The N1038S Substitution and 1153EQTRPKKSV1162 Deletion of the S2 Subunit of QX-Type Avian Infectious Bronchitis Virus Can Synergistically Enhance Viral Proliferation

Abstract

The S2 subunit of infectious bronchitis virus (IBV) plays a critical role in the process of IBV infection. A comparison between the S2 subunit sequence of chicken embryo kidney cell (CEK) adapted virulent QX-like IBV strain SczyC30 (hereafter referred to as zy30) and its CEK-attenuated strain, SczyC100, revealed an N1038S substitution in S2 subunit and a 1154EQTRPKKSV1162 residue deletion in the C-terminus of the S2 subunit. In order to explore whether these two mutations are related to changes in the biological characteristics of IBV, we firstly constructed an infectious clone of zy30 using a bacterial artificial chromosome (BAC), which combines the transcription of infectious IBV genomic RNA in non-susceptible BHK-21 cells with the amplification of rescued virus rzy30 in CEK cells. Then, three recombinant viruses, including an rzy30S2-N1038S strain that contained the N1038S substitution, an rzy30S2-CT9△ strain that contained the 1154EQTRPKKSV1162 deletion, and an rzy30S2-N1038S-CT9△ strain that contained both mutations, were constructed using rescued virus rzy30 as the backbone. The results showed that each mutation did not significantly affect the replication titer in CEK cells but reduced pathogenicity in chickens, while in combination, the N1038S substitution and 1154EQTRPKKSV1162 deletion improved the proliferation efficiency in CEK cells and reduced pathogenicity, compared to rzy30 strain. The contribution made by the 1154EQTRPKKSV1162 deletion in reducing pathogenicity was higher than that of N1038S substitution. Our results revealed that the N1038S substitution and 1154EQTRPKKSV1162 deletion in S2 subunit were deeply involved in the replication efficiency of IBV and contributed to reduction of viral pathogenicity.

The S2 Subunit of Infectious Bronchitis Virus Affects Abl2-Mediated Syncytium Formation

“…This finding could account for the discrepancy in the production of syncytia between zy100 and zy30. Our research discovered that the N1038S substitution is positioned in the HR2 region, with the 1154 EQYRPKKSV 1162 deletion in the cytoplasmic tail being essential for the function of the S2 subunit [ 8 , 49 ]. Alterations in these locations have a direct effect on S2 functioning, which causes Abl2 to become activated and strengthens the binding of actin, as well as causing the ENAH pathway to be indirectly activated.…”

“…Successive passages of Sczy3 with CEKs 100 times led to the creation of zy100, which is a variant of zy30 with a distinct S2 subunit that could generate bigger and greater numbers of syncytia when infected with CEKs. Thus, we conducted N1038S substitution and 1154 EQYRPKKSV 1162 deletion on rSczy3 to produce rSczy3S2-N1038S-CT9 ∆ (TCID 50 /mL = 10 7.23 ) [ 8 ]. The graphical representation of the contrast between CN, zy100, and zy30 with regard to amino acid composition, as well as the marked mutation sites, can be seen in Figure 1 .…”

“…[ 7 ]. Our earlier studies revealed that S2 mutated strains had dissimilar viral titers, generated irregular syncytia, and showed various levels of pathogenicity in living organism trials [ 5 , 8 ].However, the cause of this phenomenon was not fully elucidated due to a lack of investigation into the role of the S2 subunit. Gaining insight into how S2 subunit mutations affect viral infection can be facilitated through studying the contribution of the S2 subunit to syncytium formation.…”

Radix Isatidis polysaccharide (RIP) resists the infection of QX-type infectious bronchitis virus via the MDA5/TLR3/IRF7 signaling pathway

“…For the real-time RT-qPCR assays, specific primer pairs were designed to amplify the viral RNA of IBV, the mRNA of the inflammatory factors IL-6, IL-8, IL-1β, and NF-κB, the mRNA of the innate immune related genes MDA5, TLR3, STING, Myd88, IRF7, and IFN-β, as well as β-actin, using Primer Premier 5.0 software (Premier Biosoft International) ( Table 1 ). The procedure for the quantification of viral load was performed as described previously ( Li et al., 2022 ).…”

Global Emergence of Infectious Bronchitis Virus Variants: Evolution, Immunity, and Vaccination Challenges

“…The S2 subunit is a highly conserved part formed primarily by the heptad repeat regions, HRP1 and HRP2, and a fusion peptide that is responsible mainly for the viral entry and membrane fusion [48]. Research has shown that the S2 subunit is responsible for viral adaptation to various cell lines [52][53][54][55][56][57][58]. The S2 subunit also contains some minor neutralizing epitopes and contributes to the avidity and specificity of virus attachment, and thus viral host range [51].…”