Saffold virus type 3 (SAFV-3) persists in HeLa cells.

Saffold virus (SAFV) was identified as a human cardiovirus in 2007. Although several epidemiological studies have been reported, they have failed to provide a clear picture of the relationship between SAFV and human diseases. SAFV genotype 3 has been isolated from the cerebrospinal fluid specimen of patient with aseptic meningitis. This finding is of interest since Theiler's murine encephalomyelitis virus (TMEV), which is the closely related virus, is known to cause a multiple sclerosis-like syndrome in mice. TMEV persistently infects in mouse macrophage cells in vivo and in vitro, and the viral persistence is essential in TMEV-induced demyelinating disease. The precise mechanism(s) of SAFV infection still remain unclear. In order to clarify the SAFV pathogenicity, in the present study, we studied the possibilities of the in vitro persistent infection of SAFV. The two distinct phenotypes of HeLa cells, HeLa-N and HeLa-R, were identified. In these cells, the type of SAFV-3 infection was clearly different. HeLa-N cells were lyticly infected with SAFV-3 and the host suitable for the efficient growth. On the other hand, HeLa-R cells were persistently infected with SAFV-3. In addition, the SAFV persistence in HeLa-R cells is independent of type I IFN response of host cells although the TMEV persistence in mouse macrophage cells depends on the response. Furthermore, it was suggested that SAFV persistence may be influenced by the expression of receptor(s) for SAFV infection on the host cells. The present findings on SAFV persistence will provide the important information to encourage the research of SAFV pathogenicity.

Adaptation of Saffold virus 2 for high-titer growth in mammalian cells.

Saffold viruses (SAFV) are a recently discovered group of human Cardioviruses closely related to Theiler's murine encephalomyelitis viruses (TMEV). Unlike TMEV and encephalomyocarditis virus, each of which is monotypic, SAFV are genetically diverse and include at least eight genotypes. To date, only Saffold virus 3 (SAFV-3) has been grown efficiently in mammalian cells in vitro. Here, we report the successful adaptation of SAFV-2 for efficient growth in HeLa cells after 13 passages in the alpha/beta interferon-deficient human glial cell line U118 MG. Nine amino acid changes were found in the adapted virus, with single mutations in VP2, VP3, and 2B, while 6 mutations arose in VP1. Most capsid mutations were in surface loops. Analysis of SAFV-2 revealed virus growth and cytopathic effect only in human cell lines, with large plaques forming in HeLa cells, with minimal cell association, and without using sialic acid to enter cells. Despite the limited growth of SAFV-2 in rodent cells in vitro, BALB/c mice inoculated with SAFV-2 showed antibody titers of >1:10(6), and fluorescence-activated cell sorting (FACS) analysis revealed only minimal cross-reactivity with SFV-3. Intracerebral inoculation of 6-week-old FVB/n mice produced paralysis and acute neuropathological changes, including meningeal infiltrates, encephalitis, particularly of the limbic system, and spinal cord white matter inflammation.

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

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.