Guangliang Liu

Porcine epidemic diarrhea virus (PEDV) has emerged as the predominant swine enteric coronavirus, causing vomiting, diarrhea, dehydration, and high mortality in piglets. Members of the DEAD-box family play critical roles in viral infection, yet their roles in PEDV infection remain undefined. Furthermore, it is unknown whether PEDV and its proteins regulate the expression of these DEAD-box proteins. In this study, reverse transcription quantitative PCR was used to examine the gene expression changes of 15 DEAD-box family members in the intestinal tissues of one-month-old PEDV-infected piglets. We found that the expression of DEAD-box helicase 3 X-Linked (DDX3X) was upregulated following PEDV infection, a result that was similarly observed in cellular experiments. Overexpression and knockdown of DDX3X significantly inhibited or promoted PEDV replication, respectively. Moreover, the use of specific DDX3X inhibitors demonstrated that DDX3X primarily restricts PEDV RNA accumulation. Our study also showed that DDX3X enhances RIG-I-like receptor (RLR) signaling by promoting the phosphorylation of TANK-binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3), signal transducer and activator of transcription 1 (STAT1), and Janus kinase 1 (JAK1) proteins. Co-immunoprecipitation and immunofluorescence assays further demonstrated the association and colocalization of DDX3X with TBK1 and IRF3, potentially contributing to its antiviral function. We constructed all PEDV proteins and identified that NSP5 reduces DDX3X levels via ubiquitination. Additionally, we discovered that tripartite motif-containing protein 28 (TRIM28) interacts with DDX3X and mediates its K48-linked ubiquitination. Knockdown of TRIM28 significantly reduced NSP5-mediated DDX3X ubiquitination. In conclusion, this study clarified the molecular mechanism by which NSP5 antagonizes DDX3X-mediated antiviral immune responses via the ubiquitination pathway, providing new insights into PEDV-host interactions and potential targets for antiviral intervention. IMPORTANCE: Recurrent outbreaks of porcine epidemic diarrhea (PED) have led to substantial economic losses in the global swine industry. To proactively control PED, it is crucial to investigate the pathogenic mechanisms of PEDV, particularly by exploring the interactions between viral proteins and host factors. In this study, we identified a significant phenomenon: PEDV infection activates DDX3X, enhancing the antiviral activity of the RLR pathway, while PEDV NSP5 recruits TRIM28 to target DDX3X for degradation, thus suppressing immune responses. These findings shed light on the complex regulatory interplay between PEDV proteins and host factors, deepening our understanding of the role and mechanisms of PEDV NSP5 in immune suppression.

For specific detection of porcine bocavirus (PBoV) group 3 (G3), we developed a TaqMan probe-based real-time polymerase chain reaction (PCR) targeting the NS1 gene. The correlation coefficient and amplification efficiency of the PCR were 0.994 and 102.9%, respectively. There were no cross-reactions of the PCR with the genomes of classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine parvovirus, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV), and the detection limit of the PCR was 100 copies per reaction. The coefficients of variation of the inter-assay and intra-assay of the PCR were 1.38–2.71% and 0.23–0.67%, respectively. A total of 719 field samples collected from Xinjiang, Gansu, Qinghai, Ningxia, and Shaanxi Provinces were detected via the developed PCR, and the positive rates were 80.82%, 44.51%, 97.50%, 98.94%, and 93.60%, respectively. In addition, coinfections of PBoV G3 with PEDV/TGEV/PDCoV/PCV2 were determined, and dual infections, triple infections, quadruple infections, and quintuple infections were observed. Furthermore, the complete genome sequences of 10 positive samples (two per province) randomly selected from five provinces were determined, and phylogenetic analysis placed all 10 within PBoV G3, but one strain formed a distinct branch, indicating it was a novel strain in PBoV G3. The established PCR provides a reliable tool for PBoV G3 detection and facilitates the understanding of its distribution and prevalence. IMPORTANCE: Porcine bocavirus (PBoV) poses a threat to swine health and has potential public health significance. Owing to the high nucleotide diversity of PBoV, existing detection methods may yield false-negative results. A reliable detection method is needed to monitor its prevalence. Given that porcine bocavirus group 3 (PBoV G3) is the predominant group circulating in Chinese pig populations, we established a rapid and sensitive TaqMan probe-based polymerase chain reaction (PCR) for detection of PBoV G3. We detected 719 field samples collected from northwest China by the PCR and analyzed coinfections of PBoV G3 with porcine epidemic diarrhea virus/transmissible gastroenteritis virus/porcine deltacoronavirus PCV2. The results showed that the positive rate of PBoV G3 was high, and dual infections, triple infections, quadruple infections, and quintuple infections were present. Furthermore, a novel genotype was identified in PBoV G3. Taken together, the developed PCR facilitates the prevalence investigation of PBoV G3. The results enrich the data on the distribution and prevalence of PBoV G3 in China.

Porcine epidemic diarrhoea (PED) results in vomiting, diarrhoea, dehydration, and death in pigs. To analyse the relationship between intestinal development and susceptibility to porcine epidemic diarrhoea virus (PEDV), six new-born, 1-, 2-, 3-, 4-, and 8-week-old piglets were orally inoculated with 2 × 10 ⁷ copies of the PEDV strain LJX01/2014 and sacrificed 48 h after infection. The results demonstrated that PEDV infection elicits diarrhoea of diverse severity across piglets of distinct ages. Moreover, viral shedding and villous atrophy were most pronounced in neonatal piglets. With increasing age, the shedding of villi gradually decreased, resulting in significantly lower villi/crypt ratios in the duodenum of newborn, 1-week-old, and 3-week-old piglets and significantly lower villi/crypt ratios in the jejunum and ileum of newborn and 1-week-old piglets than in the control. Indirect immunofluorescence and immunohistochemistry revealed a positive correlation between intraepithelial lymphocyte counts and epithelial cells and piglet age. PEDV infection led to a significant increase in intraepithelial lymphocytes and a decrease in epithelial cells, with the most severe effects observed in newborn piglets. The numbers of goblet cells, absorptive enterocytes, and enteroendocrine cells significantly decreased, whereas the numbers of Paneth cells, stem cells, and proliferating cells significantly increased with increasing piglet age. Overall, the results indicate that intestinal tissue repair increases with increasing age. Consequently, this results in a more robust intestinal epithelial barrier and reduced PEDV-induced damage to the small intestine.

Parainfluenza virus is a significant respiratory pathogen affecting both humans and animals, capable of causing acute respiratory tract infections in infants and immunocompromised individuals. Recently, parainfluenza virus 5 (PIV5) has been frequently detected in swine diarrheal samples. However, experimental infections revealed that PIV5 alone induces only mild clinical symptoms. Further analysis suggested that co-infection with porcine sapelovirus (PSV) may underlie the observed disease severity. This hypothesis was confirmed through animal studies. Co-infection was shown to occur intracellularly, where PSV significantly enhanced PIV5 replication. Mechanistically, this effect was primarily mediated by PSV 3D, an RNA-dependent RNA polymerase (RdRp). PSV 3D directly interacted with PIV5 nucleoprotein and genomic RNA. Mini-genome assays demonstrated that PSV 3D substantially increased PIV5 genomic activity. Using an infectious cDNA clone of PIV5 and a series of mutants, we further confirmed that PSV 3D promoted transcription of both plus- and minus-strand PIV5 RNAs. Notably, RdRp (3D) proteins from two other picornaviruses also enhanced PIV5 RNA synthesis, suggesting a conserved function across picornaviruses. In summary, this study provides the first evidence that PIV5 hijacks the RdRp of a co-infecting virus from a different viral family to support its own replication. These findings advance our understanding of virus–virus interactions and offer a novel perspective in virology. IMPORTANCE: The gastrointestinal tract harbors a vast and diverse community of microorganisms, making it an ideal environment for exploring microbial interactions. While virus–bacteria interactions have been widely studied, virus–virus interactions remain largely uncharacterized. In this study, we demonstrated that porcine sapelovirus (PSV) and parainfluenza virus 5 (PIV5) co-infect cells and directly interact within the host. Specifically, the RNA-dependent RNA polymerase protein (3D) of PSV significantly promoted PIV5 replication by interacting with key components of the PIV5 ribonucleoprotein complex and enhancing the synthesis of both plus- and minus-strand viral RNAs. Similar effects were observed with the 3D proteins from two additional picornaviruses, suggesting a shared mechanism among picornaviruses in facilitating co-infecting virus replication. This work uncovers a novel cross-family polymerase hijacking event and provides important insights into virus–virus interactions, highlighting new potential targets for the control and prevention of swine enteric diseases.

Volume 100, no. 2, e01373-25, 2025, https://doi.org/10.1128/jvi.01373-25. Materials and Methods, “Virus purification” paragraph: “…and presented in sFig. 2” should read “…and results are presented in Fig. S1 at https://doi.org/10.57760/sciencedb.28945.”

Global climate change has increased the frequency and intensity of heat waves, posing a significant threat to livestock production. During heat exposure, the disruption of intestinal barrier integrity is a pivotal event in the pathogenesis of heat stress-induced intestinal injury. Endoplasmic reticulum (ER) stress and mitochondrial dysfunction are key consequences of heat stress at the cellular level. However, direct causal evidence linking ER stress to mitochondrial dysfunction in heat-stressed enterocytes remains limited. To investigate this, we used an integrated transcriptomic, metabolomic, and functional validation strategy to assess mitochondrial bioenergetics and cellular ultrastructure in porcine intestinal epithelial (IPEC-J2) cells under acute heat stress. Transcriptomic analysis revealed extensive reprogramming, highlighting the significant enrichment of pathways related to protein processing in the endoplasmic reticulum, apoptosis, and MAPK signaling. Untargeted metabolomics identified significant perturbations in amino acid and energy metabolism, as well as altered bile acid profiles. Functional assessments confirmed that heat stress severely impaired mitochondrial bioenergetics, as evidenced by reduced maximal respiration and ATP production, and induced ultrastructural damage to mitochondria. The pharmacological inhibition of ER stress by 4-phenylbutyric acid (4-PBA) significantly attenuated the mitochondrial bioenergetic impairment and ultrastructural damage, whereas ER stress induction recapitulated these defects. We demonstrate that heat stress induces profound transcriptional and metabolic remodeling characterized by ER stress activation, which critically mediates subsequent mitochondrial bioenergetic dysfunction and ultrastructural damage. Our findings suggest that targeting ER stress may represent a promising therapeutic strategy to ameliorate enterocyte mitochondrial dysfunction and mitigate heat stress-induced intestinal injury in livestock.

This study reports the development of a novel and highly sensitive electrochemical immunosensor for detecting porcine epidemic diarrhea virus (PEDV). To th

N6-methyladenosine (m ⁶ A) is the most abundant internal modification in eukaryotic RNA and plays diverse roles in RNA metabolism. Increasing evidence indicates that m ⁶ A is also present in viral RNAs, where it exerts virus-specific effects. While several studies have shown that m ⁶ A can facilitate viral replication, its antiviral mechanisms remain less understood. In this study, we used transmissible gastroenteritis virus (TGEV) as a model to investigate the inhibitory role of m ⁶ A in viral infection. We demonstrated that m ⁶ A modification is present in the TGEV genome and suppresses viral replication. The m ⁶ A reader proteins bind to viral RNA and reduce the stability of m ⁶ A-modified transcripts. Notably, TGEV infection increased global m ⁶ A levels in host RNA, particularly in interferon (IFN)-associated genes. Inhibition of m ⁶ A methylation significantly diminished IFN gene expression. Furthermore, compared to other viruses, TGEV genomic RNA displayed an abnormally higher m ⁶ A ratio, which can be distinguished by RIG-I to promote an immune response. Collectively, our findings reveal that high m ⁶ A modification enhances RIG-I-mediated sensing of TGEV RNA, leading to the activation of IFN responses and inhibition of viral replication. This study provides new insights into the complex regulatory functions of m ⁶ A during viral infection and host antiviral defense. IMPORTANCE: N6-methyladenosine (m ⁶ A) is one of the most prevalent RNA modifications in viral genomes, but its functional impact varies widely across viruses. While m ⁶ A often promotes viral replication, it can exert inhibitory effects in certain viruses, particularly within the Flaviviridae and Coronaviridae families. Despite growing evidence of this antiviral role, the underlying mechanisms remain largely unclear. Here, we used transmissible gastroenteritis virus (TGEV), a swine coronavirus, as a model to explore the inhibitory function of m ⁶ A. We show that the TGEV genome harbors a relatively high density of m ⁶ A modification compared to other viruses and host mRNA, which are efficiently detected by the host pattern recognition receptor RIG-I. This interaction enhances innate immune activation and restricts viral replication. Our findings uncover the mechanism by which abnormal m ⁶ A modification can be sensed to activate antiviral immunity and provide deeper insight into the multifaceted role of m ⁶ A in host–virus interactions.