Baby Hamster Kidney Cell Line Research Articles

A versatile cell-penetrating peptide-adaptor system for efficient delivery of molecular cargos to subcellular destinations.

Cell penetrating peptides have long held great potential for delivery of biomolecular cargos for research, therapeutic and diagnostic purposes. They allow rapid, relatively nontoxic passage of a wide variety of biomolecules through the plasma membranes of living cells. However, CPP-based research tools and therapeutics have been stymied by poor efficiency in release from endosomes and a great deal of effort has been made to solve this ‘endosomal escape problem.’ Previously, we showed that use of a reversible, noncovalent coupling between CPP and cargo using calmodulin and a calmodulin binding motif allowed efficient delivery of cargo proteins to the cytoplasm in baby hamster kidney and other mammalian cell lines. The present report demonstrates the efficacy of our CPP-adaptor scheme for efficient delivery of model cargos to the cytoplasm using a variety of CPPs and adaptors. Effective overcoming of the endosomal escape problem is further demonstrated by the delivery of cargo to the nucleus, endoplasmic reticulum and peroxisomes by addition of appropriate subcellular localization signals to the cargos. CPP-adaptors were also used to deliver cargo to myotubes, demonstrating the feasibility of the system as an alternative to transfection for the manipulation of hard-to-transfect cells.

Genotoxic and cytotoxic potential of whole plant extracts of Kalanchoe laciniata by Ames and MTT assay.

Lack of data on safety of herbal medicines have endangered human health and life. The present study evaluated the genotoxic and mutagenic effect of Kalanchoe laciniata to access the safety and usefulness of the medicinal plant. Aqua-methanolic and n-hexane extracts of K. laciniata were evaluated for the genotoxic potential using Ames assay and cytotoxicity was evaluated using MTT assay. Ames assay was conducted using two strains of Salmonella typhimurium TA-100 and TA-102 whereas MTT assay was performed on baby hamster kidney cell line BHK-21. Aqua-methanolic extract of K. laciniata exhibited significant mutagenicity when exposed to TA-102 strain with a mutagenic index of 50.66 and 54.74 at maximum dose 150 mg/plate. The extract was also mutagenic to TA-100 strain but to a lesser extent. M.I of n-hexane extract was 12.15 and 15.51 for TA-100 and TA-102 respectively. n-hexane extract was mutagenic but little difference was observed between results of two strains. Both extracts were found to be cytotoxic with an IC50 of 321.9 and 638.5 µg/mL for aqua-methanolic and n-hexane extracts respectively. On the basis of results it was concluded that aqua-methanolic and n-hexane extracts of K. laciniata possess mutagenic and cytotoxic potential. It is suggested to explore the plant further to evaluate its safety in rodents and other species.

BAY 81‐8973, a full‐length recombinant factor VIII: manufacturing processes and product characteristics

BAY 81-8973 (Kovaltry® , Bayer, Berkeley, CA, USA) is an unmodified, full-length recombinant human factor VIII (FVIII) approved for prophylaxis and on-demand treatment of bleeding episodes in patients with haemophilia A. The BAY 81-8973 manufacturing process is based on the process used for sucrose-formulated recombinant FVIII (rFVIII-FS), with changes and enhancements made to improve production efficiency, further augment pathogen safety, and eliminate animal- and human-derived raw materials from the production processes. The baby hamster kidney cell line used for BAY 81-8973 was developed by introducing the gene for human heat shock protein 70 into the rFVIII-FS cell line, a change that improved cell line robustness and productivity. Pathogen safety was enhanced by including a 20-nm filtration step, which can remove viruses, transmissible spongiform encephalopathy agents and potential protein aggregates. No human- or animal-derived proteins are added to the cell culture process, purification or final formulation. The BAY 81-8973 manufacturing process results in a product of enhanced purity with a consistently high degree of sialylation of N-linked glycans on the molecular surface. The innovative manufacturing techniques used for BAY 81-8973 yield an effective rFVIII product with a favourable safety profile for treatment of haemophilia A.

Anti-Herpes Simplex Virus Type-1 Activity and Phenolic Content of Crude Ethanol Extract and Four Corresponding Fractions of Quercus brantii L Acorn.

This research was aimed to evaluate anti–herpes simplex virus type-1 (anti-HSV-1) activity of crude ethanol extract and 4 corresponding fractions of Quercus brantii acorn in vitro. Crude ethanol extract was prepared and subjected to fractionation with different polarity. Anti-HSV-1 activity was evaluated on baby hamster kidney cell line using MTT assay. The inhibitory effect of the plant materials on adsorption and/or post-adsorption stages of HSV-1 replication cycle were determined. Regression analysis was used to determine 50% inhibitory concentration and 50% cytotoxicity concentration, from which selective index was calculated. Based on our results, the chloroform fraction and the crude extract had the highest effect against HSV-1 with selectivity indices of 53.8 and 48.4, respectively. The n-hexane, n-butanol, and chloroform fractions inhibited HSV-1 replication in postadsorption stage (P < .001). The results obtained indicated that the chloroform fraction of Q brantii acorn with high inhibitory effect against HSV-1 replication could be a new promising anti-HSV-1 agent.

Efficacy assessment of an inactivated Tembusu virus vaccine candidate in ducks

Duck Tembusu virus (TMUV) is a recently identified pathogen that causes severe egg drop and neurological disease in domestic duck and goose flocks. The infection has spread across the China mainland since its outbreak in 2010. Effective vaccines are needed to fight the disease. In this work, we describe the development and laboratory assessment of a cell culture-derived, inactivated duck TMUV vaccine. The TMUV-JXSP strain was successfully propagated on a baby hamster kidney cell line (BHK-21), inactivated with beta-propiolactone (BPL) and emulsified with mineral oil. The efficacy of different vaccination schedules was assessed in laying ducks and table ducks using virus challenge experiments. Two doses of vaccine provided efficient protection against the virus challenge to avoid the egg production drop in laying ducks. An ELISA demonstrated that 97% (39/40) of ducks seroconverted on day 21 after one dose of the inactivated vaccine and that significant increases in antibody titers against the virus were induced after the second immunization. For table ducks, a single dose of vaccine immunization resulted in a protection index of 87% and significant reduction of viral loads in tissues. Sterilizing immunity can be attained after second immunization. Our results demonstrate that BHK-21 cell culture is suitable for duck TMUV propagation and that BPL-inactivated TMUV vaccine can provide a high level of protection from virus challenge in laying ducks and table ducks. These data provide a scientific basis for the development of an inactivated vaccine for the prevention of duck TMUV infection.

Genotoxic and cytotoxic action potential of Terminalia citrina, a medicinal plant of ethnopharmacological significance.

Most herbal medicines utilized in complementary and alternative medicine lack safety evaluation setting our lives under unwarranted risks. Present study comprised of genotoxic and cytotoxic appraisal of Terminalia citrina fruits which are used as a folklore medicine for treatment of various ailments. Aqueous and ethanolic extracts of T. citrina fruit extracts were evaluated for the presence of different phytochemicals. Genotoxic potential of both the extract of T. citrina was assessed through Ames reverse mutagenicity assay in Salmonella TA 100 and 102 strains. Cytotoxic potential of T. citrina was determined in baby hamster kidney cell line (BHK-21). Statistical analysis was carried out by ANOVA following post hoc test. Phytochemical analysis showed the presence of alkaloids, carbohydrates, phenolic compounds, tannins, catechins and saponins. It was revealed that both the extracts of T. citrina exhibited significant mutagenicity in tester strains. Ethanolic extract showed higher mutagenicity in TA 100 strain, whereas aqueous extract of T. citrina exhibited higher mutagenicity in TA 102 strain than TA 100. Both the extracts of T. citrina showed dose-dependent mutagenicity. Fifty percent cell viability was exhibited by 260 and 545 µg/mL of ethanolic and aqueous extracts respectively. This study concludes that the ethanolic and aqueous fruit extracts of T. citrina may not be safe owing to their mutagenic and cytotoxic potential and it necessitates further investigation regarding its safety evaluation.

RT-PCR based detection and adaptation of foot and mouth disease virus serotype “A” in BHK-21 cell line

The economic consequences of an outbreak of Foot and Mouth Disease (FMD) in Bangladesh is devastating. A sensitive, reliable and rapid diagnosis is crucial for the effective control of FMD. The present research was conducted for quick molecular detection and adaptation of FMD virus (FMDV) in BHK-21 (Baby Hamster Kidney) cell line. A two-step RT-PCR method was applied for the detection of the FMDV. Without prior adaption into BHK-21 cell culture, it is difficult to detect FMDV directly from the field samples by RT-PCR method. Samples were collected from the tongue epithelium (N=19) and inter digital tissues (N=4) of the suspected animals in Sujanagar, Bera, Santhia of Pabna district and Kotowali, Trishal of Mymensingh districts of Bangladesh during the year 2014. BHK-21 cell subculture was done from a previously cultured bottle containing BHK-21 cells. Prepared inocula were inoculated into BHK-21 cell culture and incubated at 37ºC for 24 h. After 36 h, cytopathic effects (CPE) were observed in BHK-21 cell line characterized by rounding and flattening of the cells, multinucleated giant cells formation, breaking down of the intracellular bridges and finally cell death indicated the presence of FMDV. Clear infectious BHK-21 cell culture fluid was collected and preserved at -20ºC temperature for virus detection by RT-PCR with serotype specific primers. Viral RNA was extracted from the clear infectious cell culture fluid for cDNA synthesis and used for PCR. Out of 23 samples tested, 3(13.04%) were positive for FMDV serotype A. The findings of this study can be helpful for the selection of vaccine having specific FMDV type, and it may help in controlling FMD in Bangladesh.Progressive Agriculture 27 (1): 64-69, 2016

Safety evaluation of Acalypha wilkesiana in albino rats and BHK-21 cell line

Acalypha wilkesiana is a member of the spurge family, genus Acalypha, that is widely used in folklore medicine. The aim of this study was to screen aqueous leaf extract of A. wilkesiana for toxic effects in vitro and in vivo. We examined the phytochemical profile, cytotoxic effects on baby hamster kidney cell line (BHK-21), and oral subacute toxicity of A. wilkesiana leaf decoction in rats. Rats were given 0, 300, 600, and 1200 mg/kg body weight of A. wilkesiana leaf extract, daily, orally for 14 days. The phytochemical profile showed the presence of flavonoids, saponin, cardiac glycosides, and tannins. It caused apoptosis in BHK-21 cell line at concentrations of 0, 25, 50, 100, and 200 μg/ml. There was significant increase in the levels of serum AST, ALT, creatinine, urea, Na+, K+, and Cl− levels in all the test groups compared to the control. Histology of the liver revealed centrilobular degeneration and necrosis with sinusoidal dilatation as well as polymorphonuclear and mononuclear infiltration. The kidney showed severe glomerular and tubular degeneration and necrosis with hemorrhage at all doses administered. We conclude that the plant was toxic at the doses tested in vitro and in vivo, and care should be exercised in its use in herbal medicine.

Apoptotic events induced by prototype foamy virus infection

ABSTRACTFoamy virus infection induces cytopathology in several cell types from different species. But the exact mechanism is still unknown. In this study, we have investigated the mechanism of cell death induced by prototype foamy virus (PFV) infection in baby hamster kidney (BHK 21) cell lines. PFV induces apoptosis by exhibiting morphological alterations such as chromatin condensation, blebbing, and nuclear fragmentation. In addition, PFV infection causes chromosomal DNA fragmentation, up-regulation of Bax, and activation of caspase-3, but not caspase-8. Up-regulation of Bax initiates the translocation of cytochrome-c from mitochondria to the cytoplasm, suggesting predominantly to the mitochondrial-mediated pathway. Blocking apoptosis using caspase inhibitors increased PFV-infected BHK 21 cell viability. Although blocking apoptosis resulted in reduced progeny release, maximal accumulation of PFV was found in apoptosis-blocked cells. This report provides the first experimental evidence of apoptosis induced by PFV infection, which will provide valuable insights for foamy viral pathology.

IDENTIFICATION OF THE SIBERIAN TYPE TICK-BORNE ENCEPHALITIS VIRUS AND SEROLOGICAL SURVEILLANCE IN MONGOLIA

The goal of this study was to conduct serosurveillance for tick-borne encephalitis virus (TBEV)in Mongolia, to isolate TBEV by in vivo methods and determine subtypes of TBEV. Blood samples were collected from 750 domestic animals in Khuvsgul, Selenge, Bulgan and Tuv aimags in 2012. After sample collection, a total of 184 sera were (horse 43, cattle 41, sheep 44, goat 56respectivel) randomly chosen and tested for antibodies against TBEV by c-ELISA and virus neutralization (VN) test. The c-ELISA results showed that 17 serum samples were positive and 25 -suspicious. In order to confirm c-ELISA result all these 42 serum samples were checked with VN test. The result of VN test showed 19/42 positive serum samples. In order to detect of TBEV in Mongolia, Ixodes persulcatus ticks were collected from Eruu sum, Selenge aimag and categorized into pools. Each pool was mixed with sterile PBS and homogenized with asterile mortar and pestle. The homogenate was centrifuged and the collected supernatant was inoculated into baby hamster kidney (BHK-21) cell line. Cytopathic effects (CPE) in the cell line was observed by light microscopy daily. RNA was extracted from supernatant of cells with CPE and confirmed by RT-PCR using specific primer for the Siberian subtype. The present results were indicated that TBEV in Mongolia was belonged to the Siberian subtype.Mongolian Journal of Agricultural Sciences Vol.13(2) 2014: 19-26

Expression of Factor X in BHK-21 Cells Promotes Low Pathogenic Influenza Viruses Replication.

A cDNA clone for factor 10 (FX) isolated from chicken embryo inserted into the mammalian cell expression vector pCDNA3.1 was transfected into the baby hamster kidney (BHK-21) cell line. The generated BHK-21 cells with inducible expression of FX were used to investigate the efficacy of the serine transmembrane protease to proteolytic activation of influenza virus hemagglutinin (HA) with monobasic cleavage site. Data showed that the BHK-21/FX stably expressed FX after ten serial passages. The cells could proteolytically cleave the HA of low pathogenic avian influenza virus at multiplicity of infection 0.01. Growth kinetics of the virus on BHK-21/FX, BHK-21, and MDCK cells were evaluated by titrations of virus particles in each culture supernatant. Efficient multicycle viral replication was markedly detected in the cell at subsequent passages. Virus titration demonstrated that BHK-21/FX cell supported high-titer growth of the virus in which the viral titer is comparable to the virus grown in BHK-21 or MDCK cells with TPCK-trypsin. The results indicate potential application for the BHK-21/FX in influenza virus replication procedure and related studies.

Application Of Recent Methods For Diagnosis Of Foot And Mouth Disease In Cattle And Buffaloes

Egypt was recently exposed to a sever outbreak of Foot and Mouth Disease (FMD) in 2012 with a new introduced strain of the virus (SAT2). This study concerned with diagnosis of FMD by using some recent methods in addition to the traditional methods of diagnosis. A total of 326 animals were clinically examined in different localities at Sharkia Governorates; 301 animals showed clinical signs of FMD (diseased animals) and 25 animals were apparently healthy. The diseased animals consisted of 277 cattle and 24 buffaloes. Some of them were vaccinated with the FMD bivalent vaccine before the outbreak and others were not vaccinated. Eighty two clinical samples (73 epithelial tissues (Epi.T.) and 9 vesicular fluids (V.F)) from cattle and buffaloes were isolated on Baby Hamster kidney (BHK-21) cell line and 76 samples (92.7%) were positive. By using FMDV ELISA Kits for typing the virus only serotype SAT2 was detected. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect SAT2 serotype in 10 sample collected from 5 diseased cattle and 5 diseased buffaloes from different localities. Only one sample showed negative reaction (this sample was also negative by virus isolation and ELISA typing). Serum samples were collected from the 301 animals at different times after the infection; the antibodies against FMDV non-structural protein were detected by FMDV′CHEK Test in 270 cases (89.7%) of examined animals, while antibodies against the structural protein were detected by SNT and ELISA where antibodies against serotypes O, A and SAT2 were detected in 75.7%, 75.7% and 100% of examined animals for each serotype respectively by both tests. Mixed infection with more than one serotype was only recorded at Al-Salhia dairy farm. It is concluded that combination between recent and traditional techniques for diagnosis of FMD help in accurate and rabid diagnosis and subsequently control the disease.

Exploring the transcriptome space of a recombinant BHK cell line through next generation sequencing

Baby Hamster Kidney (BHK) cell lines are used in the production of veterinary vaccines and recombinant proteins. To facilitate transcriptome analysis of BHK cell lines, we embarked on an effort to sequence, assemble, and annotate transcript sequences from a recombinant BHK cell line and Syrian hamster liver and brain. RNA-seq data were supplemented with 6,170 Sanger ESTs from parental and recombinant BHK lines to generate 221,583 contigs. Annotation by homology to other species, primarily mouse, yielded more than 15,000 unique Ensembl mouse gene IDs with high coverage of KEGG canonical pathways. High coverage of enzymes and isoforms was seen for cell metabolism and N-glycosylation pathways, areas of highest interest for biopharmaceutical production. With the high sequencing depth in RNA-seq data, we set out to identify single-nucleotide variants in the transcripts. A majority of the high-confidence variants detected in both hamster tissue libraries occurred at a frequency of 50%, indicating their origin as heterozygous germline variants. In contrast, the cell line libraries' variants showed a wide range of occurrence frequency, indicating the presence of a heterogeneous population in cultured cells. The extremely high coverage of transcripts of highly abundant genes in RNA-seq enabled us to identify low-frequency variants. Experimental verification through Sanger sequencing confirmed the presence of two variants in the cDNA of a highly expressed gene in the BHK cell line. Furthermore, we detected seven potential missense mutations in the genes of the growth signaling pathways that may have arisen during the cell line derivation process. The development and characterization of a BHK reference transcriptome will facilitate future efforts to understand, monitor, and manipulate BHK cells. Our study on sequencing variants is crucial for improved understanding of the errors inherent in high-throughput sequencing and to increase the accuracy of variant calling in BHK or other systems.

Effect of mixed infections of Sindbis and La Crosse viruses on replication of each virus in vitro

Mixed viral infections are ubiquitous in natural populations and may have significant but unpredictable biological and epidemiological consequences. These infections may be acquired simultaneously (coinfection) or result from two single infections at different time intervals (superinfection). This study investigated the effect of mixed infections of two mosquito-borne viruses, Sindbis (SINV) and La Crosse (LACV), on the replication of each virus in vertebrate baby hamster kidney (BHK) and invertebrate Aedes albopictus (C6/36) cell lines. SINV and LACV were inoculated onto confluent monolayers of each cell type either singly, simultaneously, or with a 2-h delay between single inoculations and titers for each virus were quantified by qRT-PCR. The titers for both viruses were significantly higher in BHK compared to C6/36 cells. Mixed infections suppressed replication of both viruses in BHK cells except for one superinfection treatment where inoculation of SINV ahead of LACV did not result in a reduction in SINV titer relative to single infection treatment. In C6/36 cells mixed infections had no effect on LACV replication, while coinfection enhanced SINV replication. These results demonstrate the potential for mixed viral infections to modify arbovirus transmission and pathogenesis.

Osmotic Stress Changes the Expression and Subcellular Localization of the Batten Disease Protein CLN3

Juvenile CLN3 disease (formerly known as juvenile neuronal ceroid lipofuscinosis) is a fatal childhood neurodegenerative disorder caused by mutations in the CLN3 gene. CLN3 encodes a putative lysosomal transmembrane protein with unknown function. Previous cell culture studies using CLN3-overexpressing vectors and/or anti-CLN3 antibodies with questionable specificity have also localized CLN3 in cellular structures other than lysosomes. Osmoregulation of the mouse Cln3 mRNA level in kidney cells was recently reported. To clarify the subcellular localization of the CLN3 protein and to investigate if human CLN3 expression and localization is affected by osmotic changes we generated a stably transfected BHK (baby hamster kidney) cell line that expresses a moderate level of myc-tagged human CLN3 under the control of the human ubiquitin C promoter. Hyperosmolarity (800 mOsm), achieved by either NaCl/urea or sucrose, dramatically increased the mRNA and protein levels of CLN3 as determined by quantitative real-time PCR and Western blotting. Under isotonic conditions (300 mOsm), human CLN3 was found in a punctate vesicular pattern surrounding the nucleus with prominent Golgi and lysosomal localizations. CLN3-positive early endosomes, late endosomes and cholesterol/sphingolipid-enriched plasma membrane microdomain caveolae were also observed. Increasing the osmolarity of the culture medium to 800 mOsm extended CLN3 distribution away from the perinuclear region and enhanced the lysosomal localization of CLN3. Our results reveal that CLN3 has multiple subcellular localizations within the cell, which, together with its expression, prominently change following osmotic stress. These data suggest that CLN3 is involved in the response and adaptation to cellular stress.

CFTR mutations altering CFTR fragmentation

Most CF (cystic fibrosis) results from deletion of a phenylalanine (F508) in the CFTR {CF transmembrane-conductance regulator; ABCC7 [ABC (ATP-binding cassette) sub-family C member 7]} which causes ER (endoplasmic reticulum) degradation of the mutant. Using stably CFTR-expressing BHK (baby-hamster kidney) cell lines we demonstrated that wild-type CTFR and the F508delCFTR mutant are cleaved into differently sized N- and C-terminal-bearing fragments, with each hemi-CFTR carrying its nearest NBD (nucleotide-binding domain), reflecting differential cleavage through the central CFTR R-domain. Similar NBD1-bearing fragments are present in the natively expressing HBE (human bronchial epithelial) cell line. We also observe multiple smaller fragments of different sizes in BHK cells, particularly after F508del mutation (ladder pattern). Trapping wild-type CFTR in the ER did not generate a F508del fragmentation fingerprint. Fragments change their size/pattern again post-mutation at sites involved in CFTR's in vitro interaction with the pleiotropic protein kinase CK2 (S511A in NBD1). The F508del and S511A mutations generate different fragmentation fingerprints that are each unlike the wild-type; yet, both mutants generate new N-terminal-bearing CFTR fragments that are not observed with other CK2-related mutations (S511D, S422A/D and T1471A/D). We conclude that the F508delCFTR mutant is not degraded completely and there exists a relationship between CFTR's fragmentation fingerprint and the CFTR sequence through putative CK2-interactive sites that lie near F508.

Cultivation and comparison of BHK-21 anchorage semi-dependent cell line in different production systems

Aim: The investigation of the different culture systems in order to get high yield of baby hamster kidney (BHK) cell line. Materials and Methods: Different cultivation systems, vibrofermentor and 5-L stirred tank bioreactors, have been used for the production of BHK cell line. The production systems were evaluated with respect to pH, temperature, viable cell number, aeration rate and stirring speed. Results: BHK cells were successfully cultivated in vibrofermentor and stirred tank bioreactor (STR) systems. In addition, higher cell concentration was obtained with low aeration rate (0.005 vvm) comparing to those of 0.02 and 0.05 vvm. The cultivation in STR can be operated with low aeration rate due to decrease the expenditure and improve the cell productivity. Conclusion: In order to improve the cell yield of the anchorage semi-dependent cell line in pilot scale, it should be preferred that the cells are firstly adapted to suspension culture by vibrational stirring and then be cultivated in stirred tank bioreactor which is operated at low aeration rate. These results showed consistency with the previous studies.

Determination of in vivo toxicity and in vitro cytotoxicity of lipopolysaccharide isolated from Salmonella Enteritidis and its potential use for production of polyclonal antibody

In the present study, the effects of lipopolysaccharide (LPS) isolated from Salmonella Enteritidis were investigated by in vivo and in vitro methods. The mean lethal dose of 50% death (LD50) was determined by a Probit test as 450-µg/mouse after 72 h post-treatment of LPS. Madin–Darby bovine kidney (MDBK), baby hamster kidney (BHK), mouse fibroblastic cell-lines (L929), and hybridoma cell lines were treated with different amounts of LPS (0–100 µg/ml) cultivated for 72 h in 24 well tissue culture plates. Morphological investigation was done with inverted microscope after Giemsa staining. The results suggested that MDBK, BHK, and L929 cell lines were resistant to LPS cytotoxicity due to lack of the specific membrane receptor unlike hybridoma cells. After immunisation, one priming and seven booster's diluted sera (1:100) antibody level was found to be 2.00 ± 0.06–2.41 ± 0.07 optical density (OD) by enzyme linked immunosorbent assay (ELISA) on day 142. This result indicated that the LPS, instead of whole bacteria, have potential application in the immunisation.

Human growth hormone gene expression in vitro following rAAV2/1-mediated gene transfer

Background: Viral vectors can produce longer-lasting effects than a recombinant protein. Objective: Develop a system of sustained GH1 (a human growth hormone gene) transfer and expression in baby hamster kidney cell (BHK-21) line using recombinant adeno-associated viral vectors pseudotyped with viral capsids from serotype 1 (rAAV2/1). Methods: The expression of GH1 in vitro was examined by reverse transcription polymerase chain reaction and Western blot analysis. Effects of GH1 on cell proliferation were measured by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT) experiments. Mice serum IGF-1 and blood glucose, after injection of virus infected BHK-21 cells, were measured by enzyme-linked immunosorbent assays. Results: rAAV2/1-mediated GH1 gene transfer occurred effectively in vitro after 48 hours of transduction at 1 105 vg/mL (multiplicities of infection). MTT experiments indicated notable effects of the GH1 gene on cell proliferation in vitro. Subsequent animal experiment suggested that the injection of virus infected BHK-21 cells might induce increase of serum IGF-1. Conclusion: Our study shows the feasibility of rAAV2/1-mediated GH gene delivery in vitro, applicable for future experimental and clinical investigations. Keywords: Baby hamster kidney cell 21, growth hormone, insulin-like growth factor 1, recombinant adenoassociated virus vector

A dual reporter cell assay for identifying serotype and drug susceptibility of herpes simplex virus

A dual reporter cell assay (DRCA) that allows real-time detection of herpes simplex virus (HSV) infection was developed. This was achieved by stable transfection of cells with an expression cassette that contains the dual reporter genes, secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein (EGFP), under the control of an HSV early gene promoter. Baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cell lines were used as parental cell lines because the former is permissive for both HSV serotypes, HSV-1 and HSV-2, whereas the latter is susceptible to infection only by HSV-2. The DRCA permitted differential detection of HSV-1 and HSV-2 by observation of EGFP-positive cells, as substantiated by screening a total of 35 samples. The BHK-based cell line is sensitive to a viral titer as low as a single plaque-forming unit with a robust assay window as measured by a chemiluminescent assay. Evaluations of the DRCA with representative acyclovir-sensitive and acyclovir-resistant HSV strains demonstrated that their drug susceptibilities were accurately determined by a 48-h format. In summary, this novel DRCA is a useful means for serotyping of HSV in real time as well as a rapid screening method for determining anti-HSV susceptibilities.