Whole-genome analysis of porcine epidemic diarrhea virus (PEDV) from eastern China.

The complete genome sequence of a porcine epidemic diarrhea virus variant, strain SHQP/YM/2013, from China was determined and compared with those of other porcine epidemic diarrhea viruses. The full-length genome was 28,038 nucleotides (nt) in length without the poly (A) tail, and it was similar to that of other reported PEDV strains, with the characteristic gene order 5'-replicase (1a/1b) -S-ORF3-E-M-N-3'. Nucleotide sequence analysis based on individual virus genes indicated a close relationship between the S gene of SHQP/YM/2013 and those of the four Korean field strains from 2008-2009. Its ORF3 gene, however, fell into three groups. Recent prevalent Chinese PEDV field isolates were divided between group 1 and group 3, which suggests that the recent prevalent Chinese PEDV field isolates represent a new genotype that differs from the genotype that includes the vaccine strains. Based on phylogenetic analysis of the M gene, ORF3 gene and S gene, our study demonstrated that prevalent PEDV isolates in China may have originated from Korean strains. This report describes the complete genome sequence of SHQP/YM/2013, and the data will promote a better understanding of the molecular epidemiology and genetic diversity of PEDV field isolates in eastern China.

Epidemiological situation and genetic analysis of H7N9 influenza viruses in Shanghai in 2013.

The first reported human case of H7N9 influenza virus infection in Shanghai prompted a survey of local avian strains of influenza virus, involving the analysis of a large number of samples taken from poultry, wild birds, horses, pigs, dogs and mice. Seven instances of H7N9 virus infection were identified by real-time RT-PCR (1.47 % of samples), all in chickens sold in live-poultry markets. H7N9 antibody was not detected in serum samples collected from local poultry farms since 2006. The two H7N9 virus strains in the live-poultry markets and one H9N2 virus strain in the same market were genetically characterized. Resequencing of two of the seven isolates confirmed that they closely resembled H7N9 virus strains characterized elsewhere. Various strains co-exist in the same market, presenting a continuing risk of strain re-assortment. The closure of live-poultry markets has been an effective short-term means of minimizing human exposure to H7N9 virus.

Epidemiological survey of porcine epidemic diarrhea virus in swine farms in Shanghai, China.

An epidemiological survey of porcine diarrheal disease prevalence between September 2011 and January 2012 revealed that porcine epidemic diarrhea virus (PEDV) contributed to outbreaks of diarrhea in pig farms in Shanghai, China. The distribution profile of 10 PEDV strains revealed three distinct genotypes coexisting in the same pig farm. Two of the ten field strains that were isolated exhibited a distinct evolution from the others. In addition to PEDV, other enteric pathogens, including porcine kobuvirus, porcine teschovirus and Lawsonia intracellularis, were identified.

Epidemiological survey and genetic evolution of H9 subtype influenza viruses in Shanghai, China, from 2006 to 2010.

The H9N2 influenza virus is endemic in poultry. We report its occurrence in live-poultry markets, fair-trade markets and poultry farms in the Shanghai region between September 2006 and December 2010. An analysis of partial sequences of the HA, NA, PB1, PB2 and NP genes of eleven distinct H9N2 isolates revealed that all carried an RSSR motif at the cleavage site of HA, diagnostic of low pathogenicity in chickens. A phylogenetic analysis indicated that these isolates are derived from the lineage represented by Duck/HK/Y280/97, but they have evolved a range of reassortments. Their PB1 and NP sequences resembled those of H5N1 strains, indicating a hybrid origin involving both H9 and H5 strains. The HA and NA sequences present in all eleven isolates resembled those of the Duck/HK/Y280/97-like lineage. Infection by H9N2 is commonplace in Shanghai live-poultry markets, allowing the viruses to have evolved rapidly.

Genetic evolution of H9 subtype influenza viruses from live poultry markets in Shanghai, China.

H9N2 influenza viruses have become established and maintain long-term endemicity in poultry. The complete genomes of seven avian H9N2 influenza viruses were characterized. These seven influenza virus isolates were obtained from live poultry markets in Shanghai, China, in 2002 and from 2006 to 2008. Genetic analysis revealed that all seven isolates had an RSSR motif at the cleavage site of hemagglutinin (HA), indicating low pathogenicity in chickens. Phylogenetic analyses indicated that the seven avian H9N2 viruses belonged to the lineage represented by Duck/Hong Kong/Y280/97 (H9N2), a virus belonging to the Chicken/Beijing/1/94-like (H9N2) lineage, and that they are all quadruple reassortants consisting of genes from different lineages. The six internal genes of the isolates possessed H5N1-like sequences, indicating that they were reassortants of H9 and H5 viruses. All of the viruses had nonstructural (as well as HA and neuraminidase) genes derived from the Duck/Hong Kong/Y280/97-like virus lineage but also had other genes of mixed avian virus origin, including genes similar to those of H5N1 viruses (Gs/GD-like). The infected chickens showed no signs of disease. These results show the genetic and biological diversity of H9N2 viruses in Shanghai and support their potential role as pandemic influenza agents.

Loss of epithelial FAM20A in mice causes amelogenesis imperfecta, tooth eruption delay and gingival overgrowth.

FAM20A has been studied to a very limited extent. Mutations in human FAM20A cause amelogenesis imperfecta, gingival fibromatosis and kidney problems. It would be desirable to systemically analyse the expression of FAM20A in dental tissues and to assess the pathological changes when this molecule is specifically nullified in individual tissues. Recently, we generated mice with a Fam20A-floxed allele containing the beta-galactosidase reporter gene. We analysed FAM20A expression in dental tissues using X-Gal staining, immunohistochemistry and in situ hybridization, which showed that the ameloblasts in the mouse mandibular first molar began to express FAM20A at 1 day after birth, and the reduced enamel epithelium in erupting molars expressed a significant level of FAM20A. By breeding K14-Cre mice with Fam20A(flox/flox) mice, we created K14-Cre;Fam20A(flox/flox) (conditional knock out, cKO) mice, in which Fam20A was inactivated in the epithelium. We analysed the dental tissues of cKO mice using X-ray radiography, histology and immunohistochemistry. The molar enamel matrix in cKO mice was much thinner than normal and was often separated from the dentinoenamel junction. The Fam20A-deficient ameloblasts were non-polarized and disorganized and were detached from the enamel matrix. The enamel abnormality in cKO mice was consistent with the diagnosis of amelogenesis imperfecta. The levels of enamelin and matrix metalloproteinase 20 were lower in the ameloblasts and enamel of cKO mice than the normal mice. The cKO mice had remarkable delays in the eruption of molars and hyperplasia of the gingival epithelium. The findings emphasize the essential roles of FAM20A in the development of dental and oral tissues.

[Apoptosis and expression of Bcl-2 and caspase-3 in ciclosporin-induced gingival overgrowth of rats].

OBJECTIVE: To observe the effect of Ciclosporin (CsP) on apoptosis and expression of the associated protein Bcl-2, Caspase-3 in gingival epithelium of rats in order to approach the mechanism of CsP-induced gingival epithelium overgrowth. METHODS: Eighty SPF grade male 7-week-old Wistar rats were randomly divided into experimental group and control group, and each group was divided into 4 subgroups according to the duration of treatment (10, 20, 30 and 40 days). The experimental objects were given fresh milk including CsP intragastrically and the control ones were given only fresh milk. After perfusion of 4% paraform for internal fixation, the specimens' bucco-lingual paraffin sections at lower first molar were made. Apoptosis was detected using TdT-mediated dUT nick end labeling (TUNEL) and the expression of Bcl-2 and Caspase-3 using immunohistochemisty of PV. The apoptotic index, positive cell rate of Caspase-3 and average gray scale of Bcl-2 was measured with an image analysis system. Data were analyzed by two-way analysis of variance of factorial design. RESULTS: The apoptosis index and positive cell rate of Caspase-3 were down-regulated in the experimental group, and were significant difference from the control group (P < 0.05). The average gray scale of Bcl-2 was up-regulated in the experimental group, and was significant difference from the control group (P < 0.05). CONCLUSION: CsP-induced gingival epithelial overgrowth is likely to associated with interference to the path of mitochondrial apoptosis and inhibition apoptosis.

[Quantitative analysis the effect of Ciclosporin on gingival tissue modality of rats].

OBJECTIVE: Quantitative analysis the regional specificity and time dependence of Ciclosporin (CsP) on gingival tissue modality of rats. METHODS: Eighty SPF grade male Wistar rats of 7 weeks old were randomly divided into experimental group and control group, and each group was divided into 4 subgroups according to the duration of treatment (10, 20, 30 and 40 days). The experimental objects were given intragastric administration of fresh milk including CsP, and the control ones were given intragastric administration of fresh milk the same as the experimental objects. After giving perfusion 4% paraform trans-heart to internal fixation, the specimens were get and made bucco-lingual paraffin sections at lower first molar and made HE staining. The area of buccal and lingual gingival epithelial and connective tissue, the length of the longest rete pegs were measured with the image analysis system. Data were analyzed by two-way analysis of variance of factorial design. RESULTS: The rats of the experimental group took place gingival overgrowth, and the rete pegs of gingival epithelium of attached gingiva approach muco-gingival junction were prolonged. The area of buccal and lingual gingival epithelium and connective tissue, the length of buccal longest rete pegs of the experimental group were significantly higher than that of the control group rats (bucca P < 0.01, lingua 0.01 < P < 0.05). The length of lingual longest rete pegs of the experimental group were no difference than that of the control group rats. The area of buccal and lingual gingival epithelial, connective tissue, the length of longest rete pegs among experimental groups were no difference. CONCLUSION: CsP may have the regional specificity on the rats' gingival epithelium of buccal attached gingiva approach muco-gingival junction, but the effect of CsP on the rats' gingiva was no time dependence.

[Human hnRNP I prokaryotic expression and preliminary application.].

AIM: To express recombinant protein hnRNP I with prokaryotic expression system and assess the presence of autoantibodies against hnRNP I in systemic sclerosis (SSc) as well as other CTD. METHODS: Human hnRNP I gene was amplified by RT-PCR from HeLa cells and cloned into pET-30a vector , then pET-30a-hnRNP I plasmid was transferred into E.coli BL21 (DE3) to express recombinant protein. The sera from patients including SSc, SLE, SS, MCTD, UCTD, RA and controls were detected by ELISA with the purified recombinant hnRNP I protein. RESULTS: The recombinant protein was highly expressed in E.coli BL21(DE3) and specially reacted with clinically diagnosed SSc patients's serum. The result suggested that the autoantibodies against hnRNP I had higher positive ratio(48.72%) in SSc than other serum of AID and control. CONCLUSION: Human hnRNP I protein was successfully expressed in prokaryotic expression system. The purified hnRNP I protein can be used to diagnosis of SSc.