Myricetin, a polyhydroxyflavoid compound, was first found in bayberry bark in the late 18th century (Perkin and Hummel, 1896), and then successively isolated in other plants, such as Myricaceae, Vitaceae, Fabaceae, Ericaceae, and Euphorbiaceae (Liu and Yu, 2014). It is also one of the main ingredients of various human’s foods, such as fruits, vegetables, fruit wine, tea, and honey, thus also known as dietary flavonoids (Deepak et al., 2016). The content of myricetin varies in different plants. It is well known that large amount of myricetin is present in the red bayberry trees which up to 10.53 mg/g in the red bayberry leaves (Yuan-Dan et al., 2015), but in bayberry core only 0.05 mg/g (Zou, 1995). The content of myricetin in fresh strawberries is only 75.7 μg/g (Gema and Maria, 2016). Xanthoceras Sorbifolia Bunge is a kind of Tibetan medicine, mostly grown in Inner Mongolia, and has the content of myricetin about 0.8 mg/g (Yang et al., 2014). Vine tea is known as the “king of flavonoids” and contains 44% of total flavonoids, in which myricetin content was 6.73 mg/g (Chen et al., 2015). Zhang et al. (2021) compared 22 different sources of Xinyang Maojian tea and found that the content of myricetin reach up to 0.370 mg/g. As a natural flavonoid, myricetin has been developed as medicines, foods, health care products and cosmetics. Previous studies have shown that myricetin has a strong antioxidant activity to scavenge a series of free radicals and ions such as DPPH, NO (Rostoka et al., 2010), ROS (Di et al., 2011). It can protect the organism from oxidative damage, and has both antioxygen and promoting oxidation which depending on ascorbic acid and Fe3⁺ involvement (Chobot and Hadacek, 2011; Duthie and Morrice, 2012). Myricetin has a wide range of anti-cancer effects, which is not only cytotoxic to various cancer cells such as liver, lung, and skin, but also inhibits the enzymatic activity associated with the development of cancer (Ci et al., 2018; Li et al., 2019; Xie et al., 2019). The anti-inflammatory activity of myricetin has been demonstrated in many trials through regulation of various signaling pathways (Hou et al., 2018; Kan et al., 2019). Myricetin could inhibit the growth of both bacteria and viruses (Cai and Wu, 1996; Naz et al., 2007). It was reported that myricetin could target the viral gD protein and downregulate the cellular EGFR/PI3K/Akt pathway to inhibit HSV replication (Li et al., 2020). Myricetin could inhibit HIV infection through suppression of SEVI fibers formation (Ren et al., 2018) and a direct killing effect (Pasetto et al., 2014). It could inhibit the activation of reverse transcriptase of murine leukemia virus (RLV) and HIV (Ono et al., 2010) and the activation of SARS-CoV helicase protein (nsP13; Yu et al., 2012). In addition, myricetin also has anti-allergy, antiacid, antihypertensive, and analgesic activities.

Herpesvirus, a class of viruses with double-stranded DNA, mainly invade skin, mucosal and neural tissues (Wildy, 1985), and then cause latent infection which is one of the major features of herpesvirus (Cohen, 2020). It can infect a wide range of hosts including amphibians, mammals and primates, and also involves in the development of certain cancers and causes complications of multiple diseases (Rafferty and Keen, 1973; Burrows, 1977; Boštíková et al., 2014). Pseudorabies virus (PRV), also known as swine herpes virus type I, belongs to the α subfamily of the Herpesviridae family. It is the pathogen that causes various mammalian Aujeszky’s disease (Wittmann and Rziha, 1989). Viruses in the α subfamily were found to be highly homologous, so PRV is often served as a model virus for studying α-herpesviruses (Karlin et al., 1994). Because of its high neurophilicity, PRV is often used as the “live” tracer of neuropathways (Sun et al., 2019). Although previous studies found that there was no cross-reactive between antibodies to PRV and HSV (Robbins et al., 1987), cases of humans infected with PRV have occurred since 1914. The patients developed symptoms associated with the CNS and tested positive for antibody specificity at 5–15 months of onset (Mravak et al., 1987; Skinner et al., 2001). Recent reports showed that some patients suffered from endophthalmitis and encephalitis caused by the variant PRV strain (Ai et al., 2018). Thus, it appears that PRV is most likely to vary into the neglected zoonosis (Wong et al., 2019). Although many trials have been conducted, vaccines against the human herpes virus are still not approved (Rajčáni et al., 2018). The approved drugs, including acyclovir, valacyclovir, and penciclovir are a class of nucleoside analogues that strongly inhibit DNA polymerase, which can effectively prevent and treat primary infection of herpes virus, but cannot protect the body from the damage caused by secondary infection (Sawleshwarkar and Dwyer, 2015). Studies have found that some compounds extracted from natural plants have good anti-herpesvirus activity, especially flavonoids (Ninfali et al., 2020). They can function at various stages of the viral infection cycle, providing new strategies for the treatment of herpesviruses. In this study, the antiviral activity of myricetin against PRV was evaluated in order to developing a new treatment for herpesvirus infection from dietary flavonoids.

Myricetin is a polyhydroxy dietary flavonoid compound widely found in fruits, vegetables, and tea with antiviral activity against some viruses, such as HSV, HIV, SARS-CoV (Ono et al., 2010; Yu et al., 2012; Pasetto et al., 2014; Ren et al., 2018; Li et al., 2020). Herpesvirus hosts have a wide range and still cause serious harm to humans and livestock and poultry today. Myricetin can target the viral gD protein and downregulate the cellular EGFR/PI3K/Akt pathway to inhibit HSV infection and replication. PRV belongs to a member of the family Herpesviridae and is often regarded as a model virus to study herpesviruses (Karlin et al., 1994). In this study, myricetin can achieve antiviral effects by directly inactivating the virus and inhibiting viral adsorption and penetration. Myricetin can also inhibit PRV-induced apoptosis and regulate the body immune and inflammatory responses caused by viral infection through the NF-κB and MAPK signal pathways. Thus, myricetin exhibited the potential to developing as an antiviral drug against herpesviruses.

The proliferative process of most viruses includes adsorption, penetration, replication, and release, and blocking any stage may inhibit viral infection. The approved anti-herpesvirus drugs are mainly a class of nucleoside analogs that selectively inhibit viral DNA polymerases, such as acyclovir, valacyclovir, and penciclovir. These drugs have adverse reactions involving the kidney, nerve, and heart in different ways of administration (Arndt, 1988; Haefeli et al., 1993; Izzedine et al., 2005; Ono et al., 2010; Gill et al., 2017). And long-term use has led to the production of resistant strains, making the treatment of herpes virus infections more difficult (Kukhanova et al., 2014). In this study, myricetin has a different antiviral mechanism from approved drugs. Pre-incubation of PRV with myricetin can reduce its infective ability and also inhibit PRV adsorption, penetration, and replication phases. It has been shown that PRV progeny viruses are usually detected upon infection with 4–5 h in commonly used cell lines (Pomeranz et al., 2005). Our results suggested that inhibitory effect of myricetin after PRV entry into cells may be due to its direct inactivation and inhibiting adsorption and penetration to suppress viral transmission from cell to cell, but it does not rule out that myricetin has an effect of inhibiting PRV replication. The initial attachment of herpesviruses to cells was achieved by binding of the virus gC glycoproteins to the HSPG and CSPG receptors on the cell membrane. Although gC glycoproteins are important for viral adhesion cells, this effect can be replaced by gB when lacking (Wudunn and Spear, 1989; Herold et al., 1991; Cheshenko and Herold, 2002). In addition, the viral envelope proteins, gD, gH, and gL, are also taking part in viral entry, release, and cell-to-cell transmission, and these glycoproteins are also strongly associated with host immune defense (Mettenleiter, 1996, 2000). Myricetin may bind to glycoproteins on the herpes virus membrane to block PRV entry. Other flavonoids were found to exhibit similar antiviral activity (Ninfali et al., 2020).

Stimulation to cells caused by herpes virus infection often triggers autophagy and apoptosis, which is detrimental to viral proliferation. Therefore, most viruses in the early stage of infection can promote the production of antiapoptotic factors to delay cell death to obtain maximum viral production, and in the later stages of infection, virus will promote apoptosis to release the progeny virus (Teodoro and Branton, 1997; Roulston et al., 1999). Annexin V-FITC/PI double staining is a commonly used method to detect apoptosis (Hu et al., 2019). In this study, we found that only very few cells developed apoptosis early in PRV infection, whereas after 18 h of infection, large number of cells gradually developed apoptosis and necrosis. Apoptosis induced by viral infection was controlled after myricetin treatment. Not only that, by examining the expression of apoptosis-related cytokines as well as PRV US3 genes, it was found that myricetin can significantly inhibit the regulation of PRV infection on Bcl-2, Bcl-xl, and Bax and also inhibit US3 gene expression in PRV. It was found that in the early stages of α-herpesvirus infection, the protein kinase encoding by US3 plays an important role in promoting cell survival to improve self-production during infection of host cells (Deruelle et al., 2010). The US3 protein kinase can inhibit early apoptosis in infected cells by inhibiting the overexpression of relevant apoptotic factors such as Bcl-2 family members, as well as by activating the PI3-K/Akt and NF-κB signaling pathway (Ogg et al., 2004; Chang et al., 2013). US3 also reduces autophagy levels in cells by activating the AKT/mTOR signaling pathway (Sun et al., 2017). However, the inhibitory effect of herpes virus on apoptosis is only temporary. PRV was found to activate caspase-3 expression in neurons during late in infection. In addition, ERK1/2 was also found to be activated to regulate cell apoptosis upon PRV infection (Marcaccini et al., 2006; Schulz et al., 2014). It was found that myricetin had both anti-apoptotic and pro-apoptotic effects. Myricetin can promote apoptosis and autophagy in various cancer cells by regulating the enzymatic activity associated with the development of cancer, as well as signaling pathways (Ci et al., 2018; Li et al., 2019; Xie et al., 2019). In addition, myricetin inhibits high glucose and oxidative stress-induced apoptosis through the inhibition of CDK-5 activity and by regulating apoptosis-related proteins (Karunakaran et al., 2019; Aminzadeh and Bashiri, 2020). Myricetin also protects cells from H₂O₂-induced apoptosis by regulating PI3K/Akt and MAPK signaling pathways (Kang et al., 2010). Thus, inhibition of PRV-induced apoptosis by myricetin may act by inhibiting the gene expression of PRV US3 and regulating apoptosis-related signaling pathways.

Myricetin can regulate several signaling pathways, such as MAPK, PI3K/AKT/mTOR, and IκB/NF-κB, leading to enhancement of immunomodulatory function and inhibition of cytokine storms (Cheung et al., 2000). PRV infection leads to an uncontrollable systemic inflammatory response in the body, which is greatly related to the continuous activation of the NF-κB pathway (Romero et al., 2020). The MAPK signaling pathway plays an important role in regulating cell growth, differentiation, inflammation, apoptosis, and other stress responses (Chong et al., 2003). The MAPK and NF-κB pathways were found to be associated with viral infection (Sun et al., 2017; Xu et al., 2019). When virus invades cells, the cells will trigger a series of cellular immune responses to resist the virus invasion and the virus also produces an immune escape mechanism (Banks and Rouse, 1992). In this study, the NF-κB and MAPK signaling pathways that are closely related to viral infection were detected. NF-κB pathway dysregulation is closely linked with allergies, autoimmune disorders among other pathologies (Zhang et al., 2017). Thus, aberrant PRV-induced NF-κB activation may therefore serve as a viral immune evasion strategy. HSV-1 inhibits the NF-κB pathway by binding to IκBα through its early protein ICP 27, enabling it to escape the immune response of the host cells early in infection (Jin et al., 2008). Similarly, it was found that PRV degraded P65 through its UL24 protein to inhibit the NF-κB pathway for immune escape (Wang et al., 2020). Our results suggested that myricetin inhibited PRV infection in the early period of infection through the activation of the NF-κB pathway and regulating the immune response. However, PRV does not exert a sustained inhibition effect on the NF-κB pathway. It was found that PRV caused persistent NF-κB activation when infecting epithelial cells, thus modulating the host pro-inflammatory response for immune escape purposes, and that activation suppresses apoptosis (Romero et al., 2020). Myricetin can alleviate the inflammatory response by inhibiting the activation of PRV-induced NF-κB in the later stages of infection. Alternatively, PRV can perform immune escape by modulating the cellular antiviral immune response and inflammatory responses mediated by the JAK/STAT, MAPK, and NLRP3 pathways (Li et al., 2022). An ethyl acetate fraction of flavonoids from Polygonum hydropiper L. was found to reduce the PRV-induced inflammatory response by reducing the phosphorylation of the MAPK key proteins and the displacement of NF-κB p65 into the nucleus (Ren et al., 2020).The P38 MAPK signaling pathway delivers extracellular signals to the nucleus and plays an important role in regulating multiple cellular processes, such as the cell life cycle and the immune response (Ono and Han, 2000; Cuadrado and Nebreda, 2010). Viral infection activates the P38 MAPK pathway to promote its own replication, and drug blocking activation of P38 effectively suppresses viral infection (Cheng et al., 2020). It was found that the cell apoptosis caused by PRV infection was closely correlated with the activation of the P38 and the JNK/SAPK signaling pathways (Marcaccini et al., 2006). This is consistent with this study that the P38 MAPK pathway is continuously activated after PRV infection and PRV infection promoted apoptosis in a time-dependent manner. However, viral-induced P38 activation and apoptosis were inhibited by myricetin treatment. ERK1/2, also belonging to the MAPK pathway as P38, is an evolutionarily conserved signaling pathway that is closely related to cell proliferation, differentiation, apoptosis and the transcription and expression of certain genes (Chong et al., 2003; O’Neill and Kolch, 2004). We found that PRV infection consistently activates ERK1/2, and after myricetin treatment the activation was promoted at 4 and 8hpi, turning into inhibition at 12 hpi. The gE glycoprotein of PRV was found to induce T lymphocyte migration by inducing the phosphorylation of ERK1/2 in T lymphocytes, thereby spreading the infection to other susceptible cells (Pontes et al., 2014). Alternatively, PRV participates in mitosis and apoptosis to disrupting the nuclear membrane by activating the ERK1/2 pathway, and releasing nucleocapsids from the nucleus to the cytosol, what is a process important in the herpesvirus replication cycle (Schulz et al., 2014). These results suggested that myricetin regulates apoptosis and nuclear membrane rupture by regulating PRV-induced ERK1/2 activation to affecting viral replication.

To further validate the regulatory role of myricetin on the both pathways, we examined the expression of relevant genes in the pathway. IL-1α and IL-1β, the first described members of the IL-1 family, control the inflammatory responses to tissue damage by identifying pathogen molecules and the signaling molecules released from damaged cells. IL-1α activation by NF-κB signaling regulates the release of inflammatory cytokines, including IL-1α, IL-1β, and IL-6 (March et al., 1985; Kelly-Welch et al., 2005; Dinarello, 2009; Lawrence, 2009). PRV infection was found to cause a systemic lethal inflammatory response, increasing the contents of major pro-inflammatory cytokines such as IL-β and IL-6 (Laval et al., 2018; Ren et al., 2021). In previous experiments, we found that PRV infection could continuously activate the NF-κB pathway, and that the activation of this pathway would promote the expressions of related cytokines, such as IL-1α, IL-1β, and IL-6, which was consistent with the results of cytokine expressions as we examined. Thus, it is shown that myricetin modulates pro-inflammatory cytokine production to inhibit the inflammatory responses by inhibiting the PRV-induced NF-κB signaling. Activation of the MAPK pathway facilitates viral replication and is involved in the cellular immune response and apoptosis caused by viral infection. The c-Jun, STAT1, c-Fos and c-Myc are transcription factors in the MAPK signaling pathway and transcription of pro-apoptotic and anti-apoptotic factors including Bax is also regulated by JNK and P38 in MAPK (Green and Llambi, 2015; Kumar et al., 2018). In our study, PRV infection significantly upregulated expression levels of c-Jun, c-Fos, c-Myc, as well as STAT1, while expression levels of these transcription factors were significantly reduced upon myricetin treatment (Figure 6). PRV infection activated MAPK signaling and changed the expression level of c-Jun, c-Fos, c-Myc, and STAT1 which are the downstream target genes in the pathway. Moreover, c-Jun and c-Fos can form a AP-1 heterodimeric complex, implying that AP-1 is also involved in PRV infection and regulates cell apoptosis (Glover and Harrison, 1995). In our study, PRV infection would continuously activate the MAPK pathway, while the activation of the MAPK pathway modulates the expressions of related cytokines, which was consistent with the results of cytokine expressions. These results suggest that myricetin inhibits PRV infection and controls cell apoptosis by regulating the expression levels of relevant transcription factors in the MAPK pathway.

Pseudorabies virus hosts a wide range, and can infect a variety of other mammals in addition to primates (Woźniakowski and Samoreksalamonowicz, 2015). In vivo study, PRV-infected animal models (often mice and rabbits) through artificial infection were used. The routes for PRV infection based on the research purpose is different, mainly including nasal drip, abdominal injection, intramuscular injection, foot injection, and subcutaneous injection. No matter the infection route, PRV will lead to unnatural host severe itching (Laval and Enquist, 2020). In order to explore whether myricetin could inhibit PRV infection in vivo, we established a mouse model by intraperitoneal injection. The clinical symptoms included manifested as scratching the injection site, decreased diet and mental depression. The mice were orally treated with 100 mg/kg myricetin twice a day because the oral elimination half-life in rats was only 3.53 h (Li et al., 2021). After myricetin treatment, the survival time of the mice was prolonged; the pathological damages caused by infection were also relieved; the proliferation of PRV was also inhibited in each organ (Figures 7A–D). In vitro study, myricetin mainly acted on the direct killing of virus and inhibiting the adsorption or penetration, so it is more suitable for the prevention of virus infection or combination with other drugs which acting in the virus replication stage. Studies have found that PRV infection will lead to a series of host histopathological damage. In PRV-infected pigs, the brain appeared congestion mainly non-suppurative encephalitis and the formation of “satellitosis,” “neuronophagy” phenomenon, and the lung mainly appeared pathological changes of congestion and edema (Yang et al., 2016a). After mice infected with PRV, similar pathological changes were exhibited, such as encephalitis and pulmonary hemorrhage edema, even die in a short period of time, which were consistent with to our results (Ren et al., 2021). The rapid death in mice may attribute to pyroptosis and dysregulation of pro-inflammatory factors which induced unbalance of anti-inflammatory and pro-inflammatory cytokines, leading to a “cytokine storm” (Sun et al., 2021). Viral infection usually causes cell apoptosis and necrosis and an inflammatory response (Knipe and Howley, 2013). PRV can mediate cell apoptosis in vitro and in vivo by regulating a range of signaling pathways as well as the expression of caspase-3 (Marcaccini et al., 2006; Chang et al., 2013; Sun et al., 2017). Herpes simplex virus can also mediate the apoptosis of infected cells by regulating the expression of Bcl-2 family members (Mettenleiter, 2000). In the present study, ocular purulence in infected mice was observed which may be due to conjunctivitis caused by PRV infection, and the increased index of brain and lung organs may be due to edema and congestion. At the same time, through histopathological observation, different degrees of inflammatory responses in the test organs (Figure 7D). The expression levels of the inflammation-related cytokines, MCP-1, G-CSF, IL-1, IL-1, and IL-6, as well as the apoptosis-related cytokines, Bcl-2, Bcl-x, and Bax were altered in organs of the infected mice (Figure 7E). The congenital immune system, the first line of defense against foreign invasion, often acts by initiating the inflammatory response, but the uncontrolled inflammatory response usually leads to organ failure and dysfunction of the body and even death (Tisoncik et al., 2012). In influenza, excessive expressions of pro-inflammatory cytokines also known as “cytokine storm” is the cause of various complications and death (Liu et al., 2016). And the expression levels of interferons, tumor necrosis factors, interleukins or chemotactic factors would significantly increase (Beigel et al., 2005). Similarly, Sindbis virus infection can cause elevated expression levels of interferons, TNF-α, and IL-6 in the serum of newborn mice, thus causing a systemic inflammatory response (Klimstra et al., 1999). Myricetin has a variety of pharmacological activities, including anti-inflammatory effects. It was shown that myricetin can exert a protective effect on LPS-induced early inflammation in cardiomyocytes by inhibiting IL-6 and IL-1β expression through NF-B signaling pathway (Chen and Fan, 2018). Similarly, myricetin also inhibited the apoptosis and inflammatory response of human umbilical vein endothelial cells HUVECs by upregulating the expression of miR-29a-3p (Bai et al., 2021). In general, myricetin can enhance body immunity and inhibit cytokine storms by regulating multiple signaling pathways (Cheung et al., 2000). Based on the above studies, the protective effect of myricetin on PRV-infected mice may be due to its mitigation of cytokine storms by regulating cytokine expressions through related signaling pathways.

Myricetin can effectively inhibit the infection of herpes virus and prevent viral entry into cells at the early stage of infection. It can regulate the NF-κB and MAPK pathways to inhibit viral replication and control the inflammatory response and apoptosis caused by viral infection. Myricetin also inhibits PRV proliferation in mice and alleviates the inflammatory response induced by infection to prolong the survival of infected mice. The study provided a new strategy for the treatment of herpes virus infection with dietary flavonoids.

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

The animal study was reviewed and approved by National Institute of Ethics Committee at Sichuan Agricultural University [approval number SYXK (Sichuan) 2018-012].

HH, ZH, and YZh carried out laboratory work, wrote the manuscript, and performed the data analyses. HH and XS designed the experiment. HW, XL, LL, XZ, LY, GY, YZo, YC, and HT carried out laboratory work. ZY, RJ, HZ, and XS conceived and supervised this work. All authors contributed to the article and approved the submitted version.

This research was financially supported by the Natural Science Foundation of Sichuan Province (2022NSFSC1681) and the Program Sichuan Veterinary Medicine and Drug Innovation Group of China Agricultural Research System (SCCXTD-2020-18).

ZH was employed by Shandong New Hope Liuhe Agriculture and Animal Husbandry Technology Co., Ltd. (Dezhou, China).

The remaining 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.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.