Feline Calicivirus Can Tolerate Gross Changes of Its Minor Capsid Protein Expression Levels Induced by Changing Translation Reinitiation Frequency or Use of a Separate VP2-Coding mRNA

Caliciviruses use reinitiation of translation governed by a ‘termination upstream ribosomal binding site’ (TURBS) for expression of their minor capsid protein VP2. Mutation analysis allowed to identify sequences surrounding the translational start/stop site of the feline calicivirus (FCV) that fine tune reinitiation frequency. A selection of these changes was introduced into the infectious FCV cDNA clone to check the influence of altered VP2 levels on virus replication. In addition, full length constructs were established that displayed a conformation, in which VP2 expression occurred under control of a duplicated subgenomic promoter. Viable viruses recovered from such constructs revealed a rather broad range of VP2 expression levels but comparable growth kinetics showing that caliciviruses can tolerate gross changes of the VP2 expression level.     

In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

Small recombinant antibody fragments (e.g. scFvs and V_HHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)₂ antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama V_HHs were isolated from an immune V_HH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity V_HH (C2) was fused to a human Fc fragment to create a V_HH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our V_HH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity V_HHs (C2 and C20) and the V_HH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx V_HHs and V_HH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy.     

Genome‐wide association study identifies loci associated with liability to alcohol and drug dependence that is associated with variability in reward‐related ventral striatum activity in African‐ and European‐Americans

Genetic influences on alcohol and drug dependence partially overlap, however, specific loci underlying this overlap remain unclear. We conducted a genome‐wide association study (GWAS) of a phenotype representing alcohol or illicit drug dependence (ANYDEP) among 7291 European‐Americans (EA; 2927 cases) and 3132 African‐Americans (AA: 1315 cases) participating in the family‐based Collaborative Study on the Genetics of Alcoholism. ANYDEP was heritable (h ² in EA = 0.60, AA = 0.37). The AA GWAS identified three regions with genome‐wide significant (GWS; P < 5E‐08) single nucleotide polymorphisms (SNPs) on chromosomes 3 (rs34066662, rs58801820) and 13 (rs75168521, rs78886294), and an insertion‐deletion on chromosome 5 (chr5:141988181). No polymorphisms reached GWS in the EA. One GWS region (chromosome 1: rs1890881) emerged from a trans‐ancestral meta‐analysis (EA + AA) of ANYDEP, and was attributable to alcohol dependence in both samples. Four genes (AA: CRKL, DZIP3, SBK3; EA: P2RX6) and four sets of genes were significantly enriched within biological pathways for hemostasis and signal transduction. GWS signals did not replicate in two independent samples but there was weak evidence for association between rs1890881 and alcohol intake in the UK Biobank. Among 118 AA and 481 EA individuals from the Duke Neurogenetics Study, rs75168521 and rs1890881 genotypes were associated with variability in reward‐related ventral striatum activation. This study identified novel loci for substance dependence and provides preliminary evidence that these variants are also associated with individual differences in neural reward reactivity. Gene discovery efforts in non‐European samples with distinct patterns of substance use may lead to the identification of novel ancestry‐specific genetic markers of risk.     

Standing geographic variation in eclosion time and the genomics of host race formation in Rhagoletis pomonella fruit flies

Taxa harboring high levels of standing variation may be more likely to adapt to rapid environmental shifts and experience ecological speciation. Here, we characterize geographic and host‐related differentiation for 10,241 single nucleotide polymorphisms in Rhagoletis pomonella fruit flies to infer whether standing genetic variation in adult eclosion time in the ancestral hawthorn (Crataegus spp.)‐infesting host race, as opposed to new mutations, contributed substantially to its recent shift to earlier fruiting apple (Malus domestica). Allele frequency differences associated with early vs. late eclosion time within each host race were significantly related to geographic genetic variation and host race differentiation across four sites, arrayed from north to south along a 430‐km transect, where the host races co‐occur in sympatry in the Midwest United States. Host fruiting phenology is clinal, with both apple and hawthorn trees fruiting earlier in the North and later in the South. Thus, we expected alleles associated with earlier eclosion to be at higher frequencies in northern populations. This pattern was observed in the hawthorn race across all four populations; however, allele frequency patterns in the apple race were more complex. Despite the generally earlier eclosion timing of apple flies and corresponding apple fruiting phenology, alleles on chromosomes 2 and 3 associated with earlier emergence were paradoxically at lower frequency in the apple than hawthorn host race across all four sympatric sites. However, loci on chromosome 1 did show higher frequencies of early eclosion‐associated alleles in the apple than hawthorn host race at the two southern sites, potentially accounting for their earlier eclosion phenotype. Thus, although extensive clinal genetic variation in the ancestral hawthorn race exists and contributed to the host shift to apple, further study is needed to resolve details of how this standing variation was selected to generate earlier eclosing apple fly populations in the North.