The Phylogeography and Spatiotemporal Spread of South-Central Skunk Rabies Virus

The south-central skunk rabies virus (SCSK) is the most broadly distributed terrestrial viral lineage in North America. Skunk rabies has not been efficiently targeted by oral vaccination campaigns and represents a natural system of pathogen invasion, yielding insights to rabies emergence. In the present study we reconstructed spatiotemporal spread of SCSK in the whole territory of its circulation using a combination of Bayesian methods. The analysis based on 241 glycoprotein gene sequences demonstrated that SCSK is much more divergent phylogenetically than was appreciated previously. According to our analyses the SCSK originated in the territory of Texas ~170 years ago, and spread geographically during the following decades. The wavefront velocity in the northward direction was significantly greater than in the eastward and westward directions. Rivers (except the Mississippi River and Rio Grande River) did not constitute significant barriers for epizootic spread, in contrast to deserts and mountains. The mean dispersal rate of skunk rabies was lower than that of the raccoon and fox rabies. Viral lineages circulate in their areas with limited evidence of geographic spread during decades. However, spatiotemporal reconstruction shows that after a long period of stability the dispersal rate and wavefront velocity of SCSK are increasing. Our results indicate that there is a need to develop control measures for SCSK, and suggest how such measure can be implemented most efficiently. Our approach can be extrapolated to other rabies reservoirs and used as a tool for investigation of epizootic patterns and planning interventions towards disease elimination.     

Production of a Dominant-Negative Fragment Due to G3BP1 Cleavage Contributes to the Disruption of Mitochondria-Associated Protective Stress Granules during CVB3 Infection

Stress granules (SGs) are dynamic cytosolic aggregates containing messenger ribonucleoproteins and target poly-adenylated (A)-mRNA. A key component of SGs is Ras-GAP SH3 domain binding protein-1 (G3BP1), which in part mediates protein-protein and protein-RNA interactions. SGs are modulated during infection by several viruses, however, the function and significance of this process remains poorly understood. In this study, we investigated the interplay between SGs and Coxsackievirus type B3 (CVB3), a member of the Picornaviridae family. Our studies demonstrated that SGs were formed early during CVB3 infection; however, G3BP1-positive SGs were actively disassembled at 5 hrs post-infection, while poly(A)-positive RNA granules persisted. Furthermore, we confirmed G3BP1 cleavage by 3C^pro at Q325. We also demonstrated that overexpression of G3BP1-SGs negatively impacted viral replication at the RNA, protein, and viral progeny levels. Using electron microscopy techniques, we showed that G3BP1-positive SGs localized near mitochondrial surfaces. Finally, we provided evidence that the C-terminal cleavage product of G3BP1 inhibited SG formation and promoted CVB3 replication. Taken together, we conclude that CVB3 infection selectively targets G3BP1-SGs by cleaving G3BP1 to produce a dominant-negative fragment that further inhibits G3BP1-SG formation and facilitates viral replication.     

Kinetic Characterization of 100 Glycoside Hydrolase Mutants Enables the Discovery of Structural Features Correlated with Kinetic Constants

The use of computational modeling algorithms to guide the design of novel enzyme catalysts is a rapidly growing field. Force-field based methods have now been used to engineer both enzyme specificity and activity. However, the proportion of designed mutants with the intended function is often less than ten percent. One potential reason for this is that current force-field based approaches are trained on indirect measures of function rather than direct correlation to experimentally-determined functional effects of mutations. We hypothesize that this is partially due to the lack of data sets for which a large panel of enzyme variants has been produced, purified, and kinetically characterized. Here we report the k_cat and K_M values of 100 purified mutants of a glycoside hydrolase enzyme. We demonstrate the utility of this data set by using machine learning to train a new algorithm that enables prediction of each kinetic parameter based on readily-modeled structural features. The generated dataset and analyses carried out in this study not only provide insight into how this enzyme functions, they also provide a clear path forward for the improvement of computational enzyme redesign algorithms.     

Transstadial immune activation in a mosquito: Adults that emerge from infected larvae have stronger antibacterial activity in their hemocoel yet increased susceptibility to malaria infection

Larval and adult mosquitoes mount immune responses against pathogens that invade their hemocoel. Although it has been suggested that a correlation exists between immune processes across insect life stages, the influence that an infection in the hemocoel of a larva has on the immune system of the eclosed adult remains unknown. Here, we used Anopheles gambiae to test whether a larval infection influences the adult response to a subsequent bacterial or malaria parasite infection. We found that for both female and male mosquitoes, a larval infection enhances the efficiency of bacterial clearance following a secondary infection in the hemocoel of adults. The adults that emerge from infected larvae have more hemocytes than adults that emerge from naive or injured larvae, and individual hemocytes have greater phagocytic activity. Furthermore, mRNA abundance of immune genes—such as cecropin A, Lysozyme C1, Stat‐A, and Tep1—is higher in adults that emerge from infected larvae. A larval infection, however, does not have a meaningful effect on the probability that female adults will survive a systemic bacterial infection, and increases the susceptibility of females to Plasmodium yoelii, as measured by oocyst prevalence and intensity in the midgut. Finally, immune proficiency varies by sex; females exhibit increased bacterial killing, have twice as many hemocytes, and more highly express immune genes. Together, these results show that a larval hemocoelic infection induces transstadial immune activation—possibly via transstadial immune priming—but that it confers both costs and benefits to the emerged adults.     

The analgesic tropane analogue (+/-)-SM 21 has a high affinity for sigma2 receptors

The analgesic properties of the tropane analogue (+/-)-SM 21 have been attributed to the antagonism of presynaptic m2 receptors resulting in a potentiation of acetylcholine release. However, drugs targeting a number of other neurotransmitter receptors have been shown to enhance acetylcholine release. In the current study, in vitro studies were conducted in order to determine the affinity of (+/-)-SM 21 for serotonin 5-HT3, 5-HT4, and sigma receptors. Our results indicate that (+/-)-SM 21, and its structural congeners, have a relatively high affinity for sigma2 receptors relative to their reported affinity for muscarinic receptors. The higher affinity for sigma2 versus sigma1 receptors indicates that (+/-)-SM 21 may be a suitable lead compound for developing sigma2-selective ligands.     

Effects of transforming growth factor-alpha (TGF-alpha) in vitro and in vivo on reovirus replication

We have utilized growth factors in in vitro and in vivo systems to examine the role of cellular proliferation in reovirus replication. In vitro, proliferating RIE-1 cells can be infected with whole reovirus virions, but are relatively resistant to infection once confluent (Go arrest). It has been shown that TGF-alpha, which signals through the EGF-receptor (EGF-R), is capable of dramatically increasing the number of RIE-1 cells entering the S-phase in the presence of additional serum factors. Stimulation of the EGF-R without serum results in minimal increases in cells entering the S-phase with a restriction in reovirus replication. Therefore, other factors in serum are essential for fully permissive infection. In vivo, we used metallothionein (MT) promoter/enhancer-TGF-alpha transgenic mice to study the effect of cytokine activation on reovirus type 1 infection. Virus replication decreased following oral infection in these transgenic mice at 1 month of age, concordant with increased mucin production. Titers of reovirus obtained from the livers of 1 year old transgenic mice were approximately 10-fold higher than titers obtained in control mice. Taken together, these data indicate that while growth factor activation ultimately leads to an increase in virus infectivity, other factors may be necessary for reovirus replication.     

Linearization of a naturally occurring circular protein maintains structure but eliminates hemolytic activity

Cyclotides are a recently discovered family of disulfide rich proteins from plants that contain a circular protein backbone. They are exceptionally stable, as exemplified by their use in native medicine of the prototypic cyclotide kalata B1. The peptide retains uterotonic activity after the plant from which it is derived is boiled to make a medicinal tea. The circular backbone is thought to be in part responsible for the stability of the cyclotides, and to investigate its role in determining structure and biological activity, an acyclic derivative, des-(24-28)-kalata B1, was chemically synthesized and purified. This derivative has five residues removed from the 29-amino acid circular backbone of kalata B1 in a loop region corresponding to a processing site in the biosynthetic precursor protein. Two-dimensional NMR spectra of the peptide were recorded, assigned, and used to identify a series of distance, angle, and hydrogen bonding restraints. These were in turn used to determine a representative family of solution structures. Of particular interest was a determination of the structural similarities and differences between des-(24-28)-kalata B1 and native kalata B1. Although the overall three-dimensional fold remains very similar to that of the native circular protein, removal of residues 24-28 of kalata B1 causes disruption of some structural features that are important to the overall stability. Furthermore, loss of hemolytic activity is associated with backbone truncation and linearization.     

The CD45 locus in cattle: allelic polymorphism and evidence for exceptional positive natural selection

Cattle in Africa are a genetically diverse population that has resulted from successive introduction of Asian Bos indicus and European B. taurus cattle. However, analysis of mitochondrial genetic diversity in African cattle identified three lineages, one associated with Asian B. indicus, one with European B. taurus, and a third ascribed to an indigenous African sub-species of cattle. Due to their extended coevolution, indigenous African herbivores are generally tolerant to endemic African pathogens. We are interested in identifying alleles derived from the indigenous African cattle that may be associated with tolerance to African pathogens. An analysis of the locus which encodes the abundant plasma membrane-associated tyrosine phosphatase, CD45, identified three highly divergent allelic families in Kenya Boran cattle. Analysis of allelic distribution in a diverse range of cattle populations suggests a European B. taurus, an Asian B. indicus, and an African origin. This demonstrates not only significant allelic polymorphism at the CD45 locus in cattle but also convincing autosomal evidence for a distinct African sub-species of cattle. Furthermore, maximum-likelihood analysis of selection pressures revealed that the CD45 locus is subject to exceptionally strong natural selection which we suggest may be pathogen driven.