Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

USDA, Agricultural Research Service

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen of swine worldwide and causes considerable economic loss. The main target of infection is the porcine alveolar macrophage (PAM). Infection of PAMs by PRRSV causes significant changes in their function by mechanisms that are not understood. We have employed Serial Analysis of Gene Expression (SAGE) to examine the global expression of genes in PRRSV-infected PAMs. Total cellular RNAwas prepared from in vitro mock-infected and PRRSV strain VR-2332-infected PAMs at 0, 6, 12, 16 and 24 hours after infection, and subjected to SAGE analysis to obtain > 100,000 tags per time point. These sequences were processed to account for sequencing error before generating tag:count lists. These lists were deposited into a modified Identitag database for mapping to porcine and PRRSV genes. Identified unique mRNAtags were analyzed for their identity and relative abundance. Examination of the SAGE data indicated that there were changes in gene expression occurring in the PRRSV-infected PAMs over time post-infection. More than 400 unique tags with significantly altered expression levels were identified (p < 0.01 with Bonferroni correction). The validity and kinetics of expression of SAGE identified genes were evaluated using real-time RT-PCR.

Effect of porcine reproductive and respiratory syndrome virus on porcine alveolar macrophage function as determined using serial analysis of gene expression (SAGE).

Control of virus replication initially depends on rapid activation of the innate immune response. Toll-like receptor (TLR) ligands are potent inducers of innate immunity against viral infections. Porcine reproductive and respiratory syndrome virus (PRRSV), a positive-sense RNA virus, initiates infection in porcine alveolar macrophages (PAMs), elicits weak immune responses, and establishes a persistent infection. To understand the role of single-stranded RNA and double-stranded RNA (dsRNA) intermediates in eliciting host immunity, we sought to determine if TLRs, particularly those that respond to viral molecular patterns, are involved in PRRSV infection. Activation of TLR3 in PAMs with dsRNA increased gene expression for alpha interferon and suppressed PRRSV infectivity. In contrast, TLR4 activation by the treatment of PAMs with lipopolysaccharide did not influence PRRSV infectivity.

Role of Toll-like receptors in activation of porcine alveolar macrophages by porcine reproductive and respiratory syndrome virus.

Pseudorabies virus (PRV) is a neurotropic alphaherpesvirus that produces fatal encephalitis in newborn pigs, respiratory disorders in fattening pigs, and reproductive failure in sows. Following primary infection of the respiratory tract, PRV can develop into a systemic infection with dispersion of the virus via the lymphatic system that involves mononuclear cells in tracheobronchial lymph nodes (TBLNs). The objectives of the present study were to evaluate the pathogenesis and to determine the early immune cytokine profiles in TBLNs following experimental infection with a feral swine PRV isolate at 1, 3, 6, and 14 days postinfection (dpi). Forty healthy pigs were purchased from a PRV-negative herd. Twenty pigs received the Florida strain isolate (FS268) of feral swine PRV intranasally, and 20 uninfected controls received a sham inoculum. Compared to the levels in the controls, the levels of alpha interferon (IFN-alpha), interleukin-1beta (IL-1beta), IL-12, and IFN-gamma were increased in TBLN homogenates from PRV-infected pigs at 1 dpi, whereas the IL-18 levels were decreased from 3 to 6 dpi. The protein levels of IL-4 and IL-10 did not differ between the controls and the PRV-infected pigs at any time point. Flow cytometric analysis of TBLN homogenates of PRV-infected pigs and the controls revealed increases in the percentages of B cells at 6 dpi, CD4(+) cells at 14 dpi, and CD25 expression in TBLN homogenates (in the total mononuclear fraction and on B cells) in the PRV-infected pigs. Collectively, these findings demonstrate that a feral PRV in commercial swine can modulate the host's early immune response to allow the virus to establish an infection.

Cytokine protein expression levels in tracheobronchial lymph node homogenates of pigs infected with pseudorabies virus.

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.

Analysis of the swine tracheobronchial lymph node transcriptomic response to infection with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus.

Studies have found that a cluster of duplicated gene loci encoding the interferon-inducible transmembrane proteins (IFITMs) family have antiviral activity against several viruses, including influenza A virus. The gene family has 5 and 7 members in humans and mice, respectively. Here, we confirm the current annotation of pig IFITM1, IFITM2, IFITM3, IFITM5, IFITM1L1 and IFITM1L4, manually annotated IFITM1L2, IFITM1L3, IFITM5L, IFITM3L1 and IFITM3L2, and provide expressed sequence tag (EST) and/or mRNA evidence, not contained with the NCBI Reference Sequence database (RefSeq), for the existence of IFITM6, IFITM7 and a new IFITM1-like (IFITM1LN) gene in pigs. Phylogenic analyses showed seven porcine IFITM genes with highly conserved human/mouse orthologs known to have anti-viral activity. Digital Gene Expression Tag Profiling (DGETP) of swine tracheobronchial lymph nodes (TBLN) of pigs infected with swine influenza virus (SIV), porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus or porcine circovirus type 2 over 14 days post-inoculation (dpi) showed that gene expression abundance differs dramatically among pig IFITM family members, ranging from 0 to over 3000 tags per million. In particular, SIV up-regulated IFITM1 by 5.9 fold at 3 dpi. Bayesian framework further identified pig IFITM1 and IFITM3 as differentially expressed genes in the overall transcriptome analysis. In addition to being a component of protein complexes involved in homotypic adhesion, the IFITM1 is also associated with pathways related to regulation of cell proliferation and IFITM3 is involved in immune responses.

Evolutionary characterization of pig interferon-inducible transmembrane gene family and member expression dynamics in tracheobronchial lymph nodes of pigs infected with swine respiratory disease viruses.

Clinical disease associated with porcine epidemic diarrhea virus (PEDV) infection in naïve pigs is well chronicled; however, information on endemic PEDV infection is limited. To characterize chronic PEDV infection, the duration of infectious virus shedding and development of protective immunity was determined. On Day 0 (D0), a growing pig was challenged with PEDV and 13 contacts were commingled. On D7, 9 contact pigs (principal virus group (PG)), were selected, moved to a separate room and commingled with one sentinel pig (S1). This process was repeated weekly with S2, S3 and S4. The PG was PEDV-positive by PCR from D3-11, with some pigs intermittently positive to D42. Pigs S1 and S2 were PEDV-positive within 24 hours of commingling. Antibodies were detected in all PG by D21 and by 7 days post-contact in S1 and S2. Pigs S3 and S4 were PCR and antibody negative following commingling. To evaluate protective immunity, 5 naïve pigs (N) and the PG were challenged (N/C, PG/C) with homologous virus on D49. All N/C pigs were PEDV PCR-positive by D52 with detection out to D62 in 3/5 N/C pigs. All PG/C pigs were PEDV PCR-negative post-challenge. By D63, all N/C seroconverted. Although PEDV RNA was demonstrated in pigs after primary infection until D42, infectious PEDV capable of horizontal transmission to naïve pigs was only shed 14-16 days after infection to age-matched pigs. Homologous re-challenge 49 days post initial PEDV exposure did not result in re-infection of the pigs. This demonstrates potential for an effective PEDV vaccine. 

Evaluation of porcine epidemic diarrhea virus transmission and the immune response in growing pigs.

In April 2013, a Porcine epidemic diarrhea virus (PEDV) epidemic began in the United States. As part of the response, real-time reverse transcription polymerase chain reaction (RT-PCR) assays to detect PEDV were developed by several veterinary diagnostic laboratories. Our study evaluated RT-PCR PEDV assays that detect the N gene (gN) and S gene (gS) for their ability to detect PEDV infection and the transmission potential of pigs experimentally exposed to PEDV. Detection limits and quantification cycle (Cq) values of real-time RT-PCR were assayed for PEDV samples and positive controls for both gN and gS. The limit of detection for the gN assay was 10(-6) (mean Cq: 39.82 ± 0.30) and 10(-5) (mean Cq: 39.39 ± 0.72) for the gS assay with PEDV strain USA/Colorado/2013. Following recommended guidelines, rectal swabs (n = 1,064) were tested; 354 samples were positive by gN assay and 349 samples were positive by gS assay (Cq ≤ 34.99), 710 samples were negative by gN assay and 715 were negative by gS assay (Cq > 34.99) of which 355 and 344 were "undetermined" (i.e., undetected within a threshold of 40 RT-PCR cycles, by gN and gS assays, respectively). The coefficient of variation (intra-assay variation) ranged from 0.00% to 2.65% and interassay variation had an average of 2.75%. PEDV could be detected in rectal swabs from all pigs for ~2 weeks postinfection at which time the prevalence began to decrease until all pigs were RT-PCR negative by 5 weeks postinfection. Our study demonstrated that RT-PCR assays functioned well to detect PEDV and that the gN assay was slightly better. 

Evaluation of two real-time polymerase chain reaction assays for Porcine epidemic diarrhea virus (PEDV) to assess PEDV transmission in growing pigs.

It has been established that reduced susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV) has a genetic component. This genetic component may take the form of small non-coding RNAs (sncRNA), which are molecules that function as regulators of gene expression. Various sncRNAs have emerged as having an important role in the immune system in humans. The study uses transcriptomic read counts to profile the type and quantity of both well and lesser characterized sncRNAs, such as microRNAs and small nucleolar RNAs to identify and quantify the classes of sncRNA expressed in whole blood between healthy and highly pathogenic PRRSV-infected pigs. Our results returned evidence on nine classes of sncRNA, four of which were consistently statistically significantly different based on Fisher's Exact Test, that can be detected and possibly interrogated for their effect on host dysregulation during PRRSV infections.

Laura C. Miller

Virus and Prion Research Unit,
National Animal Disease Center,
Virus and Prion Research Unit, National Animal Disease Center

USDA, Agricultural Research Service

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

Laura C. Miller

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USDA, Agricultural Research Service

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

Virus and Prion Research Unit,
National Animal Disease Center,
Virus and Prion Research Unit, National Animal Disease Center