Comparative analysis of early immune responses induced by two strains of Newcastle disease virus in chickens

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1 Received: 22 April 2018 Revised: 28 June 2018 Accepted: 3 July 2018 DOI: /mo3.701 ORIGINAL ARTICLE Comprtive nlysis of erly immune responses induced y two strins of Newcstle disese virus in chickens Tingting Zhng 1,2 * Mengting Ren 1,2 * Chenggng Liu 1,2 Liwen Xu 1,2 Fngfng Wng 1,2 Zongxi Hn 2 Yuho Sho 2 Deying M 1 1 College of Animl Science nd Technology, Northest Agriculturl University, Hrin, Chin 2 Division of Avin Infectious Diseses, Stte Key Lortory of Veterinry Biotechnology, Hrin Veterinry Reserch Institute, Chinese Acdemy of Agriculturl Sciences, Hrin, Chin Correspondence Deying M, College of Animl Science nd Technology, Northest Agriculturl University, Hrin, Chin. Emil: mdeying@neu.edu.cn Funding informtion Reserch Progrm for Applied Technology of Heilongjing Province, Grnt/Awrd Numer: PC13S02 Astrct Newcstle disese, cused y virulent strin of Newcstle disese virus (NDV), is n cute, highly contgious disese tht is prevlent worldwide nd is responsile for serious economic losses to the poultry industry. In the current study, we compred the erly immune responses in chickens infected with two strins of velogenic NDV, duck origin, nmed GD strin (Md/CH/LGD/1/2005, genotype VIId), nd n chicken origin, F48E9 strin (genotype IX). The virl RNA level of GD strin ws significntly higher thn those of F48E9 in most tissues of chicken. Furthermore, the high level of virl RNA of the GD strin ws ssocited with stronger immune response compred to tht of F48E9, chrcterized y upregulted expression of some of vin β- defensins nd cytokines, most of toll- like receptors, nd some of the other immunerelted genes investigted. This study thus demonstrted differences in host erly immune responses to the two NDV strins. Further studies re needed to chrcterize the sic moleculr mechnisms involved in the host responses in chickens infected y the two NDV strins. KEYWORDS chicken, immune response, Newcstle disese virus, virl RNA 1 INTRODUCTION Newcstle disese (ND) is one of the most importnt virl diseses ffecting irds worldwide, nd is responsile for serious economic losses to the poultry industry due to high mortlity, decresed egg production, nd ody weight loss (Alexnder, 1997). The custive gent, virulent ND virus (NDV), elongs to genus Avulvirus of the Prmyxoviride fmily. Virulent NDV continues to e endemic in mny countries, despite the ppliction of vccines (Dimitrov, Afonso, Yu, & Miller, 2016). NDV cn infect wide vriety of irds, ut chickens remin the most importnt nd susceptile host (Dimitrov, Rmey, Qiu, Bhl, & Afonso, 2016). Genotype VII is the most prevlent NDVs found in Chin in recent yers, nd hs een considered to e minly responsile for outreks of ND in this country (Liu, Wn, Ni, Wu, & Liu, 2003; Qin et l., 2008; Yu, Wng, Jing, Chng, & Kwng, 2001). Despite the importnce of the ntiody- medited response for protecting ginst NDV infection, the innte immune response induced y NDV chllenge remins uncler. The host innte immune system provides the first line of defense ginst pthogens. The innte immune system cn recognize components of pthogens clled pthogen- ssocited moleculr ptterns vi pttern recognition receptors (Amimo et l., 2014; Yn et l., 2017). Toll- like receptors (TLRs) re fmily tht elongs to the pttern recognition receptors. *These uthors re joint first uthor. This is n open ccess rticle under the terms of the Cretive Commons Attriution License, which permits use, distriution nd reproduction in ny medium, provided the originl work is properly cited The Authors. MicroiologyOpen pulished y John Wiley & Sons Ltd. MicroiologyOpen. 2018;e of 10

2 2 of 10 So fr, pproximtely 13 TLRs hve een identified in mmmls (Pul, Brisin, Adul- Csreem, & Shrif, 2013) nd 10 TLRs hve een identified in vin species (Brownmlie & Alln, 2011). Once the pthogen- ssocited moleculr ptterns re recognized y TLRs, the TLRs my then trigger downstrem signl trnsduction through ctivtion of MyD88- dependent or - independent intrcellulr pthwys, to ctivte NF- κb trnscription fctors nd mitogen- ctivted protein kinse to induce immune- relted molecules, such s host defense peptides nd cytokines to defend ginst the invding pthogens (Akir & Tked, 2004; Seth, Sun, & Chen, 2006; Tkeuchi & Akir, 2010). In the pst severl yers, innte immune responses to different strin of virus infections hve een studied. Different expression levels of TLRs, cytokines, vin β- defensins (AvBDs) hve een detected in tissues following chllenge of chickens, geese, nd pigeon with different strins of NDV (Adel- Mgeed, Isoe, & Yoshimur, 2014; Li et l., 2015; Xu et l., 2016; Yn et l., 2017). In ddition, innte immune response to some other vin virus infection hve een lso reported, such s innte immunity of duck ginst duck heptitis virus (M et l., 2011, 2012), innte immunity of chicken ginst infectious ronchitis virus (Xu et l., 2015). Although numerous studies hve focused on the pthogenesis of different isoltes nd pthotypes of NDV, limited dt re ville regrding the host immune responses induced y different NDV strins. A recent study demonstrted tht infection with NDV strin IBS002 (genotype VII) cused n erlier cytokine pek nd higher virl lod compred to those of AF2240 strin (genotype VIII) (Rsoli et l., 2014). Furthermore, NDV strin JS5/05 (genotype VIId) produced more severe dmge to the lymphtic orgns in chickens compred to Herts/33 strin (genotype IV) nd F48E8 strin, due to the high virl lod nd strong immune response induced y JS5/05 strin (Hu et l., 2015). These findings indicted tht differentil regultion of the host immune response y different NDV strins is n importnt spect of NDV pthogenesis. In this study, we evluted the moleculr ses of the different immune responses induced y two NDV strins, GD ( genotype VIId strin,isolted from the kidney of disesed white Muscovy duck during n outrek in 2005) nd F48E9 y mesuring the virl RNA nd expression ptterns of vin β- defensins (AvBDs), TLRs, cytokines, nd other immune- relted genes in the tissue smples of chickens during the erly stge of infection. 2 MATERIALS AND METHODS 2.1 Virus strins The velogenic NDV genotype VII strin GD (Md/CH/LGD/1/2005) ws isolted from kidney smples from disesed white Muscovy duck during n outrek in 2005 (Wu et l., 2015), nd the virulent NDV genotype IX strin F48E9 ws isolted from chickens nd stored t the Lortory of Avin Infectious Diseses, Stte Key Lortory of Veterinry Biotechnology, Hrin Veterinry Reserch Institute (HVRI), Chinese Acdemy of Agriculturl Sciences (Hrin, Chin). According to pthogenicity tests in chickens, the GD strin hd n intrcererl pthogenicity index of 1.88 nd men deth time of 50 hr, compred to 1.63 nd 71 hr, respectively, in ducks (Wu et l., 2015). The intrcererl pthogenicity index for the F48E9 strin in chickens ws 2.0 (Guo et l., 2014). Viruses were purified nd propgted s descried y Xu et l. (2016). Infective llntoic fluid contining virus ws hrvested nd stored t 70 C until use. All the experiments involving viruses were conducted in purifier clss II iosfety cinet in iosfety level- 2 lortory. 2.2 Animls One- dy- old specific- pthogen- free chickens were otined from the Lortory Animl Center, HVRI. The chickens were mintined in isoltors with negtive pressure, nd food nd wter were provided d liitum until use. All these irds were negtive for NDV ntiody. 2.3 Preprtion of stndrds for rel- time reverse trnscription- polymerse chin rection (rrt- PCR) We otined stndrd DNA for quntifiction of trget gene trnscripts, including 18S rrna, TLRs (1, 2, 3, 4, 5, 7, 15 nd 21), AvBDs (1 14), inducile nitric oxide synthse (inos), MHC clss I nd II, cytokines (interleukin (IL)- 1β, IL- 2, IL- 6, IL- 8, nd IL- 18, nd interferon- γ (IFN- γ), Fs/Fs lignd (FsL), myeloid differentition primry response 88 (MyD88), interferon regultory fctor (IRF)- 7, nucler fctor- κb (NF- κb), nd the M gene from oth NDV strins, y RT- PCR mplifiction with specific primers (Supporting Informtion Tle S1). Totl RNA ws extrcted from one mrrow or spleen from helthy chickens, nd cdna ws synthesized s descried y Li et l. (2015). The plsmid products were prepred s descried previously (Li et l., 2015), nd used s respective stndrds for susequent quntittive RT- PCR. 2.4 Experimentl design nd quntittive RT- PCR of mrnas in tissues Chickens t 20 dys of ge were llotted rndomly to three groups of ten ech. Groups 1 nd 2 were inoculted intrnslly with 100 μl of the GD strin t % egg infective doses (EID 50 ), or the sme dose of F48E9 respectively. Group 3 ws inoculted with 100 μl of phosphte- uffered sline served s control. Five irds from ech group were slughtered t 24 nd 48 hr postinfection (hpi) respectively. Tissue smples of lung, trche, proventriculus, kidney, cecl tonsil, liver, spleen, Hrderin glnd, one mrrow, nd rin were collected nd used for quntittive RT- PCR nlyses of host genes nd NDV. The mrna expression levels of the trget genes were mesured using One- step Rel- time PrimeScript RT- PCR (Tkr Biotechnology, Dlin, Chin) with specific primers (Supporting Informtion Tle S1), s descried previously (Li et l., 2015; Xu et l., 2016). All mplifictions were performed in triplicte. The

3 3 of 10 1.E+12 1.E+11 Virl copy numers 1.E+10 1.E+09 1.E+08 1.E+07 1.E+06 rin trche lung kidney liver proventriculus one mrrow spleen cecl tonsil Hrderin glnds FIGURE 1 Virl repliction in tissues of chickens chllenged with Newcstle disese virus (NDV) strin GD (Md/CH/LGD/1/2005) nd NDV strin F48E9, respectively. Virl RNA copy numers in tissue smples were mesured y quntittive RT- PCR. Ech r indictes the men ± SD. c Vlues with different superscripted letters re significntly different (p < 0.05) mrna levels of ech trget gene were normlized to the levels of 18S rrna in the sme smples. 2.5 Sttisticl nlyses Dt re expressed s the men ± SD. The significnce of the results ws ssessed using SPSS, version 18 (SPSS, Armonk, NY, USA). p < 0.05 ws considered to e sttisticlly significnt. 3 RESULTS 3.1 Different virl RNA levels in tissues of chickens infected with GD nd F48E9 strins We investigted the differences in virus distriution in tissues of chickens infected with the two different NDV strins t 24 nd 48 hpi. Both NDV strins successfully estlished n erly infection, nd virl RNA ws detected in ll the tested tissues t 24 nd 48 hpi. In contrst, no virl RNA ws detected in tissues from control irds. Of these tissues investigted, GD virl RNA level were significntly higher in the kidney, proventriculus, nd spleen t 24 hpi, nd in the trche nd lung t 48 hpi, thn those of F48E9 (p < 0.05) (Figure 1). 3.2 GD infection upregulted expression of AvBDs in chicken tissues The expression ptterns of AvBDs 1 13 were investigted in chickens infected with the two NDV strins respectively. Of these AvBDs investigted, GD infection significntly upregulted AvBD2 in lung, kidney, nd Hrderin glnds t 48 hpi, AvBD3 in kidney nd spleen t 24 hpi, in cecl tonsil nd Hrderin glnds t 48 hpi, nd AvBD6 in rin, Hrderin glnds, nd lung t 48 hpi, s compred to those of the control nd F48E9 (p < 0.05) (Figure 2). In contrst, neither of NDV strins infection hd oviously effects on the expression levels of the other AvBDs (dt not shown). 3.3 GD triggered stronger cytokine response thn F48E9 GD infection inhiited IFN- γ expression in the trche, kidney, nd spleen t 24 hpi (p < 0.05) (Figure 3). In contrst, GD infection significntly upregulted expression levels of IFN- γ in the rin, lung, proventriculus, spleen, nd Hrderin glnds t 48 hpi, compred to the control (p < 0.05). Both strins of NDV decresed IL- 6 mrna expression in the one mrrow nd spleen t 24 hpi (p < 0.05). The IL- 6 expression ws remrkly upregulted in the rin, lung, nd Hrderin glnds t 48 hpi following GD infection (p < 0.05). However, neither of NDV strins hd ny significnt effect on mrna expression levels of IL- 1β, IL-2, IL-8, nd IL- 18 in ny of the tested tissues (dt not shown). Furthermore, GD infection significntly upregulted inos expression in the lung, one mrrow, nd Hrderin glnds t 48 hpi, compred to the control nd F48E9, nd spleen t 48 hpi, compred to the control (p < 0.05) (Figure 3). Overll, these dt suggested tht GD infection triggered more potent cytokine response thn infection with the F48E9 strin. 3.4 GD infection trigged strong TLR responses in chicken tissues GD infection significntly upregulted the expression of most TLRs (except TLR4 nd TLR5) in oth Hrderin glnds nd

4 4 of 10 AvBD2 Copy numers of trget gene/reference gene 1.E-06 1.E+01 rin trche lung kidney liver proventriculus one mrrow spleen cecl tonsil Hrderin glnds AvBD3 1.E-06 rin trche lung kidney liver proventriculus one mrrow spleen cecl tonsil Hrderin glnds AvBD6 1.E-06 rin trche lung kidney liver proventriculus one mrrow spleen cecl tonsil Hrderin Tissues glnds FIGURE 2 Reltive gene expression levels of AvBDs in tissues of chickens chllenged with Newcstle disese virus (NDV) strin GD (Md/CH/LGD/1/2005) nd NDV strin F48E9 respectively. The cdna copy numers in the tissue smples were mesured y quntittive RT- PCR. The mrna levels of trget genes were normlized to tht of 18S rrna in the sme smples. Ech r is the men ± SD., Vlues with different superscripted letters re significntly different (p < 0.05)

5 5 of 10 IFN-γ Copy numers of trget gene/reference gene rin trche lung kidney liver proventriculus one mrrow spleen cecl tonsil Hrderin glnds IL-6 Control-24 h F48E9-24 h GD-24 h Control-48 h F48E9-48 h GD-48 h rin trche lung kidney liver proventriculus one mrrow spleen cecl tonsil Hrderin glnds inos rin trche lung kidney liver proventriculus one mrrow spleen cecl tonsil Hrderin glnds Tissues FIGURE 3 Reltive gene expression levels of cytokines nd inducile nitric oxide synthse (inos) in tissues of chicken chllenged with Newcstle disese virus (NDV) strin GD (Md/CH/LGD/1/2005) nd NDV strin F48E9, respectively. cdna copy numers in the tissue smples were mesured y quntittive RT- PCR. The mrna levels of trget genes were normlized to tht of 18S rrna in the sme smples. Ech r is the men ± SD., Vlues with different superscripted letters re significntly different (p < 0.05) lung, nd TLR1 nd TLR2 in cecl tonsil t 48 hpi (p < 0.05), compred to the control nd F48E9 (Figure 4). In contrst, gene expression levels of TLR4 nd TLR5 in ll the tested tissues were unffected y either NDV strins infection (dt not shown). 3.5 GD infection trigged the other immune- relted moleculr responses in chicken tissues Of the immune- relted molecules investigted, MyD88 mrna expression ws significntly upregulted in one mrrow t 24 hpi

6 6 of 10 TLR1 Control-24 h F48E9-24 h GD-24 h Control-48 h F48E9-48 h GD-48 h Copy numers of trget gene / reference gene 1.E+01 rin trche lung kidney liver proventriculus one mrrow spleen cecl tonsil Hrderin glnds TLR2 rin trche lung kidney liver proventriculus one mrrow spleen cecl tonsil Hrderin glnds TLR3 rin trche lung kidney liver proventriculus one mrrow spleen cecl tonsil Hrderin glnds Tissues FIGURE 4 Reltive gene expression levels of toll- like receptors (TLRs) in tissues of chickens chllenged with Newcstle disese virus (NDV) strin GD (Md/CH/LGD/1/2005,) nd NDV strin F48E9 respectively. The cdna copy numers in the tissue smples were mesured y quntittive RT- PCR. The mrna levels of trget genes were normlized to tht of 18S rrna in the sme smples. Ech r is the men ± SD., Vlues with superscripted different letters re significntly different (p < 0.05)

7 7 of 10 TLR7 rin trche lung kidney liver proventriculus one mrrow spleen cecl tonsil Hrderin glnds reference gene / TLR15 gene trget of Copy numers rin trche lung kidney liver proventriculus one mrrow spleen cecl tonsil Hrderin glnds TLR21 rin trche lung kidney liver proventriculus one mrrow spleen cecl tonsil Hrderin glnds Tissues FIGURE 4 (Continued) nd in Hrderin glnd t oth time points following infection with oth NDVstrins (p < 0.05) (Supporting Informtion Figure S1). NF- κb 52 ws significntly upregulted in spleen t 24 hpi nd in Hrderin glnds t oth time points, nd NF- κb 65 ws significntly upregulted in one mrrow t 48 hpi (p < 0.05) in response to GD infection, while none of the isoforms were significntly ffected y F48E9 infection (Supporting Informtion Figure S2).

8 8 of 10 GD infection lso gretly incresed the expression of FsL in one mrrow, Fs in lung nd Hrderin glnds, nd MHC clss I in rin, lung, nd Hrderin glnds t 48 hpi (p < 0.05). Notly, MHC clss II expression in the kidney ws downregulted y GD infection t oth 24 nd 48 hpi nd in spleen t 48 hpi (p < 0.05) (Supporting Informtion Figure S3). These results suggested tht these molecules might involve in the innte immune response of chickens to GD infection. 4 DISCUSSION Domestic wterfowl nd chickens re trditionlly rised together in most res of Chin. However, this frming pttern my fcilitte the trnsmission of vrious viruses, including NDV, mong these domestic irds. Numerous studies hve investigted the pthogenicity of NDV isoltes, ut there is currently limited informtion regrding the ntivirl immune response in irds. In this study, we evluted the immune responses induced in chickens y two NDV strins, GD nd F48E9. Despite lower ICPI, GD induces stronger immune response, s well s higher virl RNA level in tissues t erly stge of infection, compred to tht of F48E9 in this study. In consistent with this oservtion, it ws previously reported tht two NDV strins, JS3/05 nd JS5/05 (ICPI 1.88) replicted more efficiently, nd induced stronger immune responses in splenocytes of chicken t 6, 12, nd 24 hpi in vitro (Hu et l., 2012) or in spleen, thymus, nd urs tissues of chicken t 24, 48, nd 72 hpi in vivo (Hu et l., 2015), compred to tht of F48E9 (ICPI 2.0). These oservtions reveled tht the strong immune response cused y GD strin, s well s JS3/05 nd JS5/05 strins, might e ssocited with high levels of virl lod in infected tissues or cells t the erly stge of infection. However, the underlying moleculr mechnisms need to e further investigted. In ddition, chickens nd geese chllenged y goose- or chicken- origin (VIId genotypes) of NDV strins mnifested similr pthologicl fetures, distinct from those of irds infected with Herts/33 strin (Wng et l., 2012). These results reveled tht the virl lod of NDV my e ssocited with the specific genotype, rther thn with their origin. Our results lso indicted tht GD infection upregulted the expression of three of ll of AvBDs, compred to F48E9. Fourteen AvBDs hve een identified in chickens to dte, nd re considered s key components of the host innte immune response ginst pthogens, nd potentilly provide link etween innte nd dptive immunities. In greement with the present oservtions, expression levels of AvBDs were upregulted in tissues of vrious vin species infected with NDV strins of pigeon or goose origin (Li et l., 2015; Xu et l., 2016), s well s with other vin viruses (M et l., 2011, 2012; Xu et l., 2015). However, differences in their expression levels in chickens infected with different NDV strins hve not previously een studied. Interestingly, we found tht higher virl lod ws ssocited with upregultion of these AvBDs in GDinfected chickens, compred with F48E9 infection. These results reveled tht the virl RNA level in tissues plyed pivotl role in regulting expression of these AvBDs. Considering their key roles in the regultion of immune responses, cytokine gene expression levels re used extensively s n indictor of the host immune response to virus infection. Surprisingly, the current study showed tht oth IFN- γ nd IL- 6 expression decresed in some tissues t 24 hpi following GD infection. This oservtion is different from most of previous findings (Adel- Mgeed et l., 2014; Hu et l., 2012, 2015; Kpczynski, Afonso, & Miller, 2013). Moreover, the result showed tht expression of IFN- γ in trche, kidney, nd spleen, nd IL- 6 expression in spleen ws higher t 24 hpi following F48E9 infection thn tht of GD infection. This result is lso different from their expression ptterns t 48 hpi in most tissues. In order to verify this oservtion, severl time experiments were repeted. Indeed, we could not figure out wht fctors cuse this kind of specil expression pttern, nd wht kind of roles oth cytokines would ply on NDV infection t this time point. Similrly, previous studies with Respirtory syncytil virus nd Influenz virus demonstrted tht viruses seem to suppress the production of type I IFNs y disrupting the signling pthwys of type I IFN synthesis (Oshnsky, Krunkosky, Brer, Jones, & Tripp, 2009; Xi et l., 2016). However, whether the NDV strins hve devised similr strtegies to interfere with the production of IFN- γ nd IL- 6 t the time point of infection needed to e further elucidted. In contrst, expression of IFN- γ nd IL- 6 in some tissues ws incresed t 48 hpi following oth GD nd F48E9 infection, compred to tht of the control. These findings were prtly in greement with those of Hu et l. (2012), who showed tht JS3/05 nd JS5/05 NDV strins induced more potent IFN response thn F48E9 strin. In ddition, our results, s well s those of Hu et l. (2012), lso showed tht the higher IFN- γ expression induced y GD, JS3/05, or JS5/05 infection ws ssocited with high virl lod, compred to F48E9. Cytokines could induce specific immune response to prevent infection upon virus invsion. IFN- γ medites the production of nitric oxide, resulting in the recruitment of more neutrophils nd mcrophges (Hung et l., 2012). IL- 6 regultes immune nd inflmmtory responses involved in the ctivtion, growth, nd differentition of T cells, nd contriutes to T- cell- medited inflmmtory rections (Hung et l., 2012). However, overproduction of cytokines s result of virus infection my result in cytokine storm, thus mplifying the detrimentl effect of inflmmtion on the host (Tisoncik et l., 2012). Notly, we showed tht inos expression ws upregulted in some tissues of chickens t 48 hr fter GD infection. These results showed tht high levels of cytokines induced y GD my e ssocited with the fsted virus repliction, nd then to further overcome the ntivirl effects of these cytokines. TLRs cn respond to virl infection in oth mmmls nd irds, nd produce ntivirl fctors tht help inhiit virl repliction (Kpczynski et l., 2013; Li et l., 2015; Xu et l., 2016). Consistent with this oservtion, the present study showed tht expression levels of most TLRs were significntly upregulted in some chicken tissues t 48 hr fter infection with either GD or F48E9. Consistent with this finding, TLRs expression levels were induced in different tissues of NDV- infected geese (Xu et l., 2016), while TLR3, TLR7,

9 9 of 10 nd TLR15 were induced in some tissues of pigeons infected with pigeon prmyxovirus type 1, vrint strins of NDV (Li et l., 2015). Overll, these results suggested tht these TLRs my e involved in the host ntivirl defense ginst oth NDV strins. In this study, the upregultion of MyD88, NF- κb 52, nd NF- κb 65 in response to oth GD nd F48E9 infection ws oserved. Accordingly, previous reports showed tht MyD88 nd NF- κb plyed importnt roles in innte ntivirl immunity ginst West Nile virus (Dffis, Smuel, Keller, Gle, & Dimond, 2007; Dffis et l., 2008), dengue virus (Nsirudeen et l., 2011), vin Temusu virus (Chen et l., 2016), nd vin reticuloendotheliosis virus (Mio et l., 2015) infections. The current results suggested tht these molecules my ply similr roles in innte ntivirl immunity ginst oth strins of NDV. However, further studies re needed to determine whether other trnscription fctors re involved in innte immunity ginst NDV infection. The poptotic response hs een identified s key element in the pthogenesis of virus infection. FsL is strongly stimulted y the NF- κb pthwy in response to virus infections, such s H9N2 vin influenz virus infection (Xing, Crdon, Anuncicion, Adms, & Do, 2010). Consistent with previous studies on other viruses, we showed tht Fs/FsL ws induced following GD infection, suggesting tht Fs/FsL my e involved in the immune response to GD infection. In ddition, MHC clss I nd clss II were lso upregulted in response to GD infection in this study. Consistent with this oservtion, MHC clss I ws upregulted y vin influenz H3N2 virus (Tong, Long, Li, & DeMri, 2004) nd y NDV strin of goose origin (Xu et l., 2016). MHC molecules re criticl components of the verterte dptive immune system (Fljnik & Kshr, 2010), nd the current nd previous results demonstrted criticl roles for these molecules in the host defense ginst viruses. 5 CONCLUSION We showed tht the virl RNA of GD strin in most tissues of chickens ws higher thn those of F48E9 strin t the erly stge of infection. The higher virl RNA level of the GD strin my e ssocited with stronger innte immune response compred to the F48E9 strin. However, this study only nlyzed differences in the host immune responses to infection with the two NDV strins t the erly stge, nd further investigtions re needed to elucidte how these NDV strins induce differentil host responses in chickens. ACKNOWLEDGMENTS The study ws prtly supported y Reserch Progrm for Applied Technology of Heilongjing Province (PC13S02). CONFLICT OF INTEREST The uthors declre tht they hve no competing interests. DATA ACCESSIBILITY The min dt supporting this study re provided in the results section of this pper. The others dt re provided s supplementry informtion ccompnying this pper. ETHICS STATEMENT All niml experiments were pproved y the Ethicl nd Animl Welfre Committee of Heilongjing Province, Chin (License no. SQ ). ORCID Deying M REFERENCES Adel-Mgeed, A. M., Isoe, N., & Yoshimur, Y. (2014). Effect of different TLR lignds on the expression of proinflmmtory cytokines nd vin et- defensins in the uterine nd vginl tissues of lying hens. Veterinry Immunology nd Immunopthology, 162, doi.org/ /j.vetimm Akir, S., & Tked, K. (2004). Functions of toll- like receptors: Lessons from KO mice. Comptes Rendus Biologies, 327, org/ /j.crvi Alexnder, D. J. (1997). Newcstle disese nd vin prmyxovirus infections. In B. W. Clnek, H. L. Brnes, C. W. Berd, & L. R. McDougl (Eds.), Diseses of poultry, 10th ed. (pp ). Ames: Iow Stte University Press. Amimo, J. O., Okoth, E., Jung, J. O., Ogr, W. O., Njhir, M. 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