CRISPR/Cas9-Mediated Generation of Mutant Lines in Medicago truncatula Indicates a Symbiotic Role of MtLYK10 during Nodule Formation.

CRISPR/Cas9 systems are commonly used for plant genome editing; however, the generation of homozygous mutant lines in Medicago truncatula remains challenging. Here, we present a CRISPR/Cas9-based protocol that allows the efficient generation of M. truncatula mutants. Gene editing was performed for the LysM receptor kinase gene MtLYK10 and two major facilitator superfamily transporter genes. The functionality of CRISPR/Cas9 vectors was tested in Nicotiana benthamiana leaves by editing a co-transformed GUSPlus gene. Transformed M. truncatula leaf explants were regenerated to whole plants at high efficiency (80%). An editing efficiency (frequency of mutations at a given target site) of up to 70% was reached in the regenerated plants. Plants with MtLYK10 knockout mutations were propagated, and three independent homozygous mutant lines were further characterized. No off-target mutations were identified in these lyk10 mutants. Finally, the lyk10 mutants and wild-type plants were compared with respect to the formation of root nodules induced by nitrogen-fixing Sinorhizobium meliloti bacteria. Nodule formation was considerably delayed in the three lyk10 mutant lines. Surprisingly, the size of the rare nodules in mutant plants was higher than in wild-type plants. In conclusion, the symbiotic characterization of lyk10 mutants generated with the developed CRISPR/Cas9 protocol indicated a role of MtLYK10 in nodule formation.

An extracellular ß-N-acetylhexosaminidase of Medicago truncatula hydrolyzes chitooligosaccharides and is involved in arbuscular mycorrhizal symbiosis but not required for nodulation.

Establishment of symbiosis between plants and arbuscular mycorrhizal (AM) fungi depends on fungal chitooligosaccharides (COs) and lipo-chitooligosaccharides (LCOs). The latter are also produced by nitrogen-fixing rhizobia to induce nodules on leguminous roots. However, host enzymes regulating structure and levels of these signals remain largely unknown. Here, we analyzed the expression of a ß-N-acetylhexosaminidase gene of Medicago truncatula (MtHEXO2) and biochemically characterized the enzyme. Mutant analysis was performed to study the role of MtHEXO2 during symbiosis. We found that expression of MtHEXO2 is associated with AM symbiosis and nodulation. MtHEXO2 expression in the rhizodermis was upregulated in response to applied chitotetraose, chitoheptaose, and LCOs. M. truncatula mutants deficient in symbiotic signaling did not show induction of MtHEXO2. Subcellular localization analysis indicated that MtHEXO2 is an extracellular protein. Biochemical analysis showed that recombinant MtHEXO2 does not cleave LCOs but can degrade COs into N-acetylglucosamine (GlcNAc). Hexo2 mutants exhibited reduced colonization by AM fungi; however, nodulation was not affected in hexo2 mutants. In conclusion, we identified an enzyme, which inactivates COs and promotes the AM symbiosis. We hypothesize that GlcNAc produced by MtHEXO2 may function as a secondary symbiotic signal.

RLI2 regulates Arabidopsis female gametophyte and embryo development by facilitating the assembly of the translational machinery.

Eukaryotic protein translation is a complex process that requires the participation of different proteins. Defects in the translational machinery often result in embryonic lethality or severe growth defects. Here, we report that RNase L inhibitor 2/ATP-BINDING CASSETTE E2 (RLI2/ABCE2) regulates translation in Arabidopsis thaliana. Null mutation of rli2 is gametophytic and embryonic lethal, whereas knockdown of RLI2 causes pleiotropic developmental defects. RLI2 interacts with several translation-related factors. Knockdown of RLI2 affects the translational efficiency of a subset of proteins involved in translation regulation and embryo development, indicating that RLI2 has critical roles in these processes. In particular, RLI2 knockdown mutant exhibits decreased expression of genes involved in auxin signaling and female gametophyte and embryo development. Therefore, our results reveal that RLI2 facilitates assembly of the translational machinery and indirectly modulates auxin signaling to regulate plant growth and development.

Prevalence of neutropenia in US residents: a population based analysis of NHANES 2011-2018.

AIMS: Neutrophils play a pivotal in immunity and inflammation. We aim to investigate the prevalence of neutropenia in the United States. METHODS: In this cross-sectional study, participants from the National Health and Nutrition Examination Survey (NHANES) (2011-2018) were enrolled. Demographic information, hematologic measurements, smoking status of all participants were collected for all participants. All statistical analyses were performed utilizing the NHANES survey weights. Covariate-adjusted linear regression was used to compare hematologic indices in different population grouped by age, sex, ethnicity, and smoking. We also employed multivariate-logistic regression to estimate the weighted odds ratio with a 95% confidence interval and predict the neutropenia risk among. RESULTS: 32,102 participants from NHANES survey were included, represented 286.6 million multiracial population in the United States. Black participants had lower mean leukocyte count (mean difference (MD): 0.71 × 109/L; P < 0.001) and lower neutrophil count (MD: 0.83 × 109/L; P < 0.001) compared with white participants after adjusting for age and sex. Furthermore, t a notable observation was the significant downward shift in the distribution curves of leukocyte count and neutrophil count among black participants. Smokers had a higher mean leukocyte count (MD: 1.10 × 109 cells/L; P < 0.001) and a higher mean neutrophil count (MD: 0.75 × 109 cells/L; P < 0.001) comparing with nonsmokers. The estimated prevalence of neutropenia was 1.24% (95% CI: 1.11 - 1.37%), which corresponds to approximately 35.5 million individuals in the United States. The prevalence of neutropenia in black participants was significantly higher than other races. Results of logistic regression analysis showed that black individuals, male individuals, and children younger than 5 years had a higher risk of neutropenia. CONCLUSIONS: Neutropenia is more common in the general population than we thought, especially in black individuals and children. More attention should be paid to neutropenia.

The Medicago truncatula hydrolase MtCHIT5b degrades Nod factors of Sinorhizobium meliloti and cooperates with MtNFH1 to regulate the nodule symbiosis.

Nod factors secreted by nitrogen-fixing rhizobia are lipo-chitooligosaccharidic signals required for establishment of the nodule symbiosis with legumes. In Medicago truncatula, the Nod factor hydrolase 1 (MtNFH1) was found to cleave Nod factors of Sinorhizobium meliloti. Here, we report that the class V chitinase MtCHIT5b of M. truncatula expressed in Escherichia coli can release lipodisaccharides from Nod factors. Analysis of M. truncatula mutant plants indicated that MtCHIT5b, together with MtNFH1, degrades S. meliloti Nod factors in the rhizosphere. MtCHIT5b expression was induced by treatment of roots with purified Nod factors or inoculation with rhizobia. MtCHIT5b with a fluorescent tag was detected in the infection pocket of root hairs. Nodulation of a MtCHIT5b knockout mutant was not significantly altered whereas overexpression of MtCHIT5b resulted in fewer nodules. Reduced nodulation was observed when MtCHIT5b and MtNFH1 were simultaneously silenced in RNA interference experiments. Overall, this study shows that nodule formation of M. truncatula is regulated by a second Nod factor cleaving hydrolase in addition to MtNFH1.

Effector-triggered inhibition of nodulation: A rhizobial effector protease targets soybean kinase GmPBS1-1.

Type III protein secretion systems of nitrogen-fixing rhizobia deliver effector proteins into leguminous host cells to promote or inhibit the nodule symbiosis. However, mechanisms underlying effector-triggered inhibition of nodulation remain largely unknown. Nodulation outer protein T (NopT) of Sinorhizobium sp. NGR234 is an effector protease related to the Pseudomonas effector Avirulence protein Pseudomonas phaseolicola B (AvrPphB). Here, we constructed NGR234 mutants producing different NopT variants and found that protease activity of NopT negatively affects nodulation of smooth crotalaria (Crotalaria pallida). NopT variants lacking residues required for autocleavage and subsequent lipidation showed reduced symbiotic effects and were not targeted to the plasma membrane. We further noticed that Sinorhizobium fredii strains possess a mutated nopT gene. Sinorhizobium fredii USDA257 expressing nopT of NGR234 induced considerably fewer nodules in soybean (Glycine max) cv. Nenfeng 15 but not in other cultivars. Effector perception was further examined in NopT-expressing leaves of Arabidopsis (Arabidopsis thaliana) and found to be dependent on the protein kinase Arabidopsis AvrPphB Susceptible 1 (AtPBS1) and the associated resistance protein Arabidopsis Resistance to Pseudomonas syringae 5 (AtRPS5). Experiments with Nicotiana benthamiana plants indicated that the soybean homolog GmPBS1-1 associated with AtRPS5 can perceive NopT. Further analysis showed that NopT cleaves AtPBS1 and GmPBS1-1 and thus can activate these target proteins. Insertion of a DKM motif at the cleavage site of GmPBS1-1 resulted in increased proteolysis. Nodulation tests with soybeans expressing an autoactive GmPBS1-1 variant indicated that activation of a GmPBS1-1-mediated resistance pathway impairs nodule formation in cv. Nenfeng 15. Our findings suggest that legumes face an evolutionary dilemma of either developing effector-triggered immunity against pathogenic bacteria or establishing symbiosis with suboptimally adapted rhizobia producing pathogen-like effectors.

The dietary inflammatory index and its association with the prevalence of hypertension: A cross-sectional study.

Aims: We aim to investigate the association of the Dietary Inflammatory Index (DII) with the prevalence of hypertension in a large multiracial population in the United States. Methods: Participants from the National Health and Nutrition Examination Survey (NHANES) (1999-2018) were included in this cross-sectional study. Dietary information was obtained and used to calculate DII. Blood pressures of participants were measured by experienced examiners. The NHANES used the method of "stratified multistage probability sampling," and this study is a weight analysis following the NHANES analytic guidance. Weight logistic regression analysis was adopted to investigate the association of hypertension with DII. Least Absolute Shrinkage and Selection Operator (LASSO) regression was carried out to screen the most important dietary factors associated with the risk of hypertension. Moreover, a nomogram model based on key dietary factors was established; the receiver operating characteristic (ROC) curve was used to evaluate the diagnostic power of the nomogram model for screening hypertension risk. Results: A total of 45,023 participants were included in this study, representing 191 million residents in the United States. Participants with hypertension had an elevated DII compared with those without hypertension. Weight logistic regression showed that an increment of DII was strongly associated with hypertension after adjusting for confounding factors. The nomogram model, based on key dietary factors screened by LASSO regression, showed a favorable discriminatory power with an area under the curve (AUC) of 78.5% (95% CI: 78.5%-79.3%). Results of the sensitivity analysis excluding participants who received any drug treatment were consistent with those in the main analysis. Conclusion: An increment of DII is associated with the risk of hypertension. For better prevention and treatment of hypertension, more attention should be paid to controlling dietary inflammation.

Use of the rhizobial type III effector gene nopP to improve Agrobacterium rhizogenes-mediated transformation of Lotus japonicus.

BACKGROUND: Protocols for Agrobacterium rhizogenes-mediated hairy root transformation of the model legume Lotus japonicus have been established previously. However, little efforts were made in the past to quantify and improve the transformation efficiency. Here, we asked whether effectors (nodulation outer proteins) of the nodule bacterium Sinorhizobium sp. NGR234 can promote hairy root transformation of L. japonicus. The co-expressed red fluorescent protein DsRed1 was used for visualization of transformed roots and for estimation of the transformation efficiency. RESULTS: Strong induction of hairy root formation was observed when A. rhizogenes strain LBA9402 was used for L. japonicus transformation. Expression of the effector gene nopP in L. japonicus roots resulted in a significantly increased transformation efficiency while nopL, nopM, and nopT did not show such an effect. In nopP expressing plants, more than 65% of the formed hairy roots were transgenic as analyzed by red fluorescence emitted by co-transformed DsRed1. A nodulation experiment indicated that nopP expression did not obviously affect the symbiosis between L. japonicus and Mesorhizobium loti. CONCLUSION: We have established a novel protocol for hairy root transformation of L. japonicus. The use of A. rhizogenes LBA9402 carrying a binary vector containing DsRed1 and nopP allowed efficient formation and identification of transgenic roots.

NopD of Bradyrhizobium sp. XS1150 Possesses SUMO Protease Activity.

Effectors secreted by the type III protein secretion system (T3SS) of rhizobia are host-specific determinants of the nodule symbiosis. Here, we have characterized NopD, a putative type III effector of Bradyrhizobium sp. XS1150. NopD was found to possess a functional N-terminal secretion signal sequence that could replace that of the NopL effector secreted by Sinorhizobium sp. NGR234. Recombinant NopD and the C-terminal domain of NopD alone can process small ubiquitin-related modifier (SUMO) proteins and cleave SUMO-conjugated proteins. Activity was abolished in a NopD variant with a cysteine-to-alanine substitution in the catalytic core (NopD-C972A). NopD recognizes specific plant SUMO proteins (AtSUMO1 and AtSUMO2 of Arabidopsis thaliana; GmSUMO of Glycine max; PvSUMO of Phaseolus vulgaris). Subcellular localization analysis with A. thaliana protoplasts showed that NopD accumulates in nuclear bodies. NopD, but not NopD-C972A, induces cell death when expressed in Nicotiana tabacum. Likewise, inoculation tests with constructed mutant strains of XS1150 indicated that nodulation of Tephrosia vogelii is negatively affected by the protease activity of NopD. In conclusion, our findings show that NopD is a symbiosis-related protein that can process specific SUMO proteins and desumoylate SUMO-conjugated proteins.

A method for functional testing constitutive and ligand-induced interactions of lysin motif receptor proteins.

BACKGROUND: Plant receptors with lysin motifs (LsyM) recognize microbial signals such as fungal chitin and lipo-chitooligosaccharidic Nod factors of nitrogen-fixing rhizobia. It is generally assumed that ligand-induced dimerization of LysM receptors is an essential step in activation of intracellular kinase domains and downstream signaling. Consequently, genes required for plant defense and establishment of symbiosis are expressed. We recently found that three LysM receptor proteins (namely LYK1, LYK4 and LYK5) of Arabidopsis thaliana form a tripartite receptor complex to perceive chitin. However, constitutive and ligand-induced interactions of LysM receptors generally remain difficult to be characterized. RESULTS: Interactions between ectodomains of LYK1, LYK4 and LYK5 were investigated by a chimeric receptor approach using hairy roots of the legume Lotus japonicus. Synthetic receptor pairs consisting of a LYK ectodomain and the intracellular domain of a L. japonicus Nod factor receptor (NFR1 and NFR5, respectively) were tested for their capacity to activate expression of the symbiotic NIN (nodule inception) gene. The results indicated constitutive (LYK4ED-LYK4ED, LYK4ED-LYK5ED) and chitin-induced interactions (LYK1ED-LYK1ED, LYK1ED-LYK5ED) of the examined ectodomains. CONCLUSION: We present a method to functionally analyze constitutive and ligand-induced interactions of LysM-type proteins.

LYK4 is a component of a tripartite chitin receptor complex in Arabidopsis thaliana.

LysM receptor-like kinases (LYKs) of Arabidopsis thaliana (namely LYK1, LYK4 and LYK5) play a major role in chitin perception and immunity against pathogenic fungi. Chitin-induced heterodimerization of LYK1 and LYK5 has been previously reported, but protein interaction partners of LYK4 have not yet been identified. In this study, by analysing mutants we confirmed a role of LYK4 in chitin perception, and found that the ectodomain of LYK4 homodimerizes and also interacts with the ectodomain of LYK5 in vitro. Pull-down experiments with proteins expressed in protoplasts indicated LYK4-LYK4 and LY4-LYK5 interactions in planta. When protoplasts were treated with chitoheptaose or chitin, a protein complex was immunoprecipitated that appeared to be composed of LYK1, LYK4, and LYK5. Similar experiments with proteins expressed in lyk mutant plants suggested that elicitor treatment induced a physical interaction between LYK1 and LYK5 but not between LYK1 and LYK4. Bimolecular fluorescence complementation experiments substantiated these findings. Overall, our data suggest that LYK4 functions as a LYK5-associated co-receptor or scaffold protein that enhances chitin-induced signaling in Arabidopsis.

A novel astrovirus identified in wild rhesus monkey feces in China.

The discovery and analysis of pathogens carried by non-human primates are important for understanding zoonotic infections in humans. We identified a highly divergent astrovirus (AstV) from fecal matter from a rhesus monkey in China, which has been tentatively named "monkey-feces-associated AstV" (MkAstV). The full-length genome of MkAstV was determined to be 7377 nt in length. It exhibits the standard genomic AstV organization of three open reading frames (ORFs) and is most closely related to duck AstV (28%, 49%, and 35% amino acid sequence identity in ORF1a, ORF1b, and ORF2, respectively). Coincidentally, while this report was being prepared, an astrovirus sequence from Hainan black-spectacled toad became available in the GenBank database, showing 95%, 94% and 92% aa sequence identity in ORF1a, ORF1b and ORF2, respectively, to the corresponding ORFs of MkAstV. Phylogenetic analysis of ORF1a, ORF1b, and ORF2 indicated that MkAstV and the amphibian-related astroviruses formed an independent cluster in the genus Avastrovirus. The host of MkAstV remains unknown. Epidemiological and serological studies of this novel virus should be undertaken in primates, including humans.

Gradual replacement of all previously circulating respiratory syncytial virus A strain with the novel ON1 genotype in Lanzhou from 2010 to 2017.

ON1 is a novel genotype of human respiratory syncytial virus (HRSV) subtype A, in children with acute respiratory tract infections (ARTIs). However, there is not much data on the prevalence and clinical and molecular characterization in China.Our study is based on the children who had respiratory infections positive for RSV-A admitted by Gansu Provincial Maternity and Child-care Hospital in Lanzhou (northwestern China) during the last 7 epidemic seasons from 2010 to 2017.In our study, different strains of the novel RSV-A genotype ON1, first identified in Canada in December 2010, were first detected in Gansu Provincial Maternity and Child-care Hospital in August 2012 and then followed by an abrupt expansion in the number of ON1 variants in the beginning of 2014 and eventually replaced all other RSV-A strains from 2015 to 2017. ON1 is characterized by a 72-nt duplication in the C-terminal region of the highly variable attachment glycoprotein (G), predicted to lengthen the polypeptide with 24 amino acids, including a 23-aa duplication, which likely changes antigenicity. New N-glycosylation sites occurred within the 23-aa duplication and 24-aa insertion of the ON1 viruses in our study. Notably, RSV infections occurred later, but peaked sooner from the 2014/2015 to 2016/2017 epidemic seasons, compared with the previous 4 seasons.Our study concluded that genotype ON1 has caused larger outbreaks and became the predominate genotype for HRSV subgroup A in Lanzhou from 2013 to 2017, and became the sole genotype of RSV-A in 2015/2016 and 2016/2017. Our data indicate that northwest of China and the world will eventually be dominated by the ON1 RSV-A genotype, including the possibility for vaccine development. Based on trends seen in RSV-B BA genotype, which predominated for decades, there is a possibility to develop a vaccine for children in the next 10 years.

Biochemical properties and in planta effects of NopM, a rhizobial E3 ubiquitin ligase.

Nodulation outer protein M (NopM) is an IpaH family type three (T3) effector secreted by the nitrogen-fixing nodule bacterium Sinorhizobium sp. strain NGR234. Previous work indicated that NopM is an E3 ubiquitin ligase required for an optimal symbiosis between NGR234 and the host legume Lablab purpureus Here, we continued to analyze the function of NopM. Recombinant NopM was biochemically characterized using an in vitro ubiquitination system with Arabidopsis thaliana proteins. In this assay, NopM forms unanchored polyubiquitin chains and possesses auto-ubiquitination activity. In a NopM variant lacking any lysine residues, auto-ubiquitination was not completely abolished, indicating noncanonical auto-ubiquitination of the protein. In addition, we could show intermolecular ubiquitin transfer from NopM to C338A (enzymatically inactive NopM form) in vitro Bimolecular fluorescence complementation analysis provided clues about NopM-NopM interactions at plasma membranes in planta NopM, but not C338A, expressed in tobacco cells induced cell death, suggesting that E3 ubiquitin ligase activity of NopM induced effector-triggered immunity responses. Likewise, expression of NopM in Lotus japonicus caused reduced nodule formation, whereas expression of C338A showed no obvious effects on symbiosis. Further experiments indicated that serine residue 26 of NopM is phosphorylated in planta and that NopM can be phosphorylated in vitro by salicylic acid-induced protein kinase (NtSIPK), a mitogen-activated protein kinase (MAPK) of tobacco. Hence, NopM is a phosphorylated T3 effector that can interact with itself, with ubiquitin, and with MAPKs.

Role of the Nod Factor Hydrolase MtNFH1 in Regulating Nod Factor Levels during Rhizobial Infection and in Mature Nodules of Medicago truncatula.

Establishment of symbiosis between legumes and nitrogen-fixing rhizobia depends on bacterial Nod factors (NFs) that trigger symbiosis-related NF signaling in host plants. NFs are modified oligosaccharides of chitin with a fatty acid moiety. NFs can be cleaved and inactivated by host enzymes, such as MtNFH1 (MEDICAGO TRUNCATULA NOD FACTOR HYDROLASE1). In contrast to related chitinases, MtNFH1 hydrolyzes neither chitin nor chitin fragments, indicating a high cleavage preference for NFs. Here, we provide evidence for a role of MtNFH1 in the symbiosis with Sinorhizobium meliloti Upon rhizobial inoculation, MtNFH1 accumulated at the curled tip of root hairs, in the so-called infection chamber. Mutant analysis revealed that lack of MtNFH1 delayed rhizobial root hair infection, suggesting that excess amounts of NFs negatively affect the initiation of infection threads. MtNFH1 deficiency resulted in nodule hypertrophy and abnormal nodule branching of young nodules. Nodule branching was also stimulated in plants expressing MtNFH1 driven by a tandem CaMV 35S promoter and plants inoculated by a NF-overproducing S. meliloti strain. We suggest that fine-tuning of NF levels by MtNFH1 is necessary for optimal root hair infection as well as for NF-regulated growth of mature nodules.

Human bocavirus and human metapneumovirus in hospitalized children with lower respiratory tract illness in Changsha, China.

BACKGROUND: Lower respiratory tract illness is a major cause of morbidity and mortality in children worldwide, however, information about the epidemiological and clinical characteristics of LRTIs caused by HMPV and HBoV in China is limited. OBJECTIVES: Human bocavirus (HBoV) and human metapneumovirus (HMPV) are two important viruses for children with lower respiratory tract infections (LRTI). We aimed to assay the correlation between viral load and clinical characteristics of HBoV and HMPV with LRTI in Changsha, China. METHODS: Nasopharyngeal aspirates (NPAs) from children with LRTI were collected. Real-time PCR was used to screen HBoV and HMPV. Analyses were performed using SPSS 16.0 software. RESULTS: Pneumonia was the most frequent diagnosis. There was no significant difference between HBoV- and HMPV-positive patients in age (P = .506) or hospitalization duration (P = .280); 24.1% and 18.2% were positive for HBoV and HMPV. HBoV infections peaked in summer (32.2%), and HMPV infections peaked in winter (28.9%). The HBoV-positive patients had a shorter hospitalization duration than the HBoV-negative patients (P = .021), and the HMPV-positive patients had a higher prevalence of fever than the HMPV-negative patients (P = .002). The HBoV viral load was significantly higher among patients aged <1 year (P = .006). The mean HBoV and HMPV viral loads were not significantly different between patients with single infections and coinfections. Patients infected with HBoV only were older than those coinfected with HBoV and other respiratory viruses (P = .005). No significant difference was found in the clinical characteristics of patients infected with HMPV only and those coinfected with HMPV and other respiratory viruses. CONCLUSION: Pneumonia was the most frequent diagnosis caused by HBoV and HMPV. Neither HBoV nor HMPV viral load was correlated with disease severity.

The suppression of innate immune response by human rhinovirus C.

Rhinovirus C (RV-C), a newly identified group of human rhinoviruses (RVs), is associated with exacerbation of severe asthma. The type I interferon (IFN) response induced by this virus and the mechanisms of evasion of IFN-mediated innate immunity for RV-C remain unclear. In this study, we constructed a full-length cDNA clone of RV-C (LZ651) from a clinical sample. IFN-ß mRNA and protein levels were not elevated in differentiated Human bronchial epithelial (HBE) cells at the air-liquid interface infected with RV-C, except in the early stage of infection. The ability to attenuate IFN-ß activation was ascribed to 3Cpro of RV-C, and the 40-His site of 3Cpro played an important role. Furthermore, RIG-I was degraded by 3Cpro in a caspase-dependent manner and 3Cpro cleaved MAVS at 148 Q/A, which inhibited IFN signaling. Taken together, our results demonstrate the mechanism by which RV-C circumvents the production of type I IFN in infected cells.

Clinical characteristics and viral load of respiratory syncytial virus and human metapneumovirus in children hospitaled for acute lower respiratory tract infection.

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are two common viral pathogens in acute lower respiratory tract infections (ALRTI). However, the association of viral load with clinical characteristics is not well-defined in ALRTI. To explore the correlation between viral load and clinical characteristics of RSV and HMPV in children hospitalized for ALRTI in Lanzhou, China. Three hundred and eighty-seven children hospitalized for ALRTI were enrolled. Nasopharyngeal aspirates (NPAs) were sampled from each children. Real-time PCR was used to screen RSV, HMPV, and twelve additional respiratory viruses. Bronchiolitis was the leading diagnoses both in RSV and HMPV positive patients. A significantly greater frequency of wheezing (52% vs. 33.52%, P = 0.000) was noted in RSV positive and negative patients. The RSV viral load was significant higher in children aged <1 year (P = 0.003), children without fever and wheezing (P = 0.015 and P = 0.000), days of illness <14 days (P = 0.002), children with bronchiolitis (P = 0.012) and children with RSV single infections (P = 0.000). No difference was found in the clinical features of HMPV positive and negative patients. The HMPV viral load had no correlation with any clinical characteristics. The incidences of severe disease were similar between single infection and coinfection for the two viruses (RSV, P = 0.221; HMPV, P = 0.764) and there has no statistical significance between severity and viral load (P = 0.166 and P = 0.721). Bronchiolitis is the most common disease caused by RSV and HMPV. High viral load or co-infection may be associated with some symptoms but neither has a significant impact on disease severity for the two viruses. J. Med. Virol. 89:589-597, 2017. © 2016 Wiley Periodicals, Inc.

Identification of 12 Cases of Acute Measles Encephalitis Without Rash.

Twelve cases of acute measles encephalitis without rash were identified from October 2011 to July 2013 in Changsha city, China; 5 were found to be genotype H1 and 2 were B3. Our data suggest that screening for measles virus is necessary in children with viral encephalitis, to eliminate the disease.

A single amino acid substitution in a chitinase of the legume Medicago truncatula is sufficient to gain Nod-factor hydrolase activity.

The symbiotic interaction between nitrogen-fixing rhizobia and legumes depends on lipo-chitooligosaccharidic Nod-factors (NFs). The NF hydrolase MtNFH1 of Medicago truncatula is a symbiotic enzyme that hydrolytically inactivates NFs with a C16 : 2 acyl chain produced by the microsymbiont Sinorhizobium meliloti 1021. MtNFH1 is related to class V chitinases (glycoside hydrolase family 18) but lacks chitinase activity. Here, we investigated the substrate specificity of MtNFH1-related proteins. MtCHIT5a and MtCHIT5b of M. truncatula as well as LjCHIT5 of Lotus japonicus showed chitinase activity, suggesting a role in plant defence. The enzymes failed to hydrolyse NFs from S. meliloti. NFs from Rhizobium leguminosarum with a C18 : 4 acyl moiety were neither hydrolysed by these chitinases nor by MtNFH1. Construction of chimeric proteins and further amino acid replacements in MtCHIT5b were performed to identify chitinase variants that gained the ability to hydrolyse NFs. A single serine-to-proline substitution was sufficient to convert MtCHIT5b into an NF-cleaving enzyme. MtNFH1 with the corresponding proline-to-serine substitution failed to hydrolyse NFs. These results are in agreement with a substrate-enzyme model that predicts NF cleavage when the C16 : 2 moiety is placed into a distinct fatty acid-binding cleft. Our findings support the view that MtNFH1 evolved from the ancestral MtCHIT5b by gene duplication and subsequent symbiosis-related neofunctionalization.