A comprehensive survey of bat sarbecoviruses across China in relation to the origins of SARS-CoV and SARS-CoV-2.

SARS-CoV and SARS-CoV-2 have been thought to originate from bats. In this study, we screened pharyngeal and anal swabs from 13 064 bats collected between 2016 and 2021 at 703 locations across China for sarbecoviruses, covering almost all known southern hotspots, and found 146 new bat sarbecoviruses. Phylogenetic analyses of all available sarbecoviruses show that there are three different lineages-L1 as SARS-CoV-related CoVs (SARSr-CoVs), L2 as SARS-CoV-2-related CoVs (SC2r-CoVs) and novel L-R (recombinants of L1 and L2)-present in Rhinolophus pusillus bats, in the mainland of China. Among the 146 sequences, only four are L-Rs. Importantly, none belong in the L2 lineage, indicating that circulation of SC2r-CoVs in China might be very limited. All remaining 142 sequences belong in the L1 lineage, of which YN2020B-G shares the highest overall sequence identity with SARS-CoV (95.8%). The observation suggests endemic circulations of SARSr-CoVs, but not SC2r-CoVs, in bats in China. Geographic analysis of the collection sites in this study, together with all published reports, indicates that SC2r-CoVs may be mainly present in bats of Southeast Asia, including the southern border of Yunnan province, but absent in all other regions within China. In contrast, SARSr-CoVs appear to have broader geographic distribution, with the highest genetic diversity and sequence identity to human sarbecoviruses along the southwest border of China. Our data provide the rationale for further extensive surveys in broader geographical regions within, and beyond, Southeast Asia in order to find the most recent ancestors of human sarbecoviruses.

Molecular evolution of human coronavirus-NL63, -229E, -HKU1 and -OC43 in hospitalized children in China.

Human coronaviruses (HCoVs) HCoV-NL63, HCoV-229E, HCoV-HKU1 and HCoV-OC43 have been circulated in the human population worldwide, and they are associated with a broad range of respiratory diseases with varying severity. However, there are neither effective therapeutic drugs nor licensed vaccines available for the treatment and prevention of infections by the four HCoVs. In this study, we collected nasopharyngeal aspirates of children hospitalized for respiratory tract infection in China during 2014-2018 and conducted next-generation sequencing. Sequences of four HCoVs were then selected for an in-depth analysis. Genome sequences of 2 HCoV-NL63, 8 HCoV-229E, 2 HCoV-HKU1, and 6 HCoV-OC43 were obtained. Based on the full-length S gene, a strong temporal signal was found in HCoV-229E and the molecular evolutionary rate was 6 × 10-4 substitutions/site/year. Based on the maximum-likelihood (ML) phylogenetic tree of complete S gene, we designated H78 as a new sub-genotype C2 of HCoV-HKU1, and the obtained P43 sequence was grouped into the reported novel genotype K of HCoV-OC43 circulating in Guangzhou, China. Based on the complete genome, potential recombination events were found to occur as two phenomena, namely intraspecies and interspecies. Moreover, we observed two amino acid substitutions in the S1 subunit of obtained HCoV-NL63 (G534V) and HCoV-HKU1 (H512R), while residues 534 and 512 are important for the binding of angiotensin-converting enzyme 2 and neutralizing antibodies, respectively. Our findings might provide a clue for the molecular evolution of the four HCoVs and help in the early diagnosis, treatment and prevention of broad-spectrum HCoV infection.

Genome-wide identification and functional analysis of circRNAs in Trichophyton rubrum conidial and mycelial stages.

BACKGROUND: Circular RNAs (circRNAs) are a group of noncoding RNAs that participate in gene expression regulation in various pathways. The essential roles of circRNAs have been revealed in many species. However, knowledge of circRNAs in fungi is still not comprehensive. RESULTS: Trichophyton rubrum (T. rubrum) is considered a model organism of human pathogenic filamentous fungi and dermatophytes. In this study, we performed a genome-wide investigation of circRNAs in T. rubrum based on high-throughput sequencing and ultimately identified 4254 circRNAs. Most of these circRNAs were specific to the conidial or mycelial stage, revealing a developmental stage-specific expression pattern. In addition, 940 circRNAs were significantly differentially expressed between the conidial and mycelial stages. PCR experiments conducted on seven randomly selected differentially expressed (DE-) circRNAs confirmed the circularized structures and relative expression levels of these circRNAs. Based on their genome locations, most circRNAs originated from intergenic regions, unlike those in plants and animals. Furthermore, we constructed circRNA-miRNA-mRNA regulatory networks that included 661 DE-circRNAs targeting 140 miRNAs and further regulating 2753 mRNAs. The relative expression levels of two randomly selected circRNA-miRNA-mRNA axes were investigated by qRT-PCR, and the competing endogenous RNA (ceRNA) network theory was validated. Functional enrichment analysis of the target genes suggested that they were significantly involved in posttranscriptional processes and protein synthesis as well as some small-molecule metabolism processes. CircRNAs are relatively more conserved in closely related dermatophytes but rarely conserved in distantly related species. Tru_circ07138_001 is a highly conserved circRNA that was conserved in all ten dermatophytes analyzed in our study and three distantly related species. Its host gene TERG_07138 was also highly conserved in two of these distantly related species Gallus gallus and Caenorhabditis elegans. The specific role of this circRNA deserves further exploration. CONCLUSIONS: Our study is the first to provide a global profile of circRNAs in T. rubrum as well as dermatophytes. These results could serve as valuable resources for research on circRNA regulatory mechanisms in fungi and reveal new insights for further investigation of the physical characteristics of these significant human fungal pathogens.

Genetic Characteristics of Human Parainfluenza Virus Types 1-4 From Patients With Clinical Respiratory Tract Infection in China.

Human parainfluenza viruses (HPIV1-4) cause acute respiratory tract infections, thereby impacting human health worldwide. However, there are no current effective antivirals or licensed vaccines for infection prevention. Moreover, sequence information for human parainfluenza viruses (HPIVs) circulating in China is inadequate. Therefore, to shed light on viral genetic diversity and evolution, we collected samples from patients infected with HPIV1-4 in China from 2012 to 2018 to sequence the viruses. We obtained 24 consensus sequences, comprising 1 for HPIV1, 2 for HPIV2, 19 for HPIV3, and 2 for HPIV4A. Phylogenetic analyses classified the 1 HPIV1 into clade 2, and the 2 HPIV4 sequences into cluster 4A. Based on the hemagglutinin-neuraminidase (HN) gene, a new sub-cluster was identified in one of the HPIV2, namely G1c, and the 19 HPIV3 sequences were classified into the genetic lineages of C3f and C3a. The results indicated that HPIV1-4 were co-circulated in China. Further, the lineages of sub-cluster C3 of HPIV3 were co-circulated in China. A recombination analysis indicated that a putative recombination event may have occurred in the HN gene of HPIV3. In the obtained sequences of HPIV3, we found that two amino acid substitution sites (R73K in the F protein of PUMCH14028/2014 and A281V in the HN protein of PUMCH13961/2014) and a negative selection site (amino acid position 398 in the F protein) corresponded to the previously reported neutralization-related sites. Moreover, amino acid substitution site (K108E) corresponded to the negative selection site (amino acid position 108) in the 10 F proteins of HPIV3. However, no amino acid substitution site corresponded to the glycosylation site in the obtained HPIV3 sequences. These results might help in studying virus evolution, developing vaccines, and monitoring HPIV-related respiratory diseases.

An Optimized Metagenomic Approach for Virome Detection of Clinical Pharyngeal Samples With Respiratory Infection.

Respiratory virus infections are one of the major causes of acute respiratory disease or exacerbation of chronic obstructive pulmonary disease (COPD). However, next-generation sequencing has not been used for routine viral detection in clinical respiratory samples owing to its sophisticated technology. Here, several pharyngeal samples with COPD were collected to enrich viral particles using an optimized method (M3), which involved M1 with centrifugation, filtration, and concentration, M2 (magnetic beads) combined with mixed nuclease digestion, and M4 with no pretreatment as a control. Metagenomic sequencing and bioinformatics analyses showed that the M3 method for viral enrichment was superior in both viral sequencing composition and viral taxa when compared to M1, M2, and M4. M3 acquired the most viral reads and more complete sequences within 15-h performance, indicating that it might be feasible for viral detection in multiple respiratory samples in clinical practice. Based on sequence similarity analysis, 12 human viruses, including nine Anelloviruses and three coronaviruses, were characterized. Coronavirus OC43 with the largest number of viral reads accounted for nearly complete (99.8%) genome sequences, indicating that it may be a major viral pathogen involved in exacerbation of COPD.

Metagenomic analysis of the lung microbiome in pulmonary tuberculosis - a pilot study.

The lung microbiome plays an important role in the pathophysiological processes associated with pulmonary tuberculosis (PTB). However, only a few studies using 16S rDNA amplicon sequencing have been reported, and the interactions between Mycobacterium tuberculosis (MTB) and the lung microbiome remain poorly understood. Patients with respiratory symptoms and imaging abnormalities compatible with tuberculosis (TB) were enrolled. We analyzed the lung microbiome in bronchoalveolar lavage (BAL) samples from 30 MTB-positive (MTB+) subjects and 30 MTB negative (MTB-) subjects by shotgun metagenomic sequencing. Alpha diversity tended to be lower in the MTB+ group than in the MTB- group. There was a significant difference in beta diversity between the MTB+ and MTB- subjects. MTB+ lung samples were dominated by MTB, while MTB- samples were enriched with Streptococcus, Prevotella, Nesseria, Selenomonas and Bifidobacterium, which more closely resemble the microbial composition of a healthy lung. Network analysis suggested that MTB could greatly impact the microbial community structure. MTB+ and MTB- communities showed distinct functional signatures. Fungal communities were also found to be associated with the presence or absence of MTB. Furthermore, it was confirmed that 16S rDNA amplicon sequencing underrepresents Mycobacterium. This pilot study is the first to explore the interplay between MTB and the host microbiome by using metagenomic sequencing. MTB dominates the lung microbiome of MTB+ subjects, while MTB- subjects have a Streptococcus-enriched microbiome. The 16S approach underrepresents Mycobacterium and is not the best way to study the TB-associated microbiome.

Distinct lung microbial community states in patients with pulmonary tuberculosis.

An improved understanding of the lung microbiome may lead to better strategies to diagnose, treat, and prevent pulmonary tuberculosis (PTB). However, the characteristics of the lung microbiomes of patients with TB remain largely undefined. In this study, 163 bronchoalveolar lavage (BAL) samples were collected from 163 sputum-negative suspected PTB patients. Furthermore, 12 paired BAL samples were obtained from 12 Mycobacterium tuberculosis-positive (MTB+) patients before and after negative conversion following a two-month anti-TB treatment. The V3-V4 region of the 16S ribosomal RNA (rRNA) gene was used to characterize the microbial composition of the lungs. The results showed that the prevalence of MTB in the BAL samples was 42.9% (70/163) among the sputum-negative patients. The α-diversity of lung microbiota was significantly less diverse in MTB+ patients compared with Mycobacterium tuberculosis-negative (MTB-) patients. There was a significant difference in ß-diversity between MTB+ and MTB- patients. MTB+ patients were enriched with Anoxybacillus, while MTB- patients were enriched with Prevotella, Alloprevotella, Veillonella, and Gemella. There was no significant difference between the Anoxybacillus detection rates of MTB+ and MTB- patients. The paired comparison between the BAL samples from MTB+ patients and their negative conversion showed that BAL negative-conversion microbiota had a higher α-diversity. In conclusion, distinct features of airway microbiota could be identified between samples from patients with and without MTB. Our results imply links between lung microbiota and different clinical groups of active PTB.

Neoadjuvant PD-1 Immune Checkpoint Blockade Reverses Functional Immunodominance among Tumor Antigen-Specific T Cells.

PURPOSE: Surgical resection of primary tumor with regional lymphadenectomy remains the treatment of choice for patients with advanced human papillomavirus-negative head and neck squamous cell carcinoma. However, even when pathologic disease-free margins can be achieved, locoregional and/or distant disease relapse remains high. Perioperative immunotherapy may improve outcomes, but mechanistic data supporting the use of neoadjuvant or adjuvant treatment clinically are sparse. EXPERIMENTAL DESIGN: Two syngeneic models of oral cavity carcinoma with defined T-cell antigens were treated with programmed death receptor 1 (PD-1) mAb before or after surgical resection of primary tumors, and antigen-specific T-cell responses were explored with functional and in vivo challenge assays. RESULTS: We demonstrated that functional immunodominance developed among T cells targeting multiple independent tumor antigens. T cells specific for subdominant antigens expressed greater levels of PD-1. Neoadjuvant, but not adjuvant, PD-1 immune checkpoint blockade broke immunodominance and induced T-cell responses to dominant and subdominant antigens. Using tumors lacking the immunodominant antigen as a model of antigen escape, neoadjuvant PD-1 immune checkpoint blockade induced effector T-cell immunity against tumor cells lacking immunodominant but retaining subdominant antigen. When combined with complete surgical excision, neoadjuvant PD-1 immune checkpoint blockade led to formation of immunologic memory capable of preventing engraftment of tumors lacking the immunodominant but retaining subdominant antigen. CONCLUSIONS: Together, these results implicate PD-1 expression by T cells in the mechanism of functional immunodominance among independent T-cell clones within a progressing tumor and support the use of neoadjuvant PD-1 immune checkpoint blockade in patients with surgically resectable carcinomas.

A Whole-genome Sequencing Analysis of Neisseria gonorrhoeae Isolates in China: An Observational Study.

BACKGROUND: Tracking the spread of the Neisseria gonorrhoeae strains with decreased susceptibility or resistance to cephalosporins is a major priority for global surveillance programmes. Whole-genome sequencing (WGS) has been widely used by increasing countries in North America, Europe, and Pacific to determine the decreased susceptible or resistance determinants of Neisseria gonorrhoeae, track the spread of these determinants throughout the gonococcal population at national or regional level. However, no studies to date have examined the genomic epidemiology of gonorrhea in Asia where the antimicrobial resistant strains of Neisseria gonorrhoeae appears to have emerged before disseminating the strains globally. METHODS: We obtained clinical isolates and data from the China Gonococcal Resistance Surveillance Programme (China-GRSP) from 2012 to 2013. We sequenced the genomes of 435 clinical isolates of Neisseria gonorrhoeae, including 112 (25.6%) isolates with decreased susceptibility to ceftriaxone (Cfx-DS). We assessed the association between antimicrobial resistance genotype and phenotype. We also compared our data with the whole genome data of the isolates from the USA and the UK in the GenBank. FINDINGS: The most prevalent MLST STs in our gonococcal population were MLST ST7827 (n = 74), followed by ST7365 (n = 58), ST1600 (n = 38), ST7367 (n = 35), and ST7363 (n = 29). MLST ST1901 which was reported as the predominant ST in the US was not found in our population. A total of 2512 strains, including additional 2077 published NG strains, were further included for phylogenetic analysis. It generated two distinct lineages - lineage 1 and lineage 2. Analysis of MLST ST1901 in the database indicate that most of MLST ST1901 isolates in the lineage2.6 were Cfx-DS isolates while all isolates in the lineage 2.1 were sensitive to ceftriaxone (77/110 vs. 0/13; p < 0.001). ST1901/lineage 2.6 is a ceftriaxone resistant clone which cannot distinguished by MLST genotyping. In the isolates from our study, the MICs of ceftriaxone for ST7363/lineage 2.6 isolates ranged from 0.008-0.125 mg/L (mean ±â€¯SD; 0.054 ±â€¯0.043 mg/L) while those for ST7363/lineage 2.8 isolates ranged from 0.032-0.250 mg/L (0.134 ±â€¯0.085 mg/L). All ST7363/lineage 2.8 isolates contained penA mosaic alleles. INTERPRETATION: To our knowledge, current study is the first WGS-based analysis of gonococcal population at national level in Asia. China harbors the different predominant clones associated with decreased susceptibility to ceftriaxone from those clones circulated in other regions. The findings from the study can be not only used as baseline data for future studies in China but also contributable to our understanding on spread of N. gonorrhoeae and its resistant strains at regional and global levels. FUNDING: The Chinese Academy Medical Sciences (CAMS) Initiative for Innovative Medicine.

Gut microbiota associated with pulmonary tuberculosis and dysbiosis caused by anti-tuberculosis drugs.

BACKGROUND: An improved understanding of the gut microbiota could lead to better strategies for the diagnosis, therapy and prophylaxis of tuberculosis (TB). The impact of both Mycobacterium tuberculosis (Mtb) infection and anti-TB treatment on the gut microbiota has rarely been studied. METHODS: We characterized the diversity and composition of the gut microbiota in pulmonary TB patients as well as the effects of anti-TB drugs on the gut microbiota. RESULTS: Pulmonary Mtb infection led to a minor decrease in the α diversity of the gut microbiota when compared to healthy controls, which mainly resulted from changes in the relative abundance of the members of genus Bacteroides. Anti-TB therapy caused a rapid, significant alteration in the community structure. The relative abundance of members of genus Clostridiales of the phylum Firmicutes significantly decreased during anti-TB treatment, while many members of genus Bacteroides, including Bacteroides OTU230 and Bacteroides fragilis, were among the taxa that increased. OTU8 and OTU2972 assigned to family Erysipelotrichaceae of the phylum Firmicutes showed a dramatic increase 1 week after the start of therapy, while the other members of this family decreased. CONCLUSIONS: Pulmonary TB and anti-TB treatment caused a distinct dysbiosis of the gut microbiota. Our study contributes valuable information implying potential links between the gut microbiota and TB.

The impact of combined gene mutations in inhA and ahpC genes on high levels of isoniazid resistance amongst katG non-315 in multidrug-resistant tuberculosis isolates from China.

Whole-genome sequencing was used to analyze the profiles of isoniazid (INH) resistance-related mutations among 188 multidrug-resistant strains of Mycobacterium tuberculosis (MDR-TB) and mono-INH-resistant isolates collected in a recent Chinese national survey. Mutations were detected in 18 structural genes and two promoter regions in 96.8% of 188 resistant isolates. There were high mutation frequencies in katG, the inhA promoter, and ahpC-oxyR regulator regions in INH-resistant isolates with frequencies of 86.2%, 19.6%, and 18.6%, respectively. Moreover, a high diversity of mutations was identified as 102 mutants contained various types of single or combined gene mutations in the INH-resistant group of isolates. The cumulative frequencies of katG 315 or inhA-P/inhA mutations was 68.1% (128/188) for the INH-resistant isolates. Of these isolates, 46 isolates (24.5% of 188) exhibited a high level of resistance. A high level of resistance was also observed in 21 isolates (11.2% of 188) with single ahpC-oxyR mutations or a combination of ahpC-oxyR and katG non-315 mutations. The remaining 17 mutations occurred sporadically and emerged in isolates with combined katG mutations. Such development of INH resistance is likely due to an accumulation of mutations under the pressure of drug selection. Thus, these findings provided insights on the levels of INH resistance and its correlation with the combinatorial mutation effect resulting from less frequent genes (inhA and/or ahpC). Such knowledge of other genes (apart from katG) in high-level resistance will aid in developing better strategies for the diagnosis and management of TB.

Comparative analysis of rodent and small mammal viromes to better understand the wildlife origin of emerging infectious diseases.

BACKGROUND: Rodents represent around 43% of all mammalian species, are widely distributed, and are the natural reservoirs of a diverse group of zoonotic viruses, including hantaviruses, Lassa viruses, and tick-borne encephalitis viruses. Thus, analyzing the viral diversity harbored by rodents could assist efforts to predict and reduce the risk of future emergence of zoonotic viral diseases. RESULTS: We used next-generation sequencing metagenomic analysis to survey for a range of mammalian viral families in rodents and other small animals of the orders Rodentia, Lagomorpha, and Soricomorpha in China. We sampled 3,055 small animals from 20 provinces and then outlined the spectra of mammalian viruses within these individuals and the basic ecological and genetic characteristics of novel rodent and shrew viruses among the viral spectra. Further analysis revealed that host taxonomy plays a primary role and geographical location plays a secondary role in determining viral diversity. Many viruses were reported for the first time with distinct evolutionary lineages, and viruses related to known human or animal pathogens were identified. Phylogram comparison between viruses and hosts indicated that host shifts commonly happened in many different species during viral evolutionary history. CONCLUSIONS: These results expand our understanding of the viromes of rodents and insectivores in China and suggest that there is high diversity of viruses awaiting discovery in these species in Asia. These findings, combined with our previous bat virome data, greatly increase our knowledge of the viral community in wildlife in a densely populated country in an emerging disease hotspot.

Clinically prevalent mutations in Mycobacterium tuberculosis alter propionate metabolism and mediate multidrug tolerance.

The global epidemic of drug-resistant tuberculosis is a catastrophic example of how antimicrobial resistance is undermining the public health gains made possible by combination drug therapy. Recent evidence points to unappreciated bacterial factors that accelerate the emergence of drug resistance. In a genome-wide association study of Mycobacterium tuberculosis isolates from China, we find mutations in the gene encoding the transcription factor prpR enriched in drug-resistant strains. prpR mutations confer conditional drug tolerance to three of the most effective classes of antibiotics by altering propionyl-CoA metabolism. prpR-mediated drug tolerance is carbon-source dependent, and while readily detectable during infection of human macrophages, is not captured by standard susceptibility testing. These data define a previously unrecognized and clinically prevalent class of M. tuberculosis variants that undermine antibiotic efficacy and drive drug resistance.

Proteogenomic Analysis and Discovery of Immune Antigens in Mycobacterium vaccae.

Tuberculosis (TB) is one of the leading causes of death worldwide, especially in developing countries. Neonatal BCG vaccination occurs in various regions, but the level of protection varies in different populations. Recently, Mycobacterium vaccae is found to be an immunomodulating therapeutic agent that could confer a significant level of protection against TB. It is the only vaccine in a phase III trial from WHO to assess its efficacy and safety in preventing TB disease in people with latent TB infection. However, the mechanism of immunotherapy of M. vaccae remains poorly understood. In this study, the full genome of M. vaccae was obtained by next-generation sequencing technology, and a proteogenomic approach was successfully applied to further perform genome annotation using high resolution and high accuracy MS data. A total of 3,387 proteins (22,508 unique peptides) were identified, and 581 proteins annotated as hypothetical proteins in the genome database were confirmed. Furthermore, 38 novel protein products not annotated at the genome level were detected and validated. Additionally, the translational start sites of 445 proteins were confirmed, and 98 proteins were validated through extension of their translational start sites based on N terminus-derived peptides. The physicochemical characteristics of the identified proteins were determined. Thirty-five immunogenic proteins of M. vaccae were identified by immunoproteomic analysis, and 20 of them were selected to be expressed and validated by Western blot for immunoreactivity to serum from patients infected with M. tuberculosis The results revealed that eight of them showed strong specific reactive signals on the immunoblots. Furthermore, cellular immune response was further examined and one protein displayed a higher cellular immune level in pulmonary TB patients. Twelve identified immunogenic proteins have orthologous in H37Rv and BCG. This is the first study to obtain the full genome and annotation of M. vaccae using a proteogenomic approach, and some immunogenic proteins that were validated by immunoproteomic analysis could contribute to the understanding of the mechanism of M. vaccae immunotherapy.

An elaborate landscape of the human antibody repertoire against enterovirus 71 infection is revealed by phage display screening and deep sequencing.

Enterovirus 71 (EV71) causes outbreaks of hand, foot and mouth disease (HFMD), primarily in the Asia-Pacific area, that are often associated with complications of severe to fatal neurological symptoms. There are currently no anti-viral therapies or vaccines available for the treatment of EV71 infection. Illustrating human antibody responses neutralizing EV71 infection could potentially provide valuable information for the development of effective therapies and vaccines. Here, we constructed a comprehensive phage display library based on peripheral blood of eight EV71-infected donors and identified 27 EV71-specific human antibodies, of which four have neutralizing activity in in vitro experiments. Deep sequencing analysis of the antibody heavy chains at the transcript level of another three independent EV71-infected donors and three controls demonstrates that heavy chains of the EV71-specific antibodies are conserved among EV71-infected individuals but absent in controls, suggesting convergent evolution of human antibodies against EV71.

The outer membrane phospholipase A is essential for membrane integrity and type III secretion in Shigella flexneri.

Outer membrane phospholipase A (OMPLA) is an enzyme located in the outer membrane of Gram-negative bacteria. OMPLA exhibits broad substrate specificity, and some of its substrates are located in the cellular envelope. Generally, the enzymatic activity can only be induced by perturbation of the cell envelope integrity through diverse methods. Although OMPLA has been thoroughly studied as a membrane protein in Escherichia coli and is constitutively expressed in many other bacterial pathogens, little is known regarding the functions of OMPLA during the process of bacterial infection. In this study, the proteomic and transcriptomic data indicated that OMPLA in Shigella flexneri, termed PldA, both stabilizes the bacterial membrane and is involved in bacterial infection under ordinary culture conditions. A series of physiological assays substantiated the disorganization of the bacterial outer membrane and the periplasmic space in the ΔpldA mutant strain. Furthermore, the ΔpldA mutant strain showed decreased levels of type III secretion system expression, contributing to the reduced internalization efficiency in host cells. The results of this study support that PldA, which is widespread across Gram-negative bacteria, is an important factor for the bacterial life cycle, particularly in human pathogens.

Distribution and characteristics of rodent picornaviruses in China.

Rodents are important reservoir hosts of many important zoonotic viruses. The family Picornaviridae contains clinically important pathogens that infect humans and animals, and increasing numbers of rodent picornaviruses have recently been associated with zoonoses. We collected 574 pharyngeal and anal swab specimens from 287 rodents of 10 different species from eight representative regions of China from October 2013 to July 2015. Seven representative sequences identified from six rodent species were amplified as full genomes and classified into four lineages. Three lineage 1 viruses belonged to a novel genus of picornaviruses and was more closely related to Hepatovirus than to others genera of picornaviruses based on aa homology. Lineage 2, lineage 3, and lineage 4 viruses belonged to the genera Rosavirus, Hunnivirus, and Enterovirus, respectively, representing new species. According to both phylogenetic and identity analyses, Lineage 2 viruses had a close relationship with rosavirus 2 which was recovered from the feces of a child in Gambia and Lineage 3 viruses had a close relationship with domestic animal Hunnivirus. Lineage 4 viruses provide the first evidence of these enteroviruses and their evolution in rodent hosts in China.