Abstract
BackgroundTranscriptomic approaches are relevant for studying virus-host cell dialogues to better understand the physiopathology of infection and the immune response at the cellular level. Pseudorabies virus (PrV), a porcine Alphaherpesvirus, is a good model for such studies in pig. Since PrV displays a strong tropism for mucous epithelial cells, we developed a kinetics study of PrV infection in the porcine PK15 epithelial cell line. To identify as completely as possible, viral and cellular genes regulated during infection, we simultaneously analyzed PrV and cellular transcriptome modifications using two microarrays i.e. a laboratory-made combined SLA/PrV microarray, consisting of probes for all PrV genes and for porcine genes contained in the Swine Leukocyte Antigen (SLA) complex, and the porcine generic Qiagen-NRSP8 oligonucleotide microarray. We confirmed the differential expression of a selected set of genes by qRT-PCR and flow cytometry.ResultsAn increase in the number of differentially expressed cellular genes and PrV genes especially from 4 h post-infection (pi) was observed concomitantly with the onset of viral progeny while no early global cellular shutoff was recorded. Many cellular genes were down-regulated from 4 h pi and their number increased until 12 h pi. UL41 transcripts encoding the virion host shutoff protein were first detected as differentially expressed at 8 h pi. The viral gene UL49.5 encoding a TAP inhibitor protein was differentially expressed as soon as 2 h pi, indicating that viral evasion via TAP inhibition may start earlier than the cellular gene shutoff. We found that many biological processes are altered during PrV infection. Indeed, several genes involved in the SLA class I antigenic presentation pathway (SLA-Ia, TAP1, TAP2, PSMB8 and PSMB9), were down-regulated, thus contributing to viral immune escape from this pathway and other genes involved in apoptosis, nucleic acid metabolism, cytoskeleton signaling as well as interferon-mediated antiviral response were also modulated during PrV infection.ConclusionOur results show that the gene expression of both PrV and porcine cells can be analyzed simultaneously with microarrays, providing a chronology of PrV gene transcription, which has never been described before, and a global picture of transcription with a direct temporal link between viral and host gene expression.
Highlights
Transcriptomic approaches are relevant for studying virus-host cell dialogues to better understand the physiopathology of infection and the immune response at the cellular level
Construction of the Swine Leukocyte Antigen (SLA)/Pseudorabies virus (PrV) microarray and complementarity with the Qiagen-NRSP8 microarray The 1789 DNA/cDNA probes spotted on the SLA/PrV microarray fall into four distinct probe sets: i) 420 probes localized on a segment of chromosome 7 between the loci PRL and PRIM2A (SSC7p1.1-q1.2), which includes the extended-SLA region and represents 272 unique sequences, 111 belonging to the strict SLA region between
(page number not for citation purposes) http://www.biomedcentral.com/1471-2164/9/123 the loci UBD and RING1 [16]; ii) 73 probes specific to 73 genes encoding molecules involved in immunity and localized outside the SLA region [18]; iii) 80 PrV probes specific to the 70 viral genes (Figure 1) and iv) 1170 probes randomly chosen for data normalization from porcine cDNA AGENAE library [19,20]
Summary
Transcriptomic approaches are relevant for studying virus-host cell dialogues to better understand the physiopathology of infection and the immune response at the cellular level. Pseudorabies virus (PrV), a porcine Alphaherpesvirus, is a good model for such studies in pig. Since PrV displays a strong tropism for mucous epithelial cells, we developed a kinetics study of PrV infection in the porcine PK15 epithelial cell line. Young piglets are more severely affected by PrV infection often resulting in fatal encephalitis, than older infected pigs, which can remain asymptomatic or develop mild to severe respiratory disease symptoms associated with a limited mortality. Because PrV is easy to propagate in cells of several mammalian species including rodents and is not harmful to laboratory workers, PrV is a highly relevant model to study the biology of alphaherpesviruses and their interactions with host cells in vitro [1]. Its genome has been reconstructed from sequences of six different strains (Kaplan, Becker, Rice, Indiana-Funkhauser, NIA-3, and TNL) and 70 genes encoding structural and non structural proteins have been annotated [4]
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