Cultivation and serological characterization of a human Theiler's-like cardiovirus associated with diarrheal disease

Abstract

Cardioviruses (e.g., Theiler's murine encephalomyelitis virus [TMEV]) are members of the Picornaviridae family that cause myocarditis and encephalitis in rodents. Recently, several studies have identified human cardioviruses, including Saffold virus (SAFV) and a related virus named human TMEV-like cardiovirus (HTCV). At least eight cardiovirus genotypes are now recognized, with SAFV and most strains of HTCV belonging to genotypes 1 and 2, respectively; genotype 2 strains are the most common in the population. Although a genotype 3 cardiovirus has recently been cultured (SAFV-3), the genotype 1 and 2 cardioviruses have been difficult to propagate in vitro, hindering efforts to understand their seroprevalence and pathogenicity. Here we present the isolation and characterization of a genotype 2 human cardiovirus (HTCV-UC6). Notably, successful cultivation of HTCV-UC6 from stool required the addition of cytokine-blocking antibodies to interrupt downstream antiviral pathways. Unlike SAFV-3, HTCV-UC6 exhibited slow replication kinetics and demonstrated only a moderate cytopathic effect. Serologic assays revealed that 91% of U.S. adults carry antibodies to the genotype 2 cardioviruses, of which 80% generate neutralizing antibodies, in agreement with previous data showing that cardiovirus infection is widespread in humans. We also demonstrate an acute cardiovirus seroconversion event in a child with diarrhea and vomiting, thus reporting for the first time evidence linking cardiovirus infection to diarrheal disease in humans.

FIG. 1.

Replication of HTCV-UC6 in an LLC-MK2 cell line. (A) Growth kinetics of HTCV-UC6 (stool filtrate) over 10 days with the addition of an anti-IFN-α antibody (triangles), an anti-TNF-α antibody (circles), both antibodies (diamonds), or no antibody (squares) to the culture medium. Viral titers are calculated as viral genome equivalents/ml. (B) A moderate cytopathic effect (CPE) was first visible at day 7 after infection with HTCV-UC6 and after addition of anti-IFN-α/anti-TNF-α antibodies to the culture medium. After infection with HTCV-UC6, a similar CPE was observed with the addition of the anti-IFN-α or anti-TNF-α antibody alone (data not shown).

FIG. 2.

Northern blot analysis of extracted RNA from virus-infected cells. Radioactive Northern blot analysis was performed using HTCV-UC6-infected LLC-MK2 cells, poliovirus-infected HeLa cells, and HTCV-UC1 in vitro-transcribed (IVT) genomic mRNA with cardiovirus- or poliovirus-specific probes to positive-strand (A) or negative-strand (B) RNA. β-Actin served as the loading control. Lane 6, marked (−), refers to the control uninfected LLC-MK2 cell line.

FIG. 3.

Effects of anti-cytokine antibodies on the replication of singly passaged HTCV-UC6 in LLC-MK2 cells. (A) Growth kinetics of HTCV-UC6 (P1) over 10 days with the addition of an anti-IFN-α antibody (triangles), an anti-TNF-α antibody (circles), both antibodies (diamonds), or no antibody (squares) to the culture medium. (B) Direct immunofluorescence assay for detection of cardioviruses in cells infected with HTCV-UC6 and no antibodies (right) or HTCV-UC6 with the addition of anti-IFN-α/anti-TNF-α antibodies to the culture medium (left) at days 3, 7, and 10 postinfection.

FIG. 4.

Replication of HTCV-UC6 (P1) at various cell confluences. Viral stocks were inoculated into LLC-MK2 cells at cell confluences of 100%, 90%, 70%, 50%, 40%, 20%, and 5%. The y axis shows the viral titer (viral genome equivalents/ml) in the cell culture supernatant at day 10 postinfection.

FIG. 5.

Replication of HTCV-UC6 (P1) in human, monkey, and rodent cell lines. The y axis shows the viral titer (viral genome equivalents/ml) in the cell culture supernatant at day 10 postinfection.

FIG. 6.

Nucleotide similarity plots of full-length cardiovirus sequences using HTCV-UC6 as the query sequence. Analysis was performed using a 200-nucleotide sliding window and a step size of 20 nucleotides. Data show the percentages of nucleotide identity between each cardiovirus sequence and the HTCV-UC6 sequence.

FIG. 7.

Seroprevalence of neutralizing antibodies to HTCV-UC6 among adult blood donors. The dual-axis column chart shows the virus titers (viral genome equivalents/ml) at day 10 postinfection by a cardiovirus neutralization assay (blue lines, left axis) and the absorbances at 450 nm measured by a cardiovirus VP1 ELISA (red lines, right axis) for 57 random adult-donor serum samples and negative controls. Asterisks indicate serum samples for which there was overt CPE at day 10. The horizontal dashed line indicates the 3-log-unit cutoff threshold for virus neutralization.

FIG. 8.

Acute seroconversion to cardiovirus infection in a child with diarrhea and vomiting. (A) Cardiovirus neutralization assay. Shown are the growth kinetics of HTCV-UC6 (P1) in LLC-MK2 cells over 10 days after a 24-h incubation of the virus with acute-phase (left) and convalescent-phase (right) sera at dilutions of 1:20 (diamonds), 1:320 (squares), and 1:5,120 (triangles). (B) Cardiovirus VP1 ELISA. Shown are the measured absorbances at 450 nm (OD₄₅₀) for acute- and convalescent-phase sera at serial 2-fold dilutions from 1:20 to 1:5,120.