Distributional conformal prediction.

We propose a robust method for constructing conditionally valid prediction intervals based on models for conditional distributions such as quantile and distribution regression. Our approach can be applied to important prediction problems, including cross-sectional prediction, k-step-ahead forecasts, synthetic controls and counterfactual prediction, and individual treatment effects prediction. Our method exploits the probability integral transform and relies on permuting estimated ranks. Unlike regression residuals, ranks are independent of the predictors, allowing us to construct conditionally valid prediction intervals under heteroskedasticity. We establish approximate conditional validity under consistent estimation and provide approximate unconditional validity under model misspecification, under overfitting, and with time series data. We also propose a simple "shape" adjustment of our baseline method that yields optimal prediction intervals.

Testability of high-dimensional linear models with nonsparse structures.

Understanding statistical inference under possibly non-sparse high-dimensional models has gained much interest recently. For a given component of the regression coefficient, we show that the difficulty of the problem depends on the sparsity of the corresponding row of the precision matrix of the covariates, not the sparsity of the regression coefficients. We develop new concepts of uniform and essentially uniform non-testability that allow the study of limitations of tests across a broad set of alternatives. Uniform non-testability identifies a collection of alternatives such that the power of any test, against any alternative in the group, is asymptotically at most equal to the nominal size. Implications of the new constructions include new minimax testability results that, in sharp contrast to the current results, do not depend on the sparsity of the regression parameters. We identify new tradeoffs between testability and feature correlation. In particular, we show that, in models with weak feature correlations, minimax lower bound can be attained by a test whose power has the n rate, regardless of the size of the model sparsity.

CrfP, a fratricide protein, contributes to natural transformation in Streptococcus suis.

Streptococcus suis (S. suis) is an important zoonotic pathogen that causes septicaemia, meningitis and streptococcal toxic shock-like syndrome in its host, and recent studies have shown that S. suis could be competent for natural genetic transformation. Transformation is an important mechanism for the horizontal transfer of DNA, but some elements that affect the transformation process need to be further explored. Upon entering the competent state, Streptococcus species stimulate the transcription of competence-related genes that are responsible for exogenous DNA binding, uptake and processing. In this study, we performed conserved promoter motif and qRT-PCR analyses and identified CrfP as a novel murein hydrolase that is widespread in S. suis and stimulated with a peptide pheromone in the competent state through a process controlled by ComX. A bioinformatics analysis revealed that CrfP consists of a CHAP hydrolase domain and two bacterial Src homology 3-binding (SH3b) domains. Further characterization showed that CrfP could be exported to extracellular bacterial cells and lytic S. suis strains of different serotypes, and this finding was verified by TEM and a turbidity assay. To investigate the potential effect of CrfP in vivo, a gene-deletion mutant (ΔcrfP) was constructed. Instead of stopping the natural transformation process, the inactivation of CrfP clearly reduced the effective transformation rate. Overall, these findings provide evidence showing that CrfP is important for S. suis serovar 2 competence.

The Novel Streptococcal Transcriptional Regulator XtgS Negatively Regulates Bacterial Virulence and Directly Represses PseP Transcription.

Streptococcus agalactiae (group B streptococcus [GBS]) has received continuous attention for its involvement in invasive infections and its broad host range. Transcriptional regulators have an important impact on bacterial adaptation to various environments. Research on transcriptional regulators will shed new light on GBS pathogenesis. In this study, we identified a novel XRE-family transcriptional regulator encoded on the GBS genome, designated XtgS. Our data demonstrate that XtgS inactivation significantly increases bacterial survival in host blood and animal challenge test, suggesting that it is a negative regulator of GBS pathogenicity. Further transcriptomic analysis and quantitative reverse transcription-PCR (qRT-PCR) mainly indicated that XtgS significantly repressed transcription of its upstream gene pseP Based on electrophoretic mobility shift and lacZ fusion assays, we found that an XtgS homodimer directly binds a palindromic sequence in the pseP promoter region. Meanwhile, the PseP and XtgS combination naturally coexists in diverse Streptococcus genomes and has a strong association with sequence type, serotype diversification and host adaptation of GBS. Therefore, this study reveals that XtgS functions as a transcriptional regulator that negatively affects GBS virulence and directly represses PseP expression, and it provides new insights into the relationships between transcriptional regulator and genome evolution.

Identification of an Autorepressing Two-Component Signaling System That Modulates Virulence in Streptococcus suis Serotype 2.

Streptococcus suis is one of the most important pathogens affecting the swine industry and is also an emerging zoonotic agent for humans. Two-component signaling systems (TCSs) play important roles in the adaptation of pathogenic bacteria to host environments. In this study, we identified a novel TCS, named TCS09HKRR, which facilitated Streptococcus suis serotype 2 (SS2) resistance to clearance by the host immune system and contributed to bacterial pathogenicity. Furthermore, RNA-sequencing analyses identified 79 genes that were differentially expressed between the wild-type (WT) and ΔTCS09HKRR strains, among which half of the 39 downregulated genes belonged to the capsular biosynthesis clusters. Transmission electron microscopy confirmed that the capsule of the ΔTCS09HKRR strain was thinner than that of the WT strain. Electrophoretic mobility shift assays (EMSA) showed that the regulator of TCS09HKRR (TCS09RR) could not bind the promoter regions of cps and neu clusters, which suggested that TCS09HKRR regulates capsule biosynthesis by indirect pathways. Unexpectedly, the TCS09HKRR operon was upregulated when TCS09HKRR was deleted. A specific region, ATGACATTTGTCAC, which extends from positions -193 to -206 upstream of the TCS09HKRR operon, was further identified as the TCS09RR-binding site using EMSA. These results suggested the involvement of a negative feedback loop in this regulation. In addition, TCS09RR was significantly upregulated by more than 18-fold when coincubated with RAW264.7 macrophages. Our data suggested that autorepression renders TCS09HKRR more sensitive to host stimuli, which optimizes the regulatory network of capsular biosynthesis in SS2.

The Two-Component Signaling System VraSRss Is Critical for Multidrug Resistance and Full Virulence in Streptococcus suis Serotype 2.

Streptococcus suis has received increasing attention for its involvement in severe human infections worldwide as well as in multidrug resistance. Two-component signaling systems (TCSSs) play important roles in bacterial adaptation to various environmental stimuli. In this study, we identified a novel TCSS located in S. suis serotype 2 (SS2), designated VraSRSS, which is involved in bacterial pathogenicity and susceptibility to antimicrobials. Our data demonstrated that the yvqFSS gene, located upstream of vraSRSS , shared the same promoter with the TCSS genes, which was directly regulated by VraSRSS, as shown in electrophoretic mobility shift assays. Notably, YvqFSS and VraSRSS constitute a novel multidrug resistance module of SS2 that participates in resistance to certain groups of antimicrobials. Further analyses showed that VraSRSS inactivation significantly attenuated bacterial virulence in animal models, which, coupled with the significant activation of VraSRSS expression observed in host blood, strongly suggested that VraSRSS is an important regulator of SS2 pathogenicity. Indeed, RNA-sequencing analyses identified 106 genes that were differentially expressed between the wild-type and ΔvraSRSS strains, including genes involved in capsular polysaccharide (CPS) biosynthesis. Subsequent studies confirmed that VraSRSS indirectly regulated the transcription of CPS gene clusters and, thus, controlled the CPS thickness shown by transmission electron microscopy. Decreased CPS biosynthesis caused by vraSRSS deletion subsequently increased bacterial adhesion to epithelial cells and attenuated antiphagocytosis against macrophages, which partially clarified the pathogenic mechanism mediated by VraSRSS Taken together, our data suggest that the novel TCSS, VraSRSS, plays critical roles for multidrug resistance and full virulence in SS2.

SssP1, a Streptococcus suis Fimbria-Like Protein Transported by the SecY2/A2 System, Contributes to Bacterial Virulence.

Streptococcus suis is an important Gram-positive pathogen in the swine industry and is an emerging zoonotic pathogen for humans. In our previous work, we found a virulent S. suis strain, CZ130302, belonging to a novel serotype, Chz, to be associated with acute meningitis in piglets. However, its underlying mechanisms of pathogenesis remain poorly understood. In this study, we sequenced and analyzed the complete genomes of three Chz serotype strains, including strain CZ130302 and two avirulent strains, HN136 and AH681. By genome comparison, we found two putative genomic islands (GIs) uniquely encoded in strain CZ130302 and designated them 50K GI and 58K GI. In mouse infection model, the deletion of 50K and 58K GIs caused 270-fold and 3-fold attenuation of virulence, respectively. Notably, we identified a complete SecY2/A2 system, coupled with its secretory protein SssP1 encoded in the 50K GI, which contributed to the pathogenicity of strain CZ130302. Immunogold electron microscopy and immunofluorescence analyses indicated that SssP1 could form fimbria-like structures that extend outward from the bacterial cell surface. The sssP1 mutation also attenuated bacterial adherence in human laryngeal epithelial (HEp-2) cells and human brain microvessel endothelial cells (HBMECs) compared with the wild type. Furthermore, we showed that two analogous Ig-like subdomains of SssP1 have sialic acid binding capacities. In conclusion, our results revealed that the 50K GI and the inside SecY2/A2 system gene cluster are related to the virulence of strain CZ130302, and we clarified a new S. suis pathogenesis mechanism mediated by the secretion protein SssP1.IMPORTANCEStreptococcus suis is an important zoonotic pathogen. Here, we managed to identify key factors to clarify the virulence of S. suis strain CZ130302 from a novel serotype, Chz. Notably, it was shown that a fimbria-like structure was significantly connected to the pathogenicity of the CZ130302 strain by comparative genomics analysis and animal infection assays. The mechanisms of how the CZ130302 strain constructs these fimbria-like structures in the cell surface by genes encoding and production transport were subsequently elucidated. Biosynthesis of the fimbria-like structure was achieved by the production of SssP1 glycoproteins, and its construction was dependent on the SecA2/Y2 secretion system. This study identified a visible fimbria-like protein, SssP1, participating in adhesion to host cells and contributing to the virulence in S. suis These findings will promote a better understanding of the pathogenesis of S. suis.

Functional role of ompF and ompC porins in pathogenesis of avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli is an important pathogen causes systemic infections in avian species and large economic losses in poultry industry worldwide. The functional role of porins during the infection and their mechanisms of interaction with host tissues for adhesion to and invasion are poorly understood. However, whether porins play a role in infection remains unclear. In this study we evaluated the potential of ompF and ompC outer membrane porins in the pathogenesis of avian pathogenic E. coli (APEC) strain TW-XM. The ompF and ompC were deleted to generate a series of mutants. We found that, ΔompF and ΔompC reduced significantly the adherence by 41.3% and 46.1% and invasion capabilities of APEC to mouse brain microvascular endothelial cell (BMEC) bEnd.3 cells in vitro by 51.9% and 49.7% respectively, compared with the wild strain TW-XM. In vivo experiment based on the measurement of the LD50 have also shown that, ΔompF and ΔompC reduced the bacterial virulence by 9.8-fold, 12.3-fold in ducklings and 9-fold, 10.2-fold in mouse models. Animal infection experiments further revealed that, loss of ompF and ompC reduced TW-XM colonization and invasion capacity in brains, lungs and blood compared to wild-type strain TW-XM (P > 0.01). These virulence-related phenotypes were partially recoverable by genetic complementation. The results of the quantitative real-time reverse transcription-PCR (qRT-PCR) indicated that, the loss of ompF and ompC significantly decreased the expression levels of ompA, fimC and iBeA genes in the mutant strains, compared to wild-type strainTW-XM (P < 0.01). Collectively, our data demonstrate that inactivation of these two porins decreased adhesion, invasion, colonization, proliferation capacities, possibly by reduced expression levels of ompA, fimC and iBeA, which may indicate the involvement of ompF and ompC in APEC pathogenesis.

Characterization and virulence clustering analysis of extraintestinal pathogenic Escherichia coli isolated from swine in China.

BACKGROUND: Swine extraintestinal pathogenic Escherichia coli (ExPEC) is an important pathogen that leads to economic and welfare costs in the swine industry worldwide, and is occurring with increasing frequency in China. By far, various virulence factors have been recognized in ExPEC. Here, we investigated the virulence genotypes and clonal structure of collected strains to improve the knowledge of phylogenetic traits of porcine ExPECs in China. RESULTS: We isolated 64 Chinese porcine ExPEC strains from 2013 to 14 in China. By multiplex PCR, the distribution of isolates belonging to phylogenetic groups B1, B2, A and D was 9.4%, 10.9%, 57.8% and 21.9%, respectively. Nineteen virulence-related genes were detected by PCR assay; ompA, fimH, vat, traT and iutA were highly prevalent. Virulence-related genes were remarkably more prevalent in group B2 than in groups A, B1 and D; notably, usp, cnf1, hlyD, papA and ibeA were only found in group B2 strains. Genotyping analysis was performed and four clusters of strains (named I to IV) were identified. Cluster IV contained all isolates from group B2 and Cluster IV isolates had the strongest pathogenicity in a mouse infection model. As phylogenetic group B2 and D ExPEC isolates are generally considered virulent, multilocus sequence typing (MLST) analysis was performed for these isolates to further investigate genetic relationships. Two novel sequence types, ST5170 and ST5171, were discovered. Among the nine clonal complexes identified among our group B2 and D isolates, CC12 and CC95 have been indicated to have high zoonotic pathogenicity. The distinction between group B2 and non-B2 isolates in virulence and genotype accorded with MLST analysis. CONCLUSION: This study reveals significant genetic diversity among ExPEC isolates and helps us to better understand their pathogenesis. Importantly, our data suggest group B2 (Cluster IV) strains have the highest risk of causing animal disease and illustrate the correlation between genotype and virulence.

Antibacterial effect of porcine PTX3 against Streptococcus suis type 2 infection.

Pentraxin 3 (PTX3), a soluble pattern recognition receptor, plays an important role in innate immunity and has been implicated to be a candidate resistance gene against Streptococcus suis 2 infection. To discover the antibacterial effect of porcine PTX3 against S. suis 2, the 42-kDa PTX3 protein was expressed by Chinese hamster ovary cells (CHO), and an additional eukaryotic expression vector pVAX-ptx3 was constructed. The expressed porcine PTX3 mediated a range of antibacterial activities including increasing phagocytic capacity of primary porcine alveolar macrophages (PAM) against S. suis 2 and inhibiting adhesion of S. suis 2 to human epidermoid cancer cells (Hep-2). In mouse model, pre-intramuscular injecting with pVAX-ptx3 reduced mortality and reduced bacteria loads in blood, spleen, lung and brain compared with that of control group during 2-12 h following intraperitoneal injection (i.p.) with S. suis 2. Meanwhile, the expressions of IL-6 and IL-8 in blood were increased in pVAX-ptx3 group, whereas no obvious changes about IL-10. In piglet model, bacteria load in blood of pVAX-ptx3 group was significantly lower than that of control group after i.p. with S. suis 2, correspondingly, expression of IL-6 and IL-8 were significantly increased in pVAX-ptx3 group. In contrast, white blood cell (WBC) and neutrophil cell (NEU) count of peripheral blood in pVAX-ptx3 group were lower than that of control group. These studies described a novel antibacterial role for porcine PTX3 against S. suis 2 both in vitro and in vivo and suggested that porcine PTX3 may be a potential biological agent against S. suis 2 in pig and be used for the clinical prevention and treatment of streptococcosis caused by S. suis 2.

Porcine extraintestinal pathogenic Escherichia coli delivers two serine protease autotransporters coordinately optimizing the bloodstream infection.

Extraintestinal pathogenic Escherichia coli (ExPEC) is one of the leading causes of bloodstream infections in a broad spectrum of birds and mammals, thus poses a great threat to public health, while its underlying mechanism causing sepsis is not fully understood. Here we reported a high virulent ExPEC strain PU-1, which has a robust ability to colonize within host bloodstream, while induced a low level of leukocytic activation. Two serine protease autotransporters of Enterobacteriaceae (SPATEs), VatPU-1 and TshPU-1, were found to play critical roles for the urgent blood infection of strain PU-1. Although the Vat and Tsh homologues have been identified as virulence factors of ExPEC, their contributions to bloodstream infection are still unclear. In this study, VatPU-1 and TshPU-1 were verified to interact with the hemoglobin (a well-known mucin-like glycoprotein in red blood cell), degrade the mucins of host respiratory tract, and cleave the CD43 (a major cell surface component sharing similar O-glycosylated modifications with other glycoprotein expressed on leukocytes), suggesting that these two SPATEs have the common activity to cleave a broad array of mucin-like O-glycoproteins. These cleavages significantly impaired the chemotaxis and transmigration of leukocytes, and then inhibited the activation of diverse immune responses coordinately, especially downregulated the leukocytic and inflammatory activation during bloodstream infection, thus might mediate the evasion of ExPEC from immune clearance of blood leukocytes. Taken together, these two SPATEs play critical roles to cause a heavy bacterial load within bloodstream via immunomodulation of leukocytes, which provides a more comprehensive understanding how ExPEC colonize within host bloodstream and cause severe sepsis.

Study on the Protective Immunity Induced by Pseudotyped Baculovirus Expressing the E Protein of Tembusu Virus in Ducklings.

The Duck Tembusu virus (DTMUV), a pathogenic flavivirus, has been causing significant economic losses in the Chinese poultry industry since 2010. This virus can severely decrease egg production and inhibit the growth of laying ducks and ducklings. While many vaccines have been developed to prevent DTMUV infection, fresh outbreaks continue to occur, as few effective vaccines are available. The E glycoprotein of DTMUV is the primary target for inducing protective immunity in the natural host. Therefore, we conducted an investigation and successfully developed a recombinant baculovirus containing the DTMUV E gene. Ducklings were then vaccinated with the purified protein derived from this virus as a potential vaccine candidate. Our findings demonstrated that the E glycoprotein of DTMUV was highly expressed in Sf9 cells. The vaccination of ducklings with the recombinant baculovirus Bac-E resulted in the induction of strong humoral and cellular immune responses. Most significantly, we observed that the vaccine provided 100% protective immunity against lethal challenges with the DTMUV YY5 strain.

Inactivation Performance of Pseudorabies Virus as African Swine Fever Virus Surrogate by Four Commercialized Disinfectants.

This study was based on similar physicochemical characteristics of pseudorabies virus (PRV) and African swine fever virus (ASFV). A cellular model for evaluation of disinfectants was established with PRV as an alternative marker strain. In the present study, we evaluated the disinfection performance of commonly used commercialized disinfectants on PRV to provide a reference for the selection of good ASFV disinfectants. In addition, the disinfection (anti-virus) performances for four disinfectants were investigated based on the minimum effective concentration, onset time, action time, and operating temperature. Our results demonstrated that glutaraldehyde decamethylammonium bromide solution, peracetic acid solution, sodium dichloroisocyanurate, and povidone-iodine solution effectively inactivated PRV at concentrations 0.1, 0.5, 0.5, and 2.5 g/L on different time points 30, 5, 10, and 10 min, respectively. Specifically, peracetic acid exhibits optimized overall performance. Glutaraldehyde decamethylammonium bromide is cost effective but requires a long action time and the disinfectant activity is severely affected by low temperatures. Furthermore, povidone-iodine rapidly inactivates the virus and is not affected by environmental temperature, but its application is limited by a poor dilution ratio such as for local disinfection of the skin. This study provides a reference for the selection of disinfectants for ASFV.

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

Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases of pigs. Most pig farms in China are immunized against these two diseases. Here, we describe a stabilized E2 protein as an immunogen inserted into the PRV genome as a bivalent live virus-vectored vaccine. The E2 protein has 48 variant sites, there are 2-5 candidate amino acids per variant site, and the relative energy contribution of each amino acid to E2 energy was calculated. Combined substitutions of amino acids at the neighbor variant site (neighbor substitution) were performed to obtain the E2 protein sequence with the lowest energy (stabilized E2). Multiple amino acid substitutions at 48 variant sites were performed, and the results were consistent with neighbor substitutions. The stabilized E2 sequence was obtained, and its energy decreased by 22 Rosetta Energy Units (REUs) compared with the original sequence. After the recombinant PRV expressing stabilized E2 of CSFV was constructed, the secretion efficiency of stabilized E2 was increased by 2.97 times, and the thermal stability was increased by 10.5 times. Immunization of mice resulted in a 2-fold increase in antibody production, and a balanced antibody level against subtype 1.1 and subtype 2.1d E2 was achieved. In rabbits immunized, the lethal challenge of PRV-ZJ and the fever response induced by CSFV could be prevented simultaneously. These findings suggest that rPRV-muta/287aaE2 is a promising bivalent vaccine against CSFV and PRV infections.

High expression of the classical swine fever virus (CSFV) envelope protein E2 by a single amino acid mutation and its embedded in the pseudorabies virus (PRV) vector for immunization.

Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases in pigs. Most pig farms in China are vaccinated against these two diseases. Gene-deleted pseudorabies virus (PRV) can be used to develop promising and economical multivalent live attenuated viral vector vaccines. It has been reported that recombinant PRV can express a truncated E2 protein (1-338 aa), but it has not been reported that recombinant PRV can express a full-length E2 protein. We constructed nine groups of E2 proteins with different expression forms and found that the E2 protein could be expressed in vitro only when the transmembrane region of E2 was removed and the signal peptide was added. Analysis of the transmembrane region of E2 revealed that the high hydrophobicity of the E2 transmembrane region was the main reason for its inability to express. By mutating an amino acid to reduce the hydrophobicity of the transmembrane region, it was found that the full-length mutant of E2 (E2FL-muta3 or E2FL-muta4) could be expressed. The expressed full-length mutant E2 could also localize to the cell membrane. Mice immunized with a PRV vector vaccine expressing E2FL-muta3 or E2FL-muta4 developed specific cellular immunity to the E2 protein and stimulated higher levels of E2 antibody than mice immunized with a PRV vector expressing truncated E2. After immunizing the rabbits, the lethal challenge by PRV-ZJ2013 and the febrile response elicited by CSFV were simultaneously prevented. These results suggest that rPRV-dTK/gE-E2FL-muta4 is a promising bivalent vaccine against CSFV and PRV infections.

Distinct Whole Transcriptomic Profiles of the Bursa of Fabricius in Muscovy Ducklings Infected by Novel Duck Reovirus with Different Virulence.

Novel duck reovirus (NDRV) is a newly identified reovirus that brings about more severe damage on multiple organs and mortality in various species of waterfowl. We previously characterized the transcriptomic profiles responding to NDRV in the bursa of Fabricius of Muscovy ducklings, which is a major immunological organ against virus infection. However, the molecular mechanisms of variant cell responses in the bursa of Fabricius to NDRV with different virulence is unclear. Here, we conducted a whole transcriptomic analysis to study the effects of two strains, HN10 (virulent NDRV) and JDm10 (artificially attenuated NDRV), on the bursa of Fabricius of Muscovy ducklings. We harvested a large number of differentially expressed genes (DEGs) of the bursa of Fabricius specially induced by HN10 and JDm10, and we found that HN10 induced DEGs enriched in differentiation and development in multiple organs beyond JDm10. Moreover, the ceRNA regulatory network also indicated the different connections among mRNA, lncRNA and miRNA. Interestingly, we further noticed that a population of differential expressed miRNA could particularly target to transcripts of HN10 and JDm10. We took miR-24 as an example and observed that miR-24 could reduce the transcription of GLI family zinc finger 3 (Gli3) and membrane-associated guanylate kinase, WW and PDZ domain containing 1 (Magi1) via recognition 3' UTR of these two genes by a dual luciferase reporter gene assay in vitro. However, this effect could be compromised by HN10 infection or the ectopic over-expression of the putative miR-24 targeting regions in L1 and L3 fragments of HN10. Taken together, we examined and proposed a novel regulatory competitive mechanism between transcripts of NDRV and Muscovy ducklings for miRNA. These findings may advance the understanding of the molecular pathogenesis of NDRV in Muscovy ducklings, and help provide the potential targets for vaccine and drug development against NDRV.

Development and Evaluation of a Monoclonal Antibody-Based Blocking Enzyme-Linked Immunosorbent Assay for the Detection of Antibodies against Novel Duck Reovirus in Waterfowl Species.

The novel duck reovirus (NDRV) is an emerging pathogen that causes disease in various waterfowl species. Since the outbreak, it has caused huge economic losses to the duck industry in China. A rapid, reliable, and high-throughput method is required for epidemiological investigation and evaluation of vaccine immunogenicity. A good first step would be establishing an enzyme-linked immunosorbent assay (ELISA) that could detect NDRV antibodies in different breeds of ducks and geese from the serum and egg yolk. This study used a recombinant NDRV σB protein and a corresponding horseradish peroxidase (HRP)-labeled monoclonal antibody to develop a blocking ELISA (B-ELISA). The cutoff value of the B-ELISA was 37.01%. A total of 212 serum samples were tested by the B-ELISA, and the virus neutralization test (VNT) was the gold standard test. The sensitivity and specificity of the B-ELISA were 92.17% (106/115) and 97.94% (95/97), respectively. The agreement rates between the B-ELISA and VNT were 94.81% (kappa value, 0.896). The B-ELISA could specifically recognize anti-NDRV sera without cross-reacting with other positive serums for other major diseases in ducks and geese. The inter- and intra-assay coefficients of variation (CVs) of the B-ELISA and VNT assays were acceptable. In conclusion, the novel B-ELISA could be a rapid, simple, safe, and economically attractive alternative to the VNT in assessing duck flocks' immunity status and in epidemiological surveillance in multiple waterfowl species. IMPORTANCE NDRV disease is a new epidemic disease in waterfowl that first appeared in China. Compared with the classical DRV (CDRV), NDRV is associated with more severe symptoms, a higher mortality rate, and a broader host range. NDRV has become the prevalent genotype in China. At present, there are no commercially available diagnostic products for the NDRV disease. VNT, as the gold standard serologic test, is not only time-consuming and laborious, but also has high requirements for facilities and equipment, which is not suitable for clinical application. Conventional ELISA requires specific antispecies conjugates that are not currently available. B-ELISA not only has the advantage of higher analysis specificity, but also can be used to test specific antibodies against different waterfowl species, because no species-specific conjugates are required in such detection. Therefore, it is necessary to establish a B-ELISA for the detection of antibodies against NDRV in waterfowl species.

Characterization and epidemiological analysis of Vibrio parahaemolyticus isolated from different marine products in East China.

Vibrio parahaemolyticus is a major foodborne pathogen with a wide distribution in the world that causes economic and public health problems. Here, we isolated 152 V. parahaemolyticus strains from shellfish, shrimp, crab, and snails from 5 provinces in East China, and analyzed the genetic diversity, population structure, and virulence profiles of these isolates. Our results showed that the 152 V. parahaemolyticus strains belong to 84 different sequence types (STs), of which 69 (82.14 %) STs and 60 alleles were newly identified. The thermostable direct hemolysin (TDH) and the TDH-related hemolysin (TRH) were present in 4 V. parahaemolyticus isolates (2.63 %) respectively, while toxRS/new, a distinctive toxRS sequence that was associated with pandemic V. parahaemolyticus strains, is present in 52 isolates (34.21 %). Thereinto, both the ZJ11 and ZJ12 strains measure up to the standard of toxRS/new+, tdh+, and trh-, which was widely used marker for the rapid screening of pandemic strains, and thus these strains may have the risk of infectious outbreaks. In addition, we observed that all the 152 V. parahaemolyticus isolates encode type III secretion systems 1 and type VI secretion system (T6SS) 2, while 119 isolates (78.29 %) of them also contain T6SS1. The genetic relatedness of our isolates to the human V. parahaemolyticus collection was explored, which showed that ST6, ST79, ST162, ST1060, and ST1061 were all identified in both human isolates (7 isolates) uploaded in the PubMLST database and our marine products isolates (7 isolates). Our findings expand the views of the genetic diversity of V. parahaemolyticus and will contribute to understanding the potential risk of the transboundary spread of this bacterium.

Intergrated Transcriptomic and Proteomic Analysis Revealed the Differential Responses to Novel Duck Reovirus Infection in the Bursa of Fabricius of Cairna moschata.

The bursa of Fabricius is an immunologically organ against the invasion of duck reovirus (DRV), which is a fatal bird virus belonging to the Reoviridae family. However, responses of the bursa of Fabricius of Cairna moschata to novel DRV (NDRV) infection are largely unknown. Transcriptomes and proteomes of the samples from control and two NDRV strain (HN10 and JDm10) with different virulence were analyzed. Differentially expressed genes and differential accumulated proteins were enriched in the serine protease system and innate immune response clusters. Most of the immune-related genes were up-regulated under both JDm10/HN10 infections. However, the immune-related proteins were only accumulated under HN10 infection. For the serine protease system, coagulation factor IX, three chains of fibrinogen, and complements C8, C5, and C2s were significantly up-regulated by the HN10 infection, suggesting that the serine protease-mediated immune system might be involved in the resistance to NDRV infection. For the innate and adaptive immune system, RIG-I, MDA5, MAPK20, and IRF3 were significantly up-regulated, indicating their important roles against invaded virus. TLR-3 and IKBKB were only up-regulated in the liver cells, MAPK20 was only up-regulated in the bursa of Fabricius cells, and IRAK2 was only up-regulated in the spleen samples. Coagulation factor IX was increased in the bursa of Fabricius, not in the liver and spleen samples. The data provides a detailed resource for studying the proteins participating in the resistances of the bursa of Fabricius of duck to NDRV infections.