Genetic Characterization and Variation of African Swine Fever Virus China/GD/2019 Strain in Domestic Pigs

African swine fever (ASF) was first introduced into Northern China in 2018 and has spread through China since then. Here, we extracted the viral DNA from the blood samples from an ASF outbreak farm in Guangdong province, China and sequenced the whole genome. We assembled the full length genomic sequence of this strain, named China/GD/2019. The whole genome was 188,642 bp long (terminal inverted repeats and loops were not sequenced), encoding 175 open reading frames (ORF). The China/GD/2019 strain belonged to p72 genotype II and p54 genotype IIa. Phylogenetic analysis relationships based on single nucleotide polymorphisms (SNPs) also demonstrated that it grouped into genotype II. A certain number of ORFs mainly belonging to multigene families (MGFs) were absent in the China/GD/2019 strain in comparison to the China/ASFV/SY-18 strain. A deletion of approximately 1 kb was found in the China/GD/2019 genome which was located at the EP153R and EP402R genes in comparison to the China/2018/AnhuiXCGQ strain. We revealed a synonymous mutation site at gene F317L and a non-synonymous mutation site at gene MGF_360-6L in China/GD/2019 comparing to three known Chinese strains. Pair-wise comparison revealed 165 SNP sites in MGF_360-1L between Estonia 2014 and the China/GD/2019 strain. Comparing to China/GD/2019, we revealed a base deletion located at gene D1133L in China/Pig/HLJ/2018 and China/DB/LN/2018, which results in a frameshift mutation to alter the encoding protein. Our findings indicate that China/GD/2019 is a new variant with certain deletions and mutations. This study deepens our understanding of the genomic diversity and genetic variation of ASFV.

Stem cell-derived porcine macrophages as a new platform for studying host-pathogen interactions

Background Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. Results We explored how porcine pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages. Porcine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages and were productively infected by pig pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, porcine PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied either in parental stem cells or directly in the macrophages by lentiviral vector transduction. Conclusions We show that porcine PSCdMs exhibit key macrophage characteristics, including infection by a range of commercially relevant pig pathogens. In addition, genetic engineering of PSCs and PSCdMs affords new opportunities for functional analysis of macrophage biology in an important livestock species. PSCs and differentiated derivatives should therefore represent a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in pigs, and also have wider applications in livestock.

Development of an Indirect ELISA to Detect African Swine Fever Virus pp62 Protein-Specific Antibodies

African swine fever (ASF) is a highly detrimental viral disease caused by African swine fever virus (ASFV). The occurrence and prevalence of this disease have become a serious threat to the global swine industry and national economies. At present, the detection volume of African swine fever is huge, more sensitive and accurate detection techniques are needed for the market. pp62 protein, as a protein in the late stage of infection, has strong antigenicity and a high corresponding antibody titer in infected pigs. In this study, the CP530R gene was cloned into expression vector pET-28a to construct a prokaryotic expression plasmid, which was induced by IPTG to express soluble pp62 protein. Western blot analysis showed that it had great reactivity. Using the purified recombinant protein as an antigen, an indirect ELISA method for detecting ASFV antibody was established. The method was specific only to ASFV-positive serum, 1:1600 diluted positive serum could still be detected, and the coefficients of variation (CV) of the intra assay and inter assay were both <10%. It turns out that the assays had excellent specificity, sensitivity, and repeatability. This provides an accurate, rapid, and economical method for the detection of ASFV antibody in clinical pig serum samples.

African Swine Fever Virus MGF360-14L Negatively Regulates Type I Interferon Signaling by Targeting IRF3

African swine fever (ASF) is a devastating infectious disease caused by African swine fever virus (ASFV). The ASFV genome encodes multiple structural and non-structural proteins that contribute to evasion of host immunity. In this study, we determined that the viral non-structural protein MGF360-14L inhibits interferon-β (IFN-β) promoter activity induced by cGAS-STING signaling. MGF360-14L was also found to downregulate expression of the IRF3 protein and promote its degradation through ubiquitin-meditated proteolysis. Moreover, MGF360-14L was shown to interact with and destabilize IRF3 by facilitating E3 ligase TRIM21-mediated K63-linked ubiquitination of IRF3. Overall, our study revealed that MGF360-14L promotes degradation of IRF3 through TRIM21, thereby inhibiting type I interferon production. These findings provide new insights into the mechanisms underlying ASFV immune evasion.

Efficient inactivation of African swine fever virus by a highly complexed iodine combined with compound organic acids

African swine fever (ASF) is a highly contagious disease with high morbidity and mortality caused by African swine fever virus (ASFV). Cleaning and disinfection remain one of the most effective biosecurity measures to prevent and control the spread of ASFV. In this study, we evaluated the inactivation effects of highly complexed iodine (HPCI) combined with compound organic acids (COAs) against ASFV under different conditions. The results showed that the inactivation rates of the disinfectants on the reporter ASFV increased in dose- and time-dependent manners, the best inactivation effects were obtained when the compatibility ratio of HPCI and COAs was 5:1 at 25°C. Furthermore, there were no significant differences by comparing the efficacy of HPCI combined with COAs (HPCI+COAs) in inactivating wild-type ASFV and the reporter ASFV (P > 0.05). ASFV of 104.0 TCID50/mL was completely inactivated by 0.13% HPCI (0.0065% effective iodine), 0.06% COAs or 0.13% HPCI+COAs (approximately 0.0054% effective iodine), respectively, while 106.0 TCID50/mL ASFV was completely inactivated by 1.00% HPCI (0.05% effective iodine), 0.50% COAs or 1.00% HPCI+COAs (0.042% effective iodine), respectively. Therefore, HPCI+COAs had synergistic effects to inactivate ASFV. This study demonstrated that HPCI+COAs could rapidly and efficiently inactivate ASFV and represent an effective compound disinfectant for the control of ASF.

Production and application of mouse monoclonal antibodies targeting linear epitopes in pB602L of African swine fever virus

African swine fever (ASF) is an acute hemorrhagic disease of domestic pigs. The causative agent of ASF, ASF virus (ASFV), is a double-stranded DNA virus, the sole member in the family Asfarviridae. The non-structural protein pB602L of ASFV is a molecular chaperone of the major capsid protein p72 and plays a key role in icosahedral capsid assembly. This protein is antigenic and is a target for developing diagnostic tools for ASF. To generate monoclonal antibodies (mAbs) against pB602L, a prokaryotically expressed recombinant pB602L protein was produced, purified, and used as an antigen to immunize mice. A total of eight mouse mAbs were obtained, and their binding epitopes were screened by Western blot using an overlapping set of polypeptides from pB602L. Three linear epitopes were identified and designated epitope 1 (366ANRERYNY373), epitope 2 (415GPDAPGLSI423), and epitope 3 (498EMLNVPDD505). Based on the epitope recognized, the eight mAbs were placed into three groups: group 1 (B2A1, B2F1, and B2D10), group 2 (B2H10, B2B2, B2D8, and B2A3), and group 3 (B2E12). The mAbs B2A1, B2H10, and B2E12, each representing one of the groups, were used to detect pB602L in ASFV-infected porcine alveolar macrophages (PAMs) and pig tissues, using an indirect fluorescence assay (IFA) and immunohistochemical staining, respectively. The results showed that pB602L was detectable with all three mAbs in immunohistochemical staining, but only B2H10 was suitable for detecting the proteins in ASFV-infected PAMs by IFA. In summary, we developed eight anti-pB602L mouse mAbs recognizing three linear epitopes in the protein, which can be used as reagents for basic and applied research on ASFV.

Lateral Flow Assay for the Detection of African Swine Fever Virus Antibodies Using Gold Nanoparticle-Labeled Acid-Treated p72

African swine fever is a widespread and highly contagious disease in the porcine population, which is caused by African swine fever virus (ASFV). The PCR and ELISA detection methods are the main conventional diagnostic methods for ASFV antigen/antibody detection in the field. However, these methods have limitations of expensive equipment, trained technicians, and time-consuming results. Thus, a rapid, inexpensive, accurate and on-site detection method is urgently needed. Here we describe a double-antigen-sandwich lateral-flow assay based on gold nanoparticle-conjugated ASFV major capsid protein p72, which can detect ASFV antibody in serum samples with high sensitivity and specificity in 10 min and the results can be determined by naked eyes. A lateral flow assay was established by using yeast-expressed and acid-treated ASFV p72 conjugated with gold nanoparticles, which are synthesized by seeding method. A high coincidence (97.8%) of the assay was determined using clinical serum compared to a commercial ELISA kit. In addition, our lateral flow strip can detect as far as 1:10,000 diluted clinically positive serum for demonstration of high sensitivity. In summary, the assay developed here was shown to be rapid, inexpensive, accurate and highly selective. It represents a reliable method for on-site ASFV antibody detection and may help to control the ASFV pandemic.

African Swine Fever: Prevalence, Farm Characteristics, Farmer’s Insight and Attitude toward Reporting of African Swine Fever Cases in the Northwest, West, Littoral and Southwest Regions of Cameroon

The African swine fever virus (ASFV) has been circulating in and ravaging the swine industry in Cameroon for decades. Annual ASFV epidemics occur in Cameroon from April to August. With the absence of vaccines and antiviral drugs against this virus, biosecurity has been the only effective control tool available. When properly applied, biosecurity measures allow control of the spread of ASFV and the eventual eradication of this virus. Many outbreak investigations by PCR were effected in Cameroon, with ASFV prevalences ranging from 15.23% to 42.80%. Considering that pre-outbreak studies are not available for Cameroon, the present study aimed at assessing the status of the animals before an outbreak. A two-stage cluster sampling study was conducted from January to March 2020. In this study, the primary unit was the farm and the secondary unit was the individual animals. In all, 97 farms were visited and 277 samples were collected. Pre-outbreak ASFV prevalence, as assessed by PCR, was 9.75%. In parallel, data were collected using a survey of farm characteristics, awareness and attitude of the farmers toward ASF. The survey results showed that 34.1% of the farms were backyard cemented piggeries with the majority having less than 10 pigs (54.1%). The majority of farmers (90.6%) had heard of and knew about the disease caused by ASF. Though 69.4% of the farmers were uninformed on the clinical signs of ASF, 73.6% of them did treat sick pigs presenting similar clinical signs to ASF (with no risk of mortality) with antibiotics, while 79.4% did not treat but sold the pigs presenting clinical signs similar to ASF with an increased risk of mortality. Sixty-three percent of the farmers had reported a case of ASF in the past and believed that reporting was useful and had no negative consequences on other farmers or third parties. We established that poor implementation of biosecurity measures in addition to poor training are contributing factors to the enzootic nature of ASFV in Cameroon and, thus, the spread of ASFV. Hence, pig farmers in Cameroon must be properly trained in ASFV awareness and the impact thereof on pig production. Furthermore, training will also facilitate the successful implementation of biosecurity measures to contain ASFV outbreaks.

Diversity and emergence of new variants of African swine fever virus Genotype I circulating in domestic pigs in Nigeria (2016-2018)

African swine fever (ASF) is the most lethal disease of pigs caused by ASF virus (ASFV) with severe economic implications and threat to food security in endemic countries. Between 2016 and 2018, several ASF outbreaks were reported throughout pig producing States in Nigeria. This study was designed to identify the ASFV genotypes responsible for these outbreaks and the transmission pathways of the virus during this period. Twenty-two ASFV-positive samples collected during passive surveillance in eight States of Nigeria were characterized using 3 partial genes sequences of the virus. The genes were: p72 capsid protein of the B646L, p54 envelope protein of E183L, and the central variable region (CVR) within B602L of ASFV. Phylogenetic analysis based on p72 and p54 revealed ASFV genotype I as the circulating virus. Sequence analysis of the CVR of B602L revealed genetic variations with six ASFV variants namely: Tet-15, Tet-20a, Tet-21b, Tet-27, Tet-31 and Tet-34, thus increasing the overall genetic diversity of ASFV in Nigeria. Three of these variants: Tet-21b, Tet-31 and Tet-34 were identified for the first time in Nigeria. The new variants of ASFV genotype I were identified in the States of Enugu, Imo, Plateau and Taraba, while co-circulation of multiple variants of ASFV genotype I were recorded in Plateau and Benue States. The high genetic diversity, emergence and increasing recovery of new variants of genotype I in Nigeria should be a concern given that ASFV is a relatively stable DNA virus. The epidemiological implications of these findings require further investigation.