Nephropathogenic infectious bronchitis virus (NIBV) has become the most common IBV strain in the commercial poultry industry, which is highly infectious and spreads quickly (He et al., 2016a; Kuang et al., 2020). The genome of this virus is a single plus-stranded RNA that belongs to the genus Gammacoronavirus, family Coronaviridae, in the order Nidovirales (Bayry et al., 2005). The NIBV strain was first reported in the United States and Australia (Winterfield and Hitchner, 1962; Song et al., 1998), and it is now being reported in other parts of the world, especially in Asian and Middle Eastern countries (Lim et al., 2011; Bande et al., 2017; Jiang et al., 2020). All ages of chickens can be infected, and infected chickens are characterized by coughing, sneezing, decreased egg production and intestinal symptoms; at autopsy, the kidneys are pale and enlarged. The case fatality rate can reach 5%–30%, resulting in serious economic losses for the poultry breeding industry (Bing et al., 2007; Li et al., 2020).

Chicken kidneys are the main target organ of NIBV. A large number of studies have shown that NIBV replicates in renal tubular epithelial cells and can cause renal structural changes (Han et al., 2011; Huang et al., 2017; Zhu et al., 2020). Our previous study proved that NIBV infection could lead to decreased antioxidant capacity and metabolic dysfunction in the kidney (Xu et al., 2019). Transcriptomic analysis of the kidneys of IBV-infected animals revealed local activation of several innate immune genes, including Toll receptors, cytokines and chemokines (Smith et al., 2015; He et al., 2016b). However, the relationship between NIBV and the innate immune system of the host remains unclear, as does the specific mechanism of the innate immune response to NIBV infection through the TLR7/NF-κB signaling axis.

Toll-like receptor 7 (TLR7) is localized to the endosomal compartment, where it binds to microorganisms or to self-derived single-stranded RNA (ssRNA) ligands (Petes et al., 2017). It is well known that viruses stimulate Toll receptors, leading to the nuclear translocation of nuclear factors and the activation of inflammatory cytokines and chemokines such as IL-6 and IL-8 (Li et al., 2013; Gimeno et al., 2021). Recently, it has been demonstrated that the type II IFN signaling pathway is indispensable in TLR7 for promoting autoreactive B cell development and systemic immunity (Chodisetti et al., 2020). Moreover, multiple studies have indicated that IFN-γ is critical in the development of coronaviral hepatitis (Kim et al., 2007) and is one of the major cytokines elevated in SARS patients (Yang et al., 2014). In addition, the TLR7/NF-κB signaling axis was shown to be closely related to nephritis, renal insufficiency and nephrotic syndrome (Lin et al., 2017; Zhu et al., 2020; Abaidullah et al., 2021). However, the role of the TLR7/NF-κB signaling axis in the kidney tissues of chickens infected with NIBV remains unclear.

In the present study, we established a model of NIBV infection to investigate the role of NIBV infection on the TLR7/NF-κB signaling axis of the kidney. Our findings indicated that NIBV infection induced TLR7/NF-κB signaling axis activation and promoted the expression of cytokines in the kidney. This study addresses TLR7/NF-κB signaling pathway responses to NIBV in chickens and provides essential information to further improve our understanding of the immune pathogenic mechanism of avian coronaviruses.

The kidney is the main target organ for NIBV infection, and the outbreak of COVID-19 in the past two years can also directly induce kidney disease (Santoriello et al., 2020), which further highlights the need for research on the relationship between coronavirus and innate immunity. There is also increasing evidence that the TLR7/NF-κB signaling pathway plays an important role in innate immunity, but the relationship between NIBV and the TLR7/NF-κB pathway and its precise regulatory mechanism remain unclear. In this study, we reported the results of renal tissue section observation, ultrastructural observation, immunofluorescence and related gene proteins in chickens infected with NIBV. The results suggest that NIBV activates the TLR7/NF-κB signaling axis in renal tissue, thereby promoting cytokine production. This may provide important information for future studies on how coronavirus infection induces innate immune responses.

We observed the renal tissue structure under a microscope, which showed that there were multiple coronavirus particles in the renal tissue, and the renal tubules were missing, with a large number of infiltrating inflammatory cells. Autopsy examination showed red and white spotted kidneys at 11 dpi, and these results are consistent with previous studies (Lin et al., 2015; Xu et al., 2019). This indicates that we successfully established the model of coronavirus NIBV. However, the mechanism by which NIBV mediates renal injury through the TLR7 signaling pathway is still unclear, so we detected the TLR7 signaling pathway according to the pathogenic characteristics of NIBV. The innate immune system emerged in the early stages of evolution, and Toll-like receptors play a key role in innate immunity. TLR7 is a single-stranded RNA (ssRNA) sensor in innate immunity and is one of the most important pattern recognition receptors (Mukherjee et al., 2016 , Zhang et al., 2016). The experimental results of this study showed that after NIBV infection, TLR7 mRNA levels were significantly upregulated at multiple time points, consistent with the degree of kidney injury observed at different time points. It has been suggested that NIBV infection can promote TLR7 mRNA expression, induce the immune response and mediate kidney injury in the body, which is consistent with the idea proposed by He et al. (He et al., 2016a) that the TLR7 receptor is activated after M41 IBV infection. However, in this study, the expression of TLR7 mRNA was somewhat different from that of M41 IBV in the later stage of infection, possibly due to the difference in the type of virus. Studies have shown that different types of IBV infection can induce different expression levels of inflammation-related genes (Jang et al., 2013; Okino et al., 2017), indicating that different pathogenic types of IBV infect hosts with different innate immune responses in different target organs. These results suggest that NIBV infection promotes TLR7 mRNA expression.

Tumor necrosis factor receptor associated factor 6 (TRAF6), a conjugated protein that mediates NF-κB activation, has been identified as an immunomodulatory actor downstream of the Toll receptor family (Walsh et al., 2015; Brenke et al., 2018). In addition, IKKβ is considered to be an essential regulator of NF-κB, and IKKβ forms a dimer with IKKα and plays a leading role in the regulation of NF-κB activity through protein phosphorylation (Page et al., 2017). IRF7 and NF-κB have similar functions; an important regulator of the innate immune response is activated by phosphorylation after viral infection and translocates into the nucleus after homologous dimerization or allodimerization to initiate transcription expression of inflammatory factors (Yu et al., 2018). Overall, these genes are downstream factors of the TLR7 gene and promote the expression of inflammatory cytokines. We found that the downstream genes TRAF6 and IKKβ were significantly upregulated in renal tissues throughout the experiment, and the expression trend of TLR7 was the same; however, the increase in TRAF6 was not obvious in the later stage, suggesting that SX9 inhibited the expression of TRAF6 in the later stage of kidney infection. Studies have shown that TRAF3 and TRAF6 complexes are inactivated by pathogenic microorganisms to limit or terminate proinflammatory cytokines and interferons and to evade host innate immunity (Panda et al., 2015). The mRNA level of NF-κB P65 was consistent with the mRNA expression trends of the upstream genes TRAF6 and IKKβ. At 28 dpi, however, the NF-κB P65 mRNA level was the opposite, which may be mainly dependent on the IRF7 nuclear transcription factor at the late stage or NF-κB P65 adapted to the host at the late stage. These findings were also consistent with the results of IRF7 in this experiment, which showed a significant increase in the level of IRF7 mRNA at 28 dpi. The NF-κB P65 protein level was opposite to the NF-κB P65 transcription level and decreased after NIBV infection. It may be that coronavirus infection inhibits the production of host antiviral proteins but does not affect mRNA expression (Huang et al., 2011). The fluorescence intensity of NF-κB p65 at 1 dpi and 5 dpi was lower than that of the Con group, and the target protein was mainly located in the cytoplasm. The intensity of the NF-κB P65 fluorescence signal was stronger in the middle and late stages than in the early stage, and the target protein was mainly distributed in the nucleus. These results proved that NIBV infection alters P65 gene expression and translocation to the nucleus. It was further demonstrated that NIBV infection promoted TLR7 mRNA expression, activated the downstream factors TRAF6 and IKKβ, and then activated nuclear transcription factors.

IFN-γ, as the only type II interferon that has been characterized, is a key regulatory protein for the function of the entire immune system (Wheelock, 1965; Fenimore and A, 2016). IFN-γ is mainly secreted by NK cells, is a typical representative proinflammatory factor, and can also promote the secretion of other cytokines (IL-6, IL-8) (den Hartog et al., 2020). In summary, IFN-γ, IL-8 and IL-6 are innate immune cytokines that play an important role in the body’s natural immunity. The results of this study are consistent with those of other viral infections (Pananghat et al., 2016; Li et al., 2020). IL-6 and IL-8 mRNA expression levels were significantly upregulated (consistent with the observation of H&E staining, which increased first and then decreased). In the early stage, the IL-8 and IL-6 protein levels were different from the transcription levels, mainly because the mRNA expression in the renal tissue was not upregulated after NIBV infection, so the protein expression was significantly decreased in the early stage of infection and was even significantly lower than that in the Con group. At 11 dpi, IL-8 protein levels were inhibited by high viral levels. It has also been reported that the IBV S protein can interact with the initiation factor EIF-3F, leading to the inhibition of general protein synthesis (Kint et al., 2016). These results suggest that NIBV infection activates IL-8 and IL-6. The expression of IFN-γ mRNA was inhibited at first and then significantly increased under NIBV infection and then significantly decreased at 28 dpi. In the early stage, this change may be due to the stress response caused by challenge and fasting before sampling, and in the later stage, it is mainly because the host has adapted to the environment in vivo and in vitro. The IFN-γ protein level was generally consistent with mRNA expression but with obvious hysteresis. In this study, the expression levels of the cytoinflammatory factors IFN-γ, IL-8, and IL-6 were consistent with the trends of the nuclear transcription factors (NF-κB P65 and IRF7). Moreover, we found consistent responses to changes in the TLR7/NF-κB signaling axis, innate immune cytokines and histopathology throughout the course of infection. These results suggest that NIBV infection activates the TLR7 signaling axis and further activates the downstream pathway NF-κB, thereby inducing the production of inflammatory cytokines and chemokines.

In conclusion, infection with the NIBV SX9 strain activates the TLR7 signaling axis in target tissue, activates the nuclear transcription factors IRF7 and NF-κB, induces the production of proinflammatory cytokines, causes an inflammatory response and leads to kidney damage. Our present results suggest that the natural immune response plays an important antiviral role in NIBV infection. According to this study, a signal diagram of TLR7/NF-κB was drawn (as shown in Figure 9 ).

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

The Institutional Animal Care and Use Committee of Jiangxi Agricultural University approved these animal experiments, and all animal experiments adhered rigorously to the animal care guidelines of Jiangxi Agricultural University (approval ID: JXAULL-202112; approval date: 6 August 2021). All the birds were put down using carbon dioxide euthanasia, and all attempts were made to minimize the suffering of the animals.

NL: Methodology; Writing-original draft; Writing-review and editing. CH: Methodology; Visualization; Writing-review and editing. XNG: Conceptualization; Methodology; Visualization; Writing-review and editing. XQG: Funding acquisition; Writing-original draft; Writing-review and editing. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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