The Role Of PI3K Signaling Pathway In Response To NDV Infection

Newcastle disease(ND) is a highly contagious infectious disease caused by virulent Newcastle disease virus(NDV). Virulent NDV isolates could induce severe pathological changes in infected birds,such as hemorrhage of gastrointestinal tract and respiratory tract,injury of central nerve system. Since ND was first reported from Indonesia in 1926 and in England in 1927,successive ND outbreaks were reported from different parts of the world, including Asia, Europe, Africa and South America. Secondly,many new strains(such as genotype XVII and XVIII) have been isolated predominantly from poultry in Africa;Most importantly,an increasing number of Class I NDVs and Class II(especially genotype VI and VII NDV) have been isolated from domestic poultry in worldwide in recent years. Finally, it is distributed worldwide and which is able to infect over 250 species of birds, and which spreads primarily through direct contact between infected and healthy birds, as well as has the potential to cause large economic losses in the poultry industry. However, wild birds were considered to be transmission vector and narural reservoir that contribute to spreading of the virus to new areas via long-distance migration and transmission to domestic poultry, the mode of its transmission among avian species remains largely unknown. Therefore, we conducted epidemic surveillance of NDVs in live bird markets(LBMs) in South China’s Guangdong Province since 2010 and investigated the pathogenicity in different birds.In this study we genotypically and pathotypically characterized 25 NDV isolates collected from chickens, ducks and pigeons in live bird markets(LBMs) in Guangdong Province during December 2010–February 2014. To simulate the natural transmission of different kinds of animals in LBMs, we selected five representative NDVs—namely, GM,SS-10, NH-10, YF18 and GZ289—isolated from different birds to evaluate the pathogenicity and transmission of the indicated viruses in chickens, ducks and pigeons.Furthermore, to investigate the replication and shedding of NDV in poultry, we inoculated the chickens, ducks and pigeons with 106 EID50 of each virus via intraocular and intranasal routes. Eight h after infection, the na?ve contact groups were housed with those inoculated with each of the viruses as a means to monitor contact transmission. Our results indicated that genetically diverse viruses circulate in LBMs in South China’s Guangdong Provinceand that NDV from different birds have different tissue tropisms and host ranges when transmitted in different birds.Virus infection and pathogenicity is the result of the interaction between viral and host,including the virus evade host defense mechanism change to promote virus replication, and also host for the removal and transfer the innate immune system by producing effect molecules quickly to build the body’s biological defense against a virus invasion. To elucidate the molecular mechanisms of NDV pathogenesis, we demonstrated for the first time that NDV can transiently activates the PI3K/Akt pathway in chicken cells at an early step during NDV infection. This activation was observed as early as 15 min postinfection,peaked by 60 min, and gradually weakened after 24 h postinfection. Activation of the PI3K/Akt signaling pathway could also be mediated by UV-inactivated NDV, indicating that virus replication was not required for this activation. The treatment of cells with the specific phosphatidylinositol 3-kinase(PI3K) inhibitors LY294002 or Wortmannin prior to NDV infection leads to decreased NDV virus titers and suppressed Akt phosphorylation.Additionally,Inhibition of the PI3 K activity at early but not at later time points of infection results in reduced titers of progeny NDV. Furthermore, NDV entry is probably involved in the PI3K/Akt signaling pathway, because the Akt activation triggered by GM or F48E9 was abolished by clathrin-mediated endocytosis inhibitor methyl β-cyclodextrin and chlorpromazine, whereas the treatment after La sota infection had no obvious effect.However, inhibition of PI3 K activation greatly enhanced apoptotic responses as evidenced by the cleavage of poly-ADP ribose polymerase(PARP) and Caspase-3 as well as apoptotic cells using Annexin V-FITC/PI staining during the early stage of NDV infection.Furthermore, the pancaspase inhibitor ZVAD-FMK alleviated the reduction in Akt phosphorylation levels by inhibiting PI3 K activation, indicating that the signaling pathway promotes cell survival and thereby favors viral replication. These results reveal that an antiapoptotic role for the PI3K/Akt pathway induced by NDV infection to suppress premature apoptosis for improved virus growth after infection.Based on substrate specificity and sequence homology, PI3 Ks are grouped into three classes: class I, class II, and class III. Class I PI3 Ks are composed of a p85 regulatory subunit and a p110 catalytic subunit that produces phosphatidylinositol 3,4,5-trisphosphate(PI(3,4,5)P3), which activates the Akt/m TOR-signaling pathway. It is believed that class I PI3 Ks inhibit autophagy by promoting nutrient uptake and metabolic activities through Akt/m TOR. In contrast, the class III PI3 K catalytic subunit Vps34 is bound to the regulatory subunit Vps15 and converts phosphatidylinositol(PI) to phosphatidylinositol3-phosphate(PI(3)P), which is essential for autophagy initiation. Autophagy and apoptosis are different physiological processes of the cell, however, they have a complicated relationship in terms of their interactions with each other. NDV infection is known to induce both autophagy and apoptosis. In the present study, to further investigate the molecular mechanisms of NDV pathogenesis and study about the virus–host interaction,we infected CEF cells with NDV and analyzed the effect of NDV infection on autophagy and apoptosis over time,as well as evaluated deeply about the relationship between autophagy and apoptosis. Herein, compared to the mock-infected CEF cells, we observed that autophagosome formation increased by transmission electron microscopyin NDV-infected and starvation-induced primary CEF cells at the early stage of infection, as evidenced by the conversion of LC3-I to LC3-II and the degradation of p62/SQSTM1. However, we did not observed that the conversion of LC3-I to LC3-II and the cleavage of caspase 3 and PARP in UV-inactivated NDV-infected CEF cells, indicating autophagosome formation was required for virus replication. Inhibition of autophagy in vivo and in vitro greatly enhanced apoptotic responses as evidenced by the cleavage of Caspase 3 and PARP as well as apoptotic cells using FITC-V/PI staining during the early stage of NDV infection.Similarly, induction of autophagy reduced apoptotic responses. Additionally, except for Beclin1,the expression levels of autophagy-related genes, anti-apoptosis factors(Bcl-2 and Bcl-x L) and pro-inflammatory cytokines(IL-6 and IL-1β) in NDV-infected spleen and lung were upregulated after autophagy induction by rapamycin in vivo,however, the number of apoptotic cell in NDV-infected spleen and lung were decreased when compared to mock-infected spleen and lung. Furthermore, the pancaspase inhibitor ZVAD-FMK promoted the conversion of LC3-I to LC3-II and the degradation of p62/SQSTM1 as well as virus replication by Inhibiting apoptosis, suggesting that the signaling promotes cell survival.In conclusion, these results reveal the relationship between PI3K/Akt signaling pathway, autophagy and apoptosis in response to NDV infection of host cells, it will provide important information about the molecular mechanisms of virus–host interaction and NDV pathogenesis, as well as provide a new insight to prevent and control NDV infection.