Anti-CDV monoclonal antibody, clone 95J3

Universal Verification Methodology (UVM) is a standardized approach of verifying integrated circuit designs, targeting a Coverage-Driven Verification (CDV). It combines automatic test generation, self-checking testbenches, and coverage metrics to indicate progress in the design verification. The flow of the CDV differs from the traditional directed-testing approach. With the CDV, a testbench developer, by setting the verification goals, starts with an structured plan. Those goals are targeted further by a developed testbench, which generates legal stimuli and sends them to a device under test (DUT). The progress is measured by coverage monitors added to the simulation environment. In this way, the non-exercised functionality can be identified. Moreover, the additional scoreboards indicate undesired DUT behaviour. Such verification environments were developed for three recent ASIC and FPGA projects which have successfully implemented the new work-flow: (1) the CLICpix2 65 nm CMOS hybrid pixel readout ASIC design; (2) the C3PD 180 nm HV-CMOS active sensor ASIC design; (3) the FPGA-based DAQ system of the CLICpix chip. This paper, based on the experience from the above projects, introduces briefly UVM and presents a set of tips and advices applicable at different stages of the verification process-cycle.

The paramyxovirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxovirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the Morbillivirus genus, a series of measles virus (MeV) and canine distemper virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono-and polycistronic minireplicon assays and supported efficient replication of recombinant viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbillivirus disease. The recombinant viruses displayed different stages of attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual virus attenuation for next-generation paramyxovirus vaccine design. IMPORTANCE Investigating the role of the paramyxovirus N protein tail domain (Ntail) in virus replication, we demonstrated in this study that the structurally disordered central Ntail region is a determinant for viral pathogenesis. We show that internal deletions in this Ntail region of up to 55 amino acids in length are compatible with efficient replication of recombinant viruses in cell culture but result in gradual viral attenuation in a lethal canine distemper virus (CDV)/ferret model. Mechanistically, we demonstrate a role of the intact Ntail region in the regulation of viral transcriptase activity. Recombinant viruses with Ntail truncations induce protective immunity against lethal challenge of ferrets with pathogenic CDV. This identification of the unstructured central Ntail domain as a nonessential paramyxovirus pathogenesis factor establishes a foundation for harnessing Ntail truncations for vaccine engineering against emerging and reemerging members of the paramyxovirus family.