No abstract

The goal of this study was to determine how enteric viruses persist within shellfish tissues. Several lines of novel evidence show that phagocytic blood cells (hemocytes) of Eastern oysters (Crassostrea virginica) play an important role in the retention of virus particles. Our results demonstrated an association of virus contamination with hemocytes but not with hemolymph. Live oysters contaminated overnight with hepatitis A virus (HAV) and murine norovirus (MNV) had 56% and 80% of extractable virus associated with hemocytes, respectively. Transfer of HAV-contaminated hemocytes to naïve (virus-free) oysters resulted in naïve oyster meat testing HAV positive for up to 3 weeks. Acid tolerance of HAV, MNV, poliovirus (PV), and feline calicivirus (FCV) correlated with the ability of each virus to persist within oysters. Using reverse transcription-PCR (RT-PCR) to evaluate persistence of these viruses in oysters, we showed that HAV persisted the longest (>21 days) and was most acid resistant, MNV and PV were less tolerant of acidic pH, persisting for up to 12 days and 1 day, respectively, and FCV did not persist (<1 day) within oysters and was not acid tolerant. This suggests that the ability of a virus to tolerate the acidic conditions typical of phagolysosomal vesicles within hemocytes plays a role in determining virus persistence in shellfish. Evaluating oyster and hemocyte homogenates and live contaminated oysters as a prelude to developing improved viral RNA extraction methods, we found that viruses were extracted more expediently from hemocytes than from whole shellfish tissues and gave similar RT-PCR detection sensitivities.

A recent study (Ogushi, K., Wada, A., Niidome, T., Okuda, T., Llanes, R., Nakayama, M., Nishi, Y., Kurazono, H., Smith, K. D., Aderem, A., Moss, J., and Hirayama, T. (2004) J. Biol. Chem. 279, 12213-12219) concluded that gangliosides serve as co-receptors for flagellin signaling via toll-like receptor 5 (TLR5). In view of several findings in this study that were inconsistent with a role for gangliosides as co-receptors, we re-examined this important issue. Using TLR5-negative RAW 264.7 cells and a TLR5-enhanced yellow fluorescent protein chimera, we established an assay for specific binding of flagellin to cells. Inhibition of clatherin-mediated internalization of flagellin.TLR5-enhanced yellow fluorescent protein complexes did not impair flagellin activation of IRAK-1. Thus flagellin signal occurs at the cell surface and not intracellularly. Exogenous addition of mixed gangliosides (GM1, GD1a, and GT1b) as well as GD1a itself inhibited flagellin-induced interleukin-1 receptor-associated kinase activation as well as tumor necrosis factor alpha production in HeNC2, THP-1, and RAW 264.7 cells. Gangliosides inhibited flagellin signaling in the absence of an effect on flagellin binding to TLR5. Depletion of gangliosides in RAW 264.7 cells did not alter the concentration dependence or magnitude of flagellin signaling as measured by interleukin-1 receptor-associated kinase activation or tumor necrosis factor alpha production. Our findings are consistent with the conclusions that gangliosides are not essential co-receptors for flagellin and that the inhibitory effect of gangliosides is mediated by at least one mechanism that is distinct from any effect on the binding of flagellin to TLR5.

No abstract

The family Arenaviridae includes a number of highly pathogenic viruses that are responsible for acute hemorrhagic fevers in humans. Genetic diversity among arenavirus species in their respective rodent hosts supports the continued emergence of new pathogens. In the absence of available vaccines or therapeutic agents, the hemorrhagic fever arenaviruses remain a serious public health and biodefense concern. Arenaviruses are enveloped virions that assemble and bud from the plasma membrane. In this study, we have characterized the microdomain organization of the virus envelope glycoprotein (GPC) on the cell surface by using immunogold electron microscopy. We find that Junín virus (JUNV) GPC clusters into discrete microdomains of 120 to 160 nm in diameter and that this property of GPC is independent of its myristoylation and of coexpression with the virus matrix protein Z. In cells infected with the Candid#1 strain of JUNV, and in purified Candid#1 virions, these GPC microdomains are soluble in cold Triton X-100 detergent and are thus distinct from conventional lipid rafts, which are utilized by numerous other viruses for assembly. Virion morphogenesis ultimately requires colocalization of viral components, yet our dual-label immunogold staining studies failed to reveal a spatial association of Z with GPC microdomains. This observation may reflect either rapid Z-dependent budding of virus-like particles upon coassociation or a requirement for additional viral components in the assembly process. Together, these results provide new insight into the molecular basis for arenavirus morphogenesis.