N-glycosylation on hemagglutinin head reveals inter-branch antigenic variability of avian influenza virus H5-subtypes.

H5-subtype avian influenza virus (AIV) is globally prevalent and undergoes frequent antigenic drift, necessitating regular updates to vaccines. One of the many influencing elements that cause incompatibility between vaccinations and epidemic strains is the dynamic alteration of glycosylation sites. However, the biological significance of N-glycosylation in the viral evolution and antigenic changes is unclear. Here, we performed a systematic analysis of glycosylation sites on the HA1 subunit of H5N1, providing insights into the changes of primary glycosylation sites, including 140 N, 156 N, and 170 N within the antigenic epitopes of HA1 protein. Multiple recombinant viruses were then generated based on HA genes of historical vaccine strains and deactivated for immunizing SPF chickens. Inactivated recombinant strains showed relatively closer antigenicity compared to which has identical N-glycosylation patterns. The N-glycosylation modification discrepancy highlights the inter-branch antigenic diversity of H5-subtype viruses in avian influenza and serves as a vital foundation for improving vaccination tactics.

Cryo-EM structure of severe fever with thrombocytopenia syndrome virus.

The severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne human-infecting bunyavirus, which utilizes two envelope glycoproteins, Gn and Gc, to enter host cells. However, the structure and organization of these glycoproteins on virion surface are not yet known. Here we describe the structure of SFTSV determined by single particle reconstruction, which allows mechanistic insights into bunyavirus assembly at near-atomic resolution. The SFTSV Gn and Gc proteins exist as heterodimers and further assemble into pentameric and hexameric peplomers, shielding the Gc fusion loops by both intra- and inter-heterodimer interactions. Individual peplomers are associated mainly through the ectodomains, in which the highly conserved glycans on N914 of Gc play a crucial role. This elaborate assembly stabilizes Gc in the metastable prefusion conformation and creates some cryptic epitopes that are only accessible in the intermediate states during virus entry. These findings provide an important basis for developing vaccines and therapeutic drugs.

High Levels of Antibiotic Resistance in MDR-Strong Biofilm-Forming Salmonella Typhimurium ST34 in Southern China.

Salmonella enterica subsp. enterica serovar Typhimurium (S. typhimurium) is an important zoonotic pathogen with important public health significance. To understand S. typhimurium's epidemiological characteristics in China, multi-locus sequence typing, biofilm-forming ability, antimicrobial susceptibility testing, and resistant genes of isolates from different regions and sources (human, food) were investigated. Among them, ST34 accounted for 82.4% (243/295), with ST19 ranking second (15.9%; 47/295). ST34 exhibited higher resistance levels than ST19 (p < 0.05). All colistin, carbapenem, and ciprofloxacin-resistant strains were ST34, as were most cephalosporin-resistant strains (88.9%; 32/36). Overall, 91.4% (222/243) ST34 isolates were shown to have multidrug resistance (MDR), while 53.2% (25/47) ST19 isolates were (p < 0.05). Notably, 97.8% (45/46) of the MDR-ACSSuT (resistance to Ampicillin, Chloramphenicol, Streptomycin, Sulfamethoxazole, and Tetracycline) isolates were ST34, among which 69.6% (32/46) of ST34 isolates were of human origin, while 30.4% (14/46) were derived from food (p < 0.05). Moreover, 88.48% (215/243) ST34 showed moderate to strong biofilm-forming ability compared with 10.9% (5/46) ST19 isolates (p < 0.01). This study revealed the emergence of high-level antibiotic resistance S. typhimurium ST34 with strong biofilm-forming ability, posing concerns for public health safety.

BolA affects the biofilm formation ability, outer membrane permeability and virulence, thus is required for the adaptability of Salmonella enterica serotype Typhimurium to the harsh survival environment.

Salmonella enterica serotype Typhimurium, an important foodborne pathogen with high adaptability to the host's internal and external survival environment, seriously threatens public health. Therefore, to understand the mechanism underlying the high adaptability, this study investigated the transcription factor BolA by constructing BolA deletion strain 269△BolA, complemented strain 269BolAR and overexpression strain 269BolA+ based on WT269. BolA significantly inhibited motility; at 6 h, the BolA overexpression strain (269BolA+) showed 91.2% and 90.7% lower motility than the wild type (WT269) and BolA deletion strain (269△BolA), respectively, by downregulating motility-related flagellar genes. BolA promoted biofilm formation; 269BolA+ showed 3.6-fold and 5.2-fold higher biofilm formation ability than WT269 and 269ΔBolA, respectively, by upregulation biofilm formation-related genes. BolA overexpression downregulated the outer membrane gene OmpF and upregulated OmpC, thereby regulating cell permeability, and reducing the antibacterial effect of vancomycin, which can destruct the outer membrane. BolA improved adaptability; 269△BolA showed higher susceptibility to eight antibiotics and 2.5- and 4-fold lower acid and oxidative stress tolerance, respectively, than WT269. In Caco-2 and HeLa cells, 269△BolA showed 2.8- and 3-fold lower cell adhesion ability, respectively, and 4- and 2-fold lower cell invasion ability, respectively, than WT269, through downregulation of the virulence genes. Thus, BolA expression promotes biofilm formation and balances the membrane permeability, thereby improving the resistance of the strains, and enhances its host cell invasion ability by upregulating bacterial virulence factors. Results of this study suggest that the BolA gene may serve as a potential target of therapeutic or preventative strategies to control Salmonella Typhimurium infections.

The genetic diversity, replication, and transmission of 2009 pandemic H1N1 viruses in China.

Background: The 2009 pandemic H1N1 influenza A virus (pdm09) continue to evolve, and few studies have systemically analyzed the evolution, replication, and transmission of pmd09 viruses in China. Methods: To better understand the evolution and pathogenicity of pdm09 viruses, we systematically analyzed viruses that were confirmed in 2009-2020 in China and characterized their replication and transmission ability. We extensively analyzed the evolution characteristics of pdm/09 in China over the past decades. The replication ability of 6B.1 and 6B.2 lineages on Madin-Darby canine kidney (MDCK) and human lung adenocarcinoma epithelial (A549) cells and their pathogenicity and transmission in guinea pigs were also compared. Results: In total, 3,038 pdm09 viruses belonged to clade 6B.1 (62% of all pdm09 viruses) and clade 6B.2 (4%). Clade 6B.1 pdm09 viruses are the predominant clade, with proportions of 54.1%, 78.9%, 57.2%, 58.6%, 61.7%, 76.3%, and 66.6% in the North, Northeast, East, Central, South, Southwest, and Northeast regions in China, respectively. The isolation proportion of clade 6B.1 pdm/09 viruses was 57.1%, 74.3%, 96.1%, 98.2%, 86.7%, and 78.5% in 2015-2020, respectively. A clear differentiation time point appeared in 2015 before which the evolution trend of pdm09 viruses in China was similar to that in North America but then showed a different trend after that point. To characterize pdm09 viruses in China after 2015, we further analyzed 33 pdm09 viruses isolated in Guangdong in 2016-2017, among which A/ Guangdong/33/2016 and A/Guangdong/184/2016 (184/2016) belonged to clade 6B.2, and the other 31 strains belonged to clade 6B.1. A/Guangdong/887/2017 (887/2017) and A/Guangdong/752/2017 (752/2017) (clade 6B.1), 184/2016 (clade 6B.2) and A/California/04/2009 (CA04) replicated efficiently in MDCK cells and A549 cells, as well as the turbinates of guinea pigs. 184/2016 and CA04 could transmit among guinea pigs through physical contact. Conclusion: Our findings provide novel insights into the evolution, pathogenicity, and transmission of pdm09 virus. The results show that enhancing surveillance of pdm09 viruses and timely evaluation of their virulence are essential.

Genomic Characterization of Salmonella enterica serovar Weltevreden Associated with Human Diarrhea.

Salmonella Weltevreden is an emerging pathogen associated with human diarrhea, and knowledge of the genomics and epidemiology of this serovar is still limited. In this study, we performed whole-genome sequencing of 96 S. Weltevreden isolates recovered from diarrheal patients and 62 isolates from food animals in China between 2006 and 2017. Together, with an additional 199 genome sequences of S. Weltevreden published in NCBI, we performed an analysis on all 357 S. Weltevreden genome sequences. Our results demonstrated that the majority of S. Weltevreden from diarrheal patients from China (97.92%, 94/96) and the other regions in the world (94.97%, 189/199) identified in this study were sequence type (ST) 365. The remaining types were ST3771 (n = 3), ST22 (n = 1), ST155 (n = 1), and ST684 (n = 1). In addition, ST365 was also widely recovered from animals, food, and environmental samples in different regions of the world. Phylogenetic analysis and pulsed-field gel electrophoresis (PFGE) revealed that S. Weltevreden from diarrheal patients was closely related to those recovered from food and environmental specimens. We also showed that S. Weltevreden did not exhibit severe antimicrobial resistance profiles, suggesting administering antibiotics is still effective for controlling the agent. Interestingly, we found that S. Weltevreden strains carried a number of virulence factor genes, and a 100.03-kb IncFII(S) type plasmid was widely distributed in S. Weltevreden strains. Elimination of this plasmid decreased the bacterial capacity to infect both Caco-2 cells and C57BL/6 mice, suggesting the importance of this plasmid for bacterial virulence. Our results contribute to the understanding of the epidemiology and virulence of S. Weltevreden. IMPORTANCE Salmonella Weltevreden is a pathogen associated with human diarrheal diseases found across the globe. However, knowledge of the genomics and epidemiology of this pathogen is still limited. In this study, we found S. Weltevreden sequence type (ST) 365 is commonly recovered from diarrheal patients in China and many other regions of the world, and there is no major difference between the Chinese isolates and the global isolates at the phylogenetic level. We also demonstrated that ST365 was widely recovered from animal, food, and environmental samples collected in different, global regions. Importantly, we discovered an IncFII(S) type plasmid commonly carried by S. Weltevreden strains of human, animal, and food origins, and this plasmid is likely to contribute to the bacterial pathogenesis. These findings enhance our understanding of the emergence of S. Weltevreden involved in diarrheal outbreaks and the global spread of S. Weltevreden strains.

A novel linear displacement isothermal amplification with strand displacement probes (LDIA-SD) in a pocket-size device for point-of-care testing of infectious diseases.

Nucleic acid amplification is crucial for disease diagnosis, especially lethal infectious diseases such as COVID-19. Compared with PCR, isothermal amplification methods are advantageous for point-of-care testing (POCT). However, complicated primer design limits their application in detecting some short targets or sequences with abnormal GC content. Herein, we developed a novel linear displacement isothermal amplification (LDIA) method using two pairs of conventional primers and Bacillus stearothermophilus (Bst) DNA polymerase, and reactions could be accelerated by adding an extra primer. Pseudorabies virus gE (high GC content) and Salmonella fimW (low GC content) genes were used to evaluate the LDIA assay. Using strand displacement (SD) probes, a LDIA-SD method was developed to realize probe-based specific detection. Additionally, we incorporated a nucleic acid-free extraction step and a pocket-sized device to realize POCT applications of the LDIA-SD method. The LDIA-SD method has advantages including facile primer design, high sensitivity and specificity, and applicability for POCT, especially for amplification of complex sequences and detection of infectious diseases.

Transmembrane domain of IFITM3 is responsible for its interaction with influenza virus HA2 subunit.

Interferon-inducible transmembrane protein 3 (IFITM3) inhibits influenza virus infection by blocking viral membrane fusion, but the exact mechanism remains elusive. Here, we investigated the function and key region of IFITM3 in blocking influenza virus entry mediated by hemagglutinin (HA). The restriction of IFITM3 on HA-mediated viral entry was confirmed by pseudovirus harboring HA protein from H5 and H7 influenza viruses. Subcellular co-localization and immunocoprecipitation analyses revealed that IFITM3 partially co-located with the full-length HA protein and could directly interact with HA2 subunit but not HA1 subunit of H5 and H7 virus. Truncated analyses showed that the transmembrane domain of the IFITM3 and HA2 subunit might play an important role in their interaction. Finally, this interaction of IFITM3 was also verified with HA2 subunits from other subtypes of influenza A virus and influenza B virus. Overall, our data demonstrate for the first time a direct interaction between IFITM3 and influenza HA protein via the transmembrane domain, providing a new perspective for further exploring the biological significance of IFITM3 restriction on influenza virus infection or HA-mediated antagonism or escape.

Genomic Investigation of Proteus mirabilis Isolates Recovered From Pig Farms in Zhejiang Province, China.

Proteus mirabilis is a common opportunistic zoonotic pathogen, and its ongoing acquisition of antimicrobial resistance genes poses challenges to clinical treatments. Human-sourced whole genomic sequencing of human P. mirabilis isolates has been reported, but pig-sourced isolates have not been thoroughly investigated even though these animals can serve as reservoirs for human infections. In the current study, we report a molecular epidemiological investigation to unravel the antimicrobial and virulence gene risk factors for P. mirabilis contamination in 9 pig farms in 3 different cities in Zhejiang Province, China. We collected 541 swab samples from healthy pigs and 30 were confirmed as P. mirabilis. All 30 isolates were resistant to tetracyclines, macrolides, sulfonamides, ß-lactams and chloramphenicol, and all were multiple drug-resistant and 27 were strong biofilm formers. Phylogenetic analyses indicated these 30 isolates clustered together in 2 major groups. Whole genome sequencing demonstrated that the isolates possessed 91 different antimicrobial resistance genes belonging to 30 antimicrobial classes including rmtB, sul1, qnrS1, AAC(6') - Ib - cr, blaCTX - M - 65 and blaOXA - 1. All isolates contained mobile genetic elements including integrative conjugative elements (ICEs) and integrative and mobilizable elements (IMEs). Minimum inhibitory concentration (MIC) testing indicated direct correlates between cognate genes and antimicrobial resistance. We also identified 95 virulence factors, almost all isolates contained 20 fimbrial and flagellar operons, and this represents the greatest number of these operon types found in a single species among all sequenced bacterial genomes. These genes regulate biofilm formation and represent a confounding variable for treating P. mirabilis infections. Our P. mirabilis isolates were present in healthy animals, and multiple drug resistance in these isolates may serve as a reservoir for other intestinal and environmental Enterobacteriaceae members. This prompts us to more strictly regulate veterinary antibiotic use.

The global succinylation of SARS-CoV-2-infected host cells reveals drug targets.

SARS-CoV-2, the causative agent of the COVID-19 pandemic, undergoes continuous evolution, highlighting an urgent need for development of novel antiviral therapies. Here we show a quantitative mass spectrometry-based succinylproteomics analysis of SARS-CoV-2 infection in Caco-2 cells, revealing dramatic reshape of succinylation on host and viral proteins. SARS-CoV-2 infection promotes succinylation of several key enzymes in the TCA, leading to inhibition of cellular metabolic pathways. We demonstrated that host protein succinylation is regulated by viral nonstructural protein (NSP14) through interaction with sirtuin 5 (SIRT5); overexpressed SIRT5 can effectively inhibit virus replication. We found succinylation inhibitors possess significant antiviral effects. We also found that SARS-CoV-2 nucleocapsid and membrane proteins underwent succinylation modification, which was conserved in SARS-CoV-2 and its variants. Collectively, our results uncover a regulatory mechanism of host protein posttranslational modification and cellular pathways mediated by SARS-CoV-2, which may become antiviral drug targets against COVID-19.

Vegetation Classification and Distribution Patterns in the South Slope of Yarlung Zangbo Grand Canyon National Nature Reserve, Eastern Himalayas.

Yarlung Zangbo Grand Canyon National Nature Reserve has the most complete vertical vegetation belts in China. However, identification and distribution of vertical vegetation belts is still uncertain and in debate. To explore the above issues, 190 plots were surveyed within the reserve from 2019 to 2021. Based on the vegetation plot data, cluster analysis, ordination analysis, and biodiversity statistics were performed to reveal the structure of vertical vegetation belts-the driving factors of vegetation distribution-to describe the main biodiversity patterns. Five vertical vegetation belts were identified by clustering. NMDS ordination showed that the main factor of vegetation distribution is elevation. Based on the results of the analysis and previous literature, a new scheme of vertical vegetation belts in the south slope of the reserve was proposed. There was a lower montane seasonal rainforest belt (600-1100 m), a lower montane evergreen broadleaf forest belt (1100-1800 m), a middle montane semi-evergreen broadleaf forest belt (1800-2400 m), a subalpine evergreen needleleaf forest belt (2400-3800 m), a alpine shrubland and meadow belt (3800-4400 m), an alpine sparse vegetation belt (4400-4800 m), and a nival belt (4800-7782 m). Among them, the seasonal rainforest belts are the northernmost distribution of this type, and the semi-evergreen broadleaf forest belts exist only in the Eastern Himalayas. The study showed a unimodal pattern in plant species diversity, the peak of which is about 1900 m. The middle montane semi-evergreen broadleaf forest belt had the highest species diversity in the reserve. This study settled the issues regarding the vertical vegetation belts, the main drivers of vegetation and assessment of plant species diversity in the south slope of the Yarlung Zangbo Grand Canyon National Nature Reserve. It provides essential support for the management and conservation of these ecosystems in the reserve.

Chicken Peripheral Blood Mononuclear Cells Response to Avian Leukosis Virus Subgroup J Infection Assessed by Single-Cell RNA Sequencing.

Chicken peripheral blood mononuclear cells (PBMCs) exhibit wide-ranging cell types, but current understanding of their subclasses, immune cell classification, and function is limited and incomplete. Here we performed single-cell RNA sequencing (scRNA-seq) of PBMCs in Avian leukosis virus subgroup J (ALV-J) infected and control chickens at 21 days post infection (DPI) to determine chicken PBMCs subsets and their specific molecular and cellular characteristics. Eight cell populations and their potential marker genes were identified in PBMCs. T cell populations had the strongest response to (ALV-J) infection, based on the detection of the largest number of differentially expressed genes (DEGs), and could be further grouped into four subsets: activated CD4+ T cells, Th1-like cells, Th2-like cells, and cytotoxic CD8+ T cells. Furthermore, pseudotime analysis results suggested that chicken CD4+ T cells could potentially differentiate into Th1-like and Th2-like cells. Moreover, ALV-J infection activated CD4+ T cell was probably inclined to differentiate into Th1-like cells. Compared to the control PBMCs, ALV-J infection also had an obvious impact on PBMCs composition. B cells showed inconspicuous response and their numbers decreased in PBMCs from ALV-J infected chicken. Proportions of cytotoxic Th1-like cells and CD8+ T cells increased in the T cell population of PBMCs from ALV-J infected chicken, which were potentially key mitigating effectors against ALV-J infection. More importantly, our results provide a rich resource of gene expression profiles of chicken PBMCs subsets for a systems-level understanding of their function in homeostatic condition as well as in response to viral infection.

The "LLQY" Motif on SARS-CoV-2 Spike Protein Affects S Incorporation into Virus Particles.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein mediates viral entry and membrane fusion. Its cleavage at S1/S2 and S2' sites during the biosynthesis in virus producer cells and viral entry are critical for viral infection and transmission. In contrast, the biological significance of the junction region between both cleavage sites for S protein synthesis and function is less understood. By analyzing the conservation and structure of S protein, we found that intrachain contacts formed by the conserved tyrosine (Y) residue 756 (Y756) with three α-helices contribute to the spike's conformational stability. When Y756 is mutated to an amino acid residue that can provide hydrogen bonds, S protein could be expressed as a cleaved form, but not vice versa. Also, the L753 mutation linked to the Y756 hydrogen bond prevents the S protein from being cleaved. Y756 and L753 mutations alter S protein subcellular localization. Importantly, Y756 and L753 mutations are demonstrated to reduce the infectivity of the SARS-CoV-2 pseudoviruses by interfering with the incorporation of S protein into pseudovirus particles and causing the pseudoviruses to lose their sensitivity to neutralizing antibodies. Furthermore, both mutations affect the assembly and production of SARS-CoV-2 virus-like particles in cell culture. Together, our findings reveal for the first time a critical role for the conserved L753-LQ-Y756 motif between S1/S2 and S2' cleavage sites in S protein synthesis and processing as well as virus assembly and infection. IMPORTANCE The continuous emergence of SARS-CoV-2 variants such as the delta or lambda lineage caused the continuation of the COVID-19 epidemic and challenged the effectiveness of the existing vaccines. Logically, the spike (S) protein mutation has attracted much concern. However, the key amino acids in S protein for its structure and function are still not very clear. In this study, we discovered for the first time that the conserved residues Y756 and L753 at the junction between the S1/S2 and S2' sites are very important, like the S2' cleavage site R815, for the synthesis and processing of S protein such as protease cleavage, and that the mutations severely interfered with the incorporation of S protein into pseudotyped virus particles and SARS-CoV-2 virus-like particles. Consequently, we delineate the novel potential target for the design of broad-spectrum antiviral drugs in the future, especially in the emergence of SARS-CoV-2 variants.

Highly Prevalent Multidrug-Resistant Campylobacter spp. Isolated From a Yellow-Feathered Broiler Slaughterhouse in South China.

The purpose of this study was to investigate the prevalence, antimicrobial resistance, virulence genes, and genetic diversity of Campylobacter spp. along the yellow-feathered broiler slaughtering line in Southern China from December 2018 to June 2019. A total of 157 Campylobacter spp. isolates were identified from 1,102 samples (including 53.6% (75/140) of live chicken anal swab samples, 27.5% (44/160) of defeathering samples, 18.1% (29/160) of evisceration samples, 2.1% (3/140) of washing samples, 1.4% (2/140) of chilling samples, and 1.1% (4/362) of environmental samples). The prevalence of Campylobacter spp. was 14.2%, including 43.9% Campylobacter jejuni, 53.5% Campylobacter coli, and 2.5% other Campylobacter species. The highest antimicrobial resistance rate was found to be against sulfamethoxazole (138/157, 87.9%), and 90.4% (142/157) of the isolates were multidrug resistant (MDR). Examination of resistance-related genes revealed the double base mutated Thr-86-Ile, which informed ACA-TTA, with an Arg-79-Lys substitution in gyrA. Eleven virulence-associated genes (cadF, cdtA, cdtB, ciaB, flaA, imaA, dnaJ, plaA, virB11, racR, and cdtC) were also detected by a polymerase chain reaction (PCR) analysis, and cadF (81.5%) was the most prevalent. Based on an analysis of pulsed-field gel electrophoresis (PFGE) results, we found that Campylobacter spp. could be cross-contaminated throughout the entire slaughtering line. These results show that it is imperative to study the Campylobacter spp. from the yellow-feathered broiler along the slaughtering line in China to develop preventative and treatment measures for the poultry industry, as well as food safety and public health.

Characterization of the emerging multidrug-resistant Salmonella enterica serotype Kentucky ST314 in China.

Salmonella enterica serotype Kentucky (S. Kentucky) is an important Salmonella serotype with multiple sequence types (ST) with a worldwide incidence. We identified 8 STs from 180 strains of S. Kentucky, and ST314 emerged as the most commonly encountered ST. Drug susceptibility testing revealed that ST314 had multiple resistance properties, and 75.5% of the strains were resistant to three or more classes of antimicrobials. The rate of resistance to chloramphenicol, florfenicol, sulfafurazole and tetracycline were greater than 60%. The rates of ST314 resistance to quinolones were as follows: ciprofloxacin, 32.1%; nalidixic acid, 16%; and ofloxacin, 7.5%. Investigating the mechanism of quinolone resistance of ST314 revealed that mutations in the quinolone resistance-determining regions were rare, and resistance mainly occurred due to the resistance genes carried by plasmids. Only 1.9% (2/106) of ST314 strains had mutations in the quinolone resistance-determining regions (QRDR). The drug resistance genes of ST314 were primarily of plasmid-mediated quinolone resistance (PMQR). The detection rate of Salmonella genomic island 1 (SGI1) in ST314 was 12.3%. XbaI-pulsed-field gel electrophoresis revealed that S. enterica Kentucky ST314 was capable of cross-regional and cross-host transmission in China. We found ST314 to be the dominant S. Kentucky ST in China, and it carried multidrug resistance. This is the first report about the emergence of quinolone-resistant S. enterica Kentucky ST314 in China, which is different from previous reports, and the findings of the present study suggest that the mechanism of quinolone resistance in these strains are plasmid-mediated. Notably, plasmids carrying resistance genes may promote the rapid spread of ciprofloxacin resistance.

Competitive activation cross amplification combined with smartphone-based quantification for point-of-care detection of single nucleotide polymorphism.

In this study, we firstly propose a novel smartphone-assisted visualization SNP genotyping method termed competitive activation cross amplification (CACA). The mutation detection strategy depends on the ingenious design of both a start primer and a verification probe with ribonucleotide insertion through competitive combination and perfect matching with the target DNA, Meanwhile, the RNase H2 enzyme was utilized to specifically cleave ribonucleotide insertion and achieve extremely specific dual verification. Simultaneously, the results allow both colorimetric and fluorescence product dual-mode visualization by using self-designed 3D-printed dual function cassette. We validated this novel CACA by analyzing the Salmonella Pullorum rfbS gene at the 237th site, successfully solve the current bottleneck of specific identification and visual detection of this pathogen. The concentration detection limits of the plasmid and genomic DNA were 1500 copies/µL and 3.98 pg/µL, respectively, and as low as the presence of 0.1% mutant-type can be distinguished from 99.9% wild-type. Combined with a powerful hand-warmer, which can provide heating more than 60 °C for 20 h to realize power-free, dual function cassette and smartphone quantitation, our novel CACA platform firstly realizes user-friendly, cost-effective, portable, rapid, and accurate POC detection of SNP.

Molecular Characteristics, Antigenicity, Pathogenicity, and Zoonotic Potential of a H3N2 Canine Influenza Virus Currently Circulating in South China.

Canine influenza viruses (CIVs) could be a source of influenza viruses which infect humans because canine are important companion pets. To assess the potential risk of H3N2 CIVs currently circulating in southern China to public health, biological characteristics of A/canine/Guangdong/DY1/2019 (CADY1/2019) were detected. CADY1/2019 bound to both avian-type and human-type receptors. CADY1/2019 had a similar pH value for HA protein fusion to human viruses, but its antigenicity was obviously different from those of current human H3N2 influenza viruses (IVs) or the vaccine strains recommended in the North hemisphere. CADY1/2019 effectively replicated in the respiratory tract and was transmitted by physical contact among guinea pigs. Compared to human H3N2 IV, CADY1/2019 exhibited higher replication in MDCK, A549, 3D4/21, ST, and PK15 cells. Sequence analysis indicated that CADY1/2019 is an avian-origin virus, and belongs to the novel clade and has acquired many adaptation mutations to infect other mammals, including human. Taken together, currently circulating H3N2 CIVs have a zoonotic potential, and there is a need for strengthening surveillance and monitoring of their pathogenicity.

The Biological Characteristics of Novel H5N6 Highly Pathogenic Avian Influenza Virus and Its Pathogenesis in Ducks.

Clade 2.3.4.4 H5Nx highly pathogenic avian influenza viruses (HPAIVs) have caused outbreaks in poultry in the world. Some of these viruses acquired internal genes from other subtype avian influenza viruses (AIVs) such as H9 and H6 for the generation of novel reassortant viruses and continually circulated in poultry. Here, we applied a duck-origin virus DK87 and a chicken-origin virus CK66 to assess the biological characteristics of novel reassortant H5N6 HPAIVs and its pathogenesis in ducks. A genetic analysis indicated that the HA genes of the two H5N6 HPAIVs were closely related to the H5 viruses of clade 2.3.4.4 circulating in Eastern Asia and classified into H5 AIV/Eastern Asia (EA)-like lineage. Their NA genes fell into Eurasian lineage had close relationship with those of H5N6 viruses circulating in China, Laos, Vietnam, Japan, and Korea. All internal genes of DK87 were aggregated closely with H5 AIV/EA-like viruses. The internal genes (PB1, PA, NP, M, and NS) of CK66 were derived from H9N2 AIV/SH98-like viruses and the PB2 were derived from H5 AIV/EA-like viruses. These results indicate that clade 2.3.4.4 H5N6 AIVs have continually evolved and recombined with the H9N2 viruses circulating in Southern China. Pathogenicity test showed that the two viruses displayed a broader tissue distribution in ducks and caused no clinical signs. These results indicated that ducks were permissive for the replication of the chicken-origin reassortant virus CK66 without prior adaptation, but the duck-origin virus DK87-inoculated ducks showed significantly higher viral titers in some organs than the CK66-inoculated ducks at 5 day post-inoculated (DPI). The recovery of viruses from oropharyngea and cloacal swabs of contacted ducks indicated that they transmitted in native ducks by direct contact. Quantitative reverse transcription PCR (qRT-PCR) results revealed that the immune-relative genes (PRRs, IFNs, Mx-1, IL-6, and IL-8) in the lungs of inoculated ducks were expressed regardless of virus origin, but the expression of these genes was significantly higher in response to infection with the DK87 virus compared to the CK66 virus at 3 DPI. Overall, we should provide further insights into how clade 2.3.4.4 H5N6 AIVs undergo genetic and pathogenic variations to prevent outbreaks of this disease.

A one-step closed-tube enzyme-activated blocked probe assay based on SNP for rapid detection of Salmonella Pullorum.

Salmonella enterica serovar Gallinarum biovars Pullorum (S. Pullorum) is an infectious bacterial pathogen in the poultry industry that causes systemic pullorum disease. This disease causes great losses in terms of the clinical production and quality of chicken products in breeding farms. However, an acknowledged usable rapid detection method for its specific identification has not been reported, and it is generally difficult to distinguish from fowl typhoid caused by Salmonella enterica serovar Gallinarum biovars Gallinarum. The development of a specific and rapid detection method for this pathogen is therefore needed. In the present study, we targeted the single-nucleotide mutation position 237 of the S. Pullorum rfbS gene to develop an enzyme-activated blocked probe for its clinical rapid detection. The method displayed robust specificity and reproducibility, and it achieved minimal detection limits of 21 copies/µL of copy number and 4.53 pg/µL of genomic DNA. Compared with traditional identification and PCR methods, this method performed better for the detection of 100 clinical actual samples and without false negative results. The entire process can be accomplished in a 1-step closed-tube operation, overcomes the difficulties currently associated with S. Pullorum detection, and provides a specific and rapid method with broad application potential for SNP detection.

Highly prevalent multidrug resistance and QRDR mutations in Salmonella isolated from chicken, pork and duck meat in Southern China, 2018-2019.

This study was undertaken to investigate the prevalence, serotype distribution and antimicrobial resistance in Salmonella isolated from retail meat in Southern China, and to characterize the major mechanisms that mediate the ciprofloxacin resistance of isolates. High levels of Salmonella contamination were detected in pork (67.0%), duck (50.5%) and chicken (46.2%). Thirty different serotypes were identified among 500 detected Salmonella isolates, as well as significant differences in serotypes between different retail meat samples. Notably, 405 (80.1%) isolates exhibited multidrug resistance (MDR). Meanwhile, we also found that 74 (14.8%) Salmonella isolates were resistant to ciprofloxacin and the major mechanisms underlying this resistance were investigated. The commonest mutations in gyrA S83F (40.5%) and D87N (35.1%), and in parC was T57S (71.6%) and S80I (35.1%). Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) analysis revealed that the S. Kentucky isolates that were resistant to ciprofloxacin mostly belonged to ST198 (21/23, 91.3%) and PFGE revealed the presence of various genotypes. This study identified a diversity of Salmonella serotypes and a high prevalence of multidrug resistance (MDR) among Salmonella isolated from retail meat in Southern China, which indicates that foodborne Salmonella potentially constitutes a potential food safety risk.