The gene encoding the VP28 envelope protein of White spot syndrome virus (WSSV)was cloned into expression vector pET-30a and transformed into the Escherichia coli strain BL21.After induction,the recombinant VP28 (rVP28) protein was purified and then used to immunize Balb/c mice for monoclonal antibody (MAb) production.It was observed by immuno-electron microscopy the MAbs specific to rVP28 could recognize native VP28 target epitopes of WSSV and dot-blot analysis was used to detect natural WSSV infection.Competitive PCR showed that the viral level was approximately 104 copies/mg tissue in the dilution of gill homogenate of WSSV-infected crayfish at the detection limit of dot-blot assay.Our results suggest that dot-blot analysis with anti-rVP28 MAb could rapidly and sensitively detect WSSV at the early stages of WSSV infection.

One strain of Hantavirus(HV) was isolated from patients with hemorrhagic fever with renal syndrome (HFRS) in Zhejiang province and its S-segment was cloned and submitted to nucleotide sequence analysis in order to determine the type of strain and extent of genetic variation for further study on its evolution and mutation .The HV antigen was detected from mouse lungs in endemic area by direct immunofluorescene test and the HV-positive sample of mouse lung was inoculated to Vero-E6 cells to isolate virus. The total cellular RNA was extracted from infected cell culture and the S segment gene was amplicated by RT-PCR. Then, the purified PCR product was cloned into T vector for sequencing. The result showed that the full-length sequence of the S segment was 1 700 bp with one open reading frame (ORF) encoding 429 amino acids. Comparison with Hantavirus HTN type showed 85.7%-91.9% homology at the nucleotide level. In comparison with the SEO type of viruses, the homology at nucleotide level was shown to be 71.2%-75%. This new HV strain may be typed as to HTN virus and may be a new subtype of this virus.

AIM: To observe the change of insulin receptor in rabbit kidney with acute ischemic-reperfusion injury. METHODS: 15 Japanese white rabbits were allocated randomly into control group, ischemic-reperfusion group(IR group). IR group received clamping for 1 h followed by 2 h or 48 h of reperfusion. At 2 h or 48 h after reperfusion, glucose and insulin in serum were determined. Insulin receptor in renal tissue was analyzed by radioligand binging assay(BAD). RESULTS: The level of serum glucose increased after 2 h reperfusion in 2 groups, but in IR group the value increased much more higher than those in control groups(P

Objective To study the complete genome sequence of Hantavirus ZT71 strain gene isolated in Zhejiang province and explore its evolution. nethods The total RNA was prepared from ZT71 virus infected cells and the RT-PCR products were cloned into T vector, sequenced and analyzed. Results The L, M and S segments of the strain ZT71 genome were 6530,3651 and 1753 nucleotides in length with a single open reading frame individually encoding 2151,1133 and 429 amino acids. The sequence analysis of nucleotides showed that the homology of L, M and S segments of strain ZT71 between those of other strains of Seoul virus could reach 95.5%-99.7%, 84.1%-99.6% and 88.7%-99.5%, respectively. The analysis of the deduced amino acids showed the similar result. The source of strain ZT71 could be traced from the analysis of the phylogentic trees of nucleotides and amino acids, and it should belong to Seoul type of Hantavirus which was also verified serologically. Conclusion The nucleotide sequences and deduced amino acid sequences of L, M and S segments of strain ZT71 are similar to that of those of Seoul type of Hantavirus. And Hantaan type virus used to be prevalent primarily in Zhejiang province,and it would be an endemic area of mixed type of Hantavirus since the discoveries of the viruses of Soeul type in recent years.

Objective To explore the efficacy and safety of a raltegravir (RAL)-containing regimen among patients on methadone maintenance therapy.Methods From January 2010 to November 2010, 30 virus (HIV) treatment naive patients who were on methadone maintenance therapy were enrolled from a HIV clinic in Kunming, Yunnan Province and a HIV clinic in Hengyang, Hunan Province.All patients were given RAL, tenofovir (TDF) and lamivudine (3TC) as highly active antiretroviral therapy (HARRT).Patients were followed up for 48 weeks to evaluate the adjustment of methadone dose, opiate withdrawal reaction, antiretroviral efficacy and safety.Results From January 2010 to November 2010, 30 HIV patients were enrolled from the two appointed HIV clinics.The mean age was 39±6 years, with 73.3% male patients and 97% Han population.Before the treatment, their mean CD4+T lymphocyte counts was 210 /μL.Ninety percent of patients were co-infected with hepatitis C.Twenty-nine patients who completed study follow-up were included in final analysis.Five (17.8%) patients reported opiate withdrawal symptoms and increased methadone dose 4 weeks after HARRT.At 24 weeks and 48 weeks of HARRT, the average increase of CD4+T lymphocyte counts were (136±71) /μL and (185±88)/μL, respectively.Among patients who provided valid HIV-1 RNA testing results, 82.6% (19/23) and 95.8% (23/24) of patients had undetectable viral load at week 24 and week 48.Six grade 1-2 adverse events were reported in 4 patients.Conclusions In this pilot study, the new regimen containing RAL, TDF and 3TC appears to be an ideal option for patients on methadone maintenance therapy, because of its limited impact on methadone dose and good efficacy and safety profile.

The M and S segment cDNAs of hantavirus Z5 strain was amplified by RT-PCR,and the purified PCR products were cloned into vector pGEM-T and then sequenced.It was demonstrated that the M genome segment of Z5 was found to be 3 616 nucleotides in length with a single open reading frame encoding 1 135 amino acids.And the S genome segment was 1700 nucleotides in length with a single open reading frame encoding 429 amino acids.As demonstrated by the homologous analysis of nucleotides and amino acids,it was showed that the Z5 strain belonged to hantaan viruses HTN type and was the same subtype of the Z10 strain.It is conclouded that difference in nucleotide sequence exists between Z5 strain with other Hantavirus strains but high level of homology in amino acid sequences is still present.

Objective To analyze the effect of long-term anti-viral treatment in children with acquired immune deficiency syndrome (AIDS) and investigate the factors affecting the treatment efficacy and growth and development of the children, so as to provide reference for improving the efficacy of antiviral drugs.Methods Children with AIDS receiving anti-retroviral treatment during 2004 to 2016 were retrospectively enrolled.The height, weight and CD4+ T cell counts were recorded every half year and the measurement of HIV RNA load was recorded on an annual basis.The CD4+ T cell counts and viral inhibition rates for the children who were under the treatment in the first year, 1~<5 years, 5~<10 years, and ≥10 years were compared.And their growth and development were also assessed.Treatment efficacy and growth and development of the children were compared between those who raised by social organization and by family.Children who raised by family were further divided into two groups: high-income and low-income groups.All categorical data were analyzed using chi-square test and those non-normal distribution were compared by rank-sumtest.Results After comparison between the children who have received anti-virus treatment for 1 to 5 years (including 5 year) and those for 5 to 10 year (including 10 years), the baseline CD4+ T cell counts were 436.5(265, 728)cells/μL and 334 (102, 535)cells/μL, respectively with the statistically significant difference (Z=-2.619, P<0.01).The last measured CD4+ T cell counts were 779 (622, 1 024)cells/μL and 720 (640, 977)cells/μL, respectively with no statistical significance (Z=-0.708, P>0.05);and viral inhibition rates were 92.9% and 97.6%, respectively with no statistical significance (χ2=1.071, P>0.05).The viral inhibition rate for the children receiving the treatment for 1 year was 85.7%, while that for whose treatment lasted for more than 10 years was 100.0%.A total of 5 cases developed drug-resistance (2 cases treated for 1 to 5 years and 3 cases for 5 to 10 years), and the virus replication was completely inhibited after switching to Lopinavir/ritonavir (LPV/r).The drug compliance was more than 95.0%.64.8% of children met the standard height, while 68.5% met the standard body mass.The baseline and last measured CD4+ T cell counts showed no significant differences between family-raised and social organization-raised children (Z=-1.159 and -0.523, respectively, both P>0.05).The children from high-income families had no significant differences compared with those from low-income ones in terms of the baseline and last measured CD4+ T cell counts (Z=-0.019 and -0.776, respectively, both P>0.05).Conclusions The long-term anti-retroviral treatment can effectively elevate the CD4+ T cell counts, inhibit viral replication and ensure drug compliance, which may promote the growth and development of children.However, approximately 30% children are still lower than the normal standards in terms of height and body mass.The drug-taking observer plays a central role on treatment effect.Most of the children′s family suffer from poor economic conditions.

Aim To investigate the effect and mecha-nism of quercetin combined with cisplatin on prolifera-tion and apoptosis of human osteosarcoma cell line MG-63 . Methods MG-63 cells were treated with quercetin alone or combined with cisplatin. Cellular morphologic changes were observed under inverted phase contrast microscope. The effects of proliferation inhibition were assayed by CCK-8 method. The combination effect was judged through Chou-Talaly analysis. The apoptosis ra-tios of cells were analyzed by flow cytometry. The gene expression of Bcl-2 and caspase-3 was detected by RT-PCR assay. The protein expression of Bcl-2 and caspase-3 was measured by Western blot assay. Re-sults Quercetin alone or combined with cisplatin could inhibit the proliferation, but induce the apoptosis of MG-63 cells. Combination of quercetin and cisplatin revealed a synergistic effect on cell proliferation and apoptosis as it reduced the expression of Bcl-2 but en-hanced that of caspase-3 at both gene and protein lev-els. Conclusion Synergistic effect of quercetin com-bined with cisplatin on cell proliferation and apoptosis of MG-63 cells is possibly due to reduction of Bcl-2 and enhancement of caspase-3 expression.

Objective The radiotherapy resistance is one of important causes for nasopharyngeal carcinoma(NPC) treatment failure.Junctional adhesion molecule A(JAMA)is closely correlated with the tumor poor prognosis.Thus this experiment is to in vestigate the relationship between JAMA expression and the radiosensitivity of NPC.Methods To overexpress or interfere the JAMA expression in CNE2 and HONE1 cell lines.Then different doses of X-ray were adopted to conduct irradiation.The cell clone formation capacity and cellular apoptosis change were detected after 24 h.The role of JAMA in the NPC radiotherapy was understand.The related signal pathway protein in cell lines with different JAMA expression was detected by Western blot.Results The cell lines with low JAMA expression were more sensitive to radiotherapy:After low JAMA expression,the D0 value in the CNE2 cell line was decreased from 3.26 ±0.78 to 1.92 ± 0.23;the Dq value was decreased from 46.51 ± 4.27 to 32.12 ± 3.19.The radio therapy induced apoptosis was significantly increased in the cell lines with low JAMA expression,after low JAMA expressing,thcellular apoptosis was elevated from 6.9 ％ ± 0.9 ％ to 13.7 ％ ± 1.3 ％;the HONE1 cellular apoptosis was elevated from 6.5 ％ + 1.1 ％ to 12.3 ％ ± 1.7％;JAMA overexpression cell lines were significantly decreased.The preliminary mechanism research results showed that JAMA played the effect via Akt signal pathway.Conclusion This research results verifiy that JAMA expression level is closely correlated with the radiosensitivity of NPC cell line:JAMA can increase the radiotherapy resistance of NPC cell lines,which provides a new feasible research direction for NPC enhancing radiosensitivity.

Objective To identify the role of miR-124 in regulating the radiosensitivity and the epithelial-mesenchymal transition (EMT) of nasopharyngeal carcinoma (NPC). Methods Transient transfection of cells with miR-124 mimic or inhibitor was performed and wound-healing assay was used to investigate the role of miR-124 in the EMT of NPC. The apoptosis affected by miR-124 was also measured after irradiation , followed by investigating the cell proliferation by EdU assay. Finally , proteins of Akt and ERK associated with EMT and radiosensitivity, were measured by western blot. Results The migration index from NPC cell line indicated that miR-124 repressed the EMT. The results from caspase-3 activity assay showed that caspase-3 activity after irradiation significantly increased in miR-124 mimic group compared with the control group (P < 0.01). It was also confirmed that irradiation led to a higher percentage of apoptosis in miR-124 group compared with the control group in NPC cells. Cell proliferation after irradiation was significantly decreased in MiR-124 group as compared with control group . MiR-124 inhibited the protein expression of p-Akt . Conclusion MiR-124 may repress the EMT and decrease radio-resistance of NPC via p-Akt signaling pathway , which may provide a new insight into radio-resistance in NPC.