Liu et al. Vet Res (2017) 48:47 DOI 10.1186/s13567-017-0456-z RESEARCH ARTICLE Open Access RNA‑seq comparative analysis of Peking ducks spleen gene expression 24 h post‑infected with duck plague virulent or attenuated virus Tian Liu1,2, Anchun Cheng1,2,3*, Mingshu Wang1,2,3*, Renyong Jia1,2,3, Qiao Yang1,2,3, Ying Wu1,2,3, Kunfeng Sun1,2,3, Dekang Zhu2,3, Shun Chen1,2,3, Mafeng Liu1,2,3, XinXin Zhao1,2,3 and Xiaoyue Chen2,3 Abstract Duck plague virus (DPV), a member of alphaherpesvirus sub-family, can cause signifcant economic losses on duck farms in China. DPV Chinese virulent strain (CHv) is highly pathogenic and could induce massive ducks death. Attenu- ated DPV vaccines (CHa) have been put into service against duck plague with billions of doses in China each year. Researches on DPV have been development for many years, however, a comprehensive understanding of molecular mechanisms underlying pathogenicity of CHv strain and protection of CHa strain to ducks is still blank. In present study, we performed RNA-seq technology to analyze transcriptome profling of duck spleens for the frst time to iden- tify diferentially expressed genes (DEGs) associated with the infection of CHv and CHa at 24 h. Comparison of gene expression with mock ducks revealed 748 DEGs and 484 DEGs after CHv and CHa infection, respectively. Gene path- way analysis of DEGs highlighted valuable biological processes involved in host immune response, cell apoptosis and viral invasion. Genes expressed in those pathways were diferent in CHv infected duck spleens and CHa vaccinated duck spleens. The results may provide valuable information for us to explore the reasons of pathogenicity caused by CHv strain and protection activated by CHa strain. Introduction induce massive petechial hemorrhages in parenchymal After frstly reported in Netherlands at 1923, duck plague organ, lymphoid and digestive tract and causes massive (DP) was rapidly spread around the world [1]. In China, ducks death [5–8]. To control this disease on duck farms, the DP was not suspected until 1958 and the outbreak of attenuated DPV vaccines, such as CHa, have been put DP results in a devastated hit to duck industries in China, into service against duck plague with billions of doses in one of the largest duck breeding countries in the world, China each year [9]. due to DP’s high mortality [2, 3]. Te disease is caused by Compared to other Herpesviruses, the development duck plague virus (DPV), a member of alphaherpesvirus of DPV researches, particularly in viral gene functions subfamily, which is a double-stranded DNA virus com- and virus-host interplay, undergoes a slow process. posed with capsid, tegument and envelope [4]. DPV Chi- After the frst reports of DPV highly virulent strain and nese virulent strain (CHv), which was isolated from dead attenuated strain genomes, the studies of gene structure infected ducks in China, is highly pathogenic and could and functions have been spring up. DPV highly virulent strain genes, such as capsid protein genes UL19, UL35 tegument protein genes UL16, UL51 and envelope glyco- *Correspondence: [email protected]; [email protected] protein genes like UL44, US7, US8 were well studied [10– 1 Avian Disease Research Center, College of Veterinary Medicine, Sichuan 16]. Furthermore, it has been reported that the fve ORFs, Agricultural University, Wenjiang, Chengdu 611130, People’s Republic of China including UL2, UL12, US10, UL47 and UL41, might rep- Full list of author information is available at the end of the article resent DPV virulence factors [17]. However, although © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. Vet Res (2017) 48:47 Page 2 of 14 exploring basic gene mechanisms of highly virulent DPV DPV CHv strain or attenuated DPV CHa strain at 24 hpi strains and attenuated DPV strains on genomic level is and the results may provide valuable information for us indeed helpful, it still lacks systemic and comprehensive to explore the reasons of pathogenicity caused by CHv studies on nature host duck biological processes when strain and protection activated by CHa strain. infect with DP virus. Performed works reported that DPV virulent strain conducted a latent infection follow- Materials and methods ing a primary infection in trigeminal ganglion and lym- Virus and experiment design phoid tissues including PBL and spleen [18], whereas Highly virulence DPV CHv strain and attenuated modi- how the virus processes a latent infection and what the fed vaccine DPV CHa strain used in this study were mechanisms of latency infection have not been clear. obtained from the Key Laboratory of Animal Disease Meanwhile, although Yuan observed programmed cell and Human Health of Sichuan Province. Twenty-seven death on lymphocytes after infection of highly virulent Peking ducklings were conducted from a DPV-free farm DPV strain, and the result indicated membrane proteins where vaccination against DPV was not implementa- of DPV could alter characteristics of apoptotic cells in tion. Tree experimental groups, including CHv infected duck embryo fbroblast culture [19]. However, no further group, CHa vaccinated group and normal saline intra- studies about DPV-induced apoptosis were observed. muscular injected control group, were explored in this Moreover, as we all know, the processes for virus to entry study. Each experimental group contained three biol- into a cell is critical to initiate their whole life cycles, ogy repeats and each repeat included three ducks. CHv- however, we have little information about DPV entry infected and CHa-vaccinated groups were inoculated 8 [20–22]. Although genes involved in other herpesvirus using intramuscular administration (4.1 × 10 copies entry such as glycoprotein gB and gD were well studied, CHv and CHa, respectively). Ducks were euthanized to it is still a puzzle part that whether these genes are neces- collect spleen samples at 24 hpi. Te samples were rap- sary in DPV entry or not [23, 24]. idly kept in liquid nitrogen for further experiments. In this study, with the help of RNA-seq platform we explored a bioinformatics analysis on duck spleen gene Library preparation for RNA‑seq expression for the frst time after highly virulent DPV Total RNA was isolated from spleen samples by TRIzol CHv strain and attenuated DPV CHa strain infection at extraction (Invitrogen, CA, USA) following the manu- post-infected 24 h. Previous study indicated that attenu- facturer’s instructions. RNA purity was checked by using ated DPVs were rapid distribution and proliferation in the NanoPhotometer® spectrophotometer (IMPLEN, duck’s tissues 12 h post-inoculation, however, copies of CA, USA) and its concentration was measured using attenuated DPV were dramatic decline in duck’s tissues Qubit® RNA Assay Kit in Qubit ® 2.0 Flurometer (Life 24 h post-infection (hpi), which means that attenuated Technologies, CA, USA). RNA integrity was assessed DPV could rapidly invoke host immune response and using the RNA Nano 6000 Assay Kit of the Bioana- inhibit DPV proliferation in the early stage of inocula- lyzer 2100 system (Agilent Technologies, CA, USA). A tion [6]. Tus, 24 h was chosen as a samples collection total amount of 3 μg RNA per sample was used as input time point to explore reasons of protection stimulated material for the RNA sample preparations. Sequenc- by attenuated DPV in short time after infection. In order ing libraries were generated using NEBNext® UltraTM to detect accurate biological diferences from sequenc- RNA Library Prep Kit for Illumina ® (NEB, USA) fol- ing data, the setting of multiple biological replicates and lowing manufacturer’s recommendations and index the meticulous analysis of statistic were performed. Fur- codes were added to attribute sequences to each sam- thermore, high quality reads mapping was presented by ple. Briefy, mRNA was purifed from total RNA using using a paired-end mRNA sequencing approach. Even- poly-T oligo-attached magnetic beads. Fragmentation tually, 748 diferentially expressed genes (DEGs) in CHv was carried out using divalent cations under elevated infected group and 484 DEGs in CHa vaccinated group temperature in NEBNext First Strand Synthesis Reac- were selected. Gene pathway analysis of DEGs high- tion Bufer (5×). First strand cDNA was synthesized lighted several valuable biological processes involved in using random hexamer primer and M-MuLV Reverse host immune response, cell apoptosis and viral invasion. Transcriptase (RNase H-). Second strand cDNA syn- Genes expressed in those pathways were diferent in CHv thesis was subsequently performed using DNA Poly- infected duck spleens and CHa vaccinated duck spleens. merase I and RNaseH were converted into blunt ends Overall, our fndings generated in this work frstly pro- via exonuclease/polymerase activities. After adenyla- vided a detailed view of global changes in duck spleen tion of 3′ ends of DNA fragments, NEBNext Adaptors gene expression when infected with either highly virulent with hairpin loop structure were ligated to prepare for Liu et al. Vet Res (2017) 48:47 Page 3 of 14 hybridization. In order to select cDNA fragments of Results preferentially 150–200 bp in length, the library frag- Collection of duck spleen samples and construction ments were purifed with AMPure XP system (Beckman of cDNA libraries after CHv and CHa infection Coulter, Beverly, USA). Ten 3 μL USER Enzyme (NEB, Transcriptome sequencing by RNA-seq was used to USA) was used with size-selected, adaptor-ligated cDNA detect global changes of Peking duck spleen gene expres- at 37 °C for 15 min followed by 5 min at 95 °C before sion after CHv and CHa infection.