In 2018, there was an outbreak of African swine fever (ASF) in China, which spread to other provinces in the following three years, severely damaging China’s pig industry. ASF is caused by the African swine fever virus (ASFV). Given that the genome of the African swine fever virus is very complex and whole genome information is currently inadequate, it is important to efficiently obtain virus genome sequences for genomic and epidemiological studies. The prevalent ASFV strains have low genetic variability; therefore, whole genome sequencing analysis provides a basis for the study of ASFV. We provide a method for the efficient sequencing of whole genomes, requiring only a small number of tissues. The pig spleen were crushed and homogenized, and the tissue and cell fragments were removed by low-speed centrifugation. The virus-like particles (VLPs) in the solution were purified and concentrated by filtration and over-speed centrifugation. After obtaining the relatively pure VLP sample, nucleic acids were extracted simultaneously by co-extraction of viral DNA (dsDNA, ssDNA) and RNA (ssRNA, dsRNA). Then, the database construction method was selected according to the genomic types of ASFV, and the whole ASFV genome was obtained through data filtering, host sequence removal, virus classification, data assembly, virus sequence identification, statistical analysis, gene prediction, and functional analysis. Our proposed method will facilitate ASFV genome sequencing and novel virus discovery.

On October 30, 2020, piglets and sows in the farrowing house of a pig farm in Jiangxi showed clinical symptoms such as anorexia, watery diarrhea, and vomiting. Epidemiological test, clinical necropsy, and RT-PCR test were carried out on the pig farm for diagnosis. After comprehensive considerations, the disease was judged as porcine epidemic diarrhea virus infection. Thereafter, a series of comprehensive prevention and control measures such as emergency vaccination with autogenous vaccines were adopted. Half a month after inoculation with autogenous vaccines for the farm, the mortality rate of newborn piglets in the farrowing house began to decline, and production gradually returned to being stable. The second-generation sequencing analysis and phylogenetic analysis showed that the porcine epidemic diarrhea virus (PEDV) sequence obtained from the stool and small intestine samples of the diseased pigs on the farm was 97.8% homologous to the vaccine strain. At the same time, antibody testing found that the vaccinated pigs on the pig farm had satisfactory immune response, indicating that the PEDV outbreak on the pig farm might aggravate owing to the strain being mutated and could escape the immune protection of the existing vaccine. This case has accumulated technical data for the clinical prevention and control of porcine epidemic diarrhea.

To date, there is no effective vaccine or antiviral therapy available to prevent or treat African swine fever virus (ASFV) infections. ASFV gene deletion strains have been proposed as promising anti-ASFV vaccine candidates. In recent years, most ASFV gene deletion strains worldwide have been recombinant strains expressing EGFP or mCherry as markers. However, a method for their wide range detection is still lacking. Therefore, in this study, a new triplex real-time PCR (RT-PCR) method was established for the broad and accurate differentiation of ASFV wild-type vs. gene deletion strains. We designed three pairs of primers and probes to target B646L, EGFP, and mCherry, and RT-PCR was used to detect these three genes simultaneously. The detection method prevented non-specific amplification of porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, circovirus type 2, pseudorabies virus, and classical swine fever virus genes. The minimum copy number of standard plasmid DNA detected using triplex RT-PCR was 9.49, 30.20, and 9.60 copies for B646L, EGFP, and mCherry, respectively. Importantly, of the 1646 samples analyzed in this study, 67 were positive for ASFV, all corresponding to the wild-type virus. Overall, our data show that the triplex RT-PCR method established in this study can specifically identify both ASFV wild-type and gene deletion strains.