The biological basis of severe tissue damage in the lymphoid system induced by genotype VIId NDV strains is unknown. We hypothesized that dysregulation of host response may play an important role. In this study, our findings demonstrated that two isolates (JS5/05 and JS3/05) of genotype VIId NDV elicited robust innate immune response that was evidenced by upregualtion of IFNs expression compared to genotype IX NDV F48E8 and genotype IV NDV Herts/33. In addition, JS5/05 and JS3/05 also induced enhanced apoptosis and necrosis compared to F48E8 and Herts/33. Virus replication was observed with partial association with virus-induced cell death. Therefore, our in vitro results indicate that dysregulation of host response may contribute to the severe spleen pathology caused by genotype VIId NDV infection.
Host cells mount the cytokine response to prevent infection upon virus invasion. However, overproduction of cytokines may form a cytokine storm to amplify detrimental effect of inflammation on the host . In this study, quantitative analysis of transcription levels of IFN genes showed that JS5/05 and JS3/05 increased expression levels of IFN-α, IFN-β and IFN-γ compared to F48E8 and Herts/33, indicating that genotype VIId NDV strains induce a more potent IFN response (Figure 3). Therefore, strong activation of IFN response may intensify inflammation that causes damages in tissue structures. It is notable that hyperinduction of IFN-γ, a key pro-inflammatory cytokine, may increase synthesis of nitric oxide that has toxic effect on host. IFNs are a family of cytokines that play a central role in innate immunity to viruses. The correlation of JS5/05-induced high levels of IFNs and high virus load may result from the faster virus replication to overcome anti-viral effect of IFNs. Additionally, NDV V protein serves as an antagonist of alpha IFN and contributes to viral virulence . Our data showed that at least at mRNA level, a block of IFN function did not occur during infection with JS5/05 and JS3/05. Perhaps these two isolates can activate many genes that involved in IFN pathway to overwhelm the suppression effect of V protein.
In addition to the role in inflammation and anti-virus activity, IFNs also act as key mediators of apoptosis . Many factors involved in IFN signaling pathway, such as IFN regulatory factors (IRFs), protein kinase R (PKR) and 2',5-oligoadenylate/RNaseL system, contribute to apoptosis through different mechanisms. IFNs can also induce the activation of TNF-related apoptosis-inducing ligand (TRAIL) that initiates the death receptor-mediated cell death .
Here we showed that JS5/05 and JS3/05 induced stronger apoptosis than F48E8 and Herts/33, reflected by the significantly larger amount of free nucleosomal DNA in JS5/05- and JS3/05-inucolated cells (Figure 2). In addition, splenocytes inoculated with JS3/05 and JS5/05 also underwent marked necrosis (Figure 4). One possible consequence is that stronger cell death in splenocytes infected with JS5/05 and JS3/05 may accelerate disease severity. It has been shown that apoptosis in lymphoid tissues in NDV-infected birds was followed by extensive lymphocellular necrosis [13, 14]. Harrison et al. have demonstrated that the apoptotic rate is proportional to the disease severity produced by NDV strains of varying virulence . Accumulating evidence has shown that the pathogenicity of avian influenza virus H5N1 is closely associated with virus-induced apoptosis [19–21]. Of particular interest, as an alternate form of programmed cell death, necrosis may have different effects on disease severity from that of apoptosis. Necrotic cells will release the cytoplasmic contents into the extracellular fluid to stimulate inflammatory response .
Based on our findings, we could speculate that cytokine response and cell death may act in concert to exacerbate the disease severity caused by genotype VIId NDV. Our results also indicate that viral dysregulation of host response by genotype VIId NDV strains may be a genotype-specific phenotype.
It is commonly assumed that efficiency of virus replication is an important determinant for the severity of disease. NDV can propagate in chicken lymphocytes and cause lymphocyte depletion, which lead to severe consequences for bird immune system [13, 14, 23, 24]. Replication efficiency of JS5/05 in splenocytes was significantly higher than that of F48E8 and Herts/33, whereas replication efficiency of JS3/05 was partially inhibited by the early strong apoptosis (Figure 1). Thus, high replication capacity of genotype VIId NDV in splenocytes may be virus-dependent. Our results also suggest that in vitro replication of NDV may be determined by various factors, including cell type, viral genes and virus-induced CPE. Moreover, there may be an active interplay between virus replication and virus-induced cell death. At the early stage of infection, JS5/05 induced low-level apoptosis that allowed high virus replication, whereas excessive apoptosis induced by JS3/05 inhibited virus replication. Reversely, at the late phase of infection, JS5/05-induced cell death may promote virus replication, which may result from the failure of dead lymphocytes to clear virus and active virus release from leaky necrotic cells.
Our data are consistent with some previous findings. Rue et al. have demonstrated that virulent NDV elicits a robust host response in the spleen represented by hyperinduction of many cytokines, suggesting a correlation between pathogenicity and host innate immune response . Ecco et al. have shown that the genotype VIId NDV strain ZJ1 is a stronger inducer of cytokine response than other virulent strains and the high-cytokine phenotype is consistent with viral pathogenicity and disease outcome .
Although there are many studies performed to elucidate virus pathogenicity and host response to NDV strains of varying virulence, evidence focusing on differences in virus-host interplay of different virulent isolates is limited. Our study presents in vitro evidence that differential viral modulation of host response corresponds with the contrasting histopathology phenotypes. Based on these results, we are performing animal experiments to provide supporting in vivo results to confirm this correlation.