ELECTRON MICROSCOPIC OBSERVATION OF SIMIAN SARCOMA VIRUS TRANSFORMED NIH 3T3 CELLS IN VITRO

For electronic microscopic observation, we found SSV-transformed NIH 3T3 cells were different from non-transformed cells. In SSV-transformed NIH 3T3 cells nuclei cytoplasma ratio was increased and in cytoplasma the ribosomes (polyribosomes were attached to the swollen rough endoplasmic reticulum. It was likely that ribosomes were lined together functionally and structionally to produce specific protein (PDGF-like protein).

Herpes simplex virus-1 infection or Simian virus 40-mediated immortalization of corneal cells causes permanent translocation of NLRP3 to the nuclei

AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses.METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1(HSV-1). Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40(SV40)-immortalized human corneal epithelial cell line were also examined.Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β.RESULTS: The NLRP3 activation induced by HSV-1infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore,in the SV40-immortalized human corneal epithelial cells,NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium(known as an inhibitor of NLRP3activation) effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot.· CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.

Development of Mutiplex Polymerase Chain Reaction for the Detection and Differentiation of Enteric Adenoviruses in Stool Samples

To find a technique of detecting and differentiating enteric adenoviruses (EAds) in clinical samples, a novel PCR approach was developed. EAds were able to be detected by use of a pair of subgroup F general primers (P1 and P2), and they were also be able to be differentiated from each other in the presence of another adenovirus type 40 (Ad40) specific primer (P3) in the same tube. Our results showed that there was one band for Ad41 and two bands for Ad40, respectively, on running-gel after PCR performance. PCR was performed on 40 specimens in parallel directly with dot-hybridization assay on the same diluted stool samples. 20 of 40 specimens were positive by hybridization (of them 12 were Ad41 and 8 were Ad40), whereas 26 were positive by PCR performance on the same samples with Ad41 18 and Ad40 8 positive as well. Our study indicated that this novel method could be used in clinical laboratory or in epidemic investigation for Eads

The preparation and expression of replication- deficient human interleukin-2 recombinant adenoviruses

Interleukin-2(IL-2) has been demonstrated to beone of the most effective target genes in cancerimmunogene therapy. There are more than 20 clinicalprotocols of cancer gene therapy introducing IL-2 intotumor patients to treat melanoma, renal carcinoma,prostate carcinoma, colon carcinoma,

COMBINED IL-2/IL-3 GENE THERAPY FOR G422 MOUSE GLIOBLASTOMA BY INTRATUMORAL INJECTION OF RECOMBINANT ADENOVIRUSES

Recombinant adenoviruses encoding murine IL 2 gene or IL 3 gene were directly injected into established subcutaneous tumor model of G422 glioblastoma cells. After treatment, the tumor size and survival of the glioblastoma bearing mice were observed. The splenic NK and CTL cytotoxicities were detected by standard 4 hour 51 Cr release assay. We also examined the histopathological changes of tumor by hematoxylin and eosin staining. The results showed that intratumoral injection of adenoviruses encoding murine IL 2 gene or IL 3 gene significantly inhibited the growth of G422 glioblastoma and prolonged the survival period of glioblastoma bearing mice. The CTL cytotoxicity of the gene therapy groups was significantly higher than that of the control groups, but NK activity remained unchanged, indicating that specific immunity contributes to the in vivo antitumor effect of the direct gene therapy. There were much more tumor necrosis and inflammatory cell infiltration in the tumor of the gene therapy groups. Combined IL 2/IL 3 gene therapy could induce higher level of CTL and enhance the therapeutic potential further. The results suggest that intratumoral injection of recombinant adenoviruses encoding certain kind of cytokines may be a useful approach in the treatment of a malignancy of the central nervous system.

Quantification of Simian Immunodeficiency Virus by SYBR Green RT-PCR Technique

Plasma viral RNA load is widely accepted as the most relevant parameter to assess the status and progression of Simian immunodeficiency virus （SIV） infections. To accurately measure RNA levels of the virus, a one-step fluorescent quantitative assay was established based on the SYBR green Real-time reverse transcription-polymerase chain reaction （RT-PCR）. The lower detection limit of the assay was 10 copies per reaction for the virus. This method was successfully applied to quantify SIVmac251 and SIVmac239 viruses produced in CEMx174 cells. Additionally, the performance of the SYBR green RT-PCR was assessed in a SIVmac251 infected rhesus macaque. The result demonstrated that the method could detect as little as 215 copies per milliliter of plasma and the dynamic pattern of viral load was highly consistent with previous results. With regard to convenience, sensitivity and accuracy our assay represents a realistic alternative to both branched-chain DNA （b-DNA） assays or real-time PCR assays based on TaqMan probes.

Severer nodular lesion in white matter than in gray matter in simian immunodeficiency virus-infected monkey, but not closely correlated with viral infection

Immune cell accumulation and white matter anomaly are common features of HIV(human immunodeficiency virus)-infected patients in combination antiretroviral therapy(cART) era.Neuroimaging tests on cART treated patients displayed prominent diffuse white matter lesions.Notably,immune cell nodular lesion(NL) was a conspicuous type of pathological change in HIV/SIV(simian immunodeficiency virus) infected brain before cART.Therefore,we used SIV infected brain to investigate the distribution of those NLs in gray and white matters.We found a significant higher number of NLs in white matter than that in gray matter.However,virus infection correlated with macrophage NLs but not with microglia NLs,especially in white matter.In addition,NLs interrupted white matter integrity more severely,since even tiny nodules could disconnect nerve fibers in white matter tracts.In the gray matter with dense myelinated axons,NLs obviously encroached those fibers;in the area of few myelinated axons,small nodules well co-localized with extracellular matrix between neurons.

The default mode network is affected in the early stage of simian immunodeficiency virus infection:a longitudinal study

Acquired immune deficiency syndrome infection can lead to cognitive dysfunction represented by changes in the default mode network.Most recent studies have been cross-sectional and thus have not revealed dynamic changes in the default mode network following acquired immune deficiency syndrome infection and antiretroviral therapy.Specifically,when brain imaging data at only one time point are analyzed,determining the duration at which the default mode network is the most effective following antiretroviral therapy after the occurrence of acquired immune deficiency syndrome.However,because infection times and other factors are often uncertain,longitudinal studies cannot be conducted directly in the clinic.Therefore,in this study,we performed a longitudinal study on the dynamic changes in the default mode network over time in a rhesus monkey model of simian immunodeficiency virus infection.We found marked changes in default mode network connectivity in 11 pairs of regions of interest at baseline and 10 days and 4 weeks after virus inoculation.Significant interactions between treatment and time were observed in the default mode network connectivity of regions of interest pairs area 31/V6.R and area 8/frontal eye field(FEF).L,area 8/FEF.L and caudal temporal parietal occipital area(TPOC).R,and area 31/V6.R and TPOC.L.ART administered 4 weeks after infection not only interrupted the progress of simian immunodeficiency virus infection but also preserved brain function to a large extent.These findings suggest that the default mode network is affected in the early stage of simian immunodeficiency virus infection and that it may serve as a potential biomarker for early changes in brain function and an objective indicator for making early clinical intervention decisions.

Purification and identification of simian parvovirus protein Vp2 expressed in E.coli

To purify and identify the simian parvovirus （SPV） protein Vp2 expressed in E. coli, fusion protein of SPV Vp2 was expressed in E. coli DHSα competent cells transformed with vector pThioHis AVp2, and the new bacterial protein extraction reagent was used to extract the protein. Detergents with different characteristics were used to solubilize the fusion protein, and metal chelating resin （ProBond） with a continuous elusion polyacrylamide gel electrophoresis procedure was employed to purify the fusion protein. SDS-PAGE gel stained with coomassie blue and Western-blotting probed with anti-thio and anti-SPV Vp2 antibodies were used to identify the specificity of the expressed and purified fusion proteins. It was found that the SPV Vp2 protein expressed in E. coli was highly insoluble, and could not be solublized by the commonly used detergent. However, 6 M urea could solubilize the fusion proteins and was then employed for the further purification procedure, but metal chelating resin could not be used for this procedure, because of the loss of the tertiary structure of HP-thiaoredoxin and the metal-binding domain. The technique with continuous elusion polyacrylamide gel electrophoresis yielded a homogenous protein with a single band on the gel stained with coomassie blue and retained reactivity with anti-thio or anti-SPV Vp2 antibodies. It is evident that this technique with successful purification of SPV Vp2 protein has practical significance for the further investigation on the simian parvovirus infection.

An extract from the earthworm Eisenia fetida non-specifically inhibits the activity of influenza and adenoviruses

OBJECTIVE： To test the in vitro antiviral activity of a crude tissue extract （CTE/from the earthworm Eisenia fetida, determine any effective components in the CTE, andelucidate possiblemechanismsofaction. METHODS： A CTE was made by homogenizing earthworms, followed by treatment with ammoni- um sulfate, then thermal denaturation. Inhibition of virus-induced cytopathic effect （CPE） was used to assess antiviral activity. Chromatographic analy- sis was used to identify effective components in the CTE. RESULTS： The CTE inhibited viral CPE at non-cyto- toxic concentrations. Chromatography indicated that antiviral components corresponded to three active peaks indicative of proteases, nucleases and lysozymes. For adenoviruses, reduction in viral ac- tivity occurred for 100 lag/mL CTE. The reduction in adenoviral activity for four fractions was 100%, 91.8%, 86.9%, and 94.7%. For influenza viruses, re- duction in viral activity of 100%, 86.6%, 69.1% and 88.3% was observed for 37 pg/mL CTE. In addition, three active fractions mixture had stronger antiviral activity （98.7% and 96.7%） than three fractions alone.Gel electrophoresis results indicated that nu- cleases from E. fetida could degrade the genome of influenza viruses and adenoviruses. CONCLUSION： The earthworm CTE displayed non-specific antiviral properties, possibly mediated by a combination of proteases, nucleases and lyso- zymes. Nucleases likely participate in the antiviral process, and degrade the genome of the virus thereby preventing further replication.

Construction and characterization of a new simian/human immunodeficiency viruses clone carrying an env gene derived from a CRF07_BC strain

Background The CRF07_BC recombinant strain has been one of the most predominantly circulated HIV-1 strains in China, it is therefore necessary and urgent to develop a relevant animal model to evaluate candidate vaccines targeting HIV-1 CRF07 BC. A highly replication-competent simian/human immunodeficiency viruses （SHIV） construct containing the Chinese CRF07_BC HIV-1 env gene with the ability to infect Chinese rhesus monkeys would serve as an important tool in the development of HIV vaccines. The aim of this study was to examine whether SHIV XJDC6431 with the env fragment from a Chinese HIV-1 isolate virus could infect the human and monkey peripheral blood mononuclear cell （PBMC）, establish infection in Chinese rhesus macaque. Methods A SHIV strain was constructed by replacing the rev/env genes of SHIV KB9 with the corresponding fragment derived from the HIV-1 CRF07_BC strain. The infectious activity of the SHIV clones was determined in vitro in PBMCs from both non-human primate animals and humans. Finally, one Chinese rhesus macaques （Macaca mulatta） was infected with one SHIV via intravenous infusion. Results One SHIV clone designated as SHIV XJDC6431, was generated that could infect macaque and human PBMC. The virus produced from this clone also efficiently infected the CCR5-expressing GHOST cell lines, indicating that it uses CCR5 as its coreceptor. Finally, the virus was intravenously inoculated into one Chinese rhesus macaque. Eventually, the animal became infected as shown by the occurrence of viremia within 3 of infection. The viral load reached 105 copies of viral RNA per ml of plasma during the acute phase of infection and lasted for 10 weeks post infection. Conclusions We conclude that SHIV XJDC6431 is an R5-tropic chimeric virus, which can establish infection not only in vitro but also in vivo in the Chinese rhesus macaque. Although the animal inoculated with SHIV XJDC6431 became infected without developing a pathologic phenotype, the virus efficiently replicated with a persistent level of viral load in the plasma. This suggested that the SHIV could be used as a tool to test candidate AIDS vaccines targeting the Chinese HIV-1 CRF 07BC recombinant strain.

Traditional Chinese Medicine etiology and pathogenesis of acquired immune deficiency syndrome in simian immunodeficiency virus-infected Chinese rhesus macaques

OBJECTIVE： To investigate the traditional Chinese Medicine （TCM） etiology and pathogenesis of ac- quired immune deficiency syndrome （AIDS） by 18-month observation of Chinese rhesus macaques infected with simian immunodeficiency virus （SIV） mac239. METHODS： Thirty-five healthy Chinese rhesus ma- caques were divided into a model group （n=30） and a control group （n=5）. The model was estab- lished by inoculating monkeys intravenously with SIVmac239. Changes in TCM symptoms after SIV in- fection within 18 months were then observed and recorded. Routine blood tests, SIV viral load, T-lym-phocyte subsets, plasma triiodothyronine （T3）, tet- raiodothyronine （14）, adrenocorticotropic hormone （ACTH） and cortisol （Cor） were tested periodically during the experiment. RESULTS： During the acute infection period of SIV, model monkeys temporarily showed clinical symp- toms such as diarrhea, dysphoria and slight weight loss. Decrease percentages of CD4~ T-lymphocytes were observed but levels of T3, 14, Cot, and ACTH were relatively unchanged. Monkeys in the model group during the early and middle periods of infec- tion showed no obvious symptoms, except few monkeys exhibited transient diarrhea and reduced food intake. All variables at this stage showed nor- mal fluctuations. In the middle period model group monkeys showed chronic and persistent diarrhea, weight loss, reduced food intake and low levels of 13 and Cot. In the late period, symptoms including emaciation, weight loss, listlessness, crouching in corners and low levels of T3appeared. CONCLUSION： The results suggest that the rhesus monkey SIWSAIDS model can be applied to re- search on TCM etiology and pathogenesis of AIDS. According to this model, the etiology of disease is the SIV virus. The pathogenesis manifests as the in- vasion of SIV virus, incubation of the virus, balance between virus and healthy ＂Qi＂, damage to spleen and kidney as the disease progressed, exhaustion of vitality and finally the failure of five zang and six fu organs.

High efficient generation of replication-defective adenoviruses containing thymidine kinase by homogeneous recombination in bacteria

Background Suicide gene therapy is a widely used molecular treatment for head and neck cancer. In this study, we try to use the method of homogenous recombination in bacteria to clone thymidine kinase gene （tk）-a kind of suicide gene to adenovirus backbone vectors for the construction of replication-defective adenoviruses. Methods pAdTrack-CMV/tk was constructed through subclone of a restriction endonuclease fragment including thymidine kinase gene from plasmid pCMV-tk to another plasmid pAdTrack-CMV, and then co-transfected with supercoiled pAdEasy-1, which was an adenoviral backbone vector except for deletions of E1 and E3, to competent E. coli BJ5183 for homogenous recombination using electroporation procedure. With the same method, pAdTrack-CMV was also co-transformed with pAdEasy-1 for homogenous recombination in BJ5183. Identified with restriction endonuclease Pad and polymerase chain reaction （PCR）, plasmids pAd-GFP/tk and pAd-GFP were successfully constructed. Each of them was digested with Pacl and sequently transfected into human embryo kidney 293 cells （HEK293） using Lipofectamine 2000. Results Comet-like adenovirus-producing foci of Ad-GFP/tk and Ad-GFP were observed after 5 to 7 days of cell culture After twelve days of packaging, the replication-defective adenoviruses were collected. Identified with PCR, thymidine kinase gene was successfully constructed into Ad-GFP/tk. Conclusion The replication-defective adenoviruses containing thymidine kinase can be constructed more easily by homogenous recombination in bacteria than conventional techniques.

Oncolytic adenoviruses:A thorny path to glioma cure

Glioblastoma Multiforme(GBM)is a rapidly progressing brain tumor.Despite the relatively low percentage of cancer patients with glioma diagnoses,recent statistics indicate that the number of glioma patients may have increased over the past decade.Current therapeutic options for glioma patients include tumor resection,chemotherapy,and concomitant radiation therapy with an average survival of approximately 16 months.The rapid progression of gliomas has spurred the development of novel treatment options,such as cancer gene therapy and oncolytic virotherapy.Preclinical testing of oncolytic adenoviruses using glioma models revealed both positive and negative sides of the virotherapy approach.Here we present a detailed overview of the glioma virotherapy field and discuss auxiliary therapeutic strategies with the potential for augmenting clinical efficacy of GBM virotherapy treatment.

Hairpin Probe for Sequence-specific Recognition of Double-stranded DNA on Simian Virus 40

Simian virus 40（SV40） is a polyomavirus and can induce a series of different tumors. The recognition of SV40 genome is crucial to tumor diagnosis and gene therapy. Herein, a sensitive and selective colorimetric method for sequence-specific recognition of homopyrimidine-homopurine duplex DNA（dsDNA） of SV40（4424-4440, gp6） was established with a hairpin probe based upon the formation of triplex DNA. Hairpin probe 5＇-CCC TAC CCA TTT TTT CTT CTC TTT CCT GGG TAG GGC GGG TTG GG-3＇（HP） containing G-rich sequence and 17-bp triplex-forming sequence was used as the signal probe, which was stem-loop structure alone and exhibited low catalytic activity. Upon its binding to the target duplex of SV40, hairpin probe transferred from stem-loop structttre to parallel triplex DNA, accompanied by the recovery of catalytic activity of DNAzyme and a sharp increase of absorbance. Under optimum conditions, the absorbance was increased proportionally to the concentration of dsDNA over the range from 500 pmol/L to 40.0 nmol/L with a detection limit of 433 pmol/L. Moreover, satisfied results were obtained when the assay was used to recognize the mismatched sequences.

The “Sino-India Monitor on INDCs Adequacy and Necessity”(SIMIAN) initiative

The papers in this special topic of Sino-India Monitor on NDCs,authored by a select group of researchers from both China and India,provide a perspective on areas of common interest for societies in both countries as well as a focus on common objectives defining global action.

Abnormal liver function in children hospitalized with acute respiratory infection of adenoviruses:a retrospective study

Human adenoviruses(HAdVs)can cause acute hepatitis in immunocompromised patients.However,it is unclear whether HAdVs are contributors to hepatitis in immunocompetent children.In this study,the liver function test(LFT)results were retrospectively analyzed among children hospitalized(age<14 years)between January 2016 and October 2019 for acute respiratory infection caused by adenoviruses.Alanine transaminase(ALT)and aspartate aminotransferase(AST)levels were elevated in 7.74%and 46.89%of patients,respectively.All patients with>2 folds of the upper limit of ALT or AST levels were infected with HAdV-7 or HAdV-55.Significantly higher levels of ALT,AST,γ-glutamyl transpeptidase(γ-GT),and lower albumin levels were observed in the HAdV-7 infection group than in the HAdV-3 infection group.HAdV-55 infection led to significantly higherγ-GT,total bilirubin,and direct bilirubin levels than the other infection types.The records of four patients with serial monitoring of the LFT results were further analyzed.Multiple indicators remained abnormal during the entire hospitalization in these patients.These results indicate that HAdV infection is often accompanied by abnormal liver function,and HAdV-7 and HAdV-55 might be under-recognized contributors to hepatitis among children.

重庆四面山不同林分土壤抗蚀抗冲特征

为探讨重庆四面山地区不同林分土壤抗蚀抗冲特征,采用水浸试验和冲刷试验,计算土壤抗蚀指数与抗冲系数,对四面山4种林分类型(针叶林、阔叶林、针阔混交林和楠竹林),共9个不同植物组成的林地土壤抗蚀抗冲特征进行研究。结果表明:1)阔叶林的抗蚀指数最大,楠竹林抗蚀指数最小,随着土壤深度的增加,土壤抗蚀性能减弱,天然针阔混交林土壤抗蚀指数上下层差异最大(1.92倍)。2)二次多项式函数能高度拟合不同林分土壤抗蚀指数与水浸时间的关系(R2>0.95),随着水浸时间的增加,不同林分土壤抗蚀性能下降。3)土层越深,土壤抗冲系数越大,抗冲性能越强,坡面上层土壤抗冲系数为下层的1.05~5.79倍。阔叶林的抗冲性优于其他林分。4)≤1和>1~3 mm根径的根系总根长与根长密度与土壤抗蚀指数显著正相关(P<0.05),与土壤抗冲性呈显著负相关(P<0.05)。土壤抗蚀性和抗冲系数与总根质量、根质量密度呈显著负相关(P<0.05)。研究结果可为重庆四面山水土保持措施布设、选择合理的植被恢复模式及配置方式提供理论依据。

秭归丝绵茶鲜叶非挥发性成分及丝形态结构分析

为研究丝绵茶品质形成特点,以秭归当地生产丝绵茶的茶树种‘丝绵土茶’6个嫩度部位叶片为研究对象,通过超高效液相色谱飞行时间质谱、电感耦合等离子体发射光谱和扫描电子显微镜,分析其非挥发性代谢成分、矿质元素、“丝”结构及数量特点。结果表明,丝绵茶鲜叶中的非挥发性代谢成分在嫩度较高的叶位富集较多,其中一叶和二叶(L1、L2)中氨基酸、生物碱、儿茶素和香气糖苷物质含量较高;茶氨酸、有机酸和黄酮类在第三叶(L3)富集最多。而嫩度较低的五六叶中非挥发性成分含量相对较低。不同嫩度鲜叶原料积累的各种元素具有明显差异,氮、磷、钾、锌和铜元素在嫩度较高的一、二叶位(L1、L2)含量较高,L1分别为32.41 mg/g、4.53 mg/g、15.65 mg/g、45.45μg/g、10.75μg/g,L2分别为30.60 mg/g、3.70mg/g、14.12mg/g、35.82μg/g、9.02μg/g;而铁、锰和钙在成熟叶位含量较高。通过扫描电镜观察发现丝绵茶鲜叶中“丝”结构包括三股卷曲和单股卷曲两种形式,分布在主脉和侧脉的维管束木质部内螺纹或环纹导管;且二、三、四叶中“丝”的数量较芽头和一叶多。