• Journal of Veterinary Science
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• v.21 no.4
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• pp.63.1-63.9
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• 2020

Background: Canine adenovirus type 2 (CAV-2) induces infectious laryngotracheitis in members of the family Canidae, including dogs. To date, no ELISA kits specific for CAV-2 antibody have been commercialized for dogs in Korea. Objectives: We aimed to develop new indirect enzyme-linked immunosorbent assay (I-ELISA) to perform rapid, accurate serological surveys of CAV-2 in dog serum samples. Methods: In total, 165 serum samples were collected from dogs residing in Chungbuk and Gyeongbuk provinces between 2016 and 2018. The Korean CAV-2, named the APQA1701-40P strain, was propagated in Madin-Darby canine kidney cells and purified in an anion-exchange chromatography column for use as an antigen for I-ELISA. The virus-neutralizing antibody titers of CAV-2 in the dog sera were measured by virus neutralization (VN) test. Results: We compared the results obtained between the VN and new I-ELISA tests. The sensitivity, specificity, and accuracy of new I-ELISA were 98.6%, 86.4% and 97.0% compared with VN test, respectively. New I-ELISA was significantly correlated with VN (r = 0.91). Conclusions: These results indicate that new I-ELISA is useful for sero-surveillance of CAV-2 in dog serum.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.151-158
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• 2007

The possibility of inadvertent introduction of therapeutic gene expressing viral vectors has raised safety concerns about germ-line infection. Particularly, for indications such as prostate cancer and ovarian cancer, the proximity of the point of viral administration to organs of the reproductive system raises concerns regarding inadvertent germ-line transmission of genes carried by the virus vector. To evaluate the safety of in vivo adenovirus mediated gene transfer, we explored the biodistribution, persistance and potential germ-line transmission of p53-expressing adenovirus (Ad-CMV-p53). Both male and female Balb/c mice were injected with $1{\times}10^9$ PFU of Ad-CMV-p53. The PCR analysis showed that there were detectable vector sequences in liver, kidney, spleen, seminal vesicle, epididymis, prostate, ovary, and uterus. The RT-PCR analysis for detecting inserted gene, p53 showed that Ad-CMV-p53 viral RNA were present in spleen, prostate and ovary. Direct injected male and female mice of adenovirus vector into testis and ovary were mated and their of offspring were evaluated for germ-line transmission of the adenoviral vector. The PCR and RT-PCR analysis showed no evidence of germline transmission, although vector sequences were detected in DNA extracted from gonadal tissues. Real-time PCR result confirmed a significant decrease of adenovirus in gonad tissues 1 week after injection. We have also analysed the cell specific localization of viral DNA in gonad tissues by using in-situ PCR. Positive signals were detected in interstitial tissue but not in seminiferous tubule in sperm. In the case of ovary, adenovirus signal were localized to the stromal tissue, but no follicular signals were observed. Together, these data provide strong evidence that the risk of the Inadvertent germ-line transmission of vector sequences following intraperitoneal or direct injection into genito-urinary system of adenovirus is extremely low.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.31 no.3
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• pp.199-218
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• 2005

Purpose of Study: Peripheral nerve regeneration depends on neurotrophism of distal nerve stump, recovery potential of neuron, supporting cell like Schwann cell and neurotrophic factors such as BDNF. Peripheral nerve regeneration can be enhanced by the conduit which connects the both sides of transected nerve. The conduit maintains the effects of neurotrophism and BDNF produced by Schwann cells which can be made by gene therapy. In this study, we tried to enhance the peripheral nerve regeneration by using calcium phosphate coated porous conduit and BDNF-Adenovirus infected Schwann cells in sciatic nerve of rats. Materials and Methods: Microporous filter which permits the tissue fluid essential for nerve regeneration and does not permit infiltration of fibroblasts, was made into 2mm diameter and 17mm length conduit. Then it was coated with calcium phosphate to improve the Schwann cell adhesion and survival. The coated filter was evaluated by SEM examination and MTT assay. For effective allogenic Schwann cell culture, dorsal root ganglia of 1-day old rat were extracted and treated with enzyme and antimitotic Ara-C. Human BDNF cDNA was obtained from cDNA library and amplified using PCR. BDNF gene was inserted into adenovirus shuttle vector pAACCMVpARS in which E1 was deleted. We infected the BDNF-Ad into 293 human mammary kidney cell-line and obtained the virus plaque 2 days later. RT-PCR was performed to evaluate the secretion of BDNF in infected Schwann cells. To determine the most optimal m.o.i of BDNF-Ad, we infected the Schwann cells with LacZ adenovirus in 1, 20, 50, 75, 100, 250 m.o.i for 2 hours and stained with ${\beta}$-galactosidase. Rats(n=24) weighing around 300g were used. Total 14mm sciatic nerve defect was made and connected with calcium phosphate coated conduits. Schwann cells$(1{\times}10^6)$ or BDNF-Ad infected Schwann cells$(1{\times}10^6)$ were injected in conduit and only media(MEM) was injected in control group. Twelve weeks after surgery, degree of nerve regeneration was evaluated with gait analysis, electrophysiologic measurements and histomorphometric analysis. Results: 1. Microporous Millipore filter was effective conduit which permitted the adhesion of Schwann cells and inhibited the adhesion of fibroblast. We could enhance the Schwann cell adhesion and survival by coating Millipore filter with calcium phosphate. 2. Schwann cell culture technique using repeated treatment of Ara-C and GDNF was established. The mean number of Schwann cells obtained 1 and 2 weeks after the culture were $1.54{\pm}4.0{\times}10^6$ and $9.66{\pm}9.6{\times}10^6$. 3. The mRNA of BDNF in BDNF-Ad infected Schwann cells was detected using RT-PCR. In Schwann cell $0.69\;{\mu}g/{\mu}l$ of DNA was detected and in BDNF-Adenovirus transfected Schwann cell $0.795\;{\mu}g/{\mu}l$ of DNA was detected. The most effective infection concentration was determined by LacZ Adenovirus and 75 m.o.i was found the most optimal. Conclusion: BDNF-Ad transfected Schwann cells successfully regenerated the 14mm nerve gap which was connected with calcium phosphate coated Millipore filter. The BDNF-Ad group showed better results compared with Schwann cells only group and control group in aspect to sciatic function index, electrophysiologic measurements and histomorphometric analysis.

• Archives of Plastic Surgery
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• v.37 no.5
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• pp.519-525
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• 2010

Purpose: Of various effects of relaxin, we assumed that anti-fibrotic effects, neovascularization effects and vasodilatation effects of relaxin might enhance the survival rate of skin flap. In the current study, we used adenovirus expressing relaxin genes to examine whether these genes could enhance the survival rate of a skin flap. Methods: A total of 30 Sprangue-Dawley rats were divided into three groups: RLX group (10; relaxin virus injected group), CTR group (10; no gene coded virus injection group), and PBS group (10; PBS injected group). Each group was intradermally injected with the virus ($10^7$ PFU) and PBS 48 hours before and immediately before the flap elevation. A distally based flap $3{\times}9\;cm$ in size was elevated on the dorsal aspect of each rat. Following this, a flap was placed in the original location and then sutured using a #4-0 Nylon. A surviving area of the flap was measured and then compared on postoperative days 3, 7 and 10. Using a laser Doppler, the amount of blood flow was measured. On postoperative day 10, tissues were harvested for histologic examination and the number of blood vessels was counted. Results: There was a significant increase in the area of the flap survival in the RLX group on postoperative days 3 and 7. The Doppler measurement also showed significantly increased blood flow immediately after the operation and on postoperative days 7 and 10. The number of blood vessels was significantly greater in the RLX group in the tissue harvested on postoperative day 10. The VEGF concentration was significantly higher in the RLX group than others in the tissues harvested on postoperative day 10. Conclusion: Following an analysis of the effects of relaxin-secreting adenovirus on the survival of a flap, the surviving area of the flap and the blood flow also increased. A histopathology also showed an increase in the number of blood vessels and the concentration of VEGF.

• Pediatric Infection and Vaccine
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• v.10 no.1
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• pp.87-94
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• 2003

Purpose : Respiratory viruses are one of the most infectious agent in human. Acute lower respiratory tract infection(ALRTIs) is associated with significant morbidity and mortality in children. This study is performed to investigate the etiologic organism, age and sex distribution, clinical manifestations and seasonal occurrence of ALRTIs in children. Methods : Viral agent was evaluated with nasopharyngeal aspirates, rhinorrhea and saliva collected from 568 patients. We confirmed viral agents in 54 patients who were younger than 15 year old. They had visited Maryknoll Hospital, Busan in Korea from January, 2002 to December, 2002 for ALRTIs. Results : The viral pathogens identified were Influenza A virus(59.3%), Enterovirus(33.3%), Adenovirus(5.6%), and Influenza B virus(1.9%). Parainfluenza virus and Respiratory syncytial virus were not detected. The occurrence of acute lower respiratory infections was high between 3 & 6 years old. The clinical patterns include pneumonia(51.9%), bronchitis(31.5%), croup(9.3%), bronchiolitis(7.4%). The respiratory viral agents had their characteristic seasonal patterns. Conclusion : Influenza A virus was the most common cause of acute lower respiratory tract infections in Busan area during the 2002. ALRTIs had high occurrence between 3 to 6 years old. And the most common clinical patterns were pneumonia and bronchitis.

• Journal of Life Science
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• v.27 no.3
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• pp.275-282
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• 2017

Adenovirus (ADV) and human bocavirus (hBoV) cause acute respiratory tract infections, and are often associated with increased rates of hospitalization and death, particularly in infants and young children. The aim of this study was to analyze the clinical features and molecular phylogeny of ADV and hBoV isolated in Busan, from January 2011 to November 2013. In total, 3,230 specimens (throat swabs) were collected from patients with influenza-like illnesses and acute respiratory tract infections. Multiplex real-time RT-PCR was performed to detect eight respiratory viru [rhinovirus, adenovirus, respiratory syncytial virus, human coronavirus, human metapneumovirus, human bocavirus, parainfluenza virus and influenza virus] and detected 1,485(46.0%) cases. Among 1,485 positive specimens, 257(8.0%) cases were ADV and 68(2.1%) cases of hBoV. A significant clinical feature of ADV is fever and headache whereas hBoV is wheezing. Serotypic distributions of isolated ADV and hBoV were analyzed by sequencing of hexon and VP1/VP2 gene, respectively. ADV was identified seven different serotypes(1~6, 8), revealing a high similarity among the isolates (>97%). The predominant types of ADV were type 1 in 2011, type 3 and 4 in 2012, type 3 in 2013, respectively. ADV type 3 was major causative type during outbreaks in 2013. All of the hBoV was identified as hBoV type 1.

• Pediatric Infection and Vaccine
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• v.3 no.2
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• pp.145-153
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• 1996

The incidence of Adenovirus(AV) pneumonia in children is estimated at 8%. Long-term complications reported with type 7 include hyperleucent lung syndrome, bronchiolitis obliterans and may require several months to recover. We reviewed 8 case of AV pnemonia confirmed by AV culture and sero-diagnosis among selected 19 AV pneumonia suspicious cases who were admitted to the Sanggye Paik Hospital of Inje University due to clinical pneumonia from May to July 1996. From 19 children, nasal aspirates or trachial aspirates were collected: viral agents were detected by virus isolation and virus antigen was detected by indirect immunofluorescent staining. Serologic diagnosis for type-specific AV antibody was done by neutralisation test. The results are as follows: 1) Age of the patients ranged from 6 months to 47 months with a mean of $24{\pm}9$ months. 6 were male and 2 were female(M:F= 3:1). 2) Monthly distribution showed 4 cases on June, 2 cases on May and July, respectively. 3) The mean duration of admission was 20.4 days and mean duration of fever was 11.3 days 4) Peripheral leukocyte counts were elevated in 4 cases(50%) and findings of shift to left were showed in 4 cases(50%) and serum aminotransferase activities were elevated in 6 case(75%). 5) Radiologic findings include linear and streaky infiltration(1/8 cases), patchy and lobar consolidation (7/8 cases), pleural effusion(2/8 cases) and hyperleucency was seen in 3/8 cases at time of admission and increased to 6/8 cases on hospital discharge. 6) The AV serotypes were type 7, type 3, and type 1. 7) Oxygen supply was done in 4 cases(50%) and all 8 patients include 2 cases of type 7 treated with artificial ventilator were survived. In conclusion, the serotypes of AV pneumonia outbreaks in early summer of 1996 were type 7, 3, 1 and, as showed severe clinical course, continued follow up surveillance is necessary to the long term pulmonary complications.

• Pediatric Infection and Vaccine
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• v.16 no.2
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• pp.199-204
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• 2009

Purpose : Various proteins encoded in the early region 3 (E3) of adenoviruses protect cells from being killed by cytotoxic T cells and death-inducing cytokines. We sought to find out whether the genetic heterogeneity of the E3 gene might contribute to the molecular diversity of adenoviruses. Methods : Sequences in the E3 region were analyzed for 14 adenovirus type 3 (Ad3) strains that were isolated from children with lower respiratory tract infections in the Seoul National University Children's Hospital during the period 1991-2000. Full-length adenoviral DNA was purified from the infected A549 cell lysates using a modified Hirt procedure. Results : There was 98% homology between 14 Korean Ad3 strains with a reference strain (M15952). Homology within the Korean Ad3 strains was 98.7%. Variation was found in the region of transcripts 20.1 kDa, 20.6 kDa, truncated 7.7 kDa, 10.3 kDa, 14.9 kDa, and 15.3 kDa. In particular, all 14 Korean strains showed a missense single point mutation at the start codon of the truncated 7.7 kDa. In addition, a deletion was found in the truncated 7.7 kDa region by 58 base pairs in 10 strains and 94 base pairs in 4 strains. Variations in amino acids were observed in the receptor internalization and degradation complex (10.3 kDa/14.9 kDa) which stimulates the clearance from the cell surface and subsequent degradation of the receptors for the Fas ligand and TRAIL, while no variations were observed in another immunoregulatory transcript, 19 kDa. Conclusion : Sequence analysis of the immunoregulatory region of adenovirus E3 shows that genetic heterogeneities are related to genome type patterns.

• Journal of Life Science
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• v.14 no.4
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• pp.620-626
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• 2004

Viruses causing acute conjuntivitis were isolated from 675 patients carrying eye infections for year 2001 to 2003 in Busan reagion and their antigenic properties characterized by a serological survey. In 2001, adenoviruses (serotype 8) were found in 5 of 48 cases. In 2002, the isolated viruses were 7 adenoviruses (serotype 8 and 37), 8 coxsakieviruses (serotype A24 and B3) and 1 echoviruses (serotype 6) from 324 specimens that are known as the causative agents of acute hemorrhagic conjuctivitis (AHC). In 2003, 25 case of 303 specimens were 7 adenoviruses (serotype 3, 4, 8 and 37), 7 echoviruses (serotype 6 and 7) and 4 untypable enteroviruses. Although coxsakievirus (serotype B3) and echoviruses (serotype 6 and 7) were generally known as causative agent of aseptic meningitis, it hasn't been reported until now that they were isolated from the conjunctival swabs. The out break of AC was observed from April to October in Busan. These isolated viruses showed a strong cytophatic effects on HEp-2, RD, Vero and BGM cell strains. Analysis of electron micrograph of those viruses showed that adenovirus consists of a 80 nm diameter and nonenvloped icosahedron and then echovirus and coxsackievirus were small nonenveloped and isometric-shaped viruses. Adenovirus showing a cytophatic effect was resulted in a 458 bp single band by PCR and echovirus, coxsackievirus and untypable enterovirus were detected a 437 bp products by RT-PCR.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.31 no.4
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• pp.306-311
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• 2005

Despite advances in surgery, radiotherapy, and chemotherapy, the survival of patients with oral squamous cell carcinoma has not significantly improved over the past several decades. Gene therapy is currently under investigation and shows us new possibility of cancer curing method. This experiment was undergone to find out the cell growth inhibition effect and evidence of apoptosis by HCCS-1(human cervical cancer suppressor-1), one of the candidates of tumor suppressor gene, transducted to human oral cancer cell line. To determine the efficiency of the adenovirus as a gene delivery vector cell line was transducted with LacZ gene and analysed with X-gal staining. Northern blot was performed to confirm the transfection with HSCC-1 gene and cell viability was assessed by cell cytotoxicity assay using cell count kit(CCK). To show the evidence of apoptosis, DNA fragmentation assay and flow cytometry(FACS) were performed. We had successfully construct the recombinant HSCC-1 adenovirus(Ad5CMV-HCCS-1), and importation efficiency was 20% at 2 MOI(multiplicity of infection), 80% at 20 MOI. Northern blot analysis showed that a single 0.6kb mRNA transcript was expressed in Ad5CMV-HCCS-1 transducted cell lines. As a result of CCK, when comparing to control subjects, transducted group showed 50% growth inhibition. In DNA fragmentation assay, according to increasing of MOI, DNA volume was diminished. In FACS analysis, DNA distribution showed fragmentation. This results imply that HCCS-1gene has growth inhibition effect in human oral cancer cell lines through apoptosis induction.