be more similar to bacterial proteins, indicating possible transfer events between bacteria and viruses [Together with an earlier study , all the viruses . Indeed, viruses . One of  viruses .Although CRV157 evidently contains a dsRBD, as previously reported , it was An interesting observation from the LBR assay was that the individual tested PKRs appeared to exhibit differential sensitivity to both CRV157 and E3, with human PKR being inhibited the least and chicken PKR being inhibited the most strongly, indicating that some PKRs might be more sensitive to viral dsRNA binding proteins than others. Notably, expression of the dsRBD of CRV157 alone (CRV157 del C) did not inhibit PKR as efficiently as full length CRV157, indicating that the uncharacterized C-terminal domain of CRV157 might be also involved in PKR inhibition. This is reminiscent of the situation found in VACV E3 and the variola virus E3 homolog, for which the N-terminal Zα domain was shown to be important for PKR inhibition in addition to the C-terminal dsRBD in yeast assays ,67. In tPoxviruses have evolved multiple inhibitors of antiviral host proteins, often targeting various steps in antiviral pathways . The criBecause of the negative impact of crocodilepox viruses on crocodile farming, an important goal would be to develop a vaccine for this industry. Since vaccinia virus with targeted deletion in the PKR inhibitor E3L has been shown to protect mice and rabbits from lethal challenges from ectromelia virus and rabbitpox virus, respectively ,72, CRV


Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated viruses , Grapevine virus A (GVA), Grapevine rupestris stem pitting-associated viruses (GRSPaV), and Grapevine red blotch virus (GRBV). Sixty percent of the analyzed samples showed infection with some of the analyzed viruses, except GRBV. GLRaV-3 and GFLV were the most frequent viruses, present in 34% and 21% of the positive samples, respectively. This study represents the first survey report of the presence of grapevine viruses in the region of the Calchaquíes Valleys and contributes to the knowledge to maintain the sanitary status of commercial vineyards in Argentina.This study aimed to survey the occurrence of eight grapevine viruses in commercial vineyards located in the Calchaquíes Valleys in the northwest region of Argentina. A total of 103 samples of mature canes of vines showing either none or some viral-like symptoms were randomly collected. The samples were tested by RT-PCR/PCR-based assays for the screening of the following viruses:  Vitis vinifera) is the main fruit crop in Argentina. According to the International Organization of Vine and Wine [Grapevine  and small artisanal producers (1767%). The trend is that the cultivated area, as well as the expansion and establishment of vineyards, will continue to increase. Most of the planting materials (either certified virus-free or not) used to establish vineyards in the Calchaquíes Valleys are imported from nurseries located in the provinces of Mendoza and San Juan, in the Cuyo region of Argentina, with a smaller proportion coming from foreign nurseries or being generated by the producer themselves. Thus, the competitiveness of the Calchaquíes Valleys wine sector depends, in part, on the performance of the planted vines, for which it is necessary to be certain of their quality and health aspects.Grapevine cultivars are commonly affected by a wide range of biotic factors, among which viruses are considered the most threatening due to the lack of therapeutic methods. More than 80 distinct grapevine viruses have been identified worldwide . All theThe occurrence of Grapevine leafroll-associated virus (GLRaV)-1, GLRaV-2, GLRaV-3, GLRaV-4, Grapevine fanleaf virus (GFLV), Grapevine virus A (GVA), Grapevine rupestris stem pitting-associated virus (GRSPaV), and Grapevine red blotch virus (GRBV) has been identified and reported in vineyards in the province of Mendoza, with yield losses in most of the cases ,8,9,10. Due to the variable symptomatology observed between and within cultivars, which hinders the visual diagnosis, molecular diagnosis becomes necessary for accurate identification of the etiology of viral diseases, as well as for their subsequent control and management ,14. TherOur results demonstrated that vineyards in the Calchaquíes Valleys are infected by, at least, seven of the most common grapevine viruses in the Cuyo region and worldwide  as GRBV was not detected in this study. From the 103 randomly selected samples, 61 (59%) tested positive for at least one virus . Single P. ficus) observed only in this locality , possibly due to the presence of mealybugs , Cachi (22%), Tafí del Valle (14%), and Molinos (4%) . ConsideXiphinema index, in the soils of the Calchaquíes Valleys and to determine whether it is contributing to GFLV spread.GFLV was the second most frequent virus, present in 22 samples (21%), half of them in mixed infections with other viruses detected. The localities with the highest prevalence of GFLV were Cafayate and Tafí del Valle (27%), followed by San Carlos (20%), Cachi (19%), and Molinos (15%). GFLV is one of the most widespread and damaging viruses worldwide . These rAlthough GRBV was not detected in the vine samples studied, the absence of this virus in the region cannot be confirmed with certainty, considering it has been previously reported in the country, in the province of Mendoza, by Luna and colleagues .Forty-two of the grapevines studied (41%) were negative for the eight viruses tested. This suggests that the symptoms observed in these plants could be due to other viruses not included in the present study, could have a fungal or bacterial origin, or even abiotic disorders.This survey confirmed the presence of several viral diseases, known to cause major economic losses worldwide, in the grapevines cultivars of the Calchaquíes Valleys. Taking into account that there is no cure for vines with virus infections, there is a need of proper early diagnosis and pathogen identification in the region, especially in propagation planting material, to prevent and/or manage viral diseases ,20. MoreThis study represents the first report of the presence of grapevine viruses in the region of the Calchaquíes Valleys and contributes to the knowledge of the sanitary status in commercial vineyards in Argentina, along with previous reports of viral diseases carried out in the Cuyo region ,8,10,23.P. ficus) was also recorded at the end of summer and the beginning of autumn . All plant samples used in this study were collected with authorization of the vineyard owners. The vineyards’ names and geographical locations are kept confidential to protect the anonymity of the growers who participated in the study.The survey was carried out in the grapevine-growing region of Calchaquíes Valleys, in northwestern Argentina, in the localities of Cachi, Molinos, San Carlos, and Cafayate in Salta province, and Tafí del Valle in Tucumán province, throughout the 2019 to 2020 seasons . Vines eTM  and visualized under UV light. First-strand cDNA was synthesized as described by Gambino and Gribaudo [2, and 0.5 U Taq DNA polymerase . The cycling conditions consisted of initial denaturation at 94 °C for 4 min, followed by 35 cycles at 94 °C for 30 s, 50 °C for 60 s, and 72 °C for 90 s and final extension at 72 °C for 5 min. Reaction products were resolved by electrophoresis on 1.5% agarose gels buffered in TAE 1X  and visualized under UV light after staining with GelRedTM . Positive DNA samples for each virus were included in the PCR reactions as controls.RNA was extracted following the rapid CTAB-based procedure described by Gambino and colleagues . RNA intGribaudo , and theGribaudo ,27,28,29This is the first study to identify viruses that affect vines in the Calchaquíes Valleys, the second largest wine region of Argentina. The results found warn of problems in the propagation material used in this region and the need for differentiated management strategies in localities with and without mealybugs. It is imperative to carry out more in-depth studies on the viruses that affect the vine and its vectors in the area.


It is highly desirable to develop a safe, highly efficient, and biodegradable drug carrier with an enhanced drug transport efficiency. Cellulose nanofibrils (CNF) and chitosan (CS) composite hydrogels are promising candidate carriers with biological compatibility and non-cytotoxicity. Herein, the CNF/CS composite beads were prepared by dissolving cellulose and CS in LiBr molten salt hydrate and regenerating in ethanol. This preparation method is facile and efficient, and the obtained porous CNF/CS beads with the weight ratio of 8:2 exhibited a large specific surface area, uniform micro-nano-sized pores, strong mechanical property, and water absorption-resistance. Moreover, these beads as drug  carriers showed a higher encapsulation efficiency (47.4%) at the TH concentration of 5 mg/mL in 24 h, and a higher drug loading rate (12.0%) than pure CNF and other CNF/CS beads prepared with different ratios. In addition, the TH releasing behavior of CNF/CS (8:2) beads fitted well into the zero-order, first-order, and Higuchi models under an acid condition, indicating that the drug release of these pH-sensitive beads was mainly affected by drug concentration under an acid condition. Therefore, these CNF/CS beads have great potential to be used as drug carriers for medical applications. Cellulose is a β-1,4 linked polysaccharide with good biodegradability and low production price and is widely used in the fields of packaging, papermaking, textiles , etc. OvCommonly, CNF with a large aspect ratio and specific surface area is prepared using chemical/physical treatment methods from lignocellulose, such as 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation, acid hydrolysis, mechanical process . In our However, there is no report on the fabrication of drug carrier with this regenerated CNF from MSH solution. In addition, the drug loading efficiency of pure CNF (TEMPO method) needs to be improved with the control of burst release and more sustained release of drugs. Therefore, in this work, the CNF/CS composite beads were fabricated using lithium bromide trihydrate (LBTH). Then, the structure and properties of the resultant beads were characterized, and the drug loading and releasing performance of the beads as drug carriers were analyzed. Also, tetracycline hydrochloride (TH) was used as the drug model due to its broad-spectrum antibiotic performance and low toxicity. This work will provide valuable fundamental knowledge for the simple preparation of the CNF-based beads for potential use in drug carrier applications.Microcrystalline cellulose (MCC) and lithium bromide  were purchased from Aladdin Reagents . Chitosan , and anhydrous ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. In addition, tetracycline hydrochloride (TH) and solutions of Phosphate Buffer Saline (PBS buffer) with pH of 5.0 and 7.0 were bought from Macklin Reagents (Shanghai) and Tianjin Berens Biotechnology Co., Ltd., respectively. All reagents were used without any purification. 2O), was prepared by mixing lithium bromide and deionized water with the molar ratio of 1:3. Then, the molten salt solution and CS were added to a round flask, and the mixture was heated at 140 °C for 10 min in an oil bath with a stirring rate of 500 rpm. Afterward, MCC was added and heated for another 10 min to obtain a yellow mixture solution, and then MCC/CS beads were regenerated through dripping the solution into an anhydrous ethanol bath using a pipette. Here, the total amount of MCC and CS in molten salt solution was 1 wt.%, and the mass ratios of MCC to CS were 9:1, 8:2, 7:3, 6:4, and 5:5, respectively. Finally, the resultant gel beads were immersed and rinsed with running deionized water for 4 days until the conductivity of the solution containing the beads was less than 20 μs/cm. The rinsed beads were directly used as drug carriers of TH. In addition, a part of gel beads was freeze-dried using a lyophilizer  at −80 °C for further characterization. The technical route of this work is shown in The CNF/CS beads were prepared according to the previous report . EspeciaIn the absence of CS, this process would regenerate MCC with the size of <100 nm , thus th−1. The crystal structure of raw materials and the resultant beads was tested by an X-ray diffractometer  in the scattering angle (2θ) range of 10 and 40°. The Ni-filtered Cu Ka radiation was generated at 80 mA and 40 kV. The microstructure of the resultant beads was observed by a scanning electron microscope  with an accelerating voltage of 100 kV. All samples were coated with gold under vacuum before observation. The diameter of CNF and CNF/CS nanofibers was analyzed using a software of Nano Measurer 1.2. The data was the average of twenty tests. Brunauer-Emmett-Teller (BET) specific surface area and pore volume of CNF and CNF/CS beads were determined using a surface area and pore size analyzer . Total pore volume for pores was estimated from a single point on adsorption isotherm at P/Po = 0.99, and pore size was measured using the BJH method. The compressive strength and modulus of the beads were investigated using an electronic universal material testing machine . Here, all CNF and CNF/CS beads for characterization were freeze-dried samples. The Fourier transform infrared spectroscopy-Attenuated Total Reflectance (FTIR-ATR) spectra of raw materials  and the resultant CNF and CNF/CS beads (8:2) were recorded using a spectrometer  in the wavenumber range of 4000–700 cmdM and hM are the mass of freeze-dried beads and hydrated beads, respectively, rM is the remaining mass of the hydrated beads after being immersed in PBS buffer.Freeze-dried CNF beads and CNF/CS beads were weighed and immersed in deionized water for 24 h, and then the free water on the surface was carefully removed with filter paper. The swelling ratio of bead samples at water saturation was calculated based on the Equation (1). In addition, the obtained CNF or CNF/CS gel beads were immersed in PBS buffer (pH = 5 or 7) for a certain time, and the mass loss ratio of the beads was calculated according to the Equation (2).Tetracycline hydrochloride (TH) was dissolved in 20 mL of deionized water under ultrasonic conditions to obtain TH solutions with different concentrations . Subsequently, CNF or CNF/CS composite beads were soaked in 20 mL of TH solution for a certain time . After that, the TH-loaded beads were separated through filtration, and then oven-dried at 50 °C. m and lm (mg) are the mass of beads and loaded drug (TH), respectively, and tm (mg) is the total mass of TH in the solution.As is known, the maximum absorption wavelength of TH is 357 nm. The absorbance of TH solution with different concentrations was tested using a UV spectrophotometer , and then the standard concentration-absorbance curve of TH solution was established. Subsequently, the concentration of TH solution after drug loading was measured by testing the absorbance of the supernatant, and the difference in the concentrations of TH before and after treating with the beads will be loaded drug. The loading ratio and encapsulation efficiency of the beads were calculated based on the Equations (3) and (4), respectively.m is the mass of loaded TH of beads, nC and Cn−1 (μg/mL) are the concentrations after TH releasing for n and n − 1 times, respectively. The TH-loaded beads were placed in conical flasks and 100 mL of PBS buffer with pH of 5.0 or 7.0 were added. The flasks were incubated in a water bath with stirring speed of 50 rpm at 37 °C. After a certain time, 1 mL liquid was taken out from the flasks and the same amount of PBS buffer was supplemented in the flasks to keep the total volume of the solution unchanged. The released TH amount from beads was calculated using a standard concentration-absorbance curve, and the TH released curves of the beads were obtained. All experiments were independently repeated at least three times. The cumulative release amount of TH was calculated following the Equation (5).t and the total cumulative released TH amount, respectively, t, and K0, K1, and K2 are the characteristic constants of the Equations (6)–(8), respectively.To evaluate the mechanism of TH released from the regenerated CNF or CNF/CS beads, the released data at different pH values was fitted into three commonly-used kinetic models of zero-order, first-order, and Higuchi diffusion models, respectively. The three models were described as Equations (6)–(8).2O solution, and then regenerated in anhydrous ethanol to obtain the CNF/CS composite beads. The resultant beads were used as drug carriers for TH via physical adsorption [As mentioned, MCC and CS were dissolved in LiBr·3Hsorption ,7,8, and2/g) and pore volume (0.0541 cm3/g) of CNF/CS beads (8:2) were larger, and its corresponding pore size (12.16 nm) was smaller than other beads  in the spectra of the regenerated CNF bead and CNF/CS composite beads, which is caused by the reconstruction and strengthening of hydrogen bonds between the regenerated cellulose and CS [−1 and 1592 cm−1 are attributed to amide I and amide II peaks [−1 in the spectrum of CNF/CS beads. In addition, the peak around 2906 cm−1 assigns to the stretching vibration of the saturated C-H bond, and the peak at 1052 cm−1 assigns to the primary hydroxyl group of the pyranoid ring in all spectra [As shown in spectrum . Howevere and CS . FurtherII peaks , respect spectra . TherefoFurthermore, MCC exhibits typical cellulose I crystal structure with the main characteristic diffraction peaks of 14.6°, 16.5° and 22.6° at 2θ b, corresThe swelling properties of the freeze-dried CNF/CS beads in water and the degradation properties of CNF/CS beads in PBS buffer are shown in In addition, the physical strength of the beads is a key factor to influence their application performance. As shown in Therefore, owing to the excellent stability under neutral conditions and degradability under acid conditions, the robust CNF/CS beads (8:2) have great potential to be used as a drug delivery carrier as it will be degraded in the gastrointestinal tract in the presence of gastric acid for the purpose of slow-releasing of drugs.Here, TH as a broad-spectrum antibiotic with a characteristic absorption peak at 360 nm was employed to be a drug model in vitro experiment. According to the standard concentration curve of TH a. It indAlso, the release amount of CNF/CS beads (8:2) in the first 1 h of releasing under acid conditions (14.5%) was lower than other reported TH carriers, such as polydopamine/CNF hydrogel (20%) , collage2) indicated how good the model fitting was. Results showed that the R2 of all models was over 98% under acid conditions, and the R2 of first-order and Higuchi models (>98%) was higher than that of a zero-order model (95%) under neutral conditions. The results indicated that drug release curves of the CNF/CS beads (8:2) fitted all models well under acid condition, while fitting first-order and Higuchi models under neutral conditions. Therefore, the drug release of the CNF/CS beads with pH sensitivity was affected by drug concentration and mass of beads.In addition, three release models of zero-order model, first-order model, and Higuchi model were used to fit the release curves of the CNF/CS composite beads (8:2) for further investigating the release mechanism a–c, respIn this work, the CNF/CS composite beads were prepared using a facile dissolution/regeneration method, and LiBr molten salt hydrate was employed as a high-efficiency solvent of MCC and CS. When the weight ratio of MCC to CS was 8:2, the obtained CNF/CS beads exhibited a more uniform porous structure, larger specific surface area, better shape stability and stronger mechanical properties due to the synergistic action of hydrogen bonds between CNF and CS, which was beneficial for the loading and slow releasing of the drug (TH). Moreover, the drug loading and releasing investigation of the CNF/CS beads (8:2) showed higher drug encapsulation and loading efficiency  in comparison with other beads. The total drug-releasing amount of the CNF/CS (8:2) was higher than 88% without burst release at pH 5. In addition, the releasing curves of the drug-loaded CNF/CS beads (8:2) fitted very well into the zero-order, first-order, and Higuchi models under an acid condition. Therefore, the as-prepared CNF/CS composite beads have the potential to be used for sustained drug delivery in medical applications.


Arabidopsis thaliana Co-1 ecotype. Systemic necrosis reduced virus fitness by shortening the infectious period and limiting virus multiplication; thus, this phenotype could be adaptive for the plant population as a defense against CMV. However, the low frequency (less than 1%) of this phenotype in A. thaliana wild populations argues against this hypothesis. These results expand current knowledge on the resistance mechanisms to virus infections associated with ETI in plants.Effector-triggered immunity (ETI) is one of the most studied mechanisms of plant resistance to viruses. During ETI, viral proteins are recognized by specific plant R proteins, which most often trigger a hypersensitive response (HR) involving programmed cell death (PCD) and a restriction of infection in the initially infected sites. However, in some plant–virus interactions, ETI leads to a response in which PCD and virus multiplication are not restricted to the entry sites and spread throughout the plant, leading to systemic necrosis. The host and virus genetic determinants, and the consequences of this response in plant–virus coevolution, are still poorly understood. Here, we identified an allelic version of RCY1—an R protein—as the host genetic determinant of broad-spectrum systemic necrosis induced by cucumber mosaic virus (CMV) infection in the  Plants are recurrently challenged by parasites in the wild and agroecosystems . BecauseR genes encodes a conserved nucleotide binding site (NBS), a leucine-rich repeat domain (LRR) . The sequence of the RCY1 version of Co-1 has been deposited in GenBank under Acc. No. OP991902.To obtain the complete sequence of the t 150 nt . PCR proscle 3.8 . Nucleote length  using pAVirus multiplication and the kinetics of systemic necrosis were normally distributed, and variances were homogeneous according to Kolmogorov–Smirnov and Levene’s tests, respectively. Therefore, they were fitted to a normal distribution, and differences between plant ecotypes were analyzed by general linear models (GLMs) considering the Arabidopsis ecotype or the virus isolate as fixed factors. The segregation of the CMV-induced systemic necrosis in F2 plants derived from the Co-1 x Ler crossing was analyzed using Fisher exact tests . StatistWe have reported that systemic necrosis was induced in Arabidopsis Co-1 ecotype after infection by CMV isolates Fny-CMV and De72-CMV in subgroup IA, but not by isolate LS-CMV in subgroup II . Thus, pPlants infected by Fny-CMV or by pseudorecombinants F1L2F3, L1L2F3 and L1F2F3 started to develop systemic necrosis two weeks after inoculation, which led to plant death a week later . In contF1,17 = 0.03, p = 0.857) and was much higher for the latter isolate afterward  , and the multiplication was much higher for LS-CMV than for Fny-CMV from that point to the end of the monitored period . Interestingly, the peak of Fny-CMV multiplication occurred at 18 dpi, whereas that of LS-CMV was observed at 9 dpi . Moreover, Co-1 was included in the mentioned set of 18 ecotypes, and the reported virus accumulation and ours are in the same range  . The max × 10−4) A. Similaat 9 dpi B. The obS-CMV in  and 9.1 Takahashi et al.  describeTo test if this differential response was due to differences in virus determinants, ten Co-1 plants were inoculated with two additional CMV isolates belonging to subgroup IA  and with Y-CMV, and with one subgroup IB isolate, BAR92/1 . Co-1 plN. clevelandii plants, and if an M at position 45 was present in the CP of subgroup I isolates inducing systemic necrosis in this work. For this, the nucleotide sequence of the CP gene of all these isolates, obtained by [Takahashi et al.  identifiained by , was retχ2 = 0.14, p = 0.712), indicating its control by a single dominant gene.As a first step in the analysis of Co-1 determinants of the systemic necrosis reaction, the inheritance of this trait was determined. To do so, we inoculated Fny-CMV in 174 plants of the F2 derived from a Co-1 x Ler crossing. All inoculated plants were infected, and systemic necrosis developed in 128 (73.56%), while the infection induced leaf curl and lamina reduction in 46 (26.44%). Thus, the systemic necrosis phenotype had a 3:1 segregation , with an average of 5.66 and 5.60 days for F2 and Co-1 plants, respectively. However, it should be noted that the span of days to complete systemic necrosis was narrower for Co-1 plants (3 to 8 days) than for F2 plants (2–10 days)  B.RCY1. Given that Fny-CMV infection induced the formation of NLLs well before the beginning of the systemic necrosis, that this trait was determined by a single dominant gene and that recognition involved the same genomic segment of CMV [Takahashi et al.  describet of CMV , the potRCY1 allele and of the Ler RPP8 allele was performed with primers that amplified a 343 nt (nucleotides 5044 to 5387) fragment for RCY1 and a fragment of 382 nt (from nucleotide 4151 to nucleotide 4533) for RPP8, allowing the identification of the two alleles both in homozygosity and in heterozygosity , which flank RCY1 at 6 cM at each side, with the development of systemic necrosis upon Fny-CMV infection was analyzed. The primers used for nga129 yielded amplicons of 179 nucleotides in Ler and of 170 nucleotides in Co-1, and those for CIW9 yielded amplicons of 145 nt in Ler and of 170 nt in Co-1. The difference in the amplicon size allowed identifying the two alleles both in homozygosity and in heterozygosity , indicating that they are at the same distance from the gene that controls the systemic necrosis  (Since t of C24 . Thirty-in (LRR) .To analyze the frequency of determinants of systemic necrosis in reaction to CMV infection, seven individuals of 100 Arabidopsis ecotypes from the Iberian Peninsula were inoculated with Fny-CMV. All plants developed typical symptoms of virus infection, including different degrees of leaf curl and lamina reduction in the rosettes  and stunResistance, together with tolerance , is one Our work shows that systemic necrosis in Co-1 is triggered by the recognition of a protein encoded in RNA3 of subgroup I of CMV isolates, either the movement protein or the CP. Recognition results in an HR response that fails to localize the necrosis to the initial infection foci but propagates systemically until the infected plant dies. Interestingly, the onset of systemic necrosis, at 14 dpi, is much delayed compared to the appearance of NLLs in inoculated leaves at 6 dpi, which can be explained if (i) the virus systemic spread is delayed during necrosis at inoculated leaves, later triggering necrosis in newly invaded organs, or (ii) the systemic necrosis is activated by a long-distance signal that is produced in inoculated leaves. In this regard, failure in the restriction of NLL resulting in virus-induced systemic necrosis has been associated with changes in the levels of salicylic acid (SA) , which aRCY1 and RNA3 as the host and virus determinants of systemic necrosis in Co-1. These are also the determinants of HR in ecotype C24 upon Y-CMV infection [RCY1 sequence of Co1 and C-24, particularly in the LRR domain, rather than by differences in previously described determinants of systemic necrosis between the CMV isolates considered in our work. This conclusion is consistent with point mutations in the LRR domain of C24 RCY1 determining a systemic necrosis phenotype in response to Y-CMV infection [We identified nfection ,34,50. Dnfection . Interesnfection ,55. We mRCY1-controlled systemic necrosis, and the authors of [TuNI gene that determines systemic necrosis in response to infection by turnip mosaic virus (TuMV), where virus accumulation was quantified two weeks after inoculation [Regardless of the mechanisms involved, our results suggest that systemic necrosis is a resistance reaction, as it reduces virus accumulation. In addition to the results presented in thors of , who repculation .Potato virus Y–tobacco, Soybean mosaic virus–soybean and Plantago asiatica mosaic virus–N. benthamiana interactions [As a resistance reaction, systemic necrosis reduces subgroup I CMV titer in systemically infected leaves, which will translate into a reduction in virus transmission: it is well established that the level of virus multiplication correlates positively both with aphid ,56 and wractions ,23,58,59ractions  showed tractions , which aractions . Accordiractions . A detaiIn summary, the present study, based on the characterization of the systemic necrosis phenotype of a wild ecotype of Arabidopsis in response to an ample spectrum of CMV isolates, contributes to understanding the underlying mechanisms and shows that systemic necrosis is a resistance reaction. However, the benefits of systemic necrosis for Arabidopsis fitness under CMV infection do not seem to suffice for this phenotype being frequent in wild populations of this host, which calls into question its adaptive value in this plant–virus system.


The growth of poultry farming has enabled higher spread of infectious diseases and their pathogens among different kinds of birds, such as avian infectious bronchitis virus (IBV) and avian influenza virus (AIV). IBV and AIV are a potential source of poultry mortality and economic losses. Furthermore, some pathogens have the ability to cause zoonotic diseases and impart human health problems. Antiviral treatments that are used often lead to virus resistance along with the problems of side effects, recurrence, and latency of viruses. Though target hosts are being vaccinated, the constant emergence and re-emergence of strains of these viruses cause disease outbreaks. The pharmaceutical industry is gradually focusing on plant extracts to develop novel herbal drugs to have proper antiviral capabilities. Natural therapeutic agents developed from herbs, essential oils (EO), and distillation processes deliver a rich source of amalgams to discover and produce new antiviral drugs. The mechanisms involved have elaborated how these natural therapeutics agents play a major role during virus entry and replication in the host and cause inhibition of viral pathogenesis. Nanotechnology is one of the advanced techniques that can be very useful in diagnosing and controlling infectious diseases in poultry. In general, this review covers the issue of the poultry industry situation, current infectious diseases, mainly IB and AI control measures and, in addition, the setup of novel therapeutics using plant extracts and the use of nanotechnology information that may help to control these diseases. Gammacoronavirus, is one such kind of virus. In 1931, IBV was the first coronavirus identified in poultry and was considered a very vital pathogenic virus in livestock  is another very important virus that belongs to the family industry , 16. Thiindustry . In addiindustry , 19. Theindustry . Based oindustry , 22. Theindustry . Thus, iindustry . For insindustry . Additioindustry . Furtherindustry .in vivo intrinsically instable along with multiple boosters. Hence, the development of a novel vaccine that can perform as an immunogen along with adjuvants to produce protection against the pathogen with enhanced immune response , quercetin, pseudohypericin, and quercitrin, were assessed as antiviral against IBV. It was proved that a reduction in mRNA expression of pro-inflammatory cytokines [interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)] via the nuclear factor kappa B (NF-κB) signaling pathway and upregulation of type I interferon via the MDA5 signaling pathway  have been found to be a very effective therapeutic agent against corona viruses including COVID-19 as far as the immune system is concerned , 71−9 m The antiviral effects of G-Ag nanocomposites against IBV and Feline CoV  were invSeveral uses of full NP based on magnetic and gold quantum dots (QDs) for virus detection and tracing have been described . Ahmed eglycyrrhizin diammonium (GD) and lithium chloride (LiCl) conducted by Li et al.  has an auspicious role of novel anti-influenza. Choi et al.   .in vitro, at least partly by activating the expression of the Nrf2/HO-1 pathway  of influenza viruses. During in vitro studies, they elaborated on the mechanism of Oximacro that prevented virus entry and attachment, leading to virucidal activities. In the past, the immune response, mainly humoral, in chickens during the inactivation of the vaccine for AIV has been induced by the addition of immunoadjuvants and extracts of plants in feed. Similarly, when the stems and leaves of Ginseng saponins were added to the drinking water, the humoral immune response increased to a significant level. In another such instance, serum Abs level enhancement was observed significantly when H. perforatum L. was administered orally and in the drinking water  as adjuvant in inactivated vaccines of NDV, AIV, and IBV in chickens was tested by Li et al.  supported this research.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Viruses in the aqueous phase can be easily inactivated in the suspension tests, but once attached to the materials, they can become resistant to disinfectants, and require longer times to be inactivated. This highlights the importance of thorough cleaning with detergent before disinfection, and keeping elevated contact durations of proper disinfectants to reduce viral contamination and decrease infectious diseases incidence in farms. It was also reaffirmed that the suspension and carrier tests are necessary to evaluate disinfectants and thus determine their actual use. Particularly, the mixture of QAC and FdCa(OH)2 was found to exhibit synergistic and broad-spectrum effects compared to their use alone, and is now recommended for use on livestock farms.Livestock farming is affected by the occurrence of infectious diseases, but outbreaks can be prevented by effective cleaning and disinfection along with proper farm management. In the present study, bovine coronavirus (BCoV) and bovine rotavirus A (RVA) were inactivated using food additive-grade calcium hydroxide (FdCa(OH) Disinfection is an important strategy to reduce the amount of pathogens and the risk of contaminating environments, and disinfectants are important tools for enhancement of livestock farm biosecurity ,2,3.In livestock farms, shoe soles and clothing of farm workers can serve as carriers for pathogens. Disinfection of porous and nonporous surfaces is especially important because plastics, rubber, and clothing can be a source of infection for as long as 4 days after the pathogen is attached ,4. In adEnteric viruses constitute an important and also complex community of viruses. Infections with enteric viruses can result in the excretion of large amounts of viral particles in body secretions, particularly in feces; the viral particles are extremely stable in the environment, and are spread through fecal–oral route . Bovine 2) is relatively novel compared to other materials that can inactivate pathogens [2 [2’s alkalinity that destructs cytoplasmic phospholipid membranes, thereby affecting the integrity of the cytoplasmic membrane [2 mixture, it took over 3 min to inactivate pathogens on abiotic carriers  [2 mixture for more than 3 min, since the mixture may inactivate both nonenveloped and enveloped viruses [Food additive grade calcium hydroxide (FdCa(OH)athogens ,13,14. Qathogens . Howeverogens [2 ,17. The r steel) . Therefo viruses .2 mixture until their next use  could prevent transmission of porcine epidemic diarrhea (PED) virus to sentinel pigs as a result of biosecurity procedures . In Septnext use .Virucidal activity should be tested using both suspension and carrier methods, based on established international standardization principles. In the present paper, this pertains to disinfectants that meet the suspension and carrier test standards described in the American Society for Testing and Materials (ASTM) international standards E-1052-20, E1053-20 and E2966-14 (2019) ,23,24 anIn the present experiments, contaminated suspension tests and carrier tests were conducted with 5% fetal bovine serum (FBS) or feces to compare virucidal activities of disinfectants, while simulating dirty conditions. In the carrier tests, rayon sheets were used instead of clothes or coveralls and rubber carriers instead of boots; RVA and BCoV were deposited on those carriers, and then the effects of the disinfectants on the viruses on the carriers were compared by the two following methods: (1) 500 μL of each disinfectant was dropped onto the rayon sheets or the rayon sheets were soaked in a tube containing 400 μL of each disinfectant; (2) 500 μL of each disinfectant was dropped onto the virus on the rubber carrier. g for 15 min to remove cellular debris, and the supernatants were collected, aliquoted and stored at −80 °C until used. The virus stock titers of RVA and BCoV were around 107 plaque forming units (PFU)/mL and around 107 fifty percent tissue culture infectious dose (TCID50)/mL, respectively.The RVA strain RVA/Bovine-tc/JPN/AH1041/2019/G8P , with mo®) was purchased from Scientific Feed Laboratory Co., Ltd.  and diluted 1:500 (QACx500) with redistilled water (dW2), to obtain a final concentration of 200 ppm didecyl-dimethylammonium chloride (DDAC), as recommended by the manufacturer. FdCa(OH)2 powder at pH 13 with an average diameter of 10 μm of powder particles [2 solution or as a QAC and FdCa(OH)2 mixture (Mix500) [The QAC (Rontectarticles  was purc(Mix500) ,17.A blocking solution  was used for stopping virus inactivation reactions, as previously described ,4.Disinfectant procedures were conducted according to ASTM-E1052, E1053 and E2966 standards with some modifications.2, or Mix500 were mixed with 100 μL of RVA or BCoV in a microtube and incubated for 0 s, 5 s or 1 min at room temperature (RT: 25 ± 2 °C). The virus inactivation was immediately stopped by adding 500 μL of the blocking solution [2 was used instead of disinfectants. Then, the remaining virus was titrated. To evaluate the inactivating activity of the test solutions in the presence of organic materials mimicking virus environment on farms, FBS was added to each virus to reach 5%.Four hundred microliters of QACx500, 0.17% FdCa(OH)solution . To asce® 5, Iwatsuki Co. Ltd., Tokyo, Japan) were placed in 1.5 mL microtubes and autoclaved at 121 °C for 15 min. To examine virucidal activity toward viruses on clothes, 100 μL of virus containing 5% FBS was inoculated onto the rayon sheets in microtubes and allowed to dry at RT in a biosafety cabinet for 30 min. After drying, 400 μL of Mix500 or dW2 as positive control was added by complete dipping and incubated for 0, 5, 15 and 30 s, respectively. Following incubation times, the treatment of samples was stopped by adding blocking solution  was used for determining the viral inactivation. Viral inactivation was considered effective when the RF was greater than or equal to 3 log10 .2 solution and Mix500 could inactivate RVA to the effective level within 5 s, even in the presence of 5% FBS. Mix500 inactivated RVA to undetectable levels  within 5 s, while QACx500 could not inactivate RVA to the effective level in 1 min. By contrast, QACx500, 0.17% FdCa(OH)2 solution and Mix500 were found to reduce BCoV virus titer to the effective level within 5 s in the presence of 5% FBS. When the blocking solution was added before the addition of the virus (0 s), the viral titers were similar to the positive control. Thus, the blocking solution evidently stopped the virucidal activity of the tested solutions, when mixed in equal amounts with the reaction solution.As shown in 50/mL; RF > 3.66) within 15 s. When the blocking solution was added before the addition of the virus (0 s), the viral titers were similar to the positive controls.The capacity of Mix500 to inactivate RVA and BCoV in the presence of 5% FBS on rayon sheets through dipping technique is shown in In contrast to the results of the dipping technique, the dropping techniques with Mix500 could not reduce the viral titers on the rayon sheets to the effective level even after 1 h for RVA and 10 min for BCoV, as shown in 2 from the surface of rubber carrier after drying for 1 h was measured as 40% for RVA and 1% for BCoV, respectively  within 10 min. On the other hand, BCoV on the rubber carrier was inactivated to the effective level within 1 min by the drop of Mix500  [In the suspension tests, QACx500 could not inactivate RVA in 1 min. It is indeed reasonable that a non-enveloped virus will not be inactivated with QAC ,33. FdCaus (IBV) , in contus (IBV) . These d2, was selected for the carrier tests.The spectral broadening effect of QAC in aqueous phase was observed toward RVA within 5 s at Mix500. As shown in People and vehicles can be important pathways, mechanically, for the introduction of new diseases into farms. Fomites carried by people  or even the people themselves, through contaminated skin, can spread various pathogens onto farms . CompareGenerally, enteric viruses are highly resistant, especially under environmental conditions. The viruses can survive on the surface of materials, fomites and food for long periods ,36. TherIn the present study, the rubber coupon was used for simulating farm personnel boots, and using the dropping method instead of footbath showed the effect of Mix500 toward RVA and BCoV within 10 and 1 min, respectively as shown in In the presence of organic matter such as litter and feces, disinfectants required more time to inactivate viruses and became less effective, suggesting that a cleaning step is important before disinfection . In our Demonstrating an accurate evaluation system for appraising disinfectants is very critical to prevent misleading results. The data in 2 content of 65–75% for the enforcement of biosecurity measures; however, slaked lime failed to show synergistic effects with QAC toward AIV and NDV at low temperatures [2 seems to be much safer and more effective than slaked lime, since the former has a much smaller particle size (average 10 µm) and is purer (97% Ca(OH)2) than slaked lime (less than 150 µm). FdCa(OH)2 would not damage or bleach boots, clothes and floors. These characteristics are superior to slaked lime or sodium hypochlorite (NaOCl). The findings of the present study can also help farmers to properly apply FdCa(OH)2 with QAC-based disinfectants in appropriate concentrations and exposure times in farms, in order to prevent and control infectious disease outbreaks.It is important to select disinfectants that are effective against a wide spectrum of pathogens under the conditions normally found on livestock farms. Mix500 can be applied as a disinfectant or a microbiocidal agent which can inactivate enveloped and non-enveloped viruses and bacteria in contaminated fomites, especially in mammals and poultry farms ,4,16,17.Routine disinfection in livestock farms is an important hygienic measure to prevent the transmission of pathogens from a previous flock/shed to the next production cycle ,37. It i


Flavivirus genus consists of small, positive-stranded, single-stranded RNA viruses transmitted by arthropods, especially mosquitoes and ticks. The genus contains several globally significant human/animal pathogens, such as Dengue virus, Japanese encephalitis virus, West Nile virus, Zika virus, Yellow fever virus, Tick-borne encephalitis virus, and Tembusu virus. After flavivirus invades, the expression of host miRNA changes, exerting the immune escape mechanism to create an environment conducive to its survival, and the altered miRNA in turn affects the life cycle of the virus. Accumulated evidence suggests that host miRNAs influence flavivirus replication and host–virus interactions through direct binding of viral genomes or through virus-mediated host transcriptome changes. Furthermore, miRNA can also interweave with other non-coding RNAs, such as long non-coding RNA and circular RNA, to form an interaction network to regulate viral replication. A variety of non-coding RNAs produced by the virus itself exert similar function by interacting with cellular RNA and viral RNA. Understanding the interaction sites between non-coding RNA, especially miRNA, and virus/host genes will help us to find targets for antiviral drugs and viral therapy.MicroRNAs (miRNAs) are small non-coding RNA that affect mRNA abundance or translation efficiency by binding to the 3′UTR of the mRNA of the target gene, thereby participating in multiple biological processes, including viral infection.  MicroRNAs (miRNA) is a class of small non-coding RNA, about 20–24 nucleotides in length. miRNA is first transcribed from the genome to form primary miRNA (pri-miRNA), and then cleaved by Drosha enzyme and Dicer enzyme successively to form mature miRNA. One strand of mature miRNA typically degrades mRNA or inhibits translation by binding to the 3′untranslated region (UTR) of the target gene . Not onlFlavivirus genus contains more than 50 arthropod-borne viruses (arboviruses), of which important family members include Dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), Zika virus (ZIKV), Yellow fever virus (YFV), Tick- borne encephalitis virus (TBEV) and Tambusu virus (TMUV). Flavivirus is a positive-sense RNA virus with a full length of about 11 kb. The structure of viral genome contains a single open reading frame (ORF) that encodes multiple proteins, cleaved by host and viral proteases into three structural proteins  and seven non-structural proteins , flanked by a 5′UTR and a 3′UTR, which is conserved throughout the Flavivirus genus  and circular RNA (circRNA), have attracted increasing attention for their important roles in various biological processes such as viral infection. lncRNA/circRNA, as competing endogenous RNA (ceRNA), competitively bind to miRNA through their miRNA recognition element (MRE), thereby regulating the expression level of miRNA target mRNA . Hence, Based on the character that flavivirus completes its life cycle and pathogenicity is highly dependent on the metabolism of living host cells, clarifying the effect of host miRNA as well as other non-coding RNA on host–virus interactions will help us better understand the pathogenesis of flavivirus and novel candidates for the development of therapeutic interventions . In thisFlavivirus genus and has been reported to regulate its own replication by using host miRNA  that cause very similar disease spectrum in humans. It is the tenth leading cause of death and morbidity in developing countries and the leading cause of death among children under 15 years of age in some South-East Asian countries . At presst miRNA .Several independent studies have suggested that miRNAs can complementarily bind to non-coding regions or even coding regions of DENV genome and consequently influencing viral replication, while other research results imply that DENV infection regulates cellular miRNAs expression for their own interests ; Table 1Non-coding regions of flavivirus, the 5′UTR and 3′UTR, have significant impact in translation, replication and assembly of virus. miR-484, miR-744, and miR-133a are downregulated at early stage during DENV infection in Vero cells . MoreoveMore recently, it has been achieved to control the virus tissue tropism by incorporating a specific miRNA recognition element (MRE) from the viral genome into engineered viruses. This has been implemented as a neoteric strategy to exploit secure vaccines against pathogenic viruses . For insAfter DENV infection, miRNAs can regulate series of key genes related to innate immunity. For example, miR-146a-targeted TNFR-associated factor 6 (TRAF6) and miR-155-targeted BTB domain and CNC homolog 1 (BACH1) can promote viral replication by dampening IFN-β . TRAF6, Previous studies have clarified overexpressed miR-30* and miR-424 knockdown IκBα and Siah E3 ubiquitin protein ligase 1 (SIAH1) genes related to inherent immunity. Subsequently, miR-30* mediated NF-κB/IκBα negative feedback loop is disrupted with an excessive activation of NF-κB and increases IFN-β and the downstream ISGs, eventually leading to inhibition of DENV replication . As an EJapanese encephalitis virus is a neurotropic virus that infects the Central Nervous System (CNS) and causes fatal encephalitis. Accumulating evidence points to microglia activation and bystander damage as the main pathogenesis of Japanese encephalitis and its complications . After vmiR-301a , miR-22 Persistently JEV infection increases miR-125b-5p expression and limits viral replication by dampening STAT3, MAP2K7, TRIAP1 production, which genes are associated with apoptosis and cell growth . OverexpDuring JEV infection, host miRNAs can not only regulate JEV replication, but also increase microglial activation and accelerate the production of inflammatory cytokines by influencing important genes in the pro-inflammatory pathway . Highly West Nile virus belongs to the JEV serotype, which maintains in the environment by an avian–mosquito life cycle . The neuin vivo. Upregulation of Hs_154 and subsequent suppression of CTCF and ECOP, two genes related to anti-apoptosis, cause a significant reduction in viral replication through facilitating WNV-dependent induction of apoptosis  and Integrin subunit alpha V (ITGAV), then inhibits ZIKV replication, possibly through activation of innate immune antiviral response and phagosome pathway ; Table 4Moreover, due to the close relationship between ZIKV and mosquitoes, multiple studies reported changes in miRNA expression in mosquitoes after ZIKV infection. in vitro. Bhagat et al. revealed miR-1273g-3p as well as miR-204-3p are upregulated in response to ZIKV envelope E protein, a viral component responsible for attachment and entry into host cell , this cell is the best choice to study the effects of ZIKV infection on neurogenesis ost cell . Furtherost cell . Interesost cell . This maZika virus infection was found to increase the incidence of microcephaly in fetuses born to mothers infected with ZIKV in 2015. Then the association of ZIKV infection and microcephaly was confirmed in a ZIKV-infected mouse model the following year . CurrentFlavivirus genus whose infection is also closely related to cellular miRNA. TMUV is an emerging and re-emerging zoonotic pathogen that adversely affects poultry industry in recent years, especially the duck breeding industry  was proved to be involved in promoting stability and integrity of vascular endothelial barrier during DENV infection by sponging miR-183-5p . Li et aFlavivirus genus and not found in the other two genera (Ae. aegypti cells after DENV-2 infection. As vsRNA5 has the most significant effect on virus replication, it was selected for further study. The results showed that vsRNA5 with characteristics similar to miRNAs with AGO-2 targets the viral genome NS1 and inhibits viral replication. The same research group also utilized bioinformatics analysis to identify and coin a WNV-encoded small regulatory viral RNA, KUN-miR-1, from viral 3′UTR (Ae. aegypti C6/36 cells, which seems to be necessary for efficient WNV replication.sfRNA is a product of incomplete degradation of viral genomic RNA by host cellular 5′-3′ exonuclease XRN-1 . Howevero genera . This noo genera . But theo genera . Only whal 3′UTR . The traTo understand host–virus interactions is critical to antiviral response. Based on miRNAs regulate a variety of intracellular biological pathways, including virus infection, which has been demonstrated in many viruses . Hence, Flavivirus genus members, mainly DENV, JEV, WNV, and ZIKV. Through the classification of regulatory effects, it is found that these miRNAs principally participate in the process of viral replication, neuroinflammation, neurogenesis and so on (In this view, we collected the differentially expressed miRNAs infected by nd so on . Furthernd so on . As a mend so on . Overallnd so on . Based ond so on . These hOther host-derived RNAs, such as lncRNA and circRNA, often regulate target mRNAs by forming a functional grid together with miRNA. During flavivirus infection, lncRNA-ERGAL and cirC_0000220 can regulate the biological processes of infected cells by associating with miR-183-5p and miR-326-3p, respectively . But thiFlavivirus genus members, mainly through miRNA directly binds to the viral genome or vital host genes, although the latter is more widely reported. Understanding host–virus interactions will help us discover promising targets of antiviral drugs and viral therapeutic, which will provide new thinking directions for preventing viral infection.In brief, we discussed the regulatory role of host miRNAs after infection with WC and YP contributed ideas for the review, wrote the manuscript, and produced the figures. AC, MW, ZY, and RJ edited and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


IntroductionIn this study, we aimed to compare the clinical and radiological results of patients who underwent medial retinaculum plication (MRP) combined with lateral retinacular release (LRR) or lateral retinacular lengthening (LRL) with the diagnosis of patellofemoral (PF) instability.MethodsIn our study, we retrospectively analyzed 75 knees of 75 adult patients  who underwent MRP+LRR or MRP+LRL due to PF instability without osseous pathologies. Patients were divided into two groups (MRP+LRR and MRP+LRL) according to the surgical method. The clinical and radiological results of the two groups were compared.ResultsMRP+LRL surgery was performed on 45 knees and MRP+LRR surgery on 30 knees. The mean age was 26.5 (18-43) years. There was no significant difference between the two groups in the change in patellar lateral shift (PLS) (p=0.429) and congruence angle (CA) (p=0.218) values. However, there was a significant difference between the two groups in the change in patellar tilt angle (PTA) (p=0.009) and lateral patellofemoral angle (LPFA) (p<0.001) values. The change in PTA and LPFA values was higher in the MRP+LRL group. There was no significant difference between the two groups in terms of pre-operative and post-operative Lysholm knee scoring scale , Kujala pain scale , and Tegner activity level scale values .ConclusionsMRP+LRR or MRP+LRL provided successful results for correcting the instability in PF instability without osseous pathologies such as patella alta, tibial tubercle-trochlear groove (TT-TG) dysplasia, trochlea dysplasia, genu valgus, and tibial-femoral torsion. While PTA and LPFA values improved more with the MRP-LRL method, clinical results were similar in both methods. The term patellofemoral (PF) instability refers to the abnormal position of the patella in any plane. PF stability depends on the alignment of the lower extremities; the anatomical compatibility of the articular surfaces of the patella and trochlea, which are static stabilizers; and the adequacy of the medial patellofemoral ligament (MPFL) and quadriceps muscle group, which are dynamic stabilizers . The priThe lateral retinaculum is effective in the lateral balance of the patella. Surgeries for the lateral retinaculum (LRR/LRL) aim to regulate the lateral tensile strength balance and the extensor mechanism . LRR wasOur study aimed to compare the radiologic and functional results of the cases where the adolescent development process was completed. We applied MRP+LRR or MRP+LRL due to PF instability and investigated the effect of patella and trochlea morphology on these results.Our study retrospectively evaluated 112 knees of 112 patients who underwent MRP+LRR or MRP+LRL due to PF instability between April 2015 and February 2020 and whose epiphyseal line was closed. Approval for the study was obtained from the local ethics committee with protocol number 367, dated February 22, 2021. Informed consent was obtained from all patients. Inclusion criteria for the study were patients diagnosed with chronic patellar subluxation, with at least three episodes of subluxation, positive J sign, unsuccessful conservative treatment for more than 12 months, and minimum one-year postoperative follow-up. Exclusion criteria included patients with patella alta (Caton-Deschamps index >1.3) with lower extremity rotational deformity, hereditary connective tissue laxity , genu valgus, tibial tuberosity-trochlear grove (TT-TG) distance of more than 20 mm, and those patients who had undergone previous surgery for the knee joint and who developed PF instability after severe knee trauma.Seventy-five knees  of 75 patients who could be followed up postoperatively until a minimum of 12 months were included in the study. Patients were divided into two groups (MRP+LRR and MRP+LRL) according to the surgical method. MRP+LRL surgery was performed on 45 knees and MRP+LRR surgery on 30 knees. Patients were informed about both treatment methods preoperatively. The patient and the orthopedic specialist decided together which method would be applied. Then, the clinical and radiological results of the two groups were compared. In clinical evaluation, pre-operative and post-operative first year "Kujala pain scale," "Tegner activity level," and "Lysholm knee scale" values were used. In the radiological evaluation, standard anteroposterior (AP) and lateral radiographs, merchant radiographs of the knee at 30 degrees of flexion, and CT and MRI of the knee at 15 degrees of flexion were used preoperatively and in the first year postoperatively. Patellar tilt angle (PTA) Figure , LPFA F, patellaThe knees that we performed revision surgery on were accepted as the recurrences of PF instability. A single surgeon performed all surgical procedures.Surgical techniqueFirst, knee arthroscopy was performed from the anterolateral and anteromedial portals and, if necessary, from the superolateral and superomedial portals for the diagnosis and treatment of intra-articular pathologies. The development of PF instability with knee flexion extension, chondral damage in the PF joint (Outerbridge classification was used), and menisci and cruciate ligaments were evaluated with arthroscopy. MRP+LRL and MRP+LRR surgeries were performed if there was no chondral damage in the PF joint or the damage was stage 1-2 according to the Outerbridge classification.In LRL, the lateral retinaculum was reached by applying a parapatellar longitudinal 5-6 cm incision. A vertical incision was made approximately 1 cm lateral to the patella to the superficial longitudinal fibers of the lateral retinaculum. Superficial longitudinal fibers and deep, transverse fibers were separated from this incision site. Next, a vertical incision was made to the deep, transverse fibers anterior to the femoral epicondyle attachment site. The posterior of the superficial fibers and the anterior of the deep fibers were combined with a non-absorbable suture  to lengthen the lateral retinaculum between 1 and 2 cm. In LRR, the vastus lateralis was preserved with a hook-shaped radiofrequency probe under the guidance of arthroscopy, and the lateral retinaculum between the vastus lateralis obliquus and the lateral femoral epicondyle was released full-thickness. Using the radiofrequency probe was an attempt to prevent hematoma formation, the most common complication in the literature.Surgical treatment of MRP began with a medial parapatellar longitudinal 4 cm incision. The VMO and medial retinaculum were separated along the medial border of the patella and transferred laterally with a non-absorbable suture .The tourniquet was opened, and the centralization of the patella in the trochlear groove was checked with suture tension and arthroscopic imaging. Active PF joint adjustment and ligament balance were checked by flexing the knee at 90 degrees several times.Post-surgical follow-up protocolIn both patient groups, mobilization was started in all patients with a hinged knee brace  and a single cane on the first postoperative day and with isometric quadriceps exercises. On the 15th day, the flexion angle was increased to 90 degrees. Light resistance exercises were started, and standing with a single cane was continued. In the first month, the patients began the rehabilitation program with 120 degrees of flexion, standing unsupported, and using a simple knee compression. Patients were allowed to return to normal daily life after two months and to active sports after six months.Statistical analysisFrequency, mean, and SD values were calculated by descriptive statistical methods. A T-test was used to evaluate the differences between the two groups. ANOVA test was used to evaluate the effect of patella type on the results. The analyses considered a two-tailed hypothesis, and differences were accepted as significant if the p-value was 0.05 or less. SPSS 18.0 software for Windows  was used to evaluate statistical analyses.In our study group, 57.33% (n=43) were female and 42.67% (n=32) were male. Thirty knees  treated with MRP+LRR and 45 knees  treated with MRP+LRL were compared. The mean age was 26.5 years (range: 18-43). The mean age in the MRP+LRR group was 32.24 (range: 18-43), and the mean age in the MRP+LRL group was 26.88 (range: 19-38) (p=0.343). The mean follow-up period was 40.65 months (range: 14-70). The mean follow-up time was 41.35 months (range: 16-70) in the MRP+LRR group and 39.80 months (range: 14-68) in the MRP+LRL group (p=0.807).Radiological evaluationThere was a significant difference between the two groups in terms of PTA change before and after surgery (p=0.009) Table . There wWhen the effect of patella morphology on radiological results is evaluated, according to patella morphology, 13.33% (n=10) of knees were Type 1, 33.33% n=25) of knees were Type 2, and 53.33% (n=40) of knees were Type 3. In the MRP+LRL group, 8.88% (n=4) of knees were Type 1, 35.55% (n=16) of knees were Type 2, and 55.55% (n=25) of knees were Type 3. In the MRP+LRR group, 20% (n=6) of knees were Type 1, 30% (n=9) of knees were Type 2, and 50% (n=15) of knees were Type 3. Both groups had no significant difference between patella type and pre-postoperative PLS, LPFA, CA, or PTA values  preoperatively and 74.2 (range: 56-90) postoperatively, and a significant increase was observed (p<0.001). The Kujala pain scale was found to be 46.17 (range: 40-55) preoperatively and 77 (range: 55-90) postoperatively, with a significant increase (p<0.001) observed. Tegner activity level scale was found to be 2.4 (range: 1-3) preoperatively and 5.6 (range: 3-8) postoperatively, with a significant increase (p<0.001) observed.In the MRP+LRL group, the mean Lysholm knee scale was 50.82 (range: 36-58) preoperatively and 78.82 (range: 58-92) postoperatively, and a significant increase was observed (p<0.001). The Kujala pain scale was found to be 46.56 (range: 35-55) preoperatively and 80 (range: 55-90) postoperatively, with a significant increase (p<0.001) observed. Tegner activity level scale was found to be 2.64 (range: 1-3) preoperatively and 6.18 (range: 3-8) postoperatively, with a significant increase (p<0.001) observed.A comparison of MRP+LRR and MRP+LRL groups found no significant difference between the pre-operative (p=0.205) and post-operative (p=0.228) Lysholm knee scales of both groups. There was no significant difference between the pre-operative p=0.393) and post-operative (p=0.596) Kujala pain scales of both groups. There was no significant difference between the pre-operative (p=0.121) and post-operative (p=0.899) Tegner activity level scales of both groups (Table 93 and poComplicationsRevisions were performed on two knees in the MRP+LRR group and on two knees in the MRP+LRL group. There was no significant difference in the recurrence rates of both groups (p=0.157). A revision was performed on three knees because of the increase in PLS and PTA. MRP+LRR was applied to two of these three knees, and MRP+LRL was applied to one of them. Retrospective investigation of these patients found that the TT-TG distance was at the upper limit . In revision, a tibial tuberosity anteromedialization osteotomy (Fulkerson osteotomy) was performed. In the case of the fourth patient, MRP+LRL was applied, and medial subluxation of the patella developed. Arthroscopic medial patellar release was performed in the revision. Hematoma developed in four knees with arthroscopic LRR and in one knee with LRL. No additional surgical procedure was required.Pre-operative and post-operative PTA and LPFA changes were significantly better in knees with MRP+LRL compared to knees with MRP+LRR. There was no significant difference between the two groups in terms of PLS and CA changes before and after surgery. There was no significant correlation between patella and trochlea morphology and changes in PTA, LPFA, PLS and AA in both groups. While there was a significant difference between the pre-operative and post-operative changes in the Kujala pain scale, Lysholm instability scale, and Tegner activity scale in both groups, there was no significant difference between the two groups.In patients with PF instability and without advanced osteochondral damage, treatment first provides neuromuscular control of the knee through conservative methods and physical therapy applications. It has been reported that surgical treatment yields satisfactory results in case persistent pain lasts for more than six months, despite conservative treatment lasting longer than six months . The popA cadaver study in which isolated LRR was performed in PF instability reported that LRR was effective in centralizing the patella by regulating the extensor mechanism and relieving pain associated with lateral hypercompression syndrome . A studyLRR has been used for many years to correct the extensor mechanism, and successful results have been reported. In the surgical technique, it is recommended not to extend the LRR to the proximal of VLO but to apply it from distal to proximal by checking the medial mobilization of the patella. However, it has been reported that LRL prevents medial subluxation of the patella better than LRR and is more successful in centralizing the patella to the trochlear sulcus and providing patellar tracking .Many publications in the literature compare LRR and LRL. A study investigating the approach of experienced surgeons with a special interest in PF instability to LRR reported performing isolated LRR less often than isolated LRL. This was because isolated LRL was suggested to give better results than LRR in reducing lateral PF load and preventing chondral damage in patients with moderate patellar maltracking, lateral retinaculum, and capsule tension . LRR andSatisfactory results have been reported regarding patellar re-luxation in knees with PF instability where open MRP and LRR are applied in combination therapy compared to arthroscopically applied MRP and LRR . HoweverMPFL reconstruction is the current topic of PF instability surgery. With the anatomical presentation of the medial patellar ligament structures more clearly, the tendency to reconstruct the ligaments instead of medial plication has increased . HoweverHan H et al. reported adequate results in their study applying MPFL reconstruction and LRR . HoweverIt has been reported that clinical results will be better when the adjustment angle in young male knees undergoing LRR is normalized . Our stuOur study is different in its investigation of the relationship between patella and trochlea morphology and MRP+LRR and MRP+LRL. Furthermore, few studies exist in the literature comparing the radiological and clinical results of MRP+LRL and MRP+LRR.The change in PTA and LPFA before and after surgery was significantly better in knees with MRP+LRL compared to knees with MRP+LRR. There was no significant difference between the two groups in terms of PLS and CA changes before and after surgery. We think that this difference may be due to the contraction that may develop during the healing of the lateral retinaculum defect with fibrous tissue after release. Both groups had no significant link between patella and trochlea morphology and changes in PTA, LPFA, PLS, and CA. A significant difference existed in the pre-and post-operative changes in the Kujala pain scale, Lysholm instability scale, and Tegner activity scale in both groups. However, there was no significant difference between the two groups.There are some limitations in our study. Initially, the study was retrospective and based on medical records. The last control data of the patients were used. There is also the possibility that additional complications may have developed after these data. Secondly, radiological measurements were made by a single orthopedic specialist. It would be more reliable if it were evaluated by a second person and a radiologist.In conclusion, MRP+LRR or MRP+LRL provided successful results for correcting the instability in PF instability without osseous pathologies such as patella alta, TT-TG dysplasia, trochlea dysplasia, genu valgus, and tibial-femoral torsion. PTA, LPFA, and PLS values improved with both methods, but PTA and LPFA values improved more with the MRP-LRL method. Clinical results  were similar in both methods. There was no difference between the two methods in terms of post-operative complications and the need for revision surgery.


Different genome editing approaches have been used to engineer resistance against plant viruses. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein  systems to create pinpoint genetic mutations have emerged as a powerful tool for molecular engineering of plant immunity and increasing resistance against plant viruses. This review presents (i) recent advances in engineering resistance against plant viruses by CRISPR/Cas and (ii) an overview of the potential host factors as targets for the CRISPR/Cas system-mediated broad-range resistance and immunity. Applications, challenges, and perspectives in enabling the CRISPR/Cas system for crop protection are also outlined. Virgaviridae, Tospoviridae, Geminiviridae, Bromoviridae, and Potyviridae families, such as Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV), Tomato yellow leaf curl virus (TYLCV), Cucumber mosaic virus (CMV), Potato virus Y (PVY), African cassava mosaic virus (ACMV), Plum pox virus (PPV), Brome mosaic virus (BMV), and Bean golden mosaic virus  that recognizes a DNA sequence of the genome. However, some drawbacks make the use of ZFNs and TALENs limited. Because ZFNs require an amino acid sequence to specify the target site, there is a need for multiple designs to recognize different regions in the genome. Likewise, a single TALE motif recognizes one nucleotide, and hence an array of TALENs is required to associate with longer DNA sequences  and transcription activator-like effector nucleases (TALENs) have taken a prominent place as genome editing techniques . ZFNs arequences . Furtherequences .The CRISPR/Cas system is not limited as ZFNs and TALENs. Due to the feasibility of its mechanism, CRISPR/Cas has become an alternative tool for controlling viral infections by directly editing viral genomes or host factors. CRISPR/Cas was first described as an immune system of archaea and bacteria for defense against viruses by specific interaction of short-viral sequences based on complementarity . The sysA recently developed system, the PAMless SpCas9 variant with relaxed nucleotide preference, has overcome these sequence limitations by increasing the number of possible CRISPR/Cas targets . AdvantaNicotiana benthamiana plants expressing Cas9-sgRNA to the targeted bean yellow dwarf virus (BeYDV) genome displayed reduced virus load and symptoms  in cucumber, papaya ringspot mosaic virus-W (PRSV-W) in papaya, and immunity to ipomovirus cucumber vein yellowing virus 4E from Arabidopsis thaliana by CRISPR/Cas9 provided resistance to TuMV /flavone synthase II (FNSII) genes have also been used as targets for resistance to RNA virus of the Potyviridae family. Editing coilin by the CRISPR/Cas9 system increased the resistance of edited potato lines to PVY (Glycine max (Gm)F3H1 and GmF3H2] and flavone synthase II (GmFNSII-1) genes as a metabolic engineering approach that resulted in increased isoflavone content and enhanced resistance of edited soybean plants to soybean mosaic virus  in Arabidopsis without yield penalty, an essential property for considering susceptibility genes as targets for engineering recessive resistance. Accordingly, the silenced lines display lower DNA viral load and attenuated symptoms and are phenotypically indistinguishable from the control lines under normal conditions , and NSP-interacting GTPase (NIG), which are involved in the intracellular traffic of viral DNA, may be targets for enhancing resistance in crops . Silencinditions . Likewishe virus . Silencihe virus . CollectClustered regularly interspaced short palindromic repeats/Cas systems have a central role in plant biotechnology as an accurate molecular tool for editing genomes, rapidly improving desired traits, creating new plant varieties, and enhancing plant immunity against phytopathogens. The use of CRISPR/Cas systems is suitable for mediating viral genome editing while maintaining the biological functions of cells. Additionally, CRISPR/Cas systems have the potential to edit host factors, improving plant immunity against plant viruses. Nevertheless, a drawback in CRISPR/Cas-mediated host genome editing to enhance plant immunity is the limited repertoire of well-characterized recessive resistant genes or host susceptibility genes in which mutations are not likely to cause host growth defects. A better understanding of the host-virus interactome will expand the use of CRISPR/Cas for editing host susceptibility genes, which may be more efficient targets for durable resistance against viruses.Meanwhile, advances in the next generation CRISPR editing technology variants, such as the CRISPR-MAD7 system and engineered nucleases Cas12a, increase the accuracy, range of possibilities, and applications. Engineered Cas MAD7-RR, MAD7-RVR, and M-AFID (MAD7-APOBEC fusion-induced deletion) increase the targeting range of MAD7 by creating predictable deletions from 5′-deaminated Cs to the MAD7-cleavage site. This new CRISPR-MAD7 system has an efficiency of up to 65.6%, as demonstrated in mutant rice and wheat plants . MAD7 caFDAS and EPBF wrote the drafts with input from both authors. All authors contributed to the article and approved the submitted version.This work was partially supported by the Council for Advanced Professional Training (CAPES)—88887.511855/2020-00, National Council for Scientific and Technological Development (CNPq)—441955/2019-3, and Fapemig.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This paper proposes a solution to a long-standing problem concerning the joint distribution of allelic identity by descent between two individuals at two linked loci. Such distributions have important applications across various fields of genetics, and detailed formulas for selected relationships appear scattered throughout the literature. However, these results were obtained essentially by brute force, with no efficient method available for general pedigrees. The recursive algorithm described in this paper, and its implementation in R, allow efficient calculation of two-locus identity coefficients in any pedigree. As a result, many existing procedures and techniques may, for the first time, be applied to complex and inbred relationships. Two such applications are discussed, concerning the expected likelihood ratio in forensic kinship testing, and variances in realized relatedness. For the alleles of two individuals at a single locus, common coefficients range in complexity from the simple kinship and inbreeding coefficients  and also included in the R package ribd featured in the present paper. The latter has the advantage of supporting selfing and pedigrees with inbred founders  relatedness, which is sometimes more relevant than the pedigree-based expectation  members of nontrivial, i.e. that a and b are connected with at least one path in a and b are IBD if they descend from the same allele carried by a common ancestor of a and b within A kinship coefficienta and b is the probability that a random allele from a is IBD with a random allele from b at the same autosomal locus. The inbreeding coefficientc between a and b is defined by c is inbred if completely inbred if The ications .Note about notation: Relatedness coefficients are conventionally written with the individuals as subscripts rather than superscripts. We use superscripts in this paper, reserving subscripts for other indices. When the context is clear, we may drop the superscripts and simply write φ or f.detailed identity states. The expected relative frequencies of these states in a given relationship detailed identity coefficients of a and b.condensed identity statesIf the allele ordering  within each individual is ignored, the 15 states are reduced to 9 sS1,…,S9 , with exnoninbred if neither a nor b is inbred in a and b share, respectively, 0, 1, and 2 alleles IBD. Following IBD coefficients, to distinguish them from the previously defined identity coefficients.We say that a relationship unilineal if bilineal if direct if a is a direct descendant of b or vice versa, and collateral otherwise. The following simple fact will be used in later proofs:A noninbred relationship  if κ2>0 . FurtherLemma 1Let Proof.a is an ancestor of b. If b, we can then assume w.l.o.g. that a is an ancestor of  f. In order for a must be related to m as well. But then m and f are related, contradicting the assumption that b is noninbred. The same argument applies if b is an ancestor of a, hence we conclude that For the first implication, suppose for a contradiction that a is a founder of a in a. ȃ□The second implication follows from the fact that if φ and κ at a single locus. The following well-known facts, which we include for easy reference, are straightforward from the definitions  and half-siblings (H) have different two-locus IBD functions,where Next, we show that the opposite situation is also possible. This particular example appears to be original, although it seems likely that the effect it illustrates has been known to previous authors.Example 2Consider an outbred parent-offspring relationship (PO), and compare it with half-sibs whose shared parent is completely inbred (H-i). It is easy to see that both of these satisfyfor allρ; in other words, they have a constant IBD matrixin cis, i.e. on the same haplotype, since they come from the same parent. For PO, on the other hand, the alleles are in cis in the child, but not necessarily in the parent. This difference leads to distinct A remarkable consequence of Example a and b have two different common ancestors For our final example, we return to Haldane’s problem mentioned in the introduction. In our notation this amounts to the following: Given a relationship It is possible that [these coefficients] do not give all the needful information.As it turns out, Haldane’s intuition was correct: In general, Example 3a and b are full siblings whose parents P and Q are unilineally related. Then the two-locus kinship a and b is given bySuppose In particular,cannot be expressed solely byandThe formula  is obtaiFor an explicit example, consider the case of siblings whose parents are half-siblings. Inserting the expressions for phase of the IBD alleles. He found efficient formulas for some of these extended coefficients, but had to resort to tedious path tracing for the remaining ones. One contribution of the current paper is to enable recursive calculation of all of these phased coefficients, which in turn provide the cisin  or transin  in a and Starting with the coefficient haplotype(s) in a and b, or if a recombination has happened. See Turning to bilineal relationships, the coefficients a and b emit gametes with IBD alleles at both loci. For instance, in the diagram corresponding to For each pattern in Example 4Analyzing the phase elucidates the differences between PO and H-i in Example equation  producesgiven in .generalized kinship coefficients. Recursive formulas for such coefficients were given by Karigl and further developed by other authors, allowing efficient computation of identity coefficients.A key idea introduced by K and D.Passing to the two-locus situation, it is natural to seek a similar generalization of two-locus kinship coefficients. We begin by reviewing the single-locus case.φ generalizes naturally to three or more individuals. For example, a, c are all IBD. We will adopt a more flexible notation, close to that of a, b, d, then the alleles from a and b are IBD, and the ones from c and d are IBD, but different from those in the first group. More generally, a generalized kinship pattern is a finite collection of blocks of pedigree members,generalized kinship coefficientThe pairwise kinship coefficient A few simple properties of generalized kinship coefficients are worth noticing. Firstly, ρ. If generalized two-locus kinship coefficient is the probabilityρ.Let segregation index superscript to each allele J9=Let a and b share 0 alleles IBD at both loci, i.e. that both loci are in state a and b share 2 alleles), while Switching to the two-locus situation, our aim is to give a two-locus version of . To compTheorem 2a and b are nonfounders in Suppose fined in . The twoA complete overview of the detailed two-locus states and their corresponding patterns given in κ22=ΦFor any na is a founder of a’s parents, as unrelated founders. Theorem The assumption that The algorithms described in this paper are implemented in the R package ribd , which iD. However, this number can be almost halved by exploiting linear dependencies. Let D, we have j (including 8). This procedure eliminates 104 of the 225 terms, leaving only 121 , ribd computes founder inbreeding, i.e. the assignment of nonzero inbreeding coefficients to any pedigree founders. As shown in complete inbreeding. All two-locus functions in ribd support completely inbred founders . For example, the H-i pedigree featured in Example A particular feature of the ped suite is the support of # Half siblings with completely inbred parentx = halfSibPedfounderInbreeding = 1# A two-locus kinship coefficienttwoLocusKinship[1] 0.140625# The two-locus IBD matrix (same for any rho)twoLocusIBDThe output of the last command (not shown) is the matrix K in equation .a, is a founder, then the algorithm described in Section “General relationships” requires that we extend the pedigree by adding the parents of a before applying Theorem a is completely inbred; hence in this particular case founder inbreeding is not supported in the current implementation.If either of the two individuals, say In order to check the implementation, numerical validation was performed against a wide variety of previously published two-locus probability formulas. Details and source code for these efforts can be found in the documentation of ribd, including examples from θ to denote the recombination fraction). Furthermore, Table 6 has a misprint in the entry for “First cousin and second cousin,” where the coefficient of Of particular interest is the work of Considering the enduring interest in two-locus IBD probabilities, the lack of general algorithms may seem surprising. One explanation may be that for simple relationships explicit formulas can be obtained by direct calculation. Below we briefly discuss two applications of two-locus coefficients which, with the methods of the present paper, can now be extended to larger classes of pedigrees.G denotes the genotypes at a predetermined set of markers. Egeland and Slooten discovered that if G is interpreted as a random variable, the expectation ρ is the recombination fraction between the markers. Furthermore, suppose that true relationship ρ for various relationships =En gives:Var(kj)=EThis paper presents an algorithm for computing joint IBD probabilities at two linked loci, called two-locus identity coefficients, in any pedigree. Previous work in this area have focused on simple cases of noninbred relationships, where explicit formulas can be obtained by brute force. In contrast, the method described here applies to any pairwise relationship, both noninbred and inbred. The inbred case requires as many as 81 two-locus coefficients, which may seem like a daunting task. However, they can all be expressed in terms of generalized two-locus kinship coefficients, for which a recursive algorithm is given. All methods, including numerous examples, are implemented in the R package ribd, which runs on all common platforms and is freely available from CRAN. As a result, a variety of methods and applications, previously restricted to simple, special cases, may now be applied and explored in general pedigrees.