The host innate immune response to viral infections often involves the activation of parallel pattern recognition receptor (PRR) pathways that converge on the induction of type I interferons (IFNs). Several viruses have evolved sophisticated mechanisms to attenuate antiviral host signaling by directly interfering with the activation and/or downstream signaling events associated with PRR signal propagation. Here we show that the 3Cpro cysteine protease of coxsackievirus B3 (CVB3) cleaves the innate immune adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF) as a mechanism to escape host immunity. We found that MAVS and TRIF were cleaved in CVB3-infected cells in culture. CVB3-induced cleavage of MAVS and TRIF required the cysteine protease activity of 3Cpro, occurred at specific sites and within specialized domains of each molecule, and inhibited both the type I IFN and apoptotic signaling downstream of these adaptors. 3Cpro-mediated MAVS cleavage occurred within its proline-rich region, led to its relocalization from the mitochondrial membrane, and ablated its downstream signaling. We further show that 3Cpro cleaves both the N- and C-terminal domains of TRIF and localizes with TRIF to signalosome complexes within the cytoplasm. Taken together, these data show that CVB3 has evolved a mechanism to suppress host antiviral signal propagation by directly cleaving two key adaptor molecules associated with innate immune recognition.

In previous studies, we observed that the virulent avian influenza A virus A/Turkey/Ontario/7732/66 (Ty/Ont) induced severe lymphoid depletion in vivo and rapidly killed an avian lymphocyte cell line (RP9) in vitro. In examining the mechanism of cell killing by this virus, we found that Ty/Ont induced fragmentation of the RP9 cellular DNA into a 200-bp ladder and caused ultrastructural changes characteristic of apoptotic cell death by 5 h after infection. We next determined that the ability to induce apoptosis was not unique to Ty/Ont. In fact, a variety of influenza A viruses (avian, equine, swine, and human), as well as human influenza B viruses, induced DNA fragmentation in a permissive mammalian cell line, Madin-Darby canine kidney (MDCK), and this correlated with the development of a cytopathic effect during viral infection. Since the proto-oncogene bcl-2 is a known inhibitor of apoptosis, we transfected MDCK cells with the human bcl-2 gene; these stably transfected cells (MDCKbcl-2) did not undergo DNA fragmentation after virus infection. In addition, cytotoxicity assays at 48 to 72 h after virus infection showed a high level of cell viability for MDCKbcl-2 compared with a markedly lower level of viability for MDCK cells. These studies indicate that influenza A and B viruses induce apoptosis in cell cultures; thus, apoptosis may represent a general mechanism of cell death in hosts infected with influenza viruses.

The 65 serotypes of human enteroviruses are classified into four species, Human enterovirus (HEV) A to D, based largely on phylogenetic relationships in multiple genome regions. The 39-non-translated region of enteroviruses is highly conserved within a species but highly divergent between species. From this information, species-specific RT-PCR primers were developed that can be used to rapidly screen collections of enterovirus isolates to identify species of interest. The four primer pairs were 100 % specific when tested against enterovirus prototype strains and panels of isolates of known serotype (a total of 193 isolates). For evaluation in a typical application, the species-specific primers were used to screen 186 previously uncharacterized non-polio enterovirus isolates. The HEV-B primers amplified 68?3 % of isolates, while the HEV-A and HEV-C primers accounted for 9?7 and 11?3 % of isolates, respectively; no isolates were amplified with the HEV-D primers. Twelve isolates (6?5 %) were amplified by more than one primer set and eight isolates (4?3 %) were not amplified by any of the four primer pairs. Serotypes were identified by partial sequencing of the VP1 capsid gene, and in every case sequencing confirmed that the species-specific PCR result was correct; the isolates that were amplified by more than one species-specific primer pair were mixtures of two (11 isolates) or three (one isolate) species of viruses. The eight isolates that were not amplified by the species-specific primers comprised four new serotypes (EV76, EV89, EV90 and EV91) that appear to be unique members of HEV-A based on VP1, 3D and 39-non-translated region sequences. INTRODUCTIONThe 65 human enterovirus serotypes (family Picornaviridae) are classified into four species, Human enterovirus (HEV)-A, HEV-B, HEV-C and HEV-D, largely on the basis of phylogenetic analysis of multiple genome regions (King et al., 2000). Analysis of the complete genome sequences of all the prototype strains has shown that the species remain phylogenetically coherent in all genome regions except in the 59-non-translated region (NTR) (Brown et al., 2003; Oberste et al., 2004a, d). Seven of the 13 simian enterovirus prototype strains are provisional members of two HEV species, with six strains in HEV-A and one in HEV-B (Oberste et al., 2002). The remaining six strains are proposed to define three new species in the genus Enterovirus (Oberste et al., 2002), while the bovine and porcine enteroviruses comprise two additional species (King et al., 2000). Recombination, a major mechanism of enterovirus evolution, has been observed to occur in nature only among members of the same species, except in the 59-NTR (Brown et al., 2003; Lindberg et al., 2003;Lukashev et al., 2003 Lukashev et al., , 2004 Oberste et al., 2004a, d, e;Oprisan et al., 2002). Species identity therefore appears to have some biological significance integral to virus survival.Genus-specific primers are widely used for the amplification of enteroviruses from clinical specimens by RT-PCR (Rotbart & Ro...

The importance of influenza viruses as worldwide pathogens in humans, domestic animals, and poultry is well recognized. Discerning how influenza viruses interact with the host at a cellular level is crucial for a better understanding of viral pathogenesis. Influenza viruses induce apoptosis through mechanisms involving the interplay of cellular and viral factors that may depend on the cell type. However, it is unclear which viral genes induce apoptosis. In these studies, we show that the expression of the nonstructural (NS) gene of influenza A virus is sufficient to induce apoptosis in MDCK and HeLa cells. Further studies showed that the multimerization domain of the NS1 protein but not the effector domain is required for apoptosis. However, this mutation is not sufficient to inhibit apoptosis using whole virus.