Orf virus induces complete autophagy to promote viral replication via inhibition of AKT/mTOR and activation of the ERK1/2/mTOR signalling pathway in OFTu cells.

Orf virus (ORFV) is the causative agent of contagious ecthyma, which is an important zoonotic pathogen with a widespread distribution affecting sheep, goats and humans. Our previous research showed that autophagy can be induced in host cells by ORFV infection. However, the exact mechanism of ORFV-induced autophagy remains unknown. In this study, we investigated the underlying mechanisms of autophagy induced by ORFV in OFTu cells and the impact of autophagy on ORFV replication. By using specific autophagy inhibitors and activators, Western blotting, immunofluorescence and transmission electron microscopy imaging, we confirmed that ORFV infection triggered intracellular autophagosome accumulation and the activation of autophagic flux. Moreover, ORFV-induced autophagic activity was found to rely on an increase in the phosphorylation of tuberous sclerosis complex 2 (TSC2) and a decrease in the phosphorylation of mammalian target of rapamycin (mTOR), which is mediated by the suppression of the PI3K/AKT/mTOR signalling pathway and activation of the ERK1/2/mTOR signalling pathway. Furthermore, we investigated the role of mTOR-mediated autophagy during ORFV replication using pharmacological agents and demonstrated that ORFV-induced autophagy correlated positively with viral replication. Taken together, our data reveal the pathways of ORFV-induced autophagy and the impact of autophagy on ORFV replication, providing new insights into ORFV pathogenesis.

Complete genomic sequences and comparative analysis of two Orf virus isolates from Guizhou Province and Jilin Province, China.

Orf virus (ORFV, species Orf virus) belongs to the typical species of the Parapoxvirus genus of the family Poxviridae, which infects sheep, goats, and humans with worldwide distribution. Although outbreaks of Orf have been reported sequentially in several Chinese provinces, the epidemiology of Orf and genetic diversity of ORFV strains still needs to be further characterized. To further reveal the genomic organization of the ORFV-GZ18 and ORFV-CL18 isolates, the complete genome sequences of two recently obtained ORFV isolates were sequenced using the next-generation sequencing technology and analyzed, which had been deposited in the GenBank database under accession number MN648218 and MN648219, respectively. The complete genomic sequence of ORFV-CL18 was 138,495 bp in length, including 131 potential open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) of 3481 bp at both ends, which has genomic structure typical Parapoxviruses. The overall genomic organization of the fully sequenced genome of ORFV-GZ18 was consistent with ORFV-CL18 genome, with a complete genome size of 138,446 nucleotides, containing 131 ORFs flanked by ITRs of 3469 bp. Additionally, the overall G + C contents of ORFV-GZ18 and ORFV-CL18 genome sequences were about 63.9% and 63.8%, respectively. The phylogenetic analysis showed that both ORFV-GZ18 and ORFV-CL18 were genetically closely related to ORFV-SY17 derived from sheep. In summary, the complete genomic sequences of ORFV-GZ18 and ORFV-CL18 are reported, with the hope it will be useful to investigate the host range, geographic distribution, and genetic evolution of the virus in Southern West and Northern East China.

Assisted Selection of Biomarkers by Linear Discriminant Analysis Effect Size (LEfSe) in Microbiome Data.

There is growing attention toward closed biological genomes in the environment and in health. To explore and reveal the intergroup differences among different samples or environments, it is crucial to discover biomarkers with statistical differences among groups. The application of Linear discriminant analysis Effect Size (LEfSe) can help find good biomarkers. Based on the original genome data, quality control, and quantification of different sequences based on taxa or genes are carried out. First, the Kruskal-Wallis rank test was used to distinguish between specific differences among statistical and biological groups. Then, the Wilcoxon rank test was performed between the two groups obtained in the previous step to assess whether the differences were consistent. Finally, a linear discriminant analysis (LDA) was conducted to evaluate the influence of biomarkers on significantly different groups based on LDA scores. To sum up, LEfSe provided the convenience for identifying genomic biomarkers that characterize statistical differences among biological groups.

Comammox bacteria predominate among ammonia-oxidizing microorganisms in municipal but not in refinery wastewater treatment plants.

Comammox bacteria have proved to be one dominant and significant ammonia-oxidizing microorganisms (AOMs) in municipal wastewater treatment plants (WWTPs), however, it still remains unknown about their abundance and diversity in industrial WWTPs. In this study, activated sludge samples from 8 municipal WWTPs and 6 industrial WWTPs treating refinery wastewater were taken and analyzed using qPCR and amoA gene sequencing. Intriguingly, quantitative real-time PCR (qPCR) results suggested that comammox bacteria had a higher numerical abundance compared with ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) in municipal WWTPs but did not in refinery WWTPs. Moreover, comammox amoA sequences obtained from high-throughput sequencing were retrieved from all the 8 municipal samples but only 1 industrial sample. Further phylogenetic analysis revealed that N. nitrosa cluster accounted for as high as 79.56% of the total comammox affiliated sequences, which was the most numerically abundant comammox species in municipal WWTPs. This study provided new insights into the abundance and diversity of comammox bacteria in the biological nitrification process in municipal and refinery wastewater treatment systems.

Active ammonia-oxidizing bacteria and archaea in wastewater treatment systems.

Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are two microbial groups mediating nitrification, yet little is presently known about their abundances and community structures at the transcriptional level in wastewater treatment systems (WWTSs). This is a significant issue, as the numerical abundance of AOA or AOB at the gene level may not necessarily represent their functional role in ammonia oxidation. Using amoA genes as molecular markers, this study investigated the transcriptional abundance and community structure of active AOA and AOB in 14 WWTSs. Quantitative PCR results indicated that the transcriptional abundances of AOB amoA (averaged: 1.6 × 108 copies g-1 dry sludge) were higher than those of AOA (averaged: 3.4 × 107 copies g-1 dry sludge) in all WWTSs despite several higher abundances of AOA amoA at the gene level. Moreover, phylogenetic analysis demonstrated that Nitrosomonas europaea and unknown clusters accounted for 37.66% and 49.96% of the total AOB amoA transcripts, respectively, suggesting their dominant role in driving ammonia oxidation. Meanwhile, AOA amoA transcripts were only successfully retrieved from 3 samples, and the Nitrosospaera sister cluster dominated, accounting for 83.46%. Finally, the substrate utilization kinetics of different AOA and AOB species might play a fundamental role in shaping their niche differentiation, community composition, and functional activity. This study provides a basis for evaluating the relative contributions of ammonia-oxidizing microorganisms (AOMs) to nitrogen conversions in WWTSs.

Nitrous oxide (N2O) emissions from a pilot-scale oxidation ditch under different COD/N ratios, aeration rates and two shock-load conditions.

Nitrous oxide (N2O) generated from wastewater treatment plants (WWTPs) has drawn attention due to its high emission load and significant greenhouse effect. In the present study, N2O emissions from a pilot-scale Carrousel oxidation ditch under various chemical oxygen demand (COD) to nitrogen ratio (COD/N) and aeration rates were systematically investigated. The highest N2O emission factor was 0.142 ± 0.013%, at COD/N of 5 and aeration rate of 1.8 m3 h-1, which was much lower than the majority of previous studies. The results could be attributed to the high internal recycle ratio of the oxidation ditch process which lightened the burden of influent load to the system. The profiles of N2O emissions and dissolved N2O concentration along the channels showed a distinct spatial variation that N2O emissions primarily occurred in the aeration zones due to the air stripping effect. However, both the aeration and anoxic zones contributed to N2O generation due to autotrophic nitrification (AN), which was considered to be the main N2O generation process. In addition, two simulated shock-load conditions, ammonia overload shock and aeration failure shock, were carried out to explore the response of the biological nitrogen removal (BNR) system. The results indicated that both shock-loads lead to excessive N2O emissions, especially at higher aeration rates, which could be explained by the improved N2O generation by AN process during the shock-load period. This study offered new insights into the role of operational parameters to N2O emission and the alternative approach for N2O mitigation during both the steady-state operation and shock-load conditions in the oxidation ditch process.

Role of sludge retention time in mitigation of nitrous oxide emission from a pilot-scale oxidation ditch.

Nitrous oxide (N2O) emission from wastewater treatment plants (WWTPs) has become a focus of attention due to its significant greenhouse effect. In this study, the role of sludge retention time (SRT) in mitigation of N2O emission from a pilot-scale oxidation ditch was systematically investigated. The activated sludge system that operated at SRT of 25 days demonstrated significantly lower N2O emission factor, higher resistance to ammonia overload and aeration failure shock than those obtained at SRT of 15 days no matter which hydraulic retention time (HRT) was adopted. Batch experiments revealed that nitrifier denitrification (ND) was the primary mechanism of N2O generation. However, more microbes affiliated with Nitrospira genera were harbored in the system at SRT 25 d, which could effectively avoid nitrite accumulation, a key factor promoting N2O generation by ND. PICRUSt results further suggested the system at SRT 25 d possessed higher genetic potential for N2O reduction reflected by the more abundant nitrous-oxide reductase.

Abundance and community composition of comammox bacteria in different ecosystems by a universal primer set.

Complete ammonia oxidizing bacteria (CAOB) have been recognized as a new member of ammonia-oxidizing microorganisms (AOMs) due to its single-step nitrification capability. However, the abundance and diversity of CAOB in environmental ecosystems were still far from known owing to the lack of specific molecular marker. Herein, a universal primer set specifically targeting both clades of CAOB amoA gene with high specificity and coverage was successfully designed. Intriguingly, real-time quantitative PCR tests revealed that CAOB were ubiquitous and unexpectedly abundant in agricultural soils, river sediments, intertidal zones, drinking water and wastewater treatment systems. Phylogenetic analysis indicated that clade A existed in all the five types of ecosystems, while clade B were only detected in soil and sediment samples. Four sub-clusters were further classified within clade A, in which N. nitrosa cluster dominated CAOB amoA in activated sludge samples while the new recognized soil cluster was the primary constitute in soils. Moreover, the niche specialization between different CAOB species and the environmental conditions were supposed to be the primary driven force to shape the diversity and community of CAOB. This study provided a strong evidence in support of the ubiquities and high abundances of CAOB in various environmental ecosystems and highlighted the significance of including CAOB as the new member of AOMs to re-evaluate the biogeochemical nitrogen cycle.

The effects of graphene oxide on nitrification and N2O emission: Dose and exposure time dependent.

With the extensive application of graphene oxide (GO), its leakage and release into wastewater treatment plants become inevitable. However, the toxicity of graphene oxide (GO) on nitrification process and the underlying mechanisms still remain unclear. In this study, the toxic effects of GO at concentration of 10 and 100 mg/L in 4 h and 10 days were evaluated with sealed reactors operated in sequencing batch mode. In the initial 4 h, both GO concentrations showed no negative effect on nitrogen conversion. However, the exposure to 100 mg/L GO significantly weakened the NH+ 4-N and NO- 2-N conversion capabilities and intensified the nitrous oxide (N2O) generation after 10 days. Extracellular polymeric substance (EPS) analysis suggested that 100 mg/L GO decreased the protein content of the nitrifying activated sludge. Moreover, reactive oxygen species (ROS) level was promoted by 100 mg/L GO owing to the impaired endogenous antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT), which caused oxidative stress to bacteria. Finally, quantitative PCR results confirmed that nitrite-oxidizing bacteria (NOB) and complete ammonia oxidizing bacteria (CAOB) were more sensitive to GO, which was the primary cause for the significant promotion of N2O generation in the high GO concentration. This study offered new insights in the toxicity of GO on nitrification and N2O generation in the terms of dose and exposure time.

Transcriptional activity and diversity of comammox bacteria as a previously overlooked ammonia oxidizing prokaryote in full-scale wastewater treatment plants.

The discovery of complete ammonia oxidizing bacteria (CAOB) has fundamentally overturned the traditional recognition of nitrification. However, little was known about the transcriptional activity and diversity of the newly recognized ammonia oxidizing prokaryote in engineered ecosystems. To fill this gap, transcriptional investigations of CAOB amoA genes were carried out comparatively with the canonical ammonia oxidizing bacteria (AOB) and archaea (AOA) in eight full-scale wastewater treatment plants (WWTPs). Remarkably, qPCR results revealed the transcriptional levels of CAOB amoA gene were unexpectedly high in most of samples with the highest 24-fold that of AOB amoA, suggesting CAOB were actively participating in ammonia oxidation while they were previously overlooked. This result also well explained the confusing high abundances of genus Nitrospira which were frequently detected in WWTPs. Furthermore, phylogenetic analysis based on high throughput sequencing indicated the CAOB amoA gene sequences formed three well-supported clusters and Nitrospira nitrosa cluster accounted for 97% of all the retrieved sequences, which was supposed to be the dominant taxon of CAOB in the ammonia-intensive environment due to niche partitioning. This study highlighted the significance of including the newly discovered ammonia oxidizing bacterial member when assessing the nitrification process and ecological function in the future.