Mixed Transcriptome Analysis Revealed the Possible Interaction Mechanisms between Zizania latifolia and Ustilago esculenta Inducing Jiaobai Stem-Gall Formation.

The smut fungus Ustilago esculenta infects Zizania latifolia and induces stem expansion to form a unique vegetable named Jiaobai. Although previous studies have demonstrated that hormonal control is essential for triggering stem swelling, the role of hormones synthesized by Z. latifolia and U. esculenta and the underlying molecular mechanism are not yet clear. To study the mechanism that triggers swollen stem formation, we analyzed the gene expression pattern of both interacting organisms during the initial trigger of culm gall formation, at which time the infective hyphae also propagated extensively and penetrated host stem cells. Transcriptional analysis indicated that abundant genes involving fungal pathogenicity and plant resistance were reprogrammed to maintain the subtle balance between the parasite and host. In addition, the expression of genes involved in auxin biosynthesis of U. esculenta obviously decreased during stem swelling, while a large number of genes related to the synthesis, metabolism and signal transduction of hormones of the host plant were stimulated and showed specific expression patterns, particularly, the expression of ZlYUCCA9 (a flavin monooxygenase, the key enzyme in indole-3-acetic acid (IAA) biosynthesis pathway) increased significantly. Simultaneously, the content of IAA increased significantly, while the contents of cytokinin and gibberellin showed the opposite trend. We speculated that auxin produced by the host plant, rather than the fungus, triggers stem swelling. Furthermore, from the differently expressed genes, two candidate Cys2-His2 (C2H2) zinc finger proteins, GME3058_g and GME5963_g, were identified from U. esculenta, which may conduct fungus growth and infection at the initial stage of stem-gall formation.

Identification of two novel epitopes targeting glycoprotein E of pseudorabies virus using monoclonal antibodies.

Pseudorabies virus (PRV), the agent of pseudorabies, has raised considerable attention since 2011 due to the outbreak of emerging PRV variants in China. In the present study, we obtained two monoclonal antibodies (mAbs) known as 2E5 and 5C3 against the glycoprotein E (gE) of a PRV variant (JS-2012 strain). The two mAbs reacted with wild PRV but not the vaccine strain (gE-deleted virus). The 2E5 was located in 161RLRRE165, which was conserved in almost of all PRV strains, while 5C3 in 148EMGIGDY154 was different from almost of all genotype I PRV, in which the 149th amino acid is methionine (M) instead of arginine (R). The two epitopes peptides located in the hydrophilic region and reacted with positive sera against genotype II PRV (JS-2012), which suggests they were likely dominant B-cell epitopes. Furthermore, the mutant peptide 148ERGIGDY154 (genotype I) did not react with the mAb 5C3 or positive sera against genotype II PRV (JS-2012). In conclusion, both mAb 2E5 and 5C3 could be used to identify wild PRV strains from vaccine strains, and mAb 5C3 and the epitope peptide of 5C3 might be used for epidemiological investigation to distinguish genotype II from genotype I PRV.

MOV10 inhibits replication of porcine reproductive and respiratory syndrome virus by retaining viral nucleocapsid protein in the cytoplasm of Marc-145 cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been a major threat to global industrial pig farming ever since its emergence in the late 1980s. Identification of sustainable and effective control measures against PRRSV transmission is a pressing problem. The nucleocapsid (N) protein of PRRSV is specifically localized in the cytoplasm and nucleus of virus-infected cells which is important for PRRSV replication. In the current study, a new host restricted factor, Moloney leukemia virus 10-like protein (MOV10), was identified as an inhibitor of PRRSV replication. N protein levels and viral replication were significantly reduced in Marc-145 cells stably overexpressing MOV10 compared with those in wild-type Marc-145 cells. Adsorption experiments revealed that MOV10 did not affect the attachment and internalization of PRRSV. Co-immunoprecipitation and immunofluorescence co-localization analyses showed that MOV10 interacted and co-localized with the PRRSV N protein in the cytoplasm. Notably, MOV10 affected the distribution of N protein in the cytoplasm and nucleus, leading to the retention of N protein in the former. Taken together, these findings demonstrate for the first time that MOV10 inhibits PRRSV replication by restricting the nuclear import of N protein. These observations have great implications for the development of anti-PRRSV drugs and provide new insight into the role of N protein in PRRSV biology.

The gene expression profile of porcine alveolar macrophages infected with a highly pathogenic porcine reproductive and respiratory syndrome virus indicates overstimulation of the innate immune system by the virus.

Since the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) variant emerged in 2006, it has caused death in more than 20 million pigs in China and other Southeast Asian countries, making it the most destructive swine pathogen currently in existence. To characterize the cellular responses to HP-PRRSV infection, the gene expression profile of porcine alveolar macrophage (PAM) cells, the primary target cells of PRRSV, was analyzed in HP-PRRSV-infected and uninfected PAMs by suppression subtractive hybridization. After confirmation by Southern blot, genes that were differentially expressed in the HP-PRRSV-infected and uninfected PAMs were sequenced and annotated. Genes that were upregulated mainly in HP-PRRSV-infected PAM cells were related to immunity and cell signaling. Among the differentially expressed genes, Mx1 and HSP70 protein expression was confirmed by western blotting, and IL-8 expression was confirmed by ELISA. In PAM cells isolated from HP-PRRSV-infected piglets, the differential expression of 21 genes, including IL-16, TGF-beta type 1 receptor, epidermal growth factor, MHC-I SLA, Toll-like receptor, hepatoma-derived growth factor, FTH1, and MHC-II SLA-DRB1, was confirmed by real-time PCR. To our knowledge, this is the first study to demonstrate differential gene expression between HP-PRRSV-infected and uninfected PAMs in vivo. The results indicate that HP-PRRSV infection excessively stimulates genes involved in the innate immune response, including proinflammatory cytokines and chemokines.

Complete genome sequence of a virulent porcine epidemic diarrhea virus strain.

In recent years, acute outbreaks of epizootic diarrhea have occurred on many swine farms in China. Although the putative causative virus of the disease was not isolated, the genomic sequence of porcine epidemic diarrhea virus (PEDV) was consistently detected from feces of diseased pigs by reverse transcription-PCR (RT-PCR). Here we report a complete genome sequence of PEDV which is apparently different from those of early PEDV circulated in Chinese swine herds.

Identification of nonessential regions of the nsp2 protein of an attenuated vaccine strain (HuN4-F112) of highly pathogenic porcine reproductive and respiratory syndrome virus for replication in marc-145 cell.

BACKGROUND: The regions in the middle of nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) have been shown to be nonessential for PRRSV replication, and these nonessential regions are different in various viral strains. FINDING: In this study, the nonessential regions of the nsp2 of an attenuated vaccine strain (HuN4-F112) of highly pathogenic porcine reproductive and respiratory syndrome virus were identified based on an infectious cDNA clone of HuN4-F112. The results demonstrated that the segments of nsp2 [amino acids (aa) 480 to 667] tolerated deletions. Characterization of the mutants demonstrated that those with small deletions did not affect the viral growth on Marc-145 cells, but deletion of these regions led to earlier PRRSV replication increased (before 36 h after infectious in vitro). CONCLUSION: The functional roles of nsp2 variable middle region for PRRSV HuN4-F112 replication have been identified. Our results also suggested that none-essential region might be an ideal insertion region to express foreign gene in PRRSV genome.

cGAS Restricts PRRSV Replication by Sensing the mtDNA to Increase the cGAMP Activity.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that causes great economic losses globally to the swine industry. Innate immune RNA receptors mainly sense it during infection. As a DNA sensor, cyclic GMP-AMP synthase (cGAS) plays an important role in sensing cytosolic DNA and activating innate immunity to induce IFN-I and establish an antiviral cellular state. In contrast, the role of innate immune DNA sensors during PRRSV infection has not been elucidated. In this study, we found that cGAS facilitates the production of IFN-ß during PRRSV infection. Western blot and virus titer assays suggested that cGAS overexpression suppressed the replication of multiple PRRSV strains, while knockout of cGAS increased viral titer and nucleocapsid protein expression. Besides, our results indicated that the mitochondria were damaged during PRRSV infection and leaked mitochondrial DNA (mtDNA) into the cytoplasm. The mtDNA in the cytoplasm co-localizes with the cGAS, and the cGAMP activity was increased when the cGAS was overexpressed during PRRSV infection. Furthermore, the cGAMP also possesses an anti-PRRSV effect. These results indicate for the first time that cGAS restricts PRRSV replication by sensing the mtDNA in the cytoplasm to increase cGAMP activity, which not only explains the molecular mechanism by which cGAS inhibits PRRSV replication but also provides research ideas for studying the role of the cGAS-STING signaling pathway in the process of RNA virus infection.

Recombinant pseudorabies virus expressing E2 of classical swine fever virus (CSFV) protects against both virulent pseudorabies virus and CSFV.

Both classical swine fever (CSF) and pseudorabies are highly contagious, economically significant diseases of swine in China. Although vaccination with the C-strain against classical swine fever virus (CSFV) is widely carried out and severe outbreaks of CSF seldom occur in China, CSF is sporadic in many pig herds and novel sub-subgenotypes of CSFV endlessly emerge. Thus, new measures are needed to eradicate CSFV from Chinese farms. The emergence of a pseudorabies virus (PRV) variant also posed a new challenge for the control of swine pseudorabies. Here, the recombinant PRV strain JS-2012-ΔgE/gI-E2 expressing E2 protein of CSFV was developed by inserting the E2 expression cassette into the intergenic region between the gG and gD genes of the gE/gI-deletion PRV variant strain JS-2012-ΔgE/gI. The recombinant virus was stable when passaged in vitro. A single vaccination of JS-2012-ΔgE/gI-E2 via intramuscular injection fully protected against lethal challenges of PRV and CSFV. Vaccination of piglets with the recombinant JS-2012-ΔgE/gI-E2 in the presence of high levels of maternally derived antibodies (Abs) to PRV can provide partial protection against lethal challenge of CSFV. Vaccination of the recombinant PRV JS-2012-ΔgE/gI-E2 strain did not induce the production of Abs to the gE protein of PRV or to the CSFV proteins other than E2. Thus, JS-2012-ΔgE/gI-E2 appears to be a promising recombinant marker vaccine candidate against PRV and CSFV for the control and eradication of the PRV variant and CSFV.

Nucleocapsid protein of porcine reproductive and respiratory syndrome virus antagonizes the antiviral activity of TRIM25 by interfering with TRIM25-mediated RIG-I ubiquitination.

Porcine reproductive and respiratory syndrome (PRRS) is caused by PRRS virus (PRRSV), and is characterized by respiratory diseases in piglet and reproductive disorders in sow. Identification of sustainable and effective measures to mitigate PRRSV transmission is a pressing problem. The nucleocapsid (N) protein of PRRSV plays a crucial role in inhibiting host innate immunity during PRRSV infection. In the current study, a new host-restricted factor, tripartite motif protein 25 (TRIM25), was identified as an inhibitor of PRRSV replication. Co-immunoprecipitation assay indicated that the PRRSV N protein interferes with TRIM25-RIG-I interactions by competitively interacting with TRIM25. Furthermore, N protein inhibits the expression of TRIM25 and TRIM25-mediated RIG-I ubiquitination to suppress interferon ß production. Furthermore, with increasing TRIM25 expression, the inhibitory effect of N protein on the ubiquitination of RIG-I diminished. These results indicate for the first time that TRIM25 inhibits PRRSV replication and that the N protein antagonizes the antiviral activity by interfering with TRIM25-mediated RIG-I ubiquitination. This not only provides a theoretical basis for the development of drugs to control PRRSV replication, but also better explains the mechanism through which the PRRSV N protein inhibits innate immune responses of the host.

Effects of RNA interference-induced tryptase down-regulation in P815 cells on IL-6 and TNF-alpha release of endothelial cells.

OBJECTIVE: To explore the effects of down-regulated tryptase expression in mast cells on the synthesis and release of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) of vascular endothelial cells. METHODS: Tryptase-siRNA (small-interfering RNA) vector was constructed to inhibit tryptase expression in P815 cells. The medium of P815 cells treated by the tryptase-siRNA (RNAi-P815 group) or pure vector (P815 group) was collected and used to culture bEnd.3 cells. The messenger RNAs (mRNAs) of IL-6 and TNF-alpha in bEnd.3 cells and their protein levels in the medium were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: IL-6 and TNF-alpha mRNAs in bEnd.3 cells cultured in RNAi-P815-conditioned medium decreased significantly compared to those in P815-conditioned medium. Consistently, IL-6 and TNF-alpha protein levels in the medium of bEnd.3 of RNAi-P815 group were lower than those of P815 group. CONCLUSION: Reduced tryptase expression significantly inhibited the synthesis and release of IL-6 and TNF-alpha in vascular endothelial cells. RNA interference targeting tryptase expression may be a new anti-inflammatory strategy for vascular diseases.

Process and Results of Implementing Disease Management Program in Patients with First-time Ischemic Stroke.

BACKGROUND: This study aimed to examine the effect of disease management program (DMP) on the patients with first-time ischemic stroke (IS). METHODS: A DMP with 4 parts of performance indicators (PIs, including outpatient, emergency department, inpatient and follow-up treatment) was implemented in patients with stroke in 2 hospitals (Hospital T and R) in Shanghai China from 2007-2010. The effect of DMP on the outcome of IS patients was analyzed according to the criteria of the National Institute of Health Stroke Scale (NIHSS). Furthermore, the total effective rate of DMP, average length of stay, hospitalization cost, and cost-effectiveness ratio (CER) between DMP and non-DMP patients were calculated, followed by the cost-effectiveness analysis. RESULTS: The total effective rate of DMP (T: 69.9%; R: 76.6%) was significantly (P<0.05) higher than that of non-DMP (T: 60.8%; R: 62.7%) group in the same hospital. In addition, a significant (P<0.05) difference in effective rate was observed between DMP and non-DMP at the NIHSS score ≥ 7. Furthermore, the average length of stay and hospitalization cost of the patients in DMP group were significantly (P<0.05) lower than those in non-DMP group. A superior CER was also found in DMP group than non-DMP group. CONCLUSION: The implementation of DMP for IS can effectively improve the treatment outcome and reduce the average length of stay and hospitalization cost.

The emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus with additional 120aa deletion in Nsp2 region in Jiangxi, China.

Highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS), which emerged in China in 2006, was characterized by high fever, high morbidity and high mortality. The causative agent of the disease was a highly pathogenic variant of porcine reproductive and respiratory syndrome virus (also called HP-PRRSV), which has a discontinuous deletion of 1 + 29 amino acids (aa) in the Nsp2 coding region, compared to classical PRRSV. In 2014, fattened pigs on a pig farm in Jiangxi Province suffered from clinical symptoms of high fever, dyspnoea and death. A PRRSV, termed JX2014T2, was isolated from samples of the dead pigs. Genomic analysis of the isolated PRRSV indicated that the genome of the virus was 14,960 bp in length and belonged to the North American genotype. In the Nsp2-coding region, there was a discontinuous deletion of 1 + 29 aa, similar to HP-PRRSV; however, an additional continuous deletion of 120 amino acids between aa 628 and 747 was found. Further analysis of the pathogenicity of PRRSV JX2014T2 was performed in piglets, and the results indicated that all infected piglets suffered from typical clinical symptoms of PRRS, such as high fever, cough, mental depression, anorexia, dyspnoea and palpebral swelling and died within 15 days postinfection (dpi). This demonstrated that the newly isolated PRRSV JX2014T2 strain containing an additional deletion of 120 aa is highly pathogenic to piglets, suggesting that a highly pathogenic variant with new genetic features is circulating in China.

Monoclonal Antibody Against HA Protein of the European Avian-Like H1N1 Swine Influenza Virus.

The purified whole-virus proteins derived from A/swine/Shanghai/1/2014 (H1N1) (SH1) were chosen to immunize BALB/c mice to prepare the monoclonal antibody (MAb) against hemagglutinin (HA) protein of an European avian-like (EA) H1N1 swine influenza virus (SIV). After cloning three times by limiting dilution, one strain of hybridoma cells named 3C7 secreting anti-HA protein MAb was obtained by hybridoma technique. The results of indirect immunofluorescence assay and western blot analyses showed that the MAb 3C7 specifically reacted with the HA protein of EA H1N1 SIV. This work indicated that the MAb 3C7 would be a valuable tool as a specific diagnostic reagent for SIV epidemiological surveys and identification of HA protein epitopes of the EA H1N1 SIVs in the future.

Monitoring imaging of lesions induced by high intensity focused ultrasound based on differential ultrasonic attenuation and integrated backscatter estimation.

We investigated the feasibility of two monitoring imaging methods to visualize and evaluate the high intensity focused ultrasound (HIFU) induced lesions in vitro during and after their formation, which were based on differential ultrasonic parameter estimation. Firstly, ultrasonic attenuation slope of tissue sample was estimated based on the spectral analysis of ultrasound RF backscattered signals. The differential attenuation slope maps were acquired, which were interpreted as the differences between the pretreatment image and those obtained in different stages during HIFU therapy. Secondly, ultrasonic integrated backscatter (IBS), defined as the frequency average of the backscatter transfer function over the useful bandwidth, was proposed quantitatively to evaluate the extent of lesions with the same RF signals as the first method. Differential IBS maps were also acquired to visualize temporal evolution of lesion formation. It was found in pig liver in vitro that more precise definition of the treated area was obtained from the differential IBS images than from differential attenuation slope images. Dramatic increase in both attenuation and IBS value was observed during the therapy, which may be related to dramatic enhancement of cavitation due to boiling and accompanying tissue damage. Two methods to obtain one differential image were compared and the cumulative differential image was found to be able to eliminate noises and artifacts to some extent, which was the cumulation of a series of differential images acquired from the differences between the temporally adjacent RF data frames. Moreover, we presented a bidirectional color code for identification of the artifacts due to tissue movements caused by HIFU radiation force. We conclude that cumulative differential IBS images have the potential to monitor the formation of HIFU-induced lesions.

[Characteristics of Nitrogen and Phosphorus Losses in Longhong Ravine Basin of Westlake in Rainstorm Runoff].

In order to understand the non-point source pollution status in Longhong ravine basin of Westlake, the characteristics of nutrient losses in runoff was investigated during three rainstorms in one year. The results showed that long duration rainstorm event generally formed several runoff peaks, and the time of its lag behind the peaks of rain intensity was dependent on the distribution of heavy rainfall. The first flush was related to the antecedent rainfall, and the less rainfall in the earlier period, the more total phosphorus (TP) and ammonia (NH4+ -N) in runoff was washed off. During the recession of runoff, more subsurface runoff would result in a concentration peak of total nitrogen (TN) and nitrogen (NO3- -N) . The event mean concentration (EMC) of runoff nitrogen had a negative correlation with rainfall, rainfall duration, maximum rain intensity and average rain intensity except for antecedent rainfall, whereas the change in TP EMC showed the opposite trend. The transport fluxes of nutrients increased with an elevation in runoffs, and Pearson analysis showed that the transport fluxes of TN and NO3- -N had good correlations with runoff depth. The average transport fluxes of TP, TN, NH4+ -N and NO3- -N were 34.10, 1195.55, 1006.62 and 52.38 g x hm(-2), respectively, and NO3- -N was the main nitrogen form and accounted for 84% of TN.

A novel M2e-multiple antigenic peptide providing heterologous protection in mice.

Swine influenza viruses (SwIVs) cause considerable morbidity and mortality in domestic pigs, resulting in a significant economic burden. Moreover, pigs have been considered to be a possible mixing vessel in which novel strains loom. Here, we developed and evaluated a novel M2e-multiple antigenic peptide (M2e-MAP) as a supplemental antigen for inactivated H3N2 vaccine to provide cross-protection against two main subtypes of SwIVs, H1N1 and H3N2. The novel tetra-branched MAP was constructed by fusing four copies of M2e to one copy of foreign T helper cell epitopes. A high-yield reassortant H3N2 virus was generated by plasmid based reverse genetics. The efficacy of the novel H3N2 inactivated vaccines with or without M2e-MAP supplementation was evaluated in a mouse model. M2e-MAP conjugated vaccine induced strong antibody responses in mice. Complete protection against the heterologous swine H1N1 virus was observed in mice vaccinated with M2e-MAP combined vaccine. Moreover, this novel peptide confers protection against lethal challenge of A/Puerto Rico/8/34 (H1N1). Taken together, our results suggest the combined immunization of reassortant inactivated H3N2 vaccine and the novel M2e-MAP provided cross-protection against swine and human viruses and may serve as a promising approach for influenza vaccine development.

Emergence of a Pseudorabies virus variant with increased virulence to piglets.

Pseudorabies virus (PRV) causes Pseudorabies (PR), an economically important disease in domestic swine. PR outbreaks on pig farms caused by PRV variant strains in Bartha-K61-vaccinated pigs have resulted in considerable economic losses in China since 2011. In this study, the pathogenicity of the PRV variant JS-2012 strain to pigs was investigated by experimentally inoculating piglets of different ages in comparison with a classic virulent PRV SC strain. The JS-2012 strain caused an earlier onset of clinical signs and higher mortality in 15, 30, and 60-day-old pigs, as compared with a classic virulent PRV SC strain. The Bartha-K61 vaccination provided complete protection against challenge with classical virulent PRV, but only partial protection against challenge with the JS-2012 strain in piglets. In conclusion, the increased virulence of the PRV variant may have partly contributed to the PR outbreak in China.

Characterization of three porcine reproductive and respiratory syndrome virus isolates from a single swine farm bearing strong homology to a vaccine strain.

Three porcine reproductive and respiratory syndrome viruses (PRRSV), NT1, NT2, and NT3, were isolated from three dying piglets from a single pig farm in Jiangsu Province, China. Whole genome sequencing revealed that the three isolates share the highest homology with JXA1-P80, an attenuated vaccine strain developed by serial passage of highly pathogenic PRRSV JXA1 in MARC-145 cells. More than ten amino acids residues in ORF1a, ORF1b, GP4, and GP5 that were thought to be unique to JXA1 attenuated on MARC-145 cells were each found in the corresponding locations of NT1, NT2, and NT3. In virulence assays, piglets infected with NT1, NT2, or NT3 exhibited clinical signs of disease, including high fever, anorexia, and respiratory distress, leading to the death of the majority of the piglets within two weeks. Collectively, these data indicate that NT1, NT2, and NT3 are highly pathogenic PRRSVs and they are likely to be revertants of the vaccine strain JXA1-P80.

Genomic characterization of emergent pseudorabies virus in China reveals marked sequence divergence: Evidence for the existence of two major genotypes.

Recently pseudorabies outbreaks have occurred in many vaccinated farms in China. To identify genetic characteristics of pseudorabies virus (PRV) strains, we obtained the genomic sequences of PRV strains HeN1 and JS, which were compared to 4 PRV genomes and 729 partial gene sequences. PRV strains isolated in China showed marked sequence divergence compared to European and American strains. Phylogenetic analysis revealed that for the first time PRV can be divided into 2 distinct clusters, with Chinese strains being genotype II and PRVs isolated from other countries being genotype I. Restriction fragment length polymorphism analysis confirmed differences between HeN1 and Bartha strains, as did the presence of unique insertion/deletion polymorphisms and microsatellites. This divergence between the two genotypes may have been generated from long-term, independent evolution, which could also explain the low efficacy of the Bartha vaccine in protecting pigs infected with genotype II PRV.

Treating both wastewater and excess sludge with an innovative process.

The innovative process consists of biological unit for wastewater treatment and ozonation unit for excess sludge treatment. An aerobic membrane bioreactor (MBR) was used to remove organics and nitrogen, and an anaerobic reactor was added to the biological unit for the release of phosphorus contained at aerobic sludge to enhance the removal of phosphorus. For the excess sludge produced in the MBR, which was fed to ozone contact column and reacted with ozone, then the ozonated sludge was returned to the MBR for further biological treatment. Experimental results showed that this process could remove organics, nitrogen and phosphorus efficiently, and the removals for COD, NH3-N, TN and TP were 93.17%, 97.57%, 82.77% and 79.5%, respectively. Batch test indicated that the specific nitrification rate and specific denitrification rate of the MBR were 1.03 mg NH3-N/(gMLSS x h) and 0.56 mg NOx-N/(gMLSS x h), and denitrification seems to be the rate-limiting step. Under the test conditions, the sludge concentration in the MBR was kept at 5000-6000 mg/L, and the wasted sludge was ozonated at an ozone dosage of 0.10 kgO3/kgSS. During the experimental period of two months, no excess sludge was wasted, and a zero withdrawal of excess sludge was implemented. Through economic analysis, it was found that an additional ozonation operating cost for treatment of both wastewater and excess sludge was only 0.045 RMB Yuan (USD 0.0054)/m3 wastewater.