Newcastle disease virus infection in chicken embryonic fibroblasts but not duck embryonic fibroblasts is associated with elevated host innate immune response

This experiment was conducted with the approval of the South China Agricultural University Experimental Animal Welfare Ethics Committee (permit no. 2015–08).

CEFs and DEFs were obtained from 10-d-old SPF chicken embryos and 11-d-old Pekin duck embryos (South China Agricultural University, Guangzhou, China) as previously described [22]. The CEFs and DEFs were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10 % fetal bovine serum (FBS) (Gibco), 100 U/mL penicillin and 100 ug/mL streptomycin at 37 °C with 5 % CO₂ until cell density reached approximately 80 % confluence. The two NDV strains used were Duck/CH/GD/SS/10 (SS-10) and Duck/CH/GD/NH/10 (NH-10), genotypes VII and IX, respectively, both duck-adapted viruses characterized well in the Key Laboratory of Animal Disease Control and Prevention of the Ministry of Agriculture, College of Veterinary Medicine [11]. The viruses were inoculated into the allantoic cavity of 10-d-old SPF-embryonated chicken eggs at 37 °C for 3 d according to the standard procedures of the Office International Des Epizooties [1]. Fresh allantoic fluid was collected and stored at −80 °C until used, and virus titers were quantified by plaque assay with both CEFs and DEFs [23].

To determine the multicycle growth kinetics of SS-10 and NH-10, CEFs and DEFs in triplicate wells of six-well plates were infected with either virus at a multiplicity of infection (moi) of 1. Following 1 h of adsorption, the cells were washed and covered with DMEM containing 2 % heat-inactivated FBS at 37 °C and 5 % CO₂. Cell culture supernatant samples were collected and replaced with an equal volume of fresh medium at 6, 12, 24, 36, 48 and 60 h post-infection (p.i.). Virus titers were quantified by a plaque assay on CEFs as previously described [23].

CEFs and DEFs were seeded 16 h prior to infection in triplicate wells of six-well plates at a cell density of approximately 1.7 × 10⁵ cells/well. The cells were infected with genotype VII SS-10 and genotype IX NH-10, both predominant duck-origin genotypes of NDV strains circulating in Guangdong Province, at an moi of 1 and incubated at 37 °C in a humidified atmosphere containing 5 % CO₂ for 1 h. Afterward, the growth medium was replaced with DMEM supplemented with 2 % heat-inactivated FBS. Mock-infected cells were regarded as negative controls. CEFs and DEFs were treated with the double-stranded RNA (dsRNA) analog poly(I:C) (Sigma–Aldrich) at a concentration of 20 ug/mL and used as positive controls. At 0, 6, 24 and 36 h p.i., cell monolayers were harvested and stored at −80 °C for RNA extraction.

Total RNA was extracted from the infected and negative control embryo fibroblast cells harvested at each time point using the RNeasy Mini RNA Purification Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Viral RNA was extracted from culture supernatants by using an RNA extraction kit (Takara, Japan). RNA in each sample was quantified using an Ultrospec 2000 mass spectrophotometer (Pharmacia Biotech, Uppsala, Sweden). Approximately 2 ug RNA from each sample was treated with DNase to remove genomic DNA and was later reverse-transcribed to cDNA using the SuperScript® III First-Strand Synthesis System (Clontech) according to the manufacturer’s protocol.

The qRT-PCR method was applied in a final volume of 25 uL using a QuantiFast SYBR Green PCR Master Mix Kit (Qiagen) with specific primers designed with Oligo 7 software (http://​www.​oligo.​net/​) based on published sequences [24]. Primers were developed for IL-1beta, lipopolysaccharide-induced TNF-α-factor (LITAF), IL-2, IL-6, IFN types I and II (IFN-alpha, IFN-beta and IFN-gamma), MHC class I and II molecules, TLR3, TLR7 and IL-8, all derived from published sequences. Predicted product sizes are shown in Table 1. All qRT-PCR assays were conducted using the ABI Prism 7500 Fast Real-Time PCR System (Applied Biosystems), which involved predegeneration for 2 min at 50 °C and initial denaturation for 30 s at 95 °C, followed by 40 cycles of 5 s at 94 °C and 34 s at 60 °C, as well as a melt-curve analysis to confirm the specificity of the SYBR green PCR signal. A one-step qRT-PCR assay of viral RNA using NDV P/V/W gene-specific primers was performed as previously described. Cycle threshold (CT) values were converted to viral gene copy numbers following a standard curve generated using cDNA. To rule out genomic contamination, control qRT-PCRs were performed in the absence of reverse transcriptase. Amplified products were run on a gel and extracted using a QIAEX II DNA gel extraction kit (Qiagen, Germany) according to the manufacturer’s instructions. To validate assays, purified products were subcloned into pMD19-T with a TA cloning kit (Clontech, Japan) and sequenced for verification using M13 forward and reverse primers.

The cDNA sample of each CEF and DEF was tested in triplicate. Relative expression levels were calculated according to the 2^-△△CT method, which involved using glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as the endogenous control to normalize the level of target gene expression [25]. Data were expressed as M ± SD. Growth characteristics of each group were analyzed using an unpaired Student’s t-test, and differences between the means of the CEF and DEF target genes were evaluated using two-way analysis of variance (ANOVA) followed by Duncan’s test. All p values less than .05 were considered to be statistically significant. Statistical analyses for M, two-way ANOVA and SD were conducted by using Prism 6 (GraphPad Software, Inc., San Diego, CA, USA).

The multicycle growth kinetics and replication magnitude of SS-10 and NH-10 were determined in CEFs and DEFs by using a plaque assay. CEFs and DEFs were inoculated with each virus at a moi of 1, and cell supernatants were harvested at the time points indicated. As shown in Fig. 1a and b, SS-10 replicated more efficiently and had a significantly higher titer in CEFs and DEFs than NH-10 at each time point, though both viruses achieved similar maximum titers at 36 h p.i. On the whole, the virus titers of both strains were higher in CEFs than in DEFs during the 60 h of testing (Fig. 1a and b). Additionally, CEFs and DEFs were infected with SS-10 and NH-10 at a moi of 1 over a period of 36 h. Normalized to the endogenous control, viral P/V/W gene RNA accumulation in DEFs was consistently less than that in the corresponding CEFs for two NDV isolates (Fig. 1c and d).

We compared the expression levels of TLR genes TLR3 and TLR7 in CEFs and DEFs infected with SS-10 and NH-10 at 6, 24 and 36 h p.i. Compared to those in mock-infected samples, the expression levels of TLR3 and TLR7 in CEFs were upregulated at 6 h p.i. and peaked at 24 h p.i., with the exception of TLR7’s expression level induced by SS-10 at 36 h p.i. Thereafter, the levels decreased slightly at 36 h p.i. when either infected with both viruses or treated with poly(I:C), as shown in Fig. 2a and b. In DEFs, the expression levels of TLR3 and TLR7 induced by both viruses and positive control poly(I:C) clearly exhibited patterns of expression. For TLR3, the expression level was downregulated at 6 and 24 h p.i. (0.85- and 0.75-fold, respectively) and upregulated at 36 h p.i. (1.46-fold) in response to SS-10 infection, whereas the expression level of TLR3 was upregulated at 6 h p.i. and downregulated at 24 and 36 h p.i. following infection with NH-10 or after stimulation with poly(I:C) (Fig. 2a). Meanwhile, the expression level of TLR7 was upregulated throughout the period of infection, except at 36 h p.i. induced by NH-10, in response to infection with both viruses or treatment with poly(I:C) (Fig. 2b). Notably, the expression levels of TLR genes TLR3 and TLR7 in CEFs induced by both viruses and poly(I:C) were greater than in DEFs throughout the testing period, whereas TLR7 was slightly increased in DEFs at 24 h p.i. following stimulation with dsRNA analogs (poly(I:C)).

To compare proinflammatory and Th1-associated cytokines and chemokines in CEFs and DEFs infected by SS-10 and NH-10, cytokines and chemokines such as IL-1beta, IL-6, IL-8 and LITAF were measured early during either NDV infection or treatment with poly(I:C). Compared to uninfected cells, CEFs and DEFs exhibited upregulated expression levels of IL-1beta and LITAF in response to infection with SS-10 and NH-10 or treatment with poly(I:C) during the testing period (Fig. 3a and d), though the expression levels of IL-1beta and LITAF showed different expression patterns at each time point for both infected CEFs and DEFs (Fig. 3a and d). In CEFs, the expression of both IL-1beta and LITAF was upregulated when induced by infection with SS-10 and NH-10 at all time points and peaked at 24 h p.i. (22.87- and 12.66-fold versus 319.48- and 21.13-fold, respectively), but was greater for SS-10 than NH-10. Albeit also upregulated, IL-1beta expression in DEFs was induced at a slightly weaker level and peaked at 36 h p.i. (17.93- and 7.88-fold, respectively), induced by both viruses. LITAF expression in DEFs was also induced 6.93- and 4.84-fold at 6 h p.i., respectively, decreased slightly at 24 h p.i. (3.71- and 4.25-fold, respectively) and peaked at 36 h p.i. (12.33- and 6.86-fold, respectively), when infected with SS-10 and NH-10, yet was induced at a far lower level than with CEFs during the observation period (Fig. 3d). The expression level of IL-6 was upregulated in CEFs during the period of infection, peaking at 36 h p.i. and 8 h p.i. (88.22-, 7.60- and 21.21-fold, respectively) in response to infection with SS-10 and NH-10 or treatment with poly(I:C) compared to uninfected CEFs. However, it was downregulated at 6 and 24 h p.i. and maintained baseline level at 36 h p.i. in DEFs induced by SS-10. It was moreover upregulated 2.49-fold at 6 h p.i. and downregulated between 24 and 36 h p.i. (0.56- and 0.34-fold, respectively) in DEFs induced by NH-10 (Fig. 3b). Remarkably, SS-10 induced the expression of proinflammatory cytokine IL-6 to a greater extent than NH-10 during the testing period, whereas it was not statistically significant in DEFs (Fig. 3b).

The expression level of chemokine IL-8 was suppressed at 6 and 24 h p.i., yet upregulated at 36 h p.i. in CEFs when induced by SS-10 and NH-10. However, the expression level of IL-8 showed a different pattern of increase at 6 h p.i. in DEFs and maintained the same tendency at 24 and 36 h p.i. when infected with SS-10 and NH-10, but was higher than that of CEFs at each time point. By contrast, the expression level of IL-8 was upregulated when treated with poly(I:C) in CEFs and DEFs during the testing period, but was higher in DEFs than in CEFs (Fig. 3c).

In sum, these results indicate that the expression levels of proinflammatory cytokines and chemokines IL-1beta, IL-6, IL-8 and LITAF in CEFs and DEFs showed different patterns following infection with NDVs of different pathogenicities.

As is well known, the IFN system is the most important host defense mechanism during infection with viral pathogens, for it controls and surpasses viral replication and modulates innate immune responses. In that context, in this study we compared the induction of IFN type I and II (IFN-alpha, IFN-beta and IFN-gamma) responses in CEFs and DEFs triggered by the SS-10 and NH-10 of different pathogenicities at the early stage of infection and that exhibited a similar species-dependent immune response. IFN-alpha, IFN-beta and IFN-gamma expression was upregulated in CEFs throughout the experiment period and peaked at 24 h p.i. (126.96- and 535.49-fold, 77.32- and 474.78-fold and 152.19- and 409.92-fold, respectively) induced by SS-10 and NH-10 when compared to mock-infected controls, yet was greater for NH-10 than SS-10 (Fig. 4a, b and c). In DEFs, the expression level of IFN-alpha was upregulated at 6 h p.i. (2.21- and 16.36-fold, respectively), gradually decreased to baseline level at 24 h p.i. (1.72- and 1.14-fold, respectively) and decreased further at 36 h p.i. (1.62- and 0.55-fold, respectively) after infection with SS-10 and NH-10 (Fig. 4a). The expression level of IFN-beta was upregulated during the tested period and peaked at both 6 and 24 h p.i. (7.09- and 25.28-fold, respectively) induced by both viruses (Fig. 4b). The expression level of IFN-gamma was downregulated for the duration of the study induced by SS-10; however, it was upregulated at 6 h p.i. (1.83-fold) and then downregulated at the other time points induced by NH-10 (Fig. 4c). In brief, these results demonstrate that the induction of rapid and robust type I and II IFNs in CEFs is far higher than in DEFs following challenge with virulent NDV and is lower for SS-10 than NH-10.

To compare the expression of MHC class I and II molecules, we examined their relative expression in CEFs and DEFs by qRT-PCR during early-phase NDV infection. MHC class I molecule expression was upregulated in CEFs and DEFs induced by SS-10 and NH-10 or treatment with poly(I:C) during the period of infection, except at 6 and 24 h p.i. in DEFs in response to infection with SS-10 (Fig. 5a). Importantly, the expression level of MHC class I induced by SS-10 was higher than NH-10 in CEFs, yet lower for SS-10 than NH-10 in DEFs at all time points (Fig. 5a). The expression level of MHC class II molecules was downregulated when induced by SS-10 and NH-10 or following stimulation with poly(I:C) for the duration of the study, whereas it was upregulated in CEFs at all time points when treated with poly(I:C) compared to mock-infected controls (Fig. 5b). Most importantly, MHC class I and II molecule expression in CEFs and DEFs showed different expression patterns that were associated with different pathogenicities against ND.