Replication and virulence in pigs of the first African swine fever virus isolated in China

African swine fever (ASF) entered China in August 2018 and rapidly spread across the entire country, severely threatening the Chinese domestic pig population, which accounts for more than 50% of the pig population worldwide. In this study, an ASFV isolate, Pig/Heilongjiang/2018 (Pig/HLJ/18), was isolated in primary porcine alveolar macrophages (PAMs) from a pig sample from an ASF outbreak farm. The isolate was characterized by using the haemadsorption (HAD) test, Western blotting and immunofluorescence, and electronic microscopy. Phylogenetic analysis of the viral p72 gene revealed that Pig/HLJ/18 belongs to Genotype II. Infectious titres of virus propagated in primary PAMs and pig marrow macrophages were as high as 107.2 HAD50/ml. Specific-pathogen-free pigs intramuscularly inoculated with different virus dosages at 103.5–106.5 HAD50 showed acute disease with fever and haemorrhagic signs. The incubation periods were 3–5 days for virus-inoculated pigs and 9 days for contact pigs. All virus-inoculated pigs died between 6–9 days post-inoculation (p.i.), and the contact pigs died between 13–14 days post-contact (p.c.). Viremia started on day 2 p.i. in inoculated pigs and on day 9 p.c. in contact pigs. Viral genomic DNA started to be detected from oral and rectal swab samples on 2–5 days p.i. in virus-inoculated pigs, and 6–10 days p.c. in contact pigs. These results indicate that Pig/HLJ/18 is highly virulent and transmissible in domestic pigs. Our study demonstrates the threat of ASFV and emphasizes the need to control and eradicate ASF in China.</p

Glycan binding specificity of H1N1 and H5N1 viruses.

<p>(<b>A</b>) H1N1 human influenza BC/05 virus. (<b>B</b>) DKGX/35 virus. (<b>C</b>) 35/HA-226L/228S. (<b>D</b>) 35/HA-160T.</p

Schematic representation of the negative regulation of ASFV pB318L in the cGAS-STING and JAK-STAT signaling pathways.

Following ASFV infection, host cGAS senses ASFV genomic DNA, which promotes cGAMP production. cGAMP binds to STING to promote STING activation and translocation from the ER to the Golgi apparatus. Active TBK1 phosphorylates IRF3, promotes the translocation of IRF3 to the nucleus, and initiates the transcription of IFN-I. Secreted IFN-I binds to IFNAR1 and IFNAR2, resulting in activation of JAK1 and TYK2 kinases. The phosphorylated STAT1/2 and IRF9 form a heterotrimeric complex ISGF3. Subsequently, ISGF3 enters the nucleus to bind ISRE to regulate the transcription of ISGs. ASFV pB318L interacts with STING and inhibits the transfer of STING from the endoplasmic reticulum to the Golgi apparatus, thereby inhibiting IFN-I production. On the other side, pB318L binds to IFNAR1/IFNAR2 and blocks the phosphorylation of TYK2 and JAK1, thereby negatively inhibiting IFN-mediated ISGs production. (TIF)</p

Asp129 of ASFV pB318L affects its inhibition of STING phosphorylation and translocation.

(A) HEK293T cells were transfected with an IFN-β luciferase reporter, a Renilla-TK reporter, and plasmids expressing HA-cGAS and HA-STING, together with a plasmid expressing Flag-pB318L. After 24 h, the cells were treated with Lovastatin, Lonfarnib, GGTI-286 for 12 h, then the luciferase activities were detected. (B) HEK293T cells were transfected with an IFN-β luciferase reporter, a Renilla-TK reporter, and plasmids expressing HA-cGAS and HA-STING, together with increased amount (100 ng, 200 ng, 400 ng) of a plasmid expressing Flag-pB318L-WT or Flag-pB318L-Mut, the luciferase activities were detected after 24 h. (C-D) HEK293T cells were transfected with plasmids expressing HA-cGAS and HA-STING, together with increase amount (100 ng, 200 ng, 400 ng) of a plasmid expressing Flag-pB318L-WT (100 ng, 200 ng, 400 ng) or Flag-pB318L-Mut, the mRNA levels of Ifnb1 and Isg56 were analyzed by qPCR. (E) HEK293T cells were transfected with plasmids expressing Flag-pB318L-D129A and HA-STING. Co-IP analysis was performed to detect the interaction between pB318L-D129A and STING after 24 h. (F-G) CRL2843 cells were transfected with plasmids expressing HA-STING and GFP-pB318L or GFP-pB318L-D129A as indicated. At 24 hpt, the cells were stimulated with cGAMP (10 μg/mL) for another 12 h. The subcellular localization of STING was visualized by immunofluorescence microscopy. Scale bars, 20 μm (F). The fluorescence intensity of STING was analyzed using the Zeiss processing system (G). (H-I) HeLa cells were transfected with a plasmid expressing Flag-pB318L or Flag-pB318L-D129A for 24 h, and then treated with the STING agonist for another 6 h. The cells were collected and lysed, and the phosphorylation of STING was detected by Western blotting (H). Quantitation of p-STING/STING ratio was analyzed with Image J (I). Data are representative of three independent experiments with three biological replicates (mean ± s.d.). Ns, not significantly, ** p < 0.01, *** p < 0.001 (one-way ANOVA).</p

Replication of H5N1 avian influenza viruses in guinea pigs.

a<p>Data shown are summarized from previous reports <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000709#ppat.1000709-Chen1" target="_blank">[2]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000709#ppat.1000709-Chen2" target="_blank">[7]</a>. Six-week-old BALB/c mice were infected i.n. with 10⁶ EID₅₀ of each virus in a 50-µl volume. Organs were collected on day 3 p.i., and clarified homogenates were titrated for virus infectivity in eggs at initial dilutions of 1∶10 (lung), 1∶2 (other tissues), or undiluted if negative at the lowest dilution. + and −, virus was detected or not detected, respectively, in the undiluted samples.</p>b<p>Groups of four guinea pigs were slightly anesthetized and intranasally inoculated with 10⁶EID₅₀ of test virus in a 300µl volume, 150 µl per nostril. Two animals from each group were euthanized on day 3 p.i. and samples, including nasal wash, trachea, lung, spleen, kidney, colon and brain, were collected for virus titration in eggs. The remaining two animals were observed for two weeks and sera were collected at the end of the observation period. Virus was not detected in the spleen, kidney, colon and brain of any animals inoculated with the six viruses, therefore, the data from these samples are not shown in the table. −, virus was not detected in the undiluted sample.</p>c<p>Data shown are log₁₀EID₅₀/ml.</p>d<p>Virus was only detected in one of the two animals inoculated.</p>e<p>Seroconversion was confirmed by hemagglutination inhibition (HI) assay.</p

ASFV pB318L reduces STING-mediated IFN production.

(A-C) HEK293T cells were transfected with an IFN-β luciferase reporter, a Renilla-TK reporter, a plasmid expressing HA-STING (A), HA-TBK1 (B), or HA-IRF3-5D (C), together with increase amounts (0, 100, 200, and 400 ng) of a plasmid expressing Flag-pB318L. The luciferase activities were detected after 24 h. (D-F) HEK293T cells were transfected with a plasmid expressing HA-STING (D), HA-TBK1 (E), or HA-IRF3-5D (F), together with increased amounts (0, 100, 200, and 400 ng) of a plasmid expressing Flag-pB318L. The mRNA levels of Ifnb1 were analyzed by qPCR. The expressions of STING, TBK1, IRF3-5D, pB318L, and GAPDH were detected by Western blotting. Data are representative of three independent experiments with three biological replicates (mean ± s.d.). Ns, not significantly, *** p < 0.001 (one-way ANOVA).</p

Replication and transmission of DKGX/35 HA mutants.

<p>(<b>A</b>) Groups of two guinea pigs were inoculated i.n. with 10⁶EID₅₀ of test virus and euthanized on day 3 p.i. Organs were collected for virus titration in eggs. (<b>B</b>) Transmission of 35/HA-226L/228S in guinea pigs. (<b>C</b>) Transmission of 35/HA-160T in guinea pigs. The dashed blue lines in these panels indicate the lower limit of detection.</p

Primers used to generate mutations in the HA gene of our H5N1 influenza viruses.

<p>The nucleotides that have been changed are underlined and in boldface.</p

Replication and transmission of DKGX/35 PB2 mutants.

<p>(<b>A</b>) Groups of two guinea pigs were inoculated i.n. with 10⁶EID₅₀ of test virus and then euthanized on day 3 p.i. Organs were collected for virus titration in eggs. (<b>B</b>) Transmission of the 22/PB2-701N virus in guinea pigs. (<b>C</b>) Transmission of the 35/PB2-701D virus in guinea pigs. The dashed blue lines in these panels indicate the lower limit of detection.</p

Receptor-binding preference and transmission of BHGQH/3 and its HA mutant.

<p>Receptor-binding preference of r-BHGQH/3 (<b>A</b>) and BHGQH/3-160T (<b>B</b>) were performed by dose-dependent direct binding assay as described in the text. (<b>C</b>) and (<b>D</b>) Transmisson of H5N1 duck viruses in guinea pigs. (C) r-BHGQH/3-inoculated group. (D) BHGQH/3-160T-inoculated group. The dashed blue lines in these panels indicate the lower limit of detection.</p