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Coronaviruses are widely transmissible between humans and animals, causing diseases of varying severity. Porcine enteric alphacoronavirus (PEAV) is a newly-discovered pathogenic porcine enteric coronavirus in recent years, which causes watery diarrhea in newborn piglets. The host inflammatory responses to PEAV and its metabolic regulation mechanisms remain unclear, and no antiviral studies have been reported. Therefore, we investigated the pathogenic mechanism and antiviral drugs of PEAV. The transcriptomic analysis of PEAV-infected host cells revealed that PEAV could upregulate lipid metabolism pathways. In lipid metabolism, steady-state energy processes, which can be mediated by lipid droplets (LDs), are the main functions of organelles. LDs are also important in viral infection and inflammation. In infected cells, PEAV increased LD accumulation, upregulated NF-κB signaling, promoted the production of the inflammatory cytokines IL-1β and IL-8, and induced cell death. Inhibiting LD accumulation with a DGAT-1 inhibitor significantly inhibited PEAV replication, downregulated the NF-κB signaling pathway, reduced the production of IL-1β and IL-8, and inhibited cell death. The NF-κB signaling pathway inhibitor BAY11-7082 significantly inhibited LD accumulation and PEAV replication. Metformin hydrochloride also exerted anti-PEAV effects and significantly inhibited LD accumulation, downregulated the NF-κB signaling pathway, reduced the production of IL-1β and IL-8, and inhibited cell death. LD accumulation in the lipid metabolism pathway therefore plays an important role in the replication and pathogenesis of PEAV, and metformin hydrochloride inhibits LD accumulation and the inflammatory response to exert anti-PEAV activity and reducing pathological injury. These findings contribute new targets for developing treatments for PEAV infections.

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen that targets pig intestines to cause disease. It is globally widespread and causes huge economic losses to the pig industry. PEDV N protein is the protein that constitutes the core of PEDV virus particles, and most of it is expressed in the cytoplasm, and a small part can also be expressed in the nucleus. However, the role of related proteins in host nucleotide metabolic pathways in regulating PEDV replication have not been fully elucidated. In this study, PEDV-N-labeled antibodies were co-immunoprecipitated and combined with LC-MS to screen for host proteins that interact with N proteins. Bioinformatics analyses showed that the selected host proteins were mainly enriched in metabolic pathways. Moreover, co-immunoprecipitation and confocal microscopy confirmed that the second-largest subunit of RNA polymerase II (RPB2) and uridine phosphorylase 1 (UPP1) interacted with the N protein. RPB2 is the main subunit of RNA polymerase II and plays an important role in eukaryotic transcription. UPP1 is an enzyme that catalyzes reversible phosphorylation of uridine to uracil and ribo-1-phosphate to promote catabolism and bio anabolism. RPB2 overexpression significantly promoted viral replication, whereas UPP1 overexpression significantly inhibited viral replication. Studies on interactions between the PEDV N and host proteins are helpful in elucidating the pathogenesis and immune escape mechanism of PEDV.

African swine fever (ASF), caused by the African swine fever virus (ASFV), has resulted in significant losses in the global pig industry. Considering the absence of effective vaccines, developing drugs against ASFV may be a crucial strategy for its prevention and control in the future. In this study, punicalagin, a polyphenolic substance extracted from pomegranate peel, was found to significantly inhibit ASFV replication in MA-104, PK-15, WSL, and 3D4/21 cells by screening an antiviral compound library containing 536 compounds. Time-of-addition studies demonstrated that punicalagin acted on early viral replication stages, impinging on viral attachment and internalization. Meanwhile, punicalagin could directly inactivate the virus according to virucidal assay. RT-qPCR and Western blot results indicated that punicalagin modulated the NF-κB/STAT3/NLRP3 inflammasome signaling pathway and reduced the levels of inflammatory mediators induced by ASFV. In conclusion, this study reveals the anti-ASFV activity of punicalagin and the mechanism of action, which may have great potential for developing effective drugs against ASFV.

Alphacoronaviruses are the primary coronaviruses responsible for causing severe economic losses in the pig industry with the potential to cause human outbreaks. Currently, extensive studies have reported the essential role of endosomal sorting and transport complexes (ESCRT) in the life cycle of enveloped viruses. However, very little information is available about which ESCRT components are crucial for alphacoronaviruses infection. By using RNA interference in combination with Co-immunoprecipitation, as well as fluorescence and electron microscopy approaches, we have dissected the role of ALIX and TSG101 for two porcine alphacoronavirus cellular entry and replication. Results show that infection by two porcine alphacoronaviruses, including porcine epidemic diarrhea virus (PEDV) and porcine enteric alphacoronavirus (PEAV), is dramatically decreased in ALIX- or TSG101-depleted cells. Furthermore, PEDV entry significantly increases the interaction of ALIX with caveolin-1 (CAV1) and RAB7, which are crucial for viral endocytosis and lysosomal transport, however, does not require TSG101. Interestingly, PEAV not only relies on ALIX to regulate viral endocytosis and lysosomal transport, but also requires TSG101 to regulate macropinocytosis. Besides, ALIX and TSG101 are recruited to the replication sites of PEDV and PEAV where they become localized within the endoplasmic reticulum and virus-induced double-membrane vesicles. PEDV and PEAV replication were significantly inhibited by depletion of ALIX and TSG101 in Vero cells or primary jejunal epithelial cells, indicating that ALIX and TSG101 are crucial for PEDV and PEAV replication. Collectively, these data highlight the dual role of ALIX and TSG101 in the entry and replication of two porcine alphacoronaviruses. Thus, ESCRT proteins could serve as therapeutic targets against two porcine alphacoronaviruses infection.

IntroductionPorcine epidemic diarrhea (PED) is an acute, highly contagious, and high-mortality enterophilic infectious disease caused by the porcine epidemic diarrhea virus (PEDV). PEDV is globally endemic and causes substantial economic losses in the swine industry. The PEDV E protein is the smallest structural protein with high expression levels that interacts with the M protein and participates in virus assembly. However, how the host proteins interact with E proteins in PEDV replication remains unknown.MethodsWe identified host proteins that interact with the PEDV E protein using a combination of PEDV E protein-labeled antibody co-immunoprecipitation and tandem liquid-chromatography mass-spectroscopy (LC-MS/MS).ResultsBioinformatical analysis showed that in eukaryotes, ribosome biogenesis, RNA transport, and amino acid biosynthesis represent the three main pathways that are associated with the E protein. The interaction between the E protein and isocitrate dehydrogenase [NAD] β-subunit (NAD-IDH-β), DNA-directed RNA polymerase II subunit RPB9, and mRNA-associated protein MRNP 41 was validated using co-immunoprecipitation and confocal assays. NAD-IDH-β overexpression significantly inhibited viral replication.DiscussionThe antiviral effect of NAD-IDH-β suggesting that the E protein may regulate host metabolism by interacting with NAD-IDH-β, thereby reducing the available energy for viral replication. Elucidating the interaction between the PEDV E protein and host proteins may clarify its role in viral replication. These results provide a theoretical basis for the study of PEDV infection mechanism and antiviral targets.

IntroductionAfrican swine fever virus (ASFV) is a highly contagious virus that spreads rapidly and has a mortality rate of up to 100% in domestic pigs, leading to significant economic losses in the pig industry. The major capsid protein p72 of ASFV plays a critical role in viral invasion and immune evasion.MethodsIn this study, we used yeast two-hybrid screening to identify host proteins interacting with p72 in porcine alveolar macrophages (PAMs) and verified these proteins using confocal microscopy and immunoprecipitation techniques.Results and DiscussionWe validated 13 proteins that interact with p72, including CD63, B2M, YTHDF2, FTH1, SHFL, CDK5RAP3, VIM, PELO, TIMP2, PHYH, C1QC, CMAS, and ERCC1. Enrichment analysis and protein-protein interaction network analysis of these interacting proteins revealed their involvement in virus attachment, invasion, replication, assembly, and immune regulation. These findings provide new insights into the function of p72 and valuable information for future research on the interaction between ASFV and host proteins.

Abstract African swine fever (ASF), caused by ASF virus (ASFV) infection, poses a huge threat to the pork industry owing to ineffective preventive and control measures. Hence, there is an urgent need to develop strategies, including antiviral drugs targeting ASFV, for preventing ASFV spread. This study aimed to identify novel compounds with anti-ASFV activity. To this end, we screened a small chemical library of 102 compounds, among which the natural flavonoid dihydromyricetin (DHM) exhibited the most potent anti-ASFV activity. DHM treatment inhibited ASFV replication in a dose- and time-dependent manner. Furthermore, it inhibited porcine reproductive and respiratory syndrome virus and swine influenza virus replication, which suggested that DHM exerts broad-spectrum antiviral effects. Mechanistically, DHM treatment inhibited ASFV replication in various ways in the time-to-addition assay, including pre-, co-, and post-treatment. Moreover, DHM treatment reduced the levels of ASFV-induced inflammatory mediators by regulating the TLR4/MyD88/MAPK/NF-κB signaling pathway. Meanwhile, DHM treatment reduced the ASFV-induced accumulation of reactive oxygen species, further minimizing pyroptosis by inhibiting the ASFV-induced NLRP3 inflammasome activation. Interestingly, the effects of DHM on ASFV were partly reversed by treatment with polyphyllin VI (a pyroptosis agonist) and RS 09 TFA (a TLR4 agonist), suggesting that DHM inhibits pyroptosis by regulating TLR4 signaling. Furthermore, targeting TLR4 with resatorvid (a specific inhibitor of TLR4) and small interfering RNA against TLR4 impaired ASFV replication. Taken together, these results reveal the anti-ASFV activity of DHM and the underlying mechanism of action, providing a potential compound for developing antiviral drugs targeting ASFV.

African swine fever (ASF) is a virulent infectious diseases of pigs caused by the African swine fever virus (ASFV) that can spread widely and cause high fatality rates. Currently, there is no effective way to treat the disease, and there is no effective vaccine to prevent it. Rhein, an anthraquinone compound extracted from many traditional Chinese medicines, exhibits anti-inflammatory, anti-tumor, and anti-viral activities. However, the anti-viral effects of rhein on ASFV remain unclear. Therefore, this study aimed to investigate the anti-ASFV activity of rhein in porcine alveolar macrophages (PAMs) and the underlying mechanisms. In this study, we confirmed that rhein inhibits ASFV replication significantly in a dose-dependent manner in vitro. Moreover, rhein could alter the susceptibility of PAMs to ASFV and promoted the production of superoxide in the mitochondria, which induced the loss of mitochondrial membrane potential, leading to the activation of caspase-9, caspase-3, and apoptosis. Mito-TEMPO, a mitochondria-targeted antioxidant, blocked rhein-induced mitochondrial superoxide generation and loss of mitochondrial membrane potential, prevented caspase-9 and caspase-3 activation, alleviated apoptosis, and suppressed the anti-ASFV activity of rhein. Altogether, our results suggested that rhein could play an anti-ASFV role by inducing apoptosis through the activation of the caspase-dependent mitochondrial apoptotic pathway and may provide a novel compound for developing anti-ASFV drugs.

ABSTRACTAfrican swine fever (ASF) is a highly contagious disease of domestic and wild pigs caused by the African swine fever virus (ASFV). The current research on ASF vaccines focuses on the development of naturally attenuated, isolated, or genetically engineered live viruses that have been demonstrated to produce reliable immunity. As a result, a genetically engineered virus containing five genes deletion was synthesized based on ASFV Chinese strain GZ201801, named ASFV-GZΔI177LΔCD2vΔMGF. The five-gene-deleted ASFV was safe and fully attenuated in pigs and provides reliable protection against the parental ASFV strain challenge. This indicates that the five-gene-deleted ASFV is a potential candidate for a live attenuated vaccine that could control the spread of ASFV.

Biochar has attracted increasing attention as an additive for enhancing the performance of anaerobic digestion (AD), but the effect of biochar on microbial regulatory mechanisms in enhancing AD performance is unclear. To investigate how biochar modulates the process of AD, different inoculum sources including cellulose–peptone–swine inoculum (CPI) and swine manure inoculum (SMI) were designed to determine the effect of biochar on the performance and microbial communities of anaerobic digestion of the feedstock concentration from 1 to 6%. The results showed that the methane yields of CPI seeds were higher 20.3–38.7% than those of SMI seeds without a biochar addition, whereas the biochar addition reduced 5.3 and 23.1% of the corresponding methane yield of CPI and SMI, respectively. The biochar enhances the accumulation of volatile fatty acids (VFAs) and weakens the potential ammonia inhibition by adsorption, and it can improve the degradation rate of organic content of soluble COD for different inoculum sources. Microbial community analyses showed that the biochar addition could facilitate the growth of Bacteroidetes and Clostridiales, and it enriched the relative abundance of hydrogenotrophic methanogens Methanobrevibacter and Methanobacterium. Overall, although the modulation of biochar possessed different effects on the anaerobic digestion performance, it contributed to the stability and degradation efficiency of the digestion system. The recycling implication of biochar is critical to realizing a low-carbon and renewable treatment system for organic wastes.

Porcine epidemic diarrhea (PED) is an enterophilic infectious disease caused by the porcine epidemic diarrhea virus (PEDV), which can lead to dehydration-like diarrhea in piglets with a mortality rate of up to 100%, causing huge economic losses to the global pig industry. In this study, we isolated two PEDV strains, FS202201 and JY202201, from diarrheal samples collected from two new PED outbreak farms in 2022. We performed phylogenetic analysis of the S gene and whole gene sequence. The effects of the different mutations on viral pathogenicity were investigated using piglet challenge experiments. The results showed that both strains belong to the G2c subtype, a widely prevalent virulent strain. Compared with FS202201, JY202201 harbored substitution and deletion mutations in nsp1. Both FS202201 and JY202201 infected piglets showed severe diarrhea and significant intestinal tissue lesions at an infection dose of 104 TCID50/mL, with a mortality rate of 50%; however, JY202201 required an additional day to reach mortality stabilization. An infection dose of 103 TCID50/mL reduced diarrhea and intestinal tissue lesions in piglets, with mortality rates of the two strains at 16.7% and 0%, respectively. In addition, PEDV was detected in the heart, liver, spleen, lungs, kidneys, mesenteric lymph nodes, stomach, large intestine, duodenum, jejunum, and ileum, with the highest levels in the intestinal tissues. In conclusion, this study enriches the epidemiology of PEDV and provides a theoretical basis for the study of its pathogenic mechanism and prevention through virus isolation, identification, and pathogenicity research on newly identified PED in the main transmission hub area of PEDV in China (Guangdong).

Since it was first reported in 1987, porcine reproductive and respiratory syndrome virus (PRRSV) has caused several economic crises worldwide. The current prevalence of PRRSV NADC30-like stains causing clinical disease outbreaks in Chain is highly concerning. Immunization against and the prevention of this infection are burdensome for farming organizations as the pathogen frequently mutates and undergoes recombination. Herein, the genetic characterization of a NADC30-like strain (termed BL2019) isolated from a farm in Guangdong Province, China, was analyzed and its pathogenicity for piglets and sows was assessed. Results revealed that BL2019 exhibits a nucleotide homology of 93.7% with NADC30 PRRSV and its NSP2 coding region demonstrates the same 131aa deletion pattern as that of NADC30 and NADC30-like. Furthermore, we identified two recombination breakpoints located nt5804 of the NSP5-coding region and nt6478 of NSP2-coding region, the gene fragment between the two breakpoints showed higher homology to the TJ strain(a representative strain of highly pathogenic PRRSV) compared to the NADC30 strain. In addition, BL2019 infection in piglets caused fever lasting for 1 week, moderate respiratory clinical signs and obvious visual and microscopic lung lesions; infection in gestating sows affected their feed intake and increased body temperature, abortion rates, number of weak fetuses, and other undesirable phenomena. Therefore, we report a NADC30-like PRRSV strain with partial recombination and a representative strain of HP-PRRSV, strain TJ, that can provide early warning and support for PRRS immune prevention and control.

Batch process monitoring datasets usually contain missing data, which decreases the performance of data-driven modeling for fault identification and optimal control. Many methods have been proposed to impute missing data; however, they do not fulfill the need for data quality, especially in sensor datasets with different types of missing data. We propose a hybrid missing data imputation method for batch process monitoring datasets with multi-type missing data. In this method, the missing data is first classified into five categories based on the continuous missing duration and the number of variables missing simultaneously. Then, different categories of missing data are step-by-step imputed considering their unique characteristics. A combination of three single-dimensional interpolation models is employed to impute transient isolated missing values. An iterative imputation based on a multivariate regression model is designed for imputing long-term missing variables, and a combination model based on single-dimensional interpolation and multivariate regression is proposed for imputing short-term missing variables. The Long Short-Term Memory (LSTM) model is utilized to impute both short-term and long-term missing samples. Finally, a series of experiments for different categories of missing data were conducted based on a real-world batch process monitoring dataset. The results demonstrate that the proposed method achieves higher imputation accuracy than other comparative methods.

African swine fever virus (ASFV) is a complex large DNA enveloped virus that causes African swine fever (ASF) with a fatality rate of up to 100%, seriously threatening the global swine industry. Due to the strict cell tropism of ASFV, there is no effective in vitro cell line, which hinders its prevention and control. Herein, we analyzed genome-wide transcriptional profiles of ASFV-susceptible porcine alveolar macrophages (PAMs) and non-susceptible cell lines PK15 and 3D4-21, an found that PAM surface pattern recognition receptors (PRRs) were significantly higher and common differential genes were significantly enriched in phagocytosis compared with that observed in PK15 and 3D4-21 cell lines. Therefore, endocytosis functions of host cell surface PRRs may play key roles in ASFV infection in vitro. ASFV was found to be infective to PK15 and 3D4-21 cell lines overexpressing CD163 and Siglec1, and to the PK15S1-CD163 cell line stably expressing CD163 and Siglec1. However, the PK15 and 3D4-21 cell lines overexpressing CD163 or Siglec1 alone were not infectious. Simultaneous interference of CD163 and Siglec1 in PAMs with small interfering RNA (siRNA) significantly reduced the infectivity of ASFV. However, siRNA interference of CD163 and Siglec1 respectively did not affect ASFV infectivity. ASFV significantly inhibited IFN expression levels in PAMs and PK15S1-CD163 cells, but had no effect on PK15 and 3D4-21 cell lines. These results indicate that CD163 and Siglec1 are key receptors for ASFV-infected host cells, and both play a synergistic role in the process of ASFV infection. ASFV inhibits IFN expression in susceptible cells, thereby downregulating the host immune response and evading the immune mechanism. The discovery of the ASFV receptor provides novel ideas to study ASFV and host cell interactions, pathogenic mechanisms, development of receptor blockers, vaccine design, and disease resistance breeding.

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) that causes a lethal hemorrhagic disease that is currently threatening the global pig industry. ASFV structural protein p30 is a membrane phosphoprotein that suggests it may play a regulatory role, possibly in signal transduction. Despite its significance in internalization into host cells, the interaction between p30 and host proteins is relatively unknown. In this study, we describe the application of a DUALmembrane yeast two-hybrid assay to screen a primary porcine alveolar macrophages cDNA library and analyze the interactome of p30 protein. Our data identify seven host cellular proteins (DAB2, RPSA, OAS1, PARP9, CAPG, ARPC5, and VBP1) that putatively interact with the p30. We further verified the interaction between p30 and host proteins by laser confocal microscopy, co-immunoprecipitation, and GST-pulldown assay. To further understand the relationship between host proteins and p30, we drew the interaction network diagram and analyzed the functional enrichment of each host protein. Enrichment analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes indicated that host proteins were mainly related to endocytosis, actin cytoskeleton regulation, and innate immunity. Collectively, we identified the interaction between p30 and host cell protein using a membrane protein yeast two-hybrid system, which increases our knowledge of the interaction between ASFV and the host and informs future research on antiviral strategies.

African swine fever (ASF) outbreak have caused tremendous economic loss to the pig industry in China since its emergence in August 2018. Previous studies revealed that many published sequences are not suitable for detailed analyses due to the lack of data regarding quality parameters and methodology, and outdated annotations. Thus, high-quality genomes of highly pathogenic strains that can be used as references for early Chinese ASF outbreaks are still lacking, and little is known about the features of intra-host variants of ASF virus (ASFV). In this study, a full genome sequencing of clinical samples from the first ASF outbreak in Guangdong in 2018 was performed using MGI (MGI Tech Co., Ltd., Shenzhen, China) and Nanopore sequencing platforms, followed by Sanger sequencing to verify the variations. With 22 sequencing corrections, we obtained a high-quality genome of one of the earliest virulent isolates, GZ201801_2. After proofreading, we improved (add or modify) the annotations of this isolate using the whole genome alignment with Georgia 2007/1. Based on the complete genome sequence, we constructed the methylation profiles of early ASFV strains in China and predicted the potential 5mC and 6mA methylation sites, which are likely involved in metabolism, transcription, and replication. Additionally, the intra-host single nucleotide variant distribution and mutant allele frequency in the clinical samples of early strain were determined for the first time and found a strong preference for A and T substitution mutation, non-synonymous mutations, and mutations that resulted in amino acid substitutions into Lysine. In conclusion, this study provides a high-quality genome sequence, updated genome annotation, methylation profile, and mutation spectrum of early ASFV strains in China, thereby providing a reference basis for further studies on the evolution, transmission, and virulence of ASFV.

African swine fever virus (ASFV) represents a serious threat to the global swine industry, and there are no safe or commercially available vaccines. Previous studies have demonstrated that inactivated vaccines do not provide sufficient protection against ASFV and that attenuated vaccines are effective, but raise safety concerns. Here, we first constructed a deletion mutant in which EP153R and EP402R gene clusters were knocked out. Based on the deletion mutant, a further deletion from the MGF_360-12L, MGF_360-13L to MGF_360-14L genes was obtained. The five-genes knockout virus was designated as ASFV-ΔECM3. To investigate the efficacy and safety of the ASFV-ΔECM3 virus as a vaccine candidate, the evaluation of the virus was subsequently carried out in pigs. The results showed that the ASFV-ΔECM3 virus could induce homologous protection against the parental isolate, and no significant clinical signs or viremia were observed. These results show that the contiguous deletion mutant, ASFV-ΔECM3 encompassing the EP153R/EP402R and MGF_360-12L/13L/14L genes, could be a potential live-attenuated vaccine candidate for the prevention of ASFV infection.

Since the first outbreak of ASFV genotype II in China in 2018, ASF has posed a significant threat to the swine industry. After the emergence of genotype I in China in 2020, the epidemic prevention and control have become more difficult. No effective commercial vaccine is currently available, and the disease is difficult to eradicate; therefore, the identification of the ASFV genotype is critical to establish biosafety control measures. In this study, a dual real-time PCR detection method based on B646L and E183L genes was developed to distinguish between ASFV genotypes I and II by specifically amplifying the genotype I E183L gene. The method is strongly specific, detects B646L and E183L genes simultaneously, and does not cross-react with PEDV, PCV, PRRSV, PRV, and CSFV. The double real-time PCR detection of ASFV genotypes I and II showed a B646L amplification curve, and only genotype I showed an E183L amplification curve, consistent with our expectations. The method has high sensitivity and the lowest copy numbers detected for recombinant plasmids B646L and E183L were 1.07 × 102 and 3.13 × 104 copies/μL, respectively. The method is reproducible, and the coefficient of variation for detecting the coefficient of variation (CV) values of the two recombinant plasmids was

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. 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, 15.60, 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.

Abstract Background Pseudorabies (PR) is latent and can persist in infected sows for a long time, and thus, convalescent sows can carry the virus throughout life, causing severe economic losses to farmers and posing a tremendous challenge to PR prevention and control. Here, to investigate the biological characteristics of pseudorabies virus (PRV), a variety of physical and chemical factors were analyzed under controlled conditions. Results The results showed that a high ambient temperature and dry environment led to faster virus inactivation. PRV had a certain resistance to weakly acidic or alkaline environments and was rapidly inactivated in strongly acidic or alkaline environments. The effect of ultraviolet (UV) radiation on PRV activity primarily depended on the frequency, intensity, and irradiation time of the UV exposure. Exposure to sunlight inactivated PRV via multiple factors, including temperature, sunlight intensity, UV intensity, and environmental humidity, and any shielding from sunlight strongly lowered the killing effect. Conventional disinfectants had a good disinfection effect on PRV. Conclusions The biological characteristics of different PRV strains are variable. Generally, the activity of PRV is affected by multiple factors, which can show both synergy and antagonism. Real-world conditions should be taken into consideration to guide pork production.

In 2018, there was an outbreak of African swine fever (ASF) in China, which spread to other provinces in the following 3 years and severely damaged 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, which requires only a small number of tissues. 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.

Background: 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.Results: 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.Conclusion: This case indicated 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.

Abstract Background Avian influenza A H7N9 virus has caused five outbreak waves of human infections in China since 2013 and posed a dual challenge to public health and poultry industry. The number of reported H7N9 virus human cases confirmed by laboratory has surpassed that of H5N1 virus. However, the mechanism for how H7N9 influenza virus overcomes host range barrier has not been clearly understood. Methods To generate mouse-adapted H7N9 influenza viruses, we passaged three avian-origin H7N9 viruses in mice by lung-to-lung passages independently. Then, the characteristics between the parental and mouse-adapted H7N9 viruses was compared in the following aspects, including virulence in mice, tropism of different tissues, replication in MDCK cells and molecular mutations. Results After ten passages in mice, MLD50 of the H7N9 viruses reduced >750-3,160,000 folds, and virus titers in MDCK cells increased 10-200 folds at 48 hours post-inoculation. Moreover, the mouse-adapted H7N9 viruses showed more expanded tissue tropism and more serious lung pathological lesions in mice. Further analysis of the amino acids changes revealed 10 amino acid substitutions located in PB2 (E627K), PB1 (W215R and D638G), PA (T97I), HA (H3 numbering: R220G, L226S, G279R and G493R) and NA (P3Q and R134I) proteins. Moreover, PB2 E627K substitution was shared by the three mouse-adapted viruses (two viruses belong to YRD lineage and one virus belongs to PRD lineage), and PA T97A substitution was shared by two mouse-adapted viruses (belong to YRD lineage). Conclusions Our result indicated that the virulence in mice and virus titer in MDCK cells of H7N9 viruses significantly increased after adapted in mouse model. PB2 E627K and PA T97A substitutions are vital in mouse adaption and should be monitored during epidemiological study of H7N9 virus.

Porcine enteric alphacoronavirus (PEAV) is a newly identified swine enteropathogenic coronavirus that causes watery diarrhea in neonatal piglets. The pathogenesis and host immune responses of PEAV infection are not fully characterized. The reason lies in the stomach environment, which would degrade cell-cultured live viruses via oral infection, making it difficult to establish an effective infection model to study the pathogenesis and host immune responses in pigs with a mature immune system. To solve this problem, in this study, coated PEAV-loaded microspheres were developed by centrifugal granulation-fluidized bed coating and demonstrated as an effective oral delivery system/animal infection model to protect PEAV virion against the complex gastrointestinal environment in vitro and to cause infection in weaned piglets in vivo. Weaned piglets orally inoculated with coated PEAV-loaded microspheres developed diarrhea and virus RNA was detected in rectal swabs from one to seven days post inoculation. In addition, microscopic lesions in the small intestine were observed, and viral antigens were also detected in the small intestines with PEAV immunohistochemical staining. Importantly, PEAV significantly inhibited mRNA expression of IFN-α, IFN-β, OAS, Mx1, and PKR, the genes involved in modulation of the host immune responses, in infected Peyer's patches, indicating that PEAV can overcome the antiviral response to cause damage when infection occurs. Collectively, our research successfully established a PEAV animal infection model in weaned piglets and suggested that the observed gene expression profile might help explain immunological changes associated with PEAV infection.

African swine fever virus (ASFV) mainly infects the monocyte/macrophage lineage of pigs and regulates the production of cytokines that influence host immune responses. Several studies have reported changes in cytokine production after infection with ASFV, but the regulatory mechanisms have not yet been elucidated. Therefore, the aim of this study was to examine the immune response mechanism of ASFV using transcriptomic and proteomic analyses. Through multi-omics joint analysis, it was found that ASFV infection regulates the expression of the host NF-B signal pathway and related cytokines. Additionally, changes in the NF-κB signaling pathway and IL-1β and IL-8 expression in porcine alveolar macrophages (PAMs) infected with ASFV were examined. Results show that ASFV infection activates the NF-κB signaling pathway and up-regulates the expression of IL-1β and IL-8. The NF-κB inhibitor BAY11-7082 inhibited the expression profiles of phospho-NF-κB p65, p-IκB, and MyD88 proteins, and inhibited ASFV-induced NF-κB signaling pathway activation. Additionally, the results show that the NF-κB inhibitor BAY11-7082 can inhibit the replication of ASFV and can inhibit IL-1β and, IL-8 expression. Overall, the findings of this study indicate that ASFV infection activates the NF-κB signaling pathway and up-regulates the expression of IL-1β and IL-8, and inhibits the replication of ASFV by inhibiting the NF-κB signaling pathway and interleukin-1 beta and interleukin-8 production. These findings not only provide new insights into the molecular mechanism of the association between the NF-κB signaling pathway and ASFV infection, but also indicate that the NF-κB signaling pathway is a potential immunomodulatory pathway that controls ASF.

Abstract Background Porcine epidemic diarrhea virus (PEDV) is emerging as a pathogenic coronavirus that causes a huge economic burden to the swine industry. Interaction of the viral spike (S) surface glycoprotein with the host cell receptor is recognized as the first step of infection and is the main determinant of virus tropism. The mechanisms by which neutralizing antibodies inhibit PEDV have not been defined. Isolating PEDV neutralizing antibodies are crucial to identifying the receptor-binding domains of the viral spike and elucidating the mechanism of protection against PEDV infection. Methods B cell hybridoma technique was used to generate hybridoma cells that secrete specific antibodies. E.coli prokaryotic expression system and Bac-to-Bac expression system were used to identify the target protein of each monoclonal antibody. qPCR was performed to analyze PEDV binding to Vero E6 cells with neutralizing antibody. Results We identified 10 monoclonal antibodies using hybridoma technology. Remarkably, 4 mAbs (designed 2G8, 2B11, 3D9, 1E3) neutralized virus infection potently, of which 2B11 and 1E3 targeted the conformational epitope of the PEDV S protein. qPCR results showed that both 2B11 and 2G8 blocked virus entry into Vero cells. Conclusion The data suggested that PEDV neutralizing antibody inhibited virus infection by binding to infectious virions, which could work as a tool to find the receptor-binding domains.

African swine fever virus (ASFV) poses serious threats to the swine industry. The mortality rate of African swine fever (ASF) is 100%, and there is no effective vaccine currently available. Complex immune escape strategies of ASFV are crucial factors affecting immune prevention and vaccine development. CD2v and MGF360-505R genes have been implicated in the modulation of the immune response. The molecular mechanisms contributing to innate immunity are poorly understood. In this study, we discover the cytopathic effect and apoptosis of ΔCD2v/ΔMGF360-505R-ASFV after infection in porcine alveolar macrophages (PAMs) was significantly less than wild-type ASFV. We demonstrated that CD2v- and MGF360-505R-deficient ASFV decrease the level of apoptosis by inhibiting the NF-κB signaling pathway and IL-1β mRNA transcription. Compared with wild-type ASFV infection, the levels of phospho-NF-κB p65 and p-IκB protein decreased in CD2v- and MGF360-505R-deficient ASFV. Moreover, CD2v- and MGF360-505R-deficient ASFV induced less IL-1β production than wild-type ASFV and was attenuated in replication compared with wild-type ASFV. We further found that MGF360-12L, MGF360-13L, and MGF-505-2R suppress the promoter activity of NF-κB by reporter assays, and CD2v activates the NF-κB signaling pathway. These findings suggested that CD2v- and MGF360-505R-deficient ASFV could reduce the level of ASFV p30 and the apoptosis of PAMs by inhibiting the NF-κB signaling pathway and IL-1β mRNA transcription, which might reveal a novel strategy for ASFV to maintain the replication of the virus in the host.

We identified from suckling piglets with diarrhea in China a new bat-HKU2–like porcine coronavirus (porcine enteric alphacoronavirus). The GDS04 strain of this coronavirus shares high aa identities (>90%) with the reported bat-HKU2 strains in Coronaviridae-wide conserved domains, suggesting that the GDS04 strain belongs to the same species as HKU2.

Pseudorabies, also known as Aujezsky’s disease, is an acute viral infection caused by pseudorabies virus (PRV). Swine are one of the natural hosts of pseudorabies and the disease causes huge economic losses in the pig industry. The establishment of a differential diagnosis technique that can distinguish between wild-type infection and vaccinated responses and monitor vaccine-induced immunoglobulin G(IgG) is crucial for the eventual eradication of pseudorabies. The aim of this study was to develop a rapid dual detection method for PRV gE and gB protein IgG antibodies with high specificity and sensitivity. PRV gE codons at amino acid residues (aa) 52–238 and gB codons at aa 539–741 were expressed to obtain recombinant PRV gE and gB proteins via a pMAL-c5x vector. After purification with Qiagen Ni–nitrilotriacetic acid (NTA) agarose affinity chromatography, the two proteins were analyzed via SDS-PAGE and immunoblotting assays. Two single fluorescent-microsphere immunoassays (FMIAs) were established by coupling two recombinant proteins (gE and gB) to magnetic microbeads, and an effective dual FMIA was developed by integrating the two single assays. Optimal serum dilution for each assay, correlation with other common swine virus-positive sera, and comparison with ELISA for two PRV antigens were tested for validation. Compared with ELISA, the specificity and sensitivity were 99.26% and 92.3% for gE IgG antibody detection, and 95.74% and 96.3% for the gB IgG antibody detection via dual FMIA. We provide a new method for monitoring PRV protective antibodies in vaccinated pigs and differentiating wild-type PRV infection from vaccinated responses simultaneously.

The Eurasian avian-like swine (EA) H1N1 virus has affected the Chinese swine industry, and human infection cases have been reported occasionally. However, little is known about the pathogenic mechanism of EA H1N1 virus. In this study, we compared the mouse pathogenicity of A/swine/Guangdong/YJ4/2014 (YJ4) and A/swine/Guangdong/MS285/2017 (MS285) viruses, which had similar genotype to A/Hunan/42443/2015 (HuN-like). None of the mice inoculated with 106 TCID50 of YJ4 survived at 7 days post infection, while the survival rate of the MS285 group was 100%. Therefore, a series of single fragment reassortants in MS285 background and two rescued wild-type viruses were generated by using the reverse genetics method, and the pathogenicity analysis revealed that the PB2 gene contributed to the high virulence of YJ4 virus. Furthermore, there were 11 amino acid differences in PB2 between MS285 and YJ4 identified by sequence alignment, and 11 single amino acid mutant viruses were generated in the MS285 background. We found that the R251K mutation significantly increased the virulence of MS285 in mice, contributed to high polymerase activity and enhanced viral genome transcription and replication. These results indicate that PB2-R251K contributes to the virulence of the EA H1N1 virus and provide new insight into future molecular epidemiological surveillance strategies.

Virus replication relies on complex interactions between viral proteins. In the case of African swine fever virus (ASFV), only a few such interactions have been identified so far. In this study, we demonstrate that ASFV protein p72 interacts with p11.5 using co-immunoprecipitation and liquid chromatography-mass spectrometry (LC-MS). It was found that protein p72 interacts specifically with p11.5 at sites amino acids (aa) 1-216 of p72 and aa 1-68 of p11.5. To assess the importance of p11.5 in ASFV infection, we developed a recombinant virus (ASFVGZΔA137R) by deleting the A137R gene from the ASFVGZ genome. Compared with ASFVGZ, the infectious progeny virus titers of ASFVGZΔA137R were reduced by approximately 1.0 logs. In addition, we demonstrated that the growth defect was partially attributable to a higher genome copies-to-infectious virus titer ratios produced in ASFVGZΔA137R-infected MA104 cells than in those infected with ASFVGZ. This finding suggests that MA104 cells infected with ASFVGZΔA137R may generate larger quantities of noninfectious particles. Importantly, we found that p11.5 did not affect virus-cell binding or endocytosis. Collectively, we show for the first time the interaction between ASFV p72 and p11.5. Our results effectively provide the relevant information of the p11.5 protein. These results extend our understanding of complex interactions between viral proteins, paving the way for further studies of the potential mechanisms and pathogenesis of ASFV infection. [ABSTRACT FROM AUTHOR]