African Swine Fever Virus and Host Response: Transcriptome Profiling of the Georgia 2007/1 Strain and Porcine Macrophages

Abstract

African swine fever virus (ASFV) has a major global economic impact. With a case fatality in domestic pigs approaching 100%, it currently presents the largest threat to animal farming. Although genomic differences between attenuated and highly virulent ASFV strains have been identified, the molecular determinants for virulence at the level of gene expression have remained opaque. Here we characterise the transcriptome of ASFV genotype II Georgia 2007/1 (GRG) during infection of the physiologically relevant host cells, porcine macrophages. In this study we applied Cap Analysis Gene Expression sequencing (CAGE-seq) to map the 5’ ends of viral mRNAs at 5 and 16 hours post-infection. A bioinformatics analysis of the sequence context surrounding the transcription start sites (TSSs) enabled us to characterise the global early and late promoter landscape of GRG. We compared transcriptome maps of the GRG isolate and the lab-attenuated BA71V strain that highlighted GRG virulent-specific transcripts belonging to multigene families, including two predicted MGF 100 genes I7L and I8L. In parallel, we monitored transcriptome changes in the infected host macrophage cells. Of the 9,384 macrophage genes studied, transcripts for 652 host genes were differentially regulated between 5 and 16 hours-post-infection compared with only 25 between uninfected cells and 5 hours post-infection. NF-kB activated genes and lysosome components like S100 were upregulated, and chemokines such as CCL24, CXCL2, CXCL5 and CXCL8 downregulated. African swine fever virus (ASFV) causes haemorrhagic fever in domestic pigs with case fatality rates approaching 100%, and no approved vaccines or antivirals. The highly-virulent ASFV Georgia 2007/1 strain (GRG) was the first isolated when ASFV spread from Africa to the Caucasus region in 2007. Then spreading through Eastern Europe, and more recently across Asia. We used an RNA-based next generation sequencing technique called CAGE-seq to map the starts of viral genes across the GRG DNA genome. This has allowed us to investigate which viral genes are expressed during early or late stages of infection and how this is controlled, comparing their expression to the non-virulent ASFV-BA71V strain to identify key genes that play a role in virulence. In parallel we investigated how host cells respond to infection, which revealed how the ASFV suppresses components of the host immune response to ultimately win the arms race against its porcine host.

Experimental Infections of Pigs with African Swine Fever Virus (Genotype II); Studies in Young Animals and Pregnant Sows

“…The size of the viral genome varies between strains and is about 170–190 kbp in length. Approaching 200 genes are closely spaced along the genome and are transcribed from the two different strands of the genome [ 10 , 11 , 12 , 13 ]. The functions of many of these genes are currently unknown.…”

Transcriptome profile of spleen tissues from locally-adapted Kenyan pigs (Sus scrofa) experimentally infected with three varying doses of a highly virulent African swine fever virus genotype IX isolate: Ken12/busia.1 (ken-1033)

“…Late transcription gene. [ 97 , 98 ] C84L −2.74 7.99E-03 −2.82 8.18E-03 TND TND Uncharacterised protein [ 99 ] CP204L (p30) 2.23 4.80E-02 TND TND TND TND Plays a role in virus cell tropism, and is essential for effective virus entry and replication in macrophages. [ 10 ] E184L (j12L) 2.13 4.80E-02 2.25 4.76E-02 TND TND Uncharacterised protein EP402R (CD2v) 2.08 4.81E-02 2.28 4.76E-02 TND TND Similar to host CD2 protein, needed for binding red blood cells to infected cells and extracellular virus particles; responsible for heamadsorption in infected cells; glycoprotein inserted into external virus envelope [ 100 ] I177L (k14L) 2.13 4.80E-02 2.61 3.54E-02 TND TND Uncharacterised protein [ 100 ] I196L (k15L) 2.15 4.80E-02 2.37 4.72E-02 TND TND Uncharacterised protein [ 100 ] I215L (k13L) TND TND 1.51 4.76E-02 TND TND Ubiquitin-conjugating enzyme [ 100 ] I73R (k10R) 2.24 4.80E-02 2.59 3.54E-02 TND TND Uncharacterised protein [ …”

Temporally integrated transcriptome analysis reveals ASFV pathology and host response dynamics

“…RNA sequencing (RNA-seq) has been applied to study various biological processes, such as revealing the interaction of virus infection and host response (10,13,50). However, studies using RNAseq to preform transcriptomic profiling of ASFV and infected host cells are scarce, and these studies target a single strain or time point and do not provide a comprehensive picture of host-virus interactions (8,(14)(15)(16)(17). Here, we integrated RNA-seq analysis to examine and compare the transcriptomic landscape of porcine PAMs during infection with highly (SY18) and low virulent (HuB20) ASFV strains at different stages of infection, depicting unprecedented details about the temporal host response after ASFV infection.…”

“…RNA sequencing (RNA-Seq) is a high-throughput experiment that can be applied to profile the transcriptome of host and virus during infection (10)(11)(12)(13). Using RNA-seq, researchers quantified gene expression levels in Vero cells infected with ASFV-BA71V at early (5 hour) and late (16 hour) stages, providing insights into the temporal expression of known and novel viral genes (14). However, the use of non-ASFV targeted cells is suboptimal and may introduce bias.…”