Linfang Cheng

Novel Reassortant Influenza A(H5N8) Viruses in Domestic Ducks, Eastern China

Domestic ducks are natural reservoirs of avian influenza viruses and serve as reassortant hosts for new virus subtypes. We isolated 2 novel influenza A(H5N8) viruses from domestic ducks in eastern China, sequenced their genomes, and tested their pathogenicity in chickens and mice. Circulation of these viruses may pose health risks for humans.

Novel reassortant highly pathogenic H5N6 avian influenza viruses in poultry in China.

We characterized two novel highly pathogenic H5N6 influenza viruses isolated from Chinese poultry in 2013. Genomic analysis showed that both isolates were reassortants, and derived their genes from H5 and H6 subtype viruses found in poultry in China. The virulence of the two isolates was examined in chickens and mice, and both isolates were found to be highly pathogenic in chickens and only moderately virulent for mice. Our results show that continued circulation of these viruses could endanger both avian species and humans.

Agonistic Monoclonal Antibodies Potentiate Tumorigenic and Invasive Activities of Splicing Variant of the RON Receptor Tyrosine Kinase

Ligand-dependent or independent activation of the RON receptor tyrosine kinase is essential in transducing invasive signals leading to increased tumorigenic activities. In this study, we characterized two monoclonal antibodies (mAbs) specific to the extracellular domains of human RON and studied their agonistic effect on tumorigenic activities mediated by oncogenic variant RON?160. The mAb Zt/g4 and Zt/c1 are specific to human RON. They bind to RON with high affinities and recognized different epitopes on the RON extracellular domain. Because of their reactivity with native RON, Zt/g4 and Zt/c1 are useful in various applications such as immunoprecipitation, immunofluorescent analysis, and immunohistochemical staining. Functional studies revealed that Zt/g4 and Zt/c1 are capable of inducing RON phosphorylation which activates signaling proteins such as Erk1/2 and Akt. In NIH3T3 cells expressing RON?160, both mAbs significantly enhanced RON?160-mediated tumorigenic activities including cell proliferation, focus formation, and anchorage-independent growth. Cell shape changes with increased motile and invasive activities were also observed. Studies in vivo further demonstrated that Zt/g4 and Zt/c1 increase RON?160-mediated tumor growth in nude mice with a shortened time of onset and enlarged tumor volume. Thus, by recognizing specific epitopes on the RON extracellular domains, Zt/g4 and Zt/c1 have abilities to elicit a full array of RONmediated responses. These mAbs will be useful in studying mechanisms underlying RON activation which lead to increased tumorigenic activities.

Genetic and molecular characterization of H9N2 and H5 avian influenza viruses from live poultry markets in Zhejiang Province, eastern China

Live poultry markets (LPMs) are a key source of reassorted avian influenza viruses (AIVs) because of the density of terrestrial and aquatic poultry and the frequency of AIV infection. H9N2 viruses are prevalent in terrestrial poultry throughout Asia and have been isolated from poultry outbreaks worldwide. They infect both avian and mammalian species and may be significant donors of genetic material to emerging human pathogens. LPMs in Zhejiang Province were surveyed from 2013–2014 for AIVs. Three hundred seventy-four (374) AIV strains were isolated from 3,328 samples. Whole–genome sequencing and phylogenetic analyses were performed. We identified a novel H9N2 virus genotype that had undergone reassortment with gene segments from Qa/HK/G1/97–like, Ck/BJ/1/94–like, and Dk/HK/Y439/97–like viruses. Phylogenetic analyses suggested the H9N2 viruses had undergone reassortments with other AIV subtypes. The results also suggested that two different clades (2.3.2 and 2.3.4.6) of H5 viruses were co–circulating in Zhejiang Province. Given that reassorted H5 AIVs were detected in geese and ducks, it is possible that apparently healthy birds contribute to emerging H5 AIVs. Continued surveillance is required in poultry in eastern China.

Dihydrotestosterone protects human vascular endothelial cells from H2O2-induced apoptosis through inhibition of caspase-3, caspase-9 and p38 MAPK

Oxidative stress is proved to be harmful to the vascular endothelial cells which are important in preventing the formation and progression of atheromatous plaque. This study was designed to investigate the protective effect and potential mechanisms of dihydrotestosterone (DHT) against H(2)O(2)-induced apoptosis of human umbilical vein endothelial cells (ECV-304). ECV-304 cells were pretreated with different concentrations of DHT (0.01, 0.1 and 1 microM) for 2h, followed by exposure to 100 microM H(2)O(2) for 18h. 3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate cell viability. To detect apoptosis, the cells were assessed by morphologic examination and Annexin V-propidium iodide double staining with flow cytometry. Finally, the expression of caspase-3, caspase-9 and phospho p38 MAPK was assayed by Western blot to investigate the possible molecular mechanisms. We found that H(2)O(2) treatment for 18h significantly decrease the viability of ECV-304 cells characterized by a high percentage of apoptotic cells. DHT could antagonize the apoptosis inducing effect of H(2)O(2) in a dose-dependent manner. Consistently, DHT also significantly inhibit the expression of caspase-3, caspase-9 and phospho p38 MAPK induced by H(2)O(2). In summary, pretreatment with DHT can inhibit apoptosis of ECV-304 cells induced by H(2)O(2). The protective effect of DHT was associated with the inhibition of caspase-3, caspase-9 and phospho p38 MAPK expression.

B7-H4 expression in various tumors determined using a novel developed monoclonal antibody

B7-H4, a new member of the B7 family, may participate in the negative regulation of cell-mediated immunity, while aberrant B7-H4 expression is detected in some tumors and it participates in the occurrence and development of the tumors. In this study, we developed one monoclonal antibody (mAb) whose clone No. was 4H8 against the extracellular domains of B7-H4 through immunization of Balb/c mice with the 3T3-mB7-H4 cells that expressed extrinsic B7-H4 stably. And we detected the expression characteristics of B7-H4 in various tumors using 4H8 mAb by enzyme-linked immunosorbent assay (ELISA), immunoprecipitation and immunohistochemistry (IHC) analysis. B7-H4 expression was significantly higher in the tumor tissues derived from uterus, breast, and colon than in their corresponding normal tissues. Further, the B7-H4 expression was related to the stage of the tumors. In contrast, B7-H4 expression did not differ significantly between the tumor tissues derived from the stomach and liver and the normal tissues. Different expression levels of B7-H4 in the tumors indicated that B7-H4 may be involved in tumor formation and development. Specific mAbs against B7-H4 will be useful in studying the role of B7-H4 in tumor pathogenesis and pathological process.

Parthenolide inhibits LPS-induced inflammatory cytokines through the toll-like receptor 4 signal pathway in THP-1 cells.

Parthenolide (PTL) shows potent anti-inflammatory and anti-cancer activities. In the present study, the molecular mechanisms of PTLs activities were explored in lipopolysaccharide (LPS)-induced human leukemia monocytic THP-1 cells and human primary monocytes. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay was used to analyze the effect of PTL on THP-1 cell viability. Enzyme-linked immunosorbent assay was used to determine the effect of PTL on LPS-induced inflammatory cytokine secretion. Flow cytometry and quantitative real-time polymerase chain reaction were used to assess the effect of PTL on LPS-induced toll-like receptor 4 (TLR4) expression. Phosphorylation levels of signaling molecules were determined by western blot analysis. Results showed that PTL <12.5 μM did not significantly affect THP-1 cells viability. LPS treatment led to a marked up-regulation of interleukin (IL)-6, IL-1β, IL-8, IL-12p40, tumor necrosis factor-α, IL-18, and NO in THP-1 cells. However, PTL inhibited the expression of these cytokines in a dose-dependent manner, with IC50 values of 1.091-2.620 μM. PTL blocked TLR4 expression with an IC50 value of 1.373 μM as determined by the flow cytometry analysis, and this blocking effect was verified at both protein and mRNA levels. Up-regulation of phosphorylation levels of extracellular signal-regulated kinase 1/2, Jun N-terminal kinase, p38, nuclear factor κB (NF-κB) p65, and IκBα and up-regulation of expressions of other molecules (inducible nitric oxide synthase, TLR4, and TNF receptor-associated factor 6) induced by LPS were abolished by PTL in a dose-dependent manner. The anti-inflammatory mechanisms of PTL operate partly through the TLR4-mediated mitogen-activated protein kinase and NF-κB signaling pathways. Therefore, TLR4 may be a new target for anti-inflammation therapies.

Characterization of a novel highly pathogenic H5N2 avian influenza virus isolated from a duck in eastern China

During surveillance for avian influenza viruses (AIVs) in live-poultry markets (LPMs) in eastern China in 2013, one H5N2 AIV was isolated from a duck. Phylogenetic analysis showed that the hemagglutinin of this strain belongs to clade 2.3.4 and received its genes from H5, H3 and H6 AIVs of poultry in China. The virulence of this strain was examined in chickens and mice, and it was found to be highly pathogenic in chickens but demonstrated moderate pathogenicity in mice. These results suggest that active surveillance of AIVs in LPMs should be used in an early warning system for avian influenza outbreaks.

Multiple amino acid substitutions involved in the adaptation of avian-origin influenza A (H10N7) virus in mice.

AbstractTo identify substitutions that are possibly associated with the adaptation of avian-origin H10N7 virus to mammals, adaptation of the H10N7 virus in mouse lung was carried out by serial lung-to-lung passage. Genomic analysis of the mouse-adapted virus revealed amino acid changes in the PB2 (E627K), PA (T97I), and HA (G409E) proteins, and this virus was more virulent in mice than the wild-type virus. Our results suggest that these substitutions are involved in the enhancement of the replication efficiency of avian-origin H10N7 virus, resulting in severe disease in mice. Continued poultry surveillance of these substitutions in H10N7 viruses is required.

Amino acid substitutions occurring during adaptation of an emergent H5N6 avian influenza virus to mammals.

Avian influenza viruses (AIVs) are known to cross species barriers, and emergent highly pathogenic H5N6 AIVs pose a serious threat to human health and the poultry industry. Here, we serially passaged an H5N6 virus 10 times in BALB/c mice. The pathogenicity of the wild-type 6D2 (WT-6D2) and mammal-adapted 6D2 strain (MA-6D2) were compared. The viral titer in multiple organs and the death rate for MA-6D2 were significantly higher than for WT-6D2. We provide evidence that the mutations HA A150V, NA R143K and G147E, PB2 E627K, and PA A343T may be important for adaptation of H5N6 AIVs to mammals.