Jpn.J.Infect.Dis., 52, 1999

Laboratory and Epidemiology Communications

Detection of Adeno-associated Virus Type 2 in Patients with Viral Infection

Yuko Sato1, Yasuko Asahi1, Takuya Iwasaki1, Toshihiko Matsukura2, Takeshi Kurata1 and Tetsutaro Sata1, 3, *

1Department of Pathology, 2Virology II, and 3Laboratory of Pathology, AIDS Research Center, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo

Communicated by Hiroshi Yoshikura

(Accepted May 6, 1999)

Adeno-associated virus (AAV) is a member of the Dependovirus genus in the Parvovirinae family. Among 5 known AAV serotypes, AAV-2, -3, and -5 infect humans, and AAV-1 and -4 infect monkeys. The AAV-2 genome is 4,679 bp long. It is a linear single stranded DNA with a negative sense polarity. It has two open reading frames, Rep (replication) and Cap (capsid; VP1-3). AAV replicates within the nucleus of the infected cells when a helper virus coinfects the cells. In some instances, in the absence of helper virus, the AAV genome is integrated as a single copy in the specific site of the long arm of chromosome 19 (1).

Adenovirus, herpes simplex virus type 1 or 2, cytomegalovirus, vaccinia virus, and, more recently, human papillomavirus (HPV) have been found to serve as a helper virus (2). About 80% of the normal population has been found seropositive for the AAV antibody (3). AAV infects humans horizontally, but no known diseases have yet been associated with AAV. However, the AAV genome has been frequently detected in cervical tissues, particularly in HPV-infected pathological ones (4, 5, 6). We surveyed pathological tissue specimens for the presence of AAV antigens and genomes, using immunohistochemistry and PCR, respectively.

Thirty-six cases of congenital or acquired infections by adenovirus, varicella-zoster virus (VZV), herpes simplex virus (HSV) type 1 or 2, cytomegalovirus (CMV), or parvovirus B19 were examined for AAV antigens and genomes in various visceral tissues, such as lung, liver, kidney, appendix, spleen, etc. The tissue specimens examined were all formalin-fixed and paraffin-embedded sections. The deparaffinized sections were first treated with 0.3% H2O2-methanol to inactivate any endogenous peroxidase activity. After blocking with normal goat serum for 30 min at 37 C, the sections were incubated overnight at 4 C with anti-AAV-Cap protein (VP1-3) monoclonal antibody (Progen, Heidelberg, Germany). The virus antigen was detected by using an LSAB kit (DAKO, Kyoto). One lung specimen from an infant was positive for the AAV antigen (Fig. 1). This patient was suspected of adenovirus infection, but none of adenovirus, HSV, VZV, EBV (Epstein-Barr virus), HHV6 (human herpesvirus 6), or parvovirus B19 antigens were detected.

Peripheral blood mononuclear cells (PBMC) from 210 cases of various virus infections, and cervical tissues from 96 HPV-positive cervical specimens were examined for AAV genomes by nested PCR (See Table 1 for primer pairs). The PCR amplification protocol was 30 cycles of 94 C for 30 sec, 58 C for 1 min, and 72 C for 1 min. The products were electrophoresed in 2% agarose gels. The band with the expected size (157 bp), if it appeared, was confirmed for the AAV genome by sequencing the nt 1512-1669 region of the AAV-2 genome.

Three PBMC specimens were positive for AAV DNA. PCR assay determined them EBV-positive but CMV-negative. HIV cases (96 cases) were all negative for AAV. In contrast, among 96 cervical specimens consisting of 24 cases each of CIN 1, 2, and 3, and cervical cancer, AAV was detected in six CIN 1 cases (25 %), two CIN 2 cases (8 %), five CIN 3 cases (21 %), and 15 cervical cancer cases (63 %).

The high frequency of the AAV genome in the HPV-associated cancerous cervix tissues is worth noting. In the PBMC specimens, AAV was associated with EBV but not with HIV. Our observation may suggest the presence of co-operative interaction between AAV and HPV or EBV infections.

This work was supported in part by a grant-in-aid from Research on Human Genome and Gene Therapy, Ministry of Health and Welfare, Japan.


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* Corresponding author: E-mail:, Fax: +81-3-5285-1189

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