ORF1, ORF2, and ORF17 genes, either individually or in combination. Mutant viruses were analyzed for their replication properties in cultured equine dermal cells, PBMC infection efficiency, virus-induced changes in the PBMC proteome, and cytokine and chemokine expression profiles. ORF1, ORF2, and ORF17 are not essential for virus replication, but ORF17 deletion resulted in a significant reduction in plaque size. Deletion of ORF2 and ORF17 gene significantly reduced cell-to-cell virus transfer from virus-infected PBMC to EC. EHV-1 infection of PBMC resulted in upregulation of several pathways such as Ras signaling, oxidative phosphorylation, platelet activation and leukocyte transendothelial migration. In contrast, chemokine signaling, RNA degradation and apoptotic pathways were downregulated. Deletion of ORF1, ORF2 and ORF17 modulated chemokine signaling and MAPK pathways in infected PBMC, which may explain the impairment of virus spread between PBMC and EC. The proteomic results were further confirmed by chemokine assays, which showed that virus infection dramatically reduced the cytokine/chemokine release in infected PBMC. This study uncovers cellular proteins and pathways influenced by EHV-1 after PBMC infection and provide an important resource for EHV-1 pathogenesis. EHV-1-immunomodulatory genes could be potential targets for the development of live attenuated vaccines or therapeutics against virus infection.Equine herpesvirus type 1 (EHV-1) causes encephalomyelopathy and abortion, for which cell-associated viremia and subsequent virus transfer to and replication in endothelial cells (EC) are responsible and prerequisites. Viral and cellular molecules responsible for efficient cell-to-cell spread of EHV-1 between peripheral blood mononuclear cells (PBMC) and EC remain unclear. We have generated EHV-1 mutants lacking  Alphaherpesviruses are ubiquitous pathogens affecting human and animal populations ,2,3,4. HVaricellovirus in the subfamily Alphaherpesvirinae and are important pathogens that infect horses . Ho. Ho32]. ORF1 did not affect virus transfer at any stage.Following inhalation of EHV-1, the virus primarily replicates in respiratory tract epithelial cells, subsequently passes through the basement membrane, establishes cell-associated viremia, and, finally, is transferred to the endothelial epithelium . To mimip-values ≤0.05, as calculated by Perseus software. In total, 63 cellular proteins were upregulated and 78 proteins were downregulated. KEGG-pathway enrichment analysis of differentially expressed proteins (p ≤ 0.05) was calculated using Benjamini-corrected Fisher’s exact test. In Ab4-wt-infected PBMC, upregulation of several pathways including platelet activation, Ras signaling, leukocyte transendothelial migration, endocytosis, lysosome, oxidative phosphorylation pathways, and cAMP signaling pathways was detected. On the other hand, downregulation of proteins associated with herpesvirus infection, chemokine signaling, spliceosome, RNA degradation, and apoptotic pathways was observed  out of 23 cytokines tested in the panel. All standards and quality controls were within the specified range. In general, the release of most cytokines and chemokines was strongly inhibited upon Ab4-wt infection of PBMC. Only FGF-2 was expressed in infected PBMC at 6 hpi  . Infectiytokines . All mutytokines C. IFNγ l6) were infected with Ab4-wt at an MOI of 1. At 24 hpi, infected PBMC were applied over EC, and supernatants were collected at 3 and 6 h for cytokine estimation. In PBMC-EC co-cultures, non-infected PBMC released more cytokines than virus-infected PBMC. Further, Ab4-wt infected PBMC in the presence of EC did not show much difference in terms of release of additional cytokines and chemokines, except release of IL-6  clearly altered the cytokine expression profile and restored cytokine expression partially at different time points. ORF2 gene deletion expressed higher levels of IL-1β cytokine at all time points.Taken together, infection of PBMCs with Ab4-wt mostly resulted in downregulation of cytokine expression and deletion of different viral genes ; however, they had comparable viremia to Ab4-wt [ORF2 deletion mutant was attenuated; however, it had strong immunogenicity without altering viremia in Icelandic horses [ORF1, ORF2, and ORF17) are essential for virus pathogenesis and play an important role in virus spread from PBMC to EC.EHV-1 infection of PBMC is a critical step in deciding EHV-1 pathogenesis; thereby the virus can spread from respiratory epithelium to the endothelium without being captured by the host immune system. As described earlier, the process of virus transfer is complex, requires coordinated action of viral proteins, adhesion molecules expressed by both cells, and cytokines and chemokines for facilitating inter-cellular adhesion, intra-cellular trafficking, and cellular polarity ,72. Seven ponies . Experimo Ab4-wt . In anotc horses ,76. Failc horses ,49,50. AORF1, ORF2, and ORF17 genes of EHV-1 are dispensable for virus replication in equine cells as confirmed by growth kinetics and plaques size assays. Double and triple gene deletion mutants also replicated normally as with the parental virus. Only ORF17 deletion mutant showed a significant, but slight, reduction in plaque size, ranging between 10–15%. Albeit the plaque size reductions were less, similar plaque size reductions of UL43 (ORF17 homolog) deletion mutants were observed in HSV-1 and PrV [ and PrV ,78. DespORF17 and ORF2 as single-gene deletion mutant significantly reduced virus transfer between PBMC and EC under static and flow conditions. Furthermore, ORF17 and ORF2 mutants (as double and triple gene deletions mutants in combination with ORF1) also reduced virus transfer to EC. Double and triple gene deletions showed an additive reduction in virus transfer to EC in a flow condition compared to static condition, which can be attributed to a synergetic effect on the functional dynamic rolling of infected PBMC over EC. The flow chamber system creates homogenous fluid shear stress in the endothelium similar to that observed in the blood vessel environment in vivo. Fluid flow over the endothelium results in ranges of ion fluxes, modulation of several pathways including biochemical pathways and gene and protein expression in both in vitro and in vivo. Specifically, shear forces modulate cell surface transmembrane adhesion molecules via cytoskeleton by integrin-dependent activation of MAP kinases via Ras GTPase and RhoA activation [ORF2 and ORF17 modulated the MAP kinase and Ras GTPase pathways, which might explain the reduction of virus spread.We show here that PBMC can be infected, to similar levels, with all mutant viruses without any significant differences. Co-cultivation and flow chamber assays with parental EHV-1 virus showed efficient virus spread from infected PBMC to EC as reported previously . Howevertivation . Furthertivation , which wORF2 and ORF17) are involved in modulating several pathways in PBMC, including cell signaling, adhesion, and immune pathways, thereby ORF2 and ORF17 gene deletions resulted in reduced virus transfer to EC. Our findings clearly correlate with previous in vivo findings where ORF2, ORF1/ORF71, and ORF1/ORF2 deletion mutants showed respiratory infection, comparable viremia, but no noticeable endothelial cell infection in four different experimental studies in horses [In the epithelium-PBMC contact assay, all mutant viruses transferred from infected epithelial cells to PBMC similar to the parental virus. However, subsequent flow chamber assay with infected PBMC showed reduced virus transfer to EC. It is very clear that gene deletions affected only the virus transfer step to EC, but not from the epithelium to the PBMC. The process of cell-to-cell virus transfer from infected epithelial cells to PBMC is relatively simple. EHV-1 undergoes full replication cycles in the epithelium and the released infectious virus particles result in dendritic cell infection ,82,83. Sn horses ,49,50,76It was shown before that all three PBMC subpopulations could be infected with EHV-1 ,20,85,86EHV-1 undergoes limited virus replication in PBMC ,85, but Parental EHV-1 infection upregulated proteins associated with several host pathways including MAPK, Ras signaling, endocytosis, oxidative phosphorylation, lysosomal pathways but downregulated herpesvirus and spliceosome pathways. MAPK pathway is involved in the manipulation of cellular functions such as signal transduction, cell adhesion, virus replication in target cells, and cell survival ,91. Ras-ORF1, ORF2, and ORF17 gene deletions have modulated several pathways, mainly chemokine signaling, MAPK, herpesvirus infection, and oxidative phosphorylation pathways. Ab4-wt infection downregulated herpesvirus infection pathway, while Ab4∆ORF1/ORF2/ORF17 mutant virus upregulated this pathway. Similar to the single gene deletion (Ab4∆ORF1 and Ab4∆ORF2), triple gene deletions modulated several pathways including chemokine signaling pathways. A previous in vitro study showed the potential role of MAPK pathway in EHV-1-infected PBMC. Inhibition of this pathway with a chemical inhibitor reduced virus transfer from infected monocytes to EC [ORF17 and ORF2 gene deletions downregulated the proteins associated with MAPK pathway and showed reduced virus transfer to EC. We surmise that ORF17 and ORF2 viral proteins are involved in upregulating the MAPK pathway thereby facilitating virus transfer to EC. As described earlier, MAPK is essentially involved in transmitting various extracellular signals that induce cellular proliferation, differentiation, and survival [ORF17 and ORF2 in modulating MAPK pathway will be the scope of our future studies.While comparing the proteomic profile of Ab4-wt and mutants infected PBMC, es to EC . In our survival . It has survival ,106. StuORF1 deletion showed release of more cytokines in infected PBMC. Similar findings were confirmed by mRNA expression, cytokine quantification, and migration assay [ORF1 deletion mutants. IFNγ is produced by antigen-activated leukocytes. Secreted IFNγ activates antigen-specific immunity, innate-cell mediated immunity, and cytokines/chemokines release. Ab4-wt infection blocks IFNγ release from PBMC and deleting ORF1 gene resulted in release of more cytokines/chemokines which shows the immune-modulating potential of ORF1 in Ab4 strain. ORF2 gene deletion also showed release of more cytokines in both in vitro and in vivo [ORF2 gene deletion resulted in expression of higher levels of IL-1β cytokine, which is considered as a host defense against virus infection. It is a well-known immune evasion strategy employed by HSV-1 to retain IL-1β in the intracellular space of infected macrophages without being released to the extracellular space by blocking the function of caspase-1, which blocks the pro-inflammatory activity of IL-1β [ORF2 of EHV-1 also may have a similar function, thereby Ab4-wt infection in PBMC results in decreased release of IL-1β, while ORF2 deletion restores expression. The role of ORF2 in modulating of IL-1β secretion could be the potential area for further investigation. ORF17 gene deletion did not restore cytokine/chemokine release completely, however, released more cytokines than parental EHV-1.Parental EHV-1 infection greatly reduced cytokine and chemokine release. Higher levels of FGF-2 in Ab4-wt infected PBMC can be correlated with activation of MAPK pathway as confirmed by proteomic analysis. FGF-2 results in the activation of Ras-MAPK pathway, which is essential for virus spread ,108. Preon assay . Further in vivo ,76. ORF2of IL-1β ,112. We ORF1 and ORF2 in its genome in comparison to Ab4-wt and showed release of more cytokines upon infecting PBMC [ORF1 and ORF2 genes in Ab4 restored expression of more cytokines, which clearly demonstrates that ORF1 and ORF2 have a major role in cytokine production.RacL11 strain, which is poorly spread to EC, naturally lacks ing PBMC . SimilarORF1 probably plays a major role in Ras-signaling, chemokine signaling, and cell adhesion pathways. ORF2 and ORF17 are implicated in chemokine signaling and MAPK signaling pathways thereby facilitating cell-to-cell virus spread. Furthermore, ORF1, ORF2, and ORF17 deletion mutants  were capable of stimulating strong cytokine responses in vitro. These genes could be potential targets for the development of live attenuated vaccine therapeutics against EHV-1 infection in equines. In addition, identifying possible interactions and finding interaction partners for ORF1, ORF2, and ORF17 proteins including viral and cellular proteins are the potential area of future studies.In conclusion, we have identified and confirmed immune evading and cytokine modulating properties of different viral proteins of herpesviruses. Based on our flow chamber, proteomics, and cytokine/chemokine assay, all three targeted genes were multifunctional in nature. Nevertheless, these viral proteins are involved in more than one pathway in PBMC. We presume that


Sinorhizobium species, which have diverse applications. In this study, the rheological analysis confirmed that succinoglycan produced by Sinorhizobium meliloti Rm 1021 binds weakly to various metal ions, including Fe2+ cations, to maintain a sol form, and binds strongly to Fe3+ cations to maintain a gel form. The Fe3+-coordinated succinoglycan (Fe3+-SG) hydrogel was analyzed by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, circular dichroism (CD), and field-emission scanning electron microscopy (FE-SEM). Our results revealed that the Fe3+ cations that coordinated with succinoglycan were converted to Fe2+ by a reducing agent and visible light, promoting a gel-sol conversion. The Fe3+-SG hydrogel was then successfully used for controlled drug delivery based on gel-sol conversion in the presence of reducing agents and visible light. As succinoglycan is nontoxic, it is a potential material for controlled drug delivery.Hydrogel materials with a gel-sol conversion due to external environmental changes have potential applications in a wide range of fields, including controlled drug delivery. Succinoglycans are anionic extracellular polysaccharides produced by various bacteria, including  Hydrogels are “soft” materials that can absorb large amounts of water while maintaining their form in water long term . Over th3+ cations and showed a gel-sol conversion by a reducing agent [3+ cations that were gradually contracted by light irradiation energy [3+ cations that exhibited a photoresponsive activity [3+ cations were anionic polysaccharides that can effectively bind Fe3+ cations. However, not all anionic polysaccharides and metal cations are coordinated to form hydrogels. In order for anionic polysaccharides to coordinate with metal cations to form hydrogels, the anionic polysaccharides must have a unique structure that can effectively sterically fit by combining with metal cations [Hydrogels coordinated with Fe cations as crosslinkers have been well studied ,10. As tng agent . In addin energy , and theactivity . The polRhizobium, Agrobacterium, and species of soil microorganisms that play a crucial role in the development of the root nodule symbiosis between the bacteria and legumes of Alfalfa [2+ to provide antioxidant activity through an anti-Fenton reaction, thereby effectively controlling Fe biochemistry [3+ cations.Succinoglycans are a type of exopolysaccharides (EPSs) secreted from  Alfalfa . Succino Alfalfa , emulsif Alfalfa , and pse Alfalfa , and can Alfalfa . With a  Alfalfa . Low molhemistry . HoweverSinorhizobium meliloti Rm 1021, and, to our knowledge, hereby present the first evidence that it effectively coordinates with Fe3+ cations to form a hydrogel. Fe3+-coordinated succinoglycan (Fe3+-SG) hydrogels were investigated by various methods, including rheometry, attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, circular dichroism (CD) spectropolarimetry, and field-emission scanning electron microscopy (FE-SEM). Fe3+-SG hydrogels were able to control the release of drugs through the gel-sol conversion, depending on the photoreductant concentration and a reducing agent capable of reducing Fe3+ to Fe2+.In this study, we isolated succinoglycan from Congo red and 1,10-phenanthroline were purchased from Sigma-Aldrich Chemicals Co., St. Louis, MO, USA. A sodium lactate 50% solution was obtained from Duksan Pure Chemicals Co., Ltd. Ascorbic acid was purchased from the Beijing Chemical Works Reagents Company, Beijing, China. Irradiation with visible light was performed by using a 405 nm laser.S. meliloti Rm 1021 supplied by the Microbial Carbohydrate Resource Bank (MCRB) of Konkuk University, Korea. The bacteria were grown in a production medium at 25 °C for 14 days. The production medium comprised of d-mannitol (50 g/L), glutamic acid (7.5 g/L), K2HPO4 (15 g/L), KH2PO4 (15 g/L), MgSO4.7H2O (1 g/L), and CaCl2 (0.2 g/L), and the pH was adjusted to 7.0. Microbes were then removed by centrifugation at 8000 g for 15 min; three volumes of ethanol were added to precipitate the supernatant, and the precipitate was collected using filter paper. Subsequently, the collected precipitate was purified by dialysis (MWCO 12–14 kDa membrane) with distilled water (DW) for three days and lyophilized after collection to obtain the purified succinoglycan [Succinoglycan was produced from noglycan .w/v) succinoglycan solution was first prepared and then mixed with a 40 mM metal ion solution in a 1:1 volume ratio. The storage modulus (G’) and loss modulus (G”) of hydrogels was measured at 25 °C by applying a 1.0% strain between 0.1 and 100 rad/s.The rheological experiments were performed by using a DHR-2 rheometer . To demonstrate the ability to form hydrogels through the coordination of succinoglycan with metal ions, a 1.0% (3+ and Fe2+ cations was mixed with a 1.0% (w/v) succinoglycan solution, respectively. Succinoglycan mixed with Fe2+ cations in the solution formed a sol; in contrast, the mixture with Fe3+ cations formed a gel. Three samples, including the succinoglycan, were frozen in the deep freezer, and then lyophilized. Spectra were collected in transmission mode in the range from 4000 to 500 cm−1 at a resolution of 0.5 cm−1 using 10 scans.The ATR-FTIR spectroscopy analysis was conducted by using an ATR-FTIR spectrometer  equipped with a PIKE MIRacle ATR accessory. According to the conditions mentioned in the rheological studies, a solution of Few/v) succinoglycan and 0.02 mM Fe2+ and Fe3+ cations in DW. Experiments were performed in a 0.1 cm path length cuvette at 25 °C and expressed, on average, five scans. The response time and bandwidth were 2 s and 0.2 nm, respectively.The CD spectra of samples in the wavelength range from 190 to 260 nm were recorded by using a JASCO J-810 spectropolarimeter. All samples contained 0.05% , and the resulting mixtures were stirred gently at room temperature. The vials were then turned upside down to determine whether gelation had occurred or not. In the vial tests, Fe3+-SG hydrogel beads were prepared, as follows. The prepared 1% (w/v) succinoglycan solution was added dropwise to an aqueous 30 mM Fe3+ solution through a 10 µL pipette tip. The beads were kept in a Fe3+ solution for 15 min to ensure that sufficient coordination occurred. Subsequently, the Fe3+-SG hydrogel beads were washed three times with DW to remove the excess Fe3+ cations. Next, the prepared Fe3+-SG hydrogel beads were analyzed by FE-SEM, UV–Vis spectroscopy, Congo red release, and cytotoxicity.Succinoglycan 1.0% (w/v) was dissolved in DW and placed in individual glass vials. The succinoglycan solutions were treated with different concentrations of Fe3+-SG hydrogel beads produced were rinsed three times in DW and were lyophilized overnight. The beads were cut in half to analyze their internal structures, which were visualized by using FE-SEM. The beads were coated with a platinum layer at 30 W for 30 s in a vacuum prior to the FE-SEM analysis.The Fe2+ cations were measured by UV–Vis spectrometry, as Fe2+ cations were reacted with 1,10-phenanthroline to form a stable Fe2+-1,10-phenanthroline complex (Fe2+-phen), showing a maximum absorption at 510 nm [2 + cations was measured under two conditions. First, Fe3+-SG hydrogel beads (20 mg beads) were stored in 5 mL vials. Next, 2 mL of a 10 mM ascorbic acid solution was added to the vial and gently shaken. Every 10 min, stored aliquots of the suspension of Fe3+-SG hydrogel beads were collected and equilibrated with 1,10-phenanthroline for 2 min, diluted, and the UV–Vis absorption of the resultant beads was measured in the range of 400–600 nm. In the second condition, the prepared Fe3+-SG hydrogel beads were stored in 2 mL of a 10 mM sodium lactate solution, then Fe3+-SG hydrogel beads were irradiated by a diffuse 405 nm laser beam. Irradiation tests were conducted at 10 min intervals to record the UV–Vis spectra.In this study, the reduced Fet 510 nm . Based o3+-SG hydrogel beads were ground then dissolved in a PBS buffer (pH = 7.4). The suspensions were stirred for 2 h, subsequently centrifuged at 13,500 g for 5 min. The extracted weight of Congo red was measured in the same manner as for the Congo red release studies. The Congo red loading amount was calculated as follows:For Congo red loading studies, 50 µL of a Congo red solution (1 mg/mL) was mixed with 450 µL of a 1% (w/v) succinoglycan solution for 30 min. The final concentration of Congo red was adjusted to 0.1 mg/mL . The Con3+-SG beads was estimated as follows [The encapsulation efficiency of Congo red in Fe follows ,27:Encap3+-SG beads was 11.11 mg/g beads. Congo red-loaded Fe3+-SG hydrogel beads (50 mg) in a 3 mL PBS buffer solution (pH 7.4) were treated with either ascorbic acid as a chemical reductant or visible light as a photoreductant. At specific time intervals, the release of Congo red was determined at 498 nm. The cumulative release of Congo red was calculated from the following formula [V is the release of the medium volume, The theoretical loading amount of Congo red in Fe formula :Cumulati2) [2+-SG sol, and Fe3+-SG hydrogel beads in 96-well plates containing 5 × 103 cells per well, respectively. In addition, the indirect extract cytotoxicity test was performed as described in the ISO 10993-5:2009—Biological evaluation of medical devices, part 5: Tests for in vitro cytotoxicity, by indirect contact [3+-SG hydrogel beads were immersed in a 10 mL DMEM and then incubated for 12 h at 25 °C in the dark. Thereafter, the cells were placed in a 96-well plate  containing 5000 cells/well, treated with a prepared stock solution and indirect extract, which incubated for 24 h. After 24 h, cells were washed with a PBS buffer and the WST-1 reagent  was added to each well [We used the human embryonic kidney 293 (HEK293) cell line purchased from the Bank of Korea Cell Line  . The celS. meliloti was characterized by 1H NMR and gel permeation chromatography (GPC). The chemical structure is shown in 1H NMR data of succinoglycan produced by S. meliloti Rm 1021, the peaks with chemical shifts at 1.43 ppm represented methyl protons of the 1-carboxyethylidene (pyruvate); the peaks with shifts at 2.08 ppm represented methyl protons of acetate groups; the broad peak at 2.64 ppm represented the methylene protons of the succinate groups; and the complex region from 3.3 to 4.0 ppm indicated the protons of the carbohydrate backbone constituents. 1H NMR spectroscopy showed the fully acetylated succinoglycan containing approximately one or two succinate, one acetate, and one pyruvate groups. The average molecular weight of the succinoglycan was 354,839 g/mol , all the same phenomena as the original succinoglycan solution alone were observed, except for the Fe3+ cation, in which the succinoglycan and Fe3+ cation mixture solution formed a yellowish hydrogel (2+-coordinated succinoglycan (Fe2+-SG) did not show any significant difference, with the storage modulus always lower than the loss modulus, indicating that Fe2+-SG was a liquid-like sol. Both G’ and G” were sensitive to the angular frequency and did not cross over each other in the tested range  glycoside bonds, one of which links the side chain to the main chain. This special connection may give flexibility to the side arms of succinoglycan. The fixed charges of pyruvate and succinate linked to this side chain strengthen it, and the uncharged backbone is somewhat strengthened by the entanglement of the residues of glycofuranose and galtopyranose, which create a regular and helical structure [2+ of succinoglycan did not appear to be as large as shown in the CD spectrum, and this result was similar to the previously reported Na+ ion complexation with succinoglycan [3+ cations to succinoglycan in an aqueous solution greatly reduced the flexibility of the side chains, and could also be expected to be complexed with the hydroxyl groups of a rigid backbone, as confirmed by the ATR-FTIR results. The anionic polysaccharide xanthan is known to undergo significant changes in physicochemical behavior owing to the removal of charged groups [3+ cations to form a gel. The CD spectrum of polysaccharides was used as a tool to suggest the indicator of a secondary structure . In the weakened ; inset.  studies . In contyl group . As showtructure . Owing ttructure . Structunoglycan . Howeverd groups . These r3+-SG hydrogel by using a reducing agent. The concentration at which the Fe3+-SG hydrogel was formed was checked before making the beads. In Fe3+-polysaccharide hydrogel systems, the concentration of the Fe cation is an important point in the physical state of the sample and can also determine whether it can form a uniform solid gel. The progress of the gelation was monitored by mixing different concentrations of Fe cations with succinoglycan aliquots  succinoglycan solution to a 30 mM Fe3+ cation solution were used. The diameters of Fe3+-SG hydrogel beads were measured using an optical microscope (magnification 10×) [Bead materials can be integrated into microfluidic chips, optical fibers, microwells, and tips, and the beads are a useful material in various areas, such as disease diagnostics, and biological and chemical analyses. In this study, the beads were fabricated for an examination of the behavior of the gel-sol conversion of the Fealiquots . As show of Fe2+ b, the suion 10×) . To dete3+-SG hydrogel beads, the bead surface, and a cross-section of the bead are shown in 3+-SG hydrogel beads from the coordinating succinoglycan with Fe3+ cations was observed, as shown in 3+-SG hydrogel bead shown in 3+-SG hydrogel bead outer surface showed a rough and corrugated structure. This rough, dense surface appearance was because of the strong binding of succinoglycans with Fe3+ ions on the surface of the beads when the succinoglycan is added into the Fe3+ solution to form a bead [3+ cation was effective, even in the beads. The cross-section of the Congo red-loaded Fe3+-SG hydrogel beads still appeared as pores, and after the drug was released, it became a sol and the pores disappeared  as a Congo red release media. The cumulative release of Congo red from Fe3+-SG hydrogel beads in buffers of various ascorbic acid concentrations is shown in 3+-SG hydrogel beads were released for only 6% of Congo red over 150 min in the PBS buffer without any reducing agent. This is because on the surface of the hydrogel beads, as shown in the FE-SEM results, succinoglycans are strongly bound to Fe3+ cations, which interferes with the release of the drug molecules (2+-phen complex study that the reduction of Fe3+ by light was less effective than the reduction by the reducing agent (3+-SG hydrogel beads on cargo release stimuli-responsive properties, the Congo red-loaded Fe3+-SG hydrogel beads were prestored in the PBS buffer, which, after 60 min, were subjected to ascorbic acid and visible light, with the results shown in 3+ coordinated with succinoglycan has the potential as an effective controlled drug delivery system.Congo red is a good drug model that can be used in drug release experiments because it is photo stable and nonreactive . As an aolecules c. In addng agent . To stud3+-SG hydrogel beads, and indirect extract was 90%, 95%, and 91%, respectively. DMSO-treated cells were used as a positive control for cytotoxicity, and less than 20% of cells are viable under these conditions. As shown in 2+ also did not affect cell viability. These results confirmed that Fe3+-SG hydrogel beads favored biocompatibility and cell viability, suggesting that the Fe3+-SG hydrogel has great potential as a future biomedical material. Since hydrogels have been widely used as drug carriers, in tissue engineering, etc., the cytotoxicity of hydrogels has a great impact on further research. A previous study has confirmed that succinoglycan has low toxicity to organisms . Cell viS. meliloti Rm 1021 was subjected to structural analysis using 1H NMR spectroscopy and GPC. The rheological analysis suggested that when succinoglycans were mixed with Fe2+ cations, they formed a fluid solution; however, in the presence of Fe3 + cations, strong gels could be formed through coordination bonds. CD spectra and ATR-FTIR confirmed that Fe3+ cations can establish coordination bonds with succinoglycan groups such as hydroxyl, succinate, pyruvate, and acetate to form a strong Fe3+-SG hydrogel. We confirmed that the Fe3+-SG hydrogel beads can undergo gel-sol conversion when stimulated with a reducing agent and visible light. The Fe3+-SG hydrogel has internal pores to load the drug, and because of the strong coordination between succinoglycan and Fe3+ cations, no pore formation occurs on the surface. Thus, the loaded drug is released gradually in the PBS buffer. However, in the presence of a reducing agent, the drug could be released rapidly. In addition, the nontoxicity of Fe3+-SG hydrogel beads indicated that these hydrogel beads exhibited a superior biological safety. These results reveal that the Fe3+-SG hydrogel may serve as a potential stimuli-responsive release system.Succinoglycan isolated from


Wild birds are the natural reservoir of the avian influenza virus (AIV) and may transmit AIV to poultry via direct contact or indirectly through the environment. In the Netherlands, a clinically suspected free-range layer flock was reported to the veterinary authorities by the farmer. Increased mortality, a decreased feed intake, and a drop in egg production were observed. Subsequently, an infection with low pathogenic avian influenza virus was detected. This study describes the diagnostic procedures used for detection and subtyping of the virus. In addition to routine diagnostics, the potential of two different environmental diagnostic methods was investigated for detecting AIV in surface water. AIV was first detected using rRT-PCR and isolated from tracheal and cloacal swabs collected from the hens. The virus was subtyped as H10N7. Antibodies against the virus were detected in 28 of the 31 sera tested. An intravenous pathogenicity index (IVPI) experiment was performed, but no clinical signs (IVPI = 0) were observed. Post-mortem examination and histology confirmed the AIV infection. Multiple water samples were collected longitudinally from the free-range area and waterway near the farm. Both environmental diagnostic methods allowed the detection of the H10N7 virus, demonstrating the potential of these methods in detection of AIV. The described methods could be a useful additional procedure for AIV surveillance in water-rich areas with large concentrations of wild birds or in areas around poultry farms. In addition, these methods could be used as a tool to test if the environment or free-range area is virus-free again, at the end of an AIV epidemic. In recent decades, the avian influenza virus (AIV) has caused many infections in poultry worldwide, leading to significant economic and animal welfare implications , 2. MostAnseriformes and Charadriiformes, which are the natural reservoir of AIV . RNA was extracted from the pools using the MagNA Pure 96 System  and the MagNA Pure 96 DNA and Viral NA Small Volume kit (Roche) following manufacturer's instructions. Total RNA was tested by rRT-PCR targeting the influenza M gene (M-PCR), which detects all AIV subtypes, as described previously . In addi2, 0.3 μM Rox, 0.6 μM of each primers, and 0.1 μM of the probe added to the mix. Finally, 5 μl of RNA was amplified in a volume of 25 μl by employing the following temperature profile: 50°C for 30 min and 95°C for 15 min, followed by 45 cycles of 94°C for 10 sec, 58°C for 30 sec, 72°C for 10 sec.After sequencing, the isolated RNA was tested in an H10-specific rRT-PCR. The sequences of the primers (RF5127 5′-TGTGCATCGCATGTTTCCAT-3′ and RF5133 5′-CATCATTCTCTGGTTTAGCTTCGG-3′) and probe (RF5134 5′6-FAM-TGACAACGGCTAGAAGAACAAAACATGATGCC-BHQ-3′) were kindly provided by Ron Fouchier, Erasmus MC, Netherlands. One-step rRT-PCR was accomplished with a MX3005p cycler  by use of the OneStep RT-PCR Kit . The manufacturer's instructions of the kit were followed with 1.25 mM MgClA tracheal and a cloacal swab pool were each inoculated into the allantoic cavity of four 9- to 11-day-old specific pathogenic free embryonated chicken eggs (0.2 ml/egg) following the guidelines of the OIE . The eggTwo days after virus detection, blood samples were collected from 31 randomly selected hens at the farm. At the laboratory, blood was centrifuged (20 min at 1780g) to obtain sera, which were subsequently inactivated in a water bath of 56°C for 30 min. Sera were tested for the presence of antibodies against AIV using the nucleoprotein-blocking ELISA and the Haemagglutination Inhibition (HI) assay.The employed indirect double-antibody sandwich ELISA detects antibodies against all AIV subtypes and is a modified version of the nucleoprotein-blocking ELISA described previously . The ant2 HI titres, with titres ≥3 log2 considered positive.The used HI assay is a classic laboratory procedure for the subtyping of antibodies of AIV and is based on the inhibition of the agglutination reaction by haemagglutinin subtype-specific antisera . Auto-agThe IVPI was determined for the virus (A/chicken/Netherlands/17013178-006010) that was obtained from the cloacal swabs. The IVPI was performed according to the method described in the “Diagnostic Manual for avian influenza” . Ten 6-wMycoplasma gallisepticum  and subsequently boiled in Tris (0.01 M) EDTA (0.001 M), pH 9.0 for 10 min. The binding of Fc-receptors was blocked by incubation with 10% fetal bovine serum for 20 min at RT. The immunostaining of influenza A virus-positive-cells was performed using 1:1,000 diluted anti-influenza A virus nucleoprotein monoclonal antibody  in Normal Antibody Diluent  for 30 min at RT. After three subsequent wash steps with phosphate-buffered saline, the sections were treated with anti-mouse Dako EnVision+  for 30 min at RT. Again, sections were washed three times with phosphate-buffered saline and then treated with DAB+ (Dako UK Ltd) for 5 min at RT. Finally, the sections were counter-stained using haematoxylin. Sections incubated in the absence of primary antibody were taken along as negative controls.Samples of trachea, lung, air sac, duodenum and shell gland were fixed in 4% neutral buffered formalin, embedded in paraffin, sectioned at 2 μm, and stained with hematoxylin and eosin (H&E) for light microscopic examination. In the same organs, the presence of influenza A virus antigen was investigated using immunohistochemistry (IHC). For IHC, samples were fixated for at least 24 h in buffered 10% formalin, followed by dehydration in absolute ethanol and embedding in paraffin wax, sections were cut at 4 μm and mounted on glass slides. Endogenous peroxidase activity was blocked by incubation with 1% HWater samples were collected from the free-range area and a waterway around the farm, 2 days after the virus detection in hens. The free-range area is a fenced grassland connected to the poultry house that allows the hens going outside during daylight. After severe and prolonged rainfall, puddles of water were formed in the free-range area . During After the virus was detected in hens, they were prohibited to enter the free-range area any longer. Because the hens could not excrete new virus in the free-range area, there was a possibility to examine the survival of the virus in the environment. Therefore, the farm was visited 2 weeks after the first water sampling moment. During the second visit, 14 days after the first visit, the sampling sites of the first visit were sampled again plus an additional site of the waterway  and centrifuged in a Mistral 6000  for 15 min at 1781 g at 4°C. After centrifuging, the supernatant was filtrated using a Sterifil™ 47 mm Aseptic Vacuum Filter System and Holder  and two different types of filters. The first filter, a hydrophilic Glass Fiber Filter with a pore size of 1.0 μm without binder resins , was used for liquid clarification and was replaced when the filter was constipated. The second filter, a hydrophilic mixed cellulose esters membrane  with a pore size of 5.0 μm, is able to catch the virus if present. After processing the sample, the membrane was collected from the filter system into a bead tube and frozen (−80°C) until the start of the RNA isolation. In addition, the filter system was cleaned by soaking the system in a chlorine solution for 10 min, followed by rinsing the system with demi water.RNA was isolated from the membrane with the PowerWater® RNA Isolation kit (Qiagen). Manufacturer's instructions were followed, with several modifications: (1) the prescribed vortex adapter was not used; instead the bead tubes were horizontally vortexed manually on the vortex , (2) the fluid was centrifuged for 3 min at 3000 g instead of 1 min at 4000 g at point 9, and 3) the RNA was eluted in 50 μl RNase-free water to obtain a higher concentration of RNA. The eluate was frozen (-80°C) until it was processed in the M-PCR.4 plaque-forming units/ml was added as an intern control. After these preparations, the water was filtered through a Rexeed™-25A hemodialyzer dead-end hollow fiber ultrafilter  using a peristaltic pump  and sterilized silicon hoses . After processing the sample, the filter system was rinsed with 0.5L of tap water to flush the last part of the sample from the silicon hoses into the filter. The filter was stored overnight at 4°C, when necessary. The virus was flushed from the filter during a 20-min back flush with 500 ml TGBE buffer . After back flushing, the TGBE buffer containing virus was divided over two conical centrifuge tubes  and centrifuged for 2 h at 4500 g at 4°C. After centrifuging, the pH was set to a pH of 7 by adding 5M HCL and 125 ml 5x PEG/NaCl (500 gram PEG 8000 plus 87 gram NaCl and filled with distilled water up to 500 ml). After an incubation period of 1 h (or overnight) at 4°C, the centrifuge step was repeated and the supernatant was removed. To make the pellets more compact, the tubes were centrifuged for another 10 min at 4500 g at 4°C and again the supernatant was removed. Each pellet was resuspended in 500 μl Dulbecco's Modified Eagle Medium . Finally, 500 μl of the fluid was added to 2 ml of NucliSens lysis buffer  and stored for maximum 24 h at 4°C.Water samples were stored at 4°C before filtering for a maximum of 24 h. First, large substances, such as water plants, were filtered out of the water samples by flowing the water through a clean towel. Secondly, 200 μl of Porcine Epidemic Diarrhea virus (PEDV) with a titer of 6.3 × 10RNA was isolated using the Nuclisens® Magnetic Extraction Reagents and the Nuclisens® MiniMAG® (bioMérieux) according to the manufacturer's instructions. The eluate was frozen (−80°C) until it was processed in the M-PCR.Next-generation sequencing (NGS) was performed to determine the full genome sequences of the AIV isolated from the hen (EPI_ISL_394155) and from the 1L water sample that was collected at sampling site 1 during the first visit (EPI_ISL_394156). Illumina sequencing was performed as described previously .https://www.gisaid.org), using the HA segment of A/Chicken/Netherlands/17013178-006-010/2017 as query. The collected sequences were aligned using Multiple Alignment with Fast Fourier Transformation [MAFFT v7.427; Katoh and Standley (http://tree.bio.ed.ac.uk/software/~figtree).For phylogenetic analysis, a dataset was composed of HA nucleotide sequences of the top 100 blast hits  from the GISAID EpiFLu Database  were observed swimming in the puddle of water at sampling site 1 . However, no viral antigen was demonstrated with IHC staining for influenza A virus in the tracheas. One of the four hens had a mild pneumonia, consisting of collapse of multiple atria and capillaries by moderate infiltrates of mainly macrophages, lymphocytes, less heterophils and fibrinous and necrotic debris with organization . In addiThe duodenum of all investigated hens showed mild villus atrophy and a moderate lymphoplasmacytic infiltrate with scattered heterophils within the lamina propria. The epithelium was intact, but numbers of intraepithelial lymphocytes were multifocally increased. Crypts appeared normal. No Influenza A viral antigen was demonstrated by IHC. The shell gland of two hens did not show any abnormalities, while in one hen that arrived alive scattered perivascular lymphocytic infiltrates were present in het deep stromal region of the secondary folds. The glandular and surface epithelium were not altered and Influenza A viral antigen could not be demonstrated by IHC staining.Water samples collected on both sampling days from the free-range area and waterway were examined for the presence of AIV. The results of the M-PCR and H10-specific rRT-PCR for both methods are shown in The full genome sequences of the viruses detected in the hens and in the water samples collected in the free-range area were determined by NGS. The genome sequences of these viruses were similar, and no specific mutations or adaptations which lead to any changes in amino acids sequences were identified. Therefore, it remains unclear whether the water in the free-range area was contaminated by infected wild birds, resulting in introduction of the virus into the poultry farm. Or, whether the free-range area was contaminated later, by the already infected chickens. Phylogenetic analysis of the HA gene segment was performed to study the relationship between the H10N7 virus detected at the farm and other poultry and wild bird viruses. The most closely related virus based on the HA gene segment was a H10N6 virus detected in a mallard in Denmark in 2011. No closely related H10N7 viruses were detected in poultry or wild birds in 2017 .Post-mortem examination, diagnostics and additional experimental research confirmed an LPAIV H10N7 infection in a commercial free-range layer flock in the Netherlands. The circumstances surrounding this particular outbreak enabled us to investigate the potential of two methods for detecting AIV in the environmental water samples during a natural AIV infection. The described methods can be a useful additional procedure for AIV surveillance, or can be used as a tool to decide when precautionary measures, like keeping free-range chickens indoors, could be lifted at the end of an AIV epidemic. In contrast to the waterways near the farm, in which no AIV was detected, H10N7 virus was detected in the puddles of water in the free-range area. Both methods used for detecting AIV in water allowed the detection of H10N7 virus, demonstrating the potential of these diagnostic methods. Unfortunately, full genome sequencing did not provide conclusive insight into the epidemiology of the infection. Phylogenetic analysis did not reveal circulation of similar H10 viruses in wild birds or poultry in Europe in 2017.The clinically suspect situation, as a result of the infection with the LPAIV H10N7 was observed and officially reported by the farmer. He observed an increase in mortality, a decrease in feed intake and a drop in egg production although none of these three parameters reached the official reporting thresholds described in the acting Statutory Regulation . These tStaphylococcus sp., Ornitholobacterium rhinotracheale, Mycoplasma gallisepticum, and Escherichia coli /ml, which means that the 1L method is able to detect a minimum of 2140 virus particles in 1L of water. Looking at the mean cloacal HPAIV shedding of ducks—which is 3.8 log10 EID50/ml and takes on average 6.6 days . PEDV, which was also used as an intern control, was detectable with CTs of around 20, when 200 μl of virus with a titer of 6.3 × 106.6 days —this envven days . When thven days  using siven days , and froven days .To conclude, this study showed the potential of two methods to detect AIV in water during field infections. Detecting AIV in the environment using water samples may be a good tool to improve AIV surveillance and may assist in deciding to lift precautionary measures in outbreak situations.All datasets analyzed for this study are included in the article.The animal study was reviewed and approved by the Centrale Commissie Dierproeven (CCD), Den Haag, Netherlands.EG, AE, WP, and NB: conceptualization. EG, NDB, RH, RHH, and WV: methodology. RH, RHH, WP, and WV: validation. EG: data curation. AE, WP, and NB: supervision. SB and ME: phylogenetic analysis. EG and NDB: writing—original draft preparation. EG, AE, NDB, RH, WV, SB, ME, RHH, WP, and NB: writing—review and editing.NDB was employed by the company GD-Animal Health, Deventer, Netherlands. The remaining 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.


Both GD19GZ01 and GD19GZ02 almost entirely lacked the rTM region and E element, while the latter was retained in GD19GZ03 and GD19GZ04. Moreover, the virus replication levels in GD19GZ03 and GD19GZ04were much higher than those in GD19GZ01 and GD19GZ02. And three virus recombination events in GD19GZ01 and GD19GZ02 were revealed by the results of PDR5 and SimPlot software analysis. Additionally, we found that some interferon-stimulating genes  and inflammatory mediators  were significantly upregulated in the immune system organs of clinical chickens. Taken together, these findings clarify and reveal the sequence characteristics and trends in the variation of ALV-J infection in yellow chicken flocks of South China.The aim of this study was to better understand the sequence characteristics and immune responses in avian leukosis virus subgroup J (ALV-J) infected yellow chicken flocks in South China. We isolated four strains of ALV-J virus from these flocks, which were then identified by several methods, including subtype-specific polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assay (IFA). All four viruses were sequenced for their complete genomes and named GD19GZ01, GD19GZ02, GD19GZ03, and GD19GZ04. In comparison with the reference sequence, the homology analysis showed that the  Alpharetrovirus of the Retroviridae family. According to the molecular biological characteristics of serum neutralization testing, host range, and envelope glycoproteins, the ALVs can be divided into 10 subgroups corresponding to A~J [Avian leukosis virus (ALV) is a type of avian retrovirus that belongs to the genus g to A~J . The ALVg to A~J . In Ching to A~J . Studiesg to A~J , 6. Impog to A~J , 8. Furtg to A~J . Due itsThe yellow chicken breeding industry is a crucial component of the larger poultry industry in South China. Yet, since many small farms still do not strictly screen and eradicate ALVs, coupled to the lack of effective vaccines or therapeutic drugs, the yellow chicken industry has been continuously threatened by ALV-J outbreaks in South China in recent years , 10. In A group of parental yellow chickens of the egg-type in a local farm in South China showed telltale symptoms of infection, such as a pale cockscomb, listlessness, a thin body, cluttered feathers, and obvious hemangiomas on their skin and digits. To understand the epidemiology ALVs' infection in yellow chickens, a total of 198 birds (40 weeks old) from that farm were analyzed. In short, anticoagulated blood samples were collected aseptically from each yellow chicken, whose blood was centrifuged to obtain plasma for the virus isolation. According to the previously described method , the sinFresh tissues  were collected, fixed with 10% neutral formalin buffer, then dehydrated, embedded in paraffin, sliced, and then stained with hematoxylin and eosin (HE) . The preμl of blood per chicken was taken for its DNA extraction, performed with the NRBC Blood DNA Kit . This extracted blood DNA was used as the substrate for PCR. A previous methodology was referred to and followed to synthesize specific primers used for each subgroup of ALVs, which also included specific primers for the Marek's disease virus (MDV) and reticuloendothelial hyperplasia virus (REV) [μg/ml ethylene bromide).The peripheral blood was collected aseptically in an anticoagulation tube, of which 200 us (REV) , 14. The5 cells/well), and the plate then placed at 37°C in the presence of 5% CO2. After 24 h, the culture solution was removed and replaced with fresh cell maintenance solution , 100 U/ml penicillin, and 100 U/ml streptomycin). These cells were maintained at 37°C under 5% CO2 for 5–7 days with daily monitoring. Supernatants and precipitated substrates were centrifuged and harvested after the cells had been frozen and thawed three times. According to the manufacturer's instructions, the supernatants were tested for ALV-specific antibodies (namely p27) by ELISA . Then, according to the manufacturer's instructions, the precipitated substrates underwent DNA extraction, using the Tissue DNA Kit , followed by the full-length amplification of the proviral genome by the PCR.Since DF-1 cells were only sensitive to exogenous virus particles, the interference of endogenous virus replication could be ruled out . The remα Escherichia coli competent cells. After the transformation's completion, the bacterial solution was evenly spread on an agar plate containing ampicillin and incubated at 37°C for 12–16 h. Finally, the recombinant plasmid harboring the target DNA fragment detected by the PCR was sent to TSINGKE Biotech  for sequencing. Three independent cloned plasmids were used in each group. The primer sequences are listed in According to the conserved region of the ALV-J original strain HPRS-103, four pairs of specific primers were designed , enablinAll sequences were aligned and analyzed using DNAStar version 7.1. All the ALVs' strain reference sequences are from the National Center for Biotechnology Information (NCBI) GenBank database  as the primary antibody and FITC-labeled antimouse IgG  were used. Cell morphology was observed under a fluorescence microscope, with images recorded using NIS-Elements BR analysis software .ΔΔCt- method was used to calculate relative expression changes. The primer sequences are listed in Total RNA was extracted using TRIzol reagent (Invitrogen) from tissues or cells and reversed-transcribed with a PrimeScript RT reagent kit with gDNA Eraser  according to the manufacturer's instructions. qPCR was performed on a Biorad CFX96 Real-Time Detection System using iTaq™ Universal SYBR® Green Supermix Kit reagents . The 24TCID50/0.1 ml were used to inoculate DF-1 cells, and the supernatant and cells were collected at 1, 3, 5, and 7 dpi. The virus copy number was calculated by the absolute quantitative method.For the comparison experiment of virus replication in vitro, samples with a viral load of 10t-test if the data were in Gaussian distribution and had equal variance, or by unpaired t-test with Welch's correction if the data were in Gaussian distribution but with unequal variance, or by nonparametric test (Mann–Whitney U test) if the data were not normally distributed (Prism 8.0). Data were represented as means ± SD/SEM. Differences withP < 0.05were considered significant:∗P < 0.05,∗∗P < 0.01,∗∗∗P < 0.001. Differences within groups are marked with letters . The same letter means P < 0.05, between different uppercase or lowercase letters means P < 0.01, and between uppercase and lowercase letters means P < 0.001.The data between two groups were analyzed by unpaired In this study, our experimental animals came from a group of egg-type chickens that were 40-week-old, which exhibited a pale cockscomb, bodyweight loss, mental depression, and decreased productivity. After detecting the ALVs' specificity antibody p27, it was found that a total of 15.7% (31/198) of the sampled chickens were positive for infection and the mortality rate was 3.5% (7/198). In particular, we found that the joints and diameter of the toes of these sick chickens were swollen, with blood blisters and ruptures evident, and signs of bleeding and hemangioma on their skin . We synthesized specific primers for different subgroups of ALV, which also included those for MDV and REV. The four samples tested positive when using the ALV-J specific primers H5/H7, with a specific band evident at 545 bp, while the other subgroup primers had negative results . Similargag-pol-env-LTR, but they did not contain a viral oncogene, thus indicating that the four isolates belonged to the chronic tumor-causing avian leukemia virus. The four isolates shared 85.1%–98.0% nucleotide identity when compared with the reference strains. The detailed results of these pairwise comparisons are presented in After alignment with the sequence characteristics of the original ALV-J strain HPRS-103 (GenBank accession number: Z46390), the full-length proviral genomes of GD19GZ01 to GD19GZ04 were obtained, which were 7479–7626 nucleotides (nt) in size, respectively. They all had the classic structure of a retrovirus, featuring the Long Terminal Repeated- (LTR-) leader-env gene (between bases 6187-6188). The four isolates had different degrees of insertions and deletions in their 5′U3, env, and 3′UTR regions, when compared with the original strain HPRS103  region and downstream E element. Both GD19GZ01 and GD19GZ02 lacked the rTM region and E element almost entirely; however, GD19GZ03 and GD19GZ04 lacked the rTM region but retained the E element , the GD19GZ01 virus strain had lost its CAAT Box .The phylogenetic analysis of the 26 reference sequences and four isolates revealed that the latter formed the same cluster as other isolates in South China in recent years. In this respect, GD19GZ01 through GD19GZ04 have the highest consistency with GDHN-YM2, GDHN-YM1, K243, and M180, respectively. There were also obvious regional differences among the same subgroups, and the sequence identity of strains from the same region was relatively high .gag and pol genes. The P values for the six algorithm results (from PDR5 software) are in Supplementary Table Several potential recombination events were detected by PDR5, but only three of them were accepted as being robust. The recombination sequence of event 1 was for GD19GZ01, and its parental sequences were M180 (ALV-J) and AF227 (ALV-E); that of event 2 was for GD19GZ02 and its parental sequences were GDHN-YM1 (ALV-J) and AF227 (ALV-E), and that of event 3 was GD19GZ02 and its parental sequences were GDHN-YM1 (ALV-J) and SD07LK1 (ALV-J) Figures . All posCH25H, MX, PKR, OAS, and ZAP, have been proven to block virus invasion in different ways to protect cells from infection [CH25H, MX, OAS, and ZAP was significantly upregulated in the cecal tonsils, kidneys, and livers of the four sick chickens , and this is an important line of defense for the innate immune response . These Infection –22. Therchickens . At the egulated .4TCID50/0.1 ml, after which the level of viral RNA synthesis and viral particle content were detected at different time points. The qPCR results indicated that GD19GZ02 and GD19GZ03 underwent higher levels of replication at 1 dpi (days postinoculation) than the other two strains, a difference that was an extremely significant difference. However, the virus replication levels of GD19GZ03 and GD19GZ04 became the highest at 3, 5, and 7 dpi. The results of ELISA and qPCR were basically the same. It is worth noting that GD19GZ04 had a low level of virus replication early on, at 1 dpi, but later on, the virus was clearly capable of a higher level of replication. At any time period, the virus replication level of GD19GZ01 was the lowest among the four isolates  of those virus isolates featured an E element deletion. Coincidentally, the number of strains lacking the rTM region was also 50%, so whether it was an isolate that caused either hemangioma (He) or myeloma (ML), the rTM region might still be lost. This suggests that losing the rTM region was probably not related to the type of tumor elicited in the host. Nevertheless, only 15.3% (4/26) of the isolates simultaneously lacked both the rTM region and E element; evidently, then, the deletion of these two regions at once is a relatively rare phenomenon. The specific function of the E element remains unclear, however. Some researchers believe that despite not being essential for tumorigenesis induction by viruses, the E element nonetheless contributes to oncogenicity in certain genetic lines of chicken . In the env gene, which encodes two proteins, GP85 and GP37, of which the former is highly variable while the latter is relatively conserved [env gene's variation in the four isolates was relatively large, and its consistency with the other reference strains was only 56.1%–97%. Most of these mutations occurred in the hypervariable region hr1, hr2, and the variable region vr3 of GP85.The unique pathogenicity of ALV-J is considered to be closely related to its special onserved , 29. In CH25H, MX, OAS, and ZAP were all significantly upregulated in multiple immune organs (CH25H was proven to inhibit the replication of ALV-J by producing 25HC [Chicken type-I interferons (I-IFN) play a dominant role in the chicken immune system and mediate the first line of defense against viral pathogens infecting the avian species . When IFe organs . Among ting 25HC . HoweverIL-4 and IL-6 were significantly upregulated in the liver, and both are known to have marked inhibitory effects on the expression and release of proinflammatory cytokines [TNF-α was inhibited in the liver. Although IL-10 was downregulated in the liver of chickens, it was significantly upregulated in the cecal tonsils, spleen, and heart (IL-12, IL-18, and TNF-α that were all significantly upregulated in most of their immune organs. It is worth mentioning that the expression of anti-inflammatory cytokines and proinflammatory cytokines increased abnormally in GD19GZ01. Despite complexities inherent in inflammatory cytokines, therapeutic interventions with specific cytokine inhibitors or anti-inflammatory cytokines have already been shown to confer significant clinical benefits in humans [A dynamic and ever-shifting equilibrium exists between proinflammatory cytokines and anti-inflammatory components of the immune system . Accordiytokines . Therefond heart . The invn humans . Howevergag and pol genes. In recombination events 1 and 2, the recombination fragment is composed of an exogenous retrovirus and an endogenous retrovirus. Actually, in the early days, there was a view that ALV-J is a virus obtained by mutual recombination of exogenous retrovirus and endogenous retrovirus [Finally, we used software to detect the occurrence of three viral recombination events on GD19GZ01 and GD19GZ02, all of which were found between the trovirus . It was trovirus , 36. TheIn summary, we isolated four ALV-J from yellow chickens on a farm in South China and performed detailed sequence analyses of them. The results further demonstrate the complexity of ALVS infection in chicken flocks in different regions. Our findings thus help to reveal the clinical symptoms and sequence characteristics of ALV-J' infection in yellow chickens in South China and, more generally, they provide valuable data for elucidating the evolution and variation trends of ALVs.


Plant Virus Emergence”. The issue includes a series of articles that elaborate on important plant virus diseases that are among the most recent epidemiological concerns. This Special Issue is predicated on the paradigm that plant virus epidemiology, outbreaks, epidemics, and pandemics parallel zoonotic viruses, and can be consequential to global food security [Homo sapiens: the superspreader of plant viral diseases” [Disease Pandemics and Major Epidemics Arising from New Encounters between Indigenous Viruses and Introduced Crops Viruses” [We are pleased to present in this Special Issue a series of reviews and research studies on the topic of “security . There isecurity ,3. As coiseases” . This thViruses” , Jones cViruses” , Hendra,Viruses” . ClimatePotato virus Y emergence and evolution from the Andes of South America to become a major destructive pathogen of potato and other solanaceous crops worldwide”, Torrance and Talianksly [Family Level Phylogenies Reveal Relationships of Plant Viruses within the Order Bunyavirales” [Other articles in this Special Issue describe plant viruses that are studied for their associations with “outbreak” phenomena (sudden appearance), reservoir hosts and “spillover” phenomena (transfer from wild to domesticated areas), common genetics, and reliance on “vectors” for viral spread. In “lianksly  discuss virales” , the impTransmission of the Bean-Associated Cytorhabdovirus by the Whitefly Bemisia tabaci MEAM1” [Can Winged Aphid Abundance Be a Predictor of Cucurbit Aphid-Borne Yellows Virus Epidemics in Melon Crop?” [Bemisia tabaci Middle East-Asia Minor 1 (MEAM1) transmission of bean-associated cytorhabdovirus to common bean, and with a lower efficiency to cowpea and soybean. However, not only is the particular vector important, but the timing of its occurrence in fields relative to crop age can influence emergence. For example, the abundance of A. gossypii during the first two weeks after planting is a good predictor of disease caused by CABYV. Early control of the vector is necessary to minimize the potential for CABYV epidemics in melon crops.Insects are significant drivers of virus emergence, as featured in submissions by Pinheiro-Lima et al., “i MEAM1” , and Schn Crop?” . These sRNAseq Analysis of Rhizomania-Infected Sugar Beet Provides the First Genome Sequence of Beet Necrotic Yellow Vein Virus from the USA and Identifies a Novel Alphanecrovirus and Putative Satellite Viruses” [Increasingly, scientists depend upon advanced DNA and RNA sequencing technologies to monitor the emergence and spread of new plant virus strains or species, facilitate novel virus discovery, and uncover the etiology of complex diseases. In the article by Weiland et al., “Viruses” , advanceEmergence and Full Genome Analysis of Tomato Torrado Virus in South Africa” [Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants” [SARS-CoV-2 is the third coronavirus to emerge in recent years, SARS and MERS being the other two, suggesting that virus surveillance and discovery in reservoir species is important preparatory work for modeling disease outbreaks and planning for vaccines before their emergence in humans. The discovery of emergent virus species and strains requires further molecular characterization. Moodley et al., in “ Africa” , report  Plants”  produce  Plants”  review tPlant Virus Emergence” highlight elaborate efforts in plant virology that support broad models for virus outbreaks, spillovers, genetics, reliance on “vectors” and human trade for spread, as well as the maintenance of viruses in “reservoir” hosts. In closing, we thank all of the authors for their enlightening contributions to this Special Issue.We hope that you find the collection in this Special Issue informative and of interest. As the public becomes informed of scientific theories on virus disease emergence and spread during the COVID-19 pandemic, it is timely that plant viruses are included in the discussion. We hope that the articles in this Special Issue on “


Cryptococcus bacillisporus (VGIII) in an immunocompetent patient from Santa Cruz, Bolivia. This first report of a member of the Cryptococcus gattii species complex from Bolivia suggests that C. bacillisporus (VGIII) is present in this tropical region of the country and complements our epidemiological and clinical knowledge of this group of emerging fungal pathogens in South America.We describe a case of chronic meningoencephalitis with hydrocephalus caused by  Cryptococcus gattii species complex are emerging human and veterinary pathogens found in soils and in association with trees in various parts of the world [C. neoformans, which mainly causes opportunistic infections, C. gattii causes substantial morbidity and mortality in immunocompetent individuals and has the potential to cause outbreaks [Cryptococcus, former C. gattii genotypes (VGI–VGIV) were reassigned as five distinct species with differing epidemiological features, with a possible additional genotype/species reported from Sub-Saharan Africa [C. gattii species complex nomenclature when referring to all genotypes (VGI–VGV) and use the suggested species names when referring to single genotypes.Members of the he world . Initialhe world ,3. Unlikutbreaks . Due to n Africa ,5. As thn Africa ,6,7, in C. gattii species complex is endemic in Brazil and Colombia, although clinical and environmental isolates were also reported from Venezuela, Argentina, Peru, Paraguay, Uruguay, and Chile; most of them belonging to the species C. deuterogattii (VGII) and C. bacillisporus (VGIII) [Cryptococcus species in South America are incompletely understood [C. gattii complex in Bolivia, which was from a patient with chronic meningoencephalitis caused by C. bacillisporus (VGIII) acquired in the Santa Cruz region.In South America, the  (VGIII) ,9. Howevderstood . Here, wHistoplasma capsulatum, and tuberculosis. While a chest CT scan was unremarkable, brain MRI showed leptomeningeal infiltrates and hydrocephalus, together with ganglionar and periventricular edema and cystic lesions  was positive (1:64); Ziehl Neelsen and India ink stains as well as Xpert MTB/RIF were negative. CSF cultures on Sabouraud agar showed small whitish colonies, which produced a blue color on canavanine–glycine–bromothymol blue agar, compatible with C. gattii species complex. MALDI-TOF mass spectrometry  confirmed the strain as “C. gattii” with the maximum confidence score (99.9%). PCR and subsequent restriction fragment length polymorphism (RLFP) analysis of the URA5 gene classified the strain as C. bacillisporus (VGIII). Further multilocus sequence typing (MLST) and specific PCR tests [C. bacillisporus sequence type 79 (ST79). Antifungal susceptibility testing by microdilution using the Clinical and Laboratory Standards Institute (CLSI) M27 protocol [A 48-year-old previously healthy man from Santa Cruz, Bolivia, attended Clínica Alemana de Santiago (Chile) with a 12-months history of progressive neurological problems. Symptoms had started with malaise, tremor, and headache, later accompanied by dysphagia, dysphonia, singultus, progressive severe cognitive deterioration, and a weight loss of 10 kg. The patient denied former travel outside Bolivia; his visit to Chile was for medical reasons, i.e., evaluation of the current illness. At presentation, the patient was prostrated and showed severe memory disorder, right central facial paralysis, right brachial and crural paresis, and symmetric postural tremor. Routine laboratory exams were normal, including tests for HIV, syphilis,  lesions . CerebroCR tests , performMonotherapy with liposomal AMB  was started since flucytosine is not available in Chile. After 2 weeks and clinical improvement, the patient decided to continue treatment with oral fluconazole (400 mg bid) in Bolivia. Four months later, he returned with severe headache, cognitive impairment, tremor, and severe gait disorder. A brain MRI showed decreased leptomeningeal enhancement, but persistent hydrocephalus and multiple cystic lesions of increased size. CSF samples demonstrated low glucose (11 mg/dL), elevated protein (296 mg/dL), pleocytosis , and were positive for the cryptococcal antigen by latex agglutination (1:64) and lateral flow testing . Endoscopic third ventriculocisternostomy was performed, showing cotton-like whitish material . BiopsieC. deuterogattii (VGII) reference strains. In short, 20 last instar larvae of Galleria mellonella weighing 230–330 mg without any grey marks were injected with 106 cryptococci per animal in PBS, as previously described [Cryptococcus isolates, known to be highly-virulent (R265) and avirulent (CBS7750) in a mouse model [p = 0.0009), suggesting a low virulence of this isolate in this model , which causes outbreaks related to few hypervirulent clones, clinical isolates of C. bacillisporus (VGIII) have a higher genetic diversity [C. gattii complex, leading to prolonged survival and chronic infections [CAP59 gene [Galleria mellonella model, suggesting that the Bolivian isolate was of low virulence. As this case suggests, such isolates may still cause CNS infections in non-immunocompromised individuals presenting as subacute meningoencephalitis without evident signs of infection such as fever [C. deuterogattii (VGII), often involving pulmonary manifestations. The chronic course of C. bacillisporus (VGIII) together with the prolonged incubation period may lead to misdiagnosis or significant delays including the risk of a fatal outcome [C. gattii [The taxonomy of human pathogenic riations . Howeverriations . The pre America . Most ca America ,8,19. University . This spfections . Two maifections  and seroP59 gene . The preP59 gene ,21,22,23P59 gene . This isas fever , in cont outcome ,26. The  outcome ,28. An a. gattii . The clinical management in resource-poor settings is problematic, as essential antifungals are often not accessible or of uncertain quality . FlucytoC. bacillisporus (VGIII) is present in the tropical lowlands east of the Andes Mountains in Bolivia and has to be considered as a cause of CNS infections in this region. Further studies are necessary to understand the distribution and clinical relevance of this and other C. gattii complex species in Bolivia and neighboring countries. Our case also highlights the importance of diagnostic cooperation with mycology reference laboratories across national borders, to improve individual patient management and also to understand the epidemiological landscape and risk factors regarding this group of emerging fungal pathogens.In conclusion, our report suggests that


Amalgaviridae and Partitiviridae. Furthermore, rolling circle amplification and restriction enzyme digestion revealed the complete genome of chickpea chlorosis Australia virus, a mastrevirus (family Geminiviridae) previously reported only from chickpea and French bean that was 97% identical to the chickpea isolate. The sequence data also enabled the assembly of the first complete genome (RNAs 1–3) of an Australian AMV isolate from alfalfa.Alfalfa plants in the field can display a range of virus-like symptoms, especially when grown over many years for seed production. Most known alfalfa viruses have RNA genomes, some of which can be detected using diagnostic assays, but many viruses of alfalfa are not well characterized. This study aims to identify the RNA and DNA virus complexes associated with alfalfa plants in Australia. To maximize the detection of RNA viruses, we purified double-stranded RNA (dsRNA) for high throughput sequencing and characterized the viromes of ten alfalfa samples that showed diverse virus-like symptoms. Using Illumina sequencing of tagged cDNA libraries from immune-captured dsRNA, we identified sequences of the single-stranded RNA viruses, alfalfa mosaic virus (AMV), bean leafroll virus, a new emaravirus tentatively named alfalfa ringspot-associated virus, and persistent dsRNA viruses belonging to the families  Medicago sativa L.) is a legume cultivated in more than 80 countries on about 45 million ha and used as silage for grazing livestock [Alfamovirus, family Bromoviridae), bean leafroll virus , and cucumber mosaic virus  are the most frequently detected viruses in Australian alfalfa pastures [Alfalfa or lucerne  and alfalfa enamovirus 1 , and the DNA virus alfalfa leaf curl virus  [Nucleorhabdovirus, family Rhabdoviridae), alfalfa virus F , alfalfa virus S , Medicago sativa deltapartitivirus , and alphapartitiviruses including Medicago sativa alphapatitivirus 1 and 2 (family Partitiviridae) were identified in alfalfa with HTS [In recent years, several novel viruses have been identified in alfalfa worldwide, with the help of high throughput sequencing (HTS). For example, in 2010, alfalfa dwarf disease (ADD), which affects hay yield and quality of alfalfa, was reported from Argentina . HTS of viridae) ,10,11,12with HTS ,16,17,18Non-targeted HTS is a broad-spectrum diagnostic screening tool with high sensitivity to detect even low-titre viruses. It has many advantages over serological and molecular detection methods that require specific antisera or prior knowledge of nucleotide sequences . MetagenHTS approaches to identify viruses and viroids mainly differ in (1) plant sample preparation by enriching various types of genetic materials including messenger RNA (mRNA), total RNA with or without ribosomal RNA depletion, nucleic acids extracted from purified virions, small RNAs, and double-stranded (ds) RNAs; (2) sequencing platforms; and (3) bioinformatics used for assembly of contigs and viral genomes ,30,31,32Sequencing total plant RNA generates more than 90% host transcript data, which is a challenge for detection of low-titre viruses . The abuBoth DNA and RNA viruses can be detected by sequencing of small RNAs generated by RNA silencing during plant defence ,37,38. HIn this study, we used metagenomics with Illumina sequencing of tagged cDNA libraries derived from immuno-captured dsRNA to profile the RNA viromes of ten alfalfa samples that showed diverse virus-like symptoms. We specifically sought to identify the biodiversity of alfalfa RNA viruses and to characterize viral taxa that may be new to Australia. We also investigated the possible presence of novel DNA viruses circulating within Australian alfalfa pastures, which may pose a biosecurity threat for seed and hay production. DNA viruses were detected by Sanger sequencing of cloned rolling circle amplified DNA from selected alfalfa samples with leaf curl/roll-like symptoms that had tested negative for ALCV .Emaravirus, family Fimoviridae, order Bunyavirales), and the persistent viruses Medicago sativa alphapartitivirus 1 (MsAPV1) and Medicago sativa amalgavirus 1 (MsAV1)  . Taxonom (MsAV1) . RNA vir (MsAV1) . Based o (MsAV1)  and emar (MsAV1) . Caulimoviridae family, sharing 43% and 34% nt sequence identity with the reverse transcriptase gene of soybean chlorotic mottle virus and cauliflower mosaic virus, respectively (Medicago sativa (GenBank KU321683).BLASTx searches of reads belonging to samples 1 and 8  as well ectively . Howeverectively . The remEcoRI yielded a ~3-kb band, indicative of a full-length DNA virus genome, only from sample 11  chickpea chlorosis Australia virus (CpCAV) (GenBank KC172686). Restriction enzyme digests of RCA products derived from thirty-four selected symptomatic alfalfa samples (leaf curling and minor leaf enation) using ample 11  which diample 11 . HindIIIIn all tested alfalfa samples, BLAST analysis of contigs ranging from 1706 nt to 3583 nt showed 95–100% nt sequence identity to RNAs 1, 2, and 3 of AMV isolates on GenBank . The preDue to the low number of reads (146) that mapped to BLRV, the complete sequence of BLRV genome could not be assembled from sample 10, the only sample infected with this virus in this study. Unresolved genome regions included the RNA-dependent RNA polymerase (RdRP), CP and movement protein genes, and the 3’ end of the intergenic region between open reading frames (ORF) 2 and 3 . The BLRFimoviridae, order Bunyavirales) sequences available in GenBank  RdRP (MK292286), and CP (MK292287), and Medicago sativa amalgavirus 1 , with a genome coverage of 16 to 2657-fold, 16 to 737-fold, and 8 to 836-fold, respectively  contains two partially overlapping ORFs in the positive strand . ORF1 stCicer arietinum)  was identified in alfalfa sample 11 from South Australia, which was also infected by AMV and showed leaf rolling and yellowing symptoms . The comietinum) a. The knietinum) b. Nucleoietinum) .Amalgaviridae and Partitiviridae families, previously reported in alfalfa from other countries [We identified several known and one novel RNA virus through HTS of 10 alfalfa samples in Australia that showed a range of virus-like symptoms including yellow mosaic, ring spots, and leaf curling. We also identified and determined the complete genome of the ssDNA virus CpCAV from another alfalfa sample with leaf curl/roll-like symptoms following rolling circle amplification. The identified RNA viruses included the endemic AMV and BLRV ,5,45, a ountries ,18,52,53ountries .In this study of alfalfa viromes, we sequenced cDNA libraries from immune-captured dsRNA, followed by a bioinformatics pipeline to identify and assemble viral sequences. Viral nucleic acid enrichment in metagenomics has accelerated detection of novel viruses especially in large-scale surveys ,34,35,36AMV was identified in all ten alfalfa samples confirming that this virus was wide-spread in plants grown for seed and hay . In the A small number of diagnostic BLRV reads were found in only one sample. A recent limited survey using CP-specific primers in RT-PCR detected BLRV in 70% of symptomatic samples , but thiFimoviridae, order Bunyavirales that are transmitted by eriophyid mites [Sequence reads that most closely matched emaraviruses were identified in four samples, two from seed paddocks in South Australia and two from hay paddocks in Victoria. Based on current scientific literature and available GenBank data, there are no emaraviruses known to infect alfalfa. Based on all four samples showing ringspot symptoms on the leaves, the associated virus was tentatively named alfalfa ringspot-associated virus (ARaV). Emaraviruses are enveloped, segmented negative-sense RNA plant viruses in the family id mites ,53. Receid mites ,56,57,58The sequence coverage for the negative-sense RNA virus ARaV was considerably less than for the positive-sense RNA genomes of AMV and BRLV and the dsRNA genomes of MsAV1 and MsAPV1. The percentage of reads that mapped to a consensus sequence of ARaV was also lower than for the other RNA viruses detected in this study. This concurs with the findings by Weber and collaborators  that sufMastrevirus (family Geminiviridae) have ssDNA genomes, are transmissible by leafhoppers, and can infect both mono- and eudicotyledonous plants [Although HTS data suggested a caulimo-like virus may be present in some samples, this was not confirmed by RCA and restriction enzyme digestion. The detected caulimovirus-like sequences may instead be derived from endogenous integrated elements . Howevers plants ,64. In aPartitiviridae, Amalgaviridae, Totiviridae, Endornaviridae, and Chrysoviridae are known as persistent cytoplasmic plant viruses. Persistent viruses have been shown to infect plants or plant-interacting fungi [Zea mays chrysovirus had only changed by 3% in 1000 years [Viruses that are taxonomically classified in the families ng fungi ,66,67. Tng fungi . The pre00 years . Althoug00 years .Medicago sativa deltapartitivirus 1 [Previous studies have identified and characterized dsRNA viruses from alfalfa that are associated with persistent, non-symptomatic infections including MsAPV1, MsAPV2, and ivirus 1 ,18. The ivirus 1 ,17,18. TCucumis melo cryptic virus, Cucumis melo amalgavirus 1 from melon [Raphanus sativus cryptic virus from radish [Salvia hispanica amalgavirus 1 from chia [Capsicum annuum amalgavirus and southern tomato virus, do not contain this conserved motif [Analysis of a transcriptome dataset also identified persistent dsRNA viruses in other plant species, including om melon , a novelm radish , and Salrom chia . Most ofrom chia ,72. The ed motif ,72.An HTS study of small RNAs from alfalfa in China recently detected AMV, ADV, and ALCV  that hadOur study is the first dsRNA-based HTS virome analysis reported for alfalfa in Australia. In addition to the endemic AMV and BLRV, we have identified −ssRNA genome segments associated with a new emaravirus and dsRNA genomes of an alphapartitivirus and an amalgavirus. RCA and restriction enzyme analysis also revealed alfalfa as an alternative host of chickpea chlorosis Australia virus.Alfalfa leaf samples with diverse virus-like symptoms including leaf curling, yellow mosaic, ring spots, leaf enation, and reddening were selected from a 2015–2017 collection of Australian samples from seed crops near Keith, South Australia and from fodder crops from Hamilton and Edenhope, Victoria . All sam® 20 (TBST) was added, vortexed, and centrifuged at 10,000 rpm to remove plant debris. Pierce protein L magnetic beads  (10 μg/ul) were washed two times with 150 μL and 1000 μL, respectively, of TBST as per the manufacturer’s protocol and were then coated with 500 μL of dsRNA-specific mAb 4G2 [DsRNA was purified from freeze-dried alfalfa leaf samples as described by Blouin and collaborators  with min mAb 4G2  and incuFor reverse transcription (RT), 6 μL of the resuspended dsRNA-bound beads was mixed with 3 μL of 20 μM random N6 primer (5′-CCTTCGGATCCTCCNNNNNN-3′) and 9 μL of nuclease-free ultrapure water and heated at 95 °C for 2 min followed by immediate cooling on ice for 1 min. After denaturation, 300 units (1.5 μL) of SuperScript III reverse transcriptase (Thermo Fisher Scientific), 1.5 μL of 10 mM deoxyribonucleotide triphosphate, 6 μL of 5 × buffer, and 3 μL of 100 mM dithiothreitol earlier mixed in a separate PCR tube were added to the RT-mix. The mix was kept on ice for 15 min and then incubated at 50 °C for 1 h. This was followed by treatment with 0.375 μL ribonuclease A  at room temperature for 15 min and enzyme inactivation at 85 °C for 2 min. The cDNA was column-purified using the PCR Clean-Up System  and eluted in 30 μL of ultrapure nuclease-free water.® Ultra™ DNA Library Prep Kit for Illumina®  and HTS were done by Genewiz, Inc.  on an Illumina MiSeq in a 2 × 250 bp paired-end format.cDNA was amplified by PCR and individual barcodes  incorporEcoRI, gel-purified and ligated using T4 DNA ligase (NEB) into pUC19 that had been linearized by EcoRI digestion and dephosphorylated using Antarctic Phosphatase (NEB). Three recombinant clones per amplicon were sequenced in both directions at the Australian Genome Research Facility  using M13F and M13R and internal primers. Trimmed sequence reads were assembled into contigs and a consensus sequence generated for each cloned DNA using Geneious R 9.1 . Tomato yellow leaf curl virus  was used as a positive control. Individual consensus nucleotide sequences were searched against GenBank databases via BLASTn and BLASTx and mapped to the closest reference sequence using Geneious.Viral DNA genomes were amplified by RCA from total DNA extracts  using phhttps://usegalaxy.org/). Trimmomatic was used with default parameter settings to remove low-quality reads from the Illumina sequence data, and read quality was assessed by FASTQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Using barcode splitter, (https://toolshedg2.bx.psu.edu/view/devteam/fastx_barcode_splitter), individual sequence reads were de-multiplexed. Barcodes listed in http://kaiju.binf.ku.dk/) [HTS reads were analyzed in the Galaxy web-based platform (.ku.dk/)  using th.ku.dk/) .®Taq DNA polymerase (Thermo Fisher Scientific) following the manufacturer’s protocol. RT-PCR conditions were 50 °C for 30 min, 94 °C for 2 min, 35 cycles of 94 °C for 15 s, 55 °C for 30 s, and 68 °C for 1 min, followed by a final extension at 68 °C for 5 min. Amplicons were analyzed on 1% agarose/TBE gel, DNA bands stained using Redsafe™  and visualized under UV light using a gel documentation system . DNA bands were excised, gel-purified using Wizard® DNA clean up system (Promega), and quantified by Nanodrop® spectrophotometer. Amplicons were either sequenced directly or first ligated into pGEM-T Easy (Promega) and sequenced at the AGRF using M13 forward and reverse primers.The identity of virus-like sequences detected by HTS was verified by RT-PCR using primers designed from the viral contigs and targeting RdRP and CP or NC genes of the viral genomes . RT-PCR Sequences of type isolates and representatives of phylogroups related to the viruses that were identified during HTS analysis in this study were retrieved from GenBank for phylogenetic analyses. De novo assembled contigs were subjected to bulk BLASTn and BLASTx searches. Reference assemblies of complete or partial viral genomes from reads of the individual ten barcoded libraries were done using Geneious with default parameters, and the reads were mapped against the most similar viral genomes identified in BLASTn and BLASTx. Multiple sequence alignments based on the complete viral genomes, NC, CP, and RdRP nucleotide and amino acid consensus sequences of the viruses identified in this study and published sequences available on GenBank were generated by MUSCLE, and conserved domains prediction of the CP and RdRP were done using Geneious. Phylogenetic trees were inferred from the aligned sequences using the NJ method implemented in Geneious with 1000 bootstrap replicates, and branches with less than 60% bootstrap support were collapsed. Pairwise identity analyses of the viral genome sequences were implemented by the MUSCLE-based pairwise alignment option using Sequence Demarcation Tool (SDT v1.2) . For CpChttp://rna.urmc.rochester.edu/RNAstructureWeb/. The protein structure was predicted with Emboss online software (http://www.bioinformatics.nl/cgi-bin/emboss/garnier), which is based on the Garnier Osguthorpe–Robson method [http://bat.ina.certh.gr/tools/circoletto/) based on BLASTP searches with an E-value of 1e−1 threshold [RNA secondary structure was predicted with RNA structure prediction tool  availabln method . The aa hreshold .


Pakistan is in the grip of COVID-19, due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) since 26 February 2020, and the number of infected people and mortality is rising gradually. The health workers, doctors, pathologists and laboratory staff are front line fighters who are facing the risk. Few things are important for public and health workers, human behavior is at the core of preparedness and response i.e, personal protective measures, , social distancing and travel measures because the virus spreads through the respiratory channels, eyes, nose and mouth. While working in the Pathology labs, use the personal protection equipment (PPE), during the work in the duty. Avoiding the over duties and long shifts. It is good to keep the immune system healthy by taking a healthy balanced diet, vitamin supplements, and a night of proper sleep. It is also important to avoid taking food during duties and avoid making close contact without wearing safety dress. Pakistan is a developing country with a population of more than 220 million, with low hygienic facilities and insufficient awareness about the current Outbreak of COVID-19. Due to the global available date, the virus has been widespread since December 2019. “How can we go on to make an standard operating procedure (SOP) list off “what we have to do and what are the precautions that we should adopt to save ourselves, staff working in the labs, is very important.” It is important to cut off human-to-human transmission including reducing the spread of infection among close contact and workers of the health care, ceasing the transmission boosting routes, and preventing the pandemic. The social behaviour is very important to controll the outbreak of this virus. At laboratory level, classification is done in terms of arrangements to keep any virus safe. Conjecture is that the outbreak of coronavirus was spread from Wuhan, that the SARS-CoV2 may have leaked from her institute, where a lab certified as BSL-4, the highest level for handling dangerous pathogens, opened three years ago. How it is possible that an outbreak has taken place, is this a biological terrorism? Is this a part of the biological war? All these question raise and the world should unite to search the answers. How can we save our Pathology staff, because we are the people who are directly dealing with the infected material, the outbreak has provided some common sense tips that you have likely seen elsewhere by now, it also highlighted what should be done to keep the lab safe.4The initial important points included in SOP are washing of hand for 20 seconds, after touching anything in general which has been touched or used by others in a day many times, such as door handles, stair bars, light switches, elevator buttons, ATM Machines etc. In labs pippets, microscopes, knobs and all other instruments and machinery which are used should not be touched before being cleaned properly and everything should be used wearing gloves. The PCR areas are very sensitive hence should not be used without adopting the SOP for these labs. Sample collection of the suspected people should only be collected wearing PPE protected gloves, goggles and N95 masks. These samples should be transported carefully to the PCR labs. By following the advice of National Institute of Health (NIH) and Centers for Disease Control (CDC). Everything that is associated with infected people will be contaminated and potentially infectious. The virus is on the surfaces and one will not be infected unless one has unprotected face is directly coughed or sneezed upon.6When you are in labs use disposable overalls, disposable gloves or paper towels especially when you use these things or lift the gasoline dispenser. The restrooms, grocery places, baby carts at commercial places, offices are commonly used by hundreds of people daily, at such places, when you need to open the doors, use your fist to turn the opening position and use your hip or elbow to open it to avoid grasping the handle with your hand. If there is no other way to open the door, then wash hands before starting the work. Use the latex or nitrile latex disposable gloves for use.Use alcohol-based hand sanitizer whenever you return home from your duty activity that involves locations where other people have been present. Installation of sanitizers at your home entrances, and even in your cars for use after getting gas or touching other contaminated objects when you can’t immediately wash your hands is advised. One should change clothes and take a hot bath after coming home from work. Keep the things outside which you have used at work.5When you cough or sneeze, always use disposable tissue papers and then dump them or discard properly. Avoid touching the clothing near your wrists, use the clothes up to the elbow because the clothing on your writs will contain an infectious virus that can be passed up in the next few days. Avoid shaking hands, touching your face, hugging the guests, relatives kissing the babies as a cultural routine in different countries, in Arabs and in Saraiky culture in Central Pakistan, South Punjab.7This current outbreak of COVID-19 has repeated the memories of the previous outbreaks of SARS-CoV , MERS CoV  resulting in mortalities 858 (37%) and 744 (10%) respectively. It is not yet clear whether the disease was first transmitted to humans by animals or through contamination levels by the contact.8However, it is now clear that the COVID-19 is spreading through droplets by coughing and sneezing, it cannot reach if the person is at a distance of two meters from infected person but if the air is not infecting you, but contaminating, all the surfaces where these droplets are striking can infect others trough touching the infected surface for about a week on average.10The current virus can infect the lower respiratory region; due to having cell receptors for lungs. The route of infection is through nose, mouth, and eyes, through coughing, sneezing, touching the infected surfaces and helping the virus to enter in the mucous membrane, through hands. These infections may be serious in patients whose immune system is compromised i.e., diabetes, blood pressure, Tuberculosis (TB), chronic obstructive diseses (COPDs), hepatitis or any heart disease, they need to be more cautious. Younger children and the elderly need more care. Avoid going to the fish or meat markets and stay away from pets and animals kept at home. Be careful about the family and relatives of people who have had the virus.9The use of disposable surgical masks and spectacles prevent the virus in a direct sneeze from getting into your nose or mouth as well as it can keep you safe from touching the eyes, nose or mouth, because unintentionally we touch these areas many time a day and eat things without washing the hands. There will be no drugs or vaccines available this year to protect us or limit the infection within us. Only symptomatic support is available.” But there are certain important things to boost your immunity, like vitamic C and D. The use of Remdesivir (antiebola), Favipiravir (antiflu), lopinavir (anit HIV) hyrdoxy choloquine, chroloquine phasphate,  azithromycin, (antibiotic), interferon beta, antibody therapy all are still not approved by the FDA, but the use of these drugs has shown better recovery in the patients suffering from life threatening COVID19.10haa” sound, then spit out the water after a few seconds and repeat the process 3-5 times. Because viruses or bacteria enter in the nasopharynx through the nostrils, by attaching the mucosa, the diluted salt mixed light hot water can kill them on the spot at about the temperature of 26-27ºC, thereby achieving the purpose of preventing infection. During last SARS, the method was taught to the students to prevent the attack of this virus, and none of the students in the class got cold, cough or fever. This method is simple, effective, easy to do. It was just his observation which may require perseverance.11It has been suggested that washing your mouth, throat, and the larynx with light warm salt mix water by doing gargles, every day in the morning after breakfast before leaving to your laboratories, every day may not allow the virus to attach your mucous membrane. Nanshan Z* (SARS coronavirus in 2003) suggested simple ways to prevent pneumonia by rinsing throat with light salt mix hot water before going to the hospital or other public places (and do the same after you return home), may stop the invasion of this new virus. The method is as follows: take a mouthful of dilute salty light hot water, raise the head; let the salt mixed water stay around the throat area, open the mouth slightly and exhale it slowly, let the air bubbling through the water in the throat by gargling with “During your duties in laboratories, imbibe the Zinc or vitamin C containing tablets which may help in stopping any virus and especially these days Coronavirus (and most other viruses) from multiplying in the throats and nasopharynx as they improve the immunity. The Zinc is required for the normal functioning of living cells. And vitamin C contributes to immune defense by supporting various cellular functions of the immune system. The Zinc acts as co-factor, for the enzymes involved in the cell cycle for the DNA replication and transcription, i.e., plays a significant role in the growth and tissue maintenance.12A Pathologist, Eby GA, has conducted a clinical trial on the use of zinc gluconate lozenges for the treatment of the flu, according to his observations, the zinc gluconate improves the human immune function by protecting and curing the common cold, as a nonspecific antiviral effect. In so many studies, zinc is supplemented to patients under immunosuppressive therapy especially for cancer. Once George Eby used a zinc gluconate tablet for his 3-year-old daughter, who was going under chemotherapy for acute T-cell lymphocytic leukemia. He used this to recover her from a cold within several hours after dissolving in her mouth rather than swallowing this tablet.13Now if you use these zinc lozenges several times each day when you begin to feel any “cold-like” symptoms. It is best to lie down and let the lozenge dissolve in the back of your throat and nasopharynx. Cold-Eeze lozenges could work as a ‘silver bullet’ that would kill coronavirus. The zinc will inhibit the replication of many viruses, including coronaviruses. We cannot claim but can expect that the COVID-19 may be inhibited similarly. Take vitamin A, D, and Vitamin C, because the literature shows these vitamins may support the human immune system.14The disease has become a pandemic, with lots of moralities, but there is no need to panic, because 80% of the cases, it is mild, 14-15% moderate and only 5% severe, with the mortality rate 1.5-5% in different countries. Following the precautionary measures advised by WHO and improving immune system with good diets, it can be controlled. In COVID19 so far, the death toll has not shown the trend than the other pandemics of history like Spanish flu, plague, Zika virus, and Ebola virus. What should be done to keep the immunity strong and take special care of cleanliness?16Hand washing, using face masks, adopting respiratory etiquette, and cleaning surface and objects, social distancing and travel measures can protect us from the COVID19. The identification and follow up of the contacts, to reduce the community spread. Creation of aware of the infection prevention, implementation of health measures and risk communication to the public is important in current conditions rather making lock down. The health workers, laboratory staff, pathologists should use surgical mask, and PPE kits, N95 masks during dealing with the patients or material of the infected person. Take good healthy food, optive drugs, and vitamin supplements to maintain proper health.Few things are important for public and health workers, like handwashing, face masks, respiratory etiquette, surface and objects cleansing), social distancing and travel measures because the virus spreads through the respiratory channels, eyes, nose and mouth. While working in the Pathology labs, use the personal protection equipment (PPE), during the work in the duty. Avoiding the over duties and long shifts. It is good to keep the immune system healthy by taking a healthy balanced diet, vitamin supplements, and a night of proper sleep. It is also important to avoid taking food during duties and avoid making close contact without wearing safety dress.