
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00728-4
10.1016/j.psj.2024.104149
104149
IMMUNOLOGY, HEALTH AND DISEASE
Genetic heterogeneity and potential recombination across hosts of Gyrovirus galga1 in central and eastern China during 2021 to 2024
Zhang Zhibin *
Man Yuanzhuo *
Xu Xin *
Wang Yan *
Ji Jun jijun020@126.com
*1
Yao Lunguang *
Bi Yingzuo †
Xie Qingmei †
⁎ Henan Provincial Engineering Laboratory of Insects Bio-reactor, Henan Provincial Engineering, and Technology Center of Health Products for Livestock and Poultry, Henan Provincial Engineering and Technology Center of Animal Disease Diagnosis and Integrated Control, Nanyang Normal University, Nanyang, 473061, China
† College of Animal Science, South China Agricultural University, Guangzhou 510642, China
1 Corresponding author: jijun020@126.com
02 8 2024
11 2024
02 8 2024
103 11 10414927 5 2024
27 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Gyrovirus galga1 (GyVg1), formerly known as AGV2, was initially identified in chickens in southern Brazil. The prevalence of GyVg1 from 2021 to 2024 in 28 out of the 63 poultry farms located in Jiangsu, Anhui, Henan, Hunan, Shandong, and Hubei provinces in eastern and central China was detected via PCR. The complete genomes of the 28 strains were sequenced and exhibited a full length of 2,376 bp. Similarity analysis of these strains did not suggest definite correlation with evolutionary branching and geographical distribution. Compared with the reference GyVg1 strains, HN2202 shared the highest similarity of 99.71% with HLJ1511 (chicken-originated) from northeastern China in 2015 to 2016. Recombination analysis revealed that AH2102 was a potential recombinant of peafowl-originated HN2019-PF1 and chicken-originated HLJ1506-2, whereas HN2304 was a recombinant of peafowl-originated HN2019-PF1 and the Hungarian ferret strain G13. Mutation site analysis of the capsid protein revealed that highly mutated regions occurred between sites 288 to 316 and 383 to 419. These results indicate that GyVg1 may have undergone an interspecies transmission, which involved complex mutations and recombination. This study may provide a reference for subsequent investigations targeting the molecular epidemiology and viral evolution of GyVg1.

Key words

gyrovirus galga1
avian gyrovirus 2
phylogenetic analysis
recombination analysis
mutation analysis
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pmcINTRODUCTION

The genus Gyrovirus initially consisted of only 1 member: Gyrovirus chicken anemia (GyVCA), which used to be categorized as a circovirus since it was discovered by Japanese researchers from contaminated vaccines in 1979 (Yuasa et al., 1979). However, genomic structures and sequences have lately indicated GyVCA to be closer to ssDNA viruses of the Anelloviridae family. As a result, Gyrovirus was reclassified into Anelloviridae by the International Committee on Taxonomy of Viruses in 2017 (Rosario et al., 2017). Gyrovirus galga1 (GyVg1, formerly known as AGV2) has been designated as the second species of Gyrovirus according to the identification report (Rijsewijk et al., 2011). GyVs subsequently identified in other organisms were also classified into this genus. However, the pathogenesis of these novel GyVs remains unclear, and their genetic characteristics are still poorly known.

The GyVg1 genome, having a length of 2.37 kb, harbors 3 major open reading frames, coding for VP1–3 that contain repeated regions (Rijsewijk et al., 2011). The VP1 coding region spans nucleotides 953 to 2335, encoding the viral capsid protein (461 amino acids [AAs]), and starts with a high arginine and lysine region (Bullenkamp et al., 2012; Yao et al., 2017). After the VP1 stop codon, a high GC region forms 2 stem-loop structures, which may play critical roles in the virus's replication, pathogenicity, and infectivity (Rijsewijk et al., 2011). Open reading frames for VP2 (450–1145 nt) and VP3 (577–951 nt) encode nonstructural proteins consisting of 232 and 125 AAs, respectively (Yao et al., 2017). VP2 coding regions contain a protein tyrosine phosphatase sequence that is highly conserved and crucial for the replication, cellular pathology, and pathogenicity of GyVg1. The VP3 protein contains a motif with a nuclear localization signal similar to that of VP3 to the GyVCA, and it can act as an apoptosis-inducing factor exclusively in tumor cells (Danen-Van Oorschot et al., 2003; Bullenkamp et al., 2012).

The specific manifestations related to GyVg1 infections are ambiguous. An autopsy suggested some clinical signs and symptoms, such as hemorrhage, glandular gastric erosion, mental impairment, and retardation in infected chickens (dos Santos et al., 2012). GyVg1 contamination has been detected in poultry vaccines in Brazil and may be the cause of GyVg1 epidemics (Varela et al., 2014). In 2015, GyVg1 infections with high prevalence in chicken were also reported in mainland China (Yao et al., 2017). A recent study found that the prevalence of GyVg1 in several poultry farms in southern China varied from 11.69% to 22.46% (Zhang et al., 2024). At present, GyVg1 infections have been detected in dozens of Chinese provinces, and the distribution suggests that GyVg1 is widespread in mainland China (Yao et al., 2016).

In addition to the initial host—chicken—GyVg1 has been detected in various species around the world. A human gyrovirus (HGyV), showing 96% nucleotide similarity to GyVg1, was discovered in the skin swabs of healthy French individuals, suggesting that GyVg1 carries the potential risk of infecting humans (Sauvage et al., 2011). Subsequently, GyVg1 was also identified in human fecal specimens from Tunisia, Chile, and Hong Kong in China (Chu et al., 2012; Gia Phan, 2013; Fehér et al., 2014). Because chickens are the original hosts of GyVg1, its continued study is of great importance. Herein, we conducted a targeted survey to investigate the prevalence, recent evolution, and mutation of GyVg1 in poultry flocks in various provinces of China.

MATERIALS AND METHODS

Clinical Sample Collection

An epidemiological survey was conducted in Henan, Hubei, Hunan, Anhui, Shandong, and Jiangsu provinces of central and eastern China from 2021 to 2024. In total, 630 pooled visceral tissues were collected from chickens that died naturally at 63 large-scale poultry farms (1 pooled sample per chicken and 10 birds per farm). All samples were frozen in the same manner and stored in a −80°C freezer for subsequent testing.

DNA/RNA Extraction and Detection

Viral nucleic acids were extracted from the tissues using a FastPure Viral DNA/RNA Mini Kit Pro (Vazyme Biotechnology, Inc., Nanjing, China). PCR for GyVg1 detection was performed as previously reported, with a specific primer set modified based on the alignment of various sequences that included GyVg1-1F: 5ʹ-CGT(G/C)TCCGCCAGCAGAAACGAC-3ʹ and GyVg1-1R: 5ʹ-GGTAGA(A/G)GCCAAAGCGTCC(A/G)CGA-3ʹ (Ye et al., 2015).

Sequencing of the GyVg1 Genome

Overlapping segments of the GyVg1 genome sequence were completely amplified and sequenced using partial overlapping to ensure the integrity of the results. Based on a previous report, primers for sequencing were modified to amplify the GyVg1 genome into 3 segments of sizes 802, 733, and 981 bp (Yao et al., 2017). The primers comprised GyVg1-1F: 5ʹ-ATTTCCTAGCAC(T/A) CAAAAACCCATT-3ʹ, GyVg1-1R: 5ʹ-TCTGGGCGTGCTCA(A/G)TTCTGA-3ʹ; GyVg1-2F: 5ʹ-TCACAGCCAATCAGAA(T/C)TGAGCACG-3ʹ, GyVg1-2R 5ʹ-TTCTACGCGCATATCGAAATTTACC-3ʹ; GyVg1-3F 5ʹ-TATTCCCGGAGG(G/A) GTAAATTTCGAT-3ʹ, and GyVg1-3R 5ʹ-CCCCTGTCCCCGT(G/A)ATGGAATGTT-3ʹ.

The total PCR reaction volume was 25 μL, with 0.5 μL of 1U/μL Phanta SE Super-Fidelity DNA Polymerase (Vazyme Biotechnology), 1 μL of each primer, 1 μL of template, 12.5 μL 2× of Phanta SE Buffer, and PCR-grade sterile deionized water. The mixtures were predenatured at 95°C for 3 min and then subjected to 35 cycles of predenaturation at 98°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 50 s, with a final extension at 72°C for 10 min. The PCR amplicons were purified and ligated into the pCE3 Blunt Vector (Vazyme Biotechnology) for sequencing (Syn-Biotechnology, Suzhou, China).

Sequence-Identity and Phylogenetic Analyses

The SeqMan software (DNASTAR, Lasergene, Madison, WI) was used to assemble the amplified fragments of the GyVg1 whole genome. GyVg1 genomes from the same chicken flock having identical sequences were considered to be of 1 strain. The genome sequences obtained were compared for identity with 49 reference sequences (Supplementary Table 1) downloaded from GenBank using the ClustalX v1.83 software. BioAider Version 1.314 was applied to display more intuitively the variations in sequence similarity and was demonstrated utilizing the online Chiplot (Zhou et al., 2020) (https://www.chiplot.online/). Phylogenetic analysis was conducted on the genome sequences of GyVg1 strains identified in this study and 49 reference strains using the maximum likelihood method, with the Kimura 2-parameter model and 1000 bootstrap replicates applying MEGA-X v.10.1.5 (Srivathsan and Meier, 2012; Kumar et al., 2018; Nguyen et al., 2015). The phylogenetic tree was then processed and labeled using the iTOL website (Letunic and Bork, 2021) (https://itol.embl.de/).

Recombination Analysis

The RDP4.101 software was used to predict the recombination events related to the obtained and reference strains using 6 algorithms, including BOOTSCAN, CHIMAERA, GENECONV, MaxChi, SISCAN, and 3SEQ (Boni et al., 2007; Martin et al., 2015). Only the recombination events supported by at least 4 methods were considered. Subsequently, the predicted recombinants were individually evaluated using the SimPlot v3.5.1 software.

Mutation Sites and Antigenic Epitope Prediction

The potentially dominant B-cell antigenic epitopes were predicted based on the AA variations in the VP1 protein of the 28 GyVg1 and reference strains using DNAMAN Version 5.2.2. Mutation sites in VP1, VP2, and VP3 were embellished and visualized by rose diagrams created using Chiplot (https://www.chiplot.online/polarPlot/rosePlot.html).

RESULTS

Positive Rate of Clinical Samples

In this study, 28 out of 63 (44.4%) chicken flocks from the 6 provinces were tested positive for GyVg1. Among them, 9 were from Henan, 8 from Jiangsu, 6 from Hubei, and 5 from Anhui. Notably, Henan exhibited the highest proportion of positive detections at 32.14% (9/28), whereas Anhui showed the lowest at 17.86% (5/28). The rate for each GyVg1-positive flock ranged from 70% to 100%.

Sequence Similarity

The full-length genome of the 28 GyVg1 strains consisted of 2376 bp. The sequences were submitted to GenBank under accession numbers PP826999–PP827026. As shown in Figure 1, the sequences of the 28 GyVg1 strains obtained showed 61.21% similarity with the reference human GyV3 strains identified in the United States (accession nos.: NC_017091.1 and JQ308210.1), thus exhibiting a relatively high level of interspecies sequence similarity. Compared with the reference GyVg1, the obtained strains exhibited relatively low similarities, ranging from 86.85% (139Anello-1; accession no.: OM892265.1, 2022) to 92.68% (141Anello; accession no.: OM892269.1, 2022) detected in lion samples in China.Figure 1 Heat map indicating the genome similarity of the newly obtained and reference GyVg1 strains. The gradient colors on the right side indicate the similarity values of different strains in the 95% to 100% interval.

Figure 1

Phylogenetic Analysis

The strains obtained in this study were separated from the GyVg1 strains obtained from other organisms and countries and divided into 4 major branches (Figure 2). The branching and clustering of the phylogenetic tree constructed based on the complete genomes was neither region- nor host-determined. AH2101, AH2102, HN2401, HN2101, HN2102, JS2401, JS2101, JS2102, HB2401, and HB2101, as well as the reference strains that originated from ferrets in Hungary and humans from France, were clustered in a large branch and were relatively close to each other. In contrast, AH2301 and HN2303, combined with the peafowl-originated HN2019-PF1, formed a separate cluster. However, the 2 GyVg1 strains obtained (AH2302 and JS2302) were only close to the 2 reference strains (139Anello-1 and 141Anello) isolated from lions in Jiangsu.Figure 2 A phylogenetic tree using the full-length genomes of the 28 GyVg1 strains obtained in this study and 49 reference strains was constructed. Strains from chickens are shown in yellow, and reference strains from other species are shown in green. Referring to the bootstrap test (1,000 repetitions), the red-filled circles along the branches indicate the bootstrap values. Various colored shapes around the evolutionary tree indicate different countries or regions. Among them, red balls indicate strains from China, and black stars represent the new strains obtained in this study.

Figure 2

Recombinant Analysis

Based on the results predicted using the RDP4 software, 4 representative recombination events were observed (Table 1). For the first event, AH2102 was potentially derived from multiple recombination of the first parent of HN2019-PF1 and the second parent of HLJ1506-2. In event 2, HN2304 was predicted to have been recombined from the ferret-derived G13 strain with the peacock-derived HN2019-PF1 strain. Recombination events 3 and 4 involved JS2101 and HN2302, which had HLJ1510 as the common parent (Figure 3).Table 1 Recombination analysis of GyVg1 using the RDP4 software.

Table 1Recombination Event	Recombinant Strain	Breakpoint Positions	Major Parent		Minor Parent		P-value	
		Begin	End		Similarity		Similarity		
1	AH2102	1084	2049	HN2019-PF1	96.63%	HLJ1506-2	97.52%	4.84E-15	
2	HN2304	393	1592	HN2019-PF1	97.64%	G13	96.89%	1.56E-11	
3	JS2101	2050	78	HLJ1510	98.86%	HN2019-L1	96.59%	8.19E-04	
4	HN2302	381	1032	HN2019-T1	97.85%	HLJ1510	96.46%	2.17E-06	

Figure 3 Representative recombined strains with recombination events predicted using the SimPlot software. The recombination breakpoint is indicated by the intersection point of the 2 lines. (A) Strain AH2102 was a recombinant of HN2019-PF1 and HLJ1506-2. (B) HN2304 was a recombinant of HN2019-PF1 and G13. (C) Strain JS2101 was a recombinant of HLJ1510 and the reference strain HN2019-L1. (D) Strain HN2302 was a recombinant of HLJ1510 and the reference strain HN2019-H1.

Figure 3

Antigenic Epitope Prediction

The 16 potential B-cell antigenic epitopes predicted for the 28 strains obtained are displayed in Table 2, with the highest scoring epitope located at AAs 98–112 (Table 2).Table 2 B-cell epitope prediction.

Table 2Amino acid sites	Amino acid sequence	Score	
98–112	NLTVCHVASINVNLR	1.216	
422–433	TPWCVVKVRSIW	1.207	
226–237	FSPVASLLVQND	1.201	
59–67	PGSYVVRLP	1.161	
312–331	SSRCFYSKACFPSFAALSAM	1.155	
173–180	WALLVMHP	1.135	
72–93	KLTLFFQGIVFIPEAQAFVKST	1.122	
366–373	LTLVPKGV	1.113	
388–408	TDIATLFLAQGSPVWAPYKFG	1.111	
132–143	PYPQHLQGCQWS	1.105	
292–309	QQGCSQNVAPGIYRLAGL	1.104	
207–220	LFRHVKTKFRVLAT	1.092	
245–256	EGFPVKGAPPMC	1.088	
441–446	PYPWQV	1.085	
276–284	EQWLPVNPP	1.071	
162–168	RPSVPPS	1.067	

Analysis of Mutation Sites

Based on the spatial and temporal diversity, 6 reference strains, HM590588.1, G13, S53/It, 915F06007FD, CL33 and HLJ1510 from different species or regions were selected for analyzing the AA mutation sites. A comparison of the AA sequences of the 28 GyVg1 strains obtained with those of the 5 reference strains revealed a few unique variations. Compared to HM590588.1, the 3 unique sites of VP1, namely Arg212Lys, Ala270Ser, and Gln310Glu, were detected in all the obtained and reference strains. Meanwhile, 2 hypervariable regions, which may correspond to those of GyVCA (loci 131–165), were identified in the N-terminal and central domains of VP1. They were located at loci 44–73 and 288–316, with 7 and 8 substitutions (24.14% and 28.57% of the fragments), respectively.

Compared to the VP2 of HM590588.1, 9 mutation sites, namely Gln141Arg, Arg156Gly, Arg157Lys, Gly158Arg, His161Tyr, Thr164Ala, Asp173Glu, Asp174Glu, and Val178Ala, were present in 15 of the 28 strains obtained (53.57%), whereas the remaining 13 (46.43%) exhibited consistency with the 5 reference strains at these loci. Mutations in VP3 were mainly concentrated at positions 54 to 81 and 93 to 105. Similar to GyVCA, a bipartite nuclear localization sequence was detected at 84–88 (RRPRR) and 120 to 124 (KKLRL), which was highly conserved in GyVg1. The putative nuclear export signal was also identified at 102 to 110 (EKQQKENLI). Different from G13, S53/It, 915F06007FD, CL33, HLJ1510, Lys(103), Gln(104), and Gln(105) were replaced by Arg in this motif from the 19 strains obtained and HM590588.1. The Nontranslated region (NTR) also varied partially from the previously reported strains, including the different direct repeat (DR) regions. The 2 primary DR sequences reported previously are 5ʹ-GTACAGGGGGGTACGTACCAT-3ʹ and 5ʹ-GTACAGGGGGGTACGTATCAT-3ʹ, as well as 1 terminal DR sequence 5ʹ-GTACAGGGGGGTACGTCACAGC-3ʹ. These appeared in a different order of alignment in the newly identified strains than those previously reported. These mutation sites and their frequencies are presented in Figure 4.Figure 4 Multiple mutations were found in all 3 coding region proteins of the 28 strains acquired. Rose charts illustrate the distribution of mutation sites and frequencies of the mutations in VP1 (A), VP2 (B), and VP3 (C) proteins in each strain, respectively. (1) denotes the set of mutation sites in strains with <5 mutations, and (2) denotes the set of mutation sites in strains with >5 mutations (inclusive of 5 strains).

Figure 4

DISCUSSION

Since GyVg1 was initially found in chickens from Brazil, it has subsequently been reported in several other countries, including Italy, France, Hungary, South Africa and Japan(Rijsewijk et al., 2011; Maggi et al., 2012; Biagini et al., 2013; Abolnik and Wandrag, 2014; Fehér et al., 2014; Mase et al., 2022). GyVg1 has currently been identified in various hosts besides chicken. According the available references, reports related to GyVg1 from other nations were mainly found in human and ferrets, while studies on GyVg1 identification in various animals was predominantly originated from China. GyVg1 was first detected in 2019 in snake tissues in China (Wu et al., 2019). Additionally, multiple gyroviruses, including GyVg1, were found in the feces of pet cats in northeastern China (Niu et al., 2019). In 2021, GyVg1 was found in the sera of some mammals (tigers, lions, sika deer, hippopotamuses, etc.) and birds (including silver pheasants, egrets, and peacocks) from a zoo in China (Ji et al., 2022). These reports indicate that GyVg1 may be transmit across different hosts, serving as a reminder of the need to strengthen and continue the monitoring of GyVg1.

Herein, we screened GyVg1 circulating in the poultry farms of a few major poultry farming provinces in central and eastern China. Conjoint similarity analysis of the evolutionary tree constructed using the 28 novel and reference GyVg1 strains downloaded from the GenBank exhibited a marked evolutionary divergence and varied evolutionary trends. HB2401 and HN2401 clustered with the 915-F-06-007-FD (French reference strain) in a small branch. HB2201 was closely related to 2 Japanese reference strains, KGSM/N0313-5S and KGSM/N0326-1S, forming another small branch. The sequence similarity analysis between the 3 obtained strains and the related reference strains from different countries was consistent with the phylogenetic tree clustering. The highest similarity (99.75%) was found between HN2401 and HB2401. Intriguingly, HN2202 identified in 2022 shared the highest similarity of 99.71% with the 3 reference strains HLJ1511, HLJ1603-1, and HLJ1603-2 identified in 2015–2016, implying the complexity of virus epidemics. The similarity among the 3 strains originated from Jiangsu in 2023 varied markedly from 96.38% to 99.07%. Furthermore, phylogenetic analysis revealed that the 3 strains did not belong to the same evolutionary branch, exhibiting a vast evolutionary distance.

Intra and interhost recombination events serve as significant drivers of viral evolution (Pérez-Losada et al., 2015; Stedman, 2015). AH2102 and HN2304 were recombination products of the HN2019-PF1 parent (identified in peacocks, China, 2019), with the HLJ1506-2 parent (identified in chickens, China, 2015) and G13 (derived from ferrets), respectively. HN2302 and JS2101 shared a common parent, HLJ1510 (identified in chickens, China, 2015), whereas their other parent was HN2019-H1 (identified in hippopotamuses, China, 2019) and HN2019-L1 (identified in lions, China, 2019), both of which were from a zoo in Henan. These recombination events predicted in the strains obtained were related to parent strains detected in various species, suggesting the complexity of the horizontal GyVg1 transmission mode. Meanwhile, analysis of recombination events revealed that these recombinant strains mainly recombined from Chinese strains. Although the 28 GyVg1 strains were genetically related to chicken-derived reference strains from other countries, no recombination signals were detected, suggesting a certain degree of genetic differentiation between Chinese strains and those from other countries.

Compared with the reference strains, the various strains obtained had mutation sites reported previously, and the VP1 of GyVg1 had various AA substitutions at sites 44 to 73. The prediction of the antigenic epitope of VP1 of the strains obtained revealed specific variant sites, including Leu73Pro, Ser106Phe, Arg212Lys, Gly293Gln, Arg314Lys/Gly, Ile390Val, Val401Met, and Trp433Arg. These mutations may have resulted under the host immune pressure, which implies the possibility of widespread and persistent GyVg1 infection among hosts. The residues 107 to 113 of VP2 may be similar to the protein tyrosine phosphatases of the GyVCA-VP2 (Rijsewijk et al., 2011). The phosphatase motif with the sequence “WX7HX3CXCX5H” is highly conserved in GyVCA, as well as the GyVg1 strains obtained, but with 2 mutations in the region adjacent to the Leu96Pro locus in JS2302 and Arg101His in HN2303. The previously reported strain HN2019PF1 had mutations within the motif. The present study also found 2 such mutations, but their impact on function remains to be investigated in the future. Although these mutation sites were inconsistent with the mutation identified at position 110 in HN2019PF1 in 2021, it indicates that the sequence containing the phosphatase motif in VP2 is not highly conserved, contrary to earlier reports (Yao et al., 2017; Ji et al., 2022). Leucine-rich hydrophobic stretches positioned at 38 to 51 (IQIGIGSTIITLSL) were identified in the VP3s of all the 28 novel strains, which may crucially affect the binding of the virus to cell-surface proteins (Heilman et al., 2006). Similar to GyVCA, a putative nuclear export signal was found to be located at 102–110 (EKQQKENLI) in GyVg1. However, this fragment showed 3 AA substitutions, Lys103Arg, Gln104Arg, and Gln105Arg, in the various strains obtained. This alteration requires further investigation as it may affect the charge distribution of the protein and the entry of the viral nucleic acids into the cytoplasm through the nuclear pore complex. The DR region of the UTR of GyVCA is considered a transcriptional enhancer (Miller et al., 2005). The GyVg1 NTR was situated between the canonical polyadenylation site (AATAAA) and the transcription start site. Similar DR regions exist in between, including 3 types of DR structures and their varied arrangements (Yao et al., 2017). The specific roles played by these arrangements in enhancing transcription and their particular functions during virus replication remain unclear. In-depth studies are needed in the future to validate the exact function of the DR region.

Overall, this study elucidated the complex recombination of chicken-originated GyVg1 across species and regions, potentially driving viral evolution and adaptation. The analysis of genetic heterogeneity and complicated recombination can facilitate virus research, vaccine development, and continuous surveillance of GyVg1.

AUTHOR CONTRIBUTIONS

JJ and XX conceived and designed the study; ZBZ and YZM amplified the sequences; ZBZ and YW performed the phylogenetic and recombination analyses; JJ and LGY analyzed the sequencing data and conducted statistical analyses; ZBZ compiled the manuscript; YZB and QMX critically revised the manuscript. All authors have read and approved the manuscript.

DISCLOSURES

The authors have no competing interests to declare.

Appendix Supplementary materials

Image, application 1

ACKNOWLEDGMENTS

This study was supported by the National Natural Science Foundation of China (Grant no. 31802185 ), the Program for Science & Technology Innovation Talents in Universities of Henan Province (Grant no. 22HASTIT042 ), and the Cultivation Project of Nanyang Normal University for NSFC (Grant no. 2024PY005 ).

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104149.
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