PSMB4 Degrades the Porcine Reproductive and Respiratory Syndrome Virus Nsp1α Protein via the Autolysosome Pathway and Induces the Production of Type I Interferon.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes respiratory disease in pigs of all ages and reproductive failure in sows, resulting in great economic losses to the swine industry. In this work, we identified the interaction between PSMB4 and PRRSV Nsp1α by yeast two-hybrid screening. The PSMB4-Nsp1α interaction was further confirmed by coimmunoprecipitation, glutathione S-transferase (GST) pulldown, and laser confocal experiments. The PCPα domain (amino acids 66 to 166) of Nsp1α and the C-terminal domain (amino acids 250 to 264) of PSMB4 were shown to be critical for the PSMB4-Nsp1α interaction. PSMB4 overexpression reduced PRRSV replication, whereas PSMB4 knockdown elicited opposing effects. Mechanistically, PSMB4 targeted K169 in Nsp1α for K63-linked ubiquitination and targeted Nsp1α for autolysosomal degradation by interacting with LC3 to enhance the activation of the lysosomal pathway. Meanwhile, we found that PSMB4 activated the NF-κB signaling pathway to produce type I interferons by downregulating the expression of IκBα and p-IκBα. In conclusion, our data revealed a new mechanism of PSMB4-mediated restriction of PRRSV replication, whereby PSMB4 was found to induce Nsp1α degradation and type I interferon expression, in order to impede the replication of PRRSV. IMPORTANCE In the swine industry, PRRSV is a continuous threat, and the current vaccines are not effective enough to block it. This study determined that PSMB4 plays an antiviral role against PRRSV. PSMB4 was found to interact with PRRSV Nsp1α, mediate K63-linked ubiquitination of Nsp1α at K169, and thus trigger its degradation via the lysosomal pathway. Additionally, PSMB4 activated the NF-κB signaling pathway to produce type I interferons by downregulating the expression of IκBα and p-IκBα. This study extends our understanding of the proteasome subunit PSMB4 against PRRSV replication and will contribute to the development of new antiviral strategies.

ZNF283, a Krüppel-associated box zinc finger protein, inhibits RNA synthesis of porcine reproductive and respiratory syndrome virus by interacting with Nsp9 and Nsp10.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a viral pathogen with substantial economic implications for the global swine industry. The existing vaccination strategies and antiviral drugs offer limited protection. Replication of the viral RNA genome encompasses a complex series of steps, wherein a replication complex is assembled from various components derived from both viral and cellular sources, as well as from the viral genomic RNA template. In this study, we found that ZNF283, a Krüppel-associated box (KRAB) containing zinc finger protein, was upregulated in PRRSV-infected Marc-145 cells and porcine alveolar macrophages and that ZNF283 inhibited PRRSV replication and RNA synthesis. We also found that ZNF283 interacts with the viral proteins Nsp9, an RNA-dependent RNA polymerase, and Nsp10, a helicase. The main regions involved in the interaction between ZNF283 and Nsp9 were determined to be the KRAB domain of ZNF283 and amino acids 178-449 of Nsp9. The KRAB domain of ZNF283 plays a role in facilitating Nsp10 binding. In addition, ZNF283 may have an affinity for the 3' untranslated region of PRRSV. These findings suggest that ZNF283 is an antiviral factor that inhibits PRRSV infection and extend our understanding of the interactions between KRAB-containing zinc finger proteins and viruses.

Brequinar inhibits African swine fever virus replication in vitro by activating ferroptosis.

BACKGROUND: African swine fever virus (ASFV) is one of the most fatal swine etiological agents and has a huge economic impact on the global pork industry. Given that no effective vaccines or anti-ASFV drugs are available, there remains a pressing need for novel anti-ASFV drugs. This study aimed to investigate the anti-African swine fever virus (ASFV) activity of brequinar, a DHODH inhibitor. METHODS: The anti-ASFV activity of brequinar was investigated using IFA, HAD, HAD50, qRT-PCR, and western blotting assays. The western blotting assay was used to investigate whether brequinar inhibits ASFV replication by killing ASFV particles directly or by acting on cell factors. The confocal microscopy and western blotting assays were used to investigate whether brequinar inhibits ASFV replication by activating ferroptosis. RESULTS: In this study, brequinar was found to effectively inhibit ASFV replication ex vivo in porcine alveolar macrophages (PAMs) in a dose-dependent manner. In kinetic studies, brequinar was found to maintain ASFV inhibition from 24 to 72 hpi. Mechanistically, the time-of-addition assay showed that brequinar exerted anti-ASFV activity in all treatment modes, including pre-, co-, and post-treatment rather than directly killing ASFV particles. Notably, FerroOrange, Mito-FerroGreen, and Liperfluo staining experiments showed that brequinar increased the accumulation of intracellular iron, mitochondrial iron, and lipid peroxides, respectively. Furthermore, we also found that ferroptosis agonist cisplatin treatment inhibited ASFV replication in a dose-dependent manner and the inhibitory effect of brequinar on ASFV was partially reversed by the ferroptosis inhibitor ferrostatin-1, suggesting that brequinar activates ferroptosis to inhibit ASFV replication. Interestingly, exogenous uridine supplementation attenuated the anti-ASFV activity of brequinar, indicating that brequinar inhibits ASFV replication by inhibiting DHODH activity and the depletion of intracellular pyrimidine pools; however, the induction of ferroptosis by brequinar treatment was not reversed by exogenous uridine supplementation, suggesting that brequinar activation of ferroptosis is not related to the metabolic function of pyrimidines. CONCLUSIONS: Our data confirm that brequinar displays potent antiviral activity against ASFV in vitro and reveal the mechanism by which brequinar inhibits ASFV replication by activating ferroptosis, independent of inhibiting pyrimidine synthesis, providing novel targets for the development of anti-ASFV drugs.

Dihydromyricetin inhibits African swine fever virus replication by downregulating toll-like receptor 4-dependent pyroptosis in vitro.

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.

First report and genetic diversity of porcine bufavirus in China.

BACKGROUND: Bufavirus is a newly discovered zoonotic virus reported in numerous mammals and humans. However, the epidemiological and genetic characteristics of porcine bufaviruses (PBuVs) in China remain unclear. METHODS: To detect PBuVs in China, 384 samples (92 fecal and 292 serum specimens) were collected from 2017 to 2018, covering six provinces in China, and were evaluated by nested PCR. Further, the positive samples from different provinces were selected to obtain the complete genome of Chinese PBuVs. RESULTS: The prevalence rate of PBuV was 16.7% in Chinese domestic pigs in the Guangdong, Guangxi, Fujian, Jiangxi, Anhui, and Henan provinces. Additionally, the positive rate of fecal specimens was higher than that of the serum samples. Next, we sequenced nine near-complete genomes of Chinese field PBuV strains from different provinces. Homology and phylogenetic analyses indicated that Chinese PBuVs have high genetic variation (93.3-99.2%), showed higher nucleotide identity with an Austrian PBuV strain (KU867071.1), and developed into different branches within the same cluster. CONCLUSION: To our knowledge, this is the first report on PBuV in China, expanding the geographic boundaries of PBuV circulation. Our data demonstrate that PBuVs are widely distributed in the six Chinese provinces. Moreover, these Chinese PBuVs exhibit genetic variation and continuous evolution characteristics. Taken together, our findings provide a foundation for future studies on bufaviruses.

First identification of porcine parvovirus 7 in China.

Porcine parvovirus (PPV) are small, non-enveloped and single-stranded DNA viruses, taxonomically classifiable within the family Parvoviridae. Seven PPV genotypes (PPV1 to PPV7) have been identified to date. PPV7, the most recently discovered PPV genotype, was first reported in US pigs in 2016. To explore PPV7 status in Chinese pig populations a total of 64 serum samples collected from two commercial farms in Guangdong province in 2014 were analyzed. PPV7 DNA was detected in 32.8% (21/64) of tested samples. On the porcine circovirus type 2 (PCV2) positive farm, the prevalence rate of PPV7 was 65.5% (19/29) which was significantly higher than that on the PCV2 negative farm (2/35, 5.7%), indicating a possible association between PCV2 and PPV7 infections. The sequences of three PPV7 strains were determined. Phylogenetic analysis revealed that the identified PPV7 strains circulating in China shared 98.7%-99.7% nucleotide homology with the US strain. Further sequence comparison analysis indicated that GD-2014-2 and GD-2014-3 possess a consecutive 9-nt deletion in the VP gene. This is the first report of the existence of PPV7 in China and this finding will strengthen understanding of the epidemiology of porcine parvovirus in Chinese pigs.

Genetic variation, pathogenicity, and immunogenicity of highly pathogenic porcine reproductive and respiratory syndrome virus strain XH-GD at different passage levels.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important infectious diseases of swine worldwide. Immunization with an attenuated vaccine is considered an effective method for reducing the economic losses resulting from porcine reproductive and respiratory syndrome virus (PRRSV) infection. Several studies have shown that PRRSV can be attenuated by passage in Marc-145 cells, but it is still not clear whether this attenuation influences the immunogenicity of PRRSV and what the mechanism of attenuation is. In order to study the mechanism of attenuation and immunogenicity of highly pathogenic (HP) PRRSV, the HP-PRRSV strain XH-GD was serially 122 times passaged in Marc-145 cells. Genomic sequence comparisons were made at selected passages. To explore the differences in pathogenicity and immunogenicity at different passages, three passages (P5, P62 and P122) were selected for an animal challenge experiment, which showed that passage in Marc-145 cells resulted in attenuation of the virus. After 122 passages, 35 amino acid changes were observed in the structural proteins and non-structural proteins. The animal challenge experiment showed that pathogenicity decreased with increasing passage number. The N antibody level and specific neutralizing (SN) antibody titers also decreased with increasing passage number in the late stage of the animal experiment. This study indicated that the virulence of XH-GD was decreased by passage in Marc-145 cells and that overattenuation might influence the immunogenicity of virus. These results might contribute to our understanding of the mechanism of attenuation.

Lack of exposure of H10N8 avian influenza virus among veterinarians in Guangdong Province, China.

We conducted a retrospective seroepidemiological study for H10N8 avian influenza infection among 400 veterinarians sampled from February 2013 to August 2013 in Guangdong Province, China. None of the veterinarians had evidence of previous infection with the emergent H10N8 AIV. Although there is no evidence of H10N8-infected veterinarian before the first human index case of H10N8 infection in southern China, a more rigorous and long-term surveillance remained essential for early warning of novel reassortant viruses and interspecies transmission events.

Altered splenic miRNA expression profile in H1N1 swine influenza.

Previous studies have demonstrated the key regulatory roles played by microRNAs (miRNAs) in influenza virus-host interactions. To gain more insight into the contribution of miRNAs to the host immune response, spleen tissues from mice infected with A/Swine/GD/2/12 (H1N1) virus were harvested 5 days postinfection, and miRNA deep sequencing was performed. The results showed that 50 miRNAs were modulated. Interestingly, pathway analysis of miRNAs and targets showed that upregulated miR-124-3p interacts with innate immune-related pathways such as the Toll-like receptor pathway, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway and JAK-STAT signaling pathway, and this might play a major role in the anti-inflammatory response. Further understanding of the roles played by these miRNAs in influenza virus infection will provide new insights into host-pathogen interactions.

Mapping the heterogeneous removal landscape of wastewater virome in effluents of different advanced wastewater treatment systems of swine farm.

In advanced wastewater treatment plants on pig farms, meticulous design aims to eliminate intrinsic pollutants such as organic matter, heavy metals, and biological contaminants. In our field survey across Southern China, a notable disparity in wastewater treatment procedures among various farming facilities lies in the utilization of terminal chemical oxidation post-sedimentation tank. However, recent focus in wastewater surveillance has predominantly centered on antibiotic resistance genes, leaving the efficacy of virus removal in different effluent systems largely unexplored. To profile virus composition at the effluent, assess the virus elimination efficiency of chemical oxidation at the effluent end, and the potential environmental driver of virus abundance, we deployed a meta-transcriptomics approach to first determine the total virome in effluent specimens of terminal clean water tank system (CWT) and terminal chemical oxidation system (TCO) in Southern China pig farms, respectively. From these data, 172 viruses were identified, with a median reads per million (RPM) of 27,789 in CWT and 19,982 in TCO. Through the integration of analyses encompassing the co-occurrence patterns within viral communities, the ecology of viral diversity, and a comparative assessment of average variation degrees, we have empirically demonstrated that the procedure of TCO may perturb viral communities and diminish their abundance, particularly impacting RNA viral communities. However, despite the diminished abundance, pathogenic viruses such as PEDV and PRRSV persisted in the effluent following chemical deoxidation at a moderate RPM value, indicating a substantial in situ presence at effluent. Our environmental driver modeling, employing GLM and mantel tests, substantiated the intricate nature of virus community variation within the effluent, influenced heterogeneously by diverse factors. Notably, pond temperature emerged as the foremost determinant, while fishing farming exhibited a positive correlation with virus diversity (p < 0.05). This revelation of the cryptic persistence of virus communities in wastewater effluent expands our understanding of the varied responses of different virus categories to oxidation. Such insights transcend mere virus characterization, offering valuable implications for enhancing biosafety measures in farming practices and informing wastewater-based epidemiological surveillance.

Demographic and zoological drivers of infectome diversity in companion cats with ascites.

Cats (Felidae) have become an integral part of many households. However, our understanding of the full spectrum of pathogens affecting cats (referred to as the infectome) is limited, mainly due to the inadequacy of commonly used diagnostic tools in capturing the complete diversity of potential pathogens and the prevalence of pathogen co-infections. In this study, we employed a meta-transcriptomic approach to simultaneously characterize the infectome contributing to different disease syndromes and to investigate spatial, demographic, and ecological factors influencing pathogen diversity and community composition in a cohort of 27 hospitalized cats and seven stray cats. We identified 15 species of pathogens, with Candidatus Rickettsia tarasevichiae and Tritrichomonas foetus representing potential spillover risks. Importantly, although most cases of ascites hyperplasia were explained by coinfection with multiple pathogens, we identified the potential novel clinical outcomes of M. aubagnense infection among cats. We demonstrated that the increase in infectome diversity can be explained by a variety of predictors including age growth, temperature increase, and a higher proportion of females, with age growth presenting the strongest effect. Fine-scale analysis indicated that a higher diversity of infectomes were harbored in young cats rather than adult ones. Our results demonstrated that most feline diseases are better explained by the presence of virus-bacteria or virus-virus coinfection. This study serves as a timely endorsement for clinical diagnosis by vets to consider the cause of a disease based on a panel of cryptical co-infecting pathogens rather than on individual infectious agents. IMPORTANCE: Frequent studies reported the risks of cats as an intermediate host of zoonotic pathogens (e.g., SARS-CoV-2). Cats have a physically close interaction with their owners through activities like petting, kissing, and being licked on the cheek and hands. However, there are still limited studies that systematically investigate the infectome structure of cats. In this study, we employed a meta-transcriptomics approach to characterize 15 species of pathogens in cats, with Candidatus Rickettsia tarasevichiae first characterizing infection in diseased cats. Most feline diseases were better explained by the presence of virus-bacteria or virus-virus coinfection. The increase in infectome diversity could be influenced by a variety of predictors including age growth, temperature increase, and a higher proportion of females. A higher diversity of pathogens was harbored in young cats rather than adults. Importantly, we showed the value of linking the modern influx of meta-transcriptomics with comparative ecology and demography and of utilizing it to affirm that ecological and demographic variations impact the total infectome.

African swine fever virus maintains de novo global cellular protein synthesis and inhibits stress granules formation via dephosphorylating eIF2α.

African swine fever virus (ASFV) has caused enormous economic losses since its first reported detection, and there is still no effective vaccines or drug treatment. During infection, viruses may employ various strategies, such as regulating the host endoplasmic reticulum stress/unfolded protein response or the formation of stress granules (SGs), to form an optimal environment for virus replication. However, how ASFV infection regulates host endoplasmic reticulum stress, eIF2α-regulated protein synthesis, and the formation of SGs remains unclear. Here, we evaluated the activation of ER stress and its three downstream axes during ASFV infection and identified a powerful dephosphorylation of eIF2α by ASFV ex vivo. This strong dephosphorylation property could maintain the efficiency of eIF2α-mediated de novo global protein synthesis, thus ensuring efficient viral protein synthesis at early stage. In addition, the powerful dephosphorylation of eIF2α by ASFV upon infection could also inhibit the formation of SGs induced by sodium arsenite. In addition, a specific eIF2α dephosphorylation inhibitor, salubrinal, could partially counteract ASFV-mediated eIF2α dephosphorylation and inhibit viral replication. Our results provide new insights into the areas of ASFV`s escape from host immunity and hijacking of the host protein translation system.

Genomic and pathogenicity analysis of two novel highly pathogenic recombinant NADC30-like PRRSV strains in China, in 2023.

Porcine reproductive and respiratory syndrome viruses (PRRSVs) exhibit high mutability and recombination, posing challenges to their immunization and control. This study isolated two new PRRSV strains, GD-7 and GX-3, from samples collected in Guangdong and Guangxi in 2023. Whole-genome sequencing, along with phylogenetic and recombination analyses, confirmed that GD-7 and GX-3 are natural novel recombinant strains of NADC30 PRRSV. Moreover, we established a pathogenicity model for piglets and sows based on the two isolates. The results of piglet pathogenicity revealed that both GD-7 and GX-3 caused clinical symptoms such as fever, loss of appetite, depression, and slow weight gain. Moreover, we observed that the mortality rate of GD-7-inoculated group piglets was 33.3%, which was similar to that of piglets infected with other highly pathogenic PRRSV strains and exceeded the mortality rate of most NADC30-like PRRSV. In pregnant sow models, the survival rate of sows in the GD-7 group was 75%, in contrast to the GX-3 group, where no sow mortality was observed, and both strains resulted in abortion, mummified fetuses, and stillbirths. These results highlight the elevated pathogenicity of these recombinant strains in sows, with GD-7 mainly causing sows to abort, and GX-3 mainly causing sows to give birth to mummified fetuses. This study introduces two distinct clinical recombinant PRRSV strains that differ from the prevalent strains in China. This research furthers our understanding of the epidemiology of PRRSV and underscores the significance of ongoing monitoring and research in the face of evolving virus strains. Moreover, these discoveries act as early warnings, underscoring the necessity for active control and immunization against PRRSV.IMPORTANCESince the discovery of NADC30-like PRRSV in China in 2013, it has gradually become the dominant strain of PRRSV in China. NADC30-like PRRSV exhibits high recombination characteristics, constantly recombining with different strains, leading to the emergence of numerous novel strains. Of particular importance is the observation that NADC30-like PRRSV with different recombination patterns exhibits varying pathogenicity, which has a significant impact on the pig farming industry. This emphasizes the necessity of monitoring and responding to evolving PRRSV strains to develop effective immunization and control strategies. In this paper, we conducted pathogenicity studies on the isolated NADC30-like PRRSV and analyzed the differences in the genomes and pathogenicity of the different strains by recording clinical symptoms, temperature changes, detoxification tests, and changes in viremia and histopathology in infected pigs. This was done to provide a theoretical basis for the epidemiological situation and epidemic prevention and control of PRRSV.

Untangling lineage introductions, persistence and transmission drivers of HP-PRRSV sublineage 8.7.

Despite a rapid expansion of Porcine reproductive and respiratory syndrome virus (PRRSV) sublineage 8.7 over recent years, very little is known about the patterns of virus evolution, dispersal, and the factors influencing this dispersal. Relying on a national PRRSV surveillance project established over 20 years ago, we expand the available genomic data of sublineage 8.7 from China. We perform independent interlineage and intralineage recombination analyses for the entire study period, which showed a heterogeneous recombination pattern. A series of Bayesian phylogeographic analyses uncover the role of Guangdong as an important infection hub within Asia. The spatial spread of PRRSV is highly linked with a composite of human activities and the heterogeneous provincial distribution of the swine industry, largely propelled by the smaller-scale Chinese rural farming systems in the past years. We sequence all four available modified live vaccines (MLVs) and perform genomic analyses with publicly available data, of which our results suggest a key "leaky" period spanning 2011-2017 with two concurrent amino acid mutations in ORF1a 957 and ORF2 250. Overall, our study provides an in-depth overview of the evolution, transmission dynamics, and potential leaky status of HP-PRRS MLVs, providing critical insights into new MLV development.

Pathogenicity and horizontal transmission evaluation of a novel isolated African swine fever virus strain with a three-large-fragment-gene deletion.

African swine fever has caused substantial economic losses to China`s pig industry in recent years. Currently, the highly pathogenic African swine fever virus strain of genotype II is predominantly circulating in China, accompanied by a series of emerging isolates displaying unique genetic variations. The pathogenicity of these emerging strains is still unclear. Recently, a novel ASFV strain with a distinguishable three-large-fragment gene deletion was obtained from the field specimens, and its in vivo pathogenicity and transmission were evaluated in this study. The animal experiment involved inoculating a high dose of YNFN202103 and comparing its effects with those of the highly pathogenic strain GZ201801_2. Results showed that pigs infected by YNFN202103 exhibited significantly prolonged onset and survival time, lower viremia levels, and less severe histopathological lesions compared to GZ201801_2. These findings contributed valuable insights into the pathogenicity and transmission of ASFV and its prevention and eradication strategies in practical settings.

Emodin and rhapontigenin inhibit the replication of African swine fever virus by interfering with virus entry.

Africa swine fever (ASF) is a highly pathogenic contagion caused by African swine fever virus (ASFV), which not only affects the development of domestic pig industry, but also causes huge losses to the world agricultural economy. Vaccine development targeting ASFV remains elusive, which leads to severe difficulties in disease prevention and control. Emodin (EM) and rhapontigenin (RHAG), which are extracted from the dried rhizome of Polygonum knotweed, have various biological properties such as anti-neoplastic and anti-bacterial activities, but no studies have reported that they have anti-ASFV effects. This study discovered that EM and RHAG at different concentrations had a significant dose-dependent inhibitory effect on the ASFV GZ201801 strain in porcine alveolar macrophages (PAMs), and at the specified concentration, EM and RHAG showed continuous inhibition at 24 h, 48 h and 72 h. Not only did they strongly impact virion attachment and internalization, but also inhibit the early stages of ASFV replication. Further research proved that the expression level of Rab 7 protein was reduced by EM and RHAG, and treatments with EM and RHAG induced the accumulation of free cholesterol in endosomes and inhibited endosomal acidification, which prevented the virus from escaping and shelling from late endosomes. This study summarized the application of EM and RHAG in inhibiting ASFV replication in-vitro. Similarly, EM and RHAG targeted Rab 7 in the viral endocytosis pathway, inhibited viral infection, and induced the accumulation of cholesterol in the endosomes and the acidification of the endosomes to inhibit uncoating. A reference could be made to the results of this study when developing antiviral drugs and vaccines.

Theaflavin inhibits African swine fever virus replication by disrupting lipid metabolism through activation of the AMPK signaling pathway in virto.

African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), which is one of the most harmful swine diseases in the pig industry because of its nearly 100% mortality rate in domestic pigs and results in incalculable economic loss. Ever since ASF was initially reported, scientists have worked to develop anti-ASF vaccines; however, currently no clinically effective vaccine for ASF is available. Therefore, the development of novel measures to prevent ASFV infection and transmission is essential. In this study, we aimed to investigate the anti-ASF activity of theaflavin (TF), a natural compound mainly isolated from black tea. We found that TF potently inhibited ASFV replication at non-cytotoxic concentrations ex vivo in primary porcine alveolar macrophages (PAMs). Mechanistically, we found that TF inhibited ASFV replication by acting on cells rather than interacting directly with ASFV to inhibit viral replication. Further, we found that TF upregulated the AMPK (5'-AMP-activated protein kinase) signaling pathway in ASFV-infected and uninfected cells, and treatment with the AMPK agonist MK8722 upregulated the AMPK signaling pathway and inhibited ASFV proliferation in a dose-dependent manner. Notably, the effects of TF on AMPK activation and ASFV inhibition were partially reversed by the AMPK inhibitor dorsomorphin. In addition, we found that TF down-regulated the expression of genes related to lipid synthesis and decreased the intracellular accumulation of total cholesterol and total triglycerides in ASFV-infected cells, suggesting that TF may inhibit ASFV replication by disrupting lipid metabolism. In summary, our results demonstrated that TF is an ASFV infection inhibitor and revealed the mechanism by which ASFV replication is inhibited, providing a novel mechanism and potential lead compound for the development of anti-ASFV drugs.

Porcine reproductive and respiratory syndrome virus regulates lipid droplet accumulation in lipid metabolic pathways to promote viral replication.

Porcine reproductive and respiratory syndrome (PRRS) is a severe respiratory disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) that can lead to the abortion of pregnant sows and decreased boar semen quality. However, the mechanisms of PRRSV replication in the host have not yet been fully elucidated. As lipid metabolism and lipid droplets (LDs) have been reported to play important roles in the replication of various viruses, we aimed to explore the mechanisms through which LDs affect PRRSV replication. Laser confocal and transmission electron microscopy revealed that PRRSV infection promoted intracellular LD accumulation, which was significantly reduced by treatment with the NF-κB signaling pathway inhibitors BAY11-7082 and metformin hydrochloride (MH). In addition, treatment with a DGAT1 inhibitor significantly reduced the protein expression of Phosphorylated NF-ΚB P65and PIκB and the transcription of IL-1ß and IL-8 in the NF-κB signaling pathway. Furthermore, we showed that the reduction of the NF-κB signaling pathway and LDs significantly reduced PRRSV replication. Together, the findings of this study suggest a novel mechanism through which PRRSV regulates the NF-κB signaling pathway to increase LD accumulation and promote viral replication. Moreover, we demonstrated that both BAY11-7082 and MH can reduce PRRSV replication by reducing the NF-κB signaling pathway and LD accumulation. This study lays a theoretical foundation for research on the mechanism of PRRS prevention and control, as well as the research and development of antiviral drugs.

Origin, genomic diversity and evolution of African swine fever virus in East Asia.

Since 2018, the outbreaks of genotype II African swine fever virus (ASFV) in China and several eastern Asian countries have caused a huge impact on the local swine industry, resulting in huge economic losses. However, little is known about the origin, genomic diversity, evolutionary features, and epidemiological history of the genotype II ASFV. Here, 14 high-quality complete genomes of ASFVs were generated via sequencing of samples collected from China over the course of 3 years, followed by phylogenetic and phylodynamic analyses. The strains identified were relatively homogeneous, with a total of 52 SNPs and 11 indels compared with the prototype strain HLJ/2018, among which there were four exceptionally large deletions (620-18,023 nt). Evolutionary analyses revealed that ASFV strains distributed in eastern Asia formed a monophyly and a 'star-like' structure centered around the prototype strain, suggesting a single origin. Additionally, phylogenetic network analysis and ancestral reconstruction of geographic state indicated that genotype II ASFV strains in eastern Asia likely originated from Western Europe. Overall, these results contribute to the understanding of the history and current status of genotype II ASFV strains in eastern Asian, which could be of considerable importance in disease control and prevention.

Isolation, identification, and pathogenicity of porcine epidemic diarrhea virus.

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).