PURPOSE: Gene therapy (ex vivo) has recently been used as a means of delivering bone morphogenetic proteins (BMPs) to sites of tissue regeneration. In the present study, we investigated the effect of co-transduction of adenoviruses expressing BMP-2 and BMP-7 on osteogenesisof C2C12 cells in vitro. METHODS: A replication-defective human adenovirus 5 (Ad5) containing a cDNA for BMPs in the E1 region of the virus (Ad5BMP-2 and Ad5BMP-7) was constructed by in vivo homologous recombination. Functional activity of Ad5BMP-2 and Ad5BMP-7 were evaluated in mouse stromal cells (W20-17cells). C2C12 cells are transduced with various MOI (multiplicity of infection) of Ad5BMP-2 and Ad5BMP-7 to assess most effective and stable titer. Based on this result, C2C12 cells were transduced with Ad5BMP-2 and Ad5BMP-7 alone or by combination. BMPs expression, alkaline phosphatase (ALPase) activity, cell proliferation, and mineralization were assessed. RESULTS: Ad5BMP-2 and Ad5BMP-7 are successfully transduced to W20-17 cells, and secreted BMPs stimulated cell differentiation. Also, C2C12 cells transduced with Ad5BMPs showed expression of BMPs and increased ALPaseactivity. In all groups, cell proliferation was observed over times. At 7days, cells co-transduced with Ad5BMP-2 and Ad5BMP-7 showed lower proliferation than the others. C2C12 cells co-transduced with Ad5BMP-2 and Ad5BMP-7 had greater ALPaseactivity than that would be predicted if effect of individual Ad5BMPs were additive. Little mineralized nodule formation was detected in cells transduced with individual Ad5BMPs. In contrast, Ad5BMP-2 and Ad5BMP-7 combination stimulated mineralization after culturing for 10 days in mineralizing medium. CONCLUSIONS: Present study demonstrated that adenoviruses expressing BMPs gene successfully produced BMPs protein and these BMPs stimulated cells to be differentiated into osteoblastic cells. In addition, the osteogenic activity of Ad5BMPs can be synergistically increased by co-transduction of cells with Ad5BMP-2 and Ad5BMP-7.
Adenoviridae ; Adenoviruses, Human ; Alkaline Phosphatase ; Animals ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Protein 7 ; Bone Morphogenetic Proteins ; Cell Differentiation ; Cell Proliferation ; DNA, Complementary ; Durapatite ; Genetic Therapy ; Homologous Recombination ; Mice ; Osteoblasts ; Osteogenesis ; Regeneration ; Stromal Cells ; Viruses

OBJECTIVE: Adeno associated virus (AAV) is a human DNA virus and is included in the Parvovirus family. AAV has been detected in cervical tissues as well as cervical cancer cell lines. Previous studies showed that AAV infection has some negative effects on HPV infection and that the cervical cancer cell growth is inhibited by AAV infection. The aim of this study is to determine the prevalence of AAV 2 infection and its possible roles for influencing HPV 16 and 18 infection in Korean women by analyzing adjacent normal, CIN, and invasive cervical cancer tissue samples. METHODS: CIN I (20), CIN II (24), CIN III (25), invasive cervical cancer (23) tissues were investigated by microdissection and PCR analyses using primers of HPV 16, 18 and AAV 2 as well as beta- globin as an internal control. RESULTS: AAV 2 was detected in 57 out of 92 cervical lesion biopsies. Among these, mild dysplasia, moderate dysplasia, severe dysplasia and invasive cancer showed 55% (11/20), 95.8% (23/24), 52% (13/25) and 52.2% (12/23), respectively. However, HPV 16 was detected in 14 out of 92 cervical lesion biopsies. Among these, mild dysplasia, moderate dysplasia, severe dysplasia and invasive cancer showed 0% (0/20), 8.3% (2/24), 24% (6/25) and 26.1% (6/23), respectively. HPV 18 was detected in 3 out of 92 cervical lesion biopsies. Among these, mild dysplasia, moderate dysplasia, severe dysplasia and invasive cancer showed 0% (0/20), 4.2% (1/24), 8% (2/25) and 0% (0/23), respectively. In contrast, In 92 perilesional normal biopsies, AAV 2, HPV 16 and HPV 18 were detected to be 57.6% (53/92), 3.3% (3/92) and 0% (0/92), respectively. CONCLUSION: AAV 2 was detected in CIN and invasive cervical cancer biopsies by microdissection and PCR analyses in Korean women. It is difficult to confirm any significant roles of AAV 2 infection for developing cervical cancer. However, we observe that there is some correlation between AAV 2 and HPV infection in the carcinogenesis of cervical cancer. Further research remains to be done to further elucidate AAV 2 infection and its role for HPV infection and cervical cancer.
Biopsy* ; Carcinogenesis ; Cell Line ; Dependovirus* ; DNA Viruses ; Female ; Globins ; Human papillomavirus 16 ; Human papillomavirus 18 ; Humans* ; Microdissection* ; Papilloma* ; Parvovirus ; Polymerase Chain Reaction ; Prevalence ; Satellite Viruses ; Uterine Cervical Neoplasms

To construct a recombinant replication-defective human adenovirus type 5 expressing Cap protein of PCV2 and test the immunological efficacy in mice. In this study, the recombinant replication-defective human adenovirus type 5, named as rAd5-Cap (wt-rAd5), was constructed through homologous recombination internally in the HEK293AD cells after co-transfection of the Pac I-linearized backbone plasmid and the shuttle plasmid pacAd5CMV-Cap containing the open reading frame (ORF2) of the porcine circovirus type 2 (PCV2) cap protein or pacAd5CMV without inserted fragment. Furthermore, the rAd5-Cap could induce the expression of PCV2 cap protein in the HEK293AD cells with high efficacy evaluated by the RT-PCR and indirect immunofluorescence assay (IFA). The virus titer of rAd5-Cap could reach up to 10(8.5) TCID50/mL similarly to that of wt-rAd5, indicating that there was little affect on the virus proliferation after the insertion of PCV2 cap protein gene. The humeral immune responses could be activated and detected 14 days after the inoculation of the mice with 10(7) TCID50 rAd5-Cap intramuscularly, and constantly in crease in another 14 days. These molecular biological and animal experiments results demonstrated that the PCV2 cap protein could be efficiently expressed by the recombinant adenovirus rAd5-Cap in eukaryotic cells and induce robust immune responses in mice, which laid a good foundation for the development of new type vaccine against porcine circovirus.
Adenoviruses, Human ; genetics ; Animals ; Antibodies, Viral ; blood ; Capsid Proteins ; genetics ; immunology ; Circovirus ; immunology ; Defective Viruses ; genetics ; HEK293 Cells ; Humans ; Mice ; Recombinant Proteins ; biosynthesis ; immunology ; Virus Replication

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by the human polyomavirus JC virus. PML mainly occurs in immunocompromised patients. A 36-year-old man with no evidence of immunosuppresion presented seizures. MRI scans of brain showed multifocal lesions in the cerebral white matters. JC virus DNA was positive in the cerebrospinal fluid examined by JC virus PCR. We report a rare case of PML presenting as viral encephalitis that occurred in a healthy adult person.
Adult ; Brain ; Central Nervous System ; Demyelinating Diseases ; DNA ; Encephalitis, Viral ; Humans ; Immunocompromised Host ; JC Virus ; Leukoencephalopathy, Progressive Multifocal ; Magnetic Resonance Imaging ; Polymerase Chain Reaction ; Seizures ; Viruses

Infectious complications have been considered as a major cause of morbidity and mortality after kidney transplantation, especially in the Asian population. Therefore, prevention, early detection, and prompt treatment of such infections are crucial in kidney transplant recipients. Among all infectious complications, viruses are considered to be the most common agents because of their abundance, infectivity, and latency ability. Herpes simplex virus, varicella zoster virus, Epstein–Barr virus, cytomegalovirus, hepatitis B virus, BK polyomavirus, and adenovirus are well-known etiologic agents of viral infections in kidney transplant patients worldwide because of their wide range of distribution. As DNA viruses, they are able to reactivate after affected patients receive immunosuppressive agents. These DNA viruses can cause systemic diseases or allograft dysfunction, especially in the first six months after transplantation. Pretransplant evaluation and immunization as well as appropriate prophylaxis and preemptive approaches after transplant have been established in the guidelines and are used effectively to reduce the incidence of these viral infections. This review will describe the etiology, diagnosis, prevention, and treatment of viral infections that commonly affect kidney transplant recipients.
Adenoviridae ; Allografts ; Asia ; Asian Continental Ancestry Group ; BK Virus ; Cytomegalovirus ; Diagnosis ; DNA Viruses ; Hepatitis ; Hepatitis B virus ; Herpesvirus 3, Human ; Humans ; Immunization ; Immunosuppression ; Immunosuppressive Agents ; Incidence ; Kidney Transplantation ; Kidney* ; Mortality ; Simplexvirus ; Transplant Recipients* ; Virus Diseases

OBJECTIVETo construct a recombinant adenovirus vector expressing hCD40L gene and explore it in the use of anti-tumor gene therapy.

METHODS1,900 bp gene fragment was obtained form plasmid pORF-hCD40L by Xho I/Swa I cutting and then cloned directionally into the pShuttle plasmid, finally, the resultant plasmid was digested by restriction endonnuclease PmeI and subsequently cotransformtion into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant and then the recombinant was packaged in the 293 cells. Some methods such as PCR and endonulease digestion were employed to identify the recombinant adenovirus.

RESULTSThe evidences of endonulease digestion and PCR analysis confirmed that recombinant hCD40L gene was correctly inserted into adenovirus vector.

CONCLUSIONThe adenoviral vector which expressed hCD40L gene was constructed. It provides an experimental basis for studies on it expression in the mammalian cells and in tumor gene therapy.

Apoptosis is a host defense mechanism that the cell uses to limit production of infectious virus. Although many viruses can induce apoptosis in infected cells, large DNA viruses, such as poxviruses, herpesviruses and adenoviruses, usually exhibit the ability to suppress the induction of apoptosis in the infected cells. Several publications have attested to the ability of herpesviruses to protect cells against apoptosis. We investigated the ability of the virus to protect cells in continuous cultivation from apoptosis induced by the virus itself. The gamma herpesvirus alcelaphine herpesvirus 1 (AlHV-1) has been shown to harbor genes with antiapoptotic potentialities. However, here we have demonstrated that productive infection of adherent, permissive cell lines by AlHV-1 resulted in a cytopathic effect characterized by induction of apoptosis. This phenomenon was confirmed using different techniques to detect apoptosis and using different virus strains and cell lines. Therefore, despite the presence of antiapoptotic genes in its genome, AlHV-1 could complete its cycle of productive infection while inducing apoptosis of infected cells. This finding might have implications for the pathobiology of AlHV-1 and other gamma herpesviruses in vivo.
Adenoviridae ; Apoptosis ; Cell Line* ; DNA Viruses ; Genome ; Herpesviridae ; Poxviridae

The aim of this study was to investigate viral etiology in dilated cardiomyopathy (DCM) by polymerase chain reaction (PCR) or nested reverse tanscription PCR (RT-PCR), and characterize the enteroviral RNA presented in the clinical specimens. Twenty-eight paraffin-embedded heart tissue samples were assayed to detect cytomegalovirus, herpes simplex virus type 1, type 2, parvovirus, adenovirus, and enterovirus (EV) with each specific primer. Of these 28 patients (mean age: 27, M: 24, F: 4), 26 were histologically diagnosed as DCM and 2 as myocardial infarction (MI). Nested RT-PCR detected enteroviral RNA in 7 (26.9%) of 26 patients with DCM, and none of patients with MI. And none of DNA viruses tested were detected from the samples. Amplified products were also genotyped by single-variation of EV is present in the explanted heart tissues from patients with DCM. Although most of the sequences among the wild isolates have the greatest similarity to those of coxsackievirus B3, there are specific regions of variable sequences (no 490 - no 510). The data suggest that enterovirus may be a major viral pathogen for the DCM in Korea and nucleotide sequence data indicate that coxsackievirus B3 may be a leading etiologic agent of DCM.
Adenoviridae ; Base Sequence ; Cardiomyopathy, Dilated* ; Cytomegalovirus ; DNA Viruses ; Enterovirus ; Heart* ; Herpesvirus 1, Human ; Humans ; Korea ; Myocardial Infarction ; Parvovirus ; Polymerase Chain Reaction ; RNA*

BACKGROUND: Herpes simplex virus (HSV) and varicella zoster virus (VZV) are two of the most common causes of mucocutaneous vesicular eruptions. Their diagnoses are usually made clinically, but the clinical distinction between HSV and VZV is sometimes difficult. OBJECTIVE: We investigated the usefulness of polymerase chain reaction (PCR) and Tzanck test in patients with HSV and VZV infections. METHODS: From June 2008 through June 2010, a total of 396 patients (53 patients with HSV and 343 patients with VZV) were included in this study. Wright-stained smears of scrapings from the base of skin lesions and viral DNA amplification by PCR were examined in all patients. We compared the positivity rates of Tzanck test and PCR according to virus type and duration of skin lesions. RESULTS: The overall positivity rates of Tzanck test and PCR were 56.6% and 86.9%, respectively, and the difference between the positivity rates of the two tests was statistically significant (p<0.0001). The Tzanck test was positive in 41.5% and 58.9% of the HSV and VZV cases, respectively, and the difference was statistically significant (p=0.0259). For PCR, there were no significant differences between the HSV and VZV cases. The positivity rate of the Tzanck test decreased in the old skin lesions over 7 days; however, PCR method showed no significant differences in positivity rates according to duration of the skin lesions. CONCLUSION: PCR is a more sensitive method for the diagnoses of HSV and VZV infections, and it can be utilized for diagnosis even of old skin lesions.
Chickenpox ; DNA, Viral ; Herpes Simplex ; Herpesvirus 3, Human ; Humans ; Methylmethacrylates ; Polymerase Chain Reaction ; Polystyrenes ; Simplexvirus ; Skin ; Viruses

Herpes simplex virus (HSV) is one of Herpesviridae family viruses which belong to DNA viruses. HSV-associated diseases are among the most widespread infections, affecting nearly 60% to 95% of human adults. Labial herpes typically results from infection with HSV type 1 (HSV-1), whereas most genital herpes is caused by HSV type 2 (HSV-2). They are incurable and persist during the lifetime of the host, often in latent form. Antiviral agents do not cure HSV infections, but rather modify the clinical course of the disease. Topical, oral, or intravenous antiviral agents may be used in the management of HSV infections. Acyclovir, valacyclovir hydrochloride, and famciclovir are the 3 antiviral drugs commonly used to treat symptomatic HSV infections. However, it is very difficult to choose an appropriate drug and dosing regimen.
2-Aminopurine ; Acyclovir ; Adult ; Antiviral Agents ; DNA Viruses ; Herpes Genitalis ; Herpes Simplex ; Herpesviridae ; Humans ; Methylmethacrylates ; Polystyrenes ; Simplexvirus ; Valine