Recent insights into reverse genetics of norovirus.

Norovirus is the leading cause of viral gastroenteritis globally, and poses substantial threats to public health. Despite substantial progress made in preventing norovirus diseases, the lack of a robust virus culture system has hampered biological research and effective strategies to combat this pathogen. Reverse genetic system is the technique to generate infectious viruses from cloned genetic constructs, which is a powerful tool for the investigation of viral pathogenesis and for the development of novel drugs and vaccines. The strategies of reverse genetics include bacterial artificial chromosomes, vaccinia virus vectors, and entirely plasmid-based systems. Since each strategy has its pros and cons, choosing appropriate approaches will greatly improve the efficiency of virus rescue. Reverse genetic systems that have been employed for norovirus greatly extend its life cycle and facilitate the development of medical countermeasures. In this review, we summarize the current knowledge on the structure, transmission, genetic evolution and clinical manifestations of norovirus, and describe recent advances in the studies of norovirus reverse genetics as well as its future prospects for therapeutics and vaccine development.

Long-term antibacterial composite via alginate aerogel sustained release of antibiotics and Cu used for bone tissue bacteria infection.

Bone related-bacterial diseases including wound infections and osteomyelitis (OM) remain a serious problem accompanied with amputation in most severe cases. In this work, we report an exceptional effective antibacterial alginate aerogel, which consists of tigecycline (TGC) and octahedral Cu crystal as an organo-inorganic synergy platform for antibacterial and local infection therapy applications. The alginate aerogel could greatly prolong the release of copper ions and maintain effective antibacterial concentration over 18 days. The result of in-vitro experiments demonstrated that the alginate aerogel has an exceptional effective function on antibacterial activity. Cytotoxicity tests indicated that the alginate aerogel has low biological toxicity (average cell viability >75%). These remarkable results suggested that the alginate aerogel exhibits great potential for the treatment of OM, and has a prosperous future of application in bone tissue engineering.

Muscle destruction caused by coxsackievirus A10 in gerbils: Construction of a novel animal model for antiviral evaluation.

The morbidity and mortality of coxsackievirus A10 (CVA10)-associated hand, foot, and mouth disease (HFMD) have been increasing in recent years, while few studies on the vaccine and animal model of CVA10 have been reported. Here, we first established a CVA10-infected gerbil model and employed it to evaluate the immunoprotective effect of an inactivated CVA10 vaccine. The results showed that gerbils up to the age of 14 days were fully susceptible to CVA10, and all died within five days post-infection by intraperitoneal inoculation. Lethargy, wasting, hind-limb paralysis, and even death could be observed in the CVA10-infected gerbils. Pathological examination suggested that CVA10 has a strong tropism toward muscle tissue, and muscle bundle fracture and muscular fibers necrosis were observed in the limb muscles. Additionally, active immunization results showed that gerbils immunized with the inactivated CVA10 vaccine were 100 % protected from lethal CVA10 challenge. The antisera from vaccinated gerbils also showed high neutralizing titers against CVA10. Based on these results, the CVA10-infected gerbil model was a suitable tool for analyzing the pathogenesis of CVA10 and assessing the protective efficacy of CVA10 candidate vaccines.

Two new compounds from Nigrospora sphaerica ZMT05, a fungus derivated from Oxya chinensis Thunber.

A new pyrrolidinone derivative named nigrosporamide A (1), and a new acetophenone derivative, 4-prenyloxyclavatol (2), were isolated from an endophytic fungus Nigrospora sphaerica (collection No. ZMT05) isolated from Oxya chinensis Thunberg. Their chemical structures were established on the basis of the interpretation of spectroscopic data. In primary in vitro bioassay, nigrosporamide A (1) exhibited strong antifungal activity against Colletotrichum gloeosporioides and high inhibitory activity towards α-glucosidase.

Capsule switching of Neisseria meningitidis sequence type 7 serogroup A to serogroup X.

OBJECTIVES: The bacterial pathogen Neisseria meningitidis is able to escape the currently available capsule-based vaccines by undergoing capsule switching. In this study, we investigated whether capsule switching has occurred in a recently emerged sequence type (ST) 7 serogroup X isolate in China, for which currently no vaccine is available. METHODS: To identify capsule switching breakpoints, the capsule locus and flanking regions of the ST-7 serogroup X isolate and three endemic ST-7 serogroup A isolates were sequenced and compared. To obtain further insight into capsule switching frequency and length of DNA fragments involved, capsule switching assays were performed using genomic DNA containing combinations of antibiotic selection markers at various locations in the capsule locus and flanking regions. RESULTS: Sequence analyses showed that capsule switching has occurred and involved a 8450 bp serogroup X DNA fragment spanning the region from galE to ctrC. Capsule switching assays indicate that capsule switching occurs at a frequency of 6.3 × 10-6 per bacterium per µg of DNA and predominantly involved DNA fragments of about 8.1-9.6 kb in length. CONCLUSIONS: Our results show that capsule switching in N. meningitidis occurs at high frequency and involves recombination in the flanking regions of the capsule biosynthesis genes.

[Sequence-based typing for 61 Legionella pneumophila serogroup 1 isolates in Zhejiang Province].

OBJECTIVE: To analyze the sequence characteristics of the serogroup 1 Legionella pneumophila (Lp1) strains isolated from the cooling tower water in Zhejiang province by sequence-based typing (SBT). METHODS: 61 strains of Lp1 isolated from cooling tower water in ten cities of Zhejiang Province from 2005 to 2011 were genotyped by SBT method. The nucleotide polymorphism of the sequencing results was analyzed by DnaSP 5.0, and the SBT results were cluster analyzed using SplitsTree and eBURST software. RESULTS: All 61 isolates of Lp1 were resolved into 11 STs, and 5 new STs were found in this study. The ST with the largest number of isolates was ST1 (n=50), and these ST-1 strains were found in 10 cities. The nucleotide polymorphism (Pi) of these seven housekeeping genes ranged from 0.01095 (mip) to 0.05355 (pilE). The STs of the Lpl isolates were clustered to four clonal complexes (CC), and ST-1, ST-149, ST-154 were the three main clonal complexes (CC). CONCLUSION: Genetic diversity was shown in the serogroup 1 Legionella pneumophila strains isolated from cooling tower water in Zhejiang province.

[Development of multiplex real time PCR methodology for better identification and discrimination of Vibrio cholerae and O139 serotype].

OBJECTIVE: To develop a rapid, sensitive and specific assay method, based on multiplex real time PCR for identifying Vibrio cholerae and distinguishing Vibrio cholerae O139 serotype from Vibrio cholerae. METHODS: Cholera toxin A subunit gene (ctxA) and glycosyltransferase gene (LPSgt) were chosen as targets according to Vibrio cholerae and Vibrio cholerae O139 serotype, and then the primers and TaqMan-MGB probe were designed. The 5'end of probes was labeled with FAM and VIC fluoresceins respectively while the 3'end of probes was labeled with MGB. The PCR reaction was optimized systematically. Then the specificity, sensitivity and reproducibility of multiplex real time PCR were estimated. Finally, multiplex real time PCR was applied to detect the clinical specimens. RESULTS: Vibrio cholerae was identified by multiplex real time PCR accurately and quickly, which could distinguish Vibrio cholerae O139 serotype from Vibrio cholerae. Vibrio cholerae was identified positive for primer pairs and probes from ctxA gene, and Vibrio cholerae O139 serotype tested was positive for LPSgt gene. Meanwhile, none of other bacteria was identified. Sensitivity of the test was 2 × 10(2) cfu/ml. When this assay was applied directly to identify 45 clinical specimens, results showed that 10 were positive to Vibrio cholerae, in which 4 clinical specimens were positive to Vibrio cholerae O139 serotype. All the results were the same to the one that had been obtained from the conventional assays. CONCLUSION: Our results showed that the multiplex real time PCR was a reliable, accurate and feasible method for identifying Vibrio cholerae that carrying toxin gene and distinguishing Vibrio cholerae O139 serotype from Vibrio cholerae. This method could be applied to the cholera surveillance, prevention and control system for identifying and distinguishing Vibrio cholerae in the labs.

[Establishment and application of loop-mediated isothermal amplification method for rapid detection].

OBJECTIVE: To develop a loop-mediated isothermal amplification (LAMP) method for rapid diagnosing of Legionella pneumophila in the Pathogen Detection Department(PDD) or in small-scale laboratory. METHODS: Five primers (2 Inner Primers, 2 Outer Primers and a Loop Primer) for the LAMP test were designed by targeting the mip gene of L. pneumophila and reaction system of LAMP reaction was optimized. 12 strains of L. pneumophila, 45 local strains, 6 non-L. pneumophila strains, 11 other strains and 59 environmental water samples were analyzed to evaluate the specificity and sensibility of the LAMP amplification. At the same time, the results of the LAMP were also compared with biochemical culture and quantitative PCR methods. RESULTS: The amplification products of L. pneumophila turned green by visual inspection and had ladder-like pattern on the gel, but non-L. pneumophila and other products from the strains remained orange by visual examination and had no band on the gel. The detection rate of LAMP was higher than the biochemical culture and the real-time PCR methods. Reaction time of the LAMP method was only 1.5 h and the detection limit of LAMP assay was 5 cfu/reaction. In addition, the LAMP results could be determined only by visual inspection. CONCLUSION: LAMP assay targeting the mip gene of L. pneumophila appeared to be rapid, specific, and sensitive for the detection of L. pneumophila. This method not only reduced the dependence of complicated equipment but also had a potential for wider use in the PDD, small-scale laboratory, emergency motor vehicle or for field survey.

[Detection of enterohemorrhagic Escherichia coli O157:H7 by loop-mediated isothermal amplification].

OBJECTIVE: To develop a loop-mediated isothermal amplification (LAMP) method for rapidly diagnosing of Escherichia coli (EHEC) O157:H7 in pathogen detection department or small-scale laboratories. METHODS: Primers for LAMP test were designed by targeting the antigen coding rfbE of EHEC O157:H7, the Shiga-like toxin stx2 and the fliC encoding gene of H7 flagella antigen, respectively. The reaction condition and reaction system of LAMP were optimized. 2 EHEC O157:H7 type strains, 17 local strains and 33 other enterobacteria were analyzed to evaluate the LAMP's specificity and sensitivity. The results of the LAMP reaction were also compared with routine PCR method. RESULTS: The amplification products of O157 which had the corresponding target genes turned green by visual inspection and had ladder-like pattern on the gel, but products of other enterobacteria remained orange by visual examination and had no band on the gel. The detection results of LAMP were the same as of routine PCR method. The reaction time of the LAMP method was only 1.5 hours and the detection limit of LAMP assay was 26 CFU/reaction. In addition, the LAMP results could be determined only by visual inspection. CONCLUSION: LAMP assay is rapid, specific, and sensitive for the detection of EHEC O157:H7. This method might not only reduce the dependence of complicated equipments but also be a potential method for wider use in pathogen detection department, small-scale laboratory, emergency motor vehicle or field survey.

[The emergence of candidate pathogenicity island 89K DNA sequence of Streptococcus suis isolated from sporadic patients in Zhejiang province].

OBJECTIVE: To identify the presence of candidate pathogenicity island 89K DNA sequence of Streptococcus suis serotype 2 (SS2) strains isolated from patient in Zhejiang province. METHODS: Genes and DNA fragments were amplified by PCR, using specific primers, and three amplified fragments of the 89K sequence were directly sequenced. The results were analyzed using software related to bioinformatics and epidemiology. RESULTS: 8 strains of SS2 all contained 89K sequence, cps2J and mrp virulent genes, and species-specific 16S rDNA. 3 amplified fragments of 89K candidate pathogenicity island of SS2 ZJ0501 were above 99% similar to SS2 strain identified from outbreaks in Jiangsu in 1998, and the gene fragment of coding DNA recombinant protein in the 89K sequence was highly homological with that of S. dysgalactiae and S.agalactiae. CONCLUSION: In recent years SS2 strains isolated from patients with clinical symptoms in Zhejiang province had been detected to have contained candidate pathogenic 89K DNA fragment.