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LitCovid_Glycan-Motif-Structure

{"project":"LitCovid_Glycan-Motif-Structure","denotations":[{"id":"T1","span":{"begin":5128,"end":5139},"obj":"https://glytoucan.org/Structures/Glycans/G81533KY"}],"text":"4 Discussion\nVaccines, particularly live-attenuated vaccines, remain the most effective means of protection against IBV challenge (e.g., the Mass serotype strains H120, H52, Ma5, and W93, which are still widely used in the poultry industry) [38], [39], [40]. However, due to the poor cross-protection provided between the different serotypes, these attenuated serotype-specific vaccines cannot provide complete protection, and the IB endemic in China cannot be controlled [22]. Genetic mutations and immune pressure during the replication process of the single stranded RNA virus often results in the emergence of IBV variants [41], [42]. To date, at least six genotypes of IBV strains have been identified in China, of which the QX-type (first isolated in 1999) remains the most prevalent genotype [10], [42], [43]. The poor relationship between the QX-type strains, which are abundant and display variable virulence in various parts of China, and the Mass-type vaccine strains could explain the failure of the Mass-type vaccination programs to control IBV in these flocks [19], [39]. Consequently, the development of novel vaccines against circulating IBV strains in China using local IBV strains is required [20], [43], [44].\nOptimal vaccination against circulating IBV strains in China requires the development of attenuated vaccines designed from local strains [20], [21], [22], [45]. However, due to the particularity of RNA virus replication, attenuated vaccine strains generated by continuous passage remain associated with the risk of reversion to virulence or potential recombination between vaccine strains and virulent field strains [27], [46], [47], which represents a substantial hidden concern to the poultry industry. To reduce the problems associated with vaccine reversion, researchers have explored the option of creating vaccine viruses using reverse genetic technology (e.g., Beaudette strains carrying the S1 gene of the H120 vaccine strain, virulent M41 strain, or QX-like strain) [30], [48]. However, the titer of the Beaudette strains carrying the S1 gene of the vaccine H120 strain only reaches 106.13 ± 0.23 EID50 [49], which is lower than that of the H120 backbone strain. Therefore, we aimed to develop a recombinant rH120-S1/YZ strain based on the H120 vaccine strain that carries the S1 gene of the ck/CH/IBYZ/2011 strain (a Chinese QX-like nephoropathogenic strain) using reverse genetic technology [35], [36].\nIn the present study, we isolated a nephoropathogenic strain (IBYZ) in 2011. This strain was isolated, identified, and preserved from a large-scale chicken farm where an outbreak of IBV emerged in Jiangsu province. Chickens infected with the IBYZ strain showed severe kidney damage and a high mortality rate (Fig. 3B). The complete sequence has been deposited in the GenBank database under the accession number of KF663561. The S1 gene sequencing data indicated that the IBYZ strain belonged to the QX-like genotype (Fig. 8 ), with a nucleotide homology with the attenuated vaccine H120 strain of only 77.4%, and amino acid homology of 77.1%. Moreover, the S1/S2 cleavage site of IBYZ is HRRRR/S, which differed from RRFRR/S of the H120 vaccine strain. After whole-genome sequencing, we constructed a molecular clone strain using reverse genetic technology, termed rIBYZ. In addition, another strain used in this study is the live attenuated IBV vaccine, H120, and the molecular clone strain, rH120, which was constructed using the same method.\nFig. 8 Phylogenetic tree constructed based on the S1 gene of isolate ck/CH/IBYZ/2011 and 28 published IBV reference strains using the neighbor-joining method (Mega10.1.6).\nThe spike protein is the coronavirus structural proteins, which plays an important role on pathogenicity in coronaviruses [6], [50]. However, replacing the S gene of Beaudette strain by the virulent strains does not make it virulent [51], [52]. The S protein can be cleaved into S1 and S2 subunits at the S1/S2 cleavage site [53], the S1 subunit is responsible for receptor binding to the host cells [54]. Whether the S1 gene has an effect on the virulence of the virus is unknown. In this study, the recombinant strain, rH120-S1/YZ, expressed the S1 gene of the QX-like strain, which retains the S1/S2 cleavage site of H120, maintained the ability to replicate and exhibited pathogenicity in embryos. However, providing IBYZ S1 sequences to H120 did not increase its virulence to that of rIBYZ. IBVs replicate in many epithelial cells, including respiratory tissues, the alimentary canal, kidney, gonads, and bursa [2], [6]. The QX-like rIBYZ strain is capable of considerable growth in the trachea, lung, and kidney, which leads to severe tissue damage. In addition, this strain caused cilia necrosis and loss, lung congestion and hemorrhage, nephritis, as well as inflammatory cell infiltration in these tissues in infected chickens at different time points post-infection. The ability of IBV to replicate within many respiratory, enteric, and other epithelial surfaces may be partially related to the fact that the attachment of IBV to host cells is dependent on sialic acid on the cell surface being recognized by the S1 subunit [6], [54], [55]. However, the binding of virus to the host cell is the first step in determining tropism and S2 is responsible for membrane fusion [56]. Thus, to further explore the different tissue tropism between rH120-S1/YZ and rIBYZ, we examined the viral loads and histopathology in the tissues of the rH120-S1/YZ-infected group. It was found that rH120-S1/YZ remains moderately pathogenic in the trachea (e.g., some cilia necrosis and loss and decreased secretory cells) but no lesions were observed in the lungs and kidneys of infected chickens, in contrast to chickens infected with the rIBYZ strain. This finding suggests that both the S2 and S1 genes might play an important role in viral tissue tropism as also suggested by others [57]. Another possibility is that the lack of viral replication and damage in kidney following rH120-S1/YZ is due to lower replication ability of backbone except S2 gene from its parental rH120 strain. The viral load of the respiratory-type strain, rH120 in the different tissues indicated that the viral levels in the trachea, early following infection were significantly higher than those of rIBYZ, which indicates that the ability of the virus to attached to trachea epithelial cells of the rH120 strain was stronger than that of the rIBYZ strain. However, during the later time points post-infection, the level of the rH120 strain was significantly lower than rIBYZ, which suggests that the virulence of IBV depends both on tissue invasion and viral replication ability, as well as other functions mediated by other viral proteins (e.g., non-structural, structural, and accessory proteins) [6], [29], [58], [59], [60]. The reduction of the viral load at 5 dpi in the group infected with the rIBYZ strain may be associated with the necrosis and shedding of the trachea epithelium. Moreover, the ability to replication in the different organs of chickens infected with rH120-S1/YZ as reflected by viral loads at multiple time points was lower than that exhibited in the chickens infected with the rIBYZ strain, which demonstrated that the S1 subunit was not the only determinant of viral tropism that was consistent with results by others [57], [61], [62], and the significant difference in viral load was related to differences in the viral backbone. However, when comparing the rH120-S1/YZ and rH120 strains which carry the same backbone, it was found that the viral RNA of rH120-S1/YZ in the lungs and kidneys was significantly lower than that of rH120, which may due to the replacement of the IBYZ S1 gene in the recombinant virus, whose structure on the surface contains some degree of changes, which we speculated that may affects the functionality of the S protein of coronaviruses. Although cryo-EM structure of IBV spike protein has been determined, the relation between structure and function of different IBV S1 genes is unknown. [63]. In addition to the growth in epithelial tissues observed during the early infection period, IBV can establish long-term persistent infections in chicken flocks via oral and cloacal shedding, which represents a substantial challenge to IBV control [1], [6]. In this study, we detected viral RNA in the oral and cloacal swabs isolated from infected chickens at 7, 14, 18, 22, and 26 dpi by real-time PCR. Compared with rH120 and rIBYZ, the viral load in both the oral and cloacal swabs of the rH120-S1/YZ group at different time points after infection was significantly decreased. However, while it has been reported that the persistent virus will be re-excreted at the point of lay [1], the persistence of rH120-S1/YZ strain in the egg-laying chickens needs to be further verified. In a word, due to its low virulence i presented in virus loading and lesion in different tissue, oral and cloacal shedding of host animals, rH120-S1/YZ can be considered a safe attenuated vaccine candidate for young chickens.\nIn addition to its role in tissue tropism, the S1 glycoprotein also has an important function in inducing a neutralizing antibody response [64], [65], and small differences in the S1 contribute to poor cross protectionby neutralization test [64], [66]. To evaluate the protective efficacy of this candidate vaccine, we selected the QX-like virulent rIBYZ strain to infect chickens at 14 dpv that were vaccinated with either the rH120-S1/YZ strain or rH120 strain. We found that little protection against rIBYZ virulent strain challenge in the rH120 vaccinated group was induced, with a 50% survival rate and severe clinical symptoms associated with IB. This finding could be explained by differences in the antigenicity between rH120 and rIBYZ. No death, clinical signs, or lesions were observed in the rH120-S1/YZ-vaccinated group, indicating that this strain could provide effective protection against rIBYZ challenge in young chickens. Since high humoral antibody titers can contribute to reduced viral replication in various organs and disease recovery [67], [68], the level of specific IgG antibodies remains an important standard for evaluating the immune response to an IBV vaccine. In the present study, the humoral antibody level of chickens vaccinated with the rH120-S1/YZ strain was observed to gradually increase compared to the non-vaccinated group. However, although the rH120 strain was able to induce similar levels of humoral antibodies, it provided poor protection against rIBYZ, suggesting that protection against IBV infection is not only with humoral immunity, but also with both mucosal immunity combined with the local tracheal and cellular immune response [69], [70], [71], [72]. Thus, the detection of mucosal and cellular immunity in rH120-S1/YZ-vaccinated animals requires further exploration. Nevertheless, some studies have shown that a peptide located near the amino terminal end of S2 can be recognized by neutralizing monoclonal antibodies [73], and the S2 domain also plays an important role in inducing protective immunity [28], [61], [74]. Because the S2 domain plays an important role in cell tropism [57], we also considered that expressing the S1 subunit would provide greater safety than the entire S gene, which could lead to further destruction in some organs of the vaccinated chickens; however, further experiments are required."}

LitCovid-PD-FMA-UBERON

LitCovid-PD-UBERON

LitCovid-PD-MONDO

{"project":"LitCovid-PD-MONDO","denotations":[{"id":"T36","span":{"begin":4806,"end":4815},"obj":"Disease"},{"id":"T37","span":{"begin":4927,"end":4936},"obj":"Disease"},{"id":"T38","span":{"begin":6281,"end":6290},"obj":"Disease"},{"id":"T39","span":{"begin":6531,"end":6540},"obj":"Disease"},{"id":"T40","span":{"begin":8160,"end":8169},"obj":"Disease"},{"id":"T41","span":{"begin":8217,"end":8230},"obj":"Disease"},{"id":"T42","span":{"begin":8626,"end":8635},"obj":"Disease"},{"id":"T43","span":{"begin":10630,"end":10639},"obj":"Disease"}],"attributes":[{"id":"A36","pred":"mondo_id","subj":"T36","obj":"http://purl.obolibrary.org/obo/MONDO_0001166"},{"id":"A37","pred":"mondo_id","subj":"T37","obj":"http://purl.obolibrary.org/obo/MONDO_0005550"},{"id":"A38","pred":"mondo_id","subj":"T38","obj":"http://purl.obolibrary.org/obo/MONDO_0005550"},{"id":"A39","pred":"mondo_id","subj":"T39","obj":"http://purl.obolibrary.org/obo/MONDO_0005550"},{"id":"A40","pred":"mondo_id","subj":"T40","obj":"http://purl.obolibrary.org/obo/MONDO_0005550"},{"id":"A41","pred":"mondo_id","subj":"T41","obj":"http://purl.obolibrary.org/obo/MONDO_0005550"},{"id":"A42","pred":"mondo_id","subj":"T42","obj":"http://purl.obolibrary.org/obo/MONDO_0005550"},{"id":"A43","pred":"mondo_id","subj":"T43","obj":"http://purl.obolibrary.org/obo/MONDO_0005550"}],"text":"4 Discussion\nVaccines, particularly live-attenuated vaccines, remain the most effective means of protection against IBV challenge (e.g., the Mass serotype strains H120, H52, Ma5, and W93, which are still widely used in the poultry industry) [38], [39], [40]. However, due to the poor cross-protection provided between the different serotypes, these attenuated serotype-specific vaccines cannot provide complete protection, and the IB endemic in China cannot be controlled [22]. Genetic mutations and immune pressure during the replication process of the single stranded RNA virus often results in the emergence of IBV variants [41], [42]. To date, at least six genotypes of IBV strains have been identified in China, of which the QX-type (first isolated in 1999) remains the most prevalent genotype [10], [42], [43]. The poor relationship between the QX-type strains, which are abundant and display variable virulence in various parts of China, and the Mass-type vaccine strains could explain the failure of the Mass-type vaccination programs to control IBV in these flocks [19], [39]. Consequently, the development of novel vaccines against circulating IBV strains in China using local IBV strains is required [20], [43], [44].\nOptimal vaccination against circulating IBV strains in China requires the development of attenuated vaccines designed from local strains [20], [21], [22], [45]. However, due to the particularity of RNA virus replication, attenuated vaccine strains generated by continuous passage remain associated with the risk of reversion to virulence or potential recombination between vaccine strains and virulent field strains [27], [46], [47], which represents a substantial hidden concern to the poultry industry. To reduce the problems associated with vaccine reversion, researchers have explored the option of creating vaccine viruses using reverse genetic technology (e.g., Beaudette strains carrying the S1 gene of the H120 vaccine strain, virulent M41 strain, or QX-like strain) [30], [48]. However, the titer of the Beaudette strains carrying the S1 gene of the vaccine H120 strain only reaches 106.13 ± 0.23 EID50 [49], which is lower than that of the H120 backbone strain. Therefore, we aimed to develop a recombinant rH120-S1/YZ strain based on the H120 vaccine strain that carries the S1 gene of the ck/CH/IBYZ/2011 strain (a Chinese QX-like nephoropathogenic strain) using reverse genetic technology [35], [36].\nIn the present study, we isolated a nephoropathogenic strain (IBYZ) in 2011. This strain was isolated, identified, and preserved from a large-scale chicken farm where an outbreak of IBV emerged in Jiangsu province. Chickens infected with the IBYZ strain showed severe kidney damage and a high mortality rate (Fig. 3B). The complete sequence has been deposited in the GenBank database under the accession number of KF663561. The S1 gene sequencing data indicated that the IBYZ strain belonged to the QX-like genotype (Fig. 8 ), with a nucleotide homology with the attenuated vaccine H120 strain of only 77.4%, and amino acid homology of 77.1%. Moreover, the S1/S2 cleavage site of IBYZ is HRRRR/S, which differed from RRFRR/S of the H120 vaccine strain. After whole-genome sequencing, we constructed a molecular clone strain using reverse genetic technology, termed rIBYZ. In addition, another strain used in this study is the live attenuated IBV vaccine, H120, and the molecular clone strain, rH120, which was constructed using the same method.\nFig. 8 Phylogenetic tree constructed based on the S1 gene of isolate ck/CH/IBYZ/2011 and 28 published IBV reference strains using the neighbor-joining method (Mega10.1.6).\nThe spike protein is the coronavirus structural proteins, which plays an important role on pathogenicity in coronaviruses [6], [50]. However, replacing the S gene of Beaudette strain by the virulent strains does not make it virulent [51], [52]. The S protein can be cleaved into S1 and S2 subunits at the S1/S2 cleavage site [53], the S1 subunit is responsible for receptor binding to the host cells [54]. Whether the S1 gene has an effect on the virulence of the virus is unknown. In this study, the recombinant strain, rH120-S1/YZ, expressed the S1 gene of the QX-like strain, which retains the S1/S2 cleavage site of H120, maintained the ability to replicate and exhibited pathogenicity in embryos. However, providing IBYZ S1 sequences to H120 did not increase its virulence to that of rIBYZ. IBVs replicate in many epithelial cells, including respiratory tissues, the alimentary canal, kidney, gonads, and bursa [2], [6]. The QX-like rIBYZ strain is capable of considerable growth in the trachea, lung, and kidney, which leads to severe tissue damage. In addition, this strain caused cilia necrosis and loss, lung congestion and hemorrhage, nephritis, as well as inflammatory cell infiltration in these tissues in infected chickens at different time points post-infection. The ability of IBV to replicate within many respiratory, enteric, and other epithelial surfaces may be partially related to the fact that the attachment of IBV to host cells is dependent on sialic acid on the cell surface being recognized by the S1 subunit [6], [54], [55]. However, the binding of virus to the host cell is the first step in determining tropism and S2 is responsible for membrane fusion [56]. Thus, to further explore the different tissue tropism between rH120-S1/YZ and rIBYZ, we examined the viral loads and histopathology in the tissues of the rH120-S1/YZ-infected group. It was found that rH120-S1/YZ remains moderately pathogenic in the trachea (e.g., some cilia necrosis and loss and decreased secretory cells) but no lesions were observed in the lungs and kidneys of infected chickens, in contrast to chickens infected with the rIBYZ strain. This finding suggests that both the S2 and S1 genes might play an important role in viral tissue tropism as also suggested by others [57]. Another possibility is that the lack of viral replication and damage in kidney following rH120-S1/YZ is due to lower replication ability of backbone except S2 gene from its parental rH120 strain. The viral load of the respiratory-type strain, rH120 in the different tissues indicated that the viral levels in the trachea, early following infection were significantly higher than those of rIBYZ, which indicates that the ability of the virus to attached to trachea epithelial cells of the rH120 strain was stronger than that of the rIBYZ strain. However, during the later time points post-infection, the level of the rH120 strain was significantly lower than rIBYZ, which suggests that the virulence of IBV depends both on tissue invasion and viral replication ability, as well as other functions mediated by other viral proteins (e.g., non-structural, structural, and accessory proteins) [6], [29], [58], [59], [60]. The reduction of the viral load at 5 dpi in the group infected with the rIBYZ strain may be associated with the necrosis and shedding of the trachea epithelium. Moreover, the ability to replication in the different organs of chickens infected with rH120-S1/YZ as reflected by viral loads at multiple time points was lower than that exhibited in the chickens infected with the rIBYZ strain, which demonstrated that the S1 subunit was not the only determinant of viral tropism that was consistent with results by others [57], [61], [62], and the significant difference in viral load was related to differences in the viral backbone. However, when comparing the rH120-S1/YZ and rH120 strains which carry the same backbone, it was found that the viral RNA of rH120-S1/YZ in the lungs and kidneys was significantly lower than that of rH120, which may due to the replacement of the IBYZ S1 gene in the recombinant virus, whose structure on the surface contains some degree of changes, which we speculated that may affects the functionality of the S protein of coronaviruses. Although cryo-EM structure of IBV spike protein has been determined, the relation between structure and function of different IBV S1 genes is unknown. [63]. In addition to the growth in epithelial tissues observed during the early infection period, IBV can establish long-term persistent infections in chicken flocks via oral and cloacal shedding, which represents a substantial challenge to IBV control [1], [6]. In this study, we detected viral RNA in the oral and cloacal swabs isolated from infected chickens at 7, 14, 18, 22, and 26 dpi by real-time PCR. Compared with rH120 and rIBYZ, the viral load in both the oral and cloacal swabs of the rH120-S1/YZ group at different time points after infection was significantly decreased. However, while it has been reported that the persistent virus will be re-excreted at the point of lay [1], the persistence of rH120-S1/YZ strain in the egg-laying chickens needs to be further verified. In a word, due to its low virulence i presented in virus loading and lesion in different tissue, oral and cloacal shedding of host animals, rH120-S1/YZ can be considered a safe attenuated vaccine candidate for young chickens.\nIn addition to its role in tissue tropism, the S1 glycoprotein also has an important function in inducing a neutralizing antibody response [64], [65], and small differences in the S1 contribute to poor cross protectionby neutralization test [64], [66]. To evaluate the protective efficacy of this candidate vaccine, we selected the QX-like virulent rIBYZ strain to infect chickens at 14 dpv that were vaccinated with either the rH120-S1/YZ strain or rH120 strain. We found that little protection against rIBYZ virulent strain challenge in the rH120 vaccinated group was induced, with a 50% survival rate and severe clinical symptoms associated with IB. This finding could be explained by differences in the antigenicity between rH120 and rIBYZ. No death, clinical signs, or lesions were observed in the rH120-S1/YZ-vaccinated group, indicating that this strain could provide effective protection against rIBYZ challenge in young chickens. Since high humoral antibody titers can contribute to reduced viral replication in various organs and disease recovery [67], [68], the level of specific IgG antibodies remains an important standard for evaluating the immune response to an IBV vaccine. In the present study, the humoral antibody level of chickens vaccinated with the rH120-S1/YZ strain was observed to gradually increase compared to the non-vaccinated group. However, although the rH120 strain was able to induce similar levels of humoral antibodies, it provided poor protection against rIBYZ, suggesting that protection against IBV infection is not only with humoral immunity, but also with both mucosal immunity combined with the local tracheal and cellular immune response [69], [70], [71], [72]. Thus, the detection of mucosal and cellular immunity in rH120-S1/YZ-vaccinated animals requires further exploration. Nevertheless, some studies have shown that a peptide located near the amino terminal end of S2 can be recognized by neutralizing monoclonal antibodies [73], and the S2 domain also plays an important role in inducing protective immunity [28], [61], [74]. Because the S2 domain plays an important role in cell tropism [57], we also considered that expressing the S1 subunit would provide greater safety than the entire S gene, which could lead to further destruction in some organs of the vaccinated chickens; however, further experiments are required."}

LitCovid-PD-CLO

LitCovid-PD-CHEBI

LitCovid-PD-GO-BP

LitCovid-sentences

LitCovid-PD-HP

{"project":"LitCovid-PD-HP","denotations":[{"id":"T20","span":{"begin":2712,"end":2725},"obj":"Phenotype"},{"id":"T21","span":{"begin":4806,"end":4815},"obj":"Phenotype"}],"attributes":[{"id":"A20","pred":"hp_id","subj":"T20","obj":"http://purl.obolibrary.org/obo/HP_0000112"},{"id":"A21","pred":"hp_id","subj":"T21","obj":"http://purl.obolibrary.org/obo/HP_0000123"}],"text":"4 Discussion\nVaccines, particularly live-attenuated vaccines, remain the most effective means of protection against IBV challenge (e.g., the Mass serotype strains H120, H52, Ma5, and W93, which are still widely used in the poultry industry) [38], [39], [40]. However, due to the poor cross-protection provided between the different serotypes, these attenuated serotype-specific vaccines cannot provide complete protection, and the IB endemic in China cannot be controlled [22]. Genetic mutations and immune pressure during the replication process of the single stranded RNA virus often results in the emergence of IBV variants [41], [42]. To date, at least six genotypes of IBV strains have been identified in China, of which the QX-type (first isolated in 1999) remains the most prevalent genotype [10], [42], [43]. The poor relationship between the QX-type strains, which are abundant and display variable virulence in various parts of China, and the Mass-type vaccine strains could explain the failure of the Mass-type vaccination programs to control IBV in these flocks [19], [39]. Consequently, the development of novel vaccines against circulating IBV strains in China using local IBV strains is required [20], [43], [44].\nOptimal vaccination against circulating IBV strains in China requires the development of attenuated vaccines designed from local strains [20], [21], [22], [45]. However, due to the particularity of RNA virus replication, attenuated vaccine strains generated by continuous passage remain associated with the risk of reversion to virulence or potential recombination between vaccine strains and virulent field strains [27], [46], [47], which represents a substantial hidden concern to the poultry industry. To reduce the problems associated with vaccine reversion, researchers have explored the option of creating vaccine viruses using reverse genetic technology (e.g., Beaudette strains carrying the S1 gene of the H120 vaccine strain, virulent M41 strain, or QX-like strain) [30], [48]. However, the titer of the Beaudette strains carrying the S1 gene of the vaccine H120 strain only reaches 106.13 ± 0.23 EID50 [49], which is lower than that of the H120 backbone strain. Therefore, we aimed to develop a recombinant rH120-S1/YZ strain based on the H120 vaccine strain that carries the S1 gene of the ck/CH/IBYZ/2011 strain (a Chinese QX-like nephoropathogenic strain) using reverse genetic technology [35], [36].\nIn the present study, we isolated a nephoropathogenic strain (IBYZ) in 2011. This strain was isolated, identified, and preserved from a large-scale chicken farm where an outbreak of IBV emerged in Jiangsu province. Chickens infected with the IBYZ strain showed severe kidney damage and a high mortality rate (Fig. 3B). The complete sequence has been deposited in the GenBank database under the accession number of KF663561. The S1 gene sequencing data indicated that the IBYZ strain belonged to the QX-like genotype (Fig. 8 ), with a nucleotide homology with the attenuated vaccine H120 strain of only 77.4%, and amino acid homology of 77.1%. Moreover, the S1/S2 cleavage site of IBYZ is HRRRR/S, which differed from RRFRR/S of the H120 vaccine strain. After whole-genome sequencing, we constructed a molecular clone strain using reverse genetic technology, termed rIBYZ. In addition, another strain used in this study is the live attenuated IBV vaccine, H120, and the molecular clone strain, rH120, which was constructed using the same method.\nFig. 8 Phylogenetic tree constructed based on the S1 gene of isolate ck/CH/IBYZ/2011 and 28 published IBV reference strains using the neighbor-joining method (Mega10.1.6).\nThe spike protein is the coronavirus structural proteins, which plays an important role on pathogenicity in coronaviruses [6], [50]. However, replacing the S gene of Beaudette strain by the virulent strains does not make it virulent [51], [52]. The S protein can be cleaved into S1 and S2 subunits at the S1/S2 cleavage site [53], the S1 subunit is responsible for receptor binding to the host cells [54]. Whether the S1 gene has an effect on the virulence of the virus is unknown. In this study, the recombinant strain, rH120-S1/YZ, expressed the S1 gene of the QX-like strain, which retains the S1/S2 cleavage site of H120, maintained the ability to replicate and exhibited pathogenicity in embryos. However, providing IBYZ S1 sequences to H120 did not increase its virulence to that of rIBYZ. IBVs replicate in many epithelial cells, including respiratory tissues, the alimentary canal, kidney, gonads, and bursa [2], [6]. The QX-like rIBYZ strain is capable of considerable growth in the trachea, lung, and kidney, which leads to severe tissue damage. In addition, this strain caused cilia necrosis and loss, lung congestion and hemorrhage, nephritis, as well as inflammatory cell infiltration in these tissues in infected chickens at different time points post-infection. The ability of IBV to replicate within many respiratory, enteric, and other epithelial surfaces may be partially related to the fact that the attachment of IBV to host cells is dependent on sialic acid on the cell surface being recognized by the S1 subunit [6], [54], [55]. However, the binding of virus to the host cell is the first step in determining tropism and S2 is responsible for membrane fusion [56]. Thus, to further explore the different tissue tropism between rH120-S1/YZ and rIBYZ, we examined the viral loads and histopathology in the tissues of the rH120-S1/YZ-infected group. It was found that rH120-S1/YZ remains moderately pathogenic in the trachea (e.g., some cilia necrosis and loss and decreased secretory cells) but no lesions were observed in the lungs and kidneys of infected chickens, in contrast to chickens infected with the rIBYZ strain. This finding suggests that both the S2 and S1 genes might play an important role in viral tissue tropism as also suggested by others [57]. Another possibility is that the lack of viral replication and damage in kidney following rH120-S1/YZ is due to lower replication ability of backbone except S2 gene from its parental rH120 strain. The viral load of the respiratory-type strain, rH120 in the different tissues indicated that the viral levels in the trachea, early following infection were significantly higher than those of rIBYZ, which indicates that the ability of the virus to attached to trachea epithelial cells of the rH120 strain was stronger than that of the rIBYZ strain. However, during the later time points post-infection, the level of the rH120 strain was significantly lower than rIBYZ, which suggests that the virulence of IBV depends both on tissue invasion and viral replication ability, as well as other functions mediated by other viral proteins (e.g., non-structural, structural, and accessory proteins) [6], [29], [58], [59], [60]. The reduction of the viral load at 5 dpi in the group infected with the rIBYZ strain may be associated with the necrosis and shedding of the trachea epithelium. Moreover, the ability to replication in the different organs of chickens infected with rH120-S1/YZ as reflected by viral loads at multiple time points was lower than that exhibited in the chickens infected with the rIBYZ strain, which demonstrated that the S1 subunit was not the only determinant of viral tropism that was consistent with results by others [57], [61], [62], and the significant difference in viral load was related to differences in the viral backbone. However, when comparing the rH120-S1/YZ and rH120 strains which carry the same backbone, it was found that the viral RNA of rH120-S1/YZ in the lungs and kidneys was significantly lower than that of rH120, which may due to the replacement of the IBYZ S1 gene in the recombinant virus, whose structure on the surface contains some degree of changes, which we speculated that may affects the functionality of the S protein of coronaviruses. Although cryo-EM structure of IBV spike protein has been determined, the relation between structure and function of different IBV S1 genes is unknown. [63]. In addition to the growth in epithelial tissues observed during the early infection period, IBV can establish long-term persistent infections in chicken flocks via oral and cloacal shedding, which represents a substantial challenge to IBV control [1], [6]. In this study, we detected viral RNA in the oral and cloacal swabs isolated from infected chickens at 7, 14, 18, 22, and 26 dpi by real-time PCR. Compared with rH120 and rIBYZ, the viral load in both the oral and cloacal swabs of the rH120-S1/YZ group at different time points after infection was significantly decreased. However, while it has been reported that the persistent virus will be re-excreted at the point of lay [1], the persistence of rH120-S1/YZ strain in the egg-laying chickens needs to be further verified. In a word, due to its low virulence i presented in virus loading and lesion in different tissue, oral and cloacal shedding of host animals, rH120-S1/YZ can be considered a safe attenuated vaccine candidate for young chickens.\nIn addition to its role in tissue tropism, the S1 glycoprotein also has an important function in inducing a neutralizing antibody response [64], [65], and small differences in the S1 contribute to poor cross protectionby neutralization test [64], [66]. To evaluate the protective efficacy of this candidate vaccine, we selected the QX-like virulent rIBYZ strain to infect chickens at 14 dpv that were vaccinated with either the rH120-S1/YZ strain or rH120 strain. We found that little protection against rIBYZ virulent strain challenge in the rH120 vaccinated group was induced, with a 50% survival rate and severe clinical symptoms associated with IB. This finding could be explained by differences in the antigenicity between rH120 and rIBYZ. No death, clinical signs, or lesions were observed in the rH120-S1/YZ-vaccinated group, indicating that this strain could provide effective protection against rIBYZ challenge in young chickens. Since high humoral antibody titers can contribute to reduced viral replication in various organs and disease recovery [67], [68], the level of specific IgG antibodies remains an important standard for evaluating the immune response to an IBV vaccine. In the present study, the humoral antibody level of chickens vaccinated with the rH120-S1/YZ strain was observed to gradually increase compared to the non-vaccinated group. However, although the rH120 strain was able to induce similar levels of humoral antibodies, it provided poor protection against rIBYZ, suggesting that protection against IBV infection is not only with humoral immunity, but also with both mucosal immunity combined with the local tracheal and cellular immune response [69], [70], [71], [72]. Thus, the detection of mucosal and cellular immunity in rH120-S1/YZ-vaccinated animals requires further exploration. Nevertheless, some studies have shown that a peptide located near the amino terminal end of S2 can be recognized by neutralizing monoclonal antibodies [73], and the S2 domain also plays an important role in inducing protective immunity [28], [61], [74]. Because the S2 domain plays an important role in cell tropism [57], we also considered that expressing the S1 subunit would provide greater safety than the entire S gene, which could lead to further destruction in some organs of the vaccinated chickens; however, further experiments are required."}

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