Deletion of the H240R Gene of African Swine Fever Virus Decreases Infectious Progeny Virus Production Due to Aberrant Virion Morphogenesis and Enhances Inflammatory Cytokine Expression in Porcine Macrophages

FIG 1. Biological characteristics of the H240R protein (pH240R). (A) Multiple sequence alignment of pH240R of 21 ASFV isolates. To assess the nature of the replacements at multiple residues, conservation scores based on the biological properties of each amino acid were included, with lower scores associated with more divergent replacements. Analysis was conducted on Jalview software version 2.11.1.4, using the ClustalW algorithm. (B) H240R gene transcriptional dynamics. Average cycle threshold values of the ASFV HLJ/2018-infected primary porcine alveolar macrophages (MOI of 5) at 1, 4, 7, 10, and 24 hpi were examined by quantitative reverse transcription-PCR analyses using primers against H240R, CP204L, B646L, and GAPDH.

FIG 2. The H240R protein (pH240R) was identified as a capsid protein of ASFV. (A) Intracellular localization of pH240R. HEK293T cells were transfected with plasmid pMyc-pH240R (1 μg). After 24 hpi, the cells were fixed and probed with a mouse MAb and the nuclear marker DAPI and then detected by confocal microscopy. Bars, 10 μm. (B) Subcellular localization of pH240R. HEK293T cells infected with HEK293T-adapted ASFV (ASFV-P31) were immobilized at 18 hpi and immunolabelled with rabbit anti-pH240R pAb, anti-p72 Mab, and DAPI. Bars, 2 μm. (C) Subviral localization of pH240R. ASFV HLJ/2018-infected primary porcine alveolar macrophages were fixed at 18 hpi and immunolabeled with anti-pH240R PAb, followed by conjugation to 10-nm-diameter gold particles. The arrows indicate that gold particles presenting on intracellular viruses were associated with the outer protein capsid (solid lines) outside of the inner envelope (dotted lines). (D) Identification of the interaction between pH240R and p72. To screen the pH240R-interacting protein(s) related to ASFV morphogenesis, HEK293T cells were transfected with plasmids pFlag-p17, pFlag-pE248R, pFlag-pCP312R, pFlag-pB438L, and pFlag-p72 (6 μg each),…

FIG 3. ASFV-ΔH240R showed growth defect but retained hemadsorption characteristics in primary porcine alveolar macrophages (PAMs). (A) Schematic diagram of the genome structure of the gene-deleted ASFV HLJ/2018 (ASFV-ΔH240R). The recombinant transfer vector containing the left arm, EGFP marker, and right arm was designed to sequentially delete H240R at nucleotides 155339 to 156064 of the ASFV-WT genome. ASFV-ΔH240R was finally purified by limiting dilution in 96-well plates based on the EGFP signal. (B) ASFV-ΔH240R was amplified in PAMs for downstream evaluation. (C) Identification of the H240R gene deletion in ASFV-ΔH240R. H240R was examined by PCR in both ASFV-ΔH240R (lane 1) and ASFV HLJ/2018 (ASFV-WT) (lane 3) using primers d/H240R-F/R, and the p72EGFP gene was examined by PCR in both ASFV-ΔH240R (lane 2) and ASFV-WT (lane 4) using primers 72EGFP-F/R. NC, negative control. (D and E) In vitro replication characteristics of ASFV-ΔH240R or ASFV-WT. PAMs were infected at an MOI of 0.01 (D) or 3 (E) with ASFV-ΔH240R or ASFV-WT. The cell cultures were collected at each time point, and growth curves were constructed by using the titer of each virus at each time point. The data represent…

FIG 4. ASFV-ΔH240R shows decreased viral production but normal genome replication in primary porcine alveolar macrophages (PAMs). (A and B) Intracellular (A) and extracellular (B) genome copies of ASFV-ΔH240R or ASFV HLJ/2018 (ASFV-WT). PAMs in 24-well plates were infected with either ASFV-ΔH240R or ASFV-WT at an MOI of 3. At the indicated hpi, the numbers of genome copies in the cell lysates or supernatants of infected PAMs were determined by quantitative PCR (qPCR). (C and D) Dynamics of intracellular or extracellular ASFV replication. The intracellular (C) or extracellular (D) K value represents the proliferation curve of the virus at each time point and is calculated by dividing the number of virus genome copies at each time point by that at 2 h. The viral proliferation curves were generated using the virus genome copies at 2, 6, 12, 18, 24, and 30 hpi. (E) Genome copies of progeny ASFV-ΔH240R or ASFV-WT. PAMs in 24-well plates were infected with either ASFV-ΔH240R or ASFV-WT at genome copies of 107. At the indicated hpi, the numbers of genome copies in supernatants were determined by qPCR. (F) Infectious progeny virus production of ASFV-ΔH240R or ASFV-WT. The titers of infectious…

FIG 5. Deletion of the H240R gene from ASFV causes aberrant ASFV virion morphogenesis. (A) Transmission electron microscopy (TEM) analysis of ASFV HLJ/2018 (ASFV-WT)- or ASFV-ΔH240R-infected primary porcine alveolar macrophages (PAMs) at 24 hpi. The assembly sites contain amounts of precursor viral membranes, as well as immature and mature virions in ASFV-WT-infected PAMs (white arrows). The factories contain, in addition to precursor membranes as well as a small number of mature virions (white arrows), large numbers of aberrant tubular (red arrows) and bilobulate (blue arrows) structures in the ASFV-ΔH240R-infected PAMs. (B) Ultrathin sections of ASFV-WT- or ASFV-ΔH240R-infected PAMs are shown at 48 hpi. The morphologies of virions in the viral factories from PAMs infected with ASFV-WT or ASFV-ΔH240R are consistent with those at 24 hpi; the difference is that the number of normal structures in the ASFV-WT-infected PAMs (white arrows) or aberrant structures in ASFV-ΔH240R-infected PAMs (red arrows) increased.

FIG 6. ASFV-ΔH240R displays aberrant virion structures. (A) Immunoelectron microscopy (IEM) analysis of ASFV HLJ/2018 (ASFV-WT)- or ASFV-ΔH240R-infected primary porcine alveolar macrophages (PAMs). The ASFV-infected PAMs were fixed and prepared for sectioning at 24 hpi. Sections were incubated with anti-p72 antibodies, followed by incubation with a protein A-gold conjugate (10 nm). The black arrows indicate labeling in the icosahedral capsid, while the red arrows indicate representative labeling observed with antibodies against the capsid proteins in the aberrant structures. (B) Higher magnifications of normal or aberrant structures. The outer electron-dense layers of these structures containing tubules and bilobular structures are indicated by red arrows, while those layers resembling the capsid of a normally assembling particle are indicated by black arrows. (C) The aberrant-to-normal particle ratios of ASFV HLJ/2018 (ASFV-WT) or ASFV-ΔH240R. ASFV-WT- or ASFV-ΔH240R-infected PAMs were examined ultrastructurally at 24 and 48 hpi. Aberrant or normal ASFVs were calculated from 30 infected PAMs. Error bars denote standard errors of the means. The significance of differences between groups…

FIG 7. The H240R protein is not required for ASFV binding to or entry into PAMs. (A) Genome copies of ASFV-ΔH240R or ASFV HLJ/2018 (ASFV-WT) per 105 and 106 HAD50. Genome copies were determined from 105 and 106 HAD50 ASFV-ΔH240R or ASFV-WT by quantitative PCR (qPCR) based on B646L. (B) ASFV-ΔH240R or ASFV-WT particle-to-infectious virus ratios. The particle-to-infectious virus ratios of ASFV-ΔH240R are nearly 50 times higher than those of ASFV-WT based on samples from panel A. (C and E) The attachment levels of ASFV-ΔH240R or ASFV-WT were similar. Equal numbers of genome copies (105, 106, and 107) (C) or equal titers (105 and 106) (E) of ASFV-ΔH240R or ASFV-WT were added to PAMs at 4°C and allowed to attach for 1 h. The numbers of genome copies of attached ASFVs were quantified by qPCR. (G) The fold change in attached ASFV-ΔH240R was nearly 50 times higher than that of ASFV-WT with equal titers (E). (D and F) The internalization levels of ASFV-ΔH240R and ASFV-WT were similar. Equal numbers of genome copies (105, 106, and 107) (D) or equal titers (105 and 106) (F) of ASFV-ΔH240R or ASFV-WT were added to PAMs at 37°C and allowed to internalize for 2 h. The genome copies of internalized…

FIG 8. Gene expression profiling in the ASFV-ΔH240R-infected primary porcine alveolar macrophages (PAMs) by RNA-sequencing analysis. (A) Hierarchical clustering analysis of differentially expressed genes (DEGs) in the groups of ASFV-ΔH240R versus ASFV HLJ/2018 (ASFV-WT). PAMs were mock infected or infected with ASFV-ΔH240R or ASFV-WT for 4, 12, and 20 h. Total RNA was extracted from the PAMs and sequenced using the Illumina HiSeq 2500 platform. Each group of DEGs and genes of interest is shown on the right. (B) Venn diagrams of mRNAs in different groups. The overlapping section represents the number of common mRNAs in each group, while the nonoverlapping sections represent the numbers of group-specific mRNAs. (C) Top 20 pathways from KEGG enrichment analysis of 3,002 upregulated genes. The TNF signaling pathway is indicated by a red arrow. (D) Analysis of the 46 upregulated genes in the TNF signaling pathway induced by ASFV-ΔH240R versus ASFV-WT in the three groups. Asterisks indicate upregulated expression of target genes.

FIG 9. ASFV-ΔH240R induces higher cytokine production than ASFV-WT. (A to G) Primary porcine alveolar macrophages (PAMs) were either mock infected or infected with ASFV-ΔH240R or ASFV HLJ/2018 (ASFV-WT) at equal numbers of genome copies of 107. At 6, 10, 15, or 24 hpi, the expression levels of TNF-α (A) and IL-1β (C) in the cell culture supernatants were detected by enzyme-linked immunosorbent assay kits, and the mRNA levels of TNF-α (B), IL-1β (D), IL-6 (E), CXCL8 (F), and IL-10 (G) in the cell lysates were determined by quantitative RT-PCR. (H) Cell viability was analyzed using a CCK-8 counting kit. (I) The genome copies of ASFV were measured by quantitative PCR. Error bars denote standard errors of the means. The significance of differences between groups (n = 3) was determined using Student's t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). ns, not significant.