The long-term objectives are a better understanding of the nature of human parvovirus B19 infection, the mechanisms by which B19 causes arthropathy, and those viral and host factors that modify disease expression. B19 causes a chronic rheumatoid-like arthritis in an estimated 12% of patients presenting to rheumatology clinics. It is not known whether arthropathy results from infection by specific arthritogenic strains of virus or from a unique response to wild type virus infection. Nor is it known whether B19 can cause latent or chronic infection in the immune competent host. As a first step in addressing these issues, we propose to study persistent B19 infection in chronic B19 arthropathy by using polymerase chain reaction (PCR) methodology to detect and analyze B19-specific DNA sequences amplified from tissues of patients with long-standing B19 arthropathy.
The specific aims are as follows: (1) To determine the presence and disease specificity of B19-specific DNA sequences in bone marrow and synovial tissues from patients with chronic B19 arthropathy; (2) To determine the fine structure of persistent B19 DNA and to identify any sequence variation unique to B19 persistence; and (3) To determine whether persistent B19 DNA is integrated into the host genome and, if integrated, to identify the extent and site(s) of integration.
In aim 1, B19-specific DNA from bone marrow and synovium from chronic B19 arthropathy patients will be amplified by PCR; tissues from normal donors and classic, rheumatoid arthritis patients will be controls.
In aim 2, double stranded symmetric PCR will be used to sequence amplified product obtained from chronic B19 arthropathy patients.
In aim 3, inverse PCR of circularized genomic fragments from restriction endonuclease digestion will be used to characterize host sequences flanking integrated B19 DNA. This research will help test the following hypotheses: (a) Persistence of B19 DNA in patient tissues is associated with chronic B19 arthropathy; (b) Genetic variation in B19 allows persistent virus to escape immune surveillance; (c) B19 viral DNA may persist even in the absence of productive virus infection; (d) B19 DNA integration into the host genome may allow persistence; (e) Integration of B19 DNA into the host genome may confer pathogenicity on the persistent B19 DNA; (f) The site of integration may play an important role in the pathogenicity and chronicity of B19 infection. Demonstrations of B19 latency in the immune competent host will have broad implications for models of viral arthritis.
