Guihong Zhang

Signal Transduction in Electrically Stimulated Bone Cells

Background: Electrical stimulation is used to treat nonunions and to augment spinal fusions. We studied the biochemical pathways that are activated in signal transduction when various types of electrical stimulation are applied to bone cells. Methods: Cultured MC3T3-E1 bone cells were exposed to capacitive coupling, inductive coupling, or combined electromagnetic fields at appropriate field strengths for thirty minutes and for two, six, and twenty-four hours. The DNA content of each dish was determined. Other cultures of MC3T3-E1 bone cells were exposed to capacitive coupling, inductive coupling, or combined electromagnetic fields for two hours in the presence of various inhibitors of signal transduction, with or without electrical stimulation, and the DNA content of each dish was determined. Results: All three signals produced a significant increase in DNA content per dish compared with that in the controls at all time-points (p < 0.05), but only exposure to capacitive coupling resulted in a significant, ever-increasing DNA production at each time-period beyond thirty minutes. The use of specific metabolic inhibitors indicated that, with capacitive coupling, signal transduction was by means of influx of Ca2+ through voltage-gated calcium channels leading to an increase in cytosolic Ca2+ (blocked by verapamil), cytoskeletal calmodulin (blocked by W-7), and prostaglandin E2 (blocked by indomethacin). With inductive coupling and combined electromagnetic fields, signal transduction was by means of intracellular release of Ca2+ leading to an increase in cytosolic Ca2+ (blocked by TMB-8) and an increase in activated cytoskeletal calmodulin (blocked by W-7). Conclusions: The initial events in signal transduction were found to be different when capacitive coupling was compared with inductive coupling and with combined electromagnetic fields; the initial event with capacitive coupling is Ca2+ ion translocation through cell-membrane voltage-gated calcium channels, whereas the initial event with inductive coupling and with combined electromagnetic fields is the release of Ca2+ from intracellular stores. The final pathway, however, is the same for all three signals—that is, there is an increase in cytosolic Ca2+ and an increase in activated cytoskeletal calmodulin. Clinical Relevance: Electrical stimulation in various forms is currently being used to treat fracture nonunions and to augment spinal fusions. Understanding the mechanisms of how bone cells respond to electrical signals—that is, understanding signal transduction and the metabolic pathways utilized in electrically induced osteogenesis—will allow optimization of the effects of the various bone-growth-stimulation signals.

Avian-origin H3N2 canine influenza A viruses in Southern China

This study reports four sporadic cases of H3N2 canine influenza in Southern China, which were identified from sick dogs from May 2006 to October 2007. The evolutionary analysis showed that all eight segments of these four viruses are avian-origin and phylogenetically close to the H3N2 canine influenza viruses reported earlier in South Korea. Systematic surveillance is required to monitor the disease and evolutionary behavior of this virus in canine populations in China.

Up-regulation of Chondrocyte Matrix Genes and Products by Electric Fields

This study tested the hypothesis that selective and specific capacitively coupled electrical signals could stimulate gene expression and matrix production in bovine articular chondrocytes. Starting with a capacitively coupled electric signal that previously was shown to be effective in stimulating proliferation in bovine articular cartilage chondrocytes, dose responses were done sequentially for duration, response time, amplitude, duty cycle, and frequency. Results showed that a 0.5-hour, 20 mV/cm, signal at 60 kHz up-regulated aggrecan gene expression approximately eightfold (p < 0.0003) using a 50% duty cycle, whereas Type II collagen gene expression was up-regulated approximately fivefold (p < 0.02) using an 8.3% duty cycle. Using a compound signal (a 0.5-hour continuous period plus multiple 1-hour periods of 50% duty cycle for 7 days) both proteoglycan and collagen accumulation in vitro were increased approximately fivefold (p < 0.0003) and twofold (p < 0.0008), respectively. Also, the most effective capacitively coupled electric signal was different for each of the two molecules studied (aggrecan, 50% duty cycle and 4-hour response time; Type II collagen, 8.3% duty cycle and 6-hour response time). We conclude that selective up-regulation of gene expression and matrix accumulation of cartilage structural macromolecules (such as aggrecan and Type II collagen) with specific capacitively coupled fields occurs in vitro. This may be useful in vivo as a noninvasive modality to promote cartilage healing or ameliorate the effects of osteoarthritis, or both.

Identification of an H6N6 swine influenza virus in southern China.

This is the first report of avian-like H6N6 swine influenza virus from swine in southern China. Phylogenetic analysis indicated that this virus might originate from domestic ducks. Serological surveillance suggested there had been sporadic H6 swine influenza infections in this area. Continuing study is required to determine if this virus could be established in the swine population and pose potential threats to public health.

Genetic diversity of the ORF5 gene of porcine reproductive and respiratory syndrome virus isolates in China from 2006 to 2008

Since April 2006, swine herds have experienced the outbreaks of a highly pathogenic porcine reproductive and respiratory syndrome (PRRS) in China. To explore the possible mechanism of the emergence of the highly pathogenic PRRS and more fully understand the extent of genetic diversity of PRRSV in China, we analyzed the ORF5 gene sequences of 159 representative PRRSV isolates in 16 provinces from 2006 to 2008. Sequence and phylogenetic analyses showed that all these 159 isolates belonged to the North American genotype and were further divided into six subgenotypes; 140 of 159 isolates were closely related to the highly pathogenic PRRSV with 98.5-100% nucleotide and 98.3-100% amino acid sequence identities and belonged to Subgenotype I; and 3, 8, 4, 3, 1 of 159 isolates were part of Subgenotypes II-VI, respectively. Amino acid analysis of the GP5 protein revealed that all the isolates in Subgenotypes I-III were found to be highly variable in the primary neutralizing epitope; most of the isolates in Subgenotypes I and IV had more glycosylation sites than those in Subgenotypes II, III, V and VI; and 1, 5, and 9 unique amino acid mutations were observed in Subgenotypes I, IV and VI, respectively. In conclusion, our study provides the evidence of coexistence of six different subgenotype isolates in pigs in China from 2006 to 2008, and emphasizes the importance of reinforcing PRRSV surveillance, especially after the emergence of highly pathogenic PRRS in China.

Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Subgroup J Avian Leukosis Virus

ABSTRACT Infection of breeder flocks in China with subgroup J avian leukosis virus (ALV-J) has increased recently. In this study, we have developed a loop-mediated isothermal amplification (LAMP) assay for rapid detection of ALV-J from culture isolates and clinical samples. The ALV-J-specific LAMP assay efficiently amplified the target gene within 45 min at 63°C using only a simple laboratory water bath. To determine the specificity of the LAMP assay, various subgroup ALVs and other related viruses were detected. A ladder pattern on gel electrophoresis was observed for ALV-J isolates but not for other viruses. To evaluate the sensitivities of the LAMP assay and conventional PCR, the NX0101 isolate plasmid DNA was amplified by them. The detection limit of the LAMP assay was 5 target gene copies/reaction, which was up to 20 times higher than that of conventional PCR. To evaluate the application of the LAMP assay for detection of ALV-J in clinical samples, 49 samples suspected of ALV infection from breeder flocks were tested by the LAMP assay and PCR. Moreover, virus isolation from these samples was also performed using cell culture. The positive-sample ratios were 21/49 (43%) by conventional PCR, 26/49 (53%) by the LAMP assay, and 19/46 (41%) by virus isolation. Additionally, a positive LAMP reaction can be visually ascertained by the observation of turbidity or a color change after addition of SYBR green I dye. Consequently, the LAMP assay is a simple, rapid, and sensitive diagnostic method and can potentially be developed for rapid detection of ALV-J infection in the field.

Virological and Epidemiological Evidence of Avian Influenza Virus Infections Among Feral Dogs in Live Poultry Markets, China: A Threat to Human Health?

TO THE EDITOR— Since its first detection in March 2013, the novel H7N9 avian influenza virus (AIV) has quickly spread among poultry and people in China. As of 16 February 2014, a total of 348 laboratory-confirmed human H7N9 infections in China have been confirmed by the World Health Organization [1–3]. The H7N9 virus has spread widely with little sign of infection among poultry [4]. Epidemiologic studies have identified poultry exposure as an important risk factor for human infections with H5N1 and H7N9, especially for those individuals associated with live poultry markets (LPMs) [5–8]. As dogs in China have been shown to be infected with AIVs, we sought to investigate whether dogs living in close proximity to LPMs and H7N9-affected farms might have been infected with the novel H7N9 virus or other influenza viruses. From August 2011 to August 2013, we studied a total of 2357 dogs that lived in close proximity to LPMs and poultry farms in the rural areas of Shanghai, Guangdong, Zhejiang, and Jiangsu provinces in China where novel H7N9 AIV had been previously detected (forMaterials and Methods, see Supplementary Data). Overall, 68.18% (n = 1607) of the 2357 stray dog samples were collected in rural areas, with the remaining samples collected in LPMs (Table 1). Of the 2357 nasal swab samples collected, 93 (3.9%) were positive for influenza A virus by realtime reverse transcription polymerase chain reaction (PCR), and 11 viruses were isolated from these samples (see Supplementary Data). Hemagglutination inhibition (HI) assays and hemagglutinin antigen–specific enzyme-linked immunosorbent assays against H7N9 viral antigens revealed no evidence of H7N9 infection. Results of the HI and microneutralization (MN) assays are reported in Table 1 and in the Supplementary Data. A total of 19 serum samples had HI antibody titers of ≥1:20 against H5 antigen (Table 1), and 3 of these 19 samples were also positive by MN assay. Dogs that were sampled in LPMs had a greater probability of having elevated HI antibodies against avian H9N2, avian H5N1, and canine H3N2 viruses (Table 2), compared with the dogs that were raised in poultry farms. Our study supports this premise in that, although we failed to find evidence of previous H7N9 infections among the dogs, we found the world’s first evidence of previous H5N1 and H9N2 infection among dogs by real-time PCR, HI, and MN assay. These findings were unexpected but biologically plausible. In LPMs and farms in rural China, stray dogs and cats have considerable contact with poultry or poultry products. This can occur indirectly through aerosol and fecal transmission or directly through the consumption of dead bird carcasses or entrails. LPMs are particularly problematic as they offer a mixing of animal species from often diverse geographical areas, frequent venues for contact with the public, and often nonhygienic behavior of workers who handle and process the birds for sale. Both rural farms and LPMs provide opportunities for wild aquatic birds, domestic poultry, stray dogs, and humans to closely interact and potentially share pathogens (Supplementary Figure 1). Additionally, compared

Genetic characterization of H5N1 avian influenza viruses isolated in southern China during the 2003–04 avian influenza outbreaks

Summary.The recent H5N1 avian influenza outbreaks in Asia spread over more than 8 countries. It has caused enormous economic loss and grand challenges for the public health. During these breakouts we isolated three strains of H5N1 Avian Influenza Virus (AIV) from chickens and one from duck in different farms of Southern China. We completely sequenced these four AIVs. Molecular characterization demonstrated that these strains retain the reported H5N1 AIV sequence properties relevant to virus virulence and host adaptation. Phylogeny results demonstrated that three of these isolates (except A/Chicken/Guangdong/174/04) were closely linked to other H5N1 AIVs isolated from the recent H5N1 outbreaks in Asia. Six of 8 segments (except PA and M) of A/Chicken/Guangdong/174/04 also shares a close linkage to other H5N1 AIVs isolated from the recent H5N1 outbreaks. However, the PA gene of A/Chicken/Guangdong/174/04 and another H5N1 strain forms a distinct subgroup along with an H6N1 AIV, and the M gene of A/Chicken/Guangdong/174/04 shows a close linkage to some H5N1 AIVs from aquatic species in China. Our findings suggest that a new genotype of AIV (in addition to previous reported ones) was present during the 2003–04 Asian bird flu outbreaks and that continuing virus surveillance of AIVs be conducted to monitor the evolutionary paths of the A/Chicken/Guangdong/174/04-like AIVs.

Further evidence for infection of pigs with human-like H1N1 influenza viruses in China.

Classical swine and avian-like H1N1 influenza viruses were reported widely in swine population worldwide, but human-like H1N1 swine viruses were reported occasionally. In 2006, a human-like H1N1 swine virus (A/swine/Guangdong/96/06) was isolated from pigs in Guangdong province, which was reported in China for the first time. To get further evidence for infection of pigs with human-like H1N1 influenza viruses, we analyzed eight gene segments of three human-like swine H1N1 viruses (A/swine/Guangdong/96/06, A/swine/Tianjin/01/04 and A/swine/Henan/01/06) isolated in China. All the eight genes of the three viruses are highly homologous to recent (about 2000) and early (1980s) human H1N1 influenza viruses, respectively. Phylogenetic analyses revealed that A/Swine/Guangdong/96/06 was directly derived from about 2000 human H1N1 influenza viruses, while A/swine/Tianjin/01/04 and A/swine/Henan/01/06 seemed to be descendants of human H1N1 viruses circulating in 1980s. Seroprevalence of our isolate (A/swine/Guangdong/96/06) confirmed the presence of human-like H1N1 virus in pigs in China. Existence of these influenza viruses, especially older viruses (A/swine/Tianjin/01/04 and A/swine/Henan/01/06), indicates that human-like H1N1 influenza viruses may remain invariant for long periods in pigs and provides the evidence that pigs serve as reservoirs of older influenza viruses for human pandemics.

Avian-origin H3N2 canine influenza virus circulating in farmed dogs in Guangdong, China.

Since 2006, more and more cases of the infectious H3N2 canine influenza virus (CIV) in pet dogs have been reported in southern China. However, little is known about the prevalence situation of H3N2 CIV infections in farmed dogs in China. This is the first systematic epidemiological surveillance of CIV in different dog populations in southern China. Two virus strains A/Canine/Guangdong/1/2011(H3N2) and A/canine/Guangdong/5/2011(H3N2) were isolated from canine nasal swabs collected at one dog farm in Guangzhou and the other farm in Shenzhen. Sequence and phylogenetic analysis of eight gene segments of these viruses revealed that they were most similar to the newly isolated canine H3N2 viruses in dogs and cats from Korea and China, which originated from avian strain. This indicates that H3N2 CIV may be a common pathogen for pet and farmed dog populations in southern China at present. Serological surveillance has shown that the infection rate of this avian-origin canine influenza in farmed dogs and in pet dogs were 12.22% and 5.3%, respectively; as determined by the ELISA. The data also suggested that transmission occurred, most probably by close contact, between H3N2 CIV infected dogs in different dog populations in recently years. As H3N2 outbreaks among dogs continue in the Guangdong province (located very close to Hong Kong), the areas where is densely populated and with frequent animal trade, there is a continued risk for pets H3N2 CIV infections and for mutations or genetic reassortment leading to new virus strains with increased transmissibility among dogs. Further in-depth study is required as the H3N2 CIV has been established in different dog populations and posed potential threat to public health.