Porcine circovirus type 2 infection promotes the SUMOylation of nucleophosmin-1 to facilitate the viral circular single-stranded DNA replication.

The mechanism of genome DNA replication in circular single-stranded DNA viruses is currently a mystery, except for the fact that it undergoes rolling-circle replication. Herein, we identified SUMOylated porcine nucleophosmin-1 (pNPM1), which is previously reported to be an interacting protein of the viral capsid protein, as a key regulator that promotes the genome DNA replication of porcine single-stranded DNA circovirus. Upon porcine circovirus type 2 (PCV2) infection, SUMO2/3 were recruited and conjugated with the K263 site of pNPM1's C-terminal domain to SUMOylate pNPM1, subsequently, the SUMOylated pNPM1 were translocated in nucleoli to promote the replication of PCV2 genome DNA. The mutation of the K263 site reduced the SUMOylation levels of pNPM1 and the nucleolar localization of pNPM1, resulting in a decrease in the level of PCV2 DNA replication. Meanwhile, the mutation of the K263 site prevented the interaction of pNPM1 with PCV2 DNA, but not the interaction of pNPM1 with PCV2 Cap. Mechanistically, PCV2 infection increased the expression levels of Ubc9, the only E2 enzyme involved in SUMOylation, through the Cap-mediated activation of ERK signaling. The upregulation of Ubc9 promoted the interaction between pNPM1 and TRIM24, a potential E3 ligase for SUMOylation, thereby facilitating the SUMOylation of pNPM1. The inhibition of ERK activation could significantly reduce the SUMOylation levels and the nucleolar localization of pNPM1, as well as the PCV2 DNA replication levels. These results provide new insights into the mechanism of circular single-stranded DNA virus replication and highlight NPM1 as a potential target for inhibiting PCV2 replication.

Comparison of the immune effects of the Chlamydia abortus MOMP antigen displayed in different parts of bacterial ghosts.

Bacterial ghosts (BGs) are promising vaccine platforms owing to their high adjuvant properties and delivery efficiency. Heterologous antigens can be anchored to different parts of BGs using genetic engineering strategies to prepare vaccines. However, several key issues need to be resolved, including the efficient preparation of BGs and determining the optimal anchoring position of exogenous antigens in the BGs. Here, we prepared an efficient temperature-controlled lysis system using lysis gene E of phage PhiX174 and used the major outer membrane protein (MOMP) of Chlamydia abortus (C. abortus) as a model antigen to explore the optimal display location of exogenous antigens in BGs. We demonstrated that the constructed recombinant temperature-controlled lysis plasmid can still stably inhibit E gene expression at 37°C, and the lysis efficiency of E. coli can reach above 99.9%. Four recombinant MOMP Escherichia coli (E. coli) ghost vaccines were constructed using different anchor sequences. These vaccines all induced strong specific antibody responses and secrete high levels of IFN-γ in immunized mice and significantly increased the clearance of C. abortus in a mouse infection model. Notably, the strongest immune effect was observed when MOMP was displayed on the surface of E. coli ghosts (rECG-InpN-M), which resulted in the clearance of C. abortus in mice 6 days earlier than that with the recombinant MOMP vaccine. Altogether, we constructed an efficient BG temperature-controlled lysis system and provided a feasible strategy for developing a BG delivery platform with enhanced immune effects.

Feeding Postures and Substrate Use of François' Langurs (Trachypithecus francoisi) in the Limestone Forest of Southwest China.

The feeding posture of a group of François' langurs in Fusui County, Guangxi, was studied using instantaneous scan sampling from January to December 2016 to explore how the species adapts to karst limestone forests by collecting data on feeding posture, forest strata height, and substrate use. The results showed that leaves were the main food type of the François' langurs, with young leaves accounting for 64.97% ± 19.08% of the food composition, mature leaves accounting for 11.88% ± 12.09%, fruits accounting for 12.96% ± 12.89%, flowers accounting for 4.16% ± 4.06%, and other food types, including stems, petioles, and other unknown parts of the tree, accounting for a total of 6.03% ± 9.09%. The François' langurs had four main postures during feeding, of which sitting and bipedal standing feeding accounted for the largest proportions, at 85.99% ± 5.97% and 12.33% ± 6.08% of the total records, respectively. Quadrupedal standing and suspending were rarely observed and only appeared occasionally during feeding activities at the peak resting period, the two postures together accounting for 1.39% ± 1.59% of the total records. The feeding postures of the langurs had marked seasonal variation, as evidenced by the fact that seated feeding accounted for a significantly higher proportion of the total behavioral records in the rainy season than in the dry season, whereas feeding while standing bipedally was significantly more frequent during the dry season. Correlation analyses showed that feeding posture was correlated with food composition, showing a positive correlation between the proportion of bipedal standing feeding and mature leaf consumption. François' langurs preferred to forage in the lower and middle forest layers, with the lower forest layer accounting for 55.93% ± 16.50% of the total number of recordings and the middle forest layer accounting for 33.63% ± 18.33%. Langurs were less likely to forage on the ground (rocks), accounting for only 6.79% ± 4.78% of the records. The frequency of langurs feeding in the upper part of the forest layer was the lowest at 3.65% ± 2.73%. Additionally, in the dry season, langurs utilized the lower forest layer more but used the middle forest layer less than in the rainy season. This study demonstrates that the spatial distribution of foods in the limestone forest has an important effect on the feeding posture of François' langurs and their forest layer utilization.

Interference of pseudorabies virus infection on functions of porcine granulosa cells via apoptosis modulated by MAPK signaling pathways.

BACKGROUND: Pseudorabies virus (PRV) is one of the major viral pathogens leading to reproductive disorders in swine. However, little is known about the effects of PRV infection on porcine reproductive system. Ovarian granulosa cells are somatic cells surrounding oocytes in ovary and required for folliculogenesis. The present study aimed to investigate the interference of PRV on functions of porcine ovarian granulosa cells in vitro. METHODS: Primary granulosa cells were isolated from porcine ovaries. To investigate the PRV infectivity, transmission electron microscopy (TEM) was used to check the presence of viral particles, and the expression of viral gE gene was detected by quantitative real-time PCR (qPCR) in PRV-inoculated cells. After PRV infection, cell viability was detected by MTS assay, Ki67 for proliferative status was determined by immunofluorescence assay (IFA), cell cycle and apoptosis were detected by flow cytometry, and progesterone (P4) and estradiol (E2) were determined by radioimmunoassay. The checkpoint genes of cell cycle and apoptosis-related proteins were studied by qPCR and western blotting. RESULTS: Virus particles were observed in the nucleus and cytoplasm of PRV-infected granulosa cells by TEM imaging, and the expression of viral gE gene increased in a time-dependent manner post infection. PRV infection inhibited cell viability and blocked cell cycle at S phase in porcine granulosa cells, accompanied by decreases in expression of Ki67 protein and checkpoint genes related to S phase. Radioimmunoassay revealed decreased levels in P4 and E2, and the expressions of key steroidogenic enzymes were also down-regulated post PRV-infection. In addition, PRV induced apoptosis with an increase in Bax expression and activation of caspase 9, and the phosphorylation of JNK, ERK and p38 MAPKs were significantly up-regulated in porcine ovarian granulosa cells post PRV infection. CONCLUSIONS: The data indicate that PRV causes infection on porcine ovarian granulosa cells and interferes the cell functions through apoptosis, and the MAPK signaling pathway is involved in the viral pathogenesis.

Seroprevalence of Chlamydia abortus and Brucella spp. and risk factors for Chlamydia abortus in pigs from China.

Chlamydiosis and brucellosis induced abortions have resulted in significant economic losses in the global livestock industry. Although there have been numerous reports on these two diseases in ruminants in China, limited information is available regarding the prevalence of Chlamydia abortus (C. abortus) and Brucella spp. infection in pigs. This study aimed to investigate the prevalence of C. abortus and Brucella spp. infections in pig serum using serology and to identify potential risk factors. In total, 2816 serum samples were collected from 12 provinces in China. The presence of C. abortus antibodies was determined using an enzyme-linked immunosorbent assay (ELISA), while the presence of Brucella spp. antibodies was examined using the Rose Bengal Plate Test (RBPT) and the Standard Agglutination Test (SAT). The seroprevalences of C. abortus and Brucella spp. were 8.38 % (236/2816) and 0.11 % (3/2816), respectively. Geographical location, season, and age were found to be risk factors associated with C. abortus infection in pig herds in China (p<0.01), and the seropositive rate for C. abortus in sow herds was strongly associated with the occurrence of abortion (p<0.01). Overall, in China, pigs exhibit a higher seroprevalence of C. abortus, whereas the prevalence of Brucella is limited. This study represents the first comprehensive survey of C. abortus and Brucella spp. in pig herds in China that established potential risk factors and provided data for the prevention and control of intraspecies and interspecies transmission of C. abortus to humans.

Porcine circovirus type 2 infection inhibits macrophage M1 polarization induced by other pathogens via viral capsid protein and host gC1qR protein.

Porcine circovirus type 2 (PCV2) has been proven to co-infect with a variety of pathogens and cause immunosuppression. Previously, we have reported that PCV2 infection attenuates the production of pro-inflammatory cytokines induced by other pathogens in porcine macrophages. However, whether PCV2 can affect M1-type macrophage polarization induced by other pathogens is less well reported. Herein, we found that PCV2 infection suppressed M1 macrophage production induced by porcine reproductive and respiratory syndrome virus (PRRSV) and Haemophilus parasuis (H. parasuis) in the lung and promoted the proliferation of these pathogens in the piglets. Consistently, we confirmed that PCV2 inhibits M1 macrophage production and its associated gene expression in porcine alveolar macrophages (PAMs) both ex vivo and in vitro. Meanwhile, PCV2 inhibited lipopolysaccharide (LPS)-induced pro-inflammatory cytokines in vitro in a time- and dose-dependent manner. In PCV2-infected cells, LPS-induced signal transducer and activator of transcription (STAT1) phosphorylation and its nuclear translocation were decreased. Based on these findings, we further identified a role for PCV2 capsid protein (Cap) in LPS-induced M1 macrophage-associated genes and found that PCV2 Cap can significantly reduce STAT1 phosphorylation and its nuclear translocation, as well as the production of M1 macrophage-related genes. As the binding protein of PCV2 Cap, gC1qR protein was also associated with this inhibition process. gC1qR-binding activity-deficient PCV2 Cap mutated protein (Cap RmA) appeared an attenuated inhibitory effect on other pathogen-induced polarization of M1-type macrophages, suggesting that the inhibitory effect of PCV2 infection on M1-type macrophage polarization induced by other pathogens is dependent on Cap protein and the host gC1qR protein. Altogether, our results demonstrate that PCV2 infection inhibits macrophage M1 polarization induced by other pathogens via capsid and host gC1qR protein modulating JAK/STAT signaling.

Chronic kidney disease in a giant panda (Ailuropoda melanoleuca): a case report.

BACKGROUND: Chronic kidney disease (CKD) is a common cause of morbidity and mortality in captive wildlife species. However, CKD has been rarely documented in giant pandas. CASE PRESENTATION: The following report describes a case of an eight-year-old female giant panda showing clinical signs of epistaxis, bloody diarrhea, polyuria, azotemia and anemia. The animal died despite of supportive treatments. Necropsy was performed. Grossly, both kidneys were shrunken and scarred with pallor. Subcutis edema and petechia on the epicardium of the heart were observed. The tissue samples were made into paraffin sections and stained by H.E and special staining including Periodic Acid-Schiff (PAS), von Kossa, Masson's trichrome, Phosphotungstic acid-hematoxylin (PTAH), and Congo red. Histopathology examination revealed severe chronic tubulointerstitial nephritis with marked interstitial fibrosis, glomerulosclerosis, tubular atrophy and calcification in kidneys, and acute necrotizing hemorrhagic myocarditis with calcification in heart. Other lesions included intestinal hemorrhage, hepatic fatty degeneration and necrosis with hemosiderin, and splenic hemosiderin. CONCLUSIONS: In summary, chronic kidney disease was finally diagnosed based on the association of clinical, gross, and histopathological findings. Heart failure secondary to CKD is the leading cause of death in this giant panda. The potential cause of CKD in this animal is possibly due to long term and uncontrolled hypertension. Blood pressure monitoring is essential in establishing the diagnosis and management of hypertension in giant panda.

Pseudorabies virus infection activates the NLRP3 and IFI16 inflammasomes to trigger pyroptosis.

Pseudorabies virus (PRV) preferably invades neural tissue and various organs, whereupon may result in multisystemic lesions. Pyroptosis mediated by proteolytic cleavage of gasdermin D (GSDMD) by inflammatory caspases (caspase-1/4/5/11), is closely associated with the activation of inflammasomes, a multiprotein proinflammatory complex. However, further studies on the mechanisms regarding PRV-induced pyroptosis in its natural host are required. Herein, it is demonstrated that PRV infection triggered GSDMD- not GSDME-mediated pyroptosis in porcine alveolar macrophage cells, resulting in increased secretion of IL-1ß and LDH. During this process, caspase-1 was activated and participated in the cleaving of GSDMD. Interestingly, we found that the viral replication process or protein production is required to induce pyroptotic cell death. Also, our findings showed that PRV triggered NLRP3 inflammasome activation, which was associated with the production of reactive oxygen species (ROS) and potassium efflux. In addition to the NLRP3 inflammasome, the IFI16 inflammasome was also activated. Importantly, the NLRP3- and IFI16- inflammasomes were both involved in pyroptosis during PRV infection. Finally, we observed that the cleaved GSDMD, activated caspase-1, increased IFI16 levels, and elevated NLRP3 protein in PRV-infected tissues (brain and lung), supporting the occurrence of pyroptosis and the activation of NLRP3- and IFI16- inflammasome in PRV-infected pigs. This research advances our understanding of the PRV-mediated inflammatory response and cell death pathways, providing a deeper knowledge of effective treatments for pseudorabies.

Guanylate-binding protein 1 inhibits nuclear delivery of pseudorabies virus by disrupting structure of actin filaments.

The alphaherpesvirus pseudorabies virus (PRV) is the causative agent of pseudorabies, responsible for severe economic losses to the swine industry worldwide. The interferon-inducible GTPase guanylate-binding protein 1 (GBP1) exhibits antiviral immunity. Our findings show that there is a robust upregulation in the expression of porcine GBP1 during PRV infection. GBP1 knockout promotes PRV infection, while GBP1 overexpression restricts it. Importantly, we found that GBP1 impeded the normal structure of actin filaments in a GTPase-dependent manner, preventing PRV virions from reaching the nucleus. We also discovered that viral US3 protein bound GBP1 to interfere with its GTPase activity. Finally, the interaction between US3 and GBP1 requires US3 serine/threonine kinase activity sites and the GTPase domain (aa 1 to 308) of GBP1. Taken together, this study offers fresh perspectives on how PRV manipulates the host's antiviral immune system.

Expressions of ghrelin and GHSR-1a in the corpus luteum and the stimulatory effect of ghrelin on luteal function of pregnant sows.

Studies have shown that ghrelin played direct actions in ovarian function, but the direct role of ghrelin in corpus luteum (CL) of pregnant sows has remained obscure. The study aimed to examine the expressions of ghrelin and its functional receptor (GHSR-1a) in the CL of sows during pregnancy, and evaluate the role of ghrelin in CL function of pregnant sows. Immunohistochemistry analysis showed that ghrelin and GHSR-1a are both predominantly localized in the luteal cells of pregnant sows CL. Strong immunoreactivity for ghrelin and GHSR-1a is detected at days 20 (early) and 50 (middle), but weak immunoreactivity is observed at days 90 (late) post mating. Similarly, there is a significant effect of pregnant phase on the expression (mRNA and protein) of ghrelin and GHSR-1a in the CL, with higher levels at days 20 (early) and 50 (middle), and lower values at 90 (late) post mating. In vitro, treatments of luteal cells with ghrelin (from 0.01 to 10 ng/mL) are promoted cell viability and P4 secretion in a dose-dependent manner. Ghrelin is also accelerated the LH-induced P4 secretion in luteal cells. Moreover, ghrelin is induced the release and mRNA expression of LH, and increased the release of prostaglandin (PG)E2, but reduced the secretion of PGF2α in luteal cells. In conclusion, the presences of ghrelin and GHSR-1a in the porcine CL during pregnancy, and the stimulatory effect of ghrelin on luteal cells suggest positive regulation by ghrelin in CL function of pregnant sows.

The S protein of a novel recombinant PEDV strain promotes the infectivity and pathogenicity of PEDV in mid-west China.

Porcine epidemic diarrhoea virus (PEDV) is an emerging and re-emerging swine enterovirus that causes highly contagious diarrhoea and mortality in piglets. To better understand the current prevalence of PEDV in mid-west China, and to find out the reason for the re-emergence of PEDV from the viral genomic characteristics. Herein, we firstly investigated epidemiology of PEDV in mid-west China from 2019 to 2020. A total of 62.23% (257/413) of diarrhoea samples were positive for PEDV, and the PEDV-positive cases were mainly detected in winter. Then, we selected the SXSL strain as a representative strain to study the genetic and pathogenic characterization of PEDV pandemic strains in mid-west China. The recombination analysis showed that SXSL strain was a recombinant strain, and the major and minor parent strains of the recombination are CH/SCZJ/2018 strain and GDS48 strain, respectively. Complete genome sequencing and homology analysis showed that the S protein of SXSL strain contained multiple amino acid indels and mutations compared to the PEDV representative strains. Furthermore, we evaluated the effect of S protein on the infectivity and pathogenicity of PEDV by the PEDV reverse genetics system, and results showed that SXSL S protein increased the infectivity and pathogenicity of chimeric virus. Overall, our findings provided important information for understanding the roles of S protein in the prevalence of PEDV in mid-west China and developing vaccines based on PEDV pandemic strains.

Chaperonin CCT5 binding with porcine parvovirus NS1 promotes the interaction of NS1 and COPƐ to facilitate viral replication.

Porcine parvovirus (PPV) is an important pathogen causing reproductive disorders in first pregnant sows. The non-structure protein NS1 of PPV is a multifunctional protein playing a key role in viral replication. Chaperonin-containing T-complex polypeptide complex (CCT), containing CCT1-CCT8 subunits, belongs to the type II chaperones that interact with proteins to help in folding and maintaining. In this study, CCT5, for the first time, was found to be one of the host interacting proteins of PPV NS1, and CCT5 was directly bound with NS1. Interference of CCT5 expression by specific siRNA and knockout of CCT5 expression by CRISPR/Cas9 suppressed PPV replication, while overexpression of CCT5 promoted PPV replication in PK-15 cells. The interaction of CCT5 and PPV NS1 was dependent on the 36-42 aa motif at the N-terminal end of NS1. More importantly, CCT5 was also found interacting with COPƐ, which has previously been demonstrated to promote PPV replication by regulating type I interferon. Interference and knockout of CCT5 expression significantly reduced the interaction of PPV NS1 and host protein COPƐ, and promoted the IFN-ß expression. These results show that CCT5 mediates the interaction of PPV NS1 and COPƐ to regulate viral replication, providing new insight into the mechanism of PPV replication.

The gC1qR Binding Site Mutant PCV2 Is a Potential Vaccine Strain That Does Not Impair Memory CD4+ T-Cell Generation by Vaccines.

PCV2 has been reported to reduce the protective effects of various vaccines on immunized pigs. Our previous studies showed that the interaction of Cap and host protein gC1qR mediated the PCV2 infection-induced suppression of immune response. Thus, we wondered whether the gC1qR binding site mutant PCV2RmA could be a vaccine strain and whether this mutant PCV2RmA impairs other vaccines. Herein, we showed that PCV2 infection reduced the classic swine fever virus (CSFV) vaccine-induced generation of memory CD4+ T cells through the interaction of Cap with gC1qR. PCV2RmA can effectively induce the production of PCV2-specific antibodies, neutralizing antibodies, and peripheral blood lymphocyte proliferation in piglets at the same levels as the commercial inactivated PCV2 vaccine. The PCV2RmA-induced anti-PCV2 immune responses could eliminate the serum virus and would not lead to pathological lesions like wild-type PCV2. Moreover, compared to the commercial inactivated PCV2 vaccine, PCV2RmA is capable of inducing more durable protective immunity against PCV2 that induced production of PCV2-specific antibodies and neutralizing antibodies for a longer time via stronger induction of memory CD4+ T cells. Importantly, PCV2RmA infection did not impair the CSFV vaccine-induced generation of memory CD4+ T cells. Collectively, our findings showed that PCV2 infection impairs memory CD4+ T-cell generation to affect vaccination and provide evidence for the use of PCV2RmA as an efficient vaccine to prevent PCV2 infection. IMPORTANCE PCV2 is one of the costliest pathogens in pigs worldwide. Usage of PCV2 vaccines can prevent the PCV2 infection-induced clinical syndromes but not the viral spread. Our previous work found that PCV2 infection suppresses the host type I interferon innate immune response and CD4+ T-cell-mediated Th1 immune response through the interaction of Cap with host gC1qR. Here, we showed that the gC1qR binding site mutant PCV2RmA could effectively induce anti-PCV2 immunity and provide more durable protective immunity against wild-type PCV2 infection in pigs. PCV2RmA would not impair the generation of memory CD4+ T cells induced by classic swine fever virus (CSFV) vaccines as wild-type PCV2 did. Therefore, PCV2RmA can serve as a potential vaccine strain to better protect pigs against PCV2 infection.

A new circular RNA-encoded protein BIRC6-236aa inhibits transmissible gastroenteritis virus (TGEV)-induced mitochondrial dysfunction.

Transmissible gastroenteritis virus (TGEV), a member of the coronavirus family, is the pathogen responsible for transmissible gastroenteritis, which results in mitochondrial dysfunction in host cells. Previously, we identified 123 differentially expressed circular RNAs (cRNA)from the TGEV-infected porcine intestinal epithelial cell line jejunum 2 (IPEC-J2). Previous bioinformatics analysis suggested that, of these, circBIRC6 had the potential to regulate mitochondrial function. Furthermore, mitochondrial permeability transition, a key step in the process of mitochondrial dysfunction, is known to be caused by abnormal opening of mitochondrial permeability transition pores (mPTPs) regulated by the voltage-dependent anion-selective channel protein 1 (VDAC)-Cyclophilin D (CypD) complex. Therefore, in the present study, we investigated the effects of circBIRC6-2 on mitochondrial dysfunction and opening of mPTPs. We found that TGEV infection reduced circBIRC6-2 levels, which in turn reduced mitochondrial calcium (Ca2+) levels, the decrease of mitochondrial membrane potential, and opening of mPTPs. In addition, we also identified ORFs and internal ribosomal entrance sites within the circBIRC6-2 RNA. We demonstrate circBIRC6-2 encodes a novel protein, BIRC6-236aa, which we show inhibits TGEV-induced opening of mPTPs during TGEV infection. Mechanistically, we identified an interaction between BIRC6-236aa and VDAC1, suggesting that BIRC6-236aa destabilizes the VDAC1-CypD complex. Taken together, the results suggest that the novel protein BIRC6-236aa encoded by cRNA circBIRC6-2 inhibits mPTP opening and subsequent mitochondrial dysfunction by interacting with VDAC1.

circEZH2 inhibits opening of mitochondrial permeability transition pore via interacting with PiC and up-regulating RSAD2.

Transmissible gastroenteritis virus (TGEV) infection can lead to mitochondrial damage in porcine intestinal epithelial cells-jejunum 2 (IPEC-J2) cell line. The abnormal opening of mitochondrial permeability transition pore (mPTP) is the most important factor for mitochondrial damage. We previously demonstrated that circEZH2 could inhibit the abnormal opening of mPTP by binding miR-22. However, circEZH2 binding to miR-22 cannot completely enable mPTP opening to recover to normal level compared with the control group. So, we assume that circEZH2 also regulates the mPTP opening in other ways. To prove it, we identified the differentially expressed proteins (DEPs) caused by circEZH2 and circEZH2-interacting proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). It turns out there are 123 DEPs (0.83 ≤ fold change ≥ 1.2) upon overexpression circEZH2 and 200 proteins interacted with circEZH2. The kyoto encyclopedia of genes and genomes (KEGG) analysis, gene ontology (GO) analysis, subcellular localization analysis, and protein interaction network results show that the DEPs and circEZH2-interacting proteins may involve in the regulation of mPTP opening. RNA immunoprecipitation (RIP) assay and flow cytometry (FCM) results indicate that circEZH2 can inhibit the opening of mPTP by interacting with Pi carrier (PiC, also named SLC25A3). Quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and FCM results reveal that circEZH2 can inhibit mPTP opening by promoting the expression of radical s-adenosyl methionine domain-containing protein 2 (RSAD2). In addition, PiC can promote RSAD2 expression. The data indicate that circEZH2 inhibits TGEV-induced mPTP opening by interacting with PiC and upregulating RSAD2.

Porcine parvovirus triggers autophagy through the AMPK/Raptor/mTOR pathway to promote viral replication in porcine placental trophoblasts.

Autophagy has been demonstrated to play important roles in the infection and pathogenesis of many viruses. We previously found that porcine parvovirus (PPV) infection can induce autophagy in porcine placental trophoblast cells (PTCs), but its underlying mechanism has not yet been fully revealed. In this study, we showed that PPV infection inhibited the activation of mTORC1 and promoted the expression of Beclin 1 and LC3II in PTCs. Treatment with a mTOR activator inhibited the expression of Beclin 1 and LC3II, as well as autophagy formation, and reduced viral replication in PPV-infected PTCs. Furthermore, we found that inhibition of AMPK expression, but not the inhibition of PI3K/Akt, p53, or MAPK/ERK1/2 pathway activation, can significantly increase mTOR phosphorylation in PPV-infected PTCs. Then, we found that the regulation of mTOR phosphorylation by AMPK was mediated by Raptor. AMPK expression knockout inhibited the activation of Raptor, decreased the expression of Beclin 1 and LC3II, suppressed the formation of autophagosomes, and reduced viral replication during PPV infection. Together, our results showed that PPV infection induces autophagy to promote viral replication by inhibiting the activation of mTORC1 through activation of the AMPK/Raptor pathway. These findings provide information to understand the molecular mechanisms of PPV-induced autophagy.

Porcine circovirus type 2 infection inhibits the activation of type I interferon signaling via capsid protein and host gC1qR.

Porcine circovirus 2 (PCV2) has been proved to increase the risk of other pathogens infection through antagonizing the host type I interferon (IFN) response. Previously, we have reported that PCV2 infection efficiently inhibits type I interferon production induced by other DNA viruses. However, whether PCV2 can inhibit type I interferon signaling is less reported. Herein, we found that PCV2 interfered with the activation of IFN signaling pathway, which led to a significantly reduced IFN-stimulated genes (ISGs) transcription after IFN-α stimulation both in vivo and in vitro. In PCV2-infected cells, IFN-induced tyrosine phosphorylation of STAT1 and STAT2 and their heterodimerization were decreased. Meanwhile, the nuclear translocation of phosphorylated STAT1/STAT2 was also decreased. Based on these findings, we further determined that roles of PCV2 Cap and Rep in the suppression of IFN-I signaling, and found that Cap acted as a predominant regulator in the early phase infection. PCV2 Cap could significantly reduce the phosphorylation of STAT1 and STAT2, the nuclear translocation of phosphorylated STAT1/STAT2, and IFN-stimulated response element (ISRE) promoter activity, results in a decreased ISGs transcription. As the binding protein of PCV2 Cap, gC1qR protein was also involved in this inhibition process. Knockdown of gC1qR could alleviate the inhibitory effects of either PCV2 infection or Cap on the activation of IFN signaling. These findings demonstrated that PCV2 infection interferes with the activation of type I IFNs signaling pathway depending on its Cap and host gC1qR protein.

The ultrasonically treated nanoliposomes containing PCV2 DNA vaccine expressing gC1qR binding site mutant Cap is efficient in mice.

Nowadays, vaccines are broadly used to prevent porcine circovirus type 2 (PCV2) infection-induced expenditures, but the virus is still spreading among pigs. The current PCV2 vaccines all rely on the immunogenicity of Cap, yet our previous studies found that Cap is also the major component mediating the PCV2 infection-induced immune suppression through its interaction with host gC1qR. Thereby, new vaccines are still necessary for PCV2 prevention and control. In this study, we constructed a new PCV2 DNA vaccine expressing the gC1qR binding site mutant Cap. We introduced the Intron A and WPRE elements into the vector to improve the Cap expression level, and fused the IL-2 secretory signal peptides to the N-terminal of Cap to mediate the secretion of Cap. We also screened and selected chemokines CXCL12, CCL22, and CCL25 to migrate dendritic cells. In addition, we contained the vectors with PEI and then ultrasonic them into nano size to enhance the entrance of the vectors. Finally, the animal experiments showed that the new PCV2 DNA vaccine expressing the gC1qR binding site mutant Cap could induce stronger humoral and cellular immune responses than the PCV2 DNA vaccine expressing the wild-type Cap and the non-ultrasonic treated PCV2 DNA vaccine in mice, and protect the mice from PCV2 infection and lung lesions. The results indicate the new PCV2 DNA vaccine expressing the gC1qR binding site mutant Cap has a certain development value, and provide new insight into the development of novel PCV2 vaccines.

T598 and T601 phosphorylation sites of canine parvovirus NS1 are crucial for viral replication and pathogenicity.

Canine parvovirus-2 (CPV-2) is an important pathogen causing severe diseases in dogs and other wild carnivores. Phosphorylation of NS1 may be related to CPV-2 pathogenicity, but the exact mechanism is unclear. Here, we conducted parvovirus disease surveillance in Shaanxi Province of China and 51 fecal swabs were detected to be infected with CPV-2. The 7 CPV-2 strains were identified, all of which belonged to CPV-2c. The complete genome sequence of one of the strains (CPV-2c XY) was cloned into pKQLL plasmid to construct a full-length infectious clone plasmid pX-CPV-2c, which carried a genetic marker. The plasmid pX-CPV-2c was transfected into F81 cells for virus rescue. And the rescued virus, which was designed as X-CPV-2c, showed the similar biological property to parental CPV-2c XY in vitro and in vivo. We further constructed four NS1 phosphorylation site mutant strains (X-CPV-2cT584A, X-CPV-2cS592A, X-CPV-2cT598A/T601A and X-CPV-2cT617A) on the basis of X-CPV-2c. After the analysis and comparison of biological characteristics, the low pathogenic strain X-CPV-2cT598A/T601A was further screened out, which emphasized the importance of phosphorylation sites 598 T/601 T for the pathogenicity of CPV-2. Overall, our data indicated that T598 and T601, the C-terminal phosphorylation site of CPV-2 NS1, play important roles in viral pathogenicity and laid the foundation for the development of new attenuated live vaccine vectors.