We also explored different hairpin reporter methods such as biotin/streptavidin-AP and the Alexa Fluor-488 Fluorophore. TB-HCR has applications for low-cost diagnostics for large sample numbers, rapid diagnostic deployment for new viruses, and can be performed as a preliminary triage assay prior to downstream applications.Assays for the high throughput screening of crops for virus monitoring need to be quick, easy, and low cost. One method involves using tissue blot immunoassays (TBIA), where plant stems are blotted onto nitrocellulose membrane and screened with available antibodies against a range of viruses. TBIAs are inexpensive but limited by antibody availability and specificity. To circumvent the antibody limitations, we developed the tissue blot hybridization chain reaction (TB-HCR). As with TBIA, plant stems are blotted onto a nitrocellulose membrane, however, TB-HCR involves using nucleic acid probes instead of antibodies. We demonstrated for the first time that TB-HCR can be used for plant viruses by designing and testing probes against species from several virus genera including  Techniques for virus identification over the last decade have improved significantly and allowed for more precise identification of viruses. However, most of these techniques such as reverse transcription (RT)-PCR, high throughput sequencing, loop-mediated isothermal amplification (LAMP), and real-time PCR are expensive when large numbers of plant samples need to be screened. Breeding programs aimed at developing virus resistant lines often test large numbers of plant samples  for multiple viruses. Plant surveys aimed at identifying a range of viruses across a broad range of plant species and families can also involve screening hundreds of samples for multiple viruses. One assay for the high throughput screening of plant samples is the tissue blot immunoassay (TBIA) where the stems of each plant are blotted onto nitrocellulose membranes . MembranThe application of TBIA is limited by the availability and specificity of antibodies. Begomoviruses are antigenically closely related, so the production of antibodies to specific species is difficult, if not impossible. The BLRV-5G4 monoclonal antibody (McAb) is commonly used to identify poleroviruses and luteoviruses, but there are limited antibodies that can distinguish between species within the families as they are antigenically related ,3. If anAn attractive alternative to antibodies are nucleic acid probes that can target either a specific virus or a group of viruses. The problem with using probes is the low signal for visualization of the virus–probe interaction on the membrane. This is due to the probe binding to one genome per virion compared to many binding sites per virion for antibodies. Probes can be directly labelled but the signal, especially with low titer viruses, would not be visible on a tissue blot assay, or requires more extensive detection regimes such as chemiluminescence. To improve the signal-to-background ratio for visualization of the probe targets, research has focused on in situ hybridization chain reactions (HCR) ,17,18,19In this study, we set out to determine whether the HCR system could amplify the probe–virus interactions in the virus infected plant samples blotted onto a nitrocellulose membrane. We designed and tested probes targeted against different virus genera such as begomoviruses, poleroviruses, luteoviruses, a cucumovirus, and an alfamovirus. We also explored different hairpin and reporter methods such as biotin/streptavidin-alkaline phosphatase, biotin/anti-biotin-alkaline phosphatase, digoxigenin (DIG)/anti-DIG-alkaline phosphatase, fluorescein amidites (FAM)/anti-fluorescein-alkaline phosphatase and directly labelled hairpins with the Alexa Fluor 488 fluorophore. Using the TB-HCR assay, we were able to visually distinguish between the viral positive plant blots and negative blots and show that this assay can be used as an inexpensive triage-based assay prior to downstream applications.We designed a tissue blot hybridization chain reaction assay (TB-HCR) for the detection of viruses in plants. The design of the assay is shown in Geminiviridae, genus: begomovirus; probes TYLCV-P1 to P4) and the polerovirus (family Solemoviridae) turnip yellows virus . Probes for systemic viruses were designed to target the cucumovirus, cucumber mosaic virus  and the alfamovirus, alfalfa mosaic virus  from the family Bromoviridae (DNA probes are designed to target specific viruses or a group of viruses . The DNAoviridae .Tombusviridae) to be used in place of the broad-spectrum TBIA BLRV-5G4 antibody [We also designed DNA probes or groups of probes to target all poleroviruses and luteoviruses  for 1 min before washing with 2x SSC improved the TYLCV positive signal  and biotin labelled hairpins under various conditions. Several steps or conditions were found to improve the signal, both the intensity and number of positive spots for TYLCV on the blot. First, exposing tissue blots to UV light . The TYLCV signal improved, and the reaction was quicker when the hairpin chain reaction was carried out at 37 °C compared with room temperature .To improve the strength and visualization of the TYLCV signal on the blot, three more TYLCV probes targeting different regions of the genome were designed—TYLCV-P1 was positioned in the coat protein (500 nt from the start of CP), TYLCV-P2 was in the C1 gene, TYLCV-P3 was in the movement protein, and TYLCV-P4 was 700 nt into the coat protein . All proWhen three probes targeting TYLCV were used together , the positive signal of TYLCV was markedly increased .Probes were designed as per Choi, Beck, and Pierce , with a The TB-HCR assay was tested with other low titer, phloem-limited plant viruses. The probe HCR-A1-TuYV was designed to target the polerovirus turnip yellows virus (TuYV). Using a TuYV tissue blot processed with the HCR-A1-TuYV probe and biotin HPs, TuYV was successfully detected on the blot  and CMV subgroup 2  and AMV   were desThe TB-HCR assays described above use biotin labelled hairpins (HPs) , followeTB-HCR can also be used with fluorescent hairpins. We used Alexa Fluor 488 labelled HPs 488-HPs,  on a varPositive blots of TuYV, SbDV, BLRV, and PBMYV were probed with HCR-A1, HCR-A2, and HCR-A3 as well as negative controls of cotton bunchy top virus (CBTV) and healthy blots. After the addition of 488-HPs, small fluorescent spots were observed only on the positive blots for each of the targeted viruses .For the systemic viruses, the CMV and AMV positive and negative blots were probed with all six CMV-SG probes and two AMV-CP probes, respectively. After 10—20 min of incubation with 488-HPs at 37 °C, the positive blots exhibited strong fluorescence, while the negative controls did not fluoresce .To compare TB-HCR with the traditional TBIA method, which uses antibodies, positive blots of TYLCV were probed with TYLCV-P1, TYLCV-P3, and TYLCV-P4 (TB-HCR) or the broad-spectrum African cassava mosaic virus (ACMV) SCR-20 Begomovirus McAb (TBIA). CMV positive blots were compared using the six CMV-SG probes or anti-CMV rabbit polyclonal antibody. For TYLCV, both tissue blot methods successfully stained the TYLCV positive blot, although the TBIA method stained slightly stronger and showed more positive spots than TB-HCR a. For thWe set out to determine whether the hybridization chain reaction assay could be applied to a tissue blot system, which traditionally uses antibodies for the detection of plant viruses. The TB-HCR assay works in detecting low titer phloem restricted viruses such as TYLCV, TuYV, SbDV, BLRV, and PBMYV and high titer viruses not restricted to phloem (CMV and AMV) in plant stems blotted onto a nitrocellulose membrane.Begomoviruses (TYLCV) contain a circular single-stranded DNA genome in a twin (geminate) quasi-icosahedral shell. Poleroviruses (TuYV and PBMYV) and luteoviruses (SbDV and BLRV) are also encapsidated in an icosahedral shell with a monopartite linear single-stranded RNA genome. Both CMV (bromovirus) and AMV  have a tripartite single stranded RNA genome forming icosahedral or bacilliform virions, respectively. We also used TB-HCR for the potyvirus bean yellow mosaic virus (BYMV), which has a single stranded RNA genome incorporated into filamentous flexible rods with its coat protein. However, to date, TB-HCR has not worked with this virus (data not shown). This could be due to the shape of the virion, and/or the accessibility of the genome for the probe to attach as the potyvirus genome is packed tightly with the coat proteins, which are interlocked. This suggests that potyvirus virions may need to be broken open prior to attaching to the nitrocellulose membrane, so that the genome is available for probe attachment. Therefore, TB-HCR is currently not optimized for all virus types and further testing is needed to find a way to break open potyvirus virions for probe attachment to RNA. Similarly to TBIA, this assay needs to be designed, tested and optimized for each target virus or virus family.The main reporter method used in this study was the biotin hairpins, followed by streptavidin-AP and an alkaline phosphatase colorimetric stain. The advantage of this reporter/staining method is that it is cheaper than fluorescent hairpins. However, the disadvantage is that some plants contain endogenous biotin, and some purpling was observed as background staining on the blot. In most cases, the difference between background staining and a positive stain was clearly obvious. Nonetheless, in some cases, for some plants such as hibiscus, a strong staining was observed. This has also been observed with the TBIA assay. If this occurs, then another reporter system that does not use streptavidin and alkaline phosphates is advised such as the Alexa Fluor 488 hairpins. When comparing TB-HCR with TBIA, both methods successfully detected TYLCV positive blots, although TBIA produced a stronger more visible stain . TBIA usThe TB-HCR assay was designed as a triage assay that can target individual viruses or a group of viruses without the use of antibodies. When sampling large numbers of plants for a range of viruses, the TB-HCR triage assay would reduce the costs compared to PCR/RT-PCR or real-time PCR assays. We estimated that screening three hundred plants for seven different RNA viruses would cost approximately AUD 130 to AUD 140 depending on how many probes are used for the TB-HCR assay and using the biotin/streptavidin-AP method. In comparison, TBIAs would be cheaper if antibodies are available (approx. AUD 20 to AUD 35). However, RT-PCR and real-time assays are much more expensive due to the cost of the enzymes and carrying out individual PCRs for each of the seven viruses . The main set up costs for TB-HCR are the development and synthesis of specific probes and hairpins, with fluorescent hairpins being more costly than biotin. The time-consuming part of both the TB-HCR and TBIA assays is the cutting, blotting, and recording of plant samples. It is advisable to make the blots in triplicate, at least. This can be conducted in the laboratory or in the field. Once the tissue blots are finished and dry, they can be easily transported and remain stable enough to be stored for many years prior to use. The TB-HCR assay only takes between 2.5 and 4 h to process, depending on whether the buffers are already set up and pre-heated. The time it takes is the same regardless of how many samples are blotted on the membrane: a blot of two or 200 plants will take the same amount of time as large numbers of plant samples can be screened at once. As with TBIA, this type of screening assay is a valuable qualitative test allowing for rapid narrowing down of the possible likely infected samples from large numbers, allowing for further testing as required. The development of a semi- or fully quantitative assay in this format is theoretically possible, but would require the further development of appropriate quantitated controls for each virus, possibly in an appropriately matched plant host. This would be the case for either TBIA or TB-HCR.The TB-HCR assay was successfully shown to detect both low and high titer viruses from five plant virus families and is therefore likely to also work for further viruses. This novel detection method could be used as a triage assay for virus surveys, the screening of germplasm breeding lines, or the screening of imported plant lines for the presence of exotic viruses. The TB-HCR may be useful in the event of biosecurity incursions because of the short time needed to design probes and the cheap, high throughput format. The assay could also potentially be an in-field assay. Future research will focus on increasing the signal-to-background ratio and expanding the analysis to other plant viruses from different families. Plant samples that were used for TB-HCR blots were collected from virus surveys or sent to us for diagnostics from various locations around Australia . Virus iDNA probes contain one or two 36 nt initiator(s), a 4 nt spacer separating the initiator and viral sequence, and a 50–30 nt viral recognition site . Probes Hairpin sequences used in this study were as per Choi, Beck, and Pierce , DNA HCR2 for 1 min to aid in DNA/RNA hybridizing to the membrane. Membranes were rinsed three times (5 min per wash) in 2x SSC + 1.0% SDS at room temperature (RT). Membranes were then transferred to pre-heated (55—60 °C) hybridization buffer for 2–5 min with gentle shaking. Three different hybridization buffers were used—ULTRAhyb™ Ultrasensitive Hybridization Buffer , Rapid-hyb buffer  and PerfectHyb buffer —and all worked equally well. Membranes were transferred to fresh pre-warmed (55 °C–60 °C) hybridization buffer containing probe(s) at a final concentration of 0.05 µM (each probe) and incubated at 55 °C–60 °C for a minimum of 10 min with gentle shaking. Excess probe(s) were removed by washing once with 0.5x SSC + 0.1% SDS at 55 °C, then twice with 0.2x SSC + 0.1% SDS at 37 °C for 2–5 min each. Membranes were then washed for 1–2 min in pre-warmed (37 °C) amplification buffer  then transferred to fresh pre-warmed (37 °C) amplification buffer, where pre-prepared hairpins were added to start the HCR reaction. Prior to the HCR step, each hairpin was prepared separately in 5x SSC at a final concentration of 30 µM, heated at 95 °C for 90 s, then cooled to room temp for at least 30 min prior to use. The HCR step took 10–20 min at 37 °C. Prior to washing, tissue blots were UV-crosslinked in a HL-2000 hybriLinker at 1500 kJ/cmWhen fluorescently labeled hairpins (Alexa fluor 488) were used, fluorescence on positive blots were observed after 10–20 min when the HP reaction was carried out at 37 °C with gentle shaking or 25–40 min when incubated at room temperature. Membranes were then washed in fresh amplification buffer to wash off excess hairpins and were visualized and photographed using a Nikon i80 biological compound microscope with a FITC (465/495 nm) fluorescent filter or a Leica stereo microscope with a GFP filter. Blots were stored in a dark dry place. When biotin, FAM, or DIG hairpins were used for colorimetric assays, membranes were transferred and washed twice with amplification buffer + 3% BSA  at RT. For biotin hairpins, membranes were then transferred to fresh amplification buffer +3% BSA + streptavidin-AP  for 10 min at RT with shaking. Or alternatively, biotin, FAM, or DIG hairpins were detected with antibodies. In this case, membranes were incubated in fresh amplification buffer +3% BSA and their respective antibody; anti-biotin-AP , anti-fluorescein-AP , or anti-digoxigenin-AP , respectively, for 10–30 min at RT with gentle shaking. 2·6H2O, 0.2% Tween 20, 0.01% dextran sulfate), then transferred to fresh substrate buffer containing solutions A and B from the AP conjugate substrate kit  at a concentration of 5 µL/mL. Membranes were left to develop for ~10–30 min at RT until color development was observed with a Leica dissecting microscope. Following the addition of streptavidin-AP or anti-antibody-AP, membranes were washed twice in substrate buffer  were carried out as per Makkouk and Comeau . Membran


The continued spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans necessitates evaluation of variants for enhanced virulence and transmission. We used the ferret model to perform a comparative analysis of four SARS-CoV-2 strains, including an early pandemic isolate from the United States (WA1), and representatives of the Alpha, Beta, and Delta lineages. While Beta virus was not capable of pronounced replication in ferrets, WA1, Alpha, and Delta viruses productively replicated in the ferret upper respiratory tract, despite causing only mild disease with no overt histopathological changes. Strain-specific transmissibility was observed; WA1 and Delta viruses transmitted in a direct contact setting, whereas Delta virus was also capable of limited airborne transmission. Viral RNA was shed in exhaled air particles from all inoculated animals but was highest for Delta virus. Prior infection with SARS-CoV-2 offered varied protection against reinfection with either homologous or heterologous variants. Notable genomic variants in the spike protein were most frequently detected following WA1 and Delta virus infection. Coronaviridae family causes zoonotic disease , the etiologic agent of coronavirus disease 2019 (COVID-19), has caused the most profound pandemic of modern times, resulting in millions of deaths worldwide ( disease . Sustain disease . As vari0) at the population level do not fully capture the intrinsic and relative capacity for transmission of newly emerged SARS-CoV-2 variants , each representing a different lineage and displaying key amino acid differences in the S protein see . Fever w10.1128/mbio.02421-22.2TABLE S1Table S1, PDF file, 0.01 MB.SARS-CoV-2 strains used in the study. SARS-CoV-2 strains used in the study; lineage, passage history, GISAID accession number, and amino acid mutations in the S protein are listed. Download Copyright © 2022 Pulit-Penaloza et al.2022Pulit-Penaloza et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10 PFU/mL, respectively), while infectious Beta virus was recovered at a low titer in one NW specimen only. Mean peak infectious virus titers and frequencies of detection gradually fell with each tissue that was collected deeper into the respiratory tract. Only WA1 and Delta viruses were detected in lungs and at a low titer (≤2.5 log10 PFU/g). SARS-CoV-2 RNA was much more prevalent in tissues than was infectious virus, and up to 3 to 4 orders of magnitude higher, depending on the strain and tissue (Virus dissemination throughout and beyond the respiratory tract was examined in specimens collected on day 3 postinoculation (p.i.) from 16 sites of 3 inoculated ferrets for each virus group. Infectious virus was predominately limited to the respiratory tract, with few exceptions; notably, Alpha and Delta variants were detected in olfactory bulb and brain tissues . WA1, Ald tissue . In agreConcurrent with viral quantification, histopathological examination of trachea, lung, and extrapulmonary tissues collected from three ferrets per virus on day 3 p.i. was performed. No significant differences were observed among virus groups or between virus-inoculated and naive ferrets . Trachea10.1128/mbio.02421-22.1FIG S1FIG S1, PDF file, 0.3 MB.Histopathology of the respiratory tract of SARS-CoV-2-inoculated ferrets. Trachea samples (top row) from naive and SARS-CoV-2-infected animals show intact, ciliated respiratory epithelium and no significant submucosal inflammation. Lung samples (bottom row) from all animals show bronchioles (*) free of degenerative or inflammatory changes, alveolar spaces are collapsed (tissue collection artifact), and alveolar walls are diffusely congested. Download Copyright © 2022 Pulit-Penaloza et al.2022Pulit-Penaloza et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10 PFU/mL. Ferrets inoculated with WA1 or Alpha virus reached mean peak NW titers of 4.4 to 4.5 log10 PFU/mL, with peak titers of WA1 virus delayed relative to those of Delta or Alpha viruses (10 PFU/mL), but none of the animals seroconverted , and conjunctival wash (CW) samples were collected. Ferrets inoculated with the Delta variant had the highest NW titers on day 1 p.i. at 5.1 log viruses ; Fig. 2.onverted , indicat10.1128/mbio.02421-22.3TABLE S2aAll ELISAs were performed using recombinant SARS-CoV-2 S1+S2 proteins. OD450 values for IgG were normalized by subtracting the ELISA cutoff value (negative-control mean OD [0.06 to 0.44] + 3× STDEV [standard deviation]). Each cell shows data for an individual ferret. Negative, below the ELISA cutoff value. bDirect contact transmission model (DCT); respiratory droplet transmission model (RDT). cFerrets were inoculated with 6.0 log10 PFU of SARS-CoV-2, and sera were collected on day 19 or 20 postinoculation (p.i.) or postcontact (p.c.). Download Table S2, PDF file, 0.01 MB.Analysis of ferret serum collected during SARS-CoV-2 transmission experiments. Copyright © 2022 Pulit-Penaloza et al.2022Pulit-Penaloza et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10 PFU/mL) and was an order of magnitude higher than in the inoculated group, and virus shedding persisted for 7 to 9 days p.c.; all contacts seroconverted to the challenge virus . Values for area under the curve  for time courses  revealed that overall airborne Delta virus RNA was at least 5 times higher than that of any of the other viruses. Ferrets inoculated with WA1 and Delta viruses shed virus for the full 10-day time course, while Alpha and Beta viruses were exhaled from all animals for 5 and 2 days p.i., respectively  cyclone samplers that separate airborne particles into three fractions: >4 μm, 1 to 4 μm, and <1 μm. The kinetics of virus RNA shedding into the air varied by virus  to L, buectively  to L. Aln = 2 per virus). Clinical signs observed during the secondary infections were mild with no respiratory symptoms or lethargy noted. Fever did not exceed 1.8°C above baseline, on par with primary infection (Enzyme-linked immunosorbent assay (ELISA) of sera collected 83 days following primary challenge showed that ferrets initially inoculated with WA1, Alpha, or Delta virus had IgG antibodies against homologous and heterologous SARS-CoV-2 , suggestnfection . Althoug10 copies/mL) and was highest at day 1 p.i., likely in part due to residual inoculum present in upper airways. RS samples were negative for infectious virus but positive for viral RNA only when rechallenged with WA1 or Alpha variant viruses , but none was detected from RS samples (10 copies/L of air), but not Alpha, virus rechallenge. In contrast, rechallenge with Delta virus resulted in detection of infectious virus and viral RNA in NW . Delta viral RNA, but no infectious virus, was detected in the RS of a single ferret, while both animals in this group exhaled viral RNA into air samples (mean peak 1.2 log10 copies/L), all of which cleared within a week , and viral RNA was detected until day 9 p.i. (mean peak 6.1 log10 copies/mL) (10 copies/L of air). Heterologous challenges of ferrets previously inoculated with Beta variant led to productive infection as evidenced by the detection of infectious WA1 (mean peak 4.7 log10 PFU/mL) and Alpha (mean peak 4.2 log10 PFU/mL) viruses in NW from all the rechallenged ferrets. Viral RNA was detected in NW and air samples until days 9 and 8 p.i., respectively  ; Fig. 3Cectively . ELISA rectively . The Del10 copies/mL) (10 copies/L of air) (10 PFU/mL) and viral RNA (6.0 log10 copies/mL) in NW samples until day 5 p.i. RS were positive for WA1 and Alpha virus RNA, while Alpha virus was detected in the air until day 4 p.i. Homologous challenge with Delta variant also resulted in detectable infectious virus in NW (2.9 log10 PFU/mL) from both ferrets as well as viral RNA (6.0 log10 copies/mL), and Delta virus shedding into the air was also detected from each of the challenged ferrets (2.8 log10 copies/L). Normalized IgG antibodies against the S protein of secondary challenge viruses were detectable prior to rechallenge and, in most cases, were elevated following rechallenge with any of the viruses  ; Fig. 3D of air) . Initial viruses .With few exceptions, prior exposure to any of the SARS-CoV-2 strains provided some level of protection. Nevertheless, SARS-CoV-2 RNA was detected in airborne particles collected during rechallenge from nearly all the animals  to H, su10 viral RNA copies/μL extracted RNA and variant populations detected at >5% frequency; the term variant in this context is not to be confused with a circulating variant virus . Although this part of the text focuses on major variants of the S protein (detection of >25% frequency of genome copies), full genomes were sequenced, and all variants are listed in Key amino acid substitutions were reported for the SARS-CoV-2 human isolates chosen for this study. Compared with WA1 virus, Alpha, Beta, and Delta lineage viruses acquired the D614G substitution, which has been associated with enhanced receptor binding affinity, infectivity, and transmissibility –14. Alph–10.1128/mbio.02421-22.4TABLE S3aThe amino acid(s) listed on the left of the residue position substituted for the amino acid(s) listed on the right side. An insertion (ins) or deletion (del) was found at the indicated amino acid position. Spike protein variants at amino acids 1271 to 1274 are not listed. bNT, not tested because viral RNA levels were too low. ND, no variants detected above the 5% threshold cutoff. Download Table S3, PDF file, 0.03 MB.Major and minor genomic variants detected in WA1 virus-infected ferrets. Copyright © 2022 Pulit-Penaloza et al.2022Pulit-Penaloza et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Major variants were most often observed in the S protein, especially at the receptor binding motif . Combinations of F486L, Q498H, and N501T major variants were detected in NW samples from all WA1-inoculated ferrets with detection at >75% frequency noted at positions F486L (1/6 ferrets), Q498H (3/6 ferrets), and N501T (1/6 ferrets) starting at day 3 p.i. . InteresWith few exceptions, only minor variants with low frequency were detected in Alpha and Beta virus NW specimens from inoculated animals. Exceptions were noted in the RBM of the S protein in the day 5 p.i. NW of an Alpha virus-inoculated DCT donor ferret (F486L at 57.8%) and in NW (Y453F at 69.6%) and nasal turbinate specimens (Q498R at 51.5%) collected from ferrets that were euthanized on day 3 p.i. , Alpha. Among ferrets challenged with Delta virus, variants harboring mutations in the S protein were detected in NW samples from all six inoculated ferrets. Three of these animals shed virus harboring variants in the RBM: Y453F at frequencies ranging from 41.1 to 82.6% , Delta. Due to the exceptional capacity for sustained transmission among humans displayed by SARS-CoV-2, it is becoming increasingly evident that, like seasonal coronaviruses, variants of SARS-CoV-2 will likely continue to circulate globally , 20. Sev22–25–As shown in our DCT model, all contacts cohoused with animals that were inoculated with WA1, Alpha, or Delta virus had detectable viral RNA in at least one NW sample; however, only WA1 virus (2/3) and Delta virus (3/3) contacts had infectious virus in NW and seroconverted to homologous virus. The lack of infectious virus and seroconversion following contact with Alpha virus-inoculated ferrets suggests that the sole detection of viral RNA in cohoused contacts likely represents environmental contamination. Enhanced detection of viral RNA relative to infectious virus in NW specimens is consistent with other studies using this model , 29. It Respiratory viruses capable of airborne transmission, notably influenza A viruses, have been detected in the exhaled breath of infected humans  and ferrThe size of airborne virus-laden particles dictates the deposition site, which is an important parameter of virus transmission, especially for pathogens that display tropism for specific cells or sites of the respiratory tract. In general, particles of <5 μm can reach lower respiratory tract surfaces, in contrast to larger particles, which likely are deposited in the upper and central human respiratory mucosa , 39. StuWhile most human cases of SARS-CoV-2 from 2020 through 2021 were due to primary infection, continued circulation and emergence of diverse SARS-CoV-2 variants have resulted in increased numbers of both reinfections and breakthrough infections postvaccination –43. We f–46–NGS analysis revealed signs of early adaptation of SARS-CoV-2 to the ferret host, especially in the S protein. Substantive genomic variants were more often detected in the WA1 and Delta viruses that replicated well and displayed transmission capability in the ferret model, while Alpha and Beta viruses acquired fewer major variants in the RBM of the S protein and were not capable of transmission in this model. Emergence of predominant variant populations during the course of infection suggests that such species may have better fitness or replicative advantages in the ferret host. The S protein major variants detected in samples from ferrets inoculated with WA1 or Delta  were key residues located at the cellular receptor ACE2 binding interface , 52, andin vitro and in vivo models are necessary to fully understand the pathogenesis and transmissibility dynamics of circulating strains, ultimately enabling rapid identification of emerging variants that present a threat to humans. Continued surveillance and risk assessment of emerging SARS-CoV-2 variants will be a critical component for pandemic response and preparedness.Overall, we found the ferret to be a useful model of the assessment of SARS-CoV-2 infection and transmission, revealing enhanced transmissibility of the Delta virus relative to previously detected strains. Although phenotypes displayed in ferrets did not always closely reflect those observed in humans, our comparative analysis of variants revealed clear differences in virus shedding in the air and the potential to evaluate reinfection and/or breakthrough infections using the ferret model. Similar studies using Omicron group viruses would also be beneficial. As different animal models have individual strengths and limitations, investigations of SARS-CoV-2 variants using multiple 2 atmosphere . The following day, a serologically naive ferret was placed in the same cage as each of three inoculated ferrets (direct contact transmission model) or in a cage with a perforated side wall adjacent to each of three additional inoculated ferrets (respiratory droplet transmission model) (n = 3). The 3 remaining inoculated ferrets in each virus group were euthanized on day 3 p.i. for the assessment of virus spread to pulmonary and extrapulmonary tissues and blood as previously described  were housed in Duo-Flo Bioclean mobile units  during experimentation. Nine ferrets were inoculated intranasally with 1 mL of 6.0 logn model) . Nasal wn model) , 60 wereescribed , 62. Con10 PFU of homologous or heterologous virus diluted in PBS (2 ferrets per group). Following inoculation, the ferrets were observed for clinical signs and symptoms of infection for 15 days; NW and RS samples were collected every second day. Aerosol samples were collected daily for 10 days p.i. from each of the inoculated ferrets. Serum samples were collected the day before and 28 days after rechallenge. All animal procedures were approved by the Institutional Animal Care and Use Committee  of the Centers for Disease Control and Prevention and were conducted in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility.To evaluate protection against reinfection, 6 inoculated donor ferrets from the transmission studies and up to 2 contact ferrets (contacts with detectable virus in NW samples) were maintained for 12 weeks and rechallenged with 1 mL of 6.0 logEach day, prior to other scheduled sample collections, conscious and alert ferrets were individually held in disinfected, enclosed, vented transport containers (23.9 L in size) with a perforated lid for 1 h while air was sampled inside the container at 3.5 L/min . The samplers were attached to the outside of a container holding a single ferret with the sampler inlet protruding 3 to 4 cm into the container. Air samples were collected using a National Institute for Occupational Safety and Health (NIOSH) BC 251 two-stage cyclone aerosol sampler  at 3.5 LInfectious virus was quantified using a previously described plaque assay with modifications . BrieflyMN908947.3) (Twist Bioscience) was included on each plate. Molecular-grade water was used as a negative control. Any specimen with a gene copy number of <1 per μL of extracted RNA was regarded as negative. The mean viral RNA copy was normalized and expressed as RNA copy number/milliliter or gram of specimen.Concurrent with infectious virus titration, 140 μL of each sample was inactivated in 560 μL of AVL buffer  according to the manufacturer’s protocol and stored at −80°C. Viral RNA was extracted using a QIAamp 96 viral RNA mini-extraction kit (Qiagen) and a QIAcube HT automated high-throughput nucleic acid purification platform (Qiagen) (100-μL elution volume). Each RNA sample (5 μL) was tested in duplicate by real-time reverse transcription-PCR (RT-PCR) using the CDC influenza SARS-CoV-2 multiplex assay, with nucleoprotein (N) as the target gene. To quantify viral copy number in each specimen, a 10-fold serial dilution of synthesized SARS-CoV2 viral RNA (GenBank identifier [ID] 450). Positivity cutoffs were determined based on screening of 40 naive prepandemic ferret sera for reactivity to SARS-COV-2 proteins. OD450 values for IgG were normalized by subtracting the ELISA cutoff value (negative-control mean OD [0.06 to 0.44] plus 3 times standard deviation [SD]).Enzyme-linked immunosorbent assay (ELISA) was performed as previously described  with modNecropsy tissues  were fixed in 10% neutral buffered formalin, processed for routine paraffin histology, sectioned at 4 μm, and stained by hematoxylin and eosin (H&E). Tissues were evaluated by two veterinary pathologists for inflammatory, degenerative, or other significant histopathologic changes.3 per μL of extracted RNA were selected for next-generation sequencing (NGS). In brief, viral RNAs were first reverse transcribed, and viral whole-genome cDNA was then amplified into 29 amplicons of approximately 1.2 kb in length using the SARS-CoV-2 Midnight primer panel (IDT) according to the manufacturer’s protocol. Next, the amplicons were purified with Sera-mag SpeedBeads (Fisher Scientific) followed by NGS library preparation with the Illumina Nextera DNA Flex library preparation kit at half-volume reaction size and sequenced paired-end 150 on iSeq100 (Illumina). The Fastq files obtained from iSeq runs were trimmed using BBDuk and then assembled to SARS-COV-2 reference genomes with Bowtie2  listed on the left of the residue position substituted for the amino acid(s) listed on the right side. An insertion (ins) or deletion (del) was found at the indicated amino acid position. Spike protein genomic variants at amino acids 1271 to 1274 are not listed. Alpha virus spike protein is numbered based on WA1 virus numbering upon alignment. bNT, not tested because viral RNA levels were too low. ND, no variants detected above the 5% threshold cutoff. Download Table S4, PDF file, 0.03 MB.Major and minor genomic variants detected in Alpha virus-infected ferrets. Copyright © 2022 Pulit-Penaloza et al.2022Pulit-Penaloza et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.02421-22.6TABLE S5aThe amino acid(s) listed on the left of the residue position substituted for the amino acid(s) listed on the right side. An insertion (ins) or deletion (del) was found at the indicated amino acid position. Spike protein genomic variants at amino acids 1271 to 1274 are not listed. Alpha virus spike protein is numbered based on WA1 virus numbering upon alignment. bNT, not tested because viral RNA levels were too low. ND, no variants detected above the 5% threshold cutoff. Download Table S5, PDF file, 0.03 MB.Major and minor genomic variants detected in Beta virus-infected ferrets. Copyright © 2022 Pulit-Penaloza et al.2022Pulit-Penaloza et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.02421-22.7TABLE S6aThe amino acid(s) listed on the left of the residue position substituted for the amino acid(s) listed on the right side. An insertion (ins) or deletion (del) was found at the indicated amino acid position. Spike protein genomic variants at amino acids 1271 to 1274 are not listed. Alpha virus spike protein is numbered based on WA1 virus numbering upon alignment. bNT, not tested because viral RNA levels were too low. ND, no variants detected above the 5% threshold cutoff. Download Table S6, PDF file, 0.03 MB.Major and minor genomic variants detected in Delta virus-infected ferrets. Copyright © 2022 Pulit-Penaloza et al.2022Pulit-Penaloza et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


X-ray diffraction studies suggested that the LSMO film on LAO was stabilized in a tetragonal structure, which was relaxed in-plane and strained along the out-of-plane direction. The interfacial reconstruction of the LSMO–LAO interface due to the reorientation of the Mn ion spin induced spin-glass behavior due to the presence of non-collinear Mn ion spins. Consequently, the interface effect was observed on the Curie temperature, temperature-dependent resistivity, metal-to-semiconductor transition temperature, and magnetoresistance (MR). At a magnetic field of 7 T, MR decreased from 99.8% to 7.69% as the LSMO film thickness increased from 200 Å to 500 Å. A unique characteristic of the LSMO films is the large low-field MR after a decrease in the field from the maximum field. The observed temperature-dependent magnetization and low-temperature resistivity upturn of the LSMO films grown on LAO provide direct evidence that the low-field MR is due to the non-collinear interfacial spins of Mn. The present work demonstrates the great potential of interface and large low-field MR, which might advance the fundamental applications of orbital physics and spintronics.Herein, interfacial reconstruction in a series of La 0.7Sr0.3MnO3 film grown on (001) oriented LaAlO3 at 300 mT field exhibits 60% MR, which decreases with an increase in the La0.7Sr0.3MnO3 film thickness due to a decrease in the non-collinear Mn ion spins near the interface.The 200 Å thick La Nevertheless, many other factors have been found to account for this behavior, for example, the oxygen vacancies and charge transfer at the interfaces.La8,9 It has been demonstrated that the magnetoresistance (MR) behavior in LSMO films can be obtained at much lower magnetic fields by naturally or artificially modifying the Mn–O–Mn bond angle and bond length.10 Large low-field MR (LFMR) was observed by incorporating artificial grain boundaries in ferromagnetic manganite/spacer/ferromagnetic manganite trilayers. The highest LFMR value of 45–50% was obtained below a 0.02 T field at 4.2 K for La0.67Sr0.33MnO3/SrTiO3/La0.67Sr0.33MnO3 thin film junctions.11,12 The LFMR value of the composite films such as La0.67Sr0.33MnO3/ZnO was 23.9% at 0.5 T field and 10 K.13 The contribution of the grain boundary towards 23–27% LFMR at 0.02–0.1 T field and 77 K was explained by designing a single SrTiO3 bicrystal substrate grain boundary into an epitaxial film of La0.7Ca0.3MnO3.14 However, the growth of high-quality epitaxial manganite thin films with controlled and atomically smooth artificial grain boundaries is a very challenging task. Recently, we reported a unique pulsed plasma deposition (PPD) method, where the thin films of LSMO were stabilized epitaxially, even on the native SiO2 surface of the (001) oriented Si substrate by using a home-built RF magnetron sputtering system.4,15 Herein, we report the study of the spin dynamics in LSMO grown on an LaAlO3 (LAO) substrate, which exhibited giant LFMR (∼99%) at low temperatures. This LFMR strongly depends on the LSMO thickness (tLSMO), which suggests the interfacial origin of MR due to the variation in the bond length and bond angle at the interface.Extensive efforts are being devoted to exploring the electronic transport properties of LSMO films grown on a wide variety of substrates with or without a buffer layer.15 The thin films were grown at 700  °C under 7 × 10−3 mbar of an argon and oxygen gas mixture. The chamber pressure was maintained by flowing the argon and oxygen gas at a 1 : 4 proportion during the deposition. The deposition was performed in pulsed mode by keeping the sputtering power density at 2.96 W cm−2. All the thin films were grown for different durations by altering the opening and closing of the shutter for a period of 10 s and 50 s, respectively. After the deposition of the desired thickness, the deposition chamber was filled with 50 mbar O2 followed by post-annealing for 45 min at 700  °C, and then, the film was cooled down to room temperature. LSMO was deposited on the (001) oriented LAO for six different growth periods. The LSMO films were found to be 100, 150, 200, 250, 375, and 500 Å thick, which was confirmed by optical measurements.The highly dense ceramic target of LSMO prepared by the solid-state reaction method was used to grow thin films on the (001) oriented LAO by adopting the PPD growth sputtering process. The sputtering system was designed in such a way that it (i) generates a horizontally orientated plasma, (ii) offers a larger cathode surface in front of the plasma, (iii) places the substrate heater inside the specified port, (iv) maintains a uniform temperature of the port wall using cooled water, and (v) provides an option to customize the target-to-substrate distance.M(T)) measurements for the out-of-plane orientation of the magnetic field were performed using a superconducting quantum interference device-based vibrating sample magnetometer. The electronic transport properties of the thin films were measured in the presence of a magnetic field using a physical property measurement system. The resistivity of the thin films was measured using the two-probe technique in the presence of an out-of-plane-oriented magnetic field. The temperature-dependent resistance of the films was measured while warming the films from the lowest temperature.A four-cycle X-ray diffractometer was used to record the out-of-plane X-ray diffraction (XRD) patterns and reciprocal space mappings (RSMs) of the thin films. The temperature dependence of magnetization (θ–2θ XRD patterns of the LSMO films with different thickness grown on (001) oriented LAO. The XRD patterns show only the (00l) oriented Bragg's peaks of LSMO and LAO without any signature of the other phase of LSMO. The out-of-plane pseudocubic lattice parameter (cpc) of the different LSMO films calculated from the (00l) peak positions of the XRD patterns is plotted in cpc of the LSMO films is independent of thickness and the average value is 3.99 Å, which is larger than the pseudo-cubic lattice parameter of the bulk LSMO (cpc = 3.88 Å), as shown in 16 The larger cpc value of the LSMO film compared to that of its bulk suggests that LAO provides out-of-plane tensile strain for the epitaxial growth of LSMO. The 100 Å thick LSMO film shows −2.68% tensile strain along the [001] orientation. As the LSMO film thickness increased to 150 Å, the strain also increased to −3.06%. However, the strain fluctuated between −2.65% to −3.06% as the LSMO film thickness varied from 100 to 500 Å [Fig. S2Å Fig. S2. Thus, tϕ-scans of LSMO on LAO. The ϕ-scans of LSMO show four 90°-separated film peaks [ϕ-scans of the film and substrate show four-fold symmetry with the cube-on-cube epitaxial growth of LSMO on the LAO substrate. The 150 Å thick LSMO film in the ϕ-scan showed relatively broader peaks with FWHM ∼ 1° compared to the 500 Å thick films due to the presence of in-plane misorientation of the pseudocubic unit cells.The highly aligned grains and in-plane epitaxy were confirmed by asymmetric (101) lm peaks  and are θ–ω coupling scans for different ω values of {101} for the 500 Å thick LSMO film grown on LAO. The diffraction peaks associated with the substrate and the film are located at different values of q‖, indicating that the films are relaxed. In addition, the transverse scattering vectors corresponding to the diffraction peak positions of the substrate and film are different. The angular positions of the Bragg's peaks due to diffraction along 〈101〉 of {101} of LSMO are very close to each other, which confirm the presence of the same in-plane lattice parameters irrespective of 〈101〉. The cpc extracted from the RSM is very close to that measured from the symmetric scan. The in-plane lattice parameter of LSMO is apc = bpc = 3.87 Å. As the LSMO film thickness decreased below 500 Å, the in-plane lattice parameter remained the same up to 200 Å and then decreased to 3.86 Å for the 150 Å thick film. However, the (101) peak intensity of the 100 Å thick LSMO films was not noticeable to calculate the in-plane lattice parameter. Overall the in-plane lattice parameter of LSMO is close to the pseudocubic lattice parameter of the bulk LSMO with a negligible strain of ∼0.2% [Fig. S2% Fig. S2. The sub% Fig. S2 in the LR3̄C and lattice constants 5.471 Å and αr = 60.43°.17 In this unit cell, the octahedron rotations can be described by Glazer's tilt system a−b−c−, which consists of equivalent out-of-phase rotations  and c = 7.98 Å . Thus, the tetragonal structure has the space group I4/mcm with Glazer's tilt system a0b0c−.19 The synthesis of the LSMO film on LAO introduced structural reconstruction of LSMO from a−b−c− type octahedral tilt to a0b0c− similar to PrMnO3/SrRuO3.20 Interestingly, the tetragonal LSMO thin film does not conserve the unit cell volume, in contrast to the LSMO film on LAO with a conserved volume.21The bulk LSMO possesses a rhombohedral unit cell with the space group s see ESI about thM(T) indicates the presence of randomly oriented non-collinear Mn ion spins in the LSMO film. The difference in the ZFC magnetization of these two LSMO films suggests that the 500 Å thick film has comparatively more non-collinear Mn ion spin domains with a coupling energy stronger than the Zeeman energy at 0.01 T field. The difference in ZFC and field-cooled (FC) magnetization at a temperature below the irreversible temperature and the decrease in peak temperature in the ZFC curve with an increase in applied field observed for these LSMO films [Fig. S3M(T) of the 200 Å thick LSMO indicates that the paramagnetic phase transforms into a ferromagnetic phase at a temperature of TC ≈ 196 K, which is the TC of LSMO. The monotonic increase in the 0.01 T FC magnetization at a temperature below 112 K indicates the existence of uniform exchange coupling of the non-collinear Mn ion spin domains. The distinct change in 0.01 T FC magnetization with the temperature of around 112 K was suppressed after increasing the cooling field to 0.5 T [Fig. S3TC of the LSMO films decreased from 262 K to 102 K as the film thickness decreased from 500 Å to 100 Å [Fig. S3TC values of the LSMO films are significantly lower than that of the bulk LSMO. However, the observed TC values of these films are very close to that of the reported LSMO/LAO.22 The TC of the LSMO films depends on the spin orientation of Mn, which is determined by MnO6. As seen in 6 in the LSMO film on LAO is elongated along the ‘c’ direction compared to its bulk. Thus, the reduced TC of the LSMO film on LAO compared to its bulk is attributed to possible sources of MnO6 rotation and tilting near the interfaces such as surface twining, substrate-induced strain,8 and oxygen non-stoichiometry.23 The FC M(T) of the LSMO films with a thickness of <200 Å did not show a clear paramagnetic to ferromagnetic transition, suggesting the richness of the non-collinear Mn ion spin domains [Fig. S3As shown in s Fig. S3 are the T Fig. S3 due to tÅ Fig. S3. The incs Fig. S3.ρ(T)) of the ∼2 mm wide LSMO films was measured using the two-probe method [ρ(T) was observed with an extended measurable temperature range after the application of a magnetic field along the out-of-plane direction of the 100 Å thick LSMO film. The ) of the 150 Å thick LSMO film [ρ(T) of the LSMO films with a thickness of ≤150 Å did not exhibit metal-like behavior but semiconductor-like behavior. The ρ(T) data of these films fits well with24 The ρ0 may be either independent of temperature or a slowly varying function of temperature, while T0 is a constant of the material, and is related to the rate at which the wavefunction decreases with hopping distance. Thus, the T0 of the ES-VRH is correlated inversely with the localization length (ξ) of the carriers. The T0 of the 100 Å and 150 Å thick LSMO films obtained from the ES-VRH fit decreased with an increase in the magnetic field [Fig. S4ξ [ξ is expected. The ES-VRH fit suggests the presence of interactions between the localized electrons, which may be due to the structural reconstruction, i.e., the lattice distortion [The temperature-dependent resistivity  of the 150 Å thick LSMO film indicates that the LSMO–LAO interface consists of a semiconducting phase. The semiconducting phase is rich in non-collinear Mn ion spins; thus, around an 80% drop [ρ of the 150 Å thick LSMO compared to its ρ at ∼135 K was observed. The large drop in resistivity confirms the significant contribution of the spin-dependent scattering in the absence of a magnetic field.The variation in 80% drop  in resisTP ∼ 95 K, which we marked as the metal-insulator transition temperature. Upon further cooling the 200 Å thick LSMO film below TP, the resistivity decreased gradually and became minimum at a temperature of TMin ∼ 45 K, followed by a sharp increase down to the lowest temperature [tLSMO increased above 200 Å, a qualitatively similar ρ was observed with a lower resistivity value, higher TP, and lower TMin [TP of the LSMO film increased, while the TMin decreased [TMin decreased, while the TP increased [TP of the LSMO film grown on LAO with an increase in LSMO film thickness has been reported previously.25 The variation in resistivity at temperatures TP > T > TMin can be explained by the double exchange26 and phase separation27 mechanisms in the LSMO films. The observed variation in resistivity with tLSMO is attributed to the expanded ‘cpc’, which induces a Jahn–Teller distortion along the ‘c’ direction and controls the spin reorientations and electron localization.28The resistivity of the 200 Å thick LSMO film on cooling below room temperature increased monotonically and became maximum at a temperature of perature . As tLSMwer TMin . The resecreased , which increased . The incT < TMin for the LSMO films [Fig. S529ρ0 is the residual resistivity, the second term is due to the contribution of electron–electron scattering, the third term is the spin-dependent Kondo-like effect, and the last term is related to the inelastic scattering. The fitting result as denoted by the solid lines in ρ½ and ρ1 decreased [M(T). The coefficients ρ½ and ρ1 also decreased with an increase in the magnetic field [ρ½ is smaller than the coefficient ρ1 irrespective of the thickness of the LSMO film or magnetic field value, and thus the electron–electron scattering is smaller compared to the spin-dependent Kondo-like effect. The Kondo effect is attributed to the interaction of conduction electrons with the localized spin at the non-collinear Mn ion spins domain boundary. As the temperature decreases below TMin, the lattice distortion due to structural reconstruction and magnetic disorder effect becomes dominant, and the resistance upturn emerges.30The resistivity at s Fig. S5 was fittecreased , suggestic field . The coe of the LSMO films with different thicknesses. The MR of the 100 Å thick LSMO film was positive for a 1 T field. As the field increased to 3 T, the MR of this film remained positive at room temperature; however, on cooling below room temperature, the MR decreased and became negative at ∼175 K. A similar temperature-dependent MR ) was observed at a higher magnetic field with an enhanced MR and expanded temperature range for the negative MR. At a 1 T field, the MR of the 150 Å thick LSMO was positive in the temperature range of 360 K to 207 K, while it was negative at a temperature lower than 207 K [T) curve was similar, the positive MR of the 150 Å thick LSMO decreased, while its negative MR increased. The MR of the 200 Å thick LSMO film was negative at a 1 T field. As the temperature decreased below room temperature, the MR at 1 T increased up to 80 K and remained at ∼96% until 35 K and then started decreasing down to the lowest temperature [T,H) was qualitatively similar to that of the 200 Å thick LSMO with (i) a decrease in MR, (ii) the plateau became a peak, and (iii) the peak became narrow [T,H) shifted towards a higher temperature with an increase in its sharpness upon increasing the film thickness as the TC and ferromagnetic domain size increased [Fig. S3T,H) was negligible since the films were in the paramagnetic state. The constant MR temperature window near 360 K became wider as the film thickness decreased from 500 Å because of the decrease in TC, i.e., expansion of the paramagnetic state.an 207 K . Upon inperature . Upon ine narrow . The pead Fig. S3. The varH) of the 200 Å thick LSMO film. After applying a current, the voltage was measured as a function of the magnetic field at 10 K from 0 T to 7 T, which is the virgin curve. The virgin curve of the 200 Å thick LSMO, as the magnetic field increased from zero to 7 T, shows a colossal drop in resistance to 99.8%. The MR(H) of this film exhibited several unique features such as (i) sharp drop in MR up to 60% under a 300 mT field in the virgin curve, (ii) after decreasing the field from 7 T, the MR remained ∼99% for the entire range of field variation of ±7 T, and (iii) the butterfly shape MR loop is asymmetric [inset of H) with a lower value of MR was observed at 10 K for the LSMO films of higher thicknesses [H) curve may be due to the exchange biasing between the Mn ion non-collinear spin and ferromagnetic spin in LSMO. The sharp drop in the MR with a low field (<300 mT) indicates that a fraction of the film has non-collinear Mn ion spins with weak magnetic coupling. The gradual drop in MR with an increase in the magnetic field above 300 mT demonstrates the presence of weak magnetic coupling between the Mn ions. The negligible variation in MR after applying ±7 T even at a low field attests the achievement of complete ferromagnetic ordering in the LSMO films. The virgin curve in the MR(H) of the 200 Å thick LSMO film at a 300 mT field shows MR 60%, which decreases to 0.7% for the 500 Å thick LSMO film. Similarly, at 7 T field, the 99.8% MR in the MR(H) of the 200 Å thick LSMO film decreased to 7.69% for the 500 Å thick LSMO film [tLSMO is attributed to the decrease in the non-collinear Mn ion spins.inset of . A qualicknesses . The varSMO film . The obs31ψ is the digamma function and α is a coefficient reflecting the strength of the spin–orbit coupling and magnetic scattering. The value of α is 1 if the spin–orbit interaction and the magnetic scattering are weak, α = 0 (unitary) for strong magnetic scattering, and α = −0.5 for the symplectic case. The α value obtained from the fit of Δσ(H) for the 200 Å thick LSMO film is 0.08, which increased to 0.54 for the 500 Å thick LSMO. Thus, the LSMO films exhibit weak localization, and the magnetic scattering decreases with an increase in thickness, consistent with the observed reduction in MR with an increase in thickness. The phase coherence length (lϕ) describes the quantum correction to the conductivity in 2D systems. The lϕ obtained from the fit of Δσ(H) of the different LSMO films is plotted in lϕ values of the LSMO films are larger than the film thickness, which indicates that the charge carriers are confined in 2D. In addition, these lϕ values are consistent with that for the previously reported LSMO films grown on SrTiO3.32 The increase in lϕ with the LSMO film thickness is consistent with the formation of ferromagnetic and conducting domains with an increase in the LSMO film thickness on LAO.The change in magnetoconductance (MC) at a field below 0.4 T for the LSMO films was fitted using the Hikami–Larkin–Nagaoka (HLN) theory . Accordig1 electron of Mn3+ in manganites couples ferromagnetically to the local spin of the t2g3 orbital.1 However, the thermal energy induces the spin reorientation of the eg1 electrons, which increases the magnetic disorder and requires a high magnetic field to reduce the spin-dependent scattering, i.e., colossal magnetoresistance.33,34 The spin-dependent scattering, i.e., the resistivity of LSMO, was reduced to a significant fraction by the application of a < 1000 mT magnetic field, which is associated with a large number of grain boundaries having a non-collinear Mn ion spin structure.35,36 Thus, the low field MR of the LSMO films can be attributed to the non-collinear spins of Mn near the LSMO–LAO interface. Further, the observed LFMR in LSMO/LAO is considerably larger than that in the ferromagnetic manganite/spacer/ferromagnetic manganite trilayers, which consist of artificial grain boundaries at the interface.11–13 The strain analysis indicates that the effect of strain on the LSMO films with a thickness of 100 to 500 Å is the same. However, the variation in M(T), ρ, and MR with film thickness confirms that the orientation of the Mn ion spin plays a vital role in the physical properties of these films. The spin state of the Mn ions in LSMO on LAO is controlled by MnO6, which in general can be influenced by the twin boundaries, strain, and oxygen vacancies.According to Hund's rule, the e0.7Sr0.3MnO3 thin films of different thicknesses were grown on (001) oriented LaAlO3 by adopting the pulsed plasma deposition method in a sputtering system. The reciprocal space mapping indicated that the in-plane growth of LSMO is relaxed; however, the symmetric XRD scan confirmed the strained growth along the out-of-plane direction with tetragonal structure. The consequences of the LSMO–LAO interface effect appear in the TC, temperature-dependent resistivity, metal-to-semiconductor transition temperature, and MR. At a magnetic field of 7 T, the MR decreased from 99.8% to 7.69% as the LSMO thickness increased from 200 Å to 500 Å. A unique characteristic of the LSMO films is the large low-field MR after decreasing the field from the maximum field. The observed ZFC and FC M(T) of the LSMO thin films grown on LAO provide direct evidence that the low field MR in LSMO films on LAO is associated with a large number of grain boundaries having non-collinear Mn ion spins. Thus, the present work demonstrates the great potential of interface and large low-field MR, which may advance the fundamental applications of orbital physics and spintronics.In conclusion, a series of LaThe authors declare no competing financial interest.NA-002-D0NA00287A-s001


Effective broadband antiviral platforms that can acton existingviruses and viruses yet to emerge are not available, creating a needto explore treatment strategies beyond the trodden paths. Here, wereport virus-encapsulating DNA origami shells that achieve broadbandvirus trapping properties by exploiting avidity and a widespread backgroundaffinity of viruses to heparan sulfate proteoglycans (HSPG). Witha calibrated density of heparin and heparan sulfate (HS) derivativescrafted to the interior of DNA origami shells, we could encapsulateadeno, adeno-associated, chikungunya, dengue, human papilloma, noro,polio, rubella, and SARS-CoV-2 viruses or virus-like particles, inone and the same HS-functionalized shell system. Additional virus-type-specificbinders were not needed for the trapping. Depending on the relativedimensions of shell to virus particles, multiple virus particles maybe trapped per shell, and multiple shells can cover the surface ofclusters of virus particles. The steric occlusion provided by theheparan sulfate-coated DNA origami shells can prevent viruses fromfurther interactions with receptors, possibly including those foundon cell surfaces. However, antibodies are prone to losingtheir function due to mutational drift, they take time to develop,and they will typically be effective for only one virus or virus serotypeat a time.At present, there are over 200known viral-vector borne human diseases, of which few are treatablewith current antiviral drugs.5 located on the surface of mammalian cells9 Other investigations have considered the surfacefunctionalization of nanoparticles and polymers with HS derivativesto create virus-binding complexes with antiviral activity.13 Commonly, multivalency is required to increase the strength of bindingbetween the HS nanoparticles and viruses to prevent the undesirablerelease of infectious viruses from the virus-sequestering coatings.9The interactions of heparin and heparan sulfatederivatives withviruses have already been exploited for medical purposes, for example,in virus-sequestering coatings of condoms that are based on HS-decorateddendrimers.14 The shells mechanically prevent interactionsbetween trapped viruses and host cells. For the previous proof-of-conceptexperiments, we coated the inside of the shell with antibodies tosequester virus particles in the shells. One key advantage of theshells is that the virus-binding moieties used in their interior themselvesdo not need to have a neutralizing function since this task is performedby the shell material. Nonetheless, as discussed above, the use ofantibodies in the virus trapping shells as in our previous work presentsseveral challenges that may limit the usefulness of the virus-trappingconcept. Here, we exploit the conserved background binding of HSPGto viruses to irreversibly trap viruses in HS-functionalized shells. The HS-modifiedoligonucleotide can hybridize to single-stranded DNA extensions termed“handles” that are located on the inward-facing surfacesof the DNA origami shells. For the coupling, we used azide-group modifiedHS derivatives containing either 8 or 18 saccharide monomers , including monomerssuch as N-acetyl-glucosamine and glucuronic acidwhich characterize HS polymers. As controls, we used 8-mer and 18-merpolysaccharides lacking the sulfate and sulfonate groups . The DNA oligonucleotide to beclicked to the different HS polymers was modified with a dibenzocyclooctyne(DBCO) moiety (2), and the SPAAC reaction occurred rapidlyupon mixture of both components. We analyzed the reaction products(3a–d) by polyacrylamide gel electrophoresis(PAGE), which revealed different electrophoretic mobilities for thedifferent product versions consistent with expectation , we covalently attached a heparan sulfate (HS) derivativeto a DNA oligonucleotide S1. The HT = 1 icosahedral half shell designs  featuring 40 and 85 nm wide cavities, respectively. The resulting shell has a cavity diameter of approximately 150nm. Negative stain transmission electron microscopy (TEM) images validatethe successful assembly of T3 shells .To capturedifferently sized viruses, we fabricated three DNA origamishell variants and functionalized their interior with the same HSderivative. We used the previously described octahedral and ectively1D.14 We 1 shells 1E. The Tng rules S2. The rH1), distal (H2), and branched (H3) to determinethe type and density of the HS modifications required for trappingviruses , improving from the∼30% and 84% of occupied shells achieved with H1 and H2, respectively . We therefore used the branched handle design H3,and the HS 18-mer variant (3c) henceforth, unless otherwisespecified. We confirmed that the interaction with AAV2 is due to thesulfate and sulfonate groups present in the HS structure, as the 3d HS derivative used as a negative control did not demonstrateany binding . Importantly, allAAV2 particles were trapped with O shell excess .In experiments with adeno-associated virus serotype2 (AAV2), weexplored three different DNA handle designs: proximal . We used HS-modified O shells to sequesterAAV2, poliovirus, mature dengue, and norovirus (T1 shells to trap human papilloma virus 16 (HPV 16), SARS-CoV-2,chikungunya, and rubella particles .With the HS handle design thusestablished, we tested the HS-modifiedDNA origami shells for their ability to trap a variety of exemplaryviruses and virus-like particles (VLPs).T3 shell material deformed from spherical to ellipticalaround adenovirus particles, presumably driven by maximization ofthe number of molecular interactions between the HS moieties on theshell interior surface and the viral surface, at the expense of elasticallydeforming the shell. The O shells deformed occasionallyso that up to four AAV2 particles were accommodated in its cavity, multiple target particles could besequestered. For example, we observed up to six AAV2 per T1 shell . Furthermore, multiple copies of HS-modifiedshells could also partition over and cover the surface of clustersof AAV2 particles . In fact, we could trap chikungunya evenwith the 3b negative control, which are shells with acoating lacking the sulfate and sulfonate groups, albeit at a loweryield . We attribute the finding that chikungunyaparticles can be trapped in T1 even without specificshell functionalization due to cooperative amplification of weak electrostaticinteractions between the negatively charged DNA shells and the chikungunyaparticles as they interact over extended surface areas.In the negative staining TEM images, we saw thatthe chikungunyaVLP particles appeared to completely fill the 16 The surfaces relevant for the interactions with HS are presumablyhidden in the immature dengue virus conformations, which undergo conformationalchanges to become infectious, allowing them to move between vectorand host, and/or infected and healthy cells.20 While the usage of VLPs is highlyconvenient for safety reasons, we do acknowledge some limitations.For instance, initially our dengue VLP samples contained a high percentageof immature particles which did not bind to our HS-functionalizedshells. By contrast, when we induced enzymatic maturation of the dengueVLPs, to mimic the mature dengue as would occur in vivo, we did observe binding of the matured particles .Denguevirus, as well as some other viruses, present two distinct“mature” and “immature” conformations.In brief, dengue virus infectivity depends on the ability of envelopeproteins to target cell heparan sulfates.O shells showed no binding toward hepatitis B core particlesas expected . Gold nanoparticleswith diameters comparable to whole viruses (∼30 nm) conjugatedto carboxylic acid functional groups (AuNP-COOH) showed no bindingto our O shells. In a trapping competition study withboth AuNP-COOH and AAV2, the AAV2 were selectively trapped withinthe O shell cavity, while the AuNP-COOH remained unbound. These experiments suggestthat the electrostatic binding properties of the HS modificationscan discriminate between functionalities and protein composition.The unsuccessful trapping of immature dengueparticles serves asa test for understanding the selectivity of our system. We performedfurther binding experiments with other viruses and similarly sizednanoparticles as additional negative controls. For instance, O and T1 shells, respectively . While one O shell is not sufficiently large to encapsulate an entire HPV 16particle, two O copies can coordinate and completelycover an entire VLP . Consistently, we also found that the gap distances in between O shells .We also performed cryogenic electron microscopy (cryo-EM) studiesof HPV 16 and chikungunya VLPs trapped inside ectively 4A,D. Twotire VLP 4C. 2D cltire VLP S16. Constire VLP S16. ConsO shells to a dilution series. The fraction of occupied shells remainedcomparable within margin of error prior to and after a 100-fold dilutionand incubation for 14 days in the diluted sample relative to the nondilutedsample . This observation suggeststhat the spontaneous dissociation rate for the complexes formed betweenAAV2 particles and the surrounding HS-modified shell is on the scaleof weeks under the conditions tested. The high stability is presumablyavidity-driven and can be understood by considering that a spontaneousdissociation of AAV2 from a surrounding shell requires simultaneouslybreaking of dozens of bonds formed between HS chains on the engulfingshell and the virus surface. The likelihood for such event to happendecreases exponentially with the number of HS bonds formed. Furthermore,we also performed virus trapping experiments in the presence of serumto demonstrate the capabilities of the system to bind to virus particlesin biologically relevant conditions. Exemplarily, AAV2 were successfullytrapped with O shells in the presence of cell culturemedia containing 10% FBS .To test the stability of virus trapping by theshells, we subjectedexemplarily a sample consisting of AAV2 encapsulated in invitro virus-blocking ELISA assays with AAV2  as previously established.14 We quantified the extent of AAV2 binding toantibodies immobilized on a solid surface through the binding of orthogonalAAV2-specific reporter antibodies coupled to horseradish peroxidase(HRP). Residual AAV2 particles that are bound to the surface are detectedby HRP-catalyzed production of a colorimetric signal. The AAV2 trappingabilities of HS-modified T1 shells were tested and comparedto an unfunctionalized T1 shell control. The HS-modified T1 shells demonstrated a significantly lower concentrationof AAV2 particles available for binding when compared to the unmodified T1 shell control. In addition, AAV2 particles directly incubatedwith an equivalent concentration of HS 1c (without anyshells present) were also blocked from binding to the surface butwith reduced blocking capacity compared to when using HS-modifiedshells. This finding indicates that the HS by themselves do not fullypassivate the virus surface and that the DNA origami material shieldsthe virus from its exterior by steric occlusion and block potentialbinding interactions.To examine the capacity of our shells to prevent trapped virusesfrom undergoing interactions with surfaces, we performed in vivo remains an important task forthe future. Beyond potential virus neutralization, our system mayalso serve as a sink for trapping associated viral proteins  and other side products such as subviralparticles that could potentially overwhelm the immune system.21We presented a viral trapping system thattargets features of virusesthat are conserved across many families through the usage of HS derivatives.Overall, we achieved encapsulation of nine different virus and VLPtest samples, each representing a different viral family, and differentsizes and surface complexities. Our modular shell system creates alocally curved environment within the cavity that enables multivalentbinding between HS and viral surfaces and that can be optimized accordingto size and ligand density/type. Our shells can flex and adapt toa certain degree to the shape of trapped virus particles, suggestingthat the shell system can also adapt to pleomorphic virus particles.We envision that our HS-modified DNA origami shells can act as a cellularsurface decoy, sequestering the viruses and preventing interactionswith cell surfaces, and thus reduce the effective viral load in acuteinfections. Testing the therapeutic potential of this system to reduceviral load Table S1).Staple strands for origami folding reactionswere purchased fromIntegrated DNA Technologies (IDT) and used with standard desaltingpurification unless stated otherwise. DBCO-modified handle strandswere purchased from Biomers at HPLC grade. Azide-modified heparansulfate derivatives were purchased from Glycan Therapeutics. VLPswere purchased from The Native Antigen Company, Creative Biostructure,and Creative Biolabs .22 All folding reactions were subjected to optimizedthermal annealing ramps (Table S2) in aTetrad (Bio-Rad) thermal cycling device.DNA origamistructures were folded in one-pot reactions containing 50 nM of single-strandedscaffold DNA  and 250 nM of each staple strand ina standardized “folding buffer” (FoBx) containing 2) and centrifuged for 30 min at maximum speed for residual agarosepelleting. If a concentration step was needed, ultrafiltration (AmiconUltra 500 μL with 100 kDa molecular weight cutoff) was performedprior to shell assembly. For shell assembly, the purified triangleswere mixed in a 1:1 ratio. Typical triangle subunit concentrationsranged from 5 to 400 nM, while assembly times depended on the shelltype. Table S3 summarizes and offers acomparison on the optimized salt concentrations, temperature, andself-assembly times required for all shells used in this study. Theassembled shells were UV cross-linked for 1 h at 310 nm using AsahiSpectra Xenon Light source 300 W MAX-303.23All origami structures were purified using agarose gel extraction(1.5% agarose containing 0.5× TBE and 5.5 mM MgCl1a–d) were mixed in a 4:1 ratio with DBCO-modified DNA to form the respectiveproducts (3a–d). MgCl2 was added to a 0.5 M concentration, and the mixture was left overnightat 37 °C to achieve >90% conversion. The products were runina preparative 10% PAGE gel for 2 h at 35 W. Subsequently, the productbands were cut away and were crushed. 1× TEN buffer  was added to dissolve and recoverthe modified oligonucleotides, and EtOH precipitation was used forconcentration and buffer exchange. The pure products were redissolvedand kept in double distilled H2O at either 4 °C or−20 °C. Hybridization of the DNA-HS handles to the complementaryhandles protruding from the shell’s interior was performedat r.t. overnight in FoB40. Buffer exchange to 1× PBS containing10 mM MgCl2 was then performed prior to VLP encapsulationexperiments using ultrafiltration (Amicon Ultra 500 μL with100 kDa molecular weight cutoff) or dialysis .Excess of azide-modifiedheparan sulfate derivatives ) were incubated at 37 °C for 2 h. Usual amounts of sample for TEM analysis ranged from 5to 10 μL total solution at ∼10 nM triangle origami concentration.Negative stain TEM grids were prepared immediately after the 2 h incubation.Preassembled and UV-weldedshells in 1× PBS containing 10 mM MgCl2 or T1 half shellswithout any HS modification (positive controls), a dilution seriesof the T1 shells with interior HS modifications, or theequivalent concentration of unmodified HS. 1× PBS containing10 mM MgCl2 was used as a negative/blank control. Sampleswere incubated for 2 h prior to being diluted in 1× PBS containing10 mM MgCl2 and 0.05% Tween-20) and transferred to theELISA plate . From here, the capture ELISA kit wasused as per manufacturer’s protocol. End-point absorbance measurements(OD) were made at both 450 and 650 nm . FinalOD measurements were calculated by subtraction of individual referencewavelength (650 nm) values and blank controls. Samples were repeatedin triplicate.A fixed amount of AAV2 particles was incubated with either 1×PBS containing 10 mM MgCl18 Briefly, dengue VLP sample  was added to MES buffer  and gently mixed. Next, CaCl2 (aq) (0.75 μL 0.1 M) and furin  were added and mixed, and the sample wasincubated at 30 °C for 16 h. After incubation, Tris buffer  was added to the sample,and the sample was immediately dialyzed against 1× PBS . Matured dengue VLP sample was used immediately andstored at 4 °C.Dengue VLP maturation wasadapted by methods described by Yu et al.2 concentrations. Next, the grids were stained for30 s with 2% aqueous uranyl formate containing 25 mM NaOH. Imagingwas performed with magnifications in between 10000× and 42000×in a SerialEM at a FEI Tecnai T12 microscope operated at 120 kV witha Tietz TEMCAM-F416 camera. TEM micrographs were high-pass filteredto remove long-range staining gradients, and the contrast was autoleveledusing Adobe Photoshop CS5. To obtain TEM statistics in an unbiasedfashion, automatic grid montages were acquired. For detailed informationon selected particles, negative stain EM tomography was used as avisualization technique. The tilt series were performed from −50°to +50°, and micrographs were acquired in 2° increments.Samples were incubated on glowdischarged  formvar carbon-coated Cu400 TEM grids (ElectronMicroscopy Sciences) for 90–120 s depending on origami andMgCl24 The micrographs were aligned to each other bycalculating a cross correlation of the consecutive tilt series images.The tomogram was then generated using a filtered back-projection.The Gaussian-Filter used a cutoff between 0.25 and 0.5, and a falloffof 0.035.Tilt series were processed with Etomo (IMOD) to acquire tomograms.O + HPV: 70 nM triangles; T1 + chikungunya: 200 nM triangles)were incubated 60 s on glow-discharged lacey carbon 400-mesh coppergrids with an ultrathin carbon film. Free HPV 16 VLPs were appliedto C-flat 1.2/1.3 grids (Protochip). Subsequently, the grids wereplunge frozen in liquid ethane with a FEI Vitrobot Mark V . Cryo-EM imaging was performed with a spherical-aberration(Cs)-corrected Titan Krios G2 electron microscope (Thermo Fisher)operated with 300 kV and equipped with a Falcon III 4k direct electrondetector (Thermo Fisher). Automated image acquisition was performedin EPU 2.6  and 0.29 nm (T1 + chikungunya),defocus: −1.5 to −2 μm). Micrographs were processedin RELION-325 using MotionCor226 and CTFFIND4.1.27 Particles were automatically picked with crYOLO 1.7.6.28 Extracted particle images were classified andselected by visual inspection through multiple rounds of 2D and 3Dclassifications. Initial models were generated in silico in RELION-3.3D reconstructions and multibody refinement yielded electron densitymaps with resolutions of 26 Å for O shells trappingHPV  and 36 Å for T1 shells trapping chikungunya .DNA origami shells were prepared and functionalized,and viruses were trapped as described above. Samples (


Miscanthus sinensis (silver grass) held in a post-entry quarantine facility after being imported into Western Australia, Australia. The MsiMV genome is 9604 nucleotides (nt) in length, encoding a 3071-amino-acid (aa) polyprotein with conserved sequence motifs. The MsiMV genome is most closely related to that of sorghum mosaic virus (SrMV), with 74% nt and 78.5% aa sequence identity to the SrMV polyprotein region. Phylogenetic analysis based on the polyprotein grouped MsiMV with SrMV, sugarcane mosaic virus (SCMV), and maize dwarf mosaic virus (MDMV). This is the first report of a novel monopartite ssRNA virus in Miscanthus sinensis related to members of the genus Potyvirus in the family Potyviridae.Here, we describe the full-length genome sequence of a novel potyvirus, tentatively named “Miscanthus sinensis mosaic virus” (MsiMV), isolated from  Miscanthus sinensis, commonly known as silver grass, is a member of the family Poaceae and is native to eastern Asia, where it is a keystone species in grasslands. In addition to ornamental applications, M. sinensis has been investigated for use as a biofuel crop due to its high biomass yield. Miscanthus has been found to be an alternate host for viruses infecting plants of the family Poaceae, including switchgrass mosaic virus and barley yellow dwarf virus [rf virus .Potyviruses represent the largest and most economically damaging genus of known plant RNA viruses . PotyvirAll potyviruses have a monopartite genome containing a long open reading frame (ORF) , which eMiscanthus sinensis cultivar “Morning Light” plant imported into Western Australia from the USA in 1985 was transferred to the Plant Quarantine Station, Rydalmere, New South Wales, for virus screening, as per new import conditions for clonal grasses at the time [Zea mays cv. Supagold resulted in mosaic symptoms [A symptoms . The infin silico assembly of 21- to 22-nt small RNA sequences were performed as described previously [Collection of RNA samples and eviously , yieldineviously .The completeness of the small-RNA-based genome assembly was confirmed by mapping of both Illumina and long-read Oxford Nanopore Technology (ONT reads > 500 bp) ribosomal-depleted RNA-seq data FigD. OveralThe qualifiers accepted by the International Committee on Taxonomy of Viruses for a new species of potyvirus are < 76% nt and < 82% aa ORF sequence identity [Miscanthus, corn, and sugarcane. The phylogenetic position of MsiMV, separate from SrMV, SCMV, and MDMV, further supports the classification of MsiMV as a member of a novel species in the genus Potyvirus.Phylogenetic analysis based on polyprotein sequences placed MsiMV within a clade with SrMV and MDMV, with SCMV as a sister clade Fig. . The cla