Determination of Critical Requirements for Classical Swine Fever Virus NS2-3-Independent Virion Formation.

For members of the Flaviviridae, it is known that, besides the structural proteins, nonstructural (NS) proteins also play a critical role in virion formation. Pestiviruses, such as bovine viral diarrhea virus (BVDV), rely on uncleaved NS2-3 for virion formation, while its cleavage product, NS3, is selectively active in RNA replication. This dogma was recently challenged by the selection of gain-of-function mutations in NS2 and NS3 which allowed virion formation in the absence of uncleaved NS2-3 in BVDV type 1 (BVDV-1) variants encoding either a ubiquitin (Ubi) (NS2-Ubi-NS3) or an internal ribosome entry site (IRES) (NS2-IRES-NS3) between NS2 and NS3. To determine whether the ability to adapt to NS2-3-independent virion morphogenesis is conserved among pestiviruses, we studied the corresponding NS2 and NS3 mutations (2/T444-V and 3/M132-A) in classical swine fever virus (CSFV). We observed that these mutations were capable of restoring low-level NS2-3-independent virion formation only for CSFV NS2-Ubi-NS3. Interestingly, a second NS2 mutation (V439-D), identified by selection, was essential for high-titer virion production. Similar to previous findings for BVDV-1, these mutations in NS2 and NS3 allowed for low-titer virion production only in CSFV NS2-IRES-NS3. For efficient virion morphogenesis, additional exchanges in NS4A (A48-T) and NS5B (D280-G) were required, indicating that these proteins cooperate in NS2-3-independent virion formation. Interestingly, both NS5B mutations, selected independently for NS2-IRES-NS3 variants of BVDV-1 and CSFV, are located in the fingertip region of the viral RNA-dependent RNA polymerase, classifying this structural element as a novel determinant for pestiviral NS2-3-independent virion formation. Together, these findings will stimulate further mechanistic studies on the genome packaging of pestiviruses.IMPORTANCE For Flaviviridae members, the nonstructural proteins are essential for virion formation and thus exert a dual role in RNA replication and virion morphogenesis. However, it remains unclear how these proteins are functionalized for either process. In wild-type pestiviruses, the NS3/4A complex is selectively active in RNA replication, while NS2-3/4A is essential for virion formation. Mutations recently identified in BVDV-1 rendered NS3/4A capable of supporting NS2-3-independent virion morphogenesis. A comparison of NS3/4A complexes incapable/capable of supporting virion morphogenesis revealed that changes in NS3/NS4A surface interactions are decisive for the gain of function. However, so far, the role of the NS2 mutations as well as the accessory mutations additionally required in the NS2-IRES-NS3 virus variant has not been clarified. To unravel the course of genome packaging, the additional sets of mutations obtained for a second pestivirus species (CSFV) are of significant importance to develop mechanistic models for this complex process.

Lifespan map creation enhances stream restoration design.

Research and engineering efforts are establishing a vast number of stream restoration planning approaches, design testing frameworks, construction techniques, and performance evaluation methods. A primary question arises as to the lifespan of stream restoration features. This study develops a framework to identify relevant parameters, design criteria and survival thresholds for ten multidisciplinary restoration techniques: •Parameterize relevant features, notably, (1) bar and floodplain grading; (2) berm setback; (3) vegetation plantings; (4) riprap placement; (5) sediment replenishment; (6) side cavities; (7) side channel and anabranches; (8) streambed reshaping; (9) structure removal; and (10) placement of wood in the shape of engineered logjams and rootstocks.•Identify survival thresholds for parameters, where the feature life ends when the threshold value is exceeded.•Compare parameter thresholds with spatial data of topographic change and hydrodynamic forces as a result of hydrodynamic modelling of multiple discharges. The discharge or topographic change rate that is related to the lowest (flood) return period spatially determines the feature's lifespan in years.

Hydro-morphological parameters generate lifespan maps for stream restoration management.

Anthropogenic, eco-morphological degradation of lotic waters necessitates laws, directives, and voluntary actions involving stream restoration and habitat enhancement. Research and engineering efforts are establishing a vast number of stream restoration planning approaches, design testing frameworks, construction techniques, and performance evaluation methods. As the practice of restoration scales up from an individual action at a single site to sequences of actions at many sites in a long river segment, a primary question arises as to the lifespan of such a sequence. This study develops a new framework to identify relevant parameters, design criteria and survival thresholds for ten multidisciplinary restoration techniques, adequate for site-scale to segment-scale application, in a comprehensive review: (1) bar and floodplain grading; (2) berm setback; (3) vegetation plantings; (4) riprap placement; (5) sediment replenishment; (6) side cavities; (7) side channel and anabranches; (8) streambed reshaping; (9) structure removal; and (10) placement of wood in the shape of engineered logjams and rootstocks. Survival thresholds are applied to a sequence of proposed habitat enhancement features for the lower Yuba River in California, USA. Spatially explicit hydraulic and sediment data, together with numerical model predictions of the measures, were vetted against the survival thresholds to produce discharge-dependent lifespan maps. Discharges related to specific flood-return periods enabled probabilistic estimates of the longevity of particular design features. Thus, the lifespan maps indicate the temporal stability of particular stream restoration and habitat enhancement features and techniques. Areas with particularly low or high lifespans help planners optimise the design and positioning of restoration features.

Pestivirus virion morphogenesis in the absence of uncleaved nonstructural protein 2-3.

The family Flaviviridae contains three genera of positive-strand RNA viruses, namely, Flavivirus, Hepacivirus (e.g., hepatitis C virus [HCV]), and Pestivirus. Pestiviruses, like bovine viral diarrhea virus (BVDV), bear a striking degree of similarity to HCV concerning polyprotein organization, processing, and function. Along this line, in both systems, release of nonstructural protein 3 (NS3) is essential for viral RNA replication. However, both viruses differ significantly with respect to processing efficiency at the NS2/3 cleavage site and abundance as well as functional relevance of uncleaved NS2-3. In BVDV-infected cells, significant amounts of NS2-3 accumulate at late time points postinfection and play an essential but ill-defined role in the production of infectious virions. In contrast, complete cleavage of the HCV NS2-3 counterpart has been reported, and unprocessed NS2-3 is not required throughout the life cycle of HCV, at least in cell culture. Here we describe the selection and characterization of the first pestiviral genome with the capability to complete productive infection in the absence of uncleaved NS2-3. Despite the insertion of a ubiquitin gene or an internal ribosomal entry site between the NS2 and NS3 coding sequences, the selected chimeric BVDV-1 genomes gave rise to infectious virus progeny. In this context, a mutation in the N-terminal third of NS2 was identified as a critical determinant for efficient production of infectious virions in the absence of uncleaved NS2-3. These findings challenge a previously accepted dogma for pestivirus replication and provide new implications for virion morphogenesis of pestiviruses and HCV.