Foot-and-Mouth Disease Virus Interserotypic Recombination in Superinfected Carrier Cattle

Viral recombination contributes to the emergence of novel strains with the potential for altered host range, transmissibility, virulence, and immune evasion. For foot-and-mouth disease virus (FMDV), cell culture experiments and phylogenetic analyses of field samples have demonstrated the occurrence of recombination. However, the frequency of recombination and associated virus–host interactions within an infected host have not been determined. We have previously reported the detection of interserotypic recombinant FMDVs in oropharyngeal fluid (OPF) samples of 42% (5/12) of heterologously superinfected FMDV carrier cattle. The present investigation consists of a detailed analysis of the virus populations in these samples including identification and characterization of additional interserotypic minority recombinants. In every animal in which recombination was detected, recombinant viruses were identified in the OPF at the earliest sampling point after superinfection. Some recombinants remained dominant until the end of the experiment, whereas others were outcompeted by parental strains. Genomic analysis of detected recombinants suggests host immune pressure as a major driver of recombinant emergence as all recombinants had capsid-coding regions derived from the superinfecting virus to which the animals did not have detectable antibodies at the time of infection. In vitro analysis of a plaque-purified recombinant virus demonstrated a growth rate comparable to its parental precursors, and measurement of its specific infectivity suggested that the recombinant virus incurred no penalty in packaging its new chimeric genome. These findings have important implications for the potential role of persistently infected carriers in FMDV ecology and the emergence of novel strains.

Introduction
Foot-and-mouth disease virus (FMDV; genus Aphthovirus, family Picornaviridae), the causative agent of the eponymous disease, is a rapidly evolving pathogen that impacts livestock across much of the world. Currently, there are six antigenically distinct serotypes in circulation defined by the VP1 capsid-encoding region of the genome: A, O, Asia1, SAT 1, SAT 2, and SAT 3. Multiple serotypes regularly co-circulate in endemic regions and animals have been found to be naturally coinfected with multiple serotypes [1][2][3][4]. Further, FMDV has a high rate of mutation which contributes to a need for continuous development of updated vaccines and genomic surveillance.
The clinical phase of FMDV infection in naïve cattle is characterized by fever and vesiculo-erosive lesions in the mouth and on the feet [5]. Importantly, FMDV can persist subclinically in the upper respiratory tract of cattle for months to years [6][7][8]. These FMDV carriers are defined by detection of infectious virus in oropharyngeal fluid (OPF) sampled using a probang cup at least 28 days after initial infection [9,10]. Importantly, animals with natural or vaccine-induced immunity can become subclinically infected with FMDV, regardless of the occurrence of clinical FMD [7]. In cattle, both the early (neoteric) and persistent forms of subclinical infection are restricted to the nasopharyngeal mucosa [11]. The main distinction between these two phases of FMDV infection is that neoteric infection is associated with substantial virus shedding in oronasal secretions, whereas probang sampling is necessary to detect infectious virus during the persistent phase of infection [12,13].
Historically, laboratory investigations into FMDV recombination detected genomic crosses of conditionally lethal mutants in tissue culture [14][15][16][17]. Subsequently, the detection of recombination between distinct FMDV genomes has been largely limited to broad phylogenetic studies [18][19][20][21]. FMDV recombination in cattle under controlled experimental conditions has only been described in the predecessor to this current study, which reported that recombinant FMDVs were detected in 42% of superinfected carrier cattle [22]. The same study reported that no recombinant FMDVs were detected in samples from cattle simultaneously co-infected with distinct strains of FMDV.
The mechanism by which picornavirus recombination is believed to take place is termed 'copy choice' or 'template switching', and consists of the RNA-dependent RNA polymerase (RdRp) switching from one template genome to another during replication, thereby creating a hybrid product [23,24]. While recombinants from infection within the genome of a single FMDV strain would not be readily detectable, coinfection with two distinct serotypes can produce recombinants with detectable mosaic genomes. Patterns of recombination are shared between FMDV and other picornaviruses. The most common breakpoints, or recombination 'hotspots', are reported between the structural (capsidencoding) regions and the non-structural protein coding regions [25][26][27]. For FMDV, though multiple breakpoints have been identified in all non-structural proteins as well as in the 5 -UTR, some more common hotspots are coding regions VP4, 2A, and 2C [17,19,25].
The present work builds upon the recently-published investigation that followed the clinical progression and infection dynamics of cattle coinfected with FMDV serotypes A and O [22]. Within a subset of coinfected cattle, interserotypic chimeric viruses were identified. Detailed analysis of these recombinant viruses and their associated populations is the focus of the present work.

Animal Study and Virus Identity Results
Virus samples used for the current investigation were derived from a set of experimental studies of FMDV coinfection in cattle [22]. Specifically, these viruses were isolated from oropharyngeal fluid sampled from 12 cattle that were persistently infected with FMDV A24 Cruzeiro (FMDV-A), and subsequently superinfected with FMDV O1 Manisa (FMDV-O). In brief, the animals were first inoculated with FMDV A24 Cruzeiro (serotype A) through intra-nasopharyngeal deposition. All cattle developed classical clinical signs of FMD, including fever and vesicles on the feet and mouth. Oropharyngeal fluid samples consistently contained infectious FMDV during the transitional (post-acute to persistent) phase of infection. After either 21 (eight cattle) or 35 (four cattle) days, animals were superinfected with FMDV O1 Manisa (serotype O) by intra-nasopharyngeal deposition ( Figure 1). The incidence of clinical signs upon serotype O infection was variable in the 21-day coinfection group, where two animals developed full classical FMD and six animals were subclinically infected with FMDV-O, confirmed through virus isolation (VI). In the 35-day coinfection group, all cattle developed full classical FMD upon infection with serotype O. Oropharyngeal fluid sampling was paused during the acute phase of infection to not interfere with disease pathogenesis. At 10 or 14 days after the serotype O superinfection, sampling of OPF was performed at 3-4 day intervals over the course of 14-28 days ( Figure 1, Table 1). Through this period, all samples from two cattle, animal IDs #1937 and #1940, were VI-negative, indicating clearance of infection. Samples from animals #1913, #1915, #1916, #1931, and #1939 contained single non-recombinant FMDV-A or FMDV-O viruses ( Figure 1). OPF samples from the remaining five cattle, IDs #1914, #1929, #1930, #1932, and #1938, were found to include interserotypic recombinant FMDV ( Figure 1). To the authors' knowledge, interserotypic FMDV has not previously been isolated from a host in which the recombination events took place. The prevalence (42% of superinfected cattle) and independent emergence of these recombinant viruses was remarkable.

Virus Population Sequencing and Characterization
In order to investigate the virus populations associated with the emergence of interserotypic recombinant FMDV, all VI-positive OPF samples isolated following superinfection with FMDV serotype O were Illumina-sequenced as previously described [22]. All samples were sequenced in duplicate, at minimum. Total reads mapped to FMDV-A and FMDV-O reference genomes ranged from 231,427-4,152,638 per sample (Table 1). Average coverages ranged from 3702-69,258 reads per site. Recombinant viruses were initially identified by analyzing site-wise coverage of mapped reads to each reference genome. Recombinants were identified by characteristic shifts in reference genome coverage, e.g., a 100-fold change in genome coverage of one reference paired with a commensurate opposite change in coverage of the other at homologous genomic loci (breakpoints). Paired-end reads were regularly broken at these breakpoints, with the forward read mapping to one reference and the reverse read to the other. This method of investigation also indicated that many of the samples included evidence of secondary (minority) recombinants (addressed below). Recombinants were also confirmed through targeted RT-PCR of regions crossing breakpoints followed by traditional Sanger sequencing [22].  Table 1). Additionally, the first date of OPF sampling following superinfection at 31 days post-infection with FMDV-A (dpi A )/10 days post-infection with FMDV-O (dpi O ) or 35 dpi A /14 dpi O was also the first observation of recombinant viruses for all animals in which recombinants were detected. Importantly, these timepoints fell within the early (neoteric) phase of the second FMDV infection, when transmission remains viable [28]. Further, viruses isolated from nasopharyngeal tissue samples contained recombinant viruses as early as 48 h after FMDV-O infection (data not shown). For animals #1914, #1930, and #1932, recombinants comprised the majority virus in OPF samples for at least two sampling dates spanning at least 4 days (Figures 2, 4 and 5). Samples obtained from animal #1938 included only one VI-positive OPF with evidence of only a single FMDV species (a recombinant) at 10 days after serotype O infection ( Figure 6). Viruses detected in samples from animal #1929 were exclusively recombinant from 38 dpi A /17 dpi O through study close at 56 dpi A /35 dpi O ( Figure 3). The same approach was applied to numerous acute (vesicle) samples with no identification of recombinant viruses.  Chimeric reads were defined as reads composed of both FMDV-A-derived and FMDV-O-derived sequence. Chimeric read data from OPF samples for each animal were combined and plotted along an x-axis representing coding region 2A-3D, which is homologous (i.e., each protein coding region is the same length) between both parental serotypes ( Figures 2-6). Subconsensus analysis indicated that most of the earlier OPF samples (e.g., 31 dpi A /10 dpi O and 35 dpi A /14 dpi O ) included multiple interserotypic recombinants. Minority recombinants identified commonly became dominant in the following OPF samples. For example, a minority recombinant virus in the #1929 52 dpi A /31 dpi O sample was identified as the majority recombinant virus in the subsequent 56 dpi A /35 dpi O sample ( Figure 3). All recombinants shared a similar genomic structure; while the specific breakpoints were variable, all recombinant genomes were derived from the FMDV-O parental genome 5 of coding region 2B (Figures 2-6, alignments). Thus, the 5 -UTR, leader protease, and all FMDV structural proteins (capsid-encoding regions) were derived from the FMDV-O inoculum. Chimeric reads belonging to recombinants that remained at minority levels were also consistently composed of FMDV-O-derived 5 -oriented segments and FMDV-A-derived 3 -oriented segments. The only exception to this was those chimeric reads associated with the multiple-recombinant in #1929, specifically in the 3B-3C region. Dominant recombinants had diverse breakpoints across the nonstructural coding regions including 2B, 2C, 3A, 3B 3 , and 3D (Figures 2-6). Minority recombinants also included breakpoints in region 3C (Figures 3 and 5).

Recombinant Breakpoint Analysis
The locations of interserotypic recombinant breakpoints were analyzed. The mechanism of recombination is thought to be enabled by the RdRp (encoded by 3D) switching templates due to high regional homology between the donor and acceptor templates [29]. In order to assess sites of recombination, genomic locations of chimeric reads (the midpoints of which approximate breakpoints) were correlated with average site-wise FMDV-A:FMDV-O parental sequence homology within a 35-nt sliding window. A calculated Spearman's correlation coefficient (ρ) of 0.64 (p < 0.0001) suggested that sequence homology between the parent genomes was important in recombination. RDP4 software version 4.101 [25,30] was used to identify recombination hotspots which were compared to hotspots identified in a dataset of 767 FMDV field-derived polyprotein coding sequences (CDS) [27]. Breakpoint distribution counts identified in the 767 FMDV CDS dataset within a 35-nt sliding window were extracted and compared to those observed in recombinants within the present study. Spearman's ρ of 0.153 (p < 0.0001) suggested that some of the breakpoints identified in the current study may be classified as hotspots.

Within-Host Evolution: Genomic Substitution Rates
In order to assess whether coinfection affected virus evolution and to evaluate evolution of the recombinant viruses, nucleotide substitution rates of viruses isolated from OPF samples were measured. Rates were estimated based on consensus level changes, calculated per nucleotide per day across the FMDV polyprotein CDS. Rates were also calculated for the 2A-3D coding regions when comparing non-recombinant FMDV-A genomes to each other, or to recombinants. Comparison of the FMDV-A inoculum sequence to the FMDV-A-derived segments of each animal's final VI-positive OPF (45 dpi A /24 dpi O -56 dpi A /45 dpi O ) provided an indication of the genomic change detected over the course of the study (Table 2). Additionally, comparison of the final FMDV-A sequence OPF obtained before FMDV-O inoculation (17 or 31 dpi A ) to the FMDV-A-derived segments of each animal's final VI-positive OPF (45 dpi A /24 dpi O -56 dpi A /45 dpi O ) provided an indication of the genomic change that took place after coinfection (Table 3).  (Table 2). In cattle with persistently infecting FMDV-A viruses that were transiently infected with recombinant viruses (#1914, #1930, and #1932), the substitution rates for the non-recombinant FMDV-A viruses averaged 7.20 × 10 −5 and 8.83 × 10 −5 s/n/d ( Table 2). For both groups and regions, substitution rates were consistently higher (ranging from 1.24-1.70 × 10 −4 s/n/d) during the period of coinfection than over the entire study period (Table 3). However, the sample sizes were too limited for statistical assessment.

Within-Host Evolution: Non-Synonymous Changes and Potential Selective Advantages
Changes in protein coding that took place over time were analyzed in viral genomes obtained from serial OPF samples. Specifically, this analysis focused on instances in which one virus replaced another as the dominant species. In comparing the FMDV-O inoculum to the FMDV-O-derived segments of succeeding recombinants, several nonsynonymous changes were identified in structural proteins (Table 4). Most notably, Ser134Cys in VP1 was identified in all recombinants. This residue lies in the GH loop, which is both the host integrin receptor binding site and part of the primary antigenic site [31]. The minority FMDV-O viruses that were present simultaneously with the recombinants also encoded this specific change, as evidenced by their lack of variation at the subconsensus level. Other nonsynonymous changes were detected, but were only present in a subset of recombinants and did not otherwise suggest any selective advantage. Additionally, the amino acid differences were analyzed between recombinants and FMDV-A viruses in instances when one virus outcompeted the other. A maximum of two amino acid changes were identified per animal, but none were shared between animals ( Table 4). The recombinants identified in animal #1929 did not encode any nonsynonymous differences in FMDV-A-derived segments. The sole virus isolated from animal #1938 OPF encoded only an Arg98Lys change within coding region 3A; nonsynonymous changes detected in the FMDV-O-derived segment were also seen in FMDV-O virus isolated in this animal.

In Vitro Recombinant Characterization
In order to investigate the functional characteristics of genomic changes in the chimeric viruses, one OPF sample that included an interserotypic recombinant was chosen for investigation of plaque morphology, growth rate, and specific infectivity in cell culture. The 35 dpi A /14 dpi O #1914 sample included a dominant (>99%) interserotypic recombinant composed of an FMDV-O genome from the 5 UTR through a portion of the 2C coding region and an FMDV-A-derived genome thereafter (breakpoint~4560, Figure 2). This recombinant was compared to each inoculum virus and to the 49 dpi A /28 dpi O OPF sample from animal #1914. This 49 dpi A /28 dpi O #1914 sample included only non-recombinant FMDV-A virus; this isolate was chosen because it ultimately displaced the recombinant in this animal.
Samples were serially diluted and incubated for 24 h on LFBK-αvβ6 cells [32]. Plaques were stained and measured using the ViralPlaque macro in ImageJ software version 1.53 [33] ( Figure 7A,B). The average plaque size of the 35 dpi A /14 dpi O OPF sample (~99.8% recombinant) was 2.8 mm 2 . This was substantially larger than the average for the FMDV-A inoculum, 0.6 mm 2 , and smaller than FMDV-O virus plaques, 4.4 mm 2 . Interestingly, the 49 dpi A /28 dpi O plaques (FMDV-A at study end) averaged 1.5 mm 2 , more than twice the size of the FMDV-A inoculum plaque average.
The growth rates of plaque-purified samples, confirmed by Illumina sequencing to represent the intended viruses, were similar in LFBK-αvβ6 cells ( Figure 7C). Plaque-purified samples were concentrated through polyethylene glycol (PEG) precipitation, titrated and run through a sucrose density gradient with OD 260 peak absorbances measured per fraction. Specific infectivity measures, defined as the ratio of viral particles to plaque forming units (VP/PFU) were 7.94 × 10 4 for the FMDV-A inoculum, 2.10 × 10 5 for the FMDV-O inoculum, 5.86 × 10 4 for the 35 dpi A /14 dpi O #1914 recombinant, and 1.41 × 10 5 for the 49 dpi A /28 dpi O #1914 non-recombinant FMDV-A virus ( Figure 7D).

Discussion
It is well-established that recombination is one of the major drivers of virus evolution. Recombination of FMDV has been characterized in tissue culture systems and has been documented retrospectively in field samples. However, recombination of FMDV has never previously been documented and characterized in a natural host species under controlled laboratory conditions. The current study demonstrated that FMDV recombination occurs in the upper respiratory tract of a substantial quantity (42%) of persistently infected cattle that were superinfected with a heterologous virus strain. By contrast, within the same studies, recombinant viruses were not detected in any samples from vesicle lesions [22]. Given that dual infections with multiple FMDV strains are known to occur under natural conditions [1,2], these findings suggest that superinfection of carrier animals should be considered as a source of recombinant FMDVs detected in retrospective field studies.
Virus populations present in OPF samples were found to be highly diverse. This was particularly true of those sampled relatively early following FMDV-O superinfection (Figures 2-5). Those samples commonly included non-recombinant serotypes A and O as well as interserotypic recombinants (Figures 2, 4 and 5). When recombinant viruses were detected, they always became the dominant species for at least one sampling date; in one animal, the recombinant remained the dominant virus in OPF until the end of the study. Additional recombinant viruses were also regularly identified at minority levels.
Those minority species included different breakpoints from the predominant recombinants. In some cases, the minorities became the dominant virus in subsequent OPF samples. In others, they appeared to remain at low levels. While it was not possible to estimate the full extent of recombination that these populations suggest, it is clearly a common event.
Consistent with the complexity of OPF virus populations, substitution rates were higher during the periods of superinfection compared to the periods of single virus infection. This elevated rate of within-host evolution during FMDV co-infection suggests that distinct immune mechanisms and/or selective pressures may exist during co-infection when compared to single strain infection.
Findings from in vitro assays did not indicate significant growth differences between the parental viruses and a selected interserotypic recombinant. The specific infectivity of this recombinant had a lower ratio of viral particles to plaque forming units than its parents or its successor. That specific measurement is impacted by multiple aspects of the virus infection cycle, and thus does not necessarily indicate that the recombinant is more fit overall. Nevertheless, that finding suggests that packaging an interserotypic recombinant FMDV genome into an FMDV-O capsid does not incur a fitness loss. This is consistent with a recent investigation of cell culture coinfection which demonstrated trans-encapsidation of serotypes FMDV-Asia 1 and FMDV-O genomes in heterologous capsids [34].
All recombinant viruses detected in these studies encoded FMDV-O capsids. Serotypes A and O are antigenically distinct, meaning that acquired immunity against one does not protect against infection with the other serotype. Thus, the animals' capacity to neutralize FMDV-O capsids is presumed to have been weaker directly following superinfection with that serotype, while the adaptive response to FMDV-A capsids was already wellestablished at that time [22]. This concept largely explains the emergence of recombinant viruses encoding FMDV-O capsids in the present study. However, the mechanistic advantage conferred by the FMDV-O capsid combined with FMDV-A-derived nonstructural coding regions remains undetermined. Similarly, it is undetermined why, in some cases, these recombinants emerged to dominance but were out-competed by non-recombinant viruses. Studies of human pathogens causing chronic infection such as HIV-1 and human cytomegalovirus among others have identified recombination between distinct viruses following superinfection [35][36][37]. Further, these studies implicate host immune evasion as a main driver of recombinant virus emergence. While the present findings suggest similar phenomena, future investigations into the FMDV acute phase and persistent phase viral populations are required to fully address these hypotheses.
There was no consistency in the dominance of viruses detected in final OPF samples across the 12 superinfected cattle. Amino acid analysis did not indicate any specific change related to dominance of one genome over another. Nevertheless, the success of interserotypic recombinant viruses over non-recombinant FMDV-O viruses is curious. This is because both groups encode FMDV-O capsids, presumably subject to comparable immune pressure. Analysis of nonsynonymous substitutions identified several amino acid changes in recombinants, most notably a change at the receptor binding site. However, this change was present in the non-recombinant FMDV-O genomes as well; thus, it does not suggest an advantage that was unique to recombinants. A more substantial difference between the recombinants and the non-recombinant FMDV-O viruses are the FMDV-A-derived portions of the recombinant genomes. FMDV recombination hotspots have been consistently identified to fall between the functional units of the genome, e.g., capsid, proteases, polymerase etc. [19,38]. If the (inconsistent) dominance of recombinants over nonrecombinants is in part due to higher fitness, the location of recombinant breakpoints can hint at the protein coding regions that confer this advantage. Breakpoints were identified in nearly all of the nonstructural protein coding regions. Thus, the only FMDV-A-derived segment that was present in all recombinants was the C-terminal portion of 3D. Interestingly, 3D is very highly conserved, as evidenced by only two amino acid differences between the two serotypes in the affected region. It is possible that some fitness advantages may have been conferred by differences in noncoding regions that were beyond the scope of this study.
Mechanistically, when the viral polymerase switches templates during a recombination event, it has been proposed that this movement is facilitated by elevated local sequence identity and related secondary structure elements between the donor and acceptor templates [39][40][41][42]. The breakpoints identified in the present work correlated very well (Spearman's correlation coefficient = 0.64) with pairwise sequence identity, which is consistent with the dogma that template switching may be facilitated by homology. In comparing the breakpoints identified in the present work with those identified across a dataset of 767 field-derived FMDV sequences, there was a weak but positive correlation (ρ = 0.153). This suggests that some of the breakpoints observed in the current investigation may not be as common or consistent in recombinants that have been isolated in the field. Alternatively, the diverse recombinants observed in the present study may represent examples of intermediaries (snapshots) in the emergence of more stable recombinants that might become epidemiologically relevant on a longer timeline, as has been shown in picornavirus recombination mechanism research [43,44].
The mechanisms by which FMDV maintains persistent infection are incompletely understood [7]. Studies have suggested down-regulation of the cellular immune response and apoptotic pathways in nasopharyngeal epithelium in association with persistent FMDV infection [13,[45][46][47]. Additionally, it has been demonstrated that during both primary and persistent infection, only a small subset of nasopharyngeal epithelial cells become infected with FMDV [48,49]. Further, recent work from our laboratory has revealed that during the carrier phase, contiguous specimens of nasopharyngeal epithelium contain foci of distinct FMDV variants. It is likely that interplay of this multifocal viral clonality combined with regionally variable mucosal immune responses may contribute to variant emergence and dominance more than fitness advantages being conferred by specific substitutions or segments. Further investigation of regionally specific host responses and FMDV populations in these tissues is needed to investigate the complex interplay of viral and host cellular factors involved in this process.
In all animals in which recombinants were detected, chimeric viruses were found to be the dominant virus in the first OPF sample obtained following FMDV-O infection, corresponding to 10-14 days after superinfection. Because recombinants were already dominant at these sampling time points, it is likely that these viruses were common, if not dominant, at earlier timepoints as well. However, the precise timing of onset of detection of recombination was not determined herein. Importantly, those earlier time points are when FMDV transmission is more likely during single-virus infections [28]. This is relevant to the concept that FMDV carriers are not believed to be significant sources of transmission [50,51].
The samples used in the current study were derived from persistently infected carrier animals that were superinfected with a heterologous virus. These animals thereby represent a distinct category as they were both persistently and acutely infected, regardless of their clinical status. Coinfection is not uncommon in FMD-endemic regions where multiple serotypes are in circulation [1][2][3][4] and superinfection of FMDV carriers comprises a subset of these dual infections. However, there are infinite permutations of the timing and strain involvement which could affect superinfection under natural conditions. The two timepoints and strains of superinfection investigated in the present work are plausible representatives of this diverse potential variability, however the output described herein should not be interpreted as paradigmatic for all potential iterations. FMDV coinfection separated by fewer than 21, or more than 35 days could induce different frequencies or patterns of recombination than were observed in the current investigation. Furthermore, different viral strains and host genetic backgrounds could also affect such output. Ultimately, greater understanding of the role of recombination in FMDV evolution and epidemiology will only be achieved through a combination of additional laboratory-based investigations and field-based longitudinal studies in FMDV-endemic countries.
We have previously characterized the neoteric (early) subclinical phase of FMDV infection as distinct from the more commonly recognized persistent phase of infection based on higher quantities of virus detectable in oronasal secretions. This neoteric subclinical shedding of FMDV has an increased probability of transmission compared to persistently infected carriers that are not superinfected [12,13,51]. Importantly, the period during which recombinant viruses were most common in these cattle occurred within the neoteric phase. Therefore, we hypothesize that transmission of novel recombinant FMDV in a carriersuperinfection scenario is more likely than in a typical persistent infection. However, the transmissibility of emergent viruses from superinfected carriers remains to be demonstrated, and this is a current focus of work in our laboratory. If proven experimentally, this relationship between FMDV persistence, superinfection, recombination, and transmission would have substantial implications for FMD epidemiology and control measures.

In Vivo Study and Sample Background
Virus samples used for the current investigation were derived from a previously published study investigating the pathogenesis of FMDV coinfection in cattle, using two bovine-derived and well-characterized FMDV isolates of strains A24 Cruzeiro and O1 Manisa [22]. The 12 cattle from which samples used for the current investigation were derived were first infected by intra-nasopharyngeal deposition of 10 6 TCID 50 of FMDV A24, after which all animals developed clinical FMD of expected severity. After either 21 (eight animals) or 35 (four animals) days, the animals were superinfected with FMDV O1 Manisa using the same titer and route. Details of the pathogenesis across these studies have been previously published [22]. Oropharyngeal fluid samples were taken from all animals 4-7 days prior to serotype O inoculation, all of which were confirmed to be positive for infectious FMDV. OPF sampling was not performed during acute infection, but restarted at 10-14 days following serotype O inoculation.

Illumina Sequencing and Assembly
Deep-sequencing of virus isolates analyzed in the present work was performed on the NextSeq 550 (Illumina) from OPF samples passaged once in LFBK-αvβ6 cells [32]. FMDV full-length genome sequencing was performed as previously described [22]. In short, FMDV RNA was extracted from cell culture supernatant using the MagMAX RNA Isolation Kit (ThermoFisher), reverse-transcribed using Superscript II (Invitrogen) using random hexameric primers, followed by Nextera XT (Illumina) library construction. All samples were sequenced in duplicate at minimum.
CLC Genomics Workbench v. 20-v. 21 was used to filter reads for quality and length and competitively assemble them to each of the two reference genomes corresponding to the two inocula, A24 Cruzeiro (GenBank #AY593768) and O1 Manisa (#AY593823). The identification of recombinant genomes was initially based on inconsistencies in read mapping, but later confirmed by Sanger sequencing amplicons created with breakpointcrossing primers, de novo assembly, resequencing, sequencing of un-passaged samples, chimeric read analysis and haplotype reconstruction; all methods covered in brief here are detailed in [22].

Breakpoint Association Analysis
Breakpoints were identified both at the consensus and subconsensus level through a combination of significant reference genome coverage changes, chimeric read identification and haplotype reconstruction. A subset of samples from each animal were also Sanger sequenced as noted above. Chimeric reads were mapped to the CDS of each reference genome and the site-wise coverage values were extracted. The product of site-wise chimeric read coverage between each reference was calculated such that putative chimeric reads that only mapped to one parental genome were discounted. This data set was then compared to breakpoints predicted among a large, 769 FMDV CDS data set (767 with the inoculum viruses removed) curated and published by Aiewsakun, Pamornchainavakul and Inchaisri [27] using the Breakpoint Distribution Plot (35-nt window) [25] function in RDP4 software [30]. Breakpoints were also compared to site-wise homology between the two parental genomes, FMDV-A and FMDV-O. Spearman's correlation coefficient was calculated in Prism 9.2 (GraphPad Software).

Haplotype Reconstruction
FMDV populations are comprised of numerous genomic variants, regularly called a mutant swarm or quasispecies. For FMDV in particular, variant virus genomes within FMDV populations have been found to fall into clades or haplogroups that can persist over time [52,53]. In order to computationally reconstruct the major component viral haplotypes, the software CliqueSNV, version 1.41 [54] was implemented. Reads mapped in CLC Genomics Workbench to the 2A-3D genomic region of the FMDV-O reference genome were extracted to BAM format and run in CliqueSNV with the parameters -t (20-100) and -tf (0.0001-0.005), depending on average coverage and estimated SNV frequencies (as estimated in CLC). In addition, haplotypes were also analyzed with the reconstruction program QuasiRecomb [55] with the -conservative flag.

In Vitro Virus Analysis
Each inoculum, the recombinant virus identified in the 35 dpi A /14 dpi O OPF sample from animal #1914, and the FMDV-A (non-recombinant) virus identified in the 49 dpi A /28 dpi O sample from animal #1914 were amplified on LFBK-αvβ6 cells for 24 h [32]. Next, each sample was serially diluted and incubated on LFBK-αvβ6 cells for 24 h. Plates were stained and viral plaques were measured using the ViralPlaque macro [33] in ImageJ v. 1.53 [56].
To ensure genetic homogeneity for subsequent analyses, plaques were isolated for each inoculum as well as both the 35 dpi A /14 dpi O and 49 dpi A /28 dpi O OPFs from animal #1914. Each sample was serially diluted and plated on LFBK-αvβ6 cells in 6-well cell culture plates (low-MP agar, 37 • C, 4.9% CO 2 ). Discrete plaques were picked, transferred to 500 µL PBS and stored at −70 • C. Thawed plaques were then incubated on LFBK-αvβ6 cells for 24-48 h (until cytopathic effect was observed) and stored at −70 • C. Homogeneity of each isolate was confirmed through targeted RT-PCR as well as Illumina sequencing as described above. Each virus was then amplified in LFBK-αvβ6 cells in 2 L roller bottles. Lastly, each virus was concentrated using polyethylene glycol precipitation and titrated.
Growth rates of each virus were measured in LFBK-αvβ6 cells, with aliquots titrated at 1, 2, 4, 7, and 24 h. Growth was below the level of detection for 1 and 2 h timepoints except for FMDV-O which averaged 2.17 × 10 2 TCID 50 /mL at 2 h. Specific infectivity was measured via sucrose gradient diffusion followed by spectrographic analysis [57,58]; Samples were titrated by plaque assay and OD 260 was measured for each virus within the same gradient fraction corresponding to the encapsidated virus absorbance peak.


MST1/2 inhibitor XMU‐MP‐1 alleviates the injury induced by ionizing radiation in haematopoietic and intestinal system

Abstract The Hippo signalling pathway has been considered as potential therapeutic target in self‐renewal and differentiation of stem and progenitor cells. Thus, mammalian Ste20‐like kinase 1/2 (MST1/2) as the core serine‐threonine kinases in the Hippo signalling pathway has been investigated for its role in immunological disease. However, little information of MST1/2 function in bone marrow suppression induced by ionizing radiation was fully investigated. Here, we reported that MST1/2 inhibitor XMU‐MP‐1 could rescue the impaired haematopoietic stem cells (HSCs) and progenitor cells (HPCs) function under oxidative stress condition. Also, XMU‐MP‐1 pretreatment markedly alleviated the small intestinal system injury caused by the total body irradiation 9 Gy and extended the average survival days of the mice exposed to the lethal dose radiation. Therefore, irradiation exposure causes the serious pathological changes of haematopoietic and intestinal system, and XMU‐MP‐1 could prevent the ROS production, the haematopoietic cells impairment and the intestinal injury. These detrimental effects may be associated with regulating NOX/ROS/P38MARK pathway by MST1/2.

| INTRODUC TI ON
There are constant research, ongoing innovation and technical and scientific advancements in order to tackle different forms of cancer.
Various molecules have been considered as the therapeutic targets for cancer prevention. [1][2][3][4][5][6][7] Excluding chemotherapy, radiation therapy is another cancer treatment that uses high doses of radiation to kill cancer cells and shrink tumours. However, ionizing radiation can induce irreversible damage in all radiosensitive organs. The level of damage depends on the radiation dose absorbed, radiation type and organ sensitivity. The haematopoietic system is most sensitive to radiation, especially the HSCs undergo senescence in vitro and in vivo following irradiation exposure. 8,9 Therefore, the bone marrow suppression would be observed by encompassing all cell lines causing anaemia, leucopenia, thrombocytopenia and neutropenia. 10 The ROS production is one crucial reason for radiation damage; furthermore, the persistent oxidative stress is observed in haematopoietic stem/progenitor cells (HSPCs). Previous researches revealed the persistent ROS elevation induced oxidative stress associated with the activation of NOX4 signalling. 11,12 NOX4 mediates ROS production in radiation-induced senescent cells and contributes to normal tissue damage after IR via the recruitment of inflammatory cells and the exacerbation of tissue inflammation. In addition, radiation-induced ROS production was diminished by genetic or pharmacological inhibition of NOX4. 13 High-dose irradiation (>10 Gy) can also cause severe intestinal toxicity, known as gastrointestinal syndrome. Under homeostatic conditions, rapid turnover of enterocytes is driven by ISCs located at the base of the crypts. XMU-MP-1 as an inhibitor of mammalian Ste20-like kinase 1/2 (MST1/2), displayed excellent in vivo pharmacokinetics and was able to promote mouse intestinal repair. The Hippo pathway regulates the self-renewal and differentiation of stem and progenitor cells and plays key roles in controlling organ size and regeneration. 14,15 MST1/2, mammalian homologs of Hippo, are a core pair of serine-threonine kinase in the Hippo signalling pathway that regulates the cell cycle and apoptosis. 16 A recent study showed that MST1/2 are also crucial for CD8α+ dendritic cell-mediated antigen presentation to CD8+ T cells, which requires a balance of metabolic activity and cytokine signalling depending on MST1/2 activity. 17 And it is reported that MST1/2 functioned to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. 18 Studies of MST1-deficient mice have demonstrated that MST1 regulates the production of reactive oxygen species (ROS) through FOXO3A (forkhead box O3A), thereby preventing apoptosis of peripheral T cells. 19 Meanwhile, the roles of MST1/2 among HSPCs in vivo did not reach an agreement. We hypothesized that MST1/2 could associate with the ROS production in the mitochondria of HSPCs, and XMU-MP-1 as a putative MST1/2 inhibitor was utilized as a novel radioprotectant to ameliorate TBI-induced residual BM injury. The effect of XMU-MP-1 on the intestinal system injury induced by radiation was assessed as well.  For assessing the radiation injury in the haematopoietic system, animals (n = 5 per group) were exposed to total body irradiation (TBI) at a single sublethal dose of 4.0 Gy and then monitored for 9 days. Regarding the radiation challenge to the small intestine, mice (n = 5 per group) were exposed to 9 Gy TBI, and small intestine was harvested for the histol-

| Peripheral blood cells and bone marrow nucleated cells (BMNCs) counts
Blood samples isolated by orbital sinus were kept in the tube with ethylenediaminetetraacetic acid (EDTA) and tested by Celltac E hemocytometer (Nihon Kohden). The BMNCs were collected by flushing the femur with cold PBS and analysed with the hemocytometer.

| ROS level analysis on HSCs, HPCs and BMNCs with irradiation exposure.
Reactive oxygen species level of HSCs, HPCs and BMNCs was tested by the flow cytometry with 2,7-dichlordihydrofluorescein diacetate

| The immune cells proportion analysis of the peripheral blood
For analysis of multilineage blood cells, FITC-Anti-GR-1 (BD), FITC-Anti-CD45R/B220 and FITC-Anti-CD11b antibodies were used for the test of the peripheral blood. And the proportion of positive marked cells was measured on a flow cytometer (BD Accuri™ C6 Plus).

| Histological analysis
Mice were sacrificed, and jejunum was isolated after TBI 3.5 days, fixed overnight in 10% neutral formalin and embedded in paraffin.
5μm sections were prepared and stained with haematoxylin and eosin (H&E) according to standard protocols and observed under a light microscope (Olympus Corp. Japan).

| Organ weight index detection
Animals were sacrificed organs including the thymus, spleen, heart, kidney, lung and liver were isolated. Organ index was calculated using the following equation: Organ index = organ weight/body weight.

| Statistical analysis
The results were expressed as the mean ± SD. An unpaired t-test (twotails) was used for the majority of comparisons. Significant differences between experimental groups were evaluated using one and two-way analysis of variance (ANOVA) with repeated measures followed by post hoc comparisons with Tukey's multiple paired comparison test.

| XMU-MP-1 rescues the damage of the haematopoietic system
Haematopoietic system is vulnerable to ionizing radiation, especially, WBC as a sensitive marker of irradiation effect is associated with the irradiation dose and time. The results ( Figure 1) show that the WBC of the mice exposed to 4 Gy TBI decreased to 24.7% of that in the control group (p < 0.001). XMU-MP-1 could significantly rescue the WBC impairment after IR by increasing the WBC to 56.4% of pre-irradiation (p < 0.05). The effect of radiation exposure on RBCs, HCT, PLTs and BMNCs showed the same pattern with the WBC, while the HGB level was not obviously influenced at 9 days after IR.

| XMU-MP-1 reduces the ROS level of haematopoietic system
Radiation generates ROS that can interact with a various cellular separately compared with irradiation alone. While ROS level in HPCs of the irradiated mice shows the similar pattern, but the difference is not significant. NOX4, as the phagocyte-type oxidase, is responsible for the production of ROS. As Figure 2C shown that the NOX4 expression of the mice exposed to 4 Gy TBI was induced obviously to 1.5-fold (BMNCs, p < 0.05), 1.3-fold (HPCs, p < 0.05) and 2.2fold (HSCs, p < 0.001) compared with the control groups. And NOX4 expression associated with ROS induction could be significantly decreased by XUM-MP-1 pretreatment, which recovered to 72% (BMNCs, p < 0.05), 74% (HPCs, p < 0.05) and 52% (HSCs, p < 0.001) compared with 4.0 Gy TBI. Additionally, the HSCs protein expression of γ-H2AX, Nrf2 and PP38 was measured in the mice exposed to 4 Gy TBI, the result shows the same pattern with the ROS induction in the HSCs.

| XMU-MP-1 improves the immune cells impairment induced by TBI 4 Gy
The radiation exposure can suppress or weaken the immune system. CD11b was used to label myeloid lineage cells including monocytes, neutrophils and eosinophils. As Figure 3 shown that the proportion of CD11b+ cells depleted obviously (control,

| XMU-MP-1 relieves the intestinal injury induced by 9 Gy TBI
To determine the effect of XMU-MP-1 on the intestinal system exposed to irradiation, a higher dose of 9 Gy TBI was applied to mice.
The radiation experiment was set up as the diagram Figure 4A shown. The results of haematoxylin and eosin staining ( Figure 4B

| XMU-MP-1 extends the survival days exposed to 7.5 Gy TBI
To evaluate the radioprotection of XMU-MP-1 to the acute radiation challenge, the lethal dose radiation of 7.5 Gy was applied to the mice with XMU-MP-1 treatment (shown in Figure 5A). And the results ( Figure 5B) show that there existed a turning point of the body weight of irradiated mice at the 6th day after radiation.
As Figure 5C shown, the average survival days of mice treated with

F I G U R E 3 XMU-MP-1 relieves the immune cells impair induced by 4 Gy TBI.
Mice were administrated with XMU-MP-1 for 7 days before being exposed to TBI 4 Gy. Bone marrow cells were harvested 9 days after IR and tested the cells proportion by flow cytometry. Results are expressed as Mean ± SD, n = 5 per group. (Significance: *p < 0.05, **p < 0.01 and ***p < 0.001) irradiated mice suffered from the weight loss starting at the 6th day.
Although suffering with extreme weight loss, there were more mice survived in the XMU-MP-1 pretreated group. Therefore, XMU-MP-1 could provide a tolerance of the body weight loss challenge.

| DISCUSS ION
Ionizing radiation exposure on body can activate the boost of ROS, consequently induce a various reaction on DNA, protein and lipids. Double-strand breaks (DSBs), although rare, are probably the most lethal mechanism and are commonly produced by ionizing radiation. 25 Regarding the radiosensitivity, the quiescent and slowly

ACK N OWLED G EM ENTS
This work was supported by the National Natural Science Foundation of China (81972975, 81972847); the key technologies R&D project of Tianjin (17YFZCSY00670).

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.


MicroRNAs in Serum Exosomes as Circulating Biomarkers for Postmenopausal Osteoporosis

Postmenopausal osteoporosis (PMOP) is the most common skeletal disease in postmenopausal women and has become a global public health issue. Emerging evidence demonstrated the important relationship between microRNAs and PMOP. However, miRNAs have not yet been reported in PMOP. Hence, the present study aimed to investigate the differences in miRNA expression profiles in PMOP with fragility fractures to identify the key circulating miRNAs in serum exosomes and to validate these molecules as potential biomarkers. Postmenopausal women with osteoporotic fracture and normal bone mass were enrolled. Serum exosomes were isolated by traditional differential ultracentrifugation from participants. Isolated exosomes were identified by electron microscopy, western blotting and nanoparticle-tracking analysis and then examined for exosomal small RNA sequencing. The expression of miRNAs was compared by sRNA deep sequencing and bioinformatics analysis. Three miRNAs (mir-324-3p, mir-766-3p and mir-1247-5p) were found to be associated with BMD of L1-L4, FN (femur neck) and TH (total hip), while mir-330-5p and mir-3124-5p were associated with BMD of FN and TH. Furthermore, mir-330-5p was found to promote the ALP activity of hBMSCs, while mir-3124-5p showed the opposite result. The results showed that serum exosomal miRNAs were differentially expressed in postmenopausal osteoporosis patients with fragility fractures. Our study provides the first evidence that exosomal miRNA profiling revealed aberrant circulating miRNA in postmenopausal osteoporosis. Mir-324-3p, mir-766-3p, mir-1247-5p, mir-330-5p and mir-3124-5p, which were associated with bone mineral density (BMD), may serve as candidate diagnostic biomarkers as well as potentially contribute to pathophysiology of PMOP.

INTRODUCTION
Osteoporosis (OP) is a systemic bone disorder characterized by an imbalance between bone formation and resorption, which leads to a reduction in bone mass (1). In women, postmenopausal osteoporosis (PMOP) is characterized by low bone mass and consequent fragility fractures, which have impaired quality of life and increased mortality in the population (2,3). Although the measurement of BMD by dual energy X-ray absorptiometry (DXA) has been regarded as the "gold standard" and current approaches for predicting fractures are largely based on the measurement of BMD, BMD is associated with only 30-50% patients with major fragility fractures (4). In addition, the change in bone mass by DXA is gradual, and a period of 1 or 2 years is usually necessary to identify significant changes, which is inadequate to monitor bone loss (5). It is urgent to find a more accurate way to diagnose OP and predict fracture risk.
In recent years, miRNAs have attracted extensive attention for their roles in many biological processes, such as cell proliferation, differentiation, apoptosis and migration (6)(7)(8)(9). It is known that miRNAs in serum may be associated with biological processes and play an important role in the progression of diseases. Regarding bone metabolism, some researches have shown that miRNAs were associated with bone metabolic disorders (10) and several miRNAs were proved to regulate the osteogenic differentiation of BMSCs by mediating bcatenin-dependent migration (11) or contribute to the regulation of Smad5 (12) and Runx2 (13). Additionally, it was revealed that miRNAs might be biomarkers with diagnostic and prognostic potential in cancer and other diseases (14). However, the complexity and inherent heterogeneity of miRNAs in the circulation make it difficult to develop biomarkers and let alone evaluate the prognosis of diseases.
Exosomes are cell-derived spherical lipid bilayer vesicles (EVs) with a diameter around 40-160nm, widely present in various body fluids, carrying proteins, mRNAs and miRNAs that can be transferred from donor to recipient cells via target cell membrane fusion. After release, exosomes are taken up by neighboring or distant cells, and the miRNAs contained within modulate such processes as interfering with the microenvironment, facilitating proliferation, differentiation, senenscence and apoptosis. Exosome also regulate epigenetic processes by delivering miRNAs and regulate the biological function of recipient cells in bone regeneration. A number of researches have confirmed that bone marrow mesenchymal stem cells-derived exosomes could improve bone mass by promoting osteogenesis or inhibiting osteoclastogenesis through mir-196a (15), mir-150-3p (16), mir-181a (17), mir-218 (18), and mir-29a (19). More importantly, serum exosomal miRNAs may affect bone metabolism, and can be good biomarkers based on their stability under various storage conditions. Ruchun Dai et al. reported that, serum exosomes highly expressing mir-19b-3p improved the osteogenic differentiation ability by decreasing the expression of PTEN protein (20). MiRNAs in exosomes were stable enough under different storage conditions even at 4°C for a short time (21), suggesting that serum exosome miRNA panel could be as a noninvasive biomarker for the assessment of bone loss and the detection of fragility fractures. However, there are few studies on serum exosome miRNAs in osteoporosis.
In this study we tested the potential role of these molecules as biomarkers in diagnosis and prognosis of osteoporosis and fragility fracture. We compared the serum exosomal miRNAs between osteoporosis with fragility fractures and normal BMD without fracture in postmenopausal women, to dissect the links between serum exosomal miRNAs and severe osteoporosis. The study of miRNA signatures will provide a deeper understanding of bone turnover mechanism to further identify potential diagnostic biomarkers of fragility fracture and assess fracture risk.

Patient Serum Samples
A total of 577 postmenopausal women aged 65-75 years from two communities were enrolled and history of fragility fractures were collected, BMD of the lumbar, vertebra and hip were detected by DXA. Hip fractures and spine fractures were verified by review of medical records and imaging examinations. According to the National Osteoporosis Foundation, fragility fractures are fractures resulting from any fall from a standing height or less (22). All the participants were divided into two groups: subjects of control group (CON) had no fracture history with T-score of BMD of any site >-1.0, while subjects of severe osteoporosis group (SOP) suffered from fragility fractures in the vertebral spine and/or hip with BMD T-score of any site ≤-2.5. Serum levels of calcium, phosphorus, 25-hydroxyvitamin D(25(OH)D), parathyroid hormone (PTH), Procollagen 1 N-Terminal Propeptide (P1NP) and b-carboxy-terminal collagen crosslinks (b-CTX) were obtained to rule out secondary OP. Participants using insulin, sex hormones, glucocorticoids, anti-osteoporosis drugs such as bisphosphonates, estrogen and progesterone replacement, selective estrogen receptor modulators (SERMs), parathyroid gland hormones or other drugs affecting bone metabolism, or those who suffered from diabetes, severe cardiopulmonary disease, liver and kidney disease, endocrine and metabolic diseases, autoimmune diseases, malignant tumors and hyperlipemia were excluded from the study.
We implemented the following exclusion criteria: participants without consent form (n = 52), those not finishing the DXA scan (n = 37), BMD or history of fragility fracture not meeting the requirement of the study (n = 298), and those who had medical condition excluded from the study (n = 156). Finally, there were 18 participants remained in CON, and16 participants remained in SOP. This study was approved by the Medical Ethics Committee of Huadong Hospital (2019K055) and informed consent was obtained from all participants. The participants' information was listed in Table 1.

Serum Exosomes Isolation
For this study, blood from 34 participants were sampled on weekday mornings between June 2017 and December 2017. A 10 ml tube of whole blood was collected by the trained nurse following standard procedures using a serum separator tube (367820, BD) from each participants. Serum samples were allowed to clot for 30 minutes at room temperature, and then centrifuged at approximately 1000g for 10 minutes. 3 ml peripheral serum from each participant was collected, and exosomes were isolated from serum by traditional differential ultracentrifugation in four steps. At first, serum was diluted with sterile phosphate-buffered saline to 50ml, centrifugation at 3000×g for 30 min was performed, then supernatant was centrifuged at 12,000×g for 45min followed by ultracentrifugation for 2h at 120,000 ×g in 4°C. The exosome pellet was resuspended in 100ul lysis buffer or sterile PBS, depending on subsequent experiments.

Transmission Electron Microscopy (TEM)
The suspension was mixed with an equal volume of 4% paraformaldehyde, and 25ul of the solution was taken up to the loaded copper mesh, dried at room temperature for 20 minutes, and the liquid on the filter screen was blotted from one side with a filter paper, and 30ul of phosphotungstic acid solution was added, stained for 5 min at room temperature, and then was blotted with a filter paper and dried at room temperature. The exosomes were photographed under a transmission electron microscope.
Western Blot Analysis E x o s o m e s w e r e l y s e d i n R I P A b u ff e r w i t h 1 % phenylmethylsulfonyl fluoride (PMSF) and placed on ice for 10 minutes. Protein was quantified by using BCA protein quantitative kit (Sangon Biotech, Shanghai) according to the instruction. The concentration was adjusted by appropriate amount of radioimmunoprecipitation assay (RIPA) buffer, and sodium dodecyl sulfate (SDS) loading buffer of 1/4 volume was added. Protein samples were loaded, separated on 10% SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and transferred to a polyvinylidene difluoride (PVDF) membranes, followed by blocking for 1 hour in 5% non-fat skimmed milk in tris buffered saline with tween(TBST) solution. After blocking, membranes were incubated with primary antibodies against TSG101 (1:1000 dilution, ab125011, Abcam) and CD63 (1:1000 dilution, ab216130, Abcam) respectively overnight at 4°C. Membranes were then washed using TBST for three times and incubated in secondary antibody for 1hour in room temperature. At last, membranes were washed and developed by Tanon3500 gel imaging and photographing system (Tanon Science & Technology Co, Ltd.)

Nanoparticle Tracking Analysis (NTA)
Isolated pallets were analyzed by the Nanosight NS300 System (Malvern Instruments, UK) configured with a 488 nm laser and a high sensitivity scientific CMOS camera to determine the size and quantity of particles. The exosome samples were diluted (1:300) in particle-free PBS to an acceptable concentration, according to the manufacturer recommendations. Samples were analyzed under constant flow conditions (flow rate=50) at 25°C. For bootstrapped samples, 30 s × 60 s successive videos were captured with a camera level of 16. Data were analyzed using NTA 3.1.54 software with a detection threshold of 5. For the validation cohort, 15 s × 60 s videos were captured with a camera level of 16 and a detection threshold of 10. Laserirradiated nanoparticles are captured for 60 seconds and particle were analyzed by NTA software.

MiRNA Library Construction and Sequencing
Serum exosomes were isolated, prepared and sent to BGI-Wuhan (Wuhan, China) for miRNA library construction and nextgeneration sequencing. For each sample, clean reads were obtained via removing the low quality readsand aligned with the human genome. Clean reads were further mapped to sRNA in the GenBank and Rfam to analyze their distribution and annotate small RNA sequences. After sequencing by an Illumina sequencer, image analysis, and base identification, the raw reads after quality control were harvested. Clean reads were aligned against known miRNA precursors and mature miRNAs in the miRBase to identify conserved miRNAs. We filtered out all the samples with library size (total uniquely mapped reads) <50,000 reads. We calculated miRNAs normalized counts by using Variance stabilization normalization (VSN). The resulting VSN counts were corrected for various cohorts along with the removal of the unwanted variances by using the R (v 3.2.2) package RUVSeq (v 1.14.0). We filtered out miRNAs that had a VSN read count less than 0.5 in the 95% of control and PMOP samples, respectively. Fold-change (FC) > 2(|log 2 FC|>1) and FDR < 0.05 were the criteria for differential expression.

Cell Cultures, Transfection and Osteogenic Differentiation
Bone Mesenchymal Stem Cells (BMSCs) were purchased from CyagenBioscience Inc and cultured in human bone marrow

Statistical Analysis
Numerical data was presented as the mean ± standard deviation. Significant differences between two groups were determined by Student's t test. Differences between multiple groups were compared using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Statistical analysis was performed using SPSS and correlations were analyzed using Spearman data. p<0.05 was considered statistically significant. Statistical significance is displayed as *P < 0.05, **P < 0.01, and ***P < 0.001.

Characterization of Participants
18 participants were in control group who had a normal bone mass and16 participants were in SOP group who suffered from vertebral fracture (68.75%) and/or hip fracture (43.75%). BMI, age, biochemical markers and bone metabolism markers, 25 (OH)D, PTH, and BMD between the two groups were shown in Table 1. No statistical differences in the age were observed between the two groups. However, the mean values of height and weight were lower in SOP than those in CON (p<0.05). In addition, the SOP group have significantly lower BMD at all measured sites (lumbar spine, femoral neck and total hip) compared to CON group.

Serum Exosome Characterization
Isolated exosomes from participants were identified by a combination of TEM, western blotting and NTA. In Figure 1 results on exosome isolation were only shown for control group as representative for the feasibility of the method to isolate exosomes. According to TEM results, we observed that isolated particles were approximately 80 nm in diameter and appeared to be round vesicles ( Figure 1A). To further confirm the identity of the isolated pellets as exosomes, we performed the NTA measurements and observed that the size of isolated particles was112.8 ± 2.0nm in diameter ( Figure 1B). In detail, the size of exosome was 112.8 ± 2.0nm in CON group and 175.7 ± 5.1nm in SOP group (Supplementary Figures 1A, B). The concentration of exosome was 6.07e+008 ± 3.90e+007 particles/ml in CON group and 6.52e+008 ± 1.57e+007 particles/ml in SOP group (Supplementary Figures 1C, D). Western blotting identified increased exosome-enriched protein markers CD63 and TSG101 in isolated particle samples, compared to serum samples after exosome-isolation procedure ( Figure 1C).

Serum Exosome-Associated miRNAs Profile
DEGseq was used to identify differentially expressed microRNAs between CON group and SOP group by second-generation sequencing. According to Volcano Plots, there were statistically significant regulated miRNAs between CON and SOP ( Figure 2A). We further filtered and analyzed the differentially expressed miRNAs in serum exosome with the miRBase database to obtain all known miRNA counts, and unknown miRNAs were excluded. Compared to control group, 169miRNAs were significantly upregulated (p-value<0.05 and log 2 FC [log 2 FC]>1) and 70miRNAs were downregulated in SOP group ( Figure 2B and Supplementary Table 1).

Gene Ontology Enrichment Analysis
To greater determine the role of differentially expressed miRNAs in pathological process of PMOP, we input predicted target genes of these known miRNAs to DAVID for GO functional analysis to understand the functional distribution characteristics. The items of biological process(BP), cellular component(CC) and molecular function(MF) terms were presented in Figure 2C. The top 3 significant terms from the analysis showed that in the BP category, the diverse miRNAs were involved in cellular process, singleorganism process and metabolic process. For the CC category, the different miRNAs were correlated with cell, cell part and organelle. For the MF category, the diverse miRNAs were enriched in binding, catalytic activity and nucleic acid binding transcription factor activity.

Signaling Pathway Analysis of Target Genes
We further analyzed and investigated the potential function of differently expressed top 10miRNAs with online bioinformatics data analysis tools TargetScan and DIANA. Target genes of upregulated miRNAs were mainly involved in rheumatoid arthritis, maturity onset diabetes of the young, glycosphingolipid biosynthesis-globo series, N-glycan biosynthesis, glycosphingolipid biosynthesis-lacto and neolacto series ( Figure 3A). Target genes of downregulated top 10miRNAs were mainly involved in proteoglycans in cancer, adrenergic signaling in cardiomyocytes, arrhythmogenic right ventricular cardiomyopathy (ARVC), and mucin type O-glycan biosynthesis ( Figure 3B).

Correlation Analysis of miRNAs With Bone Mineral Density
In this study, we analyzed the correlation between miRNA profiling that was filtered with FC>2(|log2FC|>1) and p-value<0.05 and BMD so as not to miss miRNAs that may have impact on the bone metabolism.To greater confirm the core exosomal miRNAs involved in the progression of PMOP, correlations between known miRNAs and BMD of lumbar L1-L4, FN, and TH were analyzed and the results were shown in  with BMD of FN and TH (mir-330-5p was positively correlated, whilemir-3124-3p was negatively correlated). Signaling pathway enrichment for target genes of these five miRNAs were investigated and the results showed that Wnt signaling pathway was the most enrichment pathway relating to bone metabolism and osteogenic differentiation as shown in Table 3. Hence, we further analyzed the potential role of these five miRNA candidates in Wnt signaling pathway and online bioinformatics tools as TargetScan and DAVID was applied to help to predict target genes that may be involved in Wnt signaling pathway ( Figure 4). Wnt family members, frizzled class receptors and dishevelled segment polarity proteins in Wnt signaling pathway were found for more than 3 times as target genes of those 5 miRNAs ( Table 4)

MiRNA Candidates Relating to BMD Could Regulate ALP Activity in hBMSCs
Signaling pathway enrichment results showed that 5 miRNA candidates relating to BMD were also involved in regulating pluripotency of stem cells. To further confirm the function of these five miRNA candidates on bone turnover imbalance, ALP activity were detected in bone mesenchymal stem cells by transfecting with miRNA mimics or vehicles. ALP activity results showed that mir-330-5psuppressed ALP activity and inhibited the osteogenic differentiation of BMSCs, while mir-3124-3p showed the opposite result ( Figure 5). In aggregate, these observations suggest that these differentially expressed miRNAs may be involved in the progression of PMOP and have potential to be novel diagnostic biomarkers of PMOP.

DISCUSSION
Osteoporosis and fracture have been strongly associated with women in post-menopausal age. Although clinical and basic research is constantly progressing, patients are still facing delayed diagnosis and fragility fractures, which indicate that exploration of circulating biomarkers is needed to provide a convenient and noninvasive diagnosis. MiRNAs are regarded as promising biomarkers to evaluate disease progression and miRNAs in the serum of OP patients has been investigated, and the patterns of circulating miRNAs are likely to be diagnostic predictors of OP (23). However, few data about miRNAs, let alone circulating exosomal miRNAs, are available in PMOP with fragility fractures. Hence, we presented, for the first time, the serum exosomal miRNAs expression profiles in postmenopausal women, and compared the differences between women suffered from severe osteoporosis and those with normal BMD and discovered exosome miRNAs with promising diagnostic values. In the DEGseq, among miRNAs with fold change >2 and p-value<0.5, 169miRNAs were found to be significantly upregulated, while 70miRNAs were downregulated in SOP group, compared to CON group. GO functional analysis was applied to describe the items of BP, CC and MF terms that provided significant clues to studying molecular functions in the progression of osteoprosis. PMOP with fragility fractures is a complex biological process that involves complicated signaling pathways. In this study, we focused on miRNAs associated with BMD and related molecular mechanisms to gain insight into the link between PMOP with fragility fractures and miRNAs. We found five exosomal miRNAs (mir-324-3p, mir-766-3p, mir-1247-5p, mir-330-5p and mir-3124-5p) were related to BMD. Moreover, predicted target genes of these five miRNAs were highly associated with Wnt signaling pathway. Wnt signaling pathway is well known for its role in regulating selfrenewal and differentiation in stem cells and bone metabolism (24)(25)(26)(27). Wnt family members, frizzled class receptors and dishevelled segment polarity proteins in Wnt signaling pathway were notable as target genes of those 5 miRNAs that may provide rewarding points for further research. Among them, mir-324-3p, mir-766-3p and mir-1247-5p were found to be associated with BMD of the lumbar spine, femoral neck and hip sites, while mir-330-5p and mir-3124-5p were found to be associated with BMD of the hip. Previous studies (28) proved that mir-324-3p was expressed at low levels in low-traumatic fractures, indicating that in elderly individuals, low expression of mir-324-3p may result in fractures by reducing bone density. Mir-766-3p could reduce the protein expression of Wnt3a (29) and NF-kB (30), which play important roles in OP. In breast tumors, mir-1247-5p promotes tumor growth via the Dishevelled1 (DVL1)/Wnt/b-catenin signaling pathway (31), which promotes the differentiation of skeletal cells and accelerates bone regeneration (32). However, not for all the five miRNAs found associated with BMD, the function in bone metabolism has been validated with in vitro study. The only two miRNAs found involved in ALP activity are mir 330-5p and mir-3124 with the highest fold change (mir-330-5p [log 2 FC=-5.84], mir-3124-5p [log 2 FC=5.72]). Mir-330-5p suppress ALP activity and inhibit the osteogenic differentiation of BMSCs, and mir-3124-5p significantly promoted osteogenic differentiation of BMSCs. Mir-330-5p was reported to be  upregulated in senescent MSCs compared with young MSCs (33). MSCs are known to have self-renewal and multi-differentiation abilities, and a reduction in osteogenic differentiation of MSCs leads to loss of bone mass and contributes to increased risk of fracture. Knockdown of mir-330-5p facilitates osteogenesis through the biglycan-induced bone morphogenetic protein (BMP)/Smad pathway and further to influence the progression of OP (34). On the other hand, mir-330-5p was found to silence SPRY2 expression and further influence the progression of tumors via Mitogenactivated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) signaling (35), which is a regulator of osteoclastogenesis and plays an important role in bone loss (36). In our study, mir-330-5p in exosomes was positively correlated to the BMD of FN and TH and in vitro suppress ALP activity and inhibit the osteogenesis. These results indicate that for those people with relatively high bone mass, the expression of mir-330-5p may be elevated to suppress the osteogenic differentiation of MSCs to maintain the balance of bone metabolism. There were few studies on mir-3124-5p before, however, in this study we found mir-3124-5p was negatively related to BMD in vivo, and significantly promoted osteogenesis in vitro. We consider that in patients with low bone mass, the expression of mir-3124-5p may be upregulated compensatively to promote the osteogenic differentiation of MSCs to prevent bone loss. These results suggested that the exosomal miRNAs candidates associated with BMD might play complex regulatory roles in the progression of PMOP by networking with cell signaling pathways, and mir-330-5p and mir-3124-5p in circulating exosomes could not only be biomarkers but also functional molecules in the progression of PMOP. However, more studies are needed to clarify the molecular mechanisms of miRNAs in circulating exosomes in PMOP with fragility fractures.
In conclusion, this study provided the first information on differential serum exosomes miRNA expression profiling between severe osteoporosis and normal BMD in postmenopausal women using second-generation sequencing. mir-324-3p, mir-766-3p, mir-1247-5p,mir-330-5p and mir-3124-5p were found to be associated with BMD, but only miR-330 and miR-3124 had been confirmed its role in bone metabolism in vitro, which may serve as circulating biomarkers as well as therapeutic targets and treatment options for PMOP. However, in order to apply these miRNA profiles in clinical practice, further studies on prospectively collected datasets are needed to validate these findings, and more reliable and reproducible analysis model are required in following studies.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are publicly available. They can be found on Figshare via the DOI 10.6084/ m9.figshare.17086307.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Medical Ethics Committee of Huadong Hospital (2019K055). The patients/participants provided their written informed consent to participate in this study.


Broadly neutralizing antibodies target the coronavirus fusion peptide

The potential for future coronavirus outbreaks highlights the need to broadly target this group of pathogens. We use an epitope-agnostic approach to identify six monoclonal antibodies that bind to spike proteins from all seven human-infecting coronaviruses. All six antibodies target the conserved fusion peptide region adjacent to the S2' cleavage site. COV44-62 and COV44-79 broadly neutralize alpha and beta coronaviruses, including SARS-CoV-2 Omicron subvariants BA.2 and BA.4/5, albeit with lower potency than RBD-specific antibodies. In crystal structures of Fabs COV44-62 and COV44-79 with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine at the S2' cleavage site. COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings highlight the fusion peptide as a candidate epitope for next-generation coronavirus vaccine development.

Binding of secreted antibody to the beads was detected in the CY5 or TRED channels by capturing images at 6 min intervals over a 30 min time course. In the second step of the assay, the MERS/OC43 beads were replaced with 7 μm streptavidin beads coated with 10 μg/mL SARS-CoV-2 spike, and antibody binding was detected as before. OEP light cages were applied to export individual cross-reactive monoclonal antibody (mAb)-secreting B cells directly into Dynabeads mRNA DIRECT lysis buffer (Life Technologies, 61011) in 96-well plates. Plates were sealed with Microseal foil film (BioRad, MSF1001) and immediately frozen on dry ice before transferring to -80 °C for long-term storage. mAb sequence analysis and expression Heavy and light chain sequences were amplified from single B cell lysates using RT-PCR (20,43,52) and resolved by Sanger Sequencing (Eurofins and Genewiz). Analyses of the heavy chain variable (VH) and (lambda/kappa variable) Vλ/Vκ genes, complementarity-determining region 3 (CDR3) sequences, and percentage of somatic mutations were carried out using Geneious Prime  Human leukocyte antigen (HLA) typing for donor identification Several mAbs were isolated from screens that involved pooling of B cells from two different individuals. To identify the source donor of these mAbs, a commercially available ScisGo ® -HLA-v6 kit (Scisco Genetics Inc., Seattle WA) employing an amplicon-based sequencing by synthesis approach was used to determine HLA types of amplified complementary DNA (cDNA) from single cell isolates. The approach uses a two-stage amplicon-based PCR for locus amplification and sample barcoding. Although designed for amplification from genomic DNA, a subset of the kit amplicons was functional in amplifying product from cDNA. Briefly, samples were sequentially applied to stage 1 (S1) and stage 2 (S2) PCR amplification according to the manufacturer supplied protocol. After amplification, the reactions were combined, purified, and applied to a MiSeq using Illumina Version 2 chemistry with 500-cycle, paired-end sequencing (Illumina, San Diego, CA). Data assembly and analysis were performed using Sciscloud ® (Scisco Genetics Inc., Seattle WA) computational tools adapted specifically to assemble HLA genomic sequences derivative from the ScisGo ® -HLA-v6 kit. Amplified portions of the HLA class I and class II genes were compared with prior typing data allowing the unambiguous identification of corresponding samples. Access to all software for data transfer and analysis was included as a component of the kit and made available through a web browser.

Recombinant mAb binding to coronavirus antigens
Recombinant mAbs were diluted 4-fold in 0.05% BSA w/v in PBS to generate a 47.7 ng/mL -200 μg/mL dilution series. Multiplexed antigen-labelled beads were incubated with mAb titrations for 30 min at room temperature, then washed and stained with 2.5 μg/mL goat anti-human IgG Alexa Fluor 647 (Jackson ImmunoResearch, 109-606-170). Samples were acquired on the iQue Screener Plus and data were analysed with FlowJo. Data points from the titration curves were interconnected without logistic regression and AUC analyses were performed with GraphPad Prism and reported after correction using the AUC of the negative control CD4 population. ThCoV-HKU12 (Uniprot accession # B6VDX8), TCoV (Uniprot accession # B3FHU5), WiCoV-HKU20 (Uniprot accession # H9BR25) were aligned using MAFFT v7 server using a BLOSUM62 scoring matrix and L-INS-i algorithm. The sequence alignment was used to generate a sequence logo plot using the Weblogo 3.0 server (56)and to color conserved amino acid residues on a prefusion stabilized spike protein (PDB 6VSB) using Chimera X.
SARS-CoV-2 spike and S2 subunit epitope binning by surface plasmon resonance (SPR) Epitope binning experiments were performed on the Carterra LSA with cross-reactive mAbs coupled to an HC30M chip (Carterra).

Imaging-based fusion inhibition assay
HeLa cell lines (Expasy CVCL_0030) stably expressing either CoV spike proteins or their cognate receptor were generated as previously reported (15)  Hoechst for 10 min and washed twice with PBS. Images were acquired in A488, A568 and DAPI channels using a BZ-X fluorescence microscope (KEYENCE) and processed using Fiji ImageJ (57).

Colorimetric fusion inhibition assay
HEKBlue cells (Invivogen, hkb-hace2tpsa) stably transfected to express human angiotensin-I- hours. Absorbance was measured using an EnSpire Multimode (Perkin Elmer) plate reader at 635 nm. Percent inhibition was calculated as described in (58), briefly (1-(E-N)/(P-N)) x 100; where "E" is absorbance of the antibody treatment group, "N" is the absorbance of vector control, and "P" is the absorbance of the no antibody treatment group.
Shotgun mutagenesis epitope mapping of antibodies by alanine scanning Epitope mapping was performed essentially as previously described (59), using a SARS-CoV-2 (Wuhan Hu-1 strain) S2 subunit shotgun mutagenesis mutation library, made using a full-length expression construct for the SARS-CoV-2 spike glycoprotein. 513 S2 residues (between residues 689 -1247) were mutated individually to alanine, and alanine residues to serine. Mutations were confirmed by DNA sequencing, and clones arrayed in a 384-well plate, one mutant per well.
Binding of mAbs to each mutant clone in the alanine scanning library was determined, in duplicate, by high-throughput flow cytometry. A plasmid encoding cDNA for each spike protein mutant was transfected into HEK-293T cells and allowed to express for 22 h. Cells were fixed in 4% (v/v) PFA (Electron Microscopy Sciences), and permeabilized with 0.1% (w/v) saponin (Sigma-Aldrich) in PBS before incubation with mAbs diluted in PBS, 10% normal goat serum (Sigma), and 0.1% saponin. mAb screening concentrations were determined using an independent immunofluorescence titration curve against cells expressing wild-type spike protein to ensure that signals were within the linear range of detection. Antibodies were detected using 3.75 μg/mL of Alexa-Fluor-488-conjugated secondary antibodies (Jackson ImmunoResearch) in 10% normal goat serum with 0.1% saponin. Cells were washed three times with PBS/0.1% saponin followed by two washes in PBS, and mean cellular fluorescence was detected using a high-throughput Intellicyt iQue flow cytometer (Sartorius). Antibody reactivity against each mutant spike protein clone was calculated relative to wild-type spike protein reactivity by subtracting the signal from mock-transfected controls and normalizing to the signal from wild-type spike-transfected controls.
Mutations within clones were identified as critical to the mAb epitope if they did not support reactivity of the test mAb but supported reactivity of other SARS-CoV-2 antibodies. This counterscreen strategy facilitates the exclusion of spike protein mutants that are locally misfolded or have an expression defect.
Where NC is the average readout of negative controls (wells without live virus), and PC is the Core and Pathology data packages were received. Animals were randomly assigned to groups to balance as closely as possible between ages (same in this experiment), weight ranges, and sex distribution prior to challenge in consultation with a NIH statistician.