An improved, rapid competitive ELISA using a novel conserved 3B epitope for the detection of serum antibodies to foot-and-mouth disease virus.

The highly contagious foot-and-mouth disease virus (FMDV) afflicts cloven-hoofed animals, resulting in significant costs because of loss of trade and recovery from disease. We developed a sensitive, specific, and rapid competitive ELISA (cELISA) to detect serum antibodies to FMDV. The cELISA utilized a monoclonal blocking antibody specific for a highly conserved FMDV nonstructural 3B epitope, a recombinant mutant FMDV 3ABC coating protein, and optimized format variables including serum incubation for 90 min at 20-25°C. Samples from 16 animals experimentally infected with one FMDV serotype (A, O, Asia, or SAT-1) demonstrated early detection capacity beginning 7 d post-inoculation. All samples from 55 vesicular stomatitis virus antibody-positive cattle and 44 samples from cloven-hoofed animals affected by non-FMD vesicular diseases were negative in the cELISA, demonstrating 100% analytical specificity. The diagnostic sensitivity was 100% against sera from 128 cattle infected with isolates of all FMDV serotypes, emphasizing serotype-agnostic results. Diagnostic specificities of U.S. cattle ( n = 1135) and swine ( n = 207) sera were 99.4% and 100%, respectively. High repeatability and reproducibility were demonstrated with 3.1% coefficient of variation in percent inhibition data and 100% agreement using 2 kit lots and 400 negative control serum samples, with no difference between bench and biosafety cabinet operation. Negative results from vaccinated, uninfected cattle, pig, and sheep sera confirmed the DIVA (differentiate infected from vaccinated animals) capability. This rapid (<3 h), select agent-free assay with high sensitivity and specificity, DIVA capability, and room temperature processing capability will serve as a useful tool in FMDV surveillance, emergency preparedness, response, and outbreak recovery programs.

Susceptibility of murine norovirus and hepatitis A virus to electron beam irradiation in oysters and quantifying the reduction in potential infection risks.

Consumption of raw oysters is an exposure route for human norovirus (NoV) and hepatitis A virus (HAV). Therefore, efficient postharvest oyster treatment technology is needed to reduce public health risks. This study evaluated the inactivation of HAV and the NoV research surrogate, murine norovirus-1 (MNV-1), in oysters (Crassostrea virginica) by electron beam (E-beam) irradiation. The reduction of potential infection risks was quantified for E-beam irradiation technology employed on raw oysters at various virus contamination levels. The E-beam dose required to reduce the MNV and HAV titer by 90% (D(10) value) in whole oysters was 4.05 (standard deviations [SD], ±0.63) and 4.83 (SD, ±0.08) kGy, respectively. Microbial risk assessment suggests that if a typical serving of 12 raw oysters was contaminated with 10(5) PFU, a 5-kGy treatment would achieve a 12% reduction (from 4.49 out of 10 persons to 3.95 out of 10 persons) in NoV infection and a 16% reduction (from 9.21 out of 10 persons to 7.76 out of 10 persons) in HAV infections. If the serving size contained only 10(2) PFU of viruses, a 5-kGy treatment would achieve a 26% reduction (2.74 out of 10 persons to 2.03 out of 10 persons) of NoV and 91% reduction (2.1 out of 10 persons to 1.93 out of 100 persons) of HAV infection risks. This study shows that although E-beam processing cannot completely eliminate the risk of viral illness, infection risks can be reduced.

Discrimination between infectious and non-infectious human norovirus using porcine gastric mucin.

Human noroviruses (NoVs) are known to bind to human histo-blood group antigens, as well as to chemically-similar porcine gastric mucins. Here, the binding ability of NoV to porcine mucin is shown to be substantially deficient after UV, thermal, and high pressure treatments. Using qRT-PCR, ≥ 68% of GI.1 NoV (Norwalk strain) bound to porcine gastric mucin-conjugated magnetic beads (PGM-MBs). Application of 600-MPa high pressure treatments reduced binding of the virus to PGM-MBs by 4.7-log10, as determined by qRT-PCR, while a 300-MPa pressure treatment, reduced binding to PGM-MBs by only 0.45-log10. This is consistent with a previously reported clinical trial (Leon et al., 2011. Appl. Environ Microbiol. 77:5476-5482.) which demonstrated inactivation of 4-log10 of GI.1 NoV at 600-MPa. After thermal treatment, binding to PGM-MBs decreased when samples were heated from 0 to 80 °C. Ultraviolet treatments of 0.5 and 2 J/cm² reduced observed PGM-MB binding of norovirus to 33% and negligible levels, respectively, from an initially observed 84% binding for untreated NoV. Although thermal and UV treatments are generally recognized to inactivate viruses, verification of NoV inactivation by these treatments may require volunteer studies. In total, these results suggest the loss of NoV binding to porcine mucin as a potential means to preferentially exclude non-infectious virus particles from subsequent RT-PCR detection.

Hemocytes are sites of enteric virus persistence within oysters.

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.

Assembly of arenavirus envelope glycoprotein GPC in detergent-soluble membrane microdomains.

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.

Vesicular stomatitis virus matrix protein mutations that affect association with host membranes and viral nucleocapsids.

Viral matrix (M) proteins bind the nucleoprotein core (nucleocapsid) to host membranes during the process of virus assembly by budding. Previous studies using truncated M proteins had implicated the N-terminal 50 amino acids of the vesicular stomatitis virus M protein in binding both membranes and nucleocapsids and a sequence from amino acids 75-106 as an additional membrane binding region. Structure-based mutations were introduced into these two regions, and their effects on membrane association and incorporation into nucleocapsid-M protein complexes were determined using quantitative assays. The results confirmed that the N terminus of M protein is involved in association with plasma membranes as well as nucleocapsids, although these two activities were differentially affected by individual mutations. Mutations in the 75-106 region affected incorporation into nucleocapsid-M complexes but had only minor effects on association with membranes. The ability of site-specific mutant M proteins to complement growth of temperature-sensitive M mutant virus did not correlate well with the ability to associate with membranes or nucleocapsids, suggesting that complementation involves an additional activity of M protein. Mutants with similar abilities to associate with membranes and nucleocapsids but differing in complementation activity were incorporated into infectious cDNA clones. Infectious virus was repeatedly recovered containing mutant M proteins capable of complementation but was never recovered with mutant M proteins that lacked complementation activity, providing further evidence for a separate activity of M protein that is essential for virus replication.

Gangliosides inhibit flagellin signaling in the absence of an effect on flagellin binding to toll-like receptor 5.

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.