Erythronate utilization activates VdtR regulating its metabolism to promote Brucella proliferation, inducing abortion in mice.

Brucella is a facultative intracellular pathogen that preferentially colonizes reproductive organs and utilizes erythritol as a preferred carbon source for its survival and proliferation. In this study, we identified a virulence-related DeoR-family transcriptional regulator (VdtR) and an erythronate metabolic pathway responsible for four-carbon acid sugar metabolism of D-erythronate and L-threonate in Brucella. We found that VdtR plays an important role in Brucella intracellular survival and trafficking to the endoplasmic reticulum in RAW 264.7 macrophages and in virulence in a mouse model. More importantly, we found that VdtR negatively regulates the erythronate metabolic pathway to promote extracellular proliferation of Brucella, depending on utilization of D-erythronate, an oxidative product of erythritol in the host. In a pregnant mouse model, the erythronate metabolic pathway was shown to cooperate with erythritol metabolism and play a crucial role in Brucella proliferation in the placenta, inducing placentitis and finally resulting in abortion or stillbirth. Our results demonstrate that, in addition to erythritol, erythronate is a preferred carbon source for Brucella utilization to promote its extracellular proliferation. This discovery updates the information on the preferential colonization of reproductive organs by Brucella and provides a novel insight into the Brucella-associated induction of abortion in pregnant animals. IMPORTANCE Brucella is an intracellular parasitic bacterium causing zoonosis, which is distributed worldwide and mainly characterized by reproductive disorders. Erythritol is found in allantoic fluid, chorion, and placenta of aborted animals, preferentially utilized by Brucella to cause infertility and abortion. However, the erythritol metabolism-defected mutant was unable to function as a vaccine strain due to its residual virulence. Here, we found that erythronate, an oxidative product of erythritol in the host, was also preferentially utilized by Brucella relying on the function of a deoxyribonucleoside regulator-family transcriptional regulator VdtR. Erythronate utilization activates VdtR regulation of the erythronate metabolic pathway to promote Brucella extracellular proliferation, inducing placentitis/abortion in mice. Double mutations on Brucella erythritol and D-erythronate metabolisms significantly reduced bacterial virulence. This study revealed a novel mechanism of Brucella infection-induced abortion, thus providing a new clue for the study of safer Brucella attenuated vaccines.

Isolation and Characterization of a Novel Recombinant Classical Pseudorabies Virus in the Context of the Variant Strains Pandemic in China.

Pseudorabies virus (PRV) variants were discovered in immunized pigs in Northern China and have become the dominant strains since 2011, which caused huge economic losses. In this study, a classical PRV strain was successfully isolated in a PRV gE positive swine farm. The complete genome sequence was obtained using a high-throughput sequencing method and the virus was named JS-2020. The nucleotide homology analysis and phylogenetic tree based on complete genome sequences or gC gene showed that the JS-2020 strain was relatively close to the classical Ea strain in genotype II clade. However, a large number of amino acid variations occurred in the JS-2020 strain compared with the Ea strain, including multiple immunogenic and virulence-related genes. In particular, the gE protein of JS-2020 was similar to earlier Chinese PRV strains without Aspartate insertion. However, the amino acid variations analysis based on major immunogenic and virulence-related genes showed that the JS-2020 strain was not only homologous with earlier PRV strains, but also with strains isolated in recent years. Moreover, the JS-2020 strain was identified as a recombinant between the GXGG-2016 and HLJ-2013 strains. The pathogenicity analysis proved that the PRV JS-2020 strain has typical neurogenic infections and a strong pathogenicity in mice. Together, a novel recombinant classical strain was isolated and characterized in the context of the PRV variant pandemic in China. This study provided some valuable information for the study of the evolution of PRV in China.

Characterization of Brucella abortus Mutant A19mut2, a Potential DIVA Vaccine Candidate with a Modification on Lipopolysaccharide.

BACKGROUND: Brucella abortus is the main causative agent for bovine brucellosis. B. abortus A19 is a widely used vaccine strain to protect cows from Brucella infection in China. However, A19 has a similar lipopolysaccharide (LPS) antigen to that of the field virulent Brucella strain, whose immunization interferes with the serodiagnosis of vaccinated and infected animals. [Aim] To develop a novel Brucella DIVA vaccine candidate. STUDY DESIGN AND METHODS: The B. abortus mutant A19mut2 with the formyltransferase gene wbkC is replaced by an acetyltransferase gene wbdR from E. coli O157 using the bacterial homologous recombination technique, generating a modified O-polysaccharide that cannot induce antibodies in mice against wild-type Brucella LPS. The biological phenotypes of the A19mut2 were assessed using a growth curve analysis, agglutination tests, Western blotting, and stress resistance assays. Histopathological changes and bacterial colonization in the spleens of vaccinated mice were investigated to assess the residual virulence and protection of the A19mut2. Humoral and cellular immunity was evaluated by measuring the levels of IgG, IgG subtypes, and the release of cytokines IFN-γ and IL10 in the splenocytes of the vaccinated mice. ELISA coated with wild-type LPS can distinguish mouse antibodies induced by A19 and A19mut2 immunization. RESULTS: The A19mut2 showed a decreased residual virulence in mice, compared to the A19 strain, but induced significant humoral and cellular immune responses, as the A19 immunization did. The protection efficacy of A19mut2 immunization against B. abortus S2308 NalR infection was similar to that of A19 immunization. CONCLUSION: The A19mut2 has potential as a novel DIVA vaccine candidate in the future.

LGP2 Promotes Type I Interferon Production To Inhibit PRRSV Infection via Enhancing MDA5-Mediated Signaling.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the global pig industry, which modulates the host's innate antiviral immunity to achieve immune evasion. RIG-I-like receptors (RLRs) sense viral RNA and activate the interferon signaling pathway. LGP2, a member of the RLR family, plays an important role in regulating innate immunity. However, the role of LGP2 in virus infection is controversial. Whether LGP2 has a role during infection with PRRSV remains unclear. Here, we found that LGP2 overexpression restrained the replication of PRRSV, while LGP2 silencing facilitated PRRSV replication. LGP2 was prone to interact with MDA5 and enhanced viral RNA enrichment and recognition by MDA5, thus promoting the activation of RIG-I/IRF3 and NF-κB signaling pathways and reinforcing the expression of proinflammatory cytokines and type I interferon during PRRSV infection. Meanwhile, there was a decreased protein expression of LGP2 upon PRRSV infection in vitro. PRRSV Nsp1 and Nsp2 interacted with LGP2 and promoted K63-linked ubiquitination of LGP2, ultimately leading to the degradation of LGP2. These novel findings indicate that LGP2 plays a role in regulating PRRSV replication through synergistic interaction with MDA5. Moreover, targeting LGP2 is responsible for PRRSV immune evasion. Our work describes a novel mechanism of virus-host interaction and provides the basis for preventing and controlling PRRSV. IMPORTANCE LGP2, a member of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), shows higher-affinity binding to RNA and work synergism with RIG-I or MDA5. However, LGP2 has divergent responses to different viruses, which remains controversial in antiviral immune responses. Here, we present the detailed process of LGP2 in positively regulating the anti-PRRSV response. Upon PRRSV infection, LGP2 was prone to bind to MDA5 and enhanced MDA5 signaling, manifesting the enrichment of viral RNA on MDA5 and the activation of downstream IRF3 and NF-κB, which results in increased proinflammatory cytokines and type I interferon expression, ultimately inhibiting PRRSV at the early stage of infection. Moreover, PRRSV Nsp1 and Nsp2 interacted with LGP2 via ubiquitin-proteasome pathways, thus blocking LGP2-mediated immune response. This research helps us understand the host recognition and innate antiviral response to PRRSV infection by neglected pattern recognition receptors, which sheds light on the detailed mechanism of virus-host interaction.

Human and animal exposure to newly discovered sand fly viruses, China.

Introduction: The Hedi virus (HEDV) and Wuxiang virus (WUXV) are newly discovered Bunyaviruses transmitted by sandflies. The geographical distribution of isolation of these two viruses continues to expand and it has been reported that WUXV causes neurological symptoms and even death in suckling mice. However, little is known about the prevalence of the two viruses in mammalian infections. Methods: In order to understand the infection status of HEDV and WUXV in humans and animals from regions where the viruses have been isolated, this study used Western blotting to detect the positive rates of HEDV and WUXV IgG antibodies in serum samples from febrile patients, dogs, and chickens in the forementioned regions. Results: The results showed that of the 29 human serum samples, 17.24% (5/29) tested positive for HEDV, while 68.96% (20/29) were positive for WUXV. In the 31 dog serum samples, 87.10% (27/31) were positive for HEDV and 70.97% (22/31) were positive for WUXV, while in the 36 chicken serum samples, 47.22% (17/36) were positive for HEDV, and 52.78% (19/36) were positive for WUXV. Discussion: These findings suggest there are widespread infections of HEDV and WUXV in mammals (dogs, chickens) and humans from the regions where these viruses have been isolated. Moreover, the positive rate of HEDV infections was higher in local animals compared to that measured in human specimens. This is the first seroepidemiological study of these two sandfly-transmitted viruses. The findings of the study have practical implications for vector-borne viral infections and related zoonotic infections in China, as well as providing an important reference for studies on the relationship between sandfly-transmitted viruses and zoonotic infections outside of China.

Metabolomics Analysis of PK-15 Cells with Pseudorabies Virus Infection Based on UHPLC-QE-MS.

Viruses depend on the metabolic mechanisms of the host to support viral replication. We utilize an approach based on ultra-high-performance liquid chromatography/Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass (UHPLC-QE-MS) to analyze the metabolic changes in PK-15 cells induced by the infections of the pseudorabies virus (PRV) variant strain and Bartha K61 strain. Infections with PRV markedly changed lots of metabolites, when compared to the uninfected cell group. Additionally, most of the differentially expressed metabolites belonged to glycerophospholipid metabolism, sphingolipid metabolism, purine metabolism, and pyrimidine metabolism. Lipid metabolites account for the highest proportion (around 35%). The results suggest that those alterations may be in favor of virion formation and genome amplification to promote PRV replication. Different PRV strains showed similar results. An understanding of PRV-induced metabolic reprogramming will provide valuable information for further studies on PRV pathogenesis and the development of antiviral therapy strategies.

Isolation and genomic characterization of a Chinese NADC34-like PRRSV isolated from Jiangsu province.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important causative agents to swine industry, which has been epidemic more than 30 years. The emergence and recombination of new virus strains bring great challenges to the prevention and control of PRRSV. In the present study, we reported and characterized a novel PRRSV strain, designated as JS2021NADC34, which was for the first time isolated from clinical samples in Jiangsu province, China. Phylogenetic analysis demonstrated that JS2021NADC34 belonging to sublineage 1.5 of PRRSV-2 and was highly related to NADC34-like strains. Genetically, JS2021NADC34 strain had a continuous 100 aa depletion in NSP2, as compared to VR-2332 strain, which was consistent with most reported NADC34-like strains. Moreover, there were several amino acid substitutions occurred in the antigenic regions of GP2-GP5. Similar to other reported NADC34-like PRRSV in China, JS2021NADC34 had no recombination with other domestic strains, which indicates this sublineage of PRRSV may be directly transported from the United States and have not undergone extensive mutation and recombination with local strains yet.

Induction of UPR Promotes Interferon Response to Inhibit PRRSV Replication via PKR and NF-κB Pathway.

Porcine reproductive and respiratory syndrome virus (PRRSV) was previously shown to induce a certain level of cellular stress during viral replication. Unfolded protein response (UPR) is a cellular stress response responsible for coping with stress and cellular survival. However, the pathway leading to the induction of UPR that may influence PRRSV replication is still unknown. Here, we found that PRRSV infection induced UPR prior to interferon response. Induction of UPR significantly enhanced the expression of interferon and interferon-related genes, thus leading to the suppression of PRRSV infection. Next, we explored the underlying mechanisms of UPR-induced antiviral response. We found that induction of UPR promoted the expression of protein kinase R (PKR), and PKR was highly correlated with the reduction of PRRSV replication. Furthermore, tunicamycin stimulation and PKR overexpression activated NF-κB and interferon response at the early stage of PRRSV infection, thus reinforcing the expression of type I interferons and proinflammatory cytokines and leading to inhibition of PRRSV. In addition, PRRSV nsp4 was shown to reduce the expression of PKR. These findings might have implications for our understandings of the host's immune mechanism against PRRSV and a new strategy of PRRSV to evade the host antiviral immunity.

Development and application of an indirect ELISA for the detection of antibody to porcine circovirus 4 in pigs.

Porcine circovirus type 4 (PCV4) was first reported in 2019 in China. So far, the viral DNA was detected from both healthy and diseased pigs in China and South Korea by using molecular techniques including PCR and real-time PCR. In contrast, a serological survey regarding the presence of PCV4 antibodies in the pig population was seldomly reported. In the present study, we describe the development of an indirect enzyme-linked immunosorbent assay (ELISA) based on capsid protein for the detection of PCV4 antibodies in swine sera. After validating the specificity and sensitivity, the ELISA was used in a retrospective serological survey for PCV4 antibodies in pig sera from Jiangsu Province of China. Note that 3.44% of analyzed samples collected between 2018 and 2021 were tested positive for PCV4 antibodies. However, PCV4 genome was absent in all ELISA-positive serum samples. Therefore, the dynamic of viremia and antibody response to PCV4 infection in pigs warrant further investigation.

Characteristics of Brucella abortus vaccine strain A19 reveals its potential mechanism of attenuated virulence.

Brucella vaccination is one of the most important strategies for controlling brucellosis in livestock. The A19 strain was the effective vaccine used to control brucellosis in China. However, the characteristics of physiological and attenuated virulence of the A19 strain are not investigated in detail. In this study, we compared the phenotypic characteristics of the A19 to the wild-type strain S2308. Virulence test showed that the A19 was significantly attenuated at chronic infection stage in infected mouse model. In growth analysis, the A19 exhibited a quick growth at exponential phase and premature at stationary phase. The inflammatory response of macrophages infected by the A19 was detected using TaqMan qPCR assay, indicating that the inflammatory level of the A19-infected macrophages was higher than that of the S2308 infection. Cell death analysis showed that the A19 was not cytotoxic for macrophages. Cell infection showed that the A19 reduced its ability to invade, survive and traffic within host cells, and the intracellular A19 hardly excludes lysosome-associated marker LAMP-1, suggesting that the A19 can't escape the lysosome degradation within host cells. In further study, the sensitivity test exhibited that the A19 is more sensitive to stress and bactericidal factors than the S2308 strain, Western blot and silver staining analysis exhibited that the A19 has a different expression pattern of OMPs and reduces LPS O-antigen expression relative to the S2308 strain. Those data give us a more detailed understanding about the A19 vaccine strain, which will be beneficial for improvement of current Brucella vaccine and overcoming its defects.

Meclizine Inhibits Pseudorabies Virus Replication by Interfering With Virus Entry and Release.

Pseudorabies virus (PRV) is a pathogen that causes substantial economic losses to the swine industry. With the emergence and widespread of PRV variants since 2011 in China, current commercial vaccines cannot provide complete protection against PRV infection. Therefore, antiviral drugs may work as an alternative way to control and prevent PRV. In this study, the inhibitory effects and underlying molecular mechanisms of meclizine against PRV were studied. Meclizine displayed a significant inhibitory effect against PRV when it was added before, simultaneously with, or after virus infection. The inhibitory effect of meclizine occurred during viral entry and cell-to-cell spreading but not at viral attachment into PK-15 cells. Meclizine also inhibited viral particle release at the late stage of infection. The antiviral effect of meclizine was tested in mice, and the results showed that meclizine reduced the severity of clinical symptoms and the viral loads in tissues, and delayed the death, after PRV challenge. The above results indicated that meclizine had an inhibitory effect on PRV. Our findings will contribute to the development of potential therapeutic drugs against PRV infection.

A rough Brucella mutant induced macrophage death depends on secretion activity of T4SS, but not on cellular Txnip- and Caspase-2-mediated signaling pathway.

Brucella is a facultative intracellular bacterium, dividing into smooth- and rough-type Brucella. Smooth-type Brucella can dissociate into rough mutants with cytotoxicity for macrophages during infection, which is critical for Brucella egress and dissemination. However, the mechanism of cytotoxicity infected by rough Brucella is incomplete. In this study, we verified that a rough-type Brucella (RB14 strain) was cytotoxic for macrophages dependent on Type IV secretion system (T4SS). Two specific T4SS VirB4 and VirB11 mutants were constructed, which affect the secretion of T4SS effectors, but not the expression of T4SS components. Cytotoxicity analysis showed that RB14- induced macrophages death depends on T4SS secretion activity. In a further study, 15 reported T4SS effectors were evaluated in inducing macrophage death using over-expression and transfection methods, the results showed that 15 recombinant strains with over-expression of respective effector were not cytotoxicity. In addition, 10 effectors transfected individually, or co-transfected with five effectors barely induced macrophage death, suggesting that all 15 effectors were not associated with macrophage death. Besides, we also evaluated endoplasmic reticulum (ER) stress, Txnip- or Caspase-2 roles in RB14-induced macrophages death. The results showed that inhibition of ER stress, Caspase or Caspase-2 activation was not associated with RB14-infected macrophages death. The casp2 and txnip knockout cells also showed death when infected by the RB14 strain. In all, the RB14-induced macrophage death depends on the secretion activity of T4SS, but not on ER stress, Txnip- or Caspase-2 signal pathway. This study provides a deep insight for rough Brucella-induced macrophage death, which favors for elucidating Brucella infection lifecycle.

Characterization of the main immunogenic proteins in Brucella infection for their application in diagnosis of brucellosis.

Brucellosis is an important zoonotic bacterial disease widespread in the world. The key step of control this disease is accurate diagnosis and elimination of diseased animals. The classic diagnostic methods, such as tube agglutination test, are inaccurate and nonspecific, because of cross-reaction with Yersinia enterocolitica serotype O:9. Previously, several proteins were reported as Brucella main immunogens. In this study, we used animal infection model to evaluate antibody production against OMP16, BP26, BLS, BCSP31, VirB12, SodC and GroEL proteins and investigated their application in diagnosis of brucellosis. The results showed that the BP26 and BLS are two best immunogenic proteins. In further study, we detected 44 clinical bovine sera using western blot, showing that the BP26 and BLS reacted with 30 Brucella-positive sera, but false-positive results were also shown in 14 Brucella-free sera. In an indirect ELISA assay, compared to lipopolysaccharide-based ELISA, the conformance of the BP26-based ELISA was 92.68 % in Brucella-positive sera, but only 52.94 % in Brucella-free sera. The BLS-based ELISA can hardly differentiate positive sera from negative sera. Besides, truncated fragments of the BP26 protein cannot exclude false-positive results in detection of Brucella-free sera. Altogether, although Brucella main immunogenic proteins have good reaction with Brucella-positive sera, false-positive reaction with Brucella-free sera may lead to misdiagnosis of brucellosis, suggesting that it should be more careful to use these immunogenic proteins as antigen targets to diagnosis of brucellosis.

Brucella Infection Regulates Thioredoxin-Interacting Protein Expression to Facilitate Intracellular Survival by Reducing the Production of Nitric Oxide and Reactive Oxygen Species.

Thioredoxin-interacting protein (TXNIP) is a multifunctional protein that functions in tumor suppression, oxidative stress, and inflammatory responses. However, how TXNIP functions during microbial infections is rarely reported. In this study, we demonstrate that Brucella infection decreased TXNIP expression to promote its intracellular growth in macrophages by decreasing the production of NO and reactive oxygen species (ROS). Following Brucella abortus infection, TXNIP knockout RAW264.7 cells produced significantly lower levels of NO and ROS, compared with wild-type RAW264.7 cells. Inducible NO synthase (iNOS) inhibitor treatment reduced NO levels, which resulted in a dose-dependent restoration of TXNIP expression, demonstrating that the expression of TXNIP is regulated by NO. In addition, the expression of iNOS and the production of NO were dependent on the type IV secretion system of Brucella Moreover, Brucella infection reduced TXNIP expression in bone marrow-derived macrophages and mouse lung and spleen. Knocked down of the TXNIP expression in bone marrow-derived macrophages increased intracellular survival of Brucella These findings revealed the following: 1) TXNIP is a novel molecule to promote Brucella intracellular survival by reducing the production of NO and ROS; 2) a negative feedback-regulation system of NO confers protection against iNOS-mediated antibacterial effects. The elucidation of this mechanism may reveal a novel host surveillance pathway for bacterial intracellular survival.

Brucella infection regulates peroxiredoxin-5 protein expression to facilitate intracellular survival by reducing the production of nitric oxide and reactive oxygen species.

Peroxiredoxin-5 (Prdx5) is a multifunctional protein involved in oxidative stress, apoptosis and inflammatory responses. However, how Prdx5 functions during microbial infections is rarely reported. In this study, we demonstrate that Brucella infection increased Prdx5 expression to promote its intracellular growth in macrophages. Further study show that B. abortus infection promoted its intracellular growth by decreasing the production of nitric oxide and reactive oxygen species. In addition, the expression of Prdx5 was independent on live Brucella and the type IV secretion system of Brucella. Instead, its expression was regulated by the lipopolysaccharide of Brucella. Moreover, Brucella infection increased Prdx5 expression in primary macrophage and mice. Collectively, these findings demonstrate for the first time that Prdx5 promotes Brucella intracellular growth by decreasing the production of NO and ROS. This finding provides new insights into the evasive strategies of Brucella and will be useful for the development of novel effective therapeutic approaches to treat Brucella infections.

Identification of a novel, small, conserved hypothetical protein involved in Brucella abortus virulence by modifying the expression of multiple genes.

Brucellosis is an important zoonotic disease worldwide, caused by Brucella spp., which are facultative intracellular bacteria with no classic virulence factors, as virulence is dependent on the ability to invade and replicate within host cells. In this study, we identified a novel gene bab_RS22045 that encodes a small highly conserved protein in Rhizobiales. To investigate the role of this gene, a deletion mutant and complement strain were constructed. Virulence testing showed that bab_RS22045 is necessary for Brucella virulence, and was designated as virulence-related hypothetical protein, VhpA. The results of a cell infection experiment showed that vhpA was not associated with Brucella adherence to and invasion of HeLa cells, or further intracellular survival within RAW264.7 cells. The results of sensitivity testing showed the vhpA mutant had similar sensitivity to hydrogen peroxide, polymyxin B, and sodium nitroprusside as the wild-type (WT) strain. Interestingly, RNA-seq analysis showed that deletion of the vhpA gene affected the expression patterns of multiple Brucella genes, and the main four up-regulated genes and five down-regulated genes were further confirmed using quantitative real-time PCR analysis. Subsequently, a series of over-expression strains were constructed, and virulence testing showed that over-expression of four up-regulated genes (bab_RS17930, bab_RS17925, bab_RS26460, and bab_RS30050) significantly reduced virulence of the WT strain, and over-expression of bab_RS18680 in the vhpA mutant partially restored virulence, suggesting that vhpA plays an important role in Brucella virulence by changing the expression patterns of multiple genes. Additionally, heterogeneous complementary analysis showed that the homologous vhpA genes of Sinorhizobium meliloti and Agrobacterium tumefaciens could not restore virulence of the vhpA mutant, although VhpA is a highly conserved protein in Rhizobiales. Overall, a novel, small, hypothetical gene was identified that is associated with B. abortus virulence, which highlights the roles of small encoding genes in Brucella virulence.