Since the 1980s, the spread of AD worldwide has inevitably caused serious economic losses . The farms in China currently rely on vaccination to prevent and treat AD, including inactivated vaccines, attenuated vaccines and gene-deleted vaccines . However, during the past decades, the PRV strain has mutated, and the existing vaccines have been unable to control all infections, which eventually led to the repeated outbreak of AD. Recent case reports of human PRV infections suggested possible transmission to the humans . Therefore, development of alternative control measures is still the main task at present. Kaempferol is a very common dietary flavonoid compound that has inhibitory effects on several viruses, such as bovine herpesvirus 1 , HSV , Japanese encephalitis virus , and enterovirus 71 .
In this study, kaempferol could effectively protect mice from PRV infection through improvement of survival rate by 22.22% (Fig. 1). In addition to pigs, many mammals are susceptible to PRV, and often died after infection. Mice would die quickly after being infected with PRV, and the mortality rate is mostly 100% within 3–7 dpi . The same result was observed in this study that the mice in the infected-untreated group had all died at 6 dpi. Acyclovir and its analogues are the most frequently used drugs approved for the treatment of HSV, which can inhibit the viral DNA polymerase . Aciclovir also showed antiviral activity against other herpesviruses, such as varicella zoster virus, Epstein-Barr virus, cytomegalovirus and human herpesvirus 6 , thus it was served as positive control. In recent human cases, acyclovir were used for antiviral treatment of PRV-induced acute human encephalitis . In this study, acyclovir exhibited antiviral activity against PRV in mice by improvement of 16.67% survival rate, but it is lower than kaempferol which suggested a potential application of kaempferol in control of PRV infections in animals and humans.
Viral load is a direct parameter in the evaluation of antiviral effects in vivo, which can reflect the virus replication in different organs [38; 39; 40; 41]. In this study, PRV reproduction in the test organs, including brain, heart, spleen, lung and kidney, were observed. The results (Fig. 2) showed that PRV replicated most in brain, it was about 50-fold than other test organs. The reason could be attributed to PRV belonging to a neurotropic alphaherpesvirus which can invade the CNS via the trigeminal nerve, as well as by sympathetic and parasympathetic pathways . The main feature of PRV infection is encephalomyelitis, which is often accompanied by inflammation of the upper respiratory tract and lungs . kaempferol could significantly inhibit virus replication in the test organs, especially in the brain that the viral gene copies were decreased by more than 700-fold. In contrast, the acyclovir showed lower ability to inhibit virus reproduction than kaempferol, and the viral gene copies were decreased by about 10-fold in the brain. The immunohistochemical study also demonstrated that the amount of progeny virus in the brain was markedly decreased after kaempferol or acyclovir treatment (Fig. 3). In the antiviral effects of resveratrol in piglets infected with PRV, resveratrol can significantly inhibit the virus titer in the brain, and the levels of viral copies in the brain were positively linked to the clinical parameters of infected piglets . Kaempferol were found effective to delay and inhibit the clinical symptoms in the PRV-infected mice which may attributed to inhibition of virus replication in the brain.
After mice were infected with PRV, histopathological analysis found that the brain, spleen, and liver tissues had different degrees of lesions . Studies have shown that PRV could cause lethal respiratory disease in an animal model of PRV-infected BALB/c mice . In the present study, mild tissue damages were observed in the PRV-infected mice after treated with kaempferol (Fig. 4), indicating that kaempferol can alleviate the histopathological changes.
Although the innate immune system is the first line of defense by triggering an inflammatory response to prevent the spread of viral infections, the main challenge is to ensure that inflammation is resolved , then the body's homeostasis can be restored to normal. When the inflammatory response is uncontrolled, it usually leads to more severe inflammation, which may cause damages to the host . In this study, the levels of pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α and IFN-γ, were elevated after infection, suggesting the innate immunity was activated by PRV. After kaempferol treatment, the levels of these pro-inflammatory cytokines were higher than the infected control at 3 dpi, and then almost declined to the normal level at 5 dpi, suggesting that kaempferol could enhance the inflammatory responses at the early stage of infection to inhibit viral replication, then recovered it to normal to avoid severe inflammation. The anti-inflammatory cytokine IL-4, produced by Th2 cells, NKT cells, basophils and mast cells, has a wide range of Immunological functions, such as regulating the function of macrophage [48; 49]. Studies have shown that IL-4 could suppress PMA-induced HIV expression at the transcriptional level in monocytic U1 cell . PRV infection increased the IL-4 level in mice. After kaempferol treatment, IL-4 level was significantly higher than that of the infected-untreated control at 3 dpi, indicating that kaempferol can suppress PRV by enhancing immune function.
After PRV enter into the host cells, the capsid is transported along microtubules to the cell nucleus and the viral DNA was injected into the nucleus. Then, the only immediate-early gene of PRV, IE180, is transcribed directly following infection . The product of IE180 gene was the transactivator required for early genes transcription [28; 10]. In this study, the transcriptional level of early genes, EPO and TK was inhibited by kaempferol in the brain of PRV-infected mice, which may indicate that the function of IE180 protein was blocked (Fig. 6). After the body is infected with the virus for the first time, the virus can establish latent infection in the sensory ganglion and brain . The latent infection of the virus can trigger a continuous inflammatory response, which can directly cause damage to the nerve tissue. LAT plays an important role in the establishment, maintenance, and reactivation of PRV latency. LAT prevents the expression of the immediate early gene IE180 during the incubation period, which could prevent the virus from entering the lytic infection period [52; 53]. The present study found that the transcriptional level of LAT in brain was inhibited, suggesting that kaempferol could inhibit PRV latency in mice.