In vitro surrogate models to aid in the development of antivirals for the containment of foot-and-mouth disease outbreaks.

Foot-and-mouth disease virus (FMDV) is a highly pathogenic member of the genus Aphthovirus (family Picornaviridae) that is only to be manipulated in high-containment facilities, thus complicating research on and discovery of antiviral strategies against the virus. Bovine rhinitis B virus (BRBV) and equine rhinitis A virus (ERAV), phylogenetically most closely related to FMDV, were explored as surrogates for FMDV in antiviral studies. Although no efficient cell culture system has been reported so far for BRBV, we demonstrate that infection of primary bovine kidney cells resulted in an extensive but rather poorly-reproducible induction of cytopathic effect (CPE). Madin-Darby bovine kidney cells on the other hand supported viral replication in the absence of CPE. Antiviral tests were developed for ERAV in Vero A cells employing a viral RNA-reduction assay and CPE-reduction assay; the latter having a Z' factor of 0.83±0.07. The BRBV and ERAV models were next used to assess the anti-aphthovirus activity of two broad-spectrum antiviral agents 2'-C-methylcytidine (2CMC) and ribavirin, as well as of the enterovirus-specific inhibitor enviroxime. The effects of the three compounds in the CPE-reduction (ERAV) and viral RNA-reduction assays (BRBV and ERAV) were comparable. Akin to 2CMC, compound A, a recently-discovered non-nucleoside pan-serotype FMDV inhibitor, also inhibited the replication of both BRBV and ERAV, whereas enviroxime was devoid of activity. The BRBV and ERAV surrogate models reported here can be manipulated in BSL-2 laboratories and may facilitate studies to unravel the mechanism of action of novel FMDV inhibitors.

The MCV MC159 protein inhibits late, but not early, events of TNF-α-induced NF-κB activation.

Tumor necrosis factor (TNF-alpha) triggers biphasic activation of the NF-kappaB transcriptional regulator. This process consists of an initial, IkappaBalpha-mediated transient phase and a later, persistent phase dependent on IkappaBbeta degradation. To presumably interfere with the fulfillment of this immunity-associated event in cells infected with the molluscum contagiosum virus (MCV), this pathogen produces the intracellular MC159 protein. To define the mode of action of MC159, the impact of TNF-alpha on HEK 293T cells ectopically expressing the MC159 protein was examined. In this regard, TNF-alpha-induced expression of an NF-kappaB-regulated luciferase reporter gene was partially inhibited by the MC159 protein. This ability was attributed to blockage of the persistent phase of TNF-alpha-induced NF-kappaB activation for the following reasons: (1) the initial phase of NF-kappaB transcriptional activation was not affected by the MC159 protein; (2) the MC159 protein inhibited TNF-alpha-directed degradation of IkappaBbeta, but not IkappaBalpha; and (3) expression of the late NF-kappaB-regulated cell genes, TNF-alpha and CCL2, was decreased in the presence of the MC159 protein while transcription of the early NF-kappaB-regulated cell gene, CXCL1, was not altered. Previously reported MC159-RIP interactions appear to be irrelevant for the MC159 inhibitory function. In contrast, MC159-TRAF2 associations are more relevant for inhibitory function since mutant MC159 proteins unable to bind TRAF2 also cannot inhibit TNF-mediated NF-kappaB activation. In vivo, the MC159 protein may act to prolong virus survival by preventing the infected cell from responding to TNF-alpha, ultimately preventing the cellular production of proinflammatory and immunoattractant molecules.