First Report of Chilli veinal mottle virus Infecting Tomato (Solanum lycopersicum) in China.

Chilli veinal mottle virus (ChiVMV), a potyvirus, is widespread over the world. In China, it was first reported in chili pepper (Capsicum annuum) in Hainan Province (south China) in 2006 (2). Subsequently, it was reported in tobacco (Nicotiana tabacum) in Yunnan Province (southwest China) in 2011 (1). Sichuan Province is one of the largest vegetable producing areas of China. In May 2012, tomatoes with leaves displaying virus-infected symptoms like mottling, mosaic, narrowing, or curling were observed in several fields of Chengdu, eastern Sichuan Province, southwest China. Of the 20 fields we investigated, four fields with 90% tomato plants were infected. During 2012 and 2013, six samples were collected from symptomatic tomato leaves based on different symptoms and locations. All six samples were assayed by western blotting using polyclonal antisera (Cucumber mosaic virus [CMV], Tobacco mosaic virus [TMV]) obtained from Agdia (Elkhart) and one antiserum to ChiVMV obtained from Yunnan Academy of Agricultural Science (China). Two samples from Pengzhou and one sample from Shuangliu exhibiting mosaic leaves were positive for TMV, one sample from Pixian exhibiting narrowing leaves was positive for CMV, and the other two samples from Shuangliu exhibiting mottle and leaf distortion were positive for ChiVMV. Total RNAs was extracted from all six samples and healthy tomato leaves using Trizol reagent (Invitrogen), First-strand cDNA synthesis primed with oligo(dT) by SuperScript III Reverse Transcriptase (Invitrogen). RT-PCR was performed using primer pairs ChiVMV-CP F (5'-GCAGGAGAGAGTGTTGATGCTG-3') and ChiVMV-CP R (5'-(T)16AACGCCAACTATTG-3'), which were designed to direct the amplification of the entire capsid protein (CP) gene and 3' untranslated region (3'-UTR) of ChiVMV (GenBank Accession No. KC711055). The expected 1,166-bp DNA fragment was amplified from the two tomato samples from Shuangliu that were positive for ChiVMV in the western blot tests, but not from the others. The obtained fragments were purified and cloned into the PMD18-T vector (TaKaRa) and sequenced. The sequencing results showed that the two ChiVMV isolates from tomato in Shuangliu were identical (KF738253). Nucleotide BLAST analysis revealed that this ChiVMV isolate shared ~84 to 99% nucleotide identities with other ChiVMV isolates available in GenBank (KC711055 to KF220408). To fulfill Koch's postulates, we isolated this virus by three cycle single lesion isolation in N. tabacum, and mechanically inoculated it onto tomato leaves. The same mottle and leaf distortion symptoms in systemic leaves were observed. Subsequent RT-PCR, fragment clone, and sequence determination tests were repeated and the results were the same. All the evidence from these tests revealed that the two tomato plants were infected by ChiVMV. To our knowledge, this is the first report of ChiVMV naturally infecting tomato in China. It shows that ChiVMV is spreading in China and is naturally infecting a new solanaceous crop in the southwest area, and the spread of the virus may affect tomato crop yields in China. Thus, it is very important to seek an effective way to control this virus. References: (1) M. Ding et al. Plant Dis. 95:357, 2011. (2) J. Wang et al. Plant Dis. 90:377, 2006.

First Report of Sweet potato chlorotic fleck virus Infecting Sweet Potato in Sichuan Province, China.

Sweet potato chlorotic fleck virus (SPCFV) is one of several viruses naturally infecting sweet potato (Ipomoea batatas L.), and it has recently been classified as a new member of the genus Carlavirus (family Flexiviridae) (1). However, SPCFV is distantly related to typical carlaviruses, as most of its putative gene products share amino acid sequence identities of <40% with those of typical carlaviruses (1). China is the largest sweet potato producing country in the world. So far, SPCFV has been reported in eastern China, such as Jiangsu, Anhui, Henan (3), and Guangdong (2) provinces, but no reports exist in western China. Sichuan Province, located in southwestern China, is the largest sweet potato producing area in the country. There are big differences between the environment and climate conditions between Sichuan and eastern China. During 2012, a survey was constructed to determine the genetic diversity and distribution of sweet potato viruses in Sichuan. Forty-seven sweet potato samples exhibiting virus-like symptoms were collected from four different geographic areas of the province. Western blotting using the antisera obtained from the International Potato Center showed that two samples were positive for SPCFV, whereas with reverse transcription (RT)-PCR, only one isolate of SPCFV was obtained from a sample exhibiting symptoms of chlorosis, leaf distortion, and vein clearing. Serological detection indicated that the plant was co-infected with SPCFV, Sweet potato feathery mottle virus (SPFMV), and Sweet potato virus G (SPVG). Total RNA was extracted from symptomatic leaves using Trizol reagent (Invitrogen) according to the manufacturer's protocol, and RT-PCR was performed by using primer pairs SPCFV-CP F (5'-ATGGCGGCGAAGGAGGCTGATA-3') and SPCFV-CP R (5'-TCACTTGCACTTCCCATTAC-3') corresponding to the entire coat protein (CP) gene of SPCFV. Expected DNA fragments of 900 bp were obtained from the symptomatic plant but not from control plants. The obtained fragments were purified and cloned into the PMD19-T vector (TaKaRa). Recombinant plasmids were then transformed into competent cells of Escherichia coli strain DH5α. Nucleotide BLAST analysis revealed that the 900-bp fragment (GenBank Accession No. KC414676) shared 87 to 91% nucleotide identities with other SPCFV isolates available in the GenBank database. To our knowledge, this is the first report of the co-infection between SPCFV and other sweet potato viruses including SPFMV and SPVG in China, and this is the first molecular report of SPCFV in Sichuan, western China. It shows that SPCFV is spreading to a new ecological area of China, and the spread of the virus may affect sweet potato crop yields in western China. Some measures must be carried out quickly to control the virus. References: (1) V. Aritua et al. Arch. Virol. 152:813, 2007. (2) V. Aritua et al. Plant Dis. 93:87, 2009. (3) Q. Wang et al. Crop. Prot. 29:110, 2010.

[Laboratory diagnosis of Acanthamoeba keratitis].

OBJECTIVE: To find a rapid method for diagnosing Acanthamoeba keratitis and identifying Acanthamoeba. METHODS: 10% potassium hydroxide (KOH) wet-mount preparations, Acanthamoeba culture, inverted phase contrast microscopy, and pathological examination using H. E. staining and PAS staining. RESULTS: Using corneal scrapings and corneal materials obtained from surgery, 7 cases and 5 cases of Acanthamoeba keratitis were diagnosed by 10% KOH wet-mount preparations. 6 strains of Acanthamoeba were isolated in corneal materials of 6 cases by protozoa culture method. The cysts, trophozoites and pseudopods on the trophozoites of Acanthamoeba were directly observed under the inverted phase contrast microscope. The cysts and trophozoites of Acanthamoeba were seen by H. E. staining and PAS staining with 20 h. CONCLUSION: Acanthamoeba keratitis could be rapidly diagnosed by 10% KOH wet-mount preparations and inverted phase contrast microscopy. Acanthamoeba organisms could be directly observed and identified under inverted phase contrast microscope.

[Approach to application of laser scanning confocal microscope in bone morphometry].

The laser scanning confocal microscope(LSCM) is the new high distinguishing microscope, which can be used in observation on fluorescence, since 1990. The bone morphometry is an important way to study metabolic bone diseases. Our study on research combination of LSCM with bone morphometry in observing microarchitecture of bone found that LSCM could make the photo of bone trabecula distinctly and its location accurately. Because of its technical advantages, LSCM could link histochemical or immunohistochemical method for the bone morphometry and bone cells at the same time and obserrate the thick-slide. It is a new method and way to study and diagnose metabolic bone diseases.

[Bone mineral density characteristics at the femoral neck and Ward's triangle for Chinese women].

The incidence of hip fracture is not as high in Chinese women compared to women from Western countries, though they usually have low bone mineral density(BMD) and get osteopenia easily. In this study, reference data(white women) supplied by the manufacturer of Hologic was compared with data obtained from healthy women in Changsha, Hunan, P.R.C. One thousand four hundred and eighty-eight Chinese women aged 15 to 95 years were randomly recruited. Measurements of BMD were taken at the hip by the dual energy x-ray absorptiometry(QDR 4,500A, Hologic Inc., USA). The BMD was somewhat lower than reference curves at all ages and all sites. But at the femoral neck and Ward's triangle, Chinese women reached their peak BMD 5 to 10 years later than the reference group, and had a lower BMD rate of decrease for about 35 years after peak BMD was reached. Whether the differences(i.e., longer time to peak BMD and a lower BMD decrease rate at the neck and Ward's triangle after the peak BMD reached) will result in a protection from hip fractures for Chinese women needs to be studied in the future.

Effects of 17beta-estradiol on the expression of membrane type 1 matrix metalloproteinase (MT1-MMP) and MMP-2 in human osteoblastic MG-63 cell cultures.

Estrogens are important regulators of bone cell function. Osteoblast-derived membrane type 1 matrix metalloproteinses (MT1-MMP) have recently been implied to play an important role in the process of bone resorption by proteolytically activating latent matrix metalloproteinase-2 (proMMP-2) at the cell surface and degrading tumor necrosis factor-alpha (TNF-alpha). In the present study, we observed the effects of 17beta-estradiol (E2) on MT1-MMP production and subsequent activation of latent matrix proMMP-2, and also proMMP-2 secretion in cultures of human osteoblastic MG-63 cells. Western immunoblot analysis showed that treatment with increasing doses of E2 in MG-63 cells caused a dose-dependent increase in expression of MT1-MMP protein. Confocal immunohistochemistry analysis also confirmed that E2 induced MT1-MMP synthesis in MG-63 cells. We found unexpectedly that although MT1-MMP synthesis was up-regulated by E2 in cultures of MG-63 cells, activation of proMMP-2 was unchanged, which can be attributed partly to the undetectable tissue inhibitor of metalloproteinase-2 (TIMP-2) protein in MG-63 cells by Western immunoblotting. ProMMP-2 production was also not influenced by E2. In conclusion, E2 induces MT1-MMP protein expression in MG-63 cells while it is not followed by proMMP-2 activation, E2 may suppress bone resorption by accentuated degradation of TNF-alpha which mediated through increasingly MT1 -MMP production in osteoblastic cells.

[Comprehensive assessment of the ovariectomized rat model of postmenopausal osteoporosis].

Dual-energy X-ray absorptiometry(DEXA), scanning electron microscopy, and bone biomechanical test were used to assess comprehensively bone quantity and quality of ovariectomized rats. In OVX rats, not only bone mineral density (BMD) of lumbar vertebrae in vivo and vitro, but also BMD of femora (except for R3 region) and proximal metaphysis(R1 region) in vitro decreased obviously (P < 0.01), whose bone loss rates of L5 and L6 were the highest and achieved 13%; the trabeculae of OVX rats were few, fine, and discontinued and there were lacunae on the surface; in OVX rats, both compressive strength of vertebral bodies and the mechanical properties of femora decreased; the falling degree of the former was greater; the maximal compressive power of lumbar vertebrae decreased with 33.32%. We conclude that after 4-month post-ovariectomy, the bone quantity and quality of six-month-old rats decreased, especially in the area of abundant cancellous bone such as vertebral bodies, distal femora; the assessment of the efficiency of new drugs to prevent and treat postmenopausal osteoporosis using rat models should include these drugs' effects to the bone mass, the bone structure, and the bone strength.

Effects of hepatocyte growth-promoting factor and panax notoginseng saponins on intrasplenic hepatocellular autotransplantation.

AIM: To increase the weight of liver tissue mass present in spleen and to shorten the regeneration period of transplanted hepatocytes by stimulating DNA synthesis and protection against ischemic-reperfusion injury. METHODS: Hepatocyte growth-promoting factor (PHGF) and panax notoginseng saponins (PNGS) were used after intrasplenic hepatocellular autologous transplantation (IHAT) with 70 % partial hepatectomy. Histological examinations were carried out under both light and electron microscopy and content of ALT in hepatized spleen homogenate was investigated 2 weeks after transplantation. Furthermore, 99mTc diethyl-iminodiacetic acid (99mTc-HIDA) splenic scintiphotography was carried out and proliferation index of transplanted hepatocytes was detected by flow cytometry at the 12th week after operation. RESULTS: (1) Hepatocellular degeneration was slightly less in group B [intrasplenic hepatocyte autologous transplantation (IHAT) + PNGS 25 mg/kg, im, qd] vs the control group (group C, IHAT without drugs) at the 2nd week after transplantation, and the ALT content of group B (928 U/g +/- 268 U/g) was higher than that of group C (639 U/g +/- 138 U/g, P < 0.01). (2) At the 12th week, hepatocellular regeneration in group A (IHAT + PHGF 5 mg/kg, im, qd) was obviously better than that in group C, and the ALT content (2325 U/g +/- 401 U/g ), the radioactivity accumulation of 99mTc-HIDA (58 Bq +/- 18 Bq), and proliferation index (3.8 % +/- 0.4 %) of group A were all higher than those of control (P < 0.05). CONCLUSION: PHGF has effects in increasing the weight of liver tissue grown in spleen and shortening the regeneration period of the transplanted hepatocytes, while PNGS has certain effects on protecting the hepatocytes against ischemic reperfusion injury in the early stage of transplantation.