Analysis of the swine tracheobronchial lymph node transcriptomic response to infection with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus
© Miller et al.; licensee BioMed Central Ltd. 2012
Received: 31 July 2012
Accepted: 15 October 2012
Published: 30 October 2012
Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen of swine worldwide. Emergence in 2006 of a novel highly pathogenic PRRSV (HP-PRRSV) isolate in China necessitated a comparative investigation into the host transcriptome response in tracheobronchial lymph nodes (TBLN) 13 days post-infection with HP-PRRSV rJXwn06, PRRSV strain VR-2332 or sham inocula. RNA from each was prepared for next-generation sequencing. Amplified library constructs were directly sequenced and a list of sequence transcripts and counts was generated using an RNAseq analysis pipeline to determine differential gene expression. Transcripts were annotated and relative abundance was calculated based upon the number of times a given transcript was represented in the library.
Major changes in transcript abundance occurred in response to infection with either PRRSV strain, each with over 630 differentially expressed transcripts. The largest increase in transcript level for either virus versus sham-inoculated controls were three serum amyloid A2 acute-phase isoforms. However, the degree of up or down-regulation of transcripts following infection with HP-PRRSV rJXwn06 was greater than transcript changes observed with US PRRSV VR-2332. Also, of 632 significantly altered transcripts within the HP-PRRSV rJXwn06 library 55 were up-regulated and 69 were down-regulated more than 3-fold, whilst in the US PRRSV VR-2332 library only 4 transcripts were up-regulated and 116 were down-regulated more than 3-fold.
The magnitude of differentially expressed gene profiles detected in HP-PRRSV rJXwn06 infected pigs as compared to VR-2332 infected pigs was consistent with the increased pathogenicity of the HP-PRRSV in vivo.
Porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of PRRS in swine, is a member of the Arteriviridae family in the order Nidovirales. PRRSV causes highly significant economic losses to the swine industry worldwide  as a result of both reproductive failure (late-term abortions and stillbirths) in pregnant sows and respiratory disease (pneumonia) in nursery and grower/finishing pigs . Infection with PRRSV also predisposes pigs to infection by bacterial pathogens as well as other viral pathogens [3–7], as such, PRRSV is a key etiological agent of the porcine respiratory disease complex (PRDC). Clinical disease caused by PRRSV is highly variable, ranging from mild, subclinical infection to acute death of adult animals . Differences in virulence have been attributed to numerous factors including host genetics, management practices, and virus strain heterogeneity [9–16]. Relatively little is known about the interactions of PRRSV and host cells. The lymph node is an anatomic site where the innate immune response and adaptive immune system interface. Tracheobronchial lymph nodes (TBLN) in swine drain the lung field and provide the focal structure that can reproducibly be identified. Although the TBLN contains a number of cell types, sampling this tissue allows study of direct and indirect effects of an infectious agent on the lung and cells within the lymph node.
In 2006 a unique syndrome with high morbidity and mortality was recognized in growing pigs in China that was originally known as porcine high fever disease (PHFD) due to its uncertain etiology . Experimental infection of pigs in China with these novel viral isolates reproduced the clinical disease providing strong evidence for the role of PRRSV as the causal agent of PHFD. However, there was still a question as to whether there was some unknown agent in the PRRSV preparations that increased the severity of the clinical disease over what was expected for a “routine” PRRSV infection. This question was resolved when PHFD was reproduced in China with virus derived from an infectious clone of the JX143 PRRSV isolate  demonstrating that PRRSV isolates with a common genetic motif had a causal role in PHFD leading to this lineage of virus being called highly pathogenic PRRSV (HP-PRRSV). We imported a plasmid containing a full-length clone of the 2006 JXwn06 HP-PRRSV isolate  from which infectious virus (rJXwn06) was rescued. An animal study was conducted comparing the pathogenicity of HP-PRRSV isolate rJXwn06 with the North American prototype strain VR-2332 PRRSV . The objective of this report was to investigate gene expression profiles in porcine tracheobronchial lymph node (TBLN) during viral infection with HP-PRRSV rJXwn06 strain alongside of US PRRSV strain VR-2332 at a snapshot of 13 days post-infection using bioinformatics.
Results and discussion
Mapping short RNA-seq reads and estimating transcript expression levels
Genomic Short-read Nucleotide Alignment Program (GSNAP) was used for alignment and genome construction, and Cufflinks to determine if differential expression and changes in transcript abundance were statistically significant [21, 22]. The RNASeq yielded 55,527,464 reads for the control, 43,263,207 reads for the HP-PRRSV, and 34,555,783 for VR-2332 libraries after quality trimming and excluding any reads less than 25 bp. Cufflinks was used to measure transcript abundances in fragments per kilobase of exon per million fragments mapped (FPKM). The Cuffdiff output contained normalized FPKM for comparison between libraries (Additional file 1). These values were used to calculate the fold change (log2 transformed) in expression between the experimental unit and the control.
Table of transcript counts
Up-regulated genes >3-fold
Down-regulated genes >3-fold
HP-PRRSV rJXwn06 vs. Control
US PRRSV VR-2332 vs. Control
This derived catalog of expressed genes represents the first comparative analysis of the HP-PRRSV rJXwn06 and VR-2332-infected TBLN transcript abundance profiles and provides a database that informs us of genes involved in normal TBLN physiology, as well as genes whose abundance is altered by PRRSV infection.
Annotation of significantly differentially expressed genes
Gene annotation of all significant hits (Additional files 1 and 2) was then carried out using a MySQL database matching the Ensmbl (Sscrofa9.56) chromosome location of aligned transcripts to gene names. Gene IDs and log2 fold-change expression values for significant hits, that had FPKM values in both the control and the infected differential expression testing for transcripts (Cuffdiff output files), were then analyzed using the Ingenuity Pathway Analysis software. When comparing the TBLN transcriptome from sham-inoculated controls vs. the HP-PRRSV rJXwn06-infected pigs, 568 of the 632 gene IDs mapped to the Ingenuity Knowledge Base and 165 were up-regulated while 148 were down-regulated. In the TBLN of control vs. VR-2332-infected pigs, 528 of the 633 gene IDs mapped to the Ingenuity Knowledge Base and only 8 were up-regulated while 235 were down-regulated.
Top Ten HGNC named genes with their fold change obtained from the RNAseq data
US PRRSV VR-2332
Fold change (log2)
Fold change (log2)
This study produced transcriptional profiles of TBLNs from non-infected, HP-PRRSV rJXwn06 and US PRRSV VR-2332-infected pigs that provides insight into immune dysregulation elicited by the virus on host transcript abundance levels necessary for a effective immune response.
This RNA-Seq compendium extends the analyses of previous gene expression atlases performed using Affymetrix GeneChip technology and provides an example of new methods to accommodate the increase in transcriptome data obtained from next generation sequencing [29, 31, 32].
It is well established that many pathogens cause changes in expression of specific genes that act to protect the host and clear the infection. PRRSV strains differ in their dysregulation of the immune response to infection and delay in development of a protective immune response in vaccinated pigs [33, 34]. A higher number of significantly differentially expressed gene instances were detected in HP-PRRSV rJXwn06 than VR-2332 when normalized to control samples at a snapshot of 13 days post inoculation (dpi). As anticipated, some of the genes (e.g., resistin) and pathways identified would be expected to be involved in the host response to a severe disease. In the case of resistin, it would be expected that adipose tissue stores are being mobilized as part of the host response to infection, which includes a high fever typical of infection with HP-PRRSV. There are specific cellular proteins that regulate a protective immune response, for example the pro-inflammatory genes that were up-regulated to a greater extent in HP-PRRSV rJXwn06 than VR-2332 when normalized to control samples as observed when comparing the pathogenicity of HP-PRRSV isolate rJXwn06 with the North American prototype strain VR-2332 PRRSV. At 13 dpi HP-PRRSV rJXwn06 inoculated pigs had an interstitial pneumonia that was significantly more severe than theVR-2332 inoculated group which appeared to be convalescing . Future studies of these differentially expressed genes, their transcript abundance, protein level, and protein function will enhance our understanding of the interaction of PRRSV with the host. Identification of new virulence mechanisms of PRRSV may improve the prospects for rational design of more effective vaccines to limit viral replication and shedding.
Cells and virus
MARC-145 cells were cultured in minimum essential medium (EMEM, SAFC 56416C) with 10% fetal bovine serum at 37°C, 5% CO2. Wild-type (wt) Type 2 PRRSV strain VR-2332 (GenBank U87392), passage 6 on MARC-145 cells, was titrated and used for the swine study. Virus (rescued JXwn06; rJXwn06) was rescued from a cloned cDNA of Chinese highly pathogenic Type 2 PRRSV strain JXwn06 [pWSK-JXwn; GenBank EF641008, ] and passaged 3 times on MARC-145 cells for use in the swine study.
The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the National Animal Disease Center-USDA-Agricultural Research Service. Thirty-two 10-week-old cross-bred pigs were obtained from a U.S. high-health herd and were found to be free of PRRSV and influenza virus antibodies using commercially available enzyme-linked immunosorbent assay (ELISA) kits (HerdChek PRRS 2XR; IDEXX Laboratories, Westbrook, Maine) and NP ELISA (MultiS ELISA, IDEXX, Westbrook, Maine), respectively. Pigs were also confirmed negative for porcine circovirus type 2 by quantitative real-time PCR . One day prior to starting the experiment, pigs were bled, weighed and randomly assigned to one of four groups. Group 1 (N = 8) consisted of negative control pigs, which received an intranasal 2 ml sham inoculum of minimum essential media (MEM) on 0 dpi. Group 2 pigs (N = 12) were challenged intranasally with 2 ml of 1 × 106 50% tissue culture infective dose (TCID50)/ml of Chinese PRRSV strain rJXwn06 in Animal Biosafety Level-3-Agriculture (ABSL-3-Ag) housing, where they remained for the duration of the experiment. Group 3 consisted of naïve pigs (N = 4) that were placed in contact with Group 2 swine on 2 dpi. Group 4 pigs (N = 8) were challenged intranasally with 2 ml of 1 × 106 TCID50/ml of Type 2 prototype strain VR-2332. Groups 1 and 2 were housed in separate isolation rooms in an ABSL2 facility. Animal care and euthanasia were conducted in accordance with the Report of the AVMA Panel on Euthansia and under the supervision of IACUC of NADC. Serum and bronchoalveolar lung lavage fluid (BALF) were tested for infectious virus as described previously . Lungs were scored for gross lesions  and sections fixed for histopathology. Swabs were collected from BALF, and various sites for bacterial isolation .
Following humane euthanasia, tracheobronchial lymph nodes (TBLN) from in vivo HP-PRRSV rJXwn06 (N = 8), US PRRSV VR-2332 (N = 7), or sham-infected pigs (N = 8) were harvested at 13 days post-infection and total cellular RNA was prepared as follows. One gram of TBLN from each pig was collected immediately upon necropsy, minced and stored in RNAlater (Life Technologies, Grand Island, NY) at −80°C until homogenized for extraction of total RNA with MagMAX™-96 for Microarrays Total RNA Isolation Kit (Applied Biosystems, Carlsbad, CA) using the manufacturer's protocol. The integrity of the RNA was confirmed with a 2100 Bioanalyzer and RNA 6000 Nano-chip (Agilent, Santa Clara, CA). The samples used had an average RNA Integrity Number (RIN) value of 7.8 and 28S:18S rRNA ratio of 1.9.
cDNA library construction
cDNA libraries were constructed from pooled total cellular RNA from the TBLN in each treatment group using TruSeq Sample Prep Kits (Illumina Inc., San Diego, CA) and sequenced by 2 × 100 paired-end sequencing on an Illumina HiSeq 2000 instrument.
RNA-Seq (Whole Transcriptome Shotgun Sequencing) pipeline
Ingenuity pathway analysis (IPA)
Datasets representing genes with altered expression profile derived from RNAseq analyses were imported into the Ingenuity Pathway Analysis Tool (IPA Tool; Ingenuity® Systems, Redwood City, CA, USA; http://www.ingenuity.com). In IPA, differentially expressed genes were mapped to genetic networks available in the Ingenuity database and then ranked by score.
The basis of the IPA program consists of the Ingenuity Pathway Knowledge Base (IPKB) that is derived from known functions and interactions of genes published in the literature. Thus, the IPA Tool allows the identification of biological networks, global functions within the host and functional pathways of a particular dataset. The program also gives the significance value of the differentially expressed genes, the other genes with which it interacts, and how the products of the genes directly or indirectly act on each other, including those not involved in the microarray analysis. The networks created are ranked depending on the number of significantly expressed genes they contain and also list diseases that were most significant (Figure 1).
We thank the following members of the Virus and Prion Research Unit at the National Animal Disease Center: J. Huegel, J. Crabtree, A. Burow, D. Adolphson, S. Anderson, M. Kappes and A. Vorwald for technical assistance. We also gratefully acknowledge D. Alt of the Genomics Unit at the National Animal Disease Center, and A. Severin of Iowa State University NGS Bioinformatics, for assistance in data analysis. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
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