Serological evidence of human klassevirus infection

Abstract

Klassevirus is a proposed new genus of picornavirus that has been associated with pediatric diarrhea. In this study, we used recombinant klassevirus 3C protease as the capture antigen for an indirect serological enzyme-linked immunosorbent assay (ELISA). Four of six klassevirus reverse transcription (RT)-PCR-positive individuals demonstrated seroconversion against the 3C protease, suggesting that klassevirus infection and replication occur in humans. Additional screening of 353 samples from an age-banded serological cohort from two St. Louis hospitals indicated a seroprevalence of 6.8%.

FIG. 1.

Purification of klassevirus 3C protease. N-terminal His-tagged klassevirus 3C protease was expressed in E. coli and purified by Ni-NTA chromatography (A and B). Ni-NTA eluates were further purified by gel filtration chromatography using a Superdex 200 column (C). The 91-ml fraction was tested via silver stain to confirm purity of klassevirus 3C protease (D). Lane M, molecular mass markers.

FIG. 2.

Klassevirus 3C ELISA for matched serum samples from klassevirus PCR-positive children from India. Endpoint OD₄₅₀ values for matched preinfection serum samples (1:100 dilution; white) and postinfection serum samples (1:100 dilution; shaded) are grouped together, along with polyclonal rabbit antisera generated against klassevirus 3C and blocking buffer (A). The dotted line indicates an OD₄₅₀ cutoff value of 0.30 that separates pre- and postinfection OD₄₅₀ values. Asterisks indicate significant increases in OD₄₅₀ (P < 0.005). Remaining serum was available from two matched serum samples (2246 and 3516) for Western confirmation, which showed a 4-fold and 8-fold increase in signal between pre- and post-klassevirus infection serum samples at a dilution of 1:200, respectively (B).

FIG. 3.

Klassevirus 3C ELISA for 353 age-banded samples from St. Louis Children's Hospital and Barnes-Jewish Hospital. Approximately 30 samples for each age band were included. A specific OD₄₅₀ cutoff value of 0.30 based on the matched Indian serum in Fig. 2 suggested 24 (6.8%) of the serum samples were seropositive for klassevirus.