Zhang et al. Vet Res (2018) 49:18 https://doi.org/10.1186/s13567-018-0512-3 RESEARCH ARTICLE Open Access Identifcation and characterization of microRNA in the lung tissue of pigs with diferent susceptibilities to PCV2 infection Ping Zhang1†, Liyuan Wang1†, Yanping Li1, Ping Jiang2, Yanchao Wang1, Pengfei Wang1, Li Kang1, Yuding Wang1, Yi Sun1* and Yunliang Jiang1* Abstract Porcine circovirus type 2 (PCV2) is the primary cause of post-weaning multisystemic wasting syndrome (PMWS) and other PCV-associated diseases. According to our previous RNA-sequencing analysis, the diferences in the suscep- tibility to PCV2 infection depended on the genetic diferences between the Laiwu (LW) and Yorkshire Landrace crossbred (YL) pigs, but the cellular microRNA (miRNA) that are diferentially expressed between the LW× and YL pigs before and after PCV2 infection remain to be determined. In this study, high-throughput sequencing was performed to determine the abundance and diferential expression of miRNA in lung tissues from PCV2-infected and PCV2-unin- fected LW and YL pigs. In total, 295 known and 95 novel miRNA were identifed, and 23 known and 25 novel miRNA were signifcantly diferentially expressed in the PCV2-infected vs. PCV2-uninfected LW pigs and/or the PCV2-infected vs. PCV2-uninfected YL pigs. The expression levels of ssc-miR-122, ssc-miR-192, ssc-miR-451, ssc-miR-486, and ssc- miR-504 were confrmed by quantitative real-time PCR (qRT-PCR). Analysis of the potential targets of the four up-reg- ulated miRNA (i.e., ssc-miR-122, ssc-miR-192, ssc-miR-451 and ssc-miR-486) identifed pathways and genes that may be important for disease resistance. Among the up-regulated miRNA, ssc-miR-122 can repress the protein expression and viral DNA replication of PCV2 and down-regulate the expression of the nuclear factor of activated T-cells 5 (NFAT5) and aminopeptidase puromycin sensitive (NPEPPS) by binding to their 3′ untranslated region (3′UTR) in PK15 cells. Therefore, ssc-miR-122 may indirectly suppress PCV2 infection by targeting genes related to the host immune system, such as NFAT5 and NPEPPS. Introduction [2, 3]. PCV2-afected pigs show wasting and progressive Porcine circovirus (PCV) was frst considered a con- weight loss, enlarged lymph nodes, and respiratory dis- taminant in a porcine kidney cell line in 1974 and was tress, jaundice, and occasional diarrhea [4]. Morbidity described in greater detail in 1982. In the late 1990s, in PCV2-afected farms is commonly 4–30% (occasion- PCV2 was found to be associated with post-weaning ally as high as 50–60%), and mortality ranges from 4 to multisystemic wasting syndrome (PMWS), which is cur- 20% [5]. Because of the increased mortality rates and rently considered one of the most important swine dis- the impact on weight gain, PCV2 has had a serious eco- eases worldwide [1]. PCV2 belongs to the Circoviridae nomic impact on the swine production industry [6]. Te family, which is characterized by a genome consisting of replication patterns of PCV2 in pulmonary alveolar mac- single-strand circular DNA with 1768-9 nucleotides (nt) rophages are diferent among macrophages derived from diferent conventional crossbred pigs [7]. MicroRNA (miRNA) are ~21–23 nt small RNA (sRNA) *Correspondence: [email protected]; [email protected] †Ping Zhang and Liyuan Wang contributed equally to this work molecules that regulate gene expression at the post- 1 Shandong Provincial Key Laboratory of Animal Biotechnology transcriptional level [8]. Currently, miRNA have been and Disease Control and Prevention, Shandong Agricultural University, 61 estimated to constitute 1–5% of animal genes and col- Daizong Street, Taian 271018, Shandong, China Full list of author information is available at the end of the article lectively regulate up to 30% of genes; therefore, miRNA © The Author(s) 2018. 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. Zhang et al. Vet Res (2018) 49:18 Page 2 of 13 are among the most abundant regulators [9]. No miRNA on both the whole genome and ORF2 sequences indi- encoded by PCV2 genomic DNA were detected in ton- cate that the genotype of PCV2-SD is PCV2b. Each pig sil and mediastinal lymph node tissues from PCV2- from LW-i and YL-i groups was intramuscularly injected 3.8 infected pigs [10]. In PK15 cells expressing PCV2 ORF1, with 3 mL PCV2-SD solution with 10 TCID50 (50% ORF2 and ORF3, 51, 74 and 32 miRNA were identifed, tissue culture infective dose)/mL. Te pigs from LW-u respectively, difering in abundance from those in the and YL-u groups were treated similarly with an identi- controls [11]. In PCV2-infected Landrace × Yorkshire cal volume of phosphate bufered saline (PBS). During pigs, miR-126-3p, 126-5p, 129a, let-7d-3p and let-7b-3p the experimental period, clinical signs were monitored were up-regulated, while miR-193a-5p, 574-5p and 34a daily and the copy number of PCV2 DNA in serum was were down-regulated in the mediastinal lymph node detected by quantitative real-time PCR (qRT-PCR) at 0, [12]. Tese diferentially expressed miRNA are mainly 4, 7, 10, 14, 21, 28 and 35 dpi. From 7 dpi, PCV2 virus involved in the regulation of the immune system and could be detected from the serums of PCV2-infected cell proliferation. Although certain PCV2 infection- pigs, indicating that the intramuscular injection was suc- associated miRNA have been identifed, pig-breed-spe- cessful. Te mean copy number of PCV2 DNA in serum cifc miRNA that are diferentially expressed and likely signifcantly increased in both PCV2-infected LW and YL account for the resistance to PCV2 infection have not pigs at 14 dpi. PCV2-infected YL pigs exhibited serious been characterized. clinical signs typifying PMWS, while the PCV2-infected Te Laiwu (LW) pig is a Chinese indigenous pig breed LW pigs showed slightly clinical symptoms. Te lung tis- from Shandong Province that is well-known for its sue of PCV2-infected YL pigs showed serious lesions, extremely high intramuscular fat content of > 10%. Te while they were not observed in PCV2-infected LW pigs LW pig also exhibits a higher resistance to certain infec- [13]. All pigs were sacrifced at 35 dpi and tissue samples tious diseases, including PCV2. In our previous study, were frozen in liquid nitrogen or preserved by immersion PCV2-infected Yorkshire × Landrace (YL) pigs exhibited in 10% neutral-bufered formalin. serious clinical features that are typical of PCV2 disease, particularly severe lesions in the lungs, such as conges- sRNA library preparation and deep‑sequencing tion, bleeding, interstitial pneumonia and lymphocyte Four sRNA libraries of LW-u, LW-i, YL-u and YL-i were infltration, while the PCV2-infected LW pigs showed constructed using the homogenized and pooled total only a few clinical symptoms; at 35 days post-infection RNA from four individuals selected from each group as (dpi), the PCV2 DNA copy in the YL pigs was signif- previously described [15–17]. Te construction of pooled cantly higher than that in the LW pigs [13]. In this study, small RNA libraries is certainly better than pooled total using Illumina/Solexa high-throughput sequencing, we RNA before Illumina sequencing. To minimize devia- identifed the diferentially expressed miRNA in the lung tion of this procedure compared with the results com- tissues between LW and YL pigs prior to and post PCV2 ing from each individual per group, more miRNA were infection and further characterized the role of miR-122 validated by qRT-PCR in this study. Total RNA was in conferring higher resistance to PCV2 infection. extracted from the lung tissues of PCV2-infected and uninfected pigs of each breed with TRIzol ® Reagent (Inv- Materials and methods itrogen Life Technologies, Carlsbad, CA, USA). Briefy, Sample collection for each sample, 20 μg of total RNA and a Small RNA Fifteen purebred LW and 15 YL weaned pigs that were Sample Prep Kit (Illumina, San Diego, CA, USA) were validated to be free of PCV2, porcine circovirus type used for library construction according to the manufac- 1 (PCV1), porcine reproductive and respiratory syn- turer’s instructions. Ten, fractions between 18 and 30 nt drome virus (PRRSV) and porcine parvovirus (PPV) were removed and purifed using 15% Tris–borate-EDTA were raised under identical conditions on the same farm. denaturing polyacrylamide gel electrophoresis (PAGE). All pigs were randomly divided into the following four Subsequently, the 3′ and 5′ RNA adaptors were ligated groups: PCV2-infected LW pigs (LW-i, n = 10), PCV2- to the purifed fragments with T4 RNA ligase in proper uninfected LW pigs (LW-u, n = 5), PCV2-infected YL order. Te cDNA from the adaptor-ligated sRNA were pigs (YL-i, n = 10) and PCV2-uninfected YL pigs (YL-u, then amplifed by RT-PCR with 15 cycles. Te products n = 5). Te procedures used for the pig management and (90-bp sRNA + adaptors) after purifcation on 4% aga- PCV2 inoculation have been previously described [13, rose gels were used for sequencing on an Illumina 1G 14]. Briefy, the PCV2 strain (named PCV2-SD) used in Genome Analyzer at Beijing Genomics Institute (BGI, this experiment was isolated from suspected PMWS pigs Shenzhen, China). After masking and removing the in the Shandong province.