Abstract
We have developed a three-phase screening procedure aimed at identifying KD-associated viral cDNA. In the first phase suppression subtractive hybridization (SSH) was carried out using cDNA which was reverse-transcribed from pooled RNA from PBMNCs of 6 acute phase KD patients as the tester, and cDNA generated from pooled RNA from 6 non-KD febrile patients as the driver. In the second phase the subtracted cDNA sequences were inserted into a plasmid expression vector (pBAD), and an E. coli library was prepared. The expression library was screened using pooled convalescent KD sera as the primary antibody which was incubated with protein A or protein G alkaline phosphatase conjugates designed to identify the immunopositive clones. In the third phase the immunopositive clones were PCR amplified using primers based on flanking vector sequences and the recombinant inserts were sequenced. Molecular analysis of the immunopositive clones yielded 2 sequences: an 800 bp fragment identical to a portion of a known human gene and a 3400 bp PCR product which had no significant homology to known sequences using standard BLASTN or BLASTX database searches. However, using position specific iterated (PSI) BLAST search,the 3400 bp insert showed extensive but low-level homology (25-33% identities and 28-45% positives) throughout its entire length to gene pB602L of the African swine fever virus (ASFV), a lymphotropic virus which replicates in monocyte-macrophage lineage cells. Experiments are in progress to test the association of the novel virus with the course of KD by measuring the relative virus load in samples from acute, subacute and convalescent samples.
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Peters, J., Kanamaru, H., Ayusawa, M. et al. Identification of a Novel Virus-specific cDNA derived from Peripheral Blood Mononuclear Cells of Acute Phase Kawasaki Disease (KD) Patients. Pediatr Res 53, 168 (2003). https://doi.org/10.1203/00006450-200301000-00090
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DOI: https://doi.org/10.1203/00006450-200301000-00090